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K M Gautvik

Publications and source records attributed to K M Gautvik.

At least 55 records · Page 3Linked to original sources
PubMed1992

Modulation of G proteins and second messenger responsiveness by steroid hormones in GH3 rat pituitary tumour cells.

We have investigated the modulation of different G protein alpha- and beta-subunit levels in prolactin (PRL) and growth hormone producing rat pituitary adenoma cells (GH3 cells) in culture after prolonged exposure (6-48 h) to the steroid hormones 17 beta-oestradiol and dexamethasone. Gi-3 alpha- and G beta-subunits were the only G protein subunits which increased in response to 10(-6) M oestradiol (to approximately 150 and 200% of controls, respectively), while the other alpha-subunits investigated (Gs alpha, Gi-2 alpha and G(o) alpha) remained relatively unchanged. Thyroliberin (TRH)--and guanosine 5'-[beta gamma-imido]trisphosphate (Gpp(NH)p)-elicited adenylyl cyclase (AC) activities were reduced during 6-12 h of oestradiol treatment (by 60 and 20%, respectively), while the inhibitory effect of somatostatin (SRIF) increased by approximately 100%. Dexamethasone (10(-6) M) increased levels of the stimulatory G protein Gs alpha (to approximately 340%) and decreased levels of Gi-3 alpha (to 25%). After 48 h, the AC response to TRH was reduced by approximately 70%, whereas the effect of the other modulators remained close to controls. We conclude that G protein subunits in GH3 cells are subject to specific regulation by steroid hormones and that this may be important in the tuning of the responsiveness of PRL secretion to hormones in the in vivo situation.

Adenylyl Cyclases↗
PubMed1992

Cloning strategies for peptide hormone receptors.

With the advancement in molecular biology in the last decade many receptors have been cloned and the understanding of their mechanism of function has improved proportionally. In addition the concurrent discovery of receptor gene families has been used to design elegant cloning strategies which in turn have facilitated the characterization of new receptors and receptor subtypes. This review covers the most currently used cloning strategies and gives some examples.

Animals↗
PubMed1991

[Determination of intact parathyroid hormone in patients with calcium metabolism disorders].

We have used an immunoradiometric assay (IRMA) to measure intact PTH. The serum-levels of PTH followed a log-normal distribution in a population of 85 healthy post-menopausal women, with a geometric mean of 1.9 pmol/l and a range of 0.8-6.1 pmol/l. The correlation between measurements performed using the IRMA and a radioimmunoassay which measured the C-terminal portion of the PTH molecule was 0.85. It was only the IRMA, however, that could measure subnormal values. Two of 27 patients with primary hyperparathyroidism had PTH-values in the upper reference interval. The rest of the patients had elevated levels. Patients with hypercalcemia due to malignancy had subnormal or low normal (less than 2.5 pmol/l) PTH values. Half of the patients with hypoparathyroidism had PTH below the limit of detection. Four subjects with familial hypercalciuric hypercalcemia had normal (3) or slightly elevated PTH-levels.

Adult↗
PubMed1991

[Malignant humoral hypercalcemia and the parathyroid hormone related protein].

Humoral hypercalcemia in malignant disease results from the production of humoral factors that act on bone to demineralize the skeleton, with subsequent release of calcium. It is characteristic of certain tumours without bony metastases. A recently discovered parathyroid hormone-related protein (PTHrP) has been implicated as a causative hypercalcemic agent. PTHrP exerts its calcium-mobilizing effects by interaction with parathyroid hormone (PTH) receptors in bone and kidney through its amino-terminal sequence, which is homologous with that of PTH. The human PTHrP gene could encode multiple isoforms of the protein due to alternative exon usage. Apart from its involvement in humoral hypercalcemia of malignancy, PTHrP has also been identified in normal tissues, such as keratinocytes and placenta, and is present in high concentration in milk. PTHrP may modulate the calcium homeostasis in some normal physiological conditions, probably acting in a paracrine fashion.

Amino Acid Sequence↗
PubMed1991

Thrombin and H64A subtilisin cleavage of fusion proteins for preparation of human recombinant parathyroid hormone.

