Alicia E. Consolini

Pharmacological basis for the empirical use of Eugenia uniflora L. (Myrtaceae) as antihypertensive

The rational basis for the use of Eugenia uniflora L. (Myrtaceae) as antihypertensive in Northeastern Argentina was assessed in normotensive rats. Intraperitoneal administration of the aqueous crude extract (ACE) decreased blood pressure (BP) of normotensive rats dose-dependently until 47.1 +/- 8.2% of control. The effective-dose 50 was 3.1 +/- 0.4 mg dried leaves/kg (d.l./kg) (yielding of ACE: 17% w/w). To determine the origin of hypotensive activity. Alpha-adrenergic antagonistic and vasorelaxant ACE activities were tested. The dose-response curve for phenylephrine on BP was inhibited non-competitively until 80% of its maximal effect (at 8 mg d.l. ACE/kg). Perfusion pressure (PP) of rat hindquarters (previously vasoconstricted by high-K+) was decreased by ACE in a concentration-dependent manner until -32.3 +/- 11.5% of tonic contraction at 1.2 g d.l. ACE/100 ml. In addition, A.C.E demonstrated diuretic activity at a dose (120 mg d.l./kg) higher than the hypotensive one. It was almost as potent as amiloride, but while amiloride induced loss of Na+ and saving of K+, ACE induced decrease in Na+ excretion. The results suggest that the empirical use of Eugenia uniflora L. (Myrtaceae) is mostly due to a hypotensive effect mediated by a direct vasodilating activity, and to a weak diuretic effect that could be related to an increase in renal blood flow.

Pharmacological effects of Eugenia uniflora (Myrtaceae) aqueous crude extract on rat's heart.

The effect of aqueous crude extract (ACE) of Eugenia uniflora L. (Myrtaceae) was studied on rats perfused ventricles. This plant is used in South American traditional medicine as an antihypertensive and we already demonstrated previously its hypotensive properties. In this paper, maximal left intraventriclular pressure (P) of rats hearts beating at 0.2 Hz firstly increased to 162.1+/-11.1% of basal value during 1-3 min of perfusing ACE 0.6%. Maximum rate of contraction (+P) also increased to duplicating +P/P ratio. Both types of effect were significantly decreased by either propranolol 0.35 microM, and pre-treatment with reserpine (5 mg/kg), suggesting that they were caused by a compound that releases cathecolamines with beta-adrenergic action. Nevertheless, after 20 min of perfusing ACE, ventricles decreased P to about 50% of their basal value, suggesting a negative-inotropic compound present in the extract. The perfusion of 1.2% ACE decreased P in a pressure-[Ca](o) curve (0.5-2 mM) in a non-competitive manner, suggesting that an irreversible Ca-blocking compound is also present in the extract. In summary, E. uniflora ACE has a dual effect on the heart related to its hypotensive action and is probably responsible for the therapeutic or adverse effects in patients under cardiac risk.

Mitochondrial role in ischemia-reperfusion of rat hearts exposed to high-K + cardioplegia and clonazepam: energetic and contractile consequences

The role of the mitochondrial Na/Ca-exchanger (mNCX) in hearts exposed to ischemia-reperfusion (I/R) and pretreated with cardioplegia (CPG) was studied from a mechano-calorimetric approach. No-flow ischemia (ISCH) and reperfusion (REP) were developed in isolated rat hearts pretreated with 10 micromol/L clonazepam (CLZP), an inhibitor of the mNCX, and (or) a high K+ - low Ca2+ solution (CPG). Left ventricular end diastolic pressure (LVEDP), pressure development during beats (P), and the steady heat release (Ht) were continuously measured and muscle contents of ATP and PCr were analyzed at the end of REP. During REP, Ht increased more than P, reducing muscle economy (P/Ht) and the ATP content. CPG induced an increase in P recovery during REP (to 90% +/- 10% of preISCH) with respect to nonpretreated hearts (control, C, to 64% +/- 10%, p < 0.05). In contrast, CLZP reduced P recovery of CPG-hearts (50% +/- 6.4%, p < 0.05) and increased LVEDP in C hearts. To evaluate effects on sarcoplasmic reticulum (SR) function, ischemic hearts were reperfused with 10 mmol/L caffeine -36 mmol/L Na (C - caff - low Na). It increased LVEDP, which afterwards slowly relaxed, whereas Ht increased (by about 6.5 mW/g). CLZP sped up the relaxation with higher DeltaHt, C - caff - low Na produced higher contracture and lower Ht in perfused than in ischemic hearts. Values of DeltaHt were compared with reported fluxes of Ca2+-transporters, suggesting that mitochondria may be in part responsible for the DeltaHt during C - caff - low Na REP. Results suggest that ISCH-REP reduced the SR store for the recovery of contractility, but induced Ca2+ movement from the mitochondria to the SR stores. Also, mitochondria and SR are able to remove cytosolic Ca2+ during overloads (as under caffeine), through the mNCX and the uniporter. CPG increases Ca2+ cycling from mitochondria to the SR, which contributes to the higher recovery of P. In contrast, CLZP produces a deleterious effect on ISCH-REP associated with higher heat release and reduced resynthesis of high energy phosphates, which suggests the induction of mitochondrial Ca cycling and uncoupling.

