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M L Toews

Publications and source records attributed to M L Toews.

At least 37 records · Page 2Linked to original sources
PubMed1993

Agonist-induced desensitization of beta-adrenoceptors of bovine bronchial epithelial cells.

1. Cultured bovine bronchial epithelial cells express a large number of beta-adrenoceptors of the beta 2 subtype which are coupled to activation of adenylate cyclase and synthesis of cyclic AMP. The mechanisms involved in agonist-induced desensitization of beta-adrenoceptor function in these cells were investigated. 2. Preincubation of cells with the beta-adrenoceptor agonist isoprenaline induced a rapid desensitization of adenylate cyclase activity stimulated by isoprenaline (40% at 30 min) with little or no decrease in forskolin stimulation or in the number of beta-adrenoceptors detected by radioligand binding, indicating that uncoupling of beta-adrenoceptors from adenylate cyclase had occurred. 3. Uncoupling of beta-adrenoceptors from the stimulatory guanine-nucleotide-binding protein Gs was indicated by the loss of GTP effects on agonist competition for radioligand binding. Results from subcellular fractionation by sucrose density gradient centrifugation suggest that rapid internalization or sequestration of beta-adrenoceptors occurred along with the desensitization. 4. Preincubation with isoprenaline for longer times led to a marked decrease in total receptor number (85% at 18 h), indicating that receptor down-regulation also occurs. Recovery of receptors after down-regulation was prevented by cycloheximide and by actinomycin D, suggesting that degradation of both receptor protein and receptor mRNA may be involved in down-regulation.

Adenylyl Cyclases↗
PubMed1993

Lysophosphatidic acid mimics serum-induced sensitization of cyclic AMP accumulation.

Pretreatment of 1321N1 human astrocytoma cells with serum induces a pronounced increase in subsequent stimulation by forskolin and other agents of intracellular cyclic AMP accumulation, a phenomenon referred to as sensitization (Mol. Pharmacol. 39, 399-406, 1991). Pretreatment of these cells with lysophosphatidic acid induced sensitization to a similar extent as that with serum (approximately fivefold for forskolin stimulation and twofold for isoproterenol and prostaglandin E1 stimulation), with half-maximal effects at approximately 30 nM lysophosphatidic acid. Phosphatidic acid was effective but less potent whereas other lipids were ineffective. Sensitization by serum and by lysophosphatidic acid were almost completely inhibited by pertussis toxin pretreatment and partially inhibited by prolonged phorbol ester exposure to induce protein kinase C down-regulation. Among nine cell lines tested, those that exhibited sensitization with serum showed comparable sensitization with lysophosphatidic acid. The effects of both lysophosphatidic acid and serum were markedly inhibited by treatment with phospholipase B but only minimally altered with phospholipases A2, D, and C. Exposure of cells to phospholipase C alone induced approximately threefold sensitization, but both serum and lysophosphatidic acid were able to induce further three- to fourfold sensitization above that induced by phospholipase C alone. In contrast, the effects of serum and lysophosphatidic acid were not additive with each other. Together these results suggest that lysophosphatidic acid or a closely related compound present in serum is the factor responsible for sensitization of the cyclic AMP pathway.

Alprostadil↗
PubMed1993

Radioligand binding methods: practical guide and tips.

Radioligand binding assays are a relatively simple but extremely powerful tool for studying receptors. They allow an analysis of the interactions of hormones, neurotransmitters, growth factors, and related drugs with the receptors, studies of receptor interactions with second messenger systems, and characterization of regulatory changes in receptor number, subcellular distribution, and physiological function. As a result, these assays are widely used (and often misused) by investigators in a variety of disciplines, including pharmacology, physiology, biochemistry, immunology, and cell biology. This article presents a broad overview of the radioligand binding assay technique, primarily for the investigator who has limited experience with this technique. Practical guidelines for setting up a new assay are presented, including the receptor preparation to be used, choice of appropriate radioligand, optimizing assay conditions, and appropriate methods for data analysis. Tips for avoiding some of the common pitfalls in application of these assays are also included. The primary focus is on radioligand binding assays of membrane-bound receptors studied in membrane preparations. However, similar assay techniques can be used to study receptors on intact cells. The unique advantages and disadvantages of these intact cell binding assays are also discussed. In particular, the occurrence of regulatory changes in receptors during the course of intact cell binding assays is considered, with approaches for circumventing these complications and for using intact cell assays to advantage in studying these regulatory changes.

