Cardiovascular Research

Cardiovascular Research 2002 54(1):152-161; doi:10.1016/S0008-6363(02)00227-4
© 2002 by European Society of Cardiology

This Article Abstract FREE Full Text (PDF) E-letters: Submit a response Alert me when this article is cited Alert me when E-letters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Request Permissions Disclaimer Google Scholar Articles by Belevych, A. E. Articles by Smirnov, S. V. Search for Related Content PubMed PubMed Citation Articles by Belevych, A. E. Articles by Smirnov, S. V. Social Bookmarking          What's this?

Copyright © 2002, European Society of Cardiology

Developmental changes in the functional characteristics and expression of voltage-gated K⁺ channel currents in rat aortic myocytes

Andriy E. Belevych^b,1, Richard Beck^b,2, Paolo Tammaro^a,b, Lucilla Poston^b,c and Sergey V. Smirnov^a,b,*

^aDepartment of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY, UK
^bCentre for Cardiovascular Biology and Medicine, King's College London, St Thomas's Campus, London SE1 7EH, UK
^cMaternal and Fetal Research Unit, Department of Women's Health, King's College London, St. Thomas* Campus, London SE1 7EH, UK

^* Corresponding author. Tel.: +44-122-582-6826x4471; fax: +44-122-582-6114 s.v.smirnov{at}bath.ac.uk

Received 1 August 2001; accepted 11 December 2001

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: Active control of the arterial diameter by vascular smooth muscle is one of the principle mechanisms by which vessels adapt to a significant rise in blood pressure after birth. Although voltage-gated K⁺ (Kv) channels play an important role in the regulation of excitation–contraction coupling in arteries, very little is known about postnatal modification of Kv channels. We therefore investigated changes in the functional characteristics and expression of Kv channels in rat aortic myocytes (RAMs) during early postnatal development. Methods: Kv currents (I_Kv) were investigated in single smooth muscle cells freshly dispersed from neonatal (1–3 days) and adult Wistar rat thoracic aorta using the whole-cell patch clamp technique. Results: I_Kv in neonates had significantly faster activation kinetics and was inactivated at more positive voltages than I_Kv in adults (half-inactivation potential –24±2 and –40±3 mV and slope factor 4.2±0.4 and 11.1±0.5 mV, respectively). No difference in the steady state activation was found. I_Kv in neonates was insensitive to a high concentration of tetraethylammonium (TEA, 10 mM) but blocked 4-aminopyridine (4-AP, IC₅₀=0.5±0.1 mM), whereas I_Kv in adult RAMs was almost completely abolished by 10 mM TEA and was relatively insensitive to low concentrations of 4-AP. I_Kv in both age groups was insensitive to charybdotoxin (300 nM) or -dendrotoxin (200 nM). Immunoblot analysis showed that the expression of Kv1.2 -protein decreased and Kv2.1 increased with development. Conclusion: Significant changes in functional characteristics of the native I_Kv and the expression of particular Kv channel proteins occurred during postnatal vascular development. These changes could play an important role in adaptation to extrauterine life.

KEYWORDS Arteries; Smooth muscle; Ion channels; K-channel; Developmental biology

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
After birth, blood vessels undergo rapid structural alterations in order to maintain cardiovascular homeostasis in the face of a significant rise in blood pressure [1]. Active control of the arterial diameter by vascular smooth muscle (VSM) is also likely to play a pivotal role and functional studies have reported altered contractility during development [2,3]. Relatively little is known, however, of analogous changes in membrane mechanisms which control Ca²⁺ homeostasis in VSM.

K⁺ channels play an important role in the regulation of VSM contractility [4]. Investigation of K⁺ channels during development of the cardiovascular system has hitherto focused predominantly on cardiac tissue, with significant alterations being observed [5–7]. Observations in the vasculature are largely limited to pulmonary circulation [8–10]. Ovine foetal pulmonary myocytes predominantly express Ca²⁺-activated K⁺ (BK_Ca) currents whereas I_Kv is the major current in adult myocytes [9], and greater expression of Kv2.1 channels has been reported in cultured ovine pulmonary myocytes from adult animals compared to those of neonatal and foetal cells [10]. In systemic vessels, however, the evidence suggests the activity of BK_Ca channels increases during development [11,12], but whether significant changes in Kv channels occur in the systemic circulation, which may be more important at earlier stages of postnatal development and may differ in this respect to the pulmonary vasculature, remains controversial. Thus, Gomez et al. [12] found no significant differences in electrophysiological and pharmacological properties of Kv channel currents in neonatal and adult rat aortic myocytes (RAMs), whereas our preliminary data, obtained under different experimental conditions, had suggested that I_Kv in the same preparation was developmentally regulated [13].

Therefore, the main purpose of this study was to characterise fully the electrophysiological and pharmacological properties of I_Kv. We have also compared the expression of Kv channel -subunits in RAMs from the thoracic aorta of neonatal and adult rats. Significant differences in the properties of I_Kv in neonates in comparison to adults were found. These were associated with a lower expression of the Kv2.1 channel protein in the neonatal aorta and reduced expression of Kv1.2 channel protein in adult rat aorta.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
2.1 Cell isolation procedure
Experiments were performed on RAMs freshly isolated from neonatal (1–3 days old) or male adult (6–8 weeks, 225–300 g) Wistar rats. Adult animals were killed by stunning and cervical dislocation, and pups were decapitated without prior anaesthesia in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Segments of aorta (1–3 mm), preincubated in physiological saline solution (PSS) containing 0.2 mM EGTA and no Ca²⁺ (Ca²⁺-free EGTA–PSS, 10 min, 37 °C), were transferred into 2 ml of Ca²⁺-free PSS containing collagenase Type XI and papain (1 and 0.3–0.5 mg/ml for neonates, and 3 and 1 mg/ml for adults, respectively). Dithiothreitol (1 mM) and PSS (10 µl/ml) were added to activate papain and increase collagenase activity, respectively. After incubation (37 °C; 15–18 min neonates, 30 min adults), segments were gently triturated in three consecutive volumes of Ca²⁺-free EGTA–PSS. The last two volumes were combined, filtered through 95-µm nylon mesh and centrifuged (1100 g for 5 min). Cells were then resuspended in PSS and maintained at +4 °C for use on the same day.

