Sensitivity to dihydropyridines, ω-conotoxin and noradrenaline reveals multiple high-voltage-activated Ca2+ channels in rat insulinoma and human pancreatic β-cells

High-voltage-activated (HVA) Ba²⁺ currents of rat insulinoma (RINm5F) and human pancreatic -cells were tested for their sensitivity to dihydropyridines (DHPs), -conotoxin (-CgTx) and noradrenaline. In RINm5F cells, block of HVA currents by nimodipine, nitrendipine and nifedipine was voltage- and dose-dependent (apparent K _D<37 nM) and largely incomplete even at saturating doses of DHPs (mean 53%, at 10 M and 0 mV). Analysis of slow tail currents in Bay K 8644-treated cells indicated the existence of Bay K 8644-insensitive channels that turned on at slightly more positive voltages and deactivated more quickly than Bay K 8644-modified channels. DHP Ca²⁺ agonists and antagonists in human -cells had similar features to RINm5F cells except that DHP block was more pronounced (76%, at 10 M and 0 mV) and Bay K 8644 action was more effective, suggesting a higher density of L-type Ca²⁺ channels in these cells. In RINm5F cells, but not in human -cells, DHP-resistant currents were sensitive to -CgTx. The toxin depressed 10–20% of the DHP-resistant currents sparing a residual current (25–35%) with similar voltage-dependent characteristics and Ca²⁺/Ba²⁺ permeability. Noradrenaline (10 M) exhibited different actions on the various HVA current components: (1) it prolonged the activation kinetics of -CgTx-sensitive currents, (2) it depressed by about 20% the size of DHP-sensitive currents, and (3) it had little or no effects on the residual DHP- and -CgTx-resistant current although intracellularly applied guanosine 5-O-(3-thiotriphosphate) (GTP--S) prolonged its activation time course. The first action was clearly voltage-dependent and most evident in RINm5F cells that displayed neuronal-like processes. The second was observed more frequently, was voltage-independent and fully blocked by saturating doses of nifedipine (10 M). Both actions were prevented by intracellular perfusion with guanosine 5-O-(2-thiodiphosphate) (GDP--S). Our data suggest that beside a majority of L-type channels, RINm5F and human pancreatic -cells may express a variable fraction of DHP-insensitive channels that may be involved in the control of insulin secretion during -cell activity.     

Voltage-dependent noradrenergic modulation of ω-conotoxin-sensitive Ca2+ channels in human neuroblastoma IMR32 cells

High-threshold (HVA) Ca²⁺ channels of human neuroblastoma IMR32 cells were effectively inhibited by noradrenaline. At potentials between –20 mV and +10 mV, micromolar concentrations of noradrenaline induced a 50%–70% depression of HVA Ba²⁺ currents and a prolongation of their activation kinetics. Both effects were relieved at more positive voltages or by applying strong conditioning pre-pulses (facilitation). Facilitation restored the rapid activation of HVA channels and recruited about 80% of the noradrenaline-inhibited channels at rest. Re-inhibition of Ca²⁺ channels after facilitation was slow ( _r 36–45 ms) and voltage-independent between –30 mV and –90 mV. The inhibitory action of noradrenaline was dose-dependent (IC₅₀=84 nM), mediated by ₂-drenergic receptors and selective for -conotoxin-sensitive Ca²⁺ channels, which represent the majority of HVA channels expressed by IMR32 cells. The action of noradrenaline was mimicked by intracellular applications of GTP[S] and prevented by GDP[S] or by pre-incubation with pertussis toxin. The time course of noradrenaline inhibition measured during fast application (onset) and wash-out (offset) of the drug were independent of saturating agonist concentrations (10–50 M) and developed with mean time constants of 0.56 s ( _on) and 3.6 s ( _off) respectively. The data could be simulated by a kinetic model in which a G protein is assumed to modify directly the voltage-dependent gating of Ca²⁺ channels. Noradrenaline-modified channels are mostly inhibited at rest and can be recruited in a steep voltage-dependent manner with increasing voltages.     

