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Biomedical subjects

L E Limbird

Publications and source records attributed to L E Limbird.

At least 91 records · Page 5Linked to original sources
PubMed1983

Removal of extraplatelet Na+ eliminates indomethacin-sensitive secretion from human platelets stimulated by epinephrine, ADP, and thrombin.

We have previously observed that removal of extraplatelet Na+ markedly diminishes human platelet aggregation and secretion in response to epinephrine. The present studies demonstrate that this effect of the removal of extraplatelet Na+ on platelet function is not unique to activation of platelets by alpha 2-adrenergic agents but represents a phenomenon also evident for other platelet stimuli. Thus, platelet aggregation and secretion in response to maximal concentrations of ADP and lower concentrations of thrombin (less than 0.04 unit/ml) were also markedly reduced in platelets in "Na+-free" medium, suggesting that these agents share an effector mechanism that is similarly inhibited by the removal of extraplatelet Na+. In contrast, platelet aggregation and secretion in response to higher concentrations of thrombin (greater than or equal to 0.04 unit/ml) and to 0.04-1.0 microM (15S)-hydroxy-11 alpha, 9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619), an endoperoxide analog, were identical in control platelets and in those suspended in "Na+-free" medium, indicating that platelets suspended in "Na+-free" medium are functionally intact, at least in response to some stimuli. Furthermore, the observation that U46619 can elicit platelet aggregation and secretion independently of extraplatelet Na+ indicates that the loss of platelet responsiveness to epinephrine, ADP, and low concentrations of thrombin cannot be attributed to a loss of sensitivity to the stimulus-provoked secondary mediator(s) of platelet function, endoperoxides or thromboxane A2. Treatment with indomethacin to block the secondary aggregation and secretion pathways of platelets reduced the aggregatory and secretory responses of control platelets induced by epinephrine, ADP, and low concentrations of thrombin to those characteristic of platelets suspended in "Na+-free" medium. In contrast, indomethacin did not alter the functional responses induced by these agents in platelets suspended in "Na+-free" medium, suggesting that "primary" aggregation is intact but that the "secondary" aggregation and secretion mediated by arachidonic acid metabolites are eliminated by removal of extraplatelet Na+. Consistent with this interpretation is the observation that the indomethacin-insensitive aggregation and secretion induced by U46619 and higher concentrations of thrombin were retained in platelets suspended in "Na+-free" medium. Thus, the responses eliminated by removal of extraplatelet Na+ are those eliminated by treating control platelets with indomethacin, suggesting a strong link between the presence of extraplatelet Na+ and the operation of platelet function mediated by the cyclooxygenase pathway.

Adenosine Diphosphate↗
PubMed1983

Luteinizing hormone receptor appearance in cultured porcine granulosa cells requires continual presence of follicle-stimulating hormone.

During the differentiation of ovarian granulosa cells, follicle-stimulating hormone (follitropin; FSH) mediates the induction of cell surface receptors for luteinizing hormone (lutropin; LH). Using primary cultures of porcine granulosa cells, we demonstrate that both the induction and maintenance of LH receptors are critically dependent upon the continual presence of FSH. The termination of FSH-promoted LH receptor induction is paralleled by the termination of FSH-induced intracellular cAMP accumulation. Changing the medium is without effect on FSH-induced appearance of LH receptors. Furthermore, 1 mM aminoglutethimide, which completely blocks FSH-stimulated progesterone biosynthesis, does not decrease the induction of LH receptors by FSH. Thus, the induction of LH receptors by FSH does not appear to require the accumulation of a factor in the medium, nor does it appear to be mediated via FSH-stimulated progesterone synthesis. These data suggest that intracellular cAMP, produced while FSH remains bound to the cell surface, mediates the induction of LH receptors and that the continual presence of FSH is required for both the induction and maintenance of cell surface LH receptors.

Animals↗
PubMed1983

Dynamic regulation of leukocyte beta adrenergic receptor-agonist interactions by physiological changes in circulating catecholamines.

beta-Adrenergic receptors on human mononuclear leukocytes were assessed using [125I]iodohydroxybenzylpindolol binding. Subjects were studied supine and after being ambulatory, a maneuver that increases plasma catecholamines approximately two-fold. beta-Receptor affinity for agonists, measured by the competition of [125I]iodohydroxybenzylpindolol binding by (-)isoproterenol was significantly reduced with ambulation and this reduction was associated with a reduction in the proportion of beta-receptors binding agonist with a high affinity from a mean (+/- SEM) of 42 +/- 5 to 24 +/- 2% (P less than 0.01). In a parallel series, beta-adrenergic-stimulated adenylate cyclase activity was also reduced with postural change from 4.6 +/- 1.1 to 2.4 +/- 0.6 pmol [32P]cAMP/min per mg protein (P less than 0.05) after ambulation. Similar reductions in the proportion of receptors binding agonist with a high affinity were seen after infusion of norepinephrine. We conclude that the maneuver of ambulation reduces leukocyte beta-receptor responsiveness and affinity for agonists, probably by the effect of increased plasma catecholamines mediating an uncoupling of the beta-receptor-adenylate cyclase complex.

