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PubMed1998

Differences in chemokine coreceptor usage between genetic subtypes of HIV-1.

HIV-1 uses chemokine coreceptors for cell entry. CXCR4 is the major coreceptor for T-cell-line-adapted isolates and CCR5 for non-T-cell-line-adapted isolates. This study investigated if coreceptor usage differs between genetic subtypes of HIV-1. Eighty-one primary isolates representing nine different genetic subtypes (A-J, except I) were tested on U87.CD4 glioma cells stably expressing chemokine receptor CCR1, CCR2b, CCR3, CCR5, or CXCR4. Coreceptor usage was compared to biological phenotype of the isolates (rapid/high, syncytium-inducing or slow/low, non-syncytium-inducing) and to clinical and immunological status of the study subjects. CXCR4 usage was perfectly correlated to the biological phenotype for all subtypes; all of 26 isolates with rapid/high phenotype and none of 55 isolates with slow/low phenotype could infect the CXCR4 expressing cell line. Importantly, the CXCR4-positive, rapid/high phenotype was underrepresented among subtype C isolates. Furthermore, dual tropism for CXCR4 and CCR5 was not found among subtype D isolates. Uni- and multivariate analyses indicated that these subtype-specific differences in coreceptor usage were not due to differences in clinical status, CD4 counts, or treatment. This study shows that CXCR4 usage determines the biological phenotype for all subtypes, but that there appear to exist subtype-dependent differences in frequency of usage of certain coreceptors. This opens up the possibility that genetic subtypes may differ in important biological properties such as virulence, tissue tropism, and transmissibility.

Genotype↗
PubMed1999

Subtype G and multiple forms of A/G intersubtype recombinant human immunodeficiency virus type 1 in Nigeria.

Multiple human immunodeficiency virus type 1 (HIV-1) genetic subtypes, intersubtype recombinants, and group O have been found in west central Africa. In Nigeria, where HIV-1 prevalence is rising rapidly, characterization of HIV-1 strains has been limited. Each of three full-length genome sequences acquired to date shows evidence of recombination: two are largely subtype G with subtype A segments in the midgenome accessory region; the third, IbNG, is subtype G with the long terminal repeats and two segments of pol from subtype A. In this study, peripheral blood mononuclear cells obtained in 1994-1995 from 10 patients hospitalized in northeastern Nigeria were evaluated by sequencing of the complete envelope and, from 7 patients, a portion of gag. Four patients harbored subtype G viruses and six patients had recombinant viruses. Two had strains sharing the A/G recombinant structure of IbNG. Two had a previously undescribed recombinant, mostly subtype A, whose carboxyl-terminal gp41 could not be classified. An A/G recombinant different from IbNG but similar to CA1, a Cameroonian strain, was found in one patient. The remaining patient had a strain that was otherwise subtype G but shared an unclassified carboxyl-terminal gp41 segment with the CA1-like strains. Other subtypes and group O were not found.

Base Sequence↗
PubMed1993

Distribution and regulation of natriuretic factor-R1C receptor subtypes in mammalian cell lines.

The differential distribution of natriuretic peptide receptor subtypes and their distinct properties were assessed in mammalian cellular models which were screened for their ability to produce cGMP upon stimulation by different natriuretic peptides. The ANF-R1A receptor subtype was distinguished by its selective activation by atrial natriuretic factor (ANF) while the ANF-R1C was characterized by preferential stimulation by C-type natriuretic peptide (CNP). AT-620 pituitary cells, bovine adrenal chromaffin cells, and NIH-3T3 fibroblasts mainly express the ANF-R1C receptor subtype. Other cell lines such as PC12, RASM and GH3 express significant but varying amounts of both ANF-R1A and ANF-R1C subtypes. A10 and NIH cells which express high density of ANF-R2 receptor subtype, also demonstrate a higher sensitivity to CNP over ANF suggesting that they express significant amounts of ANF-R1C. Studies of the regulation by ATP of guanylyl cyclase activity indicate that both ANF-R1A and ANF-R1C subtypes are modulated in the same manner. In the presence of Mn2+, ATP inhibits the CNP-stimulated guanylyl cyclase activity while in the presence of Mg2+ adenine nucleotides potentiate the stimulation by CNP. In addition, we show that like the ANF-R1A, the ANF-R1C guanylyl cyclase activity can be regulated by phosphorylation since preincubation with TPA or FKL attenuates the subsequent stimulation by CNP in cultured cells. The results presented demonstrate that specific cell types express distinct natriuretic peptide receptor subtypes and also that the newly characterized ANF-R1C subtype is regulated by ATP and serine/threonine kinases in the same way as the ANF-R1A subtype.

