High nitric oxide production in human paranasal sinuses.

Nitric oxide (NO) is present in air derived from the nasal airways. However, the precise origin and physiological role of airway-derived NO are unknown. We report that NO in humans is produced by epithelial cells in the paranasal sinuses and is present in sinus air in very high concentrations, close to the highest permissible atmospheric pollution levels. In immunohistochemical and mRNA in situ hybridization studies we show that an NO synthase most closely resembling the inducible isoform is constitutively expressed apically in sinus epithelium. In contrast, only weak NO synthase activity was found in the epithelium of the nasal cavity. Our findings, together with the well-known bacteriostatic effects of NO, suggest a role for NO in the maintenance of sterility in the human paranasal sinuses.

Calcitonin gene-related peptide is a stimulator of renin secretion.

Calcitonin gene-related peptide (CGRP) was found to stimulate renin secretion in vivo in normal human volunteers. Moreover, CGRP stimulated the release of renin in vitro from isolated rat renal juxtaglomerular cells (half-maximal effective concentration [EC50] 100 nM) concomitant with stimulation of cAMP production (EC50 60 nM). Immunoreactive CGRP was recognized in rat renal cortical nerve fibers, and intact rat CGRP was identified in extracts of the rat renal cortex. Because CGRP containing sensory nerve fibers are seen in the region of the juxtaglomerular apparatus, it would seem that the release of CGRP from these afferent nerves may be involved in the physiological control of renin secretion.

Postexercise ischemia is associated with increased neuropeptide Y in patients with coronary artery disease.

BACKGROUND: Neurohormones may influence vascular tone both during and after exercise. Neuropeptide Y (NPY), which is costored and released with norepinephrine (NE) during sympathetic activity, is a potent vasoconstrictor with a relatively long half-life. We therefore examined its possible association with the ischemic response to exercise in patients with coronary artery disease. METHODS AND RESULTS: Twenty-nine male patients with effort-induced angina pectoris underwent a symptom-limited exercise test. In addition to conventional ST-segment analysis, we examined ischemia on the basis of heart rate (HR)-adjusted ST-segment changes through calculation of the ST/HR slope during the final 4 minutes of exercise and of the ST/HR recovery loop after exercise. Blood samples were taken before, during, and after exercise for an analysis of several neurohormones. Mean ST-segment depression was -223+/-20.2 microV (P:<0.0001) just before the termination of exercise, followed by a gradual normalization, but it remained significant after 10 minutes (-49+/-8.9 microV, P:<0.0001). At the end of exercise, the ST/HR slope, which reflects myocardial ischemia, was -6.0+/-0.77 microV/HR. In most patients, ST-segment levels at a given HR were lower during recovery than during exercise, here referred to as ST "deficit." Exercise increased the plasma levels of NPY, NE, epinephrine, and N-terminal proatrial natriuretic peptide, but big endothelin remained unchanged. Although NE and epinephrine peaked at maximal exercise, the highest levels of NPY and N-terminal proatrial natriuretic peptide were observed 4 minutes after exercise. The maximal increase in the NPY correlated significantly with ST-segment depression at 3 minutes after exercise (r=-0.61, P:= 0.0005), the ST deficit at the corresponding time point (r=-0.66, P:= 0.0001), and the duration of ST-segment depression after exercise (r= 0.42, P:=0.02). In contrast, no such correlations were found for NE. CONCLUSIONS: The present study has for the first time demonstrated a correlation between plasma NPY levels and the degree and duration of ST-segment depression after exercise in patients with coronary artery disease, which suggests that NPY may contribute to myocardial ischemia in these patients.

Cyclo-oxygenase products released by low pH have capsaicin-like actions on sensory nerves in the isolated guinea pig heart.

