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  • 1
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 53 (1989), S. 0 
    ISSN: 1471-4159
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Medizin
    Notizen: Abstract: Mg2+ increased but Na+ and GTP decreased [3H]substance P (SP) binding to rat cerebral cortical membranes and to 10 mM 3-[(3-cholamidopropyl)dimethyl-ammonio]-l-propane sulfonate (CHAPS)-solubilized membrane fraction. To determine the binding parameters that are modified by the cations and GTP, inhibition experiments of [3H]SP binding by unlabeled SP were performed in both of the preparations. Nonlinear least-squares regression analysis of data in the membrane fraction indicated that optimal fitting of the inhibition curves in the presence of 10 mM MgCl2 was attained with a two-site model, corresponding to a “high-affinity (H)” and a “low-affinity (L)” state. By omitting MgCl2, or by addition of NaCl and GTP, the [3H]SP specific binding was decreased, the H state disappeared, and the L state and a new “super-low affinity (SL)” state were observed. The SP/ [3HJSP inhibition curves in the cerebral cortical membranes by in vivo treatment with pertussis toxin (islet-activating protein) were similar to that in the presence of GTP in control membranes. The effects of MgC12, NaCl, and GTP were greater in the CHAPS-solubilized fraction than in the membrane fraction. In contrast to the membrane fraction, the inhibition curves of [3HJSP binding by unlabeled SP in the presence of MgCl2 in the CHAPS-solubilized fraction were best fitted to a one-site model. The KD value was relatively close to that of the low-affinity state in the membrane fraction. Even with the addition of NaCl or GTP, or by reducing MgCI2concentration to 1 mM, although the inhibition curves consistently fit the one-site model, the KD values changed only slightly.
    Materialart: Digitale Medien
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  • 2
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 50 (1988), S. 0 
    ISSN: 1471-4159
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Medizin
    Notizen: The specific binding protein for substance P (SP) was solubilized in an active form from the crude mitochondrial (P2) fraction of bovine brainstem. After incubation with 3–[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) and 0.1 M NaCl at 0°C for 30 min, the SP binding to the supernatant fraction (100,000 g, 60 min) was determined by the glass fiber filtration method reported by Bruns et al. (1983). The specific [3H]SP binding to the solubilized fraction was highly specific for SP and was displaced by nanomolar concentrations of SP and physalaemin, but only by micromolar concentrations of eledoisin. In addition, the binding was inhibited by GTP (approximately 40% of the specific binding decreased by 10 μM GTP) in both preparations. These results were virtually identical to those of P2 membrane preparations and suggested that this high-affinity SP binding site belongs to the SP-P type. Scatchard analyses of SP binding to the solubilized fraction revealed a single saturable component with a Bmax of 22.0 ± 5.10 fmol/mg protein and a KD of 0.79 nM, and these values are almost the same as those obtained in the P2 fraction (Bmax= 31.3 ± 3.56 fmol/mg protein, KD= 0.82 nM). Gel filtration analysis showed that the detergent-SP binding protein complex has two calculated molecular weights of 〉 1,000,000 and 55,000–60,000 (a corresponding Stokes radius of 35.5 nm).
    Materialart: Digitale Medien
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  • 3
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 50 (1988), S. 0 
    ISSN: 1471-4159
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Medizin
    Notizen: Abstract: Histamine stimulated the enzymatic synthesis of phosphatidylcholine from phosphatidylethanolamine in crude synaptic membranes of rat brain containing the methyl donor S-adenosyl-l-methionine (SAM). In the presence of, but not in the absence of SAM, histamine increased cyclic AMP accumulation at the concentrations that stimulate phospholipid methylation. S-Adenosyl-l-homocysteine, an inhibitor of phospholipid methyltransferases, inhibited histamine-stimulated phospholipid methylation and histamine-induced cyclic AMP accumulation in the presence of SAM in a concentration-dependent manner. Histamine-induced [3H]methyl incorporation into phospholipids exhibited a marked regional heterogeneity in rat brain in the order of cortex 〉 medulla oblongata 〉 hippocampus 〉 striatum 〉 midbrain 〉 hypothalamus. The regional distribution of histamine-induced cyclic AMP accumulation exactly paralleled histamine-stimulated [3H]-methyl incorporation in rat brain. Histamine-induced cyclic AMP accumulation was inhibited by the addition of cimetidine or famotidine, but not by mepyramine or diphenhydramine. The accumulation of cyclic AMP in the presence of SAM was observed by the addition of impromidine or dimaprit, but not by 2-pyridylethylamine. These results indicate that phospholipid methylation is induced by histamine and may participate in H2-receptor-mediated stimulation of adenylate cyclase in rat brain.
