No abstract

Although dopamine inhibits PRL release from the normal anterior pituitary lactotroph, a conclusive demonstration of the mechanisms involved in this response has been impeded by the presence of other cell types in the anterior pituitary. To circumvent this problem, we have isolated a clonal cell line, designated MMQ, from the 7315a rat pituitary tumor. The MMQ cell is an exemplary model for our use because it only secretes PRL. Our studies show that dopamine inhibits secretagogue-induced PRL release from these cells. In addition, dopamine decreases the intracellular cAMP concentration in MMQ cells that have been exposed to forskolin, cholera toxin, or vasoactive intestinal polypeptide, each a stimulator of cAMP generation. This inhibition is, in turn, reversed by the dopamine antagonist haloperidol and by pertussis toxin, an inactivator of the GTP-binding coupling protein. Dopamine also decreases the uptake and fractional efflux of 45Ca2+ by MMQ cells that have been exposed to the calcium channel activator maitotoxin. It seems, therefore, that dopamine decreases PRL release from MMQ cells at least in part by decreasing intracellular cAMP levels and calcium uptake. In additional experiments, we have found that MMQ cells are responsive to somatostatin, estrogen, progesterone, and acetylcholine, but not to TRH, angiotensin II, neurotensin, or bombesin. Furthermore, these cells possess a functional protein kinase-C system, as evidenced by the increase in PRL release and decrease in stimulated intracellular cAMP levels that occur in response to treatment with phorbol diesters. We suggest that the MMQ cell line will prove a useful model system for study of the biochemical effects of dopamine and other factors that modify PRL release.

No abstract

Thymosin fraction 5 (TF5) is a partially purified extract of bovine thymus containing 40-60 peptides. In addition to its well documented immunopotentiating effects, TF5 reportedly modulates the secretion of some hypothalamic peptides and pituitary hormones. In this study, TF5 (10-100 micrograms/ml) stimulated PRL release from normal, MtTW15, and 7315a cells and GH release from normal and MtTW15 cells, but had no apparent effect on LH release. No changes in intracellular cAMP or cGMP levels could be correlated with these responses. Stimulation of PRL release from perifused normal anterior pituitary cells was rapid, sustained, and concentration related. Although it had no apparent effect on normal prelabeled anterior pituitary cells with respect to 45Ca2+ efflux, the calcium channel blocker D-600 inhibited TF5-mediated hormone release from these cells. Additive increases in TRH-stimulated PRL release and GRF-stimulated GH release by TF5 suggested independent mechanisms of action. Dopamine (500 nM) blocked TF5-stimulated PRL release, but somatostatin (10-100 nM) had no effect on TF5-stimulated PRL or GH release. TF5 failed to affect either basal or TRH-induced polyphosphoinositide hydrolysis. Perifused normal anterior pituitary cells prelabeled with [3H]arachidonate responded to TF5 treatment with a liberation of radioactive arachidonate and/or its metabolites. BW755c, an inhibitor of all known catabolic pathways of arachidonic acid, blocked the ability of TF5 to stimulate PRL and GH release. Reversed phase HPLC separation of TF5 into five fractions resulted in two fractions that exhibited hormone-releasing activity. These data suggest that TF5 stimulates pituitary hormone release through a mechanism different from that ascribed to TRH or GRF. The stimulus-secretion coupling mechanism involves neither polyphosphoinositide hydrolysis nor cAMP generation, but appears to be dependent on the generation of arachidonate metabolites.

A series of 1-phenyl-3-(aminomethyl)pyrroles were prepared in two steps from aniline and their affinities for D2, D3, and D4 dopamine receptor subtypes determined. A 15-fold selectivity for cloned human D4 receptors over cloned African Green monkey D2 receptors was observed with 1-(2-pyridyl)-4-[[3-(1-phenylpyrrolyl)]methyl]piperazine.

