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K M Gautvik

Publications and source records attributed to K M Gautvik.

At least 73 records · Page 4Linked to original sources
PubMed1990

Uptake and receptor binding of dexamethasone in cultured 7800 C1 hepatoma cells in relation to regulation of cell growth and peroxisomal beta-oxidation.

1. Uptake and binding of dexamethasone to glucocorticoid receptor has been studied in Morris hepatoma 7800 C1 cells in relation to its effect on cell growth and peroxisomal beta-oxidation. 2. Intact cells showed saturable, specific dexamethasone binding of limited capacity and Scatchard analysis revealed one single class of binding sites with equilibrium dissociation constant (Kd) of 0.24 nM similar to other glucocorticoid receptors. However, the binding capacity of 24 fmol/mg cell protein is less than 5% of previously reported values. 3. Uptake of [3H]dexamethasone by intact cells was temperature dependent giving a linear Arrhenius plot with a calculated energy of activation of 58.5 kJ mol-1 x degree-1. 4. Cytosol fractions had specific binding proteins for glucocorticoid hormones with sedimentation coefficient of ca 7S. No specific binding sites for [3H]dexamethasone was demonstrated in purified membrane fractions. 5. Dexamethasone and the synthetic fatty acid analogue tetradecylthio acetic acid (TTA) both inhibited the growth of the 7800 C1 cells and induced the peroxisomal acyl-CoA oxidase activity. A combination of the two compounds gave additive effects. Both these effects of dexamethasone and TTA were counteracted by insulin. 6. We conclude that dexamethasone induces growth inhibition and enzyme induction by binding to functional intracellular glucocorticoid receptors. The action of dexamethasone is consistent with a dissolution in the membrane from where it diffuses passively into the cell and binds to specific receptors in an energy dependent step. 6. The synergistic action of dexamethasone and TTA and the counteraction exerted by insulin are not due to changes in the dexamethasone receptor affinity or binding capacity.

Acyl-CoA Oxidase↗
PubMed1990

Different-sized mRNAs from GH4C1 cells induce a TRH-dependent electrical response in Xenopus laevis oocytes.

Poly(A)+ RNA from the GH4C1 rat pituitary cell line elicited a thyrotropin releasing hormone response in Xenopus laevis oocytes which could be measured as a change in membrane current by the voltage-clamp method. Oocytes injected with Poly(A)+ RNA from GH12C1 cells which do not bind thyrotropin releasing hormone or with buffer solution alone did not show this response. Size fractionation of total poly(A)+ RNA by sucrose density-gradient centrifugation shows two response maximal representing various mRNA fractions larger than 18S. These results indicate the presence of thyrotropin releasing hormone receptor mRNA heterogeneity where the smallest mRNA is at least 2 kb.

Animals↗
PubMed1989

Protein kinase C stimulates adenylate cyclase activity in prolactin-secreting rat adenoma (GH4C1) pituicytes by inactivating the inhibitory GTP-binding protein Gi.

The phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA) and thyroliberin exerted additive stimulatory effects on prolactin release and synthesis in rat adenoma GH4C1 pituicytes in culture. Both TPA and thyroliberin activated the adenylate cyclase in broken cell membranes. When combined, the secretagogues displayed additive effects. TPA did not alter the time course (time lag) of adenylate cyclase activation by hormones, guanosine 5'-[beta,gamma-imino]triphosphate or forskolin, nor did it affect the enzyme's apparent affinity (basal, 7.2 mM; thyroliberin-enhanced, 2.2 mM) for free Mg2+. The TPA-mediated adenylate cyclase activation was entirely dependent on exogenously added guanosine triphosphate. ED50 (dose yielding half-maximal activation) was 60 microM. Access to free Ca2+ was necessary to express TPA activation of the enzyme, however, the presence of calmodulin was not mandatory. TPA-stimulated adenylate cyclase activity was abolished by the biologically inactive phorbol ester, 4 alpha-phorbol didecanoate, by the protein kinase C inhibitor polymyxin B and by pertussis toxin, while thyroliberin-sensitive adenylate cyclase remained unaffected. Experimental conditions known to translocate protein kinase C to the plasma membrane and without inducing adenylate cyclase desensitization, increased both basal and thyroliberin-stimulated enzyme activities, while absolute TPA-enhanced adenylate cyclase was maintained. Association of extracted GTP-binding inhibitory protein, Gi, from S49 cyc- murine lymphoma cells with GH4C1 cell membranes yielded a reduction of basal and hormone-stimulated adenylate cyclase activities, while net inhibition of the cyclase of somatostatin was dramatically enhanced. However, TPA restored completely basal and hormone-elicited adenylate cyclase activities in the Gi-enriched membranes. Finally, TPA completely abolished the somatostatin-induced inhibition of adenylate cyclase in both hybrid and non-hybrid membranes. These data suggest that, in GH4C1 cells, protein kinase C stimulation by phorbol esters completely inactivates the n alpha i subunit of the inhibitory GTP-binding protein, leaving the n beta subunit functionally intact. It can also be inferred that thyroliberin conveys its main effect on the adenylate cyclase through activation of the stimulatory GTP-binding protein, Gs.

Adenylate Cyclase Toxin↗
PubMed1989

[Hybridization in situ. A new technic for the demonstration of viruses and gene products in histologic and cytologic specimens].

The use of in situ hybridization for light-microscopic demonstration of specific DNA or RNA-sequences is illustrated with examples. This method is useful for demonstration of viruses and gene products (mRNA) in individual cells in tissue sections or in cells in suspension. In situ hybridization technology is particularly useful in diagnostic pathology as an adjunct to the conventional methods for infectious diseases. In addition, the method is a powerful tool for analysing the interaction between viral infection and the induction and maintenance of certain human neoplasms. We discuss various aspects of tissue handling, fixation, hybridization procedures and detection of the hybridization signal. We also stress the importance of close cooperation between laboratories in the field of gene technology.

DNA, Viral↗
PubMed1989

Early liver metastases in patients with medullary carcinoma of the thyroid gland.

In 13 patients who had been thyroidectomized for medullary carcinoma (MCT), basal and pentagastrin-stimulated immunoreactive calcitonin (iCT) concentrations were measured in a peripheral vein and concomitantly in hepatic veins as well as in the superior and inferior caval veins. The basal concentrations of iCT in serum from the antecubital vein ranged from normal values (less than 0.50 micrograms/1) to grossly elevated (greater than 100 micrograms/1). Intravenous pentagastrin injections (0.5 micrograms/kg) stimulated transiently calcitonin secretion in all patients and peak concentrations occurred in peripheral venous blood and in the inferior caval vein after 3 to 5 minutes. In all patients peak iCT values in the hepatic vein occurred at 1 to 2 minutes and were much higher than in samples taken from other veins. Only three patients showed metastases to liver as judged by conventional clinical and laboratory examinations. Two patients with nonthyroid, malignant disease did not show an increase in serum iCT concentration of hepatic veins. The authors conclude that MCT may spread early to the liver. The presence of liver metastases is an important prognostic factor in patient evaluation and can be demonstrated by measuring iCT in hepatic veins before and during pentagastrin stimulation.

Adult↗
PubMed1989

Microinjection and expression of a mouse metallothionein human growth hormone fusion gene in fertilized salmonid eggs.

Using a microinjection method (Rokkones et al. 1985) deoxyribonucleic acid was introduced into fertilized salmonid eggs. The survival rate after a 28 day period was 91% for injected eggs in comparison to non-injected controls. A gene construct containing the mouse metallothionein promoter fused to the human growth hormone structural gene was microinjected either as a supercoiled plasmid or as a linear sequence. In Southern blot analysis of both 5 and 73 day old dissected rainbow trout embryos, as well as in 1 year old Atlantic salmon, the mouse metallothionein human growth hormone gene sequence was detected together with the chromosomal DNA when micro-injected as plasmid or as linear DNA. After digestion with Bam HI restriction endonuclease, the human growth hormone gene was excised from the high molecular weight DNA fraction. Transcription into human growth hormone specific RNA, as well as translation and release of human growth hormone immunoreactive protein, could be demonstrated in early embryonic stages.

