Haruhiro Kuwashima

Differential Secretion Of Adrenaline And Noradrenaline In Response To Various Secretagogues From Bovine Chromaffin Cells

1. Differential secretion of adrenaline (Adr) and noradrenaline (NA) in response to various secretagogues was studied in bovine adrenal chromaffin cells.

White-to-opaque switching is involved in the phospholipase B production of Candida dubliniensis on Price’s egg yolk agar

Measuring the production of Candida dubliniensis (C. dubliniensis) phospholipase B (PLase B) by the Price’s method has long been considered to be unattainable because the levels of PLase produced are undetectable. In this study, C. dubliniensis, C. glabrata, C. guilliermondii, C. krusei, C. parapsilosis and C. tropicalis were shown to produce PLase B and form clear white zones around their colonies when peptone, a component of the original Price’s egg yolk (OP) agar, is replaced with a yeast nitrogen base (YNB). This new medium is named modified Price’s (MP) agar. Based on this finding, we propose a new modified Price’s (NMP) agar containing 0.75% peptone and 0.25% YNB, which enabled measurement of PLase B production by C. dubliniensis and C. albicans with results consistent with those obtained for C. albicans grown on OP agar. We strongly believe that the MP and NMP agars are very useful for screening PLase B production by C. dubliniensis and non-albicans Candida spp. Moreover, the addition of several bioactive agents (the proteinase inhibitors pepstatin A and saquinavir, the calcineurin inhibitors cyclosporine A and tacrolimus, the cell-permeable cAMP analog dBcAMP, and the quorum-sensing molecule farnesol) to the OP agar enhanced PLase B production by C. dubliniensis. During the course of our study to clarify the reason why PLase B was not produced, we found that C. dubliniensis cells grown on OP agar undergo a white-to-opaque transition, which may explain why they showed minimal production of PLase B on this medium.

INVOLVEMENT OF PROTEIN KINASE C IN THE REGULATION OF Na+/Ca2+ EXCHANGER IN BOVINE ADRENAL CHROMAFFIN CELLS

1 The Na+/Ca2+ exchanger (NCX) exchanges Na+ and Ca2+ bidirectionally through the forward mode (Ca2+ extrusion) or the reverse mode (Ca2+ influx). The present study was undertaken to clarify the role of protein kinase C (PKC) in the regulation of NCX in bovine adrenal chromaffin cells. The Na+‐loaded cells were prepared by treatment with 100 µmol/L ouabain and 50 µmol/L veratridine. Incubation of Na+‐loaded cells with Na+‐free solution in the presence of the Ca2+ channel blockers nicardipine (3 µmol/L) and ω‐conotoxin MVIIC (0.3 µmol/L) caused Ca2+ uptake and catecholamine release. 2 The Na+‐dependent Ca2+ uptake and catecholamine release were inhibited by 2‐[4‐[(2,5‐difluorophenyl)methoxy]phenoxy]‐5‐ethoxyaniline (SEA0400; 1 µmol/L) and 2‐[2‐[4‐(4‐nitrobenzyloxy)phenyl]isothiourea (KB‐R7943; 10 µmol/L), both NCX inhibitors. These results indicate that the Na+‐dependent responses are mostly due to activation of the NCX working in the reverse mode. 3 In addition, we examined the effects of PKC inhibitors and an activator on the NCX‐mediated Ca2+ uptake and catecholamine release. Bisindolylmaleimide I (0.3–10 µmol/L) and chelerythrine (3–100 µmol/L), both PKC inhibitors, inhibited NCX‐mediated responses. In contrast, phorbol 12,13‐dibutyrate (0.1–10 µmol/L), a PKC activator, enhanced the responses. Bisindolylmaleimide I and chelerythrine, at effective concentrations for inhibition of Na+‐dependent catecholamine release, had a little or no effect on high K+‐induced catecholamine release in intact cells or on Ca2+‐induced catecholamine release in β‐escin‐permeabilized cells. 4 These results suggest that PKC is involved in the activation of NCX in bovine adrenal chromaffin cells.