Sodium-glucose cotransporter 2 inhibitor Dapagliflozin attenuates diabetic cardiomyopathy.

BACKGROUND: Diabetes mellitus type 2 (DM2) is a risk factor for developing heart failure but there is no specific therapy for diabetic heart disease. Sodium glucose transporter 2 inhibitors (SGLT2I) are recently developed diabetic drugs that primarily work on the kidney. Clinical data describing the cardiovascular benefits of SGLT2Is highlight the potential therapeutic benefit of these drugs in the prevention of cardiovascular events and heart failure. However, the underlying mechanism of protection remains unclear. We investigated the effect of Dapagliflozin-SGLT2I, on diabetic cardiomyopathy in a mouse model of DM2. METHODS: Cardiomyopathy was induced in diabetic mice (db/db) by subcutaneous infusion of angiotensin II (ATII) for 30 days using an osmotic pump. Dapagliflozin (1.5 mg/kg/day) was administered concomitantly in drinking water. Male homozygous, 12-14 weeks old WT or db/db mice (n = 4-8/group), were used for the experiments. Isolated cardiomyocytes were exposed to glucose (17.5-33 mM) and treated with Dapagliflozin in vitro. Intracellular calcium transients were measured using a fluorescent indicator indo-1. RESULTS: Angiotensin II infusion induced cardiomyopathy in db/db mice, manifested by cardiac hypertrophy, myocardial fibrosis and inflammation (TNFα, TLR4). Dapagliflozin decreased blood glucose (874 ± 111 to 556 ± 57 mg/dl, p < 0.05). In addition it attenuated fibrosis and inflammation and increased the left ventricular fractional shortening in ATII treated db/db mice. In isolated cardiomyocytes Dapagliflozin decreased intracellular calcium transients, inflammation and ROS production. Finally, voltage-dependent L-type calcium channel (CACNA1C), the sodium-calcium exchanger (NCX) and the sodium-hydrogen exchanger 1 (NHE) membrane transporters expression was reduced following Dapagliflozin treatment. CONCLUSION: Dapagliflozin was cardioprotective in ATII-stressed diabetic mice. It reduced oxygen radicals, as well the activity of membrane channels related to calcium transport. The cardioprotective effect manifested by decreased fibrosis, reduced inflammation and improved systolic function. The clinical implication of our results suggest a novel pharmacologic approach for the treatment of diabetic cardiomyopathy through modulation of ion homeostasis.

Cardioprotection from stress conditions by weak magnetic fields in the Schumann Resonance band.

The Schumann Resonances (ScR) are Extremely Low Frequency (ELF) electromagnetic resonances in the Earth-ionosphere cavity excited by global lightning discharges. This natural electromagnetic noise has likely existed on the Earth ever since the Earth had an atmosphere and an ionosphere, hence surrounding us throughout our evolutionary history. The purpose of this study was to examine the influence of extremely weak magnetic fields in the ScR first mode frequency range on the spontaneous contractions, calcium transients and Creatine Kinase (CK) release of rat cardiac cell cultures. We show that applying 7.8 Hz, 90 nT magnetic fields (MF) causes a gradual decrease in the spontaneous calcium transients' amplitude, reaching 28% of the initial amplitude after 40 minutes of MF application, and accompanied with a gradual decrease in the calcium transients' rise time. The mechanical spontaneous contractions cease after the ScR fields have been applied for more than 30 minutes, when the calcium transient's amplitude reached ~60% of its initial value. The influence of the ScR MF was reversible, independent of the field magnitude in the range 20 pT-100 nT, and independent of the external DC magnetic field. However, the effect is frequency dependent; the described changes occurred only in the 7.6-8 Hz range. In addition, applying 7.8 Hz, 90 nT MF for 1.5 hours, reduced the amount of CK released to the buffer, during normal conditions, hypoxic conditions and oxidative stress induced by 80 µM H2O2. We show that the ScR field induced reduction in CK release is associated with a stress response process and has a protective character.

A PARP1-ERK2 synergism is required for the induction of LTP.