Adenylyl Cyclases↗
PubMed1983

The cAMP-Dependent induction of LH receptors in primary cultures of porcine granulosa cells is not due to the expression of an intracellular pool of LH receptors.

The present studies examined whether the increase in cell surface LH receptors in primary cultures of porcine granulosa cells after exposure to FSH or cholera toxin, agents that increase intracellular cAMP, is due to de novo synthesis of the receptor or to a cAMP-dependent translocation of an intracellular pool of LH receptors to the cell surface. LH receptor induction by FSH was fully inhibited by the addition of cycloheximide to the incubation media, but resumed after cycloheximide was removed. These data suggest that FSH-induced LH receptor appearance requires protein synthesis. However, to be confident that the inhibition of LH receptor appearance did not result from lack of transit of preformed receptors requiring a rapidly turning over pool of proteins, we assayed for possible latent receptors in the cell interior by extracting granulosa cells with Triton X-100. Under conditions which detected about 74% of LH receptors in cells exposed to cholera toxin, little [125]iodo-hCG-binding activity was detected in cells not exposed to a cAMP-promoting stimulus. These findings suggest that a preformed pool of LH receptors does not exist in untreated cells, and that the cAMP-mediated induction of LH receptors requires de novo synthesis of the receptor.

Animals↗
PubMed1983

N-ethylmaleimide, elevated temperature, and digitonin solubilization eliminate guanine nucleotide but not sodium effects on human platelet alpha 2-adrenergic receptor-agonist interactions.

Three independent perturbants of the human platelet membrane were found to differentially interfere with modulation of platelet alpha 2-adrenergic receptor-agonist interactions by guanine nucleotides and Na+. Treatment of intact platelets with 1mM N-ethylmaleimide, exposure of particulate preparations to 60 degrees or digitonin solubilization of platelet membranes all eliminated guanine nucleotide regulation of alpha 2-receptor-agonist interactions without interfering with Na+-promoted decreases in receptor affinity for epinephrine. The observations with N-ethylmaleimide and elevated temperature are consistent either with separate membrane components conferring sensitivity to Na+ and guanine nucleotides or with different domains of a single protein component mediating effects of Na+ and GTP on alpha 2-receptor affinity for agonists. However, the observation that Na+ modulation of alpha 2-receptor-agonist interactions is retained in digitonin-solubilized preparations is persuasive evidence that distinct membrane components confer Na+ and GTP sensitivity to alpha 2-adrenergic receptor-agonist interactions, since previous studies (Smith, S.K. and Limbird, L.E. Proc. Natl. Acad. Sci. USA 78:4026-4030, 1981) have demonstrated that the GTP-binding protein modulating alpha 2-receptor affinity for agonists is dissociated from the alpha 2-receptor upon digitonin solubilization unless the receptor is occupied by an agonist at the time of exposure to detergent. Thus, although GTP and Na+ have similar phenomenological effects to permit or enhance alpha-adrenergic attenuation of adenylate cyclase activity and to decrease receptor affinity for agonists, these agents nonetheless appear to mediate their regulatory effects via different membrane components or at least distinct subunits of perhaps shared multisubunit proteins.

Adrenergic alpha-Agonists↗
PubMed1982

Evidence that human platelet alpha-adrenergic receptors coupled to inhibition of adenylate cyclase are not associated with the subunit of adenylate cyclase ADP-ribosylated by cholera toxin.

Exposure of the alpha-adrenergic receptor of the human platelet to agonist prior to solubilization stabilizes a receptor complex of the alpha-adrenergic receptor with the GTP-binding protein(s) which modulates receptor affinity for agonists (Smith, S. K., and Limbird, L. E. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 4026-4030). The soluble alpha-adrenergic receptor is characterized by retention of sensitivity to GTP and a faster rate of sedimentation in sucrose gradients than antagonist-occupied or unoccupied receptors. The present studies were undertaken to determine whether the alpha-adrenergic receptor, which is coupled to inhibition of adenylate cyclase, contains the same GTP-binding protein that is involved in activation of adenylate cyclase. The GTP-binding protein that is coupled to activation of adenylate cyclase was labeled with [32P]ADP-ribose using cholera toxin. Incorporation of [32]ADP-ribose into a Mr = 42,000 peptide in human platelet membranes was paralleled by an enhancement of GTP-sensitive catalytic activity in the membranes. However, cholera toxin treatment did not modify alpha-receptor-mediated inhibition of adenylate cyclase or interaction of the alpha-receptor with agonist agents. Moreover, sucrose gradient centrifugation revealed that the [32P]ADP-ribosylated Mr = 42,000 subunit of the stimulatory GTP-binding protein did not appear to associate with the agonist-alpha-receptor complex. These data suggest that the GTP-binding protein that mediates GTP activation of adenylate cyclase in the human platelet membrane is distinct from the GTP-binding protein that modulates alpha-adrenergic receptor affinity for agonist agents and which associates with the receptor in the presence of agonists.

