Autoradiographic study of peripheral benzodiazepine receptors in animal brain tumor models and human gliomas.

In vitro binding of [3H]PK-11195 (1-(2-chlorophenyl)-N-methyl-(1- methylpropyl)-3-isoquinoline carboxamide) in rodent AA ascites and C6 glioma as well as in human gliomas was investigated. The Bmax (mean +/- S.D.) of AA ascites tumor and C6 glioma is 1.39 +/- 0.15 pmol/mg tissue and 4.50 +/- 0.76 pmol/mg tissue, respectively. This Bmax is 9 and 30 times, respectively, higher than the one found in the rat cortex (0.15 +/- 0.03 pmol/mg tissue). A Bmax of 1.26 +/- 0.24 pmol/mg tissue and 0.64 +/- 0.08 pmol/mg tissue was found in human malignant and low grade gliomas respectively. This Bmax value should be compared to 0.35 +/- 0.04 pmol/mg tissue found in the normal human cortex. There are significant (P less than 0.05) differences between Bmax in tumors and normal cortex. There was no significant difference in KD between the malignant and low grade gliomas. C6 glioma has a KD significantly greater than rat cortex. In some cases of human low grade gliomas, kinetic measurements suggested the presence of two affinity receptor sites. However, at this time, heterogeneity of the tissue cannot be excluded as being at least in part a source of this.     

Effects and specific binding sites of endothelin in human lung preparations

Endothelin binding in human isolated lung membrane fractions revealed a single class of high affinity recognition sites with a K_d of 1.33±0.15 nM and a B_max of 9.61±1.44 pmol/mg protein. Endothelin inhibited [¹²⁵I]endothelin binding with a K_i of 1.90±0.15 nM whereas structurally unrelated compounds had no effect. Endothelin was a potent contractile agonist on human isolated pulmonary arterial (HPA) and venous (HPV) muscle preparations (pD₂ values: 9.64 and 10.36, respectively). Neither indomethacin (1 μM), nicardipine (0.01, 0.10, 1.0 μM) nor diltiazem (1, 10, 100 μM) altered the sensitivity of HPA to endothelin. Human isolated bronchial muscle preparations were less sensitive to endothelin than vascular tissues. These data suggest that pulmonary veins may be a major target for this constrictory peptide in the human lung.     

Methylation of captopril in human liver,kidney and intestine

1. The methylation of captopril was studied in the microsomal fraction from 20 human liver, 12 kidney, and 14 intestinal rnucosa specimens.

2. The hepatic methyltransferase activity (mean ± SD) was 477 ± 204 pmol/min per mg. Renal and intestinal methyltransferase activities were 3 and 8 times lower, respectively, than hepatic activity.

3. The kinetics of methyltransferase with captopril as substrate were studied in four specimens of liver, kidney and intestine. The maximum velocities of reaction (mean ± SD; pmol/min per?mg) were 697 ± 219 (liver), 456 ± 120 (renal cortex), 264 ± 77 (renal medulla) and 101 ± 28 (ileum mucosa). K_m values (mean ± SD; mM) were 5.2±2.3 (liver) 4.3±1.7 (renal cortex) 4.1±1.5 (renal medulla) and 5.3 ± 2.0 mM (ileum mucosa). V_max is subjected to a marked tissue dependence whereas K_m is similar in all tissues.

4. Liver is the primary site of captopril methylation whereas the intestine plays only a minor role. Kidney may contribute substantially to the hepatic methylation of captopril.     

Identification of multiple nitrobenzylthioinosine binding sites in guinea pig platelets: Comparison with binding in guinea pig erythrocytes

The novel existence of multiple binding sites for the potent nucleoside transporter Probe, [³H]nitrobenzylthioinosine, was identified in guinea pig platelet membranes and the binding characteristics compared to those of guinea pig erythrocyte membranes. Scatchard analysis of the binding in platelets reveled two high affinity binding sites with affinity constant (K_D) of 0.94 ± 0.07 nM and 83 ± 13 nM with corresponding maximal binding capacities (B_max) of 21 ± 7 and 110 ± 25 fmol/mg protein, respectively. In comparison, guinea pig erythrocyte membranes revealed a homogeneous population of the binding sites with K_D of 0.17 ± 0.04 nM and a B_max value of 73 ± 11 fmol/mg protein. Biphasic semi-log plots of the binding site heterogeneity in erythrocytes not reveled by Scatchard plots. Determination of the potencies of selected drugs in inhibiting the binding showed evidence of differential interacitons with the binding sites by various agents which may be exploited pharmacologically. © 1993 Wiley-Liss, Inc.     

