The nitric oxide and cGMP signal transduction system: regulation and mechanism of action

153

Biochimica et Biophysica Acta, 1178 (1993) 153-175 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4889/93/$06.00

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BBAMCR 13452

The nitric oxide and cGMP signal transduction system: regulation and mechanism of action Harald H.H.W. Schmidt, Suzanne M. Lohmann and Ulrich Walter Department of Clinical Biochemistry and Pathobiochemistry, Medical University Clinic, Wiirzburg (Germany) (Received 22 March 1993)

Key words: Nitric oxide; Endothelium-derived relaxing factor; cGMP; Guanylyl cyclase; Protein kinase; Phosphorylation

Contents I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

154

II. cGMP synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Membrane receptor guanylyl cyclases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Soluble guanylyl cyclases (GC-S) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

154 154 155

III. NO synthases: structure, biochemistry and regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. NO synthase isozymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. NO synthase structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Mechanism of NO synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Regulation of NOS expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Regulation of NOS activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

156 156 157 158 160 160

IV. NO: mechanisms of action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Activation of GC-S by NO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. NO-independent activation of GC-S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. cGMP-independent actions of NO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

161 161 161 163

V. cGMP: mechanisms of action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Regulation of cGMP-gated channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Regulation of phosphodiesterases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Cross activation of cAMP-dependent protein kinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Activation of cGMP-dependent protein kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. cGMP kinase localization, heterogeneity, and domain structure/function . . . . . . . . . . . . . . 2. Physiological substrates and functions of cGMP kinase (cGKII, cGKI) . . . . . . . . . . . . . . . .

163 163 163 164 165 165 167

VI. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

171

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

171

References

171

Correspondence to: U. Walter, Medizinische Universit~its Klinik, Labor fiir Klinische Biochemie, Josef Schneider StraBe 2, 97080 Wiirzburg, Germany. Abbreviations: ANP, atrial natriuretic peptide; biopterin, 6-(L-erythro-l',2'-dihydroxypropyi)pterin; BNP, brain natriuretic peptide; cAK, cAMP-dependent protein kinase; cGK, cGMP-dependent protein kinase; CNP, natriuretic peptide type C; CPR, NADPH-cytochrome P450 reductase; EDRF, endothelium-derived relaxing factor; GC-S, soluble guanylyl cyclase; InsP3, inositol trisphosphate; NO, nitric oxide; NOS, NO synthase; OHArg, N'~-hydroxy-L-arginine; PDE, phosphodiesterase; retGC, retinal guanylyl cyclase; SNP, sodium nitroprusside; STa, E. coli heat-stable enterotoxin; VASP, vasodilator-stimulated phosphoprotein. This paper is dedicated to Dr. Eycke B6hme (1943-1993), Professor of Pharmacology at the Free University of Berlin, and a pioneer in cGMP and NO research.

154 I. Introduction

A wide spectrum of ligands, including hormones, autacoids, drugs and toxins, have cGMP as their common mediator in eliciting diverse physiological responses. Signal transduction pathways leading to these physiological responses can be composed of any of multiple types of soluble and particulate guanylyl cyclases which catalyze cGMP synthesis, and any of an array of cGMP mediators, including cGMP gated ion channels, cGMP-regulated phosphodiesterases, and cGMP-dependent protein kinases which carry out cGMP actions. The spectrum of cGMP-regulated events includes the retinal rod response to light, olfactory reception, steroidogenesis, renal and intestinal ion transport, and cellular calcium movements important for platelet aggregation, and cardiac and smooth muscle contractility. Furthermore, overactivity at certain steps in the signal transduction pathway has been correlated with conditions such as endotoxic shock and secretory diarrhoea, and underactivity with hypertension. In addition to being an overview, this review will place particular emphasis on recent insights gained with respect to nitric oxide synthase, including its role in cGMP production, and cGMP-dependent protein kinase as a major mediator of cGMP action. Finally,

Agonists a

Ca2+

platelets will be discussed as a cell system in which the chain of events mediated by eGMP has been extensively elucidated from the initial activation of nitric oxide synthase in neighboring endothelial cells to determination of the site at which platelet cGMP inhibits intracellular Ca 2+ levels required for platelet aggregation. II. c G M P synthesis

II-A. Membrane-receptor guanylyl cyclases The central role of cGMP in several signaling pathways is illustrated in Fig. 1 by the convergence of different mechanisms of cGMP synthesis and the dispersal of cGMP effects via multiple mediators. The constellation of these components present in a given cell type will determine the cellular response to a given agonist. The two systems known to generate cGMP are markedly different. One is activated by peptide ligands which bind to cell membrane receptors having transmembrane domains contiguous with intracellular guanylyl cyclase. Overexpression of cloned particulate guanylyl cyclase has demonstrated that one protein contains both cyclase activity and ligand binding activity indicative of cell surface receptor function [1,2].

Cytokines b Agonists a

Transcrip.

ANP / BNP

CNP

GC-A

GC-B

STa / Guanylin

?

Ca2+

Factors

I

II

III

constitutive

inducible

particulate

[

NO Synthase ~ 1

I constitutive

I~

II inducible

Heme oxygenase _1

CO

Other Targets (see Table II)

/

GC-Sal-a ~-a

/

1

I ~ Soluble guanylyl cyclase .~1

cGMP-inhibited cAMP PDE

cGMP-stimulated cAMP PDE

Ion flux

Increased c A M P response

Decreased c A M P response

retGC

Particulate guanylyl cyclase

=:= :: cGMP-gated channels

GC-C

I

07

cGMP-dependent protein cAMP-dependent protein kinase let,13,II kinase leql3,Ilm13 Protein phosphorylation

Protein phosphorylation

Fig. t. Model of the signal transduction pathways leading to c G M P synthesis and subsequent actions, a Agonists and b cytokines which stimulate the different forms of NO synthase are described in the text (Section II-B).

155 Parallel terminology describing these proteins has evolved from the viewpoint of the receptors and their ligands versus the cyclase activity shown by the same proteins. Three mammalian membrane receptor guanylyl cyclases have been cloned and characterized in humans and rats. Atrial natriuretic peptide receptor type A (ANPR-A), also called guanylyl cyclase A (GCA), binds atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP); atrial natriuretic peptide receptor type B (ANPR-B), also called guanylyl cyclase B (GC-B), is selectively activated by natriuretic peptide type C (CNP) [3,4]. A third membrane receptor guanylyl cyclase, also called guanylyl cyclase C (GC-C), is the intestinal receptor for Escherichia coli heat-stable enterotoxin (STAR) which is activated by both STa and a recently discovered endogenous intestinal peptide designated guanylin [5,6]. At the primary amino acid sequence level, the rat intestine receptor shows significant similarity to ANPR-A and ANPR-B in the cytoplasmic domain but only 10% in the extracellular, ligand binding domain [7]. Substantial similarity is observed between the ligands ANP and BNP, or between STa and guanylin, but not between these two groups or between them and CNP [3,6]. Very recently a fourth membrane receptor-cyclase, a human retinal guanylyl cyclase (retGC), was cloned and expressed [4], for which no endogenous ligand has been identified. The physiological function of the ligands for the membrane receptor guanylyl cylases have not been fully elucidated. The natriuretic peptides ANP, BNP and CNP can cause vasodilation, natriuresis and diuresis, and inhibit aldosterone synthesis. ANP and BNP are found principally in the heart, but also in other tissues, whereas CNP appears to be brain or nervous system specific and has been suggested to be a neurotransmitter (reviewed in Ref. 3). E. coli STa, and by analogy guanylin, act via GC-C to increase cGMP and alter salt and fluid transport in the intestine, which in the case of STa can lead to secretory diarrhoea [8]. None of the known natriuretic peptides are ligands for the receptor domain of retGC, but retGC is postulated to be involved in the resynthesis of cGMP required for recovery of the dark state after phototransduction [4]. At the protein level, a candidate for the cloned retGC is a guanylyl cyclase dependent on a Ca 2÷ sensitive activator called recoverin [9,10] which also has been recently cloned [11].

II-B. Soluble guanylyl cyclases (GC-S) In contrast to the particulate cyclase which can be activated by ligands in broken cell preparations by virtue of its intrinsic ligand binding domain, the soluble guanylyl cyclase, which constitutes the other major synthetic pathway for cGMP, could not be be hormonally stimulated in broken cell systems. This led to the

hypothesis that cytosolic GC-S was activated by hormonal factors through indirect mechanisms involving Ca z÷ [12]. As shown in Fig. 1, there are indeed Ca/÷dependent mechanisms of activation of certain forms of nitric oxide synthase (NOS), producing nitric oxide (NO) which activates soluble GC [13,14], presumably by binding to its associated heme moeity and causing a conformational change in GC [15]. More detailed aspects of NOS activation, as well as carbon monoxide stimulation of GC (Fig. 1) will be discussed in later sections. Several GC-S forms composed of a a n d / o r / 3 subunits have been cloned and expressed. The presently known forms are rat and bovine lung a 1 and /31, rat kidney/32, and human fetal brain a 2 [3,16-18]. Adult human brain subunits designated a3,/33 may represent additional isoforms or species variants [19]. At the protein level a GC-S [20] and nitric oxide synthase [21] have also been demonstrated in retina. Although the kidney /32 form is considered to be a soluble form based on its similarity to/31, its cellular localization is unknown but of interest since the C-terminal end of/32 that extends beyond /31 contains amino acids conforming to the consensus sequence for post-translational modification by isoprenylation/carboxymethylation [3]. Modification of this site in other proteins appears to be a determinant for their membrane association. So far, expression of GC-S subunits in mammalian cells has demonstrated that a heterodimeric protein composed of a and/3 subunits is required for enzyme activity and stimulation by sodium nitroprusside (SNP) [22,23]. More specifically, actual coexpression of the two subunits is required, mixing of two subunits after their separate expression did not reconstitute enzyme activity. Our understanding of the physiological significance of GC-S was extended tremendously by the recognition of the widespread nature of the NO signaling system. NO is both an intracellular and intercellular mediator, and exists in several cell types either as constitutive or inducible forms which can be activated or induced respectively by a number of agents [24]. Briefly, constitutive NOS can be activated by acetylcholine, bradykinin, thrombin, Ca 2+, excitatory amino acids and leukotrienes to mention a few agents, whereas the inducible form is induced by cytokines such as interferons and tumor necrosis factor, as well as bacterial lipopolysaccharide. NO appears to have a number of cellular targets and effects (see Section IV and Table II), only one of which is GC-S activation [3,24]. Thus not all effects of NO involve cGMP. For example, cytotoxic effects of NO, as in macrophage mediated cytotoxicity or glutamate neurotoxicity, may be direct NO effects independent of cGMP. However, the endothelial derived relaxing factor (EDRF), strongly believed to be NO, can diffuse to other cell types and stimulate their

