Electrophoresis of a highly charged fluid droplet in dilute electrolyte solutions: Analytical Hückel-type solution.

An analytical formula is presented here for the electrophoresis of a dielectric or perfectly conducting fluid droplet with arbitrary surface potentials suspended in a very dilute electrolyte solution. In other words, when the Debye length (κ-1 ) is very large, or κa ≪ $\ll $ 1, where κ is the electrolyte strength and a stands for the droplet radius. This formula can be regarded as an extension of the famous Hückel solution valid for weakly charged rigid particles to arbitrarily charged fluid droplets. The formula reduces successfully to the ones obtained by Booth for a dielectric droplet, and Ohshima for a perfectly conducting droplet, both under Debye-Hückel approximation valid for weakly charged droplets. Moreover, the formula is valid for a gas bubble and a rigid solid particle as well. Classic results obtained by Hückel for a rigid particle are reproduced as well. We found that for a dielectric droplet, the more viscous the droplet is, the faster it moves regardless of its surface potential, contrary to the intuition based on the purely hydrodynamic consideration. For a perfectly conducting liquid droplet, on the other hand, the situation is reversed: The less viscous the droplet is, the faster it moves. The presence or absence of the spinning electric driving force tangent to the droplet surface is found to be responsible for it. As a result, an axisymmetric exterior vortex flow surrounding the droplet is always present for a dielectric liquid droplet, and never there for a conducting liquid droplet.

Diffusiophoresis in suspensions of highly charged soft particles.

Diffusiophoresis phenomenon of aoft particles suspended in binary electrolyte solutions is explored theoretically in this study based on the spherical cell model, focusing on the chemiphoresis component in absence of diffusion potential. Both the electrostatic and hydrodynamic aspects of the boundary confinement, or steric effect, due to the presence of neighboring particles are examined extensively under various electrokinetic conditions. Significant local extrema are found in mobility profiles expressed as functions of the Debye length in general, synchronized with the strength of the motion-inducing double layer polarization. Moreover, a seemingly peculiar phenomenon is observed that the soft particles may move faster in more concentrated suspensions. The competition between the simultaneous enhancement of the motion-inducing electric driving force and the motion-retarding hydrodynamic drag force from the boundary confinement effect of the neighboring particles is found to be responsible for it. The above findings are also demonstrated experimentally in a very recent study on the diffusiophoretic motion of soft particles through porous collagen hydrogels. The results presented here are useful in various practical applications of soft particles like drug delivery.

Analytical solution to dielectric droplet diffusiophoresis under Debye-Hückel approximation.

A simple analytical formula is obtained for the diffusiophoresis of a dielectric fluid droplet in symmetric binary electrolyte solutions under Debye-Hückel approximation valid for weakly charged droplets. The chemiphoresis is found to yield negative mobilities most of the time for droplets of constant surface charge density, which implies that the droplets tend to move away from the source releasing ionic chemicals. This is undesirable in some practical applications like drug delivery with liposomes in terms of conveying the drug-carrying liposomes to the desired area in the human body releasing specific ionic chemicals utilizing the self-guiding nature of diffusiophoresis. The further involvement of the electrophoresis component, however, may change the scenario via the oriented electric field generated by the induced diffusion potential. The lesson here is that while the impact of the chemiphoresis component is determined by nature and uncontrollable, the electrophoresis component serves as an artificially adjustable factor via choosing droplets with the surface charge of appropriate sign in practical applications. The results here have potential use in practical applications such as drug delivery. The portable simple analytical formula is a powerful asset to experimental researchers and design engineers in colloid science and technology to facilitate their works.

Feeding behavior elicited by mu opioid and GABA receptor activation in the lateral septum.

