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C J Homcy

Publications and source records attributed to C J Homcy.

At least 91 records · Page 5Linked to original sources
PubMed1986

Atrial natriuretic factor: assessment of its structure in atria and regulation of its biosynthesis with volume depletion.

Atrial natriuretic factor (ANF) is a polypeptide that has been isolated from mammalian atrial tissue with potent natriuretic, diuretic and spasmolytic properties. Details of its biosynthesis, cleavage, storage and release are not yet fully defined. Using sequence-specific anti-peptide antisera in immunocytochemical and radioimmunoassay studies, we demonstrated the presence in atrial granules of sequences corresponding to both the large molecular weight ("prohormone") form of ANF and the carboxyterminal peptide believed to be responsible for its biological activity. However, a peptide sequence spanning the proposed site of cleavage of the "signal peptide" from preproANF was undetected. In volume-depleted rats, a 28% decrease in the relative concentrations of atrial ANF mRNA was observed as compared to that of controls (P less than 0.01). There was, however, no significant difference observed between the two groups of animals with respect to atrial levels of ANF, as measured by radioimmunoassay. We conclude from these studies that proANF, but not preproANF, is stored in atrial granules. The concentration of stored ANF remains constant in volume-depleted rats despite a 28% reduction in ambient levels of ANF mRNA. The physiologic significance of these findings is discussed.

Animals↗
PubMed1986

The effect of phenothiazines upon maintenance of membrane integrity in the cultured myocardial cell.

The cultured myocardial cell provides a defined model for examining factors which are responsible for maintaining cellular viability and sarcolemmal integrity. Our data indicates that the spontaneous loss of myocyte membrane integrity is a calcium-dependent process and thus provides a method for examining the mechanism through which calcium exerts this effect. Antimyosin antibody staining and propidium iodide uptake were used to quantitate membrane integrity. The integrity of the cell membrane was inversely related to the calcium concentration in the culture medium. This loss of membrane integrity was calmodulin-dependent as demonstrated by the following: phenothiazines (trifluoperazine greater than chlorpromazine greater than promethazine) and structurally dissimilar calmodulin-inhibitors prevented the formation of sarcolemmal defects at concentrations similar to those known to inhibit calmodulin; phenothiazines and calcium demonstrated a competitive interaction with respect to this effect on membrane integrity. Electron microscopy confirmed the integrity of the sarcolemma of the cells exposed to high phenothiazine concentrations although metabolic alterations occurred in these cells as evidenced by an increased membrane permeability to the low molecular weight probe propidium iodide, degenerative changes in the fine structure of the mitochondria, the accumulation of autophagic vacuoles in the cytoplasm and the loss of contractile ability. These findings indicate that calmodulin inhibitory compounds are capable of preserving the membrane integrity of cardiac myocytes, interfering with a calcium-dependent process that is associated with the spontaneous attrition of these cells in culture. Significant intracellular alterations appear at high doses of these agents even while the sarcolemma is free of gross defects.

Animals↗
PubMed1986

Photoaffinity labeling of the porcine brain alpha 2-adrenergic receptor using a radioiodinated arylazide derivative of rauwolscine: identification of the hormone-binding subunit.

