Chapter 21 / Cardiovascular 321

21 Cardiovascular Hormones

Willis K. Samson, PhD and Meghan M. Taylor, PhD

CONTENTS VASOCATIVE FAMILIES NATRIURETIC FAMILY ENDDOTHELINS AM GENE PRODUCTS CARDIOVASCULAR HORMONES AS DIAGNOSTIC AND THERAPEUTIC TOOLS

1. VASOACTIVE HORMONE FAMILIES 1.1. The Heart as an Endocrine Organ Although the heart had long been considered merely acterized, their hallmark effects are to unload the vas- a muscular pump that performed the physical labor of cular tree via a combination of CNS, pituitary, adrenal, the circulation, it has been recognized for more than vascular, and renal actions (Fig. 1). This results in six decades that in addition to the contractile ultra- decreased venous return to the pump as a consequence structure, a secretory function was evidenced by dense- of increased renal excretion of water and solute, core granules in the myocytes. Over the past two vasorelaxation in certain vascular beds, increased cap- decades, the endocrine nature of the heart has been illary permeability, and decreased cardiac output. The established, and the physiology and pathophysiology third member of this family of hormones, although of the cardiac hormones have been extensively charac- exerting many of the same actions as ANP and BNP, is terized. In both a constitutive and regulated fashion, unique in that it is predominantly produced in the vas- myocytes produce two members of a family of hor- cular endothelium, not in the heart, and is thought to mones designated natriuretic based on their act more in a paracrine or an autocrine fashion, regu- abilities to stimulate salt and water excretion by direct lating primarily vascular tone and growth. Addition- renal actions and by actions in other tissues, including ally, this hormone, designated C-type natriuretic endocrine organs, responsible for the control of fluid peptide (CNP), exerts several CNS actions that oppose and . Two members of the natri- those of ANP and BNP. uretic peptide family, atrial (ANP) For years the central focus of vascular endocrinol- and brain natriuretic peptide (BNP, actually a misno- ogy was the renin–angiotensin system (see Chapter mer since very little if any of this peptide is produced 23); however, with the discovery of the cardiac hor- in the central nervous system) are produced in the heart mones and the realization that at least some of their and released in response to a variety of cues, many actions were expressed by a functional antagonism of typical of plasma volume overload or hyperosmolality. the actions of angiotensin, a broader view of the impor- Although numerous biologic actions have been char- tance of circulating hormones controlling vascular and renal function took shape. Then this doctrine of endo- From: Endocrinology: Basic and Clinical Principles, Second Edition crine regulation of cardiovascular and renal function (S. Melmed and P. M. Conn, eds.) © Humana Press Inc., Totowa, NJ was challenged and expanded by the realization that 321 322 Part IV / Hypothalamic–Pituitary

Fig. 1. Summary of biologic actions of ANP and CNP. AVP = vasopressin; ACTH = adrenocorticotropin; GnRH = gonadotropin- releasing hormone; CRH = corticotropin-releasing hormone; CO = cardiac output; UV = volume; UNaV = urinary sodium excretion; NO = nitric oxide; ET = ; T3/T4 = thyroid hormones.