The lateral septum (LS), a brain region typically associated with behaviors involving reward, anxiety-like behavior, learning, and memory, has recently received increased interest due to its potential role in eating behavior. Our current results showed that morphine (5 µg) microinjected into the LS produced a stable feeding response. Specifically, across five days of repeated injections, there was no increase or sensitization effect, nor a decrease in feeding or tolerance. Additionally, we found that pretreatment with the broad-spectrum opioid receptor antagonist naloxone blocked morphine-elicited feeding, further supporting a role for LS opioid receptors in the activation of feeding behaviors. We had previously found that the GABAA receptor agonist muscimol produces a similar increase in feeding when injected into the LS. Given the involvement of the LS in multiple behaviors, we next evaluated whether other behaviors might be co-occurring with feeding in response to opioid or GABAA receptor agonist injection into the LS. We assessed eating, drinking, grooming, sleeping, activity levels and resting behavior for 3 h after injection of aCSF, DAMGO, morphine, or muscimol. We found that morphine and muscimol both decreased the latency to eat, and all drugs tested increased food intake. The feeding occurred within 30 min of muscimol injection but was delayed after opioid injections. The absence of increases in other goal-oriented behavior like drinking or grooming or behavioral hyperactivity supports a primary effect of muscimol and the opioids on LS mechanisms of feeding control. SIGNIFICANCE STATEMENT: The LS is interesting because of its role in a wide range of behaviors including defensive behaviors, social behaviors, learning, memory, and motivation. Although the LS was discovered to have a role in feeding stimulation over 30 years ago, only recently has major progress begun to reveal the underlying mechanisms. The present paper contributes by suggesting that LS GABAA and µ-opioid receptors elicit eating by inhibiting LS neurons that themselves inhibit eating. Importantly, this work informs lateral septal research which may shed light on disordered eating included binge eating and anorexia.

Stimulation of GABA Receptors in the Lateral Septum Rapidly Elicits Food Intake and Mediates Natural Feeding.

The increasing prevalence of obesity and eating disorders makes identifying neural substrates controlling eating and regulating body weight a priority. Recent studies have highlighted the role of the lateral septum (LS) in eating control mechanisms. The current study explored the roles of gamma-aminobutyric acid (GABA) receptors within the LS in the control of food intake. Experiments with a rat model (n ≥ 11/group) showed that LS microinjection of the GABAA receptor agonist, muscimol, and the GABAB receptor agonist, baclofen hydrochloride (baclofen), elicited intense, dose-dependent feeding. In contrast, LS pretreatment with the GABAA receptor antagonist, picrotoxin, markedly reduced the muscimol-elicited feeding, and pretreatment injections with the GABAB receptor antagonist, 2-hydroxysaclofen (2-OH saclofen), reduced the baclofen evoked response. Next, we showed that picrotoxin injection at the beginning of the dark phase of the light-dark cycle-when rats show a burst of spontaneous eating-reduced naturally occurring feeding, whereas 2-OH saclofen was ineffective. These results indicate that the activation of LS GABAA and GABAB receptors strongly stimulates feeding and suggests potential roles in feeding control neurocircuitry. In particular, our evidence indicates that endogenous LS GABA and GABAA receptors may be involved in mediating naturally occurring nocturnal feeding.

High ambient glucose induces angiotensin-independent AT-1 receptor activation, leading to increases in proliferation and extracellular matrix accumulation in MES-13 mesangial cells.

Diabetic nephropathy is associated with mesangial ECM (extracellular matrix) accumulation. We have shown that AT-1R [Ang II (angiotensin II) type I receptor] signalling induces ECM proteins via transactivation of PI3K (phosphoinositide 3-kinase) in mesangial cells. In the present study, we examined the mechanisms underlying the effect of high ambient glucose on cell proliferation and ECM expansion in a mesangial context. High glucose induced increases in PI3K activity, proliferation and ECM accumulation in mesangial cells. These effects were abrogated by losartan, an AT-1R antagonist, but not by [Sar1,Thr8]-Ang II (Sar is sarcosine), an inactive analogue of Ang II, or by a neutralizing antibody against Ang I/II. Overexpression of a constitutively active PI3Kalpha or AT-1R alone was sufficient to induce similar changes by high glucose. In contrast, overexpression of an inactive AT-1R lowered the basal levels and rendered the cells non-responsive to high glucose. Moreover, cells overexpressing wild-type AT-1R had enhanced sensitivity to acute Ang II stimulation. These cells, however, did not respond to conditioned medium obtained from mesangial cells cultured in high glucose. We further demonstrated that iAng (intracellular Ang II) can be induced by high glucose but only under certain conditions. Efficient suppression of iAng by short hairpin RNA against angiotensinogen, however, did not affect high glucose-induced effects on MES-13 cells. These results suggest that high ambient glucose induces activation of AT-1R in an Ang II-independent manner to transactivate PI3K, resulting in proliferation and ECM accumulation in mesangial cells.

