Skip Navigation

Cardiovascular Research 2002 54(3):590-600; doi:10.1016/S0008-6363(02)00267-5
© 2002 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Ogura, T.
Right arrow Articles by Shibamoto, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ogura, T.
Right arrow Articles by Shibamoto, T.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2002, European Society of Cardiology

Sarcolemmal hydraulic conductivity of guinea-pig and rat ventricular myocytes

Toshitsugu Ogura*, Hiroyuki Matsuda, Sunao Imanishi and Toshishige Shibamoto

Second Department of Physiology, Kanazawa Medical University, 1-1, Daigaku, Uchinada-machi, Kahoku-gun, Ishikawa-ken 920-0293, Japan

* Corresponding author. Tel.: +81-76-286-2211 (ext. 3644); fax: +81-76-286-8010 physiol2{at}kanazawa-med.ac.jp

Received 19 September 2001; accepted 31 December 2001


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Osmotic gradient-induced volume change and sarcolemmal water permeability of cardiac myocytes were evaluated to characterize the mechanism of water flux across the plasma membranes. Methods: Cell surface dimensions were measured from isolated guinea-pig and rat ventricular myocytes by digital videomicroscopy, and membrane hydraulic conductivity (Lp) was obtained by analyzing the time course of cell swelling and shrinkage in response to osmotic gradients. Results: Superfusion with anisosmotic solution (0.5–4 times normal osmolality) caused a rapid (<3 min to steady states) and reversible myocyte swelling or shrinkage. Lp was {approx}1.9x10–10 l N–1 s–1 for guinea-pig myocytes and {approx}1.7x10–10 l N–1 s–1 for rat myocytes at 35 °C. Arrhenius activation energy (Ea), a measure of the energy barrier to water flux, was {approx}3.7 (guinea-pig) and {approx}3.6 kcal mol–1 (rat) between 11 and 35 °C; these values are equivalent to Ea of self-diffusion of water in bulk solution ({approx}4 kcal mol–1). Treatment with 0.1 mM Hg2+, a sulfhydryl-oxidizing reagent that blocks membrane water channels, reduced Lp by {approx}80%, and the sulfhydryl-reducing reagent dithiothreitol (10 mM) antagonized the inhibitory action of Hg2+. Inhibition of the volume-sensitive cation (30 µM Gd3+) and anion (1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonate) channels and Na+–K+ pump (10 µM ouabain) modified the size of osmotic swelling but had little effect on Lp. Conclusions: Although the observed Lp is relatively small in magnitude, the low Ea and the sulfhydryl reagent-induced modification of Lp are characteristic of channel-mediated water transport. These data suggest that water flux across the sarcolemma of guinea-pig and rat heart cells occurs through parallel pathways, i.e., the majority passing through water channels and the remainder penetrating the lipid bilayers.

KEYWORDS Ion transport; Membrane permeability/physics; Membrane transport; Myocytes; Sarcolemma


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Functional measurements of water permeability across cell membranes are important for the investigation of water transport mechanisms. Generally, transport of water into and out of the cells takes place by two distinct mechanisms: diffusional movement through the lipid bilayers, or transport through membrane aqueous pores [1–4]. The former is constrained by membrane fluidity and lipid organization, and therefore relatively small in magnitude with a high activation energy (<10 kcal mol–1). The latter, channel-mediated water transport, is large in magnitude, and characterized by a low activation energy (<5 kcal mol–1) and inhibition by mercurial sulfhydryl reagents [2,5]. Recent volumetric study on rabbit ventricular myocytes demonstrated that sarcolemmal water permeability, as assessed by hydraulic conductivity (Lp), is low and its activation energy is high ({approx}12 kcal mol–1), and concluded that the primary route for water crossing the sarcolemma is likely to be directly through the lipid bilayer itself [6]. However, water flux across cardiac plasma membranes in other species has not to date been documented.

The purpose of this study was to evaluate osmotic gradient-induced water flux across guinea-pig and rat heart cell membranes. Myocyte surface dimensions were measured by digital videomicroscopy, and Lp was obtained from the time course of cell swelling and shrinkage in response to osmotic gradients. The results indicate that Lp of guinea-pig and rat myocyte membranes is as low as the rabbit myocyte Lp [6]. However, analysis of the temperature dependence and pharmacological characteristics of Lp allows us to conclude that channel-mediated water transport has a functional significance in the osmosis of guinea-pig and rat ventricular myocytes.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Cell preparation
The investigation confirms to 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). Guinea-pigs (250–350 g) and Wistar rats (100–200 g) were anesthetized with sodium pentobarbital (50 mg kg–1, i.p.). Hearts were quickly excised, and single ventricular myocytes were enzymatically isolated as described previously [7,8]. Briefly, the excised hearts were mounted on a Langendorff column, and retrogradely perfused (37 °C) through the aorta with Ca2+-free Tyrode's solution containing collagenase (0.08–0.12 mg ml–1; Yakult, Tokyo, Japan) for 10–15 min. The cells were dispersed and maintained in a modified "KB" storage solution at 4 °C. A few drops of the cell suspension were placed in a 0.25-ml glass-bottomed chamber mounted on an inverted microscope (Diaphot-TMD, Nikon, Tokyo, Japan), and were superfused (3–4 ml min–1) with bathing solution at 35 °C, unless otherwise noted. Only rod-shaped quiescent cells with smooth contours were selected for study.

