Opposite effects of tolbutamide and diazoxide on the ATP-dependent K+ channel in mouse pancreatic β-cells

The influence of the antidiabetic sulphonylurea tolbutamide on K⁺ channels of mouse pancreatic -cells was investigated using different configurations of the patch clamp technique. The dominant channel in resting cells is a K⁺ channel with a single-channel conductance of 60 pS that is inhibited by intracellular ATP or, in intact cells, by stimulation with glucose. In isolated patches of -cells membrane, this channel was blocked by tolbutamide (0.1 mM) when applied to either the intracellular or extracellular side of the membrane. The dose-dependence of the tolbutamide-induced block was obtained from whole-cell experiments and revealed that 50% inhibition was attained at approximately 7 M. In cell-attached patches low concentrations of glucose augmented the action of tolbutamide. Thus, the simultaneous presence of 5 mM glucose and 0.1 mM tolbutamide abolished channel activity and induced action potentials. These were not produced when either of these substances was added alone at these concentrations. The inhibitory action of tolbutamide or glucose on the K⁺ channel was counteracted by the hyperglycaemic sulphonamide diazoxide (0.4 mM). Tolbutamide (1 mM) did not affect Ca²⁺-dependent K⁺ channels. It is concluded that the hypo- and hyperglycaemic properties of tolbutamide and diazoxide reflect their ability to induce the closure or opening, respectively, of ATP-regulated K⁺ channels.     

Effects of T-type, L-type, N-type, P-type, and Q-type calcium channel blockers on stimulus-induced pre-and postsynaptic calcium fluxes in rat hippocampal slices

The contribution of T-, L-, N-, P-, and Q-type Ca²⁺ channels to pre-and postsynaptic Ca²⁺ entry during stimulus-induced high neuronal activity in area CA1 of rat hippocampal slices was investigated by measuring the effect of specific blockers on stimulus-induced decreases in extracellular Ca²⁺ concentration ([Ca²⁺]₀). [Ca²⁺]₀ was measured with ion-selective electrodes in stratum radiatum (SR) and stratum pyramidale (SP), while Ca²⁺ entry into neurons was induced with stimulus trains (20 Hz for 10 s) alternately delivered to SR and the alveus, respectively. The [Ca²⁺]₀ decreases recorded in SR in response to SR stimulation represented mainly presynaptic Ca²⁺ entry (Ca_pre), while [Ca²⁺]⁰ decreases recorded in SP in response to alvear stimulation were predominantly based on postsynaptic Ca²⁺ entry (Ca_post). Ethosuximide and trimethadione were ineffective m concentrations up to 1 mM. At 10 mM, they reduced Ca_post and, much less, also Ca_pre Nimodipine (25 M) reduced Ca_post and, to a minor extent, Ca_pre. -Agatoxin IVA (0.4–1 M) and -conotoxin MVIIC (1 M) also reduced both Ca_pre and Ca_post, but with a stronger action on Ca_pre. -Conotoxin GVIA (3–8 M) reduced Ca_post without effect on Ca_pre. We conclude that during stimulus-induced, high-frequency neuronal activity Ca_post is carried by P/Q-, N-, and L-type channels and probably a further channel type different from these channels. Ca_pre includes at least P/Q-and possibly L-type channels. N-type channels did not contribute to Ca_pre in our experiments. Since ethosuximide and trimethadione were only effective in high concentrations, their action may be unspecific. Thus, T-type channels do not seem to play a major part in Ca²⁺ entry in this situation.     

Bursting electrical activity in pancreatic β-cells: evidence that the channel underlying the burst is sensitive to Ca2+ influx through L-type Ca2+ channels