3T3 Cells↗
PubMed1993

Effect of lymphoblastoid alfa-interferon in patients with chronic hepatitis C having different genotypic subtype of hepatitis C virus.

Five subtypes of hepatitis C virus (Pt[I], K1[II], K2a[III], K2b[IV] and Tr[V]) have been suggested based on the nucleotide sequence of the non-structural five region. To assess the susceptibility of each subtype to interferon therapy, we developed a one-step method which allows quick determination of subtype using polymerase chain reaction with a mixed primer set deduced from the sequence of each subtype. We were able to determine the subtype of 69 of 80 (86.3%) Japanese patients who received natural alfa-interferon treatment. The incidence of each subtype was K1: 53 (76.8%), K2a: 14 (20.3%), K2b: 3 (4.3%) and Tr: 1 (1.4%). Interferon was administered to these patients and found to be effective in 28 of 51 (54.9%) patients with K1 subtype and in 16 of 18 (88.9%) patients with other subtypes (P < 0.01). These data show that K1 is a major subtype in Japan and relatively resistant to interferon treatment.

Adult↗
PubMed2002

Actions of alpha-2 noradrenergic agonists on spatial working memory and blood pressure in rhesus monkeys appear to be mediated by the same receptor subtype.

RATIONALE: alpha-2 Noradrenergic agonists improve spatial working memory in animals and in humans. Of the three alpha-2 receptor subtypes, evidence has suggested that this cognitive improvement may be mediated by the alpha-2A receptor subtype, but this has not been established. alpha-2 Agonists are also known to decrease blood pressure significantly. Recent evidence using genetically altered mice indicates that the alpha-2A receptor subtype mediates this decrease in blood pressure. OBJECTIVES: The present study examined whether the cognitive improvement and hypotension produced by alpha-2 agonists are mediated by the same receptor subtype in rhesus monkeys. The hypotensive and cognitive-enhancing effects of clonidine and guanfacine were challenged with two alpha-2 antagonists with differing affinities for the three alpha-2 receptor subtypes: MK912, a potent antagonist which shows preferential binding to the alpha-2C receptor subtype, and idazoxan, which slightly prefers the alpha-2A receptor subtype. If alpha-2C receptors contribute to the cognitive enhancement, MK912 should reverse the cognitive-enhancing effects of alpha-2 agonists at lower doses than those needed to reverse the hypotensive effects of these compounds. Conversely, if alpha-2A receptors contribute to cognitive enhancement, MK912 and idazoxan should reverse the cognitive-enhancing effects of alpha-2 agonists at the same doses as those needed to reverse the hypotensive effects of these compounds. RESULTS: MK-912 and idazoxan dose-dependently reversed both clonidine and guanfacine-induced cognitive improvement and hypotension. Both antagonists were equally potent in reversing either the cognitive enhancement or the hypotension. CONCLUSIONS: The identical pattern of dose-dependent reversal of cognitive improvement and hypotension indicates that, in non-human primates, the same receptor subtype mediates both effects. Previous evidence suggests that the most likely candidate is the alpha-2A receptor subtype.

Adrenergic Agents↗
PubMed1983

High and low potency interferon-alpha subtypes induce (2'-5') oligoadenylate synthetase with similar efficiency.

