Protein kinase-mediated reciprocal modulatory changes in anesthetic sensitivity of (BK)-K+- and GABA-A receptor-gated conductances in guinea-pig sympathetic neurons.

(1) The interaction of substance P (SP)-mediated synaptic transmission with general anesthetics remains unknown. (2) Intracellular recordings were obtained from guinea-pig inferior mesenteric ganglion neurons to study monosynaptic responses to exogenous SP and GABA. (3) Propofol (1-100 microM) caused an increase in SP-evoked inward current responses and a concurrent decrease in peak amplitude of the afterspike hyperpolarization of intermittently evoked action potentials. These effects were occluded by the (BK)-K+-channel-selective blocker charybdotoxin (10 nM), and prevented by the protein kinase inhibitor staurosporine (100 nM). (4) Propofol also increased GABA-evoked current (I(GABA)) responses. (5) When elicited during a SP response, I(GABA) was significantly diminished compared to control. In the presence of staurosporine (100 nM), the inhibitory effect of SP upon I(GABA) was abolished, and the propofol-induced augmentation of I(GABA) was significantly increased. (6) Thus, SP-evoked protein kinase activity produced reciprocal changes in anesthetic sensitivity of (BK)-K+- and GABA A-receptor-gated currents of these sympathetic neurons.

Enteric GABA-containing nerves projecting to the guinea-pig inferior mesenteric ganglion modulate acetylcholine release.

1. The effect of GABA and GABA receptor-modulating drugs on release of [3H]acetylcholine was studied in the guinea-pig inferior mesenteric ganglion. 2. GABA caused a dose-dependent increase in [3H]acetylcholine release during stimulation of the lumbar colonic nerves. Muscimol (10 microM) and diazepam (5 microM) also increased [3H]acetylcholine release during stimulation of the lumbar colonic nerves whereas baclofen (10 microM) had no effect. 3. Bicuculline (20-100 microM) and picrotoxin (50 microM) alone reduced [3H]acetylcholine release during electrical stimulation of the lumbar colonic nerves whereas phaclofen (300 microM) had no effect. 4. Bicuculline (100 microM) significantly decreased whereas diazepam (5 microM) significantly increased distension-induced [3H]acetylcholine release. 5. Colonic distension significantly increased [3H]GABA release in the inferior mesenteric ganglion compared to basal periods when the colon was not distended. Distension-induced release of [3H]GABA resulted from active neuronal transmission from the colon to the inferior mesenteric ganglion, since perfusion of the inferior mesenteric ganglion with tetrodotoxin (1 microM) reduced basal release of [3H]GABA and abolished distension-evoked increases in the release of [3H]GABA. 6. In contrast to its excitatory effects on peripheral colonic afferent cholinergic nerves, exogenous GABA caused a dose-dependent decrease in [3H]acetylcholine release during electrical stimulation of the central lumbar splanchnic nerves. Baclofen (10 microM) also inhibited [3H]acetylcholine release whereas muscimol (10 microM) or diazepam (5 microM) had no effect. Phaclofen (300 microM) antagonized the inhibitory effects of exogenous GABA (10 microM) and of baclofen (10 microM). Bicuculline (100 microM), picrotoxin (50 microM) and phaclofen (300 microM) alone had no effect on [3H]acetylcholine release during splanchnic nerve stimulation. 7. Phaclofen (300 microM) increased [3H]acetylcholine release during simultaneous electrical stimulation of the lumbar colonic nerves and splanchnic nerves and when GABAA receptors were blocked by bicuculline (20 microM). 8. The data suggest that GABAA receptors facilitate release of acetylcholine from peripheral cholinergic mechanosensory nerves projecting from the colon to the inferior mesenteric ganglion and that GABAB receptors inhibit release of acetylcholine from central cholinergic nerves. Enteric GABA-containing nerves projecting to the inferior mesenteric ganglion are mechanosensory. Endogenous release of GABA may act on GABAA receptors to facilitate peripheral cholinergic mechanosensory transmission and/or on GABAB receptors to inhibit central cholinergic transmission.

The electrophysiological effects of endogenous GABA in the guinea-pig inferior mesenteric ganglion.

