Does adrenaline modulate the Na+-Ca2+ exchanger in isolated toad pacemaker cells?

β-Adrenergic stimulation of pacemaker cells from the sinus venosus of the cane toad (Bufo marinus) increases intracellular calcium ([Ca²⁺]_i) and firing rate. The increase in [Ca²⁺]_i could contribute to the increased firing rate by increasing the inward Na⁺-Ca²⁺ exchange current (I _Na-Ca) during diastole. In this study we measured [Ca²⁺]_i and membrane currents in single, isolated, voltage-clamped pacemaker cells. We show that I _Na-Ca increases during β-adrenergic stimulation. To test whether this increase in I _Na-Ca is caused by elevated [Ca²⁺]_i or by changes in the properties of the Na⁺-Ca²⁺ exchanger, we made rapid applications of caffeine and plotted the I _Na-Ca against [Ca²⁺]_i. This relationship was linear during the declining phase of the [Ca²⁺]_i signal caused by caffeine and was not significantly different in the presence or absence of β stimulation. These results show that I _Na-Ca is increased during β-adrenergic stimulation and will contribute to the increased firing rate. However the increase in I _Na-Ca appears to be a consequence of the increase in [Ca²⁺]_i and is not caused by changes in the intrinsic properties of the Na⁺-Ca²⁺ exchanger. Received: 18 January 1999 / Received after revision: 9 April 1999 / Accepted: 22 April 1999     

[Ca2+]i-dependent membrane currents in guinea-pig ventricular cells in the absence of Na/Ca exchange

Transient inward currents (I _ti) during oscillations of intracellular [Ca²⁺] ([Ca²⁺]_i) in ventricular myocytes have been ascribed to Na/Ca exchange. We have investigated whether other Ca²⁺-dependent membrane currents contribute to I _ti in single guinea-pig ventricular myocytes, by examining membrane currents during [Ca²⁺]_i oscillations and during caffeine-induced Ca²⁺ release from the sarcoplasmic reticulum in the absence of Na⁺. Membrane currents were recorded during whole-cell voltage clamp and [Ca²⁺]_i measured simultaneously with fura-2. In the absence of Na/Ca exchange, i.e., with Li⁺, Cs⁺ or N-methyl-D-glucamine (NMDG⁺) substituted for Na⁺, the cell could be loaded with Ca²⁺ by repetitive depolarizations to +10 mV, resulting in spontaneous [Ca²⁺]_i oscillations. During these oscillations, no inward currents were seen, but instead spontaneous Ca²⁺ release was accompanied by a shift of the membrane current in the outward direction at potentials between –40 mV and +60 mV. This [Ca²⁺]_i-dependent outward current shift was not abolished when NMDG⁺ was substituted for internal monovalent cations, nor was it sensitive to substitution of external Cl^–. It was however, sensitive to the blockade of I_Ca by verapamil. These results suggest that the transient outward current shift observed during spontaneous Ca²⁺ release represents [Ca²⁺]_idependent transient inhibition of I _Ca. Similarly, during the [Ca²⁺]_i transients induced by brief caffeine (10 mM) applications, we could not detect membrane currents attributable to a Ca²⁺-activated nonselective cation channel, or to a Ca²⁺-activated Cl^– channel; however, transient Ca²⁺-dependent inhibition of I _Ca was again observed. We conclude that neither the Ca²⁺-activated nonselective cation channel nor the Ca²⁺-activated Cl^– channel contribute significantly to the membrane currents during spontaneous [Ca²⁺]_i oscillations in guineapig ventricular myocytes. However, in the voltage range between –40 mV and +60 mV Ca²⁺-dependent transient inhibition of I _Ca will contribute to the oscillations of the membrane current.     

