Effects of chemical sympathectomy with 6-hydroxydopamine on alpha- and beta-adrenoceptors and muscarinic cholinoceptors in rat kidney

The autonomic receptors in the rat kidney were characterized using the radioligands [3H]prazosin, [3H]clonidine, [3H]dihydroalprenolol (DHA) and [3H]quinuclidinyl benzilate (QNB). The specific binding of [3H]prazosin, [3H]clonidine, [3H]DHA and [3H]QNB to rat kidney membranes was saturable and of high affinity, and showed a pharmacological specificity as well as stereospecificity which characterized renal alpha 1-, alpha 2- and beta-adrenoceptors and muscarinic cholinoceptors, respectively. There was a relatively greater density of alpha-adrenoceptors than beta-adrenoceptors or muscarinic cholinoceptors in the rat kidney. Chemical sympathectomy of rats with 6-hydroxydopamine X HBr (6-OHDA, 50 X 2 mg/kg i.v., 24 h interval) caused a significant increase (21-56%) in the Bmax values for renal [3H]prazosin, [3H]clonidine and [3H]DHA binding at 1 and 2 weeks following the treatment, without a change in the Kd values. 6-OHDA treatment had no significant effect on the Kd and Bmax values for [3H]QNB binding at 1-3 weeks after the treatment. The norepinephrine (NE) concentration was reduced (68-76%) in the 6-OHDA-treated rat kidney. In conclusion, the present study provides biochemical evidence for the possible localization of postsynaptic alpha 1-, alpha 2- and beta-adrenoceptors and muscarinic cholinoceptors in the rat kidney and also for the regulation of these adrenoceptors by the sympathetic nervous system.     

Developmental changes in beta-adrenergic and cholinergic interactions on calcium-dependent slow action potentials in rat ventricular muscles.

1. Developmental changes in the effect of isoprenaline (Iso) and acetylcholine (ACh) interactions on Ca2(+)-dependent slow action potentials (APs) were studied in the ventricular muscles of foetal (12-20 days post-gestation), neonatal (0-20 days old), and adult (2-3 months old) rats. The slow APs were recorded at 0.2 Hz in partially depolarized preparations (an extracellular K+ concentration of 25 mM). 2. Iso (1 nM to 10 microM) began to increase the Vmax of the slow APs (an approximate indicator of Ca2+ current) on foetal day 18; its potentiating effect became greater with age and reached the adult level about 2 weeks after birth. 3. ACh (10 microM) abolished the Iso (1 microM)-induced increased in the Vmax observed in the late foetal and neonatal periods. 4. The inhibitory effect of ACh on the Vmax was antagonized by atropine but not by pirenzepine, suggesting that ACh reduces Ca2+ current (in the presence of beta-adrenoceptor agonists) by stimulating muscarinic (M2) cholinoceptors. 5. These results suggest that developmental changes in the modulatory effects of beta-adrenoceptor and cholinoceptor agonists on Ca2+ channels occur from a few days before birth to 2 weeks after birth and that the functional coupling between muscarinic cholinoceptors and Ca2+ channels has already been established when the coupling between beta-adrenoceptors and Ca2+ channels starts to operate.     

Interaction of the hexamethonium derivative W84, an allosteric effector at muscarinic cholinoreceptors, with rat brain nicotinic binding sites

