• Archives of Pharmacal Research
• /
• v.14 no.2
• /
• pp.181-187
• /
• 1991

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. Chlopheniramine (CHP), histamine $H_1$ blocker, increased $K_D$ vlue 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 1.44\mu{M}$ 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 $ED_{50}$ value of $0.11\mu{M}$. CHP caused the rightward shift of the dose-response curve to carbachol. The $pA_2$ value of CHP determined from Schild analysis of carbacholinduced 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 (1.69 \mu{M)}$ of CHP as inhibitor of carbachol induced contraction in rabbit ileum. This result suggest that the binding of $H_i$ blocker. CHP, vs $([^3H]$QNB to muscarinic receptors in ileal membranes represents an interaction with a receptor of physiological relevance.

• YAKHAK HOEJI
• /
• v.39 no.6
• /
• pp.645-653
• /
• 1995

Dibenamine inhibited [$^{3}$H]quinuclidinyl benzilate ([$^{3}$H]QNB) binding in both concentration and incubation time-dependent manners. The $IC_{50}$/ value of dibenamine for the inhibition of the specific binding of 100 pM [$^{3}$'H]QNB following incubation of cerebral microsomes with dibenamine at 37.deg. C for 15 min was 20.mu.M. Dibenamine irreversibly decreased the binding site concentration for [$^{3}$H]QNB binding without affecting the affinity of [$^{3}$H]QNB for the muscarinic receptor. Analysis of the pirenzepine inhibition curve of [$^{3}$H]QNB binding to cerebral microsomes indicated the presence of two receptor subtypes with high(M$_{1}$ receptor, Ki=5nM) and low (M$_{2}$ receptor, Ki=160nM) affinity for pirenzepine. However, dibenamine(20.mu.M) treatment under the condition employed in these experiments caused steepening of the pirenzepine competition curve. The Ki value for pirenzepine in dibenamine treated-microsomes was approximately 120nM. suggesting a selective decrease in the number of M$_{1}$ receptor. Although dibenamine also inhibited [$^{3}$H]QNB binding to ventricular microsomes with $IC_{50}$/ value of 120.mu.M, the sensitivity for dibenamine in the ventricle was much lower than that in the cerebrum. These results indicate that dibenamine at low concentrations welectively inactivates the muscarinic M$_{1}$ receptor.

• Biomolecules & Therapeutics
• /
• v.2 no.4
• /
• pp.361-368
• /
• 1994

The nature of the muscarinic receptors in bovine adrenal medulla was investigated in this study. [$^3$H]Quinuclidinyl benzilate(QNB) specifically bound to a single class of muscarinic receptor with a $K_{D}$ value of about 70 pM in bovine adrenal medullary, cardiac ventricular and ileal homogenates. Pirenzepine inhibition curves of [$^3$H]QNB binding to cardiac ventricular and ileal homogenates were steep, indicating the presence of a single class of binding site for pirenzepine with a Ki value of 990 nM and 508 nM, respectively. However, pirenzepine/[$^3$H]QNB competition binding curves in adrenal medulla suggested the presence of two binding sites (Hill coefficient=0.59) with a high( $M_1$) and a low( $M_2$) affinity. Respective Ki values for pirenfepine were 16 nM and 633 nM, with 44% of total sites having a high affinity( $M_1$). Gallamine, which is selective to cardiac $M_2$-receptor, inhibited [$^3$H]QNB binding to adrenal medullary, cardiac ventricular and ileal homogenates with Ki values of 12 $\mu$M, 6 $\mu$M and 13 $\mu$M, respectively. Thus, the binding affinities of pirenzepine and gallamine for $M_2$-receptor in adrenal medulla were similar to those in ileum, which contains the $M_3$-receptor. These results indicate that the $M_1$- and $M_3$- muscarinic receptor subtypes coexist in the bovine adrenal medulla.a.

• Archives of Pharmacal Research
• /
• v.10 no.4
• /
• pp.212-218
• /
• 1987

Intact brain cell aggregates were dissociated from adult rat brains without cerebellum using a sieving technique. This proparation was used to elucidate the binding characteristics of agonist to muscarinic acetylcholine receptors (mAchR) in brain. Incubation of cells with carbamylcholine (carbachol) was shown agonist-induced receptor down-regulation depending on the concentration of agonist, not depending on the incubation time. This effect of carbachol was due to a reduction in the maximal binding capacity ($B_{max}$) to the mAchR without decreasing the affinity of the remaining receptors in incubation at 37.deg.C but was not apparent inincubation at $15^{\circ}}C$In addition, it was abolished when the receptors were blocked by atropine. The decline in ($^3H$)N-methylscopolamine (($^3H$)NMS) binding induced by agonist was reflected as a significant reduction in the receptor density with no change in receptor affinity, suggesting that 'true' receptor down-regulation takes place. Moreover, when the receptors were labeled with the lipophilic antagonist ($^3H$) quinuclidinyl benzilate (($^3H$) QNB) insted of the hydrophilic ligand ($^3H$)NMS, the magnitude of the observed receptor down-regulation was significantly lower in case of the former than the latter. This suggested that exposure of intact brain cells to muscarinic agonists might induce a slight degree of accumulation of receptors in intracellular sites before the receptors are actually degraded.

