Physiological effects of heat stress on Hawaiian picture-wing Drosophila: genome-wide expression patterns and stress-related traits.

Climate change is compounding the threats to the future of biodiversity, already impacted by habitat loss, invasive species and diseases. In the Hawaiian Islands, many of the endemic species have narrow habitat ranges that make them especially vulnerable to climate change. The Hawaiian Drosophila, a remarkably diverse group of species with 11 listed as federally endangered, are thought to be sensitive to temperature changes. To examine the species differences in sensitivity of Hawaiian picture-wing Drosophila to temperature changes, wild populations of Drosophila sproati, a relatively common species, and Drosophila silvestris, a rare species, were collected from two locations on Hawaii Island and bred in common laboratory conditions. Adult flies were exposed to hot and cold temperatures and compared with adult flies at control temperatures. Drosophila silvestris adults were less tolerant to heat stress than D. sproati for both survival and sperm mobility. In contrast, D. silvestris adults were more tolerant to cold stress than D. sproati for adult survival. The expression of 4950 Gene Ontology annotated gene transcripts was also analysed in high-temperature-treated and control males to identify candidate genes related to heat tolerance. There were more than twice as many transcripts differentially expressed after high temperature treatment for D. silvestris (246 transcripts) as for D. sproati (106 transcripts), with 13 Gene Ontology terms enriched between temperatures for D. silvestris and merely three in D. sproati. The combined results are consistent with D. sproati occurring more widely today as well as occurring at lower elevations than D. silvestris and with a genetically based temperature response, which is more severe in D. silvestris at high temperatures than that in D. sproati. These experiments demonstrate the potential for different capacities of species to adapt to future climate change conditions as well as providing an explanation for historical changes in the distribution of species.

Small cell carcinoma of the cervix in liquid-based Pap test: utilization of split-sample immunocytochemical and molecular analysis.

Small cell (neuroendocrine) carcinoma of the uterine cervix (SMCC) is a rare, highly aggressive malignant neoplasm. Both conventional and liquid-based cytology (LBC) cervical smears have low sensitivity in diagnosing SMCC, requiring immunocytochemical (ICH) confirmation. We present the first series of SMCC primarily diagnosed in cytology specimens, and ICH studies performed on the residual LBC specimens with subsequent confirmation of the diagnosis on surgical pathology specimens. Immunocytochemical stains for keratin, p16INK4, and neuroendocrine markers (synaptophysin, chromogranin, CD56) were performed on additional ThinPrep slides. HPV test used chromogenic in situ hybridization high risk HPV DNA probe. The Pap smears in all three specimens were highly cellular with a mixture of squamous cells and numerous well-preserved single or small cohesive clusters of malignant epithelial cells. Tumor cells were small, monomorphic with minimal cytoplasm and high nuclear/cytoplasmic ratio. There was significant nuclear overlap, but no nuclear molding, or smudging of nuclear chromatin. The chromatin pattern was stippled. A background tumor diathesis was prominent. Atypical squamous cells of undetermined significance (ASCUS) were noted in one case, and markedly abnormal squamous cells were seen in another case. The main cytology differential diagnoses included high-grade squamous intraepithelial lesion and an endometrial adenocarcinoma. Immunocytochemical positivity for the neuroendocrine markers supported the diagnoses of SMCC in all three cases. The morphologic features of the concurrent surgical pathology specimens were typical of SMCC. The tissue diagnoses were also confirmed by immunohistochemistry. Our study allows us to conclude that SMCC can be primarily diagnosed in LBC specimens using a panel of immunocytochemical stains.

Ischemic loss of sarcolemmal dystrophin and spectrin: correlation with myocardial injury.