CARDIAC role of the mitochondrial Ca2+ transporters in the high-[K+](o) cardioprotection of rat hearts under ischemia and reperfusion: a mechano-energetic study.

The role of mitochondrial transporters in the cardioprotection of rat hearts exposed to high [K+]-low [Ca2+]-cardioplegia (CPG) and ischemia and reperfusion (I/R) was studied through the mechano-energetic consequences of target drugs. The total heat rate (Ht) and the left intraventricular pressure (LVP) were simultaneously measured in isolated perfused hearts (30°C and 1 Hz) inside a flow-calorimeter during 45 minutes of no-flow I and 45 minutes of R. After stabilization (C) they were pretreated with CPG and 100 μM 5-hydroxidecanoate (5HD, selective mKATP blocker) without and with 10 or 30 μM clonazepam (Clzp, mNCX inhibitor), 30 μM diazoxide (Dzx, selective mKATP opener), 1 μM Ru360 (selective Ca2+-uniporter blocker), and 0.2 μM cyclosporine-A, (mPTP inhibitor, before I and during R). Before I, 5-hydroxydecanoate in CPG increased the resting heat rate (17.83 ± 3.55 mW/g) without changing the stunning. Clzp 30 μM + CPG + 5-hydroxydecanoate reduced the postischemic P with diastolic contracture and high Ht. Dzx protected C-hearts from stunning but increased it in CPG hearts with low economy (P/Ht) as well as Ru360. Cyclosporine-A did not modify the stunning of C or CPG ischemic hearts, suggesting that the mPTP was not opened. Conclusions: Mitochondria have a precise role for determining cardioprotection or stunning in high-K+ cardioplegic rat hearts under I/R. Known protective drugs, such as Dzx and Ru360, which reduce the mitochondrial Ca2+-uptake, increased the stunning of CPG-rat hearts and reduced muscle economy, whereas 5-hydroxydecanoate and Clzp together increased the stunning by inducing mitochondrial Ca2+ overload.

Cardioprotective effect of hyperthyroidism on the stunned rat heart during ischaemia–reperfusion: energetics and role of mitochondria

What is the central question of this study? Hyperthyroidism is a cardiac risk factor, but thyroid therapy is used on myocardial stunning. What is the consequence of hyperthyroidism for mitochondrial metabolism and Ca2+ handling of the postischaemic stunned heart? What is the main finding and its importance? Hyperthyroidism reduced stunning and improved muscle economy of the postischaemic rat heart. The activities of the mitochondrial sodium–calcium exchanger and mitochondrial K+ channel in hyperthyroid rat hearts were different from those in the euthyroid rat hearts. These findings contribute to the understanding of mitochondrial bioenergetics in pathology and support thyroid therapy in the stunning induced by ischaemia.

Sex differences in the mechano-energetic effects of genistein on stunned rat and guinea pig hearts.

