Density Of Muscarinic Binding Sites Research Articles

Postnatal development of muscarinic receptor-stimulated phosphoinositide metabolism in mouse cerebral cortex: sensitivity to ethanol

An enhanced coupling of cholinergic muscarinic receptors to phosphoinositide metabolism had been previously observed in brain from immature rat. This study reports that the postnatal development of muscarinic receptor-stimulated phosphoinositide metabolism is also enhanced in cerebral cortex slices from immature Swiss-Webster and Balb-c mice, as compared to adults. Response to the agonist carbachol was lower on postnatal day 3, peaked between days 5 and 12 and then declined to adult levels. Density of muscarinic binding sites, measured with the M 1 ligand [ 3H]telenzepine on postnatal day 7, was, on the other hand, only half of the adult value. Phosphoinositide hydrolysis stimulated by glutamate decreased with age, while that elicited by norepinephrine increased. These results are also similar to those previously reported in the rat. Ethanol has been found to inhibit muscarinic receptor-stimulated phosphoinositide metabolism in rat brain in an age-dependent manner. This was confirmed in mouse brain, where ethanol inhibited this response in cerebral cortex of immature but not adult animals. These results indicate that the enhanced muscarinic receptor-stimulated phosphoinositide metabolism, which coincides with the brain growth spurt, is similar in rats and mice. Mice may be a useful species in which to genetically manipulate muscarinic receptors to gain a better understanding of their potential role in brain development.

Bindung und Elektrophysiologie des muskarinischen Antagonisten QNB in der Säugetiernetzhaut

Based upon the extensive information from various laboratories on cholinergic enzymes and receptors in mammalian retina as well as cholinergic effects on retinal neurons we became interested in studying (1) retinal binding of a muscarinic antagonist, Quinuclidinyl benzilate (QNB) and (2), effects of the antagonist on retinal information processing. Eyes from deeply anesthetized cats were used for homogenate of freshly isolated retina in the binding study, or they were arterially perfused for electrophysiology in vitro. The electroretinogram (ERG) and the compound action potential of the optic nerve (optic nerve response, ONR) were recorded under rod- and cone-stimulating conditions. QNB was infused intraarterially for 10-30 min, followed by washout (avoiding recycling or extraocular metabolism). 3H-QNB revealed a high affinity to muscarinic receptors with a dissociation constant KD of 0.27 nM and a relatively high density of muscarinic binding sites of 110 fmol per mg protein. QNB enhanced the amplitude of the ERG b-wave, but decreased dose-dependently and reversibly the components of the ONR. In addition, we observed a moderate vasoconstriction as indicated by a slight, dose-related decrease in flow rate of perfusion. The biochemical data on binding of 3H-QNB in connection with the marked electrophysiological changes induced by QNB suggest a substantial contribution of muscarinic cholinergic transmission in the cat retina.

Intracerebroventricular administration of nerve growth factor affects muscarinic cholinergic receptors in the cerebral cortex of neonatal rats.

The repeated intracerebroventricular administration of nerve growth factor (5 micrograms/2.5 microliters) to neonatal rats induced the activation of choline acetyltransferase in forebrain cholinergic neurons that was paralleled by a concomitant change in the density of muscarinic cholinergic receptors in the cerebral cortex. The administration of nerve growth factor altered muscarinic binding sites in a biphasic fashion during postnatal development. A significant stimulation of the developmental increase in the density of muscarinic binding sites occurred in nerve growth factor-treated animals at days 2 and 3 after birth. Conversely, nerve growth factor induced a significant decrease in the receptor number at postnatal days 8 and 14. Muscarinic receptor number returned to control values after treatment, suggesting that nerve growth factor-induced changes to muscarinic cholinergic receptors are reversible. Nerve growth factor administration did not affect muscarinic cholinergic receptor density in striatal membranes and did not alter the relative content of cortical messenger RNAs encoding m1 and m3 muscarinic cholinergic receptor subtypes at postnatal day 14, as determined by reverse transcriptase-polymerase chain reaction. The up- and down-regulation of muscarinic cholinergic receptors induced by nerve growth factor during postnatal development may be temporally related events associated with concomitant changes in the activity of choline acetyltransferase.

