NO-sensitive Guanylyl Cyclase Research Articles

The β2 subunit of nitric oxide-sensitive guanylyl cyclase is developmentally regulated in rat kidney

We have recently shown that nitric oxide activates the beta2 subunit of soluble guanylyl cyclase. In the present study, we show developmental regulation of this subunit. Analysis of mRNA expression by RT-PCR and RNase protection analysis in kidneys revealed no expression of the beta2 subunit in neonatal and strong expression in adult rats. A reciprocal regulation with much lower expression levels was observed in rat lung. Further examination of kidneys from 3, 6, 16, 22, 25, 31 and 36-day-old rats showed that significant expression appears between postnatal day 16 and 22. Isolation of the rat beta2 promoter by genome walking and cloning into a reporter gene vector showed promoter activity for the sense but not the antisense construct providing an in vitro assay for further analysis of the developmental beta2 subunit regulation.

Guanylyl Cyclase/PSD-95 Interaction: TARGETING OF THE NITRIC OXIDE-SENSITIVE α2β1 GUANYLYL CYCLASE TO SYNAPTIC MEMBRANES

The signaling molecule nitric oxide (NO) exerts most of its effects by the stimulation of the NO-sensitive guanylyl cyclase. Two isoforms of the NO receptor molecule exist: the ubiquitously occurring alpha(1)beta(1) and the alpha(2)beta(1) with a more limited distribution. As the isoforms are functionally indistinguishable, the physiological relevance of these isoforms remained unclear. The neuronal NO synthase has been reported to be associated with PSD-95. Here, we demonstrate the interaction of the so far unnoticed alpha(2)beta(1) isoform with PSD-95 in rat brain as shown by coprecipitation. The interaction is mediated by the alpha(2) C-terminal peptide and the third PDZ domain of PSD-95. As a consequence of the PSD-95 interaction, the so far considered "soluble" alpha(2)beta(1) isoform is recruited to the membrane fraction of synaptosomes, whereas the alpha(1)beta(1) isoform is found in the cytosol. Our results establish the alpha(1)beta(1) as the cytosolic and the alpha(2)beta(1) as the membrane-associated NO-sensitive guanylyl cyclase and suggest the alpha(2)beta(1) isoform as the sensor for the NO formed by the PSD-95-associated neuronal NO synthase.

CGMP-mediated inhibition of cardiac L-type Ca(2+) current by a monoclonal antibody against the M(2) ACh receptor.

The effects of a monoclonal antibody (B8E5) directed against the second extracellular loop of the muscarinic M(2) receptor were studied on the L-type Ca(2+) currents (I(Ca,L)) of guinea pig ventricular myocytes using the whole cell patch-clamp technique. Similar to carbachol, B8E5 reduced the isoproterenol (ISO)-stimulated I(Ca,L) but did not significantly affect basal I(Ca,L). Atropine blocked the inhibitory effect of B8E5. The electrophysiological parameters of ISO-stimulated I(Ca,L) were not modified in presence of B8E5. Inhibition of I(Ca,L) by B8E5 was still observed when intracellular cAMP was either enhanced by forskolin or maintained constant by using a hydrolysis-resistant cAMP analog (8-bromoadenosine 3',5'-cyclic monophosphate) or by applying the phosphodiesterase inhibitor IBMX. The effect of B8E5 was mimicked by 8-bromoguanosine 3',5'-cyclic monophosphate, a potent stimulator of cGMP-dependent protein kinase, and prevented by a selective inhibitor of nitric oxide-sensitive guanylyl cyclase [1H-(1,2,4)oxadiazolo[4,3-a]quinoxaline-1-one]. These results indicate that the antibody B8E5 inhibits the beta-adrenergic-stimulated I(Ca,L) through activation of the M(2) muscarinic receptor and further suggest that the antibody acts not via the classical pathway of decreasing intracellular cAMP, but rather by increasing cGMP.

