Biological response to phorbol ester determined by alternative G1 pathways.

A plethora of extracellular signals is known to induce a common set of immediate early genes. The immediate early response, therefore, must not be sufficient to determine the biological outcome. An example of this is found with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). A potent activator of protein kinase C, TPA can either stimulate or inhibit cell proliferation, depending on the cell type. This cell context-dependent response to TPA is observed with two subclones of NIH 3T3 cells, the P- and the N-3T3 clones. TPA is a mitogen for the P-3T3 but an antimitogen for the N-3T3 cells. The immediate early pathway is activated by TPA in both cell types, indicating that this pathway alone does not activate DNA synthesis. The delayed induction of cyclin D1 expression by TPA is observed only in the P-3T3 cells, correlating with mitogenesis. N-Acetylcysteine does not affect the immediate early pathway but can inhibit the TPA-mediated induction of cyclin D1 and DNA synthesis. In the N-3T3 cells, TPA causes an inhibition of the cyclin E-associated kinase at the G1/S transition, correlating with growth inhibition. The growth-inhibitory activity of TPA is not affected by N-acetylcysteine. Thus, the two TPA-regulated G1 pathways can be distinguished by their sensitivity to N-acetylcysteine. These results demonstrate that TPA can activate alternative G1 pathways. Moreover, the selection of the alternative G1 pathways is determined by the cell context, which, in turn, dictates the biological response to TPA.

Citrate modulates the regulation by Zn2+ of N-methyl-D-aspartate receptor-mediated channel current and neurotransmitter release.

The effect of the two metal-ion chelators EDTA and citrate on the action of N-methyl-D-aspartate (NMDA) receptors was investigated by use of cultured mouse cerebellar granule neurons and Xenopus oocytes, respectively, to monitor either NMDA-evoked transmitter release or membrane currents. Transmitter release from the glutamatergic neurons was determined by superfusion of the cells after preloading with the glutamate analogue D-[3H]aspartate. The oocytes were injected with mRNA isolated from mouse cerebellum and, after incubation to allow translation to occur, currents mediated by NMDA were recorded electrophysiologically by voltage clamp at a holding potential of -80 mV. It was found that citrate as well as EDTA could attenuate the inhibitory action of Zn2+ on NMDA receptor-mediated transmitter release from the neurons and membrane currents in the oocytes. These effects were specifically related to the NMDA receptor, since the NMDA receptor antagonist MK-801 abolished the action and no effects of Zn2+ and its chelators were observed when kainate was used to selectively activate non-NMDA receptors. Since it was additionally demonstrated that citrate (and EDTA) preferentially chelated Zn2+ rather than Ca2+, the present findings strongly suggest that endogenous citrate released specifically from astrocytes into the extracellular space in the brain may function as a modulator of NMDA receptor activity. This is yet another example of astrocytic influence on neuronal activity.