Reoxygenation, but neither hypoxia nor intermittent ischemia, increases [125I]endothelin-1 binding to rat cardiac membranes.

Standard binding techniques were used to establish whether either hypoxia, reoxygenation, perfusion under acidotic conditions, or "stunning" of the myocardium resembles ischemia and postischemic reperfusion in increasing cardiac membrane [125I]endothelin-1 (ET-1) binding site density (Bmax). Membranes from aerobically perfused rat hearts bound [125I]ET-1 to a single population of sites, with an affinity (KD) of 0.093 +/- 0.004 nM and a Bmax of 98.8 +/- 5.2 fmol/mg of protein. Bmax was increased (p less than 0.01) after 30 min of global ischemia, and further increased upon reperfusion, without changes in KD or selectivity. Neither three 10 min episodes of ischemia separated by 15 min of perfusion, nor perfusion at pH 6.8 instead of 7.4, nor 60 min of hypoxia altered Bmax, KD, or selectivity. Reoxygenation after 60 min of hypoxia increased Bmax (p less than 0.01) and KD (p less than 0.01) without changing selectivity. These results are interpreted to mean that the ischemia-induced increase in Bmax for [125I]ET-1 cannot be explained simply in terms of the ischemia-induced acidosis, or the accompanying reduction in tissue adenosine triphosphate and creatine phosphate.     

Effect of amlodipine pretreatment on ischaemia-reperfusion-induced increase in cardiac endothelin-1 binding site density.

Endothelin-1 (ET-1) may be implicated in the pathophysiology of myocardial ischaemia. To determine whether the long-acting calcium antagonist amlodipine attenuates the ischaemia- and reperfusion-induced increase in cardiac ET-1 binding sites, hearts from rats pretreated with amlodipine (0.25 or 0.5 mg/kg) 2 or 5 h before they were killed were made ischaemic for 20 or 40 min, reperfused, and subfractionated. Twenty- and 40-min ischaemia caused a time-dependent increase in ET-1 binding site density (Bmax) identified with [125I]ET-1. Amlodipine pretreatment attenuated this increase in a time- and dose-dependent manner. 0.25 and 0.5 mg/kg amlodipine also suppressed the reperfusion-induced increase in [125I]ET-1 binding site density, even when the 0.5-mg/kg pretreatment series reperfusion was administered after 40-min ischaemia.     

Binding sites for 125I-neuropeptide Y (NPY) on membranes from bovine adrenal medulla

Bovine adrenal medulla membranes were examined for the presence of specific 125I-neuropeptide Y (125I-NPY) binding sites using rapid centrifugation to measure the amount of bound ligand. Specific binding was determined from the difference between 125I-NPY bound in the presence and absence of 10(-7) M unlabeled NPY. The binding was saturable and reached equilibrium within 5 min at 0 degrees C. Analysis of specific 125I-NPY binding using the LIGAND computer program indicated a best fit for a two site model with a Kd of 0.26 nM and a Bmax of 12 fmol/mg protein for the high affinity site and a Kd of 170 nM and a Bmax of 6 pmol/mg protein for the low affinity site. The rate of dissociation (k-1) was 0.071/min with a t1/2 of 9 min. Displacement curves for avian or human pancreatic polypeptide revealed that these peptides displaced 125I-NPY from both sites with IC50 values greater than 10 nM.     

Non-benzodiazepine, Ag-dependent, brain-specific binding sites for [3H]beta-carboline-3-carboxylic acid ethyl ester

Specific, saturable and reversible binding of [3H]beta + CCE ([3H]beta-carboline-3-carboxylic acid ethyl ester) in buffer containing 20 microM AgNO3 and 10 microM diazepam was detected in rat brain membranes. The binding of [3H]beta CCE to non-benzodiazepine binding sites is Ag+ (Cu)-dependent, stimulated by NaCl and ascorbic acid and inhibited by dithiothreitol. The concentration of non-benzodiazepine [3H]beta CCE binding sites (Bmax) determined in the brain membranes was 1180 +/- 320 pmol/g tissue, and Kd = 77 +/- 19 nM. [3H]beta CCE bound to benzodiazepine receptors in the same membranes with Bmax = 81 +/- 9 pmol/g tissue and Kd = 3.2 +/- 0.4 nM.     

