Endogenous inhibitor of GABAB and GABAA receptors

An endogenous inhibitor of γ-aminobutyric acid (GABA) receptors was partially purified from bovine brain striatum. It was obtained as a low molecular weight fraction by gel filtration on Biogel P-2 and was adsorbed to Dowex AG 50W-X8, but not to Dowex AG 1-X8. It was ninhydrin-negative, basic, heat-stable substance. It caused dose-dependent inhibition of Na⁺-independent [³H]GABA bindings. Scatchard plot analysis of the [³H]GABA binding to GABA “B” receptor recognition site showed this inhibitor increased the K_d value (24.1 nM to 3.6 nM) without changing the B_max. On the other hand, Scatchard plot analysis of the [³H]GABA binding to GABA “A” receptor recognition site showed that the inhibitor decreased number of binding sites (706 fmol/mg protein to 494 fmol/mg protein) without affecting the K_d value. These results suggest that the endogenous inhibitor functions as a modulator for GABA_B and GABA_A receptors.     

Colocalization of the (Pro)renin Receptor/Atp6ap2 with H+-ATPases in Mouse Kidney but Prorenin Does Not Acutely Regulate Intercalated Cell H+-ATPase Activity

The (Pro)renin receptor (P)RR/Atp6ap2 is a cell surface protein capable of binding and non-proteolytically activate prorenin. Additionally, (P)RR is associated with H⁺-ATPases and alternative functions in H⁺-ATPase regulation as well as in Wnt signalling have been reported. Kidneys express very high levels of H⁺-ATPases which are involved in multiple functions such as endocytosis, membrane protein recycling as well as urinary acidification, bicarbonate reabsorption, and salt absorption. Here, we wanted to localize the (P)RR/Atp6ap2 along the murine nephron, exmaine whether the (P)RR/Atp6ap2 is coregulated with other H⁺-ATPase subunits, and whether acute stimulation of the (P)RR/Atp6ap2 with prorenin regulates H⁺-ATPase activity in intercalated cells in freshly isolated collecting ducts. We localized (P)PR/Atp6ap2 along the murine nephron by qPCR and immunohistochemistry. (P)RR/Atp6ap2 mRNA was detected in all nephron segments with highest levels in the collecting system coinciding with H⁺-ATPases. Further experiments demonstrated expression at the brush border membrane of proximal tubules and in all types of intercalated cells colocalizing with H⁺-ATPases. In mice treated with NH₄Cl, NaHCO₃, KHCO₃, NaCl, or the mineralocorticoid DOCA for 7 days, (P)RR/Atp6ap2 and H⁺-ATPase subunits were regulated but not co-regulated at protein and mRNA levels. Immunolocalization in kidneys from control, NH₄Cl or NaHCO₃ treated mice demonstrated always colocalization of PRR/Atp6ap2 with H⁺-ATPase subunits at the brush border membrane of proximal tubules, the apical pole of type A intercalated cells, and at basolateral and/or apical membranes of non-type A intercalated cells. Microperfusion of isolated cortical collecting ducts and luminal application of prorenin did not acutely stimulate H⁺-ATPase activity. However, incubation of isolated collecting ducts with prorenin non-significantly increased ERK1/2 phosphorylation. Our results suggest that the PRR/Atp6ap2 may form a complex with H⁺-ATPases in proximal tubule and intercalated cells but that prorenin has no acute effect on H⁺-ATPase activity in intercalated cells.     

