Adenine nucleotides and intracellular Ca2+ regulate a voltage-dependent and glucose-sensitive potassium channel in neurosecretory cells

Effects of membrane potential, intracellular Ca²⁺ and adenine nucleotides on glucose-sensitive channels from X organ (XO) neurons of the crayfish were studied in excised inside-out patches. Glucose- sensitive channels were selective to K⁺ ions; the unitary conductance was 112 pS in symmetrical K⁺, and the K⁺ permeability (P _K) was 1.3 × 10⁻¹³ cm ⋅s⁻¹. An inward rectification was observed when intracellular K⁺ was reduced. Using a quasi-physiological K⁺ gradient, a non-linear K⁺ current/voltage relationship was found showing an outward rectification and a slope conductance of 51 pS. The open-state probability (P _o) increased with membrane depolarization as a result of an enhancement of the mean open time and a shortening of the longer period of closures. In quasi-physio- logical K⁺ concentrations, the channel was activated from a threshold of about −60 mV, and the activation midpoint was −2 mV. P _o decreased noticeably at 50 μM internal adenosine 5′-triphosphate (ATP), and single-channel activity was totally abolished at 1 mM ATP. Hill analysis shows that this inhibition was the result of simultaneous binding of two ATP molecules to the channel, and the half-blocking concentration of ATP was 174 μM. Internal application of 5′-adenylylimidodiphosphate (AMP-PNP) as well as glibenclamide also decreased P _o. By contrast, the application of internal ADP (0.1 to 2 mM) activated this channel. An optimal range of internal free Ca²⁺ ions (0.1 to 10 μM) was required for the activation of this channel. The glucose--sensitive K⁺ channel of XO neurons could be considered as a subtype of ATP-sensitive K⁺ channel, contributing substantially to macroscopic outward current. Received: 13 November 1995/Received after revision and accepted: 13 December 1995     

Effect of alkalinization of cytosolic pH by amines on intracellular Ca2+ activity in HT29 cells

The effect of secondary, tertiary and quaternary methyl- and ethylamines on intracellular pH (pH_i) and intracellular Ca²⁺ activity ([Ca²⁺]_i) of HT₂₉ cells was investigated microspectrofluorimetrically using pH- and Ca²⁺- sensitive fluorescent indicators, [i.e. 2′,7′-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) and fura-2 respectively]. Membrane voltage (V _m) was studied by the patch-clamp technique. Secondary and tertiary amines led to a rapid and stable concentration-dependent alkalinization which was independent of their pK _a value. Trimethylamine (20 mmol/l) increased pH_i by 0.78 ± 0.03 pH units (n = 9) and pH remained stable for the application time. Removal led to an undershoot of pH_i and a slow and incomplete recovery: pH_i stayed 0.26 ± 0.06 pH units more acid than the resting value. The quaternary amines, tetramethyl- and tetraethylamine were without influence on pH_i. All tested secondary and tertiary amines (dimethyl-, diethyl-, trimethyl-, and triethyl-amine) induced a [Ca²⁺]_i transient which reached a peak value within 10–25 s and then slowly declined to a [Ca²⁺]_i plateau. The initial Δ[Ca²⁺]_i induced by trimethylamine (20 mmol/l) was 160 ± 15 nmol/l (n = 17). The [Ca²⁺]_i peak was independent of the Ca²⁺ activity in the bath solution, but the [Ca²⁺]_i plateau was significantly lower under Ca²⁺-free conditions and could be immediately interrupted by application of CO₂ (10%; n = 6), a manoeuvre to acidify pH_i in HT₂₉ cells. Emptying of the carbachol- or neurotensin-sensitive intracellular Ca²⁺ stores completely abolished this [Ca²⁺]_i transient. Tetramethylamine led to higher [Ca²⁺]_i changes than the other amines tested and only this transient could be completely blocked by atropine (10⁻⁶ mol/l). Trimethylamine (20 mmol/l) hyperpolarized V _m by 22.5 ± 3.7 mV (n = 16) and increased the whole-cell conductance by 2.3 ± 0.5 nS (n = 16). We conclude that secondary and tertiary amines induce stable alkaline pH_i changes, release Ca²⁺ from intracellular, inositol-1,4,5-trisphosphate-sensitive Ca²⁺ stores and increase Ca²⁺ influx into HT₂₉ cells. The latter may be related to both the store depletion and the hyperpolarization. Received: 11 September 1995/Received after revision and accepted: 18 December 1995     

