Impact of extracellular alkalinization on the survival of human CD24-/CD44+ breast cancer stem cells associated with cellular metabolic shifts

Cancer stem cells reside in a distinct region within the tumor microenvironment that it is believed to play a fundamental role in regulating stemness, proliferation, survival, and metabolism of cancer cells. This study aimed to analyze the effect of extracellular alkalinization on metabolism and survival of human CD24-/CD44+ breast cancer stem cells (BCSCs). BCSCs were cultured in alkalinized DMEM-F12 and incubated at 37°C, 5% CO2, and 20% O2 for 30 min, 6, 24, and 48 h. After each incubation period, we analyzed the modulation of various mRNA expressions related to pH and cellular metabolic regulation using the qRT-PCR. Metabolic state was measured using colorimetric and fluorometric assays. To examine cell proliferation and apoptosis, we used trypan blue and annexin V/propidium iodide assay, respectively. This study demonstrated that alkalinization could stimulate extracellular carbonic anhydrase (CAe) activity, as well as CA9 and HIF1α expression. Under alkaline pH and HIF1α regulation, glucose consumption, extracellular lactate production, and LDH activity of BCSCs were upregulated while O2 consumption was downregulated. These metabolic shifts seemed to promote apoptosis and suppress the proliferation of BCSCs. To conclude, modulation of the extracellular environment through alkalinization could change the metabolic states of BCSCs, which in turn affect the cell survival.

Mechanisms underlying contraction of rat isolated coronary artery induced by acidosis / 中国病理生理杂志

AIM:To explore the mechanisms underlying contraction induced by extracelluar acidosis (pHex6.8) in rat isolated coronary artery (RCA).METHODS:Using the microvessel tension recorder system, the effects of acid-base transporters on RCA contraction induced by pHex6.8 were explored by applying the selective pharmacological inhibitors of Na+-H+ exchanger 1 (NHE-1) and Na+-HCO-3 cotransporter (NBC), HOE-642 and S0859, respectively.The effects of chloride channel on RCA contraction induced by pHex6.8 were explored by applying the inhibitors of chloride channel (NPPB and NFA), and by replacing the extracellular NaCl with equimolar NaBr.RESULTS:pHex6.8 augmented the resting tension of RCA, and the maximum contraction was (3.90±0.95) mN.HOE-642 at 30 μmol/L and S0859 at 100 μmol/L both inhibited the contraction of RCA induced by pHex6.8 (P<0.01).NPPB and NFA both inhibited the contraction of RCA induced by pHex6.8 or KCl (60 mmol/L) in a concentration-dependent manner.NPPB and NFA (100 μmol/L) both inhibited the contraction of RCA induced by U46619 (1 μmol/L).Replacing the extracellular NaCl with equimolar NaBr almost completely inhibited RCA contraction induced by pHex6.8 (P<0.01), but had no obvious effect on the contraction induced by KCl (60 mmol/L) or U46619 (1 μmol/L).CONCLUSION:Extracellular acidosis-induced contraction in RCA may be related to the activated NHE-1 and NBC, and it may be also related to the enhanced chloride transport across the membrane.

Efeito do bicarbonato de sódio no tratamento de vinhaça em AnSBBR operado a 55 e 35ºC / Effect of the sodium bicarbonate in the treatment of vinasse in AnSBBR operated at 55 and 35ºC

Avaliou-se a influência do bicarbonato de sódio (NaHCO3) no desempenho do reator anaeróbio batelada sequencial com biomassa imobilizada tratando vinhaça a 55 e a 35ºC. O reator foi preenchido com espuma de poliuretano e agitado a 300 rpm. A adaptação a 55ºC estendeu-se por 50 dias, tendo o reator sido alimentado com vinhaça (DQO de 0,3 a 1,0 g/L). Para a operação a 35ºC, procedeu-se inicialmente ao enriquecimento da população metanogênica, durante 21 dias, com substrato à base de etanol (2,5 gDQO/L). Posteriormente, cargas orgânicas volumétricas (COV) de 0,85 a 5,70 gDQO/L.d foram aplicadas no reator a 55ºC, com suplementação de 1,2 a 0,4 gHCO3-/gDQO. A 35ºC, o reator foi submetido a COV de 2,85 a 36,0 gDQO/L.d com 0,4 a 0,2 gHCO3-/gDQO. A remoção de DQO variou de 43 a 78 por cento a 55ºC e de 75 a 85 por cento a 35ºC. A suplementação de alcalinidade mostrou-se essencial para a estabilidade do processo, sendo requerida em menor quantidade a 35ºC.

