Evidence for galanin as an inhibitory neuropeptide on myenteric cholinergic neurons in the guinea pig small intestine

The effect of galanin on the [3H]ACh release from myenteric plexus-longitudinal muscle strips of the guinea pig small intestine was studied. While galanin did not alter the basal spontaneous efflux of ACh, it significantly depressed the ACh release evoked by electrical stimulation or caused by VIP and substance P. These results suggest an important neuromodulatory role for galanin in the enteric nervous system.     

Cholecystokinin octapeptide-evoked [3H]acetylcholine release from guinea pig gallbladder

Cholecystokinin-octapeptide (CCK-OP) produced a contractile response in isolated guinea pig gallbladder; the response consisted of scopolamine-sensitive and scopolamine-insensitive components, neither of which were affected by tetrodotoxin or hexamethonium. In the presence of tetrodotoxin, CCK-OP evoked [3H]acetylcholine (ACh) release from strips of gallbladder in a concentration-dependent manner. These results suggest that CCK-OP acts at two sites in the guinea pig gallbladder, viz. the smooth muscle cell and the postganglionic cholinergic nerve terminal.     

Ionotropic glutamate receptors regulating labeled acetylcholine release from rat striatal tissue in vitro: possible involvement of receptor modulation in magnesium sensitivity

This study evaluated the role of glutamate ionotropic receptors on the control of [3H]acetylcholine ([3H]ACh) release by the intrinsic striatal cholinergic cells. [3H]-choline previously taken up by chopped striatal tissue and converted to [3H]ACh, was released under stimulation by glutamate, N-methyl-d-aspartate (NMDA), kainate and a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA). Experiments were conducted in the absence of choline uptake inhibitors or acetylcholinesterase inhibitors. A paradigm of two stimulations was employed, the first in control conditions and the second after 9 min of perfusion with the test agents MK-801, 2-amino-5-phosphonopentanoic acid (AP-5), tetrodotoxin (TTX), 6,7-dinitroquinoxaline-2,3-dione (DNQX), 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo-[f]quinoxaline-7-sulfonamide (NBQX), glycine and magnesium. Our results support that (1) in the absence of Mg2+, NMDA is the most effective agonist to stimulate [3H]ACh release from striatal slices (2) magnesium effectively antagonized kainate and AMPA stimulation suggesting that at least part of the kainate and AMPA effects might be attributed to glutamate release (3) besides NMDA, kainate receptors showed a more direct involvement in [3H]ACh release control based on the smaller dependence on Mg2+ and less inhibition by TTX and (4) stimulation of ionotropic glutamate receptors may induce long lasting biochemical changes in receptor/ion channel function since the effects of TTX and/or Mg2+ ions on [3H]ACh release were modified by previous exposure of the tissue to agonists.     

The effect of topically applied atropine on resting and evoked cortical acetylcholine release

1. Cortical acetylcholine (ACh) output was measured in cats anaesthetized either with Dial compound (0.6 ml./kg) or with halothane-N(2)O. ACh output was found to be 1.67 ng/cm(2).min under Dial anaesthesia, and 0.30 ng/cm(2).min under halothane-N(2)O.2. Addition of atropine sulphate (1 mug/ml.) to the collection fluid increased ACh output fourfold under Dial anaesthesia but had no effect under halothane-N(2)O anaesthesia.3. Isolation of the cortex, lesions in the mesencephalon and topical application of tetrodotoxin (TTX) reduced ACh output under Dial anaesthesia to about 0.8 ng/cm(2).min. The effect of atropine on ACh output was somewhat reduced by isolation and completely abolished by mesencephalic lesions or TTX.4. ACh release evoked by reticular formation stimulation under halothane-N(2)O anaesthesia was increased fourfold by atropine but evoked release due to direct stimulation of the cortex was increased only twofold.5. ACh release due to depolarization of the cortex with KCl was not increased by atropine.6. Dihydro-beta-erythroidine (DHE) or D-tubocurarine failed to affect ACh output even in a concentration of 100 mug/ml.7. It is concluded that atropine does not increase spontaneous ACh release and only ACh release evoked by trans-synaptic stimulation of cholinergic neurones is potentiated by atropine.8. These findings are fully consistent with the hypothesis that atropine increases ACh output by blocking cortical cholinergic synapses which are a part of a circuit inhibiting cholinergic neurones.     