Stereoselective binding of niguldipine enantiomers to α1A-adrenoceptors labeled with [3H]5-methyl-urapidil

[³H]5-Methyl-urapidil, a potent antihypertensive derivative of urapidil, binds to α_1A-adrenoceptors in rat brain cortex membranes with a dissociation constant (K_D) of 0.89 nM and a B_max of 116 fmol/mg protein. The ligand does not bind to purified liver cell membranes (α_1B-adrenoceptors. [³H]5-Methyl-urapidil also labels 5-HT_1A receptors in brain membranes (K_D: 0.84 nM and B_max: 235 fmol/mg protein). (±)-Niguldipine, a novel 1,4-dihydropyridine with Ca²⁺-antagonistic as well as α_1A-adrenoceptor blocking properties, is a competitive inhibitor of [³H]5-methyl-urapidil binding to α_1A-adrenoceptors. In contrast to those for prazosin, the K_i values for niguldipine were highly dependent on the membrane protein concentration, indicating partitioning of niguldipine into hydrophobic compartments unavailable for α-adrenoceptor interaction. The extrapolated, ‘true’ K_i values were as follows: (±)-niguldipine: 0.298 nM, (−)-niguldipine: 3.12 nM, (+)-niguldipine: 0.145 nM.     

Serotonin-binding proteins in the bovine cerebral cortex: interaction with serotonin and catecholamines

The soluble serotonin-binding proteins (SBP) present in bovine frontal cortex are very similar to those reported in rat brain. Binding of [³H]serotonin to SBP, present in ammonium sulphate-precipitated proteins from bovine cortex, requires Fe²⁺ but not Fe³⁺. In the presence of an optimal concentration of Fe²⁺ (0.1 mM), bovine SBP behave as a single class of non-cooperative sites for [³H]serotonin binding (B_max = 120 ± 12 pmol/mg protein, K_D = 0.12 ± 0.04 μM, n = 3). Binding of [³H]serotonin is decreased by nucleotides and by reagents which modify sulfhydryl groups and reduce disulfide bonds and by metal ion chelators. Serotonin analogs possessing an hydroxyl group on the indole ring and catecholamine analogs possessing an intact catechol moiety are effective competitors (K_i from 0.1 to 0.3 μM). In both cases, the aliphatic amino group does not contribute to the binding, but the affinity is strongly decreased if aromatic hydroxyl groups are methoxylated. Catecholamine-SBP interactions can also be demonstrated directly by binding experiments. Binding of [³H]dopamine is greatly enhanced by Fe²⁺, Cu²⁺ and Mn²⁺, but not by Fe³⁺. The Fe²⁺-dependent binding component of [³H]dopamine is saturable (B_max = 279± 64 pmol/mg protein, K_D = 0.19 ± 0.02 μM, n = 3), and possesses the same physicochemical properties as SBP: it elutes immediately after the void volume on a Sephacryl S100 HR (1.6 × 140 cm) gel filtration column (reflecting aggregation) and it migrates with an apparent molecular weight of 57–58 kDa on native polyacrylamide gel electrophroresis. Whereas the serotonin-storing role of SBP in serotonergic neurons has already been well documented, the present data advocate that these proteins may also possess catecholamine-storing properties.     

