Atsushi Mizushima - Academia.edu (original) (raw)

Papers by Atsushi Mizushima

Research paper thumbnail of Urinary excretion of aquaporin-2 water channel protein in human and rat

Journal of the American Society of Nephrology : JASN, 1997

Previous studies by the authors demonstrated that the response of urinary aquaporin-2 (AQP2) excr... more Previous studies by the authors demonstrated that the response of urinary aquaporin-2 (AQP2) excretion to dDAVP (deamino-8-D-arginine vasopressin) infusion is an index of vasopressin action on the kidney (N Engl J Med 332: 1540-1545, 1995). In the study presented here, the characteristics of urinary excretion of AQP2 were examined further. An RIA suitable for AQP2 in the urine was established. Relatively high concentrations of detergent and bovine serum albumin in the RIA buffer allowed analysis of urine samples with a wide range of concentrations and increased the sensitivity of the assay. AQP2 in the urine existed as a high molecular weight form of approximately 190 kD by HPLC analysis. The mean urinary AQP2 concentration corrected for creatinine in spot urine samples of healthy subjects who voided in the morning was 1081 +/- 699 fmol/mg creatinine (mean +/- SD, n = 208). The amount of daily excretion of AQP2 in the urine was the same in men and women. Urinary AQP2 content was not...

Research paper thumbnail of Agonist bindings and their relationship to receptor responses in heart muscarinic receptors

Advances in experimental medicine and biology, 1988

Research paper thumbnail of Effect of urea-treatment on agonist binding affinity of the muscarinic acetylcholine receptor

Life Sciences, 1983

Urea-treatment of the microsome fraction of the heart of guinea-pigs caused selective reduction i... more Urea-treatment of the microsome fraction of the heart of guinea-pigs caused selective reduction in the apparent affinity of an agonist (carbachol), but not an antagonist (atropine), to muscarinic acetylcholine receptors (mAChR), measured as inhibition of binding of 3H-quinuclidinyl benzilate (3H-QNB). This effect was similar to that of Gpp (NH)p. The effects of urea-treatment and Gpp (NH)p were not additive. On the other hand, treatment of the microsome fraction with 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) increased the apparent affinity of agonist, but not antagonist. The effect of DTNB predominated over those of urea-treatment and Gpp (NH)p, when these treatments were combined with DTNB.

Research paper thumbnail of Alterations in muscarinic receptors of ventricular muscle in carbachol-induced short-term desensitization

The Japanese Journal of Pharmacology, 1986

Research paper thumbnail of Effect of membrane depolarization by high K+ on carbachol-stimulated phosphoinositides hydrolysis in guinea pig cerebral cortical slices

The Japanese Journal of Pharmacology, 1990

Stimulation of phosphoinositide hydrolysis by carbachol was studied in slices of guinea pig cereb... more Stimulation of phosphoinositide hydrolysis by carbachol was studied in slices of guinea pig cerebral cortex under normal conditions (4.7 mM K+) and depolarization conditions with high K+ (42 mM K+). Slices were labeled with [myo-3H]-inositol, and the effects of carbachol and high K+ on the formation of inositol-bisphosphates (IP2) and inositol-trisphosphates (IP3) were determined. Carbachol (10 mM) caused only 140% stimulation of the formations of IP2 and IP3 over the control value in normal Krebs Ringer Buffer (KRB), but about 200% stimulation in high K+ medium. Dose-response curves for the effect of carbachol on the formations of IP2 and IP3 showed that high K+ medium selectively decreased the ED50 value of carbachol for IP2 formation about 3-fold. A Ca++ channel blocker, verapamil, inhibited the synergistic effect of carbachol and high K+ on IP2 formation, and a decrease in extracellular Ca++ also inhibited IP2 formation induced by high K+, but these treatments had little, if any, effect on IP3 formation. The possibility that IP2 may be directly generated by hydrolysis of phosphatidylinositol 4-monophosphate (PIP) as well as from hydrolysis of IP3 was discussed.

Research paper thumbnail of Effects of pantoyl-GABA on GABAA and GABAB receptors in the rat brain

The Japanese Journal of Pharmacology, 1987

The interactions of the "antidementia drug" pantoyl-gamma-aminobutyric acid (pa... more The interactions of the "antidementia drug" pantoyl-gamma-aminobutyric acid (pantoyl-GABA) with gamma-aminobutyric acid (GABA) receptors were investigated by studies on bindings of radiolabelled ligands in rat brain. Pantoyl-GABA inhibited the binding of [3H]GABA to GABAA receptors and those of [3H]baclofen and [3H]GABA to GABAB receptors in the rat cerebral cortex. These data suggest that pantoyl-GABA interacts with both types of GABA-receptors in the rat brain.

