Action Of Muscarinic Agonists Research Articles

Neurons, Receptors, and Channels.

Here, I recount some adventures that I and my colleagues have had over some 60 years since 1957 studying the effects of drugs and neurotransmitters on neuronal excitability and ion channel function, largely, but not exclusively, using sympathetic neurons as test objects. Studies include effects of centrally active drugs on sympathetic transmission; neuronal action and neuroglial uptake of GABA in the ganglia and brain; the action of muscarinic agonists on sympathetic neurons; the action of bradykinin on neuroblastoma-derived cells; and the identification of M-current as a target for muscarinic action, including experiments to determine its distribution, molecular composition, neurotransmitter sensitivity, and intracellular regulation by phospholipids and their hydrolysis products. Techniques used include electrophysiological recording (extracellular, intracellular microelectrode, whole-cell, and single-channel patch-clamp), autoradiography, messenger RNA and complementary DNA expression, antibody injection, antisense knockdown, and membrane-targeted lipidated peptides. I finish with some recollections about my scientific career, funding, and changes in laboratory life and pharmacology research over the past 60 years.

Agonist- and nerve-induced phasic activity in the isolated whole bladder of the guinea pig: evidence for two types of bladder activity.

Spontaneous localised propagating waves of contraction and localised stretches have been reported to occur in the isolated whole bladder of the guinea pig. The physiological role and the cellular processes underlying these events are unknown. In order to gain insight into the mechanisms generating this complex activity, experiments were performed to examine and compare the responses of the whole bladder preparation to (i) the muscarinic agonists carbachol and arecaidine, (ii) the nicotinic ligand lobeline and (iii) nerve stimulation. High concentrations of the muscarinic agonists (>3 micro M) induced a slow rise in intra-vesical pressure upon which were superimposed pressure transients, while low concentrations (< 300 nM) induced only phasic rises in pressure. One interpretation of these data is that there are two separate mechanisms activated by muscarinic agonists: one generating contracture and the other phasic activity. Immunocytochemical staining revealed M(3) muscarinic receptors on smooth muscle cells within trabeculae and a second population of positive cells in the sub-urothelial layer. This observation raises the possibility that the actions of muscarinic agonists are a consequence of activating different cell types. Lobeline (1-60 micro M) activated phasic contractions but did not cause a rise in basal pressure. Atropine did not inhibit the lobeline-induced responses but abolished the muscarinic responses. Also, hexamethonium or tetrodotoxin did not affect the lobeline-induced responses. These observations suggest that the mechanism generating phasic activity is activated by a nicotinic stimulus that does not involve ganglia, nerves or the neuromuscular junction. Stimulation of the bladder nerve at frequencies between 20 and 30 Hz for 5 s resulted in a rapid rise in intra-vesical pressure. Prolonged nerve stimulation (10-200 s) at frequencies between 1 and 10 Hz activated phasic rises in pressure. Low frequency nerve stimulation increased the frequency of agonist-induced phasic activity. Thus, nerve stimulation can also produce two forms of activity and low frequency stimulation can augment the processes generating phasic activity. These observations suggest that there are two distinct types of bladder activity: global contractions involving most of the bladder wall and phasic contractions comprising propagating waves of contraction. The mechanisms generating these contractile events appear to be different and they may involve cells located in different regions of the bladder. The nature of these mechanisms and their possible physiological significance is discussed.

Inhibition by Ro 31-8220 of acid secretory activity induced by carbachol indicates a stimulatory role for protein kinase C in the action of muscarinic agonists on isolated rat parietal cells

The bisindolylmaleimide Ro 31-8220 is a selective inhibitor of protein kinase C. This compound was used to investigate the involvement of protein kinase C in the stimulation of gastric acid secretion by the muscarinic cholinergic receptor on rat isolated parietal cells. The accumulation of the weak base aminopyrine by both crude and enriched preparations of parietal cells was used as an index of secretory activity. Ro 31-8220 antagonized ( ic 50, 1.0 μM) the effect of the activator of protein kinase C, 12- O-tetradecanoylphorbol 13-acetate (TPA), on histamine-stimulated aminopyrine accumulation. Ro 31-8220 (0.1–2.14 μM) inhibited the aminopyrine response to 0.1 mM carbachol ( ic 50, 0.78 μM; 49% inhibition at 2.14 μM Ro 31-8220) and shifted the dose-response curve for the effect of carbachol concentration of aminopyrine accumulation downwards and to the right. No inhibition of aminopyrine accumulation induced by histamine was found with Ro 31-8220 (0.1–2.14 μM). In a preparation containing >80% parietal cells incubated with 0.1 mM carbachol, 2.14 μM Ro 31-8220 inhibited aminopyrine accumulation by 43%, but had no effect on the increase in intracellular Ca 2+ which was measured by using the fluorescent probe FURA-2. In conclusion, Ro 31-8220 (0.1–2.14 μM) produced a selective reduction in secretory activity in parietal cells by inhibition of protein kinase C. The predominant role of protein kinase C in parietal cells activated with carbachol is not to cause feedback inhibition of the response but to facilitate stimulation of secretory activity.

