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Cardiovascular Research 2000 46(3):569-578; doi:10.1016/S0008-6363(00)00055-9
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Copyright © 2000, European Society of Cardiology

An inward rectifier and a voltage-dependent K+ current in single, cultured pericytes from bovine heart

Nicolas von Beckeratha,*, Stefan Neesb, Franz-Josef Neumanna, Bjarne Krebsa, Gerd Juchemb and Albert Schömiga

aDeutsches Herzzentrum and 1.Medizinische Klinik, Technische Universität München, 80636 Munich, Germany
bPhysiologisches Institut, Ludwig-Maximilians-Universität München, 80336 Munich, Germany

* Corresponding author. Deutsches Herzzentrum München, Lazarettstr. 36, D-80636 München, Germany. Tel.: +49-89-1218-4011; fax: +49-89-1218-4593 beckerath{at}dhm.mhn.de

Received 14 April 1999; accepted 15 February 2000


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: The purpose of this study was to describe passive electrical properties and major membrane currents in coronary pericytes. Methods: 78 single, cultured bovine pericytes were studied with the patch-clamp technique in the whole-cell mode. Results: The membrane potential of the cells was –48.9±9.6 mV (mean±S.D.) with 5 mM and –23.2±2.2 mV with 60 mM extracellular K+. The membrane capacitance was 150.2±123.2 pF. The current–voltage relation of the pericytes was dominated by an inward current at hyperpolarized potentials and an outward current at depolarized potentials. Increasing extracellular K+ from 5 to 60 mM led to an increase of the inward current and to a shift of this current to more depolarized potentials. The inward current was very sensitive to extracellular barium (50 µM). The maximum slope conductance of the cells at hyperpolarized potentials was 2.9±2.8 nS. Inward rectification of whole-cell currents was steep (slope factor=6.8 mV). With elevated external K+ the outward current reversed near the potassium equilibrium potential. Onset of the outward current was sigmoid and inactivation of this current was monoexponential, slow (time constant =12.8 s) and incomplete. Voltage-dependence of outward current steady-state activation was steep (slope factor =4.6 mV). The outward current was very sensitive to 4-aminopyridine (dissociation constant {approx}0.1 mM). The maximum slope conductance at depolarized potentials was 16.6±15.6 nS. Conclusion: We report for the first time, patch-clamp recordings from coronary pericytes. An inward rectifier and a voltage-dependent K+ current were identified and characterized. Regulation of these currents may influence coronary blood flow.

KEYWORDS K-channel; Membrane potential; Microcirculation; Endothelial function; Coronary circulation


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Pericytes are specialized cells with distinct morphology and function in each tissue [1–3]. Therefore it is necessary to study local pericytes derived from the vessels of interest. In ventricular myocardium, the majority of pericytes are found at the transition from coronary arterioles to myocardial capillaries, suggesting a role for pericytes in the regulation of coronary blood flow [4,5]. In a detailed scanning electron microscopy study of chemically digested tissues, Shimada and colleagues [5] showed that the arterial sides of myocardial capillaries are densely covered with pericytes whereas more distal segments of myocardial capillaries are only sparsely covered with these mural cells.