Three major subtypes of human interferon-alpha (IFN-alpha), isolated from virus-induced leukocytes, were compared for their antiviral and anticellular activities on one hand, and for their ability to induce (2'-5') oligoadenylate synthetase on the other hand. One subtype, IFN-alpha 1, was found to have low specific antiviral (6.10(6)-5.10(7) units/mg) and anticellular activities when measured on a variety of human cells. A second subtype, exhibiting an unusually high molecular weight (26,000) by SDS-polyacrylamide gel electrophoresis (IFN-alpha 26K), was found to have the highest known specific antiviral (8.10(8)-2.10(9) units/mg) and anticellular activities. Thus, these two subtypes of IFN-alpha differ by a factor of 330 and represent the two extremes in the antiviral scale on human cells. A third subtype, IFN-alpha 2, was tested as well and was found to have intermediate antiviral and anticellular activities. The ability of these three subtypes to induce (2'-5') oligoadenylate synthetase in human cells was then measured. It was found that on a weight basis, the three subtypes were equally effective in inducing the enzyme. Since the level of (2'-5') adenylate oligomers is affected also by the interferon-induced (2'-5') phosphodiesterase, the ability of these subtypes to induce this enzyme was compared as well and was found to be very similar. We therefore conclude that the differences in potency between these IFN-alpha subtypes are not related to their ability to induce (2'-5') oligoadenylate synthetase.

2',5'-Oligoadenylate Synthetase↗
PubMed1986

Use of monoclonal antibodies to determine if HBsAg of mixed subtype is one particle or two.

Of the ten universally recognized subtypes of hepatitis B surface antigens, two contain both of what are normally mutually exclusive subtype determinants. These mixed subtypes presumably result from a concurrent infection by different viral subtypes and result in the presence of the various subtypes either on separate particles or intermixed on one particle. This study demonstrates that both of these alternatives can occur. Additionally, a mixed subtype present on one particle could result from either intermixing of separate protein subtypes into the same viral envelope (phenotypic mixing) or genetic recombination between viruses of different subtypes resulting in the production of one protein with a mixed subtype (genotypic mixing). The latter possibility would make the definition of the antigenic determinants and production of a vaccine for hepatitis B more difficult since it implies that the virus can alter its surface characteristics. Evidence is provided against genotypic mixing, demonstrating that phenotypic mixing does occur but does not 'breed true' upon viral transmission.

Antibodies, Monoclonal↗
PubMed1996

Heteroduplex mobility assay for subtyping HIV-1: improved methodology and comparison with phylogenetic analysis of sequence data.

The feasibility of using DNA heteroduplex mobility analysis (HMA) as a rapid and reproducible method for routine subtyping HIV-1 in clinical specimens was examined by comparison with subtype determination by sequencing in both the gag and env genes. The heteroduplexes formed were examined by conventional polyacrylamide gel electrophoresis (PAGE) and also by electrophoresis in the Pharmacia PhastSystem. The significance of the HMA results was determined by the Kruskal-Wallis test, a non-parametric one way analysis of variance. It was possible to obtain an HMA profile rapidly (1-2 days) using fast PCR conditions and the PhastSystem. The HMA bands were generally sharper and more satisfactory on the Phast gels than on conventional polyacrylamide gels and the use of Phast gels was an improvement over conventional PAGE. Non-B subtype viruses could be distinguished from B subtypes, but it was more difficult to distinguish between the non-B subtypes and to assign a subtype to them. Thus, HMA can be adapted to offer a rapid screening method for HIV-1 subtyping, but sequencing is still necessary to assign a definitive subtype. This reflects the empirical nature of the subtype definitions and the quasispecies nature of the HIV genome population.

DNA, Viral↗
PubMed1989

Isolation and characterisation of five histone H1 subtypes from adult rat liver.

A procedure is described for quantitative purification of H10 and five H1-1 subtypes--named H1-1a to e--from adult rat liver by reverse-phase high-pressure liquid chromatography. Milligram amounts of each fraction have been obtained. The H1-1a subtype shows a very high lysine content (34%) and H1-1d subtype has an amino-acid composition close to that of H10, but its electrophoretic mobility is different. Salt dependent folding of these subtypes has been studied by circular dichroism. In the presence of 2 or 10 mM sodium phosphate buffers at pH 7.5, H1-1a shows the lowest alpha-helix content. In phosphate-buffer containing 1 M NaCl the number of residues in alpha-helix for all the subtypes rises to 9-10%. Partial cleavage of these subtypes by endoproteinase Glu-C produce three main peptides arising from C-terminal domains. The interaction of the H1-1 subtypes with 196 basepairs linear DNA, purified from rat liver chromatin by high-pressure ion-exchange liquid chromatography, has for consequences a modification of the patterns of digestion: partial proteolysis of the H1-1a and H1-1b subtypes shows differences in the presence or in absence of DNA; on the contrary, H1-1c and H1-1d seem to have the same organization. So these subtypes may play a role in the differential packing of specific region of chromatin.