125I]iodoclonazepam, a specific high affinity radioligand for the identification of BZ1 and BZ2 sites in rat brain.

Binding of the radioligand [125I]iodoclonazepam to three different areas of rat brain (cerebellum, hippocampus and striatum) has been characterised. In all three regions binding is rapid, saturable and of high affinity (cerebellum Bmax = 1.49 +/- 0.3 pmol/mg of protein, Kd = 0.39 +/- 0.06 nM; hippocampus Bmax = 1.5 +/- 0.14 pmol/mg of protein, Kd = 0.38 +/- 0.6 nM and striatum Bmax = 0.53 +/- 0.1 pmol/mg of protein, Kd = 0.34 +/- 0.03 nm, n = 3). In all regions only one population of sites was apparent. However, competition for [125I]iodoclonazepam sites by a series of benzodiazepine agonists and antagonists showed some regional differences. The BZ1 selective compounds zolpidem and CL218,872 showed a 4.3-fold and 5.2-fold selectivity for cerebellar binding sites respectively. We conclude that [125I]iodoclonazepam is a novel, high affinity ligand which recognises both BZ1 and BZ2 classes of receptor and should be a useful addition to the panel of benzodiazepine ligands currently available.     

Characterization of endothelin receptors on a human neuroblastoma cell line: evidence for the ETA subtype.

1. Specific binding sites for synthetic endothelin (ET) isoforms were studied on intact cells of the SK-N-MC cell line, derived from a human neuroblastoma. 2. [125I]-ET-1 (2.5 x 10(-11) M) specifically bound to a single class of binding sites on these cells (Hill coefficient of 1.06 +/- 0.04, n = 3) with an apparent Kd of 1.4 +/- 0.3 x 10(-9) M and a Bmax of 3.1 +/- 1.0 pmol mg-1 protein. [125I]-ET-3 (2.5 x 10(-11) M), did not specifically bind to SK-N-MC cells. 3. The binding of [125I]-ET-1 was competitively inhibited by other ET isoforms, the order of potency being ET-1 greater than sarafotoxin S6b greater than ET-3. 4. Association of 1 nM [125I]-ET-1 at 37 degrees C reached apparent equilibrium at 60-80 min, with half-maximal binding being achieved at 12 min. 5. Dissociation was measured after both 10 min and 60 min of association with 64% and 30% respectively of specifically bound [125I]-ET-1 dissociating. The actual amounts of [125I]-ET-1 dissociated were similar in both cases. 6. Incubation of [125I]-ET-3 with SK-N-MC cells at 37 degrees C for 60 min did not result in significant degradation of this peptide. However, [125I]-ET-1 was broken down by incubation with SK-N-MC cells, the pattern of degradation of dissociable [125I]-ET-1 (and that found in the supernatant) being different from that of non-dissociable [125I]-ET-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

An effect of ischemia on myocardial dihydropyridine binding sites

The [3H]nitrendipine binding activity of sarcolemmal fragments isolated from aerobically perfused or ischemic rat hearts was studied. After 90 min aerobic perfusion, two populations of binding sites were detected--high affinity sites with KD of 0.24 +/- 0.04 nM and Bmax 313 +/- 110 fmol/mg protein, and low affinity sites with KD of 47.6 +/- 8.7 nM and Bmax 12.4 +/- 1.88 pmol/mg protein. Sixty minutes global ischemia significantly reduced the KD of the low (15.8 +/- 2.9 nM, P less than 0.03) but not of the high (0.22 +/- 0.05 nM) affinity sites. Under these same conditions the Bmax of both the high (82.4 +/- 14.5 fmol/mg protein, P less than 0.03) and low (6.1 +/- 1.7 pmol/mg protein, P less than 0.01) affinity binding sites was reduced but the sites retained their selectivity, with nifedipine displacing bound [3H]nitrendipine more potently than D600. Bay K 8644, when added upon reperfusion, promoted a dose-related increase in Ca2+ entry which was reduced by nifedipine, indicating that dihydropyridine binding sites can be activated after 60 min ischemia.     

Heterogeneity of binding sites for ICI 198,615 in human lung parenchyma.