Interaction of the synthetic immunomodulatory dipeptide bestim with murine macrophages and thymocytes

The tritium-labeled dipeptide bestim (γ-D-Glu-L-Trp) with a specific activity of 45 Ci/mmol was obtained by high-temperature solid-state catalytic isotope exchange. It was found that [³H]bestim binds with a high affinity to murine peritoneal macrophages (K _d 2.1 ± 0.1 nM) and thymocytes (K _d 3.1 ± 0.2 nM), as well as with plasma membranes isolated from these cells (K _d 18.6 ± 0.2 and 16.7 ± 0.3 nM, respectively). The specific binding of [³H]bestim to macrophages and thymocytes was inhibited by the unlabeled dipeptide thymogen (L-Glu-L-Trp) (K _i 0.9 ± 0.1 and 1.1 ± 0.1 nM, respectively). After treatment with trypsin, macrophages and thymocytes lost the ability to bind [³H]bestim. Bestim in the concentration range of 10^?10 to 10^?6 M reduced the adenylate cyclase activity in the membranes of murine macrophages and thymocytes.     

Magnesium reduces affinities of antagonists at rat cortex α2-adrenergic receptors labeled with 3H-clonidine: Evidence for heterogeneity of α2-receptor conformations with respect to antagonists

³H-clonidine labeled two binding sites in rat cortex membranes with apparent K_D values of about 1.0 and 5.9 nM. These sites appeared analogous to “super-high” (SH) and “high” (H) affinity states of the α₂-receptor described in human platelets. 10 mM magnesium increased the number of SH receptors by 30% whereas 100 μM GTP reduced SH₃receptor number by 45% with no significant change in the K_D of ³H-clonidine at α₂(SH) sites. In drug competition studies using 1.0 nM ³H-clonidine, 100 μM GTP reduced the affinity of clonidine and increased the affinity of yohimbine, whereas 10 mM magnesium increased the affinity of clonidine and reduced the affinity of yohimbine. The effect of magnesium on the affinity of several antagonists at cortex ³H-clonidine sites ranged from none (phentolamine) to a 6-fold reduction (piperoxan). These data indicate that different states of the α₂-receptor exhibit different affinities for some antagonists.     

Specific binding of [3H]nitrendipine to membranes from coronary arteries and heart in relation to pharmacological effects. Paradoxical stimulation by diltiazem

High affinity binding sites for the calcium channel inhibitor [³H]nitrendipine have been identified in microsomes from pig coronary arteries (K_D=1.6 nM; B_max=35 fmol/mg) and in purified sarcolemma from dog heart (K_D=0.11 nM; B_max=230 fmol/mg). [³H]nitrendipine binding to coronary artery microsomes was completely inhibited by nifedipine, partially by verapamil and D600 and, surprisingly, was stimulated by d-cis-diltiazem but not by 1-cis-diltiazem, a less active isomer. Half-maximal relaxation of KCl-depolarized coronary rings occurred in a slow process at 1 nM nitrendipine or 100 nM d-cis-diltiazem. In dog trabecular strips, nitrendipine caused a negative inotropic response (ED₅₀=1μM). These results suggest that there may be multiple binding sites for different “subclasses” of calcium channel inhibitors, and that drug binding sites may be different molecular entities from the putative calcium channels.     

Bovine ovarian cells have (pro)renin receptors and prorenin induces resumption of meiosis in vitro

The discovery of a receptor that binds prorenin and renin in human endothelial and mesangial cells highlights the possible effect of renin-independent prorenin in the resumption of meiosis in oocytes that was postulated in the 1980s.This study aimed to identify the (pro)renin receptor in the ovary and to assess the effect of prorenin on meiotic resumption. The (pro)renin receptor protein was detected in bovine cumulus-oocyte complexes, theca cells, granulosa cells, and in the corpus luteum. Abundant (pro)renin receptor messenger ribonucleic acid (mRNA) was detected in the oocytes and cumulus cells, while prorenin mRNA was identified in the cumulus cells only. Prorenin at concentrations of 10⁻¹⁰, 10⁻⁹, and 10⁻⁸ M incubated with oocytes co-cultured with follicular hemisections for 15 h caused the resumption of oocyte meiosis. Aliskiren, which inhibits free renin and receptor-bound renin/prorenin, at concentrations of 10⁻⁷, 10⁻⁵, and 10⁻³ M blocked this effect (P < 0.05). To determine the involvement of angiotensin II in prorenin-induced meiosis resumption, cumulus-oocyte complexes and follicular hemisections were treated with prorenin and with angiotensin II or saralasin (angiotensin II antagonist). Prorenin induced the resumption of meiosis independently of angiotensin II. Furthermore, cumulus-oocyte complexes cultured with forskolin (200 μM) and treated with prorenin and aliskiren did not exhibit a prorenin-induced resumption of meiosis (P < 0.05). Only the oocytes’ cyclic adenosine monophosphate levels seemed to be regulated by prorenin and/or forskolin treatment after incubation for 6 h. To the best of our knowledge, this is the first study to identify the (pro)renin receptor in ovarian cells and to demonstrate the independent role of prorenin in the resumption of oocyte meiosis in cattle.     

