Intracellular Na+ modulates large conductance Ca2+-activated K+ currents in human umbilical vein endothelial cells

We studied the effects of Na⁺ influx on large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels in cultured human umbilical vein endothelial cells (HUVECs) by means of patch clamp and SBFI microfluorescence measurements. In current-clamped HUVECs, extracellular Na⁺ replacement by NMDG⁺ or mannitol hyperpolarized cells. In voltage-clamped HUVECs, changing membrane potential from 0 mV to negative potentials increased intracellular Na⁺ concentration ([Na⁺]_i) and vice versa. In addition, extracellular Na⁺ depletion decreased [Na⁺]_i. In voltage-clamped cells, BK_Ca currents were markedly increased by extracellular Na⁺ depletion. In inside-out patches, increasing [Na⁺]_i from 0 to 20 or 40 mM reduced single channel conductance but not open probability (NPo) of BK_Ca channels and decreasing intracellular K⁺ concentration ([K⁺]_i) gradually from 140 to 70 mM reduced both single channel conductance and NPo. Furthermore, increasing [Na⁺]_i gradually from 0 to 70 mM, by replacing K⁺, markedly reduced single channel conductance and NPo. The Na⁺–Ca²⁺ exchange blocker Ni²⁺ or KB-R7943 decreased [Na⁺]_i and increased BK_Ca currents simultaneously, and the Na⁺ ionophore monensin completely inhibited BK_Ca currents. BK_Ca currents were significantly augmented by increasing extracellular K⁺ concentration ([K⁺]_o) from 6 to 12 mM and significantly reduced by decreasing [K⁺]_o from 12 or 6 to 0 mM or applying the Na⁺–K⁺ pump inhibitor ouabain. These results suggest that intracellular Na⁺ inhibit single channel conductance of BK_Ca channels and that intracellular K⁺ increases single channel conductance and NPo. GH Liang and MY Kim contributed equally to this publication and therefore share the first authorship.     

Small-conductance Ca2+-activated K+ channels in bovine chromaffin cells

Simultaneous whole-cell patch-clamp and fura-2 fluorescence [Ca²⁺]_i measurements were used to characterize Ca²⁺-activated K⁺ currents in cultured bovine chromaffin cells. Extracellular application of histamine (10 M) induced a rise of [Ca²⁺]_i concomitantly with an outward current at holding potentials positive to –80 mV. The activation of the current reflected an increase in conductance, which did not depend on membrane potential in the range –80 mV to –40 mV. Increasing the extracellular K⁺ concentration to 20 mM at the holding potential of –78 mV was associated with inwardly directed currents during the [Ca²⁺]_i elevations induced either by histamine (10 M) or short voltage-clamp depolarizations. The current reversal potential was close to the K⁺ equilibrium potential, being a function of external K⁺ concentration. Current fluctuation analysis suggested a unit conductance of 3–5 pS for the channel that underlies this K⁺ current. The current could be blocked by apamin (1 M). Whole-cell current-clamp recordings snowed that histamine (10 M) application caused a transient hyperpolarization, which evolved in parallel with the [Ca²⁺]_i changes. It is proposed that a small-conductance Ca²⁺-activated K⁺ channel is present in the membrane of bovine chromaffin cells and may be involved in regulating catecholamine secretion by the adrenal glands of various species.     

Localized L-type calcium channels control exocytosis in cat chromaffin cells

Depolarizing 1-s pulses to 0 mV from a holding potential of −70 mV, induced whole-cell currents through Ca²⁺ channels (I _Ca) in patch-clamped cat adrenal medulla chromaffin cells. The dihydropyridine (DHP) furnidipine (3 μM) reduced the peak current by 47% and the late current by 80%. ω-Conotoxin GVIA (CgTx, 1 μM) reduced the peak I _Ca by 42% and the late I _Ca by 55%. Pulses (10 s duration) with 70 mM K⁺/2.5 mM Ca²⁺ solution (70 K⁺/2.5 Ca²⁺), applied to single fura-2-loaded cat chromaffin cells increased the cytosolic Ca²⁺ concentration ([Ca²⁺]_i from 0.1 to 2.21 μM; this increase was reduced by 43.7% by furnidipine and by 42.5% by CgTx. In the perfused cat adrenal gland, secretion evoked by 10-s pulses of 70 K⁺/2.5 Ca²⁺ was reduced by 25% by CgTx and by 96% by furnidipine. Similar results were obtained when secretion from superfused isolated cat adrenal chromaffin cells was studied and when using a tenfold lower [Ca²⁺]_o. The results are compatible with the existence of DHP-sensitive (L-type) as well as CgTx-sensitive (N-type) voltage-dependent Ca²⁺ channels in cat chromaffin cells. It seems, howevever, that though extracellular Ca²⁺ entry through both channel types leads to similar increments of averaged [Ca²⁺]_i, the control of catecholamine release is dominated only by Ca²⁺ entering through L-type Ca²⁺ channels. This supports the idea of a preferential segregation of L-type Ca²⁺ channels to localized “hot spots” in the plasmalemma of chromaffin cells where exocytosis occurs.     

