QNB Binding Research Articles

Decreased levels of muscarinic receptors in bladders from the alcohol preferring rat line

The B max for [ 3H]QNB binding in the bladders of alcohol preferring (AA) rats was only approximately 60% of that in the alcohol non-preferring (ANA) rats. No significant change in B max for [ 3H]QNB binding in bladder was observed between alcohol insensitive (AT) and alcohol sensitive (ANT) rats. No significant change in K d for [ 3H]QNB binding in bladder was observed between the four different rat lines studied. Therefore, alcohol preference but not sensitivity is associated with a decrease in muscarinic receptor density in the rat bladder. Because all of the rats used in this study were ethanol-naive, the decrease in muscarinic receptor density in the bladders of alcohol preferring rats is associated with genetic factors inherent to this rat line. Further studies are needed to determine if these observations are tissue specific or specific to the m2 subtype, which predominates in the rat bladder.

Alterations in cortical muscarinic receptors following cholinotoxin (AF64A) lesion of the rat nucleus basalis magnocellularis

Cortical choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), tryptophan hydroxylase (TPH), muscarinic receptors and sodium-dependent, high-affinity, choline uptake (SDHACU) sites were examined in the rat brain following unilateral stereotaxic injection of the cholinotoxin, AF64A, into the nucleus basalis magnocellularis (NBM). Injection of AF64A resulted in a significant loss of presynaptic cholinergic markers in the cortex without alteration in TH and TPH activity. The binding to SDHACU sites was reduced to background values in the NBM and increased in the central amygdala (Ce) and cortex. The increase in cortical [ 3H]QNB binding was the result of a change in muscarinic receptor number (B MAX) and not a change in receptor affinity (K D). Examination of muscarinic receptor subtypes demonstrated a reduction of M 1 receptor binding in the cortex and NBM without any alteration in the Ce. Non-M 1 binding was significantly increased in all the laminae of the cortex and in the Ce, but decreased in the NBM. These data suggest that there exists a population of M 1 receptors on NBM projections to the cortex and that NBM projections influence a population of postsynaptic receptors in the cortex and Ce which are not of the M 1 subtype.

Stereoselective binding and activity of oxotremorine analogs at muscarinic receptors in rat brain.

The activities of the enantiomers of BM-5 were examined to measure muscarinic cholinergic selectivity in the central nervous system. Autoradiographic studies assessed the ability of each enantiomer to inhibit the binding of [3H]-(R)-quinuclidinyl benzilate ([3H]-(R)-QNB) to muscarinic receptors in the rat brain. (+)-(R)-BM-5 inhibited [3H]-(R)-QNB binding to rat brain sections at concentrations below 1.0 microM, while 100-fold higher concentrations of (-)-(S)-BM-5 were required for comparable levels of inhibition. Analysis of the autoradiograms indicated that both stereoisomers had a similar distribution of high affinity binding sites. Each enantiomer displayed higher affinity for muscarinic receptors in the superior colliculi and lower affinity for receptors in the cerebral cortex and hippocampus. (+)-(R)-BM-5 and oxotremorine inhibited adenylyl cyclase activity in the cerebral cortex with efficacies comparable to that for acetylcholine. (+)-(R)-BM-5 was 26-fold more potent than (-)-(S)-BM-5 in inhibiting adenylyl cyclase. Oxotremorine-M and carbamylcholine stimulated phosphoinositide turnover in the cerebral cortex. Oxotremorine had lower activity and (+)-(R)-BM-5 was essentially inactive at comparable concentrations. The difference in activity of the two enantiomers indicates a remarkable stereochemical selectivity for muscarinic receptors. The stereoselectivity index is comparable for both the autoradiographic assays (48) and measures of adenylyl cyclase activity (26) in the cerebral cortex.