Genomic and cDNA clones, encoding a protein that is a member of the guanine nucleotide-binding regulatory protein (G protein)-coupled receptor superfamily, were isolated by screening rat genomic and thoracic aorta cDNA libraries with an oligonucleotide encoding a highly conserved region of the Ml muscarinic acetylcholine receptor. Sequence analyses of these clones showed that they encode a 343-amino acid protein (named RTA). The RTA gene is single copy, as demonstrated by restriction mapping and Southern blotting of genomic clones and rat genomic DNA. Sequence analysis of the genomic clone further showed that the RTA gene has an intron interrupting the region encoding the amino terminus of the protein. RTA RNA sequences are relatively abundant throughout the gut, vas deferens, uterus, and aorta but are only barely detectable (on Northern blots) in liver, kidney, lung, and salivary gland. In the rat brain, RTA sequences are markedly abundant in the cerebellum. RTA is most closely related to the mas oncogene (34% identity), which has been suggested to be a forebrain angiotensin receptor. We cannot detect angiotensin binding to the RTA protein after introducing the cognate cDNA or mRNA into COS cells or Xenopus oocytes, respectively, nor can we detect an electrophysiologic response in the oocyte after application of angiotensin peptides. We conclude that RTA is not an angiotensin receptor; to date, we have been unable to identify its ligand.An interesting recent development in receptor biology has been the realization that many of the receptors interacting with the guanine nucleotide-binding regulatory protein (G protein) signaling system are homologues. The mammalian members of this receptor superfamily described to date by molecular cloning include ,B-adrenergic (1-3), a-adrenergic (4-6), muscarinic acetylcholine (7-10), serotonin (11-14), dopamine (15), substances K (16) and P (17), luteinizing hormone (18,19), and rhodopsin (20) receptors. The quintessential feature of these proteins is seven stretches of 20-26 hydrophobic amino acids; these regions, which are the most conserved regions among different receptors, are thought to form a-helices that span the cytoplasmic membrane. Other features common to these proteins include one or more potential N-linked glycosylation sites near the amino terminus, the lack of a discernible amino-terminal signal peptide (except the luteinizing hormone receptor) (18,19), and several invariant amino acid residues. Another feature of this family is that the coding regions of the genes often lack introns; exceptions to this trend include bovine rhodopsin (20) and D2 subtype of the dopamine receptor (15). A currently popular model proposes that these proteins have their amino terminus outside the cell and the seven hydrophobic segments spanning the cytoplasmic membrane with the connecting segments alternately extending into the cytoplasm and the extracellular space and the carboxyl terminus inside the cell. The evidence for this structure is largely an extrapolation from re...

A series of 2-phenyl-4-(aminomethyl)imidazoles were designed as conformationally restricted analogs of the dopamine D2 selective benzamide antipsychotics. The title compounds were synthesized and tested for blockade of [3H]YM-09151 binding in cloned African green monkey dopamine D2 receptor preparations. The binding affinity data thus obtained were compared against that of the benzamides and a previously described series of 2-phenyl-5-(aminomethyl)-pyrroles.

The dynamics of arachidonic acid (AA) liberation and PRL release were highly correlated in perifused rat anterior pituitary cells during stimulation by three different neuropeptides: TRH, angiotensin II (AII), and neurotensin (NT). After preincubation of these cells with 1 microCi [3H]AA, a 20-min perifusion with AII (100 nM), TRH (100 nM), or NT (1 microM) elicited a sharp initial increase in PRL release and [3H]AA efflux, which rapidly subsided (within 6 min) to less elevated levels of PRL release and AA liberation. The plateau responses were sustained throughout the remainder of the 20-min treatment period; after the cessation of neuropeptide perifusion, the responses rapidly returned to basal levels. AII and TRH elicited a greater initial stimulation of PRL release and AA liberation, whereas NT resulted in less pronounced initial responses and a greater plateau of sustained PRL release and AA liberation. Dopamine (DA; 500 nM) or calcium-depleted medium (containing 60 microM EGTA) evenly attenuated the stimulation of PRL release throughout exposure to the neuropeptides; however, the initial stimulation of AA efflux by AII and TRH was relatively resistant to inhibition by DA or calcium-depleted medium. In contrast, the stimulation of AA liberation by NT was abolished by DA or calcium-dependent medium. These results establish that the time course of AA liberation is complimentary to that of PRL release during stimulation by AII, TRH, and NT and support a possible role for AA liberation and metabolism as one of the mechanisms that participates in the regulation of PRL release. A lesser ability of NT to elicit functional and biochemical responses to intracellular calcium mobilization is postulated as an explanation for the observed differences among AII, TRH, and NT effects on PRL release and AA liberation.