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T F Martin

Publications and source records attributed to T F Martin.

At least 73 records · Page 4Linked to original sources
PubMed1985

Direct stimulation by thyrotropin-releasing hormone (TRH) of polyphosphoinositide hydrolysis in GH3 cell membranes by a guanine nucleotide-modulated mechanism.

Polyphosphoinositide hydrolysis was examined in membranes from thyrotropin-releasing hormone (TRH)-responsive GH3 pituitary cells. [3H]Inositol phosphates (IP2 and IP3) were generated upon incubation of membranes from [3H]inositol-labeled cells indicating the presence of a membrane-associated polyphosphoinositide phosphodiesterase (PPI PDE). Membrane PPI PDE activity was found to be stimulated by TRH and by GTP-gamma-S in Ca2+-modulated manner. In addition, TRH-stimulated PPI hydrolysis was potentiated by GTP. These results demonstrate direct in vitro effects of a hormone on PPI turnover and suggest the involvement of a GTP-binding component in transmembrane signalling by TRH.

Animals↗
PubMed1985

Prolactin secretion in permeable GH3 pituitary cells is stimulated by Ca2+ and protein kinase C activators.

We have used GH3 cells permeabilized by electric field discharge to examine the effects of Ca2+ and protein kinase C activators (phorbol ester and diacylglycerol) on prolactin (PRL) release. Ca2+ was found to stimulate PRL release approximately 4 fold at 3 microM Ca2+ with a half-maximal response at approximately .5 microM estimated free Ca2+. 12-O-tetradecanoyl phorbol-13-acetate and 1-oleoyl-2-acetyl-sn-glycerol stimulated PRL release throughout a range of Ca2+ concentrations (1 nM -3 microM), but stimulation was greater at higher Ca2+ concentrations (.1 microM to 1 microM). Both agents decreased by 1.8 fold the apparent [Ca2+] at which half-maximal stimulation of secretion occurred. Quin 2 was used to measure the free [Ca2+] of intact and permeable cells; PRL secretion at a free [Ca2+] corresponding to resting cytoplasmic [Ca2+] was 10% of maximal, while secretion at the [Ca2+] corresponding to the Ca2+ spike induced by thyrotropin-releasing hormone was approximately 25% of maximal.

Aminoquinolines↗
PubMed1985

Protein kinase C translocates from cytosol to membrane upon hormone activation: effects of thyrotropin-releasing hormone in GH3 cells.

The subcellular distribution of protein kinase C (PK C) was examined in thyrotropin-releasing hormone (TRH)--responsive GH3 pituitary cells. TRH treatment, which is known to stimulate polyphosphoinositide turnover and diacylglycerol generation, resulted in a rapid (less than or equal to 15 sec) and transient redistribution of the enzyme from cytosol to membrane fraction. Other agents which either stimulate PK C directly (1-oleoyl-2-acetyl-sn-glycerol and 12-O-tetradecanoyl phorbol-13-acetate) or elevate cellular diglyceride levels (phospholipase C) also promoted a redistribution of the enzyme from cytosol to membrane. These results provide evidence for the concept that cell-surface receptor-mediated phosphoinositide breakdown activates PK C. It appears that translocation of PK C to the membrane is an early step in the cellular activation of this enzyme.

Animals↗
PubMed1984

Thyrotropin-releasing hormone rapidly activates protein phosphorylation in GH3 pituitary cells by a lipid-linked, protein kinase C-mediated pathway.

Thyrotropin-releasing hormone (TRH) is a tripeptide that rapidly enhances prolactin secretion in clonal, hormone-responsive GH3 rat pituitary cells. In an effort to identify postreceptor mechanisms for TRH, protein phosphorylation studies have been conducted. Our previous studies (Drust, D.S., Sutton, C.A., and Martin, T. F. J. (1982) J. Biol. Chem. 257, 3306-3312; Drust, D.S., and Martin, T. F. J. (1982) J. Biol. Chem. 257, 7566-7573) showed that TRH rapidly (less than 15 s) increased the phosphorylation of at least six cytosolic proteins (41K (Mr = 41,000), several 59K, 65K, 82K, and 97K) and, with a 5- to 10-min latency, increased the phosphorylation of a seventh (80K). Cyclic AMP did not appear to mediate TRH-stimulation of protein phosphorylation; in contrast, Ca2+ translocation and Ca2+-dependent protein phosphorylation accounted for hormone-induced changes in 97K (and possibly 41K) phosphorylation. The studies reported here indicate that lipid (diacylglycerol) accumulation and protein kinase C activation mediate TRH-stimulated phosphorylation of the additional five cytosolic proteins (two 59K, 65K, 80K, and 82K). This conclusion is based on the findings that: 1) phospholipase C treatment, which produces diacylglycerol, mimicked several TRH effects; 2) bombesin, another peptide that induces inositol phosphatide turnover, mimicked several TRH effects; 3) phorbol esters, which were shown to activate GH3 cell protein kinase C directly, produced TRH-like effects; 4) partially purified GH3 cell cytosolic protein kinase C was activated by diacylglycerol; and 5) 59K and 82K proteins were endogenous in vitro substrates for a cytosolic lipid-stimulated protein kinase. We conclude that rapid TRH effects in promoting inositol phosphatide turnover in GH3 cells may be linked to the activation of protein phosphorylation mediated by protein kinase C. These, and previously reported studies, indicate a role for Ca2+ and lipids (diacylglycerol) as dual intracellular messengers for TRH.

