Blocking by 2,4,6-triaminopyrimidine of increased tight junction permeability induced by acetylcholine in the pancreas

1. The permeability of the paracellular pathway in the isolated rabbit pancreas has been studied with the aid of 2,4,6-triaminopyrimidine. 2. Addition of 2,4,6-triaminopyrimidine (1--10 mM) to the bathing medium has no effect on the rate of fluid secretion or on protein, Na+, K+, Ca2+ and sucrose concentrations in the secreted fluid. 3. When 1 x 10(-5) M carbachol is also added to the 2,4,6-triaminopyrimidine-containing bathing medium, there is a marked reduction in the increase of the paracellular permeability for sucrose and Ca2+ found upon addition of carbachol alone. The enzyme secretion, induced by carbachol, is not affected. 4. The minimal concentration of 2,4,6-triaminopyrimidine in the bathing medium required to reach its maximal effect on the paracellular permeability is approx. 0.55 mM at pH 7.4. 5. The effect of 2,4,6-triaminopyrimidine on the paracellular permeability after carbachol stimulation is also present when 2,4,6-triaminopyrimidine is added 5 min after the addition of 1 x 10(-5) M carbachol. 6. 2,4,6-Triaminopyrimidine has no effect on the increases in enzyme secretion and sucrose permeability caused by 1 x 10(-8) pancreozymin C octapeptide. 7. 2,4,6-Triaminopyrimidine appears in the secreted fluid at a concentration of 50% of that in the bathing medium. Upon addition of 1 x 10(5) M carbachol this concentration increases up to 80%. 8. These results indicate that: (a) the increased paracellular permeability upon stimulation with carbachol is not caused by the enzyme secretion as such and (b) addition of 2,4,6-triaminopyrimidine prevents the carbachol-induced increase in permeability of a channel in the tight junction complex.     

Pharmacology of the isolated foregut of the locust Schistocerca gregaria—IV. Characterization of a muscarinic acetylcholine receptor

1. Acetylcholine (ACh; 10⁻⁶ M—7 × 10⁻⁵ M), in the presence of neostigmine (10⁻⁵ M), caused contraction of the locust isolated foregut.2. The effect of ACh was mimicked by carbachol, propionylcholine (PCh), butyrylcholine (BCh), nicotine, SD35651, oxotremorine and muscarine.3. The contractions caused by ACh, BCh and carbachol were abolished by atropine (10⁻⁶M) and reduced by d-tubocurarine (10⁻⁵ M) and decamethonium (5 × 10⁻⁵ M). Hexamethonium and α-bungaro-toxin had no effect on contractions caused by the above agonists.4. None of the antagonists used in this study blocked the contractile effects of nicotine.5. It is concluded that the foregut contains a neuronal nicotinic receptor which, when activated, causes release of ACh which acts on a neuromuscular muscarinic receptor.     

Possible functional role of substance P on the mammalian motor nerve terminals

The effect of substance P (sP) on mammalian skeletal myoneural transmission was studied employing innervated and denervated isolated rat diaphragm preparations. sP at a concentration of 3.7 nM facilitated the indirect twitch responses of the rat diaphragm and antagonised the paralytic effect of d-tubocurarine (d-Tc). sP failed to affect the direct twitch responses as well as the contractures induced by acetylcholine (ACh) and potassium chloride (KCl) in the denervated diaphragm. The amount of ACh released into the bathing medium in response to tetanic stimulation of the phrenic nerve was doubled in presence of sP. The study illustrates a presynaptic facilitatory involvement of sP on mammalian myoneural transmission.     

Monosodium glutamate impairs the contraction of uterine visceral smooth muscle ex vivo of rat through augmentation of acetylcholine and nitric oxide signaling pathways

