Biomedical subjects

S T Crooke

Publications and source records attributed to S T Crooke.

At least 163 records · Page 9Linked to original sources

PubMed1987

Antitumor activity of bis(diphenylphosphino)alkanes, their gold(I) coordination complexes, and related compounds.

Bisphosphines related to bis(diphenylphosphino)ethane (dppe) and their gold complexes are described that are active in a spectrum of transplantable tumor models. When administered ip on days 1-5 at its maximally tolerated dose (MTD) of 40 mumol/kg, dppe reproducibly gives 100% increase in life span (ILS) in mice bearing ip P388 leukemia. Coordination of chlorogold(I) to each phosphine in dppe gave a complex that had similar activity but at a much lower dose level than dppe; the MTD for the gold(I) complex was 7 mumol/kg. Among other metal complexes of dppe, the Au(III) complex was active (greater than 50% ILS) whereas Ag(I), Ni(II), Pt(II), Pd(II), and Rh(I) complexes were inactive. Among dppe analogues, replacement of phenyl groups with ethyl or benzyl groups resulted in inactivity for both ligands and the corresponding gold complexes whereas substitution with cyclohexyl or heterocyclic ring systems yielded ligands and/or gold complexes with antitumor activity. Among substituted-phenyl dppe and dppe(AuCl)2 analogues, 3-fluoro, 4-fluoro, perdeuterio, 4-methylthio, and 2-methylthio analogues were active; 4-methyl, 3-methyl, 4-methoxy, 4-dimethylamino, and 4-trifluoromethyl analogues were marginal or inactive. Analogues in which the ethane bridge of dppe or dppe(AuCl)2 was varied between one and six carbons, unsaturated or substituted, revealed that activity was maximal with ethane or cis-ethylene. Compounds with good P388 activity were also active in other animal tumor models.

Animals↗

PubMed1987

Structure-activity relationships of beta-adrenergic receptor-coupled adenylate cyclase: implications of a redox mechanism for the action of agonists at beta-adrenergic receptors.

The present studies have tested the hypothesis that agonists at beta-adrenergic receptors activate the beta-receptors by reducing them. This was examined by analyzing the interactions of 41 beta-agonists and antagonists with the receptors. The structural features which determined binding affinity (KD) were shown to be distinct from those which determined intrinsic, activity (IA). The IA was shown to be related to the oxidation-reduction properties which were determined primarily by the nature of the substituents on the phenyl ring. Thus, the parent compound phenylethanolamine, having no phenolic substituent, acted as an antagonist (IA = 0) and was also redox inactive. All of the antagonists tested (19) exhibited EP (peak potential for the first oxidative wave) values greater than 0.75 V, suggesting that they were difficult to oxidize. Agonists, however, exhibited a wide range of EP (0.25-0.7 V) with values lower than those of the antagonists. The agonists tested include catecholamines, catecholamine analogs bearing meta-substituted amino functionalities (such as amino, methylamino, formanilide, sulfonamide, urea, and carbamate), resorcinol, and hydroxymethyl congeners. It is proposed that the oxidizing tendency of the substituent on the phenyl ring is one of the factors that influences IA. To test the hypothesis further, we electrolytically oxidized isoproterenol to adrenochrome or to the o-quinone intermediate and tested for activity. The 4e-, 4H+-oxidation product adrenochrome did not bind to or stimulate adenylate cyclase, suggesting that the reducing ability to isoproterenol is important for its agonistic activity. A cyclic redox mechanism for the action of agonists at beta-adrenergic receptors is presented. We propose that agonist are electron donors. Their interactions with receptors result in reduction leading to activation of the receptors.

Adenylyl Cyclases↗

PubMed1987

Identification and characterization of vascular (V1) vasopressin receptors of an established smooth muscle cell line.

