Characterisation and Distribution of Inositol Polyphosphate and Ryanodine Receptors in the Rat Brain

Abstract: The regional distribution of inositol 1,4,5-trisphosphate (InsP₃), inositol 1,3,4,5-tetrakisphosphate (InsP₄), and ryanodine binding sites has been characterised and compared in the rat brain using radioligand binding assays. Cortical [³H]InsP₃ binding indicated similar positional and stereospecificity as observed in other tissues, with 100-fold selectivity for lnsP₃ over InsP₄. Similarly, high-affinity [³²P]InsP₄ binding also showed a high degree of positional specificity, with a 1,000-fold selectivity for InsP₄ over InsP₃. Initial characterisation of [³H]ryanodine binding to cortical membranes demonstrated that specific binding was highly dependent on high salt and micromolar Ca²⁺ concentrations and inhibited by Ca²⁺ levels of >1 mM. [³H]-Ryanodine binding was also enhanced by β,γ-methylene-adenosine 5′-trisphosphate and caffeine and inhibited by magnesium and ruthenium red (K_i= 0.81 μM). However, dantrolene (300 μM) was ineffective on the binding. Therefore, although the results indicate a greater similarity to the binding properties of the Ca²⁺-induced Ca²⁺ release channel isoform present in skeletal, rather than cardiac, muscle, it does not appear to be identical. Detailed binding analysis of ryanodine and polyphosphate sites, with the exception of ruthenium red, indicated no interaction between binding sites. Ruthenium red markedly enhanced the binding of both [³H]InsP₃ and [³²P]InsP₄, an effect most probably due to nonspecific complex formation. Regional binding of InP₃, InsP₄, and ryanodine in the rat brain was of similar affinity for each ligand in each area, but the density profile for each ligand was clearly different. The highest density of InsP₃ sites was in the cerebellum, whereas the highest density of ryanodine sites was in the hippocampus. High-affinity InsP₄ sites showed less regional diversity, with highest densities in the cerebellum, cortex, and hippocampus. However, in each area studied the density of sites followed the order InsP₃ > InsP₄ > ryanodine.     

Embryonic and larval development of brown trout, Salmo trutta L.: exposure to aluminium, copper, lead or zinc in soft, acid water

Freshly fertilized ova, eyed ova and yolk-sac fry of brown trout, Salmo trutta L., were exposed to each of four trace metals (aluminium: 6000 nmol l^?1; copper: 80 nmol l^?1; lead: 50 nmol l^?1; zinc: 300 nmol l^?1) while held in flowing artificial soft-water media maintained at pH 4.5 or 5.6 and [Ca] 20 or 200 μmol l^?1. In continuous exposure from fertilization, survival of ova was severely affected at pH 4.5 and [Ca] 20 μmol l^?1, regardless of the presence of Cu, Pb or Zn; Al reduced embryonic mortality and improved hatching success. High ambient [Ca] at pH 4.5 increased egg survival. At ‘swim-up’, surviving fry exposed to Al or Pb had lower whole body Ca, Na and K content, irrespective of pH or ambient [Ca]. Cu reduced whole body Ca and K content at pH 5.6 and [Ca] 200 μmol^?1, and whole body Ca, Na and K content in the other media. Zn reduced whole body mineral content at pH 5.6 and [Ca] 20 μmol l^?1. Whole body Mg content was reduced by all trace metals at pH 5.6 and [Ca] 20 μmol l^?1, and by Cu at pH 5.6 and [Ca] 200 μmol l^?1. Al and Cu impaired skeletal calcification at pH 5.6 at both ambient [Ca]; Pb only at [Ca] 20 μmol I^?1. Zn enhanced calcification at pH 4.5 and [Ca] 200 μmol l^?1. In the absence of trace metals, low pH reduced body Ca, Na, K content and skeletal calcification at [Ca] 200 μmol l^?1. The uptake of Ca, Na and K, measured at regular intervals from hatching was impaired to the same extent by all treatments at pH 4.5, irrespective of ambient [Ca] or trace metal presence. At pH 5.6, irrespective of ambient [Ca], Al, Cu and Pb impaired Ca and K uptake. The rate of Na uptake was reduced by Al and Cu. Al-treated yolk-sac fry, exposed to low ambient [Ca] from 200–300° days post-hatch, suffered high mortalities regardless of pH. Ca, Na and K uptake was impaired by all treatments at pH 4.5, and by Al and Cu at pH 5.6 in a similar exposure period. The development of the early stages of brown trout in the presence of trace metals is discussed in relation to recruitment failure in areas of soft, acid water.     