OBJECTIVE: Previous work has shown that ischaemia releases calcitonin gene related peptide (CGRP) from capsaicin sensitive nerve terminals in the perfused heart. Prostacyclin (PGI2) is also released during ischaemia. The aim of this study was to investigate whether the release of CGRP by low pH and lactic acid was associated with PGI2 formation and if PGI2 mediated its effect through capsaicin receptors which could be inhibited by capsazepine. METHODS: The isolated Langendorff perfused guinea pig heart was used with a constant perfusion pressure of 70 cm H2O. Low pH was accomplished by changing the Tyrode solution to buffers with pH 7, 6, and 5, or lactic acid (5 mM with pH 6.9). The outflow of CGRP and the stable PGI2 metabolite 6-keto-PGF1 alpha was measured by radioimmunoassay. RESULTS: Low pH (pH 7, 6, 5) and lactic acid evoked release of CGRP. At moderate acidosis (pH 7 and 6) the CGRP release was dependent on extracellular Ca2+, while at pH 5 approximately half of the peptide release persisted in the absence of extracellular Ca2+. This release was attenuated by diclofenac or indomethacin, two inhibitors of prostaglandin formation, as well as by the capsaicin receptor antagonist capsazepine. Both arachidonic acid and PGI2, the predominant cyclo-oxygenase product formed during myocardial ischaemia, evoked a capsazepine sensitive release of CGRP, while capsazepine did not influence the formation of PGI2 evoked by low pH or arachidonic acid. CONCLUSIONS: In the isolated guinea pig heart, moderate acidosis is associated with CGRP release dependent on influx of extracellular Ca2+ and formation of PGI2, with subsequent stimulation of capsazepine sensitive receptors. With more severe acidosis there is an additional non-PGI2-linked CGRP release. Capsazepine represents a novel pharmacological principle for inhibiting the effects of prostanoids on sensory nerves without influencing their formation.

Protective effects of non-peptide endothelin receptor antagonist bosentan on myocardial ischaemic and reperfusion injury in the pig.

OBJECTIVE: The aim was to investigate the effects of the non-peptide endothelin receptor antagonist bosentan (Ro 47-0203) on haemodynamic variables, infarct size, myocardial overflow, and tissue content of endothelin-like immunoreactivity (ET-LI) during ischaemia and reperfusion in anaesthetised pigs, and to study the inhibitory effect of bosentan on ET-1 induced coronary constriction in vitro. METHODS: Ischaemia was induced by ligation of the left anterior descending coronary artery for 45 min, followed by 4 h of reperfusion. Bosentan was given either intravenously (5 mg.kg-1) 15 min before ischaemia or as a 25 min local coronary venous retroinfusion (10(-4) M) starting at 30 min of ischaemia. ET-LI was analysed in myocardial tissue and in plasma from the anterior interventricular coronary vein and aorta. The effect of bosentan on endothelin-1 induced vasoconstriction was evaluated in isolated diagonal branches of left anterior descending coronary artery. RESULTS: Intravenous bosentan slightly reduced arterial blood pressure (P < 0.05) but did not affect basal coronary vascular resistance. Local retroinfusion of bosentan did not change blood pressure. Intravenous and retroinfused bosentan significantly reduced infarct size by 58% and 48% respectively (P < 0.01) and enhanced the recovery of coronary blood flow by 65-90% compared to vehicle treated controls at the end of 4 h reperfusion. The basal plasma levels of ET-LI and the myocardial overflow of ET-LI during reperfusion increased twofold after bosentan. A threefold increase in the concentration of ET-LI was observed in the ischaemic/reperfused myocardium and this enhancement was significantly attenuated by bosentan. Bosentan effectively antagonised the endothelin-1 induced but not the serotonin induced, contractions of isolated coronary arteries and reversed the established contraction induced by endothelin-1. CONCLUSIONS: The non-peptide endothelin receptor antagonist bosentan markedly protects the myocardium from ischaemia/reperfusion injury and improves blood flow to the reperfused area, indicating the involvement of endogenous endothelin-1 and the therapeutic value of bosentan in the treatment of ischaemia/reperfusion injury.

Big ET-1 infusion in man causes renal ET-1 release, renal and splanchnic vasoconstriction, and increased mean arterial blood pressure.

