Search

We studied the modes of activation of the salt-secreting rectal gland of the spiny dogfish, Squalus acanthias, by the native cardiac peptide CNP. The stimulatory action of CNP in isolated perfused glands is inhibited by 10 mM procaine, presumably by blocking release of vasoactive intestinal peptide (VIP) from nerves. Procaine reduces the slope of the dose-response curve of human CNP and that of shark CNP (each P < 0.0001). CNP increases short-circuit current in cultured rectal gland cells from 4.8 +/- 1.6 to 27.0 +/- 7.8 microA/cm2. It also stimulates the secretion of chloride in isolated perfused glands in the presence of 10 mM procaine from 72 +/- 31 to 652 +/- 173 microeq. h(-1). g(-1). These results suggest that CNP has a direct cellular action not mediated by the neural release of VIP. The residual stimulation of perfused glands in the presence of procaine was almost completely inhibited by staurosporine [10 nM; an inhibitor of protein kinase C (PKC)] from 652 +/- 173 to 237 +/- 61 microeq. h(-1). g(-1). Although CNP stimulates guanylyl cyclase in shark rectal gland, chloride secretion of perfused glands was not elicited by 8-bromoadenosine-cGMP (8-BrcGMP) alone nor by the activator of PKC phorbol ester. The combination of PKC activation and 8-BrcGMP infusion, however, stimulated chloride secretion in perfused glands from 94 +/- 30 to 506 +/- 61 microeq. h(-1). g(-1), a level comparable to that observed in glands blocked with procaine. Several parallel pathways appear to be synergistic in activating chloride secretion stimulated by CNP in the rectal gland.

We investigated the influence of nitrovasodilators on DNA synthesis in cultured human aortic smooth muscle cells and explored the hypothesis that nitric oxide (NO) is directly involved in mediating the inhibitory effects of hydroxyurea on DNA synthesis. Both NO and hydroxyurea inhibited ongoing DNA synthesis and S phase progression in our cells. Exogenous deoxynucleosides partially reversed this inhibition, suggesting that ribonucleotide reductase is a primary target for both NO and hydroxyurea. Nitrovasodilators inhibited DNA synthesis by releasing NO, as detected by chemiluminescence and as shown by the reversal of DNA synthesis inhibition by NO scavengers. This inhibition appears to occur via a cGMP-independent mechanism. In contrast, hydroxyurea did not produce a detectable NO signal, and NO scavengers had no influence on its inhibition of DNA synthesis, suggesting that NO does not mediate the inhibitory action of hydroxyurea in our system. Furthermore, the action of nitrovasodilators and hydroxyurea on DNA synthesis differed according to redox sensitivity. The redox agents N-acetyl-L-cysteine and ascorbate reversed NO inhibition of DNA synthesis and had no effect on DNA synthesis inhibition caused by hydroxyurea.

Airway smooth muscle (ASM) hypertrophy and hyperplasia are important determinants of bronchial responsiveness in asthma, and agents that interfere with these processes may prevent airway remodeling. We tested the hypothesis that activators of soluble and particulate guanylyl cyclases would inhibit human ASM cell (HASMC) proliferation. We report that the nitric oxide (NO) donors S-nitroso-N-acetylpenicillamine (SNAP; 10(-6) to 10(-4) M) and sodium nitroprusside (10(-5) to 10(-3) M) and human atrial natriuretic peptide [ANP-(1-28); 10(-8) to 10(-6) M], which activate soluble and particulate guanylyl cyclases, respectively, inhibited serum- and thrombin-induced proliferation of cultured HASMCs. The antimitogenic effect of SNAP was reversed by hemoglobin (10(-5) M), an NO scavenger, suggesting that NO donation was involved. The antiproliferative effects of SNAP and ANP-(1-28) were potentiated by the cGMP-specific phosphodiesterase zaprinast and mimicked by 8-bromo-cGMP (10(-6) to 10(-3) M), suggesting that cGMP-dependent mechanisms were involved. However, first, ANP-(1-28) produced a smaller antiproliferative effect than SNAP in contrast to their abilities to elevate cGMP, and second, rat ANP-(104-126), which binds selectively to ANP clearance receptors without elevating cGMP, had a small antiproliferative effect, suggesting that cGMP-independent mechanisms were also involved. These results provide evidence for a novel antiproliferative effect of NO and ANP in HASMCs mediated through cGMP-dependent and cGMP-independent mechanisms.

Previous experimental findings have led to the suggestion that guanosine 3',5'-cyclic monophosphate (cGMP) plays a permissive role in hypercapnic cerebral vasodilation. However, we recently reported that the technique used to reveal a permissive role for cGMP [cGMP repletion in the presence of nitric oxide synthase (NOS) inhibition] created a situation where CO(2) reactivity was normalized but where different mechanisms (i.e., K(+) channels) participated in the response. In the present study, we examined whether that nascent K(+)-channel dependence is related in any way to an increase in the influence of the miconazole-inhibitable cytochrome P-450 epoxygenase pathway. Using intravital microscopy and a closed cranial window system in adult rats, we measured pial arteriolar diameters during normo- and hypercapnia, first in the absence and then in the presence of a neuronal NOS (nNOS) inhibitor [7-nitroindazole (7-NI)]. This was followed by suffusion of a cGMP analog and then cGMP plus miconazole. Separate groups of rats were used to evaluate whether miconazole either alone or in the presence of 8-bromoguanosine 3', 5'-cyclic monophosphate (8-BrcGMP) or its vehicle (0.1% ethanol) had any effect on CO(2) reactivity and whether miconazole affected K(+)-channel opener-induced dilations. Hypercapnic (arterial PCO(2), congruent with65 mmHg) pial arteriolar dilations, as expected, were reduced by 70-80% with 7-NI and restored with cGMP repletion. CO(2) reactivity was again attenuated after miconazole introduction. Miconazole, with and without 8-BrcGMP, and its vehicle had no influence on pial arteriolar CO(2) reactivity in the absence of nNOS inhibition combined with cGMP repletion. Miconazole alone also did not affect vasodilatory responses to K(+)-channel openers. Thus present results suggest that the nascent K(+)-channel dependence of the hypercapnic response found in our earlier study may be related to increased epoxygenase activity. The specific reasons why the pial arteriolar CO(2) reactivity gains a K(+)-channel and epoxygenase dependence only under conditions of nNOS inhibition and cGMP restoration remain to be identified. These findings again call into question the interpretations applied to data collected in studies evaluating potential permissive actions of cGMP or NO.

We investigated the role of nitric oxide (NO)/cGMP in the coupling of neuronal activation to regional cerebral blood flow (rCBF) in alpha-chloralose-anesthetized rats. Whisker deflection (60 s) increased rCBF by 18 +/- 3%. NO synthase (NOS) inhibition by N(omega)-nitro-L-arginine (L-NNA; topically) reduced the rCBF response to 9 +/- 4% and resting rCBF to 80 +/- 8%. NO donors [S-nitroso-N-acetylpenicillamine (SNAP; 50 microM), 3-morpholinosydnonimine (10 microM)] or 8-bromoguanosine 3', 5'-cyclic-monophosphate (8-BrcGMP; 100 microM)] restored resting rCBF and L-NNA-induced attenuation of the whisker response in the presence of L-NNA, whereas the NO-independent vasodilator papaverine (1 mM) had no effect on the whisker response. Basal cGMP levels were decreased to 35% by L-NNA and restored to 65% of control by subsequent SNAP superfusion. Inhibition of neuronal NOS by 7-nitroindazole (7-NI; 40 mg/kg ip) or soluble guanylyl cyclase by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 100 microM) significantly reduced resting rCBF to 86 +/- 8 and 92 +/- 10% and whisker rCBF response to 7 +/- 4 and 12 +/- 3%, respectively. ODQ reduced tissue cGMP to 54%. 8-BrcGMP restored the whisker response in the presence of 7-NI or ODQ. We conclude that NO, produced by neuronal NOS, is a modulator in the coupling of neuronal activation and rCBF in rat somatosensory cortex and that this effect is mainly mediated by cGMP. L-NNA-induced vasomotion was significantly reduced during increased neuronal activity and after restoration of basal NO levels, but not after restoration of cGMP.

In the glycerol model of renal injury we describe an acute rise in systemic arterial pressure which is attended by a reduced vasodilatory response to acetylcholine in vivo; vasodilatory responses to verapamil, however, were not impaired. Neither arginine nor sodium nitroprusside diminished this rise in blood pressure; N(omega)-nitro-L-arginine methyl ester (L-NAME) elevated basal mean arterial pressure and markedly blunted the rise in mean arterial pressure following the administration of glycerol. Aortic rings from the glycerol-treated rat demonstrate an impaired vasodilatory response to acetylcholine, an effect not repaired by arginine; the vasodilatory responses to nitric oxide donors, sodium nitroprusside and SIN-1, were also impaired; 8-bromo-cGMP, at higher doses, evinced a vasodilatory response comparable to that observed in the control rings. This pattern of responses was not a nonspecific effect of aortic injury, since aortic rings treated with mercuric chloride, a potent oxidant, displayed an impaired vasodilatory response to acetylcholine but not to sodium nitroprusside. We conclude that in the glycerol model of heme protein-induced tissue injury, there is an acute elevation in mean arterial pressure attended by impaired endothelium-dependent vasodilatation in vitro and in vivo. We suggest that the acute scavenging of nitric oxide by heme proteins depletes the blood vessel wall of its endogenous vasodilator and permeation of heme proteins into the blood vessel wall may contribute to such sustained effects as observed in vitro.

