Research Cell Calcium (1997) Zl(4). 291300 0 Pearson Professional Ud 1997

Calcium-activated potassium channels in cultured human endothelial cells are not directly modulated by nitric oxide

Marian Haburcdk*, Lin Wei, F&ix Viana, Jan Prenen, Guy Droogmans, Bernd Nilius Laboratorium voor Fysiologie, Campus Gasthuisberg, KU Leuven, Leuven, Belgium

Summary Nitric oxide has been proposed to directly activated large conductance Ca2+-dependent K+ channels (BK& [Bolotina V.M., Najibi S., Palacino J.J., Pagan0 P.J., Cohen R.A. Nitric oxide directly activates calcium-dependent potassium channels in vascular smooth muscle. Nature 1994; 368: 850-8531. The nitric oxide (NO) donor S- nitrosocysteine (SNOC) was used to evaluate a possible direct modulation of BK,, by NO in EAhy926 (EA cells), a cultured human umbilical vein derived endothelial cell line, using the whole-cell, cell-attached and inside-out configuration of the patch-clamp technique, together with simultaneous amperometric measurement of NO and the concentration of free intracellular calcium [Ca’+],. BK,, channels with a large conductance of -190 pS, voltage- dependent activation and a reversal potential close to -80 mV have been identified in EA cells. Exposure of EA cells in the experimental chamber to 1 mM SNOC delivered approximately 5 yM NO, as recorded by an amperometric probe in situ. SNOC produced a modest increases in [Ca’+], that was insufficient to activate BK,, channels. NO alone neither activated BK,, channels directly nor modulated preactivated BK,, channels in EA cells. These results do not support a direct modulatory effect of NO on large conductance BK,, channels in cultured endothelial cells.

INTRODUCTION

Nitric oxide (NO) is a bioregulatory messenger of the vas- cular system which causes potent vasodilation in vascu- lar smooth muscle cells (SM). Vasorelaxation seems to be mediated mainly by activation of soluble guanylyl cyclase, leading to increased intracellular levels of cGMP and subsequent activation of protein kinases. Other actions of NO include interactions with protein thiol

Received 19 December 1996 Revised 30 January 1997 Accepted 30 January 1997

Correspondence to: Or Bernd Nilius, Laboratorium voor Fysiologie, Campus Gasthuisberg, KU Leuven, Herestraat 49, B-3000 Leuven, Belgium Tel. +32 16 34 5726: Fax. +32 16 34 5991 E-mail bernd.nilius@med.kuleuven.ac.be *On leave from the Institute of Normal and Pathological Physiology, Slovak Academy of Sciences, Bratislava, Slovak Republic

groups to form S-nitrosyl compounds, thus resulting in modulation of enzymes and receptor functions [1,2]. In the vascular system, endothelium is the principle source of NO which diffuses readily to underlying and adjacent SM cells, but it might also influence neighbouring endothelial cells.

NO also affects a variety of cell functions by modulat- ing the activity of ion channels: in pancreatic B cells, NO inhibits electrical activity and ionic currents [3], in neu- rones it influences the interaction of NMDA with its receptor-channel complex [4]. L-type Ca2+ channels in cardiac cells are also regulated by NO 151. NO stimulates an ATP-sensitive K+ channel [6] and activates a high-con- ductance Ca2+-dependent K+ channel (BKcJ in vascular [7] and gastrointestinal smooth muscles [8,9]. In some cases, the modulatory effects of NO on K+ channels appear to be indirect, through a cGMP-dependent mech- anism [lo], while in other cases channels may be acti- vated directly by NO [ 111.

292 M Haburcdk, L Wei, F Viana, J Prenen, G Droogmans, B Nilius

The effects of NO on K+ channels are still controversial. It has been reported that the NO donor S-nitroso-N- acetyl penicillamine (SNAP) does not affect BKC,. chan- nels in excised patches of visceral canine colonic SM [8]. On the other hand, it was shown in the same report that NO has direct, cGMP-independent effects on voltage gated K+ channels of small and intermediate conduc- tance (4 pS and 80 pS, respectively).

The regulatory role of NO in endothelial cells is not satisfactorily understood. A direct modulation of B&, by NO could be an important feedback of NO on its own synthesis (via a modulation of the intracellular calcium level). Modulation of endothelial ion channels by NO has not been investigated previously, In this study, we inves- tigated the effects of the NO donor S-nitrosocysteine (SNOC) on BKc, channels of cultured human endothelial cells using the whole-cell, cell-attached and inside-out configurations of the patch-clamp technique, combined with simultaneous measurements of NO and the concen- tration of free intracellular calcium, [Caz+],

Here we report that SNOC elevates [Caz+], to a level insufficient to activate BK,, channels, but neither acti- vates directly large conductance BK,, channels nor mod- ulates pre-activated BK,, channels in these human endothelial cells.

