Inhibition of TASK1-like channels by muscarinic receptor stimulation in rat adrenal medullary cells

The muscarinic receptor is known to be involved in the acetylcholine-induced secretion of catecholamines in the adrenal medulla (AM) cells of various mammals. The ionic mechanisms, however, have not been elucidated yet. Thus, we investigated the issue in acutely isolated rat AM cells with the perforated patch clamp method. Bath application of 30 μM muscarine induced depolarization with the consequent generation of action potentials or an inward current at negative membrane potentials. The muscarine-sensitive current instantaneously changed in amplitude upon application of command pulses without a time-dependent component, altered the polarity as a K⁺-electrode, and showed rectification of the Goldman-Hodgkin-Katz (GHK) type. The whole-cell current at −20 mV was inhibited by external H⁺ ions with a concentration responsible for half inhibition of pH 7.09 and muscarine failed to induce a further inward current during exposure to a saline in which pH decreased to 6.5. A similar occlusion occurred in secretion when pH in muscarine-containing saline decreased to 6.6. RT-PCR, immunoblotting, and immunocytochemistry suggested that rat AM cells mainly express the TASK1 channel. This TASK channel in AM cells may directly sense a decrease in blood pH, which occurs during exercise. The muscarine action was mimicked by oxotremorine–methiodide, but not by oxotremorine. The present results indicate that activation of muscarinic receptors or a decrease in external pH in the rat AM cell induces secretion through the inhibition of TASK1-like channels.     

Muscarinic modulation of GABAergic transmission to neurons in the rat subfornical organ

Cholinergic actions on subfornical organ (SFO) neurons in rat slice preparations were studied by using whole cell voltage- and current-clamp recordings. In the voltage-clamp recordings, carbachol and muscarine decreased the frequency of GABAergic inhibitory postsynaptic currents (IPSCs) in a dose-dependent manner, with no effect on the amplitudes or the time constants of miniature IPSCs. Meanwhile, carbachol did not influence the amplitude of the outward currents induced by GABA. Furthermore, carbachol and muscarine also elicited inward currents in a TTX-containing solution. From the current-voltage relationship, the reversal potential was estimated to be -7.1 mV. These carbachol-induced responses were antagonized by atropine. In the current-clamp recordings, carbachol depolarized the membrane with increased frequency of action potentials. These observations suggest that acetylcholine suppresses GABA release through muscarinic receptors located on the presynaptic terminals. Acetylcholine also directly affects the postsynaptic membrane through muscarinic receptors, by opening nonselective cation channels. A combination of these presynaptic and postsynaptic actions may enhance activation of SFO neurons by acetylcholine.     

Ca(2+)-dependent K(+) current and exocytosis in responses to caffeine and muscarine in voltage-clamped guinea-pig adrenal chromaffin cells

We characterized changes in membrane currents and the cytosolic Ca(2+) concentration, [Ca(2+)](i), in response to caffeine, and compared them with those in response to muscarine using the perforated patch-clamp technique and fura-2 microfluorimetry in guinea-pig adrenal chromaffin cells. Catecholamine release from single voltage-clamped cells was monitored with amperometry using carbon microelectrodes. Caffeine produced a transient outward current (I(out)) at holding potentials over - 60 mV, increasing in amplitude with increasing the potentials. It also evoked a rapid increase of [Ca(2+)](i) at all potentials examined. The current-voltage relation revealed that the activation of K(+) channels was responsible for the I(out) evoked by caffeine. Both current and [Ca(2+)](i) responses were reversibly abolished by cyclopiazonic acid, an inhibitor of Ca(2+)-pump ATPase. At - 30 mV, the caffeine-induced I(out), but not [Ca(2+)](i), was partly inhibited by either charybdotoxin or apamin. In the majority of cells tested, caffeine induced a larger I(out) but a smaller [Ca(2+)](i) increase than muscarine. Caffeine and muscarine increased catecholamine release from voltage-clamped single cells concomitant with the transient increase of [Ca(2+)](i), and there was a positive correlation between them. These results indicate that caffeine activates Ca(2+)-dependent K(+) channels and catecholamine secretion due to the release of Ca(2+) from internal stores in voltage-clamped adrenal chromaffin cells of the guinea-pig. There seems to be a spatial difference between [Ca(2+)](i) increased by Ca(2+) release from caffeine-sensitive stores and that released from muscarine (inositol 1,4,5-trisphosphate)-sensitive ones.     

