Distribution of alpha-1 adrenoceptor subtypes in RNA and protein in rabbit eyes.
Ontology highlight
ABSTRACT:
SUBMITTER: Suzuki F
PROVIDER: S-EPMC1573172 | biostudies-literature | 2002 Feb
REPOSITORIES: biostudies-literature
Publications
Distribution of alpha-1 adrenoceptor subtypes in RNA and protein in rabbit eyes.
Suzuki Fumiko F Taniguchi Takanobu T Nakamura Seigo S Akagi Yoshio Y Kubota Chikara C Satoh Makoto M Muramatsu Ikunobu I
British journal of pharmacology 20020201 3
We investigated subtypes of alpha-1 adrenoceptor (AR) in rabbit ocular tissues using reverse transcription-polymerase chain reaction (RT - PCR), in situ hybridization (ISH) and binding studies. Competitive RT - PCR assays specific for the subtypes of alpha-1 AR revealed that the mRNA expression of alpha-1a AR was dominant, and that of each alpha-1b and alpha-1d was less than 10% and 0.5% of total alpha-1 ARs mRNA, respectively, in the iris, ciliary body, choroid and retina. In alpha-1a AR splice ...[more]
Similar Datasets
Project description:Two splice isoforms of rabbit alpha(1a)-adrenergic receptor (AR), (named alpha(1a)-OCU.2-AR and alpha(1a)-OCU.3-AR) have been isolated from the liver cDNA library in addition to the previously reported isoform (alpha(1a)-OCU.1-AR). Although they have the identical splice position with human alpha(1a)-AR isoforms, the C-terminal sequences are distinct from those of human isoforms. Among these rabbit alpha(1a)-AR isoforms, there are no significant differences in pharmacological properties: high affinity for prazosin, WB4101, KMD-3213 and YM617 and low affinity for BMY7378, using COS-7 cells expressing each isoform by radioligand binding assay. Competitive reverse transcription-polymerase chain reaction (RT - PCR) analysis revealed that mRNA of alpha(1a)-ARs was expressed in liver, thoracic aorta, brain stem and thalamus of rabbit. The splice isoforms exhibited a distinct distribution pattern in rabbit; alpha(1a)-OCU. 1-AR was expressed most abundantly in those tissues. CHO clones, stably expressing each isoforms with receptor density 740 fmol mg(-1) protein in alpha(1a)-OCU.1-AR, 1200 fmol mg(-1) in alpha(1a)-OCU.2-AR and 570 fmol mg(-1) in alpha(1a)-OCU.3-AR, respectively, showed a noradrenaline-induced increase in inositol trisphosphate which was suppressed by prazosin. Noradrenaline elicited a concentration-dependent increase in extracellular acidification rate (EAR) in the CHO clones with pEC(50) values of 6. 19 for alpha(1a)-OCU.1-AR, 6.49 for alpha(1a)-OCU.2-AR and 6.58 for alpha(1a)-OCU.3-AR, respectively. Noradrenaline caused a concentration-dependent increase in intracellular Ca(2+) concentration ([Ca(2+)]i) in the CHO clones with pEC(50) values of 6. 14 for alpha(1a)-OCU.1-AR, 7.25 for alpha(1a)-OCU.2-AR and 7.70 for alpha(1a)-OCU.3-AR, respectively. In conclusion, the present study shows the occurrence of three splice isoforms of rabbit alpha(1a)-AR, which are unique in C-terminal sequence and in tissue distribution. They show similar pharmacological profiles in binding studies but alpha(1a)-OCU.3-AR had the highest potency of noradrenaline in functional studies in spite of the lowest receptor density. These findings suggest that the structure of C-terminus of alpha(1a)-ARs may give the characteristic functional profile.
