Ischemic Preconditioning Protects Against Gap Junctional Uncoupling in Cardiac Myofibroblasts

Ischemic preconditioning increases the heart's tolerance to a subsequent longer ischemic period. The purpose of this study was to investigate the role of gap junction communication in simulated preconditioning in cultured neonatal rat cardiac myofibroblasts. Gap junctional intercellular communication was assessed by Lucifer yellow dye transfer. Preconditioning preserved intercellular coupling after prolonged ischemia. An initial reduction in coupling in response to the preconditioning stimulus was also observed. This may protect neighboring cells from damaging substances produced during subsequent regional ischemia in vivo, and may preserve gap junctional communication required for enhanced functional recovery during subsequent reperfusion.     

Ischemic preconditioning protects against gap junctional uncoupling in cardiac myofibroblasts

Ischemic preconditioning increases the heart's tolerance to a subsequent longer ischemic period. The purpose of this study was to investigate the role of gap junction communication in simulated preconditioning in cultured neonatal rat cardiac myofibroblasts. Gap junctional intercellular communication was assessed by Lucifer yellow dye transfer. Preconditioning preserved intercellular coupling after prolonged ischemia. An initial reduction in coupling in response to the preconditioning stimulus was also observed. This may protect neighboring cells from damaging substances produced during subsequent regional ischemia in vivo, and may preserve gap junctional communication required for enhanced functional recovery during subsequent reperfusion.     

NO produced by endothelial NO synthase is a mediator of delayed preconditioning-induced endothelial protection

Preconditioning with brief periods of ischemia-reperfusion (I/R) induces a delayed protection of coronary endothelial cells against reperfusion injury. We assessed the possible role of nitric oxide (NO) produced during prolonged I/R as a mediator of this endothelial protection. Anesthetized rats were subjected to 20-min cardiac ischemia/60-min reperfusion, 24 h after sham surgery or cardiac preconditioning (1 x 2-min ischemia/5-min reperfusion and 2 x 5-min ischemia/5-min reperfusion). The nonselective NO synthase (NOS) inhibitor l-NAME, the selective inhibitors of neuronal (7-nitroindazole) or inducible (1400W) NOS, or the peroxynitrite scavenger seleno-l-methionine were administered 10 min before prolonged ischemia. Preconditioning prevented the reperfusion-induced impairment of coronary endothelium-dependent relaxations to acetylcholine (maximal relaxation: sham 77 +/- 3; I/R 44 +/- 6; PC 74 +/- 5%). This protective effect was abolished by l-NAME (41 +/- 7%), whereas 7-NI, 1400W or seleno-l-methionine had no effect. The abolition of preconditioning by l-NAME, but not by selective nNOS or iNOS inhibition, suggests that NO produced by eNOS is a mediator of delayed endothelial preconditioning.     

Intercellular communication and carcinogenesis

Two types of intercellular communication (humoral and cell contact-mediated) are involved in control of cellular function in multicellular organisms, both of them mediated by membrane-embedded proteins. Involvement of aberrant humoral communication in carcinogenesis has been well documented and genes coding for some growth factors and their receptors have been classified as oncogenes. More recently, cell contact-mediated communication has been found to have an important role in carcinogenesis, and some genes coding for proteins involved in this type of communication appear to form a family of tumor-suppressor genes. Both homologous (among normal or (pre-)cancerous cells) as well as heterologous (between normal and (pre)cancerous cells) communications appear to play important roles in cell growth control. Gap junctional intercellular communication (GJIC) is the only means by which multicellular organisms can exchange low molecular weight signals directly from within one cell to the interior of neighboring cells. GJIC is altered by many tumor-promoting agents and in many human and rodent tumors. We have recently shown that liver tumor-promoting agents inhibit GJIC in the rat liver in vivo. Molecular mechanisms which could lead to aberrant GJIC include: (1) mutation of connexin genes; (2) reduced and/or aberrant expression of connexin mRNA; (3) aberrant localization of connexin proteins, i.e., intracytoplasmic rather than in the cytoplasmic membrane; and (4) modulation of connexin functions by other proteins, such as those involved in extracellular matrix and cell adhesion. Whilst mutations of the cx 32 gene appear to be rare in tumors, cx 37 gene mutations have been reported in a mouse lung tumor cell line. Our results suggest that aberrant connexin localization is rather common in cancer cells and that possible molecular mechanisms include aberrant phosphorylation of connexin proteins and lack of cell adhesion molecules. Studies on transfection of connexin genes into tumor cells suggest that certain connexin genes (e.g., cx 26, cx 43 and cx 32) act as tumor-suppressor genes.     