2.2 Solutions
Normal Tyrode's solution contained (mM) NaCl 140, KCl 5.4, CaCl2 1.8, MgCl2 1, glucose 10, and HEPES 5 (pH 7.4 with NaOH). Isosmotic (1T) Tyrode's solution was made by replacing 70 mM NaCl with 140 mM sucrose (319 mosmol kg–1). Hyperosmotic 1.5T, 2.2T, 2.8T and 4T solutions were made by adding 140–750 mM sucrose to the 1T solution (485, 689, 900, and 1235 mosmol kg–1, respectively); hyposmotic 0.5T solution was made by omitting sucrose (166 mosmol kg–1), and 0.75T solution was made by adding 70 mM sucrose (240 mosmol kg–1) to the 0.5T solution. The osmolalities of the solutions were verified with a freezing-point depression osmometer (OM-6050, Arkray, Kyoto, Japan), and the variation was less than 10 mosmol kg–1 for a given type of the solution.

2.3 Cell dimensions
Myocyte images were captured on-line using a high-resolution CCD camera (HV-D28S, Nikon) to a Macintosh computer via a video frame-grabber at {approx}0.25 µm pixel–1 resolution. Cell area, length and width were obtained with an image-analysis software (NIH Image, National Institutes of Health, Bethesda, MD, USA). The borders of cell images were traced using a graphics tablet, and cell area was measured by counting included pixels. Cell length was measured as the maximal distance along the long axis, and cell width was calculated as area/length. Reproducibility was assessed by repeatedly (10 times) tracing the same images recorded from 20 cells; the deviation of each measurement from the mean cell area averaged 1.15±0.06%.

Cell surface dimensions in 1T Tyrode's solution were width 23±0.5 µm and length 125±2 µm in guinea-pig myocytes (n=80), and width 21±0.4 µm and length 116±1.9 µm in rat myocytes (n=85). Relative myocyte volume was calculated as lengthx(width)2 by assuming that a myocyte is brick-shaped of which changes in width and thickness under anisosmotic conditions are proportional [8–10]. The validity of the assumption was tested by measuring the ratio of thickness to width from individual cells superfused with isosmotic and anisosmotic solutions. Cell thickness was obtained by focusing at the top and bottom surfaces of myocytes, and measured as the distance between the two focal planes by averaging 3–5 measurements for each myocyte. To unambiguously identify the top and bottom surfaces, alumina particles (diameter 1–2 µm, Sumitomo Chemical, Tokyo, Japan) placed on the myocyte and the adjacent floor of the chamber were used as a marker. The thickness-to-width ratio was 0.65±0.03 in guinea-pig cells (n=33) and 0.63±0.02 in rat cells (n=20) under isosmotic conditions, and neither osmotic swelling nor shrinkage significantly changed the ratio in both species (guinea-pigs: 0.65±0.03 in 0.5T, n=22; 0.64±0.06 in 2.2T, n=11; rats: 0.62±0.03 in 0.5T, n=20; 0.60±0.02 in 2.2T, n=20). The mean thickness-to-width ratio, 0.65 in guinea-pig cells and 0.63 in rat cells, was used to estimate absolute cell volume and surface area from width and length of each cell.

2.4 Membrane water permeability
The hydraulic conductivity Lp, a measure of the ability of water to cross biological membranes, was calculated from the time course of osmotic cell swelling and shrinkage as described by Solomon [11]. The rate of change in cell volume, dVc/dt, in response to an osmotic gradient across the sarcolemma is given by the Jacobs* equation:

Formula (1)
where A is the cell surface area, Viso is the absolute cell volume under control isosmotic conditions, Vc is the relative cell volume as a function of time, Vb is the osmotically-inactive volume fraction, and {pi} is the osmotic pressure with the subscripts "o" outside and "i" inside the cell. By assuming that {pi}i={pi}o in the steady state, {pi}i is taken as equal to {pi}o at zero time and {pi}o refers to the osmotic pressure of the test solution. Integrating and rearranging yields the following equation:

Formula (2)

A plot of the right-hand side of Eq. (2) [f(Vc)] as a function of time (t) gives a straight line of which slope is proportional to Lp (see Fig. 2), i.e., Lp=slopex(Viso{pi}i)/(A{pi}o2).


Figure 2
View larger version (38K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Analysis of osmotically-induced swelling of guinea-pig (A and B) and rat (C and D) myocytes. A and C, Time courses of cell swelling in 0.75T and 0.5T solutions (guinea-pig, n=9 cells; rat, n=7 cells). B and D, Plots of the right-hand side of the Eq. (2) [f(Vc), see Methods] against time. Straight lines are least-squares fits to the mean data, which come from the same myocytes as in A and C, except the first few points that were affected by solution mixing.

 
The Arrhenius activation energy (Ea), a measure of the energy barrier to water movement across a membrane, was determined from the slope of a semi-logarithmic plot of Lp vs. 1/temperature (Fig. 3C).


