Purinergic-independent Calcium Signaling Mediates Recovery from Hepatocellular Swelling

Abstract

Swelling of hepatocytes and other epithelia activates volume-sensitive ion channels that facilitate fluid and electrolyte efflux to restore cell volume, but the responsible signaling pathways are incompletely defined. Previous work in model HTC rat hepatoma cells has indicated that swelling elicits ATP release, which stimulates P2 receptors and activates Cl ؊ channels, and that this mechanism is essential for hepatocellular volume recovery. , which is essential for ion channel activation and volume recovery, but that this increase does not stem from activation of volume-sensitive P2 receptors. Collectively, these observations imply that regulatory responses to hepatocellular swelling involve a dual requirement for a purinergic-independent Ca 2؉ signaling cascade and a Ca 2؉ -independent purinergic signaling pathway.Epithelia face substantial osmotic stresses from the vectorial transport of solutes that cause cell swelling and challenge cellular integrity. An adaptive response to cell swelling, termed regulatory volume decrease (RVD), 1 provides a dynamic safeguard against tissue injury produced by such stresses. RVD is mediated, in part, by the opening of swelling-activated K ϩ and Cl Ϫ channels in the plasma membrane, which leads to fluid and electrolyte efflux and consequent restoration of cell volume (1).Although it has been well appreciated that activation of both K ϩ and Cl Ϫ channels is critical for RVD, the mechanisms that couple cell swelling to ion channel activation and RVD exhibit tissue diversity.Among epithelia, hepatocytes are particularly susceptible to dynamic perturbations in cell volume, given the central role of the liver in nutrient uptake and metabolism (2). Whereas emerging evidence suggests that hepatocellular volume per se is an important determinant of several critical organ level functions, including glucose metabolism and bile formation (2), the mechanisms that govern hepatocellular volume regulation remain to be defined. We and others (3, 4) have provided evidence for the involvement of purinergic signaling in this process. In both HTC rat hepatoma cells and human hepatocytes, hypotonic swelling elicits ATP release, which stimulates P2 purinoreceptors, the activation of which leads to the opening of volume-sensitive Cl Ϫ channels (3, 4). This purinergic mechanism is essential for hepatocellular RVD. Although it is known that swelling-induced ATP release requires activation of phosphatidylinositol 3-kinase (5) and involves a putative member of the ATP-binding cassette family (6), the downstream effectors that couple osmosensitive P2 receptors to ion channel opening and RVD are unknown.In many cell types, including hepatocytes, activation of P2 receptors elicits increases in cytosolic [Ca 2ϩ ] ([Ca 2ϩ ] i ) through stimulation of phospholipase C, intracellular inositol trisphosphate (IP 3 ) formation, and activation of IP 3 receptors (7,8). However, P2 receptors have also been reported to be coupled to Ca 2ϩ -independent cellular effectors (9 -12). This leaves unresolved whether Ca 2ϩ...

Calcium Mobilization Evoked by Hepatocellular Swelling Is Linked to Activation of Phospholipase Cγ

“…Measurement of [Ca 2ϩ ] i - [Ca 2ϩ ] i was determined in individual cells by dual wavelength microfluorimetry, using the Ca 2ϩ -sensitive fluorescent dyes fluo-3 and Fura Red as described previously (4). Measurements of fluo-3 and Fura Red fluorescence emission intensity were acquired every 10 s, from which the ratio R of fluo-3 to Fura Red fluorescence was calculated.…”

“…Cells and Cell Culture-HTC rat hepatoma cells were cultured as previously described (4). Cell swelling was elicited by exposure to hypotonic medium.…”

“…We have recently shown that K ϩ and Cl Ϫ channel opening evoked by hepatocellular swelling is triggered by an increase in cytosolic [Ca 2ϩ ] ([Ca 2ϩ ] i ) 1 and that this increase is critical for volume recovery (4). The initial increase in [Ca 2ϩ ] i results from Ca 2ϩ mobilization from thapsigargin-sensitive intracellular stores, which suggests the involvement of the endoplasmic reticulum in this process.…”

P2X4 Activation Modulates Volume-sensitive Outwardly Rectifying Chloride Channels in Rat Hepatoma Cells

“…Conversely, in non-pigmented ciliary epithelial cells Ca o 2ϩ removal slowed down the current onset as well as decreased the maximal current amplitude, suggesting a role for Ca o 2ϩ (21). Finally, in HTC cells, despite the fact that the regulatory volume decrease was impaired in the absence of external Ca 2ϩ , maximal VSOR Cl Ϫ current measured at steady-state remained unaffected by external Ca 2ϩ removal (18). In the same cells, it has been observed that after exposure to a hypotonic solution, these cells respond with a transient increase in NAD(P)H oxidase-dependent H 2 O 2 production (22), [Ca 2ϩ ] i (18) and release of ATP (23).…”

“…In bovine pulmonary artery cells, hypotonicity-activated Cl Ϫ currents were found to be already maximally activated at 50 -100 nM [Ca 2ϩ ] i (15), and in Ehrlich ascites tumor cells 25 nM [Ca 2ϩ ] i was reported to be sufficient (16). In contrast, in a rat hepatoma cell line (17) (HTC), it was demonstrated that hypotonicityinduced cell swelling elicits an increase in [Ca 2ϩ ] i , which proved necessary for volume recovery (18).…”