Antispasmodic, cardioprotective and blood-pressure lowering properties of Gomphrena perennis L. and its mechanisms of action.

Background: Gomphrena perennis L. is a native plant of South America whose pharmacological properties have not been studied yet. Aim: To evaluate the cardiovascular and intestinal pharmacological effects of Gomphrena perennis L. leaves tincture (GphT) and the mechanisms involved. Experimental procedure: The chromatographic profile of GphT was done. Its ex vivo effects were evaluated by contractile concentration-response curves (CRCs) obtained from the agonist carbachol or calcium found in isolated rat small intestine, as well as in the relaxant CRCs. Cardiac effects were evaluated on isolated rat hearts exposed to ischemia/reperfusion (I/R). Experiments in vivo were performed to evaluate the diuretic activity in conscious rats and the hypotensive effect in anaesthetised rats. Results: Fifteen flavonoids were identified in GphT by HPLC-UV, including diosmin. GphT induced a non-competitive inhibition in both carbachol and calcium CRCs on rat small intestine. The first was not affected by indomethacin. Moreover, GphT, unlike diosmin, relaxed the contracture produced by a high-potassium solution in a dose-dependently way. Neither propranolol nor l-NAME changed it. GphT did not show diuretic activity but induced hypotension insensitive to l-NAME. While GphT perfusion of isolated hearts increased injury consequent to I/R, oral administration was cardioprotective and reversed by l-NAME. However, diosmin did not improve the post-ischemic recovery. Conclusions: This study supports the use of Gomphrena perennis L. tincture as an antispasmodic and hypotensive agent. Moreover, it has been demonstrated to be preventive of post-ischemic cardiac dysfunction. However, diosmin would not be responsible for these effects.

Lepidium meyenii (maca) and soy isoflavones reduce cardiac stunning of ischemia-reperfusion in rats by mitochondrial mechanisms.

BACKGROUND AND AIM: Phytoestrogens are traditionally used for cardiovascular risks but direct effects on the ischemic heart remain unclear. Plants with phytoestrogens are used for reducing menopausic symptoms and they could also be cardioprotectives. Here we investigated whether maca (Lepidium meyenii) contains isoflavones and prevents cardiac stunning, in comparison to soy isoflavones. EXPERIMENTAL PROCEDURE: Both products were orally and daily administered to rats during 1 week before exposing isolated hearts to ischemia/reperfusion (I/R). Young male (YM), female (YF) and aged female (AgF) rats treated with maca (MACA, 1 g/kg/day) or soy isoflavones (ISOF, 100 mg/kg/day) were compared to acute daidzein (DAZ, 5 mg/kg i.p.) and non-treated rat groups. Isolated ventricles were perfused inside a calorimeter to simultaneously measure contractile and calorimetrical signals before and during I/R. RESULTS AND CONCLUSIONS: Maca has genistein and daidzein. MACA and ISOF improved the post-ischemic contractile recovery (PICR) and muscle economy (P/Ht) in YM and YF hearts, but not in AgF hearts. DAZ improved PICR and P/Ht more in YM than in YF. The mKATP channels blockade reduced both PICR and P/Ht in DAZ-treated YM hearts, without affecting them in ISOF or MACA-treated YM hearts. In MACA treated YF hearts, the simultaneous blockade of NOS and mKATP channels, or the mNCX blockade reduced cardioprotection. Results show that subacute oral treatment with maca or with soy isoflavones was strongly preventive of cardiac ischemic dysfunction, more than the acute administration of a pure isoflavone (daidzein, genistein). Maca induced synergistic and complex mechanisms which prevented mitochondrial calcium overload.

The mKATP Channels and protein-kinase C Are Involved in the Cardioprotective Effects of Genistein on Estrogen-Deficient Rat Hearts Exposed to Ischemia/Reperfusion: Energetic Study.