The aim of the study was to examine the toxic effects of Monosodium glutamate (MSG), an extensively used food additive, on the contraction of uterine visceral smooth muscle (UVSM) in rat and to elucidate the probable neurocrine mechanism involved in it. MSG produced significant potentiation of the force and inhibition of frequency of uterus recorded ex vivo in chronic MSG exposure and in single dose acute experiments. MSG also produced significant potentiation of force of acetylcholine induced contraction and no alterations in atropine induced contraction of uterus. Further, MSG produced significant increase in force and frequency of contraction of neostigmine incubated uterus. We have found significant potentiation of the post pause force of contraction of uterus when MSG was applied in adrenaline incubated uterus. MSG also produced significant decrease in frequency of contraction of sodium nitroprusside incubated uterus; increase in frequency of N-ω-Nitro-l-Arginine Methyl Ester incubated uterus and no significant changes in frequency of contraction of methylene blue incubated uterus. These results indicate that MSG potentiates the force of contraction of UVSM predominantly by augmenting the activity of cholinergic intrinsic efferents and inhibits the frequency of contraction probably by augmenting the activity of nitrergic efferents. In conclusion, MSG potentiates the force and inhibits the frequency of contraction of UVSM, and the MSG induced effect is probably mediated through the augmentation of acetylcholine and nitric oxide signaling pathways.     

GABA modulation of cholinergic transmission in rat oviduct

The effects of electrical stimulation, γ-aminobutyric acid (GABA), acetylcholine (ACh), norepinephrine (NE), 5-hydroxytryptamine (5-HT), GABA agonists and bicuculline were studied on spontaneous movements of isolated rat oviduct. The tissue did not respond to electrical stimulation or to GABA, NE and 5-HT when added to the incubation medium. ACh produced contractions related to its concentration which were maximal at the diestrous-1 phase when GABA caused a 20% rise in the ACh contraction. This effect was mimicked by GABA agonists whereas it was suppressed by bicuculline. β-Estradiol benzoate (EB) increased ACh contractions in diestrous-1 and in the late proestrous phases. GABA did not modify the EB effect. Progesterone did not modify ACh contractions in any of the studied phases. These findings suggest a possible modulatory role for GABA on ACh responses in the isolated rat oviduct.     

Spasmolytic activity of essential oil and various extracts of Ferula gummosa Boiss. on ileum contractions

Traditional herbal medicines such as Ferula gummosa Boiss. have been used for treatment of intestinal disorders in Iran. To date no pharmacological evidence for their effectiveness has been reported. The aim of this study was to examine the relaxant effect of essential oil, hydro-alcoholic, etheric, petrolic and methanolic extracts of Ferula gummosa and two of its components, alpha-pinene and beta-pinene, on isolated rat ileum contractions induced by KCl and acetylcholine (ACh). Ferula gummosa essential oil (FGEO) and hydro-alcoholic, etheric, petrolic and methanolic extracts all inhibited the response to 80 mM KCl in a concentration-dependent manner and attenuated the maximum attainable response of the ACh concentration-response curve. Although the effect of etheric extract on ACh contractions was less than that of petrolic extract, the overall order of effectiveness on the weight basis was the etheric, petrolic, methanolic, and hydro-alcoholic extracts, and the essential oil, respectively. A mixture of etheric and petrolic extracts together had a similar effect on KCl response to etheric extract was used alone. Alpha-pinene and beta-pinene both exhibited inhibitory effect on the contraction of rat ileum, but the inhibitory effect of beta-pinene on KCl contraction was more pronounced. The inhibitory effect of a mixture of these two compounds was, however, less than the sum of their separate effects. When a mixture of alpha-pinene and beta-pinene together were examined on the ileum, without presence of the spasmogen, they initially caused contraction of the tissue, while neither of them used alone caused a noticeable contraction of the ileum. This study shows that Ferula gummosa essential oil and its various extracts are relaxant of rat isolated ileum and that at least part of their inhibitory effect is due to alpha-pinene and beta-pinene components. As the inhibition of contractile over-activity of the ileum is the basis of the treatment of some gastero-intestinal disorders such as diarrhea, Ferula gummosa may have clinical benefits for treatment of this condition.     

Effect of Clostridium perfringens Epsilon Toxin on Rat Isolated Aorta

The effect of Clostridium perfringens epsilon toxin on rat isolated aorta was investigated. The toxin caused contraction of the isolated aorta in a dose-dependent manner. The toxin induced no contraction of the isolated aorta in low-Na medium and of the tissue stored at 4 C for 7 days. However, tetrodotoxin (TTX) had no effect on the toxin-induced contraction. The toxin-induced contraction was significantly inhibited by phentolamine and prazosine, but did not by atropine, mecamylamine, chlorpheniramine and methysergide. These data suggest that the toxin-caused contraction is mediated through nervous system in rat isolated aorta.     

