Biomedical subjects

R M Graham

Publications and source records attributed to R M Graham.

At least 19 recordsLinked to original sources

PubMed1992

Genomic organization and expression of the human alpha 1B-adrenergic receptor.

alpha 1-Adrenergic receptors (ARs) are members of the guanine nucleotide-binding protein-coupled receptor superfamily. The genes for all ARs described thus far are intronless. We report here the cloning and the nucleotide sequence of the gene for the human alpha 1B-AR. It consists of two exons and a single large intron of at least 20 kilobases which interrupts the coding region at the end of the putative sixth transmembrane domain. The deduced amino acid sequence of the encoded receptor has a high degree of homology to the cloned hamster, rat, and dog alpha 1B-ARs. To characterize the encoded protein, a fusion gene constructed by splicing together exon 1 and exon 2 was expressed transiently in COS-1 cells. The transfected gene fusion product resulted in the production of an alpha 1B-AR with ligand binding characteristics indistinguishable from those of the expressed hamster alpha 1B cDNA. Evidence that the human alpha 1B-AR gene we have isolated is indeed transcribed is the finding of similar sized (2.8-kilobase) transcripts in human heart and other tissues by Northern blot analysis when either exon 1 or exon 2 is used as a probe. Moreover, using primers designed to span the exon 1/exon 2 boundary, a polymerase chain reaction product generated from single-stranded DNA prepared from human heart mRNA had the exact size and nucleotide sequence predicted for a transcript in which exon 1 is spliced to exon 2. The 5'-flanking region (924 base pairs (bp)) of exon 1 contains neither a TATA box nor a CAAT box but is high in GC content (70%) and contains several Sp1 binding sites (GC boxes), consistent with promoters described for housekeeping genes. The 5'-untranslated region also contains a putative cyclic AMP response element. Primer extension studies and RNase protection assays suggested that there are several potential transcription start sites in most tissues with a predominant site located 173 bp upstream from the translation start site. The 3'-flanking region contains a putative polyadenylation signal (ATTAAA) 492 bp downstream from the stop codon. The genomic organization of the human alpha 1B-AR with a single large intron interrupting its coding region differs from those of other ARs as well as muscarinic and 5-hydroxy-tryptamine receptors, which are intronless. The location of the intron in the human alpha 1B-AR gene is also unique among those members of the G-protein-coupled receptor family that do possess introns.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1992

Structure and function in rhodopsin. Studies of the interaction between the rhodopsin cytoplasmic domain and transducin.

Structural requirements for the activation of transducin by rhodopsin have been studied by site-specific mutagenesis of bovine rhodopsin. A variety of single amino acid replacements and amino acid insertions and deletions of varying sizes were carried out in the two cytoplasmic loops CD (amino acids 134-151) and EF (amino acids 231-252). Except for deletion mutant delta 137-150, all the mutants bound 11-cis-retinal and displayed normal spectral characteristics. Deletion mutant delta 236-239 in loop EF caused a 50% reduction of transducin activation, whereas deletion mutant delta 244-249 and the larger deletions in loop EF abolished transducin activation. An 8-amino acid deletion in the cytoplasmic loop CD as well as a replacement of 13 amino acids with an unrelated sequence showed no transducin activation. Several single amino acid substitutions also caused significant reduction in transducin activation. The conserved charged pair Glu-134/Arg-135 in the cytoplasmic loop CD was required for transducin activation; its reversal or neutralization abolished transducin activation. Three amino acid replacements in loop EF (S240A, T243V, and K248L) resulted in significant reduction in transducin activation. We conclude that 1) both the cytoplasmic loops CD and EF are required for transducin activation, and 2) effective functional interaction between rhodopsin and transducin involves relatively large peptide sequences in the cytoplasmic loops.

Amino Acid Sequence↗

PubMed1992

Identification of 5-ethyl-5-(2-methylbutyl)barbituric acid as an impurity of manufacture in amobarbital.