2.2 Electrophysiological recordings
Cells were placed in a chamber of volume 50–100 µl and were continually superfused (1 ml/min) with PSS or test solution via a five-barrel pipette [14]. Whole cell K⁺ currents were recorded at room temperature using the standard patch-clamp technique (Axopatch 200B amplifier and PCLAMP 6 software, Axon Instruments). Glass micropipettes filled with pipette solution had a resistance range of 3–5 M. At the beginning of each experiment, the capacitive transient in response to 10 mV hyperpolarizing step depolarisation (filtered at 50 kHz and sampled at 200 kHz) was recorded and cell membrane capacitance (C_m) calculated from the area under the capacitive transient. Time constants of the capacitance transient decay were 25–60 times faster in neonatal (0.06±0.005 ms, n=42) and adult (0.15±0.01 ms, n=71) RAMs than changes in the activation kinetics of K⁺ currents and, therefore, capacitance transients were not compensated. Calculated average series resistance was similar in neonatal (13.6±0.9 M, n=42) and adult (13.7±0.8 M, n=71) RAMs. Since the whole-cell current was small (average amplitude <500 pA at +80 mV in PSS) in both neonatal and adult myocytes, the calculated maximum voltage error was <7 mV and therefore neglected. In all voltage protocols used cells were held at –80 mV and stimulated at 0.1 Hz.

2.3 Western blot analyses
Aorta segments were placed in cold lysis buffer containing a protease inhibitor cocktail (Complete, Boehringer Mannheim) and homogenised (24 000 rpm, 2x1 min; Ultra-Turrax T25 homogeniser, Janke & Kunkel, IKA-Labortechnik). Cell lysate was then agitated slowly for 1 h and debris removed by centrifugation (2500 g, 30 min). All procedures were performed at 4 °C to minimise protein breakdown. Supernatants were stored at –70 °C prior to use.

Samples (20–40 µg of protein) were mixed with SDS–gel loading buffer and separated using 6 or 8% acrylamide gels. Total protein concentration was measured by the Bradford method using bovine serum albumin as a standard and gel lanes loaded with equal amounts of protein. Proteins were blotted onto PVDF membranes and washed in phosphate buffered saline (PBS, Gibco) and PBS containing 0.05% Tween-20 (PBS–T). After blocking (5%, w/v, solution of dried skimmed milk in PBS–T, 1 h, room temperature), membranes were probed overnight (4 °C) with either anti-Kv1.1 (1:1000), anti-Kv1.2 (1:1000), anti-Kv1.5 (1:500) or anti-Kv2.1 (1:1000) antibodies (Alomone, Israel) in 1% milk in PBS–T. Proteins were labelled with a secondary horseradish-peroxidase-conjugated goat anti-rabbit antibody (1:2000; 1 h, room temperature). Bound antibody was detected by the ECL method (Amersham) and analysed using QUANTISCAN software (Biosoft, UK).

2.4 Composition of solutions and materials
PSS (mM): 130 NaCl, 5 KCl, 1.5 CaCl₂, 1.2 MgCl₂, 10 HEPES and 10 glucose, pH 7.2; Ca²⁺-free PSS: same composition, but omitting CaCl₂; pipette solution (mM): 110 KCl, 10 NaCl, 5 MgATP, 10 HEPES, 10 EGTA and 0.5 CaCl₂ (estimated free [Ca²⁺]=8 nM), pH 7.2; lysis buffer (mM): 50 Tris–Cl (pH 7.5), 250 NaCl, 5 EDTA, 5 DTT, 10 NaF, and 0.1% v/v Igepal. All chemicals and enzymes, except charybdotoxin (ChTx, Peninsula Labs., USA), were purchased from Sigma or BDH Merck.

2.5 Data analysis and statistics
Data analysis, statistics and curve fitting were performed with PCLAMP6, Origin 4.1 (Microcal Software) and Microsoft EXCEL computer software. Data are presented as mean±S.E.M. Significance was determined using unpaired Student's t-test and differences were deemed significant at P<0.05 unless stated otherwise.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
3.1 Outward K⁺ currents in neonatal and adult RAMs
Fig. 1A shows a family of whole cell outward K⁺ currents recorded from a representative neonatal RAM in response to 300 ms step membrane depolarisations. In normal PSS current appeared at membrane potentials positive to –30 mV, rapidly reached maximum amplitude and then slowly decayed during maintained depolarisation. At membrane potentials above +60 mV small fluctuations superimposed on the outward current were often observed. Application of 1 µM paxilline, a selective inhibitor of BK_Ca channels [15], did not significantly affect the outward K⁺ current, but eliminated fluctuations (Fig. 1A and B). The amplitude of the outward K⁺ current measured at test potential +60 mV in PSS ranged between 25 and 480 pA (mean 131±25 pA) in 24 neonatal myocytes.

