Cyclic-3′,5′-Nucleotide Phosphodiesterase

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Kidney International, Vol. 55 (1999), pp. 29–62

PERSPECTIVES IN BASIC SCIENCE

Cyclic-3Ј,5Ј-nucleotide phosphodiesterase isozymes in cell biology and pathophysiology of the kidney

THOMAS P. DOUSA

Renal Pathophysiology Laboratory, Department of Physiology and Biophysics, Mayo Clinic and Foundation, Mayo Medical School, Rochester, Minnesota, USA

-Cyclic-3؅,5؅-nucleotide phosphodiesterase isozymes in cell biology and through intracellular mediation of a specific adenine nu pathophysiology of the kidney. Investigations of recent years revealed cleotide, cyclic-3Ј,5Ј-adenosine monophosphate (cAMP), that isozymes of cyclic-3Ј,5Ј-nucleotide phosphodiesterase (PDE) are a critically important component of the cyclic-3Ј,5Ј-adenosine mono- and the era of second messengers and intracellular sig- phosphate (cAMP) protein kinase A (PKA) signaling pathway. The naling was born. Shortly after the discovery of cAMP, an superfamily of cyclic-3Ј,5Ј-phosphodiesterase (PDE) isozymes consists analogous guanine nucleotide, cyclic-3Ј,5Ј-GMP (cGMP), of at least nine gene families (types): PDE1 to PDE9. Some PDE families are very diverse and consist of several subtypes and numerous was identified [2], and this nucleotide turned out to play PDE isoform-splice variants. PDE isozymes differ in molecular struc- a similar—albeit not identical—role in cellular signaling ture, catalytic properties, intracellular regulation and location, and sensitivity to selective inhibitors, as well as differential expression in as cAMP [3]. Since then, a number of other second mes- various cell types. A number of type-specific “second-generation” PDE sengers and related components of intracellular signaling inhibitors have been developed. Current evidence indicates that PDE pathways have been discovered. However, the intricate isozymes play a role in several pathobiologic processes in kidney cells. In rat mesangial cells, PDE3 and PDE4 compartmentalize cAMP sig- mechanisms by which cAMP and cGMP, the archetypal naling to the PDE3-linked cAMP-PKA pathway that modulates mito- second messengers, act in cellular signaling are far from genesis and PDE4-linked cAMP-PKA pathway that modulates genera- clarified, and novel, often surprising, aspects of cyclic tion of reactive oxygen species. Administration of selective PDE isozyme inhibitors in vivo suppresses proteinuria and pathologic nucleotide signaling functions are still being revealed. changes in experimental anti-Thy-1.1 mesangial proliferative glomeru- The cellular content and, by extension, biological ef- lonephritis in rats. Increased activity of PDE5 (and perhaps also PDE9) fects of cAMP or cGMP are, in principle, defined by in glomeruli and in cells of collecting ducts in sodium-retaining states, such as nephrotic syndrome, accounts for renal resistance to atriopep- the balance between activities of synthesizing enzyme tin; diminished ability to excrete sodium can be corrected by adminis- cyclases, adenylate cyclase and guanylate cyclase, respec- tration of the selective PDE5 inhibitor zaprinast. Anomalously high tively, and catabolizing enzymes, the cyclic 3Ј,5Ј-nucleo- PDE4 activity in collecting ducts is a basis of unresponsiveness to vasopressin in mice with hereditary nephrogenic diabetes insipidus. tide phosphodiesterases (PDE; EC 3.1.4.17) that hy- Apparently, PDE isozymes apparently also play an important role in drolyze the 3Ј-phosphoester bond of cAMP and cGMP the pathogenesis of acute renal failure of different origins. Administra- to their respective biologically inactive noncyclic nucleo- tion of PDE isozyme–selective inhibitors suppresses some components of immune responses to allograft transplant and improves preservation tides 5Ј-AMP and 5Ј-GMP (Fig. 1). and survival of transplanted organ. PDE isozymes are a target for The question of how cAMP is synthesized by cyclases action of numerous novel selective PDE inhibitors, which are key and how this biosynthesis is regulated by hormonal components in the design of novel “signal transduction” pharmacotherapies of kidney diseases. agents attracted immediate and enduring attention and has been extensively studied by many investigators. On the other hand, although the existence of cyclic nucleo- More than 35 years ago, Robison, Butcher and Suther- tide PDE activities was discovered soon after the discov- land [1] discovered that hormones, autocoids, and neuro- ery of cyclic nucleotides [4], these enzymes did not attract transmitters exert their regulatory effect on cell functions as much attention. Perhaps cAMP- and cGMP-hydrolyzing enzymes have been tacitly perceived as commonplace hydrolases with rather static function, that is, inactivation Key words: cAMP, extracellular signaling, collecting ducts, protein of cAMP or cGMP in the cell when hormonal effects kinase A, mesangial proliferative nephritis, nephrogenic diabetes insipidus, PDE superfamily. are terminated and these second-messenger nucleotides are no longer needed. The hydrolytic products 5Ј-AMP Received for publication February 6, 1998 and 5Ј-GMP are then duly recycled into the general pool and in revised form April 8, 1998 Accepted for publication April 9, 1998 of purine nucleotides. Updated August 19, 1998 It has long been known, but little appreciated, that in  1999 by the International Society of Nephrology most cells and tissues the maximal catalytic capacity for

29 30 Dousa: Cyclic-3Ј,5Ј-nucleotide PDE isozymes

-Fig. 1. General scheme of cyclic 3؅,5؅-nucleo tide metabolism. Abbreviations are: AdC, adenylate cyclase; GC, guanylate cyclase; PDE1- PDE9, types (gene families) of PDE isozymes. PDE3, PDE4, and PDE7 and PDE8 hydrolyze only cAMP (cAMP-PDE). PDE5, PDE6 and PDE9 hydrolyze only cGMP (cGMP-PDE), and isozymes PDE1 and PDE2 accept both nucleotides as a substrate. The thickness of the lines reﬂects the catalytic capacity of the pathway.

cAMP and cGMP synthesis is considerably (ϳ10 times) pharmacologic agents suited for “signaling transduction less than the capacity for hydrolysis by PDEs (Fig. 1) pharmacotherapy” [21]. and is one of the key factors determining turnover of In several areas of medicine—namely, in pathophysi- cAMP [5, 6]. This feature is consonant with general ob- ology and pharmacology of the cardiovascular, thoracic/ servations that even partial inhibition of PDE results in pulmonary, and endocrine systems, in the pathobiology a many-fold increase in the accumulation of cAMP and of immune-inflammatory processes, and in neurosci- the activation of the cAMP-activated protein kinase, or ence—a keen interest has developed in the study of PDE protein kinase A (PKA), signaling pathway [7–9]. It fol- isozymes, and considerable knowledge of the PDE sys- lows that PDEs in intact cells must operate at lower tem has recently accrued in these fields. On the other than maximal capacity and that they should be tightly hand, recent progress in the field of PDE isozyme re- regulated [5, 10–12]. Furthermore, it is increasingly evi- search did not attract much interest of investigators in dent that PDE activities in cellulo serve more than one nephrology and renal physiology. Consequently, our cur- function. In addition to determining the amplitude of rent knowledge of the role of PDE isozymes in biology cyclic nucleotide–mediated hormonal responses, PDE and pathobiology of renal cells and kidney is relatively also modulates the duration of the signal, and in some limited. This review provides a concise overview of the systems they also modulate rapid oscillation of cyclic basic biology and pharmacology of PDE isozymes in nucleotides, as exemplified in regulation of PDE’s func- general, and then reviews the current knowledge and tion [8, 10]. Current evidence shows that activities of concepts pertaining to the role of PDE isozymes in biol- PDE are regulated by multiple inputs from other signal- ogy and pathophysiology of the kidney. ing systems and are a crucial point of “cross-talk” between signaling pathways [8] and an important point of SUPERFAMILY OF PHOSPHODIESTERASE integration of intracellular regulatory networks [10–12]. ISOZYMES IN VERTEBRATES Moreover, PDEs may be key factors in cellular compartmentalization of cyclic nucleotides [8, 13–16]. Taxonomy of phosphodiesterases In spite of the initially low profile of PDEs, a small, For many years, PDEs were identified and known determined cadre of investigators persisted in the study almost solely by their catalytic and regulatory properties of PDEs as a primary subject of investigation. This effort [22]. For example, there were recognized PDEs with has yielded, mainly in recent years, a veritable explosion high Km and low Km for the substrate, PDEs dependent of new and fundamental information on PDEs and has for their activity on calcium (Ca2ϩ) and calmodulin established the novel concept of PDEs as intricate, inter- (CaM), or PDEs stimulated by cGMP, etc. [10–12, 22]. related, and highly relevant components of cellular sig- Recent advances in molecular genetics and biochemistry naling systems. It is now established that vertebrate have allowed the cDNA sequences and amino acid struc- PDEs comprise a large superfamily of related isozymes tures of many currently known PDE isozymes to be and their isoforms [10–12, 17, 18] that are diverse in terms determined. This analysis has revealed that vertebrate of structure, localization, regulation, and sensitivity to PDEs constitute a large superfamily of related isozymes, selective PDE inhibitors [7, 9, 18–20]. An attractive fea- comprising several gene families (types), single gene prod- ture of PDE isozymes is the fact that PDEs are targets for ucts (subtypes), and numerous isoforms-splice variants a number of synthetic PDE isozyme–selective inhibitors [17, 19, 23], which are schematically shown in Figure 2. [7–10]. These compounds are both experimental tools The systematic classification of the PDE isozyme system singularly useful in the study of the role of PDEs in [10–12, 19, 23] is based primarily on cDNA genetic codes cyclic nucleotide signaling and represent a novel class of and primary amino acid sequences, which can virtually Dousa: Cyclic-3Ј,5Ј-nucleotide PDE isozymes 31

Fig. 2. Schematic depiction of superfamily of vertebrate PDE isozymes (class 1). The number of isoforms, currently known, is in parentheses.

unequivocally identify any mammalian PDE isozyme [5, cGMP, is not sensitive to 3-isobutyl-1-methyl-xanthine 9, 10, 17, 19, 23]. When, based on these studies, the exis- (IBMX) and other tested PDE inhibitors, and is inhibited tence of PDE gene families became recognized, the first by Ca/CaM [24]. Otherwise, very little is known about five PDE gene families were designated by Roman nu- the properties and biologic significance of the enigmatic merals as PDE-I through PDE-V [7–9, 18]. In 1994, an cCMP, including cCMP cyclase and cCMP-PDE [24]. international committee of investigators in the PDE field Most gene families (types) of vertebrate PDEs are adopted a modified, updated nomenclature of PDE iso- comprised of more than one isogene; a product of a zymes [10, 23]. In this classification system, gene families single PDE isogene is also called a “PDE subtype” (Fig. (types) of the PDE are designated by Arabic numerals 2). Some PDE gene families, namely PDE1 and PDE4, [10, 23], for example, PDE4 instead of formerly PDE- are very diverse, whereas for others, such as PDE2, IV [7, 9, 18]. This nomenclature (Fig. 3) is also in confor- PDE5, PDE7, PDE8, or PDE9, the existence of well- mity with the Gene Bank nomenclature system and iden- documented isogenes has not yet been reported (Fig. 2). tifies PDE isozymes by vertebrate species, gene family, Furthermore, most PDE gene products have one or more single gene product, and isoform-splice variant. As cur- mRNA splice variants (PDE isoforms) that are the result rently known, the superfamily of mammalian PDE iso- or either alternative splicing of mRNA or alternative zymes consists of nine gene families (also called “types”) transcription initiation sites [10–12]. PDE isoforms, denoted PDE1 through PDE9 (Fig. 2). Because this des- “splice variants,” confer an additional layer of diversity ignation of gene families (types) by Arabic numerals was and specificity to the PDE superfamily system; PDE only introduced recently [23], readers can find the use isoforms are often tissue specific and are selectively ex- of Latin numerals even in recent publications. pressed in various tissues and cell types [8, 10, 11, 25–27]. The PDE activity that hydrolyzes specifically cyclic- A number of recently discovered PDE isoforms are 3Ј,5Ј-cytidine phosphate (cCMP), that is, the cCMP- known solely by the structure of cDNA sequence, deter- PDE, remains elusive. A protein with cCMP-PDE activ- mined with the use of recombinant DNA technology, and ity was isolated from rat liver [24]. The enzyme selec- the enzymatic properties of such PDE isoforms remain tively hydrolyzes cCMP as compared with cAMP and to be determined, in particular, by elucidation of their 32 Dousa: Cyclic-3Ј,5Ј-nucleotide PDE isozymes

Fig. 3. Current nomenclature of PDE isozymes, an example [10]. The nomenclature follows criteria for naming PDE isozymes according to the entries into LOCUS field of Gene Bank [23]. The first two letters at the beginning denote the species by the initials of taxonomic classification, the following three letters (PDE) plus Arabic numeral designate gene family (type), the next letter denotes isogene product (subfamily or subtype), last numeral denotes isoform (splice variant), and the final letter on the right end allows Gene Bank to assign a unique LOCUS field designation based on when the entry was submitted and also to give different LOCUS names to conflicting or incomplete sequences [8]. In the current literature, in an abbreviated version, the first two letters (species) and the last letter (Gene Bank LOCUS field designation) often are not specified. The descriptive abbreviations for the gene families (types), based on catalytic and regulatory properties, are often used alternatively and are listed in Table 1.

catalytic and regulatory properties. Currently, more than PDE monomers that are members of one gene PDE 30 PDEs have been identiﬁed (Table 1). However, the family show 65% or more amino acid sequence identity, number of known PDE isozymes does and will undoubt- but less than 40% identity is found among PDE families edly continue to increase. [10, 11, 17, 19]. Interestingly, PDE isozymes of the same PDE gene family (type) appear to be more homologous Molecular structure of PDE isozymes across the species than between two members of the A comparison of the nucleotide sequences of the same family within one species. For example, rat isozyme cDNA of PDE isozymes shows that all PDEs—the en- PDE3A and corresponding human isozyme PDE3A zymes that hydrolyze cyclic 3Ј,5Ј-nucleotides—can be have a greater homology than rat isozyme PDE3A and distinguished as two major distinct groups [17]. One large rat isozyme PDE3B [33]. Localization of genes encoding group, “Class I,” includes all vertebrate PDEs and some PDE isozymes in human and animal chromosomes di- yeast enzymes and has as a common feature a catalytic verges widely [8, 19]. As an example, PDE4D is located core of 250 amino acids or more, whereas “Class II” on chromosome 2 in rat but on chromosome 5 in humans PDEs lack this structural feature of a “central core” (Fig. [19], and PDE4B is located on chromosome 5 of rat, but 4). Included in Class II are some PDEs found in yeasts on chromosome 1 in humans. Within the PDE3 family, (S. cerevisiae) or slime molds (Dictyostelium discoideum) human PDE3A is located on chromosome 12, whereas [16, 18]; herein, exclusively vertebrate PDEs of Class I human PDE3B is located on chromosome 11 [33]. are covered [17]. Typically, the PDE monomer (Fig. 4) consists of three Phosphodiesterases of Class I are chimeric, multido- domains [8, 10–12]. These include a central catalytic core, main proteins that are composed of several different a region that is highly conserved in all vertebrate PDEs segment-domains, each of which is associated with a (Class I) consisting of approximately 270 amino acids particular function [10–12, 17, 19]. The PDE domains and positioned close to the COOH terminus. This core are separated by “hinge” regions, points in which these region is much more similar in terms of amino acid iden- PDE domains can be experimentally separated by lim- tity within one individual PDE gene family (more than ited proteolysis [11, 12, 17]. It is believed that the great 80%), rather than between two distinct PDE gene fami- diversity of the PDE isozyme system might have been lies (ϳ25% to 40%). The central catalytic core domain formed by genetic mechanisms that result in fusion of of PDEs also contains signature consensus sequences various gene domains, which are, putatively, of an evolu- and motifs that are common for all vertebrate PDEs [5, tionary origin [17]. The common pattern of structural 57]. In this domain, a sequence, HD(L,I,V,M,F,Y)H-X- features of PDE isozyme monomers is in Figure 4. H(A,G)X-X-N-X(L,I,V,M,F,Y), and also motif, E,F As a rule, vertebrate PDEs are dimers of linear 50 to (F,W)-X-X-QGD(R,K,L)E, identify uniquely any verte- 150 kDa proteins. An exception is the PDE6 family, brate PDE [5, 57]. (The sequence is in single-letter amino a photoreceptor-speciﬁc PDE, which is a tetramer and acid symbols; X indicates that any amino acid can be consists of at least four types of subunits [10, 11, 49]. present in this position. Groups in parentheses note that Dousa: Cyclic-3Ј,5Ј-nucleotide PDE isozymes 33

