Toshio Ohnuki

Age-related changes in transmitter glutamate and NMDA receptor/channels in the brain of senescence-accelerated mouse

The senescence-accelerated mouse (SAM-P/8) is known as a murine model of aging and memory dysfunction. In the hippocampus and cerebral cortex of P/8, the contents of glutamic acid and glutamine were significantly higher than those of normal strain R/1 during 2 and 14 months. High K(+)-evoked endogenous glutamic acid release from the slices of P/8 was increased in comparison with R/1 at 9 and 11 months. In addition, the Bmax of [3H]dizocilpine (MK-801, channel blocker for N-methyl-D-aspartic acid receptor/channel) binding in the cerebral cortex was age-dependently decreased in P/8 but not in R/1. These results suggest that synaptic dysfunctions in the glutamatergic system occur in the CNS of SAM-P/8.

Senescence‐Accelerated Mouse

Senescence-accelerated mouse (SAMP8) is known as a murine model of accelerated aging and memory dysfunction. The binding activity of [3H] 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxam ide (PK-11195) as a neurochemical marker of gliosis markedly increased with aging in the cerebral cortex and hippocampus of SAMP8. Immunoreactivity for glial fibrillary acidic protein (GFAP) was also enhanced. A beta-amyloid precursor protein (APP)-like immunoreactivity and 27-kDa-carboxyl terminal fragments of APP increased in SAMP8 brain. In addition, anti-APP antibody stained reactive astrocytes surrounding spongy degeneration in brain stern of SAMP8. These results suggest that astrocytosis and production of APP-derived fragments occur markedly in SAMP8 brains.

Ligand-binding characteristics of [3H]QNB, [3H]prazosin, [3H]rauwolscine, [3H]TCP and [3H]nitrendipine to cerebral cortical and hippocampal membranes of senescence accelerated mouse.

The senescence accelerated mouse (SAM) is known as a murine model of aging and memory dysfunction. In the cerebral cortical membranes of male 9-month-old SAM mice, the Bmax values of [3H]rauwolscine and [3H]nitrendipine binding, and the values of both Kd and Bmax of [3H]TCP binding in the accelerated aging strain SAM-P/8, were significantly increased compared with the values in the control strain SAM-R/1. In hippocampal membranes, however, the Bmax values of [3H]quinuclidinyl benzilate and [3H]nitrendipine binding were significantly decreased in SAM-P/8 compared with those in SAM-R/1. These results suggest that muscarinic acetylcholine receptors, alpha 2-adrenoceptors, N-methyl-D-aspartate receptor channels and L-type Ca2+ channels are changed in cerebral cortex and hippocampus in SAM-P/8 at 9 months.

Alterations in acetylcholine, NMDA, benzodiazepine receptors and protein kinase C in the brain of the senescence-accelerated mouse: an animal model useful for studies on cognitive enhancers.

The senescence-accelerated mouse (SAMP8) is a useful murine model of accelerated aging and learning deficiency. We examined bindings of [3H]pirenzepine, [3H]dizocilpine (MK-801), [3H]flunitrazepam, [3H]8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) and [3H]phorbol 12,13-dibutylate (PDBu) in SAMP8 brains, and compared them to those of SAMR1 (control). In the hippocampus of SAMP8 at 12 months, bindings of [3H]pirenzepine, [3H]MK-801, [3H]flunitrazepam, [3H]8-OH-DPAT and [3H]PDBu were significantly lower than those in SAMR1. In the cerebral cortex, bindings of [3H]pirenzepine, [3H]flunitrazepam and [3H]8-OH-DPAT were higher in SAMP8 than in SAMR1 at 12 months. [3H]PDBu binding was decreased in both the fractions of the membrane and cytosol in the hippocampus of SAMP8. The neurochemical findings presented here support behavioral and pharmacological findings that SAMP8 is a useful model of learning dysfunction and anxiety-deficiency. The usefulness of SAMP8 in studies on cognitive enhancers is also discussed.

