miR-216b-5p promotes late apoptosis/necroptosis in trastuzumab-resistant SK-BR-3 cells.

Breast cancer is the most common cancer in women. The human epidermal growth factor receptor 2 (HER2) overexpressing subtype is related to poor prognosis with an aggressive phenotype and is reported as one of the most commonly seen subtypes. Trastuzumab is prevalently used as a treatment method for HER2+ breast cancer however, resistance to the drug frequently occurs following the treatment. MicroRNAs (miRNAs) are 19-23 nucleotide long small RNAs, which regulate gene expression at post-transcriptional level and studies show that there are differentially expressed miRNAs between drug sensitive and resistant groups, indicating that they might have some key roles in drug effectiveness. In this study, the aim is to find out the role of miR-216b-5p in trastuzumab resistance. SK-BR-3 cells developed resistance to trastuzumab after continuous treatment with increasing concentrations of the drug for 6 months. To investigate the effect of miR-216b-5p on cancer cell behavior in resistance state, proliferation, motility, and invasion capacities of these resistant cells were analyzed by xCELLigence real-time cell analyzer. To further understand the molecular mechanisms underlying the regulation of resistant SK-BR-3 cells by miR-216b-5p, microarray analysis was performed. Apoptosis analysis was also performed since the pathway enrichment analysis pointed out cell death related pathways. The proliferation, motility, and invasion capacities of the miR-216b-5p transfected resistant cells were diminished compared to sensitive cells. We identified the necroptosis signaling pathway as the result of microarray and pathway enrichment analyses. STAT1, IRF9, and PKR were validated as the significant elements of the pathway, which are also the putative targets of miR-216b-5p. Our apoptosis analysis showed that a significant amount of trastuzumab resistant SK-BR-3 cells entered to late apoptosis/necrosis stage upon miR-216b-5p overexpression, it could be concluded that reexpression of miR-216b-5p sensitizes trastuzumab resistance through necroptosis in breast cancer.

The Ca2+/CaM, Src kinase and/or PI3K-dependent EGFR transactivation via 5-HT2A and 5-HT1B receptor subtypes mediates 5-HT-induced vasoconstriction.

G protein-coupled receptors (GPCR) and receptor tyrosine kinases (RTK) modulate vascular tone and contraction via rapid and long-term processes. Sustained activation of these receptor types can change vascular structure, and the ability of vasculature to adapt to high pressure. In this study, the interaction between serotonin (5-HT) receptors and epidermal growth factor receptors (EGFR) on vasoconstriction and the mechanisms of EGFR transactivation and its downstream mediators were investigated. We measured 5-HT-induced vasoconstriction in the aorta and the mesenteric artery; and the effects of EGFR, Src and PI3K, and their downstream mediators Erk1/2 and Akt phosphorylation on 5-HT-mediated vasoconstriction in the presence or absence of pharmacological inhibitors of Ca2+/CaM, EGFR, Src, and PI3K. Furthermore, we determined the contribution of 5-HT receptor subtypes to 5-HT-induced vasoconstriction and EGFR transactivation using selective 5-HT2A and 5-HT1B receptors ligands. Our results show that EGFR, Src, and PI3K are involved in 5-HT-induced vasoconstriction both in the aorta and the mesenteric artery, and that these kinases have a more prominent role in the mesenteric artery than the aorta. With regard to EGFR transactivation by 5-HT, Ca2+/CaM, Src and PI3K are upstream mediators, and transactivation is partly mediated by Erk1/2 and Akt activation. Furthermore, Ca2+/CaM, Src, and PI3K are the main regulators for Akt activation, however Src only has a prominent role for Erk1/2 activation. 5-HT2A and 5-HT1B receptors have different EGFR transactivation profiles through Src and/or PI3K, with 5-HT2A having a greater role than 5-HT1B receptors.

miR-770-5p regulates EMT and invasion in TNBC cells by targeting DNMT3A.

