Inhibition of the sodium–calcium exchanger via SEA0400 altered manganese‐induced T1 changes in isolated perfused rat hearts

Manganese (Mn²⁺)‐enhanced MRI (MEMRI) provides the potential for the in vivo evaluation of calcium (Ca²⁺) uptake in the heart. Recent studies have also suggested the role of the sodium–calcium (Na⁺–Ca²⁺) exchanger (NCX) in Mn²⁺ retention, which may have an impact on MEMRI signals. In this study, we investigated whether MEMRI with fast T₁ mapping allowed the sensitive detection of changes in NCX activity. We quantified the dynamics of the Mn²⁺‐induced T₁ changes in isolated perfused rat hearts in response to SEA0400, an NCX inhibitor. The experimental protocol comprised 30 min of Mn²⁺ perfusion (wash‐in), followed by a 30‐min wash‐out period. There were three experimental groups: 1, NCX inhibition by 1 µ m SEA0400 during Mn²⁺ wash‐in only (SEAin, n = 6); 2, NCX inhibition by 1 µ m SEA0400 during Mn²⁺ wash‐out only (SEAout, n = 6); 3, no NCX inhibition during both wash‐in and wash‐out to serve as the control group (CNTL, n = 5). Rapid T₁ mapping at a temporal resolution of 3 min was performed throughout the perfusion protocol using a triggered saturation–recovery Look–Locker sequence. Our results showed that NCX inhibition during Mn²⁺ wash‐in caused a significant increase in relaxation rate (R₁) at the end of Mn²⁺ perfusion. During the wash‐out period, NCX inhibition led to less reduction in R₁. Further analysis of Mn²⁺ content in myocardium with flame atomic absorption spectroscopy was consistent with the MRI findings. These results suggest that Mn²⁺ accumulation and retention in rat hearts are, in part, dependent on NCX activity. Hence, MEMRI may provide an imaging method that is also sensitive to changes in NCX activity. Copyright © 2012 John Wiley & Sons, Ltd.     

Monitoring dynamic alterations in calcium homeostasis by T1‐mapping manganese‐enhanced MRI (MEMRI) in the early stage of small intestinal ischemia–reperfusion injury

Manganese‐enhanced MRI studies have proven to be useful in monitoring physiological activities associated with calcium ions (Ca²⁺) due to the paramagnetic property of the manganese ion (Mn²⁺), which makes it an excellent probe of Ca²⁺. In this study, we developed a method in which a Mn²⁺‐enhanced T₁‐map MRI could enable the monitoring of Ca²⁺ influx during the early stages of intestinal ischemia–reperfusion (I/R) injury. The Mn²⁺ infusion protocol was optimized by obtaining dose‐dependent and time‐course wash‐out curves using a Mn²⁺‐enhanced T₁‐map MRI of rabbit abdomens following an intravenous infusion of 50 mmol/l MnCl₂ (5–10 nmol/g body weight (BW)). In the rabbit model of intestinal I/R injury, T₁ values were derived from the T₁ maps in the intestinal wall region and revealed a relationship between the dose of the infused MnCl₂ and the intestinal wall relaxation time. Significant Mn²⁺ clearance was also observed over time in control animals after the infusion of Mn²⁺ at a dose of 10 nmol/g BW. This technique was also shown to be sensitive enough to monitor variations in calcium ion homeostasis in vivo after small intestinal I/R injury. The T₁ values of the intestinal I/R group were significantly lower (P < 0.05) than that of the control group at 5, 10, and 15 min after Mn²⁺ infusion. Our data suggest that MnCl₂ has the potential to be an MRI contrast agent that can be effectively used to monitor changes in intracellular Ca²⁺ homeostasis during the early stages of intestinal I/R injury. Copyright © 2015 John Wiley & Sons, Ltd.     

