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Sympathetic and angiotensinergic activation reduce splanchnic oxygen delivery during hypovolemia, which may lead to failure of the intestinal mucosal barrier and eventually multiple organ dysfunction. This study integrates sympathetic and angiotensinergic responses with splanchnic hemodynamics and duodenal mucosal function during hypovolemia and evaluates pharmacologic blockade of either system to ameliorate the impact of acute hypovolemia. Chloralose-anesthetized pigs subjected to 20 and 40% blood volume reductions were randomized to controls or administered guanethidine or enalaprilate to block sympathetic and angiotensinergic activation, as assessed by plasma norepinephrine spillover and angiotensin II levels, respectively. Mesenteric and hepatic oxygen delivery/consumption as well as duodenal mucosal alkaline secretion and potential difference were determined. Hypovolemia preferentially increased mesenteric sympathetic outflow and caused a vigorous angiotensinergic activation. Guanethidine and enalaprilate blocked effectively the sympathetic and angiotensinergic responses. Treatment with enalaprilate, but not guanethidine, prevented the reduction of mesenteric oxygenation and duodenal mucosal alkaline secretion and potential difference observed in control animals. The down-regulation of mesenteric oxygenation and duodenal mucosal function during hypovolemia can be prevented by administration of enalaprilate, whereas guanethidine is uneffective in this respect. Interference with the reninangiotensin system might be of clinical interest to support mesenteric perfusion and organ function in hypovolemia.

Tropospheric ozone exerts well-described toxic effects on the respiratory tract. Less documented, by contrast, is the ability of ozone to induce protective mechanisms against agents that are toxic to the lungs. In particular, interactions between ozone and the sympathetic nervous system have never been considered. Using a model of permeability edema in isolated perfused rabbit lungs, we report here that, immediately after exposure of rabbits to 0.4 ppm ozone for 4 hr, the pulmonary microvascular responses to acetylcholine and substance P are completely blocked. Several lines of evidence, including partial inhibition of the ozone-induced protective effect by several drugs (alpha2- and beta-adrenergic antagonists, neuropeptide Y antagonist, guanethidine), measured levels of released catecholamines in blood and urine and the in vitro response of isolated lungs exposed to 0.4 ppm ozone all seem to suggest that ozone can stimulate pulmonary adrenergic fibers and induce the local release of catecholamines and neuropeptide Y, this resulting in transient protection against pulmonary edema. We also showed that, 48 hr after the exposure, ozone increased the baseline microvascular permeability and the response to low concentrations of acetylcholine., (Copyright 1997 Academic Press.)

Unlike its effects on the rest of the GI tract, the effects of pituitary adenylate cyclase-activating peptide (PACAP) on the internal anal sphincter (IAS) are not known. We examined the actions of PACAP-38 (here PACAP) and PACAP-27 on the basal IAS tone of circular smooth muscle strips before and after the administration of different neurohumoral antagonists. PACAP caused a concentration-dependent fall in the basal tone of the IAS. Interestingly, however, at higher concentrations, PACAP caused a biphasic response: an initial contraction followed by a relaxation. Both the contractile and the relaxant responses were insensitive to atropine, guanethidine, apamin or tetrodotoxin. Both the contractile and the relaxant effects were inhibited by PACAP 6-38 (a selective antagonist of PACAP), vasoactive intestinal polypeptide 10-28 (a vasoactive intestinal polypeptide antagonist) and PACAP tachyphylaxis. The nitric oxide synthase inhibitor Nomega-nitro-L-arginine attenuated the inhibitory but not the excitatory effect of PACAP. Conversely, the contractile but not the relaxant effect of PACAP on the IAS was nearly obliterated by the substance P antagonist spantide. The N-type Ca++-channel blocker omega-conotoxin caused significant suppression of both the contractile and the inhibitory actions of PACAP. We conclude that in the IAS, PACAP has a dual effect: a contraction followed by a relaxation. The contraction of IAS by PACAP is speculated to occur via the activation of PACAP receptor at the substance P-containing nerve terminals. PACAP-induced IAS relaxation, on the other hand, appears to be mediated in large part by its direct action at the smooth muscle cells and in part by its action at the nerve terminals of the myenteric inhibitory neurons.

1. We investigated the vagal pathways mediating the gastric accommodation reflex in the rat stomach. 2. Gastric distension (6 ml) evoked an increase of 9.0 +/- 1.0 cmH2O of intragastric pressure in vivo. Pretreatment with tetrodotoxin (TTX) caused a significant pressure increase by gastric distension, reaching 17.0 +/- 1.7 cmH2O, suggesting mediation by neural pathways. 3. The pressure increase evoked by gastric distension was significantly enhanced in vivo by acute truncal vagotomy (TV), hexamethonium (C6), and NG-nitro-L-arginine methyl ester (L-NAME), but not by vasoactive intestinal polypeptide (VIP) antiserum, guanethidine, or splanchnicotomy. 4. Gastric distension (6 ml) evoked a much larger intragastric pressure in the denervated, vascularly isolated, perfused rat stomach in vitro. Intra-arterial application of TTX and L-NAME did not cause further pressure increases evoked by gastric distension. 5. The pressure increase evoked by gastric distension remained high 2 weeks after TV in vivo. However, the accommodation reflex was fully restored 4 weeks after TV in vivo. This reflex was antagonized by TTX, C6 and L-NAME, but not by VIP antiserum, guanethidine and splanchnicotomy. 6. Similar to in vivo studies, gastric distension caused a smaller increase in intragastric pressure in response to gastric distension in the denervated, vascularly isolated, perfused stomach obtained from rats 4 weeks after vagotomies in vitro. The pressure increase evoked by gastric distension was significantly enhanced by L-NAME, hexamethonium and TTX. 7. It is suggested that the vago-vagal reflex plays an important role in mediating the accommodation reflex. This involves a vagal efferent pathway that uses nitric oxide as a final neurotransmitter mediating gastric relaxation in intact rats. It is also suggested that the adaptive mechanism mediating the accommodation reflex following vagotomy occurs in the gastric myenteric plexus.

Neuropeptide Y (NPY) appears to be involved in the sympathetic regulation of vascular tone. To assess the putative role of NPY in mesenteric circulation, the release and biological effect of NPY were examined after electrical stimulation of perimesenteric arterial nerves. Nerve stimulation with trains of 2-30 Hz increased the perfusion pressure of the arterially perfused rat mesenteric bed in a frequency- and time-dependent fashion. Trains of 15-30 Hz significantly displaced to the left, approximately threefold, the noradrenaline (NA)-induced pressor concentration-response curve, in addition to increasing significantly its efficacy. Perfusion with 10 nM exogenous NPY mimicked the electrical stimulation effect, causing a threefold leftward shift of the NA concentration-response curve and increasing the maximal NA response. These effects were antagonized by 100 nM BIBP 3226, indicating the activity of NPY-Y1 receptors. Electrical stimulation of the perimesenteric nerves released immunoreactive NPY (ir-NPY) in a frequency-dependent fashion; the ir-NPY coelutes with synthetic NPY as confirmed by HPLC. Both the electrically induced pressor response and the calcium-dependent release of NPY were obliterated in preparations perfused with 1 microM guanethidine or in rats pretreated intravenously for 48 h with 6-hydroxydopamine, thus revealing the sympathetic origin of these phenomena. Only a small proportion of the total NPY content in the perimesenteric arterial nerves is released after electrical stimulation. Chromatographic studies of the physiological sources of the ir-NPY support that NPY fragments are generated via peptidase degradation. The present findings demonstrate that NPY is released from the perimesenteric arterial sympathetic nerves and acts, via the activation of NPY-Y1 receptors, as the mediator responsible for the potentiation of NA's effect on perfusion pressure in the isolated rat mesenteric bed.

1. Electrical field stimulation (EFS) (1-10 Hz, 30 V, 2 ms) of frog oesophageal body strips resulted in frequency-dependent non-adrenergic, non-cholinergic (NANC) relaxations. 2. Tetrodotoxin (TTX) (10(-6)-10(-5) M) had no effect on EFS evoked relaxations with a 2 ms pulse width. At a pulse width of 0.5 ms only the responses to the highest frequency (10 Hz) were significantly inhibited by TTX at 10(-5) M. Relaxation at 2 ms pulse width were unaffected by omega-conotoxin (10(-6) M), nifedipine (10(-6) M) or cobalt (5 x 10(-4) M). 3. NG-nitro-L-arginine (L-NOARG) (10(-6)-10(-4) M), a nitric oxide synthase (NOS) inhibitor, caused a concentration-dependent inhibition of the EFS-induced NANC relaxant responses. The inhibitory effect of L-NOARG was both prevented and reversed by L-arginine but not D-arginine (5 x 10(-3) M). 4. The phosphodiester type V inhibitor (PDE V), SK&F 96231 (10(-7)-10(-4) M), caused a concentration-dependent potentiation of both the percentage relaxation and the duration of the relaxant responses to EFS. 5. ODQ (10(-7)-10(-5) M), a guanylate cyclase inhibitor, produced a concentration-dependent inhibition of EFS-evoked NANC relaxations. 6. Oxyhaemoglobin (10(-6) M), which binds nitric oxide (NO), inhibited NANC relaxations to EFS. 7. The NO donor sodium nitroprusside (SNP) (10(-8)-10(-4) M) produced a concentration-dependent inhibition of evoked tone. L-NOARG (10(-4) M) had no effect on the SNP evoked relaxations. Preincubation with oxyhaemoglobin (10(-6) M) caused a reduction in the SNP (10(-6)-10(-5) M) induced relaxations. 8. These results suggest NO is the relaxant transmitter of the frog oesophageal body and the source of NO may be non-neuronal.

