Differences in efficacy and Na(+) sensitivity between alpha(2B) and alpha(2D) adrenergic receptors: implications for R and R* states.

The ability of Na(+) ions to modulate coupling of alpha(2B)- and alpha(2D)-adrenergic receptors to G proteins was investigated in isolated membranes from transfected PC12 and NIH 3T3 fibroblast cells. The initial rate of epinephrine-stimulated [(35)S]GTPgammaS binding was higher for alpha(2D)-receptors (the rat homolog of the alpha(2A)-receptor) in both cell types, whereas both alpha(2B)- and alpha(2D)-receptor responses were higher in PC12 cell membranes. Pertussis toxin completely blocked agonist-stimulated binding. Graded increases in Na(+) caused a progressive loss of basal GTP binding, indicative of its ability to reduce the level of the active R* state of the receptor. This inhibitory effect of Na(+) was more pronounced in PC12/alpha(2B) than PC12/alpha(2D) membranes. Epinephrine-stimulated GTP binding in PC12/alpha(2B) membranes was also more sensitive to Na(+) inhibition than in PC12/alpha(2D) membranes. In saturation [(35)S]GTPgammaS binding studies, the presence of Na(+) reduced apparent GTP affinity, and its effect was greater in PC12/ alpha(2B) membranes, consistent with a greater reduction in the active R* conformation of the receptor. The higher efficacy of epinephrine at alpha(2D) receptors and their lesser sensitivity to Na(+) are both indicative of a more stable R* state. Together these results suggest that differences in the modulatory influence of Na(+) within a family of G(i)-coupled receptors may reflect differences in the stability of the active R* state.

Bidirectional allosteric effects of agonists and GTP at alpha(2A/D)-adrenoceptors.

Agonists and GTP exert reciprocal effects on the stability of the G protein-coupled receptor/G protein complex, implying bidirectional control over the receptor/G protein interface. To investigate this relationship, we compared the ability of a series of hydroxyl-substituted phenethylamine and imidazoline agonists to stimulate [(35)S]guanosine 5'-O-(3-thio)triphosphate ([(35)S]GTPgammaS) binding in membranes from alpha(2A/D)-adrenergic receptor-transfected PC12 cells with the magnitude of the GTP-induced reduction in agonist affinity in [(3)H]rauwolscine-binding studies. Agents previously described as full and partial agonists in functional studies showed similar relative efficacies in promoting GTP binding (r = 0.97) as well as similar relative potencies (r = 0.94). Efficacy among agonists for promotion of [(35)S]GTPgammaS binding was closely correlated with the relative influence of GTPgammaS on agonist binding (r = 0.97), consistent with a bidirectional allosteric influence by agonists and GTP on receptor/G protein complexation. In an additional series of tolazoline derivatives, a range in efficacy from full agonism to strong inverse agonism was observed, depending on the presence or absence of hydroxyl substituents. Together these results suggest that agonist-induced repositioning of transmembrane helices via their hydroxyl interactions is a critical determinant of the stability of the receptor/G protein complex and therefore of agonist efficacy.

Importance of glucose-6-phosphate dehydrogenase activity in cell death.

The intracellular redox potential plays an important role in cell survival. The principal intracellular reductant NADPH is mainly produced by the pentose phosphate pathway by glucose-6-phosphate dehydrogenase (G6PDH), the rate-limiting enzyme, and by 6-phosphogluconate dehydrogenase. Considering the importance of NADPH, we hypothesized that G6PDH plays a critical role in cell death. Our results show that 1) G6PDH inhibitors potentiated H2O2-induced cell death; 2) overexpression of G6PDH increased resistance to H2O2-induced cell death; 3) serum deprivation, a stimulator of cell death, was associated with decreased G6PDH activity and resulted in elevated reactive oxygen species (ROS); 4) additions of substrates for G6PDH to serum-deprived cells almost completely abrogated the serum deprivation-induced rise in ROS; 5) consequences of G6PDH inhibition included a significant increase in apoptosis, loss of protein thiols, and degradation of G6PDH; and 6) G6PDH inhibition caused changes in mitogen-activated protein kinase phosphorylation that were similar to the changes seen with H2O2. We conclude that G6PDH plays a critical role in cell death by affecting the redox potential.

