Sodium-binding Benzofuran Isophthalate Research Articles

Condensed tannin accretions specifically distributed in mesophyll cells of non-salt secretor mangroves help in salt tolerance.

Auto-fluorescent condensed tannins specifically accumulated in mesophyll cells of non-salt secretor mangroves are involved in the compartmentation of Na+ and osmotic regulation, contributing to their salt tolerance. Salinity is a major abiotic stress affecting the distribution and growth of mangrove plants.The salt exclusion mechanism from salt secretor mangrove leaves is quite known; however, salt management strategies in non-salt secretor leaves remain unclear. In this study, we reported the auto-fluorescent inclusions (AFIs) specifically accumulated in mesophyll cells (MCs) of four non-salt secretor mangroves but absent in three salt secretors. The AFIs increased with the leaf development under natural condition, and applied NaCl concentrations applied in the lab. The AFIs in MCs were isolated and identified as condensed tannin accretions (CTAs) using the dye dimethyl-amino-cinnamaldehyde (DMACA), specific for condensed tannin (CT), both in situ leaf cross sections and in the purified AFIs. Fluorescence microscopy and transmission electron microscope (TEM) analysis indicated that the CTAs originated from the inflated chloroplasts. The CTAs had an obviousmembrane and could induce changes in shape and fluorescence intensity in hypotonic and hypertonic NaCl solutions, suggesting CTAs might have osmotic regulation ability and play an important role in the osmotic regulation in MCs. The purified CTAs were labeled by the fluorescent sodium-binding benzofuran isophthalate acetoxymethyl ester (SBFI-AM), confirming they were involved in the compartmentation of excess Na+ in MCs. This study provided a new view on the salt resistance-associated strategies in mangroves.

Camera-based Measurements of Intracellular [Na+] in Murine Atrial Myocytes

Intracellular sodium concentration ([Na+]i) is an important regulator of intracellular Ca2+. Its study provides insight into the activation of the sarcolemmal Na+/Ca2+ exchanger, the behavior of voltage-gated Na+ channels and the Na+,K+-ATPase. Intracellular Ca2+ signaling is altered in atrial diseases such as atrial fibrillation. While many of the mechanisms underlying altered intracellular Ca2+ homeostasis are characterized, the role of [Na+]i and its dysregulation in atrial pathologies is poorly understood. [Na+]i in atrial myocytes increases in response to increasing stimulation rates. Responsiveness to external field stimulation is therefore crucial for [Na+]i measurements in these cells. In addition, the long preparation (dye-loading) and experiment duration (calibration) require an isolation protocol that yields atrial myocytes of exceptional quality. Due to the small size of mouse atria and the composition of the intercellular matrix, the isolation of high quality adult murine atrial myocytes is difficult. Here, we describe an optimized Langendorff-perfusion based isolation protocol that consistently delivers a high yield of high quality atrial murine myocytes. Sodium-binding benzofuran isophthalate (SBFI) is the most commonly used fluorescent Na+ indicator. SBFI can be loaded into the cardiac myocyte either in its salt form through a glass pipette or as an acetoxymethyl (AM) ester that can penetrate the myocyte’s sarcolemmal membrane. Intracellularly, SBFI-AM is de-esterified by cytosolic esterases. Due to variabilities in membrane penetration and cytosolic de-esterification each cell has to be calibrated in situ. Typically, measurements of [Na+]i using SBFI whole-cell epifluorescence are performed using a photomultiplier tube (PMT). This experimental set-up allows for only one cell to be measured at one time. Due to the length of myocyte dye loading and the calibration following each experiment data yield is low. We therefore developed an EMCCD camera-based technique to measure [Na+]i. This approach permits simultaneous [Na+]i measurements in multiple myocytes thus significantly increasing experimental yield.

Camera-based Measurements of Intracellular [Na+] in Murine Atrial Myocytes.

Intracellular sodium concentration ([Na+]i) is an important regulator of intracellular Ca2+. Its study provides insight into the activation of the sarcolemmal Na+/Ca2+ exchanger, the behavior of voltage-gated Na+ channels and the Na+,K+-ATPase. Intracellular Ca2+ signaling is altered in atrial diseases such as atrial fibrillation. While many of the mechanisms underlying altered intracellular Ca2+ homeostasis are characterized, the role of [Na+]i and its dysregulation in atrial pathologies is poorly understood. [Na+]i in atrial myocytes increases in response to increasing stimulation rates. Responsiveness to external field stimulation is therefore crucial for [Na+]i measurements in these cells. In addition, the long preparation (dye-loading) and experiment duration (calibration) require an isolation protocol that yields atrial myocytes of exceptional quality. Due to the small size of mouse atria and the composition of the intercellular matrix, the isolation of high quality adult murine atrial myocytes is difficult. Here, we describe an optimized Langendorff-perfusion based isolation protocol that consistently delivers a high yield of high quality atrial murine myocytes. Sodium-binding benzofuran isophthalate (SBFI) is the most commonly used fluorescent Na+ indicator. SBFI can be loaded into the cardiac myocyte either in its salt form through a glass pipette or as an acetoxymethyl (AM) ester that can penetrate the myocyte's sarcolemmal membrane. Intracellularly, SBFI-AM is de-esterified by cytosolic esterases. Due to variabilities in membrane penetration and cytosolic de-esterification each cell has to be calibrated in situ. Typically, measurements of [Na+]i using SBFI whole-cell epifluorescence are performed using a photomultiplier tube (PMT). This experimental set-up allows for only one cell to be measured at one time. Due to the length of myocyte dye loading and the calibration following each experiment data yield is low. We therefore developed an EMCCD camera-based technique to measure [Na+]i. This approach permits simultaneous [Na+]i measurements in multiple myocytes thus significantly increasing experimental yield.