Amobarbital [5-ethyl-5-(3-methylbutyl)barbituric acid], USP, was found to contain an impurity that was not associated with hydrolysis and decomposition of the barbiturate ring. The impurity was isolated by semipreparative HPLC and was identified as 5-ethyl-5-(2-methylbutyl)barbituric acid (1) by MS (electron impact and chemical ionization) and 1H NMR. The substitution pattern on the alkyl side chain was verified by using the achiral NMR shift reagent tris(6,6,7,7,8,8,8-heptafluoro-2,2- dimethyl-3,5-octanedionato)europium(III). Older samples of amobarbital, USP, contained greater than 6% of 1, whereas recent samples of amobarbital, USP, contained less than 1% of 1. Because the pharmacological profiles of 1 and amobarbital in rodents are comparable, the impurity probably does not constitute a clinically significant problem for humans.

Amobarbital↗

PubMed1992

Plasma platelet-activating factor degradation in patients with severe coronary artery disease.

1. Platelet-activating factor is inactivated in plasma by the action of a specific acetylhydrolase that cleaves the acetate moiety from the sn-2 position. Degradation was determined under optimized conditions and under conditions closer to those which may occur in vivo. The latter, or platelet-activating factor half-life, was measured by a modified method that is simple, inexpensive and reliable. 2. A hyperbolic relationship was found to exist between the two measures of degradation, the values in both normal subjects and patients with coronary artery disease falling on the tail of the hyperbola. Thus, there is an increase in platelet-activating factor half-life associated with a lowering of acetylhydrolase activity, but this increase is relatively small. 3. There were significant direct linear relationships between acetylhydrolase activity and serum total cholesterol and low-density-lipoprotein-cholesterol concentrations in both subject groups. Although acetylhydrolase activity was most closely associated with the low-density-lipoprotein-cholesterol fraction, the activity for a given serum level of low-density-lipoprotein-cholesterol was higher in patients with coronary artery disease.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗

PubMed1992

Plasma platelet activating factor degradation and serum lipids after coronary bypass surgery.

OBJECTIVE: Platelet activating factor (PAF) is a potent mediator in inflammatory responses and maybe involved in various disease states. Degradation of PAF in plasma results from the action of a specific, lipoprotein associated, acetylhydrolase. The aim was to determine plasma acetylhydrolase activity under optimised conditions, PAF half life, phospholipase A2 activity, the lyso-derivative of PAF (lyso-PAF), and lipids in patients undergoing coronary artery bypass grafting. METHODS: The study variables were determined 3 d and 7 d following coronary artery surgery and compared to presurgical values in 15 males, age 55(SEM 4) years. RESULTS: Three days following coronary bypass grafting, total, LDL and HDL cholesterol fell significantly by 30%, 45%, and 15% respectively (p less than 0.001), all decreases correlating with bypass time (p less than 0.025). Concentrations remained low at 7 d (p less than 0.005). Acetylhydrolase activity fell by 38% (p less than 0.001) at 3 d post-surgery and remained depressed, but plasma PAF half life did not change after surgery. The inverse relationship between acetylhydrolase activity and plasma PAF half life preoperatively (p less than 0.01) was not evident after surgery. There was a direct linear relationship between acetylhydrolase activity and both total (p less than 0.002) and LDL cholesterol (p less than 0.001) before surgery. The fall in acetylhydrolase activity correlated with the fall in these lipids (p less than 0.01) but not with that of HDL cholesterol. Plasma lyso-PAF decreased by 65% (p less than 0.001) at 3 d and remained depressed (p less than 0.001). Plasma phospholipase A2 activity increased by 60% (p less than 0.01) and remained raised (p less than 0.05), the increase at 3 d being related to bypass time (p less than 0.05). CONCLUSIONS: The large fall in plasma acetylhydrolase activity after coronary bypass grafting is consistent with the fall in plasma lipids. However, the absence of a significant change in the measured PAF half life in plasma raises questions as to the pathophysiological significance of the decrease in acetylhydrolase activity.

Carboxylic Ester Hydrolases↗

PubMed1992

Mechanisms of alpha 1-adrenergic vascular desensitization in conscious dogs.

