Nitric oxide may be the final mediator of nonadrenergic, noncholinergic inhibitory junction potentials in the gut.

This study tested the hypothesis that the final mediator of nonadrenergic, noncholinergic (NANC) inhibitory junction potentials (ijps) and associated relaxation responses was nitric oxide (NO) or a related substance and not vasoactive intestinal polypeptide (VIP). We used opossum esophagus body circular muscle and canine intestine circular muscle. In both these tissues, ijps had reversal potentials near the potassium equilibrium potential, (EK); in esophagus the ijps were apamin insensitive, but in the intestine they were partially apamin sensitive. N omega-Nitro-L-arginine methyl ester (NAME) (10(-5) to 5 x 10(-4) M) abolished ijps in both tissues, an effect overcome by 10(-3) M L-arginine but not D-arginine. NAME increased input resistance of esophagus tissues in the double sucrose gap but caused no significant depolarization in the sucrose gap or in studies with microelectrodes. Contractions and basal tension were increased in both tissues by NAME. The apamin sensitive and insensitive ijp components in canine muscle were both abolished by NAME, but the time course of this abolition was different for the two components. Methylene blue (10-50 microM) with variable rapidity and extent inhibited ijps in both tissues, but L-arginine could not overcome this effect. Methylene blue, like NAME, did not depolarize detectably but enhanced the contractile activity. VIP (10(-6) M) had very small effects in both tissues, little or no hyperpolarization and increased input resistance in esophagus, these effects were not changed by NAME, and VIP did not affect ijps. We conclude that NO may be the final mediator of NANC-initiated inhibitory junction potentials in gastrointestinal circular smooth muscle.     

Nitrergic and purinergic regulation of the rat pylorus

The role of nitric oxide (NO) and ATP in the regulation of nonadrenergic, noncholinergic (NANC) inhibitory transmission in the pylorus remains unclear. In the presence of atropine and guanethidine, electric field stimulation induced NANC relaxations in a frequency-dependent manner (1-20 Hz) in the rat pylorus. NANC relaxations were significantly inhibited by N(G)-nitro-L-arginine methyl ester (L-NAME; 10(-4) M). P(2X) purinoceptor antagonist pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS; 3 x 10(-5) M) and P(2Y) purinoceptor antagonist reactive blue 2 (2 x 10(-5) M) had no effect on NANC relaxations. However, the combined administration of L-NAME and PPADS, but not reactive blue 2, evoked greater inhibitory effects on NANC relaxation than that evoked by L-NAME alone. alpha-Chymotrypsin and vasoactive intestinal polypeptide antagonist did not affect NANC relaxations. ATP (10(-5)-10(-3) M) and P(2X) purinoceptor agonist alpha, beta-methyleneadenosine 5'-triphosphate (10(-7)-10(-5) M), but not P(2Y) purinoceptor agonist 2-methylthioadenosine 5'-triphosphate (10(-7)-10(-5) M), induced muscle relaxations in a dose-dependent manner, and relaxations were significantly reduced by PPADS and unaffected by TTX. These studies suggest that NO and ATP act in concert to mediate NANC relaxation of the rat pylorus. ATP-induced relaxation appears to be mediated by P(2X) purinoceptors located on smooth muscle cells.     

Role of nitric oxide in lower esophageal sphincter relaxation to swallowing.

Studies were performed in the opossum to define the role of the L-arginine-nitric oxide (NO) pathway in lower esophageal sphincter (LES) relaxation to swallowing and vagal stimulation in viv and intramural nerve stimulation in vitro. In vivo, L-NAME, a water soluble NO synthase (NOS) inhibitor, caused antagonism of LES relaxation due to reflex-induced swallowing. L-NAME (20 mg/kg i.v.) reduced the amplitude of swallow induced relaxation from 88% to 28%. LES relaxation due to electrical stimulation of peripheral end of decentralized vagus nerve was also antagonized. The effects of L-NAME were reversed by L-arginine, but not by D-arginine. L-NAME treatment did not antagonize LES relaxation to intravenous administration of isoproterenol. In vitro, NO and sodium nitroprusside (SNP) caused a decrease in the sphincter tone. The relaxing effect caused by NO and SNP was not antagonized by tetrodotoxin or omega-conotoxin. Inhibitors of NO synthase, L-NMMA and L-NNA, caused slight increase in the spontaneous resting LES tone and concentration-dependent antagonism of electrical field stimulation (EFS) induced LES relaxation. L-NNA (10(-4)M) abolished EFS induced LES relaxation at low frequencies (less than 5 Hz) and antagonized the relaxation to a value 20% of the control at 20 Hz. The antagonistic action of L-NMMA and L-NNA was unaffected by D-arginine but was reversed by L-arginine. The inhibitory effect of NO, SNP, or two other putative inhibitory neurotransmitters (VIP and CGRP) on the LES was not antagonized by L-NNA. These studies show that inhibitors of NO synthase selectively antagonize LES relaxation to all three modes of intramural inhibitory nerve stimulation including physiological swallowing. These studies suggest that the L-arginine-nitric oxide pathway is involved in physiological relaxation of the LES.     

