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PubMed2005

Synapse-to-synapse variation of calcium channel subtype contributions in large mossy fiber terminals of mouse hippocampus.

Both N- and P/Q-type voltage-dependent calcium channels are involved in fast transmitter release in the hippocampus, but are differentially regulated. Although variable contributions of voltage-dependent calcium channel subtypes to presynaptic Ca2+ influx have been suggested to give a neural network of great diversity, their presence has only been demonstrated in a culture system and has remained unclear in the brain. Here, the individual large mossy fiber presynaptic terminal was labeled with Ca2+/Sr2+-sensitive fluorescent dextrans in the hippocampal slice of the mouse. The fractional contribution of voltage-dependent calcium channel subtypes to presynaptic Ca2+/Sr2+ influx was directly measured by the sensitivity of Ca2+/Sr2+-dependent fluorescent increment to subtype-selective neurotoxins, omega-conotoxin GVIA (an N-type selective blocker), omega-agatoxin IVA (a P/Q-type selective blocker) and SNX-482 (an R-type selective blocker). Synapse-to-synapse comparison of large mossy fiber terminals revealed that the contributions of N- and R-type voltage-dependent calcium channels varied more widely than that of P/Q-type. Even two large mossy fiber presynaptic terminals neighboring on the same axon differed in the fractional contributions of N- and R-type voltage-dependent calcium channels. On the other hand, these terminals were similar in the fractional contributions of P/Q-type voltage-dependent calcium channels. These results provide direct evidence that individual large mossy fiber synapses are differential in the contribution of N- and R-type voltage-dependent calcium channel subtypes to presynaptic Ca2+/Sr2+ influx. We suggest that the synapse-to-synapse variation of presynaptic voltage-dependent calcium channel subtype contributions may be one of the mechanisms amplifying diversity of the hippocampal network.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed2005

Galanin (2-11) binds to GalR3 in transfected cell lines: limitations for pharmacological definition of receptor subtypes.

The neuropeptide galanin regulates a variety of physiological and pathophysiological processes through three G protein coupled receptors, GalR1, GalR2, and GalR3. The studies on galanin receptor subtype specific effects have been hampered by the lack of high affinity subtype selective antagonist and/or agonist to any of these three galanin receptor subtypes. Since its recent introduction in 2003, galanin (2-11) has been widely used as a GalR2 selective agonist in several in vitro and in vivo studies. In the present paper, we demonstrate that galanin (2-11) binds to rat GalR3 receptors in transfected cell lines with a similar affinity as it binds to GalR2. As none of the available antagonists are galanin receptor subtype selective, as shown here for M35 and M40, more work is needed to confirm whether a galanin (2-11) effect is GalR2 mediated and there is an urgent need for high affinity galanin receptor subtype selective ligands. For now one needs to interpret the data obtained at lower galanin (2-11) concentrations as effects mediated by non-GalR1 type galanin receptors, i.e., GalR2 and/or GalR3.

Animals↗
PubMed2004

Agitated depression: a valid depression subtype?