To investigate the mechanisms of alpha 1-adrenergic vascular desensitization, osmotic minipumps containing either saline (n = 9) or amidephrine mesylate (AMD) (n = 9), a selective alpha 1-adrenergic receptor agonist, were implanted subcutaneously in dogs with chronically implanted arterial and right atrial pressure catheters and aortic flow probes. After chronic alpha 1-adrenergic receptor stimulation, significant physiological desensitization to acute AMD challenges was observed, i.e., pressor and vasoconstrictor responses to the alpha 1-adrenergic agonist were significantly depressed (p < 0.01) compared with responses in the same dogs studied in the conscious state before pump implantation. However, physiological desensitization to acute challenges of the neurotransmitter norepinephrine (NE) (0.1 micrograms/kg per minute) in the presence of beta-adrenergic receptor blockade was not observed for either mean arterial pressure (MAP) (30 +/- 7 versus 28 +/- 5 mm Hg) or total peripheral resistance (TPR) (29.8 +/- 4.9 versus 28.9 +/- 7.3 mm Hg/l per minute). In the presence of beta-adrenergic receptor plus ganglionic blockade after AMD pump implantation, physiological desensitization to NE was unmasked since the control responses to NE (0.1 micrograms/kg per minute) before the AMD pumps were now greater (p < 0.01) than after chronic AMD administration for both MAP (66 +/- 5 versus 32 +/- 2 mm Hg) and TPR (42.6 +/- 10.3 versus 23.9 +/- 4.4 mm Hg/l per minute). In the presence of beta-adrenergic receptor, ganglionic, plus NE-uptake blockade after AMD pump implantation, desensitization was even more apparent, since NE (0.1 micrograms/kg per minute) induced even greater differences in MAP (33 +/- 5 versus 109 +/- 6 mm Hg) and TPR (28.1 +/- 1.8 versus 111.8 +/- 14.7 mm Hg/l per minute). The maximal force of contraction induced by NE in the presence or absence of endothelium was significantly decreased (p < 0.05) in vitro in mesenteric artery rings from AMD pump dogs compared with saline control dogs. Furthermore, alpha 1-adrenergic receptor density, as determined by [3H]prazosin binding in membrane preparations from vessels in the mesentery, was decreased (8.2 +/- 1.0 versus 18.4 +/- 1.4 fmol/mg protein, p < 0.001) without any change in Kd in the AMD pump dogs compared with the saline pump dogs.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenergic alpha-Agonists↗

PubMed1991

Cloning of the gene and cDNA for human heart chymase.

We have recently identified and characterized a chymotrypsin-like serine proteinase in human heart (human heart chymase) that is the most catalytically efficient enzyme described, thus far, for the cleavage of angiotensin I to yield angiotensin II and the dipeptide His-Leu. Compared to other chymases, this enzyme also has an unusually high degree of specificity for the substrate angiotensin I. We report here the molecular cloning and nucleotide sequence of the gene and cDNA encoding human heart chymase, and determination of its entire deduced amino acid sequence. These data indicate that human heart chymase is highly homologous to other members of the chymase subfamily of chymotrypsin-like proteinases and, most likely, all evolved from a common ancestral gene. Potential regulatory elements found in the 5'-untranslated region of other chymases are also found in the human heart chymase gene. However, this gene lacks mast cell-specific sequences found in the 5'- and 3'-untranslated regions of the rat chymase II gene. In addition, human heart chymase contains clusters of unique amino acid sequences located at key positions likely involved in substrate binding, which may contribute to its high substrate specificity. These contrasting features of the human heart chymase gene and cDNA, and the potential determinants of its primary structure that underlie its unique functional characteristics are considered.

Amino Acid Sequence↗

PubMed1991

Effects of atrial natriuretic factor on blood flow distribution and vascular resistance in conscious dogs.

