Effects of adrenalectomy or RU-486 on rat muscle fibers during hindlimb suspension

The purpose of this study was to investigate the effects of a glucocorticoid antagonist, RU-486, and of adrenalectomy (ADX) on rat skeletal muscle structural properties after 3, 7, and 14 days of hindlimb suspension (H). After H, a significant loss in muscle weight was observed as early as 3 days in soleus (SOL; -10%) and adductor longus (AL; -14%) muscles. In SOL, after only 7 days, a reduction (-14%) in type I fiber percent distribution occurred, accompanied by an increase (+129%) in intermediate type I fibers. Fiber type changes increased depending on the duration of H. In AL muscle, no change occurred after H in the fiber type composition despite a similar degree of muscle atrophy. Treatment with RU-486 or ADX significantly reduced the loss of SOL weight observed after 14 days (-42 and -44%, respectively, vs. -50% for H rats), delayed the SOL atrophy (from 3 to 7 days), and normalized the shift in fiber type distribution induced by H. In SOL, administration of RU-486 (but not ADX) partly prevented the reduction in size induced by H of all the fibers. In AL, neither treatment affected the extent of muscle atrophy, even though the reduction in type IIa fiber size was prevented by RU-486 but not by ADX after 14 days of suspension. ADX or RU-486 administration did not prevent the extensor digitorum longus weight loss observed after 14 days of suspension but allowed a recovery of its normal fiber type composition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of spinal cord transection on N-methyl-D-aspartate receptors in the cord

Spinal cord injury can lead to an exaggeration of transmission through spinal pathways, resulting in muscle spasticity, chronic pain, and abnormal control of blood pressure and bladder function. These conditions are mediated, in part, by N-methyl-D-aspartate (NMDA) receptors on spinal neurons, but the effects of cord injury on the expression or function of these receptors is unknown. Therefore, antibodies to the NMDA-R1 receptor subunit and binding of [3H]MK-801 were used to assess NMDA receptors in the spinal cord. Receptor density in rats with intact spinal cords was compared to that in rats 1 and 2 weeks after spinal cord transection (SCT) at the mid-thoracic level. At 1 and 2 weeks after SCT, [3H]MK-801 binding was reduced in most laminae in cord segments caudal to the injury, whereas no decrease in amount of R1 subunit immunoreactivity was observed. No significant changes in [3H]MK-801 binding and NMDA-R1 immunoreactivity could be seen rostral to the transection. Since [3H]MK-801 binding requires an open ion channel, the discrepancy between [3H]MK-801 binding and immunocytochemistry may indicate a loss of functional receptors without a consistent change in their total number. Therefore, the exaggerated reflexes that are well established in rats 2 weeks after cord injury must be mediated by a mechanism that withstands attenuation of NMDA receptor function.     

Pharmacological properties of nicotinic acetylcholine receptors on adrenal chromaffin cells and their modification by hypoxic exposure

To characterize the properties of nicotinic acetylcholine receptors (nAChRs) in autonomic ganglia, we examined specific L-[3H]nicotine binding to membrane fraction prepared from cultured bovine adrenal chromaffin cells, using a modified filtration method. Binding of L-[3H]nicotine to non-treated glass fiber filters interfered with detection of specific binding to the membrane fraction. Presoaking the glass fiber filters in 3% or greater concentrations of polyethyleneimine solution (sixty times higher than the reported concentration) for more than 5 h could reduce the binding of L-[3H]nicotine to the filters to background level. Furthermore, specific L-[3H]nicotine binding to the membrane fraction was detected only when the membrane fraction was prepared in buffer containing no divalent cations such as Ca2+ and Mg2+ (EDTA and EGTA were added) and protease inhibitors. Specific binding of L-[3H]nicotine was saturable and reversible. Scatchard analysis revealed a single class of high affinity binding sites with an average Kd of 8.7 nM and a Bmax of 43.2 fmol/mg protein. Specific binding was sensitive to cholinergic agonists (carbamylcholine and L-nicotine) and ganglionic stimulating agents (lobeline and DMPP), but was resistant to neuromuscular blocking agents (alpha-bungarotoxin, d-tubocurarine) and hexamethonium. These results suggest that high-affinity nicotine binding sites on adrenal chromaffin cells are nAChRs of ganglion-type, which have properties different from nAChRs on the neuromuscular junction but similar to nAChRs in the brain.     

