Adult rats received intraperitoneal injections of guanethidine or saline for 5 weeks. Six to 8 weeks following completion of treatment, concentrations of substance P and neuropeptide Y (NPY) were measured by radioimmunoassay in the superior cervical ganglion (SCG) and thoracic spinal cord. The SCG was also immunostained for NPY and substance P. No differences were observed in thoracic spinal cord content of either NPY or substance P. We observed depletion of NPY immunoreactive neurons and NPY levels in the SCG, and pharmacologic evidence of postganglionic denervation in guanethidine-treated rats. In guanethidine-treated rats, there was a marked increase of substance P levels in the SCG, where substance P was localized in fibers, but not cell bodies. Thus, sprouting of substance P-containing sensory fibers in the sympathetic ganglia occurs following postganglionic sympathectomy in adult rats.

Chronic rather than acute changes in the autonomic innervation of the vasculature are a feature of ageing and several cardiovascular disorders. To investigate the long-term influence of perivascular innervation on the vascular endothelium, the release of vasoactive substances which have been localized in endothelial cells, namely ATP, endothelin, substance P and vasopressin, was monitored from cells isolated from adult rat thoracic aorta following neonatal guanethidine sympathectomy. The endothelial cells were initially perfused at 0.5 ml/min and exposed to two periods of increased flow at 3.0 ml/min. Cells isolated from control rats released significantly more ATP on both occasions of switching from the lower to higher flow rate and significantly more endothelin on the second exposure to the higher flow rate. In contrast, endothelial cells isolated from sympathectomised rats showed no increased release of either ATP or endothelin with increase flow, although the release of endothelin at the initial flow rate of 0.5 ml/min was higher than in the controls. Substance P and vasopressin levels in the perfusate were the same in controls and after sympathectomy. In summary, long-term sympathectomy suppresses increased flow-induced release of selected vasoactive substances from the endothelium, thus shear-stress-induced changes in local blood flow may be impaired when there are chronic disturbances in the autonomic innervation.

This study was aimed at disclosing the influence of intravesically instilled guanethidine (GUA) on the distribution, relative frequency and chemical coding of both the urinary bladder intramural sympathetic nerve fibers and their parent cell bodies in the caudal mesenteric ganglion (CaMG) in juvenile female pigs. GUA instillation led to a profound decrease in the number of perivascular nerve terminals. Furthermore, the chemical profile of the perivascular innervation within the treated bladder also distinctly changed, as most of axons became somatostatin-immunoreactive (SOM-IR), while in the control animals they were found to be neuropeptide Y (NPY)-positive. Intravesical treatment with GUA led not only to a significant decrease in the number of bladder-projecting tyrosine hydroxylase (TH) CaMG somata (94.3 ± 1.8% vs. 73.3 ± 1.4%; control vs. GUA-treated pigs), but simultaneously resulted in the rearrangement of their co-transmitters repertoire, causing a distinct decrease in the number of TH+/NPY+ (89.6 ± 0.7% vs. 27.8 ± 0.9%) cell bodies and an increase in the number of SOM-(3.6 ± 0.4% vs. 68.7 ± 1.9%), calbindin-(CB; 2.06 ± 0.2% vs. 9.1 ± 1.2%) or galanin-containing (GAL; 1.6 ± 0.3% vs. 28.2 ± 1.3%) somata. The present study provides evidence that GUA significantly modifies the sympathetic innervation of the porcine urinary bladder wall, and thus may be considered a potential tool for studying the plasticity of this subdivision of the bladder innervation.

1. The role of tachykinin NK1 receptors in the recruitment of eosinophils to airway nerves, loss of inhibitory neuronal M2 muscarinic receptor function and the development of vagal hyperreactivity was tested in antigen-challenged guinea-pigs. 2. In anaesthetized guinea-pigs, the muscarinic agonist, pilocarpine (1-100 microg kg(-1), i.v.), inhibited vagally induced bronchoconstriction, in control, but not in antigen-challenged guinea-pigs 24 h after antigen challenge. This indicates normal function of neuronal M2 muscarinic receptors in controls and loss of neuronal M2 receptor function in challenged guinea-pigs. Pretreatment of sensitized guinea-pigs with the NK1 receptor antagonists CP99994 (4 mg kg(-1), i.p.), SR140333 (1 mg kg(-1), s.c.) or CP96345 (15 mg kg(-1), i.p.) before antigen challenge, prevented M2 receptor dysfunction. 3. Neither administration of the NK1 antagonists after antigen challenge, nor pretreatment with an NK2 receptor antagonist, MEN10376 (5 micromol kg(-1), i.p.), before antigen challenge, prevented M2 receptor dysfunction. 4. Electrical stimulation of the vagus nerves caused a frequency-dependent (2-15 Hz, 10 V, 0.2 ms for 5 s) bronchoconstriction that was significantly increased following antigen challenge. Pretreatment with the NK1 receptor antagonists CP99994 or SR140333 before challenge prevented this increase. 5. Histamine (1-20 nmol kg(-1), i.v.) caused a dose-dependent bronchoconstriction, which was vagally mediated, and was significantly increased in antigen challenged guinea-pigs compared to controls. Pretreatment of sensitized animals with CP99994 before challenge prevented the increase in histamine-induced reactivity. 6. Bronchoalveolar lavage and histological studies showed that after antigen challenge significant numbers of eosinophils accumulated in the airways and around airway nerves. This eosinophilia was not altered by pretreatment with the NK1 receptor antagonist CP99994. 7. These data indicate that pretreatment of antigen-sensitized guinea-pigs with NK1, but not with NK2 receptor antagonists before antigen challenge prevented the development of hyperreactivity by protecting neuronal M2 receptor function. NK1 receptor antagonists do not inhibit eosinophil accumulation around airway nerves.

