Quinuclidinyl Benzilate Binding Research Articles

Changes in γ-aminobutyric acid, μ-opioid and neurotensin receptors in the accumbens-pallidal projection after discrete quinolinic acid lesions in the nucleus accumbens

Discrete quinolinic acid lesions in the nucleus accumbens altered [ 3H]muscimol binding to γ-aminobutyric acid receptors, [ 125I]neurotensin binding to neurotensin receptors, [ 125I]Tyr- d-Ala-Gly-NMePhe-Gly-OH binding to μ-opioid receptors, and [ 3H]quinuclidinyl benzilate binding to muscarinic receptors. Within lesions of the lateral accumbens core, [ 3H]muscimol binding increased and [ 125I]Tyr- d-Ala-Gly-NMePhe-Gly-OH, [ 125I]eurotensin and [ 3H]quinuclidinyl benzilate binding decreased. Lesions of the medial nucleus accumbens resulted in decreased [ 125I]Tyr- d-Ala-Gly-NMePhe-Gly-OH and [ 3H]quinuclidinyl benzilate binding while no alterations were observed for [ 3H]muscimol or [ 125I]neurotensin binding. These data support anatomical distinctions between medial and lateral nucleus accumbens. Destruction of intrinsic neurons in the dorsomedial nucleus accumbens core increased [ 3H]muscimol binding in the dorsal rim of the ventral pallidum and the rostral globus pallidus without altering [ 125I]Tyr- d-Ala-Gly-NMePhe-Gly-OH binding. Destruction of neurons in the lateral nucleus accumbens core or medial shell did not alter [ 3H]muscimol binding in the ventral pallidum. The lack of upregulation in γ-aminobutyric acid receptors suggests that the γ-aminobutyric acid-containing projection from the dorsomedial core to the dorsal rim of the ventral pallidum differs from the projection from the lateral accumbens core and medial shell to the more ventral regions of the pallidum. Fluoro-gold retrogade tracer histochemistry confirmed the specific projection from the dorsomedial core to the dorsal ventral pallidum; and from the shell of the nucleus accumbens to more ventral regions of the ventral pallidum.

Cholinotoxic Effects of Aluminum in Rat Brain

The in vivo and in vitro effects of Al on the cholinergic system of rat brain were studied. The amount of Al accumulated after the chronic, intraperitoneal administration of aluminium gluconate (Al-G) or AlCl3, both at a dose of 1 mg/ml/100 g of body weight, increased in the frontal and parietal cortices, the hippocampus, and the striatum. Significantly decreased choline acetyltransferase activities after chronic Al treatment were measured in the parietal cortex, the hippocampus, and the striatum, but not in the frontal cortex. The acetylcholinesterase activity was not changed significantly in any brain area investigated. Both Al-G and AlCl3 administrations resulted in a general decrease (to 40-70% of the control values) in the specific l-[3H]nicotine binding, involving all brain areas studied. The specific (-)-[3H]quinuclidinyl benzilate binding was reduced (to 40-60% of the control values) only after 25 days of Al treatment. Al-G and AlCl3 were equivalent in eliciting these reductions in vitro studies revealed different alterations of the cholinergic system in response to Al treatment. No changes were observed either in choline acetyltransferase activity or in cholinergic receptor bindings. Both Al-G and Al2(SO4)3 treatments, however, exhibited a biphasic effect on the acetylcholinesterase activity. At low Al concentrations (10(-8)-10(-6) M), the activity was slightly increased, whereas at higher concentrations (10(-6)-10(-4) M), it was inhibited by a maximum of 25% as compared to the controls. Thus, these cholinotoxic effects are probably due not to a direct interaction between the metal and the cholinergic marker proteins, but rather to a manifestation and consequence of its neurodegenerative effects.

