Nicotinic Receptor Blocker Hexamethonium Research Articles

Hydrogen sulfide stimulates catecholamine secretion in rainbow trout (Oncorhynchus mykiss)

We tested the hypothesis that endogenously produced hydrogen sulfide (H(2)S) can potentially contribute to the adrenergic stress response in rainbow trout by initiating catecholamine secretion from chromaffin cells. During acute hypoxia (water Po(2) = 35 mmHg), plasma H(2)S levels were significantly elevated concurrently with a rise in circulating catecholamine concentrations. Tissues enriched with chromaffin cells (posterior cardinal vein and anterior kidney) produced H(2)S in vitro when incubated with l-cysteine. In both tissues, the production of H(2)S was eliminated by adding the cystathionine beta-synthase inhibitor, aminooxyacetate. Cystathionine beta-synthase and cystathionine gamma-lyase were cloned and sequenced and the results of real-time PCR demonstrated that with the exception of white muscle, mRNA for both enzymes was broadly distributed within the tissues that were examined. Electrical field stimulation of an in situ saline-perfused posterior cardinal vein preparation caused the appearance of H(2)S and catecholamines in the outflowing perfusate. Perfusion with the cholinergic receptor agonist carbachol (1 x 10(-6) M) or depolarizing levels of KCl (1 x 10(-2) M) caused secretion of catecholamines without altering H(2)S output, suggesting that neuronal excitation is required for H(2)S release. Addition of H(2)S (at concentrations exceeding 5 x 10(-7) M) to the perfusion fluid resulted in a marked stimulation of catecholamine secretion that was not observed when Ca(2+)-free perfusate was used. These data, together with the finding that H(2)S-induced catecholamine secretion was unaltered by the nicotinic receptor blocker hexamethonium, suggest that H(2)S is able to directly elicit catecholamine secretion via membrane depolarization followed by Ca(2+)-mediated exocytosis.

The nicotinic receptor blocker hexamethonium alters neuronal responses to glutamate in the medial septal area of the brain of the ground squirrel in vitro

Despite extensive interest in studies of the medial septal area, the nature of the interactions of its various neurochemical systems remains largely unclear. The aim of the present work was to clarify the role of nicotinic receptors in mediating the interaction of the glutamatergic and cholinergic systems in this structure. Extracellular recording of neuron activity in living slices of ground squirrel brain was used to study the influences of L-glutamate (1 microM) during application of the nicotinic receptor blocker hexamethonium (1 mM). The responses of septal neurons to glutamate depended on the type of their initial activity and the presence of pacemaker properties. This study is the first to show that glutamate increases the frequency of volleys in rhythmic neurons in the septum. Hexamethonium induced changes in neuron activity similar to the influences of glutamate. After prior application of hexamethonium, the responses of neurons to glutamate changed: activatory responses were masked and inhibitory responses were enhanced. Cholinergic modulation of the responses of septal neurons to glutamate were shown to occur, as did modulation of the strength of the oscillatory properties of the septal network by nicotinic receptors.

Abstract no.: 4 Experimental inflammation of the colon modulates stomach motility via neuronal pathways

