Stationed or Relocating: The Seesawing EMT/MET Determinants from Embryonic Development to Cancer Metastasis.

Epithelial and mesenchymal transition mechanisms continue to occur during the cell cycle and throughout human development from the embryo stage to death. In embryo development, epithelial-mesenchymal transition (EMT) can be divided into three essential steps. First, endoderm, mesoderm, and neural crest cells form, then the cells are subdivided, and finally, cardiac valve formation occurs. After the embryonic period, the human body will be subjected to ongoing mechanical stress or injury. The formation of a wound requires EMT to recruit fibroblasts to generate granulation tissues, repair the wound and re-create an intact skin barrier. However, once cells transform into a malignant tumor, the tumor cells acquire the characteristic of immortality. Local cell growth with no growth inhibition creates a solid tumor. If the tumor cannot obtain enough nutrition in situ, the tumor cells will undergo EMT and invade the basal membrane of nearby blood vessels. The tumor cells are transported through the bloodstream to secondary sites and then begin to form colonies and undergo reverse EMT, the so-called "mesenchymal-epithelial transition (MET)." This dynamic change involves cell morphology, environmental conditions, and external stimuli. Therefore, in this manuscript, the similarities and differences between EMT and MET will be dissected from embryonic development to the stage of cancer metastasis.

An annotation-free whole-slide training approach to pathological classification of lung cancer types using deep learning.

Deep learning for digital pathology is hindered by the extremely high spatial resolution of whole-slide images (WSIs). Most studies have employed patch-based methods, which often require detailed annotation of image patches. This typically involves laborious free-hand contouring on WSIs. To alleviate the burden of such contouring and obtain benefits from scaling up training with numerous WSIs, we develop a method for training neural networks on entire WSIs using only slide-level diagnoses. Our method leverages the unified memory mechanism to overcome the memory constraint of compute accelerators. Experiments conducted on a data set of 9662 lung cancer WSIs reveal that the proposed method achieves areas under the receiver operating characteristic curve of 0.9594 and 0.9414 for adenocarcinoma and squamous cell carcinoma classification on the testing set, respectively. Furthermore, the method demonstrates higher classification performance than multiple-instance learning as well as strong localization results for small lesions through class activation mapping.

Downregulation of STK4 promotes colon cancer invasion/migration through blocking ß-catenin degradation.

Mammalian STE20-like kinase 1 (MST1/STK4/KRS2) encodes a serine/threonine kinase that is the mammalian homolog of Drosophila Hippo. STK4 plays an important role in controlling cell growth, apoptosis, and organ size. STK4 has been studied in many cancers with previous studies indicating an involvement in colon cancer lymph node metastasis and highlighting its potential as a diagnostic marker for colon cancer. However, the role of STK4 defect in promoting colon cancer progression is still understudied. Here, we found that STK4 was significantly downregulated in colon cancer and was associated with distal metastasis and poor survival. Furthermore, STK4 knockdown enhanced sphere formation and metastasis in vitro and promoted tumor development in vivo. We found that STK4 colocalized with ß-catenin and directly phosphorylated ß-catenin resulting in its degradation via the ubiquitin-mediated pathway. This may suggest that STK4 knockdown causes ß-catenin phosphorylation failure and subsequently ß-catenin accumulation, consequently leading to anchorage-independent growth and metastasis in colon cancer. Our results support that STK4 may act as a potential candidate for the assessment of ß-catenin-mediated colon cancer prognosis.

Effect of Gradient Energy Density on the Microstructure and Mechanical Properties of Ti6Al4V Fabricated by Selective Electron Beam Additive Manufacture.

Selective Electron Beam Additive Manufacturing (SEBAM) is a promising powder bed fusion additive manufacturing technique for titanium alloys that select particular area melting in different energy density for producing complexly shaped biomedical devices. For most commercial Ti6Al4V porous medical devices, the gradient energy density is usually applied to manufacture in one component during the SEBAM process which selects different energy density built on particular zones. This paper presents gradient energy density base characterization study on an SEBAM built rectangular specimen with a size of 3 mm × 20 mm × 60 mm. The specimen was divided into three zones were built in gradient energy density from 16 to 26.5 J/mm3. The microstructure and mechanical properties were investigated by means of scanning electron microscopy, X-ray diffraction, transmission electron microscopy and mechanical test. The α' martensitic and lack of fusion were observed in the low energy density (LED) built zone. However, no α' phase and no irregular pores were observed both in overlap energy density (OED) and high energy density (HED) built zones located at the middle and bottom of the specimen respectively. This implies the top location and lower energy density have positive effects on the cooling rate but negative effects on densification. The subsequence mechanical properties result also supports this point. Moreover, the intermetallic Ti3Al found in the bottom may be due to the heat transfer from the following melting layer. Furthermore, the microstructure evolution in gradient energy built zones is discussed based on the findings of the microstructure and thermal history correlation analysis.

