Cholinergic agents increase the activity of the renal Na-HCO3 cotransporter and have been shown to stimulate the production of nitric oxide (NO) in other cells. To study the role of NO in mediating the effect of carbachol on Na-HCO3 cotransporter, we measured the activity of the cotransporter in rabbit proximal tubule cells treated with carbachol (10–4 M) or the NO inhibitor, L-NAME (10–3M), or carbachol+L-NAME. The activity of NaHCO3 cotransporter was measured by recovery of intracellular pH (pHi) in cells loaded with pH-sensitive dye, BCECF. In control cells, carbachol significantly increased Na-HCO3 cotransporter activity while L-NAME did not affect the activity of the cotransporter but completely blocked the enhancement induced by carbachol. Carbachol increased NO production by proximal tubule cells. We also studied the effect of the NO donor, SNAP (10–3M), on the cotransporter incubated for 1 h in cultured proximal tubule cells. SNAP caused a similar enhancement in the activity of the cotransporter sug- gesting that a different NO donor is capable of enhancing the activity of the cotransporter to the same extent as that observed with carbachol. Because the effect of NO is thought to involve cGMP, we examined the effect of 8-Br-cGMP (10–3 M) on the cotransporter. 8-Br-cGMP caused stimulation of the Na-HCO3 cotransporter activity although to a lesser degree than carbachol. We have previously shown that carbachol increases cytosolic calcium but the role of intracellular calcium (Cai) per se on the cotransporter has not been studied. We therefore studied the role of Cai on the activity of Na-HCO3 cotransporter in rabbit proximal tubule cells by utilizing the calcium ionophore, ionomycin, the microsomal Ca-ATPase inhibitor, thapsigargin, and the calcium chelator, BAPTA. Ionomycin, 5 µM, caused a significant stimulation of Na-HCO3 cotransporter which was prevented by BAPTA. The microsomal Ca-ATPase inhibitor, thapsigargin, also increased the cotransporter activity. As expected both ionomycin and thapsigargin caused a significant increase in Cai. Calyculin A, an inhibitor of protein phosphatase 2A prevented the stimulation of the cotransporter by calcium (in pH units/min: control 1.8±0.13; Ca 2.22±0.07; p<0.05; Ca+calyculin A 1.9±0.09, p<0.025) suggesting that calcium acting through kinases/phosphatases, plays a role in the phosphorylation of the cotransporter. These results demonstrate that NO and Cai modulate the activity of the cotransporter.

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