Attenuation of stimulated Ca2+ influx in colonie epithelial (HT29) cells by cAMP

In HT₂₉ colonic epithelial cells agonists such as carbachol (CCH) or ATP increase cytosolic Ca²⁺ activity ([Ca²⁺]_i) in a biphasic manner. The first phase is caused by inositol 1,4,5-trisphophate-(Ins P ₃-) mediated Ca²⁺ release from their respective stores and the second plateau phase is mainly due to stimulated transmembraneous Ca²⁺ influx. The present study was undertaken to examine the effect of increased adenosine 3′,5′-cyclic monophasphate (cAMP) (forskolin 10 μmol/l = FOR) on the Ca²⁺ transient in the presence of CCH (100 μmol/l). In unpaired experiments it was found that FOR induced a depolarization and reduced cytosolic Ca²⁺ ([Ca²⁺]_i, measured as the fura-2 fluorescence ratio 340/380 nm) significantly. Dideoxyforskolin had no such effect. The effect of FOR was abolished when the cells were depolarized by a high-K⁺ solution. In further paired experiments utilizing video imaging in conjunction with whole-cell patch-clamp, [Ca²⁺]_i was monitored separately for the patch-clamped cell and three to seven neighbouring cells. In the presence of CCH, FOR reduced [Ca²⁺]_i uniformly from a fluorescence ratio (345/380) of 2.9 ± 0.12 to 1.8 ± 0.07 in the patch-clamped cell and its neighbours (n = 48) and depolarized the membrane voltage (V _m) of the patch-clamped cells significantly and reversibly from −54 ± 7.4 to −27 ± 5.9 mV (n = 6). In additional experiments V _m was depolarized by 15–54 mV by various increments in the bath K⁺ concentration. This led to corresponding reductions in [Ca²⁺]_i. Irrespective of the cause of depolarization (high K⁺ or FOR) there was a significant correlation between the change in V _m and change in [Ca²⁺]_i. These data indicate that the cAMP-mediated attenuation of Ca²⁺ influx is caused by the depolarization produced by this second messenger. Received: 12 March 1996/Accepted: 2 April 1996     

Modulation of Ca2+ channels by intracellular Mg2+ ions and GTP in frog ventricular myocytes

Under conditions of low intracellular [Mg²⁺] ([Mg²⁺]_i), achieved by dialysis with pipette solutions containing ethylenediamine tetraacetic acid (EDTA), 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) and adenosine triphosphate (ATP) as chelator, calcium currents through the L-type calcium channels (I _Ca) were increased in frog ventricular myocytes. Total suppression of phosphorylation by depleting the cell of ATP with a cocktail of β,γ-methyleneadenosine 5′-triphosphate (AMP-PCP) 2-deoxyglucose and carboxylcyanide-M-chlorophenylhydrazone (CCCP) did not inhibit the increase in I _Ca in the Mg²⁺-deficient medium. Thus, the involvement of phosphorylation process in the increase in I _Ca was not likely. Effective suppression of this enhancement of I _Ca was achieved by the application of guanosine triphosphate (GTP). From the dose-response curve for GTP, the GTP concentration required for half-maximal inhibition (IC₅₀) was estimated to be 4.0 μM at pMg 6. This GTP-induced suppression of I _Ca is not due to the guanine nucleotide binding protein (G-protein) cascade, because both activators and inhibitors of G-protein, which are structural analogues of GTP, suppressed I _Ca similarly. Treatment with pertussis toxin (PTX) did not affect the inhibitory action of Mg²⁺ and GTP on I _Ca. GTP is therefore assumed to bind directly to the Ca²⁺ channel. Interaction of Mg²⁺ and GTP with the Ca²⁺ channel activated in the Mg²⁺-deficient medium was examined by comparing the dose/response curves for GTP at two different [Mg²⁺]. The IC₅₀ for GTP suppression was estimated to be 5.7 μM at pMg 6 and 6.9 μM at pMg 5. The results suggest strongly that Mg²⁺ and GTP independently bind and control Ca²⁺ channels. Received: 22 December 1995/Received after revision and accepted: 11 March 1996     

Effects of the Na+/H+-exchange inhibitor Hoe 642 on intracellular pH,calcium and sodium in isolated rat ventricular myocytes