Protective role of gap junctions in preconditioning against myocardial infarction

The aim of the present study was to examine the hypothesis that acceleration of gap junction (GJ) closure during ischemia contributes to anti-infarct tolerance afforded by preconditioning (PC). First, the effects of PC on GJ communication during ischemia were assessed. Isolated buffer-perfused rabbit hearts were subjected to 5-min global ischemia with or without PC with two cycles of 5-min ischemia/5-min reperfusion or a GJ blocker (2 mM heptanol), and then the tissue excised from the ischemic region was incubated in anoxic buffer containing lucifer yellow (LY; 2.5 mg/ml), a tracer of GJ permeability, for 20 min at 37 degrees C. PC and heptanol significantly reduced the area to which LY was transported in the ischemic myocardium by 39% and by 54%, respectively. In the second series of experiments, three GJ blockers (heptanol, 18beta-glycyrrhetinic acid, and 2,3-butanedione monoxime) infused after the onset of ischemia reduced infarct size after 30-min ischemia/2-h reperfusion to an extent equivalent to that in the case of PC. In the third series of experiments, Western blotting for connexin43 (Cx43) showed that PC shortened the time to the onset of ischemia-induced Cx43 dephosphorylation but reduced the extent of Cx43 dephosphorylation during a 30-min period of ischemia. Calphostin C, a protein kinase C (PKC) inhibitor, abolished preservation of phosphorylated Cx43 but not the early onset of Cx43 dephosphorylation after ischemia in the preconditioned myocardium. These results suggest that PC-induced reduction of GJ permeability during ischemia, presumably by PKC-mediated Cx43 phosphorylation, contributes to infarct size limitation.     

A Neuroprotective Role for Gap Junctions

Glial-neuronal interactions have been implicated in both normal information processing and neuroprotection. One pathway of cellular interactions involves gap junctional intercellular communication (GJIC). In astrocytes, gap junctions are composed primarily of the channel protein, connexin43 (Cx43), and provide a substrate for formation of a functional syncytium implicated in the process of spatial buffering in the CNS. Thus gap junctional communication may be neuroprotective following a CNS insult that entails glutamate cytotoxicity (i.e. ischemia). We have shown that blocking gap junctions during a glutamate insult to co-cultures of astrocytes and neurons results in increased neuronal injury. To assess the effect of reduced Cx43 and GJIC on neuroprotection, we examined brain infarct volume in wild type and Cx43 heterozygote null mice following focal ischemia. Cx43 heterozygous null mice exhibited a significantly larger infarct volume compared to wild type. At the cellular level, a significant increase in TUNEL positive cells was observed in the penumbral region of the Cx43 heterozygote mice. These results suggest that augmentation of GJIC in astrocytes may contribute to neuroprotection following ischemic injury. These findings support the hypothesis that gap junctions play a neuroprotective role against glutamate cytotoxicity.     