Characteristics of cytosolic Ca2+ elevation induced by muscarinic receptor activation in single adrenal chromaffin cells of the guinea pig

In Fura-2 loaded-single guinea pig adrenal chromaffin cells, muscarine, nicotine and KCl all caused an early peak rise in intracellular Ca concentration ([Ca²⁺]_i) followed by a sustained rise. In Ca²⁺-free solution, muscarine, but neither nicotine nor KCl, caused a transient increase in [Ca²⁺]_i, which was partially reduced by preceding application of caffeine or by treatment with ryanodine plus caffeine. In voltage-clamped cells at a holding potential of −60 mV, the muscarine-induced [Ca²⁺]_i, rise, especially its sustained phase, decreased in magnitude. intracellular application of inositol 1,4,5-trisphosphate caused a transient increase in [Ca²⁺]_i and inhibited the following [Ca²⁺]_i response to muscarine without affecting responses to nicotine and a depolarizing pulse. Muscarine evoked membrane depolarization following brief hyperpolarization in most cells tested. There was a significant positive correlation between the amplitude of the depolarization and the magnitude of the sustained rise in [Ca²⁺]_i. Muscarine-induced sustained [Ca²⁺]_i rise was much greater in the current-clamp mode than that in the voltage-clamp mode. The sustained phase of [Ca²⁺]_i rise and Mn²⁺ influx in response to muscarine were suppressed by a voltage-dependent Ca²⁺ channel blocker, methoxyverapamil. These results suggest that stimulation of muscarinic receptors causes not only extracellular Ca²⁺ entry, but also Ca²⁺ mobilization from inositol 1,4,5-trisphosphate-sensitive intracellular stores. Voltage-dependent Ca²⁺ channels may function as one of the Ca²⁺ entry pathways activated by muscarinic receptor in guinea pig adrenal chromaffin cells.     

TASK (TWIK-related acid-sensitive K+ channel) is expressed in glomerulosa cells of rat adrenal cortex and inhibited by angiotensin II

The present study was conducted to explore the possible contribution of a recently described leak K+ channel, TASK (TWIK-related acid-sensitive K+ channel), to the high resting K+ conductance of adrenal glomerulosa cells. Northern blot analysis showed the strongest TASK message in adrenal glomerulosa (capsular) tissue among the examined tissues including heart and brain. Single-cell PCR demonstrated TASK expression in glomerulosa cells. In patch-clamp experiments performed on isolated glomerulosa cells the inward current at -100 mV in 30 mM [K+] (reflecting mainly potassium conductance) was pH sensitive (17+/-2% reduction when the pH changed from 7.4 to 6.7). In Xenopus oocytes injected with mRNA prepared from adrenal glomerulosa tissue the expressed K+ current at -100 mV was virtually insensitive to tetraethylammonium (3 mM) and 4-aminopyridine (3 mM). Ba2+ (300 microM) and Cs+ (3 mM) induced voltage-dependent block. Lidocaine (1 mM) and extracellular acidification from pH 7.5 to 6.7 inhibited the current (by 28% and 16%, respectively). This inhibitory profile is similar (although it is not identical) to that of TASK expressed by injecting its cRNA. In oocytes injected with adrenal glomerulosa mRNA, TASK antisense oligonucleotide reduced significantly the expression of K+ current at -100 mV, while the sense oligonucleotide failed to have inhibitory effect. Application of angiotensin II (10 nM) both in isolated glomerulosa cells and in oocytes injected with adrenal glomerulosa mRNA inhibited the K+ current at -100 mV. Similarly, in oocytes coexpressing TASK and ATla angiotensin II receptor, angiotensin II inhibited the TASK current. These data together indicate that TASK contributes to the generation of high resting potassium permeability of glomerulosa cells, and this background K+ channel may be a target of hormonal regulation.     