MATERIALS AND METHODS

Cells

Cultured vascular endothelial cells from a permanent human cell line, EAhy926, (EA cells) established by Edgell and co-workers [ 121 by hybridisation from human umbili- cal vein endothelial cells were used. Cells were grown at 37°C in a fully humidified atmosphere of 10% CO, in Dulbecco’s MEM medium containing 200 g/l fetal calf serum plus 10% HAT 50X supplement (Gibco BRL). The culture medium was exchanged every 48 h. Cells were detached by exposure to 0.5 g/l trypsin in a Ca2+- and Mg2+-free solution for approximately 10 min and reseeded on gelatine-coated glass coverslips. Only non-confluent cells were used (2-4 days after plating). All experiments were carried out at room temperature (18-22°C).

NO measurements

NO levels were continuously monitored throughout the experiment using an amperometric NO sensor and a commercially available NO meter @SO-NO, World Precision Instrument, Inc., Sarasota, FL, USA), which detects concentrations of NO gas up to 20 i.rIvI in aqueous solution [ 131. Briefly, NO diffuses through a semiperme- able membrane - which excludes NO+, NO- and other

charged species - and is then oxidised at a working plat- inum electrode resulting in an electric current. This redox current is proportional to the concentration of NO at the membrane’s outer surface. Output of the ISO-NO meter was connected to an A/D interface (see below). Electrode calibration was performed after each experi- ment. A calibration curve was obtained by measuring the NO generated by addition of aqueous potassium nitrite (KNO,) to a solution containing KI, H,SO,, and I(;SO, (Fig. 1A). This resulted in the immediate generation of NO by the following reaction [ 131:

2KI + 2H,SO, + 2K,SO, + 2KN0, + 2NO+ I,+ 2H,O + 4K$O,

90% of the maximum NO sensor response was achieved within 9 s, and the full response within 25 s for concentrations smaller than 5 PM (Fig. 1A). The NO cali- bration curves were always linear (r 2 0.98 in the mea- surable range; Fig. 1B).

During the experiments, the tip of the NO sensor was dipped l-2 mm into the solution in a Petri dish with or without endothelial cells, When combined with patch- clamp and [Caz+], measurements, the NO sensor was placed in the vicinity of the patched cell. Basal levels of NO release by endothelial cells were not measured. Instead, the probe was placed in the vicinity of an endothelial cell for several minutes until the current reached a stationary level which was then defined as zero. The reported changes in NO current/concentration are shown as deviations from this baseline.

Patch-clamp current measurements

The electrophysiological recording methods have been described in detail elsewhere [ 141. Whole cell membrane currents were measured using ruptured patches, moni- tored with an EPC-7 (I& Electronics, Darmstadt, Germany) or an EPC-9 (Heka Electronics, Lambrecht, Pfalz, Germany) patch clamp amplifier, and sampled at 1 ms intervals (fil- tered at 200 Hz). The following voltage protocol was repeated every 15 s: from a holding potential of 0 mV, a 0.3 s step was applied to -80 mV, thereafter the potential was stepped for 0.1 s to - 150 mV, followed by a 1.3 s linear volt- age ramp to +lOO mV

Single channel currents were measured in cell-attached and inside-out patches. They were filtered between 200 Hz and 500 Hz and sampled at 1 kHz.

intracellular calcium [Ca*+], measurements

[Caz+li was measured with the fluorescent calcium indi- cator Fura-2. The cells were loaded for 20 min at 37°C by incubation with 4 @4 of the membrane-per-meant

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NO donor effects on human endothelium 293

A

2 nA

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0.05 0.1 0.3 0.5 1 3 5 8 PM NO

B

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i 6-

2 4- 0 = 2-

Oi

b I NE , NE+SNOC

I 4 -I

I ’ I ’ I ’ I ’ I ’ I 0’7 , I , I , I , I

0 2 4 6 8 10 0 200 400 600 800 1000 NO concentration [PM] time (s)

Fig. 1 Production of nitric oxide (NO) by S-nitrosocysteine (SNOC) and effect of SNOC on precontracted rabbit aortic ring. (A) Calibration of the ISO-NO sensor. NO at indicated concentrations was generated after addition (indicated by arrows) of KNO, to the titrated mixture (KI, H,SO,, K,SO,). Stabilised level of the current corresponds to the indicated NO concentrations. (B) ISO-NO calibration curve. Relationship between the NO concentrations and current response of the NO sensor. Results from typical calibration procedure. Note the linear relationship. The slope of the linear regression curve (y = A + Bx, A = -128.7 pA, B = 1185 pN@l, r = 0.998) indicates the sensitivity of the probe. (C) Norepinephrine (NE) induced contraction and SNOUNO induced relaxation of aortic ring. 10 uM NE evoked contraction (maximum contraction taken as 100% - lower trace ). 1 mM SNOC superfused together with 10 pM NE caused relaxation of the ring to -20% of maximum. The bars above traces indicate changes of the bath solution in the experimental chamber. The upper trace shows SNOC/NO current changes in the experimental chamber measured amperometrically, simultaneously with the contractions of the ring. The maximal SNOUNO current corresponds approximately to 3.8 f.rM NO.

