Melatonin ameliorates endothelial dysfunction, vascular inflammation, and systemic hypertension in rats with chronic intermittent hypoxia.

The pathogenesis of hypertension in patients with obstructive sleep apnea (OSA) is associated with endothelial dysfunction induced by chronic intermittent hypoxia (IH). Studies have shown that administration of melatonin ameliorates oxidative injury and inflammation. This study examined the effect of melatonin on the oxidative stress, endothelial dysfunction, and inflammation during the pathogenesis of hypertension in chronic IH. Adult Sprague-Dawley rats that had received a daily injection of melatonin or vehicle were exposed to IH treatment mimicking a severe OSA condition for 14-21 days. Systolic pressure was significantly higher in the vehicle-treated (144 ± 2.7 mmHg) but not in the melatonin-treated rats (123 ± 5.1 mmHg) by 21-day IH treatment when compared with the normoxic control. Levels of malondialdehyde and the expressions of NADPH oxidase, pro-inflammatory mediators (TNF-α, inducible NO synthase, COX-2), and adhesion molecules (ICAM-1, VCAM-1, and E-selectin) of the thoracic aorta were markedly increased by 14-day IH treatment preceding the hypertensive response. Also, levels of nitric oxide (NO˙), endothelial-dependent relaxation, and the expressions of endothelial NO synthase (eNOS) and antioxidant enzymes (GPx, CAT, and Cu/Zn SOD) were significantly lowered in the IH rats. Melatonin treatment significantly mitigated the increased expression of NADPH oxidase, pro-inflammatory mediators, and adhesion molecules. Moreover, melatonin prevented the endothelial dysfunction with ameliorated levels of NO˙, endothelial-dependent relaxation, and expressions of eNOS and antioxidant enzymes. These results suggest that melatonin is protective against IH-induced hypertension and endothelial dysfunction via an antioxidant and anti-inflammatory mechanism.

Cardiac contractile dysfunction during acute hyperglycemia due to impairment of SERCA by polyol pathway-mediated oxidative stress.

Hyperglycemia is an indication of poor outcome for heart attack patients, even for nondiabetic patients with stress-induced hyperglycemia. Previous studies showed that inhibition of aldose reductase, the first and rate-limiting enzyme of the polyol pathway, attenuated contractile dysfunction in diabetic animals, but the mechanism is unclear. We therefore wanted to find out whether the polyol pathway also contributes to acute hyperglycemia-induced cardiac contractile dysfunction, and determine the mechanism involved. Rat hearts were isolated and retrogradely perfused with Krebs buffer containing either normal or high concentrations of glucose for 2 h. Short exposure to high-glucose medium led to contractile dysfunction as indicated by decreased -dP/dt(max), as well as elevation in left ventricular end-diastolic pressure. Cardiomyocytes incubated in high-glucose medium showed abnormal Ca2+ signaling, most likely because of decreased activity of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) inactivated by oxidative stress. Inhibition of aldose reductase or sorbitol dehydrogenase, the second enzyme in the polyol pathway, ameliorated contractile dysfunction, attenuated oxidative stress, and normalized Ca2+ signaling and SERCA activity caused by high glucose, indicating that the polyol pathway is the major contributor to acute hyperglycemia-induced oxidative stress leading to the inactivation of SERCA and contractile dysfunction.

Polyol pathway impairs the function of SERCA and RyR in ischemic-reperfused rat hearts by increasing oxidative modifications of these proteins.