The vasodilator effects of pituitary adenylate cyclase-activating polypeptide (PACAP)-27 are subject to tachyphylaxis in rats treated with the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME). We examined whether this tachyphylaxis could be prevented by administration of the putative endothelium-derived nitrosyl factor S-nitroso-L-cysteine (L-SNC) and whether L-SNC may exert its effects via increases in cGMP levels in vascular smooth muscle. Five doses of PACAP-27 (2 nmol/kg iv) produced pronounced vasodilator responses in saline-treated rats. These responses were not subject to tachyphylaxis. The first injection of PACAP-27 (2 nmol/kg iv) in L-NAME-treated (50 micromol/kg iv) rats produced vasodilator responses similar to those in saline-treated rats, whereas subsequent injections produced progressively smaller responses. The injection of L-SNC (1,200 nmol/kg iv) before each injection of PACAP-27 prevented tachyphylaxis to the Gs protein-coupled receptor agonist in L-NAME-treated rats, whereas equihypotensive doses of the NO donor sodium nitroprusside (100 micrograms/kg iv) did not. The injection of the membrane-permeant cGMP analog 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate (8-CPT-cGMP; 30 micromol/kg iv) to L-NAME-treated rats restored resting hemodynamic values to pre-L-NAME levels but did not prevent the development of tachyphylaxis to PACAP-27. These results suggest that nitrosyl factors prevent the development of tachyphylaxis to the hemodynamic actions of PACAP-27. These nitrosyl factors may act independently of their ability to generate cGMP in vascular smooth muscle.

In this study, an in vitro model of the blood-brain barrier, consisting of porcine brain-derived microvascular endothelial cells (BMEC), was used to evaluate the mechanism of hypoxia-induced hyperpermeability. We show that hypoxia-induced permeability in BMEC was completely abolished by a neutralizing antibody to vascular endothelial growth factor (VEGF). In contrast, under normoxic conditions, addition of VEGF up to 100 ng/ml did not alter monolayer barrier function. Treatment with either hypoxia or VEGF under normoxic conditions induced a twofold increase in VEGF binding sites and VEGF receptor 1 (Flt-1) mRNA expression in BMEC. Hypoxia-induced permeability also was prevented by the nitric oxide (NO) synthase inhibitor NG-monomethyl-L-arginine, suggesting that NO is involved in hypoxia-induced permeability changes, which was confirmed by measurements of the cGMP level. During normoxia, treatment with VEGF (5 ng/ml) increased permeability as well as cGMP content in the presence of several antioxidants. These results suggest that hypoxia-induced permeability in vitro is mediated by the VEGF/VEGF receptor system in an autocrine manner and is essentially dependent on reducing conditions stabilizing the second messenger NO as the mediator of changes in barrier function of BMEC.

Estradiol had a biphasic effect on permeability across cultures of human umbilical vein endothelial cells (HUVEC): at nanomolar concentrations it decreased the HUVEC culture permeability, but at micromolar concentrations it increased the permeability. The objective of the present study was to test the hypothesis that the changes in permeability were mediated by nitric oxide (NO)-related mechanisms. The results revealed dual modulation of endothelial paracellular permeability by estrogen. 1) An endothelial NO synthase (eNOS)-, NO-, and cGMP-related, Ca2+-dependent decrease in permeability was activated by nanomolar concentrations of estradiol, resulting in enhanced Cl- influx, increased cell size, and increases in the resistance of the lateral intercellular space (RLIS) and in the resistance of the tight junctions (RTJ); these effects appeared to be limited by the ability of cells to generate cGMP in response to NO. 2) An inducible NO synthase (iNOS)- and NO-related, Ca2+-independent increase in permeability was activated by micromolar concentrations of estradiol, resulting in enhanced Cl- efflux, decreased cell size, and decreased RLIS and RTJ. We conclude that the net effect on transendothelial permeability across HUVEC depends on the relative contributions of each of these two systems to the total paracellular resistance.

A beta-escin-permeabilized canine tracheal smooth muscle preparation was used to test the hypothesis that cGMP decreases Ca2+ sensitivity in airway smooth muscle primarily by inhibiting the membrane receptor-coupled mechanisms that regulate Ca2+ sensitivity and not by inhibiting Ca2+/calmodulin activation of the contractile proteins. 8-Bromo-cGMP (100 microM) had no effect on the free Ca2+ concentration-response curves generated in the absence of muscarinic receptor stimulation. In the presence of 100 microM ACh plus 10 microM GTP, 8-bromo-cGMP (100 microM) caused a rightward shift of the free Ca2+ concentration-response curve, significantly increasing the EC50 for free Ca2+ from 0.35 +/- 0.03 to 0.75 +/- 0.06 microM; this effect of 8-bromo-cGMP was concentration dependent from 1 to 100 microM. 8-Bromo-cGMP (100 microM) decreased the level of regulatory myosin light chain (rMLC) phosphorylation for a given cytosolic Ca2+ concentration but had no effect on the amount of isometric force produced for a given level of rMLC phosphorylation. These findings suggest that cGMP decreases Ca2+ sensitivity in canine tracheal smooth muscle primarily by inhibiting the membrane receptor-coupled mechanisms that modulate the relationship between cytosolic Ca2+ concentration and rMLC phosphorylation.

We have previously shown that PGI2 and PGE2 have a synergistic role in restoring electrical transepithelial resistance (R) in ischemia-injured porcine ileum via the second messengers Ca2+ and cAMP. Because Ca2+ and cAMP stimulate Cl- secretion, we assessed the role of PG-induced Cl- secretion in recovery of R. Mucosa from porcine ileum subjected to ischemia for 45 min was mounted in Ussing chambers and bathed in indomethacin and Ringer solution. Addition of PGs stimulated a twofold increase in R, which was preceded by elevations in short-circuit current (increase of 25 microA/cm2). The PG-induced effect on R was partially inhibited with bumetanide, an inhibitor of Cl- secretion. The remaining elevations in R were similar in magnitude to those induced in ischemic tissues by amiloride, an inhibitor of Na+ absorption. Treatment with 10(-4) M 8-bromo-cGMP or 300 mosM mucosal urea resulted in elevations in R similar to those attained with PG treatment. PGs signal recovery of R via induction of Cl- secretion and inhibition of Na+ absorption, possibly by establishing a transmucosal osmotic gradient.

In functional experiments, the nitric oxide (NO) donor N-morpholino-N-nitroso-aminoacetonitrile or the cGMP analog 8-(4-chlorophenylthio)-cGMP caused a concentration-dependent, tetrodotoxin-resistant relaxation of precontracted strips from rat small intestine. The inhibitory effect of both substances was completely blocked at lower concentrations and was significantly attenuated at higher concentrations by the selective cGMP-dependent protein kinase (cGK) antagonist KT-5823 (1 microM). cGK-I was identified by immunohistochemistry in circular and longitudinal muscle, lamina muscularis mucosae, and smooth muscle cells of the villi and in fibroblast-like cells of the small intestine. Additionally, there was staining of a subpopulation of myenteric and submucous plexus neurons. Double staining for neuronal NO synthase (nNOS) and cGK-I demonstrated a colocalization of these two enzymes. Western blot analysis of smooth muscle preparations and isolated nerve terminals demonstrated that these structures predominantly contain the cGK-Ibeta isoenzyme, whereas the cGK-Ialpha expression is about threefold less. The isoform cGK-II was entirely confined to mucosal epithelial cells. These results show that cGK-I is expressed in different muscular structures of the small intestine and participates in the NO-induced relaxation of gastrointestinal smooth muscle. The presence of cGK-I in NOS-positive enteric neurons further suggests a possible neuronal action site.

Ciliary beating is required for the maintenance of lung mucociliary transport. We investigated the role of cyclic nucleotide-dependent protein kinases in stimulating ciliary beat frequency (CBF) in bovine bronchial epithelial cells (BBECs). cAMP-dependent protein kinase (PKA) activity and cGMP-dependent protein kinase (PKG) activity were distinguished after DEAE-Sephacel chromatography of BBEC extracts. cAMP levels and PKA activity are increased in BBECs stimulated with 0.01-1 mM isoproterenol, with a corresponding increase in CBF. cGMP levels and PKG activity are increased in BBECs stimulated with 0.1-10 microM sodium nitroprusside, with a corresponding increase in CBF. Direct protein kinase-activating analogs of cAMP and cGMP (dibutyryl cAMP and 8-bromo-cGMP, respectively) also activate their specific kinases and stimulate CBF. Preincubation of BBECs with inhibitors of PKA or PKG [KT-5720 or Rp-8-(p-chlorophenylthio)-guanosine 3',5'-cyclic monophosphothioate] results in the inhibition of specific kinase activity as well as in the inhibition of CBF. These studies suggest that the activation of either PKA or PKG can lead to the stimulation of CBF in bovine airway epithelium.

Cells of a newly described, immortalized, epithelial, human transverse colonic cell line, NCM460, reach approximately 90% confluence on plastic and develop transepithelial resistances of 120-250 Omega . cm2 on porous substrates. Its utility as a model for the transverse human colon was validated by comparing second messenger-mediated Cl- transport, using the fluorescent probe 6-methoxy-quinolyl acetoethyl ester, in NCM460 cells and colonocytes isolated from human transverse crypts. Basal Cl- influx was increased (P < 0.01) by PGE1 (1 microM), forskolin (1 microM), 8-bromoadenosine 3'5'-cyclic monophosphate (100 microM), heat-stable Escherichia coli enterotoxin (STa; 1 microM), 8-bromoguanosine 3'5'-cyclic monophosphate (100 microM), histamine (1 microM), and phorbol 12,13-dibutyrate (1 microM) in both cell types. The Cl- channel blocker diphenylamine 2-carboxylic acid (50 microM) and the Na+-K+-2Cl- cotransport inhibitor furosemide (1 microM), but not the K+ channel blocker Ba2+ (3 mM), inhibited these Cl- permeabilities. These cells possess transcripts for cystic fibrosis transmembrane conductance regulator, Na+-K+-2Cl- cotransporter, STa receptor, and intestine-specific cGMP-dependent protein kinase II. Thus cAMP-, cGMP-, and Ca2+-dependent secretagogues act on NCM460 and primary colonocytes to stimulate Cl- transport. This validates the utility of NCM460 as a model for transverse colonic crypts and is the first demonstration of a colonic cell line whose origin is known.