Vasoconstrictor responses elicited by periarterial electrical nerve stimulation were analyzed pharmacologically in the canine isolated, perfused intermediate auricular artery. Phentolamine (10 microM) significantly inhibited the vasoconstrictor responses to stimulation at 5 Hz and over but not those to stimulation at frequencies below 5 Hz. Additionally administered alpha, beta-methylene ATP (1 microM) abolished the phentolamine-resistant vasoconstrictions at all frequencies used in this study. In contrast, suramin (100 microM) inhibited the vasoconstrictor responses to stimulation at 5 Hz and below but not those to stimulation at frequencies higher than 5 Hz. Phentolamine abolished the suramin-resistant vasoconstriction at all frequencies. Phentolamine and alpha, beta-methylene ATP selectively abolished the vasoconstrictor responses to exogenous noradrenaline and ATP, respectively. These results show that the co-transmission of noradrenaline and ATP exists at sympathetic nerve terminals in the canine intermediate auricular artery, and that purinergic transmission is mainly involved in the vasoconstrictor responses to low-frequency nerve stimulation.

We now report on the effects of nociceptin, the endogenous ligand for the orphan opioid-like ORL1 receptor, on cardiovascular parameters (blood pressure and heart rate) in urethane-anaesthetized rats. Nociceptin dose dependently (10-100 nmol/kg i.v.) produced a transient (< 10 min) hypotension and bradycardia. The reduction in blood pressure was inhibited by guanethidine pretreatment and unaffected by bilateral cervical vagotomy. The bradycardia elicited by nociceptin was reduced after bilateral vagotomy and by guanethidine and was abolished by the combination of the two treatments. These findings indicate that nociceptin exerts a pronounced depressant effect on cardiovascular function which is produced indirectly through a concomitant inhibition and activation, respectively, of the sympathetic and parasympathetic outflows to the cardiovascular system.

Noradrenergic contractions induced by electrical field stimulation (EFS) of the rabbit anococcygeus muscle and the human and rabbit corpus cavernosum did not occur until termination of stimulation, even when EFS was applied for long periods (10 min). After treatment with a nitric oxide synthase inhibitor, a scavenger of NO, or a specific inhibitor of the soluble guanylate cyclase, EFS-induced contraction began as soon as stimulation commenced and its magnitude and duration were increased. In the presence of a cGMP-phosphodiesterase inhibitor, the lag period between the end of EFS and the onset of contraction was longer, and the response was smaller. Even when the concentration of endogenous noradrenaline was increased with cocaine, the contraction still did not occur during EFS and the lag period was unchanged, although the response was enhanced. When tissue tone was elevated, relaxation occurred during EFS followed by a contraction. After blockade of neuronal noradrenaline release with guanethidine, contractions of the tissues to increasing concentrations of exogenous noradrenaline were significantly reduced by EFS, an effect that was reversible by inhibition of NO synthase. In contrast, in the rat and mouse anococcygeus muscles contraction began immediately with EFS, and nitrergic stimulation by EFS did not affect the responses elicited by high concentrations of exogenous noradrenaline. These results suggest that the human and rabbit genitourinary organs have a powerful nitrergic innervation that does not merely modulate, but actually controls, the sympathetic responses. Our observations may increase understanding of the balance between nitrergic and sympathetic systems in humans, disruption of which may contribute to certain pathological conditions.

The action of 16 newly synthesized metal complexes having the general structure cis-Pt-(II)-Xn-Ln have been tested in vitro against the promastigote forms of Leishmania donovani. The metal complexes at 24 h and maximum dosages inhibited growth from 0%, e.g. in cis-Pt-nifurtimox, to 100%, e.g. in cis-Pt-(2,3,4,5,6-pentafluoroaniline)2Br2 or cis-Pt-pentamidine-I2. A study of the cytotoxicty of these latter complexes on the phagocytic cell line J-774 showed neither high cytotoxicity nor cytolysis. At the maximum dosage after 24 h of permanent contact with the cells (extreme, non-physiological conditions), cytolysis did not exceed 30%. For most of the compounds, cytolysis ranged from 0%, for cis-Pt-oxamniquine-Cl2 to 27.7%, for cis-Pt-pentamidine-I2. The compound cis-Pt-(2,3,4,5,6-pentafluoroaniline)2-Br2 caused up to 1.4% cytolysis under the above conditions. Parasites exposed to cis-Pt-pentamidine-I2 showed notably reduced DNA, RNA and protein synthesis, unlike those exposed to other compounds. Parasites examined by electron microscopy showed effects mainly on the nucleus, though in some cases the mitochondria were affected, altering the internal membranes of the cytoplasmic organelles. The in-vivo activity of the complex cis-Pt-guanethidine-Cl2 was evaluated in parasitized Wistar rats, in which the number of amastigotes per gram of spleen was reduced by 75% compared with controls.

The presence of inhibitory nonadrenergic noncholinergic (NANC) intrinsic innervation of the circular muscle of the gastrointestinal sphincters of the South American (SA) opossum was investigated in vitro. Isolated circular muscle strips from the esophagogastric and ileocolonic junctions but not from the gastroduodenal(pylorus)region developed spontaneous tension. Tetrodotoxin (TTX, 1 microM) augmented the spontaneous tension only in the ileocolonic junction strips. Electrical field stimulation of esophagogastric and ileocolonic junction strips caused frequency-dependent responses consisting of a relaxation at lower frequencies (< 1 Hz) and a biphasic response or contraction field stimulation abolished the spontaneous activity at lower frequencies and induced contractions at higher frequencies. The responses elicited by electrical field stimulation in the three sphincters were abolished by TTX (1 microM). Electrical field-induced contractions were reduced while relaxations were enhanced by atropine (1 microM). In the presence of atropine (1 microM) and guanethidine (3 microM), electrical field stimulation, nicotine and ATP induced frequency- or concentration-dependent relaxations of the three sphincters that were abolished by TTX (1 microM). Isoproterenol and sodium nitroprusside caused concentration-dependent relaxations which were TTX-resistant. These findings indicate that the sphincteric circular muscle of the SA opossum gastrointestinal tract is relaxed by the activation of intrinsic NANC nerves and therefore can be used as a model for the study of the mechanisms involved in these responses.

This work examined the effects of nicotine on mean arterial pressure and heart rate in non-anesthetized spinal rats. Nicotine (200 mg/kg) was administered as a single bolus, as infusions lasting 7.5, 15 or 30 min, and as a post-infusion bolus. A nicotine bolus increased pressure and rate. These effects were less marked as the rate of infusion decreased. The infusions affected differentially the effects of a subsequent bolus. Thus, while tachycardia was decreased, the blood pressure rise was increased. An initial transient bradycardia was observed after bolus administration, but not during infusions; this effect was unchanged after post-infusion boluses. Pharmacological analysis indicated that tachycardia and bradycardia were predominantly due to ganglionic stimulation, while adrenal and sympathetic nerve catecholamine release played a major role in the pressor response. These results indicate that slow nicotine infusions do not induce tachyphylaxis for all of the cardiovascular effects of a subsequent bolus, and that development of acute tolerance appears to depend on the mechanism of action of the response.

