Perspectives
`
`Cyclic Guanosine Monophosphate as a Mediator of Vasodilation
`Ferid Murad
`Departments ofMedicine and Pharmacology, Stanford University and Veterans Administration Medical Center,
`Palo Alto, California 94304
`
`Although cyclic guanosine monophosphate (GMP)! was first
`described in biological samples more than two decadesago,its
`role in some physiological processes has only become apparent
`in the past few years (see references 1-4). This relatively slow
`developmentis probably attributable to the low concentrations
`of the nucleotide in tissues, the complex and insensitive methods
`available during the early studies, and the biases many investi-
`gators had regarding its possible functions. The latter was un-
`doubtedly influenced by the manysimilarities of the cyclic GMP
`system with that of cyclic AMP andtheattention cyclic AMP
`has received during this period. While analogies and similarities
`between these two cyclic nucleotide systems do exist, the cyclic
`GMP system presents more complexities due to the existence
`of several isoenzymes responsible forits synthesis.
`It is known that the conversion of guanosine triphosphate
`(GTP) to cyclic GMPis catalyzed by at least two isoenzyme
`forms of guanylate cyclase. The kinetic, physicochemical, and
`antigenic properties of the cytosolic and membrane-associated
`isoenzymesare quite different (see references 2, 4). Therelative
`abundanceofthe soluble and particulate enzymeis variable in
`different tissues and species. While intestinal mucosa andretina
`possess predominately the particulate isoenzyme andplatelets
`contain the soluble isoenzyme, most tissues such as vascular
`smooth muscle have both isoenzymes. Furthermore, the regu-
`lation of each of these isoenzymesis quite different. The soluble
`enzyme appears unique in that it can be activated by reactive
`free radicals such as nitric oxide (5), and probably hydroxylfree
`radical (6) and some porphyrins(7, 8). On the other hand, the
`particulate isoenzymecan be activated with agents such as Esch-
`erichia coli heat-stable enterotoxin (9-11), atriopeptins (12, 13),
`and hemin (14). Cations, thiols, other redox agents, and deter-
`gents also have complex effects on the activity of both iso-
`enzymes(2).
`Studies with the kinetic characterization of these isoenzymes
`led to the present understanding of the role of cyclic GMP in
`smooth muscle relaxation. Azide, added to inhibit GTPase ac-
`tivity in crude enzymepreparations, was foundto activate the
`enzyme(15). While some hormones, autocoids, and other agents
`wereable to increase cyclic GMP accumulation inintact tissues,
`these agents had noeffects on guanylate cyclase activity in broken
`
`
`
`Receivedfor publication 9 January 1986.
`
`1. Abbreviations used in this paper: EDRF, endothelial-derived relaxant
`factor, GMP, guanosine monophosphate; GTP, guanosinetriphosphate;
`ST, Escherichia coli heat-stable enterotoxin.
`
`J. Clin. Invest.
`© The American Society for Clinical Investigation, Inc.
`002 1-9738/86/07/0001/05
`$1.00
`Volume 78, July 1986, 1-5
`
`cell preparations. It was reasoned that an understanding of azide
`activation of guanylate cyclase could lead to understanding the
`mechanisms of hormonal regulation of the enzyme and some
`functions of cyclic GMP. Someof these predictions have been
`fulfilled. The stimulatory effects of azide were dependent upon
`the presence of heme-containing proteins in guanylate cyclase
`incubations such as catalase, peroxidase, or cytochromes(16).
`In addition to azide, other agents that are capable of generating
`nitric oxide in incubations also activated the enzyme and in-
`cluded hydroxylamine, sodium nitrite, nitroglycerin, sodium
`nitroprusside (15, 17), and nitrosamines (18). Most of these
`agents also increased cyclic GMP accumulation in variousintact
`tissue preparations including smooth muscle segments (19-22).
`The increase in cyclic GMP with these guanylate cyclase acti-
`vators was associated with relaxation of tracheal, intestinal, and
`vascular smooth muscle (20, 22, 23). These studies led to the
`first proposed role of cyclic GMP, regulation of smooth muscle
`relaxation, that has withstood the test of time (22). These and
`subsequent studies with cyclic GMP in E.coli heat-stable en-
`terotoxin-induced diarrhea (9-11) and light-induced ion con-
`ductance in the retina (24) have further stimulated the interest
`in cyclic GMP andthesearch for other functions.
