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R M Graham

Publications and source records attributed to R M Graham.

At least 127 records · Page 7Linked to original sources
PubMed1987

Structural properties of the alpha 1-adrenergic receptor: studies with membrane and purified receptor preparations.

The contribution of hydrophobic, ionic, and disulfide bonds in the alpha 1-adrenergic receptor structure was evaluated using rat hepatic plasma membrane and purified receptor preparations. In addition, the findings were compared with similar structural evaluations of the beta 2-adrenergic receptor purified from canine lung. Both the alpha 1- and beta 2-adrenergic receptors contain a disulfide bond critical for ligand binding. However, the disulfide bond in the beta-receptor structure is accessible to solvent, while the disulfide bond within the alpha-receptor structure is "masked" and inaccessible to solvent. Reduction of this disulfide bond requires initial denaturation of the alpha 1-receptor. Although the tertiary structures of both receptor proteins appear to be stabilized by strong hydrophobic interactions, differential sensitivities are observed with a variety of structural perturbants, including high concentrations of sodium chloride, guanidine hydrochloride, and ethylene glycol.

Animals↗
PubMed1987

Glycosylation of the mammalian alpha 1-adrenergic receptor by complex type N-linked oligosaccharides.

The binding subunit of the alpha 1-adrenergic receptor has been identified as an Mr = 80,000 peptide in several tissues. Adsorption of the alpha 1-adrenergic receptor to a wheat germ agglutinin lectin-agarose resin suggests that the receptor protein is glycosylated. In this study, we investigated the nature of the carbohydrate chains linked to the alpha 1-adrenergic receptor peptide. The alpha 1-adrenergic receptor from DDT2 MF-2 smooth muscle cell and rat brain membranes was photolabeled with 125I-azido-prazosin [( 125I]CP65,526) and then treated with exoglycohydrolases prior to SDS-PAGE and autoradiography. Removal of terminal sialic acid residues by neuraminidase decreased the receptor Mr by 6,000; however, alpha-mannosidase was without effect, indicating complex type glycosylation of the receptor-protein. Similar results were observed for the rat hepatic membrane alpha 1-adrenergic receptor. Removal of N-linked carbohydrates at asparagine residues by peptide-N4[N-acetyl-beta-glucosaminyl]asparagine amidase (from Flavobacterium meningosepticum) resulted in a specifically labeled peptide at Mr = 50,000-55,000 in DDT1 MF-2 membrane and solubilized receptor preparations. Treatment of DDT1 MF-2 cells with swainsonine or (+)-1-deoxymannojirimycin, inhibitors of complex type carbohydrate chain biosynthesis, caused a reduction in the apparent molecular weight of the receptor (Mr = 60,000) but did not alter the number of alpha 1-adrenergic receptors per cell or their affinity for the radioligand [3H]prazosin. These findings indicate that the alpha 1-adrenergic receptor is heavily glycosylated, the major oligosaccharide moiety being of the complex type, N-linked to asparagine residues. The peptide backbone of the receptor has an Mr less than or equal to 55,000, consistent with the predicted molecular mass of other membrane neurotransmitter receptors based on sequence analysis of isolated cDNA clones.

Animals↗
PubMed1986

Agonist interactions at hepatic alpha 1- and beta-adrenergic receptors: affinity-state regulation by guanine nucleotides and temperature.

We investigated the binding characteristics of agonists to alpha 1- and beta-adrenergic receptors of intact liver cells, broken rat liver cell membranes, and detergent-solubilized preparations under varying experimental conditions, focusing on the different "states" of the receptor for agonists and the regulation of these states by temperature and guanine nucleotides. While only low-affinity binding of agonists to both receptor subtypes was evident in studies performed at 37 degrees C with solubilized preparations, biphasic competition curves for agonists were observed in both intact cells and membrane preparations; the majority of sites were of low affinity. In membrane preparations, the nonhydrolyzable GTP analogue Gpp(NH)p caused a rightward shift of agonist competition curves and a loss of high-affinity binding. These results are consistent with the involvement of guanine nucleotide binding proteins in both alpha 1- and beta-adrenergic transduction pathways. When competition studies were performed at 4 degrees C, receptor sites existed predominantly in the high-affinity configuration, in intact cells and membranes, as well as in soluble preparations. In contrast to the studies conducted at 37 degrees C, no Gpp(NH)p-induced conversion to the lower affinity state could be demonstrated in studies performed with membrane preparations at 4 degrees C. Thus, the high-affinity state of alpha 1- and beta-adrenergic receptors is stabilized at 4 degrees C in intact cells, membranes, and soluble preparations. After incubations had been performed at 37 degrees C, high-affinity binding of agonists could not be restored by subsequent incubation at 4 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic alpha-Agonists↗
PubMed1986

Serum-mediated enhancement of ANF accumulation in the culture medium of cardiac atriocytes.

