Na⁺Ca²⁺ exchange and calcium permeability in canine basolateral membrane vesicles: The effects of dibutyryl cAMP and specific inhibitors

The role of dibutyryl 3′,5′-cyclic adenosine monophosphate (dibutyryl cAMP) as putative second messenger for parathyroid hormone (PTH) in regulating canine proximal tubular basolateral membrane Na⁺Ca²⁺ exchange and passive calcium permeability was assessed, as was the nature of this passive calcium permeability. Dibutyryl cAMP (50 mg) infused in vivo over 30 min increased fractional phosphate excretion from 4.9 ± 1.8% to 20.5 ± 4.6%, P < 0.05, n = 6, but had no effect on either passive Ca²⁺ efflux or sodium-stimulated Ca²⁺ efflux from Ca²⁺-preloaded basolateral membrane vesicles (BLMV). Both of these mechanisms have been previously shown to be stimulated by PTH. Further studies were performed to investigate the mechanism of the passive calcium flux. Calcium uptake by BLMV was blocked by lanthanum (La³⁺) but not by the calcium-channel blocker verapamil. La³⁺ blocked efflux of Ca²⁺ from preloaded vesicles when it was placed in the external solution. This La³⁺-blockable efflux was larger in potassium equivalent BLMV prepared from normal dogs than in the BLMV prepared from thyroparathyroidectomized dogs. Benzamil produced 50% inhibition of sodium-stimulated Ca²⁺ uptake at 250 μM whereas neither amiloride nor diltiazem achieved 50% inhibition at the maximal doses studied. Benzamil, 1 mM, had no effect on passive calcium efflux and neither did the substitution of sucrose for potassium, which has been shown to affect Ca²⁺Ca²⁺ exchange by the Na²⁺Ca²⁺ exchanger. This suggests that the calcium flux under potassium equivalent conditions was not mediated by Ca²⁺Ca²⁺ exchange by the Na²⁺Ca²⁺ exchanger. These results demonstrate that the basolateral membrane of proximal tubular cells possesses both a Na²⁺Ca²⁺ exchanger inhibitable by benzamil and a passive calcium permeability not inhibited by benzamil nor by verapamil but by La³⁺. Neither of these two mechanisms of calcium flux was affected by dibutyryl cAMP whereas both have been shown to be stimulated by PTH.