perhaps the largest endocrine organ in the body, by ments via factors produced in the endothelium. Much virtue of its enormous surface area and ubiquitous pres- attention has been focused on the ability of the endothe- ence, was the vasculature itself, mainly the endothe- lium to cause vasorelaxation via the generation of a lium. Not only was one member of the natriuretic soluble gas, nitric oxide (NO); however, peptidergic peptide family produced in and released from this tis- factors originating in the endothelial cells control VSMC sue, but it became apparent that numerous peptidergic, function as well. Here the role of CNP as a paracrine as well as nonpeptidergic, factors originating in the factor has been established, and the endothelial cell– endothelium controlled vascular tone and prolifera- VSMC interface was the setting for the discovery and tion. characterization of two additional, potent vasoactive hormones, endothelin (ET) and adrenomedullin (AM). 1.2. Hormones of the Endothelium The ETs are potent hypertensive agents that exert their The vascular endothelium controls access of blood- effects directly on the VSMCs. AM, on the other hand, borne factors not only to the interstitium, but also to the is a potent hypotensive agent, acting in a paracrine or an contractile and proliferative elements of the vascular autocrine fashion in the vasculature. Thus, both circu- tree, the vascular smooth muscle cells (VSMCs). Addi- lating and locally produced vasoactive hormones can tionally, the endothelial cells are positioned optimally control regional blood flow, and this cellular interface to respond themselves to circulating factors and to trans- has provided a model for the paracrine and autocrine duce those messages to the VSMCs. Many hormonal effects of these peptides in other tissues as well. Ago- messages are in fact delivered to the contractile ele- nists and antagonists selective for these peptides have Chapter 21 / Cardiovascular Hormones 323 been successfully tested in models of cardiovascular physical factors. There is a regional mismatch in the disease and some have even been approved for clinical adult in gene expression of the two peptides, with ANP use in humans. expressed primarily in the atria and BNP in the ven- tricles under basal conditions. Physical factors such as 2. NATRIURETIC PEPTIDE FAMILY cardiac overload induce transcription of both genes, 2.1. Gene Structure and Regulation with the appearance of ANP expression in the ventricles The members of the natriuretic peptide family share as well. Most striking, however, is the level of induc- structural homology but are products of unique genes. tion of the BNP gene in the ventricles, resulting in a Expression of separate genes and posttranslational pro- remarkable increase in circulating hormone levels. At cessing of the nascent hormones is very similar. The the molecular level, ANP gene transcription is regu- genes for ANP and BNP have been localized to the same lated by numerous members of the activating protein- chromosome, whereas that for CNP resides on a sepa- 1 complex, being induced by c-jun and in most cases rate chromosome, further suggesting the similar actions suppressed by c-fos. A close relative of c-fos, fra-1, of ANP and BNP and disparate effects of CNP. Cloning exerts biphasic effects, reducing the magnitude of c-jun of the cDNA complementarity to ANP mRNA revealed activation of ANP gene expression in atriocytes, while the presence of three exons in the ANP gene and the amplifying the induction of expression by c-jun in transcription of a prepro-ANP mRNA that encoded a ventriculocytes. Thus, the response of the ANP promo- 151- to 152-amino-acid preprohormone, depending on tor to these early response elements may vary under species. Removal of the N-terminal unique physiologic conditions, permitting a wider rep- results in a prohormone of 126 amino acids, which dem- ertoire of control of gene expression. onstrates extensive homology across species. This 126- amino-acid prohormone is the major form of the peptide 2.2. Hormone Secretion stored in secretory granules in the heart. Stored pro- 2.2.1. PHYSIOLOGIC RELEASE ANP is processed at secretion to a variety of smaller, Plasma levels of ANP and BNP are extremely low biologically active forms, primarily the mature 28- (5–10 and 0.5–1.0 fmol/mL, respectively) and rise in amino-acid, C-terminal fragment. In brain, on the other response to any interventions that increase venous hand, the prohormone is further processed to the mature return and, therefore, atrial pressure and stretch. Pressor peptide before packaging in secretory granules. An agents can release ANP in vivo and some even act in additional form of ANP is produced in , this form isolated tissue in vitro, suggesting direct cellular effects being 33 amino acids of the C-terminus by posttransla- independent from increased venous return. The natri- tional processing that includes four more amino acids uretic effects of ANP and BNP are mirrored by the abil- on the N-terminus. This isoform, designated urodilatin, ity of hyperosmolality to stimulate directly, and is thought to act as a paracrine regulator of tubular indirectly via volume expansion, hormone secretion. In function. Expression of the BNP gene differs in that the addition to secretion from the heart, these peptides are resultant mRNA is less stable and the final posttrans- produced in and secreted from or into a variety of other lational product is 32 amino acids long. Finally, CNP tissues where distinct biologic actions have been char- processing is quite similar to that of ANP, with the acterized. The absolute contribution of those release exception that the final posttranslational product lacks sites to circulating levels of the hormones is in all like- the C-terminal extension distal to the shared (within the lihood minor; however, the potential importance of natriuretic family) 17-membered disulfide loop, con- paracrine effects of the natriuretic peptides in those other sisting, thus, of only 22 amino acids. In humans, owing tissues makes the study of the regulation of release in to the presence of an arginine in the prohormone at noncardiac sites extremely important. Indeed, renal, position 73, a second form of CNP is present, which is CNS, gonadal, and thymic production sites suggest a N-terminally extended, consisting of 53 amino acids. diversity of function for the peptides, and the mecha- CNP-22 and CNP-53 exert similar actions in many bio- nisms responsible for the regulation of secretion first logic systems. Note that in all three isoforms, the integ- must be elucidated before the physiologic or pathologic rity of the internal disulfide loop is necessary for biologic significance of those production sites is fully under- activity. stood. Within the CNS, some of the same circulating Although little is known about the regulation of CNP factors that can stimulate ANP release from the gene transcription, mechanisms for activation of ANP myocytes (i.e., vasopressin and ET) similarly stimulate and BNP gene transcription have been extensively stud- neuronal production and release of the peptide. ied. Gene transcription is induced by glucocorticoids, Endothelial cell production of CNP has been clearly α-adrenergic agents, growth factors, calcium, and established and the control of peptide secretion partially 324 Part IV / Hypothalamic–Pituitary characterized. A variety of cytokines and growth fac- uretic peptide receptor-A (NPR-A) and NPR-B. A third tors (including interleukin-1α [IL-α] and IL-β, tumor receptor subtype, called the clearance receptor or NPR- necrosis factor-α [TNF-α], and transforming growth C, shares approx 30% homology with NPR-A and NPR- factor [TGF-β], as well as ANP and BNP) can stimulate B in the extracellular ligand-binding domain; however, CNP release from endothelial cells. Shear stress and this receptor lacks the intracellular C-terminal exten- hypoxia stimulate CNP release in the vasculature, as sion (i.e., it is missing the kinase and GC domains). This they do for ANP and BNP in the heart. Thus, the receptor was originally thought to have no biologic endothelial cell can, via CNP secretion, both transduce activity other than to sequester or clear natriuretic pep- the antimitogenic effects of circulating ANP and BNP tides from the extracellular fluid; however, it is now and buffer the proliferative effects of circulating cyto- recognized that NPR-C plays important biologic roles kines and growth factors. and signals via a reduction in cyclic adenosine mono- phosphate (cAMP) levels and possibly a stimulation of 2.2.2. STATE OF HYPERSECRETION polyinositol phosphate turnover (increased phospho- Elevations in circulating natriuretic peptides have lipase C [PLC] activity). This receptor appears to medi- been reported in a variety of pathophysiologic states. ate the antimitogenic actions of the natriuretic peptides CNP is remarkably elevated in septic shock, but not in in the CNS. A distinct hierarchy of binding affinities hypertension or congestive heart failure (CHF). This characterizes these receptors with all three forms of again points to the more likely paracrine actions of natriuretic peptides binding with equal affinity to NPR- CNP within the endothelial cell interface with VSMCs. C. NPR-A prefers ANP as a ligand (ANP > BNP >> CHF, myocardial ischemia, and hypertension all result CNP), whereas NPR-B recognizes more readily CNP in increased ANP and BNP secretion, reflecting pos- (CNP >> ANP = BNP). Thus, the sites of action of the sible compensatory mechanisms called into play during natriuretic peptides are determined by the relative dis- those conditions. Plasma BNP levels in cardiac over- tributions of the three receptors, with NPR-B predomi- load states exceed those of ANP and are used clinically nating in the brain (e.g., the hypothalamo-hypophyseal as diagnostic and prognostic tools to assess the progres- system) and muscular component of the vasculature, sion and degree of heart failure. Although elevated dur- whereas NPR-A is more abundant in the kidney, adrenal ing those overload states, the bioactivity of ANP and gland, and endothelium. The clearance (NPR-C) recep- BNP appears to be reduced owing to a possible com- tor is present throughout the body. bination of effects, including reduced renal perfusion, receptor downregulation, or the counterregulatory 2.4. Biologic Actions effects of simultaneous activation of the renin-angio- Originally CNP was thought to act only in a paracrine tensin-aldosterone system. Of these three possible fashion to regulate vascular tone and growth; however, explanations, the best case can be made for the latter. CNP can also exert cardiovascular, renal, and adrenal The increased circulating levels of ANP in critically ill actions when infused intravenously. This may simply trauma patients is thought to be a potential cause of be a reflection of the fact that CNP is produced in a suppressed adrenocorticotropic hormone levels fre- variety of tissues, and, therefore, multiple paracrine quently observed, because ANP can act at both the actions may occur. CNP levels are elevated in chronic hypothalamic and pituitary levels to inhibit corticotro- renal failure, and the peptide is produced in kidney, pin release. where it exerts and natriuretic effects. One can recognize the sites of action of the natriuretic peptides 2.3. Sites of Action by locating receptors, but the assignment of biologic Three natriuretic peptide receptor subtypes have activity is not as simple. Two reagents that have clari- been identified (Fig. 2). Two of these proteins contain fied the receptor subtype responsible for a variety of intracellular kinase homology domains (adenosine natriuretic peptide actions are the clearance receptor triphosphate binding sites) and C-terminal guanylyl ligand C-ANF4–23, which binds preferentially to NPR- cyclase (GC) domains. Activation of these receptors C, and the GC antagonist HS-142-1, which blocks the results, therefore, in elevated cellular cyclic guanosine ability of the natriuretic peptides to signal via activa- 5´-monophosphate (cGMP) levels. Their extracellular tion of GC. By using a combination of methodologic domains share 44% homology, whereas the intracellu- approaches, it has been realized that although the NPR- lar domains share 63% homology in the kinase homol- C controls the antimitogenic effects of the natriuretic ogy domain and 88% homology in the GC domains. peptides centrally, NPR-B performs a similar function These two receptors have been designated the GC-A in the vascular compartment. Within the kidney, mul- and GC-B receptors and are alternatively called natri- tiple receptors are found, explaining the ability of both Chapter 21 / Cardiovascular Hormones 325

