alpha 2-Adrenoceptors take centre stage at Nashville meeting.
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Adrenergic alpha-2 Receptor Antagonists↗
Biomedical subjects
K R Lynch
Publications and source records attributed to K R Lynch.
Characterization of a rat kidney thromboxane A2 receptor: high affinity for the agonist ligand I-BOP.
We have cloned a rat kidney thromboxane A2 receptor (TP) cDNA. This receptor was shown to be functional in that the thromboxane A2 mimetics, U46619 and I-BOP, elicited calcium transients in Xenopus oocytes and HEK293 cells expressing the TP receptor, respectively. Comparison of the affinities of the rat and human TP sites for the agonist radiolgand [125 I] BOP showed that the rat TP site has about a ten-fold higher affinity for this drug (KD approximately 0.5 vs. 4.4 nM) while the affinities of the two sites for other compounds (U46619, I-PTH-OH) were the same. Our results are significant in that they identify a cloned TP as having a picomolar affinity for [125 I] BOP.
15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
Characterization of a monoclonal antibody that neutralizes the activity of prostaglandin E2.
We describe the generation of an IgG1 mAb, 2B5, that neutralizes the biologic activity of PGE2 in vitro and in vivo. The Ab was derived from a BALB/c mouse that was immunized with a PGE2-thyroglobulin conjugate. 2B5 is one of the highest affinity and specific anti-PGE2 mAbs reported to date. The Kd for PGE2 was approximately 300 pM and crossreactivity toward eicosanoids other than PGE1 was less than 1%. The addition of 2B5 to [3H]PGE2 blocked the binding of radioligand to cell membranes transfected with the murine EP3 prostaglandin receptor. In functional studies, 2B5 neutralized the capacity of PGE2 to suppress T cell proliferation induced by a mitogenic anti-CD3 Ab in vitro. In contrast, immunosuppression by the phosphodiesterase inhibitor, isobutylmethylxanthine was not affected. In an in vivo model of nociception, 2B5 substantially reduced the dorsoflexion response of mice to phenylbenzoquinone. This response is associated with prostaglandin production and is blocked by inhibitors of prostanoid synthesis. Our findings demonstrate that this nociceptive response is largely due to PGE2. In the absence of antagonists that prevent PGE2 from activating a diverse family of receptor subtypes, neutralizing Abs to PGE2 should represent useful reagents to delineate the biologic properties of this eicosanoid in vivo.
Animals↗
Alpha-2 adrenergic receptors. Immunohistochemical localization and role in mediating inhibition of adrenergic RVLM presympathetic neurons by catecholamines and clonidine.
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Animals↗
Characterization of alpha 2A-adrenergic receptors in GT1 neurosecretory cells.
In this paper, we report the endogenous expression of functional alpha 2-adrenergic receptors (alpha 2-ARs) in the immortalized hypothalamic cell line, GT1. Membranes from GT1 cells exhibited high-affinity binding for the specific alpha 2-AR radioligand [3H]RX821002 (Kd = 0.2 +/- 0.03 nM, Bmax = 29.5 +/- 2.1 fmol/mg protein, n = 3). The Ki values for the adrenergic ligands, oxymetazoline (1.6 +/- 0.3 nM, n = 3) and prazosin (287 +/- 89 mM, n = 3), were consistent with the pharmacological properties of the A subtype of alpha 2-AR (alpha 2A-AR). The presence of mRNA encoding the alpha 2A-AR in GT1 cell extracts was confirmed by Northern blot analysis. alpha 2-ARs in GT1 cells were found to be coupled to the inhibition of adenylyl cyclase through the pertussis toxin-sensitive class of heterotrimeric G-proteins. A maximal dose of the alpha 2-AR agonist UK 14304 inhibited forskolin-stimulated cAMP production in GT1 cells by 53% (EC50 = 316 nM). Double labeling of rodent brain sections with antibodies specific for GnRH and the alpha 2A-AR indicated a large proportion of neurons labeled with the GnRH antibody also contained alpha 2A-AR-like immunoreactivity. In both GT1 cells and GnRH-immunopositive neurons in brain, clusters of alpha 2A-AR-like immunoreactivity were associated with cell bodies and occasionally with neuritic processes. Punctate alpha 2A-AR-like immunoreactivity was localized to intracellular compartments in GT1 cells as determined by confocal microscopy. Whole cell radioligand binding techniques were used to show that at least one third of the alpha 2-AR population in GT1 cells was intracellular. In view of these data, GT1 cells may serve as a representative system in which to study the biology of alpha 2A-ARs in relation to neuronal and neurosecretory functions.