Activation of Ca2+-sensitive Cl– current by reverse mode Na+/Ca2+ exchange in rabbit ventricular myocytes

 We investigated how Ca²⁺-sensitive transient outward current, I _to(Ca), is activated in rabbit ventricular myocytes in the presence of intracellular Na⁺ (Na⁺ _i) using the whole-cell patch-clamp technique at 36°C. In cells dialysed with Na⁺-free solutions,the application of nicardipine (5 μM) to block L-type Ca²⁺ current (I _Ca) completely inhibited I _to(Ca). In cells dialysed with a [Na⁺]_i≥5 mM, however, I _to(Ca) could be observed after blockade of I _Ca, indicating the activity of an I _Ca-independent component. The amplitude of I _Ca-independent I _to(Ca) increased with voltage in a [Na⁺]_i-dependent manner. The block of Ca²⁺ release from the sarcoplasmic reticulum by caffeine, ryanodine or thapsigargin blocked I _Ca-independent I _to(Ca). In Ca²⁺-free bath solution I _to(Ca) was completely abolished. The application of 2 mM Ni²⁺ or the newly synthesized compound KBR7943, a selective blocker of the reverse mode of Na⁺/Ca²⁺ exchange, or perfusion with pipette solution containing XIP (10 μM), a selective blocker of the exchanger, blocked I _Ca-independent I _to(Ca). From these results we conclude that, in the presence of Na⁺ _i, I _to(Ca) can be activated via Ca²⁺-induced Ca²⁺ release triggered by Na⁺/Ca²⁺ exchange operating in the reverse mode after blockade of I _Ca. Received: 20 January 1998 / Received after revision: 6 July 1998 / Accepted: 25 July 1998     

Calcium receptor message, expression and function decrease in differentiating keratinocytes

Calcium-sensing receptor (CaSR) expression and function were studied in proliferating and differentiating cultured human gingival keratinocytes (HGKs). CaSR mRNA and protein were present in proliferating HGKs cultured in 0.03 mM [Ca²⁺] and decreased in cells induced to differentiate by culturing in 1.2 mM [Ca²⁺] for 2 days. CaSR protein was also detected in gingival tissue. Exposure to 10 mM extracellular [Ca²⁺] activated two sequential whole-cell currents. The first was a small, transient calcium release activated calcium current I_CRAC-like current with an inwardly rectifying I-V curve. The second current was larger with a linear I-V curve. Both currents were significantly decreased in differentiating cells. Neither neomycin nor gadolinium induced changes in whole cell currents nor in intracellular [Ca²⁺], but neomycin inhibited the late large current. Extracellular Ca²⁺ increased intracellular [Ca²⁺] of proliferating HGKs in a dose-dependent fashion. Comparison of the time-courses of the whole-cell currents and the intracellular [Ca²⁺] responses indicated both induced currents supported a Ca²⁺ influx. Extracellular [Mg²⁺] changes did not affect intracellular [Ca²⁺]. La³⁺ and 2-APB inhibited the whole cell current and intracellular [Ca²⁺] changes. The results indicate that the CaSR signaling response likely plays a major role in initiating Ca²⁺ induced differentiation responses in HGKs.     

Effects of caffeine on intracellular calcium,calcium current and calcium-dependent potassium current in anterior pituitary GH3 cells

Caffeine elicits physiological responses in a variety of cell types by triggering the mobilization of Ca²⁺ from intracellular organelles. Here we investigate the effects of caffeine on intracellular Ca²⁺ concentration ([Ca²⁺]_i) and ionic currents in anterior pituitary cells (GH₃) cells. Caffeine has a biphasic effect on Ca²⁺-activated K⁺ current [I _K(Ca)]: it induces a transient increase superimposed upon a sustained inhibition. While the transient increase coincides with a rise in [Ca²⁺]_i, the sustained inhibition of I _K(Ca) is correlated with a sustained inhibition of the L-type Ca²⁺ current. The L-type Ca²⁺ current is also inhibited by other agents that mobilize intracellular Ca²⁺, including thyrotropin releasing hormone (TRH) and ryanodine, but in a matter distinct from caffeine. Unlike the caffeine effect, the TRH-induced inhibition washes-out under whole-cell patch-clamp conditions and is eliminated by intracellular Ca²⁺ chelators. Likewise, the ryanodine-induced inhibition desensitizes while the caffeine-induced inhibition does not. Simultaneous [Ca²⁺]_i and Ca²⁺ current measurements show that caffeine can inhibit Ca²⁺ current without changing [Ca²⁺]_i. Single-channel recordings show that caffeine reduces mean open time without affecting single-channel conductance of L-type channels. Hence the effects of caffeine on ion channels in GH₃ cells are attributable both to mobilization of intracellular Ca²⁺ and to a direct effect on the gating of L-type Ca²⁺ channels.     