The influence of the sodium bicarbonate (NaHCO3) on the performance of an anaerobic sequencing batch biofilm reactor treating vinasse at 55 and 35ºC was evaluated. The reactor was filled with polyurethane foam and agitated at 300 rpm. The acclimatization period at 55ºC lasted 50 days, and the reactor was fed with vinasse (COD from 0.3 to 1.0 g/L). A previous enrichment of methanogenic population was provided for the reactor operated at 35ºC, feeding it with an ethanol based substrate (2.5 gCOD/L) during 21 days. Further on, organic loading rates (OLR) ranging from 0.85 to 5.70 gCOD/L.d were applied to the reactor at 55ºC, supplemented with 1.2 to 0.4 gHCO3-/gCOD. At 35ºC the reactor was subjected to OLR ranging from 2.85 to 36.0 gCOD/L.d with 0.4 to 0.2 gHCO3-/gCOD. COD removal ranged from 43 to 78 percent at 55ºC and from 75 to 85 percent at 35ºC. Alkalinity supplementation was found to be essential for process stability, but the amount required was lower at 35ºC.

Roles of ERK and cPLA2α in biphasic regulation of renal proximal Na+-HCO3-transport by angiotensin Ⅱ / 中华肾脏病杂志

Objective To clarify the signaling mechanisms underlying angiotensin Ⅱ biphasic regulation of renal proximal Na+-HCO3- transport. Methods Different concentration Ang Ⅱ to the responses of Na+-HCO3- cotransporter (NBC) activity in isolated proximal tubules, with or without ATR, MAPK, cPLA2α, P450 blockade was compared in wild-type and Ang Ⅱ type 1a receptor (AT1aR)-deficient mice. The phospholipase of ERK was examined by Western blotting. AT1aR mRNA was examined by RT-PCR from kidney proximal tubules. Results (1)In isolated wild-type mouse, renal proximal tubules showed biphasic effects of Ang Ⅱ on NBC activity. Low concentration Ang Ⅱ (10-10 mol/L) increased NBC activity, but high concentration Ang Ⅱ (10-6 mol/L) decreased NBC activity. Olmcsartan (AT1 antagonist) blocked both stimnlatory and inhibitory effects of Ang Ⅱ on NBC activity, but PD98059 (mitogen-activated protein kinase inhibitor) blocked only the stimulatory effect of low concentration Ang Ⅱ ( 10-10 mol/L). (2)In AT1aR-deficient mice, only the stimulatory effect by high concentration of Ang Ⅱ (10-6 mol/L) was observed, which was blocked by olmesartan and PD98059. (3)In wild-type mice, pharmacological blockade of cPLA2 or P450 converted the inhibition effect by high concentration Ang Ⅱ (10-6 mol/L) to the stimulation, which was blocked by olmesanan and PD98059. These results indicated that the extracellular sigual-regulated kinase (ERK) activation via AT1 mediated only the stimulatory effect of Ang Ⅱ, while the cPLA2α/P450 activation via AT1 mediated the inhibitory effect of Ang Ⅱ independently of ERK. The analysis of ERK phosphorylation by Ang Ⅱ also supported a view that the cPLA2α/P450 pathway worked to suppress the ERK activation. Conclusions Ang Ⅱ activates ERK and cPLA2α with different concentration dependency via AT1. The balance between ERK and cPLA2α activities determines the final responses to Ang Ⅱ in intact proximal tubules.

