Objective: The aim was to further characterise an experimental model of preconditioning of isolated rabbit cardiomyocytes and to determine the role of adenosine receptor subtypes in initiation of the protective response. Methods: Isolated myocytes were subjected to 5 min preincubation in the presence or absence of glucose and various agonists and antagonists of adenosine receptors. Ischaemic pelleting was preceded by a 30 min postincubation period. Rate and extent of injury during ischaemia was determined by sequential sampling of the pelleted cells and assessment of trypan blue permeability following 85 mOsm swelling. Results: Myocytes were preconditioned with a 30-50% reduction of injury by a 5 min glucose-free preincubation. Substitution of 5 mM pyruvate for glucose during preincubation did not prevent the protective response. Protection was maintained over a 60-180 min postincubation period. Protection was blocked by 100 μM of the non-specific adenosine A1A2, antagonist SPT, both when added only during preincubation or only into the ischaemic pellet. Calphostin C, a specific protein kinase C inhibitor at 200 nM, added to the ischaemic pellet blocked protection. Preincubation with R-PIA, the adenosine A1 agonist, did not precondition at an A1 selective dose of 1 μM, but did at 100 μM. The selective A2 agonist CGS 12680 (1 μM) did not precondition. The selective A1/A3 adenosine agonist, APNEA, preconditioned at 1 μM and 200 nM dose levels. Preconditioning induced either by 200 nM APNEA or by glucose-free preincubation was not blocked by 200 nM or 10 μM of the A1 antagonist DPCPX, which has extremely low affinity for A3 receptors, but was blocked by 1 μM of the A1/A3 adenosine antagonist BW 1433U83. Conclusions: Preconditioning can be induced in isolated myocytes by a 5 min preincubation/30 min postincubation protocol, and a similar protection induced by adenosine agonists with A3, but not A1 selectivity. Preconditioning is blocked by non-selective or selective A1/A3 adenosine antagonists and a specific protein kinase C inhibitor, but not by A1 antagonists with little affinity for A3 receptors. The results suggest that preconditioning in isolated rabbit myocytes requires participation of adenosine receptors with agonist/antagonist binding characteristics of the A3 subtype, and is likely to be mediated by activation of protein kinase C.Cardiovascular Research 1994;28:1049-1056.

Objective: The aim was to discriminate among several hypotheses of preconditioning of isolated rabbit cardiomyocytes and to determine if ischaemic preincubation would evoke a protective response. Methods: Isolated myocytes were subjected to 5 min of preincubation, in the presence or absence of glucose, and incubated in the presence of 1 mM iodoacetic acid during the final sustained ischaemic period. In a second series, the protein kinase C (PKC) activators phorbol 12-myristate 13-acetate (PMA), ingenol 3, 20-dibenzoate, and thymeleatoxin were added during preincubation. In a third series, preincubation periods were substituted by brief ischaemic pelleting of cells. Final prolonged ischaemic pelleting was preceded by a 30 min postincubation period. Rate and extent of injury was determined by sequential sampling and assessment of trypan blue permeability following 85 mOsM swelling. Results: Myocytes were preconditioned by a 5 min glucose-free preincubation. Addition of iodoacetic acid into the final ischaemic pellet increased the rates of rigor contracture and injury, but did not abolish the protective response. Direct protein kinase C activation with PMA, a non-selective phorbol ester, and ingenol, an ε, δ-PKC isozyme selective activator, protected cells, but thymeleatoxin, an α,β,γ-PKC isozyme selective activator, did not. A 10 min ischaemic preincubation preconditioned, but the protection was not enhanced when ischaemia was extended to 30 min, or when PMA was included during the initial ischaemic preincubation. Adenosine partially inhibited the response. Conclusions: (1) Preconditioning of isolated myocytes is not dependent on glycolysis or glucose transport. (2) Preconditioning appears dependent on activation of the ε-PKC isoformn. (3) Ischaemia is capable of preconditioning isolated myocytes in vitro, and initiation of this effect is modified by simultaneous additional of adenosine but not by direct protein kinase C activation with PMA. Induction of protection by PMA and ingenol shows that protection requires protein kinase C activation, but direct potassium channel activation by regulatory G proteins is not critical.Cardiovascular Research 1994;28:1700-1706.