Table 1. Mammalian PDE isozymes gene families, isogenes and isoforms Isogene products Properties of products of PDE isogenes (subtypes) and isoforms (products of Gene family (type) (subtypes) Isoformsc alternative transcription, initiation or splicing) PDE1 3 (Ͼ9)a PDE1A PDE1A1 Km cAMP Ͼ Km cGMP, m.m. ෂ 50 kDa, major sources: heart; higher affinity b 2ϩ (CaM-PDE) (Kact) for Ca /CaM [10, 20] PDE1A2 Km cAMP Ͼ Km cGMP m.m. ෂ 61 kDa; source: brain, kidney; lower affinity 2ϩ (Lact) for Ca /CaM binding [10, 20]; Kact decreased by protein kinase A (PKA) phosphorylation PDE1B Km cAMP Ͼ Km cGMP, m.m. ෂ 63 kDa; source: brain, kidney, lower affinity 2ϩ (Kact) for Ca /CaM binding [10, 20] Kact decreased by phosphorylatioin via Ca/CaM-protein kinase II [10]; can be induced by PKC-␣ and PKC-⑀ [28] PDE1C PDE1C(1-5) Km cAMP Х Km cGMP; m.m. ෂ 75 kDa; source: olfactory bulb, testis, brain, at least 5 splice variants [29] PDE2 1 (3) PDE2 PDE2A1 m.m. ෂ 80 kDa, cystosolic form; source: adrenal, liver, heart [30] (cGS-PDE) PDE2A2 m.m. ෂ 105 kDa, membrane-bound form has N-hydrophobic sequence; source: heart, brain [31] PDE2A3 similar to PDE2A2; differs at N-terminal sequence [32]

PDE3 3 (Ͼ2) PDE3A lower KicGMP than PDE3B; is activated and phosphorylated by PKA [11, 33, 34] (cGI-PDE) PDE3B higher KicGMP than PDE3A; is activated by phosphorylation by PKA and also by insulin-stimulated protein kinase (PDE31K); the pathway includes phosphatidyl inositol-3 kinase (Pi-3K); N-terminal rich in proline [33, 34] PDE4 4 (Ͼ15) PDE4A PDE4A1A membrane-bound isoform, binds through N-terminal 1-25 amino acids [25, 35, 36] (cAMP-PDE) PDE4A4B when membrane-bound is 10 times more sensitive to inhibition by rolipram [69] PDE4A5 isoform that can bind via SH3 domains (class 1 sites) src tyrosine kinases or to fodrin [37, 38]; is activated by growth hormone [224] PDE4B PDE4B1 “long isoform” [39] PDE4B2A “short isoform,” de novo synthesis of the enzyme is induced by cAMP [40, 41] PDE4B2B “short isoform” [39] can be phosphorylated by MAP kinase at Ser487 close to COOH terminal region [42] PDE4B3 “long isoform” [39] PDE4C isoforms are less frequently expressed, and are less sensitive than other PDE4 subtypes to some selective inhibitors (rolipram, CDP-840, Ro-20 174) [43, 44] PDE4D PDE4D1 “short isoform” m.m. ෂ 72 kDa; de novo synthesis of the enzyme is induced by cAMP [12, 40, 41, 45]; present only in cytosol [41] PDE4D2 “short isoform” m.m. ෂ 68 kDa; de novo synthesis of the enzyme is induced by cAMP [12, 41]; present only in cytosol [46] PDE4D3 “long isoform” m.m. ෂ 93 kDa; is activated via phosphorylation by PKA at Ser54 [47]; also is activated by binding of phosphatidic acid [48]; in part membrane bound [46] PDE4D4 “long isoform,” binds to SH3 domains [38]; in rat membrane bound [46] PDE4D5 “long isoform”; in part membrane bound [46] PDE5 2 (2) PDE5 binds noncatalytically cGMP; is phosphorylated and activated by PKA and/or (cG-BPDE) PKG [10, 11, 56] PDE6 3 (2) PDE6A heterotetramer (␣, ␤, ␥␥) prenylated at COOH terminal; inhibitory ␥ subunit (photoreceptor) PDE6B phosphorylated by PKC; located in retinal rods [19, 49] cG-PDE PDE6G homotetramer (␣Ј, ␣Ј, ␥␥); located in retinal cones [19, 49] PDE7 1 (2) PDE7A PDE7A1 m.m. 57 kDa, soluble, expressed in multiple tissues [50] (HCP-7-PDE) PDE7A2 m.m. 50 kDa with N-terminal hydrophobic sequence, membrane bound, expressed in a skeletal muscle and myocardium; insensitive to rolipram, enoximone, cGMP, EHNA, zaprinast [51, 52]

PDE8 1 PDE8A PDE8A1 m.m. ෂ 83 kDa, cloned from human, cAMP-speciﬁc, low Km ϭ 55 nm, low VMAX ϭ 150 pmol/min/mg protein; insensitive to IBMX, rolipram, vinpocetine, zaprinast, enoximone, SKF94120; is inhibited by dipyridamole IC50 Ӎ 9␮m; expressed in multiple tissues, highest in gonads and intestine [53] PDE8A2 m.m. 95 kDa; cloned from mouse, expressed predominately in testis and liver; kinetics and inhibitor sensitivity similar to PDE8A1 [54]

PDE9 1 (2) PDE9A PDE9A1 m.m. 62 kDa; cloned from mouse, cGMP-speciﬁc, low KmcGMP ϭ 70 nm; insensitive to: IBMX, EHNA, rolipram, PDE3 inhibitors; inhibited by SCH 518666 Ki Ӎ 1.5 ␮m, zaprinast Ki ෂ 20 nm; expressed predominately in the kidney [55] PDE9A2 nm ෂ 69 kDa; cloned from human, low KmcGMP ϭ 170 nm;VMAX ϭ 4.9 nmol/min/ mg protein; insensitive to: IBMX, dipryidamole, rolipram, vinpocetine, PDE3 inhibitor; inhibited by: zaprinast Ki ϭ 35 ␮m [56] a Denote number of genes in the family; in parentheses number of known isoforms b In parentheses are frequently used alternative abbreviations c Species origin of isoforms is not speciﬁed; the most frequent species designations are: human, HS (homo sapiens); rat, RN (ratus norvegicus); mouse, MM (mus musculus); bovine, BT (bos taurus); for details, see Fig. 4 34 Dousa: Cyclic-3Ј,5Ј-nucleotide PDE isozymes

Fig. 4. Schematic depiction of the general structure of a PDE isozyme monomer. It consists of an N-terminal domain, central catalytic core, and C-terminal domain, connected by hinge regions. The domains are not drawn in proportion. In particular, the size of the N- terminal domain is substantially different in various PDE types [19]. Abbreviations are: PKA, protein kinase A; PKG, protein kinase G; PKC, protein kinase C; PDE3IK, insulin- dependent PDE3B protein kinase.

