Effects of magnesium ion on the interaction of atrial muscarinic acetylcholine receptors and GTP-binding regulatory proteins.

Muscarinic acetylcholine receptors (mAChR) purified from porcine atrium were reconstituted into lipid vesicles with GTP-binding regulatory proteins (G proteins, Gi, Go, or Gn) purified from porcine cerebrum. Apparent affinities of the reconstituted mAChR and G proteins for carbachol and GDP, respectively, were estimated from the effects of these ligands on the binding of [3H]-L-quinuclidinyl benzilate ([3H]QNB) to mAChR and [35S]guanosine 5'-O-(3-thiotriphosphate) ([35S]GTP gamma S) to G proteins in the presence of different concentrations of MgCl2. A total of 30-35% of reconstituted mAChRs exhibited low affinity for carbamylcholine, irrespective of the presence or absence of guanine nucleotides, and the remainder of the mAChRs showed high affinities for carbamylcholine in the absence of GTP or GDP and a low affinity in their presence. The affinity for carbamylcholine in the absence of guanine nucleotides, but not in their presence, increased with increases in MgCl2 concentration. Apparent Kd's for carbamylcholine were estimated to be approximately 100 microM in the presence of guanine nucleotides, 1.5 microM in the absence of guanine nucleotide and Mg2+ (< 0.1 microM), and 0.1 microM in the absence of guanine nucleotide and the presence of MgCl2 (10 mM). These results indicate that mAChRs may assume at least three different conformations that are characterized by different affinities for agonists. Furthermore, the data suggest that MgCl2 is not necessary for the formation of the mAChR-G protein complex, but can induce a conformational change in the complex. On the other hand, the presence of MgCl2 was necessary for carbamylcholine to influence the binding of guanine nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ascorbate Modulates 5-[3H]Hydroxytryptamine Binding to Central 5-HT3 Sites in Bovine Frontal Cortex

Ascorbate is present in millimolar concentrations in mammalian brain and can be released from cellular stores by membrane depolarization. We report here that physiologically relevant concentrations of ascorbate modulate 5-[3H]hydroxytryptamine ([3H]5-HT) binding to bovine frontal cortex membranes. Under conditions where [3H]5-HT binding is reversible and saturable, ascorbate causes a concentration-dependent increase in the affinity of [3H]5-HT for central 5-HT3 binding sites. At pH 7.4, increasing ascorbate from 0 to 5.7 mM changes the equilibrium affinity constant (KD) of binding to 5-HT3 sites from 125 nM to 30 nM, without affecting binding site number. These ascorbate-induced effects are pH dependent. At pH 7.1 binding to central 5-HT3 sites is essentially eliminated in the presence of ascorbate. These studies suggest that ascorbate and hydrogen ion concentration interactions may modulate serotonergic function.     

Prostaglandin D2 receptor of mastocytoma P-815 cells--possible regulation by phosphorylation and dephosphorylation

The 3H-labeled prostaglandin D2 [( 3H]PGD2) binding protein in the membrane fraction of mastocytoma P-815 cells was characterized. The specific binding of [3H]PGD2 to the cells or the membranes reached a maximum at pH 5.6, and was saturable, displaceable and of high affinity when incubated at 0 or 37 degrees C. The Bmax values for [3H]PGD2 binding in the two preparations at pH 5.6 were much higher at 0 degrees C than at 37 degrees C, whereas the Kd values were almost equal (85.3 nM for the cells and 80.5 nM for the membranes, respectively). High specific [3H]PGD2 binding activity in the mildly acid-treated cells was still observed when the external pH was raised from 5.6 to 7.2. Furthermore, specific [3H]PGD2 binding to the membranes (at 0 degrees C, pH 5.6) increased on addition of phosphatase inhibitors (NaF and molybdate) in the presence of 10 microM ATP, but practically disappeared on pretreatment of the membranes with phosphatase. On incubation of the membrane with [gamma-32P]ATP and molybdate, the stimulated incorporation of the [32P]phosphate into several peptides, including ones having an Mr of around 100,000-120,000, was observed. These results suggest that [3H]PGD2 binding in the mastocytoma P-815 cell membrane is controlled through phosphorylation-dephosphorylation of the receptor itself.     