Importance of glucose-6-phosphate dehydrogenase activity for cell growth.

The intracellular redox potential, which is determined by the level of oxidants and reductants, has been shown to play an important role in the regulation of cell growth. The principal intracellular reductant is NADPH, which is mainly produced by the pentose phosphate pathway through the actions of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway, and by 6-phosphogluconate dehydrogenase. Previous research has suggested that an increase in G6PD activity is important for cell growth. In this article, we suggest that G6PD activity plays a critical role in cell growth by providing NADPH for redox regulation. The results show the following: 1) inhibition of G6PD activity abrogated growth factor stimulation of [3H]thymidine incorporation in all cell lines tested; 2) overexpression of G6PD stimulated cell growth, as measured by an increase in [3H]thymidine incorporations as compared with cells transfected with vector alone; 3) inhibition of G6PD caused cells to be more susceptible to the growth inhibitory effects of H2O2; 4) inhibition of G6PD led to a 30-40% decrease in the NADPH/NADP ratio; and 5) inhibition of G6PD inhibited cell anchorage and significantly decreased the growth-related stimulation of tyrosine phosphorylation.

Evaluation of agonist efficacy and receptor reserve for alpha 2D-adrenergic receptor regulated G protein activation in PC12 cell membranes.

The receptor-coupling efficiency for epinephrine (EPI) stimulated heterotrimeric G protein activation was studied at the G protein level in membranes prepared from PC12 cells expressing cloned alpha 2D-adrenergic receptors (alpha 2D-AR). After pretreatment with different concentrations of N-ethoxycarbonyl-1,2-dihydroquinoline, which irreversibly inactivates alpha 2D-AR, the portion of alpha 2D-ARs remaining active (q) was estimated from EPI-stimulated [35S]GTP gamma S binding. This function-derived estimate was close to the actual remaining number of receptors, as determined in saturation-binding studies using the selective alpha 2-AR antagonist [3H]rauwolscine in the same membranes. The agonist dissociation constant (KA) derived from EPI-stimulated [35S]GTP gamma S binding via Furchgott analysis was similar to the EC50 of EPI in the same assay, but 40-fold lower than its Ki measured from EPI competition for [3H]rauwolscine-binding sites in the presence of GTP gamma S and Na+. The occupancy-response relationship, calculated using Ki rather than KA, was markedly nonlinear, consistent with the high expression of alpha 2D-AR in these membranes. A nonlinear occupancy-response relationship was more directly confirmed by measuring the maximal level (i.e., full occupancy level) of G protein activation at graded densities of alpha AD-AR after N-ethoxycarbonyl-1,2-dihydroquinoline treatment. Determination of the number of G-proteins activated per receptor yielded lower values at higher receptor densities, indicating that overexpression of receptors can reduce their efficiency. Our results indicate the potential utility of using GTP-binding studies to assess agonist efficacy at the G protein level under conditions where receptor occupation can also be directly measured.

Signal transduction proteins that associate with the platelet-derived growth factor (PDGF) receptor mediate the PDGF-induced release of glucose-6-phosphate dehydrogenase from permeabilized cells.

Permeabilized rat kidney cells rapidly released glucose 6-phosphate dehydrogenase (G6PD) following stimulation with peptide growth factors (Stanton, R.C., Seifter, J.L., Boxer, D.C., Zimmerman, E., and Cantley, L. C. (1991) J. Biol. Chem. 266, 12442-12448). To evaluate the signal transduction pathways mediating release of G6PD, two cell lines transfected with wild type or mutant platelet-derived growth factor (PDGF) receptors (PDGFR) were studied using two permeabilization protocols. G6PD release was evaluated by enzyme activity and Western blot analysis. PDGF caused a significant increase in G6PD release in 1 min in cells transfected with wild type PDGFR. PDGF did not stimulate G6PD release in cells transfected with tyrosine kinase-deficient PDGFR. PDGF did not stimulate G6PD release in cells transfected with partially autophosphorylation-deficient PDGFR in which four known signaling proteins do not associate with the PDGFR. The PDGF-stimulated release of G6PD was partially restored in PDGFR mutants in which either phosphatidylinositol-3-kinase or phospholipase C gamma 1 could associate with the PDGFR. Lastly, there was no basal or PDGF-stimulated phosphorylation of G6PD. We conclude that release of G6PD: 1) requires intrinsic PDGFR tyrosine kinase activity; 2) requires PDGFR autophosphorylation; 3) is mediated by signaling proteins that associate with the PDGFR; 4) is not mediated by direct phosphorylation of G6PD.

