Coupling of beta2-adrenoceptors to XLalphas and Galphas: a new insight into ligand-induced G protein activation.

Galpha(s) and extra-large Galpha(s) (XLalpha(s)) can both transduce receptor activation into intracellular cAMP generation. It is unknown, however, whether these two GNAS-locus products display distinct properties with respect to receptor coupling. Here, we show that XLalpha(s) couples to the beta2-adrenoceptor more efficiently than Galpha(s). In transfected human embryonic kidney 293 cells and mouse embryonic fibroblasts null for both Galpha(s) and XLalpha(s) (2B2 cells), basal cAMP accumulation mediated by XLalpha(s) was higher than that mediated by Galpha(s). Inverse agonist treatment reduced Galpha(s)-mediated basal activity, whereas its effect was markedly blunted on XLalpha(s)-mediated basal activity. Rank order of ligand efficacies regarding cAMP accumulation was the same when the receptor was coupled to XLalpha(s) or Galpha(s). However, ligand-induced and XLalpha(s)-mediated cAMP generation was higher than that mediated by Galpha(s). The relatively high efficiency of XLalpha(s)-mediated cAMP generation was conditional, disappearing with increased level of receptor expression or increased efficacy of ligand. Full-agonist responses in XLalpha(s)- and Galpha(s)-expressing cells were comparable even at low receptor levels, whereas partial agonist responses became comparable only when the receptor expression was increased (>3 pmol/mg). Radioligand binding studies showed that the high-affinity component in agonist binding to beta2-adrenoceptor was more pronounced in cells expressing XLalpha(s) than those expressing Galpha(s). We discuss these findings in the framework of current receptor-G protein activation models and offer an extended ternary complex model that can fully explain our observations.

Quasi-irreversible binding of agonist to beta-adrenoceptors and formation of non-dissociating receptor-G(s) complex in the absence of guanine nucleotides.

Here, we tested the hypothesis that receptor-G protein and agonist may form an irreversible complex in the absence of guanine nucleotides. We used the beta-adrenoceptor-G(s) system of guinea pig lung parenchymal membranes as a model. Two groups of membranes were used in the experiments: (1) washed with nucleotide-free buffer in the presence of isoproterenol (isoproterenol-treated), and (2) washed with buffer alone or with agonist+GDP (both were treated as control). Results were as follows: (1) the iodopindolol binding capacity of isoproterenol-treated membranes was reduced by about 30%. (2) No such reduction was observed in control membranes. (3) Addition of GDP to the isoproterenol-treated membranes completely restored the pindolol binding capacity. We interpreted this result as indicating irreversible agonist-receptor complex is formed when the receptor interacts with nucleotide-free G(salpha). (4) We observed a single peak of beta(2)-adrenoceptor activity in the control group by size-exclusion chromatography of the solubilized membranes. Inclusion of isoproterenol in the washing buffer led to an additional (heavier) peak of beta(2)-adrenoceptor activity. This peak disappeared when GDP was added to the detergent extract before high-pressure liquid chromatography (HPLC) analysis. Western blot analysis of these HPLC fractions showed that the agonist-induced heavier peak contained significantly more G(salpha) protein than did the other fractions. We interpreted this result as indicating that a practically irreversible complex of receptor and G protein is formed in the absence of GDP. We suggest that the tightly bound (nucleotide-free) receptor-G protein complex also contains the agonist, and that this complex can be reversed only by the addition of nucleotides. The implications of these results are also discussed.

Pharmacokinetics of phenprobamate after oral administration to healthy subjects.

Phenprobamate (CAS 673-31-4) is a centrally acting skeletal-muscle relaxant agent. There are only two studies in the literature about the pharmacokinetics of phenoprobamate in man. The inconsistency between the results of these studies can be attributed partly to the different analytical methodologies used. A sensitive, specific and reproducible HPLC-assay, which may increase the reliability of the pharmacokinetic studies of phenprobamate in plasma, has been developed recently. The objective of this investigation was to assess the single-dose kinetics of phenprobamate in human and to determine the pharmacokinetic parameters of clinical and regulatory concern. The plasma pharmacokinetics of phenprobamate have been investigated following single oral administration at a dose of 800 mg in eleven healthy volunteers.

Ca2+-induced inhibition of adenylyl cyclase in turkey erythrocyte membranes.