The hexamethonium derivative W84 (hexamethylene-bis-[dimethyl-(3-phthalimidopropyl)-ammonium bromide]) combined with atropine has an overadditive protective action against organophosphorus intoxications. It affects allosterically the binding of (-) [3H]N-methylscopolamine [(3H]NMS) to muscarinic cholinoceptors. Because nicotinic receptors are involved in organophosphorus intoxications, the interaction of W84 with nicotinic cholinoceptors was investigated. (-) [3H]nicotine (2.5 nM) was used to label nicotinic binding sites in rat brain membranes in 50 nM Tris, pH 7.3 at 23 degrees. Under control conditions, (-) [3H]nicotine-binding revealed a KD of 4 X 10(-9) M and a Bmax of 53 fmol/mg membrane protein. W84 inhibited (- ) [3H]nicotine-binding with an IC50 of 3 X 10(-5) M by reducing the binding affinity. The IC50 of hexamethonium was 20 X 10(-5) M. At 10(-4) M, W84 did not affect the dissociation rate of (-)[3H]nicotine, suggesting a lack of allosteric activity. For sake of comparison, the action of W84 was checked on [3H]NMS-binding (control: KD approximately 1 X 10(-9) M, Bmax approximately 500 fmol/mg prot). W84 inhibited the binding of [3H]NMS (0.5 nM) with an IC50 of 1.5 X 10(-9) M. At 10(-4) M, W84 prevented [3H]NMS-dissociation almost completely, thus displaying the allosteric action at muscarinic cholinoceptors. In conclusion, the results of the (-)[3H]nicotine-binding experiments point to a pure competitive action of W84 at nicotine cholinoceptors, lacking any allosteric effect. This competitive action may contribute to the protective effect of W84 in organophosphorus poisoning.     

新抗胆碱药[3H]三环哌酯对人脑M受体的作用

用放射配体受体结合试验法,研究了新化合物三环哌酯与人大脑皮质M受体的结合特性,并与QNB作了比较。饱和实验结果显示,[³H]三环哌酯的结合参数与[³H]QNB相近,两种配体的作用均符合单位点模型。竞争性抑制实验结果表明二者作用强度相当。[³H]三环哌酯的结合和解离速率常数均较[³H]QNB大,且其与皮质M受体的解离受季铵酚的变构调节,结果提示,两种配体与M受体有一些不同的结合特性,在M受体研究中,[³H]三环哌酯可以作为[³H]QNB的补充工具。     

Characterisation of stereospecific binding sites for inositol 1,4,5-trisphosphate in airway smooth muscle.

1. A 'P2' membrane fraction of bovine tracheal smooth muscle displays high affinity (KD 3.8 +/- 0.2 nM), saturable (Bmax 1003 +/- 170 fmol mg-1 protein) and reversible binding of D-myo[3H]-inositol 1,4,5-trisphosphate ([3H]-Ins(1,4,5)P3). 2. This binding site shows strict stereo- and positional specificity for the D-Ins(1,4,5)P3 isomer with L-Ins(1,4,5)P3, DL-Ins(1,3,4,5)P4 and D-Ins(1,3,4)P3 displacing [3H]-Ins(1,4,5)P3 with Ki values of 20 microM, 0.35 microM and 2.4 microM, respectively. 3. Specific binding of [3H]-Ins(1,4,5)P3 is enhanced at alkaline pH values (maximal at pH 7.75) and, in distinct contrast to [3H]-Ins(1,4,5)P3 binding in rat cerebellum membranes, is not inhibited by Ca2+ (5-500 microM). 4. Heparin displaces [3H]-Ins(1,4,5)P3 specific binding with an IC50 of 7.6 +/- 1.0 micrograms ml-1. 5. Comparative studies demonstrated specific [3H]-Ins(1,4,5)P3 binding in bovine cardiac atrial preparations (Bmax 75 +/- 5 fmol mg-1 protein) and very low specific [3H]-Ins(1,4,5)P3 binding in bovine cardiac ventricle and skeletal muscle membranes (less than or equal to 25 fmol mg-1 protein).     

Characteristics of 5-HT3 binding sites in NG108-15, NCB-20 neuroblastoma cells and rat cerebral cortex using [3H]-quipazine and [3H]-GR65630 binding.