In glucose-stimulated pancreatic -cells, the membrane potential alternates between a hyperpolarized silent phase and a depolarized phase with Ca²⁺ action potentials. The molecular and ionic mechanisms underlying these bursts of electrical activity remain unknown. We have observed that 10.2–12.8 mM Ca²⁺, 1 M Bay K 8644 and 2 mM tetraethylammonium (TEA) trigger bursts of electrical activity and oscillations of intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in the presence of 100 M tolbutamide. The [Ca²⁺]_i was monitored from single islets of Langerhans using fura-2 microfluorescence techniques. Both the high-Ca²⁺ and Bay-K-8644 evoked [Ca²⁺]_i oscillations overshot the [Ca²⁺]_i recorded in tolbutamide. Nifedipine (10–20 M) caused an immediate membrane hyperpolarization, which was followed by a slow depolarization to a level close to the burst active phase potential. The latter depolarization was accompanied by suppression of spiking activity. Exposure to high Ca²⁺ in the presence of nifedipine caused a steady depolarization of approximately 8 mV. Ionomycin (10 M) caused membrane hyperpolarization in the presence of 7.7 mM Ca²⁺, which was not abolished by nifedipine. Charybdotoxin (CTX, 40–80 nM), TEA (2 mM) and quinine (200 M) did not suppress the high-Ca²⁺-evoked bursts. It is concluded that: (1) the channel underlying the burst is sensitive to [Ca²⁺]_i rises mediated by Ca²⁺ influx through L-type Ca²⁺ channels, (2) both the ATP-dependent K⁺ channel and the CTX and TEA-sensitive Ca²⁺-dependent K⁺ channel are highly unlikely to provide the pacemaker current underlying the burst. We propose that the burst is mediated by a distinct Ca²⁺-dependent K⁺ channel and/or by [Ca²⁺]_idependent slow processes of inactivation of Ca²⁺ currents.     

Identification of single transiently opening (“A-type”) K channels in guinea-pig colonic myocytes

Two K⁺ channel populations were identified in depolarized cell-attached membrane patches of myocytes freshly dispersed from the circular smooth muscle of guinea-pig proximal colon. First, a large-conductance (150 pS) Ca²⁺-activated K⁺ channel which was non-inactivating and sensitive to blockade by tetraethylammonium (TEA, 0.5–5 mM); and second, a smaller conductance K⁺ channel which opened and closed within 100 ms, was insensitive to TEA (0.5–5 mM), but was blocked by 5 mM 4-aminopyridine (4-AP) or maintained depolarization, and which had a unitary conductance of 12–13 pS. The averaged time course of these smaller conductance K⁺ channels closely resembled the time course of the 4-AP-sensitive, Ca²⁺-insensitive transient outward K⁺ current recorded in the whole-cell recording mode.     

The nonselective cation channel in the basolateral membrane of rat exocrine pancreas

Nonselective Ca²⁺-sensitive cation channels in the basolateral membrane of isolated cells of the rat exocrine pancreas were investigated with the patch clamp technique. With 1.3 mmol/l Ca²⁺ on the cytosolic side, the mean openstate probabilityP _o of one channel was about 0.5. In insideout oriented cell-excised membrane patches the substances diphenylamine-2-carboxylic acid (DPC), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and 3,5-dichlorodiphenylamine-2-carboxylic acid (DCDPC) were applied to the cytosolic side. These compounds inhibited the nonselective cation channels by increasing the mean channel closed time (slow block). 100 mol/l of NPPB or DPC decreasedP _o from 0.5 (control conditions) to 0.2 and 0.04, respectively, whereas 100 mol/l of DCDPC blocked the channel completely. All effects were reversible. 1 mmol/l quinine also reducedP _o, but in contrast to the abov mentioned substances, it induced fast flickering. Ba²⁺ (70 mmol/l) and tetraethylammonium (TEA⁺; 20 mmol/l) had no effects. We investigated also the stilbene disulfonates 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS), 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) and 4,4-dinitro-2,2-stilbenedisulfonate (DNDS). 10 mol/l SITS applied to the cytosolic side increasedP _o from 0.5 to 0.7 and with 100 mol/l SITS the channels remained nearly permanently in its open state (P _o1). A similar activation of the channels was also observed with DIDS and DNDS. These effects were poorly reversible. The stilbene disulfonates acted by increasing the channel mean open time. When the channel was inactivated by decreasing bath Ca²⁺ concentration to 0.1 mol/l, addition of 100 mol/l of SITS had no effect. Similarly, reducing bath Ca²⁺ concentration from 1.3 mmol/l in presence of 100 mol/l SITS (channels are maximally activated) to 0.1 mol/l, inactivated the channels completely. These results demonstrate, that SITS can only activate the channels in the presence of Ca²⁺. SITS had no effects, when applied to the extracellular side in outside out patches. In summary, the substances DPC, NPPB and DCDPC inhibit nonselective cation channels, where DCDPC has the most potent and NPPB the smallest effect; whereas SITS, DIDS and DNDS activate the channel when applied from the cytosolic side in the presence of Ca²⁺ ions.     

