Treatment with crocin improves cardiac dysfunction by normalizing autophagy and inhibiting apoptosis in STZ-induced diabetic cardiomyopathy.

BACKGROUND AND AIM: The association of diabetes mellitus (DM) and poor metabolic control with high incidence of cardiovascular diseases is well established. The aim of this study was to investigate the potential cardioprotective effect of crocin (Crocus sativus L. extract) on diabetic heart dysfunction and to elucidate the mediating molecular mechanisms. METHODS AND RESULTS: Streptozotocin (STZ)-induced diabetic rats were treated with two different concentrations of crocin (10 or 20 mg/kg), while isolated cardiac myocytes exposed to 25 mM glucose, were treated with 1 or 10 µM of crocin. Treatment of STZ-diabetic rats with crocin resulted in normalization of plasma glucose levels, inhibition of cardiac hypertrophy and fibrosis, and improvement of cardiac contractile function. Heat Shock Response was enhanced. Myocardial AMPK phosphorylation was increased after treatment with crocin, resulting in normalization of autophagy marker proteins (LC3BII/LC3BI ratio, SQSTM1/p62 and Beclin-1), while the diabetes-induced myocardial apoptosis was decreased. Similar results regarding the effect of crocin on autophagy and apoptosis pathways were obtained in isolated cardiac myocytes exposed to high concentration of glucose. CONCLUSION: The results suggest that crocin improves the deteriorated cardiac function in diabetic animals by enhancing the heat shock response, inhibiting apoptosis and normalizing autophagy in cardiac myocytes. Thus, treatment with crocin may represent a novel approach for treating diabetic cardiomyopathy.

Mass transfer kinetics and quality attributes of osmo-dehydrated candied pumpkins using nutritious sweeteners.

The aim of this study was to investigate the mass transfer and quality properties changes during the osmotic dehydration (OD) step of the candying process in pumpkins. The goal was to obtain nutritious, low calorie candied pumpkins improving the time-consuming and inconsistent traditional technique. The osmotic agents were sucrose, oligofructose and mixture of sucrose-oligofructose (1:1), while the concentration of each solution was constant (70° Brix). The process temperature varied in three levels (75, 85 and 95 °C) and the duration was 180 min for sucrose and 240 min for the other osmotic agents. The determined parameters during OD include solid gain, water loss, water activity, chroma, hardness and compression work. An empirical model based on a first-order kinetic equation was developed to predict the products' properties, in which the rate constant is a function of the process temperature. The process temperature (Tosm ) had a significant effect on the water loss and solid gain as well as on the physiochemical characteristics of processed pumpkins. The chroma of osmo-dehydrated pumpkins was affected significantly by process parameters. Both hardness and compression work decreased until an equilibrium value was reached as time and temperature of the process increased, regardless the osmotic agent used.

Remote preconditioning as a novel "conditioning" approach to repair the broken heart: potential mechanisms and clinical applications.

Remote ischemic preconditioning (RIPC) is a novel strategy of protection against ischemia-reperfusion (IR) injury in the heart (and/or other organs) by brief episodes of non-lethal IR in a distant organ/tissue. Importantly, RIPC can be induced noninvasively by limitation of blood flow in the extremity implying the applicability of this method in clinical situations. RIPC (and its delayed phase) is a form of relatively short-term adaptation to ischemia, similar to ischemic PC, and likely they both share triggering mechanisms, whereas mediators and end-effectors may differ. It is hypothesized that communication between the signals triggered in the remote organs and protection in the target organ may be mediated through substances released from the preconditioned organ and transported via the circulation (humoral pathways), by neural pathways and/or via systemic anti-inflammatory and antiapoptotic response to short ischemic bouts. Identification of molecules involved in RIPC cascades may have therapeutic and diagnostic implications in the management of myocardial ischemia. Elucidation of the mechanisms of endogenous cardioprotection triggered in the remote organ could lead to the development of diverse pharmacological RIPC mimetics. In the present article, the authors provide a short overview of RIPC-induced protection, proposed underlying mechanisms and factors modulating RIPC as a promising cardioprotective strategy.

