Effects of cholinergic agonists on diacylglycerol and intracellular calcium levels in pancreatic β-cells

We have studied the effects of cholinegic agonists on the rates of insulin release and the concentrations of diacylglycerol (DAG) and intracellular free Ca²⁺ ([Ca²⁺]_i) in the β-cell line MIN6. Insulin secretion was stimulated by glucose, by glibenclamide and by bombesin. In the presence of glucose, both acetylcholine (ACh) and carbachol (CCh) produced a sustained increase in the rate of insulin release which was blocked by EGTA or verapamil. The DAG content of MIN6 β-cells was not affected by glucose. Both CCh and ACh evoked an increase in DAG which was maximal after 5 min and returned to basal after 30 min; EGTA abolished the cholinergic-induced increased in DAG. ACh caused a transient rise in [Ca²⁺]_i which was abolished by omission of Ca²⁺ or by addition of devapamil. Thus, cholinergic stimulation of β-cell insulin release is associated with changes in both [Ca²⁺]_i and DAG. The latter change persists longer than the former and activation of protein kinase C and sensitization of the secretory process to Ca²⁺ may underlie the prolonged effects of cholinergic agonists on insulin release. However, a secretory response to CCh was still evident after both [Ca²⁺]_i and DAG had returned to control values suggesting that additional mechanisms may be involved.     

Delayed inhibition of gap-junctional intercellular communication in the acinar cells of rat submandibular glands induced by parasympathectomy and cholinergic agonists

In this study, the effects of parasympathectomy and cholinergic agonists on gap-junctional intercellular communication and salivary secretion were investigated to clarify the involvement of salivary secretion in delayed uncoupling between acinar cells of rat submandibular glands. Gap-junctional intercellular communication was monitored as dye-coupling in the acinar cells of isolated acini by the transfer of Lucifer Yellow CH. Parasympathectomy induced dye-uncoupling in the acinar cells isolated from denervated salivary glands 12 hr after parasympathectomy-induced salivary secretion. Intraperitoneal application of carbachol (CCh), acetylcholine, pilocarpine, but not isoproterenol, stimulated salivary secretion, and then induced dye-uncoupling in the acinar cells 12 hr later. Atropine suppressed both the salivary secretion and delayed dye-uncoupling induced by parasympathectomy and CCh, when atropine was applied intraperitoneally before the induction of salivary secretion. However, atropine did not suppress the delayed dye-uncoupling by intraperitoneal application of CCh, when atropine was injected after the cessation of CCh-induced secretion. These results suggest that delayed inhibition of gap-junctional intercellular communication by parasympathectomy and cholinergic agonists in rat submandibular glands might be related to the change of secretory function after salivary secretion.     

The role of protein kinase C in cholinergic stimulation of insulin secretion from rat islets of Langerhans.

The role of the Ca2+/phospholipid-dependent protein kinase C (PKC) in cholinergic potentiation of insulin release was investigated by measuring islet PKC activity and insulin secretion in response to carbachol (CCh), a cholinergic agonist. CCh caused a dose-dependent increase in insulin secretion from cultured rat islets at stimulatory glucose concentrations (greater than or equal to 7 mM), with maximal effects observed at 100 microM. Short-term exposure (5 min) of islets to 500 microM-CCh at 2 mM- or 20 mM-glucose resulted in redistribution of islet PKC activity from a predominantly cytosolic location to a membrane-associated form. Prolonged exposure (greater than 20 h) of islets to 200 nM-phorbol myristate acetate caused a virtual depletion of PKC activity associated with the islet cytosolic fraction. Under these conditions of PKC down-regulation, the potentiation of glucose-stimulated insulin secretion by CCh (500 microM) was significantly decreased, but not abolished. CCh stimulated the hydrolysis of inositol phospholipids in both normal and PKC-depleted islets, as assessed by the generation of radiolabelled inositol phosphates. These results suggest that the potentiation of glucose-induced insulin secretion by cholinergic agonists is partly mediated by activation of PKC as a consequence of phospholipid hydrolysis.     

