JPET Fast Forward. Published on February 16, 2007 as DOI: 10.1124/jpet.106.119057 JPET FastThis Forward.article has not Published been copyedited on and February formatted. The16, final 2007 version as DOI:10.1124/jpet.106.119057may differ from this version.
JPET #119057
TITLE PAGE
CHARACTERIZATION OF RENAL ECTO-PHOSPHODIESTERASE
Edwin K. Jackson, Jin Ren, Lefteris C. Zacharia and Zaichuan Mi Downloaded from
CENTER FOR CLINICAL PHARMACOLOGY
DEPARTMENTS OF PHARMACOLOGY (EKJ) AND MEDICINE (EKJ, JR, LCZ, ZM) jpet.aspetjournals.org
UNIVERSITY OF PITTSBURGH SCHOOL OF MEDICINE
PITTSBURGH, PA 15219 at ASPET Journals on September 30, 2021
1
Copyright 2007 by the American Society for Pharmacology and Experimental Therapeutics. JPET Fast Forward. Published on February 16, 2007 as DOI: 10.1124/jpet.106.119057 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #119057 RUNNING TITLE PAGE
Running Title: Renal Ecto-Phosphodiesterase
Address for correspondence:
Edwin K. Jackson, Ph.D.
Center for Clinical Pharmacology, University of Pittsburgh School of Medicine
100 Technology Drive, Suite 450, Pittsburgh, PA 15219 Downloaded from Phone: (412) 648-1505 (1880); Fax: (412) 648-1837; E-mail: [email protected]
Document Properties:
Total Length of Manuscript: 28 pages including figures jpet.aspetjournals.org
Number of Tables: 0
Number of Figures: 4 at ASPET Journals on September 30, 2021 Number of References: 40
Number of Words in Abstract: 237
Number of Words in Introduction: 688
Number of Words in Discussion: 1074
List of Abbreviations: phosphodiesterase, PDE; ecto-phosphodiesterase, ecto-PDE; ecto-5'- nucleotidase, ecto-5'-NT; α,β-methylene-adenosine-5'-diphosphate, AMPCP; 8-methoxymethyl-
3-isobutyl-1-methylxanthine, mmIBMX; 1, 3-isobutyl-1-methylxanthine, IBMX; 1,3-dipropyl-8- p-sulfophenylxanthine, DPSPX; erythro-9-(2-hydroxy-3-nonyl)adenine, EHNA; 5-nitro-2,N,N- trimethylbenzenesulfonamide, BRL-50481; 4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one,
RO 20-1724.
2 JPET Fast Forward. Published on February 16, 2007 as DOI: 10.1124/jpet.106.119057 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #119057 ABSTRACT
In kidneys, stimulation of adenylyl cyclase causes egress of cAMP, conversion of cAMP to
AMP by ecto-phosphodiesterase and metabolism of AMP to adenosine by ecto-5'-nucleotidase.
Although much is known about ecto-5'-nucleotidase, the renal ecto-phosphodiesterase remains
uncharacterized. We administered cAMP (10 μmol/L in the perfusate) to 12 different groups of
perfused kidneys. AMP was measured in perfusate using ion trap mass spectrometry. In control Downloaded from kidneys (n=17), basal renal secretion rate of AMP was 0.49 ± 0.08 and increased to 3.0 ± 0.2
nmoles AMP per gram kidney weight per min during adminstration of cAMP. A broad spectrum
PDE inhibitor (1,3-isobutyl-1-methylxanthine, 300 :mol/L, n=6) and an ecto-phosphodiestease jpet.aspetjournals.org
inhibitor (1,3-dipropyl-8-sulfophenylxanthine, 1 mmol/L, n=6) significantly attenuated cAMP-
induced AMP secretion by 60% and 74%, respectively. Blockade of PDE1 (8-methoxymethyl- at ASPET Journals on September 30, 2021 3-isobutyl-1-methylxanthine, 100 :mol/L), PDE2 (erythro-9-(2-hydroxy-3-nonyl)adenine, 30
:mol/L), PDE3 (milrinone, 10 μmol/L; cGMP, 10 :mol/L), PDE4 (RO 20-1724, 100 :mol/L),
PDE5 and PDE6 (zaprinast, 30 :mol/L) and PDE7 (BRL-50481, 10 :mol/L) did not alter renal ecto-phosphodiesterase activity. Administration of a concentration (100 :mol/L) of
dipyridamole that blocks PDE8 inhibited ecto-phosphodiesterase activity (by 44%). However, a lower concentration of dipyridamole (3 :mol/L) that blocks PDE9, PDE10 and PDE11, but not
PDE8, did not inhibit ecto-phosphodiesterase activity. These data support the conclusion that renal ecto-phosphodiesterase activity is not mediated by PDE1, PDE2, PDE3, PDE4, PDE5,
PDE6, PDE7, PDE9, PDE10 or PDE11, and is inhibited by high concentrations of dipyridamole.
