Effect of ammonium on the expression of osmosensitive genes in Madin–Darby canine kidney cells

The cells of the kidney medulla are exposed routinely to high extracellular concentrations of various solutes including NaCl, urea and ammonium (NH₄⁺). Although it is well established that the expression of a variety of osmosensitive genes and proteins, which confer cytoprotection on renal medullary cells, is induced by high NaCl concentrations, the role of NH₄⁺ in these cellular responses is unclear. This study thus addressed the effect of NH₄⁺ on the expression of the betaine/GABA transporter (BGT-1), the sodium/ myo -inositol cotransporter (SMIT), aldose reductase (AR), and heat shock protein 70 (HSP70) in Madin–Darby canine kidney (MDCK) cells, using Northern and Western blot analyses and enzyme-linked immunosorbent assay (ELISA). The incidence of apoptosis was monitored by determining caspase-3 activity and annexin V binding. Addition of NH₄Cl (50 m m ; total osmolality 400 mosmol (kg H₂O)⁻¹ to the medium was more effective than equiosmolar NaCl in increasing BGT-1 and HSP70 mRNA abundance, but less effective in enhancing BGT-1 and HSP70 expression at the protein level. Qualitatively similar results were obtained for SMIT and AR mRNAs. Exposure to both isotonic and hypertonic, NH₄Cl-containing medium enhanced apoptosis compared with equiosmolar, NaCl-containing media. These results suggest that, in addition to NaCl, NH₄Cl may play a role in regulating the intracellular accumulation of organic osmolytes, the abundance of HSP70 and cell turnover in the renal medulla in vivo .     

Compatible osmolytes modulate the response of porcine endothelial cells to hypertonicity and protect them from apoptosis

Porcine pulmonary arterial endothelial cells accumulated myo -inositol and taurine, as well as betaine, during adaptation to hypertonic stress. The cells grew and maintained their normal morphology during culture in hypertonic (0.5 osmol (kg H₂O)⁻¹) medium that contained osmolytes such as betaine, myo -inositol or taurine at concentrations close to reported physiological values. The cells did not grow well in hypertonic medium depleted of potential compatible osmolytes. After a few days, cell density decreased by about 50 % and many cells rounded up and detached from the plates, their nuclei showing clear apoptotic morphology. The caspase-3 activity of the cells also increased dramatically under these conditions, but remained negligibly low when betaine and myo -inositol were added to the medium. Addition of betaine and myo -inositol to hypertonic medium depleted of compatible osmolytes increased the number of colonies remaining after 12 days of culture; with each solute at 30–100 μmol l⁻¹ the number increased about sixfold. In the absence of compatible osmolytes, increased mRNA levels and corresponding activities of betaine/γ-aminobutyric acid transporter (BGT1) and sodium/ myo -inositol transporter (SMIT) induced by hypertonicity remained high after 72 h incubation, whereas they were down regulated in the presence of betaine and myo -inositol. Similarly, the down regulation of the amino acid System A transporter (ATA2) was markedly slowed in the absence of compatible osmolytes. We conclude that these compatible osmolytes at concentrations close to physiological values enable the endothelial cells to adapt to hypertonic stress, protecting them from apoptosis, and also modulate the adaptation process.     

Differential expression of heat shock protein 27 and 70 in renal papillary collecting duct and interstitial cells – implications for urea resistance