 The inhibitors of the Na⁺/H⁺-exchange (NHE1) system Hoe 694 and Hoe 642 possess cardioprotective effects in ischaemia/reperfusion. It is assumed that these effects are due to the prevention of intracellular sodium (Na_i) and calcium (Ca_i) overload. The purpose of the present study was to investigate the effects of Hoe 642 on intracellular pH, Na⁺ and Ca²⁺ (pH_i, Na_i and Ca_i) in isolated rat ventricular myocytes under anoxic conditions or in cells in which oxidative phosphorylation had been inhibited by 1.5 mmol/l cyanide. In cells which were dually loaded with the fluorescent dyes 2,7-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) and Fura-2, anoxia caused acidification of the cells (from pH_i 7.2 to pH_i 6.8) and an increase in Ca_i from about 50 nmol/l to about 1 μmol/l. The decrease in pH_i began before the cells underwent hypoxic (rigor) contracture, whereas Ca_i only began to rise after rigor shortening had taken place. After reoxygenation, pH_i returned to its control value and Ca_i oscillated and then declined to resting levels. It was during this phase that the cells rounded up (hypercontracture). When 10 μmol/l Hoe 642 was present from the beginning of the experiment, pH_i and Ca_i were not significantly different from control experiments. At reoxygenation, pH_i did not recover, but Ca_i oscillated and returned to its resting level. To monitor Na_i, the cells were loaded with the dye SBFI. After adding 1.5 mmol/l cyanide or 100 μmol/l ouabain, Na_i increased from the initial 8 mmol/l to approximately 16 mmol/l. Hoe 642 or Hoe 694 (10 μmol/l) did not prevent the increase in Na_i. In contrast, the blocker of the persistent Na⁺ current R56865 (10 μmol/l) attenuated the CN^–-induced rise in Na_i. The substance ethylisopropylamiloride was not used because it augmented considerably the intensity of the 380 nm wavelength of the cell’s autofluorescence. In conclusion, the specific NHE1 inhibitor Hoe 642 did not attenuate anoxia-induced Ca_i overload, nor CN^–-induced Na_i and Ca_i overload. Hoe 642 prevented the recovery of pH_i from anoxic acidification. This low pH_i maintained after reoxygenation may be cardioprotective. Other possible mechanisms of NHE1 inhibitors, such as prevention of Ca²⁺ overload in mitochondria, cannot be ruled out. The increase in Na_i during anoxia is possibly due to an influx of Na⁺ via persistent Na⁺ channels. Received: 1 March 1996 / Received after revision: 30 April 1996 / Accepted: 12 July 1996     

Glibenclamide suppresses stretch-activated ANP secretion: involvements of K+ ATP channels and L-type Ca2+ channel modulation

 The mechanism by which glibenclamide regulates mechanically activated atrial natriuretic peptide (ANP) secretion was investigated using isolated perfused rat atria. A reduction in atrial pressure from an experimentally imposed distending pressure stimulated the secretion of ANP and caused concomitant translocation of extracellular fluid (ECF) into the atrial lumen. The activation of ANP secretion and ECF translocation were closely correlated with atrial volume changes and the increase in ANP secretion was a function of the ECF translocation. Glibenclamide (1, 10, 100 μM), an ATP-sensitive K⁺ (K⁺ _ATP) channel blocker, had no effect on the basal secretion of ANP, suppressed the stimulation of stretch-activated ANP secretion in a dose-dependent manner, but not the translocation of the ECF. Glipizide (100 μM) and tolbutamide (100 μM), other K⁺ _ATP channel blockers, had similar effects to those of glibenclamide. Suppression by glibenclamide (100 μM) of the stretch-induced ANP secretion was not observed in atria that had been pretreated with pinacidil (200 μM), an ATP-sensitive K⁺ channel opener: pinacidil alone had no effect on ECF translocation and ANP secretion. Furthermore, blocking Ca²⁺ influx by using the Ca²⁺ channel blocker diltiazem (10 nM), or a Ca²⁺-depleted medium prevented the suppression of stretch-induced ANP secretion by glibenclamide. In other experiments, atrial distension produced a slight membrane depolarization of cardiomyocytes; this was accentuated in the presence of glibenclamide. Furthermore, in single cardiomyocytes, glibenclamide increased the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in a dose-dependent manner. From these results, we suggest that glibenclamide suppresses atrial release of ANP by blocking K⁺ _ATP channels and increasing Ca²⁺ influx and that the K⁺ _ATP channels are associated with the regulation of the mechanically activated ANP secretion from the atria. Received: 13 May 1996 / Received after revision: 10 February 1997 / Accepted: 5 March 1997     

Characterization of the ATP-inhibited K+ current in canine coronary smooth muscle cells

