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4. Cell swelling triggered mitogen-activated protein (MAP) kinase cascades leading to the activation of extracellular signal-regulated kinase 1 and 2 (ERK1/ERK2) and p38 kinase. The volume-responsive ERK1/ERK2 signalling pathway linked with the activation of K + and Cl _ channels, and taurine transport. However, the volume-regulatory mechanism was independent of the activation of p38 MAP kinase.5. The phosphorylated ERK1/ERK2 expression following a hypotonic shock was up-regulated by protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and downregulated by PKC inhibitor staurosporine. The response of ERK activation to hypotonicity also required Ca 2+ entry and depended on tyrosine kinase and mitogen-activated/ERKactivating kinase (MEK) activity.6. Considering the results overall, osmotic swelling promotes the activation of tyrosine kinase and ERK1/ERK2 and raises intracellular Ca 2+ , all of which play a crucial role in the volumeregulatory mechanism of human cervical cancer cells.

Red blood cells from patients with sickle cell disease (SCD) exhibit increased electrogenic cation permeability, particularly following deoxygenation and hemoglobin (Hb) polymerisation. This cation permeability, termed P sickle , contributes to cellular dehydration and sickling, and its inhibition remains a major goal for SCD treatment. Nevertheless, its characteristics remain poorly defined, its molecular identity is unknown, and effective inhibitors have not been established. Here, patch-clamp methodology was used to record whole-cell currents in single red blood cells from healthy individuals and patients with SCD. Oxygenated normal red blood cells had a low membrane conductance, unaffected by deoxygenation. Oxygenated HbS cells had significantly increased conductance and, on deoxygenation, showed a further rise in membrane conductance. The deoxygenation-induced pathway was variable in magnitude. It had equal permeability to Na ؉ and K ؉ , but was less permeable to NMDG ؉ and Cl ؊ . Conductance to Ca 2؉ was also of a similar magnitude to that of monovalent cations. It was inhibited by DIDS (100 M), Zn 2؉ (100 M), and by Gd 3؉ (IC 50 IntroductionSickle cell disease (SCD) is caused by the presence in red blood cells of mutant hemoglobin, HbS (HbS-containing red blood cells are here called HbS cells, whereas normal HbA-containing red blood cells are called HbA cells). The reduced lifespan of HbS cells contributes to the prevailing anemia which characterizes the disease. 1 Furthermore, deoxygenated HbS polymerizes, distorting the red blood cell shape into a variety of elaborate patterns, including the eponymous sickle. Sickled cells participate in vascular occlusion and associated sequelae, including ischemia, organ dysfunction, pain, and, ultimately, death. 1 Although the molecular nature of the Hb defect underlying SCD is well established, 2,3 details of the pathophysiology are uncertain, and treatment remains largely supportive. 4 Dehydration of HbS cells, and particularly of certain HbS cell subpopulations, 5 markedly promotes polymerization by reducing the lag time to polymer formation. 6 Several membrane transport pathways promote solute loss, but a deoxygenation-induced cation permeability, called P sickle , is pre-eminent (reviewed by Joiner, 7 Gibson, 8 and Lew 9 ). P sickle increases cell membrane permeability to Ca 2ϩ (as well as to monovalent cations), thereby elevating cytosolic [Ca 2ϩ ]. Subsequent activation of the Ca 2ϩ -activated K ϩ channel (also known as the Gardos channel) mediates particularly rapid K ϩ loss, with Cl Ϫ following via separate pathways. 10 Inhibition of P sickle , which will reduce the propensity of cells to shrink, represents an immediate goal for SCD therapy. 7,9,11 Permeability studies on P sickle date from the seminal work of Tosteson and colleagues. 12 Subsequent radioactive tracer studies indicate that P sickle behaves like a conductive cation channel, lacking selectivity between alkali cations (including Na ϩ and K ϩ ), with moderate permeability to Ca 2ϩ and Mg 2ϩ , 1...

Objective. To determine the effects of varying O 2 on pH homeostasis, based on the hypothesis that the function of articular chondrocytes is best understood at realistic O 2 tensions.Methods. Cartilage from equine metacarpophalangeal/tarsophalangeal joints was digested with collagenase to isolate chondrocytes, and then loaded with the pH-sensitive fluorophore 2 ,7 -bis-2-(carboxyethyl)-5(6)-carboxylfluorescein. The radioisotope 22 Na ؉ was used to determine the kinetics of Na ؉ /H ؉ exchange (NHE) and the activity of the Na ؉ /K ؉ pump, and ATP levels were assessed with luciferin assays. Levels of reactive oxygen species (ROS) were determined using 2 ,7 -dichlorofluorescein diacetate.Results. The pH homeostasis was unaffected when comparing tissue maintained at 20% O 2 (the level in water-saturated air at 37°C) with that at 5% O 2 (which approximates the normal level in healthy cartilage); however, an O 2 tension of <5% caused a fall in intracellular pH (pH i ) and slowed pH i recovery following acidification, an effect mediated via inhibition of NHE activity (likely through acid extrusion by NHE isoform 1). The Na ؉ /K ؉ pump activity and intracellular ATP concentration were unaffected by hypoxia, but the levels of ROS were reduced. Hypoxic inhibition of NHE activity and the reduction in ROS levels were reversed by treatment with H 2 O 2 , Co 2؉ , or antimycin A. Treatment with calyculin A also prevented hypoxic inhibition of NHE activity. Conclusion.The ability of articular chondrocytes to carry out pH homeostasis is compromised when O 2 tensions fall below those normally experienced, via inhibition of NHE. The putative signal is a reduction in levels of ROS derived from mitochondria, acting via altered protein phosphorylation. This effect is relevant to both physiologic and pathologic states of lowered O 2 , such as in chronic inflammation.