Nitric oxide as an autocrine regulator of sodium currents in baroreceptor neurons.

Arterial baroreceptors are mechanosensitive nerve endings in the aortic arch and carotid sinus that play a critical role in acute regulation of arterial blood pressure. A previous study has shown that nitric oxide (NO) or NO-related species suppress action potential discharge of baroreceptors. In the present study, we investigated the effects of NO on Na+ currents of isolated baroreceptor neurons in culture. Exogenous NO donors inhibited both tetrodotoxin (TTX) -sensitive and -insensitive Na+ currents. The inhibition was not mediated by cGMP but by NO interaction with channel thiols. Acute inhibition of NO synthase increased the Na+ currents. NO scavengers (hemoglobin and ferrous diethyldithiocarbamate) increased Na+ currents before but not after inhibition of NO synthase. Furthermore, NO production in the neuronal cultures was detected by chemiluminescence and immunoreactivity to the neuronal isoform of NO synthase was identified in fluorescently identified baroreceptor neurons. These results indicate that NO/NO-related species function as autocrine regulators of Na+ currents in baroreceptor neurons. Modulation of Na+ channels may represent a novel response to NO.

Diet-induced atherosclerosis increases the release of nitrogen oxides from rabbit aorta.

We examined the hypothesis that impaired endothelium-dependent vasodilation in atherosclerosis is associated with decreased synthesis of nitrogen oxides by the vascular endothelium. The descending thoracic aortae of rabbits fed either normal diet, a high cholesterol diet for 2-5 wk (hypercholesterolemic, HC), or a high cholesterol diet for 6 mo (atherosclerotic, AS) were perfused in a bioassay organ chamber with physiologic buffer containing indomethacin. Despite a dramatic impairment in the vasodilator activity of endothelium-dependent relaxing factor (EDRF) released from both HC and AS aortae (assessed by bioassay), the release of nitrogen oxides (measured by chemiluminescence) from these vessels was not reduced, but markedly increased compared to NL. Thus, impaired endothelium-dependent relaxation in atherosclerosis is neither due to decreased activity of the enzyme responsible for the production of nitrogen oxides from arginine nor to arginine deficiency. Because the production of nitrogen oxides increased in response to acetylcholine in both hypercholesterolemic and atherosclerotic vessels, impairments in signal transduction are not responsible for abnormal endothelium-dependent relaxations. Impaired vasodilator activity of EDRF by cholesterol feeding may result from loss of incorporation of nitric oxide into a more potent parent compound, or accelerated degradation of EDRF.

5-HT activates vagal afferent cell bodies in vivo: role of 5-HT2 and 5-HT3 receptors.

Occipital artery (OA) injections of 5-HT elicit pronounced reductions in heart rate and mean arterial blood pressure (MAP) in urethane-anesthetized rats by activation of vagal afferent cell bodies in the ipsilateral nodose ganglion. In contrast, internal carotid artery (ICA) and i.v. injections elicit similar cardiovascular responses by activation of peripheral vagal afferent terminals. The aim of this study was to examine the roles of 5-HT3 and 5-HT2 receptors in the 5-HT-induced activation of vagal afferent cell bodies and peripheral afferent terminals in urethane-anesthetized rats. OA, ICA and i.v. injections of 5-HT elicited dose-dependent reductions in heart rate and MAP that were virtually abolished after i.v. administration of the 5-HT3 receptor antagonists, MDL 7222 or ICS 205-930. The responses elicited by the OA injections of 5-HT were markedly diminished after i.v. injection of the 5-HT2 receptor antagonists, xylamidine or ketanserin, whereas the responses elicited by i.v. or ICA injections of 5-HT were not affected. The present findings suggest that (1) 5-HT3 and 5-HT2 receptor antagonists gain ready access to nodose ganglion cells upon i.v. administration, and (2) functional 5-HT3 and 5-HT2 receptors exist on the cell bodies of vagal afferent neurons mediating the cardiovascular responses elicited by OA injections of 5-HT. These findings also support a wealth of evidence that 5-HT3 receptors exist on the peripheral terminals of vagal afferents, and although they do not discount the possibility that 5-HT2 receptors exist on peripheral vagal afferent terminals, it appears that activation of these receptors does not have pronounced effects on 5-HT3 receptor activity on terminals that mediate the hemodynamic responses to 5-HT.

