Detection of an mRNA polymorphism by differential display.

Differential Display (DD) technology was utilized to compare programs of gene expression in primary cultures of human skin fibroblasts from normal volunteers and patients diagnosed with melancholic depression. Polymorphic transcripts of a single gene differing by one tandem repeat sequence of four nucleotides (TGAT) in the 3' noncoding region were detected.

Lack of beta adrenoceptor desensitization in brain following the dual noradrenaline and serotonin reuptake inhibitor venlafaxine.

Venlafaxine, a dual amine reuptake inhibitor, was utilized to delineate the role of the individual aminergic components of the 'serotonin/noradrenaline link' in modifying receptor-linked second messenger cascades. Venlafaxine (20 mg/kg i.p. bid for 10 days) failed to alter in normal animals either the density of beta adrenoceptors or the response of the beta adrenoceptor-coupled adenylate cyclase system to noradrenaline but significantly decreased the cyclic AMP response to noradrenaline in the brain of rats with selective depletion of brain serotonin by p-chlorophenylalanine. The studies provide evidence for a cross-talk between noradrenergic and serotonergic receptor cascades at the level of mechanisms involved in the desensitization of the beta adrenoceptor-coupled adenylate cyclase system.

Norepinephrine-independent regulation of GRII mRNA in vivo by a tricyclic antidepressant.

Desipramine (DMI), a tricyclic antidepressant drug used in the treatment of depression, has been shown to increase steady-state levels of glucocorticoid receptor type II (GRII) mRNA in vitro and in vivo. To determine whether this effect is secondary to norepinephrine (NE) reuptake inhibition i.e., increases in synaptic NE induced by DMI, GRII mRNA levels were assayed in rat hippocampus following neurotoxic lesioning of NE neurons with DSP4. Chronic DMI treatment significantly increased GRII mRNA levels to the same degree in lesioned and non-lesioned animals. In contrast to DMI, the non-tricyclic antidepressant fluoxetine had no effect on GRII mRNA. These results provide evidence which demonstrates that a tricyclic antidepressant can regulate steady-state mRNA levels in vivo by a mechanism which is independent of its effects on synaptic monoamine levels.

Steady state levels of hepatic alpha 1- and beta 2-adrenergic receptors and gene transcripts during development of the male rat.

Metabolic events stimulated by epinephrine and norepinephrine in hepatocytes isolated from fetal and early postnatal male rats are largely mediated through the beta 2-adrenergic receptor-/cyclic AMP dependent-system, whereas the same stimuli are transduced through the alpha 1-adrenergic receptor-/phosphatidylinositol dependent-system in hepatocytes isolated from young adult male rats. This developmental transition was investigated by correlating hepatic alpha 1- and beta 2-adrenergic receptor gene transcript levels with receptor levels as determined with selective radioligands in livers from late fetal to postnatal day 120 male Sprague-Dawley rats. beta 2-Adrenergic receptor concentration, initially high in membrane preparations isolated from fetal livers (203 +/- 21 fmol/mg protein), dropped precipitously in postnatal day 6 livers (14 +/- 2 fmol/mg protein) and remained low throughout development out to postnatal day 90. beta 2-Adrenergic receptor mRNA levels were highest in fetal livers, were decreased somewhat in postnatal day 6 livers and were undetectable in livers beyond postnatal day 15. In contrast, hepatic alpha 1-adrenergic receptor concentration was relatively low in fetal livers (86 +/- 25 fmol/mg protein) and remained low until postnatal day 18. Thereafter, a steady increase in alpha 1-adrenergic receptors was observed until adult levels. (270 +/- 24 fmol/mg protein) were achieved at postnatal day 27. alpha 1-Adrenergic receptor mRNA levels increased approximately 3-fold, reaching a peak at postnatal day 24. Surprisingly, at postnatal day 30 hepatic alpha 1-adrenergic receptor mRNA levels dropped to fetal levels; but, gradually increased with continued development. Thus, hepatic alpha 1- and beta 2-adrenergic receptors appear to be under complex regulatory control which may include transcriptional, as well as post-transcriptional, mechanisms.

Detection by northern analysis of alpha 1-adrenergic receptor gene transcripts in the rat.

In Northern blots of total cellular and poly(A+) RNA isolated from rat liver, renal cortex, spleen, and brain probed with a full-length cDNA encoding the hamster alpha 1-adrenergic receptor, hybridization was observed to two distinct mRNAs, at approximately 3.3 kb and approximately 2.7 kb. Only the approximately 2.7 kb mRNA species was visualized in Northern blots of total cellular and poly(A+) RNA isolated from cardiac ventricular muscle. From screening a rat heart cDNA library with the full-length hamster alpha 1-adrenergic receptor cDNA, a 632 base pair cDNA was isolated. Based upon its high degree of identity, 86% at the nucleotide level, with the hamster alpha 1-adrenergic receptor cDNA, this cDNA was considered to include the 3' end of the rat alpha 1-adrenergic receptor. When used as a probe in Northern blots of liver RNA, both the approximately 3.3 kb and approximately 2.7 kb mRNAs were visualized. Both mRNA species were expressed in fetal as well as adult liver, but steady-state levels of each gene transcript were approximately 3-fold higher in adult compared to fetal liver. Finally, results from Southern analysis of restriction enzyme fragments of genomic DNA suggest that the two gene transcripts may be products of a single gene.