Purification and identification of pituitary cytotropic factor.

It has been shown that the pituitary contains a cytotropic factor (CTF) that stimulates the secretion of catecholamines by dopaminergic neurons of the hypothalamus. In the present study, CTF was purified from rat pituitaries and found by means of mass spectrometric analysis to be adenosine. This finding was corroborated by the observations that CTF behaves identically to adenosine when subjected to liquid chromatography, is inactivated and converted to inosine by adenosine deaminase, and is qualitatively and quantitatively indistinguishable from adenosine in its biological activity. It is concluded that pituitary adenosine is a trophic factor for hypothalamic dopaminergic neurons.

Accelerated evolution in the protein-coding regions is universal in crotalinae snake venom gland phospholipase A2 isozyme genes.

The nucleotide sequences of four genes encoding Trimeresurus gramineus (green habu snake, crotalinae) venom gland phospholipase A2 (PLA2; phosphatidylcholine 2-acylhydrolase, EC 3.1.1.4) isozymes were compared internally and externally with those of six genes encoding Trimeresurus flavoviridis (habu snake, crotalinae) venom gland PLA2 isozymes. The numbers of nucleotide substitutions per site (KN) for the noncoding regions including introns were one-third to one-eighth of the numbers of nucleotide substitutions per synonymous site (KS) for the protein-coding regions of exons, indicating that the noncoding regions are much more conserved than the protein-coding regions. The KN values for the introns were found to be nearly equivalent to those of introns of T. gramineus and T. flavoviridis TATA box-binding protein genes, which are assumed to be a general (nonvenomous) gene. Thus, it is evident that the introns of venom gland PLA2 isozyme genes have evolved at a similar rate to those of nonvenomous genes. The numbers of nucleotide substitutions per nonsynonymous site (KA) were close to or larger than the KS values for the protein-coding regions in venom gland PLA2 isozyme genes. All of the data combined reveal that Darwinian-type accelerated evolution has universally occurred only in the protein-coding regions of crotalinae snake venom PLA2 isozyme genes.

Evidence for a role of pituitary ATP receptors in the regulation of pituitary function.

Despite a rapidly increasing acceptance for a role of ATP as an extracellular mediator in several biological systems, the present report shows that ATP may mediate physiological responses in pituitary cells. We have now been able to demonstrate a specific action of ATP receptors to mediate the release of luteinizing hormone from gonadotropes and have coupled them with further studies that clearly show that ATP can be exocytotically released from cultured rat pituitary cells. Both ATP and UTP (100 microM) caused a > 14-fold increase in the rate of luteinizing hormone release from superfused cells. Adenosine 5'-[alpha, beta-methylene]triphosphate and 5'-[beta,gamma-methylene triphosphate were ineffective, and 2-methylthio-ATP had only a modest stimulatory effect. Homologous and heterologous desensitization occurred with UTP and ATP, and these did not have additive effects. Thus, nucleotides can be effective stimulators of luteinizing hormone release through a single class of ATP receptor (P2U subtype). The calcium ionophore A23187 provoked release of a substantial amount of ATP from pituitary cells in a concentration- and Ca(2+)-dependent manner, which was desensitized by pretreatment with A23187. This implies a possible paracrine and/or autocrine mechanism by which nucleotides may exert their effects on pituitary cells. In conclusion, we have provided strong evidence for a novel role of extracellular nucleotides as mediators in pituitary--in particular, in gonadotrope--function.

Estrogen regulates the expression of several different estrogen receptor mRNA isoforms in rat pituitary.

A 5.2-kb mRNA band that contains estrogen receptor (ER) sequence and exhibits sex- and tissue-specific expression has been identified in rat pituitary via Northern analysis; this band is composed of at least two distinctive ER mRNA isoforms. This mRNA is expressed in high levels in female pituitary but is absent in male pituitary and uterus, whereas the mRNA encoding the full-length receptor (6.2 kb) is expressed in all the aforementioned tissues. Estradiol treatment potently induces the expression of the 5.2-kb band in the male pituitary. Oligonucleotide hybridization and ribonuclease-protection experiments indicate that the pituitary ER variant is missing exons 1-4. Two corresponding cDNA clones, truncated estrogen receptor product 1 and 2 (TERP-1 and TERP-2), were isolated by using the anchored PCR. Both sequences contain a 31-bp segment of specific sequence upstream of exon 5; TERP-2, however, contains an additional 66 bp of specific sequence between the 31-bp segment and exon 5. On Northern analysis, probes complementary to the 31-bp segment of specific sequence hybridize only to the 5.2-kb band. Immunoblotting identified several proteins in rat pituitary that could represent the translation products of these or related transcripts. In summary, several ER isoforms have been identified that exhibit both tissue-specific expression and marked estrogen regulation and differ from full-length receptor by virtue of sequence upstream of the exon 4/5 boundary. Physiologically, the putative proteins encoded by these or similar isoforms might be important modulators of the tissue- and promoter-specific effects of estradiol.