Membrane-permeable Cyclic GMP Research Articles

NOS/sGC/cGK1 pathway in cardiac-specific differentiation of mouse embryonic stem cells

The influence of nitric oxide synthetase (NOS)/soluble guanylyl cyclase (sGC)/cGMP-dependent protein kinase I (cGKI) pathway in adult cardiac performances is well defined. Indeed, physiological amounts of NO generated by NOS1 and NOS3 or pharmacological administration of NO-donor drugs can influence cardiac contractility and increase coronary blood flow throughout the Ser/Thr phosphorylation (sGC/cGKI pathway) and directly by S-nitrosylation of several proteins. Although the role of NOS/sGC/cGKI is already extensively studied in adult cardiac cells, the influence of this pathway as trigger/modulator of cardiac differentiation of embryonic stem cells (ESCs) is less defined. Therefore, we decided to investigate the influence of NOS/sGC/cGKI in the early stage of cardiac differentiation of mouse embryonic cells, studying i) enzyme expressions and activities during the cardiac maturation and ii) the acute and chronic effect of pathway alteration. The mouse embryonic cell line CGR8 was used for these experiments: cells were cultured in undifferentiated form using LIF. Hanging drop methodology was used to promote CGR8 cardiac differentiation. This method allowed cells to aggregate and eventually form embryoid bodies (EBs). The cardiac maturation process was followed for 21 days. Beating EBs were monitored starting from 7-10 day. At different stages of cardiac maturation, mNos3, mGuCy1b and mPrkgI expressions were detected by real-time PCR (Q-PCR); western blot analysis and enzymatic activity were used for protein evaluation. Cytosolic calcium dynamics was evaluated using epifluorescence microscopy in spontaneous beating EBs from 13-16 day of differentiation. Real-time PCR showed that during differentiation the expression of the enzymes increased in a time-dependent mode and different time-courses. The peak of mNos3 expression was measured at day 8 of differentiation and then rapidly decreased. mGuCy1b and mPrkgI were detected starting from day 5 and increased until day 14, maintaining similar values until the end of observation period. Protein expressions were in line with RNA messenger measures, with detectable level of NOS3 only at the beginning of differentiation (days 5 and 8). Enzymatic activities were tested at day 15: sGC activity was increased of 3 times with the NO-donor SNAP and inhibited by 50% with ODQ. Moreover, cyclic GMP was reduced when EBs were incubated for 30 min with L-NMA, suggesting an endogenous activity of NOS. The phosphorylation of Ser/Thr residues on target proteins, were increased by membrane permeable cyclic GMP, isosorbide-5-mononitrate and SNAP and reduced by KT5823, a selective cGKI inhibitor. Functionally, cell incubation with isosorbide-5-mononitrate from the beginning of differentiation slightly increased the percentage of beating EBs at early stages of cardiac maturation (days 5 and 8). In spontaneous beating EBs, acute block of NOS endogenous activity with L-NMA (100 μmol/L) caused a 50% increase of calcium transient frequency. The effect developed progressively during 10 minute exposure and remained constant thereafter. Additionally, a 30-40% reduction of calcium transient amplitude was observed upon NOS blockade, associated with a slight speed of calcium transient decay velocity. The time-dependent expression of NOS/sGC/cGKI during cardiac differentiation suggests a potential role of these pathways to trigger cardiomyogenesis and modulate calcium handling properties in the developing cardiomyocytes. Further functional data will clarify its role in cardiac maturation.

A novel adipocytokine, nesfatin-1 modulates peripheral arterial contractility and blood pressure in rats