Amino Acids↗
PubMed1994

Molecular cloning of rat cDNAs for the zeta and theta subtypes of 14-3-3 protein and differential distributions of their mRNAs in the brain.

We isolated from the rat brain two cDNA clones encoding the zeta and theta subtypes of the 14-3-3 protein. Both clones encoded 245 amino acid sequences, which share a high sequence homology with each other and also with other subtypes of the 14-3-3 protein. The distribution of their mRNAs was determined in the developing brain, by in situ hybridization with subtype-specific oligonucleotide probes. At embryonic day 18, the zeta and theta subtype mRNAs were expressed at high levels throughout the brain and the spinal cord. Distribution patterns of the two mRNAs were distinct in the brain at postnatal day 21. The zeta subtype mRNA was distributed widely in the brain gray matter, and high levels of the transcripts were detected in various brain regions, including the neocortex, hippocampus, caudate-putamen, thalamus, cerebellar cortex, and several brainstem nuclei. On the other hand, high signal levels of the theta subtype mRNA in the gray matter were restricted to the cerebellar cortex and the hippocampus. In addition, significant signals for the theta subtype mRNA were found over the white matter, where cell bodies of glial cells are populated. The wide gene expression of the zeta and theta subtypes suggests their fundamental and essential role in the brain function, but the degrees of functional involvement by the respective subtypes would be heterogeneous between neuron and glia, and also among neuron types.

14-3-3 Proteins↗
PubMed2004

Pleural biopsy: a reliable method for determining the diagnosis but not subtype in mesothelioma.

BACKGROUND: Survival after tri-modality therapy with extrapleural pneumonectomy (EPP) and postoperative chemoradiotherapy is longer for patients with epithelial MPM versus mixed or sarcomatoid subtypes, leading some to decline aggressive therapy for patients with nonepithelial histology. However, pathologic diagnosis of malignant pleural mesothelioma (MPM) and subclassification into one of the three histologic subtypes (epithelial, mixed, sarcomatoid) can be challenging. Pleural biopsy has been proposed as the diagnostic gold standard. We investigated the accuracy of open pleural biopsy for diagnosis and subtype identification in MPM. METHODS: Patients with suspected MPM routinely undergo open pleural biopsy to establish diagnosis. Those diagnosed definitively by pleural biopsy or cytology are offered pleurectomy or EPP dependent on stage and cardiorespiratory status. We reviewed medical records for all patients undergoing EPP at our institution, comparing tissue and subtype diagnosis at initial diagnostic biopsy versus definitive resection. RESULTS: Between 1988 and 2000, 305 of 332 consecutive patients undergoing EPP had MPM. One patient diagnosed with MPM at pleural biopsy was misclassified. Subtype analysis at pleural biopsy proved correct in 80% (226/282). Most patients (174/192) with epithelial subtype at final diagnosis were diagnosed correctly at pleural biopsy. However, 44% (45/103) with pathologic diagnosis of nonepithelial subtype at resection were initially misdiagnosed with the epithelial subtype. The sensitivity of pleural biopsy for epithelial MPM was 97% with a specificity of 56%. CONCLUSIONS: Open pleural biopsy is accurate and should be considered the gold standard diagnostic method for MPM. It is less sensitive for determining histologic subclass, particularly with nonepithelial subtypes.

Biopsy↗
PubMed2006

Clinicopathologic significance of the basal-like subtype of breast cancer: a comparison with hormone receptor and Her2/neu-overexpressing phenotypes.

