Gennadi M. Kravtsov

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.

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.

Testosterone-augmented contractile responses to α1- and β1-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.

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.

L‐NAME inhibits Mg2+‐induced rat aortic relaxation in the absence of endothelium

L‐NG‐nitro‐arginine methyl ester (L‐NAME; 100 μM), a nitric oxide synthase (NOS) inhibitor, reversed the relaxation induced by 3 μM acetylcholine (ACh) and 2–10 mM Mg2+ in endothelium‐intact (+E) rat aortic rings precontracted with 1 μM phenylephrine (PE). In PE‐precontracted endothelium‐denuded (−E) rat aorta, 3 μM ACh did not, but Mg2+ caused relaxation which was reversed by L‐NAME, but not by D‐NAME. The concentration response profiles of L‐NAME in reversing the equipotent relaxation induced by 5 mM Mg2+ and 0.2 μM ACh were not significantly different. L‐NAME (100 μM) also reversed Mg2+‐relaxation of −E aorta pre‐contracted with 20 mM KCl or 10 μM prostaglandin F2α (PGF2α). L‐NG‐monomethyl‐arginine (L‐NMMA; 100 μM) was also effective in reversing the Mg2+‐relaxation. Addition of 0.2 mM Ni2+, like Mg2+, caused relaxation of PE‐pre‐contracted −E aorta, which was subsequently reversed by 100 μM L‐NAME. Reversal of the Mg2+‐relaxation by 100 μM L‐NAME in PE‐precontracted −E aorta persisted following pre‐incubation with 1 μM dexamethasone or 300 μM aminoguanidine (to inhibit the inducible form of NOS, iNOS). Pretreatment of either +E or −E aortic rings with 100 μM L‐NAME caused elevation of contractile responses to Ca2+ in the presence of 1 μM PE. Our results suggest that L‐NAME exerts a direct action on, as yet, unidentified vascular smooth muscle plasma membrane protein(s), thus affecting its reactivity to divalent cations leading to the reversal of relaxation. Such an effect of L‐NAME is unrelated to the inhibition of endothelial NOS or the inducible NOS.

Aggregation, membrane potential, and transport in platelets of spontaneously hypertensive rats

Abstract1.Aggregation, plasma membrane potential, and calcium uptake by platelets of spontaneously hypertensive rats (SHR) were studied. 2. The sensitivity of SHR platelets to the action of ADP and collagen was increased. The addition of Ca-ionophore (A-23187) resulted in aggregation of SHR platelets at a Ca2+ concentration in the incubation medium 20%–30% lower than that required for aggregation of platelets from normotensive rats. 3. The plasma membrane of SHR platelets was found to be partially depolarized (by 8–10 mV) as compared to that of normotensive controls. This difference was apparently due to a higher membrane permeability for Na+. 4. It is suggested that the increase in45Ca uptake by SHR platelets is related to an increased rate of Ca2+ diffusion along a potential-dependent channel, due to partial depolarization of plasma membrane.

Activity of protein kinase C in erythrocytes in primary hypertension.

The activity of protein kinase C and A was studied in the erythrocytes of patients with essential hypertension (EH) and in spontaneously hypertensive rats (SHR, Okamoto-Aoki strain). Protein kinase C activity was also studied in the erythrocytes of patients with hypertension of renal origin. Protein kinase C activity in the lysate of erythrocytes of patients with EH and in SHR was found to be increased 1.6-2.0-fold as compared to that in normotensive controls. No notable differences in protein kinase A activity were observed between hypertensive and normotensive groups. In erythrocytes of patients with renal hypertension, no notable changes in protein kinase C activity were revealed.

Receptor‐mediated modulation of avian caecal muscle contraction by melatonin: role of tyrosine protein kinase

Melatonin receptors in the quail caecum were studied by 2[125I]iodomelatonin binding assay and the involvement of tyrosine protein kinase in the melatonin‐induced contraction was explored. The binding of 2[125I]iodomelatonin in the quail caecum membrane preparations was saturable, reversible and of high affinity with an equilibrium dissociation constant (Kd) of 24.6 ± 1.1 pm (n=7) and a maximum number of binding sites (Bmax) of 1.95 ± 0.09 fmol (mg/protein) (n=7). The relative order of potency of indoles in competing for 2[125I]iodomelatonin binding was: 2‐iodomelatonin > melatonin > 2‐phenylmelatonin >  6‐chloromelatonin > 6‐hydroxymelatonin > N‐acetylserotonin, indicating that ML1 receptors are involved. The binding was inhibited by Mel1b melatonin receptor antagonists, luzindole and 4‐phenyl‐2‐propionamidotetralin (4‐P‐PDOT) as well as by non‐hydrolyzable analogs of GTP like GTPγS and Gpp(NH)p but not by adenosine nucleotides. The latter suggests that the action of melatonin on the caecum is G‐protein linked. Cumulative addition of melatonin (1–300 nM) potentiated both the amplitude and frequency of spontaneous contractions in the quail caecum. The potentiation of rhythmic contractions was blocked by both luzindole and 4‐P‐PDOT. Antagonists of tyrosine kinase, genistein(2 μM) and erbstatin(4 μM) suppressed the modulation of spontaneous contractions by melatonin, but not inhibitors of protein kinase C (PKC) or protein kinase A (PKA). Melatonin‐induced increment in spontaneous contraction was blocked by nifedipine (0.4 nM). Thus, we suggest that melatonin potentiates spontaneous contraction in the quail caecum via interacting with G‐protein‐coupled Mel1b receptor which may activate L‐type Ca2+ channels by mobilizing tyrosine kinases.

2[125I]Iodomelatonin binding sites in guinea pig platelets

Using 2[125I]iodomelatonin as the radioligand, we characterized 2[125I]iodomelatonin binding sites in guinea pig platelet membrane preparations. Saturation radioreceptor studies indicated that these 2[125I]iodomelatonin binding sites were of picomolar affinity and femtomolar density. The dissociation constant (Kd) and maximum number of receptor sites (Bmax) were 42.5 ± 1.79 pM and 11.8 ± 0.8 fmol/mg protein (n=6), respectively. 2[125I]Iodomelatonin competition studies with indoles or drugs indicate the following rank order of potency: 2‐iodomelatonin >melatonin ≥ 6‐chloromelatonin > 6‐hydroxymelatonin > N‐acetylserotonin >  5‐methoxytryptophol, whereas serotonin and its analogs had less than 20% inhibition at 0.1 mM. Guanosine 5′‐O‐(3‐thiotriphosphate) significantly increased the Kd by twofold suggesting that these binding sites are coupled to the guanine nucleotide binding proteins. Immunoblotting studies using anti‐MT1 IgG demonstrated one peptide blockable band with an apparent molecular mass of 37 kDa. Melatonin had no effect on prostacyclin or forskolin‐stimulated intracellular 3′,5′‐cyclic adenosine monophosphate accumulation. A diurnal variation in binding density, which was abolished after the animals were adapted to constant light conditions, was observed. Age related studies demonstrated that Bmax increased as the animal matured. Physiological melatonin concentrations potentiated whereas those at pharmacological levels inhibited adenosine diphosphate‐ or arachidonic acid‐stimulated platelet aggregation. Our study demonstrated G‐protein coupled, saturable, reversible and highly specific picomolar affinity 2[125I]iodomelatonin binding sites in guinea pig platelets. Pharmocological and physiological data indicate that they may be different from the nanomolar [3H]melatonin binding sites in human platelets previously reported.