Characterization of agonist and antagonist binding to muscarinic cholinergic receptors solubilized from rat cerebral cortex

Summary The muscarinic acetylcholine receptor was solubilized from rat brain cortex by the zwitter-ionic detergent, 3-((3-cholamidopropyl)dimethylamino)1-propane sulfonate (CHAPS). The supernatant, after centrifugation at 100,000 × g, was shown to contain molecules with binding sites for both³H-pirenzepine (³H-PZ) and³H-(-) quinuclidinyl benzilate (³H-QNB). Maximum binding values for³H-PZ and³H-QNB binding to solubilized receptors were approximately 176±24 pmol/g and 370±53 pmol/g of protein, respectively. The K_d values for³H-PZ and³H-QNB binding to solubilized receptors were 27±6.3nM and 0.17±0.03 nM, respectively. The rank order of potencies of muscarinic drugs, in terms of their ability to inhibit binding of either³H-PZ or³H-QNB, was atropine > pirenzepine > oxotremorine > carabachol. Pirenzepine inhibited³H-QNB binding with a Hill coefficient of 0.77, but inhibited³H-PZ with a Hill coefficient of 0.94. Hill coefficients for agonists were less than 1. These findings indicate that muscarinic receptors solubilized from rat brain cortex retain their abilities to interact selectively with muscarinic receptor agonists and antagonists.     

Quantitative autoradiography of muscarinic cholinergic receptors in the rat brain

Using the in vitro autoradiographic technique with tritium-sensitive LKB sheet film and the liquid scintillation counting method, the distribution and the binding parameters of the muscarinic cholinergic receptors (MChR) were determined in various discrete regions of the rat brain. The results obtained in the present study were as follows:

(1) Specific binding of [³H]QNB to the slide-mounted tissue sections increased slowly when incubated at room temperature; saturation occurred 2 h after incubation. Only 23% of [³H]QNB bound to the tissue section was dissociated 5 h after the addition of 20 μM atropine to the medium. These findings were very different from those obtained in the study using the tissue homogenates.

(2) The regional distribution of MChR was determined using both autoradiographic and liquid scintillation counting methods. The distribution of MChR was heterogeneous, with highest densities in the striatum and nucleus accumbens and lowest in the globus pallidus, nucleus interpenduncularis and nucleus septi. Moreover, MChR were unevenly distributed within the subfields of each region.

(3) In saturation binding studies using the slide-mounted tissue sections of 20 μm thickness the (K_d)_app-values were similar but not exactly identical in 5 discrete regions, i.e. the striatum, somatosensory cortex, hippocampus (the subiculum + CA1 field), nucleus accumbens and gyrus dentatus, determined in the present study. The (K_d)_app-value of each region was about 700 pM which was about 20 times higher than that obtained in the study using the tissue homogenates. However (K_d)_app-values obtained with 5 and 10 μm tissue sections were approximately 3-fold lower.

Characterization of muscarinic cholinergic receptors in human and dog cerebral arteries

Various concentrations of acetylcholine (ACh) produced dose dependent relaxations of isolated, helical preparations of human cerebral arteries and these responses were blocked by atropine. The median effective concentration (EC50) of ACh was 6.1 +/- 0.2 X 10(-6)M. ACh produced dual responses in isolated dog cerebral arteries. ACh in low concentrations produced relaxation, and contraction occurred when the concentration was raised to 1 X 10(-5)M. The EC50 of ACh which produced relaxation, in dog cerebral arteries was 7.2 +/- 0.2 X 10(-7)M. Muscarinic cholinergic receptors in these arteries were analyzed directly using 3H-QNB as the ligand. The specific 3H-QNB binding to the arteries was saturable and of KD = 1.5 nM and Bmax = 93 fmol/mg protein in human cerebral arteries and KD = 0.59 nM, Bmax = 340 fmol/mg protein in dog cerebral arteries. Specific binding of 3H-QNB was displaced by muscarinic cholinergic agents. Ki values and Hill coefficients were as follows: QNB, 1.0 X 10(-9)M, 0.89; atropine, 1.1 X 10(-8)M, 0.95; ACh, 0.8 X 10(-8)M and 2.1 X 10(-6)M, 0.54; carbachol, 1.2 X 10(-7)M and 4.3 X 10(-5)M, 0.33 in human cerebral arteries and QNB, 0.55 X 10(-9)M, 0.85; atropine 0.9 X 10(-9)M, 1.00; ACh, 0.9 X 10(-9)M and 1.1 X 10(-6)M, 0.43; carbachol 6.3 X 10(-8)M and 1.1 X 10(-5)M, 0.36 in dog cerebral arteries. Endogenous choline acetyltransferase (ChAT) activity was 1.6 +/- 0.4 nmol/mg protein/hr in human cerebral arteries and 3.7 +/- 0.1 nmol/mg protein/hr in dog cerebral arteries.(ABSTRACT TRUNCATED AT 250 WORDS)     