DNA microarray profiling studies have led to the classification of invasive breast carcinoma into luminal/estrogen receptor-positive, normal breast-like, Her2/neu-overexpressing, and basal-like types. Among these groups, the basal-like subtype is associated with the poorest clinical outcome in Western countries. To date, the clinicopathologic characteristics of the basal-like carcinomas, compared with other subtypes, have not been described in the Korean population. In this study, we used tissue microarray to examine the expression of basal cytokeratins (CK) (CK5 and CK14) and luminal CK (CK8/18), epidermal growth factor receptor, c-kit, hormone receptors (HRs), p53, and Her2/neu in 776 consecutive patients diagnosed with invasive breast carcinoma from January 1993 to December 1998 and categorized these cases into 5 subgroups (basal-like, HR-expressing, Her2/neu-overexpressing, HR and Her2/neu-expressing, and null subtypes negative for all markers), based on the immunohistochemical data. We identified cases of 114 (14.7%) basal-like, 345 (44.5%) HR-expressing, 133 (17.1%) Her2/neu-overexpressing, 61 (7.8%) HR and Her2/neu-expressing, and 123 (15.9%) null subtypes. Histologically, most basal-like breast cancers were invasive ductal carcinoma, not otherwise specified (98 cases, 86.0%), with high nuclear and/or histologic grades, and most metaplastic carcinomas (6 [75.0%] of 8 cases) were the basal-like subtype. Both basal-like and Her2/neu-overexpressing subtypes were associated with larger tumor sizes (mean, 3.6 and 3.3 cm, respectively) than the HR-expressing group (mean, 2.8 cm) (P = .001 and P = .036, respectively). Nodal stage of Her2/neu-overexpressing subtype was higher than that of basal-like subtype; however, overall stage was not different between the 2 groups (P = .010 and .123, respectively). Distant metastasis was most frequently observed in the Her2/neu-overexpressing subtype (33.8%), which was prognostically the worst subgroup of breast cancers. In contrast to previous findings from Western countries, our analyses reveal that the Her2/neu status is the most important prognostic factor of breast cancers.

Adult↗
PubMed2006

Evaluation of NucliSens EasyQ HIV-1 assay for quantification of HIV-1 subtypes prevalent in South-east Asia.

BACKGROUND: Monitoring anti-retroviral therapy requires that viral load assays for human immunodeficiency virus type 1 (HIV-1) be applicable to diverse HIV-1 subtypes. OBJECTIVES: To evaluate NucliSens EasyQ HIV-1 assay for quantitation of common HIV-1 subtypes prevalent in South-east Asia. STUDY DESIGN: One hundred and nineteen plasma samples collected in Hong Kong and Cambodia were used to compare the performance of NucliSens EasyQ HIV-1 and COBAS Amplicor HIV-1 Monitor version 1.5 assays. Viral RNA extracted from the NucliSens MiniMAG was also used for HIV-1 subtyping. RESULTS: Performance of NucliSens EasyQ correlated well with COBAS Amplicor (r=0.777, p<0.001) and the small mean difference (0.0462log(10)IU/mL) obtained in the Bland and Altman model indicated good agreement between two assays. The NucliSens EasyQ assay demonstrated a 95% sensitivity at 500IU/mL and 100% specificity. Reproducibility of this assay was within log(10)2-4IU/mL and had a coefficient of variation between 2.3% and 10.4%. Among the 109 specimens included in the analysis, HIV-1 subtyping identified 64 CRF01_AE, 38 subtype B, 3 subtype C, 3 CRF07_BC and 1 subtype G viruses. CONCLUSIONS: Performance of NucliSens EasyQ was comparable to COBAS Amplicor for HIV-1 viral load monitoring. RNA extracts from NucliSens MiniMAG could be used for HIV-1 viral load monitoring, subtyping and drug resistance mutations detection. Our findings highlight the versatility of both NucliSens EasyQ and COBAS Amplicor in monitoring prevalent subtypes and rare circulating recombinant forms (CRFs) in the South-east Asia region.

Cambodia↗
PubMed2006

In rat hepatocytes, different adenosine receptor subtypes use different secondary messengers to increase the rate of ureagenesis.

