Effects of diadenosine polyphosphates,ATP and angiotensin II on cytosolic Ca2+ activity and contraction of rat mesangial cells

Diadenosine polyphosphates (Ap _n A) are known to influence cellular Ca²⁺ activity ([Ca²⁺]_i) in several cells. Their vasoactive potency has been described in various systems including the kidney. We examined the effects of diadenosine polyphosphates, adenosine 5-triphosphate (ATP) and angiotensin II (Ang II) on cytosolic Ca²⁺ activity of mesangial cells (MC) in culture obtained from normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. [Ca²⁺]_i was measured as a fluorescence ratio F ₃₄₀/F ₃₈₀ with the fura-2 technique using three excitation wavelengths (340 nm, 360 nm and 380 nm) and a photon counting tube. Resting [Ca²⁺]_i was not significantly different in MC from WKY and SHR rats and was measured as 132±9 nmol/l (n=65) and 114±12 nmol/l (n=36), respectively. Diadenosine polyphosphates (Ap₃A–Ap₆A) increased [Ca²⁺]_i transiently with an initial peak and a secondary plateau phase comparable to the effects of ATP or Ang II. Increases in [Ca²⁺]_i induced by all these agonists were not significantly different between MC of WKY and SHR rats. ATP, Ap₃A, Ap₄A, Ap₅A, Ap₆A (each 5 mol/l) increased the fura-2 fluorescence ratio initially by 0.66±0.09 (n=33), 0.52±0.08 (n=18), 0.25±0.05 (n=16), 0.09±0.06 (n=7), 0.09±0.04 (n=11), respectively. A half-maximal initial increase in the fura-2 fluorescence ratio was reached at 22 nmol/l, 0.9 mol/l, 2.0 mol/l and 4.0 mol/l with Ang II, Ap₃A, ATP and Ap₄A, respectively. Ap₄A (100 mol/l, n=18) led to a reversible contraction of MC. Diadenosine polyphosphates increase [Ca²⁺]_i in rat MC, in a similar manner to ATP or Ang II and lead to a contraction of MC, suggesting that these nucleotides are also involved in the control of glomerular haemodynamics.     

Ruthenium red reduces the Ca2+ sensitivity of Ca2+ uptake into cardiac sarcoplasmic reticulum

 Ruthenium red inhibits mitochondrial Ca²⁺ uptake and is widely used as an inhibitor of ryanodine-sensitive Ca²⁺ channels that function to release Ca²⁺ from the sarcoplasmic reticulum (SR) of muscle cells. It also has effects on other Ca²⁺ channels and ion transporters. To study the effects of ruthenium red on Ca²⁺ transport into the SR of cardiac muscle cells, fluorescence measurements of Ca²⁺ uptake into cardiac SR vesicles were made. Ruthenium red significantly decreased the Ca²⁺ sensitivity of SR uptake in a dose-dependent manner at concentrations ranging from 5 μM to 20 μM. There were no significant effects of ruthenium red on the maximum velocity or the Hill coefficient of SR Ca²⁺ uptake. Received: 14 January 1998 / Received after revision: 12 March 1998 / Accepted: 16 March 1998     

[Ca2+]i elevation evoked by Ca2+ readdition to the medium after agonist-induced Ca2+ release can involve both IP 3-, and ryanodine-sensitive Ca2+ release