Occipital artery injections of 5-HT may directly activate the cell bodies of vagal and glossopharyngeal afferent cell bodies in the rat.

The primary objective of this study was to determine whether circulating factors gain direct access to and affect the activity of vagal afferent cell bodies in the nodose ganglia and glossopharyngeal afferents cell bodies in the petrosal ganglia, of the rat. We found that the occipital and internal carotid arteries provided the sole blood supply to the nodose ganglia, and that i.v. injections of the tracer, Basic Blue 9, elicited strong cytoplasmic staining in vagal and glossopharyngeal afferent cell bodies that was prevented by prior ligation of the occipital but not the internal carotid arteries. We also found that occipital artery injections of 5-HT elicited pronounced dose-dependent reductions in heart rate and diastolic arterial blood pressure that were (1) virtually abolished after application of the local anesthetic, procaine, to the ipsilateral nodose and petrosal ganglia, (2) markedly attenuated after transection of the ipsilateral vagus between the nodose ganglion and brain and virtually abolished after subsequent transection of the ipsilateral glossopharyngeal nerve between the petrosal ganglion and the brain, (3) augmented after ipsilateral transection of the aortic depressor and carotid sinus nerves, and (4) augmented after transection of all ipsilateral glossopharyngeal and vagal afferent nerves except for vagal cardiopulmonary afferents. These findings suggest that blood-borne 5-HT in the occipital artery gains direct access to and activates the cell bodies of vagal cardiopulmonary afferents of the rat and glossopharyngeal afferents of undetermined modalities.

Use-dependent loss of acetylcholine- and bradykinin-mediated vasodilation after nitric oxide synthase inhibition. Evidence for preformed stores of nitric oxide-containing factors in vascular endothelial cells.

In the present study, we examined the possibility that the endothelium-dependent vasodilators acetylcholine and bradykinin release preformed pools of nitric oxide-containing factors. Successive injections of selected doses of acetylcholine (1.18 +/- 0.3 micrograms/kg IV) or bradykinin (5 micrograms/kg IV) caused reproducible hypotensive and vasodilator responses within sympathetically intact and sympathetically denervated hindlimbs of conscious rats. After administration of the nitric oxide synthesis inhibitor N omega-nitro-L-arginine methyl ester (L-NAME, 25 mumol/kg IV), the first injection of acetylcholine or bradykinin produced pronounced depressor and vasodilator responses that, in the case of bradykinin, were greater than those observed before L-NAME administration. However, each successive injection of acetylcholine and bradykinin produced progressively smaller responses, such that the later injections elicited a markedly diminished hypotension and vasodilation. This "use-dependent" loss of endothelium-dependent vasodilation was not due to the diminished vasorelaxant potency of nitric oxide-containing factors because the vasodilator effects of the nitric oxide donor sodium nitroprusside (32 micrograms/kg IV) and the S-nitrosothiol compound S-nitro-socysteine (200 nmol/kg IV) were augmented in the presence of L-NAME. These results suggest that the use-dependent loss of the hemodynamic effects of acetylcholine and bradykinin in L-NAME-treated rats may be due to the release and subsequent depletion of a factor whose synthesis depends on the bioavailability of nitric oxide. Taken together, these results suggest that preformed pools of nitric oxide-containing factors exist within the endothelium of resistance vessels and that endothelium-dependent agonists exert their vasorelaxant effects at least in part by the mobilization of these performed pools.

Stabilization of the reduced halocarbon-cytochrome P-450 complex of halothane by N-alkanes.

Under anaerobic conditions, various halogenated compounds, when metabolized by cytochrome P-450, form complexes which are spectrally detectable. Previous studies have shown that halothane forms such a complex with cytochrome P-450, and the result is a strong absorption at 470 nm. Stabilization of this proposed intermediate carbanion complex has never been demonstrated in a biological system. Data are presented which show that several organic solvents (C5-C7N-alkanes) will stabilize the complex formed between halothane and cytochrome P-450. Stabilization allowed the decay of the complex to be studied, and it is demonstrated that the product of decay was chlorodifluoroethylene, which substantiates the hypothesis that the complex is a two electron-reduced carbanion. Carbon tetrachloride and benzyl bromide, which also produce spectrally visible intermediate complexes, were not stabilized by this treatment. Stabilization of the halothane complex in a biological system may facilitate studies to identify precisely the halothane-cytochrome P-450 complex and to clarify the mechanisms of halothane reduction by cytochrome P-450.