1. We have investigated the effect of nociceptin on the micturition reflex evoked by distension or topical application of capsaicin on the urinary bladder of urethane-anaesthetized rats. 2. Nociceptin produced a dose-dependent (3-100 nmol kg(-1) i.v.) transient suppression of the distension-evoked micturition reflex: its effect was not modified by guanethidine (68 micromol kg(-1) s.c.) nor by bilateral cervical vagotomy, alone or in combination, and by naloxone (1.2 micromol kg(-1) i.v.). 3. Nociceptin (100 nmol/kg i.v.) slightly (about 30%) inhibited the contractions of the rat bladder produced by pre- or postganglionic electrical stimulation of the pelvic nerve. 4. Nociceptin almost totally abolished the reflex component of the response to topical capsaicin (1 microg in 50 microl). 5. In the rat isolated bladder, submaximal contractions produced by electrical field stimulation were slightly reduced (25+/-4% inhibition) by 1 microM nociceptin. Nociceptin did not affect the contraction of the rat bladder induced by acetylcholine (10 microM) or ATP (1 mM). 6. These findings indicate that nociceptin exerts a naloxone-resistant suppression of the volume-evoked micturition reflex which involves inhibition of transmitter release from postganglionic bladder nerves. An inhibitory effect on bladder afferent nerves is also suggested.

1. The effect of antidromic stimulation of the sensory fibres of the sciatic nerve on inflammatory plasma extravasation in various tissues and on cutaneous vasodilatation elicited in distant parts of the body was investigated in rats pretreated with guanethidine (8 mg kg(-1), i.p.) and pipecuronium (200 microg kg(-1), i.v.). 2. Antidromic sciatic nerve stimulation with C-fibre strength (20 V, 0.5 ms) at 5 Hz for 5 min elicited neurogenic inflammation in the innervated area and inhibited by 50.3 +/- 4.67% the development of a subsequent plasma extravasation in response to similar stimulation of the contralateral sciatic nerve. Stimulation at 0.5 Hz for 1 h also evoked local plasma extravasation and inhibited the carrageenin-induced (1%, 100 microl s.c.) cutaneous inflammation by 38.5 +/- 10.0% in the contralateral paw. Excitation at 0.1 Hz for 4 h elicited no local plasma extravasation in the stimulated hindleg but still reduced the carrageenin-induced oedema by 52.1 +/- 9.7% in the paw on the contralateral side. 3. Plasma extravasation in the knee joint in response to carrageenin (2%, 200 microl intra-articular injection) was diminished by 46.1 +/- 12.69% and 40.9 +/- 4.93% when the sciatic nerve was stimulated in the contralateral leg at 0.5 Hz for 1 h or 0.1 Hz for 4 h, respectively. 4. Stimulation of the peripheral stump of the left vagal nerve (20 V, 1 ms, 8 Hz, 10 min) elicited plasma extravasation in the trachea, oesophagus and mediastinal connective tissue in rats pretreated with atropine (2 mg kg(-1), i.v.), guanethidine (8 mg kg(-1), i.p.) and pipecuronium (200 microg kg(-1), i.v.). These responses were inhibited by 37.8 +/- 5.1%, 49.7 +/- 9.9% and 37.6 +/- 4.2%, respectively by antidromic sciatic nerve excitation (5 Hz, 5 min) applied 5 min earlier. 5. Pretreatment with polyclonal somatostatin antiserum (0.5 ml/rat, i.v.) or the selective somatostatin depleting agent cysteamine (280 mg kg(-1), s.c.) prevented the anti-inflammatory effect of sciatic nerve stimulation (5 Hz, 5 min) on a subsequent neurogenic plasma extravasation of the contralateral paw skin. The inhibitory effect of antidromic sciatic nerve excitation on plasma extravasation in response to vagal nerve stimulation was also prevented by somatostatin antiserum pretreatment. 6. Cutaneous blood flow assessment by laser Doppler flowmetry indicated that antidromic vasodilatation induced by sciatic nerve stimulation was not inhibited by excitation of the sciatic nerve of the contralateral leg (1 Hz, 30 min) or by somatostatin (10 microg/rat, i.v.) injection. 7. Plasma levels of somatostatin increased more than 4 fold after stimulation of both sciatic nerves (5 Hz, 5 min) but the stimulus-evoked increase was not observed in cysteamine (280 mg kg(-1), s.c.) pretreated rats. 8. These results suggest that somatostatin released from the activated sensory nerve terminals mediates the systemic anti-inflammatory effect evoked by stimulating the peripheral stump of the sciatic nerve.