Research paper thumbnail of Multiple agonist binding sites of muscarinic acetylcholine receptors and their relation to the negative inotropic action of agonists in guinea-pig heart

European Journal of Pharmacology, 1985

The Kd values of the multiple agonist binding sites in cardiac muscarinic receptors (mAChR) and p... more The Kd values of the multiple agonist binding sites in cardiac muscarinic receptors (mAChR) and pD2 values for negative inotropic actions were determined independently and their relation was examined. The guinea-pig cardiac mAChR is known to have three agonist binding sites (super-high (SH), high (H) and low (L) affinity agonist binding sites) for carbachol (CCh). Pilocarpine (Pilo) and oxotremorine (Oxo) distinguished two sites (higher (Ho/p) with pKd of 5.88 and 8.20, respectively, and lower (Lo/p) affinity agonist binding sites with pKd of 5.08 and 6.17, respectively). The effects of guanine nucleotide and sulfhydryl reagent indicated that the Ho/p site corresponded with the SH site for carbachol, and the Lo/p site with the H + L sites for carbachol. The pD2 values of CCh, Pilo and Oxo for negative inotropic actions on autocontraction of right atria were 5.38, 5.30 and 6.80, respectively. The pD2 values of CCh and Oxo on electrically stimulated contraction of left atria in the presence of isoproterenol were 5.80 and 6.46, respectively, thus being closely related to H or Lo/p agonist binding sites of mAChR.

Research paper thumbnail of 1-oleoyl-2-acetyl-glycerol and phorbol diester stimulate Ca2+ influx through Ca2+ channels in neuroblastoma × glioma hybrid NG108-15 cells

European Journal of Pharmacology, 1986

The effect of 1-oleoyl-2-acetyl-glycerol (OAG) and the phorbol diester 12-O-tetradecanoyl-phorbol... more The effect of 1-oleoyl-2-acetyl-glycerol (OAG) and the phorbol diester 12-O-tetradecanoyl-phorbol-acetate (TPA) on the intracellular Ca2+ concentration ([Ca2+]i) in NG108-15 cells was studied using a Ca2+ indicator, quin 2. OAG and TPA induced an increase in [Ca2+]i from 100 +/- 19 to 187 +/- 24 nM and 192 +/- 15 nM, respectively, within 15 min. The increase in [Ca2+]i induced by activators of protein kinase C was dependent on the extracellular Ca2+ concentration [Ca2+]o) and was inhibited by the Ca2+ blockers, verapamil and nifedipine. These results indicate that the OAG- and TPA-induced [Ca2+]i increase is mediated by the influx of extracellular Ca2+ through voltage-sensitive Ca2+ channels.

Research paper thumbnail of Role of a protein regulating guanine nucleotide binding in phosphoinositide breakdown and calcium mobilization by bradykinin in neuroblastoma × glioma hybrid NG108-15 cells: Effects of pertussis toxin and cholera toxin on receptor-mediated signal transduction

European Journal of Pharmacology, 1987

The addition of bradykinin to NG108-15 cells resulted in an increase in the intracellular Ca 2+ c... more The addition of bradykinin to NG108-15 cells resulted in an increase in the intracellular Ca 2+ concentration ([Ca2+]i) and the formation of inositol monophosphate, inositol bisphosphate, and inositol trisphosphate in these cells. The bradykinin-stimulated formation of inositol polyphosphates in plasma membrane preparations was dependent on the presence of GTP or guanosine-5'-O-thiotriphosphate (GTPTS) but not of GDP. GTPyS, unlike GTP, increased the basal formation of inositol polyphosphate in NG108-15 membranes. Iontophoretic injection of GTPyS into single cells induced increases in [Ca2+]i . These effects of bradykinin and GTP-/S on [Ca2+]i and the formation of inositol phosphates in the intact cells and membranes were not affected by treatment of the cells with pertussis toxin or cholera toxin. Data on binding of bradykinin to membrane preparations indicated the presence of two classes of binding sites with K d values of 0.80 + 0.26 and 9.63 + 0.13 nM. Approximately 74% of the receptors were in the high affinity state. In the presence of guanyl-5'-yl-imidodiphosphate [Gpp(NH)p], the high affinity sites in the membrane preparations were converted to low affinity sites with no change in the total receptor number. These toxin treatments had no effect on binding of bradykinin to its receptors. Thus, these results indicate that a guanine nucleotide regulatory protein, which is not a substrate of pertussis toxin or cholera toxin, is involved in mediating the effects of bradykinin on membrane-bound phosphoinositide-specific phospholipase C to induce the increase of cytosolic calcium.