Adrenergic alpha-Antagonists↗
A1 adenosine receptors. Two amino acids are responsible for species differences in ligand recognition.
Species differences in ligand binding to A1 adenosine receptors were localized to the seventh transmembrane (TM7) region based on the binding of [8-3H]cyclopentyl-1, 3-dipropylxanthine and three other ligands to wild type and six bovine/canine interspecies receptor chimeras expressed in COS-1 cells. Subsequent site-directed mutagenesis experiments identified amino acid 270 (isoleucine/methionine, bovine/canine) as being primarily responsible for species differences in the binding of N6-adenine-substituted compounds, R-N6-phenylisopropyladenosine (R-PIA) and (S)-N6-endonorbornan-2-yl-9-methyladenine, and the C-8-substituted xanthine, [3H]cyclopentyl-1,3-dipropylxanthine. These data are consistent with the hypothesis that the N6 region of adenines and the C-8-region of xanthines bind to the same region of the receptor. A second TM7 amino acid, 277 (serine/threonine, bovine/canine), selectively influences the binding of the ribose-substituted adenosine analog, 5'-N-ethylcarboxamidoadenosine to a variable extent, depending on the nature of amino acid 270. We hypothesize that amino acid 270 of the A1 receptor interacts with the N6 region of adenosine, while amino acid 277 is important, especially in the absence of an N6 substitution, for interactions with a distinct nucleoside region, possibly on the ribose.
Adenosine↗
cDNA cloning of a G-protein-coupled receptor expressed in rat spinal cord and brain related to chemokine receptors.
A series of cDNAs and a genomic clone (named RBS11) were isolated, from a variety of rat brainstem, pituitary and/or spinal cord cDNA libraries and a genomic library by low-stringency hybridization screening with a rat angiotensin receptor cDNA. The RBS11 protein, as conceptualized from these DNAs, is a novel member of the rhodopsin family of the G-protein-coupled receptor (GCR) superfamily. Comparison of RBS11 to other members of the GCR superfamily suggests that the RBS11 protein might be a receptor for a peptide ligand in the chemokine family. The RBS11 protein sequence is unusual in that it is without N-linked glycosylation consensus sequences in the putative exofacial regions. Northern analysis indicates that the mRNA for RBS11 accumulates widely and unevenly in the adult rat, with the mRNA being most prominent in extracts of spinal cord, brain, kidney, gut, uterus and testes.
Animals↗
Alpha 2A-adrenergic receptors are present in lower brainstem catecholaminergic and serotonergic neurons innervating spinal cord.
A subtype-specific polyclonal antibody was used for the immunohistochemical detection of alpha 2A-adrenergic receptors (alpha 2A-ARs) in the rat lower brainstem (medulla and pons). Using dual-label fluorescence histochemistry, punctate alpha 2A-AR-like immunoreactivity (alpha 2A-AR-LIR) was identified in noradrenergic, adrenergic, and serotonergic neurons of the pontomedullary region. Confocal microscopic examination of material simultaneously labeled for TH-LIR and alpha 2A-LIR revealed that the clusters of alpha 2A-LIR were located intracellularly. Lower medullary neurons with spinal projections to segment T3 were retrogradely labeled using FITC-conjugated microbeads and the material was processed for simultaneous detection of alpha 2A-LIR and either TH-LIR or 5-HT-LIR. Using this triple-label approach, we found that virtually all medullary serotonergic cells (raphe pallidus, raphe obscurus and parapyramidal area) including those with identified spinal projections contain punctate alpha 2A-AR-LIR. In contrast, fewer than 10% of dorsal raphe serotonergic cells examined for comparison were immunoreactive. The triple labeling approach also indicated that more than 95% of the TH-immunoreactive cells of the dorsal and ventrolateral medulla, including those with demonstrable spinal projections (A5 noradrenergic and C1/C3 adrenergic) had detectable amounts of alpha 2A-AR-LIR. The presence of alpha 2A-ARs in a large fraction of bulbospinal pre-sympathetic neurons (noradrenergic A5, adrenergic C1 and C3 and serotonergic raphe cells) could explain the powerful and relatively selective effect of clonidine and other centrally acting alpha 2A-AR agonists on sympathetic efferent activity and hypertension.
