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Our previous study revealed that changes of the intracellular Cl - concentration ([Cl - ] i ) affected cell proliferation in cancer cells. However, the role of Cl - on cell migration and invasion in cancer cells remains unanalyzed. Therefore, the aim of the present study is to investigate whether changes of [Cl - ] i affects cell migration and invasion of cancer cells. In human prostate cancer DU145 cells, cell migration and invasion were enhanced by culturing in the low Cl - medium (replacement of Cl - by NO 3 - ). We also found that DU145 cells in the low Cl - condition caused significant transient ERK1/2 activation followed by an increase of MMP-1 mRNA levels. Inhibition of ERK1/2 activation in the low Cl - condition reduced enhancement of MMP-1 mRNA levels and decreased cell migration and invasion. These observations indicate that [Cl - ] i plays important roles in metastatic function by regulating the ERK1/2 signaling pathway in human prostate cancer cells, and intracellular Cl - would be one of the key targets for anti-cancer therapy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The onset establishment and maintenance of gonadotropin-releasing hormone (GnRH) secretion is an important phenomenon regulating pubertal development and reproduction. GnRH neurons as well as other neurons in the hypothalamus have high-energy demands and require a constant energy supply from their mitochondria machinery to maintain active functioning. However, the involvement of mitochondrial function in GnRH neurons is still unclear. In this study, we examined the role of NADH Dehydrogenase (Ubiquinone) Fe-S protein 4 (Ndufs4), a member of the mitochondrial complex 1, on GnRH neurons using Ndufs4-KO mice and Ndufs4-KO GT1-7 cells. Ndufs4 was highly expressed in GnRH neurons in the medial preoptic area (MPOA) and NPY/AgRP and POMC neurons in the arcuate (ARC) nucleus in WT mice. Conversely, there was a significant decrease in GnRH expression in MPOA and median eminence of Ndufs4-KO mice, followed by impaired peripheral endocrine system. In Ndufs4-KO GT1-7 cells, Gnrh1 expression was significantly decreased with or without stimulation with either kisspeptin or NGF, whereas, stimulation significantly increased Gnrh1 expression in control cells. In contrast, there was no difference in cell signaling activity including ERK and CREB as well as the expression of GPR54, TrkA and p75 NTR , suggesting that Ndufs4 is involved in the transcriptional regulation system for GnRH production. These findings may be useful in understanding the mitochondrial function in GnRH neuron., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Many pathological proteins related to neurodegenerative diseases are misfolded, aggregating to form amyloid fibrils during pathogenesis. One of the pathological proteins, alpha-synuclein (α-syn), accumulates in the brains of Parkinson disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), which are designated as synucleinopathies. Recently, structural properties of abnormal accumulated proteins are suggested to determine the disease phenotype. However, the biochemical and structural characteristics of those accumulated proteins are still poorly understood. We previously reported the sequence and seed-structure-dependent polymorphic fibrils of α-syn and the polymorphism was identified by proteinase K-resistant cores determined by mass spectrometry (MS) analysis. In this study, we applied this method to analyze α-syn aggregates of MSA and DLB. To perform MS analysis on proteinase K-resistant cores, we first performed amplification of α-syn aggregates by seeding reaction and protein misfolding cyclic amplification (PMCA) to obtain a sufficient amount of aggregates. Using SDS insoluble fraction of the disease brain, we successfully amplified enough α-syn aggregates for MS analysis. We differentiated between mouse and human α-syn aggregates by MS analysis on proteinase K-resistant cores of the aggregates before and after amplification. The results suggest that structural properties of amplified α-syn fibrils are preserved after PMCA and these methods can be applicable in the study of pathological proteins of the neurodegenerative disorders., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Amyotrophic lateral sclerosis (ALS), which is the most common motor neuron disease in adults, is a neurodegenerative disease that involves the selective and systematic death of upper and lower motor neurons. In addition to the motor neuron death, altered metabolic functions, such as dyslipidemia, have also been reported for ALS patients; however, the underlying mechanism remains unknown. In the present study, we investigated the effects of ALS-associated P56S-vesicle-associated membrane proteinassociated protein B (VAPB), P56S-VAPB on 3T3-L1 preadipocyte differentiation and on the expression of differentiation-associated genes and unfolded protein response (UPR)-related genes. Experiments with 3T3-L1 cells transfected with wild-type (Wt)-VAPB and P56S-VAPB expression vectors showed that the size of lipid droplets was markedly smaller in P56S-VAPB-expressing cells, although fat accumulated intracellularly. In P56S-VAPB-expressing cells, increased the expression of PPARγ2, aP2, and C/EBPα, the genes deeply involved in adipocyte differentiation, was not observed. Furthermore, the expression levels of the UPR-related ATF4 and CHOP genes were found to be enhanced in the P56S-VAPB-expressing cells. From these results, P56S-VAPB was found to suppress adipocyte differentiation by promoting the activation of the ATF4-CHOP pathway. Given previous reports showing increased ATF4 and CHOP expression levels in neurons of ALS patients, results from the present study suggest that dyslipidemia is caused by enhanced ATF4-CHOP pathway in the adipose tissue of ALS patients., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Insulin resistance in the skeletal muscle is manifested by diminished insulin-stimulated glucose uptake and is a core factor in the pathogenesis of type 2 diabetes mellitus (DM), but the mechanism causing insulin resistance is still unknown. Our recent study has shown that pH of interstitial fluids was lowered in early developmental stage of insulin resistance in OLETF rats, a model of type 2 DM. Therefore, in the present study, we confirmed effects of the extracellular pH on the insulin signaling pathway in a rat skeletal muscle-derived cell line, L6 cell. The phosphorylation level (activation) of the insulin receptor was significantly diminished in low pH media. The phosphorylation level of Akt, which is a downstream target of the insulin signaling pathway, also decreased in low pH media. Moreover, the insulin binding to its receptor was reduced by lowering extracellular pH, while the expression of insulin receptors on the plasma membrane was not affected by the extracellular pH. Finally, insulin-stimulated 2-deoxyglucose uptake in L6 cells was diminished in low pH media. Our present study suggests that lowered extracellular pH conditions may produce the pathogenesis of insulin resistance in skeletal muscle cells., (Copyright © 2014. Published by Elsevier Inc.)