Intracellular Mg2+ inhibits the IP3-activated I K(Ca) in NG108-15 cells. [Why intracellular citrate can be useful for recording I K(Ca)]

Receptor-mediated formation of inositol 1,4,5-trisphosphate (IP₃) can induce an outward Ca²⁺-activated K⁺ current [I _K(Ca)] some neural cells. We have investigated I _K(Ca) activated by intracellular injections of IP₃ in whole-cell patch-clamped neuroblastoma×glioma hybrid cells. The current could only be recorded reliably using citrate as the anion in the pipette, but not using acetate, aspartate, chloride, fluoride, gluconate or methylsulphate. This could be attributed to buffering of intracellular Mg²⁺ by citrate. Theoretical calculations suggested free [Mg²⁺] of 1.0 and 0.07 mM respectively in the acetate- and citrate-based recording solutions. Further, IP₃-activated I _K(Ca) could be recorded when the free Mg²⁺ level in the acetate, chloride or methylsulphate solutions was lowered to the range (0.05 mM) calculated for the citrate solution. Thus, raised [Mg²⁺] blocks I _K(Ca). This appeared to be due to inhibition of the response to released Ca²⁺, since high [Mg²⁺] also blocked the response to intracellular injections of Ca²⁺ ions. Mean Mg²⁺ levels in intact neuroblastoma×glioma hybrid cells measured by Mag-Indo-1/AM fluorescence were estimated to be less than 0.14 mM. We therefore conclude that IP₃-induced I _K(Ca) is expressed under normal conditions, but may be subject to regulation by intracellular Mg²⁺.     

Two distinct calcium-activated potassium currents in larval muscle fibres ofDrosophila melanogaster

The non-synaptic membrane currents of muscle fibres have been studied in late embryogenesis ofDrosophila melanogaster using the voltage-clamp technique in wild-type andShaker mutant third instar larvae. Five currents were found in the wild type muscle membrane at this embryonic stage: one fast inward Ca current (I_Ca), two fast outward K currents (I_A and I_Acd) and two slow outward K currents (I_K and I_C). I_Acd and I_C are Ca-dependent.Several procedures were used to separate I_Acd from I_A: I_Acd is present inShaker mutants which are characterized by the absence of I_A (Salkoff and Wyman 1981); I_Acd, but not I_A, is suppressed by Co²⁺ (10 mM) or La³⁺ (1 mM); I_Acd shows steady-state inactivation at more positive potentials than I_A; I_Acd, unlike I_A, is 3,4-diaminopyridine (3,4-DAP) resistant. Furthermore, tetraethylammonium (TEA, 20 mM) which is known to be uneffective on I_A, blocks I_Acd. I_Acd could not be triggered by using strontium or barium as calcium substitutes. By partial substitution of Ca by Ba or Sr ions, it was found that Ba, but not Sr, blocks the I_Acd channel.A non-inactivating, TEA sensitive, Ca-dependent K current (I_C), which gave N-shaped I-V plots, could be separated from I_K by using Ca-channel blockers. I_C and I_K activate at membrane potentials of about –25 mV and –10 mV, respectively.The participation of I_Acd and I_C to membrane electrophysiology is discussed.     