acetoxymethylester of Fura- (Fura-2/AM, Molecular Probes, Eugene, OR, USA) dissolved in Krebs’ solution. After dye loading, cells on the coverslips were washed 3 times with Krebs’ solution to remove extracellular Fura-2/AM. Changes in [Caz+li were monitored by means of a photomultiplier-based system, consisting of an inverted microscope (Zeiss IM lo), filter wheel, amplifier and controller (Luigs and Neumann, Ratingen, Germany) and a photomultiplier unit (Hamamatsu, Toyooka Vill, Japan). The technique has been described in detail previ- ously [ 15,161. In short, cells loaded with Fura-2/AM were illuminated alternatively at excitation wavelengths of 360 and 390 nm through a rotating filter wheel (2 revo- lutions/s). Emitted fluorescent light was measured between 510 and 570 nm. The background fluorescence was recorded from cell-free parts of the coverslips. After

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subtraction of background fluorescence, the apparent concentration of free [Ca*+], was calculated from the fluo- rescence ratio R according to the equation [ 171:

[Ca2+li = R - R,,

Kc8 x cR I

where Keff is the ‘effective binding constant: R0 is the flu- orescence ratio at zero Ca2+ and R that at high Ca2+. For data acquisition, an 8-channel A/D converter (Max Planck Institute of Biophysical Chemistry, Giittingen, Germany) connected to an Atari Mega 4 system was used, which allowed simultaneous sampling (2 Hz) of flu- orescence signals, transmembrane current, as well as amperometric NO current.

Cell Calcium (1997) 21(4), 291-300

294 M Haburca’k, L Wei, F Viana, J Prenen, G Droogmans, B Plilius

Preparation of rabbit aortic rings and tension recording Chemicals

Rings of rabbit aorta, 2-3 mm wide and cleaned of fat and connective tissue, were mounted horizontally on stainless steel hooks placed in an experimental chamber perfused with Krebs’ solution. The rings were allowed to equilibrate for approximately 30 mm before starting the experiments. Tension was measured isometrically, using a Shinkoh U-GAGE transducer (Minebea Co., Ltd, Japan) connected to an amplifier (Strain amplifier 6M82).

Preparation of S-nitrosocysteine (SNOC)

Equimolar solutions (200 mM) of I(+)-cysteine chloride and sodium nitrite were prepared in degassed Milli-Q water (Millipore). After mixing, they were acidified with HCl [3]. SNOC at a final concentration of 1 mM was pre- pared by dilution of 100 mM SNOC solution in degassed Krebs’ solution, and pH adjusted to 74 or 72 with NaOH or KOH, respectively,to be used as external or internal (for inside-out patches) bath solution. SNOC was prepared freshly prior to application. As a control, denitrosylated SNOC, in which NO had dissipated, was obtained by incubating the solution at room temperature for at least 2 days. The SNOC containing solutions were superfused continuously over the cultured cells via a capillary tube.

L-Noradrenaline-hydrochloride and EGTA were from Fluka (Fluka Chemie AG, Buchs, Switzerland); ATP was from Boehringer Mannheim GmbH, Germany. Sodium nitrite and I-cysteine chloride were purchased from Merck (Darmstadt, Germany).

Statistics

Paired comparisons of data are presented whenever pos- sible. In other cases, pooled data are given as mean + SEM. For significance, Student’s &test was used (level of significance, P < 0.05). Fitting routines used were from the software package Origin 4.0 (Microcal Software, Inc., Northampton, MA, USA).

RESULTS

Delivery of NO from SNOC and decay

Solutions

The standard bath Krebs’ solution had the following com- position (in mM): 150 NaCl, 1.5 CaCl,, 6 KCl, 1 MgCl,, 10 HEPES, 10 glucose, titrated to pH 24 with NaOH.

Pipette solutions for whole cell recordings contained (in mM): 100 K-aspartate, 40 KCl, 2 MgCl,, 4 Na,ATP, 10 HEPES, and 0.1 EGTA, adjusted to pH 72 with KOH. In some whole cell experiments, [Ca’+], was buffered at 2 @?I free Ca2+ (total CaCl, concentration of 4.7 n&I).

To record single channel activity from inside-out and cell-attached patches, NaCl in bath Krebs’ solution was substituted by KC1 (K+-Krebs), pH adjusted with KOH to 72 for inside-out, and 74 for cell-attached measurements, respectively. In experiments in the inside-out configura- tion, 1.5 mM EGTA was added to the bath solution together with different Ca2+ concentrations to obtain free Ca2+ concentrations from 0 to 5 @vI. In nominal Caz+-free solution, CaCl, was omitted.

