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1.
Biochem Biophys Res Commun ; 272(3): 922-8, 2000 Jun 16.
Article in English | MEDLINE | ID: mdl-10860852

ABSTRACT

We have characterized vasoactive intestinal peptide (VIP) receptor/G-protein coupling in rat alveolar macrophage (AM) membranes and find that pertussis toxin treatment and antisera against G(alphai3) and G(alphas) reduce high-affinity (125)I-VIP binding, indicating that both G(alphas) and G(alphai3) couple to the VIP-receptor. The predominant VIP-receptor subtype in AM is VPAC(1) and we examined the G-protein interactions of the human VPAC(1) that had been transfected into HEK293 cells. VPAC(1) has a molecular mass of 56 kDa; GTP analogs reduced (125)I-VIP binding to this protein demonstrating that high-affinity binding of VIP to the receptor requires coupling to G-protein. Functional VIP/VPAC(1)/G-protein complexes were captured by covalent cross-linking and analyzed by Western blotting. The transfected human VPAC(1) receptor in HEK293 was found to be coupled to G(alphas) but not G(alphai) or G(alphaq). Furthermore, pertussis toxin treatment had no effect on VPAC(1)/G-protein coupling in these cells. These observations suggest that the G-proteins activated by VPAC(1) may be dependent upon species and cell type.


Subject(s)
Heterotrimeric GTP-Binding Proteins/agonists , Heterotrimeric GTP-Binding Proteins/metabolism , Macrophages, Alveolar/drug effects , Receptors, Vasoactive Intestinal Peptide/metabolism , Vasoactive Intestinal Peptide/pharmacology , Animals , Binding Sites/drug effects , Cell Line , Cell Membrane/drug effects , Cell Membrane/metabolism , Cross-Linking Reagents/metabolism , Enzyme Activation/drug effects , GTP-Binding Protein alpha Subunits, Gi-Go/agonists , GTP-Binding Protein alpha Subunits, Gi-Go/antagonists & inhibitors , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , GTP-Binding Protein alpha Subunits, Gs/agonists , GTP-Binding Protein alpha Subunits, Gs/antagonists & inhibitors , GTP-Binding Protein alpha Subunits, Gs/metabolism , Guanosine Triphosphate/analogs & derivatives , Guanosine Triphosphate/pharmacology , Heterotrimeric GTP-Binding Proteins/antagonists & inhibitors , Humans , Immune Sera/pharmacology , Macrophages, Alveolar/cytology , Macrophages, Alveolar/metabolism , Male , Muscle, Smooth/cytology , Muscle, Smooth/drug effects , Muscle, Smooth/metabolism , Organ Specificity , Pertussis Toxin , Protein Binding/drug effects , Rats , Rats, Sprague-Dawley , Receptors, Vasoactive Intestinal Peptide/chemistry , Receptors, Vasoactive Intestinal Peptide/genetics , Receptors, Vasoactive Intestinal Polypeptide, Type I , Signal Transduction/drug effects , Species Specificity , Virulence Factors, Bordetella/pharmacology
2.
Pharmacol Ther ; 78(3): 155-66, 1998 Jun.
Article in English | MEDLINE | ID: mdl-9690816

ABSTRACT

The endothelium, lining the inner surface of blood vessels, can be considered as the main local regulator of vascular wall homeostasis. It secretes various factors in response to mechanical and hormonal stimuli, which, in turn, influence smooth muscle cell contractility, vascular structure, blood fluidity, and other cell-to-cell interactions. Within the same species, there are, however, variations in endothelial cell function, depending not only on the vascular bed of origin, but also on the size of the vessel within the same vascular territory. Differences have been observed in phenotype, antigen expression, cell size and growth, secretory function, and G-protein expression. Furthermore, some pathological processes affect endothelial function selectively; that is, some vascular beds are sensitive to atherosclerosis while others are not. Understanding these distinctions is particularly important for a rational approach to the treatment of vascular disorders.


