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R R Neubig

Publications and source records attributed to R R Neubig.

At least 55 records · Page 3Linked to original sources
PubMed1994

Multisite interactions of receptors and G proteins: enhanced potency of dimeric receptor peptides in modifying G protein function.

Synthetic peptides that activate or inhibit G proteins reveal structural determinants of receptor-G protein interactions and show promise as potential therapeutic agents. A cysteine-containing peptide from the carboxyl-terminal part of the third cytoplasmic loop of the alpha 2-adrenergic receptor (peptide Q) uncouples alpha 2-adrenergic receptors from Gi. Peptide Q readily forms disulfide-linked dimers (Qdimer), as detected by high performance liquid chromatography and mass spectrometry. Qdimer is > 100-fold more potent than monomeric Q peptide in inhibiting p-[125I] iodoclonidine binding to the human alpha 2a-adrenergic receptor in platelet membranes and transfected Chinese hamster ovary cells. In addition, Qdimer is 10-20 times more potent than monomeric Q peptide in inhibiting alpha 2 agonist-stimulated GTPase in cell membranes and in directly stimulating G(o)/Gi GTPase in lipid vesicles. The effect of Qdimer is reversible and not mimicked by cystine. Formylation of both tryptophans greatly reduces the potency of the dimer but a single formyl group is well tolerated, indicating an asymmetric interaction of the dimer with Gi in membranes. A mixed dimer of peptides from the amino- and carboxyl-terminal ends of the third cytoplasmic loop of the alpha 2-adrenergic receptor is most potent in all measures of G protein interactions, suggesting that the dimer of Q peptides mimics multiple intracellular portions of the alpha 2-adrenergic receptor with the G protein. These data confirm the importance of multiple receptor regions in G protein activation and suggest a strategy for examining the role of physically separated regions in protein-protein interactions.

Animals↗
PubMed1993

Synthesis and characterization of fluorescently labeled bovine brain G protein subunits.

G proteins play an important role in transmitting hormonal signals, and fluorescence techniques would be useful to study their cellular distribution and mechanisms. To prepare active fluorescent G protein Go/Gi or beta gamma subunits were reacted with fluorescein isothiocyanate (FITC) to label the alpha (F-alpha) and gamma (F-gamma/beta) subunits or with (iodoacetamido)tetramethylrhodamine (TMR-IAA) to label the beta subunit (TMR-beta gamma). Unreacted dye was removed from the labeled proteins by ultrafiltration, followed by further purification using HPLC gel filtration. The molar ratios of dye to protein were 0.96 +/- 0.15, 0.59 +/- 0.07, and 1.37 +/- 0.09 for labeled alpha,beta, and gamma subunits, respectively. GTP gamma S binding to F-alpha and ADP-ribosylation by pertussis toxin of F-alpha were reduced to 63% and 78% of control, respectively. F-alpha was a heterogeneous population of alpha subunits. Active F-alpha containing less than one (0.7) label/subunit (F-alpha-Mono Q) was separated from unlabeled and multiply labeled F-alpha by Mono Q anion-exchange chromatography. F-alpha-Mono Q displayed reduced GTPase activity (turnover number was 46% of control), while GTP gamma S binding and ADP-ribosylation by pertussis toxin were only decreased to 78% and 82% of control, respectively. TMR-beta gamma and F-gamma/beta retain full function compared to native beta gamma, as measured by three methods: (1) TMR-beta gamma and F-gamma/beta are able to form heterotrimers with alpha o subunits, (2) TMR-beta gamma and F-gamma/beta support the ADP ribosylation of alpha o subunits by pertussis toxin, and (3) TMR-beta gamma and F-gamma/beta inhibit forskolin-stimulated adenylyl cyclase activity. The fluorescent G protein subunits will be valuable tools to study G protein mechanisms in reconstituted membranes and intact cells.

Animals↗
PubMed1993

Resonance energy transfer between guanine nucleotide binding protein subunits and membrane lipids.