In rat hepatocytes, the role of cAMP and Ca(2+) as secondary messengers in the ureagenic response to stimulation of specific adenosine receptor subtypes was explored. Analyzed receptor subtypes were: A(1), A(2A), A(2B) and A(3). Each receptor subtype was stimulated with a specific agonist while blocking all other receptor subtypes with a battery of specific antagonists. For the A(1) and A(3) adenosine receptor subtypes, the secondary messenger was the cytoplasmic Ca(2+) concentration ([Ca(2+)](cyt)). Accordingly, the A(1) or A(3)-mediated increase in [Ca(2+)](cyt) and in ureagenic activity were both inhibited by chelating Ca(2+) with either EGTA or BAPTA-AM. Also, Gd(3+) blocked both the increase in [Ca(2+)](cyt) and ureagenesis, suggesting that a Ca(2+) channel may be involved in the response to both A(1) and A(3). A partial effect was observed with the sarcoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin. The concentration of cyclic AMP ([cAMP]) increased in response to stimulation of either the A(2A) or the A(2B) adenosine receptor subtypes, while it decreased slightly in response to stimulation of either A(1) or A(3). The stimulation of either the A(2A) or A(2B) adenosine receptor subtypes resulted in an increase in [cAMP] and an ureagenic response which were not sensitive to EGTA, BAPTA-AM, Gd(3+) or to thapsigargin. In addition, the adenylyl cyclase inhibitor MDL12,330A blocked the ureagenic response to A(2A) and A(2B), but not the response to either A(1) or A(3). Our results indicate that in the ureagenic liver response to adenosine, the secondary messenger for both, the A(1) and A(3) adenosine receptor subtypes is [Ca(2+)](cyt), while the message from the A(2A) and A(2B) adenosine receptor subtypes is relayed by [cAMP].

Adenosine↗
PubMed2005

Characteristics of HIV-1 env V3 loop sequences for subtype A1 variant spread in Eastern Europe.

OBJECTIVES: The aim of our investigation was to study the HIV-1 env V3 loop sequences of the subtype A variant of the virus actively circulating in the territory of Eastern Europe (A1-EE). DESIGN: It appears that the characteristics of HIV-1 V3 loop sequences of the thoroughly studied subtype B strains determine the viruses tropism and phenotype. We were interested to find out to what extent these trends are preserved with the subtype A isolates spread in Eastern Europe. METHODS: About 474 HIV-1 strains were isolated by cocultivation with phytohemagglutinin stimulated and interleukin-2 activated PBMC. Isolates were subtyped after distinct regions gag, pol and env were amplified by nested polymerase chain reaction. V3 sequences were studied in detail and compared with the isolates that appeared as SI or NSI in test-cultures MT-2. RESULTS: The subtype A HIV-1 strains spread in Eastern Europe appeared to have V3 loop sequences with a high level of conservatism. The consensus sequences of the SI and NSI isolates proved identical, and correspond to the phenotype markers of subtype B NSI strains. Nevertheless, the subtype A SI isolates induced active syncytium-formation processes in the MT-2 test culture. CONCLUSION: The data obtained evidenced to the following: the HIV-1 subtype A variant spread in Eastern Europe, is capable of using for interaction with receptor structures of sensitive cells the tools and mechanisms, which are significantly different from those described for subtype B.

Amino Acid Motifs↗
PubMed2003

Differential expression of IFN-alpha subtypes in human PBMC: evaluation of novel real-time PCR assays.

Studies of the human IFN-alpha subtype system have been hampered by the lack of efficient procedures to quantify and differentiate the expression of the highly homologous IFN-alpha subtypes. Here we evaluate four novel real-time PCR assays for the specific detection and quantification of IFN-alpha mRNA for the subtypes alpha(2), alpha(6), alpha(8) and alpha(1/13) in a combined assay in human peripheral blood mononuclear cells (PBMC). This included (a) the selection of beta-glucuronidase (GUS) as a suitable housekeeping gene for relative quantification; (b) verification of the specificity by using human DNA of different IFN-alpha subtypes; and (c) comparison of the amplification efficiencies among the different assays. This highly sensitive method allows the detection of low-level, constitutive IFN-alpha mRNA and shows differences in the composition of constitutive IFN-alpha subtypes compared to other cell types (HeLa and HEp-2). The in vitro stimulation of PBMC with Newcastle disease virus (NDV), Respiratory syncytial virus (RSV) or an inactivated Herpes simplex (HSV) preparation leads to the transcriptional induction of all IFN-alpha subtypes investigated but to different expression levels. Among the subtypes detected, IFN-alpha(13/1) and alpha(2) are the major transcripts followed by alpha(8), and finally alpha(6) as a minor transcribed subtype. Time-kinetics of IFN-alpha transcriptional activation also revealed variations in the course of IFN-alpha transcription between NDV, RSV or HSV. The data obtained from the real-time PCR assays correlated well with IFN-alpha(2) protein release. In conclusion, we have demonstrated the suitability and reliability of new real-time PCR assays for the rapid and efficient analysis of IFN-alpha subtype expression.

