Autoradiographic localization of muscarinic receptors within the rat kidney

We used a combination of radioreceptor binding and autoradiographic techniques to study the pharmacological characteristics and anatomical localization of [3H]quinuclidinyl benzylate (QNB) in kidney sections of Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). [3H]QNB was bound by sections of rat kidney in a manner consistent with the labeling of muscarinic acetylcholine receptors. Anatomically, these receptor sites were located primarily within the smooth muscle of the renal vascular tree and to a lesser extent within cortical and medullary tubules. The density of renal muscarinic acetylcholine receptors (both vascular and tubular) was decreased in hypertension but the affinity of the ligand for muscarinic receptors was unchanged. A possible role of muscarinic acetylcholine receptors in renal function is suggested.     

An autoradiographic study of muscarinic cholinoceptors in blood vessels: no localization on vascular endothelium

In vitro labelling and autoradiographic techniques were used to examine the localization of [3H]quinuclidinyl benzilate ([3H]QNB) and [125I]4-iodo-QNB ([125I]4IQNB) to slide-mounted sections of rabbit aorta and pulmonary artery, cat aorta, pulmonary and superior mesenteric arteries. These vessels all respond to acetylcholine (ACh) with endothelium-dependent relaxation, yet there was no evidence for endothelium-related binding of either [3H]QNB or [125I]4IQNB. Muscarinic receptors were localized over the medial smooth muscle and, in the rabbit pulmonary artery, the density of binding increased towards the adventitia. Binding of either radioligand to sections of rabbit pulmonary artery was not affected by the muscarinic M1 receptor antagonist pirenzepine (20 nM) but was markedly reduced by the muscarinic M2 antagonist 4DAMP (4-diphenylacetoxy-N-methyl-piperidine methobromide) (1 nM). This study provides evidence for muscarinic receptors located directly on smooth muscle cells, indicating that endothelium-dependent relaxation to ACh results from an indirect mechanism involving smooth muscle muscarinic receptors.     

Characterization of cholinergic muscarinic receptors in cow tracheal muscle membranes. Effect of maturation

The parasympathetic nervous system is important in the control of basal airway muscle tone and caliber. We characterized muscarinic cholinergic receptors in isolated tracheal membranes from cows of three age groups (immature, less than 2 weeks; transition, 3-5 months; and mature, greater than 5 years) using l-[3H]quinuclidinyl benzilate (l-[3H]QNB) as the radioligand. There were significant decreases in the densities of l-[3H]QNB binding sites with maturation (Bmax: 2344 +/- 169 vs 1381 +/- 85 vs 1116 +/- 80 fmol/mg protein for tissues from immature, transition and mature cows respectively). No change in the dissociation constant was observed with maturation (Kd: 0.38 +/- 0.09 vs 0.55 +/- 0.06 vs 0.50 +/- 0.07 nM for tissues from immature, transition and mature animals respectively). The association and dissociation rate constants did not vary between tissues from immature and mature animals. The specific activity of the enzyme, acetylcholinesterase, was correlated with the density of l-[3H]QNB binding sites present in the tracheal homogenates; that is, with maturation, there were significant decreases in acetylcholinesterase activity [0.28 +/- 0.01 vs 0.16 +/- 0.02 vs 0.08 +/- 0.01 mol X l-1 X min-1 X (mg protein)-1 for tissues from immature, transition and mature animals respectively]. All competition binding studies using muscarinic antagonists exhibited single site binding and did not show any differences in drug affinities between the age groups. In contrast, multiple binding sites were observed with carbachol, methacholine and muscarine, and there were significant decreases in receptor affinities for the muscarinic agonists. No changes in the proportion of high and low affinity sites were found. These results indicate that with maturation there are alterations in the properties of muscarinic receptors in tracheal smooth muscle.     

Muscarinic receptors in canine colonic circular smooth muscle. I. Coexistence of M2 and M3 subtypes.