The purpose of this study was to determine whether vasoactive intestinal peptide (VIP) modulates vasoconstriction elicited by phenylephrine and ANG II in vivo and, if so, to begin to elucidate the mechanisms underlying this phenomenon. Using intravital microscopy, we found that suffusion of phenylephrine and ANG II elicits significant vasoconstriction in the in situ hamster cheek pouch that is potentiated by VIP-(10-28), a VIP receptor antagonist, but not by VIP-(1-12) (P < 0.05). Aqueous VIP has no significant effects on phenylephrine- and ANG II-induced vasoconstriction. However, VIP on sterically stabilized liposomes (SSL), a formulation where VIP assumes a predominantly alpha-helix conformation, significantly attenuates this response. Maximal effect is observed within 30 min and is no longer seen after 60 min. Empty SSL are inactive. Indomethacin has no significant effects on responses induced by VIP on SSL. The vasodilators ACh, nitroglycerin, calcium ionophore A-23187, 8-bromo-cAMP, and isoproterenol have no significant effects on phenylephrine- and ANG II-induced vasoconstriction. Collectively, these data suggest that vasoconstriction modulates VIP release in the in situ hamster cheek pouch and that alpha-helix VIP opposes alpha-adrenergic- and ANG II-induced vasoconstriction in this organ in a reversible, prostaglandin-, NO-, cGMP-, and cAMP-independent fashion.

The purpose of this study was to determine the precise role of angiotensin subtype-1 (AT1) and -2 (AT2) receptors and the mechanisms by which they act to alter fluid transport in the rat jejunum. In rats on normal sodium intake, ANG II at low dose stimulated net jejunal fluid absorption, whereas at a high dose the peptide inhibited absorption. Low-dose ANG II-stimulated fluid absorption was blocked completely by the specific AT2 receptor antagonist PD-123319 (PD) but was unchanged by the AT1 receptor antagonist losartan (Los). The AT2 receptor agonist CGP-42112A, caused an inversely dose-dependent increase in fluid absorption, which also was totally prevented by PD but was unaltered by Los. Conversely, high-dose ANG II inhibition of absorption was blocked by Los but not by PD. In animals receiving normal sodium intake, neither Los nor PD alone altered fluid absorption. In sodium-restricted animals, however, Los alone increased absorption and PD alone inhibited absorption. In rats on normal sodium intake, low-dose ANG II increased jejunal interstitial and luminal (loop) fluid concentrations of cGMP. These increases in cGMP were blocked with PD but not with Los. 8-Bromoguanosine-3',5'-cyclic monophosphate administered via the mesenteric artery or the submucosal interstitial space markedly increased absorption, but it inhibited absorption when administered into the loop. High-dose ANG II decreased jejunal interstitial and loop fluid cAMP and increased PGE2. The increase in PGE2 was blocked by Los but not by PD. The data demonstrate that ANG II mediates jejunal sodium and water absorption by an action at the AT2 receptor involving cGMP formation. The data also show that ANG II inhibits absorption via the AT1 receptor by a mechanism that is both negatively coupled to cAMP and increases jejunal PGE2 production.

We investigated the role of K+ channels and intracellular Ca2+ stores in the relaxations induced by the NO donor 3-morpholinosydnonimine (SIN-1) and 8-bromo-cGMP (8-BrcGMP), 8-(4-chlorophenylthio)-cGMP (pCPT-cGMP), and alpha, beta-methylene-ATP in isolated segments of rat ileum. The inhibitory responses to SIN-1 and the cGMP analogs were not influenced by the K+ blockers apamin, charybdotoxin, iberiotoxin, or glibenclamide, whereas relaxations induced by alpha,beta-methylene-ATP were abolished by apamin and tetraethylammonium. The NO-donor SIN-1 and the cGMP analogs were able to inhibit contractions induced by activation of L-type Ca2+ channels (BAY-K-8644), by carbachol (CCh), and by cyclopiazonic acid (CPA), a blocker of sarcoplasmic Ca2+-ATPase. However, the inhibition of the combined CPA and CCh response was reduced and the dose-response curve of SIN-1 shifted to the right. Intracellular Ca2+ stores were emptied by incubation in Ca2+-free buffer and repetitive stimulation with CCh or BAY-K-8644. After restoration of extracellular Ca2+, the inhibitory effect of SIN-1 and pCPT-cGMP was only attenuated, whereas in the additional presence of CPA, the inhibitory effect of SIN-1 was blocked and the effect of 8-BrcGMP reduced. Thus depleting intracellular Ca2+ stores attenuated the effect of SIN-1 and 8-BrcGMP, suggesting an involvement of functional Ca2+ stores.

We previously demonstrated that nitric oxide (NO) stimulates the basolateral small-conductance K+ channel (SK) via a cGMP-dependent pathway [M. Lu and W. H. Wang. Am. J. Physiol. 270 (Cell Physiol. 39): C1336-C1342, 1996]. Because NO at high concentration has been shown to react with superoxide (O-2) to form peroxynitrite (OONO-) [W. A. Pryor and G. L. Squadrito. Am. J. Physiol. 268 (Lung Cell. Mol. Physiol. 12): L699-L722, 1995 and M. S. Wolin. Microcirculation 3: 1-17, 1996], we extended our study to examine, using patch-clamp technique, the effect of high concentrations of NO on SK in cortical collecting duct (CCD) of rat kidney. Addition of NO donors [100-200 microM S-nitroso-N-acetyl-penicillamine (SNAP) or sodium nitroprusside (SNP)] reduced channel activity, defined as the product of channel number and open probability, to 15 and 25% of the control value, respectively. The inhibitory effect of NO was completely abolished in the presence of 10 mM Tiron, an intracellular scavenger of O-2. NO donors, 10 microM SNAP or SNP, which stimulate channel activity under control conditions, can also inhibit SK in the presence of an O-2 donor, pyrogallol, or in the presence of an inhibitor of superoxide dismutase, diethyldithiocarbamic acid. The inhibitory effect of NO is still observed in the presence of exogenous cGMP, suggesting that the NO-induced inhibition is not the result of decreased cGMP production. We conclude that the inhibitory effect of NO on channel activity results from an interaction between NO and O-2.

To investigate the mechanisms whereby guanosine 3',5'-cyclic monophosphate (cGMP) modulates microvessel permeability in vivo, we measured changes in microvessel hydraulic conductivity (Lp) and endothelial cytoplasmic Ca2+ concentration ([Ca2+]i) in response to the cGMP analogs 8-bromo-cGMP (8-BrcGMP) and 8-(p-chlorophenylthio)cGMP (8-pCPT-cGMP) in the presence and absence of inflammatory stimuli in intact individually perfused microvessels in frog and rat mesenteries. The cGMP analog caused a transient increase in Lp and potentiated ATP or bradykinin-induced increases in Lp in frog and rat mesenteric microvessels, respectively. The mean peak value of the test Lp/control Lp after exposure to 8-BrcGMP was 5.3 +/- 0.5 in frog microvessels and 2.8 +/- 0.4 in rat microvessels. The ATP-induced increase in Lp in frog microvessels was further raised by 8-BrcGMP from 7.0 +/- 0.9 to 12.4 +/- 1.9 times the control. In rat mesenteric microvessels, the bradykinin-induced increase in Lp was potentiated by 8-BrcGMP from 4.8 +/- 0.4 to 8.3 +/- 1.3 times the control and was suppressed by the guanylate cyclase inhibitor LY-83583 to 2.6 +/- 0.5 times the control. A similar but larger effect was found when using 8-pCPT-cGMP. In contrast to the actions of increased cGMP on microvessel permeability, cGMP analogs had no effect on basal endothelial [Ca2+]i and did not alter the magnitude and time course of ATP or bradykinin-induced increases in endothelial [Ca2+]i. These results suggested that an elevation of cGMP levels in endothelial cells is a necessary step to increase microvessel permeability in intact microvessels, and this regulatory process occurs downstream from Ca2+ influx, which differs from that reported in large-vessel endothelium in culture and in vascular smooth muscle cells. Experiments carried on microvessels in both frog and rat mesenteries provided a direct comparison of the endothelial cell regulatory mechanisms between species.

Recent studies on the role of nitric oxide (NO) in gastrointestinal smooth muscle have raised the possibility that NO-stimulated cGMP could, in the absence of cGMP-dependent protein kinase (PKG) activity, act as a Ca(2+)-mobilizing messenger [K. S. Murthy, K.-M. Zhang, J.-G. f1p4 J. T. Grider, and G. M. Makhlouf. Am. J. Physiol. 265 (Gastrointest. Liver Physiol. 28): G660-G671, 1993]. This notion was examined in dispersed gastric smooth muscle cells with 8-bromo-cGMP (8-BrcGMP) and with NO and vasoactive intestinal peptide (VIP), which stimulate endogenous cGMP. In muscle cells treated with cAMP-dependent protein kinase (PKA) and PKG inhibitors (H-89 and KT-5823), 8-BrcGMP (10 microM), NO (1 microM), and VIP (1 microM) stimulated 45Ca2+ release (21 +/- 3 to 30 +/- 1% decrease in 45Ca2+ cell content); Ca2+ release stimulated by 8-BrcGMP was concentration dependent with an EC50 of 0.4 +/- 0.1 microM and a threshold of 10 nM. 8-BrcGMP and NO increased cytosolic free Ca2+ concentration ([Ca2+]i) and induced contraction; both responses were abolished after Ca2+ stores were depleted with thapsigargin. With VIP, which normally increases [Ca2+]i by stimulating Ca2+ influx, treatment with PKA and PKG inhibitors caused a further increase in [Ca2+]i that reverted to control levels in cells pretreated with thapsigargin. Neither Ca2+ release nor contraction induced by cGMP and NO in permeabilized muscle cells was affected by heparin or ruthenium red. Ca2+ release induced by maximally effective concentrations of cGMP and inositol 1,4,5-trisphosphate (IP3) was additive, independent of which agent was applied first. We conclude that, in the absence of PKA and PKG activity, cGMP stimulates Ca2+ release from an IP3-insensitive store and that its effect is additive to that of IP3.