PURPOSE: The diagnostic validity of agitated depression (AD, a major depressive episode (MDE) with psychomotor agitation) is unclear. It is not classified in DSM-IV and ICD-10 classification of mental and behavioural disorder (ICD-10). Some data support its subtyping. This study aims to test the subtyping of AD. METHODS: Consecutive 245 bipolar-II (BP-II) and 189 major depressive disorder (MDD) non-tertiary-care MDE outpatients were interviewed (off psychoactive drugs) with Structured Clinical Interview for DSM-IV Axis I Disorders--Clinician Version (SCID-CV), Hypomania Interview Guide (HIGH-C), and Family History Screen. Intra-MDE hypomanic symptoms were systematically assessed. AD was defined as an MDE with psychomotor agitation. Mixed AD was defined as an MDE with four or more hypomanic symptoms (including agitation). FINDINGS: AD was present in 34.7% of patients. AD was mixed in 70.1% of AD patients. AD, vs. non-AD, had significantly (at alpha = 0.05) lower age at onset, more BP-II, females, atypical depressions, bipolar-I (BP-I) and BP-II family history, and was more mixed; racing/crowded thoughts, irritability, more talkativeness, and risky behaviour were significantly more common. Mixed AD, vs. non-AD, had significantly (at alpha = 0.01) lower age at onset, more intra-MDE hypomanic symptoms, BP-II, females, atypical depressions, BP-II family history, and specific hypomanic symptoms (distractibility, racing thoughts, irritable mood, more talkativeness, risky activities). Mixed AD, vs. non-mixed AD, had significantly more intra-MDE hypomanic symptoms (by definition), more recurrences, and more specific hypomanic symptoms (by definition). Non-mixed AD, vs. non-AD, had significantly more intra-MDE hypomanic symptoms and more talkativeness. CONCLUSIONS: AD was common in non-tertiary-care depression outpatients, supporting its diagnostic utility. AD and many bipolar diagnostic validators were associated, supporting its link with the bipolar spectrum. Mixed AD, but not non-mixed AD, had differences vs. non-AD similar to those of AD, suggesting that psychomotor agitation by itself may not be enough to identify AD as a subtype. Findings seem to support the subtyping of mixed AD. This subtyping may have important treatment impact, as antidepressants alone might increase agitation.

Adult↗
PubMed2004

Serum prolactin levels in unmedicated first-episode and recurrent schizophrenia patients: a possible marker for the disease's subtypes.

Various studies indicate that we must consider schizophrenia not as a single disease but as several distinct etiological processes that give rise to characteristic symptoms. In the current study, we aimed to examine prolactin serum levels in unmedicated first-episode and recurrent schizophrenic patients. The prolactin levels were compared among the different schizophrenia subtypes, i.e. paranoid, schizoaffective and disorganized. Prolactin serum samples were assessed on the morning after the admission in 48 first-episode and 38 recurrent unmedicated hospitalized schizophrenia patients. Two psychiatrists made the diagnosis without knowledge of laboratory results and completed the rating scales. Despite all prolactin levels being within or close to the normal range, we found significant differences in prolactin serum levels among schizophrenia subtype patients: the lowest values were for the paranoid type, intermediate for the schizoaffective and the highest for the disorganized patients. The results seem to indicate a pronounced hyperdopaminergic activity in paranoid schizophrenia, suggesting differences in dopaminergic tone between the schizophrenia subtypes, and support the clinical and the neuropsychological individuality of disease subtypes. There were no significant differences in prolactin serum levels of the schizophrenia subtypes between the first-episode and the recurrent patients. It appears that there are constant patterns of dopamine bioactivity in acutely psychotic unmedicated schizophrenia patients, whether the patients are first admitted or recurrent.

Adult↗
PubMed2004

Cellular immune responses to feline immunodeficiency virus (FIV) induced by dual-subtype FIV vaccine.

Vaccine-induced T cell responses to FIV were assessed by measuring FIV-specific cytokine and cytotoxic-effector molecule production. A total of 22 cats at 10-12 weeks of age received either dual-subtype FIV vaccine (n=12), uninfected cell lysate (n=5) consisting of cells used to produce vaccine viruses, or no immunization (n=5). Significant increases in mRNA and protein production of T-helper 1 (Th1) cytokines (IL-2, IFNgamma), mRNA production of a cytotoxic-effector molecule (perforin), and lymphoproliferation response were observed in peripheral blood mononuclear cells (PBMC) from dual-subtype FIV-vaccinated cats after in vitro stimulation with inactivated FIV. In contrast, no statistically significant increase in FIV-stimulated mRNA production of Th2 cytokines (IL-4, IL-6) or other cytotoxic-effector molecules (TNFalpha, FasL) was observed in the PBMC from dual-subtype vaccinated cats. Moreover, no FIV-specific increases in the IFNgamma, IL-2, and perforin mRNA productions and in the IFNgamma bioactivity and lymphoproliferation responses were observed in the PBMC from cell-immunized cats. These observations suggest that IFNgamma induction, lymphoproliferation, and significant portion of IL-2 and perforin productions in the PBMC from dual-subtype vaccinated cats are clearly specific for viral antigens. Overall, dual-subtype FIV vaccine elicited strong Th1 response (IFN(, IL-2), which may contribute to the vaccine protection by enhancing the perforin-mediated cytotoxic-cell activity against FIV.