Developmental changes in β‐adrenoceptors, muscarinic cholinoceptors and Ca2+ channels in rat ventricular muscles

1. In an attempt to explain the previous electrophysiological data on the ontogeny of beta-adrenergic and muscarinic cholinergic interactions on cardiac Ca2+ current, biochemical studies were performed on the ontogeny of beta-adrenoceptors, muscarinic cholinoceptors and Ca2+ channels in cardiac muscle of developing rats: 16-20 days old foetuses, 0-20 days old neonates, and 2-3 months old adults. 2. Developmental changes in cardiac beta-adrenoceptors, muscarinic cholinoceptors, and Ca2+ channels were determined with the use of specific radioligands, [3H]-dihydroalprenolol (DNA), [3H]-quinuclidinyl benzilate (QNB), and [3H]-nitrendipine (NTD), respectively. 3. The Bmax value (fmol mg-1 tissue) for [3H]-DNA binding started to increase on post-gestation day 20, reached almost its maximum level on neonatal day 6, kept almost the same level until neonatal day 20, and then decreased slightly to its adult level. 4. The Bmax value (fmol mg-1 tissue) for [3H]-QNB binding started to increase on post-gestation day 16, reached almost its maximum level on neonatal day 0, remained almost constant until neonatal day 15, and then decreased to its adult level. 5. The Bmax value (fmol mg-1 tissue) for [3H]-NTD binding increased with age between post-gestation day 18 and neonatal day 15, stayed almost constant until neonatal day 20, and then decreased to its adult level. 6. The Kd values for [3H]-DHA, [3H]-QNB, and [3H]-NTD bindings remained almost constant during the developmental period examined. 7. Isoprenaline (Iso) increased the kx of slow action potentials (APs) from post-gestation day 18, and the adult level was reached at about 2 weeks after birth; this developmental time course is similar to that of Ca2+ channels. The number of beta-adrenoceptors also started to increase a few days before birth, but attained its peak about one week earlier than did the Pax of slow APs or the number of Ca2 + channels. 8. Acetylcholine (ACh) almost completely abolished the Iso-induced increase in m,,ax observed from postgestation day 18 to neonatal day 20; this developmental time course for the ACh effect is consistent with the finding that the number of muscarinic cholinoceptors started to increase on post-gestation day 16 and reached a peak on the day of birth. 9. Previous electrophysiological and the present biochemical findings strongly suggest that the functional coupling between muscarinic cholinoceptors and Ca2+ current is already established when the coupling between beta-adrenoceptors and Ca2 + current starts to operate in developing rat hearts.

Ethylchline aziridinium ion depletes acetylcholine and causes muscarinic receptor supersensitivity in rat retina

Three weeks after a single intravitreal injection of ethylcholine aziridinium ion (AF64A), there was a significant depletion of retinal acetylcholine (ACh) content. The depletion was accompanied by an increase of [ 3H]quinuclidinyl benzilate binding sites and a greater than control conversion of myo-[ 3H]inositol to inositol monophosphate following incubation with ACh. These changes are consistent with receptor supersensitivity due to AF64A-induced denervation.

Effects of WEB 1881 FU, a Novel Nootropic, on Cholinergic and Adrenergic Receptors in the Rat Brain: Action on M1-Muscarinic Receptors

AbstractEffects of 4-aminomethyl-1-benzylpyrrolidin-2-one-hemifumarate (WEB 1881 FU), a novel pyrrolldinone nootropic, on acetylcholine (ACh) receptors and adrenoceptors were investigated using crude membranes of the rat brain. The affinity order of WEB 1881 FU was: M1-muscarinic (m) ACh receptor>M2-mACh receptor>α2-adrenoceptor>β- adrenoceptor>α1-adrenoceptor>nicotinic ACh re-ceptor. The WEB 1881 FU-competition curve for [3H]pirenzepine binding in hippocampal membranes was rightward-shifted by GTPγS; such behavior was also observed in the case of oxotremorine but not with scopolamine. The effects of long-term administration of WEB 1881 FU(30 mg/kg/day, I.p.) for 21 days resulted in a significant decrease in the Bmix of [3H]quinuclidinyl benzilate binding in the cerebral cortex, hippocampusandstriatum. In addition, the Bmax of [3H] pirenzepi ne binding to hippocampal and striatal membranes and that of [3H]AF-DX 116 binding to cerebellar membranes were significantly decreased as well. From these results, WEB 1881 FU seems to act on M1-mACh receptors, and its long-term administration probably induces the down-regulation of mACh receptors, mainly M1-mACh receptors in the hippocampus and striatum and M2-mACh receptors In the cerebellum

Effects of WEB 1881 FU, a novel nootropic, on cholinergic and adrenergic receptors in the rat brain: Action on M1-muscarinic receptors.