Unexpectedly, a post-translational modification of DNA-binding proteins, initiating the cell response to single-strand DNA damage, was also required for long-term memory acquisition in a variety of learning paradigms. Our findings disclose a molecular mechanism based on PARP1-Erk synergism, which may underlie this phenomenon. A stimulation induced PARP1 binding to phosphorylated Erk2 in the chromatin of cerebral neurons caused Erk-induced PARP1 activation, rendering transcription factors and promoters of immediate early genes (IEG) accessible to PARP1-bound phosphorylated Erk2. Thus, Erk-induced PARP1 activation mediated IEG expression implicated in long-term memory. PARP1 inhibition, silencing, or genetic deletion abrogated stimulation-induced Erk-recruitment to IEG promoters, gene expression and LTP generation in hippocampal CA3-CA1-connections. Moreover, a predominant binding of PARP1 to single-strand DNA breaks, occluding its Erk binding sites, suppressed IEG expression and prevented the generation of LTP. These findings outline a PARP1-dependent mechanism required for LTP generation, which may be implicated in long-term memory acquisition and in its deterioration in senescence.

An ultra-low dose of tetrahydrocannabinol provides cardioprotection.

UNLABELLED: Tetrahydrocannabinol (THC), the major psychoactive component of marijuana, is a cannabinoid agonist that exerts its effects by activating at least two specific receptors (CB1 and CB2) that belong to the seven transmembrane G-protein coupled receptor (GPCR) family. Both CB1 and CB2 mRNA and proteins are present in the heart. THC treatment was beneficial against hypoxia in neonatal cardiomyocytes in vitro. We also observed a neuroprotective effect of an ultra low dose of THC when applied to mice before brain insults. The present study was aimed to test and characterize the cardioprotective effects of a very low dose (0.002mg/kg) of THC which is 3-4 orders of magnitude lower than the conventional doses, administered before myocardial infarction in mice in vivo. Three regimens of THC administration were tested: single THC application 2h or 48h before the induction of infarct, or 3 weeks continuous treatment before MI. All protocols of THC administration were found to be beneficial. In the case of THC treatment 2h before MI, fractional shortening was elevated (37±4% vs. 42±1%, p<0.04), troponin T leakage to the blood was reduced (14±3ng/ml vs. 10±4ng/ml, p<0.008), infarct size decreased (29±4% vs. 23±4%, p<0.02), and the accumulation of neutrophils to the infarct area declined (36±10cells/field vs. 19±4cells/field, p<0.007) in THC- compared to vehicle-pretreated mice, 24h after MI. ERK1/2 phosphorylation following infarct was also inhibited by pre-treatment with THC (p<0.01). CONCLUSION: A single ultra low dose of THC before ischemia is a safe and effective treatment that reduces myocardial ischemic damage.

Conditioned medium from hypoxic cells protects cardiomyocytes against ischemia.

The hypothesis of the present study is that cardiomyocytes subjected to prolonged ischemia, may release survival factors that will protect new cardiac cells from ischemic stress. We exposed neonatal rat cardiomyocyte primary cultures to hypoxia, collected the supernatant, treated intact cardiac cells by this posthypoxic supernatant, and exposed them to hypoxia. The results show cardioprotection of the treated cells compared with the untreated ones. We named the collected posthypoxic supernatant "conditioned medium" (CM), which acts in a dose-dependent manner to protect new cardiac cells from hypoxia: 100 or 75% of CM diluted in phosphate-buffered saline (PBS) protected cells as if they were not exposed to hypoxia (P < 0.001). When CM was removed from the cells before hypoxia, protection was not observed. CM also protected skeletal muscle cultures from hypoxia, but not cardiac cells against H(2)O(2)-induced cell damage. Finally, CM treatment protected the isolated heart in Langendorff set-up against ischemia. Smaller infarct size (9.9 ± 4.4% vs. 28.3 ± 8.5%, P < 0.05), better Rate Pressure Product (67 ± 11% vs. 48.6 ± 13.4%, P < 0.05) and better rate of contraction and relaxation were observed following ischemia and reperfusion (1341 ± 399 mmHg/s vs. 951 ± 349 mmHg/s, P < 0.05 and 1053 ± 347 mmHg/s vs. 736 ± 314 mmHg/s, P < 0.05). To conclude, there are factors that are released from the heart cells subjected to ischemia/hypoxia that protects cardiomyocytes from ischemic stress.

