PubMed Health⌕ Search

Biomedical subjects

H Rasmussen

Publications and source records attributed to H Rasmussen.

At least 19 recordsLinked to original sources
PubMed1992

Glucose-induced phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) in isolated rat pancreatic islets.

In order to further evaluate the role of protein kinase C activation in glucose-induced insulin secretion, the extent of phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) was examined in freshly isolated rat pancreatic islets prelabeled with [32P]orthophosphate. The islets were incubated with either 2.75 mM glucose alone, 2.75 mM glucose + 1 microM phorbol myristate acetate, 20 mM glucose, or 20 mM glucose + 50 nM staurosporine. After stimulation, the homogenized islets were processed by immunoprecipitation with a specific polyclonal anti-MARCKS antibody, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Densitometric analysis of autoradiograms revealed that phorbol myristate acetate caused a 3.78 +/- 0.97-fold increase in MARCKS phosphorylation over control. In the islets exposed to 20 mM glucose, an increase of 3.43 +/- 0.46-fold over control was observed. In islets exposed to G20 + 50 nM staurosporine, MARCKS phosphorylation was inhibited by 90 +/- 4% compared with control islets exposed to 20 mM glucose alone. Islets similarly treated (but incubated without 32P) were examined by immunocytochemistry using an alpha-PKC-specific monoclonal antibody and visualized by confocal immunofluorescence microscopy. The alpha-PKC redistributed from the cytosol to the plasma membrane in the beta-cells of islets exposed to 20 mM glucose. In separate experiments, unlabeled but similarly treated islets were shown to respond with a 5-7-fold increase in insulin secretion in static incubation. Thus, when freshly isolated rat pancreatic islets are exposed to stimulatory glucose concentrations, they exhibit both a translocation of alpha-PKC and a significant increase in the extent of phosphorylation of MARCKS protein. These data suggest that alpha-PKC is activated during glucose-induced insulin secretion.

Alkaloids↗
PubMed1992

Metabolic fate of plasma membrane diacylglycerols in NIH 3T3 fibroblasts.

We have examined the metabolism of three radiolabeled 1,2-diacylglycerols (DGs) in NIH 3T3 fibroblasts. Since the lipids used are not appreciably taken up by the cells, we used a phosphatidylserine (PS)-based liposome fusion system to rapidly associate the lipid species with the plasma membrane. When 1,2-[1-14C]dioleoyl-sn-3-glycerol ([14C]DOG) is delivered in this way, it is rapidly converted predominantly to phosphatidylcholine (PC) and triacylglycerol (TG) and to a lesser extent, to monoacylglycerol (MG) and fatty acids (FA), as well as phosphatidic acid (PA) and phosphatidylinositol (PI). We present evidence that [14C] DOG is largely utilized as an intact molecule rather than being broken down to FA and then incorporated to cell lipids. Examination of the metabolism of 1-stearoyl-2-[1-14C]myristoyl-sn-3-glycerol ([14C]SMG) and 1-stearoyl-2-arachidonoyl-sn-3-glycerol ([14C]SAG) reveal important differences. Both produce substantial labeling of PC but [14C]SMG gives rise to the highest proportion of TG and the lowest of PA and PI, whereas [14C]SAG yields the opposite pattern. When phosphatidic acid labeled on its glycerol backbone (1,2-dioleoyl-sn-[U-14C] glycero-3-phosphate) was supplied to the cells via the liposomes, rapid appearance of labeled DG was found which then decreased with concomitant labeling of cellular PC and TG. Only small amounts of the glycerol backbone were recovered in PI. Our experiments identify three types of processes involved in the metabolism of plasma membrane DGs: (i) transferase-catalyzed conversions to PC and TG, (ii) lipolytic breakdown to MG and FA, and (iii) phosphorylation to PA and then conversion to PI. The relative proportions of each DG species converted to these different products are strongly dependent on the fatty acyl composition of the particular DG molecular species, even though formation of PC is the major event in all cases. Since DGs are important second messengers, our study supports the view that conversion to PC and TG can play a key role in DG signal attenuation.

3T3 Cells↗
PubMed1992

Low-dose 17 beta-estradiol vaginal tablets in the treatment of atrophic vaginitis: a double-blind placebo controlled study.