A functionalized derivative of the alpha 2-selective antagonist rauwolscine formed the basis for a photoaffinity adduct that has allowed identification of the hormone-binding subunit of the brain alpha 2-adrenergic receptor protein. Rauwolscine carboxylate underwent reaction with 4-N-t-butyloxycarbonyl-aminoaniline, leading to the synthesis of rauwolscine 4-aminophenyl carboxamide (Rau-AmPC). Rau-AmPC was radioiodinated and converted to the arylazide derivative, 17 alpha-hydroxy-20 alpha-yohimban-16 beta-[N-(4-azido-3-[125I]iodo)phenyl] carboxamide (125I-Rau-AzPC), via a diazonium salt intermediate. The characterization of 125I-Rau-AzPC as a photolabile probe employed alpha 2-adrenergic receptors, which were first solubilized from porcine brain membranes and partially purified by affinity chromatography utilizing a yohimbine-agarose affinity matrix. In the partially purified receptor preparation incubated with 125I-Rau-AzPC, photolysis resulted in covalent labeling of a major (Mr, 62,000) peptide as determined by NaDodSO4/PAGE and autoradiography. Labeling of this peptide was inhibited by the alpha 2-selective antagonist, yohimbine, and the non-subtype-selective alpha-antagonist, phentolamine, but not by the alpha 1-antagonist, prazosin, or the beta-receptor antagonist, (-)-alprenolol. The alpha-adrenergic agonist epinephrine also inhibited labeling in a stereoselective manner. These data indicate that the photolabeled Mr 62,000 peptide is the hormone-binding subunit of the alpha 2-adrenergic receptor protein. The availability of this radioiodinated photoaffinity probe for the alpha 2-adrenergic receptor should facilitate further structural and biophysical characterization of the receptor protein.

Affinity Labels↗
PubMed1986

Subtypes of beta-adrenergic receptors in bovine coronary arteries.

Whether large coronary artery dilation induced by beta-adrenergic stimulation is mediated by beta 1- or beta 2-adrenergic receptors remains controversial. This problem is particularly difficult to address in vivo due to the concomitant increase in coronary blood flow with beta-adrenergic stimulation, which by itself can dilate large coronary arteries. To reconcile this problem, 5 calves were instrumented with intraaortic and intracoronary (i.c.) catheters, ultrasonic diameter transducers, Doppler flow transducers, and hydraulic occluders on the left circumflex coronary artery. Two to six weeks following surgery, beta-adrenergic agonists were administered i.c. to avoid complicating systemic effects. Isoproterenol (0.0025 micrograms/kg, a beta 1 + beta 2-adrenergic agonist) increased coronary diameter (7.1 +/- 0.8% from 5.80 +/- 0.58 mm) (p less than 0.01). Similar increases (p less than 0.01) in coronary diameter occurred with prenalterol (0.4 micrograms/kg, beta 1-adrenergic agonist) (9.5 +/- 1.4%) and pirbuterol (0.25 micrograms/kg, beta 2-adrenergic agonist) (8.1 +/- 1.2%). When coronary blood flow was prevented from rising with the hydraulic constrictor, increases in coronary diameter to all three beta-adrenergic agonists were not attenuated. Large coronary artery dilation with prenalterol and pirbuterol was abolished with beta 1- and beta 2-adrenergic receptor blockade, respectively, while neither beta 1- nor beta 2-adrenergic blockade alone abolished the large coronary artery dilation with isoproterenol. To identify the predominant subtype of beta-adrenergic receptor, competitive inhibition curves utilizing 125I-cyanopindolol (125I-CYP) as the radiolabel versus isoproterenol, epinephrine, and norepinephrine were generated in membrane preparations from calf heart (predominant beta 1), calf lung (predominant beta 2) and calf coronary artery. The coronary artery membrane preparations demonstrated an intermediate pattern. Competition curves with selective beta 1- and beta 2-adrenergic receptor agonists and antagonists again demonstrated a pattern for coronary artery intermediate to that of heart and lung, further confirming the presence of both beta-adrenergic receptor subtypes in large coronary arteries, with a ratio of beta 1: beta 2 of 1.5-2.0:1.0. Thus, large coronary arteries of the calf contain both beta 1- and beta 2-adrenergic receptors identified utilizing ligand binding techniques, and stimulation of both receptor subtypes in the intact conscious animal results in large coronary artery dilation, independent of blood-flow-mediated vasodilation.

Adrenergic beta-Agonists↗
PubMed1986

Synthesis and characterization of a high affinity radioiodinated probe for the alpha 2-adrenergic receptor.