any of the amino acids enclosed may occupy that posi- binding sites for cGMP or for phosphatidic acid. Several tion.) Furthermore, the central catalytic core contains consensus sequences that are phosphorylation sites for two consensus Zn2ϩ-binding motifs containing histidines: Ser/Thr protein kinases, another frequent mechanism for ϳ3 mol of Zn2ϩ binds to 1 mol of PDE5 monomer [58]. regulation of PDE activity, are located in the N domain Even early studies have shown that divalent cations are [10–12]. These include phosphorylation sites for: (1) important cofactors of PDE. For example, the addition Ca2ϩ-CaM protein kinase II in PDE1; (2) PKA in PDE1, of ethylenediaminetetraacetic acid almost completely PDE3, and PDE4; (3) protein kinase G (PKG) in PDE5; abolished activity of PDE from heart [4] or kidney [59], (4) insulin-stimulated protein kinase (PDE3IK) in and the activity was restored by addition of Mg2ϩ [4, 59]. PDE3B; and (5) protein kinase C (PKC) in PDE1 and The maximum cAMP-PDE activity was achieved at 10 PDE6 (Table 1 and Fig. 4) [8–10]. Finally, the N-terminal mm or more Mg2ϩ [59]. A similar effect of Mg2ϩ was found regulatory domain of some PDE isozymes, such as rat in recent studies of recombinant PDE4 isozymes [60]. PDE4A5A (RNPDE4A5A) [37], also contains Src ho- Other specific signature motifs specifically identify cGMP- mology 3 (SH3)-binding motifs [37] that confer to these binding domains [10, 11] in several PDE isozyme types: PDEs the capacity for noncovalent association with pro- PDE2 (cGMP-stimulated PDE), PDE5 (cGMP-binding teins of other signaling pathways that contain SH3 do- PDE), and PDE6 (photoreceptor PDE), where this domains, such as tyrosine protein kinases of the Src family main is located in catalytic subunits ␣, ␣Ј, and ␤ [9]. [61] or cellular structures such as subunits of nicotin- Furthermore, unique and defining for the isozymes of amide adenine dinucleotide phosphate (NADPH) oxi- the PDE3 family is a 44-amino acid insert located within dase [62]. The N-terminal domain contains sites for di- the catalytic core that is not found in any other PDEs. merization [10, 11], as well as sites for hydrophobic In contrast to the central catalytic core, the structure interactions and/or membrane-association sites [11, 25, of the NH2-terminal “regulatory domain” of PDE mole- 33, 35, 36]. cules diverges widely among PDE isozymes and isoforms Experimental maneuvers that delete the N-terminal in structure and size [17]. The N domain contains se- regulatory domain from the rest of the molecule, such quences that are target sites for various modes of regula- as limited proteolysis, result in a many-fold increase of tion: ligand-binding sites, phosphorylation sites, and sites PDE catalytic activity [10–12, 17, 35]. These findings for several modes of protein–protein interactions (Fig. 4). support a hypothesis proposing that in a basic ambient These include binding sites for Ca2ϩ-CaM and noncatalytic state the N-terminal regulatory domain of PDE exerts Dousa: Cyclic-3Ј,5Ј-nucleotide PDE isozymes 35 an autoinhibitory effect on the catalytic core domain and procedures are fastidiously avoided, reported results keeps PDEs in a low-activity state [8, 11, 12]. This feature from such fractionation studies often differ and may be is consistent with the notion that in cellulo PDE isozymes different between various tissues and, with respect to a are present in an inhibited state, and various regulatory given tissue, between vertebrate species. Generally, in mechanisms, that is, phosphorylation or binding of mod- preparations from myocardium, liver, or adipocytes, a ulators that interact with the N-terminal domain, in most considerable proportion (ϳ50%) of PDE activities are instances, relieve such autoinhibition and enhance cata- apparently associated with membranes [7, 9], whereas lytic activity of the PDE isozyme [10–12, 17, 63]. in preparations from platelets, kidney, smooth muscle, The functional significance of the rather small C-termi- or mesangial cells (MCs) [66], the majority (ϳ90%) of nal domain of PDEs is largely unknown. Perhaps it may PDE activities are in the cytosol [7, 9]. serve some regulatory or localization functions that are As recent studies show, localization of PDE isozymes analogous to the N-terminal regulatory/localization do- within the cell is highly relevant for functions of PDE main. According to a recent report, a mitogen-activated isozymes in situ. Elucidation of the primary structure of protein kinase (MAPK) can phosphorylate Ser487 in the PDE isozymes and the study of cells transfected with C-terminal portion of the PDE4B2B isoform [42], a pos- cDNAs encoding PDEs provided the first precise insights sible point of direct cross-talk between cAMP and into interactions of PDEs with intracellular structures. MAPK signaling pathways. Consensus sequences— Such investigations showed that the N-terminal hydropho- CAAX—for prenylation are present in the C terminus bic region of PDE3 is a likely site for interaction with of catalytic subunits of PDE6: in the PDE6␣ subunit for membranes [33] and that the first 25 amino acids in farnesylation and in the PDE6␤ subunit for geranylgera- the N-terminal portion of the PDE4A1A molecule are rylation, prenyl moieties represent another mode of required for attachment to plasma membranes and Golgi mechanism for anchoring PDEs to membranes [10, 11]. apparatus [63]. After deletion of this N-terminal, 25- Phosphodiesterase isozyme activities can be regulated amino acid sequence, the membrane-associated enzyme by hormones that act via genomic mechanism and modu- becomes diffusely distributed in the cytoplasm [63, 67]. late gene expression and de novo synthesis of PDE iso- Catalytic subunits of photoreceptor isozymes of PDE6 zyme proteins [12, 40]. For example, follicle stimulating are apparently anchored to membranes via posttransla- hormone (FSH), via stimulating cAMP generation, can tionally prenylated subunits, a lipophilic interaction. induce the synthesis of mRNA encoding “short forms” Some of the PDE4 isoforms, PDE4A5A [37], and per- of PDE4, such as PDE4D1 and PDE4D2 isoforms [12, haps also PDE4D4 [38] that have SH3-binding domains 19, 40]. In these responses, cAMP acts as an inducer and [61] in the N-terminal region have the potential to inter- may conceivably act via PKA phosphorylation of cAMP- act with cytoskeletal proteins [68], as well as with pro- responsive element-binding protein [64]. In studies on teins involved in signaling [32]. Recent studies indicate cultured CHO cells, agents that stimulate phosphoinosi- that an isoform(s) of PDE4 from MCs [15] is likely to tide metabolism and that activate PKC selectively in- interact with SH3 binding domains [69] and also Src duced expression of PDE1B [28]; the action of growth homology 2 (SH2) domains that allow binding on phos- hormone to increase PDE4 isoform PDE4A5 probably photyrosine (T.P. Dousa, M. Beard, M.D. Houslay, C.S. involves stimulation of p7056 kinase [65]. The up-regula- Chini, H.I. Walker, preliminary observations). Protein– tion of PDEs via a genomic mechanism is a basis for protein interactions that involve SH3 and SH2 may be long-term desensitization to effect of hormone-agonists, important for the localization of PDEs in the cell as well such as FSH [10, 40]. In contrast, some increases of PDE as for cross-talk with other signaling pathways [8, 38]. isozyme activities, with fast onset, are due to Ser/Thr phosphorylations of a site within the N-terminal domain Differential expression of PDE isozymes and are a likely basis for a short-term negative feedback in cells and tissues or desensitization of cells to action of hormones that The expression and precise location of PDE isozymes stimulate cAMP synthesis [8, 11, 12, 33]. and PDE isoforms in a number of cell types and tissues have been recently determined, mainly with the use of in Intracellular localization of PDE isozymes situ hybridization and by immunohistochemical methods Phosphodiesterases are not intrinsic membrane pro- [10, 11]. For example, Reinhardt et al mapped the distri- teins but are often associated, loosely or tightly, with bution of two isozymes, PDE3A and PDE3B, in major membranes [8, 10, 11, 35, 63]. The association of PDE rat tissues [70]. Discovery of the unique expression of isozymes with membranes is sometimes difficult to deter- PDE2 in cells of the zona glomerulosa of the adrenal mine with certainty with use of standard subcellular frac- gland [30] contributed to the delineation of the signaling tionation techniques [7], as much depends on the method pathway by which atrial natriuretic peptide (ANP) acts for disruption of the tissues or cells by mechanical forces to inhibit synthesis of aldosterone. In glomerulosa cells, or detergent treatment [7]. Even when gross disruptive ANP increases cGMP synthesis. Increased cGMP stimu- 36 Dousa: Cyclic-3Ј,5Ј-nucleotide PDE isozymes lates cAMP breakdown by PDE2A1, and the resulting should be interpreted with caution. Extracts from rat decrease of cAMP is a signal to decrease aldosterone myocardium contain several PDE types, including PDE1 synthesis and secretion [30]. Studies of the central ner- [75]. However, similar extracts prepared from isolated vous system showed that high activity of PDE1B is con- rat cardiomyocytes show analogous PDE patterns, but centrated in basal ganglia, and its presence correlates without PDE1 activity, thus suggesting that PDE1 is with sites of dopaminergic innervation [71]. Yan et al likely contained in cells other than myocytes, for exam- determined the localization of splice variants of several ple, fibrocytes [75]. PDE1C isoforms (PDE1C1 to PDE1C5) in mouse tis- Differential expression of PDE isozymes and isoforms sues, particularly in the brain, olfactory epithelium, and is a critically important determining factor that adds to testis [29]. In this context, it should be stressed that great fundamental diversity of the cell-specific PDE iso- detection of cognate mRNA by the in situ hybridization zyme systems. In principle, signal transduction via cyclic technique alone may give only partial information about nucleotide systems in a given cell type is determined by the presence and function of the PDE isozyme. In studies (a) the expression of specific cell-surface receptors for of isozyme PDE7A1, abundant mRNA was detected hormones, neurotransmitters, or autacoid that is cou- with use of in situ hybridization technique in muscle pled, positively or negatively, to adenylate cyclase or is tissue, T lymphocytes, and B lymphocytes [51]. However, part of guanylate cyclase, and (b) the pattern of PDE the catalytic activity of both PDE7 and immunoreactive isozymes and isoforms that is expressed to a different protein was found in only T-cell lymphocytes; little or degree. PDE isozymes thus confer to cAMP and cGMP no immunoreactive protein or enzymatic activity was signaling pathways, in a given cell, a second crucial point found in B lymphocytes and in muscle [51]. Such findings that determines specificity of signal transduction, which, illustrate that quantitatively high expression of cognate together with receptors and cyclases, define the “cyclic mRNA in the cell does not necessarily indicate the pres- nucleotide signaling phenotype” of a particular cell type ence of high activity of a translated and active PDE [10]. isozyme. Possibly, in some cell types, mRNA of PDE is not translated or the translated protein is either inactive CHARACTERISTICS OF PHOSPHODIESTERASE or unstable, and conceivably the expression of some PDE ISOZYME FAMILIES (TYPES) isozymes might be regulated at the translational level [51]. The key properties of seven known gene families The expression of PDE isozymes may depend on the (types) of PDE isozymes are concisely outlined here and biologic state and environment of the cell. For example, are summarized in Table 1. freshly isolated circulating monocytes/macrophages show PDE1 family high activity of PDE4 but little or no other PDEs, whereas monocytes/macrophages that reside in tissue, such as The PDE1 family (Ca/CaM-stimulated PDE) is a pulmonary alveolar macrophages, express less PDE4 group of isozymes that are dependent for their activity than circulating monocytes but have high PDE3 activity on Ca2ϩ and CaM. The binding site for CaM/Ca2ϩ is and very high activity of PDE1 [72, 73]. When isolated positioned close to the N terminus of the regulatory monocytes are grown in cell culture, they assume the domain [10, 19]. The PDE1 isozyme family, along with same PDE isozyme pattern as alveolar macrophages [72, the PDE4 family, is the most diverse and includes numer- 73]. Diverse expression of PDE1 isozymes is well docu- ous splice-variant PDE isoforms, such as PDE1C1–5 [29]. mented in a study of vascular smooth muscle [74]. Products of two genes, PDE1A and PDE1B, have higher Whereas freshly prepared quiescent human aortic vascu- affinity for cGMP than for cAMP, but some isoforms of lar smooth muscle cells contain isozymes PDE1A and PDE1C exhibit also high affinity for cAMP [10]. Further- PDE1B, in proliferating smooth muscle cells, which are more, activities of PDE1 isozymes are modulated by Ser/ grown in primary culture, PDE1C is the dominant iso- Thr phosphorylations of sites Ser131, Ser143 located in the zyme [74]. Moreover, cultured vascular smooth muscle N-terminal domain. PDE1A1 from heart and PDE1A2 cells from monkey aorta have highly expressed PDE1B from brain are phosphorylated by PKA, whereas PDE1B but no PDE1C; PDE1A was the only detectable PDE1 is phosphorylated by CaM-dependent protein kinase II, isozyme in vascular smooth muscle cells grown from rat and all of these phosphorylations can be reversed by cal- aorta [74]. Clearly, the expression of PDE1 isozymes cineurin, a Ca/CaM-dependent phosphatase [20]. Both depends on the status of the cell cycle and also differs types of phosphorylation decrease the affinity of PDE1 2ϩ between species [74]. The two members of the PDE3 for CaM and increase Ka for Ca activation. According family, PDE3A and PDE3B, are subject to develop- to a recent report, PDE1B can be induced by activation mental regulation of gene expression [70]. Results of of protein kinase C (PKC) or transfection with PKC-␣ studies when the PDE pattern is determined in extracts or PKC-⑀ [28]. Thus, PDE1 isozymes have potential for from whole tissues or heterogeneous cell populations cross-talk with several other signaling systems that in- Dousa: Cyclic-3Ј,5Ј-nucleotide PDE isozymes 37 clude, at least, Ca2ϩ and CaM, Ca2ϩ/CaM-dependent pro- pure cAMP-PDE [10, 11, 33, 64]. Products of two iso- tein kinases and phosphatases, and cAMP-PKA and genes, PDE3A and PDE3B, differ in their distribution PKC pathways. Any compounds that chelate Ca2ϩ or in organs and tissues [70], as well as in their regulation inhibit CaM activity act as effective inhibitors of PDE1 [11, 33]. PDE3A is more sensitive to inhibition by cGMP but have many other effects. Some selective inhibitors than is PDE3B. Both subtypes, PDE3A and PDE3B, act directly on the catalytic site of PDE1 isozymes, such are activated by Ser/Thr phosphorylation on Ser302 in the as 8-methoxymethyl-IBMX (8-MeoM-IBMX) and vin- N-regulatory domain, which is catalyzed by PKA [33]. pocetin [7, 9, 76]. PDE1 isozymes are present in many In addition, PDE3B is activated by phosphorylation of tissues and are abundant mainly in the central nervous the same site, Ser302, which is catalyzed by the insulin- system, heart, and kidney. stimulated protein kinase of PDE3 (PDE3IK) [33]. Al- though the insulin-dependent phosphorylation and acti- PDE2 family vation of PDE3B catalyzed by PDE3IK is amply docu- In the PDE2 family (cGMP-stimulated PDE, cGs-PDE), mented [33], the whole sequence of steps in the signaling as well as in the case of PDE1, both cAMP and cGMP mechanism by which insulin, after interaction with its are substrates for PDE2, which is specifically stimulated receptor, leads ultimately to activation of PDE3IK is not by cGMP with positive cooperativity kinetics [10, 11]. yet clarified, but it is likely that an essential step in this In the PDE2 molecule, the two specific, noncatalytic pathway involves activation of phosphatidylinositol-3 ki- cGMP-binding sites are located in the N-terminal regula- nase [33]. Thus, PDE3 is a target for negative cross-talk tory domain, upstream of the conserved central core. with cGMP pathways and positive cross-talk between Hydrolysis of both cAMP and cGMP is stimulated by insulin, as well as cAMP signaling pathways. It is postu- cGMP; PDE2 has a rather low affinity (Km Х 10 ␮m) for lated that phosphorylation of PDE3A or PDE3B by both cAMP and cGMP. Three PDE2 isoforms, probably PKA probably represents a short-term negative feed- splice variants, have been described: the cytosolic PDE2A1 back regulation. Both PDE3A and PDE3B are sensitive enzyme in adrenal cortex [30], the PDE2A2 isoenzyme to inhibition by numerous synthetic inhibitors, including in membranes from brain and cardiac tissue [31], and many “cardiotonic inhibitors,” which are effective in low most recently, isoform PDE2A3 [32]. The PDE2A2 iso- micromolar concentrations. The experimental and clini- form has a hydrophobic region in the N-terminal domain cal use of PDE3 inhibitors was extensively investigated, that likely accounts for membrane targeting [31]. Be- and some, such as cilostazol [81] or milrinone [82], are cause PDE2 was found in cells that express little or no used in clinical practice. PKG, PDE2 is considered to be one of the main receptor targets for cGMP in some cGMP-mediated signal- PDE4 isozymes transduction mechanisms, and represents a classic site of Phosphodiesterase 4 isozymes (cAMP-specific PDE; cross-talk between cAMP and cGMP signaling pathways cAMP-PDE) are the most diverse family of PDEs and [8, 10]. The only known selective inhibitor of PDE2 is hydrolyze selectively and with high affinity only cAMP. erythro-9-(2-hydroxyl-3-nonyl)-adenine (EHNA), which Therefore, PDE4 is also sometimes referred to as is also a potent inhibitor of adenosine deaminase [77]. “cAMP-specific PDE” [12]. PDE4 isozymes consist of Thus, when employed as a PDE2 inhibitor, EHNA ought four subfamilies (subtypes) encoded by isogenes PDE4A to be compared with the effect of a structurally unrelated to PDE4D, which in turn are transcribed and expressed adenosine deaminase inhibitor that does not inhibit PDE2, in numerous splice-variant PDE isoforms [10–12]. Splice- such as 1Ј-deoxycoformycin [78]. EHNA has positive ino- variant isoforms of PDE4 are distinguished by the struc- tropic [78] and thrombostatic [79] effects that are related ture in the N-terminal domain, which confers diverse to inhibition of PDE2. Some analogues of cGMP that are regulatory properties and capacity for interactions with often used to mimic cGMP effects, namely C 8-substituted other cell structures [37, 35, 39, 46, 63, 67]. Some iso- compounds, 8-pCPT-cGMP, or 8-Br-cGMP do not stim- zymes of PDE4 can be up-regulated by cAMP via two ulate PDE2 [80] and cannot be used for probing this different mechanisms. “Short isoforms” (less than 72 isozyme. kDa), such as PDE4D1 or PDE4D2, are subject to long- term up-regulation by cAMP via a transcriptional control PDE3 isozymes mechanism [8, 12, 40, 41, 43, 45]. An increase in the Phosphodiesterase 3 isozymes (cGMP-inhibited PDE) cellular cAMP level enhances synthesis of cognate display high affinity for both cAMP and cGMP but have mRNA and de novo synthesis of the enzyme molecules a far higher Vmax for cAMP than for cGMP [11, 33, 64]. [12, 40, 41, 45, 83], and the induction can be blocked cGMP in micromolar concentrations binds to the cata- by actinomycin D and cycloheximide [84]. Interestingly, lytic site of PDE3 and thereby competes with cAMP elevation of cAMP in T cells resulted in an induction of hydrolysis; however, because of the low Vmax, cGMP hy- PDE4D and a simultaneous decrease of PDE4A [84]. drolysis by PDE3 is negligible, and PDE3 behaves as a In a monocytic cell line, U937 increased cAMP-enhanced 38 Dousa: Cyclic-3Ј,5Ј-nucleotide PDE isozymes expression of PDE4A and PDE4B, whereas expression gers within the catalytic core domain of PDE5. Only one PDE4D apparently decreased [85]. gene is known so far, and some observations suggest the On the other hand, “long isoforms” of PDE4D (more existence of two splice variants [11]. PDE5 is inhibited than 93 kDa), such as PDE4D3, are activated by a cAMP- selectively by zaprinast and dipyridamol [95]; novel in- 54 reversible phosphorylation of Ser , which is located within hibitors with higher affinity (Ki ϳ1nm) include E4021 the “upstream conserved region” UCR1 close to the [96], sildenafil [97], WIN58237 [95], or DMPPO [98]. N terminus that is catalyzed by PKA [47]. This phosphorylation causes a rapid, several-fold increase in catalytic PDE6 isozymes activity of PDE4D3 and also greatly increases (more The PDE6 isozymes (photoreceptor PDE) family are than 100 times) the sensitivity of PDE4D3 to the selec- heterotetramers or homotetramers composed of varia- tive inhibitors rolipram or compound RS-33793 [86, 87]. tions of catalytic subunits (␣, ␣Ј, ␤) and inhibitory sub- Some isoforms, such as PDE4A4B, become more sensitive units (␥ and ␦) [10, 11, 19, 49]. PDE6 isozymes are ex- to rolipram when associated with membranes, and the pressed solely in retinal rods and cones and have a highly kinetics of the inhibition also change [88]. Another bioreg- specialized function in photoreceptor signaling [10, 11, ulator is phosphatidic acid, which activates PDE4D3, 19, 49]. PDE6 is a pivotal step in a pathway initiated by probably via binding on N-terminal domain [48]. It is photons that interact with rhodopsin, which is coupled postulated that up-regulation of PDE4D isoforms by via the heterotrimeric G-protein “transducin” to mem- cAMP via different mechanisms is a basis for long- and brane-associated PDE6, and results in increased PDE6 short-term desensitization of cell responses to increased activity by causing dissociation of inhibitory ␥ subunits synthesis of cAMP, as it is exemplified by desensitization from catalytic subunits. Activation of PDE6 then results of Sertoli cells to FSH or thyroid epithelial cell line to in a drop in cGMP levels and an opening of the cGMP- thyroid-stimulating hormone [12, 40, 83, 89]. gated ion channel [11, 19, 49]. The termination of the Archetypal selective inhibitors of PDE4 are rolipram activated state of PDE6 involves reassociation of ␥ sub- and Ro 20-1727. However, recently a number of newer units with catalytic PKC [11]. PKC may phosphorylate PDE4 inhibitors that display up to 1000 times higher the ␥ subunit on Thr35 and thereby increase its inhibitory affinity for PDE4 (ϳ1nm) than rolipram (ϳ1 ␮m) [8, activity [11]. Kinetic and catalytic properties and sensitiv- 44, 90, 91] have been synthesized [8, 90–92]. Rolipram ity to inhibitors of PDE6 isozymes closely resemble iso- binds with a very high affinity (1 nm) to PDE4 isozymes zymes of the PDE5 family; however, substantial differ- at the site that is probably within the core catalytic do- ences in primary structure and subunit composition, as main [43, 44]. The relationship of these “high affinity well as its highly specialized function, distinguish PDE6 rolipram-binding sites” (HARBS) [44] in the PDE4 mol- from the PDE5 family [10, 11, 19]. ecule to the inhibitory action of rolipram on cAMP hydrolysis is not clear [43, 44, 90–92]. Rolipram that is PDE7 isozyme family bound to HARBS can be displaced competitively by Recently the PDE7 isozyme family (HCP1) was dis- cAMP; however, recent studies indicate that HARBS covered. PDE7 was cloned, expressed, and identified as are not identical with the catalytic site for cAMP in the a new PDE family that is genetically distinct from others catalytic core of PDE4 [43, 44]. It is believed that [50]. The known characteristics of PDE7 include high HARBS relate to some of the pharmacologic effects of affinity and selectivity for cAMP as a substrate, similar PDE4 inhibitors that are unrelated to inhibition of to that of PDE4. Importantly, PDE7 isozyme differs cAMP hydrolysis, namely emetic action and gastric acid significantly from PDE4 and PDE3 by its lack of sensitiv- hypersecretion [44]. Several novel inhibitors of PDE4 ity to inhibition by cGMP, by inhibitors of PDE4 roli- have a higher affinity for PDE4D than for PDE4A, pram and RO 20-1724, PDE3 inhibitor enoximone, PDE4B, and PDE4C [43, 44]. PDE2 inhibitor EHNA or PDE5 inhibitor zaprinast [50– Phosphodiesterase 5 isozymes (cGMP-binding PDE) 52]. Also, PDE7 is apparently relatively insensitive to are abundant in lung, platelets, and smooth muscle [10– inhibition by nonselective IBMX [28, 99]. Cognate 12]. PDE5 isozymes are strictly specific for cGMP and mRNAs encoding PDE7 were isolated and found ex- are characterized by the presence of two high-affinity pressed in a number of tissues, including kidney [50–52], noncatalytic cGMP-binding sites in the N-terminal do- and two isoforms-splice variants, PDE7A1 and more main [5, 10, 11, 93]. Binding of cGMP to PDE5 by itself recently PDE7A2 [51, 52], have been identified. Isoform does not influence PDE activity but results in a confor- PDE7A2 has a hydrophobic sequence in the N terminal mational change of the PDE5 molecule that facilitates and is, at least in part, membrane bound [52]. phosphorylation by PKG [10, 93] and possibly also by PKA [94], both of which are associated with the up- PDE8 isozymes regulation of PDE5 activity [93]. Furthermore, PDE5 is The PDE8 isozyme family of isozymes was recently activated by Zn2ϩ ions, which probably bind to zinc fin- cloned from human [53] and murine tissues [54], and Dousa: Cyclic-3Ј,5Ј-nucleotide PDE isozymes 39 enzymes expressed in transfected cells were examined. artifacts [9, 18, 83]. It is now known that when working PDE8 is cAMP specific and has a very high (so far the with cell-free preparations from tissues or cells, all proce- highest) affinity for cAMP as a substrate: ϳ 20 times dures ought to be carried out in the presence of a mixture lower KmcAMP than PDE4. However, it has approximately of protease inhibitors, calcium chelators and sulfydryl- 10-times lower Vmax than PDE4 [53]. Another specific reducing reagents, and other agents that minimize prote- feature of PDE8 is its insensitivity (at 100 ␮m or more) olysis, as well as to fastidiously keep the temperature at to cGMP, to the PDE4 inhibitor rolipram, the PDE5 0 to 4ЊC. Fractionation and isolation procedures that are inhibitor zaprinast, the PDE1 inhibitor vinpocetine, and based on charge and size, affinity to cyclic nucleotides, the PDE3 inhibitor SKF-94120, as well as to the nonse- modulators, antibodies, and PDE inhibitors preferably lective inhibitor IBMX [53, 54]. Surprisingly, PDE8, a should be carried out with rapid separation techniques, cAMP-hydrolyzing enzyme, is inhibited by dipyridamole such as biocompatible high pressure liquid chromatogra-