Effects of 4-aminomethyl-1-benzylpyrrolidin-2-one-hemifumarate (WEB 1881 FU), a novel pyrrolidinone nootropic, on acetylcholine (ACh) receptors and adrenoceptors were investigated using crude membranes of the rat brain. The affinity order of WEB 1881 FU was: M1-muscarinic (m) ACh receptor greater than M2-mACh receptor greater than alpha 2-adrenoceptor greater than beta-adrenoceptor greater than alpha 1-adrenoceptor greater than nicotinic ACh receptor. The WEB 1881 FU-competition curve for [3H]pirenzepine binding in hippocampal membranes was rightward-shifted by GTP gamma S; such behavior was also observed in the case of oxotremorine but not with scopolamine. The effects of long-term administration of WEB 1881 FU (30 mg/kg/day, i.p.) for 21 days resulted in a significant decrease in the Bmax of [3H]quinuclidinyl benzilate binding in the cerebral cortex, hippocampus and striatum. In addition, the Bmax of [3H]pirenzepine binding to hippocampal and striatal membranes and that of [3H]AF-DX 116 binding to cerebellar membranes were significantly decreased as well. From these results, WEB 1881 FU seems to act on M1-mACh receptors, and its long-term administration probably induces the down-regulation of mACh receptors, mainly M1-mACh receptors in the hippocampus and striatum and M2-mACh receptors in the cerebellum.

Two distinct, divalent cation-sensitive, antagonist binding states of heart muscarinic receptors: Differential modulation by guanine nucleotide

1. 1. The binding of [ 3H]quinuclidinylbenzilate (QNB), a muscarinic antagonist, to cardiac muscarinic receptors was investigated in two subcellular fractions (washed particles and microsomes) isolated from rat heart atria and ventricles. 2. 2. 5′-guanylylimidodiphosphate (Gpp(NH)p, 0.1 mM), increased (2-3-fold) the binding to washed particles, but not to microsomes, whereas Mg 2+ (1–20 mM) increased (up to 5-fold) the binding to microsomes, but not to washed particles. Gpp(NH)p modestly increased the affinity while Mg 2+ decreased the affinity towards the radiolabelled antagonist. 3. 3. Treatment with N-ethylmaleimide (NEM, 2 mM) increased the antagonist binding to either fraction. The stimulatory effect of Gpp(NH)p was not evident while that of Mg 2+ survived in the NEM-treated fractions. 4. 4. The treatment of fractions with divalent cations chelators (EDTA, EGTA; 10 mM) augmented the stimulatory effect of Mg 2+ on [ 3H]QNB binding to microsomes while that of Gpp(NH)p on the washed particle [ 3H]QNB binding was decreased. Such treatment further revealed an inhibitory action (about 40%) of Mg 2+ on the washed particle binding.

Differential distribution of muscarinic receptor subtypes and their regulation by G-protein in rat brain

1. [ 3H]quinuclidinyl benzilate ([ 3H]QNB) binding in rat cerebral and cerebellar synaptosomes had different B max values, but similar K d values. 2. These bindings could be displaced by classic muscarinic agents: pilocarpine (partial agonist), and atropine (antagonist), which both had similar binding affinities in rat cerebral and cerebellar synaptosomes. 3. The new muscarinic M 1 selective agents: McN-A-343 (agonist), prienzepine and trihexyphenidyl (antagonists) and higher affinities for receptor sites in the cerebrum than in the cerebellum. 4. The muscarinic M 2 selective agents: carbachol, oxotremorine (agonists), and AF-DX-116 (antagonist) had higher affinities for receptor sites in the cerebellum than in the cerebrum. 5. GPP(NH)p (40 μM) decreased the binding affinities of carbachol and oxotremorine in the cerebellum, but not in the cerebrum. However, it did not decrease the binding affinities of all the antagonists studied in both brain regions. 6. These results reveal that more muscarinic M 1 sites are present in the cerebrum than in the cerebellum, while the opposite is true for M 2 sites. Furthermore, the regulatory role of G-protein on these muscarinic receptor subtypes in the brain is different.