Nesfatin-1 is a novel adipocytokine which exerts not only anorexigenic but also hypertensive roles through acting on hypothalamus melanocortin-3/4 receptors. Although it is logical to hypothesize that nesfatin-1 could also affect the contractile reactivity of peripheral blood vessels, it still remains to be examined. The present study was performed to test the hypothesis. In both endothelium-intact and -denuded mesenteric artery of rats, acute treatment with nesfatin-1 (10nM, 30min pretreatment) had no influence on the noradrenaline- and 5-hydroxytryptamine-induced concentration-dependent contractions. Chronic treatment of mesenteric artery with nesfatin-1 (10nM, 3days) using organ-culture method had also no influence on the agonists-induced contractions. In contrast, nesfatin-1 (10nM, 30min) significantly inhibited the sodium nitroprusside (SNP)-induced relaxations of smooth muscle in mesenteric artery. A membrane permeable cyclic GMP (cGMP) analog, 8-bromo-cGMP-induced relaxations were not affected by nesfatin-1. Consistently, the SNP-induced cGMP production in smooth muscle was impaired by nesfatin-1. Intravenous application of nesfatin-1 to rats not only increased blood pressure but also impaired the SNP-induced decreases in blood pressure. The present study for the first time reveals that nesfatin-1 affects peripheral arterial blood vessel and inhibits the nitric oxide donor-induced smooth muscle relaxations via impairing the cGMP production. The results are the first to demonstrate that nesfatin-1 modulates blood pressure through directly acting on peripheral arterial resistance.

An Unsuspected Property of Natriuretic Peptides

Background— Although natriuretic peptides are considered cardioprotective, clinical heart failure trials with recombinant brain natriuretic peptide (nesiritide) failed to prove it. Unsuspected proadrenergic effects might oppose the anticipated benefits of natriuretic peptides. Methods and Results— We investigated whether natriuretic peptides induce catecholamine release in isolated hearts, sympathetic nerve endings (cardiac synaptosomes), and PC12 cells bearing a sympathetic neuron phenotype. Perfusion of isolated guinea pig hearts with brain natriuretic peptide elicited a 3-fold increase in norepinephrine release, which doubled in ischemia/reperfusion conditions. Brain natriuretic peptide and atrial natriuretic peptide also released norepinephrine from cardiac synaptosomes and dopamine from nerve growth factor–differentiated PC12 cells in a concentration-dependent manner. These catecholamine-releasing effects were associated with an increase in intracellular calcium and abolished by blockade of calcium channels and calcium transients, demonstrating a calcium-dependent exocytotic process. Activation of the guanylyl cyclase-cyclic GMP-protein-kinase-G system with nitroprusside or membrane-permeant cyclic GMP analogs mimicked the proexocytotic effect of natriuretic peptides, an action associated with an increase in intracellular cyclic AMP (cAMP) and protein-kinase-A activity. Cyclic AMP enhancement resulted from an inhibition of phosphodiesterase type 3–induced cAMP hydrolysis. Collectively, these findings indicate that, by inhibiting phosphodiesterase type 3, natriuretic peptides sequentially enhance intracellular cAMP levels, protein kinase A activity, intracellular calcium, and catecholamine exocytosis. Conclusions— Our results show that natriuretic peptides, at concentrations likely to be reached at cardiac sympathetic nerve endings in advanced congestive heart failure, promote norepinephrine release via a protein kinase G–induced inhibition of phosphodiesterase type 3–mediated cAMP hydrolysis. We propose that this proadrenergic action may counteract the beneficial cardiac and hemodynamic effects of natriuretic peptides and thus explain the ineffectiveness of nesiritide as a cardiac failure medication.

Nitric Oxide Modulation of GABAergic Synaptic Transmission in Mechanically Isolated Rat Auditory Cortical Neurons

The auditory cortex (A1) encodes the acquired significance of sound for the perception and interpretation of sound. Nitric oxide (NO) is a gas molecule with free radical properties that functions as a transmitter molecule and can alter neural activity without direct synaptic connections. We used whole-cell recordings under voltage clamp to investigate the effect of NO on spontaneous GABAergic synaptic transmission in mechanically isolated rat auditory cortical neurons preserving functional presynaptic nerve terminals. GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) in the A1 were completely blocked by bicuculline. The NO donor, S-nitroso-N-acetylpenicillamine (SNAP), reduced the GABAergic sIPSC frequency without affecting the mean current amplitude. The SNAP-induced inhibition of sIPSC frequency was mimicked by 8-bromoguanosine cyclic 3',5'-monophosphate, a membrane permeable cyclic-GMP analogue, and blocked by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, a specific NO scavenger. Blockade of presynaptic K(+) channels by 4-aminopyridine, a K(+) channel blocker, increased the frequencies of GABAergic sIPSCs, but did not affect the inhibitory effects of SNAP. However, blocking of presynaptic Ca(2+) channels by Cd(2+), a general voltage-dependent Ca(2+) channel blocker, decreased the frequencies of GABAergic sIPSCs, and blocked SNAP-induced reduction of sIPSC frequency. These findings suggest that NO inhibits spontaneous GABA release by activation of cGMP-dependent signaling and inhibition of presynaptic Ca(2+) channels in the presynaptic nerve terminals of A1 neurons.