We investigated the effects of calcium ions (Ca2+) on the adenylyl cyclase activity in purified turkey erythrocyte membranes. Results showed the following: (i) Ca2+ inhibits cAMP accumulation stimulated by isoproterenol (1 micromol/l), NaF + AlCl3 (10 mmol/l + 20 micromol/l) or forskolin (10 micromol/l) in EGTA-washed turkey erythrocyte membranes. IC50 of free [Ca2+] is approximately 0.1 mmol/l in the presence of Mg2+ (2.5 mmol/l) and isobutylmethylxanthine (1 mmol/l). (ii) The potency of Ca2+ to inhibit cAMP accumulation is independent of the type of stimulus used to activate the adenylyl cyclase. We also evaluated the calcium sensitivity of the basal cAMP accumulation in the presence of GTP (10 micromol/l) and Mg2+ (2.5 mmol/l) which was also inhibited by Ca2+ with the same potency. (iii) The inhibition pattern of cAMP accumulation is not affected by the presence of added calmodulin (100 nmol/l). (iv) Ca2+ is ineffective on the binding of isoproterenol to the beta-adrenoceptors. (v) Increasing the concentration of Ca2+ does not induce an observable activation of cyclic nucleotide phosphodiesterase in the present experimental conditions. Thus, we concluded that the inhibition of cAMP accumulation is due to an inhibition of the adenylyl cyclase rather than the activation of phosphodiesterase(s). The presence of a yet unidentified isoform of adenylyl cyclase that can be directly inhibited by Ca2+ or a Gi protein that can be activated by Ca2+ seems to explain these results. In either case, these results provide an additional mode of cross-talk that can take place between the Ca2+- and cAMP-signaling systems.

Decrease in apparent alpha1-adrenoceptor-G protein coupling during maturation in rat aorta.

We have previously shown that the adrenoceptor agonist norepinephrine (NE) is more potent in eliciting contraction in aortas from 1-month-old Fischer 344 rats than it is in older animals. In the present study, we examined alpha1-adrenoceptor-guanine nucleotide regulatory binding protein (G protein) coupling in aortic membranes in order to investigate the mechanism for the age-dependent reduced responsiveness of aorta to NE. We used the guanosine 5'(betagamma-imido)triphosphate (Gpp[NH]p)-induced shift in agonist binding affinity as a measure of the efficiency of alpha11-adrenoceptor-G protein coupling. The binding of NE was assessed by measuring the displacement of 2-[beta-(4-hydroxy-3-[125I]iodophenyl)ethylaminomethyl] tetralone ([125]-HEAT) by NE in aortic membranes. In 1-, 6-, and 24-month-old rat aortas, two apparent binding sites were detected in the competition isotherms for NE. This heterogeneous binding pattern was independent of Gpp(NH)p at all ages, and is likely to be due to a heterogeneous receptor population (alpha(1a), alpha(1b), and alpha(1d) subtypes). In 1-month-old rats, the high affinity binding of NE to alpha,-adrenoceptors was sensitive to Gpp(NH)p, indicating a significant interaction between the receptor and G protein. This Gpp(NH)p-sensitive high affinity binding was not observed in aortas from 6- or 24-month-old animals. Despite the lack of Gpp(NH)p-sensitive high affinity binding of agonist in 6- or 24-month-old aortas, NE was still able to induce maximal contraction in these aortas, albeit, with a relatively low potency. A partial reduction in alpha1-adrenoceptor-G protein coupling between 1 and 6 months of age can explain the observed decrease in ago- nist potency and the loss of Gpp(NH)p-sensitive high affinity binding of NE. This phenomenon can be explained as a reduction of allosteric coupling between the bindings of ligand and G protein to the receptor, that has been formulated in the ternary complex model. Computer simulation using the simple ternary complex model shows that manipulating the reciprocal coupling factor alone can lead to a loss of Gpp(NH)p-sensitive high affinity agonist binding, along with a reduction in agonist potency for contraction without altering the maximal response. Thus, a change in the relative expression of different alpha,-adrenoceptor subtypes, which we have previously observed in the aorta, and which possess diverse intrinsic allosteric couplings, may be speculated to be the mechanism for the apparent reduction of alpha,-adrenoceptor-G protein coupling during maturation.

