Differential regulation of agonist-stimulated Ca2+ influx in acini of rat pancreas and submandibular gland.

In order to characterize agonist-dependent Ca2+ influx pathways, changes in cystolic Ca2+ (Ca2+i) during stimulation with multiple Ca(2+)-mobilizing agents were measured in rat pancreatic and submandibular gland acini loaded with fura-2. In pancreatic acini, maximal levels of carbachol and cholecystokinin octapeptide (CCK-8) produced virtually identical changes in Ca2+i when added alone or together: an immediate increase to 4-5 times resting Ca2+i followed by a decline to a steady-state level 2-3 times resting, which was unchanged by another stimulus. In submandibular gland acini, maximal carbachol stimulation increased Ca2+i 3-4-fold followed by a plateau at 2-3 times resting, which was further increased by epinephrine. Epinephrine alone increased steady-state Ca2+i to 53 +/- 18% (n = 21) of that observed with carbachol. Stimulation with both agents increased the steady-state plateau level of Ca2+i to 144 +/- 28% of that during exposure to carbachol alone (n = 11, p < 0.05). When changes in Ca2+i due solely to Ca2+ influx were measured, carbachol and epinephrine together increased Ca2+i during the steady-state phase to 149 +/- 31% of that measured with carbachol alone (n = 8, p < 0.05). Atropine blocked only responses to carbachol, prazosin blocked only responses to epinephrine and L 364,718 blocked only CCK-8-induced changes in Ca2+i. Thus, in pancreatic acini, a single agonist-sensitive Ca2+ influx pathway is linked independently to muscarinic cholinergic and peptidergic (CCK-8) receptors. In contrast, submandibular gland acini contain functionally separate agonist-sensitive Ca2+ influx pathways, which are independently linked to muscarinic and to alpha-1 adrenergic receptors.

Changes of cytosolic Ca2+ interfere with measurements of cytosolic Mg2+ using mag-fura-2.

In rat pancreatic and submandibular gland acini during exposure to carbachol, changes in the fluorescence emission intensity ratio (R) of acini loaded with mag-fura-2 resemble changes in cytosolic Ca2+ concentration (Ca2+i) in acini loaded with fura-2. Furthermore, changes of R depend on the presence of extracellular Ca2+ (Ca2+o) but are much less influenced by changes in extracellular Mg2+ (Mg2+o). To evaluate interference with measurement of cytosolic Mg2+ (Mg2+i) by changes in Ca2+i, we determined the dissociation constant (Kd) and Hill coefficient (NH) of the Ca(2+)-mag-fura-2 and Mg(2+)-mag-fura-2 complexes in standard solutions, in intact acini after loading with the acetoxymethyl ester of mag-fura-2 (mag-fura-2/AM), and in lysates derived from acini loaded with mag-fura-2/AM. The Kd of the Ca(2+)-mag-fura-2 complex in acini (determined with ionomycin) was 20.49 +/- 5.20 microM, and NH was 1.44 +/- 0.16 (n = 24). Kd of the Mg(2+)-mag-fura-2 complex in acini was 2.25 +/- 0.98 mM, and NH was 1.20 +/- 0.20 (n = 25). Mean Kd values were slightly lower in acinar lysates and in solutions of standard mag-fura-2. In acini from either gland, 1,2-bis(2-aminophenoxy)ethan-N,N,N',N'-tetraacetic acid (BAPTA) suppressed carbachol-induced Ca2+i transients. The R value in stimulated acini loaded with BAPTA and mag-fura-2 increased slightly when Mg2+o was increased from less than 10 nM to 1.2 mM, suggesting that Mg2+ influx contributes to the maintenance of Mg2+i during exposure to carbachol. Under these conditions, pancreatic acinar Mg2+i is 0.53 +/- 0.14 mM (n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)

Regulating transient and sustained changes of cytosolic Ca2+ in rat pancreatic acini.

The regulation of changes in cytosolic Ca2+ concentration (Cai2+) during exposure to carbachol was studied in rat pancreatic acini loaded with fura-2. With an extracellular Ca2+ concentration (Cao2+) of 2.5 mM, resting Cai2+ is 185 +/- 48 (SD) nM (n = 23), which rises to 696 +/- 222 nM, then falls over 2 min to a stable plateau of 401 +/- 106 nM. With Cao2+ less than 10 nM, carbachol produces an immediate threefold increase in Cai2+ that dissipates over 2-3 min, and Cai2+ steadily falls below prestimulation levels. Atropine prevents all responses to carbachol, and when it is added during a response to carbachol, Cai2+ drops to resting values within seconds. Ca2+ influx is required for the prolonged elevation of Cai2+ during carbachol exposure, but Ca2+ entry is not regulated by an increase in Cai2+ itself nor does Ca2+ enter via voltage-gated L-type channels. The muscarinic receptor-operated Ca2+ entry mechanism is sensitive to Cao2+, since sustained elevations in Cai2+ are maximal at 2.5 mM Cao2+ but are much less pronounced at lower external Ca2+ concentrations.

Regulation of cytosolic Ca2+ in resting and stimulated rat submandibular salivary gland acini.

To determine the range over which cytosolic Ca2+ concentration (Cai2+) is regulated, Cai2+ was measured in salivary acini loaded with Fura-2. With extracellular Ca2+ (Cao2+) = 2.5 mM, mean resting Cai2+ (+/- SD) was 103 +/- 20 nM (n = 29). Carbachol increased Cai2+ immediately to 288 +/- 81 nM, which then declined to a sustained level of 253 +/- 58 nM. When Cao2+ was less than 10 nM, the immediate response to carbachol was little affected but the sustained response was abolished. Atropine abolished all responses to carbachol. Changes in Cai2+ were not due to Ca2+-induced Ca2+ release or to Ca2+ influx via voltage-dependent channels. With Cao2+ = 2.5 mM, epinephrine raised Cai2+ initially to 196 +/- 57 nM (n = 6), which soon declined to stable levels of 178 +/- 40 nM. When Cao2+ was less than 10 nM the prolonged response to epinephrine was abolished but the initial increase was relatively unaffected. All epinephrine-induced changes in Cai2+ were abolished by prazosin. The range of Cai2+ measured corresponds to that over which non-mitochondrial Ca2+ transport is stimulated in permeabilized submandibular gland acini suggesting that such a Ca2+ transport system may regulate physiological changes in Cai2+ in these acini in the rat.