Nucleotide, c-di-GMP, c-di-AMP, cGMP, cAMP, (p)ppGpp signaling in bacteria and implications in pathogenesis.

For an organism to survive, it must be able to sense its environment and regulate physiological processes accordingly. Understanding how bacteria integrate signals from various environmental factors and quorum sensing autoinducers to regulate the metabolism of various nucleotide second messengers c-di-GMP, c-di-AMP, cGMP, cAMP and ppGpp, which control several key processes required for adaptation is key for efforts to develop agents to curb bacterial infections. In this review, we provide an update of nucleotide signaling in bacteria and show how these signals intersect or integrate to regulate the bacterial phenotype. The intracellular concentrations of nucleotide second messengers in bacteria are regulated by synthases and phosphodiesterases and a significant number of these metabolism enzymes had been biochemically characterized but it is only in the last few years that the effector proteins and RNA riboswitches, which regulate bacterial physiology upon binding to nucleotides, have been identified and characterized by biochemical and structural methods. C-di-GMP, in particular, has attracted immense interest because it is found in many bacteria and regulate both biofilm formation and virulence factors production. In this review, we discuss how the activities of various c-di-GMP effector proteins and riboswitches are modulated upon c-di-GMP binding. Using V. cholerae, E. coli and B. subtilis as models, we discuss how both environmental factors and quorum sensing autoinducers regulate the metabolism and/or processing of nucleotide second messengers. The chemical syntheses of the various nucleotide second messengers and the use of analogs thereof as antibiofilm or immune modulators are also discussed.

Second messenger regulation of biofilm formation: breakthroughs in understanding c-di-GMP effector systems.

The second messenger bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) has emerged as a broadly conserved intracellular signaling molecule. This soluble molecule is important for controlling biofilm formation, adhesion, motility, virulence, and cell morphogenesis in diverse bacterial species. But how is the typical bacterial cell able to coordinate the actions of upward of 50 proteins involved in synthesizing, degrading, and binding c-di-GMP? Understanding the specificity of c-di-GMP signaling in the context of so many enzymes involved in making, breaking, and binding the second messenger will be possible only through mechanistic studies of its output systems. Here we discuss three newly characterized c-di-GMP effector systems that are best understood in terms of molecular and structural detail. As they are conserved across many bacterial species, they likely will serve as central paradigms for c-di-GMP output systems and contribute to our understanding of how bacteria control critical aspects of their biology.

The nitric oxide-guanosine 3',5'-cyclic monophosphate pathway regulates dopamine efflux in the medial preoptic area and copulation in male rats.

Dopamine in the medial preoptic area (MPOA) plays a significant role in regulation of male copulation. One mediator of the MPOA dopamine level is nitric oxide. In the current study, we investigated the role of the nitric oxide-guanosine 3',5'-cyclic monophosphate (cGMP) pathway in the regulation of MPOA dopamine and copulation in male rats. The reverse-dialysis of a membrane-permeable analog, 8-Br-cGMP, increased, while a soluble guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ), significantly reduced basal dopamine and its metabolite levels. ODQ successfully blocked a nitric oxide donor-induced increase in dopamine levels, while a neuronal nitric oxide synthase (nNOS) inhibitor was ineffective in blocking an 8-Br-cGMP-induced increase in dopamine, indicating that cGMP is "downstream" of nitric oxide. Furthermore, 8-Br-cGMP facilitated, while ODQ inhibited copulation. Given the steroid-sensitive nature of nNOS functions and the multiple roles nitric oxide plays in the MPOA, we propose that nitric oxide provides important integration of various neurochemical and neuroendocrine signals. The involvement of the central nitric oxide-cGMP pathway in the regulation of copulation also raises an interesting therapeutic possibility, as the manipulation of the same pathway in peripheral tissue is already utilized in treatment of male sexual dysfunction.

Guanylic nucleotide starvation affects Saccharomyces cerevisiae mother-daughter separation and may be a signal for entry into quiescence.