Axonal transport of muscarinic cholinergic receptors in rat vagus nerve: high and low affinity agonist receptors move in opposite directions and differ in nucleotide sensitivity

The presence and transport of muscarinic cholinergic binding sites have been detected in the rat vagus nerve. These binding sites accumulate both proximal and distal to ligatures in a time-dependent manner. The results of double ligature and colchicine experiments are compatible with the notion that the anterogradely transported binding sites move by fast transport. Most of the sites accumulating proximal to ligatures bind the agonist carbachol with high affinity, while most of the sites accumulating distally bind carbachol with a low affinity. Also, the receptors transported in the anterograde direction are affected by a guanine nucleotide analogue (GppNHp), while those transported in the retrograde direction are less, or not, affected. The bulk of the sites along the unligated nerve trunk bind carbachol with a low affinity and are less sensitive to GppNHp modulation than the anterogradely transported sites. These results suggest that some receptors in the vagus may undergo axonal transport in association with regulatory proteins and that receptor molecules undergo changes in their binding and regulatory properties during their life cycle. These data also support the notion that the high and low affinity agonist form of the muscarinic receptor represent different modulated forms of a single receptor molecule.     

Characterization of muscarinic cholinergic receptors in the submandibular gland of the rat

The specific muscarinic ligand [3H]quinuclidinyl benzilate ([3H]QNB) was used to label acetylcholine receptors in the submandibular gland of the rat. Specific binding of [3H]QNB increased linearly with tissue concentration in the range of 0.02-0.3 mg of protein/ml. Kinetic analysis of [3H]QNB binding revealed the presence of a single population of high affinity binding sites, with a dissociation constant of 87.2 pM and a Hill coefficient of 0.95. The binding was saturable and the receptor density was 214 fmol/mg of protein. The rate constants at 37 degrees C for association and dissociation of the [3H]QNB-receptor complex were 5.98 X 10(-8) M-1 X min-1 and 6.6 X 10(-3) X min-1, respectively. The ratio k-1/k+1 gave a Kd value of 11.1 pM, similar to the Kd value (13.1 pM) determined by kinetic parameters when extrapolated at infinitely low receptor concentration. Muscarinic antagonists displaced [3H]QNB from muscarinic receptors with a Hill coefficient near to 1.0. Displacement curves for muscarinic agonists and for the atypical antagonist pirenzepine had Hill values significantly less than one. In the presence of 0.1 mM GPP(NH)P, the potency of agonists but not antagonists in displacing [3H]QNB binding decreased 2 to 3-fold. The [3H]QNB binding site was sensitive to the inhibitory effect of various sulfhydryl reagents. Repeated treatments of rats with an acetylcholinesterase inhibitor led to a decreased density of muscarinic receptors in the submandibular gland. This alteration was specific for the muscarinic recognition site and was paralleled by a reduced sensitivity to carbachol.     