Figure 3
View larger version (31K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Dependence of Lp on temperature. A and B, Rate of cell swelling in 0.5T solution. f(Vc) is plotted against time; straight lines are least-squares fits to the mean data. Rat, n=6–7 cells; guinea-pig, n=7–8 cells. C, Arrhenius plot of Lp on switching from 1T to 0.5T solution. Lp was calculated from the data shown in A and B. Arrhenius activation energy (Ea) was measured as the slope of the regression line between 11 and 35 °C.

 
Example experiments shown in Fig. 1A depict that superfusion of guinea-pig myocytes with anisosmotic solution caused a rapid and reversible cell swelling or shrinkage. To estimate Vb, steady-state volumes obtained from similar experiments on guinea-pig and rat myocytes were plotted against the reciprocal of relative osmolality (van't Hoff plot, Fig. 1B). In 1.5T, 2T and 4T solutions, relative volumes were 0.80±0.01, 0.66±0.02 and 0.51±0.02 for guinea-pig cells (n=4–18) and 0.77±0.01, 0.65±0.02 and 0.50±0.01 for rat cells (n=4–14), respectively. The van't Hoff relationships were fitted with linear regression lines (r>0.99), and extrapolations of the regression lines to the volume axis suggest the osmotically-inactive fractions of 0.35 for guinea-pig cells and 0.34 for rat cells (Fig. 1B, arrow).


Figure 1
View larger version (18K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Osmotically-induced changes in myocyte volume. A, Osmotic swelling and shrinkage of guinea-pig myocytes in 0.75T and 1.5T solutions (n=6 cells each). B, A van't Hoff plot of relative cell volume against the reciprocal of relative osmolality of superfusate. The straight lines are linear regression fits to the mean data (guinea-pig: n=4–18 cells; rat: n=4–14 cells); the volume intercepts (arrow) at 0.35 (guinea-pig) and 0.34 (rat) are estimates of osmotically-inactive fractions. The broken line illustrates the theoretical relationship for perfect osmometer behavior.

 
2.5 Statistics
Data are expressed as means±S.E.M., with n indicating the number of experiments, and comparisons were made using Student's t-test. Analysis of variance (ANOVA) with Bonferroni correction was used for multiple comparisons. Differences were considered significant when P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Lp under various osmotic gradients
Myocyte swelling and shrinkage were induced by a step change in the osmolality of extracellular solution. Fig. 2A and C, depict the time courses of cell swelling in guinea-pig and rat myocytes exposed to hyposmotic 0.75T and 0.5T solutions. Cell volume was measured every 10 s during hyposmotic superfusion, and the right-hand side of Eq. (2) [f(Vc), see Methods] was calculated from the volume data and plotted against time (Fig. 2B and D). By applying a linear least-squares fit to the f(Vc) plot, Lp was determined from a slope of the regression line. Table 1 summarizes the observed Lp in response to various osmotic gradients at 35 °C. There were no significant differences in the values of Lp between osmotic perturbations, suggesting that water permeability of cardiac membranes is independent of the magnitude and direction of osmotic gradients. The overall average of Lp was 1.88±0.16x10–10 l N–1 s–1 for guinea-pig myocytes (n=28) and 1.69±0.17x10–10 l N–1 s–1 for rat myocytes (n=26). These values are comparable to Lp reported for rabbit ventricular myocytes ({approx}1.2x10–10 l N–1 s–1: 6), but are 5 to 20 times smaller than Lp of specialized water-transporting cells, such as renal tubules [12,13] and mammalian erythrocytes [14,15].


View this table:
[in this window]
[in a new window]

  		Table 1 Sarcolemmal hydraulic conductivity and its Arrhenius activation energy in guinea-pig and rat ventricular myocytes

 
3.2 Dependence of Lp on temperature
Water movement across biological membranes is dependent on temperature [1–3,16]. Fig. 3A and B, depict the f(Vc) measured on switching from 1T to 0.5T solution at 35, 22 and 11 °C. The f(Vc) slope was less steep at lower temperature, and Lp measured at 35, 22 and 11 °C was 1.59±0.27x10–10 (n=9), 0.90±0.25x10–10 (n=8) and 0.61±0.14x10–10 (n=8) l N–1 s–1 in guinea-pig cells, and 1.70±0.37x10–10 (n=7), 0.93±0.06x10–10 (n=6) and 0.68±0.07x10–10 (n=6) l N–1 s–1 in rat cells, respectively. Fig. 3C depicts an Arrhenius plot for the temperature dependence of Lp. The activation energy Ea, given by the slope of the plot between 35 and 11 °C, was 3.5 kcal mol–1 in guinea-pig cells and 3.4 kcal mol–1 in rat cells; similar values were obtained by an osmotic shrinkage assay with 2T solution (Table 1). These values, which are equivalent to Ea of self-diffusion of water in bulk solution (Ea{approx}4 kcal mol–1), indicate that the temperature dependence of water transport across heart cell membranes is low in comparison with dissolution–diffusion of water through lipid membranes (Ea>10 kcal mol–1) [1–3,16].