Objective: The aim was to determine if isolated rat cardiomycytes could be protected from ischaemic cell death by preincubation with adenosine or adenosine agonists. Methods: Cardiomyocytes isolated from rat hearts were preincubated in the presence of adenosine, CCPA (2-chloro-N6-cyclopentyladenosine), or carbachol prior to concentration into an ischaemic slurry. Effects of glycolysis and of isoprenaline were determined by addition of iodoacetic acid or isoprenaline to the ischaemic incubates and by exclusion of glucose from all media. Rates of ischaemic contracture were determined and survival of the myocytes versus paired control preparations was determined after various times of ischaemia, following resuspension of the cells in isotonic or hypotonic media. Results: Adenosine and CCPA produced only a small reduction of the rates of contracture and death of isolated myocytes. Carbachol gave no significant protection. Neither the degree of injury of control cells nor the amount of protection by CCPA was altered in the presence of added isoprenaline. Protection was abolished by the A1 receptor blocker sulphophenyl theophylline, iodoacetic acid, and exclusion of glucose. Conclusions: Adenosine and adenosine agonists afford a minimal degree of protection to ischaemic isolated myocytes by a glucose dependent mechanism. This protection does not appear to account for the larger degree of protection seen in intact hearts, following similar preconditioning protocols. The failure of adenosine to protect may be related to the quiescent state of isolated cardiomyocytes, or be species specific in that adenosine may not be the trigger for preconditioning in rats.Cardiovascular Research 1993;27:1670-1676.

Calcium tolerant rabbit cardiomyocytes, isolated by collagenase perfusion, were preincubated for varying periods of time followed by resuspension in fresh media and centrifugation into an ischaemic pellet with restricted extracellular fluid. Pellets were incubated for 240 min under oil at 37°C to mimic severe ischaemia. Time to onset of ischaemic contracture (rod to square transformation) and trypan blue permeability following resuspension in 85 mOsm media were monitored at sequential times. The protocol of Series 1 was a 5-10 min pre-incubation, immediately followed by ischaemic pelleting. Preincubation with pinacidil (50 μm) protected cells from ischaemic insult, but pinacidil added only into the ischaemic pellet did not protect. Protection was abolished by the protein kinase (PKC) inhibitors chelerythrine (10 μm) added with pinacidil and calphostin C (200nm) added only into the ischaemic pellet. Neither PKC inhibitor had an effect on injury of untreated ischaemic myocytes (data not shown). Series 2-5 were preconditioning protocols with a 10 min intervention period, followed by a 30 min oxygenated drug-free period, prior to ischaemic pelleting. In series 2 pinacidil protected cells from ischaemic insult and this protection was abolished when glyburide (10 μm) was present during preincubation, or during post-incubation and ischaemia. Glyburide only partially inhibited the protection when glyburide was added only into the ischaemic pellet. In Series 3, 8-sulfophenyltheophyline (SPT)(100 μm) or adenosine deaminase during preincubation, or SPT only added into the ischaemic pellet abolished pinacidil’s protection. In Series 4, cardiomyocytes were ischaemically preconditioned by pelleting for 10 min followed by 30 min reoxygenation. Glyburide during initial ischaemic blocked protection, but when added during post incubation and into the final pellet protection was not reduced. In Series 5 8-cyclopentyl-1,3, dipropylxanthine (DPCPX) (10 μm) added into the final pellet abolished protection by pinacidil, but not protection following ischaemic preconditioning. In contrast to pinacidil, ischaemically preconditioned cells maintain protection in the presence of glyburide, indicating that: (1) pinacidil does not exactly mimic preconditioning and (2) ischaemically preconditioned cells do not require opened K+ATP channels for protection, although they appear to be important during initiation of the preconditioned state. It is hypothesized that pinacidil opening of K+ channels may facilitate induction of preconditioning.