Animals↗
PubMed1993

Bicarbonate induces sensitization of cyclic AMP accumulation by intact 1321N1 human astrocytoma cells.

Preincubation of 1321N1 human astrocytoma cells in medium buffered with bicarbonate induced 50 to 100% increases in subsequent stimulation of cyclic AMP accumulation by the beta adrenergic receptor agonist isoproterenol and by the direct adenylyl cyclase activator forskolin, compared to cells incubated in buffered medium without bicarbonate. This "bicarbonate-induced sensitization" of cyclic AMP accumulation occurred rapidly, was rapidly reversible and was bicarbonate concentration-dependent. Although protein kinase C activation also induces sensitization in these cells, sensitization by bicarbonate does not appear to involve protein kinase C, because neither protein kinase C down-regulation nor the kinase inhibitor staurosporine prevented sensitization. Pertussis toxin neither mimicked nor prevented the sensitization by bicarbonate, suggesting that pertussis toxin-sensitive guanine nucleotide-binding proteins are not involved. The effect of bicarbonate appears to be on synthesis of cyclic AMP rather than degradation, inasmuch as the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine did not prevent expression of sensitization and because cyclic AMP degradation rates were not altered after bicarbonate incubation. However, increased adenylyl cyclase activity was not retained in broken cell preparations from cells incubated with bicarbonate. These results suggest the occurrence of a novel mechanism through which bicarbonate exposure can regulate adenylyl cyclase activity in intact cells.

1-Methyl-3-isobutylxanthine↗
PubMed1993

Effects of antimycin A on the binding properties of beta and alpha-1 adrenergic receptors measured on intact cells.

Depletion of intracellular ATP with antimycin A has been shown to inhibit the internalization of several types of receptors, including beta adrenergic and muscarinic acetylcholine receptors. The effects of antimycin A treatment on conversion of beta and alpha-1 adrenergic receptors to a form exhibiting low apparent affinity for agonists in assays with intact cells were investigated, because conversion to the low-affinity form has been postulated to result from receptor internalization. Treatment of DDT1 MF-2 hamster smooth muscle cells with antimycin A greatly decreased conversion of beta adrenergic receptors to the form exhibiting low affinity for agonists. Binding of antagonists to intact cell beta adrenergic receptors was not altered by antimycin A. In contrast to the results with beta adrenergic receptors, conversion of alpha-1 adrenergic receptors to the low-affinity form was not inhibited by antimycin A. These results are consistent with the possible involvement of receptor internalization in conversion to the low affinity form for beta adrenergic receptors. They also provide further evidence that different mechanisms may be involved in conversion to the low-affinity form or in the internalization pathway in the case of alpha-1 adrenergic receptors.

Adenosine Triphosphate↗
PubMed1992

Neuropeptide Y inhibits forskolin-stimulated adenylate cyclase in bovine adrenal chromaffin cells via a pertussis toxin-sensitive process.

The effect of neuropeptide Y (NPY) on adenylate cyclase activity and the role of G-proteins mediating NPY's effect were investigated in cultured bovine adrenal chromaffin cells. The equilibrium binding of [125I]NPY to sucrose gradient purified bovine adrenal medulla plasma membranes revealed high- (GTP gamma S sensitive) and low-affinity binding sites with calculated IC50 values of 0.27 nM and 0.14 microM, respectively. Inhibition of forskolin-stimulated cyclic AMP accumulation was dependent upon the NPY concentration (IC50 = 0.9 nM) and independent of cyclic AMP (cAMP) phosphodiesterase activity. NPY-related peptides, except peptide YY, and NPY fragments exhibited minimal inhibitory activity. The inhibitory effect of NPY on forskolin-stimulated adenylate cyclase activity was completely abolished by pretreatment of the cells with pertussis toxin (PTX). Incubation of membranes with PTX and [32P]nicotinamide adenine dinucleotide revealed a protein band with an apparent molecular mass of 41 kDa. The time course and dose dependence of PTX pretreatment for in vitro ADP-ribosylation were similar to those for PTX to attenuate the NPY effect on forskolin-stimulated adenylate cyclase activity. The direct relation between the NPY receptor and the PTX-sensitive G-protein was further shown by the ability of NPY to inhibit PTX-catalyzed in vitro ADP-ribosylation. ADP-ribosylation of the 41-kDa protein was partially inhibited by 5'-guanylylimidodiphosphate and further inhibited by high concentrations of NPY. An antibody against Gi1/i2 alpha 1 recognized two species of which a 41-kDa protein comigrated with the PTX substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate Ribose↗
PubMed1992

Serum-induced sensitization of cyclic AMP accumulation in C62B rat glioma cells.

