Acute Activation of the Renal Betaine/GABA Transporter in Response to a Decrease in Extracellular Calcium

The betaine/GABA transporter (BGT1) is important for osmoprotection in kidney medullary cells. We previously reported an acute (30？min) increase in extracellular Ca2+ caused dose dependent inhibition of BGT-1 in renal MDCK cells. To determine if extracellular Ca2+ might be a local regulator of BGT-1, we have tested the response to low Ca2+ serum-free growth medium (LCM, 0.05？mM Ca2+). Chronic treatment (8–24？h) of MDCK cell monolayers completely blocked hypertonic adaptation of BGT1 and disrupted tight junctions. In contrast, acute treatment activated BGT1 transport within 30？min in MDCK cells previously adapted to hypertonic growth medium containing normal Ca2+ (1.6？mM). Activation was significant after 60–90？min and was independent of medium osmolarity. Peak transport was increased 50% in isotonic LCM and 100% in hypertonic (500？mOsm) LCM over controls. The activation was reversed by restoration of normal Ca2+. Perfusion of Fura-2-loaded MDCK cells with LCM decreased intracellular Ca2+ by 31% within 6-7 min. Inclusion of staurosporine (0.6？μM), a protein kinase C inhibitor, potentiated the action of LCM. We suggest that activation of BGT1 by LCM may be due in part to inhibition of protein kinase C. 1. Introduction Adaptation to chronic hypertonic stress by nephron segments in the kidney inner medulla involves cellular accumulation of organic osmolytes that do not disturb cell function. Betaine is an important osmolyte and it also may act as a protein chaperone to help protect intracellular proteins from the denaturing effects of medullary solutes such as urea and ammonia. Cell uptake of betaine is mediated by the betaine/GABA transporter (BGT1) located primarily in the basolateral plasma membrane [1]. BGT1 also can accept GABA as a substrate but betaine is the principal substrate in the kidney [2, 3]. Up- and down-regulation of BGT1 by changes in extracellular osmolarity is slow, requiring almost 24 hr, likely due to the need for de novo synthesis and intracellular trafficking. The response to hypertonic stress has been studied in great detail in the MDCK established cell line derived from the renal medulla [4, 5]. However, we have previously demonstrated relatively rapid down-regulation (within 30？min) of BGT1 transport in MDCK cells in response to extracellular ATP, adenosine, nitric oxide, and calcium [6–8]. Inhibition of BGT1 transport by calcium was reproduced by a phorbol ester (phorbol myristate 13 acetate) and a diacylglycerol analog (dioctanoglycerol), suggesting a role for protein kinase C and was accompanied by internalization of BGT1 from