Na+/K+-ATPase as a target for cardiotonic steroids and cisplatin

The sodium (Na+)/potassium (K+)-ATPase is an ion pump located on the surface of all animal cells. It pumps three sodium ions out of the cell while pumping two potassium ions into the cell, hydrolyzing one ATP molecule as the driving force for the reaction. Na+/K+-ATPase forms and maintains the electrochemical gradient in cells, which provides the basis for the excitability of nerve and muscle tissues and contributes to the osmotic regulation of cell volume. In addition, the electrochemical Na+ gradient is the driving force for the secondary transport of nutrients such as amino acids, sugars, and drugs. Recently, the Na+/K+-ATPase has been studied as an important target for cancer treatment, as it has been implicated in the development and progression of many cancers. Na+/K+-ATPase forms a phosphoenzyme intermediate (EP) during ATP hydrolysis.　Cardiotonic steroids have been used to treat congestive heart failure and arrhythmias, and recently their anti-cancer activities have been reported. Ouabain, a specific inhibitor of Na+/K+-ATPase, is a cardiotonic steroid that binds to EP, inhibiting its dephosphorylation and the release of inorganic phosphate. Cisplatin is one of the most potent anti-tumor agents. Many studies have examined the relationship between cisplatin and Na+/K+-ATPase from the viewpoint of cisplatin accumulation and the prevention of nephrotoxicity. It has been suggested that the transport of cisplatin into cells is mediated by the Na+/K+-ATPase and that Na+/K+-ATPase activity is inhibited by cisplatin, although the underlying mechanism remains unclear.　In this review, we evaluate the mechanisms underlying inhibition of Na+/K+-ATPase by cisplatin. We also summarize the structure, function, and enzymatic reaction of Na+/K+-ATPase, as well as the potential for the pump to serve as a target for ouabain and cisplatin. Finally, we will describe experiments conducted by our group showing the mechanism of Na+/K+-ATPase inhibition by cisplatin, and the combined effects of ouabain and cisplatin on cancer cell viability

Mechanism for propofol inhibition of Na＋, K＋-ATPase activity in rat brain

Propofol is one of the most widely used intravenous anesthetics, however the mechanism of the anesthetic effect is not fully understood. Na+,K+-ATPase is an enzyme present in all animal cell membranes and plays essential roles for the maintenance of neuronal excitability. There is a report of propofol inhibition of Na+,K+-ATPase activity, but the mechanism is not clearly established. To study the mechanism for propofol inhibition of Na+,K+-ATPase purified from whole brains of rats, the effects of propofol on Na+,K+-ATPase activity, Na+-ATPase, and K+-pNPPase activities, which are partial reactions of Na+,K+-ATPase were examined. Na+,K+-ATPase and Na+-ATPase activities decreased depending on the concentration of propofol, and were completely inhibited at 1.03 mM. Propofol decreased the maximum activity of Na+-, K+-, Mg2+-, and ATPdependent activation of Na+,K+-ATPase activity depending on its concentration, and changed the half maximal concentration for Na+, K+, and ATP, but not for Mg2+. Propofol also decreased the maximum activities of Na+-ATPase and K+-pNPPase, suggesting that propofol inhibits Na+,K+-ATPase activity by affecting the whole reaction process of Na+,K+-ATPase. The inhibition of Na+,K+-ATPase activity by propofol was reversible by dilution of its concentration. These results suggest that propofol reversibly inhibits Na+,K+-ATPase activity in a mixed-type inhibition pattern

How much medium do you use for cell culture? Medium volume influences mineralization and osteoclastogenesis in vitro

Bone is maintained by a balance between bone formation and resorption. This remodeling is controlled by a wide variety of systemic and local factors including hormones, cytokines and mechanical stresses. The present in vitro study examined the impact of medium volume, using 0.4, 0.6, 0.8, 1.0, 1.5 and 2.0 ml/well in a 24-well plate, on the differentiation of osteoblasts and osteoclasts. There were no differences in the alkaline phosphatase activity of osteoblasts amongst the groups; however, the area of mineral deposition was decreased in a media volume-dependent manner. A co-culture of osteoblastic cells with bone marrow cells revealed a reduction in the total number of osteoclastic tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells (>= 2 nuclei), whereas the formation of large osteoclastic TRAP-positive multinuclear cells (>= 8 nuclei) was increased, in a media volume-dependent manner. There were also no differences in receptor activator of nuclear factor-kappa B ligand mRNA and total osteoprotegerin (OPG) protein expression levels amongst the groups, however the concentration of OPG decreased in a media volume-dependent manner. In conclusion, the present study demonstrated that the suppression of mineralization in osteoblastic cells and the stimulation of osteoclast fusion are dependent on the medium volume, indicating that media volume is an important factor in in vitro cell culture systems