Inhibitory NANC nerves in human tracheal smooth muscle: a quest for the neurotransmitter.

Inhibitory nonadrenergic noncholinergic (i-NANC) nerves are the only neural bronchodilator pathway in human airways. Possible candidates for the neurotransmitter include vasoactive intestinal peptide (VIP) and nitric oxide (NO) and purines such as ATP. We have investigated the potential role of these neurotransmitters. Phosphoramidon (10(-5) M) significantly potentiated relaxations to low doses of VIP with no effect on i-NANC responses. Relaxations induced by VIp were abolished with alpha-chymotrypsin (2 U/ml), but i-NANC responses were unaffected. Reactive blue 2 had no effect on i-NANC neural responses, indicating that endogenous ATP was not involved. The NO synthase inhibitor L-NG-nitroarginine methyl ester (L-NAME, 10(-4) M) produced a concentration-dependent inhibition of the i-NANC response, producing almost complete inhibition at every frequency studied (0.5-40 Hz), whereas L-NG-monomethyl arginine was effective only at low stimulation frequencies. The inhibitory effect of L-NAME was partially reversed by L- but not D-arginine, and D-NAME was without effect. These results suggest that in human tracheal segments the neural bronchodilator response is mediated by NO, and there is no functional evidence for implicating VIP in this response.     

Pharmacological evidence for both neuronal and smooth muscular PAC1 receptors and a VIP-specific receptor in rat colon

The receptors for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP) were characterised in vitro on rat colon longitudinal smooth muscle with adherent myenteric ganglia. VIP, PACAP-38 and PACAP-27 all caused concentration-dependent relaxations. PACAP-27 and PACAP-38 were equipotent, while VIP was less potent. Tetrodotoxin (10(-6) M), L-NAME (10(-4) M), 7-NINA (10(-5) M) and ODQ (3 x 10(-6) M) reduced the amplitude of the relaxatory responses to PACAP-38 but did not affect relaxations induced by VIP or PACAP-27. Apamin (10(-6) M) almost totally abolished the PACAP-27- and PACAP-38-induced relaxations, while VIP-induced relaxations were only slightly reduced. Tetraethylammonium (TEA) reduced VIP- but not PACAP-27-induced relaxations, while charybdotoxin was ineffective. Cross-desensitisation between PACAP-27, PACAP-38 and VIP could be revealed to some extent. In conclusion: VIP, PACAP-27 and PACAP-38 are effective relaxants in rat colon longitudinal muscle. The receptors involved are classified as: (1) a neuronal PAC1 receptor localised on NO-synthesising neurones, the preferred ligand being PACAP-38. Activation of this receptor leads to an increased NO production. (2) A smooth muscle PAC1 receptor, the preferred ligand being PACAP-27. However, also PACAP-38 and, to a less extent, VIP activate this receptor. The relaxatory responses elicited by both PACAP-27 and PACAP-38 are abolished by apamin and thus mediated through small conductance Ca2+-activated K+ channels. (3) A VIP-specific receptor localised on smooth muscle cells. The mechanisms whereby this receptor elicits a relaxatory response involve, at least to some extent, TEA-sensitive K+ channels.     

Sources and implications of basal nitric oxide in spontaneous contractions of guinea pig taenia caeci

We examined in vitro the source and role of basal nitric oxide (NO) in proximal segments of guinea pig taenia caeci in nonadrenergic, noncholinergic (NANC) conditions. Using electron paramagnetic resonance (EPR), we measured the effect of the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 10(-4) M), the neuronal blocker tetrodotoxin (TTX, 10(-6) M), or both on spontaneous contractions and on the production of basal NO. Both L-NAME and TTX, when tested alone, increased the amplitude and frequency of contractions. NO production was abolished by L-NAME and was inhibited by 38% by TTX. When tested together, L-NAME in the presence of TTX or TTX in the presence of L-NAME had no further effect on the amplitude or frequency of spontaneous contractions, and the NO production was inhibited. These findings suggest that basal NO consists of TTX-sensitive and TTX-resistant components. The TTX-sensitive NO has an inhibitory effect on spontaneous contractions; the role of TTX-resistant NO is unknown.     