The availability of radioiodinated probes has facilitated the localization and molecular characterization of cell membrane receptors for hormones and neurotransmitters. However, such probes are not available for the study of the alpha 2-adrenergic receptor. This report describes the synthesis and characterization of functionalized derivatives of the selective alpha 2-adrenergic antagonists, rauwolscine and yohimbine, which can be radiolabeled to high specific activity with 125I. Following demethylation of rauwolscine or yohimbine, the resultant carboxylic acid derivatives were reacted with 4-aminophenethylamine to yield the respective 4-aminophenethyl carboxamides, 17 alpha-hydroxy-20 alpha-yohimban-16 beta-[N-4-amino-phenethyl]carboxamide (rau-pAPC) and 17 alpha-hydroxy-20 beta-yohimban-16 alpha-[N-4-aminophenethyl]carboxamide. In competitive inhibition studies using rat renal membranes and the radioligand [3H]rauwolscine, rau-pAPC (Ki = 11 +/- 1 nM) exhibited a 14-fold greater affinity than the corresponding yohimbine derivative (Ki = 136 +/- 45 nM). The higher affinity compound, rau-pAPC, was radioiodinated by the chloramine T method, and the product, 125I-rau-pAPC [17 alpha-hydroxy-20 alpha-yohimban-16 beta-(N-4-amino-3 -[125I]iodophenethyl)carboxamide], was purified by reverse phase HPLC to high specific activity (2175 Ci/mmol) and its binding characteristics were investigated in rat kidney membranes. Specific binding of 125I-rau-pAPC was saturable and of high affinity as determined by Scatchard analysis (KD = 1.8 +/- 0.3 nM) or from kinetic studies (KD = k2/k1 = 0.056 +/- 0.013 min-1)/4.3 +/- 0.2 X 10(7) M-1 min-1 = 1.3 +/- 0.3 nM). In competition studies, alpha-adrenergic antagonists and agonists inhibited the binding of 125I-rau-pAPC with a potency order consistent with an interaction at alpha 2-adrenergic receptors (rauwolscine greater than phentolamine greater than prazosin; clonidine greater than (-)-epinephrine greater than (-)-norepinephrine greater than dopamine greater than (+)-epinephrine). In rat liver and human platelet membranes, high affinity binding of 125I-rau-pAPC was also observed (liver, KD = 1.2 +/- 0.4 nM; platelet, KD = 3.2 +/- 1.5 nM). In addition, the density of alpha 2-adrenergic receptors identified from binding studies with 125I-rau-pAPC in kidney, liver, and platelet membranes was similar to that observed in parallel studies with [3H]rauwolscine. These findings indicate that 125I-rau-pAPC is a high affinity probe that selectively identifies alpha 2-adrenergic binding sites. Availability of this radioligand should facilitate the localization and biochemical characterization of this alpha-adrenergic receptor subtype.

Animals↗
PubMed1985

Biosynthesis and secretion of proatrial natriuretic factor by cultured rat cardiocytes.

Rat atrial natriuretic factor (ANF) is translated as a 152-amino acid precursor preproANF. PreproANF is converted to the 126-amino acid proANF, the storage form of ANF in the atria. ANF isolated from the blood is approximately 25 amino acids long. It is demonstrated here that rat cardiocytes in culture store and secrete proANF. Incubation of proANF with serum produced a smaller ANF peptide. PreproANF seems to be processed to proANF in the atria, and proANF appears to be released into the blood, where it is converted by a protease to a smaller peptide.

Amino Acid Sequence↗
PubMed1985

Transient high-affinity binding of agonists to alpha 1-adrenergic receptors of intact liver cells.