An efficacy-dependent effect of cardiac overexpression of beta2-adrenoceptor on ligand affinity in transgenic mice.

In previous studies, it was shown that the overexpression of beta2-adrenoceptor (beta2AR) in the hearts of transgenic mice (Tg) leads to agonist-independent activation of adenylate cyclase and enhanced myocardial function. Here, we measured the physical coupling of beta2AR and Gs by evaluating the coimmunoprecipitation of beta2AR and Gs and the ligand binding properties of beta2AR in the hearts of Tg mice to investigate the details of the interaction among ligand, receptor, and G protein. The following results were obtained: (i) coimmunoprecipitation of beta2AR and Gs was increased in the absence of agonist in Tg mice compared with the control animals. This demonstrates directly the increased interaction between unliganded beta2AR and Gs, which is consistent with increased background cAMP production and cardiac function in the hearts of Tg mice. (ii) Guanosine-5'-(beta,gamma-imido)triphosphate abolished the association of beta2AR/Gs in the immunoprecipitate. (iii) The affinities for ligands that show agonist (isoproterenol, clenbuterol, and dobutamine), neutral antagonist (alprenolol and timolol), and negative antagonist (propranolol and ICI 118551) activities in this experimental system were increased, not changed and decreased, respectively, in Tg mice compared with the controls. (iv) This efficacy-dependent alteration in ligand affinities was still observed in the presence of a guanosine-5'-(beta,gamma-imido)triphosphate concentration that abolishes beta2AR/Gs coupling. This suggests that the altered beta2AR binding affinities in Tg mice are not due to the increased interaction between beta2AR and Gs. These data cannot be explained by using ternary, quinternary, two-state extended ternary, or cubic ternary complex models. We therefore discuss the results using a "two-state polymerization model" that includes an isomerization step for the conversion of receptor between an inactive and an active form (denoted as R and R*, respectively) and a polymerization of the active state (R*n). The simplest form of this model (i.e., noncooperative dimerization of the receptor) is found to be consistent with the experimental data.

Allosteric equilibrium model explains steady-state coupling of beta-adrenergic receptors to adenylate cyclase in turkey erythrocyte membranes.

We used a simple experimental approach to clarify some contradictory predictions of the collision coupling and equilibrium models (e.g. ternary complex, two-state ternary complex or quinternary complex), which describe G-protein-mediated beta-adrenergic receptor signalling in essentially different manners. Analysis of the steady-state coupling of beta-adrenoceptors to adenylate cyclase in turkey erythrocyte membranes showed that: (1) in the absence of an agonist, Gpp(NH)p (a hydrolysis-resistant analogue of GTP) can activate adenylate cyclase very slowly; (2) this activity reaches a steady state in approx. 5 h, the extent of activity depending on the concentration of the nucleotide; (3) isoprenaline-activated steady-state adenylate cyclase can be inactivated by propranolol (a competitive antagonist that relaxes the receptor activation), in the presence of Gpp(NH)p (which provides a virtual absence of GTPase) and millimolar concentrations of Mg2+ (the rate of this inactivation is relatively fast); (4) increasing the concentration of Gpp(NH)p can saturate the steady-state activity of adenylate cyclase. The saturated enzyme activity was lower than that induced by isoprenaline under the same conditions. This additional agonist-induced activation was reversible. In the light of these results, we conclude that agonist can also activate the guanine nucleotide-saturated system in the absence of GTPase by a mechanism other than guanine nucleotide exchange. We explain these phenomena in the framework of a quinternary complex model as an agonist-induced and receptor-mediated dissociation of guanine nucleotide-saturated residual heterotrimer, the equilibrium concentration of which is not necessarily zero. These results, which suggest a continuous interaction between receptor and G-protein, can hardly be accommodated by the collision coupling model that was originally suggested for the present experimental system and then applied to many other G-protein systems. Therefore we attempt to unify the equilibrium and collision coupling approaches to provide a consistent theoretical basis for the G-protein-mediated beta-adrenergic receptor signalling in turkey erythrocyte membranes.

Pharmacokinetics of recombinant human erythropoietin in children with chronic renal failure.