Innervation of holothurian body wall muscle: inhibitory effects and localization of 5-HT

We investigated innervation to body wall muscles as well as distribution of 5-HT (serotonin) and its effects on longitudinal muscles of body wall (LMBW) of the sea cucumber Apostichopus japonicus. With serial sections we found neural branches and fibers extending from hyponeural part of radial nerve towards LMBW and circular muscles of body wall. With the aqueous aldehyde (Faglu) method yellow fluorescence indicating indolamines was observed in LMBW and in the mesentery connecting LMBW to the body wall. With indirect immunohistochemistry 5-HT-like immunoreactivity was observed in LMBW and in mesentery. These results strongly suggested that both LMBW and mesentery contained 5-HT. The effects of monoamine neurotransmitters were studied in LMBW. Putative neurotransmitters tested were 5-HT, adrenaline, noradrenaline, dopamine, and DOPA at the concentration of 10(-6) M. The application of 5-HT caused no contraction or relaxation, but it inhibited the contraction induced by 10(-6)-10(-5) M acetylcholine (ACh). Catecholamines were ineffective by themselves and had no effects on the contraction induced by ACh. The present histological, histochemical, and pharmacological studies strongly suggested that holothurian LMBW was innervated by inhibitory serotonergic neurons of the hyponeural nervous system.     

The lack of influence of some neuropeptides present in the posterior pituitary lobe on the frequency of spontaneous contraction of the isolated heart auricle.

Investigations have shown the presence of a cardiodepressant factor in the fluid incubating the posterior pituitary lobe "in situ", which decreased contraction frequency of the isolated heart auricle (Acta Physiol. Pol., 1984, 35: 460-468). The influence on the spontaneous contraction frequency of the isolated heart auricle of the following synthetic neuropeptides was determined: substance P, leu-enkephalin, met-enkephalin, angiotensin II, arg-vasopressin, oxytocin, delta sleep-inducing peptide and atrial natriuretic factor. It was found that the investigated neuropeptides had no effect on the contraction frequency of the isolated auricle of the heart right atrium of two-day-old rat in a concentration from 2.1 x 10(-7) to 1 x 10(-3) mol/l in the bathing medium and it was concluded that their biological properties differ from the cardiodepressant factor.     

Simulated microgravity upregulates an endothelial vasoconstrictor prostaglandin.

Endothelial nitric oxide contributes to the vascular hyporesponsiveness to norepinephrine (NE) observed in carotid arteries from rats exposed to simulated microgravity. The goal of the present study was to determine whether a cyclooxygenase product of arachidonic acid also influences vascular responsiveness in this setting. Microgravity was simulated in rats by hindlimb unweighting (HU). After 20 days of HU, carotid arteries were isolated from control and HU-treated rats, and vascular rings were mounted in tissue baths for the measurement of isometric contraction. Two cyclooxygenase inhibitors, indomethacin and ibuprofen, and the selective thromboxane A(2) prostanoid-receptor antagonist, SQ-29548, had no effect on the contraction to NE in control vessels but markedly reduced contraction to NE in HU vessels. When the endothelium was removed, indomethacin no longer had any effect on the NE-induced contraction in HU vessels. In endothelium-intact vessels in the presence of indomethacin, the addition of the nitric oxide synthase inhibitor, N(G)-L-nitro-arginine methyl ester, to the medium bathing HU vessels increased the contraction to NE to the level of that of the control vessels. These results indicate that HU treatment induced two endothelial changes in carotid artery that opposed each other. Nitric oxide activity was increased and was responsible for the vascular hyporesponsiveness to NE. The activity of a vasoconstrictor prostaglandin was also increased, and attenuated the vasodilating effect of nitric oxide.     

The Role of Chloride in Acetylcholine Metabolism

Abstract: The chloride dependence of acetylcholine (ACh) synthesis and release and of choline uptake was studied in synaptosomal preparations from rat brain. The substitution of propionate for chloride, in the presence of 35 m m -potassium, lowered the ACh content of the synaptosomes. However, in the presence of 5 m m -potassium, the ACh level in synaptosomes was reduced, but significantly less so. Propionate had no effect on choline acetyltransferase (EC 2.3.1.6) activity when measured in a standard chloride-containing medium. In the presence of propionate, the spontaneous release of ACh was unchanged, but potassium-stimulated release of ACh was markedly reduced as compared with a chloride-containing medium. The synthesis of ACh, as measured by the net increase in the amount of ACh in the synaptosomes and that released to the medium, was reduced with propionate at 5 m m -potassium and was totally inhibited when the potassium concentration was increased to 35 m m . Choline uptake studies revealed that with propionate only a low-affinity component of the choline transport system existed. Further, the V _max was markedly reduced when the potassium concentration was increased to 35 m m . The results suggest that under certain conditions choline transported by a low-affinity system might provide a substantial source of choline for ACh synthesis.     

