The membrane permeability of pancreatic zymogen granules was evaluated in vitro with granules isolated from rats in different secretory states: 1) untreated, 2) pretreated with a muscarinic antagonist, 3) pretreated with a muscarinic and an adrenergic antagonist, 4) pretreated as in 3 and then stimulated with the secretagogue cholecystokinin 4 min before death, and 5) pretreated as in 3 and then stimulated with the secretagogue secretin 4 min before death. Granules isolated from untreated rats had variable ionic permeabilities but in general possessed both chloride conductance and electroneutral exchange pathways with low permeabilities to alkali metal ions. In contrast, granules from animals pretreated with secretory antagonists had very low ion permeabilities to both inorganic anions, such as chloride, and alkali metal ions. Injection of the peptide secretagogues cholecystokinin or secretin resulted in a relatively fast (within 4 min) activation or induction of high chloride permeabilities through both chloride conductance and chloride/hydroxide (or chloride/bicarbonate) exchange pathways. In addition, the secretagogues increased the cation permeability of the granule membrane, which exhibited a distinct potassium selectivity. Chloride conductance has been postulated to play a major role in fluid secretion coupled to exocytosis of macromolecules [R. C. DeLisle and U. Hopfer, Am. J. Physiol. 250 (Gastrointest. Liver Physiol. 13): G489-G496, 1986]. These results demonstrate that granules may actively participate in the secretory process and suggest that some of the physiological targets in the cascade of events leading to secretion are anion and cation transporters in the zymogen granule membrane.
The regulation of Cl- conductance by cytoplasmic nucleotides was investigated in pancreatic and parotid zymogen granules. Cl- conductance was assayed by measuring the rate of cation-ionophore-induced osmotic lysis of granules suspended in iso-osmotic salt solutions. Both inhibition and stimulation were observed, depending on the type and concentration of nucleotide. Under optimal conditions, the average inhibition measured in different preparations was 1.6-fold, whereas the average stimulation was 4.4-fold. ATP was inhibitory at 1-10 microM but stimulated Cl- conductance above 50 microM. Stimulation by ATP was more pronounced in granules with low endogenous Cl- conductance. The potency of nucleotides in terms of inhibition was ATP greater than adenosine 5'-[gamma-thio]triphosphate (ATP[S]) greater than UTP much greater than or equal to CTP much greater than or equal to GTP much greater than or equal to guanosine 5'-[gamma-thio]triphosphate (GTP[S]) much greater than or equal to ITP. The potency with respect to stimulation had the following order: adenosine 5'-[beta gamma-methylene]triphosphate (App[CH2]p) greater than ATP greater than guanosine 5'-[beta-thio]diphosphate (GDP[S]). Adenosine 5'-[beta gamma-imido]triphosphate (App[NH]p) was also stimulatory, and was more potent than ATP in the parotid granules, but less potent in the pancreatic granules. Aluminium fluoride stimulated Cl- conductance maximally at 15-30 microM-Al3+ and 10-15 mM-F. F was less effective at higher concentrations. Protein phosphorylation by kinases was apparently not involved, since the nucleotide effects (1) could be mimicked by non-hydrolysable analogues of ATP and GTP, (2) showed reversibility, and (3) were not abolished by the protein kinase inhibitors 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H-7) or staurosporine. The data suggest the presence of at least two binding sites for nucleotides, whereby occupancy of one induces inhibition and occupancy of the other induces stimulation.
Zymogen granules that were stable at physiological conditions of pH, ionic strength, and temperature were isolated from the rat pancreas and parotid. The cation permeability of these granules was evaluated to characterize the mechanism of secretagogue-stimulated fluid secretion by acinar cells. Granule swelling and lysis provide a measure of the rate of cation transport, since the use of ionophore combinations such as tripropyltin and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) will render cation conductance the rate-limiting step for salt influx. This technique supplies evidence for the existence of K+ conductance in the granule membrane. The pancreatic and parotid granules have a K+-selective conductance that is not inhibited by the K+ channel blockers barium, tetraethylammonium, quinidine, cesium, or 4-aminopyridine. Furthermore, the intragranular pH of pancreatic zymogen granules was measured to be approximately 6.5 and was identified as a factor that modulates the K+ conductance. Although the pancreatic and parotid granules were qualitatively identical, quantitatively the relative K+ transport rate constant was over twofold higher for the parotid than for the pancreatic granules. The zymogen granule K+ conductance may have an important role in active K+ secretion by exocrine glands, which is prominent in the parotid after stimulation with beta-adrenergic agents.
The Cl- transport pathways in secretory granules isolated from the parotid glands of rats were characterized by the technique of ionophore-induced lysis in defined salt solutions. The granules were shown to possess a Cl- conductance that exhibited a distinct anion selectivity with a sequence I- greater than Br- greater than Cl- greater than F- greater than SO4(2-) much greater than gluconate-. This conductance could be reduced approximately 40% by the stilbene 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) from the cytoplasmic side; the half-maximal concentration for inhibition was 50 microM. Furthermore, the apparent Cl- conductance was reduced by outwardly directed granule H+ gradients and stimulated by inwardly directed gradients. An outwardly directed H+ gradient mimics the in vivo environment and may serve in a regulatory capacity, providing for a tonic inhibition of transport until the granule fuses with the luminal membrane. The granules also possessed a Cl(-)-HCO3- exchange based on electroneutrality of Cl- uptake and stimulation of this uptake by HCO3-. This pathway displayed a different anion selectivity, I- greater than Br- greater than F- greater than Cl- much greater than SO4(2-) much greater than gluconate-, and was not inhibited by SITS on the cytoplasmic side. The presence of these electrolyte transport pathways in the granule membrane is consistent with the production of primary fluid by parotid acinar cells after fusion of granules with the luminal plasma membrane.
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