We report the identification and characterization of specific vasopressin-binding sites on intact cells and membranes of the established vascular smooth muscle cell line A-10, the fate of vasopressin associated with the cells, the role of guanine nucleotides in the regulation of the affinity of the vasopressin-binding sites, and the determination of the vasopressin receptor subtype. We have found specific vasopressin-binding sites on intact cells in monolayer (110,000 sites per cell during log growth and 60,000 sites per cell in stationary culture) with a KD of 6 nM at 37 degrees. After incubation of [3H]-8-arginine vasopressin ([3H]AVP) and cells for less than 20 min, cell-associated AVP was intact; with longer incubation times, AVP was progressively degraded. The major metabolites included phenylalanine and a fraction that eluted from a C18 reverse phase high performance liquid chromatography column between AVP and 8-arginine, 9-desglycinamide vasopressin. Extensive degradation also occurred when AVP was allowed to dissociate from the cells. With increased time of incubation, the amount of specifically bound AVP that could dissociate decreased, suggesting receptor-mediated endocytosis. In saturation equilibrium binding experiments with plasma membranes, two affinity states with KD of 0.7 nM and 379 nM were observed. The number of high affinity binding sites was similar to the number of receptors found on intact cells. Guanosine 5'-(beta,gamma-imido)triphosphate decreased vasopressin binding to the high affinity sites and did not significantly affect the low affinity sites. Competition binding experiments indicated that the vasopressin-binding sites of A-10 cells belong to the vascular V1 receptor subtype. We conclude that the established vascular smooth muscle cell line A-10 expressed vasopressin receptors of the vascular V1 subtype. Vasopressin bound to the receptors reversibly, but could also be degraded by the cells presumably after receptor-mediated endocytosis. The receptors might exist in different affinity states; guanosine 5'-(beta,gamma-imido)triphosphate decreased the affinity of the high affinity binding state.

Animals↗

PubMed1987

A novel radiolabeled vasopressin antagonist: [3H-Phe]-desGlyd(CH2)5D-Tyr(Et)VAVP, [3H]-SK&F 101926.

We report the vasopressin receptor-binding properties of [3H-Phe]-desGlyd(CH2)5D-Tyr(Et)VAVP, [3H]-SK&F 101926, the first radiolabeled vasopressin receptor antagonist. We chose to radiolabel SK&F 101926 because this vasopressin analog is a potent antagonist of vascular V1 and renal V2 vasopressin receptors in all species studied. [3H]-SK&F 101926 bound with a single high affinity to intact vascular smooth muscle cells (A-10; KD = 0.5 nM), and plasma membranes A-10 cells (KD = 0.4 nM) and rat liver (KD = 0.2 nM). In competition experiments with [3H]-SK&F 101926 and [3H]arginine vasopressin ([3H]AVP) using cell and liver membranes, the affinity rank orders of vasopressin analogs were the same and were typical for the V1 receptor subtype. In competition binding experiments with [3H]-SK&F 101926 using cell and liver membranes, guanosine 5'-(beta,gamma-imido)triphosphate did not significantly alter the affinity of the V1 antagonist d(CH2)5Tyr(Me)AVP, but the affinity of AVP was decreased. These data indicate that the V1 receptor can exist in at least two affinity states that are modulated by guanine nucleotides. [3H]-SK&F 101926 also bound specifically and with high affinity to V2 receptors of MDCK cells. We conclude that [3H]-SK&F 101926 binds with high affinity to V1 and V2 vasopressin receptors and is a powerful new tool for the identification of vasopressin receptors and the study of molecular mechanisms involved in the interaction of vasopressin with its receptors.

Animals↗

PubMed1987

Solubilization and reconstitution of vasopressin V1 receptors of rat liver.

