Inhibitor Of Guanylate Cyclase Activation Research Articles

The peripheral L-arginine–nitric oxide–cyclic GMP pathway and ATP-sensitive K+ channels are involved in the antinociceptive effect of crotalphine on neuropathic pain in rats

Crotalphine, a 14 amino acid peptide first isolated from the venom of the South American rattlesnake Crotalus durissus terrificus, induces a peripheral long-lasting and opioid receptor-mediated antinociceptive effect in a rat model of neuropathic pain induced by chronic constriction of the sciatic nerve. In the present study, we further characterized the molecular mechanisms involved in this effect, determining the type of opioid receptor responsible for this effect and the involvement of the nitric oxide-cyclic GMP pathway and of K⁺ channels. Crotalphine (0.2 or 5 μg/kg, orally; 0.0006 μg/paw), administered on day 14 after nerve constriction, inhibited mechanical hyperalgesia and low-threshold mechanical allodynia. The effect of the peptide was antagonized by intraplantar administration of naltrindole, an antagonist of δ-opioid receptors, and partially reversed by norbinaltorphimine, an antagonist of κ-opioid receptors. The effect of crotalphine was also blocked by 7-nitroindazole, an inhibitor of the neuronal nitric oxide synthase; by 1H-(1,2,4) oxadiazolo[4,3-a]quinoxaline-1-one, an inhibitor of guanylate cyclase activation; and by glibenclamide, an ATP-sensitive K⁺ channel blocker. The results suggest that peripheral δ-opioid and κ-opioid receptors, the nitric oxide-cyclic GMP pathway, and ATP-sensitive K⁺ channels are involved in the antinociceptive effect of crotalphine. The present data point to the therapeutic potential of this peptide for the treatment of chronic neuropathic pain.

Additional evidence for the cyclic GMP signaling pathway resulting in the photophobic behavior of Stentor coeruleus.

We report that exo- and endogenous guanosine 3',5'-cyclic monophosphate (cGMP) specifically influenced the photophobic response. In behavioral experiments the slowly hydrolyzable and membrane-permeable analogs of cGMP (8-bromo-cGMP [Br-cGMP] and N6,2'-o-dibutyryl-cGMP) dramatically prolonged the time for ciliary stop response and decreased the duration of ciliary reversal in a dose-dependent manner. When analogs of adenosine 3',5'-cyclic monophosphate (cAMP) (8-bromo-cAMP or N6,2'-o-dibutyryl-cAMP) were used, no essential effects were detected on the kinetics of the photophobic response. Both nonspecific cyclic nucleotide phosphodiesterase (PDE) activity inhibitors (3-isobutyl-1-methylxanthine [IBMX] and 1,3-dimethylxanthine [theophylline]) and the highly specific cGMP-PDE activity inhibitor 1,4-dihydro-5-[2-propoxyphenyl]-7H-1,2,3-triazolo[4,5-d]pyrimidine-7-one (zaprinast) mimicked the effects of cGMP analogs. Treatment of cells with an inhibitor of guanylate cyclase activity (6-anilino-5,8-quinolinedione [LY 83583]) exerted an effect opposite to that of cGMP analogs and PDE activity inhibitors. The positive physiological effect of LY 83583 was significantly diminished in ciliates that were treated simultaneously with Br-cGMP. In an assay of cell cyclic nucleotide content, the exposure of dark-adapted Stentor to light evoked a transient decrease in the basal level of intracellular cGMP. Alterations in internal cGMP levels were more distinct when the intensity of applied illumination was increased. In the presence of IBMX or theophylline the basal content of cGMP was markedly enhanced, and the photoinduced changes in cGMP level were less pronounced. In this paper the possible whole molecular mechanism by which the ciliary orientation in Stentor is controlled by light is presented.