Animals↗
PubMed1984

Evidence for the role of calcium and diacylglycerol as dual second messengers in thyrotropin-releasing hormone action: involvement of diacylglycerol.

TRH stimulates the secretion of PRL by clonal GH3 pituitary cells. The studies of the accompanying paper have shown that the secretory response during the first 30-60 min is biphasic (phase I, 0-3 min; phase II, 5-60 min) and that the phase I response may be mediated through mechanisms involving Ca+2 translocation. In previous studies, it has been shown that TRH treatment rapidly induces the breakdown of inositol phospholipids with accompanying diacylglycerol accumulation. In this paper, we present evidence for a possible role for diacylglycerol as a second messenger which mediates the phase II response to TRH. A role for lipid-dependent mechanisms in regulating PRL secretion in GH3 cells was supported by the finding that phospholipase C, phorbol esters, melittin, and exogenous diacylglycerols were effective secretagogues in GH3 cells. Secretion promoted by these agents was found to be persistent, as was the phase II response to TRH. For three of the agents examined (TRH, phorbol esters, and phospholipase C), stimulated PRL release was found to be nonadditive, suggesting the presence of some common element in the pathways by which these agents exert their effects. This lipid-linked pathway of activation was distinguished from a cAMP-mediated pathway.

Animals↗
PubMed1984

Evidence for the role of calcium and diacylglycerol as dual second messengers in thyrotropin-releasing hormone action: involvement of Ca+2.

The studies reported here were directed toward establishing the mechanism by which TRH acutely stimulates PRL secretion in GH3 pituitary cells. Studies of TRH stimulation of PRL secretion were conducted on a time scale which enables comparison with other reported rapid effects of TRH on GH3 cells. TRH stimulation of secretion was found to be extremely rapid in onset (less than or equal to 10 sec) and biphasic (phase I, 0-2 min; phase II, 5-60 min). The earliest (phase I) secretory response was observed to be independent of medium Ca+2 concentration or Ca+2 influx, but to be dependent on an intracellular Ca+2 pool. The phase II response to TRH was found to depend, in part, on medium Ca+2. The phase I response to TRH could be mimicked only by agents known to influence Ca+2 translocation in GH3 cells (60 mM K+, A23187, ionomycin, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and carbonyl cyanide m-chlorophenylhydrazone). These agents failed to promote sustained PRL release characteristic of phase II. It is concluded that the ability of TRH to rapidly stimulate PRL secretion (phase I) is correlated with its ability to rapidly promote a transient cytoplasmic Ca+2 concentration rise from an intracellular Ca+2 pool.

Animals↗
PubMed1983

Thyrotropin-releasing hormone rapidly activates the phosphodiester hydrolysis of polyphosphoinositides in GH3 pituitary cells. Evidence for the role of a polyphosphoinositide-specific phospholipase C in hormone action.

The early actions of thyrotropin-releasing hormone (TRH) have been studied in hormone-responsive clonal GH3 rat pituitary cells. Previous studies had demonstrated that TRH promotes a "phosphatidylinositol response" in which increased incorporation of [32P]orthophosphate into phosphatidylinositol and phosphatidic acid was observed within minutes of hormone addition. The studies described here were designed to establish whether increased labeling of phosphatidylinositol and phosphatidic acid resulted from prior hormone-induced breakdown of an inositol phosphatide. GH3 cells were prelabeled with [32P]orthophosphate or myo-[3H]inositol. Addition of TRH resulted in the rapid disappearance of labeled polyphosphoinositides, whereas levels of phosphatidylinositol and other phospholipids remained unchanged. TRH-promoted polyphosphoinositide breakdown was evident by 5 S and maximal by 15 s of hormone treatment. Concomitant appearance of inositol polyphosphates in [3H]inositol-labeled cells was observed. In addition, TRH rapidly stimulated diacylglycerol accumulation in either [3H]arachidonic- or [3H]oleic acid-labeled cultures. These results indicate that TRH rapidly causes activation of a polyphosphoinositide-hydrolyzing phospholipase C-type enzyme. The short latency of this hormone effect suggests a proximal role for polyphosphoinositide breakdown in the sequence of events by which TRH alters pituitary cell function.

