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S Cotecchia

Publications and source records attributed to S Cotecchia.

At least 73 records · Page 4Linked to original sources
PubMed1988

Molecular cloning and expression of the cDNA for the hamster alpha 1-adrenergic receptor.

The cDNA for the Syrian hamster alpha 1-adrenergic receptor has been cloned with oligonucleotides corresponding to the partial amino acid sequence of the receptor protein purified from DDT1MF-2 smooth muscle cells. The deduced amino acid sequence encodes a 515-residue polypeptide that shows the most sequence identity with the other adrenergic receptors and the putative protein product of the related clone G-21. Similarities with the muscarinic cholinergic receptors are also evident. Expression studies in COS-7 cells confirm that we have cloned the alpha 1-adrenergic receptor that couples to inositol phospholipid metabolism.

Animals↗
PubMed1987

Agonist-dependent phosphorylation of the alpha 2-adrenergic receptor by the beta-adrenergic receptor kinase.

Desensitization of the beta-adrenergic receptor, a receptor which is coupled to the stimulation of adenylate cyclase, may be regulated via phosphorylation by a unique protein kinase. This recently discovered enzyme, known as the beta-adrenergic receptor kinase, only phosphorylates the agonist-occupied form of the beta-adrenergic receptor. To assess whether receptors coupled to the inhibition of adenylate cyclase might also be substrates, we examined the effects of beta-adrenergic receptor kinase on the partially purified human platelet alpha 2-adrenergic receptor. Phosphorylation of the reconstituted alpha 2-adrenergic receptor was dependent on agonist occupancy and was completely blocked by coincubation with alpha 2-antagonists. The time course of phosphorylation of the alpha 2-adrenergic receptor was virtually identical to that observed with the beta-adrenergic receptor with maximum stoichiometries of 7-8 mol of phosphate/mol of receptor in each case. In contrast, the alpha 1-adrenergic receptor, which is coupled to stimulation of phosphatidylinositol hydrolysis, is not a substrate for the beta-adrenergic receptor kinase. These results suggest that receptors coupled to either stimulation or inhibition of adenylate cyclase may be regulated by an agonist-dependent phosphorylation mediated by the beta-adrenergic receptor kinase.

Affinity Labels↗
PubMed1987

Regulation of adrenergic receptor function by phosphorylation. I. Agonist-promoted desensitization and phosphorylation of alpha 1-adrenergic receptors coupled to inositol phospholipid metabolism in DDT1 MF-2 smooth muscle cells.

Continuous exposure of DDT1 MF-2 smooth muscle cells to 10-100 microM norepinephrine results in a dramatic attenuation of the ability of norepinephrine to stimulate inositol phospholipid hydrolysis via alpha 1-adrenergic receptors (alpha 1-AR). In addition to the functional desensitization, norepinephrine exposure also reduces the number of accessible cell surface alpha 1-AR as assayed by [3H]prazosin binding at 4 degrees C. Desensitization of the cells with norepinephrine results in an increase in the phosphorylation of the Mr 80,000 alpha 1-AR ligand binding peptide (2.4 +/- 0.2 mol of 32P per mol of alpha 1-AR; n = 5) when compared to control cells (1.1 +/- 0.1 mol of 32P per mol of alpha 1-AR; n = 5). The time courses of these three processes are all comparable being half-maximal within 1-2 min. These norepinephrine-promoted effects can be prevented by the alpha 1-AR receptor antagonist phentolamine indicating that they are mediated via the alpha 1-AR. Treatment of cells with the vasoactive peptide bradykinin (10 microM) induces desensitization of alpha 1-AR function similar to that induced by tumor-promoting phorbol ester treatment (Leeb-Lundberg, L. M. F., Cotecchia, S., Lomasney, J. W., DeBernardis, J. F., Lefkowitz, R. J., and Caron, M. G. (1985) Proc. Natl. Acad. Sci. USA 82, 5651-5655). Both treatments also result in phosphorylation of the alpha 1-AR, with stoichiometries of 1.7 +/- 0.1 (bradykinin; n = 5) and 3.6 +/- 0.1 (PMA; n = 5) mol of 32P/mol of alpha 1-AR. However, neither phorbol esters nor bradykinin reduce the number of accessible cell surface alpha 1-AR. Similar phosphopeptide maps are obtained from tryptic phosphopeptides generated from phosphorylated alpha 1-AR derived from cells treated with norepinephrine, phorbol 12-myristate 13-acetate, and bradykinin. Phosphoamino acid analysis reveals that the various agents induce phosphorylation on both serine and threonine residues. Thus, phosphorylation of receptors linked to the inositol phospholipid/Ca2+ signaling pathway may represent an important mechanism of regulation of receptor responsiveness.

