Decreased muscarinic binding sites in small intestine from mice treated with neostigmine

It has been reported by several authors that animals given repeated sublethal doses of an organophosphate, acetylcholinesterase (AChE) inhibitor, develop tolerance to its toxicity. This phenomenon seems to be due, at least partially, to a decrease of central and peripheral cholinergic receptors. In the present study, we report that a decrease of muscarinic receptors, as measured by [³H]-quinuclidinyl benzilate (³H-QNB) binding, occurs in the small intestine of mice treated with the carbamate, AChE inhibitor, neostigmine. Male mice were given neostigmine in the drinking water at daily increasing concentrations (from 20 to 1000 ppm). Methylatropine (20mg/kg, i.p.) was administered twice a day for the same period to two groups of control and neostigmine-treated animals. AChE activity was inhibited 60–70% in small intestine and diaphragm and [³H]-QNB binding was significantly reduced in the small intestine of neostigmine-treated mice; both the number of receptors and the affinity were lower than control. This decrease was not present in the tissue of mice given methylatropine together with neostigmine. Administration of methylatropine alone caused a significant increase of [³H]-QNB binding in the small intestine.     

Analysis of the role of adrenergic receptors in the regulation of small intestine motor activity in sheep

In sheep with chronic fistulae of the small intestine and rumen the participation of alpha- and beta-adrenergic receptors in the regulation of the motor activity of the small intestine was studied by the method of pharmacological analysis. The movements of the fistulated parts of the alimentary tract were recorded by the balloon method. Slow intravenous infusion of isoprenaline inhibited the contractions of the small intestine. This inhibitory effect of isoprenaline was abolished by propranolol. Intravenous phenylephrine inhibited the motor activity of this intestinal part as well. The effect of phenylephrine was abolished by pretreatment with dihydroergotamine. In the small intestine of sheep stimulation of the alpha and beta adrenergic receptors decrease the motor activity of intestine.     

PKC-epsilon translocation in enteric neurons and interstitial cells of Cajal in response to muscarinic stimulation

Interstitial cells of Cajal in the deep muscular plexus (ICC-DMP) of the small intestine express excitatory neurotransmitter receptors. We tested whether ICC-DMP are functionally innervated by cholinergic neurons in the murine intestine. Muscles were stimulated by intrinsic nerves and ACh and processed for immunohistochemistry to determine these effects on PKC-epsilon activation. Under control conditions, PKC-epsilon-like immunoreactivy (PKC-epsilon-LI) was only observed in myenteric neurons within the tunica muscularis. Electrical field stimulation or ACh caused translocation of neural PKC-epsilon-LI from the cytosol to a peripheral compartment. After stimulation, PKC-epsilon-LI was found in spindle-shaped cells in the DMP. These cells were identified as ICC-DMP by Kit-LI and vimentin-LI. PKC-epsilon-LI in ICC-DMP and translocation of PKC epsilon-LI in neurons were blocked by tetrodotoxin or atropine, suggesting that these responses were due to activation of muscarinic receptors. Western blots also confirmed translocation of PKC-epsilon-LI. In conclusion, PKC-epsilon translocation is linked to muscarinic receptor activation in ICC-DMP and a subpopulation of myenteric neurons. These studies demonstrate that ICC-DMP are functionally innervated by excitatory motoneurons.     

Tachykinin activation of muscarinic inhibition in canine small intestine is SPP in nature

Substance P when injected intraarterially into the small intestine of the anaesthetized dog during phasic activity produces three concentration dependent responses of the circular muscle. At lowest doses (approximately 10(-12) moles) inhibition occurs via release of acetylcholine to a muscarinic auto-receptor. At slightly higher doses (10(-10) moles) inhibition is preceeded by excitation via release of acetylcholine to muscarinic receptors on the smooth muscle. At still higher doses (10(-9) moles) substance P excites the smooth muscle directly. The present study demonstrates that other members of the tachykinin family also produce inhibition in vivo. The potency sequence was found to be physalaemin greater than or equal to substance P = neuromedin K greater than kassinin greater than alpha neurokinin = eledoisin. Such a sequence suggests that substance P is a natural stimulant of this pathway and that the receptor is SPP-like. The C-terminal fragment, substance P8-11, was a weak agonist at this receptor, while substance P1-9 was ineffective.     

