Cholinergic agonists increase cell calcium in rat medullary collecting tubules. A fura-2 study

The intracellular free calcium concentration [Ca2+]i of rat medullary collecting tubules was calculated from microscope fluorescence measurements in single pieces of fura-2-loaded tubules superfused at 37 degrees C. When carbachol (10(-4)-10(-3) M) was added in the superfusate, a biphasic increase in [Ca2+]i was generally obtained, which included an early peak phase and a sustained plateau thereafter; sometimes, the peak phase was not apparent; the plateau was maintained as long as the agonist was applied. Several responses could be induced successively without a fall in responsiveness. From dose/response curves, K1/2 values of about 10(-5) M for carbachol and 10(-6) M for acetylcholine were obtained. The effects of the agonists were suppressed with 10(-4) M of atropine or pirenzepine, indicating the presence of muscarinic receptors of the M1 type. In the absence of external calcium, the peak phase of the response was preserved while the plateau phase was suppressed; thus, the peak involves the release of calcium stored in organelles, whereas the plateau involves the entry of external calcium through calcium channels which were voltage independent and insensitive to the usual calcium blockers.