`Wehavecoined the term “nitrovasodilators” for those agents
`that can lead to the formation of the reactive nitric oxide—free
`radical in incubations andincrease cyclic GMPsynthesis. These
`agents include the organic nitrates such as nitroglycerin, the in-
`organic nitrates and nitrites such as nitroprusside and sodium
`nitrite, various nitrosoureas and nitrosamines, as well as azide,
`hydroxylamine, and perhaps some hydrazines. Someof these
`nitrovasodilators require catalytic conversion to nitric oxide by
`a macromolecule or enzyme while others are nonenzymatically
`converted to nitric oxide under the appropriate oxidizing orre-
`ducing conditions. Theeffects of pH and oxygen tension on the
`formation of nitric oxide from a precursor and the metabolism
`of these nitrovasodilators may explain their apparentspecificity
`for different vascular beds and tissues. However, this hypothesis
`requires additional investigation. Activation of guanylate cyclase
`and cyclic GMP accumulation with these nitric oxide-generating
`precursors occurs at relatively low concentrations (micromolar
`to nanomolar range) and is a function of the presence of other
`compoundsthat act as sinks, or reduce or oxidize the formed
`nitric oxide. Activation of the enzymeis instantaneous without
`a temporallag andis reversible (5, 17). While the precise mech-
`anism of activation of the enzymeis not presently understood,
`critical thiol groups on the enzyme (25, 26) and heme(7, 27)
`which maybe a prosthetic group on the enzyme (28) appear to
`participate in the activation mechanism. Clearly, homogeneous
`preparationsof the
`nitrovasodilators u
`concentration of ni
`tion of purified en:
`
`Cyclic G
`
`Human Power of N Company
`EX1018
`Page 1 of 5
`
`Human Power of N Company
`EX1018
`Page 1 of 5
`
`

`

`Perspectives
`
`Cyclic Guanosine Monophosphate as a Mediator of Vasodilation
`Ferid Murad
`Departments ofMedicine and Pharmacology, Stanford University and Veterans Administration Medical Center,
`Palo Alto, California 94304
`
`Although cyclic guanosine monophosphate (GMP)! was first
`described in biological samples more than two decadesago,its
`role in some physiological processes has only become apparent
`in the past few years (see references 1-4). This relatively slow
`developmentis probably attributable to the low concentrations
`of the nucleotide in tissues, the complex and insensitive methods
`available during the early studies, and the biases many investi-
`gators had regarding its possible functions. The latter was un-
`doubtedly influenced by the manysimilarities of the cyclic GMP
`system with that of cyclic AMP andtheattention cyclic AMP
`has received during this period. While analogies and similarities
`between these two cyclic nucleotide systems do exist, the cyclic
`GMP system presents more complexities due to the existence
`of several isoenzymes responsible forits synthesis.
`It is known that the conversion of guanosine triphosphate
`(GTP) to cyclic GMPis catalyzed by at least two isoenzyme
`forms of guanylate cyclase. The kinetic, physicochemical, and
`antigenic properties of the cytosolic and membrane-associated
`isoenzymesare quite different (see references 2, 4). Therelative
`abundanceofthe soluble and particulate enzymeis variable in
`different tissues and species. While intestinal mucosa andretina
`possess predominately the particulate isoenzyme andplatelets
`contain the soluble isoenzyme, most tissues such as vascular
`smooth muscle have both isoenzymes. Furthermore, the regu-
`lation of each of these isoenzymesis quite different. The soluble
`enzyme appears unique in that it can be activated by reactive
`free radicals such as nitric oxide (5), and probably hydroxylfree
`radical (6) and some porphyrins(7, 8). On the other hand, the
`particulate isoenzymecan be activated with agents such as Esch-
`erichia coli heat-stable enterotoxin (9-11), atriopeptins (12, 13),
`and hemin (14). Cations, thiols, other redox agents, and deter-
`gents also have complex effects on the activity of both iso-
`enzymes(2).
`Studies with the kinetic characterization of these isoenzymes
`led to the present understanding of the role of cyclic GMP in
`smooth muscle relaxation. Azide, added to inhibit GTPase ac-
`tivity in crude enzymepreparations, was foundto activate the
`enzyme(15). While some hormones, autocoids, and other agents
`wereable to increase cyclic GMP accumulation inintact tissues,
`these agents had noeffects on guanylate cyclase activity in broken
`
`
`
`Receivedfor publication 9 January 1986.
