Comparison of the Effects of Ions and GTP on Substance P Binding to Membrane-Bound and Solubilized Specific Sites

Mg2+ increased but Na+ and GTP decrease [3H]substance P (SP) binding to rat cerebral cortical membranes and to 10 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS)-solubilized membrane fraction. To determine the binding parameters that are modified by the cations and GTP, inhibition experiments of [3H]SP binding by unlabeled SP were performed in both of the preparations. Nonlinear least-squares regression analysis of data in the membrane fraction indicated that optimal fitting of the inhibition curves in the presence of 10 mM MgCl2 was attained with a two-site model, corresponding to a "high-affinity (H)" and a "low-affinity (L)" state. By omitting MgCl2, or by addition of NaCl and GTP, the [3H]SP specific binding was decreased, the H state disappeared, and the L state and a new "super-low affinity (SL)" state observed. The SP/[3H]SP inhibition curves in the cerebral cortical membranes by in vivo treatment with pertussis toxin (islet-activating protein) were similar to that in the presence of GTP in control membranes. The effects of MgCl2, NaCl, and GTP were greater in the CHAPS-solubilized fraction than in the membrane fraction. In contrast to the membrane fraction, the inhibition curves of [3H]SP binding by unlabeled SP in the presence of MgCl2 in the CHAPS-solubilized fraction were best fitted to a one-site model. The KD value was relatively close to that of the low-affinity state in the membrane fraction. Even with the addition of NaCl or GTP, or by reducing MgCl2 concentration to 1 mM, although the inhibition curves consistently fit the one-site model, the KD values changed only slightly.     

Characteristics of the β-Adrenoreceptor from Neuronal and Glial Cells in Primary Cultures of Rat Brain

The cellular characteristics of the beta-adrenoreceptor in glial and neuronal cells from the newborn rat brain were determined by (-)-[125I]iodocyanopindolol binding. In membranes from both cell types, the binding was saturable and from competition assays the potency series of (-)-isoproterenol greater than (-)-epinephrine = (-)-norepinephrine greater than (+)-isoproterenol was observed. 5'-Guanylyl-imidodiphosphate reduced the affinity of (-)-isoproterenol for the beta-adrenoreceptor from glial cells but had no effect on agonist affinity in neuronal cells. Chronic treatment of both cell types with (-)-isoproterenol reduced the receptor content and the capacity of the agonist to increase the cellular cyclic AMP content. However, the receptor recovery after chronic agonist treatment was faster in glial cells (72 h) than neuronal cells (120 h) and was blocked by cycloheximide. Treatment of both types with the irreversible beta-blocker bromoacetylalprenololmentane (2 microM) reduced the receptor content by 78% but no receptor recovery was observed for 120 h after the initial receptor loss. The data indicated that the majority of beta-adrenoreceptors in both cell types are the beta-1 subtype, but show some differences in receptor-agonist interactions. Furthermore, these CNS cells may be useful models for regulatory studies on the beta-adrenoreceptor.     

Ca2+-Dependent, ATP-Induced Conversion of the [3H]Hemicholinium-3 Binding Sites from High- to Low-Affinity States in Rat Striatum: Effect of Protein Kinase Inhibitors on This Affinity Conversion and Synaptosomal Choline Transport