The adaptation of renal medullary cells to their hyperosmotic environment involves the accumulation of compatible organic osmolytes and the enhanced synthesis of heat shock proteins (HSP) 27 and 70. While the mechanisms leading to osmolyte accumulation are similar in papillary collecting duct (PCD) and papillary interstitial (PI) cells, the present data demonstrate that HSP27 and HSP70 are expressed differentially in these cells both in vivo and in vitro . HSP70 is abundant in PCD, but not expressed in PI cells in the papilla in situ , while HSP27 is expressed in both PCD and PI cells. These observations could be reproduced by non-permeant solutes in cultured cells. Osmotic stress strongly induced HSP70 in MDCK cells (as a model for PCD cells), but not in PI cells, while HSP27 was constitutively expressed in MDCK cells and was up-regulated in PI cells. Since prior hypertonic stress (NaCl addition) protects MDCK against subsequent exposure to high urea concentrations, this effect was also assessed in PI cells. In both cell lines, hypertonic pretreatment prior to urea exposure (400 m m ) strongly attenuated caspase-3 activation. Inhibition of HSP27 expression by antisense transfection diminished the protective effect of hypertonic preconditioning in PI cells, while attenuation of HSP70 expression in MDCK cells diminished the protective effect of hypertonic preconditioning in these cells. These observations indicate that PCD and PI cells employ cell-specific mechanisms for protection against high urea concentrations as present in the renal papilla during antidiuresis.     

Intrathoracic pressure changes and cardiovascular effects induced by nCPAP and nBiPAP in sleep apnoea patients

SUMMARY  The effect of nasal continuous positive airway pressure (nCPAP) and nasal bi-level positive airway pressure (nBiPAP) on intrathoracic pressure and haemodynamics during wakefulness was studied in a group of nine patients with severe sleep apnoea. No patient took cardiovascular medication.
Patients were studied with a Swan Ganz catheter, an arterial line and an oesophageal balloon. nCPAP and nBiPAP were applied in the following pressure sequence: 5, 10 and 15 cm H₂O of CPAP and 10/5 and 15/10 cm H₂O of nBiPAP. Measurements were made at the end of a 5-min period at each pressure level. Intrathoracic pressure was noted to increase to a level of approximately 50% of the pressure delivered at the mask. At a CPAP of 10 cm H₂O and above, as well as at BiPAP of 10/5 or higher, there was a decrease in cardiac output (CO) and cardiac index (CI). CI fell below the normal value in two of the patients. Transmural pulmonary artery pressure (PPA_tm) decreased at a CPAP of 15 cm H₂O and at both BiPAP levels. Transmural right atrial pressure (PRA_tm) decreased at both BiPAP levels. There were no differences in CO, CI, PPA_tm and PRA_tm between nCPAP and nBiPAP at equal inspiratory pressures. SaO₂ increased during BiPAP 15/10 cm H₂O, whereas heart rate and arterial blood pressure did not change significantly. The data presented here are consistent with the literature on positive end-expiratory pressure (PEEP) applied via intratracheal tube and are likely to be due to a reduced venous return. It is concluded that nasally applied positive pressure may have acute negative effects on cardiac function in patients with sleep apnoea.     

Enhanced susceptibility to cytotoxic T lymphocytes without increase of MHC class I antigen expression after conditional overexpression of heat shock protein 70 in target cells

Antigenic peptides have been found associated with heat shock proteins (HSP) including cytoplasmic HSP70 and heat shock cognate protein 70 as well as the endoplasmic reticulum-resident glucose-regulated protein 94. Recently, HSP70 transfection has been reported to increase MHC class I cell surface expression and antigen presentation on mouse melanoma B16 cells (Wells et al., Int. Immunol. 1998. 10: 609). To analyze the effect of HSP70 on MHC class I cell surface expression and lysability of target cells we transfected a human melanoma cell line with the rat Hsp70-1 gene using the Tet-On system for conditional overexpression of HSP70. Induction of HSP70 did not increase cell surface expression of HLA class I molecules in general or individual HLA-A and B antigens in particular. Nonetheless, induction of HSP70 enhanced susceptibility of these cells to lysis by allospecific CTL. The same effect was observed using an HLA-A2-restricted tyrosinase-specific CTL clone after pulsing the tyrosinase-negative target cells with the specific peptide. Thus, HSP70 induction can increase killing by CTL without affecting MHC class I cell surface expression or antigen processing. This effect of HSP70 appears to be different from the commonly found protection exerted by HSP70 against stress like heat shock, and might be mediated by improving CTL-induced apoptosis.     

