guanosine-triphosphate and apraclonidine

In the presence of RS-15385-197 to preclude binding to alpha 2-adrenoceptors, [3H]p-aminoclonidine labelled a low affinity high capacity site, (Kd = 127.6 +/- 19.7 nM, Bmax 978 +/- 172 fmol/mg protein) whereas [3H]idazoxan labelled a high affinity low capacity site (Kd = 1.66 +/- 0.28 nM, Bmax 45.3 +/- 11.4 fmol/mg protein). Clonidine and p-aminoclonidine showed moderate affinity for the site labelled by [3H]p-aminoclonidine, but low affinity for the site labelled by [3H]idazoxan, whereas idazoxan showed high affinity for [3H]idazoxan and low affinity for [3H]p-aminoclonidine binding. Naphazoline inhibited [3H]idazoxan in a biphasic manner suggesting that [3H]idazoxan may label an heterogeneous population of imidazoline sites. GTP inhibited [3H]idazoxan but not [3H]p-aminoclonidine binding. These results suggest that [3H]idazoxan labelled imidazoline I2 binding sites, whereas [3H]p-aminoclonidine labelled a novel subtype which showed marked differences to the imidazoline I1 binding site reported in bovine and human brainstem.

Topics: Adrenergic alpha-Antagonists; Animals; Binding, Competitive; Clonidine; Dioxanes; Guanosine Triphosphate; Idazoxan; Imidazoles; Imidazoline Receptors; In Vitro Techniques; Isoquinolines; Kidney; Male; Naphazoline; Naphthyridines; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, Drug; Tritium

The interaction between alpha 2- and beta-adrenergic receptors was investigated in rat cerebral cortical membranes. Clonidine inhibition of [3H]dihydroalprenolol ([3H]DHA) binding resulted in biphasic competition curves with a mean Hill coefficient of 0.45. The addition of 1 microM yohimbine caused a rightward shift of the first portion of the clonidine inhibition curve. In the presence of 1 microM clonidine, the maximum concentration which did not inhibit [3H]DHA binding, inhibition curves of [3H]DHA binding by isoproterenol shifted to the right. A mean Hill coefficient increased from a control value of 0.63 to 0.76. Computer modeling analysis revealed that 1 microM clonidine decreased a beta-adrenergic high-affinity state from 28% to 13%. However, the addition of 1 microM yohimbine completely prevented the clonidine-induced reduction in the beta-adrenergic high-affinity state. In the presence of 200 microM GTP, the effect of clonidine was not observed. In addition, Kd and Bmax values for [3H]p-aminoclonidine ([3H]PAC) binding were not significantly changed by the addition of 100 nM isoproterenol, the maximum concentration which did not inhibit [3H]PAC binding. Moreover, isoproterenol inhibition of [3H]PAC binding resulted in steep competition curves with a mean Hill coefficient of 0.97. The addition of 1 microM alprenolol did not affect the isoproterenol inhibition curve. These data demonstrated that clonidine caused a decrease in agonist and antagonist affinity for beta-adrenergic receptors, while isoproterenol did not modulate the binding characteristics of alpha 2-adrenergic receptors. Furthermore, these results suggest that regulation between alpha 2- and beta-adrenergic receptors is not bidirectional, but is instead unidirectional from alpha 2-adrenergic receptors to beta-adrenergic receptors.

Topics: Animals; Binding, Competitive; Cerebral Cortex; Clonidine; Dihydroalprenolol; Guanosine Triphosphate; Isoproterenol; Male; Rats; Rats, Inbred Strains; Receptors, Adrenergic, alpha; Receptors, Adrenergic, beta; Sympathomimetics; 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

A simple large-scale purification of alpha 2-adrenergic receptor-enriched membranes from human platelets is described. Binding of the antagonist [3H]yohimbine is enriched 3-5-fold compared to a crude membrane fraction. Binding of low concentrations of the partial agonist 3-H-rho-aminoclonidine is increased 15-20-fold due to a higher binding affinity for the purified membranes. A soluble inhibitor of 3H-rho-aminoclonidine binding to purified membranes is found even in thrice-washed crude platelet membranes. The guanine nucleotides GDP and GTP are found to account for this inhibitory activity. Forskolin-stimulated adenylate cyclase activity is also enriched in the purified membrane fraction. Adenylate cyclase activity is inhibited by alpha 2-agonist to a comparable extent in all membrane fractions. This membrane preparation should prove useful in studies of alpha 2-adrenergic receptor mechanisms.