    Materialart: Digitale Medien
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  • 4
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 48 (1987), S. 0 
    ISSN: 1471-4159
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Medizin
    Notizen: Abstract: Histamine stimulated [3H]methyl group incorporation into phospholipids in crude synaptic membranes of rat whole brain (without cerebellum) in modified Krebs-Ringer solution containing the methyl donor S-adenosyl-[wethyl-3H]methionine. The transient increase of [3H]-methyl incorporation into lipids peaked within 45 s after addition of histamine (5 or 10 μM) and decreased the basal level in 60 s. Histamine-stimulated [3HJmethyl incorporation was increased linearly in a protein concentration-dependent manner. The stimulation was temperature and histamine concentration dependent. TLC analysis of a chloroform/methanol extract indicated that radioactive phospholipids (phosphatidylcholine, phosphatidyl-N,N-dimethylethanolamine, and phosphatidyl-N-monomethylethanolamine) accounted for 60–65% of the total radioactivity recovered. The synaptosomal fraction had the highest specific activity of all the subfractions of crude synaptic membranes (P2). Histamine-induced [3H]methyl incorporation was inhibited by addition of cimetidine (0.01–10 μM) or famotidine (0.01–1.0 μM) in a concentration-dependent manner but not by mepyramine (0.1–10 μM) or diphenhydramine (0.1–10 μM). The stimulation of [3H]methyl incorporation was also observed by addition of impromidine (0.01–10 μM) or dimaprit (1.0 μM-1.0 mM) in a concentration-dependent manner but not by 2-pyridylethylamine (1.0 μM-1.0 mM). These results indicate that phospholipid methylation is induced by histamine acting on H2 receptors in rat brain synaptosomes.
    Materialart: Digitale Medien
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  • 5
    ISSN: 1471-4159
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Medizin
    Notizen: Abstract: The characteristics of the carbohydrate chain on the rat cerebral cortical substance P (SP) receptor were studied. We examined the effects of pretreatment with three lectins (concanavalin A, wheat germ agglutinin, lens culinaris agglutinin) on the [3H]SP binding activities. Each lectin can bind to the specific carbohydrate chain. Among these lectins, only concanavalin A inhibited specific [3H]SP binding by reducing the affinity of the binding sites. The inhibitory action of concanavalin A was dose-dependent and diminished by the addition of α-methyl-D-mannoside. The present results suggest that the rat cortical SP receptor has either a biantennary complex-type or a high mannose-type of carbohydrate chain, and that the carbohydrate chain is implicated in the SP binding activity of the SP receptor system.
    Materialart: Digitale Medien
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  • 6
    ISSN: 1471-4159
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Medizin
    Notizen: 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.
    Materialart: Digitale Medien
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  • 7
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 38 (1982), S. 0 
    ISSN: 1471-4159
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Medizin
    Notizen: Abstract: Ontogenetic development of specific [3H]spiperone binding to crude synaptic membranes and its regulation by Na+ and GTP was investigated in the rat striatum, (d)-Butaclamol more effectively inhibited [3H]spiperone binding than (l)-butaclamol. The ratio of inhibitory activity of (d)- and (l)-butaclamol for [3H]spiperone binding was not different between 1-, 7-, and 70-day-old animals but eight- to ninefold lower at 18 days of gestation than during the postnatal period. A Scatchard plot of specific binding indicated the presence of two types of binding: low-affinity (KD=1.51 nm) and high-affinity (KD= 0.09 nM) binding on day 70. Only one component (KD= 0.075 nM) was observed on days 1 and 7 and both types of binding were found on day 15. Bmax gradually increased with age and reached a peak on day 30, followed by a decline on days 70 and 360. Na+, 100 mM, significantly increased specific binding on days 1, 7, 15, and 70. GTP, 50 μM, completely reversed the Na+-induced decrease in IC50 of apomor-phine on both days 15 and 70, but not on day 7. It is suggested that receptors could recognize ligand stereospecificity on day 1. The density in dopamine receptors in the striatum reaches a peak on day 30, followed by a decrease on days 70 and 360. In addition, regulation by Na+ and GTP in agonist binding to dopamine receptors seems to become functional between 1 and 2 weeks after birth.
    Materialart: Digitale Medien
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  • 8
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 37 (1981), S. 0 
    ISSN: 1471-4159
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Medizin
    Notizen: 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.
    Materialart: Digitale Medien
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  • 9
    ISSN: 1471-4159
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Medizin
    Notizen: 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.
    Materialart: Digitale Medien
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  • 10
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 42 (1984), S. 0 
    ISSN: 1471-4159
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Medizin
    Notizen: Abstract: Cerebral cortical [3H]clonidine binding and influences of GTP and cations were investigated in developing rats. The results from Scatchard plots were compatible with the presence of two populations of binding sites [high-affinity binding (KD= 0.59 nM) and low-affinity binding (KD= 7.12 nM)] in 70-day-old rats but only high-affinity binding (KD= 0.27 nM) on day 1. Low-affinity binding was detectable on day 7. KD values in high- and low-affinity binding were not significantly changed during development after 7 days. Bmax of high-affinity binding reached a peak on day 15, and the value of low-affinity binding gradually increased with age. The addition of 10 μM GTP caused a significant reduction in Bmax of high-affinity binding after day 7. Neither KD nor Bmax of low-affinity binding was affected by 10 μM GTP during development. NaCl (10 and 100 mM) diminished the binding on days 7 and 70. MnCl2 (0.1 and 1.0 mM) markedly increased the binding on days 15 and 70 but not on day 7. It is suggested that: (1) single binding sites of α2-adrenoceptors with higher affinity seem to be present on day 1; (2) low-affinity binding appears on day 7; (3) the number of high-affinity binding sites reaches a peak on day 15, followed by changes in populations of high-affinity as well as low-affinity sites without changing affinity; (4) the regulatory mechanism in α2-receptors by guanine nucleotide reaches functional maturity between days 1 and 7; and (5) the involvement of Na+ and Mn2+ in α2-receptor binding becomes functional by days 7 and 15, respectively.
    Materialart: Digitale Medien
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