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Biomedical subjects

M Ros

Publications and source records attributed to M Ros.

At least 55 records · Page 3Linked to original sources
PubMed1990

Coupling of adenosine A1 receptors to a G-protein in coated vesicles isolated from bovine brain: presence of pertussis and cholera toxin substrates.

Adenosine A1 receptors have been described in coated vesicles isolated from bovine brain (Gonzalez-Calero et al., J. Neurochem. 1990, 55, 106-113). Addition of non hydrolyzable GTP analogue (guanyl-5-yl-imidodiphosphate) caused a transition of the receptor from the high- to the low-affinity state, without any significant change in the total binding sites. The presence of G-proteins has been investigated by pertussis and cholera toxins catalyzed ADP-ribosylation. A band of Mr = 41,000 D, similar to the alpha Gi subunit, was specifically labeled in the presence of preactivated pertussis toxin. Bands of Mr = 42,000 D and Mr = 47,000 D were specifically labeled in the presence of preactivated cholera toxin. These results confirm the presence of GTP binding proteins (alpha Gi and alpha Gs) in coated vesicles isolated from bovine brain.

Animals↗
PubMed1990

Cross-regulation between G-protein-mediated pathways. Stimulation of adenylyl cyclase increases expression of the inhibitory G-protein, Gi alpha 2.

The hormone-sensitive adenylyl cyclase system is under dual control, receiving both stimulatory and inhibitory inputs. Guanine nucleotide-binding regulatory proteins (G-proteins) transduce signals from cell surface receptors to effectors such as adenylyl cyclase. Hormonal stimulation is propagated via Gs, inhibition by Gi. Persistent (24-h) activation of the stimulatory pathway of adenylyl cyclase by the diterpene forskolin or the beta-adrenergic agonist isoproterenol in S49 mouse lymphoma cells enhanced the effects of somatostatin mediated via the inhibitory pathway of adenylyl cyclase. Stimulating cells with forskolin or isoproterenol for 24 h resulted in a 3-fold increase in the steady-state levels of Gi alpha 2 and a 25% decline in Gs alpha, as quantified by immunoblotting. Within 12 h of stimulation of adenylyl cyclase, Gi alpha 2 mRNA levels increased 4-fold, measured by DNA-excess solution hybridization. Gs alpha mRNA levels, in contrast, increased initially (25%), but then declined to 75% of control. In S49 variants that lack functional protein kinase A (kin-), stimulation by isoproterenol failed to alter Gi alpha 2 expression at either the protein or the mRNA levels. A 3-fold increase in relative synthesis rate and no change in the half-life (approximately 80 h) of Gi alpha 2 was observed in response to forskolin stimulation. Although Gs alpha synthesis increased (70%) modestly in response to forskolin stimulation, the half-life of Gs alpha actually decreased from 55 h in naive cells to 34 h in treated cells. Thus, the two G-protein-mediated pathways controlling adenylyl cyclase display "cross-regulation." Persistent activation of the stimulatory pathway increases Gi alpha 2 mRNA and expression. Transiently elevated Gs alpha mRNA levels are counterbalanced by a reduction in the half-life of the protein.

Adenylyl Cyclases↗
PubMed1990

G-protein subunit mRNA levels in rat heart, liver, and adipose tissues: analysis by DNA-excess solution hybridization.

The steady-state levels of mRNAs for the G-proteins Gi alpha 2, Go alpha, and the G beta-subunits common to each were established in rat adipose, heart and liver. Uniformly-radiolabeled, single-stranded antisense probes were constructed from cDNAs or assembled from oligonucleotides. Direct comparison of the steady-state levels of the G-protein mRNAs was performed under identical assay conditions, and on a molar basis. In adipose, liver and heart, Gs alpha mRNA was more abundant than mRNA for Go alpha, Gi alpha, and G beta. In adipose tissue, mRNA levels were as follows: 19.4, 7.6, 7.0, and 2.3 amol mRNA per micrograms total cellular RNA for Gs alpha, G beta, Gi alpha 2, and Go alpha, respectively. In heart Gs alpha mRNA was less abundant than in adipose, but the relative trend among the G-protein subunits was the same. In liver, G beta mRNA was more abundant than either Go alpha or Gi alpha 2. Go alpha mRNA levels ranged from 1.2 to 2.3 amol/micrograms total RNA in liver and adipose, respectively. The present work demonstrates the many advantages of this strategy when applied to the study of a family of homologous, low-abundance proteins and establishes for the first time the molar levels of Gi alpha 2, Gs alpha, Go alpha, and G beta-subunit mRNAs in several mammalian tissues.