Chronic treatment with 17β-estradiol increases susceptibility of smooth muscle cells to nitric oxide

The purpose of this study was to evaluate the role of estrogen as a vasodilator or relaxing modulator during vascular tonus through chronic estrogen treatment. Experiments were conducted using isolated basilar arteries from ovariectomized female rabbits divided into two groups (the with and without estrogen replacement groups, respectively). Both acetylcholine and carbachol relaxed the basilar arteries of rabbits in the with estrogen replacement group (pre-contracted by 30 mM K+) more strongly than in the without estrogen replacement group. Vasodilatation effects of (±)-(E)-4-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3 -hexenamide (NOR1) and S-nitroso-N-acetyl-penicillamine (SNAP) were greater in rabbits in the with estrogen replacement group than the without estrogen replacement both with endothelium-intact and denuded preparations. On the other hand, vasodilatation effects of nicardipine, 17β-estradiol and membrane-permeable cyclic-GMP or cyclic-AMP were the same in both groups. These results suggest that chronic administration of estradiol potentiates reactivity to nitric oxide (NO) in smooth muscle cells, which could be a therapeutic target for cardiovascular diseases in postmenopausal women.

C-Type Natriuretic Peptide Down-Regulates Expression of Its Cognate Receptor in Rat Aortic Smooth Muscle Cells

The C-type natriuretic (CNP) peptide signals through the type B natriuretic peptide receptor (NPR-B) in vascular smooth muscle cells to activate the particulate guanylyl cyclase activity intrinsic to that receptor and raise cellular cyclic GMP levels. In the present study, we demonstrate that CNP down-regulates the expression of this receptor leading to a reduction in NPR-B activity. Pretreatment of rat aortic smooth muscle cells with CNP reduces NPR-B activity, NPR-B protein levels, NPR2 (NPR-B gene) mRNA levels, and NPR2 promoter activity. The decrease in NPR2 promoter activity is dependent on DNA sequence present between -441 and -134 relative to the transcription start site. The reduction in NPR2 gene expression appears to operate through generation of cyclic GMP. 8-Bromo cyclic GMP, a membrane-permeable cyclic GMP analog, reduced NPR2 mRNA levels and NPR2 promoter activity. Atrial natriuretic peptide, which signals through the type A natriuretic peptide receptor (NPR-A) to increase cyclic GMP levels in these cells, also reduced NPR-B mRNA levels and inhibited NPR-B promoter activity; however, this inhibition was not additive with that produced by CNP, implying that the two ligands traffic over a common signal transduction pathway. This report provides the first documentation that CNP is capable of autoregulating the expression of its cognate receptor.

Up-regulation of Na +-dependent Mg 2+ transport by nitric oxide and cyclic GMP pathway in renal epithelial cells

A putative, Na +-dependent Mg 2+ transport pathway controls the intracellular free Mg 2+ concentration ([Mg 2+] i) in various mammalian cells. The characteristics of this Mg 2+ transport pathway have not been clarified. Herein, we examined the regulatory mechanism of Na +-dependent Mg 2+ efflux in renal epithelial NRK-52E cells. Mg 2+ removal from the extracellular bathing solution induced an Na +-dependent [Mg 2+] i decrease in Mg 2+ (5 mM)-loaded cells but not in control cells. Amiloride inhibited the [Mg 2+] i decrease in a dose-dependent manner (IC 50=3 μM). Similarly, atomic absorption spectrophotometry showed that Mg 2+ removal decreased intracellular Mg 2+ content, while it increased Na + content. Calphostin C (1 μM), a protein kinase C inhibitor, and genistein, a tyrosine kinase inhibitor (10 μM), blocked the [Mg 2+] i decrease. The [Mg 2+] i decrease was accompanied by an increase in intracellular nitric oxide (NO) and cyclic GMP contents. ( E)-4-methyl-2-[( E)-hydoxyimino]-5-nitro-6-methoxy-3-hexenamide (0.1 mM), an NO donor, and 8-bromo-cyclic GMP (0.1 mM), a membrane-permeable cyclic GMP analogue, accelerated the [Mg 2+] i decrease. In contrast, N G-monomethyl- l-arginine ( l-NMMA, 0.1 mM), an NO competitive inhibitor, and 1 H-[1,2,4]oxadiazolo[4,3- a]quinoxaline-1-one (ODQ, 10 μM), an NO-sensitive guanylate cyclase inhibitor, significantly blocked the [Mg 2+] i decrease. These results indicate that a decrease in extracellular Mg 2+ concentration induces the production of NO and cyclic GMP, which leads to the up-regulation of Na +-dependent Mg 2+ efflux.