Intracellular adenosine triphosphate (ATP)-inhibited K⁺ currents (I _{K, ATP} ) in canine coronary artery smooth muscle cells were characterized in the wholecell configuration using the suction pipette method. Cells dialysed internally with solutions containing 5 mM ATP (ATP_i) showed little detectable whole-cell current at potentials more negative than –30 mV. However, cells dialysed with ATP_i-free solutions developed a time- and voltage-independent current which reached a maximum of 132±25 pA at –40 mV about 10 min following patch rupture. After run-up, the current showed little run-down. Concentration-dependent inhibition by ATP_i yielded an inhibition constant (K _i of 350 M and a Hill coefficient of 2.3. In ATP_i-free solutions, the large current at –40 mV was reduced by glibenclamide with aK _i of 20 nM and a Hill coefficient of 0.95. Conversely, in 1 mM ATP_i solutions, the small current at –40 mV was increased by P-1075 from 8±2 pA to 143±33 pA, with a dissociation constant (K _d) of 0.16 M and a Hill coefficient of 1.7. The effect of P-1075 was antagonized by glibenclamide. Maximal current density elicited by either ATP_i depletion or external application of the channel opener P-1075 was similar with slope conductances of 81±10 pS/pF and 76±13 pS/pF respectively in the potential range of –90 to –40 mV. External Ca²⁺ had no effect on this current. Finally, in 1 mM ATP_i, 20 and 50 M adenosine increased the current slope conductance by 36±15% and 73±10% respectively between –90 to –40 mV. TheI _{K, ATP} although very small in these cells, was extremely effective in causing membrane potential hyperpolarization.     

Presence of two Ca2+ influx components in internal Ca2+-pool-depleted rat parotid acinar cells

The molecular mechanism(s) involved in mediating Ca²⁺ entry into rat parotid acinar and other non-excitable cells is not known. In this study we have examined the kinetics of Ca²⁺ entry in fura-2-loaded parotid acinar cells, which were treated with thapsigargin to deplete internal Ca²⁺ pools (Ca²⁺-pool-depleted cells). The rate of Ca²⁺ entry was determined by measuring the initial increase in free cytosolic [Ca²⁺] ([Ca²⁺]_i) in Ca²⁺-pool-depleted, and control (untreated), cells upon addition of various [Ca²⁺] to the medium. In untreated cells, a low-affinity component was detected with K _Ca = 3.4 ± 0.7 mM (where K _Ca denotes affinity for Ca²⁺) and V _max = 9.8 ± 0.4 nM [Ca²⁺]_i/s. In thapsigargin-treated cells, two Ca²⁺ influx components were detected with K _Ca values of 152 ±  79 μM (V _max = 5.1 ± 1.9 nM [Ca²⁺]_i/s) and 2.4 ±  0.9 mM (V _max = 37.6 ± 13.6 nM [Ca²⁺]_i/s), respectively. We have also examined the effect of Ca²⁺ and depolarization on these two putative Ca²⁺ influx components. When cells were treated with thapsigargin in a Ca²⁺-free medium, Ca²⁺ influx was higher than into cells treated in a Ca²⁺-containing medium and, while there was a 46% increase in the V _max of the low-affinity component (no change in K _Ca), the high-affinity component was not clearly detected. In depolarized Ca²⁺-pool-depleted cells (with 50 mM KCl in the medium) the high-affinity component was considerably decreased while there was an apparent increase in the K _Ca of the low-affinity component, without any change in the V _max. These results demonstrate that Ca²⁺ influx into parotid acinar cells (1) is increased (four- to five-fold) upon internal Ca²⁺ pool depletion, and (2) is mediated via at least two components, with low and high affinities for Ca²⁺. Received: 30 October 1995/Received after revisionand accepted: 13 December 1995     

Role of calcium-dependent K+ channels in the regulation of arterial and venous tone by nitric oxide in pigs