The parasympathetic neurotransmitter acetylcholine, acting postsynaptically at the smooth muscle muscarinic receptor, is a principle determinant of colonic motility. In order to elucidate the receptor signal-transduction events responsible for muscarinic receptor-induced contraction of colonic circular smooth muscle, we present here and in the accompanying work studies designed to characterize the muscarinic receptors present in colon and to determine their biochemical coupling. Muscarinic receptor subtypes in canine colonic circular smooth muscle were characterized using radioligand binding techniques. The nonselective muscarinic receptor antagonist radioligand [3H]quinuclidinyl benzilate ([3H]QNB) binds rapidly and reversibly to a single class of saturable sites in colon circular smooth muscle membranes, with an affinity (KD) for the antagonist radioligand of 79.8 +/- 12.6 pM and a density of 123.3 +/- 18.7 fmol/mg of protein. Experiments using membranes prepared from isolated cells purified from the circular smooth muscle layer of canine colon (KD = 102.4 +/- 13.5 pM) confirm the smooth muscle origin of the binding and yield a receptor density of 124,340 receptors/cell. The order of potencies of selective muscarinic receptor antagonists in competition with [3H]QNB for binding to colonic receptors is 4-diphenylacetoxy-N-methylpiperidine methobromide greater than methoctramine greater than AF-DX 116 greater than pirenzepine. Unlike other antagonists tested, pirenzepine competition of [3H]QNB binding is biphasic. The high and low affinities deduced from nonlinear fit of the binding data in colon correlate very well with affinities determined for pirenzepine in mixtures of both submandibular gland (M3) and atrium (M2), indicating the presence of two muscarinic receptor subtypes (82% M2, 18% M3) in colon circular smooth muscle. The muscarinic agonist carbachol binds to both high and low affinity sites in colon, and addition of guanine nucleotide (100 microM GTP gamma S) shifts the agonist competition curve to the right, without eliminating high affinity binding sites. Agonist competition studies with a known ratio of M2 and M3 receptors, obtained by mixing pure M2 and M3 populations, predict the result obtained in colon. cDNA probes specific for each of the muscarinic receptors m1 through m4 were hybridized to colon RNA in a Northern blot analysis. Only m2 and m3 probes hybridized to colon RNA, suggesting the presence of both M2 and M3 receptors. Our data demonstrate that the colon circular smooth muscle contains muscarinic receptors of both the M2 and M3 subtypes, which may be coupled to disparate signal transduction pathways important in the physiological actions of acetylcholine in this tissue.     

Regulation of muscarinic receptor subtypes and their responsiveness in rat brain following chronic atropine administration

Chronic administration of l-atropine to rats caused a dose-dependent increase (30%) in the density of muscarinic receptors, as measured with [3H]quinuclidinyl benzilate ([3H]QNB], in cortex (CTX), dorsal hippocampus (DH), and heart but not the corpus striatum. Serum concentrations of l-atropine reached 80 to 160 nM within 6 hr, whereas densities of binding sites for [3H]QNB did not show a significant increase until after the second day of infusion and receptor densities did not reach new steady state levels until after the fourth day of infusion. The density of binding sites for the M1-selective muscarinic receptor antagonist, [3H]pirenzepine ([3H]PZ) was also measured. As noted previously, the density of binding sites for [3H]PZ (defined as M1) was lower than the density of binding sites for [3H]QNB (defined as M1 plus M2). When the densities of binding sites for [3H]QNB and [3H]PZ in CTX plus DH were determined after 14 days of treatment, [3H]QNB binding sites showed a 28% increase, whereas [3H]PZ binding sites did not show any increase. The difference between the densities of binding sites for [3H]QNB and [3H]PZ, an estimate of the density of M2 sites, doubled. The density of binding sites for [3H]QNB appeared to be stable for at least 64 hr after the withdrawal of the drug. The increase in the density of binding sites for [3H]QNB was not reflected in the binding of [3H]oxotremorine-M ([3H]OXO-M), a muscarinic agonist, which was unchanged by l-atropine administration. Because the binding of [3H]OXO-M is sensitive to GTP, this observation suggests that the "induced" receptors may not be coupled to a guanine nucleotide-binding protein. In spite of the fact that there was a doubling of the density of M2 sites, no significant differences in dose-response curves for carbachol-induced inhibition of [3H]cAMP accumulation were observed in slices of CTX plus DH from control and l-atropine-treated rats. Similarity, acetylcholine-stimulated accumulation of [3H]inositol phosphates in slices of CTX plus DH showed no significant differences between the tissues from control and treated rats.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparative studies of muscarinic and dopamine receptors in three strains of rat

