Characterization of leukotriene B4 binding sites on guinea pig lung macrophages

Specific binding sites for [3H]leukotriene B4 (LTB4) were identified on guinea pig lung macrophages, using high specific activity [3H] LTB4 in the presence or absence of unlabeled LTB4. At 0 degrees C, [3H] LTB4 binding reached equilibrium within 30 min, and addition of a large excess of unlabeled LTB4 resulted in a rapid decrease of specific binding. Binding was saturable, reversible and dependent upon the number of lung macrophages. The KD and Bmax were found to be 3.85 +/- 0.6 nM and 35 +/- 3 fmol/10(6) cells, respectively, as determined from Scatchard analysis. In competition studies for the displacement of [3H]LTB4 from binding sites, LTB4 and its analogs had the following order of potency: LTB4 (Ki = 2.9 +/- 0.8 nM) greater than 5-epi LTB4 (Ki = 58.7 +/- 0.3 nM) greater than 5-deoxy-LTB4 (Ki = 91.7 +/- 0.3 nM) greater than 12-epi LTB4 (Ki = 4.7 +/- 1.2 microM) greater than 5,12-deoxy LTB4 (Ki = 7.6 +/- 0.01 microM) greater than or equal to 12-deoxy LTB4 (Ki = 8.9 +/- 0.01 microM). LTC4 and LTD4 did not displace the [3H] LTB4 binding at concentrations up to 10 microM. [3H]LTB4 was not metabolized during the binding process as determined by reverse-phase high performance liquid chromatography. It was suggested that this binding site might be an LTB4 receptor.     

Two distinct populations of [3H]prazosin and [3H]yohimbine binding sites in the plasma membranes of rat mesenteric artery

Postsynaptic alpha adrenoceptor subtypes have been investigated by radioligand binding studies in plasma membrane vesicles prepared from rat mesenteric arteries using [3H]prazosin and [3H]yohimbine. Both the radioligands displayed monophasic saturation in binding with a single component on Scatchard analysis. In the estrogenized female rat mesenteric artery, the specific binding of [3H]prazosin was rapid, saturable, reversible and of high affinity (0.65 +/- 0.05 nM) with a maximum binding capacity (Bmax) of 177 +/- 14 fmol/mg of protein. The maximum number of [3H]yohimbine binding sites were 427 +/- 31 fmol/mg of protein with the Kd equal to 34.5 +/- 3.8 nM. There was no evidence of cooperativity in the binding of both the ligands. The Kd values of [3H]prazosin and [3H]yohimbine, calculated from their respective kinetic analyses of binding, were in good agreement with the Kd values estimated from Scatchard plots. Prazosin was 15,000 times more potent in competing at the [3H]prazosin binding sites than at the [3H]yohimbine sites. In contrast, unlabeled yohimbine was 100-fold more potent in competing at the [3H]yohimbine binding sites than at the [3H]prazosin sites. The affinity of BE 2254 was 10,500 times higher for the [3H]prazosin binding sites than its affinity for the [3H]yohimbine binding sites. Non-alpha adrenoceptor antagonists competed poorly for both the radioligand binding sites. The Kd and Bmax of [3H]prazosin and [3H]yohimbine binding in the membranes of male rat mesenteric arteries were not significantly different from the corresponding values in the membranes of estrogenized female rat mesenteric artery.(ABSTRACT TRUNCATED AT 250 WORDS)     

3H]52770 RP, a platelet-activating factor receptor antagonist, and tritiated platelet-activating factor label a common specific binding site in human polymorphonuclear leukocytes

In human polymorphonuclear leukocytes (PMNs), the tritiated platelet activating factor ([3H]PAF) labels in a saturable manner a single class of binding sites with a Kd of 3.5 +/- 0.5 nM (n = 7) and a maximum binding capacity (Bmax) of 206 +/- 13 fmol/2.5 X 10(6) PMNs (n = 7). 52770 RP, a nonphospholipid antagonist of PAF receptors, fully and competitively displaced the [3H]PAF from its binding sites with a Ki of 7.0 +/- 0.7 nM (n = 4). The high potency and the low solubility in cellular membranes of this compound led us to prepare [3H]52770 RP. This ligand was characterized by a binding which was rapid, reversible, confined to a single site, saturable, specific and stereoselective. Its Kd and Bmax were 4.2 +/- 0.3 nM and 181 +/- 11 fmol/2.5 X 10(6) PMNs, respectively. The stereoselectivity of the binding was suggested by the 600- and 1050-fold higher potency of the d-enantiomer with respect to l-52770 RP in displacing [3H]52770 RP or [3H]PAF, respectively. Several PAF analogs (e.g., lyso-PAF, 2-O-methyl-lyso-PAF), which are poorly active as PAF receptor agonists in functional tests, were weak displacers of [3H]PAF and [3H]52770 RP. Furthermore, for a series of 14 known PAF receptor agonists or antagonists belonging to different chemical families, there was an excellent correlation (r = 0.98) between their ability to displace [3H]PAF and [3H]52770 RP. Thus, [3H]52770 RP and [3H]PAF appear to interact with the same binding site on human PMNs which is proposed to be the PAF receptor mediating functional responses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of alpha-1 adrenergic receptors in the heart using [3H]WB4101: effect of 6-hydroxydopamine treatment