Fig. 2. Three members of the natriuretic peptide family, ANP, BNP, and CNP, share 65% homology (indicated by shaded circles) in the biologically active ring structure formed by the disulfide links and vary in composition and lengths of their N- and C-terminal extensions. All three peptides are recognized by the natriuretic peptide C (clearance) receptor (NPR-C); however, the A receptor (NPR-A) prefers ANP and BNP. The B receptor (NPR-B) recognizes with relative preference CNP. Activation of the three receptors has been reported to generate the indicated changes in intracellular levels of cGMP or cAMP and/or phosphoinositol (PI) turnover.

ANP and CNP to act as diuretic and natriuretic agents. other examples where interactive effects via the shared The multiple peripheral effects of the natriuretic pep- NPR-C cannot underlie the effects observed. Thus, the tides are summarized in Fig. 1. Although not all of these ability of ANP to inhibit the behavioral (water drinking) actions seem related to fluid and electrolyte homeosta- and endocrine ( [PRL] secretion) aspects of sis, some may instead be related to the antiproliferative fluid and electrolyte homeostasis is opposed by the effects of the peptides. stimulatory effects of CNP. Certainly in these cases, Within the CNS, similar and diverging actions of activation of NPR-A must underlie the effects of ANP, ANP and CNP have been described. In most species, whereas NPR-B must be responsible for the stimulatory more CNP is produced within the brain than ANP or effects of CNP. In the absence of antagonists that can BNP and, for the most part, NPR-B and NPR-C pre- distinguish between these two GC receptor subtypes, dominate within the brain interstitium. It should be rec- other methodologies had to be created to make these ognized that ANP may therefore exert its biologic distinctions. One such approach is receptor-specific actions in the brain by displacing CNP from NPR-C cytotoxin cell targeting using the plant lectin ricin. With (clearance receptor). This has been demonstrated to be this approach, evidence for the involvement of the NPR- the case in the neuroendocrine hypothalamus. There are A in the physiologic regulation of salt appetite has been 326 Part IV / Hypothalamic–Pituitary obtained, and the importance of NPR-B in the hypotha- tion, but the condition can be rescued by simultaneous lamic mechanisms controlling neuroendocrine function targeted overexpression of a CNP transgene. These data has been established. complement the results (just discussed) in the CNP Which of the pharmacologic actions of the natri- overexpression system alone and further support a sig- uretic peptides are physiologically relevant? A combi- nificant role of CNP in normal bone development and nation of experimental approaches has provided turnover. The CNP knockout animals display early evidence for the role of the peptides in a variety of mortality; thus, other important roles of CNP must be tissues. Use of the selective clearance receptor ligand present and are certainly awaiting discovery. C-ANF4–23 established the importance of ligand bind- ing to the NPR-C on astrocyte proliferation (i.e., 2.5. Potential Therapeutic Uses antimitogenic effects). The GC receptor antagonist Although the action of the natriuretic peptides HS-142-1 was employed to demonstrate that the natri- appears to be blunted in edematous states, such as CHF, uretic peptides play important roles in the maintenance cirrhosis, and the nephrotic syndrome, therapeutic use of glomerular filtration and sodium excretion under of the peptides in these states may prove at least acutely basal conditions. Ricin cytotoxin adminstration stud- advantageous. Certainly, if the mechanism by which ies demonstrated the role of endogenous brain-derived the biologic activity of the peptides has been reduced CNP in the neuroendocrine regulation of PRL and in these states can be elucidated, strategies might be luteinizing hormone secretion, and the importance of employed that overcome those deficits. In particular, it central ANP to the control of sodium homeostasis has already been demonstrated that in CHF, adminis- (i.e., appetite). Passive immunoneutralization was tration of high doses of ANP and urodilatin can lower employed to demonstrate the physiologic relevance of preload and increase diuresis and natriuresis, providing the action of ANP to inhibit thirst. significant benefit in this life-threatening situation. Recently two molecular techniques have provided Even though plasma ANP levels are elevated in CHF, additional insight into the physiology of the natriuretic further elevation by exogenous administration has pro- peptides. Transgenic mouse models of overexpression vided salutary therapy. Like conventional , of natriuretic peptide have been created. In the case of ANP increases urine volume and urinary sodium excre- the ANP transgene, homozygotes displayed signifi- tion, at least in part by inhibiting sodium reabsorption cantly lower blood pressure under basal conditions than in the collecting duct by an action on the sodium/chlo- nontransgenic littermates; however, sodium excretion ride transporter. However, unlike current diuretic was not different. Transgene-induced overexpression agents, ANP inhibits the renin-angiotensin-aldosterone of BNP led to increased endochondral ossification and system by directly inhibiting renin secretion and bony overgrowth. These studies uncovered an action of thereby lowering aldosterone levels in plasma. In addi- BNP that was not revealed in pharmacologic studies, tion, administration of ANP lowers sympathetic tone, probably due to the chronic effect of BNP overexpres- and the combined reduction in plasma renin activity sion, something not possible to accomplish in classic and sympathetic tone effectively lowers sodium reab- pharmacologic application studies. Transgene-induced sorption in the proximal tubule. ANP also inhibits overexpression of CNP or BNP improved postischemic tubuloglomerular feedback, and the maintenance of insult neovascularization by stimulating reendothelial- glomerular filtration even in the face of decreased renal ization and suppressing neointimal formation. These blood flow may be an important reason for the peptide’s studies strongly suggest a therapeutic option for the protective effect on renal function even in low perfu- natriuretic peptides in patients with tissue ischemia. sion states such as heart failure. Finally, ANP not only The second molecular approach to the study of the inhibits arginine vasopressin (AVP) release, but it physiologic relevance of the pharmacologic effects of blocks AVP’s ability to stimulate water reabsorption in the natriuretic peptides is the generation of null muta- the collecting duct. ANP therapy in acute heart failure tions (knockouts), which results in the absence of a given was approved in Japan almost 10 yr ago, and recently peptide. ANP-null mice are more susceptible to the a synthetic BNP, nesiritide, was approved for use in the hypertensive consequences of high salt ingestion. This United States. model reveals two important things. First, ANP is not Because a role of endogenous ANP in the phenom- essential for normal embryonic and postnatal develop- enon has been suggested, perhaps a similar strategy can ment. Second, endogenous ANP must play some role in be used to induce mineralocorticoid escape. Interest in the physiologic mechanisms that protect against the the postoperative use of ANP and urodilatin to prevent development of high blood pressure. CNP knockouts acute renal failure has been stimulated by early studies are dwarfs, displaying impaired endochondral ossifica- demonstrating the ability of high pharmacologic doses Chapter 21 / Cardiovascular Hormones 327