Antioxidant potential and quality characteristics of vegetable-enriched corn-based extruded snacks.

Phenolic content, antioxidant activity and sensory characteristics of vegetable-enriched extrudates were investigated as a result of extrusion conditions, including extrusion temperature (140-180 °C), screw rotation speed (150-250 rpm) and feed moisture content (14-19 % w.b.). Broccoli flour and olive paste was used in mixtures with corn flour at a ratio of 4 to 10 % (broccoli/corn) and 4 to 8 % (olive paste/corn). A simple power model was developed for the prediction of phenolic content and antioxidant activity of extrudates by extrusion conditions and feed composition. Phenolic content and antioxidant activity of broccoli enriched extrudates increased with extrusion temperature and broccoli addition and decreased with feed moisture content. The antioxidant activity of olive paste extrudates increased with material ratio and decreased with feed moisture content and screw rotation. Sensory porosity, homogenous structure, crispness, cohesiveness and melting decreased with feed moisture content, while the latter increased the mealy flavor and hardness of extrudates. Acceptable snacks containing broccoli flour or olive paste can be produced by selecting the appropriate process conditions.

Upregulation of genes involved in cardiac metabolism enhances myocardial resistance to ischemia/reperfusion in the rat heart.

UNLABELLED: Genes encoding enzymes involved in fatty acids (FA) and glucose oxidation are transcriptionally regulated by peroxisome proliferator-activated receptors (PPAR), members of the nuclear receptor superfamily. Under conditions associated with O(2) deficiency, PPAR-alpha modulates substrate switch (between FA and glucose) aimed at the adequate energy production to maintain basic cardiac function. Both, positive and negative effects of PPAR-alpha activation on myocardial ischemia/reperfusion (I/R) injury have been reported. Moreover, the role of PPAR-mediated metabolic shifts in cardioprotective mechanisms of preconditioning (PC) is relatively less investigated. We explored the effects of PPAR-alpha upregulation mimicking a delayed "second window" of PC on I/R injury in the rat heart and potential downstream mechanisms involved. Pretreatment of rats with PPAR-alpha agonist WY-14643 (WY, 1 mg/kg, i.p.) 24 h prior to I/R reduced post-ischemic stunning, arrhythmias and the extent of lethal injury (infarct size) and apoptosis (caspase-3 expression) in isolated hearts exposed to 30-min global ischemia and 2-h reperfusion. Protection was associated with remarkably increased expression of PPAR-alpha target genes promoting FA utilization (medium-chain acyl-CoA dehydrogenase, pyruvate dehydrogenase kinase-4 and carnitine palmitoyltransferase I) and reduced expression of glucose transporter GLUT-4 responsible for glucose transport and metabolism. In addition, enhanced Akt phosphorylation and protein levels of eNOS, in conjunction with blunting of cardioprotection by NOS inhibitor L-NAME, were observed in the WY-treated hearts. CONCLUSIONS: upregulation of PPAR-alpha target metabolic genes involved in FA oxidation may underlie a delayed phase PC-like protection in the rat heart. Potential non-genomic effects of PPAR-alpha-mediated cardioprotection may involve activation of prosurvival PI3K/Akt pathway and its downstream targets such as eNOS and subsequently reduced apoptosis.

Subcellular mechanisms of adaptation in the diabetic myocardium: Relevance to ischemic preconditioning in the nondiseased heart.