To identify and characterize the cardiac alpha-adrenoceptors, a radioreceptor binding assay using the potent alpha adrenergic antagonist, [3H]WB4101 was performed in rat hearts. Specific [3H]WB4101 binding to rat left ventricular homogenates was saturable, reversible and of high affinity (Kd = 0.18 nM) with a Bmax of 2.57 fmol/mg of tissue (27.7 fmol/mg of protein). Adrenergic agonists competed for specific [3H]WB4101 binding in the order: (-)-epinephrine > (-)-norepinephrine greater than (-)-isoproterenol. Stereospecificity of the [3H]WB4101 binding sites was also demonstrated with (-)-epinephrine, (Ki = 90) nM being 270 times as potent as (+)-epinephrine, (K1 = 24 microM). Adrenergic antagonists competed for the binding in the order: WB4101 = prazosin greater than yohimbine greater than (-)-propranolol. WB4101 and prazosin exhibited a markedly greater (2000 times) affinity for [3H]WB4101 binding sites than yohimbine. The affinities (pKi) of alpha agonists and antagonists for [3H]WB4101 binding sites in the rat heart closely correlated with their pharmacological potencies in the heart. Scatchard analysis for [3H]WB4101 binding, performed in five regions from control and 6-hydroxydopamine-treated rat hearts, revealed specific [3H]WB4101 binding (Bmax) significantly greater in the ventricles and intraventricular septae than in atria. At 1 week after 6-hydroxydopamine treatment, there was a significant increase (40%) in the Bmax for [3H]WB4101 binding to ventricles and intraventricular septae without a change in Kd. We conclude: 1) [3H]Wb4101 selectively labels postsynaptic alpha-1 adrenoceptors in the rat heart; 2) there is a definite regional variation for cardiac alpha-1 adrenoceptors; and 3) 6-hydroxydopamine treatment caused a significant increase in the density of alpha-1 adrenoceptors in ventricles and intraventricular septae, compatible with a postsynaptic localization of the [3H]WB4101 binding site.     

Identification of A1 and A2 adenosine receptors in the rat spinal cord

The adenosine receptors in membranes prepared from rat ventral and dorsal lumbar spinal cord were characterized by comparing the binding characteristics of [3H]5'-N-ethylcarboxamide adenosine ([3H]NECA), an agonist with nearly equal affinities at the A1 and A2 adenosine receptor subtypes, with those of [3H]N6-[(R)-1-methyl-2-phenylethyl]adenosine ([3H]R-PIA), an A1-selective agonist. Saturation isotherms of the ventral and dorsal spinal cord yielded Kd values 1.9 to 2.3 nM for [3H]R-PIA and 18.1 to 19.5 nM for [3H]NECA. The Bmax for [3H]NeCA was approximately twice the Bmax for [3H]R-PIA in ventral and dorsal halves (267 vs. 128 fmol/mg of protein and 402 vs. 206 fmol/mg of protein, respectively). Displacement of specific [3H]NECA binding by the A2-selective agonist, 2-(phenylamino)adenosine, the relatively nonselective antagonist, theophylline and six A1-selective agonists, R-PIA, S-PIA, N6-(cyclohexyl)adenosine, N6-(cyclopentyl)adenosine, N6-(m-aminophenyl)adenosine and N6-(m-iodophenyl)adenosine, revealed two [3H]NECA binding components with the characteristics of A1 and A2 receptors. All curves best fit a two-site model when analyzed by the computer program LIGAND. R-PIA, N6-(cyclohexyl)adenosine and N6-(cyclopentyl)adenosine were the most potent displacers at the first site (Ki = 0.6-1.4 nM). All A1-selective agonists were poor displacers of [3H]NECA at the second site (Ki = 0.6-18.6 microM). The A2-selective agonist, 2-(phenylamino)adenosine, was as potent as R-PIA in displacing [3H]NECA from this site with a Ki value 0.57 microM. Finally, the A1 and A2 adenosine receptor-mediated inhibition and stimulation of adenylate cyclase were demonstrated directly in synaptic membranes prepared from the spinal cord.     