Nitric oxide generation from nitroprusside by vascular tissue. Evidence that reduction of the nitroprusside anion and cyanide loss are required.

Nitric oxide (NO) was produced from sodium nitroprusside in the presence of vascular tissue but was not released spontaneously from the nitroprusside anion. In the absence of tissue in the dark nitroprusside did not release NO. When solutions of nitroprusside alone were irradiated with visible light, nitric oxide was released at rates linearly proportional to nitroprusside concentration and light intensity. Nitric oxide was produced from solutions of nitroprusside in the dark after the addition of vascular tissue, including lengths of rabbit aorta, subcellular fractions of aorta, and human plasma. NO was also released from nitroprusside after reaction with various reducing agents including cysteine and other thiols, ascorbic acid, sodium dithionite, ferrous chloride, hemoglobin, myoglobin, and partially purified cytochrome P450 with an NADPH-regenerating system. HCN was simultaneously produced in these solutions, and addition of KCN blocked NO release. Iodine oxidized intermediate cyanoferrates and blocked nitric oxide release. KCN or iodine also blocked NO production by tissue, but had no effect upon photochemical NO release. These results show that, apart from photolysis which makes no physiological contribution, release of nitric oxide from nitroprusside, in simple solutions and in biological tissue, occurs after nitroprusside has undergone reduction and lost cyanide.

Nitric oxide inhibits aromatase activity: mechanisms of action.

NO synthase is present in human ovarian granulosa-luteal cells and NO inhibits estradiol secretion by granulosa cells in culture. These findings suggest that NO is an autocrine regulator of ovarian steroidogenesis. The purpose of this investigation was to explore the mechanisms through which NO exerts an inhibitory effect on cytochrome P450 aromatase activity. To examine the effect of NO on aromatase mRNA levels, human granulosa-luteal cells were cultured in the presence or absence of the NO donor SNAP for 16 h. Using a probe for human aromatase, Northern blots revealed a 26% decrease in aromatase mRNA in cells exposed to SNAP. Because this modest decrease in mRNA is unlikely to explain a rapid and profound reduction in estradiol secretion that we have observed, we looked for direct effects of NO on cytochrome P450 aromatase activity. Aromatase activity was assayed in placental microsomes and granulosa-luteal cells by measuring the release of 3H2O from [1 beta-3H] androstenedione. NO (10(-4)-10(-3)M), added as a saturated saline solution, reduced aromatase activity by as much as 90% in a concentration-dependent, non-competitive manner. In contrast, carbon monoxide (CO), a gas known to bind to the heme iron in aromatase, had no effect on aromatase activity when added alone nor could CO reverse the NO-induced inhibition of aromatase. These data suggest that NO binding to the heme is insufficient to inhibit aromatase activity. NO has been reported to alter protein function by reacting with the sulfhydryl group of cysteines, forming a nitrosothiol group. Because a cysteine sulfhydryl group is thought to participate in the catalytic mechanism of all P450 enzymes, experiments were designed to test whether NO might inhibit aromatase via such a mechanism. Addition of increasing amounts of mercaptoethanol, a chemical with free sulfhydryl groups, blocked the NO-induced inhibition of aromatase in microsomes. N-Ethylmaleimide, a chemical which covalently modifies sulfhydryl groups, reduced aromatase activity in a concentration-dependent manner. We conclude that NO inhibits aromatase both by decreasing mRNA for the enzyme and by an acute, direct inhibition of enzyme activity. We hypothesize that the direct inhibition occurs as a result of the formation of a nitrosothiol on the cysteine residue adjacent to the heme in aromatase.

Continuous infusion epidural analgesia with lidocaine: efficacy and influence during the second stage of labor.