Tritium-labeled hemicholinium-3 ([3H]HC-3) was used to characterize the sodium-dependent high-affinity choline carrier sites in rat striatal preparations. In an earlier study, we had shown that [3H]HC-3 labels choline carrier sites with high and low affinities and had suggested that the low-affinity sites represent "functional" carrier sites. The objective of the present study was to examine the mechanisms involved in the regulation of the two affinity states of [3H]HC-3 binding. Here, we demonstrate that these two affinity states are totally interconvertible; addition of 0.1 mM ATP in the binding assay medium quantitatively converted all the binding sites to the low-affinity state, whereas addition of 1 mM beta,gamma-methylene 5'-ATP quantitatively converted all the binding sites to the high-affinity state. Preincubation of the tissue (for 15 min at 37 degrees C) before the binding assay also converted the binding sites to the high-affinity state, whereas supplementation of the assay medium with ATP (0.5 mM) again induced expression of the low-affinity state of the binding sites. This effect of ATP was found to be selective for this nucleotide. Neither ADP (1 mM) nor cyclic AMP could mimic such an effect. Other nucleotide triphosphates--CTP (0.5 mM) and GTP (0.5 mM)--also could not substitute for ATP. GTP, however, caused nearly a 35% reduction in the number of binding sites, accompanying a loss of the low-affinity component of binding. This effect of GTP was also shared by 5'-guanylylimidodiphosphate but not by GDP or cyclic GMP. This ATP-dependent low-affinity conversion of [3H]HC-3 binding sites requires divalent metal ions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of clonidine and clonidine analogues with alpha-adrenergic receptors of neuroblastoma X glioma hybrid cells and rat brain: comparison of ligand binding with inhibition of adenylate cyclase

Clonidine and several analogues of clonidine are shown to be useful probes for alpha 2-adrenergic receptors in a comparative study of ligand binding and inhibition of adenylate cyclase. The alpha-adrenergic properties of a new potential probe, N-(4-hydroxyphenacetyl)-4-aminoclonidine hydrochloride, are described. [3H]Clonidine binds to alpha-receptors of NG108-15 neuroblastoma X glioma hybrid cell membranes with Kd values of 1.7 and 33 nM for putative high-affinity and low-affinity sites, respectively. p-Aminoclonidine and hydroxyphenacetyl aminoclonidine displace [3H]clonidine from the high-affinity sites with Kd values of 2.3 and 5.8 nM, respectively. Rat brain alpha 2-receptors also exhibit high affinity toward clonidine, p-aminoclonidine, and hydroxyphenacetyl aminoclonidine, as determined by displacement of specifically bound [3H]clonidine. Clonidine, p-amino-clonidine, and hydroxyphenacetyl aminoclonidine elicit modest inhibition (up to 24%) of NG108-125 adenylate cyclase by interaction with alpha 2-receptors (Kd,app 300, 30, and 130 nM, respectively); these compounds also partially reverse the inhibition elicited by (--)-norepinephrine. Components of the adenylate cyclase assay mixture, particularly ATP, GTP, sodium ions, and a nucleoside-triphosphate-regenerating system, decrease the high-affinity [3H]clonidine binding to NG108-15 membranes; in the presence of these components, alpha-receptors possess only low affinity (Kd 43 nM) for [3H]clonidine. The results are consistent with the concept that certain components required for the receptor-mediated inhibition of adenylate cyclase convert alpha 2-receptors from a high-affinity inactive state to a low-affinity active state.     

Developmental Changes of Cerebral Cortical [3H]Clonidine Binding in Rats: Influences of Guanine Nucleotide and Cations

Abstract: Cerebral cortical [³H]clonidine binding and influences of GTP and cations were investigated in developing rats. The results from Scatchard plots were compatible with the presence of two populations of binding sites [high-affinity binding ( K_D = 0.59 n M ) and low-affinity binding ( K_D = 7.12 n M )] in 70-day-old rats but only high-affinity binding ( K_D = 0.27 n M ) on day 1. Low-affinity binding was detectable on day 7. K_D values in high- and low-affinity binding were not significantly changed during development after 7 days. B_max of high-affinity binding reached a peak on day 15, and the value of low-affinity binding gradually increased with age. The addition of 10 μ M GTP caused a significant reduction in B_max of high-affinity binding after day 7. Neither K _D nor B_max of low-affinity binding was affected by 10 μ M GTP during development. NaCl (10 and 100 m M ) diminished the binding on days 7 and 70. MnCl₂ (0.1 and 1.0 m M ) markedly increased the binding on days 15 and 70 but not on day 7. It is suggested that: (1) single binding sites of α₂-adrenoceptors with higher affinity seem to be present on day 1; (2) low-affinity binding appears on day 7; (3) the number of high-affinity binding sites reaches a peak on day 15, followed by changes in populations of high-affinity as well as low-affinity sites without changing affinity; (4) the regulatory mechanism in α₂-receptors by guanine nucleotide reaches functional maturity between days 1 and 7; and (5) the involvement of Na⁺ and Mn²⁺ in α₂-receptor binding becomes functional by days 7 and 15, respectively.     

