Regulation of Neuronal Nitric Oxide and Cyclic GMP Formation by Ca2+

Nitric oxide (NO) acts as a messenger molecule in the CNS by activating soluble guanylyl cyclase. Rat brain synaptosomal NO synthase was stimulated by Ca2+ in a concentration-dependent manner with half-maximal effects observed at 0.3 microM and 0.2 microM when its activity was assayed as formation of NO and L-citrulline, respectively. Cyclic GMP formation was apparently inhibited, however, at Ca2+ concentrations required for the activation of NO synthase, indicating a down-regulation of the signal in NO-producing cells. Purified synaptosomal guanylyl cyclase was not inhibited directly by Ca2+, and the effect was not mediated by a protein binding to guanylyl cyclase at low or high Ca2+ concentrations. In cytosolic fractions, the breakdown of cyclic GMP, but not that of cyclic AMP, was highly stimulated by Ca2+, and 3-isobutyl-1-methylxanthine did not block this reaction effectively. The effects of Ca2+ on cyclic GMP hydrolysis and on apparent guanylyl cyclase activities were abolished almost completely in the presence of the calmodulin antagonist calmidazolium, whose effect was attenuated by added calmodulin. Thus, a Ca2+/calmodulin-dependent cyclic GMP phosphodiesterase is highly active in synaptic areas of the brain and may prevent elevations of intracellular cyclic GMP levels in activated, NO-producing neurons.     

Cyclic GMP regulates cromakalim-induced relaxation in the rat aortic smooth muscle: role of cyclic GMP in K(ATP)-channels.

Recent studies have shown that nitric oxide (NO) modulates K+-channel activity which play an important role in controlling vascular tone. The formation of cyclic guanosine 3',5'-monophosphate (cyclic GMP) has also been recognized to be associated with the vasodilatory effect of NO. Both cyclic GMP and NO increase whole-cell K+-current by activating Ca2+-activated K+-channels (K(Ca)-channels). Here, we show evidence that activators of soluble guanylyl cyclase sodium nitroprusside or 3-morpholino-sydnonimine (SIN-1), and an analogue of cyclic GMP 8-bromo-cyclic GMP enhance the relaxation induced by cromakalim which is blocked by glibenclamide (a specific inhibitor of ATP-sensitive K+-channels [K(ATP)-channels]), and partially attenuated by methylene blue (an inhibitor of cyclic GMP formation). However, this is not due to the increase of cyclic GMP level by cromakalim itself because the relaxation induced by cromakalim is not associated with the changes of cyclic GMP level formed in the aortic smooth muscle. Thus, it is most likely that cyclic GMP also modulates activity of K(ATP)-channels, in addition to K(Ca)-channels, in the rat aorta.     

Solubilization of guanylyl cyclase from bovine rod outer segments and effects of lowering Ca2+ and nitro compounds.

Guanylyl cyclase from bovine rod outer segments was solubilized using Triton X-100 and a high concentration of KCl, and its regulation was studied. The efficiency of solubilization was about 50-90% of total activity. When the Ca2+ content was lowered (less than 80 nM), guanylyl cyclase was activated about 2-fold. In the presence of higher concentrations of Ca2+ (greater than 140 nM), the activity was decreased. The regulation by Ca2+ was also demonstrated with solubilized preparations. In the presence of 186 nM Ca2+ which inhibited guanylyl cyclase, La3+ activated the enzyme about 2-fold, suggesting that the Ca2(+)-binding protein similar to other Ca2(+)-binding proteins associates with guanylyl cyclase regulation. Sodium nitroprusside and nitric oxide which are activators of soluble guanylyl cyclase in other tissues also activated the retinal guanylyl cyclase. Maximum activation by sodium nitroprusside was 20-fold using Mg2+ as a cofactor. Activation by nitric oxide and related compounds suggests that retinal guanylyl cyclase contains a heme prosthetic group that may participate in a novel regulatory mechanism for this enzyme.     

Regulation of nitric oxide-responsive recombinant soluble guanylyl cyclase by calcium.