| S-EPMC1572000 | biostudies-literature
Project description:Background and purposeMesenteric and carotid arteries from the alpha(1B/D)-adrenoceptor knockout (alpha(1B/D)-KO) were employed to isolate alpha(1A)-adrenoceptor pharmacology and location and to reveal these features in the wild-type (WT) mouse.Experimental approachFunctional pharmacology by wire myography and receptor localization by confocal microscopy, using the fluorescent alpha(1)-adrenoceptor ligand BODIPY FL-Prazosin (QAPB), on mesenteric (an 'alpha(1A)-adrenoceptor' tissue) and carotid (an 'alpha(1D)-adrenoceptor' tissue) arteries.Key resultsAlpha(1B/D)-KO mesenteric arteries showed straightforward alpha(1A)-adrenoceptor agonist/antagonist pharmacology. WT had complex pharmacology with alpha(1A)- and alpha(1D)-adrenoceptor components. alpha(1B/D)-KO had a larger alpha(1A)-adrenoceptor response suggesting compensatory up-regulation: no increase in fluorescent ligand binding suggests up-regulation of signalling. alpha(1B/D)-KO carotid arteries had low efficacy alpha(1A)-adrenoceptor responses. WT had complex pharmacology consistent with co-activation of all three subtypes. Fluorescent binding had straightforward alpha(1A)-adrenoceptor characteristics in both arteries of alpha(1B/D)-KO. Fluorescent binding varied between cells in relative intracellular and surface distribution. Total fluorescence was reduced in the alpha(1B/D)-KO due to fewer smooth muscle cells showing fluorescent binding. WT binding was greater and sensitive to alpha(1A)- and alpha(1D)-adrenoceptor antagonists.Conclusions and implicationsThe straightforward pharmacology and fluorescent binding in the alpha(1B/D)-KO was used to interpret the properties of the alpha(1A)-adrenoceptor in the WT. Reduced total fluorescence in alpha(1B/D)-KO arteries, despite a clear difference in the functionally dominant subtype, indicates that measurement of receptor protein is unlikely to correlate with function. Fewer cells bound QAPB in the alpha(1B/D)-KO suggesting different cellular phenotypes of alpha(1A)-adrenoceptor exist. The alpha(1B/D)-KO provides robust assays for the alpha(1A)-adrenoceptor and takes us closer to understanding multi-receptor subtype interactions.
| S-EPMC2795267 | biostudies-literature
Project description:α2-Adrenegic receptors (α2Rs) are important presynaptic modulators of central noradrenergic function (auto receptors) and postsynaptic mediators of many of the widespread effects of catecholamines and related drugs. Studies have shown that ruminants (such as goats and cattle) express special α2DR subtypes in addition to α2BR and α2CR. Real-time quantitative PCR and Western blotting were used to investigate the distribution and density of α2R in different nuclei of the goat central nervous system, selected regions of the spinal cord (L4-L6), and in various peripheral tissues. α2-AR subtype-specific antibodies were injected intrathecally and intracerebroventricularly into the tested goats to block the corresponding subtype of receptors. Pain threshold and physiological parameters were evaluated to explore the functional characteristics of α2BR, α2CR and α2DR in goats. Our results suggest that the expression of the mRNAs and proteins of all three α2R subtypes are widely but unevenly distributed in the goat CNS and peripheral tissues. Furthermore, α2DR plays a more important role in α2R-mediated analgesia in goats than α2BR and α2CR, whereas α2CR activation exerts a greater effect on body temperature than α2BR and α2DR.
| S-EPMC8909296 | biostudies-literature
Project description:PurposeTo investigate the intraocular distribution and kinetics of antibodies and nanoparticles in the experimental model.MethodsAntibodies (whole IgG 149kDa, antigen-binding fragments 48.39 kDa) and four kinds of nondegradable nanoparticles (25, 50, 200, and 250 nm) were intravitreally injected in the right eye of New Zealand white rabbits. The average optical density and concentration were used to measure intraocular distribution and kinetics.ResultsAfter intravitreal injection, antibodies were distributed throughout the vitreous humor and eliminated gradually into anterior and posterior routes. Fluorescence intensity decreased 1 day after injection and was not detected 25 days after injection. The nondegradable nanoparticles migrated posteriorly to the retina 7 days after injection onward and anteriorly to the aqueous humor from 1 hour to 1 day after injection. The fluorescence intensity of the nanoparticles was relatively stable in the vitreous humor, compared to antibodies. Nanoparticles accumulated on the internal limiting membrane of the retina with no penetration into deeper retinal tissue, whereas the smaller size 25 nm nanoparticles passed across the ciliary body and moved into choroid, retina, and suprachoroidal space. A gradual decrease of nanoparticles by their sizes in the vitreous after 30 days after injection was described as the percentage ratio: 61.1% (25 nm), 69.1% (50 nm), 78.6% (200nm), and 85.3% (250 nm).ConclusionsOur study revealed the in vivo intraocular distribution and kinetics of antibodies and nanoparticles with diverse sizes and the result might help to develop newer intraocular drugs and drug delivery systems to treat retinal diseases.Translational relevanceThese experimental results can be valuable data for human research.
