Cholera toxin mediated activation of adenylate cyclase in intact rat hepatocytes

Is the receptor‐mediated endocytosis of cholera toxin a pre‐requisite for its activation of adenylate cyclase in intact rat hepatocytes?

“…It has been suggested [ 181 that the binding of the B subunit to the membrane causes a conformational change in the toxin such that the A subunit is able to penetrate through the bilayer and interact with the guanine nucleotide regulatory subunit of adenylate cyclase at the cytosol side of the membrane. However, besides the obvious thermodynamic difficulties this poses, if the lag period did reflect such an event it is difficult to see why it should be affected by toxin concentration [6] and also why it is apparently unaffected by covalently cross-linking the A and B subunits together [20]. Furthermore, the A chain of cholera toxin does not appear to be inserted into the bilayer when the toxin binds to ganglioside-containing lipid vesicles [21].…”

“…However in intact cells there is a characteristic lag period before the onset of activation of adenylate cyclase [ 1,2]. The duration of this lag period in intact hepatocytes is dependent upon cholera toxin concentration and upon the fluidity of the cell plasma membrane [6]. The lag time has also been shown to be related to the cell surface redistribution of fluorescent-labelled cholera toxin [7,8].…”

“…The lag time has also been shown to be related to the cell surface redistribution of fluorescent-labelled cholera toxin [7,8]. These observations led to the suggestion [6] that the lateral redistribution of bound toxin may be of importance to the process that allows the A subunit to gain access to the cytosol surface of the plasma membrane where it can act on the guanine nucleotide regulatory unit of adenylate cyclase.…”

“…Cells (3-5 mg dry wt/ml) were pre-incubated for 20 min with lactate (10 mM) and in some instances with the test ligands prior to the addition of cholera toxin (10 E.cg/ml). Incubations, gassing, sampling and the treatment of samples were as in [6].…”

Treatment of intact hepatocytes with either the phorbol ester TPA or glucagon elicits the phosphorylation and functional inactivation of the inhibitory guanine nucleotide regulatory protein Gi

“…A washed membrane fraction was obtained from the hepatocytes as detailed previously described by us [10]. In all cases membranes were used within 2 h of their preparation.…”