Empagliflozin reduces TNFa-induced reactive oxygen species through inhibition of the human endothelial Na+/H+ exchanger 1

Abstract Background Chronic low-grade inflammation, endothelial dysfunction and oxidative stress are major interactive pathways contributing to the pathogenesis of HFpEF. The sodium/glucose cotransporter 2 (SGLT2) inhibitor Empagliflozin (EMPA) inhibits inflammation-induced ROS generation in endothelial cells. EMPA also lowers Na+/H+ exchanger 1 (NHE1) activity and cytoplasmatic Na+ levels ([Na+]c) in cardiomyocytes. Purpose It is unknown how inflammation causes oxidative stress in endothelial cells and how EMPA can mitigate this. Here we examined for human endothelial cells whether 1) inflammatory mediators activate NHE1 activity, 2) increased [Na+]c mediates the inflammation-induced ROS production, and 3) EMPA reduces inflammation-induced ROS through NHE1 inhibition. Methods Human umbilical vein endothelial cells (HUVECs) and human coronary artery endothelial cells (HCAECs) were incubated with vehicle (V), 10ng/mL TNFα, 1μM EMPA or the canonical NHE1 inhibitor 10μM Cariporide. NHE1 activity was measured using the intracellular pH sensitive seminaphtharhodafluor fluorescence, by the pH recovery rate after an ammonium pulse. Intracellular ROS were detected by fluorescent live cell imaging in the presence of 10ng/mL TNFα or under increased Na+ conditions using 100nM ouabain (partial inhibition of the Na+/K+ pump). [Na+]c was measured using the sodium-binding benzofuran isophthalate 1 fluorescent probe. Results TNFα enhanced NHE1 activity in HCAECs (Fig a, in Δ[H+]/sec, V 0.81±0.14, TNFα 1.86±0.35 p<0.05). Increasing [Na+]c by ouabain elevated ROS generation (Fig b, in mM, HUVECs; V 5.6±1.3, ouabain 25.2±1.6 p<0.001, HCAECs; V 18.5±2.4, ouabain 91.2±32.6 p<0.05). EMPA reduced NHE1 activity in HUVECs (in Δ[H+]/sec, V 3.55±0.54, EMPA 1.66±0.25 p<0.01 vs V, Cariporide 0.95±0.11 p<0.001 vs V). HCAECs showed similar results. EMPA lowered [Na+]c (HUVECs; from 8.8±0.9 to 6.0±0.5 mM, p<0.05, HCAECs; from 7.8±0.7 to 4.0±0.8 mM, p<0.001). ROS generation was reversed in the presence of EMPA and/or Cariporide indicating that the effect was mediated by NHE inhibition (Fig c). Conclusions TNFα induces oxidative stress in EC through NHE1 activation and the consequently increased [Na+]c. EMPA acutely inhibits TNFα-induced ROS generation by inhibiting NHE1 and lowering [Na+]c in human cardiac and non-cardiac endothelial cells. Our data suggest that the previously reported cardiac effects of EMPA on NHE1 and [Na+]c are also present in endothelial cells. Endothelial ion homeostasis may be a valuable target to reduce inflammation-induced ROS generation and can constitute the pathway through which SGLT2 inhibitors offer protection in HFpEF patients, as examined in the EMPEROR-Preserved trial. Funding Acknowledgement Type of funding sources: None. Figure 1

Off-target effects of sodium-glucose co-transporter 2 blockers: empagliflozin does not inhibit Na+/H+ exchanger-1 or lower [Na+]i in the heart.