Central muscarinic receptor subtypes and carrageenin-induced paw oedema in rats

In an earlier report from this laboratory, it was demonstrated that the central cholinergic system exerted a modulatory pro-inflammatory effect on carrageenin-induced paw inflammation in rats. In this study, the role of the central muscarinic receptors in cholinergic modulation of peripheral inflammation was investigated by using several M1 and M2 receptor agonists and antagonists. The M1 receptor agonists aceclidine and arecholine and the nonspecific muscarinic receptor agonist oxotremorine augmented carrageenin oedema, an effect attenuated by the M1 receptor antagonist scopolamine. Physostigmine behaved like a M1 receptor agonist at all dose levels. However, the other M2 receptor agonist, carbachol, produced a dose-dependent dual effect, with lower doses attenuating the oedema and higher doses augmenting the inflammation. While the former action appeared to be due to M2 receptor stimulation, because it was blocked by AF-DX 116--a M2 receptor antagonist, the latter action appeared to be induced by M1 receptor stimulation, because it was inhibited by scopolamine. The pro-inflammatory effect of the M2 receptor antagonists AF-DX 116 and gallamine appeared to be induced by enhanced neuronal release of acetylcholine, because the effects were not evident following pretreatment with hemicholinium, which attenuates synthesis of the amine. Muscarinic receptor binding studies with (3H)-QNB indicated that the corpus striatum has substantially higher population of M1 receptors compared with the cerebellum. In the corpus striatum, (3H)-QNB binding indicated initial up-regulation followed by down-regulation of M1 receptors during peak inflammation, which appeared to persist even after a decrease in the inflammation. In contrast, the M1 receptors in the cerebellum appeared to be down-regulated very transiently during the early phase of the inflammation. While these receptor alterations may be due to the inflammation, it is equally possible that they represent changes induced by the stress of pain and inflammation induced by carrageenin.

Muscarinic receptors in diabetic rat prostate

To investigate the effects of experimentally-induced diabetes on prostatic muscarinic cholinergic receptors, the binding characteristics of [ 3H]quinuclidinyl benzilate ([ 3H]QNB) to prostatic membrane participates were examined in four groups of rats: control, diabetic, diabetic insulin treated, and diabetic myo-inositol treated. Diabetes was induced by i.v. injection of streptozotocin (STZ), 65 mg/kg. Diabetic and diabetic myo-inositol-treated rats had hyperglycemia, hypoinsulinemia, glucosuria, polydipsia, and polyuria as well as significantly smaller prostates and lower body weights compared to control and diabetic insulin-treated animals. The densities of muscarinic receptors ( B max ) as determined by saturation studies with [ 3H]QNB in the prostatic plasma membranes of control, diabetic, diabetic insulin-treated and diabetic myo-inositol-treated rats were 80 ± 8, 51 ± 5, 78 ± 3, and 47 ± 7 fmol/mg of protein, respectively. [ 3H]QNB binding to muscarinic receptors was inhibited by muscarinic antagonists with the following rank order of K i , values: atropine ⪡ pirenzepine < AF-DX 116. The pharmacological profile of the muscarinic receptors was similar in all groups examined and was consistent with the predominance of the M 3 muscarinic receptor subtype in prostatic membrane particulates. Our data indicate that STZ-induced diabetes caused a variety of abnormalities including a down-regulation in the density of M 3 muscarinic receptors in the rat prostate and that insulin, but not myo-inositol could prevent the development of these abnormalities.

Synthesis, molecular modeling studies, and muscarinic receptor activity of azaprophen analogs

Synthesis, radioligand binding, and pharmacologic activities of a series of muscarinic receptor ligands including and related to azaprophen (6-methyl-6-azabicyclo[3.2.1]octan-3 alpha-ol 2,2-diphenylpropionate, 1) have been measured to determine activity and selectivity for muscarinic receptor subtypes. Pharmacologic affinities of antagonists were determined as pA2 values for antagonism of methacholine-induced tension responses in guinea pig ileum. Binding affinities were measured by competition against [3H]QNB binding in guinea pig ileum, rat heart and brain, and m1- or m3-transfected Chinese hamster ovary (CHO) cells. The efficacies of muscarinic agonists in brain were determined by the ratio of binding affinities against [3H]QNB or [3H]NMS and [3H]oxotremorine-M ([3H]Oxo-M). Nine muscarinic antagonists, including azaprophen, did not discriminate significantly between the subtypes of muscarinic receptors. KI values for receptor binding for azaprophen (1) were between 8.81 x 10(-11) and 4.72 x 10(-10) M in ileum, heart, brain, and m1- or m3-transfected CHO cells. The alpha- and beta-benzilate esters 5 and 6 are as potent as azaprophen, and diphenylacetate esters 3 and 4 and N-(6)-benzyl alpha-isomer 7 are less potent than azaprophen. Significant stereoselectivity was exhibited with (+)-azaprophen being approximately 200 times more potent than the (-)-enantiomers and the 3 beta-ol isomer 2 being ca. 50 times less potent than azaprophen in all systems. A molecular modeling-molecular mechanics study was conducted to account for the difference. Putative muscarinic agonists (analogues and isomers of 6-methyl-6-azabicyclo[3.2.1]octan-3-ol acetate) did not discriminate muscarinic receptor subtypes with KI values between 2.77 x 10(-6) and 4.33 x 10(-5) M without significant stereoselectivity in the systems examined. The most active analogue was (1R,3R,5S)-6-[1(R)-phenylethyl]-6-azabicyclo[3.2.1]octan-3 alpha-ol acetate. However, efficacies of these putative agonists were in general very low.