Blockade of HERG channels expressed in Xenopus oocytes by external H+

 We have investigated the effect of external H⁺ concentration ([H⁺]_o)on the human-ether-a-go-go-related gene (HERG) current (I _HERG), the molecular equivalent of the cardiac delayed rectifier potassium current (I _Kr), expressed in Xenopus oocytes, using the two-microelectrode voltage-clamp technique. When [H⁺]_o was increased, the amplitude of the I _HERG elicited by depolarization decreased, and the rate of current decay on repolarization was accelerated. The activation curve shifted to a more positive potential at lower external pH (pH_o) values (the potential required for half-maximum activation, V _1/2, was: –41.8 mV, –38.0 mV, –33.7 mV, –26.7 mV in pH_o 8.0, 7.0, 6.6, 6.2, respectively). The maximum conductance (g _max) was also affected by [H⁺]_o: a reduction of 7.9%, 14.6%, and 22.8% was effected by decreasing pH_o from 8.0 to 7.0, 6.6, and 6.2, respectively. We then tested whether this pH effect was affected by the external Ca²⁺ concentration, which is also known to block HERG channels. When the extracellular Ca²⁺ concentration was increased from 0.5 mM to 5 mM, the shift in V _1/2 caused by increasing [H⁺]_o was attenuated, suggesting that these two ions compete for the same binding site. On the other hand, the decrease in g _max caused by increasing [H⁺]_o was not significantly affected by changing external Ca²⁺ levels. The results indicate that HERG channels are inhibited by [H⁺]_o by two different mechanisms: voltage-dependent blockade (shift of V _1/2) and the decrease in g _max. With respect to the voltage-dependent blockade, the interaction between H⁺ and Ca²⁺ is competitive, whereas for the decreasing g _max, their interaction is non-competitive. Received: 12 January 1999 / Received after revision: 15 February 1999 / Accepted: 16 February 1999     

[Ca2+]i-induced augmentation of the inward rectifier potassium current (IK1) in canine and human ventricular myocardium

The inward rectifier K⁺ current (I_K1) plays an important role in terminal repolarization and stabilization of the resting potential in cardiac cells. Although I_K1 was shown to be sensitive to changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i), the nature of this Ca²⁺ sensitivity—in spite of its deep influence on action potential morphology—is controversial. Therefore, we aimed to investigate the effects of a nonadrenergic rise in [Ca²⁺]_i on the amplitude of I_K1 in canine and human ventricular myocardium and its consequences on cardiac repolarization. I_K1, defined as the current inhibited by 10 μM Ba²⁺, was significantly increased in isolated canine myocytes following a steady rise in [Ca²⁺]_i. Enhanced I_K1 was also observed when [Ca²⁺]_i was not buffered by ethylene glycol tetraacetic acid, and [Ca²⁺]_I transients were generated. This [Ca²⁺]_i-dependent augmentation of I_K1 was largely attenuated after inhibition of CaMKII by 1 μM KN-93. Elevation of [Ca²⁺]_o in multicellular canine and human ventricular preparations resulted in shortening of action potentials and acceleration of terminal repolarization. High [Ca²⁺]_o enhanced the action potential lengthening effect of the Ba²⁺-induced I_K1 blockade and attenuated the prolongation of action potentials following a 0.3-μM dofetilide-induced I_Kr blockade. Blockade of I_Ks by 0.5 μM HMR-1556 had no significant effect on APD₉₀ in either 2 mM or 4 mM [Ca²⁺]_o. It is concluded that high [Ca²⁺]_i leads to augmentation of the Ba²⁺-sensitive current in dogs and humans, regardless of the mechanism of the increase. This effect seems to be at least partially mediated by a CaMKII-dependent pathway and may provide an effective endogenous defense against cardiac arrhythmias induced by Ca²⁺ overload.     