MicroRNAs (miRNAs) are shown to regulate various processes in cancer like motility and invasion that are key features of the metastatic triple negative breast cancer (TNBCs). Epithelial-mesenchymal transition (EMT) is one of the well-defined cellular transitioning processes characterized with reduced E-cadherin expression and increased mesenchymal molecules such as Vimentin or Snail thereby gives the cells mobility and invasive character. Aberrant DNA methylation by DNA methyltransferases (DNMTs) plays an important role in carcinogenesis. It is well known that DNMTs are required for transcriptional silencing of tumor-associated genes. DNMT3A-induced promoter hypermethylation of E-cadherin has also been known to improve cancer metastasis. Our results indicated that miR-770-5p could downregulate Vimentin and Snail expression levels, while increasing or restoring the expression of E-Cadherin hence, leading to inhibition of EMT phenotypes along with motility and invasion. Specifically, we showed that overexpression of miR-770-5p restored the expression of E-Cadherin in MDA-MB-231 cells via directly targeting DNMT3A. We also observed the change in the spindled shapes showing the loss of mesenchymal characteristics and gain of epithelial phenotype in miR-770-5p overexpressing cells. When considered together, our results show that miR-770-5p could effectively inhibit invasion potential driven by EMT.

MiR-25 and KLF4 relationship has early prognostic significance in the development of cervical cancer.

Cervical squamous cell carcinoma (SCC) is one of the common cancer types among women. MicroRNAs (miRNAs) are small non-coding RNAs that play an important role in the formation and development of many cancer types by regulating expression of their targets. While many studies have investigated the relationship between miRNAs and cervical cancer, no robust miRNA biomarkers have been defined yet for diagnosis of cervical lesions. In this study, we performed a statistical meta-analysis to identify miRNAs and a class compassion analysis to evaluate mRNAs with the power to discriminate between normal, intraepithelial lesions and invasive cancer samples. Differentially expressed (DE) mRNAs were compared with the targets of meta-miRNAs. After bioinfomatics analysis and qRT-PCR validations with cytology samples and FFPE tissues, we defined miR-25 and its target KLF4 (Kruppel-like factor 4) as candidate biomarkers for in vitro studies. Our results showed that miR-25 expression was significantly higher in precancerous lesions and invasive carcinoma while presenting consistent expression patterns in both cytological and FFPE tissue samples. In line with this, its direct target KLF4 expression decreased in precancerous lesions in cytological samples and also in the invasive cancer group in FFPE tissues. Furthermore, in vitro studies showed that mir-25 inhibition decreased proliferation and motility of HeLa cells and promoted an increase in the protein level of KLF4. We conclude that inhibition of miR-25 may upregulate KLF4 expression and regulate cell proliferation and motility in cervical cancer.

Overexpression of ß-Arrestins inhibits proliferation and motility in triple negative breast cancer cells.

ß-Arrestins (ßArrs) are intracellular signal regulating proteins. Their expression level varies in some cancers and they have a significant impact on cancer cell function. In general, the significance of ßArrs in cancer research comes from studies examining GPCR signalling. Given the diversity of different GPCR signals in cancer cell regulation, contradictory results are inevitable regarding the role of ßArrs. Our approach examines the direct influence of ßArrs on cellular function and gene expression profiles by changing their expression levels in breast cancer cells, MDA-MB-231 and MDA-MB-468. Reducing expression of ßArr1 or ßArr2 tended to increase cell proliferation and invasion whereas increasing their expression levels inhibited them. The overexpression of ßArrs caused cell cycle S-phase arrest and differential expression of cell cycle genes, CDC45, BUB1, CCNB1, CCNB2, CDKN2C and reduced HER3, IGF-1R, and Snail. Regarding to the clinical relevance of our results, low expression levels of ßArr1 were inversely correlated with CDC45, BUB1, CCNB1, and CCNB2 genes compared to normal tissue samples while positively correlated with poorer prognosis in breast tumours. These results indicate that ßArr1 and ßArr2 are significantly involved in cell cycle and anticancer signalling pathways through their influence on cell cycle genes and HER3, IGF-1R, and Snail in TNBC cells.

Assessment of replication of bovine herpesvirus type 4 in human glioblastoma and breast cancer cells as a potential oncolytic virus.