A number of studies have shown that the polyol pathway, consisting of aldose reductase (AR) and sorbitol dehydrogenase (SDH), contributes to ischemia-reperfusion (I/R)-induced myocardial infarction due to depletion of ATP. In this report we show that the polyol pathway in I/R heart also contributes to the impairment of sacro/endoplasmic reticulum Ca(2+)-ATPase (SERCA) and ryanodine receptor (RyR), two key players in Ca(2+) signaling that regulate cardiac contraction. Rat hearts were isolated and retrogradely perfused with either Krebs' buffer containing 1 microM AR inhibitor, zopolrestat, or 200 nM SDH inhibitor, CP-170,711, and challenged by 30 min of regional ischemia and 45 min of reperfusion. We found that post-ischemic contractile function of the isolated perfused hearts was improved by pharmacological inhibition of the polyol pathway. I/R-induced contractile dysfunction is most likely due to impairment in Ca(2+) signaling and the activities of SERCA and RyR. All these abnormalities were significantly ameliorated by treatment with ARI or SDI. We showed that the polyol pathway activities increase the level of peroxynitrite, which enhances the tyrosine nitration of SERCA and irreversibly modifies it to form SERCAC674-SO(3)H. This leads to reduced level of S-glutathiolated SERCA, contributing to its inactivation. The polyol pathway activities also deplete the level of GSH, leading to decreased active RyR, the S-glutathiolated RyR. Thus, in I/R heart, inhibition of polyol pathway improved the function of SERCA and RyR by protecting them from irreversible oxidation.

Testosterone-augmented contractile responses to alpha1- and beta1-adrenoceptor stimulation are associated with increased activities of RyR, SERCA, and NCX in the heart.

We hypothesized that testosterone at physiological levels enhances cardiac contractile responses to stimulation of both alpha(1)- and beta(1)-adrenoceptors by increasing Ca(2+) release from the sarcoplasmic reticulum (SR) and speedier removal of Ca(2+) from cytosol via Ca(2+)-regulatory proteins. We first determined the left ventricular developed pressure, velocity of contraction and relaxation, and heart rate in perfused hearts isolated from control rats, orchiectomized rats, and orchiectomized rats without and with testosterone replacement (200 microg/100 g body wt) in the presence of norepinephrine (10(-7) M), the alpha(1)-adrenoceptor agonist phenylephrine (10(-6) M), or the nonselective beta-adrenoceptor agonist isoprenaline (10(-7) M) in the presence of 5 x 10(-7) M ICI-118,551, a beta(2)-adrenoceptor antagonist. Next, we determined the amplitudes of intracellular Ca(2+) concentration transients induced by electrical stimulation or caffeine, which represent, respectively, Ca(2+) release via the ryanodine receptor (RyR) or releasable Ca(2+) in the SR, in ventricular myocytes isolated from the three groups of rats. We also measured (45)Ca(2+) release via the RyR. We then determined the time to 50% decay of both transients, which represents, respectively, Ca(2+) reuptake by sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) and removal via the sarcolemmal Na(+)/Ca(2+) exchanger (NCX). We correlated Ca(2+) removal from the cytosol with activities of SERCA and its regulator phospholamban as well as NCX. The results showed that testosterone at physiological levels enhanced positive inotropic and lusitropic responses to stimulation of alpha(1)- and beta(1)-adrenoceptors via the androgen receptor. The increased contractility and speedier relaxation were associated with increased Ca(2+) release via the RyR and faster Ca(2+) removal out of the cytosol via SERCA and NCX.

Chronic intermittent hypoxia alters Ca2+ handling in rat cardiomyocytes by augmented Na+/Ca2+ exchange and ryanodine receptor activities in ischemia-reperfusion.