We have identified and characterized two different subclasses of binding site for the novel peptido-leukotriene (LT) antagonist, [3H]ICI 198,615, in membranes from human lung parenchyma using a receptor-ligand assay. This novel compound is representative of a new class of LT receptor antagonists and it has been demonstrated to be several orders of magnitude more potent and selective than most other LT antagonists described to date. The binding of [3H]ICI 198,615 is rapid, specific and saturable. Equilibrium was reached within 5-10 min. Non linear fitting of dissociation time courses has revealed the presence of two different components (K(off)1 = 8.3 +/- 6.8 x 10(-4) sec-1 and K(off)2 = 0.79 +/- 1.66 x 10(-3) sec-1) of the kinetic curves, suggesting heterogeneity of the binding sites. Computer analysis of equilibrium binding data obtained at 25 degrees results in a model with two classes of binding sites, a high affinity-low capacity class with Kd1 = 0.024 +/- 0.014 nM and Bmax1 = 0.015 +/- 0.004 pmol/mg protein and a low affinity-high capacity class with Kd2 = 6326 +/- 3859 nM and Bmax2 = 473 +/- 383 pmol/mg protein. In competition studies, LTD4 was also found to interact with two classes of binding site (Kd1 = 0.016 +/- 0.008 nM and Kd2 = 15195 +/- 8965 nM). On the contrary, LTE4 and LTC4 were found to interact with a homogeneous class of sites only with Kd = 7466 +/- 4629 nM and Kd = 428 +/- 73 nM, respectively. Furthermore, we have evaluated the effect of a number of LT antagonists on the binding of [3H]ICI 198,615. Ro 24-5913 (Kd = 3.0 +/- 2.1 nM), FPL55712 (Kd = 4945 +/- 2868 nM), LY171883 (Kd = 19628 +/- 12365 nM), SKF 104353 (Kd = 74.2 +/- 46 nM) and its enantiomer SKF 104373 (Kd = 13627 +/- 6813 nM) were found to interact with a single class of binding sites. The present studies indicate a heterogeneity of binding sites for ICI 198,615 in membranes from human lung parenchyma and that ICI 198,615 is a very potent and selective antagonist of LTD4 receptors in this tissue.     

Characteristics of endothelin receptors in the central nervous system of spontaneously hypertensive rats.

The binding of [125I]sarafotoxin 6b (SRT 6b) and [125I]endothelin-1 (ET-1) to endothelin (ET) receptors of neuronal membranes prepared, from regions of the brain and spinal cord of 8 week-old, spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats was determined. Spontaneously hypertensive rats had significantly higher blood pressure as compared to WKY rats. Heart rate was similar in SHR and WKY rats. [125I]SRT 6b and [125I]ET-1 bound to the membranes of the cerebral cortex, hypothalamus, ventrolateral medulla, dorsomedial medulla and spinal cord at a single, high affinity site. The Kd and Bmax values of the binding of [125I]SRT 6b were found to be similar to binding of [125I]ET-1 in all the regions. The concentration-dependent inhibition of binding of [125I]ET-1 by unlabeled ET-1, in spinal cord membranes showed an IC50 value of 2.66 +/- 0.59 nM and a Ki value of 2.35 +/- 0.52 nM in WKY rats and an IC50 value of 2.82 +/- 0.76 nM and a Ki value of 2.43 +/- 0.70 nM in SHR rats. On the other hand, the concentration-dependent inhibition of the binding of [125I]SRT 6b by unlabeled ET-1, in spinal cord membranes showed an IC50 value of 10.31 +/- 1.82 pM and a Ki value of 8.44 +/- 1.41 pM in WKY rats, while SHR rats showed an IC50 value of 10.28 +/- 1.94 pM and a Ki value of 8.89 +/- 2.00 pM. The binding of [125I]SRT 6b and [125I]ET-1 in the cerebral cortex, dorsomedial medulla and spinal cord membranes was found to be similar in SHR and WKY rats.(ABSTRACT TRUNCATED AT 250 WORDS) [corrected]     

Effects of berbamine on hemodynamics and myocardial reperfusion injury in isolated working rabbit hearts

The isolated working rabbit hearts perfused with Tyrode's buffer solution persisted working in good condition at least up to 70 min. Berbamine (Ber) 3 mumol.L-1 significantly changed the hemodynamic parameters and reduced cardiac functions. The effects of Ber appeared in a dose-dependent manner. Reperfusion following 30-min of global ischemia aggravated the myocardial damages induced by ischemia, and the parameters of cardiac functions in working hearts markedly reduced and did not restore even at 20 min of reperfusion. Ber 1 mumol.L-1 reduced the myocardial ischemic reperfused damages and restored all parameters to the level of preischemia within 10 min of reperfusion, and this situation of working hearts also lasted 40-50 min. The present results showed that Ber could protect myocardium against ischemic reperfused damages, promote the recovery of cardiac functions and prolong the efficient working period in isolated working rabbit hearts.     