The aim of the study was to investigate the effect of 25 micrograms 17 beta-estradiol administered as a small vaginal tablet (Vagifem, Novo Nordisk A/S) on the symptoms of the vagina related to atrophy. The study was designed as a double-blind randomized placebo controlled study running for 12 weeks. The women were treated once daily for 2 weeks with the active or the placebo tablet. During the subsequent 10 weeks the women were treated twice a week. One hundred and sixty-four women were included. Ten dropped out for minor reasons, most of these due to lack of effect in the placebo group. In the Vagifem group 78.8% were suffering from moderate to severe atrophy of the vagina, compared with 81.9% in the placebo group. After 2 weeks the percentages were 14.3 and 35.3, respectively. After 12 weeks of treatment, 10.7% in the Vagifem group compared with 29.9% in the placebo group had moderate to severe atrophy (P less than 0.0001). A substantial part of the women complained about subjective symptoms such as vaginal dryness and dyspareunia. After 12 weeks of treatment, a significant improvement was found in the Vagifem group (P less than 0.002). Before treatment 53.1% in the treatment group and 41.0% in the placebo group were suffering from urological symptoms. After 2 weeks, 60.5% of the women in the Vagifem group underwent a change for the better compared to 35.3% in the placebo group. After 12 weeks of treatment the percentages in the two groups feeling an improvement were 62.8% and 32.4%. In this study local low-dose treatment with 25 micrograms 17 beta-estradiol was found to have a significant effect on the postmenopausal urogenital symptoms related to atrophy.

Administration, Intravaginal↗
PubMed1992

Signal transduction mechanisms involved in carbachol-induced aldosterone secretion from bovine adrenal glomerulosa cells.

In cultured bovine adrenal glomerulosa cells, diacylglycerol content remains elevated for up to 75 min following the removal of angiotensin II. This maintained increase could provide a mechanism by which angiotensin II pretreatment may prime cells to secrete aldosterone in response to the calcium channel agonist Bay K 8644. In the present study we find that carbachol failed both to produce this persistent diacylglycerol elevation and to exert a priming effect. In addition, because carbachol was also a less potent activator of phospholipase D than angiotensin II, our results implicate phospholipase D in the maintained increase in diacylglycerol content observed following stimulation with and removal of angiotensin II. Carbachol also elicited changes in the radiolabeled levels of both myristate- and arachidonate-containing diacylglycerol. However, the rapid decline in diacylglycerol content following carbachol removal resembled the rapid fall in arachidonate-diacylglycerol; we therefore proposed that the diacylglycerol species generated with carbachol stimulation contains predominantly arachidonic acid. In summary, our results suggest that prolonged elevations in diacylglycerol content following removal of hormones such as angiotensin II, as well as the identity of the diacylglycerol species itself, may be important in the regulation of cellular responses.

Aldosterone↗
PubMed1992

Immunocytochemical localization of alpha-protein kinase C in rat pancreatic beta-cells during glucose-induced insulin secretion.

To investigate the role of protein kinase C (PKC) in the regulation of insulin secretion, we visualized changes in the intracellular localization of alpha-PKC in fixed beta-cells from both isolated rat pancreatic islets and the pancreas of awake unstressed rats during glucose-induced insulin secretion. Isolated, perifused rat islets were fixed in 4% paraformaldehyde, detergent permeabilized, and labeled with a mAb specific for alpha-PKC. The labeling was visualized by confocal immunofluorescent microscopy. In isolated rat pancreatic islets perifused with 2.75 mM glucose, alpha-PKC immunostaining was primarily cytoplasmic in distribution throughout the beta-cells. In islets stimulated with 20 mM glucose, there was a significant redistribution of alpha-PKC to the cell periphery. This glucose-induced redistribution was abolished when either mannoheptulose, an inhibitor of glucose metabolism, or nitrendipine, an inhibitor of calcium influx, were added to the perifusate. We also examined changes in the intracellular distribution of alpha-PKC in the beta-cells of awake, unstressed rats that were given an intravenous infusion of glucose. Immunocytochemical analysis of pancreatic sections from these rats demonstrated a glucose-induced translocation of alpha-PKC to the cell periphery of the beta-cells. These results demonstrate that the metabolism of glucose can induce the redistribution of alpha-PKC to the cell periphery of beta-cells, both in isolated islets and in the intact animal, and suggest that alpha-PKC plays a role in mediating glucose-induced insulin secretion.

Animals↗
PubMed1992

Ca(2+)-cyclic AMP interactions in sustained cellular responses.

As early as 1970 it was apparent that the cyclic AMP (cAMP) and Ca2+ messenger systems often interact to regulate cellular responses. Work over the past 20 years has greatly expanded our knowledge of these interactions, and has shown that these signalling systems interact in complex ways to regulate sustained cellular responses such as aldosterone secretion, smooth muscle contraction and insulin secretion. The latter system is considered in detail because it illustrates several types of interactions, both positive and negative, which help to determine the normal response of beta-cells to physiological stimuli, and how abnormalities in secretory patterns can develop as a consequence of the prolonged stimulation of a messenger system.

Animals↗
PubMed1991

Effects of the phorbol ester phorbol 12-myristate 13-acetate (PMA) on islet-cell responsiveness.