Topics: Adenylyl Cyclases; Blood Platelets; Cell Fractionation; Cell Membrane; Centrifugation, Density Gradient; Clonidine; Colforsin; Epinephrine; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Norepinephrine; Receptors, Adrenergic, beta; Tritium; Yohimbine

We have characterized the alpha 2-adrenergic receptor in membranes from the human colonic adenocarcinoma cell line, HT29, using the recently introduced alpha 2-agonist 5-bromo-6-[2-imidazolin-2-yl-amino]quinoxaline [( 3H]UK-14,304), two other radioligands, and a series of adrenergic agonists and antagonists. We also investigated alpha 2-agonist inhibition of HT29 cell adenylate cyclase and reversal of inhibition by alpha-adrenergic antagonists. [3H] Yohimbine saturation experiments indicated a single class of sites with a KD of 0.61 nM which agreed with the kinetically determined KD of 0.62 nM. Computer analysis of kinetic and saturation experiments with [3H]UK-14,304 revealed two classes of sites. From the saturation data, one site had high affinity for the radioligand (0.14 nM) and comprised 33% of the total number of sites, whereas the other site had lower affinity (6.1 nM). The total number of sites labeled by [3H]UK-14,304 (360 fmol/mg of protein) was approximately equal to the number of sites labeled by [3H]yohimbine (330 fmol/mg), whereas [3H]para-aminoclonidine labeled fewer sites of a single class. Rank order potencies of adrenergic agonists and antagonists obtained from competition binding assays indicated that: the same receptors were labeled by the three radioligands, and the receptors were of the alpha 2 subtype. UK-14,304 and epinephrine inhibited forskolin- and vasoactive intestinal peptide-stimulated adenylate cyclase in a dose-dependent manner up to 32%. Inhibition of the enzyme was reversed by yohimbine and, less potently, by phentolamine and prazosin in a dose-dependent manner. The HT29 cell line appears to be a useful model system for the investigation of the regulation and mechanism of action of alpha 2-adrenergic receptors in human tissues.

Topics: Adenocarcinoma; Adenylyl Cyclase Inhibitors; Adrenergic alpha-Agonists; Brimonidine Tartrate; Cell Line; Clonidine; Colforsin; Colonic Neoplasms; Dose-Response Relationship, Drug; Guanosine Triphosphate; Humans; Kinetics; Magnesium; Quinoxalines; Radioligand Assay; Receptors, Adrenergic, alpha; Vasoactive Intestinal Peptide; Yohimbine

The alpha-2 adrenergic antagonist [3H]yohimbine (YOH) and the alpha-2 agonist [3H]p-aminoclonidine (PAC) saturably label high-affinity binding sites in the submandibular gland from 3-week-old rats and 5-week-old pigs and in the lung from neonatal rats and 5-week-old pigs. [3H]YOH had KD values of 5.5, 1.8, 0.45 and 0.22 nM in the rat gland and lung and porcine gland and lung, respectively. KD values of 2.4, 5.3 and 1.3 nM were found for [3H]PAC in rodent and pig submandibular gland and pig lung, respectively. Both 3H-ligands labeled approximately the same density of sites within each tissue except in the rat lung in which [3H]PAC binding was too low to reliably estimate. In all cases the pharmacologic profile was indicative of an alpha-2 adrenergic receptor site. However, the Ki of yohimbine vs. [3H]PAC was 30- to 140-fold higher for the rodent relative to the porcine species. GTP decreased the affinity of (-)-epinephrine and PAC at [3H]YOH-labeled sites in the pig gland and lung, but did not shift the affinity of epinephrine in the rat gland. These results suggest the possibility of subtype or species differences for the alpha-2 receptor. The Ki values of the antagonists YOH and phentolamine were different at [3H]PAC and [3H]YOH sites. GTP caused a dose-dependent reduction in [3H]PAC binding in the porcine submandibular gland and lung. At 10 microM GTP, this loss was due to a decrease in 3H-agonist affinity, but not density.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adrenergic alpha-Agonists; Adrenergic alpha-Antagonists; Animals; Binding Sites; Clonidine; Guanosine Triphosphate; Lung; Magnesium; Rats; Rats, Inbred Strains; Receptors, Adrenergic, alpha; Species Specificity; Submandibular Gland; Swine; Tritium; Yohimbine