(IC50 ϭ 9 ␮m), a compound that specifically inhibits phy (HPLC) or fast protein liquid chromatography PDE5, an isozyme that is known to hydrolyze only cGMP (FPLC) systems. PDE isozymes are more stable when [7, 9–11]. Human [53] and murine [54] PDE8 isozymes stored at Ϫ20ЊC in a non-solid frozen state in a medium are quite similar. Analysis of mRNA encoding PDE8 containing 30% ethylene glycol, which prevents freezing indicates that this isozyme is abundant in numerous ex- [101, 102]; this storage method is often superior to rapid amined tissues, most prominently in testis, ovary, and freezing and storage at Ϫ70ЊC. Finally, methods of mo- intestine [53, 54]. lecular cloning and recombinant DNA technologies in the study of PDE isozymes are similar to those used in PDE9 isozymes other areas of investigations. The PDE9 isozyme family was discovered almost si- Unique experimental tools are selective PDE isozyme multaneously with PDE8; however, it has profoundly inhibitors that are used for probing PDE isozymes, and different characteristics [55, 56]. PDE9 hydrolyzes spe- their function in vitro and especially in vivo should be cifically cGMP, not cAMP [55, 56]. Further, compared discussed in more detail. Of equal importance is the with other cGMP-specific PDEs, PDE9 apparently lacks potential of PDE inhibitors to become unique novel noncatalytic cGMP-binding domain, which is present in pharmacologic agents [7–9]. Availability of PDE iso- PDE5, PDE6, and also in molecules of PDE2 [5, 10, 11, zyme–selective inhibitors and identification of their spe- 93]. As it is the case of PDE7 and PDE8, the activity of cific biomodulators are of singular importance for the PDE9 is insensitive to nonselective inhibitor IBMX [55, study of PDE isozymes in cell-free systems and even 56] and is also resistant to selective inhibitors of PDE1, more so for studies of PDE isozyme functions in cellulo. PDE2, PDE3, PDE4, and PDE5 [55, 60]. The only compounds identified to date that inhibit the activity of PDE9 Overview of PDE inhibitors

[55, 56] is zaprinast (IC50 ϳ30 ␮m), an inhibitor that was Nonselective inhibitors. Even in the initial phases of previously considered to be selective for PDE5, and even PDE research, it was determined that several common more potent, on a molar basis, is SCH518866 [55], a alkaloids, including methyl xanthines theophylline, theo- compound that inhibits both PDE5 and PDE1 [100]. bromine, and caffeine, as well as the isoquinoline deriva- The mRNA encoding PDE9 is well expressed in many tive papaverine, which have been widely used as pharma- examined human tissues, including spleen, small intes- ceuticals in clinical practice, were identified as PDE tine, and brain [56]. Expression of mRNA encoding mu- inhibitors, compounds that specifically inhibit the activity rine PDE9 is very prominent in the kidney and far less of PDE and not other phosphohydrolases [4, 7, 9, 102, in other examined tissues [56]. 103]. These PDE inhibitors inhibit PDE competitively with low affinity (Ki Х 1mm) and do not discriminate between PDE isozymes; both cAMP-PDE and cGMP- STUDY APPROACH TO PDE activities are inhibited [4, 103]. Nonselective PDE PHOSPHODIESTERASE ISOZYMES inhibitors are currently referred to as “first-generation AND THEIR INHIBITORS PDE inhibitors” [7, 9, 103, 104]. Besides their low affinity Except for a few specific features mentioned herein, for PDEs, the “first-generation” PDE inhibitors often the biochemical, cellular, and molecular biologic meth- have pharmacologic effects unrelated to the inhibition odologies for the investigation of PDE isozymes do not of PDE. Theophylline and other methyl xanthines are differ from those used to study other enzyme systems. potent antagonists of adenosine receptors [7, 9, 104]; In the course of studies about PDEs, it became evident caffeine can trigger or potentiate Ca2ϩ release from en- that PDE isozymes are proteins very susceptible to pro- doplasmic or sarcoplasmic reticulum through the rya- teolytic attack. Consequently, many past studies aiming nodine receptor channel [105]; and papaverin blocks to enrich and isolate PDEs, using the then standard pro- adenosine uptake [103]. One methylxanthine derivative, tein isolation techniques, very likely produced numerous IBMX, which was synthesized by Wells et al [106], has 40 Dousa: Cyclic-3Ј,5Ј-nucleotide PDE isozymes

a much higher affinity (Ki Х 1–10 ␮m) for PDEs and in should be carefully chosen to maximize specificity and low concentrations preferentially inhibits cGMP-PDE selectivity of these pharmacologic probes [7, 9, 10, 23, over cAMP-PDE [9–11, 106]. IBMX became a popular 26]. It should also be kept in mind that some second- and frequently used experimental tool and is widely em- generation PDE isozyme–specific inhibitors may have ployed in experimental studies to suppress cyclic nucleo- effects on other cellular functions that are unrelated to tide breakdown by PDE. Recent advances in PDE re- PDE. Compounds, such as antagonists of CaM or Ca2ϩ- search indicate that previously assumed nonselectivity chelating agents, do selectively inhibit PDE1 as also do of IBMX is not universal. A PDE activity insensitive to other Ca2ϩ/CaM-dependent enzymes and membrane IBMX was found in a fraction of rat liver homogenate channels. EHNA, the only known selective inhibitor of [107]. Newly identified PDE types, cAMP-specific PDE7 PDE2, was first known as a potent inhibitor of adenosine and PDE8, and cGMP-specific PDE9 are resistant to deaminase [77]. Dipyridamole, a compound that inhibits inhibition by IBMX at 0.1 mm or less. These novel find- specifically PDE5, PDE6, and PDE8, also blocks trans- ings may explain why, even with maximum inhibitory port of adenosine [7], and the methyl xanthine derivative IBMX concentration, certain cAMP-PDE and cGMP- denbufylline, a selective inhibitor of PDE1 [7], is also an PDE cannot be suppressed [35, 107]. This “residual activ- adenosine receptor antagonist. The existence of effects ity” of PDE [73] found in tissue extracts is likely caused unrelated to PDE does not preclude these compounds by activities of PDE7, PDE8, and PDE9, which are ex- from being used as selective PDE isozyme inhibitors, pressed in numerous tissues. This new information providing that these side effects are kept in mind in should be kept in mind when interpreting experiments interpreting the results and when appropriate controls with the use of IBMX, both in cell-free systems and in are included in the experimental design. intact cells. Despite obvious limitations, “first-genera- Phosphodiesterase inhibitors that discriminate be- tion” PDE inhibitors were very helpful in probing tween PDE isozymes that are products of a single isogene cAMP-mediated actions, especially in intact cells. In fact, (such as PDE subfamilies or PDE subtypes) within one one of Robison et al’s four criteria [108] to determine gene family, or even discriminate between splice variants whether hormonal response is mediated by cAMP in- of PDE isoforms (the putative “third-generation” PDE cludes the requirement that the observed functional re- inhibitors) have yet to be developed. However, because sponse is synergistically enhanced by the addition of of intense efforts by many pharmaceutical drug-discov- PDE inhibitors [108]. These inhibitors continue to be ery groups, such PDE inhibitors may soon become avail- widely used in clinical practice. able. In fact, some novel inhibitors of PDE4 show some Isozyme-selective PDE inhibitors. Over the last 15 degree of selectivity for PDE subtypes. For example, years, the search for more potent PDE inhibitors yielded compound CP77059 inhibits subtype PDE4D to approxi- a large number of compounds that were synthesized or mately a tenfold greater degree than PDE4A, PDE4B, identified as novel PDE inhibitors. These compounds and PDE4C [43]. Another novel compound, RS33793, have higher affinity (Ki Յ 1 ␮m) for PDEs than nonselec- displays high potency and selectivity for the “long” iso- tive inhibitors and, even more importantly, show selectiv- form PDE4D3 when it is phosphorylated by PKA; ity for various gene families (types) of PDE isozymes RS33793 inhibits phosphorylated PDE4D3 with approxi- [7, 9, 17, 23]. These “second-generation” PDE inhibitors mately 300 times greater potency than in the dephos- [7, 9, 17] do exhibit about 100-fold selectivity for PDE phorylated state [86, 87]. This observation indicates that isozymes of a particular type (family), as assessed by the sensitivity of PDE subtypes and isoforms to selective

IC50 when measured under comparable conditions [10]. PDE inhibitors may depend on the functional/biochemi- To date, second generation, type-selective PDE inhibi- cal state of PDE isozyme and hence on the conditions tors have been found or developed for selective inhibi- of testing. tion of PDE1, PDE2, PDE3, PDE4, and PDE5 (Table Several selective PDE inhibitors that act simultane- 2). Photoreceptor PDE6 is sensitive to the same selective ously on two distinct PDE isozymes are referred to as inhibitor as PDE5, whereas no specific inhibitor of PDE7 “hybrid,” “combination” or “dual” inhibitors [10, 137, has yet been found [50–52, 99]. Most “second-genera- 138]. Such inhibitors were developed based on the notion tion” PDE inhibitors identified to date are selective for that simultaneous inhibition of two PDE isozymes has PDE3 or PDE4 [7, 9–11, 33, 34, 44, 92]. a synergistic pharmacologic effect [137]. The most com- Even these type-selective PDE inhibitors are not abso- mon dual inhibitors simultaneously block the activities lutely specific, particularly at higher concentrations. For of both PDE3 and PDE4, PDE3/4 inhibitors [137, 138], example, zaprinast at lower concentrations inhibits selec- and were developed with the aim of suppressing two tively PDE5, and at higher concentrations, it inhibits major pathogenic components of bronchial asthma: in- also PDE1 [95, 136] and newly discovered PDE9. There- flammation and bronchoconstriction [26, 44, 90–92]. fore, in designing experiments with the use of such inhibi- Dual PDE4/5 inhibition was observed with WIN58237 tors, the proper concentration and other conditions [95]; dual PDE1/5 inhibition with zaprinast [95] and dual Dousa: Cyclic-3Ј,5Ј-nucleotide PDE isozymes 41

Table 2. Second-generation PDE isozyme inhibitors and their pharmacologic properties PDE type Pharmacotherapeutic applications, (family) Inhibitory compounds Pharmacologic actions experimental and clinical PDE1 vinpocetine nootropic Dementia, memory loss [109, 110] 8-MeoM-IBMX PDE2 EHNA (MEP-1) inhibits platelet aggregation increases L-type Ca2ϩ current in heart PDE3 milrinone, vesnarinone, inotropic Congestive heart failure [34, 82, 111, 112] cilostazol, cilostamide vasorelaxation thrombosis [113, 114] (and many others) antithrombotic pulmonary hypertension [115, 116] antiproliferative glomerulonephritis [117, 118]

PDE4 lower affinity: (Ki Ӎ 1 ␮m) antidepressive Bipolar depression [119, 120] rolipram, Ro-20-(724) immunosuppressive bronchial asthma [27, 92, 121] denbufylline anti-inflammatory atopic diseases [122, 123] high affinity: (Ki Ӎ 1nm) (suppress superoxidation and generation autoimmune enceptholomyelitis [124, 125] CDP 840 of TNF␣, IL-1␤, INF␥) organ transplantation [126–129] RP 73401 smooth muscle relaxation CP 80, 633, RS 33793 PDE5 zaprinast (M&B 22,948) vasorelaxation, natriuretic, antithrombotic Pulmonary hypertension [130, 131] dipyridamole salt retention in nephrotic syndrome [132, 133] E-4021, WIN 58237 erectile dysfunction [97, 134] sildenafil (Viagra௡) organ transplantation [135] DMPPO PDE6 zaprinast dipyridamole PDE7 none known PDE8 dipyridamole PDE9 SCH 51866 zaprinast

PDE3/5 inhibitors have recently been reported [95]. as valuable tools for in vitro and in vivo investigations Some inhibitors that were until recently considered spe- of the PDE superfamily’s function. Enhancement or po- cific (or at least relatively specific) for one PDE type tentiation of tissue response to hormones and autocoids turned out to be “hybrid” inhibitors. Dipyridamole, al- by first-generation PDE inhibitors was one of the key ready known as a potent and specific inhibitor of cGMP- criteria proving that the response is mediated by cyclic specific isozyme PDE5 [10, 11, 96], also was shown to nucleotides [108]. inhibit newly discovered cAMP-specific isozyme PDE8 Selectivity for second-generation PDE inhibitors is so [53, 54]. Compound SCH 51866, a dual inhibitor of PDE1 prominent that when used in the proper experimental and PDE5 [100], turned out to inhibit also PDE9 [55], design, the effects of these inhibitors could be considered and in view of this, it may be considered a “triple” PDE “diagnostic” for detecting the presence of PDE isozyme inhibitor. Although the rationale for the usefulness of (types) in cells and tissues, or for their involvement in such compounds remains to be seen, it is arguable functional responses that are mediated by cyclic nucleo- whether dual inhibitors with fixed ratio of activities can tides in vivo and in vitro [7, 9, 72, 73]. Two or more be superior to combination of two monospecific PDE selective PDE inhibitors of distinct chemical structure inhibitors that can be administered in various ratios of ought to be used, whenever feasible, as probes in such doses to yield optimum pharmacologic effects. experiments. According to several reports, some selective PDE in- With the use of PDE isozyme inhibitors and activators hibitors can suppress to a different extent the activities of the same PDE isozyme isolated from different tissues. of PDE modulators, it is possible to detect the presence The PDE3 inhibitor vesnarinone had approximately 10- of PDE isozymes even in crude cell-free preparations times higher potency to inhibit PDE3 from heart or kid- [9, 73]. When PDE activities are measured in cell homog- ney than PDE3 from other organs [139]. Gensenoside enates or in a fraction of the homogenate, the effect of derivatives inhibit PDE1A from myocardium but not a PDE isozyme–selective inhibitor indicates the presence PDE1B from brain [140]. of PDE type; maximum inhibition can approximate the abundance of the PDE isozyme [9, 73]. Such analysis PDE inhibitors as experimental tools and drugs of cAMP-PDE isozymes is schematically illustrated in From the very inception of cyclic nucleotide research, Figure 5. Maximum inhibition of cAMP-PDE activity by PDE inhibitors, both nonspecific and selective, served selective PDE3 and PDE4 inhibitors approximates the 42 Dousa: Cyclic-3Ј,5Ј-nucleotide PDE isozymes