Agonist-induced up-regulation of alpha4beta2 nicotinic acetylcholine receptors in M10 cells: pharmacological and spatial definition

Chronic nicotine up-regulates the number of high affinity nicotinic acetylcholine receptors (nAChRs) in mammalian brain. Here, we studied up-regulation of the nAChR composed of alpha4 and beta2 subunits in the M10 cell line by using [3H]epibatidine to measure nAChR in cells in situ and in membrane preparations. Cultures were exposed to drugs for 2 days before assay. All agonists up-regulated [3H]epibatidine binding sites with EC50 values typically 10-100-fold higher than their respective Ki values from competition binding assays. Maximum up-regulation ranged from 40% to 250% above control values. Maximally effective concentrations of the less efficacious agonists methylcarbamylcholine or (+/-)-epibatidine together with nicotine resulted in less up-regulation than that produced by nicotine alone, showing that they are partial up-regulatory agonists. The antagonists dihydro-beta-erythroidine, methyllycaconitine, d-tubocurarine, hexamethonium, decamethonium, and mecamylamine either failed to up-regulate [3H]epibatidine binding sites or up-regulated mildly at high concentrations. When tested at non-up-regulating concentrations, only d-tubocurarine significantly inhibited agonist-induced up-regulation; this inhibition seemed to be noncompetitive. Comparison of [3H]epibatidine displacement in intact M10 cells and membrane preparations by membrane-impermeant ligands indicated that 85% of [3H]epibatidine binding sites are intracellular. On chronic treatment with agonist, the proportion of surface receptors did not change significantly, indicating that most up-regulated [3H]epibatidine binding sites are internal. However, up-regulation is mediated at the cell surface because the impermeant ligand tetramethylammonium was as efficacious as nicotine in eliciting up-regulation, and methylcarbamylcholine (i.e., impermeant but with low efficacy) blocked nicotine induced up-regulation. Thus, agonists elicit up-regulation (mainly of intracellular receptors) by interacting with cell surface nAChRs that are not compatible with either an active or high affinity desensitized conformation.     

Changes in the excitability of soleus muscle short latency stretch reflexes during human hopping after 4 weeks of hopping training

Changes in the excitability of the human triceps surae muscle short latency stretch reflexes were investigated in six male subjects before and after 4 weeks of progressive two-legged hopping training. During the measurements the subjects performed 2-Hz hopping with: preferred contact time (PCT) and short contact time. The following reflex parameters were examined before and after the training period: the soleus muscle (SOL) Hoffmann-reflex (H-reflex) at rest and during hopping, the short latency electromyogram (EMG) components of the movement induced stretch reflex (MSR) in SOL and medial gastrocnemius muscle (MG), and the EMG amplitude of the SOL and MG tendon reflexes (T-reflexes) elicited at rest. The main results can be summarized as follows: the SOL T-reflex had increased by about 28% (P < 0.05) after training while the MG T-reflex was unchanged; the SOL MSR (always evident) and the MG MSR (when observable) did not change in amplitude with training, and before training the SOL H-reflex in both hopping situations was significantly depressed to about 40% of the reference value at standing rest (P < 0.05). After training the H-reflex during PCT hopping was no longer depressed. As the value of the measured mechanical parameters (the total work rate, joint angular velocity and the ankle joint work rate) was unchanged after training in both hopping situations, the reflex changes observed could not be ascribed to changes in the movement pattern. To explain the observed changes, hypotheses of changes in the excitability of the stretch reflex caused by the training were taken into consideration and discussed.     

