Mohammed Nayeem

ATP-sensitive potassium channel mediates delayed ischemic protection by heat stress in rabbit heart.

Heat shock protects against myocardial ischemia-reperfusion injury possibly via increased expression of heat shock proteins. The direct evidence of heat shock protein protection in vivo remains circumstantial, and no other new mechanism of protection has been proposed. Recent studies suggest that opening of ATP-sensitive K+ channels (KATP channels) plays an important role in ischemic preconditioning; however, it is not known whether this channel is also important in delayed protection conferred by heat shock. Anesthetized rabbits underwent heat shock treatment by raising core temperature to 42°C for 15 min. Twenty-four hours later, the animals were reanesthetized and subjected to regional ischemia-reperfusion. The specific KATP channel blockers glibenclamide (0.3 mg/kg ip) and sodium 5-hydroxydecanoate (5HD; 5 mg/kg iv) were used to block the channel function. The drugs were administered at two different times, either pre-heat stress or preischemia. Infarct size was determined by triphenyltetrazolium chloride staining. The 72-kDa heat shock protein (HSP 72) was measured by Western blots. Our results show that heat shock produced a marked reduction in infarct size (39.4 ± 8.1 to 14.3 ± 2.5% of risk area, P < 0.05). Glibenclamide and 5HD completely abolished heat shock-induced reduction in infarct size (42.3 ± 0.32 and 33.7 ± 4.8%) when given before ischemia-reperfusion; however, these antagonists failed to block protection when administered before the onset of heat shock. Furthermore, the enhanced expression of HSP 72 in heat shock groups was not diminished by glibenclamide or 5HD, suggesting a lack of a direct role of this protein in conferring cardiac protection by heat shock. The complete blockade of cardiac protection by glibenclamide and 5HD strongly suggests that opening of this channel is a very important component of heat shock-induced ischemic protection in rabbit hearts.Heat shock protects against myocardial ischemia-reperfusion injury possibly via increased expression of heat shock proteins. The direct evidence of heat shock protein protection in vivo remains circumstantial, and no other new mechanism of protection has been proposed. Recent studies suggest that opening of ATP-sensitive K+ channels (KATP channels) plays an important role in ischemic preconditioning; however, it is not known whether this channel is also important in delayed protection conferred by heat shock. Anesthetized rabbits underwent heat shock treatment by raising core temperature to 42 degrees C for 15 min. Twenty-four hours later, the animals were reanesthetized and subjected to regional ischemia-reperfusion. The specific KATP channel blockers glibenclamide (0.3 mg/kg i.p.) and sodium 5-hydroxydecanoate (5HD; 5 mg/kg i.v.) were used to block the channel function. The drugs were administered at two different times, either pre-heat stress or preischemia. Infarct size was determined by triphenyltetrazolium chloride staining. The 72-kDa heat shock protein (HSP 72) was measured by Western blots. Our results show that heat shock produced a marked reduction in infarct size (39.4 +/- 8.1 to 14.3 +/- 2.5% of risk area, P < 0.05). Glibenclamide and 5HD completely abolished heat shock-induced reduction in infarct size (42.3 +/- 0.32 and 33.7 +/- 4.8%) when given before ischemia-reperfusion; however, these antagonists failed to block protection when administered before the onset of heat shock. Furthermore, the enhanced expression of HSP 72 in heat shock groups was not diminished by glibenclamide or 5HD, suggesting a lack of a direct role of this protein in conferring cardiac protection by heat shock. The complete blockade of cardiac protection by glibenclamide and 5HD strongly suggests that opening of this channel is a very important component of heat shock-induced ischemic protection in rabbit hearts.

Adenosine A1 receptors link to smooth muscle contraction via CYP4a, protein kinase C-α, and ERK1/2.