Adipose Tissue↗
PubMed1990

Integration of lipid metabolism in the mammary gland and adipose tissue by prolactin during lactation.

Prolactin deficiency, induced by bromocryptine treatment, brought about reciprocal changes in the ability of adipocytes and acini isolated from lactating rats to synthesize lipids. The capacity to synthesize fatty acids and phospholipids decreased in the mammary gland and increased in adipocytes by bromocryptine treatment. In the mammary gland, the maximum potential activity of the pentose shunt as well as the specific activities of the pathway dehydrogenases were significantly reduced by bromocryptine treatment. Simultaneously, adipose tissue increased its lipogenic capacity but neither the maximum potential of the shunt nor the specific activities of the pentose phosphate shunt dehydrogenases were significantly changed with respect to the control lactating rats. Thus, a differential regulatory mechanism(s) of the pentose phosphate shunt activity appears to operate in these two tissues. Adipocytes from lactating rats showed a poor responsiveness to insulin in terms of lipid synthesis from glucose. In contrast, in adipocytes from bromocryptine treated rats insulin was able to increase lipid synthesis (105%). Sheep prolactin administration 'in vivo' partially reversed the effects of bromocryptine. These data suggest that prolactin mediates adipocytes resistance to insulin during lactation. Phospholipid synthesis, as occurred in fatty acid synthesis, is increased in adipose tissue and decreased in mammary gland by bromocryptine treatment. However, alpha 1-adrenergic stimulation increases phosphatidylinositol turnover to about the same percentages in both mammary gland acini and adipocytes from lactating rats independently of bromocryptine treatment.

Adipose Tissue↗
PubMed1990

Regulation of transmembrane signalling elements: transcriptional, post-transcriptional and post-translational controls.

G-protein-mediated transmembrane signalling is a common motif in biology. The actions of a populous group of G-protein-linked receptors in hormone action, olfaction and vision in vertebrates are examples in which input signals are transferred from a receptor molecule (or photopigment) to an effector unit(s) via G-proteins. The expression and functional status of the receptors, G-proteins, and effectors that constitute these transmembrane signalling systems are regulated physiologically. Altering the abundance, function, or both of these elements provides the means for modulating transmembrane signalling and integration of information among separate pathways. Recent advances in the cell and molecular biology of transmembrane signalling elements provide insight as to the mechanisms by which regulation occurs. Transcriptional control is exemplified by glucocorticoid induction of beta-adrenergic receptor expression. Agonist-induced down-regulation of beta-adrenergic receptor mRNA via message destabilization best highlights post-transcriptional control. Examples of post-translational control of transmembrane signalling elements include protein phosphorylation, thioldisulphide exchange, and altered rates of protein degradation. Simultaneous analysis of physiological regulation at the levels of the gene, mRNA, and protein provide new opportunities for understanding how information processing extends from the plasma membrane to the genome.

Cell Membrane↗
PubMed1989

G-protein mRNA levels during adipocyte differentiation.