Systemic and regional hemodynamic responses to atrial natriuretic factor (ANF) administered by either bolus or infusion were examined in conscious dogs in the presence and absence of ganglionic blockade. In intact dogs, bolus injection of ANF (9 micrograms/kg), which increased plasma ANF by 100,913 +/- 12,330 pg/ml, initially reduced mean arterial pressure (7 +/- 1%) and total peripheral (-31 +/- 2%), coronary (-28 +/- 3%), mesenteric (-19 +/- 2%), renal (-36 +/- 2%), and iliac (-24 +/- 2%) vascular resistances. During the recovery period, resistance rose in the total peripheral (+13 +/- 3%), coronary (+19 +/- 5%), mesenteric (+7 +/- 2%), and iliac (+42 +/- 8%) beds but did not rise in the renal bed. At 15 min after infusion of ANF (0.3 micrograms.kg-1.min-1 x 30 min), which increased plasma ANF by 4,296 +/- 869 pg/ml, resistance rose in the total peripheral (+19 +/- 3%), coronary (+17 +/- 2%), mesenteric (+23 +/- 7%), and iliac (+40 +/- 11%) beds but remained slightly depressed in the renal bed (-8 +/- 5%). Ganglionic blockade with hexamethonium did not modify substantially the pattern of change of regional vascular resistances to ANF. Thus in conscious dogs, ANF under steady-state conditions was found to elicit direct vasoconstriction in most regional vascular beds and minor vasodilation in the renal vascular bed. However, after bolus administration of ANF, which results in extremely high plasma ANF levels, transient vasodilation occurs in all regional vascular beds. These data suggest that the distribution of ANF receptors, which elicit vasodilation, differs in regional vascular beds, and that in most vascular beds, except those of the kidney, these receptors require extremely high levels of ANF to be activated.

Animals↗

PubMed1991

Inotropic response to norepinephrine is augmented early and maintained late in conscious dogs with perinephritic hypertension.

We studied the inotropic responses to intravenous infusions of norepinephrine in nine conscious chronically instrumented dogs before and early (2-4 weeks) in the development of perinephritic hypertension; seven conscious dogs were studied later (approximately 14 weeks), during a more stable phase of hypertension. perinephritic hypertension was associated with a 24% increase in left ventricular (LV) mass during developing hypertension; no further increase was seen during the stable hypertension phase. LV end-systolic stress was increased early (p less than 0.01) but was normalized later. The LV end-systolic stress-volume relation demonstrated an enhanced contractile response to norepinephrine during developing hypertension, which returned toward control later in the course of stable hypertension. The LV dP/dt responses to norepinephrine (0.4 microgram/kg/min) were significantly greater during developing hypertension (7,509 +/- 337 mm Hg/sec, p less than 0.05) compared with the control period (4,737 +/- 286 mm Hg/sec) and returned toward the control value during stable hypertension (5,168 +/- 465 mm Hg/sec). The enhanced inotropic responses to norepinephrine in developing hypertension were preserved in the presence of ganglionic blockade, suggesting that the augmentation was not mediated via reflex mechanisms. These physiological responses were associated with an increase in beta-adrenergic receptor density, but no significant change in basal or maximal adenylate cyclase stimulation occurred during developing hypertension. Thus, in contrast to prior studies in anesthetized animals, the inotropic response to beta-adrenergic stimulation is not depressed in conscious dogs but is enhanced selectively during the development of hypertension and maintained during stable hypertension.

Animals↗

PubMed1991

Solution-phase library screening for the identification of rare clones: isolation of an alpha 1D-adrenergic receptor cDNA.

