Intracellular Cl− Dependence of Na-H Exchange in Barnacle Muscle Fibers under Normotonic and Hypertonic Conditions

Abstract

We previously showed that shrinking a barnacle muscle fiber (BMF) in a hypertonic solution (1,600 mosM/kg) stimulates an amiloride-sensitive Na-H exchanger. This activation is mediated by a G protein and requires intracellular Cl−. The purpose of the present study was to determine (a) whether Cl− plays a role in the activation of Na-H exchange under normotonic conditions (975 mosM/kg), (b) the dose dependence of [Cl−]i for activation of the exchanger under both normo- and hypertonic conditions, and (c) the relative order of the Cl−- and G-protein-dependent steps. We acid loaded BMFs by internally dialyzing them with a pH-6.5 dialysis fluid containing no Na+ and 0–194 mM Cl−. The artificial seawater bathing the BMF initially contained no Na+. After dialysis was halted, adding 50 mM Na+ to the artificial seawater caused an amiloride-sensitive pHi increase under both normo- and hypertonic conditions. The computed Na-H exchange flux (J Na-H) increased with increasing [Cl−]i under both normo- and hypertonic conditions, with similar apparent K m values (∼120 mM). However, the maximal J Na-H increased by nearly 90% under hypertonic conditions. Thus, activation of Na-H exchange at low pHi requires Cl− under both normo- and hypertonic conditions, but at any given [Cl−]i, J Na-H is greater under hyper- than normotonic conditions. We conclude that an increase in [Cl−]i is not the primary shrinkage signal, but may act as an auxiliary shrinkage signal. To determine whether the Cl−-dependent step is after the G-protein-dependent step, we predialyzed BMFs to a Cl−-free state, and then attempted to stimulate Na-H exchange by activating a G protein. We found that, even in the absence of Cl−, dialyzing with GTPγS or AlF3, or injecting cholera toxin, stimulates Na-H exchange. Because Na-H exchange activity was absent in control Cl−-depleted fibers, the Cl−-dependent step is at or before the G protein in the shrinkage signal-transduction pathway. The stimulation by AlF3 indicates that the G protein is a heterotrimeric G protein.

Evidence for Involvement of the Putative First Extracellular Loop in Differential Volume Sensitivity of the Na+/H+ Exchangers NHE1 and NHE2

“…Furthermore, two recent studies have shown that an osmoregulated ABC transporter OpuA of Lactococcus latis (32) and a betanine carrier BetP of Corynebacterium glutamicum (33) are activated in response to hyperosmotic stress when purified proteins were reconstituted in proteoliposomes that are devoid of all other cellular proteins, strongly suggesting that transporter activation by a transmembrane osmotic gradient is mediated via changes in membrane properties such as membrane strain, curvature stress, or protein-lipid interactions. On the other hand, previous literature also reported that cell shrinkage-induced activation of NHE1 occurs via intracellular events involving several families of protein kinases (34)(35)(36)(37) or a Cl --dependent, G-protein-mediated process (38) or Ca 2+ /calmodulin-dependent process (39). It is not clear how these intracellular events are related to the membrane events that we have seen in this study.…”

Cell Volume Decrease as a Link between Azaspiracid-Induced Cytotoxicity and c-Jun-N-Terminal Kinase Activation in Cultured Neurons

“…However, the cytoprotective effect of ouabain against AZA-1-induced cytotoxicity suggests that the cytoprotective effect of anion channel blockers against AZA-induced cytotoxicity is mainly due to the preventive effect of these compounds against apoptosis (Gulbins et al, 2000;Heimlich and Cidlowski, 2006). With regard to the neuroprotective effect of amiloride against AZA-1-induced cytotoxicity, it should be pointed out that activation of the Na þ /H þ exchanger is dependent on intracellular chloride (Aharonovitz et al, 2001;Goss et al, 2001;Hogan et al, 1997) and requires JNK activation (Goss et al, 2001). Altogether, the results presented here indicate that AZA-1induced JNK activation is secondary to the decrease in cell volume, presumably during an apoptotic insult, initiated by the toxin.…”

Modulation of Na+/H+exchange activity by Cl−

“…This activation was similarly found to depend on Cl Ϫ , an effect that was attributed to coupling of NHE with a heterotrimeric GTP-binding protein (8,9). Subsequently, Hogan and colleagues (15) found that the barnacle exchanger could also be activated by a profound acidification. Because this acidinduced effect was also sensitive to anion replacement, it was suggested that regulation by the GTP-binding protein may be exerted tonically.…”