Inhibition of ω-conotoxin-sensitive Ca2+ channel currents by internal Mg2+ in cultured rat cerebellar granule neurones

The effects of changing the intracellular concentrations of either free Mg²⁺ ions ([Mg²⁺]_i) or Mg²⁺-bound adenosine triphosphate ([Mg · ATP]_i) on Ca²⁺ channel currents were assessed in cultured rat cerebellar granule neurones using the whole-cell patch-clamp technique. Raising [Mg²⁺]_i from 0.06 mM to 1.0 mM inhibited Ca²⁺ channel currents by approximately 50%. The action of -conotoxin GVIA (-CgTX), a selective inhibitor of N-type Ca²⁺ channels was also investigated. With increasing [Mg²⁺]_i, the proportion of current irreversibly blocked by -CgTX was reduced, and was negligible (approximately 5 pA of current) in the presence of [Mg²⁺]_i values of 0.5 mM or greater. Block of the -CgTX-sensitive current accounted for the reduction in total current by concentrations of [Mg²⁺]_i to 0.5 mM. Raising [Mg²⁺]_i had no effect on the rate of decay of Ca²⁺ currents, but did produce a negative shift in current activation, possibly due to a non-specific interaction with negative surface charge. Altering [Mg · ATP]_i from 0.3 to 5.0 mM caused a twofold increase in the size of currents without affecting the proportion of current sensitive to -CgTX. [Mg²⁺]_i was also effective in inhibiting the Ca²⁺ channel current following potentiation by increasing [Mg · ATP]_i. These data suggest that -CgTX-sensitive current in these cells is selectively inhibited by internal Mg²⁺ whereas both -CgTX-sensitive and -resistant components of current are potentiated by internal Mg · ATP. The mechanism by which Mg²⁺ inhibits N-type channels is unclear, but may involve an open channel block.     

Inhibition of voltage-dependent Ca2+ channels via α2-adrenergic and opioid receptors in cultured bovine adrenal chromaffin cells

Adrenal chromaffin cells secrete catecholamindes and opioids. The effects of these agents on whole-cell Ca²⁺ channel currents were studied, using bovine adrenal chromaffin cells kept in short term culture. Ca²⁺ channel currents recorded during voltageclamp pulses from a holding potential of –80 mV to 0 mV were reversibly reduced by 10 M epinephrine (in the presence of 1 M propranolol) or 5 M of the synthetic opioid, d-Ala²-d-Leu⁵-enkephalin (DADLE) by approximately 35% and 25%, respectively. The inhibitory action of epinephrine was mimicked by clonidine, reduced by yohimbine but not affected by prazosin. The DADLE-induced reduction of the Ca²⁺ channel current was antagonized by naloxone. The dihydropyridine (+)PN 200-110 (5 M) reduced the Ca²⁺ channel current by approximately 40%; the Ca²⁺ channel current inhibited by (+)PN 200-110 was not further reduced by epinephrine. Intracellular infusion of guanosine-5-O-(2-thiodiphosphate) and pretreatment of cells with pertussis toxin abolished the inhibitory effect of both epinephrine and DADLE. In membranes of adrenal chromaffin cells, four pertussis-toxin-sensitive G-proteins were identified, including G_i1, G_i2, G_o1 and another G_o subtype, possibly G_o2. The data show that activation of ₂-adrenergic and opioid receptors causes an inhibition of dihydropyridine-sensitive Ca²⁺ channels in adrenal chromaffm cells. These inhibitory modulations are mediated by pertussis-toxin-sensitive G-proteins and may represent a mechanism for a negative feedback signal by agents released from the adrenal medulla.     