Subject(s)
Endothelium, Vascular/cytology , Vascular Diseases/pathology , Endothelins/metabolism , Endothelium, Vascular/metabolism , GTP-Binding Proteins/metabolism , Humans , Major Histocompatibility Complex/physiology , Nitric Oxide/metabolism , Phenotype , Vascular Diseases/metabolism
3.
Hypertension ; 30(4): 830-6, 1997 Oct.
Article in English | MEDLINE | ID: mdl-9336380

ABSTRACT

Agonists acting on the vascular endothelium can modulate the release of a number of factors that interact with the surrounding smooth muscle cells and influence their tone. One such factor is the vasoconstricting agent endothelin-1 (ET-1), which has been implicated in several disease states, including stroke. However, very little is known about the physiological role of ET-1 in the cerebral circulation. We demonstrate that activation of alpha2-adrenoceptors in human pial artery endothelial cells reduces both constitutive and agonist-stimulated release of immunoreactive ET-1. That this has physiological relevance is supported by our demonstration that in segments of rabbit middle cerebral arteries, alpha2-adrenoceptor activation reduces the release of endothelium-derived ET-1 and causes an endothelium-dependent relaxation. The adrenoceptor-dependent relaxation was not blocked by combined addition of indomethacin and N omega-nitro-L-arginine in 25 mmol/L KCl-depolarizing physiological solution but was selectively antagonized by a subthreshold concentration of exogenous ET-1. Our data suggest that activation of endothelial alpha2-adrenoceptor would favor a decrease in ET-1 production and possibly promote vascular relaxation.


Subject(s)
Cerebral Arteries/physiology , Endothelin-1/metabolism , Endothelium, Vascular/metabolism , Receptors, Adrenergic, alpha/physiology , Vasodilation/physiology , Adolescent , Adult , Animals , Biological Factors/pharmacology , Cells, Cultured , Endothelin Receptor Antagonists , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Humans , In Vitro Techniques , Peptides, Cyclic/pharmacology , Potassium Chloride/pharmacology , Rabbits
4.
Stroke ; 28(2): 375-81, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9040693

ABSTRACT

BACKGROUND AND PURPOSE: Hormones, neurotransmitters, and autacoids play a key role in the regulation of vascular tone as a result of their interaction with the endothelium. The aim of this study was to compare selected properties of three human endothelial cell lines isolated from cerebral pial arteries (PEC) and two peripheral vessels, the superficial temporal (SEC) and omental (OEC) arteries. METHODS: Intracellular free calcium concentration ([Ca2+]i) and receptor protein expression were measured in characterized primary cultures of human endothelial cells. RESULTS: All cell lines labeled positively for factor VIII/von Willebrand factor. Growth rate and constitutive release of endothelin-1, expressed as a function of protein, were both significantly lower in cerebral cells (PEC) than in endothelial cells derived from peripheral vessels. Basal [Ca2+]i measured with the fluorescent calcium indicator fura 2-AM (2 mumol/L) did not differ in either of the three cell lines. Although PEC responded to endothelin-1 (0.1 mumol/L) and vasoactive intestinal peptide (1 mumol/L) by a twofold to threefold increase in [Ca2+]i, OEC were unresponsive to these peptides. Moreover, the calcium response to alpha-thrombin (10 nmol/L) was greater in cerebral (PEC) than in peripheral (SEC, OEC) endothelial cells, while bradykinin (100 nmol/L) increased [Ca2+]i to a similar level in all three cell types. CONCLUSIONS: This study demonstrates that endothelial cells from different sites of the vasculature exhibit different growth rates and vary in their response to agonists.


Subject(s)
Endothelium, Vascular/cytology , Omentum/blood supply , Pia Mater/blood supply , Temporal Arteries/cytology , Adolescent , Adult , Bradykinin/pharmacology , Calcium/metabolism , Cell Division/drug effects , Cells, Cultured , Cerebrovascular Circulation , Endothelin-1/metabolism , Endothelin-1/pharmacology , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Female , Humans , Male , Organ Specificity , Receptors, Vasoactive Intestinal Peptide/metabolism , Thrombin/pharmacology , Vasoactive Intestinal Peptide/pharmacology , von Willebrand Factor/metabolism
5.
Stroke ; 27(12): 2211-5, 1996 Dec.
Article in English | MEDLINE | ID: mdl-8969783