Effects of noradrenaline depletion on adrenergic and muscarinic cholinergic receptors in the cerebral cortex, hippocampus, and cerebellum

The effects of noradrenaline depletion on α- and β-adrenergic and muscarinic cholinergic receptors in the cerebral cortex, hippocampus, and cerebellum of Wistar rats were studied. Noradrenaline depletion was obtained either by chemical (6-hydroxydopamine) lesions of the locus cerulet's or by chronic reserpine treatment. Two weeks after locus ceruleus lesions and 10 days after chronic reserpine treatment, the rats were killed and the cerebral cortex, hippocampus, and cerebellum dissected. A small portion of each tissue was assayed for noradrenaline, to assess the success of locus ceruleus lesions or reserpine treatment, and the remainder was used to measure the specific binding of tritiated α- and β-adrenergic and muscarinic cholinergic receptor ligands to particulate fractions of these tissues. The effect of noradrenaline depletion on isoproterenol-induced cyclic AMP generation in cerebellar slices was also studied. Noradrenaline depletion whether by locus ceruleus lesion or chronic reserpine treatment induced reproducible and significant increases in the binding to β-adrenergic receptors in the cerebral cortex and hippocampus. Scatchard analyses revealed that this increased binding was due to an increased density of β-adrenergic receptor binding sites. In the cerebellum, however, noradrenaline depletion did not result in an increase in β-adrenergic receptor binding nor in isoproterenol-induced cyclic AMP generation. Noradrenaline depletion did not cause significant changes in the binding characteristics of α-adrenergic or muscarinic cholinergic receptors in any of the three regions of the brain that were studied.     

Repeated noise exposure affects muscarinic cholinergic receptors in the rat brain

We examined the effect of repeated exposure to 100-dB white noise (10 daily 45-min sessions) on muscarinic cholinergic receptors in different regions of the rat brain. Twenty-four hours after the last exposure session, increase in concentration (Bmax) of [3H]quinuclidinyl benzilate ([3H]-QNB) binding sites was observed in the hippocampus, but no significant change was seen in the striatum, frontal cortex, and hypothalamus. No significant effect of noise on receptor binding affinity (Kd) was found. Pretreating the rats with naltrexone (1 mg/kg, IP) before exposure blocked the noise-induced increase in cholinergic receptors in the hippocampus.     

Autoradiographic localization of high affinity GABA, benzodiazepine, dopaminergic, adrenergic and muscarinic cholinergic receptors in the rat, monkey and human retina

High affinity gamma-aminobutyric acid, benzodiazepine, strychnine (glycine), dopamine, spirodecanone, alpha 1-adrenergic, alpha 2-adrenergic, beta-adrenergic and muscarinic cholinergic binding sites were localized by semiquantitative autoradiography in rat and, in some instances, in monkey and human retinae using [3H]muscimol, [3H]flunitrazepam, [3H]strychnine, [3H]spiperone, [3H]prazosin, [3H]para-aminoclonidine, [3H]dihydroalprenolol and [3H]quinuclidinyl benzylate, respectively. In nearly every case, the inner plexiform layer (IP) contained a high receptor density. The distribution of alpha 1 sites was unusual in that binding was concentrated in the outer plexiform layer (OP). Dopaminergic and, to a lesser extent, beta-adrenergic binding was diffusely distributed in the outer nuclear layer, the OP, the inner nuclear layer and the IP. The ganglion cell layer displayed significant benzodiazepine binding. The intraretinal distribution of pre- and postsynaptic markers of these neurotransmitters is discussed.     

Cell excitation enhances muscarinic cholinergic responses in rat association cortex.

The cerebral cortex receives a prominent cholinergic innervation which is thought to play an important role in regulating its normal function. Electrophysiological studies have shown that activation of cholinergic receptors results in a marked enhancement of excitatory stimuli onto cortical neurons and it has been suggested that this effect is secondary to the blockade of several voltage- and calcium-dependent potassium conductances in these cells. It is reported here that, in addition to these effects, activation of muscarinic receptors in the prefrontal cortex elicits the appearance of a slow calcium-dependent inward current in response to the generation of action potentials. This inward aftercurrent produces a slowly decaying depolarizing afterpotential which, when activated by stimulation of the cell, can summate with the carbachol-induced depolarization greatly increasing its magnitude. As a result the ability of muscarinic receptor to elicit a depolarization and excite cells in this region can be dramatically potentiated by evoked cell activation. This effect expands the range of mechanisms by which muscarinic receptors can facilitate excitatory inputs and provides a mechanism by which the association of brief excitatory stimuli to cholinergic stimulation can selectively enhance muscarinic responses among discrete cell populations in the cerebral cortex.     