HIGH-LEVEL EXPRESSION OF RAT D1A DOPAMINE RECEPTOR cDNA IN MOUSE FIBROBLAST LTK- CELLS BY n-BUTYRATE

1. In order to develop a simple, efficient system for the high-level expression of dopamine receptors in eukaryotic cells, we have studied the effects of n-butyrate on the expression of rat D_1A dopamine receptor cDNA in mouse fibroblast LTK^- cells as compared with those of n-butyrate on endogenous D₁ receptor levels in opossum kidney cells. 2. In the transfected LTK^- cell membranes with pRc/CMV-D_1A receptor cDNA, a selective D₁ dopamine antagonist, [³H]-SCH 23390, exhibited a K₄ of 0.9 ± 0.1 nmol/L and a B_max of 0.35 ± 0.05 pmol/mg protein (n= 5). 3. Addition of n-butyrate (2–10 mmol/L) to the culture medium for 48 h dose-dependently increased the D_1A receptor level up to 1.5 ± 0.3 pmol/mg protein (n= 7), although the K₄ values were not affected. The increase in receptor level was accompanied by an elevation of selective D₁ agonist-induced adenylyl cyclase activity. 4. In contrast, n-butyrate treatment (2–10 mmol/L) did not affect either endogenous D₁ receptor levels or fendoldopam-induced adenylyl cyclase activity in opossum kidney cells. 5. These results suggest n-butyrate is a useful tool for obtaining high-level expression of D_1A dopamine receptor cDNA in mouse fibroblast LTK^- cells.     

The role of human cytochrome P450 enzymes in metabolism of acrylamide in vitro

Abstract

Objective: Acrylamide (AA), a probable human carcinogen, is present in fried and baked starch-rich food. In vivo, the substance is partly biotransformed to glycidamide (GA), which may account for carcinogenic effects. Existing data suggest an important but not exclusive contribution of CYP2E1 to GA formation. The aim of this project was to derive respective enzyme kinetic parameters for CYP2E1 and to assess a possible role of other important human CYPs for this reaction in vitro.

Methods: AA (0.2–20?mM) was incubated with human liver microsomes (HLM) and human cytochrome P450 enzymes (supersomes?). GA was quantified by a specific LC-MS/MS method. Enzyme kinetic parameters were estimated assuming a single binding site. Furthermore, inhibition experiments were performed with diethyldithiocarbamate (DDC), a potent inhibitor of CYP2E1.

Results: The mean?±?SD maximum formation rate (V_max) and Michaelis–Menten constant (K_m) for GA formation in HLM were 199?±?36?pmol GA/mg protein/min and 3.3?±?0.5?mM, respectively. In AA incubations with supersomes?, only for CYP2E1 measurable GA formation was detected in all tested AA concentrations (V_max and K_m were 5.4?nmol GA/nmol CYP2E1/min and 1.3?mM, respectively). Inhibition constant (IC₅₀) of DDC was 3.1?±?0.5?µM for HLM and 1.2?±?0.2?µM for CYP2E1 supersomes?. Therefore, relevant participation of CYPs other than CYP2E1 in the metabolism of AA to GA in humans does not seem likely.

Conclusion: Our results confirm the major role of CYP2E1 in GA formation from AA, albeit with low affinity and low capacity. Further studies are needed to identify other pathways of GA formation.     

COMPARATIVE STUDY ON α1-ADRENOCEPTOR ANTAGONIST BINDING IN HUMAN PROSTATE AND AORTA

1. Specific binding of [³H]-prazosin in prostatic and aortic membranes of humans was saturable and of high affinity (prostate: apparent dissociation constant, K_d= 0.35 ± 0.03 nmol/L; aorta: K_d= 0.26 ± 0.03 nmol/L). The density of [³H]-prazosin binding sites (B_max) for prostate and aorta was 546 ± 31 and 61.6 ± 1.6 fmol/mg protein, respectively. 2. Prazosin, YM617, naftopidil and urapidil competed with [³H]-prazosin for the binding sites in a dose-dependent manner in the prostate and aorta of humans. The binding affinities of these antagonists in both tissues were compared, based on the inhibition constant, K_i. Both prazosin and urapidil showed similar affinity to [³H]-prazosin binding sites in human tissue, whereas YM617 and naftopidil showed approximately a 12 and two times higher affinity, respectively, to α₁-adrenoceptor sites of prostate than aorta. 3. The chloroethylclonidine treatment reduced partially the B_max values for specific [³H]-prazosin binding in the prostate and aorta of humans with little effect on the K_d values. 4. These data suggest that YM617 is a relatively selective antagonist of human prostatic α₁-adrenoceptors.     