ABSTRACT

BACKGROUND AND PURPOSE: During thrombosis, alpha-thrombin becomes sequestered by fibrin and the subendothelial basement membrane, and it is available to interact with the vasculature over prolonged periods. In this study, we investigated the long-term effect of alpha-thrombin on G alpha i3 and G alpha s levels in human vascular endothelial cells (EC). Because obesity is associated with changes in receptor signaling in many animal models, we also explored the influence of this clinical risk factor. METHODS: Primary cultures of human EC were exposed to alpha-thrombin for 16 hours, and immunologically detectable G alpha i3 and G alpha s levels were measured. RESULTS: alpha-Thrombin (100 nmol/L) increased G alpha i3 levels in EC derived from the cerebral microvasculature and superficial temporal artery (4.2 +/- 1.2-fold and 2.8 +/- 0.32-fold, respectively) but had no significant effect on EC derived from omental artery (P > .6) or from the superficial temporal artery of obese (body mass index > or = 28 kg/m2) patients (P > .4). The expression of G alpha s was unchanged in all cell types (P > or = .1). Two other circulating peptides, vasoactive intestinal peptide and endothelin-1, failed to alter the expression of either G protein in EC from the cerebral microvasculature, further demonstrating the specificity of the alpha-thrombin effect. However, thrombin receptor activating protein-14 mimicked the alpha-thrombin response and increased G alpha i3 in EC derived from the cerebral microvasculature and superficial temporal artery. CONCLUSIONS: We conclude that alpha-thrombin increases G alpha i3 expression in some EC through activation of its tethered liganded receptor. Obesity appears to suppress this action of alpha-thrombin.


Subject(s)
Endothelium, Vascular/drug effects , GTP-Binding Protein alpha Subunits, Gi-Go/biosynthesis , Gene Expression Regulation/drug effects , Obesity/metabolism , Thrombin/pharmacology , Adult , Aged , Aged, 80 and over , Body Mass Index , Cells, Cultured , Cerebral Arteries/drug effects , Cerebral Arteries/metabolism , Cycloheximide/pharmacology , Endothelium, Vascular/metabolism , Female , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , GTP-Binding Protein alpha Subunits, Gs/biosynthesis , GTP-Binding Protein alpha Subunits, Gs/genetics , Hirudins/pharmacology , Humans , Male , Middle Aged , Obesity/genetics , Obesity/pathology , Omentum/blood supply , Peptide Fragments/pharmacology , Protein Synthesis Inhibitors/pharmacology , Receptors, Thrombin/drug effects , Receptors, Thrombin/physiology , Signal Transduction/drug effects , Temporal Arteries/drug effects , Temporal Arteries/pathology
7.
Mol Pharmacol ; 50(3): 624-30, 1996 Sep.
Article in English | MEDLINE | ID: mdl-8794903

ABSTRACT

Although vasoactive intestinal peptide (VIP) exerts many of its effects through stimulation of adenylyl cyclase, there is increasing evidence that other signaling pathways may contribute to its action. The role of inhibitory G proteins (Gi) in VIP-mediated signaling in the lung was assessed by a combination of equilibrium-binding and covalent cross-linking studies. Pertussis toxin treatment of rat lung membranes reduced the high affinity binding of 125I-VIP, implicating a member of the Gi family in signaling from the VIP receptor. The particular G protein involved was identified as Gi3 through capture of a VIP/receptor/ Gi3 ternary complex by covalent cross-linking. There was a progressive rise with increasing VIP concentration in formation of the complex reported by the cross-linking strategy. Guanine nucleotides and an anti-G alpha i3 antiserum suppressed formation of the VIP/receptor/Gi3 ternary complex, demonstrating its functional nature in native lung membranes. Inhibition of high affinity 125I-VIP binding by the anti-G alpha i3 antiserum verified this functionality. Taken together, these data suggest that receptor/ Gi3 coupling makes a significant contribution to VIP-mediated signaling in the lung and illustrate the value of covalent cross-linking as a strategy to define receptor/G protein complexes that arise under conditions in which the stoichiometry and microdomains of the native cell membrane are preserved.