Generating Lap Joints Via Friction Stir Spot Welding on DP780 Steel.

Friction stir spot welding (FSSW), a derivative of friction stir welding (FSW), is a solid-state welding technique that was developed in 1991. An industry application was found in the automotive industry in 2003 for the aluminum alloy that was used in the rear doors of automobiles. Friction stir spot welding is mostly used in Al alloys to create lap joints. The benefits of friction stir spot welding include a nearly 80% melting temperature that lowers the thermal deformation welds without splashing compared to resistance spot welding. Friction stir spot welding includes 3 steps: plunging, stirring, and retraction. In the present study, other materials including high strength steel are also used in the friction stir welding method to create joints. DP780, whose traditional welding process involves the use of resistance spot welding, is one of several high strength steel materials used in the automotive industry. In this paper, DP780 was used for friction stir spot welding, and its microstructure and microhardness were measured. The microstructure data showed that there was a fusion zone with fine grain and a heat effect zone with island martensite. The microhardness results indicated that the center zone exhibited a greater degree of hardness compared with the base metal. All data indicated that the friction stir spot welding used in dual phase steel 780 can create a good lap joint. In the future, friction stir spot welding can be used in high-strength steel welding applied in industrial manufacturing processes.

Hypoxia-induced epithelial-mesenchymal transition and fibrosis for the development of breast capsular contracture.

Fibrosis has been considered as a major cause of capsular contracture. Hypoxia has widely emerged as one of the driving factors for fibrotic diseases. The aim of this study was to examine the association between hypoxia-induced fibrosis and breast capsular contracture formation. Fibrosis, epithelial-mesenchymal transition (EMT), expression levels of hypoxia-inducible factor-1α (HIF-1α), vimentin, fibronectin, and matrix metalloproteinase-9 (MMP-9) in tissues from patients with capsular contracture were determined according to the Baker classification system. Normal breast skin cells in patients with capsular contracture after implant-based breast surgery and NIH3T3 mouse fibroblasts were cultured with cobalt chloride (CoCl2) to mimic hypoxic conditions. Treatment responses were determined by detecting the expression of HIF-1α, vimentin, fibronectin, N-cadherin, snail, twist, occludin, MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1) and -2, as well as phosphorylated ERK. The expression levels of HIF-1α, vimentin, fibronectin, and fibrosis as well as EMT were positively correlated with the severity of capsular contracture. MMP-9 expression was negatively correlated the Baker score. Hypoxia up-regulated the expression of HIF-1α, vimentin, fibronectin, N-cadherin, snail, twist, TIMP-1 and -2, as well as phosphorylated ERK in normal breast skin cells and NIH3T3. Nonetheless, the expression levels of MMP-9 and occludin were down-regulated in response to CoCl2 treatment. This study is the first to demonstrate the association of hypoxia-induced fibrosis and capsular contracture.

Nitinol powders generate from Plasma Rotation Electrode Process provide clean powder for biomedical devices used with suitable size, spheroid surface and pure composition.

The nickel-titanium alloy (57Ni-43Ti in wt%) was atomized by the plasma rotating electrode process (PREP). The PREP parameters such as plasma arc current, rotating electrode speed with corresponding PREP powder size range in weight percentage analysis, powder morphology and biocapability of cells were studied by scanning electron microscopies, Inductively Coupled Plasma and X-ray diffraction techniques. From the electrode of the produced powders, the composition has no obviously changes. Weight percentage up to 31.8% of the range under 300 µm while the rotation electrode speed increase to 12k rpm. Spherical and flat with smooth surface were observed in different size range. Brittle phase was not observed of XRD data. The nitinol powder has high biocapability with cells showed no cytotoxicity and well cell adhesion in the in vivo assay.

Schwann-Cell Autophagy, Functional Recovery, and Scar Reduction After Peripheral Nerve Repair.