The observation that vasopressin receptors loose their binding capacity on solubilization suggests that lipid-milieu may be a requirement for the binding of vasopressin to these receptors. To preserve the ligand binding capacity, we have developed reconstitution procedures to study solubilized rat liver vasopressin receptors. We report here that vasopressin V1 receptors were efficiently solubilized from rat liver membranes with egg lysolecithin and quantitatively reconstituted into phospholipid vesicles resulting in a 4-fold purification. Binding of [3H]arginine vasopressin to the reconstituted receptors was saturable and of high affinity (Kd = 0.6 +/- 0.1 nM). In competition binding experiments the solubilized, reconstituted receptors exhibited the same pharmacological profile as was observed with vasopressin V1 receptors in membranes.

Animals↗

PubMed1987

Leukotriene-induced hydrolysis of inositol lipids in guinea pig lung: mechanism of signal transduction for leukotriene-D4 receptors.

Addition of leukotriene D4 (LTD4) to [3H]myo-inositol-labeled guinea pig lung induced rapid breakdown of inositol lipids. Formation of [3H]inositol trisphosphate was rapid, with a peak of 140-160% of the control level, 30 sec post-treatment. Formation of [3H]inositol bisphosphate and [3H]inositol monophosphate ([3H]IP1) was also rapid in the presence of LiCl. LTD4-induced [3H]IP1 formation was concentration dependent, stereoselective, and not inhibited by the cyclooxygenase inhibitor, indomethacin. Agonist analogs of LTD4 and leukotriene E4 also induced dose-dependent increases in the synthesis of [3H]IP1. The rank order potency of the agonist-induced [3H]IP1 formation was equivalent to those reported for LTD4 receptor binding, smooth muscle contraction, and thromboxane B2 biosynthesis. Furthermore, a specific receptor antagonist, SKF 102922, inhibited LTD4-induced [3H]IP1 formation in guinea pig lung. These studies suggest that LTD4 may interact with membrane receptor and activate a phospholipase C, which in turn induces the hydrolysis of inositol lipids. The hydrolysis products, diacylglycerol and inositol trisphosphate, can be regarded as the intracellular messengers for LTD4 receptors in guinea pig lung. This concept may explain a variety of pharmacological effects of leukotrienes in different types of target cells or tissues.

Animals↗

PubMed1987

Phorbol ester-mediated inhibition of vasopressin and beta-adrenergic responses in a vascular smooth muscle cell line.

We have reported previously that in the vascular smooth muscle cell line A-10 (ATCC CRL 1476), vasopressin stimulated phosphatidylinositol turnover Ca2+ efflux and inhibited isoproterenol-stimulated cAMP accumulation. Here we report that pretreatment of these cells with phorbol dibutyrate, an activator of protein kinase C, attenuated the responses to vasopressin and isoproterenol. This effect was concentration dependent and could be observed after pretreatment for 2 min. 4 alpha Phorbol 12,13-didecanoate, which does not activate protein kinase C, did not attenuate the responses. These data suggest that activation of protein kinase C by phorbol dibutyrate attenuates the responses of vascular smooth muscle cells to isoproterenol and vasopressin. Although phorbol ester did not affect [3H]-8-arginine vasopressin binding to intact cells, it appeared to uncouple vasopressin receptors from guanine nucleotide-binding protein.

Animals↗

PubMed1987

Glucocorticoid regulation of beta-adrenergic receptors in 3T3-L1 preadipocytes.