`
`1. Abbreviations used in this paper: EDRF, endothelial-derived relaxant
`factor, GMP, guanosine monophosphate; GTP, guanosinetriphosphate;
`ST, Escherichia coli heat-stable enterotoxin.
`
`J. Clin. Invest.
`© The American Society for Clinical Investigation, Inc.
`002 1-9738/86/07/0001/05
`$1.00
`Volume 78, July 1986, 1-5
`
`cell preparations. It was reasoned that an understanding of azide
`activation of guanylate cyclase could lead to understanding the
`mechanisms of hormonal regulation of the enzyme and some
`functions of cyclic GMP. Someof these predictions have been
`fulfilled. The stimulatory effects of azide were dependent upon
`the presence of heme-containing proteins in guanylate cyclase
`incubations such as catalase, peroxidase, or cytochromes(16).
`In addition to azide, other agents that are capable of generating
`nitric oxide in incubations also activated the enzyme and in-
`cluded hydroxylamine, sodium nitrite, nitroglycerin, sodium
`nitroprusside (15, 17), and nitrosamines (18). Most of these
`agents also increased cyclic GMP accumulation in variousintact
`tissue preparations including smooth muscle segments (19-22).
`The increase in cyclic GMP with these guanylate cyclase acti-
`vators was associated with relaxation of tracheal, intestinal, and
`vascular smooth muscle (20, 22, 23). These studies led to the
`first proposed role of cyclic GMP, regulation of smooth muscle
`relaxation, that has withstood the test of time (22). These and
`subsequent studies with cyclic GMP in E.coli heat-stable en-
`terotoxin-induced diarrhea (9-11) and light-induced ion con-
`ductance in the retina (24) have further stimulated the interest
`in cyclic GMP andthesearch for other functions.
`Wehavecoined the term “nitrovasodilators” for those agents
`that can lead to the formation of the reactive nitric oxide—free
`radical in incubations andincrease cyclic GMPsynthesis. These
`agents include the organic nitrates such as nitroglycerin, the in-
`organic nitrates and nitrites such as nitroprusside and sodium
`nitrite, various nitrosoureas and nitrosamines, as well as azide,
`hydroxylamine, and perhaps some hydrazines. Someof these
`nitrovasodilators require catalytic conversion to nitric oxide by
`a macromolecule or enzyme while others are nonenzymatically
`converted to nitric oxide under the appropriate oxidizing orre-
`ducing conditions. Theeffects of pH and oxygen tension on the
`formation of nitric oxide from a precursor and the metabolism
`of these nitrovasodilators may explain their apparentspecificity
`for different vascular beds and tissues. However, this hypothesis
`requires additional investigation. Activation of guanylate cyclase
`and cyclic GMP accumulation with these nitric oxide-generating
`precursors occurs at relatively low concentrations (micromolar
`to nanomolar range) and is a function of the presence of other
`compoundsthat act as sinks, or reduce or oxidize the formed
`nitric oxide. Activation of the enzymeis instantaneous without
`a temporallag andis reversible (5, 17). While the precise mech-
`anism of activation of the enzymeis not presently understood,
`critical thiol groups on the enzyme (25, 26) and heme(7, 27)
`which maybe a prosthetic group on the enzyme (28) appear to
`participate in the activation mechanism. Clearly, homogeneous
`preparationsof the soluble isoenzymecan be activated by these
`nitrovasodilators under the appropriate conditions, and the
`concentration of nitric oxide required for half-maximal activa-
`tion of purified enzyme is estimated to be in the nanomolar
`
`Cyclic Guanosine Monophosphate and VasodilationPage 1 of 5
`
`Page 1 of 5
`
`

`

`range (5, 25). While crude particulate preparations of guanylate
`cyclase are also activated, this may be attributable to entrapped
`soluble isoenzymethatis virtually always present in the absence
`of somepurification steps to removeit. Unfortunately, detergents
`prevent activation of either isoenzyme by a variety of agents.
`Thus, the issue of contaminating soluble isoenzyme contributing
`to the apparentactivation of the particulate isoenzyme can not
`be presently resolved with purification procedures to separate
`the isoenzymes.