The role of nitric oxide in mediating non-adrenergic non-cholinergic relaxation in longitudinal muscle of human taenia coli.

Electrical field stimulation (EFS) of isolated longitudinal muscle of human taenia coli at 4Hz produced relaxation which was abolished by tetrodotoxin but not adrenergic and cholinergic blockade (NANC-relaxation). NG-nitro L-arginine (L-NOARG; 1-100 microM), an NO synthesis inhibitor, produced a concentration-dependent partial inhibition of the NANC response; 10 microM L-NOARG inhibited EFS-induced relaxation by 48.6 +/- 5.20% and 100 microM L-NOARG by 54.2 +/- 10.1%. L-Arginine (1mM), but not D-arginine (1mM) partially reversed the inhibitory effect and this was inversely proportional to the concentration of L-NOARG used. Cumulative administration of NO (acidified sodium nitrite solution; 1-100 microM) produced a concentration-dependent relaxation of the strips. L-NOARG (1 mM) did not affect either NO or isoprenaline-induced relaxations. These results provide the first preliminary evidence that NO is partially responsible for the NANC inhibitory transmission in the longitudinal muscle of the taenia coli of human colon.     

Neural control of relaxation in cat airways smooth muscle

Functional innervation of cat airways smooth muscle was examined in isolated segments of trachea and bronchi using electrical field stimulation (EFS) techniques. Field stimulation caused contraction in tissues at resting tone and biphasic responses (contraction followed by relaxation) in tissues precontracted with 5-hydroxytryptamine (5-HT). Contractions were abolished by 10(-6) M atropine. Inhibitory responses were dependent on impulse voltage, duration, and frequency. At low voltages (less than or equal to 10 V) and pulse durations (less than or equal to 0.3 ms), EFS induced relaxations were abolished by 3 X 10(-6) M tetrodotoxin (TTX). Greater stimulus parameters elicited TTX-resistant relaxations. Pretreatment of the tissues with 10(-6) M propranolol and 10(-5) M guanethidine caused rightward shifts in relaxation frequency-response curves. These findings indicate that cat airways are innervated by excitatory cholinergic, inhibitory adrenergic, and inhibitory nonadrenergic noncholinergic (NANC) nerves. Pretreatment of the tissues with hexamethonium, cimetidine, indomethacin, or nordihydroguaiaretic acid did not affect NANC relaxation responses. It is concluded that NANC inhibitory responses in cat airway smooth muscle are mediated through intrinsic postganglionic nerve fibers and occur independently of histamine H2-receptor activation and without involvement of cyclooxygenase or lipoxygenase products of arachidonic acid metabolism.     

Opposing roles of K(+) and Cl(-) channels in maintenance of opossum lower esophageal sphincter tone

The ionic basis underlying the maintenance of myogenic tone of lower esophageal sphincter circular muscle (LES) was investigated in opossum with the use of standard isometric tension and conventional intracellular microelectrode recordings in vitro. In tension recording studies, nifedipine (1 microM) reduced basal tone to 27.7 +/- 3.8% of control. The K(+) channel blockers tetraethylammonium (TEA, 2 mM), charybdotoxin (100 nM), and 4-aminopyridine (4-AP, 2 mM) enhanced resting tone, whereas apamin and glibenclamide were without affect. Cl(-) channel blockers DIDS (500 microM) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (500 microM), as well as niflumic acid (0.1-300 microM), decreased basal tone, but tamoxifen was without effect. Intracellular microelectrode recordings revealed ongoing, spontaneous, spike-like action potentials (APs). Nifedipine abolished APs and depolarized resting membrane potential (RMP). Both TEA and 4-AP significantly depolarized RMP and augmented APs, whereas niflumic acid dose-dependently hyperpolarized RMP and abolished APs. These data suggest that, in the opossum, basal tone is associated with continuous APs and that K(+) and Ca(2+)-activated Cl(-) channels have important opposing roles in the genesis of LES tone.     