156 G C - S to synthesize c G M P which t h e n inhibits s m o o t h muscle c o n t r a c t i o n a n d p l a t e l e t aggregation (discussed in s u b s e c t i o n V-D.2). Excessive activation of this pathway c a n result in the l i f e - t h r e a t e n i n g v a s o d i l a t i o n of endotoxic shock. I n contrast, deficits in e n d o t h e l i u m d e p e n d e n t v a s o d i l a t i o n m a y c o n t r i b u t e to o t h e r p a t h o logical c o n d i t i o n s such as essential h y p e r t e n s i o n , or h y p e r t e n s i o n of o t h e r etiologies such as chronic obstructive lung disease. T h e s e a n d o t h e r examples of N O f u n c t i o n a n d dysfunction are extensively s u m m a rized in Ref. 24. T h e m e c h a n i s m s of action of c G M P via c G M P - g a t e d channels, cGMP-regulated phosphodiesterases and c A M P or c G M P - d e p e n d e n t p r o t e i n kinases (Fig. 1) will be discussed in Section V.

III. NO synthases: structure, biochemistry and regulation I l i A . NO synthase isozymes I n m a m m a l i a n cells, nitric oxide ( N O ) is enzymatically f o r m e d from a t e r m i n a l g u a n i d i n o - n i t r o g e n of e - a r g i n i n e [25-27] by a gene family of N O S ( E C 1.14.23) [28]. All described N O S yield L-citrulline as a co-product of this reaction. T h e original classification of N O S into t h r e e isoforms, NOS-I, II a n d III ( T a b l e I), was b a s e d o n the physical a n d b i o c h e m i c a l characteristics of the purified enzymes, i.e., s u b c e l l u l a r location (soluble versus p a r t i c u l a t e fraction) a n d r e g u l a t i o n by the free Ca 2÷ c o n c e n t r a t i o n (see Sections I I I - A a n d I l l - E ) .

TABLE I

Isoforms of NO synthase Type

I

II

III

Source Calculated a Mr (kDa) Vmax (/~mol/mg per min) Native structure

Brain 160 0.3-3.4 Dimer

Macrophages 130 0.9-1.6 Dimer b

Endothelium 133 0.015 NR c

Identified binding sites Flavin sites Diphenylene iodonium NADPH site NADPH dialdehyde Calmodulin site Calmodulin required for activity Ca2+, ECs0 (p.M) Calmodulin, ECs0 (nM) Calmidazolium, IC50 (/xM)

(FAD, FMN, NADPH, CaM) Inhibits

Inhibits

NR

Inhibits

NR

NR

Yes 0.2-0.4 1-70 3.2

NR ~,a No dependence NR No effect

Yes 0.3 3.5 > 10

(L-Arginine,H4biopterin, heme, 0 2)

Unidentified, postulated binding sites L-Argininesite L-Arginine, K m (/xM)

NO2Arg, IC50 (/xM) MeArg, IC50 (/xM) Heme site CO NO Regulation of expression Constitutive TNF, INF, IL-1 Glucocorticoids TGF, IL-4,10 Posttranslational Modifications Phosphorylation Irreversible calmodulin binding Acylation Subcellular compartmentalization Soluble fraction

2-4.3 0.9 1.6

2.3-2.8 212 ~ 7.4 e

2.9 0.2 0.9

Inhibits Inhibits

Inhibits Inhibits

NR NR

Yes Suppress No effect

No f Induce Inhib. induct. Inhib. induct.

Yes Suppress No effect

Ser/Thr No NR

NR Yes NR

NR No Myristoylation

90-100%

85-90%

5%

Deduced from amino-acid sequence. b Tetramer postulated to contain 2 calmodulin subunits with NOS homodimer. c Not reported. a Yes for liver inducible NOS-II. e Values obtained with partially purified enzyme preparations. f Except a type II NOS that is constitutively expressed in smooth-muscle cells of colon and vas afferens from kidney. Abbreviations: TNF, tumor necrosis factor-a; TGF, transforming growth factor-/3; INF, interferon-y; IL, interleukin.

157 TABLE II

Targets and mechanisms of action of NO Target

Mechanism

Heme protein

Nitrosoheme Metheme

GC-S

SHprotein

Nitroso thio proteins

Plasma nitroso albumin t-PA

Fe-S protein

Nitroso iron sulfur proteins

Effects Activation

Inhibition

Other

Hemoglobin Myoglobin Dehydrogenases NADH : ubiquinone oxidoreductase NADH : succinate oxidoreductase

cis-aconitase Ferritin Ribonucleotide reductase FeZ ÷ and free thiols

DinitrosylFe(II) complexes

Free amine

Nitrosamine

Tumorigen

DNA

Desamination

Mutagen Carcinogen

DNA-binding proteins? NAD dehydrogenase

Auto ADPribosylation

02

Nitrite, nitrate

Iron depletion of cells

Induction of expression (TNF)

Transport form of NO

Suppression of expression? Glyceraldehyde-3phosphate dehydrogenase Inactivation of NO

(tl/2 = 5 s) "O~- (superoxide)

Peroxynitrite, OH

Superoxide scavenging

Toxification of NO

t-PA, tissue plasminogen activator; TNF, tumor necrosis factor.

This classification has been confirmed by the recent molecular cloning and expression studies of the corresponding genes [29-32]. In addition, for one NOS gene, alternate splicing products may exist [31]. Other classifications group NOS isoforms according to their regulation of expression (constitutive versus inducible) and their respective IC50 values for halfmaximal inhibition by inhibitory N~'-substituted L-arginine analogs. NOS-I and III are constitutively expressed and more readily inhibited by NO2Arg , whereas NOS-II is expressed after immunological activation of cells with different cytokines or endotoxin (see Section Ill-D) and is more readily inhibited by MeArg. III-B. N O synthase structure

biopterin, calmodulin and, of course, L-arginine, but these have not been clearly identified. NOS presents the typical CO difference spectrum of P450-type hemeproteins. Due to its CPR-like domain NOS can be viewed as a mechanistically self-contained cytochrome Pas0/CPR chimeric protein. H4biopterin , a cofactor which is utilized by several other amino-acid-hydroxyl-

cytochrome P 4 s o domain I

NADPH-cytochrome P4r,o reductmm-Ilke domain

I

heine

H®

I

I=MN

as

III

FAD NADPH ,

I

•

0 I

All NOS forms are homodimers (NOS 2) [33] of subunits which range between 130 and 160 kDa [29,31,32,34-38]. The N-terminal half of all NOS contains bindings motifs for NADPH, FAD and FMN and shows a 29-39% overall identity to NADPH-cytochrome-P450 reductase (CPR), the only other mammalian enzyme that contains both flavin nucleotides (Fig. 2). Additional binding sites within the NOS polypeptide must be postulated for heme, tetrahydro-

I

CaM

I

arginlne/H4bioptlrln binding domains?

Fig. 2. Protein domains and binding sites of NO synthases. Catalytic sites include the cytochrome P4s0-1ike (heme binding site) and CPRlike domains (FMN, FAD, NADPH binding sites) and the putative L-arginine binding site (active center). The CPR domain is sufficient for NADPH-diaphorase activity. Regulatory domains include the calmodulin binding site and various phosphorylation consensus sequences. Still unidentified is the putative H4biopterin site which may represent a quinoid-Hzbiopterin reductase domain, an allosteric activation site, or a region which protects the active center of NOS.

158 ases, is tightly associated with NOS (1-2 mol per dimer). However, unlike NOS, other H4biopterinutilizing enzymes do not bind H4biopterin with such a high affinity that it copurifies with them. This fact and some of the biochemical characteristics of NO catalysis (see Section III-C) suggest a novel H4biopterin binding site in NOS. Moreover, dimerization of NOS, a prerequisite for L-arginine turnover, is a Habiopterin-dependent process [39]. All NOS forms bind calmodulin in a process which can be either Ca2+-dependent (NOS-I and III) or Ca2+-independent (NOS-II) [40,41]. In the case of NOS-II, calmodulin and enzyme copurify, suggesting a native heterotetrameric structure (NOS2/calmodulin 2) of this isoform.

