Alpha-2 adrenergic receptors in the bovine retina. Presence of only the alpha-2D subtype.

PURPOSE: To identify and characterize the alpha-2 adrenergic receptor subtypes present in the bovine neurosensory retina. METHODS: Radioligand saturation and inhibition binding assays were performed with the antagonist radioligands [3H]RX821002 and [3H]rauwolscine. RESULTS: [3H]RX821002 bound to a single class of receptors with the characteristics of an alpha-2 adrenergic receptor with an affinity (KD) of 0.16 nM and a receptor density (Bmax) of 1500 fmol/mg protein. Correlation of the affinities (pKi values) for nine antagonists in the bovine neurosensory retina with the alpha-2D receptor of the bovine pineal gave a correlation coefficient of 0.99. The correlation coefficients for the alpha-2A (0.84), alpha-2B (0.36), and alpha-2C (0.39) subtypes were much lower. The presence of a minor population of alpha-2B or alpha-2C receptors was excluded. CONCLUSIONS: A high density of alpha-2D adrenergic receptors is present in the bovine neurosensory retina. Neither the alpha-2B nor the alpha-2C subtype is detectable.

Characterization of alpha-2D adrenergic receptor subtypes in bovine ocular tissue homogenates.

The alpha-2 adrenergic receptors are known to be present in the mammalian eye and to mediate the effects of alpha-2 agonists used in the treatment of glaucoma. Little is known, however, regarding the relative densities of the three alpha-2 subtypes in the various tissues of the eye. We used receptor binding experiments with the radioligand [3H]RX821002 to characterize the alpha-2 adrenergic receptors in three tissues of the bovine eye, the ciliary body, retinal pigment epithelium/choriocapillaris and iris. The K(D) values in the three tissues were similar (0.12-0.14 nM), and the Bmax values ranged from 100 fmol/mg of protein for the ciliary body and retinal pigment epithelium/choriocapillaris to 200 fmol/mg of protein for the iris. The pharmacological characteristics of the alpha-2 receptors in all three tissues of the bovine eye, as assessed by competition studies, were essentially identical and were similar to the characteristics of the alpha-2A/D receptors of the bovine neurosensory retina. The correlation coefficients between the logarithms of the Ki values for the three tissues and the neurosensory retina for nine adrenergic agents were .98 to .99. We conclude that the alpha-2 adrenergic receptors in the ciliary body, iris and retinal pigment epithelium/choriocapillaris of the bovine eye are mainly alpha-2D.

The cloning and expression of an OK cell cDNA encoding a 5-hydroxytryptamine1B receptor.

Serotonin (5-hydroxytryptamine or 5-HT) is an important biogenic amine that functions as both a neurotransmitter and a hormone in the central nervous system (CNS) and the periphery. We report here the isolation of a cDNA from the OK cell that encodes a serotonin receptor (OKc1). When expressed in cultured cells, it displayed the pharmacological profile and negative coupling with adenylyl cyclase characteristic of a 5-HT1B receptor subtype. Similar to the cloned rodent 5-HT1B receptors, it had high affinity for the beta-adrenergic ligand [125I]iodocyanopindolol, because of the presence of an asparagine instead of a threonine residue in the seventh transmembrane region. The ligands used displayed the following rank order of potencies: cyanopindolol > RU24969 > methiothepin > serotonin > sumatriptan > methysergide > 8-OH-DPAT > isoproterenol. This profile correlates well (r = 0.97) with the native OK cell 5-HT1B receptor. When OKc1 is compared to the rat, mouse, and human 5-HT1B receptors, it has an amino acid sequence identity of 82%, but it is only 54% identical to the human 5-HT1D receptor.

Species orthologs of the alpha-2A adrenergic receptor: the pharmacological properties of the bovine and rat receptors differ from the human and porcine receptors.

Four pharmacological subtypes of the alpha-2 adrenergic receptor have been identified; however, only three subtypes exist in any given species. Although the alpha-2A adrenergic receptor, as defined by the human platelet, and the alpha-2D receptor, as defined in the bovine pineal, have very different pharmacological characteristics, they are more similar to each other than either is to the alpha-2B or alpha-2C subtype. The human alpha-2-C10 clone (alpha-2A) and the rat RG20 clone have an 89% identity in their predicted amino acid sequence and are considered to be species orthologs. Although the expressed RG20 clone appears to have alpha-2D pharmacology, a careful comparison of its pharmacological characteristics with the bovine pineal has not been reported previously. Based on the pKi values of a panel of 13 alpha-2 adrenergic agents that have been used previously to compare the alpha-2A, alpha-2B and alpha-2C subtypes, the pharmacological characteristics of the bovine pineal alpha-2D receptor appear to be very similar to the rat RG20 clone (correlation coefficient, r, of 0.93). The porcine ortholog of the human alpha-2-C10 receptor has pharmacological characteristics identical to the human alpha-2A receptor (r = 0.99). Because of its higher affinity for the alpha-2D receptor, [3H]RX821002 is a better radioligand than [3H]rauwolscine for studying this receptor subtype.

Characterization of [3H]RX821002 binding to alpha-2 adrenergic receptor subtypes.