Resonance energy transfer was used to estimate the distances of closest approach between fluorescent labels on G protein subunits (alpha, beta, and gamma) and the phospholipid bilayer surface. Fluorescein-labeled alpha, beta, and gamma subunits were the energy transfer donors and hexadecylaminoeosin (HAE) in phospholipid vesicles was the acceptor. Bovine brain G protein (alpha o, beta, and gamma subunits) were individually labeled with fluorescein as described in the accompanying paper [Kwon et al. (1993) Biochemistry (preceding paper in this issue)]. Fluorescein-labeled subunits were combined with the appropriate unlabeled G protein subunits and reconstituted into phospholipid vesicles. HAE quenched the fluorescein emission in a concentration-dependent manner which was dependent upon the G protein subunit labeled (gamma = beta > alpha o). From steady-state quenching data with hexadecylaminofluorescein (HAF) as a standard to determine the density of HAE in the phospholipid bilayer, the calculated distances between fluorescein-alpha o, -beta, and -gamma and HAE are 46 +/- 2, 38 +/- 3, and 37 +/- 2 A, respectively. Energy transfer was identified as the means of fluorescence quenching by two methods: (1) addition of 0.1% Lubrol reversed the quenching of fluorescein-labeled G protein subunits by HAE and (2) sensitized HAE emission was observed in the presence of fluorescein-labeled G protein subunits. These results are the first physical measurements of the distances between sites on G protein subunits and the lipid bilayer. These data demonstrate that the label in the beta and gamma subunits is closer to the lipid bilayer than that in the alpha subunit.

Animals↗
PubMed1993

Compartmentation of receptors and guanine nucleotide-binding proteins in NG108-15 cells: lack of cross-talk in agonist binding among the alpha 2-adrenergic, muscarinic, and opiate receptors.

Many different types of receptors couple to the inhibitory guanine nucleotide-binding protein (G protein) Gi. In NG108-15 neuroblastoma-glioma cells, alpha 2b-adrenergic, m4 muscarinic, and delta-opiate receptors all use Gi as a transducer. According to the ternary complex model of receptor-G protein interactions, agonists bind to these receptors with high affinity only in their G protein-associated form. Conversely, G protein affinity for the receptor is increased by agonist binding. We have developed an extended ternary complex model in which multiple receptors couple to a single G protein and we have examined two consequences of the model theoretically and experimentally. First, the simple ternary complex model can account for the observed high and low affinity agonist binding only when G protein is limiting; however, measurements show a significant excess of G protein over receptor. Could this paradox be explained by other receptors competing for the same G protein and limiting the amount of free G protein so that high and low affinity agonist binding would be seen? Our theoretical simulations show that this does not occur unless the receptors and G protein are present in a precise stoichiometric ratio and have an extremely high affinity, such as when agonists for both receptors are present. The second prediction of this model is that binding of an agonist at one receptor should produce competition for G protein used by another receptor. If the G protein pool were limiting and freely mobile, this would result in an unlabeled agonist at one receptor decreasing binding of a radiolabeled agonist to another receptor. Experimentally, the G protein was made limiting by a partial pertussis toxin treatment. Radioligand binding to alpha 2b-adrenergic and m4 muscarinic receptors in these pertussis toxin-treated NG108-15 membranes showed no cross-talk with the delta-opiate or muscarinic receptors, which are present in excess. This could occur because the different receptors interact with structurally different G proteins (e.g., distinct beta or gamma subunits). More likely it is because of limitations of the mobility of G proteins in the membrane due to 1) attachment to structural elements, such as the cytoskeleton, 2) sequestration in lipid pools, or 3) organization into slowly exchanging supramolecular complexes. These results show that we must reexamine the assumptions of the collision coupling and ternary complex models.

Adrenergic alpha-Agonists↗
PubMed1993

Subsecond modulation of formyl peptide-linked guanine nucleotide-binding proteins by guanosine 5'-O-(3-thio)triphosphate in permeabilized neutrophils.