Role of cGMP as a mediator of nerve-induced motor functions of the opossum esophagus

Stimulation of esophageal nerves produces biphasic relaxation of the lower esophageal sphincter (LES) and an off response of circular esophageal muscle. Previously, we proposed that cGMP mediates nerve-induced hyperpolarization of circular LES muscle but not LES relaxation. These experiments explore whether cGMP mediates LES relaxation or the off response. Strips of muscle from the opossum esophagus and LES were connected to force-displacement transducers, placed in tissue baths containing oxygenated Krebs solution at 37 degrees C, and stimulated by an electrical field. 1H-[1,2, 4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), a selective inhibitor of guanylyl cyclase, antagonized the off response, shortened its latency, and blocked the first phase of LES relaxation. ODQ also antagonized LES relaxation by exogenous nitric oxide (NO) but not relaxations by vasoactive intestinal polypeptide (VIP). Part of the nerve-induced LES relaxation and the off response appear to be mediated by the second messenger cGMP. These studies indicate that VIP-induced LES relaxation is not mediated by cGMP and therefore do not support the hypothesis that VIP produces LES relaxation by causing the generation of NO.     

Nonadrenergic inhibitory nerves attenuate neurally mediated contraction in cat bronchi

Effects of nonadrenergic and noncholinergic (NANC) inhibitory nerves on cholinergic neurotransmission were examined in isolated bronchial segments from cats in the presence of propranolol (10(-6) M) and indomethacin (10(-6) M) by use of electrical field stimulation (EFS) techniques. EFS caused contraction alone in tissues at the baseline tension and biphasic responses (contraction and relaxation) in tissues precontracted with 5-hydroxytryptamine. Contraction was abolished by atropine (10(-6) M), and relaxation was abolished by tetrodotoxin (10(-6) M). At the baseline tension, EFS at frequencies greater than 10 Hz inhibited the subsequent (4 min later) contraction induced by EFS at 1-5 Hz. EFS-induced inhibition was stimulus frequency dependent and reached maximum at 20 Hz. However, EFS at 20 Hz did not inhibit the subsequent contractile response to acetylcholine (10(-7) to 10(-3) M). Exogenously applied vasoactive intestinal peptide mimicked EFS-induced inhibitory effects, but substance P and calcitonin gene-related peptide did not. The inhibitory effect of EFS at 20 Hz was not altered by pyrilamine, cimetidine, naloxone, methysergide, phentolamine, BW755C, AF-DX 116, or removal of epithelium. These results imply that the NANC transmitter acts via presynaptic cholinergic receptors.     

Somatostatin selectively inhibits excitatory contractile pathways of the feline lower esophageal sphincter.

Intrinsic reflexes of the feline lower esophageal sphincter (LES) have been shown to be mediated by specific arrangements of excitatory peptidergic interneurons. Inhibition of intrinsic reflexes may also be mediated by neuropeptides. The specific aims of this study were: (1) to examine the effect of somatostatin (SOM) and vasoactive intestinal peptide (VIP) on basal LES tone, and (2) to determine if these transmitters exert selective inhibitory effects on excitatory contractile pathways. Intraluminal pressures were recorded from the LES, esophagus and fundus by a fixed perfused catheter assembly in anesthetized cats. Peptides were administered via the left gastric artery. SOM had no effect on basal LES pressure with doses ranging from 10(-9) to 10(-5) g/kg. VIP induced a dose-dependent inhibition of basal LES pressure. The maximal effective dose of VIP, 10(-6) g/kg, completely inhibited basal LES pressure (34.7 +/- 6.8 to 1.0 +/- 0.6 mmHg, P less than 0.001). We have previously shown that bombesin (BN) but not substance P (SP) or bethanechol contracts the LES via tetrodotoxin-sensitive pathways. BN at the D50 (5.10(-8) g/kg) increased LES pressure by 32.1 +/- 3.6 mmHg. SOM (10(-5) g/kg) decreased this BN response to 19.2 +/- 5.0 mmHg, P less than 0.05. In contrast, while the D50 of SP (5.10(-8) g/kg) gave a similar increase in LES pressure, 28.8 +/- 5.1 mmHg, this effect was not altered by SOM (23.8 +/- 6.7 mmHg, P greater than 0.10). SOM also had no effect on bethanechol-induced LES contractions (P greater than 0.10). VIP (10(-6) g/kg) totally inhibited the LES response to the D50 of BN, SP, and bethanechol. A submaximal dose of VIP (10(-7) g/kg) partially inhibited the contractile response of all three. Conclusions: (1) VIP, but not SOM, inhibits basal LES tone. (2) SOM selectively inhibits BN but not SP- or bethanechol-induced LES contraction. (3) VIP inhibits BN, SP and bethanechol-induced LES contractions. These studies suggest that somatostatin can selectively inhibit excitatory interneurons at the LES.     