UTP reduces infarct size and improves mice heart function after myocardial infarct via P2Y2 receptor.

Pyrimidine nucleotides are signaling molecules, which activate G protein-coupled membrane receptors of the P2Y family. P2Y(2) and P2Y(4) receptors are part of the P2Y family, which is composed of 8 subtypes that have been cloned and functionally defined. We have previously found that uridine-5'-triphosphate (UTP) reduces infarct size and improves cardiac function following myocardial infarct (MI). The aim of the present study was to determine the role of P2Y(2) receptor in cardiac protection following MI using knockout (KO) mice, in vivo and wild type (WT) for controls. In both experimental groups used (WT and P2Y(2)(-/-) receptor KO mice) there were 3 subgroups: sham, MI, and MI+UTP. 24h post MI we performed echocardiography and measured infarct size using triphenyl tetrazolium chloride (TTC) staining on all mice. Fractional shortening (FS) was higher in WT UTP-treated mice than the MI group (44.7±4.08% vs. 33.5±2.7% respectively, p<0.001). However, the FS of P2Y(2)(-/-) receptor KO mice were not affected by UTP treatment (34.7±5.3% vs. 35.9±2.9%). Similar results were obtained with TTC and hematoxylin and eosin stainings. Moreover, troponin T measurements demonstrated reduced myocardial damage in WT mice pretreated with UTP vs. untreated mice (8.8±4.6 vs. 12±3.1 p<0.05). In contrast, P2Y(2)(-/-) receptor KO mice pretreated with UTP did not demonstrate reduced myocardial damage. These results indicate that the P2Y(2) receptor mediates UTP cardioprotection, in vivo.

Extracellular nucleotide derivatives protect cardiomyocytes against hypoxic stress.

RATIONALE: Extracellular nucleotides have widespread effects and various cell responses. Whereas the effect of a purine nucleotide (ATP) and a pyrimidine nucleotide (UTP) on myocardial infarction has been examined, the role of different purine and pyrimidine nucleotides and nucleosides in cardioprotection against hypoxic stress has not been reported. OBJECTIVE: To investigate the role of purine and pyrimidine nucleotides and nucleosides in protective effects in cardiomyocytes subjected to hypoxia. METHODS AND RESULTS: Rat cultured cardiomyocytes were treated with various extracellular nucleotides and nucleosides, before or during hypoxic stress. The results revealed that GTP or CTP exhibit cardioprotective ability, as revealed by lactate dehydrogenase (LDH) release, by propidium iodide (PI) staining, by cell morphology, and by preserved mitochondrial activity. Pretreatment with various P2 antagonists (suramin, RB-2, or PPADS) did not abolish the cardioprotective effect of the nucleotides. Moreover, P2Y2 -/- , P2Y4 -/-, and P2Y2 -/-/P2Y4 -/- receptor knockouts mouse cardiomyocytes were significantly protected against hypoxic stress when treated with UTP. These results indicate that the protective effect is not mediated via those receptors. We found that a wide variety of triphosphate and diphosphate nucleotides (TTP, ITP, deoxyGTP, and GDP), provided significant cardioprotective effect. GMP, guanosine, and ribose phosphate provided no cardioprotective effect. Moreover, we observed that tri/di-phosphate alone assures cardioprotection. Treatment with extracellular nucleotides, or with tri/di-phosphate, administered under normoxic conditions or during hypoxic conditions, led to a decrease in reactive oxygen species production. CONCLUSIONS: Extracellular tri/di-phosphates are apparently the molecule responsible for cardioprotection against hypoxic damage, probably by preventing free radicals formation.

Adenosine A(1) and A (3) receptor agonists reduce hypoxic injury through the involvement of P38 MAPK.