The effects of adrenergic receptor ligands on uptake and storage of the radiopharmaceutical [125I]metaiodobenzylguanidine (MIBG) were studied in the human neuroblastoma cell line SK-N-SH. For uptake studies, cells were incubated for 15 min with varying concentrations of alpha-agonist (clonidine, methoxamine, and xylazine), alpha-antagonist (phentolamine, tolazoline, phenoxybenzamine, yohimbine, and prazosin), beta-antagonist (propranolol, atenolol), beta-agonist (isoprenaline and salbutamol), mixed alpha/beta antagonist (labetalol), or the neuronal blocking agent guanethidine, prior to the addition of [125I]MIBG (0.1 microM). The incubation was continued for 2 h and specific cell-associated radioactivity was measured. For the storage studies, cells were incubated with [125I]MIBG for 2 h, followed by replacement with fresh medium with or without drug (MIBG, clonidine, or yohimbine). Cell-associated radioactivity was measured at various times over the next 20 h. Propanolol reduced [125I]MIBG uptake by approximately 30% (P<0.01) at all concentrations tested, most likely due to nonspecific membrane changes. However, incubation with the other beta-agonists or antagonists failed to elicit significant reductions in uptake. In contrast, all of the alpha-agonists significantly inhibited uptake (P<0.05); guanethidine >xylazine >clonidine=methoxamine. The alpha-antagonists demonstrated a broad range of inhibition (phenoxybenzamine >>phentolamine >prazosin >>yohimbine=tolazoline)(P< 0.05). The mixed ligand, labetolol, inhibited MIBG uptake in a dose-dependent manner with an apparent IC50 of 0.65 microM. The retention studies demonstrated that unlabeled MIBG caused profound self-inhibition (P<0.01). Clonidine produced a modest inhibition of retention and yohimbine had no effect. Labetalol, phenoxybenzamine, guanethidine, and propranolol reduced uptake of [125I]MIBG by neuroblastoma cells in culture. Although only labetalol has been reported to cause false-negative MIBG scans, our results suggest that these other drugs have the potential to interfere with MIBG imaging and therapy, particularly at high doses. Adrenergic drugs did not alter cytoplasmic retention of [125I]MIBG in neuroblastoma cells but may have potential in tumors such as phenochromocytoma, where granular storage of MIBG has been observed. Inhibition of [125I]MIBG retention by unlabeled MIBG supports the use of high specific activity radioiodinated MIBG for both diagnosis and therapy.

We aimed to assess whether calcitonin gene-related peptide (CGRP) can modulate the release of tachykinins which are the main nonadrenergic noncholinergic (NANC) excitatory transmitters to the circular muscle of the guinea-pig proximal colon. In organ bath experiments, electrical field stimulation (EFS) in the presence of atropine (1 microM) and guanethidine (3 microM) evoked twitch phasic NANC contractions which were abolished by the combined administration of tachykinin NK1 and NK2 receptor antagonists. Human alphaCGRP (CGRP, 1-100 nM) produced a concentration-dependent potentiation of the amplitude of the NANC contractions induced by EFS while salmon calcitonin (up to 1 microM) had no effect. The potentiating effect of CGRP was unaffected by in vitro capsaicin pretreatment (10 microM for 15 min), peptidase inhibitors (captopril, bestatin and thiorphan, 1 microM each), apamin (0.3 microM) plus L-nitroarginine (L-NOARG, 100 microM) and by the CGRP1 receptor antagonist, the C-terminal fragment CGRP(8-37) (1 microM). The NK2 receptor antagonist MEN 10627 which, when administered alone, had only a partial inhibitory effect on the amplitude of NANC twitches, concentration-dependently (10 nM-1 microM) inhibited the potentiating effect of CGRP. CGRP (1-100 nM) produced a concentration-dependent potentiation of the atropine-sensitive cholinergic contractions evoked by EFS in the presence of guanethidine and of tachykinin NK1 and NK2 receptor antagonists. Similar to the effect of CGRP, application of capsaicin (0.1-1 microM) potentiated the amplitude of the NANC contraction to EFS, an effect undergoing complete desensitization upon a second application of the drug. CGRP (0.1 microM) did not affect the contractile action of a submaximally effective concentration of neurokinin A (2 nM) while it inhibited that induced by substance P (2 nM). In sucrose gap, single pulse EFS in the presence of atropine (1 microM) and guanethidine (3 microM) induced an inhibitory junction potential (i.j.p.) and a small excitatory junction potential (e.j.p.). CGRP (0.1 microM) produced membrane hyperpolarization and relaxation without affecting i.j.p. amplitude but concomitantly increased the e.j.p. amplitude to induce a contraction in correspondence to each electrical pulse. In the presence of the NK1 receptor antagonist, GR 82334 (3 microM), the membrane hyperpolarization and relaxation produced by CGRP and the EFS-evoked i.j.p. were unaffected, while the potentiating effect of CGRP on the EFS-evoked NANC e.j.p. and the corresponding contraction were abolished. We conclude that, in addition to the previously characterized direct smooth muscle relaxant action via CGRP1 receptors (Maggi et al. Regulatory Peptides 61, 27-36, 1996), CGRP also induces a remarkable potentiation of excitatory neurotransmission to the circular muscle of the guinea-pig colon via CGRP2 receptors. The latter effect, documented in this study, is evidenced on both the atropine-sensitive and the atropine-resistant (tachykinin-mediated) components of excitatory transmission: this effect does not involve mediator(s) release from capsaicin-sensitive primary afferent nerves, nor inhibition of peptide degradation or modulation of NANC inhibitory transmission.

1. Positive inotropic responses to calcitonin gene-related peptide (CGRP) were evaluated in atria isolated from in vivo rat models of hyper-sensory-motor innervation (following neonatal guanethidine treatment) and hypo-sensory-motor innervation (following neonatal capsaicin treatment), to explore the hypothesis that functional responsiveness of atrial myocardium to CGRP may correlate with tissue levels of the sensory-motor neurotransmitters. Comparative of inotropic responses to CGRP following in vitro treatment of atria with guanethidine was also performed. 2. Following long-term guanethidine treatment, positive inotropic responses to CGRP were significantly attenuated, while supersensitivity to the sympathetic transmitter noradrenaline was shown. Maximal inotropic responses to CGRP (30 nM) were 214.0 +/- 28.1 (n = 8) and 146.8 +/- 21.7 mg (n = 8; P < 0.01) increase of the basal contractile tension in control and treated preparations, respectively. The pD2 values for noradrenaline were 6.71 +/- 0.12 (n = 8) and 7.26 +/- 0.13 (n = 6; P < 0.01) in control and treated atria, respectively. Acute application of guanethidine in vitro did not modify the positive inotropism by CGRP or the beta-adrenoceptor agonist isoprenaline. 3. Sensory-motor hypoinnervation following chronic treatment with capsaicin did not affect the inotropic responses to CGRP. Neither guanethidine nor capsaicin treatment affected the contractile apparatus of myocytes, as demonstrated by similar basal contractile tension as well as calcium-evoked inotropic responses in control and treated preparations. 4. In summary, increased sensory-motor innervation, following long-term sympathectomy with guanethidine, resulted in attenuation of the inotropic responses of the rat atrium to CGRP, while no changes in the inotropic responses were seen following sensory-motor denervation with capsaicin. Down-regulation of CGRP receptors or altered post-receptor signalling may be involved in the reduced responsiveness to CGRP.

Although vasodilator agents have been used to alleviate the pain of complex regional pain syndromes, the precise mechanism of pain relief is not well known. In this study the effects of various kinds of vasodilators on primary afferent nociceptors were investigated by measuring the thermal pain threshold. Evaluated in the study were the effects of guanethidine (2 mg/mL), nicardipine (0.2 mg/mL). Nitroglycerin (0.3 mg/mL), and prostaglandin E1 (1 microgram/mL) on the cutaneous pain threshold and blood flow at 7-day intervals in six healthy volunteers. Each aliquot of 0.5 mL of the test vasodilator or lidocaine (10 mg/mL) and saline (control) were intradermally injected at three sites each on both forearms. The pricking-pain threshold and skin tissue blood flow were determined using a radiant heat-stimulating system and a laser-Doppler tissue-blood flowmeter, respectively. The pain threshold increased with lidocaine, guanethidine, and nicardipine; remained unchanged with Nitroglycerin; but decreased with prostaglandin E1. In contrast, the skin tissue blood flow increased by four to nine times with all vasodilators. These results indicate that the effect of vasodilators on primary afferent nociceptors is not related to the vasodilating effect and may not involve a common mechanism of action for pain relief in complex regional pain syndromes.

