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M M Cripps, D J Bennett; Guanine nucleotide binding proteins in the dual regulation of lacrimal function.. Invest. Ophthalmol. Vis. Sci. 1992;33(13):3592-3600.
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The purpose of this study was to identify and characterize functional G proteins that couple regulatory peptides with lacrimal secretory functions. Membranes were prepared from isolated rat exorbital lacrimal gland acini, and guanosine 5'-triphosphate (GTP)-dependence of adenylyl cyclase activity, known to be coupled with regulation of secretion, was measured. The guanine nucleotide GTP produced a biphasic response in the activity of membrane-bound adenylyl cyclase during a 10 min incubation with a maximum stimulation at 10(-5) mol/l GTP. Significant inhibition occurred at a dose of 10(-3) mol/l GTP, with cyclic adenosine monophosphate (cAMP) production reduced to less than basal levels. The effect of ADP-ribosylation of membrane proteins by the toxins produced by Vibrio cholera or Bordetella pertussis on lacrimal adenylyl cyclase was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography, and laser densitometry. Cholera toxin treatment of membranes resulted in dose-(0.5-100 micrograms/ml) and time-dependent (0-45 min) adenosine diphosphate (ADP)-ribosylation of two membrane proteins with M(r) values of 42,000 and 45,000. Pertussis toxin treatment resulted in the specific ADP-ribosylation of a single protein that migrates with an M(r) value of 41,000. This also was dose (0.5-25 micrograms/ml) and time dependent (0-30 min). Incorporation of 32P into the 45,000 M(r) and 42,000 M(r) proteins in the presence of 50 micrograms/ml cholera toxin was guanine nucleotide dependent, with a two- to threefold increase in labeling when the membranes were incubated with 1 or 0.5 mmol/l GTP. This effect was enhanced in the presence of the nonhydrolyzable GTP analog GTP gamma S.(ABSTRACT TRUNCATED AT 250 WORDS)
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