Intracellular regulation of neuronal nicotinic cholinoreceptors.

Experiments on isolated superior cervical ganglia from rats were used to study the effects of substances affecting intracellular second messengers on membrane currents evoked by iontophoretic application of acetylcholine (ACh currents) and on excitatory postsynaptic currents (EPSC) induced by single discharges of preganglionic nerve fibers. These studies showed that the adenylate cyclase activator forskolin, the phosphodiesterase inhibitor isobutylmethylxanthine (IMBX), and the protein kinase C activator phorbol ester decreased the amplitude of the ACh current. Neither IMBX nor phorbol ester had any effect on the amplitude or decay time constant of EPSC, while forskolin increased the amplitude of EPSC without altering its decay time constant. Thapsigargin, which liberates intracellular calcium, not only decreased the amplitude of the ACh current, but also decreased EPSC amplitude without affecting its decay time constant. These results suggest that intracellular signaling via protein kinases A and C may affect neuronal nicotinic cholinoceptors (nAChR) only by altering receptor desensitization and not affecting receptor sensitivity to transmitters released from nerves or the kinetics of receptor ion channels. At the same time, neuronal nAChR are influenced by intracellular calcium, which decreases their ability to be activated by exogenous (perhaps acting via desensitization) and nerve-released acetylcholine without affecting the kinetics of ion channel function.

Alpha subunit composition of nicotinic acetylcholine receptors in the rat autonomic ganglia neurons as determined with subunit-specific anti-alpha(181-192) peptide antibodies.

The subunit composition of nicotinic acetylcholine receptors of rat autonomic ganglia neurons was studied by means of antibodies, which differentiated between different alpha subunits and specifically blocked acetylcholine-induced membrane currents. Polyclonal rabbit antibodies and mouse monoclonal antibodies were raised against synthetic peptides matching in sequence the alpha(181-192) region of alpha3, alpha4, alpha5, and alpha7 subunits of rat neuronal nicotinic acetylcholine receptors. The antibodies discriminated among alpha3, alpha4, alpha5, and alpha7 peptides in enzyme-linked immunosorbent assay and bound to native acetylcholine receptors expressed in PC-12 cells. By means of immunoperoxidase staining of cultured rat autonomic neurons followed by transmission, dark-field and phase-contrast microscopy, it was found that all cells of the superior cervical ganglia expressed the alpha3, alpha5, and alpha7 nicotinic acetylcholine receptors, whereas approximately half of the cells were clearly alpha4-positive. In contrast, only about one-third of the intracardiac neurons were alpha3-positive, about 50% were alpha4-positive, one-seventh were alpha5-positive, and one-fifth were alpha7-positive. All antibodies tested blocked acetylcholine-induced currents in the neurons of the superior cervical ganglia as was demonstrated by whole-cell patch-clamp studies. Although each antibody could block up to 80% of the current, the degree of inhibition varied considerably from cell to cell. It is concluded that alpha3, alpha5, and alpha7 subunits are expressed in all neurons of the superior cervical ganglion and in some intracardiac neurons, whereas alpha4 subunits are expressed in some but not all neurons of both tissues. The neurons of the superior cervical ganglion express heterogeneous acetylcholine receptors and differ in relative amounts of acetylcholine receptor subtypes expressed.

Modulation of nicotinic acetylcholine receptor activity in submucous neurons by intracellular messengers.