Binding of [3H]batrachotoxinin A-20-α-benzoate and [3h]saxitoxin to receptor sites associated with sodium channels in trout brain synaptoneurosomes

1. [³H]Batrachotoxinin A-20-α-benzoate ([³H]BTX-b) and [³H]saxitoxin ([³H]STX), radioligands that bind to distinct sites on the voltage-sensitive sodium channel, were bound specifically to saturable sites in rainbow trout (Oncorhynchus mykiss) brain synaptoneurosomes.2. Specific [³H]BTX-B binding was temperature dependent with highest levels of specific [³H]BTX-B binding observed at 7°C. Specific binding was inversely correlated with assay temperature at temperatures above 7°C.3. Saturating concentrations of scorpion (Leiurus quinquestriatus) venom (ScV) stimulated specific [³H]BTX-B binding at 27°C, but not at 7°C. The dihydropyrazole insecticide RH 3421 inhibited specific [³H]BTX-B binding at 7°C but had no effect on specific binding at 27°C. The sodium channel activators veratridine and aconitine and the local anesthetic dibucaine inhibited specific [³H]BTX-B binding at both 7°C and 27°C.4. Displacement experiments in the presence of ScV at 27°C gave an equilibrium dissociation constant (K_d) for [³H]BTX-B of 710 nM and a maximal binding capacity (B_max) of 11.3 pmol/mg protein. Kinetic experiments established the rates of association (1.17 × 10⁵min⁻¹ nM⁻¹) and dissociation (0.0514min⁻¹) of the ligand-receptor complex.5. The binding of [³H]STX reached apparent saturation at 7.5 nM. Scatchard analysis of the saturation data indicated a K_d of 3.8nM and a B_max of 1.9 pmol/mg protein.6. These studies provide evidence for high affinity, saturable binding sites for [³H]BTX-B and [³H]STX in trout brain preparations. Whereas certain neurotoxins modified the specific binding of [³H]BTX-B in trout brain synaptoneurosomes in a predictable fashion, other compounds known to affect specific [³H]BTX-B binding in mammalian brain preparations had no effect on specific [³H]BTX-B binding in the trout.     

3H-imipramine binding in membrane preparations from brains of insects (Periplaneta americana,Locusta migratoria) and mollusc (Helix pomatia)

1. The binding of [³H]-imipramine to brain membrane preparations of the insects, Periplaneta americana, Locusta migratoria and the mollusc, Helix pomatia was investigated.2. [³H]-Imipramine binding is similar in invertebrates and vertebrates although, in insects, the binding is independent of sodium.3. In the three invertebrate systems studied, [³H]-imipramine binding was inhibited by 5-hydroxytryptamine (5-HT) only at high concentrations (100 μM 5-HT caused 20–30% inhibition in insects and 10% inhibition in snail).4. Studies on binding and incorporation of [³H]-imipramine and [³H]-5-hydroxytryptamine indicate that the imipramine binding site differs from the 5-hydroxytryptamine recognition site of uptake.5. The [³H]-imipramine binding sites are not specific for 5-hydroxytryptamine and have similar affinity for dopamine.     

Detailed analysis of heterogeneity of [3H]naloxone binding sites in rat brain synaptosomes

The experiments reported in this paper address the question of heterogeneity of [³H]naloxone binding sites in rat brainstem synaptosomal preparations at 23°C in the presence of 100 mM sodium chloride. Kinetic analysis in the presence of 0.4, 4 and 10 nM [³H]naloxone gave pseudo-first order association rate of 0.9±0.04, 1.23±0.08 and 1.06±0.08 min^–1, respectively. The dissociation of a 1 nM [³H]naloxone receptor complex was biphasic with dissociation rate constants of 1.8 and 0.4 min^–1. On the other hand, dissociation of 10 nM [³H]naloxone was monophasic with ak _d of 1.1 min^–1. Two subpopulations of binding sites were also observed by steady state binding studies, with Kd values of 0.5 and 3.4 nM and a ratio of high to low affinity sites of 1:9. Heterogeneity of [³H]naloxone binding sites could be seen by displacement studies performed with opioid eptides and alkaloids. We suggest that our data best fits a model with two independent naloxone binding sites wherein either one or both undergo a multi-step interaction with ligand.     