The functional outcome after peripheral nerve repair is often unpredictable for many reasons, e.g., the severity of neuronal death and scarring. Axonal degeneration significantly affects outcomes. Post-injury axonal degeneration in peripheral nerves is accompanied by myelin degradation initiated by Schwann cells (SCs), which activate autophagy, a ubiquitous cytoprotective process essential for degrading and recycling cellular constituents. Scar formation occurs concomitantly with nerve insult and axonal degeneration. The association between SC autophagy and the mechanisms of nerve scar formation is still unknown. A rat model of peripheral nerve lesions induced by sciatic nerve transection injuries was used to examine the function of autophagy in fibrosis reduction during the early phase of nerve repair. Rats were treated with rapamycin (autophagy inducer) or 3-methyladenine (autophagy inhibitor). One week after the nerve damage, fibrosis was potently inhibited in rapamycin-treated rats and, based on gait analysis, yielded a better functional outcome. Immunohistochemistry showed that the autophagic activity of SCs and the accumulation of neurofilaments were upregulated in rapamycin-treated rats. A deficiency of SC autophagic activity might be an early event in nerve scar formation, and modulating autophagy might be a powerful pharmacological approach for improving functional outcomes.

miR-105/93-3p promotes chemoresistance and circulating miR-105/93-3p acts as a diagnostic biomarker for triple negative breast cancer.

BACKGROUND: Triple negative breast cancer (TNBC) lacks both early detection biomarkers and viable targeted therapeutics. Moreover, chemotherapy only produces 20-30% pathologic complete response. Because miRNAs are frequently dysregulated in breast cancer and have broad tissue effects, individual or combinations of circulating miRNAs may serve as ideal diagnostic, predictive or prognostic biomarkers, as well as therapeutic targets. Understanding the role and mechanism of dysregulated miRNAs in TNBC may help to develop novel diagnostic and prognostic strategy for TNBC patients. METHODS: The miRNA array profiles of 1299 breast cancer patients were collected from the Metabric database and subjected to analysis of the altered miRNAs between TNBC and non-TNBC. In Student's t-test and Kaplan-Meier analysis, four upregulated miRNAs correlated with poor survival in TNBC but not in non-TNBC. Four miRNAs were manipulated in multiple cell lines to investigate their functional role in carcinogenesis. From these results, we studied miR-105 and miR-93-3p in greater detail. The level of miR-105 and miR-93-3p were evaluated in 25 breast cancer tumor tissues. In addition, the diagnostic utility of circulating miR-105 and miR-93-3p were examined in 12 normal and 118 breast cancer plasma samples by ROC curve construction. RESULTS: miR-105 and miR-93-3p were upregulated and correlated with poor survival in TNBC patients. Both miR-105 and miR-93-3p were found to activate Wnt/ß-catenin signaling by downregulation of SFPR1. By this action, stemness, chemoresistance, and metastasis were promoted. Importantly, the combination of circulating miR-105/93-3p may serve as a powerful biomarker for TNBC, even in early-stage disease. CONCLUSIONS: miR-105/93-3p activates Wnt/ß-catenin signaling by downregulating SFRP1 and thereby promotes stemness, chemoresistance, and metastasis in TNBC cells. Most importantly, combined circulating miR-105/93-3p levels represent a prime candidate for development into a diagnostic biomarker for both early- and late-stage TNBC.

High-molecular-weight hyaluronic acid attenuated matrix metalloproteinase-1 and -3 expression via CD44 in tendinopathy.

Evidence indicates that hyaluronic acid (HA) mitigates tendinopathy, but the effect of molecular weight is unclear. We investigated the effects of different concentrations and different molecular weights of HA (350 kDa, 1500 kDa, and 3000 kDa) on matrix metalloproteinase (MMP)-1 and -3 expression in IL-1ß-stimulated rat tenocytes, and on their dynamic expression in peritendinous effusion from patients with long head of biceps (LHB) tendinopathy after high-molecular-weight (HMW)-HA treatments. Reverse transcription PCR, real-time PCR, and ELISA were used to determine MMP-1 and -3expression. Because CD44 was clearly expressed in the plasma membranes of cultured tenocytes, OX-50, a CD44 antagonist, was used to inhibit CD44 to evaluate the HA mechanism. HA (3000 kDa) significantly (p < 0.001) downregulated the mRNA and protein expression of MMP-1 and -3 in IL-1ß-stimulated tenocytes. Its attenuating effects were dose-dependent (p < 0.01). In OX-50-pretreated cells, the mRNA expression of CD44 was not significantly altered, but the mRNA expression of MMP-1 and -3 was significantly upregulated. Visual analogue scale scores were significantly lower, and MMP-1 and -3 expression was significantly (p < 0.05) lower one month posttreatment. HMW-HA attenuated tendinopathy by downregulating MMP-1 and -3 expression. Inhibiting CD44 blocked the effects of HMW-HA.

