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.

Etanercept restores vasocontractile sensitivity affected by mesenteric ischemia reperfusion.

BACKGROUND: The aim of the study is to evaluate in vivo and in vitro effects of etanercept, a soluble tumor necrosis factor receptor, on the contractile responses of superior mesenteric artery in an experimental mesenteric ischemia and reperfusion model. MATERIAL AND METHODS: After obtaining animal ethics committee approval, 24 Sprague-Dawley rats were allocated to three groups. Control group (Gr C, n = 6) underwent a sham operation, whereas ischemia/reperfusion and treatment groups underwent 90 min ischemia and 24-h reperfusion (Gr I/R, n = 12; Gr I/R+E, n = 6). The treatment group received 5 mg/kg etanercept intravenously at the beginning of reperfusion. At the end of reperfusion, all animals were sacrificed, and third branch of superior mesenteric artery was dissected for evaluation of contractile responses. In vitro effects of etanercept on vasocontractile responses were also evaluated. The excised ileums were analyzed under light microscope. Two-way analysis of variance following Bonferroni post hoc test was used for evaluation of contractile responses. RESULTS: Endothelin-1 and phenylephrine-mediated vasocontractile sensitivity were found increased in Gr I/R when compared with Gr C. Both intravenous administration and organ bath incubation of etanercept decreased the sensitivity of contractile agents for Gr I/R. Mucosal injury, lamina propria disintegration, and denuded villous tips were observed in Gr I/R, whereas the epithelial injury and the subepithelial edema were found to be milder in Gr I/R+E. CONCLUSIONS: Etanercept can be a promising agent in mesenteric ischemic reperfusion injury as it does not only inhibit inflammation by blocking tumor necrosis factor-α in circulation but also restores vascular contractility during reflow. These findings support an unexplained recuperative effect of drug beyond its anti-inflammatory effects.

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.

Role of beta-3-adrenoceptor in catecholamine-induced relaxations in gastric fundus from control and diabetic rats.

The contribution of beta-adrenoceptor subtypes to the catecholamine-mediated relaxations in gastric fundus from control and streptozotocin (STZ)-induced diabetic rats were investigated. Isolated organ bath studies and molecular techniques were used to characterize the beta-adrenoceptor subtypes mediating relaxation of rat gastric fundus. Isoprenaline-mediated relaxation was not significantly changed by nadolol (beta(1)-/beta(2)-adrenoceptor antagonist; 1 micromol/l) but only shifted to the right by SR59230A (3-(2-ethylphenoxy)-1-[[(1S)-1,2,3,4-tetrahydronaphth-1-yl]amino]-(2S)-2-propanol oxalate salt, 0.1-1 micromol/l), a selective beta(3)-adrenoceptor antagonist, in a competitive manner. Relaxant responses to noradrenaline were antagonized in a concentration-dependent manner by SR59230A (0.1-1 micromol/l), but not by metoprolol (selective beta(1)-adrenoceptor antagonist; 0.1-1 micromol/l) and ICI-118551 (1-[2,3-(dihydro-7-methyl-1Hinden- 4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol hydrochloride, selective beta(2)-adrenoceptor antagonist; 0.1-1 micromol/l). SR59230A (1 micromol/l) also caused a significant rightward shift in fenoterol-induced relaxation while ICI-118551 (1 micromol/l) did not have any effect. Selective beta(3)-adrenoceptor agonist, BRL37344 ([4-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]phenoxy]acetic acid), caused biphasic relaxation which was not affected by nadolol (1 micromol/l). SR59230A (1 micromol/l) abolished only the first phase of BRL37344 response. beta(1)-, beta(2)- and beta(3)-adrenoceptor mRNA expressions have been detected in a similar intensity in gastric fundus from control rats. Experimental diabetes caused a significant decrease in E(max) and pD(2) values of isoprenaline and noradrenaline. Diabetes also reduced E(max) but not pD(2) value of the first component of BRL37344-induced relaxation response. The band intensity of mRNA transcript of beta(3)-adrenoceptor was reduced in diabetics while no alteration has been found for beta(1)- and beta(2)-adrenoceptor mRNA transcripts between groups. These results show that functional beta-adrenoceptor subtype involved in catecholamine-mediated relaxations is beta(3)-adrenoceptor, and its function and mRNA expression are decreased in diabetes.

The effects of chronic trimetazidine treatment on mechanical function and fatty acid oxidation in diabetic rat hearts.