Subject(s)
GTP-Binding Proteins/physiology , Receptors, Vasoactive Intestinal Peptide/physiology , Vasoactive Intestinal Peptide/pharmacology , Adenylate Cyclase Toxin , Adenylyl Cyclases/metabolism , Animals , Antibody Specificity , Brain/metabolism , Cell Membrane/metabolism , Cross-Linking Reagents , GTP-Binding Proteins/isolation & purification , GTP-Binding Proteins/metabolism , Guanosine Triphosphate/pharmacology , Guanylyl Imidodiphosphate/pharmacology , Immunoblotting , Kinetics , Lung/metabolism , Pertussis Toxin , Rats , Receptors, Vasoactive Intestinal Peptide/isolation & purification , Receptors, Vasoactive Intestinal Peptide/metabolism , Signal Transduction , Vasoactive Intestinal Peptide/metabolism , Virulence Factors, Bordetella/pharmacology
8.
Eur J Pharmacol ; 268(3): 393-8, 1994 Aug 16.
Article in English | MEDLINE | ID: mdl-7805763

ABSTRACT

The expression of the alpha 1C-adrenoceptor subtype in human and rabbit blood vessels has been analyzed using the reverse transcriptase/polymerase chain reaction technique (RT/PCR). The 20 bp primers employed were designed from the bovine alpha 1C-adrenoceptor and flank a least conserved region--the putative third cytoplasmic loop. RT/PCR products generated from rabbit and human brain mRNA both had 93% homology to the bovine alpha 1C-adrenoceptor and were used as species and subtype specific probes in Southern blot analysis of vascular RT/PCR products. Poly A+ RNA was purified from the human saphenous vein and rabbit aorta, renal, pulmonary and central ear arteries and amplified by RT/PCR. Size analysis by agarose gel electrophoresis, together with Southern hybridization of the resulting cDNA products confirm the expression of the alpha 1C-adrenoceptor in these vessels.


Subject(s)
Muscle, Smooth, Vascular/chemistry , Receptors, Adrenergic, alpha-1/analysis , Animals , Arteries/chemistry , Arteries/metabolism , Base Sequence , Blotting, Southern , Humans , In Vitro Techniques , Male , Molecular Sequence Data , Muscle, Smooth, Vascular/metabolism , Polymerase Chain Reaction , RNA, Messenger/biosynthesis , Rabbits , Receptors, Adrenergic, alpha-1/genetics , Receptors, Adrenergic, alpha-1/metabolism , Saphenous Vein/chemistry , Saphenous Vein/metabolism
9.
Peptides ; 13(5): 919-26, 1992.
Article in English | MEDLINE | ID: mdl-1336189

ABSTRACT

The molecular weight of the vasoactive intestinal peptide (VIP) receptor was assessed in bovine aorta, and rat liver, lung, and brain by covalent cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The receptor in all four tissues was found to be a single polypeptide of approximate M(r) 54,000, contradicting previous claims for substantial heterogeneity in the molecular weight of this receptor. Guanine nucleotides inhibit cross-linking of 125I-VIP to its receptor, and cross-linking with ethylene glycolbis(succinimidylsuccinate) provides further evidence for complex formation between VIP, its receptor and a guanine nucleotide-binding regulatory protein (G-protein). The precise mechanism of receptor-G-protein coupling may differ between the aorta and other tissues.


Subject(s)
Aorta/metabolism , Receptors, Gastrointestinal Hormone/chemistry , Vasoactive Intestinal Peptide/metabolism , Animals , Brain/metabolism , Cross-Linking Reagents , GTP-Binding Proteins/metabolism , Liver/metabolism , Lung/metabolism , Male , Molecular Weight , Rats , Rats, Sprague-Dawley , Receptors, Gastrointestinal Hormone/metabolism , Receptors, Vasoactive Intestinal Peptide , Tissue Distribution
10.
Eur J Pharmacol ; 226(3): 191-7, 1992 Jul 01.
Article in English | MEDLINE | ID: mdl-1330629

ABSTRACT

Using a variety of detergents we find that soluble alpha 1-adrenoceptor recovery from aortic and hepatic membranes is markedly enhanced if the receptor is first prelabelled with prazosin. Moreover, prelabelling prevents the reduction in prazosin affinity induced by solubilizing concentrations of digitonin, possibly by stabilizing the receptor's conformation. 20-25% of alpha 1-adrenoceptors solubilized in sodium cholate were reconstituted into brain lipids. Specific [3H]prazosin binding to the reconstituted receptor was saturable and of high affinity (KD = 0.019 +/- 0.008 nM). We conclude that prelabelling is essential to preserve the receptor in detergents. However, once the detergent is removed brain lipids alone are able to maintain the receptor in a form with high affinity for prazosin.


