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

Cholera toxin exerts its effects on target cells by irreversibly activating the enzyme adenylate cyclase [ 1,2]. This toxin consists of two non-identical subunits which are held together by non-covalent bonds. The A subunit consists of two non-identical peptide chains, Ar and Aa, of which Ar is the active peptide responsible for catalysing the NAD-dependent ribosylation of the guanine nucleotide regulatory unit of adenylate cyclase [3]. Associated with the two A peptides are 5 identical B subunits which each can bind toa G ml ganglioside. These glycolipids act as a cell surface receptor for the toxin molecule. In isolated membranes either cholera toxin itself or the isolated A subunit can activate adenylate cyclase directly [4,5]. 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]. 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. Receptor-mediated endocytosis has been shown to be an important mechanism by which cells rapidly bind and internalise specific extracellular ligands [9-121. A number of compounds interfere with aspects of this process [ 1 I], some of which are the lysomotropic agents, which specifically elevate the