• Biomolecules & Therapeutics
• /
• v.3 no.3
• /
• pp.192-198
• /
• 1995

The affinity of mequitazine, a non-sedating antihistamine, for muscarinic receptors was evaluated in the guinea-pig ventricle and ileum by in vitro binding techniques and functional studies. In binding studies, [$^3$H]quinuclidinyl benzilate (QNB) identified a single class of muscarinic receptors with similar apparent $K_{D}$ value of about 100 pM in two tissues. Mequitazine inhibited [$^3$H]QNB binding to muscarinic receptors competitively. Analysis of the mequitazine inhibition curve of [$^3$H]QNB binding to ventricular microsome and ileal homogenate indicated the presence of a single homogeneous binding site with Ki value of 25 nM and 18 nM, respectively. In functional studies, mequitazine caused parallel rightward shifts of concentration-response curves for carbachol and histamine in the isolated guinea-pig ileum. The slope values obtained from Schild plot analysis for the antagonistic action of mequitazine on muscarinic and histamine $H_1$-receptors were not significantly different from unity. The p $A_2$values of mequitazine for muscarinic and histamine $H_1$-receptors were about 7.6 ( $K_{M}$= 25.1 nM) and 8.88 ( $K_{H}$= 1.32 nM), respectively. These results indicate that the muscarinic receptor blocking action of mequitazine is 15 times less potent than the $H_1$receptor blocking action, but high concentration of this drug may cause the peripheral muscarinic receptor blocking effect.t.t.t.

• YAKHAK HOEJI
• /
• v.32 no.2
• /
• pp.101-111
• /
• 1988

$[^3H]$ Quinuclidinyl benzilate(QNB) binding assays were performed in the dog ventricular sarcolemma fraction enriched approx. 32-fold in sarcolemma compared to the starting homogenate to elucidate the effect of antihistaminics on cardiac muscarinic receptor. Chlorpheniramine(CHP) inhibited specific binding of $[^3H]$QNB and delayed the equilibrium binding. The rate constants at $37^{\circ}C$ for formation and dissociation of the QNB receptor complex were $0.38{\times}10^9\;M^{-1}$ and $1.6{\times}10^{-2}\;min^{-1}$, respectively. The mean value for the dissociation constant from the pairs of the rate constants was 43. 2 pM and this value was similar to the value(44.8pM) determined from Scatchard analysis. CHP decreased association rate constant, indicating increase in $K_D$ value. Decrease in affinity without affecting the binding site concentration$(B_{max})$ for $[^3H]$QNB binding by CHP was also demonstrated by Scatchard analysis. $K_i$ values for $H_i$-blockers that inhibited specific $[^3H]$QNB binding were $0.02{\sim}4.8{\mu}M$. Cimetidine with $K_i$ value of $230{\mu}M$, however, was ineffective in displacing $[^3H]$QNB binding at concentration of $50{\mu}M$. The Hill coefficient for $H_1$-blockers were about one. The results indicate that $H_1$-antihistaminics inhibit $[^3H]$ QNB binding by interaction with myocardiac muscarinic cholinergic receptor and anticholinergic side effects of these drugs are mainly due to this receptor blocking mechanism.

• YAKHAK HOEJI
• /
• v.37 no.4
• /
• pp.397-407
• /
• 1993

The muscarinic antagonist l-[benzilic-4,4'-$^3H$]quinuclidinyl benzilate([$^3H$]QNB) bound to a single class of muscarinic receptor with high affinity in guinea pig ileal membranes. The $K_{D}$ and B$_{max}$ values for [$^3H$]QNB calculated from analysis of saturation isotherms were 54 pM and 156fmol/mg, respectively. H$_{1}$-blockers inhibited [$^3H$]QNB binding to ileal membranes with $K_{i}$ values ranged from 0.008 $\mu{M}$ to 1.6 $\mu{M}$. The pseudo-Hill coefficients of H$_{1}$-blockers for inhibition of [$^3H$]QNB binding to the ileal membranes were close to unit. The $K_{i}$ values for H$_{1}$-blockers were similar to the $K_{M}$ values calculated by Schild plot of functional data obtained from inhibition of the carbachol-induced contraction in guinea-pig ileum, suggesting that binding of H$_{1}$-blockers vs [$^3H$]QNB in ileal membranes represents an interaction with a receptor of physiological relevance. The $K_{H}$ values of H$_{1}$-blockers for H$_{1}$-receptor estimated from inhibition of the histamine-induced contraction were the range of 0.15 nM to 56.5 nM. The $K_{M}$/K$_{H}$ ratio of H$_{1}$-blockers varied over a wide range of 3 to 2300. Thus, the antihistaminic potencies of H$_{1}$-blockers do not correlate with their antimuscarinic potencies, which suggest that antihistamines have different antimuscarinic potencies in therapeutic blood levels causing similar antiallergic effect. Among 13 traditional antihistaminics examined in this study, drug having the highest and the lowest $K_{M}$/K$_{H}$ ratio is triprolidine and diphenidol, respectively. The present results demonstrate that the antimuscarinic property of antihistamines is not necessary for their antiallergic effect, and data on the affinity of antihistamines for muscarinic and H$_{1}$-receptors can be an important parameter in the selection and evaluation of these drugs.