Clinical and experimental evidence suggest that increased rates of fatty acid oxidation in the myocardium result in impaired contractile function in both normal and diabetic hearts. Glucose utilization is decreased in type 1 diabetes, and fatty acid oxidation dominates for energy production at the expense of an increase in oxygen requirement. The objective of this study was to examine the effect of chronic treatment with trimetazidine (TMZ) on cardiac mechanical function and fatty acid oxidation in streptozocin (STZ)-diabetic rats. Spontaneously beating hearts from male Sprague-Dawley rats were subjected to a 60-minute aerobic perfusion period with a recirculating Krebs-Henseleit solution containing 11 mmol/L glucose, 100 muU/mL insulin, and 0.8 mmol/L palmitate prebound to 3% bovine serum albumin (BSA). Mechanical function of the hearts, as cardiac output x heart rate (in (mL/min).(beats/min).10-2), was deteriorated in diabetic (73 +/- 4) and TMZ-treated diabetic (61 +/- 7) groups compared with control (119 +/- 3) and TMZ-treated controls (131 +/- 6). TMZ treatment increased coronary flow in TMZ-treated control (23 +/- 1 mL/min) hearts compared with untreated controls (18 +/- 1 mL/min). The mRNA expression of 3-ketoacyl-CoA thiolase (3-KAT) was increased in diabetic hearts. The inhibitory effect of TMZ on fatty acid oxidation was not detected at 0.8 mmol/L palmitate in the perfusate. Addition of 1 mumol/L TMZ 30 min into the perfusion did not affect fatty acid oxidation rates, cardiac work, or coronary flow. Our results suggest that higher expression of 3-KAT in diabetic rats might require increased concentrations of TMZ for the inhibitory effect on fatty acid oxidation. A detailed kinetic analysis of 3-KAT using different concentrations of fatty acid will determine the fatty acid inhibitory concentration of TMZ in diabetic state where plasma fatty acid levels are increased.

The influence of diabetes on cardiac beta-adrenoceptor subtypes.

Despite the significant developments in the treatment of diabetes mellitus, diabetic patients still continue to suffer from cardiac complications. The increase of cardiac adrenergic drive may ultimately contribute to the development and progression of diabetic cardiomyopathy. beta-Adrenoceptors play an important role in the regulation of heart function. However, responsiveness of diabetic heart to beta-adrenoceptor agonist stimulation is diminished. The chronotropic responses mediated by beta(1)-subtype, which is mainly responsible for cardiac effects of catecholamines are decreased in the atria of diabetic rats. The expression of cardiac beta(1)-subtype is significantly decreased in diabetic rats as well. beta(2)-Adrenoceptors also increase cardiac function. Although the expression of this subtype is slightly decreased in diabetic rat hearts, beta(2)-mediated chronotropic responses are preserved. On the other hand, functional beta(3)-adrenoceptor subtype was characterized in human heart. Interestingly, stimulation of cardiac beta(3)-adrenoceptors, on the contrary of beta(1)- and beta(2)-subtypes, mediates negative inotropic effect in human ventricular muscle. Cardiac beta(3)-adrenoceptors are upregulated in experimental diabetes as well as in human heart failure. These findings suggest that each beta-adrenoceptor subtype may play an important role in the pathophysiology of diabetes-induced heart disease. However, it is still not known whether the changes in the expression and/or responsiveness of beta-adrenoceptors are adaptive or maladaptive. Therefore, this review outlines the potential roles of these receptor subtypes in cardiac pathologies of diabetes.

Oral administration of liposomal insulin.

There have been several attempts published in the literature related with orally effective insulin formulations, which are increasing in popularity. Some of the results indicate that it is possible to reduce blood glucose level by orally administered liposomal insulin formulations, but there is general need to understand the mechanism and effective components of the liposome formulations. In our study, liposomal insulin formulations were prepared using insulin (Humulin R) or protamine- containing insulin (Humulin N) with cholesterol, dipalmitoyl phosphatidylcholine (egg) (DPPC)-cholesterol mixture, and mucoadhesive agent (methyl cellulose, MC)-added DPPC-cholesterol mixture. A tablet formulation of insulin was also prepared. Formulations of liposomal insulin were introduced to mice and rats orally and reduced blood glucose levels were observed. The composition of phospholipid (DPPC, cholesterol and MC mixture) was found to be quite effective in reducing blood glucose levels. The pH of the solution and the presence of the protamine sulfate were found to be important. The application site was also found to be important because liposomal insulin formulations administered through the mouth or esophagus resulted in reduced blood glucose levels. Reduced blood glucose levels were also observed when tablet formulations of insulin were administered to rats orally.

