We investigated the relationship between voltage-operated Ca2+ channel current and the corresponding intracellular Ca2+concentration ([Ca2+]i) change (Ca2+ transient) in guinea pig gastric myocytes. Fluorescence microspectroscopy was combined with conventional whole cell patch-clamp technique, and fura 2 (80 μM) was added to CsCl-rich pipette solution. Step depolarization to 0 mV induced inward Ca2+ current ( I Ca) and concomitantly raised [Ca2+]i. Both responses were suppressed by nicardipine, an L-type Ca2+ channel blocker, and the voltage dependence of Ca2+transient was similar to the current-voltage relation of I Ca. When pulse duration was increased by up to 900 ms, peak Ca2+ transient increased and reached a steady state when stimulation was for longer. The calculated fast Ca2+ buffering capacity ( B value), determined as the ratio of the time integral of I Ca divided by the amplitude of Ca2+ transient, was not significantly increased after depletion of Ca2+ stores by the cyclic application of caffeine (10 mM) in the presence of ryanodine (4 μM). The addition of cyclopiazonic acid (CPA, 10 μM), a sarco(endo)plasmic reticulum Ca2+-ATPase inhibitor, decreased B value by ∼20% in a reversible manner. When KCl pipette solution was used, Ca2+-activated K+ current [ I K(Ca)] was also recorded during step depolarization. CPA sensitively suppressed the initial peak and oscillations of I K(Ca) with irregular effects on Ca2+transients. The above results suggest that, in guinea pig gastric myocyte, Ca2+ transient is tightly coupled to I Caduring depolarization, and global [Ca2+]iis not significantly affected by Ca2+-induced Ca2+ release from sarcoplasmic reticulum during depolarization.

1 In guinea-pig gastric myocytes isolated from the antral circular layer, stimulation of muscarinic receptors by carbachol (CCh) induces a cationic current (ICch) which is known as the main mechanism of depolarization induced by muscarinic stimulation. 2 We tested the effects of a number of ion channel blockers on ACCh and focused upon quinidine which was a highly potent blocker. Externally applied quinidine suppressed ICch (IC50=0.25 JM) in a reversible and voltage-dependent manner. Applied internally, quinidine was about 100 times less potent than when applied externally. Persistent activation of G-protein by GTPyS also induced a cationic current similar to ICch and this current was also blocked by quinidine. 4-Aminopyridine and tetraethylammonium also suppressed ACCh in a dose-dependent manner (IC50=3.3 mM and 4.1 mM, respectively). 3 Pretreatment with quinidine (2 pM) selectively blocked the acetylcholine (ACh)-induced depolarization which was recorded in the multicellular tissues by a conventional intracellular microelectrode technique. 4 Voltage-dependent K-currents were also suppressed by quinidine but in a higher concentration range (IC50= 3 gM). Quinidine, 10 gM, decreased the amplitude of the voltage-dependent Ca current to only a small extent (15% decrease at 0 mV). Quinidine, 2 gM, also suppressed only a minute proportion of the Ca-activated K current (11.1% decrease at 45 mV). 5 From these experiments, it is concluded that some organic agents known as K channel blockers are able to block the CCh-activated cation channel in a non-specific manner and among them, quinidine can be used as an effective blocker for Acch in guinea-pig gastric myocytes.

Synaptic transmission between the medial nucleus of the trapezoid body (MNTB) and the lateral superior olive (LSO) was investigated in circling mice, an animal model for inherited deafness, using the voltage-clamp technique. In postnatal day 9 (P9)ϳP11 homozygous (cir/cir) circling mice, perfusion with 10 M DL-APV and 10 M CNQX reduced the 10 min average of postsynaptic currents (PSCs) to 8.8 Ϯ 3.0% compared with controls (n ϭ 6). In heterozygous (؉/cir) mice in the same age range, the 10 min PSCs average was reduced to 87.5 Ϯ 3.7% compared with controls (n ϭ 7). In P0ϳP2 homozygous (cir/cir) and heterozygous (ϩ/cir) mice, the 10 min PSCs averages were 11.0 Ϯ 2.6% (n ϭ 9) and 84.1 Ϯ 4.6% (n ϭ 11), respectively. The effects of a glutamate antagonist mixture were almost the same in single fiber stimulation of P9ϳP11 mice, reducing mean PSCs to 5.2 Ϯ 3.1% (homozygous (cir/cir) mice, n ϭ 8) and 78.3 Ϯ 4.3% (heterozygous (ϩ/cir) mice, n ϭ 12). Immunohistochemical study revealed that glycine receptor (GlyR) immunoreactivity in heterozygous (ϩ/cir) mice was more prominent than in homozygous (cir/cir) mice, while immunoreactivities of NR1 and NR2A-type NMDAR of P16 homozygous (cir/cir) mice were more prominent than in heterozygous (ϩ/cir) mice of the same age. No significant difference was found in the immunoreactivity of NR2B-type NMDAR. These results indicate that glutamatergic transmission is sustained at a later period of developing MNTB-LSO synapses in homozygous (cir/cir) mice.

In mammalian gastrointestinal myocytes, it is known that muscarinic stimulation activates nonselective cation channels through a G-protein and a Ca2+-dependent pathway. We recorded inward cationic currents following application of carbachol (ICCh) to guinea-pig gastric myocytes, which were held at -20 mV using the whole-cell patch-clamp method. ICCh was suppressed by nicardipine or removal of Ca2+ from the bath solution. The peak value of inward current induced by repetitive applications of carbachol (CCh) decreased progressively (run-down phenomenon). This run-down was significantly alleviated by the addition of calmodulin to the pipette solution (0.15 mg/ml) or by using the perforated-patch whole-cell voltage-clamp technique. Moreover, W-7[N-6(aminohexyl)-5-chloro-1-naphthalenesulphonamide], a calmodulin antagonist, was a reversible inhibitor of ICCh. However, @-7 had only a weak inhibitory effect on the same cationic current which was induced by guanosine 5'-O-(3-thiotriphosphate) (GTP¿gammaS] 0.2 mM) in the pipette solution. This GTP[gammaS]-induced cationic current was still markedly suppressed by the Ca2+-free bath solution. W-7 itself had a weak inhibitory effect on voltage-operated Ca2+ channels as well as the effects on ICCh. These data suggest that multiple Ca2+-dependent pathways are involved in the activation of CCh-gated cation channels in guinea-pig antral myocytes and a Ca2+/calmodulin-dependent pathway would be one of them.