Activation of Ca2+-dependent K+ and Cl− currents by UTP and ATP in CFPAC-1 cells

Activation of Cl^– and K⁺ conductances by nucleotide receptor-operated mobilization of intracellular Ca²⁺ was investigated in CFPAC-1 cells with the perforated-patch technique. Adenosine 5-triphosphate (ATP) and uridine 5-triphosphate (UTP) caused a dose-dependent fast and transient membrane hyperpolarization. UTP was more effective than ATP. In voltageclamped cells, two currents with different ionic permeability and kinetics were activated by the nucleotides. The first one was carried by Cl^– ions, peaked in the first few seconds after addition of nucleotides, and lasted for 1±0.3 min. Its amplitude was about 2.7 nA at –100 mV with 100 mol/l of either ATP or UTP. The second current was carried by K⁺ ions and was blocked by Cs⁺. This current peaked more slowly and had a mean duration of 4.6±0.7 min. Its amplitude was 0.9 nA and 0.5 nA at –20 mV with 100 umol/l UTP and ATP, respectively. Activation of the nucleotide receptor caused a transient increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) that was similar in the presence or absence of extracellular Ca²⁺. The ED₅₀ for UTP was 24 umol/l and that for ATP was 94 mol/l. Depletion of the inositol 1,4,5-trisphosphate-sensitive Ca²⁺ store by thapsigargin prevented both the nucleotide-induced [Ca²⁺]_i increase and the activation of membrane currents. Addition of 2 mmol/l Ca²⁺ to thapsigargin-treated cells produced a sustained increase of Cl^– and K⁺ currents, which was reversed by Ca²⁺ removal. The present study demonstrates that CFPAC-1 cells respond to nucleotide receptor activation with a transient increase in [Ca²⁺]_i that stimulates Ca²⁺-dependent Cl^– and K⁺ currents. This phenomenon is probably mediated by inositol 1,4,5-trisphosphate-dependent Ca²⁺ stores.     

Long-term modulation of Na+ and K+ channels by TGF-��1 in neonatal rat cardiac myocytes

Previous work shows that transforming growth factor-β1 (TGF-β1) promotes several heart alterations, including atrial fibrillation (AF). In this work, we hypothesized that these effects might be associated with a potential modulation of Na(+) and K(+) channels. Atrial myocytes were cultured 1-2?days under either control conditions, or the presence of TGF-β1. Subsequently, Na(+) (I(Na)) and K(+) (I(K)) currents were investigated under whole-cell patch-clamp conditions. Three K(+) currents were isolated: inward rectifier (I(Kin)), outward transitory (I(to)), and outward sustained (I(Ksus)). Interestingly, TGF-β1 decreased (50%) the densities of I(Kin) and I(Ksus) but not of I(to). In addition, the growth factor reduced by 80% the amount of I(Na) available at -80?mV. This effect was due to a significant reduction (30%) in the maximum I(Na) recruited at very negative potentials or I(max), as well as to an increased fraction of inactivated Na(+) channels. The latter effect was, in turn, associated to a -7?mV shift in V(1/2) of inactivation. TGF-β1 also reduced by 60% the maximum amount of intramembrane charge movement of Na(+) channels or Q(max), but did not affect the corresponding voltage dependence of activation. This suggests that TGF-β1 promotes loss of Na(+) channels from the plasma membrane. Moreover, TGF-β1 also reduced (50%) the expression of the principal subunit of Na(+) channels, as indicated by western blot analysis. Thus, TGF-β1 inhibits the expression of Na(+) channels, as well as the activity of K(+) channels that give rise to I(Ksus) and I(Kin). These results may contribute to explaining the previously observed proarrhythmic effects of TGF-β1.     

Relation of exocytotic release of γ-aminobutyric acid to Ca2+ entry through Ca2+ channels or by reversal of the Na+/Ca2+ exchanger in synaptosomes