Angiotensin II receptor regulation in isolated renal glomeruli

Equilibrium binding studies with angiotensin II (AII) in isolated rat renal glomeruli indicate the presence of a single population of high-affinity AII receptors. Autoradiographic studies localize these receptors to glomerular mesangial cells, which are ideally positioned to modulate glomerular capillary patency and hence the glomerular capillary ultrafiltration coefficient. Modulation of AII receptor density occurs in response to alterations of circulating AII levels, with down-regulation of receptor number in the presence of salt depletion. Kinetic studies of the ligand dissociation rate performed in the presence and absence of MgCl2 and GTP indicate multiple affinity states and suggest that this receptor is coupled to a guanyl nucleotide regulatory unit. Such coupling may provide a basis for interaction with cyclase-activating hormones in modulating the contractile state of the mesangium.     

Thiol reactivity and the molecular individuality of alpha- and beta-adrenoreceptors in rat liver plasma membranes.

Whether or not alpha- and beta-adrenoreceptors are non-identical binding sites on the same protein is still an open question. We investigated the effects of sulfhydryl reagents and dithiothreitol on the binding of [3H]dihydroalprenolol and [3H]dihydroergocryptine to beta- and alpha-adrenoreceptors of rat liver plasma membranes. Dithiothreitol inhibited the binding of [3H]dihydroalprenolol to the beta-adrenoreceptor, whereas it had no effect on the specific binding of [3H]dihydroergocryptine to the alpha-adrenoreceptor. In contrast, mersalyl, a mercurial SH reagent, readily blocked the alpha-adrenoreceptor and, although to a lesser extent the beta-adrenoreceptor. The interaction of mersalyl with the alpha-adrenoreceptors was almost instantaneous. In contrast, under the same experimental conditions, the inactivation of the beta-adrenoreceptors was much slower (t 1/2 : 7 min). Finally, a marked difference in the accessibility of the SH groups to mersalyl was observed between the alpha- and beta-adrenoreceptors. The presence of 15 microM (-)-epinephrine or 1.5 microM phentolamine was sufficient to prevent the blockade of the alpha-adrenoreceptor by mersalyl, but inactivation of the beta-adrenoreceptor by mersalyl was not modified by 500 microM (-)-epinephrine and was only slightly decreased by 50 microM (-)-propranolol. Thus, the alpha- and beta-adrenoreceptors from rat liver plasma membranes exhibited biochemical differences which may be interpreted in favor of their molecular individuality.     

Modulation of Rat Brain Cortical α-Adrenoceptors by Treatment with Hydrocortisone for 10 Days

The role of glucocorticoids in the modulation of central alpha 2-receptor mechanisms was investigated by in vitro receptor binding studies. [3H]Clonidine and [3H]idazoxan were used as radioligands. The alpha 2-receptor subtypes and guanine nucleotide sensitivity were studied in homologue and heterologue displacement experiments following hydrocortisone treatment (25 mg/kg s.c.) for 10 days. High and low agonist affinity states of the alpha 2-receptor could be identified in 3H-antagonist-agonist and 3H-agonist-antagonist displacement experiments, which may correspond to different regulatory protein-nucleotide associated forms of the receptor. In the presence of 10 microM GTP, the high-affinity binding was depressed. Following hydrocortisone treatment, there was no detectable change either in the affinity or the binding site concentration of clonidine in homologue displacement ("cold saturation") experiments. The affinity of idazoxan, however, was depressed. The effect of GTP was similar to the controls in this experimental arrangement. In contrast, in heterologue binding studies the high-affinity binding site was not demonstrable and the amount of low-affinity binding increased following the hydrocortisone treatment. The high-affinity site reappeared in the presence of GTP. The change in GTP sensitivity suggests that the nucleotide regulatory system may be involved in the action of adrenal steroids on central alpha 2-receptoral mechanisms.     

