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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+]. [ABSTRACT FROM AUTHOR]

The epithelial Na+ channel (ENaC) is tightly regulated by sodium intake to maintain whole body sodium homeostasis. In addition, ENaC is inhibited by high levels of intracellular Na+ [Na+]i, presumably to prevent cell Na+ overload and swelling. However, it is not clear if this regulation is relevant in vivo. We show here that in rats, an acute (4 h) oral sodium load decreases whole-cell amiloride-sensitive currents (INa) in the cortical collecting duct (CCD) even when plasma aldosterone levels are maintained high by infusing the hormone. This was accompanied by decreases in whole-kidney cleaved α-ENaC (2.6 fold), total β-ENaC (1.7 fold), and cleaved γ-ENaC (6.2 fold). In addition, cell-surface β- and γ-ENaC expression was measured using in situ biotinylation. There was a decrease in cell-surface core-glycosylated (2.2 fold) and maturely glycosylated (4.9 fold) β-ENaC and cleaved γ-ENaC (4.7 fold). There were no significant changes for other apical sodium transporters. To investigate the role of increases in Na+ entry and presumably [Na+]i on ENaC, animals were infused with amiloride prior to and during sodium loading. Blocking Na+ entry did not inhibit the effect of resalting on INa. However, amiloride did prevent decreases in ENaC expression, an effect that was not mimicked by hydrochlorothiazide administration. Na+ entry and presumably [Na+]i can regulate ENaC expression but does not fully account for the aldosterone-independent decrease in INa during an acute sodium load. [ABSTRACT FROM AUTHOR]

Abstract: Ankyrin-B (AnkB) loss-of-function may cause ventricular arrhythmias and sudden cardiac death in humans. Cardiac myocytes from AnkB heterozygous mice (AnkB+/−) show reduced expression and altered localization of Na/Ca exchanger (NCX) and Na/K-ATPase (NKA), key players in regulating [Na]i and [Ca]i. Here we investigate how AnkB reduction affects cardiac [Na]i, [Ca]i and SR Ca release. We found reduced NCX and NKA transport function but unaltered [Na]i and diastolic [Ca]i in myocytes from AnkB+/− vs. wild-type (WT) mice. Ca transients, SR Ca content and fractional SR Ca release were larger in AnkB+/− myocytes. The frequency of spontaneous, diastolic Ca sparks (CaSpF) was significantly higher in intact myocytes from AnkB+/− vs. WT myocytes (with and without isoproterenol), even when normalized for SR Ca load. However, total ryanodine receptor (RyR)-mediated SR Ca leak (tetracaine-sensitive) was not different between groups. Thus, in AnkB+/− mice SR Ca leak is biased towards more Ca sparks (vs. smaller release events), suggesting more coordinated openings of RyRs in a cluster. This is due to local cytosolic RyR regulation, rather than intrinsic RyR differences, since CaSpF was similar in saponin-permeabilized myocytes from WT and AnkB+/− mice. The more coordinated RyRs openings resulted in an increased propensity of pro-arrhythmic Ca waves in AnkB+/− myocytes. In conclusion, AnkB reduction alters cardiac Na and Ca transport and enhances the coupled RyR openings, resulting in more frequent Ca sparks and waves although the total SR Ca leak is unaffected. This could enhance the propensity for triggered arrhythmias in AnkB+/− mice. [Copyright &y& Elsevier]

Nicotinic acetylcholine receptors (nAChRs) are involved in the regulation of intracellular Ca-dependent processes both in normal and pathological states. α-Conotoxins from the venom of Conus marine mollusks are a valuable tool for the investigation of the pharmacological action and functional role of nAChRs. Analogues of α-conotoxin MII labeled by Bolton-Hunter reagent (BH-MII) or fluorescein isothiocyanate (FITC-MII) on the N-terminal glycine residue have been synthesized in the present work. Fluorescence microscopy studies of SH-SY5Y neuroblastoma cells loaded with Ca indicator Fura-2, or by both Ca indicator Fluo-4 and Na indicator SBFI, were used to test the effect of MII modification on its ability to block Ca and Na signals induced by nicotine. Measurements in SH-SY5Y cells showed that kinetics of the increase and recovery of the concentration of free Ca ([Ca]) upon nicotine application and washout was different from that for free Na ([Na]), this being evidence of differences in the mechanism of Ca and Na homeostasis regulation. MII suppressed the nicotine-induced increase of [Ca] and [Na] in a concentration-dependent manner. An additional tyrosine residue added to the N-terminus of one of the MII derivatives caused a significant decrease in the inhibitory action of MII; this decrease was even more pronounced when a large FITC label was introduced into MII. The BH-MII derivative had an inhibitory effect similar to that of unmodified α-conotoxin. MII and its iodinated derivatives are promising tools for radioligand assays. [ABSTRACT FROM AUTHOR]