Functional contribution of voltage-dependent and Ca2+ activated K+ (BK(Ca)) channels to the relaxation of guinea-pig aorta in response to natriuretic peptides.

We examined the relaxant effects of natriuretic peptide family on the isolated guinea-pig aorta to determine the receptor subtype which primarily mediates this vascular relaxation, with particular attention to the apparent contribution of voltage-dependent and Ca2+-activated KS (BK(Ca)) channels to the response. Three endogenous natriuretic peptide ligands (natriuretic peptide, ANP; brain natriuretic peptide, BNP; C-type natriuretic peptide, CNP) produced a concentration-dependent relaxation in de-endothelialized guinea-pig aorta pre-contracted by noradrenaline (NA), with a potency order of ANP > or = BNP >> CNP. Although the relaxations elicited by these three natriuretic peptide ligands were significantly diminished by iberiotoxin (IbTx, 10(-7) M), a selective BK(Ca) channel blocker, the inhibitory effect of IbTx was most pronounced for the CNP-induced relaxation; when estimated at 10(-7) M of each peptide, the apparent extent of BK(Ca) channel contribution to the total relaxant response was approximately 60% for CNP > approximately 20% for either ANP or BNP. Supporting the substantial role of BK(Ca) channels in the vascular responses, high-KCl (80 mM) potently suppressed the relaxations induced by these natriuretic peptide ligands. The relaxant response to 8-Bromo-cyclic GMP, a membrane permeable cyclic GMP analogue, was also diminished by IbTx (10(-7) M) and high-KCl (80 mM), which indicates the key role of cyclic GMP in the BK(Ca) channel-mediated, natriuretic peptide-elicited vascular relaxation. These results indicate that the A-type receptor (NPR-A, which is more selective for ANP and BNP) rather than the B-type receptor (NPR-B, which is more selective for CNP) predominates in the guinea-pig aorta as the natriuretic peptide receptor which mediates this vascular smooth muscle relaxation. Although activation of BK(Ca) channels substantially contributes to both NPR-A- and NPR-B-activated relaxations, particularly in the NPR-B-activated relaxation, this K channel may function as a primary relaxant mediator in this conduit artery.

C-type natriuretic peptide has a negative inotropic effect on cardiac myocytes

C-type natriuretic peptide (CNP) has vasodilatory and antimitogenic actions, but its role in the control of cardiac function is unclear. We studied the effect of CNP on cultured, beating neonatal rat cardiac myocytes. CNP caused a significant reduction in the amplitude of contraction and a significant accumulation of intracellular cyclic GMP. The effect of a membrane permeable cyclic GMP on cell contraction was similar to that of CNP. CNP caused no change in Ca 2+ transients. Blockade of natriuretic peptide receptors abolished the effects of CNP on contraction and accumulation of intracellular cyclic GMP. Blockade of cyclic GMP-dependent protein kinase abolished the effect of CNP on myocyte contraction. We conclude that CNP has a negative inotropic effect on neonatal rat cardiac myocytes. The effect of CNP is mediated via natriuretic peptide receptor(s) causing elevation of intracellular cyclic GMP which possibly activates protein kinase and causes attenuation of myofilament sensitivity to Ca 2+.