Interaction of Ca2+ agonist and depolarization on Ca2+ channel current in guinea pig detrusor cells [published erratum appears in J Gen Physiiol 1996 Jun;107(6):755]

The interaction of large depolarization and dihydropyridine Ca2+ agonists, both of which are known to enhance L-type Ca2+ channel current, was examined using a conventional whole-cell clamp technique. In guinea pig detrusor cells, only L-type Ca2+ channels occur. A second open state (long open state: O2) of the Ca2+ channels develops during large depolarization (at +80 mV, without Ca2+ agonists). This was judged from lack of inactivation of the Ca2+ channel current during the large depolarizing steps (5 s) and slowly deactivating inward tail currents (= 10-15 ms) upon repolarization of the cell membrane to the holding potential (-60 mV). Application of Bay K 8644 (in 2.4 mM Ca(2+)- containing solutions) increased the amplitude of the Ca2+ currents evoked by simple depolarizations, and made it possible to observe inward tail currents (= 2.5-5 ms at -60 mV). The open state induced by large depolarization (O2*) in the Bay K 8644 also seemed hardly to inactivate. After preconditioning with large depolarizing steps, the decay time course of the inward tail currents upon repolarization to the holding potential (-60 mV) was significantly slowed, and could be fitted reasonably with two exponentials. The fast and slow time constants were 10 and 45 ms, respectively, after 2 s preconditioning depolarizations. Qualitatively the same results were obtained using Ba2+ as a charge carrier. Although the amplitudes of the inward currents observed in the test step and the subsequent repolarization to the holding potential were decreased in the same manner by additional application of nifedipine (in the presence of Bay K 8644), the very slow deactivation time course of the tail current was little changed. The additive enhancement by large depolarization and Ca2+ agonists of the inward tail current implies that two mechanisms separately induce long opening of the Ca2+ channels: i.e., that there are four open states.     

Voltage gated ionic channels in rat cultured astrocytes, reactive astrocytes and an astrocyte-oligodendrocyte progenitor cell

Astrocytes (both type 1 and type 2), cultured from the central nervous system of newborn or 7 day old rats show voltage gated sodium and potassium channels that are activated when the membrane is depolarized to greater than -40 mV. The sodium channels in these cells have an h-infinity curve similar to that of nodal membranes but the activation (peak current-voltage) curves are shifted along the voltage axis by about +30 mV. These sodium currents are blocked only by high concentrations of tetrodotoxin. The voltage activated potassium currents in both types of astrocyte show at least two components; an inactivating component that is suppressed at holding potentials of greater than -40 mV and a persistent, non-inactivating current. Several types of single channel currents were observed in outside-out membrane patches from type 2 astrocytes. One type of potassium channel showed inactivation on depolarization and may contribute to the whole-cell inactivating current. In contrast, oligodendrocytes showed no obvious voltage gated membrane channels. The properties of the type 2 astrocyte-oligodendrocyte progenitor cell were investigated in two ways: 1) by examination of cells just beginning to differentiate along the "electrically silent" oligodendrocyte pathway or 2) by recording from progenitor cells cultured for 24 hours in the presence of cycloheximide to block the appearance of new membrane channels. In both cases, voltage gated inward (sodium) and outward (potassium) currents were noted. The outward current response showed both an inactivating and a non-inactivating component. Similar voltage activated inward and outward membrane currents were noted in reactive astrocytes freshly isolated (3-6 hours) from lesioned areas of adult rat brains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of the phospholipase C beta1 pathway in desensitization of the carbachol-activated nonselective cationic current in murine gastric myocytes