Membrane currents of coronary pericytes have not been studied yet, although, like in other coronary vascular cells [6], they are likely to have a profound influence on cell function, possibly including contraction. In addition, capillary pericytes and endothelial cells form heterocellular gap junctions allowing exchange of ions and other small molecules. This has been shown for cerebral microvessels [7–9] and an ultrastructural study suggests their presence in myocardial capillaries [10]. Therefore, membrane potential changes in coronary pericytes may affect the membrane potential of the adjacent coronary endothelium. This may result in changes of endothelial intracellular free calcium and, thus, endothelial cellular events, like the release of nitric oxide [11]. Moreover, membrane potential changes generated by coronary pericytes may be transferred to nearby arteriolar vascular smooth muscle cells [12], although the significance of the arteriolar myoendothelial gap junctions is still a matter of debate [13]. Therefore, knowledge about coronary pericyte electrophysiology could potentially be important.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Isolation and identification of coronary pericytes
Fresh bovine hearts were obtained from a local abattoir. The apex (3-cm length) was cut off from the rest of the heart. Three or four epicardial arteries of the apical myocardium were attached to perfusion cannulae. First, the apical myocardium was perfused with a nominally Ca2+-free Krebs–Henseleit solution 1 (K–H solution 1) at constant pressure (120 mmHg) for 15 min (37°C). Then, the apex was perfused with Krebs–Henseleit solution 2 (nominally Ca2+-free) containing 11 mg dispase I, 87 mg collagenase D (Böhringer Mannheim, Germany) and 194 mg bovine albumin dissolved in 100 ml. Perfusion with K–H solution 2 was carried out for 10 min at 50 mmHg and 15 min at 20 mmHg. Then, the disintegrated regions of the myocardium were removed with a sharp spoon and dissolved in freshly prepared K–H solution 2 (25 min; 37°C). The resulting suspension contained microvessel fragments and cells and was passed through five nylon nets of decreasing mesh size (300, 200, 100, 60, 30 µm). The microvessel fragments collected in the last two nets were removed, washed in phosphate-buffered saline (PBS) and transferred to a vessel containing K–H solution 2 (37°C). This suspension was gently driven in and out of a 20-ml tissue culture pipette for 30 min. The suspension was once more passed through the 30-µm mesh size nylon net to remove microvessel remnants. Then the pericyte-rich filtrate was added to 11 ml isotonic Percoll solution (Sigma, Deisenhofen, Germany) mixed and centrifuged at 400 g for 20 min. The sediment and the upper 2 ml of the suspension were discarded, the remainder diluted 1:2 with PBS and centrifuged again at 400 g for 10 min. The sediment was again suspended in PBS and fractionated on a linear Percoll density gradient (1.026–1.065 g/cm3; total volume 70 ml; centrifugation for 30 min at 1000 g; centrifuge Z323, Hermle, Wehingen, Germany). The volume range, 22–50 ml, contained almost pure pericytes. The volume range, 0–22 ml, contained endothelial cells. The cells were washed 3 times with PBS and seeded in culture medium, Dulbecco minimal essential medium (Sigma), supplemented with 10% fetal calf serum (FCS), 1 mM glutamine, 200 I.E./ml penicillin and 20 µg/ml streptomycin. Cultured pericytes proliferated rapidly and could be subcultured at least 10 times without signs of dedifferentiation like loss of the characteristic staining properties (see below). K–H solutions were equilibrated with 95% O2–5% CO2 (pH 7.4).

Fig. 1a shows cultured pericytes and residual capillary and venular endothelial cells when density gradient centrifugation was skipped. After density gradient centrifugation, almost pure cultures of pericytes (Fig. 1b, blue) and endothelial cells (1c, red) could be obtained. Staining with alkaline phosphatase (blue) and dipeptidyl(amino)peptidase IV (red) sensitive dyes was performed according to Lojda [14]. In addition, endothelial cells could be identified due to their ability to take up dye-labeled acetylated low density lipoprotein. Individual smooth muscle cells and (with less intensity) coronary pericytes stained for {alpha}-smooth muscle actin. Due to the drawbacks associated with other pericyte markers [1] and the inability to obtain recordings from cells stained with alkaline phosphatase-sensitive reagents, the experiments were performed on cultured pericytes.


Figure 1
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  		Fig. 1 Micrographs of stained coronary pericytes and endothelial cells from venules and distal capillaries. (a) Co-cultured coronary pericytes and endothelial cells from coronary venules and distal capillaries were stained with reagents for alkaline phosphatase (AP) and dipeptidyl(amino)peptidase IV (DPPIV). Pericytes stained blue, indicating the presence of AP and endothelial cells stained red indicating the presence of DPPIV. (b) Pure culture of coronary pericytes (blue). (c) Pure culture of endothelial cells (red). Bar =100 µm for a; bars =50 µm for b and c.

