Coexistence of γ-Aminobutyric Acid Type A and Type B Receptors in Testicular Interstitial Cells

The existence of specific gamma-aminobutyric acid (GABA)ergic receptors in testicular interstitial cells was investigated in the present study. Specific binding of [3H]GABA to interstitial cell membranes was found to be time- and temperature-dependent and varied according to Ca2+ concentration present in the incubation medium. We analyzed the ability of different GABAergic agonists and antagonists to displace the bound radioactivity. In the absence of Ca2+ (1 mM EDTA), GABA and the GABAergic agonist isoguvacine displaced the bound radioactivity. When the radioligand assay was performed in the presence of 2.5 mM CaCl2, the [3H]GABA specifically bound increased twofold. Under such conditions, the specific GABAergic agonist baclofen, as well as GABA and isoguvacine, displaced the [3H]GABA bound. Saturation analysis revealed the presence of a population of GABAA binding sites with a KD value of 45.2 nM and a maximal number of binding sites of 57.4 fmol/mg of protein. The maximal binding increased on addition of 2.5 mM CaCl2 to 102 fmol/mg of protein, indicating the existence of a second population of GABAergic receptors, i.e., type B, with essentially the same affinity. In addition, the incubation of testicular interstitial cells with GABA and baclofen resulted in an increase in androgen production. These results support a functional role of GABA in the neuroendocrine control of the male gonad.     

Buprenorphine: High-Affinity Binding to Dorsal Spinal Cord

The binding of the mixed opiate agonist-antagonist [3H]buprenorphine was compared with [3H]naloxone and [3H]dihydromorphine binding in membranes prepared from rat whole brain and dorsal spinal cord. Scatchard analysis of binding to whole brain yielded KD values close to 1.0 nM for all three 3H-ligands studied, although [3H]buprenorphine labelled five times as many binding sites. [3H]Naloxone and [3H]dihydromorphine bound to dorsal spinal cord with approximately the same affinity as to whole brain, although both 3H-ligands labelled fewer sites in the spinal cord. In contrast, Scatchard analysis of [3H]buprenorphine binding to spinal cord yielded curvilinear Scatchard plots, suggesting the presence of a very high-affinity (KD = 0.12 nM) binding site in addition to the high-affinity site (KD = 1.0 nM) present in the brain. Studies on the displacement of [3H]buprenorphine by opiates and D-Ala2,Met5-enkephalinamide supported the presence of two binding sites for this ligand in the spinal cord.     

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.     

[3H]8-Hydroxy-2-(Di-n-Propylamino)Tetralin Binding to Pre- and Postsynaptic 5-Hydroxytryptamine Sites in Various Regions of the Rat Brain

The specific binding of [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([ 3H]8-OH-DPAT) to 5-hydroxytryptamine (5-HT)-related sites was investigated in several regions of the rat brain. Marked differences were observed in the characteristics of binding to membranes from hippocampus, striatum, and cerebral cortex. Hippocampal sites exhibited the highest affinity (KD approximately 2 nM) followed by the cerebral cortex (KD approximately 6 nM) and the striatum (KD approximately 10 nM). Ascorbic acid inhibited specific [3H]8-OH-DPAT binding in all three regions but millimolar concentrations of Ca2+, Mg2+, and Mn2+ enhanced specific binding to hippocampal membranes, whereas only Mn2+ increased it in the cerebral cortex and all three cations inhibited specific binding to striatal membranes. Guanine nucleotides (0.1 mM GDP, GTP) inhibited binding to hippocampal and cortical membranes only. As intracerebral 5,7-dihydroxytryptamine markedly decreased [3H]8-OH-DPAT binding sites in the striatum, but not in the hippocampus, the striatal sites appear to be on serotoninergic afferent fibers. In contrast, in the hippocampus the sites appear to be on postsynaptic 5-HT target cells, as local injection of kainic acid decreased their density. Both types of sites appear to be present in the cerebral cortex. The postsynaptic hippocampal [3H]8-OH-DPAT binding sites are probably identical to the 5-HT1A subsites, but the relationship between the presynaptic binding sites and the presynaptic autoreceptors controlling 5-HT release deserves further investigation.     

