Increased expression of neuronal Src and tyrosine phosphorylation of NMDA receptors in rat brain after systemic treatment with MK-801.

We have observed that systemic treatment with the uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 increases Src expression and NMDA receptor phosphorylation in rat brain. A partial cDNA encoding rat neuronal Src was isolated and its sequence was used to design specific oligonucleotide probes. Systemically administered MK-801 (5 mg/kg for 4 h) increased by 28+/-4% mRNA expression of neuronal Src in the superficial layers of the parietal cortex. This effect was observed at doses as low as 0.2 mg/kg. A similar, although more modest, induction was observed 6 h after phencyclidine (15 mg/kg) administration, but not after high doses of memantine and ketamine. The MK-801-induced effect was not blocked by pretreatment with clozapine. Consistent with the increase in mRNA levels, cortical Src protein was increased to 186 +/- 24% of control 24 h after MK-801 treatment. Total cellular Src activity was also increased in parietal cortex homogenates 4 h after MK-801 (5 mg/kg). Moreover, MK-801 treatment (0.5 mg/kg and 5 mg/kg for 4 h) increased tyrosine phosphorylation, but not protein levels, of the NMDA receptor subunit NR2A. These results provide evidence for a contribution of Src and tyrosine phosphorylation of NMDA receptors in the pharmacological actions of uncompetitive NMDA receptor antagonists.

Skeletal muscle type ryanodine receptor is involved in calcium signaling in human B lymphocytes.

The regulation of intracellular free Ca2+ concentration ([Ca2+]i) in B cells remains poorly understood and is presently explained almost solely by inositol 1,4,5-triphosphate (IP3)-mediated Ca2+ release, followed by activation of a store-operated channel mechanism. In fact, there are reports indicating that IP3 production does not always correlate with the magnitude of Ca2+ release. We demonstrate here that human B cells express a ryanodine receptor (RYR) that functions as a Ca2+ release channel during the B cell antigen receptor (BCR)-stimulated Ca2+ signaling process. Immunoblotting studies showed that both human primary CD19(+) B and DAKIKI cells express a 565-kDa immunoreactive protein that is indistinguishable in molecular size and immunoreactivity from the RYR. Selective reverse transcription-polymerase chain reaction, restriction fragment length polymorphism, and sequencing of cloned cDNA indicated that the major isoform of the RYR expressed in primary CD19(+) B and DAKIKI cells is identical to the skeletal muscle type (RYR1). Saturation analysis of [3H]ryanodine binding yielded Bmax = 150 fmol/mg of protein and Kd = 110 nM in DAKIKI cells. In fluo-3-loaded CD19(+) B and DAKIKI cells, 4-chloro-m-cresol, a potent activator of Ca2+ release mediated by the ryanodine-sensitive Ca2+ release channel, induced Ca2+ release in a dose-dependent and ryanodine-sensitive fashion. Furthermore, BCR-mediated Ca2+ release in CD19(+) B cells was significantly altered by 4-chloro-m-cresol and ryanodine. These results indicate that RYR1 functions as a Ca2+ release channel during BCR-stimulated Ca2+ signaling and suggest that complex Ca2+ signals that control the cellular activities of B cells may be generated by cooperation of the IP3 receptor and RYR1.

Early increases in TNF-alpha, IL-6 and IL-1 beta levels following transient cerebral ischemia in gerbil brain.

The effects of transient global ischemia using bilateral carotid artery occlusion on regional cytokine levels in gerbil brain were investigated using enzyme-linked immunoassay techniques. Brain concentrations of interleukin-6 (IL-6), interleukin-1 beta (IL-1 beta), and tumor necrosis factor-alpha (TNF-alpha) were increased during the early recirculation period ( < 6 h) after 10 min of ischemia, with lesser degrees of elevation following only 5 min of ischemia. TNF-alpha levels in the hippocampus and striatum were significantly increased as early as 1 h after recirculation, declining sharply to control levels by 12 h, then transiently increasing at 24 h. Elevated levels of IL-1 beta and IL-6 were not seen until 3-6 h post-occlusion. No significant increases in cytokine concentrations were observed in the cerebellum or thalamus. These results suggest that regionally selective increases in cytokines may be involved in the pathophysiological changes in hippocampus and striatum following transient cerebral ischemia.