Mutation of the Enterohemorrhagic Escherichia coli Core LPS Biosynthesis Enzyme RfaD Confers Hypersusceptibility to Host Intestinal Innate Immunity In vivo.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important foodborne pathogen causing severe diseases in humans worldwide. Currently, there is no specific treatment available for EHEC infection and the use of conventional antibiotics is contraindicated. Therefore, identification of potential therapeutic targets and development of effective measures to control and treat EHEC infection are needed. Lipopolysaccharides (LPS) are surface glycolipids found on the outer membrane of gram-negative bacteria, including EHEC, and LPS biosynthesis has long been considered as potential anti-bacterial target. Here, we demonstrated that the EHEC rfaD gene that functions in the biosynthesis of the LPS inner core is required for the intestinal colonization and pathogenesis of EHEC in vivo. Disruption of the EHEC rfaD confers attenuated toxicity in Caenorhabditis elegans and less bacterial colonization in the intestine of C. elegans and mouse. Moreover, rfaD is also involved in the control of susceptibility of EHEC to antimicrobial peptides and host intestinal immunity. It is worth noting that rfaD mutation did not interfere with the growth kinetics when compared to the wild-type EHEC cells. Taken together, we demonstrated that mutations of the EHEC rfaD confer hypersusceptibility to host intestinal innate immunity in vivo, and suggested that targeting the RfaD or the core LPS synthesis pathway may provide alternative therapeutic regimens for EHEC infection.

MiR-193a-5p/ERBB2 act as concurrent chemoradiation therapy response indicator of esophageal squamous cell carcinoma.

Concurrent chemoradiation therapy (CCRT) is the predominant treatment in esophageal cancer, however resistance to therapy and tumor recurrence are exceedingly common. Elevated ERBB2/Her2 may be at least partially responsible for both the high rates of recurrence and resistance to CCRT. This receptor tyrosine kinase is upregulated in 10-20% of esophageal squamous cell carcinoma (ESCC) tissues, and amplification of ERBB2 has been correlated with poor prognosis in esophageal cancer. Tissues from 131 ESCC patients, along with cell and animal models of the disease were used to probe the underlying mechanisms by which ERBB2 upregulation occurs and causes negative outcomes in ESCC. We found that overexpression of ERBB2 inhibited radiosensitivity in vitro. Furthermore, miR-193a-5p reduced ERBB2 expression by directly targeting the 3'UTR. Increased miR-193a-5p enhanced radiosensitivity and inhibited tumorigenesis in vitro and in vivo. Additionally, low miR-193a-5p expression correlated with poor prognosis in ESCC patients, and ESCC patients with good CCRT response exhibited higher miR-193a-5p expression. Our data suggest that patients with high miR-193a-5p will likely benefit from CCRT treatment alone, however a combination of CCRT with Herceptin may be beneficial for patients with low miR-193a-5p expression.

Huntingtin-Interacting Protein-1 Is an Early-Stage Prognostic Biomarker of Lung Adenocarcinoma and Suppresses Metastasis via Akt-mediated Epithelial-Mesenchymal Transition.

RATIONALE: Non-small cell lung cancer (NSCLC) carries a poor survival rate mainly because of metastasis. However, the molecular mechanisms that govern NSCLC metastasis have not been described. Because huntingtin-interacting protein-1 (HIP1) is known to play a role in tumorigenesis, we tested the involvement of HIP1 in NSCLC progression and metastasis. OBJECTIVES: HIP1 expression was measured in human NSCLC tumors, and correlation with survival outcome was evaluated. Furthermore, we investigated the ability of HIP1 to suppress metastasis. The molecular mechanism by which HIP1 contributes to suppress metastasis was investigated. METHODS: We used tissue arrays containing samples from 121 patients with NSCLC to analyze HIP1 expression by immunohistochemistry. To investigate the role of HIP1 expression on metastasis, we evaluated cellular mobility, migration, and invasion using lung adenocarcinoma (AdCA) cells with modified HIP1 expression levels. The human disease mouse models with the same cells were applied to evaluate the HIP1 suppressing metastasis and its mechanism in vivo. MEASUREMENTS AND MAIN RESULTS: HIP1 expression in AdCA progression was found to be an early-stage prognostic biomarker, with low expression correlated to poor prognosis. We also found HIP1 to be a metastatic suppressor in AdCA. HIP1 significantly repressed the mobility of lung cancer cells in vitro and in vivo and regulated the epithelial-mesenchymal transition by repressing AKT/glycogen synthase kinase-3ß/ß-catenin signaling. CONCLUSIONS: HIP1 serves as an early-stage prognostic biomarker and a metastatic suppressor. Reduced expression during AdCA progression can relieve HIP1 suppression of Akt-mediated epithelial-mesenchymal transition and thereby lead to development of late metastases and poor prognosis.