Exposure of C62B rat glioma cells to fresh medium containing fetal bovine serum induced a sensitization of the subsequent ability of isoproterenol and forskolin to stimulate cyclic AMP accumulation, compared to cells exposed to fresh medium without serum. Isoproterenol stimulation was typically increased by 2- to 4-fold and forskolin stimulation by 3- to 5-fold. Sensitization occurred rapidly, was rapidly reversible and appeared to result from an increase in maximal stimulation. A commercial preparation of albumin, purified chromatographically so as to retain bound lipids and other factors, was able to mimic the effect of serum. In contrast to the effects of serum, exposure of cells to phorbol 12-myristate, 13-acetate induced little or no change in forskolin stimulation but a marked desensitization of isoproterenol stimulation that was due primarily to a decrease in potency. Neither the protein kinase C inhibitor staurosporine or overnight exposure to phorbol 12-myristate, 13-acetate to down-regulate protein kinase C prevented serum-induced sensitization. Pertussis toxin almost completely blocked serum-induced sensitization, suggesting involvement of a pertussis toxin-sensitive guanine nucleotide-binding protein in mediating the effects of serum. Sensitization was poorly retained in membrane adenylate cyclase assays. Studies with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, direct assays of cyclic AMP degradation by intact cells and assays of phosphodiesterase activity in cell lysates all indicated that degradation of cyclic AMP was decreased in serum-pretreated cells. Thus, both increased cyclic AMP synthesis and decreased cyclic AMP degradation may contribute to sensitization in these cells.

1-Methyl-3-isobutylxanthine↗
PubMed1991

Serum-induced sensitization of cyclic AMP accumulation in 1321N1 human astrocytoma cells.

Exposure of 1321N1 human astrocytoma cells to fresh medium containing fetal bovine serum induced a marked increase in the subsequent ability of isoproterenol and forskolin to stimulate cAMP accumulation in intact cells, compared with cells exposed to fresh medium without serum. This "sensitization" of cAMP accumulation by serum was dose dependent, occurred rapidly, was maintained in the continuing presence of serum, and reversed rapidly upon removal of serum. Preliminary characterization of the sensitizing factor(s) in serum has been performed, but the factor(s) remain to be identified. Sensitization appeared to result from an increase in maximal response and not from changes in the potency of isoproterenol or forskolin. The protein kinase C inhibitor staurosporine inhibited serum-induced sensitization. Furthermore, down-regulation of protein kinase C almost completely eliminated the subsequent ability of serum to induce sensitization, indicating involvement of protein kinase C in the serum effect. Pretreatment of cells with pertussis toxin also markedly reduced subsequent sensitization induced by serum, suggesting involvement of a pertussis toxin-sensitive guanine nucleotide-binding protein in the pathway for serum-induced sensitization. The rate of cAMP degradation was not changed in sensitized cells, but some increase in adenylyl cyclase activity was retained in broken cell preparations from sensitized cells, suggesting increased synthesis of cAMP by adenylyl cyclase as the mechanism for sensitization.

1-Methyl-3-isobutylxanthine↗
PubMed1990

Regulation of histamine H1 receptor-mediated phosphoinositide hydrolysis by histamine and phorbol esters in DDT1 MF-2 cells.

The regulation of histamine-stimulated phosphoinositide turnover by histamine and phorbol esters was examined in intact DDT1 MF-2 cells grown in suspension culture. Histamine increased the incorporation of 32P into phosphatidylinositol (PI) in these cells, and this stimulation was inhibited by the H1 antagonist diphenhydramine but not by the H2 antagonist cimetidine. Pretreatment of cells with histamine or with phorbol 12-myristate 13-acetate (PMA) or other activators of protein kinase C induced a marked decrease in the subsequent stimulation by histamine. PMA, but not histamine, also decreased the ability of epinephrine to stimulate PI labelling through alpha 1-adrenoceptors. Thus, histamine appears to induce homologous desensitization of histamine H1 receptor-mediated PI turnover, whereas direct activation of protein kinase C in the absence of receptor occupancy by agonist induces nonspecific heterologous desensitization of both histamine H1- and alpha 1-adrenoceptor-mediated responses.

Animals↗
PubMed1990

Protein kinase C activators sensitize cyclic AMP accumulation by intact 1321N1 human astrocytoma cells.