Quantitative measurement of calcium flux through muscle and neuronal nicotinic acetylcholine receptors

A new approach was developed to determine quantitatively the fraction of current carried by Ca2+ through an ion channel under physiological conditions. This approach entails the simultaneous measurement of membrane current and intracellular Ca2+ for single cells. Whole-cell patch-clamp techniques were used to measure current, and intracellular Ca2+ was monitored with the fluorescent indicator fura-2. To obtain a quantitative measure of the fraction of current carried by Ca2+, a cell-by-cell calibration method was devised to account for differences among cells in such factors as cellular volume and Ca2+ buffering. The method was used to evaluate the Ca2+ flux through muscle and neuronal nicotinic ACh receptors (nAChRs). In a solution containing 2.5 mM Ca2+ at a holding potential of -50 mV, Ca2+ carries 2.0% of the inward current through muscle nAChRs from BC3H1 cells and 4.1% of the inward current through neuronal nAChRs from adrenal chromaffin cells. The Ca2+ flux through neuronal nAChRs of adrenal chromaffin cells is insensitive to alpha-bungarotoxin. The influx of Ca2+ is voltage dependent, and because of the Ca2+ concentration difference across the cellular membrane, there is Ca2+ influx into the cell even when there is a large net outward current. At both muscle and neuronal cholinergic synapses, activity-dependent Ca2+ influx through nicotinic receptors produces intracellular signals that may have important roles in synaptic development, maintenance, and plasticity.     

Na(+)-K+ ATPase concentration in different adult rat skeletal muscles is related to oxidative potential

To investigate the relationship among fibre type, oxidative potential, and Na(+)-K+ ATPase concentration in skeletal muscle, adult male Wistar rats weighing 259 +/- 8 g (mean +/- SE) were sacrificed and the soleus (SOL), extensor digitorum longus (EDL), red vastus lateralis (RV), and white vastus lateralis (WV) removed. These muscles were chosen as being representative of the two major fibre type populations: slow twitch (SOL) and fast twitch (EDL, RV, WV) and exhibiting either a high (SOL, EDL, RV) or low (WV) oxidative potential. Na(+)-K+ ATPase concentration (pmol.g-1 wet weight), measured by the [3H]ouabain binding technique, differed (p < 0.01) only between the WV (238 +/- 7.9) and the SOL (359 +/- 9.6), EDL (365 +/- 10), and RV (403 +/- 12). Similarly, muscle oxidative potential as measured by the maximal activity of citrate synthase was different (p < 0.01) only between the WV and the other three muscles. Citrate synthase activity (mumol.min-1.g-1 wet weight) was 4.0 +/- 0.7, 12.3 +/- 0.9, 9.1 +/- 0.7, and 11.3 +/- 1.0 in the WV, SOL, EDL, and RV, respectively. These results indicate that Na(+)-K+ ATPase concentration is not related to the speed of contraction but to the oxidative potential of the muscle. Since chronic activity is a primary determinant of oxidative potential, it would be expected that increases in Na(+)-K+ ATPase would accompany increases in muscle utilization.     

Correlation of nicotinic binding with neurochemical markers in Alzheimer's disease

The loss of neocortical synapses that occurs in Alzheimer's disease (AD) has been shown to correlate with cognitive decline. In addition, marked losses in the cholinergic system in AD, specifically choline acetyltransferase (ChAT) activity and high affinity presynaptic neuronal nicotinic cholinergic receptors (nAChRs), have also been described. We hypothesized that in AD, the loss of [3H]-ligand binding to nAChRs, which are largely presynaptic, would correlate with changes in two other presynaptic markers: synaptophysin (Syn), a measure of synaptic density, and ChAT activity. The midfrontal (MF) cortex of 36 autopsy confirmed (NIA and CERAD criteria) AD patients (mean death age +/- SD 80.1 +/- 8.4 years) who met NINDS-ADRDA criteria for a clinical diagnosis of probable or possible AD, and 11 nondemented controls (mean death age +/- SD 77.9 +/- 8.0) were examined. Synapse counts were quantified by a dotimmunobinding assay for Syn. ChAT activity was assessed by standard biochemical assays. Nicotinic cholinergic receptor binding was assayed using the high affinity nicotinic agonist [3H]-(+/-)-epibatidine ([3H]-EPI). The mean +/- SD Syn in AD (83.4 +/- 31.9 arbitrary units (AU)/mg protein) was significantly lower than controls (126.1 +/- 19.9, p = 0.0003; t-test). The mean ChAT activity in AD (139.0 +/- 75.6 nmol ACh/hr/100 mg protein) was significantly lower than controls (219.6 +/- 70.8, p = 0.004). The mean [3H]-EPI total binding in AD (6.2 +/- 2.8 fmol/mg protein) was significantly lower than controls (14.8 +/- 3.2; p < 0.0001). Syn correlated with [3H]-EPI binding in AD (r = 0.48, p = 0.006; Pearson) but ChAT did not (r = -0.20, p = 0.34). We conclude that loss of high affinity nAChR binding correlates with loss of synapses in AD. The lack of correlation between [3H]-EPI binding and ChAT activity suggests that the targeted receptor populations may not be located exclusively on cholinergic neurons.     

