• YAKHAK HOEJI
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• v.43 no.5
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• pp.642-651
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• 1999

Nonsedating antihistamines do net cause sedation in therapeutic doses because these drugs hardly cross the blood-brain barrier. Since most of the peripheral side dffects of conventional antihistamines are related to their muscarinic receptor blocking action, the present study was performed to investigate whether nonsedating antihistamines interact with the muscarinic receptors and discriminate the muscarinic receptor subtypes in the rat cerebral microsomal fraction which containes both $M_1,{\;}M_2,{\;}M_3{\;}and{\;}M_4$ receptors. Five nonsedating antihistamines at high concentrations inhibited [$^3H$]QNB binding to the muscarinic receptor in a dose-dependent manner. The inhibition curves of these drugs except loratadine which showed positive cooperativity (nH=1.55) were steep (nH=1), indicating interaction with a single homogenous population of the binding sites. Astemizole, clemizole and mequitazine increased the $K_D$ value for [$^3H$]QNB without affecting the binding site concentrations, and this increase in the $K_D$ value resulted from the ability of these drugs to slow [$^3H$]QNB-receptor association. The Ki values of astemizole, clemizole and mequitazine for the inhibition for the inhibition of [$^3H$]QNB binding to muscarinic receptor were 0.58, 5.99 and $0.007{\;}{\mu}M$, respectively. However, loratadine and terfenadine inhibited noncompetitively [$^3H$]QNB binding with the normalized $IC_50$ value of about $2{\;}{\mu}M$. These results demonstrate that; 1) astemizole, clemizole and mequitazine interact directly with the muscarinic receptor at high concentrations; 2) muscarinic receptor blocking potency of these drugs varies widely among drugs; 3) these drugs do not discriminate between muscarinic receptor subtypes.

• YAKHAK HOEJI
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• v.37 no.4
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• pp.397-407
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• 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
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• v.14 no.3
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• pp.271-278
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• 1991

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 16.7\mu{M}$, 3.5\mu{M}$ and 15.5\mu{M}$ respectively. Saturation data of $[^3H]$QNB binding with $K_i$ VALUES OF 16.7\mu{M}$ 3.5\mu{M}$ and 15.5\mu{M}$ 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 m but atropine did not, indicating that the muscarinic receptors and the DHP bindings sites on calcium channels are distinct. The $K_i$ value of Bay K 8644 for the DHP binding sites was 4nM. 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 thes high afinity 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.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1994.04a
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• pp.290-290
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• 1994

The binding characteristic of oxomemazine to muscarinic receptor in the cerebrum, heart, and ileum were compared to those of pirenzepine to investigate whether oxomemazine could classify the muscarinic receptor subtypes. 〔$^3$H〕Quinucl idinyl benzilate(QNB) identified a single class of muscarinic receptors with apparent K$\sub$D/ value of about 60 pM in three tissues. Analysis of the pirenzepine inhibition curve of 〔$^3$H〕QNB binding to cerebral microsome indicated the presence of two receptor subtypes with high (Ki=16 nM, M$_1$-receptor) and low (Ki=400 nM, M$_2$-receptor) affinity for pirenzepine. Oxomemazine also identified two receptor subtypes with high (Ki=84 nM, On-receptor) and low (Ki=1 4 ${\mu}$M, O$\sub$L/-receptor) affinity in rat cerebral microsome, The percentage population of the M$_1$-and M$_2$-receptors to the total receptors were 61 : 39, and those of the O$\^$H/- and O$\sub$L/-receptors 39 : 61, respectively, However, the Hill coefficients of these two drugs for the inhibition of 〔$^3$H〕QNB binding to the heart and ileum were close to unity which indicated that these drugs bound to a uniform population of receptors in these two tissues. The Ki values for the low affinity sites of pirenzepine and oxomemazine in the cerebrum were similar to those of these drugs in the heart ileum. Both pirenzepine and oxomemazine increased K$\sub$D/ value for 〔$^3$H〕QNB without affecting the binding sites concentration and Hill coefficient for the 〔$^3$H〕QNB binding. Oxomemazine had a 10-fold lower affinity at Ma-receptors than at M$_1$-receptors, and pirenzepine a 8-fold lower affinity at O$\sub$L/-receptors than OH-receptors. Analysis of the shal low competition curves of oxomemazine for the H$_1$ receptors and pirenzepine for the O$\sub$L/-receptors yielded that 69% of the M$_1$-receptors were of the O$\sub$H/-receptors and the remaining 31% of the O$\sub$L/-receptors, and that 29% of the O$\sub$L/-receptors were of the M$_1$-receptors and 71% of the M$_2$-receptors. However, M$_2$ for oxomemazine and O$\sub$H/ for pirenzepine were composed of a uniform population. These results suggest that oxomemazine could discriminatethe muscarnic receptor subtypes and may subclassify the M$_1$-receptors into two subtypes.

• Biomolecules & Therapeutics
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• v.4 no.1
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• pp.36-45
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• 1996

The antihistaminic action of eighteen herbal medicines was investigated by the radioligand binding and functional assays. The hexane fractions of Trichosanthis radix, Mori cortex radicis and Evodiae fructus dosedependently inhibited [$^3$H] mepyramine binding to H$_1$, receptor in guinea-pig brain homogenates and histamine-induced contraction of isolated guinea-pig ileum. Antihistaminic action of the hexane and ethyl acetate fractions of Mori cortex radicis and the hexane fraction of Evodiae fructus was more potent than their antimuscarinic action evaluated from the inhibition of [$^3$H]QNB binding and carbachol response. The ethyl acetate and chloroform fractions from Scutellariae radix also inhibited histamine-induced contraction, but antihistaminic potencies of these fractions were almost identical with their antimuscarinic potencies. The hexane fractions of Mori cortex radicis and Evodiae fructus inhibited selectively the increase of histamine-induced cutaneous vascular Permeability in the rat dorsal skins. However, the ethyl acetate fraction from Scutellariae radix inhibited eqipotently the effects of histamine and serotonin on the vascular permeability. These results demonstrate that the hexane and ethyl acetate fractions of Mori cortex radicis and the hexane fraction of Evodiae fructus have the selective histamine H$_1$receptor blocking activity.

