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Biomedical subjects

L E Limbird

Publications and source records attributed to L E Limbird.

At least 73 records · Page 4Linked to original sources
PubMed1987

Multiple receptors linked to inhibition of adenylate cyclase accelerate Na+/H+ exchange in neuroblastoma x glioma cells via a mechanism other than decreased cAMP accumulation.

alpha 2-Adrenergic receptors, a population of receptors linked to inhibition of adenylate cyclase, accelerate Na+/H+ exchange in NG108-15 neuroblastoma x glioma cells (Isom, L. L., Cragoe, E. J., Jr., and Limbird, L. E. (1987) J. Biol. Chem. 262, 6750-6757). We now report that two other receptor populations linked to inhibition of adenylate cyclase, muscarinic cholinergic and delta-opiate receptors, also alkalinize the interior of NG108-15 cells, as measured with the pH-sensitive fluorescent probe, 2,7-biscarboxyethyl-5(6)-carboxy-fluorescein. Manipulations that block Na+/H+ exchange, i.e. removal of extracellular Na+, reduction of extracellular pH to equal that of intracellular pH, and addition of 5-amino-substituted analogs of amiloride, all block alpha 2-adrenergic, delta-opiate, or muscarinic cholinergic receptor-induced alkalinization in a parallel fashion. These data suggest that all three populations of receptors alkalinize NG108-15 cells by acceleration of Na+/H+ exchange and do so via a shared or similar mechanism. Although these three receptor populations are linked to inhibition of adenylate cyclase, decreased production of cAMP does not appear to be the mechanism responsible for receptor-accelerated Na+/H+ exchange. Thus, ADP-ribosylation of intact NG108-15 cells with Bordetella pertussis islet-activating protein prevents attenuation of prostaglandin E1-stimulated cAMP accumulation by alpha 2-adrenergic, muscarinic, and delta-opiate agonists but has no measurable effect on the ability of these agonists to accelerate Na+/H+ exchange. Similarly, manipulations that block receptor-accelerated Na+/H+ exchange influence but do not block receptor-mediated attenuation of cAMP accumulation. Thus, the present data suggest that these two receptor-mediated biochemical events, acceleration of Na+/H+ exchange and attenuation of cAMP accumulation, occur through divergent mechanisms in NG108-15 cells.

Adenosine Diphosphate Ribose↗
PubMed1987

Purification of the alpha 2-adrenergic receptor from porcine brain using a yohimbine-agarose affinity matrix.

A procedure has been developed for purification of the porcine brain alpha 2-adrenergic receptor to homogeneity. alpha 2-Adrenergic receptors were solubilized from porcine brain particulate preparations using sequential extraction into sodium cholate- and digitonin-containing buffers. The alpha 2-adrenergic receptors in the digitonin extract were identified using the alpha 2-adrenergic selective antagonist, [3H]yohimbine, and demonstrated the same specificity for interaction with adrenergic ligands as did the receptors in particulate preparations. Extraction into digitonin-containing buffers eliminated the modulation of receptor-agonist interactions by guanine nucleotides, but not by monovalent cations. A novel affinity resin, yohimbine-agarose, was synthesized and used for purification of alpha 2-adrenergic receptors. Using two sequential yohimbine-agarose affinity chromatography steps, digitonin-solubilized alpha 2-adrenergic receptors from porcine brain cortex were purified to homogeneity as assessed by radioiodination and silver stain analysis of these preparations on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified alpha 2-adrenergic receptor has an approximate Mr = 65,000, as determined by photolabeling of the adrenergic ligand-binding subunit. The yohimbine-agarose affinity resin should be useful for purifying quantities of receptor sufficient for studies of receptor structure and function.

Animals↗
PubMed1987

Regulation of porcine brain alpha 2-adrenergic receptors by Na+,H+ and inhibitors of Na+/H+ exchange.