Unilateral decortication affects muscarinic cholinergic receptor binding in rat basal forebrain

The distribution of muscarinic acetylcholine receptors (including the M(1)-receptor subtype), beta-adreno receptors, and noradrenaline uptake sites was studied in cholinergic basal forebrain nuclei (medial and lateral septum, ventral and dorsal limb of the diagonal band, substantia innominata and nucleus basalis of Meynert) of adult rats 25 days after unilateral decortication, using quantitative receptor autoradiography. Unilateral neocortex suction lesion resulted 25 days postoperatively in significant enhancements of [(3)H]quinuclidinyl benzilate binding to the total population of muscarinic acetylcholine receptors in both sides of the substantia innominata (> 55%) and of the nucleus basalis of Meynert (> 45%) compared to sham-operated control rats. Additionally, increased binding levels in the medial septum (59%) and dorsal limb of the diagonal band (48%) were found in unilaterally decorticated rats. [(3)H]pirenzepine binding to M(1)-receptors was increased only in the nucleus basalis of Meynert (by 35%), in both sides, as a consequence of hemi-decortication. Unilateral decortication did not affect either [(3)H]dihydroalprenolol binding to beta-adreno receptors, or [(3)H]desipramine binding to noradrenaline uptake sites, in any of the basal forebrain regions studied. It is suggested that the enhanced binding to muscarinic acetylcholine receptors in the substantia innominata, medial septum and dorsal limb of the diagonal band following cortical damage may reflect an adaptive response to the morphological changes seen in cholinergic neurons of the nucleus basalis of Meynert after cortical lesion, whereas the receptor binding changes in the nucleus basalis of Meynert itself cannot be easily related to the morphological alterations observed there.

Properties of a quinuclidinyl benzilate binding component in the bulb mite

1. 1. About 95% of the specific binding (defined by atropine) of the muscarinic ACh receptor antagonist [ 3H]-quinuclidinyl benzilate occurred in the low speed pellet (5500 g , 10 min) of whole bulb mite homogenates and was heat sensitive, linear with tissue concentration and saturable. Rosenthal analysis indicated that [ 3H]QNB was binding with high affinity ( K d 474 pM) to a single class of low density ( B max 202 fmol g −1) binding sites; the Hill coefficient was 1.0. 2. 2. Kinetic studies revealed that binding was rapid and reversible, with association ( K +1) and dissociation ( k −1) rate constants of 7.8 × 10 5sec −1 M −1 and 3.1 × 10 −4sec −1, respectively. The K d from kinetic data ( k −1 k −1 ) was 414 pM. 3. 3. Muscarinic drugs generally were much more potent than were nicotinic drugs as inhibitors of [ 3H]QNB binding, and muscarinic antagonists generally were more potent than were agonists. 4. 4. Binding was stereoselective because R(−)QNB was about 10,000 times more potent than S( + )-QNB, and dexetimide was about 1000 times more potent than its enantiomer levetimide as inhibitors of [ 3H]QNB binding. 5. 5. Pirenzepine, a selective antagonist of mammalian M-1 muscarinic receptors was very effective in protecting against [ 3H]QNB binding, whereas methoctramine, a selective antagonist of M-2 muscarinic receptors, was much less effective. 6. 6. Altogether these data suggest that the specific binding of [ 3H]QNB is most likely to a popoulation of putative muscarinic ACh receptors, probably of the M-1 subtype.