A neuroprotective role for gap junctions

Glial-neuronal interactions have been implicated in both normal information processing and neuroprotection. One pathway of cellular interactions involves gap junctional intercellular communication (GJIC). In astrocytes, gap junctions are composed primarily of the channel protein, connexin43 (Cx43), and provide a substrate for formation of a functional syncytium implicated in the process of spatial buffering in the CNS. Thus gap junctional communication may be neuroprotective following a CNS insult that entails glutamate cytotoxicity (i.e. ischemia). We have shown that blocking gap junctions during a glutamate insult to co-cultures of astrocytes and neurons results in increased neuronal injury. To assess the effect of reduced Cx43 and GJIC on neuroprotection, we examined brain infarct volume in wild type and Cx43 heterozygote null mice following focal ischemia. Cx43 heterozygous null mice exhibited a significantly larger infarct volume compared to wild type. At the cellular level, a significant increase in TUNEL positive cells was observed in the penumbral region of the Cx43 heterozygote mice. These results suggest that augmentation of GJIC in astrocytes may contribute to neuroprotection following ischemic injury. These findings support the hypothesis that gap junctions play a neuroprotective role against glutamate cytotoxicity.     

Preconditioning decreases ischemia/reperfusion-induced peroxynitrite formation

The role for peroxynitrite (ONOO(-)) in the mechanism of preconditioning is not known. Therefore, we studied effects of preconditioning and subsequent ischemia/reperfusion on myocardial ONOO(-) formation in isolated rat hearts. Hearts were subjected to a preconditioning protocol (three intermittent periods of global ischemia/reperfusion of 5 min duration each) followed by a test ischemia/reperfusion (30 min global ischemia and 15 min reperfusion). When compared to nonpreconditioned controls, preceding preconditioning improved postischemic cardiac performance and significantly decreased test ischemia/reperfusion-induced formation of free nitrotyrosine measured in the perfusate as a marker for cardiac endogenous ONOO(-) formation. During preconditioning, however, the first period of ischemia/reperfusion increased nitrotyrosine formation, which was attenuated after the third period of ischemia/reperfusion. We conclude that classic preconditioning inhibits ischemia/reperfusion-induced cardiac formation of ONOO(-) and that subsequent periods of ischemia/reperfusion result in a gradual attenuation of ischemia/reperfusion-induced ONOO(-) generation. This mechanism might be involved in ischemic adaptation of the heart.     

Cardioprotective action of CRF peptide urocortin against simulated ischemia in adult rat cardiomyocytes

The major objective of this study was to determine whether urocortin, a member of the corticotrophin-releasing factor (CRF) family, protects adult rat cardiomyocytes from ischemia that has been simulated by glucose deprivation and acidosis. When it was present during simulated ischemia, urocortin (0.1 microM) markedly attenuated the cellular injury, which was assessed by increases in creatine kinase and lactate dehydrogenase levels. This effect was comparable with that observed with adenosine (10 microM). The cardioprotective effect of urocortin was markedly attenuated by the protein kinase C inhibitor chelerythrine and by 5-hydroxydecanoate, an inhibitor of ATP-sensitive K(+) channels. Cardiomyocytes were also protected from injury by pretreatment with urocortin, either by incubation for 5 min with a subsequent 10-min recovery or incubation for 20 min with a 20-h recovery before simulated ischemia. Similar cardioprotective effects were observed with ischemic preconditioning protocols during both immediate and delayed phases. In conclusion, in adult cardiomyocytes, urocortin has immediate and delayed cardioprotective actions that mimic ischemic preconditioning. These actions are mediated via protein kinase C and ATP-sensitive K(+) channels.     

Gap Junctional Intercellular Communication as a Biological “Rosetta Stone” in Understanding, in a Systems Biological Manner, Stem Cell Behavior, Mechanisms of Epigenetic Toxicology, Chemoprevention and Chemotherapy