and PDE5, that is, activities in the absence of added Ca2ϩ/ CaM or cGMP. In an analogous manner, the specific activators Ca2ϩ/CaM and cGMP are used to determine PDE1 and PDE2 activities, respectively (Fig. 5B). Selec- tive inhibitors may also be used. Direct inhibitors of PDE1, such as vinpocetine or 8-MeoM-IBMX, suppress not only the Ca2ϩ/CaM-dependent component but also basal activity of PDE1. When activity of some PDE isozyme type is not detected using this approach, the results should be interpreted with caution. Possibly, the optimal conditions for detecting the PDE isozyme activity such as substrate concentration or cofactors might not have been employed. Moreover, activities of some PDE isozymes may depend on conditions such as the state of phosphorylation [11, 26, 33, 44, 86, 87]. If both PDE2 (stimulated by cGMP) and PDE3 (inhibited by cGMP) isozymes are present in comparable activities, no net effect of added cGMP on cAMP-PDE activity would be apparent [9]. Needless to say, the presence of PDE isozymes determined with the use of inhibitors and activators ought to be confirmed by direct methods. With these qualifications kept in mind, probing with selective inhibitors and modulators is a most useful approach for initial assessment of PDE isozyme patterns in unex- plored cells or tissues [9, 72, 73]. Exploration of the PDE isozyme pattern in extracts from nephron segments and cultured renal cells showed diverse expression of PDE isozymes (Table 3). Sensitivity to selective PDE isozyme inhibitors and modulators is also used for characterization of PDE isozymes in fractions of cell tissue extracts obtained by chromatography and other separation meth- Fig. 5. Example of an experimental design to detect and assess PDE ods [7, 101]. isoenzymes semiquantitatively in a tissue extract, using selective PDE Selective PDE isozyme inhibitors are also employed inhibitors and biomodulators. The values of cAMP-PDE activity and cAMP-PDE activity in the absence of modulators or inhibitors is taken in principle, using a similar approach in studies of the as 100%. (A) PDE inhibitors. The PDE3 activity and PDE4 activity whole cell in vitro to identify functions that are regulated are assessed by inhibiting cAMP-PDE with maximum dose of the PDE3- by cAMP-PKA pathways and linked to a specific PDE selective inhibitor cilostamide, and PDE4 by inhibition with the selective inhibitor PDE4 rolipram. When added together, the effects of both isozyme(s). Various preparations, such as freshly isolated inhibitors are additive. The “residual activity,” not sensitive to inhibition cells or cells grown in culture, tissue slices, etc., are incu- by cilostamide and rolipram combined, may reflect, in part, PDE7 or bated in vitro with PDE isozyme inhibitors alone or with PDE8 (which are insensitive to these inhibitors), as well as basal activities of PDE1 and PDE5 (activities measured in the absence of added adenylate cyclase stimulated either directly by forskolin Ca2ϩ/CaM or cGMP. (B) PDE biomodulators. PDE2 is determined by or by agents that act to cyclase-coupled receptors. The the cAMP-PDE activity sensitive to maximum stimulation by added effect on cAMP content, in situ activity of PKA deter- cGMP, and PDE1 is activity of cGMP-PDE maximally stimulated by Ca2ϩ with calmodulin (CaM). Maximum effective concentrations of mined by the (Ϫ cAMP/ϩ cAMP) PKA activity ratio, inhibitors and modulators are based on dose–response curves. and the specific cellular functions are determined. Using this strategy, for example, we found that in glomerular mesangial cells (MCs), a PDE3-linked cAMP pathway suppresses mitogenesis, whereas cAMP-PKA linked to relative proportion of the two PDE isozymes in the cell PDE4 selectively modulates reactive oxygen metabolite extract (Fig. 5A). The “residual activity” that remains (ROM) generation (Fig. 6) [15, 66, 143]. Signal transduc- after maximum inhibition with PDE3 and PDE4 inhibition via cGMP pathways is probed in an analogous man- tors combined (which are additive) is likely due, at least ner, except that measurement of in situ activation of in part, to activity of PDE7, an isozyme that is specific PKG is not applicable in cellular responses when cGMP for cAMP and is not sensitive to the inhibitors of PDE4 acts directly on an ion channel or PDE2 [3]. In the pres- and PDE3 [51, 52, 73], and is relatively insensitive to ence of PDE isozyme inhibitors, the activity of the cAMP IBMX [28, 99] as well as the basal activities of PDE1 PKA pathway is increased even in a state when adenylate Dousa: Cyclic-3Ј,5Ј-nucleotide PDE isozymes 43

Table 3. Approximate patterns of PDE isozyme in extracts from glomeruli, tubules and cultured renal cells. cAMP-PDE cGMP-PDE PDE1 PDE1 (cAMP) PDE2 PDE3 PDE4 (cGMP) PDE5 rat glomeruli ND ϩϩϩϩ ϩϩ ϩϩ ϩϩ ϩϩϩϩ rat glomerular epithelial cells (SGE1 cells) ND ND Ϯ ϩϩϩϩ ND ND rat mesangial cells (MC) ND ND ϩϩϩ ϩϩϩϩ ϩϩϩ ϩϩϩ rat cortical tubules (suspension) ϩ ND ϩϩ ϩϩϩ ϩϩϩ ϩϩϩ OK cells ϩ ND ND ϩϩϩϩ ϩϩϩϩ ϩ LLCPKi cells ϩϩ ϩ Ϯ ϩϩϩϩ ϩϩϩϩ ϩϩ rIMCD cell ϩϩ ND ϩ ϩϩϩ ϩϩϩϩ ϩ PDE isozyme activities were determined using the method portrayed in Fig. 5. Proportion of PDE isozyme (types) relative to total cAMP-PDE or cGMP-PDE activity; not accounted for is “residual activity” that likely includes activity of PDE7 and PDE8 that is not inﬂuenced by known inhibitors or modulators of PDE isozymes. cGMP-PDE activity insensitive to Ca2ϩ-calmodulin may also include PDE9. ND ϭ not detected.

Fig. 6. Effect of the PDE3 inhibitor cilostamide (Cs) and the PDE4 inhibitor rolipram (R), of the combination of R ϩ Cs, and of forskolin (FSK) upon activation of protein kinase A (PKA) (A and B), the mitogenesis assessed by [3H]-thymidine incorporation (C) and upon ROM generation in cultured rat mesangial cells (D). Abbreviations are: Co, controls; R, rolipram (10 ␮m); Cs, cilostamide (10 ␮m); R ϩ Cs, 10 ␮m rolipram ϩ 10 ␮m cilostamide; FSK, forskolin (10 ␮m). Each bar represents the mean Ϯ sem of 5 to 7 observations. The statistical significance is by the t-test. Black bars (right) are for values of mitogenesis; stip- pled bars (left) denote values for ROM generation. Panels A and B show in situ activation of PKA determined by increase of (– cAMP/ ϩ cAMP) PKA ratio. R, Cs, R ϩ Cs and FSK all significantly activated PKA. #Values significantly different from values with R or Cs alone or with FSK. All stimulated values (Cs, R, Cs ϩ R or FSK) were significantly different from controls. In panel C, the inhibition of mitogenesis determined by incorporation of [3H]-thymidine is shown. *Value significantly different from Cs, R ϩ Cs, and FSK; the values for Cs alone, R ϩ Cs and FSK are not different from each other. Panel D shows the inhibition of ROM generation. Values for R, R ϩ Cs, and FSK were not significantly different from each other; ϩdenotes no significant inhibitions. The content of cAMP did not increase in mesangial cells incubated with R or Cs alone, with R ϩ Cs increased 3.5 times, and with FSK, 20 times. Data are from [43, 95].

cyclase is not stimulated (Fig. 6) and even more when differ substantially, as can be demonstrated in studies of the cyclase is stimulated by submaximal concentration MCs [15, 66]. In general, the combination of submaximal of agonist or activator [15, 66, 141]. However, the extent stimulation of adenylate cyclase by agonists with addi- of the cAMP response in different compartments may tion of selective PDE isozymes inhibitors results in a 44 Dousa: Cyclic-3Ј,5Ј-nucleotide PDE isozymes pronounced increase in cAMP content, the activation of studied in a more focused way with the use of whole cAMP PKA signaling pathways, and related functional kidney parenchyma as a source [59, 144]. However, in responses [8, 9]. Because in vivo turnover rate of cyclic the investigation of adenylate and guanylate cyclases nucleotides is higher than in vitro, PDE isozyme inhibi- that are regulated by hormones, neurotransmitters and tors have accentuated effects to activate the cAMP-PKA autocoids attracted much more attention than the inves- pathway in vivo [7]. Thus, when a PDE isozyme inhibi- tigation of PDEs. Consequently, studies devoted to PDE tor is administered in vivo, it cannot be presumed that in the kidney are relatively few. Results of studies of its effects are quantitatively commensurate to dose– PDE activities prepared from whole kidney tissue pro- responses determined in vitro either in cell-free or in vided initial but limited information. In view of advances whole-cell systems [7, 9]. Cells and tissues in vivo are in renal pathophysiology, it became obvious that expres- under the tonic influence of hormones, autocoids, and sion and functional significance of PDE isozymes ought neurotransmitters that modify activities of adenylate and to be investigated in specific nephron segments, subseg- guanylate cyclases. Under these circumstances, PDE in- ments, or renal cells freshly isolated or grown in culture. hibitors will be more effective in activation of the cAMP- Only in the late 1970s and early 1980s did the enor- PKA pathway in vivo at lower doses than predicted from mous heterogeneity of the nephron regarding the regula- in vitro studies [7, 9]. Other factors that can influence tion by hormones that act through cAMP and signaling the effect of PDE inhibitors in vivo include pharmacoki- become recognized [145]. The concept of a superfamily netics of the compounds, penetration into tissues and of well-defined PDE isozymes emerged in the late 1980s, cells, and simultaneous effects in two or more organs. and “second-generation” PDE isozyme inhibitors be- Nevertheless, interpretation of results from studies in came available at that time [7, 9–11, 23]. Therefore, only vivo is less definitive than results from in vitro experi- a rather limited number of studies have focused on the ments and should be carefully weighed in the context of functions of PDE isozymes or isoforms in specific neph- other information. ron segments and homogenous renal cell populations to date. This development may be illustrated by the studies PDE isozyme inhibitors as pharmacologic agents of the cAMP-mediated signaling system in the antidiure- Many PDE isozyme inhibitors are recognized pharma- tic response to [8-Arg]-vasopressin (AVP) in a murine cologic agents (Table 2). In fact, some compounds were model of nephrogenic diabetes insipidus (NDI), the so- employed as drugs in medical practice long ago before called NDI mice [146–149]. In the first study of NDI mice, they were identified as PDE inhibitors, such as theophil- involvement of PDE was neither detected nor suspected line, papaverine, or dipyridamole. Currently, PDE iso- [146]. Later investigations on microdissected segments of zyme inhibitors, both nonspecific and PDE isozyme collecting ducts established that increased PDE activity selective, are vigorously and intensely explored as thera- plays a dominant role in the failure to elicit an increase peutic agents, experimentally and clinically [8, 10, 34], in cAMP in response to stimulation by AVP [147], and an and some PDE inhibitors are already approved for clini- even later study with the use of selective PDE inhibitors cal use, such as the “cardiotonic” inhibitor milrinone pointed to the abnormally high activity of PDE4 in col- in the treatment of congestive heart failure [34, 82] or lecting ducts as a key pathogenic factor [106–150]. cilostazol [81] as an antithrombotic agent. Many are in various phases of clinical and preclinical studies (Table PDE isozymes as studied in nephron segments 2). Currently, the most frequently available are inhibitors Information on PDEs in subdivisions of the nephron of PDE3, PDE4, and PDE5. The main potential applica- is based on studies of isolated glomeruli, suspension of tion of those PDE isozyme inhibitors, as preclinical trials tubules, and microdissected nephron segments. and observations from clinical practice show, is their use Suspensions of glomeruli and tubules. The initial study as antithrombotic, vasodilatory, and cardiotonic-inotro- of rat glomeruli showed that activities of cGMP-PDE pic agents and as central nervous system antidepressants are higher than cAMP-PDE and that cGMP stimulates and anti-inflammatory agents (Table 2). cAMP-PDE [151]. Recent studies using selective PDE inhibitors indicate the presence of PDE2 in high activity, with lower activities of PDE3 and PDE4, all of which PDE ISOZYMES IN KIDNEY are isozymes that hydrolyze cAMP modulators (Fig. 7 AND KIDNEY EPITHELIA and Table 3) [152, 153]. In glomeruli, cGMP is hy- The existence of enzymatic activity in the kidney that drolyzed by two isozymes; PDE5 is more abundant than catalyzes hydrolysis of cAMP and later cGMP was noted PDE1 [152]. Localization of PDE isozymes within the in very early studies when the cAMP system was sur- three major cell types that populate glomeruli [154] is veyed in numerous organs and tissues that were well- not yet known or is only suspected by comparison with known target tissues for hormone action [142, 143]. Sub- PDE isozyme profiles in glomerular cells grown in cul- sequently, PDE from whole kidney preparations was ture [66, 155]. Knowledge of PDE isozyme patterns in Dousa: Cyclic-3Ј,5Ј-nucleotide PDE isozymes 45

Fig. 7. Pattern of PDE isozymes in glomeruli (C and D) and in tubules (A and B) freshly prepared from rat renal cortex. Ordinates denote PDE activity fmol/min/mg protein. The PDE isozyme activities were determined by the method portrayed in Fig. 5. Data are from [108].

glomeruli provides useful basic information for studies measuring cAMP-PDE and cGMP-PDE activities in mi- of pathophysiologic changes in cyclic nucleotide signal- crodissected segments of tubules in the studies by Jack- transduction systems. For example, increased cGMP- son, Edwards and Dousa [157]. Data on PDE activities PDE activity and defect in cGMP accumulation that can in microdissected tubule segments come mostly from be corrected by the PDE5 inhibitor zaprinast was found studies done in the context of various specific topics, and in glomeruli from nephrotic rats [132, 156]. Although the thus it is sometimes difficult to compare them with each type of cells containing increased cGMP-PDE remains to other [158–164]. Activities of cAMP-PDE were explored be determined, analysis of the cGMP responses to ANP in eight tubule segments microdissected from kidneys of in whole glomeruli already points to increased PDE5 three animal species frequently employed for experi- activity as a pathogenic factor [132]. mental studies: rat, mouse [165], and rabbit (Kusano and Phosphodiesterase isozymes in extracts of tubule sus- Dousa, unpublished data). There is a tendency toward pensions from cortex show a substantially different pro- higher cAMP-PDE activity in cortical segments distal to file than from glomeruli (Fig. 7 and Table 3) [152]. PDE4 the loop of Henle, as compared with segments of proxi- has the highest activity, while PDE3 and PDE1 are much mal tubules [165]. Although in some segments cAMP- less active; all of these PDEs hydrolyze cAMP. In con- PDE activities are not different between species, distinct trast to glomeruli, where PDE2 is not detectable, cGMP differences were seen in cortical thick ascending limb is hydrolyzed equally by PDE1 and PDE5 (Fig. 7). More (CTAL) and distal convoluted tubule (DCT) of mouse nephron segment-specific information was obtained from and rabbit (unpublished observations). (Designation of 46 Dousa: Cyclic-3Ј,5Ј-nucleotide PDE isozymes tubule segments are according to the international sys- ble, cyclic nucleotide signaling and PDE isozymes may tem [166].) be well preserved in cultured cells. For example, a direct Most information about PDEs has accumulated from comparison of the cAMP signaling system in vascular studies of segments of collecting ducts; however, virtually smooth muscle cells grown in primary culture from rat all were conducted without using a selective PDE inhibi- aorta and cells from freshly dissected aortic tissue re- tor or modulator. Results of these studies show that vealed that hormones and PDE inhibitors had in princi- cGMP-PDE activity is higher than cAMP-PDE activity ple a similar effect on fresh tissue and cells cultured from in the outer medullary collecting duct (OMCD) [148, aorta, and responses of the cAMP system differed only 157], inner medullary collecting duct (IMCD) [148], and in time course and magnitude [175]. The pattern of PDE medullary thick ascending limb (MTAL) [159]. Activa- isozymes in rat MCs grown in primary culture is pre- tion of cAMP-PDE by Ca2ϩ in MTAL and in OMCD served in multiple passages [66]. The pattern of PDE indicates the presence of PDE1 [162], and effects of isozyme activities in several types of renal cultured cells, selective PDE inhibitors indicate the presence of PDE4 determined in our laboratory, is outlined in Table 3. and PDE3 in IMCD [148, 149]. There is even less infor- Cells of tubular epithelium. LLCPK1 cells are an estab- mation about PDEs in proximal cortical segments [163]. lished line of renal tubular cells with AVP-sensitive ade- Of potential interest is a report that in a strain of mice nylate cyclase and have become a frequently used model with hereditary X-linked hypophosphatemia (Hyp mice) for studies of cAMP signaling in renal epithelia. The cAMP-PDE activity in proximal convoluted tubule study of PDEs in wild LLC-PK1 cells revealed the pres- (PCT) is lower but that it is not different from controls ence of five PDE types, PDE1 to PDE5 [102]. The pres- in proximal straight tubule (PST) or DCT [167]. In rats ence of PDE7 has not been excluded, and the evidence fed a low-phosphate diet, cAMP-PDE, but not cGMP- for the presence of PDE3 is marginal, based solely on PDE, is increased in cortical extracts [168]. cAMP-PDE minor effects of PDE3 inhibitors in cell extracts [102]. activity was significantly higher in the PCT but not in It appears that PDE1 and PDE4 are the isozymes that the PST of phosphate-depleted rats [169]. Stimulation of play a role in metabolism of the cAMP pool generated PKC increased cAMP-PDE, apparently PDE4, in OMCD in response to AVP [102, 176–178]. Although PDE2 is but not in TMAL or PST of the rat nephron [170]. Obser- not abundant, its functional significance is suggested by vations based on indirect evidence reported by another the finding that atrial natriuretic peptide (ANP)-stimu- group suggest that PKC activation may increase PDE1 lated increase in cGMP reduced increases of cAMP in activity in rabbit proximal tubular cells [171]. response to arginine vasopressin (AVP) [102]. The mechanism of PDE1 may be conceivably analo- Further insights on PDEs were obtained from studies gous to the reported induction of PDE1B by PKC-␣ or of LLCPK1 cells stably transfected with PDE4D1A (in PKC-⑀ [28]. the original paper [176], the enzyme was denoted as “rat The distribution of the PDE isozyme system remains PDE3.1,” which is synonymous with the new standard to be explored with current, refined methods. The avail- abbreviation PDE4D1A [8, 23]), a “short form” of ability of selective PDE isozymes, as well as the possibil- PDE4. In cloned LLCPK1-S#16 stably transfected with ity of determining mRNA encoding various PDE proteins PDE4DA1, PDE4 activity was fivefold to sevenfold in nephron segments by reverse transcriptase-polymerase higher than sham-transfected controls, and the increase chain reaction [172, 173], by in situ hybridization [29, 51, of cAMP in response to AVP or to forskolin was virtually 70], and by immunohistochemical methods [30, 71, 174] obliterated. It was restored by the addition of the PDE4 should make the study of PDE isozyme systems in spe- inhibitor, rolipram [176]. In control cells, inhibition of cific nephron segments a rewarding subject of future PDE1 with the selective inhibitor 8-MeoM-IBMX en- investigations. hanced AVP-stimulated cAMP accumulation, as did inhi-

