Attenuation of apoptosis in vitro and ischemia/reperfusion injury in vivo in mouse skeletal muscle by P2Y6 receptor activation.

Activation of the G(q)-coupled P2Y(6) receptor heterologously expressed in astrocytes significantly attenuates apoptosis induced by tumor necrosis factor alpha (TNFalpha). We have extended the analysis of P2Y(6) receptor-induced cytoprotection to mouse skeletal muscle cells endogenously expressing this receptor. The endogenous P2Y(6) receptor agonist UDP and synthetic agonist MRS2693 protected C2C12 skeletal muscle cells against apoptosis in a concentration-dependent manner (0.1-10 nM) as determined by propidium iodide staining, histochemical analysis using hematoxylin and Hoechst 33258, and DNA fragmentation. The insurmountable P2Y(6) receptor antagonist MRS2578 blocked the protection. TNFalpha-induced apoptosis in C2C12 cells correlated with activation of the transcription factor NF-kappaB. The NF-kappaB activation was attenuated by 10nM MRS2693, which activated the antiapoptic ERK1/2 pathway. In an in vivo mouse hindlimb model, MRS2693 protected against skeletal muscle ischemia/reperfusion injury. The P2Y(6) receptor is a novel cytoprotective receptor that deserves further exploration in ameliorating skeletal muscle injury.

Regulation of death and survival in astrocytes by ADP activating P2Y1 and P2Y12 receptors.

ADP is the endogenous agonist for both P2Y(1) and P2Y(12) receptors, which are important therapeutic targets. It was previously demonstrated that ADP and a synthetic agonist, 2-methylthioadenosine 5'-diphosphate (2MeSADP), can induce apoptosis by activating the human P2Y(1) receptor heterologously expressed in astrocytoma cells. However, it was not known whether the P2Y(12) receptor behaved similarly. We demonstrated here that, unlike with the G(q)-coupled P2Y(1) receptor, activation of the G(i)-coupled P2Y(12) receptor does not induce apoptosis. Furthermore, activation of the P2Y(12) receptor by either ADP or 2MeSADP significantly attenuates the tumor necrosis factor alpha (TNFalpha)-induced apoptosis in 1321N1 human astrocytoma cells. This protective effect was blocked by the P2Y(12) receptor antagonist 2-methylthioAMP and by inhibitors of phospholipase C (U73122) and protein kinase C (chelerythrin), but not by the P2Y(1) receptor antagonist MRS2179. Toward a greater mechanistic understanding, we showed that hP2Y(12) receptor activation by 10nM 2MeSADP, activates Erk1/2, Akt, and JNK by phosphorylation. However, at a lower protective concentration of 100pM 2MeSADP, activation of the hP2Y(12) receptor involves only phosphorylated Erk1/2, but not Akt or JNK. This activation is hypothesized as the major mechanism for the protective effect induced by P2Y(12) receptor activation. Apyrase did not affect the ability of TNFalpha to induce apoptosis in hP2Y(12)-1321N1 cells, suggesting that the endogenous nucleotides are not involved. These results may have important implications for understanding the signaling cascades that follow activation of P2Y(1) and P2Y(12) receptors and their opposing effects on cell death pathways.

Leptin protects the cardiac myocyte cultures from hypoxic damage.

Leptin, a circulating hormone mainly produced by adipose tissue, regulates fatty acid metabolism and causes multiple systemic biological actions even the regulation of cardiovascular function. It is previously known that leptin is a hypoxia-inducible hormone, that hypoxic conditions increase the expression of this peptide in various tissues such as placenta, pancreas and also in the heart. Since leptin receptors are present in the heart, we hypothesized that whether leptin was a protector response for tissues especially for the heart against the deleterious effects of hypoxia. Cultured cardiomyocytes from newborn rats were initially treated with 3000 ng/ml leptin incubation for 1, 5 and 20 h separately, then subjected to 120 min of hypoxia. Hypoxic damage of myocytes was assayed using the measurements of both lactate dehydrogenase and creatine kinase releases into the medium and performing morphological observations (ultrastructural and immunocytochemical) of plates. The obtained results from leptin treated and non-treated control groups were compared to each other, and these data have demonstrated that 5 h of leptin treatment before hypoxia provides a significant protection for cardiomyocytes against hypoxia. Neither 1- nor 20-h leptin treated groups exhibited sufficient protection against hypoxia. In conclusion, leptin protects the cardiomyocyte cultures from hypoxia, but this effect is selective and evident only in the 5-h treated myocytes.