Propolis, a resinous mixture collected from plants by the Apis mellifera bee, contains high level nutrient factors including vitamins, polyphenols, and amino acids that would be expected to improve insulin sensitivity. Insulin resistance would secondarily cause elevation of blood pressure and increase the risk of cardiovascular diseases. The purpose of this study is to investigate the effect of propolis extracts on blood glucose levels and blood pressures in an early developmental stage of insulin resistance in Otsuka Long-Evans Tokushima Fatty (OLETF) rats. OLETF rats (10 weeks old) were divided into three different groups: normal diet, 0.1% propolis diet, and 0.5% propolis diet. After 8 weeks, blood glucose levels, blood pressures, plasma metabolic factors and hormones, and interstitial fluid pH were measured. Casual blood glucose levels were decreased associated with a reduction of plasma insulin levels in both propolis diet groups compared with normal diet group. Propolis decreased systolic blood pressure with no significant changes in plasma aldosterone levels. We also found that interstitial fluid pH in ascites, liver, and skeletal muscle was higher in rats fed propolis diet than rats fed normal diet. These data suggests that dietary propolis improves insulin sensitivity and blood pressures in the early stage of the process in development of insulin resistance, which may be mediated by suppression of metabolic acidosis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Circadian Clock genes are associated with the estrous cycle in female animals. Treatment with Per2 and Clock siRNAs decreased the number of granulosa cells and LHr expression in follicle-stimulating hormone FSH-treated granulosa cells. Per2 siRNA treatment did not stimulate the production of estradiol and expression of P450arom, whereas Clock siRNA treatment inhibited the production of estradiol and expression of P450arom mRNA. Per2 and Clock siRNA treatment increased and unchanged, respectively, progesterone production in FSH-treated granulosa cells. Similarly, expression of StAR mRNA was increased by Per2 siRNA and unchanged by Clock siRNA. Our data provide a new insight that Per2 and Clock have different action on ovarian granulosa cell functions., (Copyright © 2011 Elsevier Inc. All rights reserved.)

In multicellular organisms, epithelia separate and divide the internal environment maintaining appropriate conditions in each compartment. To maintain homeostasis in these compartments, claudins, major cell adhesion molecules in tight junctions (TJs), regulate movements of several substances through the paracellular pathway (barrier function). In this study, we investigated effects of the flux of several substances between apical and basolateral side on paracellular transport and TJ protein localization. NaCl flux from apical to basolateral side increased paracellular conductance (Gp) and recruited claudin-1 from lateral cell membrane to the apical end with the colocalization with occludin, one of the TJ proteins concentrated at TJ strands. Oppositely-directed flux of sucrose against NaCl flux inhibited these reactions and same directional flux of sucrose with NaCl enhanced the increase of Gp, whereas 10-kDa dextran inhibited these reactions regardless of the side of administration. Our present findings indicated that TJ protein localization and barrier function are regulated depending on the environmental differences between apical and basolateral side., (2010 Elsevier Inc. All rights reserved.)

In the epithelia and endothelia, tight junctions regulate the movement of several substances through the paracellular pathway, maintaining several gradients between apical and basal compartments including osmolality and hydrostatic pressure. In this study, we show that the change of hydrostatic pressure gradient affected tight junctions as well as actin cytoskeleton, cell height and transcellular ion transport. Hydrostatic pressure gradient from basolateral to apical side increased transepithelial conductance and altered claudin-1 localization within several tens of minutes. These changes were promptly restored by the elimination of hydrostatic pressure gradient. Hydrostatic pressure gradient also induced dynamic changes in the actin structure and cell height. We further found that hydrostatic pressure gradient from basolateral to apical side stimulates transcellular Cl(-) transport. Our present findings indicate that the epithelial cell structures and functions are regulated by the hydrostatic pressure gradient which is generated and maintained by the epithelia themselves.