Natriuretic Peptides Inhibit G Protein Activation: MEDIATION THROUGH CROSS-TALK BETWEEN CYCLIC GMP-DEPENDENT PROTEIN KINASE AND REGULATORS OF G PROTEIN-SIGNALING PROTEINS

Atrial natriuretic peptide (ANP) inhibits the proliferation of many cells, in part through interfering with signal transduction enacted by G protein-coupled growth factor receptors. Signaling interactions between ANP and the G protein-coupled growth factor receptor ligand, endothelin-3 (ET-3), regulate astrocyte proliferation at a very proximal but undefined point. Here, we find that ANP inhibits the ability of ET-3 to activate Galpha(q) and Galpha(i) in these cells. ANP stimulated the translocation of endogenous regulators of G protein-signaling (RGS) proteins 3 and 4 from the cytosol to the cell membrane, and enhanced their association with Galpha(q) and Galpha(i). ANP effects were significantly blocked by HS-142-1, an inhibitor of guanylate cyclase activation, or by ET-3. KT5823, an inhibitor of cyclic GMP-dependent protein kinase (PKG) reversed the RGS translocation induced by ANP; conversely, expression of an active catalytic subunit of PKG-I, or 8-bromo-cyclic GMP stimulated RGS translocation. ANP caused the phosphorylation of both RGS proteins in a PKG-dependent fashion, and the expressed PKG (in the absence of ANP) also stimulated RGS phosphorylation. A novel cross-talk between PKG and RGS proteins is stimulated by ANP and leads to the increased translocation and association of RGS proteins with Galpha. The rapid inactivation of G proteins provides a mechanism by which ANP inhibits downstream signaling to the cell proliferation program.

Nitrergic relaxation of the horse corpus cavernosum. Role of cGMP

The involvement of nitric oxide (NO) and the mechanisms mediating neurogenic relaxation were investigated in the horse corpus cavernosum. NADPH-diaphorase activity was expressed in nerve fibres around arteries and muscular bundles in the horse trabecular tissue. Relaxations in response to electrical field stimulation were tetrodotoxin (10 −6 M)-sensitive, indicating their neurogenic origin. The NO synthase inhibitor, l-NO-arginine ( l-NO-Arg, 3×10 −5 M), abolished the electrically induced relaxations, which were significantly reversed by l-arginine (3×10 −3 M). Exogenous NO (10 −6–10 −3 M) evoked relaxations which were unaffected by l-NO-Arg. 1 H-[1,2,4]oxadiazolo[4,3,- a]quinoxalin-1-one (ODQ, 5×10 −6 M), an inhibitor of guanylate cyclase activation by NO, reduced the relaxations in response to electrical stimulation and exogenous NO. Iberiotoxin (3×10 −8 M) or apamin (5×10 −7 M), inhibitors of large and small conductance Ca 2+-activated K + channels, respectively, and glibenclamide (3×10 −6 M), a blocker of ATP-sensitive K + channels, failed to modify the relaxations with NO. It is suggested that NO is present in nerve fibres of the horse corpus cavernosum and relaxes smooth muscle through a guanylate cyclase-dependent mechanism. Neither Ca 2+-activated nor ATP-sensitive K + channels seem to be involved in these relaxations.

Angiotensin II interacts with nitric oxide-cyclic GMP pathway in the central control of drinking behaviour: mapping with c-fos and NADPH-diaphorase