Animals↗
PubMed1998

Alpha1-adrenergic receptor subtypes in human detrusor.

PURPOSE: To identify and quantitate alpha1-adrenergic receptor (alpha1AR) subtype expression in human detrusor. MATERIALS AND METHODS: Initial studies to determine alpha1AR expression in human detrusor were performed using saturation binding with [125I]HEAT. Once the presence of alpha1ARs was documented, subtype (alpha1a, alpha1b, alpha1d) expression at the mRNA level (and comparison with rat) was determined with RNase protection assays (human detrusor) and RT-PCR (human detrusor, rat whole bladder). Competition binding analysis with the alpha1dAR-selective ligand BMY7378 was used to measure alpha1AR subtype expression at a protein level in human detrusor. RESULTS: Alpha1AR expression in human detrusor was low but reproducible (6.3 +/- 1.0 fmol./mg. total protein). RNase protection assays performed on total RNA extracted from human detrusor revealed the following alpha1AR subtype expression: alpha1d (66%) > alpha1a (34%), and no alpha1b. RT-PCR confirmed alpha1AR subtype mRNA distribution in human detrusor with alpha1d (approximately 60-70%) > alpha1a (approximately 30-40%), and a lack of alpha1b mRNA. Rat whole bladder expressed different alpha1AR subtype mRNA than human detrusor, with alpha1a approximately alpha1b approximately alpha1d. The presence of alpha1d > alpha1a expression in human detrusor was confirmed at a protein level by competition analysis utilizing BMY7378 which revealed a two-site fit, with Ki and high affinity binding (66%) consistent with the alpha1dAR subtype. CONCLUSIONS: Human detrusor contained two alpha1AR subtypes (alpha1d > alpha1a), a finding that is different from rat, another commonly used animal model. Since non-subtype selective alpha1AR antagonists ameliorate irritative bladder symptoms (in men and women with/without outlet obstruction), and Rec 15/2739 (alpha1a selective antagonist) does not improve symptom scores in BPH, our findings suggest bladder alpha1dARs may provide a potentially novel mechanism underlying these therapeutic benefits.

Animals↗
PubMed2001

Alpha1A- and alpha1B-adrenoceptors are the major subtypes in human saphenous vein.

In this study we analyzed the different alpha1-adrenoceptor (AR) subtypes present in human saphenous vein (HSV) using reverse transcription polymerase chain reaction (RT-PCR), DNA-DNA hybridization analysis and functional affinities for alpha-AR antagonists. DNA-DNA hybridization analysis of RT-PCR amplification products confirmed the presence of alpha1A- and alpha1B-ARs, and low levels of alpha1D-AR in HSV. The functional results showed: (1) prazosin, the selective alpha1-AR antagonist, phentolamine, the alpha1- and alpha2-ARs antagonist, WB 4101 and 5-MU, the selective alpha1A-AR subtype antagonists were potent, competitive antagonists of noradrenaline (NA)-induced contraction (pA2 values of 11.03, 8.06, 9.02 and 8.34, respectively). (2) Alpha1-AR-induced contraction was sensitive to the alkylating effects of CEC (the alpha1B and alpha1D-AR subtypes antagonist) and (3) The selective alpha1D-AR subtype antagonist BMY displayed low affinity (pA2 values of 6.44). This indicates that the contractile response of the HSV to alpha1-AR-induced is predominantly mediated by both alpha1A and alpha1B-AR subtypes. This was also supported by the good relationship between pA2 values from the present study and reported binding affinities (pKi) values of various alpha1-AR subtype antagonists with cloned human alpha1A- and alpha1B-AR subtypes (r=0.89 and r=0.98, respectively), but not the alpha1D-AR subtype (r=0.67). Our results indicate that alpha1A- and alpha1B-ARs are the main functional and expressed receptor subtypes in HSV.

Adrenergic alpha-Antagonists↗