OBJECTIVE: The aim was to study the vascular effects of big endothelin-1 (big ET-1) infusion and its possible conversion to ET-1. METHODS: Six healthy subjects were given an intravenous infusion of big ET-1 in a dose of 8 pmol.kg-1.min-1 for 20 min. Blood samples were taken before, during, and up to 3 h after the infusion from arterial, hepatic, and renal vein catheters for the determination of splanchnic and renal blood flows, as well as ET-1-like immunoreactivity (ET-1-LI) from these vascular beds. RESULTS: Intravenous infusion of big ET-1 was followed by a doubling of arterial ET-1-LI from 4.17(SEM 0.39) to 8.42(0.49) pmol.litre-1 (p < 0.001) and a significant increase in the renal release of ET-1-LI from 1.50(0.18) to 8.68(0.64) pmol.min-1 (p < 0.001) but no splanchnic release. Big ET-1 infusion also caused a decrease in heart rate from 57(4) to 45(3) beats.min-1 (p < 0.001) and an increase in mean arterial pressure from 86(1.3) to 106(3.2) mm Hg (p < 0.001), which lasted for at least 2 h. Renal blood flow fell from 1.38(0.06) to 0.83(0.04) litre.min-1 (p < 0.001) while splanchnic blood flow fell from 1.34(0.11) to 0.83(0.05) litre.min-1 (p < 0.001). CONCLUSIONS: Big ET-1 infusion causes a drop in heart rate, an increase in mean arterial pressure and decreases in splanchnic and renal blood flows. Arterial plasma ET-1 levels doubled and big ET-1 infusion also induced a significantly increased renal, but not splanchnic, release of ET-1-LI, suggesting a unique renal handling of circulating big ET-1. When the results of the infusion of big ET-1 are compared with our previous experiments using ET-1 infusion, more marked haemodynamic changes (as reflected in the increase in mean arterial pressure, the drop in heart rate, and the duration of renal vasoconstriction) are seen despite lower arterial plasma ET-1-LI levels.

Plasma neuropeptide Y on admission to a coronary care unit: raised levels in patients with left heart failure.

STUDY OBJECTIVE - The aim of the study was to measure plasma neuropeptide Y, which is related to sympathetic nerve stimulation, in patients admitted to a coronary care unit and to relate the findings to clinical information. DESIGN - Plasma neuropeptide Y was measured on admission and the results were related to the cause of admission and to clinical information collected prospectively and retrospectively. SUBJECTS - Plasma subjects were obtained from 377 consecutive daytime admissions to the coronary care unit at Södersjukhuset. Results of only the first sample in each patient are included in this study, so 45 cases observed more than once (readmitted patients) were omitted. Six samples were abandoned because of technical failures. The study therefore comprises 326 patients. Clinical diagnoses were defined as acute myocardial infarction, arrhythmia, angina pectoris, and miscellaneous (all other diagnoses). Heart failure was defined according to a modified Killip scheme. MEASUREMENTS and RESULTS - Neuropeptide Y like immunoreactivity was measured by radio-immunoassay. Plasma concentrations above normal (greater than 30 pmol.litre-1) were found in association with: increased age, female sex, diuretic treatment, tachycardia, arterial hypotension, increased respiratory rate, and mortality in the unit. There was a strong relationship between high neuropeptide Y concentrations and: moderate left heart failure (63%), pulmonary oedema (90%), and cardiogenic shock (100%). Of patients without heart failure only 25% had raised neuropeptide Y. In multivariate analysis, the severity of heart failure (Killip class), heart rate and respiratory rate were the only variables that were significantly and independently related to plasma neuropeptide Y. CONCLUSIONS - The presence and degree of circulatory disturbance, in particular tachycardia and left heart failure, were strongly related to increased plasma concentrations of neuropeptide Y in coronary care patients.

Release of neuropeptide Y and noradrenaline from the human heart after aortic occlusion during coronary artery surgery.