Oncolytic viruses have been extensively used in cancer treatment due to their tropism, selective replication only in tumor cells, and possible synergic interaction with other therapeutics. Different researchers have demonstrated that bovine herpesvirus 4 (BoHV-4), a member of the gammaherpesviridae family, has oncolytic potential in some human-origin cancer cell lines like glioma through the selective replication strategy. Using four apoptosis detection methods, namely MTT, LDH, TUNEL, and Annexin V assays, we evaluated the apoptotic effect of BoHV-4 Movar33/63 reference strain along with a recombinant BoHV-4 expressing EGFP in U87 MG cells (human glioblastoma cell line), MDA MB-231 (human breast cancer cell line), and MCF10a (non-tumorigenic human mammary epithelial cell line). Our findings indicate that this virus can replicate and induce apoptosis in these cell lines and hinder in vitro proliferation in a dose-dependent manner. In conclusion, BoHV-4 has in vitro potential as a novel oncolytic virus in human cancer therapy. However, its replication potential in the MCF10a cells as a non-tumorigenic human mammary epithelial cell line is a concern in using this virus in cancer therapy, at least against human mammary tumors. Further studies must therefore be conducted to examine the specific apoptotic pathways induced by this virus to move on to further experiments.

Immunological Analysis of a CCHFV mRNA Vaccine Candidate in Mouse Models.

Development of new vaccine platforms against viral diseases is considered urgent. In recent years, mRNA constructs have attracted great interest in this field due to unique advantages over conventional gene transfer platforms. In the present study, we developed a new naked conventional mRNA vaccine expressing the non-optimized small (S) segment of the Ank-2 strain of Crimean-Congo Hemorrhagic Fever virus (CCHFV). We then analyzed its single and booster dose immunogenicity and protection potential in the challenge assay in two mice models, including IFNα/ß/γR-/- and C57BL/6. The results obtained from the immunological assays, namely IL-4 and IFN-gamma ELISPOT, intracellular IFN-gamma staining, in-house sandwich ELISA, and survival data, demonstrated that our construct elicited the production of anti-nucleocapsid (N) specific immune responses in both mice models. A 100% protection rate was only obtained in the booster dose group of IFNα/ß/γR-/- mice, indicating that this platform needs further optimization in future studies. In conclusion, we assessed a novel approach in CCHFV vaccination by introducing a conventional mRNA platform which can be considered in future experiments as an efficient and safe way to battle this disease.

Involvement of miR-770-5p in trastuzumab response in HER2 positive breast cancer cells.

miRNAs may play effective roles in breast cancer so modulating their expression levels could have therapeutic benefits. Recent studies have found the combination of miRNA-based therapeutics with conventional drugs as promising. This study aimed to find drug-responsive miRNAs, and explore their anticancer activities in HER2+ breast cancer cells and regulatory role in the trastuzumab response. qRT-PCR-array analysis was performed with effective concentrations of tamoxifen and trastuzumab treated BT-474, SK-BR-3 and MCF-7 cells. Motility and invasion analyses were performed with wound healing and xCELLigence impedance-based assays respectively. Viability of cells following mimic transfection and drug treatment was assessed by WST-1 assay. Western blot analysis was used to assess miR-770-5p regulation of proteins and their phosphorylated forms. The clinical relevance of miR-770-5p was examined by TCGA data analysis. The qRT-PCR-array results indicated that miR-770-5p was responsive in a drug and cell line independent manner. Overexpression of miR-770-5p inhibited the motility and cell invasion through regulation of AKT and ERK proteins. Additionally, miR-770-5p potentiated the effectiveness of trastuzumab. Thus, regulating the expression level of miR-770-5p in combination with trastuzumab treatment may simultaneously inhibit the downstream elements of PI3K and MAPK signalling, thereby blocking the proliferation, motility and invasion capacities of HER2+ breast cancer cells.

Class III ß-tubulin Expression in Colorectal Neoplasms Is a Potential Predictive Biomarker for Paclitaxel Response.