Effects of inhibition of calcium-dependent potassium channels (K⁺ _Ca channels) on the regulation of arterial and venous tone by nitric oxide (NO) were studied in anaesthetized pigs following vagotomy and blockade of autonomic ganglia. Selective inhibition of K⁺ _Ca channels by charybdotoxin (CTX, 2 μg/kg iv) or iberiotoxin (IbTX, 1 μg/kg) significantly augmented mean total peripheral resistance (TPR) to levels 30–60% above control. Venous and pulmonary vascular tone were assessed by changes in effective compliances of the venous (EVC) and pulmonary (EPC) vascular beds as calculated from changes in central venous and diastolic pulmonary arterial blood pressure during haemorrhagia (−5 ml/kg) and hypervolaemia (+5 ml/kg). Blockade of K⁺ _Ca channels significantly decreased both EVC (−20 to −30%) and EPC (−30 to −50%). Both CTX and IbTX significantly diminished the vasodilation caused by the NO-donor S-nitroso-N-acetylpenicillamine (SNAP) both during control conditions and following experimental vasoconstriction induced by systemic inhibition of NO-synthesis by NG-nitro-L-arginine methyl ester (L-NAME) or infusion of vasoconstrictor agonists. Dilator effects of the adenosine 3′,5′-cyclic monophosphate (cAMP)-dependent agonist adenosine were only slightly reduced. However, blockade of K⁺ _Ca channels did not increase vasoconstriction induced by L-NAME significantly. These results suggest that activation of vascular K⁺ _Ca channels is an important mechanism by which NO attenuates the constrictor tone of resistance and capacitance vessels in vivo. + _Ca channel blockade during vasoconstriction by agonists The effects of CTX on the haemodynamic responses to infusions of AII, AVP, NA and ET-1, at doses producing similar increases in MAP of about 50 mmHg, are listed in Table 1. The increase in TPR caused by NA and ET-1 was significantly smaller after CTX, whereas the responses to AII and AVP were similar both before and after CTX. To characterize this effect of K⁺ _Ca channel blockade further, we constructed dose-response curves for AII and NA with and without pretreatment with IbTX. The results for TPR are shown in Fig. 5. The constrictor responses to the two lower doses of NA were significantly reduced by IbTX. Received: 12 February 1996 / Accepted: 31 March 1996     

The cAMP-regulated and 293B-inhibited K+ conductance of rat colonic crypt base cells

We have shown previously that secretagogues acting via the second messenger adenosine 3′,5′-cyclic monophosphate (cAMP) activate, besides their marked effect on the luminal Cl⁻ conductance, a K⁺ conductance in the basolateral membrane of colonic crypt cells. This conductance is blocked by the chromanol 293B. This K⁺ conductance is examined here in more detail in cell-attached (c.a.) and cell-excised (c.e.) patch- clamp studies. Addition of forskolin (5 μmol/l) to the bath led to the activation of very small-conductance (probably < 3 pS) K⁺ channels in c.a. patches (n = 54). These channels were reversibly inhibited by the addition of 0.1 mmol/l of 293B to the bath (n = 21). Noise analysis revealed that these channels had fast kinetics and produced a Lorentzian noise component with a corner frequency ( f _c) of 308 ± 10 Hz (n = 30). The current/voltage curves of this noise indicated that the underlying ion channels were K⁺ selective. 293B reduced the power density of the noise (S _o) to 46 ± 8.7% of its control value and shifted f _c from 291 ± 26 to 468 ± 54 Hz (n = 8). In c.e. patches from cells previously stimulated by forskolin, the same type of current persisted in 3 out of 18 experiments when the bath solution was a cytosolic-type solution without adenosine 5′-triphosphate (ATP) (CYT). In 15 experiments the addition of ATP (1 mmol/l) to CYT solution was necessary to induce or augment channel activity. In six experiments excision was performed into CYT + ATP solution and channel activity persisted. 293B exerted a reversible inhibitory effect. The channel activity was reduced by 5 mmol/l Ba²⁺ and was completely absent when K⁺ in the bath was replaced by Na⁺. These data suggest that forskolin activates a K⁺ channel of very small conductance which can be inhibited directly and reversibly by 293B. Received: 1 October 1995/Received after revision: 28 December 1995/Accepted: 28 December 1995     

Roles of Na+/H+ exchange in regulation of p38 mitogen-activated protein kinase activity and cell death after chemical anoxia in NIH3T3 fibroblasts