Lateral septum mu opioid receptors in stimulation of feeding.

Stimulation of mu opioid receptors using drugs like morphine can increase eating when injected into multiple brain regions including the lateral septum (LS). The LS has been classically associated with reward, anxiety and fearful behaviors but more recently has also received attention with regard to control of feeding. To investigate the role of LS opioid receptors in feeding, we injected mu, delta, and kappa opioid receptor agonists and a mu specific receptor antagonist directly into the LS of rats. We expected that if feeding is mu receptor specific then only mu receptor agonists would increase feeding. We also hypothesized that mu receptor antagonists would suppress the feeding elicited by mu receptor agonists like morphine. Further, because the LS is densely populated with GABA receptors, we used the GABAA receptor agonist muscimol to assess the effect of inhibition of LS neurons on feeding. Our results show that the mu receptor agonist morphine and the specific mu agonist DAMGO reliably and significantly increase feeding behavior across doses tested, while delta and kappa agonists were ineffective. CTAP, a specific mu receptor antagonist, at low doses unexpectedly increased morphine-elicited feeding but at high doses decreased morphine's effect, consistent with mediation by mu receptors. Finally, muscimol rapidly elicited feeding, suggesting a role for LS GABAA receptors in feeding stimulation. These findings suggest that mu opioid receptors in the LS play complex roles in feeding and that neural inhibition may be a mechanism by which they elicit feeding.

Development and performance of a gas chromatography-time-of-flight mass spectrometry analysis for large-scale nontargeted metabolomic studies of human serum.

A method for the preparation and GC-TOF-MS analysis of human serum samples has been developed and evaluated for application in long-term metabolomic studies. Serum samples were deproteinized using 3:1 methanol/serum, dried in a vacuum concentrator, and chemically derivatized in a two-stage process. Samples were analyzed by GC-TOF-MS with a 25 min analysis time. In addition, quality control (QC) samples were used to quantify process variability. Optimization of chemical derivatization was performed. Products were found to be stable for 30 h after derivatization. An assessment of within-day repeatability and within-week reproducibility demonstrates that excellent performance is observed with our developed method. Analyses were consistent over a 5 month period. Additional method testing, using spiked serum samples, showed the ability to define metabolite differences between samples from a population and samples spiked with metabolites standards. This methodology allows the continuous acquisition and application of data acquired over many months in long-term metabolomic studies, including the HUSERMET project (http://www.husermet.org/).

The role of endogenous pituitary adenylyl cyclase activating polypeptide (PACAP) in nicotine self-administration, reward and aversion.

Pituitary adenylyl cyclase activating polypeptide (PACAP) and its receptors (PAC1, VPAC1, and VPAC2) are localized in brain regions implicated in stress response, reward seeking and aversive responses, raising the possibility that PACAP may be involved in motivational effects of nicotine. To test this hypothesis, we used two-bottle choice (TBC) and place conditioning paradigms and assessed if nicotine preference or conditioned place preference (CPP) or aversion (CPA) induced by nicotine would be altered in mice lacking PACAP compared to their wild-type controls. In the TBC paradigm, mice had access to two water bottles during the first week and then one of the water bottles was switched to nicotine solution (20, 40 and then 80 µg/mL). The volume of water and nicotine consumed was measured every day. In the place conditioning paradigm, mice were tested for baseline place preference on day 1, received conditioning with saline versus a low (0.25) or high (1 mg/kg) dose nicotine and, respectively, tested for CPP or CPA 24 h following the last conditioning. We discovered that mice lacking PACAP compared to their wild-type controls exhibited more preference for nicotine over water in the TBC paradigm, particularly at the two higher concentrations of nicotine. While the rewarding action of the low dose nicotine was not altered in mice lacking PACAP, the aversive effect of the high dose nicotine was blunted in these mice compared to their wild-type controls. The present results suggest that endogenous PACAP may play a functional role in nicotine preference and its aversive effect.

Beta-adrenergic receptor mediated protection against doxorubicin-induced apoptosis in cardiomyocytes: the impact of high ambient glucose.