Tissue specificity of endothelin binding sites.

A measurement was made of the binding of 125I-labeled endothelin (125I-ET) to crude membrane fractions prepared from rat aorta, atrium, ventricle, portal vein, trachea, lung parenchyma, vas deferens, ileum, bladder, and guinea-pig taenia coli and lung parenchyma. Scatchard analysis of 125I-ET binding in all tissues indicated binding to a single class of saturable sites. The affinity and density of 125I-ET binding sites varied between tissues. The Kd of 125I-ET binding was approximately 0.5 nM for rat aorta, trachea, lung parenchyma, ventricle, bladder, and vas deferens, and guinea-pig taenia coli and lung parenchyma, 1.8 nM for rat portal vein and atrium, and 3.3 nM for ileum. The Bmax of 125I-ET binding had the following rank order of density in rat tissues: trachea greater than lung parenchyma = vas deferens much greater than aorta = portal vein = atrium greater than bladder greater than ventricle = ileum. The properties of 125I-ET endothelin binding were characterized in rat ventricular membranes. 125I-ET binding was time dependent, reaching a maximum within 45-60 min at 25 degrees C. The calculated microassociation constant was 9.67 x 10(5) s-1 M-1. Only 15-20% of 125I-ET dissociated from its binding site even when dissociation was studied as long as 3 h. Preincubation of ventricular membranes with ET prevented binding of 125I-ET. 125I-ET binding was destroyed by boiling of ventricular membranes and was temperature, pH, and cation (Ca2+, Mg2+, and Na+) dependent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of cardiac endothelin binding sites in rats: downregulation of left atrial endothelin binding sites in response to myocardial infarction.

Endothelin-1 (ET-1) is a recently described potent vasoconstrictor peptide. Plasma and myocardial tissue levels of ET-1 are increased following myocardial ischemia, however, the factors which regulate ET-1 binding sites in vivo are not well understood. ET-1 binding sites were measured by Scatchard analysis of [125I]ET-1 binding to membranes of rat myocardium. The highest number of ET-1 binding sites (2,951 fmol/mg protein) were found in right atrial tissue, and followed the rank order of right atrium greater than left atrium (2,157 fmol/mg protein), greater than right ventricle (835 fmol/mg protein), greater than septum (609 fmol/mg protein) = left ventricle (498 fmol/mg protein). Following coronary artery occlusion for 24 h, ET-1 binding sites of left atrium were decreased by 35% (p less than 0.01), without a change in the Kd (i.e., 98 pM). Other regions of the myocardium did not exhibit any change in the number of ET-1 binding sites. Similarly, no change in ET-1 binding sites were observed following coronary artery occlusion for 0.5 h followed by 24 h reperfusion. These data indicate that there exists considerable regional differences in the density of ET-1 binding sites in the myocardium, and that ET-1 sites are selectively reduced in left atrial tissue following myocardial infarction.     

Adenosine receptors in cortical-derived vesicles of the rat: studies on binding sites and accumulation of cyclic AMP

