Mechanisms of antagonistic action of internal Ca2+ on serotonin-induced potentiation of Ca2+ currents in Helix neurones

The influence of internal Ca²⁺ ions has been investigated during intracellular perfusion of isolated neurones from pedal ganglia of Helix pomatia in which serotonin (5-HT) induces a cyclic-adenosine-monophosphate-(cAMP)-dependent enhancement of high-threshold Ca²⁺ current (I _Ca). Internal free Ca²⁺ ([Ca²⁺]_i) was varied between 0.01 and 10 M by addition of Ca²⁺-EGTA [ethylenebis(oxonitrilo)tetraacetate] buffer. Elevation of [Ca²⁺]_i depressed the 5-HT effect. The dose/ effect curve for the Ca²⁺ blockade had a biphasic character and could be described by the sum of two Langmuir's isotherms for tetramolecular binding with dissociation constants K _d1=0.063 M and K _d2=1 M. Addition of calmodulin (CM) antagonists (50 M trifluoperazine or 50 M chlorpromazine), phosphodiesterase (PDE) antagonists [100 M isobutylmethylxanthine (IBMX) or 5 mM theophylline] and protein phosphatase antagonists [2 M okadaic acid (OA)] in the perfusion solution caused anticalcium action and modified the Ca²⁺ binding isotherm. Using the effect of OA and IBMX, two components of the total Ca²⁺ inhibition were separated and evaluated. In the presence of one of these blockers tetramolecular curves with K _d1=0.04 M and K _d2=0.69 M were obtained describing the activation of the retained unblocked enzyme — PDE or calcineurin (CN) correspondingly. The sum of these isotherms gave a biphasic curve similar to that in control. Leupeptin (100 M), a blocker of Ca²⁺-dependent proteases did not influence the amplitude of 5-HT effect, indicating that channel proteolysis is not involved in the depression. Our findings show that the molecular mechanism of Ca²⁺-induced suppression of the cAMP-dependent upregulation of Ca²⁺ channels is due to involvement of two Ca²⁺-CM-dependent enzymes: PDE reducing the cAMP level, and CN causing channel dephosphorylation. No other processes are involved in the investigated phenomenon at a Ca²⁺ concentration of less than or equal to 10 M.     

ATP-sensitive K+ channels regulated by intracellular Ca2+ and phosphorylation in normal (T84) and cystic fibrosis (CFPAC-1) epithelial cells

The elementary K⁺ conductance activated by the cAMP or the Ca²⁺ second messenger pathways was investigated in the model salt-secreting epithelium, the human T84 cell line. Under Cl^–-free conditions, an inwardly rectifying whole-cell K⁺ current was evoked by either forskolin 10 (mol/l) or acetylcholine 1 (mol/l) and blocked by extracellular charybdotoxin 10 (nmol/l). In the cell-attached mode, both secretory agonists induced the opening of a channel showing inward rectification with a unitary chord conductance of 36.8±2.5 pS (n=26) for inward currents. In inside-out patches, a 35-pS inwardly rectifying K⁺ channel that corresponded to the channel recorded in the cell-attached configuration was recorded in the presence of 0.3 mol/l free Ca²⁺ at the inner side of the membrane. This channel was blocked by Ba²⁺ (5 mol/l) and by charybdotoxin (50 nmol/l). Its open probability was enhanced by intracellular Ca²⁺ with and EC₅₀ of 0.25 mol/l and strongly reduced by intracellular MgATP with an IC₅₀ of 600 mol/l. In the continuous presence of ATP, the channel activity was consistently increased by 125 kU/l catalytic subunit of cAMP-dependent protein kinase. In the cystic fibrosis pancreatic duct cell line CFPAC-1, a K⁺ channel was also recorded, with similar characterictics and regulation as the 35-pS channel in T84 cells. We conclude that an ATP-sensitive K⁺ channel regulated by intracellular Ca²⁺ and phosphorylation supports the main K⁺ current activated by secretory agonists in normal cystic fibrosis cell lines. This conductance possibly represents the major pathway for K⁺ recycling at the basolateral membrane during transepithelial fluid secretion.     

