An alpha3beta4 subunit combination acts as a major functional nicotinic acetylcholine receptor in male rat pelvic ganglion neurons.

We identified major subunits of the nicotinic acetylcholine receptor (nAChR) involved in excitatory postsynaptic potential and intracellular Ca(2+) ([Ca(2+)]i) increase in the major pelvic ganglion (MPG) neurons of the male rat. ACh elicited fast inward currents in both sympathetic and parasympathetic MPG neurons. Mecamylamine, a selective antagonist for alpha3beta4 nAChR, potently inhibited the ACh-induced currents in sympathetic and parasympathetic neurons (IC(50); 0.53 and 0.22 microM, respectively). Furthermore, alpha-conotoxin AuIB (10 microM), a new selective antagonist for alpha3beta4 nAChR, blocked more than 80% of the ACh-induced currents in MPG neurons. Conversely, alpha-bungarotoxin, alpha-methyllycaconitine, and dihydro-beta-erythroidine, known as blockers of the alpha7 or alpha4beta2, did not show selective blocking effects on MPG neurons. ACh transiently increased [Ca(2+)]i which was subsequently abolished in the extracellular Ca(2+)-free environment. Simultaneous recording of [Ca(2+)]i and ionic currents revealed that ACh increased [Ca(2+)]i under the conditions of the voltage-clamped (at -80 mV) state, and this resulted from the influx through nAChR itself. ACh-induced [Ca(2+)]i increase was blocked by mecamylamine (10 microM), but was not affected by atropine (1 microM). RT-PCR analysis showed that, among subunits of nAChR, alpha3 and beta4 were predominantly expressed in MPG. We suggest that activation of alpha3 and beta4 nAChR subunits in MPG neurons induce fast inward currents and [Ca(2+)]i increase, possibly mediating a major role in pelvic autonomic synaptic transmission.

[Inhibitory effect of angiotensin II receptor antagonist on the contraction and growth of hepatic stellate cells].

BACKGROUND/AIMS: This study aimed to investigate the effects of angiotensin II (ANG II) and its receptor antagonist (losartan) on the contraction and growth of HSCs. METHODS: HSCs were isolated from Sprague Dawley rat and cultured at various conditions as follows: control, pretreatment of 10(-5) M ANG II, pretreatment of 10(-5) M endothelin, and pretreatment of 10(-5) M ANG II and 10(-6) M losartan. We conducted morphologic analysis with cellular area and length by image analysis system to estimate cell growth in each group. In addition, we measured the change of intracellular calcium currents via electrophysiological methods to evaluate the contractile effect of ANG II and losartan on HSCs. RESULTS: At the fifth day of incubation, the mean cellular area of ANG II-pretreated group and ANG II with losartan-pretreated group were 704.68+/-22.6 micro m2 and 332.90+/-32.6 micro m2, respectively. This difference was statistically significant (p<0.05). ANG II induced an increase in the intracellular calcium current by 22.0+/-3.0% compared with basal current level (p<0.05). However, when losartan was pretreated, ANG II did not cause a significant increase in calcium current (3.1+/-0.8%, p>0.05). CONCLUSION: ANG II accelerates the contraction and growth of HSCs, while its receptor blocker, losartan, inhibits the contraction and growth of HSCs.

Identification of T-type alpha1H Ca2+ channels (Ca(v)3.2) in major pelvic ganglion neurons.

Among autonomic neurons, sympathetic neurons of the major pelvic ganglia (MPG) are unique by expressing low-voltage-activated T-type Ca2+ channels. To date, the T-type Ca2+ channels have been poorly characterized, although they are believed to be potentially important for functions of the MPG neurons. In the present study, thus we investigated characteristics and molecular identity of the T-type Ca2+ channels using patch-clamp and RT-PCR techniques. When the external solution contained 10 mM Ca2+ as a charge carrier, T-type Ca2+ currents were first activated at -50 mV and peaked around -20 mV. Besides the low-voltage activation, T-type Ca2+ currents displayed typical characteristics including transient activation/inactivation and voltage-dependent slow deactivation. Overlap of the activation and inactivation curves generated a prominent window current around resting membrane potentials. Replacement of the external Ca2+ with 10 mM Ba2+ did not affect the amplitudes of T-type Ca2+ currents. Mibefradil, a known T-type Ca2+ channel antagonist, depressed T-type Ca2+ currents in a concentration-dependent manner (IC50 = 3 microM). Application of Ni2+ also produced a concentration-dependent blockade of T-type Ca2+ currents with an IC50 of 10 microM. The high sensitivity to Ni2+ implicates alpha1H in generating the T-type Ca2+ currents in MPG neurons. RT-PCR experiments showed that MPG neurons predominantly express mRNAs encoding splicing variants of alpha1H (called pelvic Ta and Tb, short and long forms of alpha1H, respectively). Finally, we tested whether the low-threshold spikes could be generated in sympathetic MPG neurons expressing T-type Ca2+ channels. When hyperpolarizing currents were injected under a current-clamp mode, sympathetic neurons produced postanodal rebound spikes, while parasympathetic neurons were silent. The number of the rebound spikes was reduced by 10 microM Ni2+ that blocked 50% of T-type Ca2+ currents and had a little effect on HVA Ca2+ currents in sympathetic MPG neurons. Furthermore, generation of the rebound spikes was completely prevented by 100 microM Ni2+ that blocked most of the T-type Ca2+ currents. In conclusions, T-type Ca2+ currents in MPG neurons mainly arise from alpha1H among the three isoforms (alpha1G, alpha1H, and alpha1I) and may contribute to generation of low-threshold spikes in sympathetic MPG neurons.