Dae-Yong Uhm

NITRIC OXIDE DIRECTLY ACTIVATES CALCIUM-ACTIVATED POTASSIUM CHANNELS FROM RAT BRAIN RECONSTITUTED INTO PLANAR LIPID BILAYER

Using the planar lipid bilayer technique, we tested whether NO directly activates calcium‐activated potassium (Maxi‐K) channels isolated from rat brain. We used streptozotocin (STZ) as NO donor, and the NO release was controlled with light. In the presence of 100–800 μM STZ, the Maxi‐K channel activity increased up to 3‐fold within several tens of seconds after the light was on, and reversed to the control level several minutes after shutting off the light. Similar activation was observed with other NO donors such as S‐nitroso‐N‐acetylpenicillamine and sodium nitroprusside. The degree of activity increase was dependent upon the initial open probability (P init). When the P init was lower, the activity increase was greater. These results demonstrate that NO can directly affect the Maxi‐K channel activity, and suggest that the Maxi‐K channel might be one of the physiological targets of NO in brain.

Effects of hypoxia and mitochondrial inhibition on the capacitative calcium entry in rabbit pulmonary arterial smooth muscle cells

We have investigated the effects of hypoxia and mitochondria inhibitors on the capacitative Ca(2+) entry (CCE) in cultured smooth muscle cells from rabbit small pulmonary arteries. Cyclopiazonic acid (CPA) depleted Ca(2+) from sarcoplasmic reticulum (SR) in Ca(2+)-free medium and subsequent addition of Ca(2+) led to the nifedipine-insensitive, La(3+)-sensitive Ca(2+) influx. The presence of CCE was further verified by the measurement of unidirectional Mn(2+) influx. During the decay phase of the CCE-induced [Ca(2+)]c transients, hypoxia (P(O2) < 50 mmHg) and the mitochondria inhibitor FCCP reversibly increased [Ca(2+)]c, that is La(3+)-sensitive. Once SR is depleted by CPA, subsequent treatment of FCCP slowed the decay of CCE-induced [Ca(2+)]c transients but it did not attenuate Mn(2+) influx. Mitochondrial uptake of incoming Ca(2+) through CCE was demonstrated by additional increase in [Ca(2+)]c with Ca(2+) ionophore after terminating CCE. Together, it is suggested that the augmentation of CCE-induced [Ca(2+)]c transients by hypoxia and FCCP reflects a net gain of [Ca(2+)]c by the inhibition of mitochondrial Ca(2+) uptake.

Slow and persistent increase of [Ca2+]c in response to ligation of surface IgM in WEHI‐231 cells

WEHI‐231 and Bal 17 B cell lines are representative models for immature and mature B cells, respectively. Their regulation of cytosolic Ca2+ concentration ([Ca2+]c) was compared using fura‐2 fluorescence ratiometry. The ligation of B cell antigen receptor (BCR) by anti‐IgM antibody induced a slow but large increase of [Ca2+]c in WEHI‐231 cells while not in Bal 17 cells. The thapsigargin‐induced store‐operated Ca2+ entry (SOCE) of Bal 17 cells reached a steady state which was blocked by 2‐aminoethoxydiphenyl borate (2‐APB). On the contrary, the thapsigargin‐induced SOCE of WEHI‐231 cells increased continuously, which was accelerated by 2‐APB. The increase of [Ca2+]c by BCR ligation was also enhanced by 2‐APB in WEHI‐231 cells while blocked in Bal 17 cells. The Mn2+ quenching study showed that the thapsigargin‐, or the BCR ligation‐induced Ca2+ influx pathway of WEHI‐231 was hardly permeable to Mn2+. The intractable increase of [Ca2+]c may explain the mechanism of BCR‐driven apoptosis of WEHI‐231 cells, a well‐known model of clonal deletion of autoreactive immature B cells.

Effects of KCNQ1 channel blocker, 293B, on the acetylcholine-induced Cl- secretion of rat pancreatic acini.

Abstract: In rat pancreatic acini (RPAs), acetylcholine (ACh) typically induces a tonic depolarization of membrane potential (Vm) via increasing cytoplasmic Ca2+ concentration and subsequent activation of Cl− channels. In this study, to investigate the role of K+ channels during the ACh-induced Cl− secretion, the intracellular Cl− concentration ([Cl−]i) of RPAs was monitored using SPQ, a fluorescent dye quenchable by Cl−, and the effects of K+ channel blockers were examined. Also, the secretion of fluid and enzyme from the whole pancreas of rat was measured. The fluorescence of RPAs loaded with SPQ (FSPQ) was slightly increased by the application of ACh (ACh-Δ FSPQ), indicating net secretion of Cl−. However, the relative change of FSPQ normalized to the control fluorescence (F/F0) of RPAs was only about 20% of the effect observed in rat submandibular gland acinus. The ACh-ΔFSPQ of RPAs was not influenced by the pretreatment with 293B (20 μmol/L), a blocker of KCNQ-type K+ channels. Even the cocktail of K+ channel blockers (10 mmol/L TEA, 3 mmol/L Ba2+, 20 μmol/L 293B) exerted only minute inhibitory effects on ACh-ΔFSPQ in RPAs. In the vascularly perfused rat pancreas, the fluid and enzyme secretion induced by ACh was directly measured. 293B and HMR-1556, both specific blockers of KCNQ1 channel, did not block but even enhanced the secretion of fluid and amylase. These results suggest that the role of KCNQ1 channels may not be essential in the Ca2+-mediated Cl− secretion in rat pancreatic acini.

Low affinity cholecystokinin receptor inhibits cholecystokinin- and bombesin-induced oscillations of cytosolic Ca2+ concentration

We have investigated whether low affinity cholecystokinin (CCK) receptors suppress agonist‐induced rises of cytosolic free Ca2+ concentration ([Ca2+]c) in pancreatic acinar cells by using properties of caffeine. A high concentration of caffeine (20 mM) completely blocked inositol 1,4,5‐trisphosphate (InsP3)‐induced [Ca2+]c rises but spared the InsP3‐independent long‐lasting [Ca2+]c oscillations. In the presence of 20 mM caffeine, only high concentrations of CCK, but not bombesin or JMV‐180, suppressed the caffeine‐resistant CCK or bombesin‐induced [Ca2+]c oscillations, indicating that low affinity CCK receptors inhibit agonist‐induced [Ca2+]c oscillations. It could be one of the underlying mechanisms by which low affinity CCK receptors suppress secretion in pancreatic acinar cells.