Search PubMed⌕ Search

Biomedical subjects

P A Insel

Publications and source records attributed to P A Insel.

At least 127 records · Page 7Linked to original sources
PubMed1989

Defining the role of protein kinase C in epinephrine- and bradykinin-stimulated arachidonic acid metabolism in Madin-Darby canine kidney cells.

Madin-Darby canine kidney cells (MDCK) are known to release free arachidonic acid and arachidonic acid metabolites (AA) in response to tumor-promoting phorbol esters, such as tetradecanoyl phorbol-13-acetate, and to agonists active at alpha 1-adrenergic and bradykinin B2 receptors. These experiments were conducted to define the role of Ca2+/phospholipid-dependent protein kinase (protein kinase C) activation in the stimulation of AA release, in the clonal isolate cell line MDCK-D1, by use of three inhibitors of protein kinase C, sphingosine, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), and staurosporine. We found that alpha 1-adrenergic- and bradykinin-stimulated [3H]AA release can be distinguished by differential dependence on protein kinase C; epinephrine-stimulated release was more dependent on protein kinase C activation than was bradykinin-stimulated release. The inhibition of bradykinin-stimulated AA release by sphingosine (20.2 +/- 6.1%) was substantially less than the inhibition observed for tetradecanoyl phorbol-13-acetate- (67.2 +/- 5.5%) and epinephrine-stimulated release (50.2 +/- 9.2%). These findings were confirmed by results using H-7 and staurosporine. The relative independence of bradykinin-stimulated AA release of protein kinase C was also demonstrated by the inability of phorbol ester-induced down-regulation of protein kinase C to eliminate bradykinin-stimulated AA release. The inhibition of alpha 1-adrenergic receptor-mediated AA release by sphingosine, H-7, and staurosporine was not due to a change in receptor number or affinity. Analysis of the products comprising [3H]AA release indicated that treatment with sphingosine did not change the composition of the released AA (34-48% prostaglandin E2, 17-27% free arachidonic acid, and 25-51% unidentified metabolites). These results indicate that two different types of hormone receptors in the same cell type can promote AA release by mechanisms that differ in their dependence on protein kinase C. The protein kinase C-dependent mechanism may represent protein kinase C-mediated activation of phospholipase A2.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1989

[3H]idazoxan and some other alpha 2-adrenergic drugs also bind with high affinity to a nonadrenergic site.

We compared the pharmacological properties of the alpha 2-adrenergic radioligand [3H]idazoxan with those of [3H]rauwolscine in rat and [3H]yohimbine in human renal cortical membranes. The density of "specific" [3H]idazoxan binding sites (defined by 100 microM tolazoline) was twice as high as that of [3H]rauwolscine in rat kidney and four times as high as that of [3H]yohimbine in human kidney. A variety of structurally different drugs fully competed for specific [3H]rauwolscine and [3H]yohimbine binding, with affinities appropriate for the interaction with alpha 2-adrenergic receptors. Specific [3H]idazoxan binding, however, was only partially competed for by the catecholamines epinephrine and norepinephrine in both tissues. Thus, [3H]idazoxan labels both alpha 2-adrenergic receptors and a nonadrenergic site. Clonidine, B-HT 920, moxonidine, phentolamine, prazosin, yohimbine, dopamine, and serotonin also could not compete for this site. However, UK 14,304, guanabenz, indanidine, tolazoline, oxymetazoline, and SK&F 104,078 competed for the additional [3H]idazoxan sites with affinities similar to those at alpha 2-adrenergic receptors. [3H]idazoxan binding substantially in excess of [3H]rauwolscine or [3H]yohimbine binding was also found in human platelets, myometrium, and erythroleukemia (HEL) cells but not in three cell lines lacking alpha 2-receptors (MDCK, BC3H1, and Jurkat cells). Although we have been unsuccessful thus far in defining the precise nature of the additional [3H]idazoxan binding sites, we hypothesize that these sites may be closely affiliated with alpha 2-adrenergic receptors but clearly distinct from the catecholamine binding site of the receptor. The results indicate that care must be taken in the use of [3H]idazoxan or drugs that are recognized at its nonadrenergic site when studying alpha 2-adrenergic effects and receptor subtypes.

