Lack of Threshold for Anisotonic Cell Volume Regulation

Abstract

Most cells possess mechanisms that are able to detect cellular volume shifts and to signal the initiation of appropriate volume regulatory responses. However, the identity and characteristics of the detecting mechanism remain obscure. In this study, we explored the influence of hypertonic and hypotonic challenges of varying magnitude on the characteristics of the ensuing regulatory volume increase (RVI) and regulatory volume decrease (RVD) of cultured bovine corneal endothelial cells (CBCECs). The main question we asked was whether a threshold of stimulation existed that would unleash a regulatory response. CBCECs (passage 1-3) were seeded on rectangular glass coverslips and grown for 1-2 days. We used a procedure based on detection of light scattering to monitor the transient volume changes of such plated cells when subjected to osmotic challenge. The osmometric responses were asymmetric: cells shrank faster than they swelled (by a factor of 3). Complete volume regulatory responses took 10-12 min. Bumetanide (50 microM) resulted in incomplete (50%) RVI. We found no threshold as the cells examined responded to hypertonic and hypotonic stimuli as low as 1%. There was some gradation as stimuli of <4% resulted in incomplete volume regulation. The degree of activation of the volume responses grew as an exponential buildup with the strength of the anisotonic challenge. We discuss how our observations are consistent with volume sensing mechanisms based on both ionic strength and the cytoskeleton.

Regulatory Volume Increase and Regulatory Volume Decrease Responses in HL-1 Atrial Myocytes

“…The most likely explanation is that in the presence of the inhibitor alternative mechanisms of RVI are still functional. As might have been expected, no effect on RVD was seen in HL-1 cells exposed to 20 % hypotonic solution in presence of 50 µM bumetanide (data not shown); such a result has been previously reported for corneal endothelium cells [37]. Moreover, there is no inhibition of post RVI - RVD in HL-1 cells when cells previously exposed to hypertonic solution are returned to isotonic solution (data not shown).…”

“…The method has been described and validated in the literature [30,31,33,34,35,36,37]. For illumination, a small laser-diode was used (model L52-265, Edmund Scientific, 4.2 mw, λ = 670 nm), generating a beam of rectangular profile reduced to a width of 1.5 mm by a variable adjustable slit.…”

“…1). After the main illuminating beam already scattered emerges from the sample, the intensity of light scattered at small angles carries information on cell volume as previously described [30,36,37]. This scattered light is captured through a lens endowed with a black plastic (1 mm wide) central band to block the laser main beam, and is measured with a photomultiplier (Hamamatsu R 928) at a fixed gain (-140 V dynode potential).…”

Fluid Transport Across Leaky Epithelia: Central Role of the Tight Junction and Supporting Role of Aquaporins

“…The other complication ensues from the value one would have to assume for the cell hypotonicity (perhaps 1% or 1.5 mM). As we have recently reported (54), that amount of anisotonicity would be enough to trigger cell volume regulation, which in turn would diminish or nullify the original gradient.…”

Methods for cell volume measurement

“…The intensity of SSC can vary dramatically within as little as 1–2 degrees . However, once a successful experimental setup is found, responses to as little as 1% osmolarity change can be detected .…”