Previous reports from this laboratory have demonstrated that alpha 2-adrenergic receptors accelerate Na+/H+ exchange in NG108-15 neuroblastoma X glioma cells and evoke platelet secretion via a pathway involving Na+/H+ exchange. The present studies were designed to examine whether agents that interact with Na+/H+ antiporters also might influence alpha 2-adrenergic receptor-ligand interactions. We observed that Na+ decreases receptor affinity for the agonists epinephrine, norepinephrine, and UK14304 and slightly increases receptor affinity for the antagonists yohimbine and idazoxan in digitonin-solubilized preparations from porcine brain cortex. Increases in [H+] also decrease receptor affinity for agonists and cause either a slight increase or no change in receptor affinity for antagonists. Amiloride analogs accelerate the rate of [3H] yohimbine dissociation from digitonin-solubilized receptors with a relative effectiveness that parallels their ability to block Na+/H+ exchange in other systems. Interestingly, these modulatory effects of Na+,H+ and 5-amino-substituted analogs of amiloride are retained in homogeneous preparations of the alpha 2-adrenergic receptor, suggesting that the allosteric-binding sites for these agents are on the receptor-binding protein itself.

Amiloride↗
PubMed1987

Alpha 2-adrenergic receptors accelerate Na+/H+ exchange in neuroblastoma X glioma cells.

The regulation of cytoplasmic pH (pHi) was examined in neuroblastoma X glioma hybrid cell-line cells (NG108-15 cells) using 2,7-biscarboxyethyl-5(6)-carboxyfluorescein. The pHi of NG108-15 cells suspended in nominally HCO-3-free, Na+-containing buffer could be reduced by the external application of acetate. The recovery of pHi to its resting value was blocked by the removal of extracellular Na+, by the addition of extra-cellular H+, and by the addition of analogs of amiloride selective for inhibition of Na+/H+ exchange. The rate of recovery of pHi from acid load exhibited an ionic selectivity of Na+ greater than Li+ much greater than K+, and no recovery was observed in N-methyl-D-glucamine+. Tetrodotoxin and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid had no effect on early pHi recovery. These data suggest that Na+/H+ exchange accounts primarily for the recovery of pHi in NG108-15 cells under our experimental conditions. Na+/H+ exchange in NG108-15 cells was accelerated by alpha 2-adrenergic receptors. Thus, (-)epinephrine, but not (+)epinephrine, elicited an intracellular alkalinization which was blocked by the alpha 2-adrenergic receptor selective antagonist yohimbine but not by the alpha 1-adrenergic receptor antagonist, prazosin, nor the beta-adrenergic antagonist, propranolol. Norepinephrine, clonidine, and the clonidine analog, UK-14304, also caused alkalinization of NG108-15 cells, whereas isoproterenol, a beta-adrenergic receptor agonist, and phenylephrine, a selective alpha 1-adrenergic receptor agonist, did not. Manipulations that blocked Na+/H+ exchange blocked the ability of alpha 2-adrenergic agonists to alkalinize the interior of NG108-15 cells without blocking the ability of these agonists to attenuate cAMP accumulation. These findings provide the first direct evidence of modulation of Na+/H+ exchange activity by a receptor linked to inhibition of adenylate cyclase and offer a possible mechanism whereby alpha 2-adrenergic receptors might influence cellular activity apart from changes in cyclic nucleotide metabolism.

Adrenergic alpha-Antagonists↗
PubMed1987

Synthesis and characterization of arylamine derivatives of rauwolscine as molecular probes for alpha 2-adrenergic receptors.

The selective alpha 2-adrenergic receptor antagonist rauwolscine was structurally modified to yield a series of arylamine carboxamide derivatives, which were investigated as potential molecular probes for the localization and structural characterization of alpha 2-adrenergic receptors. The arylamine carboxamides differ in the number of carbon atoms separating the reactive phenyl moiety from the fused ring structure of the parent compound, rauwolscine carboxylate. Competitive inhibition studies with [3H]rauwolscine in rat kidney membranes indicate that the affinity for the carboxamide derivatives is inversely related to the length of the carbon spacer arm with rauwolscine 4-aminophenyl carboxamide (zero carbon spacer arm; rau-AMPC) exhibiting the highest affinity (Kd = 2.3 +/- 0.2 nM). Radioiodination of rau-AMPC yields a ligand, 125I-rau-AMPC, which binds to rat kidney alpha 2-adrenergic receptors with high affinity, as determined by both kinetic analysis (Kd = k2/k1 = 0.016 min-1/2.1 X 10(7) M-1 min-1 = 0.76 nM) and equilibrium binding studies (Kd = 0.78 +/- 0.16 nM). 125I-rau-AMPC was quantitatively converted to the photolabile arylazide derivative 17 alpha-hydroxy-20 alpha-yohimban-16 beta-(N-4-azido-3-[125I]iodophenyl) carboxamide (125I-rau-AZPC). In a partially purified receptor preparation from porcine brain, this compound photolabels a major (Mr = 62,000) peptide. The labeling of this peptide is inhibited by adrenergic agonists and antagonists with a rank order of potency consistent with an alpha 2-adrenergic receptor binding site. Both 125I-rau-AMPC and the photolabile arylazide derivative, 125I-rau-AZPC, should prove useful as molecular probes for the structural and biochemical characterization of alpha 2-adrenergic receptors.