Intracellular Ca2+ signalling is modulated by K+ channel blockers in colonie epithelial cells (HT-29/B6)

We investigated the inhibitory action of K⁺ channel blockers on carbachol-stimulated Ca²⁺ entry into human Cl^–-secretory colonic epithelial cells (HT-29/B6). Digital imaging of the fluorescent calcium indicator dye fura-2 was performed to monitor effects of K⁺ channel blockers on cytosolic calcium in resting and carbachol-stimulated HT-29/B6 cells. Stimulation with the muscarinic agonist carbachol (100 M) caused a clearly biphasic intracellular calcium (Ca_i response: Ca_i was stimulated from resting levels (85±3 nM, n=100) to a sudden transient peak (821±44 nM) followed by a sustained plateau (317±12 nM). The maintained elevation was dependent on external Ca²⁺ and represented a new steady state between Ca²⁺ entry and exit across the plasma membrane. A monophasic Ca²⁺ response was induced in the absence of external Ca²⁺ and after the initial peak Ca_i returned to baseline. The Ca_i plateau was reduced to resting levels by either the muscarinic antagonist atropine (1 M) or the inorganic Ca²⁺ channel blocker lanthanum (effective concentration for 50% inhibition of Ca₁ plateau EC₅₀=68±18 nM), but it was unaffected by the organic Ca²⁺ channel blockers verapamil and nifedipine. Barium, lidocaine and 4-nitro-2-(3-phenylpropylamino)benzoate (NPPB), well-known blockers of basolateral K⁺ channels of HT-29/B6 cells, rapidly and reversibly reduced carbachol-stimulated Ca²⁺ entry. The Ca_i plateau was calculated to be 50% inhibited by barium (96±2 M), lidocaine (74±3 M) and NPPB (27±10 M). The Ca_i plateau was transiently increased by 1 M and 10 M NPPB to 50% and 34%, respectively, probably via hyperpolarization of the membrane potential by blockade of Cl^– channels (so that the membrane potential approached V _K). The resting Ca_i was transiently increased by 50 M and 300 M NPPB to 308±13 nM and 447±153 nM, respectively, suggesting that NPPB induced a Ca²⁺ release from internal Ca stores. We conclude that carbachol-stimulated Ca²⁺ entry into HT-29/B6 cells (a) requires muscarinic receptor occupation, (b) is highly sensitive to lanthanum and (c) is dependent on membrane potential and therefore inhibited by channel blockers that depolarize the cell potential. Also, the sensitivity of Ca_i levels to K⁺ channel blockers indicates that there are feedback relationships among rates of Ca²⁺ entry, activity of Ca²⁺-activated K⁺ and Cl^– channels and membrane potential.     

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.     

Characterisation of Ca2+-dependent inwardly rectifying K+ currents in HeLa cells

The whole-cell configuration of the patch-clamp technique was used to examine K⁺ currents in HeLa cells. Under quasi-physiological ionic gradients, using an intracellular solution containing 10^–7 mol/l free Ca²⁺, mainly outward currents were observed. Large inwardly rectifying currents were elicited in symmetrical 145 mmol/l KCl. Replacement of all extracellular K⁺ by isomolar Na⁺, greatly decreased inward currents and shifted the reversal potential as expected for K⁺ selectivity. The inwardly rectifying K⁺ currents exhibited little or no apparent voltage dependence within the range of from –120 mV to 120 mV. A square-root relationship between chord conductance and [K⁺]₀ at negative potentials could be established. The inwardly rectifying nature of the currents was unaltered after removal of intracellular Mg²⁺ and chelation with ATP and ethylenediaminetetraacetic acid (EDTA). Permeability ratios for other monovalent cations relative to K⁺ were: K⁺ (1.0)>Rb⁺ (0.86)>Cs⁺ (0.12)>Li⁺ (0.08)>Na⁺ (0.03). Slope conductance ratios measured at –100 mV were: Rb⁺ (1.66)>K⁺ (1.0)>Na⁺ (0.09)>Li⁺ (0.08)>Cs⁺ (0.06). K⁺ conductance was highly sensitive to intracellular free Ca²⁺ concentration. The relationship between conductance at 0 mV and Ca²⁺ concentration was well described by a Hill expression with a dissociation constant, K _D, of 70 nmol/l and a Hill coefficient, n, of 1.81. Extracellular Ba²⁺ blocked the currents in a concentration- and voltage-dependent manner. The dependence of the K _D for the blockade was analysed using a Woodhull-type treatment, locating the ion interaction site at 19 % of the distance across the electrical field of the membrane and a K _D (0 mV) of 7 mmol/l. Tetraethylammonium and 4-aminopyridine were without effect whilst quinine and quinidine blocked the currents with concentrations for half-maximum effects equal to 7 mol/l and 3.5 mol/l, respectively. The unfractionated venom of the scorpion Leiurus quinquestriatus (LQV) blocked the K⁺ currents of HeLa cells. The toxins apamin and scyllatoxin had no detectable effect whilst charybdotoxin, a component of LQV, blocked in a voltage-dependent manner with half-maximal concentrations of 40 nmol/l at –120 mV and 189 nmol/l at 60 mV; blockade by charybdotoxin accounts for the effect of LQV. Application of ionomycin (5–10 mol/l), histamine (1 mmol/l) or bradykinin (1–10 mol/l) to cells dialysed with low-buffered intracellular solutions induced K⁺ currents showing inward rectification and a lack of voltage dependence.     