Beta-1 receptor is the predominant beta-adrenoreceptor on rat brown adipose tissue

A new in vitro radioligand binding assay is described for brown adipose tissue using the beta adrenergic antagonist [3H]CGP 12177 (4-(3-t-butylamino-2-hydroxypropoxy)-[5,7-3H]benzimidazol-2-one). Binding was saturable and stereoselectively inhibited by propranolol. There was 60 to 80% specific binding using either 30 microM l-isoproterenol or 10 microM l-propranolol to define nonsaturable binding. [3H]CGP 12177 was bound to partially purified membranes from collagenase-separated brown adipocytes with a Kd of 0.84 nM, as determined from kinetic studies, and 1.24 +/- 0.13 nM as found by equilibrium binding studies; maximum binding was 14.2 +/- 0.9 fmol/mg of protein. Membranes from whole-pad homogenates had a similar Kd of 1.17 +/- 0.14 nM but twice the maximum number of binding sites (28.5 +/- 4.4 fmol/mg of protein). Intact brown adipocytes had a Kd of 0.55 nM and a maximum binding of 29.4 +/- 1.5 fmol X 10(-6)/cell or 17,700 sites per cell. Competitive binding studies showed about 80% of the binding sites to be of the beta-1 and 20% of the beta-2 subtype. The pA2 values derived from inhibition of isoproterenol-stimulated in vitro oxygen consumption in intact brown adipocytes by the beta-1 selective antagonist metoprolol and beta-2 selective lCl 118551 were in close agreement with their respective K1 values at the beta-1 receptor as derived from competitive binding studies. These data strongly suggest that the beta-1 adrenoreceptor on brown adipose tissue is primarily responsible for the initiation of thermogenesis in this tissue.     

Comparison of antagonist and agonist binding to the leukotriene B4 receptor intact human polymorphonuclear neutrophils (PMN).

In the present studies, the pharmacology of the leukotriene B4 (LTB4) receptor on intact human polymorphonuclear neutrophils (PMN) was characterized using radioligand binding techniques with [3H]LTB4 and a novel LTB4 receptor antagonist radioligand [3H]CGS 23131 (LY223982). Saturation studies revealed that [3H]CGS 23131 labeled a single class of recognition sites with high affinity (Kd = 13 nM) and limited capacity (apparent Bmax = 2.8 pmol/10(7) cells). In contrast, [3H]LTB4 labeled both a set of high (Kd = 0.3 nM) and lower affinity (Kd = 5 nM) recognition sites. However, the apparent density of [3H]LTB4 binding to intact human PMN (combined Bmax = 380 fmol/10(7) cells) was approximately 14% of that observed with [3H]CGS 23131. In ligand competition studies, various LTB4 agonists and antagonists were found to inhibit the binding of [3H]CGS 23131, revealing a pharmacological profile consistent with the specific labeling of the LTB4 receptor. A positive rank order correlation (r = 0.79) was observed between the ligand competition profiles obtained with [3H]CGS 23131 and [3H]LTB4. Both LTB4 and its omega oxidation product, 20-OH-LTB4, were found to inhibit the binding of 1.0 nM [3H]CGS 23131 in a biphasic fashion consistent with the existence of multiple affinity components of the LTB4 receptor. In competing for 0.5 nM [3H]LTB4 binding, these compounds were found to produce monophasic inhibition curves, which was indicative of a selective interaction at the high-affinity LTB4 receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

High affinity dextromethorphan binding sites in guinea pig brain. Effect of sigma ligands and other agents