G-protein-mediated transmembrane signaling in 3T3-L1 cells is modulated by differentiation. The regulation of G-protein expression in differentiating 3T3-L1 cells was probed at the level of mRNA by DNA-excess solution hybridization. Pertussis toxin-catalyzed ADP-ribosylation of G-protein alpha-subunits increased as fibroblasts differentiate to adipocytes. Steady-state levels of mRNA for Gi alpha 2 and Go alpha, in contrast, declined sharply. Immunoblotting with antipeptide antibodies specific for Gi alpha 2, too, revealed a decline in the steady-state expression of this pertussis toxin substrate. ADP-ribosylation of Gs alpha by cholera toxin was less in the adipocyte than fibroblast. Analysis by immunoblotting revealed only a modest decline in Gs alpha. Analysis of mRNA levels also demonstrated a decline for Gs alpha. mRNA levels for the G beta-subunits rose initially (25%) on day 1, declined from day 1 to day 3, and remained 25% lower in adipocytes than in fibroblasts. In 3T3-L1 adipocytes the molar amounts of subunit mRNAs were: 60.6 (Gs alpha); 2.1 (Gi alpha 2); and 1.5 (Go alpha) amol/microgram total cellular RNA. In rat fat cells these mRNA levels were 19.4 (Gs alpha); 7.0 (Gi alpha 2); and 2.3 (Go alpha). These data demonstrate that for Gi alpha 2 and Go alpha alike mRNA and protein expression decrease, not increase, in differentiation. A substrate for pertussis toxin other than Gi alpha 2 and Go alpha appears to be responsible for the increase in toxin-catalyzed labeling that accompanies differentiation of 3T3-L1 cells.

1-Methyl-3-isobutylxanthine↗
PubMed1989

Thyroid hormones regulate G-protein beta-subunit mRNA expression in vivo.

Thyroid hormones exert "permissive effects" on the hormone-sensitive adenylate cyclase. Regulation of the expression of Gi (Gi alpha 2) and Gs by thyroid hormones in vivo was investigated at the level of mRNA. Steady-state levels of the mRNA for Gi alpha 2 and Gs alpha, as well as the G beta-subunits, were quantified using DNA excess solution hybridization analysis. Regulation of protein and mRNA expression in adipose tissue was investigated in hypothyroid, euthyroid, and hyperthyroid rats. In euthyroid animals, steady-state levels of mRNA (amol/microgram RNA) were 13.8, 5.9, and 5.7 for Gs alpha, Gi alpha 2, and G beta 1,2, respectively. Activation of adenylate cyclase by Gs is unaffected by thyroid status. Both Gs alpha and Gs alpha mRNA levels in hypothyroid rats were the same as those of controls (euthyroid). The inhibitory control of adenylate cyclase, in contrast, is markedly potentiated in hypothyroid rats. The expression of G1 alpha s and G beta-subunits was increased in hypothyroidism. Whereas Gi alpha 2 mRNA levels remained essentially unchanged, G beta 1,2 mRNA levels were observed to increase 45% in the hypothyroid state. In the hyperthyroid state G beta 1,2 mRNA levels were observed to decline by 35%. Regulation of G-protein subunit expression, at the level of mRNA, appears to be one component of permissive hormone action on transmembrane signalling.

Adipose Tissue↗
PubMed1989

Agonist regulation of beta-adrenergic receptor mRNA. Analysis in S49 mouse lymphoma mutants.