Abstract: Adenosine A1 receptor (A1AR) activation contracts smooth muscle, although signaling mechanisms are not thoroughly understood. Activation of A1AR leads to metabolism of arachidonic acid, including the production of 20-hydroxyeicosatetraenoic acid (20-HETE) by cytochrome P4504a (CYP4a). The 20-HETE can activate protein kinase C-&agr; (PKC-&agr;), which crosstalks with extracellular signal-regulated kinase (ERK1/2) pathway. Both these pathways can regulate smooth muscle contraction, we tested the hypothesis that A1AR contracts smooth muscle through a pathway involving CYP4a, PKC-&agr;, and ERK1/2. Experiments included isometric tension recordings of aortic contraction and Western blots of signaling molecules in wild type (WT) and A1AR knockout (A1KO) mice. Contraction to the A1-selective agonist 2-chloro-N cyclopentyladenosine (CCPA) was absent in A1KO mice aortae, indicating the contractile role of A1AR. Inhibition of CYP4a (HET0016) abolished 2-chloro-N cyclopentyladenosine–induced contraction in WT aortae, indicating a critical role for 20-HETE. Both WT and A1KO mice aortae contracted in response to exogenous 20-HETE. Inhibition of PKC-&agr; (Gö6976) or ERK1/2 (PD98059) attenuated 20-HETE–induced contraction equally, suggesting that ERK1/2 is downstream of PKC-&agr;. Contractions to exogenous 20-HETE were significantly less in A1KO mice; reduced protein levels of PKC-&agr;, p-ERK1/2, and total ERK1/2 supported this observation. Our data indicate that A1AR mediates smooth muscle contraction via CYP4a and a PKC-&agr;–ERK1/2 pathway.

Time course studies on the initiation of complement activation in acute myocardial infarction induced by coronary artery ligation in rats.

This study attempted to probe the role of complement activation in promoting acute myocardial infarction (AMI) induced by coronary artery ligation (CAL) in rats. The surgical technique used in this study significantly reduced early mortality (95% survival rate) and also reduced the variation in infarct size (33± 1.87%) at 32 h after surgery. Time course studies on the initiation of AMI at various time points were carried out using physiological, biochemical, histopathological and electron microscopical techniques. Serum markers and activities of lysosomal hydrolases were found to be significantly elevated at the 8th hour post ligation. Histological studies showed polymorphonuclear cells emigration and total coagulation necrosis. Transmission electron micrograph exhibited mild distortion of muscle fibres and mitochondrial rupture with disrupted cristae. Immunoblotting studies confirmed the presence of α2-macroglobulin which supported the inflammatory response at 8th h of post ligation. The initiation of the complement (C) activation was observed by the increase in the level of the soluble form of the membrane attack complex (sC5b-9) in serum and left ventricle. Immunoexpression studies confirmed the initiation of the terminal C activation as shown by the expression of C5, C6, C7, C8, C9 and sC5b-9 complex at the 8th h of AMI. This study conclusively demonstrated that initiation of the C activation was observed to be significant at the 8th h of AMI induced by CAL in rats. (Mol Cell Biochem 268: 149–158, 2005)

Time course studies on the functional evaluation of experimental chronic myocardial infarction in rats.

In vivo models of myocardial infarction induced by coronary artery ligation (CAL) in rats usually suffer from high early mortality and a low rate of induction. This study investigated the time course initiation of chronic myocardial infarction (CMI) in albino rats and the possibility of reducing early mortality rate due to myocardial infarction by modification of the surgical technique. CAL was carried out by passing the suture through the epicardial layer around the midway of the left anterior descending coronary artery including a small area of the myocardium to avoid mechanical damage to the heart geometry. In addition, the role of endothelin-1 (ET-1) in rat heart with congestive heart failure was critically assessed. Time course initiation experiments were designed by sacrificing the animals at different time intervals and by carrying out physiological, biochemical, histopathological, electron microscopical and immunohistochemical studies. Specific markers of myocardial injury, viz. cardiac troponin-T (cTnT), high sensitivity C-reactive protein, lactate dehydrogenase and fibrinogen were measured at different time points. Serum marker enzymes and activities of lysosomal hydrolases were found to be elevated on the eighth day post-ligation. Histopathological studies demonstrated focal areas showing fibrovascular tissue containing fibroblasts, collagenous ground substance and numerous small capillaries replacing cardiac muscle fibers. Transmission electron micrographs exhibited mitochondrial changes of well-developed irreversible cardiac injury, viz. swelling, disorganization of cristae, appearance of mitochondrial amorphous matrix densities, significant distortion of muscle fibers and distinct disruption of the intercalated discs. Immunoblotting studies confirmed the presence of alpha 2-macroglobulin which supported the inflammatory response. The severity of the CMI was inferred by the measurement of the level of ET-1 in plasma and left ventricle which was significantly higher in the CMI rats than in the sham-operated rats. Immunohistochemical studies at different time intervals showed that there was a significant immunoexpression of ET-1 on the eighth day post-ligation. This study conclusively showed that ligation of left anterior descending artery minimized mortality and ET-1 was expressed during CMI. (Mol Cell Biochem 267: 47–58, 2004)

High salt diet exacerbates vascular contraction in the absence of adenosine A2A receptor