1. GABA receptor-modulating drugs and intracellular recording techniques were used to determine the functional significance of peripheral afferent GABA-containing nerves projecting from the distal colon to sympathetic neurones in the inferior mesenteric ganglion of the guinea-pig. 2. GABAA receptor-modulating drugs added selectively to the inferior mesenteric ganglion side of a two-compartment bath had pronounced effects on on-going colonic afferent cholinergic synaptic input. Bicuculline (20 microM) decreased the amplitude and frequency of fast excitatory postsynaptic potentials (EPSPs) by 40% whereas diazepam (5 microM) increased cholinergic input by 43%. Neither drug had any effect on the resting membrane potential or membrane input resistance of ganglion cells. 3. Bicuculline (20 microM) significantly reduced, whereas diazepam (5 microM) significantly enhanced, distension-induced increases in nicotinic fast EPSPs and action potentials. 4. Slow EPSPs evoked by colonic distension were not affected by bicuculline or diazepam. 5. Manual voltage clamp of the postsynaptic depolarizing response to exogenous GABA revealed GABA-induced presynaptic facilitation of colonic afferent but not central preganglionic efferent cholinergic synaptic input. 6. The data suggest that endogenously released GABA participates in on-going colo-colonic reflex activity by acting on presynaptic GABAA receptors to facilitate release of acetylcholine from colonic mechanosensory nerves.

Direct and indirect mechanosensory pathways from the colon to the inferior mesenteric ganglion.

The aims of these experiments were to determine in vitro whether colonic distension releases acetylcholine, vasoactive intestinal polypeptide (VIP), and substance P in the guinea pig inferior mesenteric ganglia (IMG) and whether cholinergic and peptidergic mechanosensory nerves projecting from the colon to the IMG receive cholinergic input from other enteric neurons. Colonic distension significantly increased the release of [3H]-acetylcholine, VIP-like immunoreactivity, and substance P-like immunoreactivity in the IMG. Nicotinic receptor blockade in the colon diminished the increase in [3H]acetylcholine release, abolished the increase in VIP-like immunoreactivity release during distension, but had no effect on the release of substance P-like immunoreactivity. Nicotinic receptor blockade in the colon also decreased fast mechanosensory input and significantly reduced by 54% the slow excitatory postsynaptic potential amplitude evoked by colonic distension. The data suggest that enteric cholinergic and VIP mechanosensory neurons that project from the colon to sympathetic neurons in the IMG receive peripheral cholinergic input from other enteric neurons. There was no evidence for enteric cholinergic input to the mechanosensory substance P pathway.

Neurotensin facilitates release of substance P in the guinea-pig inferior mesenteric ganglion.

1. Intracellular, electrophysiological techniques were combined with radio-immunological, chromatographic and pharmacological techniques to determine if nerve terminals containing substance P mediated transient depolarizing responses of principal ganglion cells induced by neurotensin. Experiments were performed in vitro on guinea-pig inferior mesenteric ganglia. 2. In 61% of principal ganglion cells tested in normal ganglia, neurotensin caused a transient membrane depolarization. In ganglia which were removed from animals which had been pre-treated with capsaicin, transient responses to neurotensin were virtually abolished. 3. In normal ganglia, neurotensin increased the amplitude and duration of noncholinergic slow EPSPs evoked by electrical stimulation of the lumbar colonic nerve. Such increases were absent in ganglia obtained from animals pre-treated with capsaicin. 4. In guinea-pigs pre-treated with capsaicin, the content of substance P-like material was significantly reduced in inferior mesenteric and coeliac ganglia, dorsal root ganglia and lumbar spinal cord, compared to control animals. The content of substance P-like material in segments of distal colon was slightly reduced. The content of vasoactive intestinal polypeptide-, cholecystokinin- and bombesin-like material in the same tissues from animals pre-treated with capsaicin was not significantly different from control animals. 5. Chromatographic analysis using HPLC (high-performance liquid chromatography) techniques revealed that the material depleted from inferior mesenteric and coeliac ganglia, dorsal root ganglia and lumbar spinal cord by capsaicin pre-treatment co-eluted with synthetic substance P. 6. Electrical stimulation of the lumbar colonic nerve released substance P-like material from isolated inferior mesenteric ganglia as determined by radioimmunoassay of samples of superfusate. Exogenous administration of neurotensin caused a significant increase in the amount of substance P-like material released during nerve stimulation. 7. Transient depolarizing responses evoked by neurotensin were markedly attenuated when ganglion cells were postsynaptically desensitized to exogenously administered substance P. 8. Taken together, these findings suggest that transient depolarizations mediated by an indirect action of neurotensin and facilitation of electrically evoked non-cholinergic slow EPSPs by neurotensin involved presynaptic release of substance P from collateral nerve terminals of primary afferent nerve fibres in the inferior mesenteric ganglion. 9. It was suggested that under normal in vivo conditions, neurotensin or a C-terminal-related peptide contained in central preganglionic nerve endings might function as an excitatory neuromodulator to enhance the release of substance P from primary afferent nerve terminals thereby facilitating non-cholinergic peripheral afferent synaptic input to prevertebral ganglion cells.