III-C. Mechanism of NO synthesis The exact structure of the endproduct of NOS catalysis is still a matter of debate. In most cases, NO was measured only indirectly, i.e., as nitrite and nitrate, stable degradation products of NO, which were formed in amounts that are equimolar to c-citrulline [39]. When selective methods were employed, e.g., gas-phase chemiluminescence without prior sample treatment, only traces of NO were detected in relation to total L-arginine turnover [42]. In addition, EPR spectroscopy frequently failed to detect the free NO radical [43,44]. Some reports demonstrate signals corresponding to iron nitrosyl, iron nitrosothiol complexes or an N~-hy -

droxy-L-arginine (OHArg) cation radical [45,46]. The latter was suggested to be the actual end product of NOS catalysis in platelets. The situation is further complicated by the fact that, subsequent to its formation, NO is highly unstable and may be stabilized by or react with free thiols [43,47], free iron, and thiol and iron-sulfur proteins, which gives rise to a whole family of secondary NO adducts and complexes including NO + and N O - [48], some of which may not be detectable as a free radicals. Nonetheless, the oxidation of a terminal guanidino nitrogen of L-arginine to NO is widely accepted as a working hypothesis for NOS catalysis [41,49-52]. Four cofactors (heine, FMN, FAD and H4biopterin) and two cosubstrates ( 0 2 and NADPH) participate in this reaction. Per tool NO formed, the electron transfer consumes 1.5 mol N A D P H / H + to yield 1.5 tool NADP + and three hydrid atoms (Eqn. 1). 3

3H+202 + C=NH

+2

' 2H20+NO+

C=O

(1)

The reaction represents an unusual odd transfer of five electrons onto the substrate guanidino nitrogen. Since electrons are usually transferred pairwise from N A D P H to flavin cofactors (e.g., N A D P H / H + ~ N A D P + + 2 H + + 2e ~ F A D H 2 ~ FMNH2), two rounds of catalysis are probably required for NOS to return to its ground state.

o2 Phase 1 1 m o l NADPH

NO.).OH.L-arginine NBT

02

Phase 2 0.5 mol F A D / F M N NADPH "NO NADPH diaphorase NADPH oxidase

0.5 tool FAD/FMN

NADPH

~ - q-BH 2

NBTformazan

H202

Fig. 3. Proposed reaction mechanism for NO synthases. Molecular oxygen is incorporated into both NO and L-citrulline in at least two phases (first two shaded areas). The flavin cofactors FAD and FMN and the cytochrome P450 domain of NOS may mediate the electron transfer from 1.5 mol N A D P H to molecular oxygen in one or both phases [54]. Habiopterin (BH 4) may also be involved in the electron transfer and both flavins and H4biopterin would then be expected to NADPH-dependently recycle. In Phase 1, L-arginine is hydroxylated to OHArg which requires 1 mol of the electron donor NADPH. In Phase 2, the oxidation of OHArg to L-citrulline and NO requires an additional 0.5 mol N A D P H [54] and is absolutely dependent on H4biopterin. The third shaded area represents (a) the uncoupling of either the putative calmodulin-independent q-H2biopterin reductase activity of NOS by the substrate nitroblue tetrazolium, NBT (i.e., NADPH-diaphorase activity which generates NBT-formazan), or (b) the uncoupling of the oxidase activity in the absence of L-arginine (i.e., N A D P H oxidase activity generating H2Oz). NBT non-competitively inhibits the conversion of OHArg and leads to its accumulation [66].

159 The oxidation of L-arginine has been dissected into two phases so far (Fig. 3). In phase 1, which consumes 1 mol of NADPH, it is suggested that OHArg is formed. Dissociation of traces of OHArg from NOS were indeed identified by HPLC and GC/MS. However, it is possible that OHArg represents a breakdown product of the actual intermediate. Subsequent to OHArg no other intermediate has been identified. As mentioned above, EPR data suggested the formation of a OHArg cation radical [53] during NOS catalysis but its precise structure was not elucidated. The reaction resembles a classical Pas0-1ike hydroxylation reaction and is therefore likely to involve the heme iron of NOS. It is not clear whether H 4biopterin is required as cofactor for this first reaction. In phase 2, which consumes an additional 0.5 mol NADPH, it is suggested that OHArg is further oxidized to NO and L-citrulline [54]. This step is clearly dependent on Habiopterin but consumes also 0.5 mol NADPH which would supply sufficient reducing equivalents (1H) to support this reaction. Thus, it has to be assumed that Habiopterin is either recycled or does not directly participate in NOS catalysis (stabilizer or allosteric activator). In all other known H4biopterinutilizing enzymes, the quinoid form of H2biopterin (q-Hzbiopterin) is the product of normal catalysis and is regenerated to H4biopterin by a q-H2biopterin reductase (dihydropteridine reductase). Purified NOS contains biopterin only in its fully reduced form (H4biopterin), indicating that NOS contains NADPHdependent q-H2biopterin reductase activity [41]. Alternatively, it was suggested that Habiopterin is not metabolized during normal catalysis but acts as an allosteric activator of NOS or stabilizes the active center of the enzyme [55]. That the q-H2biopterin/H 4 biopterin,binding site of NOS may be different from that in other known Habiopterin-metabolizing enzymes is indicated by (a) the uniquely small K a values [56,57] and high substrate specificity [55,58,59] of NOS for

H4biopterin, and (b) the resistance of NOS to inhibition by even high concentrations of methotrexate, which inhibits all known dihydrofolate and dihydropteridine reductases [55]. In the presence of L-arginine and Habiopterin, superoxide anions ( 0 2) are formed in a univalent reduction of 0 2 and transferred to OHArg (see above). At low concentrations or in the absence of L-arginine and Habiopterin, the activation of molecular oxygen uncouples from L-arginine turnover and results in a divalent reduction of 0 2 to yield hydrogen peroxide (H202). This NADPH oxidase activity of NOS is completely inhibited by OHArg or L-arginine analogs which competitively inhibit NOS. Conversely, L-arginine requires H abiopterin to induce complete inhibition of oxygen radical formation. The reductase activities of NOS extend to other substrates (Table III). Some of these reactions are related to the CPR-like domain of NOS and have higher turnover numbers than that for the conversion of L-arginine. As expected from the close similarity of NOS to CPR, NOS reduces cytochrome c in vitro. The mechanism of cytochrome-c reduction by NOS is unclear. NOS forms superoxide anions (see above), as does CPR, but also interacts directly with cytochrome c by a high affinity protein-protein interaction, whereas CPR does not bind to cytochrome c. Surprisingly, the turnover number for NOS-catalyzed cytochrome-c reduction is at least 10-times higher than that for Larginine turnover [60]. Furthermore, NOS also reduces cytochrome P450 as indicated by the fact that in vitro NOS supports the hydroxylation of N-ethylmorphine by cytochrome P450. This suggests that NOS may participate in similar electron transfer processes in vivo [60]. However, since cytochrome P450 is a membraneassociated enzyme, this characteristic may be only of physiological significance for NOS-III, a clearly membrane-associated type of NOS. Based on these data, one could argue that NOS enzymes are rather like

T A B L E III

Reactions catalyzed by NOS Electron acceptor

Substrates

Cosubstrates

Products

Reaction

0 2

L-Arginine

H4biopterin? NADPH FAD FMN ? NADPH NADPH NADPH NADPH

NO

N O Synthase

NO 02, H20 2 Reduced cytochrome c Reduced cytochrome P450 N B T diformazan

Nitrovasodilator bioactivation N A D P H oxidase Cytochrome c reductase CPR b N A D P H diaphorase

Organic nitrates? 0 2 Cytochrome c 02 NBT c

a

Cytochrome P 4 5 0

e.g., glycerol trinitrate and others. b NADPH-cytochrome P450 reductase. c Nitro blue tetrazolium. a

160 isoforms of CPR and that L-arginine turnover is merely a side track of their catalytic activities. The close similarity between NOS and the C P R / c y t o c h r o m e P450 system is underscored by the fact that cytochrome P450 catalyzes the oxidation of OHArg to NO, a reaction that is identical to Phase 2 of the NOS reaction. This again suggests that this phase of catalysis involves the P450-1ike domain of NOS. However, unlike NOS, cytochrome P450 converts the intermediate OHArg in an apparently H4biopterin-independent manner and does not convert the primary substrate of NOS, L-arginine. It remains to be established whether the P450 iron of NOS mediates the electron transfer from N A D P H onto molecular oxygen [61] and cycles between the Fe 2+ and Fe 3+ states during L-arginine turnover. Carbon monoxide (CO) binds to the heme moiety of NOS and inhibits L-arginine turnover. By a similar mechanism, the end product NO may mediate an autoinhibitory feedback control of NOS. Several artificial electron acceptors, e.g., nitro blue tetrazolium (NBT), are also NADPH-dependently reduced by NOS (Table III). NBT is converted to blue diformazan in an NADPH-diaphorase reaction which is not inhibited by superoxide dismutase. The NADPH-diaphorase activity of NOS is remarkably resistant to various protein fixatives enabling the convenient histochemical localization of NOS in tissue sections [62,63]. The utilization of NBT by NOS makes NBT also a potent non-competitive NOS inhibitor, presumably by either competing with molecular oxygen for reducing equivalents [41,64], or by reacting with intermediate superoxide radicals [65]. Interestingly, Phase 1, the formation of OHArg, appears to be less affected by NBT than Phase 2, the subsequent oxidation of OHArg to NO, for in the presence of NBT OHArg accumulates [66]. The aforementioned putative q-H2biopterin reductase activity of NOS may be closely related to the NADPH-diaphorase domain of NOS [63].

III-D. Regulation of NOS expression NOS-I and III are constitutively expressed. Additional modulation of basal expression may take place (see below). In contrast, NOS-II is not constitutively expressed in most tissues, instead its expression is induced (see Table I). Once expressed, NOS-II irreversibly binds calmodulin independently of Ca 2+ and stays maximally active, irrespective of the free Ca z+ concentration. The induction of NOS-II by cytokines is transcriptionally based. In control cells, m R N A for NOS-II is not detectable. Whether, in addition to gene transcription, m R N A stability or translation efficiency are also regulated is not known. Glucocorticoids and several interleukins suppress the apparent induction of NOS-II, whereas mineralocorticoids have no effect [67].

The sites and mechanisms of action of these immunomodulators remain to be established. Several serine protease inhibitors caused a similar inhibition of cytokine-mediated induction [68-70], however, their mechanism did not involve protease inhibition but most likely interference with protein synthesis [71] and intracellular thiol pools [72]. The same cytokines which induce NOS-II have the reverse effect on basal expression of the constitutive type-I NOS. The mechanisms which lead to reduced expression and the pathophysiological significance of this phenomenon are unclear [70,73]. Further regulation of NOS expression takes place at the translational level. As mentioned before, the subcellular distribution of the various NOS isoforms is different. Even the same isoform may be distributed at varying ratios between soluble and particulate cell fractions in different species [70]. However, none of the described NOS forms contain an amino-acid sequence suggestive of a transmembrane domain [29,37]. Co- or posttranslational modifications such as myristoylation, phosphorylation and glycosylation regulate the subcellular localization of NOS [74]. These modifications may also represent an additional mechanism of regulation of enzyme activity. The membrane-bound NOS-III is not only myristoylated but has also the lowest specific activity of all NOS [75].