Properties of beta-adrenergic receptors of cultured mammalian cells. Interaction of receptors with a guanine nucleotide-binding protein in membranes prepared from L6 myoblasts and from wild-type and cyc- S49 lymphoma cells

The radiolabeled agonist [3H]hydroxybenzylisoproterenol ([3H]HBI) and antagonist [125I]iodopindolol ([125I]IPIN) were used to investigate the properties of beta-adrenergic receptors on membranes prepared from L6 myoblasts and S49 lymphoma cells. The high affinity binding of (-)-[3H]HBI to membranes prepared from L6 myoblasts was stereoselectively inhibited by the active isomers of isoproterenol and propranolol. The density of receptors determined with (-)-[3H]HBI was less than that determined with [125I]IPIN. The binding of (-)-[3H]HBI was inhibited by guanine nucleotides, suggesting an agonist-mediated association of the receptor with a guanine nucleotide-binding protein, presumably the stimulatory guanine nucleotide-binding protein (Ns) of adenylate cyclase. Results obtained in studies with membranes prepared from wild-type S49 lymphoma cells and the adenylate cyclase-deficient variant (cyc-) were similar to those obtained in experiments carried out with membranes prepared from L6 myoblasts. Thus, the high affinity binding of (-)-[3H]HBI to membranes prepared from wild-type and cyc- S49 lymphoma cells was stereoselectively inhibited by the active isomers of isoproterenol and propranolol, and was inhibited by GTP. Moreover, the density of sites determined with (-)-[3H]HBI was less than that determined with [125I]IPIN. These results suggest either that cyc- cells contain a partially functional Ns, or alternatively, that the inhibitory guanine nucleotide-binding protein (Ni) is capable of interacting with beta-adrenergic receptors.     

A1 Adenosine Receptor-G Protein Coupling in Bovine Brain Membranes: Effects of Guanine Nucleotides, Salt, and Solubilization

The effects of guanine nucleotides, NaCl, and solubilization on the interaction of antagonists and agonists with the A1 adenosine receptor of bovine brain membranes were studied using the high-affinity antagonist radioligand [3H]xanthine amine congener ([3H]XAC). In membranes, guanine nucleotides and NaCl had no effect on [3H]XAC saturation curves. Using agonist (R)-phenylisopropyladenosine (R-PIA) competition curves versus [3H]XAC, it was demonstrated that agonists could differentiate two affinity states having high and low affinity for agonist and that guanine nucleotides shifted the equilibrium to an all-low-affinity state that was indistinguishable from the low-affinity state in the absence of guanine nucleotides. In contrast, NaCl decreased agonist affinity by a distinctly different mechanism characterized by a parallel rightward shifted agonist curve such that R-PIA still recognized two affinity states albeit of lower affinity than in the absence of salt. R-PIA competition curves in the presence of both guanine nucleotides and salt were still shallow but were shifted far to the right, and two very low affinity states were discerned. On solubilization, guanine nucleotides in a reversible, concentration-dependent manner increased antagonist ([3H]XAC) but not agonist (R-N6-[3H]phenylisopropyladenosine) binding. This was consequent to a change in maximal binding capacity. R-PIA competition curves (versus [3H]XAC) in solubilized preparations demonstrated that agonist could still differentiate two agonist specific affinity states which were modulated by guanine nucleotides. In the presence of guanine nucleotides all the receptors were shifted to a uniform low-affinity state. In contrast, NaCl had no effect on agonist affinity as determined by agonist competition curves in a solubilized receptor preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Guanine nucleotide regulation of [3H]vasopressin binding to liver plasma membranes and solubilized receptors. Evidence for the involvement of a guanine nucleotide regulatory protein.