Ecto-phosphodiesterase has some pharmacological characteristics similar to PDE8.
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JPET #119057 INTRODUCTION
Multiple biochemical pathways provide for the biosynthesis of adenosine. The well-
characterized pathways include the intracellular ATP pathway (intracellular dephosphorylation
of ATP to adenosine) (Schrader, 1991), the extracellular ATP pathway (metabolism of released
adenine nucleotides to adenosine by ecto-enzymes) (Gordon, 1986) and the transmethylation
pathway (the hydrolysis of S-adenosyl-L-homocysteine (SAH) to L-homocysteine and adenosine Downloaded from by the enzyme SAH-hydrolase) (Lloyd et al., 1988). The intracellular ATP pathway is activated
when energy demand exceeds energy supply (Schrader, 1991), the extracellular ATP pathway is
engaged when adenine nucleotides are released during sympathoadrenal activation, platelet jpet.aspetjournals.org
aggregation or activation of cardiovascular cells by clotting factors, neutrophil interactions, and
catecholamines (Pearson and Gordon, 1979; Pearson et al., 1980; LeRoy et al, 1984), and the at ASPET Journals on September 30, 2021 transmethylation pathway is triggered by methylation reactions involving S-adenosyl-L- methionine as the methyl donor (Lloyd et al., 1988; Deussen et al., 1989). These three well- described routes of adenosine biosynthesis are not well suited for physiological modulation of extracellular levels of adenosine because the intracellular and extracellular ATP pathways of adenosine production require crisis events and the transmethylation pathway is mostly constitutive.
We postulate a fourth pathway, the cAMP-adenosine pathway, for adenosine production that would be more amenable to physiological modulation of adenosine levels by hormones
(Jackson, 1991, 1997, 2001; Jackson and Dubey, 2001, 2004). In this regard, our hypothesis is that stimulation of adenylyl cyclase activates the cAMP-adenosine pathway, which has both intracellular and extracellular sites of adenosine production. The intracellular arm involves
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JPET #119057 metabolism of cAMP to AMP and AMP to adenosine via cytosolic phosphodiesterase (PDE) and
cytosolic 5'-nucleotidase (5'-NT), respectively. The adenosine thus formed would reach the
extracellular space by way of facilitated transport. However, the quantitative importance of the
intracellular cAMP-adenosine pathway would be diminished by the competition of cytosolic 5'-
NT and adenylate kinase for AMP and by the competition of transport mechanisms with
adenosine kinase for adenosine. Therefore, the extracellular limb of the cAMP-adenosine Downloaded from pathway may be quantitatively more important.
We hypothesize that the extracellular cAMP-adenosine pathway is mediated by three jpet.aspetjournals.org spatially-linked processes: egress of cAMP, metabolism of cAMP to AMP by ecto-
phosphodiesterase (ecto-PDE) and metabolism of AMP to adenosine by ecto-5'-nucleotidase
(ecto-5'-NT). Activation of adenylyl cyclase is always associated with egress of cAMP into the at ASPET Journals on September 30, 2021 extracellular space by an active transport mechanism (King and Mayer, 1974; Barber and
Butcher, 1981), and ecto- 5'-NT is a ubiquitous enzyme that is tethered to the extracellular face
of the plasma membrane via a lipid-sugar linkage (Misumi et al., 1990; Zimmermann, 1992).