Subject(s)
Prazosin/metabolism , Receptors, Adrenergic, alpha/metabolism , Animals , Aorta/metabolism , Cattle , In Vitro Techniques , Liver/metabolism , Membrane Lipids/metabolism , Microsomes/metabolism , Muscle, Smooth, Vascular/metabolism , Radioligand Assay , Rats
11.
Eur J Pharmacol ; 226(1): 29-33, 1992 May 12.
Article in English | MEDLINE | ID: mdl-1327840

ABSTRACT

The influence of the membrane environment on the alpha 1-adrenoceptor has been investigated by examining the effect of phospholipase digestion on the binding of [3H]prazosin to aortic and hepatic membranes. Membrane digestion by phospholipase A2 and phospholipase C was found to markedly reduce prazosin binding to the alpha 1-adrenoceptor whereas phospholipase D had comparatively little effect. In addition, there were differences between membrane preparations since the aortic alpha 1-adrenoceptor was less sensitive to phospholipase A2 and phospholipase C than the hepatic receptor. The results support a major role for hydrophobic groups and the negatively charged, hydrophilic phosphate moiety of phospholipids in the interaction between prazosin and the alpha 1-adrenoceptor.


Subject(s)
Membrane Lipids/chemistry , Phospholipids/metabolism , Prazosin/metabolism , Receptors, Adrenergic, alpha/metabolism , Animals , Aorta/metabolism , Binding Sites , Cell Membrane/chemistry , Liver/metabolism , Male , Membrane Lipids/metabolism , Muscle, Smooth, Vascular/metabolism , Phospholipases A/metabolism , Phospholipases A2 , Phospholipids/chemistry , Rats , Rats, Sprague-Dawley , Receptors, Adrenergic, alpha/chemistry , Type C Phospholipases/metabolism
12.
J Biol Chem ; 267(5): 3382-8, 1992 Feb 15.
Article in English | MEDLINE | ID: mdl-1310685

ABSTRACT

The molecular weight of the vasoactive intestinal peptide (VIP) receptor in rat lung and its interaction with the stimulatory guanine nucleotide-binding protein (Gs) were assessed by covalent cross-linking, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunological techniques. Studies with two cross-linking agents indicated that the VIP receptor in this tissue is a single polypeptide of Mr = 54,000. The VIP-occupied receptor could be cross-linked to neighboring proteins after detergent solubilization; higher molecular weight complexes of Mr = 114,000 and 184,000 were formed. Immunoblotting with antisera against G-protein subunits demonstrated that both complexes contained the alpha-subunit of Gs as well as the 125I-VIP cross-linked receptor whereas only the Mr = 184,000 complex contained the beta-subunit. Pretreatment with GTP reduced the prominence of these complexes, verifying the functional nature of this receptor-Gs association. Studies with a third cross-linking agent, ethylene glycol bis(succinimidyl succinate), provided direct evidence of physically associated, ternary VIP-receptor-Gs complexes actually in the membrane milieu. That these complexes were functionally associated with shown by their inhibition by anti-Gs alpha anti-serum. Since treatment of membranes with guanosine 5'-O-(3-thiotriphosphate) resulted in the separation of the VIP-cross-linked receptor from Gs such that no cross-linking could occur, we conclude that the binding of GTP analogs induces a conformational change in Gs in the membrane milieu.


Subject(s)
GTP-Binding Proteins/metabolism , Lung/metabolism , Receptors, Gastrointestinal Hormone/metabolism , Vasoactive Intestinal Peptide/metabolism , Animals , Cell Membrane/metabolism , Cross-Linking Reagents , Detergents , Electrophoresis, Polyacrylamide Gel , GTP-Binding Proteins/isolation & purification , Guanosine Triphosphate/pharmacology , Immunoblotting , Macromolecular Substances , Male , Molecular Weight , Rats , Rats, Inbred Strains , Receptors, Gastrointestinal Hormone/isolation & purification , Receptors, Vasoactive Intestinal Peptide , Succinimides , Vasoactive Intestinal Peptide/isolation & purification
13.
Eur J Pharmacol ; 188(1): 71-80, 1990 Jan 23.
Article in English | MEDLINE | ID: mdl-2155124