View larger version (26K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Outward K+ currents in neonatal and adult RAMs. (A and C) Membrane currents recorded from a representative neonatal (A, 1 day-old, Cm=7.1 pF) and adult (C, Cm=10.9 pF) myocyte in response to a 300 ms membrane depolarisation (Vm) between –40 and +80 mV (20 mV increment) from –80 mV in the absence and presence of 1 µM paxilline as indicated. B and D summarise mean I–V relationships of the outward current densities measured at 300 ms in PSS and in the presence of 1 µM paxilline in six neonatal (B) and ten adult (D) RAMs.

 
The outward K⁺ current in adult RAMs, although activated at similar membrane voltages, was characterised by larger fluctuations at positive membrane potentials (Fig. 1C). Suppression of the current by 1 µM paxilline was greater than in neonatal cells, being blocked by 35±3% (n=10), when measured at +60 mV, in contrast to 6±4% (n=6) in neonatal myocytes (P<0.0002). At more negative potentials paxilline was less effective (e.g. 17±5% at +20 mV compared with inhibition at +60 mV, P<0.002, paired t test) (Fig. 1D) suggesting that the relative contribution of BK_Ca channels to the whole cell outward current is larger in adult than in neonatal RAMs.

At +60 mV in PSS, the net outward current in adult myocytes ranged between 40 and 1160 pA (mean value 239±38 pA, n=35), approximately twice that of neonates (P<0.04). However, because the adult myocytes (C_m=11.0±0.6 pF n=72) were more than twice the size of neonatal cells (C_m=4.6±0.2 pF, n=42, P<0.0001), the current amplitude corrected for cell size was significantly smaller in adult (18±1 pA/pF, n=35) than neonatal (25±4 pA/pF, n=24, P<0.05) RAMs.

3.2 Comparison of the effect of K⁺ channel inhibitors on K⁺ currents in neonatal and adult RAMs
Fig. 2 illustrates the effect of 1 and 10 mM TEA and 5 mM 4-AP on the whole cell K⁺ currents in neonatal and adult RAMs using the voltage protocol described above. In neonatal RAMs (Fig. 2Ab) the effect of 1 mM TEA (a concentration which should eliminate most BK_Ca) was similar (13±5% inhibition at +60 mV; n=20) to that observed with 1 µM paxilline (compare Figs. 2B and 1B). In adult myocytes, the inhibitory effect of 1 mM TEA was significantly larger at positive voltages (51±3% inhibition at +60 mV, n=25, P<0.001) than that which occurred with paxilline but, as with paxilline, the degree of block was decreased at negative potentials (Fig. 2D). The addition of 300 nM ChTx, another potent inhibitor of BK_Ca, to PSS reduced the current amplitude at +60 mV in adult RAMs to a similar degree (28±8%, n=7) as with paxilline (P>0.05; not shown), suggesting that the greater effect of 1 mM TEA in adult RAMs is chiefly due to an increased sensitivity of I_Kv to this inhibitor, as described below.

View larger version (30K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Effect of TEA and 4-AP on IKv. (A and C) Families of the outward currents recorded, as described in Fig. 1, from neonatal (A, 1 day-old, Cm=8.6 pF) and adult (Cm=13.8 pF) RAMs in PSS (a) and in the presence of 1 (b) and 10 (c) mM TEA and 10 mM TEA and 5 mM 4-AP (d). (B and D) Mean I–V relationships of the current recorded at 300 ms in PSS (, n=24 and 35), 1 mM TEA (, n=20 and 25), 10 mM TEA (, n=20 and 8) and both 10 mM TEA and 5 mM 4-AP (, n=16 and 7) in neonatal (B) and adult (D) RAMs, respectively.

 
An increase of TEA concentration to 10 mM, however, caused a significantly larger decrease in the outward current amplitude in adults (Fig. 2Cc) than in neonates (Fig. 2Ac). Inhibition of the current at +60 mV was 82±2% in eight adult RAMs in comparison to 30±5% in 20 neonatal RAMs (P<0.0001). Further addition of 5 mM 4-AP completely eliminated K⁺ currents in neonates (Fig. 2Ad and B) and had little additional effect on the residual current in adult RAMs (Fig. 2Cd and D).

The current remaining in the presence of both TEA and 4-AP was small and showed an almost linear dependence on membrane voltage in both cell types (Fig. 2B and D), most likely representing a small ‘leak’ current. The mean slope resistance measured in the linear range (between –90 and –60 mV) of the I–V relationship was not significantly different in neonatal (17±3 G, n=16) and adult (8±1 G, n=35) RAMs.

Fig. 3 compares the differential sensitivity of I_Kv to 4-AP in neonatal and adult RAMs studied in the presence of 1 µM paxilline. I_Kv was potently inhibited by the drug with IC₅₀=0.5±0.1 mM in neonates, whereas in adults I_Kv was blocked only by 43.0±8.6% by 20 mM 4-AP.

View larger version (25K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 4-Aminopyridine sensitivity of IKv. IKv were recorded at +60 mV in neonatal (A, Cm=10.7 pF) and adult (B, Cm=16.8 pF) RAM in the absence (C) and subsequent addition of various concentration of 4-AP (indicated by numbers in mM). Vertical bars are 150 pA. Horizontal dashed lines in this and following figures indicate zero current. IKv amplitude, measured 150–200 s after application of 4-AP, was normalised to that in the absence of the drug and plotted in (C) against the concentration of 4-AP. Filled circles (n=7) were connected by lines and the line through open circles (n=6) is drawn according to the following equation with IC50=0.5 mM and the residual component B=0.21.