Treatment of 3T3-L1 preadipocytes (fibroblasts) with 250 nM dexamethasone for 48 hr caused a doubling of total beta-adrenergic receptors and an increase in beta 2-adrenergic receptor subtype proportion from approximately 50% in controls to 85% in treated cells. The responses to epinephrine and norepinephrine in a whole cell cAMP accumulation assay reflected these changes. The effects of dexamethasone on beta-adrenergic receptors were mediated through the glucocorticoid receptor and were time and dose dependent with an EC50 of 2.77 +/- 0.73 nM for an increase in the proportion of beta 2-adrenergic receptors. The rank order of potency of steroids to effect these changes (betamethasone = dexamethasone greater than fludrocortisone greater than hydrocortisone = triamcinolone greater than aldosterone) correlated with their glucocorticoid potency. [3H]Dexamethasone binding to intact cells yielded a KD value of 3.47 +/- 0.38 nM for binding to the glucocorticoid receptor which correlated well with the EC50 for dexamethasone to alter beta-adrenergic receptors. Inhibition of [3H]dexamethasone binding by other steroids confirmed that the ability of steroids to regulate beta-adrenergic receptors correlated with the affinity of each compound for the 3T3-L1 glucocorticoid receptor. Progesterone, which can bind to the glucocorticoid receptor but has only weak agonist activity, competitively inhibited the ability of dexamethasone to alter beta-adrenergic receptors. Protein synthesis, RNA synthesis, and N-linked glycosylation appeared to be necessary for the change in receptor subtype expression and the increase in beta-adrenergic receptor number induced by dexamethasone. The present study suggests that regulation of beta-adrenergic receptor expression in 3T3-L1 preadipocytes by dexamethasone is a glucocorticoid-specific effect which may require gene activation.

Adipose Tissue↗

PubMed1987

Characterization of a subline of P388 leukemia resistant to amsacrine: evidence of altered topoisomerase II function.

Sensitive (P388/S) and amsacrine-resistant (P388/amsacrine) sublines of P388 leukemia were cloned in vitro and tested for differential chemosensitivity against a panel of drugs. P388/amsacrine, resistant both in vivo and in vitro to amsacrine, was cross-resistant to other putative topoisomerase II inhibitors including teniposide, etoposide, bisantrene, and doxorubicin. P388/amsacrine, was however, as sensitive as cloned P388/S to camptothecin, an inhibitor of topoisomerase I. The pattern of cross-resistance suggested that an alteration in topoisomerase II may be involved in the resistance of P388/amsacrine to these drugs. No differences in the uptake of amsacrine were detected between the two sublines. Cross-resistance to vinblastine was evident in P388/amsacrine; however resistance to vinblastine was associated with alterations in uptake or efflux of the drug. The number of protein-concealed single-strand breaks induced in whole cells by amsacrine, teniposide, bisantrene, and camptothecin was measured. Diminished numbers of strand breaks in the resistant subline were consistent with decreases in DNA-protein crosslinks. In the absence of drug treatment, resistant cells sustained approximately one-half as many single-strand breaks and DNA-protein crosslinks as the sensitive cells during preparation of nuclei. As measured by the P4 phage DNA unknotting assay, 0.35 M NaCl nuclear extracts from P388/S contained approximately 2.3-fold more topoisomerase II catalytic activity than did extracts from P388/amsacrine. The amount of protein that immunoreacted with a specific antibody to calf thymus topoisomerase II was also decreased in the resistant cells. These data suggest that alterations in topoisomerase II which lead to differential drug sensitivities are partially responsible for the resistance of P388/amsacrine to a specific group of drugs.

Amsacrine↗

PubMed1987

Immuno cross-reactivity suggests that catecholamine biosynthesis enzymes and beta-adrenergic receptors may be related.

Turkey red blood cell, beta 1-adrenergic receptors (BARs) were prepared to electrophoretic homogeneity by affinity chromatography, size exclusion high performance liquid chromatography, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and used to prepare rabbit polyclonal anti-BAR antibodies. Anti-BAR activity was confirmed by immunoadsorption of [125I]cyanopindolol-labeled BAR to a protein A affinity column using the anti-BAR antibodies. BAR was compared to the catecholamine biosynthetic enzyme dopamine B-hydroxylase (DBH) by anti-BAR antibody cross-reactivity. DBH was purified from bovine adrenal medullae chromaffin vesicles by ion exchange, size exclusion, and concanavalin A-Sepharose chromatography. Final DBH specific activities were 42 +/- 4 units/mg of protein. Homogeneity was confirmed by nondenaturing polyacrylamide gel electrophoresis. Both DBH and BAR were recognized by the anti-BAR antibodies on Western transfer and immunoblotting. No interactions were observed with preimmune controls. Similar results were obtained with glycosylated and deglycosylated DBH, suggesting that the anti-BAR antibodies recognize specific portions of DBH amino acid sequence and not associated carbohydrate. DBH-cross-reactive antibodies were also purified by affinity chromatography using immobilized DBH and shown to immunoadsorb [125I]cyanopindolol-labeled BAR by protein A affinity chromatography. These results suggest that the catecholamine biosynthetic enzyme DBH and BAR may be related in structure.