Adrenergic beta-Agonists↗
PubMed1987

Evidence for a role for Na+-H+ exchange in activation of human platelets by PAF.

We have found previously that inhibitors of Na+-H+ exchange block platelet arachidonic acid release and subsequent secondary aggregation and serotonin release in response to epinephrine, ADP, and thrombin (0.004 U/ml). The present study demonstrates that the addition of ethylisopropylamiloride, an inhibitor of Na+-H+ exchange, leads to an inhibition of platelet activating factor-induced serotonin release and thromboxane B2 production in human platelets in citrated plasma. In addition, platelet activating factor-induced platelet secretion is blocked by the cyclooxygenase inhibitor indomethacin or the thromboxane antagonist SQ 29548, indicating that arachidonic acid mobilization and metabolism is required for platelet activating factor to elicit platelet activation. Our interpretation of the present findings is that platelet activating factor-induced secretion of dense granules from the human platelet requires the production of cyclooxygenase metabolites from arachidonic acid and that Na+-H+ exchange plays an important, albeit not exclusive, role in mobilization of arachidonic acid in response to platelet activating factor.

Amiloride↗
PubMed1986

Inhibitors of Na+/H+ exchange block epinephrine- and ADP-induced stimulation of human platelet phospholipase C by blockade of arachidonic acid release at a prior step.

The ability of epinephrine or ADP to cause an increase in the production of phospholipase C products (diacylglycerol and inositol phosphates) in human platelets is blocked by perturbants of Na+/H+ exchange, i.e. ethylisopropylamiloride, decreased extraplatelet pH, or removal of extraplatelet Na+. These perturbants do not, however, block inositol phosphate production in response to 0.2 unit/ml thrombin, indicating that inhibition of Na+/H+ exchange does not inhibit the phospholipase C enzyme directly. Since the cyclooxygenase inhibitor indomethacin and the endoperoxide/thromboxane antagonist SQ29548 block epinephrine- and ADP-induced inositol phosphate production, it can be concluded that these agonists activate phospholipase C secondary to mobilization of arachidonic acid and production of cyclooxygenase products. This conclusion is consistent with the observation that the endoperoxide analogue U46619 causes inositol phosphate production. Furthermore, the effect of U46619 is not blocked by inhibitors of Na+/H+ exchange. The initial pool of arachidonic acid mobilized by epinephrine can be measured using negative ion gas chromatography/mass spectrometry and is sensitive to inhibition of Na+/H+ exchange. The present data suggest that epinephrine and ADP cause mobilization of a small pool of arachidonic acid by a pathway involving Na+/H+ exchange. The cyclooxygenase products derived from this pool subsequently activate phospholipase C. Since the same treatments that block epinephrine- and ADP-induced diacylglycerol and inositol phosphate production also block epinephrine- and ADP-induced dense granule secretion, it appears that activation of phospholipase C, albeit indirectly via cyclooxygenase products, may be required for epinephrine and ADP to evoke platelet secretion.

Adenosine Diphosphate↗
PubMed1986

Evidence that Na+/H+ exchange regulates receptor-mediated phospholipase A2 activation in human platelets.

Data in the previous paper suggest that epinephrine can mobilize a small pool of arachidonic acid via an enzymatic pathway distinct from phospholipase C and that this pathway is blocked by perturbations that block Na+/H+ exchange. The present studies demonstrate that epinephrine and ADP stimulate a phosphatidylinositol-hydrolyzing phospholipase A2 activity in human platelets. This occurs even when measurable phospholipase C activation, platelet secretion, and secondary aggregation are blocked with the thromboxane A2 receptor antagonist SQ29548. Furthermore, perturbants of Na+/H+ exchange diminish lysophosphatidylinositol production in response to epinephrine, ADP, and thrombin, but not to the Ca2+ ionophore A23187. Artificial alkalinization of the platelet interior with methylamine reverses the effect of the Na+/H+ antiporter inhibitor, ethylisopropylamiloride, on thrombin-stimulated lysolipid production, suggesting that the alkalinization of the platelet interior which would occur secondary to activation of Na+/H+ exchange might play an important role in phospholipase A2 activation. In addition, treatment of platelets with methylamine increases the sensitivity of phospholipase A2 to activation by the Ca2+ ionophore A23187, suggesting that changes in pH and Ca2+ may regulate phospholipase A2 activity synergistically. Finally, epinephrine causes a prompt decrease in platelet-chlortetracyclin fluorescence even in the presence of cyclooxygenase inhibitors, suggesting that epinephrine is able to mobilize membrane-bound Ca2+ independent of phospholipase C activation. Taken together, the data suggest that epinephrine-provoked stimulation of phospholipase A2 activity may occur as a result of Ca2+ mobilization and a concomitant intraplatelet alkalinization resulting from accelerated Na+/H+ exchange.