Changes in muscarinic acetylcholine receptors of mice by chronic administrations of diisopropylfluorophosphate and papaverine.

Chronic administration of diisopropylflourophosphate (DFP) to mice induced decrease in maximal contraction and increase in the ED₅₀ of ileum to the muscarinic agonist oxotremorine. These changes were accompanied by decrease in muscarinic receptors. Papaverine prevented both the changes in contraction and the decrease in muscarinic receptors by DFP. The quantitative relationship between the decrease in receptors and contraction is discussed on the basis of the hypothesis of spare receptors.     

Enhanced muscarinic M1 receptor gene expression in the corpus striatum of streptozotocin-induced diabetic rats

Acetylcholine (ACh), the first neurotransmitter to be identified, regulate the activities of central and peripheral functions through interactions with muscarinic receptors. Changes in muscarinic acetylcholine receptor (mAChR) have been implicated in the pathophysiology of many major diseases of the central nervous system (CNS). Previous reports from our laboratory on streptozotocin (STZ) induced diabetic rats showed down regulation of muscarinic M1 receptors in the brainstem, hypothalamus, cerebral cortex and pancreatic islets. In this study, we have investigated the changes of acetylcholine esterase (AChE) enzyme activity, total muscarinic and muscarinic M1 receptor binding and gene expression in the corpus striatum of STZ – diabetic rats and the insulin treated diabetic rats. The striatum, a neuronal nucleus intimately involved in motor behaviour, is one of the brain regions with the highest acetylcholine content. ACh has complex and clinically important actions in the striatum that are mediated predominantly by muscarinic receptors. We observed that insulin treatment brought back the decreased maximal velocity (V_max) of acetylcholine esterase in the corpus striatum during diabetes to near control state. In diabetic rats there was a decrease in maximal number (B_max) and affinity (K_d) of total muscarinic receptors whereas muscarinic M1 receptors were increased with decrease in affinity in diabetic rats. We observed that, in all cases, the binding parameters were reversed to near control by the treatment of diabetic rats with insulin. Real-time PCR experiment confirmed the increase in muscarinic M1 receptor gene expression and a similar reversal with insulin treatment. These results suggest the diabetes-induced changes of the cholinergic activity in the corpus striatum and the regulatory role of insulin on binding parameters and gene expression of total and muscarinic M1 receptors.     

Galanin: an inhibitory neural peptide of the canine small intestine

Galanin injected intraarterially during phasic activity of the canine small intestine in vivo produced inhibition. Fifty percent inhibition occurred at 1.5 +/- 0.5 X 10(-10) mols lasting for 0.7 min. The inhibitory response was not decreased by treatment with atropine, hexamethonium, yohimbine or naloxone, suggesting that muscarinic, nicotinic, alpha 2 adrenergic or opiate receptors were not being stimulated. Since tetrodotoxin blockade of nerves did not reduce the response and galanin at 10(-10) mols was able to eliminate the smooth muscle response to intraarterial acetylcholine, we suggest that galanin acts to inhibit smooth muscle directly. Galanin 10(-9) M added to the muscle bath also inhibited phasic activity of the canine ileum circular muscle in vitro in the presence of tetrodotoxin. These results suggest that the neural peptide galanin may be a non-adrenergic, non-cholinergic, non-opioid neurotransmitter in the canine small intestine.     