In murine gastrointestinal myocytes muscarinic stimulation activates nonselective cation channels via a G-protein and Ca2+-dependent pathway. We recorded inward cationic currents following application of carbachol (ICCh) to murine gastric myocytes held at -60 mV, using the whole-cell patch-clamp method. The properties of the inward cationic currents were similar to those of the nonselective cation channels activated by muscarinic stimulation in other gastrointestinal smooth muscle cells. CCh-induced ICCh and spontaneous decay of ICCh (desensitization of ICCh) occurred. Unlike the situation in guinea pig gastric myocytes, desensitization was not affected by varying [EGTA]i. Pretreatment with the PLC inhibitor (U73122) blocked the activation of ICCh, and desensitization of ICCh was attenuated in PLC beta1 knock-out mice. These results suggest that the desensitization of ICCh in murine gastric myocytes is not due to a pathway dependent on intracellular Ca2+ but to the PLC beta1 pathway.     

Modulatory effects of oligodendrocytes on the conduction velocity of action potentials along axons in the alveus of the rat hippocampal CA1 region

Like neurons and astrocytes, oligodendrocytes have a variety of neurotransmitter receptors and ion channels. However, except for facilitating the rapid conduction of action potentials by forming myelin and buffering extracellular K(+), little is known about the direct involvement of oligodendrocytes in neuronal activities. To investigate their physiological roles, we focused on oligodendrocytes in the alveus of the rat hippocampal CA1 region. These cells were found to respond to exogenously applied glutamate by depolarization through N-methyl-D-aspartate (NMDA) receptors and non-NMDA receptors. Electrical stimulation of the border between the alveus and stratum oriens evoked inward currents through several routes involving glutamate receptors and inward rectifier K(+) channels. Moreover, electrical stimulation resembling in vivo activity evoked long-lasting depolarization. To examine the modulatory effects of oligodendrocytes on neuronal activities, we performed dual, whole-cell recording on CA1 pyramidal neurons and oligodendrocytes. Direct depolarization of oligodendrocytes shortened the latencies of action potentials evoked by antidromic stimulation. These results indicate that oligodendrocytes increase the conduction velocity of action potentials by a mechanism additional to saltatory conduction, and that they have active roles in information processing in the brain.     

Prostaglandin E2 Activates Ca2+ Channels in Bovine Adrenal Chromaffin Cells

We have demonstrated that prostaglandin E2 (PGE2) treatment of bovine adrenal chromaffin cells results in a sustained elevation of intracellular Ca2+ concentration ([Ca2+]i) in these cells. Because the continued elevation of [Ca2+]i was dependent on extracellular Ca2+ concentration, it can be assumed that the PGE2-induced [Ca2+]i increase is due, at least in part, to an opening of membrane Ca2+ channels. In this study, we used electrophysiological methods to examine the mechanism of the PGE2-induced [Ca2+]i increase directly. Puff application of PGE2 to the external medium resulted in a prolonged depolarization in about half of the chromaffin cells examined. In whole-cell voltage-clamp recordings, an increase in inward current was observed over a 6-7 min period following bath application of PGE2 (greater than or equal to 10 microM), even in the absence of external Na+. This inward current was abolished when the recordings were made with the cells in a Ca2(+)-free medium, but it was not inhibited by Mn2+, a blocker of voltage-dependent Ca2+ channels. In cell-attached patch-clamp configuration, PGE2 produced an increase in the opening frequency of inward currents. The reversal potential of the PGE2-induced currents was about +40 mV, which is close to the reversal potential of the Ca2+ channel. The opening frequency was not affected by membrane potential changes. In inside-out patch-clamp configuration, inositol 1,4,5-trisphosphate (2 microM) added to the cytoplasmic side activated the Ca2(+)-channel currents, but PGE2 was ineffective when applied to the cytoplasmic side. These results suggest that PGE2 activates voltage-independent Ca2+ channels in chromaffin cells through a diffusible second messenger, possibly inositol 1,4,5-trisphosphate.     