bition of PDE4 by rolipram [176]. However, in LLCPK1- PDE isozymes in renal cells grown in culture S#16 cells with overexpressed PDE4, only rolipram, but Another source of information on the function of PDE not 8-Meo-IBMX, restored the stimulatory effect of isozymes in renal cells comes from studies of either con- AVP. These results suggest that both PDE4 and PDE1 tinuous renal cell lines or renal cells grown in primary can be operant in the catabolism of the AVP-dependent culture. Inherent limitations of the cell culture approach cAMP pool, but PDE1 inhibition cannot compensate for in projecting results to in vivo cells are generally known. overexpressed PDE4, a feature that points to PDE4 as However, the homogeneity of the cell population and the key PDE isozyme that controls AVP-dependent the possibility to conduct simple and well-defined experi- cAMP metabolism in LLCPK1 cells [176–178]. mental maneuvers are advantages of these studies. Al- Because PDE4D1 is a “short form” of PDE4 [12] and though the possibility that cultured cells may dedifferen- is inducible by cAMP [45, 50], this feature allows selective tiate or change the phenotype in the course of in vitro experimental manipulation of PDE4 activity, but not of growth must be kept in mind and controlled where feasi- other PDE isozymes. Incubation of LLCPK1-S#19 clone Dousa: Cyclic-3Ј,5Ј-nucleotide PDE isozymes 47 cells with only slightly overexpressed PDE4D1A, with bilized in vitro for six days [178]. The pattern of PDE agents that increase cellular cAMP [forskolin, dibutyryl isozymes in these IMCD cells resembled that of rIMCD, cAMP (DBcAMP), or with PDE4 inhibitors Ro-20-1724 but activity of the PDE5 isozyme was also detected [178]. or rolipram] resulted in a significant increase in PDE4 PDE isozymes in glomerular cells. The pattern of activity. Exposure of LLCPK1-A#1 cells to increasing PDE isozymes has been determined in rat mesangial doses of Ro-20-1724 resulted in a gradual increment in cells grown in primary culture [66]. We found that cAMP PDE4 activity [177, 178]. The increase in PDE4 activity is hydrolyzed by PDE4 and to a somewhat lesser degree (maximum: ⌬ϩ60%) was inversely correlated (r ϭ by PDE3 (Table 3), but other isozymes with cAMP- Ϫ0.88; P Ͻ 0.05) with the decrease (maximum ⌬Ϫ70%) PDE activity were not found. Three PDE4 isoforms were in AVP-dependent cAMP levels, but basal cAMP levels identified in rat mesangial cells [69]. These include remained unchanged [177, 178]. These results further RNPDE4A5, an isoform that has proline-rich SH3-bind- support the hypothesis that PDE4 is the isozyme that is ing domains [37], RNPDE4D3, which is regulated by primarily linked to the cAMP pool generated by AVP- phosphatidic acid [48] and via phosphorylation by PKA stimulated adenylate cyclase. The finding is also conso- [47], and RNPDE4D5. Isozymes PDE7 and PDE8 most nant with the notion that anomalously high activity of likely account for “residual” cAMP-PDE activity. Activi- PDE4 can obliterate the cAMP response to AVP in NDI ties of the cGMP-hydrolyzing enzymes PDE1 and PDE5 mice [147–149]. The LLCPK1 cell line differs consider- are about the same [66], however, a portion of PDE5 ably from tubular epithelial cells in vivo. Nevertheless, activity is likely due to the presence of PDE9. In chro- this model allows investigations of basic relationships matographic fractions of mesangial cell homogenates, between AVP-dependent cAMP dynamics and PDE iso- PDE1, PDE2, and PDE4 were found, but PDE3 and zymes [178]. PDE5 were not detected [181].

Transfection of opossum kidney cells, a cell line with The glomerular epithelial cell line, “SGE1 cells” iso- many properties of proximal tubular cells, with PDE4D1A lated from rat glomeruli, retains many characteristic led to overexpression of PDE4 and down-regulation of properties of wild glomerular epithelial cells [182]. We

D1 dopamine receptors coupled to adenylate cyclase analyzed extracts from SGE1 cells and found high activity [135]. The response of the cells to dopamine was blunted, of PDE4, marginal activity of PDE3 and no PDE2 or but there was no decrease in dopamine-stimulated ade- PDE1. Surprisingly, no cGMP-hydrolyzing PDE activity nylate cyclase activity when it was assayed in isolated and no PDE1, PDE5, or PDE2 were detected in these membranes from transfected cells [179]. In two trans- cells [155]. Thus, the PDE isozyme profile in SGE1 cells fected clones of opossum kidney cells with overexpressed substantially differs from that of mesangial cells [66]. PDE4, the increase in in situ PKA activity in response Glomerular endothelial cells have not yet been investi- to dopamine was abolished. Because the in situ activation gated, but by analogy from PDE isozyme profiles re- of PKA is a sensitive index of cAMP-PKA pathway ported for endothelial cells from other organs [183, 184], activation (Fig. 6) [66], these results indicate that in- these cells are a likely source of high PDE2 activity found creased PDE4 activity can completely obliterate stimula- in extracts from whole glomeruli [152, 153]. tion of the cAMP-PKA pathway by the hormone [179]. Similarly, AVP not only increased cAMP in microdis- PDE ISOZYMES IN RENAL sected collecting ducts of NDI mice, but it also failed PATHOPHYSIOLOGY to increase in situ PKA activity. The addition of PDE isozyme inhibitors restored the PKA response [149]. Pathophysiology of glomeruli In a study of IMCD cells isolated from rat kidney and Studies of isolated glomeruli in several animal models grown in culture (rIMCD), which retain some biologic of renal disease as well as of cultured glomerular cells response of wild IMCD cells, the isozymes PDE1, PDE3, point to the role of PDE isozymes in the pathobiology and PDE4, but not PDE2 and PDE5, were detected of both noninflammatory and inflammatory glomerulo- [180]. The PDE4 inhibitor rolipram increased accumula- pathies. tion of cAMP, both basal and stimulated by AVP, Noninflammatory glomerulopathies. Several major fluid whereas the AVP-dependent cAMP pool was metabo- and electrolyte disorders are characterized by the inabil- lized primarily by PDE1 [180]. This pattern of response ity of the kidney to excrete adequate amounts of salt. is in some points similar and in others different from the Various mechanisms accounting for renal NaCl retention response of LLCPK1 cells to AVP [102, 176]. The levels are involved, including abnormal function of glomeruli of cGMP in rIMCD cells, both basal and stimulated with with inadequate delivery of ultrafiltrate to the tubules ANP, were enhanced by inhibitors of PDE1 [180]. To [132]. Glomeruli along with IMCD are major targets for compare properties of rIMCD cells (up to 20 passages) the action of ANP in the nephron [125]. Humphreys et with IMCD freshly isolated from kidney, PDE isozymes al investigated whether cGMP-mediated responses of were determined in IMCD cells freshly isolated and sta- glomeruli to ANP may be impaired in noninflammatory 48 Dousa: Cyclic-3Ј,5Ј-nucleotide PDE isozymes

Fig. 8. Cyclic GMP accumulation in glomeruli and inner medullary collecting ducts (IMCD), and cGMP-PDE activity in IMCD of control rats ( ; C) and rats with nephrotic syndrome ( ; N) due to Heymann nephritis [140]. Each bar denotes mean Ϯ sem; N ϭ 5to 10. The accumulation of cGMP was measured without or with 1 ␮m ANP, without or with 1 mm zaprinast added into the incubation. *Val- ues signiﬁcantly lower than corresponding controls; #cGMP-PDE activity in nephrotic syndrome ( ); signiﬁcantly higher than controls ( ) by the t-test. Data are from [140] with permission of the authors and the Journal of the American Society of Nephrology.

experimental nephrotic syndrome induced in rats by This is contrasted with the authors’ ﬁndings in hypergly- adriamycin, a condition marked by NaCl retention [132, cemic diabetic rats, in which ANP-stimulated cGMP ac- 133, 156]. Circulating ANP levels and ANP binding to cumulation in glomeruli is also reduced but cannot be renal receptors were not diminished, but when glomeruli corrected by inhibiting PDE5; decreased density of ANP from nephrotic kidneys were incubated in vitro with ANP, receptors likely accounts for the resistance to the hor- the accumulation of cGMP was decreased, and the ad- mone [185]. dition of the PDE5 inhibitor zaprinast restored ANP- Measurement of ANP-sensitive cGMP accumulation dependent cGMP accumulation to the normal level [156]. in glomeruli isolated from kidneys of salt-depleted rats Similar hyporesponsiveness of the glomerular cGMP sys- showed lower cGMP accumulation than in controls, and tem to ANP, which was corrected by zaprinast (Fig. this deﬁciency was corrected by the PDE5 inhibitor za- 8), was observed in rats with nephrotic syndrome that prinast [186], an inhibitor of PDE5 and PDE9 [55, 56], develops in Heymann nephritis, a model of human mem- or by cicletanine, a compound believed to inhibit PDE1 branous nephropathy [133]. These observations clearly [187]. The ANP-sensitive cGMP system was studied in show that resistance of nephrotic glomeruli to ANP is a glomeruli from kidneys of dogs with experimental con- postreceptor defect due to high cGMP-PDE activity. gestive heart failure produced by rapid ventricular pacing Dousa: Cyclic-3Ј,5Ј-nucleotide PDE isozymes 49