The specific inhibitor of the -aminobutyric acid (GABA) carrier, NNC-711, {1-[(2-diphenylmethylene) amino]oxyethyl}-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride, blocks the Ca²⁺-independent release of [³H]GABA from rat brain synaptosomes induced by 50 mM K⁺ depolarization. Thus, in the presence of this inhibitor, it was possible to study the Ca²⁺-dependent release of [³H]GABA in the total absence of carrier-mediated release. Reversal of the Na⁺/Ca²⁺ exchanger was used to increase the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) to test whether an increase in [Ca²⁺]_i alone is sufficient to induce exocytosis in the absence of depolarization. We found that the [Ca²⁺]_i may rise to values above 400 nM, as a result of Na⁺/Ca²⁺ exchange, without inducing release of [³H]GABA, but subsequent K⁺ depolarization immediately induced [³H]GABA release. Thus, a rise of only a few nanomolar Ca²⁺ in the cytoplasm induced by 50 mM K⁺ depolarization, after loading the synaptosomes with Ca²⁺ by Na⁺/Ca²⁺ exchange, induced exocytotic [³H]GABA release, whereas the rise in cytoplasmic [Ca²⁺] caused by reversal of the Na⁺/Ca²⁺ exchanger was insufficient to induce exocytosis, although the value for [Ca²⁺]_i attained was higher than that required for exocytosis induced by K⁺ depolarization. The voltage-dependent Ca²⁺ entry due to K⁺ depolarization, after maximal Ca²⁺ loading of the synaptosomes by Na⁺/Ca²⁺ exchange, and the consequent [³H]GABA release could be blocked by 50 M verapamil. Although preloading the synaptosomes with Ca²⁺ by Na⁺/Ca²⁺ exchange did not cause [³H]GABA release under any conditions studied, the rise in cytoplasmic [Ca²⁺] due to Na⁺/Ca²⁺ exchange increased the sensitivity to external Ca²⁺ of the exocytotic release of [³H]GABA induced by subsequent K⁺ depolarization. Thus, our results show that the vesicular release of [³H]GABA is rather insensitive to bulk cytoplasmic [Ca²⁺] and are compatible with the view that GABA exocytosis is triggered very effectively by Ca²⁺ entry through Ca²⁺ channels near the active zones.     

Ca2+-activated Cl– channels in Ehrlich ascites tumor cells are distinct from mCLCA1, 2 and3

Using the whole-cell patch-clamp technique, we have studied the electrophysiological and pharmacological properties of the Ca(2+)-activated Cl- current present in Ehrlich cells. The currents activated slowly upon depolarization, deactivated upon hyperpolarization, and showed strong outward rectification. An increase in [Ca2+]i activated the current with an EC50 of 165.2 nM. Extracellular application of niflumic acid (100 microM) rapidly blocked the current in a voltage-dependent manner whereas sulfhydryl-modifying agents such as dithiothreitol (DTT, 1-2 mM) and N-ethylmaleimide (NEM, 100 microM) had no effect on Ca(2+)-activated currents in Ehrlich cells. Members of the recently discovered CLCA gene family are the only molecular candidates for Ca(2+)-activated Cl- channels cloned so far. Using RT-PCR we demonstrated that the appearance of a Ca(2+)-activated Cl- current in Ehrlich cells is not associated with the expression of the murine members of the CLCA family (mCLCA1-mCLCA3). Correspondingly, the kinetic and pharmacological properties of the Ca(2+)-activated current in Ehrlich cells differ from those of CLCA-associated currents, which are time independent and DTT sensitive. Thus, phenotypic differences in combination with RT-PCR data point to the existence of different molecular species for Ca(2+)-activated Cl- channels.     

Action-potential-like depolarizations relieve opioid inhibition of N-type Ca2+ channels in NG108–15 cells

 The ability of action-potential-like waveforms (APWs) to attenuate opioid-induced inhibition of N-type Ca²⁺ channels was investigated in the neuroblastoma × glioma cell line NG108–15 using whole-cell voltage clamp methods. In in vitro differentiated NG108–15 cells, the opioid agonist [d-ala²]-methionine-enkephalin (DAME) reversibly decreased ω-conotoxin-GVIA-sensitive Ba²⁺ currents (N-type currents). Agonist-mediated inhibition of N-type currents could be transiently relieved by strong unphysiological depolarizing prepulses to +80 mV (facilitation). Significant facilitation was also achieved by conditioning the cell with a train of 15 APWs, which roughly mimicked physiological action potentials (1- to 6-ms-long depolarizations to +30 mV from a holding potential of –40 mV). The APW-induced facilitation depended on both conditioning pulse frequency and duration. Summation of the disinhibition produced by each APW was possible because reinhibition following repolarization to –40 mV was a much slower process (τ=88 ms) than the onset of facilitation at +80 mV (τ=7 ms). These results provide evidence that N-type Ca²⁺ channel facilitation may be a physiologically relevant process, and suggest that neuronal firing may relieve agonist-induced inhibition of N-type currents to an extent depending on both the shape of action potentials and the frequency of firing. Received: 14 September 1998 / Accepted: 29 September 1998     

