Histamine Increases Phospholipid Methylation and H2-Receptor-Adenylate Cyclase Coupling in Rat Brain

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 greater than medulla oblongata greater than hippocampus greater than striatum greater than midbrain greater than 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.     

Photoaffinity labeling with dihydropyridine derivatives of crude membranes from rat skeletal, cardiac, ileal, and uterine muscles and whole brain

The characteristics of photoaffinity labeling with the calcium agonist [3H]Bay K 8644 (Bay) and the calcium antagonists [3H]nitrendipine (Nit) and (+)PN200-110 (PN) of crude membranes from rat skeletal, cardiac, ileal, and uterine muscles and whole brain were investigated. In all these crude membranes, [3H](+)PN (20 nM) was mainly photoincorporated into one protein band with a molecular weight of 30,000 - 41,000 Da. It was also incorporated into some other bands of all these crude membranes. The photoincorporation of [3H](+)PN into these crude membranes was inhibited by the presence of 20 microM unlabeled (+)PN. The photoincorporation of [3H](+)PN into these crude membranes depended on its dose and on the time of UV irradiation. No incorporation of [3H](+)PN was observed in the absence of UV irradiation. The incorporation was not affected by the presence of 1 mM CaCl2 and/or 0.15 M NaCl, but was significantly decreased by 20 microM (+)PN and slightly decreased by 20 microM (-)PN, 20 microM Bay, 1 mM diltiazem, or 1 mM verapamil. Namely, enantiomers of PN caused various extents of stereoselective inhibition of photoaffinity labeling by [3H](+)PN of specific protein bands in these crude membranes. [3H]Nit was photoincorporated into these crude membranes in the same way as [3H](+)PN, but [3H]Bay was not photoincorporated. However, 20 microM unlabeled Nit did not consistently inhibit photoaffinity labeling with [3H]Nit. These findings suggested that measurement of photoaffinity of crude membranes from rat skeletal, cardiac, and uterine muscles and whole brain with [3H](+)PN by UV irradiation is a useful method for investigating the characteristics of the voltage-dependent calcium channels that are affected by 1,4-dihydropyridine derivatives.     

Phospholipid Methylation Increases [3H]Diazepam and [3H]GABA Binding in Membrane Preparations of Rat Cerebellum

The effect of phospholipid methylation on both [3H]diazepam and [3H]GABA ( [3H]gamma-aminobutyric acid) binding to crude synaptic plasma membrane from rat cerebellum has been studied. S-Adenosylmethionine (SAM) stimulates [3H]methyl group incorporation into membrane phospholipids and enhances [3H]diazepam binding by increasing the apparent Bmax. Conversely, inhibition of [3H]methyl group transfer from [3H]SAM to phospholipids by preincubation with SAM at 0 degrees C or with SAH abolishes the increase of binding. After preincubation with SAM, analysis of the GABA binding reveals the presence of binding sites with high affinity, a property absent in control membranes preincubated without SAM. Among the neurotransmitter bindings tested, only those of GABA and benzodiazepine in the cerebellum and beta-adrenergic ligands in the cerebral cortex are enhanced upon stimulation of phospholipid methyltransferase activity. [3H]Dihydromorphine, [3H]dihydro-alpha-ergokryptine and [3H]spiroperidol bindings are not affected by SAM. The present data suggest an involvement of phospholipid methylation in regulation of both [3H]GABA and [3H]-diazepam binding.     

Microsomal Opiate Receptors: Characterization of Smooth Microsomal and Synaptic Membrane Opiate Receptors