Development and properties of the secretory response in rat sweat glands: relationship to the induction of cholinergic function in sweat gland innervation

Previous studies suggest that the sympathetic innervation of the sweat glands in the rat is initially noradrenergic and during development undergoes a transition in neurotransmitter phenotype to become cholinergic. To characterize this system and its development further, we have examined the adrenergic and cholinergic components of the secretory response in adult and immature rats and have studied the onset of sweating in the plantar sweat glands of developing rats. Stimulation of the sciatic nerve in adult rats elicited a secretory response which was completely blocked by the cholinergic antagonist, atropine, and was unaffected by adrenergic antagonists, indicating that nerve-evoked secretion was cholinergic. In adult rats, the sweat glands were quite sensitive to cholinergic agonists. In addition to acetylcholine, the mature sweat gland innervation contains vasoactive intestinal peptide (VIP). In some rats, the injection of VIP alone elicited a secretory response which was blocked by atropine, suggesting that the response to VIP was mediated cholinergically. In contrast to cholinergic agonists, the glands responded relatively infrequently and with reduced volumes of sweat to the alpha- and beta-adrenergic agonists 6-fluoronorepinephrine and isoproterenol. However, when VIP, which is a potent vasodilator, was simultaneously injected with adrenergic agonists, glands in many of the injected footpads exhibited a secretory response. The response to adrenergic agonists in combination with VIP was reduced by atropine and by phentolamine plus propranolol, but was blocked completely only by a combination of the three antagonists, indicating that both adrenergic and cholinergic mechanisms were involved. In immature rats, sweating evoked by nerve stimulation first appeared at 14 days of age in 25% of the rats tested. Both the percentage of rats sweating and the number of active glands increased rapidly. At 16 days, 50% of the rats tested exhibited some active glands, and by 21 days all rats tested exhibited a secretory response. In 16-day-old rats, nerve-evoked sweating was almost completely inhibited by local injection of 1 microM atropine, but was unaffected by phentolamine and propranolol in concentrations up to 10 microM. Similarly, the glands were sensitive to 10 microM muscarine, but they exhibited no secretory response to the alpha-adrenergic agonists, clonidine and 6-fluoronorepinephrine, nor to the beta-adrenergic agonist, isoproterenol, at concentrations up to 50 microM. The simultaneous injection of VIP with adrenergic agonists did not reveal an adrenergically mediated secretory response in 16-day-old animals.(ABSTRACT TRUNCATED AT 400 WORDS)     

Contractile properties of the articular capsule or ligament,in the primary spines of the sea-urchin Eucidaris tribuloides

1. The articular capsule or ligament, of the primary spines of the sea-urchin Eucidaris tribuloides behaves as a classical excitable tissue, responding with a shortening or contracture to a variety of stimuli including the cholinergic agonists acetylcholine (ACh), methacholine (MeACh), carbamylcholine (CCh) and nicotine.2. d-Tubocurarine failed to decrease the amplitude of the acetylcholine-induced contractures, while the contractures elicited by methacholine were blocked by atropine. Neostigmine, but not eserine, increased the amplitude of ACh-induced contractures, suggesting the presence of AChE in the preparation.3. The cholinergic agonists induced contracture of the ligament, but had quite different kinetics, the rate of rise of the contracture being fastest for ACh and decreasing in the following order: CCh, MeACh and nicotine.4. Tyramine and octopamine exert an inhibitory action on “catch” and a relaxing effect on the contracting ligament.5. The time courses of the contractures elicited in the same preparation were virtually identical to those of “catch”. Therefore, we propose that “catch” and contracture are only two different aspects of the same phenomenon; namely the contracture of the muscle fibers present in the ligament.     