At alpha 1-adrenergic receptors in isolated rat liver parenchymal cells, (-)-epinephrine is potent in eliciting a maximal increase in glycogenolysis (Kact = 24 nM). This contrasts with a 100-fold lower affinity for the agonist at alpha 1-adrenergic receptors of intact hepatocytes determined from equilibrium competition assays with the alpha 1-adrenergic antagonist [3H]prazosin. We demonstrate here that agonists bind to alpha 1-adrenergic receptors of intact liver cells initially with a markedly higher affinity than under equilibrium conditions. When incubations are performed for 15 s at 37 degrees C, the affinity is more than 100-fold higher than that obtained in equilibrium (45 min) assays (IC50 = 28 +/- 3 vs 5300 +/- 400 nM for (-)-epinephrine and 32 +/- 3 vs 6100 +/- 500 nM for (-)-norepinephrine). When incubations are performed at 4 degrees C (150 min), high-affinity binding similar to that obtained in short-term incubations can also be demonstrated. In contrast, antagonist compete with similar affinities in 15 s and 45 min assays, and their dissociation constants are not affected by changes in the incubation temperature. These results indicate that agonists bind to native alpha 1-adrenergic receptors transiently with high affinity. The conversion of receptors to a state of predominantly low affinity for agonists, which occurs rapidly and irreversibly with increasing incubation at 37 degrees C, is inhibited at low incubation temperatures. It is suggested that the high-affinity configuration of the alpha 1-adrenergic receptor for agonists observed in nonequilibrium experiments or at reduced incubation temperatures represents the physiologically relevant state of the alpha 1-adrenergic receptor.

Adrenergic alpha-Agonists↗
PubMed1985

Structural similarities between the mammalian beta-adrenergic and reovirus type 3 receptors.

The mechanism by which viruses bind to and infect specific tissues to cause disease has only recently begun to be understood. The mammalian reoviruses provide an especially attractive model for studying the details of cell surface recognition. The cell and tissue tropism of reovirus is determined by a portion of the viral hemagglutinin termed the neutralization domain. We have reported previously on the generation of both monoclonal and polyclonal anti-idiotypic antibodies that mimic the viral hemagglutinin in the specificity of binding to the reovirus receptor. By using these anti-idiotypic antibodies as specific probes, we have successfully isolated the mammalian reovirus receptor from neuronal and lymphoid cells. In the present study, we report that the reovirus receptor is structurally similar to the mammalian beta-adrenergic receptor. This conclusion is based on the following observations: (i) purified beta-adrenergic receptor is immunoprecipitable by anti-reovirus receptor antibody; (ii) purified reovirus receptor obtained from murine thymoma cells and beta-adrenergic receptor obtained from calf lung exhibit identical molecular masses and isoelectric points; (iii) trypsin digests of purified reovirus and beta-adrenergic receptors display indistinguishable fragment patterns; (iv) purified reovirus receptor binds the beta-antagonist [125I]iodohydroxybenzylpindolol and this binding is blocked by the beta-agonist isoproterenol.

Animals↗
PubMed1985

Mechanisms of supersensitivity to sympathomimetic amines in the chronically denervated heart of the conscious dog.

Mechanisms of denervation supersensitivity to sympathomimetic amines were studied in conscious animals. Norepinephrine, 0.1 micrograms/kg, increased left ventricular dP/dt significantly more (4208 +/- 828 mm Hg/sec) in dogs with cardiac denervation than in intact dogs (1029 +/- 280 mm Hg/sec), P less than 0.01, whereas responses to isoproterenol were similar in both groups. Denervation supersensitivity to isoproterenol could be demonstrated only after opposing reflex effects were blocked. After ganglionic blockade, norepinephrine still induced 2- to 3-fold greater increases in left ventricular dP/dt and 3- to 7-fold greater increases in heart rate in cardiac-denervated dogs, whereas isoproterenol and prenalterol, not taken up by presynaptic nerve endings, elicited only 40%-50% greater increases in left ventricular dP/dt and heart rate in dogs with cardiac denervation. The density of beta-adrenergic receptors [( 3H]dihydroalprenolol) was elevated (P less than 0.01) in denervated left ventricles (105 +/- 6.9 fmol/mg protein, n = 8) compared to normal left ventricles (70 +/- 6.3 fmol/mg protein, n = 12). This was accompanied by enhanced isoproterenol-mediated adenylate cyclase activity. However, muscarinic cholinergic receptor density, [( 3H]quinuclidinyl benzilate), decreased from control levels of 251 +/- 11 fmol/mg protein (n = 7) to 193 +/- 14 fmol/mg protein (n = 6). Thus, chronic cardiac denervation results in upregulation of the beta-adrenergic receptor and down-regulation of the muscarinic receptor. The increased beta-adrenergic receptor density and adenylate cyclase stimulation correlated well with the amount of denervation supersensitivity to isoproterenol and prenalterol, but accounted for only a minor fraction of denervation supersensitivity to norepinephrine. The major mechanism of denervation supersensitivity to norepinephrine appears to involve lack of the norepinephrine reuptake.