In continuing studies on smooth microsomal and synaptic membranes from rat forebrain, we compared the binding properties of opiate receptors in these two discrete subcellular populations. Receptors in both preparations were saturable and stereospecific. Scatchard and Hill plots of [3H]naloxone binding to microsomes and synaptic membranes were similar to plots for crude membranes. Both synaptic membranes and smooth microsomes contained similar enrichments of low- and high-affinity [3H]naloxone binding sites. No change in the affinity of the receptors was observed. When [3H]D-ala2-D-leu5-enkephalin was used as ligand, microsomes possessed 60% fewer high-affinity sites than did synaptic membranes, and a large number of low-affinity sites. In competition binding experiments microsomal opiate receptors lacked the sensitivity to (guanyl-5'-yl)imidodiphosphate [Gpp(NH)p] shown by synaptic and crude membrane preparations. In this respect microsomal opiate receptors resembled membranes that were experimentally guanosine triphosphate (GTP)-uncoupled with N-ethylmaleimide (NEM). Agonist binding to microsomal and synaptic membrane opiate receptors was decreased by 100 mM NaCl. Like NEM-treated crude membranes, microsomal receptors were capable of differentiating agonist and antagonists in the presence of 100 mM NaCl. MnCl2 (50-100 microM) reversed the effects of 100 mM NaCl and 50 microM GTP on binding of the mu-specific agonist [3H]dihydromorphine in both membrane populations. Since microsomal receptors are unable to distinguish agonists from antagonists in the presence of Gpp(NH)p, they are a convenient source of guanine nucleotide-uncoupled opiate receptors.     

The release of 3H-1-methyl-4-phenylpyridinium from bovine adrenal chromaffin cells is modulated by somatostatin

Besides cholinergic regulation, catecholamine secretion from adrenal chromaffin cells can be elicited and/or modulated by noncholinergic neurotransmitters and hormones. This study was undertaken to investigate the influence of somatostatin and octreotide on [3H]MPP+ secretion evoked by KCl or cholinergic agents, from bovine adrenal chromaffin cells. The release of [3H]MPP+ was markedly increased by excess KCl (50 mM), acetylcholine (50 microM-10 mM) and by the nicotinic agonists, nicotine (5-100 microM) and 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP, 10-100 microM), but not by the muscarinic agonist, pilocarpine (10-100 microM). Acetylcholine-evoked release of [3H]MPP+ from these cells was mainly mediated by nicotinic receptors: a) nicotine and DMPP stimulated the release of [3H]MPP+, b) a nicotinic antagonist, hexamethonium, markedly blocked the acetylcholine-evoked response and c) pilocarpine was devoid of effect on [3H]MPP+ secretion. At all concentrations tested, somatostatin and octreotide interfered neither with [3H]MPP+ basal release nor with KCl-induced release of [3H]MPP+. However, somatostatin (0.01-0.3 microM) increased the release of [3H]MPP+ induced by a high concentration of acetylcholine (10 mM). Octreotide (1-10 microM) had no effect. These results, showing that somatostatin potentiates acetylcholine-induced [3H]MPP+ release, support the hypothesis that somatostatin may increase the release of catecholamines from adrenal medullary cells.     

GABAB Receptor-Mediated Inhibition of Histamine H1-Receptor-Induced Inositol Phosphate Formation in Slices of Rat Cerebral Cortex

Histamine-stimulated accumulation of [3H]inositol monophosphate ([3H]IP1) in lithium-treated slices of rat cerebral cortex was inhibited by gamma-aminobutyric acid (GABA) (IC50 0.30 +/- 0.03 mM). The maximum level of inhibition was 69 +/- 2%. GABA alone caused a small stimulation of basal accumulation of [3H]IP1. The inhibitory action of GABA on the response to histamine was mimicked by the GABAB agonist (-)-baclofen, IC50 0.69 +/- 0.04 microM, which was 430-fold more potent as an inhibitor than the (+)-isomer. (-)-Baclofen also inhibited histamine-induced formation of [3H]inositol bisphosphate ([3H]IP2) and [3H] inositol trisphosphate ([3H]IP3). Inhibition curves for GABA and for (-)-and and (+)-baclofen had Hill coefficients greater than unity. (-)-Baclofen, at concentrations that caused inhibition of histamine-induced [3H]IP1 accumulation, did not alter the basal level of [3H]IP1 or the incorporation of [3H]inositol into total inositol phospholipids. Isoguvacine, a GABAA agonist, had no effect on either the histamine-stimulated or basal accumulation of [3H]IP1. GABA had no effect on carbachol-stimulated [3H]IP1 formation.     