Research paper thumbnail of The SH-H subgroup of cardiac M2 receptors (M2α) inhibits adenylate cyclase activity

European Journal of Pharmacology, 1988

Muscarinic receptors in the guinea-pig heart seem to consist entirely of M 2 receptors, but are c... more Muscarinic receptors in the guinea-pig heart seem to consist entirely of M 2 receptors, but are coupled with several responses including inhibition of adenylate cyclase activity. On the other hand three affinity states (SH, H and L) can be distinguished in cardiac membranes with muscarinic agonists such as carbachol. We showed previously that the three agonist binding states were the sum of two equilibria (SH-H and H-L subgroup), both regulated by GTP-binding protein(s). In this study we determined which subgroup was responsible for the inhibitory effect of muscarinic M z receptors on adenylate cyclase activity. The EDs0 values for this response of four muscarinic agonists, acetylcholine, carbachol, pilocarpine and oxotremorine corresponded with the binding K D values of H (acetylcholine and carbachol) and Lo/p (pilocarpine and oxotremorine) sites. After alkylation of spare receptors, the EDs0 value of carbachol was changed from 4.3 to 5.6/.tM, which corresponded with the K o value of the H site. Furthermore, the four agonists were almost fully active when membrane preparations were pretreated with propylbenzilylcholine mustard (PrBCM) in the presence of carbachol to destroy the H-L subgroup, whereas after pretreatment with PrBCM and atropine, which alkylated both types of subgroups evenly, the decrease in the number of receptors was proportional to the decrease in the inhibitory effect on adenylate cyclase activity. These results suggest that only the SH-H subgroup (M2,) is responsible for the inhibitory action of muscarinic receptors on adenylate cyclase activity in the heart. Muscarinic M 2 receptors; Adenylate cyclase (inhibition of); Muscarinic acetylcholine receptor subtypes (M2, ~, M2/~) 0014-2999/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

Research paper thumbnail of Effects on guanine nucleotide and sulfhydryl reagent on subpopulations of muscarinic acetylcholine receptors in mammalian hearts: Possible evidence for interconversion of super-high and low affinity agonist binding sites

European Journal of Pharmacology, 1984

Abstract Multiple site models of muscarinic acetylcholine receptors (mAChR) for agonist binding w... more Abstract Multiple site models of muscarinic acetylcholine receptors (mAChR) for agonist binding were applied to curves for the inhibition of QNB binding by carbachol by using nonlinear least square regression analysis. The effects of a guanine nucleotide guanyl-5 ...

Research paper thumbnail of Cardiac M2 receptors consist of two different types, both regulated by GTP

European Journal of Pharmacology, 1987

Cardiac muscarinic receptors are predominantly M 2 receptors, and have three agonist binding site... more Cardiac muscarinic receptors are predominantly M 2 receptors, and have three agonist binding sites (super-high(SH), high(H) and low(L) affinity agonist binding sites). Treatment of cardiac membranes with 50 nM propylbenzilyl choline mustard (PrBCM) caused 88 % loss of binding sites for [3 H]QNB. Carbamyl choline (CCh) inhibits this alkylation dose dependently and, theoretically, generates uneven alkylation of multiple agonist binding sites. Pretreatment of the membranes with 50 nM PrBCM and 0.5 mM CCh resulted in almost complete disappearance of L sites with similar degrees of conservation of H sites and SH sites. In these pretreated membranes, guanine nucleotide and sulfhydryl reagent caused a change in the ratio of residual SH and H sites but not of L sites though previous studies showed that, in intact membranes, these reagents affected the ratio of SH and L sites without significantly changing that of the H site. These results indicate the existence of two equilibria regulated by guanine nucleotide and sulfhydryl reagent in cardiac muscarinic receptors: one between SH and H sites and the other between H and L sites. The participation of GTP binding protein(s) in all cardiac muscarinic responses is suggested. Cardiac M 2 receptors; Agonist binding sites (multiple); GTP binding proteins

Research paper thumbnail of The H-L subgroup of guinea-pig cardiac M2 receptors (M2β) regulates inositol phosphate formation

European Journal of Pharmacology, 1989

In previous studies, we showed that cardiac muscarinic receptors (M2) are composed of two subgrou... more In previous studies, we showed that cardiac muscarinic receptors (M2) are composed of two subgroups, M2a and M2t~, with different affinities for agonists and that the M2~ subgroup is coupled with inhibition of adenylate cyclase. We now studied which subgroup was responsible for the formation of inositol mono-(IP), bis-(IP2), tris-(11}3) and tetrakis-(IP4) phosphates in guinea pig heart. Carbachol (1 raM) 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 11}2, IP 3 and IP 4. However, oxotremorine (1 mM) only slightly stimulated the formation of IP2, and pilocarpine did not stimulate the formation of any IP. The pEDs0 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 o 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 (M2a) and a GTP binding protein different from those for inhibition of adenylate cyclase.

Research paper thumbnail of Regulatory mechanisms of cutaneous delayed-type hypersensitivity

Cellular Immunology, 1983

The macrophage migration inhibitory factor (MIF) fraction was prepared from the immunoadsorbent c... more The macrophage migration inhibitory factor (MIF) fraction was prepared from the immunoadsorbent column by using anti-guinea pig MIF antiserum. Suppression of cutaneous delayed-type hypersensitivity was achieved by intraperitoneal injection of the MIF fraction into the animals bearing macrophage-rich peritoneal exudates. Skin reactions induced by phytohemagglutinin (PHA) were also suppressed in these animals. Reactivity to skin reactive factor (SRF) was suppressed in these animals as well. The sera obtained from these animals exhibited the inhibitory activity against production of lymphokines from sensitized lymphocytes.