The electrophysiological effects of neurotensin on neurones of guinea-pig prevertebral sympathetic ganglia.

1. The membrane effects of neurotensin on neurons of guinea-pig prevertebral ganglia were investigated by means of intracellular recording techniques in vitro. 2. Neurotensin (2-5 microM) applied by superfusion caused depolarizing responses in fifty-seven of seventy-four neurones tested in the inferior mesenteric ganglion and thirty-seven of forty-seven neurones tested in the coeliac plexus. The remaining neurones tested showed no membrane response. 3. Responses to neurotensin could be discriminated into two different types of membrane depolarizations on the basis of their different time courses and pharmacological characteristics: a steady-state type of depolarization and a transient type of depolarization. Seven of fifty-seven responsive neurones tested in the inferior mesenteric ganglion and ten of thirty-seven responsive neurones tested in the coeliac plexus responded to neurotensin with a depolarization which was maintained constant as long as neurotensin was superfused over the preparation (steady-state type). Forty-eight of fifty-seven responsive neurones tested in the inferior mesenteric ganglion and twenty of thirty-seven responsive neurones tested in the coeliac plexus responded with a transient depolarization which was followed by a repolarization in the maintained presence of neurotensin (transient type). A combination of both types of responses was observed in two neurones tested in the inferior mesenteric ganglion and in seven neurones tested in the coeliac plexus. 4. Steady-state type responses were characterized by a slowly developing membrane depolarization which reached a plateau and lasted throughout the presence of neurotensin. Amplitude and time course of this response were not altered in a solution containing hexamethonium (10 microM) and atropine (10 microM) or by a solution low in calcium (1 mM) and high in magnesium (15 mM). 5. Transient type depolarizations evoked by neurotensin were faster in reaching their maximum and were followed by a repolarization during the maintained presence of neurotensin. Responses similar in time course and amplitude were obtained in solutions containing hexamethonium (10-100 microM) and atropine (10 microM). However, transient responses were abolished in a solution low in calcium (1 mM) and high in magnesium (15 mM) and were markedly attenuated in ganglia treated with capsaicin (3 microM). 6. Both types of depolarizations were associated with increases in membrane input resistance. Both responses converted subthreshold depolarizing electrotonic potentials and subthreshold fast EPSPs to action potentials. 7. Both types of depolarizations were observed when the C-terminal hexapeptide fragment neurotensin 8-13 was used.(ABSTRACT TRUNCATED AT 400 WORDS)

Central neurotensin nerves modulate colo-colonic reflex activity in the guinea-pig inferior mesenteric ganglion.