Animals↗
PubMed1989

Effects of recombinant human growth hormone and insulin-like growth factor 1 on body growth and blood metabolites in brook trout (Salvelinus fontinalis).

Groups of juvenile brook trout (Salvelinus fontinalis) were acclimated to 12.0-13.0 degrees dechlorified water and a photoperiod of 12 hr light: 12 hr dark. Recombinant human growth hormone (hGH) (10.0 micrograms/g body wt) or insulin-like growth factor 1 (hIGF-1), used in a wide range of dosages (0.001-10.0 micrograms/g body wt), were given weekly as intramuscular injections. The fish receiving hGH were already significantly heavier and longer than the saline-injected control fish after 3 weeks of treatment. In addition, a liver specific growth promoting effect of hGH was found. In contrast, hIGF-1 did not stimulate body growth in any dosage tested. The fish receiving the highest dosages of hIGF-1 were all seriously affected with retarded body growth and high mortality. A possible insulin-like activity of hIGF-1 was verified by measuring the plasma glucose and amino acid levels in brook trout after a single injection of hIGF-1 (2.0 micrograms/g body wt) or bovine insulin (0.01 IU/g body wt). Both hormones caused a reduction in both glucose and amino acid levels to 35% of the control levels 24-72 hr after injection. The results strongly suggest that hIGF-1 does not stimulate growth, but that in high dosages causes profound insulin-like effects in brook trout resulting in hypoglycemia and hypoaminoacidemia.

Amino Acids↗
PubMed1989

Purification and characterization of Atlantic salmon prolactin.

Prolactin was isolated from the Atlantic salmon (Salmo salar) pituitary gland by extraction with acid acetone, gel filtration, ion exchange-chromatography, and reversed-phase high-performance liquid chromatography. The yield was 0.6 mg/g wet tissue. The hormone had a molecular weight of 23.5 kDa as determined by SDS-polyacrylamide gel electrophoresis. Isoelectric focusing gave an isoelectric point of 9.2. The N-terminal sequence and the amino acid composition indicated extensive homology between Atlantic and Pacific salmon prolactin. Antiserum against Atlantic salmon prolactin cross-reacted with chum salmon prolactin, but not with human, rat, or sheep prolactin.

Amino Acid Sequence↗
PubMed1989

5-azacytidine-induced alterations in the GH12C1 cells: effects on cellular morphology, chromosome structure, DNA and protein synthesis.

Growth hormone-producing rat pituitary tumor cells (GH12C1) were cultivated in the presence of 5-azacytidine (5-AC). After cessation of treatment the cells were allowed to recover in normal cell culture medium prior to subcultivation. Within hours after the subcultivation the cells underwent several morphological alterations, later followed by changes in growth pattern: (1) cell shape was irreversibly changed from round or spindle shape to different clones of fibroblast-like cells; (2) some of these clones later formed foci; and (3) an extensive generation of multinucleated cells was seen. The demethylation was maximal approximately one week after the first subcultivation in the absence of 5-AC. Severe alterations of the chromosome structure were seen after the first subcultivation. During the following weeks the 5-AC-treated cells showed impaired chromosome condensation, and homogeneous staining with quinacrine mustard. During this period the DNA synthesis is doubled when compared to control cell DNA synthesis. The core histone synthesis increases in parallel with the DNA synthesis, but the majority of nuclear proteins, including the linker histones, remain at the control level. This results in a ratio of linker histone to core histone synthesis at approximately half the control cell level. The altered ratio of synthesis slowly decreased to control levels during a period of five weeks of continuous cultivation in the absence of the drug, and the under-condensed chromosomes could no longer be seen. The induced novel phenotypes with their pleiomorphic appearances were conserved. The growth hormone synthesis remained constant during all phases of the experiment and prolactin synthesis was not induced.