 
2.2 Electrical recordings
Cells from the third to eighth passage were studied with the patch-clamp technique in the whole-cell configuration at room temperature. Gigaseals were obtained on the cell body of the large, flat, irregularly shaped cells, most of them having long and thin cell processes. Seventy-eight cells were studied in total. In order to avoid enzymatic pretreatment of the pericytes (which may influence ion-channel function), experiments were performed on day two or three following the last passage. Thereafter, it was nearly impossible to obtain gigaseals, most likely due to the ability of pericytes to secrete large amounts of matrix molecules [1]. The cells were sparsely plated on 35-mm Petri dishes that were mounted on the stage of an inverted microscope (Leitz DM IL, Wetzlar, Germany). The cells studied were superfused (5–15 ml/h) in a recording chamber forming a channel of 8-mm length and 1.5-mm width. Solution change was performed with a hand-controlled valve. The 5 mM K+ solution contained (mM) 140 NaCl, 5 KCl, 1 MgCl2, 1 NaH2PO4, 2 CaCl2 and 10 HEPES. When the extracellular K+ concentration was elevated to 10, 20 or 60 mM to analyze K+ conductances, Na+ was replaced by the corresponding amount of K+. The pH of all extracellular solutions was adjusted to 7.4 with NaOH. The pipette solution contained in (mM): 125 KCl, 20 KOH, 11 EGTA, 1 CaCl2, 2 Na2ATP, 2 MgCl2 and 10 HEPES (adjusted pH 7.2 with NaOH). Tetraethylammonium (TEA) and 4-aminopyridine (4-AP) were obtained from Sigma. Patch pipettes were made of thin-walled glass with or without filament (Clark, Reading, UK) and were heat polished directly before use. Whole-cell currents were recorded with an EPC-9 patch-clamp amplifier (HEKA, Lamprecht, Germany) digitized with a Mac-23 interface (HEKA) at rates of 500–2000 Hz. The cut-off frequency (f) was 1/3 or more of the sampling rate. The following liquid junction potentials were determined for later correction according to Neher [15]: (1) the liquid junction potential arising between the intracellular (pipette) solution and the 5 mM K+ containing extracellular solution, (2) the liquid junction potentials arising between the different extracellular solutions ([K+]o=5, 10 and 60 mM) and the filling solution (150 mM KCl) of the bath electrode. At the beginning of each experiment, the offset potential (that is related to electrode potentials and other offsets) was cancelled after submersing the tip of the pipette in 5 mM K+ containing extracellular solution. Command voltage pulses and recorded membrane potentials were corrected off-line by –5 mV ([K+]o=5 mM), –6 mV ([K+]o=10 mM) and –7 mV ([K+]o=60 mM) following the experiments. Membrane potentials are either resting or zero-current potentials. In most cells, subtraction of capacitive currents with the procedures provided by the EPC-9 amplifier remained incomplete. This was probably related to the shape and size of the studied cells. Due to very slow inactivation of the outward current, currents recorded during slow, depolarizing voltage ramps are not steady-state currents. The voltage ramps employed for the recordings shown in Figs. 3b, 8a, bGo and Fig. 9a, b are identical to the ramp shown in Fig. 3a. Original current traces are shown without subtraction of leak currents. For quantification of current reduction induced by extracellular barium, TEA and the different concentrations of 4-AP, as well as for Fig. 4, the linear current component of the whole-cell I–V relation (that was expected to mainly represent leak current) was subtracted.


Figure 3
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  		Fig. 3 Barium-sensitivity of the inward current. (a) I–V relationship derived from slow, depolarizing voltage ramps (60 mV/s) before and after addition of 50 µM barium chloride to the extracellular solution ([K+]o=5 and 60 mM). The voltage ramp applied is illustrated above, the I–V relation is plotted below. The maximal slope conductance of the inward current was 5.8 nS ([K+]o=5 mM) and 18.2 nS ([K+]o=60 mM). With 5 mM K+ in the extracellular solution and in the absence of extracellular barium, the zero-current potential was –75.5 mV. The membrane capacitance was 53 pF. (b) Subtraction of current traces with extracellular barium from current traces without barium (data from a), yields the barium-sensitive currents ([K+]o=5 and 60 mM).