Collagenase-isolated rat islets were labelled for 2 h in myo-[2-3H]inositol solution supplemented with 2.75 mM-glucose. The phorbol ester phorbol 12-myristate 13-acetate (PMA; 0.1 or 1 microM) was also present in some experiments. After labelling, islets were washed and then perifused in 2.75 mM-glucose to establish basal [3H]inositol-efflux and insulin-secretory rates. Subsequently, the responses of these islets to stimulation with various agonists were assessed. Inositol phosphate accumulation was measured at the termination of the perifusion. In separate experiments, the cellular location of protein kinase C (PKC) after PMA pretreatment was measured by quantitative immunoblotting of membrane and cytosolic fractions. The following observations were made. (1) Labelling in 0.1-1 microM-PMA had no deleterious effect on total [3H]inositol incorporation during the 2 h labelling period. However, islets labelled for 2 h in 1 microM-PMA were unable to respond, in terms of increases in insulin release, to a 1 microM-PMA stimulus during the subsequent perifusion. (2) As compared with the responses of control islets labelled in 2.75 mM-glucose alone, islets labelled in the additional presence of 1 microM-PMA displayed a significant impairment in phosphoinositide (PI) hydrolysis, but an enhancement of both first-and second-phase insulin secretion, in response to subsequent 20 mM-glucose stimulation. (3) Decreasing extracellular Ca2+ level to 0.1 mM and including the Ca(2+)-channel antagonist nitrendipine (0.5 microM) along with 1 microM-PMA during the [3H]inositol-labelling period did not alter the response of the islets to the subsequent addition of 20 mM-glucose. Glucose-induced PI hydrolysis was still inhibited and 20 mM-glucose-induced insulin release was still enhanced. (4) A markedly amplified and sustained insulin-secretory response to 200 microM-tolbutamide in the presence of 2.75 mM-glucose was also obtained from 1 microM-PMA-pretreated islets. This contrasts sharply with the small and transient response to tolbutamide noted in control islets. (5) When present only during the perifusion phase of the experiments, nitrendipine (0.5 microM) abolished the amplified insulin-secretory responses to both 20 mM-glucose and 200 microM-tolbutamide noted in PMA-pretreated islets. (6) Prior labelling in 1 microM-PMA dramatically amplified the insulinotropic effect of 25 mM-K+ or 5 microM-A23187 stimulation. The amplified insulin-secretory response to K+, but not to A23187, was abolished by inclusion of nitrendipine during the perifusion.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Angiotensin-II-induced changes in diacylglycerol levels and their potential role in modulating the steroidogenic response.

Angiotensin-II (Ang II) not only increases aldosterone secretion from bovine adrenal glomerulosa (AG) cells, but also primes these cells to respond to a subsequent challenge with the calcium channel agonist Bay K 8644. In cultured AG cells we investigated the hypothesis that this priming effect was the result of a persistent elevation in diacylglycerol (DAG) content. Ang II elicited an increase in DAG content, which was maintained for up to 75 min after the removal of Ang II, an effect which could underlie the ability of Ang II to prime the cells to respond to Bay K 8644. We then investigated the possibility that the DAG found in bovine AG cells consists of multiple species and the potential relationship of the species to the persistent elevation. We found that [3H]arachidonate and [14C]myristate were differentially incorporated into phospholipids, with approximately 80-85% of the latter radiolabel contained in phosphatidylcholine. Ang II elicited increases in the levels of both arachidonate- and myristate-containing DAG. The subsequent addition of an Ang II antagonist resulted in a rapid decrease in [3H]arachidonate-labeled DAG levels, but a much slower decline in myristate-containing DAG. These results suggest that the species of DAG generated in response to hormonal stimulation may be important in determining the speed with which this signal is terminated. Ang II also stimulated the release of water-soluble [3H]choline metabolites, in particular choline and phosphorylcholine, from prelabeled cells. These results indicate that 1) various DAG species exhibit different turnover rates; and 2) perhaps as a result of this disparity, the increase in DAG induced by an agonist may persist for a considerable period of time after the removal of the agonist or the inhibition of its action.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1991

Parathyroid hormone modulates angiotensin II-induced aldosterone secretion from the adrenal glomerulosa cell.