Characterization of the guinea pig lung membrane leukotriene D4 receptor solubilized in an active form. Association and dissociation with an islet-activating protein-sensitive guanine nucleotide-binding protein

Membrane fractions from the guinea pig lung had high- and low-affinity binding sites for LTD4 with Kd values of 0.016 and 9.1 nM, respectively. In the presence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) or by prior treatment of the membrane with islet-activating protein (IAP), the high-affinity site shifted to a low-affinity state. Consistently, a 41-kDa protein was ADP-ribosylated by treatment of the lung membranes with IAP, and this event was inhibited by the addition of GTP gamma S. We solubilized the LTD4 receptor from the lung membranes in an active form with 5 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and 10% glycerol. On a gel filtration column, the binding activity was eluted at the volume corresponding to a Mr of 70,000 or over 500,000 in the presence or absence of Mg2+ (5-20 mM), respectively, in solubilizing buffers. The Kd value of [3H]LTD4 binding to the 70-kDa protein was similar to the low-affinity binding constant of the membrane and was insensitive to GTP gamma S. The preparation solubilized in the absence of Mg2+ showed both high- and low-affinity binding sites for LTD4, and the addition of GTP gamma S shifted the high-affinity site to a low-affinity one. Thus, 1) the LTD4 receptor is coupled to an IAP-sensitive GTP-binding protein, 2) this GTP-binding protein is dissociable from the receptor by solubilizing the lung membrane with CHAPS and Mg2+, and 3) the receptor associated to or dissociated from a GTP-binding protein exhibited a high- or low-affinity state, respectively. These data provide an insight into the molecular mechanism of regulation of the LTD4 receptor signaling process by association and dissociation with an IAP-sensitive GTP-binding protein.     

D2 Dopamine Receptors in Calf Globus Pallidus: Agonist High- and Low-Affinity Sites Not Regulated by Guanine Nucleotide

By use of the radioligand [3H]spiroperidol, D2 3,4-dihydroxyphenylethylamine (dopamine) receptor binding characteristics were studied in calf globus pallidus and compared with those of neostriatum. Antagonist competition curves were monophasic and revealed similar affinities for neostriatum and globus pallidus, suggesting a uniform receptor population with one affinity state for antagonists. In both regions, competition curves with the agonist dopamine were biphasic, distinguishing a high- and low-agonist-affinity state. In neostriatum and globus pallidus, respectively, 45% and 19% of [3H]spiroperidol binding was displaced with high affinity and the remainder with low affinity. In neostriatum, the addition of 0.4 mM GTP resulted in a partial conversion from high- to low-affinity state with a remaining high-affinity component of 15%. In globus pallidus, dopamine binding was not altered by GTP. The capability of GTP to modulate agonist binding to D2 receptors appears to be dependent on their neuroanatomical localization.     

Catecholamine-stimulated guanosine 5'-O-(3-thiotriphosphate) binding to the stimulatory GTP-binding protein of adenylate cyclase: kinetic analysis in reconstituted phospholipid vesicles

The stimulatory GTP-binding protein of adenylate cyclase, Gs, and beta-adrenergic receptors were reconstituted into unilamellar phospholipid vesicles. The kinetics of the quasiirreversible binding of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) to Gs, equivalent to Gs activation by nucleotide, was studied with respect to the stimulation of this process by beta-adrenergic agonists and Mg2+. The rate of GTP gamma S binding displayed apparent first-order kinetics over a wide range of nucleotide, agonist, and Mg2+ concentrations. In the absence of agonist, the apparent first-order rate constant, kapp, was 0.17-0.34 min-1 and did not vary significantly with the concentration of nucleotide. At 50 mM MgCl2, kapp increased somewhat, to 0.26-0.41 min-1, and remained invariant with the nucleotide concentration. In the presence of agonist, kapp was dependent on nucleotide concentration. At 10(-9) M GTP gamma S, the addition of (-)-isoproterenol caused at most a 2-fold stimulation of kapp. However, kapp measured in the presence of isoproterenol increased as an apparently saturable function of the GTP gamma S concentration, such that isoproterenol caused a 17-fold increase in kapp at 1 microM GTP gamma S. The effect of isoproterenol on kapp also appeared to saturate at high isoproterenol concentration, yielding a kapp approximately 6 min-1 at high concentrations of both nucleotide and agonist. These data suggest that the receptor-agonist complex acts by increasing the rate of conversion of a lower affinity Gs-GTP gamma S complex to the stable activated state.     