Intracellular Ca2+ inactivates an outwardly rectifying K+ current in human adenomatous parathyroid cells

We have used whole-cell patch-clamp techniques to study the conductances in the plasma membranes of human parathyroid cells. With a KCl-rich pipette solution containing Ca²⁺ buffered to a concentration of 0.1 mol/l, the zero current potential was –71.1±0.5 mV (n=19) and the whole-cell current/ voltage (I/V) relation had an inwardly rectifying and an outwardly rectifying component. The inwardly rectifying current activated instantaneously on hyperpolarization of the plasma membrane to potentials more negative than –80 mV, and a semi-logarithmic plot of the reversal potential of the inward current (estimated by extrapolation from the range in which it was linear) as a function of extracellular K⁺ concentration ([K⁺]_o) revealed a linear relation with a slope of 64 mV per decade change in [K⁺]_o, which is not significantly different from the Nernstian slope, demonstrating that the current was carried by K⁺ ions. The conductance exhibited a square root dependence on [K⁺]_o as has been observed for inward rectifiers in other tissues. The current was blocked by the presence of Ba²⁺ (1 mmol/l) or Cs⁺ (1.5 mmol/l) in the bath. The outwardly rectifying current was activated by depolarization of the membrane potential to potentials more positive than –20 mV. It was inhibited by replacement of pipette K⁺ with Cs⁺, indicating that it also was a K⁺ current: it was partially (42%) blocked when tetraethylammonium (TEA⁺, 10 mmol/l) was added to the bath. The outwardly rectifying, but not the inwardly rectifying K⁺ current, was regulated by intracellular free Ca²⁺ concentration ([Ca²⁺]_i) such that increasing [Ca²⁺]_i above 10 nmol/l inhibited the outwardly rectifying current, the half-maximum effect being seen at 1 mol/l. Since it is known that increases in [Ca²⁺]_o produce increases in [Ca²⁺]_i, and that they depolarize parathyroid cells by reducing the membrane K⁺ conductance, we suggest that it is the reduction of the outwardly rectifying K⁺ conductance by increases in [Ca²⁺]_i which is responsible for the reduction in K⁺ conductance seen when [Ca²⁺]_o is increased.     

A rapidly inactivating Ca2+-dependent K+ current in pheochromocytoma cells (PC12) of the rat

The membrane electrical properties of undifferentiated pheochromocytoma cells of the rat (PC12) were studied using both current-and voltage-clamp techniques with the use of low-resistance blunt-tipped micropipettes (patch electrodes). In the presence of tetrodotoxin (TTX, 2–3 M), a spike-like wave form with a prominent after-hyperpolarization (AHP) was recorded following brief (< 10 ms) depolarizing current pulses. The inorganic divalent cations, Cd²⁺ (0.5 mM), Mn²⁺ (4mM), and 0 mM Ca²⁺/4 mM Mg²⁺ solution prolonged the duration, attenuated the AHP, slowed the rate of repolarization, and slightly enhanced the amplitude of this wave form. A rapidly inactivating outward current was recorded in over 70% of the cells under voltage-clamp conditions. This transient current was elicited at about ±30 mV, and was blocked by tetraethylammonium (5 mM), inorganic divalent cations (Cd²⁺, 0.5 mM; Mn²⁺, 4 mM; Ba²⁺, 3 mM), and removal of Ca²⁺ (0 mM Ca²⁺/4 mM Mg²⁺) from the local perfusion medium. In addition, 4-aminopyridine (5 mM), which blocks the transient outward K⁺ current I_A in a variety of excitable cells, did not have any appreciable effect on this rapidly inactivating current. Moreover, it was possible to elicit the current at a holding potential of ±40 mV. The reversal potential of this current was ±90 mV, and shifted positively when extracellular K⁺ concentrations were elevated. It is concluded that PC12 cells have a rapidly inactivating Ca²⁺ -dependent K⁺ current. A possible explanation for the transient nature of this current may be the presence of an effective intracellular Ca²⁺ buffering (uptake or extrusion) system.     

