Louise Larose

Distribution of Muscarinic Receptors in the Digestive Tract Organs

We have studied the binding of the potent muscarinic antagonist, (3H)-quinuclidinyl benzilate [(3H)-QNB], in order to characterize muscarinic receptors of the digestive tract organs and evaluate their population. In each tissue examined, the binding sites were saturable and stereoselective; (3H)-QNB could also be displaced by known muscarinic agonists and antagonists. Along the digestive tract, the stomach (for stomach, fundus, antrum) and the colon possess the highest number of binding sites while the secretory (parotid, mandibular and pancreas), with a comparable amount of sites, are among those with less receptors. This study presents for the first time a complete distribution of the muscarinic receptors in organs of the digestive system evaluated under identical experimental conditions.

Muscarinic receptor of rat pancreatic acini: [3H]QNB binding and amylase secretion

Binding studies were performed on rat pancreatic dispersed acini with (--)-[3H]QNB, in parallel with secretory studies to establish that the muscarinic receptors characterized are involved in enzyme secretion. Scatchard analysis of [3H]QNB saturation isotherm gave an apparent KD value of 3.09 X 10(-10) M with binding capacities of 2605 fmol/mg of DNA. The true KD calculated from the ratio of the kinetic parameters was 64 pM. The receptor population can be divided into two classes for the agonist binding sites: a population of high affinity sites and one of low affinity sites. With carbamylcholine, maximal stimulation of amylase release occurred with 40% receptor occupation while occupation of the remaining 60% caused a progressive decrease in stimulation of amylase release. Moreover, the dissociation constant of the high affinity sites for carbamylcholine (1.78 X 10(-6) M) is very close to the ED50 of carbamylcholine (5.81 X 10(-7) M) found for the stimulation of enzyme secretion. These data suggest that the physiological response of the pancreatic acini to a muscarinic agonist would involve the high affinity site population.

Muscarinic cholinergic inhibition of cyclic AMP formation and adrenocorticotropin secretion in mouse pituitary tumor cells

Cholinergic muscarinic receptors were identified in AtT-20/D16-16 (AtT-20) cell membranes by receptor binding techniques and the effect of carbachol on basal and stimulated cyclic AMP formation and ACTH release was investigated. Carbachol markedly decreased the stimulatory effect of the adenylate cyclase activator, forskolin, on both cyclic AMP formation and ACTH secretion. Carbachol also reduced forskolin-stimulated adenylate cyclase activity. The stimulatory effects of (-) isoproterenol on cyclic nucleotide formation and ACTH secretion were also blocked by carbachol. The inhibitory effects of carbachol on (-) isoproterenol-stimulated cyclic AMP synthesis and ACTH secretion were reversed by the muscarinic antagonist, atropine, and not by the nicotinic antagonist, gallamine. These data suggest that in AtT-20 cells, inhibition of ACTH secretion may be regulated by activation of muscarinic receptors coupled negatively to adenylate cyclase.

PARALLEL MATURATION OF THE PANCREATIC SECRETORY RESPONSE TO CHOLINERGIC STIMULATION AND THE MUSCARINIC RECEPTOR POPULATION

1 The appearance of pancreatic muscarinic receptors during development has been measured by use of the specific ligand [3H]‐quinuclidinyl benzilate ([3H]‐QNB). 2 QNB binding sites are present in foetal pancreas; their maximal concentration is attained at the age of 30 days and a significant decrease is observed in one year old animals. 3 Affinity of [3H]‐QNB for the muscarinic receptor does not change with age. 4 An evaluation of the pancreatic secretory response to a cholinoceptor agonist as a function of age indicates that the development of this response parallels that of the receptor population. 5 It is suggested that, at all ages from 3 days after birth onwards, the maximal secretory response of the exocrine pancreas to a cholinoceptor agonist mobilizes the same proportion of the total population of QNB binding sites.

Modulation of rat pancreatic amylase secretion and muscarinic receptor populations by chronic bethanechol treatment

F8P6effect of a chronic bethanechol treatment (12 mg X kg-1 X day-1 i.p., for 14 days) was investigated on pancreatic amylase secretion and muscarinic cholinergic receptors in the rat. Dispersed pancreatic acini were used to evaluate enzyme secretion and binding of [3H]N-methylscopolamine, [( 3H]NMS). The bethanechol treatment caused a 4 fold decrease in sensitivity of the pancreas for amylase release in the presence of carbamylcholine, the EC50 being shifted from 0.69 microM to 2.9 microM. Receptor concentration was reduced by 42%, from 3360 to 1930 fmol/mg DNA. The equilibrium dissociation constant (KD) of the receptors for the ligand remained unchanged at 0.17 nM. Binding analysis of carbachol on the muscarinic receptors in terms of two classes of binding sites indicated that the shift in the dose-response curve of amylase secretion was accompanied by modifications to the high and low affinity forms. The maximal number of high affinity sites remained the same while their affinity was greatly decreased from 0.24 to 6.1 microM. The low affinity form showed a moderate decrease in affinity from 34 to 150 microM but a large drop in their numbers from 2620 to 890 fmol/mg DNA. These results suggest that the shift in the amylase dose-response curve to carbamylcholine, noted following bethanechol treatment in vivo, could be coupled with the observed change in affinity of the two agonist forms of muscarinic receptor in the pancreas. However, gradual occupancy or formation of the high affinity form of muscarinic receptors by cholinergic agonist corresponds well with the gradual stimulation of amylase release.