Recognition of the role of nitric oxide in cell-to-cell communication has changed the concept of traditional neurotransmission. We have shown previously that N-methyl- d-aspartate receptors mediate dipsogenic responses and c-Fos expression induced by intracerebroventricular infusion of angiotensin II. Since these receptors are known to be linked to the nitric oxide-cyclic GMP pathway, the present study explores the contribution of this path to the behavioural and cellular effects of intracerebroventricular angiotensin II by using behavioural testing, NADPH-diaphorase histochemistry and immunocytochemical staining for the immediate-early gene, c-fos. N G-nitro- l-arginine methyl ester (125 and 250 μg, intracerebroventricular), an inhibitor of nitric oxide synthase, and Methylene Blue (100 μg), an inhibitor of guanylate cyclase activation, antagonized water intake induced by intracerebroventricular injection of 25 pmol angiotensin II. The effects of N G-nitro- l-arginine methyl ester were reversed by co-injection of l-arginine, the substrate for nitric oxide synthase. However, N G-nitro- l-arginine methyl ester did not alter the pattern of angiotensin II-induced c-fos expression in the organum vasculosum of the lamina terminalis, median preoptic nucleus, hypothalamic paraventricular nucleus and supraoptic nucleus. Double staining with NADPH-diaphorase histochemistry and c-Fos immunocytochemistry showed that neurons staining for both were localized to the anterior third ventricle. However, only 19–25% of the c-Fos-positive neurons expressed NADPH. There were also substantial numbers of neurons in which angiotensin II induced c-Fos that were NADPH-negative. Extensive co-distribution of NADPH-diaphorase-stained cells and those expressing c-fos in response to intracerebroventricular injection of angiotensin II, especially in the median preoptic nucleus, imply that nitric oxide might participate in the mechanism of angiotensin II-induced drinking behaviour. However, a low rate of co-localization of the two markers to individual cells suggests that angiotensin II stimulated the production of nitric oxide and c-Fos in different populations of neurons. Since our previous results showed that glutamate blockade, but not nitric oxide synthase inhibition, suppressed angiotensin II-induced c-Fos, the experiments reported here further suggest that nitric oxide release is not an essential requirement for the expression of c-fos elicited by angiotensin II. They also provide evidence that the dipsogenic and c-Fos responses to angiotensin II are dissociated at a cellular level.

Role of nitric oxide/cyclic GMP pathway in the inhibitory effect of GABA and dopamine on prolactin release.

The anterior pituitary gland is a site of nitric oxide (NO) production and action, suggesting a local regulatory function. We recently reported that NO inhibits in vitro prolactin release. The aim of the present study was to establish the mechanism of action of NO on prolactin release and to determine whether NO is involved in the inhibitory effect of GABA on prolactin release. Since NO exerts its action through cGMP by activating guanylate cyclase in different tissues, we examined the effect of sodium nitroprusside (NP), a NO releaser, on intrapituitary cGMP levels. Incubation of anterior pituitary glands with 0.5 mM NP 4-fold increased intrapituitary cGMP content, but decreased intrapituitary cAMP levels. In addition, we studied the effect of NP on prolactin release in the presence of LY 83583, an inhibitor of guanylate cyclase activity and 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterase activity. 10 microM LY 83583 and 0.5 mM IBMX blocked the inhibitory effect of NP on prolactin release. (10(-3) M) 8Br-cGMP, an analogue of cGMP, mimicked the effect of NP on prolactin release. On the other hand, NO seems to be involved in the inhibitory effect of GABA on prolactin release since hemoglobin, a scavenger of NO, and Nw-nitro-L-arginine methyl ester, an inhibitor of NO synthase (NOS), blocked the pituitary response to GABA. Moreover, GABA (10(-6) M) stimulated NOS activity by almost 50%. GABA increased intrapituitary cGMP levels and decreased cAMP. Dopamine stimulated NOS activity weakly. These observations suggest that NO, acting through the guanylate cyclase-cGMP pathway, inhibits prolactin secretion. In addition, NO may be involved in the inhibitory effect of GABA and dopamine on prolactin release.

Nitric oxide and drinking behaviour

The role of brain l-arginine/nitric oxide biochemical pathway in the regulation of drinking behaviour was investigated. Drinking was induced by water deprivation or by intracerebroventricularly (i.c.v.) injected angiotensin II. l-Arginine, the amino-acid precursor of nitric oxide, i.c.v. injected, caused a dose-dependent reduction of the intake of water induced both by water deprivation and i.c.v. angiotensin II ( P < 0.001). l-NAME, inhibitor of nitric oxide synthase, reverted l-arginine antidipsogenic effect. l-Arginine, given into the preoptic area (POA) caused a potent antidipsogenic effect ( P < 0.001). Either methylene blue (inhibitor of guanylate cyclase activation) or acetylsalicylic acid (ASA), injected into the POA, antagonized the antidipsogenic effect of i.c.v. injected l-arginine. The results indicate that nitric oxide acts as an inhibitory mechanism into the POA and that its antidipsogenic effect requires prostaglandin synthesis.