Carbachol regulates pacemaker activities in cultured interstitial cells of Cajal from the mouse small intestine

We studied the effect of carbachol on pacemaker currents in cultured interstitial cells of Cajal (ICC) from the mouse small intestine by muscarinic stimulation using a whole cell patch clamp technique and Ca²⁺-imaging. ICC generated periodic pacemaker potentials in the current-clamp mode and generated spontaneous inward pacemaker currents at a holding potential of–70 mV. Exposure to carbachol depolarized the membrane and produced tonic inward pacemaker currents with a decrease in the frequency and amplitude of the pacemaker currents. The effects of carbachol were blocked by 1-dimethyl-4-diphenylacetoxypiperidinium, a muscarinic M₃ receptor antagonist, but not by methotramine, a muscarinic M₂ receptor antagonist. Intracellular GDP-β-S suppressed the carbachol-induced effects. Carbachol-induced effects were blocked by external Na⁺-free solution and by flufenamic acid, a non-selective cation channel blocker, and in the presence of thapsigargin, a Ca²⁺-ATPase inhibitor in the endoplasmic reticulum. However, carbachol still produced tonic inward pacemaker currents with the removal of external Ca²⁺. In recording of intracellular Ca²⁺ concentrations using fluo 3-AM dye, carbachol increased intracellular Ca²⁺ concentrations with increasing of Ca²⁺ oscillations. These results suggest that carbachol modulates the pacemaker activity of ICC through the activation of non-selective cation channels via muscarinic M₃ receptors by a G-protein dependent intracellular Ca²⁺ release mechanism.     

Voltage-gated potassium channels in brown fat cells

We studied the membrane currents of isolated cultured brown fat cells from neonatal rats using whole-cell and single-channel voltage-clamp recording. All brown fat cells that were recorded from had voltage-gated K currents as their predominant membrane current. No inward currents were seen in these experiments. The K currents of brown fat cells resemble the delayed rectifier currents of nerve and muscle cells. The channels were highly selective for K+, showing a 58-mV change in reversal potential for a 10-fold change in the external [K+]. Their selectivity was typical for K channels, with relative permeabilities of K+ greater than Rb+ greater than NH+4 much greater than Cs+, Na+. The K currents in brown adipocytes activated with a sigmoidal delay after depolarizations to membrane potentials positive to -50 mV. Activation was half maximal at a potential of -28 mV and did not require the presence of significant concentrations of internal calcium. Maximal voltage-activated K conductance averaged 20 nS in high external K+ solutions. The K currents inactivated slowly with sustained depolarization with time constants for the inactivation process on the order of hundreds of milliseconds to tens of seconds. The K channels had an average single-channel conductance of 9 pS and a channel density of approximately 1,000 channels/cell. The K current was blocked by tetraethylammonium or 4-aminopyridine with half maximal block occurring at concentrations of 1-2 mM for either blocker. K currents were unaffected by two blockers of Ca2+-activated K channels, charybdotoxin and apamin. Bath-applied norepinephrine did not affect the K currents or other membrane currents under our experimental conditions. These properties of the K channels indicate that they could produce an increase in the K+ permeability of the brown fat cell membrane during the depolarization that accompanies norepinephrine-stimulated thermogenesis, but that they do not contribute directly to the norepinephrine-induced depolarization.     