 

Figure 8
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  		Fig. 8 Inhibition of outward current by TEA and 4-AP. (a) I–V relation obtained from slow, depolarizing voltage ramps (60 mV/s) in the presence and absence of 5 mM extracellular TEA ([K+]o=5 mM). Membrane capacitance was 85 pF. (b) Effect of 5 mM extracellular 4-AP on the I–V relation of the same cell after complete restoration of the outward current due to wash-out of TEA ([K+]o=5 mM and 60 mM).

 

Figure 9
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  		Fig. 9 Dose–response relation for 4-AP-induced inhibition of outward current. (a) Effect of low concentrations of 4-AP (0.001, 0.01, 0.1 mM) on the I–V relation, obtained from slow, depolarizing voltage ramps (60 mV/s). Membrane capacitance was 235 pF. (b) Effect of higher concentrations of 4-AP (0.05, 0.5, 5 mM) on the I–V relation. Membrane capacitance of this cell was 128 pF. Note that this cell exhibited barely any inward rectifier current. (c) Dose–response relation obtained from experiments like that shown in (a) and (b). The data represents mean current amplitudes of outward current at a defined potential (0 mV). Mean current amplitudes in the presence of 0.001, 0.01 and 0.1 mM 4-AP were obtained from five cells, whereas values for 0.05, 0.5 and 5 mM were obtained from seven cells. The data was described with the Hill equation: Fractional I=1/1+([4-AP]/Kd)n, where Kd is the apparent dissociation constant and n is the Hill coefficient.

 

Figure 4
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  		Fig. 4 Chord conductance–voltage relationship. The chord conductance, gK', was calculated from the trace of Fig. 3a without barium and with 5 mM K+ in the extracellular solution [gK'=IK/(V–EK)]. The linear component of the I–V relation had been subtracted. The chord-conductance was normalized to the value at –120 mV. The chord conductance–voltage relationship was fitted with a Boltzmann function of the form: gK'=[1+exp((V–V')/k)]–1, with k being the slope factor indicating the steepness of the relation and V' being the potential at which gK' was reduced to 0.5. The values giving the best fit were k=8.5 mV and V'=–90.5 mV.

 
2.3 Statistical analysis
Where appropriate, results in the text are given as mean±standard deviation (S.D.); n denotes the number of cells from which the data were obtained. All quantitative data (membrane potentials, capacitances, current sizes, conductances, resistances) have been obtained from experiments with single pericytes. Since most of the data were not normally distributed, Wilcoxon's test was used for all statistical analysis. P<0.05 was considered to indicate significance. Statistical analysis was performed with SPSS 9.0 software.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
When whole-cell recordings were obtained from pericytes that made contact to other surrounding pericytes, recorded currents and capacitive transients were much larger than when truly single cells were studied. These early observations indicated electrical coupling of the cultured pericytes. Subsequently, only truly single pericytes were studied. The current–voltage (I–V) relation obtained obtained during application of slow, depolarizing ramps was characterized by: (1) a large, low noise outward current, that was activated at potentials more depolarized than –55 mV; and (2) an inward current at hyperpolarized potentials (Fig. 2a). We found the outward current to be the most characteristic current of the cells. To elucidate the contribution of K+ conductances to the whole-cell, I–V relation the extracellular K+ concentration was varied. Increasing extracellular K+ to 10 and 60 mM (EK=–71 mV and –23 mV) led to an increase of inward currents and to a shift of inward currents with EK to more depolarized potentials. With 60 mM external K+ the whole-cell I–V relation showed an inflection between –50 and –20 mV with whole-cell currents reversing near EK, suggesting that the majority of the outward current is carried by K+ ions. These initial experiments suggested that a voltage-dependent K+ (KV) and an inward rectifier K+ (KIR) current are major membrane currents of cultured coronary pericytes.