Characterization of a surface metalloprotease from Herpetomonas samuelpessoai and comparison with Leishmania major promastigote surface protease

It has been suggested that the thymic polypeptide thymopoietin interacts with skeletal muscle nicotinic acetylcholine receptors (nAChRs) and brain nicotinic receptors labeled by alpha-bungarotoxin (alpha-BuTX). In this study, the effects of neonatal thymectomy on the density of skeletal muscle nAChRs and the density of brain alpha-BuTX binding sites at 7 and 45 days postnatal were investigated. In addition, the densities of receptors in the skeletal muscle and brain of the athymic nu/nu mouse at 42 days postnatal were measured. We found little evidence that the absence of the thymus alters the density of nicotinic receptors in either skeletal muscle or brain.     

A new alpha-conotoxin which targets alpha3beta2 nicotinic acetylcholine receptors

We have isolated a 16-amino acid peptide from the venom of the marine snail Conus magus which potently blocks nicotinic acetylcholine receptors (nAChRs) composed of alpha3beta2 subunits. This peptide, named alpha-conotoxin MII, was identified by electrophysiologically screening venom fractions against cloned nicotinic receptors expressed in Xenopus oocytes. The peptide's structure, which has been confirmed by mass spectrometry and total chemical synthesis, differs significantly from those of all previously isolated alpha-conotoxins. Disulfide bridging, however, is conserved. The toxin blocks the response to acetylcholine in oocytes expressing alpha3beta2 nAChRs with an IC50 of 0.5 nM and is 2-4 orders of magnitude less potent on other nAChR subunit combinations. We have recently reported the isolation and characterization of alpha-conotoxin ImI, which selectively targets homomeric alpha7 neuronal nAChRs. Yet other alpha-conotoxins selectively block the muscle subtype of nAChR. Thus, it is increasingly apparent that alpha-conotoxins represent a significant resource for ligands with which to probe structure-function relationships of various nAChR subtypes.     

Rat alpha3/beta4 subtype of neuronal nicotinic acetylcholine receptor stably expressed in a transfected cell line: pharmacology of ligand binding and function