alpha 1-Adrenergic receptor (alpha 1-AR) subtypes (alpha 1A and alpha 1B) play a critical role in vascular smooth muscle contraction and circulatory homeostasis. Transcripts for these guanine nucleotide-binding protein-coupled receptors are extremely low in abundance, however, and isolation of their cDNAs is difficult. We have developed a novel technique for identifying rare clones in a cDNA library, which has been used successfully to isolate a cDNA clone encoding an alpha 1D-AR. A 564-bp polymerase chain reaction product encoding a region between the third and sixth transmembrane domains of the alpha 1D-AR was first generated using rat brain mRNA as template and highly degenerate primers. The primers corresponded to those domains but contained mismatches to the alpha 1B-AR sequences. A 3-kb transcript was identified with this polymerase chain reaction probe, by Northern analysis of rat hippocampus. However, traditional plaque hybridization failed to identify a cDNA in a rat hippocampus lambda gt10 library. By solution-phase screening of virtually the entire library, a cDNA containing a 3-kb insert was identified, amplified, and purified. This insert encodes a 560-amino acid protein corresponding to the topology of guanine nucleotide-binding protein-coupled receptors. This receptor has approximately 71% amino acid identity, in the transmembrane regions, to the hamster and rat alpha 1B-ARs. Characterization of the receptor expressed in COS-7 cells, by ligand binding and photoaffinity labeling, revealed some of the characteristics of an alpha 1A-AR. However, unlike alpha 1A-ARs characterized previously in membrane preparations or in solubilized partially purified preparations, the expressed receptor could be extensively inactivated by chlorethylclonidine. In addition, it displays ligand-binding properties that are not consistent with an alpha 1A-AR. This indicates that the cDNA clone that we have isolated encodes a novel alpha 1-AR subtype, which we classify as the alpha 1D-AR.

Animals↗

PubMed1990

A novel guanine nucleotide-binding protein coupled to the alpha 1-adrenergic receptor. I. Identification by photolabeling or membrane and ternary complex preparation.

The G-protein involved in alpha 1-adrenergic receptor signaling was identified using two different approaches. First, purified rat liver membranes were incubated with [alpha-32P]GTP in the absence or presence of the adrenergic agonist (-)-epinephrine, or in the presence of GTP. After UV irradiation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography, covalent labeling of a number of proteins was apparent and could be blocked by unlabeled GTP. In the preparation treated with (-)-epinephrine alone, labeling of a 74-kDa species was markedly enhanced. Enhanced labeling of 40-50-kDa species was also observed. Labeling of the 74-kDa protein was also evident in similarly treated membranes prepared from FRTL-5 thyroid cells, which contain abundant alpha 1-adrenergic receptors, but not in those prepared from turkey erythrocytes or NIH 3T3 fibroblasts, which are essentially devoid of alpha 1-receptors. Second, alpha 1-agonist-receptor-G-protein ternary complex formation was induced by incubating purified rat liver membranes with (-)-epinephrine. Rauwolscine (10(-7) M) and (+/-)-propranolol (10(-6) M) were included to prevent activation of alpha 2- and beta-adrenergic receptors by (-)-epinephrine. The ternary complex of hormone, receptor, and G-protein was solubilized, partially purified using heparin- and wheat germ agglutinin-agarose, and reconstituted into phospholipid vesicles. The vesicles displayed agonist-stimulated guanosine 5'-O-3-thiotriphosphate (GTP gamma S) binding that was blocked by phentolamine (10(-4) M). By contrast, stimulation of GTP gamma S binding was not evident when the vesicles were incubated with the beta-agonist, isoproterenol. Incubation of the vesicles with [alpha-32P]GTP or [alpha-32P]azido-GTP in the presence of (-)-epinephrine, followed by photolysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography, resulted in the covalent labeling of a 74-kDa protein. Labeling of this protein could be blocked by preincubation with phentolamine or unlabeled GTP. These findings provide direct evidence for the coupling of the alpha 1-adrenergic receptor to a previously uncharacterized G-protein (termed Gh), which has an apparent molecular mass of approximately 74 kDa.

Animals↗

PubMed1990

A novel guanine nucleotide-binding protein coupled to the alpha 1-adrenergic receptor. II. Purification, characterization, and reconstitution.