A randomized double-blind study evaluated the analgesic efficacy and influence of maintaining a continuous epidural infusion of 0.75% lidocaine during the second stage of labor in nulliparous women. When the cervix was 8 cm or more dilated, unidentified study solution was substituted for the known 0.75% lidocaine solution and continued until delivery. The study solution for 26 patients was 0.75% lidocaine; 27 subjects received saline. During the first stage of labor, 88% of women in the lidocaine group and 81% of women in the saline group had analgesia of excellent or good quality, a nonsignificant difference. During the second stage, there was a tendency (not statistically significant) toward improved analgesia quality in the lidocaine patients, but there was no significant difference in the frequency of perineal anesthesia (23% lidocaine, 7% saline). There was no difference between the groups in the duration of the second stage of labor (73 +/- 63 versus 76 +/- 48 minutes). Operative delivery frequency was similar (31 and 37%), as were umbilical cord blood acid-base values. It is concluded that maintenance of the continuous epidural infusion of 0.75% lidocaine did not prolong the second stage of labor, but it also did not significantly differ from saline in quality of second stage analgesia or frequency of perineal anesthesia.

Effect of heating on vascular reactivity in rat mesenteric arteries.

Vasoconstriction in the viscera is one of the primary cardiovascular adjustments to heating. Local temperature can influence vascular responsiveness to catecholamines and sympathetic nerve activity. Therefore, we hypothesized that heating would alter vascular reactivity in rat mesenteric arteries. Concentration-response curves to norepinephrine, phenylephrine, potassium chloride (KCl), calcium, acetylcholine, and sodium nitroprusside were obtained in vascular ring segments from rat mesenteric arteries at 37 and 41 degrees C. In some rings, basal tension increased slightly during heating. Heating to 41 degrees C did not alter the contractile responses to norepinephrine in endothelium-intact or -denuded rings but augmented the responses to KCl and calcium in endothelium-intact rings. The potentiating effect of heating on the responses to KCl and calcium was eliminated after endothelium removal. In contrast, the relaxant responses to acetylcholine and sodium nitroprusside were significantly attenuated at 41 degrees C. Collectively, these results demonstrate that heating alters vascular reactivity in rat mesenteric arteries. Furthermore, these data imply that heating reduces the ability of vascular smooth muscle to relax, possibly due to a decrease in sensitivity to nitric oxide.

Modulation of temperature-induced tone by vasoconstrictor agents.

One of the primary cardiovascular adjustments to hyperthermia is a sympathetically mediated increase in vascular resistance in the viscera. Nonneural factors such as a change in vascular tone or reactivity may also contribute to this response. Therefore, the aim of this study was to determine whether vascular smooth muscle tone is altered during heating to physiologically relevant temperatures >37 degrees C. Gradually increasing bath temperature from 37 degrees C (normothermia) to 43 degrees C (severe hyperthermia) produced graded contractions in vascular ring segments from rat mesenteric arteries and thoracic aortae. In untreated rings these contractions were relatively small, whereas hyperthermia elicited near-maximal increases in tension when rings were constricted with phenylephrine or KCl before heating. In phenylephrine-treated mesenteric arterial rings, the contractile responses to heating were markedly attenuated by the Ca2+ channel antagonists nifedipine and diltiazem. Diltiazem also blocked the contractile responses to heating in thoracic aortic rings. These results demonstrate that hyperthermia has a limited effect on tension generation in rat vascular smooth muscle in the absence of vascular tone. However, in the presence of agonist-induced tone, tension generation during heating is markedly enhanced and dependent on extracellular Ca2+. In conclusion, these data suggest that local regulation of vascular tone can contribute to the hemodynamic adjustments to hyperthermia.

Actions of S-nitrosocysteine in the nucleus tractus solitarii are unrelated to release of nitric oxide.

Cardiovascular effects elicited by microinjection of L-S-nitrosocysteine in the nucleus tractus solitarii (NTS) were compared and contrasted with those produced by the dextroisomer, other nitric oxide donors and nitric oxide itself. L-S-nitrosocysteine produced dose-related decreases of arterial pressure and heart rate. In contrast, D-S-nitrosocysteine, S-nitrosoglutathione, glyceryl trinitrate, and sodium nitroprusside produced minimal responses that were not dose-related. Likewise, injection of cystine and nitric oxide, two products of S-nitrosocysteine breakdown, produced no significant response. Headspace analysis using chemiluminescence revealed that L- and D-S-nitrosocysteine released identical amounts of nitric oxide when exposed to homogenates of whole rat brain. Responses to L-S-nitrosocysteine were not affected by local injection of oxyhemoglobin or the nitric oxide synthase inhibitor L-nitroarginine methylester. Although injection of L-cysteine into the NTS produced responses similar to those seen with injection of L-S-nitrosocysteine, blockade of excitatory amino acid receptors with kynurenic acid inhibited responses to cysteine but not those to the nitrosothiol. The study demonstrates that S-nitrosocysteine is biologically active in the NTS. Its action is independent of release of nitric oxide from the nitrosothiol but may be mediated through stereoselective sites on target neurons.