In vertebrates, craniofacial formation is accomplished by synergistic interaction of many small elements which are generated independently from distinct germ layers. Because of its complexity, the imbalance of one signaling cascade such as Wnt/beta-catenin pathway easily leads to craniofacial malformation, which is the most frequent birth defect in humans. To investigate the developmental role of a newly identified activator of Wnt/beta-catenin signaling, Rspo2, we generated and characterized Rspo2(-/-) mice. We found CLP with mild facial skeletal defects in Rspo2(-/-) mice. Additionally, Rspo2(-/-) mice also exhibited distal limb loss and lung hypoplasia, and died immediately after birth with respiratory failure. We showed the apparent reduction of Wnt/beta-catenin signaling activity at the branchial arch and the apical ectodermal ridge in Rspo2(-/-) mice. These findings indicate that Rspo2 regulates midfacial, limb, and lung morphogenesis during development through the Wnt/beta-catenin signaling.

The aim of the present study is to investigate whether the chloride affects cell growth and cell-cycle progression of cancer cells. In human gastric cancer MKN28 cells, the culture in the Cl(-)-replaced medium (replacement of Cl(-) by NO(3)(-)) decreased the intracellular chloride concentration ([Cl(-)](i)) and inhibited cell growth. The inhibition of cell growth was due to cell-cycle arrest at the G(0)/G(1) phase caused by diminution of CDK2 and phosphorylated Rb. The culture of cells in the Cl(-)-replaced medium significantly increased expressions of p21 mRNA and protein without any effects on p53. These observations indicate that chloride ions play important roles in cell-cycle progression by regulating the expression of p21 through a p53-independent pathway in human gastric cancer cells, leading to a novel, unique therapeutic strategy for gastric cancer treatment via control of [Cl(-)](i).

The purpose of the present study is to characterize the ENaC-mediated Na+ absorption in human upper airway epithelia, nasal cavity, and paranasal sinus. To perform the purpose, we obtained epithelial cells from human nasal polyp (NP) and paranasal sinus mucosa (PSM) by endoscopic surgery. We measured the short-circuit current (I(sc)) sensitive to benzamil (a specific ENaC blocker). The benzamil-sensitive I(sc) (Na+ absorption) in NP was larger than that in PSM. The mRNA expression of three subunits of ENaC was as follows: alpha>beta>gamma in both tissue, NP and MS. The mRNA expression of gamma subunit of ENaC in NP was larger than that in PSM, but no difference of mRNA expression of alpha or beta ENaC subunit between NP and PSM was detected. We found correlation of the Na+ absorption to mRNA expression of gamma ENaC in NP and PSM. Forskolin diminished the Na+ absorption associated with an increase in Cl- secretion. These observations suggest that: (1) human NP absorbs more ENaC-mediated Na+ than PSM, (2) expression of gamma ENaC in plays a key role in the ENaC-mediated Na+ absorption in NP and PSM, and (3) cAMP diminishes the ENaC-mediated Na+ absorption by stimulating Cl- secretion (diminution of driving force for Na+ absorption) in NP and PSM.

In the present study, we investigated if Cl(-) regulates the proliferation of the MC3T3-E1 osteoblastic cells. The proliferation of MC3T3-E1 osteoblastic cells was diminished by lowering the extracellular Cl(-) concentration ([Cl(-)](o)) in the culture medium. The lowered in [Cl(-)](o) increased the periods of the G(0)/G(1) and the G(2)/M phases in cell cycle. We further studied the effects of [Cl(-)](o) on the key enzymes, Rb and cdc2, playing key roles in checking points of the G(0)/G(1) and the G(2)/M phases in cell cycle. The lowered in [Cl(-)](o) diminished the active forms of enzymes, Rb and cdc2. We further found that the action of lowered [Cl(-)](o) on the cell proliferation, the cell cycle, Rb and cdc2 was abolished by the presence of 2mM glutamine, but not by that of pyruvate as another Krebs cycle substrate. Taken together, these observations indicate here for the first time that Cl(-) modulates Rb and cdc2, enhancing the proliferation of the MC3T3-E1 osteoblastic cells.

Processing of APP by BACE1 plays a crucial role in the pathogenesis of Alzheimer disease (AD). Recently, the voltage-gated sodium channel (Na(v)) beta4 subunit (beta4), an auxiliary subunit of Na(v) that is supposed to serve as a cell adhesion molecule, has been identified as a substrate for BACE1. However, the biological consequence of BACE1 processing of beta4 remains illusive. Here, we report the biological effects of beta4 processing by BACE1. Overexpression of beta4 in Neuro2a cells promoted neurite extension and increased the number of F-actin rich filopodia-like protrusions. While coexpression of BACE1 together with beta4 further accelerated neurite extension, the number of filopodia-like protrusions was reduced. Overexpression of C-terminal fragment of beta4 that was generated by BACE1 (beta4-CTF) partially recapitulated the results obtained with BACE1 overexpression. These results suggest that the processing of beta4 by BACE1 regulates neurite length and filopodia-like protrusion density in neurons.