Changes in Choline Acetyltransferase Activity and High-Affinity Choline Uptake, but Not in Acetylcholinesterase Activity and Muscarinic Cholinergic Receptors, in Rat Somatosensory Cortex after Sciatic Nerve Injury

Selected cholinergic markers (choline acetyltransferase, acetylcholinesterase, muscarinic acetylcholine receptor, high-affinity choline uptake) were studied in the hindlimb representation areas of the rat somatosensory cortex and within the visual cortex 1 to 63 days after unilateral transection of the sciatic nerve. In the contralateral somatosensory cortex, peripheral deafferentation resulted in a significant reduction of choline acetyltransferase activity (by 15%) 3 days after sciatic nerve injury, and in a significant reduction of high-affinity choline uptake (by 30%) 1 day after nerve transection, in comparison to untreated control rats. Investigations in individual cortical layers revealed that the decrease of both choline acetyltransferase activity and high-affinity choline uptake sites was mainly due to reductions in cortical layer V. Acetylcholinesterase activity and [3H]quinuclidinyl benzilate binding to muscarinic acetylcholine receptors were not affected by unilateral transection of the sciatic nerve. In the ipsilateral somatosensory cortex, as well as in the visual cortex at both cortical hemispheres, no significant changes in the cholinergic parameters studied could be detected. The data indicate that peripheral deafferentation of the somatosensory cortex results in a transient change of presynaptic cholinergic parameters within the affected somatosensory area as early as 1 to 3 days after the lesion; thus, they emphasize the involvement of cholinergic mechanisms in cortical reorganizational events.

Functional alterations in striatal cholinergic and striato-nigral gabaergic neurons following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration

Neurochemical changes in cholinergic neurons in the striatum and ?-aminobutyric acid (GABA)ergic neurons in the midbrain, especially in the basal ganglia, have been examined following the intraperitoneal administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which is known to cause behavioral changes resembling those in Parkinson's disease due to the destruction of nigro-striatal dopaminergic neurons. Although the adminstration of MPTP (50 mg/kg, once a day for 5 days) to male mice of dd strain did not induce the changes in choline and acethylcholine contents in the striatum, a decrement of GABA content in the midbrain, especially in the substantia nigra, was found in addition to a significant decrease of dopamine content in the striatum. This decrement in GABA content was accompanied by a decrease of turnover rate of GABA in the substantia nigra. Furthermore, it was found that MPTP treatment induced an increase of the GABA(A) receptor-benzodiazepine receptor-chloride channel complex in the substantia nigra in addition to that of dopaminergic D(2) receptor in the striatum. Although the MPTP treatment did not induce significant changes in acetylcholinesterase and choline acetyltransferase activities as well as high affinity choline uptake, this treatment induced a decrease of [(3)H]quinuclidinyl benzilate binding to muscarinic receptor in the striatum. These results suggest that the occurrence of behavioral changes induced by MPTP administration may involve functional alterations in cholinergic interneuron in the striatum as well as those in the striato-nigral GABAergic neuron, which are associated with the destruction of nigro-striatal dopaminergic neuron with this agent.

Effect of cold stress on cholinergic receptors in the rat adrenal gland

The effects of cold exposure on cholinergic binding sites in the rat adrenal gland were assessed by examining the binding of [ 125I]α-bungarotoxin (BTX), a nicotonic receptor antagonist and [ 3H]quinuclidinyl benzilate (QNB), a muscarinic receptor antagonist, to adrenal tissue homogenates. Cold exposure resulted in significant alterations in both nicotinic and muscarinic binding. Exposure to cold for 4 and 7 days resulted in a significant decrease in QNB binding. Scatchard analysis indicates that this alteration is due to a decrease in binding sites ( B max) rather than a change in ligand affinity ( K d). In contrast, chronic cold exposure produced a significant increase in BTX binding sites. These results indicate that adrenal cholinergic receptors are altered in reciprocal fashion by chronic cold exposure, and that this change may represent a key event in the sympathoadrenal system's adaptive response to chronic cold stress.

Evidence for cholinergic innervation in dog renal tissue.

