The human proximal tubular HK-2 cell line is an immortalized cell line commonly used for studying proximal tubular toxicity. Even as their use is presently increasing, there unfortunately are no studies focused on functional changes in HK-2 cells associated with passaging. The aim of the present study, therefore, was to evaluate the functional stability of HK-2 cells during 13 weeks of continuous passaging after 6 and 24 h of treatment with model nephrotoxic compounds (i.e., acetaminophen, cisplatin, CdCl(2)). Short tandem repeat profile, the doubling time, cell diameter, glutathione concentration, and intracellular dehydrogenase activity were measured in HK-2 cells at each tested passage. The results showed that HK-2 cells exhibit stable morphology, cell size, and cell renewal during passaging. Mean doubling time was determined to be 54 h. On the other hand, we observed a significant effect of passaging on the susceptibility of HK-2 cells to toxic compounds. The largest difference in results was found in both cadmium and cisplatin treated cells across passages. We conclude that the outcomes of scientific studies on HK-2 cells can be affected by the number of passages even after medium-term cultivation and passaging for 13 weeks.

• MeSH
• antitumorózní látky toxicita   MeSH
• buněčné kultury metody   MeSH
• buněčné linie MeSH
• cisplatina toxicita   MeSH
• kadmium toxicita   MeSH
• lidé MeSH
• neopioidní analgetika toxicita   MeSH
• paracetamol toxicita   MeSH
• proximální tubuly ledvin účinky léků   patologie   MeSH
• viabilita buněk MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antitumorózní látky MeSH
• cisplatina MeSH
• kadmium MeSH
• neopioidní analgetika MeSH
• paracetamol MeSH

Regulation of Na+/K+-ATPase in bipolar disorder and lithium therapy has been investigated for more than 40 years. Contradictory results in this area may be caused by the difference between acute and long-term Li effects on cell metabolism and variance in responsiveness of different cell types. We compared the time-course of Li action focusing on Na+/K+-ATPase and lipid peroxidation in two widely different cell models-Jurkat and HEK293. Na+/K+-ATPase expression level was determined in cells cultivated in the absence or presence of 1 mM Li for different time spans (1, 7, and 28 days) using [3H] ouabain binding and immunoblot assay of α-subunit. In parallel samples, the formation of malondialdehyde (MDA) was quantified by HPLC, and 4-hydroxy-2-nonenal (4-HNE) protein adducts were determined by immunoblot. Cultivation of Jurkat cells in 1 mM Li medium resulted in downregulation of Na+/K+-ATPase (decrease of [3H] ouabain-biding sites and intensity of immunoblot signals) in all Li-groups. In HEK293 cells, the decrease of Na+/K+-ATPase was observed after the acute, 1-day exposure only. The long-term treatment with Li resulted in Na+/K+-ATPase upregulation. MDA and 4-HNE modified proteins were decreased in Jurkat cells in all Li-groups. On the other hand, in HEK293 cells, MDA concentration was decreased after the acute, 1-day Li exposure only; the long-term cultivations, for 7 or 28 days, resulted in a significant increase of lipid peroxidation products. The Li-induced decrease of lipid peroxidation products was associated with the decrease of Na+/K+-ATPase level and vice versa.

• Klíčová slova
• HEK293 cells, Jurkat cells, Lipid peroxidation products, Lithium, Na+/K+-ATPase,
• MeSH
• bipolární porucha farmakoterapie   metabolismus   MeSH
• časové faktory MeSH
• HEK293 buňky MeSH
• Jurkat buňky MeSH
• lidé MeSH
• lipidové peroxidy metabolismus   MeSH
• peroxidace lipidů účinky léků   MeSH
• regulace genové exprese účinky léků   MeSH
• sloučeniny lithia aplikace a dávkování   metabolismus   farmakologie   MeSH
• sodíko-draslíková ATPasa genetika   metabolismus   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• lipidové peroxidy MeSH
• sloučeniny lithia MeSH
• sodíko-draslíková ATPasa MeSH

The effect of sodium, potassium, and lithium on δ-opioid receptor ligand binding parameters and coupling with the cognate G proteins was compared in model HEK293 cell line stably expressing PTX-insensitive δ-OR-Gi1α (Cys(351)-Ile(351)) fusion protein. Agonist [(3)H]DADLE binding was decreased in the order Na(+) ≫ Li(+) > K(+) > (+)NMDG. When plotted as a function of increasing NaCl concentrations, the binding was best-fitted with a two-phase exponential decay considering two Na(+)-responsive sites (r (2) = 0.99). High-affinity Na(+)-sites were characterized by Kd = 7.9 mM and represented 25 % of the basal level determined in the absence of ions. The remaining 75 % represented the low-affinity sites (Kd = 463 mM). Inhibition of [(3)H]DADLE binding by lithium, potassium, and (+)-NMDG proceeded in low-affinity manner only. Surprisingly, the affinity/potency of DADLE-stimulated [(35)S]GTPγS binding was increased in a reverse order: Na(+) < K(+) < Li(+). This result was demonstrated in PTX-treated as well as PTX-untreated cells. Therefore, it is not restricted to Gi1α(Cys(351)-Ile(351)) within the δ-OR-Gi1α fusion protein, but is also valid for stimulation of endogenous G proteins of Gi/Go family in HEK293 cells. Biophysical studies of interaction of ions with polar head-group region of lipids using Laurdan generalized polarization indicated the low-affinity type of interaction only proceeding in the order: Cs(+) < K(+) < Na(+) < Li(+). The results are discussed in terms of interaction of Na(+), K(+) and Li(+) with the high- and low-affinity sites located in water-accessible part of δ-OR binding pocket. We also consider the role of negatively charged Cl(-), Br(-), and I(-) counter anions in inhibition of both [(3)H]DADLE and [(35)S]GTPγS binding.

• MeSH
• buněčná membrána metabolismus   MeSH
• guanosin 5'-O-(3-thiotrifosfát) metabolismus   MeSH
• HEK293 buňky MeSH
• leucin-2-alanin-enkefalin metabolismus   MeSH
• lidé MeSH
• lipidové dvojvrstvy metabolismus   MeSH
• lithium farmakologie   MeSH
• proteiny vázající GTP - alfa-podjednotky Gi-Go metabolismus   MeSH
• receptory opiátové delta metabolismus   MeSH
• rekombinantní proteiny metabolismus   MeSH
• sodík metabolismus   MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• guanosin 5'-O-(3-thiotrifosfát) MeSH
• leucin-2-alanin-enkefalin MeSH
• lipidové dvojvrstvy MeSH
• lithium MeSH
• proteiny vázající GTP - alfa-podjednotky Gi-Go MeSH
• receptory opiátové delta MeSH
• rekombinantní proteiny MeSH
• sodík MeSH