Characterization of the ribosomal properties required for formation of a GTPase active complex with the eukaryotic elongation factor 2

The binding stability of the different nucleotide-dependent and -independent interactions between elongation factor 2 (EF-2) and 80S ribosomes, as well as 60S subunits, was studied and correlated to the kinetics of the EF-2- and ribosome-dependent hydrolysis of GTP. Empty reconstituted 80S ribosomes were found to contain two subpopulations of ribosomes, with approximately 80% capable of binding EF-2.GuoPP[CH2]P with high affinity (Kd less than 10(-9) M) and the rest only capable of binding the factor-nucleotide complex with low affinity (Kd = 3.7 x 10(-7) M). The activity of the EF-2- and 80S-ribosome dependent GTPase did not respond linearly to increasing factor concentrations. At low EF-2/ribosome ratios the number of GTP molecules hydrolyzed/factor molecule was considerably lower than at higher ratios. The low response coincided with the formation of the high-affinity complex. At increasing EF-2/ribosome ratios, the ribosomes capable of forming the high-affinity complex was saturated with EF-2, thus allowing formation of the low-affinity ribosome.EF-2 complex. Simultaneously, the GTPase activity/factor molecule increased, indicating that the low-affinity complex was responsible for activating the GTP hydrolysis. The large ribosomal subunits constituted a homogeneous population that interacted with EF-2 in a low-affinity (Kd = 1.3 x 10(-6) M) GTPase active complex, suggesting that the ribosomal domain responsible for activating the GTPase was located on the 60S subunit. Ricin treatment converted the 80S particles to the type of conformation only capable of interacting with EF-2 in a low-affinity complex. The structural alteration was accompanied by a dramatic increase in the EF-2-dependent GTPase activity. Surprisingly, ricin had no effect on the factor-catalyzed GTP hydrolysis in the presence of 60S subunits alone.     

Irreversible inactivation of the beta-adrenoreceptor by a partial agonist. Evidence for selective loss of the agonist high affinity binding sites

The catecholamine derivatives aminomenthylnorepinephrine (compound 1) and bromoacetylaminomenthylnorepinephrine (compound 2) were synthesized and their interaction with the rat lung beta-adrenoreceptor was characterized. Compared to (-)-isoproterenol, compounds 1 and 2 were 10 and 280 times less potent, respectively, at inhibiting (-)-[3H]dihydroalprenolol binding. At pH 7.4, all 3 compounds induced a loss of receptors (40-60%) which could be recovered by treatment with guanyl-5'-yl imidodiphosphate (Gpp(NH)p). However, at pH 8.1 Gpp(NH)p treatment did not recover those receptors lost by compound 2 only. The compound 2-induced receptor loss at pH 8.1 was time-dependent, prevented by propranolol but unaffected by Gpp(NH)p or after membrane heating at 50 degrees C which prevented the formation of the agonist high affinity binding state. Although, the maximal receptor loss as measured by [3H]dihydroalprenolol was 40-60%, more than 80% of the receptors were lost when measured by direct agonist binding, and the receptors left showed little agonist high affinity binding state formation. In rat reticulocyte membranes, compounds 1 and 2 stimulated adenylate cyclase activity with intrinsic activities of 0.55 and 0.31, respectively. However, at pH 8.1, compound 2 initially stimulated the enzyme followed by a blockade. These data indicated that both compounds 1 and 2 were partial beta-adrenoreceptor agonists and, at pH 8.1, compound 2 appeared to bind irreversibly only to those lung receptors able to form the agonist high affinity binding state. Furthermore, after irreversible binding, compound 2 appeared to act as an antagonist.     