A guanine nucleotide regulatory protein may be involved in vasopressin-receptor-mediated polyphosphoinositide breakdown in rat liver. Therefore we examined the effects of the non-hydrolysable guanine nucleotide guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) on [3H]vasopressin ([3H]AVP) binding to hepatic plasma membranes and detergent extracts. [3H]AVP bound to a single set of high-affinity binding sites in membranes. Addition of p[NH]ppG decreased the affinity of receptor binding without altering the maximal binding capacity. The rate of dissociation of [3H]AVP from membrane-bound receptors was also enhanced by p[NH]ppG. Solubilization of [3H]AVP-prelabelled membranes with dodecyl beta-D-maltoside resulted in a [3H]AVP-receptor complex that was unstable in solution. Incubation of these extracts for 5 min at 30 degrees C resulted in a 40% loss of bound [3H]AVP, whereas in the presence of p[NH]ppG there was a 54% loss. However, when membranes were prelabelled with [3H]AVP and p[NH]ppG and then solubilized, the resulting hormone-receptor complex was still temperature-labile but insensitive to the further addition of p[NH]ppG. The molecular size of soluble vasopressin receptors was estimated by gel filtration. The [3H]AVP-receptor complex was eluted as a single peak with an apparent molecular size of 258 kDa. However, no peak was detected when solubilized extract was made from membranes prelabelled with [3H]AVP and p[NH]ppG, suggesting that this receptor complex had dissociated during chromatography. It is possible therefore that the high-Mr complex contains the hormone, its receptor and a guanine nucleotide binding protein.     

Muscarinic receptors in pancreatic islets of the rat: Demonstration and dependence on long-term glucose environment

The presence of muscarinic receptors in islets of Langerhans was assessed by measurement of specific binding of [³H]methylscopolamine. Specific binding was defined as total binding minus binding obtained in the presence of 1000-fold or higher excess of unlabeled methylscopolamine. At 37°C specific binding was significant after 1 min and plateaued after 10 min of incubation. Displacement of label by increasing concentrations of unlabeled methylscopolamine indicated a dissociation constant of 1.5·10^?12 M. Effects of methylscopolamine on insulin release were evaluated from the inhibitions of cholinergic-induced insulin release. 4·10^?10 M methylscopolamine inhibited acetylcholine (20 μM)-induced insuliln release more than 60%. Binding was not influenced by the following variations during binding incubations: changing the glucose concentration from 0 to 83 mM, adding rotenon (1 μM) or omitting calcium from the incubation medium. Islets kept in tissue culture exhibited higher binding when cultured at 11.1 than at 3.3 mM glucose for 96 h. It is concluded that islets contain muscarinic receptors, the binding to which can be subject to alteration by the long-term glucose environment.     

Depolarization-induced changes in the muscarinic receptor in rat brain and heart are mediated by pertussis-toxin-sensitive G-proteins

Muscarinic receptor properties in rat cortical and brain stem synaptoneurosomes and in heart myocytes were examined at resting potential and at depolarization. Depolarization induced the conversion of agonist-binding sites of the receptor from a high to a low affinity state, which could be reversed by a return to resting potential. No effect was observed on the affinity of the receptor for antagonists. Pertussis-toxin (PTX)-catalyzed ADP-ribosylation of all substrates in both synaptoneurosomal and myocyte membranes, when conducted at resting potential, prevented depolarization-induced conversion of the receptor affinity in these preparations. The target substrates were identified by [32P]ADP-ribosylation of membranes prepared from brain stem synaptoneurosomes. Autoradiography revealed labeling of a 39-kDa protein band, which reacted mainly with antibodies to the alpha-subunit of Go-proteins. The possible involvement of G-proteins in depolarization-induced changes in the receptor activity was further investigated by examining the effect of membrane potential on the PTX-sensitive binding of di- and triphosphated guanine nucleotides to synaptoneurosomal membranes. Brain stem synaptoneurosomes were made permeable to guanine nucleotides ([3H]GTP, [3H]GDP, [3H]5'-guanylyl imidodiphosphate) by treatment with ATP. After the synaptoneurosomes had been loaded with labeled GTP/GDP, resealed, and then subjected to either resting potential of short depolarization, binding of [3H]GDP to the membranes of depolarized synaptoneurosomes was 4.0 +/- 0.3 (n = 20) times higher than to the membranes of synaptoneurosomes at resting potential. Repolarization reversed this effect. Enhancement of [3H]GDP binding to the synaptoneurosomal membranes was induced also by muscarinic activation, although the increase obtained was only 30-40% (n = 5) relative to [3H]GDP binding at resting potential. Both the depolarization-induced and the muscarinically-induced enhancement of [3H]GDP binding were prevented following PTX-catalyzed ADP-ribosylation of G-proteins in the synaptoneurosomal membrane. Our results suggest that the depolarization-induced enhancement in the binding of [3H]GTP/[3H]GDP may be attributable to activation of PTX-sensitive G-proteins, which mediate the depolarization-induced alteration of the affinity of the muscarinic receptor for agonists.     