Rapid activation of guanine nucleotide-binding protein (G protein)-mediated signal transduction mechanisms occurs in many tissues. The human neutrophil provides a useful model for studying the mechanisms of these fast processes. Fluorescent chemotactic tetrapeptide and pentapeptide exhibit 30-50% quenching of fluorescence upon binding to the neutrophil formyl peptide receptor, and their binding affinity is strongly regulated by the G protein Gi. We used rapid kinetic spectrofluorometric methods to study the assembly and disassembly of the ternary complex of ligand, receptor, and G protein in digitonin-permeabilized human neutrophils. Binding was studied up to 20 nM ligand, where the half-time for association was 1.2 sec. The rate constant of association was near that for diffusion-limited reactions of ligands and proteins, 2 x 10(7) M-1 sec-1. The rate of uncoupling of formyl peptide receptor from G protein in the presence of high concentrations of guanine nucleotide was > or = 5 sec-1 (i.e., t1/2 of 0.14 sec). Thus, disassembly of the formyl peptide receptor-G protein complex occurs in the millisecond time domain and may be faster than the next step in the signal transduction process.

GTP-Binding Proteins↗
PubMed1992

A threshold level of coupled G-proteins is required to transduce neutrophil responses.

Chemoattractant-induced activation of human polymorphonuclear leukocytes involves receptor coupling to guanine nucleotide binding proteins (G-proteins). Treatment of polymorphonuclear leukocytes with pertussis toxin, which ADP-ribosylates neutrophil G-proteins and uncouples G-proteins from receptors, causes a conversion of cells from responders to nonresponders rather than a gradual decrease in the ability of all cells to respond (Omann, G. M., and J. M. Harter. 1991. Cytometry 12:252; Omann, G. M., and M. M. Porasik-Lowes. 1991. J. Immunol. 146:1303). Flow-cytometric methods were used to measure N-formylpeptide-induced cytosolic Ca2+ elevation and actin polymerization over a wide range of ADP-ribosylation levels and showed that although the percentage of responding cells varied markedly, the responding cells were stimulated equivalent to controls. The conditions of pertussis toxin (PT) treatment did not interfere with non-G-protein-mediated pathways as assessed by measurement of phagocytosis, a complex process involving the cytoskeleton. We tested the explanation that the all-or-none effect may have been due to heterogeneous insertion of the catalytic subunit of PT into the cells such that responders had no ADP-ribosylation and nonresponders were completely ADP-ribosylated. Measurement of the binding of fluorescent N-formylpeptides to permeabilized cells, which allows the distinction between completely ribosylated and normal cells, showed that all cells treated with a submaximal concentration of PT had intermediate levels of receptor-coupled G-proteins. Thus, partial ADP-ribosylation had occurred in all cells and the all-or-none insertion of the catalytic subunit of PT was ruled out. Thus, there is a threshold of coupled G-proteins required to transduce responses. The ability of PT to inhibit N-formylpeptide-induced actin polymerization and cytosolic calcium elevation was compared and showed that both responses have essentially the same threshold of G-proteins required to transduce the responses. Thus, the pathways regulating actin polymerization and calcium elevation appear to be coupled with equal efficiency to the G-proteins.

Actin Cytoskeleton↗
PubMed1992

Calmodulin binding distinguishes between beta gamma subunits of activated G proteins and transducin.

The interactions between guanine nucleotide regulatory proteins and the Ca(2+)-binding protein calmodulin were studied using calmodulin-Sepharose affinity chromatography. Purified bovine brain beta gamma subunits bound to calmodulin-Sepharose in a Ca(2+)-dependent manner. On the contrary, beta gamma subunits produced in an activated Go/Gi preparation did not bind to calmodulin-Sepharose. The effect was independent of the type of bovine brain G protein (Go/Gi, Gs), method of activation and the presence of magnesium. To distinguish whether the binding of purified beta gamma subunits to calmodulin was unique to brain beta gamma or to the method of purification, similar experiments were performed using transducin. In contrast to bovine brain G proteins, both purified transducin beta gamma subunits and beta gamma released from rhodopsin-activated transducin bound to calmodulin-Sepharose in a Ca(2+)-dependent manner. To assess the functional significance of the binding of bovine brain beta gamma subunits to calmodulin, the ability of purified beta gamma and of beta gamma in unactivated and activated Go/Gi to inhibit partially purified calmodulin-sensitive adenylate cyclase was determined. Purified beta gamma was highly effective in inhibiting calmodulin-stimulated adenylate cyclase activity. However, unactivated Go/Gi and preactivated Go/Gi inhibited calmodulin-stimulated adenylate cyclase activity to the same extent. This Go/Gi-mediated inhibition also occurred in the presence of a 500-fold molar excess of calmodulin over added G protein. These results demonstrate: (1) that beta gamma subunits may not be completely released upon G protein activation, and (2) that inhibition of calmodulin-stimulated adenylate cyclase by beta gamma subunits does not appear to be mediated by a direct beta gamma-calmodulin interaction. Differences in the binding properties of activated bovine brain G proteins versus those of transducin could be explained by differences in the gamma subunit between the proteins, or by differences in affinities of the alpha and beta gamma subunits for each other and for calmodulin. The different functional properties of purified beta gamma subunits and beta gamma subunits produced in situ by activation of G proteins indicates that extrapolation from the effects of purified subunits to events occurring in membranes should be done with caution.