Physicochemical Properties of Serotonin 5-HT3 Binding Sites Solubilized from Membranes of NG 108-15 Neuroblastoma-Glioma Cells

Specific binding sites with pharmacological properties typical of serotonin 5-HT3 receptors were identified in membranes of the murine hybridoma cell line NG 108-15, using [3H]zacopride as a ligand. Optimal solubilization of these sites (yield, 50%) could be achieved using the detergent 3-[3-(cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) at 24 mM plus 0.5 M NaCl in 25 mM Tris-HCl, pH 7.4. Specific [3H]zacopride binding to soluble sites in the 100,000-g CHAPS extract was saturable and showed characteristics (Bmax = 425 +/- 81 fmol/mg of protein; KD = 0.19 +/- 0.02 nM) closely related to those of membrane-bound sites (Bmax = 932 +/- 183 fmol/mg of protein; KD = 0.60 +/- 0.03 nM). Determination of association (k+1 = 0.17 nM min-1) and dissociation (k-1 = 0.02 min-1) rate constants for the soluble sites gave a KD value of 0.12 nM, a result consistent with that calculated from saturation studies. As assessed from the displacement potencies (IC50) of 10 different drugs, the pharmacological profile of [3H]zacopride specific binding sites was essentially the same (r = 0.99) in the CHAPS-soluble extract and in cell membranes, although some increase in the affinity for 5-HT3 antagonists (zacopride, ICS 205-930, and MDL 72222) and decrease in the affinity for 5-HT3 agonists (2-methyl-5-hydroxytryptamine and phenylbiguanide) were noted for the soluble sites. Sucrose density gradient sedimentation of the CHAPS-soluble extract gave a Svedberg coefficient of 12S for the material with [3H]zacopride specific binding capacity. Chromatographic analyses using Sephacryl S-400 and wheat germ agglutinin-agarose columns indicated marked enrichment (by 2.5- and 10-fold, respectively) in [3H]zacopride specific binding activity in the corresponding eluates compared with the starting soluble extract, a finding suggesting that both steps are of potential interest for the partial purification of solubilized 5-HT3 receptors. Two soluble materials with apparent molecular masses of approximately 600 and approximately 36 kDa were found to bind [3H]zacopride specifically in the Sephacryl S-400 eluate. Interestingly, molecular mass determination by radiation inactivation of [3H]zacopride binding sites in frozen NG 108-15 cells gave a value of approximately 35 kDa.     

Binding of [3H]batrachotoxinin A-20-alpha-benzoate to a high affinity site associated with house fly head membranes

1. [3H]Batrachotoxinin A-20-alpha-benzoate (BTX-B), a radioligand that labels the alkaloid activator recognition site of the voltage-sensitive sodium channel, was bound specifically to high affinity, saturable sites in a subcellular preparation from house fly (Musca domestica L.) heads that was shown previously to contain binding sites for other sodium channel-directed ligands. 2. Specific binding of [3H]BTX-B was observed in the presence of 140 mM sodium or potassium and was inhibited by choline ion. 3. Saturating concentrations of scorpion (Leiurus quinquestriatus) venom stimulated the specific binding of [3H]BTX-B four-fold, increasing the proportion of specific binding of 10 nM [3H]BTX-B from less than 15% to 40%. Equilibrium dissociation studies in the presence of scorpion venom gave an equilibrium dissociation constant (KD) for [3H]BTX-B of 80 nM and a maximal binding capacity (Bmax) of 1.5 pmol/mg protein. 4. Parallel experiments in the absence of venom gave a KD value of 140 nM and a Bmax of 1.3 pmol/mg protein, indicating that scorpion venom stimulated [3H]BTX-B binding by increasing the affinity of this site approximately two-fold. 5. The specific binding of [3H]BTX-B was inhibited by the sodium channel activators aconitine and batrachotoxin and, to a lesser extent, by the anticonvulsant diphenylhydantoin. However, several other sodium channel-directed neurotoxins known to exert allosteric effects on the binding of [3H]BTX-B to mammalian brain preparations did not affect the binding of [3H]BTX-B to house fly head membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Solubilized Rat Brain Adenosine Receptors Have Two High-Affinity Binding Sites for l, 3-Dipropyl-8-Cyclopentylxanthine