The aims of this study were to determine if neural pathways between the duodenum and sphincter of Oddi are intramural, activated by duodenal electrical field stimulation (EFS) in vitro, and contain capsaicin-sensitive primary afferents. The possible involvement of cholinergic (muscarinic and/or nicotinic) and adrenergic receptors in these pathways were also investigated. Duodenal EFS (5-60 Hz, 70 V, 0.5 ms duration, 10 s train) at sites 2 cm oral and 2 cm anal to the sphincter of Oddi-duodenal junction produced frequency-dependent excitatory responses in the sphincter of Oddi, measured by manometry (n = 3). Excitatory responses from duodenal circular muscle were also evident. Tetrodotoxin (1 microM; n = 7) pretreatment abolished both sphincter of Oddi and duodenal responses to duodenal EFS. Crushing the duodenum between the site of stimulation and the sphincter of Oddi-duodenal junction also abolished sphincter of Oddi response. The sphincter of Oddi responses to duodenal EFS at the oral and anal sites were reduced by pretreatment with (i) atropine (100 nM: n = 7) to 19 +/- 6% (P < 0.05) and 22 +/- 8% (P < 0.05) of control respectively. (ii) hexamethonium (100 microM: n = 9) to 10 +/- 2% (P < 0.01) and 6.0 + 2.5% (P < 0.01) of control respectively and (iii) guanethidine (1 microM; n = 6) to 75 +/- 6% (P < 0.05) and 78 +/- 10% (P < 0.05) of control, respectively. Combined pretreatment with phentolamine and propranolol (both 1 microM; n = 7) was without effect, as was capsaicin (1 microM; n = 12) pretreatment. Excitatory intramural pathways between the sphincter of Oddi and the duodenum are primarily cholinergic in nature and contain an adrenergic component. Capsaicin-sensitive primary afferents are not involved.

The aim of this study was to investigate the interaction between the K+ channel opener levcromakalim and several quaternary ions. Cumulative vasorelaxant-response curves to levcromakalim were constructed in the absence and in the presence of the quaternary ions, in the pig coronary artery. The most potent compounds (based on 'apparent pKB' values) were: propyltriphenylphosphonium (7.33), butyltriphenylphosphonium (7.04), tetraphenylarsonium (6.86), tetraphenylphosphonium (6.81), ethyltriphenylphosphonium (6.70), and hexyltriphenylphosphonium (6.63). Tetrabutylphosphonium (6.06), tetrabutylammonium (5.12), methyltriphenylphosphonium (5.25), clofilium (5.66) and guanethidine (5.61) were significantly less potent. Tetrapropylammonium, tetrapentyltin and tetraphenylboron were inactive at the maximum concentrations used (30 microM). Tetraphenylboron (10-100 microM) fully reversed tetraphenylphosphonium, tetraphenylarsonium (both at 3 microM), tetrabutylammonium (30 microM) and clofilium (10 microM) and partially reversed guanethidine (10 microM) antagonism of levcromakalim responses indicating a similarity in the mechanism of action of these chemically distinct compounds. The results show that quaternary ions similar in structure to tetraphenylphosphonium, i.e., containing phosphonium ion centre and phenyl side chains, are the most potent antagonists of levcromakalim, in pig coronary artery. It is also apparent that marked changes can be made in the substitution on the phosphonium ion (ethyl to hexyl) with little or no effect on their potency.

Transmural electrical field stimulation (EFS, 4-32 Hz) produced a biphasic contractile response consisting of a rapid and transient contraction (first phase) followed by a slow contraction (second phase) in ring preparations of guinea pig portal veins. Both contractions were enhanced by the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 30 microM). In the presence of L-NAME, tetrodotoxin (1 microM) and guanethidine (3 microM) inhibited both contractions and phentolamine (10 microM), and reserpine treatment abolished the first-phase contraction without affecting the second-phase contraction. These results suggest that the first-phase contraction is caused by norepinephrine released from the perivascular nerves. In the presence of phentolamine and L-NAME, the second-phase contraction was inhibited by the nonselective P2-purinoceptor antagonist suramin (30-300 microM) and the P(2Y)-purinoceptor antagonist reactive blue 2 (RB2; 10-100 microM). alpha,beta-Methylene-adenosine triphosphate (alpha,beta-mATP; 3-30 microM), which desensitizes P(2X)-purinoceptors, and the P(2X)-purinoceptor antagonist 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS; 1-10 microM) had a little effect. Exogenous ATP (0.1-3 mM) and UTP (0.1-3 mM) in the presence of L-NAME produced contractions in a concentration-dependent manner. The ATP-induced contraction was enhanced by suramin, RB2, and DIDS but unaltered by alpha,beta-mATP. The UTP-induced contraction was inhibited by suramin and RB2 but unaltered by alpha,beta-mATP and DIDS. These results indicate that in the guinea pig portal vein, the classic P(2X)-purinoceptors do not contribute to the nonadrenergic component of sympathetic neurotransmission. Furthermore, the pharmacology of the nonadrenergic component of neurotransmission resembles that of vasoconstrictor responses to exogenous UTP rather than to ATP.

1. Alteration in the flow of blood to the penis is thought to be the most frequent organic cause of erectile dysfunction or impotence. However, information concerning the penile small arteries (helicine arteries) which control blood flow between the arterial systemic circulation and the cavernous sinusoids is scarce. Therefore, the purpose of the present study was to investigate the involvement of nitric oxide, which is considered to be the main neurotransmitter in corpus cavernosum, in both the non-adrenergic non-cholinergic inhibitory neurotransmission and the endothelium-dependent responses in human penile small arteries. 2. Penile small arteries (lumen diameter 200-700 microns), which were branches of the human deep penile arteries obtained either from patients undergoing penile surgery or from organ donors, were mounted in microvascular myographs for isometric tension recording and electrical field stimulation was performed in the presence of guanethidine and atropine to block adrenergic neurotransmission and muscarinic receptors, respectively. 3. In phenylephrine-contracted penile small arteries, electrical field stimulation (0.5-32 Hz) induced frequency-dependent relaxations of both endothelium-intact and -denuded preparations. The inhibitor of nitric oxide synthase, NG-nitro-L-arginine (3 x 10(-5) mol/l), abolished the relaxations at the lowest frequencies, while slow-developing relaxations were still observed at high frequencies (16 and 32 Hz). The inhibitory effect of NG-nitro-L-arginine was reversed in the presence of L-arginine (3 x 10(-3) mol/l). Tetrodotoxin totally abolished the relaxations to electrical field stimulation. In contracted small penile arteries in the presence of NG-nitro-L-arginine, the nitrovasodilator sodium nitroprusside induced potent relaxations. 4. The endothelium-dependnet vasodilator acetylcholine induced relaxations of penile small arteries, which were only partially reduced in the presence of NG-nitro-L-arginine. In contrast, the relaxations to acetylcholine of trabecular corpus cavernosum preparations were almost abolished in the presence of NG-nitro-L-arginine. 5. The present study suggests that relaxations of human intracavernosal small penile arteries induced by non-adrenergic non-cholinergic nerve stimulation partially involve nitric oxide and also another inhibitory transmitter causing relaxations resistant to nitric oxide synthase blockade. In addition, endothelium-dependnet relaxations in human small penile arteries are mediated by both nitric oxide and a factor resistant to NG-nitro-L-arginine.

1. Vasoconstrictions induced by transmural electrical field stimulation were frequency-dependent from 2 to 32 Hz in the rabbit isolated splenic artery. All contractions were abolished in the presence of tetrodotoxin 1 microM or guanethidine 100 microM. Stimulation at a frequency of more than 32 Hz induced both neurogenic and myogenic responses. 2. Prazosin (1 microM) did not significantly affect vascular contractions to electrical stimulation. Desensitization of P2X-purinoceptors with alpha, beta-methylene ATP (alpha, beta-meATP, 3 microM) abolished the contractions to stimulation at 2-8 Hz and inhibited more than 80% of the vascular response at 16 Hz, but it did not significantly change the responses at 32 Hz. Contractile responses at 32 Hz were inhibited by a combination of prazosin and alpha, beta-meATP. Effects of pyridoxal-phosphate-6-azophenyl-2', 4'-disulphonic acid tetrasodium salt (a selective P2X-purinoceptor antagonist) and suramin (a competitive P2-purinoceptor antagonist) on the neurogenic responses were investigated in this study. 3. 2,2'-Pyridylisatogen tosylate (PIT, 0.3-3 microM) significantly potentiated the vasoconstrictions to electrical stimulation at 2-32 Hz in a concentration-dependent manner. Potentiated responses were restored to the control level 30 min after washing. Concentration-dependent response curves for noradrenaline (NA) or alpha, beta-meATP were not significantly changed by 3 microM PIT, and vasoconstriction by adenosine 5'-triphosphate (ATP, 300 microM) was unaffected by PIT. Coomassie brilliant blue-G (1 microM), which shares the potentiating effect on a recombinant P2Y-purinoceptor with PIT (King et al., 1996), did not inhibit or potentiate the purinergically-mediated component of the response to sympathetic nerve stimulation. The selective alpha 2-adrenoceptor antagonist yohimbine (1 microM) also potentiated the vascular responses to electrical stimulation. 4. The present results indicate that ATP evokes postjunctional contractile responses at low and high frequency electrical stimulation of sympathetic nerves supplying the rabbit splenic artery. PIT potentiates the responses to sympathetic (purinergic) nerve stimulation; this appears to be mainly via prejunctional rather than postjunctional actions.

