STIMULUS-SECRETION COUPLING IN PANCREATIC ß-CELLS

STIMULUS-SECRETION COUPLING IN PANCREATIC ß-CELLS: A patch-clamp study using caged compounds. CARINA AMMÄLÄ, Department of Medical Physics, Göteborg University, Medicinaregatan 11, S-413 90 Göteborg, Sweden. The regulation of the stimulus-secretion coupling in single mouse pancreatic ß-cells was investigated using the patch-clamp technique in combination with microfluorimetry to record changes in the intracellular Ca2+-concentration ([Ca2*];), capacitance measurements to monitor exocytosis and flash photolysis of caged compounds to produce step increases in the concentration of the substances. Activation of G-proteins by intracellular application of GTPyS produced a slowly developing inhibition of L-type Ca2+-currents manifested as a slowed time course of activation and a reduction of the peak amplitude. Inclusion of cAMP in the pipette solution failed to counteract the GTPyS-induced inhibition excluding protein kinase A (PKA) as a mediator of the effects. The interaction between the activated G-protein and the Ca2+-channel involves the entry of the Ca2+-channel into distinct closed state from which the channel opens with a slower time-course than in the absence of GTPyS. A fraction of the channels exists in the modified closed state even in the absence of GTPyS as suggested by the finding that pretreatment with pertussis toxin increased the current amplitude under basal conditions. Rapid membrane potential oscillations, similar to those observed in cells within intact islets, could be recorded from large clusters and were then associated with a transient oscillating outward current. In single ß-cell, such rapid oscillations were usually not observed but could be evoked by extracellular application of carbamylcholine (CCh) or dibutyryl-cAMP (db-cAMP), compounds functionally related to hormones and neurotransmitters normally present in the intact islet, or by intracellular infusion with GTPyS. The oscillatory conductance is selective to K+ and activated by an increase in [Ca2+]j. Tetraethylammonium, charybdotoxin, apamin and tolbutamide, blockers of K+-channels previously known to be present in the ßcell, had no effect on the current suggesting it flows through a novel type of K+-channel. The estimated single-channel conductance is 0.1 pS under physiological conditions. The properties of the K+-channel makes it a possible candidate for a channel involved in the generation of membrane potential oscillations. The Ca2+-dependent action potentials are associated with a rise in [Ca];. Although lns(l,4,5)P3 elicited a [Ca]rtransient 20-30 times larger than the average [Ca]rtransients evoked by voltage-clamp depolarizations, the rate of exocytosis produced by the different maneuvers were the same. This suggests that exocytosis, in response to membrane depolarization, requires much higher concentrations than suggested by microfluorimetry and implies the existence of steep Ca2+-gradients within the ß-cell. The coupling between an elevation in [Ca2+]j and the stimulation of the secretion involves activation of Ca27calmodulindependent protein kinase II and inhibitors of this enzyme suppress exocytosis. In addition, secretion is markedly potentiated by cAMP through activation of PKA. A minor part (20%) of this effect is mediated by an increase in Ca2+-influx through L-type voltage-dependent Ca2+channels. The major (80%) potentiating effect results from a direct interaction with the secretory machinery, possibly exerted by increasing the Ca2+-sensitivity of the secretory machinery, thus extending the distance from the Ca2+channel over which granules can be recruited for release.