Activation of recombinant h 5-HT1B and h 5-HT1D receptors stably expressed in C6 glioma cells produces increases in Ca2+-dependent K+ current

The putative coupling between stably expressed recombinant h 5-HT_1B or h 5-HT_1D receptors and K⁺ channels which regulate excitability was investigated in C6 glioma cells. Outward K⁺ currents (I _K) were examined in non-transfected C6 glioma cells and in cells expressing cloned h 5-HT_1B or h 5-HT_1D receptors using the patch-clamp technique in the whole-cell configuration. I _K was elicited by a depolarizing step from a holding potential of –60 mV. In C6 glioma cells expressing either recombinant h 5-HT_1B or h 5-HT_1D receptors, sumatriptan similarly increased I _K in a concentration-dependent manner (maximum increase 19.4±7.2%, n=8, P<0.05 and 25.1±3.9%, n=6, P<0.001, respectively) with EC₅₀ values (geometric mean with 95% confidence intervals in parentheses) of 56.3 nM (7.9–140 nM) and 68.7 nM (16–120 nM), respectively. Sumatriptan failed to elicit increases in I _K in non-transfected cells, confirming a specific involvement of the respective membrane h 5-HT_1B and h 5-HT_1D receptors in transfected C6 cells. In the presence of the mixed 5-HT_1B/D receptor antagonist GR 127935 (0.1 μM), sumatriptan (1 μM) failed to significantly increase I _K in C6 cells expressing h 5-HT_1B receptors (–7.5±3.5%, P=NS, n=6), although a higher concentration of GR 127935 (1 μM) was required to significantly inhibit sumatriptan-evoked increases in I _K in C6 cells expressing h 5-HT_1D receptors (–1.8±3.5%, P=NS, n=6), confirming that sumatriptan-evoked responses were indeed mediated by h 5-HT_1B and h 5-HT_1D receptors, respectively. In C6 cells expressing either cloned h 5-HT_1B or h 5-HT_1D receptors, sumatriptan-induced increases in I _K were prevented by the calcium chelator EGTA (5 mM) when included in the patch pipette (maximum increase 0.57±0.6%, n=3, P=NS and –2.8±1.6%, n=5, P=NS, respectively). In C6 cells expressing cloned h 5-HT_1B receptors, sumatriptan (1 μM) similarly failed to significantly increase I _K in the presence of dibutyryl cAMP (10 μM) or when a nominally Ca²⁺-free medium was included in the patch pipette (–19.4±5.1%, n=5 and –5.2±4.3%, n=5, respectively, P=NS in each case). In addition, the Ca²⁺-dependent K⁺ channel blockers iberiotoxin (0.1 μM) and tetraethylammonium (TEA, 1 mM) abolished sumatriptan-induced increases in I _K (–0.5±1.0%, n=4 and –3.9±3.1%, n=4, respectively, P=NS in each case) in C6 cells expressing h 5-HT_1B receptors, confirming the involvement of Ca²⁺-dependent K⁺ channels. In C6 cells expressing cloned h 5-HT_1B receptors, sumatriptan (1 μM) similarly failed to significantly increase I _k after 30-min incubation with thapsigargin (1 μM) or when heparin (2 mg/ml) was included in the patch pipette (1.1±0.4%, n=5 and 1.2±2.4%, n=5, respectively, P=NS). In conclusion, evidence is provided that both recombinant h 5-HT_1B and h 5-HT_1D receptors stably transfected in C6 glioma cells are positively coupled to Ca²⁺-dependent K⁺ channels, and the outward hyperpolarizing current mediated by these channels is dependent upon IP₃ receptor-mediated intracellular Ca²⁺ release. Received: 15 April 1998 / Accepted: 9 September 1998     