 Sustained Ca²⁺ elevation (”Ca²⁺ response”), caused by subsequent readdition of Ca²⁺ to the medium after application of adenosine 5’-triphosphate (ATP, 15 μM) in a Ca²⁺-free medium, was studied using single bovine aortic endothelial (BAE) cells. In cells in which the resting intracellular Ca²⁺ concentration ([Ca²⁺]_i) was between about 50 and 110 nM, a massive Ca²⁺ response occurred and consisted of phasic and sustained components, whereas cells with a resting [Ca²⁺]_i of over 110 nM displayed small plateau-like Ca²⁺ responses. An increase of internal store depletion resulted in loss of the phasic component. When the store was partly depleted, the dependence of the Ca²⁺ response amplitude on resting [Ca²⁺]_i was biphasic over the range of 50 to 110 nM. The greatest degree of store depletion was associated with small monophasic Ca²⁺ responses, the amplitudes of which were almost constant and in the same range as resting [Ca²⁺]_i. Ni²⁺, known to partly block Ca²⁺ entry, caused no change in the half-decay time of [Ca²⁺]_i down to the level of the sustained phase [57 ± 4 s in control and 54 ± 3 s (n = 13) in the presence of 10 mM Ni²⁺] when added at the peak of the phasic component of the Ca²⁺ response. However, it lowered the sustained phase of the Ca²⁺ response by 42%. When applied at the start of the readdition of Ca²⁺, Ni²⁺ blocked the phasic component of the Ca²⁺ response, there being a threefold decrease in the initial rate of [Ca²⁺]_i rise. In cells with a resting [Ca²⁺]_i of 75–80 nM, pre-treatment with ryanodine (10 μM) did not affect the peak amplitude of the Ca²⁺ response, but it did increase the level of the sustained component. In some cells, ryanodine caused an oscillatory Ca²⁺ response. In conclusion, partial depletion of the inositol 1,4,5-trisphosphate-(IP ₃-) sensitive store by a submaximal concentration of agonist (in Ca²⁺-free medium) was followed, on readdition of Ca²⁺, by Ca²⁺ entry, which appeared to trigger IP ₃-sensitive Ca²⁺ release (IICR) which, in turn, initiated Ca²⁺-sensitive Ca²⁺ release (CICR), thus resulting in a massive elevation of [Ca²⁺]_i. Received: 3 July 1996 / Received after revision and accepted: 9 September 1996     

Involvement of Ca2+-induced Ca2+ release in the biphasic Ca2+ response evoked by readdition of Ca2+ to the medium after UTP-induced store depletion in A431 cells

 We have recently shown that the Ca²⁺ response in endothelial cells evoked by readdition of Ca²⁺ to the medium after store depletion caused by a submaximal concentration of agonist can involve Ca²⁺ release from Ca²⁺ stores sensitive to both inositol 1,4,5-trisphosphate and ryanodine. The present experiments were performed to determine whether this mechanism might also exist in other types of cell. For this purpose, we used the human carcinoma cell line A431, which has a varied resting [Ca²⁺]_i. We found that the amplitude of the Ca²⁺ response evoked by Ca²⁺ readdition did not correlate with the amplitude of the preceding UTP-evoked Ca²⁺ release, but did positively correlate with the initial [Ca²⁺]_i. An inspection of the two patterns of response seen in this study (the large biphasic and small plateau-shaped Ca²⁺ responses) revealed that there is an accelerating rise in [Ca²⁺]_i during the biphasic response. Application of ryanodine during the plateau-shaped Ca²⁺ response reversibly transformed it into the biphasic type. Unlike ryanodine, caffeine did not itself evoke Ca²⁺ release, but it caused a further [Ca²⁺]_i rise when [Ca²⁺]_i had already been elevated by thapsigargin. These data suggest that in A431 cells, as in endothelial cells, the readdition of Ca²⁺ after agonist-evoked store depletion can evoke Ca²⁺-induced Ca²⁺ release. This indicates that Ca²⁺ entry may be overestimated by this widely used protocol. Received: 28 July 1997 / Received after revision: 25 November 1997 / Accepted: 26 November 1997     

氟伐他汀调节SHR大鼠血管平滑肌细胞钙泵的表达对血管重构的影响及作用机制

目的探讨氟伐他汀（fluvastatin,Flu）对小牛血清（CS）干预的高血压大鼠胸主动脉血管平滑肌细胞增殖及血管重构的影响并初步探讨其可能的机制。方法CS、Flu和甲羟戊酸（mevalonic acid,Meva）处理血管平滑肌细胞4d,将细胞分为SHR组、SHR＋20％CS组、SHR＋20％CS＋Flu组和SHR＋20％CS＋Flu＋Meva组,采用MTT检测细胞的增殖能力,Transwell检测细胞的迁移能力,体外管样实验、天狼星红染色以及免疫荧光的方法分别检测细胞的管样结构的生成能力、胶原纤维以及F-actin的表达,Western印迹检测PMCA、SERCA和LTCC的表达。结果Flu能够抑制CS促进的VSMCs增殖效应、迁移能力,管样结构生成,胶原蛋白表达,以及骨架蛋白的聚合,并下调VSMCs中PMCA的表达,上调SERCA和LTCC的表达,而Meva则对Flu的作用进行逆转。结论氟伐他汀对CS诱导的VSMCs增殖及血管重构产生抑制,该过程可能是通过调节钙泵的表达实现的,而Meva代谢途径可能在其中发挥了重要的作用。     