Characterization of the muscarinic and serotoninergic receptors of the intestine of the rainbow trout (Salmo gairdneri)

The ability of carbachol and 5-hydroxytryptamine (5-HT) to contract isolated segments of rainbow trout intestine in a concentration-dependent manner indicates the presence of muscarinic and serotoninergic receptors in this tissue. The activity of these agonists appears to be directly on the smooth muscle, since ganglionic blockers and inhibitors of neurotransmission did not inhibit contractions. The carbachol-induced contractions were selectively inhibited by atropine and (+-)-3-quinuclidinyl xanthene-9-carboxylate hemioxalate hydrate, an M-2 muscarinic receptor antagonist. However, the inhibition was not competitive. McN-A-343, an M-1 muscarinic agonist had no effect on intrinsic tone. The 5-HT-induced contractions were selectively inhibited by methysergide and the 5-HT2 receptor blockers, ketanserin and 1-(1-naphthyl)piperazine. Again, the inhibition by these agents was not competitive. 5-HT1 and 5-HT3 receptor antagonists did not inhibit contractions. The results thus suggest that the smooth muscle of the rainbow trout intestine contains M-2 muscarinic and 5-HT2 receptors.     

Effects of electrical stimulation of the parasympathetic nerve on the levels of cholinergic muscarinic and beta-adrenergic receptors and cyclic nucleotides in rat salivary glands

Density of muscarinic receptors of rat parotid gland, although unchanged after 5 or 10 min of stimulation of the parasympathetic nerve to the gland, showed a decrease of 23% following a 15-min period of stimulation. After 30 min the decrease was 19% but by 60 min density of receptors returned to within 5% of receptor density of the unstimulated gland; there was virtually no change in density of beta adrenoceptors at any time during the 60 min of stimulation. Markedly elevated (30-fold increase) levels of cyclic GMP appeared within 5 min after initiation of nerve stimulation and remained at this level at 10 min, but dropped from 90 to 46 pmol/mg total protein by 15 min, the time at which a decrease in muscarinic receptors first was evident. GMP levels continued to decrease but were still four times basal levels after 60 min of stimulation and did not return to normal concentration until 120 min. Cyclic AMP was generally unchanged. These changes in muscarinic receptors and cyclic GMP are apparently closely related to the kind of neural stimulation, unlike the condition when stimulation of the sympathetic nerve was employed.     

The effects of ovarian hormones on beta-adrenergic and muscarinic receptors in rat heart

The in vitro and in vivo effects of estrogen and progesterone on muscarinic and beta-adrenergic receptors of cardiac tissue were studied in ovariectomized (OVX) rats. The binding assay for muscarinic receptors was performed under a nonequilibrium condition; whereas the binding assay for beta-adrenergic receptors, under an equilibrium condition. Estrogenic compounds and progesterone were found to have no effect on the binding of the radioligand, [3H]-dihydroalprenolol, to beta-adrenergic receptors in vitro. However, progestins but not estrogenic compounds inhibited the binding of the radioligand, [3H]-quinuclidinyl benzilate, to muscarinic receptors in vitro, with progesterone as the most potent inhibitor (IC50 = 37 microM, apparent Ki = 13 microM). Progesterone was found to decrease the apparent affinity of muscarinic receptors for [3H](-)QNB in vitro. Daily treatment of OVX rats with estradiol benzoate (4 micrograms) or progesterone (2.5 mg) for 4 days had no effect on the muscarinic or beta-adrenergic receptors with respect to the binding affinity and receptor density. However, administrations of these hormones together for 4 days caused an increase in the receptor density of muscarinic receptors without a significant effect on their apparent binding affinity; also these hormones induced a decrease in the binding affinity and an increase in the receptor density of beta-adrenergic receptors. The results of this study demonstrate that progestins are capable of interacting with the cardiac muscarinic receptors in vitro, and indicate that estrogen and progesterone have a synergistic effect to increase the receptor densities of muscarinic and beta-adrenergic receptors as well as to cause a decrease in the binding affinity of beta-adrenergic receptors in vivo.     

The effects of ethylenediamine in the rat small intestine: a powerful relaxant of the muscularis.