Although hyperglycemia is one factor that determines the outcome of myocardial ischemic insult, it is still not clear whether it is causally related to decreased ischemic tolerance in diabetic patients. In contrast to clinical and epidemiological studies demonstrating a higher risk of cardiovascular disorders in diabetic patients, experimental data are not unequivocal and suggest that, aside from higher myocardial vulnerability, diabetes mellitus may be associated with the triggering of adaptive processes leading to paradoxically lower susceptibility to ischemia. It has been proposed that this phenomenon shares some molecular pathways with short-term preconditioning and other forms of endogenous protection against ischemia/reperfusion injury in the nondiseased heart. The present article reviews some controversial findings of enhanced resistance to ischemia in the diabetic heart that stem from experimental studies in different models of myocardial ischemia/reperfusion injury. Specifically, it addresses the issue of potential mechanisms of increased resistance to ischemia in an experimental model of streptozotocin-induced diabetes, particularly with respect to the role of reactive oxygen species, hyperglycemia as one of the stress factors, and cell-signalling mechanisms mediated by 'prosurvival' cascades of protein kinases in relation to the mechanisms of classical ischemic preconditioning. Finally, mechanisms involved in the suppression of protection in the diabetic myocardium including the effect of concomitant pathology, such as hypercholesterolemia, are discussed.

Dopamine mimics the cardioprotective effect of ischemic preconditioning via activation of alpha1-adrenoceptors in the isolated rat heart.

The aim of the present study was to clarify whether pharmacological preconditioning with dopamine protects the heart against ischemia and whether this effect is mediated through dopaminergic receptors (D1 and D2) or alpha1-adrenoceptors. Isolated perfused rat hearts were either non-preconditioned, preconditioned with 5 min ischemia, or treated for 5 min with dopamine (1, 5 or 10 microM) before being subjected to 45 min of sustained ischemia followed by 60 min reperfusion. Postischemic functional recovery and infarct size were used as indices of the effects of ischemia. Treatment with the lower concentration of dopamine (1 microM), did not provide any protection to the ischemic myocardium. On the other hand, treatment with 5 microM dopamine resulted in significantly improved functional recovery, whereas administration of dopamine (10 microM) resulted in significantly improved functional recovery as well as reduction of infarct size. Pretreatment with the mixed D1/D2 dopaminergic receptor antagonist haloperidol or the beta-adrenoceptor selective antagonist propranolol did not attenuate the protective effect of pharmacological preconditioning with 10 microM dopamine with respect to both functional recovery and infarct size reduction. On the other hand, the cardioprotective effect of dopamine was blocked when the alpha1-adrenoceptor selective antagonist, prazosin, was administered. In conclusion, pharmacological preconditioning with dopamine protects the myocardium against ischemia and this effect seems to be mediated through activation of alpha1-adrenoceptors.

The supportive value of pre-bypass L-glutamate loading in patients undergoing coronary artery bypass grafting.

AIM: Experimental studies have demonstrated that an exogenous supply of glutamate improves mechanical function and recovery of ischemic myocardium. The aim of the present study was to investigate the effect of myocardial pre-bypass loading with glutamate on myocardial protection during global ischemia and reperfusion of patients undergoing coronary artery bypass grafting (CABG). METHODS: The study was double blinded. Twenty patients undergoing elective CABG were randomized to receive L-glutamate (n = 10) or normal saline (n = 10). Intracellular levels of glutamate, ATP and lactate were measured in left ventricular biopsies collected 10 min after aortic clamp release. Hemodynamic data, and postoperative release of CK-MB and troponin T were also measured. RESULTS: Pre-bypass administration of glutamate resulted in myocardial glutamate loading since glutamate levels were significantly higher in the glutamate group of patients than in controls (18.6 +/- 3.1 versus 8.7 +/- 1.2 microg/g tissue, P < 0.001). In the same group ATP levels were also significantly higher (2.4 +/- 0.7 versus 1.5 +/- 0.4 microg/g tissue, P < 0.05) and lactate levels significantly less than in controls (6.9 +/- 1.9 versus 12.0 +/- 2.1 microg/g tissue, P < 0.001). Glutamate patients had statistically significantly superior post-bypass hemodynamic performance (cardiac index, left ventricular stroke work index, systemic vascular resistance and pulmonary vascular resistance). Statistically significantly lower levels of CK-MB (6 h postoperative), total and peak CK-MB, troponin T (24 h postoperative), and total troponin T were found in the glutamate group. CONCLUSIONS: The results of this preliminary study indicate that pre-bypass intravenous administration of glutamate in patients undergoing CABG has a supportive effect on myocardial metabolism during global ischemia and reperfusion, improves patients' postoperative hemodynamic performance and reduces postoperative cardiac enzyme release.

