Tak Ming Wong

Calcium-Activated Potassium Channel Triggers Cardioprotection of Ischemic Preconditioning

We tested the hypothesis that the high-conductance calciumactivated potassium (KCa) channel is involved in the cardioprotection of preconditioning with ischemic insults. In the isolated perfused rat heart subjected to ischemia/reperfusion, effects of ischemic preconditioning (IPC) on infarct size and lactate dehydrogenase (LDH) release were abolished by 1 μM paxilline (Pax), an inhibitor of the KCa channel, administered 30 min before, but not during, ischemia. In isolated ventricular myocytes subjected to metabolic inhibition and anoxia (MI/A), preconditioning with MI/A increased their viability, and the effect was abolished by administering Pax before MI/A. Like IPC, 10 μM NS1619 (1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-trifluoromethyl-2Hbenzimidazol-2-one; NS), an opener of KCa channels, reduced infarct size and LDH release, effects attenuated by Pax. The harmful and protective effects of blockade and activation of the KCa channel were accompanied by impaired and improved left ventricular contractile functions, respectively. In addition, the effect of NS was not altered by 100 μM 5-hydroxydecanoate, an inhibitor of the KATP channel. Neither was the effect of 100 μM diazoxide, an activator of the KATP channel, altered by Pax. Furthermore, opening of the mitochondrial permeability transition pore (mPTP) with 20 μM atractyloside abolished the beneficial effects of IPC or NS in the isolated rat heart and myocyte. Inhibition of mPTP opening with 0.2 μM cyclosporin A decreased the infarct size and LDH release and improved the contractile function, effects not attenuated by Pax. In conclusion, the study provides evidence that the KCa channel triggers cardioprotection of IPC, which involves mPTP.

Calcium antagonistic and antiarrhythmic actions of CPU-23, a substituted tetrahydroisoquinoline

1 The effects of CPU‐23 (1‐{1‐[(6‐methoxyl)‐naphth‐2‐yl]}‐propyl‐2‐(1‐piperidine)‐acetyl‐6,7‐dimethyoxy‐1,2,3,4‐tetra‐hydroisoquinoline) were studied on mechanical and electrical activities, and intracellular free calcium ([Ca2+]i) of isolated cardiac tissues in order to investigate its spectrum and mechanisms of action in the heart. Its antiarrhythmic and haemodynamic effects in pentobarbitone‐anaesthetized rats subjected to coronary artery ligation were also evaluated. 2 CPU‐23 at 10−6‐10−4 m markedly inhibited slow action potential characteristics in guinea‐pig papillary muscles and pace‐maker action potential of rabbit sinoatrial node. It affected fast action potential only at 10−4 M. None of the effects of CPU‐23 was reversed by washout for up to 2 h. 3 Like nifedipine and diltiazem, CPU‐23 decreased the heart rate of the isolated perfused heart of the rat. However, in contrast to these two classical calcium antagonists which dose‐dependently inhibited the force of contraction, CPU‐23 inhibited and stimulated the force of contraction at 10−7‐3 × 10−6 m and 10−5 m, respectively. 4 CPU‐23 at 10−6‐10−5 m inhibited the KCl‐induced [Ca2+]i increase in the Ca2+ medium, but did not affect the caffeine‐induced [Ca2+]i increase in the Ca2+‐free medium in isolated ventricular myocytes. 5 CPU‐23 at 1–5 mg kg−1 reduced dose‐dependently ventricular arrhythmias including ventricular ectopic beats, VT and VF as well as mortality during coronary artery ligation. At 2.5–5 mg kg−1 it even abolished VF, which was accompanied by 100% survival. 6 It is suggested that CPU‐23 has calcium antagonistic properties in cardiac tissues. It selectively blocks the transmembrane influx of extracellular Ca2+ through Ca2+ channels, thus reducing the heart rate and developed tension, altering the slow action potential characteristics and producing antiarrhythmic effect against ischaemic arrhythmias.