We stably transfected human kidney embryonic 293 cells with the rat neuronal nicotinic acetylcholine receptor (nAChR) alpha3 and beta4 subunit genes. This new cell line, KXalpha3 beta4R2, expresses a high level of the alpha3/beta4 receptor subtype, which binds (+/-)- [3H]epibatidine with a Kd value of 304+/-16 pM and a Bmax value of 8942 +/- 115 fmol/mg protein. Comparison of nicotinic drugs in competing for alpha3/beta4 receptor binding sites in this cell line and the binding sites in rat forebrain (predominantly alpha4/beta2 receptors) revealed marked differences in their Ki values, but similar rank orders of potency for agonists were observed, with the exception of anatoxin-A. The affinity of the competitive antagonist dihydro-beta-erythroidine is >7000 times higher at alpha4/beta2 receptors in rat forebrain than at the alpha3/beta4 receptors in these cells. The alpha3/beta4 nAChRs expressed in this cell line are functional, and in response to nicotinic agonists, 86Rb+ efflux was increased to levels 8-10 times the basal levels. Acetylcholine, (-)-nicotine, cytisine, carbachol, and (+/-)-epibatidine all stimulated 86Rb+ efflux, which was blocked by mecamylamine. The EC50 values for acetylcholine and (-)-nicotine to stimulate 86Rb+ effluxes were 114 +/- 24 and 28 +/- 4 microM, respectively. The rank order of potency of nicotinic antagonists in blocking the function of this alpha3/beta4 receptor was mecamylamine > d-tubocurarine > dihydro-beta-erythroidine > hexamethonium. Mecamylamine, d-tubocurarine, and hexamethonium blocked the function by a noncompetitive mechanism, whereas dihydro-beta-erythroidine blocked the function competitively. The KXalpha3 beta4R2 cell line should prove to be a very useful model for studying this subtype of nAChRs.     

The power of support groups: influence on infant feeding trends in New Zealand

The effect of streptozotocin (STZ)-induced diabetes and a combination of chronic treatment with haloperidol (HPD) on dopamine (DA)D2, serotonin (5-HT) 5-HT1A and 5-HT2A receptors was investigated in rat brain. Rats were randomly assigned to one of four groups: vehicle-vehicle, STZ-vehicle, vehicle-HPD, and STZ-HPD groups. Four weeks after single administration of STZ (65 mg/kg IV) or vehicle (citrate buffer), rats received depot HPD (4 mg/kg IM) or vehicle (sesame oil) once a week for 4 weeks. Sixteen days after the last injection of HPD or vehicle, rats were sacrificed, and the density of binding sites was determined using [3H]spiperone as ligand in the striatum (D2),[3H]8-hydroxy-2-(di-n-propyl)-aminotetraline in the hippocampus (5-HT1A), and [3H]ketanserin in the frontal cortex (5-HT2A). The density of D2 receptors was significantly increased in the vehicle-HPD compared to vehicle-vehicle controls. However, striatal D2 receptor density of the STZ-HPD and the STZ-vehicle were not significantly different from the vehicle-vehicle group. A significant increase in cortical 5-HT2A receptor density was observed only in the group of STZ-vehicle. Treatment with STZ, HPD, or the combination thereof, did not affect the density of 5-HT1A receptors. The affinity constants for D2, 5-HT1A, and 5-HT2A receptors were not affected by any treatment. These results suggest that diabetic state may affect brain serotonergic activity via an increase in the density of 5-HT2A receptors. This may indicate an increased vulnerability to major depression in patients with diabetes. The lack of an effect of the combined chronic treatment with STZ and HPD on the D2 receptor density may correspond to the increased risk to develop tardive dyskinesia in patients with diabetes.     

Selective visualization of rat brain 5-HT2A receptors by autoradiography with [3H]MDL 100,907

The recently developed 5-HT2A receptor selective antagonist [3H]MDL100,907 ((+/-)2,3-dimethoxyphenyl-1-[2-(4-piperidine)-methanol]) has been characterized as a radioligand for the autoradiographic visualization of these receptors. [3H]MDL100,907 binding to rat brain tissue sections was saturable, had sub-nanomolar affinity (Kd = 0.2-0.3 nM), and presented a pharmacological profile consistent with its binding to 5-HT2A receptors (rank order of affinity for [3H]MDL100,907-labelled receptors: MDL100,907 > spiperone > ketanserin > mesulergine). The distribution of receptors labelled by [3H]MDL100,907 was compared to the autoradiographical patterns obtained with [3H]Ketanserin, [3H]Mesulergine, and [3H]RP62203 (N-[3-[4-(4-fluorophenyl)piperazin-1-y1]propyl]-1,8-naphtalenes ultam) and to the distribution of 5-HT2A receptor mRNA as determined by in situ hybridization. As opposed to the other radioligands, [3H]MDL100,907 labelled a single population of sites (5-HT2A receptors) and presented extremely low levels of non-specific binding. The close similarity of the distributions of [3H]MDL100,907-labelled receptors and 5-HT2A mRNA further supports the selectivity of this radioligand for 5-HT2A receptors and suggests a predominant somatodendritic localization of these receptors. The present results point to [3H]MDL100,907 as the ligand of choice for the autoradiographic visualization of 5-HT2A receptors.     