Identification of binding sites of prazosin, tamsulosin and KMD-3213 with α1-adrenergic receptor subtypes by molecular modeling

This investigation was performed to assess the importance of interaction in the bindings of selective and nonselective alpha(1)-antagonists to alpha(1)-adrenergic receptor (alpha(1)-AR) subtypes using molecular modeling. The alpha(1)-antagonists used in this study were prazosin, tamsulosin and KMD-3213. Molecular modeling was performed on Octane 2 workstation (Silicon Graphics) using Discover/Insight II software (Molecular Simulations Inc.). Through molecular modeling, possible binding sites for these drugs were suggested to lie between transmembrane domains (TM) 3, 4, 5 and 6 of the alpha(1)-AR subtypes. In prazosin, the 4-amino group, 1-nitrogen atom and two methoxy groups of quinazoline ring possibly interact with the amino acids in TM3, TM5 and TM6 of alpha(1)-ARs. In tamsulosin, amine group of ethanyl amine chain, methoxy group of benzene ring and sulfonamide nitrogen of benzene ring interacts in TM3, TM4 and TM5 of alpha(1)-ARs. In KMD-3213, amine of ethyl amine chain and indoline nitrogen of this compound possibly interact within TM3 and TM5 of alpha(1)-ARs. Amide nitrogen of KMD-3213 also interacts within TM4 of alpha(1A)-AR. The results of the present study suggested that prazosin has similar binding sites in all the alpha(1)-AR subtypes while tamsulosin interacts at higher number of sites with alpha(1D)-subtype than other alpha(1)-AR subtypes. KMD-3213 being an alpha(1A)-AR selective ligand, binds to higher number of sites of alpha(1A) subtype than to other subtypes. All these amino acids are located near the extracellular loop. These findings are consistent with the previous studies that antagonists bind higher in the pocket closer to the extracellular surface unlike agonist binding.

A Novel 5-HT2 Antagonist, Sarpogrelate Hydrochloride, Shows Inhibitory Effects on Both Contraction and Relaxation Mediated by 5-HT Receptor Subtypes in Porcine Coronary Arteries

In isolated porcine coronary arteries, concentrations of 5-HT (10^–8 to 3 × 10^–5 mol/l), α-methylserotonin (α-Me-5-HT, 10^–8 to 3 × 10^–5 mol/l) and ergonovine (10^–9 to 3 × 10^–4 mol/l) produced contraction, whereas high concentrations (10^–5 to 10^–4 mol/l) of these drugs produced relaxation. Both sarpogrelate and ketanserin produced rightward shifts of contraction concentration-response curves induced by 5-HT and α-Me-5-HT at the concentration from 10^–9 to 3 × 10^–5 mol/l, and only sarpogrelate inhibited the relaxation at high concentrations of 5-HT and displayed 155% of maximal contraction at 10^–4 mol/l 5-HT. On the other hand, sarpogrelate and ketanserin did not show any inhibitory effects on the relaxation induced by high concentrations of ergonovine. These results suggested that sarpogrelate and ketanserin show different inhibitory effects on the relaxation induced by high concentrations of 5-HT, indicating that these two drugs may have different affinities to 5-HT receptor subtypes that may be involved in relaxation.

Effects of Chronic Administration of Sarpogrelate on Systolic Blood Pressure of Spontaneously Hypertensive Rats: Comparison with Quinapril

Effects of long-term sarpogrelate (5-HT2 antagonist) administration on the systolic blood pressure of Wistar-Kyoto normotensive rats (WKYs) and spontaneously hypertensive rats (SHRs) were studied and compared with those of quinapril (ACE-I). Sarpogrelate and quinapril were administered orally for 12 weeks and body and heart weights, systolic blood pressure and the relationships between heart weight and systolic blood pressure were determined. Although both drug treatments caused decreases in the body weight of WKYs and SHRs, only quinapril induced a decrease in the heart weight of SHRs. In addition, quinapril induced a dose-dependent decrease in systolic blood pressure in WKYs and SHRs while sarpogrelate had no effect on systolic blood pressure. Thus, quinapril showed hemodynamic effects on WKYs and SHRs, but the 5-HT2 antagonists sarpogrelate did not shown such effects, suggesting that 5-HT2 receptor antagonists may not be important for controlling systolic blood pressure.