A Na+-activated K+ current (I K,Na) is present in guinea-pig but not rat ventricular myocytes

 The effects of removing extracellular Ca²⁺ and Mg²⁺ on the membrane potential, membrane current and intracellular Na⁺ activity (a ⁱ _Na) were investigated in guinea-pig and rat ventricular myocytes. Membrane potential was recorded with a patch pipette and whole-cell membrane currents using a single-electrode voltage clamp. Both guinea-pig and rat cells depolarize when the bathing Ca²⁺ and Mg²⁺ are removed and the steady-state a ⁱ _Na increases rapidly from a resting value of 6.4± 0.6 mM to 33±3.8 mM in guinea-pig (n=9) and from 8.9±0.8 mM to 29.3±3.0 mM (n=5) in rat ventricular myocytes. Guinea-pig myocytes partially repolarized when, in addition to removal of the bathing Ca²⁺ and Mg²⁺, K⁺ was also removed, however rat cells remained depolarized. A large diltiazem-sensitive inward current was recorded in guinea-pig and rat myocytes, voltage-clamped at –20 mV, when the bathing divalent cations were removed. When the bathing K⁺ was removed after Ca²⁺ and Mg²⁺ depletion, a large outward K⁺ current developed in guinea-pig, but not in rat myocytes. This current had a reversal potential of –80±0.7 mV and was not inhibited by high Mg²⁺ or glybenclamide indicating that it is not due to activation of non-selective cation or adenosine triphosphate (ATP)-sensitive K channels. The current was not activated when Li⁺ replaced the bathing Na⁺ and was blocked by R-56865, suggesting that it was due to the activation of K_Na channels. Received: 15 October 1998 / Received after revision: 22 January 1999 / Accepted: 5 February 1999     

Mechanisms of reduced mitochondrial Ca2+ accumulation in failing hamster heart

Mitochondrial Ca²⁺ plays important roles in the regulation of energy metabolism and cellular Ca²⁺ homeostasis. In this study, we characterized mitochondrial Ca²⁺ accumulation in Syrian hamster hearts with hereditary cardiomyopathy (strain BIO 14.6). Exposure of isolated mitochondria from 70 nM to 30 μM Ca²⁺ ([Ca²⁺]_o) caused a concentration-dependent increase in intramitochondrial Ca²⁺ concentrations ([Ca²⁺]_m). The [Ca²⁺]_m was significantly lower in cardiomyopathic (CMP) hamsters than in healthy hamsters when [Ca²⁺]_o was higher than 1 μM and a decrease of about 52% was detected at [Ca²⁺]_o of 30 μM (916 ± 67 nM vs 1,932 ± 132 nM in control). A possible mechanism responsible for the decreased mitochondrial Ca²⁺ uptake in CMP hamsters is the depolarization of mitochondrial membrane potential (Δψ _m). Using a tetraphenylphosphonium (TPP⁺) electrode, the measured Δψ _m in failing heart mitochondria was −136 ± 1.5 mV compared with −159 ± 1.3 mV in controls. Analyses of mitochondrial respiratory chain demonstrated a significant impairment of complex I and complex IV activities in failing heart mitochondria. In summary, a less negative Δψ _m resulting from defects in the respiratory chain may lead to attenuated mitochondrial Ca²⁺ accumulation, which in turn may contribute to the depressed energy production and myocardial contractility in this model of heart failure. In addition to other known impairments of ion transport in sarcoplasmic reticulum and plasma membrane, results from this paper on mitochondrial dysfunctions expand our understanding of the molecular mechanisms leading to heart failure.     