Inhibitory effect of valves on endothelium-dependent relaxations to calcium ionophore in canine saphenous vein

The present study was designed to evaluate endothelium-dependent relaxation to the calcium ionophore A-23187 in isolated canine saphenous veins. Isometric force recordings and cGMP measurements using isolated veins with and without valves were performed. During contractions to U-46619 (3 x 10(-7) M), endothelium-dependent relaxations to A-23187 (10(-9)-10(-6) M) were significantly reduced in rings with valves compared with rings without valves. Endothelial removal abolished A-23187-induced relaxation. Relaxations to forskolin (FK; 10(-8)-10(-5) M) and diethylaminodiazen-1-ium-1,2-dionate; DEA-NONOate, 10(-9)-10(-5) M) were identical in rings with and without valves. In rings without valves, a nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME; 3 x 10(-4) M), and a cyclooxygenase inhibitor, indomethacin (10(-5) M), partially reduced A-23187-induced relaxation. However, in rings with valves, L-NAME had no effect, whereas indomethacin abolished the relaxation to A-23187. A selective soluble guanylate cyclase inhibitor, 1H-[1,2,4]-oxadiazolo [4,3-a]quinoxalin-1-one (ODQ; 3x10(-6) M), had no effect on the relaxation to A-23187 in either group. In contrast, ODQ abolished the A-23187-induced increase in cGMP levels, suggesting that relaxation to nitric oxide released by A-23187 is independent of increases in cGMP. These results demonstrate that endothelium-dependent relaxation to A-23187 is reduced in regions of veins with valves compared with relaxation in the nonvalvular venous wall. Lower production of nitric oxide in endothelial cells of valvular segments appears to be a mechanism responsible for reduced reactivity to A-23187.     

Inhibition of acetylcholine-induced EDHF response by elevated glucose in rat mesenteric artery

The effects of high glucose on endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxations of isolated rat mesenteric artery and the possible involvement of reactive oxygen species in these responses were investigated. After precontraction with phenylephrine (3 x 10(-8)-10(-7) M), acetylcholine (10(-8)-3 x 10(-6) M) and A 23187 (10(-8)-3 x 10(-6) M), a calcium ionophore, induced concentration-dependent relaxations in the presence of N(W)-nitro-l-arginine methyl ester (L-NAME) (10(-4) M) and indomethacin (10(-5) M). These relaxations were abolished in the presence of charybdotoxin (2 x 10(-7) M) plus apamin (10(-7) M) and were assumed to be mediated by EDHF. Effects of elevated glucose were examined by incubating the arterial rings for 6 h in Krebs-Henseleit solution containing 22.2 mM glucose. Under these conditions relaxation to acetylcholine was significantly attenuated but was unchanged when the tissues were incubated for 6 h in solution containing 11.1 mM mannitol used as hyperosmotic control. Addition of superoxide dismutase (SOD) (75 U/ml) and combination of SOD with catalase (200 U/ml) during incubation with high glucose significantly preserved the impairment of EDHF-mediated relaxations to acetylcholine. A 23187-induced endothelium-dependent relaxation was not affected by high glucose. Similarly, relaxations to pinacidil (10(-10)-10(-5) M) and to sodium nitroprusside (SNP) (10(-10)-3 x 10(-7) M) were also unchanged in the rings exposed to high glucose. These results suggest that in rat mesenteric arteries exposed to elevated glucose receptor-dependent EDHF-mediated relaxations (acetylcholine-induced) are impaired whereas receptor-independent ones (A 23187-induced) and responses to smooth muscle relaxants that exert their effects through mechanisms independent of endothelium are unaffected. Our findings lead us to propose that reactive oxygen species like superoxide ((.)O(2)(-)) and hydrogen peroxide (H(2)O(2)) do seem to play a role in the impairment of EDHF-mediated relaxations in the presence of elevated glucose.     

