Tomio Segawa

Modifications of central 5-hydroxytryptamine binding sites in synaptic membranes from rat brain after long-term administration of tricyclic antidepressants

Abstract The effects of single and long-term administration of tricylic antidepressants, imipramine, desmethylimipramine (desipramine), amitriptyline and dimetacrine on 5-hydroxytrptamine (5-HT) binding sites in synaptic membranes from rat brain were studied. There were two types of specific [ 3 H]-5-HT binding sites; one with high affinity and the other with low affinity. Intraventricular injection of 5,6-dihydroxytryptamine did not modify the binding parameters. In in vitro experiments, 5-HT, tryptamine derivatives and 5-HT antagonists were found to be effective displacers of [ 3 H]-5-HT binding while all antidepressants studied were ineffective in displacing the binding at 10 −6 M. Single i.p. administration of antidepressants did not affect significantly the specific binding. In contrast, administration of antidepressants for 3 weeks produced a significant decrease in the maximal numbers of binding sites, without affecting the dissociation constants of the specific binding. It is suggested that the modification of central 5-HT binding sites caused by long-term administration of tricylclic antidepressants is due to persistent exposure of the binding sites to elevated concentrations of 5-HT, a consequence of the 5-HT uptake inhibition by the antidepressants. These observations could have some implication for the new theory proposed recently that in depression, there is an involvement of synaptic 5-HT with a hypersensitive receptor in the postsynaptic membranes.

Substance P: regional distribution and specific binding to synaptic membranes in rabbit central nervous system.

Abstract Regional distribution of endogenous substance P and the specific [ 3 H]substance P binding to synaptic membranes in rabbit central nervous system were investigated. The highest level of substance P was found in mesencephalon, followed by diencephalon, corpus striatum, hippocampus, pons-medulla and cortex. In spinal cord, much higher amount of substance P existed in dorsal half than in ventral half. Most of the substance P present in the areas enriched in substance P was located in crude mitochondrial P 2 fractions containing nerve endings. A saturable, high affinity, specific binding of [ 3 H]substance P in synaptic membranes was found. The apparent maximal number of binding sites was 95.7 fmole/mg protein, while the dissociation constant (K D ) was 2.74 nM. The binding was displaced by substance P sequence fragments and the related peptides with relative potencies generally parallelizing their pharmacological activities. The distribution of such specific binding generally correlated with endogenous substance P. The presence of such binding sites for substance P in synaptic membranes suggests a possible role for substance P as a transmitter or modulator of neural function.

Active Uptake of Substance P Carboxy-Terminal Heptapeptide (5–11) into Rat Brain and Rabbit Spinal Cord Slices

Abstract: We previously reported that nerve terminals and glial cells lack an active uptake system capable of terminating transmitter action of substance P (SP). In the present study, we demonstrated the existence of an active uptake system for SP carboxy‐terminal heptapeptide, (5–11)SP. When the slices from either rat brain or rabbit spinal cord were incubated with [3H](5–11)SP, the uptake of (5–11)SP into slices was observed. The uptake system has the properties of an active transport mechanism: it is dependent on temperature and sensitive to hypoosmotic treatment and is inhibited by ouabain and dinitrophenol (DNP). In the brain, (5–11)SP was accumulated by means of a high‐affinity and a low‐affinity uptake system. The Km and the Vmax values for the high‐affinity system were 4.20 × 10−8m and 7.59 fmol/10 mg wet weight/min, respectively, whereas these values for the low‐affinity system were 1.00 × 10−6m and 100 fmol/10 mg wet weight/min, respectively. In the spinal cord, there was only one uptake system, with a Km value of 2.16 × 10−7m and Vmax value of 26.2 fmol/10 mg wet weight/min. These results suggest that when SP is released from nerve terminals, it is hydrolysed into (5–11)SP before or after acting as a neurotransmitter, which is in turn accumulated into nerve terminals. Therefore, the uptake system may represent a possible mechanism for the inactivation of SP.

Pharmacological characterization of central α-adrenoceptors which mediate clonidine-induced locomotor hypoactivity in the developing rat

SummaryThe present experiment was designed to pharmacologically characterize receptors which mediate the clonidine-induced locomotor change in the developing rat. A subcutaneous injection of clonidine (0.78 μmol/kg) produced locomotor hyperactivity in 7-day-old rats but hypoactivity in 20-day-old rats. Phenoxybenzamine (1.5 μmol/kg, 5.9 μmol/kg and 15 μmol/kg) decreased spontaneous activity in a dosedependent manner but did not antagonize clonidineinduced hypoactivity in 20-day-old rats. By contrast, the significant reversal of the clonidine-induced hypoactivity by pretreatment with phentolamine (1.6 μmol/kg and 6.3 μmol/kg), yohimbine (1.3 μmol/kg and 5.1 μmol/kg) and piperoxan (7.4 μmol/kg) was observed at such doses when the blockers did not cause and hypoactivity by themselves. It is suggested that clonidine could induce locomotor hypoactivity by activating presynaptic (α1-type) α-adrenoceptors in the CNS of 20-day-old rat.

Supersensitivity to substance P after dorsal root section

Abstract Two to three weeks after unilateral dorsal root section, specific [ 3 H] substance P binding in synaptic membranes from rabbit spinal cord increased significantly compared with that in intact side, while non-specific [ 3 H] substance P binding was unchanged. Scatchard analysis indicates that the development of such supersensitivity is due to an increase in a number of receptor sites, not to an increase in a receptor affinity. These results strongly support the notion that substance P exerts a transmitter role for primary afferent fibers.

Activation of adenylate cyclase by dopamine, GTP, NaF and forskolin in striatal membranes of neonatal, adult and senescent rats.