Pharmacological sensitivity of the articular capsule of the primary spines of Eucidaris tribuloides

1. This paper describes the effects of several cholinergic agonists and antagonists, and of β-phenylethylamine (PEA) and some of its derivatives, on the articular capsule, or ligament, of the primary spines of Eucidaris tribuloides.2. Carbamylcholine (CCh), methacholine (MeACh), nicotine, and muscarine exert a stiffening effect similar to that of acetylcholine (ACh), although the time course of their actions varies widely.3. Atropine induced stiffening and blocked and responses to muscarine and MeACh. The responses to MeACh were blocked also by 4-diphenylacetoxy-N-methylpiperidine, suggesting the presence in the ligament of type M₃ muscarinic receptors, in addition to nicotinic ones. d-Tubocurarine induced stiffness of the ligament and failed to block the responses to ACh and nicotine.4. While ACh induced only a slight desensitization, CCh caused a long-lasting blockade of the stiffening effects of the cholinergic agonists. This shows that the receptors for ACh have a site or sites that recognize the ester moieties of these molecules.5. Eserine and neostigmine potentiate the responses to acetylcholine, indicating the presence of aeetyl-cholinesterase in the ligament.6. β-Phenylethy lamine, epinephrine, norepinephrine, and dopamine induce diphasic responses; usually a brief softening followed by a slow and irreversible stiffening of the ligament.7. In contrast to the above, tyramine and octopamine elicit a simple softening of ligaments which are stiff as a result of handling or by exposure to cholinergic agonists. However, tyramine and octopamine do not soften ligaments which become stiff as a result of exposure to adrenergic agonists.     

Stimulation of Guanosine 5'-O-(3-[35S]Thiotriphosphate) Binding by Cholinergic Muscarinic Receptors in Membranes of Rat Olfactory Bulb

Abstract: In membranes of rat olfactory bulb, a brain region in which muscarinic agonists increase cyclic AMP formation, the muscarinic stimulation of guanosine 5'- O -(3-[³⁵S]thiotriphosphate) ([³⁵S]GTPγS) binding was used as a tool to investigate the receptor interaction with the guanine nucleotide-binding regulatory proteins (G proteins). The stimulation of the radioligand binding by carbachol (CCh) was optimal (threefold increase) in the presence of micromolar concentrations of GDP and 100 m M NaCl. Exposure to N -ethylmaleimide and pertussis toxin markedly inhibited the CCh effect, whereas it increased the relative stimulation of [³⁵S]GTPγS binding elicited by pituitary adenylate cyclase-activating polypeptide (PACAP). On the other hand, membrane treatment with cholera toxin curtailed the PACAP stimulation of [³⁵S]GTPγS binding but did not affect the response to CCh. Like CCh, a number of cholinergic agonists stimulated [³⁵S]GTPγS binding in a concentration-dependent and saturable manner. The antagonist profile of the muscarinic stimulation of [³⁵S]GTPγS binding was highly correlated with that displayed by the muscarinic stimulation of adenylyl cyclase. These data indicate that the olfactory bulb muscarinic receptors couple to G_i/G_o, but not to G_s, and support the possibility that activation of G_i/G_o mediates the stimulatory effect on adenylyl cyclase activity.     

Alpha-adrenoceptor blockade by phentolamine inhibits beta-adrenergically and cholinergically induced glucagon secretion in the mouse

Glucagon secretion is known to be stimulated by activation of the alpha-adrenoceptors. In this study, we investigated whether alpha-adrenoceptor blockade by phentolamine affects basal and stimulated glucagon secretion in the mouse. Phentolamine was injected intraperitoneally to mice at dose levels varying from 2.6 to 260 mumol/kg. It was found that, while decreasing plasma glucose levels, phentolamine did not over this wide dose range affect basal glucagon concentrations indicating an inhibition of the hypoglycaemia-induced glucagon secretion. Further, phentolamine clearly inhibited the glucagon secretory response to beta-adrenergic or cholinergic stimulation. Thus, phentolamine (2.6 mumol/kg), impaired the glucagon secretory response to the beta 2-adrenoceptor agonist terbutaline by 51% (P less than 0.01), and to the cholinergic agonist carbachol by 44% (P less than 0.02). We conclude that alpha-adrenoceptor blockade by phentolamine inhibits the glucagon secretion following hypoglycaemia or stimulation by beta-adrenergic and cholinergic agonists. Thus, the alpha-adrenoceptors seem to be of great importance for glucagon secretion in the mouse.     