Diabetes decreases mRNA levels of calcium-release channels in human atrial appendage.

Patients with chronic diabetes mellitus usually develop reductions in rate and force of cardiac contractions. Since calcium-release channels (ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP(3)Rs)) play integral roles in effecting these processes, we rationalize that alterations in their expression may underlie these defects. To test this hypothesis, right atrial appendages were obtained from diabetic (65.0 +/- 4.5 years) and nondiabetic (56.2 +/- 2.6 years) patients undergoing coronary arterial by-pass grafting and reverse transcription-polymerase chain reactions were used to compare steady state levels of mRNA encoding the three major isoforms of RyRs and IP(3)Rs. In this study we did not detect either RyR1 or RyR3 in human atrial appendage. When compared with nondiabetic patients, mRNA encoding RyR2 from diabetic patients decreased by 74.2 +/- 6.2% (p < 0.01). Diabetes also significantly decreased steady-state levels of mRNA encoding the IP(3)Rs in human atrial appendage. IP(3)R1 decreased by 24.2 +/- 4.6%, IP(3)R2 decreased by 63.0 +/- 4.6% and IP(3)R3 decreased by 55.5 +/- 6.5%. Since a reduction in steady-state mRNA is usually indicative of a decrease in protein levels, these data suggest that the decrease in chronotropy and inotropy seen in chronic diabetic patients may be due in part to a decrease in expression of calcium-release channels.

Diabetes decreases mRNA levels of calcium-release channels in human atrial appendage.

Patients with chronic diabetes mellitus usually develop reductions in rate and force of cardiac contractions. Since calcium-release channels (ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP3Rs)) play integral roles in effecting these processes, we rationalize that alterations in their expression may underlie these defects. To test this hypothesis, right atrial appendages were obtained from diabetic (65.0 ± 4.5 years) and nondiabetic (56.2 ± 2.6 years) patients undergoing coronary arterial by-pass grafting and reverse transcription-polymerase chain reactions were used to compare steady state levels of mRNA encoding the three major isoforms of RyRs and IP3Rs. In this study we did not detect either RyR1 or RyR3 in human atrial appendage. When compared with nondiabetic patients, mRNA encoding RyR2 from diabetic patients decreased by 74.2 ± 6.2% (p< 0.01). Diabetes also significantly decreased steady-state levels of mRNA encoding the IP3Rs in human atrial appendage. IP3R1 decreased by 24.2 ± 4.6%, IP3R2 decreased by 63.0 ± 4.6% and IP3R3 decreased by 55.5 ± 6.5%. Since a reduction in steady-state mRNA is usually indicative of a decrease in protein levels, these data suggest that the decrease in chronotropy and inotropy seen in chronic diabetic patients may be due in part to a decrease in expression of calcium-release channels. (Mol Cell Biochem 263: 143-150, 2004).

Decreased expression of beta1- and beta2-adrenoceptors in human diabetic atrial appendage.

BACKGROUND: Using the streptozotocin-induced diabetic rat model, we have recently showed that the expression and function of beta1-adrenoreceptor were decreased in the diabetic rat heart. However, the effect of diabetes on expression of beta-adrenoreceptors in human cardiac tissue remains undefined. Therefore, the focus of the present study was to investigate the effect of diabetes on mRNA encoding beta1- and beta2-ARs in human atrial tissues. METHODS: Right atrial appendages from five diabetic (mean age 65 +/- 4.5; 4 female, 1 male) and five nondiabetic patients (mean age 56.2 +/- 2.8; 4 male, 1 female) undergoing coronary artery bypass grafting were collected and assayed using reverse transcriptase-polymerase chain reaction (RT-PCR) for their mRNA content. No patient from these two groups suffered from acute myocardial infarction and/or failure. All diabetic patients received insulin for at least two years and had been diagnosed as diabetics for at least five years. RESULTS: When compared with levels in nondiabetics, steady state levels of mRNA encoding beta1-adrenoreceptor decreased by 69.2 +/- 7.6% in diabetic patients while beta2-adrenoreceptor mRNA decreased by 32.2 +/- 5.5% (p < 0.001). CONCLUSIONS: Our findings show a decreased expression of beta1- and beta2-adrenoreceptors in human diabetic atrial appendage.