ATP-induced Ca2+ signals in bronchial epithelial cells

 Ca²⁺-dependent Cl^– secretion in the respiratory tract occurs physiologically or under pathophysiological conditions when inflammatory mediators are released. The mechanism of intracellular Ca²⁺ release was investigated in the immortalized bronchial epithelial cell line 16HBE14o-. Experiments on both intact and permeabilized cells revealed that only inositol 1,4,5-trisphosphate (InsP ₃) receptors and not ryanodine receptors are involved in intracellular Ca²⁺ release. The expression pattern of the three InsP ₃ receptor isoforms was assessed both at the mRNA and at the protein level. The level of expression at the mRNA level was type 3 (92.5%) >> type 2 (5.4%) > type 1 (2.1%) and this rank order was also observed at the protein level. The ATP-induced Ca²⁺ signals in the intact cell, consisting of abortive Ca²⁺ spikes or fully developed [Ca²⁺] rises and intracellular Ca²⁺ waves, were indicative of positive feedback of Ca²⁺ on the InsP ₃ receptors. Low Ca²⁺ concentrations stimulated and high Ca²⁺ concentrations inhibited InsP ₃-induced Ca²⁺ release in permeabilized 16HBE14o- cells. We localized a cytosolic Ca²⁺-binding site between amino acid residues 2077 and 2101 in the type-2 InsP ₃ receptor and between amino acids 2030 and 2050 in the type-3 InsP ₃ receptor by expressing the respective parts of these receptors as glutathione S-transferase fusion proteins in bacteria. We conclude that the InsP ₃ receptor isoforms expressed in 16HBE14o- cells (mainly type-3 and type-2) are stimulated by Ca²⁺ and that this phenomenon contributes to the ATP-induced Ca²⁺ signals in intact 16HBE14o- cells. Recieved: 11 September 1997 / Received after revision: 2 January 1998 / Accepted: 21 January 1998     

Effect of acidosis on Ca2+-activated K+ channels in cultured porcine coronary artery smooth muscle cells

 Although acidosis induces vasodilation, the vascular responses mediated by large-conductance Ca²⁺-activated K⁺ (K_Ca) channels have not been investigated in coronary artery smooth muscle cells. We therefore investigated the response of porcine coronary arteries and smooth muscle cells to acidosis, as well as the role of K_Ca channels in the regulation of muscular tone. Acidosis (pH 7.3–6.8), produced by adding HCl to the extravascular solution, elicited concentration-dependent relaxation of precontracted, endothelium-denuded arterial rings. Glibenclamide (20 μM) significantly inhibited the vasodilatory response to acidosis (pH 7.3-6.8). Charybdotoxin (100 nM) was effective only at pH 6.9–6.8. When we exposed porcine coronary artery smooth muscle cells to a low-pH solution, K_Ca channel activity in cell-attached patches increased. However, pretreatment of these cells with 10 or 30 μM O, O′-bis(2-aminophenyl)ethyleneglycol-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl)ester (BAPTA-AM), a Ca²⁺ chelator for which the cell membrane is permeable, abolished the H⁺-mediated activation of K_Ca channels in cell-attached patches. Under these circumstances H⁺ actually inhibited K_Ca channel activity. When inside-out patches were exposed to a [Ca²⁺] of 10^–6 M [adjusted with ethyleneglycolbis(β-aminoethylester)-N,N,N′,N′-tetraacetic acid (EGTA) at pH 7.3], K_Ca channels were activated by H⁺ concentration dependently. However, when these patches were exposed to a [Ca²⁺] of 10^–6 M adjusted with BAPTA at pH 7.3, H⁺ inhibited K_Ca channel activity. Extracellular acidosis had no significant direct effect on K_Ca channels, suggesting that extracellular H⁺ exerts its effects after transport into the cell, and that K_Ca channels are regulated by intracellular H⁺ and by cytosolic free Ca²⁺ modulated by acute acidosis. These results indicate that the modulation of K_Ca channel kinetics by acidosis plays an important role in the determination of membrane potential and, hence, coronary arterial tone. Received: 20 January 1998 / Received after revision: 9 April 1998 / Accepted: 22 April 1998     