Dopamine inhibits renal Na+:HCO3- cotransporter in rabbits and normotensive rats but not in spontaneously hypertensive rats. Background. Dopamine (DA) is thought to regulate renal proximal transport through the inhibition of the Na+,K+-ATPase and/or Na+/H+ exchanger. Defects in this dopaminergic system are proposed to be a pathogenic factor of genetic hypertension. However, microperfusion studies have not consistently confirmed direct tubular effects of DA. Methods. Isolated proximal straight tubules were perfused peritubularly with Dulbecco's modified Eagle's tissue culture medium (DMEM) containing norepinephrine (NE) to improve incubation conditions. Intracellular Na+ concentrations ([Na+]i) and cell pH (pHi) were measured with fluorescence probes. Results. When incubated in DMEM plus NE, DA increased [Na+]i in rabbit tubules. Inhibition of Na+,K+-ATPase could not explain this response, as it was not suppressed by ouabain. An analysis of pHi responses to bath HCO3- reduction revealed that DA, SKF 38393 (a DA1 agonist), and adenosine 3′,5′-cyclic monophosphate (cAMP) inhibited the basolateral Na+:HCO3- cotransporter in rabbit and Wistar-Kyoto rat (WKY), if its transport stoichiometry was converted to 3 HCO3-:1 Na+ by DMEM plus NE incubation. The inhibitory effect of DA was abolished by SCH 23390, a DA1 antagonist, but not by (-)-sulpiride, a DA2 antagonist. In spontaneously hypertensive rats (SHRs), however, DA and SKF 38393 failed to inhibit the cotransporter, although the inhibitory effects of cAMP and parathyroid hormone were comparable to those in WKY. Conclusion. These results indicate that DA inhibits the Na+:HCO3-... [ABSTRACT FROM AUTHOR]

Increased passive Na+ ,K+ fluxes necessitate an efficient activation of the Na+ ,K+ pump in working muscles to limit the rundown of the Na+ ,K+ chemical gradients and ensuing loss of excitability. Several studies have demonstrated an increase in Na+ ,K+ -pump rate in working muscles, and in electrically stimulated muscles up to a 22-fold increase in active Na+ ,K+ transport has been observed. Excitation-induced increase in intracellular Na+ is believed to be the primary stimulus for Na+ ,K+ pumping in a contracting muscle. In muscles recovering from electrical stimulation, however, the activity of the pump may stay elevated even after intracellular Na+ has been reduced to below the resting level. Moreover, in rat soleus muscles 10-s stimulation at 60 Hz induced a 5-fold increase in the activity of the Na+ ,K+ pump although mean intracellular [Na+ ] was unchanged. These findings strongly suggest that a substantial part of the excitation-induced increase in Na+ ,K+ -pump activity is caused by mechanisms other than increased intracellular [Na+ ]. The mechanism behind this activation is not clear, but may involve a change in the affinity of the Na+ ,K+ pump for intracellular Na+ . In addition to intracellular [Na+ ], the Na+ ,K+ pump may be stimulated in contracting muscles by other factors such as catecholamines, calcitonin gene-related peptide (CGRP), free fatty acids and cytoskeletal links. Together, this activation may form a feed forward mechanism protecting muscles from loss of excitability during periods of contraction by increasing Na+ ,K+ -pump activity prior to erosion of the Na+ ,K+ chemical gradients. During exercise of high intensity, however, intracellular [Na+ ] increases substantially constituting an additional stimulus for the pump. [ABSTRACT FROM AUTHOR]