Carbachol-stimulated transactivation of epidermal growth factor receptor and mitogen-activated protein kinase in T(84) cells is mediated by intracellular ca(2+), PYK-2, and p60(src)

Ca(2+)-dependent agonists, such as carbachol (CCh), stimulate epidermal growth factor receptor (EGFR) transactivation and mitogen-activated protein kinase activation in T(84) intestinal epithelial cells. This pathway constitutes an antisecretory mechanism by which CCh-stimulated chloride secretion is limited. Here, we investigated mechanisms underlying CCh-stimulated epidermal growth factor receptor (EGFR) transactivation. Thapsigargin (TG, 2 microM) stimulated EGFR and extracellular signal-regulated kinase (ERK) phosphorylation in T(84) cells. Inhibition of either EGFR or ERK activation, with tyrphostin AG1478 (1 microM) and PD 98059 (20 microM), respectively, potentiated chloride secretory responses to TG, as measured by changes in short-circuit current (I(sc)) across T(84) cells. CCh (100 microM) stimulated tyrosine phosphorylation and association of the Ca(2+)-dependent tyrosine kinase, PYK-2, with the EGFR, which was inhibited by the Ca(2+) chelator, BAPTA (20 microM). The calmodulin inhibitor, fluphenazine (50 microM) inhibited CCh-stimulated PYK-2 association with the EGFR and phosphorylation of EGFR and ERK. CCh also induced tyrosine phosphorylation of p60(src) and association of p60(src) with both PYK-2 and the EGFR. The Src family kinase inhibitor, PP2 (20 nM-20 microM) attenuated CCh-stimulated EGFR and ERK phosphorylation and potentiated chloride secretory responses to CCh. We conclude that CCh-stimulated transactivation of the EGFR is mediated by a pathway involving elevations in intracellular Ca(2+), calmodulin, PYK-2, and p60(src). This pathway represents a mechanism that limits CCh-stimulated chloride secretion across intestinal epithelia.     

Visualization of the secretory process involved in Ca2+-activated fluid secretion from rat submandibular glands using the fluorescent dye, calcein

The central feature of fluid and electrolyte secretion by salivary acinar cells is transepithelial Cl- movement as a driving force for the secretion. However, little is known about the membrane localization and regulation by agonists of various anion channels. To characterize the anion transport and fluid secretion, we visualized the secretory process induced by the cholinergic agonist, carbachol (CCh), using the anionic fluorescent dye, calcein, under a confocal laser scanning microscope. The fluorescence of calcein loaded into the isolated acini was spread diffusely throughout the cytoplasm and was less intense in the secretory vesicles which occupied the apical pole. Cytoplasmic calcein was released into intercellular canaliculi just after the addition of CCh, depending upon a rise in [Ca2+]i by Ca2+ release from intracellular stores. Thereafter, the formation of watery vacuoles connected with intercellular canaliculi was visualized in the calcein-loaded acini, depending upon external Ca2+. Both the calcein release and vacuole formation were inhibited by suppressing the Ca(2+)-activated K+ efflux. The calcein release was also affected by the external anion substitution, suggesting that calcein is released through an anion channel. In the isolated, perfused glands, CCh-induced fluid secretion was sustained in two phases, whereas the loaded calcein was initially and transiently released into the saliva. By revealing the [Ca2+]i dependence and sensitivities to channel blockers, our results suggest that the initial phase of CCh-induced fluid secretion was evoked in association with the release of the organic anion, calcein, and the late phase of fluid secretion, during which calcein is less permeable, was associated with the formation of watery vacuoles. Thus, the anion channels possessing the distinct property of anion permeation may be activated in the initial phase and late phase. These results indicate that the anionic fluorescent dye, calcein, is useful for visualizing the process of Ca(2+)-dependent fluid secretion, and for clarifying the relation between fluid secretion and anion transport.     