Sarcolemmal blebbing and rupture are prominent features of irreversible ischemic myocardial injury. Dystrophin and spectrin are sarcolemmal structural proteins. Dystrophin links the transmembrane dystroglycan complex and extracellular laminin receptors to intracellular F-actin. Spectrin forms the backbone of the membrane skeleton conferring an elastic modulus to the sarcolemmal membrane. An ischemic loss of membrane dystrophin and spectrin, in ischemically pelleted rabbit cardiomyocytes or in vivo 30--45 min permanently ischemic, LAD-ligated hearts, was detected by immunofluorescence with monoclonal antibodies. Western blots of light and heavy microsomal vesicles and Triton-extracted membrane fractions from ischemic myocytes demonstrated a rapid loss of dystrophin coincident with sub-sarcolemmal bleb formation, subsequent to a hypotonic challenge. The loss of spectrin from purified sarcolemma of autolysed rabbit heart, and both isolated membrane vesicles and Triton solubilized membrane fractions of ischemic cardiomyocytes correlated linearly with the onset of osmotic fragility as assessed by membrane rupture, subsequent to a hypotonic challenge. In contrast to the ischemic loss of dystrophin and spectrin from the membrane, the dystrophin-associated proteins, alpha-sarcoglycan and beta-dystroglycan and the integral membrane protein, sodium-calcium exchanger, were maintained in the membrane fraction of ischemic cells as compared to oxygenated cells. Preconditioning protected cells, but did not significantly alter ischemic dystrophin or spectrin translocation. This previously unrecognized loss of sarcolemmal dystrophin and spectrin may be the molecular basis for sub-sarcolemmal bleb formation and membrane fragility during the transition from reversible to irreversible ischemic myocardial injury.

Sarcolemmal blebs and osmotic fragility as correlates of irreversible ischemic injury in preconditioned isolated rabbit cardiomyocytes.

The hypothesis that irreversible ischemic injury is related to sub-sarcolemmal blebbing and an inherent osmotic fragility of the blebs was tested by subjecting isolated control and ischemically preconditioned (IPC) or calyculin A (CalA)-pretreated (protected) rabbit cardiomyocytes to ischemic pelleting followed by resuspension in 340, 170 or 85 mosmol medium containing trypan blue. At time points from 0-240 min, osmotic fragility was assessed by the percentage of trypan blue permeable cells. Membrane blebs were visualized with India ink preparations. Bleb formation, following acute hypo-osmotic swelling, developed by 75 min and increased with longer periods of ischemia. Osmotic fragility developed only after 75 min. Cells resuspended in 340 mosmol media did not form blebs and largely retained the ability to exclude trypan blue, even after 240 min ischemia. Although the latent tendency for osmotic blebbing preceded the development of osmotic fragility, most osmotically fragile cells became permeable without evident sarcolemmal bleb formation. The onset of osmotic fragility was delayed in protected cells, but protection did not reduce the bleb formation. It is concluded that blebbing and osmotic fragility are independent manifestations of ischemic injury. The principal locus of irreversible ischemic injury and the protection provided by IPC may lie within the sarcolemma rather than at sarcolemmal attachments to underlying adherens junctions.

Differential translocation or phosphorylation of alpha B crystallin cannot be detected in ischemically preconditioned rabbit cardiomyocytes.

Alpha B Crystallin (alpha BC) is a putative effector protein of ischemic preconditioning (IPC), that is phosphorylated on Ser 45 by ERK1/2 and Ser 59 by the p38 MAPK substrate, MAPKAPK-2. Translocation and phosphorylation of alpha BC was determined in cytosolic and cytoskeletal fractions by 1D SDS-PAGE and IEF, or using Ser 45 and Ser 59 phospho-specific antibodies in: (1) control rabbit cardiomyocytes; (2) cells preconditioned by 10 min in vitro ischemia; or after pre-treatment with specific inhibitors of (3) Ser/Thr protein phosphatase 1/2A (calyculin A); (4) p38 MAPK (SB203580); or (5) ERK 1/2 (PD98059); all prior to 180 min ischemia. Ischemia induced a cytosolic to cytoskeletal translocation of alpha BC, which was similar in all the groups. Highly phosphorylated isoforms (D1/2) of alpha BC were present in cytosolic but not cytoskeletal fractions at 0 min ischemia. By 60-90 min ischemia, D1/2 isoforms had translocated to the cytoskeletal fraction. Calyculin A maintained D1/2 levels throughout prolonged ischemia. SB203580 decreased alpha BC phosphorylation. Neither PD98059 nor IPC altered alpha BC phosphorylation during prolonged ischemia. It is concluded that alpha BC phosphorylation during ischemia is regulated by p38 MAPK but not by ERK 1/2. The inability to detect a correlation between IPC protection and either alpha BC translocation or phosphorylation suggests that the proteins in the highly phosphorylated isoform bands of alpha BC quantitated in this study are not protective end effectors of classical IPC.

Phosphorylation state of hsp27 and p38 MAPK during preconditioning and protein phosphatase inhibitor protection of rabbit cardiomyocytes.