Denervation procedures that affect the sympathetic system of the kidney, as demonstrated by norepinephrine depletion of renal tissue, increased urine volume, fractional sodium excretion, and free water clearance in anesthetized water-loaded dogs. These increases were reduced by atropine, which also blocked the increase above those basal functional levels produced by acetylcholine in both innervated and denervated kidneys. An in vitro tubular cell preparation of innervated kidneys corresponding to the outer cortex showed [3H]quinuclidinyl benzilate (QNB) binding parameters characteristic of muscarinic receptors. Denervation did not change either [3H]QNB binding parameters or the ability of inner and outer cortex cells to perform the hemicholinium-3-inhibitable, sodium-dependent choline uptake and conversion of [3H]choline to [3H]acetylcholine. This cell membrane behavior is similar to that observed in tissues where cholinergic neuronal structures are present, suggesting the existence of a cholinergic innervation of the kidney, independent of the integrity of vessel-traveling nerves. Similarly, the finding of choline acetyltransferase in renal tissue, unaffected by sympathetic denervation, seems to confirm the presence of cholinergic nerve terminals. The cholinergic system may thus contribute to the regulation of tubular reabsorption of sodium and water in some conditions.

Modulation of central muscarinic receptor binding in vitro by ultralow levels of the organophosphate paraoxon

Homogenates of calf caudate nuclei were found to contain at least three distinct subclasses of cholinergic, muscarinic receptors. These subtypes, labeled with [ 3H]quinuclidinyl benzilate (QNB), can be separated by rapid filtration with the use of the selective ligands, pirenzepine, AF-DX116, and 4-DAMP which have high affinity for the M1, M2, and M3 subtypes, respectively. Paraoxon was found to modulate [ 3H]QNB binding in a noncompetitive manner at concentrations below those needed to affect acetylcholinesterase activity. Pretreatment of the membrane protein with high concentrations of both the M2 selective antagonist, AF-DX116, and the M3 selective antagonist, 4-DAMP, protected against paraoxon inhibition of [ 3H]QNB binding, while the M1 selective antagonist pirenzepine did not. Paraoxon sensitive sites, M2 and M3, are found predominantly on presynaptic neurons in the central nervous system. It is postulated that blockade of these sites may interfere with negative feedback inhibition of acetylcholine release and facilitate the development of behavioral and motor deficits that may be associated with chronic exposure to low levels of organophosphates.

Autonomic Receptors and Cyclic Nucleotides of Rat Parotid Gland following Simultaneous Electrical Stimulation of Its Parasympathetic and Sympathetic Nerves

[3H]dihydroalprenolol and [3H]quinuclidinylbenzilate binding of membranes of rat parotid gland were generally unchanged after 10, 15, 30, or 60 min of simultaneous electrical stimulation of the parasympathetic and sympathetic nerves to the gland, although stimulation of either nerve separately caused nerve-specific changes in both. Concentrations of cyclic nucleotides of the gland were, however, increased significantly from levels of the unstimulated parotid gland. Cyclic GMP showed a 10-fold increase after 10 min of stimulation, whereas only a 2-fold increase in cyclic AMP was found at this time. The increases were maintained, albeit at reduced levels, at 15 and 30 min also but by 60 min both were not different from levels of the unstimulated gland. The increases induced by separate stimulation of each nerve were greater but nerve specific, and the changes induced with simultaneous stimulation tended to reflect a reigning influence of one nerve on the other.

The H-L subgroup of guinea-pig cardiac M 2 receptors (M 2β) regulates inositol phosphate formation

In previous studies, we showed that cardiac muscarinic receptors (M 2) are composed of two subgroups, M 2α and M 2β, with different affinities for agonists and that the M 2α subgroup is coupled with inhibition of adenylate cyclase. We now studied which subgroup was responsible for the formation of inositol mono- (IP), bis- (IP 2), tris- (IP 3) and tetrakis- (IP 4) phosphates in guinea pig heart. Carbachol (1 mM) significantly stimulated the formation of all four IPs in [ 3H]myoinositol-preloaded slices of guinea-pig ventricles. Acetylcholine (1 mM) also stimulated the formation of IP 2, IP 3 and IP 4. However, oxotremorine (1 mM) only slightly stimulated the formation of IP 2, and pilocarpine did not stimulate the formation of any IP. The pED 50 values of carbachol for IP 2 and IP 3 formation were 3.76 and 4.23, respectively, which coincided with the pK d values of the low-affinity agonist binding site (L site) measured by competition of carbachol with [ 3H]quinuclidinyl benzilate ([ 3H]QNB) binding while the pK d value for inhibition of adenylate cyclase coinceded with the pK d value of the high-affinity agonist binding site (H site). Treatment of animals with pertussis toxin decreased the formation of IP 2 and IP 3 by carbachol to 66 and 54%, respectively, but resulted in complete inhibition of adenylate cyclase. These results suggested that muscarinic stimulation of the formation of IPs was manifested through a different receptor subgroup (M 2β) and a GTP binding protein different from those for inhibition of adenylate cyclase.

