RBCK1 contributes to chemoresistance and stemness in colorectal cancer (CRC).

Chemotherapy is an essential treatment for colorectal cancer (CRC). However, colorectal cancer cells often develop resistance to chemotherapeutic drugs, resulting in relapse and poor patient prognosis. Growing studies indicate that tumor cells with stem cell-like features could promote resistance to chemotherapeutic agents. In this study, we demonstrated that RANBP2-type and C3HC4-type zinc finger-containing 1 (RBCK1) expression was markedly increased by cancer-associated fibroblasts (CAFs). First, we found that RBCK1 was over-expressed in chemoresistant CRC tumors and promoted chemoresistance in CRC cells. High RBCK1 expression was significantly correlated with poor prognosis in CRC patients. RBCK1 inhibition promoted sensitivity to chemotherapeutic drugs, and prevented migration and invasion in CRC cells. In addition, RBCK1 knockdown reduced cancer stemness by decreasing the expression of Nanog, Oct4, Sox2 and Klf4 in CRC cell lines. Furthermore, RBCK1 expression was significantly up-regulated in CRC cells cultured with conditioned medium (CM) derived from CAFs. Moreover, CAF-derived CM enhanced stemness and chemoresistance in CRC cells by over-expressing RBCK1. The in vivo experiments confirmed that RBCK1 knockdown promoted the chemotherapeutic drug sensitivity in a xenograft model. Taken together, these finding indicated that RBCK1 modulated chemosensitivity in CRC, and could be served as a promising therapeutic target for CRC prevention.

Downregulation of LncRNA-RP11-317J10.2 promotes cell proliferation and invasion and predicts poor prognosis in colorectal cancer.

OBJECTIVES: Using microarray analysis, we previously showed that many lncRNAs are differentially expressed in colorectal cancer (CRC) tissues compared with normal tissues, suggesting that lncRNAs may be involved the initiation and progression of CRC. In this study, we investigated the expression and function of lncRNA-RP11-317J10.2 in human CRC tissues and cell lines. METHODS: LncRNA-RP11-317J10.2 expression level was analyzed in 52 colon cancer and cell lines. We used shRNA to knock-down the expression of RP11-317J10.2, and then proliferation assay, colony formation assay, Boyden chamber assay, FACS and Kaplan-Meier survival analysis were performed to explore the biological effect of RP11-317J10.2. Cyclin D1 protein level was detected by Western blot. RESULTS: LncRNA-RP11-317J10.2 is downregulated in CRC and decreased expression is significantly associated with advanced tumor stage, larger tumor size and poor prognosis. RNA interference-mediated knockdown of lncRNA-RP11-317J10.2 in CRC cells promotes G1-to-S cell cycle transition, enhances invasiveness and facilitates cell growth in vitro and in mouse tumor xenograft models. Cyclin D1 was upregulated by lncRNA-RP11-317J10.2 knockdown, and co-expression of cyclin D1-targeting siRNA abrogates the pro-tumorigenic effects of lncRNA-RP11-317J10.2 knockdown. CONCLUSIONS: This study reveals a crucial role for lncRNA-RP11-317J10.2 in CRC growth and invasion via upregulation of cyclin D1 expression and suggests that expression of this lncRNA may be a potential prognostic biomarker for CRC.

Effect of CYP3A4*1G, CYP3A5*3, POR*28, and ABCB1 C3435T on the pharmacokinetics of nifedipine in healthy Chinese volunteers.