Potassium ions potentiate the muscarinic receptor-stimulated phosphoinositide metabolism in cerebral cortex slices: A comparison of neonatal and adult rats

Activation of cholinergic muscarinic receptors results in an increased turnover of membrane inositol phospholipids. In rat cerebral cortex slices, carbachol- and acetylcholine-induced inositol phosphates ([3H]InsPs) accumulation is maximal in 7 day-old rats and lowest in adults, while the density of muscarinic binding sites increases gradually with age, suggesting the presence of a more effective receptor-effector coupling during neonatal life. In the process of investigating the nature of such differential stimulation, we have studied the effects of potassium ions on muscarinic receptor-stimulated phosphoinositide metabolism during development. Increasing the concentration of K+ from 6 to 12 mM potentiated the stimulating effect of carbachol by 80-100% in adult animals, as previously shown, but only 10-20% in 7 day-old animals, without altering its EC50 values. The differential potentiation by K+ at these two ages was specific for muscarinic receptors, since norepinephrine-stimulated accumulation was potentiated only 18% and 12% in adult and 7 day-old rats, respectively. Two other monovalent cations, rubidium and cesium, had the same effect as K+ on carbachol-stimulated [3H]-InsPs accumulation. The effect of K+ was not antagonized by the K+ channel blocker 4-aminopyridine, but was antagonized by tetraethylammonium (TEA). TEA, however, also interacted with muscarinic binding sites. Omission of calcium from the incubation medium did not influence the potentiating effect of K+ during development was inversely proportional to the stimulation of phosphoinositide metabolism induced by carbachol. These results suggest that the mechanism responsible for the potentiating effect of K+ in adult rats might be already operating in neonatal animals.

Formula omitted] 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 ([ 3 H]QNB) and [ 3]N- methylscopolamine ([ 3 H]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 < 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.

A brain regional analysis of morphologic and cholinergic abnormalities in Alzheimer's disease.

In the brains of 21 patients with Alzheimer's disease (AD) and 10 nondemented controls, senile plaques (SPs), neurofibrillary tangles (NFTs), and three indexes of cholinergic function were quantified in the middle frontal (MF) and superior temporal (ST) cortex, the entorhinal cortex (HEN), and the prosubiculum (HPR) of the hippocampus. Control brains contained few SPs without preferential distribution in any of the brain regions examined, while NFTs were found almost exclusively in the HPR. In brains from patients with AD, an inverse relationship of SPs and NFTs was found in the brain regions examined; SPs were preferentially in the neocortex and NFTs preferentially in the hippocampus. The specific activities of choline acetyltransferase and acetylcholinesterase were reduced in all regions examined, while no significant change in the density of muscarinic binding sites was observed in any region. Numerous NFTs were associated with an earlier age at onset, while the presence of SPs was related to the cholinergic deficit in AD. Earlier-onset (less than 67 years) AD was also associated with a qualitative difference in the regional distribution of NFTs compared with cases with a later onset. In the latter group, most NFTs were observed in the hippocampus, a distribution pattern similar to that observed with normal aging. In AD cases with an earlier onset, NFTs were more globally distributed in the neocortex and allocortex.

Bilateral Symmetry of Cholinergic Deficits in Alzheimer's Disease

The specific activities of the cholinergic enzymes, choline acetyltransferase and acetylcholinesterase, as well as the density of muscarinic binding sites, were determined in five corresponding left and right regions of 16 brains obtained at autopsy from patients with histologically confirmed Alzheimer's disease. While a significant proportion of the individual specimens exhibited left-right asymmetries in cholinergic deficits, bilateral symmetry was the rule for frontal and temporal cortex, basal nucleus, and the prosubiculum of the hippocampus. In contrast to these four regions, left-right asymmetries in choline acetyltransferase activity and muscarinic receptor density appeared to be typical in the entorhinal cortex of the hippocampus.

Muscarinic stimulation of inositol phosphate formation in rat retina: Developmental changes

The stimulation of inositol phosphates by various cholinergic agonists and the effect of antagonists was studied in rat retinal slices of different ages. The potency order of muscarinic agonists and antagonists for receptors linked to inositol phosphate production was the same for retinas of all ages. The maximum stimulation of inositol phosphate production varied between retinas of different ages, being greatest in the younger animals and lowest in the adult. However, no difference was found in the EC 50 values. This contrasts with the neurotransmitters, noradrenaline and serotonin, both known to stimulate phosphoinositide metabolism; neither was found to have the same developmental profile of carbachol-induced stimulation of inositol phosphates. In contrast to the muscarinic receptors linked to phosphoinositide hydrolysis, the density of muscarinic binding sites measured by [ 3H]QNB binding increased in retinas during development. The affinity constants ( K d) remained constant, however. The results are interpreted as suggesting that muscarinic receptors linked to inositol phosphate production are more efficiently coupled (to a guanine regulator protein) in retinas of young animals than in the adult.