Muscarinic receptor localization and function in rabbit carotid body

Acetylcholine and muscarinic agonists inhibit chemosensory activity in the rabbit carotid sinus nerve (CSN). Because the mechanism of this inhibition is poorly understood, we have investigated the kinetics and distribution of muscarinic receptors in the rabbit carotid body with the specific muscarinic antagonist [ 3H]quinuclidinylbenzilate ([ 3H]QNB). Equilibrium binding experiments identified displaceable binding sites (1 μM atropine) with a K d = 71.46pM and a B max = 9.23pmol/g tissue. These binding parameters and the pharmacology o the displaceable [ 3H]QNB binding sites are similar to specific muscarinic receptors identified in numerous other nervous, muscular and glandular tissues. Comparisons of specific binding in normal and chronic CSN-denervated carotid bodies suggest that muscarinic receptors are absent on afferent terminals in the carotid body; however, nearly 50% of the specific [ 3H]QNB binding is lost following chronic sympathectomy, suggesting the presence of presynaptic muscarinic receptors on the sympathetic innervation supplying the carotid body vasculature. Autoradiographic studies have localized the remainder of [ 3H]QNB binding sites to lobules of type I and type II parenchymal cells. In separate experiments, the muscarinic agonists, oxotremorine (100 μM) and bethanechol (100 μM) were shown to inhibit both the release of catecholamines and the increased CSN activity evoked by nicotine (50 μM) stimulation of the in vitro carotid body. Our data suggest that muscarinic inhibition in the rabbit carotid body is mediated by receptors located on type I cells which are able to modulate the excitatory actions of acetylcholine at nicotinic sites.

Effect of bay K 8644, a calcium channel agonist, on dog cardiac muscarinic receptors

To investigate further whether the effects of the dihydropyridine (DHP) drugs on calcium channels are related to those of these drugs on muscarinic receptors, the binding characteristics of the DHP calcium channel agonist, Bay K 8644, on muscarinic receptors and calcium channels were compared to those of the DHP calcium channel antagonists, nicardipine and nimodipine in the dog cardiac sarcolemma. Bay K 8644, nicardipine and nimodipine inhibited the specific [3H]QNB binding with K i values of 16.7μM, 3.5μM and 15.5μM respectively. Saturation data of [3H]QNB binding in the presence of these DHP drugs showed this inhibition to be competitive. Bay K 8644, like nicardipine and nimodipine, blocked the binding of [3H]nitrendipine to the high affinity DHP binding sites, but atropine did not, indicating that the muscarinic receptors and the DHP binding sites on calcium channels are distinct. The K i value of Bay K 8644 for the DHP binding sites was 4 nM. Nicardipine and nimodipine (K i :0.1–0.2 nM) were at least 20 times more potent than Bay K 8644 in inhibiting [3H]nitrendipine binding. Thus, the muscarinic receptors were about 4000 times less sensitive than these high affinity DHP binding sites to Bay K 8644. These results suggest that the DHP calcium agonist Bay K 8644 binds directly to the muscarinic receptors but its interaction with the muscarinic receptors is not related to its binding to the DHP binding sites on calcium channels.