Pretreatment of 1321N1 human astrocytoma cells with phorbol 12-myristate-13-acetate or other activators of protein kinase C led to 2.5- to 5-fold increases (sensitization) in subsequent stimulation by forskolin of intracellular cyclic AMP accumulation. These compounds caused much smaller or no increases in receptor-mediated stimulation of cyclic AMP accumulation induced by isoproterenol and by prostaglandin E1. Carbachol and histamine, agonists acting at receptors coupled to polyphosphoinositide turnover in these cells, induced less sensitization of subsequent stimulation by forskolin but greater sensitization of stimulation by isoproterenol and by prostaglandin E1. The specificities of various analogs of phorbol 12-myristate-13-acetate, for induction of sensitization of forskolin stimulation were consistent with involvement of protein kinase C. The effects of protein kinase inhibitors and of down-regulation of protein kinase C activity also indicated involvement of protein kinase C in sensitization of forskolin stimulation, although additional mechanisms are likely to be involved in sensitization of isoproterenol stimulation. Neither pertussis toxin pretreatment nor inclusion of isobutylmethylxanthine during assays of cyclic AMP accumulation were able to prevent or mimic these sensitization phenomena, suggesting that the primary site of modification responsible for sensitization is neither the inhibitory guanine nucleotide-binding protein nor cyclic AMP phosphodiesterase. Sensitization was only observed in assays with intact cells. These results, together with those from our previous study describing protein kinase C-mediated desensitization of broken cell adenylate cyclase activity, indicate that activation of protein kinase C leads to multiple changes in the receptor-stimulated adenylate cyclase signal transduction pathway of these cells.

1-Methyl-3-isobutylxanthine↗
PubMed1990

Activation of protein kinase C inhibits internalization and downregulation of muscarinic receptors in 1321N1 human astrocytoma cells.

The effects of the protein kinase C activator phorbol 12-myristate, 13-acetate (PMA) on muscarinic receptor downregulation and internalization in 1321N1 human astrocytoma cells were determined. Downregulation was assessed by measuring [3H] quinuclidinyl benzilate binding to intact cells. PMA alone did not induce muscarinic receptor downregulation but instead decreased markedly both the rate and final extent of downregulation induced by the agonist carbachol. The specificity of various analogs for inhibiting carbachol-induced downregulation indicated involvement of protein kinase C. Furthermore the protein kinase C inhibitor staurosporine prevented the inhibitory effect of PMA on downregulation. In contrast, staurosporine did not inhibit agonist-induced downregulation. Neither agonist-induced downregulation nor the inhibitory effect of PMA were blocked by cycloheximide, indicating that protein synthesis is not required for these effects. Muscarinic receptor internalization was assessed both by sucrose density gradient centrifugation assays of receptor subcellular distribution and by measuring binding of the hydrophilic radioligand N-[3H]methylscopolamine to intact cells at reduced temperature. PMA did not induce muscarinic receptor internalization but rather inhibited internalization induced by the agonist carbachol. Together these results suggest that activation of protein kinase C leads to inhibition of an agonist-induced increase in the rates of muscarinic receptor internalization and degradation that are presumably responsible for receptor redistribution and eventual downregulation.

Astrocytoma↗
PubMed1989

Evidence for an agonist-induced, ATP-dependent change in muscarinic receptors of intact 1321N1 cells.

The binding of muscarinic agonists, partial agonists and antagonists to muscarinic receptors of intact 1321N1 human astrocytoma cells and of cell lysates was studied. Partial agonists and antagonists exhibited similar apparent affinities in intact cell competition binding assays with either the lipophilic radioligand [3H]quinuclidinyl benzilate [( 3H]QNB) or the hydrophilic radioligand [3H]N-methyl scopolamine [( 3H]NMS). In contrast, full agonists exhibited markedly lower apparent affinities in intact cell competition binding assays with [3H]QNB than with [3H]NMS. The affinities of agonists and antagonists in competition for [3H] NMS binding to intact cells were slightly higher than those observed in competitions for [3H]QNB binding in cell lysates. In contrast, the affinities of agonists in competition for [3H]QNB binding to intact cells were considerably lower than those in competition for [3H]QNB binding in cell lysates. Treatment of cells with antimycin A to deplete intracellular ATP prevented agonist-induced internalization of muscarinic receptors as assessed by sucrose density gradient assays of receptor subcellular distribution. In ATP-depleted cells, the apparent affinities of full agonists vs. [3H]QNB were markedly higher and were similar to their apparent affinities vs. [3H]QNB in cell lysates. The apparent affinities of partial agonists and of antagonists were unaffected by ATP depletion. ATP depletion decreased slightly the apparent affinity of the full agonist carbachol in competition for [3H]NMS binding to intact cells, whereas the apparent affinity of atropine was unchanged. These results provide evidence for an agonist-specific, ATP-dependent low affinity state of intact cell muscarinic receptors that may be similar to those observed previously for both beta and alpha-1 adrenergic receptors and that may be related to receptor internalization/sequestration.