Polyol pathway mediates iron-induced oxidative injury in ischemic-reperfused rat heart.

Recent studies have shown that the polyol pathway is involved in ischemia-reperfusion (I/R)-induced myocardial infarction, but the mechanism is unclear. We previously found that lack of aldose reductase (AR), the first enzyme of the polyol pathway, attenuated the increase in transferrin (Tf) level in I/R brain, suggesting that AR contributes to iron-catalyzed free radical-induced damage. We therefore investigated if this mechanism occurs in I/R hearts. We found that inhibition of AR or sorbitol dehydrogenase (SDH), the second enzyme of the polyol pathway, both attenuated the I/R-mediated increases in HIF-1alpha, Tf, TfR, and intracellular iron content and reduced the I/R-induced infarct area of the heart. Further, administration of niacin, which replenishes NAD+, the cofactor for SDH, also normalized TfR and HIF-1alpha levels in I/R hearts. These results suggest that during I/R polyol pathway activity increases the cytosolic NADH/NAD+ ratio. This activates HIF-1alpha that induces the expression of TfR, which in turn increases Tf uptake and iron accumulation and exacerbates oxidative damage that increases the lipid peroxidation. This was confirmed by the fact that administration of the iron chelator deferoxamine attenuated the I/R-induced myocardial infarction.

A new view of K+-induced contraction in rat aorta: the role of Ca2+ binding

Strong, K+-induced contractions of rat aorta in Ca-free, Mg-free media were not accompanied by increased intracellular calcium concentration, [Ca2+]i, whereas such contractions in the presence of the divalent cations were correlated with rising [Ca2+]i as assessed by fura-2. At the same time, calcium channel blockers, a modulator of Ca2+-binding proteins, and a modulator of actin polymerization, inhibited all types of K+-induced contractions. Increasing the K+ in isotonic medium evoked a rise of 45Ca2+ binding to the plasma membrane of freshly isolated aortic cells. Although Ca2+-dependent events underlie the mechanism of K+-induced vascular contractions in both the presence and absence of Ca2+, in contrast to the view that [Ca2+]i is a key regulator of excitation-contraction coupling in smooth muscle, we suggest that the modulation of Mg2+-dependent Ca2+ binding, probably within/at the L-type calcium channel by K+, is a trigger for aortic contraction. This Ca2+ binding may then activate actin-myosin interaction.

Increased PKA activity and its influence on isoprenaline‐stimulated L‐type Ca2+ channels in the heart from ovariectomized rats

1 We previously showed that oestrogen confers cardioprotection by downregulating the cardiac β1‐adrenoceptor (β1‐AR). The present study examined the effect of oestrogen on the post β1‐AR signalling cascade, with particular emphasis on the activity of protein kinase A (PKA) and its influence on the L‐type Ca2+ channel. 2 Three groups of adult female Sprague–Dawley rats were used: sham‐operated controls, bilaterally ovariectomized (Ovx) rats, and Ovx rats with oestrogen replacement (Ovx+E2), which restored the oestrogen concentration to normal. 3 The electrically induced intracellular Ca2+ transient (E[Ca2+]i), 45Ca2+‐uptake through cardiac L‐type Ca2+ channels (Ca2+ channels), heart rate and force of contraction in response to β‐AR stimulation with 10 nM isoprenaline (Iso) in hearts from Ovx rats were significantly greater than those of control and Ovx+E2 rats. The basal and Iso‐induced PKA activities were also higher in hearts from Ovx rats. KT5720, a selective PKA inhibitor, completely inhibited its potentiating effect on basal Ca2+ channel activity in the Ovx rat heart. On the other hand, expression of G proteins (Gαs and Gαi1−3), basal and forskolin‐stimulated cAMP accumulation, and responsiveness of PKA to cAMP, were not altered by Ovx. 4 Interestingly, the PKA inhibitor at the same concentration significantly reduced the increases in PKA activity and Ca2+ channel activity upon β‐AR stimulation in all three groups of rats and the inhibitions were significantly greater in the Ovx rat than in the other two groups of rats. 5 This study provides the first evidence that, in addition to downregulation of β1‐AR shown previously, suppression of PKA activity, which is partly responsible for the suppressed Ca2+ channel activity, also determines the E[Ca2+]i and cardiac contractility following β‐AR stimulation in the female rat.

