Modulatory effect of protein kinase C on thapsigargin-induced calcium entry in thyroid FRTL-5 cells

The aim of the present study was to investigate the regulation of calcium influx in thyroid FRTL-5 cells. Stimulating Fura 2-loaded cells with thapsigargin rapidly increased the cytosolic Ca2+ concentration ([Ca2+]i), which then stabilized at a new elevated plateau level. The initial increase in [Ca2+]i consisted mainly of the release of sequestered Ca2+. The plateau phase was totally dependent on extracellular Ca2+. The influx of Ca2+ was blocked by Ni2+ and was decreased in depolarized cells. The importance of protein kinase C in regulating influx of Ca2+ was then evaluated. Addition of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate prior to thapsigargin significantly decreased the influx of extracellular Ca2+. Studies with bisoxonol to measure membrane potential showed that TPA depolarized the plasma membrane in FRTL-5 cells. In cells where protein kinase C was downregulated or was inhibited by staurosporine, the thapsigargin-induced influx of Ca2+ was enhanced. The results indicate that emptying intracellular Ca2+ pools is sufficient to induce influx of Ca2+ in FRTL-5 cells, and that protein kinase C has a modulatory effect on this process.

Electrophysiological evidence for Na+-coupled bicarbonate transport in cultured rat hepatocytes

Recent observations suggest that hepatocytes exhibit basolateral electrogenic Na+-coupled HCO3- transport. In these studies, we have further investigated this transport mechanism in primary culture of rat hepatocytes using intracellular microelectrodes to measure membrane potential difference (PD) and the pH-sensitive fluorochrome 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein to measure intracellular pH (pHi). In balanced media containing 25 mM HCO3-, PD averaged -32.1 +/- 0.6 (SE) mV and pHi averaged 7.22 +/- 0.03. PD became more negative (hyperpolarized) when extracellular [HCO3-] was increased and less negative (depolarized) when extracellular HCO3- was decreased. Acute replacement of extracellular Na+ by choline also resulted in membrane depolarization of 18.0 +/- 1.6 mV, suggesting net transfer of negative charge. This decrease in PD upon Na+ removal was HCO3- -dependent, amiloride insensitive, and inhibited by the disulfonic stilbene 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS). PD also decreased upon acute exposure to SITS. The degree of depolarization seen with removal of Na+ or HCO3- correlated directly with resting PD (r = 0.81 and 0.95, respectively), suggesting a voltage-dependent mechanism. Removal of extracellular Na+ also decreased pHi to 7.06 +/- 0.02, and this acidification was decreased in the absence of HCO3- or in the presence of SITS or amiloride. These studies provide direct evidence for electrogenic Na+-coupled HCO3- transport in rat hepatocytes. Further, they suggest that it represents a major pathway for conductive movement of Na+ across the membrane and that it contributes, along with Na+-H+ exchange, to the intracellular acidification observed upon removal of extracellular Na+.(ABSTRACT TRUNCATED AT 250 WORDS)

Potassium channel selectivity in mouse pancreatic B cells

High-resistance microelectrodes were used to measure membrane potential changes in response to increased extracellular K+ concentration ([K+]o; or a test cation X+ such as Li+, Rb+, Cs+, NH+4) in B cells from mouse islets of Langerhans. In the absence of glucose, a sudden increase in [K+]o (or [X+]o), keeping the sum [Na+]o + [K+]o constant (or [Na+]o + [K+]o + [X+]o), induced a rapid depolarization of the membrane. The membrane potential changes were essentially unchanged in the presence of 20 mM tetraethylammonium (TEA). The Goldman-Hodgkin-Katz equation was fitted to the experimental relationship between membrane potential and [K+]o (or [X+]o), and permeability (P) ratios were estimated. In the absence of TEA, P Na/PK was estimated to be approximately 0.046. In the presence of TEA the following ratios were estimated: P Rb/PK = 0.74, P Cs/PK = 0.62, and P NH4/PK = 0.36. From these ratios the following sequence of permeabilities was obtained, PK greater than P Rb greater than P Cs greater than P NH4 greater than P Na. It is proposed that this sequence reflects the selectivity of the intracellular [Ca2+]-activated K+ channel of the pancreatic B cell.