OBJECTIVE: Nifedipine is a calcium channel blocker that is widely used in the treatment of cardiovascular disease. However, significant individual variances in the disposition of nifedipine have been reported, and genetic factors are considered to play an important role. The aim of the present study was to investigate the effect of CYP3A4*1G, CYP3A5*3, ABCB1-C3435T, and POR*28 genetic polymorphisms on nifedipine pharmacokinetics in healthy Chinese volunteers. METHODS: 45 healthy Chinese volunteers enrolled in this study received a single oral dose of 90 mg nifedipine after providing written informed consent. Volunteers were genotyped for CYP3A4*1G, CYP3A5*3, POR*28, and ABCB1-C3435T. The blood concentrations of nifedipine were determined by high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) method. RESULTS AND DISCUSSION: There were significant differences of AUC00-∞ and AUC0-48h in the different CYP3A5*3 genotype groups (p = 0.043 and p = 0.048, respectively). The CYP3A5*3 GG group and POR*28 CT/TT group were found to have lower AUC00-∞ and Cmax compared with the POR*28 CC group (p = 0.046 and p = 0.002, respectively). In addition, the POR*28 CT/TT group was found to have longer t1/2 but lower Cmax than the CYP3A4*1G GG group (p = 0.032 and p = 0.002, respectively) as well as the CYP3A4*1G GG and the CYP3A5*3 GG group (p = 0.038 and p = 0.036, respectively) compared with the POR*28 CC group. No significant associations were found between CYP3A4*1G/ABCB1-C3435T polymorphism and pharmacokinetics of nifedipine. CONCLUSION: Both CYP3A5*3 and POR*28 polymorphisms are found to be associated with the difference in disposition of nifedipine; POR*28 is considered to have an impact on CYP3A4 activity.

Effects of resveratrol on the protein expression of survivin and cell apoptosis in human gastric cancer cells.

PURPOSE: This study aimed to investigate the effects of resveratrol on cell proliferation, apoptosis and protein expression of survivin in human gastric cancer (SGC7901) cell line. METHODS: SGC7901 cell proliferation and G0/G1 phase of the cell cycle induced by resveratrol (treatment group) and phosphate buffer solution (PBS) (control group) were assessed by flow cytometry. In addition, protein expression of survivin was assessed by immunohistochemistry after treatment with resveratrol. RESULTS: SGC7901 cell apoptosis rates were 0.00% and 3.45% in the control and treatment groups, respectively. Furthermore, cell cycles were significantly changed in the resveratrol group; for example, the proportion of the G0/G1 phase increased, whereas the proportion of the S and G2/M phases decreased. Survivin protein expression was significantly reduced (p<0.01) in the treatment group compared with that in the control group. CONCLUSION: Resveratrol inhibited the proliferation of SGC7901 cancer cells by inducing cell apoptosis and down-regulating survivin expression.

Urine flow acceleration is superior to Qmax in diagnosing BOO in patients with BPH.

We performed a retrospective, case-control study to evaluate whether the urine flow acceleration (UFA, mL/s(2)) is superior to maximum uroflow (Qmax, mL/s) in diagnosing bladder outlet obstruction (BOO) in patients with benign prostatic hyperplasia (BPH). In this study, a total of 50 men with BPH (age: 58±12.5 years) and 50 controls (age: 59±13.0 years) were included. A pressure-flow study was used to determine the presence of BOO according to the recommendations of Incontinence Control Society (ICS). The results showed that the UFA and Qmax in BPH group were much lower than those in the control group [(2.05±0.85) vs. (4.60±1.25) mL/s(2) and (8.50±1.05) vs. (13.00±3.35) mL/s] (P<0.001). According to the criteria (UFA<2.05 mL/s(2), Qmax<10 mL/s), the sensitivity and specificity of UFA vs. Qmax in diagnosing BOO were 88%, 75% vs. 81%, 63%. UFA vs. Omax, when compared with the results of P-Q chart (the kappa values in corresponding analysis), was 0.55 vs. 0.35. The prostate volume, post void residual and detrusor pressure at Qmax between the two groups were 28.6±9.8 vs. 24.2±7.6 mL, 60.4±1.4 vs. 21.3±2.5 mL and 56.6±8.3 vs. 21.7±6.1 cmH2O, respectively (P<0.05). It was concluded that the UFA is a useful urodynamic parameter, and is superior to Qmax in diagnosing BOO in patients with BPH.

Small bowel preparations for capsule endoscopy with mannitol and simethicone: a prospective, randomized, clinical trial.