1. The biochemical and pharmacological properties of 5-HT3 receptors in homogenates of NG108-15 and NCB-20 neuroblastoma cells and rat cerebral cortex have been ascertained by the use of [3H]-quipazine and [3H]-GR65630 binding. 2. In NG108-15 and NCB-20 cell homogenates, [3H]-quipazine bound to a single class of high affinity (NG108-15: Kd = 6.2 +/- 1.1 nM, n = 4; NCB-20: Kd = 3.0 +/- 0.9 nM, n = 4; means +/- s.e.means) saturable (NG108-15: Bmax = 1340 +/- 220 fmol mg-1 protein; NCB-20: Bmax = 2300 +/- 200 fmol mg-1 protein) binding sites. In rat cortical homogenates, [3H]-quipazine bound to two populations of binding sites in the absence of the 5-hydroxytryptamine (5-HT) uptake inhibitor, paroxetine (Kd1 = 1.6 +/- 0.5 nM, Bmax1 = 75 +/- 14 fmol mg-1 protein; Kd2 = 500 +/- 300 nM, Bmax2 = 1840 +/- 1040 fmol mg-1 protein, n = 3), and to a single class of high affinity binding sites (Kd = 2.0 +/- 0.5 nM, n = 3; Bmax = 73 +/- 6 fmol mg-1 protein) in the presence of paroxetine. The high affinity (nanomolar) component probably represented 5-HT3 binding sites and the low affinity component represented 5-HT uptake sites. 3. [3H]-paroxetine bound with high affinity (Kd = 0.02 +/- 0.003 nM, n = 3) to a site in rat cortical homogenates in a saturable (Bmax = 323 +/- 45 fmol mg-1 protein, n = 3) and reversible manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alpha 2-adrenoceptor subtypes and imidazoline-like binding sites in the rat brain.

1. The binding of [3H]-yohimbine and [3H]-idazoxan to rat cortex and hippocampus is rapid, reversible and of high affinity. Saturation data indicate that a single population of binding sites exist for [3H]-yohimbine in the cortex (Bmax 121 +/- 10 fmol mg-1, protein; Kd 5.2 +/- 0.9 nM) and hippocampus (Bmax 72 +/- 6 fmol mg-1 protein; Kd 5.8 +/- 0.7 nM). [3H]-idazoxan labels one site in the cortex (Bmax 87 +/- 8 fmol mg-1 protein; Kd 4.1 +/- 0.9 nM) and hippocampus (Bmax 30 +/- 6 fmol mg-1 protein; Kd 3.5 +/- 0.5 nM), when 3 microM phentolamine is used to define non-specific binding. A second distinct [3H]-idazoxan binding site (Bmax 110 +/- 21 fmol mg-1 protein; Kd 3.6 +/- 0.07 nM) is identified in rat cortex if 0.3 microM cirazoline is used to define non-specific binding and 3 microM yohimbine is included to prevent binding to alpha 2-adrenoceptors. 2. Displacement studies indicate that the alpha 1-adrenoceptor antagonist prazosin and the 5-HT1 ligands 8-OH-DPAT, RU 24969 and methysergide differentiate [3H]-yohimbine binding into two components; a high and low affinity site. In contrast the displacement of [3H]-idazoxan by each ligand was monophasic. 3. The affinities of 8-OH-DPAT, RU 24969 and methysergide determined against [3H]-idazoxan binding to the cortex and hippocampus correlate significantly with the binding site displaying low affinity for prazosin and previously designated alpha 2A. In contrast, a poor correlation exists for the high affinity site for prazosin designated alpha 2B.(ABSTRACT TRUNCATED AT 250 WORDS)     

Absence of [3H]SCH 23390 binding sites in the rat adrenal gland

The binding of D2-dopamine receptor ligand [3H]spiperone and selective D1-ligand [3H]SCH 23390 to the rat adrenal gland and striatum has been compared. [3H]Spiperone showed specific binding in both tissues revealing a Bmax of 887 fmol mg-1 protein and KD of 0.38 nM, and B of 34 fmol mg-1 protein and KD of 0.66 nM in the striatum and adrenal gland, respectively. On the other hand, [3H]SCH 23390 showed a specific binding to the striatal tissue with Bmax of 747 fmol mg-1 protein and KD of 0.70 nM, while in the adrenal tissue no specific binding was observed. These results apparently indicated only D2-dopamine receptor binding sites being present in the rat adrenal gland.     

Equipotent allosteric effect of W84 on [3H]NMS-binding to cardiac muscarinic receptors from guinea-pig, rat, and pig.