The relaxant effects of electrical field stimulation (EFS) and exogenously applied acetylcholine (ACh) or acidified NaNO2 (a-NaNO2) were investigated in the isolated mouse corpus cavernosum precontracted with phenylephrine hydrochloride (PE). Tetrodotoxin (TTX) blocked the relaxant effects of EFS completely, whereas it had no effect on the responses to ACh or a-NaNO2. Guanethidine and indomethacin failed to affect the electrically or ACh-induced relaxations. Atropine completely blocked the effect of ACh; however, it caused a slight reduction in the relaxation evoked by EFS. NG-Nitro-L-arginine (L-NOARG) reduced the effects of EFS and ACh significantly, but it was ineffective on the relaxations induced by a-NaNO2. The inhibitory action of L-NOARG was partly restored by L-arginine, but not by D-arginine. Methylene blue (MB) and hydroxocobalamin (HC) exhibited significant inhibition on the relaxations evoked by EFS, ACh and a-NaNO2. Hydroquinone (HQ) reduced relaxation due to a-NaNO2, but did not affect that of EFS and ACh. Our findings suggest that EFS-induced relaxations of mouse cavernosal tissue are mediated by a transmitter which probably resembles an organic nitrate.

To determine the role of VIP-like peptides on neurotransmission of vagus nerve, we evaluated the effects of helodermin, helospectin, and vasoactive intestinal peptide (VIP) on the contraction of guinea pig tracheal smooth muscle evoked by electrical field stimulation (EFS) or the exogenous application of actylcholine (ACh). Isometric tension of tracheal strips was measured in the presence of indomethacin (10(-6) M) and of guanethidine (10(-6) M). VIP (10(-9) M to 10(-7) M) significantly suppressed the contraction evoked by EFS. VIP, at concentrations of 10(-9) M and 10(-8) M, did not affect the ACh-evoked contraction, but a concentration of 10(-7) M suppressed ACh-evoked contraction. Helospectin and helodermin (10(-8) M and 10(-7) M) significantly suppressed the EFS-evoked contraction, but 10(-9) M showed no effect. Helospectin and helodermin had no effect on the ACh sensitivity of smooth muscle up to 10(-8) M, but a concentration of 10(-7) M suppressed the ACh-evoked contraction. These results indicate that helodermin, helospectin, and VIP exert both pre- and post-junctional inhibitory effects on the airway smooth muscle of guinea pigs. These peptides, thus, inhibited tracheal smooth muscle contraction prejunctionally at low concentrations, and acted postjunctionally at higher concentrations.

In view of the extensive use of Pterodon species in Brazilian folk medicine, the present investigation was performed to examine the involvement of biogenic amines in antinociceptive by a vouacapan (6 alpha-7 beta-dihydroxy vouacapan-17 beta-oate), extracted from seeds of Pterodon polygalaeflorus Benth), using acetic acid writhing test in mice. The alpha 2-adrenergic (yohimbine) and D2-dopaminergic (domperidone) antagonists and the pretreatment with the peripheral noradrenergic depletor, guanethidine partially inhibited the antinociceptive effect of vouacapan. Dopamine and D2 dopaminergic agonist (Ly 17155) caused antinociceptive that was not antagonized by naloxone but by domperidone, whereas noradrenaline induce pain. A synergistic analgesic effect was obtained when vouacapan was associated with clonidine or dopamine. These results indicate that vouacapan acts, at least in part, through activation of the catecholaminergic system.

Brain ischemia is often accompanied by acute gastric lesions. To clarify the underlying mechanism, the influence of acute ischemic insult to the brain on gastric hemodynamics and mucosal integrity was examined in spontaneously hypertensive rats. One hour after brain ischemia, gastric mucosal blood flow decreased to 71% of the preischemic levels in the control rats but was preserved significantly better, at 94 and 108%, in the prazosin-treated and guanethidine-treated rats, respectively. Vagotomy almost abolished the decrease in gastric mucosal blood flow during cerebral ischemia. Intragastric 0.6 N hydrochloric acid administered just after reperfusion induced more severe hemorrhagic ulcers in the control than in the prazosin-treated and vagotomized groups. These results suggest that noradrenergic neurons acting through alpha1-adrenoceptors contributes to the decrease in gastric mucosal blood flow, and the subsequent disturbed integrity of the gastric mucosa, through the vagal adrenergic pathway during brain ischemia in spontaneously hypertensive rats.

Nitric oxide (NO) mediates neurogenic relaxations of gastrointestinal (GI) smooth muscle. NO synthase (NOS) inhibitors also alter neurogenic contractions, suggesting NO modulates excitatory neurotransmitter release. In circular muscle-myenteric plexus preparations, guanethidine and either scopolamine or CP-96,345, a neurokinin-1 (NK1) receptor antagonist, were used to isolate nonadrenergic, noncholinergic (NANC) or cholinergic contractions, respectively. NOS inhibitors and hemoglobin potentiated neurogenic NANC but not cholinergic contractions and did not affect NK1 receptor agonist [substance P methyl ester (SPME)]-induced contractions. Sodium nitroprusside (SNP), a NO donor, attenuated NANC and cholinergic neurogenic contractions, but cholinergic contractions were less sensitive to SNP. SNP partially attenuated SPME-induced contractions, and apamin reduced inhibition of NANC contractions by SNP. Bethanechol responses were not affected by SNP. These data indicate NANC but not cholinergic contractions are inhibited by endogenous NO, suggesting differential regulation of release of tachykinins and acetylcholine from enteric nerves. NK1 receptor-but not muscarinic receptor-activated postjunctional pathways are also inhibited by NO. Therefore, prejunctional and postjunctional modulation of NANC contractions are mechanisms for inhibition of GI motility by endogenous NO.

Nonadrenergic noncholinergic (NANC) vasodilator mechanisms may contribute to the maintenance of low vascular resistance characteristic of the pulmonary circulation. Previous studies have demonstrated that nitric oxide (NO) is the principal NANC neurotransmitter in guinea pig pulmonary arteries. We examined whether NANC relaxation could be demonstrated in human pulmonary arteries, and the role of NO in this phenomenon. Fresh human pulmonary artery rings, with and without an intact endothelium, were mounted in organ baths containing Krebs' solution and precontracted with U44069 (0.3 microM). Adrenergic and cholinergic neurotransmitter pathways were blocked with guanethidine and atropine, respectively (10 microM each). In both endothelium-intact and -denuded vessels, electrical field stimulation (EFS, 1 to 10 Hz, 100 V) resulted in a frequency-dependent relaxation (maximal relaxation of 25 +/- 4% and 15 +/- 2% in endothelium-intact and -denuded vessels, respectively). Tetrodotoxin (0.3 microM) abolished the EFS-induced relaxation. L-NG-nitro-arginine methyl ester (L-NAME, 10 microM), was used to block enzymatic synthesis of NO. In both endothelium-intact and -denuded vessels, L-NAME reduced NANC relaxation to approximately 50% of control values. This reduction was reversible with the application of L-arginine (100 microM). We conclude that NANC relaxation exists in human pulmonary arteries and that it is partly mediated through NO.

The effect of capsaicin and resiniferatoxin on gastrointestinal blood flow was studied in anaesthetized rats by laser Doppler flowmetry. Resiniferatoxin injected into the jugular vein (0.08-1.6 nmol/kg) produced a marked and dose-dependent increase in gastric blood flow, while the effect of capsaicin (0.33-19.6 nmol/kg) was transient, variable and accompanied by profound systemic blood pressure changes. After acute bilateral cervical vagotomy combined with sympathetic neurone blockade (guanethidine 16 mumol/kg) or alpha-adrenoceptor blockade (phentolamine 1.6 mumol/kg), the vasodilator response to injected resiniferatoxin was more pronounced, indicating that the resiniferatoxin-induced gastric vasodilatation is not due to reflexes via parasympathetic or sympathetic efferent fibres. Resiniferatoxin given i.v. (0.08-0.64 nmol/kg) evoked a similar increase in the blood flow of the jejunum. Capsaicin (0.33-33 microM) or resiniferatoxin (0.16-1.6 microM) applied topically to the serosal surface of the stomach or jejunum produced a pronounced and long-lasting increase in blood flow after vagotomy and guanethidine treatment. The blood flow and blood pressure responses to capsaicin and resiniferatoxin were absent in rats desensitized with systemic capsaicin pretreatment. These laser Doppler data provide the first evidence for the effect of resiniferatoxin on gastrointestinal microcirculation and indicate the advantages of this agent and technique to study the sensory-efferent function of capsaicin-sensitive fibres.