3-Isobutyl-1-methylxanthine (IBMX) affects potassium permeability in rat sensory neurones via pathways that are sensitive and insensitive to [Ca2+]in

The effects of externally applied 3-isobutyl-1-methylxanthine (IBMX), in millimolar concentrations, on the membrane currents in dorsal root ganglia (DRG) neurones isolated from newborn rats were investigated using the amphotericin-based perforated patch-clamp technique. In some experiments, simultaneous measurements of intracellular Ca²⁺ concentration ([Ca²⁺]_in) were performed using fura-2 microfluorimetry. Applications of IBMX induced elevation of [Ca²⁺]_in resulting from Ca²⁺ release from caffeine-ryanodine-sensitive internal stores. In addition to Ca²⁺ release, IBMX produced a biphasic membrane current response comprised of an inward current transiently interrupted by outward current. The onset of the inward current slightly preceded the onset of the [Ca²⁺]_in transient, while the interrupting outward current developed synchronously with the [Ca²⁺]_in rise. The development of IBMX-induced outward current ultimately needed the [Ca²⁺]_in elevation. After the depletion of Ca²⁺ stores by IBMX or caffeine exposure, the subsequent IBMX challenge failed to produce both the [Ca²⁺]_in transient and outward membrane current, although the inward current remained unchanged. Both components of the IBMX-induced membrane current response had a reversal potential close to the K⁺ equilibrium potential and the IBMX-induced membrane current response disappeared while dialysing the cell interior with K⁺-free, Cs⁺-containing solutions suggesting their association with K⁺ channel activity. External administration of 10 mM tetraethylammonium chloride (TEA-Cl) evoked an inward current similar to that observed in response to IBMX; in the presence of TEA-Cl, IBMX application was almost unable to induce additional inward current. IBMX (5 mM) effectively (50%) inhibited K⁺ currents evoked by step depolarizations of membrane potential. We suggest that IBMX affects membrane permeability via activation of Ca²⁺-regulated K⁺ channels and direct inhibition of TEA-sensitive K⁺ channels.     

Decrease in sodium–calcium exchange and calcium currents in diabetic rat ventricular myocytes

This study was designed in order to gain insight into possible changes in the inward sodium–calcium exchange current (I_Na–Ca) and the L -type calcium current (I_Ca), in ventricular myocytes isolated from streptozotocin-induced diabetic rats. Recordings were made using the nystatin-perforated patch technique which minimizes interference with the normal intracellular Ca²⁺ buffering mechanisms. The averaged I_Na–Ca current density elicited by Ca²⁺ current was smaller in diabetic than in normal myocytes at all potentials tested. I_Na–Ca activated by rapid application of caffeine was significantly reduced and the decay phase was prolonged. The density of I_Ca was also significantly reduced by diabetes in the range of test potentials between –10 and +50 mV. In addition, the fast time constant of I_Ca inactivation, which represents mainly the sarcoplasmic reticulum (SR) Ca²⁺ release-induced inactivation, was significantly higher in diabetic than in normal myocytes. The decrease in I_Ca, which is the main source of trigger Ca²⁺ for SR Ca²⁺ release, may explain the significantly lowered peak systolic [Ca²⁺]_i previously shown in diabetic myocytes. As activation of I_Ca is essential for subsequent stimulation of I_Na–Ca, reduced I_Ca may contribute to decreasing activation of the Na⁺–Ca²⁺ exchanger.     