Altered Regulation of type 3 Na+/H+ exchanger, type 1 Na+/HCO3- cotransporter, and Na+,K+-ATPase in the Kidney of Rats with Experimental Rhabdomyolysis

Metabolic acidosis was shown to correlate with deterioration of renal function in patients with rhabdomyolysis. The present study was aimed to investigate whether the changes of type 3 Na+/H+ exchanger (NHE3), type 1 Na+/HCO3- cotransporter (NBC1), and Na+,K+-ATPase alpha1 subunit may play a role in the pathogenesis of metabolic acidosis in glycerol-induced experimental rhabdomyolysis. Male Sprague-Dawley rats were deprived of fluid intake for 24 hours, and then were injected with 50% glycerol in normal saline (10 mL/kg, intramuscularly). At 24 hours after the glycerol injection, rats were sacrificed by decapitation. Control rats were injected with normal saline. The protein expression of NHE3, NBC1 and Na+,K+-ATPase alpha1 subunit was determined in the cortex of the kidney by immunoblotting and immunohistochemistry. Following the treatment of glycerol, creatinine clearance was significantly decreased, and high anion gap metabolic acidosis developed. In the experimental group, the expression of Na+,K+-ATPase alpha1 subunit was significantly decreased in the cortex of the kidney. On the contrary, the expression of NHE3 and NBC1 was significantly increased. Immunohistochemical analyses confirmed the immunoblotting data. In conclusion, the coordinate up-regulation of NHE3 and NBC1 may play an adaptive role against the metabolic acidosis in glycerol-induced rhabdomyolysis.

Regulations of Bicarbonate Ions in Pregnant Rat Kidney / 대한해부학회지

The marked hemodynamic and hormonal changes of normal pregnancy are associated with striking alterations in renal physiology involving structure, dynamics, tubular function, and volume homeostasis. A number of acid-base or electrolyte disorders are associated with decreased or increased HCO3-reabsorption in the renal tubules. The present study was to examine the alterations of expression and distribution of Na+/HCO3-cotransporter (NBC), Na+/H+ exchanger-3 (NHE-3), and carbonic anhydrase I and II (CA I, II) proteins in the kidneys of non-pregnant (NP) and pregnant rats using Western blot analysis and immunohistochemistry. Sprague-Dawley female rats were studied on days 10 (P 10), 12 (P 12), 14 (P 14), 17 (P 17), and 19 (P 19) of pregnancy. Western blot analysis demonstrated that the expression of NBC, ~110 kDa at molecular mass, was increased in pregnant rats, particularly P 12, compared with NP rat. The expression of NHE-3, ~83 kDa at molecular mass, was increased in pregnant rats, particularly P 12 and P 14. The expression of CA I, ~30 kDa at molecular mass, was decreased in pregnant rats, particularly P 14, but, CA II protein, ~30 kDa at molecular mass, was similar NP rat. In immunohistochemistry, strong immunoreactivity of NBC of NP rat was exclusively detected in the basolateral membranes of S1 and S2 segment of proximal tubules whereas not in S3 segment. In pregnant rats, the pattern of cellular labeling of NBC was identical to that of NP rat, but signal intensity was increased, particularly P 12. In NHE-3, strong immunoreactivity was detected in apical membranes and brush borders of S3 segments and moderate in S1 and S2 segments. In pregnant rats, the pattern of cellular labeling was identical to that of NP rat, but the signal intensity was increased, particularly P 12 and P 14. Expression of CA I and II proteins was detected in entire collecting duct. Signal intensity was prominent in type A intercalated cells and moderate in type B intercalated cells. In pregnant rats, the pattern of cellular labeling of CA I and II proteins was identical to that of non-pregnant rat, but the signal intensity of CA I was decreased in cortical collecting duct, particularly P 14 and CA II was identical to that of NP rat. These results suggest that the regulation of NBC and NHE-3 expressions in the proximal tubules and CA I expression in cortical collecting duct may maintain HCO3-concentration during the pregnancy.