3.3 Inhibition of osmotic swelling by Hg2+
The weak dependence of the myocyte Lp on temperature suggests that water transverses the sarcolemma through water-filled pores. To obtain additional information on this point, the size and time course of osmotic swelling were investigated in myocytes treated with Hg2+, an inhibitor of pore-mediated water transport across cell membranes [2,4,5]. A 5-min treatment with 0.1 mM Hg2+ under 1T conditions had no significant effects on relative cell volume (guinea-pig: 1.00±0.01, n=6; rat: 0.99±0.03, n=17). However, cell swelling induced by the subsequent exposure to 0.5T+0.1 mM Hg2+ solution for 10 min was significantly smaller in both guinea-pigs (relative volume 1.15±0.02, n=5) and rats (relative volume 1.18±0.07, n=6) than observed without Hg2+ treatment (Fig. 4A and D). The f(Vc) slope of 0.5T-induced swelling was less steep in Hg2+-treated myocytes (Fig. 4B and E), and the calculated Lp was 0.24±0.05x10–10 l N–1 s–1 (n=5) for guinea-pig cells and 0.37±0.10x10–10 l N–1 s–1 (n=8) for rat cells at 35 °C (P<0.01 vs. control Lp; Fig. 4C and F). Ea measured from Hg2+-treated myocytes was {approx}threefold larger (guinea-pig: 11.4 kcal mol–1; rat: 8.6 kcal mol–1) than measured from non-Hg2+-treated myocytes, revealing a blockade of membrane water-filled pores by the metal molecules. The inhibitory effects of Hg2+ were not reversed by 60-min superfusion with Hg2+-free solution.


Figure 4
View larger version (34K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Hg2+ inhibition of Lp in guinea-pig (A–C) and rat (D–F) myocytes. A and D, Effects of 0.1 mM Hg2+ on relative cell volume under 1T and 0.5T conditions. Guinea-pig, n=5–9 cells; rat, n=6–17 cells. B and E, Plots of f(Vc) for myocytes exposed to 0.5T, 0.5T+0.1 mM Hg2+, and 0.5T+0.1 mM Hg2++10 mM DTT solutions. Straight lines are least-squares fits to the mean data. Guinea-pig, n=5–9 cells; rat, n=6–7 cells. C and F, Lp calculated from the f(Vc) plots shown in B and E. * P<0.005 vs. 1T; {dagger} P<0.05 vs. 0.5T without Hg2+; {ddagger} P<0.005 vs. 0.5T without Hg2+.

 
Mercurials are sulfhydryl-oxidizing reagents, and their biological actions can be antagonized by sulfhydryl-reducing reagents [2,4,5]. To examine the effect of reducing reagent, swelling assay was performed in myocytes treated with 0.1 mM Hg2+ for 5 min and then 10 mM dithiothreitol (DTT) for 10 min. As indicated by the f(Vc) slopes in Fig. 4B and E (closed symbols), the inhibition of cell swelling by 0.5T+Hg2+ treatment was reversed by DTT. Lp in guinea-pig and rat myocytes exposed to 0.5T+Hg2++DTT solution (guinea-pig: 1.58±0.29x10–10 l N–1 s–1, n=5; rat: 1.67±0.43x10–10 liter N–1 s–1, n=6) was not significantly different from that in control myocytes (Fig. 4C and F). These results suggest that Hg2+ inhibition of Lp is due to binding of the metal to sulfhydryl groups in proteins associated with water-traversing membrane pores.

3.4 Effects of swelling-activated channel blockers
Mechanical stretch or cell swelling induces stretch-activated nonselective cation current (Ins) in many cell preparations including rat [17,18] and guinea-pig [19,20] ventricular myocytes, and influx of cation via the stretch-activated channels is likely to exert volume-increasing influence during hyposmotic stress. The latter possibility was tested by analyzing the time course of 0.5T-induced swelling in myocytes treated with Gd3+, a blocker of the stretch-activated Ins [18,19,21,22]. Superfusion with 1T solution containing 30 µM Gd3+ for 5 min caused minimal changes in relative cell volume (guinea-pig: 1.01±0.02, n=5, not significant; rat: 0.98±0.01, n=6, P=0.02 vs. pre-Gd3+), but cell swelling induced by the subsequent 10-min exposure to 0.5T+30 µM Gd3+ solution was significantly smaller (guinea-pig: 1.30±0.02, n=6; rat: 1.32±0.01, n=5) than observed in the absence of Gd3+ (Fig. 5A and D). On the other hand, the inclusion of Gd3+ had little effect on the initial rate of cell swelling in 0.5T solution, giving nearly unchanged f(Vc) slope (Fig. 5B and E), and therefore Lp in Gd3+-treated myocytes (guinea-pig: 1.44±0.23x10–10 l N–1 s–1, n=6; rat: 1.65±0.22x10–10 l N–1 s–1, n=5) was not significantly different from control values (Fig. 5C and F).


Figure 5
View larger version (37K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Modification of osmotic swelling by Gd3+ and DIDS in guinea-pig (A–C) and rat (D–F) myocytes. A and D, Relative cell volume after 10 min in 0.5T, 0.5T+30 µM Gd3+, and 0.5T+1 mM DIDS solutions. Guinea-pig, n=6–7 cells; rat, n=5–7 cells. * P<0.0001 vs. pre-0.5T, {dagger} P<0.05 vs. control 0.5T, {ddagger} P<0.005 vs. control 0.5T. B and E, Plots of f(Vc). Straight lines are least-squares fits to mean data. Guinea-pig, n=6–9 cells; rat, n=5–7 cells. C and F, Lp calculated from the data shown in B and E. There were no significant differences in Lp among the three groups.