IP3-induced Ca2+ release in A7r5 vascular smooth-muscle cells represents a partial emptying of the stores and not an all-or-none Ca2+ release of separate quanta

 There is still no agreement on the mechanism of the intracellular action of low concentrations of inositol 1,4,5-trisphosphate (IP₃). Intracellular Ca²⁺ stores may transiently release some Ca²⁺ before they become insensitive to IP₃. Alternatively, stores with a low IP₃ threshold may lose all their Ca²⁺ and the others none. We now report that the IP₃ threshold was not correlated with the extent of Ca²⁺ release in permeabilized A7r5 smooth-muscle cells. In contrast, the maximum rate of release, which was changed either by varying the level of IP₃ receptor (IP₃R) activation, or by changing the concentration of IP₃R at a constant level of IP₃R activation, was directly related to the extent of Ca²⁺ release. We conclude that IP₃-induced Ca²⁺ release reflects partial emptying of the stores and not all-or-none Ca²⁺ release of separate quanta. Received: 22 October 1998 / Received after revision: 15 December 1998 / Accepted: 11 January 1999     

An examination of the role of intracellular ATP in the activation of store-operated Ca2+ influx and Ca2+-dependent capacitance increases in rat basophilic leukaemia cells

 The role of ATP in both the activation of store-operated Ca²⁺ current I _CRAC and in Ca²⁺-dependent vesicular fusion was examined in a study of rat basophilic leukaemia (RBL) cells using the whole-cell patch-clamp technique. Fusion was monitored via changes in plasma membrane capacitance. Following a decrease in the levels of intracellular ATP, achieved using the mitochondrial poison antimycin and the ATP synthase inhibitor oligomycin, as well as a reduction of glycolysis by removal of external glucose, I _CRAC activated in a manner similar to control cells when stores are depleted by dialysis with a pipette solution containing either inositol 1,4,5-trisphosphate (InsP ₃) or ionomycin together with a high concentration of EGTA. Dialysis of cells for 150 s with the non-hydrolysable ATP analogue 5′-adenylylimidodiphosphate (AMP-PNP) (2 mM) in addition to the mitochondrial inhibitors also failed to prevent activation of I _CRAC following external application of ionomycin and thapsigargin, when compared with control recordings obtained with 2 mM ATP instead. Ca²⁺-dependent vesicular fusion was triggered by dialysing cells with 10 μM Ca²⁺ and guanosine-5′-O-(3-thiotriphosphate (GTP[γ-S]). The capacitance increase was unaffected by inhibition of glycolysis, mitochondrial inhibitors or dialysis with either AMP-PNP or adenosine 5′-O-(3-thiotriphosphate) (ATP[γ-S]) instead of ATP. We conclude that ATP hydrolysis does not seem to be necessary for the activation of I _CRAC or for the capacitance increases elicited by high concentrations of intracellular Ca²⁺. Received: 1 May 1998 / Received after revision: 16 July 1998 / Accepted: 16 July 1998     

Estimation of myofibrillar responsiveness to Ca2+ in isolated rat ventricular myocytes

 To estimate myofibrillar responsiveness to Ca²⁺, we used the relation between cell length and intracellular [Ca²⁺] ([Ca²⁺]_i) during tetanic contractions of isolated ventricular myocytes. Enzymatically isolated rat ventricular myocytes were loaded with fura-2 AM (4 μM for 10 min) and excited alternately at 340 nm and 380 nm. The ratio (R) of fura-2 fluorescence at these wavelengths [F(340)/F(380), an index of [Ca²⁺]_i] and cell length (L) were measured simultaneously. Following treatment with thapsigargin (0.2 μM), myocytes were stimulated at 10 Hz for 10 s to produce a tetanic contraction every min and an instantaneous plot of R vs L (R-L trajectory) was constructed. The R-L trajectory followed the same path during cell shortening and re-lengthening, suggesting that dynamic equilibrium between R and L was achieved during tetanus. Increasing the extracellular [Ca²⁺] from 1 to 8 mM extended the R-L trajectory without a substantial shift of the relation. The Ca²⁺-sensitizing thiadiazinone derivative, EMD57033 (1 μM), shifted the R-L trajectory to the left (sensitization of the myofibrils to Ca²⁺), whereas the non-selective phosphodiesterase inhibitor, 3-isobutyl-1-methylxantine (200 μM), shifted the R-L trajectory to the right (desensitization of the myofibrils to Ca²⁺), in agreement with previous results obtained using skinned preparations. We conclude that the R-L trajectory is useful for estimating the myofibrillar responsiveness to Ca²⁺ in isolated myocytes and may be beneficial for the evaluation of inotropic agents. Received: 13 March 1998 / Received after revision: 4 May 1998 / Accepted: 2 June 1998     

