Microspectrofluorimetry was used to measure cytosolic free Ca, Cai, in single parietal cells of intact rabbit gastric glands loaded with the Ca-sensitive fluorescent dye, fura-2. Cells were repeatedly stimulated with the cholinergic agonist carbachol to gain insights into the membrane mechanisms involved in hormonally stimulated Ca metabolism. In either Ca-containing or Ca-free solutions, carbachol (100 microM) caused a rapid (within 30 s) elevation of Cai from a resting level of 100 nM to greater than 600 nM. After the spike, Cai decreased within 3 min to a lower level that was somewhat elevated (greater than 200 nM) over base line. This plateau was dependent on both carbachol and extracellular Ca (Cao) and could be blocked by the addition of atropine (1 microM) or lanthanum ion (La, 50 microM). The spike is due to the release of Ca from internal stores, whereas the plateau is due to Ca entry across the plasma membrane through agonist-controlled, La-inhibitable channels. After a carbachol stimulation of 3 min or longer, reloading of the internal store was absolutely dependent on Cao. Under these conditions, reloading occurred through a La-sensitive (but nifedipine- and verapamil-insensitive) pathway in the plasma membrane. No significant change in Cai was detectable during the reloading. In contrast to the longer treatments, if carbachol stimulation was terminated with atropine while Cai was still elevated, significant reloading occurred from the cytosol.(ABSTRACT TRUNCATED AT 250 WORDS)
Evaluation of Pancreatic Amylase mRNA upon Cholinergic Stimulation of Secretion