Potassium conductance of smooth muscle cells from rabbit aorta in primary culture

Vascular smooth muscle cells were obtained from rabbit aorta and were studied in primary culture on days 1–7 after seeding with electrophysiological techniques. In impalement experiments a mean membrane potential difference (PD) of –50±0.3 mV (n=387) was obtained with Ringer-type solution in the bath. PD was depolarized by 6±0.3 mV (n=45) and 16±2 mV (n= 5) when the bath K⁺ concentration was increased from the control value of 3.6 mmol/l to 13.6 and 23.6 mmol/l, respectively. Ba²⁺ (0.1–1 mmol/l) depolarized PD. Tetraethylammonium (TEA, 10 mmol/l) depolarized PD only slightly but significantly. Verapamil (0.1 mmol/l) and charybdotoxin (10 nmol/l) had no effect on PD. The conductance properties of these cells were further examined with the patch-clamp technique. K⁺ channels were spontaneously present in cell-attached patches. When the pipette was filled with 145 mmol/l KCl, a mean conductance (g _K) of 209.6±4.6 mV (n=17) was read from the current/voltage curves at a clamp voltage (V _c) of 0 mV. After excision K⁺ channels were found in 129 patches with inside-out and in 50 with outside-out configuration. With KCl on one and NaCl on the other side the mean g _K at a V _c of 0 mV was 134.6±3.9 pS (n=179). The mean permeability was 0.89±0.03×10^–12 cm³/s. With symmetrical KCl solution the mean g _K was 227±6 pS (n=17). The conductance sequence was g _K g _Rb= g _Cs=g _Na=0. TEA blocked dose-dependently only from the outside.(1–10 mmol/l). Lidocaine (5 mmol/l) quinidine (0.01–1 mmol/l) and quinine (0.01–1 mmol/l) blocked from both sides. Charybdotoxin (0.5–5 nmol/l) blocked only from the extracellular side. Ba²⁺ blocked from the cytosolic side and the inhibition was increased by depolarization and reduced by hyperpolarization. At a V _c of 0 mV a half-maximal inhibition (IC₅₀) of 2 mol/l was obtained. Verapamil and diltiazem blocked from both sides, verapamil with an IC₅₀ of 2 mol/l and diltiazem with an IC₅₀ of 10 mol/l. The open probability of this channel was increased by Ca²⁺ on the cytosolic side at activities > 0.1 mol/l. Half-maximal activation occurred at Ca²⁺ activities exceeding 1 mol/l. The present data indicate that the vascular smooth muscle cells of rabbit aorta in primary culture possess a K⁺ conductance. In excised patches only a maxi K⁺ channel was detected. This channel has properties different from the macroscopic K⁺ conductance. Hence, it is likely that the K⁺ conductance of the intact cell is dominated by yet another and thus far not detected K⁺ channel.Supported by DFG Gr 480/10     

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.     

Effects of intracellular Ca2+ chelating compounds on inward currents caused by Ca2+ release from sarcoplasmic reticulum in guinea-pig atrial myocytes

Ca²⁺ release from the sarcoplasmic reticulum (SR) of mammalian cardiac myocytes occuring either due to activation by a depolarization or the resulting transmembrane Ca²⁺ current (I _Ca), or spontaneously due to Ca²⁺ overload has been shown to cause inward current(s) at negative membrane potentials. In this study, the effects of different intracellular Ca²⁺ chelating compounds on I _Ca-evoked or spontaneous Ca²⁺-release-dependent inward currents were examined in dialysed atrial myocytes from hearts of adult guinea-pigs by means of whole-cell voltage-clamp. As compared to dialysis with solutions containing only a low concentration of a high affinity ethylene glycol-bis(-aminoethylether) N,N,N,N-tetraacetic acid (EGTA) like chelator (50–200 M), inward membrane currents (at –50 mV) due to evoked Ca²⁺ release, spontaneous Ca²⁺ release or Ca²⁺ overload following long-lasting depolarizations to very positive membrane potentials are prolonged if the dialysing fluid contains a high concentration of a low affinity Ca²⁺ chelating compound such as citrate or free adenosine 5-triphosphate (ATP). Without such a non-saturable Ca²⁺ chelator in the dialysing fluid, Ca²⁺-release-dependent inward currents are often oscillatory and show an irregular amplitude. With a low affinity chelator in a non-saturable concentration, discrete inward currents with constant properties can be recorded. We conclude that the variability in Ca²⁺-release-dependent inward current seen in single cells arises from spatial inhomogeneities of intracellular Ca²⁺ concentration ([Ca²⁺]_i) due to localized saturation of endogenous and exogenous high affinity Ca²⁺ buffers (e.g. [2]). This can be avoided experimentally by addition of a non-saturable buffer to the intracellular solution. This condition might be useful, if properties of Ca²⁺ release from the SR and/ or the resulting membrane current, like for example arrhythmogenic transient inward current, are to be investigated on the single cell level.     

