Joong-Woo Lee

Expression profiles of high voltage-activated calcium channels in sympathetic and parasympathetic pelvic ganglion neurons innervating the urogenital system.

Among the autonomic ganglia, major pelvic ganglia (MPG) innervating the urogenital system are unique because both sympathetic and parasympathetic neurons are colocalized within one ganglion capsule. Sympathetic MPG neurons are discriminated from parasympathetic ones by expression of low voltage-activated Ca2+ channels that primarily arise from T-type α1H isoform and contribute to the generation of low-threshold spikes. Until now, however, expression profiles of high voltage-activated (HVA) Ca2+ channels in these two populations of MPG neurons remain unknown. Thus, in the present study, we dissected out HVA Ca2+ channels using pharmacological and molecular biological tools. Reverse transcription-polymerase chain reaction analysis showed that MPG neurons contained transcripts encoding all of the known HVA Ca2+ channel isoforms (α1B, α1C, α1D and α1E), with the exception of α1A. Western blot analysis and pharmacology with ω-agatoxin IVA (1 μM) confirmed that MPG neurons lack the α1A Ca2+ channels. Unexpectedly, the expression profile of HVA Ca2+ channel isoforms was identical in the sympathetic and parasympathetic neurons of the MPG. Of the total Ca2+ currents, ω-conotoxin GVIA-sensitive N-type (α1B) currents constituted 57 ± 5% (n = 9) and 60 ± 3% (n = 6), respectively; nimodipine-sensitive L-type (α1C and α1D) currents made up 17 ± 4% and 14 ± 2%, respectively; and nimodipine-resistant and ω-conotoxin GVIA-resistant R-type currents were 25 ± 3% and 22 ± 2%, respectively. The R-type Ca2+ currents were sensitive to NiCl2 (IC50 = 22 ± 0.1 μM) but not to SNX-482, which was able to potently (IC50 = 76 ± 0.4 nM) block the recombinant α1E/β2a/α2δ Ca2+ currents expressed in human embryonic kidney 293 cells. Taken together, our data suggest that sympathetic and parasympathetic MPG neurons share a similar but unique profile of HVA Ca2+ channel isoforms.

Nerve injury alters profile of receptor-mediated Ca2+ channel modulation in vagal afferent neurons of rat nodose ganglia.

Although nerve injury is known to up- and down-regulate some metabotropic receptors in vagal afferent neurons of the nodose ganglia (NG), the functional significance has not been elucidated. In the present study, thus, we examined whether nerve injury affected receptor-mediated Ca2+ channel modulation in the NG neurons. In this regard, unilateral vagotomy was performed using male Sprague-Dawley rats. One week after vagotomy, Ca2+ currents were recorded using the whole-cell variant of patch-clamp technique in enzymatically dissociated NG neurons. In sham controls, norepinephrine (NE)-induced Ca2+ current inhibition was negligible. Following vagotomy, however, the NE responses were dramatically increased. This phenomenon was in accordance with up-regulation of alpha2A/B-adrenergic receptor mRNAs as quantified using real-time RT-PCR analysis. In addition, neuropeptide Y (NPY) and prostaglandin E2 responses were moderately augmented in vagotomized NG neurons. The altered NPY response appears to be caused by up-regulation of Y2 receptors negatively coupled to Ca2+ channels. In contrast, nerve injury significantly suppressed opioid (tested with DAMGO)-induced Ca2+ current inhibition with down-regulation of micro-receptors. Taken together, these results demonstrated for the first time that the profile of neurotransmitter-induced Ca2+ channel modulation is significantly altered in the NG neurons under pathophysiological state of nerve injury.

Afterhyperpolarization induced by the activation of nicotinic acetylcholine receptors in pelvic ganglion neurons of male rats

The electrophysiological mechanism underlying afterhyperpolarization induced by the activation of the nicotinic acetylcholine receptor (nAChR) in male rat major pelvic ganglion neurons (MPG) was investigated using a gramicidin-perforated patch clamp and microscopic fluorescence measurement system. Acetylcholine (ACh) induced fast depolarization through the activation of nAChR, followed by a sustained hyperpolarization after the removal of ACh in a dose-dependent manner (10 microM to 1mM). ACh increased both intracellular Ca(2+) ([Ca(2+)](i)) and Na(+) concentrations ([Na(+)](i)) in MPG neurons. The recovery of [Na(+)](i) after the removal of ACh was markedly delayed by ouabain (100 microM), an inhibitor of Na(+)/K(+) ATPase. Pretreatment with ouabain blocked ACh-induced hyperpolarization by 67.2+/-5.4% (n=7). ACh-induced hyperpolarization was partially attenuated by either the chelation of [Ca(2+)](i) with BAPTA/AM (20 microM) or the blockade of small-conductance Ca(2+)-activated K(+) channels by apamin (500 nM). Taken together, the activation of nAChR increases [Na(+)](i) and [Ca(2+)](i), which activates Na(+)/K(+) ATPase and Ca(2+)-activated K(+) channels, respectively. Consequently, hyperpolarization occurs after the activation of nAChR in the autonomic pelvic ganglia.

Changes in Inward Rectifier K + Channels in Hepatic Stellate Cells During Primary Culture

Purpose This study examined the expression and function of inward rectifier K+ channels in cultured rat hepatic stellate cells (HSC). Materials and Methods The expression of inward rectifier K+ channels was measured using real-time RT-PCR, and electrophysiological properties were determined using the gramicidin-perforated patch-clamp technique. Results The dominant inward rectifier K+ channel subtypes were Kir2.1 and Kir6.1. These dominant K+ channel subtypes decreased significantly during the primary culture throughout activation process. HSC can be classified into two subgroups: one with an inward-rectifying K+ current (type 1) and the other without (type 2). The inward current was blocked by Ba2+ (100 µM) and enhanced by high K+ (140 mM), more prominently in type 1 HSC. There was a correlation between the amplitude of the Ba2+-sensitive current and the membrane potential. In addition, Ba2+ (300 µM) depolarized the membrane potential. After the culture period, the amplitude of the inward current decreased and the membrane potential became depolarized. Conclusion HSC express inward rectifier K+ channels, which physiologically regulate membrane potential and decrease during the activation process. These results will potentially help determine properties of the inward rectifier K+ channels in HSC as well as their roles in the activation process.