Nitric Oxide Activates the β2 Subunit of Soluble Guanylyl Cyclase in the Absence of a Second Subunit

Previously characterized mammalian soluble guanylyl cyclases form alpha/beta heterodimers that can be activated by the gaseous messenger, nitric oxide, and the novel guanylyl cyclase modulator YC-1. Four mammalian subunits have been cloned named alpha(1), beta(1), alpha(2), and beta(2). The alpha(1)/beta(1) and alpha(2)/beta(1) heterodimeric enzyme isoforms have been rigorously characterized. The role of the beta(2) subunit has remained elusive. Here we isolate a novel variant of this subunit and show that the beta(2) subunit does not need to form heterodimers for catalytic activity because enzyme activity can be measured when it is expressed alone in Sf9 cells. In analogy to the beta(3) subunit recently isolated from the insect Manduca sexta, activity was dependent on the presence of 4 mm free Mn(2+). The EC(50) values for the NO-donor diethylamine/NO were shifted to the left by 1 order of magnitude as compared with the alpha(1)/beta(1) heterodimeric form. In the presence of the detergent Tween, NO sensitivity of beta(2) was abolished, but the enzyme could be activated by protoporphyrin IX, indicating removal of a prosthetic heme group and exchange for the heme precursor. We suggest that the beta(2) subunit is the first mammalian NO-sensitive guanylyl cyclase lacking a heterodimeric structure.

N-acetyl-L-cysteine exerts direct anti-aggregating effect on human platelets.

N-acetyl-L-cysteine, a thiol compound, has been shown to potentiate the inhibition of platelet aggregation exerted by organic nitrates and to increase the anti-aggregating effect of L-arginine, which promotes endogenous synthesis of nitric oxide (NO) acting as substrate of platelet constitutive nitric oxide synthase (NOS). It is not known whether this thiol can exert direct effects on platelet aggregability. 14 healthy male volunteers provided platelet samples to investigate whether N-acetyl-L-cysteine directly influences platelet function and intraplatelet levels of 3',5' cyclic guanosine monophosphate (cGMP), which represents the second messenger involved in NO-induced antiaggregation. Some experiments were repeated in the presence of NOS inhibitor NG-monomethyl-L-arginine (L-NMMA), of nitric oxide-sensitive guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), of the selective cGMP phosphodiesterase inhibitor zaprinast and of calcium ionophores (A23187, ionomycin). N-acetyl-L-cysteine at 3000-6000 micromol L-1 decreases the responses of human platelets both in platelet-rich plasma (aggregation induced by adenosine 5-diphosphate) and in whole blood (aggregation induced by collagen). The anti-aggregating effect was prevented by preincubation with L-NMMA and guanylyl cyclase inhibitor ODQ. In resting platelets, N-acetyl-L-cysteine increased the levels of cGMP starting from a concentration of 3000 micromol L-1. Permeabilized platelets exhibited an increased sensitivity to the anti-aggregating effect of N-acetyl-L-cysteine. Also, cGMP phosphodiesterase inhibition or the increase in calcium availability, enhanced N-acetyl-L-cysteine effects on platelets. N-acetyl-L-cysteine exerts direct anti-aggregating effects through an increased bioavailability of platelet nitric oxide.

Developmental Changes of Nitric Oxide-Sensitive Guanylyl Cyclase Expression in Pulmonary Arteries

Inhaled nitric oxide (NO) is known to influence the contractile state of pulmonary arteries most likely by activation of soluble guanylyl cyclase (sGC) in smooth muscle cells. However, the cellular distribution of sGC has not been determined empirically, due to a lack of specific antibodies. Here, we describe a novel antibody directed against the β1 subunit of sGC to study the cellular distribution of sGC in lung during development. Using the novel antibody, the enzyme was demonstrated in fetal, neonatal, and adult lungs by Western blot, showing maximum expression in neonatal lung. These data were confirmed by measurements of sGC activity. In pulmonary arteries of fetal lung sGC-β1 immunoreactivity was present in smooth muscle cells and absent in endothelial cells. With postnatal development an increase in immunoreactivity in endothelial cells and a reciprocal decrease in smooth muscle cells was apparent. The reported changes in sGC expression likely contribute to the known age-dependent differences in response to inhaled NO.