Animals↗
PubMed1988

A 64 kDa protein is a candidate for a thyrotropin-releasing hormone receptor in prolactin-producing rat pituitary tumor cells (GH4C1 cells).

A thyrotropin-releasing hormone (TRH) binding protein of 64 kDa has been identified by covalently crosslinking [3H]TRH to GH4C1 cells by ultraviolet illumination. The crosslinkage of [3H]TRH is UV-dose dependent and is inhibited by an excess of unlabeled TRH. A 64 kDa protein is also detected on immunoblots using an antiserum raised against GH4C1 cell surface epitopes. In a closely related cell line (GH12C1) which does not bind [3H]TRH, the 64 kDa protein cannot be demonstrated by [3H]TRH crosslinking nor by immunoblotting. These findings indicate that the 64 kDa protein is a candidate for a TRH-receptor protein in GH4C1 cells.

Affinity Labels↗
PubMed1988

Hormone-sensitive adenylate cyclase of prolactin-producing rat pituitary adenoma (GH4C1) cells: molecular organization.

Hormonal activation and inhibition of the GH4Cl1 cell adenylate cyclase complex is delineated. In the presence of the guanyl nucleotide GTP, enzyme activity was enhanced twofold by thyroliberin, sixfold by vasoactive intestinal peptide (VIP), twofold by prostaglandin E2 and twofold by isoproterenol. The diterpene, forskolin, increased, the activity 14-fold. In the presence of high GTP (400 microM) and NaCl (150 mM) concentrations, somatostatin inhibited (ED50 = 0.5 microM) the cyclase activity by 40%. In the presence of 10 microM somatostatin, the ED50 values (5 nM) for thyroliberin- and VIP-stimulated adenylate cyclase activities were shifted to 20 nM. Forskolin-elicited activation was, however, not affected by somatostatin. Cholera-toxin and pertussis-toxin pretreatment of the enzyme brought about some 20-fold and twofold activation, respectively. Inhibition by somatostatin was abolished upon pre-exposure to pertussis toxin. Mild alkylation by N-ethylmaleimide increased basal and hormone-activated adenylate cyclase while somatostatin again failed to express its inhibitory potential. Further alkylation caused a gradual decline and convergence of hormone-modulated cyclase activities towards zero. The N-ethylmaleimide-induced attenuation of thyroliberin-elicited activity was paralleled by a decrease in [3H]thyroliberin binding. Trifluoperazine and an anti-calmodulin serum reduced basal and net thyroliberin-, VIP- and forskolin-enhanced cyclase activities by some 30%, 100%, 70% and 80%, respectively. The Vmax of basal and thyroliberin-stimulated adenylate cyclase was diminished by 65%, leaving the apparent Km values (7.2 mM and 2.6 mM, respectively) for Mg2+ unaltered. Finally, the phorbol ester 12-O-tetra-decanoyl-phorbol 13-acetate (TPA) doubled the activity. This effect was counteracted by the protein kinase C inhibitor, polymyxin B, while thyroliberin-enhanced adenylate cyclase remained unaffected. In summary, we have described an adenylate cyclase with stimulatory (Rs) and inhibitory (Ri) receptors coupled to a calmodulin-sensitive holoenzyme through the Gs and Gi type of GTP-binding proteins. The ratio of the Gs to Gi is high. It appears that the GH4C1 cell adenylate cyclase is also activated by protein kinase C by interference with Gi. Apparently, thyroliberin activates the cyclase both directly through Gs and indirectly via protein kinase C stimulation.

Adenylyl Cyclases↗
PubMed1988

Phorbol esters and thyroliberin have distinct actions regarding stimulation of prolactin secretion and activation of adenylate cyclase in rat pituitary tumour cells (GH4C1 cells).

The phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) enhances the effects of TRH on phase II of prolactin secretion as well as on hormone synthesis at both low and high TPA receptor occupancy. Furthermore TPA, but not the biologically inactive substance 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), stimulates the particulate bound adenylate cyclase with a time course paralleling that of TRH activation. However, the combined additions of TRH and TPA activate this cyclase in an additive manner while the Gpp(NH)p- and the forskolin-sensitive enzyme are unaffected by TPA addition. Polymyxin B, which inhibits protein kinase C, abolishes activation of adenylate cyclase by TPA without interfering with the stimulatory action of TRH. Also, when phosphatase activity is preferentially inhibited by pretreatment of the cells with sodium vanadate, the TRH-sensitive cyclase is unaltered, while TPA activation is obliterated. Maximal stimulation of adenylate cyclase by cholera toxin pretreatment, obliterated the actions of TRH and TPA. Cells pretreated with pertussis toxin retained their TRH-sensitive cyclase, however, TPA-responsiveness was lost. We therefore suggest that the action of TPA as it relates to activation of adenylate cyclase, is probably mediated via the Gi component of the adenylate cyclase complex, while TRH stimulates the enzyme via the classical pathway involving the stimulatory GTP binding protein (Gs).

Adenylyl Cyclases↗
PubMed1988

Distribution of Thyroliberin (TRH)- and 12-O-tetradecanoylphorbol 13-acetate (TPA)-activated adenylyl cyclase in normal and neoplastic tissue with special reference to the prostate.

Tissue specificity of the Thyroliberin (TRH)- and 12-O-tetradecanoylphorbol 13-acetate (TPA)-sensitive adenylyl cyclase has been studied using normal or neoplastic organ samples or cells from the pituitary gland, stomach, prostate, myocardium, liver and bone. It appeared that TRH stimulates the adenylyl cyclase in both normal (basal cells), hyperplastic and adenocarcinomatous prostate as well as in the pituitary and stomach. TPA also stimulated the enzyme from the prostate and other organs/cells, but to a greater extent in neoplastic tissue. Functional links from protein kinase C to adenylyl cyclase and from protein kinase C to tyrosine kinase/oncogene expression have been established. Hence it is believed that TRH, which stimulates the adenylyl cyclase and protein kinase C in the pituitary, may serve as a factor contributing to transformation of prostatic cells or enhanced cell proliferation in prostatic cancer.

Adenylyl Cyclases↗
PubMed1988

A longitudinal study of pre- and postmenopausal changes in calcium metabolism.

Longitudinal changes in the serum concentration of calcium, phosphate, alkaline phosphatase, PTH, calcitonin and the renal handling of calcium and phosphate were studied in 19 normal women of the same age before and after the menopause. Significant increase in serum calcium, phosphate and calcitonin and urine calcium/creatinine and TmPO4/GF were shown to precede the premenopause. After cessation of the menstruation, no statistically significant further changes were observed in these variables. Changes in PTH were not observed neither during the premenopausal nor the post-menopausal period. Alkaline phosphatase increased in the postmenopausal period suggesting an increase in bone turn-over.

Calcium↗
PubMed1988

Calcium status and calcium-regulating hormones in alcoholics.

To elucidate effects of chronic ethanol consumption on clinical chemical parameters reflecting overall calcium homeostasis 34 hospitalized male alcoholics and 35 age-matched controls were studied during the winter season. Serum concentrations of 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 were reduced by 28% (p less than 0.01) and 24% (p less than 0.02) among the alcoholics as compared to the controls, respectively. Dietary intake of vitamin D3 did not differ significantly between the groups. The calcium level was below lower limit of reference in nine alcoholics (26%). Serum concentrations of parathyroid hormone and phosphorus were within normal ranges in both groups, and no differences were observed in levels of magnesium, vitamin D-binding protein, calcitonin, or alkaline phosphatase. In conclusion, it is possible that the activities of enzymes crucial in vitamin D3 metabolism may be altered in alcoholics.

Adult↗
PubMed1988

Somatostatin inhibits prolactin secretion by multiple mechanisms involving a site of action distal to increased cyclic adenosine 3',5'-monophosphate and elevated cytosolic Ca2+ in rat lactotrophs.