BACKGROUND AND OBJECTIVE: There is no consensus concerning small bowel preparation before capsule endoscopy (CE). This study evaluated the effects of 4 regimens on small bowel cleansing and diagnostic yield. METHODS: Patients were randomly divided into 4 groups. Group A consumed a clear liquid diet after lunch on the day before CE, followed by overnight fasting. Group B took 250 mL 20% mannitol and 1 L 0.9% saline orally at 05:00 hours on the day of the procedure. In group C, the same regimen was taken at 20:00 hours on the day before and at 05:00 hours on the day of CE. In group D, in addition to the group C regimen, 20 mL oral simethicone was taken 30 minutes before CE. RESULTS: Two hundred patients were prospectively enrolled, and 7 were excluded from the final analysis because of incomplete small bowel transit. No significant difference was noted among the 4 groups for small bowel transit time. Bowel preparation in group D was significantly better than for the other regimens for overall cleansing of the proximal small bowel, and showed improved overall cleansing of the distal small bowel when compared with 10-hours overnight fasting. Pathological lesions of the proximal and distal small bowel were, respectively, achieved in 82 and 74 patients, mostly distributed in group D. CONCLUSIONS: Small bowel preparation that involves split-dose oral mannitol plus single-dose simethicone for CE can improve mucosal visualization and subsequent diagnostic yield when compared with 10-hours overnight fasting.

[Analysis of alcoholic extracts of Cistanche deserticola by tri-step identification of Fourier transform infrared spectroscopy].

Tri-step identification of Fourier transform infrared spectroscopy (FTIR) integrated with second derivative spectroscopy and two dimensional correlation infrared spectroscopy (2D-IR) were applied to analyze and evaluate the alcoholic extracts and corresponding residues of cistanche deserticola from the surface to what lies behind. It was found that active compounds including phenylethanoid glycosides were enriched effectively after alcoholic extraction and the extract by using 70% of alcohol had the highest concentration compared to the others. The technique of the tri-step identification holistically disclosed the profile of active compounds in cistanche deserticola extracted by a series of concentrations of ethanol and validated the rationality of the traditional alcoholic extraction method. It not only could be used in monitoring the process of the alcoholic extraction and the compositions of the extracts and residues, discriminating micro-differences among them, but also could provide a macroscopic guidance for medicinal and pharmacological studies.

[Analysis of different parts of scorpio by Fourier transform infrared spectroscopy].

Fourier transform infrared (FTIR) spectroscopy and second derivative IR spectroscopy were applied to analyze and evaluate different parts of Scorpio. The second derivative IR spectra show clear differences while the origin spectra are quite similar. It was found that proteins are the dominant components in each part and the tail has distinct proteins compared to the others; fats are mainly stored in the trunk; sulfates are ubiquitous in all parts. Interestingly, the back part of the trunk of degenerative Scorpio contains some purine. It was demonstrated that FTIR spectroscopy integrated with second derivative IR spectroscopy not only can offer a fast, comprehensive and objective methodology for analyzing and evaluating the micro-differences among the various parts of same medicinal materials, but also can provide a rational guidance for medicinal and pharmacological studies.

Clinical Research on the Relation Between Body Mass Index, Motilin and Slow Transit Constipation.

BACKGROUND: Constipation is a common clinical symptom but its etiology remains unknown. The aims of the study are to discuss the relation between body mass index (BMI), motilin and the slow transit constipation (STC). METHODS: A total of 178 patients with STC and 123 healthy volunteers as controls were divided into three groups according to the BMI, group A (BMI <20), group B (BMI 20-25), and group C (BMI > 25). Fasting and one hour postprandial plasma motilin were measured and the results were analyzed. RESULTS: There was significant difference in the constituent ratio between STC patients and healthy controls (p < 0.05). The percentage of group A, B and C in STC patients was 49.4% (88/178), 23.0% (41/178) and 27.6% (49/178), respectively; and group A had a higher percentage. Plasma motilin of fasting and one hour postprandial in STC patients of group A was significantly lower than that of group B and C (p < 0.05), but there was no difference between group B and C (p > 0.05). There was no significant difference in the results of plasma motilin of fasting and one hour postprandial among the three groups of healthy controls (p > 0.05). Plasma motilin of fasting and one hour postprandial in STC patients of group A was significantly lower than those healthy controls of group A (p < 0.05). The same results of plasma motilin of fasting and one hour postprandial could be seen in group B and C, respectively (p < 0.05). CONCLUSIONS: A higher proportion of low BMI sufferers was found in the STC patients. The reason may be related to the lower release of the plasma motilin.