RNA: a method to specifically inhibit PCR amplification of known members of a multigene family by degenerate primers.

The polymerase chain reaction (PCR) is a versatile method to amplify specific DNA with oligonucleotide primers. By designing degenerate PCR primers based on amino acid sequences that are highly conserved among all known gene family members, new members of a multigene family can be identified. The inherent weakness of this approach is that the degenerate primers will amplify previously identified, in addition to new, family members. To specifically address this problem, we synthesized a specific RNA for each known family member so that it hybridized to one strand of the template, adjacent to the 3'-end of the primer, allowing the degenerate primer to bind yet preventing extension by DNA polymerase. To test our strategy, we used known members of the soluble, nitric oxide-sensitive guanylyl cyclase family as our templates and degenerate primers that discriminate this family from other guanylyl cyclases. We demonstrate that amplification of known members of this family is effectively and specifically inhibited by the corresponding RNAs, alone or in combination. This robust method can be adapted to any application where multiple PCR products are amplified, as long as the sequence of the desired and the undesired PCR product(s) is sufficiently distinct between the primers.

G protein-mediated inhibitory effect of a nitric oxide donor on the L-type Ca2+ current in rat ventricular myocytes.

1. The role of the cGMP pathway in the modulation of the cardiac L-type Ca2+ current (ICa,L) by nitric oxide (NO) was examined in rat ventricular myocytes. 2. The NO donors DEANO, SIN-1, SNP, SNAP and GSNO had no significant effects on basal ICa,L. However, DEANO (100 microM) inhibited ICa,L after the current had been previously stimulated by either isoprenaline (Iso, 1-10 nM), a beta-adrenergic agonist, or isobutylmethyl-xanthine (IBMX, 10-80 microM), a wide spectrum phosphodiesterase (PDE) inhibitor. 3. The anti-adrenergic effect of DEANO on ICa,L was not mimicked by other NO donors (SIN-1, SNAP and SPNO). 4. The NO-sensitive guanylyl cyclase inhibitor ODQ (10 microM), antagonized the inhibitory effect of DEANO on ICa,L. Likewise, inhibitors of the cGMP-dependent protein kinase (cG-PK), Rp-8-chloro-phenylthio-cGMP (10 microM) and KT5823 (0.1 and 0.3 microM), also abolished the inhibitory effect of DEANO on Iso (1-10 nM)-stimulated ICa,L. 5. Intracellular dialysis with exogenous cAMP (10-100 microM) blunted the inhibitory effect of DEANO (10 and 100 microM) on ICa,L. SNAP and SNP also had no effect on the cAMP-stimulated ICa,L. 6. Pre-treatment of the myocytes with pertussis toxin (0.5 microg ml-1, 4-6 h at 37 degrees C) eliminated the inhibitory effect of DEANO (100 microM) on ICa,L, in the presence of either Iso (0.01 and 1 nM) or IBMX (10-80 microM). 7. These results demonstrate that DEANO produces anti-adrenergic effects in rat ventricular myocytes. This effect of DEANO occurs in a cGMP-dependent manner, and involves activation of cG-PK and regulation of a pertussis toxin-sensitive G protein.

Cloning and functional expression of the rat α 2 subunit of soluble guanylyl cyclase

Nitric oxide-sensitive guanylyl cyclase is a heterodimeric enzyme consisting of one α and one β subunit. Here, we clone the first α 2 subunit ortholog and functionally express the cDNA in Sf-9 cells. Our data indicate a high degree of conservation of the primary sequence and functional activity of the rat α 2 subunit.