In the experiments where we tested the effects of cys- teine on BK, channels in the inside-out configuration, EGTA was omitted from bath solution. The pipette for inside-out experiments was filled with Krebs’ solution.

SNOC spontaneously generates NO. A freshly prepared 1 mM SNOC solution contained NO at concentrations ranging between 15 and 20 l.tM (the highest level detectable by the NO meter). The decay of the NO cur- rent was monitored in an open glass vial containing 1 mM SNOC. In the absence of any perturbations (neither mixing nor perfusion) and any protection against influ- ence of atmospheric oxygen (no NO, bubbling during NO measurement), this current could be fitted with a sin- gle exponential. The time constant of this exponential gives an estimate of the decay of NO continuously pro- ducedbylmMSNOCof21~2min(n=7).

Exchange of a SNOC-free solution in the experimental chamber with one containing SNOC resulted in a rapid increase of NO in the bath (the initial rate of NO increase was -4-l 2 i.tM per min for about 0.5-l min), followed by a relatively stable sustained elevation/plateau (lasting for the duration of SNOC perfusion). The maximal concen- tration of NO generated by 1 mM SNOC in our continu- ously perfused experimental chamber was about one quarter (5.4 f 0.8 PM, n = 15) of the maximal NO values produced by 1 mM SNOC when measured directly in a glass vial. This indicates that some NO has been lost in the perfusion system. Denitrosylated SNOC solution, which had lost its red-brown colour after several days at room temperature, contained no measurable NO. L-cys- teine and sodium nitrite alone had no effect on the cur- rent measured by the ISO-NO sensor.

ATP was applied to the bath solution at a concentration of 10 I&I. SNOC, ATP and the other solutions were added SNOC induced relaxations of pre-contracted aortic rings

to the bath by a. fast perfusion system. For preparation of The physiological effects of the NO donor SNOC were the solutions Mill&Q water (Millipore, France) was used. evaluated by measuring its vasorelaxing actions on aortic

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NO donor effects on human endothelium 295

rings precontracted with 10 uM norepinephrine (NE). Addition of 1 mM SNOC in the continuous presence of NE (Fig. 1C) relaxed the contracted ring within 5 min to 25.4 + Z4% (n = 3) of the maximum contractile response. During these experiments, the maximal level of NO cur- rent reached approximately 4.0 f 0.6 ).&I NO, n = 3.

Characterisation of BK,, channels in EAhy926 cells

Activity of large conductance K+ channels was observed in 39 out of 45 patches (Fig. 2, see also [29]). Single chan- nel current amplitude increased with depolarisation (Fig. 2A,B). The measured single-channel conductance was

A WI +80

186 f 12 pS (calculated at 40 mV, 12 = 8 patches) and the reversal potential for the used inside out configuration was -82 mV (Fig. 2B) with 140 mM external K+ and 6 mM K+ in pipette. In inside-out patches, the open probability of the channels was enhanced by increasing the free Ca*+ concentration in the bathing solution (Fig. 2C,D). Open probability was also increased by depolarisation: at a free Ca2+ concentration of 5 @!I, the channel was closed at -100 mV (Fig. 2A), but the open probability increased to 0.4 at +80 mV (Fig. 2A). Figure 2D represents the mean values of the BK, channel open probability at different free Ca2+ concentrations in inside-out patches at +80 mV (n = 2-4). The results indicate that the channel is not very active at free Ca*+ concentrations below 0.5 uM.

C OmV +80 mV

[Ca’+J bM1

+40 0.5 I.__. 1 1 I

0 _..

1 .-A&LA-..-

-20 ssssl_mral.rlur..

-100 5 -....

20 pA L 1OOms

B D

30 , 1 [PAI 0.6

-100 -50 1 50 100

0.4

0.2

0.0

1 0

0 1 2 3 4 5

Fig. 2 Characterisation of calcium-activated potassium channel BK,, in EAhy926 cells. (A) Dependence of the single BK,, channel current amplitude on the holding membrane potential. Single channel traces in an inside-out membrane patch. Free Ca2+ concentration in Krebs’ solution is 5 PM. (6) Single channel I-V curve. Conductance of channel at +40 mV is about 195 pS considering the reversal potential of the channel close of -62 mV under the chosen conditions. Data are collected from 11 cells and fitted with the Goldman-Hodgkin-Katz equation. Different symbols correspond to data collected from different cells. (C) Ca2+-dependence of channel activity in an inside-out membrane patch (normal Krebs’ solution in the pipette, bath solution is K+-Krebs’ with the adjusted Ca*+-concentrations). V means the true potentials sensed by the channels (-V ). Channel activity was measured at two potentials and four different buffered CaZ+ concentrations as indicated above and at the lgR”h”and side of (C). Depolarisation of the channel from 0 to 80 mV clearly increased the single channel current amplitude and the open probability at non-saturating Ca*+ concentrations below 5 PM. (D) Dependence of the open probability (n x p,) of the channel on the free Ca2+ concentration at holding potential of +80 mV where N is the number of channels in the patch. Pooled data from 2-4 patches at each concentration. The data are fit with the Hill equation:

Y = (n x P,),, / [t + (kW1 with the parameters k = 1 .l PM, nH = 1.7, and n x p,,,,, = 0.55.