Animals↗
PubMed2005

T-cell vaccination against anti-CD4 autoimmunity in HIV-1 subtypes B and C-infected patients--an extended open trial.

This study is an extended clinical trial of the one initiated and reported in the Journal of Clinical Virology 2004;31S:S48-54. Thirteen HIV-1 patients (eight subtype B and five subtype C) that manifested T-cell autoimmunity to recombinant human CD4 (rCD4) were treated with T-cell vaccine composed of glutaraldehyde-treated autologous anti-CD4 reactive T-cells and compared to historical seven non-vaccinated HIV-1-infected subjects. This study proved to be feasible and safe. Follow-up study revealed that 7/8 subtype B and 2/4 subtype C patients (one has just received the first TCV injection) responded with a persistent increase in their blood CD4 T-cell levels and four subtype B patients manifested decreased anti-CD4 autoimmunity. Despite highly active antiretroviral therapy (HAART), the persistence of CD4 T-cell lymphopenia may be associated with anti-CD4 autoimmunity. T-cell vaccination (TCV) may decrease such autoimmunity and elevate CD4 T-cell numbers.

Adult↗
PubMed2004

Genetic relationships, serological cross-reaction and cross-protection between H1N2 and other influenza A virus subtypes endemic in European pigs.

This study examines the genetic relationships between the recently emerged H1N2 swine influenza virus and viruses of H1N1 and H3N2 subtypes, and the extent of protection against H1N2 challenge in pigs immune after infection or vaccination with the other subtypes. There was low amino acid homology (70.4-71.9%) in the haemagglutinin (HA) gene between H1N1 viruses used for primary infection or vaccination and the H1N2 challenge strain, with 94-99 amino acid changes between these viruses involving all five antigenic sites. The NA genes of H3N2 viruses used for primary infection or vaccination showed higher amino acid homology with H1N2 (88.3-92.6%), while nucleoprotein (95.5-96.3% nucleotide identity) and matrix (96.8-98.4%) genes were most conserved between the three subtypes. Pigs immune as a result of intranasal inoculation with either H1N1 or H3N2 showed partial clinical protection against H1N2 challenge, and nasal virus excretion was 2 days shorter than in naive pigs. Moreover, dually infected (H1N1 + H3N2)-immune pigs showed complete clinical protection and H1N2 virus replication in the lungs and nasal secretions was either undetectable or markedly reduced. In contrast, a double vaccination with a commercial H1N1 and H3N2-based vaccine did not protect against H1N2 challenge. Haemagglutination inhibition (HI) or virus neutralisation (VN) tests of swine sera revealed little if any antigenic cross-reactivity between subtypes. These data suggest that serum HI or VN antibodies are not essential in heterosubtypic protection, but that mucosal or cellular immunity are probably involved. It is still unknown whether this type of cross-subtype protection will also occur in infection-immune pigs in the field.

Animals↗
PubMed2006

Distribution and heterogeneity of small ruminant lentivirus envelope subtypes in naturally infected French sheep.

Small ruminants lentiviruses (SRLV) nucleotide sequences spanning the V1V2 variable regions of the env gene were amplified by nested-PCR from 38 blood samples collected from 16 naturally infected sheep flocks in France. For the rapid SRLV group determination of field isolates, the PCR-amplified fragments were subjected to a SRLV-adapted heteroduplex mobility assay (HMA). All viral sequences were clearly assignable to the SRLV group B by HMA analysis. Twenty-seven SRLV isolates were selected for DNA sequence analysis. In each case, nucleotide comparison and phylogenetic analyses confirmed the genetic relationships inferred by HMA. Six SRLV isolates belonged to subtype B1, and 21 pertained to subtype B2, one flock being infected with both subtypes. Subtypes B1 and B2 were found with different frequencies and geographic spread, but exhibited similar genetic diversities. These results give a more complete picture of the distribution and heterogeneity of SRLV env subtypes in sheep and confirmed that multiple interspecies transmission occurred in the past. Furthermore, HMA appeared to be a rapid and reliable method to differentiate caprine arthritis encephalitis virus from maedi-visna virus.