Mechanisms of leukotriene-induced contractions of guinea pig airways: leukotriene C4 has a potent direct action whereas leukotriene B4 acts indirectly

The leukotrienes (LT's) are a group of arachidonic acid derivatives implicated as mediators of allergic bronchoconstriction and acute inflammation. Tracheal spirals and strips of lung parenchyma from guinea pigs were used under non-flow conditions to characterize the contractions caused by LTA4, LTB4 and LTC4. Cumulative administrations of leukotrienes desensitized the lung strip, whereas non-cumulative dose-response relationships for the leukotrienes and histamine were reasonably parallel. Half maximal contractions of the lung strips were obtained at a final bath concentration of 1 nM for LTC4 and 300 nM for LTA4 or LTB4, as compared with 6 000 nM for histamine. In the trachea, LTC4 was approximately 100 times more potent than LTA4 and histamine. Leukotrienes B4 and C4, but not acetylcholine or histamine, elicited release of the bronchoconstrictive thromboxane A2 from the lung under non-flow conditions. Indomethacin blocked the contractile response to LTB4, whereas the contractile effect of LTC4 remained unaltered. The beta-adrenoceptor agonist isoproterenol and the LTC4 antagonist FPL 55712 attenuated the contraction, but not the release of thromboxane A2, induced by LTC4. Changing to a perifusion technique rendered the lung strips less sensitive to the direct action of LTC4, and released thromboxane A2 now contributed significantly to the contractile response. In addition, the perifusion experiments indicated that LTB4 released histamine as well. We conclude that the chemoattractant LTB4 is an indirectly acting bronchoconstrictor, whereas the slow reacting substance LTC4 contracts the airway muscle by a predominantly direct mechanism. The exquisite bronchoconstrictive activity of LTC4 may be unrelated to its ability to induce formation of thromboxane A2.     

Tetrodotoxin-resistant contractions induced by electrical stimulation of bladder muscle from man, rabbit and rat

Isolated detrusor preparations from man, rabbit and rat were suspended in an organ bath and isometric tension was recorded. The preparations were stimulated electrically in the presence of Bay K8644 and nifedipine before and after neuronal blockade with tetrodotoxin. Transmural electrical stimulation produced frequency-dependent contractions in all preparations. Bay K8644 significantly increased and nifedipine decreased these contractions. TTX effectively suppressed the response to electrical field stimulation in all species. When Bay K8644 was added to TTX blocked preparations, the responses to electrical stimulation were partly restored in bladder strips from man and rat. No increase in response was seen in the rabbit preparations. However, if the extracellular K+-concentration was increased to 10 mM (which per se did not affect the response) Bay K8644 significantly increased the contractions. All responses elicited by electrical stimulation in the presence of TTX were abolished by nifedipine. It is concluded that if the bladder smooth muscle is exposed to factors that can increase its sensitivity to contractile agents, this may result in uncontrolled (unstable) bladder contractions. Such contractions may use the 'normal' transmitter substances, but may be triggered at a lower stimulus intensity than normal. As a non-specific increase in membrane excitability seems to be associated with an influx of calcium through voltage-sensitive calcium channels, calcium antagonists, together with agents specifically blocking relevant transmitter substances, would offer an effective therapy against the unstable bladder.     