Temperature-sensitive high affinity [3H]tryptamine binding sites in rat brain

The influence of prior incubation on [3H]tryptamine binding was investigated in rat brain synaptic plasma membranes. A 55 min preincubation of the membranes at 37 degrees C induced an approx. 2.4-fold increase in the specific binding of [3H]ligand to the subsequently washed preparations and this phenomenon was quite temperature-dependent. On the other hand, the proportion of nonspecific binding sites was significantly decreased by 70% of the original sites within 20 min of the start of preincubation. Pargyline, ascorbic acid, EGTA, metal ions (Ca2+, Mg2+, Na+) and guanine nucleotides, included in the preincubation buffer, were all inactive on the stimulation of [3H]tryptamine binding, while the pretreatment of membranes with glutaraldehyde antagonized the augmentation of this binding. Furthermore, it was revealed that the Scatchard plot of the [3H]tryptamine binding preincubated at 0 degree C conformed to a straight line (KD = 33.1 nM, Bmax = 543 fmoles/mg protein), whereas a curvilinear Scatchard plot was obtained at 37 degrees C preincubation. Nonlinear regression analysis of the latter resulted in apparent KD (nM) & Bmax (fmoles/mg protein) values of 0.45 & 102.7 and 33.7 & 603.4 for the high and low affinity sites, respectively. All these observations lead to the inference that the preincubation-induced increase in [3H]tryptamine binding (i.e., nearly high affinity proportion of sites) may occur as a result of temperature-sensitive interconvertible conformational changes.     

The interaction of a kainate receptor from goldfish brain with a pertussis toxin-sensitive GTP-binding protein.

Kainate receptors are present in high concentrations in goldfish brain (Henley and Oswald, 1988a and b; Ziegra et al., 1990), possibly in neuronal and glial cells. In a number of systems, the kainate receptor has been assumed to be an integral ion channel (Watkins and Evans, 1981); but, for some kainate receptors, ion channel activity has not been demonstrated (Wada et al., 1989). This study presents evidence that a portion of the [3H]kainate-binding sites in goldfish brain is sensitive to guanine nucleotides, with a loss of high affinity binding in the presence of nonhydrolyzable GTP analogs. Pertussis toxin pretreatment of membranes causes a loss of high affinity [3H]kainate binding and of the guanine nucleotide-sensitive binding. Pertussis toxin catalyzes the specific [32P]ADP-ribosylation of a 40-kDa substrate in a kainate-sensitive manner. In addition, incorporation of [alpha-32P]GTP-gamma-azidoanilide by photoaffinity labeling was enhanced in the presence of kainate. These results indicate that a subpopulation of [3H]kainate-binding sites in goldfish brain may be coupled to G proteins.     

Artifactual High-Affinity and Saturable Binding of [3H]5-Hydroxytryptamine Induced by Radioligand Oxidation

The binding of [3H]5-hydroxytryptamine (5-HT, serotonin) to cerebellar membranes was examined after preincubation of [3H]5-HT in the presence or absence of ascorbate. The tissue preparation was identical in all experiments and consisted of rat cerebellar homogenates in Tris-HCl buffer with 0.1% ascorbate. Cerebellar membranes were used because of their low density of 5-HT1 binding sites. In the presence of ascorbate during a 4-h preincubation period, minimal specific binding of 2 nM [3H]5-HT is detected. Similar results are obtained with equimolar concentrations of other antioxidants (butylated hydroxytoluene, sodium dithionite, and sodium metabisulfite). Apparent specific binding increases 14-fold following a 4-h preincubation of [3H]5-HT in the absence of ascorbate. The increase in apparent specific [3H]5-HT binding is time-dependent and plateaus after 4-6 h of preincubation. When ascorbate is present during the 4-h preincubation, Scatchard analysis of [3H]5-HT binding reveals a KD value of 3.0 +/- 0.3 nM and a Bmax value of 1.9 +/- 0.2 pmol/g tissue. When ascorbate is absent during the preincubation, the KD is essentially unchanged at 3.6 +/- 0.1 nM but the Bmax is significantly increased to 36.5 +/- 7 pmol/g tissue. Drug competition studies reveal that the apparent specific "[3H]5-HT binding" in the absence of ascorbate appears to be displaced by nanomolar concentrations of hydroxylated tryptamines (5-HT, bufotenine) but not by nonhydroxylated tryptamines (5-methoxytryptamine, tryptamine). HPLC analysis demonstrates that [3H]5-HT is essentially destroyed by a 4-h incubation at 22 degrees C in the absence of ascorbate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dopamine D1 receptors characterized with [3H]SCH 23390. Solubilization of a guanine nucleotide-sensitive form of the receptor