Aims Emipagliflozin (EMPA) is a potent inhibitor of the renal sodium-glucose co-transporter 2 (SGLT2) and an effective treatment for type-2 diabetes. In patients with diabetes and heart failure, EMPA has cardioprotective effects independent of improved glycaemic control, despite SGLT2 not being expressed in the heart. A number of non-canonical mechanisms have been proposed to explain these cardiac effects, most notably an inhibitory action on cardiac Na+/H+ exchanger 1 (NHE1), causing a reduction in intracellular [Na+] ([Na+]i). However, at resting intracellular pH (pHi), NHE1 activity is very low and its pharmacological inhibition is not expected to meaningfully alter steady-state [Na+]i. We re-evaluate this putative EMPA target by measuring cardiac NHE1 activity.Methods and results The effect of EMPA on NHE1 activity was tested in isolated rat ventricular cardiomyocytes from measurements of pHi recovery following an ammonium pre-pulse manoeuvre, using cSNARF1 fluorescence imaging. Whereas 10 µM cariporide produced near-complete inhibition, there was no evidence for NHE1 inhibition with EMPA treatment (1, 3, 10, or 30 µM). Intracellular acidification by acetate-superfusion evoked NHE1 activity and raised [Na+]i, reported by sodium binding benzofuran isophthalate (SBFI) fluorescence, but EMPA did not ablate this rise. EMPA (10 µM) also had no significant effect on the rate of cytoplasmic [Na+]i rise upon superfusion of Na+-depleted cells with Na+-containing buffers. In Langendorff-perfused mouse, rat and guinea pig hearts, EMPA did not affect [Na+]i at baseline nor pHi recovery following acute acidosis, as measured by 23Na triple quantum filtered NMR and 31P NMR, respectively.Conclusions Our findings indicate that cardiac NHE1 activity is not inhibited by EMPA (or other SGLT2i’s) and EMPA has no effect on [Na+]i over a wide range of concentrations, including the therapeutic dose. Thus, the beneficial effects of SGLT2i’s in failing hearts should not be interpreted in terms of actions on myocardial NHE1 or intracellular [Na+].

Sodium influx through cerebral sodium-glucose transporter type 1 exacerbates the development of cerebral ischemic neuronal damage

We recently reported that cerebral sodium-glucose transporter type 1 (SGLT-1) plays a role in exacerbation of cerebral ischemia. However, the mechanism by which cerebral SGLT-1 acts remains unclear. Here we demonstrated that sodium influx through cerebral SGLT-1 exacerbates cerebral ischemic neuronal damage. SGLT-specific sodium ion influx was induced using α-methyl-D-glucopyranoside (α-MG). Intracellular sodium concentrations in primary cortical neurons were estimated using sodium-binding benzofuran isophthalate fluorescence. SGLT-1 knockdown in primary cortical neurons and mice was achieved using SGLT-1 siRNA. The survival rates of primary cultured cortical neurons were assessed using biochemical assays 1 day after treatment. Middle cerebral artery occlusion (MCAO) was used to generate a focal cerebral ischemic model in SGLT-1 knockdown mice. The change in fasting blood glucose levels, infarction development, and behavioral abnormalities were assessed 1 day after MCAO. Treatment with 200mM α-MG induced a continuous increase in the intracellular sodium concentration, and this increase was normalized after α-MG removal. Neuronal SGLT-1 knockdown had no effect on 100µM H2O2-induced neuronal cell death; however, the knockdown prevented the neuronal cell death induced by 17.5mM glucose and the co-treatment of 100µM H2O2/8.75mM glucose. Neuronal SGLT-1 knockdown also suppressed the cell death induced by α-MG alone and the co-treatment of 100µM H2O2/0.01mM α-MG. Our in vivo results showed that the exacerbation of cerebral ischemic neuronal damage induced by the intracerebroventricular administration of 5.0µg α-MG/mouse was ameliorated in cerebral SGLT-1 knockdown mice. Thus, sodium influx through cerebral SGLT-1 may exacerbate cerebral ischemia-induced neuronal damage.

Sodium transport through the cerebral sodium-glucose transporter exacerbates neuron damage during cerebral ischaemia.

We recently demonstrated that the cerebral sodium-glucose transporter (SGLT) is involved in postischaemic hyperglycaemia-induced exacerbation of cerebral ischaemia. However, the associated SGLT-mediated mechanisms remain unclear. Thus, we examined the involvement of cerebral SGLT-induced excessive sodium ion influx in the development of cerebral ischaemic neuronal damage. [Na+]i was estimated according to sodium-binding benzofuran isophthalate fluorescence. In the in vitro study, primary cortical neurons were prepared from fetuses of ddY mice. Primary cortical neurons were cultured for 5 days before each treatment with reagents, and these survival rates were assessed using biochemical assays. In in vivo study, a mouse model of focal ischaemia was generated using middle cerebral artery occlusion (MCAO). In these experiments, treatment with high concentrations of glucose induced increment in [Na+]i, and this phenomenon was suppressed by the SGLT-specific inhibitor phlorizin. SGLT-specific sodium ion influx was induced using a-methyl-D-glucopyranoside (a-MG) treatments, which led to significant concentration-dependent declines in neuronal survival rates and exacerbated hydrogen peroxide-induced neuronal cell death. Moreover, phlorizin ameliorated these effects. Finally, intracerebroventricular administration of a-MG exacerbated the development of neuronal damage induced by MCAO, and these effects were ameliorated by the administration of phlorizin. Hence, excessive influx of sodium ions into neuronal cells through cerebral SGLT may exacerbate the development of cerebral ischaemic neuronal damage.