Guanosine 3',5'-cyclic monophosphate (cGMP), a second messenger of nitric oxide (NO), regulates myocardial contractility. It is not known whether this effect is accompanied by a change in heart metabolism. We report here the effects of 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP), a cGMP analog, on regulatory steps of glucose metabolism in isolated working rat hearts perfused with glucose as the substrate. When glucose uptake was stimulated by increasing the workload, addition of the cGMP analog totally suppressed this stimulation and accelerated net glycogen breakdown. 8-BrcGMP did not affect pyruvate dehydrogenase activity but activated acetyl-CoA carboxylase, the enzyme that produces malonyl-CoA, an inhibitor of long-chain fatty acid oxidation. To test whether glucose metabolism could also be affected by altering the intracellular concentration of cGMP, we perfused hearts with NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase, or with S-nitroso-N-acetylpenicillamine (SNAP), a NO donor. Perfusion with L-NAME decreased cGMP and increased glucose uptake by 30%, whereas perfusion with SNAP resulted in opposite effects. None of these conditions affected adenosine 3',5'-cyclic monophosphate concentration. Limitation of glucose uptake by SNAP or 8-BrcGMP decreased heart work, and this was reversed by adding alternative oxidizable substrates (pyruvate, beta-hydroxybutyrate) together with glucose. Therefore, increased NO production decreases myocardial glucose utilization and limits heart work. This effect is mediated by cGMP, which is thus endowed with both physiological and metabolic properties.

This study aimed to characterize the cellular pathways along which nitric oxide (NO) influences the secretion of renin from the kidney. Using the isolated perfused rat kidney model, we found that the NO donor sodium nitroprusside (SNP) (1-30 mumol/l) induced a prompt, concentration-dependent fourfold increase of basal renin secretion. The membrane-permeable cGMP analogs 8-bromo-cGMP and 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP; each 5-50 mumol/l) inhibited basal renin secretion and attenuated the stimulation of renin secretion by SNP. Conversely, the renin stimulatory effect of SNP was enhanced in the presence of the G kinase inhibitor Rp-8-CPT-cGMPS (10 mumol/l). The renin stimulatory effect of SNP was amplified in nominally calcium-free perfusate and was abolished in the presence of angiotensin II (1 nmol/l). Renin secretion stimulated by SNP was clearly attenuated by the A kinase inhibitor Rp-8-CPT-cAMPS (25 mumol/l). These findings indicate that the renin stimulatory effect of NO donors in renal juxtaglomerular cells cannot be explained by activation of G kinase and is also less likely to be causally related to the regulation of renin secretion by calcium. Because A kinase activity is required for the stimulation of renin secretion by SNP, it appears as if the renin stimulatory effect is causally related to the cAMP pathway controlling renin secretion.

Increases in guanosine 3',5'-cyclic monophosphate (cGMP) induced by nitric oxide (NO), nitrovasodilators, and atrial peptides correlate with relaxation of vascular smooth muscle. Relaxation of myometrial smooth muscle by increases in cGMP, however, has required unusually high concentrations of the cyclic nucleotide. We tested the hypothesis that the sensitivity of myometrium to relaxation by cGMP is increased during pregnancy. Aortic smooth muscle was more sensitive to relaxation by cGMP than myometrial tissues, and, contrary to our hypothesis, myometrium from pregnant rats was least sensitive. Although levels of cGMP were elevated after treatment with the NO donor, S-nitroso-N-acetylpenicillamine, relaxation of myometrial tissues obtained from pregnant rats occurred only at extraordinarily high concentrations. The levels of cGMP-dependent protein kinase (PKG) were significantly decreased in myometrium from pregnant rats compared with myometrium from nonpregnant cycling animals or aortic smooth muscle. Administration of estradiol to ovariectomized rats increased myometrial PKG expression, and progesterone antagonized this response. We conclude that 1) myometrial tissues from pregnant rats are not sensitive to relaxation by cGMP and 2) this insensitivity to cGMP is accompanied by progesterone-mediated decreases in the level of PKG expression.

Guanosine 3',5'-cyclic monophosphate (cGMP)-binding, cGMP-specific phosphodiesterase (PDE5) is abundant in vascular smooth muscle, and this enzyme is a potent substrate for cGMP-dependent protein kinase (PKG) in vitro. Binding of cGMP to the allosteric sites of PDE5 is required for this phosphorylation to occur. Vascular smooth muscle cells (VSMC) were used to determine if PDE5 is phosphorylated in intact cells when cGMP is increased. With the use of anti-PDE5 antibodies, a phosphorylated 93-kDa protein band was immunoprecipitated from early passaged primary cultures of VSMC that had been preincubated with 32(Pi) to label cellular ATP and then treated with atrial natriuretic factor (ANF). In the absence of ANF, there was no detectable incorporation of radiolabeled phosphate into this band. Phosphorylation of the 93-kDa protein was augmented by pretreating cells with 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) to activate PKG before addition of ANF. 8-BrcGMP, which interacts poorly with the allosteric sites of PDE5, had no effect on PDE5 phosphorylation in the absence of ANF. Phosphorylation of PDE5 in response to treatment of cells with ANF was associated with a two- to fourfold increase in PDE activity in immunoprecipitates. Multiple-passaged VSMC, which are deficient in PKG but retain PDE5, demonstrated no ANF-dependent increase in phosphorylation or catalytic activity of PDE5. However, incubation of immunoprecipitated PDE5 from these cells with purified PKG, cGMP, and a phosphorylation mixture containing [gamma-32P]ATP resulted in 32(Pi) incorporation into PDE5 that was correlated with increased catalytic activity. These studies are the first to demonstrate phosphorylation of PDE5 in intact cells, thus suggesting a physiological role for this enzyme in smooth muscle regulation.

Nitric oxide (NO) is important in modulating increased pulmonary vascular tone. Whereas in other systems it is believed that the action of NO is mediated through guanosine 3',5'-cyclic monophosphate (cGMP) and protein kinase G (PKG), the validity of this pathway in the pulmonary circulation has not been established. Using isolated salt-perfused normotensive and hypertensive rat lungs, we studied the effects of the soluble guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), and the PKG inhibitors, KT5823, Rp-8-pCPT-cGMPS, and (N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide) (H-8), on pulmonary vascular resistance. In isolated normotensive lungs, ODQ-mediated inhibition of soluble guanylyl cyclase augmented hypoxic pulmonary vasoconstriction, whereas the PKG inhibitors had no effect. Despite the marked differences in the physiological effect, ODQ and Rp-8-pCPT-cGMPS inhibited PKG activity to a similar degree as determined by a back-phosphorylation assay showing decreased PKG-mediated phosphorylation of serine 1755 on the D-myo-inositol 1,4,5-trisphosphate receptor. In hypertensive lungs, inhibition of soluble guanylyl cyclase by ODQ increased perfusion pressure by 101 +/- 20% (P < 0.05), an increase similar to that seen with inhibition of NO synthase (NOS), confirming an essential role for cGMP. In contrast, KT5823, Rp-8-pCPT-cGMPS, and H-8 (used in doses 5- to 100-fold in excess of their reported inhibitory concentrations for PKG) caused only a small increase in baseline perfusion pressure (14 +/- 2%, P = not significant from vehicle control). Effectiveness of PKG inhibition in the hypertensive lungs was also confirmed with the back-phosphorylation assay. These studies suggest that whereas NO-mediated modulation of vascular tone in the normotensive and hypertensive pulmonary circulation is dependent on cGMP formation, activation of PKG may not be essential.

Cyclic nucleotide-gated cation channels in skeletal muscle are responsible for insulin-activated sodium entry into this tissue (J. E. M. McGeoch and G. Guidotti. J. Biol. Chem. 267: 832-841, 1992). These channels have previously been isolated from rabbit skeletal muscle by 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) affinity chromatography, which separates them into two populations differing in nucleotide affinity [L. C. Santy and G. Guidotti. Am. J. Physiol. 271 (Endocrinol. Metab. 34): E1051-E1060, 1996]. In this study, a polymerase chain reaction approach was used to identify skeletal muscle cyclic nucleotide-gated channel cDNAs. Rabbit skeletal muscle expresses the same cyclic nucleotide-gated channel as rabbit aorta (M. Biel, W. Altenhofen, R. Hullin, J. Ludwig, M. Freichel, V. Flockerzi, N. Dascal, U. B. Kaupp, and F. Hofmann. FEBS Lett. 329: 134-138, 1993). The entire cDNA for this gene was cloned from rabbit skeletal muscle and an antiserum to this protein produced. Expression of this cDNA produces a 63-kDa protein with cyclic nucleotide-gated channel activity. A similarly sized immunoreactive protein is present in sarcolemma. Purification of the expressed channels reveals that this single gene produces both native skeletal muscle channel populations.