Dextromethorphan (DM), a non-narcotic antitussive, binds in the guinea pig brain to specific high- and low-affinity sites with Kd values of 57 nM and 24 microM, respectively (Musacchio et al., 1988). The antitussives carbetapentane, caramiphen, butamirate and dimethoxanate competed with the high-affinity binding of [3H]DM at pH 7.4 with nanomolar Ki values. Sigma site ligands showed high affinity for [3H]DM binding sites. The rank order of potency was: haloperidol greater than (+)-pentazocine greater than (+)-cyclazocine greater than 3-(-3-hydroxyphenyl)-N-(1-propyl)piperidine greater than (+)-N-allylnormetazocine greater than (-)-butaclamol much greater than (+)-butaclamol (-)-N-allylnormetazocine. The antipsychotic perphenazine competed with low nanomolar Ki values, whereas rimcazole was weaker. The antidepressant opipramol and the benzomorphan (+)2'methoxyphenazocine were the most effective drugs tested, with Ki values of 0.4 nM. By contrast, MK-801 and phencyclidine hydrochloride were very weak competitors for [3H]DM binding. The diphenylalkylamines were the most effective competitors of the calcium channel blocking agents: prenylamine and cinnarizine had Ki values of 17 and 22 nM, respectively. Lidoflazine and hydroxyzine were slightly less potent, but nifedipine and the benzothiazepine diltiazem were much weaker. Potassium channel blockers inhibited DM binding in pharmacologically relevant concentrations: primaquine was the most effective with a Ki of 0.5 microM. Other antimalarial potassium channel blockers tested inhibited binding in the micromolar range. 4-Aminopyridine and tetraethylammonium had Ki values of 0.76 and 1.40 mM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific plasma membrane binding sites for polyphenols, including resveratrol, in the rat brain

Using [3H]resveratrol (3,5,4'-trihydroxy-trans-stilbene) as radioligand, we investigated the possible existence of specific polyphenol binding sites at the level of the cellular plasma membrane in rat brain. Specific [3H]resveratrol binding sites were found to be enriched in the plasma membrane pellet with lower levels in the nuclear and cell debris fraction. Specific [3H]resveratrol binding to the plasma membrane fraction was sensitive to trypsin digestion and protein denaturation but not to DNase and RNase treatment. Saturation binding experiments revealed that specific [3H]resveratrol recognized a single class of sites with an apparent affinity (KD) of 220+/-45 nM and a maximal capacity (Bmax) of 1060+/-120 fmol/mg protein. Various polyphenols and resveratrol derivatives competed against specific [3H]resveratrol binding in rat brain plasma membrane homogenates with the tea catechin gallates (epigallocatechin gallate and epicatechin gallate) displaying the highest affinities (Ki=25-45 nM) followed by resveratrol (Ki=102 nM). Quantitative autoradiographic studies revealed that specific [3H]resveratrol binding sites are broadly distributed in the rat brain, with highest levels of labeling seen in the choroid plexus and subfornical organ. Finally, the potency of various polyphenols and resveratrol analogs in protecting hippocampal cells against beta-amyloid-induced toxicity correlated well (r=0.74) with their apparent affinity in the [3H]resveratrol binding assay. Taken together, these results suggest that the neuroprotective action of various polyphenols and resveratrol analogs could be mediated by the activation of common "receptor" binding sites particularly enriched at the level of the cellular plasma membrane in the rat brain.     

Identification of a high-affinity peripheral-type benzodiazepine binding site in rat aortic smooth muscle membranes

The existence of a benzodiazepine binding site in rat aortic smooth muscle membranes was explored employing [3H]Ro5-4864 as radioligand. The binding site was concentrated in the mitochondrial fraction enriched with cytochrome c oxidase and semicarbazide-insensitive monoamine oxidase. [3H]Ro5-4864 binds to the membranes in the mitochondrial fraction with high affinity. The dissociation constant (KD) determined by saturation binding was 2.8 +/- 0.7 nM (n = 5). The association rate constant (k1) was 4.7 +/- 0.8 x 10(6) M1 min-1, and the dissociation rate constant (k-1) was 0.028 +/- 0.005 min-1 (n = 3). The kinetically determined KD was 6.0 +/- 0.8 nM (n = 3) at 0.5 nM [3H]Ro5-4864. The density of binding determined from saturation binding experiments was 14.0 +/- 1.2 pmol/mg protein (n = 5). The Hill coefficient of binding was 0.94 +/- 0.02 (n = 5) indicating that [3H] Ro5-4864 binds to a single site. The [3H]Ro5-4864 binding was inhibited by Ro5-4864 (Ki = 6.1 +/- 1.9 nM), PK 11195 (Ki = 8.9 +/- 1.8 nM), diazepam (Ki = 87.3 +/- 3.4 nM), flunitrazepam (Ki = 94.6 +/- 1.8 nM), clonazepam (Ki = 6.3 +/- 1.3 microM) and Ro15-1788 (Ki = 16.8 +/- 1.5 microM). The rank order of potency of the competitive inhibition of [3H]Ro5-4864 binding (Ro5-4864 = PK 11195 greater than diazepam = flunitrazepam much greater than clonazepam greater than Ro15-1788) is characteristic of the peripheral-type benzodiazepine binding site. The data indicate an abundant high affinity peripheral-type benzodiazepine binding site of unknown function in rat aortic smooth muscle cells.     