Fig. 3. Three members of the mammalian ET peptide family have been identified, each sharing remarkable homology in amino acid composition. Shaded circles indicate differing amino acids. Three receptor subtypes have been characterized. The ET-A receptor binds ET with a relative preference indicated by the thickness of the arrows (ET-1 Ն ET-2 > ET-3). The ET-B receptor recognizes equally all three forms of ET. The third receptor, ET-C, found thus far only in nonmammals, prefers ET-3. Sites of receptor expression are indicated.

of the peptide to reduce the need for hemodialysis/ of the ETs (ET-1, ET-2, and ET-3), all 21 amino acid hemofiltration in these patients. peptides differing by only 2–5 amino acids in the 15- Most promising in a therapeutic sense is the poten- membered ring structure formed by two internal disul- tial use of the natriuretic peptides as antiproliferative fide bonds (Fig. 3). Each are products of unique genes agents. In a rabbit model of vascular lesions caused by and are first processed similarly into a prohormone balloon catheter injury, administration of CNP signifi- form of 203 amino acids, in the case of ET-1, and then cantly lowered the resultant intima-to-media ratios, posttranslationally modified into the 39-amino-acid providing direct evidence for a paracrine action of the prohormone intermediate, big ET. In states of hyper- peptide to suppress intimal thickening. Furthermore, secretion, the prohormone forms appears in plasma; local CNP antagonizes the growth-promoting effects however, under normal conditions, the mature 21- of angiotensin II (Ang II) and the vascular conse- amino-acid form is the major secretory product. The quences of cyclosporine A induction of ET release and final cleavage of the prohormone is thought to occur at subsequent mitogenesis, again predicting a significant secretion and to be catalyzed by a - avenue for the prevention of vascular lesions. sensitive metalloproteinase designated ET-converting 3. (EC 3.4.24.11). Knowledge of this important conversion enzyme’s presence has led to potential 3.1. Gene Structure therapeutic intervention strategies for interruption of In addition to the production of endothelial-derived ET action in states of hypersecretion, because the relaxing factors in the vasculature, it had been known prohormone big ET has limited biologic activity. for some time that the cells lining the blood vessels The human ET-1 gene has five exons and four introns, produce potent vasoconstrictive substances as well. In with the peptide coded in the second exon. The gene is 1988, the sequence of a powerful, endogenous vaso- transcriptionally regulated via cis elements, including a constrictor substance produced by endothelial cells was GATA-2 protein-binding site and an AP-1 site that is identified. This 21-amino-acid peptide was named ET. activated by thrombin, angiotensin II, epidermal growth It is now recognized that there are at least three forms factor (EGF), basic fibroblast growth factor (bFGF), 328 Part IV / Hypothalamic–Pituitary -like growth factor, and TGF-β. Other transcrip- of hypersecretion, pulmonary hypertension (Fig. 4). In tional regulators include vasopressin, the ILs, TNF-α, VSMCs, ET stimulates contraction and mitogenesis and NO, which apparently mediates the ability of hep- via multiple signaling pathways, including activation arin to stimulate ET production. Physical factors also of PLC with the resultant formation of diacylglycerol activate transcription, including pressure and anoxia. (DAG) and inositol triphosphate (IP3). The DAG Translational regulation is exerted by a variety of fac- formed activates the kinase cascade via protein kinase tors that also regulate secretion, since little hormone is C (PKC), and IP3 mobilizes intracellular calcium, ini- stored intracellularly. High-density lipoproteins stimu- tiating the contractile event. ET-A receptor activation late production and secretion, whereas insulin not only also in these cells has been reported to open potassium stimulates production and secretion, but also augments channels and to activate adenylyl cyclase. In the myo- ET binding and action. Negative regulation of produc- cardium, the ET-A receptor is thought to be activated tion and secretion is exerted at the transcriptional level by endogenous ET released in response to ischemia by NO, and at the translational event by prostaglandins, following myocardial infarction. The resultant open- prostacyclin, AM, and ANP. ing of potassium channels causes a decrease in the elec- trical activity of the myocyte and closes the chloride 3.2. Hormone Secretion channel, resulting in a suppression of catecholamine Fortunately, the majority of the ET produced is activation of contractile function. secreted abluminally, away from the vessel lumen, to act The ET-B receptor predominates in the endothe- in a paracrine or autocrine fashion. Although levels of lium itself and in the CNS. This receptor binds all three the hormone do rise in certain pathologic conditions, in isoforms equally and is responsible for the activity of general, reflecting tissue damage in most cases, this pep- circulating ET to stimulate a transient vasodilatory tide should be kept out of the circulation because of its response in the periphery, via acute release of vasodila- potent vasoconstrictive properties and because in exper- tors such as NO and CNP. The signaling cascade that imental animals, elevation in circulating ET results in follows activation of the ET-B receptor is multifaceted. respiratory failure and/or cerebral vasospasms and G protein–coupled activation of PLC results in PKC aneurysms. Again, knowledge of the production sites activation and mobilization of intracellular calcium. NO predicts biologic activities. The major site of ET-1 pro- synthase activity is stimulated with the resultant pro- duction is the endothelium, where on release it causes duction of the potent vasodilator NO, which can act vasoconstriction. Additional production sites include the within the endothelial cell to activate GC or diffuse brain, uterus, kidney mesangial cells, Sertoli cells, and across to the smooth muscle cells to perform the same breast epithelial cells. ET-3 production occurs mainly function. Additionally, ET-B activation results in open- within the CNS, where a role for the peptide in neuronal ing of the sodium-hydrogen antiporter and inhibition of and astroglial development and proliferation has been adenylyl cyclase. The mitogenic effects of ET are suggested. What little ET-2 is produced in the body is thought to be transduced via PKC activation and tyro- found in kidney, intestine (hence, the alternative name sine phosporylation–intiated activation of the mitogen- vasoactive intestinal constrictor), myocardium, and activated protein kinase (MAPK) system. In mesangial uterus. The ETs have a relatively short half-life in cells, the mitogenic effect of ET is mediated via tran- plasma, about 4–7 min, and are released primarily in scriptional activation of immediate early genes. Activa- response to hypoxia, ischemia, and shear stress. tion of Ras proteins and downstream induction of the kinase activity of Raf-1 result in transcriptional induc- 3.3. Site and Mechanisms of Action tion of the c-fos serum response element, perhaps pro- Two mammalian ET receptor subtypes have been viding a mechanism for the mitogenic effect of ET. One cloned, and they are members of the G protein–linked, hallmark characteristic of the biologic effects of the ETs seven-transmembrane-spanning domain superfamily is their profound tachyphylaxis. Although some data of biologic receptors (Fig. 3). The ET-A receptor dis- indicate this to be the result of chronic membrane effects plays a rank order of binding affinity with ET-1 being or overloading of the cytosolic calcium pool, evidence the preferred ligand (ET-1 Ն ET-2 >> ET-3). This also exists for rapid internalization of the ligand-recep- receptor predominates in VSMCs and cardiac tor complex and continued signaling from the internal- myocytes. Activation of the receptor results, depend- ized aggregate. ing on tissue site, in the activation of a variety of sig- naling cascades, including in the lung the production 3.4. Biologic Actions of prostanoids via stimulation of PLD and PLA2 activi- Although multiple pharmacologic effects of the ETs ties resulting in bronchoconstriction and in the scenario have been reported, there is a need to establish which of Chapter 21 / Cardiovascular Hormones 329