Glycoside-Induced Collapse of the Sodium and Calcium Gradients Leads to a Biphasic Effect on Cardiac Cell Pacemaker Function

A major postulate of a modern coupled-clock pacemaker-cell theory is based upon electrochemical driving force for sodium and calcium (ENa and ECa). However, [sodium] gradients and their changes induced by glycosides (inhibiting sodium/potassium-ATPase) have never been measured. We measured time-dependent changes in intracellular [sodium](Nai), [calcium](Cai), and action-potentials (AP) [using Sodium-Binding-Benzofuran-Isophthalate (SBFI), Indo-1, and perforated patch-clamp, respectively] induced by cardiac glycosides (digoxegenin, 10μM) in single, isolated rabbit sinoatrial node cells. During 5 minutes of drug application spontaneous AP firing rate underwent biphasic changes, i.e. an increase followed by a decrease. During this time Nai monotonically increased from 8.5±0.6 mM to 13.1±1.1 mM (n=7 cells). The diastolic Cai also monotonically increased 124±6 nM to 149±11 nM (n=5). This Cai increase and biphasic drug effect on AP firing rate were closely reproduced by a coupled-clock Maltsev-Lakatta model simulations when the experimentally measured Nai time course was included in the model as an independent parameter. Model simulations also reproduced the initial AP rate increase via a moderate Cai increase (predicted by the model and validated by our Cai measurements). Specifically, when more cell calcium is available for pumping and release, it boosts the calcium clock, driving membrane clocks and the entire coupled-clock system. According to model predictions, subsequent marked increases in Nai and Cai collapse ENa and ECa and lead to AP rate decrease and dysrhythmic beating as sodium/calcium-exchanger current decreases, resulting in clock uncoupling. Thus, our results not only explain the complex glycoside effects on pacemaker cell AP-firing rate, but also provide new evidence supporting the idea that the normal automaticity of SANC emerges from coupled-clock functions, i.e. Cai and membrane ionic currents, that are controlled by sodium and calcium gradients to regulate sodium/calcium-exchanger current.

A technique for quantifying intracellular free sodium ion using a microplate reader in combination with sodium-binding benzofuran isophthalate and probenecid in cultured neonatal rat cardiomyocytes

BackgroundIntracellular sodium ([Na+]i) kinetics are involved in cardiac diseases including ischemia, heart failure, and hypertrophy. Because [Na+]i plays a crucial role in modulating the electrical and contractile activity in the heart, quantifying [Na+]i is of great interest. Using fluorescent microscopy with sodium-binding benzofuran isophthalate (SBFI) is the most commonly used method for measuring [Na+]i. However, one limitation associated with this technique is that the test cannot simultaneously evaluate the effects of several types or various concentrations of compounds on [Na+]i. Moreover, there are few reports on the long-term effects of compounds on [Na+]i in cultured cells, although rapid changes in [Na+]i during a period of seconds or several minutes have been widely discussed.FindingsWe established a novel technique for quantifying [Na+]i in cultured neonatal rat cardiomyocytes attached to a 96-well plate using a microplate reader in combination with SBFI and probenecid. We showed that probenecid is indispensable for the accurate measurement because it prevents dye leakage from the cells. We further confirmed the reliability of this system by quantifying the effects of ouabain, which is known to transiently alter [Na+]i. To illustrate the utility of the new method, we also examined the chronic effects of aldosterone on [Na+]i in cultured cardiomyocytes.ConclusionsOur technique can rapidly measure [Na+]i with accuracy and sensitivity comparable to the traditional microscopy based method. The results demonstrated that this 96-well plate based measurement has merits, especially for screening test of compounds regulating [Na+]i, and is useful to elucidate the mechanisms and consequences of altered [Na+]i handling in cardiomyocytes.

The Effects of Age on Lens Transport

Age-related nuclear cataracts involve denaturation and aggregation of intracellular proteins. We have documented age-dependent changes in membrane transport in the mouse lens to see what might initiate changes in the intracellular milieu. Microelectrode-based intracellular impedance studies of intact lenses were used to determine gap junction coupling conductance, fiber and surface cell membrane conductances, effective extracellular resistivity, and intracellular voltage. Fiber cell connexin expression was detected by Western blotting. Intracellular hydrostatic pressure was measured with a microelectrode/manometer system. Concentrations of intracellular sodium and calcium were measured by intracellular injection of sodium-binding benzofuran isophthalate and Fura2, respectively. In adult lenses, as age increased: fiber cell gap junction coupling conductance declined significantly, correlating with decreases in Cx46 and Cx50 labeling in Western blots; fiber and surface cell membrane conductances did not change systematically; effective extracellular resistivity increased monotonically; center to surface gradients for intracellular pressure, sodium, calcium, and voltage all increased, but in an interdependent manner that moderated changes. In newborn pup lenses, there were changes that did not simply fit with the above paradigm. In newborn pup lenses, the observed changes may relate to growth factors that are not related to age-dependent changes seen in adult lenses. The major change in adult lenses was an age-dependent decrease in gap junction coupling, probably due to oxidative damage leading to degradation of connexin proteins. These changes clearly lead to compromise of intracellular homeostasis and may be a causal factor in age-related nuclear cataracts.