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.     

Multiple calcium channel subtypes in isolated rat chromaffin cells

By using the whole-cell configuration of the patch-clamp technique we have investigated the pharmacological properties of Ca²⁺ channels in short-term cultured rat chromaffin cells. In cells held at a membrane potential of –80 mV, using 10 mM Ba²⁺ as the charge carrier, only high-voltage-activated (HVA) Ca²⁺ channels were found. Ba²⁺ currents (I _Ba) snowed variable sensitivity to dihydropyridine (DHP) Ca²⁺ channel agonists and antagonists. Furnidipine, a novel DHP antagonist, reversibly blocked the current amplitude by 22% and 48%, at 1 M and 10 M respectively, during short (15–50 ms) depolarizing pulses to 0 mV. The L-type Ca²⁺ channel agonist Bay K 8644 (1 M) caused a variable potentiation of HVA currents that could be better appreciated at low rather than at high depolarizing steps. Increase of I _Ba was accompanied by a 20-mV shift in the activation curves for Ca²⁺ channels towards more hyperpolarizing potentials. Application of the conus toxin -conotoxin GVIA (GVIA; 1 M) blocked 31% of I _Ba; blockade was irreversible upon removal of the toxin from the extracellular medium, -Agatoxin IVA (IVA; 100 nM) produced a 15% blockade of I _Ba. -Conotoxin MVIIC (MVIIC; 5 M) produced a 36% blockade of I _Ba; such blockade seems to be related to both GVIA-sensitive (N-type) and GVIA-resistant Ca²⁺ channels. The sequential addition of supramaximal concentrations of furnidipine (10 M), GVIA (1 M), IVA (100 nM) and MVIIC (3 M) produced partial inhibition of I _Ba, which were additive. Our data suggest that the whole cell I _Ba in rat chromaffin cells exhibits at least four components. About 50% of I _Ba is carried by L-type Ca²⁺ channels, 30% by N-type Ca²⁺channels and 15% by P-type Ca²⁺ channels. These figures are close to those found in cat chromaffin cells. However, they differ considerably from those found in bovine chromaffin cells where P-like Ca²⁺channels account for 45% of the current, N-type carry 35% and L-type Ca²⁺ channels are responsible for only 20–25% of the current. These drastic differences might have profound physiological implications for the relative contribution of each channel subtype to the regulation of catecholamine release in different animal species.     

Glucose dependent K+-channels in pancreaticβ-cells are regulated by intracellular ATP

The resting conductance of cultured-cells from murine pancreases was investigated using the whole-cell, cell-attached and isolated patch modes of the patch-clamp technique. Whole-cell experiments revealed a high input resistance of the cells (>20 G per cell or>100 k·cm²), if the medium dialysing the cell interior contained 3 mM ATP. The absence of ATP evoked a large additional K⁺ conductance. In cell-attached patches single K⁺-channels were observed in the absence of glucose. Adition of glucose (20 mM) to the bath suppressed the channel activity and initiated action potentials. Similar single-channel currents were recorded from isolated patches. In this case the channels were reversibly blocked by adding ATP (3 mM) to the solution at the intracellular side of the membrane. The conductances (51 pS and 56 pS for [K⁺]₀=145 mM, T=21° C) and kinetics (at –70 mV: _open=2.2 ms and _closed=0.38 ms and 0.33 ms) of the glucose- and ATP-dependent channels were found to be very similar. It is concluded that both channels are identical. The result suggests that glucose could depolarize the-cell by increasing the cytoplasmic concentration of ATP.     

Forskolin-induced block of delayed rectifying K+ channels in pancreaticβ-cells is not mediated by cAMP

K⁺ channels in the membrane of murine pancreatic -cells were studied using the patch-clamp technique. The delayed outward current was activated in whole-cell experiments by depolarizing voltage pulses to potentials between –30 mV and 0 mV. Forskolin blocked the current rapidly (<5 s) and reversibly with 50% inhibition at 13 M. The inhibition did not depend on a stimulation of the adenylate cyclase since it occurred even in presence of 1 mM cAMP in the pipette solution which replaced the cytoplasm. Membrane permeant cAMP analogues and phosphodiesterase inhibitors did not influence the delayed outward current. In experiments on outside-out patches forskolin (100 M) shortened the openings of a channel of about 10 pS conductance at 0 mV and a time course of activation and inactivation similar to the whole-cell current. Another smaller, slowly activating channel and the Ca²⁺- and ATP-dependent K⁺ channels were influenced only weakly or not at all. It is therefore concluded that the 10-pS channel generates most of the delayed outward K⁺ current in murine pancreatic -cells. The Ca²⁺-independent part of the delayed outward current in bovine adrenal chromaffin cells was also blocked by forskolin (100 M).     