BACKGROUND/AIM: The challenges of cololorectal cancer (CRC) management include prediction of outcome and drug response or chemoresistance. This study aimed at examining whether ßIII-tubulin (TUBB3), present in various types of normal tissues and cancer, is a biomarker for the response of colorectal neoplasms to paclitaxel. MATERIALS AND METHODS: Six tissue microarrays (TMAs) including 14 colon mucosa, 78 polyps and 202 CRCs were constructed. Assessment of TUBB3 expression was performed by immunohistochemistry, and it was scored as negative, focal and positive. In the HCT116 cell line, TUBB3 expression was silenced with siRNA. Paclitaxel toxicity was evaluated in TUBB3-silenced and control HCT116 cell lines. RESULTS: The non-neoplastic colon mucosa was negative for TUBB3, while some of colon adenomas and CRCs expressed TUBB3 in various levels from focal to diffuse. TUBB3-expressing CRCs tended to have poor prognosis and silencing of TUBB3 sensitized the cells to paclitaxel. CONCLUSION: TUBB3 was expressed in a subgroup of colorectal neoplasms. Suppression of TUBB3 potentialy sensitizes neoplastic cells to taxanes.

Partial agonistic effect of cetuximab on epidermal growth factor receptor and Src kinase activation in triple­negative breast cancer cell lines.

Cetuximab is a monoclonal antibody developed to inhibit the binding of growth factors and the subsequent activation of epidermal growth factor receptor (EGFR). Triple­negative breast cancer (TNBC) is resistant to cetuximab treatment. The aim of the present study was to examine the partial agonistic properties of cetuximab, which not only blocks ligand binding, but also partially triggers EGFR activation, which may lead to cetuximab resistance in TNBC. The phosphorylation of growth factor receptors and their signalling pathways were evaluated by determining the phosphorylation of EGFR, insulin­like growth factor receptor (IGF­1R), vascular endothelial growth factor receptor (VEGFR)­2, Src kinase, phosphoinositide­3­kinase (PI3K), extracellular signal­regulated kinase (ERK1/2) and serine/threonine­specific protein kinase (Akt) and the degradation of EGFR, and by assessing the morphology and proliferation of MDA­MB­231 and MDA­MB­468 cells. Cetuximab treatment led to the phosphorylation of EGFR, VEGFR­2, IGF­1R and downstream signalling molecules, Src kinase and PI3K in these cells, as well as Akt in the MDA­MB­231 cells. The cetuximab­mediated phosphorylation of IGF­1R, VEGFR­2 and Akt was inhibited by the EGFR kinase inhibitor, AG1478, and the Src kinase inhibitor, PP2. Cetuximab treatment led to the degradation of EGFR. The cetuximab­induced phosphorylation and EGFR degradation were less prominent compared with those induced by EGF. Cetuximab partially inhibited EGF­mediated responses. Cetuximab, similar with EGF, altered cellular morphology in a serum­free medium. In both cell lines, the Src kinase inhibitor enhanced the cetuximab­induced anti­proliferative response. These results indicate that cetuximab exerts a partial agonistic effect on EGFR, which activates Src kinase and subsequently transactivates IGF­1R and VEGFR­2. This partial agonistic property is likely one of the mechanisms underlying the resistance of TNBC to cetuximab.

The role of dual­specificity phosphatase 1 and protein phosphatase 1 in ß2­adrenergic receptor­mediated inhibition of extracellular signal regulated kinase 1/2 in triple negative breast cancer cell lines.

