Environmental signals such as ultraviolet radiation, heat shock, and osmotic stress cause p38 MAP kinase activation in several cell types (4
). While many of the physiologic effects of p38 are mediated by transcriptional regulation, there is increasing evidence that p38 can interact with and modulate other cytoplasmic and membrane proteins in a phosphorylation-dependent manner. In these studies of HTC cells, observations using a variety of techniques support a broader role for p38 MAP kinase as an early and important signal in coordinating changes in cell volume and membrane Na+
The principal findings of these studies are that (a) there is constitutive activity of p38 MAP kinase under basal (isotonic) conditions; (b) inhibition of constitutive p38 MAP kinase results in increased membrane Na+ permeability and increases in cell volume; (c) intracellular dialysis with purified p38α, and its upstream activator MEK-6, inhibits volume-sensitive channel opening; and (d) exposure to hypotonicity to increase cell volume results in large increases in p38 activity. Collectively, these findings suggest that p38 MAP kinase plays an important regulatory role governing membrane Na+ permeability and cell volume regulation through inhibitory effects on NSC channels.
Previous biophysical studies have identified NSC channels in liver cells that are regulated in part by cytosolic [Ca2+
). While channel proteins are abundant, with approximately 2,000 channels/cell, they are generally closed under basal conditions. Channels open in response to vasopressin or other agents known to mobilize Ca2+
), and in response to cell shrinkage induced by osmotic (17
) or oxidative stress (20
). The resulting influx of Na+
favors water movement into the cell and restoration of cell volume toward basal values. Interestingly, p38 MAP kinase is known to be constitutively active in hepatocytes, and its activity is selectively decreased during oxidative stress (38
), consistent with a potential role as an inhibitor of Na+
These studies provide further evidence that NSC channels are crucial to the maintenance of cell volume and RVI in hepatocytes. The conductance is characterized by equal permeability for Na+ and K+ and a linear I-V relationship, and shows no time dependence. Partial substitution of extracellular Na+ with Tris+ causes a decrease in inward currents and a negative shift in reversal potential, as anticipated for an NSC conductance. It should be noted, however, that a slight decrease in the amplitude of outward currents was also observed with Tris+ substitution. The possibility that Tris+ is also a partial channel blocker, or has other nonspecific effects on cell volume, intracellular pH, or other parameters that may affect channel open probability cannot be excluded.
The partial inhibition of volume-sensitive conductance by amiloride is consistent with previous studies of rat hepatocytes (16
). Previous reports in other cell types have demonstrated variable effects of amiloride on NSC currents (39
). This variability may reflect different channel types; the molecular identity of NSC channels in liver cells has not been defined. The basic biophysical properties of the NSC channels described here appear similar to cyclic nucleotide–gated channels described in other cell types (43
) that demonstrate amiloride sensitivity (45
). The finding that amiloride only partially inhibits NSC conductance has several potential explanations. First, amiloride sensitivity may be modulated by [Ca2+
), mechanical stress (49
), or other factors. Alternatively, more than one Na+
channel type may contribute to volume-regulated Na+
influx. In fact, the epithelial Na+
channel (ENaC), which is also inhibited by amiloride, has recently been implicated in volume-stimulated Na+
influx in rat hepatocytes (33
). However, the NSC conductance of HTC cells does not exhibit the pore or regulatory properties anticipated for ENaC. Thus, it will be important to define the molecular mechanisms responsible for volume-sensitive Na+
influx, and assess the specific regulatory pathways involved for each.
Two primary observations support a role for constitutive p38 MAP kinase activity in the regulation of cell volume. First, under isotonic conditions, inhibition of p38 with SB203580 increased membrane Na+ permeability. Currents activated by SB203580 demonstrated biophysical properties that were identical to those of currents activated by hypertonic exposure, including a linear I-V relationship, equal permeability to Na+ and K+, Ca2+-dependent regulation, and amiloride sensitivity. Second, exposure to SB203580 was followed by an increase in cell volume (~6%), consistent with an important role for p38 MAP kinase in the maintenance of resting cell volume under isotonic conditions.