Involvement of nitric oxide in the non-adrenergic non-cholinergic neurotransmission of horse deep penile arteries: role of charybdotoxin-sensitive K(+)-channels.

1. The involvement of nitric oxide (NO) and the signal transduction mechanisms mediating neurogenic relaxations were investigated in deep intracavernous penile arteries with an internal lumen diameter of 600-900 microns, isolated from the corpus cavernosum of young horses. 2. The presence of nitric oxide synthase (NOS)-positive nerves was examined in cross and longitudinal sections of isolated penile arteries processed for NADPH-diaphorase (NADPH-d) histochemistry. NADPH-d-positive nerve fibres were observed in the adventitia-media junction of deep penile arteries and in relation to the trabecular smooth muscle. 3. Electrical field stimulation (EFS) evoked frequency-dependent relaxations of both endothelium-intact and denuded arterial preparations treated with guanethidine (10(-5) M) and atropine (10(-7) M), and contracted with 10(-6) M phenylephrine. These EFS-induced relaxations were tetrodotoxin-sensitive indicating their non-adrenergic non-cholinergic (NANC) neurogenic origin. 4. EFS-evoked relaxations were abolished at the lowest frequency (0.5-2 Hz) and attenuated at higher frequencies (4-32 Hz) by the NOS inhibitor, NG-nitro-L-arginine (L-NOARG, 3 x 10(-3) M). This inhibitory effect was antagonized by the NO precursor, L-arginine (3 x 10(-3) M). NG-nitro-D-arginine (10(-4) M) did not affect the relaxations to EFS. 5. Incubation with either the NO scavenger, oxyhaemoglobin (10(-5) M), or methylene blue (10(-5) M), an inhibitor of guanylate cyclase activation by NO, caused significant inhibitions of the EFS-evoked relaxations, and while oxyhaemoglobin abolished the relaxations to exogenously added NO (acidified sodium nitrite, 10(-6) - 10(-3) M), there still persisted a relaxation to NO of 24.4 +/- 5.1% (n = 6) in the presence of methylene blue. 6. Glibenclamide (3 x 10(-6) M), an inhibitor of ATP-activated K(+)-channels, did not alter the relaxations to either EFS-stimulation or NO, while the blocker of Ca(2+)-activated K(+)-channels, charybdotoxin (3 x 10(-8) M), caused a significant inhibition of both the electrically-induced relaxations and the relaxations to exogenously added NO. Furthermore, charybdotoxin blocked relaxations induced by the cell permeable analogue of cyclic GMP, 8-bromo cyclic GMP (8 Br-cyclic GMP). 7. These results suggest that relaxations of horse deep penile arteries induced by NANC nerve stimulation involve mainly NO or a NO-like substance from nitrergic nerves. NO would stimulate the accumulation of cyclic GMP followed by increases in the open probability of Ca(2+)-activated K(+)-channels and hyperpolarization leading to relaxation of horse penile arteries.

Viral respiratory infection increases alveolar macrophage cytoplasmic motility in rats: role of NO