Antifibrotic effects of interleukin-10 on experimental hepatic fibrosis.

BACKGROUND/AIMS: To study the effects of interleukin-10 on hepatic stellate cells and liver tissue in experimental rats hepatic fibrosis. METHODOLOGY: Rat hepatic fibrosis model induced by carbon tetrachloride was established. Liver tissues were harvested from the rats administered CCl4 with or without IL-10 treatment and the animals of the control group. The expression of TGF-beta1, MMP-2 and TIMP-1 in the liver tissues was measured by S-P immunohistochemistry. In addition, another model was established; HSCs in rats in each group were isolated. RT-PCR was employed to analyze TGF-beta1, MMP-2 and TIMP-1 mRNA expression in cells and immunocytochemistry was performed to detect protein expression of alpha-SMA, NF-kappaB, TGF-beta1, MMP-2 and TIMP-1 in HSCs. RESULTS: Rat hepatic fibrosis was developed successfully. The fibrosis changes were partially reversed by simultaneous administration of IL-10. The positive signals of TGF-beta1, MMP-2 and TIMP-1 were observed more frequently (P<0.05) in the CCl4-treated group compared to those in the IL-10-treated group and the control group. HSCs were successfully isolated. TGF-beta1, MMP-2 and TIMP-1 mRNA in HSCs increased obviously during the course of hepatic fibrosis, and their levels were decreased after the treatment with IL-10 (P<0.05). The immunocytochemistry positive levels for TGF-beta1, MMP-2, TIMP-1, alpha-SMA and NF-kappaB in the fibrogenesis group were increased significantly compared to the normal group (P<0.01). The positive signals decreased significantly (P<0.05) after the treatment with IL-10. CONCLUSIONS: The expression of TGF-beta1, MMP-2 and TIMP-1 increased in liver or in HSC of hepatic fibrosis rats and decreased after treatment with IL-10. The IL-10 could inhibit the activation of HSCs and make an antifibrogenic process come into effect in this way.

Apoptosis and its pathway in X gene-transfected HepG2 cells.

AIM: To investigate the effect of hepatitis B virus (HBV) X gene on apoptosis and expressions of apoptosis factors in X gene-transfected HepG2 cells. METHODS: The HBV X gene eukaryon expression vector pcDNA3-X was transiently transfected into HepG2 cells by lipid-media transfection. Untransfected HepG2 and HepG2 transfected with pcDNA3 were used as controls. Expression of HBx in HepG2 was identified by RT-PCR. MTT and TUNEL were employed to measure proliferation and apoptosis of cells in three groups. Semi-quantified RT-PCR was used to evaluate the expression levels of Fas/FasL, Bax/Bcl-xL, and c-myc in each group. RESULTS: HBV X gene was transfected into HepG2 cells successfully. RT-PCR showed that HBx was only expressed in HepG2/pcDNA3-X cells, but not expressed in HepG2 and HepG2/pcDNA3 cells. Analyzed by MTT, cell proliferation capacity was obviously lower in HepG2/pcDNA3-X cells (0.08910+/-0.003164) than in HepG2 (0.14410+/-0.004927) and HepG2/pcDNA3 cells (0.12150+/-0.007159) (P<0.05 and P<0.01). Analyzed by TUNEL, cell apoptosis was much more in HepG2/pcDNA3-X cells (980/2,000) than HepG2 (420/2,000), HepG2/pcDNA3 cells (520/2,000) (P<0.05 and P<0.01). Evaluated by semi-quantified RT-PCR, the expression level of Fas/FasL was significantly higher in HepG2 cells transfected with HBx than in HepG2 and HepG2/pcDNA3 cells (P<0.05 and P<0.01). Bax/Bcl-xL expression level was also elevated in HepG2/pcDNA3-X cells (P<0.05 and P<0.01). Expression of c-myc was markedly higher in HepG2/pcDNA3-X cells than in HepG2 and HepG2/pcDNA3 cells (P<0.05 and P<0.01). CONCLUSION: HBV X gene can impair cell proliferation capacity, improve cell apoptosis, and upregulate expression of apoptosis factors. The intervention of HBV X gene on the expression of apoptosis factors may be a possible mechanism responsible for the change in cell apoptosis and proliferation.