Guanyl-5'-yl-lmidodiphosphate regulation of ligand binding to LTD4 receptors on guinea pig lung membranes

We investigated the mechanism of guanyl-5'-yl-imidodiphosphate (GppNHp) regulation of peptidoleukotrienes (LTs) and LT-antagonists binding to LTD4 receptors on guinea pig lung membranes (GPLMs). In saturation experiments, [3H]LTD4 saturable (maximum binding = 943 +/- 39 fmol/mg of protein) binding to GPLM was significantly (P less than .01) inhibited by GppNHp (60 nM, maximum binding = 446 +/- 113 fmol/mg of protein) in a concentration-dependent manner. No significant change in the affinity (Kd = 0.29 +/- 0.02 nM vs. 0.43 +/- 0.12 nM for control and treated GPLM, respectively) for [3H]LTD4 was observed. The binding affinity for the selective LTD4 antagonist ICI 198,615 (Ki = 0.13 +/- 0.04 nM) as determined by competition against [3H]LTD4, was not changed by GppNHp. Saturation analysis of [3H]ICI 198,615 binding confirmed that GppNHp did not change the apparent affinity or site-density for this ligand. In competition experiments against [3H]-ICI 198,615, GppNHp (1 microM) caused a significant (P less than .01) rightward shift of the inhibition by agonists (94-, 50- and 8-fold shifts for LTD4, LTE4 and YM-17690, respectively). In contrast, inhibition of [3H]ICI 198,615 by four LTD4 antagonists (ICI 198,615, 4-[5-cyclopentylcarbonylamino-1-[3-cyanobenzyl] indol-3-yl-methyl]3-methoxybenzoic acid, 4-[5-cyclopentylcarbonylamino-3-chloroindol-1-y-methyl]3-met hoxybenzoic acid and FPL55712) was not affected by GppNHp. Taken together the data suggest that LTD4 receptors are coupled to a G-protein that modulates the affinity of agonists but not antagonists binding.     

D-Pen2,4'-125I-Phe4,D-Pen5]enkephalin: a selective high affinity radioligand for delta opioid receptors with exceptional specific activity.

[D-Pen2,4'-125I-Phe4,D-Pen5]enkephalin ([125I]DPDPE) is a highly selective radioligand for the delta opioid receptor with a specific activity (2200 Ci/mmol) that is over 50-fold greater than that of tritium-labeled DPDPE analogs. [125I]DPDPE binds to a single site in rat brain membranes with an equilibrium dissociation constant (Kd) value of 421 +/- 67 pM and a receptor density (Bmax) value of 36.4 +/- 2.7 fmol/mg protein. The high affinity of this site for delta opioid receptor ligands and its low affinity for mu or kappa receptor-selective ligands are consistent with its being a delta opioid receptor. The distribution of these sites in rat brain, observed by receptor autoradiography, is also consistent with that of delta opioid receptors. Association and dissociation binding kinetics of 1.0 nM [125I] DPDPE are monophasic at 25 degrees C. The association rate (k + 1 = 5.80 +/- 0.88 X 10(7) M-1 min-1) is about 20- and 7-fold greater than that measured for 1.0 nM [3H DPDPE and 0.8 nM [3H] [D-Pen2,4'-Cl-Phe4, D-Pen5]enkephalin, respectively. The dissociation rate of [125I]DPDPE (0.917 +/- 0.117 X 10(-2) min-1) measured at 1.0 nM is about 3-fold faster than is observed for either of the other DPDPE analogs. The rapid binding kinetics of [125I]DPDPE is advantageous because binding equilibrium is achieved with much shorter incubation times than are required for other cyclic enkephalin analogs. This, in addition to its much higher specific activity, makes [125I]DPDPE a valuable new radioligand for studies of delta opioid receptors.     