Research paper thumbnail of Induction of macrophage disappearance reaction by immunoadsorbed MIF

Cellular Immunology, 1982

Research paper thumbnail of Effect of Antisense Oligonucleotides against Cholesteryl Ester Transfer Protein on the Development of Atherosclerosis in Cholesterol-fed Rabbits

Cholesteryl ester transfer protein (CETP) is the enzyme that facilitates the transfer of choleste... more Cholesteryl ester transfer protein (CETP) is the enzyme that facilitates the transfer of cholesteryl ester from high density lipoprotein (HDL) to apolipoprotein B (apoB)-containing lipoproteins. However, the exact role of CETP in the development of atherosclerosis has not been determined. In the present study, we examined the effect of the suppression of increased plasma CETP by intravenous injection with antisense oligodeoxynucle-otides (ODNs) against CETP targeted to the liver on the development of atherosclerosis in rabbits fed a cholesterol diet. The ODNs against rabbit CETP were coupled to asialoglycoprotein (ASOR) carrier molecules, which serve as an important method to regulate liver gene expression. Twenty-two male Japanese White rabbits were used in the experiment. Eighteen animals were fed a standard rabbit chow supplemented with 0.3% cholesterol throughout the experiment for 16 weeks. At 8 weeks, they were divided into three groups (six animals in each group), among which the plasma total and HDL cholesterol concentrations did not significantly change. The control group received nothing, the sense group were injected with the sense ODNs complex, and the antisense group were injected with the antisense ODNs complex, respectively, for subsequent 8 weeks. ASORpoly(L-lysine) ODNs complex were injected via the ear veins twice a week. Four animals were fed a standard rabbit diet for 16 weeks. The total cholesterol concentrations and the CETP mass in the animals injected with antisense ODNs were all significantly decreased in 12 and 16 weeks compared with those injected with sense ODNs and the control animals. The HDL cholesterol concentrations measured by the precipitation assay did not significantly change among the groups fed a cholesterol diet, and triglyceride concentrations did not significantly change in the four groups. However, at the end of the study, when the HDL cholesterol concentrations were measured after the isolation by ultracentrif-ugation and a column chromotography, they were significantly higher in the animals injected with antisense ODNs than in the animals injected with sense ODNs and in the control animals. A reduction of CETP mRNA and an increase of LDL receptor mRNA in the liver were observed in the animals injected with antisense ODNs compared with those injected with sense ODNs and the control animals. Aortic cholesterol contents and the aor-tic percentage lesion to total surface area were significantly lower in the animals injected with antisense ODNs than in the animals injected with sense ODNs and in the control animals. These findings showed for the first time that suppression of increased plasma CETP by the injection with antisense ODNs against CETP coupled to ASOR carrier molecules targeted to the liver could thus inhibit the atherosclerosis possibly by decreasing the plasma LDL very low density lipoprotein (VLDL) cholesterol in cholesterol-fed rabbits. Cholesteryl ester transfer protein (CETP) 1 is a plasma gly-coprotein that catalyzes the transfer of cholesteryl ester and triglyceride among lipoproteins (1, 2). The homozygotes for CETP deficiency demonstrated markedly elevated HDL cholesterol and plasma apolipoprotein A-I (apoA-I) levels as well as decreased LDL cholesterol and plasma apoB levels (3, 4). CETP deficiency may be associated with protection against ischemic heart disease, based on the observed longevity (5) as well as the lack of any evidence of coronary heart disease (4). However, recently it was reported that in some patients with CETP deficiency there were increased coronary heart diseases (6 – 8). Even in the study using CETP transgenic mice, the exact role of CETP in the development of atherosclerosis has yet to be clarified. Marotti et al. (9) demonstrated that transgenic mice expressing cynomolgus monkey CETP had significantly more early atherosclerotic lesions in the proximal aorta than controls when fed a high cholesterol diet. On the other hand, more recently Hayek et al. (10) concluded that transgenic mice expressing human CETP and apoCIII showed the inhibition of the development of early atherosclerotic lesions. To determine how CETPs affect atherosclerosis in clinical situations, plasma CETP levels must be changed in the experimental models because the studies in the patients with CETP deficiency or CETP transgenic mice go from one extreme to another. We have showed that intravenous injection with antisense oligode-oxynucleotides (ODNs) against CETP coupled to asialoglyco-protein carrier molecules targeted to the liver could inhibit the plasma CETP activity, and as a result, induced a decrease in the plasma LDL VLDL cholesterol and an increase in the plasma HDL cholesterol in cholesterol-fed rabbits (11). In the present study using an intravenous injection with antisense