Heme oxygenase (HO)-mediated heme degradation is the primary mechanism for production of cellular carbon monoxide (CO). Analogous to nitric oxide (NO), CO mediates physiological and cellular functions such as vasodilation, stimulation of guanylate cyclase, and neuronal transmission. In view of accumulating data demonstrating a correlation between the activity of these two gaseous molecules and that the predominant source of CO is via HO catalysis, we hypothesized that NO regulates HO expression. We demonstrate that the NO donor spermine NONOate (SNN) increases steady-state levels of HO-1 mRNA in aortic vascular smooth muscle cells (aSMC) in both a time- and dose-dependent manner. The accumulation of HO-1 mRNA that correlated with increased HO-1 protein synthesis resulted from both an increased rate of gene transcription and a decreased rate of mRNA turnover. Inhibition of the NO-induced HO-1 mRNA expression by cycloheximide suggests that new protein synthesis is required for increased HO-1 gene expression. Induction of HO-1 expression by SNN occurs in a guanosine 3',5'-cyclic monophosphate (cGMP)-independent manner because exposure of cells to 8-bromoguanosine 3',5'-cyclic monophosphate, a cGMP analog, did not increase HO-1 mRNA levels, and pretreatment of cells with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a selective guanylate cyclase inhibitor, did not prevent SNN-induced HO-1 mRNA accumulation. The antioxidant N-acetyl-L-cysteine markedly inhibited SNN-induced HO-1 mRNA expression, whereas peroxynitrite did not induce HO-1 expression in aSMC. Interestingly, CO did not attenuate NO-induced HO-1 expression through an autocrine negative feedback mechanism as had been observed for hypoxia-induced HO-1 expression. These data provide evidence for an important regulatory network between NO and CO via HO-1.

The role of Ca(2+)-activated K+ channel modulation and cyclic nucleotide second messenger signal transduction in the canine pulmonary vascular response to serotonin was determined in the isolated blood-perfused dog lung. Pulmonary vascular resistances and compliances were measured using vascular occlusion techniques. Serotonin (10(-5) M) significantly increased precapillary and postcapillary resistance and significantly decreased total vascular compliance by decreasing large vessel compliance and middle compartment compliance. Tetraethylammonium ions (TEA+; 1 mM), an inhibitor of Ca(2+)-activated K+ channels, significantly potentiated the pressor effect to serotonin on both the pulmonary arteries and pulmonary veins. Pretreatment with the guanosine 3',5'-cyclic monophosphate (cGMP)/adenosine 3',5'-cyclic monophosphate (cAMP) phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (10(-5) M), the cell membrane-permeable analog of cAMP, dibutyryl-cAMP (10(-5) M), or the cAMP-dependent vasodilator isoproterenol (10(-5) M) inhibited the serotonergic response on both the arteries and veins, which was reversed by TEA+. In contrast, the stable membrane-permeable analog of cGMP, 8-bromo-cGMP (10(-5) M), had no effect on serotonin. These results indicate that there is a basal level of vasorelaxation in canine pulmonary blood vessels that is mediated by Ca(2+)-activated K+ channel activity and that inhibition of these K+ channels increases pulmonary vascular tone and potentiates the pulmonary vasoactive response to serotonin. Also, these data suggest that cAMP-induced pulmonary vasodilation is mediated primarily by Ca(2+)-activated K+ channels and that activation of these specific K+ channels attenuates the pressor response to serotonin. Thus an important relationship appears to exist between the cAMP second messenger system and Ca(2+)-activated K+ channels in canine pulmonary vasoreactivity.

Despite intense study of neurotransmitters mediating hypothalamic controls of food intake, little is known about which second messengers are critical for these mechanisms. To determine whether adenosine 3',5'-cyclic monophosphate (cAMP) might participate in these mechanisms, we injected the membrane-permeant cAMP analog 8-bromo-cAMP (8-BrcAMP) hypothalamically in satiated rats. Injection of 8-BrcAMP (10-100 nmol) into the perifornical (PFH) and lateral hypothalamus (LH) dose dependently stimulated food intake of up to 15.7 g in 2 h. Significantly smaller responses were obtained with thalamic injections. In contrast to the strong stimulatory effects of PFH and LH 8-BrcAMP, cAMP and 8-bromo-guanosine 3',5'-cyclic monophosphate (100 nmol) were ineffective, suggesting a chemically specific, intracellular action. Consistent with this, combined PFH injection of 7-deacetyl-7-O-(N-methylpiperazino)-tau-butyryl-forskolin dihydrochloride and 3-isobutyl-1-methylxanthine, agents that increase endogeneous cAMP, stimulated eating of up to 9.9 g in 2 h. These results demonstrate that increases in PFH/LH cAMP can elicit complex, goal-oriented behavior, suggesting an important role for cAMP in hypothalamic mechanisms stimulating food intake.

It was investigated whether atrial natriuretic peptide (ANP) or the related peptide urodilatin can be used for protecting cardiomyocytes against reoxygenation-induced hypercontracture. Isolated ventricular cardiomyocytes (from adult rats) were used as the experimental model. When the cells were submitted to substrate-free anoxia (135 min) and subsequent reoxygenation (30 min), the onset of reoxygenation provoked their hypercontracture. It was studied whether the temporary presence of ANP or urodilatin (1 nM to 1 microM) or 8-bromo-guanosine 3',5'-cyclic monophosphate (8-BrcGMP; 1 microM to 1 mM) during the last 15 min of anoxia and the first 15 min of reoxygenation prevented hypercontracture. It was found that ANP (1 microM) prevented hypercontracture in 82 +/- 8% (SD), urodilatin (1 microM) in 80 +/- 9%, and 8-BrcGMP (1 mM) in 72 +/- 10% of the cells (n = 40 cells). When ANP (1 microM) was added during the last 15 min of anoxia and the first 15 min of reoxygenation, the cellular concentration of cGMP increased from 0.41 +/- 0.04 to 2.80 +/- 0.81 pmol/mg protein (n = 6 cultures). The results show that the reoxygenation-induced hypercontracture in cardiomyocytes can be attenuated by the temporary presence of the stimulators of particulate guanylate cyclase, ANP or urodilatin.

Nitric oxide (NO) released from endothelial cells or exogenous nitrates is a potent dilator of arterial smooth muscle; however, the molecular mechanisms mediating relaxation to NO in the microcirculation have not been characterized. The present study investigated the relaxant effect of nitrovasodilators on microvessels obtained from the rat mesentery and also employed whole cell and single-channel patch-clamp techniques to identify the molecular target of NO action in myocytes from these vessels. Both sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) relaxed phenylephrine-induced contractions by approximately 80% but were significantly less effective in relaxing contractions induced by 40 mM KCl. Relaxation to SNP was also inhibited by the K(+)-channel blocker tetraethylammonium or by inhibition of the activity of the guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase (PKG). These results suggest that SNP stimulated K+ efflux by opening K+ channels via PKG-mediated phosphorylation. Perforated-patch experiments revealed that both SNP and SNAP increased outward currents in microvascular myocytes, and single-channel studies identified the high-conductance Ca(2+)- and voltage-activated K+ (BKCa) channel as the target of nitrovasodilator action. The effects of nitrovasodilators on BKCa channels were mimicked by cGMP and inhibited by blocking the activity of PKG. We conclude that stimulation of BKCa-channel activity via cGMP-dependent phosphorylation contributes to the vasodilatory effect of NO on microvessels and that a direct effect of NO on BKCa channels does not play a major role in this process. We propose that this mechanism is important for the therapeutic effect of nitrovasodilators on peripheral resistance and arterial blood pressure.

Sodium nitroprusside (SNP) has been shown to elicit a guanosine 3',5'-cyclic monophosphate (cGMP)-mediated, indomethacin-sensitive contraction of the opossum esophageal longitudinal muscle. We examined the role of tyrosine phosphorylation in the signal transduction pathway of contractions induced by SNP and cGMP in longitudinal muscle strips in vitro. Force of isometric contractions was expressed as the percentage of responses to KCl (73 mM). SNP (100 microM)-induced contractions were 75 +/- 5% before and 3 +/- 2% after 50 microM genistein (P < 0.005) and 86 +/- 16% before and 0 +/- 0% after 50 microM tyrphostin B46. Contractions in response to 8-bromo-cGMP (8-BrcGMP; 1 mM) were 74 +/- 15% before and 3 +/- 2% after genistein (P < 0.01) and 63 +/- 15% before and 18 +/- 4% after tyrphostin B46 (P < 0.05). In contrast, KCl-induced contractions were 82 +/- 8% and 96 +/- 9% of the control value after genistein and tyrphostin B46 treatments, respectively (P > 0.05 for both). Carbachol contractions were partially suppressed by genistein (106 +/- 8% vs. 79 +/- 8%; P < 0.05) but unaffected by tyrphostin B46 (114 +/- 10% vs. 107 +/- 12%; P > 0.05). Western blot analysis revealed a 116-kDa phosphotyrosine protein in the control muscle strips. The level of this protein was increased to 206 +/- 15% of control after SNP treatment. Both genistein and tyrphostin B46 blocked this increase. These studies show that contractions of the esophageal longitudinal muscle induced by SNP and cGMP utilize a signal transduction pathway different from that used by the depolarizing agent KCl and the muscarinic agonist carbachol. Contractions induced by SNP and cGMP involve tyrosine phosphorylation of a protein, possibly identified as a 116-kDa protein, as a key step in the signaling pathway.