 
3.3 Activation and inactivation of I_Kv in neonatal and adult RAMs
Since it appeared that the sensitivity of I_Kv to TEA and 4-AP, under conditions when the residual BK_Ca currents were eliminated, differed in neonatal and adult myocytes, the biophysical characteristics of I_Kv in these cells in the presence of 1 µM paxilline were considered in more detail.

As shown in Figs. 1 and 2, the rate of I_Kv onset at the beginning of the membrane depolarisation appeared to be higher in neonatal than in adult RAMs. Therefore, voltage-dependence of I_Kv activation was quantified by measurement of the onset of I_Kv activation between –20 and +80 mV. This fitted well to a single exponential function in both cell types (Fig. 4A). The rate of activation for I_Kv between 0 and +80 mV was 3–14 times lower in adult than in neonatal cells (P<0.003, Fig. 4B).

View larger version (28K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Differences in the kinetics of IKv activation. (A and B) Time course of the onset of IKv between –20 and +80 mV (20 mV increment) in neonatal (1-day-old, Cm=4.2 pF) and adult (Cm=13.6 pF) RAMs, respectively. Smooth lines show monoexponential fit with time constant () equal to 54.1, 8.2, 4.8, 2.8, 2.6 and 1.9 ms in neonatal and 61.8, 29.3, 18.7, 10, 9.8 and 8.7 ms in adult myocyte at –20, 0, +20, +40, +60 and +80 mV, respectively. (C) Voltage dependence of the mean in six neonatal () and ten adult () RAMs (0.003>P>0.0001 between 0 and +80 mV).

 
Voltage-dependent inactivation of I_Kv was investigated using the voltage protocol described in Fig. 5. In neonatal RAMs I_Kv was relatively stable between –100 and –50 mV but decreased rapidly between –40 and –10 mV and was completely inactivated at potentials above –10 mV (Fig. 5Aa and B). In adult RAMs I_Kv inactivation began at more negative potentials (between –80 and –60 mV), achieving maximum inhibition between –10 and 0 mV (Fig. 5Ab and C). Interestingly, further increasing the conditioning potential resulted in a progressive increase in the current amplitude during the test pulse in adults, but not in neonates (Fig. 5A and B). The normalised I_Kv, fitted to the Boltzmann function (Fig. 5B), demonstrated a significant negative shift in voltage-dependence of inactivation in adult RAMs compared to neonatal cells (V_0.5=–23.6±1.8 and –39.6±3.3 mV respectively, P<0.003). The slope of inactivation for I_Kv in adults (k=11.1±0.5 mV, n=8) was also less than half that of neonates (k=4.2±0.4 mV, n=6, P<0.0001). Whilst the fraction A of the non-inactivating current remaining at positive potentials tended to be larger in adults (0.35±0.3, n=8) than in neonates (0.27±0.02, n=6, P<0.04, one-tailed t test), accurate comparison was complicated by a tendency for the current in adult RAMs to increase at these voltages.

View larger version (22K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Availability and steady-state activation of IKv in neonatal and adult RAMs. Availability of IKv was measured using a two-pulse protocol with 10 s conditioning potentials (Vc) and 120 ms test potential to +60 mV, as shown in (A). Interpulse interval 10 ms. (Aa and Ab) IKv recorded during the test pulse after Vc varied from –100 and +30 mV in 10 mV increment in neonatal (1-day-old, Cm=8.6 pF) and adult (Cm=22.6 pF) RAMs, respectively. Arrows indicate IKv recorded after Vc equal to +30 (a) and –10 (b) mV. (B) Dependence of the normalised IKv on Vc. The current amplitude was normalised with respect to the mean current measured between –100 and –70 mV in six neonatal () and –100 and –90 mV in eight adult () RAMs. The mean data were then fit according to the Boltzmann equation (solid lines) where V0.5, the half-inactivation potential, was equal to –23 and –39 mV (vertical dashed lines), k, the slope factor, was 4.7 and 11.9 mV and A, the noninactivating component, was 0.28 and 0.33 for neonates and adults, respectively. (C) Steady-state activation for IKv calculated from I–V relationships shown in Fig. 1 and corrected for a residual leak current calculated from the mean slope resistance measured as described in the text. Solid lines were drawn according to the Boltzmann function described above with A=0 and half-activation potentials equal to 1.3 and 4.9 mV and the slope factors of 14.8 and 15.7 mV in neonatal and adult RAMs, respectively.

 
In contrast to I_Kv availabilities no significant differences in voltage-dependence of steady-state activation of I_Kv calculated from I–V relationships were observed (Fig. 5C). The mean half-activation potential and slope factor were 1.1±2.2 and 14.3±0.7 mV (n=6) in neonatal and 5.2±3.3 and 15.2±0.8 mV (n=10) in adult RAMs, respectively.

3.4 Effect of -dendrotoxin and charybdotoxin on I_Kv currents in neonatal and adult RAMs
Some TEA-sensitive delayed rectifier currents are additionally characterised by sensitivity to nanomolar concentrations of certain toxins, including ChTx and -dendrotoxin (DTx) [16]. Therefore, the effects of 300 nM ChTx and 200 nM DTx on I_Kv, measured at +60 mV, were investigated under conditions when BK_Ca was blocked either by 1 µM paxilline or 1 mM TEA. Neither ChTx nor DTx inhibited I_Kv in neonatal and adult RAMs (P>0.05; Fig. 6).