Adenosine Diphosphate↗
PubMed1986

Photoaffinity labeling of the porcine brain alpha 2-adrenergic receptor using a radioiodinated arylazide derivative of rauwolscine: identification of the hormone-binding subunit.

A functionalized derivative of the alpha 2-selective antagonist rauwolscine formed the basis for a photoaffinity adduct that has allowed identification of the hormone-binding subunit of the brain alpha 2-adrenergic receptor protein. Rauwolscine carboxylate underwent reaction with 4-N-t-butyloxycarbonyl-aminoaniline, leading to the synthesis of rauwolscine 4-aminophenyl carboxamide (Rau-AmPC). Rau-AmPC was radioiodinated and converted to the arylazide derivative, 17 alpha-hydroxy-20 alpha-yohimban-16 beta-[N-(4-azido-3-[125I]iodo)phenyl] carboxamide (125I-Rau-AzPC), via a diazonium salt intermediate. The characterization of 125I-Rau-AzPC as a photolabile probe employed alpha 2-adrenergic receptors, which were first solubilized from porcine brain membranes and partially purified by affinity chromatography utilizing a yohimbine-agarose affinity matrix. In the partially purified receptor preparation incubated with 125I-Rau-AzPC, photolysis resulted in covalent labeling of a major (Mr, 62,000) peptide as determined by NaDodSO4/PAGE and autoradiography. Labeling of this peptide was inhibited by the alpha 2-selective antagonist, yohimbine, and the non-subtype-selective alpha-antagonist, phentolamine, but not by the alpha 1-antagonist, prazosin, or the beta-receptor antagonist, (-)-alprenolol. The alpha-adrenergic agonist epinephrine also inhibited labeling in a stereoselective manner. These data indicate that the photolabeled Mr 62,000 peptide is the hormone-binding subunit of the alpha 2-adrenergic receptor protein. The availability of this radioiodinated photoaffinity probe for the alpha 2-adrenergic receptor should facilitate further structural and biophysical characterization of the receptor protein.

Affinity Labels↗
PubMed1985

Inhibitors of Na+/H+ exchange block stimulus-provoked arachidonic acid release in human platelets. Selective effects on platelet activation by epinephrine, ADP, and lower concentrations of thrombin.

We have observed previously that removal of extraplatelet Na+ blocks platelet secretion of dense granule contents in response to epinephrine, ADP, and 0.004 unit/ml thrombin, all agents which must mobilize arachidonic acid for its subsequent conversion to cyclooxygenase products in order to elicit platelet secretion. The present studies demonstrate that removal of extraplatelet Na+ blocks arachidonic acid mobilization in response to epinephrine, ADP, and 0.004 unit/ml thrombin without altering arachidonic acid conversion to thromboxane A2. The data also provide several lines of evidence which suggest that the blockade of arachidonic acid release due to removal of extraplatelet Na+ is a manifestation of blockade of Na+/H+ exchange system. 1) There is a concentration-dependent effect of extraplatelet Na+ (EC50 congruent to 55 mM) on [3H]arachidonic acid release such that mobilization is observed when [Na+]o greater than [Na+]i. 2) Increasing extraplatelet [H+] (i.e. decreasing extraplatelet pH from pH 7.35 to 6.8) causes a concentration-dependent decline in stimulus-provoked [3H]arachidonic acid release. 3) Ethylisopropylamiloride and other potent 5-amino analogs of amiloride block [3H]arachidonic acid release with a potency that parallels their effects on Na+/H+ exchange in other cellular systems. None of the above manipulations alter primary aggregation induced by epinephrine, ADP, or 0.004 unit/ml thrombin, indicating that stimulus-receptor binding, subsequent exposure of fibrinogen receptors, and fibrinogen-mediated platelet-platelet cross-linking are not significantly inhibited by [3H]arachidonic acid release in response to greater than 0.1 unit/ml thrombin, a stimulus that can elicit platelet secretion in the absence of products of the cyclooxygenase pathway. Therefore, Na+/H+ exchange may selectively modulate arachidonic acid mobilization in response to the so-called "weak agonists," agonists that require this mobilization to effect vigorous platelet aggregation and dense granule secretion.