The effects on acetylcholine-induced membrane currents (ACh currents), produced by agents known to modify the activity of intracellular messengers, were studied in the neurons of the guinea-pig ileum submucous plexus (SMP) using a whole-cell patch clamp recording method. The ACh currents were not affected by forskolin, the adenylate cyclase activator, regardless of whether or not ATP and GTP were present in the intracellular solution, and by phorbol 12-myristate 13-acetate, the protein kinase C activator. The ACh currents were strongly suppressed by thapsigargin, the microsomal calcium ATPase inhibitor, and genistein, the tyrosine protein kinase inhibitor. They were also suppressed by 3-isobutyl-1-methylxanthine, the cyclic-AMP phosphodiesterase inhibitor, regardless of the presence of forskolin in the extracellular solution and ATP and GTP in the intracellular solution. In addition, the currents were suppressed by activation of P2 purinoceptors with ATP, which could not be explained by a direct effect of ATP on nicotinic acetylcholine receptors (nAChRs). Reactive blue 2, the P2y purinoceptor antagonist, did not abolish inhibition of the ACh current by ATP. Alpha,beta-Imido-ATP and adenosine caused no membrane current responses and did not influence the ACh currents. These results suggest that the activity of the nAChRs in the SMP neurons is strongly suppressed by raised intracellular Ca2+ level, without involvement of protein kinases A and C, and may involve the participation of tyrosine kinase. The activity of nAChRs is also influenced by the activity of P2 purinoceptors; the mechanisms responsible for this influence are not yet clear. So, the activity of the SMP neuronal nAChRs is relatively independent on the intracellular signaling known to influence many other groups of transmitter-gated receptors of neuronal membrane.

[Intracellular regulation of neuronal nicotinic cholinergic receptors]. / Vnutrikletochnaia reguliatsiia neironal'nykh nikotinovykh kholinoretseptorov.

Effects of substances affecting intracellular secondary messengers on the membrane currents evoked by ionophoretic application of acetylcholine (ACh currents) and on the excitatory postsynaptic currents (EPSC) evoked by single stimuli applied to preganglionic nerve fibres, were studied in neurones of the rat isolated superior cervical ganglion. Forskolin, the protein kinase A activator, and isobutyl-methyxanthine, the phosphodiesterase inhibitor, decreased the ACh currents. Neither forskolin nor isobutyl-methylxanthine affected the EPSC amplitude or the EPSC decay time constant. Phorbol ester, the protein kinase C activator, decreased the ACh current but did not affect either EPSC amplitude or the EPSC decay time constant. Thapsigargin, the intracellular calcium releaser, decreased the ACh current and the EPSC amplitude but did not affect the EPSC decay time constant. The data obtained suggest that nicotinic acetylcholine receptors (nAChRs) of ganglion neurones are not modulated through the pathways involving protein kinase A or protein kinase C. The nAChRs sensitivity to both exogenous and nerve-released acetylcholine is reduced by intracellular calcium without affecting kinetics of their ionic channels.

Acetylcholine receptors in rat cardiac neurones.

Membrane potential changes produced by acetylcholine (ACh), and their underlying mechanisms, were studied in neurones of isolated cardiac ganglia of the rat by means of intracellular microelectrodes. Five components of membrane potential change could be detected in cardiac neurones following 1-5 s micro-application of ACh: (i) fast depolarization resulting from an activation of nonselective cationic conductance; (ii) slow depolarization associated with a decreased membrane conductance, presumably for potassium ions; slow hyperpolarization which consisted of (iii) early and (iv) late parts resulting from an activation of calcium-sensitive potassium current and from inhibition of steady-state inward current, respectively; and (v) delayed slow hyperpolarization associated with an increased conductance, most likely for potassium ions. Components (i), (iii) and (iv) persisted in the presence of atropine and were inhibited by nicotinic antagonists. Thus they were due to activation of nicotinic ACh receptors. However, the sensitivity of component (i) to ganglion-blocking agents appeared to be rather low: IC50s for inhibiting (i) were 226 +/- 34.2 microM, 31.2 +/- 4.31 microM and 15.3 +/- 3.27 microM for hexamethonium, d-tubocurarine, and trimetaphan, respectively. Components (ii) and (v) were abolished by atropine (1 microM) and mimicked by muscarine (component (ii) also persisted in d-tubocurarine), hence they resulted from activation of muscarinic ACh receptors. It is concluded that cardiac neurones are endowed with both nicotinic and muscarinic ACh receptors. Their activation leads to membrane depolarization and discharges followed by hyperpolarization and inhibition of discharges.