In spite of the early speculation by Loewenstein that one of the critical distinguishing phenotypes of cancers from normal cells was the dysfunction of gap junctional intercellular communication (GJIC), this hypothesis has not captured the attention of most birth defects and cancer researchers. Moreover, even with later demonstrations that factors that influence normal development and carcinogenesis by modulating GJIC, such as chemical teratogens and tumor-promoting chemicals, inflammatory factors, hormones and growth factors, antisense connexin genes, knockout mouse models, human inherited mutated connexin genes, si-connexin RNA, chemopreventive and chemotherapeutic chemicals, it is rare that one sees any reference to these studies by the mainstream investigators in these fields. Based on the assumption that the evolutionarily conserved connexin genes found in metazoans are needed for normal development and the maintenance of health and T. Dobzhansky's statement "Nothing in biology makes sense except in the light of evolution," a short review of the roles of endogenous and exogenous modulators of GJIC will be made in the context of the multistage, multimechanism process of carcinogenesis, the stem cell theory of carcinogenesis, the discovery and characterization of normal adult stem "cancer stem" cells and the observation that two distinct classes of GJIC-deficient cancer cells are known. The implications of these observations to a "systems biological" view of the role of gap junctions and the nutritional prevention and treatment of several chronic diseases and cancer will be discussed.     

The role of C-reactive protein in ischemia/reperfusion injury and preconditioning in a rat model of myocardial infarction

For the first time the involvement of C-Reactive protein (CRP) in early (acute) and delayed ischemic (IPC) and pharmacological (chemical) preconditioning (CPC) in an in vivo model of rat myocardial infarction was presented. Acute IPC was produced by three 5 minute occlusion (ischemia) periods interspersed with 5 minute reperfusion, followed by 30 minute occlusion of the left coronary artery and 2 hour reperfusion injury. Acute CPC was produced by a k-opioid receptor agonist U50488H (5 mg/kg) applied i.v. 15 minutes before 30 minute ischemia/ 2 hour reperfusion. Delayed preconditioning was produced by 30 minute ischemia/ 2 hour reperfusion, induced 24 hour after either ischemic or pharmacological preconditioning. The myocardial ischemia/reperfusion injury was evaluated on the basis of total and cardiac creatine kinase isoenzyme activity, functional recovery of the heart (ECG), infarct size (% IS/RA) and mortality at the end of the experiments. The results obtained showed that: k-opioid receptor agonist U50488H mimics both the acute and delayed IPC in the above experimental protocol; Both acute IPC and most probably CPC act by opening of K(ATP) channels (the effects were blocked by nonspecific ATP-sensitive K channel blocker glybenclamide), and via activation of protein kinase C (a selective protein kinase C inhibitor chelerythrine blocked the efects); C-reactive protein (CRP) was significantly elevated by 54% in non-preconditioned acute ischemia/reperfusion injury. The elevation was more pronounced (82% increase) 24 hour after non-preconditioned ischemia/reperfusion injury. It reflected very well the increase in cardiac isoenzymes, infarct size and mortality of the rats, and can be used as a marker of the severity of myocardial injury in this model; The increase of CRP was prevented by both IPC and CPC in early, and especially in late preconditioning. This confirms the involvement of CRP as a marker in cardiac ischemic/reperfusion injury. It was concluded that in addition to the established involvement of adenosine, bradykinin, opioid and other receptors, a suppression of myocardial CRP/complement production might be involved in the biological mechanism of preconditioning. This could be a promising perspective in clinical interventions against ischemia/reperfusion injuries of the heart.     

Up-regulation of connexin 43 and gap junctional intercellular communication by Coleusin Factor is associated with growth inhibition in rat osteosarcoma UMR106 cells

Gap junctions, formed by connexin (Cx) family proteins, permit direct exchange of regulatory ions and small signal molecules between neighbouring cells. Gap junctional intercellular communication (GJIC) plays an important role in maintaining the homeostasis and preventing cell transformation. Most of the tumour cells feature deficient or aberrant connexin expression and GJIC level, and restoration of connexin expression and GJIC is correlated with cell growth control. Numerous researches has suggested the possibility of connexins as potential anti-tumour targets for chemoprevention and chemotherapy. We investigated the ability of Coleusin Factor (CF, also named FSK88) to regulate the Cx43 expression and GJIC level in rat osteosarcoma UMR106 cells. The results have demonstrated that CF increased the mRNA and protein expression of Cx43 in both in a dose- and timedependent manner, and concomitant with up-regulation of Cx43, CF treatment up-regulated the diminished GJIC level in UMR106 cells as assayed by dye transfer experiments. In addition, Cx43 distribution at the plasma membrane was also enhanced dramatically by CF treatment. Furthermore, we discovered that CF was potent to inhibit the growth and proliferation of UMR106 cells. These results provide the first evidence that CF can regulate connexin and GJIC, indicating that Cx43 may be a target of CF to exert its anti-tumour effects.     