Adenosine Diphosphate Ribose↗
PubMed1981

Mn2+-uncoupling of the catecholamine-sensitive adenylate cyclase system of rat reticulocytes. Parallel effects on cholera toxin-catalyzed ADP-ribosylation of the system.

High concentrations of Mn2+ interfere with functional interactions between the GTP-binding regulatory protein (G) and the catalytic moiety (C) of adenylate cyclase without perturbing interactions between receptor (R) and component G in rat reticulocyte membranes. The ability of cholera toxin to ADP-ribosylate component G and to enhance GTP-stimulated adenylate cyclase activity also appears to be correlated with the efficacy of the communication of component G with the adenylate cyclase system. Thus, increasing the concentration of Mn2+ in rat reticulocyte membrane during in vitro incubations causes a parallel loss of Gpp(NH)p-stimulated adenylate cyclase activity, cholera toxin-catalyzed [32P]ADP-ribosylation of the 42 000 Mr subunit of component G and cholera toxin-catalyzed enhancement of GTP-sensitive adenylate cyclase activity. Removal of Mn2+ by washing the membranes completely restores the sensitivity of adenylate cyclase activity. Removal of Mn2+ by washing the membranes completely restores the sensitivity of adenylate cyclase to all effectors, including cholera toxin. The data suggest that exposure of membranes to Mn2+ provides a useful tool for reversibly uncoupling catecholamine-sensitive adenylate cyclase systems. The data also suggest that the extent of cholera toxin-catalyzed ADP.-ribosylation of membrane substrates, i.e., the G component may rely on functional communication among the various components of the adenylate cyclase system. A corollary of the latter is that the amount of [32P]ADP-ribose-product detected in a membrane may reflect both the quantity and coupling efficiency of component G.

Adenosine Diphosphate Ribose↗
PubMed1981

Evidence that a beta-adrenergic receptor-associated guanine nucleotide regulatory protein conveys guanosine 5'-O-(3-thiotriphosphate)- dependent adenylate cyclase activity.

The guanine nucleotide regulatory protein component (N) of the frog erythrocyte membrane adenylate cyclase system appears to form a stable complex with the beta-adrenergic receptor (R) in the presence of agonist (H). This agonist-promoted ternary complex HRN can be solubilized with Lubrol. The guanine nucleotide regulatory protein associated with the solubilized complex can be adsorbed either to GTP-Sepharose directly or to wheat germ lectin-Sepharose via its interaction with the receptor which is a glycoprotein. Guanosine 5'-O-(3-thiotriphosphate)(GTP gamma S) can be used to elute the guanine nucleotide regulatory protein from either Sepharose derivative. The resulting N.GTP gamma S complex conveys nucleotide-dependent adenylate cyclase activity when combined with a Lubrol-solubilized extract of turkey erythrocyte membranes. The ability to observe GTP gamma S-dependent reconstitution of adenylate cyclase activity in the eluate from either resin required the formation of the HRN complex prior to solubilization. The N protein can be identified by its specific [32P]ADP ribosylation catalyzed by cholera toxin in the presence of [32P]NAD+. The existence of a stable HRN intermediate complex is supported by the observation that agonist pretreatment of frog erythrocyte membranes results in a 100% increase in the amount of 32P-labeled N protein eluted from the lectin-Sepharose in the presence of GTP gamma S compared to membranes pretreated with either antagonist or agonist plus GTP. Our results therefore provide evidence that the same guanine nucleotide-binding protein that associates with the beta-adrenergic receptor in the presence of agonist mediates adenylate cyclase activation.

Adenosine Diphosphate Ribose↗
PubMed1981

Solubilization of human platelet alpha-adrenergic receptors: evidence that agonist occupancy of the receptor stabilizes receptor--effector interactions.

The alpha-adrenergic receptors of human platelet membranes can be directly identified by both a radiolabeled agonist, [3H]epinephrine, and a radiolabeled antagonist, [3H]yohimbine. Digitonin solubilizes a binding component from the membrane that is indistinguishable from the alpha-receptor identified in the native platelet membrane as assessed by (i) order of potency of agonists and antagonists and (ii) affinity of the receptor for [3H]-yohimbine and competing antagonists. However, the solubilized receptor demonstrates a reduced affinity for agonists and a loss of the ability of guanine nucleotides to modulate receptor affinity for agonists. Prelabeling of human platelet membranes with [3H]-epinephrine results in a guanine nucleotide-sensitive agonist-receptor complex that sediments more rapidly in sucrose gradients than do unoccupied or antagonist-occupied receptors. Thus, agonist occupancy of the alpha-receptor prior to membrane solubilization may promote or stabilize receptor interaction with effector components in the membrane, one of which may be the GTP regulatory protein responsible for modulation of receptor affinity.