NUCLEOSIDE PRODRUGS OF A3 ADENOSINE RECEPTOR AGONISTS AND ANTAGONISTS.

9-(ß-D-Ribosfuranosyluronamide)adenine derivatives that are selective agonists and antagonists of the A3 adenosine receptor (AR) have been derivatized as prodrugs for in vivo delivery. The free hydroxy groups at the 2' and 3' positions of the agonists 2-chloro-N 6-(3-iodobenzyl)-9-(N-methyl-(ß-D-ribosfuranosyluronamide)adenine 2b, the corresponding 4'-thio nucleoside 2c, and antagonists 4a and 4b (5'-N,N-dimethylamides related to 2b and 2c, respectively) were derivatized through simple acylation reactions. The prodrug derivatives were tested in radioligand binding assays at ARs and in a functional assay of adenylate cyclase at the A3AR and found to be considerably less active than the parent drugs. The hydrolysis of nucleoside 2',3'-diesters to regenerate the parent compound in the presence of human blood was demonstrated. 2',3'-Dipropionate esters of 2b and 4a were readily cleaved in a two-step reaction to regenerate the parent drug, on a time scale of two hours. The cleavage of a 2',3'-dihexanoate ester occurred at a slower rate. This indicates that the prodrugs are suitable as masked forms of the biologically active A3AR agonists and antagonists for future evaluation in vivo.

Role of adenosine A1 and A3 receptors in regulation of cardiomyocyte homeostasis after mitochondrial respiratory chain injury.

Activation of either the A(1) or the A(3) adenosine receptor (A(1)R or A(3)R, respectively) elicits delayed cardioprotection against infarction, ischemia, and hypoxia. Mitochondrial contribution to the progression of cardiomyocyte injury is well known; however, the protective effects of adenosine receptor activation in cardiac cells with a respiratory chain deficiency are poorly elucidated. The aim of our study was to further define the role of A(1)R and A(3)R activation on functional tolerance after inhibition of the terminal link of the mitochondrial respiratory chain with sodium azide, in a state of normoxia or hypoxia, compared with the effects of the mitochondrial ATP-sensitive K(+) channel opener diazoxide. Treatment with 10 mM sodium azide for 2 h in normoxia caused a considerable decrease in the total ATP level; however, activation of adenosine receptors significantly attenuated this decrease. Diazoxide (100 muM) was less effective in protection. During treatment of cultured cardiomyocytes with hypoxia in the presence of 1 mM sodium azide, the A(1)R agonist 2-chloro-N(6)-cyclopentyladenosine was ineffective, whereas the A(3)R agonist 2-chloro-N(6)-iodobenzyl-5'-N-methylcarboxamidoadenosine (Cl-IB-MECA) attenuated the decrease in ATP level and prevented cell injury. Cl-IB-MECA delayed the dissipation in the mitochondrial membrane potential during hypoxia in cells impaired in the mitochondrial respiratory chain. In cells with elevated intracellular Ca(2+) concentration after hypoxia and treatment with NaN(3) or after application of high doses of NaN(3), Cl-IB-MECA immediately decreased the elevated intracellular Ca(2+) concentration toward the diastolic control level. The A(1)R agonist was ineffective. This may be especially important for the development of effective pharmacological agents, because mitochondrial dysfunction is a leading factor in the pathophysiological cascade of heart disease.

Enhanced connexin-43 and alpha-sarcomeric actin expression in cultured heart myocytes exposed to triiodo-L-thyronine.