Dissociation of stress-activated protein kinase (p38-MAPK and JNKs) phosphorylation from the protective effect of preconditioning in vivo.

The aim of the present study was to examine and compare the role of the stress-activated protein kinases in ischemic and stretch-induced preconditioning. A model of anesthetized rabbits was used, and the preconditioning protocol included one or three cycles of short ischemia/reperfusion, or short mechanical stretch with acute pressure overload without or with the addition of the stretch blocker gadolinium. Infarct size was determined after 2h reperfusion and p38 MAPK and JNKs phosphorylation was determined after 20 min of prolonged ischemia. Preconditioning stimuli were equally effective in reducing the infarct size (14.2+/-3.4%, 12.9+/-3.0%, 15.9+/-3.3%, P<0.01 vs control). The addition of the stretch channel blocker gadolinium abrogated the effect of stretch preconditioning only, without any effect on ischemic preconditioning. Comparing p38-MAPK and p46/p54 JNKs phosphorylation in the ischemic and non-ischemic regions of the heart at the time of sustained ischemia, activation was observed in the ischemic or mechanically preconditioned groups compared with the control. The addition of gadolinium abolished this activation. The above results indicate that the phosphorylation of p38-MAPK and p46/p54 JNKs is increased in preconditioning but this effect can be dissociated from the protective effect of ischemic preconditioning. Activation of the stress-activated protein kinases may be related to the increased contracture, a characteristic of ischemic preconditioning.

Activation of alpha 1-adrenoceptors is not essential for the mediation of ischaemic preconditioning in rat heart.

1. The aim of the present study was to clarify the role of alpha1-adrenoceptors in the mechanism of ischaemic preconditioning (IP). 2. Rat isolated perfused hearts were either non- preconditioned, preconditioned with 5 min ischaemia or treated for 5 min with alpha1-adrenoceptor agonists (50 micromol/L phenylephrine; 0.1, 0.5 and 1 micromol/L methoxamine) before being subjected to 45 min of sustained ischaemia followed by 60 min reperfusion. 3. Within each of the above protocols, hearts were divided into groups to which alpha1-adrenoceptor antagonists (prazosin, 5'-methyl urapidil and chloroethylclonidine (CEC)) were administered. Functional recovery and infarct size were used as indices of the effects of ischaemia. Ischaemic contracture characteristics and maximal diastolic pressure during reflow were also assessed. 4. Blockade of alpha(1)-adrenoceptors with prazosin or the subtype-selective antagonists 5'-methyl urapidil and CEC did not abolish the protective effect of IP with respect to both functional recovery and infarct size reduction. 5. Protection afforded by phenylephrine was attenuated in hearts treated with prazosin or the alpha(1B)-adrenoceptor- selective antagonist CEC, but not in those treated with the alpha(1A)-adrenoceptor-selective antagonist 5'-methyl urapidil. 6. Treatment with low concentrations of methoxamine, considered to be alpha(1A)-adrenoceptor selective, did not confer any protection to the ischaemic myocardium. 7. A close relationship between accelerated ischaemic contracture and enhanced cardioprotection was observed. 8. The results suggest that alpha1-adrenoceptor stimulation mimics IP, but it is not an essential component in the mechanism behind the protective effect of IP in rat heart. In addition, the present study demonstrates that stimulation of the alpha(1B)- but not the alpha(1A)-adrenoceptor subtype is responsible for the catecholamine-induced protection of ischaemic myocardium in rat.

alpha(1)-adrenergic stimulation mediates Ca(2+)-dependent inositol phosphate formation through the alpha(1B)-like adrenoceptor subtype in adult rat cardiac myocytes.