Because cAMP transport is robust and ecto-5'-NT efficiently metabolizes AMP to adenosine,
activation of adenylyl cyclase would trigger the extracellular metabolism of cAMP to AMP and
hence to adenosine, provided that sufficient levels of ecto-PDE exist. Because these reactions
would take place in a highly localized environment, this newly formed adenosine could then act
in an autocrine and/or paracrine fashion to amplify, inhibit, and/or expand the local response to
hormonal stimulation of adenylyl cyclase. In this regard, modest increases in cAMP production could significantly increase adenosine concentrations at the cell surface. Indeed, our studies
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JPET #119057 demonstrate that hormone stimulation increases adenosine via the extracellular cAMP-adenosine
pathway (Mi and Jackson, 1998; Dubey et al., 2000, 2001; Jackson et al., 2003, 2006).
Our studies in the perfused rat renal vascular bed demonstrate that infusion of cAMP
causes a concentration-dependent increase in the renal secretion rates of AMP, adenosine, and
inosine, and the increases in AMP and adenosine secretion are inhibited by 1,3-isobutyl-1-
methylxanthine (IBMX; broad spectrum PDE inhibitor) and 1,3-dipropyl-8-p- Downloaded from
sulfophenylxanthine (DPSPX; ecto-PDE inhibitor at high concentrations), whereas the increases
in adenosine, but not AMP, secretion are blocked by α,β-methylene-adenosine-5'-diphosphate jpet.aspetjournals.org (AMPCP; ecto-5'-NT inhibitor) (Mi and Jackson, 1995). Thus, our previous studies indicate that the kidney contains ecto-PDE. However, the renal ecto-PDE that mediates the conversion of extracellular cAMP to AMP remains uncharacterized. at ASPET Journals on September 30, 2021 It is conceivable that ecto-PDE is one of the currently know 11 different families of
PDE. These PDE families can be distinguished by sensitivity to PDE inhibitors (Bender and
Beavo, 2006). Therefore, the purpose of this study was to characterize pharmacologically the renal ecto-PDE by comparing the effects of a panel of PDE inhibitors on ecto-PDE activity in the isolated, perfused rat kidney.
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JPET #119057 METHODS
Seventy-six adult male Sprague-Dawley rats obtained from Charles River (Wilmington,
MA) were housed at the University of Pittsburgh Animal Facility and fed Prolab RMH 3000
(PMI Feeds, Inc., St. Louis, MO) containing 0.26% sodium and 0.82% potassium. All studies
received prior approval by the University of Pittsburgh Animal Care and Use Committee.
Rats were anesthetized (sodium pentobarbital 45 mg/kg, intraperitoneal injection), a Downloaded from midline incision was made, and the left kidney, left renal artery, abdominal aorta, and left ureter
were dissected free from surrounding tissue. The left ureter was cannulated with polyethylene
(PE)-10 tubing, the abdominal aorta below the left kidney was cannulated (PE-50 tubing), the jpet.aspetjournals.org
suprarenal aorta was ligated, and the left kidney was flushed with 2.5 ml/min of oxygenated
Tyrode's solution containing 100 units/ml of heparin. While maintaining perfusion, the left at ASPET Journals on September 30, 2021 kidney was isolated and mounted in a water-jacketed organ chamber. The organ chamber was
maintained at 37oC with a thermostatically-controlled water circulation (Thermocirculator,
Harvard Apparatus, South Natick, MA). Kidneys were perfused (5 ml/min) using a Harvard
model 1210 peristaltic pump with Tyrode's solution (composition in mM: NaCl, 137; KCl, 2.7;
0 CaCl2, 1.8; MgCl 2, 1.1; NaHCO3, 12; NaH 2PO4, 0.42; D(+)-glucose, 5.6) that was heated to 37
C with a warming coil, gassed with 95 % O2 and 5 % CO 2 and a passed through a bubble trap.