Adrenergic alpha-Antagonists↗
PubMed1989

Adrenergic receptors. Evolving concepts on structure and function.

During the past 15 years there has been a striking increase in the understanding of the molecular basis of cellular response to catecholamines. In addition to the two principal subtypes of beta-adrenergic receptors (beta 1 and beta 2), there are at least two (alpha 1, alpha 2) and very likely additional subtypes of alpha-adrenergic receptors. The discovery of guanine nucleotide binding (G) proteins as transducers of receptor occupancy to activation of second messenger systems provides a common theme in cellular regulation by catecholamines. Application of techniques such as radioligand binding and photoaffinity labeling have facilitated the direct identification, quantitation, and ultimately purification of alpha 1, alpha 2, beta 1, and beta 2 receptors. Each is a plasma membrane glycoprotein with a subunit molecular weight (without the carbohydrate portion of the glycoprotein) of 40,000 to 55,000 kDa. The recent cloning and sequencing of cDNAs for alpha 2-, beta 1-, and beta 2-adrenergic receptors has revealed that although each has a unique molecular structure, they appear to share several common features, including extracellular amino terminus, seven plasma membrane spanning domains, and intracellular carboxy terminus. The application of molecular biological techniques together with antireceptor antibodies, which will allow studies of adrenergic receptors independent of binding or functional properties, should help in answering the many unresolved questions related to activation and regulation of adrenergic receptors. Foremost among these is whether diseases such as hypertension are characterized by alterations in one or more adrenergic receptor subtypes.

Cell Membrane↗
PubMed1988

Subunit dissociation is the mechanism for hormonal activation of the Gs protein in native membranes.

We have recently reported (Ransnäs, L.A., and Insel, P.A. (1988) J. Biol. Chem. 263, 9482-9485) development of antipeptide antibodies to the alpha s protein of the stimulatory guanine nucleotide binding regulatory protein, Gs, and use of one of these antibodies, GS-1, to quantitate Gs levels in S49 lymphoma cell membranes. Another of these antibodies, termed GS-2, appears to detect only dissociated alpha s, but not the heterotrimer alpha s beta gamma. Using a competitive enzyme-linked immunosorbent assay, we have found that the guanine nucleotides GTP and guanosine 5'-O-(thiotriphosphate) (GTP gamma S) (but not GDP) and the beta-adrenergic receptor agonist isoproterenol activate Gs in native S49 cell membrane by subunit dissociation. Evidence for this includes detection of dissociated alpha s in membrane extracts and release of alpha s from S49 cell membranes treated with GTP gamma S or isoproterenol. Moreover, the estimates of apparent stoichiometry for this dissociation indicate that each beta-adrenergic receptor is able to activate greater than or equal to 100 molecules of Gs in native membranes. Thus, receptor-mediated dissociation of Gs is likely to be the major site of amplification of signal transduction by agonists active at hormone receptors that link to Gs.

Animals↗
PubMed1988

Phorbol ester and neomycin dissociate bradykinin receptor-mediated arachidonic acid release and polyphosphoinositide hydrolysis in Madin-Darby canine kidney cells. Evidence that bradykinin mediates noninterdependent activation of phospholipases A2 and C.