BACKGROUND: Guanylic nucleotides are both macromolecules constituents and crucial regulators for a variety of cellular processes. Therefore, their intracellular concentration must be strictly controlled. Consistently both yeast and mammalian cells tightly correlate the transcription of genes encoding enzymes critical for guanylic nucleotides biosynthesis with the proliferation state of the cell population. RESULTS: To gain insight into the molecular relationships connecting intracellular guanylic nucleotide levels and cellular proliferation, we have studied the consequences of guanylic nucleotide limitation on Saccharomyces cerevisiae cell cycle progression. We first utilized mycophenolic acid, an immunosuppressive drug that specifically inhibits inosine monophosphate dehydrogenase, the enzyme catalyzing the first committed step in de novo GMP biosynthesis. To approach this system physiologically, we next developed yeast mutants for which the intracellular guanylic nucleotide pools can be modulated through changes of growth conditions. In both the pharmacological and genetic approaches, we found that guanylic nucleotide limitation generated a mother-daughter separation defect, characterized by cells with two unseparated daughters. We then showed that this separation defect resulted from cell wall perturbations but not from impaired cytokinesis. Importantly, cells with similar separation defects were found in a wild type untreated yeast population entering quiescence upon nutrient limitation. CONCLUSION: Our results demonstrate that guanylic nucleotide limitation slows budding yeast cell cycle progression, with a severe pause in telophase. At the cellular level, guanylic nucleotide limitation causes the emergence of cells with two unseparated daughters. By fluorescence and electron microscopy, we demonstrate that this phenotype arises from defects in cell wall partition between mother and daughter cells. Because cells with two unseparated daughters are also observed in a wild type population entering quiescence, our results reinforce the hypothesis that guanylic nucleotide intracellular pools contribute to a signal regulating both cell proliferation and entry into quiescence.

Cyclic guanosine monophosphate role in human carcinoma pathogenesis.

In order to examine the cyclic nucleotides (cGMP) role in carcinoma growth and invasivity. We analyzed two cell lines, LSHT29 and 17GT, and tissues in patients with carcinoma and malignant tissues with (N+) and without (N-) lymph node metastases. Higher cGMP levels in pathological samples suggest a strong correlation between intracellular cGMP concentration and carcinoma progression.

Nitric oxide reduces blood pressure in the nucleus tractus solitarius: a real time electrochemical study.

Increasing evidence has demonstrated that nitric oxide (NO) is involved in central cardiovascular regulation. In this study, we directly measured extracellular NO levels, in real-time, in the nucleus tractus solitarius (NTS) of anesthetized cats using Nafion/Porphyrine/o-Phenylenediamine-coated NO sensors. We found that local application of L-arginine (L-Arg) induced NO overflow in NTS and hypotension. These responses were potentiated in the vagotomized animals. Pretreatment with NO synthase (NOS)/guanylate cyclase inhibitor methylene blue, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one or NO scavenger hemoglobin attenuated L-Arg-induced hypotension, suggesting that exogenous supplement of NO suppressed cardiac functions through the NOS/cyclic guanosine monophosphate mechanism. The role of endogenous NO was examined after local application of N(G)-nitro-L-arginine methyl ester (L-NAME). We found that L-NAME suppressed endogenous NO levels in NTS and elicited hypertension and tachycardia. Taken together, our data suggest that NO is tonically released in the NTS to inhibit blood pressure.

Adenosine inhibits thrombin-induced expression of tissue factor on endothelial cells by a nitric oxide-mediated mechanism.

Enhanced expression of tissue factor (TF) is associated with the occurrence of coronary disease, strokes and arterial thrombosis. We demonstrated previously that adenosine inhibits TF expression in human umbilical vein endothelial cells (HUVECs) stimulated with inflammatory mediators. In the present study, we evaluated the mechanism of adenosine-induced inhibition of TF expression in HUVECs. The adenosine inhibitory activity on thrombin-induced TF expression in HUVECs was potentiated by the NO precursor, l-arginine, but it was significantly suppressed by the NO scavenger, 2(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide, and by endothelial NO synthase inhibitors, N(G)-monomethyl-l-arginine and N(G)-nitro-l-arginine methyl ester, in a dose-dependent manner. The concentrations of nitrites, cGMP and cAMP in the culture medium of HUVECs treated with a mixture of thrombin and adenosine were significantly higher compared with the culture medium of HUVECs treated with thrombin alone. Northern blotting showed that thrombin decreases and adenosine increases the eNOS mRNA expression in HUVECs. A cAMP-dependent protein kinase inhibitor suppressed NO-mediated TF inhibition in a dose-dependent manner. Overall, these results suggest that adenosine inhibits thrombin-induced TF expression in endothelial cells by a NO-mediated mechanism, and that increased intracellular formation of cAMP is implicated in this inhibitory activity of NO.