A Statham pressure transducer (model P23ID, Statham Division, Gould Inc., Oxnard, CA) was
connected to an access port located in the perfusion line immediately before the kidney so that
perfusion pressure could be continuously monitored (Grass model 79D polygraph, Grass
Instruments, Quincy, MA). The perfused kidneys were allowed to equilibrate for 105 minutes to obtain a stable baseline renal venous AMP secretion rate. Next, 100 :mol/L of AMPCP, a
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JPET #119057 specific inhibitor of ecto-5'-nucleotidase (Zimmermann, 1992), was added to the perfusate to prevent the metabolism of AMP to adenosine. Our previously published results demonstrate that this concentration of AMPCP blocks AMP metabolism to adenosine in the isolated, perfused rat kidney (Mi and Jackson, 1995).
The kidneys were assigned to one of twelve groups: 1) control group (n=17); 2) group treated with IBMX [300 :mol/L, n=6, a broad spectrum PDE inhibitor (Beavo et al., 1970)]; 3) Downloaded from group treated with DPSPX [1 mmol/L, an inhibitor of ecto-PDE, but not intracellular PDEs (Mi and Jackson, 1995)]; 4) group treated with 8-methoxymethyl-3-isobutyl-1-methylxanthine
[mmIBMX, 100 :mol/L, n=6, a selective PDE1 inhibitor (Wells and Miller, 1988)]; 5) group jpet.aspetjournals.org treated with erythro-9-(2-hydroxy-3-nonyl)adenine [EHNA, 30 :mol/L, n=6, selective inhibitor of PDE2 (Benter and Beavo, 2006)]; 6) group treated with cGMP [10 μmol/L, n=6, selective at ASPET Journals on September 30, 2021 inhibitor of PDE3 (Benter and Beavo, 2006]; 7) group treated with milrinone [10 μmol/L, n=6, a selective PDE3 inhibitor (Harrison et al., 1986)]; 8) group treated with RO 20-1724 [100
:mol/L, n=6, a selective PDE4 inhibitor (Beavo and Reifsnyder, 1990)]; 9) group treated with zaprinast [30 :mol/L, n=6, a selective PDE5 (Benter and Beavo, 2006) and PDE6 inhibitor
(Ballard et al., 1998)]; 10) group treated with 4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one
[BRL-50481, 10 :mol/L, n=6, selective inhibitor of PDE7 (Benter and Beavo, 2006)]; 11) group treated with high concentration of dipyridamole [100 :mol/L, n=7, inhibits PDE8, PDE9,
PDE10 and 11 (Fawcett et al., 2000, Gamanuma et al., 2003)]; and 12) group treated with low concentration of dipyridamole [3 :mol/L, n=6, inhibits PDE9, PDE10 and PDE11, but not PDE8
(Fawcett et al., 2000, Gamanuma et al., 2003)]. The concentrations of mmIBMX, EHNA,
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JPET #119057 milrinone, RO 20-1724, zaprinast and BRL 40418 were selected on the basis of their IC50s for their respective PDE subtype targets. In this regard, a concentration approximately 30 times the
IC50 was employed. The concentrations of AMPCP, IBMX and DPSPX were selected on the
basis of our previous studies (Mi and Jackson, 1995). The low concentration of dipyridamole
was selected to be 1 to 30 times above the IC50 for PDE9, PDE10 and PDE11, while being 0.1 to
0.3 of the IC50 for PDE8. The high concentration of dipyridamole was selected to be Downloaded from
approximately three times the IC50 for PDE8. Higher concentrations of dipyridamole were precluded by lack of solubility.
In each kidney, the treatment was added directly to the perfusate, and four minutes later jpet.aspetjournals.org the perfusate exiting the renal vein was collected for one minute. Then cAMP (10 μmol/L) was added to the perfusate, and four minutes later, the venous perfusate was again collected for one at ASPET Journals on September 30, 2021 minute. Perfusate was immediately placed on ice and then frozen at -40EC for later analysis of
AMP.
The concentration of AMP in the perfusate were measured using a ThermoFinnigan high pressure liquid chromatographic system coupled to a ThermoFinnigan LCQ Duo ion trap mass spectrometer equipped with an electrospray ionization source (Thermo Electron Corporation,
Waltham, MA) as recently described (Jackson et al., 2006). The renal venous secretion rate of
AMP was calculated by multiplying the concentration of each substance in the venous perfusate by the perfusion rate.