Adenosine Triphosphate↗
PubMed1988

Vasoactive intestinal peptide receptor/adenylate cyclase system: differences between agonist- and protein kinase C-mediated desensitization and further evidence for receptor internalization.

In this study we have characterized and compared the regulation of the HT29 cell vasoactive intestinal peptide receptor/adenylate cyclase system (VIP-R/AC) by the VIP-R agonist peptide histidineisoleucineamide (PHI) and by activators of protein kinase C (PKC) including phorbol 12-myristate, 13-acetate (PMA) and mezerein. Preincubation with either PHI or PKC activator decreased maximum VIP-stimulated AC activity and decreased the number of cell surface VIP-R. A [125I]VIP binding assay using solubilized VIP-R of the plasma membrane and light vesicle fractions from sucrose density step gradients was developed as a more direct measure of VIP-R internalization. Preincubation with PHI or PMA decreased plasma membrane fraction [125I]VIP binding and increased binding in the light vesicle fraction, thus providing the most direct evidence to date for translocation of VIP-R per se from the plasma membrane to another, presumably intracellular, compartment. Two experimental approaches differentiated between agonist and PKC activator regulation of VIP-R/AC. The protein kinase inhibitors H-7 and staurosporine blocked mezerein-, but not PHI-, induced losses of cell surface VIP-R. Also, down-regulation of PKC did not block PHI-induced loss of cell surface VIP-R. Thus, although both agonist and PKC activators can lead to desensitization and internalization of VIP-R, PKC is apparently not involved in the mechanisms of agonist-induced desensitization.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1988

Evidence for alpha 1-adrenergic receptor internalization in DDT1 MF-2 cells following exposure to agonists plus protein kinase C activators.

Agonist-induced sequestration and internalization of alpha 1-adrenergic receptors were examined in DDT1 MF-2 cells. Pretreatment of cells with epinephrine or norepinephrine alone, but not with phorbol 12-myristate 13-acetate alone, resulted in a marked decrease in [3H]prazosin binding to intact cells at 4 degrees. These pretreatments resulted in little or no change in the fraction of alpha 1-adrenergic receptors exhibiting limited accessibility to the hydrophilic competing ligand epinephrine in short-time competition binding assays with intact cells and little or no change in the subcellular distribution of alpha 1-adrenergic receptors between the plasma membrane and light vesicle compartments as assessed by sucrose density gradient centrifugation assays. Pretreatment with a combination of agonist plus phorbol 12-myristate 13-acetate resulted in a greater decrease in [3H]prazosin binding at 4 degrees than was observed when cells were pretreated with agonist alone, induced the conversion of about half of cell surface alpha 1-adrenergic receptors to a form exhibiting limited accessibility to epinephrine in short-time assays, and induced a shift of about half of alpha 1-adrenergic receptors from the plasma membrane fraction to a light vesicle fraction on sucrose density gradients. A similar shift of alpha 1-adrenergic receptors was observed on sucrose density gradients after exposure to agonist plus either mezerein or beta-phorbol didecanoate, but not with agonist plus alpha-phorbol didecanoate, indicating involvement of protein kinase C. These results suggest that pretreatment with agonist alone induces the sequestration of alpha 1-adrenergic receptors into a compartment that is inaccessible to [3H]prazosin at 4 degrees but that is accessible to hydrophilic ligands at 37 degrees and remains associated with the plasma membrane. In contrast, alpha 1-adrenergic receptors are apparently internalized from the plasma membrane to a separate compartment, presumably an intracellular vesicle, when cells are pretreated simultaneously with a combination of agonist plus a protein kinase C activator.

Adrenergic alpha-Agonists↗
PubMed1987

Alpha 2-adrenergic receptor-mediated sensitization of forskolin-stimulated cyclic AMP production.