Nitric oxide-sensitive guanylyl cyclase activity inhibition through cyclic GMP-dependent dephosphorylation.

The soluble form of guanylyl cyclase (sGC) plays a pivotal role in the transduction of inter- and intracellular signals conveyed by nitric oxide. Here, a feedback inhibitory mechanism triggered by cyclic guanosine-3',5'-monophosphate (cGMP)-dependent protein kinase (PKG) activation is described. Preincubation of chromaffin cells with C-type natriuretic peptide, which increased cGMP levels and activated PKG, or with cGMP-permeant analogue (which also activates PKG), in the presence of a broad-spectrum phosphodiesterase inhibitor, resulted in a decrease in subsequent sodium nitroprusside (SNP)-dependent cGMP elevations. This inhibitory effect was mimicked by activating a protein phosphatase and counteracted by the selective PKG inhibitor KT-5823 and by different protein phosphatase inhibitors. Immunoprecipitation of sGC from cells submitted to different treatments followed by immunodetection with antiphosphoserine antibodies (clone 4A9) showed changes in phosphorylation levels of the beta subunit of sGC, and these changes correlated well with differences in SNP-elicited cGMP accumulations. Pretreatment of cells with several PKG inhibitors or protein phosphatase inhibitors produced an enhancement of SNP-stimulated cGMP rises without changing the SNP concentration required to produce half-maximal or maximal responses. Taken together, these results indicate that the catalytic activity of sGC is closely coupled to the phosphorylation state of its beta subunit and that the tonic activity of PKG or its stimulation regulates sGC activity through dephosphorylation of the beta subunit.

Expression of nitric oxide-sensitive guanylyl cyclase subunits in human corpus cavernosum

The muscles of the corpus cavernosum of the penis relax in response to stimulation of non-adrenergic, non-cholinergic nerves or nitric oxide (NO)-donating drugs to elicit erection. It is generally assumed that NO mediates this effect via activation of soluble guanylyl cyclase and a subsequent increase in cyclic guanosine 3′,5′-monophosphate concentration. However, there are no data on the expression of this enzyme in human corpus cavernosum. The purpose of the present study was the molecular characterization of NO-sensitive guanylyl cyclase in human corpus cavernosum. RNA was extracted from tissue samples obtained from seven patients undergoing penile prosthetic surgery or correction of penile deviation. Reverse transcriptase–polymerase chain reaction (RT–PCR) with specific primers for the subunits of NO-sensitive guanylyl cyclase was performed, and PCR products were subcloned and sequenced. Specific amplification products encoding the α 1, β 1, α 2, and β 2 subunits were detected. In addition, we isolated a transcript encoding a novel variant β 2 subunit. To test whether this novel transcript arises by alternative splicing or whether it is encoded by a separate gene, a 4000-bp clone of the corresponding genomic DNA sequence was isolated. Sequence analysis suggests that the novel β 2 variant arises by alternative splicing from the same gene as the β 2 subunit on chromosome 13. In conclusion, our findings suggest the presence of different subunit mRNAs of NO-sensitive guanylyl cyclase in human corpus cavernosum.

The role of nitric oxide in motoneuron spike activity and muscarinic-evoked changes in cGMP in the CNS of larval Manduca sexta.

Nitric oxide and muscarinic agonists both stimulate motoneuron spike activity and cGMP production in the central nervous system of larval Manduca sexta. The possible role of nitric oxide in mediating muscarinic changes in excitability was examined by measuring cGMP accumulation and proleg motoneuron activity while blocking or mimicking the production of nitric oxide. All the muscarinic-induced changes in cGMP are blocked by the nitric oxide-synthase inhibitor, nitro-l-arginine, an effect that is partially prevented by co-incubation with arginine. Action potential blockage with tetrodotoxin revealed that muscarinic increases in cGMP production have both spike-dependent and spike-independent mechanisms. Furthermore, nitric oxide donors can increase proleg motoneuron activity and this stimulation is blocked by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one suggesting that it is mediated by a nitric oxide-sensitive guanylyl cyclase. In contrast, nitro-l-arginine and a variety of other nitric oxide-synthase inhibitors and nitric oxide scavengers have no significant effect on muscarinic stimulation of motoneuron activity. Therefore, although a nitric oxide sensitive guanylyl cyclase is capable of elevating spike activity and muscarinic agonists can increase cGMP, this mechanism is not necessary for the normal muscarinic increase in excitability. It is concluded that muscarinic receptors are coupled to nitric oxide and cGMP production in neurons other than those controlling the prolegs.