The pharmacology of ethylenediamine (EDA) actions in the rat small intestine was examined using isolated gut-bath preparations of proximal segments of the duodenum, jejunum, and ileum. EDA evoked concentration-dependent tetrodotoxin-insensitive relaxations of the intestine, evidently by direct action on the muscularis. Such actions were simultaneous on the longitudinal and circular muscle layers. Investigation of EDA actions on the circular muscle showed that EDA actions were unrelated to any intrinsic GABAergic mechanisms. Moreover, EDA interacted with muscle sites distinct from ATP, histamine, bradykinin, muscarinic, and adrenergic receptors. The ability of EDA to relax the intestinal musculature was generally greater than the smooth muscle relaxant papaverine and substantially better than nicotinic stimulation of the intrinsic inhibitory neurones. It would appear that EDA may be useful as a direct acting smooth muscle relaxant for the study of the physiology-pharmacology of the rodent small intestine.     

Motor effects of gastrin releasing peptide (GRP) and bombesin in the canine stomach and small intestine

Close intraarterial injections of synthetic porcine gastrin releasing peptide (GRP) or bombesin stimulated contractions in the stomach and inhibited ongoing contractile activity in the small intestine of anaesthetized dogs. Contractile activity of the circular muscle was recorded by serosal strain gauges and phasic activity when desired was elicited by local field stimulation or intraarterial motilin injections. In the stomach (corpus and antrum) following tetrodotoxin blockade of field-stimulated contractions, the contractile response to either peptide was not present, suggesting that stimulation of receptors on nerves initiated contractions in the stomach. Similarly, in the small intestine, the inhibitory response was eliminated by tetrodotoxin suggesting a neural receptor. Pre-treatment with reserpine did not alter the inhibitory response, either in the presence or absence of atropine, therefore, adrenergic inhibitory mechanisms did not appear to be involved. The concentration of bombesin producing 50% inhibition of field stimulation (ED50) was increased following treatment with the putative M1 muscarinic antagonist, pirenzipine suggesting activation of M1 cholinergic inhibitory receptors by bombesin. After blockade by atropine of field-stimulated contractions and the contractile response to intraarterial acetylcholine, the ED50 for bombesin inhibition of motilin contractions was increased. After muscarinic blockade, the residual inhibitory response of GRP/bombesin may involve activation of a neural non-cholinergic non-adrenergic inhibitory mechanism. These results suggest that GRP and bombesin act to alter motility in the dog in vivo by affecting neural activity.     

The rebound excitation triggered by anticholinergic drugs from ovine pyloric antrum, small bowel and gallbladder.

The effect of anticholinergic drugs on gastrointestinal motility is complex and incompletely recognized. Accordingly, in 6 adult sheep bipolar electrodes and strain gage force transducers were surgically attached to the antral, small intestinal and gallbladder wall at the serosal side. During chronic experiments the myoelectric and mechanical recordings were performed in fasted and non-fasted animals before and after various doses of hexamethonium, atropine and pirenzepine given intravenously. Hexamethonium administration triggered rebound excitation after an inhibitory period almost in all the recording sites. Administration of atropine and pirenzepine evoked these secondary contractions mostly in the small intestine and gallbladder. No rebounds were observed when the anticholinergic drugs were given during feeding. In fasted animals, rebound excitation arrived later but more frequently than in non-fasted animals. The excitatory changes were dose-dependent. In the gallbladder, these values were lower than in the small intestine. The frequency of the recurrent pattern was dependent upon the dose of the anticholinergic drug used. It is concluded that nicotinic receptors are more important than muscarinic receptors in the initiation of the rebound excitation in pyloric antrum while in the small bowel and gallbladder the role of both cholinergic receptors is similar. The anticholinergic drugs should be used with caution in all these clinical situations, where the enhancement of gastrointestinal motility must be avoided.     