STUDY OBJECTIVE: The aim was to study the influence of complete myocardial ischaemia during aortic occlusion on release of neuropeptide Y and noradrenaline from the human myocardium. DESIGN: Coronary sinus neuropeptide Y and noradrenaline were measured after 46(SEM 7) min of aortic occlusion with cold cardioplegia in patients undergoing coronary artery surgery. Patients - Seven patients (all male), aged 64(SEM 3) years, were studied. All were undergoing coronary artery bypass grafting. MEASUREMENTS AND MAIN RESULTS: Reperfusion was associated with an increase in coronary sinus blood flow as determined by thermodilution. Simultaneously there was cardiac release of both neuropeptide Y and noradrenaline during the first two sampling periods: 3(0.6) and 7(0.6) min after the start of reperfusion. The outflow of neuropeptide Y and noradrenaline returned to preischaemic values by 14(1) min after reperfusion. Coronary sinus blood lactate and pyruvate concentrations were also increased at the start of reperfusion, while the lactate/pyruvate ratio remained unchanged. Myocardial oxygen uptake was not influenced by cardiac ischaemia. CONCLUSIONS: Ischaemia of the human heart in vivo is associated with an enhanced outflow of neuropeptide Y and noradrenaline from the heart. Since arterial blood concentrations of these substances were also increased on reperfusion, their release is probably due to increased sympathetic nerve activity, though other mechanisms such as temperature change and local metabolite formation could also participate. Local release of neuropeptide Y during cardiac ischaemia may be involved in the regulation of coronary vascular tone as well as in the release of noradrenaline and acetylcholine.

Local overflow and enhanced tissue content of endothelin following myocardial ischaemia and reperfusion in the pig: modulation by L-arginine.

OBJECTIVE: The local myocardial overflow and tissue content of endothelin-like immunoreactivity (ET-LI) during ischaemia and reperfusion as well as the coronary vascular effects of endothelin were characterised in anaesthetised pigs. METHODS: Ischaemia was induced by ligation of the left anterior descending coronary artery for 45 min followed by 4 h of reperfusion. ET-LI was analysed in plasma from the anterior interventricular coronary vein and aorta for estimation of local overflow and in myocardial tissue. Endothelin analogues were given in the coronary artery for determination of local vascular effects. RESULTS: During reperfusion, but not during ischaemia, the veno-arterial concentration difference of ET-LI increased, resulting in a significantly increased overflow at between 10 and 120 min of reperfusion. The tissue concentration of ET-LI in the left ventricle was seven times higher in the ischaemic/reperfused area than in the non-ischaemic area: 161(SEM 30.5) v 25.3(3.8) fmol.g-1, P < 0.05. The increase in myocardial ET-LI was attenuated by 70% (P < 0.01) by coronary venous retroinfusion of the nitric oxide substrate L-arginine, whereas the overflow was unaffected. Chromatographic characterisation of the myocardial ET-LI showed that it was similar to endothelin-1. Intracoronary administration of endothelin-1, endothelin-3, and the endothelin ETB receptor agonist [Ala1,3,11,15]ET-1 evoked dose dependent coronary vasoconstriction, and reductions in left ventricular dP/dt and arterial blood pressure. Endothelin-1 was two times more potent than endothelin-3 and 10 times more potent than [Ala1,3,11,15]ET-1. CONCLUSIONS: Myocardial ischaemia/reperfusion evokes enhanced local overflow of ET-LI during the reperfusion period combined with an increased tissue concentration of ET-LI which is is attenuated by L-arginine. Endothelin evokes potent coronary vasoconstriction via activation of both ETA and ETB receptors.

Specific receptor and cardiovascular effects of calcitonin gene-related peptide.

Specific binding sites for calcitonin gene-related peptide (CGRP) were demonstrated in the rat heart and spleen. Autoradiography revealed rat [125I]iodo CGRP binding associated with the intima and media of the aorta, the coronary arteries and the heart valves, and the red pulp of the spleen. Half-maximal inhibition of rat [125I]iodo-CGRP binding to membranes of the rat atria and the spleen was obtained with, respectively, 5 and 0.35 nM unlabeled rat CGRP; these values correspond to EC50 values of 3 and 0.14 nM for activation of adenylate cyclase by CGRP. In the isolated, spontaneously beating right atrium, the EC50 values of stimulation of the force and rate of contraction by rat CGRP were 120 and 70 nM, respectively. Rat CGRP caused relaxation of splenic strips, precontracted with noradrenaline; the EC50 was 50 nM. The beta-adrenergic blocking agent metoprolol, while obliterating the increase in the force and rate of contraction evoked by noradrenaline in the right atrium, did not significantly change the action of CGRP. Similarly, preserved action of CGRP in the presence of indomethacin as well as mepyramine and cimetidine argues against a role of prostaglandins or histamine in the functional responses of CGRP. Much like CGRP, capsaicin, which releases mediators from sensory neurons, caused stimulation of the force and rate of contraction of the isolated right rat atrium. After tachyphylaxis to CGRP, the response to noradrenaline was intact, while the positive chronotropic and inotropic effects of capsaicin were suppressed. The results indicate that the cardiac effects of capsaicin may be due to the release of endogenous CGRP through a local mode of action.