Animals↗
Trypsin induces Ca(2+)-activated Cl- currents in X. laevis oocytes.
The protease trypsin induces Ca(2+)-activated Cl- currents when applied in concentrations as low as 0.1 mg/ml to defolliculated, voltage clamped X. laevis oocytes. The response is dose-dependent and specific, as other proteases (chymotrypsin, Lys-C and Arg-C), or trypsin pretreated with soybean trypsin inhibitor, did not induce currents. Intracellular trypsin injection did not induce responses. The current does not appear to result from proteolytic activation of the endogenous receptor for lysophosphatidic acid, the only known Ca(2+)-mobilizing receptor consistently present in oocytes. These results suggest the presence on the oocyte membrane of a specific receptor for trypsin.
Animals↗
Apical membrane and intracellular distribution of endogenous alpha 2A-adrenergic receptors in MDCK cells.
The purpose of the current studies was to characterize the endogenous alpha 2-adrenergic receptor (AR) subtypes present in Madin-Darby canine kidney (MDCK) cells and to determine their level of expression and pattern of distribution. By saturation binding analysis with [3H]MK-912, MDCK cells expressed high levels of alpha 2-ARs with a maximum receptor density (Bmax) of 798 +/- 55 fmol/mg protein and an equilibrium dissociation constant (Kd) of 0.98 +/- 0.32 nM. Competitive binding studies using prazosin, oxymetazoline, phentolamine, and epinephrine to displace [3H]MK-912 demonstrated inhibition constant (Ki) values of 1,270 +/- 250, 5.0 +/- 0.4, 5.5 +/- 0.3, and 392 +/- 150 nM (n = 3), respectively. In Northern blot analysis we found that MDCK cells expressed transcripts encoding alpha 2A-AR and not alpha 2B-AR or alpha 2C-AR. Surface binding experiments suggested that approximately 60% of alpha 2A-ARs are distributed at the cell surface domain. Specific binding of [3H]MK-912 to soluble apical and basolateral surface proteins isolated by surface biotinylation indicated the expression of surface alpha 2A-ARs was limited to the apical domain of MDCK cells. No alpha 2A-ARs were detected on the basolateral surface. We conclude that endogenous alpha 2A-ARs are targeted to the apical domain of MDCK cells and that the intracellular compartment may contain ARs as a reservoir for de novo cell surface expression or, alternatively, may represent internalized receptors.
Adrenergic alpha-Antagonists↗
Molecular cloning of a novel rat G-protein-coupled receptor gene expressed prominently in lung, adrenal, and liver.
A gene and cDNA (named G10d) encoding a novel member of the G-protein-coupled receptor (GCR) superfamily has been isolated from a rat genomic library and a rat liver cDNA library by tandom polymerase chain reaction (PCR) amplification and hybridization screening. Comparison of the sequence of the encoded protein to other members of the rhodopsin family of the GCR superfamily suggests that this new member is a peptide receptor. The G10d gene is present as a single copy in the rat genome and its translational open reading frame is intronless. Northern analysis demonstrates a wide and uneven distribution of G10d mRNA in the adult rat, with the highest mRNA abundance in lung, liver and adrenal gland extracts.
Adrenal Glands↗
Molecular structure of a protein-tyrosine/threonine kinase activating p42 mitogen-activated protein (MAP) kinase: MAP kinase kinase.
MAP kinases p42mapk and p44mapk participate in a protein kinase cascade(s) important for signaling in many cell types and contexts. Both MAP kinases are activated in vitro by MAP kinase kinase, a protein-tyrosine and threonine kinase. A MAP kinase kinase cDNA was isolated from a rat kidney library by using peptide sequence data we obtained from MAP kinase kinase isolated from rabbit skeletal muscle. The deduced sequence, containing 393 amino acids (predicted mass, 43.5 kDa), is most similar to byr1 (Bypass of ras1), a yeast protein kinase functioning in the mating pathway induced by pheromones in Schizosaccharomyces pombe. An unusually large insert is present in MAP kinase kinase between domains IX and X and may contribute to protein-protein interactions with MAP kinase. Major (2.7 kilobases) and minor (1.7 kilobases) transcripts are widely expressed in rat tissues and appear to be derived from a single gene.