The effect of PTH on aldosterone secretion from isolated bovine adrenal glomerulosa cells was examined. PTH binding was autoradiographically localized to the adrenal cortex, suggesting a specific effect. This binding of PTH was displaceable by cold PTH, but not by ACTH. No binding was observed in the adrenal medulla. In addition, PTH was shown to stimulate aldosterone secretion in a dose-dependent manner and to potentiate aldosterone secretion in response to angiotensin-II, such that PTH (10(-9)M) elevated the secretory rate from 58.6 +/- 6.8 to 110.9 +/- 19 pg/min.million cells in the presence of 10 nM angiotensin-II. The magnitude of the synergism between the two hormones depended on the concentrations of PTH and angiotensin-II as well as the time during which aldosterone secretion was measured. Within the first 15 min of stimulation, PTH increased the sensitivity to angiotensin-II, shifting the Ka for activation from 1.0 to 0.3 nM. In contrast, between 30-45 min of angiotensin-II stimulation, PTH elevated the maximal secretory response to angiotensin-II from 109 +/- 3.4 to 219 +/- 13.3 pg/min.million cells. By itself PTH elicited only a small increase in the intracellular Ca2+ concentration, as measured by aequorin luminescence in glomerulosa cells. In cells pretreated with angiotensin-II or 15 mM potassium, the intracellular calcium response to PTH was markedly potentiated. PTH was also found to cause a small increase in the cellular cAMP content. Thus, PTH stimulates aldosterone secretion from adrenal glomerulosa cells, both alone and in combination with angiotensin-II.

Adrenal Cortex↗
PubMed1990

A potential role for phospholipase-D in the angiotensin-II-induced stimulation of aldosterone secretion from bovine adrenal glomerulosa cells.

The mechanism by which angiotensin-II (Ang II) stimulates aldosterone secretion from adrenal glomerulosa cells involves a phospholipase-C-mediated increase in phosphoinositide turnover and diacylglycerol (DAG) production. Because agonist-induced activation of phospholipase-D (PLD) also contributes to elevations in DAG in other cell types, the ability of Ang II to stimulate PLD activity in cultured bovine adrenal glomerulosa cells was examined. Ang II elicited significant increases in the levels of phosphatidic acid and, in the presence of ethanol, of phosphatidylethanol, a more specific marker for PLD activation. The potential role of this increased PLD activity in the regulation of aldosterone secretion was examined by investigating the ability of exogenous PLD to alter secretory rates. PLD alone dose-dependently increased aldosterone secretion from 5.9 +/- 0.5 to 135 +/- 48 pg/min.mg protein. In the presence of the calcium channel agonist Bay K 8644, which by itself had only a modest effect on aldosterone production, the stimulatory action of PLD was enhanced, yielding a secretory rate (442 +/- 119 pg/min.mg protein) that was approximately 60% of that elicited by 10 nM Ang II (763 +/- 182 pg/min.mg protein). Exogenous PLD also induced a significant increase in DAG levels (from 0.76 +/- 0.03 to 1.10 +/- 0.1 nmol/mg protein), which was not altered by the addition of Bay K 8644. However, PLD did not stimulate inositol phosphate production. These data indicate that 1) Ang II activates PLD; 2) exogenous PLD can elevate aldosterone secretory rates and DAG levels without eliciting phosphoinositide hydrolysis; and 3) the stimulatory action of exogenous PLD on aldosterone secretion is enhanced in the presence of Bay K 8644. Thus, PLD-induced DAG production may play an important role in the Ang II-mediated stimulation of aldosterone secretion from the adrenal zona glomerulosa.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1979

Isolation of specific bone cell types by free-flow electrophoresis.

The bone cells and fibroblasts from fetal rat calvaria can be isolated by collagenase digestion of the collagen matrix and separated into specific cell types by free-flow electrophoresis. The method involves injection of a specially prepared suspension of cells into a stream of buffer across which is maintained an electric field of 60 V/cm. The fetal bone cell types are differentially deflected toward the anode where they can be collected. Free-flow electrophoresis of this heterogenous cell preparation yields three distinguishable peaks which can be identified by morphologic, morphometric, and enzymatic characteristics. All three cell peaks have greater than 95% viability as judged by trypan blue exclusion and will grow to confluent monolayers in culture. The data indicate that these cell peaks may be comprised of osteoclasts and/or preosteoclasts, osteoblasts and/or preosteoblasts, and fibroblasts.

Acetazolamide↗
PubMed1979

Parathyroid function and vitamin D metabolism during human growth hormone replacement.

Changes in calcium and phosphorus metabolism were studied in nine children with GH deficiency before and during human GH replacement therapy. Parathyroid function and serum concentrations of physiologically important vitamin D metabolites were examined to determine their relationship to changes in mineral metabolism. By comparison with pretreatment values, the GH-treated children showed significant increases in growth rate and renal tubular phosphate reabsorption and a significant decrease in urinary calcium excretion after a standardized oral load. There was no significant change in serum concentrations of parathyroid hormone, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, or 24,25-dihydroxyvitamin D or in nephrogenous cAMP excretion. Although GH has been proposed as a regulator of vitamin D metabolism, the present study demonstrates that the anabolic changes in calcium and phosphorus metabolism accompanying GH therapy are not mediated via changes in parathyroid hormone or vitamin D status.

Adolescent↗