Human parathyroid hormone, hPTH, an 84 amino acid polypeptide, was produced intracellularly in Escherichia coli as a fusion protein, linked to the C-terminus of a 15 kD IgG-binding protein. Approximately 100 mg fusion protein was obtained per liter fermentation medium. To test the efficiency of two alternative enzymatic cleavage methods, two fusion proteins differing only in the linker region were constructed. Cleavage of a Phe-Phe-Pro-Arg linker was obtained with bovine thrombin and cleavage of a Phe-Ala-His-Tyr linker with recombinant H64A subtilisin. Both enzymes yielded the correct N-terminus and cleaved their respective linkers quantitatively, although additional internal cleavage sites in hPTH were detected and characterized. The linker cleavage conditions were optimized and hPTH was purified to homogeneity. Thrombin cleavage resulted in a final yield of 5 mg hPTH/L, while H64A subtilisin cleavage was more specific and gave 8 mg/L. The purified recombinant product was identical to native hPTH and exhibited full biological activity in an adenylate cyclase assay.

Amino Acid Sequence↗
PubMed1991

Cell specific distribution of guanine nucleotide-binding regulatory proteins in rat pituitary tumour cell lines.

To investigate the effects of guanine nucleotide-binding regulatory proteins (G proteins) on hormonal regulation of prolactin (PRL) synthesis and secretion, the qualitative distribution of G protein alpha-subunits and their mRNAs was studied in three functionally different pituitary tumour cell lines (GH cells) and normal rat pituitary tissue. Levels of basal and modulated adenylyl cyclase (AC) and phospholipase C (PLC) activities are also included. GH cells and pituitary tissue contained various amounts of mRNAs and protein for Gs alpha, Gi-2 alpha, Gi-3 alpha and Go alpha, while mRNA for Gi-1 alpha was only detected in normal pituitary tissue. Gz alpha/Gx alpha mRNA was expressed in all pituitary cell lines as well as in pituitary tissue. Go alpha mRNA and Gz alpha/G x alpha mRNA displayed size heterogeneity. These findings may have importance in the understanding of hormone regulation of second messenger systems.

Adenylyl Cyclases↗
PubMed1991

Regulation of G protein mRNA levels by thyroliberin, vasoactive intestinal peptide and somatostatin in prolactin-producing rat pituitary adenoma cells.

We have investigated the regulation of mRNA levels of alpha- and beta-subunits of guanine nucleotide-binding regulatory proteins (G proteins) by peptide hormones in prolactin producing rat pituitary adenoma cells (GH3 cells) in culture. The cells were treated with thyroliberin (1 microM), vasoactive intestinal peptide (1 microM) or somatostatin (10 microM) for 6 to 48 hours. Thyroliberin and vasoactive intestinal peptide increased the levels of Gs alpha Go alpha, Gi-2 alpha, Gi-3 alpha, Gx alpha, G beta 36 and mRNAs. The effect of vasoactive intestinal peptide was however earlier and more pronounced. Gi-2 alpha mRNA levels showed the quantitatively largest alterations. Somatostatin upregulated Gs alpha and downregulated Go alpha and Gi-2 mRNAs. G protein mRNAs for Gi-2 alpha and Go alpha were increased by exposure of the cells to a medium devoid of serum. We conclude that G protein mRNA levels are subjected to alterations by hormones that act through the corresponding G proteins in the regulation of prolactin synthesis and secretion.

Adenoma↗
PubMed1991

Signal transduction alterations in GH(1)2C1 rat pituitary tumour cells following treatment with 5-azacytidine.

In GH(1)2C1 rat pituitary cells treated with 5-azacytidine, the stimulatory effects exerted by vasoactive intestinal peptide (VIP), the GTP analogue guanyl-5'-yl imidodiphosphate (Gpp(NH)p), 12-O-tetradecanoyl phorbol 13-acetate, cholera toxin and pertussis toxin on the membrane-bound adenylyl cyclase were almost completely abolished. The corresponding inhibitory effect of somatostatin was increased. Alterations in adenylyl cyclase responsiveness began at the end of the drug treatment, and were most pronounced on day 5 after removal of 5-azacytidine. The cells subsequently and completely recovered after 10 days in the absence of the drug. Measurements of cholera toxin- and VIP-enhanced cyclic AMP levels in intact cells confirmed these results, and VIP appeared to have no stimulatory effect on GH secretion after 5-azacytidine treatment. Down-regulation of G alpha s RNA also occurred on day 5 after cessation of drug treatment. ADP-ribosylation subsequent to stimulation with pertussis toxin was markedly increased, indicating an enhancement of G alpha i and/or G alpha o. Furthermore, both basal and Gpp(NH)p-stimulated phospholipase C activities were augmented by pre-exposure to 5-azacytidine. Treatment of GH(1)2C1 rat pituitary tumour cells with 5-azacytidine therefore causes a marked but temporary increase in the ratio of G alpha i/G alpha s protein levels.