Role of different calcium pools in the contraction of respiratory smooth muscle

Contractions were evoked in isolated strips of rat trachea by potassium and acetylcholine (ACh) respectively. Extracellular calcium (Ca) was removed by EGTA and Ca influx was inhibited by diethylstilboestrol (DES). Contractions evoked by potassium were completely inhibited in the presence of EGTA or DES. ACh-induced contractions were rapidly abolished by EGTA whereas DES did not completely block these responses when a high concentration of ACh (10(-4) M) was used. Repeated activation by ACh in the presence of DES displayed two different phases of the contractile response; one initial brief contraction that decreased only slowly in the presence of DES, and a second phase with sustained contraction that was more readily depressed by DES. It is suggested that the smooth muscle in rat trachea is activated by Ca originating both intra- and extracellularly. As EGTA almost immediately blocked even the initial contractile response, the intracellular store is apparently located in or very close to the plasma membrane.     

Diabetes and reactivity of isolated human saphenous vein

Helical strips of saphenous veins from diabetic (n = 8) and non-diabetic (n = 18) humans were studied in vivo for their responsiveness to several vasoactive agents. Following application of passive force (approximately 20.0 mN), venous strips from non-diabetic humans often developed spontaneous phasic contractile activity (12 out of 18 patients; 2-5 contractions/min). These intrinsic changes in force were seen in venous strips from only one diabetic patient. The phasic contractions were not altered by treatment with phentolamine, whereas the calcium channel blocker, D-600, and calcium-free solution (1.0 mM EGTA) inhibited the phasic contractions. Saphenous veins from diabetic patients developed less maximal, active tension in response to norepinephrine than those from non-diabetic patients. Contractile responses to serotonin, angiotensin II, and elevated potassium concentration in saphenous veins from diabetic patients were not different from those in veins from non-diabetic patients. These observations demonstrate attenuated development of active tension in response to alpha-adrenergic receptor activation and reduced spontaneous contractile activity in venous smooth muscle from diabetic patients.     

Different effects of electrical stimulation of the mesencephalic and pontine reticular formation on the release of dopamine and acetylcholine in the cat caudate nucleus

The effects of unilateral electrical stimulation of the pontine (PRF) and mesencephalic (MRF) reticular formation on the release of acetylcholine (ACh) and of [3H]dopamine continuously synthesised from [3H]tyrosine were examined in both caudate nuclei of halothane-anaesthetised cats implanted with push-pull cannulae. Stimulation of PRF led to a prolonged bilateral increase in the release of [3H]dopamine, whereas a significant reduction in [3H]amine release was observed in the ipsilateral caudate nucleus following stimulation of the MRF. Changes in ACh release were also seen, but they seemed to be independent from those in dopamine release: the release of ACh was enhanced markedly in both caudate nuclei following stimulation of the MRF, whereas a more moderate increase in the release of ACh occurred ipsilaterally following stimulation of the PRF. These data indicate that both the MRF and the PRF are involved in the control of dopaminergic and cholinergic transmission in the basal ganglia.     

Chloride activity in cells of isolated perfused cortical thick ascending limbs of rabbit kidney

Muscle strips were excised from the circular and longitudinal layers of the fundus, corpus and antrum of canine stomach, and from the inner portion of the pyloric ring (inner pylorus). Substance P (SP) induced strong contractions, with the greatest sensitivity in fundus and circular corpus preparations (threshold near 10 -10 mol/l). The sensitivity to SP decreased in the sequence circular corpus - longitudinal antrum - circular antrum (threshold near 10(-7) mol/l); it re-increased towards the pylorus, and in the inner pylorus was nearly as high as in the fundus. The SP responses of fundus and longitudinal corpus were purely tonic, similar to acetylcholine (ACh) and histamine (H) responses. In circular corpus, SP induced a combined phasic-tonic response. SP induced in antrum strips purely phasic-rhythmical contractions of low frequency (similar to the H response), whereas ACh induced phasic contractions of high frequency, and in addition an increase of tone in the longitudinal antrum strips. The SP responses of the inner pylorus were not uniform; in some preparations purely tonic contractions were observed, and large phasic fluctuations of low frequency occurred in others. The phasic components of all the responses were completely suppressed by nifedipine (10(-6) mol/l). Tetrodotoxin as well as blockade of adrenoceptors, of ACh and of H receptors had no effect on the SP responses. Comparative studies with preparations from guinea-pig showed that species differences exist in the SP responses of gastric muscle.     

