Life Sciences 77 (2005) 1993–2003

www.elsevier.com/locate/lifescie

pH effects on high conductance Ca2+-activated K+ channels (BKCa)

in human internal mammary artery smooth muscle cells

Jesica Raingoa, Alejandro Rebolledoa, Angela O. Grassi de Gendea, Nora Sanzb,

Juan Tommasib, Veronica Milesia,TaCatedra de Fisiologıa, Departamento de Ciencias Biologicas, Facultad de Ciencias Exactas,

Universidad Nacional de La Plata (UNLP). Calles 47 y 115, La Plata (1900), ArgentinabHospital San Juan de Dios, La Plata, Argentina

Received 4 January 2005; accepted 14 March 2005

Abstract

Introduction: In vascular smooth muscle cells, different types of K+ channels participate in the regulation of

membrane potential and consequently in the contractile behavior of the vessel. There is little information

about the properties and role of K+ channels in human internal mammary artery (HIMA), the vessel of

choice for coronary revascularization.

Methods: Patch-clamp technique on isolated HIMA smooth muscle cells was used.

Results: This work presents for the first time single-channel properties of the high conductance Ca2+-

activated K+ channel (BKCa) of HIMA. It presents a single-channel conductance of 228F4 pS (n =44,

8 cells), is sensitive to 100 nM iberiotoxin, and its open probability is Ca2+- and voltage-dependent. Inside-

out results show that BKCa channels in HIMA are directly activated by increasing the pH of intracellular

media (NPo=0.096F0.032 at pH 7.4 and NPo=0.459F0.111 at pH 7.6, n =12 cells, pb0.05) and inhibited

by lowering this pH (NPo=0.175F0.067 at pH 7.4 and NPo=0.051F0.019 at pH 6.8, n =13 cells,

p b0.05).

Conclusions: The evidences presented about single-channel properties and intracellular pH sensitivity of BKCa

from HIMA smooth muscle cells provide useful information to elucidate physiological or pathological

0024-3205/$ -

doi:10.1016/j.l

T Correspond

E-mail add

see front matter D 2005 Elsevier Inc. All rights reserved.

fs.2005.03.015

ing author. Tel.: +54 221 425 0497x30; fax: +54 221 489 0354.

ress: veronica@biol.unlp.edu.ar (V. Milesi).

J. Raingo et al. / Life Sciences 77 (2005) 1993–20031994

mechanisms in this vessel, as well as for future studies where drugs could have BKCa channels as targets for

pharmacological therapies.

D 2005 Elsevier Inc. All rights reserved.

Keywords: BKCa channels; Human mammary artery; Patch-clamp; pH

Introduction

The human internal mammary artery (HIMA) is the vessel of choice for coronary artery bypass

grafting. In the last decade, the HIMA represented a superior long-term survival vessel compared to the

traditionally used saphenous vein bypass graft (Loop et al., 1986). HIMA spasms are frequent during

surgical manipulation, and were also reported to occur in the postoperative period (Green, 1979; Dincer

and Borman, 1983). Knowledge of regulatory mechanisms of the contractile state of this vessel is

important to understand the development of vasospasm. In HIMA, the production of endothelium-

derived factors and the reactivity of smooth muscle to them and other vasoactive substances has been

extensively studied in organ bath experiments (Verma et al., 2001; Cracowski et al., 1999, 2000; Pearson

et al., 1998). In order to avoid the vasospasms, several pharmacological strategies are used to induce

vasorelaxation and lower reactivity of the vessel (Rosenfeldt et al., 1999). Among these pharmacological

tools, the ATP-sensitive potassium channel openers (i.e., aprikalim or pinacidil) have been demonstrated

to be vasorelaxing drugs in HIMA (He and Yang, 1997; Gojkoviic Bukarica et al., 1997; Liu et al., 2001)

suggesting a possible role for this kind of channel in the regulation of contractile state of this vessel.

However, more than one type of potassium channels participate in the regulation of membrane

potential in vascular smooth muscle cells and consequently in the contractile behavior of blood vessels.

In particular, the high conductance calcium-activated K+ channel (BKCa) has been described as playing a

role in the regulation of membrane potential of human vascular smooth muscle cells (Zhang et al., 1996;

Milesi et al., 1999; Gokina et al., 1996), as well as being a target for endogenous and exogenous

vasoactive agonists (Bychkov et al., 1998; Gruhn et al., 2002). Their main electrophysiological

properties have been described in different tissues (Kaczorowski and Garcia, 1999), but these properties

and regulatory mechanisms may vary depending on the specific tissue considered. Description of these

channels at the single-channel level is sparse in human vascular smooth muscle, and absent in the case of

the HIMA.

