Supachoke Mangmool

Gi/o Protein-Dependent and -Independent Actions of Pertussis Toxin (PTX)

Pertussis toxin (PTX) is a typical A-B toxin. The A-protomer (S1 subunit) exhibits ADP-ribosyltransferase activity. The B-oligomer consists of four subunits (S2 to S5) and binds extracellular molecules that allow the toxin to enter the cells. The A-protomer ADP-ribosylates the α subunits of heterotrimeric Gi/o proteins, resulting in the receptors being uncoupled from the Gi/o proteins. The B-oligomer binds proteins expressed on the cell surface, such as Toll-like receptor 4, and activates an intracellular signal transduction cascade. Thus, PTX modifies cellular responses by at least two different signaling pathways; ADP-ribosylation of the Gαi/o proteins by the A-protomer (Gi/o protein-dependent action) and the interaction of the B-oligomer with cell surface proteins (Gi/o protein-independent action).

β1-Adrenergic receptors stimulate cardiac contractility and CaMKII activation in vivo and enhance cardiac dysfunction following myocardial infarction

The beta-adrenergic receptor (betaAR) signaling system is one of the most powerful regulators of cardiac function and a key regulator of Ca(2+) homeostasis. We investigated the role of betaAR stimulation in augmenting cardiac function and its role in the activation of Ca(2+)/calmodulin-dependent kinase II (CaMKII) using various betaAR knockouts (KO) including beta(1)ARKO, beta(2)ARKO, and beta(1)/beta(2)AR double-KO (DKO) mice. We employed a murine model of left anterior descending coronary artery ligation to examine the differential contributions of specific betaAR subtypes in the activation of CaMKII in vivo in failing myocardium. Cardiac inotropy, chronotropy, and CaMKII activity following short-term isoproterenol stimulation were significantly attenuated in beta(1)ARKO and DKO compared with either the beta(2)ARKO or wild-type (WT) mice, indicating that beta(1)ARs are required for catecholamine-induced increases in contractility and CaMKII activity. Eight weeks after myocardial infarction (MI), beta(1)ARKO and DKO mice showed a significant attenuation in fractional shortening compared with either the beta(2)ARKO or WT mice. CaMKII activity after MI was significantly increased only in the beta(2)ARKO and WT hearts and not in the beta(1)ARKO and DKO hearts. The border zone of the infarct in the beta(2)ARKO and WT hearts demonstrated significantly increased apoptosis by TUNEL staining compared with the beta(1)ARKO and DKO hearts. Taken together, these data show that cardiac function and CaMKII activity are mediated almost exclusively by the beta(1)AR. Moreover, it appears that beta(1)AR signaling is detrimental to cardiac function following MI, possibly through activation of CaMKII.

Pectin nanoparticle enhances cytotoxicity of methotrexate against hepG2 cells

Objective: This work has aimed to develop methotrexate-conjugated pectin nanoparticle for delivering a cytotoxic drug to hepatic cancer cell. Methods: Methotrexate was conjugated to pectin by carbodiimide chemistry. Nanoparticles of pectin conjugated with methotrexate (MTX) were then fabricated by using ionotropic gelation. The size, shape and surface charge were characterized by dynamic light scattering and microscopic method. Cytotoxicity of MTX-pectin nanoparticle was monitored by MTT assay. Results: Methotrexate-pectin nanoparticle was successfully formulated. Drug release study indicated that MTX-NP exhibited sustained drug release at pH 7.4. Sustained release of methotrexate may enable methotrexate-pectin nanoparticle as a controlled drug delivery system. Cytotoxicity study confirmed the activity of the drug incorporated in nanoparticles. In addition, the cytotoxicity of methotrexate was increased when conjugated to pectin nanoparticles, compared to free methotrexate. Conclusions: This study verified that pectin can deliver methotrexate to hepatic cancer cell and provide sustained drug delivery. The cytotoxicity of methotrexate was enhanced when methotrexate was conjugated to pectin indicating the improved drug delivery to cancer cell.