Research paper thumbnail of Urinary excretion of aquaporin-2 water channel protein in human and rat

Journal of the American Society of Nephrology : JASN, 1997

Previous studies by the authors demonstrated that the response of urinary aquaporin-2 (AQP2) excr... more Previous studies by the authors demonstrated that the response of urinary aquaporin-2 (AQP2) excretion to dDAVP (deamino-8-D-arginine vasopressin) infusion is an index of vasopressin action on the kidney (N Engl J Med 332: 1540-1545, 1995). In the study presented here, the characteristics of urinary excretion of AQP2 were examined further. An RIA suitable for AQP2 in the urine was established. Relatively high concentrations of detergent and bovine serum albumin in the RIA buffer allowed analysis of urine samples with a wide range of concentrations and increased the sensitivity of the assay. AQP2 in the urine existed as a high molecular weight form of approximately 190 kD by HPLC analysis. The mean urinary AQP2 concentration corrected for creatinine in spot urine samples of healthy subjects who voided in the morning was 1081 +/- 699 fmol/mg creatinine (mean +/- SD, n = 208). The amount of daily excretion of AQP2 in the urine was the same in men and women. Urinary AQP2 content was not...

Research paper thumbnail of Agonist bindings and their relationship to receptor responses in heart muscarinic receptors

Advances in experimental medicine and biology, 1988

Research paper thumbnail of Effect of urea-treatment on agonist binding affinity of the muscarinic acetylcholine receptor

Life Sciences, 1983

Urea-treatment of the microsome fraction of the heart of guinea-pigs caused selective reduction i... more Urea-treatment of the microsome fraction of the heart of guinea-pigs caused selective reduction in the apparent affinity of an agonist (carbachol), but not an antagonist (atropine), to muscarinic acetylcholine receptors (mAChR), measured as inhibition of binding of 3H-quinuclidinyl benzilate (3H-QNB). This effect was similar to that of Gpp (NH)p. The effects of urea-treatment and Gpp (NH)p were not additive. On the other hand, treatment of the microsome fraction with 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) increased the apparent affinity of agonist, but not antagonist. The effect of DTNB predominated over those of urea-treatment and Gpp (NH)p, when these treatments were combined with DTNB.

Research paper thumbnail of Alterations in muscarinic receptors of ventricular muscle in carbachol-induced short-term desensitization

The Japanese Journal of Pharmacology, 1986

Research paper thumbnail of Effect of membrane depolarization by high K+ on carbachol-stimulated phosphoinositides hydrolysis in guinea pig cerebral cortical slices

The Japanese Journal of Pharmacology, 1990

Stimulation of phosphoinositide hydrolysis by carbachol was studied in slices of guinea pig cereb... more Stimulation of phosphoinositide hydrolysis by carbachol was studied in slices of guinea pig cerebral cortex under normal conditions (4.7 mM K+) and depolarization conditions with high K+ (42 mM K+). Slices were labeled with [myo-3H]-inositol, and the effects of carbachol and high K+ on the formation of inositol-bisphosphates (IP2) and inositol-trisphosphates (IP3) were determined. Carbachol (10 mM) caused only 140% stimulation of the formations of IP2 and IP3 over the control value in normal Krebs Ringer Buffer (KRB), but about 200% stimulation in high K+ medium. Dose-response curves for the effect of carbachol on the formations of IP2 and IP3 showed that high K+ medium selectively decreased the ED50 value of carbachol for IP2 formation about 3-fold. A Ca++ channel blocker, verapamil, inhibited the synergistic effect of carbachol and high K+ on IP2 formation, and a decrease in extracellular Ca++ also inhibited IP2 formation induced by high K+, but these treatments had little, if any, effect on IP3 formation. The possibility that IP2 may be directly generated by hydrolysis of phosphatidylinositol 4-monophosphate (PIP) as well as from hydrolysis of IP3 was discussed.

Research paper thumbnail of Effects of pantoyl-GABA on GABAA and GABAB receptors in the rat brain

The Japanese Journal of Pharmacology, 1987

The interactions of the "antidementia drug" pantoyl-gamma-aminobutyric acid (pa... more The interactions of the "antidementia drug" pantoyl-gamma-aminobutyric acid (pantoyl-GABA) with gamma-aminobutyric acid (GABA) receptors were investigated by studies on bindings of radiolabelled ligands in rat brain. Pantoyl-GABA inhibited the binding of [3H]GABA to GABAA receptors and those of [3H]baclofen and [3H]GABA to GABAB receptors in the rat cerebral cortex. These data suggest that pantoyl-GABA interacts with both types of GABA-receptors in the rat brain.