The specific binding of L-N6-[3H]phenylisopropyladenosine (L-[3H]PIA) to solubilized receptors from rat brain membranes was studied. The interaction of these receptors with relatively low concentrations of L-[3H]PIA (0.5-12.0 nM) in the presence of Mg2+ showed the existence of two binding sites for this agonist, with respective dissociation constant (KD) values of 0.24 and 3.56 nM and respective receptor number (Bmax) values of 0.28 +/- 0.03 and 0.66 +/- 0.05 pmol/mg of protein. In the presence of GTP, the binding of L-[3H]PIA also showed two sites with KD values of 24.7 and 811.5 nM and Bmax values of 0.27 +/- 0.09 and 0.93 +/- 0.28 pmol/mg of protein for the first and the second binding site, respectively. Inhibition of specific L-[3H]PIA binding by 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) (0.1-300 nM) performed with the same preparations revealed two DPCPX binding sites with Ki values of 0.29 and 13.5 nM, respectively. [3H]DPCPX saturation binding experiments also showed two binding sites with respective KD values of 0.81 and 10.7 nM and respective Bmax values of 0.19 +/- 0.02 and 0.74 +/- 0.06 pmol/mg of protein. The results suggest that solubilized membranes from rat brain possess two adenosine receptor subtypes: one of high affinity with characteristics of the A1 subtype and another with lower affinity with characteristics of the A3 subtype of adenosine receptor.     

Characterization of Two Classes of Opioid Binding Sites in Drosophila melanogaster Head Membranes

Opioid receptors have been characterized in Drosophila neural tissue. [3H]Etorphine (universal opioid ligand) bound stereospecifically, saturably, and with high affinity (KD = 8.8 +/- 1.7 nM; Bmax = 2.3 +/- 0.2 pmol/mg of protein) to Drosophila head membranes. Binding analyses with more specific ligands showed the presence of two distinct opioid sites in this tissue. One site was labeled by [3H]dihydromorphine ([3H]DHM), a mu-selective ligand: KD = 150 +/- 34 nM; Bmax = 3.0 +/- 0.6 pmol/mg of protein. Trypsin or heat treatment (100 degrees C for 15 min) of the Drosophila extract reduced specific [3H]DHM binding by greater than 80%. The rank order of potency of drugs at this site was levorphanol greater than DHM greater than normorphine greater than naloxone much greater than dextrorphan; the mu-specific peptide [D-Ala2,Gly-ol5]-enkephalin and delta-, kappa-, and sigma-ligands were inactive at this site. The other site was labeled by (-)-[3H]ethylketocyclazocine ((-)-[3H]EKC), a kappa-opioid, which bound stereospecifically, saturably, and with relatively high affinity to an apparent single class of receptors (KD = 212 +/- 25 nM; Bmax = 1.9 +/- 0.2 pmol/mg of protein). (-)-[3H]EKC binding could be displaced by kappa-opioids but not by mu-, delta-, or sigma-opioids or by the kappa-peptide dynorphin. Specific binding constituted approximately 70% of total binding at 1 nM and approximately 50% at 800 nM for all three radioligands ([3H]etorphine, [3H]EKC, and [3H]DHM). Specific binding of the delta-ligands [3H][D-Ala2,D-Leu5]-enkephalin and [3H][D-Pen2,D-Pen5]-enkephalin was undetectable in this preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of Neurotensin Binding Sites in Intact and Solubilized Bovine Brain Membranes

Analysis of the equilibrium binding of [3H]-neurotensin(1-13) at 25 degrees C to its receptor sites in bovine cortex membranes indicated a single population of sites with an apparent equilibrium dissociation constant (KD) of 3.3 nM and a density (Bmax) of 350 fmol/mg protein (Hill coefficient nH = 0.97). Kinetic dissociation studies revealed the presence of a second class of sites comprising less than 10% of the total. KD values of 0.3 and 2.0 nM were obtained for the higher and lower affinity classes of sites, respectively, from association-dissociation kinetic studies. The binding of [3H]neurotensin was decreased by cations (monovalent and divalent) and by a nonhydrolysable guanine nucleotide analogue. Competition studies gave a potency ranking of [Gln4]neurotensin greater than neurotensin(8-13) greater than neurotensin(1-13). Smaller neurotensin analogues and neurotensin-like peptides were unable to compete with [3H]neurotensin. Stable binding activity for [3H]neurotensin in detergent solution (Kd = 5.5 nM, Bmax = 250 fmol/mg protein, nH = 1.0) was obtained in 2% digitonin/1 mM Mg2+ extracts of membranes which had been preincubated (25 degrees C, 1 h) with 1 mM Mg2+ prior to solubilization. Association-dissociation kinetic studies then revealed the presence of two classes of sites (KD1 = 0.5 nM, KD2 = 3.6 nM) in a similar proportion to that found in the membranes. The solubilized [3H]-neurotensin activity retained its sensitivity to cations and guanine nucleotide.     