A vesicular preparation derived from the cerebral cortex of the rat was used to obtain, under the same experimental condition, binding parameters and stimulation data for cyclic AMP. Two analogues of adenosine were employed in the binding studies: [3H]NECA, a mixed A1/A2 agonist and [3H]CHA, a more selective A1 agonist. The [3H]CHA seemed to bind to a single high affinity site (Kd = 1.31 nM, Bmax = 0.327 pmol bound); saturation data for [3H]NECA were resolved for the presence of a high and a low affinity binding site (Kd1 = 3.08 nM, Bmax1 0.115 pmol bound; Kd2 = 204 nM, Bmax2 1.59 pmol bound), but only when calcium ions were omitted from the incubation medium. At 0 degree C, [3H]NECA bound to a single, low affinity site; the presence of calcium ions (1 mM) significantly reduced the affinity of [3H]NECA (Kd 419 nM), with respect to the absence of calcium (Kd 208 nM), without affecting the Bmax value. The influence of calcium ions was also investigated on the binding of [3H]CHA and a reduction of the Bmax value (36%) was found. Regardless of the presence or the absence of calcium ions, NECA stimulated accumulation of cyclic AMP in a dose-dependent way with an EC50 of 2.79 microM; this value did not correlate with the Kd of the low affinity binding site for [3H]NECA. Thus, the purpose of establishing a correlation between binding sites for analogues of adenosine and the site in the cerebral cortex through which the accumulation of cyclic AMP is induced, was not achieved. It is concluded that the stimulatory effect of analogues of adenosine on adenylate cyclase might not be a receptor-mediated effect. The complex influence of calcium ions on affinity and binding capacity of analogues of adenosine is discussed.     

Evidence for the AT1 subtype of the angiotensin II receptor in the rat submandibular gland

We have characterized angiotensin II (Ang II) receptor subtypes on rat submandibular gland membranes using a radioligand binding assay. [3H]Ang II binding to the membrane fractions exhibited both high (Kd =0.08 nm, Bmax =2.19 fmol/mg protein) and low (Kd =4.19 nm, Bmax = 13.7 fmol/mg protein) affinity. Ang 11, Ang III and saralasin completely displaced the [3H]Ang II binding, whereas CV-11974, an AT1 receptor antagonist and PD123319, an AT2 receptor antagonist maximally displaced up to approximately 87 and 13% of the total binding, respectively. [3H]DuP753 binding to the membrane fractions exhibited a single population of binding site with a Kd of 4.22 nM and Bmax of 3.77 pmol/mg protein. Ang II, Ang III and CV-11974 completely displaced the [3H]DuP753 binding with slope factors near unity, but PD123319 did not. These findings suggest that rat submandibular gland membranes contain predominantly the AT1 receptor subtype.     

Contribution of nitric oxide in big endothelin-1-induced cardioprotective effects on ischemia/reperfusion injury in rat hearts

We have recently shown that an appropriate amount of exogenous big endothelin-1 (ET-1) has beneficial effects on ischemia-/reperfusion-induced norepinephrine overflow and cardiac dysfunction in rat hearts and that these effects occur through a conversion to ET-1 by endothelin-converting enzyme and following stimulation of ETB receptor. In this study, we examined the possible involvement of nitric oxide (NO) in the big ET-1-induced cardioprotective effects. According to the Langendorff technique, isolated rat hearts were subjected to 40-minute global ischemia followed by 30-minute reperfusion. Exogenous big ET-1 (0.3 nM) significantly increased NOx (NO2/NO3) level in the coronary effluent after onset of reperfusion. This effect was markedly attenuated by treatment with SM-19712 (selective endothelin-converting enzyme inhibitor), A-192621 (selective ETB receptor antagonist), or NG-nitro-l-arginine (nonselective NO synthase inhibitor), respectively. In addition, N-nitro-l-arginine blunted big ET-1-induced suppression of norepinephrine overflow and improvement of cardiac dysfunction after ischemia/reperfusion. These findings suggest that NO produced by ETB receptor activation plays an important role in exogenous big ET-1-induced actions.     

Binding and degradation of angiotensin II by mesenteric artery subcellular membranes

Rat mesenteric artery microsomes were previously reported to degrade 125I-angiotensin II (AII). It is now shown here that washing the membranes with EDTA and including EGTA in the assay media reduces the 125I-AII degradation to very low levels without reducing the specific binding of 125I-AII. Using EDTA wash and including 5 mM MgCl2 and 0.2 mM EGTA the following characteristics of the binding were observed: microscopic association rate constant (k1) = 1.3 to 2.2 X 10(5) M-1 s-1, microscopic dissociation rate constant (k-1) = 3.8 to 6.4 X 10(-4) s-1, equilibrium dissociation constant (Kd) = 1.8 nM and number of binding sites (Bmax) = 193 fmol/mg protein. The subcellular distribution of the specific binding of 125I-AII at 0.16 nM and 1.63 nM was studied along with the distribution of the marker enzymes. The specific binding paralleled the plasma membrane marker (5'-nucleotidase), but not the putative endoplasmic reticulum marker (NADPH-cyt. c-reductase) or the inner mitochondrial marker (cyt. c-oxidase). Thus the binding to the plasma membrane-enriched fraction F2 occurred with a similar affinity (Kd = 2.2 nM) but with higher number of binding sites (420 fmol/mg protein). This study establishes the 125I-AII binding method suitable for determining the changes in the angiotensin receptor characteristics in the pathophysiology of the vascular smooth muscle.     

