Respective Bmax Values Research Articles

Characterization of 5-hydroxytryptamine receptors on the isolated pig basilar artery by functional and radioligand binding studies.

5-Hydroxytryptamine (5-HT)-receptor subtypes on pig basilar arteries were investigated by measuring the contractile responses to 5-HT agonists, the effects of antagonists on the responses and by carrying out a radioligand binding assay with [3H]5-HT. The rank order of contractile agonist potency (according to the pEC50 values) was 5-carboxamidotryptamine > or = 5-HT > alpha-methyl-5-HT > (+/-)-8-hydroxy-dipropylaminotetralin. The contractile responses were not affected by endothelial denudation, and the 5-HT-induced contractions were antagonized competitively by ketanserin. Methiothepin shifted the 5-HT concentration-response curves to the right and downwards in a concentration-dependent manner. In the presence of ketanserin (10(-6) M), however, methiothepin antagonized the 5-HT-induced contractions competitively. Specific [3H]5-HT binding to 5-HT receptors was saturable, reversible and showed high (Kd, 2.5 nM) and low (Kd, 710 nM) affinities, with respective Bmax values of 29.5 and 1950 fmol/mg protein. These results indicate that both 5-HT1 and 5-HT2 receptors are present on pig basilar arterial smooth muscle cells, and their stimulation results in contraction.

Identification du récepteur à l'angiotensine II et détermination de ses sous-types dans les annexes foetales du lapin et du cobaye

Membrane angiotensin II receptors were measured in trophoblastic tissues using a 2-step procedure. The first step consisted of the relative measurement performed at a fixed 125I[Sar1 Ile8]AII concentration of 0.15 nM in order to determine which tissues had a sufficient number of binding sites for studying the competition curves. The second consisted of determining the maximal binding (Bmax) and the dissociation constant (Kd) for [Sar1 Ile8] AII and the receptor subtypes in these tissues. The relative binding measurement revealed a significant number of occupied sites in rabbit fetal placenta and chorion (159 +/- 17 and 51 +/- 10 fmol/mg proteins) and in guinea pig chorion (132 +/- 12). The mean values of the other trophoblastic tissues were 3-10-fold lower in the 2 species. The competition curves obtained from tissues with high angiotensin II binding receptors showed the predominance of the AT2 subtype in rabbit fetal placenta (AT1/AT2 = 25/75) and of the AT1 receptor in guinea pig chorion (97/3) and in rabbit chorion (90/10). The [SAR1 Ile8] AII affinity (Kd) obtained from Scatchard plot analysis was 1.2 +/- 0.2 nM (n = 5) in fetal placenta and 1.2 (n = 1) in rabbit chorion and 0.5 +/- 0.1 (n = 3) in guinea pig chorion. In these tissues, the respective Bmax values were 1,281 +/- 115 (n = 5), 263 (n = 1) and 1,188 +/- 134 fmol/mg proteins (n = 3). These findings indicate that rabbit fetal placenta and chorion and guinea pig chorion are the most important sites of action for the renin-angiotensin system present in trophoblastic tissues.

Solubilized Rat Brain Adenosine Receptors Have Two High‐Affinity Binding Sites for l, 3‐Dipropyl‐8‐Cyclopentylxanthine

The specific binding of L-N6-[3H]phenylisopropyladenosine (L-[3H]PIA) to solubilized receptors from rat brain membranes was studied. The interaction of these receptors with relatively low concentrations of L-[3H]PIA (0.5-12.0 nM) in the presence of Mg2+ showed the existence of two binding sites for this agonist, with respective dissociation constant (KD) values of 0.24 and 3.56 nM and respective receptor number (Bmax) values of 0.28 +/- 0.03 and 0.66 +/- 0.05 pmol/mg of protein. In the presence of GTP, the binding of L-[3H]PIA also showed two sites with KD values of 24.7 and 811.5 nM and Bmax values of 0.27 +/- 0.09 and 0.93 +/- 0.28 pmol/mg of protein for the first and the second binding site, respectively. Inhibition of specific L-[3H]PIA binding by 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) (0.1-300 nM) performed with the same preparations revealed two DPCPX binding sites with Ki values of 0.29 and 13.5 nM, respectively. [3H]DPCPX saturation binding experiments also showed two binding sites with respective KD values of 0.81 and 10.7 nM and respective Bmax values of 0.19 +/- 0.02 and 0.74 +/- 0.06 pmol/mg of protein. The results suggest that solubilized membranes from rat brain possess two adenosine receptor subtypes: one of high affinity with characteristics of the A1 subtype and another with lower affinity with characteristics of the A3 subtype of adenosine receptor.