In the previous paper, we reported the identification of a 74-kDa G-protein that co-purifies with the alpha 1-adrenergic receptor following ternary complex formation. We report here on the purification and characterization of this 74-kDa G-protein (termed Gh) isolated de novo from rat liver membranes. After solubilization of rat liver membranes with the detergent sucrose monolaurate, Gh was isolated by sequential chromatography using heparin-agarose, Ultrogel AcA 34, hydroxylapatite, and heptylamine-Sepharose columns. The protein, thus isolated, is not a substrate for cholera or pertussis toxin but displays GTPase activity (turnover number, 3-5 min-1) and high-affinity guanosine 5'-O-3-thiotriphosphate (GTP gamma S) binding (half-maximal binding = 0.25-0.3 microM), which is Mg2(+)-dependent and saturable. The relative order of nucleotide binding by Gh is GTP gamma S greater than GTP greater than GDP greater than ITP much much greater than ATP greater than or equal to adenyl-5'-yl imidodiphosphate, which is similar to that observed for other heterotrimeric G-proteins involved in receptor signaling. Moreover, specific alpha 1-agonist-stimulated GTPase (turnover number, 10-15 min-1) and GTP gamma S binding activity could be demonstrated after reconstitution of purified Gh with partially purified alpha 1-adrenergic receptor into phospholipid vesicles. The alpha 1-agonist stimulation of GTP gamma S binding and GTPase activity was inhibited by the alpha-antagonist phentolamine. A 50-kDa protein co-purifies with the 74-kDa G-protein. This protein does not bind guanine nucleotides and may be a subunit (beta-subunit) of Gh. These findings indicate that Gh is a G-protein that functionally couples to the alpha 1-adrenergic receptor.

Animals↗

PubMed1990

Atrial natriuretic factor-induced systemic vasoconstriction in conscious dogs, rats, and monkeys.

This study addresses the hypothesis that atrial natriuretic factor (ANF) is a primary vasodilator, which reduces arterial pressure directly and increases total peripheral resistance secondarily by reflex mechanisms. The effects of 30-minute infusions of ANF (0.3 micrograms/kg/min i.v.) were examined in conscious dogs, rats, and monkeys before and after ganglionic blockade with hexamethonium. In seven intact, conscious dogs, ANF reduced mean arterial pressure by 7 +/- 1% and cardiac output by 19 +/- 3% and increased total peripheral resistance by 15 +/- 3%. After ganglionic blockade, ANF reduced mean arterial pressure by 7 +/- 2% but still increased total peripheral resistance by 15 +/- 3%. Similar results were observed in four dogs with total cardiac denervation and in six dogs with arterial baroreceptor denervation. Furthermore, in two dogs, combined ganglionic and alpha 1-adrenoceptor blockades failed to alter the rise in total peripheral resistance observed with ANF. In six intact, conscious rats, ANF reduced mean arterial pressure by 8 +/- 2% and cardiac output by 27 +/- 2% and increased total peripheral resistance by 27 +/- 5%. After ganglionic blockade, ANF still increased total peripheral resistance by 13 +/- 3%. In six intact, conscious monkeys, ANF reduced mean arterial pressure by 14 +/- 2% and cardiac output by 26 +/- 3% and increased total peripheral resistance by 17 +/- 3%. However, after ganglionic blockade. ANF decreased total peripheral resistance by 11 +/- 2%. These data provide evidence for a fundamental species difference in the vascular actions of ANF. In conscious dogs, ANF elicits "direct" vasoconstriction, which increases total peripheral resistance, even in the presence of denervation of reflexes or autonomic blockade. In conscious rats, ANF elicits both direct and reflexly mediated vasoconstriction. In conscious monkeys, although a component of direct vasoconstriction may also be present, the most prominent component appears to be reflexly mediated, since it was abolished by ganglionic blockade.

Adrenergic alpha-Antagonists↗

PubMed1990

Phosphorylation state of pro-atrial natriuretic factor in rat atrial secretory granules.