Adenylyl Cyclase Inhibitors↗
PubMed1991

Two peptides from the alpha 2A-adrenergic receptor alter receptor G protein coupling by distinct mechanisms.

Peptides derived from various regions of the alpha 2A-adrenergic receptor (alpha 2A-AR) were used to study receptor-G protein interactions. Binding of the partial agonist [125I]-p-iodoclonidine and the full agonist [3H]bromoxidine (UK14,304) to membrane preparations from human platelet was potently reduced by peptides (12-14 amino acids) from the second cytoplasmic loop (A) and the C-terminal side of the third cytoplasmic loop (Q). Binding of the antagonist [3H]yohimbine was significantly less affected. Five other peptides had no significant effects on ligand binding at concentrations less than 100 microM. The IC50 values for peptides A and Q were 7 and 27 microM for [125I]-p-iodoclonidine binding at the platelet alpha 2A receptor, 15 and 71 microM for the neuroblastoma-glioma (NG108-15) alpha 2B receptor, and greater than 300 microM for yohimbine binding at both alpha 2A and alpha 2B receptors. Competition studies demonstrate that at concentrations of 100 microM, peptides A and Q reduce the affinity of bromoxidine for the platelet alpha 2A-AR and this effect was abolished in the presence of guanine nucleotide. Alpha 2A-AR-stimulated GTPase activity in platelet membranes was inhibited by peptide Q with an IC50 of 16 microM but A was inactive. These data suggest that both the second cytoplasmic loop and the C-terminal part of the third cytoplasmic loop of the alpha 2A-AR are important in the interaction between the alpha 2-AR and Gi protein. Peptide Q appears to destabilize the high affinity state of the alpha 2-AR by binding directly to Gi thus preventing it from coupling to the receptor under both binding and GTPase assay conditions. The peptide from the second cytoplasmic loop (A) also reduces high affinity agonist binding in a G protein-dependent manner but its interaction with receptor and G protein is distinct in that it does not prevent activation of the G protein. These results provide new information about regions of the alpha 2-adrenergic receptor involved in G protein coupling and high affinity agonist binding.

Amino Acid Sequence↗
PubMed1991

Multiple Gi protein subtypes regulate a single effector mechanism.

alpha 2-Adrenergic receptor (alpha 2-AR) responses are mediated by the pertussis toxin-sensitive guanine nucleotide-binding protein (G protein) Gi. Because all three known Gi subtypes are inactivated by pertussis toxin, it has been difficult to determine which of the subtypes are involved in alpha 2-AR responses. In order to investigate alpha 2-AR/Gi coupling, we performed binding and adenylyl cyclase experiments in membranes from CHO-K1 cells transfected with the human alpha 2A-AR. Antisera directed against the carboxyl-terminal region of the Gi1/Gi2 or the Gi3 proteins were used to determine which subtypes were important for high affinity agonist binding and inhibition of adenylyl cyclase. The CHO-K1 cell membranes exhibited immunoreactivity at an apparent molecular mass of 40-41 kDa for both Gi1/Gi2 and Gi3 antisera. Western blot analysis, using purified bovine brain G proteins for comparison, demonstrated that the transfected CHO-K1 cells possess Gi2 and Gi3. High affinity guanosine 5'-(beta,gamma-imido) triphosphate-sensitive binding of the alpha 2-AR agonists [3H]bromoxidine and p-[125I]iodoclonidine ([125I]PIC) was reduced by 30-50% by either the Gi1/Gi2 or Gi3 antiserum. Bromoxidine (1 microM) and PIC (1 microM) inhibited membrane adenylyl cyclase by 34 and 27%, respectively. Gi3 antiserum reduced the inhibition by 26% and 67% for bromoxidine and PIC, respectively. The Gi1/Gi2 antiserum reduced the inhibition by 56% and 63% for bromoxidine and PIC, respectively. Furthermore, when both antisera were used together, there was a complete reversal of alpha 2-AR-mediated inhibition. These observations provide evidence of alpha 2A-AR coupling to at least two subtypes of Gi proteins and the first evidence of functional involvement of Gi3 in the inhibition of adenylyl cyclase.