The release of prolactin (PRL) from a clonal cell-line of anterior pituitary cells (GH4C1) was inhibited by somatostatin (SRIH) in a dose-dependent manner (ED50 nM). The inhibition (20% of control levels) was detectable within 50 s and maximal within 90 s. Thyroliberin (TRH) enhancement of PRL secretion was biphasic. SRIH inhibited both phases equally. Ionomycin in combination with the phorbol ester, TPA, mimics the TRH-elicited PRL release, and SRIH partly inhibited this effect. SRIH had no effect on TRH-stimulated formation of inositol trisphosphate, and only small effects on TRH-activated adenylate cyclase. Vasoactive intestinal peptide (VIP) and forskolin stimulated cAMP formation and PRL release potently. SRIH inhibited both effects of VIP and forskolin, and there was a close correlation between the inhibition of PRL secretion and cAMP accumulation. 8-Bromo-cAMP enhanced PRL release, an effect that was also partly reduced by SRIH. The Ca2+ channel activator, BAY-K-8644 and high extracellular K+ increased PRL release, and SRIH caused a partial reduction in the release response to both secretagogues. SRIH lowered [Ca2+]i, and markedly reduced the rise in [Ca2+]i elicited by TRH, VIP and K+. SRIH did not influence the Ca2+ spikes recorded in Na+-free solution, and had no effect on the TRH-induced membrane potential changes. Our results demonstrate that SRIH may inhibit PRL release from GH4C1 cells by (1) inhibiting hormone-sensitive adenylate cyclase, (2) blocking the effect of cAMP and (3) lowering [Ca2+]i. None of these effects is, however, sufficient to explain all the effects of SRIH, suggesting that SRIH also exerts a major action at a step subsequent to cAMP accumulation and [Ca2+]i elevation. Since the GH4C1 cells possess one single class of binding sites, this implies that the same SRIH receptor is coupled to several cellular signalling systems.

Animals↗
PubMed1987

On the involvement of cyclic AMP and extracellular Ca2+ in the regulation of hormone release from rat pituitary tumour (GH3) cells in culture.

Thyroliberin (TRH), dibutyryl cyclic AMP (db-cAMP), and 3-isobutyl-1-methylxanthine (MIX) had a stimulatory effect on prolactin (PRL) and growth hormone (GH) release from GH3 cells. Half-maximal and maximal effects were observed for TRH at 2.5 nM and 10 nM; for db-cAMP at 0.6 mM and 5 mM, respectively. MIX (0.1 mM-1 mM) induced a dose-dependent accumulation of cellular cyclic AMP, while the hormone release was already maximally stimulated at 0.1 mM MIX. The maximal effects on hormone release of TRH and db-cAMP, but not of TRH and MIX, were additive. The Ca2+ channel blockers Co2+ (5 mM) and verapamil (100 microM) and the Ca2+ chelator EGTA (4 mM) abolished the stimulatory effect of TRH (1 microM) on hormone release. Co2+ and verapamil, but not EGTA, inhibited the stimulatory effect of db-cAMP (5 mM) on hormone release. The inhibitory effects of Co2+ and verapamil on GH release were counteracted by the combination of TRH and db-cAMP. For PRL release Co2+, but not verapamil, was able to inhibit the combined action of TRH and db-cAMP. Co2+, verapamil, and EGTA eliminated the stimulatory effect of MIX (1 mM) on PRL release while only Co2+ and EGTA affected the GH release. Hormone release in the presence of MIX plus verapamil or EGTA, but not Co2+, was increased by TRH. The calmodulin antagonist trifluoperazine (TFP) at 30 microM inhibited basal hormone release and hormone release stimulated by TRH (1 microM), db-cAMP (5 mM), and MIX (1 mM). The Ca2+ ionophore A23187 (5 microM) had a stimulatory effect on basal hormone release which was abolished by 30 microM TFP.

1-Methyl-3-isobutylxanthine↗