Nitric oxide and cyclic GMP induce vesicle release at Drosophila neuromuscular junction.

Nitric oxide (NO) diffuses as short-lived messenger through the plasma membrane and serves, among many other functions, as an activator of the cGMP synthesizing enzyme soluble guanylyl cyclase (sGC). In view of recent genetic investigations that postulated a retrograde signal from the larval muscle fibers to the presynaptic terminals, we looked for the presence of an NO/cGMP signaling system at the neuromuscular junction (NMJ) of Drosophila melanogaster larvae. Application of NO donors induced cGMP immunoreactivity in the presynaptic terminals but not the postsynaptic muscle fibers at an identified NMJ. The NO-induced cGMP immunoreactivity was sensitive to a specific inhibitor (ODQ) of the sGC. Since presynaptic terminals which were surgically isolated from the central nervous system are capable of synthesizing cGMP, we suggest that an NO-sensitive guanylyl cyclase is present in the terminal arborizations. Using a fluorescent dye that is known to stain recycling synaptic vesicles, we demonstrate that NO donors and membrane permeant cGMP analogues cause vesicle release at the NMJ. Moreover, the NO-induced release could be blocked by the specific inhibitor of the sGC. A destaining of synaptic terminals after NO exposure in Ca2+-free solution in the presence of cobalt chloride as a channel blocker suggested that NO stimulates Ca2+-independent vesicle release at the NMJ. The combined immunocytochemical and exocytosis imaging experiments imply the involvement of cGMP and NO in the regulation of vesicle release at the NMJ of Drosophila larvae.

Estradiol coupling to endothelial nitric oxide stimulates gonadotropin-releasing hormone release from rat median eminence via a membrane receptor.

The median eminence (ME), which is the common termination field for adenohypophysiotropic systems, has been shown to produce nitric oxide (NO), a signaling molecule involved in neuroendocrine secretion. Using an ex vivo technique, 17beta-estradiol exposure to ME fragments, including vascular tissues, stimulated NO release within seconds in a concentration-dependent manner, whereas 17alpha-estradiol or testosterone had no effect. 17Beta-estradiol conjugated to BSA (E2-BSA) also stimulated NO release, suggesting mediation by a membrane surface receptor. Tamoxifen, an estrogen receptor inhibitor, antagonized the action of both 17beta-estradiol and E2-BSA. Furthermore, estradiol-stimulated NO stimulates GnRH release. This was demonstrated by hemoglobin (a NO scavenger), N(omega)-nitro-L-arginine methyl ester, and L-N5-(1-iminoethyl)ornithine (nitric oxide synthase inhibitors) inhibition of estradiol stimulated NO and GnRH release. In this regard, L-N5-(1-iminoethyl)ornithine, specific for endotheliol constitutive nitric oxide synthase, was significantly more potent, suggesting that the estradiol-stimulated NO release arose from vascular endothelial cells. Additionally, the NO-stimulated GnRH release occurs via guanylyl cyclase activation in GnRH nerve terminals, as ODQ, a potent and selective inhibitor of NO-sensitive guanylyl cyclase, abolished the estradiol-stimulated GnRH release. The results suggest that at physiological concentrations, 17beta-estradiol may have immediate actions on ME endothelial cells via nongenomic signaling pathways leading to NO-stimulated GnRH release.