A Trifluoromethylphenyl Piperazine Derivative with High Affinity for 5-Hydroxytryptamine-1A Sites in Rat Brain

The inhibition of [3H]5-hydroxytryptamine [( 3H]5-HT) binding in rat brain by 1-[2-(3-bromoacetamidophenyl)ethyl]-4-(3-trifluoromethylphenyl) piperazine (BrAcTFMPP) and that by spiperone were compared. Spiperone inhibition of [3H]5-HT binding in cortex was consistent with displacement from two sites with dissociation constants (KD) of 24 nM (5-HT-1A site) and 19 microM (5-HT-1B site) for spiperone. BrAcTFMPP also discriminated two subpopulations of [3H]5-HT binding sites with dissociation constants of 0.5 nM and 146 nM for the compound. The proportion of high-affinity sites for each compound represented about 35% of the specific [3H]5-HT binding. In the presence of 1 microM spiperone, a concentration that saturates the 5-HT-1A sites while having a minimal effect on 5-HT-1B sites, BrAcTFMPP displaced [3H]5-HT from a single site with a KD for BrAcTFMPP of 145 nM. The inhibition of [3H]5-HT binding by spiperone in the presence of 30 nM BrAcTFMPP was best fit by a single-site model with a KD of 21 microM for spiperone. In corpus striatum, 5-HT-1A sites, as defined with spiperone, represented 15% of the specific [3H]5-HT binding and 30 nM BrAcTFMPP also blocked about 15% of the binding. A significant difference between spiperone and BrAcTFMPP was their affinity for 5-HT-2 receptors. BrAcTFMPP (KD = 41 nM) had an 80-fold lower affinity for these sites than spiperone (KD = 0.5 nM). Thus, BrAcTFMPP and spiperone discriminate the same two subpopulations of [3H]5-HT binding sites and BrAcTFMPP displays a high affinity and a selectivity for 5-HT-1A sites versus both 5-HT-1B and 5-HT-2 sites.     

Cooperative Modulation of [3H]MK-801 Binding to the N-Methyl-d-Aspartate Receptor-Ion Channel Complex by l-Glutamate, Glycine, and Polyamines

In extensively washed rat cortical membranes [3H](+)-5-methyl-10,11-dihydro-5 H-dibenzo [a,d]cyclohepten-5,10-imine ([3H]MK-801) labeled a homogeneous set of sites (Bmax = 1.86 pmol/mg protein) with relatively low affinity (KD = 45 nM). L-Glutamate, glycine, and spermidine produced concentration-dependent increases in specific [3H]MK-801 binding due to a reduction in the KD of the radioligand. In the presence of high concentrations of L-glutamate, glycine, or spermidine, the KD values for [3H]MK-801 were reduced to 11 nM, 18 nM, and 15 nM, respectively. Maximally effective concentrations of combinations of the three compounds further increased [3H]MK-801 binding affinity as follows: L-glutamate + glycine, KD = 6.2 nM; L-glutamate + spermidine, KD = 2.2 nM; glycine + spermidine, KD = 8.3 nM. High concentrations of spermidine did not inhibit either [3H]glycine orf [3H]3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid binding to the N-methyl-D-aspartate (NMDA) receptor complex. The concentration of L-glutamate required to produce half-maximal enhancement (EC50) of [3H]MK-801 binding was reduced from 218 nM to 52 nM in the presence of 30 microM glycine and to 41 nM in the presence of 50 microM spermidine. The EC50 value for glycine enhancement of [3H]MK-801 binding was 184 nM. This was lowered to 47 nM in the presence of L-glutamate and to 59 nM in the presence of spermidine. Spermidine enhanced [3H]MK-801 binding with an EC50 value of 19.4 microM which was significantly reduced by high concentrations of L-glutamate (EC50 = 3.9 microM) or glycine (EC50 = 6.2 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of trypsin on binding of [3H]epinephrine and [3H]-dihydroergocryptine to rat liver plasma membranes. Evidence for interconversion of binding sites