 
Hyposmotic swelling of cardiomyocytes also activates swelling-induced Cl– current (ICl,swell) [7,21–23], and efflux of Cl– via the anion channels is expected to exert volume-decreasing influence during hyposmotic stress. This was investigated in myocytes treated with 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS), a blocker of ICl,swell [23], and exposed to 0.5T solution. Under isosmotic conditions, a 5-min treatment with 1 mM DIDS caused no (rat) or small (guinea-pig) decrease in relative cell volume (guinea-pig: 0.97±0.01, n=7, P<0.01; rat: 0.99±0.01, n=14, not significant). Exposure of guinea-pig cells to 0.5T+1 mM DIDS solution for 10 min elicited swelling that was significantly larger (relative volume 1.43±0.01, n=6, P<0.05) than observed in the absence of DIDS (Fig. 5A). However, the rate of volume change in the early phase of swelling was unaffected, and therefore Lp was not significantly changed by the inclusion of DIDS (1.52±0.18x10–10 l N–1 s–1, n=6) (Fig. 5B and C). In rat cells, the inclusion of 1 mM DIDS had no significant effects on relative cell volume (1.44±0.03, n=6), the f(Vc) slope, and Lp (1.65±0.27x10–10 l N–1 s–1, n=6) during 0.5T superfusion (Fig. 5D–F).

3.5 Volume-regulatory processes and Lp
The foregoing results with Gd3+ and DIDS indicate that block of the swelling-activated channels changed the steady-state cell volume but not Lp. To further investigate the volume-regulatory processes in response to osmotic stress, 0.5T-induced swelling was assayed in rat myocytes treated with 0.1 mM Hg2+ and/or 30 µM Gd3++1 mM DIDS mixture. The results shown in Fig. 6A and B indicate that Hg2+ significantly decreased swelling (relative volume 1.12±0.02, n=7) and Lp (0.34±0.04x10–10 l N–1 s–1, n=7) in the presence of the channel blockers, as well as in their absence. This suggests that Hg2+-sensitive water transport exerted a stronger influence on cell swelling than water flux mediated by the swelling-activated channels did. In non-Hg2+-treated myocytes, the Gd3++DIDS mixture significantly decreased swelling (relative volume 1.32±0.02, n=6) but had little effect on Lp (1.64±0.13x10–10 l N–1 s–1, n=6), and both relative cell volume and Lp in these myocytes were not significantly different from those in rat myocytes treated with Gd3+ alone (Fig. 5D and F). The latter results, together with comparison of cell volume responses to DIDS between guinea-pigs and rats (Fig. 5A and D), suggest that volume-sensitive Cl– channels play a minor role in the regulation of rat myocyte volume.


Figure 6
View larger version (35K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Volume-regulatory processes in rat myocytes. A and B, Effects of 0.1 mM Hg2+ on relative cell volume and Lp in 0.5T or 0.5T+30 µM Gd3++1 mM DIDS solution. n=6–8 cells; * P<0.01, with vs. without Hg2+; {dagger} P<0.005 (unpaired t test), with vs. without Gd3++DIDS. C–E, Effects of 10 µM ouabain on relative cell volume, f(Vc), and Lp in 0.5T or 0.5T+30 µM Gd3+ solution. n=5–8; * P<0.05, with vs. without ouabain; {dagger} P<0.05, with vs. without Gd3+. There were no significant differences in Lp among the four groups (E).

 
The activity of cardiac Na+–K+ pump is increased by cell swelling [24,25], and the enhanced Na+ pumping is expected to exert volume-decreasing influence under hyposmotic conditions. To study whether the smaller swelling of rat myocytes in the presence of Gd3+ (Figs. 5D and 6A) was related to increased Na+–K+ pump activity, cell swelling assays were performed after inhibiting the pump with ouabain. Fig. 6C depicts the time courses of 0.5T-induced swelling in rat myocytes treated with 10 µM ouabain and/or 30 µM Gd3+. Whereas relative cell volume under 1T conditions was not significantly changed by ouabain treatment for 10 min (1.01±0.02, n=6), cell swelling in both normal and Gd3+-treated myocytes were significantly enlarged by ouabain (relative volume 1.60±0.03 for 0.5T+ouabain vs. 1.49±0.02 for 0.5T, n=7 each). Thus, the pump inhibitor reversed the volume-decreasing action of Gd3+ during hyposmotic stress (relative volume 1.44±0.03 for 0.5T+Gd3++ouabain, n=8, not significant vs. control 0.5T). On the other hand, ouabain had no significant effects on Lp in these myocytes (e.g., 1.77±0.26x10–10 l N–1 s–1 for 0.5T+ouabain vs. 1.70±0.37x10–10 l N–1 s–1 for 0.5T, n=7 each) (Fig. 6D and E). These results suggest that rat myocyte volume is regulated by the compensatory interaction between volume-increasing (Gd3+-sensitive Ins) and volume-decreasing (ouabain-sensitive Na+ pumping) ion transporters, and a major factor in determining the rapidity of swelling is water flux through Hg2+-inhibitable aqueous channels.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The purpose of this study was to evaluate osmotically-induced water flux of guinea-pig and rat ventricular myocytes and to characterize the mechanism of water transport across cardiac cell membranes. These experiments were motivated by the conclusion of a recent volumetric study that the majority of osmotic water flux in rabbit ventricular myocytes penetrates the lipid bilayer itself rather than passing through membrane water channels [6]. In contrast to the finding on rabbit myocytes, water channel proteins are diffusely detectable in rat heart preparations [26–28], although the functional implications of that presence have not been established yet. The results here offer evidence that channel-mediated water transport is involved in the osmosis across plasma membranes of guinea-pig and rat cardiac myocytes.