Characterization of cholecystokinin receptors in canine intestinal circular muscle

We localized and characterized the binding of [3H](+/-)-L364,718 in canine small intestine circular muscle. The highest densities of [3H]L364,718 binding were located in the fraction enriched in deep muscular plexus synaptosomal membranes. In this fraction [3H]L364,718 binding was of high density (Bmax 136.78 +/- 53.66 fmol/mg) and high affinity (Kd 1.67 +/- 0.74 nM). Kinetics studies revealed that binding was reversible and yielded a similar Kd value. L364,718, CCK-8-S, and L365,260 fully displaced [3H]L364,718 binding, but ligands at CCKB receptors, gastrin-17, and YM022 did not. Therefore, CCKA receptors in canine intestine circular muscle are located on nerve endings.     

Alpha 5 subunit forms functional alpha 3 beta 4 alpha 5 nAChRs in transfected human cells

nAChRs heterologously expressed in human cells after transient transfection with alpha 3 beta 4 alpha 5 or alpha 3 beta 4 subunit cDNAs exhibited similar sensitivities to antagonists and comparable functional channel profiles. However, the sum of two Hill equations was required for best fitting the ACh dose-current response curves after co-expression of alpha 5, alpha 3 and beta 4 subunits. One component was comparable to that obtained in alpha 3 beta 4-transfected cells, while the additional component, putatively attributed to an alpha 3 beta 4 alpha 5 nAChR population, showed a Hill coefficient > 2 and a nine-fold greater half-maximal ACh concentration (EC50). These results suggest that the alpha 5 subunit participates in the assembly of alpha 3 beta 4 alpha 5 nAChRs complexes in human cells, adding a new member to the family of neuronal nicotinic receptors.     

The heterocyclic substituted pyridine derivative (+/-)-2-(-3-pyridinyl)-1-azabicyclo[2.2.2]octane (RJR-2429): a selective ligand at nicotinic acetylcholine receptors

The present report describes in vitro studies demonstrating that the heterocyclic substituted pyridine compound (+/-)-2-(3-pyridinyl)-1-azabicyclo[2.2.2]octane (RJR-2429) is extremely potent in activating human muscle nicotine ACh receptor (nAChR) (EC50 = 59 +/- 17 nM; Emax = 110 +/- 09% vs. nicotine). RJR-2429 is markedly less potent in activating nAChRs in the clonal cell line PC12, with EC50 = 1100 +/- 230 nM and Emax = 85 +/- 20% when compared with nicotine. The activation of a putative alpha 3 beta 4-containing nAChR in PC12 by RJR-2429 reveals a potency intermediate between nicotine and epibatidine (EC50 of 20,000 nM for nicotine and 30 nM for epibatidine). Dose-response curves for agonist-induced ileum contraction indicate that RJR-2429 is equipotent with nicotine, having an EC30 of approximately 2 microM. RJR-2429 binds with high affinity to alpha 4 beta 2 receptor subtype (Ki = 1.0 +/- 0.3 nM), and chronic exposure results in significant up-regulation of the high-affinity [3H]nicotine binding sites. In addition, RJR-2429 does not activate nAChRs present in rat thalamic preparations but is a potent inhibitor of this receptor subtype. It antagonizes nicotine-stimulated ion flux in thalamic synaptosomes with an IC50 of 154 +/- 37 nM. It also is a potent partial agonist at nAChRs mediating dopamine release from rat synaptosomal preparations (EC50 = 2 +/- 1 nM; Emax = 40%; epibatidine and nicotine have EC50 values of 0.4 and 100 nM, respectively). A model for the structure-activity profile of RJR-2429, nicotine and epibatidine was derived by molecular forcefield and quantum mechanics calculations and may provide important clues for the development of ligands selective for nAChR subtypes as probes in the life sciences or as potential therapeutic tools.     