Regional differences in cholinergic innervation and drug sensitivity in the smooth muscles of pig stomach

1 To evaluate the regional differences in cholinergic mechanisms in the stomach, innervation and receptor distribution were investigated by isometric tension recording, receptor binding assay and measurement of nerve-related enzyme activity in longitudinal and circular smooth muscle layers isolated from various regions of the pig stomach. 2 The response to transmural nerve stimulation (TMS) varied with the muscle layer and portion of stomach tested. Contraction was predominant in the smooth muscle from the pyloric antrum, relaxation in the corpus region. 3 The contraction to TMS was abolished by atropine (0.1-0.5 microM) and potentiated by physostigmine (1-2 microM). On the other hand, relaxation to TMS was unaffected by a combination of phentolamine and carteolol but was abolished by tetrodotoxin (TTX) (0.67-1.54 microM). In all preparations from various portions, physostigmine unmasked the contraction and atropine revealed the relaxation to TMS. 4 The activity of choline acetyltransferase (ChAT) and cholinesterase (ChE) was higher in longitudinal than in circular muscle layers and was also higher in the fundus than in any other portion. 5 The sensitivity (pD2 value) to acetylcholine (ACh) was higher in the longitudinal than in the circular muscle layers but did not differ largely among different regions of the stomach. The maximum response induced by ACh was also highest in longitudinal muscle of the fundus. In contrast, the population of muscarinic receptors, estimated from [3H]-QNB binding, increased from the fundic to pyloric portions. 6 These results suggest that there are regional differences in the responses to nerve stimulation in pig stomach, which are likely to depend partly on the quantitative differences in cholinergic nerve supply and in the responsiveness to ACh.

Reduced D2 dopamine and muscarinic cholinergic receptor densities in caudate specimens from fluctuating parkinsonian patients.

Binding of spiperone and 3-quinuclidinyl benzilate (QNB), both labeled with hydrogen 3 (3H), were measured in caudate tissue obtained from 8 living parkinsonian patients at the time of cerebral transplantation. This was clinically homogeneous group of patients. All remained predominantly responsive to levodopa, although with marked disability secondary to clinical fluctuations (short-duration responses) and medication-induced dyskinesias; all were receiving substantial doses of levodopa and 6 of the 8 patients were additionally receiving bromocriptine or pergolide. Binding densities of dopamine D2 receptors, as measured by [3H]spiperone binding, were reduced in this group of patients, compared to caudate specimens from autopsy control subjects. This findings may reflect medication-induced receptor downregulation. Parallel changes occurred with muscarinic cholinergic receptors; [3H]QNB binding was significantly reduced, compared to autopsy control values. This reduction of muscarinic receptors might be due to loss of nigrostriatal terminals that are known to contain muscarinic receptors. Alternatively, muscarinic receptors may have been downregulated by increased corticostriatal glutamatergic input to cholinergic cells, inferred to be present based on the prominent levodopa-induced dyskinesias. Finally, receptor deficits could have been a reflection of more widespread degenerative cerebral disease, although levodopa-refractory symptoms were generally not pronounced in these patients.

Neurochemistry of aging. 2. Design, synthesis and biological evaluation of halomethyl analogs of choline with high affinity choline transport inhibitory activity

The design, synthesis, and testing of several halomethyl analogues of choline and acetylcholine as potential cholinotoxins is described. The compounds were evaluated for their ability to inhibit high-affinity choline transport and their affinity toward postsynaptic muscarinic receptors. Among the analogues tested, bromomethyl and iodomethyl analogues of choline were found to be the most potent inhibitors of the high affinity choline transport system. Introduction of a beta-methyl group in the halomethyl analogues drastically reduced their potencies. The bromomethyl and iodomethyl analogues were further investigated for their effects on choline acetyltransferase activity, acetylcholinesterase activity and QNB binding. Neither compound possesses significant ability to alter any of the above cholinergic markers, except at very high concentrations. These results suggest that the bromomethyl and iodomethyl choline analogues may be used as specific inhibitors of the presynaptic high-affinity choline transport system.

Muscarinic receptors and mucus secretion in swine tracheal epithelium: Effects of subacute organophosphate treatment

This swine trachea study was undertaken to examine the effects of nerve agents on mucus gland cell function. Subacute treatment of swine with soman, sarin, and VX inhibits acetylcholinesterase and leads to down-regulation of muscarinic receptors in tracheal submucosal gland cells. Muscarinic receptor density in isolated cells as determined by [ 3H]QNB binding was reduced by 60–65% and that measured using [ 3H]NMS was decreased by 65–73%. Subacute treatment of swine for 7 days with soman and VX caused a small, significant increase in the fraction of receptors with high affinity for carbachol. The decrease in receptor density was accompanied by a decrease in acetylcholine-induced mucus secretion. The decrease in mucus secretion was not due to a decrease in the ability of the cells to produce or release mucus since cross-tolerance did not develop to methoxamine-induced mucus secretion. Therefore, we conclude that in mucus gland cells tolerance development can be linked functionally to muscarinic receptor loss.