Abstract: High salt (4% NaCl, HS) diet modulates adenosine-induced vascular response through adenosine A2A receptor (A2AAR). Evidence suggests that A2AAR stimulates cyp450-epoxygenases, leading to epoxyeicosatrienoic acids (EETs) generation. The aim of this study was to understand the vascular reactivity to HS and underlying signaling mechanism in the presence or absence of A2AAR. Therefore, we hypothesized that HS enhances adenosine-induced relaxation through EETs in A2AAR+/+, but exaggerates contraction in A2AAR−/−. Organ bath and Western blot experiments were conducted in HS and normal salt (NS, 0.18% NaCl)–fed A2AAR+/+ and A2AAR−/− mice aorta. HS produced concentration-dependent relaxation to non-selective adenosine analog, NECA in A2AAR+/+, whereas contraction was observed in A2AAR−/− mice and this was attenuated by A1AR antagonist (DPCPX). CGS 21680 (selective A2AAR agonist) enhanced relaxation in HS-A2AAR+/+ versus NS-A2AAR+/+, which was blocked by EETs antagonist (14,15-EEZE). Compared with NS, HS significantly upregulated the expression of vasodilators A2AAR and cyp2c29, whereas vasoconstrictors A1AR and cyp4a in A2AAR+/+ were downregulated. In A2AAR−/− mice, however, HS significantly downregulated the expression of cyp2c29, whereas A1AR and cyp4a were upregulated compared with A2AAR+/+ mice. Hence, our data suggest that in A2AAR+/+, HS enhances A2AAR-induced relaxation through increased cyp-expoxygenases–derived EETs and decreased A1AR levels, whereas in A2AAR−/−, HS exaggerates contraction through decreased cyp-epoxygenases and increased A1AR levels.

Effect of Soluble Epoxide Hydrolase on the Modulation of Coronary Reactive Hyperemia: Role of Oxylipins and PPARγ.

Coronary reactive hyperemia (CRH) is a physiological response to ischemic insult that prevents the potential harm associated with an interruption of blood supply. The relationship between the pharmacologic inhibition of soluble epoxide hydrolase (sEH) and CRH response to a brief ischemia is not known. sEH is involved in the main catabolic pathway of epoxyeicosatrienoic acids (EETs), which are converted into dihydroxyeicosatrienoic acids (DHETs). EETs protect against ischemia/reperfusion injury and have numerous beneficial physiological effects. We hypothesized that inhibition of sEH by t-AUCB enhances CRH in isolated mouse hearts through changing the oxylipin profiles, including an increase in EETs/DHETs ratio. Compared to controls, t-AUCB–treated mice had increased CRH, including repayment volume (RV), repayment duration, and repayment/debt ratio (p < 0.05). Treatment with t-AUCB significantly changed oxylipin profiles, including an increase in EET/DHET ratio, increase in EpOME/DiHOME ratio, increase in the levels of HODEs, decrease in the levels of mid-chain HETEs, and decrease in prostanoids (p < 0.05). Treatment with MS-PPOH (CYP epoxygenase inhibitor) reduced CRH, including RV (p < 0.05). Involvement of PPARγ in the modulation of CRH was demonstrated using a PPARγ-antagonist (T0070907) and a PPARγ-agonist (rosiglitazone). T0070907 reduced CRH (p < 0.05), whereas rosiglitazone enhanced CRH (p < 0.05) in isolated mouse hearts compared to the non-treated. These data demonstrate that sEH inhibition enhances, whereas CYP epoxygenases-inhibition attenuates CRH, PPARγ mediate CRH downstream of the CYP epoxygenases-EET pathway, and the changes in oxylipin profiles associated with sEH-inhibition collectively contributed to the enhanced CRH.

Angiotensin II stimulation alters vasomotor response to adenosine in mouse mesenteric artery: role for A1 and A2B adenosine receptors.

Stimulation of the A1 adenosine receptor and angiotensin II receptor type‐1 (AT1 receptor) causes vasoconstriction through activation of cytochrome P450 4A (CYP4A) and ERK1/2. Thus, we hypothesized that acute angiotensin II activation alters the vasomotor response induced by the non‐selective adenosine receptor agonist, NECA, in mouse mesenteric arteries (MAs).