| S-EPMC7409074 | biostudies-literature
Project description:BACKGROUND AND PURPOSE: This study investigates the role of alpha(2)-adrenoceptor subtypes, alpha(2A), alpha(2B) and alpha(2C), on catecholamine synthesis and catabolism in the central nervous system of mice. EXPERIMENTAL APPROACH: Activities of the main catecholamine synthetic and catabolic enzymes were determined in whole brains obtained from alpha(2A)-, alpha(2B)- and alpha(2C)-adrenoceptor knockout (KO) and C56Bl\7 wild-type (WT) mice. KEY RESULTS: Although no significant differences were found in tyrosine hydroxylase activity and expression, brain tissue levels of 3,4-dihydroxyphenylalanine were threefold higher in alpha(2A)- and alpha(2C)-adrenoceptor KO mice. Brain tissue levels of dopamine and noradrenaline were significantly higher in alpha(2A) and alpha(2C)KOs compared with WT [WT: 2.8 +/- 0.5, 1.1 +/- 0.1; alpha(2A)KO: 6.9 +/- 0.7, 1.9 +/- 0.1; alpha(2B)KO: 2.3 +/- 0.2, 1.0 +/- 0.1; alpha(2C)KO: 4.6 +/- 0.8, 1.5 +/- 0.2 nmol.(g tissue)(-1), for dopamine and noradrenaline respectively]. Aromatic L-amino acid decarboxylase activity was significantly higher in alpha(2A) and alpha(2C)KO [WT: 40 +/- 1; alpha(2A): 77 +/- 2; alpha(2B): 40 +/- 1; alpha(2C): 50 +/- 1, maximum velocity (V(max)) in nmol.(mg protein)(-1).h(-1)], but no significant differences were found in dopamine beta-hydroxylase. Of the catabolic enzymes, catechol-O-methyltransferase enzyme activity was significantly higher in all three alpha(2)KO mice [WT: 2.0 +/- 0.0; alpha(2A): 2.4 +/- 0.1; alpha(2B): 2.2 +/- 0.0; alpha(2C): 2.2 +/- 0.0 nmol.(mg protein)(-1).h(-1)], but no significant differences were found in monoamine oxidase activity between all alpha(2)KOs and WT mice. CONCLUSIONS AND IMPLICATIONS: In mouse brain, deletion of alpha(2A)- or alpha(2C)-adrenoceptors increased cerebral aromatic L-amino acid decarboxylase activity and catecholamine tissue levels. Deletion of any alpha(2)-adrenoceptor subtypes resulted in increased activity of catechol-O-methyltransferase. Higher 3,4-dihydroxyphenylalanine tissue levels in alpha(2A) and alpha(2C)KO mice could be explained by increased 3,4-dihydroxyphenylalanine transport.
| S-EPMC2765593 | biostudies-literature
Project description:Rabbits have been widely used for studying ocular physiology and pathology due to their relatively large eye size and similar structures with human eyes. Various rabbit ocular disease models, such as dry eye, age-related macular degeneration, and glaucoma, have been established. Despite the growing application of proteomics in vision research using rabbit ocular models, there is no spectral assay library for rabbit eye proteome publicly available. Here, we generated spectral assay libraries for rabbit eye compartments, including conjunctiva, cornea, iris, retina, sclera, vitreous humor, and tears using fractionated samples and ion mobility separation enabling deep proteome coverage. The rabbit eye spectral assay library includes 9,153 protein groups, and 107,898 peptides. We present the data as a freely available community resource for proteomic studies in the vision field.
2021-11-24 | PXD029818 | Pride
Project description:BACKGROUND AND PURPOSE: alpha(1)-Adrenoceptors in the rabbit prostate have been studied because of their controversial pharmacological profiles in functional and radioligand binding studies. The purpose of the present study is to determine the native profiles of alpha(1)-adrenoceptor phenotypes and to clarify their relationship. EXPERIMENTAL APPROACH: Binding experiments with [3H]-silodosin and [3H]-prazosin were performed using intact tissue segments and crude membrane preparations of rabbit prostate and the results were compared with alpha(1)-adrenoceptor-mediated prostate contraction. KEY RESULTS: [3H]-Silodosin at subnanomolar concentrations bound specifically to intact tissue segments of rabbit prostate. However, [3H]-prazosin at the same range of concentrations failed to bind to alpha(1)-adrenoceptors of intact segments. Binding sites of [3H]-silodosin in intact segments were composed of alpha(1L) phenotype with low affinities for prazosin (pKi=7.1), 5-methyurapidil and N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-alpha,alpha-dimethyl-1H-indole-3-ethamine hydrochloride (RS-17053), and alpha(1A)-like phenotype with moderate affinity for prazosin (pKi=8.8) but high affinity for 5-methyurapidil and RS-17053. In contrast, both radioligands bound to a single population of alpha(1)-adrenoceptors in the membrane preparations at the same density with a subnanomolar affinity, showing a typical profile of 'classical' alpha(1A)-adrenoceptors (pKi for prazosin=9.8). The pharmacological profile of alpha(1)-adrenoceptor-mediated prostate contraction was in accord with the alpha(1L) phenotype observed by intact segment binding approach. CONCLUSIONS AND IMPLICATIONS: Three distinct phenotypes (alpha(1L) and alpha(1A)-like phenotypes in the intact segments and a classical alpha(1A) phenotype in the membranes) with different affinities for prazosin were detected in rabbit prostate. It appears that the three phenotypes are phenotypic subtypes of alpha(1A)-adrenoceptors, but are not genetically different subtypes.