Solubilization of peripheral benzodiazepine-binding sites from rat kidney

The ability of a variety of detergents to solubilize peripheral benzodiazepine-binding sites from rat kidney was tested. Of all the detergents tested, only digitonin was found to be suitable for solubilization. This detergent solubilized 21% of the binding activity; 47% was inactivated, and 32% remained in the pellet. Specific binding of [3H]Ro 5-4864 to membrane-bound and solubilized peripheral benzodiazepine-binding sites was saturable, yielding a linear Scatchard plot (r = 0.96). KD values obtained for the membrane-bound and solubilized peripheral benzodiazepine binding sites were 3.9 +/- 0.4 nM and 5.4 +/- 0.4 nM, respectively. Respective Bmax values were 4.6 +/- 0.5 and 1.9 +/- 0.2 pmol/mg of protein. The KD value for the solubilized material obtained from kinetic experiments was 5.3 +/- 0.6 nM. The potency of PK 11195, Ro 5-4864, diazepam, flurazepam, chlordiazepoxide, Ro 15-1788, methyl-beta-carboline-3-carboxylate, and clonazepam to displace bound [3H]Ro 5-4864 from peripheral binding sites was similar in the membrane-bound and the soluble states. Most of the binding activity of the solubilized binding sites was destroyed by heating at 60 degrees C for 30 min or by treatment with 2 M guanidinium chloride or 4 M urea. More than 95% of the binding activity of the solubilized binding sites was retained after 18 hr at 4 degrees C, and more than 60% was retained after 4 days at the same temperature. These results indicate that the binding characteristics of peripheral benzodiazepine-binding sites extant in the membrane-bound state are retained after solubilization.

Bradykinin receptor-mediated cyclic GMP formation in a nerve cell population (murine neuroblastoma clone N1E-115).

A clone of murine neuroblastoma (N1E-115) was shown to have functional receptors for the nonapeptide bradykinin. These receptors mediated a large, rapid (about 1 min to peak) and calcium-dependent increase in cyclic GMP. The median effective concentration (EC50) averaged 1.4 nM. In addition, this event was inhibited by quinacrine, 5,8,11,14-eicosatetraynoic acid, and nordi-hydroguaiaretic acid, suggesting involvement of phospholipase A2 with subsequent formation of lipoxygenase metabolities of arachidonic acid. [3H]Bradykinin binding to intact cells, investigated under conditions nearly identical to those used in the cyclic GMP assay, yielded binding sites with KDS of 0.83 pM, 1.0 nM, and 4.9 nM with respective Bmax values of 12, 160, and 250 fmol/10(6) cells. Apparently, the cyclic GMP response was associated with the binding site in which the KD = 1.0 nM. Peptide analogs of bradykinin stimulated cyclic GMP with EC50S nearly identical to their respective KDS determined in binding assays with [3H]bradykinin, thus providing evidence for receptor specificity of this response. This finding of a biochemical response of bradykinin promises to make N1E-115 cells a convenient model system for study of neuronal bradykinin receptors.

Characterization of adenosine receptors in brain using N 6 cyclohexyl [ 3H]adenosine

The presence of distinct binding sites for adenosine in both the CNS and PNS has been proposed in numerous studies. The recent availability of stable adenosine analogues such as cyclohexyladenosine, 2-chloroadenosine and diethylphenylxanthine has made the characterization of such a receptor feasible. In the present report the binding of N6 cyclohexyl [3H]adenosine ([3H]CHA) to rat brain synaptosomal membranes is characterized. [3H]CHA binding is saturable and exhibits a biphasic kinetic saturation profile characteristic of 2 binding sites. The high affinity site has a Kd of 0.7 nM and the low affinity site 2.4 nM. The respective Bmax values are 230 and 120 fmol/mg protein in fat forebrain. The highest density of binding sites is found in the hippocampus and subcellular distribution studies indicate that the [3H]CHA site is predominantly synaptosomal. [3H]CHA binding is highly dependent in the presence of adenosine deaminase since only 30% of the binding capacity is observed in synaptosomal membranes not treated with this enzyme. Of the many cations and anions tested only copper and zinc have effects on [3H]CHA binding. Both metals are potent inhibitors of binding with copper having an IC50 of 30 microM and zinc 150 microM. Sulfhydryl reducing and alkylating agents also inhibit binding indicating that the binding site is a sulfhydryl-dependent protein.