Dopamine D1 receptors were solubilized from canine and bovine striatal membranes with the detergent digitonin. The receptors retained the pharmacological characteristics of membrane-bound D1 receptors, as assessed by the binding of the selective antagonist [3H]SCH 23390. The binding of [3H]SCH 23390 to solubilized receptor preparations was specific, saturable, and reversible, with a dissociation constant of 5 nM. Dopaminergic antagonists and agonists inhibited [3H]SCH 23390 binding in a stereoselective and concentration-dependent manner with an appropriate rank order of potency for D1 receptors. Moreover, agonist high affinity binding to D1 receptors and its sensitivity to guanine nucleotides was preserved following solubilization, with agonist dissociation constants virtually identical to those observed with membrane-bound receptors. To ascertain the molecular basis for the existence of an agonist-high affinity receptor complex, D1 receptors labeled with [3H] dopamine (agonist) or [3H]SCH 23390 (antagonist) prior to, or following, solubilization were subjected to high pressure liquid steric-exclusion chromatography. All agonist- and antagonist-labeled receptor species elute as the same apparent molecular size. Treatment of brain membranes with the guanine nucleotide guanyl-5'-yl imidodiphosphate prior to solubilization prevented the retention of [3H]dopamine but not [3H]SCH 23390-labeled soluble receptors. This suggests that the same guanine nucleotide-dopamine D1 receptor complex formed in membranes is stable to solubilization and confers agonist high affinity binding in soluble preparations. These results contrast with those reported on the digitonin-solubilized dopamine D2 receptor, and the molecular mechanism responsible for this difference remains to be elucidated.     

Agonist binding promotes a guanine nucleotide reversible increase in the apparent size of the bovine anterior pituitary dopamine receptors

Dopamine receptors, solubilized from bovine anterior pituitary membranes with the detergent digitonin, retained a typical dopaminergic specificity for the binding of both agonists and antagonists. The affinities of antagonists for binding to the soluble receptors are virtually identical with those observed with the membrane-bound receptors. The affinities of agonists however, correspond to those for the form of the receptors in the membranes having low affinity for those agonists (De Lean, A., Kilpatrick, B. F., and Caron, M. G. (1982) Mol. Pharmacol. 22, 290-297). Thus, after solubilization, agonist high affinity interactions with the receptor and their sensitivity to modulation by guanine nucleotides are lost. However, high affinity agonist binding and its sensitivity to guanine nucleotides can be preserved if the membrane-bound receptors are prelabeled with the agonist [3H]n-propylapomorphine prior to solubilization. In order to investigate the molecular basis for these changes in the properties of agonist binding, the solubilized receptors were characterized by chromatographic procedures. Using molecular exclusion high pressure liquid chromatography, [3H]n-propylapomorphine-prelabeled receptors elute as an apparent larger molecular species than either unlabeled or antagonist [( 3H]spiroperidol)-pre-labeled receptors. Moreover, incubation of the pooled agonist-prelabeled receptor peak with guanine nucleotides effects a decrease in the apparent size of the receptors such that upon rechromatography they elute in a position coincidental with the 3H-antagonist-pre-labeled receptor peak. Thus, occupancy of the receptors by agonists promotes the formation of a guanine nucleotide-sensitive agonist high affinity form of the receptor which is of larger apparent size presumably due to the association of the receptor with a guanine nucleotide regulatory protein.     

Guanine Nucleotide Effects on 8-Cyclopentyl-1,3-[3H]Dipropylxanthine Binding to Membrane-Bound and Solubilized A1 Adenosine Receptors of Rat Brain