Muscarinic receptors in pineal

The presence of muscarinic receptors in sheep and rat pineals was detected by binding of [³H]quinuclidinyl benzilate ([³H]QNB), a potent and specific muscarinic antagonist. [³H]QNB binding to sheep pineal membrane resuspensions was saturable and reversible, with a rate constant for association at 37°C of 6×10⁸M^?1min^?1 and a rate constant for dissociation of 1×10^?2min^?1. Kinetic and saturation experiments yielded an equilibrium dissociation constant of 13–18 pM and a concentration of binding sites equivalent to 1.1 pmol/g of original wet weight. This is only about 5% of the level of β-adrenergic receptors. Competition by a variety of cholinergic drugs confirmed the muscarinic nature of the binding sites. Experiments in rats failed to detect a significant decrease in pineal [³H]QNB binding following bilateral superior cervical ganglionectomy, suggesting that the binding sites are not localized exclusively on sympathetic terminals.     

Similar Ca dependences of [H]ryanodine binding to α- and β-ryanodine receptors purified from bullfrog skeletal muscle in an isotonic medium

To understand the functions of the two ryanodine receptor isoforms (α- and β-RyRs) in nonmammalian skeletal muscles, we determined [³H]ryanodine binding to these isoforms purified from bullfrog skeletal muscle. In 0.17 M-NaCl medium, both isoforms demonstrated similar Ca²⁺ dependent ryanodine binding activities, while the Ca²⁺ sensitivity for activation of β-RyR was increased in 1 M-NaCl medium. This enhancement in Ca²⁺ sensitivity depended on the kind of salts used. These results imply that α- and β-RyRs may have similar properties as Ca²⁺-induced Ca²⁺ release channels in bullfrog skeletal muscle.     

Two Saturable Recognition Sites for (-)[125I]Iodo-N6-(4-Hydroxyphenyl-Isopropyl)-Adenosine Binding on Purified Cardiac Sarcolemma

Abstract

Analysis of (-)[¹²⁵]iodo-N⁶-(4-hydroxyphenylisopropyl)-adenosine ([¹²⁵I]HPIA) binding to purified sarcolemmal preparations of guinea pig and bovine hearts revealed two classes of binding sites when unlabeled iodo-HPIA (100 μmol/1) was used as non-specific binding marker. In the presence of 1 mmol/1 theophylline, however, only the high affinity component was detected. Adenosine receptor agonists caused biphasic displacement of [¹²⁵I]HPIA binding, with a high affinity potency rank order typical of interaction with A₁-adenosine receptors. Biphasic competition curves were also observed with 8-phenyltheophylline and isobutylmethylxanthine, whereas the theophylline curve was monophasic up to 1 mmol/1. In brain membranes, specific binding of [¹²⁵I]HPIA as well as of [³H]PIA was further reduced when unlabeled iodo-HPIA replaces theophylline as the non-specific binding marker. These results suggest the presence of two [¹²⁵I]HPIA binding sites on cardiac sarcolemma and brain membranes, but receptor function can only be ascribed to the high affinity sites. The low affinity site probably represents an artefact, which is often observed when non-specific binding is defined with the unlabeled counterpart or a structurally related ligand of the radioligand used.     