Activation of either the A(1) adenosine receptor (A(1)R) or the A(3) adenosine receptor (A(3)R), by their specific agonists CCPA and Cl-IB-MECA, respectively, protects cardiac cells in culture against ischemic injury. Yet the full protective mechanism remains unclear. In this study, we therefore examined the involvement of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases (ERK) phosphorylation in this protective intracellular signaling mechanism. Furthermore, we investigated whether p38 MAPK phosphorylation occurs upstream or downstream from the opening of mitochondrial ATP-sensitive potassium (K(ATP)) channels. The role of p38 MAPK activation in the intracellular signaling process was studied in cultured cardiomyocytes subjected to hypoxia, that were pretreated with CCPA or Cl-IB-MECA or diazoxide (a mitochondrial K(ATP) channel opener) with and without SB203580 (a specific inhibitor of phosphorylated p38 MAPK). Cardiomyocytes were also pretreated with anisomycin (p38 MAPK activator) with and without 5-hydroxy decanoic acid (5HD) (a mitochondrial K(ATP) channel blocker). SB203580 together with the CCPA, Cl-IB-MECA or diazoxide abrogated the protection against hypoxia as shown by the level of ATP, lactate dehydrogenase (LDH) release, and propidium iodide (PI) staining. Anisomycin protected the cardiomyocytes against ischemic injury and this protection was abrogated by SB203580 but not by 5HD. Conclusions Activation of A(1)R or A(3)R by CCPA or Cl-IB-MECA, respectively, protects cardiomyocytes from hypoxia via phosphorylation of p38 MAPK, which is located downstream from the mitochondrial K(ATP) channel opening. Elucidating the signaling pathway by which adenosine receptor agonists protect cardiomyocytes from hypoxic damage, will facilitate the development of anti ischemic drugs.

Acute, nongenomic effect of thyroid hormones in preventing calcium overload in newborn rat cardiocytes.

In this study, we examined the acute effects of thyroid hormones (TH) T(3) and T(4), leading to improvement of myocardial function through activation of Ca(2+) extrusion mechanisms and, consequently, prevention of intracellular calcium overload. Extracellular calcium elevation from 1.8 to 3.8 mM caused immediate increase in intracellular calcium level ([Ca(2+)](i)) in newborn cardiomyocyte cultures. Administration of 10 or 100 nM T(3) or T(4) rapidly (within 10 sec) decreased [Ca(2+)](i) to its control level. Similar results were obtained when [Ca(2+)](i) was elevated by decreasing extracellular Na(+) concentration, causing backward influx of Ca(2+) through Na(+)/Ca(2+) exchanger, or by administration of caffeine, releasing Ca(2+) from the sarcoplasmic reticulum (SR). Under these conditions, T(3) or T(4) decreased [Ca(2+)](i). T(3) and T(4) also exhibited protective effects during ischemia. T(3) or T(4) presence during hypoxia for 120 min in culture medium restricted the increase of [Ca(2+)](i) and prevented the pathological effects of its overload. An inhibitor of SR Ca(2+)-ATPase (SERCA2a), thapsigargin, increases [Ca(2+)](i) and in its presence neither T(3) nor T(4) had any effect on the [Ca(2+)](i) level. The reduction of [Ca(2+)](i) level by T(3) and T(4) was also blocked in the presence of H-89 (a PKA inhibitor), and by calmodulin inhibitors. The effect of TH on the reduction of [Ca(2+)](i) was prevented by propranolol, indicating that the hormones exert their effect through interaction with adrenergic receptors. These results support our hypothesis that TH prevent calcium overload in newborn rat cardiomyocytes, most likely by a direct, acute, and nongenomic effect on Ca(2+) transport into the SR.

Differential aspects in ratio measurements of [Ca(2+)](i) relaxation in cardiomyocyte contraction following various drug treatments.