This study examined whether triiodo-L-thyronine (T3) affects the expression of the major intercellular channel protein, connexin-43, and contractile protein alpha-sarcomeric actin. Cultured cardiomyocytes from newborn rats were treated on day three in culture with 10 or 100 nM T3 and examined 48 and 72 h thereafter. Treated and untreated cells were examined by immunofluorescence and electron microscopy. Expression levels of Cx43 and sarcomeric alpha-actin were monitored by Western blot analysis. Immunofluorescence labeling showed cell membrane location of Cx43 in punctuate gap junctions, whereby fluorescence signal area was significantly higher in cultured cardiomyocytes exposed to T3. This correlated with electron microscopical findings showing increased numbers and size of gap junction profiles, as well as with a significant dose-dependent increase of Cx43 expression detected by Western blot. Immunofluorescence of sarcomeric a-actin was enhanced and its expression increased dose- and time-dependently in T3-treated cultured heart myocytes. However, exposure to the higher dosage (100 nM) of T3 caused mild disintegration of sarcomeric a-actin in some myocytes, suggesting an over-dosage. The results indicate that T3 up-regulates Cx43 and accelerates gap junction formation in cultured neonatal cardiomyocytes. They suggest that thyroid status cannot only modulate the mechanical function of cardiomyocytes but also cell-to-cell communication essential for myocardial electrical and metabolic synchronizations.

2-Substituted adenosine derivatives: affinity and efficacy at four subtypes of human adenosine receptors.

The affinity and efficacy at four subtypes (A(1), A(2A), A(2B) and A(3)) of human adenosine receptors (ARs) of a wide range of 2-substituted adenosine derivatives were evaluated using radioligand binding assays and a cyclic AMP functional assay in intact CHO cells stably expressing these receptors. Similar to previous studies of the N(6)-position, several 2-substituents were found to be critical structural determinants for the A(3)AR activation. The following adenosine 2-ethers were moderately potent partial agonists (K(i), nM): benzyl (117), 3-chlorobenzyl (72), 2-(3-chlorophenyl)ethyl (41), and 2-(2-naphthyl)ethyl (130). The following adenosine 2-ethers were A(3)AR antagonists: 2,2-diphenylethyl, 2-(2-norbornan)ethyl, R- and S-2-phenylbutyl, and 2-(2-chlorophenyl)ethyl. 2-(S-2-Phenylbutyloxy)adenosine as an A(3)AR antagonist right-shifted the concentration-response curve for the inhibition by NECA of cyclic AMP accumulation with a K(B) value of 212 nM, which is similar to its binding affinity (K(i) = 175 nM). These 2-substituted adenosine derivatives were generally less potent at the A(1)AR in comparison to the A(3)AR, but fully efficacious, with binding K(i) values over 100 nM. The 2-phenylethyl moiety resulted in higher A(3)AR affinity (K(i) in nM) when linked to the 2-position of adenosine through an ether group (54), than when linked through an amine (310) or thioether (1960). 2-[2-(l-Naphthyl)ethyloxy]adenosine (K(i) = 3.8 nM) was found to be the most potent and selective (>50-fold) A(2A) agonist in this series. Mixed A(2A)/A(3)AR agonists have been identified. Interestingly, although most of these compounds were extremely weak at the A(2B)AR, 2-[2-(2-naphthyl)ethyloxy]adenosine (EC(50) = 1.4 microM) and 2-[2-(2-thienyl)-ethyloxy]adenosine (EC(50) = 1.8 microM) were found to be relatively potent A(2B) agonists, although less potent than NECA (EC(50) = 140 nM).

Diisothiocyanate derivatives as potent, insurmountable antagonists of P2Y6 nucleotide receptors.