1. Twitch-like contractions and non-adrenergic non-cholinergic (NANC) relaxations evoked by electrical field stimulation (EFS) of the cat bronchiole were used to examine the voltage-activated calcium channels involved in excitatory and inhibitory neurotransmission in the cat bronchiole. 2. Nifedipine (50 microM), the L-type calcium channel antagonist, did not affect the twitch-like contraction and NANC relaxations. However, low concentrations of the N-type calcium channel blocker omega-conotoxin GVIA (omega-CgTX GVIA) (0.1 microM) irreversibly abolished twitch-like contractions evoked by trains of EFS

1. The effect of incubation with the nitric oxide (NO) donor nitroglycerin and of long-term electrical stimulation on relaxations induced by non-adrenergic, non-cholinergic nerve stimulation, exogenous NO, vasoactive intestinal polypeptide (VIP) and lemakalim was investigated in the pig gastric fundus. 2. In physiological salt solution containing 10(-6) M atropine and 4 x 10(-6) M guanethidine, electrical field stimulation (40 V, 0.1 ms, 0.5-8 Hz) for periods of 10 s at 5 min intervals (train stimulation) and administration of NO (2 x 10(-6)-10(-4) M) at 5 min intervals (NO boli) induced frequency- and concentration-dependent transient relaxations, respectively. Continuous electrical field stimulation with stepwise increase of the frequency (0.5-8 Hz, cumulative stimulation) induced frequency-dependent sustained relaxations. VIP (10(-7) M), lemakalim (10(-5) M) and an infusion of NO induced a sustained relaxation. 3. Pretreatment for 30 min with 5 x 10(-4) M nitroglycerin reduced the relaxations induced by train and cumulative stimulation, but also the relaxant responses to NO, both when given in boli or as an infusion. The relaxations to VIP and lemakalim were not influenced by pretreatment with nitroglycerin. 4. Long-term electrical stimulation at 4 Hz for 40 min induced a sustained relaxation of the tissues. Administration of 3 x 10(-4) M N(G)-nitro-L-arginine methyl ester after 10, 20 or 30 min reversed the relaxation to a similar extent (approximately 70%). Previous long-term electrical stimulation at 4 Hz for 30 min did not affect the responses to stimulation, NO and VIP. 5. These results illustrate that nitroglycerin can induce a postjunctional tolerance to nitrergic stimuli in the pig gastric fundus but evidence for a prejunctional inhibition of neuronal NO synthase by NO was not obtained.