`Agents that interfere with the conversion of the nitrovaso-
`dilator precursorto nitric oxide, react with nitric oxide, or inhibit
`guanylate cyclase activation decrease cyclic GMP accumulation
`in smooth muscle and shift dose-response (relaxation) curves to
`the right. This list includes various nonspecific as well as relatively
`specific antagonists such as cystamine, cystine, methylene blue,
`hemoglobin, methemoglobin, and cyanide (15, 17, 27). While
`these agents have been useful in manyin vitro systems to im-
`plicate cyclic GMP in some process, their nonspecific effects
`limit their usefulness at present in most in vivo systems. Agents
`that inhibit cyclic GMP hydrolysis by cyclic nucleotide phos-
`phodiesterases predictably shift the dose-response curves of these
`nitrovasodilators to theleft (20, 29). These antagonistic and syn-
`ergistic effects should be important in predicting various drug
`interactions and modifying the therapeutic responses to the ni-
`trovasodilators. For example, studies in dogs have shown that
`someof the cardiovascular effects of nitroprusside can be en-
`hanced with aminophylline (30). The effects of ouabain to inhibit
`nitrovasodilator-induced cyclic GMP synthesis and vascularre-
`laxation (31) should alert us to potentially important drug in-
`teractions between nitrovasodilators and cardiac glycosides which
`are commonlyused together. The precise mechanisms by which
`inhibition of the Na-K pump with cardiac glycosides decreases
`cyclic GMP synthesis as well as cyclic GMP-induced vascular
`relaxation are unknown (31). Calcium availability for these pro-
`cesses, however, should be suspected as a candidate in these
`effects. Although the activation of guanylate cyclase and the ac-
`cumulation of cyclic GMP in tissues with nitrovasodilators is
`calcium-independent (20), calcium antagonists may modify
`some of the physiological processes regulated by cyclic GMP
`and its rate of hydrolysis by phosphodiesterase. Indeed, the
`smooth muscle relaxanteffects of the nitrovasodilators and cal-
`cium antagonists would be expected to be additive.
`Theincrease in cyclic GMPin tracheal and vascular smooth
`muscleis associated with the activation ofcyclic GMP-dependent
`protein kinase and the phosphorylation of numerous smooth
`muscle proteins (32-35). One of the smooth muscle proteins in
`which phosphorylation is decreased with nitrovasodilators or
`cyclic GMP analogues is myosin light chain (3, 35, 36). While
`the phosphorylation of other smooth muscle proteins is also
`altered, their identities and roles are presently unknown. The
`mechanism of cyclic GMP-induced dephosphorylation of
`myosin light chainis also presently unknown. These effects could
`result from the decreased activity of myosin light chain kinase,
`a calcium and calmodulin-dependent enzyme,increased activity
`of the phosphatase, or a combination ofthese effects. The recent
`demonstration of an effect of nitroglycerin on Quin-2 fluores-
`cence in smooth muscle cells (37) suggests that these effects could
`be due to decreased cytosolic calcium concentrations.
`Although azide, nitroprusside, and other nitrovasodilators
`have been useful agents in some systemsto evaluate the role of
`cyclic GMPin someprocess, their inhibition of metabolic pro-
`
`2
`
`=F. Murad
`
`cesses with high concentrations and their oxidation of someli-
`gands and hormonescanalso lead to artifacts and erroneous
`conclusions regarding functions of cyclic GMP.
`The recent studies of Furchgott and his associates (38) have
`permitted us to understand the mechanism ofaction of another
`class of vascular relaxants, the endothelium-dependent vasodi-
`lators. Relaxation of blood vessels from a variety of species by
`agents such as acetylcholine, histamine, bradykinin, ionophore
`A23187, thrombin, and ATP requires the integrity ofthe vascular
`endothelium (see references 3, 38). These endothelium-depen-
`dent vasodilators either have no effect or may cause vasocon-
`striction in the absence of the endothelium. Thelatter effects
`are dependent upon the vessel and species being examined. These
`agents interact with specific receptors on endothelial cells to in-
`duce the formation and release of a factor(s) called endothelial-
`derived relaxant factor (EDRF). Thestructure of this reactive
`and labile material is not known dueto its short half-life and
`probably low concentration (38, 39). While studies with inhib-
`itors of phospholipase and lipoxygenase have suggested that the
`factor may be an oxidized ecosinoid product (see references 3,
`38), the lack of specificity of these inhibitors limits such conclu-
`sions. Furthermore,it is not presently knownif the various en-
`dothelium-dependent vasodilators induce the release of the same
`or different factors.
`Relaxation of vascular segments with this class of vasodilators
`is also associated with guanylate cyclase activation and cyclic
`GMPaccumulation in vascular smooth muscle (35, 36, 40-42).