Two-Photon Sodium Imaging in Dendritic Spines

Neuronal activity causes substantial postsynaptic Na(+) transients. However, the physiological consequences of such Na(+) transients remain largely unknown. High-resolution Na(+) imaging is pivotal to study these questions, and, up to now, two-photon imaging with the fluorescent Na(+) indicator sodium-binding benzofuran isophthalate (SBFI) has been the method of choice. When introduced into individual neurons in acute tissue slices, SBFI enables measurements of [Na(+)](i) transients in dendrites and spines. This technique is also suitable for the determination of [Na(+)](i) transients in other cell types, such as glial cells, with high spatial resolution, and is likely to be useful for measurement of Na(+) signals in vivo. This protocol provides guidelines and tips for measuring dynamic Na(+) concentrations within neurons, as well as an in situ calibration procedure. Because the basic properties of SBFI inside a cell differ significantly from the same properties in vitro, in situ calibrations are required to obtain meaningful experimental results.

Sodium influx induced by external calcium chelation decreases human sperm motility

Calcium removal from the medium promptly reduces human sperm motility and induces a Na(+)-dependent depolarization that is accompanied by an increase in intracellular sodium concentration ([Na(+)](i)) and a decrease in intracellular calcium concentration ([Ca(2+)](i)). Sodium loading activates a Na(+)/K(+)-ATPase. Membrane potential (Vm) and [Ca(2+)](i) were simultaneously detected in human sperm populations with the fluorescent probes diSC(3)(5) and fura 2. [Na(+)](i) and was measured independently in a similar fashion using sodium-binding benzofuran isophthalate. Motility was determined in a CASA system, ATP was measured using the luciferin-luciferase assay, and cAMP was measured by radioimmunoassay. Human sperm motility reduction after calcium removal is related to either Na(+)-loading or Na(+)-dependent depolarization, because, under conditions that inhibit the calcium removal-induced Na(+)-dependent depolarization and [Na(+)](i) increase, sperm motility was unaffected. By clamping sperm Vm with valinomycin, we found that the motility reduction associated with the calcium removal was related to sodium loading, and not to membrane potential depolarization. Mibefradil, a calcium channel blocker, markedly inhibited the Na(+)-dependent depolarization and sodium loading, and also preserved sperm motility. In the absence of calcium, both ATP and cAMP concentrations were decreased by 40%. However ATP levels were unchanged when calcium removal was performed under conditions that inhibit the calcium removal-induced Na(+)-dependent depolarization and [Na(+)](i) increase. Human sperm motility arrest induced by external calcium removal is mediated principally by sodium loading, which would stimulate the Na(+)/K(+)-ATPase and in turn deplete the ATP content.

Additivity of pyrethroid actions on sodium influx in cerebrocortical neurons in primary culture.

Background: Pyrethroid insecticides bind to voltage-gated sodium channels and modify their gating kinetics, thereby disrupting neuronal function. Although previous work has tested the additivity of pyrethroids in vivo, this has not been assessed directly at the primary molecular target using a functional measure.Objectives: We investigated the potency and efficacy of 11 structurally diverse food-use pyrethroids to evoke sodium (Na+) influx in neurons and tested the hypothesis of dose additivity for a mixture of these same 11 compounds.Methods: We determined pyrethroid-induced increases in Na+ influx in primary cultures of cerebrocortical neurons using the Na+-sensitive dye sodium-binding benzofuran isophthalate (SBFI). Concentration-dependent responses for 11 pyrethroids were determined, and the response to dilutions of a mixture of all 11 compounds at an equimolar mixing ratio was assessed. Additivity was tested assuming a dose-additive model.Results: Seven pyrethroids produced concentration-dependent, tetrodotoxin-sensitive Na+ influx. The rank order of potency was deltamethrin > S-bioallethrin > β-cyfluthrin > λ-cyhalothrin > esfenvalerate > tefluthrin > fenpropathrin. Cypermethrin and bifenthrin produced modest increases in Na+ influx, whereas permethrin and resmethrin were inactive. When all 11 pyrethroids were present at an equimolar mixing ratio, their actions on Na+ influx were consistent with a dose-additive model.Conclusions: These data provide in vitro relative potency and efficacy measurements for 7 pyrethroid compounds in intact mammalian neurons. Despite differences in individual compound potencies, we found the action of a mixture of all 11 pyrethroids to be additive when we used an appropriate statistical model. These results are consistent with a previous report of the additivity of pyrethroids in vivo.