Possible mechanism for hepatitis B virus X gene to induce apoptosis of hepatocytes.

AIM: To investigate the possible mechanism for HBV X gene to induce apoptosis of hepatocyte HL-7702 cells. METHODS: HBV X gene eukaryon expression vector pcDNA3-X was established and transfected into HL-7702 cells by lipid-mediated transfection, including transient and stable transfection. Positive clones were screened by incubating in the selective medium with 600 microg/mL G418 and named HL-7702/HBV-encoded X protein (HBx) cells. The expressions of Fas/FasL, Bax/Bcl-2, and c-myc mRNA were measured by semi-quantitative RT-PCR in HL-7702/HBx and control group, respectively. RESULTS: RT-PCR analysis confirmed that HBV X gene was transfected into HL-7702 cells successfully. By semi-quantitative RT-PCR analysis, Bax and c-myc mRNA levels in HL-7702/HBx cells of transient transfection were significantly higher than those in control, FasL and c-myc mRNA levels in HL-7702/HBx cells of stable transfection were significantly higher than those in control, whereas the Bcl-2 mRNA levels in HL-7702/HBx cells of transient and stable transfection were significantly lower than those in control. CONCLUSION: HBV X gene may promote the apoptosis of hepatocytes by regulating the expressions of Fas/FasL, Bax/Bcl-2, and c-myc gene in a dose-dependent manner.

Effects of platelet-derived growth factor and interleukin-10 on Fas/Fas-ligand and Bcl-2/Bax mRNA expression in rat hepatic stellate cells in vitro.

AIM: To investigate the effects of platelet-derived growth factor(PDGF) and interleukin-10 (IL-10) on Fas/Fas-ligand and Bcl-2/Bax mRNA expressions in rat hepatic stellate cells. METHODS: Rat hepatic stellate cells (HSCs) were isolated and purified from rat liver by in situ digestion of collagenase and pronase and single-step density Nycodenz gradient. After activated by culture in vitro, HSCs were divided into 4 groups and treated with nothing (group N), PDGF (group P), IL-10 (group I) and PDGF in combination with IL-10 (group C), respectively. Semi-quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis was employed to compare the mRNA expression levels of Fas/FasL and Bcl-2/Bax in HSCs of each group. RESULTS: The expression levels of Fas between the 4 groups had no significant differences (P>0.05). FasL mRNA level in normal culture-activated HSCs (group N) was very low. It increased obviously after HSCs were treated with IL-10 (group I) (0.091+/-0.007 vs 0.385+/-0.051, P<0.01), but remained the low level after treated with PDGF alone (group P) or PDGF in combination with IL-10 (group C). Contrast to the control group, after treated with PDGF and IL-10, either alone or in combination, Bcl-2 mRNA expression was down-regulated and Bax mRNA expression was up-regulated, both following the turn from group P, group I to group C. Expression of Bcl-2 mRNA in group C was significantly lower than that in group P (0.126+/-0.008 vs 0.210+/-0.024, P<0.01). But no significant difference was found between group C and group I, as well as between group I and group P (P>0.05). Similarly, the expression of Bax in group C was higher than that in group P (0.513+/-0.016 vs 0.400+/-0.022, P<0.01). No significant difference was found between group I and group P (P>0.05). But compared with group C, Bax expressions in group I tended to decrease (0.449+/-0.028 vs 0.513+/-0.016, P<0.05). CONCLUSION: PDGF may promote proliferation of HSCs but is neutral with respect to HSC apoptosis. IL-10 may promote the apoptosis of HSCs by up-regulating the expressions of FasL and Bax and down-regulating the expression of Bcl-2, which may be involved in its antifibrosis mechanism.