Animals↗
PubMed1982

Thyrotropin-releasing hormone and cyclic AMP activate distinctive pathways of protein phosphorylation in GH pituitary cells.

The studies reported here were undertaken to clarify the cellular mechanism of the hypothalamic tripeptide, thyrotropin-releasing hormone (TRH), in clonal, hormone-responsive GH pituitary cells and to assess the possibility of a role for cyclic AMP as a mediator of TRH action. We investigated patterns of protein phosphorylation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of high speed supernatant and pellet fractions from untreated and treated GH cells. Brief treatment of cells with agents which elevate or mimic cellular cyclic AMP (8-bromo cyclic AMP, dibutyryl cyclic AMP, vasoactive intestinal polypeptide or cholera toxin) stimulated the phosphorylation of five supernatant peptides (41, 45, 47, 72, and 82 kilodaltons) and one pellet peptide (135 kilodaltons) and decreased the phosphorylation of one supernatant peptide (55 kilodaltons). In contrast, TRH promoted the phosphorylation of four different supernatant peptides (two 59, 65, and 80 kilodaltons). In addition, TRH also stimulated the phosphorylation of cyclic AMP-responsive 41-, 45-, and 82-kilodalton supernatant peptides and 135-kilodalton pellet protein and decreased the phosphorylation of 55-kilodalton supernatant peptide. Altered labeling of 47- and 72-kilodalton supernatant peptides, however, was not observed with TRH. Time course studies, as well as the overlapping biological action of TRH and vasoactive intestinal polypeptide, lead us to conclude that these peptide hormones utilize distinct, parallel pathways which converge at some late step. Furthermore, the results indicate that effects of TRH are mediated by a cyclic AMP-independent pathway.

Animals↗
PubMed1982

Thyrotropin-releasing hormone mobilizes Ca2+ from endoplasmic reticulum and mitochondria of GH3 pituitary cells: characterization of cellular Ca2+ pools by a method based on digitonin permeabilization.

Treatment of 45Ca2+-loaded GH3 pituitary cells with various concentrations of digitonin revealed discrete pools (I and II) of cellular 45Ca2+ defined by differing detergent sensitivities. Markers for cytosol and intracellular organelles indicated that the two 45Ca2+ pools were correlated with the two major cellular Ca2+-sequestering organelles, endoplasmic reticulum (I) and mitochondria (II). Studies with various inhibitors were consistent with these assignments. Mitochondrial uncouplers preferentially depleted 45Ca2+ pool II while trifluoperazine selectively depleted 45Ca2+ pool I. Control experiments indicated that translocation of in situ organellar 45Ca2+ during and after permeabilization was negligible. We used the digitonin-permeabilization method to examine the effect of thyrotropin-releasing hormone (TRH) treatment on intracellular Ca2+ pools of GH3 pituitary cells. TRH was found to rapidly deplete both endoplasmic reticulum and mitochondrial exchangeable Ca2+ by 25-30%. The 45Ca2+ loss from both pools was maximal by 1 min after TRH addition and was followed by a recovery phase; mitochondrial 45Ca2+ content returned to control levels by 30 min. Previous treatment of cells with the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxy-phenylhydrazone blocked TRH-induced 45Ca2+ efflux from mitochondria, while previous treatment with valinomycin, an agent that depleted both 45Ca2+ pools, blocked any additional effect of TRH on these pools. We conclude that TRH rapidly promotes a net loss of exchangeable Ca2+ from GH3 cells as a result of hormone-induced mobilization of Ca2+ from endoplasmic reticulum and mitochondria.

Calcium↗
PubMed1982

Thyrotropin-releasing hormone (TRH) selectively and rapidly stimulates phosphatidylinositol turnover in GH pituitary cells: a possible second step of TRH action.

TRH was found to rapidly influence 32PO4 incorporation into phospholipids of PRL-secreting GH pituitary cells. Analogs of TRH were found to exert similar effects, with potencies related to receptor-binding affinity. Additional PRL-releasing agents were also tested. Bombesin exerted a similar effect, whereas vasoactive intestinal polypeptide, 8-bromo cAMP, phosphodiesterase inhibitors, 50 mM K+, and scorpion venom toxin had no influence. Cationophore A23187 stimulated phospholipid labeling in a manner distinguishable from that of TRH. Chromatographic analysis showed the action of TRH to be restricted to the labeling of phosphatidylinositol and phosphatidic acid. Kinetic studies indicated a rapid influence of TRH on phosphatidylinositol breakdown, with subsequent accelerated 32PO4 incorporation into phosphatidylinositol and phosphatidic acid. These studied identified a rapid, receptor-mediated, cAMP-independent action of TRH on phospholipid metabolism. Similar effects of other hormones are believed to be involved in promoting cellular Ca2+ translocation. The rapid onset of the response reported here suggests that this event may play a role in mediating the PRL-releasing effects of TRH and bombesin in GH cells.