4.1 Lp of cardiac membranes
Lp of guinea-pig and rat ventricular myocytes ({approx}1.8x10–10 l N–1 s–1 at 35 °C) is comparable to that of rabbit ventricular myocytes (1.2x10–10 l N–1 s–1 [6]). These values are considerably lower than Lp of specialized water-transporting cells, such as renal tubules (14–18x10–10 l N–1 s–1 [12,13]) and mammalian erythrocytes (9–39x10–10 l N–1 s–1 [14,15]). The low Lp is not unexpected because heart cells normally serve a non-water-transporting role that does not require rapid water movement across cell membranes.

In the present study, Ea of guinea-pig and rat myocytes ({approx}3.6 kcal mol–1) is much lower than the values for diffusional water movement through lipid bilayers, liposomes and biological membranes not containing functioning water channels (<10 kcal mol–1 [1–3,16]), and indicates that the energy barrier of water crossing the cardiac sarcolemma is as low as the value for free water diffusion ({approx}4 kcal mol–1 [2,4]). The participation of membrane pores in the water transport across guinea-pig and rat myocyte membranes could be derived from the results that treatment with Hg2+ reduced Lp by {approx}80% and increased Ea to {approx}10 kcal mol–1 in both guinea-pig and rat myocytes. Because mercurials do not affect water movement through lipid bilayers but through water channels [2,4], and the low Lp accompanied by high Ea is indicative of a lipid bilayer-mediated non-pore mechanism, our results suggest a blockade of membrane water channels by the metal molecules.

Suleymanian and Baumgarten [6] obtained higher Ea (12 kcal mol–1) in rabbit ventricular myocytes, and thereby concluded that the primary route for water crossing cardiac membranes is directly through the lipid bilayers rather than passing through membrane pores. There are no substantial differences in experimental conditions, data acquisition, and analytical methods between their study and ours, and thus the divergence is most likely to be due to species difference in the expression of sarcolemmal water channels. From the single-channel permeability of the water-transporting protein aquaporin-1 ({approx}10–13 cm3 s–1 [29]) and the Hg2+-inhibited Lp of {approx}0.8x10–10 l N–1 s–1 (equivalent to {approx}11x10–4 cm s–1) at room temperature, a membrane density of aquaporin-1 monomer is estimated to be 110 µm–2 for guinea-pig and rat myocyte membranes. This estimate is roughly an order of magnitude greater than the membrane density of cardiac ionic channels, but much less than the density of aquaporin-1 in erythrocytes ({approx}2x105 monomers per erythrocyte, corresponding to {approx}1500 µm–2 [4,29]). Because of the low intrinsic permeability of water channels that requires the expression of many aquaporins for functional significance, it has been predicted that aquaporin mRNA and protein should be abundant at tissues of channel-mediated water transport [27,30]. If the density of aquaporin in cardiac membranes is 10 µm–2 rather than 110 µm–2, it would make a physiologically insignificant contribution to myocyte Lp ({approx}0.07x10–10 l N–1 s–1). In this regard, Suleymanian and Baumgarten [6] argued that the molecular identification of water channel proteins by highly sensitive biochemical techniques does not establish the physiological role of aqueous pores in the myocyte function.

4.2 Roles of volume-sensitive ionic channels and Na+–K+ pump
Osmotic swelling of heart cells stimulates a variety of membrane channels and transporters, and redistribution of osmolytes via the swelling-activated pathways can affect the magnitude of cell swelling. As expected from earlier findings on rabbit ventricular myocytes [21,22], block of the stretch-activated Ins by Gd3+ inhibited the 0.5T-induced swelling of guinea-pig and rat myocytes, whereas block of ICl,swell by DIDS had the opposite effect in guinea-pig myocytes. Furthermore, inhibition of Na+–K+ pump by ouabain enlarged the swelling of rat myocytes and thereby reversed the volume-decreasing action of Gd3+. In contrast, none of these drugs significantly affected the initial rate of swelling, and therefore myocyte Lp remained unchanged in the two species. These observations are consistent with the idea that activation of the volume-sensitive channels and pump modifies cell swelling by evoking cation and anion fluxes that alter transmembrane osmotic gradient. The osmolyte flux mediated by these transporters, however, may be too small in the early phase of swelling to significantly change the net osmotic gradient by amount that can be detected with Lp. Alternatively, the activation of the volume-sensitive channels and pump may lag behind the onset of swelling after a step change in the osmotic gradient [7,22,24,31], i.e., redistribution of osmotically-obliged water due to the volume-regulatory osmolyte flux follows the instantaneous osmotic gradient-induced water flux. The lack of effects of Gd3+, DIDS and ouabain on Lp is in agreement with earlier observation that erythrocyte water permeability is insensitive to most transport reagents [2,15], and indicates that the strong inhibition of Lp by Hg2+ cannot be explained by a nonspecific blockade of the volume-sensitive ion transporters.