Human brain imidazoline receptors: further characterization with [3H]clonidine

The aim of the present study was to further characterize [³H]clonidine binding in the ventrolateral medulla of the human brainstem, the region involved in the vasodepressor effect of imidazoline drugs of the clonidine type. Under basal conditions, [³H]clonidine can bind both to the imidazoline receptors and to the α-adrenoceptors. The latter represent only a small part of the total [³H]clonidine binding with a B_max of 61 ± 13 fmol/mg proteins and a K_D of 4.9 ± 2.2 nM. Most of the binding was associated with imidazoline receptors with a K_D of 67 ± 13 nM and a B_max of 677 ± 136 fmol/mg protein. α-Adrenoceptor binding of [³H]clonidine could be completely prevented when membranes were either treated during preparation with the aIkylating agent phenoxybenzamine or incubated in the presence of 30 μM (−)-noradrenaline or in the presence of the non-hydrolysable analogue of GTP, guanylyl imidodiphosphate (Gpp(NH)p). When the α-adrenoceptors binding was prevented, we demonstrated the insensitivity of [³H]clonidine binding to Gpp(NH)p and showed that the competition between clonidine and idazoxan for imidazoline receptors was insensitive to Gpp(NH)p suggesting that imidazoline receptors are not G protein coupled receptors. The specificity of [³H]cloniding binding to imidazoline receptors in the human ventrolateral medulla indicates that these receptors are different from imidazole receptors as defined with p-aminoclonidine in the bovine brainstem.     

Pharmacological characterization and autoradiographic localization of a putative dopamine D4 receptor in the heart

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Acute ethanol intoxication may not alter α2-adrenoceptors in the human brain

The specific binding of the ₂-adrenoceptor agonists [³H]clonidine and [³H]bromoxidine (UK 14304) was measured in the postmortem brain of ethanol intoxicated nonalcoholic subjects (blood ethanol concentration: 1.37±0.30 g/l) and matched controls. In the frontal cortex, the density of binding sites for [³H]clonidine (B_max=58±7 fmol/mg protein) and [³H]bromoxidine (UK 14304) (B_max=49±7 fmol/mg protein) in ethanol intoxicated subjects did not differ from those in the control groups (B_max=68±4 and 56±8 fmol/mg protein for the respective radioligand). The dissociation constants (K_D) were also similar in both groups. The binding capacities (B_max) and K_D values for both radioligands also were found unchanged in the hypothalamus, amygdala, head of caudate, hippocampus and cerebellum. The results demonstrate that, contrary to the -adrenoceptor, the presence of ethanol in the human brain does not alter the high-affinity state of the ₂-adrenoceptor in the frontal cortex and possibly also in other brain regions.     

Molecular cloning and functional expression of the murine noradrenaline transporter

The cDNA of the murine noradrenaline transporter (mNAT) was cloned from the RNA of the placenta of a C57BL/6 mouse. The cloned mNAT differs from a previously published sequence in two amino acids within the C-terminal region. A cDNA obtained from an inbred mouse strain showed a further amino acid exchange (Ile⁵⁰⁵Val) within the fifth intracellular loop. The pharmacological properties of both, the wild-type mNAT and the variant (mNAT-I⁵⁰⁵V), were studied in human embryonic kidney HEK293 cells transfected with the corresponding cDNA. The kinetic constants for transport (K _m, V _max) of [³H]noradrenaline ([³H]-NA) and binding (K _D, B _max) of the selective NAT inhibitor [³H]nisoxetine were not different between the two isoforms; the mean kinetic constants amounted to about 4μM and 120pmol/mg protein for K _m and V _max and 6nM and 18pmol/mg protein for K _D and B _max, respectively. [³H]-NA transport by both isoforms showed the typical properties of an NAT because it was dependent on sodium and chloride and inhibited with almost identical K _i values by various NAT substrates and inhibitors. The only significant pharmacological difference identified between the two mNAT isoforms was an about threefold higher affinity for cocaine of the very rare mNAT-I⁵⁰⁵V variant. Andrea Muck and Ralf Gilsbach contributed equally to this work.     

In‐vitro sulfation of piceatannol by human liver cytosol and recombinant sulfotransferases