Research paper thumbnail of Multiple agonist binding sites of muscarinic acetylcholine receptors and their relation to the negative inotropic action of agonists in guinea-pig heart

European Journal of Pharmacology, 1985

The Kd values of the multiple agonist binding sites in cardiac muscarinic receptors (mAChR) and p... more The Kd values of the multiple agonist binding sites in cardiac muscarinic receptors (mAChR) and pD2 values for negative inotropic actions were determined independently and their relation was examined. The guinea-pig cardiac mAChR is known to have three agonist binding sites (super-high (SH), high (H) and low (L) affinity agonist binding sites) for carbachol (CCh). Pilocarpine (Pilo) and oxotremorine (Oxo) distinguished two sites (higher (Ho/p) with pKd of 5.88 and 8.20, respectively, and lower (Lo/p) affinity agonist binding sites with pKd of 5.08 and 6.17, respectively). The effects of guanine nucleotide and sulfhydryl reagent indicated that the Ho/p site corresponded with the SH site for carbachol, and the Lo/p site with the H + L sites for carbachol. The pD2 values of CCh, Pilo and Oxo for negative inotropic actions on autocontraction of right atria were 5.38, 5.30 and 6.80, respectively. The pD2 values of CCh and Oxo on electrically stimulated contraction of left atria in the presence of isoproterenol were 5.80 and 6.46, respectively, thus being closely related to H or Lo/p agonist binding sites of mAChR.

Research paper thumbnail of 1-oleoyl-2-acetyl-glycerol and phorbol diester stimulate Ca2+ influx through Ca2+ channels in neuroblastoma × glioma hybrid NG108-15 cells

European Journal of Pharmacology, 1986

The effect of 1-oleoyl-2-acetyl-glycerol (OAG) and the phorbol diester 12-O-tetradecanoyl-phorbol... more The effect of 1-oleoyl-2-acetyl-glycerol (OAG) and the phorbol diester 12-O-tetradecanoyl-phorbol-acetate (TPA) on the intracellular Ca2+ concentration ([Ca2+]i) in NG108-15 cells was studied using a Ca2+ indicator, quin 2. OAG and TPA induced an increase in [Ca2+]i from 100 +/- 19 to 187 +/- 24 nM and 192 +/- 15 nM, respectively, within 15 min. The increase in [Ca2+]i induced by activators of protein kinase C was dependent on the extracellular Ca2+ concentration [Ca2+]o) and was inhibited by the Ca2+ blockers, verapamil and nifedipine. These results indicate that the OAG- and TPA-induced [Ca2+]i increase is mediated by the influx of extracellular Ca2+ through voltage-sensitive Ca2+ channels.

Research paper thumbnail of Role of a protein regulating guanine nucleotide binding in phosphoinositide breakdown and calcium mobilization by bradykinin in neuroblastoma × glioma hybrid NG108-15 cells: Effects of pertussis toxin and cholera toxin on receptor-mediated signal transduction

European Journal of Pharmacology, 1987

The addition of bradykinin to NG108-15 cells resulted in an increase in the intracellular Ca 2+ c... more The addition of bradykinin to NG108-15 cells resulted in an increase in the intracellular Ca 2+ concentration ([Ca2+]i) and the formation of inositol monophosphate, inositol bisphosphate, and inositol trisphosphate in these cells. The bradykinin-stimulated formation of inositol polyphosphates in plasma membrane preparations was dependent on the presence of GTP or guanosine-5'-O-thiotriphosphate (GTPTS) but not of GDP. GTPyS, unlike GTP, increased the basal formation of inositol polyphosphate in NG108-15 membranes. Iontophoretic injection of GTPyS into single cells induced increases in [Ca2+]i . These effects of bradykinin and GTP-/S on [Ca2+]i and the formation of inositol phosphates in the intact cells and membranes were not affected by treatment of the cells with pertussis toxin or cholera toxin. Data on binding of bradykinin to membrane preparations indicated the presence of two classes of binding sites with K d values of 0.80 + 0.26 and 9.63 + 0.13 nM. Approximately 74% of the receptors were in the high affinity state. In the presence of guanyl-5'-yl-imidodiphosphate [Gpp(NH)p], the high affinity sites in the membrane preparations were converted to low affinity sites with no change in the total receptor number. These toxin treatments had no effect on binding of bradykinin to its receptors. Thus, these results indicate that a guanine nucleotide regulatory protein, which is not a substrate of pertussis toxin or cholera toxin, is involved in mediating the effects of bradykinin on membrane-bound phosphoinositide-specific phospholipase C to induce the increase of cytosolic calcium.