Fig. 4. Summary of biologic actions of ETs.

those have biologic significance and physiologic rel- hypotension. Unexpected results accrued from these evance. In this case, multiple pharmacologic tools are null mutation strategies. Mice lacking expression of the available, such that selective antagonism of the ET-A normal ET-1 gene are born with severe craniofacial receptor is possible and isoform-specific activation of malformations, suggesting a developmental role of the the ET-B receptor can now be accomplished. Also avail- peptide in pharyngeal arch structures. Additionally, able are antagonists that affect both the ET-A and ET- these animals succumb to respiratory failure, suggest- B receptor, and a new generation of relatively specific ing an important embryonic role of the peptide in the ET-B antagonists. Much interest continues regarding preparation of respiratory structures for postnatal life. the possible existence of a unique, ET-3-selective ET-C Knockouts of the genes encoding the ET-B receptor or receptor in mammals similar to that found in frog mel- ET-3 itself result in a postnatal phenotype similar to anophores, and it is hoped that eventual cloning of that that observed in Hirschsprung disease (congenital protein will permit generation of similarly selective megacolon), suggesting the importance of ET in the antagonists. development of the intrinsic nervous system of the gut Surprising results from molecular approaches have and the regulation of gastrointestinal smooth muscle provided new insight into the biology of the ETs. As function. Multiple CNS actions of the ETs have been discussed below, it was anticipated that these potent reported, ranging from mitogenic effects on astrocytes vasoconstrictive peptides would be found to play an mediated via the ET-B receptor to effects on descend- important role in the development of hypertension; ing sympathetic activity. The presence of the ET pep- however, gene knockout homozygotes have, if any- tide and ET receptors during fetal development thing, slightly elevated blood pressure, not the expected indicates a potential embryonic role in CNS structures, 330 Part IV / Hypothalamic–Pituitary which may mirror the situation uncovered by the ET-3 ET antagonists improve renal function in a genetic and ET-B receptor knockouts in the intrinsic nervous model of hypertension, the spontaneously hypertensive system of the gut. Loss of the neurotropic effects of the rat, and ET has been invoked in the pathogenesis of ETs may be responsible in part for the respiratory fail- acute renal failure (postischemia renal failure). Cyclo- ure seen in the immediate postpartum interval. Regard- sporine A–induced nephrotoxicity has been identified ing neuromodulatory effects of the ETs, antagonist to be owing at least in part to ET-induced vasoconstric- studies have revealed the physiologic relevance of the tion of the afferent arteriole, since the renal toxicity of antidipsogenic effects of ET; however, the day-to-day immunosuppressant therapy was blocked by ET antago- significance of the neuroendocrine actions of the pep- nist pretreatment. The mitogenic actions of ET are tide (including activation of the hypothalamic mecha- thought to underlie the development of graft arterio- nisms controlling anterior pituitary function and sclerosis in cardiac allografts, the establishment of vasopressin secretion) and the effects of the peptides on atherosclerotic plaques, and the development of dia- sympathetic function have yet to be established. betes-related vascular lesions. Its role in vasospasm A final CNS consequence of the administration of ET secondary to subarachnoid hemorrhage has been estab- is potentially disastrous. In some species, ET infusion lished in animal models. Within the lung, roles for ET results in rupture of the basal artery on the ventral sur- in asthma and pulmonary hypertension have been pro- face of the Pons, and a role for endogenous ET in vasos- posed. The current literature favors the therapeutic use pasm subsequent to subarachinoid hemorrhage has been of ET antagonists in a variety of pathologic situations. established by the observation that pretreatment with an However, clinical trials have not always been success- ET antagonist can prevent this event. This has led to the ful in establishing their clinical value. hypothesis that ET antagonists may prove therapeuti- Several patient trials were conducted to determine cally advantageous to prevent or lessen ischemic dam- the efficacy of ET antagonists in the setting of pulmo- age downstream from damaged, hypoxic endothelium nary arterial hypertension (PAH). The rational for these of the cerebral vessels after thrombosis or infarct. trials was that lung levels of ET mRNA are increased in PAH, and a positive correlation between serum ET lev- 3.5. Pathophysiology of the ETs els and measured pulmonary vascular resistance had Because of the multiple pharmacologic activities of been described. Although the nonselective ET antago- the ETs, their involvement in numerous pathologic nist, , was effective in reducing pulmonary states has been hypothesized. These hypotheses were artery pressure and pulmonary vascular resistance in based largely on elevated circulating ET levels or one study when administered with another agent (a pros- responses seen in these conditions and, under many taglandin included to increase pulmonary blood flow), circumstances, it remains unclear whether the associa- deaths occurred in the treatment group. In another trial, tions are causal or coincidental. The most predicted bosentan did improve exercise tolerance in patients with role for the ETs in pathophysiology was that in hyper- severe PAH. This led to the approval by the Food and tensive states. Indeed, plasma levels are not consistently Drug Administration of the use of low-dose bosentan in found to correlate with blood pressure, and although PAH; however, liver function tests were abnormal in a they may be elevated in some animal models of hyper- significant portion of the treatment group, and, thus, the tension, null mutant mice actually have elevated blood possible negative side effect of hepatotoxicity must be pressures. Similarly controversial is the potential role weighed against the benefit obtained. Hepatotoxicity for ET in reperfusion injury; one group using an iso- was also a problem in trials employing the selective ET- lated perfused rat heart model argued against a caus- A antagonist sitaxsentan. ative role and another utilizing isolated ventricular Again in human trials, this time in the setting of CHF, myocytes argued in favor. In vivo evidence favoring a bosentan treatment resulted in a significant incidence of role for endogenous ET in reperfusion injury comes liver abnormalities. On the other hand, , a from studies in pig in which both myocardial ischemia nonselective ET antagonist, showed some benefit in and infarction resulted in significantly elevated ET acute heart failure patients in one trial but failed to pro- production and release. In rats receiving infusion of an vide protection in others. The selective ET-A antagonist antiserum directed against ET-1 prior to coronary artery improved cardiac index in patients with New ligation, damage distal to the ligation was markedly York Heart Association stage III heart failure, but there reduced. Similar results were obtained with an ET-A were more adverse events in the treatment group than in receptor antagonist in a canine myocardial infarction placebo control subjects. Thus, in heart failure trials, ET model. These data provide the best evidence for a role antagonists have not been proven to be safe alternatives for ET in reperfusion injury and ischemia. to conventional therapies. The same can be said of trials Chapter 21 / Cardiovascular Hormones 331