M2 muscarinic receptors mediate pressor responses to cholinergic agonists in the ventrolateral medullary pressor area

Microinjections of cholinergic agonists into the ventrolateral medullary pressor area (VLPA) evoke increase in blood pressure (BP) and heart rate (HR). Recently two major subtypes of muscarinic receptors (M1 and M2) have been identified. This investigation was designed to study the role of these muscarinic receptor subtypes in pressor responses of cholinergic agonists in the VLPA. Male Wistar rats were anesthetized with pentobarbital or decerebrated at mid-collicular level. The rats were artificially ventilated and BP and HR were recorded. Ventral medulla was exposed and the VLPA identified bilaterally by microinjections of L-glutamate. Microinjections of cis-methyldioxolane (CD, a specific agonist of M2 receptors) in the doses of 0.004-4 nanomol (nmol)/site into the VLPA evoked an increase in BP (13-56 mm Hg) and HR (7-24 bpm) which lasted for 10-50 min. Intravenous injections of the same doses of this agent failed to evoke a response. AFDX-116 (a specific M2 muscarinic receptor antagonist) microinjected into the VLPA (0.2-1.6 nmol-/site) evoked depressor responses (6-20 mm Hg). Microinjections of this agent into the VLPA prevented the pressor responses to subsequent microinjections of CD at the same sites, indicating that AFDX-116 blocked M2 receptors. AFDX-116 rendered neurons in the VLPA unresponsive to L-glutamate but this effect lasted for 30-40 min while the hypotensive and M2 receptor blocking effect lasted for 60-150 min. McN-A343 (a specific agonist for M1 receptors) or pirenzepine (PZ, a specific antagonist of M1 receptors) injected into the VLPA (0.4-4 nmol/site) failed to evoke any response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrastructural localization of cholinergic muscarinic receptors in rat brain cortical capillaries

Cholinergic innervation of the cerebrovasculature is known to regulate vascular tone, perfusion rate and permeability of the microvascular wall. Notably the cholinergic innervation of cerebral capillaries is of interest since these capillaries form the blood-brain barrier. Although there is a general consensus as to the presence of nicotinic and muscarinic receptors in the domain of the capillary wall, their precise anatomical position is unknown. The subcellular localization of muscarinic receptors in rat cortical capillaries was approached by way of immunocytochemistry at the ultrastructural level using monoclonal antibody M35 against muscarinic receptor protein. Binding of this antibody in the microvascular domain was found in 5% of the capillaries studied and was exclusively present in perivascular astroglia, and never in endothelium or pericytes. Combined with reported data on presynaptic cholinergic innervation the results indicate a cholinergic innervation pattern of non-directed presynaptic terminal structures in apposition to cholinoceptive perivascular astroglia with muscarinic receptor positive endfeet embracing the capillary basement membrane. The possible functional significance of such a cholinergic vascular innervation pattern is discussed with respect to capillary dynamics and barrier function.     

Time-dependent changes in the binding parameters of muscarinic cholinergic receptors in the rat brain

Using quantitative receptor autoradiography, effects of several incubation times on the binding parameters for [³H]quinuclidinyl benzilate ([³H]QNB) binding were investigated in 5 discrete regions of the rat brain. There were no differences in B_max-values between 3 incubation times. On the other hand, K_d(appl-values markedly depended on the duration of incubation time. Furthermore, Scatchard plots at low [³H]QNB concentrations showed an abnormal binding behavior, i.e. deviation from the straight line representation a single population of the binding site.     

Regional responses of rat brain muscarinic cholinergic receptors to immobilization stress

The effect of immobilization stress (IM-stress) on the muscarinic cholinergic (m-Ch) receptor binding was determined in 8 brain regions using [³H]quinuclidinyl benzilate (QNB). IM-stress produced an increase in specific QNB binding in the septum, striatum, hippocampus and pons + medulla oblongata. Scatchard panalyisis revealed that IM-stresss produced an increase in the affinity of m-Ch receptors in the septum, hippocampus andm pons + medulla oblangata, but did not alter the maximum number of binding sites (B_max). In the striatum, an increase in specific QNB binding was due to both the increase in B_max and reduction of the dissociation constant (K_d). The present study suggest that IM-stress induces supersensitivity of postsynaptic m-Ch receptors probably due to a deacrease in presynaptic cholinergic activities in the septum, striatum, hippocampus and pons + medulla oblongata. As the m-Ch receptors in the striatum and pons + medulla oblongata. Scatchard analysis revealed that IM-stress produced an increase in the affinity of m-Ch receptors stressful situations in this regions as well as in the septum, hippocampus and cerebral cortex.     