Activation of Na⁺/H⁺ exchange (NHE) plays a major role in cell death following ischemia/hypoxia in many cell types, yet counteracts apoptotic cell death after other stimuli. To address the role of NHE activity in regulation of cell death/survival, we examined the causal relationship between NHE, p38 mitogen-activated protein kinase (MAPK), ERK1/2, p53, and Akt activity, and cell death, after chemical anoxia in NIH3T3 fibroblasts. The NHE1 inhibitor 5′-(N-ethyl-N-isopropyl) amiloride (EIPA) (5 μM), as well as removal of extracellular Na⁺ [replaced by N-methyl-d-glucamine (NMDG⁺)], prevented recovery of intracellular pH (pH_i) during chemical anoxia (10 mM NaN₃ ± 10 mM glucose), indicating that activation of NHE was the dominating mechanism of pH_i regulation under these conditions. NHE activation by chemical anoxia was unaffected by inhibitors of p38 MAPK (SB203580) and extracellular signal-regulated kinase (ERK) (PD98059). In contrast, chemical anoxia activated p38 MAPK in an NHE-dependent manner, while ERK1/2 activity was unaffected. Anoxia-induced cell death was caspase-3-independent, mildly attenuated by EIPA, potently exacerbated by SB203580, and unaffected by PD98059. Ser¹⁵ phosphorylation of p53 was increased by anoxia in an NHE- and p38 MAPK-independent manner, while Akt activity was unaffected. It is suggested that after chemical anoxia in NIH3T3 fibroblasts, NHE activity is required for activation of p38 MAPK, which in turn protects the cells against anoxia-induced death. In spite of this, NHE inhibition slightly attenuates anoxia-induced cell death, likely due to the involvement of NHE in other anoxia-induced death pathways.     

Functional relevance of anaerobic metabolism in the isolated respiratory network of newborn rats

Respiratory (C₃–C₅) activity and extracellular K⁺, pH and Ca²⁺ (aK_e, pH_e, [Ca]_e, respectively) in the ventral respiratory group (VRG) were measured in vitro. In brainstem-spinal cord preparations from 0- to 1-day-old rats, lowering of bath glucose content from 30 to 10 mM for 1 h did not affect aK_e or rhythmic activity. In preparations from 2- to 3-day-old animals, however, an aK_e rise by about 1 mM and disturbance of rhythm occurred after a delay of 50 min. Glucose-free saline resulted, after about 30 min, in reversible blockade of respiratory rhythm and an aK_e rise by more than 8 mM, whereas pH_e remained unaffected. Exposure to anoxia for 30 min after 1 h of pre-incubation in 10 mM glucose led to a progressive rise of aK_e, and a fall of [Ca]_e. The concomitant suppression of rhythm was irreversible in preparations from 2- to 3-day-old animals. Similar effects on aK_e and [Ca]_e and irreversible blockade of rhythm were revealed during anoxia in glucose-free solution, or by addition of 2–5 mM iodoacetate to oxygenated or hypoxic solutions. Iodoacetate led to a slow increase of pH_e by more than 0.2 pH units, which was accelerated by anoxia. Our findings show that normal respiratory network functions in the en bloc medulla, in particular from rats older than 1 day, depend on high bath glucose levels, necessary for effective utilization of anaerobic metabolism. Received: 18 December 1995/Accepted: 2 April 1996     

钾通道对大鼠肺动脉平滑肌细胞[Ca2+]i的调节

目的:探讨在常氧、低氧条件下钾通道对大鼠肺动脉平滑肌细胞(PASMCs)[Ca²⁺]_i的调节。方法:采用钙荧光探针(Fura-2/AM)负载培养的大鼠PASMCs,观察常氧、低氧培养后3种钾通道抑制剂(4AP,TEA、Glib)对PASMCs[Ca²⁺]_i的调节,同时用四唑盐(MTT)比色法比较4AP、TEA、Glib对大鼠PASMCs增殖的影响。结果:(1)常氧状态下,PASMCs[Ca²⁺]_i为(156.91±8.60)nmol/L,低氧时为(294.01±16.81)nmol/L(P＜0.01)。(2)常氧状态下,4AP可引起PASMCs[Ca²⁺]_i升高,达(280.52±23.21)nmol/L(P＜0.01),而TEA、Glib无此作用。(3)低氧时,4AP和TEA都可引起PASMCs[Ca²⁺]_i的升高,分别为(422.41±24.28)nmol/L、(380.84±11.02)nmol/L(P<0.01),Glib无作用。(4)MTT比色法中,常氧和低氧状态下4AP均引起吸光度(A)值升高,分别是0.582±0.062,0.873±0.043(P＜0.01)。TEA仅在低氧时A值升高(0.729±0.041,P＜0.05),而Glib无论常氧还是低氧均无影响。结论:无论常氧还是低氧条件下,电压依赖性钾通道(K_V)对PASMCs[Ca²⁺]_i及其增殖起主要作用。钙激活的钾通道(K_Ca)在常氧条件下对[Ca²⁺]_i不起调节作用,而在低氧下使[Ca²⁺]_i降低,反应性地调节PASMCs增殖。ATP敏感性钾通道(K_ATP)无论在常氧还是低氧情况下对[Ca²⁺]_i的调节不起作用。     