Recent studies have demonstrated that the beta2-adrenergic receptor (beta2AR)-Galphai signaling pathway exerts a cardiac antiapoptotic effect. The goals of this study were to determine the intracellular signaling factors involved in beta2AR-mediated protection against doxorubicin-induced apoptosis in H9c2 cardiomyocyte and explore the impact of high ambient glucose on the antiapoptotic effect. Under physiological glucose environment (100 mg/dl), beta2AR stimulation prevented doxorubicin-induced apoptosis, which was attenuated by cotreatment with wortmannin, a phosphoinositide 3-kinase (PI3K) inhibitor, or transfection of a dominant-negative Akt. Inhibition of Src kinase with 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine or cSrc small interfering RNA 32 also attenuated the antiapoptotic effect. Inhibition of platelet-derived growth factor receptor (PDGFR) with AG1296 reversed the beta2AR-induced antiapoptotic effect. Transfection of an active Src cDNA (Y529F) alone was sufficient to render the cells resistant to apoptosis, and the resistance was blocked by wortmannin. Transfection of an active PI3K minigene (iSH2-p110) alone also induced resistance to apoptosis, and the resistance was reversed by an Akt-inhibitor but not by AG1296. High ambient glucose (450 mg/dl) caused two major effects: 1) it significantly reduced betaAR-induced PDGFR phosphorylation, Src kinase activity, and activation of PI3K signaling pathway; and 2) it partially attenuated beta2AR-induced antiapoptotic effect. These data provide in vitro evidence supporting a signaling cascade by which beta2AR exerts a protective effect against doxorubicin-induced apoptosis via sequential involvement of Galphai, Gbetagamma, Src, PDGFR, PI3K, and Akt. High ambient glucose significantly attenuates beta2AR-mediated cardioprotection by suppressing factors involved in this cascade including PDGFR, Src, and PI3K/Akt.

Targeting human CD34+ hematopoietic stem cells with anti-CD45 x anti-myosin light-chain bispecific antibody preserves cardiac function in myocardial infarction.

We have previously shown that targeting human CD34(+) hematopoietic stem cells (HSC) with a bispecific antibody (BiAb) directed against myosin light chain (MLC) increases delivery of cells to the injured hearts and improves cardiac performance in the nude rat. In this study, we have sought to validate our previous observations and to perform more detailed determination of ventricular function in immunocompetent mice with myocardial infarction (MI) that were treated with armed CD34(+) HSC. We examined whether armed CD34(+) HSC would target the injured heart following MI and restore ventricular function in vitro. MI was created by ligation of the left anterior descending artery. After 48 h, adult ICR mice received either 0.5 x 10(6) human CD34(+) HSC armed with anti-CD45 x anti-MLC BiAb or an equal volume of medium through a single tail vein injection. Two weeks after stem cell administration, ventricular function of hearts from mice receiving armed CD34(+) HSC was significantly greater compared with the same parameters from control mice. Immunohistochemistry confirmed the accumulation of CD34(+) HSC in MI hearts infused with stem cells. Angiogenesis was significantly enhanced in CD34(+) HSC-treated heart as determined by vascular density per area. Furthermore, histopathological examination revealed that the retained cardiac function observed in CD34(+) HSC-treated mice was associated with decreased ventricular fibrosis. These results suggest that peripheral administration of armed CD34(+) HSC results in localization of CD34(+) HSC to injured myocardium and restores myocardial function.

MeMo: a hybrid SQL/XML approach to metabolomic data management for functional genomics.

BACKGROUND: The genome sequencing projects have shown our limited knowledge regarding gene function, e.g. S. cerevisiae has 5-6,000 genes of which nearly 1,000 have an uncertain function. Their gross influence on the behaviour of the cell can be observed using large-scale metabolomic studies. The metabolomic data produced need to be structured and annotated in a machine-usable form to facilitate the exploration of the hidden links between the genes and their functions. DESCRIPTION: MeMo is a formal model for representing metabolomic data and the associated metadata. Two predominant platforms (SQL and XML) are used to encode the model. MeMo has been implemented as a relational database using a hybrid approach combining the advantages of the two technologies. It represents a practical solution for handling the sheer volume and complexity of the metabolomic data effectively and efficiently. The MeMo model and the associated software are available at http://dbkgroup.org/memo/. CONCLUSION: The maturity of relational database technology is used to support efficient data processing. The scalability and self-descriptiveness of XML are used to simplify the relational schema and facilitate the extensibility of the model necessitated by the creation of new experimental techniques. Special consideration is given to data integration issues as part of the systems biology agenda. MeMo has been physically integrated and cross-linked to related metabolomic and genomic databases. Semantic integration with other relevant databases has been supported through ontological annotation. Compatibility with other data formats is supported by automatic conversion.