Amino Acid Sequence↗
Signalling properties of lysophosphatidic acid.
Lysophosphatidic acid (LPA) is the simplest natural phospholipid, primarily known as a membrane component and metabolic intermediate. However, a remarkable variety of biological effects of this compound have come to light, seemingly pointing to an additional role for LPA as a signalling molecule. In this review, Marcel Durieux and Kevin Lynch integrate the recent information that indicates that LPA could be an intercellular messenger, possibly acting through a G protein-coupled receptor, and with a role in cell growth and motility.
Animals↗
Immunohistochemical localization of alpha 2A-adrenergic receptors in catecholaminergic and other brainstem neurons in the rat.
alpha 2-Adrenergic receptors mediate a large portion of the known inhibitory effects of catecholamines on central and peripheral neurons. Molecular cloning studies have established the identity of three alpha 2-adrenergic receptor genes from several species that encode the A, B and C subtypes of the receptor. The rat alpha 2A-adrenergic receptor, as defined by sequence similarity, is the orthologue of the human alpha 2A-adrenergic receptor. In this paper, we report the development of rabbit antisera directed against a portion of the third intracellular loop of the rat alpha 2A-adrenergic receptor and the histochemical localization of alpha 2A-adrenergic receptor-like immunoreactive material in the brainstem and spinal cord of the adult rat. Our antisera detected alpha 2A-adrenergic receptor-specific punctate staining associated with neuronal perikarya. alpha 2A-adrenergic receptor-like immunoreactivity was widely, but heterogeneously, distributed in the brainstem and spinal cord, predominantly in areas involved in the control of autonomic function. Double labelling with antisera to tyrosine hydroxylase or phenylethanolamine-N-methyl-transferase revealed that alpha 2A-adrenergic receptor-like immunoreactivity is present in most, perhaps all, noradrenergic and adrenergic cells of the brainstem. alpha 2A-Adrenergic receptor-like immunoreactivity was detected in a small percentage of the dopaminergic cells of the A9 and A10 groups. This study provides the first description of the specific immunohistochemical localization of alpha 2A-adrenergic receptors using a subtype-specific polyclonal antibody. The results support the view that alpha 2-adrenergic receptors are involved in central cardiovascular control and suggest that the catecholaminergic autoreceptors of central noradrenergic and adrenergic neurons are the A subtype of the alpha 2-adrenergic receptors.
Animals↗
Identification and characterization of a new mammalian mitogen-activated protein kinase kinase, MKK2.
Mitogen-activated protein (MAP) kinases are serine/threonine protein kinases activated by dual phosphorylation on threonine and tyrosine residues. A MAP kinase kinase (MKK1 or MEK1) has been identified as a dual-specificity protein kinase that is sufficient to phosphorylate MAP kinases p42mapk and p44mapk on the regulatory threonine and tyrosine residues. Because of the multiplicity of MAP kinase isoforms and the diverse circumstances and agonists leading to their activation, we thought it unlikely that a single MKK could accommodate this complexity. Indeed, two protein bands with MKK activity have previously been identified after renaturation following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We now report the molecular cloning and characterization of a second rat MAP kinase kinase cDNA, MKK2. MKK2 cDNA contains an open reading frame encoding a protein of 400 amino acids, 7 residues longer than MKK1 (MEK1). The amino acid sequence of MKK2 is 81% identical to that of MKK1, but nucleotide sequence differences occur throughout the aligned MKK2 and MKK1 cDNAs, indicating that MKK2 is the product of a distinct gene. MKK1 and MKK2 mRNAs are expressed differently in rat tissues. Both cDNAs when expressed in COS cells displayed the ability to phosphorylate and activate p42mapk and p44mapk, both MKK1 and MKK2 were activated in vivo in response to serum, and both could be phosphorylated and activated by the v-Raf protein in vitro. However, differences between MKK1 and MKK2 in sites of phosphorylation by proline-directed protein kinases predict differences in feedback regulation.
Amino Acid Sequence↗
Responses to sphingosine-1-phosphate in X. laevis oocytes: similarities with lysophosphatidic acid signaling.