Phosphorylation of the cardiac ryanodine receptor by cAMP-dependent protein kinase

The phosphorylation of canine cardiac and skeletal muscle ryanodine receptors by the catalytic subunit of cAMP-dependent protein kinase has been studied. A high-molecular-weight protein (Mr 400,000) in cardiac microsomes was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. A monoclonal antibody against the cardiac ryanodine receptor immunoprecipitated this phosphoprotein. In contrast, high-molecular-weight proteins (Mr 400,000-450,000) in canine skeletal microsomes isolated from extensor carpi radialis (fast) or superficial digitalis flexor (slow) muscle fibers were not significantly phosphorylated. In agreement with these findings, the ryanodine receptor purified from cardiac microsomes was also phosphorylated by cAMP-dependent protein kinase. Phosphorylation of the cardiac ryanodine receptor in microsomal and purified preparations occurred at the ratio of about one mol per mol of ryanodine-binding site. Upon phosphorylation of the cardiac ryanodine receptor, the levels of [3H]ryanodine binding at saturating concentrations of this ligand increased by up to 30% in the presence of Ca2+ concentrations above 1 microM in both cardiac microsomes and the purified cardiac ryanodine receptor preparation. In contrast, the Ca2+ concentration dependence of [3H]ryanodine binding did not change significantly. These results suggest that phosphorylation of the ryanodine receptor by cAMP-dependent protein kinase may be an important regulatory mechanism for the calcium release channel function in the cardiac sarcoplasmic reticulum.     

A calcium antagonist drug binding site in skeletal muscle sarcoplasmic reticulum: Evidence for a calcium channel

The sarcoplasmic reticulum (S.R.) of rabbit skeletal muscle has been found to contain a single, high affinity binding site for the Ca antagonist drug [³H] -nitrendipine. Two subfractions of the reticulum were studied, the heavy (HSR) and light (LSR) preparations, which exhibited similar nitrendipine equilibrium dissociation constants (K_D) of 1nM. Crude cardiac and brain membranes assayed under the same conditions exhibited K_D values of 0.2–0.3nM. The concentration of binding sites per mg. protein (B_max) in HSR was found to be very high, namely 6.7 picomoles/mg, some four times greater than that of LSR. [³H] -nitrendipine binding to HSR was reversible and inhibited by the Ca antagonists flunarizine and verapamil, and by the intracellular Ca release antagonist TMB-8 (8-diethylamino-octyl 3,4,5- trimethylbenzoate hydrochloride). However, unlabelled nitrendipine at 2 × 10^?5M had no effect on contraction of isolated electrically stimulated rabbit lumbrical or rat diaphragm muscles, nor did it affect the neuromuscular junction as studied in rat phrenic nerve-diaphragm preparations. Also, little effect of 2 × 10^?5M nitrendipine was seen on net ⁴⁵Ca uptake by HSR. These results suggest that [³H] -nitrendipine binding to skeletal muscle S.R. resembles that of brain membranes, which also contain a high affinity binding site for [³H] -nitrendipine and which similarly are pharmacologically insensitive to this dihydropyridine type of Ca channel blocking agent. Since HSR is also enriched in calsequestrin and terminal cysternae from which Ca is released in vivo, it seems likely that the [³H]- nitrendipine binding sites in S.R. are associated with Ca channels in the S.R.     

Caffeine analogs: effects on ryanodine-sensitive calcium-release channels and GABAA receptors

1. Caffeine at 0.3–10 mM enhanced the binding of [³H]ryanodine to calcium-release channels of rabbit muscle sarcoplasmic reticulum. A variety of other xanthines were as efficacious as caffeine or nearly so, but none appeared markedly more potent.2. Caffeine at 1 mM markedly inhibited binding of [³H]diazepam to GABA_A receptors in rat cerebral cortical membranes.3. Other xanthines also inhibited binding with certain dimethylpropargylxanthines being nearly fivefold more potent than caffeine.4. Caffeine at 1 mM stimulated binding of [³⁵S]TBPS to GABA_A receptors as did certain other xanthines.5. The dimethylpropargylxanthines had little effect. 1,3-Dipropy1-8-cyclopentylxan- thine at 100 M had no effect on [³H]diazepam binding, but markedly inhibited [³⁵S]TBPS binding.6. Structure–activity relationships for xanthines do differ for calcium-release channels and and for different sites on GABA_A receptors, but no highly selective lead compounds were identified.     