Inhibition of carbachol-stimulated phosphoinositide turnover by U-50,488H in rat hippocampus — involvement of GTP-binding protein

The effect of U-50,488H, a selective kappa-opioid agonist, on carbachol-stimulated phosphoinositide (PI) turnover response in rat hippocampal slices was examined. U-50,488H which stimulates PI turnover response in this preparation (Periyasamy and Hoss, 1990, Life Sci. 47, 219), inhibited carbachol-stimulated PI turnover in a concentration-dependent manner with an IC 50 value of 33±9.0 μM. The inhibitory effect of U-50,488H was not blocked by the kappa-selective antagonists, e.g., nor-binaltorphimine (10 μM), and MR2266 (10 μM), or tetrodotoxin (1 μM) suggesting that the effect of U-50,488H was mediated neither through the kappa-receptors nor through the release of an endogenous neurotransmitter(s). A Lineweaver-Burke plot of the stimulation of PI turnover by carbachol in the presence and absence of U-50,488H showed that the K m was not changed (11.4±3.4 and 11.5±2.6 μM) whereas the V max was reduced from 3849±460 to 1534±31 cpm indicating that the inhibition was non-competitive. U-50,488H also inhibited guanosine 5′-[β,γ-imido]triphosphate (Gpp[NH]p)-stimulated PI turnover in rat hippocampal membranes in a concentration-dependent manner with an IC 50 value of 33±12 μM. However, U-50,488H had no effect on exogenously added phospholipase-C-stimulated PI turnover. In receptor binding studies. U-50,488H displaced the non-selective muscarinic antagonist [ 3H]quinuclidinyl benzilate ([ 3H]QNB) binding in a concentration-dependent manner with an IC 50 value of 4.5±1.5 μM. A Scatchard plot of [ 3H]QNB binding in the presence and absence of U-50,488H showed that the K d was changed from 883±250 to 364±147 pM whereas the B max remained the same (1.66±0.16 and 1.65±0.18 pmol/mg protein) suggesting that the inhibition was competitive. These data indicate that the inhibitory effect of U-50,488H on carbachol-stimulated PI turnover was mediated through the interaction with the G-protein but not through kappa-receptors or release of neurotransmitter(s).

Continuous cocaine administration produces persisting changes in brain neurochemistry and behavior

Rats were administered either continuous cocaine, daily injections of cocaine, continuous amphetamine, or no drug for 5 days and then given a 30 day drug-free recovery period. When subsequently tested in open field, the daily cocaine injection animals were the most hyperactive whereas the cocaine pellet animals were the most fearful. In vitro autoradiography was then utilized to examine persisting changes in receptor binding for D 2 ([ 3H]spiperone), D 1 ([ 3H]SCH23390), benzodiazepine ([ 3H]flunitrazepam), 5-HT 1 ([ 3H]5-HT), 5-HT 2 ([ 3H]ketanserin), and muscarinic acetylcholine (ACh) receptors ([ 3H]QNB; quinuclidinyl benzilate). In the amphetamine pellet animals, there were large increases in [ 3H]spiperone binding in several dopamine (DA)-rich regions; these were accompanied by conversely decreased [ 3H]SCH23390 binding. Cocaine pellet animals showed a completely different pattern, with appreciable increases in [ 3H]flunitrazepam binding in DA-rich areas, cortex, and amygdala but decreased [ 3H]QNB binding in DA-rich areas, hippocampus and amygdala. While cocaine injection animals showed elevated [ 3H]spiperone binding in caudate and substantia nigra, they had generally smaller changes in most brain regions than the other drug groups. These findings replicate and extend previous reports that continuous drug administration induces long-lasting alterations in brain chemistry, but indicate that continuous cocaine has enduring effects on different neurochemical systems from continuous amphetamine.