We studied the effects of increased Ca(2+) influx on alpha(1)-adrenoceptor-stimulated InsP formation in adult rat cardiac myocytes. We further examined if such effects could be mediated through a specific alpha(1)-adrenoceptor subtype. [(3)H]InsP responses to adrenaline were dependent on extracellular Ca(2+) concentration, from 0.1 microM to 2 mM, and were completely blocked by Ca(2+) removal. However, in cardiac myocytes preloaded with BAPTA, a highly selective calcium chelating agent, Ca(2+) concentrations higher than 1 microM had no effect on adrenaline-stimulated [(3)H]InsP formation. Taken together these results suggest that [(3)H]InsP formation induced by alpha(1)-adrenergic stimulation is in part mediated by increased Ca(2+) influx. Consistent with this, ionomycin, a calcium ionophore, stimulated [(3)H]InsP formation. This response was additive with the response to adrenaline stimulation implying that different signaling mechanisms may be involved. In cardiac myocytes treated with the alpha(1B)-adrenoceptor alkylating agent, CEC, [(3)H]InsP formation remained unaffected by increased Ca(2+) concentrations, a pattern similar to that observed when intracellular Ca(2+) was chelated with BAPTA. In contrast, addition of the alpha(1A)-subtype antagonist, 5'-methyl urapidil, did not affect the Ca(2+) dependence of [(3)H]InsP formation. Neither nifedipine, a voltage-dependent Ca(2+) channel blocker nor the inorganic Ca(2+) channel blockers, Ni(2+) and Co(2+), had any effect on adrenaline stimulated [(3)H]InsP, at concentrations that inhibit Ca(2+) channels. The results suggest that in adult rat cardiac myocytes, in addition to G protein-mediated response, alpha(1)-adrenergic-stimulated [(3)H]InsP formation is activated by increased Ca(2+) influx mediated by the alpha(1B)-subtype.

Stimulation of multiple MAPK pathways by mechanical overload in the perfused amphibian heart.

The mitogen-activated protein kinase (MAPK) signal transduction pathway activated by mechanical stress was investigated in the isolated perfused amphibian (Rana ridibunda) heart. High perfusion pressure induced the rapid (30 s) and prolonged (30 min) phosphorylation of a p43-extracellular regulated kinase, a response almost completely inhibited by 25 microM PD-98059. c-Jun NH2-terminal kinase (JNK) was also phosphorylated with maximal values attained at 15 min and remained elevated over 30 min. In-gel kinase assays verified that phosphorylated JNKs are active, phosphorylating the transcription factor c-Jun. Furthermore, pressure overload rapidly stimulated the p38-MAPK phosphorylation (30 s), a transient process (5 min) abolished by 1 microM SB-203580. In-gel kinase assays revealed that with phosphorylation, active p38-MAPKs phosphorylate their substrate MAP kinase-activated protein kinase 2. Biochemical analysis along with immunohistochemical studies showed that with activation, the three MAPK subfamily members examined are localized not only in the cytoplasm but in the nucleus as well. Present results therefore demonstrate for the first time in an amphibian species the involvement of multiple MAPK pathways in the mechanical overload-induced adaptive responses of the heart as well as their possible physiological roles.

Activation of multiple MAPK pathways (ERKs, JNKs, p38-MAPK) by diverse stimuli in the amphibian heart.

We investigated the expression and activation of three MAPK subfamilies in the isolated perfused amphibian heart. ERK was detected as a 43 kDa band; p38-MAPK was detected as a band corresponding to 38 kDa and JNKs were detected as two bands corresponding to 46 and 52 kDa, respectively. PMA induced the activation of the ERK pathway as assessed by determining the phosphorylation state of ERK and the upstream component MEK1/2. PD98059 abolished this activation. p38-MAPK was phosphorylated by sorbitol (almost 12-fold, maximal within 10-15 min) and JNKs were phosphorylated and activated by sorbitol or anoxia/reoxygenation (approximately 4- and 2.5-fold, respectively). SB203580 completely blocked the activation of p38-MAPK by sorbitol. These results indicate that the MAPK pathways activated by phorbol esters, hyperosmotic stress or anoxia/ reoxygenation in the amphibian heart may have an important role in this experimental system.

alpha(1D)-Adrenoceptors do not contribute to phosphoinositide hydrolysis in adult rat cardiac myocytes.