View larger version (21K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Effect of DTx and ChTx on IKv in neonatal (A) and adult (B) RAMs in the presence of 1 µM paxilline or 1 mM TEA, respectively, as indicated in parenthesis. Current amplitude was normalised to that in the absence of DTx or ChTx. Number of cells studied shown inside the bars.

 
3.5 Developmental changes in the expression of Kv1.2, Kv1.5 and Kv2.1 channel proteins in rat aorta
The differences in the I_Kv characteristics of neonatal and adult RAMs could be indicative of differential expression of the Kv channel isoforms. We have focused on three Kv -subunits, Kv1.2, Kv1.5 and Kv2.1, which encode a delayed rectifier K⁺ current similar to the native I_Kv in neonates and adults. To study the expression of Kv -subunits, Western blot analysis was performed on total protein isolated from rat aorta, heart and brain (positive control). These were loaded on the same gel and developed under identical conditions (Fig. 7A). The expression of Kv1.2 was significantly decreased and that of Kv2.1 significantly increased with development (Fig. 7B). For Kv1.5 two bands, (molecular masses of 95 and 76 kDa) were detected in rat heart. Both were specific to binding of the anti-Kv1.5 antibody, as they were not detected when the primary antibody was preincubated with the corresponding antigen (not shown). No Kv1.5 bands were detectable in neonatal aorta, and only a 95 kDa protein was apparent in the adult aorta. Qualitatively similar results were obtained in another two experiments.

View larger version (33K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7 Changes in the expression of K+ channel -subunits in rat tissues with development. (A) Representative Western blot analyses of Kv1.2 (75 kDa), Kv1.5 (two bands of 95 and 76 kDa) and Kv2.1 (125 kDa) -subunit proteins. Numbers indicate molecular mass standards. H, A and B are abbreviations for heart, aorta and adult brain, respectively. (B) Statistical analysis of Kv1.2 (n=4) and Kv2.1 (n=5) immunoblots for heart and aorta from neonatal and adult rats. Band densities presented in arbitrary units. *, P<0.03.

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
4.1 Isolation and characterisation of voltage-gated K⁺ currents in neonatal and adult RAMs
Despite using a low Ca²⁺-containing pipette solution, large fluctuations of the outward current, attributed to BK_Ca channel activity, were observed at positive membrane potentials in both neonatal and adult RAMs (Figs. 1 and 2). These were sensitive to known BK_Ca blockers, paxilline, TEA and ChTx, and were larger in adults than in neonates. This observation echoes previous reports from studies of foetal and adult human aorta [11], rat aortic [12] cells and also from neonatal and adult porcine pulmonary arteries [8]. Since this study was focused on investigation of developmental changes in Kv currents, BK_Ca currents were eliminated using 1 µM paxilline. Inhibition of the net outward current with paxilline was not significantly different from that achieved with 1 mM TEA (neonates) or 300 nM ChTX (adults), although 1 mM TEA was more effective in adults. This effect could be explained by the greater sensitivity of I_Kv to TEA in adult RAMs and the subsequent underestimation of the current amplitude in the presence of 1 mM TEA. Nonetheless, these results strongly suggest that the outward K⁺ currents recorded in the presence of either inhibitor represent predominantly Kv channel currents. No evidence for inward rectifier or fast inactivating A-type or A-like K⁺ currents in adult or neonatal RAMs was found.

I_Kv thus recorded showed clear differences in electrophysiological properties and pharmacological sensitivity between neonatal and adult rat aorta. In adult RAMs (1) the activation of I_Kv was slower, (2) I_Kv inactivated at more negative membrane potentials and demonstrated a U-shape dependence on the conditioning potential in contrast to neonates, (3) the slope of I_Kv inactivation was 2-fold greater than in neonatal RAMs, and (4) I_Kv was almost completely blocked by 10 mM TEA and relatively insensitive to 4-AP in contrast to relative TEA insensitivity and 4-AP sensitivity of that in neonates. However, I_Kv in both cell types was not inhibited by high concentrations of ChTx and DTx.

The differences in electrophysiological and pharmacological properties in I_Kv in neonatal and adult RAMs could explain the differences in the sensitivity of contraction of intact rat aorta to 4-AP and TEA described previously by Gomez et al. [12]. However, in contrast to our findings voltage-dependent inactivation and 4-AP sensitivity of I_Kv in neonatal and adult RAMs were found to be similar [12]. Differing experimental conditions may provide an explanation. Thus, we directly blocked the residual BK_Ca with paxilline, whilst a Ca²⁺-free PSS containing 2 mM Co²⁺ to block Ca²⁺ entry and BK_Ca currents was employed in [12]. We avoided the use of divalent ions as they can directly affect I_Kv; at least in adult RAMs we found that addition of 0.2 mM Cd²⁺ shifted the I_Kv availability by 10 mV to more positive voltages (V_0.5=–30±2 mV) and significantly increased the slope (k=8±1 mV, n=5, P<0.03) compared to that measured in control solutions.