Sexual differences and effects of castration on secretory mode and intracellular calcium ion dynamics of golden hamster Harderian gland

The aim of the present work was to study the sexual differences in secretory mechanisms and intracellular calcium ion dynamics in the Harderian gland of the golden hamster. In both sexes the Harderian gland consisted of small and large lobes. In the intact control male glands the secretory portions of both lobes showed wide lumina that contained secretory material and cytoplasmic fragments, suggestive of the occurrence of exocytosis and apocrine secretion. After perfusion with HEPES-buffered Ringer's solution containing 10 microM carbamylcholine (CCh), the glandular cells showed features of enhanced secretion and a rise in intracellular calcium concentration ([Ca2+]i). In the intact control female gland the lumina of most secretory portions in the large lobe contained porphyrin accretions, and exocytosis was the sole secretory mechanism. Stimulation of the large lobe with 10 microM CCh did not raise [Ca2+]i or cause enhanced secretion. The small lobe in females resembled the male gland in secretory functions, and CCh administration caused enhanced secretion and a rise in [Ca2+]i. Castration in males abolished apocrine secretion; exocytosis became the sole secretory mechanism, and stimulation of the glandular cells with CCh did not cause enhanced secretion or induce a rise in [Ca2+]i. To the contrary, in females, castration restored apocrine secretion and CCh administration caused enhanced secretion and a rise in [Ca2+]i. Castration did not affect the secretory mechanisms and the effect of CCh on the glandular cells in the small lobes of both male and female glands. The present study points to the possibility that sex hormones may control the functioning or expression of muscarinic receptors in the Harderian gland of the golden hamster.     

Transactivation of the epidermal growth factor receptor mediates muscarinic stimulation of focal adhesion kinase in intestinal epithelial cells

We have previously shown that the Gq protein coupled receptor (GqPCR) agonist, carbachol (CCh), transactivates and recruits epidermal growth factor receptor (EGFr)-dependent signaling mechanisms in intestinal epithelial cells. Increasing evidence suggests that GqPCR agonists can also recruit focal adhesion-dependent signaling pathways in some cell types. Therefore, the aim of the present study was to investigate if CCh stimulates activation of the focal adhesion-associated protein, focal adhesion kinase (FAK), in intestinal epithelia and, if so, to examine the signaling mechanisms involved. Experiments were carried out on monolayers of T84 cells grown on permeable supports. CCh rapidly induced tyrosine phosphorylation of FAK in T84 cells. This effect was accompanied by phosphorylation of another focal adhesion-associated protein, paxillin, and association of FAK with paxillin. CCh-stimulated FAK phosphorylation was inhibited by a chelator of intracellular Ca2+, BAPTA/AM (20 microM), and was mimicked by thapsigargin (2 microM), which mobilizes intracellular Ca2+ in a receptor-independent fashion. CCh also induced association of FAK with the EGFr and FAK phosphorylation was attenuated by an EGFr inhibitor, tyrphostin AG1478, and an inhibitor of Src family kinases, PP2. The actin cytoskeleton disruptor, cytochalasin D (20 microM), abolished FAK phosphorylation in response to CCh but did not alter CCh-induced EGFr or ERK MAPK activation. In summary, these data demonstrate that agonists of GqPCRs have the ability to induce FAK activation in intestinal epithelial cells. GqPCR-induced FAK activation is mediated by via a pathway involving transactivation of the EGFr and alterations in the actin cytoskeleton.     

Thapsigargin potentiates histamine-stimulated HCl secretion in gastric parietal cells but does not mimic cholinergic responses.