Ingested ferrimagnetic (Fe3O4) particles were used to estimate noninvasively the motion of organelles in alveolar macrophages (AM) in intact rats during viral respiratory infection by parainfluenza type 1 (Sendai) virus. Four days after instillation of Fe3O4 particles (3 mg/kg) into the lung, remnant field strength (RFS) was measured at the body surface immediately after magnetization of Fe3O4 particles by an externally applied magnetic field. RFS decreases with time, due to particle rotation (relaxation) which is related to cytoplasmic motility of AM. Viral infection increased the relaxation rate (lambda o per min), and increases in lambda o reached a maximum 3 days after nasal inoculation (day 3). Viral infection (day 3)-induced increases in lambda o were dose dependently inhibited by either the L-arginine analogue N-nitro-L-arginine or by methylene blue, an inhibitor of guanylate cyclase activity. Bronchoalveolar lavage fluid obtained from infected rats contained significantly higher levels of nitrite than that from control rats (P < 0.01). In in vitro experiments, AM from infected rats showed significantly higher lambda o, nitrite production, and intracellular guanosine 3',5'-cyclic monophosphate levels than those from control rats (P < 0.01). Sodium nitroprusside, known to release nitric oxide concentration dependently, increased lambda o of AM from noninfected rats in vitro. These results suggest that nitric oxide plays an important role in AM cytoplasmic motility during viral respiratory infection.

Mediation by nitric oxide formation in the preoptic area of endotoxin and tumour necrosis factor-induced inhibition of water intake in the rat.

1. Drinking was induced in rats by 24 h of water deprivation. Water intake (ml) was evaluated for a 1 h period. 2. NG-nitro-L-arginine methyl ester (L-NAME, 5-10 micrograms, i.c.v., 50-100 ng into the preoptic area (POA)), an inhibitor of nitric oxide (NO) synthase, and methylene blue (50-100 ng into POA), an inhibitor of guanylate cyclase activation, antagonized the inhibition of drinking induced by E. coli endotoxin (LPS, 640 micrograms kg-1, i.v.) and tumour necrosis factor (TNF alpha, 40 ng, i.c.v.) in 24 h water-deprived rats. 3. L-Arginine (25, 50 and 100 ng), the precursor amino acid of NO, but not the stereoisomer D-arginine (100 ng), inhibited drinking induced by water deprivation when injected into the POA 30 min before water presentation (74.4% of inhibition with the highest dose). A dose of 12.5 ng L-arginine into the POA did not exhibit antidipsogenic effects. 4. TNF alpha (20 and 40 ng, i.c.v.; 1.25, 2.5 and 5 ng into the POA) showed a dose-dependent and powerful inhibition of drinking behaviour in water-deprived rats (70.4% and 80.8%, i.c.v. and into POA, with the highest doses, respectively). A dose of 10 ng of TNF alpha given i.c.v. had no effect on the intake of water. 5. LPS and TNF alpha, given at doses (160 micrograms kg-1, i.v. and 10 ng, i.c.v., respectively) that did not influence drinking in water-deprived rats, exhibited a strong antidipsogenic effect in water-deprived rats treated with a dose of L-arginine (12.5 ng, into the POA) which did not modify drinking by itself. (LPS + L-arginine:53.6% of inhibition; TNFalpha + L-arginine: 52.0% of inhibition).6. These results suggest that NO into the POA: (1) acts as an inhibitory mechanism on thirst and (2)plays a role in the antidipsogenic effect of LPS and TNFalpha.

Nitric oxide

In the present study, we examine the involvement of the L-arginine-nitric oxide pathway in seizure activity termination. Convulsions were induced reproducibly by intracerebroventricular administration of N-methyl-D-aspartate to conscious mice. The duration of the seizure activity was increased by inhibition of the NO-pathway or by intracerebroventricular injection of methylene blue, an inhibitor of guanylate cyclase activity. This increased duration in seizure activity was reversed by co-administration of L-arginine or by intracerebroventricular injection of guanosine 3':5' cyclic monophosphate (cGMP). These results suggest that nitric oxide produced in response to NMDA receptor activation leads to an increase in cGMP which induces the seizure activity termination.

Effect of methylene blue on vasoreactivity in dog lung.