Galanthamine, an acetylcholinesterase inhibitor: A time course of the effects on performance and neurochemical parameters in mice

The time course of the effects of the long-acting acetylcholinesterase (AChE) inhibitor, galanthamine, on a spatial navigation task and on AChE and acetylcholine (ACh) levels were investigated in mice. Mice received either saline or ibotenic acid injections into the nucleus basalis magnocellularis (nBM). The control and nBM group were then trained to perform a modified Morris swim task and the time to find the hidden platform was recorded. The nBM group took significantly longer to find the platform than the control group in the reversal phase of testing. Galanthamine attenuated the performance deficit in the nBM-lesioned group in a time-dependent manner, with peak performance at four hours after injection of 5.0 mg/kg galanthamine IP. This dose impaired performance of the task in control mice, with the most severe deficits observed at two hours after injections when motor activity was severely reduced. Galanthamine (5.0 mg/kg IP) significantly decreased cortical AChE activity and significantly increased cortical ACh content in control mice in a time-dependent manner. The time courses of the neurochemical effects, however, did not correlate precisely with the behavioral time course. Galanthamine concentrations up to 1 × 10 −5 M did not affect choline acetyltransferase (ChAT) activity, [ 3H]hemicholinium-3 (HCh-3) binding to the choline carrier, [ 3H]quinuclidinylbenzilate (QNB) binding to muscarinic receptors, or [ 3H]acetylcholine binding to nicotinic receptors in cortical homogenates. AChE activity was inhibited by galanthamine in cortical homogenates with an IC 50 of 4.1 × 10 −7 M. Galanthamine's ability to reverse cognitive deficits induced by nBM lesions, its relatively long half-life and its specificity of effects suggest that this drug may be effective in treating the central cholinergic deficits in Alzheimer's disease and related disorders.

Muscarinic agents modify kinetics properties of membrane-bound guanylyl cyclase activity

Plasma membranes from bovine tracheal smooth muscle show guanylyl cyclase activity, which can be stimulated by muscarinic agonists such carbamylcholine and oxotremorine and blocked by atropine. This stimulation was observed in the presence of 150 mM NaCl. In the absence of this salt, guanylyl cyclase activity was considerably higher but was not affected by muscarinic agonists. Carbamylcholine decreased the apparent K m but did not change the V max of this enzyme. When plasma membrane fractions were extracted with 1% octylglucoside, guanylyl cyclase activity was preserved, however the muscarinic activation was abolished, despite a muscarinic receptor capable of [ 3H]quinuclidinylbenzilate binding being present in the extract. The detergent extraction changed the affinity of guanylyl cyclase for GTP but the Mn 2+ kinetics was unaltered. Based on these findings and on current information in the literature, we propose that another component is required to restore the link between the muscarinic receptor and guanylyl cyclase, however the nature of this component remains to be established.

Blockade of ACh receptors by PrBCM causes deficits in shuttle avoidance performance

Studies were made examining the effect of blockade of muscarinic acetylcholine (mACh) receptors in the cerebral cortex of rats on their shuttle avoidance after training. Rats were given a session of shuttle avoidance tests once a day for 12 days. Then the irreversible antagonist of mACh receptors, propylbenzilylcholine mustard (PrBCM), was injected bilaterally into the cerebral cortex of rats showing avoidance rates of more than 75% in the last session, and avoidance rates were examined 24 hr later. The avoidance rates of the rats treated with 100 μg PrBCM were lower than those in the last session before treatment. The amount of mACh receptors in the cerebral cortex was decreased by PrBCM treatment, as shown by [ 3H]quinuclidinyl benzilate (QNB) binding studies performed just after measurement of the avoidance response. The present study indicates that cholinergic neurotransmission in rat cerebral cortex is involved in performing a learned shuttle avoidance.