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

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 microM 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 microM-1 mM caused a 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 less than 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 Mr 41,000 couples to alpha 2-adrenoceptors to regulate binding affinity of agonists and antagonists in membranes of the rat cerebral cortex.     

Manganese stimulates incorporation of [3H]inositol into an agonist-insensitive pool of phosphatidylinositol in brain membranes

In a particulate preparation from rat brain, manganese ions stimulate the incorporation of [3H]inositol into inositol phospholipids in a concentration-dependent manner. Incubation with CDP-diacylglycerol (0.5 mM) alone had no effect on the incorporation of [3H]inositol but potentiated the stimulatory effect of manganese. Despite the increase in [3H]inositol incorporation into phosphatidylinositol, the carbachol-induced accumulation of [3H]inositol-1-phosphate was unaltered in membranes preincubated with manganese but when coincubated with CDP-diacylglycerol the carbachol-induced accumulation of [3H]inositol-1-phosphate was increased. These data suggest that manganese stimulates the incorporation of [3H]inositol into an agonist-insensitive pool of phosphatidylinositol.     

The effects of fatty acids on phosphoinositide synthesis and myo-inositol accumulation in exocrine pancreas

The effects of arachidonic acid (20:4) on phosphoinositide turnover were examined in rat pancreatic acinar cells prelabeled with myo-[3H]inositol. Arachidonic acid (50 microM) increased the accumulation of myo-[3H]inositol, but not that of [3H]inositol monophosphate, [3H]inositol bisphosphate, or [3H]inositol trisphosphate. By contrast, 10 microM carbamoylcholine increased the accumulation of all four compounds. A combination of arachidonic acid plus carbamoylcholine caused a selective and marked accumulation of myo-[3H]inositol, which was abolished by 10 mM LiCl. Arachidonic acid (10-100 microM) produced a concentration-dependent inhibition of myo-[3H]inositol incorporation into phosphoinositides and markedly depressed carbamoylcholine-induced increases in myo-[3H]inositol incorporation into inositol phospholipids. Several other unsaturated and saturated fatty acids failed to elicit a synergistic response with carbamoylcholine in stimulating myo-[3H]inositol accumulation and did not retard the incorporation of myo-[3H]inositol into phosphoinositides. The fact that eicosapentaenoic acid (20:5), but not arachidic acid (20:0), mimicked the depressant effect of arachidonate on phosphoinositide labeling suggests that the degree of unsaturation of the fatty acid, rather than chain length, is important for inhibition of phosphoinositide synthesis. The arachidonate-induced decrease in myo-[3H]inositol incorporation was accompanied by a reduction in the steady state level of [32P]phosphatidylinositol 4,5-bisphosphate. The mass of arachidonic acid liberated in response to carbamoylcholine was measured by gas chromatography-mass spectrometry, and the time course of stimulated arachidonate accumulation paralleled that of inositol phosphate accumulation and amylase release. These observations suggest that in exocrine pancreas, endogenous arachidonic acid serves as a negative feedback regulator of phosphoinositide turnover.     

Inhibition by Calmodulin Antagonists of [3H]MK-801 Binding in Brain Synaptic Membranes

In brain synaptic membranes not extensively washed, (+)-5-[3H]methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5, 10-imine ([3H]MK-801) binding was markedly inhibited in a concentration-dependent manner (at concentrations above 1 microM) by several compounds having antagonistic activity at the Ca(2+)-binding protein calmodulin. Scatchard analysis revealed that N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibited the binding through a significant decrease in the density of binding sites without affecting the affinity at 10 microM. In membranes extensively washed and treated with a low concentration of Triton X-100, L-glutamic acid (Glu) drastically accelerated the initial association rate of [3H]MK-801 binding with glycine (Gly), almost doubling the initial association rate found in the presence of Glu alone. The addition of W-7 invariably reduced the initial association rate observed in the presence of either Glu alone or both Glu and Gly, without significantly altering the dissociation rate of bound [3H]-MK-801, irrespective of the presence of the two stimulatory amino acids. The maximal potencies of Glu, Gly, and spermidine in potentiating the binding were all attenuated by W-7. These results suggest that calmodulin antagonists may interfere with opening processes of an ion channel associated with an N-methyl-D-aspartate-sensitive subclass of excitatory amino acid receptors in rat brain.     