VIP augments cholinergic-induced glycoconjugate secretion in tracheal submucosal glands

Using isolated submucosal glands from feline trachea, we examined the effect of vasoactive intestinal peptide (VIP) on mucus glycoprotein secretion and glandular contraction by measuring released radiolabeled glycoconjugates and induced tension, respectively. VIP (10(-10) to 10(-6) M) produced a dose-dependent increase in [3H]glycoconjugate release of up to 300% of controls, which was inhibited by VIP antiserum and not inhibited by atropine, propranolol, or phentolamine. VIP at a low concentration (10(-9) M), which did not produce any significant increases over controls, produced a 2.4- to 5-fold augmentation of the glycoconjugate release induced by 10(-9) to 10(-7) M methacholine (MCh). Atropine or VIP antiserum abolished the augmentation. VIP did not produce any alteration in isoproterenol- or phenylephrine-evoked glycoconjugate secretion. VIP (up to 10(-5) M) did not produce any alteration in the tension, even when the gland had contracted with MCh, or any augmentation of contraction induced by MCh (10(-9) to 10(-7) M). These results indicate that VIP induces mucus glycoprotein release from secretory cells and also that it potentiates the secretion induced by cholinergic stimulation.     

Effect of exogenous ATP on canine jejunal smooth muscle

Intracellular recordings were made from the circular smooth muscle cells of the canine jejunum to study the effect of exogenous ATP and to compare the ATP response to the nonadrenergic, noncholinergic (NANC) inhibitory junction potential (IJP) evoked by electrical field stimulation (EFS). Under NANC conditions, exogenous ATP evoked a transient hyperpolarization (6.5 +/- 0.6 mV) and EFS evoked a NANC IJP (17 +/- 0.4 mV). Omega-conotoxin GVIA (100 nM) and a low-Ca(2+), high-Mg(2+) solution abolished the NANC IJP but had no effect on the ATP-evoked hyperpolarization. The ATP-evoked hyperpolarization and the NANC IJP were abolished by apamin (1 microM) and N(G)-nitro-L-arginine (100 microM). Oxyhemoglobin (5 microM) partially (38.8 +/- 5.5%) reduced the amplitude of the NANC IJP but had no effect on the ATP-evoked hyperpolarization. Neither the NANC IJP nor the ATP-evoked hyperpolarization was affected by P2 receptor antagonists or agonists, including suramin, reactive blue 2, 1-(N, O-bis-[5-isoquinolinesulfonyl]-N-methyl-L-tyrosyl)-4-phenylpiperazine , pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid, alpha, beta-methylene ATP, 2-methylthioadenosine 5'-triphosphate tetrasodium salt, and adenosine 5'-O-2-thiodiphosphate. The data suggest that ATP evoked an apamin-sensitive hyperpolarization in circular smooth muscle cells of the canine jejunum via local production of NO in a postsynaptic target cell.     

Nitric oxide-mediated nonadrenergic noncholinergic relaxation of piglet pulmonary arteries decreases with postnatal age.

Nonadrenergic noncholinergic (NANC) vasodilator mechanisms may contribute to the maintenance of adult pulmonary and systemic vascular tone. However, their actions in the neonatal circulation have not been studied. We aimed to investigate NANC vasorelaxation in neonatal and 2-week-old piglet pulmonary and mesenteric arteries and to examine the potential role of nitric oxide (NO) in this phenomenon. Responses to electric field stimulation (EFS, 50V, 0.25-32 Hz) were investigated in pulmonary and mesenteric artery rings (external diameter 150-200 microm) precontracted with the thromboxane A2 mimetic U46619, in the presence of guanethidine (10 microM) and atropine (10 microM). Under these conditions, EFS resulted in a frequency dependent relaxation of newborn pulmonary (maximal relaxation of 53+/-9.1%), mesenteric (68.8.2+/-7.1%) and 2-wk-old mesenteric (46 6.3%) arteries but this relaxation was significantly reduced (4.5+/-2.2%) in 2-week-old pulmonary arteries. In neonatal pulmonary arteries, the neurotoxin tetrodotoxin (0.3 muM), the NO synthase inhibitor L-NAME (0.1 mM), and the guanylyl cyclase inhibitor ODQ (10 microM) abolished EFS-induced relaxations, suggesting that NANC relaxation of porcine neonatal pulmonary arteries is mediated by NO, which is probably neuronal in origin. However, The expression in pulmonary arteries of the neuronal NO synthase (nNOS), as determined by Western-blot analysis, increased with postnatal age whereas the expression of the endothelial NOS (eNOS) did not change. In conclusion, NANC relaxation is present in neonatal pulmonary and mesenteric arteries and it is, at least partially, mediated through NO. NANC relaxation of porcine pulmonary and mesenteric arteries decreases with postnatal maturation.     