W84 (hexamethylene-bis-[dimethyl-(phthalimidopropyl)-ammonium bromide]) is an experimental antidote against organophosphorus poisoning and has been found to affect muscarinic cholinoceptors allosterically. The attempt was made to test whether the W84-action on muscarinic cholinoceptors depends on the species. For this purpose, the effect of W84 on the binding of [3H]N-methylscopolamine ([3H]NMS) was investigated in membrane-suspensions from the hearts of guinea pigs, rats, and pigs in 3 mM MgHPO4, 50 mM Tris, pH 7.3, at 23 degrees. W84 inhibited [3H]NMS-binding in the three membrane suspensions with similar potency (half-inhibitory concentration IC50: 2-5 microM). To evaluate the allosteric activity of W84, its effect on the dissociation of [3H]NMS was determined. At 3 microM, W84 diminished the rate of [3H]NMS-dissociation to about 20% of the control in three suspensions. At 100 microM of W84, [3H]NMS-dissociation was almost prevented. In conclusion, W84 acted equally on the cardiac cholinoceptors of guinea pigs, rats, and pigs, respectively. It can be anticipated that M2-cholinoceptors of other species would likewise be affected by W84.     

Regulation by chronic drug administration of neuronal and cardiac calcium channel, beta-adrenoceptor and muscarinic receptor levels

Chronic administration of atropine (40-100 mg/kg, 23 days) produced a 29-33% increase in muscarinic receptors, measured by [3H]quinuclidinyl benzilate binding, in rat brain. Diisopropyl phosphorofluoridate (0.9 mg/kg, 14 days) produced a 35% decrease in muscarinic receptors. Propranolol administration (800 micrograms/kg/hr, 10 days) increased beta-adrenoceptors, measured by [3H]dihydroalprenolol binding, by 69 and 50% in brain and heart respectively. Isoproterenol administration (800 micrograms/kg/hr, 10 days) produced a 50% reduction in cardiac beta-adrenoceptors but did not alter brain receptors. These drug treatments were without effect on binding of the Ca2+ channel ligands, [3H]nimodipine and [3H]nitrendipine, to brain or heart respectively. However, chronic administration of nifedipine for 20 days (36 and 360 micrograms/kg/hr) did produce down-regulation of both cardiac and neuronal Ca2+ channels and a similar down-regulation of beta-adrenoceptors. Co-regulation of Ca2+ channels and neurotransmitter receptors may occur but may not be an automatic consequence of either receptor or channel regulation.     

Characterization of the thromboxane (TP-) receptor subtype involved in proliferation in cultured vascular smooth muscle cells of rat.

1. The effects of the thromboxane A2 (TxA2)-mimetic, U-46619, on the proliferation of vascular smooth muscle cells (VSMCs) were examined in a clonal smooth muscle cell line, A10, which was derived from foetal rat aorta. 2. [3H]-U-46619 bound to A10 cells of passages 18-20 (p18-20) with two classes of sites. The high affinity site showed a Bmax of 3.0 +/- 1.8 fmol mg-1 protein with a KD value 1.0 +/- 0.1 nM, while the low affinity site showed a Bmax of 43.0 +/- 6.0 fmol mg-1 protein and KD value of 129.0 +/- 7.9 nM. However, [3H]-U-46619 bound to A10 cells from passages 28-30 (p28-30) at a single class of site with a Bmax 111.0 +/- 9.0 fmol mg-1 protein and a KD value of 175.4 +/- 22.0 nM. 3. Cinnamophilin and SQ29548 inhibited specific [3H]-U-46619 binding to p18-20 A10 cells in a concentration-dependent manner with Ki values of 390.0 +/- 3.2 and 4.6 +/- 1.0 nM, respectively at a high affinity site, and 2.6 +/- 0.2 microM and 310.0 +/- 6.4 nM, respectively at the low affinity site. 4. U-46619 produced isometric contractions of rat aorta in a concentration-dependent manner with an EC50 7.0 +/- 1.2 nM. Cinnamophilin and SQ29548 antagonized U-46619-induced aortic contractions with pA2 values 6.3 +/- 0.1 and 8.2 +/- 0.2, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in [3H]-PK 11195 and [3H]-8-OH-DPAT binding following forebrain ischaemia in the gerbil.