MicroRNA-296-5p (miR-296-5p) functions as a tumor suppressor in prostate cancer by directly targeting Pin1.

Upregulation of Pin1 was shown to advance the functioning of several oncogenic pathways. It was recently shown that Pin1 is potentially an excellent prognostic marker and can also serve as a novel therapeutic target for prostate cancer. However, the molecular mechanism of Pin1 overexpression in prostate cancer is still unclear. In the present study, we showed that the mRNA expression levels of Pin1 were not correlated with Pin1 protein levels in prostate cell lines which indicated that Pin1 may be regulated at the post-transcriptional level. A key player in post-transcriptional regulation is represented by microRNAs (miRNAs) that negatively regulate expressions of protein-coding genes at the post-transcriptional level. A bioinformatics analysis revealed that miR-296-5p has a conserved binding site in the Pin1 3'-untranslated region (UTR). A luciferase reporter assay demonstrated that the seed region of miR-296-5p directly interacts with the 3'-UTR of Pin1 mRNA. Moreover, miR-296-5p expression was found to be inversely correlated with Pin1 expression in prostate cancer cell lines and prostate cancer tissues. Furthermore, restoration of miR-296-5p or the knockdown of Pin1 had the same effect on the inhibition of the ability of cell proliferation and anchorage-independent growth of prostate cancer cell lines. Our results support miR-296-5p playing a tumor-suppressive role by targeting Pin1 and implicate potential effects of miR-296-5p on the prognosis and clinical application to prostate cancer therapy.

Carboxyl-terminal modulator protein positively regulates Akt phosphorylation and acts as an oncogenic driver in breast cancer.

Akt activation has been implicated broadly in tumorigenesis, but the basis for its dysregulation in cancer cells is incompletely understood. In this study, we sought to clarify a regulatory role for the Akt-binding carboxy-terminal modulator protein (CTMP), which has been controversial. In evaluating CTMP expression in paired normal-tumor specimens of 198 patients with breast cancer, we found that CTMP was upregulated in breast tumors, where it was associated with poor patient survival. Notably, CTMP expression also correlated positively with Akt phosphorylation in breast cancer clinical specimens and cell lines. Furthermore, ectopic expression of CTMP promoted cell proliferation and enhanced the tumorigenic properties of estrogen-dependent breast cancer cells. This effect was correlated with increased sensitivity to insulin-induced Akt phosphorylation, which is mediated primarily by the phosphoinositide 3-kinase-Akt pathway. In contrast, short hairpin RNA-mediated silencing of endogenous CTMP decreased the proliferation of estrogen-dependent or estrogen-independent breast cancer cells. Mechanistic investigations defined the N-terminal domain of CTMP at amino acids 1 to 64 as responsible for Akt binding. Taken together, our results firmly corroborate the concept that CTMP promotes Akt phosphorylation and functions as an oncogenic molecule in breast cancer.

Inhibitory action of methadone and its metabolites on erg-mediated K+ current in GH3 pituitary tumor cells.

Methadone (Mtd) is a widely used opioid drug associated with the side effect of hyperprolactinemia. The mechanism of how Mtd induces prolactin secretion remains unclear. The effects of Mtd and its two main metabolites (EDDP: (±)-2-ethyl-1,5-dimethyl-3,3-diphenylpyrrolinium percholarate and EMDP: 2-ethyl-5-methyl-3,3-dipnehyl-1-pyrroline) on ion currents were investigated in GH3 pituitary tumor cells. Hyperpolarization-elicited K+ currents in GH3 cells bathed in a high-K(+), Ca(2+)-free solution were studied to evaluate the effects of Mtd and other related compounds on the ether-à-go-go-related-gene (erg) K(+) current (I(K(erg))). Mtd suppressed the amplitude of I(K(erg)) in a concentration-dependent manner with an IC(50) value of 10.4 µM. With the aid of a minimal binding scheme, the inhibitory action of Mtd on I(K(erg)) was estimated with a dissociation constant of 8.2 µM. Mtd tended to increase the rate of I(K(erg)) deactivation in a voltage-dependent fashion. EDDP (10 µM) had no effect on I(K(erg)), while EMDP (10µM) slightly suppressed it. In GH3 cells incubated with naloxone (30 µM), the Mtd-induced inhibition of I(K(erg)) remained unaltered. Under cell-attached voltage-clamp recordings, Mtd increased the frequency of spontaneous action currents with no change in current amplitude. Similarly, Mtd can suppress I(K(erg)) in differentiated NG108-15 cells; dynorphin A(1-13) did not reverse Mtd-induced inhibition of I(K(erg)). This study shows that Mtd has a depressant effect on I(K(erg)), and suggests its ability to affect membrane excitability and prolactin secretion. The cyclization of Mtd, in which EDDP and EMDP are formed, tends to be critical in removal of the Mtd binding to erg K+ channel.