Figure 2
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  		Fig. 2 I–V relationship derived from slow, depolarizing voltage ramps (44.5 mV/s). The shape of the voltage ramp is shown above, the I–V relation is plotted below. The cell was superfused with solutions containing 5, 10 and 60 mM K+. Zero-current potentials were –45, –40 and –19 mV, respectively. Inward currents increased with elevation of extracellular K+ (slope conductances 2.5, 3.2, 5.4 nS). The minimal slope of the I–V relation was 0.9 nS and the maximal slope conductance at depolarized potentials was 40.1 nS ([K+]o=5 mM). The capacitance of the cell was 127 pF. At 0 mV, the current density of the outward current was 9.5 pA/pF [K+]o=5 mM.

 
The mean membrane capacitance of the cells was 150±123 pF (n=72). The measured membrane potential was –48.9±9.6 mV with 5 mM extracellular K+ and –23.2±2.2 mV with 60 mM extracellular K+ (n=40, P<0.001). In a few experiments, fluctuations of the membrane potential between –85 and –50 mV were observed. Slope conductances of the pericytes were obtained for three distinct segments of the I–V relation during application of slow, depolarizing voltage ramps and superfusion with 5 mM K+ containing solution. At hyperpolarized potentials (potential range –120 to –100 mV) the maximum slope conductance was 2.9±2.8 nS whereas at depolarized potentials the maximum slope conductance was 16.6±15.6 nS (P<0.001, n=78). The minimal slope conductance was found in the potential range between –90 to –60 mV, where the I–V relation was almost linear (1.2±1.3 nS; n=78 — corresponding to a slope resistance of 0.83±0.77 G{Omega}). In some experiments, the minimal slope of the I–V relation was as low as 0.1 nS (corresponding to an input resistance of 10 G{Omega}). The electrode seal resistance obtained during slow voltage ramps in the cell-attached configuration was 12.2±8.9 G{Omega} (n=61) and thus similar to the highest values for membrane resistance found in whole-cell recordings. We assumed that a significant proportion of the linear current component of the whole-cell I–V relation was due to a leak current between pipette and bath.

The inward current at hyperpolarized potentials was studied in more detail. As expected for an inward rectifier K+ current, addition of barium (50 µM) to the extracellular solution led to an almost complete and reversible inhibition of the inward current in the presence of either 5 or 60 mM extracellular K+ (Fig. 3a). At –127 mV and in the absence of extracellular barium, the size of the inward current was 416±548 pA, whereas at the same potential, after addition of barium (50 µM), the inward current was 7±10 pA ([K+]o=60 mM, n=7, P=0.02). Barium-sensitive currents calculated from current traces of Fig. 3a are shown in Fig. 3b. The potential range over which these currents stabilize the membrane potential depends on the steepness of inward rectification. Steepness of inward rectification was assessed for the cell of Fig. 3 by analyzing the relationship between chord conductance and voltage. This relationship was described with a Boltzmann function (Fig. 4). In five cells, the mean value for the slope factor k was 6.8±1.6 mV.

Subsequently the outward current which was suspected to be a voltage-dependent K+ (KV) current was analyzed in more detail. First, whole-cell currents in response to step-like depolarizing voltage commands of 100-ms duration were recorded to study activation of this current (Fig. 5A). Current onset on sudden depolarization was sigmoid, as known for other types of KV currents. Time to peak of the outward current was 12 ms, when the cell was depolarized to –5 mV. Fig. 5b shows end-pulse current amplitudes normalized to cell capacitance that were plotted against potential (data from Fig. 5a). Due to the presence of residual capacitive currents, fitting current onset with a Boltzmann function (of any power), in order to obtain a quantitative description of the activation process, was impossible.


Figure 5
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  		Fig. 5 Activation of pericyte outward current. (a) Currents were elicited by depolarizing voltage steps of 100-ms test potentials between –55 and +35 mV (steps of 10 mV). [K+]o was 5 mM. Membrane capacitance was 40 pF. (b) Averaged end-pulse currents (normalized to cell capacitance) plotted against potential.