Neuropeptide Y--a cotransmitter with noradrenaline and adenosine 5'-triphosphate in the sympathetic nerves of the mouse vas deferens? A biochemical, physiological and electropharmacological study.

UNLABELLED: A combination of biochemical, physiological and electropharmacological methods was employed to examine the occurrence of neuropeptide Y and the pre- and postjunctional effects of this peptide on sympathetic neuromuscular transmission in the mouse vas deferens. This tissue had a high content of neuropeptide Y-like immunoreactive material, suggesting a dense innervation by neuropeptide Y-containing nerve fibres. Addition of neuropeptide Y at concentrations from 5 X 10(-9) to 5 X 10(-7) M induced both pre- and postjunctional effects in vitro. Neuropeptide Y per se induced a rise in the resting tension, and "instantly" potentiated the contractile effects of exogenous noradrenaline and of the stable adenosine 5'-triphosphate (ATP) analogue, alpha,beta-methylene ATP. Neuropeptide Y reduced the secretion of [3H]noradrenaline evoked by electrical nerve stimulation, and selectively depressed the stimulus-evoked, but not the spontaneously occurring excitatory junction potentials in smooth muscle cells. Further, neuropeptide Y reduced the amplitudes of the twitch contractions evoked by electrical field stimulation with short stimulus trains at 10 Hz, and also (although to a smaller extent) the delayed contractile response to longer trains of nerve stimuli. The pre- and postjunctional effects of neuropeptide Y were not changed by alpha- or beta-adrenoceptor blocking agents, or by tachyphylaxis to the effects of ATP, or by the calcium channel blocker nifedipine. IN CONCLUSION: sympathetic neuromuscular transmission in the mouse vas deferens may be mediated not only by noradrenaline and ATP, but also by neuropeptide Y.(ABSTRACT TRUNCATED AT 250 WORDS)

Nitric oxide synthase in the pig autonomic nervous system in relation to the influence of NG--nitro-L-arginine on sympathetic and parasympathetic vascular control in vivo.

Nitric oxide synthase, the enzyme responsible for the formation of nitric oxide, was demonstrated by an indirect immunofluorescence technique to be present in both the sympathetic and parasympathetic nervous system of the domestic pig. In the sympathetic nervous system, nitric oxide synthase was mainly present in preganglionic neurons projecting to postganglionic neurons, some of which contained neuropeptide Y in the superior cervical, the coeliac and the lumbar ganglia of the sympathetic chain. A minor population of postganglionic sympathetic neurons contained nitric oxide synthase, vasoactive intestinal polypeptide and peptide histidine isoleucine. In the densely sympathetically innervated vascular beds such as the spleen, kidney and skeletal muscle, many neuropeptide Y- but no nitric oxide synthase-positive fibres were found. The nitric oxide synthase inhibitor NG-nitro-L-arginine reduced cardiac output by 40% and caused profound vasoconstriction in a variety of vascular beds. Furthermore, no or minor changes in plasma catecholamines, neuropeptide Y or endothelin-1 were observed up to 20 min after NG-nitro-L-arginine. Milrinone (a phosphodiesterase III inhibitor) prevented this NG-nitro-L-arginine-induced reduction in cardiac output, and the regional vasoconstriction was reduced, whereas some elevation of the blood pressure was still observed. Sympathetic nerve stimulation, with single impulses of 10 Hz for 1 s in the presence of NG-nitro-L-arginine, evoked vasoconstrictor responses which were largely in the same range as in control conditions. Parasympathetic postganglionic neurons to the submandibular salivary gland contained nitric oxide synthase, vasoactive intestinal polypeptide, peptide histidine isoleucine and neuropeptide Y. The vasodilatation evoked by parasympathetic nerve stimulation (10 Hz for 1 s) in the presence as well as in the absence of atropine was, on the other hand, markedly reduced by NG-nitro-L-arginine administration. Milrinone attenuated the inhibitory effect of NG-nitro-L-arginine on the parasympathetic vasodilation. In conclusion, nitric oxide synthase can be demonstrated in preganglionic sympathetic and postganglionic parasympathetic neurons. The main effect of nitric oxide synthase inhibition seems to be related to attenuation of basal endothelial nitric oxide production and parasympathetic transmission. Inhibition of phosphodiesterase counteracts both the haemodynamic and the neuronal effects of NG-nitro-L-arginine.