Comparison of muscarinic and alpha-adrenergic receptors in rat atria based on phosphoinositide turnover

The effects of muscarinic and alpha-adrenergic receptor stimulation on phosphoinositide turnover in rat atria have been compared. Despite the similar densities of muscarinic receptors in rat left and right atria, 0.1 mM carbachol increased [32P]phosphate incorporation into phosphatidylinositol (PI) by 35% (p less than 0.05) in left atria but had no effect in right atria. By contrast to the small muscarinic receptor effect, stimulation of alpha 1-adrenergic receptors by 0.1 mM methoxamine produced a more than two fold increase in [32P]phosphate incorporation into PI in both left and right atria, despite the reported smaller density of alpha-adrenergic receptors in rat atria compared to muscarinic receptors. Enhanced phosphate labelling by methoxamine did not occur in phospholipids other than PI, and was blocked by the alpha-adrenergic antagonist, phentolamine (20 microM). The results indicate that the majority of the muscarinic receptors in rat atria are not coupled to phosphoinositide turnover. If indeed the observed enhancement in [32P]-phosphate labelling by carbachol reflects phosphoinositide turnover, and assuming equal coupling efficiencies of muscarinic and adrenergic receptors, it is calculated that not more than 2% of the muscarinic receptors in rat left atria are coupled to this response.     

Mechanisms of inhibition of the contractile activity in the ileo-caecal zone in rabbits under psychogenic stress

In experiments on unanaesthetized rabbits, myoelectric activity (contractile activity index) of distal ileum, caecum, and proximal colon in two sites was studied under stress induced by fastening a rabbit to the table in supine position. The stress caused sharp decrease (up to complete disappearance) of the contractile activity in all studied compartments of the ileocaecal intestine with partial or complete restoration after release of the animal. Nonselective blockade of pre- and postsynaptic alpha-adrenoceptor with dihydroergotoxin abolished the initial component of the specified inhibitory response. The latter was caused by "adrenergic inhibition" as a result of action of catecholamines circulating in blood on inhibitory smooth muscle alpha-adrenoceptor. Against the background of muscarinic cholinoceptor blockade, the stressor inhibition of ileocaecal contractile activity observed in control experiments was completely preserved. The periods of supression of ileoceacal contractile activity under stress resistant to blockade of alpha-, beta-adrenoceptor and muscarinic cholinoceptor, are caused by the mechanism of "nonadrenergic noncholinergic inhibition", which is realized at the expence of activation of the enteric inhibitory neurones.     

Increase in muscarinic acetylcholine receptors in mouse intestine by hexamethonium treatment

The effects of hexamethonium (C6) administration on muscarinic acetylcholine receptors (mACh-R) in the intestine and brain of mice were investigated. Mice were treated with C6 with an osmotic mini-pump (330 mg/kg/day) for one week and then the binding of 3H-quinuclidinylbenzilate (3H-QNB) in the intestine and brain were assayed. This treatment increased the maximum specific binding (Bmax) of 3H-QNB from 160 to 320 fmoles/mg protein in the ileum and from 190 to 340 fmoles/mg protein in the rectum, without affecting the KD values in these regions. On the contrary, C6 treatment did not change the Bmax or KD value in brain tissues. This C6 treatment increased the sensitivity of the contractile response of the intestine to muscarinic agonists, possibly by increasing mACh-R.     

Similarities and differences in the effect of cocaine on α-adrenergic and muscarinic response

Similarities and differences in the effect of cocaine on [alpha]-adrenergic and muscarinic receptors were shown in three experimental models. The postsynaptic stimulating effect of cocaine, mediated by [alpha]-adrenergic receptors was revealed in uninnervated chick amnion and innervated rat vas deferens. In vas deferens cocaine caused an increase of the amount of active [alpha]-adrenergic receptors, the appearance of an additional receptor pool, and change in the dimerization level. Cocaine acted as an antagonist on muscarinic receptors of the chick amnion. The inhibition by cocaine of muscarinic receptors in the rat brain cortex membranes led to a decrease in the number of receptors and their partial monomerization. Thus, cocaine influences both the [alpha]-adrenergic and the muscarinic response at the receptor level. Experiments on various objects have shown that cocaine activates the [alpha]-adrenergic response and inhibits the muscarinic one.     