Basolateral K+ efflux is largely independent of maxi-K+ channels in rat submandibular glands during secretion

The involvement of large-conductance, voltage- and Ca²⁺-activated K⁺ channels (maxi-K⁺ channels) in basolateral Ca²⁺-dependent K⁺-efflux pathways and fluid secretion by the rat submandibular gland was investigated. Basolateral K⁺ efflux was monitored by measuring the change in K⁺ concentration in the perfusate collected from the vein of the isolated, perfused rat submandibular gland every 30 s. Under conditions in which the Na⁺/K⁺-ATPase and Na⁺-K⁺-2Cl^– cotransporter were inhibited by ouabain (1 mmol/l) and bumeta-nide (50 mol/l) respectively, continuous stimulation with acetylcholine (ACh) (1 mol/l) caused a transient large net K⁺ efflux, followed by a smaller K⁺ efflux, which gradually returned to the basal level within 10 min. These two components of the K⁺ efflux appear to be dependent on an increase in cytosolic Ca²⁺ concentration. The initial transient K⁺ efflux was not affected by charybdotoxin (100 nmol/l) or tetraethylammonium (TEA) (5 mmol/l) but the smaller second component was strongly and reversibly inhibited by charybdotoxin (100 nmol/l) and TEA (0.1 and 5 mmol/l). The initial K⁺ efflux transient induced by ACh was inhibited by quinine (0.1–3 mmol/l), quinidine (1–3 mmol/l) and Ba²⁺ (5 mmol/l), but not by verapamil (0.1 mmol/l), lidocaine (1 mmol/l), 4-aminopyridine (1 mmol/l) or apamin (1 mol/l). Ca²⁺-dependent transient large K⁺ effluxes induced by substance P (0.01 mol/l) and A23187 (3 mol/l) were not inhibited by TEA (5 mmol/l or 10 mmol/l). A23187 (3 mol/l) evoked a biphasic fluid-secretory response, which was not inhibited by TEA (5 mmol/l). Patch-clamp studies confirmed that the whole-cell outward K⁺ current attributable to maxi-K⁺ channels obtained from rat submandibular endpiece cells was strongly inhibited by the addition of TEA (1–10 mmol/l) to the bath. It is concluded that maxi-K⁺ channels are not responsible for the major part of the Ca²⁺-dependent basolateral K⁺ efflux and fluid secretion by the rat submandibular gland.     

Ca2+-activated K+ channels in airway smooth muscle are inhibited by cytoplasmic adenosine triphosphate

Large-conductance Ca²⁺-activated K⁺ channels were studied in membranes of cultured rabbit airway smooth muscle cells, using the patch-clamp technique. In cell-attached recordings, channel openings were rare and occurred only at very positive potentials. Bradykinin (10 M), an agonist which releases Ca²⁺ from the sarcoplasmic reticulum, transiently increased channel activity. The metabolic blocker 2,4-dinitrophenol (20 M), which lowers cellular adenosine triphosphate (ATP) levels, induced a sustained increase of channel activity in cell-attached patches. In excised patches, these channels had a slope conductance of 155 pS at 0 mV, were activated by depolarization and by increasing the Ca²⁺ concentration at the cytoplasmic side above 10^–7 mol/l. ATP, applied to the cytoplasmic side of the patches, dose-dependently decreased the channel's open-state probability. An inhibition constant (K _i) of 0.2 mmol/l was found for the ATP-induced inhibition. ATP reduced the Ca²⁺ sensitivity of the channel, shifting the Ca²⁺ activation curve to the right and additionally reducing its steepness. Our results demonstrate that cytoplasmic ATP inhibits a large-conductance Ca²⁺-activated K⁺ channel in airway smooth muscle. This ATP modulation of Ca²⁺-activated K⁺ channels might serve as an important mechanism linking energy status and the contractile state of the cells.     