Alpha-2 adrenergic receptors have been divided into four pharmacological subtypes based on their differences in affinity for several drugs. Previous studies showed that [3H]RX821002 has a high affinity for the alpha-2A subtype. The current study characterized the binding properties of [3H]RX821002 [2-(2-methoxy-1,4- benzodioxan-2yl)-2-imidazoline] to the alpha-2A receptor in CHO-C10 cells, alpha-2B in neonatal rat lung, alpha-2C in OK cells and alpha-2D in bovine pineal gland. Membrane binding studies of [3H]RX821002 were done in 25 mM glycylglycine buffer at room temperature. The nonspecific binding rates at the KD concentration were 4.9%, 20%, 14% and 8.3% of the total for CHO-C10, neonatal rat lung, OK cells and bovine pineal, respectively, which were determined by adding 100 microM norepinephrine. Saturation curves indicate that [3H]RX821002 has a high affinity for all alpha-2 adrenergic subtypes. The KD values were 0.29, 1.05, 0.37 and 0.19 nM for CHO-C10, neonatal rat lung, OK cells and bovine pineal, respectively. [3H]Rauwolscine has affinities of 0.34, 0.55 and 0.24 nM for the alpha-2A, -2B and -2C subtypes. By contrast, [3H]rauwolscine has a much lower affinity for alpha-2D subtype with a KD value of 5.2 nM. The binding site density for [3H]RX821002 was significantly lower in the neonatal rat lung compared with [3H]rauwolscine. The correlation coefficients of pKi values of adrenergic compounds against [3H]RX821002 versus [3H]rauwolscine were close to unity for each tissue. These data clearly show that the two ligands label the same alpha-2 adrenergic receptor population.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacological characteristics of alpha 2-adrenergic receptors: comparison of pharmacologically defined subtypes with subtypes identified by molecular cloning.

On the basis of extensive radioligand data and more limited functional data, three pharmacological subtypes of alpha 2-adrenergic receptors have been identified. More recently, three human genes or cDNAs for alpha 2-adrenergic receptors have been identified by molecular cloning. The relationship, however, among the pharmacologically defined subtypes and those identified by molecular cloning has not been clear. In order to resolve this issue, we have compared the pharmacological characteristics of the receptors identified by molecular cloning and expressed in COS-7 cells with the characteristics of the pharmacologically defined receptors in their respective prototypic tissue or cell line. The affinities (Ki values) of 12 subtype-selective alpha 2-adrenergic antagonists were determined for the alpha 2 receptor in the six preparations, by radioligand binding. Correlation analyses of the pKi values indicate that the alpha 2A subtype, as defined in the HT29 cell line, the alpha 2B receptor of the neonatal rat lung, and the alpha 2C subtype, as defined in an oppossum kidney cell line, correspond to the cloned human alpha 2-C10, alpha 2-C2, and alpha 2-C4 receptor subtypes, respectively.

Cloning and expression of the alpha 2C-adrenergic receptor from the OK cell line.

The alpha 2-adrenergic receptors have been divided into four pharmacological subtypes, alpha 2A, alpha 2B, alpha 2C, and alpha 2D. The OK cell line, a cell line derived from an opossum kidney, expresses the alpha 2C-adrenergic receptor and is the prototypical cell line for the alpha 2C receptor subtype. The cloned human alpha 2C-C4 and rat RG10 receptors have been shown to express alpha 2C pharmacology. Here we report the cloning and expression of the OK alpha 2C-adrenergic receptor, OKc2. The receptor has 64% deduced amino acid identity and 21% similarity to the alpha 2-C4 receptor, giving an overall similarity of 85%. The clone, expressed in Chinese hamster ovary cells, has a pharmacology that correlates very well (r = 0.97) with that of the native OK cell alpha 2C-adrenergic receptor, and it is negatively coupled to adenylyl cyclase.

Bolus intravenous injection of phosphorothioate oligonucleotides causes hypotension by acting as alpha(1)-adrenergic receptor antagonists.

Bolus intravenous injections of phosphorothioate oligonucleotides (PS-ODN) into primates cause profound hypotension, which has been attributed to complement activation, the biochemical pathway leading to acute inflammatory response. Because the hypotension was not accompanied by peripheral or pulmonary edema and epinephrine was not effective, but administration of 200 ml Ringer's lactate was effective, we examined the possibility that the 15-base PS-ODN interferes with sympathetic tone. We administered doses ranging from 3.3 to 10 mg/kg of a 15-base PS-ODN as a 30-60 s iv bolus into the right atrium of conscious Macaca mulatta. Blood pressure fell to 27 mm Hg following a 5.0 mg/kg dose, but no hypotension was observed after a 3.3 mg/kg dose; 10 mg/kg was lethal. Adrenergic receptor binding was evaluated in radioligand binding assays using rat cerebral cortex membranes with radiolabeled prazosin. The 15-base PS-ODN competes with prazosin for the alpha(1)-adrenergic receptor with an IC50 of 14 microM, which favors binding over serum albumin (K(d) = 37 to 48 microM). Admixing serum albumin with 5.0 mg/kg 15-base PS-ODN prior to injection prevented hypotension, suggesting that unbound PS-ODN interferes with sympathetic tone before binding to plasma proteins. Interactions of the 15-base PS-ODN with the alpha(1)-adrenergic receptor in vivo were confirmed by a decreased response to phenylephrine. Reducing the length from 15 to 9 or 5 bases abolished alpha(1)-adrenergic receptor binding in vitro and bolus infusion of 5.0 mg/kg of 9-base PS-ODN no longer produced hypotension. In conclusion, the 15-base PS-ODN shows cooperative binding to the alpha(1)-adrenergic receptor, which produces cardiovascular effects that are oligomer length, dose, and formulation dependent.