Many types of peptide hormone and neurotransmitter receptors mediate hydrolysis of phosphoinositides (PI) and arachidonic acid and arachidonic acid metabolite (AA) release, but the relation between these responses is not clearly defined. We have characterized bradykinin (BK)-mediated AA release and PI hydrolysis in clonal Madin-Darby canine kidney cells (MDCK-D1). Both responses occurred over a similar dose range in response to the B1 and B2 receptor agonist, BK, but not in response to the B1 receptor-selective agonist des-Arg-BK. To test whether AA release occurs via a mechanism which is sequential to and dependent upon PI hydrolysis, we used the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), which activates protein kinase C. TPA treatment blocked BK-mediated PI hydrolysis in MDCK-D1 cells, while at the same time and at similar concentrations enhancing BK-mediated AA release. Thus, TPA treatment dissociated BK-mediated AA release from PI hydrolysis. In addition, treatment of MDCK-D1 cells with neomycin blocked BK-mediated hydrolysis of phosphatidylinositol bisphosphate without reducing BK-mediated AA release. BK treatment increased formation of lysophospholipids with a time course in accord with BK-mediated AA release, indicating that at least part of the BK-mediated AA release was likely derived from activation of phospholipase A2. BK-mediated lysophospholipid production was enhanced by pretreatment with TPA, suggesting that the mechanism of AA release before and after treatment with TPA was the same. BK-mediated AA release and lysophospholipid production was dependent on the presence of extracellular calcium, while the enhanced responses to BK in the presence of TPA were not dependent on the presence of extracellular calcium. TPA treatment also enhanced AA release and lysophospholipid production in response to the calcium ionophore A23187. From these data we propose that BK, acting at B2 receptors, promotes AA release in MDCK cells via a mechanism which is 1) independent of polyphosphoinositide hydrolysis by phospholipase C, 2) dependent upon influx of extracellular calcium and activation of phospholipase A2, and 3) enhanced by activation of protein kinase C.

Animals↗
PubMed1988

Alpha 1-adrenergic receptors promote phosphatidylcholine hydrolysis in MDCK-D1 cells. A mechanism for rapid activation of protein kinase C.

Hydrolysis of phosphoinositides can lead to mobilization of calcium and production of diacylglycerol, which together are proposed to activate protein kinase C. We have shown that phosphoinositide hydrolysis mediated by alpha 1-adrenergic receptors on Madin-Darby canine kidney cells (MDCK-D1) occurred with an early lag and increased over a prolonged time course (Slivka, S.R., and Insel, P.A. (1987) J. Biol. Chem. 262, 4200-4207). In this study we characterize another type of receptor-mediated phospholipid hydrolysis in MDCK-D1 cells, alpha 1-adrenergic receptor-mediated hydrolysis of phosphatidylcholine. The predicted products of this hydrolysis, phosphorylcholine and diacylglycerol, were detectable as early as 0.5 min after alpha 1-adrenergic receptor stimulation by epinephrine. This hydrolysis appears to be a primary event after receptor occupancy because it occurred in the presence of neomycin, an inhibitor of polyphosphoinositide hydrolysis, and the protein kinase C inhibitors, sphingosine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7). In addition, we demonstrate alpha 1-adrenergic receptor-mediated activation of protein kinase C in MDCK-D1 cells. This activation was measured as a rapid translocation (0.5 min) of protein kinase C activity from the cytosolic fraction to the membrane fraction. This translocation also was not inhibited by neomycin. The time course and agonist concentration dependence of both phosphatidylcholine hydrolysis and protein kinase C activation by alpha 1-adrenergic receptors were similar. Thus, we propose that agonists acting at alpha 1-adrenergic receptors promote hydrolysis of phosphatidylcholine which results in rapid generation of diacylglycerol for the activation of protein kinase C.

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

The oligosaccharide component of alpha 1-adrenergic receptors from BC3H1 and DDT1 muscle cells. Studies with glycosidases and photoaffinity labelling of intact cells.