0 Pearson Professional Ltd 1997 Cell Calcium (1997) 21(4), 291300

296 M Habudk, L Wei, F Viana, J Prenen, G Droogmans, f3 Nibs

~OPA L

100 ms

0.4 -

a” *c

0.2 -

0.0 -

pM Ca2’ 1 mM SNOC

1 PM Ca2’

I ’ I ’ I ’ I ’ I ’ I ’

0 5 10 15 20 25 time [min]

Fig. 3 Single channel activity in inside-out patches during S-nitrosocysteine (SNOC) administration. (A) Current traces from an inside-out patch at a holding potential of 0 mV. Free Ca*+ concentration in the extracellular solution (pH = 7.2), and administration of SNOC is indicated on the left. (B) Simultaneous measurement of channel activity at 0 mV and NO concentration. Open probability (n x p,) was not significantly changed after administration of SNOC. The maximal concentration of SNOC/NO current corresponds approximately to a concentration of 6 PM NO.

NO neither directly nor indirectly activates/modulates BK,, channels

In order to study direct effects of NO on BK, channels which are not mediated by activation of guanylyl cyclase, we have applied SNOC to cell-free membrane patches that show BK, charmel activity. Figure 3 shows single channel activity at 0 mV recorded in an inside-out patch. Reducing the free Ca2+ concentration in extracellular solution to 1 PM drastically reduced the channel open probability. Perfusion with 1 mhJ SNOC did not affect the activity of the channel, suggesting that there is no direct action of SNOC derived NO on the Bq, channel. Figure 3B illus- trates a simultaneous measurement of NO and the changes in open probability, assessed from the average current in successive traces of 512 points divided by the single channel current amplitude (n x pd. Channel open probability (n x pJ before and during application of SNOC was not significantly different (n x p, in control and during SNOC application were identical 0.16 + 0.05, n = 13).

To test for indirect effects of NO on BKc, channels, chan- nel activity was monitored in cell-attached patches. The cell was superfused with K+-Krebs’ solution, and single channel activity was monitored at 0 mV in cell-attached mode, as shown in Figure 4. This patch, like others, showed spontaneous transient periods of enhanced chan- nel activity. However, n x p, of the channels in the patch was not significantly changed during SNOC perfusion. On the average, n x p, in control (prior to administration of SNOC) was 0.10 * 0.04 and 0.14 + 0.10 during administra- tion of SNOC, respectively (n = 4).

Effects of NO on [Caz+], and whole-cell current

In additional experiments, we combined measurements of NO and [Ca2+li with whole-cell current measurements during SNOC administration. The level of [Ca2+], upon SNOC administration was modestly, but significantly, increased, but this increase was much smaller compared to that evoked by 10 uM ATP (Fig. 5A). The average

Cell Calcium (1997) 21(4), 291300 0 Pearson Professional Lfd 1997

NO donor effects on human endothelium 297

4 0 - I

:$I 1 mM SNOC

I

-10 PAI 0 5 10 15

200 ms time [min]

Fig. 4 Single channel activity in cell-attached patches during S-nitrosocysteine (SNOC) administration. (A) Selected current traces from cell-attached patch recorded at a holding potential of 0 mV. Administration of SNOC is indicated on the left. (B) Simultaneous measurement of channel activity (at 0 mV) and NO. Open probability did not change after administration of SNOC (open probability before and during SNOC administration was 0.001). The maximal concentration of SNOG/NO current corresponds to a concentration of approximately 3.5 PM NO.

increase in [Caz+], induced by 1 mM SNOC was 71 f 0.5 % of the maximal agonist response (20 uM LITP), or 14.5 + 4.7% above the resting level of [Caz+], (n = 12 cells).

We also tested the effect of SNOC on the whole cell cur- rent (n = 7 cells). Figure 5B(i) shows the time course of the current measured at +lOO mV in the presence or absence of 1 mM SNOC or 10 @l All? These current values were obtained from voltage ramps from -150 to +lOO mV applied every 15 s. Instantaneous I-V curves for these var- ious experimental conditions were derived from these voltage ramp responses and are shown in Figure 5 B(ii). Neither the reversal potential (at about -60 mV) nor the outward current amplitude were significantly affected by administration of 1 mM SNOC. The mean outward current density at + 100 mV during perfusion with Krebs’ solution was 30.7 f 70 pA/pF (n = 7) compared to 29.7 + 6.5 pAlpF in the presence of 1 mM SNOC. In contrast, application of 10 FM ATP markedly increased the outward current (mean outward current density was 108.9 f 26.5 pA/pF, n = 4). However, perfusion with 1 mM SNOC in the presence of ATP did not significantly affect the current: the mean outward current density for SNOC plus ATP at 100 mV was 79.1 + 16.5 pA/pF, n = 3). Apparently, the increase in

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[Ca2+li induced by SNOC is below the threshold for acti- vating BKc, channels in EA cells.