Objectives The aim of this study was to investigate the concentration‐dependent sulfation of piceatannol, a dietary polyphenol present in grapes and wine and known for its promising anticancer and anti‐inflammatory activity. Methods Sulfation of piceatannol was investigated in human liver cytosol as well as using a panel of recombinant sulfotransferase isoforms. Furthermore, the chemical structures of novel sulfates were identified by liquid chromatography/mass spectrometry (LC/MS). Key findings In the presence of 3′‐phosphoadenosine‐5′‐phosphosulfate, three metabolites could be detected whose structures were identified by LC/MS/MS as piceatannol disulfate (M1) and two monosulfates (M2, M3). The kinetics of M1 formation exhibited a pattern of substrate inhibition with a K_i of 21.8 ± 11.3 μM and a V_max/K_m of 7.63 ± 1.80 μl/mg protein per min. Formation of M2 and M3 showed sigmoidal kinetics with apparent K_m and V_max values of 27.1 ± 2.90 μM and 118.4 ± 4.38 pmol/mg protein per min, respectively, for M2; and 35.7 ± 2.70 μM and 81.8 ± 2.77 pmol/mg protein per min, respectively, for M3. Incubation in the presence of human recombinant sulfotransferases (SULTs) demonstrated that M1 was formed equally by SULT1A1*1 and SULT1B1 and to a lesser extent by SULT1A1*2. M2 was preferentially catalysed by SULT1A1*2, 1A3 and 1E1. The formation of M3, however, was mainly catalysed by SULT1A2*1 and SULT1A3. Conclusions Our results elucidate the importance of piceatannol sulfation in human liver, which must be taken into account in humans after dietary intake of piceatannol.     

Platelet [3H]-paroxetine binding in obsessive-compulsive disorder

Platelet [³H]-paroxetine binding was analysed in 26 patients (13F, 13M) with obsessive compulsive disorder and 26 normal controls (13F, 13M). For patients with obsessive compulsive disorder, B_max was 2127 ± 480 fmol/mg protein (mean±SD) compared to control Bmax values of 1926 ± 696 fmol/mg protein. The mean K_d value for the patients was 0.075 ± 0.025 nM and for the controls was 0.076 ± 0.032 nM. Analysis of covariance indicated a significant effect of sex on both K_d and B_max but no effect of diagnosis (obsessive compulsive disorder versus normal controls) or season of sampling. The data provide no evidence for an abnormality of the platelet uptake mechanism as assessed by the measurement of [₃H]-paroxetine binding to the platelet transporter in obsessive compulsive disorder.     

Identification of cytochrome P450 enzymes involved in the metabolism of 3′,4′-methylenedioxy-α-pyrrolidinopropiophenone (MDPPP), a designer drug,in human liver microsomes

The metabolism of 3′,4′-methylenedioxy-α-pyrrolidinopropiophenone (MDPPP), a novel designer drug, to its demethylenated major metabolite 3′,4′-dihydroxy-pyrrolidinopropiophenone (di-HO-PPP) was studied in pooled human liver microsomes (HLM) and in cDNA-expressed human hepatic cytochrome P450 (CYP) enzymes. CYP2C19 catalysed the demethylenation with apparent K_m and V_max values of 120.0?±?13.4?µM and 3.2?±?0.1?pmol/min/pmol?CYP, respectively (mean?±?standard deviation). CYP2D6 catalysed the demethylenation with apparent K_m and V_max values of 13.5?±?1.5?µM and 1.3?±?0.1 pmol/min/pmol?CYP, respectively. HLM exhibited a clear biphasic profile with an apparent K_m,1 value of 7.6?±?9.0 and a V_max,1 value of 11.1?±?3.6?pmol/min/mg?protein, respectively. Percentages of intrinsic clearances of MDPPP by specific CYPs were calculated using the relative activity factor (RAF) approach with (S)-mephenytoin-4′-hydroxylation or bufuralol-1′-hydroxylation as index reactions for CYP2C19 or CYP2D6, respectively. MDPPP, di-HO-PPP and the standard 4′-methyl-pyrrolidinohexanophenone (MPHP) were separated and analysed by liquid chromatography-mass spectrometry in the selected-ion monitoring (SIM) mode. The CYP2D6-specific chemical inhibitor quinidine (3?µM) significantly (p?相似文献   

Histamine receptors in the heart—Molecular characteristics,physiology and pharmacology

Histamine is a normal constituent of mammalian heart. It affects cardiac function mainly through stimulating histamine H1- and H2-receptor subtypes. The simultaneous activation of H1- and H2-receptors in the heart results in: a positive inotropic and chronotropic effect, a negative dromotropic effect, increased automaticity and increased coronary blood flow. H1- and H2-receptors have already been cloned from different, but not yet from cardiac, tissue. They are two independent molecular entities differing in the length of their amino acid sequence, pathways of transmembrane and intracellular signalling, characteristics of their binding sites and selectivity for the specific agonists and/or antagonists. Our results of radioligand binding studies show the presence in the heart of a high-affinity (K _D 0.4 nmol/L andB _max 100 fmol/mg of protein) and a low-affinity (K _D 4.5 nmol/L,B _max 466 fmol/mg of protein) H1-receptor-binding site and only a single population of less-abundant high-affinity H2-receptor binding sites (K _D 1.0 nmol/L andB _max 27 fmol/mg of protein). The role of the histamine in cardiac pathophysiology is well established but the physiological role is unclear. The only proposed physiological role of histamine in the heart is the modulation of noradrenaline release from sympathetic nerve terminals, where H3-receptor subtypes might be involved.     