Animals↗
PubMed1992

HLA-A29.2 subtype associated with birdshot retinochoroidopathy.

Birdshot retinochoroidopathy is strongly associated with HLA-A29. This antigen can be divided into two subtypes, A29.1 and A29.2, using an immunoprecipitation method succeeded by one-dimensional electrofocusing gel electrophoresis. We reviewed the HLA typings of 58 white French patients who had birdshot retinochoroidopathy. Of these 58 subjects, 54 (93.1%) had HLA-A29 with a relative risk of 157.30. We further analyzed the HLA-A29 subtypings of 33 patients with birdshot retinochoroidopathy. Evaluation of the results showed that HLA-A29.2 subtype was present in all patients (100%). We concluded that the absence of HLA-A29.1 subtype is statistically significant (P less than .01) in this study of HLA-A29 subtyping.

Adolescent↗
PubMed1997

Characterization of CCK receptors in stomach smooth muscle: evidence for two subtypes.

Cholecystokinin (CCK) and related peptides such as gastrin are important regulators of gastric smooth muscle contraction. Several studies have shown that these effects of CCK and gastrin are mediated by CCK(B) receptors. However, recent studies suggest the expression of an additional CCK receptor subtype distinct from CCK(B) receptors in this tissue. This study was designed to distinguish between CCK(A) and CCK(B) receptors on guinea-pig stomach smooth muscle cells and to evaluate these cells for additional receptor subtypes. We cloned these receptors by hybridization screening of a guinea-pig smooth muscle cDNA library using 32P random primed labeled cDNA probes from the recently cloned rat CCK(A) and CCK(B) receptor coding regions. In addition to clones representing the CCK(B) subtype, clones of CCK(A) receptor subtype, but no additional CCK receptor subtypes, could be identified. All isolated clones displayed highly homologous nucleotide sequences in comparison to previously characterized CCK(A) and CCK(B) receptors from different species. The results of cDNA hybridization at different levels of stringency and Southern blot analysis using guinea-pig genomic DNA suggest that it is unlikely that additional CCK receptors despite CCK(A) and CCK(B) receptors exist in stomach smooth muscle.

Animals↗
PubMed2002

Subtype- and species-specific knockdown of PKC using short interfering RNA.

RNA interference (RNAi), the targeted mRNA degradation induced by double-stranded RNA (dsRNA), is a powerful tool for analyzing gene function in many organisms. Recently, it has been shown that RNAi is also applicable to cultured mammalian cells by using short interfering RNA (siRNA) [Nature 411 (2001) 494]. To examine whether this siRNA method is useful for analyzing the subtype-specific functions of protein kinase C (PKC), we first prepared siRNAs which target human alphaPKC and human deltaPKC and applied them into mammalian cells to suppress the expression of endogenous alphaPKC and deltaPKC, respectively. Each siRNA for alpha or deltaPKC specifically suppressed the endogenous expression of corresponding PKC subtype in human-derived cell lines such as HEK-293 and HeLa cells, but not in cells derived from rat species. The suppression level of deltaPKC reached maximum 48-72h after the transfection of siRNA. In addition, the siRNA targeting rat deltaPKC suppressed endogenous and exogenous rat deltaPKCs but not human deltaPKC, suggesting that siRNAs targeting PKCs effectively knocked down endogenous/exogenous PKCs in mammalian cells, in subtype- and species-specific manner. Furthermore, we also developed the method to discriminate the siRNA-transfected cells using the antibody recognizing thymine dimer. Our present results strongly suggest that siRNA method enable us to examine the subtype-specific function of PKC, not only by knockdown of the endogenous target PKC subtype, but also by subsequent compensation with the exogenous corresponding wild/mutant PKC derived from other species.