Cholinesterase activities and characteristics of muscarinic and dopamine receptors from 9 week old male Sprague-Dawley (SD), Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) were studied. Plasma cholinesterase activity in WKY was significantly lower (50%) than activity in the other strains. In studies of muscarinic receptors, the number of [3H]QNB binding sites in striata from SD rats was lower (18%) than those from WKY and SHR. However, muscarinic receptor properties (Kd and Bmax) were the same in hypothalami. Studies of dopamine receptors revealed that the densities of both D-1 and D-2 receptors in both striata and hypothalami were significantly higher in SHR than in other strains. However, there were no differences in the affinity constant (Kd). The higher densities in hypothalami from SHR were mainly due to the high population of D-1 and D-2 receptors in the posterior hypothalamus. In the anterior hypothalamus, there was no difference in the population of D-2 receptors. These results provide a substantive basis, i.e. demonstration of alterations in drug metabolizing enzymes and receptor populations, on which to build an understanding of the genetic predisposition to the actions of xenobiotic agents.     

Positive cooperativity in the binding of alcuronium and N-methylscopolamine to muscarinic acetylcholine receptors

The effect of the neuromuscular blocker alcuronium on the binding of N-[3H]-methylscopolamine [( 3H]NMS) and l-[3H]quinuclidinylbenzilate ([3H]QNB) to muscarinic binding sites in rat heart atria, longitudinal smooth muscle of the ileum, cerebral cortex, cerebellum, and submaxillary glands was measured using filtration techniques. In the presence of 10(-5) M alcuronium, the binding of [3H]NMS (which was present at a subsaturating concentration of 2 x 10(-10) M) was increased 5.3-fold in the atria and smooth muscle and 3-fold in the cerebellum; no increase was observed in the brain cortex and salivary glands. The binding of [3H]NMS was inhibited at 10(-3) M and higher concentrations of alcuronium. The rates of [3H]NMS association to and dissociation from muscarinic binding sites in the atria were diminished by 10(-5) M alcuronium. Scatchard plots of [3H]NMS binding data obtained with and without 10(-5) M alcuronium indicated that the maximum number of binding sites was not altered by the drug, whereas the apparent Kd for [3H]NMS was diminished. In contrast to [3H] NMS, the effects of alcuronium on the binding of [3H]QNB were only inhibitory. The concentration of alcuronium required to diminish the binding of [3H]QNB by 50% (IC50) was 4-7 microM in the atria, ileal smooth muscle, and the cerebellum, 140 microM in the brain cortex, and 1200 microM in the parotid gland. The results suggest that the binding of low concentrations of alcuronium to muscarinic receptors in the heart, ileal smooth muscle, and cerebellum allosterically increases the affinity of muscarinic receptors towards [3H]NMS, although not [3H]QNB. At high concentrations, alcuronium inhibits the binding of muscarinic ligands, presumably by competition for the classical muscarinic binding site. Positive cooperativity induced by alcuronium appears to be specific for the m2 (cardiac) subtype of muscarinic receptors.     