Within each group, the secretion rates of AMP were compared in the absence and presence of AMP with a paired Student’s t-test. The secretion rates of AMP during administration of cAMP were compared among the 12 groups by one-factor ANOVA. Multiple
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JPET #119057 comparisons were performed by a Fisher LSD test if the overall ANOVA indicated that some of the means were different. All statistical analyses were performed using the Number Cruncher
Statistical System (Kaysville, Utah), and all values in the text and figures refer to means ± SEM. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 30, 2021
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JPET #119057 RESULTS
Basal perfusion pressure was similar in all 12 groups (approximately 50 mm Hg), and
cAMP infusions did not alter perfusion pressure (data not shown). Figure 1 illustrates the effects
of cAMP on renal AMP secretion in control kidneys (top panel), in kidneys pretreated with
IBMX (a broad-spectrum PDE inhibitor; middle panel) and in kidneys treated with DPSPX
(inhibitor of ecto-PDE; lower panel). Both IBMX and DPSPX significantly attenuated the Downloaded from cAMP-induced increase in renal secretion of AMP by 60% and 74%, respectively.
As shown in Figure 2, inhibition of PDE1 with mmIBMX (top panel) or PDE2 with
EHNA (middle panel) did not alter cAMP-induced renal AMP secretion. Also, inhibition of jpet.aspetjournals.org
PDE3 with either cGMP (Figure 2, bottom panel) or milrinone (Figure 3, top panel), PDE4 with
RO 20-1724 (Figure 3, middle panel), PDE5 and PDE6 with zaprinast (Figure 3, bottom panel) at ASPET Journals on September 30, 2021 or PDE7 with BRL-50481 (Figure 4, top panel) did not alter cAMP-induced renal AMP
secretion.
Importantly, administration of a concentration of dipyridamole (100 :mol/L) that inhibits
PDE8, PDE9, PDE10 and PDE11 significantly reduced cAMP-induced renal AMP secretion by
44% (Figure 4, middle panel). However, a concentration of diypridamole (3 :mol/L) that inhibits PDE9, PDE10 and PDE11, but not PDE8, did not significantly attenuate cAMP-induced renal AMP secretion.
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JPET #119057 DISCUSSION
The extracellular cAMP-adenosine pathway may participate importantly in a number of physiological processes. For example, Hong et al. (1999) report that the extracellular cAMP- adenosine pathway exists in rat pial arteries and contributes to the regulation of cerebral vascular dilation in response to hypotension. Finnegan and Carey (1998) demonstrate the existence of the extracellular cAMP-adenosine pathway in adipocytes and suggest a possible role for this Downloaded from pathway in the regulation of fat metabolism. Our own work implicates the extracellular cAMP- adenosine pathway in the regulation of vascular smooth muscle cell (Dubey et al., 1996) and cardiac fibroblast (Dubey et al., 2001) growth, in the regulation of renin release (Jackson, 1991) jpet.aspetjournals.org and as a mediator of some of the cellular effects of 17β-estradiol (Dubey et al., 2000). These findings indicate the importance of elucidating the nature of the ecto-PDE that mediates the at ASPET Journals on September 30, 2021 cAMP-adenosine pathway.
The purpose of this investigation was to determine whether ecto-PDE is pharmacologically similar to one or more of the known PDEs. The PDEs represent a group of enzymes with considerable molecular diversity. In mammals there are more than 20 different
PDE genes and, due to splice variants and alternative start sites, more than 50 different molecularly distinct PDEs (Lugnier, 2006). However, PDEs can be classified into 11 different families according to their structure, substrate preference, modalities of regulation and sensitivity to pharmacological inhibitors (Bender and Beavo, 2006). The PDE1 family is stimulated by calcium-calmodulin and inhibited by mmIBMX (Wells and Miller, 1988). Some isoforms of
PDE1 hydrolyze cGMP more efficiently than cAMP, whereas at least one isoform hydrolyzes cAMP and cGMP with equal efficiency (Rybalkin and Bornfeldt, 1999). PDE2 is a cGMP-
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JPET #119057 stimulated PDE that hydrolyzes both cAMP and cGMP. In contrast, PDE3 is a cGMP-inhibited
PDE that hydrolyzes cAMP and cGMP and is potently inhibited by drugs such as milrinone
(Harrison et al., 1986). PDE4 is a cAMP-specific PDE that is not regulated by calcium- calmodulin nor by cGMP and is specifically inhibited by drugs such as RO 20-1724 (Beavo et al., 1990). PDE5 is a cGMP-preferring enzyme that is regulated by phosphorylation, and PDE6 is also a cGMP-preferring enzyme but is regulated by transducin (Lugnier, 2006). PDE7 and Downloaded from PDE8 are cAMP-preferring and insensitive to rolipram, whereas PDE9 hydrolyzes preferably cGMP. PDE10 and 11 hydrolyze both cAMP and cGMP (Lugnier, 2006).