The present experiments were designed to investigate the influence of the sympathoadrenal system on the hyperglycemic action of angiotensin II in freely moving rats divided into four experimental groups: (1) sham-operated animals submitted to intravenous administration of angiotensin II (1.9 nmol) which caused a rapid increase in plasma glucose reaching the highest values at 5 min after the injection (26.5% of the initial values; P < 0.01); (2) Sham-operated animals submitted to blockade of sympathetic noradrenergic pathways by treatment with guanethidine (10 mg/100 g body weight), which greatly decreased the baseline value of plasma glucose (85 +/- 5.5 mg% vs 136 +/- 5.1 mg% P < 0.01), and increased the hyperglycemic response to angiotensin II by 96% (P < 0.01); (3) Animals submitted to adrenodemedullation that did not alter the hyperglycemic response to angiotensin II; (4) Adrenodemedullated animals submitted to sympathetic blockade by guanethidine treatment which caused a 40.5% decrease in the hyperglycemic response to angiotensin II (P < 0.05). These data indicate that angiotensin II has a direct hyperglycemic effect in addition to its action on sympathetic nervous system activation and adrenomedullary secretion.

1. The aim of this study was a pharmacological characterization of the multiple NANC inhibitory transmission systems producing relaxation of the circular muscle of guinea-pig proximal colon. In the presence of atropine (1 microM), guanethidine (3 microM) and of the tachykinin NK1 and NK2 receptor antagonists, SR 140333 (0.3 microM) and MEN 10627 (1 microM), respectively, electrical field stimulation (EFS) produced a frequency-dependent (0.1-3 Hz) relaxation. During a cumulative frequency-response curve, the maximal relaxant effect was produced at 3 Hz and approached the maximal relaxation to 1 microM isoprenaline. In the presence of both apamin (0.3 microM) and L-nitroarginine (L-NOARG, 100 microM), EFS failed to evoke relaxation up to 1 Hz; at 1-10 Hz, a slowly developing relaxation ensured which approached 50% of the Emax to isoprenaline. The EFS-evoked NANC relaxation, either in the presence or absence of apamin and L-NOARG, was unaffected by in vitro capsaicin pretreatment (10 microM for 15 min). 2. Three protocols of EFS were developed for further pharmacological analysis: (a) EFS at 1 Hz for 5 s in the presence of L-NOARG, producing a transient fast apamin-sensitive relaxation; (b) EFS at 1 Hz for 5 s in the presence of apamin, producing a transient fast L-NOARG-sensitive relaxation; and (c) EFS at 10 Hz for 5 s in the presence of both apamin and L-NOARG, producing a transient but slowly developing and more sustained relaxation. 3. The neutral endopeptidase inhibitor, thiorphan (1-10 microM), enhanced and prolonged the apamin- and L-NOARG-resistant NANC relaxation produced by EFS at 10 Hz, without affecting that evoked at 1 Hz in the presence of apamin or L-NOARG. The angiotensin converting enzyme inhibitor, captopril (1-10 microM) was without effect. 4. The cAMP analogue inhibitor of protein kinase A, Rp-cAMPs (100-300 microM) significantly reduced and shortened the NANC relaxation produced by 10 Hz EFS in the presence of L-NOARG without affecting that produced by 1 Hz EFS in the presence of apamin or L-NOARG. 5. The inhibitor of sarcoplasmic reticulum Ca-ATPase, cyclopiazonic acid (CPA, 3-10 microM for 60 min) abolished the 1 Hz EFS-induced relaxation in the presence of L-NOARG, and greatly inhibited that produced by 10 Hz EFS in the presence of both apamin and L-NOARG. The relaxation produced by 1 Hz EFS in the presence of apamin was inhibited by about 32% at 10 microM only. 6. Nifedipine (1 microM) did not affect the EFS-induced NANC relaxations. In the presence of nifedipine, tetraethylammonium (TEA, 1 mM) enhanced the 1 Hz EFS-induced relaxation in the presence of L-NOARG (158% of control) and that produced by 10 Hz EFS in the presence of apamin and L-NOARG (215% of control) while that evoked by 1 Hz EFS in the presence of apamin was slightly affected (109% of control). 7. In the presence of atropine, guanethidine, SR 140333 and MEN 10627, bath application of human vasoactive intestinal polypeptide (VIP, 0.1 nM-10 nM) produced a concentration-dependent, slowly developing relaxation of colonic strips. The relaxation to VIP was unaffected by apamin (0.3 microM), L-NOARG (100 microM), nifedipine (1 microM) or nifedipine plus TEA (1 mM); it was inhibited by CPA (10 microM) and Rp-cAMPs (100 microM) and was potentiated by thiorphan (10 microM). 8. The putative VIP receptor antagonist, VIP(10-28) (10 microM) did not affect the VIP-induced relaxation nor the NANC relaxation to 10 Hz EFS in the presence of apamin and L-NOARG. 9. The present findings provide evidence that three distinct NANC inhibitory mechanisms mediate relaxation of the circular muscle of the guinea-pig proximal colon. The first system provides a fast relaxation in response to low frequency of stimulation and may involve the action of a transmitter(s) (possibly ATP) which mobilizes intracellular Ca2+ from sarcoplasmic reticulum leading to the activation of apamin-sensitive K+ channels. The second system likewise provides a fast relaxation of the colon in

Both electrical field stimulation and nicotine produced non-adrenergic non-cholinergic (NANC) relaxation of the circular muscle strips from the cat lower esophageal sphincter in the presence of 5 microM guanethidine and 5 microM scopolamine. Low-frequency stimulation (2 Hz, 0.2 ms duration, supramaximal current intensity, 20-s train) provoked a transient relaxation, while at high-frequency stimulation (20 Hz) a slow restoration to the resting tone was observed. Blockade of nitric oxide (NO) synthesis by 1 mM N omega -nitro-L-arginine decreased by 20% the amplitude of the 20 Hz-induced relaxation and changed the pattern of relaxation, making it similar to the sustained relaxation evoked by exogenously applied vasoactive intestinal peptide (VIP). After chymotrypsin (4 U/ml), the pattern of the high-frequency-induced relaxation resembled that of the low-frequency-induced relaxation. Similarly, chymotrypsin changed the shape of nicotine-provoked relaxation, increasing the speed of restoration to the resting tone. We suggest that the fast relaxation elicited in cat lower esophageal sphincter by electrical field stimulation or nicotine is initiated by NO. The slow restoration to the resting tone in the case of high-frequency- or nicotine-induced relaxation seems to be due to the release of VIP or VIP-like peptides. The possibility of participation of another transmitter(s) involved in NANC relaxation should not be excluded.

In urethane-anaesthetized rats, moderate colonic distention (0.5 ml) induced reflex rhythmic contractions (5 mm Hg amplitude and 1.1 cycles/min frequency). Senktide (1-10 nmol/kg, i.v.), a tachykinin NK3 receptor selective agonist, transiently suppressed distension-induced contractions. SR 142,801 (1-10 mumol/kg i.v.), a non-peptide tachykinin NK3 receptor antagonist, had no effect on distension-induced contractions but prevented the inhibitory effect of senktide. Infusion of N-omega-nitro-1-arginine methyl esther hydrochloride (L-NAME, 20 mumol/ml/h, i.v) increased the amplitude of colonic contractions and decreased the inhibitory effect of senktide. Hexamethonium (15 mumol/ml/h, i.v.) or atropine (1 mumol/ml/h, i.v.) inhibited the distension-induced contractions. In hexamethonium- or atropine-treated rats, senktide (10 nmol/kg) transiently and selectively enhanced the amplitude of contractions. Also SR 142,801 (10 mumol/kg), but not its inactive enantiomer SR 142,806, increased both amplitude and frequency of contractions. During continuous infusion of L-NAME and hexamethonium or atropine both frequency and amplitude of distension-induced colonic contractions were higher than when in hexamethonium or atropine only. Senktide (10 nmol/kg) had no effect and SR 142,801 (10 mumol/kg) produced a slight enhancement of colonic contractions. Infusion of sodium nitroprusside (3 mumol/ml/h, i.v.) decreased amplitude and frequency of distension-induced contractions. SR 142,801 had no effect in the presence of the nitric oxide (NO) donor. We conclude that tachykinins acting through NK3 receptors exert at least four different actions on colonic motility activated by distension: 1) a hexamethonium-resistant, NO-dependent, suppressant effect on contractions; 2) a hexamethonium-sensitive, NO-independent inhibitory effect on the amplitude of contractions; 3) a hexamethonium-resistant, NO-independent inhibitory effect on the amplitude of contractions and 4) a hexamethonium resistant and L-NAME-sensitive excitatory effect on amplitude of contractions. The prevalent inhibitory effect evoked in normal conditions along with the excitatory activity induced by SR 142,801 on hexamethonium-resistant colonic motility indicates that tachykinins, acting through neuronal NK3 receptors, activate NO-dependent and NO-independent inhibitory neurotransmission in the rat colon.