Basal erythropoietin (Epo) levels and single dose-pharmacokinetics of recombinant human erythropoietin (rhuEpo) were investigated in 8 predialysis (PD) patients (mean age 11.6 +/- 1.4 years) and in 8 patients on continuous ambulatory peritoneal dialysis (CAPD) (mean age 12.7 +/- 0.6 years). Basal Epo levels were found to be 1.0 +/- 0.0 mu/ml in PD group, 1.6 +/- 0.7 mu/ml in CAPD group and 8.5 +/- 1.8 mu/ml in control group. Following administration of 50 mu/kg rhuEpo (s.c.) serum Epo concentration (Cmax) was 23.2 +/- 2.5 mu/ml in 18.5 +/- 2.6 hours (tmax) in PD patients and 9.9 +/- 0.8 mu/ml in 26.8 +/- 7.7 hours in CAPD patients. Mean elimination half-lives (t1/2) were 13.3 +/- 1.9 hours and 13.5 +/- 3.0 hours in PD patients and CAPD patients, respectively. The volume of distribution (Vd) was 840.0 +/- 100.0 ml/kg; the clearance (Epo Cl) was 37.0 +/- 5.5 ml/kg/hour in PD patients. These values were significantly lower in PD patients than in CAPD patients (p < 0.05) (Vd; 1500 +/- 240.0 ml/kg; Epo Cl 110 +/- 30.0 ml/kg/hour). During the course of CAPD, more efficient clearance of uraemic toxins that inhibit erythropoiesis and more rapid extraction of erythropoietin by erythroid precursors may cause higher Vd in CAPD patients than in PD patients.

Beta gamma subunits of guanine nucleotide-binding proteins and regulation of spontaneous receptor activity: thermodynamic model for the interaction between receptors and guanine nucleotide-binding protein subunits.

We used a thermodynamic model to examine the interactions between receptor, guanine nucleotide-binding protein (G protein), and their ligands. The model describes the interactions as multiple equilibria occurring between three distinct protein species (receptor, G alpha subunit, and G beta gamma complex) and two small ligands, i.e., agonist (which interacts with receptor) and guanine nucleotide (which binds to G alpha). The equilibrium distribution of free and complexed species is determined by the total concentration of the components, the affinities that govern the biomolecular reactions, and the allosteric interactions that ligands exert on each other when they are simultaneously bound to the same species. These allosteric factors are given in terms of free energy coupling. The model explains a number of experimental observations, as follows. (i) Both GTP and GDP can reduce agonist affinity, whereas the agonist enhances the net binding of GTP and diminishes that of GDP. (ii) G beta gamma is more effective in reducing agonist-independent than agonist-dependent receptor activity. (iii) Removal of guanine nucleotides increases the ratio between agonist-independent and -dependent activation of G protein. The model leads to a number of interesting predictions. (i) Not only G alpha but also G beta gamma has effects on hormone binding. (ii) As long as the distribution of protein species is [G beta gamma] > [G alpha] > [receptor] (as often observed in the cell membrane), small changes in the concentration of G beta gamma do not alter the overall response induced by agonist. (iii) Agonist activity examined at low concentrations of guanine nucleotide is inevitably different from that observed at high concentrations, typical of intact systems. (iv) Differences in potencies and maximal effects for various guanine nucleotide analogues may reflect differences in their coupling constants that are experimentally measurable. The present model suggests several experimentally testable hypotheses that could be important in elucidating the activation mechanism and regulatory flexibility of G protein-dependent transduction systems.

Lysolipids reversibly inhibit Ca(2+)-, GTP- and pH-dependent fusion of biological membranes.

Membrane fusion in exocytosis, intracellular trafficking, and enveloped viral infection is thought to be mediated by specialized proteins acting to merge membrane lipid bilayers. We now show that one class of naturally-occurring phospholipids, lysolipids, inhibits fusion between cell membranes, organelles, and between organelles and plasma membrane. Inhibition was reversible, did not correlate with lysis, and could be attributed to the molecular shape of lysolipids rather than to any specific chemical moiety. Fusion was arrested at a stage preceding fusion pore formation. Our results are consistent with the hypothesis that biological fusion, irrespective of trigger, involves the formation of a highly bent intermediate between membranes, the fusion stalk.

Acidic pH induces fusion of cells infected with baculovirus to form syncytia.