The role of calcium in control of HCl secretion by the gastric parietal cell was examined using a recently available intracellular calcium-releasing agent, thapsigargin, which has been shown, in some cell types, to induce sustained elevation of intracellular calcium ([Ca2+]i), an action that appears to be independent of inositol lipid breakdown and protein kinase C activation and to be mediated, at least partially, by selective inhibition of endoplasmic reticulum Ca2(+)-ATPase. Using the calcium-sensitive fluorescent probe, fura-2, in combination with digitized video image analysis of single cells as well as standard fluorimetric techniques, we found that thapsigargin induced sustained elevation of [Ca2+]i in single parietal cells and in parietal cells populations. Chelation of medium calcium led to a transient rise and fall in [Ca2+]i, indicating that the sustained elevation in [Ca2+]i in response to thapsigargin was due to both intracellular calcium release and influx. Although thapsigargin appeared to affect the same calcium pool(s) regulated by the cholinergic agonist, carbachol, and the pattern of thapsigargin-induced increases in [Ca2+]i were similar to the plateau phase of the cholinergic response, thapsigargin did not induce acid secretory responses of the same magnitude as those initiated by carbachol (28 vs 600% of basal). The protein kinase C activator, 12-O-tetradecanoyl phorbol-13-acetate (TPA) potentiated the secretory response to thapsigargin but this combined response also did not attain the same magnitude as the maximal cholinergic response. In the presence but not the absence of medium calcium, thapsigargin potentiated acid secretory responses to histamine, which elevate both cyclic AMP (cAMP) and [Ca2+]i in parietal cells, as well as forskolin and cAMP analogues but had no effect on submaximal and an inhibitory effect on maximal cholinergic stimulation. Furthermore, thapsigargin did not fully mimic potentiating interactions between histamine and carbachol, either in magnitude or in the pattern of temporal response. Assuming that the action of thapsigargin is specific for intracellular calcium release mechanisms, these data suggest that 1) sustained influx of calcium is necessary but not sufficient for cholinergic activation of parietal cell HCl secretion and for potentiating interactions between cAMP-dependent agonists and carbachol; 2) mechanisms in addition to elevated [Ca2+]i and protein kinase C activation may be involved in cholinergic regulation; and 3) increases in [Ca2+]i in response to histamine are not directly involved in the mechanism of histamine-stimulated secretion.     

Cholinergic inhibition of electrogenic sodium absorption in the guinea pig distal colon

Submucosal cholinergic and noncholinergic neurons in intestines have been shown to be involved in regulating epithelial transport functions, particularly stimulating Cl(-) secretion. This study investigates the role of submucosal cholinergic neurons in regulating electrogenic Na(+) absorption in distal colon. Amiloride-sensitive short-circuit current (I(sc)) and (22)Na(+) flux were measured in mucosal and mucosal-submucosal preparations mounted in Ussing chambers. In the mucosal preparation, carbachol (CCh) added to the serosal side inhibited amiloride-sensitive I(sc) and amiloride-sensitive (22)Na(+) absorption. The inhibitory effect of CCh was observed at approximately 0.1 microM, and maximum inhibition of approximately 70% was attained at approximately 30 microM (IC(50) = approximately 1 microM). CCh-induced inhibition of amiloride-sensitive I(sc) was almost totally abolished by 10 microM atropine. Treatment of the tissue with ionomycin markedly reduced amiloride-sensitive I(sc), but a subsequent addition of CCh further decreased it. Also, CCh still had an inhibitory effect, although significantly attenuated, after the tissue had been incubated with a low-Ca(2+) solution containing ionomycin and BAPTA-AM. Applying electrical field stimulation to submucosal neurons in the mucosal-submucosal preparation resulted in inhibition of amiloride-sensitive I(sc), approximately 33% of this inhibition being atropine sensitive. Physostigmine inhibited amiloride-sensitive I(sc), this effect being abolished by atropine. In conclusion, submucosal cholinergic and noncholinergic neurons were involved in inhibiting electrogenic Na(+) absorption in colon. This inhibition by cholinergic neurons was mediated by muscarinic receptor activation.     

Isolated rat gastric parietal cells: Cholinergic response and pharmacology

Isolated, partially purified or enriched rat gastric muscosal parietal cells were shown to respond to carbamycholine (EC₅₀ = 2 μM) and other muscarinic cholinergic agonists as measured by an increased accumulation of ¹⁴C-aminopyrine, an indirect measure of acid secretion. The secretory response to carbamylcholine was shown to be inhibited stereoselectively and reversibly by nanomolar concentrations of muscarinic cholinergic antagonists. Non-muscarinic antagonists, including cimetidine, were either ineffective or very weak inhibitors. The affinity constants calculated for cholinergic antagonist inhibition of ¹⁴C-aminopyrine accumulation induced by carbamylcholine were similar to those previously calculated from direct binding studies on purified parietal cell particulate fractions using ³H-QNB (1). These studies support the existence of specific parietal cell muscarinic cholinergic receptors with which the natural secretagogue acetylcholine interacts to regulate gastric acid secretion.     