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

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

Effects of calcium release from sarcoplasmic reticulum on membrane currents in guinea pig atrial cardioballs

(1) Ca current (I _Ca) and membrane currents related to Ca-entry during activation ofI _Ca have been studied in cultured atrial myocytes from hearts of adult guinea pigs by means of patch clamp pipettes. The pipettes were filled with solutions containing citrate (65 mM) as major Ca-chelating compound and Cs ions in order to block K currents. (2) In myocytes dialysed with such solutions a monophasic time course of inactivation ofI _Ca is observed, which is 1–2 orders of magnitude slower as compared to studies on intact cardiac cells or cells dialysed with EGTA as only Ca-chelating compound. (3) During long-lasting or repetitive depolarization a second component ofI _Ca inactivation, apart from the slow decay observed in cells dialysed with such solutions, can be seen. This component of inactivation is not related to the depolarization as such but to loading of the cells with Ca²⁺. Whenever the rapid component of inactivation occurs, a transient inward current (I _ti) after repolarization to the holding potential (–40 to –50 mV) is recorded. Both,I _Ca inactivation andI _ti can be mimicked by extracellular application of caffeine (5–10 mM), suggesting both current changes to be caused by a rise in Ca_i due to Ca release from sarcoplasmic reticulum. In the presence of caffeine the rapid component ofI _Ca-inactivation andI _ti are abolished. (4) In addition toI _Ca inactivation and activation ofI _ti sarcoplasmic Ca release causes openings of a novel ion channel with large conductance (>200 pS), the function of which is unknown. (5) The results are consistent with the concept of Ca_i-dependent inactivation of Ca current, which can be caused either by Ca-entry or by Ca-release from the SR. The transient inward current is likely to reflect a process of Ca-removal from the cell, namely Na–Ca exchange.     

Intracellular calcium and electrical restitution in mammalian cardiac cells

The role of calcium current and changes in intracellular calcium concentration ([Ca²⁺]_i) in regulation of action potential duration (APD) during electrical restitution process was studied in mammalian ventricular preparations. Properly timed action potentials were recorded from multicellular preparations and isolated cardiomyocytes using conventional microelectrodes and EGTA-containing patch pipettes. APD increased monotonically in canine and guinea pig ventricular preparations with increasing diastolic interval (DI), while in rabbit papillary muscles the restitution process was biphasic: APD first lengthened, then shortened as the DI increased. When the restitution process was studied in single cardiomyocytes using EGTA-containing patch pipettes, the restitution pattern was similar in the three species studied. Similarly, no difference was observed in the recovery time constant of calcium current (I_Ca-L) measured under these conditions in voltage clamped myocytes. Loading the myocytes with the [Ca²⁺]_i-chelator BAPTA-AM had adverse effects in rabbit and canine cells. In rabbit myocytes steady-state APD lengthened and the late shortening component of restitution was abolished in BAPTA-loaded cells. In canine myocytes BAPTA-load shortened steady-state APD markedly, and during restitution, APD decreased with increasing DI. The late shortening component of restitution, observed in untreated rabbit preparations, was greatly reduced after nifedipine treatment, but remained preserved in the presence of 4-aminopyridine or nicorandil. Beat to beat changes in APD, peak I_Ca-L and [Ca²⁺]_i, measured using the fluorescent dye, Fura-2, were monitored in rabbit ventricular myocytes after a 1-min period of rest. In these cells, the shortening of APD was accompanied by a gradual reduction of the peak I_Ca-L and elevation of diastolic [Ca²⁺]_i during the initial eight post-rest action potentials. It is concluded that elevation of [Ca²⁺]_i shortens, while reduction of [Ca²⁺]_i lengthens APD in rabbit, but not in canine ventricular myocytes. These differences may probably be related to different distributions of [Ca²⁺]_i-dependent ion currents and/or to differences in calcium handling between the two species.     