In this study, we clarify the structural aspects of the oligosaccharides associated with the alpha 1-adrenergic receptor in two muscle cell lines. Photoaffinity labelling of intact BC3H1 or DDT1 muscle cells with 2-[4-(4-azido-3-[125I]iodobenzoyl)piperazin-1-yl]-4-amino-6, 7-dimethoxyquinazoline ([125I]azidoprazosin) followed by SDS/polyacrylamide-gel electrophoresis (PAGE) and autoradiography revealed specifically labelled proteins of molecular mass = 87,000 and 81,000, respectively. Treatment of photoaffinity-labelled receptors in DDT1 cells with 33 u. of endoglycosidase F/ml for 24 h resulted in the loss of the 81 kDa receptor and the appearance of a 52.5 kDa protein. When lower concentrations of glycosidase or shorter incubation times were used, the 81 kDa receptor was converted to a 66 kDa protein. Treatment of the photoaffinity-labelled BC3H1 receptor with endoglycosidase F resulted in the appearance of a 50.5 kDa protein. Neither alpha-mannosidase nor endoglycosidase H had an effect on the photoaffinity labelling patterns of the receptor from the two cell types. alpha 1-Adrenergic receptors, solubilized from membranes prepared from BC3H1 and DDT1 cells, bound to wheat germ agglutinin-Sepharose and were displaced by N-acetylglucosamine. Taken together, these results indicate that alpha 1-adrenergic receptors in BC3H1 and DDT1 cells contain complex, but not high, mannose oligosaccharide chains; differences in the composition or number of chains partially accounts for the different molecular mass of the receptor in the two cell lines. The results further indicate that the oligosaccharide chains contribute substantially to the apparent molecular mass of alpha 1-adrenergic receptors, as detected by SDS/PAGE, and that the protein backbone of these receptors is likely to be approximately 50 kDa.

Acetylglucosaminidase↗
PubMed1988

Quantitation of the guanine nucleotide binding regulatory protein Gs in S49 cell membranes using antipeptide antibodies to alpha s.

Polyclonal antibodies reactive against the guanine nucleotide binding stimulatory protein, Gs, were affinity-purified from two rabbits immunized with a synthetic peptide corresponding to amino acids 28-42 in the alpha-subunit, alpha s. On immunoblots, these antibodies recognized alpha s, but not alpha-subunits from two other guanine nucleotide binding regulatory proteins, Gi and Go. A competitive enzyme-linked immunosorbent assay was developed in which inhibition of antibody binding to peptide-coated microtiter plates was used to quantitate purified Gs or Gs in cholate extracts of cell membranes. Plasma membranes derived from wild type S49 lymphoma cells contained 18.9 +/- 2.3 pmol/mg of membrane protein of alpha s. The same membranes bound 169 +/- 12 fmol/mg of protein of [125I]iodocyanopindolol to beta-adrenergic receptors, indicating that the amount of Gs is far in excess of the amount of beta-adrenergic receptors. Thus, even if every beta-adrenergic receptor molecule were to activate 10 Gs molecules, in order for Gs to be limiting for the receptors to reach their high affinity state, it is likely that compartmentation exists for target cell membrane receptors and Gs. Moreover, a comparison of beta-adrenergic receptor number and Gs levels in several different S49 lymphoma cell mutants having lesions in receptors or Gs argues against a coordinate regulation of beta-adrenergic receptors and Gs.

Animals↗
PubMed1988

Molecular mechanism of beta-adrenergic receptor blockers with intrinsic sympathomimetic activity.

beta-Adrenergic receptor (beta AR) blocking agents with intrinsic sympathomimetic activity (ISA) can induce modest increases in beta AR-stimulated activity, such as rate and force of contraction in cardiac tissue. The molecular basis for this activity has been elusive. Previous studies have suggested that these compounds do not stimulate cyclic AMP (cAMP) formation even though activation of adenylate cyclase is the generally accepted mechanism for beta AR promotion of target cell response. In the current studies, we show that several beta AR antagonists with ISA (dichloroisoproterenol, pindolol, and celiprolol) stimulate cAMP accumulation five-, two-, and threefold, respectively, in S49 lymphoma cells, but only if cells are simultaneously incubated with the diterpene forskolin. The KI values observed for inhibition of isoproterenol-stimulated cAMP accumulation or of beta AR [( 125I]iodocyanopindolol) binding for each of the beta blockers with ISA were comparable in magnitude to their respective EC50 values for forskolin-potentiated cAMP accumulation. The forskolin-potentiated responses of these compounds were abolished by the beta AR-antagonist propranolol. These results indicate that the ISA of beta-blocking drugs most likely results from a modest beta AR-mediated stimulation of adenylate cyclase activity. The results further suggest that treatment of target cells with forskolin provides a means to define partial agonism at receptors that are linked to stimulation of adenylate cyclase.

Adenylyl Cyclases↗
PubMed1988

Certain beta-blockers can decrease beta-adrenergic receptor number: II. Down-regulation of receptor number by alprenolol and propranolol in cultured lymphoma and muscle cells.