This study examined Ca(2+) handling mechanisms involved in cardioprotection induced by chronic intermittent hypoxia (CIH) against ischemia-reperfusion (I/R) injury. Adult male Sprague-Dawley rats were exposed to 10% inspired O(2) continuously for 6 h daily from 3, 7, and 14 days. In isolated perfused hearts subjected to I/R, CIH-induced cardioprotection was most significant in the 7-day group with less infarct size and lactate dehydrogenase release, compared with the normoxic group. The I/R-induced alterations in diastolic Ca(2+) level, amplitude, time-to-peak, and the decay time of both electrically and caffeine-induced Ca(2+) transients measured by spectrofluorometry in isolated ventricular myocytes of the 7-day CIH group were less than that of the normoxic group, suggesting an involvement of altered Ca(2+) handling of the sarcoplasmic reticulum (SR) and sarcolemma. We further determined the protein expression and activity of (45)Ca(2+) flux of SR-Ca(2+)-ATPase, ryanodine receptor (RyR) and sarcolemmal Na(+)/Ca(2+) exchange (NCX) in ventricular myocytes from the CIH and normoxic groups before and during I/R. There were no changes in expression levels of the Ca(2+)-handling proteins but significant increases in the RyR and NCX activities were remarkable during I/R in the CIH but not the normoxic group. The augmented RyR and NCX activities were abolished, respectively, by PKA inhibitor (0.5 microM KT5720 or 0.5 microM PKI(14-22)) and PKC inhibitor (5 microM chelerythrine chloride or 0.2 microM calphostin C) but not by Ca(2+)/calmodulin-dependent protein kinase II inhibitor KN-93 (1 microM). Thus, CIH confers cardioprotection against I/R injury in rat cardiomyocytes by altered Ca(2+) handling with augmented RyR and NCX activities via protein kinase activation.

Altered Ca(2+) handling by ryanodine receptor and Na(+)-Ca(2+) exchange in the heart from ovariectomized rats: role of protein kinase A.

Our previous study has demonstrated that ovariectomy (Ovx) significantly increased the left ventricular developed pressure (LVDP) and the maximal rate of developed pressure over time (+/-dP/dt(max)) in the isolated perfused rat heart and the effects were reversed by female sex hormone replacement. In the present investigation, we studied the effects of Ovx for 6 wk on Ca(2+) homeostasis that determines the contractile function. Particular emphasis was given to Ca(2+) handling by ryanodine receptor (RyR) and Na(+)-Ca(2+) exchange (NCX). (45)Ca(2+) fluxes via the RyR, NCX, and Ca(2+)-ATPase (SERCA) were compared with their expression in myocytes from Ovx rats with and without estrogen replacement. Furthermore, we correlated the handling of Ca(2+) by these Ca(2+) handling proteins with the overall Ca(2+) homeostasis by determining the Ca(2+) transients induced by electrical stimulation and caffeine, which reveals the dynamic changes of cytosolic Ca(2+) concentration ([Ca(2+)](i)) in the heart. In addition, we determined the expression and contribution of protein kinase A (PKA) to the regulation of the aforementioned Ca(2+) handling proteins in Ovx rats. It was found that after Ovx there were 1) increased Ca(2+) fluxes via RyR and NCX, which were reversed not only by estrogen replacement, but more importantly by blockade of PKA; 2) an increased expression of PKA; and 3) no increase in expression of NCX and SERCA. We suggest that hyperactivities of RyR and NCX are a result of upregulation of PKA. The increased release of Ca(2+) through RyR and removal of Ca(2+) by NCX are believed to be responsible for the greater contractility and faster relaxation after Ovx.

Further study on the role of HSP70 on Ca2+ homeostasis in rat ventricular myocytes subjected to simulated ischemia.

We hypothesized that activation of heat shock protein 70 (HSP70) by preconditioning, which is known to confer delayed cardioprotection, attenuates the impaired handling of Ca(2+) at multiple sites. To test the hypothesis, we determined how the ryanodine receptor (RyR), sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA), and Na(+)/Ca(2+) exchanger (NCX) handled Ca(2+) in rat ventricular myocytes preconditioned with a kappa-opioid receptor agonist, U50488H (UP), followed by blockade of HSP70 with a selective antisense oligonucleotide and subsequently subjected to simulated ischemia. We determined the following: 1) the Ca(2+) transients induced by electrical stimulation and caffeine, which provide the overall picture of Ca(2+) homeostasis; 2) expression of RyR, SERCA, and NCX; and 3) Ca(2+) fluxes via NCX by the use of (45)Ca(2+) in the rat ventricular myocyte. We found that UP increased the activity of RyR, SERCA, and NCX and the expression of RyR and SERCA. These effects led to increases in the release of Ca(2+) from the sarcoplasmic reticulum via RyR and in the removal of Ca(2+) from the cytoplasm by reuptake of Ca(2+) to the SR via SERCA and by extrusion of Ca(2+) out of the cell via NCX. UP also reduced mitochondrial Ca(2+) accumulation. All of the effects of UP were either abolished or significantly attenuated by blockade of HSP70 synthesis with a selective antisense oligonucleotide. The results are evidence that activation of HSP70 by preconditioning improves the ischemia-impaired Ca(2+) homeostasis at multiple sites in the heart, which may be responsible, at least partly, for attenuated Ca(2+) overload, improved recovery in contractile function, and cardioprotection.