Effects of Heat Shock Protein 70 Activation by Metabolic Inhibition Preconditioning or κ-Opioid Receptor Stimulation on Ca2+ Homeostasis in Rat Ventricular Myocytes Subjected to Ischemic Insults

Heat shock protein 70 (HSP70) mediates delayed cardioprotection of preconditioning. Cytosolic calcium ([Ca2+])i overload precipitates injury, whereas attenuation of [Ca2+]i overload is believed to be responsible for cardioprotection. There is evidence suggesting a link between HSP70 and [Ca2+]i homeostasis. We hypothesize that activation of HSP70 by preconditioning may restore [Ca2+]i homeostasis altered by ischemic insults. To test the hypothesis, we determined the effects of preconditioning with metabolic inhibition or pretreating with U50,488H [trans-(+)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide (a κ-opioid receptor agonist)] on viability and injury, HSP70 expression, and [Ca2+]i in ventricular myocytes subjected to metabolic inhibition and anoxia (MI/A), with blockade of HSP70 synthesis. In myocytes with vehicle pretreatment, the percentage of dead cells determined by trypan blue exclusion, the injury reflected by release of lactate dehydrogenase, and the resting [Ca2+]i measured by spectrofluorometry significantly increased, whereas the amplitude of electrically induced [Ca2+]i transient decreased, after 10 min with 10 mM 2-deoxy-d-glucose and 10 mM sodium dithionite, known to cause MI/A. However, when myocytes were subjected for 30 min to either 20 mM lactate and 10 mM 2-deoxy-d-glucose (MIP) or 30 μM U50,488H (UP) 20 h before MI/A, the changes in viability and injury, and [Ca2+]i responses were significantly attenuated. These were accompanied by a significantly increased HSP70 expression. Furthermore, blockade of HSP70 synthesis with selective antisense oligonucleotides abolished the beneficial effects of MIP or UP. This study provides first evidence that activation of HSP70 induced by preconditioning, which conferred delayed cardioprotection, restored partially the [Ca2+]i homeostasis altered by ischemic insults.

Morphine as a drug for stress ulcer prevention and healing in the stomach

Morphine pretreatment protects against stress-induced gastric ulceration, however, the exact mechanism is still undefined. Interestingly, the effect of morphine on ulcer healing has not been investigated. In this report, we would like to study these effects in a defined stress ulcer model and to delineate a new implication for morphine to promote stress ulcer healing in rats. Our study showed that cold-restraint stress for 3 h induced hemorrhagic lesions and increased myeloperoxidase activity in the gastric mucosa. Stress also reduced the dimension of layer of periodic acid-Schiff reagent-stained cells in the gastric mucosa by about 50%. Morphine pretreatment (2 or 8 mg/kg, given intraperitoneally) at the time of stress dose-dependently reversed stress-induced gastric ulceration, increase of myeloperoxidase activity and reduction of thickness of mucus-stained cells in the gastric mucosa. Morphine treatment after stress (given at the end of a 3-h stress and also at 3 h thereafter) increased ulcer healing by reducing the ulcer size measured 24 h later. Such action was blocked by naloxone (8 mg/kg) given intraperitoneally 15 min before morphine treatment. Morphine also increased the number of cell proliferation and dimension of layer of cells stained for mucus but not the number of microvessels in the gastric mucosa. Moreover, the number of apoptotic cells was less evidenced in the morphine-treated rats. This study reports for the first time that morphine not only prevents stress ulceration but also promotes healing of stress ulcer through a defined mechanism.