Research paper thumbnail of The SH-H subgroup of cardiac M2 receptors (M2α) inhibits adenylate cyclase activity

European Journal of Pharmacology, 1988

Muscarinic receptors in the guinea-pig heart seem to consist entirely of M 2 receptors, but are c... more Muscarinic receptors in the guinea-pig heart seem to consist entirely of M 2 receptors, but are coupled with several responses including inhibition of adenylate cyclase activity. On the other hand three affinity states (SH, H and L) can be distinguished in cardiac membranes with muscarinic agonists such as carbachol. We showed previously that the three agonist binding states were the sum of two equilibria (SH-H and H-L subgroup), both regulated by GTP-binding protein(s). In this study we determined which subgroup was responsible for the inhibitory effect of muscarinic M z receptors on adenylate cyclase activity. The EDs0 values for this response of four muscarinic agonists, acetylcholine, carbachol, pilocarpine and oxotremorine corresponded with the binding K D values of H (acetylcholine and carbachol) and Lo/p (pilocarpine and oxotremorine) sites. After alkylation of spare receptors, the EDs0 value of carbachol was changed from 4.3 to 5.6/.tM, which corresponded with the K o value of the H site. Furthermore, the four agonists were almost fully active when membrane preparations were pretreated with propylbenzilylcholine mustard (PrBCM) in the presence of carbachol to destroy the H-L subgroup, whereas after pretreatment with PrBCM and atropine, which alkylated both types of subgroups evenly, the decrease in the number of receptors was proportional to the decrease in the inhibitory effect on adenylate cyclase activity. These results suggest that only the SH-H subgroup (M2,) is responsible for the inhibitory action of muscarinic receptors on adenylate cyclase activity in the heart. Muscarinic M 2 receptors; Adenylate cyclase (inhibition of); Muscarinic acetylcholine receptor subtypes (M2, ~, M2/~) 0014-2999/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

Research paper thumbnail of Effects on guanine nucleotide and sulfhydryl reagent on subpopulations of muscarinic acetylcholine receptors in mammalian hearts: Possible evidence for interconversion of super-high and low affinity agonist binding sites

European Journal of Pharmacology, 1984

Abstract Multiple site models of muscarinic acetylcholine receptors (mAChR) for agonist binding w... more Abstract Multiple site models of muscarinic acetylcholine receptors (mAChR) for agonist binding were applied to curves for the inhibition of QNB binding by carbachol by using nonlinear least square regression analysis. The effects of a guanine nucleotide guanyl-5 ...

Research paper thumbnail of Cardiac M2 receptors consist of two different types, both regulated by GTP

European Journal of Pharmacology, 1987

Cardiac muscarinic receptors are predominantly M 2 receptors, and have three agonist binding site... more Cardiac muscarinic receptors are predominantly M 2 receptors, and have three agonist binding sites (super-high(SH), high(H) and low(L) affinity agonist binding sites). Treatment of cardiac membranes with 50 nM propylbenzilyl choline mustard (PrBCM) caused 88 % loss of binding sites for [3 H]QNB. Carbamyl choline (CCh) inhibits this alkylation dose dependently and, theoretically, generates uneven alkylation of multiple agonist binding sites. Pretreatment of the membranes with 50 nM PrBCM and 0.5 mM CCh resulted in almost complete disappearance of L sites with similar degrees of conservation of H sites and SH sites. In these pretreated membranes, guanine nucleotide and sulfhydryl reagent caused a change in the ratio of residual SH and H sites but not of L sites though previous studies showed that, in intact membranes, these reagents affected the ratio of SH and L sites without significantly changing that of the H site. These results indicate the existence of two equilibria regulated by guanine nucleotide and sulfhydryl reagent in cardiac muscarinic receptors: one between SH and H sites and the other between H and L sites. The participation of GTP binding protein(s) in all cardiac muscarinic responses is suggested. Cardiac M 2 receptors; Agonist binding sites (multiple); GTP binding proteins

Research paper thumbnail of The H-L subgroup of guinea-pig cardiac M2 receptors (M2β) regulates inositol phosphate formation

European Journal of Pharmacology, 1989

In previous studies, we showed that cardiac muscarinic receptors (M2) are composed of two subgrou... more In previous studies, we showed that cardiac muscarinic receptors (M2) are composed of two subgroups, M2a and M2t~, with different affinities for agonists and that the M2~ subgroup is coupled with inhibition of adenylate cyclase. We now studied which subgroup was responsible for the formation of inositol mono-(IP), bis-(IP2), tris-(11}3) and tetrakis-(IP4) phosphates in guinea pig heart. Carbachol (1 raM) 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 11}2, IP 3 and IP 4. However, oxotremorine (1 mM) only slightly stimulated the formation of IP2, and pilocarpine did not stimulate the formation of any IP. The pEDs0 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 o 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 (M2a) and a GTP binding protein different from those for inhibition of adenylate cyclase.

Research paper thumbnail of Regulatory mechanisms of cutaneous delayed-type hypersensitivity

Cellular Immunology, 1983

The macrophage migration inhibitory factor (MIF) fraction was prepared from the immunoadsorbent c... more The macrophage migration inhibitory factor (MIF) fraction was prepared from the immunoadsorbent column by using anti-guinea pig MIF antiserum. Suppression of cutaneous delayed-type hypersensitivity was achieved by intraperitoneal injection of the MIF fraction into the animals bearing macrophage-rich peritoneal exudates. Skin reactions induced by phytohemagglutinin (PHA) were also suppressed in these animals. Reactivity to skin reactive factor (SRF) was suppressed in these animals as well. The sera obtained from these animals exhibited the inhibitory activity against production of lymphokines from sensitized lymphocytes.