We examined the effect of methylene blue (MB), a putative inhibitor of guanylate cyclase (GC) activation by endothelium-derived relaxing factor (EDRF) and nitrovasodilator compounds, on vascular tone and reactivity to vasoactive substances in the isolated, blood-perfused canine lower left lung lobe. Lobar vascular resistance was partitioned into arterial and venous segments by venous outflow occlusion. Because MB did not alter vasoconstriction to either serotonin or acetylcholine (P greater than 0.05) except after cyclooxygenase inhibition (COI), we determined the effectiveness of MB as an inhibitor of GC activation by nitrovasodilators. Lobes were given graded bolus doses of nitroglycerin (GTN), sodium nitroprusside (SNP), and bradykinin (BK) at baseline vascular tone, after COI, and after vascular tone was raised by either U-46619, a thromboxane analogue, or MB infusion. GTN and BK but not SNP induced dose-dependent vasodilation when vascular tone was raised by U-46619. However, when vascular tone was increased to a similar level by 30 mg MB and 0.5 mg/min infusion, vasodilation to GTN, SNP, and BK was enhanced from U-46619 infusion. In contrast to MB, NG-nitro-L-arginine, a putative inhibitor of EDRF synthesis, diminished vasodilation to BK in cyclooxygenase-inhibited lobes with elevated vascular tone. Because MB potentiated vasodilation to GTN, SNP, and BK, it is questionable whether MB is an effective inhibitor of vasodilation to nitrovasodilators or BK in the isolated, blood-perfused canine lung.

Role of cGMP mechanisms in response of rat pulmonary arteries to hypoxia

We have demonstrated previously that in response to hypoxia, isolated rat pulmonary arteries show an initial endothelium-dependent relaxation followed by an endothelium-independent transient contraction. In the presence of increased extracellular Ca2+, both of these responses were enhanced in endothelium-intact arteries. Nitro-L-arginine, a blocker of the biosynthesis of endothelium-derived relaxing factor (EDRF), abolished the initial endothelium-dependent relaxation and Ca(2+)-induced enhancement of hypoxic contraction in endothelium-intact arteries but did not alter responses in endothelium-denuded vessels. Inhibition of prostaglandin formation with indomethacin had no effect on the hypoxia-elicited responses. Preincubation with LY 83583, an inhibitor of guanylate cyclase activation, abolished the initial hypoxia-elicited relaxation and subsequent contraction. M & B 22948, a guanosine 3',5'-cyclic monophosphate (cGMP) phosphodiesterase inhibitor, decreased tone under O2 but not under N2, causing an apparent enhancement of the contraction to hypoxia. Thus the modulation of hypoxic responses by the endothelium is dependent on changes in EDRF production, and a decrease in smooth muscle cGMP not involving an EDRF mechanism appears to mediate the endothelium-independent hypoxic contraction observed in the isolated rat pulmonary artery.

Endothelium-dependent and independent cGMP mechanisms appear to mediate O2 responses in calf pulmonary resistance arteries.

Our laboratory has previously described in isolated 1- to 4-mm calf pulmonary arteries, an endothelium-independent contraction to hypoxia that appears to involve the removal of a H2O2-elicited guanosine 3',5'-cyclic monophosphate (cGMP)-mediated relaxation. In this study, we examined the effects of changes in O2 tension (PO2) on isolated endothelium-intact and endothelium-denuded calf pulmonary resistance arteries of approximately 200 microns in diameter. Resistance arteries precontracted with U46619 were found to undergo a contraction when exposed to a PO2 of 24-27 Torr (hypoxia) from a Po2 of 150 Torr (O2 atmosphere). This contraction was significantly larger in endothelium-intact than endothelium-removed arteries. In the intact artery, 30 microM nitro-L-arginine (NLA), an inhibitor of the biosynthesis of nitric oxide-like activators of guanylate cyclase, increased tone under O2 atmosphere and reduced the contraction to hypoxia to the level observed in the endothelium-removed artery. Reoxygenation caused a relaxation, which was not dependent on the endothelium or inhibited by NLA. The inhibitor of guanylate cyclase activation, LY83583 (10 microM), increased tone under O2 atmosphere, eliminated the contraction to hypoxia, and inhibited the relaxation to reoxygenation, whereas indomethacin (10 microM) did not alter these responses. Thus modulation of a cGMP mechanism, not involving the endothelium or metabolism of arginine, is a primary mediator of responses to changes in O2 tension, and the endothelium appears to cause an enhancement of the contraction to hypoxia via suppression by hypoxia of the tonic generation of an arginine-derived relaxing factor.

