Chronic fluoxetine treatment in socially-isolated rats modulates the prefrontal cortex synaptoproteome.

Exposure to chronic social isolation (CSIS) and synapse dysfunction have been implicated in the etiology of major depressive disorder (MDD). Fluoxetine (Flx) has been widely used to treat MDD, but its mechanisms of action remain elusive. We employed comparative synaptoproteomics to investigate the changes in the levels of proteins and molecular signaling pathways in prefrontal cortical samples of adult male Wistar rats exposed to CSIS, a rat model of depression, and CSIS rats treated with chronic Flx and controls, using liquid chromatography coupled to tandem mass spectrometry. Flx-treated control rats showed a decreased level of proteins involved in vesicle-mediated transport, and a predominantly increased level of exocytosis-associated proteins. CSIS significantly reduced the level of proteins involved in the ATP metabolic process, clathrin-dependent endocytosis, and proteolysis. Flx treatment in CSIS rats stimulated synaptic vesicle trafficking by increasing the regulation of exo/endocytosis-associated proteins, proteins involved in synaptic plasticity including neurogenesis, Cox5a, mitochondria-associated proteins involved in oxidative phosphorylation, and ion transport proteins (Slc8a2, Atp1b2). Flx treatment resulted in an increased synaptic vesicle dynamic, plasticity and mitochondrial functionality, and a suppression of CSIS-induced impairment of these processes. BIOLOGICAL SIGNIFICANCE: Identifying biomarkers of MDD and treatment response is the goal of many studies. Contemporary studies have shown that many molecular alterations associated with the pathophysiology of MDD reside within the synapse. As part of this research, a growing importance is the use of proteomics, as monitoring the changes in protein levels enables the identification of (possible) biochemical pathways and processes of importance for the development of depressive-like behavior and the efficacy of antidepressant treatments. We profiled proteomic changes representative of the development of CSIS-induced depressive-like behavior and the antidepressant effects of Flx. Our study has identified synaptosomal proteins and altered molecular pathways that may be potential markers of prefrontal cortical synaptic dysfunction associated with depressive-like behavior, and further clarified the mechanisms of depressive-like behavior and mode of action of Flx. Our findings indicate potential PFC synaptic targets for antidepressant treatment.

A hydroalcoholic extract of Senecio nutans SCh. Bip (Asteraceae); its effects on cardiac function and chemical characterization.

ETHNOPHARMACOLOGY RELEVANCE: The plant Senecio nutans SCh. Bip. is used by Andean communities to treat altitude sickness. Recent evidence suggests it may produce vasodilation and negative cardiac inotropy, though the cellular mechanisms have not been elucidated. PURPOSE: To determinate the mechanisms action of S. nutans on cardiovascular function in normotensive animals. METHODS: The effect of the extract on rat blood pressure was measured with a transducer in the carotid artery and intraventricular pressure by a Langendorff system. The effects on sheep ventricular intracellular calcium handling and contractility were evaluated using photometry. Ultra-high-performance liquid-chromatography with diode array detection coupled with heated electrospray-ionization quadrupole-orbitrap mass spectrometric detection (UHPLC-DAD-ESI-Q-OT-MSn) was used for extract chemical characterization. RESULTS: In normotensive rats, S. nutans (10 mg/kg) reduced mean arterial pressure (MAP) by 40% (p < 0.05), causing a dose-dependent coronary artery dilation and decreased left ventricular pressure. In isolated cells, S. nutans extract (1 µg/ml) rapidly reduced the [Ca2+]i transient amplitude and sarcomere shorting by 40 and 49% (p < 0.001), respectively. The amplitude of the caffeine evoked [Ca2+]i transient was reduced by 24% (p < 0.001), indicating reduced sarcoplasmic reticulum (SR) Ca2+ content. Sodium-calcium exchanger (NCX) activity increased by 17% (p < 0.05), while sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) activity was decreased by 21% (p < 0.05). LC-MS results showed the presence of vitamin C, malic acid, and several antioxidant phenolic acids reported for the first time. Dihydroeuparin and 4-hydroxy-3-(3-methylbut-2-enyl) acetophenone were abundant in the extract. CONCLUSION: In normotensive animals, S. nutans partially reduces MAP by decreasing heart rate and cardiac contractility. This negative inotropy is accounted for by decreased SERCA activity and increased NCX activity which reduces SR Ca2+ content. These results highlight the plant's potential as a source of novel cardio-active phytopharmaceuticals or nutraceuticals.