Modulation of GABA Receptor Binding by Ca+2

In frozen-thawed repeatedly washed rat cortical synaptic membranes, Ca2+ (1-5 mM) decreased the binding of [3H]muscimol whereas it increased the binding of [3H]gamma-aminobutyric acid (GABA). However, the binding of [3H]GABA was decreased by the same extent as the binding of [3H]muscimol when the membranes were incubated with baclofen (a selective ligand for the GABAB binding site) and Ca2+. Scatchard analysis of [3H]muscimol binding revealed that Ca2+ reduced the density of GABA binding sites without affecting the dissociation constant. Ca2+ was more potent than Ba2+, Mg2+ was ineffective, and the Ca2+ antagonist La3+ stimulated [3H]muscimol binding. The inhibition of [3H]muscimol binding by Ca2+ was not influenced by calmodulin (50 micrograms/ml), trifluoperazine (10(-5) M), verapamil (10(-6) M), quinacrine (10(-4) M), cordycepin (0.1 mM), leupeptin (20 microM), or soybean trypsin inhibitor (0.1 mg/ml). Moreover, the effect of Ca2+ was additive to that of GABA-modulin. These results indicate that Ca2+ decreases the number of GABAA binding sites while unveiling GABAB binding sites.     

Uncoupling of Rat Cerebral Cortical α2-Adrenoceptors from GTP-Binding Proteins by N-Ethylmaleimide

Pretreatment of membranes from rat cerebral cortex with N-ethylmaleimide (NEM) decreased [3H]-clonidine binding in a concentration-dependent manner. The Bmax values of high-affinity sites for [3H]clonidine were reduced by 50 microM NEM treatment. Treatment with 500 microM NEM diminished the sum of Bmax of both high- and low-affinity components. GTP, Na+, and Mn2+ exerted little effect on [3H]clonidine binding in NEM-treated membranes. The addition of purified GTP-binding proteins caused an increase in the binding to the membranes pretreated with 50 microM NEM, but did not increase [3H]-clonidine binding in membranes treated with 500 microM NEM. In contrast, NEM pretreatment inhibited islet activating protein (IAP)-catalyzed ADP ribosylation of membrane-bound (41,000-dalton) and purified (39,000/41,000-dalton) GTP-binding proteins. From these results, it is suggested that two or three categories of essential sulfhydryl groups are involved in the coupling between agonist, alpha 2-adrenoceptor, and GTP-binding protein. One is a highly sensitive site to NEM (a concentration range of 1-50 microM), which is probably a cysteine residue, IAP-catalyzed ADP-ribosylating site on the alpha-subunit of GTP-binding protein. Other sites have low sensitivity to NEM (a concentration range of 0.1-1 mM), and are the binding domain of agonist and/or the coupling domain of GTP-binding protein on the alpha 2-adrenoceptor. In addition, Ki-ras p21 protein may lack the capacity to couple with the alpha 2-adrenoceptor.     

Effects of Angiotensin II on [3H]Noradrenaline Release and Phosphatidylinositol Hydrolysis in the Parietal Cortex and Locus Coeruleus of the Rat

Angiotensin II (ANGII) (3-100 nM) facilitated the potassium-evoked (22.5 mM) release of [3H]-noradrenaline ([3H]NA) from slices of parietal cortex in a concentration-dependent manner, but did not significantly alter the release of [3H]NA evoked in a similar manner from locus coeruleus slices. The facilitatory action of ANGII was blocked by saralasin (0.1-3 microM). Neither nimodipine (10-30 microM) nor phenylmethylsulphonyl fluoride (1 mM) altered either [3H]NA release or the facilitatory action of ANGII in the parietal cortex. Carbachol (0.01-3 mM) and raised potassium (22.5 mM), but not ANGII (3-100 nM), stimulated the production of inositol phosphates in parietal cortex slices. The potassium-evoked increase in inositol phosphate production was unaffected by ANGII (3-100 nM). In the locus coeruleus, ANGII (3-100 nM) did not stimulate inositol phosphate production. The mechanism underlying the ANGII facilitation of [3H]NA release from the parietal cortex does not appear to involve either nimodipine-sensitive calcium channels, or, as far as we have been able to determine, the release of calcium from intracellular stores following the breakdown of phosphoinositides.     