Agonist-promoted down-regulation of beta-adrenergic receptor mRNA was investigated in S49 mouse lymphoma variants with mutations in elements of hormone-sensitive adenylate cyclase. In wild-type cells steady-state levels of beta-adrenergic receptor mRNA were established by DNA-excess solution hybridization to be 1.72 +/- 0.08 (n = 8) amol/microgram total cellular RNA. Receptor mRNA levels declined 35-45% in response to stimulation by the beta-adrenergic agonist (-)isoproterenol or forskolin as described previously in DDT1 MF-2 cells (Hadcock, J. R., and Malbon, C. C. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 5021-5025). Agonist-promoted cAMP accumulation and down-regulation of receptor mRNA were analyzed in three variants with mutations in Gs alpha (H21a, unc, cyc-) and a single variant lacking cAMP-dependent protein kinase activity (kin-). H21a (Gs alpha coupled to receptor, but not to adenylate cyclase), unc (Gs alpha uncoupled from receptor), and cyc- (lacking Gs alpha) variants accumulated cAMP and down-regulated beta AR mRNA in response to forskolin. In unc and cyc- cells isoproterenol failed to stimulate cAMP; accumulation and down-regulation of receptor mRNA was not observed. H21a cells, in contrast, displayed agonist-promoted regulation of beta-adrenergic receptor mRNA but only basal levels of cAMP accumulation in response to isoproterenol. The kin- cells displayed cAMP accumulation in response to forskolin as well as to isoproterenol but no down-regulation of receptor mRNA or receptor expression. Taken together these data demonstrate several features of agonist-promoted down-regulation of mRNA: (i) cAMP-dependent protein kinase activity is required for down-regulation of mRNA (kin-), although elevated cAMP accumulation is not (H21a); (ii) functional receptor-Gs coupling is required (H21a), and clones lacking Gs alpha (cyc-) or receptor Gs coupling (unc) lack the capacity to down-regulate mRNA in response to agonist; and (iii) in the presence of basal levels of cAMP and cAMP-dependent protein kinase activity, functional receptor-Gs coupling (H21a) to some other effector other than adenylate cyclase may be propagating the signal.

Animals↗
PubMed1989

Glucocorticoids modulate mRNA levels for G-protein beta-subunits.

Adrenalectomy decreases, whereas glucocorticoid treatment increases, the steady-state levels of G-protein beta-subunits (G beta) in rat fat-cells. A DNA-excess solution-hybridization assay was established to define the steady-state mRNA levels for G beta [5.8 +/- 0.4 amol/micrograms of RNA (n = 5) in control fat-cells]. G beta mRNA levels decrease by 20% after adrenalectomy; dexamethasone treatment reverses the decline. Dexamethasone treatment itself increases G beta mRNA levels by 50%.

Adipose Tissue↗
PubMed1989

Adipocyte G-proteins and adenylate cyclase. Effects of adrenalectomy.

Steroid hormones modulate the ability of cells to respond to hormones that act via cyclic AMP. In adipocytes of adrenalectomized rats, cyclic AMP accumulation and lipolysis in response to adrenaline are attenuated. However, the mechanism(s) of these effects are poorly understood. The effects of altered glucocorticoid status in vivo on the steady-state amounts of components of the hormone-sensitive adenylate cyclase were analysed in rat adipocytes. beta-Adrenergic receptors were analysed by using radioligand binding and immunoblotting with an anti-receptor antiserum. Neither the amount of radioligand binding nor the amount of beta-adrenergic-receptor peptide (Mr 67,000) was altered by adrenalectomy, whereas treatment of adrenalectomized rats with dexamethasone was found to increase both parameters by more than 25% with respect to the control. Forskolin-stimulated adenylated cyclase activity was unchanged in membranes isolated from adipocytes of adrenalectomized rats, but was decreased (50%) in those from dexamethasone-treated rats. The alpha-subunit of Gs was probed by using cholera-toxin-catalysed ADP-ribosylation. Immunoblotting was used to analyse the steady-state amounts of G-protein beta-subunits (beta-G35/36). Adrenalectomy was associated with decreases in the steady-state amounts of alpha-Gs (30%) and beta-G35/36 (50%). Dexamethasone treatment of adrenalectomized animals partially restored the lipolytic response of adipocytes to adrenaline and the amounts of alpha-Gs, increased the amounts of beta-G35/36 subunits from 50% to 150% of control values, increased beta-adrenergic receptors by more than 25% and decreased adenylate cyclase activity (50%). These results suggest that the steady-state amounts of components of hormone-sensitive adenylate cyclase are differentially regulated by glucocorticoids.

Adenylyl Cyclases↗
PubMed1988

Steady-state levels of G-proteins and beta-adrenergic receptors in rat fat cells. Permissive effects of thyroid hormones.