Adenosine Diphosphate↗
PubMed1991

Medullary carcinoma of the thyroid. An update of diagnostic and prognostic factors.

Medullary carcinoma of the thyroid gland (MCT) is a disease which shows an early tendency to spread both locally as well as by the blood stream. It is to day possible to make an early diagnosis in patients who have the familial form of the disease as well as perform an organ specific localization regarding possible spread of the disease. Calcitonin is a major product of MCT cells and represent the most used tumour marker for diagnosis and evaluation of prognosis. Basal calcitonin levels are not suited for making neither an accurate diagnostic or prognostic evaluation. Calcitonin provocation test (pentagastrin injections, calcium infusion/injection or a combination) are recommended for both early diagnosis as well as follow-up studies for mapping of the extent of disease. An early diagnosis can be made in kindred of a patient with familial MCT. Selective venous catheterization will in a significant way aid the localization of the disease, and together with calcitonin provocation tests reveal the presence of micro-metastases.

Adult↗
PubMed1990

Electroporation of rat pituitary (GH) cell lines: optimal parameters and effects on endogenous hormone production.

An efficient electroporation procedure was established for the genetic transformation of two clonal strains of hormone producing rat pituitary cells (GH12C1 and GH3). We used the bacterial chloramphenicol acetyltransferase (CAT) gene as reporter gene to determine optimal conditions for electroporation. The conditions found to be optimal, measured as expression of the highest CAT activity, were 240-300 V and a DNA concentration of 30-60 micrograms/ml in sucrose buffer. Cell viability was then about 50 per cent. Maximum CAT activity was seen 24 hours after electroporation. The electroporation procedure, in the presence or absence of DNA, caused a transient decrease in endogenous growth hormone (GH) and prolactin (PRL) production.

Animals↗
PubMed1990

1,25-dihydroxyvitamin D-3 and 24,25-dihydroxyvitamin D-3 affect parathormone (PTH) -sensitive adenylate cyclase activity and alkaline phosphatase secretion of osteoblastic cells through different mechanisms of action.

In UMR 106 rat osteosarcoma cells, parathormone (1-34hPTH) and calcitonin (sCT) stimulated adenylate cyclase (AC) activity 5.5-and 2.8-fold, respectively. AC in osteoblasts (OB) from collagenase-treated calvaria of 3-day-old rats responded similarly to 1-34hPTH. In contrast, fibroblasts (mouse fibroblastomas) displayed a marginal 1-34hPTH sensitive AC. Osteoclasts (OC) of collagenase-treated rat calvariae, rat monocytes and mouse macrophages did not demonstrate 1-34hPTH inducable AC activity. Physiological concentrations of 24,25-dihydroxyvitamin D-3 attenuated PTH-sensitive AC in OB and UMR 106 cells within 20 min, while 1,25-dihydroxyvitamin D-3 showed no such immediate effect. In contrast, the AC response to Gpp(NH)p was unaffected by 24,25-(OH)2D3, indicating that 24,25-(OH)2D3 interrupts the coupling of the PTH receptor to the GTP binding protein Gs. OB and UMR 106 cells were also subjected to long-term (48 h) incubation with vitamin D-3 metabolites, 1-34hPTH or 20% serum from patients with secondary hyperparathyroidism (sHBT-serum), respectively. PTH-sensitive AC was markedly attenuated by pre-exposure to both 1-34hPTH and 1,25-(OH)2D3, while minimally affected by corresponding 24,25-(OH)2D3 and 20% sHPT-serum treatment. The secretion of alkaline phosphatase (Alphos) from the two cell types was strongly increased by 1-34hPTH, the effect being abolished by the presence of 24,25-(OH)2D3. Iliac crest biopsies of normal individuals exhibited a clear negative correlation between PTH-sensitive AC and corresponding serum 24,25-(OH)2D3 levels. Basal AC activity was, however, negatively correlated to serum 1,25-(OH)2D3 concentrations. In summary, the results show that 24,25-(OH)2D3 reduces PTH-stimulated AC activity in and Alphos secretion from osteoblastic bone cells by rapidly and directly interfering with the plasma membrane. These data reinforce the probable in vivo significance of 24,25-(OH)2D3. Moreover, the negative correlation between basal AC activity and serum 1,25-(OH)2D3 levels indicates a possible role for 1,25-(OH)2D3 in regulating bone cell synthesis of AC components in vivo.

