Search

RhoA exerts a permissive effect on volumeregulated anion channels in vascular endothelial cells. Am J Physiol Cell Physiol 283: Cl15-C125, 2002. First published February 20, 2002; 10.1152/ajpcell.00038.2001.--Cell swelling triggers in most cell types an outwardly rectifying anion current, [I.sub.Cl.swell], via volume-regulated anion channels (VRACs). We have previously demonstrated in calf pulmonary artery endothelial (CPAE) cells that inhibition of the Rho/Rho kinase/myosin light chain phosphorylation pathway reduces the swelling-dependent activation of [I.sub.Cl.swell]. However, these experiments did not allow us to discriminate between a direct activator role or a permissive effect. We now show that the Rho pathway did not affect VRAC activity if this pathway was activated by transfecting CPAE cells with constitutively active isoforms of G[alpha] (a Rho activating heterotrimeric protein subunit), Rho, or Rho kinase. Furthermore, biochemical and morphological analysis failed to demonstrate activation of the Rho pathway during hypotonic cell swelling. Finally, manipulating the Rho pathway with either guanosine 5'-O-(3-thiotriphosphate) or C3 exoenzyme had no effect on VRACs in caveolin-l-expressing Caco-2 cells. We conclude that the Rho pathway exerts a permissive effect on VRACs in CPAE cells, i.e., swelling-induced opening of VRACs requires a functional Rho pathway, but not an activation of the Rho pathway. cytoskeleton; actin; myosin phosphorylation; RhoA pathway

McLean, Lee Anne, Jane Roscoe, Nanna K. Jorgensen, Fredric A. Gorin, and Peter M. Cala. Malignant gliomas display altered pH regulation by NHE1 compared with nontransformed astrocytes. Am J Physiol Cell Physiol 278: C676-C688, 2000.--Malignant gliomas exhibit alkaline intracellular pH (p[H.sub.i]) and acidic extracellular pH (p[H.sub.e]) compared with nontransformed astrocytes, despite increased metabolic [H.sup.+] production. The acidic p[H.sub.e] limits the availability of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII], thereby reducing both passive and dynamic [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]-dependent buffering. This implies that gliomas are dependent upon dynamic [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]-independent [H.sup.+] buffering pathways such as the type 1 [Na.sup.+]/[H.sup.+] exchanger (NHE1). In this study, four rapidly proliferating gliomas exhibited significantly more alkaline steady-state p[H.sub.i] (p[H.sub.i] = 7.31-7.48) than normal astrocytes (p[H.sub.i] = 6.98), and increased rates of recovery from acidification, under nominally [CO.sub.2]/[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]-free conditions. Inhibition of NHE1 in the absence of [CO.sub.2]/[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] resulted in pronounced acidification of gliomas, whereas normal astrocytes were unaffected. When suspended in [CO.sub.2]/ [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] medium astrocyte [pH.sub.i] increased, yet glioma [pH.sub.i] unexpectedly acidified, suggesting the presence of an [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]-dependent acid loading pathway. Nucleotide sequencing of NHE1 cDNA from the gliomas demonstrated that genetic alterations were not responsible for this altered NHE 1 function. The data suggest that NHE1 activity is significantly elevated in gliomas and may provide a useful target for the development of tumor-selective therapies. sodium/hydrogen antiport; tumor pH; astrocytomas; glioblastomas

KCNQ1 potassium channels are expressed in many epithelial tissues as well as in the heart. In epithelia KCNQ1 channels play an important role in salt and water transport and the channel has been reported to be located apically in some cell types and basolaterally in others. Here we show that KCNQ1 channels are located basolaterally when expressed in polarised MDCK cells. The basolateral localisation of KCNQ1 is not affected by co-expression of any of the five KCNE bb-subunits. We characterise two independent basolateral sorting signals present in the N-terminal tail of KCNQ1. Mutation of the tyrosine residue at position 51 resulted in a non-polarized steady-state distribution of the channel. The importance of tyrosine 51 in basolateral localisation was emphasized by the fact that a short peptide comprising this tyrosine was able to redirect the p75 neurotrophin receptor, an otherwise apically located protein, to the basolateral plasma membrane. Furthermore, a di-leucine-like motif at residues 38–40 (LEL) was found to affect the basolateral localisation of KCNQ1. Mutation of these two leucines resulted in a primarily intracellular localisation of the channel. [ABSTRACT FROM AUTHOR]

Ca2+-activated K+ channels of big (hBK), intermediate (hIK) or small (rSK3) conductance were co-expressed with aquaporin 1 (AQP1) in Xenopus laevis oocytes. hBK channels were activated by depolarization, whereas hIK and rSK3 channels were activated by direct injection of Ca2+ or Cd2+ into the oocyte cytoplasm, before the oocytes were subjected to hyperosmolar or hypoosmolar (±50 mOsm mannitol) challenges. In all cases, the oocytes responded rapidly to the osmotic changes with shrinkage or swelling and the effects on the K+ currents were measured. hIK and rSK3 currents were highly sensitive to volume changes and increased immediately to 178% (hIK) or 165% (rSK3) of control in response to swelling and decreased to 64% (hIK) or 61% (rSK3) of control after shrinkage. These responses were dependent on the channels being pre-activated and were almost totally abolished after injection of cytochalasin D into the oocyte cytoplasm (final concentration: 1 µM). In contrast, hBK channels showed only a minor sensitivity to volume changes; the hBK channel activity decreased approximately 20% after swelling and increased approximately 20% after shrinkage. The opposite effects of volume changes on hIK/rSK3 and hBK channels suggest that the significant stimulation of hIK and rSK3 channels during swelling is not mediated by changes in intracellular Ca2+, but rather through interactions with the cytoskeleton, provided that a sufficient basal concentration of intracellular Ca2+ or Cd2+ is present. [ABSTRACT FROM AUTHOR]