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Biomedical subjects

S M Shreeve

Publications and source records attributed to S M Shreeve.

14 recordsLinked to original sources

Reconstitution of alpha 1-adrenoceptors having high affinity for prazosin.

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.

Animals

The alpha 1-adrenoceptor is inactivated by alterations in membrane phospholipids.

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.

Animals

Evidence for the formation of a functional complex between vasoactive intestinal peptide, its receptor, and Gs in lung membranes.

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.

Animals

Effect of detergent solubilization on the affinity of some quinazoline derivatives for the alpha 1-adrenoceptor.

[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.

Animals

Variable receptor affinity hypothesis.

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.

Adrenergic alpha-Antagonists

Evolution of neurotransmitter receptor systems.

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)

Animals

Classification of bovine aortic alpha-1 adrenoceptors by the criteria of ligand affinity and molecular mass.

[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.

Affinity Labels

Activation of central muscarinic receptors inhibit Ca2+/Mg2+ ATPase and ATP-dependent Ca2+ transport in synaptic membranes.

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.

Animals

Molecular comparison of alpha 1- and alpha 2-adrenergic receptors suggests that these proteins are structurally related "isoreceptors".

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.

Adrenergic alpha-Agonists

Molecular properties of the vasoactive intestinal peptide receptor in aorta and other tissues.

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.

Animals