Na Anemia Falciforme em situações de baixa tensão de oxigênio, a hemoglobina mutante S (HbS) sofre polimerização promovendo a falcização das hemácias, que podem aderir ao endotélio vascular, causando a oclusão de vasos (VO) e isquemia tecidual (crises dolorosas) que caracterizam o quadro clínico da doença. Além disso, os pacientes falciformes apresentam outras manifestações clínicas como o priapismo, alterações ósseas, certas complicações pulmonares entre outros. Além das células eritróides, células endoteliais, leucócitos e plaquetas também desempenham um papel fundamental na fisiopatologia da anemia falciforme. A hidroxiuréia (HU), na anemia falciforme, aumenta a produção de hemoglobina fetal (HbF) em células eritróides, reduzindo a polimerização da HbS, diminuindo os sintomas clínicos dos pacientes. O aumento da HbF, no entanto, não implica necessariamente na melhora clínica, indicando desta forma a potencial ação da HU sobre outros processos. Estudos recentes vêm relacionando priapismo e asma com elevados níveis de adenosina. Devido a esta importância da adenosina relacionada a patologias comuns a AF, tivemos como objetivo identificar polimorfismos em genes de receptores de adenosina e na adenosina deaminase e verificar a possível associação entre as manifestações clínicas, além de investigar o papel da HU na modulação de marcadores envolvido na síntese e degradação da adenosina. Foram analisados diversos sítios polimórficos nos genes que codificam ADORA1, ADORA 2b, ADORA 3 e ADA, seguindo com a genotipagem em pacientes com AF, comparando afetados e não afetados. Em adição foi avaliada a expressão diferencial de mRNA de ADA pela HU em monócitos destes pacientes, comparando tratados e não tratados e também avaliamos por citometria de fluxo a modulação de marcadores de superfície CD39, CD73 e CD26, pela HU. As análises estatísticas foram realizadas utilizando os softwares GenePop 3.4 para análises de associação, cálculo do HWE, GraphPad Prism 5, Arlequin para identificação de desequilíbrio de ligação, haplótipos, heterozigozidade e SAS 9.13 para associação dos haplótipos as características. Os resultados mostraram que os pacientes sob tratamento com HU apresentaram um aumento da expressão de mRNA de ADA, aumento da expressão de CD26 em monócitos e diminuição de CD39 em linfócitos. Sem alterações significativas em relação a CD73. Encontramos também um aumento da freqüência do alelo T do SNP (rs1685103) presente no gene de ADORA 1 associado com pacientes afetados com síndrome torácica aguda. Apesar de não ter sido estatisticamente significante, concorda com dados da literatura. No gene ADORA 2B, verificamos associação do SNP 1007 C>T no desenvolvimento de STA indicando o alelo T como fator de risco e o alelo C para alterações ósseas. Para o SNP 968 G>T houve associação com alterações ósseas. Na análise haplotípica entre os SNPs 968 G>T e 1007 C>T encontramos associação dos haplótipos ht2 e ht3 com STA, como fator de risco, ht2 para hipertensão pulmonar. ht1 para priapismo, alterações ósseas e estenose/AVC. Os haplótipos formados pelos três SNPs 968 G>T, 1007 C>T e rs16851030, encontramos associação entre ht1, ht3 e ht4 entre os afetados com priapismo, caracterizando-o como haplótipo de risco e também ht1 e ht6 associados à estenose/AVC. Concluímos, que a hidroxiuréia participa na modulação da expressão da adenosina deaminase, de CD26 em monócitos e CD39 em linfócitos. Além disso, mostrou-se a importância de sítios polimórfico presente no gene ADORA 2B e ADORA1 envolvido na fisiopatologia das manifestações clínicas da doença falciforme. Associações dos SNPs em ADORA 1 e ADA, devem ser melhor estudados em um número maior de pacientes. A determinação destes polimorfismos associados com diferentes características clínicas pode levar a um melhor entendimento dos processos fisiopatológicos da anemia falciforme, levando à identificação de pacientes de risco, possibilitando um manejamento racional dos mesmos, em termos de cuidados