Nitric Oxide Inhibits the Tumor Necrosis Factor α-regulated Endocytosis of Human Dendritic Cells in a Cyclic GMP-dependent Way

Tumor necrosis factor-alpha (TNFalpha)-induced maturation of dendritic cells (DC), with down-regulation of their endocytic ability, has been reported to be mediated by the accumulation of the lipid messenger ceramide. We have now studied the effects and mechanisms of action of NO on endocytosis, investigated with fluorescein isothiocyanate-labeled dextran using human monocyte-derived DC, both immature and after treatment with TNFalpha. Exposure of DC to NO, released by either bystander phagocytes or NO donors, reversed the inhibition of endocytosis induced by TNFalpha. The intracellular accumulation of ceramide induced by TNFalpha was also inhibited by NO. In addition, NO was found to exert an inhibitory effect downstream of the TNFalpha-triggered ceramide accumulation, because NO donors reversed the inhibition of endocytosis induced by the cell-permeant C(2)-ceramide. These effects of NO were mimicked by the membrane-permeant cyclic GMP analogue, 8-Br cyclic GMP, and prevented by inhibition of the soluble guanylyl cyclase. At variance with rodents, the inducible isoform of the NO synthase was expressed neither in immature human DC nor after cell treatment with TNFalpha, interferon-gamma, and lipopolysaccharide, suggesting that regulation of these cells depends on exogenous NO. NO, working through cyclic GMP, might therefore prolong the ability of human DC to internalize antigens at the site of inflammation and thus modulate the initial steps leading to antigen-specific immune responses.

Nitric Oxide Inhibits Inducible Nitric Oxide Synthase mRNA Expression in RAW 264.7 Macrophages

Using cultured murine RAW 264.7 macrophages, the present study investigates the influence of nitric oxide (NO) on the expression of the inducible NO synthase (iNOS) enzyme at the transcriptional level. Incubation of cells with lipopolysaccharide (LPS) and interferon-γ (IFN-γ) led to a marked increase in iNOS mRNA levels. Inhibition of LPS/IFN-γ-induced NO synthesis with the L-arginine analogue NG-monomethyl-L-arginine (L-NMMA) was accompanied by a significant up-regulation of iNOS mRNA that was reversed in the presence of the NO donor sodium nitroprusside (SNP). Treatment of cells with SNP alone decreased LPS/IFN-γ-induced iNOS mRNA levels in a concentration-dependent manner. The inhibitory effect of SNP on iNOS mRNA expression was not prevented by 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ), a selective inhibitor of the soluble guanylyl cyclase. In agreement with this finding, incubation of cells with the membrane-permeable cyclic GMP analogue 8-bromo cyclic GMP left LPS/IFN-γ-induced iNOS mRNA expression virtually unaltered. Together, our results demonstrate that both iNOS-derived and exogenous NO exert an inhibitory effect on the expression of iNOS by a mechanism independent of the soluble guanylyl cyclase/cyclic GMP pathway. In conclusion, NO may control the extent of iNOS mRNA expression by a negative autoregulatory feedback.

Second messengers mediate increases in cytosolic calcium in tobacco protoplasts

Addition of membrane-permeable cyclic GMP (cGMP) and cyclic AMP (cAMP) were shown to cause elevation of cytosolic Ca2+ concentration ([Ca2+]cyt) in tobacco (Nicotiana plumbaginofolia) protoplasts. Under the same conditions these cyclic nucleotides were shown to provoke a physiological swelling response in the protoplasts. Nonmembrane-permeable cAMP and cGMP were unable to trigger a detectable [Ca2+]cyt response. Cyclic-nucleotide-mediated elevations in [Ca2+]cyt involved both internal and external Ca2+ stores. Both cAMP- and cGMP-mediated [Ca2+]cyt elevations could be inhibited by the Ca2+-channel blocker verapamil. Addition of inhibitors of phosphodiesterases (isobutylmethylxanthine and zaprinast) and the adenylate cyclase agonist forskolin to the protoplasts (predicted to elevate in vivo cyclic-nucleotide concentrations) caused elevations in [Ca2+]cyt. Addition of the adenylate cyclase inhibitor 2', 5'-dideoxyadenosine before forskolin significantly inhibited the forskolin-induced [Ca2+]cyt elevation. Taken together, these data suggest that a potential communication point for cross-talk between signal transduction pathways using cyclic nucleotides in plants is at the level of Ca2+ signaling.