III-E. Regulation of NOS acticity Once expressed, binding of the Ca2+-binding protein calmodulin or phosphorylation are established mechanisms by which enzyme activity of different isoforms of NOS is regulated (Table I). All NOS forms bind calmodulin and have conserved consensus sequences for calmodulin binding. In the case of NOS-I and III, interaction with calmodulin depends on elevated intracellular free Ca 2+ concentrations [Ca2+] i. At resting [Ca2+] i ( < 100 riM), these NOS forms are calmodulin-free and inactive. They bind calmodulin and become fully active at increased [Ca2+]i ( > 500 nM). Calmodulin antagonists, e.g., calmidazolium and trifluoperazine, block calmodulin binding and inhibit Ca2+-induced NO formation. Both compounds more potently inhibit NOS-I than NOS-III, which probably reflects differences in the respective calmodulin binding domains. In various systems, transmembrane Ca 2+ flux is initiated by the binding of a receptor agonist to its membrane receptor. Thus, receptor occupation and increased [Ca2+]i can be linked to increased NO formation in cells. Two forms of NOS-II have been isolated, one from liver [76] and one from a macrophage cell line [39,52]. At < 100 nM free Ca z+, the liver NOS-II is calmodulin-free and inactive. Similar to NOS-I and III, activity increases upon reconstitution of liver NOS-II with

161 calmodulin, but, unlike NOS-I and III, this activation is Ca2+-independent. Macrophage NOS-II has calmodulin constitutively bound and is constitutively active. So far, attempts to isolate the macrophage NOS-II in a native and calmodulin-free form were not successful. Phosphorylase kinase, a cyclic nucleotide phosphodiesterase, and Bordetella pertussis adenylyl cyclase are three other proteins which bind calmodulin constitutively, i.e., in an apparently CaZ+-independent manner [40]. Calmodulin binding emerges as a common activation principle for all NOS, whereas their dependence on the free intracellular Ca 2+ concentration distinguishes them. The predicted amino-acid sequence of NOS-I contains consensus sites for phosphorylation by cAMP-dependent protein kinases [29]. Forskolin-induced increases in intracellular cAMP levels [70] and cAMP-dependent protein kinases [77] do not regulate NOS, whereas protein kinase C, CaZ+/calmodulin-dependent kinase [78] and the phosphatase inhibitor okadaic acid [70] do. In vitro, Ca2+/calmodulin-dependent protein kinase II phosphorylates NOS on both serine and threonine [78]. Phosphorylation is Ca 2÷- and calmodulin-dependent and results in a marked decrease of NOS activity. Thus, frequent increases in [Ca2+]i and phosphorylation of NOS by Ca2+/ calmodulin-dependent kinase II could cause a negative feedback regulation of NOS activity. Because Ca2+/ calmodulin kinase II becomes Ca2+-independent upon autophosphorylation, it is conceivable that phosphorylation and inactivation of NOS proceed even after [Ca2+]i has returned to basal levels. However, these in vitro observations need to be confirmed in experiments using intact cells. IV. NO: mechanisms of action

NO can act as a paracrine substance and very likely also as an intracellular messenger molecule [79,80]. The short half life of NO (tl/2 ~ 5 S) argues against a humoral role of NO. At its site of action, NO is a 'double-edged sward'. The main if not sole effect of low NO concentrations is stimulation of the heine protein GC-S, soluble guanylyl cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) [16,81]. GC-S forms guanosine cyclic 3',5'-monophosphate (cGMP) which in turn specifically regulates protein phosphorylation, ion channel conductivity and phosphodiesterase activity (see Section V). Other effects of NO have only been observed when NO is formed in large quantities and reaches exceedingly high concentrations (see Section IV-B), e.g., by immunologically activated macrophages.

IV-A. Activation of GC-S by NO GC-S is a family of heterodimeric [82,83] heme proteins exhibiting a pyridine hemochrome visual ab-

sorption spectrum typical for ferroprotoporphyrine IX [84] and, in addition to iron, contain copper as another transition metal [84]. Two putative heme binding domains have been suggested for GC-S: (1) an invariant cystein (e.g., Cys-78 in the /31 subunit) which is conserved both in the a and /3 subunit of GC-S and has flanking sequences that are identical to those which coordinate heme in fish cytochrome P450 [85], and (2) a histidine which is, however, only conserved in the /3subunits of GC-S (e.g., His-105 in the/31 subunit) and has a conserved leucine in position - 4 [86]. Although some aspects of its mechanism of GC-S activation still need experimental support, NO apparently binds to the heme moiety of GC-S and, by dislocating the heme-iron, induces a conformational change in GC-S. This either de-inhibits or activates the catalytic site of GC-S [87]. NO is the most potent and effective activator of GC-S. Some studies suggest that overstimulation of GC-S by NO can result in reversible or irreversible refractoriness of the enzyme. Heme-free GC-S no longer responds to NO [84,87], although basal GC-S activity is independent of heine content.

IV-B. NO-independent activation of GC-S Recent data suggest that, in addition to NO, GC-S is regulated by a family of enzymatically formed guanylyl cyclase-activating-factors (GAF) [33] that includes also CO and OH. Although, of all the GAF synthesizing enzymes, NOS has probably the widest distribution in the body and has a clearly established role in GC-S regulation, other GAF synthesizing enzymes, e.g., heme oxygenase, colocalize better with GC-S in the central nervous system than does NOS [88,89]. CO is generated from two precursors, fatty acids or heme. NADPH-dependent enzymatic peroxidation of microsomal membrane lipids, e.g., methyl linolenate [90] and other polyunsaturated fatty acids, can produce a carbon-chain cleavage [91] and eventually CO [92,93]. As an alternate pathway, at least three enzyme systems exist that generate CO by oxidative heme destruction. All three heme-metabolizing enzymes require NADPH, 0 2 and CPR. The bulk of in vivo heme metabolism is provided by heme oxygenases, which convert iron protoporphyrine IX (FePPIX) and several other hemoproteins to biliverdin IXa and CO. Smaller amounts of CO arise from P450-catalyzed microsomal lipid peroxidation [93] and P450-catalyzed FePPIX destruction [94,95]. Because of the numerous similarities between NOS and CPR, some isoforms of NOS may substitute for CPR and also support heme degradation by heme oxygenases. OH formation can be enzymatic or non-enzymatic. Non-enzymatic OH formation can take place either by the iron-catalyzed Haber-Weiss reaction [96] or the iron-independent peroxynitrite pathway [97]. Both pathways require superoxide anions, which inter-

162 TABLE IV Mechanisms of action of cGMP and cAMP

Cyclic nucleotide

Effector

Regulated function

Tissue

References b

cGMP

cGMP-gated channel

Non-specific cation channel ( 1") a Non-specific cation channel ( $ ) Amiloride-sensitive Na +-channel ( $ )

Retinal rod photoreceptor Olfactory cells Renal inner medull, coll. duct.

109 108 113

cGMP-inhibited cAMP-PDE ( l i d

cAMP-response ( 1") Ca 2+ channel ($) Na + and CI - absorption ($)

Mammalian heart Distal colon

194 186

cAMP response ( ~ ) Ca 2+ channel ( $ ) Steroidogenesis ( $ )

Frog heart Adrenal glomerulosa cells

140 139

cGMP-stimulated cAMP-PDE (II)

Photoreceptor PDE (V1)

PDE inactivation ( ,~), prolonged photoresponse

Amphibian retinal rods

141

cGMP-dependent protein kinase (I)

Ca 2+ channel ( j, ) Ca 2. channel ( 1") Amiloride-sensitive Na + channel ( $ ) Ca2+-dependent K* channel ( 1")

Mammalian heart Snail neurons Renal inner medull, coll. duct. GH4C ~ pituitary tumor

192 207 113 205

Intracellular Ca 2+ release ($), Secondary Ca 2+ influx ( ], ), In vivo VASP phosphorylation ( 1' )

Platelets

224

Smooth muscle Smooth muscle

214 215

240-kDa protein Smooth muscle phosphorylation ( 1"), plasma membrane Ca2 +-ATPase ( 1")

216

Inositol phosphates ( + )

Smooth muscle

217

PLB phosphorylation ( 1"), SR Ca2 +-ATPase activity ( 1")

Smooth muscle

218

Ca2+-activated K + channels ( 1")

Smooth muscle

220

Cerebellum

200

Small intestine epithel.

191

Intracellular Ca 2 + ( $ ) Voltage-gated Ca 2+ channels ( $ ) Na +-Ca 2 + exchanger ( 1")

Phosphorylation of phosphatase inhibitor ( ? )

cAMP

cGMP-dependent protein kinase (II)

? CI ? C1

cAMP-gated channel

Hyperpolariz.-activated channel ( 1")

Mammalian heart S-A node~ pacemaker cells

Non-specific cation channel ( t )

Olfactory cells

Metabolism ( ~, $ ) Ion channels ( L, t ) Gene expression ( $, 'f )

Ubiquitous Ubiquitous Ubiquitous

cGMP-specific PDE phosphorylation ( 1`), cGMP-hydrolysis ( 1")

Lung

211

Intracellular Ca 2+ ( ,~)

Smooth muscle

152 153

cAMP-dependent protein kinases (I a,/3 II o~,/3)

cGMP-dependent protein kinase (1)

absorption ($), secretion (T)

~' ( $ ) ( 1' ) = decrease and increase in activity, respectively. b Due to space limitations, only a few representative references or reviews are listed. PBL, phospholamban; SR, sarcoplasmic reticulum; S-A, sino-atrial node.

159 (review)

163 act either with Fe 3+ or NO, respectively. In the latter case, 0 2 and NO form peroxynitrite (ONO 2) [97], which homolytically decomposes to yield O H and NO 2 [97]. Under certain conditions, xanthine oxidase [98] can form O H enzymatically. Other mechanisms may exist. The mechanism by which CO activates GC-S is probably identical to that of NO [99]; moreover, CO and NO have similar effects on platelet aggregation [99], vascular smooth muscle function [100] and intracellular cGMP levels [57]. The mechanism of activation of GC-S by O H is unclear but appears to be different from that of NO and CO. The O H may interact with regulatory thiol groups of GC-S [101], which function as a cellular redox sensor and effector protein. O H was suggested to function as a physiological mediator of endothelium-dependent relaxation in mouse cerebral arterioles [102]. Other oxygen radicals inhibit ( O y ) or have no effect ( H 2 0 2) on GC-S. The relative importance of O H for GC-S regulation and of the different pathways for O H generation may thus depend on the oxygen tension. The peroxynitrite pathway, for example, is likely to operate in a given biological system, e.g., in reperfused tissue [103] only when sufficient concomitant NO and superoxide formation takes place [97]. Similar to NO, O H and CO are also 'double-edged swords'. Depending on their concentration, they can serve signalling functions or be highly cytotoxic.