The Ba2+ block of the ATP-sensitive K+ current of mouse pancreaticβ-cells

We studied the block of whole-cell ATP-sensitive K⁺ (K_ATP) currents in mouse pancreatic-cells produced by external Ba²⁺. Ba²⁺ produced a time- and voltage-dependent block of K_ATP currents, both the rate and extent of the block increasing with hyperpolarization. With 5.6 mM [K⁺]_o, the relationship between the steady-state KATP current and [Ba²⁺]_o, was fit by the Hill equation with aK _d of 12.5 ± 2.8 M at –123 mV and of 0.18 ± 0.02 mM at –62 mV The Hill coefficient (n) was close to 1 at all potentials indicating that binding of a single Ba²⁺ ion is sufficient to block the channel. When [K⁺]_o was raised to 28 mM the K_d was little changed (12.4 ± 4.1 gM at –123 mV 0.27 ± 0.05 mM at –62 mV) and n was unaffected, suggesting that K⁺ does not interact with the Ba²⁺ binding site. The kinetics of Ba²⁺ block were slow, 10 M Ba²⁺ blocking the K_ATP current with a time constant of 20 ms at –123 mV in 28 mM [K⁺]_o. The blocking rate constant was calculated as 1.7 mM^–1 ms^–1 and the unblocking rate as 0.02 ms^–1, at –123 mV The data are discussed in terms of a model in which Ba²⁺ binds to a site at the external mouth of the channel to inhibit the K_ATP channel.     

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.     

Modulation of single slow (L-type) calcium channels by intracellular ATP in vascular smooth muscle cells

Involvement of ATP in the regulation of slow (L-type) Ca²⁺ channels of vascular smooth muscle cells was investigated by recording single Ca²⁺ channel currents (single-channel conductance of 18 pS) using a patch clamp technique. In the cell-attached configuration, intracellular composition was modified by permeabilizing the cell membrane with mechanical disruption at one end of the cell. Single cells were freshly isolated from guinea-pig portal vein by collagenase treatment. For the channel recordings, the pipette solution contained 100 mM Ba²⁺ and the bath contained K⁺-rich solution (with 5 mM EGTA) to depolarize the membrane to near 0 mV. The channel activity decreased usually within 3 min after permeabilizing the cell end and exposure to ATP-free bath solution. If ATP (1–5 mM) was applied to the bath (access to cell interior) before complete disappearance of channel activity, channel activity was partially recovered. ATP did not change the current amplitude (i) or the mean open time of the channels, whereas the number of channels available for opening and/or the probability of their being open (NP _o) were increased by ATP. A non-hydrolyzable analogue of ATP, AMP-PNP, did not exert an ATP-like effect; ATP--S had a weak effect. With 1 M Bay-K-8644 (Ca²⁺ channel agonist) in the pipette, the activity of the Ca²⁺ channel was high; such activity persisted for more than 10 min after permeabilizing the cell and exposting to ATP-free solution containing KCN (1 mM) and 2-deoxy-d-glucose (10 mM). These results indicate that activation of slow Ca²⁺ channels requires ATP. The effect of ATP may be exerted by phosphorylation and/or an energy-requiring step. Bay-K-8644 may change the nature of the slow Ca²⁺ channel, making it resistant to rundown.     