Treatment of liver plasma membranes with trypsin at low concentrations (1 to 2 microgram/mg of protein) caused at 3- to 4-fold increase in alpha-specific [3H]epinephrine binding. The change was due to an increase in the number of high affinity binding sites, with no change in the dissociation constant. With increasing trypsin concentrations, the dissociation constant was decreased and there was a progressive loss of binding. Elastase, papain, and thermolysin caused similar effects, whereas the thrombin, leucine aminopeptidase, phospholipase A2, phospholipase C, phospholipase D, and detergents did not cause an increase in [EH]epinephrine binding. The increase in epinephrine high affinity binding sites was correlated with a loss of high affinity [3H]-dihydroergocryptine binding sites which also bind [3H]epinephrine with low affinity (El-Refai, M. F., Blackmore, P. F., and Exton, J. H. (1979) J. Biol. Chem. 254, 4375-4386). Incubation of membranes with the alpha blockers dihydroergocryptine (50 nM) and phenoxybenzamine (20 nM) prior to protease treatment diminished the increase in [3H]epinephrine binding induced by trypsin (1.5 microgram/mg). The concentration dependence and time course of trypsin actions on 70 nM [3H]epinephrine binding and 10 nM [3H]dihydroergocryptine binding are consistent with a trypsin-mediated conversion of low affinity epinephrine binding sites to high affinity epinephrine binding sites.     

Opiate binding sites and endogenous opioids in Bufo viridis oocytes

Binding sites with high affinity for [3H]naloxone, but not for [3H]morphine and [3H] (D-Ala2, D-Leu5) enkephalin, have been found in membranes of Bufo viridis oocytes. The binding is reversible and saturable. Bound [3H]naloxone is easily displaced both by unlabeled naloxone and bremazocine, much worse by morphine and SKF 10,047; (D-Ala2, D-Leu5) enkephalin and beta-endorphin practically fail to displace [3H]naloxone. Scatchard analysis is consistent with the existence of two classes of binding sites with Kd 15 nM and 10(3) nM. The number of binding sites with high affinity for naloxone is 16 pmol/mg protein of homogenized oocytes which is 20-50-fold higher than in, toad or rat brain. Oocyte extract displaces [3H]naloxone bound with oocytes' membranes and inhibits electrically evoked contractions of the rabbit vas deferens. This inhibition is reversed by naloxone. It is suggested that compounds similar to opiate kappa-agonists exist in oocytes. It cannot be ruled out that they participate via specific receptors in the regulation of oocyte maturation and egg development.     

Localization and Characterization of Binding Sites with High Affinity for [3H]Ouabain in Cerebral Cortex of Rabbit Brain Using Quantitative Autoradiography

[3H]Ouabain binding was studied in sections of rabbit somatosensory cortex by quantitative autoradiography and in rabbit brain microsomal membranes using a conventional filtration assay. KD values of 8-12 nM for specific high-affinity binding of [3H]ouabain were found by both methods. High-affinity binding was not uniformly distributed in somatosensory cortex and was localized predominantly to laminae 1, 3, and 4. [3H]Ouabain binding in tissue sections was stimulated by the ligands Mg2+/Pi or Mg2+/ATP/Na+ and was inhibited by K+ (IC50 = 0.7-0.9 mM), N-ethylmaleimide, 5,5'-dithiobis(2-nitrobenzoic acid), and erythrosin B. We conclude that [3H]ouabain is reversibly and specifically bound with high affinity in rabbit brain tissue sections under conditions that favor phosphorylation of Na+,K+-ATPase. Quantitative autoradiography is a powerful tool for assessing the affinity and number of specific ouabain binding sites in brain tissue.     