Relaxing action of sodium nitroprusside independent of membrane potential in the CCh-induced contracture of the guinea pig stomach muscle.

Carbachol (CCh, 10(-6) M) induced biphasic contraction of longitudinal muscle of the guinea pig stomach, consisting of rapid phasic contraction and contracture. The contracture was almost completely inhibited by sodium nitroprusside (SNP, 10(-6) M) and S-nitroso-N-acetyl penicillamine (SNAP, 10(-6) M). A membrane permeable analogue of cyclic GMP, 8Br-cGMP (10(-4) M), also inhibited the CCh induced contracture. Although a heme site inhibitor of nitric oxide-sensitive guanylyl cyclase, 1-H-[1, 2, 4] oxadiazolo-[4, 3 a]quinoxalin-1-one (ODQ; 10(-6) M), reduced the inhibitory action of SNP, it did not affect the inhibitory action of 8Br-cGMP, indicating that the effect of SNP was developed via cyclic GMP production in the presence of D600. Charybdotoxin (10(-7) M), an inhibitor of Ca2+ activated K+ channel, did not influence on the CCh induced contracture. On the other hand, CCh induced a depolarization of the longitudinal muscle cell membrane (from -60 mV to -45 mV) in the presence of 10(-6) M D600, but SNP did not affect the depolarization. These results suggest that in the presence of D600 SNP induces relaxation of CCh induced contracture of the longitudinal muscle of the guinea pig stomach via cyclic GMP but not membrane potential dependent mechanism.

Biphasic nature of inotropic action of nitric oxide donor NOC7 in guinea-pig ventricular trabeculae.

The effects of nitric oxide (NO) donor on the contractility of guinea-pig ventricular trabeculae were explored to clarify whether NO affects the function of sarcoplasmic reticulum (SR) and the contractile elements. NO donor, 3-(2-hydroxy-1-methyl-2-nitroso-hydrazino)-N-methyl-1-propanamine (NOC7), increased monotonically the amplitude of the twitch tension induced by electrical stimulation at a concentration of 20 microM. A higher concentration of NOC7 (200 microM) caused a biphasic response: transiently increased the amplitude of twitch and then decreased it. On wash-off of the higher concentration of NOC7, a rebound increase of the twitch amplitude was observed. An inhibitor of NO-sensitive guanylyl cyclase, 1H-[1,2,4]oxadiazolo-[4, 3-a]quinoxalin-1-one (ODQ), abolished the mono-tonic increase and rebound increase in the amplitude of tension but did not affect the decrease in the amplitude of tension at the higher concentration of NOC7. Oscillatory contractions developed by beta-escin-skinned muscle fibers were not changed by NOC7 at either concentration. Caffeine-induced tension transients indicating the Ca(2+)-accumulating and -releasing functions of intracellular Ca(2+) stores were not affected by NOC7. NOC7 did not change the steady tension developed in 1.6 microM Ca(2+) containing solution with and without ODQ. These results suggest that the biphasic inotropic effects by NOC7 were not caused by modifying the function of SR and the Ca(2+) sensitivity of myofilaments of the guinea-pig ventricular trabecula, but at least the positive inotropic effect was mediated through cGMP-dependent mechanisms.