SINUSOIDAL STENOSIS AS THE CAUSE OF PORTAL HYPERTENSION IN CHOLINE DEFICIENT DIET INDUCED FATTY CIRRHOSIS OF THE RAT LIVER

The anatomical lesion of the liver is evaluated as being responsible for development of portal hypertension through measurement of portal vein pressures, hemodynamical analysis of Isolated perfused livers, diameter measurement of intrahepatic vascular trees, and histological as well as his-tometrical examinations of the liver in rats fed with choline deficient diet. The mean portal vein pressure was elevated from the normal level, 132 mm H₂O to 175 mm H₂O In fatty liver, to 179 mm H₂O In fatty liver with fibrosis, and to 218 mm H,0 in fatty cirrhosis of liver. The hepatic blood flow of the isolated perfused rat liver definitely decreased from the normal level, 3.0 ml/g/min to 1.2 ml/g/min in fatty liver, 0.4 ml/g/min in fatty liver with fibrosis and 0.7 ml/g/min in fatty cirrhosis of liver under the condition of Ht 33.5% and perfusion pressure 135 mm H₂O. There was no abnormality in the pre-and post sinusoidal vessel, but the space of sinusoid was markedly diminished, reciprocally, to the enlargement of the hepatic cells. An intimate correlationship existed between portal hypertension and reduction of sinusoidal space. Connective tissue proliferation did not have much to do with the increase in hepatic vascular resistance. The present experiment suggested that reduction of sinusoidal space was the anatomical lesion related to development of portal hypertension in choline deficient diet induced fatty liver as well as fatty cirrhosis of the liver (sinusoidal hypertension).     

Histaminergic Regulation of NK-Cells: Protection Against Monocyte-Induced Apoptosis

Human natural killer (NK) cells (with CD3⁻/56⁺ phenotype) acquired features characteristic of apoptosis after incubation with autologous monocytes, as revealed by apoptotic nuclear morphology and degradation of DNA into oligonucleosomal fragments. The monocyte-induced apoptosis in NK-cells was prevented by the biogenic amine histamine at concentrations exceeding 0.1 μ M . The protective effect of histamine was blocked by the H₂-receptor (H₂R) antagonist ranitidine but not by AH202399 A, a chemical control to ranitidine devoid of H₂R affinity. It is concluded that histaminergic mechanisms may serve to protect NK cells from damage inflicted by products of the oxidative metabolism of monocytes.     

Expression of stress proteins HSP 72 & HSP 32 in response to endotoxemia

Pretreatment with heat decreases mortality and acute lung injury in the rat septic shock model, presumably by the production of heat shock proteins (HSP). However, endotoxin, a severe cell stresser, has not been shown to induce HSP 70. We investigated the effects of severe endotoxemia on the expression of specific protective stress proteins, including HSP 72 (inducible HSP 70), HSP 32 (heme oxygenase-1), and HSP 90. Fifteen rats received intravenously either 3 mg/kg of endotoxin (E. coli O127:B8 lipopolysaccharide, LPS) (n=9) or saline (n=6). Two hr later the spleen was removed and splenocytes were separated into three groups and analyzed for specific HSP by Western blot. In Group 1, both endotoxin-treated and saline-treated splenocytes were incubated for 3 hr at 37 degrees C. In Group 2, the splenocytes were washed twice, then heat shocked for 30 min at 42 degrees C and subsequently incubated for 2.5 hr at 37 degrees C. In Group 3, splenocytes were washed twice, then incubated for 3.0 hr at 37 degrees C. HSP 90 & HSP 70c (constitutive) were present in all groups. Consistent with observations by others, HSP 72 was not induced in Group 1. HSP 72 was induced in both the saline-treated and endotoxin-treated splenocytes after heating (Group 2). However, in the absence of heat stress, HSP 72 was present in endotoxin-treated but not in saline-treated splenocytes after incubation (Group 3). Conversely, HSP 32, while present in Group 1 splenocytes, was not detected in the endotoxin-treated splenocytes of Group 2 and Group 3, but was present in the saline-treated cells. In conclusion, endotoxemic shock results in induction of HSP 72 and depletion of HSP 32, but only after the cells have been washed and further incubated.     