Nitric oxide depolarizes Type II paraventricular nucleus neurons in vitro

Nitric oxide is a labile gas which has been implicated in neuronal signalling. The enzyme responsible for the production of this molecule is present in the paraventricular nucleus of the hypothalamus, yet a specific role for nitric oxide in neurotransmission within this nucleus remains unclear. Using whole-cell patch-clamp recordings from paraventricular nucleus neurons in a coronal hypothalamic slice, we have assessed the acute effects of nitric oxide on membrane potential and ionic conductance. Recordings were obtained from 78 neurons with a mean resting membrane potential of −57.8±0.6 mV and a mean input resistance of 972±46 M Ω. Cells were electrophysiologically classified into Type I or Type II according to previously established criteria. Bath application of nitric oxide (delivered either as a gas dissolved in solution, or liberated from the donor compound, N-acetyl- S-nitroso- d-penicillamine) elicited reversible membrane depolarizations (>3 mV) in 14 of the 19 Type II cells tested. These cells also exhibited a decrease in input resistance following nitric oxide application. Similar effects were observed in response to bath application of l-arginine, with 11 of 14 cells displaying depolarizations and accompanying decreases in input resistance. Inhibition of nitric oxide synthase abolished the responses to l-arginine ( n=2). The nitric oxide effects persisted when voltage-activated Na + channels were blocked by tetrodotoxin ( n=6). The depolarizations observed in Type II cells were mimicked by bath application of a membrane permeable cyclic GMP analogue (8-bromo-cyclic GMP) ( n=8). Furthermore, nitric oxide depolarizations were abolished by pre-treatment of the slice with the guanylate cyclase inhibitor, LY83583 ( n=4). Type I cells did not depolarize in response to nitric oxide ( n=11). It is concluded that nitric oxide specifically depolarizes parvocellular neurons within the paraventricular nucleus via a mechanism that requires activation of guanylate cyclase and subsequent production of cyclic GMP. These findings provide the first insight into the cellular mechanisms underlying the acute effects of nitric oxide on neurons in the paraventricular nucleus.

Nitric oxide and excitatory postsynaptic currents in immature rat sympathetic preganglionic neurons in vitro

Neuronal nitric oxide synthase immunoreactivity was localized to sympathetic preganglionic neurons of the intermediolateral cell column and cyclic GMP immunoreactivity to nerve fibers projecting into the intermediolateral cell column of 20–25-day-old rats. Whole-cell patch-clamp recordings were made from sympathetic preganglionic neurons in spinal cord slices of immature rats and the role of nitric oxide and cyclic GMP on excitatory postsynaptic currents was studied. Superfusing the slices with the nitric oxide precursor l-arginine (300 μM) increased the amplitude of evoked excitatory postsynaptic currents as well as the frequency of spontaneous miniature excitatory postsynaptic currents in some neurons from minutes to over 1 h. The nitric oxide synthase inhibitor N W-nitro- l-arginine (100 μM) and the nitric oxide scavenger hemoglobin (100 μM) antagonized the potentiating effect of l-arginine. The nitric oxide donor sodium nitroprusside (100 μM) potentiated the synaptic currents in a manner similar to that of l-arginine and this effect was blocked by hemoglobin. The membrane-permeable cyclic GMP analogue dibutyryl guanosine 3′,5′-cyclic monophosphate (350 μM), in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (750 μM), potentiated the evoked excitatory postsynaptic currents and increased the frequency of miniature excitatory postsynaptic currents; these effects were not prevented by hemoglobin. The results indicate that nitric oxide may facilitate the release of excitatory transmitters, possibly through a presynaptic cyclic GMP-dependent mechanism.