específicos, ou mesmo à determinação de alvos para o desenvolvimento de terapias alternativas. / In sickle cell disease in low oxygen tension, mutant hemoglobin S (HbS) undergoes polymerization promoting sickling of red blood cells that can adhere to vascular endothelium, causing vessel occlusion (VO) and tissue ischemia (painful crises) that characterize the clinical disease. In addition, sickle cell patients have other clinical manifestations such as priapism, bone disorders, certain pulmonary complications among others. In addition to the erythroid cells, endothelial cells, white cells and platelets also play a key role in the pathophysiology of sickle cell anemia. Hydroxyurea (HU) in sickle cell anemia, increases the production of fetal hemoglobin (HbF) in erythroid cells, reducing the HbS polymerization, reducing the clinical symptoms of patients. The increase in HbF, however, does not necessarily imply clinical improvement, thus indicating the potential effects of HU on other processes. Recent studies relating asthma and priapism with high levels of adenosine. Due to this importance of adenosine-related pathologies common to AF, we aimed to identify gene polymorphisms in adenosine receptors and adenosine deaminase and verify the possible association between clinical manifestations, and to investigate the role of HU in the modulation of markers involved synthesis and degradation of adenosine. We analyzed several polymorphic sites in genes that encode ADORA1, ADORA 2b, 3 and ADORA ADA, according to the genotype in patients with AF, comparing affected and unaffected. In addition we assessed the differential expression of ADA mRNA by HU in monocytes of these patients, comparing treated and untreated, and also evaluated by flow cytometry modulation of surface markers CD39, CD73 and CD26 by HU. Statistical analysis was performed using the software GenePop 3.4 for association analysis, calculation of HWE, GraphPad Prism 5, Arlequin for identification of linkage disequilibrium, haplotypes, heterozygosity and SAS 9.13 for association of haplotypes features. The results showed that patients treated with HU showed an increase in mRNA expression of ADA, increased expression of CD26 on monocytes and decreased CD39 on lymphocytes. No significant changes in relation to CD73. We also found an increased frequency of allele T (SNP rs1685103) present in a gene associated with ADORA affected patients with acute chest syndrome. Although not statistically significant, agrees with literature data. ADORA 2B gene, we found association of the SNP 1007 C> T in the development of STA indicating the T allele as a risk factor for the C allele and bone changes. For the SNP 968 G> T was associated with bone disorders. In haplotype analysis between SNPs 968 G> T and 1007 C> T found association of haplotypes ht2 and HT3 with STA as a risk factor for pulmonary hypertension ht2. ht1 for priapism, stenosis and bone disorders / stroke. The three haplotypes formed by SNPs 968 G> T, 1007 C> T and rs16851030, we found association between ht1, HT3 and HT4 among those affected with priapism, characterizing it as a risk haplotype and also ht1 ht6 associated with renal and / AVC. We conclude that hydroxyurea participates in modulating the expression of adenosine deaminase of CD26 on monocytes and CD39 on lymphocytes. Moreover, he showed the importance of polymorphic sites in this gene and ADORA 2B ADORA1 involved in the pathophysiology of clinical manifestations of sickle cell disease. Associations of SNPs in ADORA 1 and ADA should be better studied in a larger number of patients. The determination of these polymorphisms associated with different clinical characteristics can lead to a better understanding of the pathophysiological processes of sickle cell anemia, leading to the identification of patients at risk, enabling a rational handling of the same in terms of specific care, or even the determination of targets for the development of alternative therapies.