Adenylyl Cyclases↗
PubMed1985

Loss of high affinity cardiac beta adrenergic receptors in dogs with heart failure.

We studied the alterations in myocardial beta-adrenergic receptor-adenylate cyclase activity and muscarinic receptor density in a canine model of left ventricular (LV) failure. LV failure was characterized by a doubling of LV weight/body weight ratio (3.3 +/- 0.1 to 6.9 +/- 0.4 g/kg) and an elevation of LV end-diastolic pressure, 32 +/- 4.5 mmHg, compared with 7.7 +/- 0.6 mmHg in normal dogs. Despite a 44% increase in receptor density as measured by antagonist binding studies with [3H]dihydroalprenolol, there was a twofold decrease in receptor affinity, i.e., an increase in the dissociation constant (Kd) (5.6 +/- 0.7 to 12 +/- 1.6 nM) in heart failure. Agonist displacement of [3H]dihydroalprenolol binding with isoproterenol in the presence and absence of 5'-guanylylimidodiphosphate [Gpp(NH)p] demonstrated a striking loss of high affinity binding sites in heart failure (51 +/- 16 to 11 +/- 5%). Beta-Adrenergic receptor-mediated stimulation of adenylate cyclase and maximal stimulation with Gpp(NH)p or sodium fluoride was reduced in heart failure. There was a concomitant marked, P less than 0.01, reduction in muscarinic receptor density (242 +/- 19 vs. 111 +/- 20 fmol/mg). Thus, while muscarinic receptor density fell, beta-adrenergic receptor density actually increased in LV failure. However, a larger portion of the beta-adrenergic receptors are not functionally coupled to the GTP-stimulatory protein (Ns), as evidenced by a decrease in the fraction of receptors that bind agonist with high affinity.

Adenylyl Cyclases↗
PubMed1985

Characterization of monoclonal antibodies to the beta-adrenergic antagonist alprenolol as models of the receptor binding site.