Animals↗
PubMed1986

Mammalian alpha 1-adrenergic receptor. Purification and characterization of the native receptor ligand binding subunit.

alpha 1-Adrenergic receptors from a cultured smooth muscle cell line (DDT1 MF-2) have been solubilized with digitonin and purified to apparent homogeneity by sequential chromatography on a biospecific affinity support (Sepharose-A55453 (4-amino-6,7-dimethoxy-2-[4-[5-(4-amino-3-phenyl) pentanoyl]-1-piperazinyl]-quinazoline), an alpha 1 receptor-selective antagonist), a wheat germ agglutinin-agarose gel, and a high performance steric exclusion liquid chromatography column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of iodinated purified receptor preparations reveals a peptide with an apparent Mr = 80,000 that co-migrates with the peptide labeled by the specific alpha 1-adrenergic receptor photoaffinity probe 4-amino-6,7-dimethoxy-2-[4-[5-(4-azido-3-[125I]iodophenyl)pentanoyl] -1-piperazinyl] quinazoline. The specific activity (approximately 13,600 pmol of ligand binding/mg of protein) of purified receptor preparations is consistent with that expected for a pure peptide of Mr = 80,000 containing a single ligand binding site. Overall yields approximate 14% of initial crude particulate binding. The purified receptor preparations bind agonist and antagonist ligands with appropriate alpha 1-adrenergic specificity, stereoselectivity, and affinity. Peptide maps of the pure alpha 1-adrenergic receptor and the pure human platelet alpha 2-adrenergic receptor (Regan, J.W., Nakata, H., DeMarinis, R.M., Caron, M.G., and Lefkowitz, R.J. (1986) J. Biol. Chem. 261, 3894-3900) using several different proteases suggest that these two receptors show little if any structural homology.

Animals↗
PubMed1986

Norepinephrine decreases EGF binding in primary rat hepatocyte cultures.

Norepinephrine (NE) produced a dose-dependent inhibition of 125I-epidermal growth factor (EGF) binding to adult rat hepatocytes in primary culture. This effect was maximal after 1 hr of incubation with NE and could be blocked by the presence of an alpha 1-specific adrenergic receptor antagonist. The inhibition of binding correlates with the ability of NE to enhance hepatocyte DNA synthesis in the presence of EGF and appears to be mediated by a reduction in EGF receptor number, without a significant change in receptor affinity.

Animals↗
PubMed1986

Stress induced desensitization of lymphocyte beta-adrenoceptors in young and aged rats.

The effects of different times of immobilization stress on intact lymphocyte beta-adrenoceptors and plasma corticosterone were compared in 3-month and 24-month-old rats. In young animals after 30 min restraint 3H-dihydroalprenolol specific binding was significantly reduced (61% of control value) and plasma corticosterone significantly raised (186% of control). The effect on beta-adrenoceptors was due changes in receptor number (Bmax) without any effect on affinity (KD). In aged rats both effects were only seen after 180 min restraint and were less pronounced. Isoproterenol treatment in vitro reduced beta-adrenoceptors on lymphocytes. This effect was less pronounced in lymphocytes from aged rats. Corticosterone in vitro increased 3H-dihydroalprenolol specific binding. We therefore suggest that the decrease of beta-adrenoceptors reflects an adaptive response to the stress-induced catecholamine release and that corticosterone could play a role in reversing this effect. This adaptive response to stress seems to be impaired in aged animals.

Age Factors↗
PubMed1986

Agonist-induced beta-adrenergic receptor internalization on intact human mononuclear leukocytes: effect of temperature of mononuclear leukocyte separation.