Blood Platelets↗
PubMed1980

Agonist-promoted coupling of the beta-adrenergic receptor with the guanine nucleotide regulatory protein of the adenylate cyclase system.

Binding of the beta-adrenergic agonist [3H]hydroxybenzylisoproterenol to the beta-adrenergic receptor of rat reticulocyte membranes results in the coupling of the receptor to the guanine nucleotide regulatory protein associated with the adenylate cyclase system. This regulatory component, referred to as the G-protein, was identified by its specific [32P]-ADP-ribosylation catalyzed by cholera toxin. Incubation of [32P]ADP-ribosylated rat reticulocyte membranes with the [3H]hydroxybenzylisoproterenol agonist prior to membrane solubilization and gel exclusion chromatography resulted in the coelution of the 42,000 Mr [32P]ADP-ribosylated G-proteins with the agonist-occupied beta-adrenergic receptors. The receptor-G-protein complex was not formed when receptors were unoccupied or occupied with antagonists at the time of solubilization. Incubation of rat reticulocyte membranes with [3H]hydroxybenzylisoproterenol in the presence of guanine nucleotides reversed or prevented the formation of this receptor-G-protein complex. These data provide direct evidence for the molecular interactions promoted by agonist occupancy of beta-adrenergic receptors. It is probable that the formation of a receptor-G-protein complex is crucial for catecholamine stimulation of the adenylate cyclase enzyme and, hence, transmembrane information transfer.

Adenosine Diphosphate Ribose↗
PubMed1979

Biochemical characterization of the beta-adrenergic receptor of the frog erythrocyte.

The beta-adrenergic receptor which is coupled to adenylate cyclase in the frog erythrocyte plasma membrane provides a convenient model system for probing the molecular characteristics of an adenylate cyclase coupled hormone receptor. Direct radioligand binding studies with beta-adrenergic agonists and antagonists such as [3H]hydroxybenzylisoproterenol and [3H]dihydroalprenolol have shed new light on the biochemical properties of the receptor as well as on its mode of interaction with other components of the adenylate cyclase system. Agonist binding to the receptor induces a high affinity state of the receptor which can be selectively reverted to a low agonist affinity state by guanyl nucleotides. This agonist-induced high affinity state of the receptor appears to correspond to a receptor moiety which has larger apparent molecular weight and which is probably a complex of the beta-adrenergic receptor and nucleotide regulatory binding protein. Antagonists do not appear capable of inducing or stabilizing the formation of this high affinity receptor-nucleotide site complex. The beta-adrenergic receptors have been solubilized using the plant glycoside digitonin as the detergent and have been highly purified by biospecific affinity chromatography on an alprenolol-agarose affinity support. These highly purified receptor preparations retain all of the binding characteristics observed in the unpurified soluble receptor preparations. Remarkably, antibodies raised in rabbits against affinity chromatography purified preparations of the receptor, themselves bind beta-adrenergic ligands with typical beta-adrenergic specificity. Such antibodies which possess binding sites similar to those of physiological receptors provide useful model systems for further probing the molecular characteristics of beta-adrenergic binding sites.

Adenylyl Cyclases↗
PubMed1979

Differential effects of GTP on the coupling of beta-adrenergic receptors to adenylate cyclase from frog and turkey erythrocytes. Application of new methods for the analysis of receptor-effector coupling.

A detailed comparison of the interaction of beta-adrenergic receptors with adenylate cyclase stimulation and modification of this interaction by guanine nucleotides has been made in two model systems, the frog and turkey erythrocyte. Objective analysis of the data was facilitated by the development of new graphical methods which involve the use of logit-logit transformations of percent receptor occupancy versus percent enzyme stimulation plots (coupling curves). Receptor-cyclase coupling in turkey erythrocyte membranes demonstrates a proportional relationship between receptor occupancy and adenylate cyclase activation and is unaffected by exogenous guanine nucleotides. By comparison, the proportional relationship of receptor occupancy and adenylate cyclase activation observed in frog erythrocyte membranes in the absence of guanine nucleotides is modified by the addition of exogenous guanine nucleotides such that a greater fractional enzyme stimulation is elicited by low receptor occupancy. Methodological criteria crucial for valid comparison of receptor occupancy and adenylate cyclase activity are delineated. In addition, the possible molecular mechanisms of receptor-cyclase coupling which might give rise to the coupling curves observed are discussed.

Adenylyl Cyclases↗