The thapsigargin-evoked increase in [Ca2+]i involves an InsP3-dependent Ca2+ release process in pancreatic acinar cells

In pancreatic acinar cells, as in many other cell types, the tumour promoter thapsigargin (TG) evokes a significant increase of intracellular free Ca²⁺ ([Ca²⁺]_i). The increases of [Ca²⁺]_i evoked by TG was associated with significant changes of plasma membrane Ca²⁺ permeability, with [Ca²⁺]_i values following changes in extracellular [Ca²⁺]. Plasma membrane Ca²⁺ extrusion is activated rapidly as a consequence of the rise in [Ca²⁺]_i evoked by TG and the rate of extrusion is linearly dependent on [Ca²⁺]_i up to 1 μM Ca²⁺. In contrast, the activation of the Ca²⁺ entry pathway is delayed and the apparent rate of Ca²⁺ entry is independent of [Ca²⁺]_i. In the presence of 20 mM caffeine, which reduces the resting levels of inositol trisphosphate (InsP₃), the increase of [Ca²⁺]_i evoked by TG was significantly reduced. The reduction was manifest both as a decrease of the amplitude of the [Ca²⁺]_i peak (30% reduction) and, more importantly, as a reduction of the apparent maximal rate of [Ca²⁺]_i increase (from 12.3±1.0 to 6.1±0.6 nM Ca²⁺/s). The inhibition evoked by caffeine was reversible and the removal of caffeine in the continuous presence of TG evoked a further increase of [Ca²⁺]_i. The amplitude of the [Ca²⁺]_i increase upon caffeine removal was reduced as a function of the time of TG exposure. Addition of TG in the presence of 1 mM La³⁺, which is known to inhibit the plasma membrane Ca²⁺-activated adenosine triphosphatase, induced a much higher peak of [Ca²⁺]_i. This increase was associated with an augmentation of the apparent rate of [Ca²⁺]_i increase (from 12.3±1.2 to 16.1±1.9 nM Ca²⁺/s). The inhibitory effect of caffeine, as well as the increase in [Ca²⁺]_i observed on caffeine removal was not affected by the presence of 1 mM La³⁺. These data indicate that an important component of the TG-evoked [Ca²⁺]_i increase is due to InsP₃-sensitive Ca²⁺ release which is probably mediated by the resting levels of InsP₃.     

Slow spontaneous [Ca2+]i oscillations reflect nucleotide release from renal epithelia

Renal epithelia can be provoked mechanically to release nucleotides, which subsequently increases the intracellular Ca²⁺ concentration [Ca²⁺]_i through activation of purinergic (P2) receptors. Cultured cells often show spontaneous [Ca²⁺]_i oscillations, a feature suggested to involve nucleotide signalling. In this study, fluo-4 loaded Madin–Darby canine kidney (MDCK) cells are used as a model for quantification and characterisation of spontaneous [Ca²⁺]_i increases in renal epithelia. Spontaneous [Ca²⁺]_i increases occurred randomly as single cell events. During an observation period of 1 min, 10.9 ± 6.7% (n = 23) of the cells showed spontaneous [Ca²⁺]_i increases. Spontaneous adenosine triphosphate (ATP) release from MDCK cells was detected directly by luciferin/luciferase. Scavenging of ATP by apyrase or hexokinase markedly reduced the [Ca²⁺]_i oscillatory activity, whereas inhibition of ecto-ATPases (ARL67156) enhanced the [Ca²⁺]_i oscillatory activity. The association between spontaneous [Ca²⁺]_i increases and nucleotide signalling was further tested in 132–1N1 cells lacking P2 receptors. These cells hardly showed any spontaneous [Ca²⁺]_i increases. Transfection with either hP2Y₆ or hP2Y₂ receptors revealed a striking degree of oscillations. Similar spontaneous [Ca²⁺]_i increases were observed in freshly isolated, perfused mouse medullary thick ascending limb (mTAL). The oscillatory activity was reduced by basolateral apyrase and substantially lower in mTAL from P2Y₂ knock out mice (0.050 ± 0.020 events per second, n = 8) compared to the wild type (0.147 ± 0.018 events per second, n = 9). These findings indicate that renal epithelia spontaneously release nucleotides leading to P2-receptor-dependent [Ca²⁺]_i oscillations. Thus, tonic nucleotide release is likely to modify steady state renal function. C. S. Geyti and E. Odgaard contributed equally to the publication.     

[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.     