Regulation of Ryanodine Receptor Calcium Release Channels by Diadenosine Polyphosphates

Abstract: [³H]Ryanodine binding to, as well as functions of, ryanodine receptor intracellular Ca²⁺ release channel complexes are modulated by several adenosine-based compounds. In this study, we determined the effects of endogenous compounds termed diadenosine polyphosphates (Ap_nAs; n = 2–6 phosphate groups) on [³H]ryanodine binding to membranes prepared from rat brain and skeletal and cardiac muscle. Under low ionic strength buffer conditions, [³H]ryanodine binding to brain membranes was significantly increased by 171% with 333 µMP¹,P⁵-di(adenosine-5′) pentaphosphate (Ap₅A) and by 209% with the same concentration of the metabolism-resistant ATP analogue βγ-methyleneadenosine 5′-triphosphate (AMP-PCP) compared with control values for [³H]ryanodine binding of 9.6 ± 1.8 fmol/mg of protein. Dose-related increases in [³H]ryanodine binding were observed for all five Ap_nAs tested [P¹,P²-di(adenosine-5′) pyrophosphate (Ap₂A), P¹,P³-di(adenosine-5′) triphosphate (Ap₃A), P¹,P⁴-di(adenosine-5′) tetraphosphate (Ap₄A), Ap₅A, and P¹,P⁶-di(adenosine-5′) hexaphosphate (Ap₆A)] as well as AMP-PCP; oxidized salts of Ap_nAs stimulated [³H]ryanodine binding to a greater degree than did nonoxidized Ap_nAs. The apparent rank order for the capacity of these agents to increase [³H]-ryanodine binding was oxidized Ap₄A = oxidized Ap₅A > oxidized Ap₃A > Ap₆A > AMP-PCP > Ap₅A > Ap₂A. Addition of the approximate EC₅₀ dose of oxidized Ap₄A (37 µM) increased the affinity (K_D) of ryanodine receptors from 34 ± 7 to 12 ± 2 nM; the apparent binding site density (B_max) was not significantly different from control values of 107 ± 33 fmol/mg of protein. Increases in [³H]-ryanodine binding by either oxidized Ap₄A or nonoxidized Ap₅A were not further enhanced by coincubation with AMP-PCP, which suggests a similar site of action for the Ap_nAs and AMP-PCP. [³H]Ryanodine binding to skeletal and cardiac muscle membranes was enhanced by addition of oxidized Ap₄A, Ap₅A, and AMP-PCP. Oxidized Ap₄A increased the specific binding by ninefold in skeletal muscle and by threefold in cardiac muscle. These results suggest that Ap_nAs, at physiologically relevant concentrations, may serve as endogenous modulators of ryanodine receptor-gated Ca²⁺ release channels.     

Physiological differences between the alpha and beta ryanodine receptors of fish skeletal muscle.

Two isoforms of the sarcoplasmic reticulum Ca2+ release channel (ryanodine receptor or RYR) are expressed together in the skeletal muscles of most vertebrates. We have studied physiological properties of the two isoforms (alpha and beta) by comparing SR preparations from specialized fish muscles that express the alpha isoform alone to preparations from muscles containing both alpha and beta. Regulation of channel activity was assessed through [3H]ryanodine binding and reconstitution into planar lipid bilayers. Distinct differences were observed in the calcium-activation and -inactivation properties of the two isoforms. The fish alpha isoform, expressed alone in extraocular muscles, closely resembled the rabbit skeletal muscle RYR. Maximum [3H]ryanodine binding and maximum open probability (Po) of the alpha RYR were achieved from 1 to 10 microM free Ca2+. Millimolar Ca2+ reduced [3H]ryanodine binding and Po close to zero. The beta isoform more closely resembled the fish cardiac RYR in Ca2+ activation of [3H]ryanodine binding. The most prominent difference of the beta and cardiac isoforms from the alpha isoform was the lack of inactivation of [3H]ryanodine binding and Po by millimolar free Ca2+. Differences in activation of [3H]ryanodine binding by adenine nucleotides and inhibition by Mg2+ suggest that the beta and cardiac RYRs are not identical, however. [3H]ryanodine binding by the alpha RYR was selectively inhibited by 100 microM tetracaine, whereas cardiac and beta RYRs were much less affected. Tetracaine can thus be used to separate the properties of the alpha and beta RYRs in preparations in which both are present. The distinct physiological properties of the alpha and beta RYRs that are present together in most vertebrate muscles support models of EC coupling incorporating both directly coupled and Ca(2+)-coupled channels within a single triad junction.     