We have used the alpha(1D)-adrenoceptor selective antagonist, BMY 7378, to investigate the presence of alpha(1D)-adrenoceptor subtype in adult rat heart by radioligand binding assays. We also determined the role of this subtype in stimulating phosphoinositide (PI) hydrolysis in adult rat cardiac myocytes. BMY 7378 inhibited [(3)H]prazosin binding to cardiac membranes in a biphasic mode with a pK(i) of 9.19 +/- 0.26 for high affinity sites and 6.64 +/- 0.09 for low affinity sites. The inhibition of the adrenaline-induced stimulation of PI hydrolysis by BMY 7378 fitted a one-site model and the calculated pK(b) value (6.92 +/- 0.28) was consistent with the involvement of alpha(1A) and alpha(1B) adrenoceptors. In addition, BMY 7378, at concentrations up to 100 nM, did not significantly affect the concentration-response curves for the adrenaline-induced stimulation of PI hydrolysis. Taken together, these data suggest that alpha(1D)-adrenoceptors are expressed in adult rat heart but this subtype is not involved in the adrenaline-induced stimulation of PI hydrolysis.

Differential effect of preconditioning on post-ischaemic myocardial performance in the absence of substantial infarction and in extensively infarcted rat hearts.

OBJECTIVES: There is controversy concerning the beneficial effects of ischaemic preconditioning during short periods of ischaemia (stunning). The aim of the study was to investigate post-ischaemic myocardial performance after various periods of ischaemia in both non-preconditioned and preconditioned hearts and to compare these results with infarct volume estimation. METHODS: Isolated perfused rat hearts were subjected to various periods of sustained ischaemia (15, 20, 30, and 45 min). Haemodynamic parameters, infarct size and lactate dehydrogenase (LDH) leakage were recorded in both preconditioned and non-preconditioned hearts. RESULTS: After 15 min of ischaemia, preconditioned hearts revealed significantly lower developed pressure than non-preconditioned hearts (80+/-4.1 vs. 95+/-0.3%, P=0.02). In the 20 min ischaemia group, preconditioning resulted in non-significantly lower developed pressure (76+/-3.1% in preconditioned hearts vs. 87+/-5.3% in non-preconditioned hearts, P=0.11). In these groups infarct volume was small and not different between non-preconditioned and preconditioned hearts. After 30 min of ischaemia, preconditioning significantly improved developed pressure (66+/-3.1% in preconditioned and 44+/-5% in non-preconditioned hearts, P=0.002). LDH leakage was significantly higher in non-preconditioned hearts compared with preconditioned hearts (16+/-2.3 vs. 9.0+/-1.3, P=0.04), whereas infarct volume was not (12.5+/-0.8 and 9.8+/-1.5, respectively, P=0.1). Non-preconditioned hearts of this group, subjected to inotropic stimulation at the end of reperfusion, responded poorly. Significantly higher developed pressure was attained by preconditioned hearts (150+/-3.1 vs. 123+/-7.5%, P=0.01). After 45 min of ischaemia, preconditioning resulted in 69% limitation of infarct volume (P<0.0001) and 53% reduction in LDH release (P=0.009). Developed pressure was 57+/-8.5% in preconditioned hearts and 32+/-4.5% in non-preconditioned hearts (P=0.02). CONCLUSIONS: When ischaemic insult results in minimally lethal injuries, preconditioned hearts do not have the advantage of not being prone to stunning rather than non-preconditioned. If ischaemic insult is potentially able to produce extensive infarction, improvement in post-ischaemic myocardial function is mainly due to infarct size limitation evoked by preconditioning.

Oxidative status and anti-oxidant enzyme activity during calcium paradox in the rat isolated heart.