This study is concerned with the analysis of the time dependency of [Ca(2+)](i), monitored by indo-1-AM, via the ratiometric time response curve R(t) as measured during contractions of spontaneous or electrical stimulated cardiomyocytes (in culture). A mathematical formulation which describes the relaxation phase of R(t) was developed. By fitting formulation to the measured data of R(t), the extraction of characteristic parameters is feasible, which may reflect the factors regulating intracellular Ca concentration. The usefulness of the suggested formulation was examined by monitoring changes induced in those parameters following the exposure of the myocytes to different drugs, among which are: caffeine, ryanodine, thapsigargin db, cyclic AMP, isoprenaline, doxorubicin, and Cl-IB-MECA.

Angiotensin II-induced apoptosis in rat cardiomyocyte culture: a possible role of AT1 and AT2 receptors.

OBJECTIVES: To investigate the mechanism of angiotensin II-induced apoptosis in cultured cardiomyocytes by determining which receptor subtype is involved, and what is the relationship between intracellular Ca2+ changes and apoptosis. DESIGN AND METHODS: Neonatal rat cardiomyocytes were pretreated with either the AT1 antagonist irbesartan or the AT2 antagonist PD123319 before exposure to angiotensin II. Apoptosis was evaluated using morphological technique, staining nuclei by Feulgen and Hoechst methods followed by image analysis and by in situ terminal deoxynucleotidyl transferase nick-end (TUNEL) labelling. TUNEL-positive cardiocytes were distinguished from other cells by double staining with alpha-sarcomeric actin. Intracellular Ca2+ changes were assessed by indo-1 fluorescence microscopy, and the effect of Ca2+ on angiotensin II-induced apoptosis was tested using the calcium channel blocker verapamil. RESULTS: Exposure to angiotensin II (10 nmol/l) resulted in cell replication and a three-fold increase in programmed cell death (P < 0.05). Pretreatment with either irbesartan (an AT1receptor antagonist, 100 nmol/l) or PD123319 (an AT2 receptor antagonist, 1 micromol/l) prevented the angiotensin II-induced apoptosis, indicating the presence of both AT1 and AT2receptors on cardiomyocytes. Exposure of myocytes to angiotensin II caused an immediate and dose-dependent increase in the concentration of intracellular free Ca2+ that lasted 40-60 s. The effect was sustained in a Ca2+ free medium. Pretreatment of cells with irbesartan (100 nmol/l) and PD123319 (10 micromol/l) blocked Ca2+ elevation. Pretreatment with verapamil (10 micromol/l) prevented angiotensin II-induced apoptosis. CONCLUSIONS: Angiotensin II-induced apoptosis in rat cardiomyocytes is mediated through activation of both AT1 and AT2 receptors. The apoptotic mechanism is not related to the immediate angiotensin II-induced Ca2+ rise from intracellular stores. However, it is accompanied by cardiomyocyte proliferation and requires Ca2+ influx through L-type channel activity.

Dispersion of cell-to-cell uncoupling precedes low K+-induced ventricular fibrillation.

We hypothesize that hypokalemia-related electrolyte imbalance linked with abnormal elevation of intracellular free Ca2+ concentration can cause metabolic disturbances and subcellular alterations resulting in intercellular uncoupling, which favor the occurrence of malignant arrhythmias. Langendorff-perfused guinea pig heart (n = 44) was subjected to a standard Tyrode solution (2.8 mmol/l K+) followed by a K+-deficient solution (1.4 mmol/l K+). Bipolar ECG of the left atria and ventricle was continuously monitored and the incidence of ventricular fibrillation was evaluated. Myocardial tissue sampling was performed during stabilization, hypokalemia and at the onset of fibrillation. Enzyme activities of succinic dehydrogenase, glycogen phosphorylase and 5-nucleotidase were determined using in situ catalytic histochemistry. The main gap junction protein, connexin-43, was labeled using mouse monoclonal antibody and FITC conjugated goat antimouse antibody. Ultrastructure was examined by transmission electron microscopy. The free Ca2+ concentration was measured by the indo-1 method in ventricular cell cultures exposed to a K+-free medium. The results showed that sustained ventricular fibrillation appeared within 15-30 min of low K+ perfusion. This was preceded by ectopic activity, episodes of bigeminy and tachycardia. Hypokalemia induced moderate reversible and sporadically irreversible subcellular alterations of cardiomyocytes and impairment of intercellular junctions, which were heterogeneously distributed throughout myocardium. Patchy areas with decreased enzyme activities and diminished immunoreactivity of connexin-43 were found. Furthermore, lack of external K+ was accompanied by an increase of intracellular Ca2+. The prevention of Ca2+ overload by either 1 mmol/l Ni2+ (Na+/Ca2+ inhibitor), 2.5 micromol/l verapamil, 10 micromol/l d-sotalol or 10 micromol/l tedisamil was associated with the protection against fibrillation. The results indicate that hypokalemia induces Ca2+ overload injury and disturbances in intercellular coupling. Dispersion of these changes throughout the myocardium may serve as the basis for microreentry circuits and thus favor fibrillation occurrence.