Among the many different modulating factors of BKCa channels behavior (Dimitropoulou et al., 2001;

Thomson et al., 1998; Carvajal et al., 2000; Lang et al., 2000), changes in intracellular pH have been

shown to modify the channel activity. Inhibition of BKCa channels induced by intracellular acidification

has been reported in vascular smooth muscle cells (Schubert et al., 2001; Petrova-Kirova et al., 2000),

and a direct interaction between protons and constitutive or regulatory channel protein subunits could be

a mechanism responsible for these effects (Austin and Wray, 2000). Intracellular pH changes are

involved in the physiological responses to shear-stress (Stamatas et al., 1997), vasoactive agonists such

as angiotensin II (Hatori et al., 1987) and endothelin (Hubel and Highsmith, 1995), and pathological

states, such as cellular ischemia, which modify the contractile state of vascular smooth muscle (Smith,

G.L. et al., 1998).

In this study, we present for the first time single-channel properties of BKCa channels in freshly

dispersed human smooth muscle cells from internal mammary artery and their main electrophysiological

J. Raingo et al. / Life Sciences 77 (2005) 1993–2003 1995

properties. We show that these channels are directly regulated by internal pH and discuss its possible

implications on the mechanical response of HIMA to pH.

Materials and methods

Sample procurement and preparation

The vessels used in this study were obtained from 57 patients undergoing primary coronary artery

bypass surgery at the Service of Cardiothoracic Surgery of the Hospital San Juan de Dios, Ministerio de

Salud de la Provincia de Buenos Aires, La Plata, Argentina. The segments of HIMA used for this study

were classified as surgical discard specimens and thus exempted from patient consent requirements.

Additionally, the Scientific Research Commission of our institution reviewed and approved all the

experimental protocols involved in this study.

After the bypass surgery was finished, the remnant segments were transported to our laboratory in ice-

cold 0.9% NaCl solution and were studied within 3 h of arrival. The vessels were placed in a Petri dish

filled with 0.9% NaCl solution, cleaned of adherent connective tissue, and cut into small pieces for

enzymatic cell isolation.

Cell isolation procedure

Cell isolation procedure was based on the method described by Klockner (Klockner, 1993) and

currently used in our laboratory to obtain isolated smooth muscle cells from human saphenous vein

(Milesi et al., 1999) or, with slight modifications, to obtain cells from human umbilical artery (Milesi et

al., 2003). Briefly, small strips were placed in a dissociation medium (DM) containing: 140 mM NaCl, 5

mM KH2PO4, 5 mM MgCl2, 20 mM glucose, 5 mM HEPES, 2 mM dithiothreitol, 2 mg ml�1

collagenase, 2 mg ml�1 papain and 0.5 mg ml�1 protease type-I; pH 7.4 at 30 8C, and mechanically

stirred for 30 min. After the incubation period, the strips were washed with DM without enzymes and

single HIMA smooth muscle cells were obtained by gentle dispersion of the treated tissue using a

Pasteur pipette. The remaining tissue and the supernatant containing isolated cells were stored at room

temperature until used.

Patch-clamp recording

HIMA smooth muscle cells were allowed to settle onto the coverglass bottom of a 0.5 ml

experimental chamber. The cells were observed with a mechanically stabilized, inverted microscope

(Zeiss-Telaval3, Jena, Germany) equipped with a 40� objective lens. The chamber was perfused for

15 min with bath saline solution (BSS, see composition later) at 0.5 ml min�1 (by gravity) before

the patch-clamp experiment was started. Application of test solutions was performed through a

multibarreled pipette positioned close to the cell investigated. After each experiment on a single

cell, the experimental chamber was replaced by another one containing a new sample of cells. Only

well-relaxed, spindle-shaped smooth muscle cells were used for electrophysiological recording. Data

were collected within 4–6 h after cell isolation. All experiments were performed at room

temperature.

J. Raingo et al. / Life Sciences 77 (2005) 1993–20031996

The standard tight-seal cell-attached and inside-out configurations of the patch-clamp technique

(Hamill et al., 1981) were used. Glass pipettes were drawn from WPI PG52165-4 glass on a two-

stage vertical micropipette puller (PP-83, Narishige Scientific Instrument Laboratories, Tokyo, Japan)

and coated with Sylgard (Dow Corning Corporation, Midland, MI, USA) to reduce recording noise.