Insulin resistance plays a key role in the pathogenesis of type 2 diabetes and is also related to other health problems like obesity, hypertension, and metabolic syndrome. Imbalance between insulin vascular actions via the phosphatidylinositol 3-Kinase (PI3K) and the mitogen activated protein kinase (MAPK) signaling pathways during insulin resistant states results in impaired endothelial PI3K/eNOS- and augmented MAPK/endothelin 1 pathways leading to endothelial dysfunction and abnormal vasoconstriction. The role of PI3K, MAPK, and protein kinase C (PKC) in Ca2+ handling of resistance arteries involved in blood pressure regulation is poorly understood. Therefore, we assessed here whether PI3K, MAPK, and PKC play a role in the Ca2+ signaling pathways linked to adrenergic vasoconstriction in resistance arteries. Simultaneous measurements of intracellular calcium concentration ([Ca2+]i) in vascular smooth muscle (VSM) and tension were performed in endothelium-denuded branches of mesenteric arteries from Wistar rats mounted in a microvascular myographs. Responses to CaCl2 were assessed in arteries activated with phenylephrine (PE) and kept in Ca2+-free solution, in the absence and presence of the selective antagonist of L-type Ca2+ channels nifedipine, cyclopiazonic acid (CPA) to block sarcoplasmic reticulum (SR) intracellular Ca2+ release or specific inhibitors of PI3K, ERK-MAPK, or PKC. Activation of α1-adrenoceptors with PE stimulated both intracellular Ca2+ mobilization and Ca2+ entry along with contraction in resistance arteries. Both [Ca2+]i and contractile responses were inhibited by nifedipine while CPA abolished intracellular Ca2+ mobilization and modestly reduced Ca2+ entry suggesting that α1-adrenergic vasoconstriction is largely dependent Ca2+ influx through L-type Ca2+ channel and to a lesser extent through store-operated Ca2+ channels. Inhibition of ERK-MAPK did not alter intracellular Ca2+ mobilization but largely reduced L-type Ca2+ entry elicited by PE without altering vasoconstriction. The PI3K blocker LY-294002 moderately reduced intracellular Ca2+ release, Ca2+ entry and contraction induced by the α1-adrenoceptor agonist, while PKC inhibition decreased PE-elicited Ca2+ entry and to a lesser extent contraction without affecting intracellular Ca2+ mobilization. Under conditions of ryanodine receptor (RyR) blockade to inhibit Ca2+-induced Ca2+-release (CICR), inhibitors of PI3K, ERK-MAPK, or PKC significantly reduced [Ca2+]i increases but not contraction elicited by high K+ depolarization suggesting an activation of L-type Ca2+ entry in VSM independent of RyR. In summary, our results demonstrate that PI3K, ERK-MAPK, and PKC regulate Ca2+ handling coupled to the α1-adrenoceptor in VSM of resistance arteries and related to both contractile and non-contractile functions. These kinases represent potential pharmacological targets in pathologies associated to vascular dysfunction and abnormal Ca2+ handling such as obesity, hypertension and diabetes mellitus, in which these signaling pathways are profoundly impaired.

Background We previously reported that pralidoxime facilitated restoration of spontaneous circulation by potentiating the pressor effect of epinephrine. We determined the optimal dose of pralidoxime during cardiopulmonary resuscitation and evaluated the involvement of α-adrenoceptors in its pressor action. Methods and Results Forty-four pigs randomly received 1 of 3 doses of pralidoxime (40, 80, or 120 mg/kg) or saline placebo during cardiopulmonary resuscitation, including epinephrine administration. Pralidoxime at 40 mg/kg produced the highest coronary perfusion pressure, whereas 120 mg/kg of pralidoxime produced the lowest coronary perfusion pressure. Restoration of spontaneous circulation was attained in 4 (36.4%), 11 (100%), 9 (81.8%), and 3 (27.3%) animals in the saline, 40, 80, and 120 mg/kg groups, respectively (P<0.001). In 49 rats, arterial pressure response to 40 mg/kg of pralidoxime was determined after saline, guanethidine, phenoxybenzamine, or phentolamine pretreatment, and the response to 200 mg/kg pf pralidoxime was determined after saline, propranolol, or phentolamine pretreatment. Pralidoxime at 40 mg/kg elicited a pressor response. Phenoxybenzamine completely inhibited the pressor response, but guanethidine and phentolamine did not. The pressor response of pralidoxime was even greater after guanethidine or phentolamine pretreatment. Pralidoxime at 200 mg/kg produced an initial vasodepressor response followed by a delayed pressor response. Unlike propranolol, phentolamine eliminated the initial vasodepressor response. Conclusions Pralidoxime at 40 mg/kg administered with epinephrine improved restoration of spontaneous circulation rate by increasing coronary perfusion pressure in a pig model of cardiac arrest, whereas 120 mg/kg did not improve coronary perfusion pressure or restoration of spontaneous circulation rate. The pressor effect of pralidoxime was unrelated to α-adrenoceptors and buffered by its vasodepressor action mediated by sympathoinhibition.