L2 cells, a cloned pneumocyte-derived cell line, express voltage-dependent L-type Ca2+ channels, causing transient depolarizing spikes of the membrane potential (Vm) [P. Dietl, T. Haller, B. Wirleitner, H. Völkl, F. Friedrich, and J. Striessing. Am. J. Physiol. 269 (Lung Cell. Mol. Physiol. 13): L873-L883, 1995]. In this study, we examined the effect of nitric oxide (NO)- and guanosine 3',5'-cyclic monophosphate (cGMP)-dependent cell signaling on the activity of L-type Ca2+ channels. Using conventional microelectrodes, spontaneous depolarizations (SD) of Vm by activation of these channels are regularly seen in the presence of 10 mM bath Sr2+. The NO donors sodium nitroprusside (SNP; 1 mM), 3-morpholinosydnonimine (SIN-1; 100 microM), as well as S-nitroso-N-acetyl-D,L-penicillamine (SNAP; 10 microM) caused a significant reduction of the frequency of Sr(2+)-induced SD. These effects were completely reversed by 6-anilino-5,8-quinolinequinone (10 microM), an inhibitor of the soluble guanylyl cyclase, and could be mimicked by 8-bromoguanosine 3'5'-cyclic monophosphate (8-BrcGMP; 100 microM). Perforated patch-clamp experiments revealed that 8-BrcGMP exerted a significant decrease of the depolarization-induced L-type Sr2+ current in the majority of tested cells. Consistent with the dependency of these NO-mediated effects on cGMP, incubation of L2 cells with SNP, SIN-1, and SNAP lead to a pronounced increase of cellular cGMP concentration. We conclude that the NO donors inhibit the activity of L-type Ca2+ channels in L2 cells via a cGMP-dependent pathway. In the alveoli, this might occur under conditions associated with the release of NO.

This study aimed to investigate whether carbon monoxide (CO), a product of heme oxygenase that degrades protoheme IX, serves as an endogenous modulator for biliary transport. To that end, effects of zinc protoporphyrin IX (ZnPP), a heme oxygenase inhibitor, on the biliary transport were tested in perfused rat liver. Perfusion of 1 microM ZnPP abolished detectable levels of CO in the venous perfusate and increased bile acid-dependent bile output accompanying an increased secretion of bile salts. The ZnPP-induced choleresis coincided with a reduction of tissue guanosine 3',5'-cyclic monophosphate (cGMP) levels and a decrease in vascular conductance. On administration of 2.5 microM CO, ZnPP-elicited choleresis, decreases in vascular conductance, and cGMP levels were all attenuated. Treatment with 1 microM 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) partly attenuated the ZnPP-induced choleresis in concert with repression of vascular conductance. Furthermore, treatment of the liver with methylene blue, a guanylate cyclase inhibitor, evoked a choleresis similar to that induced by ZnPP. Thus endogenous CO suppression stimulates the biliary transport in part through a cGMP-dependent mechanism.

We characterized the cell cycle block induced by nitric oxide (NO) on smooth muscle cells (SMC). We hypothesized that the inhibition of SMC proliferation by NO was due to a specific block in cell cycle progression. Treatment of cultured rat aortic SMC with the NO donors S-nitroso-N-acetylpenicillamine or S-nitrosoglutathione (0.1 mM for 48 h) resulted in a 50% decrease (P < 0.05) in the fraction of cells in the S and G2 + M phases and a corresponding increase in the G1 fraction, suggesting that NO inhibits entry into S phase, causing accumulation of cells in G1 phase. Application of both NO donors to cycling SMC resulted in a short-term, concentration-dependent (0.06-0.3 mM) inhibition of ongoing DNA synthesis as measured by radiothymidine incorporation, demonstrating that NO causes an S-phase arrest. The S-phase arrest by NO was not mimicked by exogenous guanosine 3',5'-cyclic monophosphate (cGMP, 10 mM) and was associated with, but not due to, a 20% inhibition of RNA synthesis. The S-phase block was completely reversed within 2 h of removal of the NO donors, similar to inhibition by the ribonucleotide reductase inhibitor hydroxyurea. Prolonged treatment of SMC with either NO donor (0.1 mM) did not synchronize cells at the G1-S boundary as expected after a prolonged S-phase arrest, but instead induced a quiescent G0-like state characterized by a 12- to 18-h lag before DNA synthesis returned to normal levels after NO removal. These findings demonstrate that NO inhibition of SMC proliferation is associated with two distinct and reversible cell cycle arrests, an immediate cGMP-independent S-phase block followed by a shift back in the cell cycle from the G1-S boundary to a quiescent G0-like state.

We used the patch-clamp technique in the split-open cortical collecting duct (CCD) to investigate the effect of nitric oxide (NO) on the low-conductance (6-pS) Na+ channel that can be blocked by 1 microM amiloride. We confirmed that the number of Na+ channels increased significantly in CCDs of rats on a low-Na+ diet (17). Application of 100 microM N(G)-nitro-L-arginine methyl ester (L-NAME), an agent that blocks endogenous NO synthase, reduced NPo [the product of channel number (N) and open probability (Po)] to 45% of the control value. The effect of L-NAME was specific, since addition of D-NAME, which does not inhibit NO synthase, did not change the activity of the Na+ channel. That the effect of L-NAME results from inhibition of NO synthase is further confirmed by experiments in which addition of an exogenous NO donor, either 10 microM S-nitroso-N-acetyl penicillamine or sodium nitroprusside (SNP), restored the Na+ channel activity when it had been blocked by L-NAME. The action of NO involves a guanosine 3',5'-cyclic monophosphate (cGMP)-dependent pathway, since 100 microM 8-bromo-cGMP (8-BrcGMP) mimicked the effect of SNAP on K+ channels. However, 100 microM 8-BrcGMP did not alter the activity of Na+ channels in inside-out patches, suggesting an indirect action. Because the Na+ channel is activated by hyperpolarization (19) and NO stimulates basolateral K+ channels (16), we tested whether hyperpolarization mediated the effect of NO. In perforated whole cell recordings, addition of L-NAME depolarized the cell membrane from -73 to 51 mV, and application of 10 microM SNP repolarized the membrane to -68 mV. Furthermore, the L-NAME-induced decrease in NPo was effectively restored by 25 mV hyperpolarization of the patch membranes, and addition of 2 mM Ba2+ also abolished the effect of L-NAME. We concluded that the stimulatory effect of NO on the Na+ channel is an indirect effect mediated by a NO-induced increase of basolateral K+ conductance.

It has been previously observed that nitric oxide (NO) contributes to hypoxic pial artery dilation and that both sodium nitroprusside (SNP), a releaser of NO, and hypoxia elicit dilation via activation of ATP-sensitive K+ channels in the newborn pig. Other studies, however, have shown that NO activates calcium-sensitive K+ (K(Ca)) channels. The present study, therefore, was designed to investigate the role of K(Ca)-channel activation in NO and hypoxic dilation and to relate this mechanism to the previously observed role of NO in hypoxic dilation in newborn pigs equipped with closed cranial windows. SNP (10(-8) and 10(-6) M) elicited pial artery dilation that was unchanged in the presence of the K(Ca)-channel antagonist iberiotoxin (10(-7) M; 10 +/- 1 and 20 +/- 1 vs. 9 +/- 1 and 20 +/- 2% for 10(-8) and 10(-6) M SNP in the absence and presence of iberiotoxin, respectively). Responses to S-nitroso-N-acetylpenicillamine and 8-bromoguanosine 3',5'-cyclic monophosphate were similarly unchanged by iberiotoxin. In contrast, iberiotoxin attenuated the dilation resulting from moderate and severe hypoxia (arterial PO2 approximately 35 and 25 mmHg, respectively; 27 +/- 1 vs. 21 +/- 2 and 34 +/- 1 vs. 16 +/- 2% for moderate and severe hypoxia in the absence and presence of iberiotoxin, respectively). Iberiotoxin blocked responses to the K(Ca)-channel agonist NS-1619, whereas responses to the ATP-sensitive K+ agonist cromakalim were unchanged (8 +/- 1 and 15 +/- 1 vs. 1 +/- 1 and 1 +/- 1% for 10(-8) and 10(-6) M NS-1619 in the absence and presence of iberiotoxin, respectively). These data show that NO and guanosine 3',5'-cyclic monophosphate do not elicit dilation via K(Ca)-channel activation. However, activation of K(Ca) channels does contribute to hypoxic pial dilation. Finally, these data suggest that substances other than NO are involved in the contribution of K(Ca)-channel activation to hypoxic pial artery dilation.

Electrical stimulation of cerebellar parallel fibers (PF) increases cerebellar blood flow (BFcrb), a response that is attenuated by glutamate receptor antagonists and NO synthase (NOS) inhibitors. We investigated whether administration of NO donors could counteract attenuation by NOS inhibitors of vasodilation produced by PF stimulation. In halothane-anesthetized rats the cerebellar cortex was exposed and superfused with Ringer solution. PF were stimulated with microelectrodes (100 microA, 30 Hz), and BFcrb was recorded by a laser-Doppler probe. During Ringer superfusion, PF stimulation increased BFcrb by 56 +/- 7% and hypercapnia by 72 +/- 5% (n = 5). Superfusion with the nonselective NOS inhibitor N-nitro-L-arginine (L-NNA, 1 mM) reduced resting BFcrb and attenuated the response to PF stimulation (-47 +/- 5%) and hypercapnia (-46 +/- 7%; PCO2 = 50-60 mmHg). After L-NNA, superfusion with the NO donors 3-morpholinosydnonimine (100 microM, n = 5) or S-nitroso-N-acetyl-penicillamine (5 microM, n = 5) reestablished resting BFcrb (P > 0.05 vs. before L-NNA) and reversed L-NNA-induced attenuation of the response to hypercapnia (P > 0.05 vs. before L-NNA) but not PF stimulation (P > 0.05 vs. after L-NNA). Similar results were obtained when NOS activity was inhibited with the inhibitor of neuronal NOS 7-nitroindazole (50 mg/kg i.p.). Like NO donors, the guanosine 3',5'-cyclic monophosphate analog 8-bromoguanosine 3',5'-cyclic monophosphate (n = 5), administered after L-NNA, restored resting BFcrb and counteracted inhibition of the response to hypercapnia but not PF stimulation. In contrast to NO donors and 8-bromoguanosine 3',5'-cyclic monophosphate, the NO-independent vasodilator papaverine (100 microM, n = 5) had no effect on attenuation of responses to PF stimulation or hypercapnia. Thus NO donors are unable to reverse the effect of NOS inhibition on vasodilation produced by PF stimulation. The data support the hypothesis that the vascular response to PF stimulation, at variance with hypercapnia, requires NOS activation and NO production. Thus NO plays an obligatory role in vasodilation produced by increased functional activity in cerebellar cortex.