The Na(+)/K(+)/2Cl(-) cotransporter (NKCC) mediates electroneutral transport of 2Cl(-) coupled with Na(+) and K(+) across the plasma membrane, and plays crucial roles in Cl(-) uptake into the cells, homeostasis of cellular Cl(-), and cell volume regulation. However, we have very limited information on the roles of ion transporters in neurite outgrowth in neuronal cells. In the present study, we report the role of NKCC1 (an isoform of NKCC) in NGF-induced neurite outgrowth of rat pheochromocytoma PC12D cells. The expression level of NKCC1 protein was increased by NGF treatment. Knock-down of NKCC1 by RNA interference (RNAi) drastically diminished the NGF-induced neurite outgrowth. Transfection of enhanced green fluorescent protein (EGFP)-tagged rat NKCC1 into cells for clarification of intracellular localization of NKCC1 revealed that the EGFP-rNKCC1 was mainly localized in the plasma membrane at growth cone during neurite outgrowth. These observations suggest that NKCC1 plays a fundamental role in NGF-induced neurite outgrowth of PC12D cells.

Neltenexine has been applied to human lung diseases such as chronic obstructive pulmonary disease (COPD) as a mucolytic agent. However, we have no information on the neltenexine action in bronchial epithelial cells. We studied the neltenexine action on the ion transport in human submucosal serous Calu-3 cells. Under a hyper-secreting condition caused by terbutaline (a beta2-adrenergic agonist), neltenexine diminished anion secretion by inhibiting the Cl- and HCO3- uptake via Na+/K+/2Cl- cotransporter and Na+/HCO3- cotransporter without blockade of the cystic fibrosis transmembrane conductance regulator (CFTR) channel, and also diminished anion secretion via stimulation of Cl-/HCO3- exchanger, which facilitates the extrusion of more CFTR-permeant anion, Cl-, with the uptake of less CFTR-permeant anion, HCO3-. Thus, neltenexine reduced the hyper-secretion to keep an appropriate fluid level in the airway, providing a possibility that neltenexine can be an effective drug in airway obstructive diseases by decreasing the airway resistance under a hyper-secreting condition.

Since our experimental results suggest that UT-B1 functions as active water transporter against osmotic gradient in C6 glial cells, we report here for the first time the evidence for the active water transport. Exposure of C6 cells to a hyperosmotic solution containing glycerol or sucrose produced cell shrinkage due to water efflux according to osmotic gradient for water movement. On the other hand, C6 cells show cell swelling against osmotic gradient for water movement just after exposure to a hyperosmotic solution containing urea, indicating that water influx against osmotic gradient for water movement is accelerated by urea; i.e., urea performs active water transport. A specific inhibitor of UT-B, pCMBS, blocked the urea-induced swelling. The urea-induced cell swelling was significantly suppressed in the siRNA-induced UT-B1-knockdown C6 cells. Taken together, these observations indicate that UT-B1 acts as an active water transporter, providing a new model on active water transport.

Aldosterone plays a crucial role in controlling mineral balance in our body. The mechanism of aldosterone has been reported to elevate renal Na+ reabsorption by stimulating expression of epithelial Na+ channel (ENaC) and also activate an ENaC-regulating protein kinase, serum and glucocorticoid-regulated kinase 1 (SGK1). However, it is unknown whether aldosterone shows its stimulatory action on ENaC and SGK1 under an abnormal, salt-sensitive hypertensive condition. To clarify this point, we studied how aldosterone regulates expression of ENaC and SGK1 in Dahl salt-sensitive (DS) rat that shows hypertension with high salt diet. RNA and protein were extracted from the kidney 6 h after application of aldosterone (1.5 mg/kg body weight) subcutaneously injected into adrenalectomized DS and Dahl salt-resistant (DR) rats. Aldosterone decreased mRNA expression of beta- and gamma-ENaC in DS rat unlike DR rat, while aldosterone increased alpha-ENaC mRNA expression in DS rat similar to DR rat. Further, we found that aldosterone elevated SGK1 expression in DR rat, but not in DS rat. These observations indicate that ENaC and SGK1 are abnormally regulated by aldosterone in salt-sensitive hypertensive rats, suggesting that disturbance of the aldosterone regulation would be one of factors causing salt-sensitive hypertension.