Animals↗
PubMed1997

Differential regulation of expression of rat hippocampal muscarinic receptor subtypes following fimbria-fornix lesion.

Quantitative RNase protection assays were performed to determine the levels of muscarinic receptor subtype (m1-m5) mRNAs in rat hippocampi. Results showed that the m1, m3, and m4 subtype mRNAs were expressed at relatively high levels, but the levels of the m2 and m5 subtype were very low. Three weeks following aspiration lesions of the fimbria-fornix to produce cholinergic denervation of the hippocampus, non-M1 receptors (non-pirenzepine displaceable [3H]quinuclindinyl benzilate binding sites) in the hippocampus were increased significantly, which correlated with increases in the levels of hippocampal m3 and m4 receptor mRNAs (m3: +24% and m4: +41%). These findings indicate that multiple muscarinic receptor subtypes are expressed in the hippocampus with the m3 and m4 subtypes predominantly postsynaptic to the septohippocampal cholinergic terminals.

Animals↗
PubMed2000

Pharmacological delta1- and delta2-opioid receptor subtypes in the human neuroblastoma cell line SK-N-BE: no evidence for distinct molecular entities.

The two pharmacological delta-opioid receptor subtypes, delta1 and delta2, have been defined on the basis of pharmacological tools but remain to be characterized at the molecular level, since only a single cDNA has been cloned. The present study aimed to investigate the pharmacological properties of delta1- and delta2-opioid subtypes expressed in the human neuroblastoma cell line SK-N-BE and to characterize their putative corresponding mRNAs. Binding experiments using "selective" delta1- and delta2-opioid agonists and antagonists revealed the presence of two binding sites, demonstrating the presence of these delta1-opioid subtypes as they were previously described. The activation of these pharmacological subtypes by the selective agonists induced the incorporation of [alpha-(32)P]azidoanilide-GTP into Galpha(i2)/Galpha(0) subunits with the same efficiency and potency and inhibited adenosine 3', 5'-cyclic monophosphate (cAMP) accumulation with similar efficiency, while their sustained activation for 15 min induced a cross-desensitization. The "selective" delta1 and delta2 antagonists, 7-benzylidenenaltrexone and naltrindole benzofuran, respectively, were found to be as potent in blocking the inhibition of cAMP accumulation induced by both [D-Pen(2,5)]enkephalin and Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH(2). The possibility that delta-opioid subtypes could arise from alternative splicing was ruled out by reverse transcription-polymerase chain reaction (RT-PCR) experiments and the sequencing of PCR products, which revealed the presence of a single transcript encoding for the delta-opioid receptor. Different possibilities which could account for the delta-opioid receptor heterogeneity observed in the SN-N-BE cell line are discussed.

Adenylyl Cyclases↗
PubMed2000

In vitro and in vivo m2 muscarinic subtype selectivity of some dibenzodiazepinones and pyridobenzodiazepinones.

Alzheimer's disease (AD) involves selective loss of muscarinic m2, but not m1, subtype receptors in cortical and hippocampal regions of the human brain. Emission tomographic study of the loss of m2 receptors in AD has been limited by the absence of available m2-selective radioligands, which can penetrate the blood-brain barrier. We now report on the in vitro and in vivo m2 muscarinic subtype selectivity of a series of dibenzodiazepinones and pyridobenzodiazepinones determined by competition studies against (R)-3-quinuclidinyl (S)-4-iodobenzilate ((R,S)-[125I]IQNB) or [3H]QNB. Of the compounds examined, three of the 5-[[4-[(4-dialkylamino)butyl]-1-piperidinyl]acetyl]-10, 11-dihydro-5-H-dibenzo[b,e][1,4]diazepin-11-ones (including DIBA) and three of the 11-[[4-[4-(dialkylamino)butyl]-1-phenyl]acetyl]-5, 11-dihydro-6H-pyrido [2,3-b][1,4]benzodiazepin-6-ones (including PBID) exhibited both high binding affinity for the m2 subtype (</=5 nM) and high m2/m1 selectivity (>/=10). In vivo rat brain dissection studies of the competition of PBID or DIBD against (R,S)[125I]IQNB or [3H]QNB exhibited a dose-dependent preferential decrease in the binding of the radiotracer in brain regions that are enriched in the m2 muscarinic subtype. In vivo rat brain autoradiographic studies of the competition of PBID, BIBN 99, or DIBD against (R,S)[125I]IQNB exhibited an insignificant effect of BIBN 99 and confirmed the effect of PBID and DIBD in decreasing the binding of (R,S)[125I]IQNB in brain regions that are enriched in the m2 muscarinic subtype. We conclude that PBID and DIBD are potentially useful parent compounds from which in vivo m2 selective derivatives may be prepared for potential use in positron emission tomographic (PET) study of the loss of m2 receptors in AD.