With real-time confocal microscopy, the effect of three nitric oxide (NO) donors, S-nitroso-N-acetylpenicillamine, S-nitrosoglutathione, and diethylamine NO adduct, on the dynamic intracellular Ca2+ concentration ([Ca2+]i) response of porcine tracheal smooth muscle (TSM) cells to acetylcholine (ACh) was examined. ACh initiated propagating [Ca2+]i oscillations in TSM cells, which were inhibited by NO donors. 8-Bromoguanosine 3',5'-cyclic monophosphate slowed the frequency of [Ca2+]i oscillations but did not completely inhibit oscillations, suggesting that the effects of NO donors are only partially mediated via guanosine 3',5'-cyclic monophosphate-dependent mechanisms. After preexposure to NO donors, ACh induced a small biphasic [Ca2+]i response that was blocked by nifedipine, suggesting a lack of effect on Ca2+ influx through voltage-gated channels. In addition, NO donors did not inhibit Ca2+ influx induced by BAY K 8644. The [Ca2+]i response to caffeine was inhibited by NO donors, indicating inhibition of sarcoplasmic reticulum (SR) Ca2+ release. When Ca2+ influx and SR Ca2+ reuptake were blocked, basal [Ca2+]i increased, and this was inhibited by NO donors, suggesting enhanced Ca2+ efflux. These results indicate that NO donors inhibit [Ca2+]i oscillations by blocking SR Ca2+ release and enhancing Ca2+ extrusion.

We have previously demonstrated that agonists increase microvascular permeability through a phospholipase C-nitric oxide synthase-guanylate cyclase cascade. The aim of this study was to further investigate the downstream end of the signaling pathway with a focus on myosin light chain (MLC) phosphorylation. The apparent permeability coefficient to albumin was measured in isolated coronary venules. Under control conditions, the nitric oxide donor sodium nitroprusside, as well as the guanosine 3',5'-cyclic monophosphate-dependent protein kinase (PKG) activator 8-bromoguanosine 3',5'-cyclic monophosphate, increased venular permeability two- to threefold. Similarly, activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate significantly elevated permeability. Inhibition of MLC phosphorylation with ML-7 significantly attenuated the hyperpermeability responses to the agonists. Furthermore, ML-7 dose dependently reduced basal venular permeability. Consistently, inhibition of dephosphorylation with the protein phosphatase inhibitor calyculin dramatically increased basal permeability. These results suggest that 1) PKG and PKC play an important signaling role in the regulation of endothelial barrier function and 2) MLC phosphorylation contributes to basal and agonist-stimulated microvascular permeability.

This study was designed to test the hypothesis that active thermoregulatory vasodilation (AVD) is the result of a neurotransmitter-induced adenosine 3',5'-cyclic monophosphate (cAMP) pathway interacting with a nitric oxide-induced guanosine 3',5'-cyclic monophosphate (cGMP) pathway. Rabbits were instrumented for measurement of arterial pressure and ear blood flow (EBF) and the infusion of drugs. In four groups of conscious animals, whole-body heating increased EBF from 0.5 +/- 0.3 to 8.3 +/- 1.3 kHz. In group 1 (n = 6), N(omega)-nitro-L-arginine methyl ester (L-NAME, a nitric oxide synthase inhibitor, 10-40 mg) reduced EBF from 7.1 +/- 0.9 to 1.9 +/- 0.5 kHz. Subsequent infusion of 8-bromo-cGMP (a cGMP analog, 5-10 mg) returned EBF to 6.2 +/- 0.7 kHz. In group 2 (n = 3), (R)-p-adenosine 3',5'-cyclic monophosphothioate (a cAMP-dependent protein kinase inhibitor, 10 mg) reduced EBF to 1.6 +/- 0.4 kHz. In group 3 (n = 6), nerve blockade of the ear (procaine, 20 mg/ml, 1.5 ml) reduced EBF from 8.6 +/- 1.3 to 1.6 +/- 0.3 kHz. Subsequent infusion of 8-bromo-cAMP (a cAMP analog, 5-10 mg) returned EBF to 8.3 +/- 2.0 kHz. In group 4 (n = 6), the infusion of L-NAME caused EBF to fall from 9.0 +/- 1.1 to 1.2 +/- 0.3 kHz. Infusion of the cAMP phosphodiesterase inhibitor Ro 20-1724 (0.2-0.5 mg) raised EBF to 5.5 +/- 0.7 kHz. These results suggest that cGMP plays a permissive role in AVD and indicate that the transmitter acts through cAMP.

We investigated the mechanisms of NO-induced antimitogenesis in primary aortic smooth muscle cells from newborn rats. S-nitroso-N-acetylpenicillamine (SNAP), an NO-releasing agent, decreased basal and growth factor-stimulated DNA synthesis with a threshold effectiveness of 0.3-3 microM. A second NO-releasing agent, 3-morpholinosydnonimine-N-ethylcarbamide, a hydrolysis-resistant cyclic nucleotide, 8-bromo-guanosine 3',5'-cyclic monophosphate (8-BrcGMP), and atrial natriuretic peptides elicited a similar effect, whereas 8-bromo-adenosine 3',5'-cyclic monophosphate (8-BrcAMP) was ineffective, supporting the view that NO and cGMP, but not cAMP, mediated at least some of SNAP's antimitogenic effect. SNAP and 8-BrcGMP decreased the levels of phosphotyrosine, especially in proteins of 70-85 kDa and approximately 215 kDa molecular mass. SNAP decreased protein phosphotyrosine levels with a threshold effectiveness similar to that of its antimitogenic effect. Moreover, SNAP increased protein tyrosine phosphatase (PTPase) activity in cell homogenates, indicating that phosphotyrosine dephosphorylation was likely to be the result of increased PTPase activity. Peroxovanadate, a selective PTPase inhibitor, blocked the antimitogenic effect of 8-BrcGMP, suggesting that loss of protein phosphotyrosine and antimitogenesis were causally linked. These findings describe a potential mechanism for NO-induced antimitogenesis in aortic smooth muscle cells in primary culture.

Conducted vasodilation has been proposed as an important component of local vascular control. Because conducted vasomotor responses have previously been studied only in response to short pulses (<500 ms) of agonist, this study examined conducted vasodilation in response to sustained stimuli. In addition, we examined the contribution of nitric oxide (NO) to initiation and maintenance of conducted responses induced by acetylcholine (ACh). Responses to 2-min applications of ACh, sodium nitroprusside, and 8-bromoguanosine 3',5'-cyclic monophosphate were obtained in cannulated, perfused hamster cheek pouch arterioles (approximately 60 microm in diameter). Changes of luminal diameter in response to pressure ejection of agonists from a micropipette placed close to the downstream end of the vessel were observed at the site of stimulation ("local") as well as 570 and 1,140 microm upstream. At the local site, ACh stimuli produced large changes in diameter (approximately 70% of the maximum response) that peaked within 45 s before declining slowly to levels of approximately 50% of the maximum response. A similar response pattern was observed at both upstream sites, with the conducted responses being maintained for the duration of the stimulus. Local responses of similar magnitude were found with sodium nitroprusside and 8-bromoguanosine 3',5'-cyclic monophosphate, but only minimal responses were observed at the conducted sites. In a separate set of arterioles, ACh responses were obtained before and during perfusion with 10 microM N(omega)-nitro-L-arginine. Inhibition of NO synthesis diminished the local response to ACh, but the initial phase of the conducted response was unaffected. Furthermore, the conducted responses faded more rapidly in the presence of N(omega)-nitro-L-arginine. We conclude from these results that local NO synthesis alone is insufficient to initiate conducted responses but that NO synthesis contributes to maintenance of sustained conducted responses.

High concentrations of nitric oxide (NO) inhibit bone resorption by mature osteoclasts. We examined the effects of low NO concentrations on osteoclast formation in mouse bone marrow cultures. The NO releasers sodium nitroprusside (SNP) and S-nitroso-N-acetyl-DL-penicillamine inhibited the formation of multinucleated cells expressing tartrate-resistant acid phosphatase (a marker for osteoclasts) when administered during the last 3 days of 6-day cultures (differentiation stage) but not during the first 3 days (proliferation stage). SNP (1 microM) completely inhibited pit formation on dentine wafers when added to cultures during osteoclast formation, but 100 microM SNP was required to inhibit pitting by mature osteoclasts. Conversely, the NO synthase inhibitors aminoguanidine and nitro-L-arginine methyl ester both increased osteoclast formation. Inhibition of osteoclast formation by NO likely was guanosine 3',5'-cyclic monophosphate (cGMP) dependent, as SNP increased cGMP in marrow cultures, and 1 mM 8-bromo-cGMP or dibutyryl-cGMP reduced osteoclast formation when administered during the differentiation stage. The cGMP-specific type V phosphodiesterase inhibitor, zaprinast (M & B 22948) also inhibited osteoclast formation (half-maximal inhibitory constant, 100 microM) only when added during the differentiation stage. We conclude that the differentiation stage of osteoclast formation is inhibited by increases in cGMP levels elicited by NO.

