Abstract
Global (all cells in an acinus) and focal (1-2 out of 10-15 cells) stimulation of pancreatic acini with bombesin or t-butyloxycarbonyl-Tyr(SO3)-Nle-Gly-Tyr-Asp-2-phenylethyl ester (CCKJ) together with modulation of gap junction (GJ) permeability by octanol and NO2- was used to study the role of GJ permeability in controlling [Ca2+]i oscillations and enzyme secretion. GJ permeability was quantitated by measuring fluorescence recovery after photobleaching. Octanol at 0.5 mM markedly reduced, whereas 15 mM NO2- increased GJ permeability. Focal application of bombesin caused synchronized oscillations in the entire acinus, whereas global stimulation resulted in asynchronous oscillations. Increasing GJ permeability with NO2- had no effect on bombesin-evoked [Ca2+]i oscillations. Octanol inhibited ongoing oscillations evoked by focal or global bombesin stimulation. However, when GJ were blocked prior to stimulation, subsequent global stimulation with bombesin induced long-lasting oscillations in all cells. Re-establishing GJ communication for as little as 37.5 s conferred GJ dependence on the order and time of [Ca2+]i spiking evoked by global bombesin stimulation. Focal and global stimulation with CCKJ gave different patterns of [Ca2+]i oscillations. However, in contrast to bombesin, inhibition of GJ with octanol had no effect on oscillations induced by global CCKJ stimulation. Increasing GJ permeability with NO2- synchronized CCKJ-stimulated oscillations by equalizing the amplitude and increasing the frequency in all cells within an acinus. These observations suggest that amplitude and frequency of [Ca2+]i oscillations can be regulated independently of each other, and that GJ permeable molecules modulate the frequency of [Ca2]i oscillation in an agonist-specific manner. Regardless of the agonist, increasing the frequency of oscillations by modulation of GJ permeability correlated with an increased enzyme secretion.
Highlights
We examined the role of gap junction (GJ) in theregulation of [Ca2+Ioi scillations in pancreatic acinar cells
Modulation of GJ Permeability-In previous studies with pancreatic acini, the techniques used to evaluate GJ permeability involved impaling the cells with microelectrodes and dye or current injection
A new noninvasive technique for measurement of GJ permeability based on fluorescence recovery after photobleaching (FRAP) has been reported recently [27, 28]
Summary
Global (all cells in an acinus) and focal In contrast to bombesin, inhibition of GJ with octanol had no effect on oscillations induced by global agonists reduces GJ permeability [8,9,10,11,12] which would suggest that GJ do not participate in controlling the Ca2+signaling. When focal stimulation was used, at the end of Fura-2 recording, the fluorescent set up was changed to monitor the CF image. Measurements of Amylase Release-Acini from one rat pancreas were suspended in 20mlof solution A and incubated for 10 min at 37 "C prior to stimulation. Amylase release is expressed as a percent of total activity present prior to agonist stimulation
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