Alzheimer Disease↗
PubMed1996

Evidence for receptor subtype cross-talk in the mitogenic action of serotonin on human small-cell lung carcinoma cells.

We previously reported a significant mitogenic effect of serotonin (5-hydroxytryptamine, 5-HT) on human small-cell lung carcinoma cells (SCLC, GLC-8), mediated by both 5-HT1D and 5-HT1A receptors. Here we investigate possible interactions between the two receptor subtypes. Dose-effect curves obtained by simultaneously applying equipotent concentrations of the selective 5-HT1A agonist 8-OH-DPAT and the selective 5-HT1D receptor agonist sumatriptan are shifted to the right, although maximal effects are additive. The nonselective 5-HT antagonist metergoline displays higher potency when both receptor subtypes are activated. The 5-HT1D receptor antagonist GR127935 is markedly more potent against sumatriptan than against the sensitive portion of 5-HT effect. Indeed, both GR127935 and the 5-HT1A antagonist spiperone shift the EC50 for the residual effect of 5-HT from approximately 300 to 120-150 nM, suggesting that blocking one receptor subtype may facilitate activation of the other. Preincubation with either 8-OH-DPAT or sumatriptan suppresses the mitogenic response to the other specific receptor agonist; suppression is complete within 10 min at 37 degrees C, and is not observed when the preincubation is done at 4 degrees C. Measurements of adenylate cyclase activity do not help in interpreting the results. Conversely, measurements of MAP kinase activity reveals biphasic activation with a delayed activation at 1 h, and reproduce the suppression of the effect of the second drug by 15 min preincubation. These findings constitute the first evidence of a reciprocal negative interference between human 5-HT1A and 5-HT1D receptors, and indicate that SCLC GLC-8 cells simultaneously express both receptor subtypes. Mere reciprocal antagonism of the drugs employed cannot account for these data. We suggest that in this cell system cross-talk occurs in the transduction pathways of the two receptor subtypes.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1997

Angiotensin receptor subtypes in the uterine artery during ovine pregnancy.

This study was undertaken to determine if changes in receptor density or affinity could account for the reduced vascular sensitivity to angiotensin II seen during pregnancy. Angiotensin receptor subtypes in the uterine arteries of non-pregnant, pregnant and postpartum ewes were investigated using saturation and competition receptor binding techniques with the specific receptor antagonists, losartan (DuP-753) and PD-123319 (S)1-[[4-(dimethylamino)-3-methylphenyl]-methyl]-5-(diphenylacetyl )-4,5,6,7-tetrahydro-1H-imidazo(4,5-c)pyridine-6-carboxylic acid, ditrifluoroacetate, monohydrate). Receptor density and affinity of total angiotensin receptors, as well as the angiotensin AT1 and AT2 receptor subtypes in uterine arteries were compared with those in the mesenteric artery and aorta. The uterine artery contains both AT1 and AT2 receptor subtypes, whereas the mesenteric artery and aorta contain primarily the AT1 receptor subtype. In uterine arteries from pregnant sheep, angiotensin receptor density was increased because AT2 receptors were increased. AT1 receptor density was not altered. This change was not seen in the aorta. In the uterine artery, receptor affinity for [Sar1,Ile8]angiotensin II decreased in mid-gestation (IC50 7.7 +/- 1.2 x 10(-9) M) compared with non-pregnant ewes (IC50 3.0 +/- 0.6 x 10(-9) M, P = 0.006), and there was decreased affinity of angiotensin AT1 receptors for losartan during pregnancy (IC50 2.8 +/- 1.0 x 10(-4) M) compared with non-pregnant ewes (IC50 2.2 +/- 1.3 x 10(-6) M, P = 0.025). Our results show changes in the density and affinity of the angiotensin receptor subtypes in the uterine artery which could explain its reduced responsiveness to circulating angiotensin II during pregnancy.