Dopamine (DA) caused a significant activation of striatal adenylate cyclase in neonatal and adult but not in senescent rats. GTP activated cyclase at the adult stage but not at both neonatal and senescent stages. NaF and forskolin activated cyclase at every stage. The coupling mechanism between DA1 receptors and catalytic units of cyclase seems to become functional at the neonatal stage but GTP recognition and/or binding sites lack in stimulatory GTP binding protein in neonatal and senescent membranes.

Possible Involvement of Inhibitory GTP Binding Regulatory Protein in α2‐Adrenoceptor‐Mediated Inhibition of Adenylate Cyclase Activity in Cerebral Cortical Membranes of Rats

Abstract: Influences of α2‐adrenoceptor stimulation on adenylate cyclase activity were investigated in cerebral cortical membranes of rats. Pretreatment of the membranes with islet‐activating protein and NAD resulted in a significant increase in basal activity as well as in GTP‐or forskolin/GTP‐induced elevation of adenylate cyclase activity. Strong activation of adenylate cyclase was also caused in membranes pretreated with cholera toxin together with NAD in comparison to that in control membranes, suggesting that adenylate cyclase activity is perhaps regulated by stimulatory and inhibitory GTP binding regulatory protein existing in synaptic membranes. In addition, adrenaline (with propranolol) or clonidine significantly reduced adenylate cyclase activity stimulated by pretreatment with forskolin and GTP. The inhibitory effects of adrenaline were also observed in membranes pretreated with cholera toxin and NAD. Moreover, the inhibition by adrenaline or clonidine was completely abolished by treatment with (a) yohimbine or (b) islet‐activating protein and NAD. It is suggested that α‐receptor stimulation causes inhibitory influences on adenylate cyclase activity mediated by the inhibitory GTP binding regulatory protein in synaptic membranes of rat cerebral cortex.

THE EFFECT OF α‐ADRENOCEPTOR ANTAGONISTS AND METIAMIDE ON CLONIDINE‐INDUCED LOCOMOTOR STIMULATION IN THE INFANT RAT

1 Subcutaneous injections of clonidine (3.9 × 10−8 mol/kg to 3.9 × 10−6 mol/kg) produced forward locomotion and wall climbing in 7‐day‐old rats in a dose‐dependent manner. 2 The effect was reduced significantly by a preceding intraperitoneal injection of phentolamine (7.9 × 10−6 mol/kg), phenoxybenzamine (7.4 × 10−6 mol/kg), yohimbine (1.3 × 10−6 mol/kg) or piperoxan (7.4 × 10−6 mol/kg). 3 The pA2‐values of the antagonists to the clonidine‐induced locomotor hyperactivity were: 5.1 (phenoxybenzamine), 5.2 (phentolamine), 6.4 (yohimbine) and 6.0 (piperoxan). 4 Metiamide (2.5 × 10−4 mol/kg, 5.0 × 10−4 mol/kg and 1.0 × 10−3 mol/kg), a histamine H2‐receptor blocker, did not affect the clonidine‐induced locomotor stimulation. 5 It is suggested that the receptors which mediate clonidine‐induced locomotor stimulation could be α‐adrenoceptors but not histamine H2‐receptors in the central nervous system of the infant rat.

α2-adrenoceptor-GTP binding regulatory protein-adenylate cyclase system in cerebral cortical membranes of adult and senescent rats

The characterization of [3H]clonidine binding and effects of GTP, forskolin, islet-activating protein (IAP) and cholera toxin on adenylate cyclase activity were investigated in cerebral cortical membranes from 70-day-old and 2-year-old rats. Neither Kd nor Bmax values in [3H]clonidine binding were changed between day 70 and year 2. The activation of adenylate cyclase by forskolin was significantly higher in senescent than in adult animals. The inhibitory effect of adrenaline, which was completely abolished by the pretreatment with IAP/NAD on forskolin/GTP-stimulated cyclase activity, was low in senescent rats compared to that in adult ones. The stimulatory effect of cholera toxin/NAD was also low at the senescent stage compared to that at the adult stage. It is suggested that ligand binding affinity and the density in alpha 2-adrenoceptors do not change between day 70 and year 2 but that GTP binding and/or coupling activity of inhibitory as well as stimulatory GTP binding regulatory protein to catalytic units decrease in synaptic membranes of 2-year-old compared to those of 70-day-old rat brain.

Decrease of Clonidine Binding Affinity to α2-Adrenoceptor by ADP-Ribosylation of 41,000-Dalton Proteins in Rat Cerebral Cortical Membranes by Islet-Activating Protein

Abstract: The IC50 value for inhibition of specific [3H]yohimbine binding to rat cerebral cortical membranes by clonidine was increased, and the Hill coefficient (nH) approached unity in the presence of 150 μM GTP. Pretreatment of membranes with islet‐activating protein (IAP) in the presence of NAD caused an increase in IC50 and nH values for clonidine compared with control membranes in the absence of GTP, the addition of which was without effect. Scatchard analysis showed that the Bmax value of the high‐affinity component in [3H]clonidine binding was decreased by pretreatment with IAP/NAD. GTP in a concentration range of 0.1 μM‐ I mM caused significant elevation of [3H]yohimbine binding. In IAP/NAD‐pretreated membranes, however, [3H]yohimbine binding was no longer affected by GTP, although IAP/NAD significantly (p < 0.01) increased [3H]yohimbine binding compared to control. IAP ADP‐ribosylated 41,000 dalton proteins of cerebral cortical membranes. From these results, it can be suggested that inhibitory guanine nucleotide regulatory protein with MI 41,000 couples to α2‐adrenoceptors to regulate binding affinity of agonists and antagonists in membranes of the rat cerebral cortex.