Diabetes mellitus and responses of the urinary bladder to acetylcholine: an in vitro study

This study was prompted by the inconsistent reports and apparent controversies that exist in the biomedical literature on the responses of diabetic bladder strips to cholinergic nerve stimulation or exogenously-administered muscarinic agonists, especially acetylcholine (ACh). In the present study, acetylcholine-induced contractions of urinary bladders isolated from normoglycaemic (normal) and streptozotocin-treated, diabetic Wistar rats were examined under physiological conditions. Mechanical contractile changes of the isolated urinary bladders of STZ-treated, diabetic rats in response to bath-applied acetylcholine were compared with those obtained from isolated urinary bladders of normal, age-matched, control rats. Results obtained show that urinary bladders from diabetic rats were always more spontaneously active after mounting, than those of the age-matched normal, control rats. ACh (10(-8)-10 (-4) M) provoked concentration-related, atropine-sensitive contractions of the isolated urinary bladders of both diabetic and age-matched normal, control rats. However, acetylcholine always induced more powerful and greater contractions of the diabetic bladders compared with bladders from the age-matched normal, control rats. The magnitude and/or intensity of the diabetic bladder enhanced contractile responses to ACh continued to increase as the diabetic state of the animals progressed.     

Differential blockade by nifedipine and omega-conotoxin GVIA of alpha 1- and beta 1-adrenoceptor-controlled calcium channels on motor nerve terminals of the rat

Electrically evoked release of [3H]acetylcholine ([3H]ACh) from the rat phrenic nerve and its facilitation by stimulation of presynaptic alpha 1- and beta 1-adrenoceptors were investigated in the absence and presence of nifedipine and omega-conotoxin GVIA. Both calcium channel antagonists did not modify electrically evoked [3H]ACh release, but selectively blocked the effect triggered by both facilitatory adrenergic receptors. The increase in [3H]ACh release mediated via beta 1-adrenoceptor activation was abolished by low concentrations (1 nM) of omega-conotoxin GVIA, whereas nifedipine (100 nM) abolished the facilitatory effect mediated via alpha 1-adrenoceptor stimulation. Therefore, the beta 1-adrenoceptor is apparently coupled to a calcium channel that can be regarded as of the N-type, and the alpha 1-adrenoceptor is apparently coupled to a calcium channel that appears as a subtype of the L-type which is not sensitive to omega-conotoxin GVIA.     

Short-chain fatty acids decrease the frequency of spontaneous contractions of longitudinal muscle via enteric nerves in rat distal colon

Short-chain fatty acids (SCFAs) produced by the bacterial fermentation of carbohydrates in the cecum and proximal colon are reported to modify colonic motility as a luminal factor. Besides the physical stimuli in the distal colon, SCFAs in the intestinal lumen also seem to affect colonic motility under physiological concentrations. This study therefore used fasted rats to investigate the effect of SCFAs on the spontaneous contractions of longitudinal muscle (LM) in rat distal colon, including mucosa in vitro. The frequency of spontaneous contractions of LM strips from the distal colon was 9.4 +/- 0.5 contractions/20 min. The exogenous addition of >5 mM SCFAs decreased the frequency of spontaneous contractions of the LM to 6.1 +/- 0.8 contractions/20 min. Among SCFAs, only acetate elicited this inhibitory response. TTX and the combination of hexamethonium and granisetron abolished SCFA-induced inhibitory response, suggesting that this inhibitory response is mediated via the ENS, including nicotinic and 5-HT(3) receptors. In conclusion, it is suggested that SCFAs in rat distal colon decrease the frequency of spontaneous contractions of the LM and that SCFAs may contribute to colonic motility, including the peristaltic reflex, by regulating the frequency of spontaneous contractions of the LM through the enteric nervous system (ENS).     