Antibodies to receptor ligands have been valuable in understanding the nature of receptor-ligand interactions. We have developed four monoclonal antibodies to the beta-adrenergic receptor antagonist alprenolol by immunizing A/J mice with (-)-alprenolol coupled to keyhole limpet hemocyanin. The antisera from these mice displayed specific [3H]dihydroalprenolol ([3H]DHA) binding that was inhibited by alprenolol, propranolol, and isoproterenol. Somatic cell fusion of spleen cells from the immunized mice to SP2/0 myeloma cells, followed by limited dilution subcloning, resulted in the isolation of four hybridomas (1B7, 5B7, 5D9, and 2G9) demonstrating three different classes of ligand binding characteristics. 1B7 had the highest binding affinity for antagonists based on Scatchard analysis (Kd [125I]- CYP = 1.4 X 10(-10) M; Kd [3H]DHA = 6.5 X 10(-9) M), and was the only antibody to demonstrate agonist-inhibition of [3H]DHA binding. Ki values computed from competitive inhibition curves of [3H]DHA binding to 1B7 resulted in a rank order of potency similar to that of beta-2-adrenergic receptors: (-)-propranolol greater than acebutolol amine greater than isoproterenol greater than (+)-propranolol greater than epinephrine greater than norepinephrine. 5B7 and 5D9 exemplified a second class of antibody. This pair had lower antagonist binding affinities (Kd [3H]DHA = 2 X 10(-8) M and 2.5 X 10(-7) M, respectively) and was stereoselective in binding receptor antagonists: (-)-propranolol greater than (+)-propranolol greater than acebutolol amine. Agonist inhibition of [3H]DHA binding to these antibodies could only be observed at very high concentrations (greater than 10(-4) M agonist), and was not dose-dependent. Finally, the class of anti-alprenolol monoclonal antibodies represented by 2G9 had the lowest antagonist binding affinity of all (IC50 alprenolol = 1 X 10(-5) M), did not demonstrate ligand stereoselectivity, and did not recognize agonists. We propose that antibodies raised against beta-adrenergic receptor ligands demonstrating stereoselective agonist binding will also demonstrate high affinity antagonist binding, and that they will closely parallel the binding characteristics of the receptor. According to this "agonist best-fit hypothesis," anti-idiotypic antibodies raised against the binding site of these idiotypes might contain true mirror images of the beta-adrenergic receptor binding site.

Alprenolol↗
PubMed1985

Rapid reciprocal changes in adrenergic receptors in intact isolated hepatocytes during primary cell culture.

In hepatocytes freshly isolated from adult female rat livers, catecholamine-stimulated glycogenolysis is mediated predominantly by alpha 1-adrenergic receptors, and to only a minimal extent by beta 2 receptors. Primary cell culture of these hepatocytes results in a switch in the adrenergic control of glycogenolysis from an alpha 1 to a predominant beta 2 type of response. To investigate whether this switch is due to an alteration in the plasma membrane receptor composition, we characterized alpha 1 and beta 2-adrenergic receptors in both freshly isolated and cultured hepatocytes, using radioligand-binding techniques. Binding of the selective alpha 1-adrenergic antagonist [3H]prazosin and the beta-adrenergic antagonist [125I]pindolol to intact freshly isolated hepatocytes was of high affinity, saturable, and of appropriate specificity for an alpha 1- and beta 2-adrenergic receptor, respectively. Equilibrium binding studies evaluated by a computer-assisted curve-fitting procedure indicated interaction with a single class of high affinity sites for radiolabeled prazosin (KD = 126 +/- 10 pM; Bmax = 93,000 +/- 5,500 sites/cell) and pindolol (KD = 66 +/- 6 pM; Bmax = 2,000 +/- 700 sites/cell). In intact hepatocytes and in membranes prepared from these hepatocytes, competitive inhibition curves revealed the coexistence of two different sites with high and low affinities for agonists at both alpha 1- and beta 2-adrenergic receptors. When isolated hepatocytes were kept in monolayer cell culture for up to 72 hr, the switch in adrenergic control of glycogenolysis (phosphorylase a activation) from an alpha to a beta pathway was confirmed and was associated with a progressive decrease in the number of alpha 1 receptors and an increase in beta 2-adrenergic receptor density, without marked change in the affinity of agonists or antagonists. To investigate the mechanism(s) of this reciprocal change, a number of perturbations were examined including alterations in the composition of the culture medium and the influence of various hormones and inhibitors of cellular function. De novo protein synthesis is implicated in both receptor alterations as the inhibitors cycloheximide and actinomycin D prevented the increase in beta- and attenuated the decrease in alpha-adrenergic sites. The other perturbations were without effect. Thus, these studies provide evidence for a coupling of the functional alteration in glycogenolysis to changes at the receptor level per se. The mechanism underlying the reciprocal changes in hepatocyte adrenergic receptors during culture remains undefined.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1984

Photoaffinity labeling of the alpha 1-adrenergic receptor using an 125I-labeled aryl azide analogue of prazosin.