The effects of guanine nucleotides on binding of 8-cyclopentyl-1,3-[3H]dipropylxanthine ([3H]DPCPX), a highly selective A1 adenosine receptor antagonist, have been investigated in rat brain membranes and solubilized A1 receptors. GTP, which induces uncoupling of receptors from guanine nucleotide binding proteins, increased binding of [3H]DPCPX in a concentration-dependent manner. The rank order of potency for different guanine nucleotides for increasing [3H]DPCPX binding was the same as for guanine nucleotide-induced inhibition of agonist binding. Therefore, a role for a guanine nucleotide binding protein, e.g., Gi, in the regulation of antagonist binding is suggested. This was confirmed by inactivation of Gi by N-ethylmaleimide (NEM) treatment of membranes, which resulted in an increase in [3H]DPCPX binding similar to that seen with addition of GTP. Kinetic and equilibrium binding studies showed that the GTP- or NEM-induced increase in antagonist binding was not caused by an affinity change of A1 receptors for [3H]DPCPX but by an increased Bmax value. Guanine nucleotides had similar effects on membrane-bound and solubilized receptors, with the effects in the solubilized system being more pronounced. In the absence of GTP, when most receptors are in a high-affinity state for agonists, only a few receptors are labeled by [3H]DPCPX. It is suggested that [3H]DPCPX binding is inhibited when receptors are coupled to Gi. Therefore, uncoupling of A1 receptors from Gi by guanine nucleotides or by inactivation of Gi with NEM results in an increased antagonist binding.     

Effect of anions on folate binding by isolated brush border membranes from rat kidney

The characteristics of folate binding by brush border membranes from rat kidney homogenates were investigated. At pH 7.4, binding of [3′, 5′, 9-³H]-pteroylglutamic acid to membranes containing endogenous folate is inhibited by anions, with chloride being most effective followed by bromide, thiocyanate, iodide, phosphate and sulfate. A maximum inhibition of 70–75% is attained at a concentration of 0.1 M chloride and an incubation time of 30 min. The inhibition diminishes with increased incubation time and at 24 h is negligible. The binding of [3′,5′,9-³H]pteroylglutamic acid to brush border membranes stripped of endogenous folate by acid treatment is not inhibited by anions. Anion sensitivity can be restored to these treated membranes by reconstitution with membrane-derived folate, particularly 5-methyltetrahydropteroylglutamic acid, or by preincubation with synthetic 5-methyltetrahydropteroylglutamic acid. Inhibition of [3′,5′,9-³H]pteroylglutamic acid binding by anions in membranes with endogenous folate is best explained by an anion-induced stabilization of endogenous folate-binding protein complex resulting in a decreased rate of exchange with exogenous [3′,5′,9-³H]pteroylglutamic acid.     

Agonist-induced changes in the modulation of K+ permeability and beating rate by muscarinic agonists in cultured heart cells

The correlation between number of muscarinic cholinergic receptor sites as measured by binding of the muscarinic antagonist [3H]methylscopolamine ([3H]MS) and the ability of muscarinic agonists to mediate a physiologic response was determined in intact heart cells cultured from chick embryos 10 d in ovo. The increase in K+ permeability and the decrease in beating rate mediated by the muscarinic agonist carbamylcholine were the responses studied. Exposure to 10(-3) M carbamylcholine caused a 15% decrease in beating rate and a 33% increase in the rate of 42K+ efflux from cells labeled to equilibrium. An assay for binding of [3H]MS to intact cells was developed. [3H]MS bound specifically to intact heart cells (185 fmol/mg protein) with a Kd of 0.48 nM. Exposure of cells for various times to 10(-3) M carbamylcholine followed by binding of [3H]MS to intact cells demonstrated that a gradual loss of 70% of [3H]MS binding sites took place over the next 6 h with a T 1/2 of 30 min. A decrease in the ability of carbamylcholine to stimulate K+ efflux and to decrease beating rate was observed after pre-exposure of cells to muscarinic agonists. A close correlation was found between the loss of the subclass of muscarinic receptors subject to agonist control and the loss of physiologic responsiveness after agonist exposure. The data suggest the absence of significant numbers of "spare" receptors within this group.     

Muscarinic receptors in pancreatic islets of the rat. Demonstration and dependence on long-term glucose environment

The presence of muscarinic receptors in islets of Langerhans was assessed by measurement of specific binding of [3H]methylscopolamine. Specific binding was defined as total binding minus binding obtained in the presence of 1000-fold or higher excess of unlabeled methylscopolamine. At 37 degrees C specific binding was significant after 1 min and plateaued after 10 min of incubation. Displacement of label by increasing concentrations of unlabeled methylscopolamine indicated a dissociation constant of 1.5 x 10(-12) M. Effects of methylscopolamine on insulin release were evaluated from the inhibitions of cholinergic-induced insulin release. 4 x 10(-10) M methylscopolamine inhibited acetylcholine (20 microM)-induced insulin release more than 60%. Binding was not influenced by the following variations during binding incubations: changing the glucose concentration from 0 to 8.3 mM, adding rotenon (1 microM) or omitting calcium from the incubation medium. Islets kept in tissue culture exhibited higher binding when cultured at 11.1 than at 3.3 mM glucose for 96 h. It is concluded that islets contain muscarinic receptors, the binding to which can be subject to alteration by the long-term glucose environment.     