4.2 Possible molecular equivalents of I_Kv in neonatal and adult RAMs
The different properties of I_Kv in adult and neonate suggested possible alterations in expression of the Kv channel proteins. In both cell types I_Kv was characterised by a relatively low rate of inactivation and can be classified as a delayed rectifier current. Genes, which encode slowly-inactivated delayed rectifiers, corresponding to Kv1.1, Kv1.2, Kv1.5, Kv1.6, Kv2.1 and to Kv3.1b channels have previously been found in VSM [17–19]. Kv1.1, Kv1.6 and Kv3.1b homomultimers, which encode a rapid delayed rectifier current similar to that observed in neonatal RAMs, are inhibited by low concentrations of TEA (<1 mM) and, in addition, Kv1.1 and Kv1.6 channel currents are also blocked by nanomolar concentrations of DTx [20,16]. These pharmacological characteristics are however not shared with I_Kv of neonatal RAMs. Moreover, no detectable expression of Kv1.1 -protein in both neonatal and adult aorta and Kv3.1b expression in adult tissue was found (not shown). Currents of Kv1.2 and Kv1.5 channels however are TEA-insensitive (in common with neonatal I_Kv) but unlike the neonatal I_Kv, currents through Kv1.2 homomultimers are inhibited by DTx and ChTx [21,22]. The neonatal I_Kv, therefore would seem to have characteristics more similar to the Kv1.5 channel. However it also shares characteristics with a Kv1.2/Kv1.5 heteromultimer since DTx and ChTx sensitivity is known to be lost in this heteromultimer. Indeed, the presence of only one Kv1.5 -subunit in the tetramer is sufficient for this loss of toxin sensitivity [21]. The loss of the toxin sensitivity may be unique for Kv1.2/Kv1.5 heteromultimers as e.g. Kv1.1/Kv1.5 heteromultimers retain sensitivity to DTx [23].

Kv2.1 channel currents are characterised by a relatively slow kinetic of activation and demonstrate a moderate sensitivity to TEA with half block between 1 and 10 mM [24,19,25]. In addition, a U-shape dependence of inactivation observed in adult RAMs was previously demonstrated for Kv2.1 channels [26]. These properties of Kv2.1 match well those of the I_Kv in adult RAMs. Also relevant, an increased Kv2.1 gene and protein expression with postnatal development has been reported in cultured ovine pulmonary myocytes [10].

If the predictions above, based on electrophysiological and pharmacological characteristics are correct, we would expect the expression of Kv1.2 and Kv1.5 -proteins to decrease and the expression of Kv2.1 -subunit to increase during postnatal development. Indeed, increased expression of Kv2.1 protein and a reciprocal decrease in expression of Kv1.2 protein was observed from neonate to adult. However, one anomaly occurred as we were not able to detect the Kv1.5 -protein in neonatal tissue, whereas the corresponding 95 kDa band was apparent in adult rat aorta. This was unlikely to be due to protein degradation in the neonatal tissue since the isolation procedure was performed on ice or at +4 °C in the presence of protease inhibitors. Secondly, the absence of signal was unlikely to result from use of an inappropriate antibody since, in preliminary experiments, using adult tissue, qualitatively similar results were obtained using anti-Kv1.5 antibody from three sources (Alomone, Chemicon and Upstate Biotechnology). Neither was unequal loading of proteins likely to be contributory as no significant differences between lanes was found in Coumassie blue stained gels run in parallel experiments (standard marker proteins, e.g. β-actin could not be used because expression of both - and β-actins changes during vascular development [27]). Furthermore, postnatal changes in Kv channel expression observed in the heart corresponded to the developmental changes in the expression of Kv1.2 and Kv2.1 channels previously described [6,28].

Whilst it is possible that the level of expression of the Kv1.5 -subunit is low in neonatal RAMs and below the level of detection, this cannot explain the appearance of the 95 kDa Kv1.5 band in adult aorta without a corresponding current. Possible contamination with endothelial cells cannot be excluded, but the relative cell mass is small compared to smooth muscle and therefore the contribution to Kv proteins should be small. Also, Kv channels are not predominant in endothelial cells [29]. Nerve cells, which increase in number with development, may, theoretically, contribute to the 95 kDa band as both 90 and 65 kDa Kv1.5 bands were found in cultured Schwann cells [30].

In the whole rat heart 95 and 76 kDa bands were detected (Fig. 7A) and the density of both increased with development. In the mouse heart, at least three alternatively spliced Kv1.5 isoforms have been found [31]. Interestingly, the carboxyl-truncated isoform was not functional, but inhibited the expression of the functionally active Kv1.5 isoform [31]. If this were to occur in the rat aorta it could provide an explanation for the expression of the Kv channel without any functional current. Studies of differential expression of these isoforms may provide an answer to this apparent paradox.

4.3 Functional relevance of developmental changes in I_Kv
Appreciation of developmental changes in ion channels may impact upon investigations of the ‘foetal programming’ hypothesis [32] which suggests that adulthood cardiovascular disease can arise from permanent modification of the cardiovascular system in utero. Arrested development of the normal pathways of control of vascular contractility e.g. the K⁺ channels described here, or permanent alteration of function, could lead to ultimate failure of cardiovascular homeostasis. We have recently described abnormalities in vascular function of the offspring of animals subjected to dietary modification in pregnancy [33,34] and it would be of considerable interest to determine whether abnormal development of K⁺ channels may play a role in the abnormalities observed.

Time for primary review 36 days.

	Acknowledgements

 
This work was supported by the British Heart Foundation (grants BS/95001, PG/96151 and FS/2000013) and Tommy's the Baby Charity.

	Notes

 
¹ Permanent address: Department of Neuromuscular Physiology, Bogomoletz Institute of Physiology, Kiev-24, Ukraine.