Estrogenic deficiency is considered a risk of coronary disease in women. The phytoestrogen genistein could be a safe preventive strategy. The first aim of this work was to validate a model of cardiac stunning in which natural estrogenic deficiency rats, ie, adult young male (YM) and aged female (AgF), are compared with young female rats (YF). The second aim was to study whether the in vivo administration of genistein prevents the stunning in estrogenic deficiency rats. The third aim was to evaluate whether in our estrogenic deficiency model exists a synergy between genistein and estradiol. The fourth aim was to characterize the underlying mechanisms of genistein. Stunning was induced by ischemia/reperfusion (I/R) in isolated hearts inside a calorimeter. The left ventricular pressure (P) and total heat rate (Ht) were simultaneously measured, while diastolic contracture and muscle economy (P/Ht) were calculated. During R, P/Ht and P recovered less in AgF and YM than in YF rat hearts. Genistein through i.p. (GST-ip) improved P and P/Ht in AgF and YM, but not in YF. In YM, the cardioprotections of GST-ip and estradiol were synergistic. After ischemia, GST-ip increased SR Ca leak causing diastolic contracture. The GST-ip cardioprotection neither was affected by blockade of PI3K-Akt, NO synthases, or phosphatases, but it was sensitive to blockade of protein-kinase C and mKATP channels. Results suggest that (1) estrogenic deficiency worsens cardiac stunning, (2) GST-ip was more cardioprotective in estrogenic deficiency and synergistic with estradiol, and (3) cardioprotection of GST-ip depends on the protein-kinase C and mKATP channel pathway activation.

Cardioprotective Mechanisms of Hypothyroidism on Ischemia/Reperfusion in Rats and Effects of Carvedilol: Energetic Study.

Hypothyroidism is considered a cardiac risk factor, but there is controversial evidence about its effects on coronary disease. The aim of this work was to evaluate the influence of hypothyroidism in rat hearts exposed to 2 degrees of stunning due to ischemia and reperfusion (I/R) as well as the underlying mechanisms. Hypothyroid (HypoT) rats were obtained by drinking 0.02% methimazole during 15 days. Isolated hearts were perfused and introduced in a flow calorimeter to measure contractile performance (P), total heat rate (Ht), and muscle economy (P/Ht). Hearts were exposed to 2 models of I/R, moderate and severe (respectively 20 or 30 minutes I/45 minutes R). Moreover, free cytosolic and mitochondrial calcium changes were measured by confocal fluorometry on cardiomyocytes. Comparison to euthyroid (EuT) hearts was done. Hypothyroidism was cardioprotective, but HypoT hearts were more sensitive than EuT hearts to the preischemic blockade of mitochondrial transporters mNCX and mKATP channels. Moreover, the postischemic recovery of P and P/Ht in HypoT hearts was strongly reduced by inhibition of the cellular pathways of PI3K/Akt and protein kinase C (PKC), and it was increased by nitric oxide synthase (NOS) inhibition. However, physiological concentrations of adrenaline reduced the cardioprotection of HypoT, but oral treatment with 20 mg/kg/day carvedilol prevented it. Results show that hypothyroidism reduces the mitochondrial Ca2+ overload during I/R by mKATP channel activation and Ca2+ extrusion through mNCX, while the PI3K/Akt and PKC pathways are involved in that cardioprotection. Contrarily, NOS activation and adrenaline blunt such cardioprotection, but carvedilol prevented the adrenergic dysfunction. These results would explain why hypothyroidism is a clinical risk factor in angor patients under adrenergic exacerbation but reduced the incidence of acute episodes of coronary syndrome in hospitalized patients. Results suggest that a treatment with carvedilol could be a potential therapeutic agent to prevent cardiac postischemic dysfunction in hypothyroid patients.

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 ±â€¯0.09 in intestine and 4.60 ±â€¯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 ±â€¯0.1 in intestine, and 4.32 ±â€¯0.07 in bladder. In uterus isoespintanol reduced, completely and non-competitively, the contraction produced by 5-HT with pK of 5.05 ±â€¯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.