24,25-Dihydroxyvitamin D 3↗
PubMed1990

Specific antisense RNA inhibition of growth hormone production in differentiated rat pituitary tumour cells.

An expression vector that carried an inverted 800 base pair insert of the rat growth hormone (rGH) cDNA downstream of the SV40 promotor was used to transfect two different growth hormone (GH) producing rat pituitary cell strains, GH12C1 and GH3. This resulted in a specific transient inhibition of growth hormone production up to 75 percent in the course of 72 hours. GH synthesis reduction occurred parallel to a decrease of GH cytoplasmic mRNA levels. Levels of beta-actin and guanine nucleotide-binding regulatory protein (G protein) mRNAs were unaltered, but PRL mRNA levels were increased. Transfection with a control vector did not affect GH production.

Animals↗
PubMed1990

Dexamethasone inhibition of rat hepatoma cell growth and cell cycle traverse is reversed by insulin.

(1) The growth of 7800 C1 Morris hepatoma cells was inhibited by dexamethasone. The inhibition was detectable at 1 nM and half-maximal effect was obtained with approx. 13 nM dexamethasone. About 80% growth inhibition was obtained with 250 nM of the hormone and the growth rate was normalized on cessation of treatment. (2) These hepatoma cells contain dexamethasone receptors with equilibrium dissociation constant of 0.24 nM and a capacity of 24 fmol/mg cell protein. Treatment of the cells with insulin did not change these dexamethasone binding properties. Binding experiments showed that 2, 10 and 100% of the receptors were occupied when the cells were incubated with 1 nM, 7 nM and 250 nM dexamethasone, respectively. (3) Insulin completely counteracted the growth inhibition by dexamethasone and antagonized the induction of peroxisomal acyl-CoA oxidase and tyrosine aminotransferase caused by the glucocorticoid. (4) Micro-flow fluorometry showed that the cultures had a major diploid DNA stem line and a minor tetraploid stem line. Changes in diploid, tetraploid and S phase cells of the diploid stem line were scored. Dexamethasone reduced the proportion of cells in S phase and of tetraploid cells. Insulin partly reversed the action of dexamethasone in S phase, but prevented the reduction in tetraploid cells caused by dexamethasone. (5) The mitotic rate was significantly reduced by dexamethasone and this effect was reversed by insulin. (6) Continuous [3H]methyl-thymidine labelling showed a growth fraction of unity in all treatment groups. (7) It is concluded that dexamethasone induces growth inhibition by reducing the G1-S transition. Insulin is able to counteract this effect and increase the rate of DNA synthesis.

Animals↗
PubMed1990

Expression of human parathyroid hormone in Escherichia coli.

Human parathyroid hormone (hPTH) is a peptide hormone consisting of 84 amino acids. Using the expression plasmid pKK223-3 with the strong tacpromoter, we have produced a variant of hPTH in E. coli. From the expression plasmid construct the expected product was hPTH with an N-terminal extension of Met-Gly. The peptide was extracted from E. coli cells and purified by high performance liquid chromatography. In two different gel electrophoresis systems including identification by immunoblotting the product behaved exactly as an hPTH standard. N-terminal amino acid sequence analysis of the purified product showed traces of Gly-hPTH. At least 90% of the expressed product was N-terminally blocked, suggesting the presence of N-formyl-methionine. This variant of hPTH did not stimulate adenylate cyclase activity in rat osteosarcoma cell membranes.

Adenylyl Cyclases↗
PubMed1990

The mechanisms by which vasoactive intestinal peptide (VIP) and thyrotropin releasing hormone (TRH) stimulate prolactin release from pituitary cells.