Although the phytoestrogen genistein (Gen) is considered protective in cardiovascular diseases, its direct effects on stunned hearts after transient ischemia‐reperfusion (I/R) are unknown. This report studied the effects of 20 μmol/L Gen on the mechano‐calorimetric behaviour during I/R of rat and guinea pig hearts to evaluate the energetics of Ca2+ homeostasis. Isolated beating hearts were perfused with control Krebs solution inside a calorimeter with or without perfusion of Gen before a transient period of I/R. Left ventricular pressure development (P) and total heat rate (Ht) were continuously measured. At 37°C, Gen did not change post‐ischemic contractile recovery (PICR), but it increased the relaxation rate. However, PICR was reduced in hearts of male rats and guinea pigs at 30°C. Total muscle economy (P/Ht) showed the same behaviour as P at each temperature. Inhibition of phosphatases with orthovanadate during Gen perfusion prevented a decrease in PICR in male rat hearts, suggesting that this effect is due to tyrosine kinase inhibition. Reperfusing ischemic hearts with 10 mmol/L caffeine‐36 mmol/L Na+‐Krebs induced contracture dependent on the sarcoreticular Ca2+ content. Contracture relaxation depends on mitochondrial Ca2+ uptake and Gen reduced the relaxation rate. Moreover, Gen prevented the increase in Rhod‐2 fluorescence (free [Ca2+]m) of rat cardiomyocytes. In guinea pig hearts, Gen maintained ischemic preconditioning, but was reduced by 5‐hydroxydecanoate, suggesting the participation of mitochondrial adenosine triphosphate (ATP)‐dependent K channels. Results suggest that Gen acts on several mechanisms that regulate myocardial calcium homeostasis and energetics during I/R, which differ in a temperature‐ and sex‐dependent manner.

Mitochondrial Bioenergetics During Ischemia and Reperfusion

During ischemia and reperfusion (I/R) mitochondria suffer a deficiency to supply the cardiomyocyte with chemical energy, but also contribute to the cytosolic ionic alterations especially of Ca2+. Their free calcium concentration ([Ca2+]m) mainly depends on mitochondrial entrance through the uniporter (UCam) and extrusion in exchange with Na+ (mNCX) driven by the electrochemical gradient (ΔΨm). Cardiac energetic is frequently estimated by the oxygen consumption, which determines metabolism coupled to ATP production and to the maintaining of ΔΨm. Nevertheless, a better estimation of heart energy consumption is the total heat release associated to ATP hydrolysis, metabolism, and binding reactions, which is measurable either in the presence or the absence of oxygenation or perfusion. Consequently, a mechano-calorimetrical approach on isolated hearts gives a tool to evaluate muscle economy. The mitochondrial role during I/R depends on the injury degree. We investigated the role of the mitochondrial Ca2+ transporters in the energetic of hearts stunned by a model of no-flow I/R in rat hearts. This chapter explores an integrated view of previous and new results which give evidences to the mitochondrial role in cardiac stunning by ischemia o hypoxia, and the influence of thyroid alterations and cardioprotective strategies, such as cardioplegic solutions (high K-low Ca, pyruvate) and the phytoestrogen genistein in both sex. Rat ventricles were perfused in a flow-calorimeter at either 30 °C or 37 °C to continuously measure the left ventricular pressure (LVP) and total heat rate (Ht). A pharmacological treatment was done before exposing to no-flow I and R. The post-ischemic contractile (PICR as %) and energetical (Ht) recovery and muscle economy (Eco: P/Ht) were determined during stunning. The functional interaction between mitochondria (Mit) and sarcoplasmic reticulum (SR) was evaluated with selective mitochondrial inhibitors in hearts reperfused with Krebs-10 mM caffeine-36 mM Na+. The caffeine induced contracture (CIC) was due to SR Ca2+ release, while relaxation mainly depends on mitochondrial Ca2+ uptake since neither SL-NCX nor SERCA are functional under this media. The ratio of area-under-curves over ischemic values (AUC-ΔHt/AUC-ΔLVP) estimates the energetical consumption (EC) to maintain CIC. Relaxation of CIC was accelerated by inhibition of mNCX or by adding the aerobic substrate pyruvate, while both increased EC. Contrarily, relaxation was slowed by cardioplegia (high K-low Ca Krebs) and by inhibition of UCam. Thus, Mit regulate the cytosolic [Ca2+] and SR Ca2+ content. Both, hyperthyroidism (HpT) and hypothyroidism (HypoT) reduced the peak of CIC but increased EC, in spite of improving PICR. Both, CIC and PICR in HpT were also sensitive to inhibition of mNCX or UCam, suggesting that Mit contribute to regulate the SR store and Ca2+ release. The interaction between mitochondria and SR and the energetic consequences were also analyzed for the effects of genistein in hearts exposed to I/R, and for the hypoxia/reoxygenation process. Our results give evidence about the mitochondrial regulation of both PICR and energetic consumption during stunning, through the Ca2+ movement.