Cholinergic receptors in renal medullary collecting duct cells

Intrarenal administration of cholinergic agents produces diuresis. However, neither cholinergic innervation or specific cholinergic receptors have been shown to be present in the kidney. Recently, we have demonstrated that carbachol, a cholinergic agent, stimulates phosphoinositide hydrolysis in the inner medullary collecting duct (IMCD) cells. The effect was blocked by atropine (a cholinergic antagonist), suggesting that phosphoinositide hydrolysis occurs through the interaction of carbachol with specific cholinergic receptors in these cells. Therefore, we examined the cholinergic receptors in IMCD cells by measurement of radioligand binding of a cholinergic receptor antagonist, I-quinuclidinyl (phenyl-4-3H)benzilate([3H]QNB). The IMCD cells were prepared from rabbit kidneys by incubating the inner medullary slices with collagenase and treating the isolated cells with hypotonic solution to lyse cells other than IMCD cells. Binding of [3H]QNB to IMCD cells was measured at 37 degrees C for 60 min in the absence (total binding) and the presence (nonspecific binding) of 100 microM atropine (a muscarinic receptor antagonist). The specific binding (the difference between total and nonspecific binding) of [3H]QNB to IMCD cells was saturable with a Bmax (maximum binding sites) of 27.5 fmol/mg of protein and Kd (dissociation constant) of 0.27 nM. Atropine, but not hexamethonium (a nicotinic antagonist), was able to displace [3H]QNB from IMCD cells with a Ki of 0.1 microM. It is, therefore, concluded that specific high affinity muscarinic receptors are present in IMCD cells. These receptors may play a role in producing the pharmacologic actions of cholinergic agents on the kidney.     

5-HT 2 Serotonin Receptor on Blood Platelet of Migraine Patients

Several lines of evidence have previously suggested that platelets might play a primary or secondary role in migraine pathogenesis, and that in addition serotonin receptors on human platelets might be involved in those processes. By using [3H]-spiperone as a radioligand and ketanserin to determine the non-specific binding, the numbers of 5-HT2 serotonin receptors on platelet membrane obtained from normal healthy and migraine subjects were determined. We found a significant decrease (p < 0.02) in the maximal receptor numbers (Bmax) on platelet membrane of migraine patients (Bmax = 33.01 +/- 5.57 fmol/mg protein) when compared to the normal healthy group (Bmax = 86.59 +/- 9.09 fmol/mg protein) whereas the dissociation equilibrium constant (Kd) values (2.47 +/- 0.44 nM and 3.41 +/- 0.95 nM for the normal and migraine subjects, respectively) remained unchanged. The platelet response showed a higher degree of aggregability in migraine subjects, whereas the platelet counts were the same. This finding implies that serotonin receptors on the platelet may reflect some aspect of the pathogenesis of migraine headache.     

Quantitative film autoradiography of opiate agonist and antagonist binding in rat brain

The binding of a radiolabeled opiate agonist ([3H]etorphine) and antagonist ([3H]naloxone) was studied using quantitative film autoradiography of rat-brain sections labeled by in vitro dipping methods. The binding activities of both [3H]naloxone and [3H] etorphine were saturable in three brain regions: noncluster striatum, nucleus accumbens and cingulate cortex. Eadie-Hofstee analysis of these regions yielded the following binding affinities and capacities: noncluster striatum binding affinity (KD) +/- S.E. = 1.59 +/- 0.23 nM, maximal binding capacity (Bmax) +/- S.E. = 28.3 +/- 1.9 fmol/mg, S.D. error of the raw data (Erad) = 6.4%; nucleus accumbens, KD +/- S.E. = 1.74 +/- 0.28 nM, Bmax +/- S.E. = 73.3 +/- 5.2 fmol/mg, S.D. (Erad) = 6.2%; cingulate cortex, KD +/- S.E. = 1.44 +/- 0.15 nM, Bmax +/- S.E. = 37.6 +/- 1.4 fmol/mg, S.D. (Erad) = 2.5%. A KD +/- S.E. = 1.72 +/- 0.29 nM, Bmax +/- S.E. = 74.1 +/- 5.3 fmol/mg, S.D. (Erad) = 5.0% was found for [3H]etorphine binding in the noncluster striatum. Hill plots of both [3H]naloxone and [3H]etorphine binding in noncluster striatum demonstrated an absence of cooperativity with slopes of 1.01 and 1.07, respectively. Stereospecificity of binding was confirmed by competition for 2.0 nM [3H]naloxone in the noncluster striatum with a levorphanol IC50 = 5.5 nM and a dextrorphan IC50 greater than 1000 nM. Rank order potency for competition for 2.0 nM [3H]naloxone binding in noncluster striatum was etorphine greater than naloxone greater than levorphanol greater than morphine greater than dextrorphan. The regional order of binding activities (femtomoles per milligram +/- S.D.) for 2.0 nM [3H]naloxone was as follows: striatal clusters (111.1 +/- 24.5) greater than interpeduncular nucleus (77.8 +/- 10.1) greater than central nucleus of amygdala (64.5 +/- 9.7) greater than nucleus accumbens (34.4 +/- 6.9) greater than median raphe (24.4 +/- 6.1) greater than striatal noncluster (23.3 +/- 3.5) greater than superior colliculus striatum grieseum (22.2 +/- 4.0). Thus, quantitative film autoradiography of brain sections labeled in vitro may be used to characterize the pharmacological binding properties of ligands in many small brain regions not amendable to study in membrane preparations.     