Branchial musculature of a venerid clam: pharmacology,distribution, and innervation

This study was meant to analyze the neural control of the branchial muscles of the clam Mercenaria mercenaria. Gills isolated from the animal contract in response to 5-hydroxytryptamine (5HT), dopamine (DA), and acetylcholine (ACh); but the ACh contraction occurred only if the gills had been pretreated with the cholinesterase inhibitor eserine. The 5HT antagonists cyproheptadine and mianserin blocked the contractile effects of all of the agonists. However, gills exposed to the 5HT antagonists and eserine relaxed in response to ACh. The DA antagonist SCH-83566 inhibited the effects of DA, but had no effect on contractions induced by 5HT and ACh. The ACh antagonist hexamethonium inhibited both the excitatory and inhibitory effects of ACh, but had no effect on contractions induced by 5HT and DA. 5HT and DA in gill tissue were visualized by using immunohistochemistry. Within each gill filament are dorsoventral neurons running adjacent to the epithelium and containing immunoreactive 5HT and DA. A complex network of 5HT-positive fibers is associated with the septa, blood vessels, and muscles, whereas DA-positive fibers are restricted to the septa. We propose that 5HT is the excitatory transmitter to the gill muscles, and that DA and ACh exert their excitatory effects by stimulating 5HT motor nerves. ACh may also be an inhibitory transmitter of the muscles.     

Isolation, partial purification and pharmacodynamics of a slow contractile substance in the venom sac extract of the wasp Vespa cincta Fabr

The venom of V. cincta contains acetylcholine (ACh), histamine and 5-hydroxytryptamine (5-HT). Blockers of these agonists did not block completely the hypotensive and smooth muscle contractile activity of venom. On smooth muscle, there was a residual slow contraction. The active substance which produced this slow contraction was separated by solvent extraction, gel filtration and TLC. The purified material (which has been provisionally designated "Vecikinin") lowered cat, rat and guinea pig blood pressure, increased amplitude of cardiac contraction, and increased capillary permeability. Vecikinin contracted several smooth muscle preparations (rat uterus, rat ascending colon, guinea pig ileum, guinea pig colon and rat ileum), while relaxing rat duodenum. Its contractile activity was not lost on boiling, but acid or alkali-boiling reduced its contractile activity. It was inactivated on incubation with chymotrypsin and carboxypeptidase but not with trypsin, pepsin or leucine aminopeptidase. It is a peptide, appears to be of low molecular weight, and could be distinguished from substance P, angiotensin, bradykinin and hornet or wasp kinin.     

Alpha-adrenergic receptor blocking effect of Cleistanthus collinus (Roxb.) Benth. and Hook f. leaf extract on guinea pig isolated smooth muscle preparations

Aqueous extract of C. collinus leaves inhibited norepinephrine induced contraction in guinea pig vas deferens and aortic strip in a dose-dependent manner. Inhibition of acetylcholine induced contraction in ileum was dose independent. C. collinus extract per se had no effect on isolated guinea pig vas deferens and aortic strip, but inhibited norepinephrine induced contraction in a dose-dependent manner probably by its antagonist action on alpha-adrenergic receptor. It had inconsistent effect on guinea pig ileum in vitro preparation.     

In vitro myometrial inhibition by the partitioned aqueous fraction of Anthocleista djalonensis leaves

This study investigated the effect on the uterus of the aqueous fraction of the partitioned methanol crude extract of the leaves of Anthocleista djalonensis (AD) and the possible mechanism of AD activity. AD inhibited the concentration-response curves induced by oxytocin and CaCl2 on the rat uterus in vitro and significantly reduced the EC50 in a concentration-dependent manner (p?< 0.05). A similar effect was observed with salbutamol and verapamil on the concentration-response curves obtained for oxytocin and CaCl2. The inhibitory effect of AD was not attenuated in the presence of propranolol. AD, salbutamol, and verapamil also produced a concentration-dependent relaxation on K+-induced sustained uterine contraction. In Ca2+-free medium, AD and salbutamol similarly inhibited oxytocin-induced contraction, but verapamil failed to produce this effect. The present results suggest that AD, being a mixture of phytochemicals, probably exerts inhibitory activity on in vitro uterine contractions of the nonpregnant, diethylstilboestrol-treated rat by multiple mechanisms that do not involve interaction with β-adrenergic receptors and do not solely depend on inhibition of calcium influx.     