Vascular smooth muscle actions of carnosine as its zinc complex are mediated by histamine H(1) and H(2) receptors

The endogenous dipeptide carnosine (beta-alanyl-L-histidine), at 0.1-10 mM, can provoke sustained contractures n rabbit saphenous vein rings with greater efficacy than noradrenaline. The effects are specific; anserine and homocarnosine are ineffective, as are carnosine's constituent amino acids histidine and beta-alanine. Zinc ions enhance the maximum carnosine-induced tension (to 127 +/- 13% of control at 10 microM Zn(total)) and muscle sensitivity is potentiated (mean K(0.5) reduced from 1.23 mM to 17 microM carnosine with 15 microM Zn(total)). The dipeptide acts as a Zn-carnosine complex (Zn. Carn). The effects of carnosine at 1 microM-10 mM (total) in the presence of 1-100 microM Zn(2+) (total) can be described as a unique function of [Zn.Carn] with an apparent K(0.5) for the complex of [7.4)(10(-8)] M. Contractures are reduced at low [Ca(2+)], unaffected by adrenoceptor antagonists, but can be blocked by antagonists to several receptor types. The most specific effect is by mepyramine, the H(1) receptor antagonist. With Zn present, carnosine can inhibit the H(1)-specific binding of [(3)H]mepyramine to isolated Guinea pig cerebella membranes. This effect of carnosine can be described as a function of the concentration of Zn.Carn with an apparent IC(50) of 2.45 microM. Like histamine, carnosine evoked an H2-mediated (cimetidine-sensitive) relaxation in the presence of mepyramine, but was less potent (10.8 +/- 3.1% of initial tension remaining at 10 mM carnosine compared with 13.4 +/- 7.5% remaining at 0.1 mM histamine). Preliminary studies with a Zn-selective fluorescent probe indicate that functionally significant levels of Zn can be released from adventitial mast cells that could modulate actions of carnosine in the extravascular space as well as those of histamine itself. We conclude that carnosine can act at the smooth muscle H(1)-receptor to provoke vasoconstriction and that it also has the potential to act at H(1)-receptors in the central nervous system. Carnosine's mode of action is virtually unique: a vascular muscle receptor apparently transduces the action of a dipeptide in the form of a metal chelate. The functional relationship of carnosine with histamine and the possible physiological relevance of Zn ions for the activity of both agents have not previously been reported.     

Amine uptake into intact mast cell granules in vitro

Histamine, the principal amine of rat peritoneal mast cells, is taken up into isolated granules with intact membranes. Uptake is pH- and concentration-dependent and is not stimulated by the addition of Mg2+-ATP. The saturable uptake has a Km of 91.1 microM and a Vmax of 95.4 pmol (mg of protein)-1 min-1. Uptake is abolished by 5 mM ammonium ion. 5-HT, the other endogenous amine of the granules, and dopamine and tyramine, which do not occur naturally in rat mast cells, each competitively inhibits [3H]-histamine uptake with Ki's close to 1 microM. Reserpine, a putative amine carrier blocker, inhibits uptake at nanomolar concentrations. At high concentrations, uptake of [3H]-5-HT is nonsaturable; at low concentrations, a saturable component is observed with a Km of 1.6 microM. Uptake of [3H]-5-HT is not enhanced by Mg2+-ATP. It is pH-dependent but with a lower apparent pKa than that of histamine. [3H]-5-HT uptake can be completely inhibited by ammonium ions. Amine inhibition of [3H]-5-HT gives nonlinear Dixon plots, and high concentrations of the competing amines or reserpine cannot completely block uptake. We propose a model consistent with these results in which amine uptake occurs by several distinct saturable transport systems. According to the model, histamine is transported by a single system, which also transports 5-HT and dopamine. 5-HT and dopamine are transported by one or more other systems.     

