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In Dictyostelium discoideum cells the enzyme adenylate cyclase is functionally coupled to cell surface receptors for cAMP. Coupling is known to involve one or more G-proteins. Receptor-mediated activation of adenylate cyclase is subject to adaptation. In this study we employ an electropermeabilized cell system to investigate regulation of D. discoideum adenylate cyclase. Conditions for selective permeabilization of the plasma membrane have been described by C.D. Schoen, J. C. Arents, T. Bruin, and R. Van Driel (1989, Exp. Cell Res. 181, 51-62). Only small pores are created in the membrane, allowing exchange of exclusively low molecular weight substances like nucleotides, and preventing the loss of macromolecules. Under these conditions functional protein-protein interactions are likely to remain intact. Adenylate cyclase in permeabilized cells was activated by the cAMP receptor agonist 2'-deoxy cAMP and by the nonhydrolyzable GTP-analogue GTP gamma S, which activates G-proteins. The time course of the adenylate cyclase reaction in permeabilized cells was similar to that of intact cells. Maximal adenylate cyclase activity was observed if cAMP receptor agonist or GTP-analogue was added just before cell permeabilization. If these activators were added after permeabilization adenylate cyclase was stimulated in a suboptimal way. The sensitivity of adenylate cyclase activity for receptor occupation was found to decay more rapidly than that for G-protein activation. Importantly, the adenylate cyclase reaction in permeabilized cells was subject to an adaptation-like process that was characterized by a time course similar to adaptation in vivo. In vitro adaptation was not affected by cAMP receptor agonists or by G-protein activation. Evidently electropermeabilized cells constitute an excellent system for investigating the positive and negative regulation of D. discoideum adenylate cyclase.

Dictyostelium discoideum cells synthesize and secrete the chemoattractant cAMP within minutes after chemotactic stimulation. During development, this signal-relay process is instrumental in cell aggregation, pattern formation, and differentiation. Cyclic AMP is known to accumulate inside the cell before secretion. In this study we investigated the subcellular localization of the nascent cAMP. After chemotactic stimulation at 0 degrees C and subsequent accumulation of intracellular cAMP, the newly synthesized chemoattractant could be released by gently opening cells in two different ways. Both methods make the cytosolic compartment accessible, whereas intracellular compartments surrounded by a membrane remain largely intact. The first method involved rapid lysis by forced passage through a 5-micron pore-size Nuclepore filter. The second technique was electropermeabilization under carefully controlled conditions that ensured the formation of small, stable pores in the plasma membrane. These pores allowed the passage of small molecules, such as cAMP, but not of macromolecules. To confirm the selectivity for the plasma membrane of both methods, we showed that a typical vesicular cell compartment, the lysosome, remained intact. Both procedures immediately released all intracellularly accumulated cAMP. We interpret our results as strong evidence for accumulation of nascent cAMP in the cytosolic compartment rather than in a vesicular compartment before it is secreted. This implies that cAMP secretion takes place via a trans-membrane transport mechanism, rather than by exocytosis.