Regulation of gonadotropin-releasing hormone gene expression by the excitatory amino acids kainic acid and N-methyl-D,L-aspartate in the male rat.

Abstract

The glutamate analogs N-methyl-D,L-aspartate (NMA) and kainic acid are involved in the regulation of GnRH and LH release in mammals. It has recently been reported that the increase in GnRH release induced by NMA is accompanied by an increase in GnRH mRNA levels, as measured by in situ hybridization. In the present study we assessed the effects of NMA and kainic acid on cytoplasmic mRNA levels using the more quantitative solution hybridization/RNase protection assay. To address the mechanism responsible for these mRNA changes, we also examined changes in heteronuclear RNA transcripts as a reflection of gene transcription. Adult male rats were implanted with a jugular catheter, and 1-2 days later, NMA (14 mg/kg BW), kainic acid (2 mg/kg BW), both NMA and kainic acid, or saline vehicle were injected through the cannula. Rats were killed 15 min or 1 h later by decapitation, blood samples were collected for RIA of LH, brains were removed, and the preoptic area was dissected and frozen. Cytoplasmic and nuclear RNA were extracted and assayed separately by RNase protection assay. Treatment with NMA or NMA plus kainic acid resulted in significantly elevated cytoplasmic mRNA levels 15 min and 1 h later compared to saline control values, with no differences between the two drug treatments observed. Kainic acid stimulated mRNA levels 1 h, but not 15 min after injection. Nuclear RNA transcripts were unaffected by all drug or vehicle treatments. As nuclear primary transcript levels presumably reflect GnRH gene transcription, and these levels are unaltered, the present study indicates that the regulation of GnRH gene expression by excitatory amino acids occurs at a posttranscriptional level. The increase in cytoplasmic GnRH mRNA levels also does not result from an increased translocation of the relatively large nuclear GnRH mRNA pool into the cytoplasm, because nuclear GnRH mRNA levels are also unchanged. Therefore, the elevation of cytoplasmic mRNA levels after excitatory amino acid treatment is probably due to an increase in mRNA stability.