1. A high density of [3H]-PK 11195 binding sites was present in gerbil cortical membranes (Bmax [3H]-PK 11195 1360 +/- 71 fmol mg-1 protein) in comparison to rat cortical membranes (254 +/- 21 fmol mg-1 protein). This effect was species-specific as similar findings were obtained with hippocampal membranes (Bmax 1430 +/- 111 fmol mg-1 protein in gerbil, compared to 196 +/- 31 in rat). 2. RO 5-4864, also a peripheral type benzodiazepine compound, displayed low affinity for the [3H]-PK 11195 site in the gerbil (pKi 6.57 +/- 0.02 and 6.70 +/- 0.12 in hippocampus and cortex respectively) compared to rat (pKi 8.16 +/- 0.07 and 8.48 +/- 0.02). Central benzodiazepine compounds, diazepam and flunitrazepam, also displayed this trend. 3. RO 5-4864 displaced [3H]-PK 11195 binding from gerbil and rat cortical membranes through a competitive interaction with Hill slopes close to unity. In both tissues, saturation isotherms of [3H]-PK 11195 binding indicated that the presence of RO 5-4864 caused changes in Kd without any effect on Bmax. In kinetic experiments, the presence of RO 5-4864 failed to modify the rate of dissociation of [3H]-PK 11195 from equilibrium in both rat and gerbil cortical membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship between beta-adrenoceptors and calcium channels in human ventricular myocardium.

The stoichiometric relationship between adrenoceptors and saturable binding sites for 1,4-dihydropyridines in calcium channels was investigated in human ventricular myocardium. Membrane particles were prepared from heart specimens of patients undergoing open heart surgery. The patients suffered from hypertrophic obstructive cardiomyopathy (HOCM) or mitral valve disease. Using [3H]-prazosin and [125I]-2-beta-hydroxy-3-iodiphenyl-ethyl-aminoethyl tetralone ([125I]-HEAT) as labels we detected only a marginal density of alpha 1-adrenoceptors, regardless of disease. No alpha 2-adrenoceptors were detected with [3H]-rauwolscine. In HOCM patients we estimated 72 +/- 10 fmol mg-1 (n = 12) beta-adrenoceptors labelled with [3H]-(-)-dihydroalprenolol and 74 +/- 5 fmol mg-1 (n = 2) beta-adrenoceptors labelled with [125I]-(-)-iodocyanopindolol; the equilibrium dissociation constants KD, were 1.2 +/- 0.2 nmol l-1 for [3H]-(-)-dihydroalprenolol and 7 +/- 1 pmol l-1 for [125I]-(-)-iodocyanopindolol. In patients with mitral valve disease we estimated 84 +/- 11 fmol mg-1 (n = 3) labelled with [3H]-(-)-dihydroalprenolol and 66 +/- 13 fmol mg-1 (n = 2) labelled with [125I]-(-)-iodocyanopindolol. The KD values were 1.8 +/- 0.6 nmol l-1 for [3H]-(-)-dihydroalprenolol and 8 +/- 2 pmol l-1 for [125I]-(-)-iodocyanopindolol. In 14 HOCM patients we estimated 107 +/- 12 fmol mg-1 calcium channel sites labelled with [3H]-nimodipine with a KD of 280 +/- 4 pmol l-1. In 5 patients with mitral valve disease the density of calcium channel sites labelled with [3H]-nimodipine was 78 +/- 5 fmol mg-1 with a KD of 290 +/- 20 pmol l-1, In HOCM patients the density of calcium channel sites labelled with the benzoxadiazol 1,4-dihydropyridine ([3H]-(+)-PN 200-110) was 1.6 fold of that labelled with [3H]-nimodipine with a KD of 84 +/- 11 pmol l-1. In a group of 4 HOCM patients in which calcium channels were labelled with [125I]-iodipine, the density of sites was 1.37 +/- 0.07 fold the density of sites labelled by [3H]-(+)-PN 200-11-. The KD value of [125I]-iodipine was 246 +/- 16 pmol-1. (+)-PN 200-110 was approximately 100 fold more potent than (-)-PN 200-110 as a competitor of [125I]-iodipine binding. For the HOCM group a significant correlation was found between beta-adrenoceptor density and calcium channel density, whereas in the mitral valve group no such correlation was found.(ABSTRACT TRUNCATED AT 400 WORDS)     