Activation of A(3)adenosine receptor protects against doxorubicin-induced cardiotoxicity.

Adenosine exerts a marked protective effect on the heart during cardiac ischemia. This protection is mediated by binding to the A(1)and A(3)subtypes of adenosine receptor (A(1)R and A(3)R, respectively). The objective of the present study was to investigate whether activation of A(1)and A(3)adenosine receptors may reduce doxorubicin-induced damage to cardiomyocytes in culture. Cultured cardiomyocytes from newborn rats were treated with 0.5--5 microm doxorubicin (DOX) for 18 h and then incubated in drug-free medium for an additional 24 h. This treatment resulted in cell damage and lactate dehydrogenase release, even after low (0.5 microm) doses of the drug, and increased in a concentration-dependent manner. Activation of A(3)-subtype but not A(1)-subtype receptors attenuated doxorubicin-cardiotoxicity after drug treatment for 18 h followed by 24 h incubation in drug-free medium. Modulation of intracellular calcium mediated by activation of A(3)R, but not by A(1)R, in cultured myocytes suggested an important pathophysiological significance of this subtype of adenosine receptors. Protection by A(3)R agonist Cl-IB-MECA (2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide) following DOX treatment is evident in: (1) decreases in intracellular calcium overloading and abnormalities in Ca(2+)transients; (2) reduction of free-radical generation and lipid peroxidation; (3) attenuation of mitochondrial damage by protection of the terminal link (COX-complex) of respiratory chain; (4) attenuation of the decrease in ATP production and irreversible cardiomyocyte damage. Cardioprotection caused by Cl-IB-MECA was antagonized considerably by the selective A(3)adenosine receptor antagonist MRS1523.

Increased cardiac alpha-1-adrenoceptor density in rats following treatment with amiodarone.

This study was undertaken to investigate the interaction between amiodarone and alpha-1-adrenoceptors in rat cardiac cells. The level (Bmax) and affinity (Kd) of alpha-1-adrenoceptors in heart cells were determined by [3H]prazosin radioligand binding following amiodarone treatment. In cultured intact cardiocytes treated for 48 h with 10 microM amiodarone, [3H]prazosin binding increased by 31% compared with the control cells (p<0.05). The increase was both dose and time dependent and was found to be specific because no significant change occurred in creatine kinase activity. Additionally, under the same conditions, an increase in [3H]prazosin binding to cultured cardiocyte cell membranes was also obtained. Oral gavage of amiodarone to rats for 8 d resulted in a 25% increase in [3H]prazosin binding to isolated ventricle membranes compared with control rats (p<0.05). We conclude that amiodarone treatment can increase the response to alpha-1-adrenoceptors agonist in the heart due to an increase in the density of alpha-1-adrenoceptors.

Cardioprotective effects of adenosine A1 and A3 receptor activation during hypoxia in isolated rat cardiac myocytes.