Research paper thumbnail of Induction of macrophage disappearance reaction by immunoadsorbed MIF

Cellular Immunology, 1982

Research paper thumbnail of Effect of Antisense Oligonucleotides against Cholesteryl Ester Transfer Protein on the Development of Atherosclerosis in Cholesterol-fed Rabbits

Cholesteryl ester transfer protein (CETP) is the enzyme that facilitates the transfer of choleste... more Cholesteryl ester transfer protein (CETP) is the enzyme that facilitates the transfer of cholesteryl ester from high density lipoprotein (HDL) to apolipoprotein B (apoB)-containing lipoproteins. However, the exact role of CETP in the development of atherosclerosis has not been determined. In the present study, we examined the effect of the suppression of increased plasma CETP by intravenous injection with antisense oligodeoxynucle-otides (ODNs) against CETP targeted to the liver on the development of atherosclerosis in rabbits fed a cholesterol diet. The ODNs against rabbit CETP were coupled to asialoglycoprotein (ASOR) carrier molecules, which serve as an important method to regulate liver gene expression. Twenty-two male Japanese White rabbits were used in the experiment. Eighteen animals were fed a standard rabbit chow supplemented with 0.3% cholesterol throughout the experiment for 16 weeks. At 8 weeks, they were divided into three groups (six animals in each group), among which the plasma total and HDL cholesterol concentrations did not significantly change. The control group received nothing, the sense group were injected with the sense ODNs complex, and the antisense group were injected with the antisense ODNs complex, respectively, for subsequent 8 weeks. ASORpoly(L-lysine) ODNs complex were injected via the ear veins twice a week. Four animals were fed a standard rabbit diet for 16 weeks. The total cholesterol concentrations and the CETP mass in the animals injected with antisense ODNs were all significantly decreased in 12 and 16 weeks compared with those injected with sense ODNs and the control animals. The HDL cholesterol concentrations measured by the precipitation assay did not significantly change among the groups fed a cholesterol diet, and triglyceride concentrations did not significantly change in the four groups. However, at the end of the study, when the HDL cholesterol concentrations were measured after the isolation by ultracentrif-ugation and a column chromotography, they were significantly higher in the animals injected with antisense ODNs than in the animals injected with sense ODNs and in the control animals. A reduction of CETP mRNA and an increase of LDL receptor mRNA in the liver were observed in the animals injected with antisense ODNs compared with those injected with sense ODNs and the control animals. Aortic cholesterol contents and the aor-tic percentage lesion to total surface area were significantly lower in the animals injected with antisense ODNs than in the animals injected with sense ODNs and in the control animals. These findings showed for the first time that suppression of increased plasma CETP by the injection with antisense ODNs against CETP coupled to ASOR carrier molecules targeted to the liver could thus inhibit the atherosclerosis possibly by decreasing the plasma LDL very low density lipoprotein (VLDL) cholesterol in cholesterol-fed rabbits. Cholesteryl ester transfer protein (CETP) 1 is a plasma gly-coprotein that catalyzes the transfer of cholesteryl ester and triglyceride among lipoproteins (1, 2). The homozygotes for CETP deficiency demonstrated markedly elevated HDL cholesterol and plasma apolipoprotein A-I (apoA-I) levels as well as decreased LDL cholesterol and plasma apoB levels (3, 4). CETP deficiency may be associated with protection against ischemic heart disease, based on the observed longevity (5) as well as the lack of any evidence of coronary heart disease (4). However, recently it was reported that in some patients with CETP deficiency there were increased coronary heart diseases (6 – 8). Even in the study using CETP transgenic mice, the exact role of CETP in the development of atherosclerosis has yet to be clarified. Marotti et al. (9) demonstrated that transgenic mice expressing cynomolgus monkey CETP had significantly more early atherosclerotic lesions in the proximal aorta than controls when fed a high cholesterol diet. On the other hand, more recently Hayek et al. (10) concluded that transgenic mice expressing human CETP and apoCIII showed the inhibition of the development of early atherosclerotic lesions. To determine how CETPs affect atherosclerosis in clinical situations, plasma CETP levels must be changed in the experimental models because the studies in the patients with CETP deficiency or CETP transgenic mice go from one extreme to another. We have showed that intravenous injection with antisense oligode-oxynucleotides (ODNs) against CETP coupled to asialoglyco-protein carrier molecules targeted to the liver could inhibit the plasma CETP activity, and as a result, induced a decrease in the plasma LDL VLDL cholesterol and an increase in the plasma HDL cholesterol in cholesterol-fed rabbits (11). In the present study using an intravenous injection with antisense