The physiological role of the P2Y(6) nucleotide receptor may involve cardiovascular, immune and digestive functions based on the receptor tissue distribution, and selective antagonists for this receptor are lacking. We have synthesized a series of symmetric aryl diisothiocyanate derivatives and examined their ability to inhibit phospholipase C (PLC) activity induced by activation of five subtypes of recombinant P2Y receptors. Several derivatives were more potent at inhibiting action of UDP at both human and rat P2Y(6) receptors expressed in 1321N1 human astrocytes than activation of human P2Y(1), P2Y(2), P2Y(4) and P2Y(11) receptors. The inhibition by diisothiocyanate derivatives of 1,2-diphenylethane (MRS2567) and 1,4-di-(phenylthioureido) butane (MRS2578) was concentration-dependent and insurmountable, with IC(50) values of 126+/-15 nM and 37+/-16 nM (human) and 101+/-27 nM and 98+/-11 nM (rat), respectively. A derivative of 1,4-phenylendiisothiocyanate (MRS2575) inhibited only human but not rat P2Y(6) receptor activity. MRS2567 and MRS2578 at 10microM did not affect the UTP (100nM)-induced responses of cells expressing P2Y(2) and P2Y(4) receptors, nor did they affect the 2-methylthio-ADP (30nM)-induced responses at the P2Y(1) receptor or the ATP (10microM)-induced responses at the P2Y(11) receptor. Other antagonists displayed mixed selectivities. The selective antagonists MRS2567, MRS2575 and MRS2578 (1microM) completely blocked the protection by UDP of cells undergoing TNFalpha-induced apoptosis. Thus, we have identified potent, insurmountable antagonists of P2Y(6) receptors that are selective within the family of PLC-coupled P2Y receptors.

Glycogen metabolism in rat heart muscle cultures after hypoxia.

Elevated glycogen levels in heart have been shown to have cardioprotective effects against ischemic injury. We have therefore established a model for elevating glycogen content in primary rat cardiac cells grown in culture and examined potential mechanisms for the elevation (glycogen supercompensation). Glycogen was depleted by exposing the cells to hypoxia for 2 h in the absence of glucose in the medium. This was followed by incubating the cells with 28 mM glucose in normoxia for up to 120 h. Hypoxia decreased glycogen content to about 15% of control, oxygenated cells. This was followed by a continuous increase in glycogen in the hypoxia treated cells during the 120 h recovery period in normoxia. By 48 h after termination of hypoxia, the glycogen content had returned to baseline levels and by 120 h glycogen was about 150% of control. The increase in glycogen at 120 h was associated with comparable relative increases in glucose uptake (approximately 180% of control) and the protein level of the glut-1 transporter (approximately 170% of control), whereas the protein level of the glut-4 transporter was decreased to < 10% of control. By 120 h, the hypoxia-treated cells also exhibited marked increases in the total (approximately 170% of control) and fractional activity of glycogen synthase (control, approximately 15%; hypoxia-treated, approximately 30%). Concomitantly, the hypoxia-treated cells also exhibited marked decreases in the total (approximately 50% of control) and fractional activity of glycogen phosphorylase (control, approximately 50%; hypoxia-treated, approximately 25%). Thus, we have established a model of glycogen supercompensation in cultures of cardiac cells that is explained by concerted increases in glucose uptake and glycogen synthase activity and decreases in phosphorylase activity. This model should prove useful in studying the cardioprotective effects of glycogen.

Mechanism of glycogen supercompensation in rat skeletal muscle cultures.

A model to study glycogen supercompensation (the significant increase in glycogen content above basal level) in primary rat skeletal muscle culture was established. Glycogen was completely depleted in differentiated myotubes by 2 h of electrical stimulation or exposure to hypoxia during incubation in medium devoid of glucose. Thereafter, cells were incubated in medium containing glucose, and glycogen supercompensation was clearly observed in treated myotubes after 72 h. Peak glycogen levels were obtained after 120 h, averaging 2.5 and 4 fold above control values in the stimulated- and hypoxia-treated cells, respectively. Glycogen synthase activity increased and phosphorylase activity decreased continuously during 120 h of recovery in the treated cells. Rates of 2-deoxyglucose uptake were significantly elevated in the treated cells at 96 and 120 h, averaging 1.4-2 fold above control values. Glycogenin content increased slightly in the treated cells after 48 h (1.2 fold vs. control) and then increased considerably, achieving peak values after 120 h (2 fold vs. control). The results demonstrate two phases of glycogen supercompensation: the first phase depends primarily on activation of glycogen synthase and inactivation of phosphorylase; the second phase includes increases in glucose uptake and glycogenin level.