The interaction of radioiodinated thyrotropin with plasma membranes: Evidence for high affinity binding sites in the thyroid

The binding of biologically active [¹²⁵I]thyrotropin to purified plasma membranes prepared from bovine thyroid glands was studied. At 4°C, specific binding reached a maximum after 2 h of incubation and a plateau was maintained for up to 20 h. Degradation of [¹²⁵I]thyrotropin was undetectable after 2 h of incubation and was only 10% of the total after 20 h.At pH 6.0, at which binding was maximal, a single class of binding sites, having a dissociation constant of approx. 25 nM, was evident. Dissociation studies revealed first order kinetics with a half-time of 2–3 min. At pH 7.5, binding curves were complex, suggesting two orders of binding sites with dissociation constants of approx. 200 nM and 80 pM. Further, at this pH, dissociation of the thyrotropin from its receptor was also complex, suggesting the presence of two first order reactions, one with a half-time similar to that seen at pH 6.0 and another with a half-time of 4 h. At both pH 6.0 and 7.5, insulin, glucagon, growth hormone, and prolactin were without effect on [¹²⁵I]thyrotropin binding.Similar high affinity and low affinity binding sites were seen with porcine thyroid membranes, but only low affinity sites were seen with either rat liver membranes or human cultured lymphocytes.     

Modulation of 5-Hydroxytryptamine1A Receptor Density by Nonhydrolyzable GTP Analogues

Co-incubation of rat cortical membranes with 10(-4) M GTP results in a competitive inhibition of 5-hydroxytryptamine1A (5-HT1A) receptor binding sites labeled by [3H]8-hydroxy-2-(di-n-propylamino)tetralin [( 3H]8-OH-DPAT). Preincubation of cortical membranes with 10(-4) M GTP does not significantly change either KD or Bmax values, indicating that the effect of GTP is reversible. By contrast, GTP gamma S and 5'-guanylylimidodiphosphate (GppNHp) are nonhydrolyzable analogues of GTP which lengthen the time course of guanine nucleotide activation of guanine nucleotide binding proteins (G proteins) and thereby alter G protein-receptor interactions. These nonhydrolyzable GTP analogues were used to characterize the effects of persistent alterations in G proteins on [3H]8-OH-DPAT binding to 5-HT1A receptors. Co-incubation of rat cortical membranes with either 10(-4) M GTP gamma S or GppNHp results in a decrease in both the affinity and apparent density of 5-HT1A binding sites. Co-incubation with the nonhydrolyzable nucleotides reduces the affinity of [3H]8-OH-DPAT binding by 65-70% and lowers the density of the binding site by 53-61%. Similarly, preincubation of membranes with a 10(-4) M concentration of either GTP gamma S or GppNHp significantly increases the KD value and reduces the Bmax value of [3H]8-OH-DPAT binding. These results indicate that GTP gamma S and GppNHp induce persistent changes in 5-HT1A receptor-G protein interactions that are reflected as a decrease in the density of binding sites labeled by [3H]8-OH-DPAT.     

Biochemical characterization of [3H]tryptamine binding sites from rat brain

Rat brain membranes were treated with different protein modifying reagents, all of which were able to reduce [3H]tryptamine binding. However, inactivation by N-ethylmaleimide and iodoacetamide only was counteracted by coincubation with tryptamine. Thus, the [3H]tryptamine binding molecule is a membrane protein with an essential sulfhydryl group at the binding site. After incubation of digitonin-solubilized membranes with seven different lectins, no precipitation of [3H]tryptamine binding sites was observed. On concanavalin A and wheat germ agglutinin affinity chromatography, no [3H]tryptamine binding activity was found to be specifically bound. Therefore, the [3H]tryptamine binding protein appears to be devoid of lectin binding carbohydrate residues.