Manganese‐enhanced MRI (MEMRI) via topical loading of Mn2+ significantly impairs mouse visual acuity: a comparison with intravitreal injection

Manganese‐enhanced MRI (MEMRI) with topical loading of MnCl₂ provides optic nerve enhancement comparable to that seen by intravitreal injection. However, the impact of this novel and non‐invasive Mn²⁺ loading method on visual function requires further assessments. The objective of this study is to determine the optimal topical Mn²⁺ loading dosage for MEMRI and to assess visual function after MnCl₂ loading. Intravitreal administration was performed to compare the two approaches of MnCl₂ loading. Twenty‐four hours after topical loading of 0, 0.5, 0.75, and 1 M MnCl₂, T₁‐weighted, T2‐weighted, diffusion tensor imaging and visual acuity (VA) assessments were performed to determine the best topical loading dosage for MEMRI measurements and to assess the integrity of retinas and optic nerves. Mice were perfusion fixed immediately after in vivo experiments for hematoxylin and eosin and immunohistochemistry staining. Topical loading of 1 M MnCl₂ damaged the retinal photoreceptor layer with no detectable damage to retina ganglion cell layers or prechiasmatic optic nerves. For the topical loading, 0.75 M MnCl₂ was required to see sufficient enhancement of the optic nerve. At this concentration the visual function was significantly affected, followed by a slow recovery. Intravitreal injection (0.25 μL of 0.2 M MnCl₂) slightly affected VA, with full recovery a day later. To conclude, intravitreal MnCl₂injection provides more reproducible results with less adverse side‐effects than topical loading. Copyright © 2014 John Wiley & Sons, Ltd.     

MEMRI and tumors: a method for the evaluation of the contribution of Mn(II) ions in the extracellular compartment

The purpose of the work was to set‐up a simple method to evaluate the contribution of Mn²⁺ ions in the intra‐ and extracellular tumor compartments in a MEMRI experiment. This task has been tackled by “silencing” the relaxation enhancement arising from Mn²⁺ ions in the extracellular space. In vitro relaxometric measurements allowed assessment of the sequestering activity of DO2A (1,4,7,10‐tetraazacyclododecane‐1,7‐diacetic acid) towards Mn²⁺ ions, as the addition of Ca‐DO2A to a solution of MnCl₂ causes a drop of relaxivity upon the formation of the highly stable and low‐relaxivity Mn‐DO2A. It has been proved that the sequestering ability of DO2A towards Mn²⁺ ions is also fully effective in the presence of serum albumin. Moreover, it has been shown that Mn‐DO2A does not enter cell membranes, nor does the presence of Ca‐DO2A in the extracellular space prompt migration of Mn ions from the intracellular compartment. On this basis the in vivo, instantaneous, drop in SE% (percent signal enhancement) in T₁‐weighted images is taken as evidence of the sequestration of extracellular Mn²⁺ ions upon addition of Ca‐DO2A. By applying the method to B16F10 tumor bearing mice, T₁ decrease is readily detected in the tumor region, whereas a negligible change in SE% is observed in kidneys, liver and muscle. The relaxometric MRI results have been validated by ICP‐MS measurements. Copyright © 2015 John Wiley & Sons, Ltd.     

Fractionated manganese injections: effects on MRI contrast enhancement and physiological measures in C57BL/6 mice

Manganese‐enhanced MRI (MEMRI) is an increasingly used imaging method in animal research, which enables improved T₁‐weighted tissue contrast. Furthermore accumulation of manganese in activated neurons allows visualization of neuronal activity. However, at higher concentrations manganese (Mn²⁺) exhibits toxic side effects that interfere with the animals' behaviour and well‐being. Therefore, when optimizing MEMRI protocols, a compromise has to be found between minimizing side effects and intensifying image contrast. Recently, a low concentrated fractionated Mn²⁺ application scheme has been proposed as a promising alternative. In this study, we investigated effects of different fractionated Mn²⁺ dosing schemes on vegetative, behavioural and endocrine markers, and MEMRI signal contrast in C57BL/6N mice. Measurements of the animals' well‐being included telemetric monitoring of body temperature and locomotion, control of weight and observation of behavioural parameters during the time course of the injection protocols. Corticosterone levels after Mn²⁺ application served as endocrine marker of the stress response. We compared three MnCl₂ · 4H₂O application protocols: 3 times 60 mg/kg with an inter‐injection interval of 48 h, six times 30 mg/kg with an inter‐injection interval of 48 h, and 8 times 30 mg/kg with an inter‐injection interval of 24 h (referred to as 3 × 60/48, 6 × 30/48 and 8 × 30/24, respectively). Both the 6 × 30/48 and the 8 × 30/24 protocols showed attenuated effects on animals' well‐being as compared to the 3 × 60/48 scheme. Best MEMRI signal contrast was observed for the 8 × 30/24 protocol. Together, these results argue for a fractionated application scheme such as 30 mg/kg every 24 h for 8 days to provide sufficient MEMRI signal contrast while minimizing toxic side effects and distress. Copyright © 2010 John Wiley & Sons, Ltd.     