Characterization of recombinant human renin: Kinetics,pH-stability,and peptidomimetic inhibitor binding

The kinetic behavior andpH-stability of recombinant human renin was analyzed using a new fluorogenic substrate based on the normal P₆-P_3′ renin cleavage sequence in human angiotensinogen. The design of this fluorogenic substrate makes possible, for the first time, direct monitoring of the kinetics of proteolytic conversion of prorenin to renin. ThepH-stability profile for renin, measured with the substrate at 25°C, indicated a broad plateau of stability betweenpH 6.0 and 10.0. Analysis of thepH-activity profile of renin for the substrate indicated a minimumK _m (～1.8 µM) atpH ～7.4 and a maximumV _m betweenpH 7.4 and 8.0. The thermodynamics of the binding of a novel, soluble, peptidomimetic inhibitor to renin indicated it is possible to retain the tight-binding characteristics and enthalpy contributions to binding of larger peptide-derived inhibitors, while reducing inhibitor size and entropic contributions to binding. A novel derivative of the fluorogenic substrate, containing a 3-methyl histidine substitution at the P₂ site, was used to test the recent hypothesis that renin functions by virtue of substrate-directed catalysis.     

A new form of renin in normal human plasma: “big renin” is a mixture of inactive prorenin and the new active high molecular weight renin

A new form of active renin was separated from inactive prorenin in normal human plasma by a new affinity chromatographic method on a column of Cibacron Blue F3GA-agarose. This active renin has a molecular weight of 54,000, considerably higher than the hitherto recognized active renin of 40,000 dalton in human plasma. The molecular weight of inactive prorenin was 56,000±2,000. Active renin produced from the inactive prorenin by trypsin or pepsin digestion or by acid treatment in $\text{in vitro}$ experiments showed a molecular weight of 54,000±2,000. Active renin with a molecular weight of 40,000 was not found in 6 samples of untreated plasma of normal human subjects nor was it formed by treatment with trypsin, pepsin, or acid pH. It is concluded that a large form of active renin (54,000 dalton) exists in normal human plasma which is distinct from a smaller form and that the activatable “big renin” is a mixture of this active renin and totally inactive prorenin. This explains the absence of molecular weight change during the activation of “big renin”.     

The platelet P2Y12 receptor under normal and pathological conditions. Assessment with the radiolabeled selective antagonist [3H]PSB-0413