1. We investigated the potentiating effect of low concentrations of neuropeptide Y (NPY) on the vasoconstriction induced by transmural nerve stimulation (TNS) and noradrenaline (NA) in human saphenous veins. The effects of (i) endothelium removal; (ii) the addition of the NO pathway precursor L-arginine; (iii) the ET(A)/ET(B) endothelin receptor antagonist Ro 47-0203; (iv) the cyclo-oxygenase inhibitor, indomethacin; (v) the selective thromboxane A2 (TxA2) receptor antagonists Bay u3405 and ifetroban, and (vi) the TxA2 synthase inhibitor, UK 38485, were studied in order to gain information about the mechanisms of NPY-induced potentiation. 2. Contractile response curves for TNS (0.5-8 Hz) and for exogenously administered NA (0.1-3 microM) were obtained in superfused saphenous vein rings. The contractions induced by both TNS and NA at all tested frequencies and concentrations, respectively, were significantly potentiated by 50 nM NPY in endothelium intact veins. Conversely, in endothelium-denuded vessel rings the contractile-response curves to TNS and NA overlapped both in the absence and presence of NPY, thus suggesting that a release of vasoactive substances from endothelial cells could account for the noradrenergic NPY-induced potentiation. 3. In vessels with intact endothelium, the potentiating action of NPY on TNS and NA was unaffected by the presence of high concentrations of the NO precursor L-arginine (3-10 mM) or the non-selective ET(A)/ET(B) endothelin receptor antagonist, Ro 47-0203 (10 microM). These data indicate that the NPY-induced effect does not involve either the endothelium-derived vasodilator nitric oxide or the vasoconstrictor endothelin. Conversely, in the presence of the cyclo-oxygenase inhibitor, indomethacin (30 microM), NPY failed to potentiate the vasoconstrictions produced by either nerve stimulation or by exogenous NA, thus providing evidence that arachidonic acid metabolites through the cyclo-oxygenase pathway are mainly responsible for the potentiation evoked by NPY. 4. When the TxA2 receptor antagonists, Bay u 3405 (1 microM) and ifetroban (1 microM) were added to the superfusing medium, NPY did not alter either the frequency- or the concentration-response curves for either TNS or NA. Accordingly, both TNS- and NA-induced contractions were not potentiated by NPY in the presence of the TxA2 synthase inhibitor, UK 38485 (10 microM). This clearly demonstrates the pivotal role of TxA2 in NPY-induced potentiation. 5. In superfused vein rings with endothelium, a subthreshold concentration (0.2 nM) of the TxA2 mimetic U 46619 potentiated both TNS- and NA-induced vasoconstrictions. This potentiation was higher at low stimulation frequencies and low NA concentrations, and resembled that produced by NPY. 6. Our results indicate that in the human saphenous vein NPY potentiates the contractions produced by sympathetic nerve stimulation acting at the postjunctional level, primarily on endothelial cells. In particular, the NPY-induced release of a cyclo-oxygenase metabolite, namely TxA2, may have a synergistic effect on the vasoconstriction induced by the noradrenergic mediator. Thus, such a mechanism may play a key role in the maintenance of the sympathetic tone of large human capacitance vessels.

1. The aim of the present study was to characterize neurogenic and pharmacological responses of human penile deep dorsal vein and to determine whether the responses are mediated by nitric oxide from neural or endothelial origin. 2. Ring segments of human penile deep dorsal vein were obtained from 22 multiorgan donors during procurement of organs for transplantation. The rings were suspended in organ bath chambers for isometric recording of tension. We then studied the contractile and relaxant responses to electrical field stimulation and to vasoactive agents. 3. Electrical field stimulation (0.5-2 Hz) and noradrenaline (3 x 10(-10)-3 x 10(-5) M) caused frequency- and concentration-dependent contractions that were of greater magnitude in veins denuded of endothelium. The inhibitor of nitric oxide synthesis NG-nitro-L-arginine methyl ester hydrochloride (L-NAME, l0(-4) M) increased the adrenergic responses only in rings with endothelium. 4. In preparations contracted with noradrenaline in the presence of guanethidine (10(-6) M) and atropine (10(-6) M), electrical stimulation induced frequency-dependent relaxations. This neurogenic relaxation was prevented by L-NAME, methylene blue (3 x 10(-5) M) and tetrodotoxin (10(-6) M), but was unaffected by removal of endothelium. 5. Acetylcholine (10(-8)-3 x 10(-5) M) and substance P (3 x 10(-11) -3 x 10(-7) M) induced endothelium-dependent relaxations. In contrast, sodium nitroprusside (10(-9)-3 x 10(-5) M) and papaverine (10(-8) 3 x 10(-5) M) caused endothelium-independent relaxations. 6. The results provide functional evidence that the human penile deep dorsal vein is an active component of the penile vascular resistance through the release of nitric oxide from both neural and endothelial origin. Dysfunction in any of these sources of nitric oxide should be considered in some forms of impotence.