Guanine nucleotide regulation of agonist binding to muscarinic cholinergic receptors. Relation to efficacy of agonists for stimulation of phosphoinositide breakdown and Ca2+ mobilization.

The efficacies of a series of six muscarinic cholinergic receptor agonists for stimulation of phosphoinositide breakdown and unidirectional efflux of 45Ca2+ in 1321N1 human astrocytoma cells were compared with the relative capacity of these agonists for formation of a GTP-sensitive high-affinity binding state in washed membranes. Carbachol and methacholine were 'full' agonists as regards phosphoinositide breakdown and Ca2+ mobilization, whereas bethanechol, arecoline and oxotremorine were 'partial' agonists for these two responses. Pilocarpine was the least efficacious of the six drugs tested. Except for pilocarpine, competition curves generated with the agonists and [3H]quinuclidinyl benzilate did not follow the Law of Mass Action for ligand interaction at a single site. Non-linear regression analyses of these data indicated that the data significantly better fit a two-, rather than a single-, site model with a high- and a low-affinity binding component. Competition curves generated in the presence of GTP were shifted to the right, and the extent of receptors in the high-affinity agonist-binding state was decreased. The relative efficacies of the six agonists for stimulation of phosphoinositide breakdown and Ca2+ mobilization were significantly correlated with the difference in affinities (KL/KH) between the two affinity states for each agonist. The relative efficacy of the agonists for stimulation of Ca2+ mobilization also was significantly correlated with the extent of receptors in the high-affinity state (%H) for each agonist. The results suggest that interaction with an as-yet unidentified guanine nucleotide regulatory protein is important in the mechanism whereby muscarinic receptors stimulate phosphoinositide breakdown in 1321N1 astrocytoma cells.     

Characterization of a Labile Naloxone Binding Site (λ Site) in Rat Brain

A high-affinity binding site selective for naloxone and other 4,5-epoxymorphinans (lambda site) has been previously described in rat brain. Following homogenization of freshly dissected brain, the lambda sites convert from a high-affinity to a low-affinity state. When measured with [3H]naloxone, the decay is very rapid at 20 degrees C (t 1/2 less than 2 min), whereas it is progressively slowed at lower temperatures. Proteinase inhibitors, antoxidants, and sulfhydryl group-protecting agents failed to prevent this conversion. Kinetic measurements of mu and lambda binding at varying temperatures demonstrated that the decrease in lambda binding does not coincide with the concurrent increase in mu binding and that the loss of high-affinity lambda binding at 20 degrees C can be partially restored when the temperature is lowered to 0 degrees C. The low-affinity state of the lambda site is rather stable in the Tris buffer homogenates and is susceptible to digestion by a protease. The (-)-isomer of WIN 44,441, a benzomorphan drug, binds to lambda sites with moderate affinity (dissociation constant, KD = 63 nM), whereas the (+)-isomer does not (KD greater than 10,000 nM), thus establishing stereoselectivity of the binding process. Neither the high-affinity nor the low-affinity state of lambda binding is significantly affected by the presence of 100 mM sodium chloride or 50 microM Gpp(NH)p, (a GTP analog), which is in contrast to the dramatic effect of these agents on the established opioid receptor system. Naltrexone, naloxone, nalorphine, and morphine (in this order of decreasing potency) bind to the lambda site in vivo in intact rat brain over dosage ranges that are commonly employed in pharmacological studies.     