ABSTRACT

[3H]Prazosin bound to a single class of high-affinity sites in both bovine aortic and rat hepatic membranes. The absolute affinity values of displacing ligands (prazosin greater than doxazosin much greater than trimazosin greater than yohimbine) were the same for both tissues. After solubilization of the alpha 1-adrenoceptors with digitonin and 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate, an identical rank order potency was observed. However, solubilization significantly reduced ligand affinity. In both tissues the affinity of prazosin was reduced 10- to 13-fold, whereas the affinities of doxazosin, trimazosin and yohimbine were reduced two- to six-fold. There appeared to be no relationship between the lipophilicities of the ligands and the degree to which affinity is affected by solubilization. The results suggest that the reductions in affinity are the consequence of a conformational change in the alpha 1-adrenoceptor and appear to support the hypothesis that the alpha 1-adrenoceptor is so constructed that the spatial configuration of the binding site can change in response to an alteration in its microenvironment.


Subject(s)
Detergents/pharmacology , Quinazolines/metabolism , Receptors, Adrenergic, alpha/metabolism , Surface-Active Agents/pharmacology , Animals , Aorta, Thoracic/drug effects , Aorta, Thoracic/metabolism , Cattle , Chemical Phenomena , Chemistry, Physical , Electrophoresis, Polyacrylamide Gel , In Vitro Techniques , Kinetics , Lipids/analysis , Liver/metabolism , Membranes/drug effects , Membranes/metabolism , Microsomes/drug effects , Microsomes/metabolism , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/metabolism , Prazosin/metabolism , Rats , Receptors, Adrenergic, alpha/drug effects
14.
FASEB J ; 3(6): 1696-704, 1989 Apr.
Article in English | MEDLINE | ID: mdl-2564831

ABSTRACT

Measurements of the contractile response to norepinephrine (NE) of a variety of arteries of three mammalian species that are commonly used in the laboratory provide evidence that tissue sensitivity and affinity of the alpha 1-adrenoceptor for NE covary over a range of several orders of magnitude. The quantitative relationship suggests that variation in affinity can, to a great extent, account for the variation in sensitivity found in a number of circumstances. Furthermore, it is argued that the variation in affinity appears to be continuous and thus does not provide a basis for receptor type subdivision. There is also evidence that adrenergic antagonist affinity can vary significantly in tissues. The factors that might account for this variation include differences in receptor chemical structure, in the local membrane microenvironment, and in a number of intracellular processes. A hypothesis of variable receptor affinity has been proposed. If it is correct, then variation in receptor affinity is an important functionally relevant variable that could account for selectivity of tissue responses to circulating hormones and may represent a mechanism of change in the intact organism and in disease.


Subject(s)
Models, Biological , Receptors, Adrenergic, alpha/metabolism , Adrenergic alpha-Antagonists/metabolism , Animals , Blood Vessels/metabolism , Cell Membrane/metabolism , Chemical Phenomena , Chemistry, Physical , Humans , Norepinephrine/metabolism , Species Specificity , Tissue Distribution
16.
J Pharmacol Exp Ther ; 245(3): 1060-7, 1988 Jun.
Article in English | MEDLINE | ID: mdl-2838601

ABSTRACT

[3H]Prazosin bound to a single class of alpha-1 adrenoceptors in bovine aortic membranes with a Kd of 25 pM. Digitonin solubilized 30% of the receptors as assayed by specific [3H] prazosin binding. The rank order potency of displacing ligands was the same for both membrane-bound and soluble alpha-1 adrenoceptors [prazosin greater than phentolamine greater than yohimbine and (-)-epinephrine = (-)-norepinephrine much greater than (+)-norepinephrine]. Prazosin had a significantly lower affinity for the soluble receptor, whereas the other adrenergic ligands had the same affinity for both forms of the receptor. The alpha-1 adrenoceptor was affinity-labeled with 2-[4-(4-azido-3-[125I]iodobenzoyl)piperazin-1-yl]-4-amino-6,7- dimethoxyquinazoline and analyzed by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass of the receptor binding subunit in both bovine aorta and rat liver was found to be 86,000 MW. It is concluded from this study that bovine aortic alpha-1 adrenoceptors have pharmacologic and biochemical characteristics similar to those in other tissues.