Vasopressin-stimulated Ca2+ reabsorption in rabbit cortical collecting system: effects on cAMP and cytosolic Ca2+

 The effect of arginine vasopressin (AVP) on transepithelial Ca²⁺ transport in primary cultures of rabbit cortical collecting system cells was examined. Addition of AVP to the basolateral side of the monolayer dose-dependently (EC₅₀ = 0.7 nM) increased active Ca²⁺ reabsorption from a basal value of 85 ± 2 nmol·h^–1·cm^–2 to a maximum value of 124 ± 3 nmol·h^–1·cm^–2. This was paralleled by a dose-dependent (EC₅₀ = 1.1 nM) increase in cellular adenosine 3′,5′-cyclic monophosphate (cAMP) content. Both effects of AVP were mimicked by the V₂ agonist deamino-Cys,D-Arg⁸-vasopressin (dDAVP) and forskolin. Addition of either AVP or dDAVP to the basolateral side evoked a sustained increase in cytosolic free Ca²⁺ concentration, which resulted from both Ca²⁺ entry and release from internal stores. Only the effect on Ca²⁺ entry was mimicked by forskolin, demonstrating that cAMP acts by activating a Ca²⁺ influx pathway. The present findings demonstrate that AVP stimulates transcellular Ca²⁺ transport in the cortical collecting system through activation of basolateral V₂ receptors coupled to adenylyl cyclase to increase the cellular cAMP content. Received: 4 July 1996 / Received after revision and accepted: 3 September 1996     

Ca2+ entry through cardiac L-type Ca2+ channels modulates β-adrenergic stimulation in mouse ventricular myocytes

 β-adrenergic receptor (β-AR) stimulation increases cardiac L-type Ca²⁺ channel (CaCh) currents via cAMP-dependent phosphorylation. We report here that the affinity and maximum response of CaCh to isoproterenol (Iso), in mouse ventricular myocytes were significantly higher when Ba²⁺ was used as the charge carrier (I _Ba) instead of Ca²⁺ (I _Ca). The EC₅₀ and maximum increase of peak currents were 43.7 ± 7.9 nM and 1.8 ± 0.1-fold for I _Ca and 23.3 ± 4.7 nM and 2.4 ± 0.1-fold for I _Ba. When cells were dialyzed with the faster Ca²⁺ chelator, BAPTA, both sensitivity and maximum response of I _Ca to Iso were significantly augmented compared to cells with EGTA (EC₅₀ of 23.1 ± 5.2 nM and maximal increase of 2.2 ± 0.1-fold). Response of I _Ca to forskolin was also significantly increased when cells were dialyzed with BAPTA or when currents were measured in Ba²⁺. In contrast, depletion of the sarcoplasmic reticulum (SR) Ca²⁺ stores by ryanodine did not alter sensitivity of I _Ca to Iso or forskolin. These results suggest that the Ca²⁺ entering through CaCh regulates cAMP-dependent phosphorylation, and such negative feedback may play a significant role in cellular Ca²⁺ homeostasis and contraction in cardiac cells during β-AR stimulation. Received: 10 December 1997 / Received after revision: 19 January 1998 / Accepted: 21 January 1998     

Effect of uncoupling NO/cGMP pathways on carbachol- and CCK-stimulated Ca2+ entry and amylase secretion from the rat pancreas