Na Anemia Falciforme em situações de baixa tensão de oxigênio, a hemoglobina mutante S (HbS) sofre polimerização promovendo a falcização das hemácias, que podem aderir ao endotélio vascular, causando a oclusão de vasos (VO) e isquemia tecidual (crises dolorosas) que caracterizam o quadro clínico da doença. Além disso, os pacientes falciformes apresentam outras manifestações clínicas como o priapismo, alterações ósseas, certas complicações pulmonares entre outros. Além das células eritróides, células endoteliais, leucócitos e plaquetas também desempenham um papel fundamental na fisiopatologia da anemia falciforme. A hidroxiuréia (HU), na anemia falciforme, aumenta a produção de hemoglobina fetal (HbF) em células eritróides, reduzindo a polimerização da HbS, diminuindo os sintomas clínicos dos pacientes. O aumento da HbF, no entanto, não implica necessariamente na melhora clínica, indicando desta forma a potencial ação da HU sobre outros processos. Estudos recentes vêm relacionando priapismo e asma com elevados níveis de adenosina. Devido a esta importância da adenosina relacionada a patologias comuns a AF, tivemos como objetivo identificar polimorfismos em genes de receptores de adenosina e na adenosina deaminase e verificar a possível associação entre as manifestações clínicas, além de investigar o papel da HU na modulação de marcadores envolvido na síntese e degradação da adenosina. Foram analisados diversos sítios polimórficos nos genes que codificam ADORA1, ADORA 2b, ADORA 3 e ADA, seguindo com a genotipagem em pacientes com AF, comparando afetados e não afetados. Em adição foi avaliada a expressão diferencial de mRNA de ADA pela HU em monócitos destes pacientes, comparando tratados e não tratados e também avaliamos por citometria de fluxo a modulação de marcadores de superfície CD39, CD73 e CD26, pela HU. As análises estatísticas foram realizadas utilizando os softwares GenePop 3.4 para análises de associação, cálculo do HWE, GraphPad Prism 5, Arlequin para identificação de desequilíbrio de ligação, haplótipos, heterozigozidade e SAS 9.13 para associação dos haplótipos as características. Os resultados mostraram que os pacientes sob tratamento com HU apresentaram um aumento da expressão de mRNA de ADA, aumento da expressão de CD26 em monócitos e diminuição de CD39 em linfócitos. Sem alterações significativas em relação a CD73. Encontramos também um aumento da freqüência do alelo T do SNP (rs1685103) presente no gene de ADORA 1 associado com pacientes afetados com síndrome torácica aguda. Apesar de não ter sido estatisticamente significante, concorda com dados da literatura. No gene ADORA 2B, verificamos associação do SNP 1007 C>T no desenvolvimento de STA indicando o alelo T como fator de risco e o alelo C para alterações ósseas. Para o SNP 968 G>T houve associação com alterações ósseas. Na análise haplotípica entre os SNPs 968 G>T e 1007 C>T encontramos associação dos haplótipos ht2 e ht3 com STA, como fator de risco, ht2 para hipertensão pulmonar. ht1 para priapismo, alterações ósseas e estenose/AVC. Os haplótipos formados pelos três SNPs 968 G>T, 1007 C>T e rs16851030, encontramos associação entre ht1, ht3 e ht4 entre os afetados com priapismo, caracterizando-o como haplótipo de risco e também ht1 e ht6 associados à estenose/AVC. Concluímos, que a hidroxiuréia participa na modulação da expressão da adenosina deaminase, de CD26 em monócitos e CD39 em linfócitos. Além disso, mostrou-se a importância de sítios polimórfico presente no gene ADORA 2B e ADORA1 envolvido na fisiopatologia das manifestações clínicas da doença falciforme. Associações dos SNPs em ADORA 1 e ADA, devem ser melhor estudados em um número maior de pacientes. A determinação destes polimorfismos associados com diferentes características clínicas pode levar a um melhor entendimento dos processos fisiopatológicos da anemia falciforme, levando à identificação de pacientes de risco, possibilitando um manejamento racional dos mesmos, em termos de cuidados