Calcium (Ca2+) and cyclic GMP (cGMP) subserve antagonistic functions that are reflected in their coordinated reciprocal regulation in physiological systems. However, molecular mechanisms by which Ca2+ regulates cGMP-dependent signaling remain incompletely defined. In this study, the inhibition of recombinant nitric oxide (NO)-stimulated soluble guanylyl cyclase (SGC) by Ca2+ was demonstrated. The alpha- and beta-subunits of recombinant rat SGC were heterologously coexpressed in HEK 293 cells which do not express NO synthase, whose Ca2+-stimulated activity can confound the effects of that cation on SGC. Ca2+ inhibited basal and NO-stimulated SGC in a concentration- and guanine nucleotide-dependent fashion. This cation inhibited SGC in crude cell extracts and immunopurified preparations. Ca2+ lowered both the Vmax and Km of SGC via an uncompetitive mechanism through direct interaction with the enzyme. In intact HEK 293 cells, increases in the intracellular Ca2+ concentration induced by ionomycin, a Ca2+ ionophore, and thapsigargin, which releases intracellular stores of that cation, inhibited NO-stimulated intracellular cGMP accumulation. Similarly, carbachol-induced elevation of the intracellular Ca2+ concentration inhibited NO-stimulated intracellular cGMP accumulation in HEK 293 cells. These data demonstrate that SGC behaves as a sensitive Ca2+ detector that may play a central role in coordinating the reciprocal regulation of Ca2+- and cGMP-dependent signaling mechanisms.     

Regulation of synthesis of guanosine 3'':5''-cyclic monophosphate in neuroblastoma cells.

The increase in intracellular cyclic GMP concentrations in response to muscarinic-receptor activation in N1E-115 neuroblastoma cells is dependent on extracellular Ca2+ ion. The calcium ionophore A23187 can also evoke an increase in cyclic GMP in the presence of Ca2+ ion. Most (about 85%) of the guanylate cyclase activity of broken-cell preparations is found in the soluble fraction. The soluble enzyme can utilize MnGTP (Km = 55 micrometer), MgGTP (Km = 310 micrometer) and CaGTP (Km greater than 500 micrometer) as substrates. Free GTP is a strong competitive inhibitor (Ki approximately 20 micrometer). The enzyme possesses an allosteric binding site for free metal ions (Ca2+, Mg2+ and Mn2+). The membrane-bound guanylate cyclase is qualitatively similar to the soluble form, but has lower affinity for the metal-GTP substrates. Entry of Ca2+ into cells may increase cyclic GMP concentration by activating guanylate cyclase through an indirect mechanism.     

Norepinephrine Increases Cyclic GMP Levels in Cerebellar Cells from Neuronal Nitric Oxide Synthase Knockout Mice

Abstract: Cyclic GMP is an important intracellular messenger in the nervous system that may mediate cellular forms of neuronal plasticity. Previous studies show that most neurotransmitters stimulate cyclic GMP levels by the activation of nitric oxide synthase (NOS). In this study, we report that in primary cell cultures from the cerebellum of neuronal NOS knockout mice, norepinephrine stimulates an increase in cyclic GMP content. This increase is seen in both granule cell and astrocyte cultures and is not blocked by inhibitors of NOS or by inhibition of soluble guanylyl cyclase. These results suggest a novel pathway by which norepinephrine enhances cyclic GMP levels in the nervous system.     

A Kainate Receptor Linked to Nitric Oxide Synthesis from Arginine

In slices of young rat cerebellum, the glutamate analogue kainate induced a large accumulation of cyclic GMP, which was inhibited by non-N-methyl-D-aspartate antagonists. Quisqualate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate evoked only small cyclic GMP responses and inhibited the effect of kainate. When tested in cerebellar cell suspensions, glutamate was also a potent antagonist of the cyclic GMP response to kainate. Superoxide dismutase enhanced the response in the isolated cells, whereas haemoglobin and methylene blue were inhibitory. The response in slices was Ca2+ dependent, augmented by arginine, and inhibited by L-NG-monomethylarginine in a manner that could be reversed by additional arginine. It is concluded that stimulation of kainate receptors leads to activation of the enzyme that synthesizes nitric oxide from arginine and that activation of soluble guanylate cyclase by the released nitric oxide accounts for the cyclic GMP generation.     