Fig. 5. Posttranslational processing of the preproadrenomedullin protein results in the formation of two biologically active peptides, AM and PAMP.

examining the efficacy of the ET antagonist in essential brain, kidney, endothelial cells, and VSMCs. Post- hypertension. Only recent, preliminary trials in patients translational processing of the 185-amino-acid pro- with coronary artery disease are promising. Because ET hormone results in the production and secretion of is a vasoconstrictor and stimulates smooth muscle pro- the mature 52-amino-acid form, designated AM, and a liferation, neutrophil adhesion, and platelet aggregation, 20-amino-acid fragment from the N-terminus desig- it was hypothesized that ET antagonists would be effec- nated proadrenomedullin N-terminal 20 peptide tive in the treatment of coronary artery disease. Indeed, (PAMP), a peptide that shares some, but not all, of the ET antagonists increased coronary artery diameter and biologic activities of AM. Although activation of prevented vasoconstriction after percutaneous coronary adenylyl cyclase was used as a screening bioassay in angiography. However, these trials need to be repeated the initial phases of discovery, the hallmark bioassay and extended with a careful examination of liver func- for AM is the potent hypotensive action when infused tion in patients before any conclusions can be made. intravenously (Fig. 6). AM production in VSMCs and endothelial cells is 4. ADRENOMEDULLIN GENE PRODUCTS regulated at the transcriptional level by a variety of cyto- 4.1. Gene Structure and Regulation kines, including IL-1α, IL-1β, TNF-α, and TNF-β. Pro- Utilizing a bioassay system that monitored accumu- duction of AM in these cells is also stimulated by lation of cAMP in platelets, Japanese investigators iden- thrombin, aldosterone, cortisol, retinoic acid, and thy- tified in 1993 a novel vasoactive hormone in extracts of roid hormones. To a lesser degree, stimulation of pro- a human pheochromocytoma. The peptide (Fig. 5) is duction in VSMCs was reported also in response to Ang produced in normal chromaffin cells of the adrenal II, epinephrine, platelet-derived growth factor, EGF, and gland, as well as a variety of other tissues, including FGF. TGF-β and cAMP inhibit production. 332 Part IV / Hypothalamic–Pituitary

Fig. 6. Summary of biologic actions of AM. CRH = corticotropin-releasing hormone; ACTH = adrenocorticotropin; AVP = vaso- pressin; OT = oxytocin; CO = cardiac output; HR = heart rate; ANP = atrial natriuretic peptide; UV = urine volume; UNaV = urinary sodium excretion; ET = endothelin.

4.2. Hormone Secretion action of the peptide, since PAMP acted as an anti- Many of the factors that stimulate hormone produc- cholinergic inhibiting sodium influx and reducing the tion also stimulate secretion in isolated cell systems, magnitude of catecholamine response to carbachol. leading to the hypothesis that AM may be responsible for the hypotension of inflammation, septic shock, and 4.3. Sites of Action atherosclerosis. Circulating levels in humans are simi- Some of the pharmacologic actions of AM can be lar to those of other vasoactive hormones (about 3 fmol/ blocked by the calcitonin gene–related peptide (CGRP) mL of plasma), suggesting that like other vasoactive receptor blocker CGRP8–37, which is not surprising substances, the actions of AM may be predominantly because AM and CGRP share considerable structural autocrine or paracrine in nature. One group failed to homology. Both activate adenylyl cyclase in a variety of observe elevations in plasma AM concentrations during tissues and can displace each other in binding assays. hypertensive attacks in patients with pheochromocyto- Mesenteric vasodilatory responses to AM are blocked mas; however, cosecretion of AM and catecholamines by CGRP8–37 in vitro, but the in vivo vasodilatory has been observed from cultured, bovine adrenal med- responses are not. Additionally, the increase in cAMP ullary cells. In fact, the cosecretion of PAMP and cat- levels observed in response to AM in endothelial cells in echolamines from these cells is calcium dependent and culture is not blocked byCGRP8–37. Both AM and CGRP induced by carbachol activation of nicotinic receptors. bind the calcitonin receptor–like receptor (CRLR), Those studies also pointed to an autocrine or paracrine which is a unique G protein–coupled receptor. When Chapter 21 / Cardiovascular Hormones 333