[188]. Accumulation of cGMP in glomeruli in response tent as forskolin, without measurably increasing cAMP to ANP was greatly reduced, and the activities of PDE1 content. However, rolipram increased the in situ activity to PDE5 were increased in glomeruli [188]. The dynam- of PKA to a far lesser extent than forskolin, and the ics of cGMP were examined in glomeruli from kidneys of inhibitory effect of rolipram on ROM generation was rats with high-output heart failure caused by aortocaval attenuated by a PKA inhibitor [153]. Cilostamide, an fistula. After prolonged incubation of glomeruli with inhibitor of PDE3, had much less suppressive effect on ANP, accumulation of cGMP in glomeruli from heart- ROM generation, and inhibitors of PDE1 and PDE5 failure kidneys was less than in controls, and rather para- had no effect at all [153]. In general, these findings indi- doxically, the glomerular cGMP-PDE activity was also cate that the cAMP-PKA signaling pathway can be acti- lower [189]. The same authors observed that cGMP- vated by a decrease in PDE4 isozyme activity without a PDE activity is decreased in glomeruli from rats with detectable increase in cAMP content as measured by unilateral ureteral obstruction [190]. standard radioimmunoassay, yet this subtle activation of All these data indicate that cGMP metabolism, in par- the cAMP-PKA pathway produces a functional response ticular the ANP-dependent cGMP pool, is dysfunctional of the same magnitude as the response of “flooding” of in glomeruli stressed by electrolyte-fluid disturbances in cells by cAMP in response to forskolin or DBcAMP different pathophysiologic models. The initial evidence [153]. Results show that activation of the cAMP-PKA points to increased PDE5 (and perhaps also PDE1 and pathway in glomeruli decreases ROM generation in one PDE9) as a major factor in blunted signaling via cGMP or more types of glomerular cells [153]. [178]. Studies of cultured rat MC show that ANP-induced Mesangial cells comprise approximately 25% of the cGMP accumulation is markedly decreased in the pres- cellular volume of glomerulus [154] and are often the focal ence of angiotensin II, and this inhibition is abolished point of pathobiologic processes involved in immune-in- by IBMX and by the blocking of CaM [191]. Calcium flammatory injury that results in glomerulonephritis inophore mimics the effect of angiotensin II. These find- [198]. In various types of glomerulonephritis, growth fac- ings suggest that angiotensin II, which is increased in tors and cytokines, formed both in glomerular cells and salt-retaining states, may activate PDE1 by increasing in infiltrating inflammatory cells, stimulate rapid prolifer- intracellular Ca2ϩ and may contribute to increased hy- ation of mesangial cells. The involvement of PDE iso- drolysis of ANP-stimulated cGMP pool in situ [191]. zymes in the control of proliferation and reactive oxygen According to recent reports, PDE3 [192] and perhaps metabolite (ROM) generation has been studied in rat also PDE4 [193] may play a role in the regulation of mesangial cells grown in primary cell culture [66, 141]. renin secretion from the juxtaglomerular apparatus in It has long been known that increases in cAMP in some the kidney, and thus, by extension, in the pathogenesis cell types accelerates, but in other cell types inhibits, the of pathophysiologic states associated with the anomalous rate of cell proliferation [199]. First, we observed that renin-angiotensin system. major increases in cAMP elicited by incubating mesan- Inflammatory glomerulopathies. Recent studies of non- gial cells with forskolin or with DBcAMP were sup- renal cell types and tissues have shown that some inhibi- pressed by mesangial cell proliferation [66]. Incubation tors of PDE3 and PDE4 have anti-inflammatory effects of mesangial cells with selective inhibitors of PDE iso- both in vivo and in vitro [73, 89, 103, 108, 136]. Several zymes, without added stimulators of adenylate cyclase, recent studies have addressed the question of whether showed that PDE3 inhibitors cause a profound decrease PDE isozymes are involved in the inflammatory pro- of mitogenesis (Fig. 6) comparable to that elicited by cesses in glomeruli. forskolin and DBcAMP [66]. At higher concentrations The pathogenic role of ROM in renal diseases, particu- (3 ␮m), PDE4 inhibitors also caused a minor inhibition larly glomerulonephritis, is now widely recognized [194], of mesangial cell proliferation that was to a much lesser and glomeruli are major sites of ROM generation within extent than PDE3 inhibitors [66]; at a lower concentra- the kidney [195]. Recent studies show that PDE isozymes tion (1 ␮) PDE4 inhibitors had no antiproliferative effect are involved in the regulation of the ROM burst in neu- [15, 141]. Inhibitors of PDE1 and PDE5 were without trophils [196] and macrophages [197]. In view of this, effect on mitogenesis. Several PDE3 inhibitors of differ- we examined the potential modulatory effect of PDE ent chemical structure [15, 66, 141] and also the dual isozymes on ROM generation in rat glomeruli [153] and PDE3/PDE4 inhibitor zardaverine [66] showed a strong in cultured MCs [66]. Incubation of isolated glomeruli antiproliferative effect [66]. The efficacy of PDE isozyme with forskolin increased the cAMP content many-fold, inhibitors (IC50) to decrease cAMP-PDE activity closely and increased at maximum the in situ activity of PKA correlated with their potency for inhibition (IC50) of mi- and suppressed ROM generation [153]. When glomeruli togenesis [141]. PDE3 inhibitors suppressed mesangial were incubated with PDE isozyme inhibitors, without cell mitogenesis not only in the ambient state, that is, stimulation of adenylate cyclase, the PDE4 inhibitor roli- when under the autocrine/paracrine influence of endoge- pram suppressed ROM generation to a comparable ex- nous growth factors and cytokines generated by mesan- 50 Dousa: Cyclic-3Ј,5Ј-nucleotide PDE isozymes gial cells, but also when stimulated by maximum doses whereas PDE3 inhibitors cilostamide and lixazinone had of added growth factors, such as epidermal growth factor no effect [15, 141]. The IC50 value for cAMP-PDE inhibi- and platelet-derived growth factor [66]. Inhibition of tion corresponded to the IC50 for suppression of ROM PDE3 suppressed mitogenesis to a comparable degree generation. The maximum inhibition of ROM genera- as forskolin, but without a detectable increase in cAMP tion by rolipram was of a similar magnitude as inhibition (Fig. 6); however, the in situ activity of PKA measured by forskolin or DBcAMP [141], in spite of the fact that by the (Ϫ cAMP/ϩ cAMP) PKA activity ratio was in- rolipram activated in situ PKA far less than forskolin creased, and H89, a specific inhibitor of PKA, reversed (Fig. 9). These results also suggest that a minor inhibitory the effect of the PDE3 inhibitor cilostamide on mesan- effect of cilostamide on ROM generation in whole glo- gial cell proliferation [66]. meruli was likely because of its action in cell types other The question was whether and how the increase in than mesangial cells [153]. The mechanism by which the PDE3-linked cAMP pool in mesangial cells might selective activation of the PDE4-linked cAMP-PKA sys- have interfered with signaling pathways that increase tem in mesangial cells inhibits ROM generation is not yet the rate of mesangial cell proliferation. In response to known. The major site of ROM generation is NADPH incubation of mesangial cells with PDE isozyme inhibi- oxidase, a multicomponent enzyme within the plasma tors, the activity of extracellular signal-regulated protein membrane that consists of seven subunits that are assem- kinase (ERK), a key enzyme in one of the currently bled in an active state [62, 117]. We hypothesize that known family of MAPK phosphorylation pathways [200, PKA, by phosphorylating Rap 1a, one of the subunits, 201], was suppressed in both the ambient state and when prevents assembly and activation of NADPH oxidase in stimulated by epidermal growth factor [66]. Our working plasma membranes of mesangial cells (Fig. 10). How- hypothesis is that PKA activated by an increase in the ever, an alternative mechanism(s) by which cAMP inhib- cAMP pool linked to PDE3 phosphorylates c-raf-1 pro- its ROM generation in mesangial cells is not excluded. tein kinase on Ser43 [203] and perhaps also on Ser621 Rolipram and denbufylline are inhibitors that do not [204], and thereby inhibits its activity on the whole ERK distinguish between isoforms and splice variants of the signaling pathway, which ultimately results in a de- PDE4 family. Conceivably, a PDE4 isoform such as ro- creased rate of mitogenesis (Fig. 9). Such negative cross- dent RNPDE4A5 or human homologue HSPDE4A4B, talk between the PKA and ERK signaling pathways has which have an N-terminal region with an SH3-binding been shown in nonrenal cell types [205]. MAPK phos- domain [37, 38], may associate with subunits of NADPH phorylation cascades, including ERK, are “progressively oxidase, which possess multiple SH3 domains [62, 117] ultrasensitive” systems in which the initial signal can be in mesangial cells as it does in other cell types. If this is highly amplified [206]. Therefore, inhibition of c-raf-1 the case, only a fraction of all PDE4 isozymes that are protein kinase, the first step in the ERK cascade, may present in mesangial cells may be functionally linked to have a profoundly suppressive effect on ERK activation modulation of ROM generation [15]. Recent results and mitogenesis (Fig. 9). It cannot be excluded that show that RNPDE4A5 is, indeed, expressed in rat mes- PDE3-linked activated PKA might conceivably block angial cells [69]. mitogenic signaling systems at one or more other points, Actions of PDE isozyme inhibitors on ROM genera- and the exact mechanism remains to be elucidated. An- tion and mitogenesis in cultured mesangial cells indicate other mechanism by which cyclic nucleotide signal trans- that PDE3 and PDE4 isozymes may function in mesan- duction pathways may decrease ERK activity involves gial cells to compartmentalize cAMP-PKA signal trans- cGMP (Fig. 9). ANP, an agonist of guanylate cyclase, duction pathways for separate regulation of two distinct inhibits mitogenesis in mesangial cells [197]. Incubation functional responses, both within one single mesangial of mesangial cells with ANP decreased MAPK activation cell (Fig. 11). This notion is in accord with the observa- in response to endothelin and to phorbol 12,13-dibuty- tion that increases in in situ PKA activity after incubation late, an activator of PKC [207]. Another study of cultured of mesangial cells with the PDE4 inhibitor rolipram and rat mesangial cells has shown that cGMP increased in the PDE3 inhibitor cilostamide are additive (Fig. 6), response to ANP or nitric oxide results in activation of and the inhibitors selectively block ROM generation and mitogen kinase phosphatase that dephosphorylates and mitogenesis [15, 66, 141]. The activities of PDE3 and thus decreases the activity of ERK [202]. I have observed PDE4 in cell-free extracts from mesangial cells are com- that this antimitogenic effect can be accentuated by in- parable [66]. However, when intact mesangial cells are hibitors of PDE1 and PDE5 (unpublished observations). incubated in the presence of forskolin, the maximum Because a PDE4 inhibitor decreased ROM generation inhibitory doses of PDE4 inhibitors caused more than in whole glomeruli [153], we investigated whether the 200-fold increase in cAMP content, whereas in contrast, PDE4-linked cAMP pool can cause suppression of ROM maximum inhibitory doses of PDE3 inhibitors barely generation in mesangial cells. The PDE4 inhibitors roli- doubled cAMP levels [66, 141]. Apparently, in intact pram and denbufylline blocked ROM generation in them, mesangial cells, as yet unknown factors cause the cAMP Dousa: Cyclic-3Ј,5Ј-nucleotide PDE isozymes 51

Fig. 9. PDE isozymes in regulation of mitogenesis in mesangial cells (MC). Schematic depiction of negative crosstalk between extracellular receptor kinase (ERK) pathway (also referred to as mitogen-activated protein kinase pathway, MAPK) and cAMP-PKA pathway linked to PDE3 in MC. Growth factors bind to their respective receptors and, via adaptive proteins Shc, Grb, and GTP/GDP exchange factor Sos, activate Ras attached to the plasma membrane. Once Ras is activated, it proceeds to interact with and stimulates Raf- 1 protein kinase. In ERK pathway Raf-1 protein kinase phosphorylates and activates mitogen extracellular kinase (MEK), which in turn phosphorylates and activates ERK kinase that translocates to nucleus and, directly or indi- rectly, activates nuclear transcription factors. Selective inhibition of PDE3 increases cAMP and activates PKA and activated PKA, phosphorylates and thereby inhibits Raf-1 protein kinase activity. A decrease in Raf-1 activity consequently results in decreased activity and subsequent phosphorylation steps in the pathway (MEK and ERK protein kinases) and ultimately in decreased phosphorylation of nuclear transcription factors and decreased the rate of DNA synthesis. ERK protein kinase can be also dephosphorylated and inacti- vated by dual-speciﬁcity mitogen kinase phosphatase (MKP), an enzyme that may be induced by increased cGMP in response to atrial natriuretic peptide (ANP) [162]. In MC the cAMP is hydrolyzed by several of PDE isozymes; when PDE3 activity is decreased, cAMP accumulates in a speciﬁc pool and activates PKA that, in turn, inhibits raf-1 protein kinase and the whole ERK cascade. Adenyl- ate cyclase (AC) that catalyzes cAMP synthesis is regulated by hormones and autocoids that act on receptors (REC) coupled to AC by tetrameric G-proteins (G). Possibly, PKA may act on some other steps in the mitogenic signal transduction pathways, however, there is little evidence for such action. Details are in the text.

pool metabolized by PDE4 to be far larger than the pool mesangial cell population are similar to those found in metabolized by PDE3. This finding is consistent with human glomerular diseases involving the mesangium, the existence of putative PDE isozyme-linked cAMP namely IgA nephropathy [198]. Rats with antithymic compartments in mesangial cells (Fig. 11). Conceivably, serum-induced MPGN received the PDE3 inhibitor lixa- various specific isoforms of PDE4 detected in these cells zinone and the PDE4 inhibitor rolipram continually via [69] may have additional roles in functional compartmen- osmotic minipumps [117]. Administration of PDE iso- talization of cAMP-PKA signaling pathways [15]. zyme inhibitors prevented the development of protein- Studies on models of glomerulonephritis. Observa- uria, a major functional change in the acute phase of tions in vivo led us to explore whether PDE isozyme MPGN [117]. Histologic and immunohistologic examina- inhibitors can also suppress pathobiologic processes in tion of the kidneys showed marked suppression of patho- glomeruli in vivo. The study was conducted on the exper- logic changes in glomeruli of MPGN rats treated with imental model of mesangial proliferative glomerulone- the PDE isozyme inhibitors (Fig. 12). Analysis using phritis (MPGN) induced in rats by injection of antithymic immunohistochemical methods showed a reduced num- serum containing anti-Thy-1.1-antibodies [117]. This ani- ber of proliferating cells (Fig. 12), less infiltration by mal model MPGN is characterized by a phase of rapid macrophages, and decreased myofibroblastic transfor- proliferation and phenotypic transformation of mesan- mation, assessed by expression of smooth muscle ␣-actin gial cells. In many ways, the pathologic changes in the [117]. In subsequent and still continuing studies, we have 52 Dousa: Cyclic-3Ј,5Ј-nucleotide PDE isozymes

Fig. 10. Proposed mechanism by which the cAMP pool linked to PDE4 inhibits activation of NADPH oxidase in plasma membranes of mesangial cells (MC). NADPH oxidase consists of four cytoplasmic subunits p47phox, p67phox, p40phox and the GTP-binding protein rac, and three subunits within the membrane including gp91phox, p22phox and Rap-1a, another GTP-binding protein. In response to stimuli, some of which involve phosphorylation of p47phox by PKC, cytoplasmic and membrane subunits assemble into an active NADPH oxidase complex that catalyzes the generation of oxygen superoxide anion from molecular oxygen. Phosphorylation of Rap-1a subunit by PKA inhibits its interaction with other subunits and consequently the assembly of NADPH oxidase into an active complex [33]. Inhibition of PDE4 activity, but not other PDE isozymes, results in the accumulation of cAMP in a speciﬁc pool linked to PDE4 and activates PKA that, in turn, phosphorylates Rap-1a and impedes the NADPH oxidase assembly and activation.

Fig. 11. Schematic depiction of proposed compartmentalization of cAMP-PKA signaling pathway by PDE isozymes in mesangial cell (MC). The intracellular cAMP pool hydrolyzed by PDE3 regulates mitogenesis in MC [43]. Inhibition of PDE3 by selective inhibitors (cilostamide, lixazinone) causes an increase in this PDE3-specific cAMP pool and activates adjacent PKA. The protein kinase A (PKA) phosphorylates c-raf-1 and thereby inhibits the mitogen-stimulated ERK phosphorylation cascade at the initial point (see Fig. 6), which leads to suppression of mitotic synthesis of DNA. On the other hand, cAMP hydrolyzed specifically by PDE4 regulates reactive oxygen metabolite (ROM) generation in MC [95]. Inhibition of PDE4 by selective inhibitors (rolipram, denbufylline) leads to an increase of another distinct cAMP pool and activates adjacent PKA. PKA, probably via phosphorylating Rap-1a, then decreases the NADPH oxidase assembly and ROM generation (see Fig. 7). It is not yet determined whether PKAs adjacent to PDE3 and PDE4 are two distinct isozymes, PKA-I and PKA- II, respectively, or isoforms of the same PKA isozyme that are localized by anchoring to specific intracellular structures. Dousa: Cyclic-3Ј,5Ј-nucleotide PDE isozymes 53

state, the effects of PDE inhibitors are greatly accentuated [7, 9]. Along with many other hormones and autocoids that act on glomeruli [210], the recently discovered peptide adrenomedullin, which is synthesized in mesangial cells, is a potent stimulator of cAMP synthesis in mesangial cells [211]. As a recent study shows, activation of the cAMP-PKA pathway by adrenomedullin in mesangial cells and in the macrophage cell line inhibits both mitogenesis and ROM generation [212]. With respect to other types of glomerulonephritis, of major interest is a recent preliminary report that administration of the PDE4 inhibitor rolipram may have a beneficial effect on nephrotoxic glomerulonephritis in rats [118]. The authors found that the administration of rolipram had both a preventive and a therapeutic effect in terms of abated albuminuria, less histopathologic Fig. 12. Effect of PDE inhibitors upon mesangial cell (MC) proliferation in glomeruli of rats with experimental mesangial proliferative glo- changes in glomeruli, and also a decrease in the urinary merulonephritis (MPGN) induced by single injection of ATS (anti- excretion of tumor necrosis factor-␣ (TNF-␣). A full Thy-1.1 nephritis). The content of proliferating cell nuclear antigen report of this study will be of major interest. Rolipram (PCNA) in glomeruli was determined immunohistochemically by count- ing of positively stained cells per glomerulus (each value is based on and other PDE4 inhibitors were shown to selectively analysis of 50 glomeruli). The control rats without MPGN or treat- suppress de novo synthesis, and to release TNF-␣ and ment(ᮀ), rats with MPGN( ) and MSGN rats treated with a combina- other proinflammatory cytokines in macrophages and tion of lixazinone and rolipram ( ) are compared; each group consists of mean ϩ sem of N ϭ 3 to 5 rats. P values for significance of differences other inflammatory and immunocompetent cells [90, are by the t-test. Data are from [157]. 213–217]. Administration of PDE4 inhibitors in vivo suppressed or prevented experimental arthritis [216, 217] and several other types of inflammatory lesions [217]. In these in vivo models, PDE4 inhibitors decreased de novo found that administration of the same dose of the PDE3 synthesis and tissue accumulation of the proinflamma- inhibitor lixazinone alone blocked mesangial cell prolif- tory cytokines TNF-␣, interleukin-␤, and interferon-␥ eration to a similar degree as in the first study [15, 208], (INF-␥) [213–217]. The therapeutic effect of PDE4 inhib- whereas the PDE4 inhibitor rolipram alone did not show itors in an animal model of experimental autoimmune significant suppression of pathologic changes in MSGN encephalomyelitis, a disease that resembles multiple [208]. Higher doses of rolipram than those used in the sclerosis, was also attributed to decreased TNF-␣ genera- subsequent study [117, 208] given subcutaneously de- tion [124, 125]. creased mesangial cell proliferation and reduced protein- Taken together, these observations suggest that PDE uria, albeit to a lesser extent than with lixazinone (T.P. isozymes in renal cells and/or inflammatory cells that Dousa and J.P. Grande, preliminary observations). Thus, infiltrate renal tissue could be targets for PDE isozyme administration of PDE isozyme-selective inhibitors in inhibitors aimed at suppressing various pathobiological vivo suppressed pathobiologic processes in mesangial processes in glomeruli by interfering with signal trans- cells in vivo, as might have been expected based on the duction mechanisms. Various PDE inhibitors, alone or studies of cultured mesangial cells in vitro [66]. According in a combination, can be selectively targeted to suppress to a recent study, glomerular ERK activity is dramati- different types of immune-inflammatory injury and rep- cally increased in experimental proliferative glomerulo- resent a novel “signal transduction pharmacotherapy” nephritis in rats [209]. Results also suggest that in MSGP [21] of glomerulonephritis. and similar types of glomerulonephritis, the inhibition of PDE3 may be a basis for a novel mode of pharmaco- PDE isozymes in tubular disorders therapy [117]. It should be stressed, however, that these Studies of several models of experimental diseases initial studies were conducted with the use of a single suggest involvement of PDE isozymes in the pathophysi- dose of inhibitors, and different doses, or a different ology of renal tubular dysfunction. ratio of doses of PDE3 and PDE4 inhibitors, might have Mice with hereditary nephrogenic diabetes insipidus a qualitatively or quantitatively different effect. Further- (NDI). As mentioned previously, hereditary NDI in mice more, lower doses of PDE inhibitors might be sufficient was identified as the first hereditary renal disease be- to produce a therapeutic effect [7, 9, 15]. As in all other cause of abnormal activity of PDE isozymes [147, 148]. cell types, adenylate cyclase in MCs in vivo is under Briefly, these in vitro studies indicate that increased ac- the influence of many stimulators, and in this functional tivity of cAMP-PDE, namely PDE4, in IMCD accounts 54 Dousa: Cyclic-3Ј,5Ј-nucleotide PDE isozymes