Adenylyl Cyclases↗
PubMed1990

Subcellular distribution of alpha 2-adrenergic receptors, pertussis-toxin substrate and adenylate cyclase in human platelets.

The subcellular distribution of the alpha 2-adrenergic receptor, pertussis-toxin substrates (Gi, the inhibitory G-protein) and adenylate cyclase was determined in human platelets. The alpha 2-adrenergic receptor and pertussis-toxin substrate activity codistribute with surface membranes identified by a novel fluorescent-lectin method. The platelet granule fractions did not contain detectable Gi. Only 2-4% of the total pertussis-toxin substrate activity appears in soluble fractions, and this amount was not increased upon addition of purified beta gamma units or after pretreatment of platelets with adrenaline. There is no evidence for compartmentation of the alpha 2-adrenergic receptor or Gi to account for the low-affinity component of agonist binding to the alpha 2-adrenergic receptor in human platelet membranes. Translocation of Gi from plasma membrane to platelet cytosol or granules does not appear to play any significant role in the mechanism of alpha 2-receptor-mediated platelet activation.

Adenylate Cyclase Toxin↗
PubMed1990

Sensitization of human alpha 1- and alpha 2-adrenergic venous responses by guanadrel sulfate.

The alpha 1- and alpha 2-adrenergic venoconstriction in dorsal hand veins of normal subjects was determined by infusion of phenylephrine or clonidine. Oral administration of prazosin reduced the constriction response to phenylephrine but not to clonidine. Subjects were treated for 3 weeks in a randomized crossover design with placebo or guanadrel sulfate. Guanadrel reduced sympathetic tone (i.e., plasma norepinephrine and norepinephrine release rate), whereas venous responses to phenylephrine and clonidine were both augmented during guanadrel treatment. The effect on phenylephrine responses was primarily attributable to a decrease in the median effective concentration with a small increase in maximum response. Clonidine showed a markedly increased maximum response with a small increase in the median effective concentration. Platelet alpha 2-adrenergic receptors increased slightly but there was no change in the amount of platelet pertussis toxin substrate during guanadrel treatment. Thus reduction in sympathetic tone in normal young men results in increased venous responses to both alpha 1- and alpha 2-agonists.

Adolescent↗
PubMed1990

p-[125I]iodoclonidine is a partial agonist at the alpha 2-adrenergic receptor.