Structural basis for nucleotide-dependent regulation of membrane-associated guanylate kinase-like domains.

CASK is a member of the membrane-associated guanylate kinases (MAGUK) homologs, a family of proteins that scaffold protein complexes at particular regions of the plasma membrane by utilizing multiple protein-binding domains. The GK domain of MAGUKs, which shares high similarity in amino acid sequence with yeast guanylate kinase (yGMPK), is the least characterized MAGUK domain both in structure and function. In addition to its scaffolding function, the GK domain of hCASK has been shown to be involved in transcription regulation. Here we report the crystal structure of the GK domain of human CASK (hCASK-GK) at 1.3-A resolution. The structure rationalizes the inability of the GK domain to catalyze phosphoryl transfer and strongly supports its new function as a protein-binding module. Comparison of the hCASK-GK structure with the available crystal structures of yGMPK provides insight into possible conformational changes that occur in hCASK upon GMP binding. These conformational changes may act to regulate hCASK-GK function in a nucleotide-dependent manner.

[Physiology and pharmacology of lacrymal and salivary secretions]. / Physiologie et pharmacologie des sécrétions lacrymale et salivaire.

Salivary and lacrymal glands have secretory mechanisms similar to those of other exocrine glands. Saliva results from two different but integrated processes i.e. hydroelectrolytic transport and protein secretion by regulated exocytosis. Both cellular processes are regulated by the autonomic nervous system with complementary effects without antagonism, and parasympathetic innervation predominates. Signal transduction mechanisms in salivary cells include: increases in cytosolic calcium, cyclic AMP and cyclic GMP. The tear film consists of three layers: mucous inner layer, middle aqueous layer, and outer lipid layer. Each layer secretion is strongly regulated. Aqueous layer secretion is controlled by autonomic nervous system and signal transduction depends from cyclic AMP and intracellular calcium levels. A review of drugs used in France modulating lacrymal and salivary secretions is proposed.

[Pharmacological profiles of mycophenolate mofetil (CellCept), a new immunosuppressive agent].

Mycophenolate mofetil (MMF, CellCept), a semisynthetic derivative of mycophenolic acid (MPA) produced by a fungus, is an inhibitor of the inosine monophosphate dehydrogenase (IMPDH) enzyme (IC50 = 25 nM) that catalyzes the synthesis of guanosine monophosphate (GMP) from inosine. GMP is an essential nucleoside for purine synthesis during cell division. As T and B-lymphocytes almost exclusively use the de novo pathway of purine synthesis, these cells are particularly sensitive to the inhibitory action of MMF. It has a mechanism of action distinct from cyclosporine and tacrolimus. Although MMF does not affect cytokine production, by inhibiting the rate-limiting enzyme IMPDH in the de novo synthesis of purines, it inhibits the proliferation of T and B-lymphocytes, the production of antibodies, and the generation of cytotoxic T lymphocytes. Reversal of acute allograft rejection and increased survival of kidney, heart and bone marrow cell allograft has been shown in several animal studies. Moreover, it was suggested that MMF combined with CsA prevented the acute rejection, and approximately half of the animals became long-term survivors. The Ministry of Health and Welfare approved MMF in 1999 for use for rejection treatment in renal transplantation based on several prospective, randomized and blind efficacy trials.

Renovascular actions of adenosine in the isolated perfused rat kidney: possible underlying mechanisms.

We studied the renovascular action of adenosine on isolated perfused rat 10 min after drug injections. Adenosine was applied intraarterially as a single bolus injection in logarithmically increasing doses (0.3-30 microg). Adenosine treatment induced a biphasic vascular-response, namely, an initial vasoconstriction followed by a long-lasting vasodilation. Pretreatment with 0.1. 0.3, or 1.0 mM theophylline or quinidine (2 microg/ml) significantly depressed both components of the adenosine response. The vasoconstrictor response to adenosine was not affected by either 0.5 or 1.0 microg/ml dihydroergocristine. whereas the vasodilatory response was dose-dependently reduced. The biphasic response to adenosine was markedly depressed by 10 microg/ml indomethacin and was augmented by combining this agent with quinidine. We studied the possible roles of the platelet activating factor (PAF) and nitric oxide-cGMP systems in the renovascular actions of adenosine. Tebokan (a PAF antagonist) antagonized both components of the response, but methylene blue (MM) reduced only the pressory part Electron-microscopic examination of kidneys exposed for 15 min to MM showed some acute degenerative alterations and constriction in the glomeruli. From these findings, we conclude that the P1/A1, and P2x purinoceptors, the prostaglandins, PAF, and the NO-cGMP systems have a share in the renovascular actions of adenosine.