The enveloped baculovirus insect cell system has been used extensively for expression of recombinant proteins, including viral fusion proteins. We tested wild-type baculovirus for endogenous fusion protein activity. Syncytia formation, dye transfer, and capacitance changes were observed after incubating infected Spodoptera frugiperda cells in acidic media, consistent with fusion protein activity. Only a short acidic pulse of 10 s is needed to trigger syncytia formation. Identical results were obtained with recombinant baculovirus. This new system is convenient for studying pH activated cell-cell fusion. However, using this enveloped virus to study the mechanism of recombinant fusion proteins requires caution.

Drug efficacy at guanine nucleotide-binding regulatory protein-linked receptors: thermodynamic interpretation of negative antagonism and of receptor activity in the absence of ligand.

The mutual effects that a hormonal ligand (H) and a guanine nucleotide regulatory protein (G protein) exert on each other when simultaneously occupying distinct sites of the receptor molecule (R) can be viewed as the molecular mechanism of drug efficacy. These effects are predictable on the basis of a model assuming that the ternary complex between the three partners (HRG) reaches equilibrium in the membrane [J. Biol. Chem. 255:7108-7117 (1980)]. Ligands can be classified as agonists, neutral antagonists, or negative antagonists, depending on whether they enhance, leave unchanged, or reduce, respectively, the spontaneous tendency of R to interact with G. Using this model and the assumption that the G protein response observed in membranes reflects the sum of ligand-independent (RG) and ligand-dependent (HRG) receptor-G protein complexes, we can explain virtually all the phenomenology reported earlier for opioid receptor-mediated stimulation of GTPase, i.e., 1) existence of ligands with both "positive" and "negative" intrinsic activity (the latter termed negative antagonists), 2) equipotency of neutral antagonists for the competitive blockade of the responses elicited both by agonists and by negative antagonists, and 3) apparent heterogeneity of binding sites for the binding isotherms of negative antagonists. The ternary complex model can also explain the differential effects of sodium on ligand binding and ligand-dependent GTPase activity, if we assume that this ion reduces the stability constant between receptor and G protein in membranes. Computer simulations predict that a negative antagonist exhibits a discrepancy between "biological" Ki (obtained by Schild plots) and true dissociation constant for the receptor, which increases as the fraction of "precoupled" receptors in the membrane increases. The demonstration of negative antagonism is definitive evidence for the existence of receptor coupling (hence activity) in the absence of ligand. Using this experimental paradigm, we show here that spontaneous receptor activity occurs in isolated membranes but not in intact NG108-15 cells.

Role of alpha-adrenoceptors in the effects of buspirone and 5-carboxamidotryptamine in rabbit isolated thoracic aorta.

1. The role of alpha-adrenoceptors in the vascular effects of buspirone (BUS) and 5-carboxamidotryptamine (5-CT) was investigated in rabbit thoracic aorta. 2. Buspirone produced a concentration-dependent contraction. The non-selective 5-HT1 and 5-HT2-receptor antagonist methysergide and the 5-HT2 receptor antagonist ketanserin did not alter the contractile effect of buspirone. However, the competitive antagonist of alpha 1-adrenoceptors, prazosin, shifted the concentration-response curve of buspirone to the right without changing the maximal response. 3. Benextramine tetrahydrochloride monohydrate (BHC), a noncompetitive antagonist of alpha 1-adrenoceptors, inhibited the contraction induced by buspirone in a noncompetitive manner. After pretreatment with two different concentrations of BHC, the estimated apparent dissociation constants of buspirone were found to be identical. 4. In addition, buspirone antagonized the concentration-response curve of phenylephrine again showing a similar dissociation constant, suggesting a partial agonistic action of buspirone at the level of alpha 1-adrenoceptors. 5. The concentration-response curve of 5-HT showed two components in the thoracic aorta obtained from reserpine treated and untreated animals as verified by different pD2 values. The second component was observed with relatively higher concentrations of 5-CT and could be blocked by prazosin or BHC. Neither of these compounds altered the first component. After Pretreatment with BHC, the first component of 5-CT was competitively antagonized by methysergide and ketanserin, having pA2 values of 8.81 and 9.1 respectively. 6. These results suggest that the contraction induced by buspirone is mainly mediated by alpha 1-adrenoceptors, while the higher concentrations of 5-CT caused contraction via alpha 1-adrenoceptor stimulation in addition to its 5-HT2 agonistic effect.