Pipette resistances ranged from 2 to 4 MV measured in BSS. Ionic currents were measured with an

Axopatch 200A amplifier (Axon Instruments, Foster City, CA, USA). Single channel currents were

filtered at 2 kHz and digitized at 16 kHz. Voltage-clamp 30–60 s recordings were obtained at

different membrane potential values. Open probability (Po) is expressed as NPo, where N is the

number of channels present in each patch. NPo values were calculated using the following expression

(Kajioka et al., 1991):

NPo ¼ A1 þ 2A2 þ 3A3 þ N þ nAnÞ= Ao þ A1 þ A2 þ N þ AnÞððwhere A0 is the area under the curve of the amplitude histogram which corresponds to the closed

state, and A1, A2. . . An are the areas corresponding to the n open-state levels of current. The

histogram parameters were obtained from least-squares Gaussian fits of experimental data.

The bath saline solution (BSS) used for single-channel experiments was (in mM): 140 KCl, 0.5

MgCl2, 1 EGTA, 10 HEPES, 10 glucose, pH was adjusted to 7.4 with NaOH. Free Ca2+

concentration in each solution was estimated by calculations made with an appropriate software

which considers total Ca2+ content, EGTA concentration, pH, presence of other salts and

temperature. For control BSS, a free Ca2+ concentration of 10�8 M was estimated considering

Ca2+ impurities from other salts (50 AM). Solutions with a free Ca2+ concentration of 10�7 and

10�6 M were prepared adding appropriate amounts of CaCl2 to the BSS. In the experiments

exploring intracellular pH effects on channel activity, a BSS with 10�7 M free Ca2+ and pH 7.4 was

used as control solution, and when pH was modified to 6.8 and 7.6, taking into account pH effects

on EGTA binding, free Ca2+ was maintained at the same concentration by changing the amount of

CaCl2 in this BSS.

The composition of the pipette solution (PS) was (in mM): 140 KCl, 0.5 MgCl2, 1 CaCl2, 10 HEPES

and 10 glucose, pH was adjusted to 7.4 with NaOH.

Reagents

Iberiotoxin (IBTX), ethylene glycol-bis(h-aminoethyl ether) N,N,NV,NV,-tetraacetic acid (EGTA) and

all the enzymes used for cell isolation were purchased from Sigma Chemical Co. All other reagents were

of analytical grade. When required by the experimental protocol, a fresh aliquot of a stock aqueous

solution of IBTX was added to the PS on the day of the experiment.

Statistics

The results are expressed as meansFSEM. Paired or unpaired Student’s t-tests were used to establish

statistically significant differences between two experimental groups. Chi-squared test was used to study

independence between groups of cells treated or untreated with IBTX. We used a Kruskal–Wallis one-

way ANOVA on ranks with Dunn’s multiple comparison procedure to test for differences between

groups of data that were not normally distributed (see Results). The level of statistically significant

differences was set to pb0.05.

J. Raingo et al. / Life Sciences 77 (2005) 1993–2003 1997

Results

BKCa characterization

Current recordings in cell-attached patches were performed on isolated HIMA smooth muscle cells

using the pipette solution (PS) containing 140 mM of K+ and perfused with the depolarizing high K+

solution (BSS). In such conditions, we observed a high conductance channel at different applied

membrane potentials (Vm) in the majority of the tested patches. The relationship between unitary current

and Vm (I–V) was linear and showed a zero current point close to 0 mV, as expected for K+ channels in

symmetric K+ concentrations. We obtained a single channel conductance of 228F4 pS (n=44, 8 cells)

from the slope of an all-points I–V curve (Fig. 1A). The channel open probability (NPo) increased

exponentially with Vm in each of the studied cells (data not shown), confirming that this is a voltage-

operated channel.

When the patch was excised from the cell-attached condition into the inside-out configuration, hence

exposing the cytosolic face of the membrane to the BSS ([Ca2+]=10�8 M), the activity of the high

conductance channel (at the same Vm) decreased to a NPo value near 0 (data not shown). Based on this

effect, we studied the internal Ca2+ dependency of NPo in inside-out patches recording the single-

channel activity at +20 mV with different free Ca2+ concentrations in the bath solution. We observed that

the NPo value was significantly augmented when free Ca2+ concentration was increased from 10�8 M

(control BSS) to 10�7 or 10�6 M (Fig. 2).