Adenosine (ADO) is a well-known regulator of a variety of physiological functions in the heart. In stress conditions, like hypoxia or ischemia, the concentration of adenosine in the extracellular fluid rises dramatically, mainly through the breakdown of ATP. The degradation of adenosine in the ischemic myocytes induced damage in these cells, but it may simultaneously exert protective effects in the heart by activation of the adenosine receptors. The contribution of ADO to stimulation of protective effects was reported in human and animal hearts, but not in rat hearts. The aim of this study was to evaluate the role of adenosine A1 and A3 receptors (A1R and A3R), in protection of isolated cardiac myocytes of newborn rats from ischemic injury. The hypoxic conditions were simulated by exposure of cultured rat cardiomyocytes (4-5 days in vitro), to an atmosphere of a N2 (95%) and CO2 (5%) mixture, in glucose-free medium for 90 min. The cardiotoxic and cardioprotective effects of ADO ligands were measured by the release of lactate dehydrogenase (LDH) into the medium. Morphological investigation includes immunohistochemistry, image analysis of living and fixed cells and electron microscopy were executed. Pretreatment with the adenosine deaminase considerably increased the hypoxic damage in the cardiomyocytes indicating the importance of extracellular adenosine. Blocking adenosine receptors with selective A1 and A3 receptor antagonists abolished the protective effects of adenosine. A1R and A3R activation during the hypoxic insult delays onset of irreversible cell injury and collapse of mitochondrial membrane potential as assessed using DASPMI fluorochrom. Cardioprotection induced by the A1R agonist, CCPA, was abolished by an A1R antagonist, DPCPX, and was not affected by an A3R antagonist, MRS 1523. Cardioprotection caused by the A3R agonist, Cl-IB-MECA, was antagonized completely by MRS 1523 and only partially by DPCPX. Activation of both A1R and A3R together was more efficient in protection against hypoxia than by each one alone. Our study indicates that activation of either A1 or A3 adenosine receptors in the rat can attenuate myocyte injury during hypoxia. Highly selective A1R and A3R agonists may have potential as cardioprotective agents against ischemia or heart surgery.

Induction of apoptosis in rat cardiocytes by A3 adenosine receptor activation and its suppression by isoproterenol.

The purpose of the present study was to investigate the mechanisms involved in the induction of apoptosis in newborn cultured cardiomyocytes by activation of adenosine (ADO) A3 receptors and to examine the protective effects of beta-adrenoceptors. The selective agonist for A3 ADO receptors Cl-IB-MECA (2-chloro-N6-iodobenzyl-5-N-methylcarboxamidoadenosine) and the antagonist MRS1523 (5-propyl-2-ethyl-4-propyl-3-(ethylsulfanylcarbonyl)-6-phenylpy rid ine-5-carboxylate) were used. High concentrations of the Cl-IB-MECA (> or = 10 microM) agonist induced morphological modifications of myogenic cells, such as rounding and retraction of cell body and dissolution of contractile filaments, followed by apoptotic death. In addition, Cl-IB-MECA caused a sustained and reversible increase in [Ca2+]i, which was prevented by the selective antagonist MRS1523. Furthermore, MRS1523 protected the cardiocytes if briefly exposed to Cl-IB-MECA and partially protected from prolonged (48 h) agonist exposure. Apoptosis induced by Cl-IB-MECA was not redox-dependent, since the mitochondrial membrane potential remained constant until the terminal stage of cell death. Cl-IB-MECA activated caspase-3 protease in a concentration-dependent manner after 7 h of treatment and more effectively after 18 h of exposure. Bcl-2 protein was readily detected in control cells, and its expression was significantly decreased after 24 and 48 h of treatment with Cl-IB-MECA. Beta-adrenergic stimulation antagonized the pro-apoptotic effects of Cl-IB-MECA, probably through a cAMP/protein kinase A-independent mechanism, since addition of dibutyryl-cAMP did not abolish the apoptosis induced by Cl-IB-MECA. Incubation of cultured myocytes with isoproterenol (5 microM) for 3 or 24 h almost completely abolished the increase in [Ca2+]i. Prolonged incubation of cardiomyocytes with isoproterenol and Cl-IB-MECA did not induce apoptosis. Our data suggest that the apoptosis-inducing signal from activation of adenosine A3 receptors (or counteracting beta-adrenergic signal) leads to the activation of the G-protein-coupled enzymes and downstream pathways to a self-amplifying cascade. Expression of different genes within this cascade is responsible for orchestrating either cardiomyocyte apoptosis or its protection.