The interaction of McN-A-343 with muscarine receptors in cardiac and smooth muscle

The interaction of the muscarine receptor partial agonist (4-m-chlorophenylcarbamoyloxy)-2-butynyltrimethylammonium chloride (McN-A-343) was investigated at muscarine receptors in the atria and taenia caeci of the guinea-pig to compare its interaction at the muscarine M2 receptor in the two tissues. In the smooth muscle, the muscarine M3 receptor subtype is responsible for the contractile response but the major subtype detected in binding or antibody experiments is the M2 subtype. In guinea pig atria the dissociation constant of McN-A-343 at muscarine receptors was 15.2 microM determined in functional experiments on left atria in McEwen's solution or 14.8 microM in binding experiments with [3H]-(-)-quinuclidinyl benzilate ([3H]QNB) in the same medium containing 5'-guanylylimododiphosphate (50 microM). In the taenia caeci, the dissociation constant estimated for McN-A-343 at the M3 receptor from functional experiments based on the contractile response to the agonist in McEwen's solution was 4.6 microM. This value was similar to the dissociation constant (6.2 microM) estimated from binding studies versus [3H]QNB conducted in the same medium although studies with 11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H- pyrido[2,3-b][1,4]benzodiazepine 6-one (AF-DX 116) versus [3H]-(-)-N-methylscopolamine suggested that 70% of the receptors were the M2 subtype. The presence of the M2 subtype in the taenia caeci was also confirmed by the ability of oxotremorine to inhibit the increase in cAMP produced by isoprenaline (10 microM) since apparent pKB values for AF-DX 116 and hexahydrosiladiphenidol were 6.95 and 6.75, respectively. McN-A-343 (100 microM) failed to inhibit the response to isoprenaline and did not antagonize the inhibitory response to oxotremorine. It is concluded that the apparent affinity of McN-A-343 for muscarine M2 receptors in the atria and the taenia caeci differs and a number of explanations are discussed.     

Solubilized rabbit striatal A2a-adenosine receptors: stability and antagonist binding

The A2a-adenosine binding subunit from rabbit striatal membranes was solubilized using 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and was characterized using the antagonist radioligand [3H]8-[4-[[[[2-aminoethyl)amino]carbonyl]methyl]oxy] phenyl]-1,3-dipropylxanthine (XAC). The solubilized receptor was very stable, with 55% of the specific [3H]XAC binding remaining after storage for 15 days at 4 degrees C. The dissociation constant (Kd) for binding of [3H]XAC to solubilized A2 receptors was determined in saturation studies to be 4.0 nM, with a Bmax of 600 fmol/mg protein. Xanthine inhibitors displaced the specific binding of the adenosine antagonist [3H]XAC (in the presence of 50 nM 8-cyclopentyl-1,3-dipropylxanthine) at 25 degrees C, with Ki values consonant with the expected affinities at A2a receptors. Binding of [3H]XAC (1 nM) or the adenosine agonist [3H]2-(carboxyethylphenylethylamino)adenosine-5'-N-ethyl carboxamide (5 nM) to A2a receptors was diminished in the presence of 0.1 M Na+ in both membranes and solubilized preparations. Agonist binding was increased (by 280% for membranes and 180% for solubilized receptors), and antagonist binding was decreased in the presence of 10 mM Mg2+. Displacement of [3H]XAC by the agonist (R)-N6-phenylisopropyladenosine was biphasic, corresponding to high (IC50 = 188 nM, RH = 30%) and low (IC50 = 9730 nM, RL = 70%) affinity sites. Preincubation with 100 microM GTP (10 mM Mg2+) converted the high affinity binding to low affinity, suggesting that receptor and G-protein are dissociated by the guanine nucleotide. The solubilized receptor was more easily inactivated by exposure to the reducing agent dithiothreitol (IC50 = 3 mM) than in membranes (IC50 = 220 mM), suggesting increased accessibility of structurally essential disulfide bridges.