Expression and localization of heat shock proteins in rat basophilic leukemia cells: differential modulation by degranulation,thermal or oxidative stress

BACKGROUND: Rat basophilic leukemia (RBL-2H3) cells are well characterized in terms of morphological and biochemical changes upon activation, and have been extensively used as a model system for studying the mechanisms of the immediate hypersensitivity reaction. To investigate whether overexpression of heat shock/stress proteins (HSP) is involved in the mast cell-dependent reactivity, we examined the adaptive responses of RBL-2H3 cells to classical stress conditions such as heat shock or oxidative injury produced by an aqueous extract of tobacco smoke. METHODS: HSP were determined by flow cytometry and immunocytochemistry. Degranulation was confirmed as the release of beta-hexosaminidase, determined spectrophotometrically, and by electron microscopy experiments. RESULTS: We found that RBL-2H3 cells respond to heat shock or oxidative injury by the synthesis of both the inducible 72 kDa HSP (Hsp70), and the oxidation-specific 32 kDa heme oxygenase (HO)-1. Heat shock induced mainly Hsp70 in a cell growth-dependent manner, whereas oxidative stress induced mainly HO-1 in a cell growth-independent manner. However, heat shock or oxidative stress had no significant effects on degranulation. CONCLUSION: Stress-mediated synthesis of HSP was not associated with RBL-2H3 degranulation and likewise, degranulation did not induce HSP.     

Induction of amyloid precursor protein mRNA after heat shock in cultured human lymphoblastoid cells.

In an attempt to examine a possible relationship between heat shock stress and an induction of amyloid precursor protein, cultured lymphoblastoid cells established from 12 human subjects were treated with heat shock at 42 degrees C for 30 min. The levels of mRNA for amyloid precursor protein (APP), heat shock protein (HSP) 70, and actin were examined by Northern blot at 1, 3, 8, 24, and 48 h after the heat shock treatment. HSP70 mRNA was induced at 1 and 3 h, and became undetectable again by 8 h. APP mRNA was also induced at 3 and 8 h, and recovered to the steady level by 48 h. No induction was observed in actin mRNA. These results indicate that APP mRNA is induced by heat shock treatment after the induction of HSP70 mRNA, suggesting a role of heat shock response in an induction of APP.     

Heat shock proteins HSP25, HSP60, HSP72, HSP73 in isoosmotic cortex and hyperosmotic medulla of rat kidney

The distribution of heat shock proteins (HSP) HSP60, HSP73, HSP72 and HSP25 in the isoosmotic cortex and the hyperosmotic medulla of the rat kidney was investigated using Western blot analysis and immunohistochemistry. HSP73 was homogeneously distributed throughout the whole kidney. The level of HSP60 was high in the renal cortex and low in the medulla. HSP25 and HSP72 were present in large amounts in the medulla. Only low levels of HSP25 and almost undetectable amounts of HSP72 were found in the cortex. HSP25 exists in one nonphosphorylated and several phosphorylated isoforms. Western blot analysis preceded by isoelectric focussing showed that HSP25 predominates in its nonphosphorylated form in the outer medulla but in its phosphorylated form in cortex and inner medulla. Although this intrarenal distribution pattern was not changed during prolonged anaesthesia (thiobutabarbital sodium), a shift from the nonphosphorylated to the phosphorylated isoforms of HSP25 occurred in the medulla. The characteristic intrarenal distribution of the constitutively expressed HSPs (HSP73, HSP60, HSP25) may reflect different states of metabolic activity in the isoosmotic (cortex) and hyperosmotic (medulla) zones of the kidney. The high content of inducible HSP72 in the medulla most likely is a consequence of the osmotic stress imposed upon the cells by the high urea and salt concentrations in the hyperosmotic medullary environment.     