Characterization of K Opioid Receptors in Neurosecretosomes from Bovine Posterior Pituitary

The binding properties of opioid receptors on isolated nerve terminals (neurosecretosomes) from bovine posterior pituitaries were characterized. Both [3H]etorphine and [3H]ethylketocyclazocine ([3H]EKC) showed high-affinity binding with complex binding isotherms, consistent with the presence of multiple classes of binding sites. [D-Ala2,D-Leu5]enkephalin showed no specific binding and failed to displace [3H]etorphine at high concentrations, indicating the absence of mu, delta, or benzomorphan (kappa 2) sites. Mathematical modelling of the data suggested the presence of three classes of binding sites. The first was of high affinity with Kd values of 0.9 and 2.0 nM for etorphine and EKC, respectively. The second class of sites appeared to bind etorphine with a KD of 150 nM, and EKC with extremely low affinity (unmeasurable binding). The third class of sites was characterized by KD values of 7 and 2 microM for etorphine and EKC, respectively. These results indicate that the nerve terminals of bovine posterior pituitary contain opioid binding sites of the kappa type. Furthermore, these binding sites appear heterogeneous, consisting of at least two and possibly more subtypes or states.     

Effect of bradykinin on Na-K-2Cl cotransport and bumetanide binding in aortic endothelial cells

Simultaneous measurements of potassium influx and binding of [3H]bumetanide were performed in endothelial cells cultured from bovine aortas to determine how bradykinin regulates Na-K-2Cl cotransport. [3H]Bumetanide displayed saturable binding and was displaced by low concentrations of unlabeled bumetanide. All three transported ions were required for binding and high concentrations of chloride inhibited binding, consistent with binding of bumetanide to the second chloride site of the transporter. Scatchard analysis of binding under maximal conditions (100 mM sodium, 30 mM potassium, 30 mM chloride) revealed a single class of binding sites with a binding constant of 112 nM and a density of 22 fmol/cm2 or approximately 122,000 sites/cells. Na-K-2Cl cotransport, measured as bumetanide-sensitive potassium influx, was stimulated 118 +/- 30% by bradykinin (p less than 0.01) at physiologic ion concentrations. Stimulation was inhibited by increased potassium or decreased external chloride concentrations and was not seen in conditions required for maximal binding of bumetanide. Simultaneous measurement of the binding of tracer [3H]bumetanide and its inhibition of potassium influx in medium containing 10 mM potassium and 130 mM chloride revealed a turnover number for the cotransporter of 293 +/- 68 s-1 which increased to 687 +/- 105 s-1 with bradykinin (p less than 0.001). There was no change in cell volume and only a 5.6 mM increase in intracellular sodium concentration associated with this stimulation. Bradykinin also increased the affinity of the cotransporter for bumetanide as indicated by a decrease in the Ki for potassium influx from 464 +/- 46 nM to 219 +/- 19 nM (p less than 0.005). Our results show that [3H]bumetanide can be used to quantitate Na-K-2Cl cotransporter sites in aortic endothelial cells and to determine the mechanism by which cotransport is regulated. The stimulation of cotransport in aortic endothelial cells by bradykinin is due to an increase in the activity of existing transporters rather than to an increase in the number of transporters. This, together with the increased affinity for bumetanide, strongly suggests that a change in cotransporter structure is occurring in response to bradykinin.     

α-[3H]Amino-3-Hydroxy-5-Methylisoxazole-4-Propionic Acid Binding to Human Cerebral Cortical Membranes: Minimal Changes in Postmortem Brains of Chronic Schizophrenics

The binding of alpha-[3H]amino-3-hydroxy-5-methylisoxazole-4-propionic acid ([3H]AMPA), a selective ligand for the ion channel-linked quisqualate receptor, was evaluated in Triton X-100-treated membranes of human cerebral cortex. The presence of chaotropic ions produced divergent effects on specific [3H]AMPA binding: A twofold increase in the binding was observed with thiocyanide at 100 mM, although iodide (100 mM) and perchlorate (100 mM) reduced the binding. Chemical modifications of the sulfhydryl group with p-chloromercuriphenylsulfonic acid (PCMBS) produced threefold increases in specific [3H]-AMPA binding in the absence of KSCN as well as in the presence of KSCN. Treatment with dithiothreitol restored the enhanced specific [3H]AMPA binding by PCMBS to the basal level. Although specific [3H]AMPA binding in the absence of KSCN showed a single site (KD = 220 nM, Bmax = 235 fmol/mg of protein), curvilinear Scatchard plots of specific [3H]AMPA binding in the presence of 100 mM KSCN can be resolved into two binding sites with the following parameters: KD1 = 5.82 nM, Bmax1 = 247 fmol/mg of protein; KD2 = 214 nM, Bmax2 = 424 fmol/mg of protein. Quisqualate and AMPA were the most potent inhibitors of the [3H]AMPA binding in the presence of KSCN. Potent inhibitors of the binding included beta-N-oxalylamino-L-alanine (L-BOAA), cysteine-S-sulfate, L-glutamate, 6-cyano-7-nitroquinoxaline-2,3-dione, and 6,7-dinitroquinoxaline-2,3-dione. Kainate, L-homocysteine sulfinic acid, and L-homocysteic acid were active with an IC50 value of a micromolar concentration, whereas L-cysteic acid and L-cysteine sulfinic acid were weakly active.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a thyroid-hormone-binding site on nuclear envelopes and nuclear matrices of the male-rat liver