Two-photon microscopy and fluorescence lifetime imaging reveal stimulus-induced intracellular Na+ and Cl− changes in cockroach salivary acinar cells

The intracellular ion homeostasis in cockroach salivary acinar cells during salivation is not satisfactorily understood. This is mainly due to technical problems regarding strong tissue autofluorescence and ineffective ion concentration quantification. For minimizing these problems, we describe the successful application of two-photon (2P) microscopy partly in combination with fluorescence lifetime imaging microscopy (FLIM) to record intracellular Na(+) and Cl(-) concentrations ([Na(+)](i), [Cl(-)](i)) in cockroach salivary acinar cells. Quantitative 2P-FLIM Cl(-) measurements with the dye N-(ethoxycarbonylmethyl)-6-methoxy-quinolinium bromide indicate that the resting [Cl(-)](i) is 1.6 times above the Cl(-) electrochemical equilibrium but is not influenced by pharmacological inhibition of the Na(+)-K(+)-2Cl(-) cotransporter (NKCC) and anion exchanger using bumetanide and 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid disodium salt. In contrast, rapid Cl(-) reuptake after extracellular Cl(-) removal is almost totally NKCC mediated both in the absence and presence of dopamine. However, in physiological saline [Cl(-)](i) does not change during dopamine stimulation although dopamine stimulates fluid secretion in these glands. On the other hand, dopamine causes a decrease in the sodium-binding benzofuran isophthalate tetra-ammonium salt (SBFI) fluorescence and an increase in the Sodium Green fluorescence after 2P excitation. This opposite behavior of both dyes suggests a dopamine-induced [Na(+)](i) rise in the acinar cells, which is supported by the determined 2P-action cross sections of SBFI. The [Na(+)](i) rise is Cl(-) dependent and inhibited by bumetanide. The Ca(2+)-ionophore ionomycin also causes a bumetanide-sensitive [Na(+)](i) rise. We propose that a Ca(2+)-mediated NKCC activity in acinar peripheral cells attributable to dopamine stimulation serves for basolateral Na(+) uptake during saliva secretion and that the concomitantly transported Cl(-) is recycled back to the bath.

Reactive Oxygen Species–Activated Ca/Calmodulin Kinase IIδ Is Required for Late I Na Augmentation Leading to Cellular Na and Ca Overload

In heart failure Ca/calmodulin kinase (CaMK)II expression and reactive oxygen species (ROS) are increased. Both ROS and CaMKII can increase late I(Na) leading to intracellular Na accumulation and arrhythmias. It has been shown that ROS can activate CaMKII via oxidation. We tested whether CaMKIIδ is required for ROS-dependent late I(Na) regulation and whether ROS-induced Ca released from the sarcoplasmic reticulum (SR) is involved. 40 μmol/L H(2)O(2) significantly increased CaMKII oxidation and autophosphorylation in permeabilized rabbit cardiomyocytes. Without free [Ca](i) (5 mmol/L BAPTA/1 mmol/L Br(2)-BAPTA) or after SR depletion (caffeine 10 mmol/L, thapsigargin 5 μmol/L), the H(2)O(2)-dependent CaMKII oxidation and autophosphorylation was abolished. H(2)O(2) significantly increased SR Ca spark frequency (confocal microscopy) but reduced SR Ca load. In wild-type (WT) mouse myocytes, H(2)O(2) increased late I(Na) (whole cell patch-clamp). This increase was abolished in CaMKIIδ(-/-) myocytes. H(2)O(2)-induced [Na](i) and [Ca](i) accumulation (SBFI [sodium-binding benzofuran isophthalate] and Indo-1 epifluorescence) was significantly slowed in CaMKIIδ(-/-) myocytes (versus WT). CaMKIIδ(-/-) myocytes developed significantly less H(2)O(2)-induced arrhythmias and were more resistant to hypercontracture. Opposite results (increased late I(Na), [Na](i) and [Ca](i) accumulation) were obtained by overexpression of CaMKIIδ in rabbit myocytes (adenoviral gene transfer) reversible with CaMKII inhibition (10 μmol/L KN93 or 0.1 μmol/L AIP [autocamtide 2-related inhibitory peptide]). Free [Ca](i) and a functional SR are required for ROS activation of CaMKII. ROS-activated CaMKIIδ enhances late I(Na), which may lead to cellular Na and Ca overload. This may be of relevance in hear failure, where enhanced ROS production meets increased CaMKII expression.