The lipid connection–regulation of voltage-gated Ca2+ channels by phosphoinositides

Recent findings have revealed a pivotal role for phospholipids phosphatidylinositol -4,5-biphosphate (PIP₂) and phosphatidylinositol -3,4,5-trisphosphate (PIP₃) in the regulation of high voltage-activated (HVA) Ca²⁺ channels. PIP₂ exerts two opposing actions on HVA Ca²⁺ channels: It stabilizes their activity but also produces a voltage-dependent inhibition that can be antagonized by protein kinase A (PKA) phosphorylation. PIP₂ depletion and arachidonic acid together mediate the slow, voltage-independent inhibition of HVA Ca²⁺ channels by G _q/11 -coupled receptors in neurons. A sufficient level of plasma membrane PIP₂ also appears to be necessary for G _βγ -mediated inhibition. On the other hand, increased production of PIP₃ by PI-3 kinases promotes trafficking of HVA Ca²⁺ channels to the plasma membrane. This review discusses these findings and their implications.     

Dual effect of nitric oxide on ATP-sensitive K+ channels in rat pancreatic β cells

We have previously shown that NO has stimulatory and inhibitory effects on insulin secretion at low and high concentrations, respectively. The present study investigated effects of NO on K_ATP channels of rat β cells by patch clamp analysis to elucidate the mechanism for the dual effect. NOC7 at 0.5 μM suppressed K_ATP channels activated by diazoxide in the cell-attached and perforated whole-cell modes but failed to suppress them in the inside-out mode. The inhibitory effect in the cell-attached mode was abolished by the soluble guanylate cyclase inhibitor ODQ and by the protein kinase G inhibitor KT5823. Moreover, 0.5 μM NOC7 failed to suppress the channel activity in the presence of the mitochondrial uncoupler FCCP. In contrast, 10 μM NOC7 activated K_ATP channels in the cell-attached and perforated whole-cell modes, although it had no effect on the channels in the inside-out mode. The K_ATP currents evoked by 10 μM NOC7 in the cell-attached mode were not inhibited by ODQ. The dual effect of NOC7 at 0.5 and 10 μM was observed in the same patch. Taken together, these results suggest that low-concentration NO exerts an inhibitory effect on K_ATP channels of β cells, which is induced through the cGMP/protein kinase G pathway, whereas high-concentration NO activates K_ATP channels through the mechanism independent of cGMP.     

Regulation of L- and N-types of Ca2+ channels by intracellular ATP4– in frog dorsal root ganglion neurons

The roles of free Mg²⁺ ions, ATP^4– ions and Mg-ATP complexes in the regulation of N- and L-types of Ca²⁺ channels were studied in frog dorsal root ganglion (DRG) neurons using the whole-cell patch-clamp technique. Because Mg²⁺ ions interact with ATP^4– ions to form Mg-ATP complexes, addition of one species can influence the concentrations of the other two. In this study their concentrations were carefully controlled by varying the concentrations of two constituents at a time while keeping the third constant. The effects of each of the three species on barium currents through L-type (I _BaL) and N-type (I _BaN) Ca²⁺ channels were plotted against its concentrations. The dose-response curves for ATP^4– show that I _BaL and I _BaN proportionally increased with ATP^4– concentrations up to 1 mM at three different Mg²⁺ concentrations. At a fixed concentration of ATP^4–, I _BaL and I _BaN remained unchanged even when pMg changed from 3 to 5. Dose-response curves for I _BaL and I _BaN plotted against Mg-ATP concentration did not show a consistent pattern. H-7 and Mg²⁺ ions did not exert any blocking effect on the activity of either Ca²⁺ channel type, and neither dibutyryl-cAMP nor NKH-477 had any stimulating effect, suggesting that phosphorylation is not likely to be involved in ATP-induced potentiation. From these observations, it is concluded that L-type and N-type Ca²⁺ channels in frog DRG neurons are regulated by ATP^4– ions alone, and that the neuronal Ca²⁺ channels are regulated by mechanisms that are different from those regulating the cardiac Ca²⁺ channels. Received: 30 October 1998 / Received after revision: 19 February 1999 / Accepted: 8 March 1999     