ology of any variants of human NDI syndromes. The genetic defects in NDI documented thus far are at the AVP receptor or post-cAMP components of the signaling pathway [219]. Nevertheless, the NDI-mouse model serves as a paradigm of a renal disease with hereditary unresponsiveness to a hormone that is, by all present evidence, caused by increased activity of a PDE isozyme. In microdissected IMCD from adrenalectomized rats with a urinary concentrating defect, cAMP-PDE activity was increased and AVP-stimulated cAMP accumulation was decreased, whereas adenylate cyclase activity was not changed [164]. All biochemical and functional abnor- malities were corrected by administration of dexametha- sone. The findings point to the importance of cAMP-PDE in AVP action and suggest that increased cAMP-PDE may be a pathogenic factor also in acquired forms of vasopressin resistance [164]. Increased activity of cGMP-PDE in collecting ducts in nephrotic syndrome. Atrial natriuretic peptide is a potent regulator of Naϩ transport in IMCD. The mechanism of ANP action in IMCD cells involves a stimulation of guanylate cyclase and an increase in cGMP that inhibits the Naϩ channel in luminal membrane of IMCD cells. Seminal observations by Humphreys et al point to a role of cGMP-PDE in the blunted ability of ANP to enhance Fig. 13. Activity of cAMP-PDE (A) and arginine vasopressin (AVP)- Na excretion in IMCD from rats with nephrotic syn- dependent cAMP accumulation (B) in inner medullary collecting ducts drome of different origins [132, 133, 156]. In rats with microdissected from the kidneys of normal mice (controls) and mice with hereditary nephrogenic diabetes insipidus (NDI-mice). The sam- nephrotic syndrome produced either by adriamycin [156] ples were incubated without (controls, ) or with either 0.1 mm roli- or by immune-complex Heymann nephritis [133], levels pram, a PDE4 inhibitor ( ), or with 0.1 mm cilostamide, a PDE3 of circulating ANP and its binding to receptors in IMCD inhibitor ( ). In A, *significantly different from all other values, #values significantly lower than that without inhibitors. In B, *significantly cells were not diminished, but incubation of collecting different from controls and R, #not significantly different from controls. duct segments with ANP in vivo resulted in low accumu- Whereas both R and Cs inhibited cAMP-PDE activity, only R restored lation of cGMP (Fig. 8). The activity of cGMP-PDE cAMP accumulation in IMCD of NDI mice. measured in homogenates of IMCD segments was increased [156, 133]. Urinary excretion of cGMP in response to acute volume expansion in vivo was also de- for the failure of AVP to increase cAMP [148] and to creased [132, 133, 156]. The selective inhibitor of PDE5 activate in situ PKA [149], and to elicit an antidiuretic zaprinast (which may also inhibit PDE9) restored cGMP effect [218]. Both the PDE4 inhibitor rolipram and the accumulation in response to ANP in IMCDs incubated PDE3 inhibitor cilostamide decrease cAMP-PDE activ- in vitro and, given in vivo restored the ability to increase ity in IMCD microdissected from NDI mice, but only salt and cGMP excretion in response to a volume load the PDE4 inhibitor rolipram restores AVP-stimulated [132, 133, 156]. Thus, increased PDE5 activity (and per- cAMP accumulation (Fig. 13). Although in the initial haps also PDE9 activity) in cells of collecting ducts in studies in vivo rolipram was administered together with nephrotic syndrome of different etiologies may account cilostamide, later experiments indicate that the PDE4 for hyporesponsiveness to ANP and blunted natriuresis. inhibitor alone can restore the functional response in The finding suggests that decreased responsiveness to vitro and in vivo [218]. The increased PDE activity is ANP both in glomeruli and tubules (Fig. 8) is due to due to a higher Vmax [150], a finding compatible with enhanced cGMP catabolism. Furthermore, the investiga- the possibility that PDE4 is overexpressed in IMCD as tors have shown that in the abnormal sodium metabolism similarly demonstrated in studies of LLCPK1 cells [105, resulting from hepatic injury due to ligation of the com- 176–178]. It will be important to determine which sub- mon bile duct in rats [220], a condition that does not type and isoform of PDE4 is overexpressed or whether involve primary renal disease, the blunted response of a mutant enzyme accounts for high PDE4 activity in the IMCD to ANP can be corrected, at least in part, by collecting ducts of NDI mice. The pathogenesis of mu- administration of the PDE5 inhibitor zaprinast in vivo rine NDI syndrome may be unrelated to the pathophysi- and in vitro [220]. Unlike the case of NDI mice, increased Dousa: Cyclic-3Ј,5Ј-nucleotide PDE isozymes 55 activity of PDE5 in these salt-retaining states is not likely ure, the administration of Ro-20-1724 attenuated renal hereditary and may be caused by extrarenal factors. Fu- vasoconstriction, reduced the decline of renal blood flow ture studies should show if increased activity of PDE5 is and glomerular filtration rate, and improved overall sur- due to overexpression of PDE5 or whether an abnormal vival rate [223]. The mechanisms accounting for the ben- PDE5 isoform is induced in the nephrotic state, and by eficial effect of this PDE4 inhibitor on endotoxemic acute which mechanism. Because it is now known that zaprinast renal failure are yet to be determined. Endotoxin is known also inhibits PDE5 besides PDE9 [55, 56], another iso- to stimulate synthesis and release of proinflammatory zyme selective for cGMP that is highly expressed in the cytokines such as TNF-␣, interleukin-1␤, or INF-␥, and kidney [55], the possible role of PDE9 should be investi- blockade of synthesis and release of cytokines [213–217] gated as well. The role of other cGMP-hydrolyzing PDE either in tubular epithelial cells [224] or in peritubular isozymes, namely that of PDE1, activated in situ also cells by PDE4 inhibitors might have been the basis of the should be clarified. Needless to say, nephrotic syndrome observed beneficial effect. Relaxation of vascular smooth and other states of sodium retention are another area muscle [225] and enhancement of kidney perfusion by ready for development of “signal transduction pharma- the PDE4 inhibitor may be another beneficial factor. cotherapy” targeted to PDE5 and other cGMP-hydrolyz- These stimulating findings await in-depth analysis of the ing isozymes. mechanism. PDE isozymes and acute renal failure. The possible In ischemic acute renal failure, according to a recent role of PDE isozymes in the pathogenesis or treatment report, administration of the PDE5 inhibitor zaprinast of acute renal failure has recently been approached with greatly accelerated recovery from established ischemic the use of selective PDE inhibitors. acute renal failure caused by clamping the renal arteries Injection of folic acid causes acute renal failure, which in rats [226]. Infusion of zaprinast dramatically improved is characterized by exceedingly rapid proliferation of postischemic glomerular filtration rate and increased cor- tubular cells without detectable changes in glomeruli tical and outer medullary blood flow. In contrast to ef- [221]. We examined whether accelerated proliferation fects of the PDE5 inhibitor, treatment with ANP, a po- of tubular epithelial cells may be modulated by changes tent stimulator of cGMP synthesis in glomeruli and in activity of PDE isozymes [152]. Administration of collecting ducts [173], had no effect. The therapeutic the PDE3 isozyme inhibitors cilostamide or cilostazol, effect of the PDE5 inhibitor was attributed primarily to together with the PDE4 inhibitor rolipram or the potent a favorable action on the renal circulation, especially in inhibitor of PDE3 lixazinone alone, almost completely blocked the burst of tubular cell proliferation in response the medulla, and such an interpretation is in accord with to folic acid, whereas rolipram by itself had no effect the presence of PDE5 in vascular smooth muscle cells [152]. The antimitogenic effect of the PDE inhibitors of nonrenal origin and its role in vasorelaxation [227]. was comparable in extent to the effect of the potent One feature with respect to the action PDE isozyme DNA transcription inhibitor actinomycin-D [152, 221]. inhibitors in vitro and in vivo should be kept in mind Cortical tubules contain significant PDE3 activity, al- when considering their use as pharmacotherapeutics. As though the exact localization is not yet determined. Con- shown in several examples here, the decrease of cAMP ceivably, the mechanism by which PDE inhibitors block or cGMP hydrolysis by specific PDE isozymes has a mitogenesis is similar to that proposed to occur in rapidly prolonged and sustained enhancing effect on signaling proliferating MC in vitro and in vivo [15, 66, 117], that pathways, even in situations when large doses of agonists is, via negative cross-talk between PDE3-cAMP-PKA that stimulate cAMP or cGMP synthesis are without and ERK signaling pathways (Fig. 9). It is plausible that effect [132, 149, 226]. It is reasonable to expect that the only those renal cells, tubular or glomerular, that prolif- effect of PDE inhibitors on PDE isozymes will not be, erate at an accelerated rate are susceptible to the inhibi- in most instances, subject to desensitization as is the tory effect of the PDE3-linked cAMP pool. This would case after prolonged stimulation of receptors coupled to be an advantage for the design of targeted pharmaco- adenylate cyclase with high doses of the hormone. therapy with PDE isozyme inhibitors. Acute renal failure that develops in the course of endo- CONCLUSIONS AND PERSPECTIVES toxic shock has a prominent vascular component. In studies of rats with acute renal failure induced by infusion Current information pertaining to the system of PDE of E. coli lipopolysaccharide the administration of the isozymes in kidney and renal cells, although still limited PDE4 inhibitor Ro-20-1724 prior to injection of endo- and fragmentary, shows that this key component of cyclic toxin prevented the decrease of renal blood flow, low- nucleotide signaling pathways is crucial to understand ered renal vascular resistance, and also prevented the the pathobiology of renal diseases and the design of decline of glomerular filtration rate [222]. In rats with novel “signal transduction” pharmacotherapeutic inter- already developed endotoxin-induced acute renal fail- ventions. There are major areas in the spectrum of renal 56 Dousa: Cyclic-3Ј,5Ј-nucleotide PDE isozymes diseases, yet untouched, in which the study of PDE iso- and/or cGMP synthesis. These findings from studies of zymes should be a high priority. nonrenal tissues constitute a compelling rationale for the Multiple studies have accrued evidence that abnormal study of PDE isozymes and the effects of their selective cAMP metabolism plays a significant role in the patho- inhibitors in various facets of renal transplantation. genesis of polycystic kidney diseases [228]. As implied In conclusion, this evidence clearly shows that PDE in a recent editorial comment [229], it is reasonable to isozymes are involved in the modulation of biological and consider that cAMP’s effect on fluid secretion or cell pathobiological processes in renal cells, and demonstrate proliferation in the course of cystogenesis may be regu- that PDE-specific inhibitors may favorably influence the lated by cAMP pathways that are related to specific PDE skein of pathophysiological entities from glomerulone- isozymes, and this may clarify some apparently paradoxi- phritis through an impaired renal handling of sodium in cal findings pertaining to the role of cAMP. Likewise, the nephrotic syndrome, to various forms of acute renal elucidation of the PDE isozyme superfamily’s role in the failure. Well-designed and focused studies of PDE iso- development of polycystic kidney disease may provide zymes and the effects of selective PDE inhibitors in renal a rational basis for novel therapeutic avenues for the use cells and kidney tissue are bound to elucidate the regula- of selective PDE inhibitors. tion of renal function by cyclic nucleotide signaling that Another major area in which PDE isozymes may play have eluded detection thus far, and point to novel targets an important role is in the theory and practice of renal for “signal transduction” pharmacotherapies [21]. transplantation. Drawing on analogies from studies of transplantation of nonrenal tissues, involvement of PDE ACKNOWLEDGMENTS isozymes and intervention by selective inhibitors should This study was supported by the National Institutes of Health grants be seriously considered. With respect to organ preserva- DK-16105; American Heart Association, Minnesota Affiliate; and by tion and survival, in the time period between harvesting the Mayo Foundation. Ms. Carol A. Davidson provided secretarial assistance. and grafting, pathophysiological changes of cAMP and cGMP signaling apparently play an important role [230, Reprint requests to: Thomas P. Dousa, M.D., 921B Guggenheim 231]. Studies of the preservation of heart and lung allo- Building, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA. grafts indicate that declining cAMP content in the period E-mail: [email protected] of hypoxia and a drop in cGMP levels in the course of subsequent reperfusion in the transplanted organ [230, 231] may be fundamental mechanisms that account for APPENDIX vascular dysfunction and increased capillary permeabil- Abbreviations used in this article are: ANP, atrial natriuretic pep- ity and decrease in survival of grafts. Inhibitors of PDE3 tide; AVP, [8-Arg]-vasopressin; CaM, calmodulin; cAMP, cyclic-3Ј,5Ј- adenosine monophosphate; cCMP, cyclic-3Ј,5Ј-cytidine phosphate; and PDE4 or dual PDE3/PDE4 inhibitors blocked the cGMP, cyclic-3Ј,5Ј-GMP; DBcAMP, dibutyryl cAMP; EHNA, erythro- increase in endothelial permeability in vitro [126], and 9-(2-hydroxyl-3-nonyl)-adenine; ERK, extracellular signal-regulated application of PDE3 and PDE4 inhibitors in vivo that protein kinase; FSH, follicle stimulating hormone; HARBS, high affinity rolipram-binding sites; IBMX, 3-isobutyl-1-methyl-xanthine; IMCD, in- diminish or prevent a drop in cAMP content improve ner medullary collecting ducts; MAPK, mitogen-activated protein ki- vascular homeostasis and enhance organ preservation nase; MC, mesangial cells; 8-MeoM-IBMX, 8-methoxymethyl-IBMX; [127, 128]. Inhibition of the cGMP breakdown via PDE5 MSGN, mesangial proliferative nephritis; NDI, nephrogenic diabetes insipidus; PDE, phosphodiesterase; PKA, protein kinase A; PKC, pro- by the selective PDE5 inhibitor zaprinast significantly tein kinase C; PKG, protein kinase G; rIMCD, rat IMCD; ROM, improved the preservation of heart and lung tissues in the reactive oxygen metabolites; SH2, Src homology 2; SH3, Src homology 3; course of and immediately after transplantation [135]. TNF-␣, tumor necrosis factor-␣. With respect to the immunological mechanism of renal allograft rejection, PDE3 and PDE4 inhibitors suppressed REFERENCES the mitogenic response of lymphocytes to mismatched 1. Robison GA, Butcher RW, Sutherland EW: Cyclic AMP. New HLA-DR alloantigens in mixed lymphocytic culture [129]. York, Academic Press, 1971 2. 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