Nuclear envelopes and nuclear matrices were isolated from the male-rat liver. Incubation of 125I-labelled 3,3',5-tri-iodothyronine (T3) with the nuclear-envelope fraction resulted in specific binding of T3 to the membranes. Maximum specific binding occurred at 30 degrees C after 2h incubation. Storage for 1 week at -80 degrees C resulted in no loss of binding. Scatchard analysis revealed a class of binding sites with KD 86 nM. 3,3',5'-Tri-iodothyronine was as effective a competitor of [125I]T3 binding to nuclear envelopes as was L-T3 itself, and tri-iodothyroacetic acid was 70% as potent as T3. L- and D-thyronine did not compete for [125I]T3 binding. Incubation of nuclear envelopes with 0.6 M-NaCl before addition of T3 resulted in the complete loss of specific binding sites, whereas exposure of the membranes to 2.0 M-NaCl after incubation with T3 did not extract binding sites. Nuclear matrices, after incubation with [125I]T3 under the same conditions, were shown to possess a class of binding sites with a similar KD but with approx. 30% of the maximum binding capacity. Nuclear envelopes from hypothyroid animals may possess slightly lower numbers of binding sites compared with nuclear envelopes from the intact animal, whereas nuclear matrices from hypothyroid animals have the same number of binding sites as do nuclear envelopes from the intact animal. In conclusion, nuclear envelopes and nuclear matrices have a class of binding sites with relatively high affinity for T3. It is distinct from nuclear and cytosolic binding sites.     

Presynaptic Cholinergic Mechanisms in the Rat Cerebellum: Evidence for Nicotinic, but Not Muscarinic Autoreceptors

The present study shows that N-[3H]methylcarbamylcholine ([3H]MCC) binds to a single population of high-affinity/low-density (KD = 5.0 nM; Bmax = 8.2 fmol/mg of protein) nicotinic binding sites in the rat cerebellum. Also, there exists a single class of high-affinity binding sites (KD = 4.8 nM; Bmax = 24.2 fmol/mg of protein) in the cerebellum for the M1 specific muscarinic ligand [3H]pirenzepine. In contrast, the M2 ligand, [3H]AF-DX 116, appears to bind to two classes of binding sites, i.e., a high-affinity (KD = 3 nM)/low-capacity (Bmax = 11.7 fmol/mg of protein) class, and a second class of lower affinity (KD = 28.4 nM) and higher capacity (Bmax = 36.3 fmol/mg of protein) sites. The putative M3 selective ligand [3H]4-diphenylacetoxy-N-methylpiperidine also binds to two distinct classes of binding sites in cerebellar homogenates, one of high affinity (KD = 0.5 nM)/low capacity (Bmax = 19.5 fmol/mg of protein) and one of low affinity (KD = 57.5 nM)/high capacity (Bmax = 140.6 fmol/mg of protein). In experiments which tested the effects of cholinergic drugs on acetylcholine release from cerebellar brain slices, the nicotinic agonist MCC enhanced spontaneous acetylcholine release in a concentration-dependent manner, and the maximal increase in acetylcholine release (59.0-68.0%) occurred at 10(-7) M. The effect of MCC to increase acetylcholine release was Ca2+-dependent and tetrodotoxin-insensitive, suggesting an action on cholinergic terminals. Also, the MCC-induced increase in acetylcholine release was effectively antagonized by dihydro-beta-erythroidine, d-tubocurarine, and kappa-bungarotoxin, but was insensitive to either atropine or alpha-bungarotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)