guanylyl-imidodiphosphate and apraclonidine

In nonsurgical management of benign prostatic hyperplasia, drugs which interfere with prostate contraction mediated through the alpha-1 adrenergic receptor are used. Clonidine acts at alpha adrenergic and I1-imidazoline receptors. In the present study, we found the Kd for [3H]clonidine binding to I1 sites in canine prostate to be 4 +/- 1 nM; the Bmax was 18 +/- 2 fmol/mg of protein. Inhibition of binding by imidazolines and by brain extracts containing putative endogenous ligand confirmed the identity of these sites as I1-imidazoline. Autoradiographic studies showed localization of both I1 and alpha-2 sites to the glandular epithelium. We sought to determine whether in vivo activation of the I1-imidazoline sites by clonidine mediates its contractile action in canine prostate. Dose-response curves were generated for para-aminoclonidine in the presence of vehicle alone, yohimbine (alpha-2 antagonist), idazoxan (alpha-2/I1/I2 antagonist) and prazosin (alpha-1 antagonist). Prazosin was the most effective antagonist. Yohimbine was less effective and did not effectively discriminate between para-aminoclonidine and phenylephrine, an alpha-1-selective agonist. Idazoxan antagonized para-aminoclonidine, but by not more than 50% at any dose. These results suggest that clonidine is active primarily at alpha-1 receptors on prostate smooth muscle in vivo. Thus the function of the I1 and alpha-2 receptors in the prostate remains to be determined; however, they may be involved in epithelial cell function.

Topics: Animals; Autoradiography; Azepines; Binding Sites; Clonidine; Dogs; Guanylyl Imidodiphosphate; Imidazoles; Imidazoline Receptors; Male; Prostate; Prostatic Hyperplasia; Receptors, Adrenergic, alpha; Receptors, Drug

The pharmacological identification and characterization of subtypes of alpha 2-adrenergic receptors have been confirmed by molecular biological investigations. Using receptor autoradiographic techniques, it has been possible to show regions of the brain where alpha 2 agonist binding ([3H]para-aminoclonidine) is preferentially labeling the presumed guaninenucleotide-sensitive, high-affinity conformations of the alpha 2 receptor. Careful examination of autoradiograms generated using the tritiated antagonists yohimbine, idazoxan, and rauwolscine also indicates some disparity in the regions occupied by these radiolabeled ligands. Inhibition of [3H]rauwolscine binding with the subtype selective compounds, ARC-239, or oxymetazoline demonstrates that there are discrete regions of the brain where one receptor subtype predominates over the other. These studies indicate that previous investigations utilizing the agonist para-aminoclonidine as the ligand for obtaining labeling of alpha 2 receptors have overlooked some regions of binding due to the subtype selectivity of this ligand. A more complete localization of alpha 2-adrenergic receptors can be obtained using the tritiated antagonist rauwolscine, and the differential distribution of at least two subtypes of the alpha 2 receptor can be obtained by selective inhibition of this binding.

Topics: Adrenergic alpha-Agonists; Adrenergic alpha-Antagonists; Animals; Autoradiography; Brain Chemistry; Clonidine; Dioxanes; Guanylyl Imidodiphosphate; Idazoxan; Male; Perfusion; Rats; Rats, Inbred Strains; Receptors, Adrenergic, alpha; Tritium; Yohimbine