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

We previously showed that oestrogen confers cardioprotection by downregulating the cardiac beta1-adrenoceptor (beta1-AR). The present study examined the effect of oestrogen on the post beta1-AR signalling cascade, with particular emphasis on the activity of protein kinase A (PKA) and its influence on the L-type Ca2+ channel. 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. The electrically induced intracellular Ca2+ transient (E[Ca2+]i), 45Ca(2+)-uptake through cardiac L-type Ca2+ channels (Ca2+ channels), heart rate and force of contraction in response to beta-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 (G(alpha)s and G(alpha)i1-3)), basal and forskolin-stimulated cAMP accumulation, and responsiveness of PKA to cAMP, were not altered by Ovx. Interestingly, the PKA inhibitor at the same concentration significantly reduced the increases in PKA activity and Ca2+ channel activity upon beta-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. This study provides the first evidence that, in addition to downregulation of beta1-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 beta-AR stimulation in the female rat.

The inhibitory effect of adrenomedullin in the rat ileum: cross-talk with beta3-adrenoceptor in the serotonin-induced muscle contraction.

In contrast to vascular muscles, the contribution of a hypotensive peptide adrenomedullin (AM) to the regulation of visceral smooth muscles is obscure. The content, synthesis, and effects of AM on the muscular tone in rat ileum were explored. It was found that there was immunoreactive AM (301 pg/mg of protein) and AM mRNA expression (162 fg/pg actin mRNA) in the ileum and that AM evoked relaxation in ileal strips (Ki = 0.85 nM) precontracted with serotonin. Antagonists of both AM (AM(22-52)) and calcitonin gene-related peptide (CGRP(8-37)) receptors did not affect this AM-induced relaxation, whereas it was suppressed by a selective blocker of beta3-adrenoreceptor (SR 59230A). The AM-induced relaxation was accompanied by a production of cAMP. Antagonists of protein kinases A (KT 5720 and H-7) and an inhibitor of the ATP-dependent K(+)-channels (glibenclamide) attenuated the effect of AM. We suggest that AM is a local regulator of the ileal tone, with an inhibitory action on muscle contraction. AM may activate the beta3-adrenoceptors, resulting in protein kinase A activation, which in turn opens the ATP-dependent K(+)-channels.

Calcium homeostasis in rat cardiomyocytes during chronic hypoxia: a time course study.

The present study determined Ca2+ handling in the hearts of rats subjected to chronic hypoxia (CH). Spectrofluorometry was used to measure intracellular Ca2+ concentration ([Ca2+]i) and its responses to electrical stimulation, caffeine, and isoproterenol in myocytes from the right ventricle of rats breathing 10% oxygen for 1, 3, 7, 14, 21, 28, and 56 days and age-matched controls. The protein expression of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) and its ryanodine receptor (RyR) were measured. The uptake of 45Ca2+ by SERCA, release by RyR, and extrusion by Na+/Ca2+ exchange (NCX) were determined. It was found that Ca2+ homeostasis and Ca2+ responses to beta-adrenoceptor stimulation reached a new equilibrium after 4 wk of CH. Ca2+ content in the sarcoplasmic reticulum (SR) was reduced, but cytosolic Ca2+ remained unchanged after CH. Expression of SERCA and its Ca2+ uptake, Ca2+ release via RyR, and NCX activity were suppressed by CH. The results indicate impaired Ca2+ handling, which may be responsible for the attenuated Ca2+ responses to beta-adrenoceptor stimulation in CH.

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 (45)Ca2+ 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.