Measuring alcohol-related consequences and motives among students attending Historically Black Colleges and Universities.

INTRODUCTION: College student drinkers have the propensity to engage in heavy alcohol consumption. These consumption patterns can be problematic given the well-established relationship between heavy drinking and negative consequences of alcohol consumption. Though the research on college student drinking is abundant, much of the work conducted has been with Caucasian samples and less so with African American samples or at Historically Black Colleges and Universities (HBCUs). METHODS: The current paper assessed the internal reliability and concurrent validity of two popular measures of alcohol related negative consequences (CAPS-r and YAACQ) and a measure of drinking motives (DMQ-R) within a HBCU sample. RESULTS: Total scores for the CAPS-r and YAACQ and all five subscales of the DMQ-R were internally reliable as determined by Cronbach's alpha. Correlations and regressions established concurrent validity for both measures of negative consequences as well as the subscales for the DMQ-R. CONCLUSIONS: Findings support the use of these measures in the assessment of negative consequences and motivations for alcohol consumption within a HBCU population.

Molecular phenotyping of a UK population: defining the human serum metabolome.

Phenotyping of 1,200 'healthy' adults from the UK has been performed through the investigation of diverse classes of hydrophilic and lipophilic metabolites present in serum by applying a series of chromatography-mass spectrometry platforms. These data were made robust to instrumental drift by numerical correction; this was prerequisite to allow detection of subtle metabolic differences. The variation in observed metabolite relative concentrations between the 1,200 subjects ranged from less than 5 % to more than 200 %. Variations in metabolites could be related to differences in gender, age, BMI, blood pressure, and smoking. Investigations suggest that a sample size of 600 subjects is both necessary and sufficient for robust analysis of these data. Overall, this is a large scale and non-targeted chromatographic MS-based metabolomics study, using samples from over 1,000 individuals, to provide a comprehensive measurement of their serum metabolomes. This work provides an important baseline or reference dataset for understanding the 'normal' relative concentrations and variation in the human serum metabolome. These may be related to our increasing knowledge of the human metabolic network map. Information on the Husermet study is available at http://www.husermet.org/. Importantly, all of the data are made freely available at MetaboLights (http://www.ebi.ac.uk/metabolights/).

The role of endogenous beta-endorphin and enkephalins in ethanol reward.

Substantial evidence has implicated the endogenous opioid system in alcohol reinforcement. However, the role of each opioid peptide in alcohol reinforcement and, particularly, reward is not fully characterized. In this study, using the conditioned place preference (CPP) paradigm as an animal model of reward, we determined the role of endogenous ß-endorphin and enkephalins in the rewarding action of ethanol. Female mice lacking beta-endorphin and/or the proenkephalin gene as well as their respective wild-type controls were tested for baseline place preference on day 1, conditioned with ethanol versus saline on days 2-4 and were then tested under a drug-free state for postconditioning place preference on day 5. On each test day, mice were placed in the central neutral chamber and allowed to freely explore all three CPP chambers. The amount of time that mice spent in each chamber was recorded. We also studied the effect of naloxone, a non-selective opioid receptor antagonist, on ethanol CPP, in which wild-type mice were treated with saline or naloxone 10 min prior to ethanol or saline conditioning. Our results showed that the absence of ß-endorphin or enkephalins alone failed to alter the acquisition of ethanol-induced CPP. However, the absence of both ß-endorphin and enkephalins significantly reduced the CPP response. Interestingly, high but not low dose naloxone blunted ethanol CPP. These findings provide the first evidence illustrating that ethanol induces its rewarding action, at least in part, via a joint action of ß-endorphin and enkephalins, possibly involving both mu and delta opioid receptors.

The role of endogenous dynorphin in ethanol-induced state-dependent CPP.