Evidence for some signal transduction elements involved in UV-light-dependent responses in parsley protoplasts

The signalling pathways used by UV-light are largely unknown. Using protoplasts from a heterotrophic parsley (Petroselinum crispum L.) cell culture that exclusively respond to UV-B light between 300 and 350 nm with a fast induction of genes encoding flavonoid biosynthetic enzymes, information was obtained about the UV-light signal transduction pathway for chalcone synthase (CHS) and phenylalanine ammonia-lyase (PAL) gene expression. Pharmacological effectors which influence intracellular calcium levels, calmodulin and the activity of serine/ threonine kinases also changed the UV-light-dependent expression of these genes. This evaluation indicated the participation of these components on the UV-B-mediated signal transduction cascade to CHS. In contrast, neither membrane-permeable cyclic GMP nor the tyrosine kinase inhibitor genistein affected CHS or PAL expression. Similar results were obtained in protoplasts, which have been transiently transformed with CHS-promoter/GUS β-glucuronidase) reporter fusion constructs. The involvement of calcium and calmodulin was further indicated in a cell-free light-responsive in vitro transcription system from evacuolated parsley protoplasts. In conclusion, there is evidence now that components of the UV-light-dependent pathway leading to the CHS-promoter are different from the previously characterized cGMP-dependent pathway to CHS utilized by phytochrome in soybean (Glycine max) and tomato seedlings (Lycopersicon esculentum).

Nitric oxide regulates substance P release from rat spinal cord synaptosomes.

In order to determine whether nitric oxide (NO) acts directly upon nerve terminals to regulate the synaptic transmission at the level of spinal cord, effects of NO-donors on release of substance P (SP) and glutamic acid (Glu) were investigated by superfusion of synaptosomes prepared from the rat spinal cord. Basal levels of endogenous SP and Glu release were 5.99 +/- 2.50 fmol/min/mg of protein and 26.2 +/- 4.8 pmol/min/mg of protein, respectively. Exposure to a depolarizing concentration of KCI evoked 2.7- and 3.8-fold increases in SP and Glu release in a calcium-dependent manner, respectively. Sodium nitroprusside (NP) caused a reduction in the depolarization-evoked overflow of SP in a concentration-dependent manner without affecting its basal release, although it failed to affect either basal or evoked release of Glu. The reduction in SP overflow was also observed by the perfusion with S-nitroso-N-acetyl-penicillamine or membrane-permeable cyclic GMP, but not with cyclic AMP. NP caused the concentration-dependent increases in cyclic GMP levels in synaptosomes. Together with reports that excitatory amino acids stimulate NO synthase and release NO in the spinal cord, these data suggest that there may be an interaction between nerve terminals containing Glu and SP, and that NO may directly participate in the regulation of synaptic transmission in SP-containing nerve terminals, which may be mediated through the activation of guanylate cyclase and the increase in cyclic GMP levels.

Nucleotide receptor-site on the labellar sugar receptor cell of the blowfly Phormia regina

Previously we reported that, among nucleotides applied extracellularly to the chemosensillum of the blowfly Phormia regina, a membrane-permeable cyclic GMP analogue, dibutyryl cyclic GMP, was particularly effective, inducing much impulse-discharge. From the results obtained, we discussed the possibility that a nucleotide site exists on the sugar receptor cell. Here we report results of our electrophysiological experiments to investigate this putative nucleotide receptor-site. As previously reported, when dibutyryl cyclic GMP was applied with sucrose, the impulse-discharge was depressed. Cyclic GMP or another membrane-permeable cyclic GMP analogue, 8-bromo cyclic GMP, enhanced the impulse frequency when applied to a chemosensillum with sucrose. However, 8-bromo cyclic GMP partly depressed the impulse-discharge evoked by dibutyryl cyclic GMP. If we hypothesize that the sugar receptor-site has a nucleotide receptor-site separated from the receptor site for sucrose (P-site) on the membrane and that the cell uses cyclic GMP as an intrinsic intracellular messenger mediating the taste transduction, then, these results can be explained as follows. Dibutyryl cyclic GMP inhibits the P-site with its butyryl groups. On the other hand, it binds the nucleotide receptor-site and stimulates the cell like other nucleotides. However, 8-bromo cyclic GMP, which does not affect the P-site, binds the nucleotide receptor-site like a competitive inhibitor. Thus, the impulses induced by 8-bromo cyclic GMP are due to the 8-bromo cyclic GMP acting in place of cyclic GMP as the intracellular messenger. As for the responses induced by dibutyryl cyclic GMP, a part which can be reduced by 8-bromo cyclic GMP is due to the dibutyryl cyclic GMP binding the nucleotide receptor-site, and other part is due to the dibutyryl cyclic GMP acting as the intracellular messenger.

Nicorandil reduces the basal level of cytosolic free calcium in single guinea pig ventricular myocytes.