The effect of extracellular Ca2+ concentration on the negative staircase of Ca2+ transient in field-stimulated rat ventricular cells

We performed experiments using the Ca²⁺ indicator dye, fura-2 to investigate the effect of extracellular Ca²⁺ concentration ([Ca²⁺]_o) on sarcoplasmic reticulum (SR) Ca²⁺ release and loading in single rat ventricular cells. In normal Tyrode solution (1.8 mM [Ca²⁺]_o) repetitive stimulation (0.5 Hz) resulted in a gradual decrease in calcium transients (the negative staircase phenomenon) without being accompanied by a gradual decrease in diastolic intracellular Ca²⁺ concentration. The rate of the slow decline in calcium transient was faster in lower [Ca²⁺]_o. However, the peak of the first calcium transient was relatively invariant over a wide range of [Ca²⁺]_o (0.5–5 mM). The size of the calcium transient elicited by field stimulation was proportional to that induced by 10 mM caffeine, applied following the field stimulation. These results suggest that the size of calcium transients depends mainly on the Ca²⁺ content of the SR. The quiescent period favoured the replenishment of the SR and this effect was promoted further by increasing the driving force for Ca²⁺ entry across the sarcolemma during this period. We conclude that in low [Ca²⁺]_o, short stimulation interval may limit Ca²⁺ influx across the sarcolemma during the quiescent period to cause a gradual reduction in calcium content of the SR and thus the calcium transient.     

Regulation of Ca channel by intracellular Ca2+ and Mg2+ in frog ventricular cells

The effects of changing the intracellular concentrations of Ca²⁺ or Mg²⁺ ([Ca²⁺]_i, [Mg²⁺]_i) on Ca current (I _Ca) was studied in frog ventricular myocytes using the whole-cell and cell-attached patch clamp techniques. In the physiological range of [Mg²⁺]_i an increase in [Ca²⁺]_i enhancedI _Ca whereas at lower [Mg²⁺]_i I _Ca was suppressed. The increase inI _Ca caused by Ca²⁺ loading was not mediated by phosphorylation since the kinase inhibitors H-8 {N-[2-(methylamino)-ethyl]-5-isoquinolinesulphonamide dihydrochloride}, staurosporine and KN-62 {1-[N,O-bis(5-isoquinoline-sulphonyl)-N-methyl-1-tyrosyl]-4-phenylpiperazine} and a non-hydrolysable adenosine 5-triphosphate analogue ,-methyleneadenosine 5-triphosphate did not prevent the Ca²⁺-inducedI _Ca increase.I _Ca was dramatically increased from 10 ± 6 (n = 4) to 71 ± 7 nA/nF (n = 4) when [Mg²⁺]_i was lowered from 1.0 × 10^–3 to 1.0 × 10^–6 M at a [Ca²⁺]_i of 10^–8 M. The concentration response relation for inhibition of Ca channels by [Mg²⁺]_i is modulated by [Ca²⁺]_i. To account for the experimental results it is postulated that competitive binding of Ca²⁺ or Mg²⁺ to the Ca channel accelerates the transition of the channel from an active to a silent mode. Single-channel recordings support this hypothesis. The regulation may have clinical relevance in cytoprotection during cardiac ischaemia.     

Cytosolic free calcium in single microdissected rat cortical collecting tubules

Cytosolic free Ca²⁺ ([Ca²⁺]_i) was measured in single fragments of rat cortical collecting tubule (CCT) by using fura-2 and a tubule superfusion device. Under basal conditions, i.e. with 1 mM of external Ca²⁺ ([Ca²⁺]_o), the average steady state [Ca²⁺]_i was 179±16 nM (n=44 tubules). Random alterations of [Ca²⁺]_o between 0 mM and 4 mM led to corresponding variations in steady state [Ca²⁺]_i levels, which were linearly correlated with [Ca²⁺]_o (average slope 93±34 nM [Ca²⁺]_i per 1 mM [Ca²⁺]_o for six tubules). In contrast, [Ca²⁺]_i was little affected by decreasing external Na⁺ concentration. Cell membrane depolarization with 100 mM of external K⁺ induced a sustained drop in [Ca²⁺]_i (21% as an average). The data suggest that steady state [Ca²⁺]_i in CCT cells resulted from a non-saturable passive entry of calcium ions across cell membranes balanced with an active extrusion by calcium ATPase (pump and leak mechanism). The passive component cannot be accounted for either by Na⁺/Ca²⁺ exchangers nor by voltage-dependent calcium channels; it is best explained by the presence of voltage-independent calcium channels in cell membranes.     

