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Abstract
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.
Highlights
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
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. e response to hypertonic stress has been studied in great detail in the MDCK established cell line derived from the renal medulla [4, 5]
Long-term exposure (8 or 24 hrs) to hypertonic growth medium (500 mOsm) containing low Ca2+concentration (0.05 mM, LCM) disrupted the tight junctions in MDCK cells, as expected. e calcium switch technique is a standard procedure for disrupting tight junctions in MDCK cells [14, 15]. e cells were rounded and many had detached from the substrate. e distribution of E-cadherin protein, normally present in tight junctions at the plasma membrane of epithelial cells [16], was primarily intracellular (Figure 1, right), in contrast to cells in normal hypertonic growth medium (Figure 1, le )
Summary
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. Acute treatment activated BGT1 transport within 30 min in MDCK cells previously adapted to hypertonic growth medium containing normal Ca2+ (1.6 mM). Long-term exposure (8 or 24 hrs) to hypertonic growth medium (500 mOsm) containing low Ca2+concentration (0.05 mM, LCM) disrupted the tight junctions in MDCK cells, as expected.
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