Relationship between blood and myocardium manganese levels during manganese‐enhanced MRI (MEMRI) with T1 mapping in rats

Manganese ions (Mn²⁺) enter viable myocardial cells via voltage‐gated calcium channels. Because of its shortening of T₁ and its relatively long half‐life in cells, Mn²⁺ can serve as an intracellular molecular contrast agent to study indirect calcium influx into the myocardium. One major concern in using Mn²⁺ is its sensitivity over a limited range of concentrations employing T₁‐weighted images for visualization, which limits its potential in quantitative techniques. Therefore, this study assessed the implementation of a T₁ mapping method for cardiac manganese‐enhanced MRI to enable a quantitative estimate of the influx of Mn²⁺ over a wide range of concentrations in male Sprague‐Dawley rats. This MRI method was used to compare the relationship between T₁ changes in the heart as a function of myocardium and blood Mn²⁺ levels. Results showed a biphasic relationship between ΔR₁ and the total Mn²⁺ infusion dose. Nonlinear relationships were observed between the total Mn²⁺ infusion dose versus blood levels and left ventricular free wall ΔR₁. At low blood levels of Mn²⁺, there was proportionally less cardiac enhancement seen than at higher levels of blood Mn²⁺. We hypothesize that Mn²⁺ blood levels increase as a result of rate‐limiting excretion by the liver and kidneys at these higher Mn²⁺ doses. Copyright © 2010 John Wiley & Sons, Ltd.     

Manganese‐enhanced MRI optic nerve tracking: effect of intravitreal manganese dose on retinal toxicity

The aim of this study was to provide data on the dose dependence of manganese‐enhanced MRI (MEMRI) in the visual pathway of experimental rats and to study the toxicity of MnCl₂ to the retina. Sprague–Dawley rats were intravitreally injected with 2 μL of 0, 10, 25, 50, 75, 100, 150 and 300 mm MnCl₂, respectively_. The contrast‐to‐noise ratio (CNR) of MEMRI for optic nerve enhancement was measured at different concentrations of MnCl₂. Simultaneously, the toxicity of manganese was evaluated by counting retinal ganglion cells and by retinal histological examination using light microscopy and transmission electron microscopy. The CNR increased with increasing concentration of MnCl₂ up to 75 mm . Retinal ganglion cell densities were reduced significantly when the concentration of MnCl₂ in the intravitreal injection was equal to or greater than 75 mm . Increasing numbers of ribosomes in retinal ganglion cells were first detected at 25 mm of MnCl₂. The retinal toxicity of MnCl₂ at higher concentration also included mitochondrial pathology and cell disruption of retinal ganglion cells, as well as abnormalities of photoreceptor and retinal pigment epithelium cells. It can be concluded that intravitreal injection of MnCl₂ induces retinal cell damage that appears to start from 25 mm . The concentration of MnCl₂ should not exceed 25 mm through intravitreal injection for visual pathway MEMRI in the rat. Copyright © 2012 John Wiley & Sons, Ltd.     

Manganese‐enhanced MRI visualizes V1 in the non‐human primate visual cortex

MRI at 7 Tesla has been used to investigate the accumulation of manganese in the occipital cortex of common marmoset monkeys (Callithrix jacchus) after administering four fractionated injections of 30 mg/kg MnCl₂ · 4H₂O in the tail vein. We found a statistically significant decrease in T₁ in the primary (V1) and secondary (V2) areas of the visual cortex caused by an accumulation of manganese. The larger T₁ shortening in V1 (ΔT₁ = 640 ms) relative to V2 (ΔT₁ = 490 ms) allowed us to robustly detect the V1/V2 border in vivo using heavily T₁‐weighted MRI. Furthermore, the dorso‐medial (DM) and middle‐temporal (MT) areas of the visual pathway could be identified by their T₁‐weighted enhancement. We showed by comparison to histological sections stained for cytochrome oxidase (CO) activity that the extent of V1 is accurately identified throughout the visual cortex by manganese‐enhanced MRI (MEMRI). This provides a means of visualizing functional cortical regions in vivo and could be used in longitudinal studies of phenomena such as cortical plasticity, and for non‐destructive localization of cortical regions to guide in the implementation of functional techniques. Published in 2009 by John Wiley & Sons, Ltd.     