Antimonial-induced increase in intracellular Ca2+ through non-selective cation channels in the host and the parasite is responsible for apoptosis of intracellular Leishmania donovani amastigotes

The capability of the obligate intracellular parasites like Leishmania donovani to survive within the host cell parasitophorous vacuoles as nonmotile amastigotes determines disease pathogenesis, but the mechanism of elimination of the parasites from these vacuoles are not well understood. By using the anti-leishmanial drug potassium antimony tartrate, we demonstrate that, upon drug exposure, intracellular L. donovani amastigotes undergo apoptotic death characterized by nuclear DNA fragmentation and externalization of phosphatidylserine. Changes upstream of DNA fragmentation included generation of reactive oxygen species like superoxide, nitric oxide, and hydrogen peroxide that were primarily concentrated in the parasitophorous vacuoles. In the presence of antioxidants like N-acetylcysteine or Mn(III) tetrakis(4-benzoic acid)porphyrin chloride, an inhibitor of inducible nitric-oxide synthase, a diminution of reactive oxygen species generation and improvement of amastigote survival were observed, suggesting a close link between drug-induced oxidative stress and amastigote death. Changes downstream to reactive oxygen species increase involved elevation of intracellular Ca2+ concentrations in both the parasite and the host that was preventable by antioxidants. Flufenamic acid, a non-selective cation channel blocker, decreased the elevation of Ca2+ in both the cell types and reduced amastigote death, thus establishing a central role of Ca2+ in intracellular parasite clearance. This influx of Ca2+ was preceded by a fall in the amastigote mitochondrial membrane potential. Therefore, this study projects the importance of flufenamic acid-sensitive non-selective cation channels as important modulators of antimonial efficacy and lends credence to the suggestion that, within the host cell, apoptosis is the preferred mode of death for the parasites.     

Endothelium-derived NO, but not cyclic GMP, is required for hypoxic augmentation in isolated porcine coronary arteries

The present study investigated the mechanism underlying the transient potentiation of vasoconstriction by hypoxia in isolated porcine coronary arteries. Isometric tension was measured in rings with or without endothelium. Hypoxia (Po(2) <30 mmHg) caused a transient further increase in tension (hypoxic augmentation) in contracted (with U46619) preparations. The hypoxic response was endothelium dependent and abolished by inhibitors of nitric oxide synthase [N(ω)-nitro-L-arginine methyl ester (L-NAME)] or soluble guanylyl cyclase (ODQ and NS2028). The addition of DETA NONOate (nitric oxide donor) in the presence of L-NAME restored the hypoxic augmentation, suggesting the involvement of the nitric oxide pathway. However, the same was not observed after incubation with 8-bromo-cyclic GMP, atrial natriuretic peptide, or isoproterenol. Assay of the cyclic GMP content showed no change upon exposure to hypoxia in preparations with and without endothelium. Incubation with protein kinase G and protein kinase A inhibitors did not inhibit the hypoxic augmentation. Thus the hypoxic augmentation is dependent on nitric oxide and soluble guanylyl cyclase but independent of cyclic GMP. The hypoxic augmentation persisted in calcium-free buffer and in the presence of nifedipine, ruling out a role for extracellular calcium influx. Hypoxia did not alter the intracellular calcium concentration, as measured by confocal fluorescence microscopy. This observation and the findings that hypoxic augmentation is enhanced by thapsigargin (sarco/endoplasmic reticulum calcium ATPase inhibitor) and inhibited by HA1077 or Y27632 (Rho kinase inhibitors) demonstrate the involvement of calcium sensitization in the phenomenon.     