CRLR associates with an accessory protein (receptor terbalance to the renin-angiotensin-aldosterone sys- activity modifying protein-1 [RAMP-1]), it functions as tem. Indeed, this may be the case because both AM and a selective CGRP receptor, one that can be blocked by PAMP inhibit Ang II–stimulated aldosterone secre- CGRP8–37. On the other hand, when CRLR associates tion in vivo and block activation of the hypothalamic- with the homologous RAMP-2 and RAMP-3, the com- pituitary-adrenal axis at the level of the anterior plex is more selective for AM binding. This may explain pituitary gland. AM is produced in cardiomyocytes and the apparent similarity in action of CGRP and AM in acts locally to inhibit fibrosis stimulated by increased some systems. Not all of the biologic activities of AM Ang II and aldosterone levels. It acts to increase coro- can be explained by the presence of the CRLR/RAMP- nary blood flow, a function that is thought to have clini- 2 or RAMP-3 receptor complexes; thus, additional yet- cal relevance in recovery of the ischemic myocardium to-be described receptors may exist. In bovine aortic following infarction. This is one area where a thera- endothelial cells, AM binding leads to activation of peutic action of AM may hold promise. Additionally, adenylyl cyclase via a cholera toxin–sensitive G pro- AM exerts positive inotropic and chronotropic actions tein mechanism, with concomitant stimulation of PLC in the heart, effects that may be beneficial in patients activity. As a result, cyctosolic calcium levels increase in CHF. Finally, AM upregulates expression of the and NO is generated. In mesangial cells, AM inhibits vasodilatory peptide ANP in the heart, while down- MAPK, an effect that may underlie the peptide’s regulating ET expression in the vasculature. antimitogenic actions. In VSMCs, AM activates a pro- The AM gene is expressed abundantly in kidney, line-rich tyrosine kinase. Very little is understood about where the peptide exerts numerous effects. It exerts the identity of the PAMP receptor. PAMP inhibits cat- direct natriuretic and diuretic actions (prostacyclin echolamine release induced by periarterial nerve stimu- mediated) in renal tubule and is an important determi- lation, possibly by a direct membrane action mediated nant of renal perfusion pressure. Even when adminis- via a pertussis toxin–sensitive G protein. Pertussis toxin tered intravenously, AM maintains renal blood flow in also blocks the ability of PAMP to inhibit opening of the face of profound decreases in mean arterial pres- voltage-gated (N-type) calcium channels in pheochro- sure. AM accomplishes this paradox by exerting a mocytoma cells (PC-12 cells), a cell line in which PAMP vasodilatory effect (NO dependent) on the afferent also opens inwardly rectifying potassium channels. A arteriole, thus maintaining glomerular filtration and potassium channel also appears to mediate the ability of urine flow. Although in pharmacologic studies the PAMP to inhibit corticotropin-releasing hormone– threshold dose in humans for the renal effects exceeded stimulated adrenocorticotropin release from cultured that required to observe the cardiovascular actions, it is anterior pituitary cells in vitro. unlikely that circulating AM explains the observed in vitro or in vivo actions of AM. Locally produced pep- 4.4. Biologic Actions tide instead appears to act in an autocrine/paracrine AM and PAMP exert profound effects on cardio- fashion to control sodium and water handling by the vascular function and fluid and electrolyte homeo- tubule and perhaps even glomerular filtration. The stasis by actions in a variety of tissues (Fig. 6). The same pharmacologic administration of AM in patients hallmark action of AM is by an action on displaying already high levels of plasma AM (renal endothelial cells to generate NO and by direct activa- failure) did improve renal function in one study, and tion of adenylyl cyclase in VSMCs. PAMP, on the other it appears that administration of exogenous AM can hand, exerts its hypotensive action not by an action on improve survival in septic crisis (a hypotensive state in the endothelial cells or VSMCs, but, instead, by pre- which endogenous AM levels are already elevated). synaptic inhibition of electrical activity in the sympa- The AM gene is highly expressed in the CNS, and thetic fibers that innervate the blood vessels. These significant effects of the peptide in brain have been vascular effects appear to be physiologically relevant demonstrated, many related to the regulation of fluid because heterozygote AM gene knockout mice (miss- and electrolyte homeostasis. The natriuretic and diuretic ing one copy of the AM gene) display only 50% of the actions of AM in kidney appear to be mirrored by CNS normal circulating levels of AM and are hypertensive effects to inhibit thirst and sodium appetite. Both of compared with wild-type (normal two-gene copy) these CNS actions have been demonstrated to be physi- mice. The vasodilatory effect of AM is even more pro- ologically relevant. AM also acts in brain to stimulate nounced in circumstances of high levels of Ang II, sympathetic tone and to stimulate the release of vaso- such as in preconstricted vessels in vitro or human pressin and oxytocin. Although the actions to elevate hypertensives in vivo. This has led to the hypothesis peripheral blood pressure and circulating levels of AVP that the physiologic action of AM is to act as a coun- may seem counter to the peptide’s renal and vascular 334 Part IV / Hypothalamic–Pituitary effects (natriurestis, diuresis, and vasodilation), they compared with control subjects, as were the blood pres- may reflect brain actions that are cardioprotective in sure–lowering effects. Later it was determined that the nature, just as those exerted by AM in the heart may failure of AM in these models was owing to an impair- have evolved to protect against cardiovascular collapse. ment in NO generation in these patients. However, more Other actions of AM include a stimulatory effect on promising were the observations that administration of progesterone secretion and a quiescent effect in uterus. AM decreased pulmonary capillary wedge pressure as The fact that the knockout of the AM gene resulted in well as pulmonary arterial pressure in patients with CHF. embryonic death at approx d 14 of mouse gestation Furthermore, AM infusion increased heart rate, stroke points to significant effects of the peptide during volume, ejection fraction, and cardiac index in patients embryogeneis as well. Indeed, the cause of embryo with CHF and in another study increased urine volume demise appeared to be a constriction of the umbilical and sodium excretion. These were short-term infusion arteries and veins. The knockout also provided insight protocols; longer-term clinical studies are needed. How- into