We previously reported that alpha 2-adrenergic agonists enhance absorption and inhibit secretion of electrolytes in small intestine. The present study was undertaken to characterize and localize the relevant receptors. Plasma membranes derived from isolated rabbit ileal epithelial cells were incubated with either [3H]yohimbine (Yo), an alpha 2-antagonist, or p-[3H]aminoclonidine (PAC), an alpha 2-agonist. Scatchard analysis of [3H]Yo binding suggests a single receptor. Competitive displacement of Yo from this receptor by other ligands had a potency order characteristic for alpha 2-receptors in other tissue systems. A Scatchard plot of [3H]PAC binding was curvilinear and best fit by assuming two independent site. Competitive displacement of [3H]PAC by PAC in the presence of 140 mM Na+ or 0.1 mM GTP increased the IC50 for PAC binding from 10 nM to 100 and 105 nM, respectively, and the Hill coefficient from 0.7 to 1.2 and 1.0, respectively. The ED50 for PAC effect on short-circuit current (200 nM) does not differ significantly from these values. We conclude that alpha 2-receptors are present on ileal enterocytes and that these receptors mediate enterocyte fluid and electrolyte transport function.

Topics: Animals; Binding, Competitive; Biological Transport; Cell Membrane; Clonidine; Dose-Response Relationship, Drug; Electrolytes; Electrophysiology; Guanosine Triphosphate; Ileum; Male; Rabbits; Receptors, Adrenergic; Receptors, Adrenergic, alpha; Sodium; Yohimbine

The labelling of rat cerebral cortex alpha 2-adrenoceptors with [3H]-yohimbine ([3H]-YOH) was investigated. At 25 degrees C, binding equilibrium was reached in about 10 min and dissociation occurred with a half time of about 1 min. Saturation experiments gave an equilibrium KD value of 10.13 +/- 1.95 nM and a maximum number of sites of 254 +/- 22 fmol/mg protein. The [3H]-YOH binding sites exhibited alpha 2-adrenergic receptor specificity; the order of potency for the antagonists was rauwolscine greater than yohimbine much greater than prazosin greater than corynanthine. For the agonists, the order was: oxymetazoline greater than clonidine greater than (-)-adrenaline greater than (-)-noradrenaline much greater than (-)-phenylephrine. Agonists exhibited shallow curves in inhibiting [3H]-YOH binding, with pseudo-Hill coefficients (nH) of less than 1.0. These curves were shifted to lower overall affinity and steepened in the presence of 100 microM GTP. Antagonist competition curves were also shallow but GTP had no significant effect. Divalent cations at millimolar concentrations decreased the [3H]-YOH binding: IC50 values were about 6.0, 6.8 and 0.3 mM for Ca2+, Mg2+ and Mn2+ respectively. The maximal number of [3H]-YOH binding sites in the cortex was close to that labelled by the agonist [3H]-paraaminoclonidine ([3H]-PAC). The regional distribution of these sites in the brain, examined at a single concentration of [3H]-YOH and [3H]-PAC, showed a similar pattern except in the striatum. Taken together, the results indicate that like [3H]-PAC, [3H]-YOH labels alpha 2-adrenoceptors in rat brain cortex. They also show that [3H]-YOH is a useful tool for the study of the high and low affinity sites.

Topics: Animals; Binding Sites; Brain; Calcium; Clonidine; Guanosine Triphosphate; In Vitro Techniques; Kinetics; Male; Rats; Rats, Inbred Strains; Receptors, Adrenergic; Receptors, Adrenergic, alpha; Tritium; Yohimbine

Topics: Adenylyl Cyclases; Animals; Binding, Competitive; Blood Platelets; Brain; Cattle; Cell Line; Cell Membrane; Clonidine; Erythrocytes; Glioma; Guanosine Triphosphate; Humans; Muscle, Smooth, Vascular; Neuroblastoma; Norepinephrine; Rats; Receptors, Adrenergic; Receptors, Adrenergic, alpha; Synaptic Membranes