In the present study we used the Na-sensitive fluorescent dye SBFI and optical measurement of endpiece volume to investigate the transport of Na in sheep parotid secretory cells. Sheep parotid endpiece cells bathed in a HCO-free Cl-rich solution had a resting intracellular Na concentration ([Na]) of 17±2 mmol/l ( n=39). Exposure of the cells to a 2-min pulse of acetylcholine (ACh) (3×10 mol/l) in a HCO-free bathing solution produced no change in [Na] or in cell volume. Changing from a Cl-containing HCO-free bath solution to a Cl solution containing 25 mmol/l HCOcaused the endpieces to swell by 8±2 % ( n=11) and the [Na] to increase by 10±2 mmol/l ( n=14). Subsequent exposure of the cells to ACh led to shrinkage of the cells by 12±2 % from the volume in the HCO-containing solution prior to ACh exposure, with the maximum decrease occurring after 29±7 s ( n=9). This shrinkage was accompanied by a rapid and transient increase in [Na], the [Na] reaching a peak at 70±5 mmol/l above the unstimulated level ( n=9). Substitution of gluconate for Cl did not significantly alter the effects of HCOon unstimulated [Na] or endpiece volume, nor did it significantly inhibit the effects of ACh on these two parameters when HCOwas present. Addition of 200 μmol/l dihydrogen-4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (H-DIDS) to the gluconate/HCOsolution significantly reduced the peak of the ACh-induced increase in [Na] to 34±10 mmol/l ( n=4), but did not have any significant effect on the magnitude of the ACh-induced shrinkage. At 500 μmol/l, H-DIDS abolished the ACh-induced increase in [Na] and also significantly reduced the shrinkage due to ACh. Finally, we found that the rate of endpiece shrinkage following ACh stimulation did not depend on the presence of Cl. We interpret these results as indicating that sheep parotid secretory cells do not contain significant Na-K-2Cl co-transport activity and do not actively accumulate Cl. Rather, the mechanism of spontaneous basal secretion by these cells, in the presence of extracellular HCO, is based on the accumulation of HCOby the Na-H exchanger. During ACh stimulation, the concentration of HCOin the cytosol is also maintained by the operation of a H-DIDS-sensitive Na-HCOco-transporter. HCOefflux across the apical membrane occurs via a HCOconductance pathway rather than by the coupled operation of a Cl channel and a Cl-HCOexchanger. [ABSTRACT FROM AUTHOR]

Kinetic properties of the Na-H antiport in the acinar cells of the isolated, superfused mouse lacrimal gland were studied by measuring intracellular pH (pH) and Na activity ( aNa) with the aid of double-barreled H- and Na-selective microelectrodes, respectively. Bicarbonate-free solutions were used throughout. Under untreated control conditions, pH was 7.12±0.01 and aNa was 6.7±0.6 mmol/l. The cells were acid-loaded by exposure to an NHsolution followed by an Na-free N-methyl- d-glucamine (NMDG) solution. Intracellular Na and H concentrations were manipulated by changing the duration of exposure to the above solutions. Subsequent addition of the standard Na solution rapidly increased pH. This Na-induced increase in pH was almost completely inhibited by 0.5 mmol/l amiloride and was associated with a rapid, amiloride-sensitive increase in aNa. The rate of pH recovery induced by the standard Na solution increased in a saturable manner as pH decreased, and was negligible at pH 7.2-7.3, indicating an inactivation of the Na-H antiport. The apparent K for intracellular H concentration was 105 nmol/l (pH 6.98). The rate of acid extrusion from the acid-loaded cells increased proportionally to the increase in extracellular pH. Depletion of aNa to less than 1 mmol/l by prolonged exposure to NMDG solution significantly increased the rate of Na-dependent acid extrusion. The rate of acid extrusion increased as the extracellular Na concentration increased following Michaelis-Menten kinetics ( V was 0.55 pH/min and the apparent K was 75 mmol/l at pH 6.88). The results clearly showed that the Na-H antiport activity is dependent on the chemical potential gradient of both Na and H ions across the basolateral membrane, and that the antiporter is asymmetric with respect to the substrate affinity of the transport site. The data agree with the current model of activation and inactivation of the antiporter by an intracellular site through changes in the intracellular Na and H concentrations. [ABSTRACT FROM AUTHOR]

The effect of various intracellular Na concentrations ( c) and membrane potentials on the Na pump current ( I) was studied in isolated, cultured sheep cardiac Purkinje cells ('cardioballs'). I was identified as cardiac steroid sensitive current. The dependence of I on c was investigated at a membrane potential of −40 mV by means of whote-cell recording from cardioballs internally perfused with media containing various Na concentrations. Internal perfusion with a Na free solution abolished I. The amplitude of I as a function of c displayed saturation kinetics. Half maximal activation of I occured at a c of about 9 mM. The maximal I density was estimated to be 1.1μA/cm. The potential dependence of I was studied by conventional whole-cell recording under various ionic conditions. Generally I displayed little voltage dependence at membrane potentials positive to −20 mV. I declined at more negative potentials. The pump cycle probably includes only one voltage sensitive step. The potential dependence of was more pronounced at lower c or lower concentrations of the external pump activator Cs. The findings are in line with the idea that increasingly steeper ionic gradients against which the cations are pumped strengthen the voltage dependence of I in the potential range studied. Other factors probably affecting the pump current-voltage ( I- V) relation are discussed. The results suggest that I varies during electrical activity. [ABSTRACT FROM AUTHOR]