Cytokine effects on gap junction communication and connexin expression in human bladder smooth muscle cells and suburothelial myofibroblasts

Background

The last decade identified cytokines as one group of major local cell signaling molecules related to bladder dysfunction like interstitial cystitis (IC) and overactive bladder syndrome (OAB). Gap junctional intercellular communication (GJIC) is essential for the coordination of normal bladder function and has been found to be altered in bladder dysfunction. Connexin (Cx) 43 and Cx45 are the most important gap junction proteins in bladder smooth muscle cells (hBSMC) and suburothelial myofibroblasts (hsMF). Modulation of connexin expression by cytokines has been demonstrated in various tissues. Therefore, we investigate the effect of interleukin (IL) 4, IL6, IL10, tumor necrosis factor-alpha (TNFα) and transforming growth factor-beta1 (TGFβ1) on GJIC, and Cx43 and Cx45 expression in cultured human bladder smooth muscle cells (hBSMC) and human suburothelial myofibroblasts (hsMF).

Methodology/Principal Findings

HBSMC and hsMF cultures were set up from bladder tissue of patients undergoing cystectomy. In cytokine stimulated cultured hBSMC and hsMF GJIC was analyzed via Fluorescence Recovery after Photo-bleaching (FRAP). Cx43 and Cx45 expression was assessed by quantitative PCR and confocal immunofluorescence. Membrane protein fraction of Cx43 and Cx45 was quantified by Dot Blot. Upregulation of cell-cell-communication was found after IL6 stimulation in both cell types. In hBSMC IL4 and TGFβ1 decreased both, GJIC and Cx43 protein expression, while TNFα did not alter communication in FRAP-experiments but increased Cx43 expression. GJ plaques size correlated with coupling efficacy measured, while Cx45 expression did not correlate with modulation of GJIC.

Conclusions/Significance

Our finding of specific cytokine effects on GJIC support the notion that cytokines play a pivotal role for pathophysiology of OAB and IC. Interestingly, the effects were independent from the classical definition of pro- and antiinflammatory cytokines. We conclude, that connexin regulation involves genomic and/or post-translational events, and that GJIC in hBSMC and hsMF depend of Cx43 rather than on Cx45.     

Role of connexin (gap junction) genes in cell growth control and carcinogenesis

Gap junctional intercellular communication (GJIC) is considered to play a key role in the maintenance of tissue independence and homeostasis in multicellular organisms by controlling the growth of GJIC-connected cells. Gap junction channels are composed of connexin molecules and, so far, more than a dozen different connexin genes have been shown to be expressed in mammals. Reflecting the importance of GJIC in various physiological functions, deletion of different connexin genes from mice results in various disorders, including cancers, heart malformation or conduction abnormality, cataract, etc. The possible involvement of aberrant GJIC in abnormal cell growth and carcinogenesis has long been postulated and recent studies in our own and other laboratories have confirmed that expression and function of connexin genes play an important role in cell growth control. Thus, almost all malignant cells show altered homologous and/or heterologous GJIC and are often associated with aberrant expression or localization of connexins. Aberrant localization of connexins in some tumour cells is associated with lack of function of cell adhesion molecules, suggesting the importance of cell-cell recognition for GJIC. Transfection of connexin genes into tumorigenic cells restores normal cell growth, supporting the idea that connexins form a family of tumour-suppressor genes. Some studies also show that specific connexins may be necessary to control growth of specific cell types. We have produced various dominant-negative mutants of Cx26, Cx32 and Cx43 and showed that some of them prevent the growth control exerted by the corresponding wild-type genes. However, we have found that connexins 32, 37 and 43 genes are rarely mutated in tumours. In some of these studies, we noted that connexin expression per se, rather than GJIC level, is more closely related to growth control, suggesting that connexins may have a GJIC-independent function. We have recently created a transgenic mouse strain in which a mutant Cx32 is specifically overexpressed in the liver. Studies with such mice indicate that Cx32 plays a key role in liver regeneration after partial hepatectomy. A decade ago, we proposed a method to enhance killing of cancer cells by diffusion of therapeutic agents through GJIC. Recently, we and others have shown that GJIC is responsible for the bystander effect seen in HSV-tk/ganciclovir gene therapy. Thus, connexin genes can exert dual effects in tumour control: tumour suppression and a bystander effect for cancer therapy.     