Testing the calcium hypothesis of aging in the rat hippocampus in vivo using manganese-enhanced MRI

In this study, we noninvasively tested the hypothesis that Mn²⁺-enhanced magnetic resonance imaging (MEMRI) is sensitive to age-related changes in Ca²⁺ influx occurring in the hippocampal region CA1. Uptake of Mn²⁺, an MRI contrast agent and Ca²⁺ surrogate with low cellular efflux rates (days to weeks), was measured in longitudinal MEMRI studies involving 2 separate groups of male Long-Evans rats: one group was studied at 2.5 and 7 months of age, whereas the other was studied at 7 and 19 months of age. Separate or combined analysis revealed that the extent of Mn²⁺ accumulation in CA1 significantly increased with age (p < 0.05). These results provide first-time in vivo confirmation of the calcium hypothesis of aging and justify future longitudinal studies combining MEMRI with behavioral testing to investigate mechanisms of age-related cognitive decline.     

Regional specificity of manganese accumulation and clearance in the mouse brain: implications for manganese‐enhanced MRI

Manganese‐enhanced MRI has recently become a valuable tool for the assessment of in vivo functional cerebral activity in animal models. As a result of the toxicity of manganese at higher dosages, fractionated application schemes have been proposed to reduce the toxic side effects by using lower concentrations per injection. Here, we present data on regional‐specific manganese accumulation during a fractionated application scheme over 8 days of 30 mg/kg MnCl₂, as well as on the clearance of manganese chloride over the course of several weeks after the termination of the whole application protocol supplying an accumulative dose of 240 mg/kg MnCl₂. Our data show most rapid accumulation in the superior and inferior colliculi, amygdala, bed nucleus of the stria terminalis, cornu ammonis of the hippocampus and globus pallidus. The data suggest that no ceiling effects occur in any region using the proposed application protocol. Therefore, a comparison of basal neuronal activity differences in different animal groups based on locally specific manganese accumulation is possible using fractionated application. Half‐life times of manganese clearance varied between 5 and 7 days, and were longest in the periaqueductal gray, amygdala and entorhinal cortex. As the hippocampal formation shows one of the highest T₁‐weighted signal intensities after manganese application, and manganese‐induced memory impairment has been suggested, we assessed hippocampus‐dependent learning as well as possible manganese‐induced atrophy of the hippocampal volume. No interference of manganese application on learning was detected after 4 days of Mn²⁺ application or 2 weeks after the application protocol. In addition, no volumetric changes induced by manganese application were found for the hippocampus at any of the measured time points. For longitudinal measurements (i.e. repeated manganese applications), a minimum of at least 8 weeks should be considered using the proposed protocol to allow for sufficient clearance of the paramagnetic ion from cerebral tissue. Copyright © 2012 John Wiley & Sons, Ltd.     

Development of manganese‐enhanced magnetic resonance imaging of the rostral ventrolateral medulla of conscious rats: Importance of normalization and comparison with other regions of interest

Spinally projecting neurons in the rostral ventrolateral medulla (RVLM) are believed to contribute to pathophysiological alterations in sympathetic nerve activity and the development of cardiovascular disease. The ability to identify changes in the activity of RVLM neurons in conscious animals and humans, especially longitudinally, would represent a clinically important advancement in our understanding of the contribution of the RVLM to cardiovascular disease. To this end, we describe the initial development of manganese‐enhanced magnetic resonance imaging (MEMRI) for the rat RVLM. Manganese (Mn²⁺) has been used to estimate in vivo neuronal activity in other brain regions because of both its paramagnetic properties and its entry into and accumulation in active neurons. In this initial study, our three goals were as follows: (1) to validate that Mn²⁺ enhancement occurs in functionally and anatomically localized images of the rat RVLM; (2) to quantify the dose and time course dependence of Mn²⁺ enhancement in the RVLM after one systemic injection in conscious rats (66 or 33 mg/kg, intraperitoneally); and (3) to compare Mn²⁺ enhancement in the RVLM with other regions to determine an appropriate method of normalization of T₁‐weighted images. In our proof‐of‐concept and proof‐of‐principle studies, Mn²⁺ was identified by MRI in the rat RVLM after direct microinjection or via retrograde transport following spinal cord injections, respectively. Systemic injections in conscious rats produced significant Mn²⁺ enhancement at 24 h (p < 0.05). Injections of 66 mg/kg produced greater enhancement than 33 mg/kg in the RVLM and paraventricular nucleus of the hypothalamus (p < 0.05 for both), but only when normalized to baseline scans without Mn²⁺ injection. Consistent with findings from our previous functional and anatomical studies demonstrating subregional neuroplasticity, Mn²⁺ enhancement was higher in the rostral regions of the RVLM (p < 0.05). Together with important technical considerations, our studies support the development of MEMRI as a potential method to examine RVLM activity over time in conscious animal subjects.     