Recent studies have demonstrated that phosphorylation of atrial natriuretic factor (ANF) (99-126) in vitro modulates the bioactivity of this hormone. The potential physiological relevance of this observation was revealed in latter studies showing that endogenous proANF can be 32PO4-biosynthetically labeled by primary cultured atrial myocytes and by atrial appendage explants. The site and extent of proANF phosphorylation were different, however, in these two model systems. Whereas proANF extracted from atrial explants was phosphorylated on the bioactive, carboxy (C)-terminal portion of the molecule [ANF(99-126)], cultured atrial myocytes phosphorylated proANF on the amino (N)-terminal portion of the prohormone molecule [ANF(1-98)]. It was the goal of this study, therefore, to determine whether the bioactive region of proANF, ANF(99-126), is phosphorylated in vivo. ProANF was obtained by acid extraction of isolated rat atrial secretory granules followed by purification using reverse phase-HPLC. Analysis of purified 125I-labeled proANF by isoelectric focusing (IEF) revealed two bands with isoelectric points of 5.3 and 5.0. The more acidic band comigrated on IEF gels with 32PO4-biosynthetically labeled proANF obtained from primary cultures of atrial myocytes, suggesting that this species of proANF represented endogenously phosphorylated proANF. The more acidic band accounted for only 15-25% of the total proANF found in the mature atrial secretory granule. The phosphorylation state of ANF(99-126) produced by thrombin cleavage of secretory granule proANF was examined using three complementary methods: 1) cation-exchange HPLC, 2) amino-terminal amino acid sequence analysis and 3) anti-ANF(99-105) antibody immunoreactivity. Evidence from these three independent approaches indicated that proANF is not phosphorylated on the C-terminal portion of the molecule in vivo. Therefore, phosphorylation is not a physiological regulator of ANF(99-126) bioactivity.

Animals↗

PubMed1990

Identification and structural characterization of alpha 1-adrenergic receptor subtypes.

Rat liver and brain membrane alpha 1-adrenergic receptors were purified greater than 500-fold by successive chromatographic steps using heparin-agarose, an affinity matrix constructed by coupling a novel derivative of the alpha 1-selective antagonist prazosin to Affigel-102 and wheat germ agglutinin-agarose. Several lines of evidence were obtained for the existence in brain of an alpha 1-adrenergic receptor subtype that is structurally distinct from that previously characterized in liver and other tissues using photoaffinity labeling, protein purification, and DNA cloning techniques. The alpha 1-selective ligand chlorethylclonidine (CEC) (an alkylating agent) irreversibly inactivates 100% of [3H]prazosin binding sites in partially purified preparations of rat liver. Under identical conditions, only 50% of brain receptors are irreversibly inactivated. Computer modeling of data obtained from the competition by the alpha antagonists WB4101 and phentolamine for [3H]prazosin binding to partially purified preparations of rat liver is best fit by assuming a single class of low affinity sites for both ligands. However, analysis of partially purified brain preparations indicates the presence of two binding sites with different affinities for these antagonists. Additionally, prior alkylation of brain receptors with CEC results in the loss of low affinity phentolamine and WB4101 binding sites. The CEC-insensitive site in brain, which displays high affinity for phentolamine and WB4101, is resistant to photoaffinity labeling by [125I]azidoprazosin. This is not due to a markedly lower affinity of the CEC-insensitive sites for the photoaffinity label, because competition studies with [127I]azidoprazosin revealed a single class of high affinity sites in partially purified brain samples. Photoaffinity labeling of partially purified liver and brain samples not treated with CEC results in the specific labeling of a single protein of Mr 80,000. No specifically labeled protein is observed for partially purified brain samples that had previously been incubated with CEC. Treatment of photoaffinity-labeled liver and brain receptors with N-glycanase to cleave N-linked oligosaccharides results in a single Mr 55,000 protein. Taken together, these data provide evidence for the existence of a single receptor subtype (alpha 1b) in rat liver and for two subtypes (alpha 1a and alpha 1b) in rat brain. Furthermore, the insensitivity of the alpha 1a subtype to CEC and the resistance of the alpha 1a subtype to covalent labeling by an alpha 1b-selective photoaffinity probe suggest that the primary structures of the two receptor subtypes differ, such that an amino acid(s) in the alpha 1b subtype that incorporates CEC and the photoaffinity label is lacking in the alpha 1a subtype.

Affinity Labels↗

PubMed1989

Selective alpha 1-adrenergic blockade, lipids, and coronary heart disease risk. Considerations in the treatment of mild hypertension.