Angiotensin I↗
PubMed1997

Molecular cloning and characterization of the four rat prostaglandin E2 prostanoid receptor subtypes.

We have characterized the rat prostanoid EP1, EP2, EP3alpha and EP4 receptor subtypes cloned from spleen, hepatocyte and/or kidney cDNA libraries. Comparison of the deduced amino acid sequences of the rat EP receptors with their respective homologues from mouse and human showed 91% to 98% and 82% to 89% identity, respectively. Radioreceptor binding assays and functional assays were performed on EP receptor expressing human embryonic kidney (HEK) 293 cells. The KD values obtained with prostaglandin E2 for the prostanoid receptor subtypes EP1, EP2, EP3alpha and EP4 were approximately 24, 5, 1 and 1 nM, respectively. The rank order of affinities for various prostanoids at the prostanoid receptor subtypes EP2, EP3alpha and EP4 receptor subtypes was prostaglandin E2 = prostaglandin E1 > iloprost > prostaglandin F2alpha > prostaglandin D2 > U46619. The rank order at the prostanoid EP1 receptor was essentially the same except that iloprost had the highest affinity of the prostanoids tested. Of the selective ligands, butaprost was selective for prostanoid EP2, M&B28767 and sulprostone were selective for EP3alpha and enprostil displayed dual selectivity, interacting with both prostanoid receptor subtypes EP1 and EP3alpha. All four receptors coupled to their predominant signal transduction pathways in HEK 293 cells. Notably, using a novel aequorin luminescence assay to monitor prostanoid EP1 mediated increases in intracellular calcium, both iloprost and sulprostone were identified as partial agonists. Finally, by Northern blot analysis EP3 transcripts were most abundant in liver and kidney whereas prostanoid EP2 receptor mRNA was expressed in spleen, lung and testis and prostanoid EP1 receptor mRNA transcripts were predominantly expressed in the kidney. The rat prostanoid EP1 probes also detected additional and abundant transcripts present in all the tissues examined. These were found to be related to the expression of a novel protein kinase gene and not the prostanoid EP1 gene [Batshake, B., Sundelin, J., 1996. The mouse genes for the EP1 prostanoid receptor and the novel protein kinase overlap. Biochem. Biophys. Res. Commun. 227. 1329-1333].

Aequorin↗
PubMed1998

Alpha1-adrenoceptor subtype activation increases proto-oncogene mRNA levels. Role of protein kinase C.

Noradrenaline increased the mRNA levels of c-fos and c-jun in rat-1 fibroblast lines stably expressing the cloned alpha1-adrenoceptor subtypes. The efficacy to induce the expression of c-fos mRNA was similar for the three cell lines (alpha1d = alpha1b = alpha1a) but different for c-jun (alpha1a > or = alpha1b > alpha1d). The EC50 values were also different: approximately 5 nM (c-fos) and approximately 300 nM (c-jun) for cells transfected with the alpha1a subtype, approximately 30 nM (c-fos) and approximately 300 nM (c-jun) for cells transfected with the alpha1b subtype and approximately 300 nM (c-fos and c-jun) for those transfected with the alpha1d subtype. Staurosporine and protein kinase C down-regulation blocked such effects, indicating a role of this protein kinase. Endothelin-1 (10 nM) also increased the levels of c-fos and c-jun mRNAs. These actions of endothelin-1 were unaffected by staurosporine and protein kinase C down-regulation. It is concluded that activation of any of the three cloned subtypes can increase the levels of c-fos and c-jun mRNAs and that protein kinase C plays a major role in mediating such effects.

Adrenergic alpha-Agonists↗