Adenosine activating A(2A)-receptors coupled to adenylate cyclase/cyclic AMP pathway downregulates nicotinic autoreceptor function at the rat myenteric nerve terminals

In addition to the somatodendritic region, myenteric motoneuron terminals are endowed with nicotinic autoreceptors. We aimed at investigating the effect of nicotinic receptor (nAChR) activation on [3H]-acetylcholine ([3H]-ACh) release from longitudinal muscle-myenteric plexus of the rat ileum and to evaluate whether this could be modulated by adenosine, an endogenous neuromodulator typically operating changes in intracellular cyclic AMP. The nAChR agonist, 1,1-dimethyl-4-phenylpiperazinium (DMPP, 1-30 microM, 3 min) increased [3H]-ACh release in a concentration-dependent manner. DMPP (30 microM)-induced [3H]-ACh outflow was attenuated by hexamethonium (0.1-1 mM), tubocurarine (1-5 microM), or by removing external Ca2+ (plus EGTA, 1 mM). In contrast to veratridine (0.2-10 microM)-induced [3H]-ACh release, the DMPP (30 microM)-induced outflow was resistant to tetrodotoxin (1 microM) and cadmium (0.5 mM). Pretreatment with adenosine deaminase (0.5 U/mL) or with the adenosine A(2A)-receptor antagonist, ZM 241385 (50 nM), enhanced nAChR-induced transmitter release. Activation of A(2A) receptors with CGS 21680C (3 nM) reduced the DMPP-induced release of [3H]-ACh. CGS 21680C (3 nM) inhibition was prevented by MDL 12,330A (10 microM, an adenylate cyclase inhibitor) and by H-89 (10 microM, an inhibitor of protein kinase A), but was potentiated by rolipram (300 microM, a phosphodiesterase inhibitor). DMPP-induced transmitter release was decreased by 8-bromo-cyclic AMP (1 mM, a protein kinase A activator), rolipram (300 microM), and forskolin (3 microM, an activator of adenylate cyclase). Both MDL 12,330A (10 microM) and H-89 (10 microM) facilitated DMPP-induced release of [3H]-ACh. The results indicate that nAChR-induced [3H]-ACh release is triggered by the influx of Ca2+, independent of voltage-sensitive calcium channels, presumably directly through nAChRs located on myenteric axon terminals. It was also shown that endogenous adenosine, activating A(2A) receptors coupled to the adenylate cyclase/cyclic AMP transducing system, is tonically downregulating this nAChR-mediated control of [3H]-ACh release.     

Stimulation by ATP of [3H]dopamine binding to synaptic membrane fractions of canine caudate nucleus

The rate of [³H]dopamine binding to crude synaptic membranes from canine caudate nucleus was considerably increased by 2 mM ATP, 5′-adenylylimidodiphosphate and GTP or by 1 mM 5′-guanylyl-imidodiphosphate, while strongly inhibited by 2 mM ADP and GDP. Half maximal concentrations of [³H]dopamine to bind to the membranes were 1.11 × 10^?7M and 8.75 × 10^?6M in the absence of 4 mM ATP, indicating a negative cooperativity of the dopamine receptor, and 9.25 × 10^?7 M in its presence. Hill coefficient was increased from 0.70 to 1.04 by addition of 4 mM ATP. The optimal concentration of ATP for [³H]dopamine binding was in the range of 0.5 to 5 mM.     