Effect of acidosis on Ca2+-activated K+ channels in cultured porcine coronary artery smooth muscle cells

 Although acidosis induces vasodilation, the vascular responses mediated by large-conductance Ca²⁺-activated K⁺ (K_Ca) channels have not been investigated in coronary artery smooth muscle cells. We therefore investigated the response of porcine coronary arteries and smooth muscle cells to acidosis, as well as the role of K_Ca channels in the regulation of muscular tone. Acidosis (pH 7.3–6.8), produced by adding HCl to the extravascular solution, elicited concentration-dependent relaxation of precontracted, endothelium-denuded arterial rings. Glibenclamide (20 μM) significantly inhibited the vasodilatory response to acidosis (pH 7.3-6.8). Charybdotoxin (100 nM) was effective only at pH 6.9–6.8. When we exposed porcine coronary artery smooth muscle cells to a low-pH solution, K_Ca channel activity in cell-attached patches increased. However, pretreatment of these cells with 10 or 30 μM O, O′-bis(2-aminophenyl)ethyleneglycol-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl)ester (BAPTA-AM), a Ca²⁺ chelator for which the cell membrane is permeable, abolished the H⁺-mediated activation of K_Ca channels in cell-attached patches. Under these circumstances H⁺ actually inhibited K_Ca channel activity. When inside-out patches were exposed to a [Ca²⁺] of 10^–6 M [adjusted with ethyleneglycolbis(β-aminoethylester)-N,N,N′,N′-tetraacetic acid (EGTA) at pH 7.3], K_Ca channels were activated by H⁺ concentration dependently. However, when these patches were exposed to a [Ca²⁺] of 10^–6 M adjusted with BAPTA at pH 7.3, H⁺ inhibited K_Ca channel activity. Extracellular acidosis had no significant direct effect on K_Ca channels, suggesting that extracellular H⁺ exerts its effects after transport into the cell, and that K_Ca channels are regulated by intracellular H⁺ and by cytosolic free Ca²⁺ modulated by acute acidosis. These results indicate that the modulation of K_Ca channel kinetics by acidosis plays an important role in the determination of membrane potential and, hence, coronary arterial tone. Received: 20 January 1998 / Received after revision: 9 April 1998 / Accepted: 22 April 1998     

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.     

The effect of L-type Ca2+ channel blockers on anoxia-induced increases in intracellular Ca2+ concentration in rabbit proximal tubule cells in primary culture

Ca²⁺ channel blockers (CCB) have been shown to be protective against ischaemic damage of the kidney, suggesting an important role for intracellular Ca²⁺ ([Ca²⁺]_i) in generating cell damage. To delineate the mechanism behind this protective effect, we studied [Ca²⁺]_i in cultured proximal tubule (PT) cells during anoxia in the absence of glycolysis and the effect of methoxyverapamil (D600) and felodipine on [Ca²⁺]_i during anoxia. A method was developed whereby [Ca²⁺]_i in cultured PT cells could be measured continuously with a fura-2 imaging technique during anoxic periods up to 60 min. Complete absence of O₂ was realised by inclusion of a mixture of oxygenases in an anoxic chamber. [Ca²⁺]_i in PT cells started to rise after 10 min of anoxia and reached maximal levels at 30 min, which remained stable up to 60 min. The onset of this increase and the maximal levels reached varied markedly among individual cells. The mean values for normoxic and anoxic [Ca²⁺]_i were 118±2 (n=98) and 662±22 (n=160) nM, respectively. D600 (1 M), but not felodipine (10 M), significantly reduced basal [Ca²⁺]_i in normoxic incubations. During anoxia 1 M and 100 M D 600 significantly decreased anoxic [Ca²⁺]_i levels by 22 and 63% respectively. Felodipine at 10 M was as effective as 1 M D600. Removal of extracellular Ca²⁺ and addition of 0.1 mM La³⁺ completely abolished anoxia-induced increases in [Ca²⁺]_i. We conclude that anoxia induces increases in [Ca²⁺]_i in rabbit PT cells in primary culture, which results from Ca²⁺ influx. Since this Ca²⁺ influx is partially inhibited by low doses of CCBs, Ltype Ca²⁺ channels may be involved.     