The binding properties of p-[125I]iodoclonidine [( 125I]PIC) to human platelet membranes and the functional characteristics of PIC are reported. [125I]PIC bound rapidly and reversibly to platelet membranes, with a first-order association rate constant (kon) at room temperature of 8.0 +/- 2.7 x 10(6) M-1 sec-1 and a dissociation rate constant (koff) of 2.0 +/- 0.8 x 10(-3) sec-1. Scatchard plots of specific [125I]PIC binding (0.1-5 nM) were linear, with a Kd of 1.2 +/- 0.1 nM. [125I]PIC bound to the same number of high affinity sites as the alpha 2-adrenergic receptor (alpha 2-AR) full agonist [3H] bromoxidine (UK14,304), which represented approximately 40% of the sites bound by the antagonist [3H]yohimbine. Guanosine 5'-(beta, gamma-imido)triphosphate greatly reduced the amount of [125I]PIC bound (greater than 80%), without changing the Kd of the residual binding. In competition experiments, the alpha 2-AR-selective ligands yohimbine, bromoxidine, oxymetazoline, clonidine, p-aminoclonidine, (-)-epinephrine, and idazoxan all had Ki values in the low nanomolar range, whereas prazosin, propranolol, and serotonin yielded Ki values in the micromolar range. Epinephrine competition for [125I]PIC binding was stereoselective. Competition for [3H]bromoxidine binding by PIC gave a Ki of 1.0 nM (nH = 1.0), whereas competition for [3H]yohimbine could be resolved into high and low affinity components, with Ki values of 3.7 and 84 nM, respectively. PIC had minimal agonist activity in inhibiting adenylate cyclase in platelet membranes, but it potentiated platelet aggregation induced by ADP with an EC50 of 1.5 microM. PIC also inhibited epinephrine-induced aggregation, with an IC50 of 5.1 microM. Thus, PIC behaves as a partial agonist in a human platelet aggregation assay. [125I]PIC binds to the alpha 2B-AR in NG-10815 cell membranes with a Kd of 0.5 +/- 0.1 nM. [125I]PIC should prove useful in binding assays involving tissues with a low receptor density or in small tissue samples and in studies of cloned and expressed alpha 2-AR.

Adenylyl Cyclases↗
PubMed1990

Nonadrenergic [3H]idazoxan binding sites are physically distinct from alpha 2-adrenergic receptors.

We have recently demonstrated that the alpha 2-adrenergic radioligand [3H]idazoxan also labels additional sites that do not recognize catecholamines but bind with high affinity several chemically distinct drugs previously assumed to be highly selective for alpha 2-adrenergic receptors [Mol. Pharmacol. 35:324-330 (1989)]. We now have used three approaches to distinguish the nonadrenergic [3H]idazoxan sites from alpha 2-adrenergic receptors. (a) No nonadrenergic [3H]idazoxan binding sites were found in COS-7 cells transfected with the genes for the two known alpha 2-adrenergic receptor subtypes. (b) The ratio of alpha 2-adrenergic and nonadrenergic [3H]idazoxan sites in human platelet membranes varied considerably between various donors. (c) Highly purified platelet plasma membranes were enriched for alpha 2-adrenergic receptors but did not contain any nonadrenergic [3H]idazoxan binding sites. We conclude that the nonadrenergic [3H]idazoxan binding sites are not co-expressed with alpha 2-adrenergic receptors and at least in human platelets may be located in an intracellular compartment.

Animals↗
PubMed1989

Rapid kinetics of alpha 2-adrenergic inhibition of adenylate cyclase. Evidence for a distal rate-limiting step.

Activation and inhibition of adenylate cyclase in the presence of GTP, the natural guanine nucleotide regulator, are too fast to study by standard biochemical methods. In order to identify the rate-limiting steps in adenylate cyclase regulation, we measured the kinetics of stimulation and inhibition of the enzyme on a subsecond to second time scale using a novel rapid-mix quench technique. Even using our rapid-mix quench method, activation by PGE1 and forskolin was instantaneous (cAMP accumulation was linear between 0.5 and 30 s). In contrast, we found a lag period of 1.2-10 s for epinephrine-mediated inhibition. The length of the lag depended on the concentration of GTP and monovalent cations present. In the absence of NaCl, the rate constant for the onset of inhibition (kinh) increased only slightly with GTP concentration saturating at a value of 0.16 s-1 (t1/2 4.3 s) at 1 microM GTP. In the presence of 100 mM NaCl, kinh was strongly dependent on GTP concentration, reaching a maximum value of 0.57 s-1 (t1/2 1.2 s) at 100 microM GTP. Thus, activation of both Gi and Gs in intact platelet membranes is much faster (t1/2 less than 5 s) than previously reported for reconstituted systems. Also, the strong dependence of the rate of adenylate cyclase inhibition on GTP concentration implies that the rate-limiting step in inhibition is distal to GTP binding. The effect of NaCl to increase the maximal rate of inhibition is specific for sodium since KCl has no effect on kinh.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclase Inhibitors↗
PubMed1989

How does a key fit a flexible lock? Structure and dynamics in receptor function.