Potentiation of serotonin-induced contractility of gastric fundus strips in lactating rats

Gastric fundus strips isolated from lactating, non-lactating and pregnant rats were used to obtain agonist-induced contractions in vitro and the mechanism of alteration in gastric contractility to serotonin during lactation was investigated. The gastric contractile responses to 5-HT expressed as area per unit mass of muscle (cm² g^-1 muscle) were 60–65 % greater in lactating rats compared with non-lactating rats. However, responses evoked by acetylcholine (ACh) or histamine were not different. The EC₅₀ values for 5-HT were not different in either group indicating that there was no alteration in the 5-HT affinity for its receptors in lactating rats. In pregnant rats, 5-HT-induced gastric responses were significantly (P < 0.05) lower than those of non-lactating rats. Pre-treatment of non-lactating rats with haloperidol (which increases plasma prolactin levels) enchanced the gastric contractile response to 5-HT (P < 0.05). On the other hand bromocriptine administration (which lowers plasma prolactin levels) in lactating or immature rats, decreased the contractile response to 5-HT significantly, while bath application of bromocriptine (0.1 μM) had no effect. Incubation of fundus strips in physiological solution containing prolactin (10 μg ml^-1) for 24 h decreased the 5-HT evoked contractions, but not the ACh evoked responses. The results in this study indicate that prolactin modulates the intrinsic contractile activity of the gastric smooth muscles to 5-HT.     

Endogenous glutamatergic synaptic activity elicits acetylcholine release from rat cultured septal cells

We tested the characteristics of acetylcholine (ACh) release from cultured rat septal cells. The spontaneous release was inhibited by treatment with tetrodotoxin (TTX) and omega-conotoxin (GVIA), indicating that the release was elicited by synaptic activity. The release was also inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor blocker, in both the absence and presence of nerve growth factor (NGF), suggesting that endogenously released glutamate produced the ACh release by stimulating AMPA receptors.This is the first report of detection of the release of ACh by endogenous spontaneous synaptic activity conducted by glutamate AMPA receptor activation in cultured septal cells. This in vitro experimental system is useful for the study of cholinergic functions.     

Acetyl-L-carnitine enhances acetylcholine release in the striatum and hippocampus of awake freely moving rats

The effect of acetyl-L-carnitine (ALC) on the spontaneous release of acetylcholine (ACh) in the striatum and hippocampus of freely moving rats was investigated using brain microdialysis coupled with HPLC-electrochemical detection. Systemic administration of ALC, in a dose-dependent manner, stimulated ACh release in both areas, while the D-enantiomer was substantially ineffective. The effect of ALC was strongly Ca2+ dependent and tetrodotoxin (TTX) sensitive. These features of an exocytotic and impulse flow-dependent mechanism suggest that the increase in ACh release is the result of ALC activation of a physiological mechanism in cholinergic neurons.     

Mechanisms underlying pre- and postjunctional effects of neuropeptide Y in sympathetic vascular control