Adenosine Diphosphate↗
PubMed1985

The resolution of agonist alpha 2-adrenergic receptor complexes from unoccupied receptors or antagonist-alpha 2 receptor complexes using DEAE chromatography.

Alpha 2-adrenergic receptors linked to inhibition of adenylate cyclase activity in human platelet membranes can be isolated using DEAE chromatography following solubilization into digitonin-containing buffers. This procedure permits resolution of agonist-occupied receptors from antagonist-occupied receptors. Antagonist-occupied receptors co-elute with unoccupied receptors. Adenylate cyclase activity elutes independently of all alpha 2-receptor activities. A greater resolution of agonist-receptor complexes from antagonist-receptor complexes is obtained using DEAE chromatography than reported earlier using approaches that rely entirely on agonist-stabilized increases in apparent receptor size. Consequently, DEAE chromatography may be of considerable value in isolating these agonist-receptor complexes to permit identification of the membrane component(s) which are more stably associated with the receptor subsequent to agonist occupancy and thus might be involved in receptor-cyclase coupling.

Adenylyl Cyclase Inhibitors↗
PubMed1984

A model system for the biochemical study of luteinizing hormone/chorionic gonadotropin receptor synthesis.

A model system for the biochemical study of LH/CG receptor synthesis has been developed. Culture conditions for porcine granulosa cells were adapted that maximized the selective induction of LH/CG receptors by cAMP-inducing stimuli with an elimination of background LH/CG receptor appearance. It was found that the addition of FSH (1.5 micrograms/ml) or cholera toxin (10 ng/ml) 1 day after plating resulted in optimal induction of the LH/CG receptor (20-60 pg [125I]CG bound/micrograms DNA 72 h after addition) with virtually no LH/CG receptor appearance in the absence of added stimuli. Later additions of FSH or cholera toxin required insulin (1.0 microgram/ml) which alone caused background LH/CG receptor appearance in the absence of any additional stimuli. Furthermore, insulin increased the general rate of cellular protein synthesis, whereas FSH or cholera toxin each decreased it. Thus, the use of FSH or cholera toxin, without insulin, may enable one to detect the synthesis of the LH/CG receptor by metabolic labeling techniques where background protein synthesis has been lowered.

Animals↗
PubMed1984

Alterations in leukocyte beta-receptor affinity with aging. A potential explanation for altered beta-adrenergic sensitivity in the elderly.

The elderly have reduced beta-adrenergic sensitivity to agonists, but no change in receptor density. We investigated the relation between beta-receptor affinity for agonists and age in beta-receptors on lymphocytes from 20 healthy men 21 to 74 years old. As an index of beta-receptor affinity for agonists, we determined the IC50 of isoproterenol--the concentration of isoproterenol required to inhibit 50 per cent of iodohydroxybenzylpindolol binding in vitro. We found that receptor affinity for agonists was correlated with age and plasma norepinephrine concentration. Twelve subjects (six 21 to 29 years old and six 55 to 74 years old) were also studied in both the supine and upright positions. In samples obtained in the supine position, the proportion of receptors binding agonist with a high affinity was decreased in the older subjects as compared with the young subjects (22 +/- 1 per cent vs. 38 +/- 3 per cent; P less than 0.05). With upright posture and the associated acute elevation of endogenous plasma catecholamines, the proportion of receptors binding agonist with a high affinity was reduced in the young; no such changes were seen in the older subjects. The data suggest that reduced beta-receptor affinity for agonists may be an explanation for altered beta-adrenergic sensitivity in the elderly.

Adrenergic beta-Agonists↗
PubMed1984

GTP and Na+ modulate receptor-adenyl cyclase coupling and receptor-mediated function.

