ClC-2 Cl- channels in human lung epithelia: activation by arachidonic acid, amidation, and acid-activated omeprazole.

ClC-2 Cl- channels represent a potential target for therapy in cystic fibrosis. Key questions regarding the feasibility of using ClC-2 as a therapeutic target are addressed in the present studies, including whether the channels are present in human lung epithelia and whether activators of the channel can be identified. Two new mechanisms of activation of human recombinant ClC-2 Cl- channels expressed in HEK-293 cells were identified: amidation with glycine methyl ester catalyzed by 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) and treatment with acid-activated omeprazole. ClC-2 mRNA was detected by RT-PCR. Channel function was assessed by measuring Cl- currents by patch clamp in the presence of a cAMP-dependent protein kinase (PKA) inhibitor, myristoylated protein kinase inhibitor, to prevent PKA-activated Cl- currents. Calu-3, A549, and BEAS-2B cell lines derived from different human lung epithelia contained ClC-2 mRNA, and Cl- currents were increased by amidation, acid-activated omeprazole, and arachidonic acid. Similar results were obtained with buccal cells from healthy individuals and cystic fibrosis patients. The ClC-2 Cl- channel is thus a potential target for therapy in cystic fibrosis.

Activation of human CIC-2 Cl- channels: implications for cystic fibrosis.

1. The CIC-2 Cl- channels are present in the adult human lung epithelia and, therefore, are a potential target for therapy in cystic fibrosis. 2. Activators of CIC-2 Cl- channels that may have physiological relevance include activation by reduced external pH, protein kinase A and arachidonic acid. 3. Activators of CIC-2 Cl- channels that have therapeutic potential include amidation and omeprazole and, perhaps, effectors of arachidonic acid metabolism.

PKA and arachidonic acid activation of human recombinant ClC-2 chloride channels.

An HEK-293 cell line stably expressing the human recombinant ClC-2 Cl(-) channel was used in patch-clamp studies to study its regulation. The relative permeability P(x)/P(Cl) calculated from reversal potentials was I(-) > Cl(-) = NO(3)(-) = SCN(-)>/=Br(-). The absolute permeability calculated from conductance ratios was Cl(-) = Br(-) = NO(3)(-) >/= SCN(-) > I(-). The channel was activated by cAMP-dependent protein kinase (PKA), reduced extracellular pH, oleic acid (C:18 cisDelta9), elaidic acid (C:18 transDelta9), arachidonic acid (AA; C:20 cisDelta5,8,11,14), and by inhibitors of AA metabolism, 5,8,11,14-eicosatetraynoic acid (ETYA; C:20 transDelta5,8,11,14), alpha-methyl-4-(2-methylpropyl)benzeneacetic acid (ibuprofen), and 2-phenyl-1,2-benzisoselenazol-3-[2H]-one (PZ51, ebselen). ClC-2 Cl(-) channels were activated by a combination of forskolin plus IBMX and were inhibited by the cell-permeant myristoylated PKA inhibitor (mPKI). Channel activation by reduction of bath pH was increased by PKA and prevented by mPKI. AA activation of the ClC-2 Cl(-) channel was not inhibited by mPKI or staurosporine and was therefore independent of PKA or protein kinase C activation.

Injury-induced vesiculation and membrane redistribution in squid giant axon.

Injury of isolated squid giant axons in sea water by cutting or stretching initiates the following unreported processes: (i) vesiculation in the subaxolemmal region extending along the axon several mm from the site of injury, followed by (ii) vesicular fusions that result in the formation of large vesicles (20-50 micron diameter), 'axosomes', and finally (iii) axosomal migration to and accumulation at the injury site. Some axosomes emerge from a cut end, attaining sizes up to 250 microns in diameter. Axosomes did not form after axonal injury unless divalent cations (Ca2+ or Mg2+) were present (10mM) in the external solution. The requirement for Ca2+ and the action of other ions are similar to that for cut-end cytoskeletal constriction in transected squid axons (Gallant, P.E. (1988) J. Neurosci. 8, 1479-1484) and for electrical sealing in transected axons of the cockroach (Yawo, H. and Kuno, M. (1985) J. Neurosci. 5, 1626-1632). Axosomes probably consist of membrane from different sources (e.g., axolemma, organelles and Schwann cells); however, localization of axosomal formation to the inner region of the axolemma and the formation dependence on divalent cations suggest principal involvement of cisternae of endoplasmic reticulum. Patch clamp of excised patches from axosomes liberated spontaneously from cut ends of transected axons showed a 12-pS K+ channel and gave indications of other channel types. Injury-induced vesiculation and membrane redistribution seem to be fundamental processes in the short-term (minutes to hours) that precede axonal degeneration or repair and regeneration. Axosomal formation provides a membrane preparation for the study of ion channels and other membrane processes from inaccessible organelles.

A computational method for calculating distensibility of the blood vessel in vivo.

A computational method for calculating the dynamic distensibility of the vessel wall in vivo, developed on the basis of the pressure pulse transmission, is proposed. Distensibilities of descending thoracic aorta, abdominal aorta, and femoral artery in normal dogs, and of femoral artery of a typical dog under the action of vasoactive drugs, have been calculated. In femoral artery it is compared with the values of the diameter change/pressure change. Comparison of the results clearly indicate the feasibility of the proposed method. The order of distensibility found is: descending thoracic aorta greater than abdominal aorta greater than femoral artery.