In the present investigation, we tested, using an array of pharmacological inhibitors, jpet.aspetjournals.org whether ecto-PDE activity in the kidney is pharmacologically similar to one or more of the known families of PDEs. There are several important conclusions that can be deduced from this at ASPET Journals on September 30, 2021 experimental series. First, renal ecto-PDE activity is inhibited by high concentrations of IBMX, a finding that supports the concept that renal ecto-PDE activity is related to the classical PDE family. Second, inhibition of ecto-PDE by DPSPX is in accordance with our previous observations (Mi and Jackson, 1995), and confirms that ecto-PDE activity is indeed an extracellular PDE since DPSPX is negatively charged at physiological pH and does not penetrate cell membranes to any significant extent (Tofovic et al., 1991). Third, ecto-PDE activity is not inhibited by high concentrations (approximately 30 times the IC50s of the inhibitors for their respective PDE targets) of mmIBMX, EHNA, milrinone, cGMP, RO 20-1724, zaprinast or BRL-
50481. These findings virtual exclude the possibility that PDE1, PDE2, PDE3, PDE4, PDE5,
PDE6 or PDE7 contribute to ecto-PDE activity in the kidney. Fourth, a concentration of dipyridamole that blocks PDE9, PDE10 and PDE11 does not inhibit renal ecto-PDE activity, a
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JPET #119057 finding that also eliminates these PDE isoforms as candidates for ecto-PDE activity.
Our findings in the perfused rat kidney are entirely consistent with the findings of Zacher
and Carey (1999). These investigators reported that swine adipocyte plasma membranes
metabolized cAMP to AMP and adenosine via PDE and 5'-NT, respectively. Although maximal
activity of microsomal membrane PDE was seven times greater than that of plasma membrane
PDE, microsomal membrane PDE was completely inhibited by the PDE3B inhibitor cilostamide, Downloaded from while plasma membrane PDE was unaffected by this PDE3B inhibitor. Moreover, PDE1, PDE2,
PDE4 and PDE5 inhibitors did not attenuate plasma membrane PDE activity. As in our study,
Zacher and Carey found that 1 mM DPSPX inhibited plasma membrane PDE activity by 72%. jpet.aspetjournals.org
Our previous studies demonstrate in isolated perfused rat kidneys that stimulation of adenylyl cyclase with the beta-adrenoceptor agonist isoproterenol increases renal cAMP at ASPET Journals on September 30, 2021 secretion and that this response is enhanced by RO 20-1724, but not by milrinone nor by mmIBMX (Jackson et al., 1997). Thus, the intracellular metabolism of endogenous cAMP in the renal vasculature appears to be mediated mostly by PDE4, whereas the metabolism of extracellular cAMP is mediated by a non-PDE4 enzyme.
Another conclusion from this study is that ecto-PDE in the kidney is pharmacologically similar to PDE8. Although our results do not imply that ecto-PDE is identical to PDE8, it is conceivable that ecto-PDE activity is mediated in part by PDE8 because PDE8 is expressed in the kidney (Bender and Beavo, 2006). Even though PDE8 does not contain putative transmembrane spanning domains, it contains a PAS domain which mediates binding to other proteins which could tether PDE8 to the cell membrane (Lugnier, 2006). Indeed, even though
PDE is a cytosolic enzyme, PDE8 is partially localized to the membrane compartment (Bender
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JPET #119057 and Beavo, 2006). Adenosine deaminase serves as an important example of how an enzyme
without membrane anchors can function as an ecto-enzyme. Although adenosine deaminase is
considered an intracellular enzyme, it is secreted by cells and binds avidly to CD26 (dipeptidyl
peptidase IV) on cell surfaces (Resta et al., 1998). Nonetheless, it is also possible that ecto-PDE is not authentic PDE8 but another PDE that is pharmacologically similar to PDE8. For example, the slime mold Dictyostelium discoideum secretes cAMP as a signaling molecule. Importantly, a Downloaded from recent study identified and characterized an ecto-PDE in Dictyostelium discoideum that has a catalytic domain with a high degree of homology with that of mammalian PDE8 (Bader et al.,
2006). A mammalian enzyme similar to the Dictyostelium discoidem ecto-PDE remains to be jpet.aspetjournals.org identified.