Transepithelial Cl(-) secretion in polarized renal A6 cells is composed of two steps: (1) Cl(-) entry step across the basolateral membrane mediated by Na(+)/K(+)/2Cl(-) cotransporter (NKCC) and (2) Cl(-) releasing step across the apical membrane via cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. We estimated CFTR Cl(-) channel activity and transcellular Cl(-) secretion by measuring 5-nitro 2-(3-phenylpropylamino)benzoate (NPPB, a blocker of CFTR Cl(-) channel)-sensitive transepithelial conductance (Gt) and short-circuit current (Isc), respectively. Pretreatment with 1 microM insulin for 24 h had no effects on NPPB-sensitive Gt or Isc. On the other hand, in A6 cells treated with carbobenzoxy-L-leucyl-leucyl-L-leucinal (MG132; 100 microM for 2 h) that inhibits endocytosis of proteins at the plasma membrane into the cytosolic space, insulin pretreatment increased the NPPB-sensitive Isc with no effects on NPPB-sensitive Gt. Genistein (100 microM) induced sustained increases in NPPB-sensitive Gt and Isc, which were diminished by brefeldin A (a blocker of protein translocation to Golgi apparatus from endoplasmic reticulum). Co-application of insulin and genistein synergically stimulated the NPPB-sensitive Isc without any effects on NPPB-sensitive Gt. These observations suggest that: (1) insertion and endocytosis of NKCC are stimulated by insulin, (2) the insulin-induced stimulation of NKCC insertion into the basolateral membrane is offset by the stimulatory action on NKCC endocytosis from the basolateral membrane, (3) genistein stimulates insertion of both CFTR Cl(-) channel into the apical membrane and NKCC into the basolateral membrane, and (4) insulin and genistein synergically stimulated NKCC insertion into the basolateral membrane.

The epithelial Na(+) transport via an epithelial Na(+) channel (ENaC) expressed in the lung epithelium would play a key role in recovery from lung edema at acute lung injury by removing the fluid in lung luminal space. The lung edema causes dysfunction of gas exchange, decreasing oxygen pressure level of artery [P(aO(2))]. To study if ENaC plays a key role in recovering P(aO(2)) from a decreased level to a normal one in acute lung injury, we applied benzamil (20microM, a specific blocker of ENaC) to the lung luminal space in acute lung injury treated with high frequency oscillation ventilation (HFOV) that is a lung-protective ventilation with a lower tidal volume and a smaller pressure swing than conventional mechanical ventilation (CMV). Benzamil facilitated the recovery of P(aO(2)) in acutely injured lung with HFOV but not CMV. The observation suggests that in acutely injured lung treated with HFOV an ENaC blocker, benzamil, can be applied as a therapeutic drug for acute lung injury combing with HFOV.

The cell growth, survival, and migration of vascular endothelial cells (ECs) are positively regulated by several protein tyrosine kinase receptors. Therefore, protein tyrosine phosphatases (PTPs) must also be important for these processes. The present study found that transmembranal PTPepsilonM, but not cytoplasmic PTPepsilonC, is expressed in porcine ECs and in rat smooth muscle cells, both of which were prepared from the aorta. The overexpression of wild-type PTPepsilonM promoted cell survival and migration in porcine aortic ECs even in medium without and with 1% serum, respectively. A catalytically inactive, substrate-trapping mutant of PTPepsilonM, respectively, did not affect and conversely suppressed cell survival and migration. Interestingly, the forced expression of wild-type PTPepsilonC reduced cell viability in contrast to PTPepsilonM in ECs lacking endogenous PTPepsilonC, indicating the biological significance of selective expression of PTPepsilon isoforms in the vasculature. PTPepsilonM activated c-Src kinase probably by directly dephosphorylating phospho-Tyr527, a negative regulatory site of c-Src. The increases in cell survival and migration induced by overexpressed PTPepsilonM were suppressed by the c-Src inhibitor SU6656. Considering the behaviors of vascular ECs in the pathogenesis of atherosclerosis, these data suggest that PTPepsilonM negatively regulates the development of this disease by activating c-Src.

The formation of glucose-derived methylglyoxal (MG), a highly reactive dicarbonyl compound, is accelerated under diabetic conditions. We examined whether MG was capable of inducing apoptosis in Schwann cells (SCs), since recent studies have suggested a potential involvement of apoptotic cell death in the development of diabetic neuropathy. MG induced apoptosis in SCs in a dose-dependent manner, accompanied by a reduction of intracellular glutathione content and activation of the p38 MAPK. Inhibiting the p38 MAPK activation by SB203580 successfully suppressed the MG-induced apoptosis in SCs. Aminoguanidine and N-acetyl-L-cysteine also inhibited the MG-induced p38 MAPK activation and apoptosis along with restoration of the intracellular glutathione content. These results suggest a potential role for MG in SC injury through oxidative stress-mediated p38 MAPK activation under diabetic conditions, and it may serve as a novel insight into therapeutic strategies for diabetic neuropathy.

Flavonoid, a plant extract, exhibits various biological actions. Dietary flavonoid intake is reported to reduce an elevated blood pressure, however the mechanism is unknown. The epithelial Na+ channel (ENaC) in the kidney plays a key role in the regulation of blood pressure by contributing to the Na+ reabsorption in renal tubules. Thus, we investigated the effect of quercetin, a flavonoid, on ENaC mRNA expression in the kidney of hypertensive Dahl salt-sensitive rats. Dahl salt-sensitive rats of 8 weeks were acclimated for 1 week in a metabolic cage and were subsequently kept for 4 weeks under four different conditions: (1) normal salt diet (0.3% NaCl), (2) normal salt diet with quercetin (10 mg/kg/day), (3) high-salt diet (8% NaCl), and (4) high-salt diet with quercetin. Quercetin diminished the alphaENaC mRNA expression in the kidney associated with reduction of the systolic blood pressure elevated by high-salt diet, suggesting that one of the mechanisms of the flavonoid's antihypertensive effect on salt-sensitive hypertension would be mediated through downregulation of ENaC expression in the kidney.