Mechanisms of Action and Targets of Nitric Oxide in the Oculomotor System

Nitric oxide (NO) production by neurons in the prepositus hypoglossi (PH) nucleus is necessary for the normal performance of eye movements in alert animals. In this study, the mechanism(s) of action of NO in the oculomotor system has been investigated. Spontaneous and vestibularly induced eye movements were recorded in alert cats before and after microinjections in the PH nucleus of drugs affecting the NO-cGMP pathway. The cellular sources and targets of NO were also studied by immunohistochemical detection of neuronal NO synthase (NOS) and NO-sensitive guanylyl cyclase, respectively. Injections of NOS inhibitors produced alterations of eye velocity, but not of eye position, for both spontaneous and vestibularly induced eye movements, suggesting that NO produced by PH neurons is involved in the processing of velocity signals but not in the eye position generation. The effect of neuronal NO is probably exerted on a rich cGMP-producing neuropil dorsal to the nitrergic somas in the PH nucleus. On the other hand, local injections of NO donors or 8-Br-cGMP produced alterations of eye velocity during both spontaneous eye movements and vestibulo-ocular reflex (VOR), as well as changes in eye position generation exclusively during spontaneous eye movements. The target of this additional effect of exogenous NO is probably a well defined group of NO-sensitive cGMP-producing neurons located between the PH and the medial vestibular nuclei. These cells could be involved in the generation of eye position signals during spontaneous eye movements but not during the VOR.

Alpha2-adrenoceptor-mediated contractions of the porcine isolated ear artery: evidence for a cyclic AMP-dependent and a cyclic AMP-independent mechanism.

1. The aim of this study was to determine the conditions under which the alpha2-adrenoceptor agonist UK14304 produces vasoconstriction in the porcine isolated ear artery. 2. UK14304 (0.3 microM) produced a small contraction of porcine isolated ear arteries which was 7.8+/-3.3% of the response to 60 mM KCl. Similar sized contractions were obtained after precontraction with either 30 nM angiotensin II, or 0.1 microM U46619 (8.2+/-1.8% and 10.2+/-2.6% of 60 mM KCl response, respectively). However, an enhanced alpha2-adrenoceptor response was uncovered if the tissue was precontracted with U46619, and relaxed back to baseline with 1-2 microM forskolin before the addition of UK14304 (46.9+/-9.6% of 60 mM KCl response). 3. The enhanced responses to UK14304 in the presence of U46619 and forskolin were not inhibited by the alpha1-adrenoceptor antagonist prazosin (0.1 microM), but were inhibited by the alpha2-adrenoceptor antagonist rauwolscine (1 microM), indicating that the enhanced responses were mediated via postjunctional alpha2-adrenoceptors. 4. In the presence of 0.1 microM U46619 and 1 mM isobutylmethylxanthine (IBMX), 1 microM forskolin produced an increase in [3H]-cyclic AMP levels in porcine isolated ear arteries. Addition of 0.3 microM UK14304 prevented this increase. 5. The enhanced UK14304 response was dependent upon the agent used to relax the tissue. After relaxation of ear arteries precontracted with 10 nM U46619 and relaxed with forskolin the UK14304 response was 46.9+/-9.6% of the 60 mM KCl response, and after relaxation with sodium nitroprusside (SNP) the response was 24.8+3.3%. However, after relaxation of the tissue with levcromakalim the UK14304 response was only 8.2+/-1.7%, which was not different from the control response in the same tissues (12.2+/-5.6%). An enhanced contraction was also obtained after relaxation of the tissue with the cyclic AMP analogue dibutyryl cyclic AMP (23.2+/-1.3%) indicating that at least part of the enhanced response to UK14304 is independent of the ability of the agonist to inhibit cyclic AMP production. 6. Relaxation of U46619 contracted ear arteries with SNP could be inhibited by the NO-sensitive guanylyl-cyclase inhibitor 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) indicating that production of cyclic GMP is necessary for the relaxant effect of SNP. However, ODQ had no effect on the relaxation of tissue by forskolin, suggesting that this compound does not act via production of cyclic GMP. Biochemical studies showed that while forskolin increases the levels of cyclic AMP in the tissues, SNP had no effect on the levels of this cyclic nucleotide. 7. In conclusion, enhanced contractions to the alpha2-adrenoceptor agonist UK14304 can be uncovered in porcine isolated ear arteries by precontracting the tissue with U46619, followed by relaxation back to baseline with forskolin, SNP or dibutyryl cyclic AMP before addition of UK14304. There was a greater contractile response to UK14304 after relaxation with forskolin than with SNP or dibutyryl cyclic AMP, suggesting that cyclic AMP-dependent and- independent mechanisms are involved in the enhancement of the UK14304 response.