Patients with IBD often suffer from impaired gastrointestinal motility, even at sites remote from the inflammation. We previously reported diminished gastric emptying associated with reduced gastric fundus contractility in rats with TNBS‐induced colitis. The aims of the present study were (1) to exclude local gastric inflammation using MPO measurements and to rule out direct systemic influences on gastric neuromuscular function by examining the effect of TNBS‐induced colitis on contractions of aortic vascular rings. Secondly (2) we tested the involvement of neuronal ganglionic (nicotinic) transmission and of extrinsic reflex pathways in impaired gastric emptying in rats with TNBS‐induced colitis. Distal colitis was induced by rectal instillation of 30 mg trinitrobenzene sulphate (TNBS) in 50% ethanol 72 h prior to experiments. Gastric emptying (GE) and the geometric center (GC) of intestinal transit were measured 30 min after intragastric injection of a semiliquid bolus of Evans blue. TNBS colitis reduced gastric emptying from 38.4 ± 3.6% in controls to 22.7 ± 4.4% but had no effect on small intestinal transit. (1) Myeloperoxidase (MPO) measurements revealed no acute inflammatory changes in the stomach whereas MPO was significantly increased in the colon of TNBS rats. The study of aortic ring contractility showed no differences between control rats and rats with TNBS colitis. (2) The nicotinic receptor blocker hexamethonium (20 mg/kg ip 1 h prior to experiment) inhibited GE by 31% in controls (P < 0.05) whereas it increased GE by 37% in TNBS rats (P < 0.05). The GC of gastrointestinal transit was similarly affected. Extrinsic afferent innervation was abolished by capsaicin pre‐treatment (50 mg/kg SC 14 days prior to experiment). Capsaicin reduced GE by 71% in control rats (P < 0.05) while in TNBS rats its effect on GE was no longer significant (40% reduction). Capsaicin pre‐treatment had comparable effects on the GC of gastrointestinal transit in controls and TNBS. In conclusion, we showed that TNBS colitis induces a pronounced gastroparesis which is not due to local gastric inflammation nor to systemic impairment of neuromuscular function. Moreover, it involves nicotinic synapses embedded in autonomic reflexes. A role for extrinsic afferent pathways sensitized by inflammatory mediators seems plausible.

Cardiovascular effects of hypocretin-1 in nucleus of the solitary tract.

Experiments were done in male Wistar rats to investigate the effects of microinjection of hypocretin-1 (Hcrt-1) into the nucleus of the solitary tract (NTS) on mean arterial pressure (MAP), heart rate (HR), and the baroreflex. In the first series, the distribution of Hcrt-1-like immunoreactivity (Ir) was mapped within the region of NTS. Hcrt-1 Ir was found throughout the NTS region, predominantly within the caudal dorsolateral (Slt), medial (Sm), and interstitial subnuclei of the NTS. In the second series, in alpha-chloralose or urethane-anesthetized rats, microinjection of Hcrt-1 (0.5-5 pmol) into the caudal NTS elicited a dose-dependent decrease in MAP and HR. A mapping of the caudal NTS region showed that the largest depressor and bradycardia responses elicited by Hcrt-1 were from sites in the Slt and Sm. In addition, doses >2.5 pmol at a small number of sites localized to the caudal commissural nucleus of NTS elicited pressor and tachycardia responses. Intravenous administration of the muscarinic receptor blocker atropine methyl bromide abolished the bradycardia response and attenuated the depressor response, whereas subsequent administration of the nicotinic receptor blocker hexamethonium bromide abolished the remaining MAP response. Finally, microinjection of Hcrt-1 into the NTS significantly potentiated the reflex bradycardia to activation of arterial baroreceptors as a result of increasing MAP by systemic injections of phenylephrine (2-4 microg/kg). These results suggest that Hcrt-1 in the NTS activates neuronal circuits that increases vagal activity to the heart, inhibits sympathetic activity to the heart and vasculature, and alters the excitability of NTS neuronal circuits that reflexly control the circulation.

The Effects of C-Type Natriuretic Peptide on Catecholamine Release in the Pacific Spiny Dogfish (Squalus acanthias)