Triple negative breast cancer cell lines express high levels of ß2-adrenergic receptor, which have a significant influence on the activity of extracellular signal­regulated kinase (ERK)1/2. Therefore, it is important to understand the link between ß2­adrenergic receptor signaling and ERK1/2 activity in terms of cancer cell regulation and cancer progression. Although the molecular mechanisms are not completely clarified, ß2­adrenergic receptor stimulation appears to reduce the basal levels of phosphorylated (p)ERK1/2 in MDA­MB­231 breast cancer cells. The aim of the current study was to determine the mechanism of ß2­adrenergic receptor­mediated ERK1/2 dephosphorylation by investigating the role of dual­specificity phosphatase (DUSP)1/6 and protein phosphatase (PP)1/2, which are established regulators of ERK1/2 phosphorylation, in MDA­MB­231 and MDA­MB­468 breast cancer cell lines. (E)­2­benzylidene­3­(cyclohexyl​amino)­2,3­ dihydro­1H­inden­1­one (BCI) and calyculin A were employed as DUSP1/6 and PP1/PP2 inhibitors, respectively. Subsequently, the protein levels of DUSP1, PP1, pPP1, ERK1/2 and pERK1/2 were measured by western blot analysis. Cells were transfected with DUSP1 small interfering (si)RNA or PP1 siRNA to inhibit their expression. The results demonstrated that ß2­adrenergic receptor agonists led to the dephosphorylation of basal pERK1/2 in MDA­MB­231 and MDA­MB­468 cells. The DUSP1/6 inhibitor, BCI, and the PP1/PP2 inhibitor, calyculin A, antagonized the ß2­adrenergic receptor­mediated dephosphorylation of ERK1/2. Furthermore, ß2­adrenergic receptor stimulation increased the protein expression level of DUSP1, with no effects on DUSP6, PP1 and PP2 expression, and enhanced the expression of the active form of PP1. Downregulation of the expression of DUSP1 or PP1 led to a decline in the ß2­adrenergic receptor­mediated dephosphorylation of ERK1/2. The results of the present study indicate that ß2­adrenergic receptor­mediated dephosphorylation of ERK1/2 may be associated with the activity of DUSP1 and PP1 in MDA­MB­231 and MDA­MB­468 triple negative breast cancer cell lines. The clinical importance of ß2­adrenergic receptor­mediated inactivation of ERK1/2 as well as the activation of DUSP1 and PP1 should be carefully evaluated in future studies, particularly when ß2­adrenergic blockers are used in patients with triple negative breast cancer.

Intracellular transactivation of epidermal growth factor receptor by α1A-adrenoceptor is mediated by phosphatidylinositol 3-kinase independently of activation of extracellular signal regulated kinases 1/2 and serine-threonine kinases in Chinese hamster ovary cells.

Transactivation of epidermal growth factor receptor (EGFR) by α1-adrenoceptor (α1-AR) is implicated in contraction and hypertrophy of vascular smooth muscle (VSM). We examine whether all α1-AR subtypes transactivate EGFR and explore the mechanism of transactivation. Chinese hamster ovary (CHO) cells stably expressing one subtype of α1-AR were transiently transfected with EGFR. The transactivation mechanism was examined both by coexpression of a chimeric erythropoietin (EPO)-EGFR with an extracellular EPO and intracellular EGFR domain, and by pharmacologic inhibition of external and internal signaling routes. All three α1-AR subtypes transactivated EGFR, which was dependent on the increase in intracellular calcium. The EGFR kinase inhibitor AG1478 [4-(3'-chloroanilino)-6,7-dimethoxyquinazoline] abrogated α1A-AR and α1D-AR induced phosphorylation of EGFR, but both the inhibition of matrix metalloproteinases by GM6001 [(R)-N4-hydroxy-N(1)-[(S)-2-(1H-indol-3-yl)-1-methylcarbamoyl-ethyl]-2-isobutyl-succinamide] or blockade of EGFR by cetuximab did not. Stimulation of α1A-AR and α1D-AR also induced phosphorylation of EPO-EGFR chimeric receptors. Moreover, α1A-AR stimulation enhanced phosphorylation of extracellular signal regulated kinase (ERK) 1/2 and serine-threonine kinases (Akt), which were both unaffected by AG1478, indicating that ERK1/2 and Akt phosphorylation is independent of EGFR transactivation. Accordingly, inhibitors of ERK1/2 or Akt did not influence the α1A-AR-mediated EGFR transactivation. Inhibition of calcium/calmodulin-dependent kinase II (CaMKII), phosphatidylinositol 3-kinase (PI3K), and Src, however, did block EGFR transactivation by α1A-AR and α1D-AR. These findings demonstrate that all α1-AR subtypes transactivate EGFR, which is dependent on an intracellular signaling route involving an increase in calcium and activation of CaMKII, PI3K, and Src, but not the of ERK1/2 and Akt pathways.

Epidermal growth factor receptor inhibitor PKI-166 governs cardiovascular protection without beneficial effects on the kidney in hypertensive 5/6 nephrectomized rats.