Exercise increases utilization of lipids and carbohydrates in skeletal muscles. After exercise, replenishment of glycogen and triglyceride occurs in skeletal muscles. To elucidate the mechanism of lipid filling effect after exercise training, expression patterns of genes related to triglyceride synthesis were examined under several exercise conditions. Mice exercised by 2-week swimming had 1.4-2.0-fold increases of sterol regulatory element-binding protein 1 (SREBP-1) mRNA in skeletal muscles after the last swimming, with increases of lipogenic genes, such as acetyl-CoA carboxylase-1 (ACC-1), stearoyl-CoA desaturase-1 (SCD-1), and acyl CoA:diacylglycerol acyltransferase-1 (DGAT-1) mRNAs. An increase of SREBP-1 mRNA was observed after the 6-h treadmill running training but not after 1-h single treadmill running. Increase of SREBP-1 mRNA was due to the increase of SREBP-1c isoform but not of SREBP-1a. These data indicate that SREBP-1c, a key transcription factor of liver triglyceride synthesis, might also be responsible for skeletal muscle triglyceride synthesis after chronic exercise training.

Vascular smooth muscle cell (VSMC) migration and growth are positively regulated by protein tyrosine phosphorylation. Therefore, a dephosphorylation process controlled by protein tyrosine phosphatases (PTPs) must also be critical. The present study identified six cytoplasmic PTPs expressed in VSMCs: low M(r) protein tyrosine phosphatase (LMW-PTP), SHP-2, PTP36, PTP2, PTP1B, and FAP1. We further examined the functions of LMW-PTP in VSMCs using the adenovirus-mediated gene transfer of recombinant LMW-PTP. PDGF-induced activation of p38, but not of ERK MAP kinase, was blocked by LMW-PTP. LMW-PTP as well as the p38 inhibitor SB203580 inhibited DNA synthesis and cell migration upon PDGF stimulation. LMW-PTP dephosphorylated activated PDGF receptors in NIH3T3 cells, but not in VSMCs. Thus, LMW-PTP negatively regulates PDGF functions by inhibiting the p38 pathway in VSMCs although its substrate is unclear. These findings strongly demonstrate that PTPs are important as negative regulators for VSMC growth and migration, processes that are closely related to the progression of atherosclerosis.

Oncogenic ras upregulates the expression of VEGF through the activation of the transcriptional enhancer hypoxia inducible factor-1alpha (HIF-1alpha) by a still poorly understood mechanism. Here, we demonstrate that both the Raf/MEK/MAPK and the PI3 kinase/Akt signaling pathways potently and additively stimulate the expression from a hypoxia response element (HRE) within the 5'flanking region of the VEGF promoter. Interestingly, while MAPK appears to specifically upregulate the transactivation activity of HIF-1alpha through direct phosphorylation of its regulatory/inhibitory domain, GSK-3, a downstream target of Akt, directly phosphorylates the HIF-1alpha oxygen-dependent degradation domain. These results suggest a novel mechanism whereby two divergent signaling pathways emerging from Ras may cooperatively but independently regulate the activity of a HIF-1alpha, thereby promoting the expression of a potent angiogenic mediator.

A cDNA encoding a novel human CYP4F enzyme (designated CYP4F12) was cloned by PCR from a human small intestine cDNA library. RT-PCR analysis demonstrated that CYP4F12 is expressed in human small intestine and liver. This cDNA contains an entire coding region of a 524-amino-acid protein that is 81.7, 78.3, and 78.2% identical to CYP4F2, CYP4F3, and CYP4F8, respectively. When expressed in Saccharomyces cerevisiae, the P450 catalyzes leukotriene B(4) omega-hydroxylation and arachidonic acid omega-hydroxylation, typical reactions of CYP4F isoforms. Their activity levels are, however, much lower than those of CYP4F2. Interestingly, CYP4F12 catalyzes the hydroxylation of the antihistamine ebastine with significantly higher catalytic activity relative to CYP4F2 (385 vs 5 pmol/min/nmol P450). These results indicate that CYP4F12 has a different profile of substrate specificity from other CYP4F isoforms, enzymes responsible for metabolizing endogenous autacoids, therefore suggesting that it may play an important role in xenobiotic biotransformation in the human small intestine., (Copyright 2001 Academic Press.)