Molecular and electrophysiological characteristics of K+ conductance sensitive to acidic pH in aortic smooth muscle cells of WKY and SHR

Changes in K(+) conductances and their contribution to membrane depolarization in the setting of an acidic pH environment have been studied in myocytes from aortic smooth muscle cells of spontaneously hypertensive rats (SHR) compared with those from Wistar-Kyoto (WKY) rats. The resting membrane potential (RMP) of aortic smooth muscle at extracellular pH (pH(o)) of 7.4 was significantly more depolarized in SHR than in WKY rats. Acidification to pH(o) 6.5 made this difference in RMP between SHR and WKY rats more significant by further depolarizing the SHR myocytes. Large-conductance Ca(2+)-activated K(+) (BK) currents, which were markedly suppressed by acidification, were larger in aortic myocytes of SHR than in those of WKY rats. In contrast, acid-sensitive, non-BK currents were smaller in SHR. Western blot analyses showed that expression of BK-alpha- and -beta(1) subunits in SHR aortas was upregulated and comparable with those in WKY rats, respectively. Additional electrophysiological and molecular studies showed that pH- and halothane-sensitive two-pore domain weakly inward rectifying K(+) channel (TWIK)-like acid-sensitive K(+) (TASK) channel subtypes were functionally expressed in aortas, and TASK1 expression was significantly higher in WKY than in SHR. Although the background current through TASK channels at normal pH(o) (7.4) was small and may not contribute significantly to the regulation of RMP, TASK channel activation by halothane or alkalization (pH(o) 8.0) induced significant hyperpolarization in WKY but not in SHR. In conclusion, the larger depolarization and subsequent abnormal contractions after acidification in aortic myocytes in the setting of SHR hypertension are mainly attributable to the larger contribution of BK current to the total membrane conductance than in WKY aortas.     

Modulation of the heteromeric Kir4.1-Kir5.1 channels by P(CO(2)) at physiological levels

Several inward rectifier K(+) (Kir) channels are pH-sensitive, making them potential candidates for CO(2) chemoreception in cells. However, there is no evidence showing that Kir channels change their activity at near physiological level of P(CO(2)), as most previous studies were done using high concentrations of CO(2). It is known that the heteromeric Kir4.1-Kir5.1 channels are highly sensitive to intracellular protons with pKa value right at the physiological pH level. Such a pKa value may allow these channels to regulate membrane potentials with modest changes in P(CO(2)). To test this hypothesis, we studied the Kir4.1-Kir5.1 currents expressed in Xenopus oocytes and membrane potentials in the presence and absence of bicarbonate. Evident inhibition of these currents (by approximately 5%) was seen with P(CO(2)) as low as 8 torr. Higher P(CO(2)) levels (23-60 torr) produced stronger inhibitions (by 30-40%). The inhibitions led to graded depolarizations (5-45 mV with P(CO(2)) 8-60 torr). Similar effects were observed in the presence of 24 mM bicarbonate and 5% CO(2). Indeed, the Kir4.1-Kir5.1 currents were enhanced with 3% CO(2) and suppressed with 8% CO(2) in voltage clamp, resulting in hyper- (-9 mV) and depolarization (16 mV) in current clamp, respectively. With physiological concentration of extracellular K(+), the Kir4.1-Kir5.1 channels conduct substantial outward currents that were similarly inhibited by CO(2) as their inward rectifying currents. These results therefore indicate that the heteromeric Kir4.1-Kir5.1 channels are modulated by a modest change in P(CO(2)) levels. Such a modulation alters cellular excitability, and enables the cell to detect hypercapnia and hypocapnia in the presence of bicarbonate.     

Identifying nicotinic and muscarinic cholinoreceptors at the soma of neuron RPa4 inHelix lucorum

Cholinoreceptors were identified at the somatic membrane of theHelix lucorum RPa4 neuron using intracellular recording techniques. Application of specific agonists of nicotinic (nicotine, cytisine) and muscarinic (muscarine, arecoline) cholinoreceptors to the soma produced neuronal depolarization. The depolarization produced by applying acetylcholine to the cell was of short duration and was often replaced by hyperpolarization. Both selective desensitization of receptors by nicotine and muscarine as well as receptor occupancy by cytisine and arecoline reduced acetylchloline-induced response. The nicotinic cholinoblocker d-tubocurarine substantially inhibited responses to nicotinic cholinomimetics, while atropine, a muscarinic cholinoblocker, depressed response to muscarinic cholinomimetics. Acetylcholine-induced response was inhibited by both cholinoblockers more or less equally. The presence at the RPa4 neuronal somatic membrane is postulated of standard nicotinic and muscarinic cholinoreceptors similar to those found in vertebrates.M. V. Lomonsov State University, Moscow. Translated from Neirofiziologiya, Vol. 20, No. 2, pp. 203–212, March–April, 1988.     