IF-C. cGMP-independent actions of NO A major action of NO is on the cGMP signal transduction system, however high concentrations of NO which are generated by the immunologically induced and Ca2+-independent NOS-II can interact with almost every cellular prot,qn. Under in vivo conditions, reactions with NO have also been described for oxygen, oxygen radicals, iron-, heine- and thiol-containing proteins, and primary amines. They are summarized in Table II. In many cases these reactions lead to loss of function of the entire cell or particular enzymes. Activated macrophages which possess a high-output NOS-II utilize some of these as non-specific immune defense mechanisms against bacterial, protozoan and possibly viral infections. V. cGMP: mechanisms of action

The effector targets for cGMP and some functions they appear to regulate are shown in Table IV. For comparison, the effectors for cAMP are shown, however, the numerous functions they regulate are shown only very abbreviated and cannot be discussed within the scope of this review.

V-A. Regulation of cGMP-gated channels The vertebrate rod photoreceptor cell response to light leads to cGMP hydrolysis, closing of a plasma

membrane cGMP-gated cation channel, transient hyperpolarization of the cell and eventually a neural response. The bovine rod channel directly regulated by cGMP was cloned and functionally expressed [104]. The genomic D N A for the human equivalent of this rod channel has been cloned and shown to be derived from chromosome 4 [105]. The cGMP-gated cone photoreceptor channel has been shown to be derived from a gene distinct from that of the rod photoreceptor channel [240]. Chick pineal ceils are also photosensitive, and contain a cation channel activated by cGMP but not cAMP in excised inside-out patches [106]. In another sensory system, that of olfaction, a channel with high affinity for cGMP compared to cAMP [107,108] was shown to be highly related to the rod channel; 60% of their amino acids were identical and 14% represented conservative changes (reviewed in Ref. 109). The retinal and olfactory cGMP regulated proteins have one cGMP binding site compared to the two sites per monomer or four sites per homodimer of the soluble form of cGMP-dependent protein kinase discussed later. The odorant stimulatory pathway for cGMP elevation is not yet clear, however some members of a multigene family of putative odorant receptors which act via G proteins to stimulate inositol trisphosphate (InsP 3) or activate adenylate cyclase and increase cAMP have been cloned and characterized [110]. More recently, a cloned odorant receptor was expressed in Sf9 cells and shown to respond to specific odorants by increasing InsP 3 as do isolated olfactory cilia [111]. In olfactory cilia, InsP 3 and cAMP activate non-specific cation channels that allow Ca 2÷ to enter. Large doses of odorants cause Ca 2÷ stimulation of NOS to form NO which diffuses to neighboring clusters of receptor neurons to stimulate their guanylyl cyclase to increase cGMP which then activates nearby Ca 2÷ channels [112]. A cGMP-mediated response to strong odorants lends a physiological basis to the observation that cGMP can activate the channel for which it has a higher affinity than does cAMP. The h i g h e r cGMP affinity is consistent with the fact that cGMP is present at much lower concentrations than cAMP in the olfactory cilia [112]. In the renal inner medullary collecting duct, the probability of a single amiloride-sensitive cation channel being open was decreased by ANP both directly via cGMP and via cGMP-dependent protein kinase (see Section V-D.2) [113]. Mouse M-1 cortical collecting duct cells also show nonselective cation channels whose open probability is decreased by cGMP, and partial clones for cDNAs related to the cGMP-gated rod photoreceptor channel have been obtained from these cells [114]. The amino-acid sequences derived from all clones were identical to the corresponding region of the rat photoreceptor channel. Evidence for related proteins in rat heart and kidney was obtained by

164 Northern analysis using a probe from the rod photoreceptor channel [115]. Preliminary reports of cDNAs cloned from aorta [116] and testis (Kaupp, U.B., personal communication) which have sequence similarity to the cGMP regulated olfactory channel and cone photoreceptor channel, respectively, indicate that these tissues contain endogenous R N A for such channels. Other channels not regulated directly by cGMP but via cGMP-dependent protein kinase are discussed in Section V-D.2.

V-B. Regulation of phosphodiesterases (PDEs) Only certain phosphodiesterases are regulated by cGMP, however it is relevant to discuss them within the framework of the spectrum of PDEs, a brief review of which will be presented. Primary protein and cDNA sequences have been used to classify PDEs which degrade cAMP a n d / o r cGMP into six general families composed of subfamilies of similar (70-90% identity) but distinct genes [117]. Representatives have been cloned from almost all the various families including family I, the CaZ+-calmodulin dependent P D E family [118,119]; family II, the cGMP-stimulated PDE (cGSPDE) [120,121]; family III, the cGMP-inhibited P D E (cGI-PDE) [122]; family IV, the low K m cAMP-specific PDE, including the Drosophila dunce gene product and rat dunce homologs [123-127]; family V, the cGMP specific PDEs, including the lung form, also called cGMP-binding P D E (cGB-PDE) (purified, in Ref. 128; cloned in Ref. 129), and the platelet and smooth muscle forms; and family VI, the photoreceptor membrane-associated rod form composed of a [130], /3 [131], and inhibitory z subunits [132] in a complex of a/3z z, and the photoreceptor cone form composed of a ; [133,134] and three low molecular mass subunits. Most of the enzymes in the above six families belong to class-! PDEs which have in common a conserved catalytic region of about 250 amino acids and are distinct from those designated class II which include a Saccharomyces cerevisiae PDE1 gene product and a Dictyostelium P D E [135-137]. The phosphodiesterases exert part of the cellular control over the levels and duration of action of cAMP and cGMP and they are themselves differentially regulated by various means. Relevant to the subject of this review is that some are regulated by cGMP itself. Phosphodiesterases in families II, V and VI contain so-called conserved non-catalytic cGMP binding sites, although there are considerable differences in the cGMP binding properties of the various PDEs [136]. The cAMP hydrolyzing cGI-PDE (family III) is inhibited by cGMP, although it is not clear whether this results from cGMP binding to the non-catalytic or catalytic site. The regulatory function of the non-catalytic sites is only partially understood. In the case of

family II, cGMP binding stimulates cAMP hydrolysis by the cGS-PDE. This has been shown to occur in response to ANP stimulation of PC 12 cells [138] and adrenal glomerulosa cells [139]. In the latter case, ANP elevation of cGMP inhibited the effects of ACTH, i.e., by stimulating the hydrolysis of cAMP and thus lowering aldosterone. In frog heart, cGS-PDE has been shown to inhibit cAMP activated inward Ca 2+ current [140]. In family VI, the amphibian rod PDE a and /3 subunits each display one to two non-catalytic binding sites which control the interaction of a and /3 with the inhibitory subunit z [141]. The presence of cGMP in these sites causes a slower inactivation of the PDE, causing a long photoresponse characteristic of a darkadapted cell. The two conserved cGMP binding regions are also present in the cone a ' subunit, and possibly in the c G B - P D E (family V). A hypothetical model of the evolution of P D E domains by duplication of an ancestral gene and its subsequent divergence into the catalytic and non-catalytic cGMP binding sites has been presented [135]. Amino-acid sequence comparisons indicate that the putative cGMP binding domains of cGMP-dependent protein kinases and the rod and olfactory cGMP-gated channels, which have certain similarities in common [109], are structurally different from the non-catalytic cGMP binding domains of cGSPDE [121]. The profound physiological importance of PDEs is exemplified by certain P D E deficiencies or excesses. Mutations in the Drosophila dunce gene coding for a low-K m cAMP-specific P D E (family IV) disrupt learning, memory and fertility in Drosophila [123]. Mice with hereditary nephrogenic diabetes insipidus which are resistent to the antidiuretic effect of vasopressin have anomalously high activities of low-K m cAMP-PDE and they fail to increase cAMP levels in their collecting ducts in response to vasopressin [142]. This is in contrast to human congenital nephrogenic diabetes insipidus (CNDI), an X-linked recessive disease which in one family has been ascribed to a deletion in the vasopressin V2-receptor gene [143]. Retinal degeneration in the rd mouse is caused by a defect in the /3 subunit of rod c G M P - P D E (family VI). Linkage mapping has localized the /3 subunit gene to the same region on mouse chromosome 5 as the rd locus [144] and retinal degeneration has been rescued in transgenic rd mice by expression of the P D E /3 subunit [145].