Various radioligands have been used to characterize and quantify the platelet P2Y₁₂ receptor, which share several weaknesses: (a) they are metabolically unstable and substrates for ectoenzymes, (b) they are agonists, and (c) they do not discriminate between P2Y₁ and P2Y₁₂. We used the [³H]PSB-0413 selective P2Y₁₂ receptor antagonist radioligand to reevaluate the number of P2Y₁₂ receptors in intact platelets and in membrane preparations. Studies in humans showed that: (1) [³H]PSB-0413 bound to 425 ± 50 sites/platelet (K_D = 3.3 ± 0.6 nM), (2) 0.5 ± 0.2 pmol [³H]PSB-0413 bound to 1 mg protein of platelet membranes (K_D = 6.5 ± 3.6 nM), and (3) competition studies confirmed the known features of P2Y₁₂, with the expected rank order of potency: AR-C69931MX > 2MeSADP ≫ ADPβS > ADP, while the P2Y₁ ligand MRS2179 and the P2X₁ ligand α,β-Met-ATP did not displace [³H]PSB-0413 binding. Patients with severe P2Y₁₂ deficiency displayed virtually no binding of [³H]PSB-0413 to intact platelets, while a patient with a dysfunctional P2Y₁₂ receptor had normal binding. Studies in mice showed that: (1) [³H]PSB-0413 bound to 634 ± 87 sites/platelet (K_D = 14 ± 4.5 nM) and (2) 0.7 pmol ± 0.3 [³H]PSB-0413 bound to 1 mg protein of platelet membranes (K_D = 9.1 ± 5.3 nM). Clopidogrel and other thiol reagents like pCMBS or DTT abolished the binding both to intact platelets and membrane preparations. Therefore, [³H]PSB-0413 is an accurate and selective tool for radioligand binding studies aimed at quantifying P2Y₁₂ receptors, to identify patients with P2Y₁₂ deficiencies or quantify the effect of P2Y₁₂ targeting drugs.     

Clinical mutants of human glucose 6-phosphate dehydrogenase: Impairment of NADP+ binding affects both folding and stability

Human glucose 6-phosphate dehydrogenase (G6PD) has both the “catalytic” NADP⁺ site and a “structural” NADP⁺ site where a number of severe G6PD deficiency mutations are located. Two pairs of G6PD clinical mutants, G6PD_Wisconsin (R393G) and G6PD_Nashville (R393H), and G6PD_Fukaya (G488S) and G6PD_Campinas (G488V), in which the mutations are in the vicinity of the “structural” NADP⁺ site, showed elevated K_d values of the “structural” NADP⁺, ranging from 53 nM to 500 nM compared with 37 nM for the wild-type enzyme. These recombinant enzymes were denatured by Gdn-HCl and refolded by rapid dilution in the presence of l-Arg, NADP⁺ and DTT at 25 °C. The refolding yields of the mutants exhibited strong NADP⁺-dependence and ranged from 1.5% to 59.4% with 1000 μM NADP⁺, in all cases lower than the figure of 72% for the wild-type enzyme. These mutant enzymes also displayed decreased thermostability and high susceptibility to chymotrypsin digestion, in good agreement with their corresponding melting temperatures in CD experiments. Taken together, the results support the view that impaired binding of “structural” NADP⁺ can hinder folding as well as cause instability of these clinical mutant enzymes in the fully folded state.     

Impact of phloem girdling on leaf gas exchange and hydraulic conductance in hybrid aspen

We investigated phloem-xylem interactions in relation to leaf hydraulic capacity in hybrid aspen (Populus tremula L. × P. tremuloides Michx.) by using phloem girdling method. Removal of bark tissues (phloem girdling) at the branch base resulted in a substantial decline in stomatal conductance (g_S), net photosynthetic rate (P_N), and leaf hydraulic efficiency, and in increase of leaf water potential (Ψ_L). Although gS declined more than P_N (83 versus 78 %), the ratio of intercellular to ambient CO₂ concentrations (c_i/c_a) increased from 0.67 to 0.87 in three days after girdling. Girdling induced a decrease in leaf hydraulic conductance (K_L) on average by 43 % (P = 0.006). The changes in gS and leaf conductance to water vapour were co-ordinated with K_L only in girdled branches whereas intrinsic water-use efficiency was invariant to K_L. The declines in K_L with girdling were not accompanied by changes in potassium ion concentration ([K⁺]), electrical conductivity, or pH of xylem sap. The results suggest that phloem girdling at the branch base does not influence the recirculation of ions between the phloem and xylem in hybrid aspen and the decrease of K_L in response to the manipulation is not related to changes in [K⁺] and total ionic content of xylem sap.     