Maturation of muscarinic agonist receptors in rat developing pancreas and its relation to maximal enzyme secretion

Abstract The true K D s of [ 3 H] (−) QNB binding to muscarinic receptors were found to be 4.13 and 6.43 × 10 −11 M in pancreas of 21 day fetal and adult rats. The competition curves of specific [ 3 H] (−) QNB bound by two antagonists have shown that the affinity of these drugs did not change with age with Hill coefficients near unity. However, with the agonist carbamylcholine as the competitive drug, a more flat curve was obtained with a Hill coefficient below unity. At least two populations of carbamylcholine binding sites were found with different K D s: a K H around 7 × 10 −7 M and a K L around 3 × 10 −5 M. These two populations were present during all the developmental periods studied. The ED 50 of bethanechol stimulated amylase secretion did not change within the age limit studied (from 11 to 365 days). The high affinity sites for carbamylcholine would seem to be the receptor implicated in the physiological response of the pancreas.

Cytological effects of urecholine stimulation on the rat pancreas

SummaryStimulation of the exocrine pancreas by the secretagogue urecholine causes degranulation of the acinar cells. Under in vivo conditions, this degranulation is not uniform throughout the tissue. Indeed some of the acini are almost completely depleted of their granules while others display the appearance of resting acini. A noticeable feature is that all the cells of the same acinus display a comparable degree of degranulation. Moreover, groups of neighbouring acini seem to respond simultaneously suggesting that the secretory stimulus is propagated from one acinus to the other. In vitro stimulation of dispersed acini also showed that some of the acini are more responsive than others indicating that this phenomenon can not be attributed to accessibility of the secretagogue to its receptor. These observations lead us to the concept that the response of the pancreatic acinar cell is controlled at the level of the acinus.

Effect of Early Weaning of the Neonatal Rat on Pancreatic Acinar Cell Responsiveness to Urecholine

Pancreatic response to urecholine was studied in rats weaned prematurely on the morning of their 13th, 15th, 17th and 19th day and killed 2 1/2 days later. In pups kept with their mother and weaned at 21 days, amylase chymotrypsin and lipase concentrations increased gradually. Weaning after 12, 14 and 16 complete days is associated with significant increases in pancreatic amylase and chymotrypsin concentrations; if it occurs after 16 and 18 days, a significant delay in lipase development is observed. Premature weaning is associated with modifications in the basal release of the three enzymes and their secretion in response to urecholine. Amylase and chymotrypsin secretions are increased if weaning occurred before day 18. Lipase secretion, however, is decreased in rats weaned after 16 days. If, however, secretion is expressed in percentages, as the amount of enzyme released over the total tissue content, premature weaning does not seem to modify the capacity of the tissue to secrete enzymes under basal and urecholine stimulation. It thus seems that solid food intake as early as 12 days is not an important factor in the maturation of the pancreatic response to urecholine.

Carbamylcholine and phorbol esters desensitize muscarinic receptors by different mechanisms in rat pancreatic acini

Pretreatment of rat pancreatic acini with phorbol 12-myristate, 13-acetate (PMA), a protein kinase C (PK-C) activator, caused the desensitization of carbamylcholine (CBC)-induced amylase release in a concentration- and time-dependent fashion. The less potent phorbol-12, 13-dibutyrate (PDBu) also provoked a desensitization, but the inactive 4-α-phorbol-12,13-didecanoate had no effect. PMA or PDBu also significantly reduced subsequent amylase release induced by caerulein or secretin in contrast to CBC, which only reduced amylase release induced by CBC or secretin. Preincubation of acini with PMA did not lead to a decrease in PMA or A23187-stimulated amylase release. A 3 h resting period did not restore the desensitization induced by PMA or PDBu. Pretreatment with PMA did not cause changes in muscarinic receptor high- and low-affinity populations as observed with CBC pretreatment. The PK-C inhibitor H-7 completely prevented the desensitization induced by PDBu but not that induced by CBC. TMB-8, another PK-C inhibitor, also completely prevented the desensitization induced by PDBu but only partially that induced by CBC. These results suggest that phorbol esters can induce desensitization of muscarinic receptor-stimulated amylase release by a different mechanism than that involved in muscarinic agonist-induced desensitization.

Muscarinic cholinergic induced secretin subsensitivity in rat isolated pancreatic acini. Effects on amylase release, cyclic adenosine monophosphate and inositol phosphate formation

In this study, dispersed rat pancreatic acini exhibited secretin sub-sensitivity in their capacity to release amylase after preexposure to increasing concentrations of the muscarinic cholinergic agonist carbamylcholine. The present study also explores the potential mechanisms involved in this cellular desensitization phenomenon. Secretin subsensitivity of pancreatic acini preexposed to l0−4M carbamylcholine was observed only at secretin concentrations above 10−8 M. The desensitized cells had not recovered 3 h after the cholinergic agonist exposure. In these acini, the adenylate cyclase pathway remained unaltered because cholera toxin, forskolin, and 8-Br-CAMP still induced weak, but normal, amylase release when compared with control acini. In vivo administration of pertussis toxin failed to protect the dispersed pancreatic acini against carbamylcholine-induced secretin subsensitivity. Moreover, CAMP production by these acini in response to secretin, cholera toxin, and forskolin was similar to that observed in control acini. Secretin stimulation of inositol phosphate (InsP1, InsP2, InsP3) production after carbamylcholine preexposure remained equivalent to that observed in acini that had never been exposed to the cholinergic agonist. Thus, after muscarinic cholinergic agonist exposure, pancreatic acini showed secretin subsensitivity in their capacity to release enzyme. This phenomenon appears to result from modifications at post-second messenger loci.