Leukotriene B4 and leukotriene B5 have binding sites on lung membranes and cause contraction of bullfrog lung.

Leukotriene (LT)B4 and LTB5 cause contraction of isolated bullfrog lung. LTB4 receptors were characterized in membranes prepared from bullfrog lung. Binding of [3H]LTB4 was maximal at 5 min and was reversible with the addition of 1000-fold excess LTB4. Scatchard analysis indicated a single class of binding sites with a Kd of 2.22 nM and a Bmax of 1228.86 fmol/mg protein. The Kd and the Bmax values in the presence of guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) were 2.76 nM and 1289.61 fmol/mg protein, respectively. The Ki values for LTB4, LTB5 and 20(OH)-LTB4 were 5.5, 30.5 and 144.0 nM, respectively, whereas 20(COOH)-LTB4 was ineffective in preventing binding of [3H] LTB4 from 10(-9) to 10(-5) M. The peptide leukotrienes LTC4, LTD4 and LTE4 failed to inhibit the specific binding of [3H]LTB4. GTP gamma S in concentrations from 10(-10) to 10(-4) M did not affect the binding of 5 nM [3H]LTB4. Neither the mammalian LTD4 antagonist LY171883 nor the mammalian LTB4 antagonist LY255283 was an effective competitor for the bullfrog lung LTB4 receptor. In addition, sulfhydryl-modifying reagents NEM and PCMP did not affect LTB4 binding as they do in mammalian membrane preparations. The LTB4 receptor shows some differences from the described mammalian receptor. The cell type containing the LTB4 receptor remains to be determined.     

Effect of Hoe 140, a new B2 noncompetitive antagonist, on guinea pig tracheal bradykinin receptors.

We investigated the effect of the new B2 antagonist D-Arg0[Hyp3,Thi5, D-Tic7, Oic8]bradykinin (BK) (Hoe 140) on the binding of [3H]BK to membranes from guinea pig trachea with respect to the presence of the epithelium. Scatchard analysis of equilibrium data with [3H]BK revealed a single class of binding sites in the epithelium denuded trachea membrane preparation (E-) with a dissociation constant (Kd) of 55 pM and a Bmax of 0.71 fmol.mg tissue-1. When intact trachea (E+) was used, two binding sites were detected: a saturable high-affinity one (Kd of 40 pM and Bmax of 0.69 fmol.mg tissue-1) and a low-affinity one, not really saturable, with a Kd over 180 nM and a Bmax over 18 fmol.mg tissue-1. In guinea pig ileum, a tissue thought to contain B2 receptors, one class of binding sites was detected with a Kd of 209.3 pM and a Bmax of 16.2 fmol.mg tissue-1. In competition experiments ([3H]BK from 0.3 to 0.5 nM), similar results were obtained in (E +/-) and in ileum membrane preparations. B1 ligands did not displace [3H]BK binding, demonstrating the lack of B1 receptors. BK and B2 antagonist, except Hoe 140, fully displaced [3H]BK with Hill coefficients close to the unity. In competition studies only the high-affinity site was labeled by [3H]BK, in the (E+) preparation, as suggested by the inhibition constant value of unlabeled BK. Hoe 140 fully displaced [3H]BK in competition experiments, but with a Hill coefficient significantly less than unity, suggesting the presence of two binding sites for this compound in the three preparations used.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serotonergic neurons do not influence the regulation of beta adrenoceptors induced by either desipramine or isoproterenol.