Membrane properties of isolated mudpuppy taste cells

The voltage-dependent currents of isolated Necturus lingual cells were studied using the whole-cell configuration of the patch-clamp technique. Nongustatory surface epithelial cells had only passive membrane properties. Small, spherical cells resembling basal cells responded to depolarizing voltage steps with predominantly outward K+ currents. Taste receptor cells generated both outward and inward currents in response to depolarizing voltage steps. Outward K+ currents activated at approximately 0 mV and increased almost linearly with increasing depolarization. The K+ current did not inactivate and was partially Ca++ dependent. One inward current activated at -40 mV, reached a peak at -20 mV, and rapidly inactivated. This transient inward current was blocked by tetrodotoxin (TTX), which indicates that it is an Na+ current. The other inward current activated at 0 mV, peaked at 30 mV, and slowly inactivated. This more sustained inward current had the kinetic and pharmacological properties of a slow Ca++ current. In addition, most taste cells had inwardly rectifying K+ currents. Sour taste stimuli (weak acids) decreased outward K+ currents and slightly reduced inward currents; bitter taste stimuli (quinine) reduced inward currents to a greater extent than outward currents. It is concluded that sour and bitter taste stimuli produce depolarizing receptor potentials, at least in part, by reducing the voltage-dependent K+ conductance.     

Pharmacological parameters of muscarinic cholinoreceptors in skeletal muscles

It has been shown that bath application of muscarine delayed the early post-denervation depolarization in the muscle fibers incubated for 3 h in culture medium. The greatest reduction of the post-devervation depolarization was observed with 50 nmol/l muscarine. Atropine, a muscarinic antagonist, clozapine, a specific inhibitor of M1/M5-cholinergic receptors, and nitrocaramiphen, a M1-antagonist, completely removed the hyperpolarizing effect of muscarine. 4-DAMP, a specific inhibitor of M3-cholinergic receptors, himbacine, an antagonist of M2-cholinergic receptors, and tropicamide, a specific inhibitor of M2/M4-cholinergic receptors, failed to prevent the effect of muscarine. A M1/M2 muscarine agonists propargyl and but-2-ynyl esters of arecaidine had apparent muscarine-like effect. Nitrocaramiphen, and not himbacine, prevented the hyperpolarizing effect of these cholinomimetics. It is concluded that muscarine and esters of arecaidine delay the development of early postdenervation depolarization in M1-cholinergic receptors of skeletal muscle.     

McN-A-343, a specific agonist of M1-muscarinic receptors, exerts antinicotinic and antimuscarinic effects in the rat adrenal medulla

Pirenzepine, McN-A-343 and oxotremorine were used to determine the subtypes of muscarinic receptors involved in the secretion of catecholamines from the isolated perfused adrenal gland of the rat. In the presence of 0.1 microM pirenzepine, the concentration-secretion curve for muscarine was shifted in parallel to the right by almost one log unit. With 0.5 microM the shift was over two log units. The apparent dissociation constant for pirenzepine was about 1.12 X 10(-8) M. Perfusion with McN-A-343 (1-30 microM) did not evoke the secretion of catecholamines. A further increase to very high concentrations (100-1000 microM) caused only a modest secretion (about 50 ng/5 min with 300 microM as compared to the same amount of secretion obtained with 1 microM muscarine). Secretion evoked by nicotine was significantly reduced (30%) by 3 microM McN-A-343, and the inhibition increased (90%) with higher concentrations (100 microM). McN-A-343 also produced concentration-dependent inhibition of catecholamine secretion evoked by muscarine. A significant effect was observed at 30 microM and reached a maximum level at 300 microM. Oxotremorine, like McN-A-343 was a partial agonist on the muscarinic receptors; but unlike McN-A-343, did not block the stimulatory effects of nicotine. Although the pirenzepine data suggest that M1 receptors are responsible for the secretion of catecholamines in the rat adrenal medulla, this conclusion is not supported by the results obtained with the M1-receptor agonist, McN-A-343, which proved to be an effective blocker of muscarinic as well as nicotinic receptors.     

