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Abstract

The effect of p-chloromercuribenzoate (pCMB) on secretin stimulated pancreatic fluid secretion in vivo was investigated and compared with its effect on secretin binding and secretin stimulated adenylate cyclase in isolated pancreatic plasma membranes in vitro. A biphasic effect of pCMB “was observed. At low concentrations (10^–5 –5 × 10^-8M) pCMB stimulated adenylate cyclase activity, secretin binding and secretin stimulated pancreatic fluid secretion by ~ 50, 25 and 100%, respectively. At higher concentrations (10^-7 – 10^-5M) pCMB inhibited secretin binding by 50%. In the same range of pCMB concentrations secretin stimulated adenylate cyclase was inhibited in a dose dependent fashion. Basal adenylate cyclase activity was much less susceptible to the inhibition by pCMB since about 50 times greater concentration of pCMB is required for half-maximal inhibition (5 × 10^-5M and 10^-6M, respectively).

To restrict the effect of the SH group reagent to the outer mem­brane surface a large Dextrgn-linked derivative of pCMB was used in in vivo experiments. At 10^-8M this compound inhibited secretin induced fluid secretion by 47%. About one half of this inhibition is due to the blocking of SH groups involved in glucose transport since it is abolished by replacing glucose in perfusion fluid by substrates of the Krebs-cycle. The other half of inhibition is directly related to the secretin action since it is abolished by replacing secretin by dibutyryl cAMP and theophylline.

The data show that accessible SH groups located at the cell surface are directly involved in secretin binding and adenylate cyclase stimulation. Since the apparent K_m for secretin stimulation of adenylate cyclase and the Kd for secretin binding were in agreement and since pCMB stimulated and inhibited both secretin binding and secretin stimulated adenylate cyclase activity in the same concentration range it-is suggested that the binding of secretin to its receptor is the rate determining step in the stimulation of adenylate cyclase by this hormone. The biphasic action of pCMB can be best interpreted with the assumption that several categories of SH-groups are present in the plasma membrane.