`Since nitroglycerin-tolerant vessels have decreased cyclic GMP
`accumulation andrelaxation to the endothelium-dependent va-
`sodilators as well as the nitrovasodilators (43), it appears that
`activation of soluble guanylate cyclase also mediates the effects
`of EDRF.Effects of EDRF ontheparticular isoenzyme, however,
`can not be excluded. The increased synthesis of cyclic GMP
`with endothelium-dependentvasodilators is also associated with
`cyclic GMP-dependent protein kinase activation and dephos-
`phorylation of myosin light chain (32, 36). Indeed, the profiles
`of altered protein phosphorylation with two-dimensional poly-
`acrylamidegel electrophoresis of extracts from vascular smooth
`muscle after treatment with nitrovasodilators, endothelium-de-
`pendentvasodilators, and 8-bromo cyclic GMParesimilar (34-
`36). While these effects with nitrovasodilators and 8-bromocyclic
`GMPare endothelium-independent,theeffects ofendothelium-
`dependent vasodilators require the endothelium. Thus, the cas-
`cade of events distal to guanylate cyclase activation and cyclic
`GMPsynthesis appear to be identical with both classes of va-
`sodilators (see Fig. 1). These studies suggest that EDRF could
`be viewed as the endogenousequivalentof the nitrovasodilators
`or the “endogenous nitrate”. The effects of nitric oxide and
`EDRFon guanylate cyclase activity and the inhibition of their
`effects with similar classes of agents such as hemoglobin, meth-
`ylene blue, cyanide, and various reducing agents suggest that
`these reactive materials may also activate the enzymebysimilar
`mechanisms (2-4, 44, 45). However, additional kinetic and
`physicochemical studies with purified enzyme andactivators are
`required.
`In the presence of the endothelium the basal levels of cyclic
`GMPandtheactivity of cyclic GMP-dependentprotein kinase
`are increased, which suggests that basal release of EDRF occurs
`and can influence vascular motility (32, 36, 40, 41). The de-
`creased basal or stimulated release of EDRF with endothelial
`damagecould obviously have importantclinical and therapeutic
`
`Page 2 of 5
`
`Page 2 of 5
`
`

`

`CYCLIC GMP
`
`
`
`PAATIC.
`
`cGMP “~~ cGMP
`
`
`GUANYLATE
`KINASE W\__-/ KINASE
`
`
`CYCLASE
`(Active)
`
`
`
`RECEPTOR
`GTP
`
`
`ANF-1
`
`
`ATRIOPEPTINS
`
`
`PROTEIN wR P-PROTEIN
`RECEPTOR
`
`
`ANF-2
`MYOSIN
`
`P-MYOSIN
`
`LIGHT CHAIN ®_. LIGHT CHAIN
`
`
`Ca**
`
`SMOOTH MUSCLE
`
`
`SOLUBLE
`
` GUANYLATE CYCLASE
`
`
`L_»no
`
`EORF
`
`
` RELAXATION
`
`? FUNCTION
`
`
`CONTRACTION
`
`
`
`NITROVASODILATORS
`
`ENDOTHELIUM
`
`
`
`
`
`
`
`ENDOTHELIUM
`DEPENDENT
`VASODILATORS
`ACETYLCHOLINE.
`\ HISTAMINE,
`THROMBIN.
`ATP,
`Etc
`
`
`
`
`Figure 1. Proposed mechanism ofaction
`of some vasodilators on cyclic GMP
`synthesis and vascularrelaxation.
`Through specific receptors on endothe-
`lial cells, a variety of endothelium-de-
`pendentvasodilators lead to the synthe-
`sis and release of EDRF. Thestructure
`of EDRFis unknown.Nitric oxide
`(NO)derived from the nitrovasodilators,
`and EDRFactivate the soluble isoen-
`zyme form of guanylate cyclase and re-
`sult in increased cyclic GMP synthesis
`and cyclic GMP-dependentprotein ki-
`nase activation. These events result in
`the dephosphorylation of myosin light
`chain and relaxation. Atriopeptins,
`through a specific receptor designated
`ANF-1, result in the activation of the
`membrane-associated isoenzyme form
`of guanylate cyclase, cyclic GMP syn-
`thesis, and cyclic GMP-dependent pro-
`tein kinase activation. The function of
`the receptor that is not coupled to cyclic GMP synthesis (ANF-2) is unknown. While the atriopeptins relax vascular smooth muscle in the absence
`of the endothelium, the endothelial cells also possess atriopeptin receptors that are coupled to cyclic GMP synthesis. The function of cyclic GMP
`in endothelial cells is presently unknown.