Thyroid hormones exert a permissive influence on the ability of cells to respond to other hormones. In hypothyroidism, stimulation of adenylate cyclase by beta-adrenergic agonists is impaired in rat fat cells, whereas inhibition by adenosine is potentiated. The effects of thyroid status on steady-state levels of the G-protein subunits alpha-Go, alpha-Gi, and beta-G35/36 were investigated using specific antibodies and quantitative immunoblotting of rat fat cell membranes. The amount of alpha-Go (Mr 39,000, alpha-G39) detected in fat cell membranes of euthyroid rats was 44 +/- 5 pmol/mg of membrane protein (n = 5). In the hypothyroid state, the amount of the alpha-subunits of Gi (Mr 41,000, alpha-G41) and Go were found to be markedly increased in comparison to the control. The steady-state level of alpha-G41 and alpha-G39 increased more than 50 and 70%, respectively, in the hypothyroid state. The beta-subunit of G-proteins of rat fat cells appears as a doublet of proteins with Mr = 35,000/36,000 on sodium dodecyl sulfate-polyacrylamide gels. The amount of beta-G35/36 detected in fat cell membranes of euthyroid rats was 0.20 +/- 0.03 nmol/mg of protein (n = 5) and was found to increase by about 60% in the hypothyroid state. Administration of triiodothyronine in vivo (short term hyperthyroidism) resulted in a decrease in the amounts of alpha-G41 and alpha-G39 subunits (25 and 20%, respectively). In contrast to these effects of thyroid hormones on Go and Gi, the steady-state level of beta-adrenergic receptors was not significantly altered by changes in thyroid status. Thus, thyroid status in vivo can modulate the steady-state levels of specific G-proteins.

Adipose Tissue↗
PubMed1987

Glycerogenic pathway in the rat mammary gland.

The conversions of the isotope from [1-14C]acetate, [1-14C]glucose and [6-14C]glucose to CO2 and fatty acids in acini isolated from the mammary gland at the peak of lactation were studied. The incorporation of [9,10-3H]oleate into triacylglycerol synthesis as single substrate or in combination with substrates that potentially may supply trioses-phosphate was also determined. The rate of fatty acid synthesis paralleled the activity of the hexose monophosphate shunt and the data obtained reveal that little carbon from triose stage enters the phosphohexose pool via reversal of glycolytic pathway. The results are interpreted in terms of the NADPH producing systems and phosphoenolpyruvate carboxykinase activities as well as the possible implications in lipogenic and glyceroneogenic pathways.

Acetates↗
PubMed1986

Nutritional and hormonal regulation of malic enzyme synthesis in rat mammary gland.

Cytosolic malic enzyme was purified from rat mammary gland by L-malate affinity chromatography. The pure enzyme obtained was used to produce a specific antiserum in a rabbit. Relative synthesis of malic enzyme in the mammary gland of mid-lactating rats was 0.097%, measured by labelling the enzyme in isolated acini. When food was removed, malic enzyme synthesis decreased to 35% and 20% of the control value at 4 and 6 h respectively. Incorporation of [3H]leucine into soluble proteins was constant during the first 6 h of starvation. When lactating rats (maintained with their pups) were starved for 24 h and then re-fed, the relative rate of enzyme synthesis increased 2.5-, 4-, and 4.5-fold at 3 h, 6 h and 18 h respectively after initiation of re-feeding. The relative rate of malic enzyme synthesis was about 50% of normal at 15 h after weaning, whereas the rate of synthesis of soluble proteins did not change. Administration of bromocriptine or adrenalectomy of lactating rats decreased the relative rate of synthesis of malic enzyme by 40% or 30% respectively; these effects were counteracted by hormone supplementation. Hormone therapy also caused an increase in the rate of incorporation of [3H]leucine into soluble proteins and in malic enzyme activity.

Adrenalectomy↗
PubMed1985

Effect of prolactin and glucocorticoids on P-enolpyruvate carboxykinase activity in liver and mammary gland from diabetic and lactating rats.