The interaction between homologous C-type natriuretic peptide (dfCNP) and catecholamine release in cardiovascular control was assessed in the marine dogfish (Squalus acanthias). This was accomplished by evaluation of the dynamics of the dfCNP-elicited secretion of catecholamines in situ and in vivo. With an in situ saline-perfused postcardinal sinus preparation, it was demonstrated that perfusion with saline containing dfCNP (10−9 mol L−1) did not affect the secretion of either noradrenaline or adrenaline. However, the presence of dfCNP in the perfusate significantly enhanced carbachol-evoked secretion of noradrenaline. In vivo, intravascular injection of dfCNP (10−9 mol kg−1) caused a biphasic pressor–depressor response consisting of a brief increase in caudal artery blood pressure (PCA) followed by a prolonged reduction in PCA. Furthermore, although systemic resistance initially increased, it was subsequently maintained at baseline values in the face of persistent decreases in both PCA and cardiac output. Bolus injection of dfCNP elicited significant increases in plasma noradrenaline levels that peaked within 10 min; plasma adrenaline levels were unaffected. The release of noradrenaline elicited by dfCNP was unaffected by prior blockade of the renin–angiotensin system (RAS) (with the angiotensin converting enzyme inhibitor lisinopril) or by pretreatment with the nicotinic receptor blocker hexamethonium. The delayed decrease in PCA was not observed in the hexamethonium-treated fish. Prior blockade of β-adrenoreceptors (with sotalol) or α-adrenoreceptors (with prazosin) either significantly reduced (sotalol) or abolished (prazosin) the increase in plasma noradrenaline levels after dfCNP injection. The results of this investigation demonstrate that the elevation of plasma noradrenaline levels observed in vivo following dfCNP injection is not caused by a direct effect of dfCNP on catecholamine secretion from axillary body chromaffin cells. Furthermore, the dfCNP-mediated increase of plasma noradrenaline appears to be unrelated to changes in PCA and is insensitive to blockade of the RAS or nicotinic receptors. However, stimulation of adrenergic receptors, in particular the α-adrenoreceptors, appears to be a key mechanism underlying the dfCNP-elicited secretion of noradrenaline.

Cardiovascular depressor responses to stimulation of substantia nigra and ventral tegmental area.

Experiments were done in alpha-chloralose-anesthetized, paralyzed, and artificially ventilated rats to investigate the effect of L-glutamate (Glu) stimulation of the substantia nigra (SN) and ventral tegmental area (VTA) on arterial pressure (AP) and heart rate (HR). Glu stimulation of the SN pars compacta (SNC) elicited decreases in both mean AP (MAP; -18.9 +/- 1.3 mmHg; n = 52) and HR (-26.1 +/- 1.6 beats/min; n = 46) at 81% of the sites stimulated. On the other hand, stimulation of the SN pars lateralis or pars reticulata did not elicit cardiovascular responses. Stimulation of the adjacent VTA region elicited similar decreases in MAP (-18.0 +/- 2.6 mmHg; n = 20) and HR (-25.4 +/- 3.8 beats/min; n = 17) at approximately 74% of the sites stimulated. Intravenous administration of the dopamine D2-receptor antagonist raclopride significantly attenuated both the MAP (70%) and the HR (54%) responses elicited by stimulation of the transitional region where the SNC merges with the lateral VTA (SNC-VTA region). Intravenous administration of the muscarinic receptor blocker atropine methyl bromide had no effect on the magnitude of the MAP and HR responses to stimulation of the SNC-VTA region, whereas administration of the nicotinic receptor blocker hexamethonium bromide significantly attenuated both the depressor and the bradycardic responses. These data suggest that dopaminergic neurons in the SNC-VTA region activate a central pathway that exerts cardiovascular depressor effects that are mediated by the inhibition of sympathetic vasoconstrictor fibers to the vasculature and cardioacceleratory fibers to the heart.

Cardiovascular responses to glutamate stimulation of diagonal band of Broca.

Experiments were done in alpha-chloralose-anesthetized, paralyzed, and artificially ventilated rats to investigate the effect of L-glutamate stimulation of the diagonal band of Broca (DBB) on arterial pressure (AP) and heart rate (HR). Stimulation of the horizontal limb of the DBB (hDB) elicited decreases in both mean AP (MAP; -32.3 +/- 2.5 mmHg; n = 37) and HR (-32.5 +/- 3.8 beats/min; n = 33) at 84% of the sites stimulated. Stimulation of the vertical limb of the DBB (vDB) elicited significantly smaller decreases in MAP (-9.7 +/- 1.0 mmHg; n = 19) and HR (-13.5 +/- 1.8 beats/min; n = 7) at approximately 13% of the sites stimulated. Intravenous administration of the muscarinic receptor blocker atropine methylbromide had no effect on the magnitude of the MAP and HR responses to stimulation of the hDB. In contrast, administration of the nicotinic receptor blocker hexamethonium bromide abolished both the depressor and the bradycardic responses elicited by stimulation of the hDB. These data indicate that the hDB contains neurons that exert cardiovascular depressor effects and that these circulatory effects are mediated by the inhibition of sympathetic vasoconstrictor fibers to the vasculature, and cardioacceleratory fibers to the heart.