Transactivation of epidermal growth factor receptor (EGFR) signaling by G protein-coupled receptors has been implicated in several cardiovascular (CV) conditions, including hypertension, heart failure, and cardiac and vascular hypertrophy. However, the therapeutic potential of EGFR inhibition in these conditions is currently unknown. The main objective of the present study was to investigate cardiac, vascular, and renal effects of EGFR inhibition by 4-[4-[[(1R)-1-phenylethyl]amino]-7H-pyrrolo[2,3-d]pyrimidin-6-yl]phenol (PKI-166) in the hypertensive chronic kidney disease model. Rats underwent 5/6 nephrectomy (5/6Nx) and were treated with PKI-166, lisinopril or vehicle from week 6 after disease induction until week 12. Sham animals received either PKI-166 or vehicle. Treatment with PKI-166 did not affect the development of the characteristic renal features in 5/6Nx, including proteinuria, diminished creatinine clearance, and increased glomerulosclerosis, whereas these were attenuated by lisinopril. Despite absence of effects on progressive renal damage, PKI-166 attenuated the progression of hypertension and maintained cardiac function (left ventricle end-diastolic pressure) to a similar extent as lisinopril. Also, PKI-166 attenuated the increase in phosphorylated EGFR in the heart as induced by 5/6Nx. Moreover, PKI-166 and lisinopril restored the impaired contraction of isolated thoracic aortic rings to phenylephrine and angiotensin II and impaired myogenic constriction of small mesenteric arteries in 5/6Nx rats. Blockade of the EGFR displays a CV benefit independent of limiting the progression of renal injury. Our findings extend the evidence on EGFR signaling as a target in CV disorders.

Selenium restores defective beta-adrenergic receptor response of thoracic aorta in diabetic rats.

Increased oxidative stress is one of the basic contributors to the development of the cardiovascular complications in diabetes. Both endothelial and vascular smooth muscle cell dysfunctions are the main sign involved in the pathogenesis of diabetic cardiovascular dysfunction. Matrix metalloproteinases (MMPs) are expressed in the vasculature, and participate in tissue remodeling under pathological conditions such as increased oxidative stress, whereas little is known about effect of hyperglycemia on regulation of MMPs in vascular system. Therefore, we aimed to evaluate the effect of an antioxidant, sodium selenate treatment (0.3 mg/kg for 4 weeks) on function of streptozotocin-diabetic rat aorta. Sodium selenate treatment improved significantly impaired isoproterenol-induced relaxation responses and contraction responses of the aortic strips, and exhibited marked protection against diabetes-induced degenerative changes in the smooth muscle cell morphology. Biochemical data showed that sodium selenate treatment induced a significant regulation of MMP-2 activity and protein loss as well as normalization of increased levels of tissue nitrite and protein thiol oxidation. In addition, this treatment restored diabetes-induced increased levels of endothelin-1, PKC, and cAMP production in the aortic tissue. Taken together, our data demonstrate that these beneficial effects of sodium selenate treatment in diabetics are related to be not only inhibition of increased oxidative stress but also prevention of both receptor- and smooth muscle-mediated dysfunction of vasculature, in part, via regulation of MMP-2. Such an observation provides evidence for potential therapeutic usage of selenium compounds for the amelioration of vascular disorders in diabetes.

Effects of beta-adrenergic receptor blockers on cardiac function: a comparative study in male versus female rats.