The conductance value along with the voltage and Ca2+ sensitivity suggested us the presence of BKCa

channel in HIMA smooth muscle cells. This was confirmed by cell-attached recordings in the presence

of 100 nM IBTX in the pipette solution, which showed that this specific inhibitor of BKCa blocked the

high conductance channels. In these experiments, the tips of glass microelectrodes were filled with the

PS already described, while the back part of the electrodes were filled with the same solution plus 100

nM IBTX, which slowly diffused towards the patch. In such condition, 22 cells were tested observing

that 15 cells showed no activity of BKCa channels after the seal was obtained, while 7 cells presented

some initial activity which then declined with time (5–20 min); Fig. 3B shows a typical recording of the

A B

2mVm (mV)

I(pA

)

-60 -40 -20 0 20 40 60

15

10

5

0

-5

-10

-15

c

c

co

c

o

c

o

30 mV

20 mV

10 mV

0 mV

-20 mVo 5

pA

20 ms

Fig. 1. Characteristics of the BKCa channel in isolated HIMA smooth muscle cells. (A) All-points I–V curve whose slope gives

a value of 228F4 pS which corresponds to the BKCa channel conductance (n =44, 8 cells, r =0.9932). (B) Typical cell-attached

recordings of the BKCa channel at different Vm values (from �20 to+30 mV).

c1 min

10 min

25 min

“cell-attached”“cell-attached”

PS

5pA

100 ms

c

c

100 nM IBTX PS

PS

o1

o2

o1

o2

o1

o2

c

o1

o2

o3

c

o1

c

o1

Fig. 3. IBTX blocking effect. (A) Typical recording in cell-attached configuration at +30 mV representing the group of cells

without IBTX in PS (n =37 cells) in which BKCa activity persisted in time. (B) Typical recording in cell-attached configuration

at +30 mV representing the group of cells with IBTX in PS in which the initially observed BKCa activity declined with time

(n =7 cells).

A

10-8 10-7 10-6 Ca2+ (M)

NP

o

*

*

*B

n=15

n=18

n=120.35

0.30

0.25

0.20

0.15

0.10

0.05

0

150 ms 3p

A

10-8 M Ca2+

10-6 M Ca2+

Fig. 2. Calcium dependence of the BKCa channel in isolated HIMA smooth muscle cells. (A) Typical inside-out recordings of

BKCa channel showing an increase in NPo when free Ca2+ is risen from 10�8 M (control BSS) to 10�6 M at +20 mV. (B) Bars

represent the mean NPoFSEM value of the BKCa channel at different free Ca2+ concentrations at+20 mV. The asterisk symbol

(*) indicates statistical difference ( p b0.05) between groups by Kruskal–Wallis one-way ANOVA on ranks with Dunn’s

multiple comparison procedure to test for differences between groups of data that were not normally distributed.

J. Raingo et al. / Life Sciences 77 (2005) 1993–20031998

J. Raingo et al. / Life Sciences 77 (2005) 1993–2003 1999

attenuation of channel activity with time in one of these 7 cells. Conversely, when IBTX was absent

from the PS, BKCa activity was present in 37 out of 51 cells, without observing changes in activity with

time (Fig. 3A). In order to find statistically significant differences in the frequency of appearance of the

BKCa channel activity between the control group and the group with IBTX, we used a parametric Chi-

squared test. This analysis showed that the classificative variables (presence or absence of IBTX, and

activity or no activity of the BKCa channel) are not independent (Chi-squared test, pb0.001, n=73

cells), allowing us to conclude that the presence of IBTX statistically reduced the frequency of

appearance of the high conductance channel activity.

pH effects on BKCa activity

The effects of bath alkalinization (pH 7.6) or acidification (pH 6.8) were tested in steady-state inside-

out recordings, maintaining a constant free Ca2+ concentration (10�7 M) and Vm (+30 mV). As it is

shown in Fig. 4, intracellular alkalinization induced a significant increase in the NPo of BKCa, while

acidification significantly reduced the NPo of the BKCa channel. The effects were completely reversed

NP

o

pH 7.4 pH 7.6

*

NP

o

pH 7.4 pH 6.8

*

O2

O1

C

C

A

B

pH 7.4

pH 6.8

pH 7.6

pH 7.4

5pA

250 ms

5pA

250 ms

0.6

0.5

0.4

0.3

0.2

0.1

0

0.6

0.5

0.4

0.3

0.2

0.1

0

O2

O1

C

O3

O2

O1

C

O1

Fig. 4. pH dependence of BKCa activity. (A) Typical inside-out recordings showing BKCa activity at control (7.4) and alkaline

(7.6) pH facing the intracellular side of the channel. Bath solution free Ca2+ concentration was 10�7 M in both cases. Bars are

mean NPo valuesFSEM. *Indicates statistical difference from the control group, Student’s paired t-test (n =12, p b0.05). (B)

Typical inside-out recordings showing BKCa activity at control (7.4) and acid (6.8) pH facing the intracellular side of the

channel. Bath solution free Ca2+ concentration was 10�7 M in both cases. Bars are mean NPo valuesFSEM. *Indicates

statistical difference from the control group, Student’s paired t-test (n =13, p b0.05).