4.3 Physiologic and pathophysiologic implications
The low water permeability of cardiac membranes can restrict the rapidity of osmotic cell swelling and shrinkage. This seems less important in physiological cell volume regulation because heart cells normally do not experience significant osmotic perturbations. However, the low permeability may have functional significance under certain pathophysiological conditions, e.g., cell swelling and membrane disruption during ischaemia-reperfusion episode [32], transient osmotic disturbance induced by the intracoronary injection of hyperosmotic radiocontrast medium [33]. Recently, channel-mediated water transport has been proposed as a potential therapeutic target for a variety of disease states, such as congestive heart failure, hypertension and brain edema, and challenges to discover pharmacologically useful water channel modulators are now in progress [30,34]. Further studies are required to address the question of whether cardiac water transport is eligible for potential drug target.

Time for primary review 23 days.


   Acknowledgements
 
This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (#13670751 to T.O., #11670717 to S.I.).


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

  1. Fettiplace R., Haydon D.A. Water permeability of lipid membranes. Physiol Rev (1980) 60:510–550.[Free Full Text]
  2. Macey R.I. Transport of water and urea in red blood cells. Am J Physiol (1984) 246:C195–203.[Web of Science][Medline]
  3. Finkelstein A. Water movement through lipid bilayers, pores, and plasma membranes: theory and reality. (1987) New York: Wiley.
  4. Agre P., Preston G.M., Smith B.L., et al. Aquaporin CHIP: the archetypal molecular water channel. Am J Physiol (1993) 265:F463–476.[Web of Science][Medline]
  5. Preston G.M., Jung J.S., Guggino W.B., Agre P. The mercury-sensitive residue at cysteine 189 in the CHIP28 water channel. J Biol Chem (1993) 268:17–20.[Abstract/Free Full Text]
  6. Suleymanian M.A., Baumgarten C.M. Osmotic gradient-induced water permeation across the sarcolemma of rabbit ventricular myocytes. J Gen Physiol (1996) 107:503–514.[Abstract/Free Full Text]
  7. Shuba L.M., Ogura T., McDonald T.F. Kinetic evidence distinguishing volume-sensitive chloride current from other types in guinea-pig ventricular myocytes. J Physiol (1996) 491:69–80.[Abstract/Free Full Text]
  8. Ogura T., You Y., McDonald T.F. Membrane currents underlying the modified electrical activity of guinea-pig ventricular myocytes exposed to hyperosmotic solution. J Physiol (1997) 504:135–151.[Abstract/Free Full Text]
  9. Roos K.P. Length, width, and volume changes in osmotically stressed myocytes. Am J Physiol (1986) 251:H1373–1378.[Web of Science][Medline]
  10. Drewnowska K., Baumgarten C.M. Regulation of cellular volume in rabbit ventricular myocytes: bumetanide, chlorothiazide, and ouabain. Am J Physiol (1991) 260:C122–131.[Web of Science][Medline]
  11. Solomon A.K. Water channels across the red blood cell and other biological membranes. Methods Enzymol (1989) 173:192–222.[Web of Science][Medline]
  12. Hebert S.C., Andreoli T.E. Water movement across the mammalian cortical collecting duct. Kidney Int (1982) 22:526–535.[Web of Science][Medline]
  13. Verkman A.S., Ives H.E. Water permeability and fluidity of renal basolateral membranes. Am J Physiol (1986) 250:F633–643.[Web of Science][Medline]
  14. Farmer R.E., Macey R.I. Perturbation of red cell volume: rectification of osmotic flow. Biochim Biophys Acta (1970) 196:53–65.[Medline]
  15. Tsai S.T., Zhang R.B., Verkman A.S. High channel-mediated water permeability in rabbit erythrocytes: characterization in native cells and expression in Xenopus oocytes. Biochemistry (1991) 30:2087–2092.[CrossRef][Web of Science][Medline]
  16. Deamer D.W., Bramhall J. Permeability of lipid bilayers to water and ionic solutes. Chem Phys Lipids (1986) 40:167–188.[CrossRef][Web of Science][Medline]
  17. Craelius W. Stretch-activation of rat cardiac myocytes. Exp Physiol (1993) 78:411–423.[Abstract]
  18. Zeng T., Bett G.C., Sachs F. Stretch-activated whole cell currents in adult rat cardiac myocytes. Am J Physiol Heart Circ Physiol (2000) 278:H548–557.[Abstract/Free Full Text]
  19. Bustamante J.O., Ruknudin A., Sachs F. Stretch-activated channels in heart cells: relevance to cardiac hypertrophy. J Cardiovasc Pharmacol (1991) 17:S110–113.[CrossRef][Web of Science][Medline]
  20. Sasaki N., Mitsuiye T., Noma A. Effects of mechanical stretch on membrane currents of single ventricular myocytes of guinea-pig heart. Jpn J Physiol (1992) 42:957–970.[CrossRef][Web of Science][Medline]
  21. Suleymanian M.A., Clemo H.F., Cohen N.M., Baumgarten C.M. Stretch-activated channel blockers modulate cell volume in cardiac ventricular myocytes. J Mol Cell Cardiol (1995) 27:721–728.[Web of Science][Medline]
  22. Clemo H.F., Baumgarten C.M. Swelling-activated Gd3+-sensitive cation current and cell volume regulation in rabbit ventricular myocytes. J Gen Physiol (1997) 110:297–312.[Abstract/Free Full Text]
  23. Vandenberg J.I., Yoshida A., Kirk K., Powell T. Swelling-activated and isoprenaline-activated chloride currents in guinea pig cardiac myocytes have distinct electrophysiology and pharmacology. J Gen Physiol (1994) 104:997–1017.[Abstract/Free Full Text]
  24. Whalley D.W., Hool L.C., Ten Eick R.E., Rasmussen H.H. Effect of osmotic swelling and shrinkage on Na+–K+ pump activity in mammalian cardiac myocytes. Am J Physiol (1993) 265:C1201–1210.[Web of Science][Medline]
  25. Sasaki N., Mitsuiye T., Wang Z., Noma A. Increase of the delayed rectifier K+ and Na+–K+ pump currents by hypotonic solutions in guinea pig cardiac myocytes. Circ Res (1994) 75:887–895.[Abstract/Free Full Text]
  26. Bondy C., Chin E., Smith B.L., Preston G.M., Agre P. Developmental gene expression and tissue distribution of the CHIP28 water-channel protein. Proc Natl Acad Sci USA (1993) 90:4500–4504.[Abstract/Free Full Text]
  27. Hasegawa H., Lian S.C., Finkbeiner W.E., Verkman A.S. Extrarenal tissue distribution of CHIP28 water channels by in situ hybridization and antibody staining. Am J Physiol (1994) 266:C893–903.[Web of Science][Medline]
  28. Ma T., Yang B., Verkman A.S. Cloning of a novel water and urea-permeable aquaporin from mouse expressed strongly in colon, placenta, liver, and heart. Biochem Biophys Res Commun (1997) 240:324–328.[CrossRef][Web of Science][Medline]
  29. van Hoek A.N., Verkman A.S. Functional reconstitution of the isolated erythrocyte water channel CHIP28. J Biol Chem (1992) 267:18267–18269.[Abstract/Free Full Text]
  30. Verkman A.S., Mitra A.K. Structure and function of aquaporin water channels. Am J Physiol Renal Physiol (2000) 278:F13–28.[Abstract/Free Full Text]
  31. Sorota S., Du X.Y. Delayed activation of cardiac swelling-induced chloride current after step changes in cell size. J Cardiovasc Electrophysiol (1998) 9:825–831.[Web of Science][Medline]
  32. Garcia-Dorado D., Oliveras J. Myocardial oedema: a preventable cause of reperfusion injury? Cardiovasc Res (1993) 27:1555–1563.[Free Full Text]
  33. Lieberman P.L., Seigle R.L. Reactions to radiocontrast material. Anaphylactoid events in radiology. Clin Rev Allergy Immunol (1999) 17:469–496.[CrossRef][Web of Science][Medline]
  34. Beitz E., Schultz J.E. The mammalian aquaporin water channel family: a promising new drug target. Curr Med Chem (1999) 6:457–467.[Web of Science][Medline]