Activation of particulate guanylyl cyclase by endothelins in cultured SV-40 transformed cat iris sphincter smooth muscle cells

We investigated the effects of endothelins (ETs) on cGMP production in cultured SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells. ET-3 increased cGMP formation in a concentration-dependent manner (EC50 = 98nM), which was 2.5 times higher than that of ET-1. The ET(B)receptor agonists sarafotoxin-S6c and IRL 1620 also increased cGMP production, mimicking the effects of the ETs. The ET(B) receptor antagonist BQ 788, but not the ET(A) receptor antagonist BQ610, dose-dependently blocked ET-3-stimulated cGMP formation (IC50=10nM). The phorbol ester, Phorbol 12, 13-dibutyrate (PDBu), which inhibits particulate guanylyl cyclase in smooth muscle, dose-dependently inhibited ET-3-stimulated cGMP accumulation (IC50=66nM). LY83583 and ODQ, inhibitors of soluble guanylyl cyclases, as well as inhibitors of the nitric oxide cascade and of intracellular Ca2+ elevation had no appreciable effect on ET-3-induced cGMP production. ET-3 markedly inhibited carbachol-induced intracellular Ca2+ mobilization. We conclude that ET-3 increases intracellular cGMP levels in SV-CISM-2 cells through activation of the ET(B) receptor subtype and subsequent stimulation of the membrane-bound guanylyl cyclase. Elevation of cGMP by ET and the subsequent inhibition of muscarinic stimulation of intracellular Ca2+ mobilization by the cyclic nucleotide could serve to modulate the contractile effects of Ca2+-mobilizing agonists in the iris sphincter smooth muscle.     

Guanylyl cyclases, a growing family of signal-transducing enzymes.

Guanylyl cyclases, which catalyze the formation of the intracellular signal molecule cyclic GMP from GTP, display structural features similar to other signal-transducing enzymes such as protein tyrosine-kinases and protein tyrosine-phosphatases. So far, three isoforms of mammalian membrane-bound guanylyl cyclases (GC-A, GC-B, GC-C), which are stimulated by either natriuretic peptides (GC-A, GC-B) or by the enterotoxin of Escherichia coli (GC-C), have been identified. These proteins belong to the group of receptor-linked enzymes, with different NH2-terminal extracellular receptor domains coupled to a common intracellular catalytic domain. In contrast to the membrane-bound enzymes, the heme-containing soluble guanylyl cyclase is stimulated by NO and NO-containing compounds and consists of two subunits (alpha 1 and beta 1). Both subunits contain the putative catalytic domain, which is conserved in the membrane-bound guanylyl cyclases and is found twice in adenylyl cyclases. Coexpression of the alpha 1- and beta 1-subunit is required to yield a catalytically active enzyme. Recently, another subunit of soluble guanylyl cyclase was identified and designated beta 2, revealing heterogeneity among the subunits of soluble guanylyl cyclase. Thus, different enzyme subunits may be expressed in a tissue-specific manner, leading to the assembly of various heterodimeric enzyme forms. The implications concerning the physiological regulation of soluble guanylyl cyclase are not known, but different mechanisms of soluble enzyme activation may be due to heterogeneity among the subunits of soluble guanylyl cyclase.     

NO., CO and .OH. Endogenous soluble guanylyl cyclase-activating factors.

Several low molecular weight compounds are capable of activating soluble guanylyl cyclase. Recent evidence suggests that some of these are formed under physiological conditions: the nitric oxide radical, carbon monoxide and the hydroxyl radical. Thus, multiple signal transduction pathways appear to exist that form a family of guanylyl cyclase activating factors and thereby regulate the intracellular cyclic guanosine 3',5'-monophosphate level.     