Co-release of neuropeptide Y and noradrenaline from pig spleen in vivo: importance of subcellular storage, nerve impulse frequency and pattern, feedback regulation and resupply by axonal transport.

The importance of subcellular storage, nerve impulse rate and pattern, and feedback regulation, as well as resupply by axonal transport for the release of noradrenaline and neuropeptide Y-like immunoreactivity, was studied in the blood perfused pig spleen in vivo. Vasoconstrictor responses were recorded as perfusion pressure changes. Subcellular fractionation experiments using sucrose density gradients showed a bimodal distribution of noradrenaline (peak concentrations at 0.8 and 1.1 M sucrose) while only one main peak of neuropeptide Y was present (at 1.1 M sucrose). Overflow suggesting release of noradrenaline and neuropeptide Y-like immunoreactivity could be detected after 10 s stimulation at 10 Hz. The ratio for the output of noradrenaline and neuropeptide Y upon continuous nerve stimulation in control animals decreased with frequency. After inhibition of noradrenaline reuptake by desipramine the vasoconstrictor response and noradrenaline output were enhanced while the corresponding overflow of neuropeptide Y was reduced by 50% at 0.5 Hz. Stimulation with the irregular or regular bursting patterns at high frequencies caused larger perfusion pressure increase and relative enhancement of neuropeptide Y output compared to noradrenaline than a continuous stimulation both before and after desipramine treatment. A similar fractional release per nerve impulse was calculated both for [3H]noradrenaline (5.6 +/- 1.0 x 10(-5) and neuropeptide Y (7.3 +/- 0.3 x 10(-5). After reserpine treatment combined with preganglionic denervation the vasoconstrictor responses were more long-lasting, neuropeptide Y release was enhanced while noradrenaline content and release were reduced by 99%. The difference in neuropeptide Y overflow between continuous and bursting types of stimulation was smaller after reserpine treatment. After prolonged intermittent stimulation with regular bursts (20 Hz) for 1 h the splenic content of neuropeptide Y was reduced by 58%, while no change was observed for noradrenaline. The maximal perfusion pressure increase upon prolonged nerve stimulation after reserpine was similar in control and reserpine-treated animals, but after reserpine the vasoconstrictor response and neuropeptide Y release were subjected to fatigue. Ligation experiments of the splenic nerves revealed the splenic neuropeptide Y content was resupplied by axonal transport with a calculated total tissue turnover time of 11 days. In contrast, axonal transport contributed only to a marginal extent for the resupply of noradrenaline.(ABSTRACT TRUNCATED AT 400 WORDS)

Neurokinin A in capsaicin-sensitive neurons of the guinea-pig inferior mesenteric ganglia: an additional putative mediator for the non-cholinergic excitatory postsynaptic potential.

