Dual modulation of chloride conductance by nucleotides in pancreatic and parotid zymogen granules

Abstract

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.

Chloride and Potassium Conductances of Mouse Pancreatic Zymogen Granules Are Inversely Regulated by a ≈80-kDa mdr1a Gene Product

“…1C). AMP-PCP (0.5 mM), which increases rat pancreatic ZG Cl Ϫ conductance (15,17), enhanced lysis both in (ϩ/ϩ) and (Ϫ/Ϫ) mice. The increase in Cl Ϫ conductance was, however, more pronounced in (ϩ/ϩ) mice than in (Ϫ/Ϫ) mice.…”

“…A nonselective cation conductance is not affected by adenine nucleotides and blocked by micromolar concentrations of Ba 2ϩ and also by flufenamic acid (15). A Cl Ϫ conductance is activated by ATP and nonhydrolyzable ATP analogs and is blocked by DIDS (16,17).…”

Regulation of the water channel aquaporin‐1: isolation and reconstitution of the regulatory complex

“…Studies using isolated ZGs from the exocrine pancreas and parotid glands demonstrate the presence of Cl and ATP-sensitive K + selective ion channels at the vesicle membrane, whose activities have been implicated in vesicle swelling (Fuller et al, 1989a,b;Gasser et al, 1988;Gasser and Hopfer, 1990;Segawa et al, 1991;Takuma et al, 1993;Thevenod et al, 1990Thevenod et al, , 1992Thevenod et al, , 1996. Furthermore, secretion of ZG contents in permeabilized pancreatic acinar cells requires the presence of both K + and Cl ions (Curran and Brodwick, 1991).…”

GTP-Binding Proteins and Regulated Exocytosis

“…GTP and GTP analogs have also been demonstrated to modulate, in vitro, the Cl-conductance found to be present in ZGs isolated from rat pancreatic acini (DeLisle and Hopfer, 1986;Gasser et al, 1988;Thevenod et al, 1990) and SGs isolated from parotid acini (Gasser et al, 1988;Thevenod et al, 1990;Watson et al, 1997). Further, AIF4-also increased Cl-conductance in ZGs and SGs (Thevenod et al, 1990;Watson et al, 1997), suggesting a role for a G-protein.…”

“…Further, AIF4-also increased Cl-conductance in ZGs and SGs (Thevenod et al, 1990;Watson et al, 1997), suggesting a role for a G-protein. The Cl-conductance of isolated SGs, determined by measuring the decrease in absorbance with time at 540 nm, was considered to be a measure of granule lysis and, therefore, exocytosis.…”