Muscarinic receptors of rat parotid gland enlarged by gland ablation and bulk diet. Effects of denervation

The density of muscarinic binding sites was increased 10% in the rat parotid gland enlarged (2 times control) as a result of ablation of the submandibular-sublingual glands and maintenance of rats on bulk diet (50% inert cellulose plus 50% solid chow) for 4 weeks. When either the parasympathetic or sympathetic innervation to the gland was unilaterally removed at the time of submandibular-sublingual ablation and introduction of the bulk diet, the density of muscarinic receptors showed an even greater increase from levels of innervated glands of chow-fed controls (29%); with removal of both nerves, the increase was 39%. A 36% increase in cyclic guanosine monophosphate levels accompanied the increase in receptors of the enlarged gland, but when the parotid was denervated, there was no change in cyclic GMP. Absence of either or both nerves led to a maximal decrease of 24–29% in density of muscarinic receptors of parotid gland of chow-fed controls, but to no change in cyclic GMP levels. While autonomic influences mediate the changes in density of muscarinic receptors of parotid gland of chow-fed rats, some additional factor is apparently involved in their increase in the enlarged gland.

Divergent regulation of muscarinic binding sites and acetylcholinesterase in discrete regions of the developing human fetal brain.

The expression of muscarinic acetylcholine binding sites and of cholinesterases was studied in extracts prepared from discrete regions of the human fetal brain, between the gestational ages of 14 and 24 weeks. The specific binding of [3H]N-methyl-4-piperidyl benzilate [( 4H]-4NMPB) to muscarinic binding sites ranged between 0.05 and 1.30 pmol/mg protein in the different brain regions, with Kd values of 1.2 +/- 0.2 nM. Binding of the cholinergic agonist oxotremorine fitted, in most of the brain regions examined, with a two-site model for the muscarinic binding sites. The density of muscarinic binding sites increased with development in most regions, with different rates and onset times. It was higher by about sixfold in some areas destined to become cholinergic, such as the cortex and midbrain, than in noncholinergic areas such as the cerebellum. In other areas destined to become cholinergic, such as the hippocampus and the caudate putamen, the receptor density remained low. Average density values increased from 0.1 +/- 0.1 at 14 weeks up to 0.7 +/- 0.4 pmol/mg protein at 24 weeks. The variability in the specific activities of cholinesterase was relatively low, and extracts from different brain regions hydrolyzed from 5 to 30 nmol of [3H]acetylcholine/min/mg protein. These were mostly "true" acetylcholinesterase (EC 3.1.1.7) activities, inhibited by 10(-5) M BW284C51, with minor pseudocholinesterase (EC 3.1.1.8) activities, inhibited by 10(-5) M iso-OMPA. The enzyme from different brain regions and developmental stages displayed similar Km values toward [3H]acetylcholine (ca. 4 X 10(-4) M-1). The ontogenetic changes in cholinesterase specific activities had no unifying pattern and/or relationship to the cholinergic nature of the various brain areas. In most of the brain regions, the arbitrary ratio between the specific activity of cholinesterase and the density of muscarinic binding sites decreased with development, with average values and variability ranges of 83 +/- 50 and 19 +/- 19 at 14 and 24 weeks, respectively. Our findings suggest divergent regulation for cholinergic binding sites and cholinesterase in the fetal human brain and imply that the expression of muscarinic receptors is related to the development of cholinergic transmission, while acetylcholinesterase is also involved in other functions in the fetal human brain.

Muscarinic binding sites in small homogenates and in autoradiographic sections from rat and human spinal cord

Binding of labelled l-quinuclidinylbenzylate was studied in cryosections and homogenates of human and rat spinal cord. For the cryosections an autoradiographic method was used. With both techniques a higher density of muscarinic binding sites was found in rat than in human spinal cord. In the autoradiograhs very high densities of muscarinic binding sites were observed in the motor neurone area and in the apical part of the dorsal horn. The latter high density region was not always found in homogenates from dissected tissue samples. The autoradiographic technique has a better resolution for detecting discrete regional variations in the receptor content of the spinal cord.