Swelling of rat mesangial cells induces a Ca2+-dependent Cl− conductance

Membrane voltage (V _m) and ion currents of rat mesangial cells in primary culture were measured with the patch-clamp technique in the fast whole-cell configuration.V _m was –44 ± 1 mV (_n = 138). A reduction of the osmolality from 290 to 190 mosmol/kg depolarizedV _m from –44 ± 1 to –29 ± 1 mV (_n = 118) and increased the inward and outward conductances (Gm) from 14±2 to 39 ± 4 nS and 13±2 to 37 ± 4 nS (_n = 84), respectively. During the hypotonicity-induced depolarization the cell capacitance increased significantly from 33 ± 3 to 42 ± 4 pF (_n = 40). The effect of hypotonic cell swelling onV _m was increased in a bath with a reduced extracellular Cl^– of 32 mmol/l (by 71 ± 4%,_n = 23), indicating that a Cl^– conductance was activated. The permselectivity of this conductance was I^– Br^– > Cl^–. TheV _m response was not affected in the presence of a reduced extracellular Na⁺ of 5 mmol/l (n = 13) and was inhibited in a solution with reduced extracellular Ca²⁺ concentration (by 63 ± 9%,n = 14). In microfluorescence measurements with the Ca²⁺-sensitive dye fura-2 hypotonic cell swelling induced a sustained increase of the intracellular Ca²⁺ activity, [Ca²⁺]_i (_n = 19). The increase of [Ca²⁺]_i was completely inhibited when the extracellular solution was free of Ca²⁺. TheV _m response to hypotonic cell swelling was not attenuated in the presence of the L-type Ca²⁺ channel blockers nicardipine (n = 5), nifedipine (n = 5) and verapamil (n = 5) (all at 1 mol/l). The data indicate that in rat mesangial cells, osmotic swelling induces a Ca²⁺ influx from extracellular space. This Ca²⁺ influx activates a Cl^– conductance resulting in a depolarization ofV _m. The enhanced Cl^– conductance may lead to KCl extrusion and hence regulatory volume decrease.     

Dimebon and Tacrine Inhibit Neurotoxic Action of β-Amyloid in Culture and Block L-type Ca2+ Channels

Dimebon, a Russian-made drug, inhibited toxic effects of beta -amyloid on cultured neurons. Excessive accumulation of beta-amyloid in the brain is characteristic of Alzheimer dementias. Antialzheimer preparations tacrine and dimebon improve survival of cerebellar granule cells during long-term incubation with Ab25-35, the neurotoxic fragment of beta-amyloid. Both preparations can block potential-dependent Ca²⁺ entry into neurons by about 20%, which is explained by their selective action on L-type Ca²⁺ channels. It was assumed that the neuroprotective effect of dimebon and tacrine against Ab25-35 partially depends on inhibition of potential-dependent Ca²⁺ entry.     

ω-Grammotoxin blocks action-potential-induced Ca2+ influx and whole-cell Ca2+ current in rat dorsal-root ganglion neurons

Field-potential stimulation of rat dorsal-root ganglion (DRG) neurons evoked action-potential-mediated transient increases in intracellular free calcium concentration ([Ca²⁺]_i) as measured by indo-1-based microfluorimetry. Field-potential-evoked [Ca²⁺]_i transients were abolished by tetrodotoxin, and their dependence on stimulus intensity exhibited an abrupt threshold. -Conotoxin GVIA (-CgTx, 100 nM) inhibited action-potential-mediated Ca²⁺ influx by 79%, while nitrendipine (1 M) had little effect. -Grammotoxin SIA (-GsTx, 267 nM), a peptide toxin purified from the venom of the tarantula spider, Grammostola spatulata, blocked action-potential-mediated Ca²⁺ influx as effectively as did -CgTx, suggesting that -GsTx blocks N-type Ca²⁺ channels. In contrast to block by -CgTx, the block produced by -GsTx reversed upon washout of the peptide. -GsTx (270 nM) blocked 80%, and -CgTx (1 M) blocked 64%, of whole-cell Ca²⁺ current (I _Ca) elicited by step depolarization to 0 mV from a holding potential of –80 mV. -GsTx completely occluded inhibition of I _Ca by -CgTx. However, when applied after -CgTx, -GsTx produced an additional inhibition of 27%, indicating that -GsTx also blocked a non-N-type Ca²⁺ channel. BayK8644 (1 M) elicited an increase in I _Ca in the presence of maximally effective concentrations of -GsTx, suggesting that -GsTx does not block L-type channels. Thus, -GsTx displays a selectivity for Ca²⁺ channel subtypes which should prove useful for studying Ca²⁺ channels and Ca²⁺-channel-mediated processes.     