The preceding five years have brought remarkable advances in our understanding of the primary structure of drug receptors. The roles of certain amino acid residues in binding drugs and effecting receptor function have been proposed. As even more detailed structures become available, the goal of rational design of drug molecules based on predicted fits between the drug and its receptor will be near at hand. Although none of the classical receptors has yet yielded to X-ray crystallographic analysis, the methods of molecular biology facilitate the production of the large amounts of these rare proteins necessary for crystallization. Receptor proteins share one fundamental characteristic with allosterically regulated enzymes. Both have the structural flexibility that allows information to be transmitted to distant parts of the molecule. We will discuss recent observations about receptor structure and the dynamic nature of drug receptors, and pose questions about the significance of receptor dynamics for drug design.

Amino Acid Sequence↗
PubMed1989

Effects of low-sodium diet on regulation of platelet alpha 2-adrenergic receptors in young and elderly humans.

To examine whether there are age differences in agonist-mediated alpha 2-adrenergic receptor (alpha 2-AR) regulation, we studied the effect of a sustained increase in plasma norepinephrine (pNE) during a 7-day 10-meq Na+/day diet on platelet alpha 2-AR binding and its linked adenylate cyclase (AC) activity in 11 elderly and 11 young healthy subjects. In the young, a 41% increase in mean pNE after a low-sodium diet was correlated with a decline in receptor density (Bmax; r = -0.816; P less than 0.01) and was accompanied by a reduction in the maximal percent inhibition of sodium fluoride-stimulated AC activity by epinephrine (%AC INH; 33 +/- 4 vs. 24 +/- 4%, mean +/- SE; P less than 0.05 vs. normal diet). Despite a comparable 39% increase in mean pNE in the elderly, neither Bmax nor %AC INH was significantly reduced after a low-sodium diet. The amount of pertussis toxin substrate (Gi protein) was similar in both groups before and after dietary sodium restriction. At comparable pNE, %AC INH in the groups was similar (young, 24 +/- 4 vs. elderly, 18 +/- 4%; P = NS). We postulate that higher basal pNE levels in the elderly on normal diet may account for the lack of further downregulation of platelet alpha 2-AR density and response after low-sodium diet.

Adult↗
PubMed1988

Temperature effects on alpha 2-adrenergic receptor-Gi interactions.

The effect of temperature on the binding of alpha 2-adrenergic agonists and antagonists to human platelet membranes was studied. Equilibrium binding of the alpha 2 antagonist, [3H]yohimbine, was affected minimally, whereas the rate of dissociation changed 40-fold over a temperature range of 5-35 degrees. The antagonist dissociation rates were characterized by a linear Arrhenius plot and an activation energy of 20.5 kcal/mol. The equilibrium binding of the full alpha 2 agonist, [3H]UK 14,304 [5-bromo-6-N-2-4,5-dihydroimidazolyl)quinoxaline tartrate] showed a 50% decrease in Bmax at 5 degrees as well as a 2-fold decrease in affinity. The kinetics of [3H]UK 14,304 binding were affected more significantly by decreases in temperature. The agonist exhibited fast and slow phases of binding. The fast binding was minimally sensitive to temperature in the range of 0-30 degrees with only a 6-fold change in rate. The slow binding rates changed nearly 100-fold over the same temperature range. Also, the slow rate of agonist binding was characterized by a nonlinear Arrhenius plot with a "break" at approximately 17 degrees, which was found previously to be the phase transition temperature of platelet membrane lipids [Lohse et al., Molec. Pharmac. 29, 228 (1986)]. Despite the reduction of high affinity [3H]UK 14,304 binding at 5 degrees, approximately half of the binding remained sensitive to guanine nucleotides. The data are interpreted in the context of a model in which the fast agonist binding represents a bimolecular interaction of ligand with two pre-existing conformations of the alpha 2 receptor, one coupled to Gi and the other permanently uncoupled. The slow binding of agonist appears to require protein diffusion in the lipid membrane or a protein conformational change which is dependent on the lipid environment.

Adenylyl Cyclase Inhibitors↗