Intracellular Na content (Na) in the perfused rat mandibular gland was measured by using aNa NMR spectroscopy at 24°C. An aqueous chemical shift reagent, dysprosium triethylenetetramine-N,N,N′,N″,N‴N‴-hexaacetic acid [Dy(TTHA)] was used in order to discriminate between the intracellular and the extracellular Na signal. The mandibular gland of rat was perfused arterially with a modified Krebs solution containing 10 mM Dy(TTHA). At rest, Na was not changed by blocking the Na/K ATPase with ouabain (1 mM) and atropine (3 μM), implying that, in the absence of stimulation, the spontaneous Na influx across the plasma membrane must have been negligibly small. Following onset of stimulation with acetylcholine (1 μM), Na increased by 9.1±1.5 mmol/l intracellular fluid (mean±SEM, n=13), and remained at this level during stimulation. In the initial phase of secretion (0-5 min), about 50 mmol/min/l intracellular fluid of Na was secreted into the luminal space (estimated from the secretory rate by assuming an isotonic primary secretion) but, in spite of the higher secretion rate, Na increased only at an initial rate of 4.1 mmol/min/l intracellular fluid. During the steady phase of secretion (15-30 min) evoked by acetylcholine (1 μM), ouabain (1 mM) caused an increment of Na of 44±8 mmol/l intracellular fluid (mean±SEM, n=4). From the rate of Na increment, the Na influx rate at the steady phase was estimated as 4.5 mmol/min/l intracellular fluid. These results suggest that the influx of Na is caused by stimulation with acetylcholine. The observed Na influx rate was about 50% of the Na secretory rate at the steady phase of secretion, estimated from the secretory rate by assuming an isotonic primary secretion. This is fully compatible with the operation of Na−K-2Cl contransport system for which one would expect a Na influx rate exactly half of the rate of Na and Cl secretion. [ABSTRACT FROM AUTHOR]

The intracellular Na activity, aNa, and the intracellular Ca activity, aCa, were measured with double-barrelled neutral carrier Na- and Ca-sensitive microelectrodes in Retzius neurones in the central nervous system of the leech Hirudo medicinalis. The aNa was measured to be 8.0 mM (corrected for Ca interference), which corresponds to a cytoplasmic Na concentration of 10.7 mM, assuming a Na activity coefficient of 0.75. The calculated Na equilibrium potential was 59 mV, giving a total Na electrochemical gradient of approximately 102 mV. The aCa was found to range between 1 and 5×10 M, from which a Ca equilibrium potential near +120 mV was estimated. When the Na−K pump was inhibited by lowering the external K concentration or by adding the glycoside ouabain (5×10 M), the aNa reversibly increased severalfold. When aNa increased to high levels following complete pump inhibition, the aCa increased above 10 M, and the membrane input resistance decreased. After removal of ouabain, aNa, aCa and the membrane resistance recovered within 30 min after a delay of 20-40 min. Our results suggest that a large increase of aNa produces a rise in aCa, possibly by means of a Na−Ca exchange across the cell membrane. The elevation of the aCa may be responsible for the decrease in membrane resistance, and may also be related to the uncoupling of the paired Retzius neurones observed in the presence of Na−K pump inhibitors. [ABSTRACT FROM AUTHOR]