Muscarinic receptors of the vascular bed: radioligand binding studies on bovine splenic veins

Despite an obvious lack of parasympathetic innervation to the spleen, pharmacological evidence suggests the presence of cholinergic receptors in isolated bovine splenic veins. We therefore studied muscarinic cholinergic binding sites in a bovine splenic vein preparation by direct radioligand binding techniques using [3H]quinuclidinyl benzilate ([3H]QNB) as radioactive probe. Saturation experiments indicated one homogeneous class of high-affinity binding sites, with a KD of 0.11 nM and a binding site density Bmax of 55 fmol/mg protein. The rate constants at 37 degrees C for formation and dissociation of the [3H]QNB receptor complex were 2.7 X 10(9) M-1 h-1 and 0.38 h-1, respectively, yielding a KD of 0.14 nM. The binding sites showed a high stereospecificity, which was evident from competition experiments with dexetimide (KI = 1.3 nM) and levetimide (KI = 4.6 microM). In competition experiments with muscarinic and nicotinic antagonists and some antidepressants, only one binding site was found, whereas with muscarinic agonists, two binding sites were detected. In the presence of 0.1 mM guanyl-imido-diphosphate, only one binding site could be identified with the muscarinic agonist carbamylcholine. The affinity of [3H]QNB, on the other hand, was slightly decreased, and Bmax values were unchanged. It is concluded that specific, saturable, high-affinity muscarinic binding sites in the bovine splenic vein have been identified and characterized that exhibit properties similar to cholinergic receptors of brain and peripheral tissues and probably mediate acetylcholine-induced relaxation of splenic veins.     

Muscarinic binding sites on bovine pulmonary arterial endothelial cells in culture.

We have investigated the presence and nature of muscarinic binding sites on membranes from cultured bovine pulmonary arterial endothelial cells (BPAE). BPAE were harvested and subcultured nonenzymatically; experiments were performed 3-5 days postconfluence and between 10 and 25 passage numbers. Utilizing radioligand binding techniques with the muscarinic receptor antagonists [3H]3-quinuclidinyl benzilate ([3H]QNB) and [3H]N-methylscopolamine ([3H]MS) as probes, we identified a small population of atropine-sensitive muscarinic sites (1,800-2,000 sites/cell or 7-8 fmol/mg protein). Muscarinic binding sites on BPAE membranes resembled classical muscarinic receptors in that (a) the binding of 2 nM [3H]QNB was inhibited by muscarinic agonists and antagonists, (b) [3H]QNB binding was 30 times more sensitive to R(-)- than to S(+)-QNB, (c) binding of the muscarinic receptor agonist carbamylcholine involved high and low affinity components, (d) the stable GTP analog, Gpp(NH)p (100 microM) shifted agonist binding curves to the right by a factor of three, and (e) the high affinity binding of the agonist [3H]oxotremorine-M to muscarinic receptors was depressed by Gpp(NH)p. On the other hand, gallamine, which allosterically regulates muscarinic receptor binding in other tissues, did not affect the rates of dissociation of [3H]QNB, [3H]MS or [3H]oxotremorine-M from BPAE binding sites. We concluded that BPAE in culture exhibit muscarinic binding sites which possess many but not all of the properties associated with classical muscarinic receptors.     

Muscarinic receptor subtype in porcine coronary artery

The specific binding of (-)-[³H]QNB (quinuclidinyl benzilate) in membrane fractions of porcine coronary artery was saturable, of high affinity and stereoselective. It has been shown that there exist (-)-[³H]QNB binding sites with high (K_i = 12 nM)- and low(K_i = 1010 nM)-affinity for pirenzepine in the coronary artery but predominantly low-affinity sites in cardiac muscle. AF-DX 116 and gallamine showed a lower affinity to ( - )-[³H]QNB binding sites in the coronary artery compared to cardiac muscle. Thus, the present study suggests that porcine coronary artery contains a significant number of muscarinic receptors, probably both M₁ and M₂ subtypes.     