Mitochondrial localization and photodamage during photodynamic therapy with tetraphenylporphines.

The subcellular localization sites of TPPS4 and TPPS1 and the subsequent cellular site damage during photodynamic therapy were investigated in CT-26 colon carcinoma cells using spectroscopic and electron microscopy techniques. The association of both porphyrins with the mitochondria was investigated and the implications of this association on cellular functions were determined. Spectrofluorescence measurements showed that TPPS4 favors an aqueous environment, while TPPS1 interacts with lipophilic complexes. The subcellular localization sites of each sensitizer were determined using spectral imaging. Mitochondrial-CFP transfected cells treated with porphyrins revealed localization of TPPS1 in the peri-nuclear region, while TPPS4 localized in the mitochondria, inducing structural damage and swelling upon irradiation, as shown by transmission electron microscopy. TPPS4 fluorescence was detected in isolated mitochondria following irradiation. The photodamage induced a 38% reduction in mitochondrial activity, a 30% decrease in cellular ATP and a reduction in Na(+)/K(+)-ATPase activity. As a result, cytosolic concentrations of Na(+) and Ca(2+) increased, and the level of K(+) decreased. In contrast, the lipophilic TPPS1 did not affect mitochondrial structure or function and ATP content remained unchanged. We conclude that TPPS4 induces mitochondrial structural and functional photodamage resulting in an altered cytoplasmic ion concentration, while TPPS1 has no effect on the mitochondria.

Involvement of K(+) channels in the relaxant effects of YC-1 in vascular smooth muscle.

This study addresses the question whether K(+) channels are involved in the vasorelaxant effects of 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl-indazole (YC-1 ). In rat aorta, guinea pig aorta, and guinea pig a. carotis, YC-1 inhibited contractions induced by phenylephrine (3 microM) more potently than those induced by K(+)(48 mM). In rat aorta, tetraethylammonium (10 mM), charybdotoxin (0.2 microM), and iberiotoxin (0.1 microM), but not glibenclamide (10 microM), attenuated the relaxant effects of YC-1. In guinea pig a. carotis, YC-1 (30 microM) induced a hyperpolarisation which was antagonised by 1H-[1,2,4]oxadiazolo[4, 3-a]quinoxalin-1-one (ODQ; 50 microM). In rat aorta, YC-1 (30 microM) increased the rate constant of 86Rb-efflux. The effect of YC-1 was potentiated by zaprinast (10 microM), but inhibited by ODQ (50 microM) or charybdotoxin (0.2 microM). In smooth muscle cells from rat aorta, YC-1 (10 microM) increased BK(Ca) channel activity. It is suggested that YC-1-induced vasorelaxation is partially mediated by the activation of K(+) channels.

Characterization of "mini-nucleotides" as P2X receptor agonists in rat cardiomyocyte cultures. An integrated synthetic, biochemical, and theoretical study.

The design and synthesis of "mini-nucleotides", based on a xanthine-alkyl phosphate scaffold, are described. The physiological effects of the new compounds were evaluated in rat cardiac cell culture regarding Ca(2+) elevation and contractility. The results indicate biochemical and physiological profiles similar to those of ATP, although at higher concentrations. The biological target molecules of these "mini-nucleotides" were identified by using selective P2-R and A(1)-R antagonists and P2-R subtype selective agonists. On the basis of these results and of experiments in Ca(2+) free medium, in which [Ca(2+)](i) elevation was not observed, we concluded that interaction of the analogues is likely with P2X receptor subtypes, which causes Ca(2+) influx. Theoretical calculations analyzing electronic effects within the series of xanthine-alkyl phosphates were performed on reduced models at quantum mechanical levels. Calculated dipole moment vectors, electrostatic potential maps, and volume parameters suggest an explanation for the activity or inactivity of the synthesized derivatives and predict a putative binding site environment for the active agonists. Xanthine-alkyl phosphate analogues proved to be selective agents for activation of P2X-R subtypes, whereas ATP activated all P2-R subtypes in cardiac cells. Therefore, these analogues may serve as prototypes of selective drugs aiming at cardiac disorders mediated through P2X receptors.