1. Using conventional microelectrode techniques, membrane potentials were recorded from smooth muscle cells of guinea-pig choroidal arterioles. 2. Transmural stimulation initiated excitatory junction potentials (EJPs) which were abolished by either guanethidine or alpha,beta-methylene-ATP but not by phentolamine, indicating that they resulted from activation of purinoceptors. 3. Trains of stimuli evoked EJPs which were followed by a slow depolarization, an inhibitory junction potential (IJP) or a biphasic membrane potential change which consisted of an IJP and a subsequent slow depolarization. 4. Slow depolarizations were abolished by either phentolamine or guanethidine, indicating that they resulted from activation of alpha-adrenoceptors. 5. IJPs were abolished by atropine but not by guanethidine, and were reduced by 50 % by apamin with the residual response being abolished by charybdotoxin, indicating that they resulted from the activation of muscarinic receptors which open two sets of Ca2+-activated K+ channels. 6. Most responses were followed by slow hyperpolarizations. These were almost abolished by L-nitroarginine, an effect which was partly overcome by L-arginine, and were abolished by glibenclamide, indicating that they resulted from the release of NO and activation of ATP-sensitive K+ channels. 7. Immunohistochemical analysis showed that arterioles were densely innervated by adrenergic nerve fibres. A population of fibres, likely to be cholinergic, was also identified. Furthermore, populations of nerve fibres immunoreactive to antibodies against either nitric oxide synthase (NOS) or substance P (SP) were also identified. 8. These findings indicate that choroidal arterioles of the guinea-pig are innervated by at least three different populations of nerves, adrenergic nerves which evoke excitatory responses, cholinergic nerves which evoke inhibitory responses and a population of nerves which cause the release of NO.

The experiments were carried out on guinea pig mesenteric nerve-ileal preparations (ileal segments with mesenteric nerves originating from the superior mesenteric ganglion) isolated at various distances from the ileocecal junction (ICJ). Contractile activity was recorded in the presence of hexamethonium (50 microM). On the background of electrical field stimulation (EFS; 0.1 Hz, 0.5 ms, supramaximal current intensity)-induced twitch contractions, the mesenteric nerve stimulation (MNS; frequency of 2-30 Hz, 0.5 ms, supramaximal current intensity, 20-s trains) exerted two types of effects, depending on the distance from ICJ at which the preparations were isolated and on the pulse frequency. In preparations isolated from the ileum at a distance of 20 cm from ICJ, MNS at all the frequencies studied inhibited the EFS-induced twitch contractions, reaching the maximum at 30 Hz. In preparations isolated from the terminal ileum at a distance of 10 cm from ICJ, MNS at 20 Hz and 30 Hz decreased the twitch contraction amplitude, whereas MNS at 2-10 Hz produced an increase in the tone on which twitch contractions with reduced amplitude were superimposed. The finding that guanethidine (5 microM) eliminated the MNS twitch inhibition provides evidence for the adrenergic origin of the latter. The nitric oxide synthase inhibitor Nomega-nitro-L-arginine (100 microM) was efficient in reducing the MNS twitch inhibition but only at low-frequency (5 Hz) MNS (p < 0.05). Our results suggest the participation of nitric oxide in the nervous control exerted by the superior mesenteric ganglion over the ileal contractile activity.

The functional roles of M(2) and M(3) muscarinic receptors in neurogenic cholinergic contractions in gastrointestinal tracts remain to be elucidated. To address this issue, we studied cholinergic nerve-induced contractions in the ileum using mutant mice lacking M(2) or M(3) receptor subtypes.

Application of electrical field stimulation (EFS; trains of 10 Hz, 0.25 ms pulse width, supramaximal voltage for 60 s) to the guinea-pig isolated common bile duct pretreated with atropine (1 microM), produced a slowly-developing contraction ('on' response) followed by a quick phasic 'off' contraction ('off peak' response) and a tonic response ('off late' response), averaging 16+/-2, 73+/-3 and 20+/-4% of the maximal contraction to KCl (80 mM), n=20 each, respectively. Tetrodotoxin (1 microM; 15 min before) abolished the overall response to EFS (n 8). Neither in vitro capsaicin pretreatment (10 microM for 15 min), nor guanethidine (3 microM, 60 min before) affected the excitatory response to EFS (n 5 each), showing that neither primary sensory neurons, nor sympathetic nerves were involved. Nomega-nitro-L-arginine (L-NOARG, 100 microM, 60 min before) or naloxone (10 microM, 30 min before) significantly enhanced the 'on' response (294+/-56 and 205+/-25% increase, respectively; n=6-8, P<0.01) to EFS. The combined administration of L-NOARG and naloxone produced additive enhancing effects (655+/-90% increase of the 'on' component, n = 6, P<0.05). The tachykinin NK2 receptor-selective antagonist MEN 11420 (1 microM) almost abolished both the 'on' and 'off late' responses (P<0.01: n=5 each) to EFS, and reduced the 'off-peak' contraction by 55+/-8% (n=5, P<0.01). The subsequent administration of the tachykinin NK1 receptor-selective antagonist GR 82334 (1 microM) and of the tachykinin NK3 receptor-selective antagonist SR 142801 (30 nM), in the presence of MEN 11420 (1 microM), did not produce any further inhibition of the response to EFS (P>0.05; n=5 each). At 3 microM, GR 82334 significantly reduced (by 68+/-9%, P<0.05, n=6) the 'on' response to EFS. The contractile 'off peak' response to EFS observed in the presence of both MEN 11420 and GR 82334 (3 microM each) was abolished (P<0.01; n=6) by the administration of the P2 purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 30 microM). PPADS (30 microM) selectively blocked (75+/-9 and 50+/-7% inhibition, n = 4 each) the contractile responses produced by 100 and 300 microM ATP. Tachykinin-containing nerve fibres were detected by using immunohistochemical techniques in all parts of the bile duct, being distributed to the muscle layer and lamina propria of mucosa. In the terminal part of the duct (ampulla) some labelled ganglion cells were observed. In conclusion, this study shows that in the guinea-pig terminal biliary tract tachykinins, released from intrinsic neuronal elements, are the main NANC excitatory neurotransmitters, which act by stimulating tachykinin NK2 (and possibly NK1) receptors. ATP is also involved as excitatory neurotransmitter. Nitric oxide and opioids act as inhibitory mediators/modulators in this preparation.