BIOCHEMICAL CHARACTERISTICS OF MUSCARINIC CHOLINORECEPTORS IN SWINE TRACHEAL SMOOTH MUSCLE

• 1 The tritiated muscarinic cholinoreceptor antagonist quinuclidinyl benzilate, [³H]QNB, was used to characterize the muscarinic receptors associated with homogenized membrane of the smooth muscle from swine trachea. Based on receptor binding assays, the homogenate had specific, saturable, high-affinity receptors for [³H]QNB.
• 2 Specific binding was time- and temperature-dependent. The association of [³H]QNB with the muscarinic receptor reached equilibrium much sooner at 37°C than 25°C at a [³H]QNB concentration of 180 pM (30 min and 2 h, respectively). Equilibrium at both temperatures was attained within 5 min at a [³H]QNB concentration of 1800 pM. All remaining experiments were performed at 37°C.
• 3 Binding was saturable with respect to [³H]QNB and tissue concentrations. Analysis of binding isotherms yielded an apparent equilibrium dissociation constant (K_D) of 51±20 pM and a maximum receptor density (B_max) of 2.17±0.27 pmole/mg protein. The Hill coefficient for [³H]QNB binding was 1.07±0.16. The association (K₁) and dissociation (K_-1) rate constants were determined to be (5.51±0.16) × 10⁸ M^?1 min^?1 and (1.41±0.18) × 10^?2 min^?1, respectively. K_D calculated from the ratio of K₁ and K_-1 was 26.3±3.8 pM; this value is close to the value of K_D calculated from Scatchard plots of binding isotherms.
• 4 The density of muscarinic receptor binding sites was 10-fold greater in tracheal smooth muscle than in tracheal epithelium (0.20±0.03 pmole/mg protein). There is no difference between weanling and young adult swine in the density of muscarinic receptors in tracheal smooth muscle.
• 5 The nonselective muscarinic antagonists atropine, scopolamine and quinuclidinyl benzilate (QNB) competitively inhibited [³H]QNB binding to the homogenate with Hill coefficients of 0.9-1.0 and inhibition constants (K_i) of nanomolar range.
• 6 Competition with selective muscarinic antagonists pirenzepine and 3-quinuclidinyl xanthene-9-carboxylate (QNX) gave K_i values, 0.26 M and 0.78 nM, respectively, and Hill coefficients of approximately 1. There was a single population of [³H]QNB binding sites of the M₂ subtype for all tested muscarinic antagonists.
• 7 Competition with selective muscarinic agonists pilocarpine and carbachol yielded K_i values of micromolar range, Hill coefficients of less than 1, and revealed the existence of two binding sites (P < 0.01).

     

Parainfluenza virus type 1 reduces the affinity of agonists for muscarinic receptors in guinea-pig lung and heart

Membrane preparations of guinea-pig lung (containing multiple muscarinic receptor subtypes) and heart (containing M2 receptors only) were incubated with either neuraminidase, parainfluenza virus (which contains neuraminidase), or virus plus 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, a neuraminidase inhibitor. None of these treatments affected [3H]quinuclidinyl benzilate [( 3H]QNB) binding. In the lung and heart, carbachol displaced 0.2 nM [3H]QNB from two sites. After treatment with either neuraminidase or virus the high affinity site was shifted to the right, and carbachol displaced QNB from one site with low affinity in the lung. In contrast, neuraminidase or virus decreased the affinity of carbachol for both sites in the heart. The neuraminidase inhibitor completely blocked virus-induced changes in carbachol affinity in both tissues. These results suggest that parainfluenza virus decreases the affinity of agonists for some of the muscarinic receptors in the lung, and for all of the muscarinic receptors in the heart due to its neuraminidase activity, which results in removal of sialic acid. The decreased agonist affinity in the lung may be responsible for the increased vagally induced bronchoconstriction seen in viral respiratory infections.     

Increased 45Ca influx in response to alpha 1-adrenoceptor stimulation in spontaneously hypertensive rat caudal artery