Hypoxia increases the activity of Ca2+-sensitive K+ channels in cat cerebral arterial muscle cell membranes

The cellular mechanisms mediating hypoxia-induced dilation of cerebral arteries have remained unknown, but may involve modulation of membrane ionic channels. The present study was designed to determine the effect of reduced partial pressure of O₂, PO ₂, on the predominant K⁺ channel type recorded in cat cerebral arterial muscle cells, and on the diameter of pressurized cat cerebral arteries. A K⁺-selective single-channel current with a unitary slope conductance of 215 pS was recorded from excised inside-out patches of cat cerebral arterial muscle cells using symmetrical KCl (145 mM) solution. The open state probability (NP _o) of this channel displayed a strong voltage dependence, was not affected by varying intracellular ATP concentration [(ATP]_i) between 0 and 100 M, but was significantly increased upon elevation of intracellular free Ca²⁺ concentration ([Ca²⁺]_i). Low concentrations of external tetraethylammonium (0.1–3 mM) produced a concentration-dependent reduction of the unitary current amplitude of this channel. In cell-attached patches, where the resting membrane potential was set to zero with a high KCl solution, reduction of O₂ from 21% to < 2% reversibly increased the NP _o, mean open time, and event frequency of the Ca²⁺-sensitive, high-conductance single-channel K⁺ current recorded at a patch potential of + 20 mV. A similar reduction in PO₂ also produced a transient increase in the activity of the 215-pS K⁺ channel measured in excised inside-out patches bathed in symmetrical 145 mM KCl, an effect which was diminished, or not seen, during a second application of hypoxic superfusion. Hypoxia had no effect on [Ca²⁺]_i or intracellular pH (pH_i) of cat cerebral arterial muscle cells, as measured using Ca²⁺- or pH-sensitive fluorescent probes. Reduced PO₂ caused a significant dilation of pressurized cerebral arterial segments, which was attenuated by pre-treatment with 1 mM tetraethylammonium. These results suggest that reduced PO₂ increases the activity of a high-conductance, Ca²⁺-sensitive K⁺ channel in cat cerebral arterial muscle cells, and that these effects are mediated by cytosolic events independent of changes in [Ca²⁺]_i and pH_i.     

Direct measurements of Ca2+-activated K+ currents in inner hair cells of the guinea-pig cochlea using photolabile Ca2+ chelators

Intracellular photorelease of Ca²⁺ from caged Ca²⁺ (DM-nitrophen or nitr5) and the patch-clamp technique in the whole-cell configuration were used to investigate Ca²⁺-activated currents in inner hair cells (IHCs) of the mammalian cochlea. Photoliberation of intracellular Ca²⁺ activated outward currents with a mean amplitude of 260±110 pA when IHCs were voltage-clamped, near the resting membrane potential, at –50 mV. The photoactivated currents were reversibly blocked by extracellular application of tetraethylammonium (TEA, 10 mM), neomycin (1 mM) and charybdotoxin (1 M), but not by apamin. The voltage dependence of membrane currents activated by photolysis of DM-nitrophen demonstrated a reversal potential near the K⁺ equilibrium potential (E _k) and saturation near 0 mV. The presence of Ca²⁺-activated currents was further confirmed by the effects of extracellular adenosine 5-triphosphate (ATP, 10 M) and the Ca²⁺ ionophore ionomycin (10 M). Both agents raised intracellular Ca²⁺ and simultaneously activated outward currents when IHCs were voltage-clamped near the resting membrane potential. In experiments where currents were activated by depolarizing voltage steps, nifedipine (50 M) and Cd²⁺ (1 mM) reduced significantly (20–50%) the whole-cell outward currents, suggesting the presence of L-type Ca²⁺ currents activating K⁺ currents. These results are the first direct evidence for Ca²⁺-activated K⁺ currents in mammalian IHCs, these currents being potentially important for cell repolarization during sound-induced depolarization and synaptic transmission.     

Intracellular calcium in mammalian brain cells: fluorescence measurements with quin2

Summary Dispersed brain cells from 12–14 day old mouse embryos were loaded with the Ca²⁺-sensitive fluorescent probe, quin2 and shown to have a resting intracellular Ca²⁺ concentration ([Ca²⁺]_i) of 158 nM (SE ± 5) in the presence of 1 mM [Ca²⁺]_o. When external [Ca²⁺] was raised from 0 to 1 mM there was an increase of [Ca²⁺]_i of 70 nM; with further additions of Ca to >10 mM [Ca²⁺]_o the level of [Ca²⁺]_i increased by <25 nM. Releasable intracellular Ca²⁺ stores, estimated from the increase in [Ca²⁺] produced by 4Br A23187 in the absence of extracellular Ca²⁺, were 24 fmol/10⁶ cells. A small increase in [Ca²⁺]_i could be produced by the mitochondrial inhibitor, carbonyl cyanide m-chlorophenylhydrazone (CCCP). When extracellular K⁺ was raised by 10–20 mM, intracellular Ca²⁺ levels increased from 152 (SE ± 7) to 204 nM (SE ± 10). These K⁺-induced increases in [Ca²⁺]_i were blocked by verapamil, did not occur in the absence of extracellular Ca²⁺, and presumably reflect the activation of voltage-dependent Ca²⁺ channels. N-methyl-D-aspartic acid (NMDA) evoked an increase in [Ca²⁺]_i, while the kainate-like lathyrus sativus neurotoxin, L-3-oxalyl-amino-2aminopropionic acid (L-3,2-OAP) did not; this is consistent with previous observations of different and respectively Ca²⁺-dependent and -independent mechanisms of action of these excitatory amino acids.     