Reactive oxygen and nitrogen intermediates increase transforming growth factor-beta1 release from human epithelial alveolar cells through two different mechanisms.

Transforming growth factor (TGF)-beta1 is a growth factor involved in the mechanisms of lung repair and fibrosis that follow inflammatory processes. We sought to examine the link between the generation of reactive oxygen intermediates (ROI) or reactive nitrogen intermediates (RNI) by inflammatory cells and the expression of TGF-beta1 by alveolar epithelial cells. Exposure of the A549 lung epithelial cell line to either an ROI generating system (xanthine and xanthine oxidase) or an RNI donor (S-nitroso-N-acetyl-penicillamine [SNAP]) promoted a time- and dose-dependent increase in TGF-beta1 release, as measured by a specific enzyme-linked immunosorbent assay. At the peak, the levels of TGF-beta1 were twice the control values. The induction of TGF-beta1 release by ROI was blunted by catalase and unaffected by superoxide dismutase, indicating the involvement of hydrogen peroxide. The response was also blunted by 5, 6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB), a specific RNA polymerase II inhibitor, and accompanied by a corresponding increase in TGF-beta1 messenger RNA, as measured by quantitative/competitive reverse transcription polymerase chain reaction, suggesting the involvement of transcriptional mechanisms and possibly other downstream mechanisms. In contrast, RNI-induced TGF-beta1 release was unaffected by DRB and blunted by the protein synthesis inhibitor cycloheximide, suggesting the involvement of translational and post-translational mechanisms. This response required cyclic guanosine monophosphate (cGMP)- mediated processes because (1) immunoreactive cGMP accumulated in the culture medium of SNAP-treated cells; (2) SNAP-induced TGF-beta1 release was blunted by KT 5823, an inhibitor of cGMP-dependent protein kinase; and (3) similar increase in TGF-beta1 release was obtained by cell exposure to membrane-permeable dibutyryl-cGMP or to atrial natriuretic factor, a known agonist of particulate guanylate cyclase. These data suggest that in vitro exposure of human alveolar epithelial cells to ROI and RNI enhances TGF-beta1 release through different mechanisms. In vivo, this control may constitute a molecular link between inflammatory and fibrotic processes.

L-arginine modulates aggregation and intracellular cyclic 3,5-guanosine monophosphate levels in human platelets: direct effect and interplay with antioxidative thiol agent.

Platelet nitric oxide is involved in the control of aggregability via cyclic 3',5'-guanosine monophosphate synthesis. Since L-arginine provides a guanidino nitrogen group for nitric oxide synthesis through nitric oxide synthase activity, we tried to clarify whether an increased availability of this amino acid can directly modulate the response of human platelets. In our conditions, L-arginine (at 100-6000 micromol/L) was able to influence the response of human platelets stimulated with adenosine 5-diphosphate and collagen both in PRP and in whole blood. The anti-aggregating effect was not present when D-arginine was used. Permeabilized platelets exhibited an increased sensitivity to L-arginine. Also, an increased availability of Ca2+ enhanced L-arginine effect. L-arginine (at 120-500 micromol/L) increased cyclic 3',5'-guanosine monophosphate levels in resting platelets; the amino acid also determined an increase of cyclic 3',5'-guanosine monophosphate in platelets at the end of adenosine 5-diphosphate-induced aggregation. Nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine prevented L-arginine effects on aggregation and cyclic 3',5'-guanosine monophosphate synthesis. Phosphodiesterase III inhibitor milrinone and antioxidative thiol N-acetyl-L-cysteine enhanced the effect of L-arginine on cyclic 3',5'-guanosine monophosphate. In conclusion, L-arginine exerts inhibitory effects on human platelet response through a nitric oxide-dependent synthesis of cyclic 3',5'-guanosine monophosphate. A positive interplay on platelet response between L-arginine and milrinone or antioxidative thiol N-acetyl-L-cysteine was evidenced.

Guanine nucleotides, including GMP, antagonize kainate responses in Xenopus oocytes injected with chick cerebellar membranes.