Na+-independentl-aspartate binding sites in chick brain

1. One binding component with aK _d value of 200×10^–9 M and half-life of the ligand binding component of 30 min was found. 2. Chloride ions produced a significant increase ofl-[³H]aspartate andl-[³H]glutamate binding. 3.l-Glutamate,l-ibotenate,l-quisqualate, anddl-homocysteic acid were potent inhibitors ofl-[³H]aspartate binding. 4. In all brain regions major increases of binding were observed during the third week of the in ovo period of life.     

Ryanodine as a functional probe of the skeletal muscle sarcoplasmic reticulum Ca2+ release channel

Ryanodine is a neutral plant alkaloid which functions as a probe for an intracellular Ca²⁺ release channel (ryanodine receptor) in excitable tissues. Using [³H]ryanodine, a 30 S protein complex comprised of four polypeptides of M_r 565,000 has been isolated and functionally reconstituted into planar lipid bilayers. The effects of salt concentration and divalent cations on skeletal muscle sarcoplasmic reticulum [³H]ryanodine binding and Ca²⁺ release channel activity have been compared. These studies suggest that ryanodine is a good probe for investigating the function of the release channel.     

Calcium ions inhibit the allosteric interaction between the dihydropyridine and phenylalkylamine binding site on the voltage-gated calcium channel in heart sarcolemma but not in skeletal muscle transverse tubules

Calcium channel blockers bind with high affinity to sites on the voltage-sensitive Ca2+ channel. Radioligand binding studies with various Ca2+ channel blockers have facilitated identification and characterization of binding sites on the channel structure. In the present study we evaluated the relationship between the binding sites for the Ca2+ channel blockers on the voltage-sensitive Ca2+ channel from rabbit heart sarcolemma and rabbit skeletal muscle transverse tubules. [3H]PN200-110 binds with high affinity to a single population of sites on the voltage-sensitive Ca2+ channel in both rabbit heart sarcolemma and skeletal muscle transverse tubules. [3H]PN200-110 binding was not affected by added Ca2+ whereas EGTA and EDTA noncompetitively inhibited binding in both types of membrane preparations. EDTA was a more potent inhibitor of [3H]PN200-110 binding than EGTA. Diltiazem stimulates the binding of [3H]PN200-110 in a temperature-sensitive manner. Verapamil inhibited binding of [3H]PN200-110 to both types of membrane preparations in a negative manner, although this effect was of a complex nature in skeletal muscle transverse tubules. The negative effect of verapamil on [3H]PN200-110 binding in cardiac muscle was completely reversed by Ca2+. On the other hand, Ca2+ was without effect on the negative cooperativity seen between verapamil and [3H]PN200-110 binding in skeletal muscle transverse tubules. Since Ca2+ did not affect [3H]PN200-110 binding to membranes, we would like to suggest that Ca2+ is modulating the negative effect of verapamil on [3H]PN200-110 binding through a distinct Ca2+ binding site.(ABSTRACT TRUNCATED AT 250 WORDS)     

(Na + K)-ATPase activity of crude homogenates of rat skeletal muscle as estimated from their K-dependent 3-O-methylfluorescein phosphatase activity

A highly sensitive fluorimetric assay using 3-O-methylfluorescein phosphate as substrate was used in the determination of K⁺-dependent phosphatase activity in preparations of rat skeletal muscle. The gastrocnemius muscle was chosen because of mixed fibre composition. Crude, detergent treated homogenate was used so as to avoid loss of activity during purification. K⁺-dependent phosphatase activities in the range 0.19–0.37 μmol · (g wet weight)⁻¹ · min⁻¹ were obtained, the value decreasing with age and K⁺-deficiency. Complete inhibition of the K⁺-dependent phosphatase was obtained with 10⁻³ M ouabain. Using a KSCN-extracted muscle enzyme the intimate relation between K⁺-dependent phosphatase activity and (Na⁺ + K⁺)-activated ATP hydrolysis could be demonstrated. A molecular activity of 620 min⁻¹ was estimated from simultaneous determination of K⁺-dependent phosphatase activity and [³H]ouabain binding capacity using the partially purified enzyme preparation. The corresponding enzyme concentration in the crude homogenates was calculated and corresponded well with the number of [³H]ouabain binding sites measured in intact muscles or biopsies hereof.     