High-salt diet accelerates bone loss accompanied by activation of ion channels related to kidney and bone tissue in ovariectomized rats.

Excessive salt intake can induce a variety of diseases, such as hypertension, cardiovascular disease, kidney disease and so on，it is also one of the factors promoting bone resorption. The mechanism of osteoporosis-induced exacerbations of high salt diet is not well-defined. In this study, we used ovariectomized 6-month-old Sprague Dawley rats to construct a high bone turnover model, and then administrated with high sodium chloride diet (2.0% w/w NaCl, 8.0% w/w NaCl) for 12 weeks to observe the effect of high salt diet on bone metabolism. The results showed that high salt diet could lead to the destruction of bone microstructure, promote the excretion of urinary calcium and phosphorus and accelerate the bone turnover, as well as cause the pathologic structural abnormalities in renal tubular. At the same time, it was accompanied by the up-regulated expression of the epithelial sodium channel (ENaCα), voltage-gated chloride channels (ClC)- 3 and the down-regulated expression of Na-Cl cotransporter (NCC), sodium calcium exchanger (NCX1) in femoral tissue and renal tubules. These findings confirm that high salt diet can destroy the microstructure of bone by increasing bone resorption and affect some ion channels of bone tissue and renal tubule in ovariectomized rats.

Safranal Induces Vasorelaxation by Inhibiting Ca2+ Influx and Na+/Ca2+ Exchanger in Isolated Rat Aortic Rings.

Introduction: Safranal, which endows saffron its unique aroma, causes vasodilatation and has a hypotensive effect in animal studies, but the mechanisms of these effects are unknown. In this study, we investigated the mechanisms of safranal vasodilation. Methods: Isolated rat endothelium-intact or -denuded aortic rings were precontracted with phenylephrine and then relaxed with safranal. To further assess the involvement of nitric oxide, prostaglandins, guanylate cyclase, and phospholipase A2 in safranal-induced vasodilation, aortic rings were preincubated with L-NAME, indomethacin, methylene blue, or quinacrine, respectively, then precontracted with phenylephrine, and safranal concentration-response curves were established. To explore the effects of safranal on Ca2+ influx, phenylephrine and CaCl2 concentration-response curves were established in the presence of safranal. Furthermore, the effect of safranal on aortic rings in the presence of ouabain, a Na+-K+ ATPase inhibitor, was studied to explore the contribution of Na+/Ca2+ exchanger to this vasodilation. Results: Safranal caused vasodilation in endothelium-intact and endothelium-denuded aortic rings. The vasodilation was not eliminated by pretreatment with L-NAME, indomethacin, methylene blue, or quinacrine, indicating the lack of a role for NO/cGMP. Safranal significantly inhibited the maximum contractions induced by phenylephrine, or by CaCl2 in Ca2+-free depolarizing buffer. Safranal also relaxed contractions induced by ouabain, but pretreatment with safranal totally abolished the development of ouabain contractions. Discussion/Conclusion: Inhibition of Na+-K+ ATPase by ouabain leads to the accumulation of Na+ intracellularly, forcing the Na+/Ca2+ exchanger to work in reverse mode, thus causing a contraction. Inhibition of the development of this contraction by preincubation with safranal indicates that safranal inhibited the Na+/Ca2+ exchanger. We conclude that safranal vasodilation is mediated by the inhibition of calcium influx from extracellular space through L-type Ca2+ channels and by the inhibition of the Na+/Ca2+ exchanger.

Massive calcium-activated endocytosis without involvement of classical endocytic proteins.