 Nitric oxide (NO) production reportedly regulates guanosine 3′,5′-cyclic monophosphate (cGMP) formation and Ca²⁺ influx in pancreatic acini. We have investigated the functional roles of the NO/cGMP messenger system in rat pancreatic acini. In dispersed acini, the levels of amylase secretion, cytosolic [Ca²⁺]([Ca²⁺]_i), NO synthase, and cGMP were measured. The NO synthase inhibitor N ^G-nitro-L-arginine methyl ester (L-NAME, 0.01–100 μM) had no effect on amylase secretion induced by various concentrations of carbachol, cholecystokinin octapeptide (CCK-8) or the high affinity CCK agonist, JMV-180. Similarly, L-NAME up to 100 μM did not affect the changes in Ca²⁺ spiking evoked by these secretagogues; nor was Ca²⁺ entry, refilling or oscillation altered by L-NAME. Sub- and supramaximal concentrations of these secretagogues did not change NO synthase activities compared with basal levels. While sodium nitroprusside (SNP), a NO donor, caused a 9.4-fold increase in cGMP levels compared with basal levels, carbachol, CCK-8 and JMV-180 had no effect. In addition, the guanylate cyclase inhibitor LY 83583 (10 nM to 10 μM) altered neither amylase secretion nor Ca²⁺ signaling induced by these secretagogues. These findings indicate that the stimulatory action of carbachol or CCK-8 is not mediated by NO or cGMP. To investigate whether cGMP stimulates pancreatic secretion we showed that both SNP and a cell-permeant cGMP analog at 0.1–1 mM stimulated amylase secretion and Ca²⁺ transients to a level equal to 10–15% and 13–24%, respectively, of those observed with maximal concentrations of secretagogues. The guanylate cyclase activator guanylin (1–10 μM), which increased cGMP levels 2.4-fold compared with basal levels, elicited a small amount of amylase secretion and a small Ca²⁺ transient. In conclusion, exogenous NO is capable of increasing endogenous cGMP, which results in a modest increase in the [Ca²⁺]_i transient and pancreatic amylase secretion. However, the NO/cGMP system does not appear to be involved significantly in the mediation of Ca²⁺ signaling and amylase secretion stimulated by carbachol and CCK-8. Received: 30 October 1996 / Received after revision and accepted: 13 January 1997     

Effect of chloride on Ca2+ release from the sarcoplasmic reticulum of mechanically skinned skeletal muscle fibres

 The effect of intracellular Cl^– on Ca²⁺ release in mechanically skinned fibres of rat extensor digitorum longus (EDL) and toad iliofibularis muscles was examined under physiological conditions of myoplasmic [Mg²⁺] and [ATP] and sarcoplasmic reticulum (SR) Ca²⁺ loading. Both in rat and toad fibres, the presence of 20 mM Cl^–in the myoplasm increased Ca²⁺ leakage from the SR at pCa (i.e. –log₁₀ [Ca²⁺]) 6.7, but not at pCa 8. Ca²⁺ uptake was not significantly affected by the presence of Cl^–. This Ca²⁺-dependent effect of Cl^– on Ca²⁺ leakage was most likely due to a direct action on the ryanodine receptor/Ca²⁺ release channel, and could influence channel sensitivity and the resting [Ca²⁺] in muscle fibres in vivo. In contrast to this effect, acute addition of 20 mM Cl^– to the myoplasm caused a 40–50% reduction in Ca²⁺ release in response to a low caffeine concentration both in toad and rat fibres. One possible explanation for this latter effect is that the addition of Cl^– induces a potential across the SR (lumen negative) which might reduce Ca²⁺ release via several different mechanisms. Received: 20 October 1997 / Received after revision: 1 December 1997 / Accepted: 2 December 1997     

Intracellular Ca2+ distribution in migrating transformed renal epithelial cells

 Migration of transformed Madin-Darby canine kidney (MDCK-F) cells depends on the polarized activity of a Ca²⁺-sensitive K⁺ channel. We tested whether a gradient of intracellular Ca²⁺ concentration ([Ca²⁺]_i) underlies the horizontal polarization of K⁺ channel activity. [Ca²⁺]_i was measured with the fluorescent dye fura-2/AM. Spatial analysis of [Ca²⁺]_i indicated that a horizontal gradient exists, with [Ca²⁺]_i being higher in the cell body than in the lamellipodium. Resting and maximal levels during oscillations of [Ca²⁺]_i in the cell body were found to be 135 ± 34 and 405 ± 59 nmol/l, respectively, whereas they were 79 ± 18 and 307 ± 102 nmol/l in the lamellipodium. This gradient can partially explain the preferential activation of K⁺ channels in the plasma membrane of the cell body. We applied a local superfusion technique during migration experiments and measurements of [Ca²⁺]_i to test whether its maintenance is due to an uneven distribution of Ca²⁺ influx into migrating MDCK-F cells. Locally superfusing the cell body of migrating MDCK-F cells with La³⁺ alone or together with charybdotoxin, a specific blocker of Ca²⁺-sensitive K⁺ channels, slowed migration to 47 ± 10% and 9 ± 5% of control, respectively. Local blockade of Ca²⁺ influx into the cell body and the lamellipodium with La³⁺ was followed by a decrease of [Ca²⁺]_i at both cell poles. This points to Ca²⁺ influx occurring over the entire cell surface. This conclusion was confirmed by locally superfusing Mn²⁺ over the cell body and the lamellipodium. Fura-2 fluorescence was quenched in both areas, the decrease of fluorescence being two to three times faster in the cell body than in the lamellipodium. However, this difference is insufficient to account for the observed gradient of [Ca²⁺]_i. We hypothesize that the polarized distribution of intracellular Ca²⁺ stores contributes significantly to the generation of a gradient of [Ca²⁺]_i. Received: 22 July 1996 / Received after revision: 17 December 1996 / Accepted: 10 January 1997     