Transfection and expression of hepatitis B virus x gene and its effect on apoptosis in HL-7702 cells.

AIM: To investigate the effects of hepatitis B virus x gene and its protein product HBxAg on apoptosis in hepatocyte line HL-7702. METHODS: The reconstituted plasmid pcDNA3-x was established through recombination DNA technique; pcDNA3-X was transfected into HL-7702 cells by lipid-mediated trasfection. Positive clones were screened by G418, and HL-7702/HBx cells were analysed by the RT-PCR to confirm the steady expression of X gene in HL-7702 cells. The apoptosis rate in HL-7702 cells was determined by flow cytometry, TUNEL technology, electronic microscope. At the mean time, pcDNA3-X was transfected transiently into HL-7702 cells, and total RNA from HL-7702 cells was extracted 24, 48, 72, 96 and 120 h after the transient transfection, and semi-quantitative analysis was performed by RT-PCR to detect the expression of HBV X gene. Furthermore, apoptosis rate in HL-7702 cells was determined by flow cytometry analysis at the different times. RESULTS: RT-PCR analysis showed that HBV X gene could be expressed stably in HL-7702 cells. Both flow cytometry and TUNEL technology revealed that the apoptosis rates of HL-7702/HBx cells were much higher than those of HL-7702/pcDNA3 and HL-7702 cells. Furthermore, the apoptotic phenomena and apoptotic body were observed in HL-7702/HBx cells under electronic microscope, but not in HL-7702/pcDNA3 and HL-7702 cells. In the experiment of transient transfection, RT-PCR reveals that X gene was expressed most at 72 h after transfection; and the apoptosis rate reached the highest at the same time. After that, the apoptosis rate was reduced with the decrease of the X gene expression. CONCLUSION: HBV X gene and X protein can promote the apoptosis in hepatocyte. And there exist a quantity-effect relationship between the X gene expression and apoptosis rate in hepatocyte.

The third intracellular loop and the carboxyl terminus of beta2-adrenergic receptor confer spontaneous activity of the receptor.

It is well established that the beta2-adrenergic receptor (beta2-AR) exhibits a robust ligand-independent activity, whereas this property is considerably weaker in the closely related beta1-AR subtype. To identify the potential domain(s) of beta2-AR responsible for the spontaneous receptor activation, we created three chimeras in which the third intracellular loop (beta1/beta2-Li3) or the carboxyl terminus (beta1/beta2-CT) or both domains (beta1/beta2-Li3CT) of beta1-AR are replaced by the corresponding parts of the beta2-AR. Using adenoviral gene transfer, we individually expressed these beta1/beta2-AR chimeras in mouse cardiomyocytes lacking both native beta1-AR and beta2-AR (beta1/beta2 double knockout), and examined their possible spontaneous activities. Overexpression of these beta1/beta2-AR chimeras markedly elevated basal cAMP accumulation and myocyte contractility in the absence of agonist stimulation compared with those infected by a control adenovirus expressing beta-galactosidase or an adenovirus expressing wild type beta1-AR. These effects were fully reversed by a beta2-AR inverse agonist, (+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol (ICI 118,551; 5 x 10-7 M), regardless of inhibition of Gi with pertussis toxin, but not by a panel of beta1-AR antagonists, including [2-(3-carbamoyl-4-hydroxyphenoxy)-ethylamino]-3-[4-(1-methyl-4-trifluormethyl-2-imidazolyl)-phenoxy]-2-propanolmethanesulfonate (CGP20712A), betaxolol, bisoprolol, and metoprolol. Furthermore, we have shown that the C-terminal postsynaptic density 95/disc-large/ZO-1 (PDZ) motif of beta1-AR is not responsible for the lack of beta1-AR spontaneous activation, although it has been known that the beta1-AR PDZ motif prevents the receptor from undergoing agonist-induced trafficking and Gi coupling in cardiomyocytes. Taken together, the present results indicate that both the third intracellular loop and the C terminus are involved in beta2-AR spontaneous activation and that either domain seems to be sufficient to confer the receptor spontaneous activity.