Small heat shock proteins (hsp) have been implicated in mediation of classic preconditioning in the rabbit, Hsp27 is a terminal substrate of the p38 MAPK cascade. One and 2D gel electrophoresis and immunoblotting of cell fractions was used to determine p38 MAPK and hsp27 phosphorylation levels, respectively, during in vitro ischemia in control, calyculin A (Cal A)-treated (protein phosphatase inhibitor), SB203580-treated (p38MAPK inhibitor) and preconditioned (IPC) isolated adult rabbit cardiomyocytes. The dual phosphorylation of p38 MAPK was increased by early ischemia (30-60 min), after which there was a loss of total cytosolic p38 MAPK. The ischemic increase of p38 MAPK dual phosphorylation was enhanced by IPC. Cal A strongly activated dual phosphorylation of p38 MAPK in oxygenated cells and this was maintained into early ischemia, SB203580 inhibited the dual phosphorylation of p38 MAPK and attenuated the loss of total cytosolic p38 MAPK. In each protocol, ischemia translocated hsp27 from the cytosolic fraction to the cytoskeletal fraction at similar rates and extents, Hsp27 phosphorylation was quantitated as the fraction of diphosphorylated hsp27, based on IEF mobility shifts of hsp27 phosphorylation isoforms. In oxygenated control cells, cytosolic and cytoskeletal hsp27 was highly phosphorylated. After 90 min ischemia, cytoskeletal hsp27 was markedly dephosphorylated. Cal A slightly increased control cytoskeletal hsp27 phosphorylation. During ischemic incubation, Cal A blocked ischemic dephosphorylation, SB203580 accelerated ischemic hsp27 dephosphorylation and injury, IPC insignificantly decreased the initial rate of ischemic dephosphorylation of hsp27, but not the extent of dephosphorylation in later ischemia. Phosphorylation is regulated by both kinase and phosphatase activities. IPC protection was not correlated with a significant increase in cytosolic or cytoskeletal hsp27 phosphorylation levels during prolonged (> 60-90 min) ischemia.

Fostriecin, an inhibitor of protein phosphatase 2A, limits myocardial infarct size even when administered after onset of ischemia.

BACKGROUND: The role of protein phosphatases (PPs) during ischemic preconditioning in the rabbit heart was examined. METHODS AND RESULTS: Fostriecin, a potent inhibitor of PP2A, was administered to isolated rabbit hearts starting either 15 minutes before or 10 minutes after the onset of a 30-minute period of regional ischemia and continuing until the onset of reperfusion. After 2 hours of reperfusion, infarct size was measured with triphenyltetrazolium chloride. In a second study with isolated rabbit cardiomyocytes, the effect of fostriecin pretreatment was assessed by measuring changes in cell osmotic fragility during simulated ischemia. PP1 and PP2A activities of isolated control and ischemically preconditioned cells were also measured. In a third series of experiments, left ventricular biopsies of isolated rabbit hearts were obtained before and at selected times during 60 minutes of global ischemia, and the tissue was assayed for PP1 and PP2A activities. In isolated hearts pretreated with fostriecin, only 8% of the ischemic zone infarcted, significantly less than that in untreated control hearts (33%; P<0.001) but comparable to that in ischemically preconditioned hearts (9%; P<0.001 versus control). Significant protection was also observed in the hearts treated only after the onset of ischemia (18% infarction; P<0.05 versus control). In isolated myocytes, fostriecin also provided protection comparable to that produced by metabolic preconditioning. Preconditioning had no apparent effect on the activity of either PP1 or PP2A in isolated ventricular myocytes or ventricular tissue obtained from heart biopsies. CONCLUSIONS: Fostriecin, a potent inhibitor of PP2A, can protect the rabbit heart from infarction even when administered after the onset of ischemia. But inhibition of either PP1 or PP2A does not appear to be the mechanism of protection from ischemic preconditioning.

Protein phosphatase inhibitors calyculin A and fostriecin protect rabbit cardiomyocytes in late ischemia.