Effect of calmodulin on Ca2+-induced Ca2+ release of skeletal muscle from mutant mice expressing either ryanodine receptor type 1 or type 3

 We analyzed the effects of calmodulin (CaM) on Ca²⁺-induced Ca²⁺ release (CICR) in mouse skeletal muscle cells expressing only ryanodine receptor type 1 (RyR-1) or type 3 (RyR-3) following targeted disruption of one of the RyR genes. Under Mg²⁺-free conditions, CaM potentiated CICR via RyR-3 at low Ca²⁺ concentrations (pCa≥6) but inhibited CICR at high Ca²⁺ concentrations (pCa≤5). On the other hand, CaM potentiated CICR via RyR-1 between pCa 7 and pCa 5. Greater concentrations of CaM were required for potentiation of CICR at pCa 6 than for the inhibition at pCa 5 in the RyR-3-expressing cells. Similarly, higher concentrations of CaM were required for the potentiation of CICR via RyR-1 at pCa 6 than potentiation at pCa 5. In the presence of Mg²⁺ and β,γ-methyleneadenosine 5′-trisphosphate (AMPOPCP), the same differential effects of CaM on the CICR via the different subtypes of RyR were observed. These data suggest that multiple CaM-binding sites are involved in the differential effects on RyR-1 and RyR-3. These effects of CaM are important for the evaluation of the physiological roles of RyRs. Received: 5 May 1998 / Received after revision: 14 August 1998 / Accepted: 3 September 1998     

Effects of cytosolic Ca2+ on the Ca2+ content of the sarcoplasmic reticulum in saponin-permeabilized rat ventricular trabeculae

 Rat ventricular trabeculae were mounted for isometric tension recording, and then permeabilized with saponin. The Ca²⁺ concentration ([Ca²⁺]) within the permeabilized preparation (cytosolic [Ca²⁺]) was monitored continuously using Indo-1 and the integrals of Ca²⁺ transients resulting from brief caffeine application used as an index of the sarcoplasmic reticulum (SR) Ca²⁺ content. The relationship between SR Ca²⁺ content and cytosolic [Ca²⁺] was studied within the reported physiological range (i.e. 50–250 nmol · l^–1 Ca²⁺). Increasing cytosolic [Ca²⁺] from 50 nmol · l^–1 to 250 nmol · l^–1 increased the steady-state SR Ca²⁺ content about threefold. However, increasing [Ca²⁺] above 250 nmol · l^–1 typically resulted in spontaneous SR Ca²⁺ release, with no further increase in SR Ca²⁺ content. The SR Ca²⁺ content increased only slowly when cytosolic [Ca²⁺] was increased; it was unchanged 20 s after a rapid increase in cytosolic [Ca²⁺], but increased progressively to a new steady-state level during the following 1–2 min. In a parallel series of experiments using intact papillary muscles, increasing extracellular [Ca²⁺] (from 0.5 to 5 mmol · l^–1) significantly increased twitch tension within 20 s of the solution change. These results support previous suggestions that the SR Ca²⁺ content may increase when diastolic cytosolic [Ca²⁺] rises during inotropic interventions such as increased stimulus rate or extracellular [Ca²⁺]. However, the rate at which SR Ca²⁺ responds to changes in cytoplasmic [Ca²⁺] within the diastolic range does not appear rapid enough to explain the early potentiation of twitch tension in intact preparations after an increase in extracellular [Ca²⁺]. Received: 26 August 1997 / Accepted: 28 October 1997     