Determinants of alpha 2-adrenergic receptor activation of G proteins: evidence for a precoupled receptor/G protein state.

The ability of agonist-occupied alpha 2D-adrenergic receptors to activate G proteins was measured in membranes from PC-12 cells stably expressing the cloned receptor, using guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTP gamma S) binding as an endpoint. Epinephrine (EPI) stimulated [35S]GTP gamma S binding in a Mg(2+)-dependent manner, showing both micromolar and millimolar cation affinities. Prior treatment of cells with pertussis toxin completely eliminated the EPI stimulation. The presence of GDP decreased basal [35S]GTP gamma S binding and increased the proportion of EPI-stimulated binding. Increasing concentrations of Na+ also reduced basal [35S]GTP gamma S binding but had less effect on EPI-stimulated binding, such that the agonist response was proportionately greater at higher Na+ levels. In saturation binding studies with [35S]GTP gamma S only low affinity binding was observed in the presence of 100 mM Na+, whereas in the absence of Na+ a high affinity component was also present, indicating a Na(+)-regulated receptor/G protein interaction. EPI induced high affinity [35S]GTP gamma S binding in the presence of Na+ and increased the affinity of the high affinity component under Na(+)-free conditions. The selective alpha 2-adrenergic antagonist rauwolscine produced rightward shifts of EPI dose-response curves and decreased the basal level of [35-S]GTP gamma S binding across the same range of concentrations. The extent of decrease was dependent upon the alpha 2-adrenergic receptor expression level, indicating that alpha 2-adrenergic receptors contribute to basal G protein activation in the absence of agonist. The ability of rauwolscine to decrease basal [35S]GTP gamma S binding was diminished as the level of Na+ was increased, suggesting that both agents act to reduce receptor/G protein interaction, by distinctive mechanisms. alpha 2-Adrenergic receptor antagonists reduced basal G protein activation with a rank order for maximal effectiveness that was different from their receptor binding affinities. These results support the existence of precoupling between alpha 2D-adrenergic receptors and G proteins; coupling can be diminished by both Na+ and antagonists, whereas agonists increase the efficiency of receptor/G protein coupling.

Precoupling of Gi/G(o)-linked receptors and its allosteric regulation by monovalent cations.

The ability of receptors (R) to activate G-proteins (G) is promoted by the binding of agonists, reflecting their induction of a receptor conformation which facilitates both the formation of a RG complex and guanine nucleotide exchange. Recent evidence from isolated membrane studies has indicated, however, that some receptors have the inherent ability to form RG complexes and promote GDP/GTP exchange in their unoccupied state. These receptors preferentially activate pertussis toxin-sensitive G-proteins (i.e. Gi/G(o)) and the interactions of R and G are modulated by monovalent cations (most notably Na+) both in the unoccupied and agonist-occupied states. Basal G-protein activation by such receptors is reduced both by increasing levels of cation and by antagonists which may act by inducing receptor conformations which are less favorable for RG complexation. This behaviour conforms to the predictions of a ternary complex model of receptor function and can be considered in structural terms for those receptors such as the alpha-2 adrenergic receptor where ligand binding and G-protein activation regions have been proposed.

Agonist-induced modulation of agonist binding to alpha 1-adrenoceptors in bovine aorta.