The effects of nucleotides and potassium channel openers on the SUR2A/Kir6.2 complex K+ channel expressed in a mammalian cell line, HEK293T cells

 The effects of potassium channel opening drugs and intracellular nucleotides on the ATP-sensitive K+ (K_ATP) channel composed of SUR2A and Kir6.2 in HEK293T cells were examined using the patch-clamp technique. The SUR2A/Kir6.2 channel was activated effectively by pinacidil, marginally by nicorandil but not by diazoxide. The pinacidil-activated channel currents were inhibited by glibenclamide with a K _i value of 160 nM. Upon formation of inside-out (I-O) patches, spontaneous openings of the channels appeared, which were inhibited by intracellular ATP (ATP_i) equipotently in the presence and in the absence of intracellular Mg²⁺ (Mg²⁺ _i). The channel activity ran-down gradually in I-O patches. The run-down channels could be reactivated by ATP_i only in the presence of Mg²⁺ _i. Uridine 5’-diphosphate (UDP) antagonized the ATP_i-mediated inhibition of the channel activity before run-down. After run-down, UDP activated the channel without antagonizing ATP_i-mediated channel inhibition. Thus, the SUR2A/Kir6.2 reproduced the major properties of the native cardiac K_ATP channel well in terms of nucleotide regulation and pharmacology, and therefore can be a useful tool with which to elucidate the molecular mechanisms characterizing the K_ATP channel. Received: 24 October 1997 / Received after revision and accepted: 4 December 1997     

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.     

Effect of caffeine on K+ efflux in frog skeletal muscle

 The exposure of frog skeletal muscle to caffeine (3–4 mM) generates an increase of the K⁺ (⁴²K⁺) efflux rate coefficient (k _K,o) which exhibits the following characteristics. First it is promoted by the rise in cytosolic Ca²⁺ ([Ca²⁺]_i), because the effect is mimicked by ionomycin (1.25 μM), a Ca²⁺ ionophore. Second, the inhibition of caffeine-induced Ca²⁺ release from the sarcoplasmic reticulum (SR) by 40 μM tetracaine significantly reduced the increase in k _K,o (Δk _K,o). Third, charybdotoxin (23 nM), a blocker of the large-conductance Ca²⁺-dependent K⁺ channels (BK_Ca channels) reduced Δk _K,o by 22%. Fourth, apamin (10 nM), a blocker of the small-conductance Ca²⁺-dependent K⁺ channels (SK_Ca channels), did not affect Δk _K,o. Fifth, tolbutamide (800 μM), an inhibitor of K_ATP channels, reduced Δk _K,o by about 23%. Sixth, Ba²⁺, a blocker of most K⁺ channels, did not preclude the caffeine-induced Δk _K,o. Seventh, omitting Na⁺ from the external medium reduced Δk _K,o by about 40%. Eight, amiloride (5 mM) decreased Δk _K,o by 65%. It is concluded that the caffeine-induced rise of [Ca²⁺]_i increases K⁺ efflux, through the activation of: (1) two channels (BK_Ca and K_ATP) and (2) an external Na⁺-dependent amiloride-sensitive process. Received: 13 March 1998 / Received after revision: 17 June 1998 / Accepted: 14 September 1998     

Biophysical,pharmacological and developmental properties of ATP-sensitive K+ channels in cultured myotomal muscle cells from Xenopus embryos

Unlike mammalian muscle cells in culture, cultured myotomal muscle cells of Xenopus embryos express ATP-sensitive K⁺ (K_ATP) channels. The K_ATP channels are blocked by internal ATP (half-maximal inhibition K _0.5 = 16 M) and to a lesser extent by internal ADP, are voltage independent, have an inward rectification at positive potentials and are inhibited by glibenclamide (K _0.5 = 2 M). Surprisingly, these K_ATP channels are not sensitive to K⁺ channel openers such as cromakalim. Opening of these K_ATP channels does not occur under normal physiological conditions. It is elicited by metabolic exhaustion of the muscle cell and it precedes the development of an irreversible rigor state. Neither intracellular acidosis nor an increase of intracellular Ca²⁺ are involved in K_ATP channel opening. Different types of K⁺ channels are successively expressed after plating of myotomal muscle cells: (1) sustained delayed-rectifier K⁺ channels; (2) K_ATP channels; (3) inward-rectifier K⁺ channels; (4) transient delayed-rectifier K⁺ channels. The current density associated with K_ATP channels far exceeds that of voltage-dependent K⁺ channels. Innervation controls the expression of these K_ATP channels. Co-culture of muscle cells with neurons from the neural tube decreases the number of active K_ATP channels per patch. Similarly, in situ innervated submaxillaris muscle of tadpoles at stage 50–55 has a very low density of K_ATP channels.