白细胞介素-2对大鼠心肌收缩功能的影响涉及一氧化氮机制

The aim of the present study was to investigate the effect of interleukin-2 (IL-2) on the contractility in cardiomyocytes and the underlying mechanisms. Ventricular myocytes were isolated from adult male Sprague-Dawley rats. Contractile responses were evaluated by use of the video tracking system. Contractile parameters in cardiomyocytes electrically stimulated at 0.2 Hz included peak velocity of cell shortening (+dL/dtmax), peak velocity of cell relengthening (-dL/dtmax), contractile amplitude (dL), and end-diastolic cell length. Calcium transients of ventricular myocytes were determined by the spectrofluorometric techniques. Dose-dependent inhibition in + dL/dtmax, -dL/dtmax, dL and end-diastolic cell length were induced by IL-2 at 2-1000 U/ml. Pretreatment with the nitric oxide synthase inhibitor N(w)-nitro-L-arginine methyl ester (L-NAME, 100 micromol/L) and soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo [4,3a]quinoxalin-1-one (ODQ, 10 micromol/L) attenuated IL-2-induced inhibition of contractility. Aminoguanidine, an inhibitor of inducible nitric oxide synthase, had no effect on the inhibition by IL-2. IL-2 at 200 U/ml decreased the amplitude of electrically induced [Ca2+]i transients of ventricular myocytes. Pretreatment with ODQ diminished IL-2-induced inhibition of amplitude of the calcium transient. In conclusion, the present study indicates a direct action of IL-2 on cardiomyocyte contraction, possibly through an increased NO production, activation of soluble guanylyl cyclase and inhibition in intracellular Ca2+ level.     

Guanylyl cyclase structure, function and regulation

Nitric oxide, bicarbonate, natriuretic peptides (ANP, BNP and CNP), guanylins, uroguanylins and guanylyl cyclase activating proteins (GCAPs) activate a family of enzymes variously called guanyl, guanylyl or guanylate cyclases that catalyze the conversion of guanosine triphosphate to cyclic guanosine monophosphate (cGMP) and pyrophosphate. Intracellular cyclic GMP is a second messenger that modulates: platelet aggregation, neurotransmission, sexual arousal, gut peristalsis, blood pressure, long bone growth, intestinal fluid secretion, lipolysis, phototransduction, cardiac hypertrophy and oocyte maturation. This review briefly discusses the discovery of cGMP and guanylyl cyclases, then nitric oxide, nitric oxide synthase and soluble guanylyl cyclase are described in slightly greater detail. Finally, the structure, function, and regulation of the individual mammalian single membrane-spanning guanylyl cyclases GC-A, GC-B, GC-C, GC-D, GC-E, GC-F and GC-G are described in greatest detail as determined by biochemical, cell biological and gene-deletion studies.     

Inhibition of nitric oxide and soluble guanylyl cyclase signaling affects olfactory neuron activity in the moth, <Emphasis Type="Italic">Manduca sexta</Emphasis>

Nitric oxide is emerging as an important modulator of many physiological processes including olfaction, yet the function of this gas in the processing of olfactory information remains poorly understood. In the antennal lobe of the moth, Manduca sexta, nitric oxide is produced in response to odor stimulation, and many interneurons express soluble guanylyl cyclase, a well-characterized nitric oxide target. We used intracellular recording and staining coupled with pharmacological manipulation of nitric oxide and soluble guanylyl cyclase to test the hypothesis that nitric oxide modulates odor responsiveness in olfactory interneurons through soluble guanylyl cyclase-dependent pathways. Nitric oxide synthase inhibition resulted in pronounced effects on the resting level of firing and the responses to odor stimulation in most interneurons. Effects ranged from bursting to strong attenuation of activity and were often accompanied by membrane depolarization coupled with a change in input resistance. Blocking nitric oxide activation of soluble guanylyl cyclase signaling mimicked the effects of nitric oxide synthase inhibitors in a subset of olfactory neurons, while other cells were differentially affected by this treatment. Together, these results suggest that nitric oxide is required for proper olfactory function, and likely acts through soluble guanylyl cyclase-dependent and -independent mechanisms in different subsets of neurons.     