 
Then, current decay during prolonged depolarization (inactivation) was studied with application of long (>20 s duration) depolarizing voltage steps (Fig. 6). Outward current inactivation varied considerably from cell to cell. In most cases, however, currents were only reduced by 50% or less during prolonged depolarization to –5 mV. At less depolarized potentials which are likely to occur physiologically, little inactivation was observed. Inactivation could be described with a single exponential function. When the cells were depolarized to –5 mV from a holding potential of –75 mV, the time constant for monoexponential current decay was 12.8±3.6 s (n=4).


Figure 6
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  		Fig. 6 Inactivation of outward current. Long (25 s) depolarizing voltage steps were applied and currents recorded. Holding potential was –75 mV. Potentials denote test potentials in mV (right). During depolarisation to –5 mV, outward current declined form 182 (peak) to 95 (end-pulse) pA. At depolarization to –5 mV, the value for the time constant {tau}, describing the exponential current decay was 15.9 s. Capacitance was 235 pF.

 
Voltage-dependence of KV current activation was studied by tail current analysis. For this purpose, depolarizing voltage steps from a holding potential of –90 mV were applied and tail currents evoked upon repolarization to –60 mV. Normalized tail current amplitudes were assumed to indicate the fraction of open KV channels and, thus, current activation. Representative tail currents recorded form a pericyte are shown in Fig. 7a. Normalized tail current amplitudes derived from five cells were plotted against the potential of the preceding depolarization for Fig. 7b. Voltage-dependence of steady state activation was described with a Boltzmann function. The slope factor, k, describing the steepness of voltage-dependence for the combined curve (Fig. 7b) was 4.6 mV.


Figure 7
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  		Fig. 7 Voltage-dependence of activation. (a) Tail currents evoked by repolarization to –60 mV from depolarized potentials (interval 5 mV). The duration of the depolarizing voltage steps was 2 s. The voltage-clamp protocol is shown above. Amplitudes of tail currents were assessed by eye. Membrane capacitance was 102 pF. (b) Mean, normalized amplitudes of tail currents plotted against the preceding, depolarizing test potential. Error bars denote standard deviation. The continuous line indicates a Boltzmann function derived from the equation: Y{infty}={1+exp[(V–V')/k]}–4, with V' indicating the position on the abscissa (–48.5 mV) and k indicating the steepness of the relation (4.6 mV). The potential of half maximal activation (V1/2) was –40.9 mV.

 
As a first approach to a pharmacological characterization of the pericyte outward current, the effect of tetraethylammonium (TEA) and 4-aminopyridine (4-AP) on this current was tested. As shown in Fig. 8a, adding TEA (5 mM) to the extracellular solution led to an almost complete block of the outward current which was reversible within a few minutes. At +25 mV, extracellular TEA (5 mM) reduced the outward current from 349±245 pA to 31±25 pA ([K+]o=5 mM, n=5, P=0.04). Fig. 8b demonstrates inhibition of outward current by extracellular 4-AP (5 mM) during superfusion with 5 and 60 mM K+ solution. The 4-aminopyridine-induced block (5 mM) was incompletely reversible when the cells were superfused for 10 to 15 min with extracellular solution that did not contain 4-AP. At +25 mV, the outward current was 931±840 pA before application of 4-AP and it was reduced to 65±67 pA in the presence of 5 mM 4-AP ([K+]o=5 mM, n=8, P=0.01).

High sensitivity to 4-AP is an important characteristic of KV currents. Therefore, the dosage-dependence of 4-AP-induced inhibition of the outward current was examined. Always three concentrations of 4-AP were tested in one cell, as shown in Fig. 9a, b. The current traces reveal that extracellularly applied 4-AP leads to a reduction of slope conductance at depolarized potentials and to a shift of outward current activation to less negative potentials. In order to estimate the dose–response relationship from recordings like the ones shown in Fig. 9a, b, mean outward current amplitudes at 0 mV were plotted against the extracellular 4-AP concentration (Fig. 9c). The data was described with a Hill equation. The value for the dissociation constant, Kd, was 0.09 mM and the value for the Hill coefficient, n, was 0.81.