Comparison of angiotensin II (Ang II) effects in the internal anal sphincter (IAS) and lower esophageal sphincter smooth muscles

Studies were performed to compare the actions of Ang II in the internal anal sphincter (IAS) vs. lower esophageal sphincter (LES) smooth muscles in vitro, in opossum and rabbit. Studies also were carried out in isolated smooth muscle cells. In opossum, Ang II produced no discernible effects in the IAS, but did produce a concentration-dependent contraction in the LES. Conversely, in the rabbit, while Ang II caused a modest response in the LES, it caused a significant contraction in the IAS. The contractile responses of Ang II in the opossum LES were mostly resistant to different neurohumoral antagonists but were antagonized by AT1 antagonist losartan. AT2 antagonist PD 123,319, rather than inhibiting, prolonged the contractile action of Ang II. The contractile actions of Ang II in the opossum LES were not modified by the tyrosine kinase inhibitors (genistein and tyrphostin 1 x 10(-6) M) but were partially attenuated by the PKC inhibitor H-7 (1 x 10(-6) M), Ca2+ channel blocker nicardipine (1 x 10(-5) M), Rho kinase inhibitor HA-1077 (1 x 10(-7) M) or p(44/42) MAP kinase inhibitor PD 98059 (5 x 10(-5) M). The combination of HA-1077 and H-7 did not cause an additive attenuation of Ang II responses. Western blot analyses revealed the presence of both AT1 and AT2 receptors. We conclude that Ang lI-induced contraction of sphincteric smooth muscle occurs primarily by the activation of AT1 receptors at the smooth muscle cells and involves multiple pathways, influx of Ca2+, and PKC, Rho kinase and p(44/42) MAP kinase.     

Endothelium-dependent responses in small human mesenteric arteries

The aim of the present study was to investigate the endothelial function in human mesenteric arteries with specific reference to defining the role of endothelium-derived nitric oxide (EDNO) and the endothelium-derived hyperpolarizing factor (EDHF). Isolated segments of small human mesenteric arteries (225-450 microm inner diameter) were mounted in organ baths for recording isometric tension. In arteries precontracted with U46619 (thromboxane A(2) analogue, 10(-7) M), endothelium-dependent relaxations were induced in a concentration-dependent manner by substance P and histamine. In normal Krebs solution the relaxations to substance P (10(-9) M) and histamine (10(-7) M) were not significantly affected by preincubation with N(omega)-nitro-L-arginine (L-NNA, 10(-4) M) or indomethacin (10(-5) M). When the preparations were exposed to a solution containing 60 mM KCl, stable contractions were induced, but relaxations could still be induced by substance P and histamine. When the arteries were further preincubated with L-NNA, the relaxations were almost abolished. A combination of apamin (3 x 10(-7) M) and charybdotoxin (10(-9) M) almost abolished relaxations in normal Krebs solution. It is concluded that isolated human mesenteric arteries respond to substance P and histamine with relaxations that are endothelium-dependent. Synthesis of both EDNO and EDHF seem important for these relaxations, whereas prostaglandins seem to be of minor importance.     

The effects of antagonists of vasoactive intestinal peptide on nonadrenergic noncholinergic inhibitory responses in feline airways

Relaxations of the feline intrapulmonary bronchus (IPB) induced by VIP or nonadrenergic noncholinergic (NANC) inhibitory nervous stimulation were unaffected by the VIP receptor antagonist [Ac-Tyr1,D-Phe2]-GRF (1-29) (30 microM). A second VIP antagonist, [pCl-D-Phe6,Leu17]-VIP (30 microM), also had no effect on NANC relaxation responses or IPB sensitivity to VIP. However, responses to three of the four highest VIP concentrations were inhibited by this antagonist. These results indicate that [Ac-Tyr1,D-Phe2]-GRF (1-29) and [pCl-D-Phe6,Leu17]-VIP are not effective competitive antagonists of VIP receptors in feline airways and, hence, have but limited applicability in determining the role of VIP in mediating airway NANC inhibitory responses in this tissue.