Sphingosine-1-phosphate (S1P, 50 microM) induces inward currents in Xenopus laevis oocytes voltage clamped at -70 mV. The currents are Ca(2+)-activated Cl- currents, as shown by a reversal potential of -20 mV and absence of the response after intracellular injection of ethylene glycolbis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA; 10 mM). The response is nearly indistinguishable from that to the related compound lysophosphatidic acid (LPA), and complete cross-desensitization occurs between LPA and S1P responses. Both the LPA and S1P responses are inhibited by suramin (2 mM) and dithiothreitol (5 mM). These responses appear mediated by a specific membrane receptor, since intracellular injection of S1P (5 microM) does not induce currents, and sphingosine and the related compounds sphingosylphosphorylcholine and N,N-dimethylsphingosine, all at 100 microM, neither induce currents nor block the response to S1P. HEK-293 and COS-1 cells respond with intracellular Ca2+ release to both 50 microM S1P and 10 microM LPA; K-562 cells do not. No cross-desensitization was noted in the responsive cells. Our findings indicate that S1P and LPA might act through the same mechanism, probably a membrane receptor.
Animals↗
Leukocytes synthesize angiotensinogen.
To determine whether leukocytes express the angiotensinogen gene, we subjected circulating rat leukocytes and murine bone marrow cells to Northern blot analysis and hybridization with homologous angiotensinogen complementary DNA. Angiotensinogen messenger RNA sequences were detected in circulating adult rat leukocytes, in murine-irradiated and nonirradiated bone marrow stromal cells, and in an adherent stromal cell line (preadipocyte). Western blot analysis of rat leukocyte homogenate showed that rat leukocytes contain two main angiotensinogen isoforms with approximate molecular weights of 46.5 and 53.9 kd. Synthesis and release of angiotensinogen protein by rat leukocytes was confirmed by immunoprecipitation of radiolabeled angiotensinogen from cell lysate and media of rat leukocytes that were metabolically labeled with 35S-L-methionine. In addition, the angiotensinogen protein present in media of rat leukocytes was enzymatically cleaved by hog renin, resulting in generation of angiotensin I (305 +/- 47 pg angiotensin I per milliliter of media per hour). We conclude that circulating rat leukocytes express the angiotensinogen gene and synthesize and release angiotensinogen with the capability to generate angiotensin. Expression of angiotensinogen by leukocytes may provide a mobile angiotensin-generating system of potential importance in the regulation of local inflammatory responses, tissue injury (i.e., myocardial infarction), and arterial hypertension.
Angiotensin I↗
Genomic analysis and expression patterns reveal distinct genes for endothelial and brain nitric oxide synthase.
Constitutively active nitric oxide synthases (NOS) are a unique class of NADPH-dependent, calcium/calmodulin-dependent enzymes that catalyze the conversion of L-arginine to nitric oxide and L-citrulline. However, little is known about the molecular similarities or differences between the two prototypical constitutive NOS enzymes, endothelial NOS (ECNOS) and brain NOS (bNOS). The aims of this study were to begin characterizing the gene structure and tissue distribution of messenger RNAs (mRNAs) for ECNOS and bNOS and to examine the immunological resemblance of the proteins by Western blotting. Full-length complementary DNAs (cDNAs) encoding bovine ECNOS and rat bNOS hybridized, under high stringency, to different-sized fragments of endonuclease-digested bovine, rat, and human genomic DNA. In addition, more than one fragment was detected with both cDNAs, suggesting that ECNOS and bNOS genes contained multiple introns. Tissue distribution of ECNOS mRNA (4.4 kb) and bNOS mRNA (9.5 kb) in the rat was detected by Northern blotting. Patterns among tissue extracts were strikingly different, with ECNOS mRNA being most abundant in aorta, heart, lung, kidney, adrenal gland, spinal cord, and urogenital tissues and bNOS mRNA most prominent in brain regions, intestine, stomach, spinal cord, adrenal gland, and aorta. Interestingly, ECNOS cDNA detected two equally abundant RNA transcripts (4.4 and 4.0 kb) in most brain regions tested, suggesting an alternative splicing of the ECNOS pre-mRNA. Western blotting, using an ECNOS monoclonal antibody, recognized ECNOS protein from native bovine endothelial cells, cultured bovine endothelial cells, and COS cells transfected with ECNOS cDNA but did not recognize purified bNOS.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Oxidoreductases↗