An AF-DX 116 sensitive inhibitory mechanism modulates nicotinic and muscarinic transmission in cat superior cervical ganglion in the presence of anticholinesterase.

The effect of the muscarinic receptor antagonist AF-DX 116 on the inhibitory action of muscarinic agonists and on responses mediated by nicotinic or muscarinic ganglionic transmission was studied in the superior cervical ganglion of the anesthetized cat. The postganglionic compound action potential evoked by cervical sympathetic trunk stimulation was depressed by methacholine or acetylcholine (ACh) injected into the ganglionic arterial supply. The depression was blocked by AF-DX 116. The compound action potentials evoked by preganglionic stimulus trains were also depressed when the intratrain frequency was 2 Hz or greater. This intratrain depression was, however, insensitive to AF-DX 116. The anticholinesterase drug physostigmine markedly enhanced the intratrain depression of the compound action potential. This effect was reversed by AF-DX 116. During nicotinic receptor block with hexamethonium, preganglionic stimulus trains with intratrain frequencies of 5 Hz or greater produced nicitating membrane contractions that could be blocked by the M1 muscarinic receptor antagonist pirenzepine. The amplitude of the contractions increased with frequency and reached a maximum at 20-40 Hz. AF-DX 116 had no effect on these responses. After administration of physostigmine, the amplitude of the nictitating membrane responses decreased with increasing intratrain frequency. AF-DX 116 reversed this effect. The data suggest that, in the superior cervical ganglion, AF-DX 116 sensitive muscarinic receptors which depress synaptic transmission are activated by exogenous agonists but not by the ACh released by the preganglionic axon terminals unless cholinesterase activity is inhibited.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence Against a Role for Protein Kinase C in the Inhibition of the Calcium-activated Potassium Current IAHP by Muscarinic Stimulants in Rat Hippocampal Neurons.

The possible role of protein kinase C activation in the inhibitory action of cholinergic transmitters on the slow Ca-dependent afterhyperpolarizing current (IAHP) in hippocampal CA3 pyramidal neurons was investigated using hippocampal slice cultures. IAHP was inhibited reversibly by methacholine (100 - 600 nM) and irreversibly by the protein kinase C activator, phorbol-12,13-dibutyrate (PDBu, 10 nM to 1 microM). The inhibitory action of PDBu was antagonized by prior (15 - 60 min) exposure to staurosporin (1 microM). In contrast, the inhibitory effect of methacholine on IAHP was not reduced after up to 3 h of exposure to this compound. In addition, methacholine produced a reversible inward current at the holding potential, which was augmented by staurosporin. However, prior exposure to PDBu reduced the effect of methacholine on IAHP and occluded the methacholine-induced inward current. This effect of PDBu was also observed in the presence of staurosporin, suggesting that it might be exerted through a protein kinase C-independent pathway. Noradrenalin (2 - 5 microM) and 8-bromo cyclic adenosine 3',5'monophosphate (8-Br-cAMP, 1 mM) also produced a reversible block of IAHP. Their action was antagonized by staurosporin, probably via its effect on protein kinase A. Thus the present experiments suggest that the action of muscarinic agonists on IAHP cannot be explained by an effect on protein kinase C, but support a role for protein kinase A in mediating the action of noradrenalin.

Functionally distinct muscarinic receptors on gastric somatostatin cells

The present study was designed to examine the mode of action of muscarinic agonists on somatostatin secretion in intact gastric tissues, i.e., mucosal segments from the fundus and antrum of rat and the isolated luminally perfused mouse stomach. Methacholine caused similar decreases in somatostatin secretion in segments from the fundus (35 +/- 3%; P less than 0.001) and antrum (35 +/- 2%; P less than 0.001) of rat stomach, and in whole mouse stomach (43 +/- 3%; P less than 0.001). The decrease was the net effect of a dominant inhibition and a lesser stimulation of somatostatin secretion. Pretreatment with the permeant derivative of the acetomethoxy ester form of the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA/AM, 15 microM) caused a further decrease in methacholine-induced somatostatin secretion, implying that a stimulatory component existed that was mediated by intracellular calcium. Pretreatment with pertussis toxin (125 ng/ml) for 60 min converted the decrease in somatostatin secretion to an increase above basal levels. The increase induced by pretreatment with pertussis toxin was abolished by additional pretreatment with BAPTA/AM. Procaine (5 mM), which blocks release of calcium from intracellular stores, produced an effect on somatostatin secretion similar to that of BAPTA/AM. The results indicate that 1) methacholine exerts dual inhibitory and stimulatory effects on somatostatin cells of rat and mouse stomach, 2) the dominant effect is inhibitory and sensitive to pertussis toxin, and 3) a concurrent stimulatory effect, mediated by calcium, is unmasked after blockade of the inhibitory effect with pertussis toxin.