The isometric tension development and 45Ca influx in response to norepinephrine (NE) and methoxamine stimulation were investigated in caudal arteries of spontaneously hypertensive rats (SHR) and Wistar Kyoto normotensive rats (WKY). The maximum isometric tension developed as well as 45Ca influx in response to NE and methoxamine stimulation were significantly increased (p less than 0.05) in SHR caudal arteries as compared with WKY. On the other hand, neither the isometric tension developed nor the 45Ca influx in response to K+ depolarization were different between WKY and SHR caudal arteries. Estimation of [3H]prazosin binding to the membranes isolated from caudal artery of WKY and SHR showed a single class of high-affinity binding sites with Kd values for SHR 128 +/- 14 pM and for WKY 141 +/- 19 pM, and Bmax values for SHR 108 +/- 14 fmol/mg protein and for WKY 113 +/- 21 fmol/mg protein. From these results, we conclude: (a) Increased contractile response of SHR caudal artery rings to alpha 1-adrenoceptor stimulation appears at least in part to be due to an increased Ca2+ influx through receptor-operated Ca2+ channels; (b) the affinity or density of alpha 1-adrenoceptors estimated by [3H]prazosin binding is not altered in the SHR caudal artery.     

Presynaptic and postjunctional muscarinic receptors in dog ileum: binding studies

[3H]N-Methylscopolamine identified two distinct populations of muscarinic receptors in membranes derived from the longitudinal smooth muscle/myenteric plexus of dog ileum. In isolated axonal varicosities, the half-maximal saturation of binding sites occurred at 2.38 +/- 0.39 nM [3H]N-methylscopolamine, with maximal binding capacity 140 +/- 35 fmol/mg protein (mean +/- S.D., n = 8). In purified smooth muscle plasma membranes, the Kd value was 16 +/- 3 nM with Bmax 1960 +/- 494 fmol/mg. The displacement potencies of subtype-selective muscarinic antagonists in the fraction of axonal varicosities followed the order 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) methiodide much greater than pirenzepine = methoctramine greater than AF-DX 116 with pKi values 7.38, 5.67, 5.70 and 5.13, respectively. Both 4-DAMP methiodide and pirenzepine were approximately 4-fold less potent in displacing the ligand from the receptors in smooth muscle plasma membranes as compared to varicose receptors. The potency ratios of cardioselective antagonists methoctramine and AF-DX 116 on varicose and smooth muscle receptors were 1 and 1.7. It is concluded that presynaptic receptors located on isolated axonal varicosities have pharmacological properties similar to glandular (M3) subtype of muscarinic receptors. The binding properties of receptors present in smooth muscle plasma membranes were found incompatible with those of any of the M1, M2 or M3 subtypes.     

Inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate binding sites in smooth muscle.

1. We have previously demonstrated that activation of M3 muscarinic receptors increases inositol 1,4,5-trisphosphate (InsP3) and inositol 1,3,4,5-tetrakisphosphate (InsP4) accumulation in colonic smooth muscle. 2. In the present study, we demonstrate the existence of InsP3 and InsP4 binding sites in colonic circular smooth muscle by use of radioligand binding methods. Both [3H]-InsP3 and [3H]-InsP4 bound rapidly and reversibly to a single class of saturable sites in detergent-solubilized colonic membranes with affinities of 5.04 +/- 1.03 nM and 3.41 +/- 0.78 nM, respectively. The density of [3H]-InsP3 binding sites was 335.3 +/- 19.3 fmol mg-1 protein which was approximately 2.5 fold greater than that of [3H]-InsP4 sites (127.3 +/- 9.1 fmol mg-1 protein). 3. The two high affinity inositol phosphate binding sites exhibited markedly different pH optima for binding of each radioligand. At pH 9.0, specific [3H]-InsP3 binding was maximal, whereas [3H]-InsP4 binding was only 10% that of [3H]-InsP3. Conversely, at pH 5.0, [3H]-InsP4 binding was maximal, while [3H]-InsP3 binding was reduced to 15% of its binding at pH 9.0. 4. InsP3 was about 20 fold less potent (KI = 50.7 +/- 8.3 nM) than InsP4 in competing for [3H]-InsP4 binding sites and could compete for only 60% of [3H]-InsP4 specific binding. InsP4 was also capable of high affinity competition with [3H]-InsP3 binding (KI = 103.5 +/- 1.5 nM), and could compete for 100% of [3H]-InsP3 specific binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Down-regulation of muscarinic receptors in the striatum of organophosphate-treated swine