Calcium-tolerant rabbit cardiomyocytes were isolated using retrograde aortic perfusion with a nominally calcium-free, collagenase buffer. In vitro ischemic preconditioning was induced by a 10-min episode of ischemic pelleting, followed by a 15-min post-incubation and a prolonged period of ischemic pelleting. Injury was assessed by determination of cell contracture and trypan blue permeability following hypotonic swelling and correlated with metabolic assays of lactate and adenine nucleotides. The protein phosphatase PP1/2A inhibitor calyculin A and PP2A-selective fostriecin protected isolated rabbit cardiomyocytes from lethal injury after a 10-min pre-incubation and when added late into ischemic pellets after a delay of 75 min. At the time of late drug addition, cells were severely ATP-depleted and in rigor contracture. Protection with Calyculin A from 1 nM to 1 microM was dose-related. Cells pre-incubated with 10 nM to 10 microM fostriecin 10 min prior to ischemic pelleting were protected with an EC50 approximating 71 nM, implying protection at a PP2A-selective dose. The selective protein kinase C inhibitor, calphostin C, blocked ischemic preconditioning protection but not protection from 1 microM calyculin A. Protection of severely ischemic cardiomyocytes following protein phosphatase inhibition appears not to require PKC activity or ATP conservation. Pre-incubation of cells with calyculin A induced high levels of phosphorylation in p38 mitogen activated protein kinase (MAPK), as compared to the ischemia-induced phosphorylation observed in the untreated group only at 30 min of ischemia, providing evidence of protein phosphatase activity in cardiomyocytes. Pharmacological protection in late ischemia has been demonstrated, but the mechanism of protection is undetermined.

Preconditioning of isolated rabbit cardiomyocytes: no evident separation of induction, memory and protection.

Cardiomyocytes isolated from rabbit hearts were preconditioned in vitro by 10 min of ischemia or treatment with 100 microM adenosine. Protection was assessed as average integrated mortality following osmotic swelling and determination of viability by trypan blue exclusion over 60-180 min ischemia. Repetitive sub-maximal stimulations with 1 microM adenosine amplified the protective response. Treatment with adenosine only at the onset of prolonged ischemia afforded a dose-dependent protection. The PKC inhibitor calphostin C (500 nm) blocked preconditioning and, when added during ischemic incubation of non-preconditioned cells, significantly increased injury. The memory of adenosine-induced preconditioning decayed over a 60 min post-incubation period. Light activation of calphostin C initially added to preconditioned ischemic cells in the dark indicated that a 10 min period of PKC activity at the onset of ischemia affords full protection. The reversible PKC inhibitors chelerythrine (5 microM) or staurosporine (100 nM) added only to bracket induction of ischemia, reduced but did not abolish protection. Protection was abolished when either drug was present during induction and a subsequent 30 min post-incubation period. Staurosporine included during initiation and post-incubation but washed out in the final 5 min of post-incubation allowed significant protection to occur. It is concluded that a single adenosine receptor-stimulation induces protection as it preconditions, and PKC activity appears to be required for both induction and protection. Memory may reside in post-receptor amplification of an initial protective response.

Comparison of in vitro preconditioning responses of isolated pig and rabbit cardiomyocytes: effects of a protein phosphatase inhibitor, fostriecin.

Calcium tolerant pig and rabbit cardiomyocytes were isolated using retrograde aortic perfusion of nominally calcium-free collagenase. Preconditioning protocols used 1 or 3x10-min episodes of ischemic pelleting or pre-incubation with 100 micro M adenosine, followed by a 15-min post-incubation and 180-240-min ischemic pelleting. Control cells were incubated and washed in parallel with the experimental groups. Injury was assessed by determination of cell morphology, trypan blue permeability following osmotic swelling, lactate and HPLC analysis of adenine nucleotides. Preconditioned pig cardiomyocytes had a reduced rate of ischemic contracture, but protection occurred without conservation of ATP. Preconditioned rabbit cardiomyocytes were protected without significant changes in rates of ischemic contracture or ATP depletion. Incubation of ischemic cells with the protein phosphatase inhibitor, fostriecin, at PP2A-selective concentrations (0.1-10 micro M), mimicked preconditioning in both rabbit and pig cardiomyocytes. In rabbits, the KATP channel blocker, 5-hydroxydecanoate (5-HD), did not block preconditioning or fostriecin protection. In the pig, 5-HD blocked both preconditioning and fostriecin protection, with return of the rates of ischemic contracture to control. However, 5-HD was an effective blocker of protection only in early ischemia. Fostriecin mimicked preconditioning in the rabbit and the early responses of the preconditioned pig. Preconditioning appears associated with protein phosphorylation in both the rabbit and the pig, but major pathways leading to protection may differ in the two species.

Translocation of PKC, protein phosphatase inhibition and preconditioning of rabbit cardiomyocytes.