Up-regulation of the G3PDH ʻhousekeeping’ gene by estrogen

Proteomic and genomic studies commonly involve the assessment of mrna levels using reverse transcription- polymerase chain reaction (Pcr) and real-time quantitative Pcr. an internal standard rna is fundamentally analyzed along with the investigated mRNA to document the specificity of the effect(s) on mrna and to correct for inter-sample variations. in our studies implementing estrogen treatments on different cell lines, we initially used glyceraldehyde-3- phosphate dehydrogenase (G3PdH) as an internal standard. However, the results of PCR amplification demonstrated that 17β-estradiol enhanced the expression of the G3PdH gene, rendering it impossible to use G3PdH as an unbiased com- parative control

Optimal compressive force accelerates osteoclastogenesis in RAW264.7 cells

Mechanical stress produced by orthodontic forces is a factor in the remodeling of periodontal ligaments (PDLs) and alveolar bone. It has been reported that the expression of a number of cytokines associated with osteoclastogenesis is upregulated when compressive forces act on osteoblasts and PDL cells. The present study investigated the effects of compressive forces on the formation of osteoclasts from the macrophage cell line RAW264.7. Compressive forces on osteoclasts were exerted using layers of 3, 5, 7, 9 or 14 glass cover slips on the 4th day of culture for 24 h. The number of osteoclasts was determined by counting the number of cells positive for tartrate-resistant acid phosphatase staining. Osteoclastogenesis advanced rapidly on days four and five. The number of osteoclasts with >8 nuclei peaked when the force of 7 slips was applied, which was therefore regarded as the optimal compressive force. Alterations in the expression of osteoclast-associated genes are associated with changes in the differentiation and fusion of macrophages in response to compressive forces; therefore, osteoclast-associated genes were assessed by reverse transcription quantitative polymerase chain reaction in the present study. The mRNA expression of osteoclast-associated genes increased significantly after 3 h of optimal compression, whereas mRNA expression increased after 24 h in the control group. These findings suggested that osteoclastogenesis of macrophages was accelerated when an optimal compressive force was applied

Novel effect of estrogen on RANK and c-fms expression in RAW 264.7 cells

Temporomandibular disorder (TMD), a progressive disease entity, and osteoarthrosis preferentially affect females, denoting a possible role of estrogen. Using RAW 264.7 cells, the expression of estrogen receptors (ERs) α and ß and the consequent effect of estrogen was investigated. We present the novel detection of ER ß expression in RAW 264.7 cells. Furthermore, we innovatively demonstrated the increase in expression of both ER α and ß, as well as RANK and c-fms, with estrogen treatment. However, a decrease in expression of c-fms, RANK and ER ß, and nearly no change in the expression of ER α were experienced upon further increase in estrogen concentrations. These findings lead us to hypothesize a new mechanism of inflammation in TMD

Effect of estrogen on bone resorption and inflammation in the temporomandibular joint cellular elements

Several epidemiological studies have reported that temporomandibular disorder is more prevalent in women, which suggests the involvement of sex hormones, such as estrogen, in the pathogenesis of this disease. PCR amplification and Western blotting were employed to target the expression of estrogen receptors (ERs) in human fibroblast-like synovial and ATDC5 cells. The effect of estrogen was investigated through the expression of RANKL, osteoprotegerin (OPG), M-CSF/CSF-1 and c-fms. We showed expression of M-CSF/ CSF-1 and c-fms, with time-dependent increase in both after the addition of estrogen. Based on previous studies reporting that M-CSF/CSF-1 regulates the proliferation and differen- tiation of hemopoietic progenitor cells into mature macro- phages, we put forward a new hypothesis based on the increased inflammation and tendency of females to suffer more from temporomandibular disorder (TMD) in the presence of external exacerbating factors. Detection of RANKL and OPG in ATDC5 and expression of both in HFLS was confirmed with complete disappearance of the RANKL band, and marked increase in the expression of OPG after 1 h from the addition of estrogen