Stimulation of Adenosine A2B Receptor Inhibits Endothelin-1-Induced Cardiac Fibroblast Proliferation and α-Smooth Muscle Actin Synthesis Through the cAMP/Epac/PI3K/Akt-Signaling Pathway

Background and Purpose: Cardiac fibrosis is characterized by an increase in fibroblast proliferation, overproduction of extracellular matrix proteins, and the formation of myofibroblast that express α-smooth muscle actin (α-SMA). Endothelin-1 (ET-1) is involved in the pathogenesis of cardiac fibrosis. Overstimulation of endothelin receptors induced cell proliferation, collagen synthesis, and α-SMA expression in cardiac fibroblasts. Although adenosine was shown to have cardioprotective effects, the molecular mechanisms by which adenosine A2 receptor inhibit ET-1-induced fibroblast proliferation and α-SMA expression in cardiac fibroblasts are not clearly identified. Experimental Approach: This study aimed at evaluating the mechanisms of cardioprotective effects of adenosine receptor agonist in rat cardiac fibroblast by measurement of cell proliferation, and mRNA and protein levels of α-SMA. Key results: Stimulation of adenosine subtype 2B (A2B) receptor resulted in the inhibition of ET-1-induced fibroblast proliferation, and a reduction of ET-1-induced α-SMA expression that is dependent on cAMP/Epac/PI3K/Akt signaling pathways in cardiac fibroblasts. The data in this study confirm a critical role for Epac signaling on A2B receptor-mediated inhibition of ET-1-induced cardiac fibrosis via PI3K and Akt activation. Conclusion and Implications: This is the first work reporting a novel signaling pathway for the inhibition of ET-1-induced cardiac fibrosis mediated through the A2B receptor. Thus, A2B receptor agonists represent a promising perspective as therapeutic targets for the prevention of cardiac fibrosis.

Blockade of the Renin-Angiotensin System with Delphinidin, Cyanin, and Quercetin

Overactivation of the renin-angiotensin system is one of the most important risk factors for the development of hypertension. The use of the crude extracts and/or active compounds, such as anthocyanins and quercetin, of herbal plants that have antihypertensive effects is beneficial for decreasing of blood pressure level. However, the molecular mechanisms by which anthocyanins (delphinidin and cyanin) and quercetin regulate the renin-angiotensin system are not completely understood. In this study, we demonstrate that delphinidin, cyanin, and quercetin interrupt the renin-angiotensin system signaling pathway by inhibiting the angiotensin-converting enzyme activity and decreasing its mRNA production. Furthermore, treatment with either delphinidin or cyanin significantly inhibited renin mRNA production. However, delphinidin, cyanin, and quercetin did not act as the angiotensin II type 1 receptor antagonist and did not play roles in the regulation of its internalization. The direct inhibition of components of the renin-angiotensin system advances our understanding of the antihypertensive effects of these compounds.

Sustained βAR Stimulation Mediates Cardiac Insulin Resistance in a PKA-Dependent Manner

Insulin resistance is a condition in which cells are defective in response to the actions of insulin in tissue glucose uptake. Overstimulation of β-adrenergic receptors (βARs) leads to the development of heart failure and is associated with the pathogenesis of insulin resistance in the heart. However, the mechanisms by which sustained βAR stimulation affects insulin resistance in the heart are incompletely understood. In this study, we demonstrate that sustained βAR stimulation resulted in the inhibition of insulin-induced glucose uptake, and a reduction of insulin induced glucose transporter (GLUT)4 expression that were mediated by the β2AR subtype in cardiomyocytes and heart tissue. Overstimulation of β2AR inhibited the insulin-induced translocation of GLUT4 to the plasma membrane of cardiomyocytes. Additionally, βAR mediated cardiac insulin resistance by reducing glucose uptake and GLUT4 expression via the cAMP-dependent and protein kinase A-dependent pathways. Treatment with β-blockers, including propranolol and metoprolol antagonized isoproterenol-mediated insulin resistance in the heart. The data in this present study confirm a critical role for protein kinase A in βAR-mediated insulin resistance.