V-C. Cross-activation of cAMP-dependent protein kinase In vitro K a values for cAMP and cGMP activation of a given cyclic nucleotide kinase differ by one to two orders of magnitude (reviewed in Ref. 146). If this were also the case in vivo, then physiological levels of cAMP would preferentially activate cAMP-dependent

165 protein kinase, and cGMP would preferentially activate cGMP-dependent protein kinase. This could change in situations in which very high levels of a cyclic nucleotide are produced and cellular compartmentalization limits access to the preferred target kinase. Some possible examples of this have been proposed. The E. coli heat-stable toxin STa increases cGMP and net transepithelial secretion of C1- in intestine [8]. T84, a human colon carcinoma cell line was shown to contain predominantly the type-II isoform of cAMPdependent protein kinase and little or no cytosolic cGK. However, STa caused a marked increase in cGMP and C1- secretion, and an increase in the cAK activity ratio, an indicator of cAK activation [147]. Conversely, cGMP analogs known to be potent activators of soluble cGK were ineffective in producing CI- secretion. How representative these findings in a cultured cell line are of the physiological situation in intestine is not clear since the kinase composition of these cells may not genuinely reflect that in normal tissue. In particular, another type of membrane-bound cGK that has been proposed to mediate the effects of STa in intestine [8,148] may be missing. This kinase and its proposed function is discussed further in Sections V-D.1 and V-D.2. Retinal rods are an example of a normal cell type in which there are high concentrations of cGMP and type II cAK but no detectable level of cGK, suggesting that cGMP might possibly act via cAK (discussed in Ref. 149), besides acting on the cGMP-gated channel. However, cGMP channel opening in the dark indicates that the free cGMP concentration in rods is only 2-4/xM, much less than the total extractable cGMP of 50-60 /zM [150], although still in the lower range of the in vitro Ka of cGMP for type-II cAK. cGK-deficient platelets obtained from human chronic myelogenous leukemia patients, represent another example of cGMP effects perhaps being mediated by cAK. Treatment of these platelets with sodium nitroprusside (SNP) caused an approx. 50-fold increase (0.2-10 /zM) in cGMP, no increase in cAMP, but increased phosphorylation of a substrate protein presumably via cAK [151]. Action of cGMP on any residual amount of cGK present was not considered likely, since cGMP analogs that normally produce functional effects in normal platelets were inactive in the cGK-deficient platelets. Evidence for the converse, cAMP actions being mediated by cGK, has also been presented. In rat aortic smooth muscle ceils passaged in culture, the effect of forskolin, an adenylate cyclase activator, on cellular Ca 2+ concentrations was observed to be dependent on the presence of cGK in the cells [152]. Passaging of ceils resulted in both loss of cGK and the forskolin effect on Ca 2÷, both of which were restored by introducing cGK, but not cAK catalytic subunit back into

the cells using a hypo-osmotic shock method. In another study using pig coronary arteries, isoproterenol elevated cAMP and not cGMP, but both cAK and cGK activity ratios (an indication of kinase activation) were increased 2.3- and 1.6-fold, respectively [153].

V-D. Activation of cGMP-dependent protein kinases V-D. 1. cGMP kinase localization, heterogeneity, and domain structure/function Currently, two major types of vertebrate cGMP-dependent protein kinase, the soluble type I (GKI) and the membrane-bound type II (GKII) have been recognized. Furthermore, the soluble type-I cGK has been shown to exist in two isoforms designated a and /3, which have been purified from bovine lung and aorta [154-156] and cloned from bovine trachea and human placenta [157,158]. The cDNA-derived a and /3 protein sequences were essentially identical in their cGMP binding and catalytic domains, but were only 36% identical in their amino-terminal ends. Certain evidence suggests that the site of abrupt termination of protein sequence identity corresponds to an intron-exon junction, indicating that a and /3 are splice products from a single gene (reviewed in Ref. 159). The most highly related protein to mammalian cGK is a Drosophila gene product, DG2;T3b, derived from 8 exons [160], one of which ends at a site coinciding with the proposed site of an intron-exon junction used for splicing the type-I a//3 forms. Drosophila has two genes, DG1 and DG2, which appear to code for 4-7 cGK products. Further cGK enyzmes have been purified from a varity of organisms including Ascaris suum, the silk worm, Dictyostelium, Tetrahymena, and Paramecium (reviewed in Ref. 161). A striking and distinguishing characteristic of all purified and cloned cGKs is their variable existence as monomer and dimer forms, including the mammalian soluble GKI which is a dimer and the membrane-bound cGKII which is a monomer. Besides differing with respect to their monomeric or dimeric composition, the soluble cGKI and membrane-bound cGKII differ in many fundamental aspects (reviewed in Ref. 159). Not only do they differ with respect to their major cellular distribution, but their apparent functions are also divergent. Whereas the highest concentrations of cGKI are in cerebellar Purkinje cells, smooth muscle cells, and platelets, cGKII has its predominant localization in the epithelial brush border of small intestine (reviewed in Ref. 159). So far cGKI/3 has been detected in smooth muscles such as bovine aorta, trachea, stomach, uterus [154157,162] and human uterus and placenta [158]. Evidence suggests that cGKI controls intracellular Ca 2÷, whereas cGKII regulates intestinal CI- absorption and secretion (discussed in the following section). The most structural information is available for mammalian soluble cGKI. Since the structure/function

166 of cGK protein domains has been recently extensively reviewed [159,161], only some recent aspects will be emphasized here. A wide assortment of characteristics has been ascribed to the first amino-terminal domain of cGK. This domain is collectively called the dimerization domain but contains not only the region of dimerization of the holoenzyme, but also autophosphorylation sites, an autoinhibitory region, a designated hinge region, and a region which determines cooperativity between the c G M P binding sites, in that approximate order going toward the carboxy-terminal end. In the dimerization domain, the first 39 amino acids of cGKI was recently shown by circular dichroic spectral analysis (CD) to form a strong a-helical region containing a periodic repeat of leucines (or isoleucines) at every seventh position [163,164]. The leucine/isoleucine repeat was reminiscent of a leucine zipper motif which has been shown to promote dimerization in a number of other proteins [165], and ultracentrifugation determination of the molecular mass of a peptide of the first 39 amino acids confirmed that it could exist as a dimer in solution [164]. N M R data for the cGKI peptide 1-39 furthermore indicated that dimer formation occurs by a head-to-head parallel arrangement of monomers [164], accomodating another feature of cGK conformation which permits intrachain inhibition of the catalytic site by the autoinhibitory region [154]. The autoinhibitory region, located between amino acids 54-67 for c G K a , inhibits cGK activity in the absence of cGMP. Trypsinization to remove the first 77 amino acids of bovine lung cGK yielded a constituitively active cGK, independent of cGMP for its activity [166,167]. Further evidence suggests that a part of the dimerization domain influences the cooperative interaction between the two cGMP binding sites [168]. Except for the dimerization domain, all other domains, including the two cGMP binding domains, are identical in the bovine tracheal a and /3 forms of cGKI. Nevertheless, the apparent affinity of cGMP for binding site 1 was 10 fold lower in the I/3 than in the I a enzyme. Since previous results indicated that high affinity binding to site 1 depends on cooperative interaction between sites 1 and 2, it was concluded that different cGK a and /3 amino-termini could produce different protein conformations and cooperativity. Autophosphorylation sites also differ for a and /3 forms, Ser-50, Ser-72, Thr-58 and Thr-84 being identified in bovine lung cGK (corresponding to the cloned a form) [169], of which only Ser-50 is conserved in the cloned human /3 form [158]. Autophosphorylation has been shown to increase the rate of cGMP dissociation from the high-affinity cGMP binding site 1 [170] and increase the affinity of cAMP for this site [171,172]. This could play a role in cross activation of kinases. The cGMP binding sites of cGK have been analyzed by comparison to those of cAMP in the bacterial

catabolite activator protein (CAP) for which the crystal structure is known [173] and for which the identity of the amino acids involved in cAMP binding was determined in mutagenesis studies (reviewed in Ref. 174). These data and cGMP-analog binding and activation constants [175] were used to predict a model of the cGMP-binding pocket [176]. New cyclic nucleotide analogs have been developed which have relative advantages over previous ones [177,178]. In general, useful characteristics sought in such analogs are their specificity for cGK over cAK, their lack of effect on other targets such as phosphodiesterases or ion channels, their lipophilicity for entry into cells for intact cell studies, and their resistence to degradation. Some analyses are still incomplete, since certain kinase and phosphodiesterase isoforms have only recently become available from cloning and expression studies, and others are still unavailable. Of course, a given cell may not necessarily contain all possible cGMP targets but such information is often lacking. Two cGMP analogs, 8-(p-chiorophenylthio)-cGMP (8-pCPT-cGMP) and /3-phenyl-l,NZ-etheno-cGMP (1,N2-PET-cGMP), which are useful cGK activators can be mentioned here. Whereas 1 /zM 8-pCPT-cGMP. 8-bromo-cGMP and cGMP selectively activated cGK and not cAK in human platelet membranes, the lipophilicity of 8-pCPT-cGMP made it superior to the other two nucleotides for intact cell studies [177]. Furthermore, 8-pCPT-cGMP was shown not to activate the cGS-PDE or inhibit the cGI-PDE, and was itself not hydrolyzed by these PDEs nor the Ca 2+-calmodulin PDE or platelet homogenates containing high levels of the cGMP-specific cGMP-binding PDE. The usefulness of 8-pCPT-cGMP in studies of cGK physiological functions will be discussed further in Section V-D.2. It should however be noted that C-8 substituted analogs bind preferentially to the high-affinity cGMP binding site 1 [175], thus their affinity for c G K I a is much higher than for cGKI/3 which has two low-affinity sites, apparently due to an influence of the dimerization domain on the cooperativity of the two cGMP binding sites, as discussed earlier [155,168]. Thus, additional derivatives may need to be analyzed to ascertain a cGKI/3-mediated effect. Our unpublished results show low in vitro K a values of activation of 1,N 2-PET-cGMP for both purified bovine lung c G K I a and recombinant human placenta cGKI/3 derived from a mammalian expression vector. The effectivity of 1,N2-PET-cGMP derivatives for purified bovine aorta cGK1/3, as well as purified bovine lung c G K I a , was also observed in a large comparative study of 40 analogs [178], however these derivatives have yet to be investigated with regard to several other desired properties. Furthermore, useful membrane-permeable antagonists of cGK activation have been described [179]. These are Rp-diastereomer analogs (Rp-cGMPS and Rp-8-Cl-cGMPS)

167 in which the important equatorial exocyclic oxygen of the phosphate group of cGMP has been substituted with sulfur. Particularly the Rp-8-CI-cGMPS inhibition is useful for discriminating cGK from cAK in vitro. Intact cell studies with these and other more lipophilic antagonists are currently being conducted (Butt, E. and Walter, U., unpublished data). The carboxy-terminal segment of cGK includes the Mg/ATP-binding site and also the substrate binding site/catalytic domain which has similarity to the highly-conserved catalytic domain of the extensive protein kinase family to which cGK belongs [180]. Investigation of protein and synthetic peptide substrates of cGK do not indicate a very clear phosphorylation site consensus sequence [181,182]. It has been suggested for the bovine lung cGMP-binding, cGMP-specific PDE which is a relatively specific substrate for cGK compared to cAK, that a phenylalanine near the phosphorylation site was a negative determinant for cAK but not cGK [183]. These studies were however performed in in vitro assays and need to be verified by intact cell experiments.