The effects of porcine neuropeptide Y (NPY) regarding sympathetic vascular control were studied in vitro on isolated rat blood vessels. The 10(-9)M NPY enhanced (about two-fold) the contractile responses to transmural nerve stimulation (TNS), noradrenaline (NA) and adrenaline (about two-fold) in the femoral artery. Higher concentrations of NPY (greater than 10(-8)M) caused an adrenoceptor-resistant contraction per se. The TNS-evoked [3H]NA efflux was significantly reduced by NPY in a concentration-dependent manner (threshold 10(-9)M). The calcium antagonist, nifedipine, abolished the contractile effects of NPY and the NPY-induced enhancement of NA contractions but did not influence the prejunctional inhibition of [3H]NA release. Receptor-binding studies showed that the ratio of alpha 1-to alpha 2-adrenoceptors in the femoral artery was 30:1. The NPY did not cause any detectable change in the number of alpha 1-or alpha 2-adrenoceptor binding sites or in the affinity of alpha 2-binding sites, as revealed by prazosin- and clonidine-binding, respectively. The NPY also inhibited the TNS-evoked [3H]NA release (by 42-86%) in the superior mesenteric and basilar arteries and in femoral and portal veins. The NPY still depressed TNS-evoked [3H]NA secretion from the portal vein in the presence of phentolamine. The NPY caused a clear-cut contraction in the basilar artery, increased the contractile force of spontaneous contractions in the portal vein, while only weak responses were observed in the superior mesenteric artery and femoral vein. The NA-induced contraction was markedly enhanced by NPY in the superior mesenteric artery, only slightly enhanced in the portal vein and uninfluenced in the femoral vein. In conclusion, in all blood vessels tested, NPY depresses the TNS-evoked [3H]NA secretion via a nifedipine-resistant action. Furthermore, NPY exerts a variable, Ca2+-dependent vasoconstrictor effect and enhancement of NA and TNS contractions.     

Insulin-like growth factor 1 stimulates the release of acetylcholine from rat cortical slices

The effect of somatomedin, or insulin-like growth factors (IGF-1 and IGF-2), on the basal and potassium induced release of [3H]acetylcholine ([3H]Ach) from rat cortical slices, previously preincubated with [3H]choline ([3H]Ch), was studied in vitro. IGF-1 (1.4 x 10(-9) to 1.4 x 10(-8) M) had no effect on the basal release of [3H]ACh, while IGF-1 (1.4 x 10(-9) to 4.3 x 10(-8) M) increased the potassium induced release of [3H]ACh from rat brain slices in a concentration-dependent manner. However IGF-2 (1.4 x 10(-8) M) had no effect. Insulin (1.8 x 10(-8) to 5.3 x 10(-8) M), similarly, did not have any influence on the release of [3H]ACh, demonstrating that the facilitatory effect of IGF-1 on [3H]ACh release is not mediated via insulin receptors. This report demonstrates for the first time that IGF-1 has an effect on neurotransmission in the adult brain.     

Different effects of neuropeptide Y on electrically induced contractions in the longitudinal and circular smooth muscle layers of the female rabbit urethra

The effects of neuropeptide Y (NPY) on preparations of isolated longitudinal and circular smooth muscle from rabbit urethra were studied. In both types of muscle, electrically induced contractions and relaxations could be abolished by tetrodotoxin, (TTX). In the longitudinal muscle preparations the contraction was slightly reduced by prazosin, but markedly reduced by scopolamine and NPY. The NPY effect was not influenced by pretreatment with rauwolscine. Pretreatment with NPY had no effect on contractions induced by noradrenaline (NA) or carbachol and the peptide did not relax preparations contracted by these agents. In circular muscle an initial, fast response, not sensitive to prazosin or scopolamine was occasionally observed following electrical stimulation. A slow contraction component was regularly seen; this response was abolished by prazosin. Neuropeptide Y did not influence any of these responses. The preparations were concentration-dependently contracted by NA, whereas carbachol had no effect. Pretreatment with NPY did not affect contractions induced by NA, nor did the peptide relax NA-contracted preparations. In neither longitudinal nor circular muscle strips did NPY affect the electrically induced TTX sensitive relaxation of NA-contracted preparations. The results suggest that in the rabbit urethra NPY reduces contractions in the longitudinal muscle layer by selectively inhibiting the release of acetylcholine from cholinergic nerves. Neuropeptide Y did not appear to have any significant postjunctional effects nor to interfere with the release, or effects of NA or other transmitter agents. The physiological importance of the urethral effects of NPY remains to be established.