While the inhibitory effects of SB203580 are detectable at low micromolar concentrations and appear to be specific for p38 (50
), it is acknowledged that SB203580 could have unanticipated effects on other signaling pathways. Consequently, an alternative strategy was used to evaluate the roles of recombinant p38α and the upstream MAP kinase kinase MEK-6 on channel regulation. Intracellular delivery of these kinases inhibited volume-sensitive current activation. Indeed, intracellular delivery of active p38α resulted in a large shift in the activation curve, so that much higher degrees of hypertonicity were required before channel opening was observed. Dialysis with either p38α or MEK-6 alone resulted in only partial inhibition of currents, and dialysis with heat-inactivated p38α had no effect. Thus, p38α, when present with its specific activator, is capable of channel inhibition. The partial effect observed with either p38α or MEK-6 alone may be related to activation of these proteins by endogenous cellular kinases. It is important to note that at higher degrees of hypertonicity (50 mM sucrose), NSC conductance could be activated even in the presence of p38α and MEK-6. Thus, the inhibitory effect of p38 MAP kinase can be overcome by positive regulatory signals. In fact, there was no observable decrease in p38 activity with hypertonic exposure. Consequently, it will be important to define the additional regulatory pathways (regulatory proteins, cytoskeletal elements, insertion of new channels into the membrane, etc.) that are able to overcome the inhibitory effects of p38 and stimulate channel opening during hypertonic conditions.
The effects of p38 MAP kinase on membrane Na+
permeability have several implications. First, while p38 has previously been shown to be an important regulator of transcription, these effects on Na+
permeability imply that p38 MAP kinase has plasma membrane targets as well. Indeed, recent evidence suggests that p38 may modulate the Na+
exchanger (NHE-1) in vascular smooth muscle cells (15
-activated, voltage-gated channels in neuronal cells (14
), and acid secretion from gastric parietal cells (52
). In all cases, p38 appears to have an inhibitory role, as observed here. However, whether p38 regulates channel function through a direct phosphorylation event or through downstream kinase pathways is yet to be determined. Second, while MAP kinase pathways were thought to mediate the effects of growth factors and hormones on sustained cellular events such as proliferation and differentiation, recent evidence has now emerged that MAP kinase pathways can also be activated by heterotrimeric G proteins (53
) for rapid regulation of effector pathways (55
). For example, p38β appears to be involved in the regulation of N-type calcium currents by bradykinin in a neuronal cell line, a response that occurs within seconds (14
). Additionally, in neutrophils, p38 is activated within 2 minutes of exposure to formyl-methionyl-leucyl-phenylalanine (fMLP), an inflammatory stimulus, and modulates the response to hypertonic exposure (57
). These studies support roles for p38 MAP kinase in rapid regulation of cellular events that are not necessarily related to gene transcription.
Assuming that p38 functions as a primary signal governing Na+
influx and resting cell volume, several additional points merit further investigation. First, because the molecular identity of the NSC channel has not been established, the cellular site(s) of action of p38 is not clear. p38 may modulate channel activity by direct phosphorylation, or conversely, through effects on downstream kinases or phosphatases. Second, functional interactions between p38 and other kinases are likely to be operative. For example, preliminary evidence suggests that tyrosine kinase activity is also important in the response to cell volume changes (58
), and Src
tyrosine kinase has been shown to be a direct regulator of ion channel function in different cell types (60
). The sequence of action and relative importance of p38 versus other kinases has not been established, and is likely to be cell-type specific. For example, it should be noted that while PD98059 did not inhibit the response to hypertonic exposure, a role of ERK in channel regulation through MEK-1–independent pathways cannot be excluded. Lastly, a broad range of physiologic and pathologic stimuli modulate NSC channel activity, though the role of p38 in the mediation of these responses is largely unknown. However, p38 has been shown to play an important role in the insulin signaling pathway (61
) as well as in responses to oxidative stress (38
) and the initiation of apoptosis (62
), processes that are associated with alterations in cell volume (20
Taken together, these findings indicate that in HTC cells, p38 MAP kinase plays a key role in tonic inhibition of Na+ permeability and maintenance of cell volume. It is clear, however, that the inhibitory effects of p38 MAP kinase are opposed by intracellular [Ca2+] and presumably other stimulatory signals that work in concert to modulate Na+ permeability in response to changing physiologic demands. Definition of the complex signaling pathways involved may provide new strategies for modulating liver cell function through effects on cell volume, and for minimizing cell injury caused by sustained Na+ influx.