V-D.2. Physiological substrates and functions of cGMP kinase (cGKll, cGKI) Intestine. In comparison to the soluble cGKI, less is known about both the structure and the physiological function of the membrane-bound cGKII (reviewed in Refs. 148,159,184). Furthermore, cGKII has so far been reported only in intestinal epithelia brush border, and the only substrates observed to be phosphorylated by it were the 86-kDa cGKII itself and a 25-kDa protein [184,185]. E. coli heat-stable enterotoxin (STa) activates the intestinal particulate guanylyl cyclase GC-C and increases cGMP which has been linked to STa effects on ion and fluid transport in the intestine leading to secretory diarrhoea [8]. The actions of STa are mimicked by 8-Br-cGMP, causing decreased Na + and C1- absorption in the distal colon, and decreased Na ÷ absorption and activated CI- secretion in the proximal colon [186]. Further results indicated that the mechanism of cGMP action in the distal, but not the proximal, colon may involve inhibition of the cGI-PDE to increase cAMP [186]. Other cGMP effects could possibly be mediated by the soluble cGKI, by cAK (see cross activation, Section V-C), or by the membranebound cGKII. Experiments related to these cGMP effects have, however, not been carried out directly in intestine, but rather in a human colon carcinoma cell line called T84. In these cells STa also increases cGMP and the net transepithelial secretion of CI-, however T84 cells contain type-II cAK but very little soluble cGKI. These and other data led to the suggestion (discussed in Section V-C) that cGMP could mediate the effects of STa by cross activation of cAK, whose activity ratio was shown to be increased [147]. The

possibility of cGMP inhibition of cGI-PDE to increase cAMP and thus activate cAK, as described above, was not addressed. Also in T84 cells a 10 pS C1- channel was characterized in excised inside-out patches and shown be be regulated by cGMP [187]. In the absence of ATP, cGMP appeared not to have any direct effects indicative of the presence of a cGMP-gated channel. In the presence of ATP, cGMP caused CI- channel opening via an endogenous membrane-bound cGK presumed to be present, and additionally via exogenously added soluble cGK. It is feasible that in vivo the membrane-bound cGKII in intestine mediates cGMP effects in this mechanism since several other components, i.e., receptor-cyclase, and channels or transporters are intrinsic to the membrane. The T84 cell low-conductance C1- channel regulated by cGMP has a linear current-voltage ( I - V ) relationship, anion selectivity, and other properties similar to those of the CI- channel expressed when the cystic fibrosis (CF) gene is transfected into non-epithelial ceils [187]. The cystic fibrosis gene product CFTR (cystic fibrosis transmembrane conductance regulator) has been shown to code for a CI- channel both when put into lipid bilayers or expressed from cDNA in cells [188]. However, in contrast to the CI- channel opening caused by the combination of cGMP, ATP and soluble cGK added to T84 cell patches, no C1- channel opening was detected in similar patch clamp experiments with NIH 3T3 fibroblasts transfected with CFTR [241]. Thus, no evidence was found for regulation of this CFTR CI- channel by the soluble cGK, although this could be due to an absence of the appropriate kinase substrate protein in these cells. A role for the membrane cGK, which is most likely absent in 3T3 cells, was not studied. Humans affected with CF and transgenic mice expressing a disrupted CFTR gene have intestinal abnormalities, in addition to many other well-known problems (reviewed in Ref. 189). It has been speculated that CF patients are resistent to the dehydrating action of microbial toxins and protected against secretory diarrhoea [190], perhaps due to altered STa-regulated C1- secretion in such patients. Recent short-circuit current measurements across stripped ileal mucosa and rectal biopsies from control and CF patients have revealed CF defects in the cGMP and Ca2+-provoked C1- secretion, which are not found in airway epithelium and sweat gland coil [191]. In the latter two tissues (and in intestine) there is of course also a well-known defect in cAMP-provoked CI- secretion. The cGMPregulated C1- secretion may be mediated by endogenous membrane cGK which is found in the intestinal brush border [184], rather than the soluble cGK which is associated with smooth muscle and other cell types [159]. Besides stimulation of cGMP production via the

168 membrane receptor guanylyl cyclase (STAR or GC-C), there are indications for stimulation of cGMP production via a soluble guanylyl cyclase since neuronal localization of NOS for the synthesis of NO has been demonstrated. So-called nitrinergic or nitroxidergic nerves have been identified throughout the gastrointestinal tract [230]. In the stomach, NO mediates not only the motility required for accomodation of food and fluid, but also appears at low concentrations to be a cytoprotective factor, possibly by stimulating bicarbonate and mucous secretion, and at high concentrations to be a cytotoxic factor [231]. Studies for distinguishing the cGMP target in these functions are needed. Heart. Patch-clamp experiments on purified cardiac ventricular myocytes indicate that the mechanism of action of cGMP in this single cell type can be multifaceted. It was already mentioned in Section V-B that cAMP-stimulated Ca 2+ influx through a channel in frog heart is decreased by c G M P activation of a cAMPP D E to lower cAMP levels [140]. In mammalian heart, cGMP was shown to inhibit the slow inward Ca 2+ current (/Ca) by stimulation of cGKI [192]. In order to conclusively demonstrate an effect of cGK on Ica, it was necessary to perfuse patch-clamped myocytes with a constitutively active cGKI which did not require cGMP for stimulation, in order to eliminate any other effects of cGMP. This was achieved by using a cGK subjected to partial trypsinization to remove the first 77 amino acids of the dimerization domain that contains the region for inhibiting enzyme activity in the absence of cGMP (see discussion of cGK domain struct u r e / f u n c t i o n in Section V-D.1). This treatment reduced the 150-kDa cGK holoenzyme dimer to a monomer of 65 kDa. Perfusion with very low concentrations of either cGK (10-100 nM) or cGMP or 8-Br-cGMP (0.1-1 ~zM) caused a 30-70% reversal of the Ica elevated by cAMP or 8-Br-cAMP, suggesting that there may be very low levels of endogenous cGKI in cardiac myocytes, as our Western blot studies of purified myocytes also indicated. Experiments with A T P - r S indicated that cGMP did not activate a phosphatase or inhibit cAK to reverse the cAMP effects. Other evidence indicated that cGMP can also inhibit a cGI-PDE in both frog [193] and guinea pig [194] myocytes, causing an increase in the /Ca response to cAMP. Evidence for the presence of cGS-PDE, cGI-PDE and cGK in isolated rat cardiac myocytes has been reviewed in Ref. 159. It should be emphasized that it is unclear whether cGK affects Ica by directly phosphorylating the C a 2+ channel or some other regulatory protein, cGK has been shown to stoichiometrically phophorylate the a~ and /3 subunits of the skeletal muscle Ca 2+ channel in vitro [195], although experiments must be extended to the cardiac Ca 2+ channel and to more intact cell analyses in the future. Furthermore, although cGMP

exerts negative inotropic effects on the heart, the mechanism of cGMP elevation in heart tissue, by cholinergic agents or ANP for example, is not completely explained (reviewed in Ref. 196). Recently, several indications for the production of NO by purified neonatal and adult rat cardiac myocytes were presented [197]. Another report showed that some intracardiac neurons from newborn guinea-pig atria and interatrial septum contain NOS-I and arc capable of synthesizing NO, raising the possibility of NO involvement in neural control of cardiac function [198]. Furthermore, proinflammatory cytokines induce NOSII expression [232] resulting in negative inotropic responses that are completely reversed by NOS inhibitors [233]. Moreover, NOS inhibitors attenuate the negative chronotropic effect of carbachol and increase the inotropic effect of the fl-adrenergic agonist isoproterenol [197]. Cerebellum. Recently, NOS-I localization in the cerebellum was found to be restricted primarily to granule cells and basket cells [199], suggesting that NO liberated from these cells could act on other cellular targets in the cerebellum by increasing cGMP. Guanylyl cylase, cGK, and the only nerve cell specific substrate so far identified for cGK (G-substrate) are greatly enriched in cerebellar Purkinje cells, and phosphorylated G-substrate was found to inhibit protein phosphatases 1 and 2A (reviewed in Ref. 200). In the cerebellum, cGK immunolabeling was very pronounced in Purkinje cell bodies and dendrites, and their axon innervation of deep cerebellar nuclei [201,202]. As discussed in Ref. 201, other reports indicated that cerebellar cGK levels were greatly reduced in mutant mice specifically lacking Purkinje cells. Since Purkinje cells receive both excitatory and inhibitory inputs from several neuronal pathways and represent the only efferent pathway from the cerebellum, cGK function may be important for the integrative activities of these cells in motor coordination or other functions. Kidney. In kidney, immunohistochemical labeling demonstrated that cGK was particularly concentrated in contractile cells of the kidney vasculature, including intra- and extra-glomerular mesangial cells, vascular smooth muscle cells and microvascular pericytes, as well as in interstitial myofibroblasts [203]. NOS-I was predominantly located in the macula densa from which it was shown to regulate glomerular capillary pressure, presumably via vasodilation, and to control renal blood flow [204]. In contrast, so far the functional studies in kidney showed a potential role for cGK in the inner medullary collecting duct [113]. There, cGMP appeared to decrease the open probability of an amiloride-sensitive Na + channel by both a direct effect on the channel in the absence of MgZ+/ATP (Section V-A), and via cGK in their presence. Furthermore NO, and eventually cGMP, appear to

169 be involved in the regulation of renin secretion [234] and pressure-induced autoregulation of filtration and natriuresis [235]. Under pathophysiological conditions, such as Heymann's nephritis [236] and glomerulonephritis [237], renal NO formation is increased or occurs at unphysiological sites. NO in this case is most likely the product of NOS-II induction and involved in activating NO targets unrelated to the cGMP cascade. Various cell types and tissues. Nystatin perforated patch clamp studies on GH4C 1 rat pituitary tumor cells, showed that either 8-Br-cGMP or cGK could stimulate a large conductance, Ca 2+- and voltageactivated K ÷ channel [205]. The mechanism was concluded to be via cGK activation of a protein phosphatase. Perforated patch clamp studies in rabbit corneal epithelium showed a cGMP increase in the open probability of a K ÷ channel [206]. Microinjection of cGK into snail neurons increased an inward Ca 2+ current [207]. Studies with perforated cell patches of endothelial cells indicated that bradykinin activated a small conductance, Ca2+-activated K ÷ channel causing membrane hyperpolarization, promotion of Ca 2+ entry and cGMP formation [208]. Additionally, a suggestion has been made that cGK may regulate another cGMP effector, a PDE. cGK has been shown to phosphorylate the bovine lung cGMPbinding PDE in vitro, but no effect on PDE activity has been demonstrated [209,210]. In contrast, phosphorylation of guinea pig lung cGB-PDE by the catalytic subunit of cAK was reported to increase the Vmax of cGMP hydrolysis [211].