Blood pressure is elevated and pressor response to angiotensin II (Ang II) is exaggerated in AT2 null mice. The purpose of the present study was to elucidate the mechanism for the increased responsiveness to Ang II in the mice. The contraction of aortic strips generated by Ang II was significantly greater in the AT2 gene-deleted mice than the control, which was completely abolished by AT1 antagonist losartan. The aortic content of AT1 receptor was significantly increased (P < 0.05, n = 5) in the AT2 null mice (212 +/- 58.2 fmol/mg protein) compared with the control (98.2 +/- 55.9 fmol/mg protein). While both AT1 and AT2 mRNAs were expressed in the aorta of the control mice, only AT1 mRNA was expressed in the AT2 knockout mice. The expression of AT1 mRNA in the AT2 knockout mice was significantly higher (1.5-fold, P < 0.05, n = 5) than that in the control. The present study clearly demonstrated that the increased vascular reactivity to Ang II in AT2 knockout mice is at least partly due to an increased vascular AT1 receptor expression and suggested that AT2 counteracts AT1-mediated vascular action of Ang II through downregulation of AT1 receptor by a crosstalk between these receptors by some as yet unknown mechanisms., (Copyright 1999 Academic Press.)

We have cloned two members of the CLC chloride channel family (OmCLC-3 and OmCLC-5) from gill cDNA libraries of the euryhaline tilapia Oreochromis mosammbicus. At the amino acid level, OmCLC-3 is 90.5% identical to rat CLC-3 and OmCLC-5 is 79.2% identical to rat CLC-5. Ribonuclease protection assay revealed that OmCLC-5 was mainly expressed in the gill, kidney, and intestine in both freshwater- (FW) and seawater- (SW) adapted tilapia. Although the mRNA of OmCLC-3 was broadly expressed in tissues of FW- and SW-adapted tilapia, the most intense signals were observed in the gill, kidney, intestine, and brain. Injection of OmCLC-3 and OmCLC-5 cRNAs into Xenopus oocytes did not elicit chloride currents, but these clones did functionally complement the gef1 phenotype of YPH250(gef), a yeast strain in which a single CLC channel (GEF1) has been disrupted by homologous recombination. These results clearly indicated that CLC channels closely related to the mammalian CLC-3, -4, and -5 subfamily exist also in tilapia and that OmCLC-3 and OmCLC-5 function as intracellular chloride channels., (Copyright 1999 Academic Press.)

Lysophosphatidic acid (LPA) is a lipid mediator which acts on its putative G protein-coupled receptor (GPCR). Recently, activation of signal transducers and activators of transcription (STATs) mediated by GPCR has been reported. In this study, we examined the effect of LPA on STAT activation using the electrophoretic mobility shift assays and the heterologous promoter analysis in human epidermoid carcinoma A431 cells. We found that LPA inhibited epidermal growth factor (EGF)-induced Stat1 activation in a concentration-dependent manner. Other phospholipase C (PLC)-coupled GPCR agonists, bradykinin and ATP, also inhibited Stat1 activation. This inhibitory effect of LPA was completely mimicked by an activator of protein kinase C (PKC), a PLC-downstream effector. These findings suggest that the inhibitory effect on EGF-induced Stat1 activation may be a general characteristic of PLC-coupled GPCRs and PKC pathway may be mainly associated with this inhibitory effect. This is the first evidence showing that GPCR agonists inhibit the Janus kinase-independent Stat1 activation induced by receptor tyrosine kinase.

Thrombopoietin (TPO) is the major regulator of the proliferation and differentiation of megakaryocyte precursors through interaction with its receptor encoded by the c-mpl protooncogene. We established the human TPO-dependent leukemia cell line, UT-7/TPO (Blood 87, 4552, 1996). In these cells, TPO activated protein kinase C (PKC) in a time dependent manner. Subsequently, the c-myc gene was transiently induced to a maximal level 60-90 minutes after TPO exposure. In addition, we found that stimulating UT-7/TPO cells with TPO rapidly induces the significant accumulation of inositol 1, 4, 5-trisphosphate (Ins-P3), leading to the mobilization of calcium from intracellular stores. Taken together, the activation of PKC and subsequent c-myc gene induction are involved in the TPO-induced cellular response(s), presumably through the activation of PLC.

We examined the effects of angiotensin II (Ang II) and its analogue CGP42112 on cell growth through the Ang II type 2 receptor (AT2) and the involvement of pertussis toxin (PTX)-sensitive G proteins in the AT2-mediated pathway. Both Ang II and CGP42112 inhibited DNA synthesis in serum-stimulated NIH3T3 fibroblast cells stably expressing recombinant AT2 in a dose-dependent manner with almost the same potency. This effect was abolished by the AT2-selective antagonist PD123319, but not by the Ang II type 1 receptor-specific antagonist DuP753, suggesting that AT2 mediates the event. The anti-proliferative effect was also blocked by PTX treatment. Together, these data suggest that CGP42112 as well as Ang II primarily has an AT2-mediated inhibitory activity on serum-stimulated cell growth, and PTX-sensitive G proteins are essential in this AT2-induced event.

Previous mutagenesis studies of angiotensin II (Ang II) receptor type 1 (AT1) have focused on determining the regions responsible for Gq coupling using the rat AT1 receptor. We created human AT1 receptor mutants, expressed them in COS-7 cells, and identified the domains crucial for Gi coupling as well as for Gq coupling. Substitution of Asp125, Arg126, Tyr127, and Met134 by Gly, Gly, Ala, and Ala in the highly conserved sequence of the second intracellular loop in most G protein-coupled receptors provided a mutant AT1 receptor which lost the ability to couple to both Gq and Gi with no impairment in its binding to Ang II. A truncated mutant lacking the carboxyl terminal 50 residues was completely deficient in coupling to Gi, whereas it retained full ability to bind to Gq, in contrast to the rat AT1 receptor. These findings demonstrate that the cytoplasmic tail in the human AT1 receptor is the determinant of specific Gi coupling.