Subject(s)
Aorta/analysis , Receptors, Adrenergic, alpha/analysis , Affinity Labels , Animals , Cattle , Electrophoresis, Polyacrylamide Gel , In Vitro Techniques , Molecular Weight , Prazosin/metabolism , Protein Conformation , Radioligand Assay
17.
Prog Neurobiol ; 30(2-3): 105-69, 1988.
Article in English | MEDLINE | ID: mdl-2830635

ABSTRACT

The presence of hormones, neurotransmitters, their receptors and biosynthetic and degradative enzymes is clearly not only associated with the present and the recent past but with the past several hundred million years. Evidence is mounting which indicates substantial conservation of protein structure and function of these receptors and enzymes over these tremendous periods of time. These findings indicate that the evolution and development of the nervous system was not dependent upon the formation of new or better transmitter substances, receptor proteins, transducers and effector proteins but involved better utilization of these highly developed elements in creating advanced and refined circuitry. This is not a new concept; it is one that is now substantiated by increasingly sophisticated studies. In a 1953 article discussing chemical aspects of evolution (Danielli, 1953) Danielli quotes Medawar, "... endocrine evolution is not an evolution of hormones but an evolution of the uses to which they are put; an evolution not, to put it crudely, of chemical formulae but of reactivities, reaction patterns and tissue competences." To also quote Danielli, "In terms of comparative biochemistry, one must ask to what extent the evolution of these reactivities, reaction patterns and competences is conditional upon the evolution of methods of synthesis of new proteins, etc., and to what extent the proteins, etc., are always within the synthetic competence of an organism. In the latter case evolution is the history of changing uses of molecules, and not of changing synthetic abilities." (Danielli, 1953). Figure 4 outlines a phylogenetic tree together with an indication of where evidence exists for both the enzymes that determine the biosynthesis and metabolism of the cholinergic and adrenergic transmitters and their specific cholinergic and adrenergic receptors. This figure illustrates a number of important points. For example, the evidence appears to show that the transmitters and their associated enzymes existed for a substantial period before their respective receptor proteins. While the transmitters and enzymes appear to exist in single cellular organisms, there is no solid evidence for the presence of adrenergic or cholinergic receptors until multicellular organisms where the receptors appear to be clearly associated with specific cellular and neuronal communication (Fig. 4). One can only speculate as to the possible role for acetylcholine and the catecholamine in single cell organisms.(ABSTRACT TRUNCATED AT 400 WORDS)


Subject(s)
Biological Evolution , Neurons/physiology , Receptors, Adrenergic/physiology , Receptors, Cholinergic/physiology , Receptors, Neurotransmitter/physiology , Animals , Guanine Nucleotides/physiology , Humans , Neurons/metabolism , Receptors, Adrenergic/metabolism , Receptors, Cholinergic/metabolism , Receptors, Neurotransmitter/metabolism , Sequence Homology, Nucleic Acid
18.
Proc Natl Acad Sci U S A ; 82(14): 4842-6, 1985 Jul.
Article in English | MEDLINE | ID: mdl-2862630

ABSTRACT

The structures of human platelet alpha 2-adrenergic receptors and rat liver alpha 1-adrenergic receptors were compared by utilizing isoelectric focusing, NaDodSO4/PAGE, and monoclonal antibody crossreactivity. Digitonin-solubilized alpha 1- and alpha 2-adrenergic receptors have an identical isoelectric point of 4.6. Under reducing conditions in NaDodSO4/polyacrylamide gels, the alpha 1-adrenergic receptor has an apparent molecular mass of 85 kDa. Similarly, the alpha 2-adrenergic receptor, which had been affinity-labeled with [3H]phenoxybenzamine and partially purified by isoelectric focusing or photoaffinity-labeled with p-[3,5-3H]azidoclonidine, was also found to have an apparent molecular mass of 85 kDa. One hybridoma, developed from a fusion between SP2/O myeloma cells and splenic lymphocytes from BALB/c mice immunized with human platelet alpha 2-adrenergic receptors, secreted a monoclonal antibody (alpha 2-116p) against the ligand binding site of alpha 2-adrenergic but not alpha 1-adrenergic receptors. In contrast, three monoclonal antibodies raised against the alpha 1-receptor polypeptide backbone but not the ligand binding site were found to specifically immunoprecipitate human platelet alpha 2-adrenergic receptors. These data suggest that the alpha 1- and alpha 2-adrenergic receptors are "isoreceptors," sharing immunogenic and, by implication, structural determinants that most likely evolved as a result of gene duplication.