Interaction of antihistaminics with muscarinic receptor (III)

The muscarinic antagonist 1-[benzilic 4,4′-3H]quinuclidinyl benzilate ([3H] QNB) bound to a single class of muscarinic receptors with high affinity in rabbit ileal membranes. The K D and B max values for [3H]QNB calculated from analysis of saturation isotherms were 52.5 pM and 154 fmol/mg, respectively. Chlorpheniramine (CHP), histamine H1 blocker, increased K D value for [3H]QNB without affecting the binding site concentrations and Hill coefficient. The K i value of CHP for inhibition of [3H]QNB binding in ileal membranes was 1.44μM and the pseudo-Hill coefficient for CHP was close to unit. In the functional assay carbachol, muscarinic agonist, increased the contractile force of ileum with ED50 value of 0.11μM. CHP caused the rightward shift of the dose-response curve to carbachol. The pA2 value of CHP determined from Schild analysis of carbachol-induced contraction was 5.77 and the slope was unity indicating competitive antagonism with carbachol. The dissociation constant (K i ) of CHP obtained in competitive experiments with [3H]QNB was similar to the K A value (1.69μM) of CHP as inhibitor of carbachol-induced contraction in rabbit ileum. This result suggests that the binding of H1 blocker, CHP, vs [3H]QNB to muscarinic receptors in ileal membranes represents an interaction with a receptor of physiological relevance.

Regulation of Brain Nicotinic Receptors by Chronic Agonist Infusion

Several studies have demonstrated that chronic treatment with nicotine elicits an increase in the number of brain nicotinic receptors. To determine whether this effect is elicited by other nicotinic agonists found in tobacco, the effects of chronic infusion with nicotine on brain nicotinic receptors were compared with those after anabasine and lobeline. C57BL/6 mice were infused with saline or equimolar doses (18.5 mumol/kg/h) of nicotine, anabasine, or lobeline for 8 days. Nicotinic receptors, quantified by the binding of [3H]nicotine and [125I]iodo-alpha-bungarotoxin (alpha-[125I]BTX), and muscarinic receptors, quantified by the binding of [3H]quinuclidinyl benzilate ([3H]QNB), were then assayed in eight brain regions. An increase in [3H]nicotine binding was observed in all regions except cerebellum following chronic infusion with nicotine and anabasine, whereas lobeline did not alter the number or affinity of these binding sites. This increase was due to changes in Bmax and not in the affinity of the receptor for the ligand (KD). A slight increase in alpha-[125I]BTX binding was observed in cortex following chronic anabasine infusion. [3H]QNB binding sites were largely unaltered following chronic infusion with any of the nicotinic analogs. The levels of the agonists in the brain were also determined after chronic treatment, and the amounts of lobeline and anabasine were found to be higher than that of nicotine. Thus, the failure of lobeline to elicit changes in nicotine binding is not due to reduced brain concentrations.

Muscarinic acetylcholine receptor subtype mRNA expression and ligand binding in the aged rat forebrain

Previous studies indicate that a 20–30% decline in muscarinic acetylcholine receptor binding occurs in localized areas of rat brain during aging. In this study, reduced [ 3H]-quinuclidinyl benzilate binding was observed in striata from 24–25-month-old rats relative to 5–6-month-old animals using homogenate binding assays. To determine if the decline in receptor concentration occurs as a result of decreased receptor synthesis, the expression of the m 1, m 3, and m 4 muscarinic receptor mRNAs as well as [ 3H]-QNB binding were determined in adjacent sections of young and old male rats using in situ hybridization and in vitro receptor autoradiography respectively. A significant decline in collective muscarinic receptor binding as assessed by [ 3H]-QNB was observed in the caudate putamen, olfactory tubercle, nucleus accumbens, and several frontal and parietal cortical areas. The only difference observed in muscarinic mRNA expression for any of the three subtypes examined was a decline in m 1 hybridization in the olfactory tubercle. The results of this study demonstrate that the regional brain areas displaying age-related decreases in receptor binding do not correlate with those areas showing a decrease in muscarinic receptor expression. Apparently, the decline in muscarinic acetylcholine receptor density with age does not result from a decline in receptor gene expression.