The administration of 2 bromo-alpha-ergocryptine, to reduce serum prolactin decreased the activity of cytosolic P-enolpyruvate carboxykinase (GTP) (EC 4.1.1.32) about 50% in both liver and mammary gland of lactating animals. Adrenalectomy had similar effects to those of bromo-alpha-ergocryptine. In contrast, there was a 50% increase in enzyme activity in the mammary gland of diabetic, lactating rats and a 10-fold increase in liver as compared with normal rats. P-enolpyruvate carboxykinase activity in mammary gland as liver is coordinately regulated by prolactin, glucocorticoids and insulin.

Adrenal Glands↗
PubMed1985

Influence of starvation/refeeding transition on lipogenesis and NADPH producing systems in adipose tissue, mammary gland and liver at mid-lactation.

Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and malic enzyme are enzymes involved in NADPH synthesis. Their specific activities and glucose utilization by isolated cell systems have been measured in adipose tissue and mammary gland from mid-lactating rats during starvation/refeeding transition. Starvation for 24 h produced a 75-90% decrease in the specific activities of these NADPH producing systems in mammary gland. Acinis isolated from the gland of starved rats had a lower production of CO2, fatty acids and triacylglycerols from (1-14C)glucose and (6-14C)-glucose than did gland from control rats. The activities of these enzymes in adipose tissue were very low and did not undergo any measurable alteration with starvation. The ability of adipocytes from well fed lactating rats to synthesize fatty acids from (1-14C)glucose was completely blocked. However, starvation is accompanied by a marked decrease in glucose incorporation into triacylglycerols. All the variations observed "in vivo" and "in vitro" in mammary gland returned almost to normal values by refeeding the starved lactating rats.

Adipose Tissue↗
PubMed1984

Fatty acyl-CoAs as feedback regulators of hexose monophosphate shunt in rat adipocytes.

The high basal glucose utilization through hexose monophosphate shunt found in our experimental conditions were almost completely inhibited by oleate, octanoate and caproate. However, the inhibition of glucose oxidation due to butyrate was about 50% whereas ketone bodies and acetate did not inhibit. The rate of triacylglycerol formation was not significantly modified with the above organic acids except oleate that presented a 5-fold increase on labeling incorporation into lipids. Oleate inhibition of glucose oxidation was completely prevented by the NADPH oxidant menadione. There was no inhibition by octanoate, caproate, butyrate or ketone bodies of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase or malic enzyme in adipose tissue homogenates. In contrast, specifically glucose-6-phosphate dehydrogenase was inhibited by oleoyl-CoA. The oleoyl-CoA inhibition was prevented by enzyme preincubation with low NADP concentration. The data lend further support for the hypothesis that fatty acids and NADP fulfill an important role in the modulation of the hexose monophosphate shunt activity.

Acyl Coenzyme A↗
PubMed1983

Coordination of glucose metabolism and NADPH formation in the adipose tissue and mammary gland during the lactation-weaning transition.

The capacity of fat cells from 48-hour weaned mid-lactating rats to synthesize fatty acids from (1-14C)-glucose and (6-14C)-glucose was markedly increased compared to adipocytes isolated from mid-lactating rats kept with their pups. Pentose shunt activity and glycolytic flux were also markedly increased in fat cells from weaned mid-lactating rats. Isolated fat cells from mid-lactating weaned rats, incubated with oleate and substrates that potentially may supply triosephosphate, showed a significantly stimulated triglyceride synthesis as measured by (1-14C)-oleate incorporation into the acyl moiety. The specific activities of malic enzyme, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were measured in adipose tissue and mammary gland extracts during pregnancy, lactation and weaning. These enzyme activities in the mammary gland increased in parallel from the beginning of pregnancy to the end of lactation. After weaning there was a sharp decrease in enzyme activities to levels similar to those found in mammary glands from virgin rats. In contrast, the activities of the lipogenic enzymes in adipose tissue were very low during lactation but increased rapidly after weaning.

Adipose Tissue↗