Cholinergic receptors and catecholamine secretion from adrenal chromaffin cells of the toad.

1. The effects of cholinergic drugs on catecholamine (CA) secretion from adrenal chromaffin tissue of the toad were studied. 2. CA secretion was induced by ACh or nicotine, but not by muscarine. 3. Hexamethonium inhibited the CA release evoked by ACh or nicotine, while d-tubocurarine only affected the nicotinic response. Atropine did not prevent the secretory response. 4. Muscarine abolished the secretion induced by the agonists, this effect being prevented by atropine or gallamine, but not by pirenzepine. 5. In conclusion, CA secretion in the toad is stimulated by activation of nicotinic receptors. Inhibitory muscarinic receptors are present, most likely of type M2, which may play a regulatory function.     

Mechanisms of carbacholine and GABA action on resting membrane potential and Na+/K+-ATPase of Lumbricus terrestris body wall muscles

This work was aimed to identify the action of several ion channel and pump inhibitors as well as nicotinic, GABAergic, purinergic and serotoninergic drugs on the resting membrane potential (RMP) and assess the role of cholinergic and GABAergic sensitivity in earthworm muscle electrogenesis. The nicotinic agonists acetylcholine (ACh), carbacholine (CCh) and nicotine depolarize the RMP at concentrations of 5 μM and higher. The nicotinic antagonists (+)tubocurarine, α-bungarotoxin, muscarinic antagonists atropine and hexamethonium do not remove or prevent the CCh-induced depolarization. Verapamil, tetrodotoxin, removal of Cl(-) and Ca(2+) from the solution also cannot prevent the depolarization by CCh. In a Na(+)-free medium, however, CCh lost this depolarization ability and this indicates that the drug opens the sodium permeable pathway. Serotonin, glutamate, glycine, adenosine triphosphate (ATP) and cis-4-aminocrotonic acid (GABA(C) receptor antagonist) had no effect on the RMP. On the other hand, isoguvacin, γ-aminobutyric acid (GABA) and baclofen (GABA(B) receptor agonist) hyperpolarized the RMP. Ouabain, bicucullin (GABA(A) antagonist) and phaclofen (GABA(B) antagonist), as well as the removal of Cl(-), suppressed the effect of GABA and baclofen. CCh did not enhance the depolarization generated by ouabain but, on the other hand, hindered the hyperpolarizing activity of baclofen both in the absence and presence of atropine and (+)tubocurarine. The long-term application of CCh depolarizes the RMP primarily by inhibiting the Na(+)/K(+)-ATPase. The muscle membrane also contains A and B type GABA binding sites, the activation of which increases the RMP at the expense of increasing the action of ouabain- and Cl(-) -sensitive electrogenic pumps.     

不同信号分子参与M胆碱能受体激动剂对不同胚胎发育阶段小鼠心肌细胞起搏电流的调控

应用全细胞膜片钳技术,研究了M胆碱能对不同孕期的胚胎小鼠心肌细胞的起搏电流(If)的调节。我们发现,在胚胎发育的早期阶段,M胆碱能受体激动剂(muscarinic agonist carbachol,CCh)明显抑制If,但在胚胎发育的晚期阶段,CCh对If的抑制作用消失。腺苷酸环化酶(adeinylate cyclase,AC)激动剂毛喉素Forskolin和非选择性磷酸二酯酶(PDE)抑制剂IBMX均可增强发育早期阶段和晚期阶段的If。但有趣的是,尽管,Forskolin和IBMX可增加基础If,它们对CCh抑制的If的作用却大不相同。在胚胎发育的早期阶段,Forskolin不能拮抗CCh对If的抑制作用,但IBMX可以。在发育的中期阶段Forskolin可以拮抗CCh的抑制作用,但IBMX不可以。因此,我们推断,CCh可能是通过调控细胞内的CAMP水平来调节If的。但是在胚胎发育的早期阶段和中期阶段,CCh可能通过不同的信号转导通路来实现对胞内cAMP的水平调控。在发育的早期阶段,CCh主要是通过增强PDE的活性,加速cAMP的降解而实现对f的调控。而在发育的中期阶段,CCh则主要通过与AC耦联,抑制其活性,通过减慢cAMP的合成而实现对If的调控。     