Expression and function of Na+/Ca2+ exchangers 1 and 3 in human macrophages and monocytes

The Na⁺/Ca²⁺ exchanger (NCX) is a membrane transporter that can switch Na⁺ and Ca²⁺ in either direction to maintain the homeostasis of intracellular Ca²⁺. Three isoforms (NCX1, NCX2, and NCX3) have been characterized in excitable cells, e.g. neurons and muscle cells. We examined the expression of these NCX isoforms in primary human lung macrophages (HLM) and blood monocytes. NCX1 and NCX3, but not NCX2, are expressed in HLM and monocytes at both mRNA and protein levels. Na⁺‐free medium induced a significant increase in intracellular calcium concentration ([Ca²⁺]_i) in both cell types. This response was completely abolished by the NCX inhibitor 5‐(N‐4‐chlorobenzyl)‐20,40‐dimethylbenzamil (CB‐DMB). Moreover, inhibition of NCX activity during Ca²⁺‐signaling induced by histamine caused a delay in restoring baseline [Ca²⁺]_i. Na⁺‐free medium induced TNF‐α expression and release in HLM comparable to that caused by LPS. TNF‐α release induced by Na⁺‐free medium was blocked by CB‐DMB and greatly reduced by RNAi‐mediated knockdown of NCX1. These results indicate that human macrophages and monocytes express NCX1 and NCX3 that operate in a bidirectional manner to restore [Ca²⁺]_i, to generate Ca²⁺‐signals, and to induce TNF‐α production. Therefore, NCX may contribute to regulate Ca²⁺ homeostasis and proinflammatory functions in human macrophages and monocytes.     

Calcium homeostasis and glucose uptake of murine myotubes exposed to insulin,caffeine and 4‐chloro‐m‐cresol

The modulation of glucose uptake by cytosolic calcium and the role of insulin on calcium homeostasis in insulin‐target cells are incompletely understood and results are contradictory. To address this issue, we used the C2C12 murine skeletal muscle cell line model and examined the influence of caffeine and 4‐chloro‐m‐cresol, two ryanodine receptor agonists known to mobilize intracellular calcium stores and increase cytosolic free calcium concentration. We followed ⁴⁵calcium efflux, a validated indicator of cytosolic calcium concentration, and 3‐O‐methyl‐[1–³H]‐d ‐glucose uptake in parallel. We also determined if insulin incubation affected ⁴⁵calcium influx rate. A 30‐min treatment by 1 μm insulin highly significantly increased ⁴⁵calcium efflux by 8.5% (P = 0.0014), despite a significant reduction of ⁴⁵Ca²⁺ influx already measurable after 20 and 30 min of insulin stimulation (?16.6%, P = 0.0119 and ?21.3%, P = 0.0047, respectively). Caffeine (1–20 mm ) and 4‐chloro‐m‐cresol (0.05–10 mm ) concentration‐dependently increased ⁴⁵calcium efflux, the latter being more potent and efficacious. These agents, in a concentration‐dependent manner, inhibited both basal and, more potently, insulin‐stimulated glucose uptake. This resulted in a negative correlation of glucose uptake and ⁴⁵calcium efflux (r > 0.95, P < 0.001). This effect was ～5 times greater for caffeine than for 4‐chloro‐m‐cresol, suggesting a calcium‐independent part of the glucose uptake inhibition by caffeine. In our in vitro model of cultured muscle cells, insulin appears to prevent calcium overload by both stimulating efflux and inhibiting cell storage. This effect, taken together with the observed inhibitory, inverse relationship between ⁴⁵calcium efflux and glucose uptake, contributes to describing the complex insulin–calcium interplay involved in target cells.     