Animals↗
PubMed1981

Growth inhibition by adenosine 3',5-monophosphate derivatives does not require 3',5' phosphodiester linkage.

Analogs of adenosine 3',5'-monophosphate (cyclic AMP) inhibit the growth of cultured cell lines. The effects of 8-bromo- and N6-butyryl-substituted analogs of cyclic and noncyclic AMP on six cell lines were examined and were equally inhibitory. Variant cell lines with altered cyclic AMP-dependent protein kinase were more resistant to both cyclic and noncyclic nucleotides. We conclude that growth inhibition by analogs of cyclic AMP (i) does not require a 3',5' phosphodiester bond and (ii) may be mediated by a pathway involving endogenous cyclic AMP.

Animals↗
PubMed1981

Multiple mechanisms of growth inhibition by cyclic AMP derivatives in rat GH1 pituitary cells: isolation of an adenylate cyclase-deficient variant.

GH pituitary cells have been widely utilized for studies of hormone response mechanisms. Studies reported here were motivated by the desirability of isolating characterized GH clones defective in cyclic AMP synthesis or action. Spontaneously occurring GH1 cell variants resistant to the growth-inhibitory effects of cyclic AMP analogs were isolated. Characterization of four variants showed that these were deficient in adenosine kinase and had acquired resistance to the cytotoxic effects of purine nucleoside derivatives formed in the culture medium. A second-stage selection was undertaken with mutagenized adenosine kinase-deficient cells. One 8 Br cAMP-resistant variant was found to have normal cyclic AMP-dependent protein kinase activity but exhibited altered adenylate cyclase activity. Activation of cyclase activity by fluoride, guanyl nucleotides, cholera toxin, and hormone (VIP) was subnormal in the variant. Mn-dependent cyclase activity was also subnormal, suggesting that the 8 Br cAMP-resistant variant may have a deficiency in the catalytic moiety of adenylate cyclase. Surprisingly, adenosine 3':5'-monophosphate and 5'-monophosphate derivatives were found to be equally potent in growth-inhibiting adenosine kinase-deficient cells. Cross-resistance to 8 Br AMP was observed in the 8 Br cAMP-resistant variant. We conclude that cyclic AMP derivatives inhibit growth of GH cells by an unanticipated mechanism that is, nonetheless, related to endogenous cyclic AMP synthesis.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1980

Paradoxical effects of protein synthesis inhibitors on uridine uptake in cultured cells: possible role of uncharged tRNA in regulating metabolism.

Previous studies (J. Biol. Chem, 253: 99--105, 1978) showed that thyrotropin-releasing hormone (TRH) acutely stimulated uridine uptake in pituitary cell (GH 4Cl)) cultures. Studies on the role of protein synthesis in this response to TRH led to the finding that an inhibitor of ribosomal translation, cycloheximide, also stimulated uridine uptake acutely. Studies reported here attempt to determine the mechanism of cycloheximide action and whether cycloheximide and hormone stimulation of uridine uptake occurred by similar pathways. The experiments presented indicate that: (1) seven inhibitors of ribosomal translation stimulated uridine uptake; (2) in contrast, inhibition of protein synthesis at tRNA aminoacylation resulted in reduced rates of uridine uptake; (3) inhibition of tRNA aminoacylation blocked cycloheximide but not TRH stimulation of uptake; (4) cycloheximide stimulation of uptake was restricted to amino acid-depleted cultures; (5) amino acid supplementation stimulated uridine uptake with a time-course identical to that of cycloheximide; (6) cycloheximide and amino acid supplementation promoted reacylation of cellular tRNAs in amino acid-depleted cultures; and (7) cycloheximide stimulation of uridine uptake resulted from enhanced nucleoside phosphorylation rather than increased uridine transport. We conclude that cycloheximide and amino acid stimulation of uridine phosphorylation may be mediated through a common pathway involving the extent of amino-acylation of cellular tRNAs. Furthermore, cycloheximide and TRH stimulate uridine phosphorylation by pathways that are distinguishable. It is apparent that not all cellular effects of cycloheximide can be attributed solely to inhibition of the synthesis of proteins.

Adenosine Triphosphate↗