The opioids methionine enkephalin and leucine enkephalin contribute to hypoxic pial artery dilation in the newborn pig, and adenosine 3',5'-cyclic monophosphate (cAMP) analogs have been shown to elevate cerebrospinal fluid (CSF) opioid concentration. The present study was designed to investigate the contribution of cAMP to hypoxic dilation and to determine whether an endogenous activator of adenylate cyclase, pituitary adenylate cyclase-activating peptide (PACAP), could modulate the cAMP-induced release of opioids to contribute to hypoxic pial dilation in piglets equipped with closed cranial windows. An alpha level of P < 0.05 was considered significant in all statistical tests. Moderate and severe hypoxia (PO2 approximately 35 and 25 mmHg, respectively) induced pial artery dilation that was attenuated by the Rp diastereomer of 8-bromoadenosine 3',5'-cyclic monophosphothioate (Rp-8-BrcAMPS), a cAMP antagonist (24 +/- 1 and 36 +/- 2% vs. 21 +/- 1 and 30 +/- 1% for moderate hypoxia and 34 +/- 1 and 46 +/- 2% vs. 24 +/- 1 and 32 +/- 1% for severe hypoxia before and after Rp-8-BrcAMPS, respectively). These responses were associated with an increased CSF cAMP (1,046 +/- 25, 1,366 +/- 28, and 1,735 +/- 47 fmol/ml for control, moderate, and severe hypoxia, respectively). Hypoxic pial dilation was also accompanied by an increase in CSF methionine enkephalin (1,101 +/- 62, 3,283 +/- 119, and 3,835 +/- 129 pg/ml for control, moderate, and severe hypoxia, respectively). Hypoxic dilation additionally increased CSF PACAP (1,727 +/- 86, 2,268 +/- 157, and 7,980 +/- 238 pg/ml for control, moderate, and severe hypoxia, respectively). PACAP (10(-8) and 10(-6) M) elicited pial dilation that was associated with increased CSF cAMP and blunted by Rp-8-BrcAMPS. PACAP-induced dilation was also accompanied by increases in the opioid methionine enkephalin (1,059 +/- 23, 1,483 +/- 34, and 2,108 +/- 77 pg/ml for control and 10(-8) and 10(-6) M PACAP, respectively). These data show that cAMP contributes to hypoxic pial artery dilation. Hypoxia increases CSF PACAP, whereas PACAP elevates CSF opioid concentration. These data, therefore, suggest that PACAP modulates cAMP-induced opioid release, thereby contributing to hypoxic pial dilation.

The effect of guanosine 3',5'-cyclic monophosphate (cGMP) on cytosolic Ca2+ dynamics and associated alterations in macromolecule permeability was investigated in cultured monolayers of aortic endothelial cells. Addition of the membrane-permeable cGMP analogue 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP, 5 x 10(-4)M) or activators of the soluble (3-morpholinosydnonimine, 10(-5) M) or the particulate guanylyl cyclase (atrial natriuretic peptide, 10(-7) M) to unstimulated monolayers led to a decrease in permeability (8-BreGMP: 62 +/- 8% of control) without affecting low basal cytosolic Ca2+ concentration ([Ca2+]i, 87 +/- 8 nM). In contrast, under conditions of elevated [Ca2+]i (503 +/- 95 nM) and increased permeability (155 +/- 7% of control) induced by 10(-6) M ionomycin, 8-BrcGMP, 3-morpholinosydnonimine, or atrial natriuretic peptide provoked a further increase in permeability (8-BrcGMP: 255 +/- 27%). These agents failed to increase permeability when added before or after the ionomycin-triggered transitory rise in [Ca2+]i. The increase in permeability in response to 8-BrcGMP was due to a secondary further rise in [Ca2+]i (758 +/- 87 nM), which was abolished in the absence of extracellular Ca2+, indicating influx of exogenous Ca2+ as the cause. Changes in [Ca2+]i and permeability were inhibited, in the presence of the Rp diastereomer of 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphothioate (2 x 10(-5) M), an inhibitor of the cGMP-dependent protein kinase. These findings show that, depending on [Ca2+]i, cGMP can play opposite roles in endothelial permeability in one and the same cell preparation.

In the present study, effects of the nitric oxide donor, S-nitroso-N-acetylpenicillamine (SNAP), on sarcoplasmic reticulum (SR) Ca2+ release were examined in freshly dissociated porcine tracheal smooth muscle (TSM) cells. Fura 2-loaded TSM cells were imaged using video fluorescence microscopy. SR Ca2+ release was induced by acetylcholine (ACh), which acts principally through inositol 1,4,5-trisphosphate (IP3) receptors, and by caffeine, which acts principally through ryanodine receptors (RyR). SNAP inhibited ACh-induced SR Ca2+ release at both 0 and 2.5 mM extracellular Ca2+. Degraded SNAP had no effect on ACh-induced SR Ca2+ release. SNAP also inhibited caffeine-induced SR Ca2+ release. ACh-induced Ca2+ influx was not affected by SNAP when SR reloading was blocked by thapsigargin. SNAP also did not affect SR Ca2+ reuptake. The membrane-permeant analogue of guanosine 3',5'-cyclic monophosphate (cGMP), 8-bromo-cGMP, mimicked the effects of SNAP. These results suggest that, in porcine TSM cells, SNAP reduces the intracellular Ca2+ response to ACh and caffeine by inhibiting SR Ca2+ release through both IP3 and RyR, but not by inhibiting influx or repletion of the SR Ca2+ stores. These effects are likely mediated via cGMP-dependent mechanisms.

It has been observed that prostaglandins (PG) PGE2 and PGI2 increased cortical periarachnoid cerebrospinal fluid (CSF) methionine enkephalin (Met-enk) and leucine enkephalin (Leu-enk) concentrations in the newborn pig. It was also observed that PG-induced pial artery dilation was associated with elevated CSF guanosine 3',5'-cyclic monophosphate (cGMP) and adenosine 3',5'-cyclic monophosphate (cAMP) levels in the piglet. However, other studies have not always supported a role for cGMP in PG dilation. The present study used a pharmacological approach to test the hypothesis that both cGMP and cAMP contribute to PG-induced pial dilation and associated elevated CSF opioid concentration. PGE2 produced pial vasodilation that was blunted by the Rp diastereomer of bromoguanosine 3',5'-cyclic monophosphothioate [Rp-8-BrcGMPS (10(-5)M)], a cGMP antagonist (9 +/- 1, 16 +/- 1, and 23 +/- 1 vs. 4 +/- 1, 6 +/- 1, and 9 +/- 1% for 1, 10, and 100 ng/ml PGE2 before and after Rp-8-BrcGMPS, respectively). PGE2 elevated CSF Met-enk concentration, and these biochemical changes were also blunted by Rp-8-BrcGMPS (1,001 +/- 23, 1,424 +/- 54, and 1,973 +/- 56 vs. 804 +/- 41, 988 +/- 52, and 1,222 +/- 21 pg/ml for control, 10, and 100 ng/ml PGE2 in the absence and presence of Rp-8-BrcGMPS, respectively). Similar biochemical and vascular effects of Rp-8-BrcGMPS were observed for PGI2. Additionally, the Rp diastereomer of bromoadenosine 3',5'-cyclic monophosphothioate [Rp-8-BrcAMPS (10(-5)M)], a cAMP antagonist, blunted PGE2 dilation (10 +/- 1, 15 +/- 1, and 24 +/- 1 vs. 5 +/- 1, 8 +/- 1, and 12 +/- 1% for 1, 10, and 100 ng/ml PGE2 before and after Rp-8-BrcAMPS, respectively). PGE2-associated increases in CSF Met-enk and Leu-enk were similarly blunted by Rp-8-BrcAMPS. These data show that both cGMP and cAMP contribute to PG-induced pial dilation and that PG-associated elevated CSF cGMP and cAMP levels result in increased CSF Met-enk and Leu-enk concentration.

The objective of this study was to determine whether nitric oxide (NO) could function as a negative feedback modulator of endothelium-dependent vasodilation in vivo. To this end, the influence of exogenous NO on vasodilator responses in the rabbit hindquarters vascular bed was determined. Previous in vitro studies have demonstrated that NO inhibits both neuronal NO synthase from rat cerebellum as well as NO synthase derived from bovine aortic endothelial cells. The present study was conducted in the rabbit hindquarters vascular bed under conditions of constant blood flow so that changes in pressure directly reflected changes in vascular resistance. Under these in vivo conditions, the NO donor agent S-nitroso-N-acetylpenicillamine (SNAP) reversibly attenuated responses to the endothelium-dependent vasodilators, acetylcholine and bradykinin. In contrast, SNAP did not influence the endothelium-independent vasodilator response to SNAP itself or to 8-bromoguanosine 3',5'-cyclic monophosphate. These observations indicate clearly that NO interferes with endothelium-dependent vasodilator action and support the view that endogenous NO may actually play a physiological role in regulating vascular tone.

Synchronization of circadian rhythms is thought to be accomplished primarily through daily phase delays and advances of the endogenous circadian clock that, in mammals, is located in the hypothalamic suprachiasmatic nucleus (SCN). In the SCN, numerous second messenger pathways may participate in photic signal transduction. In these studies, the involvement of cyclic nucleotide-dependent kinases was examined in vivo using inhibitors of adenosine 3',5'-cyclic monophosphate (cAMP)- and guanosine 3',5'-cyclic monophosphate (cGMP)-dependent kinase (PKA and PKG, respectively). In constant dark, selective and nonselective inhibitors of PKG injected near the SCN of hamsters had no effect on phase delays produced by light pulses given in the early subjective night (early in the animals' active period) but significantly attenuated phase advances induced late in the subjective night. PKA inhibition had no effect at either time point. In addition, cGMP agonists had no effect on rhythmicity in the absence of light. The results suggest that PKG activity is necessary, but not sufficient, for normal photic responsiveness and that PKA activity is not required. The phase dependence of the effect of PKG inhibition supports the notion that photic entrainment is influenced by biochemical pathways that differentially regulate sensitivity in a phase-dependent manner.