The purpose of the study was to compare the role of Na ions in the damage caused by cardioplegic ischemia in fast (rat) and slow (pig) hearts. Changes in intracellular Na (Na), high energy phosphates, and contractile function were assessed during ischemia (36°C) and reperfusion in KCl-arrested perfused hearts usingP-NMR and shift reagent (DyTTHA)-aidedNa-NMR spectroscopy. In the pig hearts the rates of decrease of phosphocreatine (PCr), ATP and intracellular pH (pHi) were 3-4 times slower than the rates observed in the rat hearts. In the pig hearts PCr was observable (∼10%) during first 80 min of the ischemic period (90 min). Comparable decreases in ATP (32.0±6 vs. 38±15% of initial) and pH, (to 6.14±0.06 vs. 6.10±0.15) observed after 90 and 20 min ischemia in pig and rat hearts, respectively, were associated with a smaller Na increase in the pig hearts (to 175±31%) than in the rat hearts (to 368±62%). This Na increase in the rat hearts preceded development of ischemic contracture (41±6 mmHg at 23.6±0.7 min) while no contracture was observed in pig hearts. Reperfusion of the rat hearts (at 30 min ischemia) was followed by partial recovery of PCr (44±3%) and Na (234±69%) and poorer recovery of the pressure-rate product (PRP, 9±4%) and end-diastolic pressure (EDP, 72±5 mm Hg) compared to the pig hearts (PCr, 106±25%; Na, 82±17%; PRP, 24±3%; EDP, 4.6±2.5 mmHg). The loss of function in the pig hearts was reversed by increasing Ca in the perfusate from 1 to 2.3 mM and resulted in a rise in both PRP and oxygen consumption rate, V(O), from 24±3.3 to 64.5±5.8% and from 55±10 to 74±10% of the control levels, respectively. The PRP/ΔV(O) ratio was halved in the post-ischemic pig hearts and returned to the pre-ischemic level following Ca stimulation. It is suggested that the higher stability of Na homeostasis to ischemic stress in the pig heart may result from: 1) a lower ratio of the rate of ATP hydrolysis to glycolytic ATP production; 2) differences in the kinetic properties of the Na transporters. Reduced Na accumulation during ischemia and reperfusion is benefical for metabolic and functional preservation of cardiomyocytes. [ABSTRACT FROM AUTHOR]

The characterization of various ion transport systems has led to a better understanding of the effects, which seem to take part in the impairment of ischemic and reperfused cardiac tissue. This review discusses the role of the Na/H exchange system in the pathophysiology of ischemia and reperfusion and the beneficial effects of its inhibition. At the onset of ischemia intracellular pH (pH) decreases due to anaerobic metabolism and ATP hydrolysis, leading to an activation of Na/H exchange. This in turn increases intracellular Na (Na) and activates Na/K ATPase, with a consecutive increase of energy consumption. Since cellular Na and Ca transport are coupled by the Na/Ca exchange system, which depends on the Na gradient, the high Na leads to increased intracellular Ca (Ca). After a certain period, Na/H exchange is inactivated by a decrease of extracellular pH. In case of reperfusion the acid extracellular fluid is washed out, which reactivates Na/H exchange, leading to an unfavourably fast restoration of pH and a second time to Na and Ca overflow. High Ca is assumed to be one of the main reasons for ischemic and reperfusion injury, like arrhythmias, myocardial contracture, stunning and necrosis. It seems that the inhibition of Na/H exchange can interrupt this process at an early phase and prevent or delay the consequences of ischemia and reperfusion as demonstrated by numerous investigators. [ABSTRACT FROM AUTHOR]

Isolated rat hearts were made ischemic for 25 min after an initial recirculating perfusion, followed by 30 min of reperfusion. In some hearts, interventions including administration of ouabain and/or high [K] in the buffer were performed during the first 10 min of reperfusion. During ischemia, intracellular Na (Na) increased from 15 to 64 [μmol/g dry weight (dwt). During reperfusion, Na declined rapidly (at 10 min of reperfusion: 48 μnol/g dwt, at 30 min: 25 μ mol/g dwt) and regular rhythm was recovered within 10 min in hearts without any intervention during reperfusion.Ca uptake increased from 0.8 to 7.5 μmol/g dwt after 30 min of reperfusion. Ventricular function recovered by 45 %. A 10-min perfusion with 10 or 50 μM of ouabain increased Na (17 to 21 or 27 μmol/g dwt) with increased left-ventricular (LV) contractile function, but these effects were reversed by combination of high perfusate [K] (20 mM) in non-ischemic hearts. A 10-min reperfusion with ouabain retarded or stopped the decline in Na (at 10 min of reperfusion: 54 or 63 μmol/g dwt, at 30 min: 32 or 40 μmol/g dwt). These amounts of ouabain also increased the incidence of ventricular tachyarrhythmias during reperfusion to 30 % or 50 %, and increased the duration of ventricular fibrillation from 6.5 to 11.5 or 18.0 min.Ca uptake reached to 8.8 or 10.0 μmol/g dwt, and function recovered only 35 % or 28 %. When high perfusate [K] was combined with ouabain during reperfusion, the retarded decline in Nai, augmentedCa uptake, and reduced recovery of function caused by ouabain alone were attenuated. These results suggest that digitalis has toxic effects on reperfused ischemic hearts by inhibition of rapid active outward transport of previously elevated Na and potentiation of Ca overload. [ABSTRACT FROM AUTHOR]