The aim of this study was to determine the role of the endogenous dynorphin/kappa opioid receptor (DYN/KOP) system in ethanol-induced state-dependent conditioned place preference (CPP). To this end, mice lacking the pro-DYN gene and their wild-type littermates/controls were tested for baseline place preference on day 1, received 15-min morning and afternoon conditionings with saline or ethanol (2g/kg) each day for three consecutive days and were then tested for CPP under a drug-free state on day 5 and following a saline or ethanol (1 or 2g/kg) challenge on day 8. Given that compensatory developmental changes may occur in knockout mice, the effect of nor-binaltorphimine (nor-BNI), a KOP antagonist, on state-dependent CPP induced by ethanol was also studied in wild-type mice. On day 1, mice were tested for baseline place preference and, 4h later, treated with saline or nor-BNI (10mg/kg). On days 2-4, mice received 15-min morning and afternoon conditionings and were tested for CPP under a drug-free state on day 5 and following an ethanol (1g/kg) challenge on day 8. A comparable CPP was observed in mice lacking the pro-DYN gene and their wild-type littermates/controls as well as in wild-type mice treated with nor-BNI and their saline-treated controls. However, these mice compared to their respective controls exhibited a greater CPP response following an ethanol (1g/kg) challenge, suggesting that the endogenous DYN/KOP system may negatively regulate ethanol-induced state-dependent CPP.

Stress-induced analgesia and endogenous opioid peptides: the importance of stress duration.

Stress is known to elicit pain relief, a phenomenon referred to as stress-induced analgesia. Based on stress parameters, opioid and non-opioid intrinsic pain inhibitory systems can be activated. In the present study, we assessed whether changing the duration of stress would affect the involvement of endogenous opioids in antinociception elicited by swim in warm water (32 °C), known to be opioid-mediated. Using mice lacking beta-endorphin, enkephalins or dynorphins and their respective wild-type littermates, we assessed the role of each opioid peptide in antinociception induced by a short (3 min) vs. long (15 min) swim. Mice were tested for baseline hot plate latency, exposed to swim (3 or 15 min) in warm water (32 °C) and then tested for antinociception at 5, 15 and 30 min. Our results revealed that both swim paradigms induced significant antinociception in wild-type mice. However, the short swim failed to induce antinociception in beta-endorphin-deficient mice, illustrating that beta-endorphin is important in this form of stress-induced antinociception. On the other hand, antinociception elicited by the long swim was only slightly reduced in beta-endorphin-deficient mice despite pretreatment with naloxone, a non-selective opioid receptor antagonist, significantly attenuated the antinociception elicited by the long swim. Nevertheless, a delayed hyperalgesic response developed in mice lacking beta-endorphin following exposure to either swim paradigm. On the other hand, mice lacking enkephalins or dynorphins and their respective wild-type littermates expressed a comparable antinociceptive response and did not exhibit the delayed hyperalgesic response. Together, our results suggest that the endogenous opioid peptide beta-endorphin not only mediates antinociception induced by the short swim but also prevents the delayed hyperalgesic response elicited by either swim paradigm.

Effect of disruption of Akt-1 of lin(-)c-kit(+) stem cells on myocardial performance in infarcted heart.

AIMS: We have demonstrated an important role of bone marrow-derived stem cells in preservation of myocardial function. We investigated whether Akt-1 of lin(-)c-kit(+) stem cells preserves ventricular function following myocardial infarction (MI). METHODS AND RESULTS: Isolated lin(-)c-kit(+) cells were conjugated with anti-c-kit heteroconjugated to anti-vascular cell adhesion molecule to facilitate the attachment of stem cells into damaged tissues. Female severe combined immunodeficient mice were used as recipients. MI was created by ligation of the left descending artery. After 48 h, animals were divided into four groups: (i) sham (n = 5): animals underwent thoracotomy without MI; (ii) MI (n = 5): animals underwent MI and received medium; (iii) MI + wild-type (Wt) stem cells (n = 6): MI animals received 5 x 10(5) Wt lin(-)c-kit(+) stem cells; (iv) MI + Akt-1(-/-) stem cells (n = 6): MI animals received 5 x 10(5) Akt-1(-/-) lin(-)c-kit(+) stem cells. Two weeks later, left ventricular function was measured in the Langendorff mode. The peripheral administration of Wt armed stem cells into MI animals restored ventricular function, which was absent in animals receiving Akt-1(-/-) cells. Real-time PCR indicates a decrease in SRY3, a Y chromosome marker in hearts receiving Akt-1(-/-) cells. An increase in angiogenic response was demonstrated in hearts receiving Wt stem cells but not Akt-1(-/-) stem cells. CONCLUSION: Our results demonstrate that the peripheral administration of Wt lin(-)c-kit(+) stem cells restores ventricular function and promotes angiogenic response following MI. These benefits were abrogated in MI mice receiving Akt-1(-/-) stem cells, suggesting the pivotal role of Akt-1 in mediating stem cells to protect MI hearts.