| S-EPMC2597239 | biostudies-other
Project description:alpha(1)-adrenoceptor subtypes in human skeletal muscle resistance arteries were characterized using agonists noradrenaline (non-selective) and A61603 (alpha(1A)-selective), the antagonists prazosin (non-selective), 5-methyl-urapidil (alpha(1A)-selective) and BMY7378 (alpha(1D)-selective) and the alkylating agent chloroethylclonidine (preferential for alpha(1B)). Small arteries were obtained from the non-ischaemic skeletal muscle of limbs amputated for critical limb ischaemia and isometric tension recorded using wire myography. Prazosin antagonized responses to noradrenaline with a pA(2) value of 9.18, consistent with the presence of alpha(1)-adrenoceptors, although the Schild slope (1.32) was significantly different from unity. 5-Methyl-urapidil competitively antagonized responses to noradrenaline with a pK(B) value of 8.48 and a Schild slope of 0.99, consistent with the presence of alpha(1A)-adrenoceptors. In the presence of 300 nM 5-methyl-urapidil, noradrenaline exhibited biphasic concentration response curves, indicating the presence of a minor population of a 5-methyl-urapidil-resistant subtype. Contractile responses to noradrenaline were not affected by 1 microM chloroethylclonidine suggesting the absence of alpha(1B)-adrenoceptors. Maximum responses to noradrenaline and A61603 were reduced to a similar extent by 10 microM chloroethylclonidine, suggesting an effect of chloroethylclonidine at alpha(1A)-adrenoceptors at the higher concentration. BMY7378 (10 and 100 nM) had no effect on responses to noradrenaline. BMY7378 (1 microM) poorly shifted the potency of noradrenaline giving a pA(2) of 6.52. These results rule out the presence of the alpha(1D)-subtype. These results show that contractile responses to noradrenaline in human skeletal muscle resistance arteries are predominantly mediated by the alpha(1A)-adrenoceptor subtype with a minor population of an unknown alpha(1)-adrenoceptor subtype.
| S-EPMC1572837 | biostudies-other
Project description:BACKGROUND:Rabbit Hemorrhagic Disease Virus (RHDV) belongs to the Caliciviridae family, is a highly lethal pathogen to rabbits. Increasing numbers of studies have demonstrated the existence of antigenic variation in RHDV, leading to the emergence of a new RHDV isolate (RHDVb). However, the underlying factors determining the emergence of the new RHDV and its unpredictable epidemiology remain unclear. To investigate these issues, we selected more than 184 partial and/or complete genome sequences of RHDV from GenBank and analyzed their phylogenetic relationships, divergence, and predicted protein modification sites. RESULTS:Phylogenetic analysis showed that classic RHDV isolates, RHDVa, and RHDVb formed different clades. It's interesting to note that RHDVa being more closely related to classic RHDV than RHDVb, while RHDVb had a closer genetic relationship to Rabbit Calicivirus (RCV) than to classic RHDV isolates. Moreover, divergence analysis suggested that the accumulation of amino acid (aa) changes might be a consequence of adaptive diversification of capsid protein (VP60) during the division between classical RHDV, RHDVa, RHDVb, and RCV. Notably, the prediction of N-glycosylation sites suggested that RHDVb subtypes had two unique N-glycosylation sites (aa 301, 362) but lacked three other N-glycosylation sites (aa 45, 308, 474) displayed in classic RHDV and RHDVa VP60 implying this divergence of N-glycosylation sites in RHDV might affect viral virulence. Analysis of phosphorylation sites also indicated that some phosphorylation sites in RHDVa and RHDVb differed from those in classic RHDV, potentially related to antigenic variation in RHDV. CONCLUSION:The genetic relationship between RHDVb and RCV was closer than classic RHDV isolates. Moreover, compared to RHDV and RHDVa, RHDVb had two unique N-glycosylation sites but lacked three sites, which might affect the virulence of RHDV. These results may provide new clues for further investigations of the origin of new types of RHDV and the mechanisms of genetic variation in RHDV.
| S-EPMC6882040 | biostudies-literature
Project description: Not available
| S-EPMC10364772 | biostudies-literature