Seiichi Komori

GTP‐binding protein involvement in membrane currents evoked by carbachol and histamine in guinea‐pig ileal muscle.

1. Single smooth muscle cells obtained by enzymic dispersion of the longitudinal muscle layer of guinea‐pig ileum were used for recording membrane currents under whole‐cell voltage clamp in response to carbachol (100 microM, unless otherwise stated) or histamine (100 microM) applied extracellularly. 2. At a holding potential of 0 mV, a transient outward current was evoked by carbachol and histamine. Responses to the two agonists were very similar in size and time course to the current response to caffeine (10 mM). The response to carbachol was virtually absent in the presence of histamine, and vice versa. Caffeine was without effect in the presence of either of these agonists. Inclusion of EGTA (10 or 20 mM) in the pipette abolished the responses to carbachol, histamine and caffeine. Thus, the outward current responses were considered to represent opening of Ca(2+)‐activated K+ channels in response to a massive release of Ca2+ from the same stores by these three agents. 3. An inward current was evoked by carbachol and histamine, but not by caffeine at a holding potential of ‐40 mV, which was considered to represent opening of cationic channels. The carbachol‐induced inward current was much longer in duration and larger in size than the histamine‐induced inward current. 4. Inclusion of GDP beta S (2 mM) in the pipette abolished the inward and outward current responses to histamine, but inhibited only part of those to carbachol. 5. When the holding potential was held at 0 mV with inclusion of GTP gamma S (0.1‐1 mM) in the pipette, spontaneous transient outward currents appeared immediately after break‐through but disappeared a few minutes later. Under these conditions, caffeine (10 mM) was almost without effect, suggesting that GTP gamma S had released Ca2+ stores. When the holding potential was held at ‐40 mV and GTP gamma S (0.1 or 0.2 mM) was present in the pipette, an inward current developed a few minutes after break‐through. During the GTP gamma S‐induced inward current, application of carbachol or histamine produced no further inward current. However, when 0.01 mM‐GTP gamma S was included in the pipette solution, carbachol‐ and histamine‐induced inward currents were potentiated. 6. Pretreated with 2‐5 micrograms/ml pertussis toxin (PTX) did not change noticeably the outward current responses to carbachol and histamine, but abolished or markedly reduced the inward current responses.(ABSTRACT TRUNCATED AT 400 WORDS)

M2 and M3 muscarinic receptor-mediated contractions in longitudinal smooth muscle of the ileum studied with receptor knockout mice

Isometric contractile responses to carbachol were studied in ileal longitudinal smooth muscle strips from wild‐type mice and mice genetically lacking M2 or M3 muscarinic receptors, in order to characterize the mechanisms involved in M2 and M3 receptor‐mediated contractile responses. Single applications of carbachol (0.1–100 μM) produced concentration‐dependent contractions in preparations from M2‐knockout (KO) and M3‐KO mice, mediated via M3 and M2 receptors, respectively, as judged by the sensitivity of contractile responses to blockade by the M2‐preferring antagonist methoctramine (300 nM) or the M3‐preferring antagonist 4‐DAMP (30 nM). The M2‐mediated contractions were mimicked in shape by submaximal stimulation with high K+ concentrations (up to 35 mM), almost abolished by voltage‐dependent Ca2+ channel (VDCC) antagonists or depolarization with 140 mM K+ medium, and greatly reduced by pertussis toxin (PTX) treatment. The M3‐mediated contractions were only partially inhibited by VDCC antagonists or 140 mM K+‐depolarization medium, and remained unaffected by PTX treatment. The contractions observed during high K+ depolarization consisted of different components, either sensitive or insensitive to extracellular Ca2+. The carbachol contractions observed with wild‐type preparations consisted of PTX‐sensitive and ‐insensitive components. The PTX‐sensitive component was functionally significant only at low carbachol concentrations. The results suggest that the M2 receptor, through PTX‐sensitive mechanisms, induces ileal contractions that depend on voltage‐dependent Ca2+ entry, especially associated with action potential discharge, and that the M3 receptor, through PTX‐insensitive mechanisms, induces contractions that depend on voltage‐dependent and ‐independent Ca2+ entry and intracellular Ca2+ release. In intact tissues coexpressing M2 and M3 receptors, M2 receptor activity appears functionally relevant only when fractional receptor occupation is relatively small.