Nonselective cationic currents activated by acetylcholine in swine tracheal smooth muscle cells

Membrane currents in isolated swine tracheal smooth muscle cells were investigated using a pipette solution containing BAPTA-Ca2+ buffer and Cs+ as the major cation. With a pipette solution containing 100 nM free Ca2+, acetylcholine (ACh; 1-100 microM), in a concentration-dependent manner, activated a current without inducing shortening of cells, although neither 1 mM histamine nor 1 microM leukotriene D4 activated the current (n = 7, n is the number of cells). The effect of 100 microM ACh was suppressed by pretreatment with 100 microM atropine (n = 6) or intracellular application of preactivated pertussis toxin at a concentration of 0.1 microg x mL(-1) (n = 8). Genistein (0.1-100 microM), in a concentration-dependent manner, suppressed the activation of the inward current by 100 microM ACh, whereas it did not significantly suppress that of the outward current (n = 6-8). With a pipette solution containing 50 nM free Ca2+, outward current, but not inward current, was activated by 100 microM ACh (n = 10). When the pipette solution had free Ca2+ concentrations greater than 50 nM, the inward current together with the outward current was activated. The ratio between the amplitude of the inward and outward currents was significantly increased as the free Ca2+ concentration in the pipette solution increased. The steady-state activation curve of the ACh-activated current with the 50 nM free Ca2+ pipette solution was fitted by a single Boltzmann distribution (Vh = +69.8 mV, k = -11.9 mV, n = 10). The activation time constant became smaller as the membrane potential was more depolarized (164.3+/-5.9 ms at +40 mV to 92.4+/-6.3 ms at +120 mV, n = 10). The reversal potential was not significantly changed by reducing extracellular Cl- concentration to one-tenth of the control (n = 8), suggesting that the current is a nonselective cationic current. These results suggest that ACh activates an outward nonselective cationic current via pertussis toxin-sensitive G-protein(s) coupled with muscarinic receptors. Involvement of genistein-sensitive tyrosine kinase in the activation process of the current is unlikely.     

A calcium-permeable channel activated by muscarinic acetylcholine receptors and InsP3 in developing chick ciliary ganglion neurons

The electrical responses elicited by the muscarinic cholinergic pathway have been studied in cultured embryonic chick ciliary ganglion (CG) neurons. Neurons obtained from E7-E8 ganglia were maintained in serum-free medium for 1 to 3 days. Stimulation with 50 microM muscarine induced depolarizing responses in about 30% of the cells tested. In voltage clamp experiments at a holding potential of -50 mV, an inward current could be recorded in the same percentage of cells in response to muscarinic stimulation. In single channel experiments, with standard physiological solution in the pipette, muscarine transiently activated an inward conducting channel. Cell-attached recordings with 100 mM CaCl(2) in the pipette provided evidence that muscarinic agonists can activate a cationic calcium-permeable channel. Two main conductance levels could be detected, of 2.3+/-0.6 and 5.6+/-0.6 pS, respectively. In excised patches, addition of 5-20 microM inositol 1,4,5-trisphosphate (InsP(3)) to the bath reactivated a channel that could be blocked by heparin and whose characteristics were very similar to those of the channel seen in response to muscarinic stimulation. A channel with similar properties has been previously shown to be activated by basic fibroblast growth factor (bFGF) and InsP(3) in the same preparation.