Myofibroblasts and inflammatory cells as players of cardiac fibrosis

On myocardial infarction, many cells are injured or died owing to arterial occlusion. Intracellular molecules released from injured or dead cells initiate inflammatory responses that play important roles in cardiac remodeling including fibrosis. Fibrosis is an excess accumulation of extracellular collagen. Currently, drugs used to treat cardiac fibrosis are not commercially available. Myofibroblasts are responsible for the production and secretion of collagen. Infiltrating inflammatory cells interact with fibroblasts or other cells and promote myofibroblast formation. Inflammatory cells also modulate the activities of myofibroblasts. Regulation of collagen production is critical for modulating the progression of fibrosis. Hence, the manipulation of activities of inflammatory cells and myofibroblasts will provide promising therapeutic targets for treatment of cardiac fibrosis.

Thai Fruits Exhibit Antioxidant Activity and Induction of Antioxidant Enzymes in HEK-293 Cells.

The cellular antioxidant enzymes play the important role of protecting the cells and organisms from the oxidative damage. Natural antioxidants contained in fruits have attracted considerable interest because of their presumed safety and potential nutritional value. Even though antioxidant activities of many fruits have been reported, the effects of phytochemicals contained in fruits on the induction of antioxidant enzymes in the cells have not been fully defined. In this study, we showed that extracts from Antidesma ghaesembilla, Averrhoa bilimbi, Malpighia glabra, Mangifera indica, Sandoricum koetjape, Syzygium malaccense, and Ziziphus jujuba inhibited H2O2-induced intracellular reactive oxygen species production in HEK-293 cells. Additionally, these Thai fruit extracts increased the mRNA and protein expressions of antioxidant enzymes, catalase, glutathione peroxidase-1, and manganese superoxide dismutase. The consumption of Thai fruits rich in phenolic compounds may reduce the risk of oxidative stress.

Phosphorylation of Src by phosphoinositide 3-kinase regulates beta-adrenergic receptor-mediated EGFR transactivation.

β2-Adrenergic receptors (β2AR) transactivate epidermal growth factor receptors (EGFR) through formation of a β2AR-EGFR complex that requires activation of Src to mediate signaling. Here, we show that both lipid and protein kinase activities of the bifunctional phosphoinositide 3-kinase (PI3K) enzyme are required for β2AR-stimulated EGFR transactivation. Mechanistically, the generation of phosphatidylinositol (3,4,5)-tris-phosphate (PIP3) by the lipid kinase function stabilizes β2AR-EGFR complexes while the protein kinase activity of PI3K regulates Src activation by direct phosphorylation. The protein kinase activity of PI3K phosphorylates serine residue 70 on Src to enhance its activity and induce EGFR transactivation following βAR stimulation. This newly identified function for PI3K, whereby Src is a substrate for the protein kinase activity of PI3K, is of importance since Src plays a key role in pathological and physiological signaling.

β-Adrenergic Receptor and Insulin Resistance in the Heart

Insulin resistance is characterized by the reduced ability of insulin to stimulate tissue uptake and disposal of glucose including cardiac muscle. These conditions accelerate the progression of heart failure and increase cardiovascular morbidity and mortality in patients with cardiovascular diseases. It is noteworthy that some conditions of insulin resistance are characterized by up-regulation of the sympathetic nervous system, resulting in enhanced stimulation of β-adrenergic receptor (βAR). Over-stimulation of βARs leads to the development of heart failure and is associated with the pathogenesis of insulin resistance in the heart. However, pathological consequences of the cross-talk between the βAR and the insulin sensitivity and the mechanism by which βAR over-stimulation promotes insulin resistance remain unclear. This review article examines the hypothesis that βARs over-stimulation leads to induction of insulin resistance in the heart.