The hydrophilic ligand 3H-CGP 12177 was used to measure beta-adrenergic receptors on intact human mononuclear leukocytes (MNLs). A single homogeneous class of receptor sites was found, with KD value of 0.71 +/- 0.04 nmol/L and Bmax of 3.0 +/- 0.4 fmol/10(6) cells (mean +/- SEM; n = 12). The receptor affinity (KD) and density (Bmax) were similar when measured on MNLs, purified lymphocytes, and a T-lymphocyte-enriched population from the same individual. Preincubation of intact MNLs with 1 mumol/L isoproterenol at 37 degrees C for 20 minutes reduced the number of surface receptors, measured by 3H-CGP 12177 binding at 4 degrees C for 20 hours, by approximately 70% (receptor internalization) without affecting KD. This effect was reversible, and surface receptors completely reappeared when binding was investigated at 37 degrees C for 40 minutes. Receptor internalization was similar when either isolated MNLs or whole blood was incubated with isoproterenol. Agonist-induced receptor internalization was stable during MNL isolation from whole blood at 4 degrees C but was partially or completely lost from MNLs prepared at 20 degrees C.

Adrenergic beta-Agonists↗
PubMed1985

Phorbol ester effects on alpha 1-adrenoceptor binding and phosphatidylinositol metabolism in cultured vascular smooth muscle cells.

Tumor promoting phorbol esters stimulate Ca++ phospholipid-dependent protein kinase C. It has been suggested that this enzyme regulates the functional properties of different cell membrane receptors. In this study we investigated the effect of phorbol esters on alpha 1-adrenoceptor binding and phosphatidylinositol metabolism in cultured smooth muscle cells derived from rabbit aorta. Treatment of these cells with biologically active phorbol esters for 15 min. to 2 hours caused a marked decrease of norepinephrine stimulation of inositol phospholipid metabolism and a 3 fold decrease in agonist affinity for 125I-HEAT binding to alpha 1-adrenoceptors in the intact smooth muscle cells. The ability of phorbol esters to modulate alpha 1-adrenoceptor responsiveness suggests that activation of protein kinase C may represent an important mechanism regulating alpha 1-adrenergic receptor functional properties.

Animals↗
PubMed1985

Phorbol esters promote alpha 1-adrenergic receptor phosphorylation and receptor uncoupling from inositol phospholipid metabolism.

DDT1 MF-2 cells, which are derived from hamster vas deferens smooth muscle, contain alpha 1-adrenergic receptors (54,800 +/- 2700 sites per cell) that are coupled to stimulation of inositol phospholipid metabolism. Incubation of these cells with tumor-promoting phorbol esters, which stimulate calcium- and phospholipid-dependent protein kinase, leads to a marked attenuation of the ability of alpha 1-receptor agonists such as norepinephrine to stimulate the turnover of inositol phospholipids. This turnover was measured by determining the 32P content of phosphatidylinositol and phosphatidic acid after prelabeling of the cellular ATP pool with 32Pi. These phorbol ester-treated cells also displayed a decrease in binding affinity of cellular alpha 1 receptors for agonists with no change in antagonist affinity. By using affinity chromatography on the affinity resin Affi-Gel-A55414, the alpha 1 receptors were purified approximately equal to 300-fold from control and phorbol ester-treated 32Pi-prelabeled cells. As assessed by NaDodSO4/polyacrylamide gel electrophoresis, the Mr 80,000 alpha 1-receptor ligand-binding subunit is a phosphopeptide containing 1.2 mol of phosphate per mol of alpha 1 receptor. After phorbol ester treatment this increased to 3.6 mol of phosphate per mol of alpha 1 receptor. The effect of phorbol esters on norepinephrine-stimulated inositol phospholipid turnover and alpha 1-receptor phosphorylation showed the same rapid time course with a t1/2 less than 2 min. These results indicate that calcium- and phospholipid-dependent protein kinase may play an important role in regulating the function of receptors that are coupled to the inositol phospholipid cycle by phosphorylating and deactivating them.

Animals↗
PubMed1984

Glucocorticoids increase beta-adrenoceptors on human intact lymphocytes in vitro.

A modification of beta-adrenoceptor binding has been observed in different human and animal tissues after glucocorticoid administration. In this study we observed that hydrocortisone increased human intact lymphocyte beta-adrenoceptors labelled with 3H-dyhydroalprenolol in vitro, in physiological and pharmacological concentrations. In saturation experiments hydrocortisone (10(-6)M) significantly raised beta-adrenoceptor Bmax and KD. Intact living lymphocytes seem to be a useful model for further investigation in man of the regulation of beta-adrenoceptors by glucocorticoids in physiological conditions.