Effects of ranolazine, a novel anti-anginal drug, on ion currents and membrane potential in pituitary tumor GH(3) cells and NG108-15 neuronal cells.

Ranolazine, a piperazine derivative, is currently approved for the treatment of chronic angina. However, its ionic mechanisms in other types of cells remain unclear, although it is thought to be a selective blocker of late Na(+) current. This study was conducted to evaluate the possible effects of ranolazine on Na(+) current (I(Na)), L-type Ca(2+) current (I(Ca,L)), inwardly rectifying K(+) current (I(K(IR))), delayed-rectifier K(+) current (I(K(DR))), and Ca(2+)-activated K(+) current (I(K(Ca))) in pituitary tumor (GH(3)) cells. Ranolazine depressed the transient and late components of I(Na) with different potencies. This drug exerted an inhibitory effect on I(K(IR)) with an IC(50) value of 0.92 microM, while it slightly inhibited I(K(DR)) and I(K(Ca)). It shifted the steady-state activation curve of I(K(IR)) to more positive potentials with no change in the gating charge of the channel. Ranolazine (30 microM) also reduced the activity of large-conductance Ca(2+)-activated K(+) channels in HEK293T cells expressing alpha-hSlo. Under current-clamp conditions, low concentrations (e.g., 1 microM) of ranolazine increased the firing of action potentials, while at high concentrations (>or=10 microM), it diminished the firing discharge. The exposure to ranolazine also suppressed I(Na) and I(K(IR)) effectively in NG108-15 neuronal cells. Our study provides evidence that ranolazine could block multiple ion currents such as I(Na) and I(K(IR)) and suggests that these actions may contribute to some of the functional activities of neurons and endocrine or neuroendocrine cells in vivo.

Analytical studies of rapidly inactivating and noninactivating sodium currents in differentiated NG108-15 neuronal cells.

The rapidly inactivating (I(Naf)) and noninactivating Na(+) currents (I(Na)(()(NI)())) were characterized in NG108-15 neuronal cells differentiated with dibutyryl cyclic AMP in this study. Standard activation and inactivation protocols were used to evaluate the steady-state and kinetic properties of the I(Naf) present in these cells. The voltage protocols with a slowly depolarizing ramp were implemented to examine the properties of I(Na)(()(NI)()). Based on experimental data and computer simulations, a window component of the rapidly inactivating sodium current (I(Naf)(()(W)())) was also generated in response to the slowly depolarizing ramp. The I(Naf)(()(W)()) was subtracted from I(Na)(()(NI)()) to yield the persistent Na(+) current (I(Na)(()(P)())). Our results demonstrate the presence of I(Na)(()(P)()) in these cells. In addition to modifying the steady-state inactivation of I(Naf), ranolazine or riluzloe could be effective in blocking I(Naf)(()(W)()) and I(Na)(()(P)()). The ability of ranolazine and riluzole to suppress I(Na)(()(P)()) was greater than their ability to inhibit I(Naf)(()(W)()). In current-clamp recordings, current-induced voltage oscillations were applied to elicit action potentials (APs) through a gradual transition between spontaneous depolarization and upstroke. Ranolazine or riluzole at a concentration of 3 microM then effectively suppressed the AP firing generated by oscillatory changes in membrane current. The data suggest that a small rise in I(Na)(()(NI)()) facilitates neuronal hyper-excitability due the decreased threshold of AP initiation. The underlying mechanism of the inhibitory actions of ranolazine or riluzole on membrane potential in neurons or neuroendocrine cells in vivo may thus be associated with their blocking of I(Na)(()(NI)()).