Effect of duramycin on chloride transport and intracellular calcium concentration in cystic fibrosis and non-cystic fibrosis epithelia

Oliynyk I, Varelogianni G, Roomans GM, Johannesson M. Effect of duramycin on chloride transport and intracellular calcium concentration in cystic fibrosis and non‐cystic fibrosis epithelia. APMIS 2010; 118: 982–90. The lantibiotic duramycin (Moli1901, Lancovutide) has been suggested as a drug of choice in the treatment for cystic fibrosis (CF). It has been proposed that duramycin may stimulate chloride secretion through Ca²⁺‐activated Cl^? channels (CaCC). We investigated whether duramycin exhibited any effect on Cl^? efflux and intracellular Ca²⁺ concentration ([Ca²⁺]_i) in CF and non‐CF epithelial cells. Duramycin did stimulate Cl^? efflux from CF bronchial epithelial cells (CFBE) in a narrow concentration range (around 1 μM). However, 100 and 250 μM of duramycin inhibited Cl^? efflux from CFBE cells. An inhibitor of the CF transmembrane conductance regulator (CFTR_inh‐172) and a blocker of the capacitative Ca²⁺ entry, gadolinium chloride, inhibited the duramycin‐induced Cl^? efflux. No effect on Cl^? efflux was observed in non‐CF human bronchial epithelial cells (16HBE), human airway submucosal gland cell line, human pancreatic epithelial cells, CF airway submucosal gland epithelial cells, and CF pancreatic cells. The [Ca²⁺]_i was increased by 3 μM duramycin in 16HBE cells, but decreased after 1, and 3 μM of duramycin in CFBE cells. The results suggest that the mechanism responsible for the stimulation of Cl^? efflux by duramycin is mainly related to unspecific changes of the cell membrane or its components rather than to effects on CaCC.     

Alginate‐coated magnetic nanoparticles for noninvasive MRI of extracellular calcium

Nanoparticles (NPs) have great potential to increase the diagnostic capacity of many imaging modalities. MRI is currently regarded as the method of choice for the imaging of deep tissues, and metal ions, such as calcium ions (Ca²⁺), are essential ingredients for life. Despite the tremendous importance of Ca²⁺ for the well‐being of living systems, the noninvasive determination of the changes in Ca²⁺ levels in general, and extracellular Ca²⁺ levels in particular, in deep tissues remains a challenge. Here, we describe the preparation and contrast mechanism of a flexible easy to prepare and selective superparamagnetic iron oxide (SPIO) NPs for the noninvasive determination of changes in extracellular Ca²⁺ levels using conventional MRI. We show that SPIO NPs coated with monodisperse and purified alginate, having a specific molecular weight, provide a tool to selectively determine Ca²⁺ concentrations in the range of 250 µm to 2.5 mm , even in the presence of competitive ions. The alginate‐coated magnetic NPs (MNPs) aggregate in the presence of Ca²⁺, which, in turn, affects the T₂ relaxation of the water protons in their vicinity. The new alginate‐coated SPIO NP formulations, which have no effect on cell viability for 24 h, allow the detection of Ca²⁺ levels secreted from ischemic cell cultures and the qualitative examination of the change in extracellular Ca²⁺ levels in vivo. These results demonstrate that alginate‐coated MNPs can be used, at least qualitatively, as a platform for the noninvasive MRI determination of extracellular Ca²⁺ levels in myriad in vitro and in vivo biomedical applications. Copyright © 2014 John Wiley & Sons, Ltd.     

Cyanide inhibits the Na+/Ca2+ exchanger in isolated cardiac pacemaker cells of the cane toad