Animals↗

PubMed1987

SKF 104353, a high affinity antagonist for human and guinea pig lung leukotriene D4 receptor, blocked phosphatidylinositol metabolism and thromboxane synthesis induced by leukotriene D4.

SKF 104353 (2(S)-hydroxyl-3(R)-carboxyethylthio)-3-[2-(8-phenyloctyl) phenyl] propanoic acid) is a synthetic structural analog of leukotrienes D4 and E4 (LTD4, LTE4). This compound binds to guinea pig and human lung LTD4 receptors with affinities (Ki) of 5 +/- 2 and 10 +/- 3 nM, respectively. The Ki values of a reference compound, FPL 55712, were 2200 and 4500 nM, respectively, approximately 400- and 500-fold less effective than SKF 104353. LTD4- and LTE4-induced biosynthesis of thromboxane B2 has been shown to be mediated by LTD4 receptors in guinea pig lung in vitro. SKF 104353 did not induce synthesis of TxB2 in this system at concentrations of 1-20 microM. When SKF 104353 and increasing concentrations of LTD4 were incubated with guinea pig lung, the dose response curve of LTD4-induced TxB2 biosynthesis was shifted to the right with a -log[KB] = 8.4 +/- 0.2. LTD4-induced phosphatidylinositol (PI) hydrolysis in guinea pig lung has been shown to be the major signal transduction mechanism. In this system, SKF 104353 (1-20 microM) did not promote PI hydrolysis. Pretreatment of the [3H]myo-inositol-labeled guinea pig lung with SKF 104353 shifted the LTD4-induced PI hydrolysis dose response curve to the right, indicating that SKF 104353 inhibited LTD4 receptor-mediated intracellular second messenger formation. These results demonstrate that SKF 104353 is a high affinity, specific LTD4 receptor antagonist. It inhibited LTD4-induced PI hydrolysis and TxB2 biosynthesis in guinea pig lung. SKF 104353 may prove to be an important research tool for research on the activities of leukotrienes and of value therapeutically in the treatment of leukotriene-mediated diseases.

Animals↗

PubMed1987

Effects of N-ethylmaleimide on arginine vasopressin-induced responses in an established smooth muscle cell line.

We have previously reported that 8-arginine vasopressin (AVP) stimulates phosphatidylinositol turnover and Ca++ mobilization in rat aortic smooth muscle cells (A10). In the present study, N-ethylmaleimide (NEM) was used to further characterize the putative guanine nucleotide binding protein that transduces the V1 receptor effects on phosphatidylinositol turnover and Ca++ efflux in these cells. Pretreatment of the cells with low concentrations of NEM did not affect the basal levels of the inositol phosphates and Ca++ efflux but significantly inhibited the AVP-induced increases. NEM pretreatment did not significantly affect [3H]AVP binding to intact cells. Guanyl-5'-yl imidodiphosphate reduced the apparent binding affinity of AVP to cell membranes. NEM pretreatment abolished this guanyl-5'-yl imidodiphosphate effect. AVP stimulated a specific GTPase activity in cell membranes; this effect was also abolished by NEM pretreatment. The results suggest that in A10 cells a guanine nucleotide binding protein sensitive to NEM couples vasopressin receptors to phospholipase C.

Animals↗

PubMed1987

Effect of auranofin and other gold complexes on the activity of phospholipase C.