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

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

An analysis of the delayed outward current in single ventricular cells of the guinea-pig

Properties of the delayed outward current (I _K) in ventricular myocytes of the guinea-pig were studied using the whole cell clamp method. The experiments were performed under conditions in whichI _K was enhanced by application of isoproterenol while the Ca²⁺ current was eliminated by Ca²⁺-removal and by the addition of Cd²⁺. The reversal potential (E _rev) ofI _K, determined from the current tails, was about 10 mV less negative than the K⁺ equilibrium potential. This was estimated by examining the reversal potential of the inward rectifier K⁺ current in Ba²⁺-containing solution, or from the Nernst equation. TheE _rev-log[K⁺]₀ relationship had a slope of 49 mV per tenfold change in [K⁺]₀. In Na⁺-free solution,E _rev became more negative. Thus, although the major charge carriers inI _K are K⁺ ions, Na⁺ ions may also contribute in part to this current. TheP _Na/P _K ratio inI _K, calculated by applying a Goldman-Hodgkin-Katz relation to the reversal potential, was 0.016. The activation ofI _K during depolarization showed a sigmoidal time course at the onset, while the time course of the current tails was monoexponential at voltages more negative than –50 mV, but biexponential at more positive voltages. These observations can be explained by the conductance equation of the Hodgkin-Huxley type in which the kinetic variable is raised to the second power. These and other features ofI _K observed in the ventricular cells are discussed in comparison to the properties of similar current systems reported in other cardiac preparations.     

Regulation of membrane excitability by intracellular pH (pHi) changers through Ca2+-activated K+ current (BK channel) in single smooth muscle cells from rabbit basilar artery

Employing microfluorometric system and patch clamp technique in rabbit basilar arterial myocytes, regulation mechanisms of vascular excitability were investigated by applying intracellular pH (pH_i) changers such as sodium acetate (SA) and NH₄Cl. Applications of caffeine produced transient phasic contractions in a reversible manner. These caffeine-induced contractions were significantly enhanced by SA and suppressed by NH₄Cl. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was monitored in a single isolated myocyte and based the ratio of fluorescence using Fura-2 AM (R _340/380). SA (20 mM) increased and NH₄Cl (20 mM) decreased R _340/380 by 0.2 ± 0.03 and 0.1 ± 0.02, respectively, in a reversible manner. Caffeine (10 mM) transiently increased R _340/380 by 0.9 ± 0.07, and the ratio increment was significantly enhanced by SA and suppressed by NH₄Cl, implying that SA and NH₄Cl may affect [Ca²⁺]_i (p < 0.05). Accordingly, we studied the effects of SA and NH₄Cl on Ca²⁺-activated K⁺ current (IK_Ca) under patch clamp technique. Caffeine produced transient outward current at holding potential (V _h) of 0 mV, caffeine induced transient outward K⁺ current, and the spontaneous transient outward currents were significantly enhanced by SA and suppressed by NH₄Cl. In addition, IK_Ca was significantly increased by acidotic condition when pH_i was lowered by altering the NH₄Cl gradient across the cell membrane. Finally, the effects of SA and NH₄Cl on the membrane excitability and basal tension were studied: Under current clamp mode, resting membrane potential (RMP) was −28 ± 2.3 mV in a single cell level and was depolarized by 13 ± 2.4 mV with 2 mM tetraethylammonium (TEA). SA hyperpolarized and NH₄Cl depolarized RMP by 10 ± 1.9 and 16 ± 4.7 mV, respectively. SA-induced hyperpolarization and relaxation of basal tension was significantly inhibited by TEA. These results suggest that SA and NH₄Cl might regulate vascular tone by altering membrane excitability through modulation of [Ca²⁺]_i and Ca²⁺-activated K channels in rabbit basilar artery.