The effect of inhibitors of nitric oxide biosynthesis and cyclic GMP formation on nerve-evoked relaxation of human cavernosal smooth muscle.

1. The inhibitory transmission in isolated preparations of cavernosal smooth muscle from human penis has been studied. 2. Electrical field stimulation (EFS; 2-64 pulses/train, 0.8 ms pulse duration, 10 Hz) evoked relaxation of preparations treated with guanethidine (50 microM). The EFS-evoked relaxations were atropine-resistant and tetrodotoxin-sensitive indicating their origin to be non-adrenergic, non-cholinergic (NANC) nerve stimulation. 3. EFS-evoked relaxation was attenuated dose-dependently by the nitric oxide (NO)-synthase inhibitor, L-NG-nitro arginine (L-NOARG; 0.3-100 microM) but not by D-NG-nitro arginine. The inhibitory effect of L-NOARG on transmission was antagonized by L-arginine (100 microM), a NO precursor, but not by D-arginine. 4. Incubation with methylene blue (10-50 microM), a known inhibitor of guanylate cyclase activation by NO, caused a concentration-related inhibition of EFS-evoked relaxation. 5. It is concluded that NANC nerve-evoked relaxation of human cavernosal smooth muscle is mediated by NO or a NO-like substance.

Interactions between nitroglycerin and endothelium in vascular smooth muscle relaxation

To evaluate the role of endothelium in nitroglycerin (NTG)-mediated vascular relaxation, epinephrine-contracted rat thoracic aortic segments with and without intact endothelium were exposed to NTG (10(-10) to 10(-5) M). Aortic segments with intact (endo+, n = 15) and denuded endothelium (endo-, n = 9) exhibited typical NTG-induced relaxation. However, the mean effective concentration of NTG was lower for endo- than for endo+ segments (P less than 0.001). To determine if this phenomenon related to nitric oxide (NO) generation by endothelium, six endo+ segments were treated with NG-monomethyl-L-arginine (L-NMMA), an inhibitor of NO production. These endo+ segments exhibited greater (P less than 0.001) relaxation in response to NTG than the untreated endo+ segments. Oxyhemoglobin, an inhibitor of guanylate cyclase activation, greatly diminished NTG-mediated relaxation of all aortic segments. To determine if the enhanced NTG-mediated relaxation of endo- segments was unique to the guanosine 3',5'-cyclic monophosphate-dependent vasodilator NTG, other endo+ and endo- segments were exposed to adenosine 3',5'-cyclic monophosphate-dependent vasodilator papaverine (10(-8) to 10(-4) M), and no difference in EC50 was noted between endo+ and endo- segments. Thus endothelium attenuates NTG-mediated vasorelaxation, and this attenuation is abolished by inhibition of endothelial NO production with L-NMMA. These observations indicate that endothelium is a dynamic modulator of vascular smooth muscle relaxant effects of NTG. This modulation appears to result from a competitive interaction between endothelial NO and NTG.

Lack of Second Messenger Function of Cyclic GMP in Acetylcholine-Induced Negative Inotropism

As cyclic 3',5'-guanosine monophosphate (cGMP) is still discussed as a possible mediator of the negative inotropic effects of cholinergic agents, the influence of acetylcholine (ACh) on force of contraction and cGMP tissue levels was studied in isolated, electrically driven guinea pig auricles in the presence of methylene blue, an inhibitor of guanylate cyclase activation, as well as of M & B 22,948, an inhibitor of cGMP breakdown. Nitroprusside-Na (NP), a potent stimulator of guanylate cyclase, was tested for comparison under the same conditions. ACh at concentrations of 10(-7)-5 X 10(-6) M dose-dependently diminished force of contraction down to cardiac arrest, whereas NP only had a slight negative inotropic effect that was maximum at 10(-5) M and reduced force of contraction to 89% of control. Although ACh was much more effective in reducing force of contraction than NP, only NP significantly increased myocardial cGMP levels. The rise in cGMP produced by NP was attenuated by methylene blue (5 X 10(-5) M) and augmented by M & B 22,948 (3.7 X 10(-4) M), whereas the contractile effects (similar as those of ACh) remained unchanged. These results suggest that the negative inotropic action of ACh is not mediated by cGMP.