Membrane currents and cytoplasmic sodium transients generated by glutamate transport in Bergmann glial cells

Effects of glutamate and kainate (KA) on Bergmann glial cells were investigated in mouse cerebellar slices using the whole-cell configuration of the patch-clamp technique combined with SBFI-based Na⁺ microfluorimetry. l-Glutamate (1 mM) and KA (100 μM) induced inward currents in Bergmann glial cells voltage-clamped at −70 mV. These currents were accompanied by an increase in intracellular Na⁺ concentration ([Na⁺]_i) from the average resting level of 5.2 ± 0.5 mM to 26 ± 5 mM and 33 ± 7 mM, respectively. KA-evoked signals (1) were completely blocked in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 μM), an antagonist of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/KA ionotropic glutamate receptors; (2) reversed at 0 mV, and (3) disappeared in Na⁺-free, N-methyl-D-glucamine (NMDG⁺)-containing solution, but remained almost unchanged in Na⁺-free, Li⁺-containing solution. Conversely, l-glutamate-induced signals (1) were marginally CNQX sensitive (∼10% inhibition), (2) did not reverse at a holding potential of +20 mV, (3) were markedly suppressed by Na⁺ substitution with both NMDG⁺ and Li⁺, and (4) were inhibited by d,l-threo-β-benzyloxyaspartate. Further, d-glutamate, l-, and d-aspartate were also able to induce Na⁺-dependent inward current. Stimulation of parallel fibres triggered inward currents and [Na⁺]_i transients that were insensitive to CNQX and MK-801; hence, we suggested that synaptically released glutamate activates glutamate/Na⁺ transporter in Bergmann glial cells, which produces a substantial increase in intracellular Na+ concentration.     

Intracellular Ca2+ signals in human-derived pancreatic somatostatin-secreting cells (QGP-1N)

Single-cell microfluorimetry techniques have been used to examine the effects of acetylcholine (0.1–100 M) on the intracellular free calcium ion concentration ([Ca²⁺]_i) in a human-derived pancreatic somatostatin-secreting cell line, QGP-1N. When applied to the bath solution, acetylcholine was found to evoke a marked and rapid increase in [Ca²⁺]_i at all concentrations tested. These responses were either sustained, or associated with the generation of complex patterns of [Ca²⁺]_i transients. Overall, the pattern of response was concentration related. In general, 0.1–10 M acetylcholine initiated a series of repetitive oscillations in cytoplasmic Ca²⁺, whilst at higher concentrations the responses consisted of a rapid rise in [Ca²⁺]_i followed by a smaller more sustained increase. Without external Ca²⁺, 100 M acetylcholine caused only a transient rise in [Ca²⁺]_i, whereas lower concentrations of the agonist were able to initiate, but not maintain, [Ca²⁺]_i oscillations. Acetylcholine-evoked Ca²⁺ signals were abolished by atropine (1–10 M), verapamil (100 M) and caffeine (20 mM). Nifedipine failed to have any significant effect upon agonist-evoked increases in [Ca²⁺]_i, whilst 50 mM KCl, used to depolarise the cell membrane, only elicited a transient increase in [Ca²⁺]_i. Ryanodine (50–500 nM) and caffeine (1–20 mM) did not increase basal Ca²⁺ levels, but the Ca²⁺-ATPase inhibitors 2,5-di(tert-butyl)-hydroquinone (TBQ) and thapsigargin both elevated [Ca²⁺]_i levels. These data demonstrate for the first time cytosolic Ca²⁺ signals in single isolated somatostatin-secreting cells of the pancreas. We have demonstrated that acetylcholine will evoke both Ca²⁺ influx and Ca²⁺ mobilisation, and we have partially addressed the subcellular mechanism responsible for these events.     