The effect of vasoactive intestinal peptide (VIP) on prolactin (PRL) secretion from pituitary cells is reviewed and compared to the effect of thyrotropin releasing hormone (TRH). These two peptides induced different secretion profiles from parafused lactotrophs in culture. TRH was found to increase PRL secretion within 4 s and induced a biphasic secretion pattern, while VIP induced a monophasic secretion pattern after a lag time of 45-60 s. The secretion profiles are compared to changes in adenylate cyclase activity, production of inositol polyphosphates, changes in intracellular calcium concentrations and changes in electrophysiological properties of the cell membrane.

Adenylyl Cyclases↗
PubMed1990

Long-term administration of vitamin D3 metabolites alters PTH-responsive osteoblastic adenylate cyclase in rats.

We have examined the effect of induced hyper D3 vitaminosis on bone-related variables in the rat with special reference to the parathyroid (PTH)-sensitive adenylate cyclase (AC) in rat calvariae. Subcutaneous injections three times a week of doses theoretically corresponding to about 10 times the average physiological serum levels of either 25 hydroxyvitamin D3 (25OHD3), 1,25 dihydroxyvitamin D3 (1,25(OH)2D3), or 24,25 dihydroxyvitamin D3 (24,25(OH)2D3) for 12 weeks gave the following results: At 12 weeks of treatment, 24,25(OH)2D3 levels in the groups receiving 25OHD3 or 24,25(OH)2D3 increased significantly, whereas 1,25(OH)2D3 levels remained unaffected. Correspondingly, PTH-sensitive AC activities in crude calvarial membrane fractions from 25OHD3- and 24,25(OH)2D3-treated animals were obliterated. This effect was apparent after 4 weeks of treatment. In the group receiving 25OHD3, both basal, plus Gpp(NH)p-, and forskolin-sensitive AC activities were significantly reduced after 4 weeks of treatment. Similar effects in crude kidney membrane fractions were, however, not observed. Liver membranes from 25OHD3- or 24,25(OH)2D3-treated animals showed insignificant changes in the isoprenalin-, PGE1-, Gpp(NH)p-, or forskolin-sensitive AC activities. Finally, the significance of reduced PTH-sensitive bone AC activity has been assessed. 25OHD3 treatment yielded normocalcemic and hypercalciuric rats, whereas 1,25(OH)2D3 enhanced both serum and urine Ca2+ levels. 24,25(OH)2D3-treated and control animals were undiscernible in this respect. However, the 24,25-(OH)2D3 treatment caused reductions in both serum alkaline phosphatase levels and urinary hydroxyproline/creatinine ratio.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1990

Purification and characterization of Atlantic salmon growth hormone and evidence for charge heterogeneity.

Highly purified growth hormone (GH) has been isolated from Atlantic salmon (Salmo salar) pituitaries by extraction with acid acetone, acidic precipitation, and reversed-phase high-performance liquid chromatography (HPLC). The yield was 2.5 mg/g wet tissue. The Atlantic salmon GH (sGH) emerged as a single symmetrical peak after HPLC on a reverse phase C18 column. SDS-gel electrophoresis revealed only one band with an estimated molecular weight of 23,000. Atlantic sGH showed a uniform molecular weight, but two-dimensional (2D) gel electrophoresis of the purified sGH revealed charge heterogeneity with pI's ranging from 6.5 to 8.2. Treatment of the purified sGH with alkaline phosphatase concentrated these different forms into a single more alkaline position (pI 8.2) indicating removal of acidic groups. These results were documented using both silver- and immunostaining of the 2D SDS gels. The purified sGH was phosphorylated in vitro by a calmodulin-dependent protein kinase. Phosphorylation of sGH may be a post-translational modification resulting in several molecular forms with variable acidity. Analysis of the amino acid composition of Atlantic sGH revealed homology with GHs isolated from other teleost species and the amino-terminal sequence showed only three different amino acids within the first 25 residues compared to GH isolated from chum salmon (Oncorhynchus keta) and coho salmon (Oncorhynchus kisutch) pituitaries. Atlantic sGH had a methionine as the amino-terminal residue. Antibodies against chum sGH cross-reacted with Atlantic sGH. Antibodies against either Atlantic or chinook (Oncorhynchus tschawytscha) salmon prolactin or human GH did not cross-react with Atlantic sGH. Atlantic sGH was shown to have a slight growth-promoting activity in the rat tibia assay.

Alkaline Phosphatase↗