Inhibition of gap junctional intercellular communication in heptachlor- and heptachlor epoxide-treated normal human breast epithelial cells

Based on the concern of organochlorides in the environment and in human tissue, this study was designed to determine whether various noncytotoxic levels of heptachlor and heptachlor epoxide could inhibit, reversibly, gap junctional intercellular communication in human breast epithelial cells (HBEC). Cytotoxicity and gap junctional intercellular communication (GJIC) were evaluated by lactate dehydrogenase assay and fluorescence redistribution after photobleaching analysis, respectively. Both heptachlor and heptachlor epoxide were noncytotoxic up to 10 μg/ml. At this concentration, heptachlor and heptachlor epoxide inhibited GJIC of normal human breast epithelial cells after 1 h treatment. Within a 24 h treatment with heptachlor and heptachlor epoxide at 10 μg/ml, recovery of GJIC had not returned. GJIC completely recovered after a 12 h treatment of 1 μg/ml heptachlor epoxide, but it did not recover after a 24 h treatment of 1 μg/ml heptachlor. RT-PCR and Western blots were analyzed to determine whether the heptachlor or heptachlor epoxide might have altered the steady-state levels of gap junction mRNA and/or connexin protein levels or phosphorylation state. No significant difference in the level of connexin 43 (Cx43) message between control and heptachlor-treated cells was observed. Western blot analyses showed hypophosphorylation patterns in cells treated with 10 μg/ml heptachlor and heptachlor epoxide for 1 h with no recovery within 24 h. Immunostaining of Cx43 protein in normal HBEC indicated that heptachlor and heptachlor epoxide caused a loss of Cx43 from the cell membranes at noncytotoxic dose levels. Taken together, these results suggest that heptachlor and heptachlor epoxide can alter GJIC at the post-translational level, and that, under the conditions of exceeding a threshold concentration in the breast tissue containing ‘initiated’ cells for a long time and not being counteracted by anti-tumor-promoting chemicals, they could act as breast tumor promoters.     

Involvement of gap junctions in tumor suppression: analysis of genetically-manipulated mice

Accumulating evidence indicates that gap junctions play an important role in the maintenance of normal cell growth, so that genes for the connexin gap junction proteins form a family of tumor-suppressor genes. Although mice from which nine types of connexin gene are deleted have been established, little information from carcinogenesis experiments with these mice is available. We have previously found several mutant forms of connexin 32 (Cx32) to be able to inhibit, in a dominant-negative manner, gap junctional intercellular communication (GJIC) exerted by wild-type Cx32. By introducing a gene for such a dominant-negative Cx32 mutant expressed under the control of a liver-specific albumin gene promoter, we have generated transgenic mouse lines in which the function of Cx32 is down-regulated only in the liver. Although GJIC was diminished in the transgenic liver as expected, the reduced GJIC did not affect viability nor the number of spontaneous liver tumors. Although susceptibility to diethylnitrosamine-induced hepatocarcinogenesis was significantly elevated in the transgenic mice, liver regeneration after partial hepatectomy was delayed compared with wild-type mice, suggesting that gap junctions function not only to suppress excessive cell growth but also to promote cell proliferation when necessary for normal function of tissues. Although the phenotype of Cx32-deficient mice was similar to that of the transgenic mice, the former showed more drastically altered phenotypes, i.e. increased BrdU incorporation in the quiescent liver and development of spontaneous liver tumors. We also established 3T3 fibroblasts from embryos lacking the Cx43 gene and characterized their growth. These fibroblasts showed no difference from the wild type in growth characteristics. From these and other studies, we suggest that gap junctions do not necessarily suppress cell growth but support an optimal growth rate.     