Testosterone and cardioprotection against myocardial ischemia.

Male gender is a risk factor for cardiovascular diseases. Testosterone being the main male sex hormone is therefore believed to be responsible for the deleterious effect of the male. However, there are recent studies showing that testosterone level is lower in patients with ischemic heart diseases, and testosterone treatment alleviates the symptoms. Earlier studies showed that functional androgen receptors are present in the heart and that testosterone acts directly at the myocardium. There is increasing evidence to suggest testosterone confers cardioprotection by direct action on the myocardium. Here, we review the recent literature on association between testosterone and myocardial ischemia in males, and the signal transduction mechanisms that mediate the action of testosterone in the heart. The studies reviewed in this article provide evidence that testosterone may confer protection via a varieties of mechanisms, which may be both genomic and non-genomic. Further studies are warranted to further delineate the integration of signaling mechanisms and to explore the possibility of using testosterone in the aging male population with ischemic heart diseases.

Effects of HSP70 activation by metabolic inhibition preconditioning or κ-OR stimulation on Ca2+ homeostasis in rat ventricular myocytes subjected to ischemic insults

Heat shock protein 70 (HSP70) mediates delayed cardioprotection of preconditioning. Cytosolic calcium ([Ca2+])i overload precipitates injury, whereas attenuation of [Ca2+]i overload is believed to be responsible for cardioprotection. There is evidence suggesting a link between HSP70 and [Ca2+]i homeostasis. We hypothesize that activation of HSP70 by preconditioning may restore [Ca2+]i homeostasis altered by ischemic insults. To test the hypothesis, we determined the effects of preconditioning with metabolic inhibition or pretreating with U50,488H [trans-(+)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide (a κ-opioid receptor agonist)] on viability and injury, HSP70 expression, and [Ca2+]i in ventricular myocytes subjected to metabolic inhibition and anoxia (MI/A), with blockade of HSP70 synthesis. In myocytes with vehicle pretreatment, the percentage of dead cells determined by trypan blue exclusion, the injury reflected by release of lactate dehydrogenase, and the resting [Ca2+]i measured by spectrofluorometry significantly increased, whereas the amplitude of electrically induced [Ca2+]i transient decreased, after 10 min with 10 mM 2-deoxy-d-glucose and 10 mM sodium dithionite, known to cause MI/A. However, when myocytes were subjected for 30 min to either 20 mM lactate and 10 mM 2-deoxy-d-glucose (MIP) or 30 μM U50,488H (UP) 20 h before MI/A, the changes in viability and injury, and [Ca2+]i responses were significantly attenuated. These were accompanied by a significantly increased HSP70 expression. Furthermore, blockade of HSP70 synthesis with selective antisense oligonucleotides abolished the beneficial effects of MIP or UP. This study provides first evidence that activation of HSP70 induced by preconditioning, which conferred delayed cardioprotection, restored partially the [Ca2+]i homeostasis altered by ischemic insults.

Decreased affinity of k-receptor binding during reperfusion following ischaemic preconditioning in the rat heart

The effects of ischaemic preconditioning with three cycles of ischaemia of 3 min and reperfusion of 5 min each cycle on ventricular fibrillation threshold (VFT) and ventricular fibrillation (VF), and binding properties of tritiated U69,593, a selective kappa opioid-receptor (k-receptor) agonist, during subsequent ischaemia and/or reperfusion were studied in the rat heart. It was found that ischaemic preconditioning significantly enhanced the VFT values during ischaemic and reperfusion. VF during the subsequent reperfusion period was also significantly reduced. The Kd of the [3H]U69,593 binding sites in the sarcolemma of the heart at 5 min of reperfusion was significantly increased following ischaemic preconditioning. The Bmax was, however, not altered after the preconditioning. The study provides evidence for the first time suggesting that the cardioprotective effects of ischaemic preconditioning may be related to a reduction in affinity of the K-receptor binding.