Regulations of Bicarbonate Ions in Hypokalemic Rat Kidney / 대한해부학회지

A number of acid-base or electrolyte disorders are associated with decreased or increased HCO3- reabsorption in the renal tubules. There has been a general agreement that potassium depletion induces and maintains metabolic alkalosis in rats. However, many researchers have approached such issue only from functional studies to investigate Na+/H+ exchanger (NHE-3) and Na+/HCO(3-) cotransporter (NBC) activity which closely relates to potassium depletion. In addition the results obtained vary according to their researchers. Thus the present study was employed Western blot analysis and immunohistochemistry together, to examine the alterations of expression and distribution of NHE-3 and NBC-1 with reference to HCO3- reabsorption in the kidneys of rats fed potassium free diets according to the periods. Western blot analysis demonstrated that NHE-3 protein, ~83 kDa at molecular mass, was abundantly expressed in normal group. All potassium-depleted groups showed significantly increased NHE-3 protein compared to normal group. NBC-1 protein, ~110 kDa at molecular mass, was moderately expressed in normal group. All potassium-depleted groups had much higher amounts of the protein than normal group. There was a highly increased amount of NBC-1 protein especially in K-depleted 1 week group. Immunohistochemistry showed positive immunoreactivity of NHE-3 in the apical membranes and brush borders of proximal renal tubule cells. Its reactivity was most prominent in the S3.S1 and S2 had moderate immunoreactivity. Potassium-depleted groups had an identical pattern of cellular labeling of NHE-3 protein compared with that of normal group. However the signal intensity of NHE-3 protein in potassium-depleted groups was much higher than that of normal group. Immunoreactivity of NBC-1 was observed exclusively in the basolateral plasma membranes of proximal tubule cells. There was a strong reactivity in the S1 and S2, whereas S3 did not show any reactivity. Potassium-deprived rats exhibited an identical pattern of cellular labeling of NBC-1 protein compared with that of normal rats. However, the signal intensity of NBC-1 protein was markedly increased in potassium-deprived rats. These results suggest that increased NHE-3 and NBC-1 expression resulted from potassium depletion in the renal proximal tubules, enhances HCO3-reabsorption and consequently maintains metabolic alkalosis.

Rocuronium Injection Pain is Attenuated by NaHCO3 / 대한마취과학회지

BACKGROUND: Rocuronium administration is associated with a severe burning pain during injection. However, the mechanistic cause of the pain has not been well established. The purpose of this study was to determine whether adjusting the pH of the rocuronium with NaHCO3 would ameliorate the pain. METHODS: We examined mixtures using microscope after NaHCO3 was mixed with rocuronium to exam solubility. Sixty of 80 patients scheduled for elective gynecologic surgery were randomly allocated to one of three groups as follows: group 1 (rocuronium only, n = 20), group 2 (rocuronium 50 mg/5 ml mixed with 0.9% NaCl 3 ml, n = 20), group 3 (rocuronium 50 mg/5 ml mixed with NaHCO3 3 ml, n = 20). All patients received 0.6 mg/kg of rocuronium over 10 sec and were asked to assess pain using a visual analogue scale (VAS) followed by injection of propofol 1.5 mg/kg and fentanyl 100 mcg. The onset and duration of rocuronium were measured in three groups. Twitch responses to cumulative incremental doses of rocuronium were measured in another 20 patients, allocated to group A (rocuronium only, n = 10) or group B (rocuronium 50 mg/5 ml mixed with NaHCO3 3 ml, n = 10). RESULTS: Over 24 hours, no precipitation or particles were found after mixing NaHCO3 with rocuronium. The VAS was significantly lower in group 3 (0.5+/-0.9) than in group 1 (5.4+/-3.2) or in group 2 (4.9+/-2.1) (P < 0.05). Eighteen of 20 patients in group 3 had no pain and only 2 had mild pain, but all patients in groups 1 and 2 had mild to severe pain. There were no differences in onset or duration between the three groups and in twitch responses between group A and B. CONCLUSIONS: NaHCO3 mixed with rocuronium attenuates rocuronium injection pain, and there were no problems or complications.