Is there a role for an endothelium-derived relaxing factor in nociception?

Many of the circulating algesic agents released in response to ischemia produce a profound vasodilatation possibly through the release of an endothelium-derived relaxing factor (EDRF) as well as pain. We report here that intravenously administered S-nitrosocysteine, a putative EDRF, and not the nitric oxide liberating compound sodium nitroprusside produces significant alterations in nociceptive behavior that are abolished by bilateral vagotomy. These results are consistent with a role for EDRF in peripheral nociceptive mechanisms.

Redox regulation of S-nitrosocysteine-mediated vasodilation in vivo.

This study examined the effects of the lipophobic electron acceptor, nitroblue tetrazolium (2x5 micromol/kg, i.v.) on the vasodilation produced by the putative endothelium-derived S-nitrosothiol, L-S-nitrosocysteine (400 nmol/kg, i.v.), and the nitric oxide (NO) donor, (Z)-1-N-methyl-N-[6(N-methylammoniohexyl)amino]&z. sfnc;diazen-1-ium-1,2-diolate (MAHMA NONOate, 25 nmol/kg, i.v.), in anesthetized rats. The administration of nitroblue tetrazolium resulted in delayed but long-lasting increases in vascular resistances. The L-S-nitrosocysteine-induced vasodilator responses were markedly diminished whereas the MAHMA NONOate-induced responses were not affected by nitroblue tetrazolium. These results support the possibility that L-S-nitrosocysteine interacts with membrane thiols that are subject to nitroblue tetrazolium-induced oxidation (i.e., disulfide-bridge formation) and that nitroblue tetrazolium-induced vasoconstriction may involve a loss of potency of endothelium-derived S-nitrosothiols.

In vivo evidence that L-S-nitrosocysteine may exert its vasodilator effects by interaction with thiol residues in the vasculature.

This study examined the effects of the lipophobic thiol chelator, para-hydroxymercurobenzoic acid (25 and 50 micromol/kg, i.v.) on the falls in mean arterial blood pressure and regional vascular resistances produced by L-S-nitrosocysteine (400 nmol/kg, i.v.) and the nitric oxide (NO)-donors, (Z)-1-&z. sfnc;N-methyl-N-[6(N-methylammoniohexyl)amino]&z.sfnc; diazen-1-ium-1, 2-diolate (MAHMA NONOate, 25 nmol/kg, i.v.) and sodium nitroprusside (10 microg/kg, i.v.), in urethane-anesthetized rats. The L-S-nitrosocysteine-induced responses were markedly diminished whereas the MAHMA NONOate- and sodium nitroprusside-induced responses were minimally affected by para-hydroxymercurobenzoic acid. These results suggest that the vasodilator actions of L-S-nitrosocysteine involves the interaction with membrane thiols in vascular smooth muscle of resistance arteries and that para-hydroxymercurobenzoic acid does not markedly affect NO-mediated vasodilation.

Stereoselective S-nitrosocysteine recognition sites in rat brain.

We examined the effects of intracisternal (i.c.) injections (10-250 nmol) of the L- and D-isomers of S-nitrosocysteine (L- and D-S-nitrosocysteine) on the mean arterial blood pressure and heart rate of conscious rats, and the decomposition of L- and D-S-nitrosocysteine to nitric oxide (NO) upon addition to brain homogenates. The i.c. injection of L-S-nitrosocysteine produced initial falls in mean arterial blood pressure and heart rate which were followed by increases in these parameters. The i.c. injection of D-S-nitrosocysteine did not produce initial falls in mean arterial blood pressure or heart rate but produced the subsequent increases in these parameters. L- and D-S-nitrosocysteine decomposed equally to NO. These results suggest that the initial effects of L-S-nitrosocysteine may be due to the activation of stereoselective recognition sites on brain neurons.