Cardiomyocyte resistance to doxorubicin mediated by A(3) adenosine receptor.

Recently, we reported that the activation of A(3) adenosine receptor (A(3)R) in newborn cultured cardiomyocytes by highly selective agonist Cl-IB-MECA (2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide) induces protection against the anthracycline antibiotic doxorubicin (DOX) cardiotoxicity. The present study was undertaken to further characterize the cardioprotective action of A(3)R activation by revealing the structural changes in cardiomyocytes elicited upon exposure to DOX. Morphological observations (ultrastructural and immunocytochemical) indicate that after DOX treatment, the cardiomyocytes undergo destructive alterations, and protective action of A(3)R is not connected with its anti-apoptotic activity. A(3)R activation appeared to prevent destructive alterations of cardiomyocyte mitochondria and dissipation of mitochondrial membrane potential. In DOX-treated cardiomyocytes, appearance of disorganized desmin and contractile filaments was related to detrimental alterations in the mitochondrial structure, in particular their position and transmembrane potential. In intact cardiomyocytes, diazoxide, a selective mitochondrial K(ATP) channel opener, induced an increase in ATP synthesis within 15 min of application. Similar effect was obtained by activation of adenosine A(1)R. However, A(3)R agonist Cl-IB-MECA did not affect ATP synthesis. Neither A(1)R agonist CCPA (2-chloro-N(6)-cyclopentyladenosine) nor diazoxide protected cardiomyocytes from the detrimental effects of DOX. Thus, the opening of mitochondrial K(ATP) channels does not seem to be effective during the slow development of anthracycline cytotoxicity. Our results indicate that DOX increases the activity of lysosomes, which may contribute to cell injury in an "oncotic" manner and also demonstrate the proinflammatory potency of the drug. Furthermore, the decreased acidification of cytoplasm upon activation of A(3)R may attenuate the ongoing inflammatory response. The present study identifies a novel role for A(3)R selective agonist Cl-IB-MECA and suggests its importance in regulating cardiac cellular function.

Insights into the cardioprotective function of adenosine A(1) and A(3) receptors.

OBJECTIVES: Cardioprotection (delaying of irreversible damage in hypoxia or prevention of doxorubicin [DOX] toxicity) is achieved by increasing the energy supply, or decreasing the energy demand in the cell and may be regulated through adenosine (ADO) receptor (AR) signalling. The aim of this study was to define of the protective role of ADO A(1)R and A(3)R against these two different kinds of stress conditions via direct action on isolated cardiomyocytes. Effects of A(1) and A(3) adenosine receptors were assessed by comparing morphological-functional tolerance, cellular energy state and contribution of the mitochondrial K(ATP) channels during development of hypoxia and DOX cytotoxicity. METHODS: The primary cardiac myocyte cultures were treated in a hypoxic chamber of N(2) (100%) in glucose-free media. A second group of cells were treated on day 4 in culture with 0.5 to 5 muM DOX for 18 h and then incubated in drug-free growth medium for an additional 24 h or 72 h. The hypoxic and cytotoxic damage was characterized by morphological and biochemical evaluations. RESULTS: The A(1)R and A(3)R selective agonists (CCPA and Cl-IB-MECA, respectively) significantly decreased damage to cardiac myocytes under hypoxic conditions. Activation of both A(1)R and A(3)R together (100 nM) was more efficient in protection against hypoxia than by each one alone. The A(3)R agonist Cl-IB-MECA (100 nM) shows cardioprotective activity to the DOX-treated cells; however, the A(1)R agonist CCPA (10 nM to 10 muM) was not effective in protection against DOX toxicity. CONCLUSION: Activation of both the ADO receptors (A(1)R and A(3)R) leads to positive beneficial effects in cultured cardiomyocytes in 90 min hypoxia, but only A(3)R activation renders positive response against slowly developed DOX toxicity. Hence, the cascade of events involved in cardioprotection appears to be distinct for A(1) and A(3) receptor signalling.