Biophysical properties of Ca2+- and Mg-ATP-activated K+ channels in pulmonary arterial smooth muscle cells isolated from the rat

A novel class of Ca²⁺-activated K⁺ channel, also activated by Mg-ATP, exists in the main pulmonary artery of the rat. In view of the sensitivity of these K_Ca,ATP channels to such charged intermediates it is possible that they may be involved in regulating cellular responses to hypoxia. However, their electrophysiological profile is at present unknown. We have therefore characterised the sensitivity of K_Ca,ATP channels to voltage, intracellular Ca²⁺ ([Ca²⁺]_i) and Mg-ATP. They have a conductance of 245 pS in symmetrical K⁺ and are approximately 20 times more selective for K⁺ ions than Na⁺ ions, with a K⁺ permeability (P _K) of 4.6×10^–13cm s^–1. Ca²⁺ ions applied to the intracellular membrane surface of K_Ca,ATP channels causes a marked enhancement of their activity. This activation is probably the result of simultaneous binding of at least two Ca²⁺ ions, determined using Hill analysis, to the channel or some closely associated protein. This results in a shift of the voltage activation threshold to more hyperpolarized membrane potentials. The activation of K_Ca,ATP channels by Mg-ATP has an EC₅₀ of approximately 50 M. Although the EC₅₀ is unaffected by [Ca²⁺]_i, channel activation by Mg-ATP is enhanced by increasing [Ca²⁺]_i. One possible interpretation of these data is that Mg-ATP increases the sensitivity of K_Ca,ATP channels to Ca²⁺. It is therefore possible that under hypoxic conditions, where lower levels of Mg-ATP may be encountered, the sensitivity of K_Ca,ATP channels to Ca²⁺ and therefore voltage is reduced. This would tend to induce a depolarising influence, which would favour the influx of Ca²⁺ through voltage-activated Ca²⁺ channels, ultimately leading to increased vascular tone.     

Intracellular ADP activates ATP-sensitive K+ channels in vascular smooth muscle cells of the guinea pig portal vein

Vasodilatation following tissue ischemia is assumed to partially result from activation of ATP-dependent K⁺ channels (K_ATP). To assess the effect of cytosolic adenosine nucleotides, the balance of which depends on tissue pO₂, on K_ATP, we have measured steady state outward currents (SSC) by the whole-cell clamp technique in smooth muscle cells of the guinea pig portal vein at different concentrations of ATP and ADP in the pipette solution. Glibenclamide, a selective inhibitor of K_ATP, was used as a pharmacological tool. — With no nucleotides in the pipette solution (Ca²⁺-free), the SSC determined at +20 mV was unaffected by glibenclamide, while with 0.1 mM ATP or with 0.1 mM ADP, the SSC exhibited a glibenclamide-sensitive component indicating activation of K_ATP. At 5 mM ATP and no ADP, hardly any effect of glibenclamide on the SSC was detected, suggesting inhibition of K_ATP by this high concentration of ATP. With 0.1 mM ADP at 5 mM ATP however, activation of K_ATP was achieved. — At 10^–7 M Ca²⁺ in the pipette solution, an increased SSC was measured, but the responses to the nucleotides and/or glibenclamide were not modified. — These findings suggest that in vivo, ADP may be involved in the regulation of vascular K_ATP, linking tissue pO₂ with vascular tone and tissue perfusion.     

Tetraethylammonium ion sensitivity of a 35-pS Ca2+-activated K+ channel in GH3 cells that is activated by thyrotropin-releasing hormone

Single Ca²⁺-activated K⁺ channels were studied in membrane patches from the GH₃ anterior pituitary cell line. We have previously demonstrated the coexistence of large-conductance and small-conductance (280 pS and 11 pS in symmetrical 150 mM K⁺, respectively) Ca²⁺-activated K⁺ channels in this cell line (Lang and Ritchie 1987). Here we report the existence of a third type of Ca²⁺-activated K⁺ channel that has a conductance of about 35 pS under similar conditions. In excised inside-out patches, this channel can be activated by elevations of the internal free Ca²⁺ concentration, and the open probability increases as the membrane potential is made more positive. In excised patches, the sensitivity of this 35-pS channel to internal Ca²⁺ is low; at positive membrane potentials, this channel requires Ca²⁺ concentrations greater than 10 M for activation. However, 35-pS channels have a much higher sensitivity to Ca²⁺ in the first minute after excision (activated by 1 M Ca²⁺ at –50 mV). Therefore, it is possible that the Ca²⁺ sensitivity of this channel is stabilized by intracellular factors. In cell-attached patches, this intermediate conductance channel can be activated (at negative membrane potentials) by thyrotropin-releasing hormone-induced elevations of the intracellular Ca²⁺ concentration and by Ca²⁺ influx during action potentials. The intermediate conductance channel is inhibited by high concentrations of external tetraethylammonium ions (K _d=17 mM) and is relatively resistant to inhibition by apamin.     