Dihydroalprenolol↗
PubMed1984

Drugs: guide and caveats to explanatory and descriptive approaches--II. Drugs in psychiatric research.

Over the last two decades animal models, mediators and drugs have produced a mass of experimental data difficult to relate to the epidemiological and clinical side of psychiatric disorders. Antidepressant drugs are chosen as a model case to describe a more general scene: far from being a specific tool--a guide--in a complex situation, these drugs are acquiring a role which must be interpreted with caution (a caveat), in view of ample clinical evidence of responders to placebo, responders to any drugs, responders to many drugs, etc. Studies of biochemical descriptors of drug action as a marker of the disease and its outcome have drawn puzzling pictures, often contradictory and unstable in terms of the populations to whom they can be applied. Controlled clinical trials with antidepressant drugs over the last ten years have persisted in looking for short-term pharmacological effects rather than the medium- or long-term impact of medication in large populations. To establish a positive role for antidepressant drugs, they must be studied in a "natural" context where depressed patients are treated with all necessary follow-up.

Animals↗
PubMed1982

Selective changes of receptor binding in brain regions of aged rats.

Binding to several receptors was compared in brain regions of 3 and 21-23 month-old rats. In crude membrane preparations of aged rats the number of dopamine antagonist receptors in striatum was much reduced (-53%). beta-Noradrenergic receptors (cortex) and benzodiazepine receptors (hippocampus and cerebellum) were less but significantly reduced and serotonergic receptors, alpha 1 noradrenergic receptors (both in cortex) and dopamine agonist receptors (striatum) were unchanged. For each receptor binding the KD values were the same in young and old animals. GABA receptor binding (hippocampus and cerebellum) evaluated at only one 3H-GABA concentration (8 nM) was similar in both groups when expressed per protein content but significantly reduced in aged rats when expressed per tissue wet weight because of the partial purification of the synaptic membranes used for 3H-GABA binding. In our experimental conditions age-related changes of specific binding sites in the central nervous system were selective for some receptors studied and did not seem to be due to general non-specific modification of brain tissue composition.

Adrenergic alpha-Antagonists↗
PubMed1982

Brain levels of tofizopam in the rat and relationship with benzodiazepine receptors.

The effect of tofizopam on 3H-flunitrazepam binding was studied in rat hippocampus and cerebellum. Tofizopam (at a concentration of 10(-7) M) increased 3H-Flu binding through a 30% rise in the Bmax with no modification of Kd in either brain area. Similar results were obtained when the binding was measured in tofizopam (50 mg/kg p.o.) pretreated rats. Even though tofizopam has no anticonvulsive action against pentetrazol-induced convulsions, it significantly potentiated the action of diazepam but with no modification of brain diazepam levels and metabolism. The brain levels of tofizopam are reported and compared to plasma levels after oral administration of 5 and 50 mg/kg to rats.

Animals↗
PubMed1982

d-Amphetamine-induced anorexia and motor behavior after chronic treatment in rats: relationship with changes in the number of catecholamine receptor sites in the brain.

No tolerance to the anorectic activity of d-amphetamine was found in rats given two daily injections of 1.25 mg/kg for 28 days. Such rats also presented no changes in the number of binding sites for serotonin and catecholamines in various brain areas. A 28-day treatment with 10 mg/kg d-amphetamine twice daily caused a significant reduction in the number of binding sites for 3H-dihydroalprenolol (beta adrenergic receptor ligand) in hippocampus, and 3H-spiroperidol (dopamine receptor ligand) in striatum and nucleus accumbens. This treatment did not modify the anorectic effect of 1.25 mg/kg of amphetamine but it markedly reduced the motor hyperactivity induced by this treatment and completely blocked the stereotyped licking and biting induced by apomorphine. Amphetamine stereotypy was not significantly reduced but disappeared faster in chronically treated rats. The possible reasons for the different results (tolerance or sensitization) reported by various authors on changes in amphetamine's effects on motor behaviour during chronic treatment are discussed.

Animals↗