In heart disease, differences exist between women and men with respect to the impact of risk factors, symptoms, and therapeutic responses. The use of beta-adrenergic receptor blockers is now well established in the treatment of mild and moderate systolic heart failure. Although there are significant differences among agents, their clinical effects are predictable. To address the question of sex disparities in the heart, however, we investigated the effect of treatment with the nonselective beta-blockers timolol and propranolol on mechanical and electrical function of heart preparations from male and female rats. We examined the long-term effects of intragastric treatment with timolol (5 mg/kg per day) or propranolol (25 mg/kg per day) for 7 months on the hemodynamic and intracellular action potential parameters of the heart. Chronic administration of timolol but not propranolol produced a significant increase in the baseline activity of the left ventricular developed pressure (LVDP) in both male and female rats with no significant effect on the left ventricular end-diastolic pressure. Timolol or propranolol treatment of male rats and timolol but not propranolol treatment of female rats induced significant shortening in the repolarization phases of action potentials recorded from left ventricular papillary muscle strips of the hearts. The responses of LVDP to beta-adrenergic stimulation were similar in timolol- or propranolol-treated or untreated male rats. On the other hand, timolol treatment markedly increased, and propranolol treatment significantly decreased, the responses of increase in LVDP in female rats. Our results suggest that although treatment with beta-blockers for 7 months confirmed the role of the beta-adrenergic pathway in heart function, there are marked differences in the effects of individual beta-blockers on heart physiology. Sex differences should be taken into consideration when using beta-blockers during experimental studies and clinical therapy.

The role of gender differences in beta-adrenergic receptor responsiveness of diabetic rat heart.

Since the mechanisms responsible for gender differences in cardiac contractile function have not been fully elucidated, we focused to determine the effect of gender difference on beta-adrenergic receptors (beta-ARs) signal transduction in ventricular cardiomyocytes from insulin-dependent diabetic (streptozotocin-induced) rats. Dose-response curves of left ventricular developed pressure (LVDP) to isoproterenol (ISO) in females showed that there was only a approximately 30% decrease in the maximum response without a significant shift in EC50 in diabetic females. On the other hand, diabetes induced a clear rightward shift in the potency (5-10 folds) without a significant change in the maximum response in the males. In order to further determine of the underlying mechanism for this difference, we measured cAMP production and obtained dose-response curves with ISO stimulation in isolated cardiomyocytes. In diabetic females, there was no obvious change in the cAMP dose-response curve. On the other hand, there was a significant decrease in the maximum response without any apparent change in the potency of diabetic males. Our findings indicate that male and female rats are affected differently by diabetes in terms of LVDP responses to beta-ARs stimulation. Also, the difference between their beta-ARs induced cAMP responses may underlie this disparity.

The role of Gi proteins in reduced vasorelaxation response to beta-adrenoceptor agonists in rat aorta during maturation.

Beta-adrenoceptor mediated vasorelaxation and cAMP production decline during maturation and aging in rat aorta. beta-adrenoceptor-stimulated vasorelaxation is mainly triggered by Gsalpha-mediated activation of adenylyl cyclase. beta(2)-adrenoceptors can also activate Gi protein which inhibits adenylyl cyclase activity. In this study, we examined the role of Gi proteins in the decreased beta-adrenoceptor mediated responses during maturation. Pertussis toxin treatment of aortic rings to inhibit Gialpha activation completely restored age related decline in isoproterenol-stimulated maximal vasorelaxation in 3-month old rats. This treatment increased the potency, but not the maximal response of isoproteronol to produce vasorelaxation in 6 month old rats. The maximal isoproteronol stimulated cAMP responses were also partially restored in pertussis toxin-treated rings from 3 or 6-month old rats. We also examined beta-adrenoceptor stimulated binding of (35)[S]GTPgammaS to Gsalpha and Gialpha1/2 in aortic membranes from 1, 3 and 6-month old rats. In 1-month old rats, isoproterenol-stimulated (35)[S]GTPgammaS binding to Gsalpha was significantly higher than that of 3 or 6-month old rats. Isoproterenol-stimulated (35)[S]GTPgammaS binding to Gialpha1/2 was found to be significantly increased in 3 or 6-month old rats compared to 1-month old rats. The results of this study showed that beta-adrenoceptor-mediated activation of Gs and Gi proteins was declined and increased, respectively, and inhibition of the Gi mediated activity by pertussis toxin treatment partially restored impaired vasorelaxation and cAMP response to beta-adrenoceptor stimulation during maturation in rat aorta. The decrease in beta-adrenoceptor mediated activation of Gs gradually increased during maturation. All together these results indicated that beta-adrenoceptor mainly activates Gs protein in aorta from 1-month old rats, while it activates Gi and with a certain degree of decline it also activates Gs in aorta from 3 and 6-months old rats and not only the increase in beta-adrenoceptor coupling to Gi but also the decrease in its coupling to Gs play a role in the impaired beta-adrenoceptor responses in rat aorta during maturation.