In summary, the present study indicates that the renal ecto-PDE enzyme is not PDE1, at ASPET Journals on September 30, 2021 PDE2, PDE3, PDE4, PDE5, PDE6, PDE7, PDE9, PDE10 or PDE11. Moreover, taken together with our previous published work in the perfused rat kidney, the present results demonstrate that the ecto-PDE responsible for hydrolyzing extracellular cAMP to AMP is not the same as the intracellular enzyme found in the renal circulation. We conclude that ecto-PDE has pharmacological characteristics similar to PDE8, although the exact identify of ecto-PDE remains undefined.
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JPET #119057 FOOTNOTES
This study was supported by grants from the National Institutes of Health (HL69846 and
DK68575). Downloaded from jpet.aspetjournals.org at ASPET Journals on September 30, 2021
-22- JPET Fast Forward. Published on February 16, 2007 as DOI: 10.1124/jpet.106.119057 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #119057 LEGENDS FOR FIGURES
Figure 1: Effect of cAMP (10 :mol/L) on AMP secretion by isolated, perfused rat kidneys in the absence and presence of 1,3-isobutyl-1-methylxanthine (IBMX; broad spectrum PDE inhibitor) and 1,3-dipropyl-8-sulfophenylxanthine (DPSPX; ecto-phosphodiesterase inhibitor).
Kidneys were pretreated with α,β-methylene-adenosine-5'-diphosphate (AMPCP; ecto-5'- Downloaded from nucleotidase inhibitor; 100 :mol/L). AMPCP and the PDE inhibitors were added to the perfusate beginning 5 minutes before adding cAMP to the perfusate. Basal AMP secretion was jpet.aspetjournals.org determined during the one minute before adding cAMP, and AMP secretion in the presence of cAMP was determined from four to five minutes after adding cAMP. The horizontal line indicates the level of AMP achieved in the control group. ap<0.05 compared with basal; bp<0.05 at ASPET Journals on September 30, 2021 compared with cAMP in control group.
Figure 2: Effect of cAMP (10 :mol/L) on AMP secretion by isolated, perfused rat kidneys in the presence of 3,8-methoxymethyl-3-isobutyl-1-methylxanthine (mmIBMX; selective PDE1 inhibitor), erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA; selective PDE2 inhibitor) and cGMP
(PDE3 inhibitor). See Legend to Figure 1 for details. ap<0.05 compared with basal.
Figure 3: Effect of cAMP (10 :mol/L) on AMP secretion by isolated, perfused rat kidneys in the presence of milrinone (selective PDE3 inhibitor), 4-(3-butoxy-4- methoxybenzyl)imidazolidin-2-one (RO 20-1724; selective PDE4 inhibitor) and zaprinast
(selective PDE5 and PDE6 inhibitor). See Legend to Figure 1 for details. ap<0.05 compared
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JPET #119057 with basal.
Figure 4: Effect of cAMP (10 :mol/L) on AMP secretion by isolated, perfused rat kidneys in the presence of 5-nitro-2,N,N-trimethylbenzenesulfonamide (BRL-50481; selective PDE7 inhibitor), a high concentration of dipyridamole (inhibits PDE8, PDE9, PDE10 and PDE11) and a low concentration of dipyridamole (inhibits PDE9, PDE10 and PDE11). See Legend to Figure Downloaded from 1 for details. ap<0.05 compared with basal; bp<0.05 compared with cAMP in control group. jpet.aspetjournals.org at ASPET Journals on September 30, 2021
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5 Control
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