We describe rapid massive endocytosis (MEND) of >50% of the plasmalemma in baby hamster kidney (BHK) and HEK293 cells in response to large Ca transients. Constitutively expressed Na/Ca exchangers (NCX1) are used to generate Ca transients, whereas capacitance recording and a membrane tracer dye, FM 4-64, are used to monitor endocytosis. With high cytoplasmic adenosine triphosphate (ATP; >5 mM), Ca influx causes exocytosis followed by MEND. Without ATP, Ca transients cause only exocytosis. MEND can then be initiated by pipette perfusion of ATP, and multiple results indicate that ATP acts via phosphatidylinositol-bis 4,5-phosphate (PIP(2)) synthesis: PIP(2) substitutes for ATP to induce MEND. ATP-activated MEND is blocked by an inositol 5-phosphatase and by guanosine 5'-[γ-thio]triphosphate (GTPγS). Block by GTPγS is overcome by the phospholipase C inhibitor, U73122, and PIP(2) induces MEND in the presence of GTPγS. MEND can occur in the absence of ATP and PIP(2) when cytoplasmic free Ca is clamped to 10 µM or more by Ca-buffered solutions. ATP-independent MEND occurs within seconds during Ca transients when cytoplasmic solutions contain polyamines (e.g., spermidine) or the membrane is enriched in cholesterol. Although PIP(2) and cholesterol can induce MEND minutes after Ca transients have subsided, polyamines must be present during Ca transients. MEND can reverse over minutes in an ATP-dependent fashion. It is blocked by brief ß-methylcyclodextrin treatments, and tests for involvement of clathrin, dynamins, calcineurin, and actin cytoskeleton were negative. Therefore, we turned to the roles of lipids. Bacterial sphingomyelinases (SMases) cause similar MEND responses within seconds, suggesting that ceramide may be important. However, Ca-activated MEND is not blocked by reagents that inhibit SMases. MEND is abolished by the alkylating phospholipase A(2) inhibitor, bromoenol lactone, whereas exocytosis remains robust, and Ca influx causes MEND in cardiac myocytes without preceding exocytosis. Thus, exocytosis is not prerequisite for MEND. From these results and two companion studies, we suggest that Ca promotes the formation of membrane domains that spontaneously vesiculate to the cytoplasmic side.

Dimethyl amiloride improves glucose homeostasis in mouse models of type 2 diabetes.

Dimethyl amiloride (DMA) enhances insulin secretion in the pancreatic beta-cell. DMA also enhances time-dependent potentiation (TDP) and enables TDP to occur in situations where it is normally absent. As we have demonstrated before, these effects are mediated in part through inhibition of neuronal nitric oxide synthase (nNOS), resulting in increased availability of arginine. Thus both DMA and arginine have the potential to correct the secretory defect in diabetes by enabling or enhancing TDP. In the current study we have demonstrated the ability of these agents to improve blood glucose homeostasis in three mouse models of type 2 diabetes. The pattern of TDP under different conditions indicates that inhibition of NOS is not the only mechanism through which DMA exerts its positive effects. Thus we also have explored another possible mechanism through which DMA enables/enhances TDP, via the activation of mitochondrial alpha-ketoglutarate dehydrogenase.

Hyposmolarity evokes norepinephrine efflux from synaptosomes by a depolarization- and Ca2+ -dependent exocytotic mechanism.

Osmolarity reduction (20%) elicited 3H-norepinephrine (NE) efflux from rat cortical synaptosomes. The hyposmotic NE release resulted from the following events: (i) a Na+-dependent and La3+-, Gd3+- and ruthenium red-sensitive depolarization; (ii) a cytosolic Ca2+ ([Ca2+]i) rise with contributions from external Ca2+ influx and internal Ca2+ release, probably through the mitochondrial Na+-Ca2+ exchanger; and (iii) activation of a [Ca2+]i-evoked, tetanus toxin (TeTX)-sensitive, PKC-modulated NE efflux mechanism. This sequence was established from results showing a drop in the hyposmotic [Ca2+]i rise by preventing depolarization with La3+, and by the inhibitory effects of Ca2+-free medium (EGTA; 50%), CGP37157 (the mitochondrial Na+-Ca2+ exchanger blocker; 48%), EGTA + CGP37157 or by EGTA-AM (> 95% in both cases). In close correspondence with these effects, NE efflux was 92% decreased by Na+ omission, 75% by La3+, 47% by EGTA, 50% by CGP37157, 90% by EGTA + CGP37157 and 88% by EGTA-AM. PKC influenced the intracellular Ca2+ release and, mainly through this action, modulated NE efflux. TeTX suppressed NE efflux. The K+-stimulated NE release, studied in parallel, was unaffected by Na+ omission, or by La3+, Gd3+ or ruthenium red. It was fully dependent on external Ca2+, insensitive to CGP37157 and abolished by TeTX. These results suggest that the hyposmotic events, although different from the K+-evoked depolarization and [Ca2+]i rise mechanisms, are able to trigger a depolarization-dependent, Ca2+-dependent and TeTX-sensitive mechanism for neurotransmitter release.