We have used two different cultured cell lines--S49 lymphoma cells and BC3H-1 muscle cells--to examine the regulation of beta-adrenergic receptors by receptor antagonists. Rather than an increase ("up-regulation") of receptor number that such antagonists often produce, we found that certain beta-blockers elicit a decrease ("down-regulation") of beta-adrenergic receptors. Alprenolol and propranolol, but not sotalol or ICI 118,551, at concentrations of 10-100 nM down-regulated beta-adrenergic receptors 20-70% following 16-20 hours of treatment of S49 or BC3H-1 cells. Several observations suggest that this phenomenon depends upon beta-receptor interaction, including stereoselectivity [(-)-enantiomers more potent than (+)-enantiomers], blockade of the effect by ICI 118,551, absence of down-regulation of alpha-adrenergic receptors in BC3H-1 cells, and lack of a decrease in beta-adrenergic receptor-independent (forskolin-stimulated) cyclic AMP accumulation in S49 cells. The possibility of retained antagonist interfering with receptor measurement was precluded by the fact that the antagonist-induced decrease in receptor number required several hours incubation and occurred without a prominent change in receptor affinity. The ability of the beta-blockers to elicit down-regulation did not correlate with hydrophobicity of the drugs. Antagonist-induced down regulation of beta-adrenergic receptors did not occur in S49 lymphoma cells that lack the alpha-subunit of Gs, the guanine nucleotide-binding regulatory protein, thus implying a requirement for receptor-alpha s interaction in eliciting beta-receptor down-regulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic beta-Antagonists↗
PubMed1988

In vivo regulation of beta-adrenergic receptors on mononuclear leukocytes and heart. Assessment of receptor compartmentation after agonist infusion and acute aortic constriction in guinea pigs.

In animals injected with a bolus of isoproterenol, beta-adrenergic receptors in both mononuclear leukocytes (MNL) and heart were sequestered away from the cell surface, and the time course (0-120 min) and dose-response patterns were similar in the two tissues. In guinea pigs given a constant infusion of isoproterenol, 0.15 mg/(kg.h), down-regulation of total receptor number occurred more quickly and to a greater extent in the MNL than in the heart. We also compared receptor sequestration after aortic constriction-induced acute heart failure. Negligible sequestration (9%) of beta-adrenergic receptors occurred in the MNL of animals treated in this manner, whereas the number of receptors in the sarcolemmal fraction decreased 61%. This selective sequestration of cardiac receptors may result from the action of high concentrations of norepinephrine (which is selective for beta 1 over beta 2 receptors) present at sympathetic nerve-cardiac cell synapses. We conclude that although receptor redistribution occurs similarly in MNL and heart in response to a circulating nonselective agonist, beta-adrenergic receptor redistribution may occur selectively in the heart in response to such stimuli as aortic constriction-induced acute heart failure that activate the sympathetic nervous system.

Adenylyl Cyclases↗
PubMed1988

Noncoordinate regulation of cardiac Gs protein and beta-adrenergic receptors by a physiological stimulus, chronic dynamic exercise.

We used a physiological stimulus, chronic dynamic exercise, in pigs to examine resultant changes in chronotropic responsiveness to catecholamine and biochemical features of cardiac beta-adrenergic receptors and the stimulatory guanine nucleotide-binding protein, GS. Long-term treadmill running resulted in a substantial (44%) down-regulation of right atrial beta-adrenergic receptors, but the dose of isoproterenol yielding a 50% maximal increase in heart rate was decreased by 57% (from 0.07 +/- 0.03 to 0.03 +/- 0.01 microgram/kg; P less than 0.02) despite this decrease in receptor number. This disparity between receptor number and physiological responsiveness suggested altered signal transduction. We therefore quantitated GS in myocardial membranes obtained before and after chronic exercise in a competitive ELISA based on an antipeptide antibody developed to the alpha S portion of GS. We found a 42% increase in the amounts of GS in right atrial membranes (from 11.4 +/- 0.8 to 16.2 +/- 2.0 pmol/mg; P less than 0.05) and a 76% increase in the amounts of GS in left ventricular membranes (from 15.6 +/- 2.6 to 27.4 +/- 5.2 pmol/mg; P = 0.02) after chronic running. These data suggest that in the heart physiological perturbations can result in changes in the levels of GS, that GS and beta-adrenergic receptor number are not coordinately regulated, and that GS may contribute to altered adrenergic responsiveness independently of changes in beta-adrenergic receptor number.