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
To our knowledge, we report the first whole-cell recordings from coronary pericytes. In terms of current size, an inward rectifier and a voltage-dependent K+ current are the dominant membrane currents of coronary pericytes in the presence of physiological extracellular ion concentrations. Accordingly, almost all inward current was abolished in the presence of micromolar concentrations of extracellular barium and almost all outward current was inhibited by addition of millimolar concentrations of 4-AP to the extracellular solution. The recordings were obtained from cultured cells which is a limitation, because ion channel expression may change during cell culture. The inward current shared the distinct properties of currents mediated by classical, steep inward rectifier K+ (KIR) channels. These channels carry large inward currents at hyperpolarized potentials and small outward currents at potentials slightly positive to EK [16]. Inward rectification is a result of a voltage-dependent block of outward currents by intracellular Mg2+ and polyamines and possibly an intrinsic gating mechanism [17]. In a few cells, however, no appreciable inward current was recorded (Fig. 9b). Such variability of the KIR current has also been observed in coronary artery vascular smooth muscle cells [18,19]. The outward current was identified as a delayed rectifier type, voltage-dependent K+ (KV) current. Compared to native KV currents in arterial and venous vascular smooth muscle cells [20], the potential of half maximum activation, V1/2 (–40.9 mV) was rather negative and voltage-dependence of steady-state activation was very steep (slope factor k=4.6 mV). Since the experiments were performed at room temperature, voltage-dependence of steady-state activation may be even steeper and activation may occur at more negative potentials with physiological temperature [21]. In coronary pericytes, inactivation of outward current was slow (mean {tau}=12.8 s) and rather incomplete. At physiological membrane potentials, almost no inactivation was observed, resulting in a substantial steady-state current. The pericyte KV-current was very sensitive to external 4-AP (Kd{approx}0.1 mM). The KV-current reported here shares properties with the KV-current recently described in freshly isolated coronary capillaries from guinea pig heart [22].

Ionic currents have been studied in retinal pericytes, mesangial pericytes and hepatic stellate (Ito) cells [23–25]. Spontaneous spike-like depolarizations have been recorded from cultured retinal pericytes in the presence of extracellular barium or norepinephrine [23]. They did not occur in the absence of extracellular Ca2+ or the presence of nifedipine, indicating the involvement of voltage-dependent Ca2+ currents [23]. Such currents have also been recorded from mesangial and hepatic stellate cells [24,25]. Neither spontaneous depolarizations nor transient inward currents have been observed in cultured coronary pericytes. This does not exclude, though, the presence of voltage-dependent Ca2+ currents in these cells, as the experimental conditions were not directed at recording such currents.

During experiments with little leak current, membrane fluctuations in the potential range between –55 to –85 mV were observed. Hyperpolarizations to potentials near EK most likely reflect the influence of KIR outward current on membrane potential. K+ conductance mediated by KIR channels increases with elevation of extracellular K+. Therefore the (hyperpolarizing) influence of the KIR current may be even more important in myocardial ischemia, where extracellular K+ reaches 10 mM [26]. The voltage-dependent outward K+ current in coronary pericytes limited membrane depolarization already at rather negative potentials (V1/2=–40.9 mV). Any alteration of this current [27,28] is likely to have a profound influence on pericyte membrane potential. Since endothelial cells are electrically tight, especially at depolarized potentials [29], the pericyte KV-current could have a substantial influence on the membrane potential of the coronary endothelium.

Time for primary review 22 days.


   Acknowledgements
 
We gratefully acknowledge the invaluable contribution of Alessandra Moretti, Sinje Weisner and Gisela Pogatsa-Murray to this work


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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 Am. J. Physiol. Heart Circ. Physiol.Home page
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