Gap junctional intercellular communication is required to maintain embryonic stem cells in a non-differentiated and proliferative state

Pluripotent embryonic stem (ES) cells are capable of maintaining a self-renewal state and have the potential to differentiate into derivatives of all three embryonic germ layers. Despite their importance in cell therapy and developmental biology, the mechanisms whereby ES cells remain in a proliferative and pluripotent state are still not fully understood. Here we establish a critical role of gap junctional intercellular communication (GJIC) and connexin43 (Cx43) in both processes. Pharmacological blockers of GJIC and Cx43 down-regulation by small interfering RNA (siRNA) caused a profound inhibitory effect on GJIC, as evidenced by experiments of fluorescence recovery after photobleaching. This deficient intercellular communication in ES cells induced a loss of their pluripotent state, which was manifested in morphological changes, a decrease in alkaline phosphatase activity, Oct-3/4 and Nanog expression, as well as an up-regulation of several differentiation markers. A decrease in the proliferation rate was also detected. Under these conditions, the formation of embryoid bodies from mouse ES cells was impaired, although this inhibition was reversible upon restoration of GJIC. Our findings define a major function of GJIC in the regulation of self-renewal and maintenance of pluripotency in ES cells.     

Development of a prototype reporter-based assay for the evaluation of gap junctional intercellular communication

A new reporter-based assay for the evaluation of gap junctional intercellular communication (GJIC) is presented. This assay was applied to the study of endogenous GJIC as well as to the evaluation of cell-to-cell communication exogenously induced in non-coupling cells by transfection with connexin 32. The results obtained with 18--glycyrrhetinic acid indicate that this assay system can be used to monitor the GJIC induced by transport of cAMP induced by activation of the dopamine 1 receptor cascade.     

Activation of adenylate cyclase system in the preconditioned rat heart

Ischemic preconditioning (IP) protects the heart against subsequent prolonged ischemia. Whether the beta-adrenoceptor/adenylate cyclase pathway contributes to this cardioprotection is not yet fully known. Using enzyme catalytic cytochemistry we studied the adenylate cyclase activity and its distribution in the preconditioned rat heart. Adenylate cyclase activity was examined in Langendorff-perfused rat hearts subjected to the following conditions: control perfusion; 30 min regional ischemia; 5 min occlusion and 10 min reperfusion (IP); IP followed by ischemia. Ischemia-induced arrhythmias and the effect of ischemic preconditioning on the incidence of arrhythmias were analyzed. At the end of experiment the heart was shortly prefixed with glutaraldehyde. Tissue samples from the left ventricle were incubated in a medium containing the specific substrate AMP-PNP for adenylate cyclase and then routinely processed for electron microscopy. Adenylate cyclase activity was cytochemically demonstrated in the sarcolemma and the junctional sarcoplasmic reticulum (JSR) in control hearts, while it was absent after test ischemia. The highest activity of the precipitate was observed after ischemic preconditioning. In the preconditioned hearts followed by test ischemia, adenylate cyclase activity in the precipitate was preserved in sarcolemma and even more in JSR. Protective effect of ischemic preconditioning was manifested by the suppression of severe arrhythmias. These results indicate the involvement of the adenylate cyclase system in mechanisms underlying ischemic preconditioning.