Quantification of in vivo spiperone binding in the rat striatum after lesions produced by kainate or decortication

The potential of in vivo spiperone binding as a tool for the detection and quantitative analysis of striatal dopamine (DA) receptor alterations was studied in rat brain lesioned in several ways. Two weeks after kainate (KA) injection a significantly higher radioactivity accumulation was observed in the lesioned striatum than in the contralateral structure after a tracer dose of [3H]spiperone. The difference was maximal 2 days after surgery and it was present for at least 4 weeks while it was reversed 11 weeks after KA injection. The radioactivity uptake (tracer dose of [3H]spiperone) measured 2 weeks after surgery could be specifically prevented in both KA-lesioned and contralateral striatum by haloperidol and N-n-propylnorapomorphine while non-dopaminergic drugs were almost without effect. More than 80% of the radioactivity accumulation was saturable in both contralateral (unlesioned) and KA-lesioned striatum, leaving a slightly higher non-saturable radioactivity level in the latter. One week after unilateral ablation of the cerebral cortex overlying the striatum only minor bilateral differences in striatal radioactivity content were found after a tracer dose of [3H]spiperone. No differences were present after 6-OHDA lesion of the nigrostriatal pathway. Striatal DA receptor densities (Bmax) were determined from the dose-dependency of total striatal spiperone accumulation. This relationship was assessed using cerebellar spiperone accumulation instead of dose. Thus a Bmax of about 75 fmol X mg-1 tissue was found in the striatum of control (unoperated) rats and contralateral to the striatal KA lesion while 2 weeks after surgery it was approximately 33 fmol X mg-1 in the KA-lesioned striatum. One week after unilateral decortication Bmax values of about 50 and 65 fmol X mg-1 were found ipsi- and contralaterally to the lesion respectively.     

AUTORADIOGRAPHIC LOCALIZATION OF RECEPTORS IN THE MAMMALIAN CARDIOVASCULAR SYSTEM

1. Autoradiographic techniques have been used to examine the location of beta-adrenoceptors in the heart and beta-adrenoceptors, substance P receptors and muscarinic cholinoceptors in blood vessels. 2. Both beta 1-adrenoceptors and beta 2-adrenoceptors were present in guinea-pig and human heart, on the myocardium and associated with the cardiac nerves and blood vessels. 3. Nerves on the vasculature and vascular smooth muscle contained beta-adrenoceptors and muscarinic cholinoceptors. 4. Receptors for substance P and beta-adrenoceptors, but not muscarinic cholinoceptors were present on endothelial cells.     

Molecular characterization of muscarinic receptor subtypes in bovine cerebral cortex by radiation inactivation and molecular exclusion h.p.l.c.