the physiologic relevance of another pharmacologic ever, chronic infusion studies in experimental animals effect of AM. Heterozygote (one gene copy) animals have demonstrated the ability of AM to reduce renin develop a late-onset diabetic phenotype, suggesting that levels in models of renovascular hypertension and to the ability of AM to inhibit insulin secretion has physi- decrease renal injury in hypertensive animals. Similarly, ologic relevance. animal models have demonstrated that administra- tion of AM improves blood pressure in spontaneously 4.5. Pathophysiology of AM hypertensive (SHR) rats, exerting a more profound The multiple pharmacologic effects of AM have pre- effect in those animals than in their normotensive dicted that the peptide or its analogs would be benefi- (Wistar-Kyoto) control counterparts. This should not cial in a variety of disease states. The ability of AM to be surprising because the vasodilatory effect of AM is inhibit Ang II–mediated cardiomyocyte hypertrophy more pronounced in preconstricted vessels. In one clini- and fibroblast proliferation predicted a role for the pep- cal trial in patients with essential hypertension, admin- tide in protection against hypertension-induced ven- istration of AM reduced both systolic and diastolic tricular hypertrophy and interstitial fibrosis of the heart. blood pressure and also reduced total peripheral resis- In addition, AM exerts antimigratory and antiprolif- tance. erative effects in VSMCs, promising a possible role in Therapeutically, AM is already being employed for the prevention of atherosclerosis and angiogenesis. the treatment of septic crisis. It had been observed that This is supported by observations in heterozygote plasma AM levels correlate positively with the sever- knockout animals in which perivascular fibrosis and ity of sepsis, which initially suggested that AM was the intimal hyperplasia were exaggerated compared with causative agent for the observed hypotension, since wild-type controls following salt loading or chronic proinflammatory cytokines stimulate AM production administration of Ang II. The protective effect of AM and release. However, it was subsequently reported that is also supported by results from transgenic mice in there was also a direct, positive correlation between which overexpression of AM has been engineered. absolute levels of plasma AM and survival, in that Much less interstitial fibrosis and a milder form of patients with highest AM levels in circulation had a hypertrophy were observed in these animals compared greater chance of surviving the septic event. This was with controls in several models of hypertension. In observed to be owing to a preservation of renal func- experimental animals, overexpression of AM reduces tion in the high AM group, mirroring the ability of the magnitude of arterial thickening and promotes exogenous AM to maintain renal perfusion even in the reendothelialization following balloon angioplasty. In face of profound hypotension. one study, administration of AM immediately follow- ing myocardial infarction enhanced ejection fraction 5. CARDIOVASCULAR HORMONES AS and improved coronary sinus blood flow. Thus, admin- DIAGNOSTIC AND THERAPEUTIC TOOLS istration of AM may provide both acute and chronic The three families of cardiovascular hormones dis- benefit in this patient population. cussed in this chapter have proven to be potent regula- Plasma AM levels are elevated in CHF. Because of tors of cardiovascular and renal function. Acting as the peptide’s vasodilatory and renotropic effects, it was either true endocrine or autocrine/paracrine hormones, thought that exogenous administration might provide they exert a wide variety of physiologically and patho- some benefit in these patients. Initial results were disap- logically relevant actions. Although the effects of the pointing. Increases in forearm blood flow to iv admin- ETs are predominantly pathologic in nature, this still istration of AM were attenuated in CHF patients provides promise for the use of antagonists to block Chapter 21 / Cardiovascular Hormones 335 those deleterious actions in a variety of disease states tive, systems biology. These peptides promise to open characterized by overproduction or secretion of the pep- new avenues into cellular and molecular control tides. Results from the initial trials with these antago- mechanisms underlying other normal and pathologic nists are discussed above. More promising are the systems as well. beneficial effects of long-term administration of the natriuretic peptides or AM because their actions in gen- SELECTED READINGS eral appear organ specific and protective. In addition, Ando K, Fujita T. Lessons from the adrenomedullin knockout mouse. pathologic secretion of these two classes of peptides Regul Pept 2003;112:185–188. Charles CJ, Lainchbury JG, Nicholls MG, Rademaker MT, Richards may reflect the recruitment of compensatory mecha- AM, Troughton RW. Adrenomedullin and the renin-angiotensin- nisms within the body that can hallmark the onset of aldosterone system. Regul Pept 2003;112:41–49. disease and therefore provide diagnostic advantage. Just Costello-Borrigter LC, Boerrigter G, Burnett JC. Revisting salt and as important are the basic biomedical lessons learned water retention: new diuretics, aquaretics, and natriuretics. Med Clin North Am 2003;87:475–491. from the discovery and characterization of the actions of Eto T, Kato J, Kitamura K. Regulation of production and secretion these peptide hormones. Emerging now is an integrated of adrenomedullin in the cardiovascular system. Regul Pept view of how these hormones can coordinate endocrine, 2003;112:61–69. cardiovascular, and renal mechanisms that protect Moreau P, Schiffrin EL. Role of endothelins in animal models of hypertension: focus on cardiovascular protection. Can J Physiol against postischemia proliferative disease and tissue Pharmacol 2003;81:511–521. damage caused by volume over- or underload. The roles Rich S, McLaughlin VV. blockers in cardiovas- played by these peptides in inflammatory disease are cular disease. Circulation 2003;108:2184–2190. now being recognized and their importance in normal Stoupakis G, Klapholz M. Natriuretic peptides: biochemistry, physi- ology, and therapeutic role in heart failure. Heart Dis 2003;5: glucose metabolism and bone health is better under- 215–223. stood. Taylor MM, Samson WK. Adrenomedullin and the integrative physi- In summary, the roles played by these potent car- ology of fluid and electrolyte balance. Microsc Res Tech 2002; diovascular hormones in the maintenance of cardio- 57:105–109. Taylor MM, Shimosawa T, Samson WK. Endocrine and metabolic vascular function and fluid and electrolyte homeostasis actions of adrenomedullin. The Endocrinologist 2001;11:171– have taught investigators a great deal about integra- 177.