Effects of pyruvate on the energetics of rat ventricles stunned by ischemia-reperfusion.

Pyruvate (Pyr) was proposed as an additive to cold high-K(+)-low-Ca(2+) cardioplegia (CPG) to protect the heart during surgery. We explored whether Pyr and CPG would work synergistically to protect rat hearts from stunning during ischemia-reperfusion (I/R). We measured the heat release and contractility of perfused ventricles during I/R, and the cytosolic and mitochondrial [Ca(2+)] in cardiomyocytes by confocal microscopy. We found that under cold-CPG (30 °C), 10 mmol·L(-1) Pyr reduced the post-ischemic contractile recovery (PICR) as well as muscle economy, when added either before ischemia or during I/R, which was reversed by blockade of UCam. In noncardioplegic hearts, Pyr was cardioprotective when it was present during I/R, more so at 37 °C than at 30 °C, with improved economy. In cardiomyocytes, the addition of Pyr to CPG slightly increased the mitochondrial [Ca(2+)] but decreased cytosolic [Ca(2+)]. The results suggest that Pyr only protects hearts from stunning when present before ischemia and during reperfusion, and that it dampens the cardioprotective properties of CPG. The mechanisms underlying such different behavior depend on the dynamic balance between Pyr stimulation of the energetic state and mitochondrial Ca(2+) uptake. Our results support the use of Pyr in stunned hearts, but not in cold high-K(+) cardioplegia.

Intestinal, urinary and uterine antispasmodic effects of isoespintanol, metabolite from Oxandra xylopioides leaves

BACKGROUND Isoespintanol is a monoterpene isolated from the leaves of Oxandra xylopioides Diels. (Annonaceae) with antioxidant and antiinflammatory effects. It was of interest to know whether it has antispasmodic effects such as other known drugs, phloroglucinol and trimethoxybenzene, used in therapeutics for treating biliary, urinary and uterine spasms. PURPOSE To assess whether isoespintanol possesses antispasmodic effects on intestine, uterus and bladder. STUDY DESIGN A preclinical study was performed in which isoespintanol, phloroglucinol and trimethoxybenzene were evaluated with concentration-contractile response curves (CRC) of carbachol in isolated rat intestine and bladder, and with CRC of serotonin (5-HT) in rat uterus. Moreover, it was assessed whether isoespintanol interferes with Ca2+ influx by making CRC of Ca2+ in high-K+ medium in intestine and bladder. RESULTS Isoespintanol non-competitively inhibited the CRC of carbachol with affinity constant (pK) of 4.78 ± 0.09 in intestine and 4.60 ± 0.09 in bladder. Phloroglucinol and trimethoxybenzene were also non-competitive antagonists, but isoespintanol was 8 times more potent than trimethoxybenzene and similarly potent than phloroglucinol in intestine. In bladder, isoespintanol resulted 8 times more potent than trimethoxybenzene. The maximal inhibition of contraction followed the order of isoespintanol > trimethoxybenzene > phloroglucinol in intestine, and isoespintanol > trimethoxybenzene in bladder. Moreover, isoespintanol also completely and non-competitively inhibited the CRC of Ca2+, with a pK of 5.1 ± 0.1 in intestine, and 4.32 ± 0.07 in bladder. In uterus isoespintanol reduced, completely and non-competitively, the contraction produced by 5-HT with pK of 5.05 ± 0.07. CONCLUSION Results demonstrate that isoespintanol is a very good intestinal, urinary and uterine antispasmodic, with higher potency than the other drugs used in therapeutics. The mechanism of action of isoespintanol is the interference with Ca2+ influx, at a difference of trimethoxybenzene and phloroglucinol.

Low-flow ischaemia and reperfusion in rat hearts: energetic of stunning and cardioprotection of genistein

Low‐flow ischemia (LFI) is consequent to coronary disease and produces cardiac stunning during reperfusion (R). Energetic performance and mechanisms of Ca2+ handling during LFI/R are not known. Moreover, cardioprotection of the phytoestrogen genistein (Gen) remains to be demonstrated in LFI/R. The aim was to study the mechanisms of the stunning consequent to LFI/R and the effects of Gen on both sexes.