On the high affinity binding site for [3H]-1,3-dipropyl-8-cyclopentylxanthine in frog brain membranes.

1. Radioligand binding properties of the adenosine receptor ligands, [3H]-1,3-dipropyl-8-cyclopentylxanthine ([3H]-DPCPX), and [3H]-R-phenylisopropyladenosine ([3H]-R-PIA) were investigated in frog brain membranes. 2. The specific binding of the adenosine antagonist, [3H]-DPCPX to frog brain membranes showed one binding site with Kd and Bmax values of 43.8 nM and 0.238 +/- 0.016 pmol mg-1 protein, respectively. Guanosine 5'-triphosphate (GTP, 100 microM) decreased to 72 +/- 7% and Mg2+ (8 mM) increased to 121 +/- 3% [3H]-DPCPX (40 nM) binding to frog brain membranes. 3. [3H]-DPCPX saturation binding experiments performed in the presence of Mg2+ (8 mM), or in the presence of GTP showed that Mg2+ ions decreased the Kd value of [3H]-DPCPX to 14 nM, and GTP increased this value to 65.6 nM. Bmax values were not significantly (P > 0.05) modified (0.261 +/- 0.018 pmol mg-1 protein, with Mg2+, and 0.266 +/- 0.026 pmol mg-1 protein, in presence of GTP) by the presence of Mg2+ or GTP. 4. The specific binding of [3H]-R-PIA (15 nM) was decreased to 37 +/- 6% by GTP (100 microM) and increased to 123 +/- 4% by Mg2+ (8 mM). [3H]-R-PIA saturation binding experiments performed in the presence of Mg2+ (8 mM) showed one binding site with Kd and Bmax values of 0.9 nM and 0.229 +/- 0.008 pmol mg-1 of protein, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific [3H]neurotensin binding to rat spinal cord membranes

The binding of [3H]neurotensin to membranes prepared from rat spinal cord has been studied in vitro. Scatchard analysis of saturation binding data indicated that [3H]neurotensin binds with high affinity (Kd = 6.3 nM) to a single, saturable population of binding sites (Bmax = 12.4 pmol/g tissue). Neurotensin1-13 (IC50 = 5.9 nM) and neurotensin8-13 (IC50 = 3.7 nM) were potent inhibitors of [3H]neurotensin binding whereas neurotensin1-8 was virtually inactive at concentrations up to 10(-5) M. Sodium chloride (150 mM) significantly inhibited binding, while potassium chloride (5 mM), magnesium chloride (10 mM), manganese chloride (1 mM) and GMP-PNP (0.1 mM) were without effect. The characteristics of the binding of [3H]neurotensin obtained in this study are consistent with this ligand binding to a physiologic neurotensin receptor in rat spinal cord membranes.     

Characterization of [3H]estradiol-17 beta-(beta-D-glucuronide) binding sites in basolateral and canalicular liver plasma membranes