Synthesis and structure-activity relationships of benzyloxyphenyl derivatives as a novel class of NCX inhibitors: effects on heart failure.

In the context of heart failure and myocardial ischemia reperfusion, the activity of the sodium-calcium exchanger can lead to calcium overload, which in turn can lead to contractile dysfunction and arrhythmia. Therefore, NCX is an attractive target for treatment of heart failure and myocardial ischemia reperfusion. We have designed and synthesized a series of benzyloxyphenyl derivatives as potential NCX inhibitors, based on compound 4. These derivatives have been evaluated for their inhibitory activity against both the reverse and forward modes of NCX, and two novel potent NCX inhibitors (7i, 10a) were discovered. Compound 7i was evaluated for its efficacy on ouabain-induced tonotropy and arrhythmia in a heart-failure model.

SEA0400: a novel sodium-calcium exchange inhibitor with cardioprotective properties.

The cardiac sodium-calcium exchanger (NCX) plays an important role in calcium homeostasis. It is the primary mechanism for removing calcium ions that enter myocytes through L-type calcium channels on a beat-to-beat basis. Its direction of transport is determined by the membrane potential and the ionic concentrations of Na+ and Ca2+, with the forward (or Ca2+-efflux) mode of transport being the dominant mode under physiological conditions. In contrast, the Ca2+-influx mode (or reverse mode) of NCX becomes important in certain pathophysiological conditions, such as myocardial ischemia and reperfusion. Recent discovery of compounds that inhibit the Ca2+-influx mode (or reverse mode) of NCX has generated intense research interest in the pharmacology of NCX. Among the newer NCX inhibitors described to date, 2-[4-[(2,5-difluorophenyl)methoxy]-phenoxy]-5-ethoxyaniline (SEA0400) appears particularly promising in attenuating cardiac, renal, and cerebral ischemia/reperfusion injuries in various experimental models. Moreover, the mixed results that have emerged from clinical trials evaluating the efficacy and safety of inhibitors of the sodium-hydrogen exchanger (an upstream target in relation to the sodium-calcium exchanger) in myocardial protection stimulated interest in evaluating NCX as an alternative therapeutic target. This article reviews the pharmacological profile of SEA0400, as presented in the published literature, and discusses the therapeutic potential of this compound in attenuating myocardial ischemia/reperfusion injury.

Pharmacology of brain Na+/Ca2+ exchanger: from molecular biology to therapeutic perspectives.

In the last two decades, there has been a growing interest in unraveling the role that the Na+/Ca2+ exchanger (NCX) plays in the function and regulation of several cellular activities. Molecular biology, electrophysiology, genetically modified mice, and molecular pharmacology have helped to delve deeper and more successfully into the physiological and pathophysiological role of this exchanger. In fact, this nine-transmembrane protein, widely distributed in the brain and in the heart, works in a bidirectional way. Specifically, when it operates in the forward mode of operation, it couples the extrusion of one Ca2+ ion with the influx of three Na+ ions. In contrast, when it operates in the reverse mode of operation, while three Na+ ions are extruded, one Ca2+ enters into the cells. Different isoforms of NCX, named NCX1, NCX2, and NCX3, have been described in the brain, whereas only one, NCX1, has been found in the heart. The hypothesis that NCX can play a relevant role in several pathophysiological conditions, including hypoxia-anoxia, white matter degeneration after spinal cord injury, brain trauma and optical nerve injury, neuronal apoptosis, brain aging, and Alzheimer's disease, stems from the observation that NCX, in parallel with selective ion channels and ATP-dependent pumps, is efficient at maintaining intracellular Ca2+ and Na+ homeostasis. In conclusion, although studies concerning the involvement of NCX in the pathological mechanisms underlying brain injury during neurodegenerative diseases started later than those related to heart disease, the availability of pharmacological agents able to selectively modulate each NCX subtype activity and antiporter mode of operation will provide a better understanding of its pathophysiological role and, consequently, more promising approaches to treat these neurological disorders.

Inotropic and lusitropic effects induced by the inhibitory factor of the Na/Ca exchanger are not mediated by the beta-adrenergic activation.