Blockade of voltage-sensitive Ca(2+)-channels markedly diminishes nitric oxide- but not L-S-nitrosocysteine- or endothelium-dependent vasodilation in vivo.

The aim of this study was to determine the hemodynamic responses elicited by systemic injections of (i) the nitric oxide (NO)-donors, sodium nitroprusside (10 nmol/kg, i.v.) and (Z)-1-(N-methyl-N-(6(N-methylammoniohexyl)amino))diazen-1-ium-1, 2-diolate (MAHMA NONOate, 25 nmol/kg, i.v.), (ii) the endothelium-derived S-nitrosothiol, L-S-nitrosocysteine (100 nmol/kg, i.v.), and (iii) the endothelium-dependent agonist, acetylcholine (1.0 microg/kg, i.v.), in anesthetized rats, before and after injection of the voltage-sensitive Ca(2+)-channel (Ca(VS)(2+)-channel) blocker, nifedipine (500 nmol/kg, i.v.). Before injection of nifedipine, the agents produced similar falls in mean arterial blood pressure, and in hindquarter and mesenteric vascular resistances. The depressor and vasodilator responses elicited by sodium nitroprusside and MAHMA NONOate were markedly attenuated by nifedipine. The falls in mean arterial blood pressure and mesenteric resistance elicited by L-S-nitrosocysteine and acetylcholine were not attenuated but the falls in hindquarter resistance were slightly attenuated by nifedipine. The cyclooxygenase inhibitor, indomethacin (10 mg/kg, i.v.), did not affect the actions of sodium nitroprusside, MAHMA NONOate, L-S-nitrosocysteine or acetylcholine or the effects of nifedipine on the hemodynamic actions of these compounds. The decomposition of sodium nitroprusside (0.2 nmol/ml), MAHMA NONOate (0.5 nmol/ml) and L-S-nitrosocysteine (2 nmol/ml) to NO upon addition to rat blood was not affected by nifedipine (10 microM). These findings suggest that (i) exogenously applied NO relaxes resistance arteries in vivo by inhibition of Ca(VS)(2+)-channels whereas L-S-nitrosocysteine and the non-prostanoid endothelium-derived relaxing factor (EDRF) released by acetylcholine acts by additional mechanisms, and (ii) this EDRF may be an S-nitrosothiol which acts independently of its decomposition to NO.

L- and D-S-nitroso-beta,beta-dimethylcysteine differentially increase cGMP in cultured vascular smooth muscle cells.

We examined the effects of the L- and D-isomers of S-nitroso-beta,beta-dimethylcysteine (L- and D-S-nitrosopenicillamine, 10(-7)-10(-5) M) on the guanosine 3',5'-cyclic monophosphate (cGMP) content of cultured porcine aortic smooth muscle cells and the decomposition of these stereoisomers to nitric oxide (NO). L-S-nitrosopenicillamine was a more potent generator of cGMP than D-S-nitrosopenicillamine although both stereoisomers equally decomposed to NO. The 10(-7) M concentration of L- or D-S-nitrosopenicillamine did not generate detectable amounts of NO although 10(-7) M L-S-nitrosopenicillamine but not D-S-nitrosopenicillamine generated significant amounts of cGMP. This study shows that the stereoisomeric configuration of S-nitrosopenicillamine is an important factor in its biological potency. The data suggest that the extracellular or intracellular generation of NO is not the only mechanism by which this S-nitrosothiol generates cGMP in vascular smooth muscle.

Effect of intravenous administration of Ringer's lactate on maternal capillary blood glucose before elective cesarean section.

Capillary blood glucose concentrations were determined before and after intravenous infusion of 20 mL/kg of Ringer's lactate (RL) in 35 women scheduled for elective cesarean section. Mean (+/- SD) capillary blood glucose concentrations before and after infusion of RL were 84.7 +/- 13.2 and 89.0 +/- 16.5 mg/dL, respectively (P = NS). No parturient had a glucose level less than 60 mg/dL before or after infusion of RL. There were no instances of neonatal hypoglycemia. We conclude that large volumes of RL without dextrose may be rapidly infused into healthy pregnant women undergoing elective cesarean section without risk of dilutional hypoglycemia in the mother or neonate.