Oscillations of receptor-operated cationic current and internal calcium in single guinea-pig ileal smooth muscle cells

In single cells isolated from guinea-pig ileal smooth muscle, held under voltage clamp at −40 mV or −50 mV by patch pipette in the whole-cell recording mode, carbachol (CCh) evoked an oscillatory inward cationic current. The frequency of current oscillations increased with increasing CCh concentration. CCh-evoked current oscillations were followed very closely by oscillations in intracellular free Ca2+ estimated from the Indo-1 signal, and were abolished by inclusion of EGTA in the pipette solution. Ryanodine and heparin, but not nifedipine, blocked the generation of current oscillations. CCh-evoked current oscillations were abolished upon withdrawal of extracellular calcium and restored upon its reintroduction. Inclusion of GTP[γS] in the pipette solution caused the generation of an oscillatory inward current, which was blocked by ryanodine. The present results are consistent with the hypothesis that CChevoked cationic current is gated by activation of a G protein and is steeply dependent on [Ca2+]i, fluctuations in the release of Ca2+ from stores during carbachols action produce oscillations in [Ca2+]i which cause similar oscillations in the cationic current.

Modification of membrane currents in mouse neuroblastoma cells following infection with rabies virus

The effect on membrane currents of infection of mouse neuroblastoma NA cells with rabies virus was studied by using the whole‐cell patch clamp technique. Three types of membrane currents, namely voltage‐dependent Na+ current (INa), delayed rectifier K+ current (IK‐DR) and inward rectifier K+ current (IK‐IR) were elicited in uninfected cells. In cells 3 days after infection with the virus, no detectable change was observed in morphology and membrane capacitance, but INa and IK‐IR were significantly decreased in amplitude without any appreciable difference in the time course of current activation and inactivation. The voltage‐dependence of INa activation was significantly shifted in the positive direction along the voltage axis with a decreased slope. IK‐DR remained almost unaltered after the viral infection. The resting membrane potential, measured with a physiological K+ gradient across the cell membrane, was decreased (depolarized) after the viral infection. The depolarization was associated with the decreased amplitude of IK‐IR. These results suggest that infection of mouse neuroblastoma NA cells with rabies virus causes reduction of functional expression of ion channels responsible for INa and IK‐IR, and provide evidence for possible involvement of the change in membrane properties in the pathogenesis of rabies disease.

INHIBITORY EFFECT OF MUSCARINIC RECEPTOR ACTIVATION ON CA2+ CHANNEL CURRENT IN SMOOTH MUSCLE CELLS OF GUINEA-PIG ILEUM

1. The effect of muscarinic receptor stimulation on voltage‐gated calcium channel currents was examined in whole‐cell voltage‐clamped smooth muscle cells of the guinea‐pig ileum. 2. In cells voltage clamped at ‐60 mV and in which calcium channel currents (ICa) were elicited repeatedly by depolarizing pulses (25 ms duration, 0.25 Hz frequency) to 0 mV, carbachol (CCh, 10 microM) induced an inward current (ICCh) and were suppressed ICa, in a biphasic manner; an initial transient component was followed by a more sustained one. 3. A calcium channel current (IBa), when Ba2+ was used as a charge carrier, was also suppressed by CCh in a biphasic manner, as with ICa. The sustained phase of the IBa suppression was significantly smaller than that of the ICa suppression, suggesting that Ca2+ entry exerts a potentiating effect on the current suppression. 4. CCh had little or no effect on calcium channel currents (ICa and IBa) in cells dialysed with a pipette solution containing EGTA (20 mM). 5. Inclusion of GDP‐beta‐S (1 mM) in the pipette solution abolished ICCh and the suppression of IBa. With GTP‐gamma‐S (10 microM) in the pipette, the sustained phase of the IBa suppression remained almost unchanged even after removal of CCh. 6. Pretreatment with 2 micrograms ml‐1 pertussis toxin (PTX), which abolished ICCh, did not change noticeably the initial transient and sustained phases of IBa suppression. 7. Neomycin (100 microM) or heparin (5 mg ml‐1) in the pipette each abolished the initial transient component of ICCh as well as the initial transient phase of IBa suppression. 8. The biphasic effect of CCh on IBa was observed in the presence of either staurosporine (1 microM) or 1‐(5‐isoquinolinesulphonyl)‐2‐methylpiperazine (100 microM). Phorbol 12‐myristate 13‐acetate and phorbol 12,13‐dibutyrate (up to 10 microM) had no inhibitory effect on ICa and IBa. 9. The results suggest that stimulation of the muscarinic receptor causes a biphasic suppression of the voltage‐gated calcium channel currents through a PTX‐insensitive G protein in guinea‐pig ileal smooth muscle cells. The initial transient phase may be brought about by the release of Ca2+ from internal storage sites, and the sustained phase by a Ca(2+)‐dependent mechanism which is independent of the phosphatidylinositol pathway.