Investigation of increasing efficacy of aterial infusion of NaHCO3 to solid malignant tumors in interventional chemotherapy / 中国临床药理学与治疗学

Aim To observe the effect of interventional therapy of 5-fluorouracil(5-Fu), mitomycin C (MMC) and adriamycin (ADM) on solid malignant turmors in association with aterial infusion of NaHCO3.Methods Patients were randomly divided into two groups.With Seldinger technique,through the femoral atery to tumor atery.The patients in the control group were infused by anticarcinoma agents simply ,and patients in the treatment group were initially infused by NaHCO3,and then by NS 30 ml and anticarcinoma agents seperately. Results Partial remission (PR) in the group treated with NaHCO3 and anticarcinoma agents was significantly higher than in the group treated simply with anticarcinoma agents.Conclusion Aterial infusion of NaHCO3 into malignant tumors can increase the efficacy of ADM,MMC and 5-Fu.

Analysis of causes and management of bronchus-pleural fistula after pneumonectomy of lung cancer / 中国癌症杂志

Purpose: To evaluate the causes and management of bronchial pleural fistula after pneumonectomy. Methods: Retrqspective analysis for Bronchus-pleural fistula( BPF) of 16 cases after pneumonectomy of 820 cases of lung cancer in our hospital. BPF occurred in right peumonectomy( 13/320) is more than in left pneumonectomy(3/500) . BPF occurred in the positive stump of bronchus ( 10/41) more than in negative stump of bronchus (6/779) ; BPF occurred in preoperative chemotherapy cases( 5/110) more than in non-preoperative chemotherapy cases( 11/710), No BPF occurred in the 70 cases in which the bronchial stump was covered by autogenous tissue. The management principle in early stage is thoracocentesis and wash with antibiotics; after identification of the infection in thoracic cavity or BPF, closed drainage for thorax was done. If the results of drainage are not very good, open drainage is necessary. Results: 2 cases were discharged with completely healing, (the cavity of 1 case was washed again and again with 5% NaHC03and urokinase , another case was operated again to cover the BPF using muscle flaps 3 days after the first operation), 8 cases were discharged with closed drainage, 4 cases were discharged with open drainage changing the wound covering every day, the BPF did not heal for a long time after open drainage in 1 case, 1 case died of function failure of body organs. Conclusions: BPF is related to management of the bronchial stump and radical resection for tumor, It is a useful method to cover the bronchial stump with autogenous tissues to decrease BPF's, especially for right pneumonectomy and preoperative chemotherapy or radiotherapy cases. The management principle of BPF is thoracocentesis for early cases, especially washing with antibiotics and 5% NaHC03and urokinase repeatedly, closed drainage when necessary is also a good method for curing empyema and BPF.

Studies on the effect of NaHCO3 intake on intracellular pH and PCr concentration during exercise by 31P NMR / 体力科学

To evaluate the changes in muscle energetics following NaHCO<SUB>3</SUB> intake, we measured the phosphorus-31 nuclear magnetic resonance (<SUP>31</SUP>P NMR) spectra of human muscle <I>in vivo</I> during exercise. Seven male subjects performed two trials, a NaHCO<SUB>3</SUB> (Alka, Tr.) and a NaCI trial (Cont. Tr.), on two occasions. <SUP>31</SUP>P NMR spectra were obtained serially during leg-elevating exercises. Before and during exercise, the intracellular phosphocreatine (PCr), inorganic phosphate (Pi) and pH were determined from the NMR spectra. The decrease of intracellular pH during exercise showed a tendency to be inhibited by NaHCO<SUB>3</SUB> intake, and the intracellular pH at the end of the exercise was 6.69 for Alka. Tr, and 6.51 for Cont. Tr. The decline of the PCr/ (PCr+Pi) ratio during exercise was not influenced by NaHCO<SUB>3</SUB> intake. The PCr/ (PCr+Pi) ratio was related exponentially to the intracellular pH. A remarkable decline of PCr/ (PCr+Pi) ratio occurred until the intracellular pH fell to about 6.7, but did not decrease below that. It was suggested that the intake of NaHCO<SUB>3</SUB> could decrease the rate of fall in the intracellular pH during exercise, and that the PCr store could be influenced by the intracellular pH when the pH was above 6.7, but not below that level.