Muscarinic receptor subtypes in bovine cerebral cortex were investigated by means of radiation inactivation and molecular exclusion high performance liquid chromatography (h.p.l.c.). The functional molecular size of the muscarinic receptor in situ was determined by the radiation inactivation method. The value for the muscarinic receptor labelled with [3H]-quinuclidinylbenzilate ([3H]-QNB) was 91,000 daltons, while that labelled with [3H]-pirenzepine [( 3H]-PZ) was 157,000 daltons. The muscarinic receptor solubilized with digitonin could be labelled with [3H]-PZ as well as with [3H]-QNB. 3-[(3-Cholamidopropyl)-dimethylammonio] - propane sulphonate (CHAPS) solubilized the muscarinic receptor labelled with [3H]-QNB but not that labelled with [3H]-PZ, in agreement with the low affinity of pirenzepine for inhibiting [3H]-QNB binding in CHAPS-solubilized preparations. The size of the muscarinic receptor in solution was estimated by molecular exclusion h.p.l.c. The digitonin-solubilized muscarinic receptor had a molecular weight of 290,000 and the [3H]-QNB and [3H]-PZ binding activities behaved identically. The CHAPS-solubilized muscarinic receptor labelled with [3H]-QNB was apparently of high molecular weight (greater than 1,000,000 Mr), indicating the formation of aggregates and/or micelles. In the presence of digitonin this form was dissociated into a lower molecular weight species (580,000 Mr). These data indicate that the ligand binding component of the muscarinic receptor species labelled by both [3H]-QNB and [3H]-PZ exists on the same receptor protein, but that the [3H]-PZ binding component in situ is probably coupled to other components in the membrane.     

Characterization of muscarinic receptors in human submandibular salivary glands

We studied the binding of the muscarinic antagonist [3H]N-methyl-scopolamine [( 3H]NMS) in order to characterize muscarinic receptors located in human submandibular salivary glands obtained from intrasurgical biopsy. [3H]NMS bound with a Kd value of 1.56 nM to a single class of muscarinic receptors (Bmax 37.3 fmol/mg protein) since pirenzepine exhibited a homogeneous binding profile.     

Separate [3H]-nitrendipine binding sites in mitochondria and plasma membranes of bovine adrenal medulla.

1. Two binding sites for the 1,4-dihydropyridine (DHP) derivative [3H]-nitrendipine have been found in the bovine adrenal medulla. The high-affinity site (Kd = 0.48 nM and Bmax = 128 fmol mg-1 protein) was specifically located in purified plasma membranes. The low-affinity site (Kd = 252 nM and Bmax = 169 pmol mg-1 protein) was located only in mitochondria. Chromaffin granule membranes lacked specific binding sites for [3H]-nitrendipine. 2. Kinetic analysis of the rates of association and dissociation of [3H]-nitrendipine, saturation isotherms and displacement experiments with unlabelled nitrendipine and PN200-110 revealed single, homogeneous populations of high- and low-affinity sites in plasma and mitochondrial membranes, respectively. 3. The high affinity site was sensitive to Ca2+ deprivation and heating; it was practically unaffected by changes in ionic strength of the medium and its optimal pH was slightly alkaline. This site exhibited a strong DHP stereoselectivity; diltiazem increased and verapamil decreased the affinity of [3H]-nitrendipine. 4. In contrast, binding of [3H]-nitrendipine to the low affinity site was more heat resistant and less affected by Ca2+ removal. Its optimal pH was slightly acid and the increase in ionic strength enhanced the number of available sites. The site had no DHP stereoselectivity. Verapamil decreased the dissociation constant of [3H]-nitrendipine acting in a non-competitive manner; diltiazem did not affect equilibrium binding parameters of [3H]-nitrendipine. 5. These results suggest that both biding sites reflect different receptor entities. The high-affinity binding site corresponds to the dihydropyridine receptor associated with the L-type calcium channel. The function of the mitochondrial, low-affinity binding site is, at present, unknown.     

Testing the specificity of allosteric modulators of muscarinic receptors in phylogenetically closely related histamine H1-receptors