Using fluorescent Ca2+ indicator fura-2 and whole-cell patch-clamp techniques, we examined the effect of 2-nicotinamidoethyl nitrate (nicorandil) on the intracellular free Ca2+ concentration ([Ca2+]i) and electrical properties in single guinea pig ventricular myocytes. Nicorandil (10 nM approximately 1 mM) reduced the resting level [Ca2+]i monitored as fura-2 fluorescence ratio in a concentration-dependent manner. Dibutyryl guanosine 3':5'-cyclic monophosphate (cyclic GMP), a membrane permeable cyclic GMP analogue, mimicked the nicorandil action. Neither application of caffeine (10 mM) nor deprivation of extracellular Na+ ions could prevent the nicorandil action on [Ca2+]i. In contrast, the nicorandil effect was virtually blocked by sodium orthovanadate (40 microM), a Ca2+ pumping ATPase inhibitor. During electrophysiological experiments, nicorandil shortened action potential durations (205 +/- 80 ms to 153 +/- 76 ms) by increasing a glibenclamide-sensitive outward K+ conductance. However, the drug produced little hyperpolarization (approximately 2 mV) because the resting potential of ventricular myocytes was close to the K+ equilibrium potential. The involvement of voltage-dependent Ca-channel current and Na-Ca exchanger was considered to be minimal under physiological conditions. It is thus concluded that nicorandil decreases basal [Ca2+]i via cyclic GMP-mediated activation of the plasma membrane Ca2+ pump in guinea pig ventricular myocytes.

Cyclic GMP inhibits protein kinase C-mediated secretion in rat pancreatic acini.

Dibutyryl cyclic GMP (Bu2cGMP) inhibited agonist-induced secretion of amylase from isolated rat pancreatic acini. In contrast to previous studies, this inhibitory action was not confined to butyryl derivatives of cyclic GMP, since the membrane-permeant cyclic GMP analogues Bu2cGMP and cyclic 8-bromo-GMP (8-Br-cGMP) were equipotent (IC50 2 nM) in their inhibition of amylase secretion stimulated by cholecystokinin-(26-33)-octapeptide (CCK8): at extracellular concentrations up to 1 mM, cyclic GMP itself was devoid of inhibitory activity. Both Bu2cGMP and 8-Br-cGMP also potently inhibited secretion stimulated by 4 beta-phorbol 12-myristate 13-acetate (PMA) (IC50 6 nM), but only partially inhibited responses elicited by bethanechol or bombesin and were without effect on A23187-evoked secretion. Furthermore, agents that are known to raise intracellular cyclic GMP levels (MB22948 (2-o-propoxyphenyl-8-azapurin-6-one) or nitroprusside) or antagonize the actions of protein kinase C (4 alpha-PMA or staurosporine), also inhibited CCK8- or PMA-stimulated secretion but not secretion elicited by bombesin, bethanechol, or A23187. It is concluded from these and other observations reported here that protein kinase C is the major intracellular mediator of amylase secretion stimulated by CCK8 and that this pathway may be regulated by cyclic GMP at a step that follows protein kinase C activation.

Prevention of natural motoneurone cell death by dibutyryl cyclic GMP.

Natural neuronal cell death is a well-described developmental phenomenon common to many nerve centres in a variety of animal species. Neuronal survival has been shown to depend on the presence and size of the available target tissue and it has been suggested that neuronal survival is dependent on successful competition for either a limited number of synaptic sites or a limited amount of trophic factor(s). In the lateral motor column of the lumbar spinal cord in the chick embryo, the period of axon elongation and innervation of the periphery has been shown to precede that of natural motoneurone cell death. While muscle contractile activity appears to regulate the extent of motoneurone death, to date the intracellular molecular events that initiate and regulate the developmental process of natural neuronal cell death or, more importantly, neuronal survival are unknown. Our earlier studies suggested that either contact or association between spinal cord processes and muscle cells during neuromuscular junction formation in vivo leads to an increase in cyclic GMP in whole spinal cord. We now show that treatment of chick embryos with the membrane-permeable cyclic GMP analogue, dibutyryl cyclic GMP during the period of natural motoneurone cell death prevents greater than 58% of natural motoneurone cell death in the lumbar lateral motor column.