Influenza A virus (IAV) matrix protein 2 (M2), an ion channel, is crucial for virus infection, and therefore, an important anti-influenza drug target. Adamantanes, also known as M2 channel blockers, are one of the two classes of Food and Drug Administration-approved anti-influenza drugs, although their use was discontinued due to prevalent drug resistance. Fast emergence of resistance to current anti-influenza drugs have raised an urgent need for developing new anti-influenza drugs against resistant forms of circulating viruses. Here we propose a simple theoretical criterion for fast virtual screening of molecular libraries for candidate anti-influenza ion channel inhibitors both for wild type and adamantane-resistant influenza A viruses. After in silico screening of drug space using the EIIP/AQVN filter and further filtering of drugs by ligand based virtual screening and molecular docking we propose the best candidate drugs as potential dual inhibitors of wild type and adamantane-resistant influenza A viruses. Finally, guanethidine, the best ranked drug selected from ligand-based virtual screening, was experimentally tested. The experimental results show measurable anti-influenza activity of guanethidine in cell culture.

Cross-sections of the vas deferens taken from control adult male rats showed positive histochemical reactivity to acetylcholinesterase and immunoreactivity for antibodies to protein gene product 9.5, tyrosine hydroxylase, neuropeptide Y, vasoactive intestinal polypeptide, nitric oxide synthase and calcitonin gene-related peptide. Immunoreactivity to substance P was very sparse. Histochemical reactivity to acetylcholinesterase and immunoreactivity to vasoactive intestinal polypeptide and nitric oxide synthase was concentrated in the subepithelial lamina propria and inner smooth muscle layers. Complete surgical denervation resulting from transection of the nerve arising from the pelvic ganglion which supplies the vas deferens totally abolished the immunoreactivity to all of the antibodies tested as well as the histochemical reactivity to acetylcholinesterase. In sections of the prostatic end of the vas deferens taken from rats neonatally pretreated with capsaicin, immunoreactivity to calcitonin gene-related peptide and substance P was reduced by 75 and 83%, respectively. Immunoreactivity to neuropeptide Y, vasoactive intestinal polypeptide and nitric oxide synthase was similar in tissue sections taken from capsaicin-treated rats and those taken from control tissues. Pretreatment of rats with guanethidine or 6-hydroxydopamine decreased immunoreactivity to tyrosine hydroxylase and neuropeptide Y by 60-70%, but immunoreactivity to substance P, vasoactive intestinal polypeptide and nitric oxide synthase was unchanged, while immunoreactivity to calcitonin gene-related peptide and acetylcholinesterase staining was increased by guanethidine but not by 6-hydroxydopamine treatment. Triple labelling experiments showed nitric oxide synthase, vasoactive intestinal polypeptide and acetylcholinesterase all to be co-localized in some nerve fibres. These results indicate that the nitric oxide synthase contained in the nerve fibres innervating the rat vas deferens is unaffected by pretreatment of rats with capsaicin, 6-hydroxydopamine or guanethidine but is abolished by surgical denervation, of postganglionic parasympathetic, sympathetic and sensory nerves. Therefore it appears that nitric oxide synthase is co-localized with vasoactive intestinal polypeptide in the postganglionic parasympathetic nerves which innervate the rat vas deferens.