Recently, an endogenous inhibitory factor (NCXIF) of the cardiac Na/Ca exchanger (NCX1) has been isolated, purified, and preliminary characterized. Here, we demonstrate that low doses of NCXIF (10(-7)10(-8) M) induce strong inotropic effects in the guinea and rat ventricle strips, while having no detectable effects in the atria even at 10(-5) M. The inotropic effects of NCXIF are species-specific; the rat ventricle muscle is 20 to 50 times more sensitive to varying doses of NCXIF than the guinea pig. On the other hand the extent of maximal inotropic response is more prominent in the guinea pig model (up to 6-fold enhancement) than in the rat (up to 2-fold enhancement). The NCXIF accelerates the single-twitch relaxation (lusitropic effect) in dose-dependent manner, reaching approximately 2-fold shortening of twitch width at saturating doses. The dose-dependence curves of lusitropic and inotropic effects exhibit a reciprocal relationship, meaning that these two effects might share common mechanisms. To test a possible involvement of catecholamines, the effects of NCXIF were examined in the presence or absence of beta-adrenergic blocker, deralin. The saturating doses of deralin (1- 3 microM) do not alter either the NCXIF-induced acceleration of relaxation or twitch enhancement, meaning that the NCXIF effects cannot be mediated by occasional release of endogenous catecholamines. The capacity of NCXIF to modulate the ventricle contractility unconnectedly to the beta-adrenergic activation may provide new rational clues for future pharmacological interventions.

Ca(2+) oscillations regulated by Na(+)-Ca(2+) exchanger and plasma membrane Ca(2+) pump induce fluctuations of membrane currents and potentials in human mesenchymal stem cells.

Human bone marrow-derived mesenchymal stem cells (hMSCs) have the potential to differentiate into several types of cells. We have demonstrated spontaneous [Ca(2+)](i) oscillations in hMSCs without agonist stimulation, which result primarily from release of Ca(2+) from intracellular stores via InsP(3) receptors. In this study, we further investigated functions and contributions of Ca(2+) transporters on plasma membrane to generate [Ca(2+)](i) oscillations. In confocal Ca(2+) imaging experiments, spontaneous [Ca(2+)](i) oscillations were observed in 193 of 280 hMSCs. The oscillations did not sustain in the Ca(2+) free solution and were completely blocked by the application of 0.1mM La(3+). When plasma membrane Ca(2+) pumps (PMCAs) were blocked with blockers, carboxyeosin or caloxin, [Ca(2+)](i) oscillations were inhibited. Application of Ni(2+) or KBR7943 to block Na(+)-Ca(2+) exchanger (NCX) also inhibited [Ca(2+)](i) oscillations. Using RT-PCR, mRNAs were detected for PMCA type IV and NCX, but not PMCA type II. In the patch clamp experiments, Ca(2+) activated outward K(+) currents (I(KCa)) with a conductance of 170+/-21.6pS could be recorded. The amplitudes of I(KCa) and membrane potential (V(m)) periodically fluctuated liked to [Ca(2+)](i) oscillations. These results suggest that in undifferentiated hMSCs both Ca(2+) entry through plasma membrane and Ca(2+) extrusion via PMCAs and NCXs play important roles for [Ca(2+)](i) oscillations, which modulate the activities of I(KCa) to produce the fluctuation of V(m).

Modulation of intracellular Na+ and Ca2+ induced by the cardiac Na+-Ca2+ exchanger in guinea pig ventricular myocytes.