Preincubation of HT29 human colonic adenocarcinoma cells with alpha 2-adrenergic agonists resulted in a 10- to 20-fold increase in forskolin-stimulated cyclic AMP production as compared to cells preincubated without agonist. Similar results were obtained using either a [3H]adenine prelabeling assay or a cyclic AMP radioimmunoassay to measure cyclic AMP levels. This phenomenon, which is termed sensitization, is alpha 2-adrenergic receptor-mediated and rapid in onset and reversal. Yohimbine, an alpha 2-adrenergic receptor-selective antagonist, blocked norepinephrine-induced sensitization, whereas prazosin (alpha 1-adrenergic) and sotalol (beta-adrenergic) did not. The time for half-maximal sensitization was 5 min and the half-time for reversal was 10 min. Only a 2-fold sensitization of cyclic AMP production stimulated by vasoactive intestinal peptide was observed, indicating that sensitization is relatively selective for forskolin. Sensitization reflects an increased production of cyclic AMP and not a decreased degradation of cyclic AMP, since incubation with a phosphodiesterase inhibitor and forskolin did not mimic sensitization. Increasing the levels of cyclic AMP during the preincubation (using a phosphodiesterase inhibitor) had no effect on sensitization, indicating that sensitization is not caused by decreased cyclic AMP levels during the preincubation. This rapid and dramatic sensitization of forskolin-stimulated cyclic AMP production is a previously unreported effect that can be added to the growing list of alpha 2-adrenergic responses that are not mediated by a decrease in cyclic AMP.

Adenine↗
PubMed1987

Effects of agonist and phorbol ester on adrenergic receptors of DDT1 MF-2 cells.

The effects of the adrenergic agonist epinephrine (EPI) and of phorbol 12-myristate 13-acetate (PMA) on the regulation of alpha-1 adrenergic receptors (AAR) and beta adrenergic receptors (BAR) were compared in DDT1 MF-2 cells grown in suspension culture. Pretreatment of cells with 10 microM EPI for 30 min at 37 degrees C resulted in homologous desensitization of BAR-coupled adenylate cyclase activity assayed in membranes and induced internalization or sequestration of BAR. Pretreatment of cells with PMA did not alter BAR-coupled adenylate cyclase activity or induce internalization of BAR. EPI pretreatment caused a 50% decrease in the subsequent ability of EPI to stimulate AAR-mediated incorporation of 32P into phosphatidylinositol, whereas PMA pretreatment inhibited incorporation by 95%. Neither EPI nor PMA induced the internalization of AAR. Neither EPI nor PMA altered agonist binding properties of AAR in short-time competition binding assays on intact cells, indicating that pretreatment of cells with these agents does not alter the affinity of AAR for agonist. In control cells, agonists converted AAR from a form exhibiting predominantly high affinity for agonists, detected in short-time assays, to a form, exhibiting low apparent affinity for agonist during the course of equilibrium competition binding assays. PMA pretreatment increased the extent of this subsequent agonist-induced conversion to the low affinity form. These results indicate that PMA can mimic agonist-induced desensitization of AAR, but not BAR, and that the desensitization of AAR-coupled phosphatidylinositol turnover induced by EPI and by PMA is not due to altered receptor affinity for EPI or due to receptor internalization.

Adenylyl Cyclases↗
PubMed1987

Comparison of agonist-induced changes in beta- and alpha 1-adrenergic receptors of DDT1 MF-2 cells.

Agonist-induced changes in beta- and alpha 1-adrenergic receptors (BARs and AARs) were compared in the DDT1 MF-2 smooth muscle cell line. During equilibrium competition binding assays with intact cells at 37 degrees, agonists induced conversion of both BARs and AARs from a native form with high affinity for agonists to a form with much lower affinity for agonists. The native high affinity form of both receptors could be detected either in short-time competition binding assays at 37 degrees or in equilibrium competition binding assays on ice. Conversion to the low affinity form was nearly complete for BARs, but only about half of the AARs were converted to the low affinity form. For BARs, the high affinity form of the receptor observed in short-time assays with intact cells was similar to that observed in membrane preparations, whereas for AARs this form exhibited much higher affinity than was seen in membrane assays. None of these changes were observed during competition binding assays with antagonists. Both short-time competition binding assays with hydrophilic competing ligands and sucrose density gradient centrifugation assays were consistent with the occurrence of agonist-induced internalization of BARs. These same assays for AARs were consistent with the presence of some AARs in an intracellular compartment in the native state, but no agonist-induced increases in intracellular AARs were detected. During more prolonged exposure (13 hr) to agonists, about 80% down-regulation of BARs occurred, whereas only about 20% down-regulation of AARs was detected. These results may indicate that internalization and down-regulation are not involved in conversion of these receptors to the low affinity form observed in intact cell binding assays.

Adrenergic alpha-Agonists↗