Gallamine, alcuronium and W84 (hexane-1,6-bis[dimethyl-3'-phthalimidopropyl-ammonium bromide]) are prototype allosteric modulators of the G-protein coupled muscarinic acetylcholine receptor family, especially of the M2-subtype. In order to probe the specificity of muscarinic allosteric modulation, we checked whether these agents interact with histamine H1-receptors which have a high homology with muscarinic receptors. Binding experiments (38 mM Na2HPO4, 12 mM KH2PO4, pH 7.5) were performed with the H1-receptor antagonist [3H]mepyramine ([3H]MEP) in guinea pig cerebellar homogenates. For the sake of comparison, binding of [3H]N-methylscopolamine ([3H]NMS) at muscarinic M2-receptors was measured in porcine cardiac homogenates under identical conditions. The modulators retarded [3H]NMS dissociation (t1/2 control=1.3 min) concentration-dependently indicating their allosteric action with half-maximum effects for gallamine at EC50,discs=27 microM, for alcuronium at EC50,diss=53 nM, and for W84 at EC50,diss=170 nM. In contrast, [3H]MEP dissociation from H1-receptors (t1/2,control=2.6 min) remained unchanged up to concentrations of 1 mM of the modulators. Equilibrium binding of [3H]NMS (KD=0.46 nM, Bmax=98 fmol/mg protein) was inhibited by gallamine, elevated by alcuronium and left almost unchanged by W84, indicating negative, positive and nearly neutral cooperativity, respectively, with the radioligand. The ternary complex model of allosteric actions yielded the equilibrium dissociation constants K(A) for the binding of the allosteric modulators to free M2-receptors: K(A,gallamine)=100 nM, K(A,alcuronium)=450 nM, K(A,W84)=69 nM. In H1-receptors, more than 1,000-fold higher concentrations than in M2-receptors were required to elicit an effect on the binding of [3H]MEP (KD=1.2 nM, Bmax=205 fmol/mg protein). Half-maximal reduction was observed at 10 mM for gallamine, 1 mM for alcuronium and 92 microM for W84. In conclusion, the muscarinic modulators have little effect on the histamine H1-receptors.     

The effect of oxygen free radicals on calcium current and dihydropyridine binding sites in guinea-pig ventricular myocytes.

1. We used electrophysiological and binding techniques to determine the effects of oxygen free radicals (OFRs) generated by dihydroxyfumaric acid (DHF, 5 mM) on calcium current and dihydropyridine binding sites in guinea-pig isolated ventricular myocytes. 2. Binding of [3H]-PN200-110 to isolated ventricular myocytes revealed one population of binding sites with a KD of 0.11 +/- 0.01 nM and Bmax of 139.1 +/- 6.9 fmol mg-1 protein (n = 24). After 15 min of exposure to DHF, the density, but not the affinity of [3H]-PN200-110 binding sites was significantly (P < 0.01) reduced to 35% of the control value (Bmax = 49.4 +/- 3.7 fmol mg-1 protein, KD = 0.11 +/- 0.01 nM, n = 15). In the presence of superoxide dismutase (SOD) and catalase (CAT) the reduction in [3H]-PN200-110 binding sites was almost completely prevented (Bmax = 120.5 +/- 7.4 in control, n = 4 and 98.8 +/- 7.4 fmol mg-1 protein in DHF plus SOD and CAT, n = 4). KD values were not modified (0.08 +/- 0.01 in control and 0.09 +/- 0.01 nM in DHF plus SOD and CAT). 3. The time-course of the reduction of [3H]-PN200-110 binding sites by OFRs was paralleled by the decrease in L-type calcium current (Ica,L) measured in patch-clamped guinea-pig ventricular myocytes either in the absence or in the presence of EGTA in the patch pipette. In the former conditions OFRs induced the appearance of calcium-dependent alterations, i.e. the transient inward current, within 10 min. After 30 min of incubation with DHF, [3H]-PN200-110 binding sites were reduced to 25% of the control value. 4. In myocytes incubated with the antilipoperoxidant agent, butylated hydroxytoluene (BHT, 50 microM), the decrease in [3H]-PN200-110 binding sites caused by DHF was partially prevented (Bmax values after 30 min exposure to DHF were 55.5 +/- 1.9 and 23.7 +/- 5.9 fmol mg-1 protein in the presence and in the absence of BHT respectively, P < 0.05). BHT did not affect the decrease in [3H]-PN200-110 binding sites during the first 15 min of exposure to DHF, but was able to prevent completely the further decrease occurring during the following 15 min of incubation with OFRs. 5. Our results demonstrate that the OFR-induced decrease in calcium current is associated with a reduction in DHP binding sites. The decrease in calcium current and in calcium channels may be implicated in the mechanical dysfunction associated with oxidative stress.