Pharmacological block of Ca2+-activated Cl− current in rat vascular smooth muscle cells in short-term primary culture

Ca²⁺-activated Cl^– currents were studied in isolated cells from rat portal vein smooth muscle in short-term primary culture using the whole-cell patch-clamp technique. Cl^– currents can be activated separately by Ca²⁺ release from intracellular stores (in response to external applications of caffeine or noradrenaline) and by Ca²⁺ influx through voltage-dependent Ca²⁺ channels. The effects of several Cl^– channel blockers and of spironolactone (a substance known to reduce internal Ca²⁺ loading) on both Cl^– and Ca²⁺ currents were examined. Diisothiocyanostilbene-2,2-disulfonic acid (DIDS), anthracene-9-carboxylic acid (9-AC) and diphenylamine-2,2-dicarboxylic acid (DPC) inhibited the Ca²⁺-activated Cl^– current (IC₅₀ values between 16.5 and 306 M) with no effects on the inward Ca²⁺ current and on internal Ca²⁺ loading (tested by measuring the Ca²⁺-activated K⁺ current). These results indicate that the inhibition of Cl^– current by these compounds is due to a direct interaction with the Cl^– channel. In contrast, spironolactone inhibited both K⁺ and Cl^– currents (IC₅₀=7.6 M) by reducing the amount of Ca²⁺ located in the internal stores, whereas the Cl^– current activated by Ca²⁺ current through T-type Ca²⁺ channels was unchanged. This preparation and the protocols developed in this study appears to be appropriate for analysis of substances interfering with Cl^– channels or intracellular Ca²⁺ stores.     

Small-conductance Cl− channels in HT29 cells: activation by Ca2+, hypotonic cell swelling and 8-Br-cGMP

The present study demonstrates the activation of Cl^– channels in HT₂₉ cells by agonist (ATP, neurotensin, carbachol) increasing cytosolic Ca²⁺, by hypotonic cell swelling and by cGMP. Cell-attached nystatin patch-clamp (CAN) as well as slow and fast wholecell recordings were used. The cell membrane potential was depolarized in a dose-dependent manner with halfmaximal effects at 0.4 umol/l for ATP, 60 pmol/l for neurotensin and 0.8 mol/l for carbachol. The depolarization, which was caused by Cl^– conductances increases, occurred within 1 s and was accompanied by a simultaneous and reversible increase of the input conductance of the cell-attached membrane from 295±32 pS to 1180±271 pS (ATP; 10 mol/l, n=21) and 192±37 pS to 443±128 pS (neurotensin; 1 nmol/l, n=8). The effects of the agonists could be mimicked by ionomycin (0.2 umol/l), suggesting that an increase in intracellular Ca²⁺ was responsible for the activation of Cl^– channels. The depolarization was followed by a secondary hyperpolarization. Hypotonic cell swelling also depolarized the cells and induced an increase in the membrane conductance. With 120 mmol/l NaCl the depolarization was 10±0.8 mV and the cell-attached conductance increased from 228±29 pS to 410±65 (n=26) pS. NaCl at 90 mmol/l and 72.5 mmol/l had even stronger effects. Comparable conductance increases were also obtained when the different agonists or hypotonic cell swelling were examined in whole cell experiments.5-Nitro-2-(3-phenylpropylamino)-benzoate (1 mol/l) did not prevent the effects of Ca²⁺-increasing hormones and of hypotonic solutions. An increase in Cl^– conductance was also induced by 8-Br-cGMP (1 mmol/l) but not by heat-stable Escherichia coli toxin. In contrast to their conductance-increasing effects in CAN patches, the different agonists and cell swelling did not activate resolvable single channels in these cell-attached membranes. This indicates that the Cl^– channels involved have a single-channel conductance too small ( 4 pS, 150 Hz) to be resolved by our techniques.     