The Na+-Ca2+ exchanger current was measured in single guinea pig ventricular myocytes, using the whole-cell voltage-clamp technique, and intracellular free calcium concentration ([Ca2+](i)) was monitored simultaneously with the fluorescent probe Indo-1 applied intracellularly through a perfused patch pipette. In external solutions, which have levels of Ca2+ (approximately 66 microM Ca2+) thought low enough to inhibit exchanger turnover, the removal of external Na+ (by replacement with Li+) induced both an outward shift of the holding current and an increase in [Ca2+](i), even though the recording pipette contained 30 mM bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), sufficient to completely block phasic contractions. The effects of Na+ removal were blocked either by the extracellular application of 2 mM Ni2+ or by chelating extracellular Ca2+ with 1 mM EGTA. In the presence of 10 microM Ryanodine, the effects of external Na+ substitution with Li(+) on both membrane current and [Ca2+](i) were attenuated markedly in amplitude and at a much slower time course. Reversal potentials were estimated by using ramp pulses and by defining exchange currents as the Ni2+-sensitive components. The experimental values of the reversal potential and [Ca2+](i) were used to calculate cytosolic Na+ ([Na+](i)) by assuming an exchanger stoichiometry of 3Na+ : 1Ca2+. These calculations suggested that in the nominal absence of external Ca2+ ( approximately 66 microM under our experimental conditions), the exchanger operates at -40 mV as though approximately 40 mM Na+ had accumulated in the vicinity of the intracellular binding sites. We conclude that under the conditions of low extracellular Ca2+ and high intracellular Ca2+ buffering, the Na+-Ca2+ exchanger can still generate sufficient Ca2+ influx on the removal of external Na+ to markedly increase cytosolic free Ca2+.

Phospholemman modulates Na+/Ca2+ exchange in adult rat cardiac myocytes.

Previous studies have shown that overexpression of phospholemman (PLM) affected contractile function and Ca(2+) homeostasis in adult rat myocytes. We tested the hypothesis that PLM modulated Na(+)/Ca(2+) exchanger (NCX1) activity. PLM was overexpressed in adult rat myocytes by adenovirus-mediated gene transfer. After 72 h, the half-time of relaxation from caffeine-induced contracture, an estimate of forward NCX1 activity, was prolonged 1.8-fold (P < 0.003) in myocytes overexpressing PLM compared with control myocytes overexpressing green fluorescent protein alone. Reverse NCX1 current (3 Na(+) out:1 Ca(2+) in) was significantly (P < 0.0001) lower in PLM myocytes, especially at more positive voltages. Immunofluorescence demonstrated colocalization of PLM and NCX1 to the plasma membrane and t-tubules. Resting membrane potential, action potential amplitude and duration, myocyte size, and NCX1 and calsequestrin protein levels were not affected by PLM overexpression. At 5 mM extracellular [Ca(2+)] ([Ca(2+)](o)), the depressed contraction amplitudes in PLM myocytes were increased towards normal by cooverexpression with NCX1. At 0.6 mM [Ca(2+)](o), the supranormal contraction amplitudes in PLM myocytes were reduced by cooverexpression with NCX1. We conclude that PLM modulated myocyte contractility partly by inhibiting Na(+)/Ca(2+) exchange.

Coupling of calcium homeostasis to axonal sodium in axons of mouse optic nerve.

Axonal populations in neonatal and mature optic nerves were selectively stained with calcium dyes for analysis of calcium homeostasis and its possible coupling to axonal Na. Repetitive nerve stimulation causes a rise in axonal [Ca(2+)](i) the posttetanus recovery of which is impeded by increasing the number of action potentials in the tetanus. This effect is augmented in 4-aminopyridine (4-AP; 1 mM), which dramatically increases the calcium and presumably sodium load during the tetanus. Increasing axonal [Na](i) with the Na-ionophore monensin (4-50 microM) and ouabain (30 microM) retards posttetanus calcium decline, suggesting that efficient calcium clearance depends on a low level of axonal [Na](i). Posttetanus calcium clearance is not affected by K-mediated depolarization. To further examine coupling between axonal [Na](i) and [Ca(2+)](i), the resting axonal [Ca(2+)](i) was monitored as axonal [Na(+)](i) was elevated with ouabain, veratridine, and monensin. In all cases, elevation of axonal [Na(+)](i) evokes a calcium influx into axons. This influx is unrelated to activation of calcium channels but is consistent with calcium influx via reversal of the Na/Ca exchanger expected as a consequence of axonal [Na(+)](i) elevation. In conclusion, this study demonstrates that calcium homeostasis in the axons of the optic nerve is strongly coupled to axonal [Na(+)](i) in a manner consistent with the Na/Ca exchanger playing a major role in extruding calcium following nerve activity.

Enhancement of Ca(2+) transients and contraction of single ventricular myocytes of rats by 5-(N,N-dimethyl) amiloride.

