Characterization of chloride transport pathways in cultured human keratinocytes (original) (raw)
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Release of lipoxygenase products of arachidonic acid from freshly isolated human keratinocytes
Archives of Dermatological Research, 1984
Lipoxygenation of arachidonic acid (aa) has previously been shown to generate potent chemoattractants for neurophils, eosinophils, monocytes and fibroblasts , the most potent being 5(S), 12(R)-dihydroxyeicosa-6,14-cis-8,10 trans-tetraenoic acid (leukotriene B4, LTB4) and 5(S)-hydroxyeicosatetraenoic acid (5-HETE). Other monoHETEs [13] and co-oxidation products of LTB4 [5] are less potent chemoattractants.
1992
The CI-transport mechanism responsible for the stimulation of 3f'Cl-efflux after exposure to hypotonic medium (210 mosmol/kg) was investigated in human keratinocytes. The involvement of the anion exchanger and of the CI-/cation cotransporters was ruled out by the finding that replacement of extraceUular CI-by the poorly permeant anion gluconate, and the addition of bumetanidc and furosemide, inhibitors of the Na+/K~/CI-and K+/CI -cotransporters, respectively, failed to significantly reduce the activation of CI-efflux by hypotonic medium. 'Whole cell' configuration of the patch clamp technique directly revealed the presence of a macroscopic CI-current, which was evoked by incubation with hypotonic medium and was reversed by elevation of the extracellular osmolality. Volume-sensitive current showed outward rectification of the current-voltage relationship and time-dependent inactivation at depolarizing voltages. This current was Ci-selective, because the zero-current reversal potential approached the Ci-equilibrium potential, when extracellular CI-was replaced by gluconate. 0.1 mM 1,9-dideoxyforskolin significantly reduced either 36CI-efflux and the CI-current, suggesting that the CI-efflux and the macroscopic current activated after exposure to hypotonic medium are mediated by the same pathway. Electronic cell sizing showed that in keratinocytes hypotonic swelling was not followed by a significant regulatory volume decrease response.
Regulation of chloride transport in cultured normal and cystic fibrosis keratinocytes
Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 1992
Cultured normal (N) and cystic fibrosis (CF) keratinocytes were evaluated for their CI -transport properties by patch-clamp-, Ussing chamber-and isotopic effiux-measurements. Special attention was paid to a 32 pS outwardly rectifying CI channel which has been reported to be activated upon activation of cAMP-dependent pathways in N, but not in CF cells. This depolarization-induced C1-channel was found with a similar incidence in N and CF apical keratininocyte membranes. However, activation of this channel in excised patches by protein kinase (PK)-A or PK-C was not successfull in either N or CF keratinocytes. Forskolin was not able to activate CI-channels in N and CF cell-attached patches. The CaZ+-ionophore A23187 activated in cell-attached patches a linear 17 pS C1 channel in both N and CF cells. This channel inactivated upon excision. No relationship between the cell-attached 17 pS and the excised 32 pS channel could be demonstrated. Returning to the measurement of CI transport at the macroscopic level, we found that a drastic rise in intracellular cAMP induced by forskolin did in N as well as CF cells not result in a change in the short-circuit current (I~c) or the fractional effiux rates of 36CI and 125I-. In contrast, addition of A23187 resulted in an increase of the Isc and in the isotopic anion effiux rates in N and CF cells. We conclude that CI -transport in cultured human keratinocytes can be activated by Ca 2+, but not by cAMP-dependent pathways.
Multiple biological effects of inhibitors of arachidonic acid metabolism on human keratinocytes
2002
Background: Various compounds that inhibit processing of arachidonic acid (AA) are being intensively tested for their antitumour activity. However, the mechanisms responsible for such activity remain rather elusive. To approach this issue, we examined the effects of several structurally different inhibitors of AA metabolism in the human keratinocyte HaCaT cell line. Methods: Several parameters were determined in HaCaT cells exposed to increasing concentrations of the inhibitors for 24 and/or 48 h. These included (1) oxidoreductase activity, total protein mass and cell cycle distribution to assess cell proliferation, (2) degradation of PARP protein to assess apoptosis, and (3) cell morphology, distribution of F-actin and expression of cytokeratins and E-cadherin to evaluate changes in differentiation status. Results: While eicosatetraynoic acid (ETYA), nordihydroguaiaretic acid (NDGA), esculetin and MK-886 reduced proliferation of HaCaT cells, the cyclooxygenase inhibitors indomethacin and piroxicam had no such effects. Esculetin and NDGA arrested cells in S phase, and ETYA and MK-886 delayed cell progression through G 1 phase. Higher concentrations of NDGA, MK886 and/or ETYA caused cleavage of PARP. No changes in the expression of cytokeratins and E-cadherin were observed upon treatment with any of the inhibitors. However, esculetin induced redistribution of F-actin accompanied by increased cell adhesion and size. Conclusion: Our findings indicate that, in addition to their ability to inhibit cell pro-liferation and to induce apoptosis, lipoxygenase inhibitors and/or ETYA may also elicit other important physiological responses in HaCaT keratinocytes.
Conversion of linoleic acid into arachidonic acid by cultured murine and human keratinocytes
2000
The origin of arachidonic acid (AA) found in the epidermis is not known. Two possibilities exist: either de novo syn- thesis within the epidermal keratinocyte, or transport of AA formed at distant tissue sites. The current study examined the ability of cultured murine and human keratinocytes to metabolize exogenously added linoleic acid (LA). Conversion of radiolabeled substrate ("C-LA) into 18:3(n-6),
The Journal of Membrane Biology, 1974
Prostaglandins (El, E 2 and F2~ ) stimulated the chloride transport of the frog corneal epithelium with maximal effects at 10 -5 M in the aqueous side. This stimulation does not occur in Cl-free solutions and the net 36C1 flux increased proportionally to the short-circuit current. Polyphloretin phosphate (PPP) and diphloretin phosphate (DPP) inhibited the response if added within 3 rain before PGE 1. The maximal response to epinephrine 10-SM and dibutyryl cyclic AMP 10-3M was not changed by further addition of prostaglandins, but these drugs produced their full effect when administered at the peak of the response of prostaglandins. The maximal response to theophylline 10 -5 M was increased by PGE 1. PPP and DPP did not modify the response to epinephrine. Prostaglandin stimulation of the chloride transport was accompanied by increased light transmission through partially opaque corneas. The known release of prostaglandins in the aqueous humor can be associated to a direct action on the corneal epithelium manifested in the activation described herein.