Autoradiographic evidence of muscarinic cholinergic receptors in the corpora cavernosa penis of the rat

Recent studies have questioned the role of acetylcholine in the physiology of penile erectile tissue. The responsiveness of penile erectile tissue to acetylcholine would depend, in part, on the presence of cholinergic receptors on the smooth muscle. The specific binding of [3H]quinuclidinyl benzilate (QNB) to cholinergic receptors in sections of penile crura of the rat was analyzed by in vitro neurotransmitter autoradiography. Silver grain density measurements indicated that muscarinic cholinergic receptor binding sites are located almost entirely over the corpora cavernosa penis. Virtually no specific [3H]QNB binding was present in the tunica albuginea or adjacent skeletal muscle tissue. Within the erectile tissue, specific binding occurred both over the columns of intrinsic smooth muscle which form the walls of the cavernous spaces and around the more distal branches of the penile arteries. The high concentration of muscarinic cholinergic receptors in the corpora cavernosa penis is consistent with the suggestion that acetylcholine has an important, albeit undefined role in the function of penile erectile tissue.     

Brain muscarinic cholinergic receptors in Huntington's disease

Summary Muscarinic cholinergic receptors and choline acetyltransferase (ChAT) activity were studied in postmortem brain tissue from patients with Huntington's disease and matched control subjects. In comparison with controls, reductions in ChAT activity were found in the hippocampus, but not in the temporal cortex in Huntington's disease. Patients with Huntington's disease showed reduced densities of the total number of muscarinic receptors and of M-2 receptors in the hippocampus while the density of M-1 receptors was unaltered. Muscarinic receptor binding was unchanged in the temporal cortex. These results indicate a degeneration in Huntington's disease of the septo-hippocampul cholinergic pathway, but no impairment of the innominato-cortical cholinergic system.     

Immunolocalization of muscarinic M1 receptor in the rat medial prefrontal cortex

The medial prefrontal cortex (mPFC) has been considered to participate in many higher cognitive functions, such as memory formation and spatial navigation. These cognitive functions are modulated by cholinergic afferents via muscarinic acetylcholine receptors. Previous pharmacological studies have strongly suggested that the M1 receptor (M1R) is the most important subtype among muscarinic receptors to perform these cognitive functions. Actually, M1R is abundant in mPFC. However, the proportion of somata containing M1R among cortical cellular types, and the precise intracellular localization of M1R remain unclear. In this study, to clarify the precise immunolocalization of M1R in rat mPFC, we examined three major cellular types, pyramidal neurons, inhibitory neurons, and astrocytes. M1R immunopositivity signals were found in the majority of the somata of both pyramidal neurons and inhibitory neurons. In pyramidal neurons, strong M1R immunopositivity signals were usually found throughout their somata and dendrites including spines. On the other hand, the signal strength of M1R immunopositivity in the somata of inhibitory neurons significantly varied. Some neurons showed strong signals. Whereas about 40% of GAD67‐immunopositive neurons and 30% of parvalbumin‐immunopositive neurons (PV neurons) showed only weak signals. In PV neurons, M1R immunopositivity signals were preferentially distributed in somata. Furthermore, we found that many astrocytes showed substantial M1R immunopositivity signals. These signals were also mainly distributed in their somata. Thus, the distribution pattern of M1R markedly differs between cellular types. This difference might underlie the cholinergic modulation of higher cognitive functions subserved by mPFC.     

Disinhibition in the rat septum mediated by M1 muscarinic receptors

Acetylcholine (ACh) has been documented as an important central neurotransmitter. We have investigated the actions of ACh within the dorsolateral septal nucleus of the rat to examine its actions within this nucleus, specifically how it may interact to modulate the inhibitory action of gamma-aminobutyric acid (GABA), the known inhibitory transmitter in this area. Our results demonstrate that ACh, acting on M1 muscarinic receptors leads to disinhibition by decreasing GABA release.