alpha 1-Adrenergic receptor probes, which can be radioiodinated to yield high specific activity radioligands, have been synthesized and characterized. 2-[4-(4-Amino-benzoyl)piperazin-1-yl]-4-amino-6,7-dimethoxyquin azoline (CP63,155), an arylamine analogue of the selective alpha 1-adrenergic antagonist prazosin, and its iodinated derivative, 2-[4-(4-amino-3-[125I]iodobenzoyl)piperazin-1-yl]-4-amino-6, 7-dimethoxyquinazoline [( 125I]CP63,789), bind reversibly and with high affinity (KD = 1 nM and 0.6 nM, respectively) to rat hepatic membrane alpha 1-adrenergic receptors. Conversion of [125I]CP63,789 to the aryl azide yields a photolabile derivative, 2-[4-(4-azido-3-[125I]iodobenzoyl)piperazin-1-yl]-4-amino-6, 7-dimethoxyquinazoline [( 125I]CP65,526), which prior to photolysis binds competitively and with high affinity (KD = 0.3 nM). Binding of [125I]CP63,789 and [125I]CP65,526 (prior to photolysis) is rapid and saturable. Both ligands identify similar alpha 1-adrenergic receptor binding site concentrations as the parent probe, [3H]prazosin. Specific binding by these iodinated ligands is stereoselective and inhibited by a variety of adrenergic agents with a specificity typical of the alpha 1-adrenergic receptor. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography of [125I]CP65,526-labeled rat hepatic membranes reveal major protein species with molecular weights of 77K, 68K and 59K. Each protein binds adrenergic ligands with stereoselectivity and with a specificity typical of the alpha 1-adrenergic receptor. Inclusion of multiple protease inhibitors during membrane preparation prior to SDS-PAGE does not alter the labeling of these peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1984

Effects of pressure overload, left ventricular hypertrophy on beta-adrenergic receptors, and responsiveness to catecholamines.

Pressure overload left ventricular (LV) hypertrophy was produced by banding the ascending aorta of puppies and allowing them to grow to adulthood. LV free wall weight per body weight increased by 87% from a normal value of 3.23 +/- 0.19 g/kg. Hemodynamic studies of conscious dogs with LV hypertrophy and of normal, conscious dogs without LV hypertrophy showed similar base-line values for mean arterial pressure, heart rate, and LV end-diastolic pressure and diameter. LV systolic pressure was significantly greater, P less than 0.01, and LV stroke shortening was significantly lss, P less than 0.01, in the LV hypertrophy group. In both normal and LV hypertrophy groups, increasing bolus doses of norepinephrine or isoproterenol produced equivalent changes in LV dP/dt. beta-adrenergic receptor binding studies with [3H]-dihydroalprenolol ( [3H]DHA) indicated that the density of binding sites was significantly elevated, P less than 0.01, in the hypertrophied LV plasma membranes (111 +/- 8.8, n = 8), as compared with normal LV (61 +/- 5.6 fmol/mg protein, n = 11). The receptor affinity decreased, i.e., disassociation constant (KD) increased, selectively in the LV of the hypertrophy group; the KD in the normal LV was 6.8 +/- 0.7 nM compared with 10.7 +/- 1.8 nM in the hypertrophied LV. These effects were observed only in the LV of the LV hypertrophy group and not in the right ventricles from the same dogs. The plasma membrane marker, 5' -nucleotidase activity, was slightly lower per milligram protein in the LV hypertrophy group, indicating that the differences in beta-adrenergic receptor binding and affinity were not due to an increase in plasma membrane protein in the LV hypertrophy group. The EC50 for isoproterenol-stimulated adenylate cyclase activity was similar in both the right and left ventricles and in the two groups. However, maximal-stimulated adenylate cyclase was lower in the hypertrophied left ventricle. Plasma catecholamines were similar in the normal and hypertrophied groups, but myocardial norepinephrine was depressed in the dogs with LV hypertrophy (163 +/- 48 pg/mg) compared with normal dogs (835 +/- 166 pg/mg). Thus, severe, but compensated LV hypertrophy, induced by aortic banding in puppies, is characterized by essentially normal hemodynamics in adult dogs studied at rest and in response to catecholamines in the conscious state. At the cellular level, reduced affinity and increased beta-adrenergic receptor number characterized the LV hypertrophy group, while the EC50 for isoproterenol-stimulated adenylate cyclase activity was normal. By these mechanisms, adequate responsiveness to catecholamines is retained in conscious dogs with severe LV hypertrophy.