The importance of hypertension and hyperlipidemia as independent and interactive risk factors for the development of premature cardiovascular disease, and particularly for the development of atherosclerotic coronary heart disease, is becoming increasingly apparent both from epidemiologic data and from therapeutic trials. Nevertheless, therapeutic trials of patients with mild hypertension have not demonstrated benefits from lowering arterial pressure, in terms of reduced mortality rates from coronary events. This may, in part, be due to the fact that many of the antihypertensive agents used in these trials adversely influence lipid and lipoprotein levels. Thus, agents that are lipid-neutral or that favorably influence the lipid profile, such as selective alpha 1-inhibitors, are receiving increasing attention for the treatment of mild hypertension. Recent insights into these issues are considered.

Adrenergic alpha-Antagonists↗

PubMed1989

Characterization of an imidazoline/guanidinium receptive site distinct from the alpha 2-adrenergic receptor.

alpha 2-Adrenergic receptors recognize a number of molecules with diverse chemical structures, including the yohimban diastereoisomers yohimbine and rauwolscine, catecholamines, guanidinium analogs, and imidazolines, such as clonidine. The affinity of the receptor protein for some of these ligands can vary by 10-100-fold among various tissues and species, suggesting a heterogeneous class of binding sites. Certain cellular effects elicited by the compounds possessing an imidazoline or guanidinium moiety may actually be mediated by a membrane receptor distinct from the alpha 2-adrenergic receptor. To determine whether this imidazoline/guanidinium receptive site (IGRS) and the alpha 2-adrenergic receptor represent distinct proteins, we solubilized and partially characterized the two binding sites in rabbit kidney. This tissue expresses both alpha 2-adrenergic receptors and high affinity imidazoline/guanidinium binding sites, the latter which are rauwolscine-insensitive but can be identified with the benzodioxan [3H]idazoxan. The IGRS and alpha 2-adrenergic receptor in rabbit kidney exhibit distinct ligand recognition properties, which are maintained after solubilization and partial purification. In addition, the two receptors can be physically separated by heparin-agarose or lectin affinity chromatography indicating that the two binding sites are distinct entities. [3H]Idazoxan binding is trypsin-sensitive, indicating that the IGRS is a protein rather than a lipid component of the plasma membrane. [3H]Idazoxan binding is not inhibited by endogenous agonists for known neurotransmitter receptors. However, the IGRS does recognize clonidine-displacing substance, a small non-catechol compound isolated from calf brain, suggesting the existence of a previously uncharacterized hormonal/neurotransmitter receptor system.

Animals↗

PubMed1989

Glycerol, sodium phosphate, and sodium chloride permit the solubilization and partial purification of rat hepatic alpha 1-receptors by 3-(3-cholamidylpropyl)-dimethylammonio-1-propanesulfonate.

CHAPS [3-(3-cholamidylpropyl)-dimethylammonio-1-propanesulfonate], a zwitterionic detergent, has been used to solubilize the rat hepatic alpha 1-adrenergic receptor. Although the use of this detergent alone permitted a poor receptor solubilization, the inclusion of sodium phosphate, sodium chloride, and glycerol to the medium allowed 30% of the binding activity observed in plasma membranes to be recovered. Binding of the selective alpha 1-adrenergic antagonist, [3H]prazosin, by the solubilized preparation was saturable and of high affinity. In addition, binding of the radioligand was inhibited by a variety of adrenergic agents with affinity, specificity, and stereoselectivity comparable to that observed in plasma membranes. The use of glycerol in the solubilization medium permitted recovery of the solubilized receptor in a stable form (T1/2 = 72 h at 4 degrees C). Sequential affinity and size-exclusion gel chromatography allowed a 1000-fold purification of the solubilized receptor. The Stokes' radius and the apparent molecular mass of the purified receptor-Chaps complex (48.4 A and 160,000 Da, respectively), determined by gel filtration chromatography, were similar to those previously obtained for the rat hepatic alpha 1-receptor purified after solubilization with the nonionic detergent digitonin. These data indicate that the combination of Chaps, sodium phosphate, sodium chloride, and glycerol permitted the solubilization and partial purification of hepatic alpha 1-receptor in an active and stable form. The use of this technique might be useful for the solubilization of other membrane-bound proteins by Chaps whose biophysical characteristics make it an ideal detergent for reconstitution experiments.

Animals↗