Insulin hypersensitivity in mice lacking the V1b vasopressin receptor

We have reported that [Arg⁸]-vasopressin-stimulated insulin release is blunted in islet cells isolated from V1b receptor-deficient ( V1bR ^−/−) mice. In this study, we used V1bR ^−/− mice to examine the physiological role of the V1b receptor in regulating blood glucose levels in vivo , and we found that the fasting plasma glucose, insulin and glucagon levels were lower in V1bR ^−/− mice than in wild-type ( V1bR ^+/+) mice. Next, we evaluated glucose tolerance by performing an intraperitoneal glucose tolerance test (GTT). The plasma glucose and insulin levels during the GTT were lower in V1bR ^−/− mice than in V1bR ^+/+ mice. An insulin tolerance test (ITT) revealed that, after insulin administration, plasma glucose levels were lower in V1bR ^−/− mice than in V1bR ^+/+ mice. In addition, a hyperinsulinaemic–euglycaemic clamp study showed that the glucose infusion rate was increased in V1bR ^−/− mice, indicating that insulin sensitivity was enhanced at the in vivo level in V1bR ^−/− mice. Furthermore, we found that the V1b receptor was expressed in white adipose tissue and that insulin-stimulated phosphorylation of Akt as an important signaling molecule was increased in adipocytes isolated from V1bR ^−/− mice. Thus, the blockade of the V1b receptor could result, at least in part, in enhanced insulin sensitivity by altering insulin signalling in adipocytes.     

An important role of the heat shock response in infected cells for replication of baculoviruses

Baculoviruses serve as a stress factor that can activate both death-inducing and cytoprotective pathways in infected cells. In this report, induction of heat shock proteins (HSPs) of the 70-kDa family (HSP/HSC70) in Sf-9 cells after infection with AcMNPV was monitored by Western blot analysis. Two-dimensional electrophoresis in polyacrylamide gel revealed changes in the cellular pattern of HSP/HSC70s and synthesis of a new member of the HSP/HSC70 family in the infected cells. Although infection with AcMNPV moderately increased the HSP/HSC70 content in cells under standard conditions, the infection potentiated the response to heat shock boosting the HSP/HSC70s content in infected cells several-fold in comparison with uninfected cells. Addition of KNK437, a known inhibitor of inducible HSPs, decreased the rate of viral DNA synthesis in infected cells more than one order of magnitude and markedly suppressed the release of budded viruses indicating the importance of the heat shock response for baculovirus replication.     

热休克蛋白抑制慢性心理应激引起的焦虑样行为增加

目的：采用HSFI基因敲除小鼠模型探讨热休克蛋白（heat shock protein，HSP）对慢性心理应激引起的焦虑样行为增加的抑制作用。方法：将热休克因子1（heat shock factor 1）基因野生型小鼠（HSFI＋／＋）和热休克因子1基因敲除小鼠（HSF1-／-）暴露于2个月的慢性心理应激，部分小鼠在暴露于慢性心理应激前给予热休克预处理，2个月后采用高架十字迷宫和旷场实验检测各组小鼠焦虑样行为，Western blots检测和比较HSP72，HSC70表达，采用多因素析因设计方差分析对各组小鼠焦虑样行为进行比较。结果：慢性心理应激引起焦虑样行为增加．HSF1基因敲除导致焦虑样行为增加更加明显。热休克预处理使野生型小鼠海马组织中HSP70。HSC70明显升高，并明显减轻了心理应激所致的焦虑样行为的增加。HSF1基因敲除废除了热休克所致的HSP诱导表达及其对心理应激所致焦虑样行为增加的抑制作用。结论：诱导型HSP在抑制心理应激引起的焦虑样行为增加中起着重要的作用。     

Erythroid lineage-specific expression and inducibility of the major heat shock protein HSP70 during avian embryogenesis

We have studied the expression of the major heat shock protein HSP70 during maturation of avian erythroid cells. Primitive and definitive erythroid cells were isolated from staged day 3-8 chicken embryos, and the levels of HSP70 mRNA and protein synthesis were examined. The highest levels of HSP70 are detected in polychromatic cells of the day 3-4 primitive erythroid cell. After the initial burst of HSP70 expression the levels of HSP70 mRNA and protein synthesis decline. Although HSP70 is constitutively expressed, neither HSP70 synthesis nor HSP70 mRNA levels were heat shock inducible in primitive red cells. In contrast, definitive red cells respond to heat shock by a 10- to 20-fold increase in HSP70 protein synthesis with little change in HSP70 mRNA levels. These studies reveal that HSP70 expression in erythroid cells is lineage specific, that the levels of HSP70 mRNA are not induced by heat shock, and finally, that the increased expression of HSP70 in definitive cells is due to increased translatability of HSP70 mRNA.     