The cellular regulation mechanism of Na-K-Cl cotransport was studied in dispersed acinar cells of the guinea pig nasal gland by a microfluorimetric imaging method using the Na-sensitive dye sodium-binding benzofuran isophthalate. Addition of 1 μ m acetylcholine (ACh) induced an immediate increase in intracellular Na concentration ([Na]i) by 36.7±9.9 m m, which was almost completely abolished by the addition of atropine. The increased [Na]i after cholinergic stimulation was due to the external Cl-dependent cotransport system (about 80% of the total Na influx) and the dimethyl amiloride-sensitive Na-H exchange system (of about 20%). The ACh-induced increase in [Na]i was dependent on extracellular Ca and was prevented by pretreatment with 8-(N, N-diethylamino)octyl-3,4,5-trimethoxybenzoate or O-O′-bis(2-aminophenyl)ethyleneglycol-N, N, N′, N′-tetraacetic acid tetraacetoxymethyl ester. Addition of 1μ m ionomycin mimicked the ACh-induced increase in [Na]i which was dependent on external Cl. Moreover, both a calmodulin antagonist trifluoperazine and a myosin light chain kinase inhibitor ML-7 reduced the ACh-induced response in [Na]i. However, the following treatment did not affect the basal [Na]i nor the ACh-induced increase in [Na]i: (i) addition of dibutyryl cAMP, 8-Br-cGMP, or phorbol 12-myristate 13-acetate, (ii) pretreatment of protein kinase inhibitors, H-89, H-8, H-7 or chelerythrine, (iii) prevention of cytosolic Cl efflux by the addition of diphenylamine-2-carboxylic acid or, (iv) prevention of cytosolic K efflux by the addition of charybdotoxin. The present results suggest that the ACh-induced increase in [Na]i, mainly responsible for activation of Na-K-Cl cotransport, is mediated by a Ca/calmodulin-dependent phosphorylation. [ABSTRACT FROM AUTHOR]

The present studies were conducted to investigate the mechanisms underlying the 1,25-dihydroxycholecalciferol (1,25(OH)2D3)-induced increase in intracellular Ca2+ ([Ca2+]i) in individual CaCo-2 cells. In the presence of 2 mM Ca2+, 1,25(OH)2D3-induced a rapid transient rise in [Ca2+]i in Fura-2-loaded cells in a concentration-dependent manner, which decreased, but did not return to baseline levels. In Ca(2+)-free buffer, this hormone still induced a transient rise in [Ca2+]i, although of lower magnitude, but [Ca2+]i then subsequently fell to baseline. In addition, 1,25(OH)2D3 also rapidly induced 45Ca uptake by these cells, indicating that the sustained rise in [Ca2+]i was due to Ca2+ entry. In Mn(2+)-containing solutions, 1,25(OH)2D3 increased the rate of Mn2+ influx which was temporally preceded by an increase in [Ca2+]i. The sustained rise in [Ca2+]i was inhibited in the presence of external La3+ (0.5 mM). 1,25(OH)2D3 did not increase Ba2+ entry into the cells. Moreover, neither high external K+ (75 mM), nor the addition of Bay K 8644 (1 microM), an L-type, voltage-dependent Ca2+ channel agonist, alone or in combination, were found to increase [Ca2+]i. 1,25(OH)2D3 did, however, increase intracellular Na+ in the absence, but not in the presence of 2 mM Ca2+, as assessed by the sodium-sensitive dye, sodium-binding benzofuran isophthalate. These data, therefore, indicate that CaCo-2 cells do not express L-type, voltage-dependent Ca2+ channels. 1,25(OH)2D3 does appear to activate a La(3+)-inhibitable, cation influx pathway in CaCo-2 cells. [ABSTRACT FROM AUTHOR]

Single Na-activated K-channels were recorded from intact guinea-pig ventricular myocytes perfused with Ca-free, Mg-free Tyrode, to increase [Na]. The activity of these channels increases with the time of perfusion with this solution and decreases when the bathing sodium is reduced. The activity of the Na-activated K-channel is greater in the cell attached patches than from excised cell free patches, at the same [Na]. It is suggested that this K channel is significantly active in intact cells within the physiological range of [Na]. [ABSTRACT FROM AUTHOR]