A comparison of the subcellular distribution of 5′-nucleotidase, (Na+K+)-ATPase and adenyl cyclase in beef thyroid gland

The validity of 5′-nucleotidase as a plasma membrane marker enzyme in beef thyroid has been tested by comparing the subcellular distribution of its activity to that of (Na⁺K⁺)-activated ATPase and adenyl cyclase. The specific activity and total activity of (Na⁺K⁺)-ATPase and adenyl cyclase were greatest in the 1000 × g (“nuclear”) and 33 000 × g (“mitochondrial and lysosomal”) fractions. In contrast, 5′-nucleotidase activity was concentrated in the 165 000 × g (“microsomal”) pellet and supernatant. Partially purified plasma membranes were separated from the 1000 (N₂), 30 000 (M₂) and 165 000 × g (P₂) pellets by discontinuous sucrose gradient centrifugation. Again a discordant distribution of these enzyme activities was observed. (Na⁺K⁺)-ATPase specific activity was increased approximately 30-fold over the homogenate in Fractions N₂ and M₂. Basal, thyroid-stimulating hormone-and fluoride-stimulated adenyl cyclase activities were concentrated in the same fractions. 5′-Nucleotidase activity was preferentially located in M₂ and P₂. These differences in distribution pattern suggest that 5′-nucleotidase activity is not uniquely located in the plasma membrane in the thyroid.     

Design and synthesis of potent macrocyclic renin inhibitors

Two types of P1-P3-linked macrocyclic renin inhibitors containing the hydroxyethylene isostere (HE) scaffold just outside the macrocyclic ring have been synthesized. An aromatic or aliphatic substituent (P3sp) was introduced in the macrocyclic ring aiming at the S3 subpocket (S3sp) in order to optimize the potency. A 5-6-fold improvement in both the K_i and the human plasma renin activity (HPRA)IC₅₀ was observed when moving from the starting linear peptidomimetic compound 1 to the most potent macrocycle 42 (K_i = 3.3 nM and HPRA IC₅₀ = 7 nM). Truncation of the prime side of 42 led to 8-10-fold loss of inhibitory activity in macrocycle 43 (K_i = 34 nM and HPRA IC₅₀ = 56 nM). All macrocycles were epimeric mixtures in regard to the P3sp substituent and X-ray crystallographic data of the representative renin macrocycle 43 complex showed that only the S-isomer buried the substituent into the S3sp. Inhibitory selectivity over cathepsin D (Cat-D) and BACE-1 was also investigated for all the macrocycles and showed that truncation of the prime side increased selectivity of inhibition in favor of renin.     

Functional expression of the murine D2, D3 and D4 dopamine receptors in Xenopus laevis oocytes

The different murine D2-type dopamine receptors (D_2L, D_2S, D_3L, D_3S, and D₄) were expressed in Xenopus laevis oocytes. The D2-type receptors were all similarly and efficiently expressed in Xenopus oocytes and were shown to bind the D2 antagonist [¹²⁵I]sulpride. They were all shown to activate Cl⁻ influx upon agonist stimulation. Using the diagnostic inhibitor bumetanide, we were able to separate the Na⁺/K⁺/2Cl⁻ cotransporter component of the Cl⁻ influx from the total unidirectional Cl⁻ influx. The D_3L subtype was found to operate exclusively through the bumetanide-insensitive Cl⁻ influx whereas the other D2-type receptors acted on the Na⁺/K⁺/2Cl⁻ cotransporter as well. The pertussis toxin sensitivity of the receptor-activated chloride influx via the Na⁺/K⁺/2Cl⁻ cotransporter varied between the various D2-type receptors showing that they may couple to different G proteins, and activate different second messenger systems.     