Injection of chick cerebellar membranes, rich in kainate binding sites, into Xenopus oocytes resulted in the structural integration of chick membrane patches into the oocyte plasma membrane that could be easily identified by specific immunofluorescent staining. Application of kainate to the oocyte perfusion medium, under voltage-clamp conditions, induced dose-dependent (EC50 = 87+/-14 microM) inward currents, confirming the functional incorporation to the oocyte of kainate-driven channels. Responses to kainate were consistently nondesensitizing and strongly potentiated by cyclothiazide, suggesting the selective involvement of alpha-amino-3-hydroxy-5-methyl-4isoxazolepropionate (AMPA)-preferring receptors. Binding experiments with (S)-[3H]AMPA confirmed the presence in the chick membrane preparation of low-affinity AMPA receptors (K(D) = 278 nM) amounting to <2% of the total population of kainate binding sites. A tenfold concentration of guanine nucleotides, with different degrees of phosphorylation, blocked the responses to 100 microM kainate by approximately 90%. In the case of GMP, additional concentration-inhibition studies yielded an IC50 of 180+/-11 microM. Our results illustrate the apparent failure of kainate-binding proteins to form functional channels, even when maintaining their own native membrane environment, and confirm the antagonistic behavior of guanine nucleotides, including GMP, toward glutamate receptors, in agreement with previous results of ligand-binding experiments and, more interestingly, with the marked neuroprotective effects of some guanine nucleotides in different excitotoxicity experimental paradigms.

Dual effect of nitric oxide on the hyperpolarization-activated inward current (I(f)) in sino-atrial node cells of the rabbit.

Using the whole-cell voltage-clamp technique, we have investigated the effect of nitric oxide (NO) donor (sodium nitroprusside, SNP) on hyperpolarization-activated inward current, I(f), in isolated rabbit sinoatrial node (SAN) cells. I(f) in the basal state increased when NO was applied but decreased when I(f) was pre-stimulated by isoproterenol (ISO) or by adding cAMP to the pipette solution. Both the stimulatory and the inhibitory effects of NO were abolished by guanylyl cyclase inhibitor, methylene blue (MB), suggesting that the effect of NO is mediated by cGMP. The inhibitory effect of NO was abolished when I(f) was pre-stimulated by 3-isobutyl-1-methylxanthine (IBMX), which is a phosphodiesterase (PDE) inhibitor, or by adding 8Br-cAMP (which is resistant to PDE) to the pipette solution. An analogue of cGMP, 8Br-cGMP, which is a potent stimulator of cGMP-dependent protein kinase (PKG) but has little effect on PDE, did not inhibit I(f) when I(f) was pre-stimulated by ISO. In its basal state, I(f) was still increased by 8Br-cGMP, and this effect was not prevented by the pretreatment with H-7, PKG inhibitor. The effect of acetylcholine (ACh) was not identical to that of NO: I(f) decreased when pre-stimulated not only by ISO, but also by IBMX. The above results suggest that via cGMP, NO exerts a dual effect on I(f): the inhibitory effect is mediated by cGMP-stimulated PDE, and the stimulatory effect may be attributable to direct binding of cGMP to I(f) channels.

Introductory remarks on umami taste.

Psychophysical and electrophysiological studies indicated that the umami substances have no enhancing activity on other primary tastes. Experiments using amiloride clearly show that the umami component of canine chorda tympani nerve response to umami substances is independent of the salt component. Single fiber analysis of the responses of the mouse glossopharyngeal nerve and the monkey primary taste cortex neuron show that the responses to umami substances are independent of other primary tastes. A large synergism between monosodium glutamate (MSG) and disodium 5'-inosinate (IMP) or disodium 5'-guanylate (GMP) is observed in dogs and is explained in terms of allosteric effect. The order of intensity of umami taste induced by a mixture of 0.5 mM GMP and 1.5 mM of various agonists for the glutamate receptors was glutamate > ibotenate > DL(+)-2-amino-4-phosphonobutyric acid (DL-AP4)-(+)-1- aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD). Kainate, N-methyl-D-aspartate (NMDA) and (RS)--amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), which are agonists for ionotropic receptors, have no umami taste. It was concluded that the umami receptor is not identical to any of known glutamate receptors, and there seems to be a unique receptor for umami.

Identification of IL-6 as one of the important cytokines responsible for the ability of mononuclear cells to stimulate Sertoli cell functions.