Stimulatory actions of di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate (di-8-ANEPPS), voltage-sensitive dye, on the BKCa channel in pituitary tumor (GH3) cells

Di-8-ANEPPS (4-{2-[6-(dibutylamino)-2-naphthalenyl]-ethenyl}-1-(3-sulfopropyl)pyridinium inner salt) has been used as a fast-response voltage-sensitive styrylpyridinium probe. However, little is known regarding the mechanism of di-8-ANEPPS actions on ion currents. In this study, the effects of this dye on ion currents were investigated in pituitary GH₃ cells. In whole-cell configuration, di-8-ANEPPS (10 μM) reversibly increased the amplitude of Ca²⁺-activated K⁺ current. In inside-out configuration, di-8-ANEPPS (10 μM) applied to the intracellular surface of the membrane caused no change in single-channel conductance; however, it did enhance the activity of large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels with an EC₅₀ value of 7.5 μM. This compound caused a left shift in the activation curve of BK_Ca channels with no change in the gating charge of these channels. A decrease in mean closed time of the channels was seen in the presence of this dye. In the cell-attached mode, di-8-ANEPPS applied on the extracellular side of the membrane also activated BK_Ca channels. However, neither voltage-gated K⁺ nor ether-à-go-go-related gene (erg)-mediated K⁺ currents in GH₃ cells were affected by di-8-APPNES. Under current-clamp configuration, di-8-ANEPPS (10 μM) decreased the firing of action potentials in GH₃ cells. In pancreatic βTC-6 cells, di-8-APPNES (10 μM) also increased BK_Ca-channel activity. Taken together, this study suggests that during the exposure to di-8-ANEPPS, the stimulatory effects on BK_Ca channels could be one of potential mechanisms through which it may affect cell excitability.     

Free cytosolic calcium during spontaneous contractions in smooth muscle of the guinea-pig mesotubarium

The free intracellular calcium ion concentration ([Ca²⁺]_i) was measured simultaneously with isometric force in strips of guinea-pig mesotubarium using the Fura-2 technique. During the relaxed period (5–15 min) between spontaneous contractions [Ca²⁺]_i continues to decrease after full mechanical relaxation to reach a minimal level of 86±8 nM (n=9) just before the start of the next contraction. During the spontaneous contractions (5–15 min) [Ca²⁺]_i reached a maximum of 211±19 nM and then oscillated between 155±16 nM and 194±9 nM. Increased extracellular Ca²⁺ concentration to 10 mM from the standard concentration of 1.5 mM caused a decreased frequency of spontaneous contractions and an increase in [Ca²⁺]_i both in the relaxed and contracted states. In 10 mM extracellular Ca²⁺, addition of AlF₄ ^–, as 1 mM NaF + 10 M AlCl₃, caused a sustained increase in [Ca²⁺]_i and maintained force. Addition of verapamil (10 M) in this situation decreased [Ca²⁺]_i to the resting level. The results suggest that the cyclic appearance of trains of action potentials is related to variation in [Ca²⁺]_i, possibly via inactivation of Ca²⁺-dependent K⁺ channels.     

Dependence of intracellular free calcium and tension on membrane potential and intracellular pH in single crayfish muscle fibres

The dependence of intracellular free calcium ([Ca²⁺]_i) and tension on membrane potential and intracellular pH (pH_i) was studied in single isolated fibres of the crayfish claw-opener muscle using ion-selective microelectrodes. Tension (T) was quantified as a percentage of the maximum force, or as force per cross-sectional area (N/cm²). In resting fibres, pH_i had a mean value of 7.06. Contractions evoked by an increase extracellular potassium ([K⁺]₀) produced a fall in pH_i of 0.01–0.05 units. The lowest measured levels of resting [Ca²⁺]_i corresponded to a pCa_i (= –log [Ca²⁺]_i) of 6.8. Intracellular Ca²⁺ transients recorded during K⁺-induced contractions did not reveal any distinct threshold for force development. Both the resting [Ca²⁺]_i and resting tension were decreased by an intracellular alkalosis and increased by an acidosis. The sensitivity of resting tension to a change in pH_i (quantified as –dT/ dpH_i) showed a progressive increase during a fall in pH_i within the range examined (pH_i 6.2–7.5). The pH_i/[Ca²⁺]_i and pH_i/tension relationships were monotonic throughout the multiphasic pH_i change caused by NH₄Cl. A fall of 0.5–0.6 units in pH_i did not produce a detectable shift in the pCa_i/tension relationship at low levels of force development. The results indicate that resting [Ca²⁺]_i is slightly higher than the level required for contractile activation. They also show that the dependence of tension on pH_i in crayfish muscle fibres is attributable to a direct H⁺ and Ca²⁺ interaction at the level of Ca²⁺ sequestration and/or transport. Finally, the results suggest that in situ, the effect of pH on the Ca²⁺ sensitivity of the myofibrillar system is not as large as could be expected on the basis of previous work on skinned crustacean muscle fibres.     