Animals↗
PubMed1988

Receptor-induced degradation of atrial natriuretic peptide by a rabbit carotid artery smooth muscle cell.

We have used a smooth muscle cell line isolated from rabbit carotid artery (RCA) as a model system with which to study the expression of atrial natriuretic peptide (ANP) receptors and, in addition, the receptor-mediated degradation of ANP. RCA cells bind rat alpha ANP-(1-28) reversibly at 37 C, apparently to a single class of high affinity (Kd approximately equal to 50 pM) binding sites (approximately equal to 20,000 sites per cell). Binding of rat alpha ANP-(1-28) elicits rapid accumulation of intracellular cGMP. However, the concentrations of rat alpha ANP-(1-28) and related peptides, abbreviated at the N- and C-terminals, required to stimulate cGMP synthesis are substantially greater than those required for binding. Analysis by HPLC of 125I-labeled rat alpha ANP-(1-28) bound to RCA cells at 37 C illustrates the rapid and continuous degradation of the radioiodinated rat alpha ANP-(1-28) to two radiolabeled products, one of which, 125I-labeled tyrosine is the major radiolabeled component that dissociates from the cells. Measurement of rat alpha ANP-(1-28) interaction with RCA cells by radioligand binding techniques therefore subsumes several processes. One of these processes is the rapid and continuous degradation of specifically bound ANP by these cells and perhaps also other target cells that respond to ANP.

Animals↗
PubMed1988

Selective blockade and recovery of cell surface alpha 2-adrenergic receptors in human erythroleukemia (HEL) cells. Studies with the irreversible antagonist benextramine.

alpha 2-Adrenergic receptors are present on human erythroleukemia (HEL) cells, both on the cell surface and in a sequestered compartment. In the current study we show that benextramine, a hydrophilic irreversible antagonist, can be used to investigate alpha 2-adrenergic receptor compartmentation in these cells. In membranes prepared from HEL cells, benextramine competed for all alpha 2-adrenergic receptors ( [3H]yohimbine sites). In intact cells, at 4 degrees, benextramine exhibited a biphasic competition curve for alpha 2-adrenergic receptors, with EC50 values of approximately 10 microM and greater than 1 mM for the high and low affinity components, respectively. We propose that the alpha 2-adrenergic receptors preferentially blocked by benextramine are those on the surface of the cell, whereas those with low affinity are sequestered receptors because: 1) only epinephrine-accessible sites [i.e., cell surface sites; McKernan et al., Mol. Pharmacol. 32:258-265 (1987)] are removed by prior treatment of cells with benextramine, 2) a preparation enriched with surface membranes is also enriched in receptors with a high affinity for benextramine; and 3) after blockade of cell surface receptors (54 +/- 6% of total sites, n = 7) by benextramine, the ability of the alpha 2-adrenergic agonists epinephrine and UK-14,304 to inhibit forskolin-stimulated cAMP accumulation is lost. The latter result implies that only cell surface and not sequestered receptors are functionally coupled to adenylate cyclase. The return of receptors from the sequestered compartment to the cell surface and the recovery of alpha 2-adrenergic receptor function were measured after HEL cells were treated with benextramine (50 microM for 1 hr at 4 degrees). The recovery of receptor binding (t1/2 = 25 min) was somewhat slower than the recovery of function (t1/2 approximately 8 min). This is consistent with the existence of "spare receptors" and also suggests that the sequestered compartment of alpha 2-adrenergic receptors can rapidly exchange with those on the surface. When all alpha 2-adrenergic receptors were blocked by incubation of HEL cells with benextramine for 1 hr at 37 degrees, repopulation of surface and sequestered receptors was much slower (t1/2 = 9 hr for recovery of total receptors). Surface receptors recovered even more slowly than did total cellular receptors consistent with the idea that alpha 2-adrenergic receptors must traverse through intracellular locations before insertion into the cell surface.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding, Competitive↗
PubMed1988

Use of 1-deoxymannojirimycin to show that complex oligosaccharides regulate cellular distribution of the alpha 1-adrenergic receptor glycoprotein in BC3H1 muscle cells.