Molecular mechanisms of regulation of neuronal muscarinic receptor sensitivity

Like other neurotransmitter receptors, muscarinic acetylcholine receptors are subject to regulation by the state of receptor activation. Prolonged increases in the concentration of muscarinic agonists result in a decrease in receptor density and loss of receptor sensitivity, both in vivo and in vitro. On the other hand, when the receptor is deprived of acetylcholine for a long duration in vivo, the receptor becomes more sensitive in responding to muscarinic agonists. However, it has been more difficult to demonstrate increases in receptor concentration that accompany this supersensitive state. The purpose of this review is to provide current information related to the characteristics of muscarinic receptor regulation and the molecular mechanisms underlying this phenomenon, regarding both the density of receptors and their transduction mechanisms. Furthermore, possible feedback regulatory roles of different second messenger signals are discussed. Particular emphasis is dedicated to molecular mechanisms of regulation of neuronal muscarinic receptors.     

The role of insulin-like growth factors in small intestinal cell growth and development.

IGF-I and IGF-II receptors are expressed in the small intestine of mammalian species, as are the genes to synthesize both peptides. IGF binding proteins are also expressed in the intestine. IGF-I and IGF-II mRNA are highest in fetal and newborn tissues and decrease with age. IGF-I mRNA is present in the adult small intestine, and is associated with the submucosal regions and crypt cells. IGF-I and IGF-II receptor binding to the small intestine is higher in newborn animals and decreases with age. Both receptors are more concentrated in the crypt than villus regions, but IGF-II binding is higher than IGF-I in all regions. IGF-I receptors are associated with the submucosal region of the small intestine, whereas IGF-II receptors are more abundant in the mucosal cells. Administration of IGF-I either orally or by osmotic pump generally has no affect on small intestinal weight or length, but does increase mucosal cellularity. LR3-IGF-I administration by osmotic pump affects the small intestine similarly to IGF-I, although with a higher potency. In the few studies in which IGF-II was administered, increased gut mass was observed in adult rats, but not newborn rats or pigs. Significant effects on mucosal expression of disaccharidases was achieved with either oral or subcutaneous IGF-I or oral IGF-II. Administration of IGF in models of intestinal hypertrophy and atrophy are also reviewed.     

Muscarinic receptors on bovine chromaffin cells mediate a rise in cytosolic calcium that is independent of extracellular calcium

Although the mechanism by which nicotinic receptors on adrenal chromaffin cells regulate catecholamine secretion is reasonably well understood, that of the muscarinic receptors remains obscure. The effects of both acetylcholine and specific muscarinic agonists on cytosolic free calcium in isolated bovine adrenal chromaffin cells have been measured using the fluorescent probe Quin-2. Acetylcholine (0.1 mM) evokes a large increase in cytosolic free calcium from resting levels near 100 nM into the microM range, most of which is blocked by hexamethonium (0.5 mM) or removal of extracellular calcium. A small component of the acetylcholine-evoked rise in cytosolic free calcium (approximately 50-100 nM) is independent of extracellular calcium and is unaffected by 0.5 mM hexamethonium, but is totally blocked by 0.5 microM atropine. The muscarinic nature of this component is further confirmed by the fact that the muscarinic agonists, muscarine (0.1 mM) and methacholine (0.3 mM), stimulate a 50-100 nM rise in chromaffin cell cytosolic calcium which is blocked by 0.5 microM atropine and is largely independent of extracellular calcium. These results suggest that muscarinic receptors regulate cytosolic calcium in chromaffin cells by a new mechanism different from that of nicotinic receptors, a mechanism utilizing an intracellular calcium source. The small size of the muscarinic-induced rise in cytosolic calcium in the bovine chromaffin cell would explain why no secretion is evoked by muscarinic agonists in this species.