This study was designed to test the hypothesis that induction of the preconditioned state results in a sustained translocation of protein kinase C (PKC) which accounts for the memory associated with preconditioning. Isolated rabbit cardiomyocytes were subjected to established preconditioning protocols using either adenosine or transient ischemia. At timed intervals during induction of preconditioning (PC), post-incubation or final sustained ischemia, cells were harvested, subjected to digitonin lysis and separated into cytosolic and particulate fractions. Samples were evaluated by Western blot analysis with monoclonal antibodies to alpha, epsilon, zeta and gamma PKC isozymes, and bands were qualified by densitometry. Internal controls for each experiment included oxygenated cardiomyocytes and cell with PKC translocation evoked by treatment with phorbol 12-myristate 13-acetate (PMA). For control oxygenated cells, the particulate fraction contained about 30% of PKC epsilon, 5-10% of PKC alpha and 60-70% of PKC zeta. Preconditioning with adenosine (100 microM) or 10 min ischemia had no significant effect on these percentages. Furthermore, the relative amounts of PKC isozymes associated with the particulate fraction of control and preconditioned cells did not differ after a postincubation in oxygenated buffer or during a final ischemic incubation. PMA and ingenol completely translocated the epsilon and alpha isoforms, while thymeleatoxin totally translocated PKC alpha but only partially (50%) translocated PKC epsilon. The distribution of PKC zeta between fractions was not affected by any drug. The protein phosphatase inhibitor calyculin A protected cells mimicking preconditioning. This protection was blocked by preincubation with the selective PKC inhibitor calphostin C but was largely retained if calphostin C was added only during the final ischemic period. It is concluded that PKC activity is required for preconditioning, but a sustained translocation of PKC above basal levels is not necessary for protection of rabbit cardiomyocytes in vitro.

Concentration-response relationships for adenosine agonists during preconditioning of rabbit cardiomyocytes.

Although adenosine receptors have been implicated in the induction of preconditioning in a variety of experimental models, there is controversy concerning the specific adenosine receptor subtypes mediating this effect. Concentration-protection relationships for adenosine and adenosine agonists in rabbit cardiomyocytes were used to characterize the role of adenosine receptor subtypes in preconditioning. Isolated cells were ischemically preconditioned or pre-incubated for 10 min with increasing concentrations of adenosine, CCPA (2-chloro-N6-cyclopentyladenosine), APNEA (N6-2-(4-aminophenyl)ethyladenosine), or BNECA (N6-benzyl-5'-N-ethyl-carboxamidoadenosine) in the presence or absence of 1 or 10 microM of the selective A1-adenosine antagonist DPCPX (8-Cyclopentyl-1,3-dipropylxanthine). Following a 30-min post-incubation period, cells were pelleted, layered with oil and ischemically incubated for 180 min. Injury was assessed by osmotic swelling and trypan blue exclusion of sequential samples, and determination of the areas beneath the mortality curves. Adenosine produced a broad concentration-protection curve which was displaced to the right by DPCPX. The curve for A1-selective agonist CCPA was biphasic, with an initial response below 1 nM and a second above 1 microM. DPCPX abolished the early response leaving a steep monophasic curve between 0.1 and 10 microM CCPA. The APNEA curve appeared moriophasic, the major slope occurring between 1-100 nM; DPCPX (1 microM) shifted the concentration-response curve approximately 30-fold and decreased the slope. Adenosine receptor agonist BNECA produced preconditioning characterized by a shallow monophasic concentration-protection curve with a maximal effect of 49% and an EC50 of approximately 5 nM; DPCPX shifted the BNECA concentration-protection relationship approximately 40-fold with only a modest increase in slope. Analysis of the data suggests that induction of preconditioning results from interaction of agonists with the A1 receptor and a second adenosine receptor having properties consistent with the A3 receptor. Adenosine, CCPA, APNEA, BNECA and DPCPX each appear to be selective for the A1 adenosine receptor subtype in isolated rabbit cardiomyocytes.

Potassium channels and preconditioning of isolated rabbit cardiomyocytes: effects of glyburide and pinacidil.

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 degrees 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 microM) 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 microM) 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 microM) 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 microM) 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 microM) 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.

In vitro ischaemic preconditioning of isolated rabbit cardiomyocytes: effects of selective adenosine receptor blockade and calphostin C.