The specific binding of [3H]estradiol-17 beta-(beta-D-glucuronide) ([3H]E217G) was examined in isolated basolateral (bLPM) and canalicular (cLPM) liver plasma membranes. Two distinct binding sites were identified in each membrane fraction by competition and saturation experiments. Binding parameters obtained from competition studies were: Kd1 = 26 nM, Bmax1 = 0.26 pmol/mg protein; Kd2 = 2.6 microM, Bmax2 = 27 pmol/mg protein for bLPM; and Kd1 = 81 nM, Bmax1 = 0.61 pmol/mg protein; Kd2 = 6.7 microM, Bmax2 = 79 pmol/mg protein for cLPM. Binding parameters obtained from saturation experiments were not significantly different. There was no Na+ requirement for binding. Kinetic dissociation experiments showed that binding was reversible and revealed two components. The dissociation rate constants did not vary with the method of dilution of radioligand, i.e. by "infinite" volume, or excess unlabeled ligand, thus ruling out the possibility of cooperativity. The ability of a series of compounds to inhibit the binding of [3H]E217G was also examined. In bLPM, taurocholate (TC), estrone sulfate (E1SO4) and bromosulfophthalein (BSP) were able to compete with both binding sites, whereas estriol-17 beta-(beta-D-glucuronide) (E317G), estriol-16 alpha-(beta-D-glucuronide) (E316G), testosterone glucuronide (TG), estradiol-3-(beta-D-glucuronide) (E23G), estriol-3-(beta-D-glucuronide) (E(3)3G), cholate and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) were able to inhibit binding to only the low-affinity site. In cLPM, only the cholestatic steroid D-ring glucuronides (E(3)17G, E(3)16G and TG) and TC were able to compete with both sites, whereas the non-cholestatic steroid A-ring glucuronides (E(2)3G and E(3)3G), BSP and DIDS competed for only the low-affinity site. Based on the observed substrate specificities, the low-affinity sites in bLPM and cLPM are postulated to represent multispecific organic anion carriers. The high-affinity site in cLPM may play a role in mediating steroid D-ring glucuronide-induced cholestasis.     

Interaction of aminoalkylcarbamates of forskolin with adenylyl cyclase: synthesis of an iodinated derivative of forskolin with high affinity for adenylyl cyclase.

7-(2-Aminoethyl)aminocarbonyl-7-desacetylforskolin (7-AEC-Fsk) and 6-(2-aminoethyl)aminocarbonylforskolin (6-AEC-Fsk) were synthesized and tested for their ability to activate adenylyl cyclase and inhibit the high affinity binding of [3H]forskolin to bovine brain membranes. Forskolin and 7-AEC-Fsk were equipotent in activating adenylyl cyclase, with EC50 values of about 4 microM, whereas 6-AEC-Fsk had an EC50 of about 2 microM. 6-AEC-Fsk and 7-AEC-Fsk stimulated adenylyl cyclase about 7-fold over basal levels at 100 microM, whereas forskolin produced a 5-fold stimulation. Forskolin and 6-AEC-Fsk inhibited the binding of [3H]forskolin to bovine brain membranes with Kd values of 41 nM and 28 nM, respectively, whereas 7-AEC-Fsk had a Kd of 83 nM. The 3-(3-iodo-4-hydroxyphenyl)propionamide derivative of 6-AEC-Fsk (6-I-HPP-Fsk) was more potent than forskolin in inhibiting [3H]forskolin binding to bovine brain membranes, with a Kd of 14 nM. 6-AEC-Fsk was reacted with 125I-labeled Bolton-Hunter reagent to produce 6-125I-HPP-Fsk with a specific activity of 2175 Ci/mmol. 6-125I-HPP-Fsk bound to bovine brain membranes with a Kd of 13 nM and a Bmax of 3.8 pmol/mg of protein. Forskolin inhibited the binding of 6-125I-HPP-Fsk to bovine brain membranes with a Kd of 31 nM, whereas 1,9-dideoxyforskolin only slightly inhibited the binding at 10 microM. The binding of 6-125I-HPP-Fsk was not inhibited by agents that inhibit forskolin binding to the glucose transporter, such as D-glucose or cytochalasin B. There was no displaceable binding of 6-125I-HPP-Fsk to red blood cell membranes, which contain a large concentration of the glucose transporter. Pretreatment of bovine brain membranes with an alkylating derivative of forskolin, 7-bromoacetyl-7-desacetylforskolin (BrAcFsk), led to an irreversible decrease in the binding of [3H]forskolin and 6-125I-HPP-Fsk. The time dependence and concentration dependence for the BrAcFsk-induced decrease in [3H]forskolin binding sites were identical to those observed for the decrease in 6-125I-HPP-Fsk binding sites. 6-125I-HPP-Fsk binding was determined in human platelet membranes in the presence of Mg2+ alone and in combination with guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) or AIF4-. The presence of GTP gamma S or AIF4- increased the binding of 6-125I-HPP-Fsk by 4.5-fold and 4-fold, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)