Adenosine A1 receptor signaling inhibits BK channels through a PKCα‐dependent mechanism in mouse aortic smooth muscle

Adenosine receptors (AR; A1, A2A, A2B, and A3) contract and relax smooth muscle through different signaling mechanisms. Deciphering these complex responses remains difficult because relationships between AR subtypes and various end‐effectors (e.g., enzymes and ion channels) remain to be identified. A1AR stimulation is associated with the production of 20–hydroxyeicosatetraenoic acid (20–HETE) and activation of protein kinase C (PKC). 20–HETE and PKC can inhibit large conductance Ca2+/voltage‐sensitive K+ (BK) channels that regulate smooth muscle contraction. We tested the hypothesis that activation of A1AR inhibits BK channels via a PKC‐dependent mechanism. Patch clamp recordings and Western blots were performed using aortae of wild type (WT) and A1AR knockout (A1KO) mice. There were no differences in whole‐cell K+ current or α and β1 subunits expression between WT and A1KO. 20–HETE (100 nmol/L) inhibited BK current similarly in WT and A1KO mice. NECA (5′–N–ethylcarboxamidoadenosine; 10 μmol/L), a nonselective AR agonist, increased BK current in myocytes from both WT and A1KO mice, but the increase was greater in A1KO (52 ± 15 vs. 17 ± 3%; P < 0.05). This suggests that A1AR signaling negatively regulates BK channel activity. Accordingly, CCPA (2–chloro–N(6)‐cyclopentyladenosine; 100 nmol/L), an A1AR‐selective agonist, inhibited BK current in myocytes from WT but not A1KO mice (81 ± 4 vs. 100 ± 7% of control; P < 0.05). Gö6976 (100 nmol/L), a PKCα inhibitor, abolished the effect of CCPA to inhibit BK current (99 ± 3% of control). These data lead us to conclude that, in aortic smooth muscle, A1AR inhibits BK channel activity and that this occurs via a mechanism involving PKCα.

Exploring Adenosine Receptor Ligands: Potential Role in the Treatment of Cardiovascular Diseases

Cardiovascular diseases remain the number one diseases affecting patients’ morbidity and mortality. The adenosine receptors are G-protein coupled receptors which have been of interest for drugs target for the treatment of multiple diseases ranging from cardiovascular to neurological. Adenosine receptors have been connected to several biological pathways affecting the physiology and pathology of the cardiovascular system. In this review, we will cover the different adenosine receptor ligands that have been identified to interact with adenosine receptors and affect the vascular system. These ligands will be evaluated from clinical as well as medicinal chemistry perspectives with more emphasis on how structural changes in structure translate into ligand potency and efficacy. Adenosine receptors represent a novel therapeutic target for development of treatment options treating a wide variety of diseases, including vascular disease and obesity.

Deletion of soluble epoxide hydrolase enhances coronary reactive hyperemia in isolated mouse heart: role of oxylipins and PPARγ

The relationship between soluble epoxide hydrolase (sEH) and coronary reactive hyperemia (CRH) response to a brief ischemic insult is not known. Epoxyeicosatrienoic acids (EETs) exert cardioprotective effects in ischemia/reperfusion injury. sEH converts EETs into dihydroxyeicosatrienoic-acids (DHETs). Therefore, we hypothesized that knocking out sEH enhances CRH through modulation of oxylipin profiles, including an increase in EET/DHET ratio. Compared with sEH+/+, sEH-/- mice showed enhanced CRH, including greater repayment volume (RV; 28% higher, P < 0.001) and repayment/debt ratio (32% higher, P < 0.001). Oxylipins from the heart perfusates were analyzed by LC-MS/MS. The 14,15-EET/14,15-DHET ratio was 3.7-fold higher at baseline (P < 0.001) and 5.6-fold higher post-ischemia (P < 0.001) in sEH-/- compared with sEH+/+ mice. Likewise, the baseline 9,10- and 12,13-EpOME/DiHOME ratios were 3.2-fold (P < 0.01) and 3.7-fold (P < 0.001) higher, respectively in sEH-/- compared with sEH+/+ mice. 13-HODE was also significantly increased at baseline by 71% (P < 0.01) in sEH-/- vs. sEH+/+ mice. Levels of 5-, 11-, 12-, and 15-hydroxyeicosatetraenoic acids were not significantly different between the two strains (P > 0.05), but were decreased postischemia in both groups (P = 0.02, P = 0.04, P = 0.05, P = 0.03, respectively). Modulation of CRH by peroxisome proliferator-activated receptor gamma (PPARγ) was demonstrated using a PPARγ-antagonist (T0070907), which reduced repayment volume by 25% in sEH+/+ (P < 0.001) and 33% in sEH-/- mice (P < 0.01), and a PPARγ-agonist (rosiglitazone), which increased repayment volume by 37% in both sEH+/+ (P = 0.04) and sEH-/- mice (P = 0.04). l-NAME attenuated CRH in both sEH-/- and sEH+/+ These data demonstrate that genetic deletion of sEH resulted in an altered oxylipin profile, which may have led to an enhanced CRH response.