Purification and characterization of a cytostatic factor with anti-viral activity from the bitter melon.

We have previously reported that a crude aqueous extract of the bitter melon (Momordica charantia) has both cytostatic and cytotoxic activities, and is a competitive inhibitor of guanylate cyclase activity. This crude preparation kills human leukemic lymphocytes in a dose-dependent manner while not affecting the viability of normal human lymphocytes at these same doses. In this report we describe the purification and characterization of one of these cytostatic factors which also exhibits anti-viral activity. The partially purified factor was both cytostatic to BHK-21 cells and inhibitory to VSV plaque formation in a dose-dependent manner. This preparation was inhibitory to both viral and host cell RNA and protein synthesis as early as 30 min after addition to these samples. As determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), this purified factor is a single component with a molecular weight corresponding to 40,000 daltons. The factor is sensitive to boiling and to pre-treatments with trypsin, but not ribonuclease (RNAse), or deoxyribonuclease (DNAse). As determined by radioactive precursor uptake and incorporation studies, the purified factor inhibits both RNA and protein synthesis in intact tissue culture cells and inhibits protein synthesis in a cell-free wheat germ system. DNA synthesis was slightly stimulated. The purified factor is cytostatic for both BHK-21 and for the IM9 leukemic cell lines for at least 120 h. The cytostatic component had no effect on cellular cyclic GMP metabolism.

Enzymatic formation of inosine 3′,5′-monophosphate and of 2′-deoxyguanosine 3′,5′-monophosphate: Inosinate and deoxyguanylate cyclase activity

Enzymes in particulate fractions from sea urchin sperm and in soluble fractions from rat lung were shown to catalyze the formation of inosine 3′,5′-monophosphate (cyclic IMP) and of 2′-deoxyguanosine 3′,5′-monophosphate (cyclic dGMP) from ITP and dGTP, respectively. With sea urchin sperm particulate fractoins, Mn 2+ was an essential metal cofactor for inosinate, deoxyguanylate, guanylate and adenylate cyclase activities. Heat-inactivation studies differentiated inosinate and deoxyguanylate cyclase activities from adenylate cyclase, but indicated an association of these activities with guanylate cyclase. Preincubation of sea urchin sperm particulate fractions with trypsin altered in a very similar manner guanylate, inosinate, and deoxyguanylate cyclase activities, and various metals and metal-nucleotide combinations protected the three cyclase activities to comparable degrees against trypsin. The relative guanylate, deoxyguanylate and inosinate cyclase activities at 0.1 mM nucleoside triphosphate were 1.0, 0.5 and 0.08, respectively. With these three cyclase activities, plots of reciprocal velocities against reciprocal Mn 2+-nucleoside triphosphate concentrations were concave upward, suggesting positive homotropic effects. With rat lung soluble preparations, relative guanylate, deoxyguanylate, inosinate and adenylate cyclase activities at 0.09 mM nucleoside triphosphate were 1.0, 1.7, 0.1 and 0, respectively. MnGTP was a competitive inhibitor of deoxyguanylate cyclase activity ( K i = 12.2 μ M ) and MndGTP was a competitive inhibitor of guanylate cyclase activity ( K i = 16.2 μ M ). Inhibition studies using ITP were not conducted. When soluble fractions from rat lung were applied to Bio-Gel A 1.5 m columns, elution profiles of guanylate, deoxyguanylate and inosinate cyclase activities were similar. These results suggest that deoxyguanylate, guanylate and inosinate cyclase activities reside within the same protein molecule.