Nicotinic acetylcholine receptors on a cholinergic nerve terminal in the cockroach,Periplaneta americana

Summary Intracellular microelectrode recording and ionophoretic application of carbamylcholine (CCh) were used to compare the cholinergic sensitivity of postsynaptic dendrites of an identified neurone with that of an identified presynaptic cholinergic axon.The axon of the lateral filiform hair sensory neurone (LFHSN) in the first-instar cockroachPeriplaneta americana was found to be as sensitive to CCh as the dendritic regions of giant interneurone 3 (GI 3). The CCh response of both neurones was unaffected by replacing Ca²⁺ with Mg²⁺, confirming that the ACh receptors are present on the neurones under test. The CCh response of both neurones was mimicked by ionophoretic application of nicotine. The responses were blocked by 10^–5 M mecamylamine and 10^–6 M d-tubocurarine and were not affected by muscarinic antagonists, suggesting that the ACh receptors present on GI 3 and LFHSN are predominantly nicotinic.The muscarinic agonist oxotremorine and the antagonists atropine and quinuclidinyl benzilate had no modulatory effect on LFHSN-GI 3 synaptic transmission.The latency of the LFHSN response to CCh was consistent with the hypothesis that ACh receptors are situated on the main axon/terminal within the neuropil of the ganglion. It has previously been shown that this region of the axon does not form output synapses (Blagburn et al. 1985a). This indirect evidence indicates that presynaptic or extrasynaptic ACh receptors are present in the membrane of a cholinergic axon.LFHSN was depolarized by synaptically-released ACh after normal or evoked spike bursts, suggesting that the nicotinic ACh receptors act as autoreceptors. However, it was not possible to obtain direct evidence to support the hypothesis that these receptors modulate ACh release.Abbreviations CCh carbamylcholine - GI giant interneurone - FHSN filiform hair sensory neurone - LFHSN lateral filiform hair sensory neurone - R _in input resistance - V depolarization - V _m resting potential     

Insulin-stimulated protein synthesis in submandibular acinar cells: interactions with adrenergic and cholinergic agonists

The effects of insulin and secretory agonists on amino acid incorporation into submandibular gland proteins were studied using isolated acinar cell aggregates. Insulin stimulated the incorporation of 3H-leucine into TCA-precipitable proteins in a rapid, dose-dependent manner (half-maximal response at 1 nM). Isoproterenol, a beta-adrenergic agonist, also stimulated amino acid incorporation, and this effect was mimicked by both dibutyryl cAMP and IBMX, a phosphodiesterase inhibitor. Although insulin further stimulated incorporation in the presence of isoproterenol and IBMX, no additional increase in the rate of synthesis was observed after stimulation by dibutyryl cAMP. High concentrations of carbamylcholine, a cholinergic agonist, inhibited both basal and insulin-stimulated incorporation. At low concentrations, however, carbamylcholine stimulated synthesis, and the effects of insulin and carbamylcholine were additive. A23187, a calcium ionophore, also inhibited 3H-leucine incorporation and insulin stimulation, but in contrast to carbamylcholine, low concentrations of A23187 neither inhibited nor enhanced the rate of synthesis. Thus, protein synthesis in the rat submandibular gland is regulated by both insulin and neurotransmitters. Whereas beta-adrenergic stimulation appears to be mediated through cAMP, the intracellular signals mediating the actions of insulin and cholinergic agonists remain to be elucidated.