Depolarization Increases Chloride-Dependent Glutamate Sequestration in Synaptic Membranes of Rat Cerebral Cortex

To assess the functions of Cl- -dependent glutamate "binding" (Cl- -dependent glutamate uptake) in synaptic membranes, possible effects of depolarization on the uptake were examined. When rat cerebral cortical slices were preincubated with depolarizing agents such as veratrine (7 micrograms/ml), 10 microM aconitine, 56 mM K+, and 50 microM monensin, [3H]glutamate uptake by the crude synaptic membranes, which were subsequently prepared from the pretreated slices, was increased by 60-85%. Stimulation of the glutamate uptake by predepolarization was dependent on Na+ but not on Ca2+. The bindings of gamma-[3H]aminobutyric acid and 5-[3H]hydroxytryptamine were not significantly affected by the predepolarization. Veratrine pretreatment increased the maximal density of the glutamate uptake sites without affecting the affinity for glutamate. Several characteristics of the uptake sites increased by the veratrine pretreatment coincided with those of Cl- -dependent glutamate uptake sites. Na+-dependent glutamate binding (Na+-dependent glutamate uptake) to the membranes was not affected by pretreatment with veratrine. The content of endogenous glutamate and the noninulin space in the membrane fractions were not changed by the predepolarization. The increase in the glutamate uptake induced by pretreatment with high K+ was reversible: it returned to the control level after a second incubation of the slices in control medium. These results suggest that the Cl- -dependent glutamate sequestration system in synaptic membranes is regulated by the membrane potential.     

Involvement of Ni protein in the functional coupling of the atrial natriuretic factor (ANF) receptor to adenylate cyclase in rat lung plasma membranes

In the presence of 1 microM atrial natriuretic factor (ANF) and low (0.1 mM) Mg2+ concentrations, the initial rate of binding of [3H]guanosine 5'-[beta, gamma-imido)triphosphate [( 3H]p[NH]ppG) to rat lung plasma membranes was increased twofold to threefold. ANF-dependent stimulation of the initial rate of [3H]p[NH]ppG binding was reduced at high (5 mM) Mg2+ concentrations. Preincubation of membranes with p[NH]ppG (5 min at 37 degrees C) eliminated the ANF-dependent effect on [3H]p[NH]ppG binding whereas ANF-dependent [3H]p[NH]ppG binding was unaffected by similar pretreatment with guanosine 5'-[beta-thio]diphosphate (GDP[beta S]). An increase in ANF concentration from 10 pM to 1 microM caused a 40% decrease in forskolin-stimulated or isoproterenol-stimulated adenylate cyclase activities (IC50 5 nM) in rat lung plasma membranes. GTP (100 microM) was obligatory for the ANF-dependent inhibition of adenylate cyclase, which could be completely overcome by the presence of 100 microM GDP[beta S] or the addition of 10 mM Mn2+. Reduction of Na2+ concentration from 120 mM to 20 mM had the same effect. Pertussis toxin eliminated ANF-dependent inhibition of adenylate cyclase by catalyzing ADP-ribosylation of membrane-bound Ni protein (41-kDa alpha subunit of the inhibitory guanyl-nucleotide-binding protein of adenylate cyclase). The data support the notion that one of the ANF receptors in rat lung plasma membranes is negatively coupled to a hormone-sensitive adenylate cyclase complex via the GTP-binding Ni protein.     

Solubilization and Characterization of a [3H]Hemicholinium-3 Binding Site in Rat Brain

A sodium-dependent high-affinity [3H]-hemicholinium-3 ([3H]HCh-3) binding site was solubilized from rat striatal synaptic plasma membranes by 0.2% deoxycholate. Deoxycholate solubilization of the [3H]HCh-3 binding site was dependent upon both detergent concentration and ionic strength of the solubilization medium. Specific [3H]HCh-3 binding to the solubilized preparation was both sodium- and chloride-dependent and saturable, exhibiting an affinity of 14.2 nM and a capacity (Bmax) of 695 fmol/mg protein. Choline and other analogs inhibited specific [3H]HCh-3 binding to the solubilized preparation in a concentration-dependent manner with the similar rank order of potency observed in crude synaptic membranes. Treatments known to disrupt both protein and lipid moieties resulted in diminished specific [3H]HCh-3 binding. These results suggest that the characteristics of the solubilized [3H]HCh-3 binding site are similar to those of the membrane-bound site.