Cultured cardiac myocytes provide a useful system for investigating ANF biosynthesis and regulation. It has previously been demonstrated that the predominant form of ANF stored in and released from this myocyte model is the 17 kD prohormone, proANF. We report here that as quantitated both by radioimmunoassay and by SDS-PAGE of intrinsically labelled ANF released from these cardiocytes, the addition of serum promotes a 5-6 fold increase in ANF accumulation in the medium of these cells as compared to ANF accumulation in the presence of a defined chemical medium alone. The stimulating effect of serum is immediate and persists in a linear manner for at least 120 minutes. This effect of serum can be reproduced by the addition of albumin or other proteins to the medium but not by alterations in osmolality. Whether this phenomenon represents enhanced release of proANF or is secondary to inhibition of proANF degradation has yet to be determined.

Atrial Natriuretic Factor↗
PubMed1986

NADP improves the efficiency of cholera toxin catalyzed ADP-ribosylation in liver and heart membranes.

Guanine nucleotide binding proteins (G-proteins) can be identified by their ability to be ADP-ribosylated using [32P]NAD as the substrate and bacterial toxins as catalysts. This labelling, when performed in liver and sarcolemma membrane preparations, can be complicated by competing enzymes which degrade NAD, making it unavailable to participate in the desired reaction. The addition of NADP in reaction mixtures markedly slows the degradation of NAD, but does not compete with NAD in cholera toxin labelling of stimulatory G-protein. The efficiency of cholera toxin labelling is improved to the extent that saturation curves may be constructed, allowing the quantitation of ADP-ribosylation sites in membranes.

Animals↗
PubMed1986

Agonist-induced isomerization of the alpha 1-adrenergic receptor: kinetic analysis using broken-cell and solubilized preparations.

The affinity of agonists but not antagonists at hepatic membrane alpha 1-adrenergic receptors is temperature dependent; a 100-fold higher affinity is observed at 4 degrees C than at 37 degrees C. The relationship between these two agonist affinity states was investigated by using a strategy that allows the kinetics of this transition to be examined under equilibrium conditions. When competition assays are performed at 37 degrees C for varying intervals and the reaction mixture is then rapidly cooled by freezing, allowed to thaw, and further equilibrated at 4 degrees C, a rapid and progressive decrease (t1/2 of 1-2 min) in agonist affinity occurs, the extent of which is directly related to the incubation time at 37 degrees C. This decrease in agonist affinity is sustained as long as agonist is present but can be reversed by its subsequent removal. In contrast, no change in affinity is seen in identical experiments when antagonists are employed as the competing ligand. High-affinity binding of agonists is also demonstrated in short-term nonequilibrium experiments, indicating that the low-temperature incubations do not induce, but rather stabilize, a receptor conformation of high affinity for agonists. These findings suggest that the predominantly low-affinity binding of agonists to alpha 1-adrenergic receptors demonstrated in equilibrium studies at physiological temperatures may be the result of a ligand-driven decrease in affinity. Since the transition in receptor affinity for agonists occurs not only in broken-cell preparations but also after detergent solubilization of the membrane receptor, it most likely is due to an agonist-induced change in the conformation of the receptor protein per se.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Phorbol esters inhibit alpha 1-adrenergic effects and decrease the affinity of liver cell alpha 1-adrenergic receptors for (-)-epinephrine.