alpha-Adrenoceptors in spinal cord appear to play a role in a number of physiologic processes including the control of blood pressure, pain and motor function. In order to evaluate more clearly these potential roles, the characteristics of binding of [3H]prazosin ([3H]PRZ) to spinal alpha 1 adrenoceptors and [3H]p-aminoclonidine ([3H]PAC) to spinal alpha 2 adrenoceptors were determined. Binding of each ligand to their respective adrenoceptors was saturable and Scatchard analysis revealed binding of each to a single class of adrenoceptors with characteristics of [3H]PRZ binding of Bmax = 78 fmol/mg protein and Kd = 0.75 nM and [3H]PAC binding Bmax = 70 fmol/mg protein and Kd = 1.39 nM. Whereas [3H]PRZ specific binding (Bmax) was unaltered by guanine nucleotides. [3H]PAC binding was increased with addition of 10 microM guanosine triphosphate (GTP) (P less than 0.05) and decreased with either 50 microM GTP or guanyl-5'-yl-imidodiphosphate [Gpp(NH)p] (P less than 0.01). Competition for specific [3H]PRZ and [3H]PAC binding by various alpha 1 and alpha 2 adrenoceptor agonists and antagonists of known pharmacologic activity revealed that [3H]PRZ defines alpha 1 adrenoceptors (Ki = 2.1 nM for prazosin vs 4300 nM for yohimbine) and [3H]PAC defines alpha 2 adrenoceptors (Ki = 1.06 nM for yohimbine vs 15480 nM for prazosin). Regional spinal cord studies demonstrated that dorsal spinal cord in the lumbar region contains the highest density of both [3H]PRZ (Bmax = 93 +/- 14 fmol/mg protein) and [3H]PAC (Bmax = 101 +/- 6 fmol/mg protein) binding. In contrast, lowest binding was evident in thoracic cord with equal levels in both dorsal and ventral regions (Bmax = 44-48 fmol/mg protein). The regional distribution of both alpha 1 and alpha 2 adrenoceptors in spinal cord compares to the localization previously classified functionally utilizing various pharmacological agonists and antagonists at norepinephrine receptors.

Topics: Animals; Binding, Competitive; Cell Membrane; Clonidine; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Kinetics; Male; Prazosin; Rats; Rats, Inbred Strains; Receptors, Adrenergic, alpha; Spinal Cord

The agonist- and antagonist-binding properties of the alpha 2-adrenergic receptor in a purified plasma membrane preparation from human platelets were determined both by direct binding of radiolabeled ligands and by competition with the labeled alpha 2-antagonist, [3H] yohimbine. Binding of [3H]yohimbine was characterized by a single high affinity binding site (Kd = 6.2 +/- 1.4 nM, Bmax = 507 +/- 53 fmol/mg). In direct binding studies, the imidazoline full alpha 2-agonist, [3H]-5-bromo-6-N(2-4,5-dihydroimidazolyl)quinoxaline ([3H] UK 14,304), bound to only one quantifiable high affinity site (Kd = 0.88 +/- 0.17 nM), representing 65 +/- 6% of the number of [3H]yohimbine sites. Binding of the partial agonist [3H]-p-aminoclonidine (PAC) showed nonlinear Scatchard plots. Analysis according to a model of multiple independent binding sites showed the data to be consistent with two sites (Kd1 = 0.62 +/- 0.18 nM and Kd2 = 7.9 +/- 1.4 nM). The high affinity site corresponded to 15 +/- 6% and the low affinity site corresponded to 39 +/- 6% of the number of [3H]yohimbine sites. Competition for binding of the alpha 2-antagonist, [3H]yohimbine, with nonradiolabeled ligands revealed a single affinity for yohimbine. In contrast, competition for [3H]yohimbine binding by the full agonist UK 14,304 and epinephrine is best fit by a model with two independent binding sites. The partial agonist PAC was best characterized by a model with three distinct binding sites. The full agonists UK 14,304 and epinephrine inhibited adenylate cyclase approximately 30%, whereas PAC produced only 12% inhibition. The inhibitory guanine nucleotide-binding protein (Ni) with Mr 40,700 was the sole pertussis toxin substrate in the purified membranes. It was quantitated by pertussis toxin-catalyzed [32P]ADP ribosylation in cholate extracts. There is a 20- to 100-fold excess of Ni over alpha 2-adrenergic receptors. Comparisons made between the experimental data for agonist binding and theoretical predictions of the simple ternary complex model suggest that there is compartmentalization of Ni and/or that the alpha 2 receptors are heterogeneous.

Topics: Adenosine Diphosphate Ribose; Adenylyl Cyclase Inhibitors; Adrenergic alpha-Agonists; Adrenergic alpha-Antagonists; Blood Platelets; Brimonidine Tartrate; Clonidine; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Humans; In Vitro Techniques; NAD; Quinoxalines; Receptors, Adrenergic, alpha; Yohimbine