The basolateral Ca2+-dependent K+ channel in rat colonic crypt cells

 Previous studies have indicated that a 16-pS K⁺ channel (KC_ca) in the basolateral membrane is responsible for the acetylcholine-induced whole-cell K⁺ conductance in these cells. In the present study we have examined this channel in excised inside-out patches of the basolateral membrane. Over a wide voltage range this channel showed inward rectification. The Ca²⁺ sensitivity was very marked, with a Hill coefficient of three and with half-maximal activation at 330 nmol/l. After several minutes most channels showed a slow run-down. Channel activity could be refreshed by addition of ATP (1 mmol/l) to the bath solution. The non-metabolizable derivative 5’-adenylylimidodiphosphate (AMP-PNP) had no such effect. In contrast, it inhibited channel activity by some 50%. ATP and its derivatives had no effect on the Ca²⁺ sensitivity. Channels activated by ATP were subsequently studied in the presence of alkaline (10 kU/l) or acidic (1 kU/l) phosphatase. Both phosphatases reduced channel activity significantly. These data suggest that the 16-pS K⁺ channel is directly controlled by cytosolic Ca²⁺. This regulatory step is probably distal to an activation produced by protein-kinase-C-dependent phosphorylation. As is the case for several other K⁺ channels, high concentrations of non-metabolizable ATP analogues inhibit this channel. Received: 23 July 1997 / Accepted: 17 September 1997     

The nuclear envelope of resting C6 glioma cells is able to release and uptake Ca2+ in the absence of chemical stimulation

 Many agonists evoke events in the cell nucleus through the control of Ca²⁺ signals. Recent studies using isolated nuclei have indicated that the nuclear envelope is a store for nuclear Ca²⁺. However, the release of Ca²⁺ directly from the nuclear envelope in living cells has never been reported. In the present study, we have investigated the changes of Ca²⁺ signals at the cyto-nucleoplasmic interface of rat C6 glioma cells using confocal microscopy. Digital imaging indicates that fluo-3, a Ca²⁺-sensitive fluorescent probe, was concentrated in or around the nuclear envelope. Our experiments also revealed that C6 cells at rest produced spontaneous Ca²⁺ spikes in the absence of chemical stimulation. The amplitude of the repetitive Ca²⁺ spikes was higher at the nuclear envelope than in the whole cell or cytosol. After image subtraction, circular rims of Ca²⁺ release and uptake were seen at the outer boundary of the nucleus. When the cells were treated with thapsigargin (2 μM), a specific Ca²⁺-ATPase inhibitor, a long-lasting Ca²⁺ release was observed at the nuclear envelope. Moreover, most of the released Ca²⁺ was directed inwardly to the nucleoplasm with little outward diffusion. Our results thus indicate: (1) that the nuclear envelope is a Ca²⁺ store that possesses the ability to discharge and sequestrate Ca²⁺; and (2) the Ca²⁺-releasing channels are present in the inner nuclear membrane. Received: 21 July 1997 / Received after revision: 22 September 1997 / Accepted: 23 September 1997     

Intracellular cAMP potentiates voltage-dependent activation of L-type Ca2+ channels in rat islet β-cells

 Intracellular cAMP-dependent modulation of L-type Ca²⁺ channel activation in cultured rat islet β-cells has been investigated using the patch-clamp whole-cell current recording mode. The L-type voltage-dependent Ca²⁺ current (I _Ca) showed a fast activation followed by a slow inactivation, and was sensitive to Ca²⁺ channel blockers, for example nifedipine. Application of a cAMP analogue, dibutyryl cyclic AMP (db-cAMP), increased the magnitude of the peak I _Ca in a concentration-dependent manner. Values of the half-activation potentials (V _1/2), taken from activation curves for I _Ca, were –16.7 ± 1.8 and –21.9 ± 3.4 mV (P < 0.05) before and after application of db-cAMP, respectively, with no change of the slope factor (k) or the reversal potential. Pretreatment with a specific protein kinase A antagonist, Rp-cAMP, prevented the potentiating effect of db-cAMP. These results indicate that in rat islet β-cells, phosphorylation of cAMP-dependent kinase potentiates the voltage-dependent activation of L-type Ca²⁺ channels. Received: 9 September 1997 / Received after revision: 19 November 1997 / Accepted: 21 November 1997