We have previously shown that alpha 1-adrenergic receptors in BC3H1 muscle cells are glycoproteins containing complex but not high mannose oligosaccharides. In the present study we investigated the role of the complex sugars in functional aspects of the receptor by treating BC3H1 cells with 1-deoxymannojirimycin (dMM), which blocks conversion of high mannose oligosaccharides to complex chains. Receptors were photoaffinity labeled in intact cells with 125I-azido prazosin; drug treatment with dMM resulted in conversion of the 87-kDa receptor to 62 kDa. The 62-kDa protein was sensitive to mannosidase, indicating loss of complex sugars. Radioligand ([3H]prazosin) binding analysis carried out at 37 degrees to intact cells indicated that dMM treatment increased the affinity of the alpha 1-receptors for [3H]prazosin 2-fold and decreased the number of total cellular receptors by 15%. In order to distinguish between surface and sequestered receptors, we assessed [3H]prazosin binding to intact cells at 4 degrees using competition by the hydrophilic agonist epinephrine to define surface receptors and by nonradioactive antagonists (prazosin and phentolamine) to define total receptors. In control cells, epinephrine competed for 90% of the total receptors, whereas for dMM-treated cells this value was only 60%. In addition, dMM treatment caused a 40% reduction in epinephrine-stimulated phosphatidylinositol turnover when compared with untreated cells. The results indicate that dMM treatment reduces the number of functional alpha 1-adrenergic receptors on the cell surface while increasing the number of sequestered receptors. We conclude that complex oligosaccharides are important for cellular localization and function of alpha 1-adrenergic receptors in BC3H1 cells.

1-Deoxynojirimycin↗
PubMed1988

Characterization of a bromoacetylated derivative of pindolol as a high affinity, irreversible beta adrenergic antagonist in cultured cells.

We have utilized cultured cell lines to test the utility of N8-(bromoacetyl)-N1-[3-(4-indolyoxy)-2-hydroxypropyl]-(Z)-1,8-diam ino- p-menthane, BIM, a recently synthesized, irreversible beta adrenergic antagonist. Previously available irreversible antagonists of beta adrenergic receptors have generally exhibited low affinity (typical IC50 values greater than or equal to 1 microM). By contrast, S49 lymphoma cells incubated with 10 nM BIM for 120 min and then washed extensively showed a 70% loss in beta adrenergic receptors, as measured by [125I]iodocyanopindolol binding. This loss, which could be prevented by propranolol, represented a decrease in receptor number without a change in affinity of the remaining receptors for [125I]iodocyanopindolol. The BIM-induced decrease in binding sites was persistent in membranes incubated for several hours after BIM treatment. BIM did not inactivate alpha-1 adrenergic receptors on Madin Darby canine kidney cells, alpha-2 adrenergic receptors on human erythroleukemia cells, nor did BIM treatment alter guanyl-5'-yl-imidodiphosphate-mediated regulation of agonist binding to the beta adrenergic receptors in S49 cell membranes. BIM treatment decreased cyclic AMP (cAMP) accumulation in S49 cells in response to the beta adrenergic agonist isoproterenol, but increased prostaglandin E1-stimulated cAMP accumulation (P = .09) without altering cAMP production in response to forskolin. The inactivation of beta receptors in S49 cells by BIM (IC50 = 0.30 nM) correlated closely with the loss in beta adrenergic receptor-mediated cAMP accumulation in these cells (IC50 = 0.59 nM), implying the absence of substantial receptor reserve for this response. We conclude that BIM is a potent, irreversible, selective beta adrenergic antagonist for the study of beta adrenergic receptors in cultured cells.

Adrenergic beta-Antagonists↗