Variable affinity of the (Na+ + K+)-dependent adenosine triphosphatase for potassium: Studies using beryllium inactivation

Inactivation of the (Na⁺ + K⁺)-dependent ATPase by 50 μm BeCl₂ occurred during brief incubations in the presence of both Mg²⁺ and K⁺. Inactivation followed, initially, a first-order time course, with rate constants sensitive to the concentration of K⁺ (other components held constant). From these data dissociation constants can be calculated for K⁺ binding to sites controlling inactivation. Comparisons of relative affinities for K⁺ analogs (T1⁺ and NH₄⁺), and of sensitivity to reagents altering K⁺ activation (phlorizin and dimethylsulfoxide) indicate that the same K⁺ sites operate for both Be²⁺ inactivation and enzyme activation. With 3 mm MgCl₂ the dissociation constant, K_D, for K⁺ was 1.4 mm, but decreased 20-fold on addition of both Na⁺ and CTP. Alone, Na⁺ increased the apparent K_D for K⁺, either by direct competition or indirectly from its own site, with a K_D of 7 mm. The data suggest a model for K⁺ transport with K⁺ sites on the outer membrane surface that increase in affinity after formation of the phosphorylated enzyme intermediate, sufficiently to bind K⁺ in a high Na⁺ environment. Translocation may occur by an “oscillating pore” mechanism discharging K⁺ at the inner surface, while leaving demonstrable sites of moderate affinity at the outer end of the pore (which preclude attempts to document low-affinity discharge sites).     

Characterization of the α1B-adrenergic receptors of chicken hepatocytes. Signal transduction and actions

1. In chicken hepatocytes, α₁-adrenoceptor activation increased: (a) phosphatidylinositol labeling; (b) production of inositol trisphosphate; (c) cytosol calcium; and (d) phosphorylase activity.2. Prazosin (K_i ≈ 0.2–0.4 nM) was more potent in inhibiting these actions than 5-methyl-urapidil (K_i ≈ 30–60 nM); these actions were sensitive to chlorethylclonidine suggesting the involvement of α₁-adrenoceptors.3. The stimulation of phosphoinositide turnover was insensitive to pertussis toxin.4. In chicken liver membranes, [³H]prazosin binding sites (B_max 872 fmol/mg protein) with high affinity for prazosin (K_D 0.3 nM; K_i 0.4 nM) and lower affinity for 5-methyl-urapidil (K_i 46 nM) were detected, consistent with the presence of α_1B-adrenoceptors.     

Kinetic regulation of the binding of prothrombin to phospholipid membranes

A wide range of equilibrium and kinetic constants exist for the interaction of prothrombin and other coagulation factors with various model membranes from a variety of techniques. We have investigated the interaction of prothrombin with pure dioleoylphosphatidylcholine (DOPC) membranes and dioleoylphosphatidlyserine (DOPS)-containing membranes (DOPC:DOPS, 3:1) using surface plasmon resonance (SPR, with four different model membrane presentations) in addition to isotheral titration calorimetry (ITC, with suspensions of phospholipid vesicles) and ELISA methods. Using ITC, we found a simple low-affinity interaction with DOPC:DOPS membranes with a K _D = 5.1 μM. However, ELISA methods using phospholipid bound to microtitre plates indicated a complex interaction with both DOPC:DOPS and DOPC membranes with K _D values of 20 and 58 nM, respectively. An explanation for these discrepant results was developed from SPR studies. Using SPR with low levels of immobilised DOPC:DOPS, a high-affinity interaction with a K _D of 18 nM was obtained. However, as phospholipid and prothrombin concentrations were increased, two distinct interactions could be discerned: (i) a kinetically slow, high-affinity interaction with K _D in the 10^?8 M range and (ii) a kinetically rapid, low-affinity interaction with K _D in the 10^?6 M range. This low affinity, rapidly equilibrating, interaction dominated in the presence of DOPS. Detailed SPR studies supported a heterogeneous binding model in agreement with ELISA data. The binding of prothrombin with phospholipid membranes is complex and the techniques used to measure binding will report K _D values reflecting the mixture of complexes detected. Existing data suggest that the weaker rapid interaction between prothrombin and membranes is the most important in vivo when considering the activation of prothrombin at the cell surface.     