4 beta-Phorbol 12-myristate 13-acetate (PMA) modified the metabolic actions of three calcium-dependent hormones in different ways. The stimulations of glycogenolysis ureogenesis and phosphatidylinositol labeling produced by alpha 1-adrenergic agonist was blocked by the phorbol ester. In contrast, PMA slightly increased the stimulation of ureogenesis produced by low concentration of angiotensin II without modifying the maximal response. No effect of PMA was observed on the stimulation of ureogenesis induced by vasopressin. The stimulation of phosphatidylinositol labeling induced by vasopressin was decreased by PMA, whereas that induced by angiotensin II was not affected. In intact freshly isolated hepatocytes, [3H]prazosin binds with high affinity to a site which displays the characteristics of alpha 1-adrenergic receptor. Competitive inhibition studies with (-)-epinephrine reveal two different sites for this agonist: a high affinity site (Kd 9 nM) and a low affinity site (Kd 2 microM). In the presence of phorbol esters, (-)-epinephrine binding data now show the presence of a single class of low affinity sites, with similar affinity to those present in control cells. Thus, the inhibition of hepatocyte alpha 1-adrenergic action by PMA may be related to the loss of high affinity binding sites caused by the tumor promoter.

Angiotensin II↗
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

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

Bartter's syndrome and the atrial natriuretic factor gene.

We investigated whether the gene for atrial natriuretic factor, a recently discovered peptide hormone with potent natriuretic, diuretic, and vasorelaxant properties, was pathogenetically linked to an uncommon but well-defined fluid and electrolyte disorder, Bartter's syndrome. Restriction fragment length polymorphisms in the atrial natriuretic factor gene were sought in a large kindred with six of 23 family members being affected. A Bgl II polymorphism, identified in two of 40 (5%) apparently normal subjects, was found in one of two first-generation family members. This polymorphism was also present in five of seven unaffected second-generation siblings but in only three of six affected siblings. The failure of the absence or the presence of the polymorphism to cosegregate with the disease clearly indicates that in this kindred, the gene for atrial natriuretic factor is not linked to Bartter's syndrome.

Atrial Natriuretic Factor↗
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

Single and combined therapy for systemic hypertension with propranolol, hydralazine and hydrochlorothiazide: hemodynamic and neuroendocrine mechanisms of action.

The antihypertensive mechanisms of single and combined therapy with a beta-adrenergic antagonist (propranolol) and a vasodilator (hydralazine) were investigated in 9 patients with moderately severe hypertension, who were receiving maintenance diuretic (hydrochlorothiazide) treatment. Hemodynamic and neuroendocrine responses were determined at rest and during lower body negative pressure, and dynamic and static exercise stress after the chronic administration of propranolol and hydralazine, given alone or in combination. All 3 drug regimens, each administered for at least 10 weeks, reduced blood pressure (p less than 0.05) compared with diuretic-only therapy in patients at rest, in both the supine and standing position, and during lower body negative pressure and dynamic exercise. There was a significant additive antihypertensive effect when propranolol and hydralazine were combined. Only combination therapy effectively lowered pressure during static exercise. The regimens produced divergent effects on the supine cardiac output: a decrease with propranolol (p less than 0.05), no change with combination therapy and an increase with hydralazine (p less than 0.05). Both hydralazine and combination therapy significantly reduced supine total peripheral resistance (p less than 0.05), whereas propranolol produced no change. All 3 drug treatments significantly reduced total peripheral resistance during upright rest and dynamic exercise (p less than 0.05), without changing cardiac output or maximal exercise capacity. During exercise, cardiac output was maintained in patients treated with propranolol and in those treated with combined therapy by increases in stroke volume (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1985

Differences in response of black hypertensives to alpha vs. beta adrenergic antagonists: preliminary findings.

We tested our clinical impression that black hypertensives in our clinic population responded better to alpha-adrenergic blocking agents (clonidine and prazosin) than to beta-adrenergic blockers (atenolol, nadolol and propranolol). Compared to no effect from eight weeks of therapy with beta-blockers, clonidine significantly decreased erect mean arterial pressure (MAP) when assessed weekly for four weeks (p = 0.027 to 0.046). However, the decrease in supine MAP was not significant. The effects of prazosin were more modest. Supine MAP was significantly less than with beta-blockade (p = 0.032) at two weeks but not at four weeks and decrements in erect MAP were not significant. In this preliminary study, black hypertensives appeared to be more responsive to alpha-adrenergic antagonists than to beta-blockers, with clonidine more effective than prazosin. Elucidation of possible mechanisms of the difference and of its clinical importance warrant further study.

Adrenergic alpha-Antagonists↗