Effects of acetylcholine and substance P on electrical activity of intact toad gastric muscles

By using isolated gastric muscle cells of Bufo marinus, others have studied the mechanism of action of muscarinic agonists and substance P (SP). To compare responses of isolated cells with those of intact muscles, we have studied the effects of acetylcholine (ACh) and SP on membrane potentials of circular muscle cells in strips of intact muscle from the toad gastric corpus region. These cells had average resting potentials of -69 +/- 0.7 mV. Membrane potential rhythmically depolarized, producing slow waves at an average frequency of 1/min and average amplitude of 25 +/- 2.2 mV. The major effect of ACh (10(-7) to 10(-4) M) was chronotropic; the frequency of slow waves was increased by 88 +/- 11% by 10(-6) M ACh. The amplitudes and rates of rise of slow waves were decreased by ACh. SP had effects similar to ACh; its major effect was chronotropic. The data suggest that ACh and SP primarily affect the pacemaker mechanism in gastric muscles. Since rhythmicity is apparently not expressed in isolated gastric myocytes, it is possible that this effect of these agonists may have been missed in studies of dispersed cells. Our data suggest that the excitatory effects of ACh and SP on contractions may be due to summation of Ca2+ signals, a partial tetanus-like effect.

The effect of different calcium antagonists on histamine-and carbachol-induced acid secretion in the isolated mouse stomach

Earlier experiments suggested that the action of muscarinic agonists in gastric acid secretion might be partly mediated by endogenous histamine release. This possibility could be tested by use of various calcium antagonists provided that they show different activity on histamine- and carbachol-induced secretion. In the present work, three calcium antagonists, verapamil, nifedipine and sodium nitroprusside were used on the isolated mouse stomach where acid secretion induced by histamine and by carbachol was measured. Verapamil (100 microM) did not affect either histamine- or carbachol-induced acid secretion. Nifedipine (100 microM) reduced histamine-induced acid secretion, whereas the effect of carbachol remained unchanged. Sodium nitroprusside (10-100 microM) reduced both histamine- and carbachol-induced acid secretion. The differences in action of calcium antagonists in histamine- and carbachol-induced acid secretion could exclude the possibility that the effect of carbachol is partly mediated by histamine release from mast cells present in the mouse stomach.

Modulation of Synaptic Transmission in Autonomic Ganglia Mediated via the Activation of Postganglionic Muscarinic Ml Receptors

The facilitatory actions of muscarine on synaptic transmission were measured in rat superior cervical ganglia in vitro. Muscarine (300 nmol/l) induced increases in submaximal population action potentials, which were mimicked by the M1 receptor agonist McN-A-343 (1 mumol/l) and antagonized by the M1 receptor antagonist pirenzepine (100 nmol/l) and telenzepine (100 nmol/l), but not by the M2 receptor antagonist AF-DX 116 (1 mumol/l). Slow excitatory postsynaptic potentials recorded from curarized rabbit isolated superior cervical ganglia were also blocked by telenzepine (300 nmol/l) but not by AF-DX 116 (3 mumol/l). It is concluded that M1 receptors mediate the excitatory actions of muscarinic agonists in these sympathetic ganglia.

Enhanced cAMP accumulation after termination of cholinergic action in the heart

Cholinergic agents decrease myocardial contractility in part by inhibiting adenylate cyclase (EC 4.6.1.1) activity. We have found that after a prolonged preincubation period (greater than 6 h), washout of cholinergic agents from embryonic chick hearts or cultured heart cells results in a persistent increase in their basal and catecholamine-stimulated cAMP content. Membranes prepared from pretreated cells have elevated basal, forskolin-, and catecholamine-stimulated adenylate cyclase activities. This myocardial adaptation to cholinergic agents is analogous to changes in nerve cells and other cell types after prolonged exposures to narcotics or other inhibitors of adenylate cyclase, respectively. A rapid (less than 5 min) adaptation response to cholinergic agents can also be demonstrated in heart cells by quickly blocking agonist action with atropine. Atropine alone has no effect, but after a brief preincubation period with agonists (methacholine or oxotremorine), the addition of atropine transiently enhances catecholamine-stimulated cAMP accumulation by 2.5-fold. These responses are absent in heart cells pretreated with pertussis toxin. The data indicate that the response is not mediated by the phosphoinositide pathway, which has been demonstrated to be insensitive to pertussis toxin in chick heart. Enhanced cAMP accumulation after termination of muscarinic agonist action may provide an explanation for the observation that acetylcholine sometimes produces biphasic contractile responses.