The presence of neurokinin-A-like immunoreactivity in guinea-pig inferior mesenteric ganglia was detected by radioimmunoassay procedures. Pretreating the animals with capsaicin 7 days prior to experimentations reduced the mean content of neurokinin-A-like immunoreactivity by 85% from its control value of 150 +/- 31.3 fmol per ganglion. High-performance liquid chromatography revealed that neurokinin-A-like immunoreactivity was heterogenous as in addition to neurokinin A, peaks corresponding to the amphibian tachykinin eledoisin and to neuropeptide K were detected, and they too were depleted by capsaicin. Electrophysiological studies showed that neurokinin A applied either by superfusion or by pressure ejection evoked a slow depolarization in the majority of inferior mesenteric ganglia neurons in vitro. Neurokinin-A-evoked depolarizations in the majority of cells tested were associated with a small increase in membrane input resistance. However, the responses were increased by membrane hyperpolarization: the extrapolated mean equilibrium potential of neurokinin-A-induced depolarization was -36 mV. Removal of extracellular sodium but not chloride ions suppressed the neurokinin-A-induced depolarization. The slow depolarization elicited either by exogenously applied substance P or by repetitive stimulation of hypogastric nerves was reversibly eliminated in the presence of neurokinin A. Collectively, our studies suggest that neurokinin-A-like immunoreactivity may coexist with substance-P-like immunoreactivity in capsaicin-sensitive fibers in the guinea-pig prevertebral ganglia and that the similarity of the actions of neurokinin A on the one hand and substance P on the other raises the possibility that non-cholinergic excitatory potentials elicited in the inferior mesenteric ganglia may be generated by not one but a number of closely related tachykinins.

Capsaicin induced release of multiple tachykinins (substance P, neurokinin A and eledoisin-like material) from guinea-pig spinal cord and ureter.

The release of tachykinins from isolated slice preparations of the guinea-pig spinal cord and ureter was studied in vitro. Capsaicin (10 microM) caused release of substance P, neurokinin A and an eledoisin-like component from both the spinal cord and ureter. The release of tachykinins induced by capsaicin or potassium (60 mM) was calcium dependent. No detectable release of neurokinin B or neuropeptide K, an N-terminally extended form of neurokinin A, was induced by capsaicin. No detectable release of tachykinins could be demonstrated after exposure to agents which are known to activate C-fibre afferents, such as histamine, bradykinin, serotonin, prostaglandins E1, E2 or acetylcholine. Protein extravasation in the ureter, as determined by the Evans Blue extravasation technique was used as a functional correlate to the tachykinin release. Protein extravasation was induced in vivo by local intraluminal injections of capsaicin at several hundred-fold lower concentrations than those required to induce a detectable release of tachykinins in vitro. The difference may, however, partly depend on the experimental conditions and the detection limit of the tachykinin assay used. The protein extravasation response to capsaicin was absent after systemic capsaicin pretreatment, which causes a marked depletion of tachykinins in the ureter. In conclusion, capsaicin evokes release of several tachykinins from both central and peripheral endings of primary afferent neurons. The peptides released from sensory nerves in the periphery may induce effects such as protein extravasation and smooth muscle contraction.

Evidence for involvement of nitric oxide in the regulation of hypothalamic portal blood flow.

Vasoactive intestinal polypeptide and peptide histidine isoleucine, two peptides with a common precursor and with strong vasodilatory actions, have been suggested to be involved in control of blood flow through the hypothalamic portal blood vessels, in this way regulating the amounts of releasing and inhibitory factors reaching the anterior pituitary. Using the indirect immunofluorescence technique, we now show that this system also contains the enzyme nitric oxide synthase, as well as acetylcholinesterase. It is therefore likely that the control of blood flow through the portal vessels is mediated via relaxation of smooth muscle cells with a high myogenic tone by neuronal release of four vasodilatory compounds, acetylcholine, vasoactive intestinal polypeptide, peptidine histidine isoleucine, and nitric oxide, i.e. a classic neurotransmitter, two neuropeptides and a gas.

Cigarette smoke-induced airway oedema due to activation of capsaicin-sensitive vagal afferents and substance P release.

