Search

Kidney proximal tubule Na/H exchange is inhibited by PTH. To analyze further the cellular mechanisms involved in this regulation we have used MCT cells (a culture of SV-40 immortalized mouse cortical tubule cells) grown on permeant filter supports. Na/H exchange was measured using single cell fluorescence microscopy (BCECF) and phosphate transport (measured for comparisons) by tracer techniques. MCT cells express apical and basolateral Na/H exchangers which respond differently to inhibition by ethylisopropylamiloride and by dimethylamiloride, the basolateral membrane transporter being more sensitive. Apical membrane Na/H exchange was inhibited by PTH (10(-8) M; by an average of 25%); similar degrees of inhibition were observed when cells were exposed either to forskolin, 8-bromo-cAMP or phorbol ester. Basolateral membrane Na/H exchange was stimulated either by incubation with PTH (to 129% above control levels) or by addition of phorbol ester (to 120% above control levels); it was inhibited after exposure to either forskolin or 8-bromo-cAMP. The above effects of PTH and phorbol ester (apical and basolateral) were prevented by preincubation of cells with protein kinase C antagonists, staurosporine and calphostin C; both compounds did not affect forskolin or 8-bromo-cAMP induced effects. PTH also inhibited apical Na-dependent phosphate influx (29% inhibition at 10(-8) M); it had no effect on basolateral phosphate fluxes (Na-dependent and Na-independent). Incubation with PTH (10(-8) M) resulted in a rapid and transient increase in [Ca2+]i (measured with the fluorescent indicator, fura-2), due to stimulation of a Ca2+ release from intracellular stores. Exposure of MCT cells to PTH did not elevate cellular levels of cAMP. Taken together, these results suggest that PTH utilizes in MCT cells the phospholipase C/protein kinase C pathway to differently control Na/H exchangers (apical vs. basolateral) and to inhibit apical Na/Pi cotransport. [ABSTRACT FROM AUTHOR]

The cellular distribution (apical vs. basolateral) of parathyroid hormone (PTH) signal transduction systems in opossum kidney (OK) cells was evaluated by measuring the action of PTH on apically located transport processes (Na/Pi cotransport and Na/H exchange) and on the generation of intracellular messengers (cAMP and IP3). PTH application led to immediate inhibition of Na/H-exchange without a difference in dose/response relationships between apical and basolateral cell-surface hormone addition (half-maximal inhibition at approximately 5 x 10(-12) M). PTH required 2-3 hr for maximal inhibition of Na/Pi cotransport with a half-maximal inhibition occurring at approximately 5 x 10(-10) M PTH for basolateral application and approximately 5 x 10(-12) M for apical application. PTH addition to either side of the monolayer produced a dose-dependent production of both cAMP and IP3. Half-maximal activation of IP3 was at about 7 x 10(-12) M PTH and displayed no differences between apical and basolateral hormone addition, while cAMP was produced with a half maximal concentration of 7 x 10(-9) M for apical PTH application and 10(-9) M for basolateral administration. The PTH analog [nle8.18,tyr34]PTH(3-34), (nlePTH), produced partial inhibition of Na/Pi cotransport (agonism) with no difference between apical and basolateral application. When applied as a PTH antagonist, nlePTH displayed dose-dependent antagonism of PTH inhibition of Na/Pi cotransport on the apical surface, failing to have an effect on the basolateral surface. Independent of addition to the apical or basolateral cell surface, nlePTH had only weak stimulatory effect on production of cAMP, whereas high levels of IP3 could be measured after addition of this PTH analog to either cell surface. Also an antagonistic action of nlePTH on PTH-dependent generation of the internal messengers, cAMP and IP3, was observed; at the apical and basolateral cell surface nelPTH reduced PTH-dependent generation of cAMP, while PTH-dependent generation of IP3 was only reduced by nlePTH at the apical surface. Pertussis toxin (PT) preincubation produced an attenuation of both PTH-dependent inhibition of Na/Pi cotransport and 1P3 generation while producing an enhancement of PTH-dependent cAMP generation; these effects displayed no cell surface polarity, suggesting that PTH action through either adenylate cyclase or phospholipase C was transduced through similar sets of G-proteins at each cell surface. [ABSTRACT FROM AUTHOR]