específicos, ou mesmo à determinação de alvos para o desenvolvimento de terapias alternativas. / In sickle cell disease in low oxygen tension, mutant hemoglobin S (HbS) undergoes polymerization promoting sickling of red blood cells that can adhere to vascular endothelium, causing vessel occlusion (VO) and tissue ischemia (painful crises) that characterize the clinical disease. In addition, sickle cell patients have other clinical manifestations such as priapism, bone disorders, certain pulmonary complications among others. In addition to the erythroid cells, endothelial cells, white cells and platelets also play a key role in the pathophysiology of sickle cell anemia. Hydroxyurea (HU) in sickle cell anemia, increases the production of fetal hemoglobin (HbF) in erythroid cells, reducing the HbS polymerization, reducing the clinical symptoms of patients. The increase in HbF, however, does not necessarily imply clinical improvement, thus indicating the potential effects of HU on other processes. Recent studies relating asthma and priapism with high levels of adenosine. Due to this importance of adenosine-related pathologies common to AF, we aimed to identify gene polymorphisms in adenosine receptors and adenosine deaminase and verify the possible association between clinical manifestations, and to investigate the role of HU in the modulation of markers involved synthesis and degradation of adenosine. We analyzed several polymorphic sites in genes that encode ADORA1, ADORA 2b, 3 and ADORA ADA, according to the genotype in patients with AF, comparing affected and unaffected. In addition we assessed the differential expression of ADA mRNA by HU in monocytes of these patients, comparing treated and untreated, and also evaluated by flow cytometry modulation of surface markers CD39, CD73 and CD26 by HU. Statistical analysis was performed using the software GenePop 3.4 for association analysis, calculation of HWE, GraphPad Prism 5, Arlequin for identification of linkage disequilibrium, haplotypes, heterozygosity and SAS 9.13 for association of haplotypes features. The results showed that patients treated with HU showed an increase in mRNA expression of ADA, increased expression of CD26 on monocytes and decreased CD39 on lymphocytes. No significant changes in relation to CD73. We also found an increased frequency of allele T (SNP rs1685103) present in a gene associated with ADORA affected patients with acute chest syndrome. Although not statistically significant, agrees with literature data. ADORA 2B gene, we found association of the SNP 1007 C> T in the development of STA indicating the T allele as a risk factor for the C allele and bone changes. For the SNP 968 G> T was associated with bone disorders. In haplotype analysis between SNPs 968 G> T and 1007 C> T found association of haplotypes ht2 and HT3 with STA as a risk factor for pulmonary hypertension ht2. ht1 for priapism, stenosis and bone disorders / stroke. The three haplotypes formed by SNPs 968 G> T, 1007 C> T and rs16851030, we found association between ht1, HT3 and HT4 among those affected with priapism, characterizing it as a risk haplotype and also ht1 ht6 associated with renal and / AVC. We conclude that hydroxyurea participates in modulating the expression of adenosine deaminase of CD26 on monocytes and CD39 on lymphocytes. Moreover, he showed the importance of polymorphic sites in this gene and ADORA 2B ADORA1 involved in the pathophysiology of clinical manifestations of sickle cell disease. Associations of SNPs in ADORA 1 and ADA should be better studied in a larger number of patients. The determination of these polymorphisms associated with different clinical characteristics can lead to a better understanding of the pathophysiological processes of sickle cell anemia, leading to the identification of patients at risk, enabling a rational handling of the same in terms of specific care, or even the determination of targets for the development of alternative therapies.

Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished

Accumulation of osmotically active metabolites, which create an osmotic gradient estimated at ∼60 mOsM, and cell swelling are prominent features of ischemic myocardial cell death. This study tests the hypothesis that reduction of ischemic swelling by enhanced cell volume regulation is a key mechanism in the delay of ischemic myocardial cell death by ischemic preconditioning (IPC). Experimental protocols address whether: (i) IPC triggers a cell volume regulation mechanism that reduces cardiomyocyte swelling during subsequent index ischemia; (ii) this reduction in ischemic cell swelling is sufficient in magnitude to account for the IPC protection; (iii) the molecular mechanism that mediates IPC also mediates cell volume regulation. Two experimental models with rabbit ventricular myocytes were studied: freshly isolated pelleted myocytes and 48-h cultured myocytes. Myocytes were preconditioned either by distinct short simulated ischemia (SI)/simulated reperfusion protocols (IPC), or by subjecting myocytes to a pharmacological preconditioning (PPC) protocol (1 μM calyculin A, or 1 μM N6-2-(4-aminophenyl)ethyladenosine (APNEA), prior to subjecting them to either different durations of long SI or 30 min hypo-osmotic stress. Cell death (percent blue square myocytes) was monitored by trypan blue staining. Cell swelling was determined by either the bromododecane cell flotation assay (qualitative) or video/confocal microscopy (quantitative). Simulated ischemia induced myocyte swelling in both the models. In pelleted myocytes, IPC or PPC with either calyculin A or APNEA produced a marked reduction of ischemic cell swelling as determined by the cell floatation assay. In cultured myocytes, IPC substantially reduced ischemic cell swelling (P < 0.001). This IPC effect on ischemic cell swelling was related to an IPC and PPC (with APNEA) mediated triggering of cell volume regulatory decrease (RVD). IPC and APNEA also significantly (P < 0.001) reduced hypo-osmotic cell swelling. This IPC and APNEA effect was blocked by either adenosine receptor, PKC or Cl- channel inhibition. The osmolar equivalent for IPC protection approximated 50-60 mOsM, an osmotic gradient similar to the estimated ischemic osmotic load for preconditioned and non-preconditioned myocytes. The results suggest that cell volume regulation is a key mechanism that accounts for most of the IPC protection in cardiomyocytes.

Objective: The aim was to determine if isolated rabbit cardiomyocytes could be preconditioned. Methods: Cardiomyocytes isolated from rabbit hearts were subjected to 15 min oxygenated preincubation, with and without substrate, prior to concentration into an ischaemic slurry, with or without glucose present. The effects of an adenosine agonist (CCPA), an adenosine receptor blocker (SPT), and the protein kinase C blocker, calphostin C, on rates of ischaemic contracture and survival of the myocytes were determined after various times of ischaemia, following resuspension of the cells in hypotonic media. Results: A glucose-free preincubation period protected myocytes from subsequent ischaemic injury, with a 40% reduction of cell death at 90-120 min and 1-2 h delay in cell death. CCPA added during preincubation and during the ischaemic period also tended to protect from injury, but the differences were not significant and protection was less than with a glucose-free preincubation. Although preincubation with CCPA did not precondition, SPT added to the preincubation medium only, or to both the preincubation medium and the ischaemic pellet, inhibited the preconditioning effect of a glucose-free preincubation period. Calphostin C, added only into the ischaemic pellet, inhibited the preconditioning effect of glucose-free preincubation. Conclusions: Glucose-free preincubation protects ischaemic isolated myocytes from subsequent ischaemia. The degree of protection is great enough to account for protection seen in intact hearts, following preconditioning protocols. Protection is blocked by SPT and a highly specific protein kinase C inhibitor, calphostin C. Protection from ischaemic injury that seems to mimic ischaemic preconditioning can be induced in isolated cardiomyocytes, and appears dependent on adenosine receptors and activation of protein kinase C.Cardiovascular Research 1994;28:72-77.

This study investigated the role of opioid, adenosine, bradykinin, and calcitonin-gene related peptide (CGRP) receptors, and potential ‘cross-talk’ among suspected G-protein coupled receptors in a humoral model of remote ischemic preconditioning (rIPC) cardioprotection. Compared to Control dialysate (from non-preconditioned donor rabbit blood), rIPC dialysate (from remotely preconditioned blood) reduced cell death in rabbit cardiomyocytes following simulated ischemia and reperfusion. Non-selective, δ-, or κ-opioid receptor blockade and non-selective adenosine receptor blockade abolished rIPC dialysate protection; whereas, bradykinin B2 and CGRP receptor blockade had no effect. Non-selective adenosine receptor blockade fully and partially abolished protection by κ- and δ-opioid receptors, respectively. Multiple reaction monitoring mass spectrometry detected low levels of adenosine, and other preconditioning substances, in the dialysate. An increase in extracellular adenosine was not detected during opioid-induced preconditioning to explain this cross-talk. These results suggest that δ-opioid, κ-opioid, adenosine receptors, and opioid-adenosine cross-talk are involved in rIPC of freshly isolated cardiomyocytes.