• Archives of Pharmacal Research
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• v.10 no.4
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• pp.212-218
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• 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
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• v.7 no.4
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• pp.307-314
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• 1999

The human muscarinic acetylcholine receptor (mAChR) subtypes Hml, Hm2 and Hm3 have been expressed in insect cells (Spodoptera frugiperda, Sf9) using the baculovirus expression system. Expression of relevant DNA, transcript and receptor proteins was identified by PCR, Northern blotting and [$^{3}H$]QNB binding, respectively. As assessed by [$^{3}H$]QNB binding sites, yields of muscarinic receptors in membrane preparations in this study were as about 5-20 times high as those in mammalian cells reported in previous studies. The [$^{3}H$]QNB competition binding studies with well-known subtype-selective mAChR antagonists showed that the receptors expressed in Sf9 cells retain the pharmacological characteristics expected for the ml , m2 and m3 muscarinic receptors. The ml-selective antagonist, pirenzepine, displayed a considerably higher affinity for Hml by 110-fold and 35-fold than for Hm2 and Hm3, respectively, The m2-selective methoctramine displayed a significantly higher affinity for Hm2 than for Hml and Hm3 (10- and 26-fold, respectively). p-F-HHSiD exhibited high affinity for Hm3 that is not significantly different from those for Hml, but 66-fold higher than its affinity for Hm2. The functional coupling of the recombinant receptors to second messenger systems was also examined. While both Hml and Hm3 stimulated phosphoinositide hydrolysis upon activation by carba-chol, Hm2 produced no response. On the other hand, activation of mAChRs induced the inhibition of forsko-lin-stimulated cyclic AMP formation in Hm2-expressing cells, whereas the significant dose-dependent increase in or poor response on cyclic AMP formation were produced in Hml or Hm3-expressing cells, respectively. These results indicate the differential coupling of recombinant Hml, Hm2 and Hm3 receptors expressed in SF9 cells to intracellular signalling system.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1995.04a
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• pp.87-87
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• 1995

The muscarinic effects of carbachol were compared on the isolated ileums of guinea-pig, rat and rabbit to elucidate the underlying mechanism of species differences in sensitivity for carbachol. The ED$\_$50/ value estimated on the guinea-pig ileum was 4 to 6-fold lower than those obtained on the rat and rabbit ileums, but the K$\_$A/ values of carbachol determined by functional assays were almost identical with 12-l7 ${\mu}$M in all of three ileums. The competition data of carbachol for [$^3$H]QNB binding were best described by a two-site model yielding the Ki values of 0.4-0.6${\mu}$M and 12-16${\mu}$M for high(K$\_$H/) and low(K$\_$L/) affinity sites, respectively. The low affinity dissociation constants(K$\_$L/) of carbachol determined from receptor binding studies thus were not significantly different from the K$\_$A/ values estimated from functional studies. The percentage of receptor occupation that carbachol requires for half-maximal response was approximately 3 to 5-fold lower in guinea-pig compared to rat and rabbit whereas the density of muscarinic binding sites per gram of ileum measured by [$^3$H]QNB saturation isotherms was two-fold higher in guinea-pig than that in rat and rabbit. Therefore, the numbers of muscarinic receptors occupied at ED$\_$50/ values of carbachol were about two-fold lower in guinea-pig, suggesting two-fold greater intrinsic efficacy. These results indicate that the guinea-pig ileum has higher muscarinic receptor density and greater intrinsic efficacy for carbachol than the rat and rabbit ileums.

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

• Proceedings of the Korean Society of Applied Pharmacology
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• 1996.04a
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• pp.202-202
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• 1996

The antihistaminic action of eighteen herbal medicines was investigated by the radioligand binding and functional assays. The hexane fractions of Trichosanthis radix, Mori cortex radicis and Evodiae fructus dose-dependently inhibited [$^3$H]mepyramine binding to H$_1$ receptor and histamine-induced contraction in guinea-pig brain homogenates and isolated guinea-pig ilea, respectively. Antihistaminic action of the hexane and ethylacetate fractions of Mori cortex radicis and the hexane fraction of Evodiae fructus was more potent than their antimuscarinic action evaluated from the inhibition of [$^3$H]QNB binding and carbachol response. The ethylacetate and chloroform fractions and six known flavonoids from Scutellariae radix also inhibited histamine-induced contraction, but antihistaminic potencies of these fractions and compounds were almost identical with their antimuscarinic potencies. The hexane fractions of Mori cortex radicis and Evodiae fructus, as shown in ketotifen, inhibited selectively the increase of cutaneous vascular permeability induced by histamine. However, wogonin (SC-1) from Scutellariae radix was a nonselective inhibitor for the effect of histamine and serotonin on the vascular permeability. These results demonstrate that the hexane and ethylacetate fractions of Mori cortex radicis and the hexane fraction of Evodiae fructus have the selective histamine H$_1$ receptor blocking activities.