INCREASE IN CYTOPLASMIC FREE CALCIUM IN MURINE SPLENOCYTES FOLLOWING STIMULATION WITH ANTI-CD3 ANTIBODY IN THE PRESENCE OF DELTA-9-TETRAHYDROCANNABINOL

It has been previously shown that when mitogens such as concanavalin A (Con A) or phytohemagglutinin (PHA), are used to stimulate lymphoid cells which are treated with varying doses of DELTA-9-tetrahydrocannabinol (THC), the proliferation of splenocytes from mice of different ages is suppressed. In contrast, when these cells were stimulated with anti-CD3 antibody in combination with THC, lower doses of THC stimulated proliferation of the splenocytes. This stimulation occurred only if the spleens were obtained from adult (2 month) mice as opposed to cells from young (2 week) or aged (24 month) mice. In order to more completely understand this age related differential effect, mobilization of cytosolic free Ca2+ was studied in this system, using fluorescent Ca2+ probes and spectrofluorometry. It was found that adult splenocytes pretreated with anti-CD3 antibody responded to cross-linking by anti-IgG antibody with a further rise in intracellular free Ca2+. Such an increase in Ca2+ was not seen with cells derived from either young or old mice. A similar phenomenon occurred when 5 mug/ml THC was used in place of the anti-IgG antibody. Thus, adult spleen cells exposed to both DELTA-9-THC and anti-CD3 antibody displayed an increase in intracellular free calcium whereas spleen cells from very young mice failed to respond in this manner. Interestingly, when 11-hydroxy-THC, another metabolite of marijuana, was used instead of the DELTA-9-THC, no rise in intracellular Ca2+ influx was seen in any age group of mice tested. These results emphasize the differential effect of THC on splenocytes from individuals of different ages. Further, these findings demonstrate a contrasting effect of two metabolites of marijuana, DELTA-9-THC and 11-OH-THC, on calcium mobilization in murine splenocytes.