The effects of 5-(N,N-dimethyl)amiloride (DMA) (a blocker of Na(+)/H(+) exchanger or Na(+)/Ca(2+) exchanger) on calcium transient and cell contraction in isolated ventricular myocytes in normal rats and rats with myocardial hypertrophy were examined using ion imaging system with a charge coupled digital camera (CCD camera). Loading myocytes with Fura-2, electrically triggered Ca(2+) transients and cell shortening were measured simultaneously. The results showed that 10 micromol/L DMA increased Ca(2+) transient and cell shortening from 209.60+/-54.96 and 3.07+/-0.97 micrometer to 238.50+/-80.41 and 4.07+/-1.02 micrometer, respectively (P<0.05), which was completely abolished by application of KB-R7943, a specific reverse mode Na(+)/Ca(2+) exchanger blocker. After blocking L-type Ca(2+) channels by nicardipine, DMA also enhanced Ca(2+) transient and cell shortening. In rats with myocardial hypertrophy, DMA showed the common pharmacologic profile as in normal rats but more intense stimulating effects on Ca(2+) transient and cell contraction. The results suggest that DMA increase Ca(2+) transient and cell contraction via stimulating reverse mode Na(+)/ Ca(2+) exchange, and the stimulating effect is more pronounced in rats with myocardial hypertrophy than in normal ones.

Reverse mode of the Na+-Ca2+ exchange after myocardial stretch: underlying mechanism of the slow force response.

This study was designed to gain additional insight into the mechanism of the slow force response (SFR) to stretch of cardiac muscle. SFR and changes in intracellular Na(+) concentration ([Na(+)](i)) were assessed in cat papillary muscles stretched from 92% to approximately 98% of L(max). The SFR was 120+/-0.6% (n=5) of the rapid initial phase and coincided with an increase in [Na(+)](i). The SFR was markedly depressed by Na(+)-H(+) exchanger inhibition, AT(1) receptor blockade, nonselective endothelin-receptor blockade and selective ET(A)-receptor blockade, extracellular Na(+) removal, and inhibition of the reverse mode of the Na(+)-Ca(2+) exchange by KB-R7943. KB-R7943 prevented the SFR but not the increase in [Na(+)](i). Inhibition of endothelin-converting enzyme activity by phosphoramidon suppressed both the SFR and the increase in [Na(+)](i). The SFR and the increase in [Na(+)](i) after stretch were both present in muscles with their endothelium (vascular and endocardial) made functionally inactive by Triton X-100. In these muscles, phosphoramidon also suppressed the SFR and the increase in [Na(+)](i). The data provide evidence that the last step of the autocrine-paracrine mechanism leading to the SFR to stretch is Ca(2+) entry through the reverse mode of Na(+)-Ca(2+) exchange.

The Na+ -Ca2+ exchanger is essential for embryonic heart development in mice.

The cardiac Na+ -Ca2+ exchanger 1 (NCX1) is thought to be the major calcium extrusion mechanism and to play an important role in the regulation of intracellular calcium in the heart. The Na+ -Ca2+ exchanger is particularly abundant in the heart, although it is found in a variety of other tissues. To investigate the role of NCX1, we have generated NCX1-deficient mice. Mice heterozygous for the NCX1 mutation showed no discernable phenotype, grew normally, and were fertile; however, no viable homozygote was observed among 175 offspring obtained from intercrosses of heterozygotes. All the homozygous mutant mice died in utero before E10.5. Morphological analysis indicated that homozygotes of NCX1 mutation at E9.5 died with an underdeveloped heart with a dilated pericardium. Microscopic analysis of these embryos showed myocardial cell loss due to apoptosis. The apoptosis was first observed in E8.5 mutant heart. Areas outside the heart appeared normal in the mutant embryos at E8.5. In contrast, at E9.0, various regions of mutant embryos showed extensive cell loss. These results suggest that mutant embryos die owing to cardiac abnormalities caused by apoptotic cell loss, indicating that NCX1 is essential for normal development of the heart.

Is ouabain produced by the adrenal gland?

1. Ouabain or a related stereoisomer, termed endogenous ouabain, has been identified in adrenal cortex tissue and culture medium from adrenocortical cells. 2. Angiotensin II and adrenocorticotropin, the main activators of aldosterone secretion from adrenal glomerulosa cells appear to increase the production of this compound. 3. The purpose of this review is to briefly discuss recent available experimental evidence suggesting that endogenous ouabain is secreted by the zona glomerulosa of the adrenal gland.