Activation and inhibition of adenylate cyclase activity is mediated via two distinct GTP-binding proteins. In both stimulatory and inhibitory systems, receptor occupancy by a hormone or agonist drug stabilizes receptor interactions with its functionally associated GTP-binding protein and, as a consequence, facilitates GTP occupancy of the regulatory protein. Activation of cyclase proceeds until GTP is hydrolyzed to GDP. Although hormonal inhibition of cyclase is elicited by a sequence of molecular events seemingly parallel to those for activation of cyclase, it is not known whether hormonal inhibition of cyclase involves the direct interaction of the GTP-occupied inhibitory regulatory protein with the catalytic subunit or results indirectly from destabilization of the interaction of the stimulatory GTP-binding protein with the catalytic moiety. Sodium ion also modulates receptor-mediated inhibition of adenylate cyclase, although apparently via a component distinct from the GTP-binding subunit of the inhibitory GTP-binding protein. Sodium also influences the physiological functions elicited by receptors that mediate inhibition of cyclase, at least for alpha 2-adrenergic-promoted platelet secretion. The relationship between sodium effects on hormonal attenuation of cAMP accumulation and elicited physiological effects is discussed.

Adenylate Cyclase Toxin↗
PubMed1984

Leukocyte beta-receptor alterations in hypertensive subjects.

It has been suggested that beta-adrenergic responsiveness is reduced in hypertension. To evaluate a possible alteration in human beta-receptors that might account for diminished beta-adrenergic responsiveness, we studied leukocytes from hypertensive and normotensive subjects after an overnight rest supine, and then after being ambulatory, a maneuver that increases plasma catecholamines approximately twofold. In supine samples, beta-receptor affinity for the agonist isoproterenol was significantly reduced in hypertensives and was associated with a reduction in the proportion of beta-receptors binding agonist with a high affinity from 42 +/- 6% in normotensive subjects to 25 +/- 2% in hypertensives (P less than 0.05). Alterations in beta-adrenergic-mediated adenylate cyclase activity parallelled the differences seen in the beta-receptor affinity for agonist. In normotensive subjects, beta-receptor density and the proportion of receptors binding agonist with high affinity were reciprocally correlated with plasma catecholamines. However, in the hypertensive subjects these correlations were not evident. Thus, our data suggest an alteration in leukocyte beta-receptor interactions in hypertensive subjects, and may represent a generalized defect in beta-receptor function in hypertension.

Adenylyl Cyclases↗
PubMed1983

5'-p-Fluorosulfonylbenzoyl guanosine as a probe for the GTP-binding protein in alpha 2-adrenergic receptor-adenylate cyclase systems.

An analog of GTP, 5'-p-fluorosulfonylbenzoyl guanosine (5'-p-FSO2BzGuo), appears to interact irreversibly with guanine nucleotide-binding sites on human platelet membranes. This conclusion is based on the observation that incubation of human platelet membranes with 1.4 mM 5'-p-FSO2BzGuo followed by extensive membrane washing results in a reduction in the density of binding sites for [3H]guanylylimidodiphosphate (Gpp(NH)p), a hydrolysis-resistant analog of GTP. The alpha 2-adrenergic receptor of human platelets is felt to interact with a GTP-binding protein that modulates alpha 2-receptor-agonist interactions and mediates inhibition of adenylate cyclase. The present data suggest that 5'-p-FSO2BzGuo attains saturating, or near saturating, occupancy of this alpha 2-receptor-associated GTP-binding protein, since incubation of human platelet membranes with 5'-p-FSO2BzGuo mimics the effects of optimal concentrations of Gpp(NH)p (0.1 mM) in reducing alpha 2-receptor affinity for agonist agents: 5'-p-FSO2BzGuo increases the EC50 for epinephrine competition for [3H]yohimbine antagonist binding to alpha 2-receptors from 0.15 to 1.5 microM and promotes a time- and concentration-dependent decrease in high affinity [3H]epinephrine agonist binding. The persistent effects of 5'-p-FSO2BzGuo on alpha 2-receptor-agonist interactions following extensive washing of the platelet membranes suggest that 5'-p-FSO2BzGuo modification of the alpha 2-receptor-associated GTP-binding protein is irreversible. Taken together, the above findings suggest that 5'-p-FSO2BzGuo may be the appropriate reagent to prepare in a radiolabeled form to affinity label the GTP-binding proteins in human platelet membranes and compare the properties of alpha 2-adrenergic receptor-associated GTP-binding protein(s) with those of the presumably distinct GTP-binding protein that mediates stimulation of adenylate cyclase.

Adenylyl Cyclases↗