² Present address: Division of Protein Structure, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

1. Stehbens W.E. Structural and architectural changes during arterial development and the role of hemodynamics. Acta Anatom. (1996) 157:261–274.[CrossRef]
2. Pearce W.J., Ashwal S. Developmental changes in thickness, contractility, and hypoxic sensitivity of newborn lamb cerebral arteries. Pediatr. Res. (1987) 22:192–196.[Web of Science][Medline]
3. Belik J., Halayko A.J., Rao K., Stephens N.L. Pulmonary and systemic vascular smooth muscle mechanical characteristics in newborn sheep. Am. J. Physiol. (1992) 263:H881–H886.[Web of Science][Medline]
4. Nelson M.T., Quayle J.M. Physiological roles and properties of potassium channels in arterial smooth muscle. Am. J. Physiol. (1995) 268:C799–C822.[Web of Science][Medline]
5. Guo W., Kamiya K., Toyama J. Roles of the voltage-gated K⁺ channel subunits, Kv 1.5 and Kv 1.4, in the developmental changes of K⁺ currents in cultured neonatal rat ventricular cells. Pflügers Arch. (1997) 434:206–208.[CrossRef][Web of Science][Medline]
6. Xu H., Dixon J.E., Barry D.M., et al. Developmental analysis reveals mismatches in the expression of K⁺ channel subunits and voltage-gated K⁺ channel currents in rat ventricular myocytes. J. Gen. Physiol. (1996) 108:405–419.[Abstract/Free Full Text]
7. Wang L., Duff H.J. Developmental changes in transient outward current in mouse ventricle. Circ. Res. (1997) 81:120–127.[Abstract/Free Full Text]
8. Evans A.M., Osipenko O.N., Haworth S.G., Gurney A.M. Resting potentials and potassium currents during development of pulmonary artery smooth muscle cells. Am. J. Physiol. (1998) 275:H887–H899.[Web of Science][Medline]
9. Reeve H.L., Weir E.K., Archer S.L., Cornfield D.N. A maturational shift in pulmonary K⁺ channels, from Ca²⁺ sensitive to voltage dependent. Am. J. Physiol. (1998) 275:L1019–L1025.[Web of Science][Medline]
10. Cornfield D.N., Saqueton C.B., Porter V.A., et al. Voltage-gated K⁺-channel activity in ovine pulmonary vasculature is developmentally regulated. Am. J. Physiol. (2000) 278:L1297–L1304.[Web of Science]
11. Bregestovski P.D., Printseva O.Y., Serebryakov V., et al. Comparison of Ca²⁺-dependent K⁺ channels in the membrane of smooth muscle cells isolated from adult and foetal human aorta. Pflügers Arch. (1988) 413:8–13.[CrossRef][Web of Science][Medline]
12. Gomez J.P., Ghisdal P., Morel N. Changes of the potassium currents in rat aortic smooth muscle cells during postnatal development. Pflügers Arch. (2000) 441:388–397.[CrossRef][Web of Science][Medline]
13. Belevych A.E., Poston L., Smirnov S.V. Developmental switch in the expression of voltage-gated K⁺ channels in rat aortic smooth muscle cells. Biophys. J. (1999) 76:A293. (Abstract).
14. Smirnov S.V., Aaronson P.I. Modulatory effects of arachidonic acid on the delayed rectifier K⁺ current in rat pulmonary arterial myocytes. Structural aspects and involvement of protein kinase C. Circ. Res. (1996) 79:20–31.[Abstract/Free Full Text]
15. Li G., Cheung D.W. Effects of paxilline on K⁺ channels in rat mesenteric arterial cells. Eur. J. Pharmacol. (1999) 372:103–107.[CrossRef][Web of Science][Medline]
16. Grissmer S., Nguyen A.N., Aiyar J., et al. Pharmacological characterization of five cloned voltage-gated K⁺ channels, types Kv1.1, 1.2, 1.3, 1.5, and 3.1, stably expressed in mammalian cell lines. Mol. Pharmacol. (1994) 45:1227–1234.[Abstract]
17. Yuan X.-J., Wang J., Juhaszova M., Golovina V.A., Rubin L.J. Molecular basis and function of voltage-gated K⁺ channels in pulmonary arterial smooth muscle cells. Am. J. Physiol. (1998) 274:L621–L635.[Web of Science][Medline]
18. Osipenko O.N., Tate R.J., Gurney A.M. Potential role for Kv3.1b channels as oxygen sensors. Circ. Res (2000) 86:534–540.[Abstract/Free Full Text]
19. Patel A.J., Lazdunski M., Honoré E. Kv2.1/Kv9.3, a novel ATP-dependent delayed-rectifier K⁺ channel in oxygen-sensitive pulmonary artery myocytes. EMBO J. (1997) 16:6615–6625.[CrossRef][Web of Science][Medline]
20. Kirsch G.E., Drewe J.A., Verma S., Brown A.M., Joho R.H. Electrophysiological characterization of a new member of the RCK family of rat brain K⁺ channels. FEBS Lett. (1991) 278:55–60.[CrossRef][Web of Science][Medline]
21. Russell S.N., Overturf K.E., Horowitz B. Heterotetramer formation and charybdotoxin sensitivity of two K⁺ channels cloned from smooth muscle. Am. J. Physiol. (1994) 267:C1729–C1733.[Web of Science][Medline]
22. Hulme J.T., Coppock E.A., Felipe A., Martens J.R., Tamkun M.M. Oxygen sensitivity of cloned voltage-gated K⁺ channels expressed in the pulmonary vasculature. Circ. Res. (1999) 85:489–497.[Abstract/Free Full Text]
23. Hatton W.J., Mason H.S., Carl A., et al. Functional and molecular expression of a voltage-dependent K⁺ channel (Kv1.1) in interstitial cells of Cajal. J. Physiol. (2001) 533:315–327.[Abstract/Free Full Text]
24. Shi G., Kleinklaus A.K., Marrion N.V., Trimmer J.S. Properties of Kv2.1 K⁺ channels expressed in transfected mammalian cells. J. Biol. Chem. (1994) 269:23204–23211.[Abstract/Free Full Text]
25. Roe M.W., Worley J.F., Mittal A.A., et al. Expression and function of pancreatic β-cell delayed rectifier K⁺ channels. Role in stimulus–secretion coupling. J. Biol. Chem. (1996) 271:32241–32246.[Abstract/Free Full Text]
26. Klemic K.G., Shieh C.C., Kirsch G.E., Jones S.W. Inactivation of Kv2.1 potassium channels. Biophys. J. (1998) 74:1779–1789.[Web of Science][Medline]
27. Owens G.K., Thompson M.M. Developmental changes in isoactin expression in rat aortic smooth muscle cells in vivo. Relationship between growth and cytodifferentiation. J. Biol. Chem. (1986) 261:13373–13380.[Abstract/Free Full Text]
28. Nakamura K., Iijima T. Postnatal changes in mRNA expression of the K⁺ channel in rat cardiac ventricles. Jpn. J. Pharmacol. (1994) 66:489–492.[Medline]
29. Nilius B., Droogmans G. Ion channels and their functional role in vascular endothelium. Physiol. Rev. (2001) 81:1415–1459.[Abstract/Free Full Text]
30. Sobko A., Peretz A., Shirihai O., et al. Heteromultimeric delayed-rectifier K⁺ channels in Schwann cells: developmental expression and role in cell proliferation. J. Neurosci. (1998) 18:10398–10408.[Abstract/Free Full Text]
31. Attali B., Lesage F., Ziliani P., et al. Multiple mRNA isoforms encoding the mouse cardiac Kv1-5 delayed rectifier K⁺ channel. J. Biol. Chem. (1993) 268:24283–24289.[Abstract/Free Full Text]
32. Barker D.J.P. Fetal programming influences on development and disease in later life, 1. O'Brien P.M.S., Wheeler T., Barker D.J.P., eds. (1999) London: RCOG Press. 3–11.
33. Koukkou E., Ghosh P., Lowy C., Poston L. Offspring of normal and diabetic rats fed saturated fat in pregnancy demonstrate vascular dysfunction. Circulation (1998) 98:2899–2904.[Abstract/Free Full Text]
34. Ghosh P., Bitsanis D., Ghebremeskel K., Crawford M., Poston L. Abnormal aortic fatty acid composition and small artery function in offspring of rats fed a high fat diet in pregnancy. J. Physiol. (2001) 533:815–822.[Abstract/Free Full Text]

