[CA2-REDUCING ACTION OF CAMP IN RAT PANCREATIC BETA-CELLS - INVOLVEMENT OF THAPSIGARGIN-SENSITIVE STORES(](I))

In the present study, we examined the ability of adenosine 3',5'-cycli c monophosphate (cAMP) to reduce elevated levels of cytosolic Ca2+ con centration ([Ca2+](i)) in pancreatic beta-cells. [Ca2+](i) and reduced pyridine nucleotide, NAD(P)H, were measured in rat single beta-cells by fura 2 and autofluorescence microfluorometry. Sustained [Ca2+](i) e levation, induced by high KCl (25 mM) at a basal glucose concentration (2.8 mM), was substantially reduced by cAMP-increasing agents, dibuty ryl cAMP (DBcAMP, 5 mM), an adenylyl cyclase activator forskolin (10 m u M), and an incretin glucagon-like peptide-1-(7-36) amide (10(-9) M), as well as by glucose (16.7 mM). The [Ca2+](i)-reducing effects of cA MP were greater at elevated glucose (8.3-16.7 mM) than at basal glucos e (2.8 mM). An inhibitor of protein kinase A (PKA), H-89, counteracted [Ca2+](i)-reducing effects of cAMP but not those of glucose. Okadaic acid, a phosphatase inhibitor, at 10-100 nM also reduced sustained [Ca 2+](i) elevation in a concentration-dependent manner. Glucose, but not DBcAMP, increased NAD(P)H in beta-cells. [Ca2+](i) reducing effects o f cAMP were inhibited by 0.3 mu M thapsigargin, an inhibitor of the en doplasmic reticulum (ER) Ca2+ pump. In contrast, [Ca2+](i)-reducing ef fects of cAMP were not altered by ryanodine, an ER Ca2+-release inhibi tor, Na+-free conditions, or diazoxide, an ATP-sensitive K+ channel op ener. In conclusion, the cAMP-PKA pathway reduces [Ca2+](i) elevation by sequestering Ca2+ in thapsigargin-sensitive stores. This process do es not involve, but is potentiated by, activation of beta-cell metabol ism. Together with the known [Ca2+](i)-increasing action of cAMP, our results reveal dual regulation of beta-cell [Ca2+](i) by the cAMP-sign aling pathway and by a physiological incretin.