Influence of extracellur Ca2+ on endogenous Cl− channels in Xenopus oocytes

Removal of Ca²⁺ from the external bath solution evoked marked depolarization and large currents (up to several microamperes) in voltage-clamped defolliculated oocytes of Xenopus laevis. The resulting current was not carried by a cation influx but was due to a huge Cl^– efflux, which could be strongly inhibited by the Cl^– channel blockers flufenamic acid and niflumic acid. Removal of Mg²⁺ or Ba²⁺ from the solutions had the same effects as removing Ca²⁺. The reversal potential of –12 mV also indicated that Cl^– channels were responsible for the large currents. Patch-clamp studies revealed a single-channel slope conductance of 90 pS. During oocyte maturation these channels remained active. The half-maximal Ca²⁺ concentration of about 20 M showed that quite low doses of extracellular Ca²⁺ profoundly influence the electrical properties of the oocyte membrane.     

β-Adrenoceptor stimulation activates large-conductance Ca2+-activated K+ channels in smooth muscle cells from basilar artery of guinea pig

We studied the effect of isoproterenol on the Ca²⁺-activated K⁺(BK) channel in smooth muscle cells isolated from the basilar artery of the guinea pig. Cells were studied in a whole-cell configuration to allow the clamping of intracellular Ca²⁺ concentration, [Ca²⁺]_i. Macroscopic BK channel currents were recorded during depolarizing test pulses from a holding potential (V _H) of 0 mV, which was used to inactivate the outward rectifier. The outward macroscopic current available from aV _H of 0 mV was highly sensitive to block by external tetraethylammonium·Cl (TEA) and charybdotoxin, and was greatly augmented by increasing [Ca²⁺]_i from 0.01 to 1.0 M. With [Ca²⁺]_i between 0.1 and 1.0 M, 0.4 M isoproterenol increased this current by 58.6±17.1%, whereas with [Ca²⁺]_i at 0.01 M a sixfold smaller increase was observed. With [Ca²⁺]_i0.1 M, 100 M dibutyryl-adenosine 3:5: cyclic monophosphate (cAMP) and 1 M forskolin increased this current by 58.5±24.1% and 59.7±10.3%, respectively. The increase with isoproterenol was blocked by 4.0 M propranolol extracellularly, and by 10 U/ml protein kinase inhibitor intracellularly. Single-channel openings during depolarizing test pulses from aV _H of 0 mV recorded in the whole-cell configuration under the same conditions (outside-outwhole-cell recording) indicated a slope conductance of 260 pS. In conventional outside-out patches, this 260-pS channel was highly sensitive to block by external TEA, and in inside-out patches, its probability of opening was greatly augmented by increasing [Ca²⁺]_i from 0.01 to 1.0 M. Outside-out-whole-cell recordings with [Ca²⁺]_i0.1 M indicated that 100 M dibutyryl-cAMP increased the probability of opening of the 260-pS channel by 152±115%. In inside-out patches, the catalytic subunit of protein kinase A increased the probability of opening, and this effect also depended on [Ca²⁺]_i, with a 35-fold larger effect observed with 0.1–0.5 M Ca²⁺ compared to 0.01 M Ca²⁺. We conclude that the BK channel in cerebrovascular smooth muscle cells can be activated by-adrenoceptor stimulation, that the effect depends strongly on [Ca²⁺]_i, and that the effect is mediated by cAMP-dependent protein kinase A with no important contribution from a direct G-protein or phosphorylation-independent mechanism. Our data indicate that the BK channel may participate in-adrenoceptor-mediated relaxation of cerebral vessels, although the importance of this pathway in obtaining vasorelaxation remains to be determined.