Antillatoxin is a sodium channel activator that displays unique efficacy in heterologously expressed rNav1.2, rNav1.4 and rNav1.5 alpha subunits

BackgroundAntillatoxin (ATX) is a structurally unique lipopeptide produced by the marine cyanobacterium Lyngbya majuscula. ATX activates voltage-gated sodium channel α-subunits at an undefined recognition site and stimulates sodium influx in neurons. However, the pharmacological properties and selectivity of ATX on the sodium channel α-subunits were not fully characterized.ResultsIn this study, we characterized the pharmacological properties and selectivity of ATX in cells heterologously expressing rNav1.2, rNav1.4 or rNav1.5 α-subunits by using the Na+ selective fluorescent dye, sodium-binding benzofuran isophthalate. ATX produced sodium influx in cells expressing each sodium channel α-subunit, whereas two other sodium channel activators, veratridine and brevetoxin-2, were without effect. The ATX potency at rNav1.2, rNav1.4 and rNav1.5 did not differ significantly. Similarly, there were no significant differences in the efficacy for ATX-induced sodium influx between rNav1.2, rNav1.4 and rNav1.5 α-subunits. ATX also produced robust Ca2+ influx relative to other sodium channel activators in the calcium-permeable DEAA mutant of rNav1.4 α-subunit. Finally, we demonstrated that the 8-demethyl-8,9-dihydro-antillatoxin analog was less efficacious and less potent in stimulating sodium influx.ConclusionsATX displayed a unique efficacy with respect to stimulation of sodium influx in cells expressing rNav1.2, rNav1.4 and rNav1.5 α-subunits. The efficacy of ATX was distinctive inasmuch as it was not shared by activators of neurotoxin sites 2 and 5 on VGSC α-subunits. Given the unique pharmacological properties of ATX interaction with sodium channel α-subunits, decoding the molecular determinants and mechanism of action of antillatoxin may provide further insight into sodium channel gating mechanisms.

Temporary Sequestration of Potassium by Mitochondria in Astrocytes

Increases in extracellular potassium concentration ([K(+)](o)), which can occur during neuronal activity and under pathological conditions such as ischemia, lead to a variety of potentially detrimental effects on neuronal function. Although astrocytes are known to contribute to the clearance of excess K(+)(o), the mechanisms are not fully understood. We examined the potential role of mitochondria in sequestering K(+) in astrocytes. Astrocytes were loaded with the fluorescent K(+) indicator PBFI and release of K(+) from mitochondria into the cytoplasm was examined after uncoupling the mitochondrial membrane potential with carbonyl cyanide m-chlorophenylhydrazone (CCCP). Under the experimental conditions employed, transient applications of elevated [K(+)](o) led to increases in K(+) within mitochondria, as assessed by increases in the magnitudes of cytoplasmic [K(+)] ([K(+)](i)) transients evoked by brief exposures to CCCP. When mitochondrial K(+) sequestration was impaired by prolonged application of CCCP, there was a robust increase in [K(+)](i) upon exposure to elevated [K(+)](o). Blockade of plasmalemmal K(+) uptake routes by ouabain, Ba(2+), or a mixture of voltage-activated K(+) channel inhibitors reduced K(+) uptake into mitochondria. Also, reductions in mitochondrial K(+) uptake occurred in the presence of mito-K(ATP) channel inhibitors. Rises in [K(+)](i) evoked by brief applications of CCCP following exposure to high [K(+)](o) were also reduced by gap junction blockers and in astrocytes isolated from connexin43-null mice, suggesting that connexins also play a role in K(+) uptake into astrocyte mitochondria. We conclude that mitochondria play a key role in K(+)(o) handling by astrocytes.

Phorbol ester-evoked Ca2+ signaling in human platelets is via autocrine activation of P(2X1) receptors, not a novel non-capacitative Ca2+ entry.

Platelets are reported to possess a protein kinase C (PKC)-dependent non-capacitative Ca(2+)entry (NCCE) pathway. The phorbol ester, phorbol, 12-myristate, 13-acetate (PMA) has been suggested to stimulate platelet NCCE. Recently we demonstrated important roles in store-operated Ca(2+)entry in human platelets for Na(+)/Ca(2+) exchangers (NCXs) and autocrine signaling between platelets after dense granule secretion. As PMA evokes dense granule secretion, we have investigated the role of NCXs and autocrine signaling in PMA-evoked Ca(2+)entry. To investigate the roles of NCXs and dense granule secretion in PMA-evoked Ca(2+)signaling in human platelets. Fura-2- or sodium-binding benzofuran isophthalate (SBFI)-loaded platelets were used to monitor cytosolic Ca(2+)or Na(+) concentrations. Dense granule secretion was monitored as ATP release using luciferin-luciferase. The NCX inhibitors KB-R7943 or SN-6, and removal of extracellular Na(+), significantly reduced PMA-evoked Ca(2+)entry. PMA-evoked dense granule secretion was almost abolished by pretreatment with the PKC inhibitor Ro-31-8220 and significantly slowed by KB-R7943. The P(2X1) antagonists Ro-0437626 or MRS-2159, or desensitization of P(2X1) receptors by prior treatment with alpha,beta-Methylene-ATP or omitting apyrase from the medium, reduced PMA-evoked Ca(2+)entry. Ro-0437626 or chelation of extracellular Ca(2+) slowed but did not abolish PMA-evoked ATP release, indicating that PMA-evoked dense granule secretion does not require P(2X1) receptor activation but is accelerated by P(2X1)-mediated Ca(2+)entry. The presence of NCX3 in human platelets was demonstrated by Western blotting. PMA-evoked Ca(2+)entry results from an NCX3-dependent dense granule secretion and subsequent P(2X1) receptor activation by secreted ATP, rather than activation of a novel NCCE pathway.