1. The interaction between the cholinergic and nitrergic innervation was investigated in circular muscle strips of the pig gastric fundus. 2. In physiological salt solution containing 4 x 10(-6) M guanethidine, electrical field stimulation (EFS; 40 V, 0.5 ms, 0.5-32 Hz, 10 s at 4 min intervals) induced small transient relaxations at 0.5-4 Hz, and large frequency-dependent contractions, sometimes followed by off-relaxations, at 8-32 Hz. 3. In the presence of L-NG-nitroarginine methyl ester (L-NAME; 3 x 10(-4) M) or physostigmine (10(-6) M), relaxations were reversed into contractions and contractions were enhanced. Physostigmine added to L-NAME further enhanced contractions, while addition of L-NAME to physostigmine had no additional effect. Off-relaxations were enhanced in the presence of L-NAME and physostigmine. L-NAME and physostigmine consistently increased basal tone. 4. Tissues contracted by 5-hydroxytryptamine or by acetylcholine responded to EFS in a similar way as in basal conditions and L-NAME reversed the relaxations at the lower stimulation frequencies into contractions and enhanced the contractions at the higher stimulation frequencies. 5. Off-relaxations in the presence of L-NAME were partially reduced by alpha-chymotrypsin (10 U ml(-1)). 6. In the absence of physostigmine, the concentration-response curve to exogenous acetylcholine was not influenced by L-NAME. 7. Contractions of the same amplitude induced by EFS at 4 Hz and by exogenous acetylcholine were either decreased or enhanced to the same extent by sodium nitroprusside (SNP; 10(-5) M), depending upon the degree of relaxation by SNP. 8. These experiments suggest that endogenous nitric oxide interferes with cholinergic neurotransmission in the pig gastric fundus by functional antagonism at the postjunctional level. The interaction is independent of the degree of contraction.

1. The effect of cilostazol, an inhibitor of phosphodiesterase type III (PDE III), on the contraction induced by histamine was studied by making simultaneous measurements of isometric force and the intracellular concentration of Ca2+ ([Ca2+]i) in endothelium-denuded muscle strips from the peripheral part of the middle cerebral artery of the rabbit. 2. High K+ (80 mM) produced a phasic, followed by a tonic increase in both [Ca2+]i and force. Cilostazol (10 microM) did not modify the resting [Ca2+]i, but it did significantly decrease the tonic contraction induced by high K+ without a corresponding change in the [Ca2+]i response. 3. Histamine (3 microM) produced a phasic, followed by a tonic increase in both [Ca2+]i and force. Cilostazol (3 and 10 microM) significantly reduced both the phasic and tonic increases in [Ca2+]i and force induced by histamine, in a concentration-dependent manner. 4. Rp-adenosine-3':5'-cyclic monophosphorothioate (Rp-cAMPS, 0.1 mM), a PDE-resistant inhibitor of protein kinase A (and as such a cyclic AMP antagonist), did not modify the increases in [Ca2+]i and force induced by histamine alone, but it did significantly decrease the cilostazol-induced inhibition of the histamine-induced responses. 5. In Ca2+-free solution containing 2 mM EGTA, both histamine (3 microM) and caffeine (10 mM) transiently increased [Ca2+]i and force. Cilostazol (1-10 microM) (i) significantly reduced the increases in [Ca2+]i and force induced by histamine, and (ii) significantly reduced the increase in force but not the increase in [Ca2+]i induced by caffeine. 6. In ryanodine-treated strips, which had functionally lost the histamine-sensitive Ca2+ storage sites, histamine (3 microM) slowly increased [Ca2+]i and force. Cilostazol (3 and 10 microM) lowered the resting [Ca2+]i, but did not modify the histamine-induced increase in [Ca2+]i, suggesting that functional Ca2+ storage sites are required for the cilostazol-induced inhibition of histamine-induced Ca2+ mobilization. 7. The [Ca2+]i-force relationship was obtained in ryanodine-treated strips by applying ascending concentrations of Ca2+ (0.16-2.6 mM) in Ca2+-free solution containing 100 mM K+. Histamine (3 microM) shifted the [Ca2+]i-force relationship to the left and increased the maximum Ca2+-induced force. Under the same conditions, whether in the presence or absence of 3 microM histamine, cilostazol (3-10 microM) shifted the [Ca2+]i-force relationship to the right without producing a change in the maximum Ca2+-induced force. 8. It is concluded that, in smooth muscle of the peripheral part of the rabbit middle cerebral artery, cilostazol attenuates the histamine-induced contraction both by inhibiting histamine-induced Ca2+ mobilization and by reducing the myofilament Ca2+ sensitivity. It is suggested that the increase in the cellular concentration of cyclic AMP that will follow the inhibition of PDE III may play an important role in the cilostazol-induced inhibition of the histamine-contraction.