Different inhibitions of the voltage-dependent K+ current by Ca2+ antagonists in the smooth muscle cell membrane of rabbit small intestine

Actions of Ca²⁺ antagonists (verapamil, nicardipine and diltiazem) on the voltage-dependent K⁺ current, obtained from the fragmented smooth muscle cell membrane (smooth muscle ball; SMB) of the rabbit small intestine, were investigated using voltage clamp techniques. To eliminate the influence of the Ca²⁺-dependent K⁺ current, the voltage-dependent K⁺ current was recorded in 2.5 mM Mn²⁺ (Ca²⁺ omitted) solution. These three Ca²⁺ antagonists inhibited the peak amplitude of the K⁺ current, in a dose-dependent manner. During application of a long command pulse (duration, 3 s), the amplitude of the voltage-dependent K⁺ current decreased slowly with time. Diltiazem inhibited the K⁺ current with a slight prolongation of the 20% decay time, while TEA (tetraethyl-ammonium), a K⁺ channel blocker, inhibited the current, without affecting the decay. By contrast, verapamil and nicardipine accelerated inactivation. In the control, the voltage-dependent inactivation was also seen in the K⁺ current. This inactivation curve below 0 mV was not modified by 10 M diltiazem, 5 M verapamil nor 3 M nicardipine. These results indicate that inhibition of the voltage-dependent K⁺ current by verapamil or nicardipine differed from that by diltiazem.An abstract of this work was reported at the 59th general meeting of Japan Pharmacological Society (Terada et al. 1986).     

The benzazepine/benzothiazepine binding domain of the cardiac L-type Ca2+ channel is accessible only from the extracellular side

The whole-cell tight seal recording technique was used to investigate location of benzothiazepine binding site of the cardiac L-type Ca²⁺ channel. For this we utilized a permanently charged compound, SQ 32.428, out of a series of benzazepine drugs which have been characterized as competitive inhibitors of diltiazem binding. The non-permanently charged derivative SQ 32.910 was initially tested to electrophysiologically establish Ca²⁺ antagonistic properties of benzazepines. Upon extracellular application, either compound was able to completely block Ca²⁺ currents. At a stimulating frequency of 0.2 Hz IC₅₀ concentrations of SQ 32.910 and SQ 32.428 were determined as 35 nM and 15 M, respectively. Intracellular application of SQ 32.428 was then compared to control experiments in the absence of drug. Initially, adequate drug dialysis was confirmed with 100 M D890, which produced a progressive inhibition of Ca²⁺ currents within 10 min after whole-cell access. In contrast, internal application of 100 M SQ 32.428 did not change time-course of Ca²⁺ currents compared to control run-down. These results show that the benzazepine/benzothiazepine binding domain of the cardiac L-type Ca²⁺ channel is accessible only from the extracellular side and therefore suggest an extracellular location on the ₁-subunit of the Ca²⁺ channel protein.     

Properties of two calcium transport systems of isolated rat ileal epithelial cells: effects of Ca2+ channel modulators and membrane potential examined with fluorescent dye,fura-2

Calcium transport systems of isolated ileal epithelial cells were investigated. The concentration of cytosolic free calcium ions, [Ca²⁺]_i, was monitored with a fluorescent Ca²⁺ dye, fura-2. The fluorescence intensity ratio (I ₃₄₀/I ₃₈₀) was used as an index of [Ca²⁺]_i. [Ca²⁺]_i of the cells suspended in the nominally Ca²⁺-free solution was estimated at 52±3 nM. Ca²⁺ uptake was followed for as long as 5 min in the presence of 100–1000 M added CaCl₂. Most of the experiments were performed at 200 M CaCl₂. The Ca²⁺ uptake was abolished by 0.8 mM Ni²⁺ and 50 M Mn²⁺ and partitally antagonized by 50 M verapamil and 50 M diltiazem but not affected by 20 M nifedipine. The Ca²⁺ entry was reduced by increasing concentrations of extracellular K⁺ in the presence of valinomycin, suggesting a voltage-dependent nature of the uptake. On the other hand, the Ca²⁺ transport doubled in the presence of Bay K8644 (8 M), a Ca²⁺ channel agonist. The Bay-K-8644-induced uptake was inhibited by either 10 M nifedipine, 10 M verapamil or 10 M diltiazem and was relatively independent of extracellular K⁺ concentration. These results suggest that there are at least two distinct Ca²⁺ transport systems in the rat ileal epithelial cells, one resistant to organic Ca²⁺ channel blockers but relatively sensitive to membrane potential (basal uptake) and another inducible by Bay K 8644 and sensitive to the channel blockers but relatively independent of membrane potential.     