`
`implications in many cardiovascular disorders. Indeed, increased
`contraction or spasm atsites of intimal damage would be ex-
`pected to occur due to the loss of EDRF and decreased cyclic
`GMPsynthesis. Furthermore, with intimal damage, endothe-
`lium-dependent vasodilators should also be less effective than
`therapeutic agents that act
`in an endothelium-independent
`mannersuch as nitrovasodilators and atriopeptins (see below).
`Since the synthesis and release of EDRF as well as smooth muscle
`contraction is calctum-dependent, the interactions of calcium
`antagonists with these agents on cyclic GMPlevels and relaxation
`are more complicated. We hope that the ability to assay EDRF
`in media from endothelial cell cultures (39) will facilitate the
`characterization and identification of this important substance
`and could lead to a new class of therapeutic vasodilators.
`A third class of vasodilators that appear to mediate their
`effects through cyclic GMPsynthesis is the atriopeptins (12, 13,
`45). This newly described group of peptide hormones has a
`numberofcardiovascular and renal effects including natriuresis,
`diuresis, vasodilation, and inhibition of aldosterone secretion
`(46-48). This family of peptide hormonesis derived from a pre-
`cursor peptide or preprohormoneof 152 aminoacids found in
`granules in the cardiac atria (49, 50). The presence of the hor-
`mone and/or messenger RNAforits synthesis in other tissues
`as well as atria suggest that these peptides may have many other
`functions not presently appreciated. For example,it is suspected
`that these peptides may also serve as neurotransmitters as well
`as peripheral hormones.
`Theeffects of atriopeptins on ion and water transport in the
`kidney are reminiscent of the effects of E. coli heat-stable en-
`terotoxin (ST) in intestinal mucosa. Since the effects of ST are
`mediated through guanylate cyclase activation and cyclic GMP
`accumulation (9-11), we were prompted to examinetheeffects
`of atriopeptins on cyclic GMP metabolism in renal tissue, vas-
`cular tissue, and othertissues (12, 13, 51, 52). This class of peptide
`hormonesspecifically activates the particulate isoenzyme form
`
`of guanylate cyclase in manytissues including blood vessels,
`kidney, adrenal, and cultures of endothelial and smooth muscle
`cells (12, 13, 51, 52). The effects of atriopeptins on particulate
`guanylate cyclase are mediated through a specific receptor and
`they do notalter the activity of the soluble isoenzyme form of
`guanylate cyclase. Thus,the effects ofatriopeptins are analogous
`in somerespects to the effects of ST except that they alter cyclic
`GMPsynthesis in numeroustissues due to the ubiquitouspres-
`ence ofthe receptor.
`Binding studies with radiolabeled atriopeptin analogues have
`given linear Scatchard plots with manytissue preparations, which
`suggests thata single class of receptors is present in most tissues.
`However, the comparison of peptide binding with cyclic GMP
`accumulation in our structure-activity studies clearly demon-
`strates that manytissues possess at least two distinct populations
`of binding sites (51-53). While the apparentaffinities of these
`receptors to most atriopeptin analogues are similar (dissociation
`constantof 0.1 to 1 nM), the relative abundance of each receptor
`varies in different preparations. Cloned endothelial and smooth
`musclecells also possess two classes of bindingsites, indicating
`that the data are not dueto cellular heterogeneity of preparations
`(51, 52). Interestingly, cross-linking studies with radiolabeled
`atriopeptins also reveal two binding sites that are ~ 60,000 and
`120,000 D in size (54, 55). Our current data indicate that only
`oneofthe receptors, designated ANF-1, is coupled to guanylate
`cyclase activation and cyclic GMPsynthesis (52, 53, 55). While
`the mechanism of atriopeptin receptor coupling to particulate
`guanylate cyclase activation is not known, we have foundthat
`the 120,000-D binding site copurifies with particulate guanylate
`cyclase through numerousprocedures (53). At present we suspect
`that ANF binding and cyclic GMP synthesis reside in the same
`membrane glycoprotein, and presumably this macromolecule
`spans the membrane(53).