CiteULike    Connotea    Del.icio.us    What's this?

This article has been cited by other articles:

				M. Saito, Y. Murai, H. Sato, Y.-C. Bae, T. Akaike, M. Takada, and Y. Kang Two Opposing Roles of 4-AP-Sensitive K+ Current in Initiation and Invasion of Spikes in Rat Mesencephalic Trigeminal Neurons J Neurophysiol, October 1, 2006; 96(4): 1887 - 1901. [Abstract] [Full Text] [PDF]

				E. Miguel-Velado, A. Moreno-Dominguez, O. Colinas, P. Cidad, M. Heras, M. T. Perez-Garcia, and J. R. Lopez-Lopez Contribution of Kv Channels to Phenotypic Remodeling of Human Uterine Artery Smooth Muscle Cells Circ. Res., December 9, 2005; 97(12): 1280 - 1287. [Abstract] [Full Text] [PDF]

				A. Cogolludo, L. Moreno, F. Lodi, J. Tamargo, and F. Perez-Vizcaino Postnatal maturational shift from PKC{zeta} and voltage-gated K+ channels to RhoA/Rho kinase in pulmonary vasoconstriction Cardiovasc Res, April 1, 2005; 66(1): 84 - 93. [Abstract] [Full Text] [PDF]

				P. Tammaro, A. L. Smith, B. L. Crowley, and S. V. Smirnov Modulation of the voltage-dependent K+ current by intracellular Mg2+ in rat aortic smooth muscle cells Cardiovasc Res, February 1, 2005; 65(2): 387 - 396. [Abstract] [Full Text] [PDF]

				G. C. Amberg, C. F. Rossow, M. F. Navedo, and L. F. Santana NFATc3 Regulates Kv2.1 Expression in Arterial Smooth Muscle J. Biol. Chem., November 5, 2004; 279(45): 47326 - 47334. [Abstract] [Full Text] [PDF]

This Article Abstract FREE Full Text (PDF) E-letters: Submit a response Alert me when this article is cited Alert me when E-letters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Request Permissions Disclaimer Google Scholar Articles by Belevych, A. E. Articles by Smirnov, S. V. Search for Related Content PubMed PubMed Citation Articles by Belevych, A. E. Articles by Smirnov, S. V. Social Bookmarking          What's this?

Online ISSN 1755-3245 - Print ISSN 0008-6363

Copyright © 2009 European Society of Cardiology

Oxford Journals Oxford University Press

• Site Map
• Privacy Policy
• Frequently Asked Questions

Other Oxford University Press sites: Oxford University Press Oxford Journals China Oxford Journals Japan American National Biography Booksellers' Information Service Children's Fiction and Poetry Children's Reference Corporate & Special Sales Dictionaries Dictionary of National Biography Digital Reference English Language Teaching Higher Education Textbooks Humanities International Education Unit Law Medicine Music Online Products Oxford English Dictionary Reference Rights and Permissions Science School Books Social Sciences Very Short Introductions World's Classics