Auranofin (AF) is an orally active chrysotherapeutic agent used for the treatment of rheumatoid arthritis, a self-perpetuating inflammatory disease. Because of reports suggesting that AF and other gold complexes can, under certain circumstances, exacerbate rheumatoid inflammatory lesions in humans and adjuvant arthritic rats and that phospholipase C (PLC) and phospholipase A2 activities are increased in rheumatoid patients, the effects of AF and a related gold complex on in situ mammalian and purified Bacillus cereus PLC were examined. Results of our studies show that 1) AF and triethylphosphine gold chloride (TEPG), an AF analog, stimulated PLC activity in the sonicate of RAW 264.7 macrophages; 2) AF and TEPG stimulated B. cereus PLC activity in a concentration-dependent manner, but the pattern of stimulation and concentrations of drugs required to stimulate the purified enzyme differ from those seen with the macrophage PLC; 3) metals (cobalt and zinc) and sulfhydryl reagents (N-ethylmaleimide, iodoacetic acid, and glutathione), tested at the same concentrations of AF that enhanced PLC activity, had no effect on the enzyme. These data suggest that stimulation of PLC may be a generic phenomenon since two divergent PLCs are affected by gold complexes. Additionally, these studies may provide one potential explanation for rheumatoid lesion flares seen in patients and animals on chrysotherapy.

Animals↗

PubMed1987

Inhibition of phosphoinositide-specific phospholipase C by manoalide.

Manoalide is a novel sesterterpenoid which has previously been shown to be a potent inhibitor of venom phospholipases A2. To determine whether manoalide inhibited other phospholipases, the sensitivity of phosphoinsitide-specific phospholipase C (PI-PLC) to inactivation by manoalide was examined using crude cytosolic PI-PLC and a PI-PLC purified to homogeneity from guinea pig uterus cytosol (PI-PLC I). Manoalide inhibited both cytosolic and purified PI-PLC I in a concentration-dependent fashion, exhibiting an IC50 of 3-6 microM. Inactivation of PI-PLC I was calcium- and pH-dependent, with greater inactivation occurring at alkaline pH. Manoalide inhibited hydrolysis of all three phosphoinositides by purified PI-PLC I. The substrate kinetics of PI-PLC I suggest that manoalide does not inhibit purified PI-PLC I by simple competitive or noncompetitive inhibition. Enzyme activity was not recovered after dialysis of manoalide-treated PI-PLC I, indicating that inactivation of PI-PLC I was irreversible. To determine whether manoalide inhibited PI-PLC in cells, the effects of manoalide on norepinephrine (NE)-stimulated phosphoinositide hydrolysis and calcium mobilization were investigated in a smooth muscle-like cell line, DDT1MF-2. Manoalide inhibited NE-induced inositol 1,4,5-trisphosphate and inositol 1-phosphate formation in a concentration-dependent manner. The IC50 for inhibition of inositol 1-phosphate formation was 1.5 microM. Manoalide also inhibited NE-induced calcium transients in DDT1MF-2 cells, exhibiting an IC50 of 2 microM. These data suggest that inhibition of PI-PLC may account, in part, for the anti-inflammatory actions of manoalide.

Animals↗

PubMed1987

Induction of metallothionein is correlated with resistance to auranofin, a gold compound, in Chinese hamster ovary cells.

Metallothioneins (MTs) are low molecular weight, thiol-rich, metal-binding proteins. Auranofin (AF) is a gold compound active in the treatment of rheumatoid arthritis. The effects of AF on regulation of MT gene expression in Chinese hamster ovary cells were studied. AF-resistant cells accumulated substantial amounts of MT mRNA and protein, whereas no induction was observed in AF-sensitive cells. Cells capable of inducing MT in the presence of AF were much less sensitive to AF-mediated cytotoxicity. Induction of MT by low concentrations of Cd protected cells from subsequently administered doses of AF. The level of protection correlated with the level of induced MT. These findings indicate that MT plays a central role in the mechanisms underlying cellular resistance to gold compounds.

Animals↗

PubMed1986

Yeast metallothionein function in metal ion detoxification.