`Although atriopeptins bind to receptors on both endothelial
`cells and smooth muscle cells and increase cyclic GMP accu-
`
`Cyclic Guanosine Monophosphate and Vasodilatio"Page 3 of 5
`
`Page 3 of 5
`
`

`

`mulation, their effects on cyclic GMP synthesis and relaxation
`of vascular smooth muscle are endothelium-independent (13,
`45). Analogous to the nitrovasodilators, their effects are also
`independentof extracellular Cat* (13). The increase in cyclic
`GMPlevels in vascular smooth muscle with atriopeptinsis as-
`sociated with increased activity of cyclic GMP-dependentprotein
`kinase (56). While the vasorelaxant effects of the atriopeptins
`correlate with cyclic GMP synthesis, other effects of the atrio-
`peptins can not be excluded. Furthermore,therole ofthe receptor
`that is not coupled to cyclic GMP synthesis designated ANF-2
`is not presently known.It is of interest that atriopeptins have
`been found to inhibit adenylate cyclase activity in some prep-
`arations (57). The physiological relevance of this effect is un-
`known;noris it known to which receptorthis effect is coupled.
`It is unlikely that inhibition of cyclic AMP synthesis in vascular
`preparations with atriopeptins is associated with vasodilation
`since cyclic AMPsynthesis, like cyclic GMP accumulation,cor-
`relates with vascular relaxation. While atriopeptins activate par-
`ticulate guanylate cyclase in preparations of kidney, adrenal,
`and othertissues, additional studies are required to establish the
`roles of cyclic GMP, cyclic AMP,or perhaps other second mes-
`sengers in the physiological effects of atriopeptins in these tissues.
`Although the marked effects of atriopeptins on cyclic GMPsyn-
`thesis in endothelial cells suggest that other vascular effects of
`atriopeptins should be expected, the nature of these effects is
`also unknown.Atpresent, it is almost certain that one class of
`atriopeptin receptors on vascular endothelial and smooth muscle
`cells is associated with the activation of particulate guanylate
`cyclase and the accumulation of cyclic GMP (52, 55). Also, the
`accumulation of cyclic GMP with atriopeptins as with the effects
`of nitrovasodilators and endothelium-dependent vasodilators is
`associated with vascular relaxation. Since these three classes of
`vasodilators result in the activation of either the soluble or
`membrane-associated isoenzyme forms of guanylate cyclase and
`presumably increase different intracellular pools of cyclic GMP,
`some differences on vascular metabolism and function might
`be expected with additional studies. Although nitroglycerin-tol-
`erant vessels have decreased cyclic GMP accumulation andre-
`laxation when challenged with nitrovasodilators or endothelium-
`dependentvasodilators, the effects of atriopeptins are not altered
`(43). Interestingly, nitroglycerin-tolerant vessels have a relatively
`stable modification in the properties of soluble guanylate cyclase,
`while the particulate isoenzyme appears unaltered (58).
`While cyclic GMP accumulation in vascular smooth muscle
`mediates the effects of these three classes of vasodilators, other
`effects of these agents should not be overlooked. Furthermore,
`other second messengers are known to participate in vasodilation
`induced with other vasorelaxants. Although the studies sum-
`marized here permit us to develop a framework for understand-
`ing the mechanism ofaction of these vasodilators (see Fig. 1),
`additional experiments are required to develop some innovative
`therapeutic approaches to cardiovascular disorders.
`
`Acknowledgments
`
`The author thanks Sherry Oppenheim and Beverly Lyonsfor typing the
`manuscript.
`Someofthe studies summarized in this review were supported with
`grants from the National Institutes of Health (AM 30787 and HL 28474),
`the Veterans Administration, and the Council for Tobacco Research-
`USA,Inc.
`
`4
`
` F. Murad
`
`References
`
`1, Murad,F., W. P. Arnold, C. K. Mittal, and J. M. Braughler. 1979.
`Adv. Cyclic Nucleotide Res. 11:175-204,
`2. Mittal, C. K., and F. Murad. 1982. Jn Handbook of Experimental
`Pharmacology. Vol. 58-1. J. A. Nathanson and J. W. Kebabian,editors.
`Springer-Verlag, Berlin. 225-260.
`3. Rapoport, R. M., and F. Murad. 1983. J. Cyclic Nucleotide Protein
`Phosphorylation Res. 9:281-296.
`4. Murad,F., and G. D. Aurbach. 1978. In The Year in Metabolism.