Binding of endomorphin‐2 to μ‐opioid receptors in experimental mouse mammary adenocarcinoma

Abstract: Endomorphin‐2 (Tyr‐Pro‐Phe‐Phe‐NH₂) binds with high affinity and selectivity to the μ‐opioid receptor. In the present study, [¹²⁵I]endomorphin‐2 has been used to characterize μ‐opioid‐binding sites on transplantable mouse mammary adenocarcinoma cells. Cold saturation experiments performed with [¹²⁵I]endomorphin‐2 (1 nm ) show biphasic binding curves in Scatchard coordinates. One component represents high affinity and low capacity (K_d = 18.79 ± 1.13 nm , B_max = 635 ± 24 fmol/mg protein) and the other shows low affinity and higher capacity (K_d = 7.67 ± 0.81 μm , B_max = 157 ± 13 pmol/mg protein) binding sites. The rank order of agonists competing for the [¹²⁵I]endomorphin‐2 binding site was [d ‐1‐Nal³]morphiceptin > endomorphin‐2 ? [d ‐Phe³]morphiceptin > morphiceptin > [d ‐1‐Nal³]endomorphin‐2, indicating binding of these peptides to μ‐opioid receptors. The uptake of ¹³¹I‐labeled peptides administered intraperitoneally to tumor‐bearing mice was also investigated. The highest accumulation in the tumor was observed for [d ‐1‐Nal³]morphiceptin, which reached the value of 8.19 ± 1.14% dose/g tissue.     

Heterogenous Binding of 3H-Remoxipride to Membranes of Rat Liver and Brain

Abstract: The binding of the antipsychotic agent ³H-remoxipride to membranes of rat liver and brain (whole brain and cerebellum) was studied with filtration technique. Saturable high affinity binding was obtained for all three types of tissue preparations. Heterogenous binding sites were found since various types of compounds inhibited the binding of ³H-remoxipride with shallow dose-response curves. In the liver preparation it was possible to differentiate between two different binding sites. One site, called rem₁, was defined with 100 nM alaproclate and bound σ₂ ligands with high affinity, e.g. haloperidol, GBR 12909, DTG and (+)-3-PPP. High correlation (r=0.93) was obtained between the inhibition of the binding of ³H-remoxipride to the rem₁ site and the inhibition of the binding of the σ ligand ³H-DTG reported previously (Ross 1991), indicating that the rem₁ site is identical to a σ₂-like binding site. The other ³H-remoxipride binding site in the rat liver, rem₂, appears to be identical to the site binding alaproclate, proadifen and cocaine with high affinity. Cd²⁺ and Zn²⁺ were potent inhibitors of both binding sites, Cd²⁺ particularly of rem₂ binding and Zn²⁺ preferably of rem₁ binding. The apparent B_max (pmol/g original tissue) and K_D (nM) values for rem₁ binding were 441±43 and 80±14, and for rem₂ binding 727±116 and 95±6. The binding of ³H-remoxipride to the brain preparations was also inhibited with shallow dose-response curves. The B_max values for the preparations of whole brain and cerebellum were 67±15 and 71±9 pmol/g original tissue, respectively and the K_D values were 171±19 and 176±6 nM. The IC₅₀ of the compounds were highly correlated to the IC₅₀ values for the rem₁ binding in the liver (r=0.96) and to IC₅₀ values for the inhibition of ³H-DTG binding in rat brain reported previously (Ross 1991). Cd²⁺ and Zn²⁺ ions also inhibited the binding of ³H-remoxipride in the brain preparation but at higher concentrations compared with the liver preparations. The present results indicate that the main part of the binding in the brain preparations occurs to σ-like recognition sites.