Modulation of Ca2+-activated K+ channels by Mg2+ and ATP in frog oxyntic cells

Ca²⁺-activated K⁺ channels in the basolateral plasma membrane of bullfrog oxynticopeptic cells are intimately involved in the regulation of acid secretion. Patch-clamp techniques were applied to study the regulating mechanism of these channels. In the excised inside-out configuration, intracellular Mg²⁺ decreased channel activity in a dose-dependent manner. In the absence of Mg²⁺, administration of adenosine 5 triphosphate (ATP) to the cytoplasmic side also inhibited channel activity. On the other hand, in the presence of Mg²⁺, addition of ATP markedly increased channel activity. At a fixed concentration of free Mg²⁺ the Mg-ATP complex caused channel activation and shifted the dose response relationship between channel activity and the intracellular Ca²⁺ concentration to the left. A nonhydrolysable ATP analogue, adenosine 5-[,-imido]triphosphate (AMP-PNP) adenylyl [,-methylene]diphosphate (AMP-PCP), could not substitute for ATP in channel activation, but a hydrolysable ATP analogue, adenosine 5-O-(3-thiotriphosphate) (ATP[S]) could do so. Furthermore, application of alkaline phosphatase to the cytoplasmic side inhibited channel activity. These results demonstrate that Ca²⁺-activated K⁺ channels are regulated by Mg²⁺ and ATP, and suggest that a phosphorylation reaction may be involved in the regulation mechanism of these channels.     

Effects of 2,3-butanedione monoxime on smooth-muscle contraction of guinea-pig portal vein

Effects of 2,3-butanedione-2-monoxime (BDM) on the contraction of intact and skinned smooth muscles from guinea-pig portal vein were examined. In intact preparations loaded with fura-2, 5–10 mM BDM markedly suppressed Ca²⁺ transients and force developments induced by 154 mM potassium and by phenylephrine (0.1 mM). On the other hand, in Ca²⁺-free depolarizing solution, BDM did not suppress phenylephrine (0.1 mM)-induced Ca²⁺ transient and force development. In skinned preparations obtained with Staphylococcus aureus -toxin treatment, BDM did not markedly affect active force development. The above results indicate that BDM suppresses contraction of the portal vein mainly by the inhibition of voltage-dependent cytosolic Ca²⁺ transients. An additional result suggests that BDM suppresses the force-enhancing effect of ₁-adrenergic agents on the contractile elements.     

Expression of calcium channel subunits in the normal and diseased human myocardium

We investigated the expression of ₁ and subunits of the L-type Ca²⁺ channel on the protein level in cardiac preparations from normal human heart ventricles and from the hypertrophied septum of patients with hypertrophic obstructive cardiomyopathy (HOCM). 1,4-Dihydropyridine (DHP) binding and immunorecognition by polyclonal antibodies directed against the C-terminal amino acid sequences of the ₂ and ₃ subunits were used for detection and quantification of ₁, ₂, and ₃ subunits. B_max of high-affinity DHP binding was 35±2 fmol/mg protein in HOCM and 20±2 fmol/mg protein in normal human hearts (P<0.05). In rabbit hearts the anti-₂ subunit antibody immunoprecipitated 80% of the total amount of DHP-labeled Ca²⁺ channels present in the assay. Under identical experimental conditions 25% of labeled Ca²⁺ channels were recovered in the immunoprecipitates of both normal and HOCM ventricles. A similar partial immunoprecipitation was observed in pig hearts. Immunoblot analysis demonstrated that the ₂ subunit was associated with the DHP receptor/Ca²⁺ channel in cardiac muscle of rabbit, pig, and human heart. In neither of these purified cardiac Ca²⁺ channels was the ₃ subunit isoform detected. Our results suggest that both ₁ and ₂ subunit expression is upregulated in HOCM in a coordinate manner.Abbreviations B _max Maximal number of binding sites - DHP 1,4-Dihydropyridine - HOCM Hypertrophic obstructive cardiomyopathy - NH Normal human heart     