In another protocol, we loaded the cells via the patch pipette with a Ca2+ buffered intracellular solution. Figure 6 shows such an experiment in which the increase in the whole cell current monitors changes in intracellular Ca2+. Application of SNOC resulted again in release of NO (Fig. 6A). Activation of a current was monitored from the volt- age ramps. Depicted are the current values at +lOO mV (Fig. 6B). Under these conditions (elevated but constant [Ca2+li), NO failed to modulate the preactivated BK,. (Fig. 6C, traces b and c).

DISCUSSION

We have studied the effects of NO on BIQ, channel activ- ity in a human endothelial cell line (EAhy926) which is derived from umbilical vein cells using simultaneous measurements of NO, intracellular free calcium, and transmembrane currents. The primary finding of this study is that NO/SNOC neither directly nor indirectly modulated the activity of these channels at the single- channel or whole cell level.

Cell Calcium (1997) 21(4), 29 l-300

298 M HaburcAk, L Wei, F Viana, J Prenen, G Droogmans, 13 Nilius

NO

A(ii)

A - < I __.

B(i) A-W ATP SNOC -

P v! 0

00 mV

B(ii)

Fig. 5 The effect of SNOC and ATP on [Ca’+], and K+ current in whole cell mode. (A) Simultaneous measurement of [Ca2;],, A(i), and NO, A(ii), in a single EA cell. Effect of 1 mM SNOC and 10 pM ATP on [Ca’+],. Application of extracellular ATP induced a clear increase in [Ca’+],. The removal of ATP is followed by [Ca’+], recovery to the resting values A(i). The perfusion with SNOC results in an increase of SNOUNO current, A(ii), which is accompanied by a small and gradual increase in [Ca2+li, A(i). Maxima of SNOClNO current correspond to 6.3 and 5.9 uM NO, respectively. Administration of ATP and SNOC is indicated by bar. (B) Effect of 1 mM SNOC on whole cell K+ current, in another EA cell, measured at 100 mV, B(i), on current-voltage relations reconstructed from voltage ramps from -150 to 100 mV, B(ii). The current response of the cell during perfusion with Krebs’ solution (a), 10 pM ATP (b), 1 mM SNOC (c), and 10 uM ATP together with 1 mM SNOC (d). Perfusion with SNOC did not activate a K+ current. Administration of 10 uM ATP increased K+ current. Application of 10 uM ATP together with 1 mM SNOC did not evoke additional changes in the K+ current.

We have used the nitrovasodilator SNOC as a stable NO donor. The half-life of NO in a concentrated aqueous solu- tion of SNOC is of the order of minutes [ 181. As demon- strated, SNOC generates NO concentrations in the @‘I range for a relatively long period. The NO production by 1 mM SNOC is in good agreement with previous work [3], that showed a half-life of 1 mM SNOC of about 25 mm at 37°C by direct spectrophotometric measurement of SNOC denitrosylation. The modest discrepancy with our results might be due to the extreme instability of the NO radical, differences in temperature, pH, and/or a different oxygen level present in the solution. The NO concentration pro- duced by 1 mh4 SNOC is quite high, but the therapeutic plasma levels of nitrovasodilators (e.g. in congestive heart failure or angina pectoris) are also often maintained at rel- atively high levels [ 191. We also used precontracted aortic rings as a biosensor to verity the production of NO by

Cell Calcium (1997) 21(4), 291-300 0 Pearson Professional Lfd 1997

SNOC. The relaxation effect of 1 mM SNOC on aortic rings confirms its physiological action.

EA cells have been extensively used in biological stud- ies of vascular endothelium, but they have not been characterised electrophysiologically so far. Here we describe the existence of a Ca2+ activated K+ channel with a conductance of about 190 pS. Large conductance B&, channels in different endothelial cells are charac- terised by conductance between 165 and 220 pS (reviewed in [20,21]). This channel shows Caz+ and volt- age-dependent activation, characteristic also for the same type of channel in the other cells [22,23].