Calcium influx into endothelial cells and formation of endothelium-derived relaxing factor is controlled by the membrane potential

We studied the role of the membrane potential in the control of the intracellular free calcium concentration ([Ca²⁺]_i) and release of the two autacoids endothelium-derived relaxing factor (EDRF = nitric oxide) and prostaglandin I₂ in endothelial cells. ATP (3 mol/l) and bradykinin (1 nmol/l) evoked rapid increases (sixfold) in [Ca²⁺]_i in cultured endothelial cells. [Ca²⁺]_i remained elevated over several minutes. When the cells were depolarized, either by K⁺ (70–90 mmol/l) or by preincubation with the blocker of K⁺ channels tetraethylammonium (3 mmol/l), the initial peak of [Ca²⁺]_i remained unaffected but [Ca²⁺]_i returned significantly faster to resting levels, indicating a reduction in Ca²⁺ influx. In native, freshly isolated endothelial cells, K⁺ abolished increases in [Ca²⁺]_i induced by acetylcholine (3 mol/l). Release of EDRF in response to bradykinin (cultured cells) and acetylcholine (native cells) was inhibited by K⁺ (by 70%), whereas release of prostaglandin I₂ was not significantly reduced. Preincubation of cultured endothelial cells with the receptor-independent stimulus thimerosal (5 mol/l, 40 min) evoked a long-lasting release of EDRF and small elevations of [Ca²⁺]_i (twofold) after washout of the drug. Depolarization with K⁺ decreased thimerosal-induced EDRF release and [Ca²⁺]_i in a reversible manner. In patch-clamped endothelial cells, bradykinin (1 nmol/l) induced transient hyperpolarizations that were significantly prolonged by BRL 34915 (1 mol/l), an activator of K⁺ channels. BRL 34915 also elicited increases in [Ca²⁺]_i, particularly in thimerosal-stimulated endothelial cells. These effects were abolished by K⁺. We conclude that the initial rise in [Ca²⁺]_i in response to receptor-binding agonists, caused by mobilization of Ca²⁺ from intracellular stores, activates K⁺ channels, thereby inducing hyperpolarization. This hyperpolarization provides the driving force for transmembrane Ca²⁺ influx into endothelial cells and is thus an important signal for synthesis and release of EDRF.     

Ca2+- and swelling-induced activation of ion conductances in bronchial epithelial cells

The present study was performed to examine Ca²⁺-dependent and cell-swelling-induced ion conductances in a polarized bronchial epithelial cell line (16HBE14o-). Whole-cell currents were measured in fast and slow whole-cell patch-clamp experiments in cells grown either on filters or on coated plastic dishes. In addition the transepithelial voltage (V _te) and resistance (R _te) were measured in confluent monolayers. Resting cells had a membrane voltage (V _m) of –36±1.1 mV (n=137) which was mainly caused by K⁺ and Cl^– conductances and to a lesser extent by a Na⁺ conductance. V _te was apical-side-negative after stimulation. Equivalent short-circuit current (I _sc = V _te/R _te) was increased by the secretagogues histamine (0.1 mmol/l), bradykinin (0.1–10 mol/l) and ATP (0.1–100 mol/l). The histamine-induced I _sc was blocked by either basolateral diphenhydramine (0.1 mmol/l, n=4) or apical cimetidine (0.1 mmol/l, n=4). In fast and slow whole-cell recordings ATP and bradykinin primarily activated a transient K⁺ conductance and hyperpolarized V _m. This effect was mimicked by the Ca²⁺ ionophore ionomycin (1 mol/l, n=11). Inhibition of the bradykinin-induced I _sc by the blocker HOE140 (1 mol/l, n=3) suggested the presence of a BK₂ receptor. The potency sequence of different nucleotide agonists on the purinergic receptor was UTP ATP > ITP > GTP CTP [,-methylene] ATP 2-methylthio-ATP = 0 and was obtained in I _sc measurements and patch-clamp recordings. This suggests the presence of a P_2u receptor. Hypotonic cell swelling activated both Cl^– and K⁺ conductances. The Cl^– conductance was only slightly inhibited by 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (0.5 mmol/ l, n=3). These data indicate that 16HBE140- bronchial epithelial cells, which are known to express high levels of cystic fibrosis transmembrane conductance regulator protein, form a secretory epithelium. While hypotonic cell swelling activates both K⁺ and Cl^– channels, the Ca²⁺-induced Cl^– secretion is due mainly to activation of basolateral K⁺ channels.     