OBJECTIVES: 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. METHODS: Rat ventricles were perfused inside a calorimeter to measure maximal pressure development (P) and total heat rate (Ht) before and during exposition to LFI/R. The mechanisms of stunning were evaluated with selective drugs. KEY FINDINGS: Female hearts (FH) developed higher postischemic contractile recovery (PICR) and muscle economy (P/Ht) than males (MH). Cardioprotection was sensitive to blockade of mKATP channels, UCam and NOS. Perfusion of 20 µmol/l Gen reduced PICR and P/Ht during LFI/R in FH, and dysfunction was increased by mNCX blockade with mPTP opening. However, intraperitoneal 5 mg/kg Gen (Gen-ip) was cardioprotective in both sexes, and the beneficial effect of Gen-ip was blocked by 100 µmol/l 5-HD. CONCLUSIONS: FH are more protected than MH against the LFI/R dysfunction, which involves mitochondrial Ca2+ loss; Gen-ip was more cardioprotective in MH than in FH, mainly by activation of the mKATP channels.

Cardioprotection of stevioside on stunned rat hearts: A mechano-energetical study.

BACKGROUND: The sweetener and hypoglycemic properties of stevioside (STV) are well known, as the main component of the plant Stevia rebaudiana. Given its extensive use in diabetic patients, it was of interest to evaluate its effects on the most frequent cardiovascular disease, the coronary insufficiency. PURPOSE: To study whether STV could be cardioprotective against ischemia-reperfusion (I/R) in a model of "stunning" in rat hearts. STUDY DESIGN: A preclinical study was performed in isolated hearts from rats in the following groups: non-treated rats whose hearts were perfused with STV 0.3 mg/ml and their controls (C) exposed to either moderate stunning (20 min I/45 min R) or severe stunning (30 min I/45 min R), and a group of rats orally treated with STV 25 mg/kg/day in the drink water during 1 week before the experiment of severe stunning in the isolated hearts were done. METHODS: The mechano-calorimetrical performance of isolated beating hearts was recorded during stabilization period with control Krebs perfusion inside a calorimeter, with or without 0.3 mg/ml STV before the respective period of I/R. The left ventricular maximal developed pressure (P) and total heat rate (Ht) were continuously measured. RESULTS: Both, orally administered and perfused STV improved the post-ischemic contractile recovery (PICR, as % of initial control P) and the total muscle economy (P/Ht) after the severe stunning, but only improved P/Ht in moderate stunning. However, STV increased the diastolic pressure (LVEDP) during I/R in both stunning models. For studying the mechanism of action, ischemic hearts were reperfused with 10 mM caffeine-36 mM Na+-Krebs to induce a contracture dependent on sarcorreticular Ca2+ content, whose relaxation mainly depends on mitochondrial Ca2+ uptake. STV at 0.3 mg/ml increased the area-under-curve of the caffeine-dependent contracture (AUC-LVP). Moreover, at room temperature STV increased the mitochondrial Ca2+ uptake measured by Rhod-2 fluorescence in rat cardiomyocytes, but prevented the [Ca2+]m overload assessed by caffeine-dependent SR release. CONCLUSIONS: Results suggest that STV is cardioprotective against I/R under oral administration or direct perfusion in hearts. The mechanism includes the regulation of the myocardial calcium homeostasis and the energetic during I/R in several sites, mainly reducing mitochondrial Ca2+ overload and increasing the sarcorreticular Ca2+ store.

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.

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 Ca(2+) 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 Ca(2+) content. Contracture relaxation depends on mitochondrial Ca(2+) uptake and Gen reduced the relaxation rate. Moreover, Gen prevented the increase in Rhod-2 fluorescence (free [Ca(2+)]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.

