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
• Cell Culture Techniques methods   MeSH
• Cell Line MeSH
• Cisplatin toxicity   MeSH
• Cadmium toxicity   MeSH
• Humans MeSH
• Analgesics, Non-Narcotic toxicity   MeSH
• Acetaminophen toxicity   MeSH
• Antineoplastic Agents toxicity   MeSH
• Kidney Tubules, Proximal drug effects   pathology   MeSH
• Cell Survival MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cisplatin MeSH
• Cadmium MeSH
• Analgesics, Non-Narcotic MeSH
• Acetaminophen MeSH
• Antineoplastic Agents 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.

• Keywords
• HEK293 cells, Jurkat cells, Lipid peroxidation products, Lithium, Na+/K+-ATPase,
• MeSH
• Bipolar Disorder drug therapy   metabolism   MeSH
• Time Factors MeSH
• HEK293 Cells MeSH
• Jurkat Cells MeSH
• Humans MeSH
• Lipid Peroxides metabolism   MeSH
• Lipid Peroxidation drug effects   MeSH
• Gene Expression Regulation drug effects   MeSH
• Lithium Compounds administration & dosage   metabolism   pharmacology   MeSH
• Sodium-Potassium-Exchanging ATPase genetics   metabolism   MeSH
• Dose-Response Relationship, Drug MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Lipid Peroxides MeSH
• Lithium Compounds MeSH
• Sodium-Potassium-Exchanging ATPase 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
• Cell Membrane metabolism   MeSH
• Guanosine 5'-O-(3-Thiotriphosphate) metabolism   MeSH
• HEK293 Cells MeSH
• Enkephalin, Leucine-2-Alanine metabolism   MeSH
• Humans MeSH
• Lipid Bilayers metabolism   MeSH
• Lithium pharmacology   MeSH
• GTP-Binding Protein alpha Subunits, Gi-Go metabolism   MeSH
• Receptors, Opioid, delta metabolism   MeSH
• Recombinant Proteins metabolism   MeSH
• Sodium metabolism   MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Guanosine 5'-O-(3-Thiotriphosphate) MeSH
• Enkephalin, Leucine-2-Alanine MeSH
• Lipid Bilayers MeSH
• Lithium MeSH
• GTP-Binding Protein alpha Subunits, Gi-Go MeSH
• Receptors, Opioid, delta MeSH
• Recombinant Proteins MeSH
• Sodium MeSH