A genetic approach was taken to test the function of yeast metallothionein in metal ion detoxification. A yeast strain was constructed in which the metallothionein locus was deleted (cup1 delta). The cup1 delta strain was complemented with normal or mutant metallothionein genes under normal or constitutive regulatory control on high copy episomal plasmids. Metal resistance of the cup1 delta strain with and without the metallothionein-expressing vectors was analyzed. The normally regulated metallothionein gene conferred resistance only to copper (1000-fold); constitutively expressed metallothionein conferred resistance to both copper (500-fold) and cadmium (1000-fold), but not to mercury, zinc, silver, cobalt, nickel, gold, platinum, lanthanum, uranium, or tin. Two mutant versions of the metallothionein gene were constructed and tested for their ability to confer metal resistance in the cup1 delta background. The first had a deletion of a highly conserved amino acid sequence (Lys-Lys-Ser-Cys-Cys-Ser). The second was a hybrid gene consisting of the sequences coding for the first 20 amino acids of the yeast protein fused to the monkey metallothionein gene. Expression of these genes under the CUP1 promoter provided significant protection from copper, but none of the other metals tested. These results demonstrate that there is significant flexibility in the structural requirements for metallothionein to function in copper detoxification and that yeast metallothionein is also capable of detoxifying cadmium under conditions of constitutive expression.

Amino Acid Sequence↗

PubMed1986

Induction of functional beta-adrenergic receptors in rat aortic smooth muscle cells by sodium butyrate.

Rat aortic smooth muscle cells in culture (A-10; ATCC CRL 1476) exhibited low levels of beta-adrenergic receptors as determined by specific binding of [125I]cyanopindolol ([125I]CYP) and marginal stimulation of adenylate cyclase in plasma membranes by (-)isoproterenol. When these cells were exposed to 5 mM sodium butyrate, the number of beta-adrenergic receptors and the beta-agonist-stimulated adenylate cyclase activity increased markedly. However, basal, GTP, Gpp(NH)p, and fluoride-stimulated activities did not change. The induction of beta-adrenergic receptors and beta-agonist stimulated adenylate cyclase activity was time- and dose-dependent, and was relatively specific for sodium butyrate. Propionate and valerate were less effective than butyrate, while isobutyrate, succinate, and malonate were ineffective. The induction involved RNA and protein synthesis because induction was prevented by treatment with cycloheximide, puromycin, and actinomycin D. Butyrate did not cause a general increase in cell surface receptors, because the number of vasopressin receptors did not change. The sustained presence of butyrate appeared to be necessary for the maintenance of the induced beta-receptors. When butyrate was removed, receptor number and beta-agonist-stimulated adenylate cyclase activity were decreased by 90% over 24 hr. We conclude that the poor response of rat aortic smooth muscle cell plasma membranes to beta-adrenergic agonists is due to the presence of a low number of beta-adrenergic receptors. Butyrate markedly increased the number of beta-receptors which resulted in a proportional increase in beta-agonist-stimulated adenylate cyclase activity. The increase in receptor number was dependent on RNA and protein synthesis. Butyrate treatment did not affect the activity of the cyclase unit and the efficiency of coupling between the receptors and the guanine nucleotide regulatory protein, Ns.

Adenylyl Cyclases↗

PubMed1986

Evidence for tyrosine at the ligand binding center of beta-adrenergic receptors.

Beta 1- and beta 2-adrenergic receptor radioligand antagonist binding activities in plasma membrane preparations from mammalian lung, as well as amphibian and avian red blood cells, have been shown to be inactivated by agents which specifically alkylate tyrosine. In membrane preparations, protection against inactivation was afforded by both agonists and antagonists. In soluble purified preparations, antagonists but not agonists protected against inactivation. These results suggest that tyrosine is located at or near the ligand binding site of both beta 1- and beta 2-adrenergic receptors.

Adrenergic beta-Antagonists↗