A direct modulating effect of NO on Bq, channels, as has been previously reported for these channels in smooth muscle cells [ 1 l] or for cyclic nucleotide-gated channels in salamander olfactory neurones [l], was not detected in F,A cells. Possible reasons for this discrepancy

NO donor effects on human endothelium 299

A

NO bM1 - 4

I 3

2

1

u 1

0 a

kJ ’ t [min]

- - SNOC

I I I I I I 1

0 10 20 30 40

B

5 min

Fig. 6 Effect of S-nitrosocysteine (SNOC) in Ca*+-loaded cells on the whole cell current. (A) [Ca*+], was increased and buffered to 0.5 FM free Ca2+ concentration from patch pipette (arrow represents rupture of the cell membrane by patch pipette to get whole-cell configuration). SNOC (1 mM, application indicated by bars) evoked NO currents measured by the NO probe positioned in the vicinity of the patched cell. The arrow indicates rupture of the patch. (B) Whole cell current changes following patch rupture (arrow) during Ca2+- loading and SNOC perfusion measured from the voltage ramps at +lOO mV. (C) Current-voltage relationships indicating activation of the K+ current. Records were taken at time a before elevation of [Ca*+],, at time b during and time c after SNOC application.

may lie in the different experimental conditions: different sources of NO (SNOC as a NO donor versus direct NO; different multiple and interconvertible redox-forms of

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NO have different biological actions [24-261, change in intracellular pH as a result of different source used (appli- cation of SNOC may critically influence pH and thus Ca2+ buffering), temperature, etc. Our data are, however, in agreement with the findings in a number of tissues where no direct action of NO on BK, channels could be observed. It has already been reported, that SNAP does not activate BK, channels in colonic smooth muscle cells recorded in inside-out patches [8]. Application of NO donors to the internal surface of inside-out patches of rabbit coronary artery myocytes does not affect Bq, channel activity, suggesting that there is no direct effect of SNOC or SNAP [lo]. Recently, it has been shown that another NO donor - sodium nitroprusside - also does not directly affect BK, channel activity in inside-out patches of porcine tracheal smooth muscle cells [27]. Surprisingly, also no indirect effects of NO/SNOC on BK, channel activity, as reported in smooth muscle cells [8,10,27], could be detected. A feasible explanation for this discrepancy could be that these channels in endothelial cells might be less sensitive to NO than those in smooth muscle cells. On the other hand, EA cells, rep- resenting a hybrid cell-line, might lack some of the essen- tial components of the biochemical cascade to activate BK, channels through the activation of cGMP, stimula- tion of G kinase, phosphorylation of BKc, channels or associated regulatory proteins as suggested for the acti- vation of smooth muscle B&, channels [27,28]. However, also under these conditions, a direct effect of channel modulation should be detectable. In excised patches, we have seen that application of an NO-donor needs a criti- cal adjustment of the buffered [CaZ+],. Small changes in pH due to application of the donor induced already sig- nificant changes in [Ca2+li.

Stimulation with 10 PM ATP increases [Caz+li and con- comitantly activates an outward K+ current. Similarly, buffering intracellular Caz+ at a high level (2 FM) also activates this current. NO/SNOC also increased [Caz+li slightly, however only to a level which was insufficient to activate BK, channels. In addition, NO does not modu- late BK, channels in EA cells even under conditions where [Ca’+], is already elevated by other mechanisms.

In summary, our experiments provide no indication for the activation by NO of B&, channels in a human endothelial cell line. If other endothelial cell types exhibit a similar insensitivity of B&, channels to NO, this may correlate with a natural protection against NO.

ACKNOWLEDGEMENTS

MH was supported by a fellowship from DWTC, Belgium. FV was supported by contract ERB-CHBI-CT94-1137 of the European Commission. We would also like to thank Dr Edge11 (University of North Carolina, USA) for provid-

Cell Calcium (1997) 21(#), 291-300

300 M Haburcdk, L Wei, F Viana, J Prenen, G Droogmans, B Nilius

ing EAhy926 cells. Part of the work was supported by BMH4-CT96-0602.

1. Broillet M-C., Firestein S. Direct activation of the olfactory cyclic nucleotide-gated channel through modification of sulfhydryl groups by NO compounds. Neuron 1996; 16: 377-385.

2. Schulz R., Triggle CR. Role of NO in vascular smooth muscle and cardiac muscle function. Trends Phurmacol Sci 1994; 15: 255-259.

3. Krippeit-Drews P., Krijncke K.D., Welker S. et al. The effects of nitric oxide on the membrane potential and ionic currents of mouse pancreatic B cells. Endocrinology 1995; 136: 5363-5369.

4. Lei S.Z., Pan Z-H., Aggatwal S.K. et al. Effect of nitric oxide production on the redox modulatory site of the NMDA receptor-channel complex. Neuron 1992; 8: 1087-1099.

5. Mery P.F., Pavoine C., Belhassen L., Pecker F., Fischmeister R. Nitric oxide regulates cardiac Ca*+ current. Involvement of cGMP-inhibited and cGMP-stimulated phosphodiesterases through guanylyl cyclase activation. J Biol Ckem 1993; 268: 26286-26295.

6. Miyoshi H., Nakaya Y., Moritoki H. Nonendothelial-derived nitric oxide activates the ATP-sensitive K+ channel of vascular smooth muscle cells. FEBS Lett 1994; 345: 47-49.