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

NEW FINDINGS: 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 Ca(2+) 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. Transient ischaemia and hyperthyroidism are cardiovascular risk factors. Nevertheless, 3,5,3'-triiodothyronine/thyroxine therapy has been used to revert myocardial stunning. We studied the influence of hyperthyroidism on the role played by mitochondria in myocardial stunning consequent to ischaemia-reperfusion. Rats were injected s.c. daily with 20 µg kg(-1) triiodothyronine for 15 days (HpT group). Isolated ventricles from either HpT or euthyroid (EuT) rats were perfused in a calorimeter, and left intraventricular pressure (in millimetres of mercury) and heat release (Ht; in milliwatts per gram) were measured. Stunning was evoked by 20 min of no-flow ischaemia and 45 min reperfusion. The HpT hearts developed higher postischaemic contractile recovery (PICR) and improved total muscle economy (P/Ht) with lower diastolic contracture (ΔLVEDP) than EuT hearts. Release of Ca(2+) from the sarcoplasmic reticulum during reperfusion with 10 mm caffeine in low-[Na(+) ] Krebs solution evoked a higher contracture in EuT than in HpT hearts. Blockade of the mitochondrial sodium-calcium exchanger with clonazepam increased ΔLVEDP and reduced P/Ht and PICR in HpT but not in EuT hearts. The clonazepam-induced dysfunction in HpT hearts was reduced by ciclosporin, suggesting a dependance on activation of the mitochondrial permeability transition pore. Blockade of the mitochondrial Ca(2+) uniporter with Ru360 reduced P/Ht and PICR to ∼10% in both HpT and EuT hearts. Blockade of mitochondrial K(+) channels with 5-hydroxydecanoate increased LVEDP and reduced PICR and P/Ht in HpT hearts, while it only increased LVEDP in EuT hearts. The results suggest that hyperthyroidism prevents the stunning with high dependence on the mitochondrial sodium-calcium exchanger and mitochondrial K(+) channels. Both HpT and EuT hearts showed a similar and critical role of the uniporter. The HpT hearts have a slow sarcoplasmic reticulum Ca(2+) loss and low mitochondrial Ca(2+) uptake.

Antispasmodic effects and composition of the essential oils from two South American chemotypes of Lippia alba.

ETHOPHARMACOLOGY RELEVANCE: Lippia alba (Mill.) N. E. Brown (Verbenaceae) is an aromatic species used in Central and South America as eupeptic for indigestion. In Argentina, it is used by the "criollos" from the Chaco province. There are several chemotypes which differ in the chemical composition of the essential oils. Nowadays, it is experimentally cultivated in some countries of the region, including Argentina. AIM OF THE STUDY: To compare the chemical composition and pharmacology of the essential oils from two chemotypes: "citral" (CEO) and "linalool" (LEO), in isolated rat duodenum and ileum. METHODS: Contractile concentration-response curves (CRC) of acetylcholine (ACh) and calcium in 40mM K(+)-medium (Ca(2+)-CRC) were done in isolated intestine portions, in the absence and presence of CEO or LEO at different concentrations. RESULTS: Likewise verapamil, CEO and LEO induced a non-competitive inhibition of the ACh-CRC, with IC50 of 7.0±0.3mg CEO/mL and 37.2±4.2mg LEO/mL. l-NAME, a NO-synthase blocker, increased the IC50 of CEO to 26.1±8.7mg CEO/mL. Likewise verapamil, CEO and LEO non-competitively inhibited the Ca(2+)-CRC, with IC50 of 6.3±1.7mg CEO/mL, 7.0±2.5mg LEO/mL and 0.24±0.04mg verapamil/mL (pIC50: 6.28). CEO was proved to possess limonene, neral, geranial and (-)-carvone as the major components, while LEO was rich in linalool. CONCLUSIONS: Results suggest that CEO has five times more potency than LEO to inhibit muscarinic contractions. The essential oils of both chemotypes interfered with the Ca(2+)-influx, but with an IC50 about 28 times higher than that of verapamil. Moreover, CEO partially stimulated the NO production. These results show the medicinal usefulness of both Lippia alba chemotypes, thus validating its traditional use, potency and mechanism of action.