It has been suggested that 5-hydroxytryptamine (serotonin)-containing neurons influence the regulation of central beta adrenoceptors caused by antidepressants. [3H]Dihydroalprenolol ( [3H] DHA) was the radioligand used in these previous studies to measure beta adrenoceptors. In this study, we compared the binding characteristics of [3H]DHA with those of [125I]iodopindolol ( [125I]PIN) and used [125I]IPIN to study effects of lesioning serotonergic nerves on the regulation of beta adrenoceptors. A comparison was made in homogenates prepared from rat frontal cortex of the specific binding of [3H]DHA with that of [125I]IPIN to beta adrenoceptors. Nonlinear regression analysis of saturation experiments of [3H]DHA binding to cortical homogenates indicated that a two-component binding model fit the data significantly better than a one-component model. A dissociation constant value of 0.47 +/- 0.16 nM and a Bmax value of 62 +/- 7 fmol/mg protein were obtained for the high-affinity site. The low-affinity site was poorly defined. Rosenthal transformations of the saturation isotherms for [3H]DHA binding were clearly curvilinear. By contrast, nonlinear regression analysis of saturation experiments of the binding of [125I]IPIN indicated that the binding of this radioligand was described adequately by a one-component model and yielded a dissociation constant value of 147 +/- 10 pM with a Bmax of 80 +/- 5 fmol/mg protein. Rosenthal transformations of the [125I]IPIN data were linear. From such data, it was inferred that [3H]DHA binds to some site in addition to beta adrenoceptors, whereas [125I]IPIN does not.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of prolonged phenylephrine infusion on cardiac adrenoceptors and calcium channels

Effects of prolonged in vivo infusion of phenylephrine upon receptor binding and cardiac contractility were studied in Sprague-Dawley rats. A 1-hr i.v. infusion of phenylephrine (3 mg/kg/hr) resulted in a sustained 50% increase in diastolic blood pressure and 5% increase in heart rate. Chronic (6-day) infusion (3 mg/kg/hr) utilizing Alzet mini-osmotic pumps maintained plasma concentrations of phenylephrine at 1.0 microgram/ml, depleted myocardial norepinephrine stores 8-fold and resulted in a modest cardiac hypertrophy. Density and affinity of myocardial adrenoceptors and calcium channels were quantified by analyzing saturation isotherms of radioligand binding. [3H]Prazosin, [3H]dihydroalprenolol and [3H]nitrendipine bound specifically and with high affinity to cardiac alpha-1 and beta adrenoceptors and calcium channels, respectively. As measured by Scatchard analyses, phenylephrine infusion significantly decreased the maximum number (Bmax) of specific [3H]prazosin binding sites by 39% (430 +/- 20 vs. 263 +/- 16 fmol/mg of protein; P less than .05). Chronic phenylephrine treatment also decreased the Bmax for [3H]dihydroalprenolol binding by 31% (124 +/- 3.3 vs. 86 +/- 6.6 fmol/mg of protein; P less than .05) and the Bmax for [3H]nitrendipine binding by 32% (342 +/- 8.8 vs. 235 +/- 6.7 fmol/mg of protein; P less than .05). Binding affinities (Kd) of [3H]prazosin, [3H]dihydroalprenolol and [3H]nitrendipine remained unchanged. Administration of vehicle alone or surgical manipulation due to osmotic pump implantation did not affect either the density or affinity of [3H]prazosin, [3H]dihydroalprenolol or [3H]nitrendipine binding. Contractile responses to phenylephrine were studied in isolated ventricular strips to determine the functional significance of alpha-1 adrenoceptor down-regulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional validation of platelet-activating factor receptor sites characterized biochemically by a specific and reproducible [3H]platelet-activating factor binding in human platelets

In human platelet membranes, [3H]platelet-activating factor(PAF)-C18 binding sites exhibited high affinity (Kd 0.074 +/- 0.005 nM, n = 28 healthy volunteers), saturability, elevated stereoselectivity, marked pharmacological specificity and small intersubject variability. The maximal binding capacity was 215 +/- 12 fmol/mg protein. Saturation of [3H]PAF binding was obtained with 0.3 nM ligand, and its isotherm was compatible with a single class of binding sites. The stereoselectivity for [3H]PAF was clearly indicated by the low displacing potency of enantio-PAF-C16 (the synthetic enantiomer of PAF) that was 5000-fold less potent than PAF. Specific [3H]PAF binding attained 65% with 0.1 nM ligand and was displaced fully not only by cold PAF but also by RP 59227 (Ki = 6.2 +/- 1.3 nM, n = 7), a novel, potent and specific PAF receptor antagonist in a pure enantiomeric form and several other antagonists such as CV-6209, WEB 2086, L-652,731 and BN 52021. Various classical pharmacological agents did not interfere with the [3H]PAF binding. In intact platelets, [3H]PAF binding shared the same properties as those just described for membrane preparations. A functional role for these binding sites was suggested by the high correlation (r = 0.94, P less than .001) between the Ki values for several known PAF antagonists determined in [3H]PAF binding and the IC50 values obtained against PAF-induced aggregation in whole platelets. Thus, the present [3H]PAF binding in human platelet membranes may be a useful pharmacological tool to study possible changes in [3H]PAF binding parameters induced by pathological states for which PAF may be directly or indirectly responsible.