Localization and function of histamine H3 receptor in the nasal mucosa

Background Histamine is an important chemical mediator of allergic rhinitis (AR). Histamine H₃ receptors (H₃R) are located on cholinergic and NANC neurons of the myenteric plexus, and activation of H₃R regulates gastric acid secretion. However, little is known about the localization and function of H₃R in the upper airway.
Objective The objective of this study was to examine the localization and possible function of H₃R in the nasal mucosa.
Methods We extracted total RNA from the inferior turbinate mucosa of patients with AR. H₃R mRNA and β-actin mRNA were amplified by RT-PCR. We used immunohistochemistry to examine localization of H₃R protein in the inferior turbinate mucosa excised during clinically indicated surgery. We used alcian blue/periodic acid-shiff staining to examine the effects of the H₃R agonist (R)-α-methylhistamine and the H₃R antagonist thioperamide on secretion from rat submucosal glands.
Results H₃R protein was expressed around submucosal gland cells. Thioperamide induced degranulation in the submucosal gland in the nasal septum.
Conclusion The present results suggest that H₃R is localized mainly around submucosal glands, and that H₃R plays an important role in the secretion of submucosal glands in the nose.     

Expressions of heat shock proteins under heat-stress and interferon-treatment: An in vitro study on osteosarcoma

This study examined expressions of heat shock proteins (HSP) under heat-stress and/or interferon (IFN)-, ß, or γ treatment using a human osteosarcoma cell line, NY cells. IFN alone as well as heat-stress at 43–45°C for 60 min suppressed cell growth, and their combination resulted in synergistic suppression. When primary heat stress was given, cells acquired thermotolerance at the second heat-stress. Autoradiography revealed that HSP70 was mainly induced in the cells under heat-stress. In Western blot analysis, heat (44°C)-induced HSP70 was suppressed by IFN treatment. This suggests that synergistic suppression effects of the combination treatment on osteosarcoma cells would be attributable to the suppression of HSP70 expressions. Optimum combination of heat-stress and IFN is expected to increase the cure rate in osteosarcoma treatment.     

Proteasome inhibitors differentially affect heat shock protein response in cancer cells

The heat shock proteins (HSPs) are molecular chaperones that are emerging as biochemical regulators of cell growth, apoptosis, protein homeostasis and intracellular targeting of peptides. The immunological function of the HSPs are imparted by tissue specific peptides associated with the HSPs and as such autologous cancer derived HSP-peptide complexes are unique therapeutic agents. Since a majority of the intracellular peptides are generated by the proteasome, we examined the consequence of abrogation of proteasome function by proteasome inhibitors (PIs) such as Lactacystin, MG-132 and LLM on the growth and induction profile of HSP70 and gp96 using hematopoietic, lymphoid, and epithelial derived cancer cell lines. The effect on growth was measured by the XTT assay and induction of the heat shock proteins by western blot analyses using HSP70 and gp96 specific antibodies. Of the PIs tested, cancer cells, were most sensitive to MG-132 and least sensitive to LLM. MG-132 also showed a 10-fold differential sensitivity between estrogen receptor positive, (ER+) MCF-7 cells and negative cells, (ER-) MDA-MB-231. Induction of heat shock proteins, gp96 and HSP70 was, however, noted in response to LLM. Since LLM exhibited minimal cytotoxic effect, metabolic stress that results in induction of HSPs may not be translated in cell growth inhibition and that there may exist a cell-type specific phenomenon in the HSP response to PI mediated metabolic stress.