Inhibition of muscarinic cholinergic receptors by disulfide reducing agents and arsenicals: Differential effect on locust and rat

Muscarinic receptors are altered by sulfhydryl reagents. Arsenic compounds, which have been used as insecticides, exert their toxic effects by combining with sulfhydryl groups. We compared the action of arsenicals and other sulfhydryl reagents on the muscarinic receptor from invertebrate and vertebrate species (locust and rat). Disulfide-reducing reagents dithiothreitol (DTT) and British Anti-Lewisite (BAL), but not arsenicals, inhibited [ 3H]quinuclidinyl benzilate ([ 3H]QNB) binding. However, after disulfide reduction, arsenicals caused a further inhibition of muscarinic binding. The effect of DTT + arsenicals was largely irreversible. The locust receptors were more sensitive to the action of both disulfide reagents either in the absence or presence of arsenicals than the rat receptors. The sulfhydryl reagent p-chloromercuric benzoate (PCMB) was more effective at inhibiting the locust receptors than the rat receptors, but addition of arsenicals did not cause further inhibition in either the locust or rat receptors. In locust, DTT + cacodylate and DTT + arsenite caused a reduction in the number of sites without modifying the affinity of [ 3H]QNB binding. In rat, DTT + arsenite caused a decrease in the affinity, while DTT + cacodylate caused a decrease in the affinity of [ 3H]QNB binding and its number of sites. Competition experiments after DTT + cacodylate showed that the ic 50 and the Hill coefficient ( n h ) remained unchanged in the locust. In the rat, the ic 50 for atropine was increased without alteration in the n h , and both parameters were increased for carbachol. These results are explained assuming that the binding site of the locust receptor has a disulfide group similar to that of the mammalian receptor, but that the hydrophobic interactions within the binding site are weaker in the locust receptor. The higher sensitivity of the insect receptor to sulfhydryl reagents could be of interest for developing methods of pest control.

Development of D1 and D2 dopamine receptors and associated second messenger systems in fetal striatal transplants

Stereotactic implantation of fetal brain regional anlage into adult host brain (“brain transplantation”) appears to be an increasingly viable strategy for therapy of neurodegenerative diseases. We have studied implantation of fetal striatum into adult striatum, previously lesioned by neurotoxic amino acid injection, as a model for transplantation therapy of Huntington's disease. The beginning of behavioral recovery to apomorphine is not apparent until 6.5 months after implantation. By 4 months after implantation cerebral blood flow through the implants appears equal to that in the intact contralateral striatum. At this time, cerebral glucose utilization is reduced in the implants but increases following apomorphine treatment. The development of D1 and D2 dopamine (DA) receptors is markedly deficient in the striatal grafts at both 4 and 6.5 months after implantation. Very little D2 radioligand binding was observed in the grafts at either time point; D1 receptors appeared in a patchy fashion by 6.5 months at densities approaching normal striatum. In situ hybridization of D2 dopamine receptor mRNA demonstrated robust hybridization signal in normal striatum and accumbens but no signal in 6.5-month-old striatal grafts. Adenylate cyclase (AC) activity, examined with high-affinity [ 3H]forskolin binding, also appeared in patches similar to D1 receptors at 6.5 months. In contrast, protein kinase C activity, labeled with [ 3H]phorbol ester, was very apparent in the grafts at both time points. Higher and generally homogenous densities of muscarinic cholinergic receptors, assessed with [ 3H]QNB binding, develop in the grafts, but there appear to be few functioning cholinergic terminals, as measured by [ 3H]hemicholinium binding. Our data demonstrate that very few D1 or D2 DA receptors develop in fetal striatal neurons removed from E15–16 embryos and subsequently implanted into adult ibotenic acid-lesioned striata. The lack of D2 receptor development appears to derive from lack of transcription of the D2 receptor gene. By 6.5 months, D1 receptor sites and AC activity both appear in discrete patches, consistent with the known in vitro association of D1 receptors with stimulation of AC activity. In this model, the majority of graft protein kinase C activity, to the extent that it is labeled with [ 3H]phorbol ester, is not associated with DA receptors. DA receptor development in fetal striatal implants may require coimplantation of DA neurons.

Effects of Ca 2+ channel ligands on [ 3H]QNB binding at m 1 and m 3 muscarinic receptors

1. 1. The effects of Ca 2+ channel ligands on [ 3H]QNB binding in m 1- or m 3-transfected Chinese hamster ovary (CHO) cells have been studied. 2. 2. The IC 50 values of Ca 2+ channel ligands for the inhibition of [ 3H]QNB binding were between 10 −6 and 10 −4 M and the rank order of potency was HOE 166 > McN 6186 > nicardipine > tiamdipine > verapamil > diltiazem > Bay K 8644 > nifedipine at m 1 and m 3 receptors. 3. 3. The results indicate that Ca 2+ channel ligands employed in this experiment do not distinguish subtypes of muscarinic receptors.