Selective chemical sympathectomy of the internal genital organs of prepubertal to mature male Wistar rats was performed by chronic treatment with low doses of guanethidine. Sympathetic denervation caused an increase in intratesticular progesterone levels in prepubertal and early pubertal rats in addition to a decrease in androstenedione and testosterone levels in prepubertal animals, thus indicating a decrease in the conversion of progesterone into androgen, probably by blocking the steroidogenic enzymatic pathway at the 17 alpha-hydroxylase/17, 20 desmolase level. A lower degree of testicular maturation, probably related to reduced androgen activity, was observed in prepubertal and early pubertal sympathectomized rats. Concentration of spermatozoa, on the other hand, was increased in the enlarged cauda epididymidis of late pubertal and mature denervated animals. This result is discussed in terms of the impairment of epididymal mechanisms of seminal emission, fluid resorption and spermatozoal disposal.

Purpose: This study was designed to investigate whether nitric oxide mediates inhibitory innervation in human rectal circular smooth muscle., Methods: Tissue was obtained from the midrectum of patients undergoing anterior resection for carcinoma. Adjacent strips of circular muscle were dissected and mounted in superfusion organ baths for isometric tension recording and initially loaded with 1 g of weight. Strips were continuously bathed with standard Krebs solution (37 degrees C, bubbled with 97 percent O2/3 percent CO2) containing 3 X X 10(-6) M guanethidine and 3 X 10(-6) M atropine sulfate to block adrenergic and muscarinic cholinergic neurotransmission. After equilibration, strips had no intrinsic tone, and reproducible and stable tension was, therefore, induced by the addition of 3 X 10 M histamine for five-minute "test" periods, during which electrical field stimulation (EFS) and additional drugs were applied., Results: EFS elicited frequency-dependent, neurogenic (tetrodotoxin-sensitive) relaxations of precontracted strips. Addition of N-w-nitro-L-arginine, a powerful competitive inhibitor of nitric oxide synthase, reduced the relaxant response to EFS in a dose-dependent fashion, and effect reversed by addition of s X 10(-4) M L-arginine but not by D-arginine. Addition of exogenous nitric oxide (sodium nitroprusside) mimicked the relaxant response induced by EFT., Conclusions: Human rectal circular smooth muscle receives an intrinsic inhibitory innervation mediated by nitric oxide. The presennce of a residual response following blockade of the enzyme nitric oxide synthase suggests the involvement of additional neurotransmitters.

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.

The present investigation was designed to examine the effects of sympathetic nervous system (SNS) inhibition on sexual behavior in ovariectomized, steroid-treated female rats. Clonidine, an alpha2-adrenergic agonist, guanethidine, a postganglionic noradrenergic blocker, and naphazoline, an alpha2-adrenoreceptor agonist were used to inhibit SNS activity. Intraperitoneal injections of either 33 micrograms/ml or 66 micrograms/ml clonidine significantly decreased receptive (lordosis) and proceptive (ear wiggles) behaviors and significantly increased rejection behaviors (vocalization, kicking, boxing). Either 25 mg/ml or 50 mg/ml guanethidine significantly decreased receptive and proceptive behavior and had no significant effect on rejection behaviors. Naphazoline significantly inhibited lordosis behavior at either 5 mg/ml or 10 mg/ml doses, significantly inhibited proceptive behavior at 5 mg/ml, and had no significant effect on rejection behaviors. These findings support the hypothesis that SNS inhibition decreases sexual activity in the female rat.

The present study investigated the effects of the alpha 2-adrenoceptor agonist guanabenz and the adrenergic neurone blocking drug guanethidine on the resting and stimulation-induced (S-I) effluxes of radioactivity from rat isolated mesenteric artery preparations in which the noradrenergic transmitter stores had been radiolabelled with [3H]-noradrenaline. The efflux of radioactivity evoked by electrical field stimulation of periarterial sympathetic nerves (60 s trains of 1 ms pulses, 2 Hz, 12 V) was taken as an index of transmitter noradrenaline release. Guanabenz (0.1-10 microM) decreased, in a concentration-dependent manner, both the resting and S-I effluxes of radioactivity. Guanethidine (0.1 and 1 microM) also decreased S-I efflux but increased resting efflux, both effects being concentration dependent. The inhibitory effects of guanabenz on both resting and S-I effluxes were reduced by blockade of the neuronal amine carrier with desipramine (1 microM). The inhibitory effect of guanabenz on resting efflux was prevented by inhibition of monoamine oxidase with pargyline (100 microM). The inhibitory effect of guanabenz on S-I efflux was not due to activation of prejunctional alpha 2-adrenoceptors since the inhibition was not blocked by the selective alpha 2-adrenoceptor antagonist idazoxan (0.1 microM). However, the inhibitory effect of guanabenz and guanethidine on S-I efflux was reduced by the inhibitor of Ca(2+)-activated potassium channels apamin (0.1 microM). The findings suggest that guanabenz, like guanethidine, enters noradrenergic nerve terminals by the neuronal amine carrier. The inhibition of resting efflux produced by guanabenz may be due to inhibition of neuronal monoamine oxidase. The enhancement of resting efflux produced by guanethidine is attributable to its indirect sympathomimetic action. Finally, guanabenz and guanethidine may inhibit transmitter noradrenaline release by activating potassium channels on sympathetic noradrenergic nerve terminals. These findings may be relevant to the mechanism of adrenergic neurone blockade.

Immature female rats (21 days of age) were chronically intraperitoneally treated with guanethidine or muscarinic agents. The effects on the timing of puberty and ovarian wet weight, protein and total RNA and DNA contents were studied. While guanethidine (20.0 mg/kg/day) was ineffective, trihexyphenidyl and especially propantheline (15.0 mg/kg/day) delayed vaginal opening (by 23%) and the first vaginal oestrus (by 28%), and lowered ovarian weight (by 37%) and other ovarian growth parameters. Carbachol (0.2 mg/kg/day) reversed the effects of propantheline. Thus, in contrast to the adrenergic system, the cholinergic system appears to substantially contribute to the accurate onset of female puberty and ovarian growth in the rat.

Adrenomedullin (AM) is a vasorelaxant peptide that was recently isolated from human pheochromocytoma. In contrast to human (h) AM, which has vasodepressor activity, a synthetic N-terminal fragment of hAM, hAM-(1-25)-NH2 showed vasopressor activity in the anesthetized rat. The N-terminal peptides hAM-1-31)-NH2, hAM-(1-25)-OH, hAM-(1-21)-NH2, acetyl-hAM-(16-21)-NH2, and acetyl-hAM-(16-36)-OH all showed vasopressor activities. The potency of hAM-(1-21)-NH2, acetyl-hAM-(16-21)-NH2 was greater than that of hAM-(1-25)-NH2. Pretreatment with phenoxybenzamine, guanethidine, or reserpine attenuated vasopressor activities of these peptides. These data suggested that vasopressor activity of N-terminal fragment of hAM is due to a stimulation of endogenous catecholamine release.

When methylene blue (10(-4) M) was applied to the rat stomach fundus precontracted with 10(-6) M serotonin in the presence of 10(-6)M atropine and 10(-6) M guanethidine, slowly developing relaxation was induced. Methylene blue (10(-4) M) increased cyclic AMP but not cyclic GMP levels in the stomach fundus. These results suggest that methylene blue may relax the rat stomach fundus through cyclic AMP formation.

1. In the presence of atropine (1 microM) and guanethidine (5 microM), electrical field stimulation (EFS, 120 mA, 1 ms, 0.5-16.0 Hz, trains of 2 min) induced frequency-dependent relaxations of 5-hydroxytryptamine (3 microM)-precontracted longitudinal muscle strips from the rat gastric fundus. 2. L-Citrulline concentrations were measured in the incubation medium of precontracted strips before and after EFS to investigate nitric-oxide (NO) synthase activity and its possible relation to non-adrenergic non-cholinergic (NANC) relaxation. 3. Basal NO synthase activity was reflected by the finding of prestimulation levels of L-citrulline of approximately 30 nM. These levels were unaffected by tetrodotoxin (3 microM) and NG-nitro-D-arginine methyl ester (D-NAME, 100 microM), slightly reduced by a calcium-free medium and halved by NG-nitro-L-arginine methyl ester (L-NAME, 100 microM). 4. EFS evoked significant, frequency-dependent increases in bath levels of L-citrulline at all frequencies tested. The increases evoked by 16-Hz EFS were abolished by tetrodotoxin (3 microM), a calcium-free medium and L-NAME (100 microM) but not by D-NAME (100 microM). 5. L-NAME (0.1 microM-1.0 mM) produced significant reduction of 4-Hz EFS-induced L-citrulline production (100% inhibition at 10 microM), but had less marked effects on basal production (approximately 50% reduction at 100 microM) and 4-Hz EFS-induced NANC relaxation (approximately 50% reduction at 1 mM). 6. L-Arginine (1 mM), but not D-arginine (1 mM), increased basal L-citrulline levels and reversed the inhibitory effect of L-NAME (10 microM). 7. These findings represent clear biochemical evidence of both basal and EFS-stimulated NO synthase activity in the rat gastric fundus.