Nitric oxide-sensitive guanylyl cyclase inhibits acetylcholine release and excitatory motor transmission in the guinea-pig ileum

This study examined the mechanism through which nitric oxide inhibits the release of acetylcholine and excitatory motor neurotransmission in the guinea-pig ileum. The selective inhibitor of nitric oxide-sensitive guanylyl cyclase, 1 H-[1,2,4]oxadiazolo[4,3- a]quinoxalin-1-one (ODQ), concentration-dependently enhanced both basal release (−log EC 50: 6.8) and electrically (10 Hz) -evoked release (−log EC 50: 6.0) of [ 3H]acetylcholine from longitudinal muscle-myenteric plexus preparations preincubated with [ 3H]choline. The increase by ODQ of basal release appeared to be exocytotic since it was prevented by tetrodotoxin (300 nM) and absence of calcium from the superfusion medium. In addition, ODQ (1 μM) increased the electrically-evoked tachykininergic and cholinergic muscle contractions as measured in the presence of scopolamine (100 nM) or of the neurokinin-1 receptor antagonist CP 99994 (100 nM), respectively. The nitric oxide synthase inhibitor l- N G-nitro-arginine (100 μM) behaved similar to ODQ and increased cholinergic and tachykininergic motor neurotransmission. The nitric oxide-independent activator of soluble guanylyl cyclase, 3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole, concentration-dependently inhibited the electrically evoked acetylcholine release (−log EC 50: 6.0) and longitudinal muscle contractions (−log EC 50: 5.7). ODQ (10 μM) antagonized the effects of 3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole. The results suggest that endogenous nitric oxide tonically activates soluble guanylyl cyclase in myenteric neurons which leads to inhibition of the release of the excitatory transmitters acetylcholine and substance P. ODQ prevents the effects of nitric oxide and thus facilitates cholinergic and tachykininergic motor neurotransmission in the guinea-pig ileum.

Role of nitric oxide, cyclic GMP and superoxide in inhibition by adenosine of calcium current in rabbit atrioventricular nodal cells.

To study the intracellular pathways which mediate the inhibitory actions of adenosine on isoprenaline-stimulated calcium current (ICa) in atrioventricular (AV) nodal myocytes. The whole-cell patch-clamp technique was used to record ICa from rabbit AV nodal cells, isolated by enzymatic and mechanical dispersion. Isoprenaline, 0.1 microM, increased peak ICa from 0.58 +/- to 1.23 +/- 0.1 nA, and this increase was reversibly inhibited by adenosine, 10 microM (83 +/- 6%), which we have previously shown to be mediated by nitric oxide (NO) production. A membrane-permeable analogue of cyclic GMP, 8-Br-cGMP (300 microM), an inhibitor of cGMP-stimulated phosphodiesterase, prevented the effect of adenosine on ICa-Methylene blue (10 microM), an inhibitor of NO-sensitive guanylyl cyclase and a generator of superoxide (.02-), did not prevent, but increased, the inhibiting action of adenosine (49.5 +/- 6.6%, P < 0.01). Methylene blue (50 microM) caused a reduction of ICa, with further inhibition when combined with adenosine. A .O(2-)-generating system, xanthine oxidase (0.02 U/ml) and purine (2.3 mM), also increased the inhibitory action of adenosine on ICa. Inhibition of ICa by adenosine in the presence of xanthine oxidase was not prevented by 8-Br-cGMP (300 microM) and was not influenced by pre-incubation of cells with a NO synthase inhibitor, L-NAME (0.5 mM). The inhibitory effect of adenosine on ICa in rabbit AV nodal myocytes can be mediated by two mechanisms--stimulation of cGMP-stimulated phosphodiesterase by NO-induced cGMP, and a mechanism which involves interaction with .O2- production.