Smooth muscle. In smooth muscle cells (as well as in platelets, discussed below), nitrovasodilators and other cGMP-elevating agents inhibit agonist-induced Ca/+ elevation via cGK [212,213]. Several mechanisms for cGK reduction of cytosolic Ca 2+ in smooth muscle have been described which would either prevent Ca/+ entry or stimulate its efflux across the plasma membrane, or alternatively inhibit its release from the endoplasmic reticulum or pump it back into this intracellular store. These include inhibition of voltage-gated Ca 2+ channels [214], activation of a N a + / C a 2+ exchanger [215] and phosphorylation of a 240-kDa protein regulator of Ca/+ ATPase at the plasma membrane level [216]; or inhibition of inositol phosphate formation important for Ca z+ release [217] and phosphorylation of phospholamban, a regulator of the endoplasmic reticulum Ca 2+ ATPase [218], respectively (for reviews, see Refs. 159,161,219). More recently, reports have shown that NO stimulates Ca2+-activated K ÷ channels in colon smooth muscle, leading to cell hyperpolarization [220]. Nitrovasodilators appeared to act via a similar mechanism to reduce intracellular Ca 2+ and relax arterial muscle [221]. Multiple mechanisms of Ca 2÷ reduction by cGK may exist and vary for smooth muscle cells from different tissue types which have different properties. This and the fact that primary cultures of smooth muscle cells display altered properties, including loss of cGK expression itself [222], may contribute to the complexity of analyzing Ca 2+ regulation by cGK in smooth muscle. In contrast, studies of Ca 2+ regulation in platelets (see next section)

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Fig. 4. Model illustrating the intra- and intercellular signaling between endothelial cells and platelets which involve NO and cGMP. NO may also have effects on endothelial cell soluble guanylyl cyclase and may have non-cGMP mediated effects (not shown). Effects of cAMP and cGMP additional to the ones shown are not excluded. TXA2, thromboxane A2; ACh, acetylcholine, BK, bradykinin; ROC, receptor operated channel; R, receptor; PLC, phospholipase C; AC, adenylyl cyclase; DAG, diacyiglycerol; IP3, inositol trisphosphate; GC-S, guanylyl cyclase; PGI2, prostaglandin I2; PLA2, phosopholipase A2; CaM, calmodulin; NOS, nitric oxide synthase; L-arg, L-arginine.

170 a r e s i m p l i f i e d by the fact that p l a t e l e t s r e p r e s e n t o n e cell t y p e which can be freshly o b t a i n e d for e a c h experiment. B o t h c G K I a a n d /3 have b e e n d e t e c t e d in various s m o o t h m u s c l e cells [162]. S t u d i e s a t t e m p t i n g to c o r r e late a n a l o g p o t e n c i e s for activation of a given isoform with r e l a x a t i o n effects on pig c o r o n a r y a r t e r i e s w e r e inconclusive [178]. B e c a u s e of t h e h i g h e r K a of c G M P for c G K I / 3 , it has b e e n p o s t u l a t e d that the /3 e n z y m e m a y b e e x p r e s s e d w h e n i n t r a c e l l u l a r c G M P levels a r e g r e a t l y i n c r e a s e d by h o r m o n e s or drugs, a n d that t h e

relative expression of a a n d /3 e x p r e s s i o n m a y c h a n g e in d i s e a s e states [223]. F u r t h e r m o r e , it has b e e n sugg e s t e d t h a t a d r e n e r g i c r e l a x a t i o n o f s m o o t h m u s c l e via c A M P results from cross-activation of c G K by c A M P (see Section V-C). E n d o t h e l i a l , e p i t h e l i a l and n e u r o n a l l y d e r i v e d N O ( e n d o t h e l i u m - d e r i v e d relaxing factor [238] b e i n g the prototype) regulate vascular and non-vascular smooth m u s c l e function [230]. N O f o r m a t i o n is g e n e r a l l y m e d i a t e d by t h e C a 2 + - d e p e n d e n t N O S - I o r N O S - I I I in the f o r m e r cells, which i n c r e a s e i n t r a c e [ l u l a r N O in re-

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Fig. 5. Effects of 8-pCPT-cGMP on ADP-stimulated intracellular C a 2+ release in (A) platelets from a normal control person F.F., or in (B) cGK deficient platelets from patient F.B. with chronic myelogenous leukemia. The FURA-2 experiments shown here [151] were performed with 1 mM EGTA in the extracellular medium to exclude ADP-receptor operated Ca 2+ entry into the platelets which is not inhibited by cGMP (see text) [224]. The inset in panel A is a Western blot comparing standard amounts (ng) of purified bovine lung cGK to the amount of cGK in 2.10 7 platelets from the same samples used in A and B for Ca 2+ determinations.

171 sponse to flow or other mechanical stimulation and receptor agonists. Under pathopbysiological conditions (e.g., septic shock) NOS-II can be induced in macrophages, endothelial cells, and smooth muscle cells, resulting in massive local NO formation, inhibition of smooth muscle function, and extreme hypotension. Platelets. Platelet adhesion, aggregation, and secretion are inhibited by both cAMP and cGMP, as well as agents which elevate them. Stimulation of cAK and cGK are closely associated with inhibition of an early step in the human platelet activation cascade, most likely at the level of phospholipase C activation and subsequent mobilization of Ca 2+ from intracellular stores. Studies with Fura-2-1oaded platelets demonstrated that cGMP-elevating nitrovasodilators and cGMP analogs inhibited both calcium mobilization from intracellular stores and the secondary store related Ca 2+ influx in response to either thrombin or ADP, but did not inhibit the ADP receptor operated cation channel [224], schematically shown in Fig. 4. The effect of cGMP was believed to be mediated by cGK and not other effectors, since the 8-pCPT-cGMP analog used was a specific activator of cGK which neither stimulates cGS-PDE nor inhibits cGI-PDE (discussed in Section V-D.1). This interpretation was confirmed by more recent studies with cGK-deficient platelets from chronic myelogenous leukemia (CML) patients. Whereas 8-pCPT-cGMP inhibited the rise in intracellular Ca 2+ in normal activated platelets, it was unable to do so in activated cGK-deficient CML platelets, as shown in Fig. 5 [151]. Furthermore, coincubation of endothelial ceils and platelets demonstrated that the concentration of endothelial derived relaxing factor (EDRF) or NO released from endothelial cells is sufficient to stimulate cGMP-dependent protein phosphorylation [225] and the cGMP response of Ca 2÷ inhibition in platelets [242]. The possible presence of an endogenous NOS pathway in platelets [239] needs further investigation. Thus the entire sequence of intra- and intercellular events illustrated in Fig. 4 can be reconstituted ex vivo with intact cells. The major substrate protein phosphorylated by cGK in intact platelets, and its role in inhibition of platelet activation and intracellular Ca 2+ is under study. This protein of 46-50 kDa, designated vasodilator-stimulated phosphoprotein (VASP), has recently been shown to be an actin-binding protein associated with stress fibers and focal contact sites or adhesion plaques [226]. VASP association with the cytoskeleton suggests two sites at which it could inhibit platelet activation. Thrombin stimulation of platelets causes an increase in the activity of certain enzymes, including phospholipase C, associated with the platelet cytoskeleton [227]. If VASP phosphorylation were able to inhibit this process, it might potentially prevent elevation of intra-

cellular Ca 2+ by phospholipase C. The cytoskeleton also plays an important role in adhesion which precedes platelet aggregation. In addition to phospholipase C stimulation, thrombin also stimulates binding of fibrinogen to its receptor (an integrin, glycoprotein GPanbfl3). This initiates stimulation of a focal adhesion kinase (FAK kinase), c-src and other non-receptor tyrosine kinases which phosphorylate a number of cytoskeletal proteins prior to adhesion [228,229]. The location of VASP with respect to other actin binding proteins in the adhesion plaque (Fig. 3 in Ref. 228) is not yet precisely known, but this, as well as determination of whether VASP phosphorylation can regulate adhesion plaque function, is of great interest.

VI. Concluding remarks The N O / c G M P signaling system is a fascinating example of the ways in which cells combine different elements from an array to construct distinct signaling pathways leading to different responses. The NO system is furthermore designed for both intra- and intercellular regulation and only some of its effects are mediated via cGMP. Whereas selection of a signal transduction pathway may depend in part on the particular cell-specific subset of pathways present, in other cases many pathways may coexist. For example, some cells contain both ANP membrane receptor-guanylyl cyclase and soluble guanylyl cyclase for cGMP synthesis, and many types of cGMP effectors such as cGMP regulated phosphodiesterases, channels and kinases (cAK and cGK). In such cases, use of a certain pathway may depend on additional factors such as intensity of the initial signal, affinity of mediators for various effectors, cell compartmentation, and variable expression levels of individual pathway components. A spectrum of alternative mechanisms is important to enable cells to execute tailored responses to a wide variety of signals, but with perhaps some inbuilt overlap or redundancy of functions so that a defect within a given pathway can be compensated for by overdevelopment of another pathway when necessary. An understanding of many of these facets of the N O / c G M P system and its weak points leading to pathology is lacking but can perhaps now be examined based on the current foundation (see also Ref. 24). A few abnormalities were mentioned here which appear to derive from an unfortuitous defect in a critical component of a certain pathway, however, analysis of certain diseases may require a more rigorous search for an accumulation of defects for which compensation by means of even such a versatile signaling system is not possible.

Acknowledgements The original work performed in the laboratory of the authors was supported by the Deutsche For-

172

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