Cardiovascular effects of neurotensin microinjections into the nucleus of the solitary tract

Neurotensin (NT) immunoreactivity and binding sites have been demonstrated to be extensively distributed throughout the caudal nucleus of the solitary tract (NTS). In this study, the cardiovascular effects of microinjecting the tridecapeptide neurotensin (NT) or its analogues NT 1–8 and [ d-Trp 11]NT into NTS were investigated in the chloralose-anesthetized, paralyzed and artificially ventilated rat. Microinjection of NT (10 pmol) elicited decreases in arterial pressure (AP) (−34 ± 3 mm Hg) and heart rate (HR) (−28 ± 2 beats/min), whereas microinjection of equimolar amounts of the NT fragment NT 1–8 elicited a significantly smaller depressor response (−14 ± 3 mm Hg), but the bradycardic (−22 ± 4 beats/min) response was similar in magnitude to that elicited by NT. On the other hand, microinjection of [ d-Trp 11]NT did not elicit cardiovascular responses from sites in NTS. In addition, the prior injection of [ d-Trp 11]NT into cardiovascular responsive sites in the NTS did not significantly reduce the AP or HR response to NT. The depressor response elicited by NT was not affected by bilateral vagotomy but was abolished by either C1–C2 spinal cord transection or the i.v. administration of the nicotinic receptor blocker hexamethonium bromide. The cardiac slowing was partially attenuated by either bilateral vagotomy (−19 ± 2 beats/min, i.v. administration of atropine methyl bromide (−17 ± 4 beats/min), i.v. administration of hexamethonium bromide (−11 ± 4 beats/min) or by spinal cord transection (−12 ± 3 beats/min), and completely abolished after total autonomic blockade or by combined bilateral vagotomy and spinal cord transection. These data have demonstrated that within a restricted region of the caudal NTS NT activates neurons that contribute to vasodepressor responses as a result of sympatho-inhibition and to bradycardia responses as a result of vagal excitation and sympatho-inhibition. Furthermore, these data suggest that NT may act as a neurotransmitter or modulator in central cardiovascular reflex pathways.

Calcium signalling in isolated single chromaffin cells of the rainbow trout (Oncorhynchus mykiss)

Chromaffin cells were isolated from the posterior cardinal vein of rainbow trout (Oncorhynchus mykiss) to assess their suitability as a model system for studying mechanisms of catecholamine secretion in fish and to evaluate intracellular calcium changes associated with cholinoreceptor stimulation. Immunocytochemistry in concert with fluorescence microscopy was employed to identify characteristic chromaffin cell proteins and thus to confirm the presence of these specific cells in suspensions and cultures. Dopamine-β-hydroxylase, an enzyme of the catecholamine-synthesising Blaschko pathway, was identified in cytoplasmic vesicles of the isolated chromaffin cells. The actin filament-severing protein, scinderin, was co-localized with actin in the sub-plasmalemmal membrane of these chromaffin cells. Intracellular calcium [Ca2+]i was measured in single chromaffin cells by microspectrofluorometry using the fluorescent dye Fura-2. Significant increases in [Ca2+]i were observed in chromaffin cells in response to depolarisation of the cell membrane by high concentrations of K+ or by the stimulation of the cell by the cholinergic receptor agonists, nicotine, acetylcholine or carbachol. The response to the reversible agonist, nicotine, was attenuated following addition of the nicotinic receptor blocker hexamethonium. Such attenuation, however, did not occur when hexamethonium was added after stimulation with the non-specific irreversible cholinergic agonist, carbachol. These results demonstrate the presence of functional cholinoreceptors, linked to intracellular calcium signalling, on isolated trout chromaffin cells and reveal the potential of these cells as a model system for studying aspects of catecholamine secretion in fish.