ATP-Dependent inhibition of Ca2+-activated K+ channels in vascular smooth muscle cells by neuropeptide Y

Neuropeptide Y(NPY) inhibits Ca²⁺-activated K⁺ channels reversibly in vascular smooth muscle cells from the rat tail artery. NPY (200 M) had no effect in the absence of intracellular adenosine 5triphosphate (ATP) and when the metabolic poison cyanide-M-chlorophenyl hydrozone (10 M) was included in the intracellular pipette solution. NPY was also not effective when ATP was substituted by the non-hydrolysable ATP analogue adenosine 5-[, -methylene]-triphosphate (AMP-PCP). NPY inhibited Ca²⁺-activated K⁺ channel activity when ATP was replaced by adenosine 5-O-(3-thiotriphosphate) (ATP [-S]) and the inhibition was not readily reversed upon washing. Protein kinase inhibitor (1 M), a specific inhibitor of adenosine 3, 5-cyclic monophosphatedependent protein kinase, had no significant effect on the inhibitory action of NPY. The effect of NPY on single-channel activity was inhibited by the tyrosine kinase inhibitor genistein (10 M) but not by daidzein, an inactive analogue of genistein. These observations suggest that the inhibition by NPY of Ca²⁺-activated K⁺ channels is mediated by ATP-dependent phosphorylation. The inhibitory effect of NPY was antagonized by the tyrosine kinase inhibitor genistein.     

Potassium channels in the basolateral membrane of the rectal gland ofSqualus acanthias

The present study examines the influences of pH and Ca²⁺ and several putative inhibitors on the basolateral K⁺ channel of the rectal gland ofSqualus acanthias. Excised membrane patches were examined using the patch clamp technique. It is shown that reduction of the calcium activity on the cytosolic side to less than 10^–9 mol/l has no detectable inhibitory effect on this channel. Conversely, increase in calcium activity to some 10^–3 mol/l reduced the activity of this channel. Variations in cytosolic pH had only a moderate effect on the current amplitude: alkalosis by one pH unit increased and acidosis reduced the single current amplitude by some 15%. Several inhibitors were tested in excised patches when added to the cytosolic side. Ba²⁺ (5·10^–3 mol/l), quinine (10^–3 mol/l), quinidine (10^–4 mol/l), lidocaine (1 mmol/l), tetraethylammonium (10 mmol/l), Cs⁺ (10 mmol/l), and Rb⁺ (20 mmol/l) all blocked this K⁺ channel reversibly. We conclude that the basolateral K⁺ channel of the rectal gland is distinct from other epithelial K⁺ channels inasmuch as it is not stimulated by Ca²⁺ directly, but that it is qualitatively similar to many other known K⁺ channels with respect to its sensitivity towards blockers.This study was supported by Deutsche Forschungsgemeinschaft Gr 480/8 and by NSF and NIH grants to the Mount Desert Island Biological Laboratory     

Neuropeptide Y2-type receptor-mediated activation of large-conductance Ca2+-sensitive K+ channels in a human neuroblastoma cell line

We have proposed recently that a pertussistoxin-insensitive Ca²⁺ influx stimulated by Y₂-type receptor activation in CHP-234 human neuroblastoma cells underlies increases in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) induced by neuropeptide Y (NPY), which were strictly dependent on extracellular Ca²⁺ and independent of internal Ca²⁺ stores. We describe here the actions of NPY in these same cells, using the activity of Ca²⁺-activated K⁺ channels as an indicator of [Ca²⁺]_i. The elementary slope conductance of these channels was 110±3 pS (with an asymmetrical K⁺gradient), their activity was greatly increased by application of ionomycin, and they were reversibly blocked by 1 mM tetraethylammonium (TEA) and 100 nM charybdotoxin. Application of 100 nM NPY, in the presence but not in the absence of extracellular Ca²⁺, increased the channel open probability. ATP applied in the absence of external Ca²⁺ caused rises both in channel open probability and [Ca²⁺]_i. Inositol trisphosphate production was stimulated by ATP but not by NPY. In outside-out patches, NPY increased channel open probability, indicating that NPY-associated Ca²⁺ influx does not require all the intracellular machinery present in intact cells. Channel activation by NPY was unaffected by the replacement of guanosine 5-triphosphate (GTP) by (guanosine 5-O-(2-thiodiphosphate) (GDP[S]), a non-hydrolysable GDP analogue, in the pipette internal solution, consistent with the lack of involvement of G-proteins in the coupling of Y₂-type receptors to Ca²⁺ influx in CHP-234 cells.     