1. The effect of calcium paradox on oxidative status and the activity of anti-oxidant enzymes were studied in the rat isolated heart. Glutathione status, sulphydryl group contents and lipid peroxidation in the myocardium, as well as the release of oxidized and reduced glutathione from the heart, were taken as indices of oxidative events. 2. Reperfusion with calcium after calcium-free perfusion induced a significant decrease in the myocardial content of reduced and oxidized glutathione and non-protein sulphydryl groups. At the same time, a significant release of both forms of glutathione from the heart was observed. However, the ratio of oxidized to reduced glutathione remained unchanged and was not different from control. Increased lipid peroxidation was observed only after 30 min of reperfusion with calcium. 3. Increased anti-oxidant activity during the reperfusion period was observed. Mitochondrial Mn-superoxide dismutase (SOD) activity was increased throughout the reperfusion period, while cytoplasmic Cu,Zn-SOD and glutathione peroxidase activity showed a transient increase at 5 min reperfusion. 4. The results do not support an important role of oxygen free radicals in cell damage observed during calcium paradox in the rat isolated heart. Production of oxygen free radicals may occur during the reperfusion period, but the quantity produced is insufficient to exceed the anti-oxidant capacity of the heart.

Activation of mitogen-activated protein kinases (p38-MAPKs, SAPKs/JNKs and ERKs) by the G-protein-coupled receptor agonist phenylephrine in the perfused rat heart.

We investigated the ability of phenylephrine (PE), an alpha-adrenergic agonist and promoter of hypertrophic growth in the ventricular myocyte, to activate the three best-characterized mitogen-activated protein kinase (MAPK) subfamilies, namely p38-MAPKs, SAPKs/JNKs (i.e. stress-activated protein kinases/c-Jun N-terminal kinases) and ERKs (extracellularly responsive kinases), in perfused contracting rat hearts. Perfusion of hearts with 100 microM PE caused a rapid (maximal at 10 min) 12-fold activation of two p38-MAPK isoforms, as measured by subsequent phosphorylation of a p38-MAPK substrate, recombinant MAPK-activated protein kinase 2 (MAPKAPK2). This activation coincided with phosphorylation of p38-MAPK. Endogenous MAPKAPK2 was activated 4-5-fold in these perfusions and this was inhibited completely by the p38-MAPK inhibitor, SB203580 (10 microM). Activation of p38-MAPK and MAPKAPK2 was also detected in non-contracting hearts perfused with PE, indicating that the effects were not dependent on the positive inotropic/chronotropic properties of the agonist. Although SAPKs/JNKs were also rapidly activated, the activation (2-3-fold) was less than that of p38-MAPK. The ERKs were activated by perfusion with PE and the activation was at least 50% of that seen with 1 microM PMA, the most powerful activator of the ERKs yet identified in cardiac myocytes. These results indicate that, in addition to the ERKs, two MAPK subfamilies, whose activation is more usually associated with cellular stresses, are activated by the Gq/11-protein-coupled receptor (Gq/11PCR) agonist, PE, in whole hearts. These data indicate that Gq/11PCR agonists activate multiple MAPK signalling pathways in the heart, all of which may contribute to the overall response (e.g. the development of the hypertrophic phenotype).

The role of colonoscopy in the differential diagnosis of acute, severe hemorrhagic colitis.