There is increasing evidence that locally produced cytokines may play an important role in the control of testicular function. In a previous report we demonstrated that medium conditioned by activated human peripheral blood mononuclear cells (PBMC-CM), which is a rich source of cytokines, has extremely potent effects on Sertoli cell transferrin and cGMP secretion. Part of this activity could be explained by interleukin-1beta (IL-1beta) but additional cytokines were evidently involved. In the present study we tried to characterize and purify additional components active on Sertoli cells from PBMC-CM. To this end PBMC-CM was subjected to a purification procedure involving successively: adsorption to silicic acid, affinity chromatography with an antiserum recognizing a mixture of cytokines except IL-1beta, gel-filtration, reversed-phase HPLC and cation-exchange FPLC. Throughout this protocol a Sertoli cell bioassay was used to monitor the effects on transferrin and cGMP production. After cation-exchange FPLC, SDS-PAGE using silver staining showed a single protein band in the bioactive fractions. NH2-terminal amino-acid sequencing revealed that the active principle(s) in this band corresponded to four truncated forms of IL-6 missing the first 13, 14, 17 and 18 N-terminal amino-acids, respectively. The truncated IL-6 molecules were as active as intact IL-6 in the Sertoli cell bioassay. Since neither IL-1beta nor IL-6 alone or in combination could account for the extremely potent effect of PBMC-CM, we tested a series of additional cytokines (IL-1alpha, INF-alpha, IL-4, TGF-beta, IFN-gamma) alone and in combination with IL-1beta and IL-6. These data suggest that IL-1beta, IL-6 and TNF-alpha display more than additive effects on Sertoli cell transferrin and cGMP secretion and that the combination of these cytokines may explain the major part of the effects observed with crude PBMC-CM. The observation that the latter effects could be observed with murine as well as human IL-1beta, IL-6 and TNF-alpha further supports the potential physiological relevance of these findings.

Neuropeptide Y in human hand veins: pharmacologic characterization and interaction with cyclic guanosine monophosphate-dependent venodilators in vivo.

The dorsal hand vein compliance technique was used to study direct vascular effects of human neuropeptide Y in vivo. Human neuropeptide Y is an endogenous vasoconstrictor peptide that is costored with norepinephrine in sympathetic nerve endings and coreleased with the catecholamine under various physiologic and pathologic conditions. Compared with the alpha 1-adrenergic agonist phenylephrine (geometric mean dose-rate that produces the half-maximal response [ED50]: 1.05 nmol/min; maximum venoconstriction [Emax] +/- SEM, expressed as a percentage of baseline compliance: 91% +/- 3%), human neuropeptide Y was nine times more potent (geometric mean ED50: 0.122 nmol/min; p < 0.001) but markedly less efficacious (Emax: 58% +/- 4%; n = 12; p < 0.001). Venoconstrictor effects of human neuropeptide Y lasted several hours and were unchanged by simultaneous administration of alpha-adrenergic antagonists but were readily reversed by nitroglycerin or bradykinin. The high responsiveness of subcutaneous veins to human neuropeptide Y indicates that human neuropeptide Y may regulate venous compliance and filling of the venous subcutaneous capacitance bed in vivo.

The ineffectiveness of the NO-cyclic GMP signaling pathway in the atrial myocardium.

1. This study was performed to determine whether nitric oxide (NO) has direct effects on force of contraction (Fc) in atrial myocardium from rats, rabbits, guinea-pigs, frogs, and man. 2. Glyceryl trinitrate, isosorbide dinitrate, 3-morpholino-sydnonimine hydrochloride (SIN-1), and S-nitroso-N-acetylpenicillamine (SNAP) did not significantly reduce Fc in the various preparations investigated, either given alone or after stimulation of alpha- or beta-adrenoceptors. 3. SNAP did not change the time course of contractions in rat, guinea-pig and human preparations. 4. 8-Bromo-guanosine-3':5'-cyclic monophosphate (8-Br-cyclic GMP) produced a negative inotropic effect in rat, guinea-pig and human atrial preparations and shortened time to peak tension and relaxation time in human preparations. 5. High K+ (85 mmol l-1)-induced contracture in rat heart muscle was reduced by 8-Br-cyclic GMP but not by SIN-1. 6. N-monomethyl-L-arginine (L-NMMA), an inhibitor of NO synthase, failed to influence muscarinic effects on Fc or frequency from rat and guinea-pig hearts. 7. We conclude that NO, under the experimental conditions described here, has no direct effects on the heart, although cyclic GMP may be involved in the regulation of myocardial contraction.