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.

Distribution of zinc metallothionein I mRNA in rat brain using in situ hybridization.

Metallothionein (MT) isoforms I and II were first identified and characterized in our laboratories in several regions of brain, in hippocampal neurons in primary culture, and in retinoblastoma and neuroblastoma cell lines. In this study, by having employed the MT-I cDNA as a probe, we sought to gain additional insight about the function of MT by discerning the regional distribution of its mRNA. Northern blot analyses of brain mRNA revealed that the administration of zinc enhanced dramatically MT-I mRNA (570 bp). The in situ hybridization study revealed that MT-I mRNA was located in several areas of brain, with the highest concentrations found in the cerebellum, hippocampus, and ventricles. The results of these studies are interpreted to suggest that zinc enhances the synthesis of MT mRNA and MT in turn may participate in zinc associated functions in neurons.

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)

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.

Expression of alpha 2-adrenergic receptor genes in rat tissues.

Alpha-2 adrenergic receptor subtypes are coded for by three genes. Pharmacologically alpha 2 adrenergic receptors can be classified into four subtypes: alpha 2A, alpha 2B, alpha 2C, and alpha 2D. Although pharmacologically distinct, the amino acid sequences of the alpha 2A and alpha 2D subtypes are approx 90% identical and have not been detected in a single species. Thus, they should be considered species orthologs and may be referred to as alpha 2A/D. The tissue distribution of the mRNAs for the rat alpha 2A/D, alpha 2B, and alpha 2C was analyzed utilizing RNase protection assays with probes directed to the third cytoplasmic loops. Alpha-2 adrenergic receptor mRNA was found in all tissues tested. Kidney, brain, and spinal cord had transcripts for all three subtypes. Only one mRNA subtype was detected in several tissues. Aorta and spleen had only alpha 2A/D mRNA, whereas heart and liver had only alpha 2B mRNA. All other tissues had two alpha 2 adrenergic subtype transcripts present. In contrast to the rat CNS, which contains predominantly alpha 2A/D and alpha 2C mRNA with little alpha 2B mRNA, peripheral tissues contain predominantly alpha 2A/D and alpha 2B mRNA with little alpha 2C mRNA.

Molecular determinants of the alpha-2D adrenergic receptor subtype.

The alpha-2 adrenergic receptor in the bovine pineal gland and the rodent homologues of the human alpha-2-C10 receptor express alpha-2D subtype pharmacological characteristics. The alpha-2 adrenergic receptor in the chicken pineal expresses characteristics similar to the alpha-2A subtype found in human and pig. The rodent receptors (alpha-2D) contain a serine residue at position 201 whereas the human and porcine receptors (alpha-2A) have a cysteine at this position. Our results indicate that the bovine pineal receptor has a serine at position 201, supporting the alpha-2D classification. However, the chicken pineal receptor also contains a serine at position 201 suggesting that other amino acids may be responsible for the differences in pharmacological characteristics.

Alpha-2A is the predominant alpha-2 adrenergic receptor subtype in human spinal cord.

alpha-2 Adrenergic receptors can be subdivided into four subtypes based on their pharmacologic properties. The subtype of alpha-2 adrenergic receptor present in human spinal cord has not been reported previously. The affinities of nine alpha-2 subtype-selective drugs for the alpha-2 adrenergic receptor in human spinal cord homogenates were determined using [3H]rauwolscine and [3H]RX821002. These drug affinities (pKi values) were highly correlated with those obtained in a tissue or cell line containing only the alpha-2A adrenergic subtype (correlation coefficient of 0.99 and 0.98 for human platelet and HT29 cells, respectively). In contrast, the correlation of pKi values for the human spinal cord with tissues or cell lines containing other adrenergic receptor subtypes was poor. The correlation coefficients for alpha-2B (neonatal rat lung), alpha-2C (OK cell), and alpha-2D (bovine pineal gland) were 0.15, 0.68, and 0.81, respectively. These data suggest that the predominant alpha-2 adrenergic subtype present in human spinal cord is the alpha-2A subtype. Both [3H]rauwolscine and [3H]RX821002 appeared to label a single class of binding sites. The alpha-2 adrenergic receptor density was significantly greater in the sacral region of the cord as compared to either the lumbar or thoracic regions.

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.

Characterization of the alpha-2C adrenergic receptor subtype in the opossum kidney and in the OK cell line.

The alpha-2 adrenergic receptors have been subdivided into two pharmacological subtypes known as alpha-2A and alpha-2B. The OK cell, a cell line derived from an opossum kidney, expresses an alpha-2 adrenergic receptor which has pharmacological characteristics different from both the alpha-2A and alpha-2B subtypes, and thus may be a third subtype. To test this hypothesis, we compared the affinities (Ki values from radioligand binding) of 49 drugs in the OK cell with their affinity for the alpha-2A (HT-29 cells) and alpha-2B (neonatal rat lung) adrenergic receptor subtypes. Eight drugs (spiroxarine, prazosin, WY 27127, L-657,743, ARC 239, akuammigine, rauwolscine and oxymetazoline) were identified whose Ki differed by 10-fold or more between the OK cells and HT29 cells. Five drugs (BAM 1303, raubasine, WB 4101, akuammigine and rauwolscine) differentiated, by at least 10-fold, between OK cells and neonatal rat lung. Correlations of pKi values between the OK cell and tissues or cell lines expressing either the alpha-2A or alpha-2B subtypes were poor, confirming that the OK cell receptor could not be classified as either alpha-2A or alpha-2B. In contradistinction, there was a good correlation between pKi values for the OK cell line and opossum kidney showing that they express the same subtype. In addition, ratios of subtype-selective drug Ki values were similar for the OK cell and opossum kidney, but different from other tissues or cell lines expressing either the alpha-2A or alpha-2B subtypes. We conclude that the OK cell line and opossum kidney express a novel subtype of alpha-2 adrenergic receptor which we term the alpha-2C subtype.

Reserpine-induced post-receptor reduction in muscarinic-mediated airway smooth muscle contraction.

Radioligand binding was conducted on airways of the rat and human, surgically subdivided into trachea, lung airways, and parenchyma. 3H-QNB bound uniformly to receptors in separate sections of the rat and human airway. Receptor densities generally were ranked: lung airways greater than trachea greater than parenchyma. Receptor subtypes were identified mostly by pirenzepine displacement of bound 3H-QNB. The rat trachea, and rat and human lung airways had a uniformly low affinity for pirenzepine while rat and human parenchyma demonstrated both high and low affinity pirenzepine binding. Inhibition of methacholine-stimulated smooth muscle contraction by the M1 receptor antagonist, pirenzepine, and M2 receptor antagonist, gallamine, was studied in rat trachea and bronchus in vitro. Schild plot pA2 values were compatible with low potency antagonism, thereby favoring the presence of M3 receptors at these smooth muscle sites. Reserpine treatment of rats (0.5 mg kg-1 day-1 for 7 days) produced a decrease in peak tension in response to methacholine without changing the muscarinic receptor character (Kd 3H-QNB), population density (Bmax in fmol mg-1 protein), or function (methacholine EC50). These results indicate that muscarinic receptor heterogeneity exists in the airway of both laboratory rat and man. While the muscarinic receptor subserving airway smooth muscle contraction appears to be the M3 subtype, decreased contractile responses to methacholine by trachea and bronchus from reserpine-treated rats were receptor independent.