The putative M1 muscarinic receptor agonist McN-A-343 evoked a dose-dependent increase in mean arterial pressure (MAP) when administered intravenously to conscious freely-moving rats pretreated with the ganglionic nicotinic receptor antagonist pentolinium. A tachycardia accompanied the increase in MAP which was blocked by the beta-adrenergic receptor antagonist propranolol. The increase in MAP was attenuated by the alpha 1-adrenergic receptor antagonist prazosin combined with the alpha 2-adrenergic receptor antagonist yohimbine. Adding propranolol to alpha-adrenergic receptor blockade uncovered a latent pressor response. Replacing prazosin with benextramine (which blocks NPY in addition to alpha-adrenergic receptors) attenuated the pressor response unmasked by propranolol. This attenuation was comparable to that provided by benextramine of the pressor response to intravenous administration of NPY. Adrenal demedullation only slightly attenuated the pressor response while having no effect on the tachycardia. The catecholamine depletor guanethidine greatly attenuated the McN-A-343-evoked increase in MAP and heart rate. The combination of adrenal demedullation and guanethidine did not further attenuate the increase in MAP but did provide better attenuation of the tachycardia than guanethidine alone. These results show that McN-A-343-evokes an increase in MAP and heart rate of conscious freely-moving rats primarily by causing the release of catecholamines, and possibly NPY, from sympathetic neurons with the adrenal glands playing a minor role.

Pigeon oesophageal smooth muscle in vitro has spontaneous electromechanical activity. In the presence of atropine and guanethidine, electrical field stimulation evokes a transient TTX-sensitive response comprising inhibition of electric bursting activity and muscular relaxation. This NANC inhibitory response was analysed using the K+ channel blockers TEA and apamin, TEA perfusion (0.1-5 mM) induced a concentration-dependent reduction in amplitude of EFS-evoked relaxation. Responses to higher stimulation frequencies were more sensitive to TEA than those to lower ones. The maximum reduction in amplitude (29% of control) was obtained on 30 Hz EFS evoked responses during 5 mM TEA perfusion. In a similar way, apamin (0.01-10 microM) perfusion reduced NANC relaxation, up to 30% of control. These results suggest that in the pigeon oesophagus, NANC intramural neurons are responsible for muscular relaxation. We speculate that an increase in K+ conductance might be the main mechanism involved, although the residual response after K+ channel blockade indicates the existence of an additional ionic mechanism.

1. Orbital parasympathetic innervation normally provides prejunctional muscarinic inhibition of sympathetic neurotransmission without activation of excitatory muscarinic receptors located on the innervated smooth muscle. The present study examines the role of acetylcholinesterase (AChE) in limiting the effects of parasympathetically released acetycholine to prejunctional receptors. 2. Urethane anaesthetized rats were placed in a stereotaxic frame, and parasympathetic activation was achieved by electrical stimulation (20 Hz, < 2.0 V) of the ipsilateral superior salivatory nucleus. Drugs were administered through a femoral venous cannula. Superior tarsal smooth muscle responses were measured by recording eyelid tension. 3. Parasympathetic stimulation alone caused a small decrease in resting tension; previous studies have shown this to be attributable to attention of resting sympathetic tone. Parasympathetic activation following physostigmine administration, however, evoked a large contractile response. Contractions were resistant to atropine but were blocked by gallamine, guanethidine, and phentolamine. 4. We conclude that AChE inhibition results in conversion of orbital parasympathetic nerve function from inhibition of sympathetic neurotransmission to smooth muscle excitation. This occurs as a result of cholinergic activation of excitatory nicotinic receptors on sympathetic varicosities, which elicit the release of noradrenaline.

Nonadrenergic, noncholinergic (NANC) contraction has been demonstrated in animal urinary bladder. However, the exact nature of the NANC innervation is still unclear. 1,1-Dimethylphenylpiperazinium (DMPP), which generates action potentials in the cell body of the postganglionic neuron and causes neurotransmitter release (both acetylcholine and noradrenaline), was given intravenously (0.1-0.7 mg/kg) to 3-month-old female Wistar rats under anesthesia (n = 20). Intravesical pressure, heart rate and blood pressure of the rats were monitored on Gould polygraph. Monophasic dose-dependent contractile response was observed upon administration of DMPP in 12 of 20 rats. After total adrenergic and cholinergic blockade with atropine, guanethidine, phentolamine and propranolol, the contractile response was reduced, not completely, in the animals. At the dose of 0.7 mg/kg, the contraction was reduced to about 48% of the original response. The study provides in vivo evidence for NANC contraction in the rat urinary bladder, moreover, the neurotransmitter is released from the postganglionic neurons.

The present experiments were designed to further investigate the action of an angiotensin II antagonist on the hyperglycemic response to hemorrhage (1.2 ml/100 g b.wt./2 min). The animals were divided into 3 experimental groups; (1) sham-operated animals submitted to intravenous administration of [1-Sar,8-Thr]-angiotensin II (sarthran), an antagonist of angiotensin II (750 ng/100 g b.wt. as a bolus plus an infusion of 25 ng/100 g b.wt./min over 30 min), which greatly attenuated (51.8% lower than controls; P < 0.01) the hyperglycemic response to hemorrhage; (2) animals submitted to adrenodemedullation which decreased the hyperglycemic response to hemorrhage by 64% (P < 0.01). However, sarthran infusion into adrenodemedullated rats caused a 38.5% further decrease in hyperglycemic response to hemorrhage (P < 0.01); and (3) intact animals submitted to blockade of sympathetic noradrenergic pathways by treatment with guanethidine (10 mg/100 g b.wt.), which greatly decreased the baseline value of plasma glucose (64.1 +/- 3.5 mg% vs. 125.3 +/- 4.5 mg%, P < 0.01), and reduced the hyperglycemic response to hemorrhage by 34% (P < 0.01). Sarthran infusion into guanethidine-treated rats caused a further 34% decrease in hyperglycemic response to hemorrhage (P < 0.01). These data indicate that angiotensin II has a direct hyperglycemic effect in addition to its action on sympathetic nervous system activation and adrenomedullary secretion.

This study was designed to investigate (1) whether norepinephrine is released in response to glucopenia in vitro, thereby stimulating glucagon secretion and, (2) the modulating effects of norepinephrine on insulin and glucagon secretion, using isolated perfused rat pancreas preparations. Simultaneous addition of the adrenergic receptor antagonists yohimbine, prazosin and propranolol, each at a concentration of 10-(5) mol/l, significantly potentiated glucose-stimulated insulin secretion (6.23 +/- 0.76 vs. 2.11 +/- 0.72 (control) nmol/min, P < 0.01), and suppressed glucopenia-induced glucagon secretion (0.59 +/- 0.10 vs. 1.34 + 0.18 (control) ng/min, P < 0.05). Also, 10-(5) mol/l yohimbine alone significantly potentiated glucose-stimulated insulin secretion (4.86 +/- 0.50 nmol/min, P < 0.05). The norepinephrine release inhibitor, guanethidine, significantly inhibited tyramine-induced secretion of both norepinephrine (7.86 +/- 0.77 vs. 49.7 +/- 2.3 nmol/min, P < 0.01) and glucagon (0.31 +/- 0.08 vs. 1.21 +/- 0.15 ng/min, P < 0.01), but exerted no effects on glucopenia-induced secretion of either norepinephrine or glucagon. We conclude that these results further support the concept that the neurotransmitter norepinephrine is released in response to glucopenia in vitro, and modulates insulin and glucagon secretion. Our data do not, however, provide evidence indicating that glucopenia-induced glucagon secretion is mainly mediated by activation of sympathetic nerve terminals around the alpha-cells in the isolated perfused rat pancreas.

The role of adenosine in the hypotensive action of morphine was examined in the pentobarbitone anaesthetized or pithed rat preparations. Adenosine (10-300 micrograms/kg) induced dose-dependent decreases in diastolic blood pressure in the anaesthetized rat preparation. These effects were attenuated by infusion of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 50 micrograms/kg/min), 8-phenyltheophylline (8PT; 50 micrograms/kg) also induced dose-dependent decreases in diastolic blood pressure. Guanethidine (16 micrograms/kg/min), atropine (1 mg/kg), DPCPX and 8 PT reduced the effect of morphine on diastolic blood pressure, whilst DMPX (50 micrograms/kg/min) was inactive. In the pithed rat preparation morphine was inactive at doses up to 10 mg/kg. The results suggest that the hypotensive effect of morphine is mediated at least in part by the release of adenosine which then acts on centrally located adenosine receptors to induce changes in autonomic control of blood pressure.