`N.Freinkel, editor. Plenum Publishing Corp., New York. 1-32.
`5. Amold, W. P., C. K. Mittal, S. Katsuki, and F. Murad. 1977.
`Proc. Natl. Acad. Sci. USA. 74:3203-3207.
`6. Mittal, C. K., and F. Murad. 1977. Proc. Natl. Acad. Sci. USA.
`74:4360-4364.
`7. Craven, P. A., F. R. DeRubertis, and D. W. Pratt. 1979. J. Biol.
`Chem. 254:8213-8222.
`8. Ignarro, L. J., B. Ballot, and K. S. Wood. 1984. J. Biol. Chem.
`259:6201-6207.
`9. Hughes, J., F. Murad, B. Chang, and R. Guerrant. 1978. Nature
`(Lond.). 271:755-756.
`10. Field, M., L. H. Graf, W. J. Laird, and P. L. Smith. 1978. Proc.
`Natl. Acad. Sci. USA. 75:2800-2804.
`11. Guerrant, R. L., J. M. Hughes, B. Chang, D. C. Robertson, and
`F. Murad. 1980. J. Infect. Dis. 142:220-228.
`12, Waldman,S. A., R. M. Rapoport, and F. Murad. 1984. J. Biol.
`Chem. 259:14332-14334.
`13. Winquist, R. J., E. P. Faison, S. A. Waldman, K. Schwartz, F.
`Murad, and R. M. Rapoport. 1984. Proc. Natl. Acad. Sci. USA. 81:
`7661-7664.
`
`14, Waldman,S.A., M.S. Sinacore, J. A. Lewicki, L. Y. Chang, and
`F. Murad. 1984. J. Biol. Chem. 259:4038-4042.
`15. Kimura, H., C. K. Mittal, and F. Murad. 1975. J. Biol. Chem.
`250:80 16-8022.
`16. Mittal, C. K., H. Kimura, and F. Murad. 1977. J. Biol. Chem.
`252:4348-4390.
`17. Katsuki, S., W. Arnold, C. K. Mittal, and F. Murad. 1977. J.
`Cyclic Nucleotide Res. 3:23-25.
`18. DeRubertis, F. R., and P. A. Craven. 1976. Science (Wash. DC).
`193:897-899.
`19, Kimura, H., C. K. Mittal, and F. Murad. 1975. Nature (Lond).
`275:700-702.
`20. Katsuki, S., and F. Murad. 1977. Mol. Pharmacology. 13:330~-
`341.
`
`21. Schultz, K. D., K. Schultz, and G. Schultz. 1977. Nature (Lond.).
`265:750-751.
`22. Katsuki, S., W. P. Arnold, and F. Murad. 1977. J. Cyclic Nu-
`cleotide. Res. 3:239-247.
`23. Diamond,J., and K.S. Blisard. 1976. Mol. Pharmacol. 12:688-
`692.
`24. Stryer, L. 1986. Annual Review ofNeuroscience 9: In press.
`25. Braughler, J. M., C. K. Mittal, and F. Murad. 1979. J. Biol.
`Chem. 254:12450-12454.
`26. Brandwein, H. J., J. A. Lewicki, and F. Murad. 1981. J. Biol.
`Chem. 256:2958-2962.
`27. Mittal, C. K., W. P. Arnold, and F, Murad. 1978. J. Biol. Chem.
`253:1266-1271.
`28. Gerzer, R., E. Bohme, F. Hoffman, and G. Schultz. 1981. FEBS
`(Fed. Eur. Biochem. Soc.) Lett. 123:71-74.
`29. Kukovetz, W. R., S. Holzmann, A. Wurm,and G. Poch. 1979.
`Naunyn-Schmeideberg’s Arch. Pharmacol. 310:129~-138.
`30. Pearl, R. G., M. H. Rosenthal, F. Murad, and J. A. Ashton. 1984.
`Anesthesiology. 61:712-715.
`31. Rapoport, R. M., K. Schwartz, and F. Murad. 1985. Circulation
`Res. 57:164-170.
`32. Fiscus, R. R., R. M. Rapoport, and F. Murad. 1983. J. Cyclic
`Nucleotide Protein Phosphorylation Res. 9:415-425.
`
`Page 4 of 5
`
`Page 4 of 5
`
`

`

`33. Fiscus, R. R., T. J. Torphy, and S. E. Mayer. 1984. Biochim.
`Biophys Acta. 80