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


This article has been cited by other articles:


Home page
 J. Thorac. Cardiovasc. Surg.Home page
T. L. Butler, J. R. Egan, F. G. Graf, C. G. Au, A. C. McMahon, K. N. North, and D. S. Winlaw
Dysfunction induced by ischemia versus edema: Does edema matter?
J. Thorac. Cardiovasc. Surg., July 1, 2009; 138(1): 141 - 147.
[Abstract] [Full Text] [PDF]

Home page
 J. Thorac. Cardiovasc. Surg.Home page
J. R. Egan, T. L. Butler, A. D. Cole, A. Aharonyan, D. Baines, N. Street, M. Navaratnam, O. Biecker, C. Zazulak, C. G. Au, et al.
Myocardial ischemia is more important than the effects of cardiopulmonary bypass on myocardial water handling and postoperative dysfunction: A pediatric animal model.
J. Thorac. Cardiovasc. Surg., November 1, 2008; 136(5): 1265 - 1273.e2.
[Abstract] [Full Text] [PDF]

Home page
 JGPHome page
A. Takeuchi, S. Tatsumi, N. Sarai, K. Terashima, S. Matsuoka, and A. Noma
Ionic Mechanisms of Cardiac Cell Swelling Induced by Blocking Na+/K+ Pump As Revealed by Experiments and Simulation
J. Gen. Physiol., November 1, 2006; 128(5): 495 - 507.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
T. L. Butler, C. G. Au, B. Yang, J. R. Egan, Y. M. Tan, E. C. Hardeman, K. N. North, A. S. Verkman, and D. S. Winlaw
Cardiac aquaporin expression in humans, rats, and mice
Am J Physiol Heart Circ Physiol, August 1, 2006; 291(2): H705 - H713.
[Abstract] [Full Text] [PDF]

Home page
 Phil Trans R Soc AHome page
K Terashima, A Takeuchi, N Sarai, S Matsuoka, E.B Shim, C.H Leem, and A Noma
Modelling Cl- homeostasis and volume regulation of the cardiac cell
Phil Trans R Soc A, May 15, 2006; 364(1842): 1245 - 1265.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Ogura, T.
Right arrow Articles by Shibamoto, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ogura, T.
Right arrow Articles by Shibamoto, T.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?