The effect of secretagogues on ion conductances of in vitro perfused,isolated rabbit colonic crypts

Several secretagogues were used in this study, including those which enhance intracellular cyclic adenosine monophosphate (cAMP) production, as well as others which elevate intracellular Ca²⁺ activity and are known to increase Cl^– secretion in the intact colon and in colonic carcinoma cell lines. They were examined with respect to their effects on electrophysiological properties in isolated rabbit distal colonic crypts. Crypts were dissected manually and perfused in vitro. Transepithelial voltage (V _te), transepithelial resistance (R _te), membrane voltage across the basolateral membrane (V _bl), and fractional basolateral membrane resistance (FR _bl), were estimated. Basolateral prostaglandin E₂ (PGE₂, 0.1 mol/l), vasoactive intestinal peptide (VIP, 1 nmol/l) and adenosine (0.1 mmol/l) induced an initial depolarisation and a secondary partial repolarisation of (V _bl). In the case of adenosine, the initial depolarization of (V _bl) was by 31±2 mV (n=47).R _te fell significantly from 16.4±3.6 to 14.2±3.7 ·cm² (n= 6), andFR _blincreased significantly from 0.11±0.02 to 0.51±0.10 (n=6). In the second phase the repolarisation of (V _bl) amounted 11±2 mV (n=47) and a steadystate (V _bl) of –51±2 mV (n=47) was reached.R _te fell further and significantly to a steady-state value of 12.4±3.8 ·cm² (n=6) andFR _bl fell significantly to 0.42±0.13 (n=6). In 30% of the experiments, a transient hyperpolarisation of (V _bl) by 8±2 mV (n=14) was seen during wash out of adenosine. In the presence of adenosine, but not under control conditions, lowering of luminal Cl^– concentration from 120 to 32 mmol/l depolarised (V _bl) significantly by 8±1 mV (n=9). Basolateral ATP and ADP (0.1 mmol/l) led to a short initial depolarisation followed by a sustained and significant hyperpolarisation by 6±2 mV (n=27) and 5±4 mV (n=8), respectively. Carbachol (CCH) hyperpolarised (V _bl) in a concentration-dependent manner. At 100 mol/l (bath) the hyperpolarisation was by 14±2 mV (n=11) andFR _bl fell slightly. Neurotensin (10 nmol/l), isoproterenol (10 mol/l) and uridine 5-triphosphate (UTP, 0.1 mmol/l) had no effect. It is concluded that PGE₂, VIP and adenosine upregulate sequentially a luminal Cl^– conductance and a basolateral K⁺ conductance by increasing intracellular cAMP concentration. Ca²⁺ mobilising hormones such as ATP, ADP, and CCH increase the basolateral K⁺ conductance, while the effect on luminal Cl^– conductance appears to be very limited.     

Electrogenic Na+/K+-transport in human endothelial cells

Na⁺/K⁺ pump currents were measured in endothelial cells from human umbilical cord vein using the whole-cell or nystatin-perforated-patch-clamp technique combined with intracellular calcium concentration ([Ca²⁺]_i) measurements with Fura-2/AM. Loading endothelial cells through the patch pipette with 40 mmol/l [Na⁺] did not induce significant changes of [Ca²⁺]_i. Superfusing the cells with K⁺-free solutions also did not significantly affect [Ca²⁺]_i. Reapplication of K⁺ after superfusion of the cells with K⁺-free solution induced an outward current at a holding potential of 0 mV. This current was nearly completely blocked by 100 mol/l dihydroouabain (DHO) and was therefore identified as a Na⁺/K⁺ pump current. During block and reactivation of the Na⁺/K⁺ pump no changes in [Ca²⁺]_i could be observed. Pump currents were blocked concentration dependently by DHO. The concentration for half-maximal inhibition was 21 mol/l. This value is larger than that reported for other tissues and the block was practically irreversible. Insulin (10–1000 U/l) did not affect the pump currents. An increase of the intracellular Na⁺ concentration ([Na⁺]_i) enhanced the amplitude of the pump current. Half-maximal activation of the pump current by [Na⁺]_i occurred at about 60 mmol/l. The concentration for half-maximal activation by extracellular K⁺ was 2.4±1.2 mmol/l, and 0.4±0.1 and 8.7±0.7 mmol/l for Tl⁺ and NH₄ ⁺ respectively. The voltage dependence of the DHO-sensitive current was obtained by applying linear voltage ramps. Its reversal potential was more negative than –150 mV. Pump currents measured with the conventional whole-cell technique were about four times smaller than pump currents recorded with the nystatin-perforated-patch method. If however 100 mol/l guanosine 5-O-(3-thiotriphosphate) (GTPS) were added to the pipette solution, the currents measured in the ruptured-whole-cell-mode were not significantly different from the currents measured with the perforated-patch technique. We suppose that the use of the perforated-patch technique prevents wash out of a guanine nucleotide-binding protein (G-protein)-connected intracellular regulator that is necessary for pump activation.