Synaptically induced sodium signals in hippocampal astrocytes in situ

Astrocytes are in close contact to excitatory synapses and express transporters which mediate the sodium-dependent uptake of glutamate. In cultured astrocytes, selective activation of glutamate transport results in sodium elevations which stimulate Na(+)/K(+)-ATPase and glucose uptake, indicating that synaptic release of glutamate might couple excitatory neuronal activity to glial sodium homeostasis and metabolism. Here, we analysed intracellular sodium transients evoked by synaptic stimulation in acute mouse hippocampal slices using quantitative sodium imaging with the sodium-sensitive fluorescent indicator dye SBFI (sodium-binding benzofuran isophthalate). We found that short bursts of Schaffer collateral stimulation evoke sodium transients in the millimolar range in both CA1 pyramidal neurons and in SR101-positive astrocytes of the stratum radiatum. At low stimulation intensities, glial sodium transients were confined to one to two primary branches and adjacent fine processes and only weakly invaded the soma. Increasing the number of activated afferent fibres by increasing the stimulation intensity elicited global sodium transients detectable in the processes as well as the somata of astrocytes. Pharmacological analysis revealed that neuronal sodium signals were mainly attributable to sodium influx through ionotropic glutamate receptors. Activation of ionotropic receptors also contributed to glial sodium transients, while TBOA-sensitive glutamate transport was the major pathway responsible for sodium influx into astrocytes. Our results thus establish that glutamatergic synaptic transmission in the hippocampus results in sodium transients in astrocytes that are mainly mediated by activation of glutamate transport. They support the proposed link between excitatory synaptic activity, glutamate uptake and sodium signals in astrocytes of the hippocampus.

Metabolic Inhibition Strongly Inhibits Na +-Dependent Mg 2+ Efflux in Rat Ventricular Myocytes

We measured intracellular Mg 2+ concentration ([Mg 2+] i) in rat ventricular myocytes using the fluorescent indicator furaptra (25°C). In normally energized cells loaded with Mg 2+, the introduction of extracellular Na + induced a rapid decrease in [Mg 2+] i: the initial rate of decrease in [Mg 2+] i (initial Δ[Mg 2+] i/Δ t) is thought to represent the rate of Na +-dependent Mg 2+ efflux (putative Na +/Mg 2+ exchange). To determine whether Mg 2+ efflux depends directly on energy derived from cellular metabolism, in addition to the transmembrane Na + gradient, we estimated the initial Δ[Mg 2+] i/Δ t after metabolic inhibition. In the absence of extracellular Na + and Ca 2+, treatment of the cells with 1 μM carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, an uncoupler of mitochondria, caused a large increase in [Mg 2+] i from ∼0.9 mM to ∼2.5 mM in a period of 5–8 min (probably because of breakdown of MgATP and release of Mg 2+) and cell shortening to ∼50% of the initial length (probably because of formation of rigor cross-bridges). Similar increases in [Mg 2+] i and cell shortening were observed after application of 5 mM potassium cyanide (KCN) (an inhibitor of respiration) for ≥90 min. The initial Δ[Mg 2+] i/Δ t was diminished, on average, by 90% in carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone-treated cells and 92% in KCN-treated cells. When the cells were treated with 5 mM KCN for shorter times (59–85 min), a significant decrease in the initial Δ[Mg 2+] i/Δ t (on average by 59%) was observed with only a slight shortening of the cell length. Intracellular Na + concentration ([Na +] i) estimated with a Na + indicator sodium-binding benzofuran isophthalate was, on average, 5.0–10.5 mM during the time required for the initial Δ[Mg 2+] i/Δ t measurements, which is well below the [Na +] i level for half inhibition of the Mg 2+ efflux (∼40 mM). Normalization of intracellular pH using 10 μM nigericin, a H + ionophore, did not reverse the inhibition of the Mg 2+ efflux. From these results, it seems likely that a decrease in ATP below the threshold of rigor cross-bridge formation (∼0.4 mM estimated indirectly in the this study), rather than elevation of [Na +] i or intracellular acidosis, inhibits the Mg 2+ efflux, suggesting the absolute necessity of ATP for the Na +/Mg 2+ exchange.