Control of Trypanosoma cruzi Epimastigote Motility through the Nitric Oxide Pathway

ABSTRACT. Trypanosoma cruzi epimastigote motility can be enhanced by addition of L-arginine, to the culture. This effect is blocked by N-methyl-L-arginine, a competitive inhibitor of the nitric oxide synthase. N-methyl-D-aspartate and L-glutamate, two agonists of the NMDALglutamate receptor, also enhanced motility. This stimulation is blocked by MK-801 a noncompetitive antagonist of the NMDA receptor. In addition, sodium nitroprusside, a guanylyl cyclase stimulator and 8-Br-cyclic GMP, an analog of cyclic GMP, also stimulated epimastigote motility. It is suggested that an increase of intracellular cyclic GMP levels mediated by nitric oxide may be responsible for the increase in epimastigote motility.     

Purification of a Ca2+/calmodulin-dependent nitric oxide synthase from porcine cerebellum. Cofactor-role of tetrahydrobiopterin.

L-Arginine-derived nitric oxide acts as an inter- and intracellular signal molecule with cytosolic guanylyl cyclase as the effector system. Two NO synthase isoenzymes are postulated: a cytokine-inducible enzyme in macrophages and a constitutive, Ca2(+)-regulated enzyme in various other cells. An NO synthase was isolated from porcine cerebellum by ammonium sulfate precipitation and affinity chromatography on 2',5'-ADP-Sepharose. The enzyme was identified as an NO synthase with a specific NO-chemiluminescence method and with purified cytosolic guanylyl cyclase as an NO-sensitive detection system. The purified NO synthase was, besides Ca2+/calmodulin and NADPH, largely dependent on tetrahydrobiopterin as a cofactor.     

Mechanism of stimulation of cyclic-GMP level in a neuronal cell line mediated by serotonin (5-HT3) receptors. Involvement of nitric oxide, arachidonic-acid metabolism and cytosolic Ca2+

The mechanism by which serotonin (5-HT3) receptors mediate a rise in cyclic-GMP level was investigated in a neuronal cell line. Inhibitors of phospholipase A2 (mepacrine) and of lipoxygenase (eicosatetraynoic acid or nordihydroguaiaretic acid) suppressed the action of serotonin. On the other hand, inhibition by hemoglobin indicates a role for nitric oxide which could be in part responsible for the cyclic-GMP effect as an intercellular stimulant. The suppression of the serotonin effect by the arginine analogues N omega-methyl-L-arginine and canavanine is consistent with the notion that nitric oxide could be released from arginine. The serotonin-induced rise of cyclic-GMP level depends on the presence of extracellular Ca2+ with half-maximal stimulation at 0.3 mM Ca2+. The serotonin-stimulated rise of cyclic GMP was inhibited by (a) addition of inorganic blockers of Ca2(+)-permeable channels (La3+, half-maximal inhibitory concentration (IC50) 0.04 mM; Mn2+, IC50, 0.4 mM; Co2+, IC50, 0.9 mM; Ni2+, IC50, 1.2 mM) and (b) of organic blockers (diltiazem: IC50, 6 microM, methoxyverapamil: IC50, 3 microM and (c) intracellular application of the Ca2+ chelator bis-(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (IC50, 2 microM). Thus, two pathways for the activation of soluble guanylate cyclase by serotonin are possible: (a) via lipoxygenase products of arachidonic acid and/or (b) via nitric oxide or a related nitroso compound. Serotonin mediates a rise of cytosolic Ca2+ activity due to entry of extracellular Ca2+. It still has to be investigated which step depends on a rise of cytosolic Ca2+ activity that appears to be a prerequisite for activation of guanylate cyclase.     

Soluble guanylate cyclases in the retina

Soluble guanylate cyclase catalyzes the formation of cyclic GMP using GTP as substrate. It is now well established that soluble guanylate cyclase is highly activated by nitric oxide, and that many of the effects of nitric oxide on various cells and tissues are mediated through increased production of cyclic GMP. This review discusses the evidence for the presence of soluble guanylate cyclases in different classes of cells in vertebrate retina and the role of these enzymes in retinal physiology. It is concluded that the enzyme is present in nearly every class of cells in the retina and that it may be involved in signal transmission between some cells and in the modulation of signal transmission between others.