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Treatment of mouse lymphocytes with very low concentrations of alamethicin or Lubrol PX induces spontaneous permeabilization of the plasma membrane to ATP and allows determination of adenylate cyclase activity in whole cells. The permeabilized cells retain responsiveness to hormones (isoproterenol, adenosine analogs) and to fluoride. The main advantage of this new method is that it does not require any homogenization step, and thus adenylate cyclase activities can be accurately and reproducibly measured with very low amounts of cells. It should be especially useful for the study of purified lymphocyte subpopulations.

A large-scale purification of plasma membranes from pig lymph node lymphocytes is described. Centrifugation on a discontinuous sucrose density gradient was performed in a zonal rotor. Adenylate cyclase activity of untreated fractions displayed a profile different from that of plasma membrane enzymatic markers and was maximal at higher density. However, when latent adenylate cyclase was unmasked by Lubrol PX treatment, its maximum was shifted to lower density and was no longer significantly different from that of plasma membrane markers. These results are discussed in terms of cell surface topography.

Adenosine-cyclic AMP relationships have been studied in pig mesenteric lymph node lymphocytes. The early 2--3-fold increase in cyclic AMP accumulation elicited by adenosine and 2-chloroadenosine, an adenosine deaminase-resistant analogue, could not be correlated to similar effects on the adenylate cyclase activity of disrupted cell preparations, but rather to the competitive inhibition of the low Km (0.17 muM) cyclic AMP phosphodiesterase. The existence of adenosine receptors coupled to lymphocyte adenylate cyclase, which had been proposed by several authors, could not be confirmed by this study Adenosine-cyclic AMP relationships do not appear to be involved in concanavalin A stimulation of pig lymphocytes.

Lymphocytes possess membrane adenosine receptors through which this nucleoside or analogs activate adenylate cyclase. Several adenosine analogs - 5'-N-ethylcarboxamide adenosine (NECA), N6-(L-2-phenyl-isopropyl) adenosine (PIA), 2-chloro adenosine (Cl-Ado) - were tested on the adenylate cyclase of mouse thymocyte homogenates; the order of potencies: NECA greater than Cl-Ado greater than PIA is consistent with the order which has been assumed to be characteristic of Ra type adenosine receptors. The stimulation of thymocyte adenylate cyclase by NECA was biphasic while the responses to Cl-Ado and PIA were not, which suggests the existence of Ra sub-types of adenosine receptors. Moreover, these adenosine receptors were found to be present in several lymphocyte subclasses: mature and immature mouse thymocytes, B and T mouse splenocytes, human peripheral blood lymphocytes, and also in lymphoblastoid cell lines. No crucial differences were observed between the adenylate cyclase stimulations of the various population by NECA.