Exposure of rats to smoke from one cigarette caused local oedema due to a marked increased in vascular permeability from the epiglottis down to bronchioli, as indicated by extravasation of Evans blue in the airway mucosa. The cigarette smoke-induced extravasation of Evans blue was still present after removal of the tar and nicotine content of the smoke, suggesting that chemical irritants in the vapour phase were the main mediators of the vascular permeability response. Local or systemic pretreatment with capsaicin or [D-Arg1, D-Pro2, D-Trp7,9, Leu11] SP, a substance P antagonist, abolished or significantly reduced the airway oedema induced by cigarette smoke or vagal nerve stimulation. No reduction of the cigarette smoke or vagally induced tracheal oedema was seen upon pretreatment with mepyramine plus cimetidine, fentanyl, disodiumchromoglycate, methylprednisolone or terbutaline. The results thus indicate that the cigarette smoke or vagally induced tracheal oedema is most likely to be due to substance P release from local capsaicin-sensitive afferent neurons in the airway mucosa. Local administration of substance P antagonists may be considered as a pharmacological means of inhibiting local mucosal oedema in the airways caused by airway irritants such as cigarette smoke.

Immunologically induced sympathectomy of preganglionic nerves by antibodies against acetylcholinesterase: increased levels of peptides and their messenger RNAs in rat adrenal chromaffin cells.

Systemic administration of murine monoclonal acetylcholinesterase antibodies to rats has been shown to cause selective degeneration of sympathetic preganglionic neurons. In the present study rats were subjected to a single i.v. injection of these acetylcholinesterase antibodies, or to normal IgG or saline for control. Exophthalmos, piloerection and eyelid-drooping (ptosis) were observed within 1 h after administration of the antibodies. Rats were killed at different time-points after antibody administration, and the adrenal glands were analysed by means of indirect immunohistochemistry and in situ hybridization histochemistry. As soon as 3 h after the antibody treatment, a marked increase in the number of chromaffin cells expressing mRNA encoding, respectively, enkephalin, calcitonin gene-related peptide, galanin, neurotensin and substance P was seen. At 12 h the peptide mRNA levels were still elevated and there was a concomitant increase in the number of peptide-immunoreactive cells. All peptide levels remained high for at least 48 h; however, 77 days after the antibody treatment only enkephalin-immunoreactive cells could be encountered. A disappearance of acetylcholinesterase- and enkephalin-immunoreactive cells could be encountered. A disappearance of acetylcholinesterase- and enkephalin-positive fibers was already seen 3 h after the antibody treatment, and after 24 h no fibers were encountered. In contrast, up until 48 h there was no apparent change in the number or intensity of immunofluorescent fibers expressing calcitonin gene-related peptide, galanin, neurotensin or substance P. However, 77 days after the antibody treatment the number of calcitonin gene-related peptide- and substance P-immunoreactive fibers was increased as compared to controls. In addition, reappearance of acetylcholinesterase- and enkephalin-immunoreactive fibers was seen 77 days after antibody administration, although their number was still low as compared to controls. Double-labeling immunohistochemistry revealed that the chromaffin cells expressing peptides after the antibody treatment preferentially were adrenaline storing cells (noradrenaline-negative). The majority of these cells expressed only one peptide. Both surgical transection of the splanchnic nerve as well as treatment with acetylcholine receptor antagonists mimicked the effects seen after the acetylcholinesterase-antibody treatment, although changes were less pronounced. The present results show that interruption of splanchnic transmission induces fast, marked, and selective increases in peptide expression in rat adrenal chromaffin cells.

Coexistence of somatostatin- and avian pancreatic polypeptide (APP)-like immunoreactivity in some forebrain neurons.

The indirect immunofluorescence technique was used to demonstrate the coexistence of somatostatin together with avian pancreatic polypeptide-like immunoreactivity within certain neurons of the rat forebrain. Numerous neurons containing these peptides were observed in the neocortex, hippocampus, olfactory tubercle, striatum, nucleus accumbens and lateral septum. In studies of serial sections stained alternately for these two peptides, and in restaining experiments, It could be determined that in many neurons in these areas these two peptides coexisted. In other brain areas such as the anterior periventricular hypothalamus, somatostatin cells were never found to contain avian pancreatic polypeptide-like immunoreactivity. Also, within the pancreas these two peptides were never found to coexist in the same cells. The findings represent a further example of the coexistence of more than one neuropeptide within a single neuron.