Enhanced G(i) signaling selectively negates beta2-adrenergic receptor (AR)--but not beta1-AR-mediated positive inotropic effect in myocytes from failing rat hearts.

BACKGROUND: Myocardial contractile response to beta1- and beta2-adrenergic receptor (AR) stimulation is severely impaired in chronic heart failure, in which G(i) signaling and the ratio of beta2/beta1 are often increased. Because beta2-AR but not beta1-AR couples to G(s) and G(i) with the G(i) coupling negating the G(s)-mediated contractile response, we determined whether the heart failure-associated augmentation of G(i) signaling contributes differentially to the defects of these beta-AR subtypes and, if so, whether inhibition of G(i) or selective activation of beta2-AR/G(s) by ligands restores beta2-AR contractile response in the failing heart. METHODS AND RESULTS: Cardiomyocytes were isolated from 18- to 24-month-old failing spontaneously hypertensive (SHR) or age-matched Wistar-Kyoto (WKY) rat hearts. In SHR cardiomyocytes, either beta-AR subtype-mediated inotropic effect was markedly diminished, whereas G(i) proteins and the beta2/beta1 ratio were increased. Disruption of G(i) signaling by pertussis toxin (PTX) enabled beta2- but not beta1-AR to induce a full positive inotropic response in SHR myocytes. Furthermore, screening of a panel of beta2-AR ligands revealed that the contractile response mediated by most beta2-AR agonists, including zinterol, salbutamol, and procaterol, was potentiated by PTX, indicating concurrent G(s) and G(i) activation. In contrast, fenoterol, another beta2-AR agonist, induced a full positive inotropic effect in SHR myocytes even in the absence of PTX. CONCLUSIONS: We conclude that enhanced G(i) signaling is selectively involved in the dysfunction of beta2- but not beta1-AR in failing SHR hearts and that disruption of G(i) signaling by PTX or selective activation of beta2-AR/G(s) signaling by fenoterol restores the blunted beta2-AR contractile response in the failing heart.

p38 Mitogen-activated protein kinase mediates a negative inotropic effect in cardiac myocytes.

p38 Mitogen-activated protein kinase (MAPK) is one of the most ancient signaling molecules and is involved in multiple cellular processes, including cell proliferation, cell growth, and cell death. In the heart, enhanced activation of p38 MAPK is associated with ischemia/reperfusion injury and the onset of heart failure. In the present study, we investigated the function of p38 MAPK in regulating cardiac contractility and its underlying mechanisms. In cultured adult rat cardiomyocytes, activation of p38 MAPK by adenoviral gene transfer of an activated mutant of its upstream kinase, MKK3bE, led to a significant reduction in baseline contractility, compared with uninfected cells or those infected with a control adenoviral vector (Adv-beta-galactosidase). The inhibitory effect of MKK3bE on contractility was largely prevented by coexpressing a dominant-negative mutant of p38 MAPK or treating cells with a p38 MAPK inhibitor, SB203580. Conversely, inhibition of endogenous p38 MAPK activity by SB203580 rapidly and reversibly enhanced cell contractility in a dose-dependent manner, without altering L-type Ca(2+) currents or Ca(2+)(i) transients. MKK3bE-induced p38 activation had no significant effect on pH(i), whereas SB203580 had a minor effect to elevate pH(i). Furthermore, activation of p38 MAPK was unable to increase troponin I phosphorylation. Thus, we conclude that the negative inotropic effect of p38 MAPK is mediated by decreasing myofilament response to Ca(2+), rather than by altering Ca(2+)(i) homeostasis and that the reduced myofilament Ca(2+) sensitivity is unlikely attributable to troponin I phosphorylation or alterations in pH(i). These findings reveal a novel function of p38 MAPK and shed a new light on our understanding of the coincidence of p38 MAPK activation and the onset of heart failure.