Z Archer S.L., Huang J.M., Hampl V., Nelson D.P., Shuhz PJ., Weir E.K. Nitric oxide and cGMP cause vasorelaxation by activation of a charybdotoxin-sensitive K channel by cGMP-dependent protein kinase. Proc Nat1 Acad Sci USA 1994; 91: 7583-7587

8. Koh SD., Campbell J.D., Car] A., Sanders K.M. Nitric oxide activates multiple potassium channels in canine colonic smooth muscle. J Pkysioll995; 489: 735-743.

9. Thombury K.D., Ward S.M., Dalziel H.H., Carl A., Westfall D.P., Sanders K.M. Nitric oxide and nitrosocysteine mimic nonadrenergic, noncholinergic hyperpolarization in canine proximal colon. AmJPkysioll991; 261: G553-G557

10. George M J., Shibata E.F. Regulation of calcium-activated potassium channels by S-nitrosothiol compounds and cyclic guanosine monophosphate in rabbit coronary artery myocytes. JInvestMed 1995; 43: 451-458.

11. Bolotina V.M., Najibi S., Palacino J J., Pagan0 PJ., Cohen R.A. Nitric oxide directly activates calcium-dependent potassium channels in vascular smooth muscle. Nature 1994; 368: 850-853.

12. Edge11 C-J.S., McDonald CC., Graham J.B. Permanent cell line expressing human factor VIII-related antigen established by hybridization. Proc Nat1 Acad Sci USA 1983; 80: 3734-3732

13. Tsukahara H., Gordienko D.V., Goligorsky M.S. Continuous monitoring of nitric oxide release from human umbilical vein

endothelial cells. Biochem Biophys Res Commun 1993; 193: 722-729.

14. Nilius B., Viana F., Droogmans G. Ion channels in vascular endothelium. Annu Rev Physioll997; 59: 145-170.

15. Neher E. Combined fura- and patch clamp measurements in rat peritoneal mast cells. In: Sellin C., Libelius R., Ihesleff S. (Eds) Neuromz~cz&zr /u&ion. Amsterdam: Elsevier, 1989: 65-76.

16. Schwarz G., Callewaert G., Droogmans G., Nilius B. Shear stress induced calcium transients in human endothelial cells from umbilical cord veins. J PhysioZl992; 458: 527-538.

1% Grynkiewicz G., Poenie M., Tsien R.Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 1985; 260: 3440-3450.

18. Ignarro L J. Biosynthesis and metabolism of endothelium- derived nitric oxide. Annu Rev Pkurmacol Toxicoll990; 30: 535-560.

19. Matthews J.S., McWilliams P.J., Key B.J., Keen M. Inhibition of prostacyclin release from cultured endothelial cells by nitrovasodilator drugs. Biochim Biophys Acta 1995; 1269: 237-242.

20. Ling B.N., O’Neill W.C. CaZ+-dependent and Caz+-permeable ion channels in aortic endothelial cells. Am J PhysioZl992; 263: H1827-H1838.

2 1. Nilius B., Oike M., Zahradnik I., Droogmans G. Activation of a Cl- current by hypotonic volume increase in human endothelial cells. J Gen PhysioZl994; 103: 787-805.

22. McCobb D.P., Fowler N.L., Featherstone T. et al. A human calcium-activated potassium channel gene expressed in vascular smooth muscle. Am / Pkysioll995; 269: H767-H777

23. McManus O.B., Magleby K.L. Accounting for the Caz+- dependent kinetics of single large-conductance Caz+-activated K+ channels in rat skeletal muscle. JPkysioZ 1991; 443: 739-772

24. Lipton S.A., Stamler IS. Actions of redox-related congeners of nitric oxide at the NMDA receptor. Neuruphatvnucology 1994; 33: 1229-1233.

25. Stamler J.S. S-nitrosothiols and the bioregulatory actions of nitrogen oxides through reactions with thiol groups. Curr Top Microbial Immunoll995; 196: 19-36.

26. Stamler J.S., Singe1 DJ., Loscalzo J. Biochemistry of nitric oxide and its redox-activated forms. Science 1992; 258: 1898-1902.

27. Yamakage M., Hirshman CA., Croxton T.L. Sodium nitroprusside stimulates CaZ+ activated K+ channels in porcine tracheal smooth muscle cells. Am J PkysioZ1996; 270: L338-L345.

28. Robertson B.E., Schubert R., Hescheler J., Nelson M.T. cGMP- dependent protein kinase activates Ca2+-activated K channels in cerebral artery smooth muscle cells. AmJPkysioZ 1993; 265: c299-c303.

29. Viana F., Missiaen L., Droogmans G., Nilius B. Modulation of agonist-evoked calcium oscillations in human vascular endothelial cells [abstract]. BiopkysJ 1996; 70: A231.

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