Subject(s)
Blood Platelets/metabolism , Receptors, Adrenergic, alpha/metabolism , Adrenergic alpha-Agonists/metabolism , Antibodies, Monoclonal , Binding, Competitive , Cell Membrane/metabolism , Electrophoresis, Polyacrylamide Gel , Humans , Immunoassay , Kinetics , Molecular Weight , Structure-Activity Relationship
19.
Brain Res ; 329(1-2): 39-47, 1985 Mar 11.
Article in English | MEDLINE | ID: mdl-3156660

ABSTRACT

Preparations of lysed synaptosomes exhibit a high affinity Ca2+/Mg2+ ATPase and ATP-dependent Ca2+ accumulation activity, with a Km for Ca2+ congruent to 0.5 microM, close to the cytosolic concentration of Ca2+. When these membrane suspensions were incubated with cholinergic agonists muscarine or oxotremorine (1-20 microM), both Ca2+/Mg2+ ATPase and ATP-dependent CA2+ uptake were inhibited in a concentration-dependent fashion. Atropine alone (0.5-1.0 microM) had no effect on either enzyme or uptake activity, but significantly inhibited the actions of both muscarine and oxotremorine. No significant effects by cholinergic agonists or antagonists were seen on fast or slow phase voltage-dependent Ca2+ channels or Na+-Ca2+ exchange. These results suggest that activation of presynaptic muscarinic receptors produce inhibition of two processes required for the buffering of optimal free Ca2+ by the nerve terminal. Activation of presynaptic muscarinic receptors have been reported to reduce the release of ACh from nerve terminals. Alterations in intracellular free Ca2+ may contribute to a reduction in transmitter (ACh) release seen following activation of cholinergic receptors.


Subject(s)
Brain/physiology , Calcium-Transporting ATPases/physiology , Calcium/metabolism , Receptors, Muscarinic/physiology , Synaptic Membranes/metabolism , Animals , Ca(2+) Mg(2+)-ATPase , Cell Membrane Permeability , Male , Parasympathomimetics/pharmacology , Rats , Rats, Inbred Strains , Sodium/physiology , Synaptosomes/metabolism
20.
J Biol Chem ; 259(20): 12398-402, 1984 Oct 25.
Article in English | MEDLINE | ID: mdl-6490621

ABSTRACT

Digitonin-solubilized cardiac muscarinic receptors were reconstituted by dialysis into human erythrocyte acceptor membranes which lack high-affinity muscarinic receptors. The number of receptors reconstituted was proportional to the quantity of soluble receptors added to the reconstitution system. Specific [3H](-)-quinuclidinyl benzilate binding to the reconstituted receptor was found to be saturable with a Kd (dissociation constant) equal to 48 +/- 4 pM and a Bmax (maximal density of binding sites) equal to 50 +/- 5 fmol/mg of protein. Competitive binding studies indicated that the reconstituted receptors showed stereoselectivity and drug specificity consistent with a high-affinity muscarinic receptor. Agonist binding to the reconstituted receptor was decreased by the addition of guanyl-5'-yl imidodiphosphate. Sixty per cent of the reconstituted receptors were found to be integral membrane proteins. The molecular weight of the reconstituted receptor as determined by sodium dodecyl sulfate-gel electrophoresis was 76,000 +/- 2,000 and was identical to the molecular weight of the muscarinic receptor in the original cardiac membranes. The data indicate that a partially functional, intact muscarinic receptor was reconstituted into human erythrocyte acceptor membranes and that membrane constituents may be required to stabilize the receptor in a high-affinity state for antagonists.


Subject(s)
Myocardium/metabolism , Receptors, Muscarinic/metabolism , Binding, Competitive , Digitonin , Humans , Kinetics , Quinuclidinyl Benzilate/metabolism , Receptors, Muscarinic/isolation & purification , Solubility
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