A single non-L-, non-N-type Ca2+ channel in rat insulin-secreting RINm5F cells

Single high-voltage-activated (HVA) Ca²⁺ channel activity was recorded in rat insulinoma RINm5F cells using cell-attached and outside-out configurations. Single-channel recordings revealed three distinct Ca²⁺ channel subtypes: one sensitive to dihydropyridines (DHPs)-(L-type), another sensitive to -conotoxin (CTx)-GVIA (N-type) and a third type insensitive to DHPs and -CTx-GVIA (non-L-, non-N-type). The L-type channel was recorded in most patches between –30 and +30 mV The channel had pharmacological and biophysical features similar to the L-type channels described in other insulin-secreting cells (mean conductance 21 pS in control conditions and 24 pS in the presence of 5 M Bay K 8644). The non-L-, non-N-type channel was recorded in cells chronically treated with -CTx-GVIA in the presence of nifedipine to avoid the contribution of N- and L-type channels. Channel activity was hardly detectable below –10 mV and was recruited by negative holding potentials (< –90 mV). The channel open probability increased steeply from –10 to +40 mV Different unitary current sublevels could be detected and the current voltage relationship was calculated from the higher amplitude level with a slope conductance of 21 pS. Channel activity lasted throughout depolarizations of 300–800 ms with little sign of inactivation. Above 0 mV the channel showed a persistent flickering kinetics with brief openings (₀ 0.6 ms) and long bursts (_burst 60 ms) interrupted by short interburst intervals. The third HVA Ca²⁺ channel subtype, the N-type, had biophysical properties similar to the non-L-, non-N-type and was best identified in outside-out patches by its sensitivity to -CTx-GVIA. The channel was detectable only above –10 mV from a –90 mV holding potential, exhibited a fast flickering behaviour, persisted during prolonged depolarizations and had a slope conductance of about 19 pS. The present data provide direct evidence for a slowly inactivating non-L-, non-N-type channel in insulin-secreting RINm5F cells that activates at more positive voltages than the L-type channel and indicate the possibility of identifying unequivocally single HVA Ca²⁺ channels in cell-attached and excised membrane patches under controlled pharmacological conditions.     

Contribution of epoxyeicosatrienoic acids to the hypoxia-induced activation of Ca-activated K channel current in cultured rat hippocampal astrocytes

Brief hypoxia differentially regulates the activities of Ca²⁺-activated K⁺ channels (K_Ca) in a variety of cell types. We investigated the effects of hypoxia (<2% O₂) on K_Ca channel currents and on the activities of cytochrome P450 2C11 epoxygenase (CYP epoxygenase) in cultured rat hippocampal astrocytes. Exposure of astrocytes to hypoxia enhanced macroscopic outward K_Ca current, increased the open state probability (NPo) of 71 pS and 161 pS single-channel K_Ca currents in cell-attached patches, but failed to increase the NPo of both the 71 pS and 161 pS K_Ca channel currents recorded from excised inside-out patches. The hypoxia-induced enhancement of macroscopic K_Ca current was attenuated by pretreatment with tetraethylammonium (TEA, 1 mM) or during recording using low-Ca²⁺ external bath solution. Exposure of astrocytes to hypoxia was associated with generation of superoxide as detected by staining of cells with the intracellular superoxide detection probe hydroethidine (HE), attenuation of the hypoxia-induced activation of unitary K_Ca channel currents by superoxide dismutation with tempol, and as quantitated by high-pressure liquid chromatography/fluorescence assay using HE as a probe. In cultured astrocytes in which endogenous CYP epoxygenase activity has been inhibited with either miconazole or N-methylsulfonyl-6-(2-propargyloxyphenyl) hexanamide (MSPPOH) hypoxia failed to increase the NPo of both the 71 pS and 161 pS K_Ca currents and generation of superoxide. Hypoxia increased the level of P450 epoxygenase protein and production of epoxyeicosatrienoic acids (EETs) from cultured astrocytes, as determined by immunohistochemical staining and LC/MS analysis, respectively. Exogenous 11,12-EET increased the NPo of both the 71 pS and 161 pS K_Ca single-channel currents only in cell-attached but not in excised inside-out patches of cultured astrocytes. These findings indicate that hypoxia enhances the activities of two types of unitary K_Ca currents in astrocytes by a mechanism that appears to involve CYP epoxygenase-dependent generation of superoxide and increased production or release of EETs.