BACKGROUND AND STUDY AIMS: This study assesses the diagnostic value of colonoscopy performed at an early stage of a first attack of acute, severe hemorrhagic colitis. PATIENTS AND METHODS: One hundred fourteen consecutive patients were prospectively studied. The colonoscopic diagnosis was compared with the final diagnosis of the colitis, which was based on clinical, microbiological, endoscopic, and histological criteria during the acute illness, but also on the results of a thirty-month follow-up of the patients aiming to confirm whether the colitis was relapsing or nonrelapsing in nature. RESULTS: The colonoscopic diagnosis was ulcerative colitis (UC) in 40, Crohn's disease in four, and infective colitis (IC) in 70 patients. The endoscopic diagnosis was finally confirmed in all 40 UC patients and in 68 of 70 (97.1%) IC patients. Two patients with an initial endoscopic and histological diagnosis of IC presented with typical attacks of UC 28 and 30 months later, respectively. Prominent endoscopic appearances in IC were mucosal edema, erythematous areas, hemorrhagic spots, bleeding, microaphthoid ulcers, and luminal exudate. Although rectal sparing was occasionally seen, endoscopic lesions were continuous and severe in the distal colon, but were patchily and unevenly distributed in other parts of the colon in IC. In UC, prominent colonoscopic findings were bleeding, mucosal friability, granularity, and ulceration; lesions were continuously distributed in the involved area. CONCLUSIONS: Colonoscopy is a useful procedure in the differential diagnosis of severe bloody diarrhea of unknown cause.

Expression of protein kinase C isoforms during cardiac ventricular development.

The expression of protein kinase C (PKC) isoforms (PKC-alpha, PKC-beta 1, PKC-delta, PKC-epsilon, and PKC-zeta) was studied by immunoblotting in whole ventricles of rat hearts during postnatal development (1-26 days) and in the adult. PKC-alpha, PKC-beta 1, PKC-delta, PKC-epsilon, and PKC-zeta were detected in ventricles of 1-day-old rats, although PKC-alpha and PKC-beta 1 were only barely detectable. All isoforms were rapidly downregulated during development, with abundances relative to total protein declining in the adult to < 25% of 1-day-old values. PKC-beta 1 was not detectable in adult ventricles. The specific activity of PKC was also downregulated. The rat ventricular myocyte becomes amitotic soon after birth but continues to grow, increasing its protein content 40- to 50-fold between the neonate and the 300-g adult. An important question is thus whether the amount of PKC per myocyte is downregulated. With the use of isolated cells, immunoblotting showed that the contents per myocyte of PKC-alpha and PKC-epsilon increased approximately 10-fold between the neonatal and adult stages. In rat ventricles, the rank of association with the particulate fraction was PKC-delta > PKC-epsilon > PKC-zeta. Association of these isoforms with the particulate fraction was less in the adult than in the neonate. In primary cultures of ventricular myocytes prepared from neonatal rat hearts, 1 microM 12-O-tetradecanoylphorbol-13-acetate (TPA) elicited translocation of PKC-alpha, PKC-delta, and PKC-epsilon from the soluble to the particulate fraction in < 1 min, after which time no further translocation was observed. Prolonged exposure (16 h) of myocytes to 1 microM TPA caused essentially complete downregulation of these isoforms, although downregulation of PKC-epsilon was slower than for PKC-delta. In contrast, PKC-zeta was neither translocated nor downregulated by 1 microM TPA. Immunoblotting of human ventricular samples also revealed downregulation of PKC relative to total protein during fetal/postnatal development.

Deferoxamine cardioplegia reduces superoxide radical production in human myocardium.

Recent studies have demonstrated enhanced myocardial protection during ischemia using the oxygen free radical scavenger, deferoxamine. This effect of deferoxamine may be related either to its iron-chelating property or to intervention in an iron-independent mechanism. We tested the latter by determining the rate of superoxide anion production and the degree of lipid peroxidation in human myocardial tissue after including deferoxamine in cardioplegic solution. Fourteen patients who underwent aortic, mitral, or double valve replacement were included in the study. The mean value for superoxide radical production was 59.8 +/- 17.0 nmol.min-1.g-1 for the control group (group C; n = 7) and 21.3 +/- 8.1 (p < 0.001) for the deferoxamine-treated group (group D; n = 7). The mean value for thiobarbituric reactive substances was 80.00 +/- 23.4 in group C and 38.7 +/- 23.8 nmol.min-1.g-1 in group D (p < 0.01). In conclusion, deferoxamine appears to have a moderating effect on the biochemical markers of ischemia reperfusion injury. Its scavenging effect on superoxide anion could play a role in the cellular defense against oxygen radicals during cardiac operations.