Atypical Schild plots with histamine H1 receptor agonists and antagonists in the rabbit aorta.

Competitive antagonists of histamine H1 receptor were investigated for their effects on histamine-induced responses in the rabbit aorta. Antazoline-induced antagonism gave linear Schild plots with slope equal 1, while the other antagonists [+)-brompheniramine, (+/-)-chlorpheniramine, diphenhydramine and mepyramine) produced 'atypical' plots with slopes generally less than unity in the thoracic aorta. Schild plots obtained with these antagonists were evaluated using a two independent component model. The high affinity parameters thus estimated were compatible with those that have been reported for these antagonists. No such heterogeneity was observed in the abdominal aorta when diphenhydramine was investigated with different H1 agonists. The results suggest the presence of at least two components in H1-mediated responses in the thoracic aorta; these components are equally antagonized by antazoline, but differentially antagonized by the other antagonists used.

Regional histaminergic potencies in rabbit systemic circulation.

1. Regional activity of histamine was studied in various rabbit arteries, including segments of the aorta, aortic branches, branchout regions and arteries to fore and hind limbs by dose-response experiments. 2. Local histaminergic potencies were found to be heterogenceous. Circular and longitudinal smooth muscle responsivity in the aorta increased in the distal direction; aortic branches have their own histaminergic response characteristics beginning from their branchouts. Paired arteries (right and left subclavia, renal and iliac) possess matching histaminergic activities. 3. The observed characteristics of the responses suggest predetermined distribution of histaminergic activities along systemic circulation which may be regulatory in the distribution of blood.

Kinetics of antagonism at histamine-H1 receptors in isolated rabbit arteries.

Kinetics of antagonist-induced decrease of histamine-H1 receptor-mediated steady-state responses in isolated rabbit arteries were studied in the presence of histamine-H2 receptor antagonist famotidine. Data were fitted using a model which describes competition kinetics at the receptor level. Estimated rate and equilibrium constants were evaluated for their dependence on tissue, agonist and antagonist concentrations, using (+)-brompheniramine as antagonist. In large arteries (thoracic and arcus aorta), rate constants were observed to be modified by agonist and/or antagonist concentrations, suggesting a diffusion-controlled process. In relatively small (common carotid and iliac) arteries, estimated equilibrium constants (and consequently the rate constants) were found to diverge despite the invariance of equilibration times between arteries, leading us to include the effects of spare receptors in our evaluation. A model describing the effects of receptor reserve on the estimated equilibrium dissociation constant was developed and stimulated and the results then compared with those that had been experimentally estimated. The reserve hypothesis was experimentally verified in common iliac artery (where EC50 much less than KA) using the irreversible antagonist phenoxybenzamine. A rationalized rule for the optimization of experimental design for in-vitro disequilibrium-competition experiments was proposed. Common carotic artery was found to be favorable for the present design in view of its reserve properties. In addition, competition reaction seems to be the rate-determining step in this artery. Rate and equilibrium constants of mepyramine, (+)-brompheniramine, diphenhydramine and antazoline were therefore determined in the common carotid artery and were compared with those obtained from independent experiments. Results suggest that the estimated parameters reflect drug-receptor interaction.

Heterogeneity of histamine H2-receptor mediated responses in the rabbit aorta.

Distribution and properties of histamine H2-receptor mediated responses in segments of rabbit aorta was studied with histamine and H2-receptor stimulating drugs, dimaprit and impromidine. All agonists produced concentration-dependent tonus decreased in precontracted vascular strips, which were antagonised by selective H2-receptor antagonists, cimetidine and oxmetidine. Activities of the agonists were segment-dependent, and increased caudally along the aorta. A nonspecific smooth muscle relaxant, papaverine had homogeneous activity along the vessel, suggesting receptor specific nature of the observed heterogeneity.

A semiiterative method for derivation of concentration-response parameters.

A method for the analysis of concentration-response curves is described. The parameters of these curves, namely, maximal response, apparent affinity, and slope factor, are calculated by means of a semiiterative approach using a microcomputer. Considerably less computer processing time is required than with other iterative nonlinear curve fitting methods. The method is particularly helpful in cases in which accurate observation of maximal response is limited.