Prostacyclin synthesis elicited by cholinergic agonists is linked to activation of M 2α and M 2β muscarinic receptors in the rabbit aorta

This study was conducted to investigate the subtypes of muscarinic receptors involved in the action of cholinergic agents on prostacyclin synthesis in the rabbit aorta. Prostacyclin production measured as 6-keto-PGF 1α was assessed after exposing the aortic rings to different cholinergic agents. Acetylcholine (ACh) (M 1 and M 2 agonist) (1–10 μM) and arecaidine proparagyl ester (APE) (M 2 selective agonist) (1–10 μM) enhanced 6-keto-PGF 1α output in a concentration-dependent manner. A selective M 1 receptor agonist, McN-A-343, at 1 μM-1 mM did not alter 6-keto-PGF 1α output. ACH- and APE induced increases in 6-keto-PGF 1α output were attenuated by the M 1 M 2 antagonist atropine (0.1 μM), M 2α antagonist (AF-DX 116), (0.1–1.0 μM), and by selective M 2β antagonist, hexahydro-sila-difendiol (HHSiD) (0.1–10 μM), but not by the M 1 antagonist pirenzepine (1.0 μM). 6-Keto-PGF 1α output elicited by ACh- or APE was not altered by the adrenergic receptor antagonist phentolamine and propranolol or by the nicotinic receptor blocker hexamethonium. Similarly, the arachidonic acid- or norepinephrine induced 6-keto-PGF 1α accumulation was not altered by these muscarinic receptor antagonists. Indomethacin, a cyclooxygenase inhibitor, prevented arachidonic acid, ACh- or APE induced 6-keto-PGF 1α output. Removal of the endothelium abolished the production of 6-keto-PGF 1α elicited by ACH, APE, bradykinin, and calcium ionophore A 23187, but not that induced by angiotensin II, K + or norepinephrine. These data suggests that vascular prostaglandin generation elicited by cholinergic agonists is mediated via activation of M 2α and M 2β but not M 1 muscarinic receptors, which are most likely located on the endothelium.

Immunocytochemical and pharmacological evidence for an intrinsic cholinomimetic system modulating prolactin and growth hormone release in rat pituitary.

Pituitary cells were cultured as three-dimensional reaggregates in serum-free chemically defined medium supplemented with different concentrations of dexamethasone. Immunostaining of the cells using a polyclonal antiserum and three monoclonal antibodies raised against choline acetyl transferase (CAT), revealed the presence of CAT immunoreactivity in 4-10% of anterior pituitary cells depending on the antibody used. CAT immunoreactivity was also found in freshly dispersed anterior pituitary cells. CAT-immunoreactive cells could be enriched on BSA and Percoll gradients and codistributed with ACTH-immunoreactive cells in these gradients. Perifusion of the aggregates with the potent muscarinic receptor antagonist atropine (Atr) resulted in a dose-dependent (0.1-100 nM) increase in both basal PRL and GH secretion; the response was dependent on the dexamethasone concentration in the culture medium. A similar response to Atr was observed in organ-cultured pituitaries. The specificity of the Atr effect was supported by the findings that the potent and highly specific muscarinic receptor blocker dexetimide showed a similar action, whereas its inactive enantiomer levetimide and the nicotinic receptor blocker hexamethonium failed to do so. Two other muscarinic antagonists, benzatropine and pirenzepine, showed a dose-dependent hormone-releasing action similar to that of Atr, but were less potent than the latter. Pirenzepine was only effective at high molar concentrations, suggesting that an M2 muscarinic receptor subtype was mediating the present phenomenon. Atr also potentiated GH release stimulated by the beta-adrenergic agonist isoproterenol and PRL release stimulated by vasoactive intestinal peptide, but had no effect on GRF-stimulated GH release. The choline uptake blocker hemicholinium abolished the effect of Atr on GH and PRL release. These data suggest that certain pituitary cells can express CAT activity and that these cells exert a tonic inhibitory activity on GH and PRL release which is mediated by a cholinomimetic substance, possibly acetylcholine, through a muscarinic receptor.