Roles of M2 and M3 muscarinic receptors in cholinergic nerve‐induced contractions in mouse ileum studied with receptor knockout mice

The functional roles of M2 and M3 muscarinic receptors in neurogenic cholinergic contractions in gastrointestinal tracts remain to be elucidated. To address this issue, we studied cholinergic nerve‐induced contractions in the ileum using mutant mice lacking M2 or M3 receptor subtypes.

Effects of G-protein-specific antibodies and Gβγ subunits on the muscarinic receptor-operated cation current in guinea-pig ileal smooth muscle cells

The effects on the whole‐cell carbachol‐induced muscarinic cationic current (mIcat) of antibodies against the α‐subunits of various G proteins, as well as the effect of a Gβγ subunit, were studied in single guinea‐pig ileal smooth muscle cells voltage‐clamped at −50 mV. Ionized intracellular calcium concentration, [Ca2+]i, was clamped at 100 nM using a 1,2‐bis(2‐aminophenoxyl‐ethane‐N,N,N′,N′‐tetraacetic acid)/Ca2+ mixture. Application of ascending concentrations of carbachol (1–300 μM) activated mIcat (mean amplitude 0.83 nA at 300 μM carbachol; EC50 8 μM; Hill slope 1.0). A 20 min or longer intracellular application via the pipette solution of Gi3/Go or Go antibodies resulted in about a 70% depression of the maximum response without change in the EC50 value. In contrast, antibodies against α‐subunits of Gi1, Gi1/Gi2, Gi3, Gq/G11 or Gs protein over a similar or longer period did not significantly reduce mIcat. Antibodies to common Gβ or infusion of the Gβγ subunit itself had no effect on mIcat. If cells were exposed briefly to carbachol (50 or 100 μM) at early times (<3 min) after infusion of antibodies to Gαi3/Gαo or to Gαo had begun, carbachol responses remained unchanged even after 20–60 min; that is, the depression of mIcat by these antibodies was prevented. These data show that Gαo protein couples the muscarinic receptor to the cationic channel in guinea‐pig ileal longitudinal smooth muscle and that Gβγ is not involved. They also show that prior activation of the muscarinic receptor presumably causes a long‐lasting postactivation change of the G protein, which is not reflected in mIcat, but acts to hinder antibody binding.

Characteristics of ropivacaine block of Na+ channels in rat dorsal root ganglion neurons

UNLABELLED When used for epidural anesthesia, ropivacaine can produce a satisfactory sensory block with a minor motor block. We investigated its effect on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) Na(+) currents in rat dorsal root ganglion (DRG) neurons to elucidate the mechanisms underlying the above effects. Whole-cell patch-clamp recordings were made from enzymatically dissociated neurons from rat DRG. A TTX-S Na(+) current was recorded preferentially from large DRG neurons and a TTX-R Na(+) current preferentially from small ones. Ropivacaine shifted the activation curve for the TTX-R Na(+) channel in the depolarizing direction and the inactivation curve for both types of Na(+) channel in the hyperpolarizing direction. Ropivacaine blocked TTX-S and TTX-R Na(+) currents, but its half-maximum inhibitory concentration (IC(50)) was significantly lower for the latter current (116 +/- 35 vs 54 +/- 14 microM; P: < 0.01); similar IC(50) values were obtained with the (R)-isomer of ropivacaine. Ropivacaine produced a use-dependent block of both types of Na(+) channels. Ropivacaine preferentially blocks TTX-R Na(+) channels over TTX-S Na(+) channels. We conclude that because TTX-R Na(+) channels exist mainly in small DRG neurons (which are responsible for nociceptive sensation), such selective action of ropivacaine could underlie the differential block observed during epidural anesthesia with this drug. IMPLICATIONS Whole-cell patch-clamp recordings of tetrodotoxin-sensitive and tetrodotoxin-resistant Na(+) currents in rat dorsal root ganglion neurons showed ropivacaine preferentially blocked tetrodotoxin-resistant Na(+) channels over tetrodotoxin-sensitive Na(+) channels. This could provide a desirable differential sensory blockade during epidural anesthesia using ropivacaine.