Subacute (daily) administration of diisopropylfluorophosphate (DFP) to male swine (Yorkshire white) resulted in a 97% inhibition of cholinesterase and a decrease of [3H]quinuclidinyl benzilate [( 3H]QNB) binding sites in homogenates of striata by approximately 50% after 14 days. The maximal density of receptors (Bmax) decreased from 2.1 +/- 0.3 to 1.0 +/- 0.2 pmole/mg protein. There was no significant change in the dissociation constant (Kd) for [3H]QNB binding (control: 52.6 +/- 10.7 pM; 7-day: 57 +/- 2.8 pM). Carbachol displacement of [3H]QNB binding yielded data best fit by a two-binding site model. The dissociation constants were KiL = 115 +/- 62 microM (55 +/- 3%) and KiH = 1.8 +/- 0.7 microM (45 +/- 3%), respectively, for the low- and high-affinity states. Seven-Day treatment with DFP reduced the percentage of high-affinity receptors to 22 +/- 8.6%, but affected neither the low- nor the high-affinity Kd (100 +/- 20 and 2 +/- 0.6 microM). With the addition of Mg2+, striatal homogenates had low- and high-affinity receptors in the proportion of approximately 1 to 1. In the presence of Gpp(NH)p + Mg2+ the ratio of high- to low-affinity receptors was 3:1 in homogenates of control tissue (to 26 +/- 5%). This treatment had no effect on this ratio in homogenates of tissue from 7-day DFP-treated swine (3:1) since it was already 3:1. Pirenzepine displacement of [3H]QNB binding was best described by a two-binding site model, with Ki values of 38 +/- 14 and 201 +/- 78 nM, which represent 74 and 26% of the binding sites, respectively. The high affinity Kd value was unchanged following 7 days of DFP treatment (24 +/- 5 nM). There appears to be little change in the displacement curves for pirenzepine inhibition of [3H]QNB binding. This suggests that about 75% of the receptors are of the M1 subtype. Thus, subacute administration of DFP causes not only a decrease in the number of receptors, but also a change in the proportion of agonist affinity states which is related to the interaction of the guanine nucleotide binding protein and the muscarinic receptor.     

A possible mechanism for diisopropylfluorophosphate-induced memory loss in rats

Passive avoidance retention and cortical [H3]-quinuclidinyl benzilate (QNB) binding were examined in rats that were chronically treated with diisopropylfluorophosphate (DFP), an irreversible acetylcholinersterase inhibitor. Retention of a passive avoidance response in DFP-treated rats was significantly lower when compared to vehicle-treated controls. Passive-avoidance retention decreased from 93% in control animals to 68% in DFP-treated rats. QNB binding studies revealed the density of muscarinic receptors in cortical homogenates was significantly reduced from 0.95 +/- 0.04 pmole/mg protein in controls to 0.72 +/- 0.04 pmole/m protein in DFP-treated rats. Scatchard analysis of QNB binding curves did not reveal a decrease in affinity of muscarinic receptors for QNB. Based on data that DFP causes a reduction in cholinergic receptors, this study supports the hypothesis that central cholinergic receptors are associated with mechanisms involved in memory storage.     

3H]N-methylscopolamine binding to muscarinic receptors in human peripheral blood lymphocytes: characterization, localization on T-lymphocyte subsets and age-dependent changes

The properties of the binding of the muscarinic receptor ligands, [3H]quinuclidinyl benzilate ([3H]QNB) and [3H]N-methylscopolamine ([3H]NMS) in human mononuclear cells were compared. The binding of [3H]QNB showed a high, non-specific component and lack of saturability in both intact mononuclear cells and preparations of lysed mononuclear cell membranes. Conversely the specific binding of [3H]NMS had a high affinity and was saturable at concentrations greater than 30 nM in both intact and broken cells. Classical muscarinic receptor antagonists displaced specific binding of [3H]NMS binding according to the law of mass action, while displacement curves for pirenzepine and muscarinic agonists were very shallow (nH less than 1), suggesting the presence of more than one subtype of muscarinic receptor on mononuclear cell membranes. Binding studies with [3H]NMS to purified mononuclear cell subpopulations demonstrated that muscarinic binding sites were mainly localized on thymus-derived (T) lymphocytes and large granule lymphocytes. Moreover evidence is presented of an age-dependent increase of the density of muscarinic binding sites on T-lymphocytes. The results are discussed in terms of the usefulness of the binding of [3H]NMS in studying the physiological function of muscarinic receptors on human T-lymphocytes and their possible changes in patients with neurological diseases.     