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.     

Regulation of muscarinic receptor binding by guanine nucleotides and N-ethylmaleimide

The regulation of muscarinic receptor binding by guanine nucleotides and N-ethylmaleimide (NEM) was investigated using the agonist ligand, [³H] cis methyldioxolane ([³H] CD). Characterization studies on rat forebrain homogenates showed that [³H] CD binding was linear with tissue concentration and was unaffected by a change in pH from 5.5 to 8.0. The regional variation in [³H] CD binding in the rat brain correlated generally with [³H] (?)3-quinuclidinyl benzilate ([³H] (?)QNB) binding, although the absolute variation in binding was somewhat less. At a concentration of 100 μM, the GTP analogue, guanyl-5′-yl imidodiphosphate [Gpp(NH)p], caused a 43–77% inhibition of [³H] CD binding in the corpus striatum, ileum, and heart. The results of binding studies using several Gpp(NH)p concentrations demonstrated that the potency of this guanine nucleotide for inhibition of [³H] CD binding was greater in the heart than in the ileum. In contrast to its effects on [³H] CD binding, Gpp(NH)p caused an increase in [³H] (?)QNB binding in the heart heart and ileum and no change in [³H] (?)QNB binding in the corpus striatum. When measured by competitive inhibition of [³H] (?)QNB binding to the longitudinal muscle of the ileum, Gpp(NH)p (100 μM) caused an increase in the IC₅₀ values of a series of agonists in a manner that was correlated with the efficacy of these compounds. The results of binding studies on NEM treated forebrain homogenates revealed an enhancement of [³H] CD binding by NEM.     

Characterisation of the Binding of [3H]WAY-100635, a Novel 5-Hydroxytryptamine1A Receptor Antagonist, to Rat Brain

Abstract: The specific binding of [³H]WAY-100635 {N-[2-[4-(2-[O-methyl-³H]methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexane carboxamide trihydrochloride} to rat hippocampal membrane preparations was time, temperature, and tissue concentration dependent. The rates of [³H]WAY-100635 association (k₊₁ = 0.069 ± 0.015 nM^?1 min^?1) and dissociation (k_?1 = 0.023 ± 0.001 min^?1) followed monoexponential kinetics. Saturation binding isotherms of [³H]WAY-100635 exhibited a single class of recognition site with an affinity of 0.37 ± 0.051 nM and a maximal binding capacity (B_max) of 312 ± 12 fmol/mg of protein. The maximal number of binding sites labelled by [³H]WAY-100635 was ～36% higher compared with that of 8-hydroxy-2-(di-n-[³H]-propylamino)tetralin ([³H]8-OH-DPAT). The binding affinity of [³H]WAY-100635 was significantly lowered by the divalent cations CaCl₂ (2.5-fold; p < 0.02) and MnCl₂ (3.6-fold; p < 0.05), with no effect on B_max. Guanyl nucleotides failed to influence the K_D and B_max parameters of [³H]WAY-100635 binding to 5-HT_1A receptors. The pharmacological binding profile of [³H]WAY-100635 was closely correlated with that of [³H]8-OH-DPAT, which is consistent with the labelling of 5-hydroxytryptamine_1A (5-HT_1A) sites in rat hippocampus. [³H]WAY-100635 competition curves with 5-HT_1A agonists and partial agonists were best resolved into high- and low-affinity binding components, whereas antagonists were best described by a one-site binding model. In the presence of 50 µM guanosine 5′-O-(3-thiotriphosphate) (GTPγS), competition curves for the antagonists remained unaltered, whereas the agonist and partial agonist curves were shifted to the right, reflecting an influence of G protein coupling on agonist versus antagonist binding to the 5-HT_1A receptor. However, a residual (16 ± 2%) high-affinity agonist binding component was still apparent in the presence of GTPγS, indicating the existence of GTP-insensitive sites.