1. The effects of neurotensin and of stimulation of preganglionic nerves on peripheral afferent synaptic input from segments of distal colon to neurones in the inferior mesenteric ganglia of guinea-pigs were studied using intracellular recording techniques in vitro. 2. Electrical stimulation of colonic afferent nerve fibres evoked fast, nicotinic synaptic responses (fast EPSPs or action potentials) followed by a slow depolarizing response (slow EPSP). 3. Neurotensin (1 microM) increased the amplitude and duration of slow EPSPs evoked by stimulation of colonic afferents. 4. Distention of a segment of distal colon left attached to an inferior mesenteric ganglion evoked a slow depolarization. Neurotensin (1 microM) increased the amplitude and duration of distention-induced depolarizations. 5. Electrical stimulation of central preganglionic nerve fibres present in the third and fourth lumbar ventral roots increased the amplitude and duration of slow EPSPs evoked by electrical stimulation of colonic afferent nerves. This facilitatory effect was abolished after desensitization to neurotensin. 6. Slow depolarizations evoked by neurotensin and by stimulation of central preganglionic nerves converted subthreshold fast EPSPs due to mechanosensory synaptic input from an attached segment of distal colon to action potentials. This increase in firing rate of sympathetic ganglion cells led to a decrease in colonic intraluminal pressure. 7. Taken together these data support the hypothesis that neurotensin or a closely related substance contained in central preganglionic nerves facilitated release of a non-cholinergic excitatory transmitter from colonic mechanosensory nerves. The slow depolarization evoked by the non-cholinergic transmitter converted on-going subthreshold fast EPSPs to action potentials thereby increasing sympathetic output to the colon. 8. It is suggested that under normal in vivo conditions, central preganglionic fibres containing neurotensin or a closely related peptide modulate peripheral reflex activity through prevertebral ganglia in guinea-pigs.

Effect of naloxone on vagally-induced gastric acid secretion in rats.

Previous studies in man and dogs have demonstrated that endogenous opioids participate in the stimulation of cephalic phase gastric acid secretion during indirect activation of the vagus. Since the effect of naloxone during direct vagal activation is unknown gastric acid secretion was assessed during electrical stimulation of the distal cut ends of both vagal nerves. In overnight fasted anesthetized rats the distal ends of the bisectioned cervical vagi were stimulated with 10V, 5Hz, 5 msec for 15 min. Vagal stimulation elicited an increase of gastric acid secretion by 5.6 mumol/min. Naloxone (1 mumol/kg.h) augmented gastric acid secretion significantly. Since this effect of naloxone was in contrast to previous data possible mechanisms of action of naloxone were examined that might help to explain this apparently inhibitory action of endogenous opioids on vagally-induced gastric acid secretion. The additional infusion of atropine or hexamethonium abolished the stimulatory effect of naloxone on vagally-induced acid secretion completely indicating that the action of naloxone depends on cholinergic background activity. Combined blockade of alpha- and beta-adrenergic receptors with phetolamine and propanolol reduced vagally-induced acid secretion in controls and during naloxone to a similar degree. Both adrenergic blocking agents also reduced the residual acid secretion observed during atropine or atropine + naloxone infusion. Measurements of plasma gastrin levels suggested that the naloxone-induced changes of acid secretion were not due to alterations of gastrin secretion. In summary these data demonstrate that vagally-induced acid secretion in anesthetized rats is largely due to cholinergic mechanisms with a small but separate contribution of adrenergic mechanisms. Endogenous opioids are activated during peripheral vagal stimulation attenuating vagally-induced acid secretion by modulation of cholinergic but not adrenergic mechanisms.

Effect of atrial peptide on gastric acid secretion in rats.

The effect of synthetic rat atriopeptin (AP) II was examined on basal, vagally and carbachol-induced gastric acid secretion in anesthetized rats. AP II infusion, at stepwise increasing doses of 2, 20 and 100 ng/kg/min, had no effect on basal acid secretion. At doses of 2 and 20 ng/kg/min, AP II augmented vagally induced acid secretion significantly. The secretory response to vagal stimulation + AP II 20 ng/kg/min was completely abolished by atropine. In contrast a higher dose of AP II (50 ng/kg/h) reduced vagally induced acid secretion significantly. This dose of AP II also reduced acid secretion during direct cholinergic stimulation by carbachol, while the lower dose of 20 ng/kg/min had no effect on carbachol-induced acid secretion. The present data demonstrate for the first time an effect of atrial peptide on gastric acid secretion. At lower doses AP II augments the vagal influence on parietal cell function perhaps by augmenting vagally induced acetylcholine release. At higher doses AP II exerts an inhibitory effect on parietal cell function during vagally and carbachol-induced acid secretion, suggesting different and as yet unknown mechanisms of action. These results raise the possibility that the heart can exert a hormonally mediated influence on the regulation of gastric acid secretion.

Effect of continuous and oscillatory portal vein insulin infusion upon glucose-induced insulin release in rats.