The effects of the metabolic inhibition on the activity of the Na⁺/Ca²⁺ exchanger (NCX) were studied in single isolated pacemaker cells from the cane toad. Ca²⁺ influx on NCX (reverse mode) was estimated by measuring the increase in intracellular calcium concentration ([Ca²⁺]_i) in response to extracellular Na⁺-free solution. After application of 2 mM sodium cyanide for 3–5 min, the peak [Ca²⁺]_i in Na⁺-free solution was significantly decreased from 377±42 nM to 260±46 nM, suggesting inhibition of NCX. To study Ca²⁺ efflux on NCX (forward mode), we recorded the tail currents on repolarization which were abolished by Ni²⁺ and by Na⁺-free solution. Cyanide decreased the amplitude of tail currents by 36±3%. To investigate the intrinsic properties of NCX during the metabolic inhibition, we used rapid application of caffeine to trigger sarcoplasmic reticulum Ca²⁺ release, which then stimulates NCX current (I_NCX ). Both the caffeine-induced peak [Ca²⁺]_i and the peak I_NCX were reduced by cyanide exposure. When I_NCX was plotted against [Ca²⁺], the slope of the decay phase was decreased in the presence of CN^– to 44±8% of control, indicating that for a given [Ca²⁺]_i there was less I_NCX produced. These results show that cyanide (CN^–) inhibits NCX activity at least partly through changes in the intrinsic properties of NCX. The inhibition of NCX probably contributes to the slower firing rate of pacemaker cells in CN^–.     

Multiple mechanisms of manganese-induced quenching of fura-2 fluorescence in rat mast cells

Whole-cell patch-clamp recordings of membrane currents and fura-2 measurements of free intracellular calcium concentration ([Ca²⁺]_i) were used to study Mn²⁺ influx in rat peritoneal mast cells. The calcium-selective current, activated by depletion of intracellular calcium stores (I _CRAC for calcium release-activated calcium current), supports a small but measurable Mn²⁺ current. In the presence of intracellular BAPTA, a Mn²⁺ current through I _CRAC was recorded in isotonic MnCl₂ (100 mM) without a significant quenching of fura-2 fluorescence. Its amplitude was 10% of that measured in physiological solution containing 10 mM Ca²⁺. However, following store depletion, a significant quenching of fura-2 fluorescence could be measured only when intracellular BAPTA was omitted, so that all the incoming Mn²⁺ could be captured by the fluorescent dye. Two other ionic currents activated by receptor stimulation also induced Mn²⁺ quenching of fura-2 fluorescence: a small current through non-specific cation channels of 50-pS unitary conductance and a distinct cationic current of large amplitude. In addition to these influx mechanisms, Mn²⁺ was taken up into calcium stores and was subsequently co-released with Ca²⁺ by Ca²⁺-mobilizing agonists.     

Endothelin‐1‐induced proliferation of human endothelial cells depends on activation of K+ channels and Ca2+ influx

Aims: Endothelin‐1 (ET‐1) promotes endothelial cell growth. Endothelial cell proliferation involves the activation of Ca²⁺‐activated K⁺ channels. In this study, we investigated whether Ca²⁺‐activated K⁺ channels with big conductance (BK_Ca) contribute to endothelial cell proliferation induced by ET‐1. Methods: The patch‐clamp technique was used to analyse BK_Ca activity in endothelial cells derived from human umbilical cord veins (HUVEC). Endothelial proliferation was examined using cell counts and measuring [³H]‐thymidine incorporation. Changes of intracellular Ca²⁺ levels were examined using fura‐2 fluorescence imaging. Results: Characteristic BK_Ca were identified in cultured HUVEC. Continuous perfusion of HUVEC with 10 nmol L^?1 ET‐1 caused a significant increase of BK_Ca open‐state probability (n = 14; P < 0.05; cell‐attached patches). The ET_B‐receptor antagonist (BQ‐788, 1 μmol L^?1) blocked this effect. Stimulation with Et‐1 (10 nmol L^?1) significantly increased cell growth by 69% (n = 12; P < 0.05). In contrast, the combination of ET‐1 (10 nmol L^?1) and the highly specific BK_Ca blocker iberiotoxin (IBX; 100 nmol L^?1) did not cause a significant increase in endothelial cell growth. Ca²⁺ dependency of ET‐1‐induced proliferation was tested using the intracellular Ca²⁺‐chelator BAPTA (10 μmol L^?1). BAPTA abolished ET‐1 induced proliferation (n = 12; P < 0.01). In addition, ET‐1‐induced HUVEC growth was significantly reduced, if cells were kept in a Ca²⁺‐reduced solution (0.3 mmol L^?1), or by the application of 2 aminoethoxdiphenyl borate (100 μmol L^?1) which blocks hyperpolarization‐induced Ca²⁺ entry (n = 12; P < 0.05). Conclusion: Activation of BK_Ca by ET‐1 requires ET_B‐receptor activation and induces a capacitative Ca²⁺ influx which plays an important role in ET‐1‐mediated endothelial cell proliferation.     