Microsomal Opiate Receptors: Characterization of Smooth Microsomal and Synaptic Membrane Opiate Receptors

In continuing studies on smooth microsomal and synaptic membranes from rat forebrain, we compared the binding properties of opiate receptors in these two discrete subcellular populations. Receptors in both preparations were saturable and stereospecific. Scatchard and Hill plots of [3H]naloxone binding to microsomes and synaptic membranes were similar to plots for crude membranes. Both synaptic membranes and smooth microsomes contained similar enrichments of low- and high-affinity [3H]naloxone binding sites. No change in the affinity of the receptors was observed. When [3H]D-ala2-D-leu5-enkephalin was used as ligand, microsomes possessed 60% fewer high-affinity sites than did synaptic membranes, and a large number of low-affinity sites. In competition binding experiments microsomal opiate receptors lacked the sensitivity to (guanyl-5'-yl)imidodiphosphate [Gpp(NH)p] shown by synaptic and crude membrane preparations. In this respect microsomal opiate receptors resembled membranes that were experimentally guanosine triphosphate (GTP)-uncoupled with N-ethylmaleimide (NEM). Agonist binding to microsomal and synaptic membrane opiate receptors was decreased by 100 mM NaCl. Like NEM-treated crude membranes, microsomal receptors were capable of differentiating agonist and antagonists in the presence of 100 mM NaCl. MnCl2 (50-100 microM) reversed the effects of 100 mM NaCl and 50 microM GTP on binding of the mu-specific agonist [3H]dihydromorphine in both membrane populations. Since microsomal receptors are unable to distinguish agonists from antagonists in the presence of Gpp(NH)p, they are a convenient source of guanine nucleotide-uncoupled opiate receptors.     

The effects of diphenyleneiodonium on mitochondrial reactions. Relation of binding of diphenylene[125I]iodonium to mitochondria to the extent of inhibition of oxygen uptake.

1. Several ring-substituted derivatives of diphenyleneiodonium catalyse the exchange of Cl- and OH- ions across the inner membrane of rat liver mitochondria. They also inhibit state 3 and state 3u oxidations of glutamate plus malate in the presence of Cl- more than in its absence. Most have activities similar to diphenyleneiodonium, although 2,4-dichlorodiphenyleneiodonium is up to 50 times more active. 2. Diphenyleneiodonium inhibits soluble rat liver NADH dehydrogenase and NADH oxidation by rat liver sub-mitochondrial particles directly; 2,4-dichlorodiphenyleneiodonium is only about twice as inhibitory. 3. Liver mitochondria contain two classes of binding sites for diphenylene[125I]iodonium, namely high-affinity sites with an affinity constant of 3 X 10(5) M-1 (1--2 nmol/mg of protein), and low-affinity sites with an affinity constant of 1.3 X 10(3) M-1 (80 nmol/mg of protein). Both sites occur in hepatocytes with a relative enrichment of the low-affinity site. Nadh dehydrogenase preparations only apparently contain high-affinity binding sites. Only low-affinity sites occur in erythrocytes. 4. 2,4-Dichlorodiphenyleneiodonium competes with diphenylene[125I]iodonium for both low- and high-affinity sites, whereas tri-n-propyltin only competes for the low-affinity sites. 5. The high-affinity sites are apparently associated with NADH dehydrogenase and the low-affinity sites probably represent electrostatic binding of diphenylene[125I]iodonium to phospholipids. The high-affinity site does not appear to be associated with a rate-limiting stage of NADH oxidation.     

Characterization of a serotonin transporter and an adenylate cyclase-linked serotonin receptor in the cestode Hymenolepis diminuta

[3H]5-HT exhibited specific binding in membrane preparations of Hymenolepis diminuta. The specific binding was saturable, reversible and temperature dependent. A non-linear Scatchard plot was obtained in a concentration range of 11 nM - 1000 nM [3H]5-HT, which could be resolved into sites having apparent dissociation constants (KD) of 0.10 microM and 6.25 microM for the high-affinity and low-affinity components, respectively. The latter could be selectively eliminated by binding [3H]5-HT to H. diminuta membranes in the presence of 10(-3) M nitroimipramine. Drug displacement studies, using 0.20 microM and 2.0 microM [3H]5-HT, revealed that while low-affinity [3H]5-HT binding was displaced by unlabelled 5-HT and inhibitors of 5-HT uptake, high affinity [3H]5-HT binding was affected only by tryptamine derivatives and, to a lesser extent, methysergide. In addition, high-affinity binding was stimulated by MgCl2 while low-affinity binding showed sodium-dependency. The data implicate the low-affinity site as a putative 5-HT transporter and the high-affinity site as a putative 5-HT 1 receptor. Exposure of H. diminuta membranes to 5-HT resulted in a 3-4 fold stimulation of cAMP levels. The EC 50 for the 5-HT-induced activation of adenylate cyclase (0.76 microM) was of the same order of magnitude as the apparent KD for high-affinity binding. Furthermore, the order of drug potency for the elevation of cAMP levels by 5-HT agonists and reversal by 5-HT antagonists was identical to the order of drug potency for the inhibition of high-affinity binding, suggesting linkage of the putative 5-HT 1 receptor to adenylate cyclase in H. diminuta.     