Discrimination of α1-Adrenoceptor Subtypes in Rat Aorta and Prostate

This study was designed to further discriminate α1-adrenoceptor subtypes in rat aorta and prostate using functional experiments. Responses induced by phenylephrine were equilibrated in both tissues. The pA2 values and slope factors of several α1-antagonists were assessed using concentration-response curves. The antagonists used were prazosin, WB-4101, 5-methylurapidil (5-MU), HV-723, and tamsulosin. In addition, the effects of chloroethylclonidine (CEC) and nifedipine on phenylephrine-induced contractions were investigated. A high pA2 value for prazosin was observed in both tissues (aorta 9.84, prostate 9.19) and the ranking of each drug’s pA2 value is as follows: tamsulosin > prazosin > WB-4101 > HV-723 > 5-MU in the aorta, and tamsulosin > prazosin > 5-MU > WB-4101 = HV-723 in the prostate. A significant difference between the pA2 value of each drug except for tamsulosin in the aorta and in prostate was observed (p < 0.01). Inhibition of contraction by pretreatment with CEC was 83.9 ± 2.42% in the aorta, and 6.17 ± 0.94% in the prostate. On the other hand, inhibition of maximal response by pretreatment with nifedipine (1 µmol/l) was 35.1 ± 2.2% in the aorta and 24.5 ± 3.1% in the prostate. A good correlation between these pA2 values and pKi values for recombinant human α1b-adrenoceptor expressed in CHO cells (aorta) and α1a-subtypes of CEC pretreated rat hippocampus (prostate) were observed. In conclusion, these results suggest that: (1) the contraction of these two tissues is mediated by α1H-adrenoceptor with a high affinity for prazosin; (2) α1H-adrenoceptors correspond to α1b-(aorta) and α1a-subtypes (prostate), and (3) each α1-adrenoceptor subtype in the aorta and prostate may be α1b-(aorta) and α1a-subtypes (prostate), respectively.

Studies on relationships between chemical structure and β-blocking potency of bopindolol and its two metabolites

The structure-activity relationships of bopindolol and its two metabolites (18-502 and 20-785) and their beta-blocking potencies in the human beta2-adrenoceptor (AR) were assessed using molecular modeling on an INDIGO2 workstation (SGI Co., Ltd.) and DISCOVER/INSIGHT II (Biosym Co., Ltd.). Through modeling, possible binding sites for these agents were hypothesized to involve the 3rd, 4th, 5th and 6th helices of the beta2-AR, and these shared a common interaction site at Asp113 in helix 3. The different chemical structure of these three agents, however, showed binding to different binding sites (amino acids). This study therefore suggests that different beta-blocking potencies of these agents may be due to different chemical structure.

Identification of Binding Sites of Bopindolol and Its Two Metabolites with β1-Adrenoceptors by Molecular Modeling: Comparison with β2 Adrenoceptors

This study was designed to examine the importance of interaction in the bindings of nonselective β-blockers to β1-adrenoceptors (β1-ARs) as compared with β2-ARs, using molecular modeling. The β-blockers used in this study were bopindolol [4-(benzoyloxy-3-t- butylaminopropyl)-2-methylindol hydrogen malomate], its two metabolites [18-502 – hydrolyzed bopindolol or 4-(3-t-butylamino-2-hydroxypropoxy)-2-methyl indole – and 20-785 – 4-(3-t-butylaminopropoxy)-2-carboxyl indole], and propranolol. Molecular modeling was performed on an Indigo2 workstation (Silicon Graphic) using Discover/Insight II (Molecular Simulations) software. Through molecular modeling, possible binding sites for these drugs were suggested to lie between helices 3, 4, 5, and 6 of the β1-AR. The amine, benzoic acid, indole methyl, t-butyl, phenyl, and indole functional groups of bopindolol possibly interact with Asp138 (transmembrane – TM – 3), Ser190 (TM 4), Ala343 (TM 6), Val137 (TM 3), Pro339 (TM6), Cys336 (TM 4), Leu237 (TM 5), and Pro236 (TM 5) of β1-AR, respectively, by either hydrogen bonding or hydrophobic interactions. In addition, 18-502, 20-785, and propranolol also interacted with sites at the same positions as those of β2-ARs. Thus, the results of the present study suggested that although Ala343 and Val137 of β1-AR among these amino acids were different from those of β2-AR, the interactions at the same sites between ligands and amino acids of β1-AR as those of β2-ARs may occur because these drugs are nonselective.