Endothelial function in aorta segments of apolipoprotein E-deficient mice before development of atherosclerotic lesions

Acetylcholine (ACh)-induced relaxation declines in apolipoprotein E-deficient (apoE^−/−) mouse aortas, but only after atherosclerotic plaque formation. This study investigated intracellular calcium concentrations [Ca²⁺]_i and changes in phenylephrine-induced contractions as index of baseline nitric oxide (NO) bioavailability before plaque development. Isometric contractions of thoracic aorta rings of young (4 months) apoE^−/− and C57BL/6J (WT) mice were evoked by phenylephrine (3 × 10⁻⁹–3 × 10⁻⁵ M) in the presence and absence of endothelial cells (ECs) or NO synthase (NOS) inhibitors. [Ca²⁺]_i (Fura-2 AM) and endothelium-dependent relaxation were measured at baseline and after ACh stimulation. Segments of apoE^−/− mice were significantly more sensitive and developed more tension than WT segments in response to phenylephrine. The differences disappeared after NOS inhibition or EC removal or upon increasing [Ca²⁺]_i in apoE^−/− strips with 10⁻⁶ M cyclopiazonic acid or 10⁻⁷ M Ca²⁺-ionophore A23187. Expression of endothelial NOS (eNOS) mRNA was similar in apoE^−/− and WT aorta segments. Basal [Ca²⁺]_i was significantly lower in apoE^−/− than in WT strips. Relaxation by ACh (3 × 10⁻⁹–10⁻⁵ M) was time- and dose-dependently related to [Ca²⁺]_i, but neither ACh-induced relaxation nor Ca²⁺ mobilization were diminished in apoE^−/− strips. In conclusion, basal, but not ACh-induced NO bioavailability, was compromised in lesion-free aorta of apoE^−/− mice. Decreased basal NO bioavailability was not related to lower eNOS expression, but most likely related to lower basal [Ca²⁺]_i. These findings further point to important differences between basal and stimulated eNOS activity.     

Factors affecting SOCE activation in mammalian skeletal muscle fibers

Enzymatically dissociated mouse FDB muscle fibers, loaded with Fura-2 AM, were used to study the effect of mitochondrial uncoupling on the capacitative Ca²⁺ entry, SOCE. Sarcoplasmic reticulum (SR) Ca²⁺ stores were depleted by repetitive exposures to high K⁺ or 4-chloro-m-Cresol (4-CmC) in the absence of extracellular Ca²⁺. SR Ca²⁺ store replenishment was substantially reduced using 5 μM cyclopiazonic acid (CPA). Readmission of external Ca²⁺ (5 mM) increased basal [Ca²⁺]_i under two modalities. In mode 1 [Ca²⁺]_i initially increased at a rate of 0.8 ± 0.1 nM/s and later at a rate of 12.3 ± 2.6 nM/s, reaching a final value of 477.8 ± 36.8 nM in 215.7 ± 25.9 s. In mode 2, [Ca²⁺]_i increased at a rate of 0.8 ± 0.1 nM/s to a value of 204.9 ± 20.6 nM in 185.4 ± 21.1 s. FCCP, 2 μM, reduced this Ca²⁺ entry. In nine FCCP-poisoned fibers, the initial rate of Ca²⁺ increase was 0.34 ± 0.1 nM/s (mean ± SEM), reaching a plateau of 149.2 ± 14.1 nM in 217 ± 19 s. The results may likely be explained by the hypothesis that SOCE is inhibited by mitochondrial uncouplers, pointing to a possible mitochondrial role in its activation. Using time-scan confocal microscopy and the dyes CaOr-5N AM or Rhod-2 AM to label mitochondrial Ca²⁺, we show that during depletion [Ca²⁺]_mito initially increases and later diminishes. Finally, we show that the increase in basal [Ca²⁺]_i, associated with SOCE activation, diminishes upon external Na⁺ withdrawal. Na⁺ entry through the SOCE pathway and activation of the reversal of Na⁺/Ca²⁺ exchanger could explain this SOCE modulation by Na⁺.