Characterization of cholinergic muscarinic receptors in the rabbit iris

The sphincter smooth muscle of the iris is innervated by excitatory parasympathetic nerve fibers, and the activation of these fibers results in the breakdown of phosphatidylinositol 4,5-bisphosphate into its derived second messengers, myosin light chain phosphorylation and muscle contraction. The present study characterizes the muscarinic acetylcholine receptors (mAChRs) of the rabbit iris employing [3H]N-methylscopolamine ([3H]NMS) and L-[3H]quinuclidinyl benzilate ([3H]QNB) as probes. Binding studies indicated that [3H]NMS and [3H]QNB bound to homogeneous populations of mAChRs with apparent Bmax values of 0.67 and 1.09 pmol/mg protein respectively. Binding of radioligands was rapid, saturable, stereospecific, reversible, and inhibited by specific muscarinic agonists and antagonists in a competitive manner. [3H]NMS displayed a lower amount of nonspecific binding and a faster association and dissociation rate than [3H]QNB. The relative potencies for displacement of both radioligands, based on their Ki values, were (-)QNB greater than atropine greater than (+)QNB greater than pirenzepine greater than pilocarpine. Antagonist displacement of the radioligands appeared to obey the law of mass action, indicating interaction with a single binding site. However, displacement of the radioligands by the agonists carbamylcholine and methacholine indicated interaction with both high and low affinity binding sites. Comparison of the displacement of [3H]NMS and [3H]QNB by pirenzepine in microsomal fractions from rabbit iris, ileal muscle and cerebral cortex revealed the presence of a single subtype of mAChR in the iris which had an affinity for PZ that was slightly higher than that of ileal M2 receptors, but lower than that of brain M1 receptors. This suggests that the mAChRs in the iris may represent a subclass of receptors within the M2 subtype, or they may constitute an entirely different subtype of mAChRs.     

Characteristics of endothelin binding sites in the spinal cord of spontaneously hypertensive rats.

The binding of [125I]sarafotoxin 6b (SRT 6b) and [125I]endothelin-1 (ET-1) to endothelin (ET) receptors of neuronal membranes prepared from cerebral cortex and spinal cord of 8-week-old spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) was determined. SHR had significantly higher blood pressure as compared to WKY. Heart rate was similar in SHR and WKY. [125I]SRT 6b and [125I] ET-1 bound to the membranes of cerebral cortex and spinal cord at a single, high affinity site. The binding of [125I]SRT 6b and [125I]ET-1 in the cerebral cortex and spinal cord membranes was found to be similar in SHR and WKY. Concentration-dependent inhibition of [125I]ET-1 binding in spinal cord membranes by unlabeled ET-1, ET-2 and ET-3 was performed. The Ki values were found to be 2.35 +/- 0.68, 0.29 +/- 0.08 and 24.10 +/- 5.90 nM for ET-1, ET-2 and ET-3, respectively in WKY. The Ki values of ET-1 and ET-2 were found to be similar in WKY and SHR. However, the Ki value of ET-3 were found to be significantly lower (P less than 0.004) in SHR as compared to WKY. Concentration-dependent inhibition of [125I]SRT 6b binding in spinal cord membranes by unlabeled ET-1, ET-2 and ET-3 were also performed. The Ki values were found to be 9.50 +/- 2.10 pM, 0.17 +/- 0.04 nM and 31.20 +/- 6.00 nM for ET-1, ET-2 and ET-3, respectively in WKY. The Ki values of ET-1 and ET-2 were found to be similar in WKY and SHR.(ABSTRACT TRUNCATED AT 250 WORDS)