The present study was designed to determine the effect of low dose continuous and oscillatory intraportal insulin infusions upon subsequent glucose-induced insulin release. In overnight-fasted and anesthetized rats with indwelling catheters in the jugular vein, carotid artery, and mesenteric vein insulin was infused intraportally for 3 h via the mesenteric vein catheter at a continuous rate of 45 microU/kg X min, or the same amount of insulin was administered at alternating high (72 microU/kg X min) and low infusion rates (18 microU/kg X min), respectively, in 2-, 4-, 8-, and 16-min cycles (oscillatory infusions). Another group received a continuous infusion of saline. Glucose (0.4 g/kg) was given i.v. 30 min after the end of the insulin or saline infusion. During the 3-h infusion of insulin or saline the peripheral glucose level remained unchanged in all groups. In response to the i.v. glucose load peripheral arterial plasma insulin levels were significantly elevated after preceding oscillatory infusions compared to the continuous insulin infusion. As compared to the group receiving saline the glucose-induced insulin response after continuous insulin infusion was significantly reduced. The plasma glucose responses were not different except for inexplicably elevated glucose levels in the 4-min cycle group. No difference was observed for plasma glucagon levels in all groups. The present data demonstrate an augmented responsiveness of the beta-cell to glucose after a preceding oscillatory infusion of insulin and an impaired responsiveness to glucose after continuous insulin infusion. This indicates that an oscillatory insulin release might be of importance for an adequate regulation of beta-cell function.

Effect of acetylcholine on the release of pancreatic and gastric somatostatin-like immunoreactivity in normal, chemically sympathectomized and indomethacin-treated dogs.

The present study was designed to determine the effects of intravenously infused acetylcholine on the release of pancreatic and gastric somatostatin-like immunoreactivity (SLI), in anesthetized normal, chemically sympathectomized and Indomethacin-treated prostaglandin deficient dogs. In normal dogs acetylcholine infusion (500 micrograms/min) elicited a significant rise in pancreatic vein, inferior vena cava, fundic and antral vein SLI levels. In the sympathectomized animals the rise in pancreatic and antral vein SLI was not different from the controls, while the rise in fundic vein SLI was abolished, and inferior vena cava plasma SLI levels were reduced. During Indomethacin-induced prostaglandin deficiency, basal SLI levels were reduced significantly, and the acetylcholine-induced stimulation was completely abolished from both stomach and pancreas. It is concluded that in anesthetized dogs the intravenous infusion of acetylcholine stimulates pancreatic and gastric SLI release, and this stimulatory effect depends on the presence of prostaglandins and is modulated by adrenergic mechanisms.

Effect of histamine H2-receptor stimulation on postprandial pancreatic and gastric endocrine function in dogs.

The effect of histamine H2-receptor stimulation via the infusion of impromidine was assessed with regard to postprandial plasma insulin, pancreatic polypeptide (PP), somatostatin, and gastrin levels. The effect of impromidine was assessed in the postprandial state during a liver extract/sucrose test meal which had a buffer capacity to maintain the intragastric pH at a constant level for the time impromidine was infused. Postprandial plasma insulin and gastrin levels were not changed by impromidine (10 micrograms/kg X h-1). Plasma somatostatin levels rose significantly, whereas the postprandial increase of plasma PP levels was attenuated. The effects on somatostatin and PP were antagonized by the infusion of cimetidine, a specific histamine H2-receptor blocker. In conclusion the present data demonstrate that in the postprandial state activation of H2-receptors stimulates somatostatin and inhibits PP release while insulin and gastrin release are not affected.

Effect of physiological increments of blood glucose on plasma somatostatin and pancreatic polypeptide levels in dogs.

The present study was designed to determine the effects of physiological increments of plasma glucose levels upon basal and stimulated plasma somatostatin and pancreatic polypeptide levels. In seven conscious dogs the elevation of plasma glucose levels by 30-40 mg/dl did not change basal somatostatin and pancreatic polypeptide levels. During stimulation of these two hormones by acetylcholine and the octapeptide of cholecystokinin intravenous infusion of glucose elicited a significant decrease of somatostatin levels by 30 pg/ml and of pancreatic polypeptide levels by 300 pg/ml. The present data demonstrate that a physiological elevation of plasma glucose levels inhibits stimulated but not basal somatostatin and pancreatic polypeptide levels which may be of importance for nutrient entry and metabolism.