OBJECTIVE: The aim was to determine if in vitro ischaemic preincubation can precondition cardiomyocytes and if the responses to adenosine receptor antagonists are similar to those previously determined during "metabolic" preconditioning with glucose deprivation or adenosine agonists. METHODS: Isolated rabbit cardiomyocytes were preconditioned with 10 min of ischaemic preincubation, followed by a 30 min postincubation before the final sustained ischaemic period. The protein kinase C inhibitor calphostin C or the adenosine receptor antagonists 8-sulphophenyltheophylline (SPT), BW 1433U, and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) were added either during the preincubation or into the final ischaemic pellet. Adenosine deaminase (10 U.ml-1) was added during ischaemic preincubation. Rates of contracture and extent of injury were determined by sequential sampling and assessment of trypan blue permeability following 85 mOsM swelling. RESULTS: Myocytes were preconditioned by a 10 min in vitro ischaemic preincubation. Preincubation with 100 microM SPT or with adenosine deaminase, or addition of 200 nM calphostin C into the final ischaemic pellet did not alter rates of rigor contracture but nearly abolished protection. A significant degree of protection was maintained following ischaemic preincubation with the highly selective adenosine A1 receptor blocker DPCPX (10 microM), while the A1/A3 antagonist BW 1433U (1 microM) severely limited protection. SPT and BW 1433U added only into the final ischaemic pellet of preconditioned cells significantly blocked protection, while protection was maintained in the presence of DPCPX. CONCLUSIONS: Ischaemic preconditioning of cardiomyocytes is blocked by adenosine receptor antagonists known to bind to A3 receptors but not by DPCPX which has high affinity for A1 receptors, but little affinity for A3 receptors. Maintenance of protection during the final ischaemic phase has a similar receptor specificity. Blockade of protein kinase C activity abolishes protection. Ischaemic and metabolic preconditioning in vitro appear to occur through similar pathways.

Preconditioning of isolated rabbit cardiomyocytes: effects of glycolytic blockade, phorbol esters, and ischaemia.

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 epsilon, delta-PKC isozyme selective activator, protected cells, but thymeleatoxin, an alpha, beta, gamma-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 epsilon-PKC isoform. (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.

Adenosine receptor specificity in preconditioning of isolated rabbit cardiomyocytes: evidence of A3 receptor involvement.

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 microM of the non-specific adenosine A1/A2 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 microM, but did at 100 microM. The selective A2 agonist CGS 12680 (1 microM) did not precondition. The selective A1/A3 adenosine agonist, APNEA, preconditioned at 1 microM 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 microM of the A1 antagonist DPCPX, which has extremely low affinity for A3 receptors, but was blocked by 1 microM 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.

Preconditioning of isolated rabbit cardiomyocytes: induction by metabolic stress and blockade by the adenosine antagonist SPT and calphostin C, a protein kinase C inhibitor.

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.

Adenosine and A1 selective agonists offer minimal protection against ischaemic injury to isolated rat cardiomyocytes.

OBJECTIVE: The aim was to determine if isolated rat cardiomyocytes 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.

Ischaemia and the myocyte cytoskeleton: review and speculation.

The cardiomyocyte cytoskeleton is composed of a highly organised complex array of specific proteins, arranged to transmit mechanical forces within the cell, to adjacent cells and the extracellular matrix, as well as to maintain internal organisation of cellular organelles. Although most of the published reports on cytoskeletal proteins refer to non-myocyte and smooth muscle cells, there seem significant homologies with cardiac structures. The specific interactions of some proteins in certain cytoskeletal structures are established and may be analogous to interactions in cardiac myocytes, but the roles of many proteins are uncertain, and the list of proteins that compose the cytoskeleton is likely to be incomplete. Some proteins may serve a dual role, contributing to signal transduction as well as to organisation and mechanical stability of the cell. Phosphorylation of cytoskeletal proteins, and elaborate cellular systems to control protein phosphorylation levels, suggest phosphorylation as a potential mechanism of controlling cytoskeletal assembly and remodelling. Disturbances of the cytoskeleton during ischaemia may produce alterations in cell structural integrity that could account for cell injury and death. Although mechanisms both of cytoskeletal assembly in normal cells and of cytoskeletal injury in ischaemic cells are currently poorly understood, research into the interactions of cytoskeletal proteins during ischaemia includes new approaches that may increase our understanding of the pathophysiology of the cardiac myocyte.