Reconstitution of the purified porcine atrial muscarinic acetylcholine receptor with purified porcine atrial inhibitory guanine nucleotide binding protein

Purified porcine atrial muscarinic receptor (mAcChR) was reconstituted with purified porcine atrial inhibitory guanine nucleotide binding protein (Gi) in a lipid mixture consisting of phosphatidylcholine, phosphatidylserine, and cholesterol (1:1:0.1 w/w). 5'-Guanylyl imidodiphosphate (0.1 mM) had no effect on the binding of the muscarinic antagonist L-quinuclidinyl benzilate but converted high-affinity carbachol binding sites (Kd equal to 1 microM) in the reconstituted preparation to the low-affinity state (Kd equal to about 100 microM). Steady-state kinetic measurements of GTPase activity showed that the turnover number was increased from 0.19 min-1 in the presence of the muscarinic antagonist L-hyoscyamine to 2.11 min-1 for the agonist carbachol. The affinity of Gi for GDP was reduced by about 50-fold upon interaction with the carbachol-mAcChR complex, and the observed rate constant for GDP dissociation was increased by 38-fold from 0.12 to 4.5 min-1. Thus, the increase in steady-state GTPase activity observed for muscarinic agonists is largely, if not exclusively, due to the increase in GDP dissociation from Gi--probably the rate-limiting step in the steady-state mechanism. Carbachol-stimulated GTPase was sensitive to ADP-ribosylation of the reconstituted Gi by pertussis toxin, but the high-affinity agonist binding was uncoupled only when the reconstituted preparation was treated with pertussis toxin in the presence of GTP and the agonist acetylcholine. These results suggest that association with the mAcChR protects Gi from ADP-ribosylation by pertussis toxin.     

Differential Regulation by Guanine Nucleotides of Opiate Agonist and Antagonist Receptor Interactions

Abstract: Guanine nucleotides differentiate binding of tritium-labeled agonists and antagonists to rat brain membranes. In the absence of sodium, GTP (50 μM) decreased binding of [³H]-labeled agonists by 20–60% and [³H]-labeled antagonists by 0–20%. In the presence of 100 mM-NaCl, GTP had no effect on antagonist binding, but decreased agonist binding by 60–95%. GMP was less potent than either GTP or GDP in decreasing agonist binding. GTP (50 μM) reduced high-affinity [³H]dihydromorphine sites by 52% and low-affinity sites by 55%. Without sodium, GTP reduced high-affinity [³H]-naloxone sites by 36%; in the presence of 100 mM-NaCl, GTP had no effect on either high- or low-affinity [³H]naloxone sites. GTP increased the association rate of [³H]dihydromorphine twofold and the dissociation rate by fourfold, while having no effect on association or dissociation rates of the antagonist [³H]diprenorphine. The affinities of uniabeled antagonists in inhibiting [³H]-diprenorphine binding were not affected by GTP or sodium, but the affinities of agonists were reduced 40- 120-fold, with met- and leu-enkephalin affinities reduced by the greatest degree. GTP and sodium lowered [³H]dihydromorphine binding in an additive fashion, while divalent cations, especially manganese, reversed the effects of GTP on [³H]-labeled agonist binding by stimulating membrane-bound phosphatases that hydrolyze GTP to GMP and guanosine. These results suggest that by affecting binding of agonists, but not antagonists, GTP may regulate opiate receptor interactions with their physiological effectors.