To examine whether the subpopulation of the rat type 1 angiotensin II (AII) receptor (AT1A) couples with a single or multiple signal transduction pathways, we constructed Chinese hamster ovary (CHO) cell lines producing the recombinant receptor. The expressed AT1A receptor exhibits typical pharmacological characteristics of the AT1 receptor, known to mediate the main physiological function of AII. Addition of AII to the CHO cells induced a rapid, transient increase in intracellular free Ca2+ concentrations ([Ca2+]i) followed by a lower, sustained phase. Nicardipine, a blocker of voltage-dependent L-type Ca2+ channels, attenuated the transient [Ca2+]i response and abolished the sustained phase. The transient phase was also reduced dose-dependently by the phospholipase C inhibitor neomycin. Furthermore, AII inhibited forskolin-evoked cAMP accumulation. These data suggest, although another subpopulation named AT1B is present, that the rat AT1A receptor can independently couple with all three signal transduction pathways known to be induced by AII: i.e., i) activation of phospholipase C resulting in InsP3 generation with a subsequent release of intracellularly stored Ca2+, ii) activation of dihydropyridine-sensitive voltage-dependent Ca2+ channels, and iii) inhibition of adenylate cyclase activity.

Monoclonal antibodies reactive with endothelin (ET) receptors have been prepared by immunization of mice with rat lung membranes. Of four clones isolated, three clones preferentially recognized 32,000-dalton ET receptor and the other has a higher affinity for the 45,000-dalton receptor. The binding of 125I-ET-1 to detergent-solubilized ET receptors which were adsorbed to the antibodies was displaced by increasing concentrations of unlabeled ET isopeptides. These results demonstrate that the four clones specific for the receptor have the potential to be a useful tool in the characterization of ET receptors.

Competitive displacement experiments of 125I-endothelin (ET)-1, -2, or -3 binding to chick cardiac membranes were performed with unlabeled ET-1, -2, -3, and sarafotoxin S6b (STX) as competitors. 125I-ET-1 and -2 binding was competitively inhibited by increasing concentrations of these unlabeled peptides in the same order; i.e. ET-2 greater than or equal to ET-1 greater than ET-3 greater than STX. In contrast, the order of potency in displacing 125I-ET-3 binding was ET-3 greater than ET-2 greater than or equal to ET-1 greater than STX. Affinity labeling of the membranes by cross-linking with 125I-ET-1 and -2 via disuccinimidyl tartarate yielded one major specific band with an apparent Mr = 53,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. On the other hand, affinity labeling with 125I-ET-3 showed that two major and one minor bands of Mr = 34,000, 46,000, and 53,000, respectively, were specifically labeled. These results indicate the presence of two distinct types of ET receptors, one of which has higher affinity for ET-1 and -2 than ET-3 and the other is conversely ET-3-preferring.

A sequence-specific antibody that recognizes a portion of the prosegment of human renin precursor was raised and used to provide direct evidence that plasma inactive renin contains the prosequence of renal renin and is therefore probably prorenin rather than an inactivated form of previously active renin. The information may help not only to resolve a major controversy concerning the nature of inactive renin in human plasma but also to elucidate its exact physiological role.

The regulation of renin and renin messenger RNA (mRNA) in the rat ovary was examined to test the hypothesis that the expression of renin gene and the secretion of renin in the ovary is the estrogen-mediated process that responds to follicle-stimulating hormone (FSH). In the ovary of the immature 25-day female rats, the concentration of renin mRNA was comparatively low, but 36 h after injection of FSH, the renin mRNA content showed a three-fold increase compared to the basal level. This increase was consistent with the stimulation of the total renin concentration in the ovary. On the other hand, the total renin concentration in the rat uterus gradually decreased, suggesting that the enhancement of the contents of renin and renin mRNA by FSH is an ovary-specific phenomenon. In hypophysectomized rats, the total renin concentration in the rat ovary was stimulated by the estrogen as well as FSH. These findings suggest that the production of ovarian renin is regulated by the pituitary hormone, particularly FSH.

An increase in plasma prorenin during pregnancy suggests that prorenin might be synthesized in the ovary and the secretion of renin or prorenin may be stimulated by an ovarian steroid-mediated process. Recently, renin and angiotensinogen have been identified in human ovarian follicular fluid. However, there is considerable controversy over whether renin is synthesized in the ovary or derived from circulation. In the present study, we confirmed the presence of renin and renin mRNA in rat ovary and uterus by Northern blot analysis with rat renin cRNA as a hybridization probe. Our data show that ovarian or uterine renin is synthesized in the same cells. This suggests that the function of renin might be closely linked to the reproductive process.