Effect of thyrotropin-releasing hormone on rat myometrium

The effect of TRH in vitro was observed on electromyograms and isometric tension changes in the uterine horn isolated from the rat. TRH induced transient prolongation of the duration of spike bursts in the electromyogram and an increased tension in contraction of diestrous uterine horns. No distinct response to TRH was elicited in preparations from rats during other estrous stages. TRH produced a contraction associated with a burst of spike potentials in the quiescent horn from the estrogen-primed ovariectomized rat. Priming with progesterone was not a prerequisite for responsiveness to TRH. In a medium with a high Ca concentration, diestrous uteri were quiescent but a transient contraction associated with a burst of spike potentials was induced by TRH. In a Ca-free medium, TRH failed to elicit any response in the diestrous uterus but acetylcholine induced a contraction without associated spike potentials. It appears that TRH stimulates Ca-influx into the uterine muscle in which responsiveness is dependent on estrogen priming.     

Increase of cardiodepressant activity in medium incubating the posterior pituitary lobe in situ during vagal nerve stimulation in rat.

Previous studies have indicated that there is a cardiodepressant factor in the medium incubating the posterior pituitary lobe in situ. The cardiodepressant activity of the medium incubating the posterior pituitary lobe before and during stimulation of the vagus nerves was tested on isolated auricles of the right heart atrium of a two-day-old rat. It was found that the medium incubating the posterior pituitary lobe collected before stimulation decreased the contraction rate of the auricle by 34%, while that collected during the intermittent stimulation of the central ends of the cut vagus nerves caused a decrease of the auricle contractions frequency by 52%. The addition of cholinergic, serotoninergic, histaminergic receptor blockers or prostaglandin synthetase into Ringer-Lock's solution bathing the auricle has no effect on the changes of the contraction rate caused by the incubation medium.     

h-ANF does not play a role in the regulation of myocardial force of contraction

In the present study, h-ANF failed to reduce myocardial force of contraction alone and in the presence of isoprenaline. This lack of effect was observed in electrically driven, isolated human papillary muscle strips and rat papillary muscles. In contrast, adenosine and carbachol exerted antiadrenergic actions in rat and human myocardium. It is concluded that h-ANF obviously does not play a role in the regulation of human and rat myocardial force of contraction, whereas adenosine and carbachol are capable to produce antiadrenergic effects in the human and rat myocardium.     

Effect of the ionic environment and of various neurotransmitters on spontaneous motility in snail intestine (author's transl)]

A spontaneous rhythmic motility of the isolated intestine in snail, Chryptomphalus hortensis, has been registered at 30 degrees C with the aid of an electronic transductor and an appropriate amplification. These slow regular movements are affected by ionic composition changes in the suspension medium. Na+, K+ and Ca++ ions prove to be important in this motility, and the addition of Ba++ markedly stimulates it. ACh produces hypermotility from 1.8 X 10(-11) g/ml. Its effect decreases in the presence of atropine and increases in that of pyridostigmine. The intestine is sensitive to 5-HT from 10(-10) g/ml, which stimulates its activity. The effect of histamine is weak. Low concentrations of adrenaline tend to increase the amplitude, whereas concentrations from 10(-5) g/ml onward produce a cease of motility in relaxation.     

Pharmacological reactivity in cricetus aureus uterus (author's transl)]

The motility of the isolated Cricetus auratus uterus was studied and compared to that of other species. Oxitocyn, epinephrine, norepinephrine, histamine, 5-hydroxy-tryptamine and acetylcholine were used as spasmogen agents. There was not contractil response with epinephrine or nor-epinephrine. Histamine reduced basal tonus. There was contraction with acetylcholine, oxytocin and 5-hydroxy-tryptamine. Cricetus auratus uterus appeared more sensitive when the contraction was registered by the isometric method. No taquifilaxy was produced by 5-hydroxy-tryptamine, as opposed to such effect in rat uterus. The Cricetus auratus uterus has, therefore, shown similar reactivity to that of rat, but different from rabbit and guinea-pig.