J. Raingo et al. / Life Sciences 77 (2005) 1993–20032000

after restoring control conditions (BSS at pH 7.4). In neither of the cases, we observed any changes in

the value of single-channel conductance.

Discussion

There is little information in the literature about the properties and role of K+ channels in HIMA,

the vessel of choice for myocardial revascularization. In this paper, we describe for the first time the

single-channel properties of BKCa channels present in cell-attached and inside-out patches obtained

from freshly dispersed HIMA smooth muscle cells. We observed a high frequency of occurrence of

BKCa activity (~73% of all patches studied) suggesting that these channels are highly expressed in this

vessel. In a symmetrical K+ concentration gradient, they show a single-channel conductance of 228

pS, are sensitive to 100 nM IBTX, and their open probability is Ca2+- and voltage-dependent. These

data are in accordance with the known properties of BKCa channels described in other vascular

smooth muscle cells.

An increase of BKCa activity can be an important mechanism to induce relaxation in response to

different physiological stimuli, such as shear-stress, vasoactive agonists or endothelial-derived factors

(Thomson et al., 1998; Carvajal et al., 2000; Lang et al., 2000). Archer et al. have recently demonstrated

that the 11,12-epoxyecosatrienoic acid is an endothelium-derived factor that hyperpolarizes and relaxes

HIMA by enhancing the activity of BKCa channels in this vessel (Archer et al., 2003). Moreover, as it

was reported by Rusch and Liu (1997), in pathological conditions such as hypertension BKCa channels

are up regulated providing a protective effect during acute or chronic episodes of high blood pressure.

Age also seems to influence the expression of BKCa channels: it has been recently reported that in human

coronary artery smooth muscle aging induces a reduction in the density of the alpha-subunit of these

channels (Toro et al., 2002).

The evidences presented in our work about single-channel BKCa activity in HIMA show that

changes in H+ concentration could directly modify the channel protein or its environment inducing an

increase or a decrease of BKCa open probability when intracellular pH augments or diminishes,

respectively. Kume et al. (1990) have also reported that in tracheal smooth muscle cells acidosis

inhibits Ca2+-activated K+ channels resulting in membrane depolarization, and Schubert et al. (2001)

have also given a detailed description showing that in rat vascular smooth muscle cells a decrease in

intracellular pH inhibits BKCa channels in inside-out patches and depresses whole-cell currents in

isolated cells. Then, changes in intracellular pH produced by physiological stimuli (i.e., vasoactive

agonists) or pathological conditions may trigger relaxing or contractile effects by modifications of K+

flux through BKCa channels.

The bibliographic references about pH and mechanical behavior of smooth muscle are extensive and

controversial in some aspects, results appearing to be dependent on the kind of smooth muscle tested and

the experimental conditions used in each case. Changes in H+ concentration may modify several cell

membrane Ca2+ transport pathways (Smith, G.L., et al., 1998; Smith, R.D. et al., 1998; Eto et al., 2003),

K+ channel activity (Smith et al., 1998), mobilization of Ca2+ from intracellular pools and Ca2+

sensitivity of contractile proteins (Smith, G.L. et al., 1998), as well as the activity of exchangers, i.e., Na/

H and Na/HCO3 (Neylon et al., 1990). Hence, the final effects of intracellular pH on the contractile state

of HIMAwill surely reflect the balance of different cellular mechanisms able to be modified directly or

indirectly by pH. Based on our electrophysiological results, we could speculate that BKCa channels

J. Raingo et al. / Life Sciences 77 (2005) 1993–2003 2001

would be inhibited or activated by low or high intracellular pH, respectively, contributing to determining

the final level of vessel contraction.

Conclusion

We characterized the single-channel properties of BKCa present in the HIMA and demonstrated its

direct activation by high and its inhibition by low intracellular pH. These data contribute to increase the

knowledge on the behavior of a vessel commonly used in myocardial revascularization.

Acknowledgements

The authors wish to thank Ms. Silvia Salemme for her excellent technical assistance. This work was

supported by grant PIP 4708 from the CONICET and PICT 0263 from the ANPCYT.

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