Airway smooth muscle relaxations induced by dipyrone.

BACKGROUND: Dipyrone differs from other nonsteroidal anti-inflammatory drugs with a distinctive spasmolytic effect; however, the mechanism of action is not clear yet. The possible mechanism behind this effect was investigated on airway smooth muscle tone in vitro. METHOD: The effect of dipyrone on inositol phosphate (IP) accumulation was evaluated on guinea pig trachea smooth muscle. Changes in intracellular free calcium levels and IP accumulation were evaluated in LTK8 cells. RESULTS: Dipyrone (0.01, 0.1, 1 mmol/l) had a relaxing effect on histamine (0.02 mmol/l)- and adenosine triphosphate- (ATP) (0.01 mol/l)-induced contractions, but not potassium chloride (KCl)-stimulated contractions. This relaxing effect was not observed with indomethacin. Indomethacin did not inhibit the relaxation response of dipyrone. In isolated tracheal smooth muscle, histamine- (0.02 mmol/l) and ATP (0.01 mmol/l)-induced IP accumulation was significantly inhibited by dipyrone (1 mmol/l). ATP (0.01 mmol/l)-induced IP accumulation was also significantly inhibited by dipyrone (0.01 mmol/l) in LTK8 cells. Dipyrone inhibited ATP-induced (0.01 and 0.1 mmol/l) intracellular calcium increase in LTK8 cells. CONCLUSION: Inhibition of the release of intracellular Ca(2+) may play a role in the smooth muscle relaxing effect of dipyrone. The inhibitor effect of dipyrone on IP accumulation may be due to direct inhibition of phospholipase C (PLC) or impairment of the activation of PLC by G protein-coupled receptor (GPCR).

Effects of diabetes on ryanodine receptor Ca release channel (RyR2) and Ca2+ homeostasis in rat heart.

The defects identified in the mechanical activity of the hearts from type 1 diabetic animals include alteration of Ca2+ signaling via changes in critical processes that regulate intracellular Ca2+ concentration. These defects result partially from a dysfunction of cardiac ryanodine receptor calcium release channel (RyR2). The present study was designed to determine whether the properties of the Ca2+ sparks might provide insight into the role of RyR2 in the altered Ca2+ signaling in cardiomyocytes from diabetic animals when they were analyzed together with Ca2+ transients. Basal Ca2+ level as well as Ca2+-spark frequency of cardiomyoctes isolated from 5-week streptozotocin (STZ)-induced diabetic rats significantly increased with respect to aged-matched control rats. Ca2+ transients exhibited significantly reduced amplitude and prolonged time courses as well as depressed Ca2+ loading of sarcoplasmic reticulum in diabetic rats. Spatio-temporal properties of the Ca2+ sparks in cardiomyocytes isolated from diabetic rats were also significantly altered to being almost parallel to the changes of Ca2+ transients. In addition, RyR2 from diabetic rat hearts were hyperphosphorylated and protein levels of both RyR2 and FKBP12.6 depleted. These data show that STZ-induced diabetic rat hearts exhibit altered local Ca2+ signaling with increased basal Ca2+ level.

Serotonergic activity and the Beck Depression Inventory in the premenstrual syndrome.

OBJECTIVE: To investigate the relationship between platelet [3H]-imipramine binding and Beck Depression Inventory scores in the premenstrual syndrome (PMS). STUDY DESIGN: Thirty-one patients with PMS and 26 healthy female volunteers participated in the study. Imipramine binding was measured by saturation binding of [3H]-imipramine. The Beck Depression Inventory form was completed. RESULTS: The number of [3H]-imipramine binding sites (Bmax) was significantly lower in the study group (p = 0.003). Beck Depression Scores were significantly higher in the study group as compared with the control group (p = 0.001). No correlation was found between platelet [3H]-imipramine binding and Beck Depression Inventory scores (r = 0.100, p = 0.591). CONCLUSION: A significantly lower number of Bmax wasfound in patients with PMS as compared to controls, but no correlation between platelet [3H]-imipramine binding and Beck Depression Inventory scores was found.