• Archives of Pharmacal Research
• /
• v.17 no.6
• /
• pp.443-451
• /
• 1994

The binding characteristics of pirenzepine and oxomemazine to muscarinic receptor were studied to evaluate the selectivity of oxomemazine for the muscarinic receptor subtypes in rat cerebral microsomes. Equililbrium dissociation constant $(K_D){\;}of{\;}(-)[^3H]$quinuclidinyl benzilate$([^3H)QNB)$ determined from saturation isotherms was 64-pM. Analysis of the pirenzepine inghibition curve of [$^3H$]QNB binding to cerebral microsome indicatd the presence of two receptor subtypes with high $(K_1 = 16 nM, M_1 receptor)$two receptor subypes with about 20-fold difference in the affinity for high $(k_1 = 84nM, {\;} O_H receptor){\;} and {\;}low{\;} (K_1{\;} ={\;} 1.65\muM, {\;} O_L receptor$) affinity sites. The percentage populations of $M_1{\;} and M_3$, /TEX> receptors to the total receptors were 61 : 39, and those of $O_H{\;} and{\;} O_L$ receptors 39 : 61, resepectively. Both pirenzepine and oxomemazine increaed the $K_D$ value for $[^3H]QNB$ without affecting the binding site concentrations and Hii coefficient for the $[^3H]QNB$ without affecting the binding site concentractions and Hill coefficient for the [$^{3}$H]QNB binding. Oxomemazine had a 10-fold higher affinity at $M_1$ receptors than at $M_3$ receptors, and pirenzepine a 8-fold higher affinity at $O_H$ receptors were of $O_H$ receptors and 71% of $M_3$ receptors. However, $M_3$for oxomemazine and $O_H$for pirenzepine were composed of a uniform population. These results suggest that oxomemazine could be classified as a selective drug for $M_1$ receptors and also demonstrate that rat cerebral microsomes contain three different subtypes of $M_1{\;} M_3$ and the other site which is different from $M_1, {\;} M_2$, /TEX> receptors.

• YAKHAK HOEJI
• /
• v.36 no.3
• /
• pp.220-229
• /
• 1992

The muscarinic acetylcholine receptors of the dog unpregant uterus were characterized using $[^3H]quinuclidinyl$ benzilate(QNB) as a radioligand and the binding of muscarinic receptor agonists and antagonists in the uterus was compared to that in the urinary bladder which contains almost exclusively the M2 receptors in order to determine the receptor subtypes in the uterus. $[^3H]QNB$ binding to uterus and bladder was rapid, saturable and reversible. Scatchard analysis of the saturation data gave linear plots and the Hill coefficients were close to unit, which indicated that each preparation contained a single population of specific binding sites for $[^3H]QNB$. The KD values(120 pM) for QNB were almost identical in both organs, whereas the $B_{max}$ value of 256 fmol/mg protein in the uterus was significantly different from that of 563 fmol/mg protein in the bladder. Muscarinic agonists and antagonists inhibited in a competitive manner the $[^3H]QNB$ binding to the same extent in both organs. The competition binding studies using antagonists(atropine and pirenzepine) exhibited a single binding site and this site had a low affinity for pirenzepine with the Ki value of about 330 nM. However, high and low affinity binding sites were observed with carbachol, methacholine and oxotremorine. These binding studies with agonists and antagonists did not show any differences in drug affinities between uterus and bladder. These results indicate that the muscarinic receptors in the uterus are M2 receptors which have a low affinity for pirenzepine.

• The Korean Journal of Nuclear Medicine
• /
• v.27 no.1
• /
• pp.28-34
• /
• 1993

Using in vitro autoradiography with a digital autoradiography system and radioreceptor assay, the distribution and the binding characteristics of the muscarinic cholinergic receptors (mAChR) were studied in regions of rat brain. Radioreceptor assay revealed that mAChR could be measured with saturation binding assay in the brain and heart homogenates: No difference in Kd or Bmax of the brain or heart was found between the normal Wistar rats and SHR rats. Specific binding of $^3H$ quinuclidinyl benzilate (QNB) increased and saturation was reached by 2 hours after incubation with slide-mounted brain tissue. The distribution of mAChR was heterogeneous along the fields of brain. Affinity (Kd) of mAChR was not different significantly among cortex, hippocampus and caudate-putamen. No difference was found between normal rats and SHR strain. More receptors (Bmax) were found in the cortex and hippocampus than in the caudate-putamen in normal rats. More receptors were found in the cortex and caudate-putamen in SHR rats than in normal rats. Radioreceptor assay and digital autoradiographic analysis of affinity and number of mAChR gave the same results. With the above findings, we concluded that we could use digital autoradiographic system with $^3H$-QNB in the characterization of mAChR of rats and that the cortex and caudate-putamen of SHR strain rats have more receptors than those of normal rats.