Animals↗
PubMed1984

The lung beta-receptor in the spontaneous hypertensive rat.

The basic mechanism underlying the abnormal systemic vascular resistance in the spontaneously hypertensive rat (SHR) is poorly understood. To categorize the beta-receptor system in the lungs of these animals, we evaluated receptor number and affinity as an index of this subclass of beta receptors. The lung was chosen because it is not directly affected by the alteration in blood pressure and provides a ready supply of beta-receptors. The aortic blood pressure and heart rate were measured directly through cannulation of the carotid artery. Twenty-four hours later, lung membranes from the SHR and control animals were extracted and partially purified. The affinity and number of receptors were measured by saturation analysis employing 3H-dihydroalprenolol (3H-DHA). Scatchard analysis demonstrated that the number of receptors was identical and of a single class in both sets of animals. However, the beta-receptor from the SHR had a higher affinity than that of normal rats. It is not clear whether this represents an adaptive mechanism following the development of hypertension in the SHR or it represents a basic alteration in this strain of rat. Further studies aimed at categorizing the development of this defect during ontogeny as well as its physiologic consequences should prove valuable in understanding the mechanism of spontaneous hypertension.

Animals↗
PubMed1983

Photoaffinity label for the alpha 1-adrenergic receptor: synthesis and effects on membrane and affinity-purified receptors.

An azide analog, 2-[4-(4-azidobenzoyl)piperazin-1-yl]-4-amino-6, 7-dimethoxyquinazoline (CP59,430), of the highly selective alpha 1-adrenergic receptor antagonist prazosin was synthesized and its effects on rat hepatic membrane and affinity-purified alpha 1-adrenergic receptor preparations were examined. CP59,430 behaved as a competitive antagonist before photolysis. When the membrane or purified preparations pretreated with CP59,430 were irradiated with UV light, CP59,430 behaved as a noncompetitive antagonist. Labeling of membrane alpha 1-adrenergic receptors was irreversible; repeated dialysis or washing could not reverse the photolysis-induced inactivation by CP59,430, whereas dialysis completely reversed the antagonism by the same concentration of the label prior to photolysis. Additionally, photolabeling of purified receptors was resistant to Sephadex G-50 chromatography, whereas in the absence of photolysis the same concentration of CP59,430 or prazosin (10 microM) could be readily removed by this procedure. CP59,430 appears to label specifically only alpha 1-adrenergic receptors because prazosin protected the membrane and purified receptors from photolysis-induced inactivation by CP59,430. Furthermore, specific [3H]dihydroalprenolol and [3H]yohimbine binding to membrane beta- and alpha 2-adrenergic receptors, respectively, was unchanged by CP59,430 at 1 microM, a concentration that decreased specific [3H]prazosin binding to alpha 1-adrenergic receptors by 72%. In additional studies, the photolysis-induced receptor inactivation by CP59,430 remained unchanged in the presence of the scavenger p-aminobenzoic acid. It is likely, therefore, that receptor labeling by CP59,430 occurs via a true photoaffinity mechanism. CP59,430, which specifically and irreversibly labels the alpha 1-adrenergic receptor after photolysis, should thus be uniquely valuable for the molecular characterization of this receptor.

Affinity Labels↗