Temporally controlled overexpression of cardiac-specific PI3Kalpha induces enhanced myocardial contractility--a new transgenic model.

The phosphatidylinositol 3-kinase (PI3K) signaling pathway regulates multiple cellular processes including cell survival/apoptosis and growth. In the cardiac context, PI3Kalpha plays important roles in cardiac growth. We have shown that cardiac PI3K activity is highly regulated during development, with the highest levels found during the fetal-neonatal transition period and the lowest levels in the adult. There is a close relationship between cardiomyocyte proliferation and cardiac PI3K activity. In adult transgenic mice, however, the prolonged constitutive activation of PI3Kalpha in the heart results in hypertrophy. To develop a strategy to allow temporally controlled overexpression of cardiac PI3Kalpha, we engineered a tetracycline (tet) transactivator tet-off controlled transgenic mouse line with a conditional overexpression of a cardiac-specific fusion protein of the SH2 domain of p85 and p110alpha. Cardiac PI3K activity and Akt phosphorylation were significantly increased in adult mice after transgene induction following the removal of doxycycline for 2 wk. The heart weight-to-body weight ratio was not changed, and there were no signs of cardiomyopathy. The overexpression of PI3Kalpha resulted in increased left ventricular (LV) developed pressure and the maximal and minimal positive values of the first derivative of LV pressure, but not heart rate, as assessed in Langendorff hearts. Mice overexpressing PI3Kalpha also had increases in the levels of Ca(2+)-regulating proteins, including the L-type Ca(2+) channels, ryanodine receptors, and sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a. Thus the temporally controlled overexpression of cardiac PI3Kalpha does not induce hypertrophy or cardiomyopathy but results in increased contractility, probably via the increased expression of multiple Ca(2+)-regulating proteins. These distinct phenotypes suggest a fundamental difference between transgenic mice with temporal or prolonged activation of cardiac PI3Kalpha.

A novel signaling pathway for beta-adrenergic receptor-mediated activation of phosphoinositide 3-kinase in H9c2 cardiomyocytes.

Stimulation of cardiac beta-adrenergic receptors (beta-AR) activates both the G(s)- and G(i)-coupled signaling cascades, including the phosphoinositide 3 kinase (PI3K) pathway, that have important physiological implications. Multiple isoforms of PI3K exist in the heart. The goals of this study were to examine the intracellular signaling pathways linking beta-AR to PI3K and to identify the PI3K isoform mediating this transactivation in a cardiac context. Acute beta-AR stimulation with isoproterenol resulted in increased tyrosine kinase-associated PI3K activity and phosphorylation of Akt and p70S6K in H9c2 cardiomyocytes. Cotreatment with ICI-118,551, but not CGP-20712, abolished the increase in PI3K activity, suggesting a beta(2)-AR-mediated event. PI3K activation was also abrogated by cotreatment with pertussis toxin, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolol[3,4-d]pyrimidine (PP2, a selective Src-family tyrosine kinases inhibitor), or AG-1296 [selective platelet-derived growth factor receptor (PDGFR) inhibitor] but not with an inhibitor for protein kinase A, protein kinase C, Ras, adenylyl cyclase, epidermal growth factor receptor, or insulin-like growth factor-1 receptor. beta-AR stimulation induced an increase in tyrosine phosphorylation of PDGFR, which was abolished by inhibition of Src either by PP2 or small interfering RNA. Moreover, H9c2 cardiomyocytes stably transfected with a vector expressing a Gbetagamma sequestrant peptide derived from the COOH-terminus of beta-AR kinase-1 failed to activate PI3K after beta-AR stimulation, suggesting Gbetagamma is required for the transactivation. Furthermore, acute beta-AR stimulation in vivo resulted in increases in PDGFR-associated PI3K and PI3Kalpha isoform activities but not the activities of other isoforms (PI3Kbeta, -delta, -gamma) in adult mouse heart. Taken together, these data provide in vitro and in vivo evidence for a novel mechanism of beta-AR-mediated transactivation of cardiac PI3Kalpha via sequential involvement of Galpha(i)/Gbetagamma, Src, and PDGFR.