Muscarinic agonist potencies at three different effector systems linked to the M2 or M3 receptor in longitudinal smooth muscle of guinea-pig small intestine

The abilities of muscarinic agonists (arecoline, bethanechol, carbachol, McN‐A343, methacholine, pilocarpine) to inhibit isoprenaline‐induced cyclic AMP production in chopped fragments (via M2 receptors), and to evoke cationic current (Icat) (via M2 receptors) or calcium store release (via M3 receptors) in enzyme‐dispersed, single voltage‐clamped cells from longitudinal smooth muscle of the guinea‐pig small intestine were examined. All muscarinic agonists (1 – 300 μM) examined inhibited isoprenaline (1 μM)‐induced accumulation of cyclic AMP, the IC50 varying from 52 to 248 μM. However, their relative potencies to evoke this M2 effect were not significantly correlated with their ability to evoke Icat, also a M2 effect, whether or not calcium stores were depleted; pilocarpine and McN‐A343 inhibited the Icat response to carbachol. Muscarinic agonists (concentration 300 or 1000 μM), except pilocarpine and McN‐A343 which were ineffective, evoked Ca2+‐activated K+ current (IK‐Ca) resulting from Ca2+ store release (M3 effect). Their effectiveness was tested by estimating residual stored calcium by subsequent application of caffeine (10 mM). The relative potencies to evoke Ca2+ store release (M3) and for Icat activation (M2) were closely correlated (P<0.001). These data might be explained if M2‐mediated adenylyl cyclase inhibition and Icat activation involve different G proteins, or involve different populations of M2 receptors. The observed correlation of agonist potency between Icat activation and Ca2+ store release supports the proposal ( Zholos & Bolton, 1997 ) that M3 activation can potentiate M2‐cationic channel coupling through Ca2+‐independent mechanisms.

Receptor signaling mechanisms underlying muscarinic agonist-evoked contraction in guinea-pig ileal longitudinal smooth muscle

In guinea‐pig ileal longitudinal muscle, muscarinic partial agonists, 4‐(N‐[3‐chlorophenyl]‐carbomoyloxy)‐2‐butynyl‐trimethylammonium (McN‐A343) and pilocarpine, each produced parallel increases in tension and cytosolic Ca2+ concentration ([Ca2+]c) with a higher EC50 than that of the full agonist carbachol. The maximum response of [Ca2+]c or tension was not much different among the three agonists. The Ca2+ channel blocker nicardipine markedly inhibited the effects of all three agonists The contractile response to any agonist was antagonized in a competitive manner by M2 receptor selective antagonists (N,N′‐bis[6‐[[(2‐methoyphenyl)methyl]amino]hexyl]‐1,8‐octanediamine tetrahydrochloride and 11‐[[2‐[(diethlamino)methyl]‐1‐piperidinyl]acetyl]‐5,11‐dihydro‐6H‐pyrido[2,3‐b][1,4] benzodiazepine‐6‐one), and the apparent order of M2 antagonist sensitivity was McN‐A343>pilocarpine>carbachol. M3 receptor selective antagonists, 1,1‐dimethyl‐4‐diphenylacetoxypiperidinium iodide and darifenacin, both severely depressed the maximum response for McN‐A343, while darifenacin had a similar action in the case of pilocarpine. Both M3 antagonists behaved in a competitive manner in the case of the carbachol response. McN‐A343 failed to release Ca2+ from the intracellular stores, and the Ca2+‐releasing action of pilocarpine was very weak compared with that of carbachol. All three agonists were capable of increasing Ca2+ sensitivity of the contractile proteins. McN‐A343 rarely produced membrane depolarization, but always accelerated electrical spike discharge. Pilocarpine effect was more often accompanied by membrane depolarization, as was usually seen using carbachol. The results suggest that muscarinic agonist‐evoked contractions result primarily from the integration of Ca2+ entry associated with the increased spike discharge and myofilaments Ca2+ sensitization, and that Ca2+ store release may contribute to the contraction indirectly via potentiation of the electrical membrane responses. They may also support the idea that an interaction of M2 and M3 receptors plays a crucial role in mediating the contraction response.