Gaba stimulation of 3H-diazepam binding in aged mice

Specific ³H-diazepam binding to washed brain membranes from C57BL/6 mice of different age groups (3, 6, 12, 24 and 36 months) was studied in the absence and presence of 30 μM GABA. GABA treatment was found to be effective in decreasing the K_D of ³H-diazepam binding of approximately 50% in all age groups tested (mean control K_D = 6.5 nM, mean GABA-treated K_D = 3.2 nM). No significant changes with age were observed in benzodiazepine receptor K_D or B_max in the presence or absence of GABA.     

Characterization of new G protein-coupled adenine receptors in mouse and hamster

The nucleobase adenine has previously been reported to activate G protein-coupled receptors in rat and mouse. Adenine receptors (AdeR) thus constitute a new family of purine receptors, for which the designation “P0-receptors” has been suggested. We now describe the cloning and characterization of two new members of the AdeR family from mouse (MrgA10, termed mAde1R) and hamster (cAdeR). Both receptors were expressed in Sf9 insect cells, and radioligand binding studies were performed using [³H]adenine. Specific binding of the radioligand was detected in transfected, but not in untransfected cells, and K _D values of 286 nM (mAde1R, B _max 1.18 pmol/mg protein) and 301 nM (cAdeR, B _max 17.7 pmol/mg protein), respectively, were determined. A series of adenine derivatives was investigated in competition binding assays. Minor structural modifications generally led to a reduction or loss of affinity, with one exception: 2-fluoroadenine was at least as potent as adenine itself at the cAdeR. Structure–activity relationships at all AdeR orthologs and subtypes investigated so far were similar, but not identical. For functional analyses, the cAdeR was homologously expressed in Chinese hamster ovary (CHO) cells, while the mAde1R was heterologously expressed in 1321N1 astrocytoma cells. Like the previously described AdeRs from rat (rAdeR) and mouse (mAde2R), the mAde1R (EC₅₀ 9.77 nM) and the cAdeR (EC₅₀ 51.6 nM) were coupled to inhibition of adenylate cyclase. In addition, the cAdeR from hamster expressed in CHO cells produced an increase in intracellular calcium concentrations (EC₅₀ 6.24 nM) and was found to be additionally coupled to G_q proteins.     

Characterization of the Binding of the GABA Agonist [3H]Piperidine-4-Sulphonic Acid to Bovine Brain Synaptic Membranes

Abstract: The binding of radioactive piperidine-4-sulphonic acid ([³H]P4S) to thoroughly washed, frozen, and thawed membranes isolated from cow and rat brains has been studied. Quantitative computer analysis of the binding curves for four regions of bovine brain revealed the general presence of two binding sites. In these brain regions less satisfactory computer fits were obtained for receptor models showing one or three binding sites or negative cooperativity. With the use of Tris-citrate buffer at 0°C the two affinity classes for P4S in bovine cortex membranes revealed the following binding parameters: K_D= 17 ± 7 nM (B_max= 0.15 ± 0.07 pmol/mg protein) and K_D= 237 ± 100 nM (B_max= 0.80 ± 0.20 pmol/mg protein). Heterogeneity was also observed for association and dissociation rates of [³H]P4S. The slow binding component (k_on= 5.6 × 10⁷ or 8.8 × 10⁷ M^-1 min^-1, k_Off= 0.83 min^-1, and K_D= 14.7 or 9.4 nM, determined by two different methods in phosphate buffer containing potassium chloride) corresponds to the high-affinity component of the equilibrium binding curve (K_D= 11 nM, B_max= 0.12 pmol/mg protein in the same buffer system). The association and dissociation rates for the subpopulation of rapidly dissociating sites, apparently corresponding to the low-affinity sites, were too rapid to be measured accurately. The binding of [³H]P4S appears to involve the same two populations of sites with B_max values similar to those for [³H]GABA binding to the same tissue, although the kinetic parameters for the two ligands are somewhat different. Furthermore, comparative studies on the inhibition of [³H]P4S and [³H]GABA binding by various GABA analogues, strongly suggest that P4S binds to the GABA receptors. The different effects of P4S and GABA on benzodiazepine binding are discussed.