Prolonged exposure of tissues to hormone agonists results in a subsequent reduction in the sensitivity of the tissue through a process known as desensitization. The desensitization response, either homologous or heterologous, has been shown to be correlated with receptor phosphorylation. Recently we have provided evidence that protein kinase C, when activated by a phorbol ester, regulates alpha 1-adrenoceptor coupling to a G-protein. In the present study, the effects of epinephrine (10 microM) pretreatment on the binding behavior of the alpha 1-adrenoceptor were determined from radioligand binding assays at 25 degrees and 2 degrees C. Pretreatment of tissues with epinephrine for 25 min moderately decreased [3H]prazosin binding by 12% (Bmax 121.5 fmol/mg) in comparison to control (139.3 fmol/mg) with no change in its affinity. The second consequence of desensitization by epinephrine is a decrease in the affinity of agonist binding to alpha 1-adrenoceptors associated with uncoupling of the receptors from the G-protein. In control membranes, at 25 degrees C, epinephrine defined two different affinity states of the receptor, viz. high affinity (KDH 16.5 nM, % RH 21) and low affinity (KDL 710 nM, % RL 79). The high affinity state formed at 25 degrees C is stabilized by forming a ternary complex with a G-protein. Addition of guanylylimidodiphosphate (Gpp(NH)p) reduced the stability of this complex resulting in a loss of high affinity sites in control membranes. On the other hand, epinephrine treated membranes exhibited only a single class of low affinity agonist binding (KDH 659 nM) and further, Gpp(NH)p had no significant effect on binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental changes in alpha 1-adrenoceptor coupling to G-protein in bovine aorta.

Differences in epinephrine binding to alpha 1-adrenoceptors, epinephrine-induced contractile potency, susceptibility to phorbol ester (PDBu) modulation, and differences in membrane fluidity were studied in bovine aorta from young (3-8 weeks) and adult or mature (6-8 years) animals. Membranes prepared from aorta of adult animals exhibited a two-fold higher receptor density while [3H]prazosin affinity was unchanged. Epinephrine displacement studies revealed both high- and low-affinity binding in membranes from the aortas of young animals whereas, preparations from adult animals exhibited only a single class of low-affinity sites. In low-temperature binding studies, membranes prepared from aortas of adult animals exhibited both high- and low-affinity agonist binding, in proportions about equal to those of young animals. The ability of PDBu to uncouple alpha 1-adrenoceptor from G-protein interaction is demonstrable in young but not in adult animals which raises the possibility of prior phosphorylation of receptors in the latter tissues. Aortas from young animals showed increased contractile potency to epinephrine and, in addition, were significantly more fluid as compared to aortas from adult animals. Alterations in the membrane environment or phosphorylation state of the alpha 1-adrenoceptor may thus provide age-dependent modulation of its function.

Species differences in chlorethylclonidine antagonism at vascular alpha-1 adrenergic receptors.

Chloroethylclonidine (CEC) inhibition of norepinephrine-induced contractile response was studied in rat and rabbit aorta. CEC inhibition was irreversible, but displayed a competitive pattern characterized by rightward shifts of the norepinephrine EC50 with little or no decrease in maximum response. CEC appeared to shift response to a discrete but lower affinity state. The time course for irreversible inhibition by 10 microM CEC was faster for rat aorta (T1/2 = 18 min) than for rabbit aorta (T1/2 = 118 min) indicating a difference either in receptor occupation or in the rate of alkylation after occupation. Measurements of reversible CEC affinity in rabbit aorta yielded a pKB of 6.3 and the rate of alkylation was not significantly increased at 100 microM CEC so that lower occupation could not account for the difference. Pretreatment with phenoxybenzamine significantly increased the sensitivity of rabbit aorta to CEC indicating a role for receptor reserve in determining the extent of CEC inhibition. Membrane fluidity measured by diphenylhexatriene fluorescence was lower in rabbit than rat aorta; however, dimethylsulfoxide or ethanol failed to alter the rate of irreversible CEC inhibition in the rabbit. Protein kinase C activation with phorbol dibutyrate failed to alter CEC alkylation in rabbit aorta. WB 4101 [2-(2,6-dimethoxyphenoxyethyl)-aminomethyl-1,4-benzodioxane] affinity was higher in rabbit aorta (pKB = 8.9) than rat aorta (pKB = 8.3) consistent with a contribution of alpha-1A and alpha-1B adrenergic receptor subtypes to differences in CEC sensitivity, although the difference was not as great as would be predicted.(ABSTRACT TRUNCATED AT 250 WORDS)