Effect of α1‐adrenergic stimulation of Cl− secretion and signal transduction in exocrine glands (Rana esculenta)

In the present work, the effect of stimulation of α‐adrenergic receptors on Cl^? secretion via exocrine frog skin glands was investigated. The α‐adrenergic stimulation was performed by addition of the adrenergic agonist noradrenaline in the presence of the β‐adrenergic antagonist propranolol. In the presence of propranolol, noradrenaline had no effect on the cellular cAMP content. The Cl^? secretion was measured as the amiloride‐insensitive short circuit current (I_SC). Addition of noradrenaline induced a biphasic increase in the I_SC. The increase in I_SC coincided with an increase in the net ³⁶Cl^? secretion. The noradrenaline‐induced increase in I_SC was dose‐dependent with an EC₅₀ of 13 ± 0.3 μM . Epifluorescence microscopic measurements of isolated, fura‐2‐loaded frog skin gland acini were used to characterize the intracellular calcium ([Ca²⁺]_i) response. Application of noradrenaline induced a biphasic [Ca²⁺]_i response, which was dose‐dependent with an EC₅₀ of 11 ± 6 μM . The Ca²⁺ plateau unlike the peak‐response was sensitive to removal of Ca²⁺ from the extracellular medium. The noradrenaline‐induced increase in the Cl^? secretion as well as in [Ca²⁺]_i was sensitive to the α₁‐adrenergic antagonist prazosine. Ryanodine and caffeine had no effect on [Ca²⁺]_i indicating that the release was independent of ryanodine‐sensitive Ca²⁺ stores. Noradrenaline mediated a significant increase in the cellular inositol 1,4,5‐trisphosphate (IP₃) content suggesting that the signal transduction pathway leading to the noradrenaline‐induced increase in Ca²⁺ involved IP₃ and a release of Ca²⁺ from IP₃‐sensitive stores.     

Accurate identification of myocardial viability after myocardial infarction with novel manganese chelate‐based MR imaging

We developed a novel manganese (Mn²⁺) chelate for magnetic resonance imaging (MRI) assessment of myocardial viability in acute and chronic myocardial infarct (MI) models, and compared it with Gadolinium‐based delay enhancement MRI (Gd³⁺‐DEMRI) and histology. MI was induced in 14 rabbits by permanent occlusion of the left circumflex coronary artery. Gd³⁺‐DEMRI and Mn²⁺ chelate‐based delayed enhancement MRI (Mn²⁺ chelate‐DEMRI) were performed at 7 days (acute MI, n = 8) or 8 weeks (chronic MI, n = 6) after surgery with sequential injection of 0.15 mmol/kg Gd³⁺ and Mn²⁺ chelate. The biodistribution of Mn²⁺ in tissues and blood was measured at 1.5 and 24 h. Blood pressure, heart rate (HR), left ventricular (LV) function, and infarct fraction (IF) were analyzed, and IF was compared with the histology. The Mn²⁺ chelate group maintained a stable hemodynamic status during experiment. For acute and chronic MI, all rabbits survived without significant differences in HR or LV function before and after injection of Mn²⁺ chelate or Gd³⁺ (p > 0.05). Mn²⁺ chelate mainly accumulated in the kidney, liver, spleen, and heart at 1.5 h, with low tissue uptake and urine residue at 24 h after injection. In the acute MI group, there was no significant difference in IF between Mn²⁺ chelate‐DEMRI and histology (22.92 ± 2.21% vs. 21.79 ± 2.25%, respectively, p = 0.87), while Gd³⁺‐DEMRI overestimated IF, as compared with histology (24.54 ± 1.73%, p = 0.04). In the chronic MI group, there was no significant difference in IF between the Mn²⁺ chelate‐DEMRI, Gd³⁺‐DEMRI, and histology (29.50 ± 11.39%, 29.95 ± 9.40%, and 29.00 ± 10.44%, respectively, p > 0.05), and all three were well correlated (r = 0.92–0.96, p < 0.01). We conclude that the use of Mn²⁺ chelate‐DEMRI is reliable for MI visualization and identifies acute MI more accurately than Gd³⁺‐DEMRI.