Expression of stress proteins (HSP-70 and HSP-90) in the rabbit urinary bladder subjected to partial outlet obstruction

Partial obstruction of the rabbit bladder outlet induces a rapid hypertrophy characterized by increased bladder mass, increased smooth muscle content, and increased collagen deposition. In addition, partial outlet obstruction induces decreased contractile responses to both field stimulation and postsynaptic receptor stimulation. Although the morphological and contractile responses to partial outlet obstruction have been well characterized, there is little information on the cellular and molecular mechanisms of these changes. In a previous study, we demonstrated that one of the earliest genes to be expressed following partial outlet obstruction in rabbits was the gene expressing stress protein-70 (HSP-70). In order to further define the genetic and molecular basis of these responses, the expression of stress gene products HSP-70 and HSP-90 in rabbit urinary bladder subjected to partial outlet obstruction has been quantitatively evaluated by Western blot coupled with laser densitometry using anti-HSP-70 and-90 monoclonal antibodies. The data show that stress gene products HSP-70 and HSP-90 are constitutively expressed in control rabbit bladder tissue and transiently increased following partial outlet obstruction. Increased content of HSP-70 was detected at 6 hr after obstruction and reached a maximum (2.7-fold over the control level) at 24 hr. Increased HSP-90 was also detected at 6 hr but reached a maximum (4.5-fold over the control level) at 12 hr. By 7 day post-obstruction, the content of these two proteins returned to the control levels. This study suggests that alterations of stress gene expression resulting in increased HSP-70 and 90 may play an important role in the response of the bladder to partial outlet obstruction.     

Heat shock proteins of chicken lens

The presence of heat shock proteins HSP-40, HSP-70, and HSc-70 in adult and embryonic chicken lenses were determined. The epithelium, cortex, and nucleus of adult chicken lens were separated and tested for the presence of heat shock proteins (hsps) by western blot, using specific antibodies for HSP-40, HSP-70, and HSc-70. Water soluble (WSF) and water insoluble fractions (WIF) of embryonic chicken lenses were isolated and tested for the presence of HSP-40, HSP-70, and HSc-70 by immunoblot. Embryonic chicken lens sections were also analyzed for the presence of heat shock proteins by immunofluorescence technique. Data obtained from these experiments revealed that HSP-40, HSP-70, and HSc-70 are present in all areas of both adult and embryonic chicken lens. Presence of hsps protein in the deep cortex and nucleus is intriguing as no detectable metabolic activities are reported in this area. However it can be proposed that hsps HSP-40, HSP-70, and HSc-70 can interact with protein of these areas and protect them from stress induced denaturation.     

Gene expression analysis following hypoxia–reoxygenation in rat gastric epithelial cells using a high-density oligonucleotide array

Abstract

Recent investigations have demonstrated that the signaling of hypoxia–re-oxygenation is a major contributing pathway leading to gastric mucosal injury induced by stress, non-steroidal anti-inflammatory drugs, and Helicobacter pylori. The aim of the present study was to perform a gene expression analysis on the gastric mucosal cellular response to hypoxia–reoxygenation using a high-density oligonucleotide array. Cells were subjected to hypoxia with 95% N₂ and 5% CO₂ at 37°C for 2 h. Reoxygenation was initiated by placing the cells in an environment of normoxia for 2 h. Total RNA was extracted, and differences in gene expression profiles between the normoxia and hypoxia–reoxygenation groups were investigated using a GeneChip of Rat Toxicology U34 array (Affymetrix). Hypoxia–reoxygenation up-regulated the stress-related genes (heat shock protein-70 [HSP-70], catalase). The enhanced expression of HSP-70 was confirmed by Western blot analysis. In conclusion, these results suggest that up-regulation of the HSP-70 gene after reoxygenation may play a role in maintaining cell survival and supporting cell function as a molecular chaperone.     

The effect of partial outlet obstruction on prostaglandin generation in the rabbit urinary bladder

Partial outlet obstruction of the urinary bladder has been demonstrated to induce specific dysfunctions in cellular and sub-cellular membrane structures within the bladder's smooth muscle and mucosal compartments. Recent studies have linked these membrane dysfunctions to alterations in phospholipid metabolism leading to mobilization of free arachidonic acid, the precursor for synthesis of prostaglandins (PG). The purpose of this study was to determine if partial outlet obstruction of the urinary bladder induces changes in the capacity of bladder smooth muscle and mucosa to generate PG. PG were isolated from control and partially obstructed urinary bladder smooth muscle and mucosa of male New Zealand White (NZW) rabbits. PG concentrations (PGE2, PGF2alpha and PGI2, as its stable metabolite 6-keto-PGF1alpha) were determined after 30 minute incubations using enzyme-linked immunoassay (ELISA) kits. In both control and obstructed rabbit urinary bladders, PG generation was significantly higher in isolated mucosa than muscle tissues. A significantly higher concentration of PGF2alpha, and 6-keto-PGF1alpha was measured in obstructed muscle tissue relative to controls. The concentration of 6-keto-PGF1alpha was also significantly higher than the concentrations measured for PGE2 and PGF2alpha in both control and obstructed smooth muscle samples. The generation of PGE2 was significantly higher in rabbit urinary bladder mucosa than either PGF2alpha or 6-keto-PGF1alpha in both control and obstructed samples. The capacity of obstructed mucosal tissue to generate 6-keto-PGF1alpha was significantly higher than control tissue, while no significant differences in PGE or PGF2alpha generation were noted. These data suggest obstruction of the urinary bladder induce specific elevations in PG in both smooth muscle and mucosal tissues.     

Gene expression analysis following hypoxia-reoxygenation in rat gastric epithelial cells using a high-density oligonucleotide array

Recent investigations have demonstrated that the signaling of hypoxia-re-oxygenation is a major contributing pathway leading to gastric mucosal injury induced by stress, non-steroidal anti-inflammatory drugs, and Helicobacter pylori. The aim of the present study was to perform a gene expression analysis on the gastric mucosal cellular response to hypoxia-reoxygenation using a high-density oligonucleotide array. Cells were subjected to hypoxia with 95% N(2) and 5% CO(2) at 37 degrees C for 2 h. Reoxygenation was initiated by placing the cells in an environment of normoxia for 2 h. Total RNA was extracted, and differences in gene expression profiles between the normoxia and hypoxia-reoxygenation groups were investigated using a GeneChip of Rat Toxicology U34 array (Affymetrix). Hypoxia-reoxygenation up-regulated the stress-related genes (heat shock protein-70 [HSP-70], catalase). The enhanced expression of HSP-70 was confirmed by Western blot analysis. In conclusion, these results suggest that up-regulation of the HSP-70 gene after reoxygenation may play a role in maintaining cell survival and supporting cell function as a molecular chaperone.     

Over-expression of hsp-70 inhibits bacterial lipopolysaccharide-induced production of cytokines in human monocyte-derived macrophages

Cytokines released from monocytes and macrophages are major mediators of inflammation. Heat shock significantly inhibits cytokine production from these cells. To investigate whether this inhibitory effect was mediated by heat-shock proteins (HSP), we transfected human peripheral blood monocyte-derived macrophages (MDM) with HSP-70 cDNA and examined Brucella melitensis lipopolysaccharide (LPS)-induced cytokine production in transfected cells. Over-expression of HSP-70 protein in the gene-transfected MDM had no effect on cytokine synthesis unless LPS was added. LPS-induced increases in production of tumour necrosis factor alpha (TNF-alpha), interleukin 1beta (IL-1beta), IL-10 and IL-12 were significantly inhibited by the over-expression of HSP-70. However, over-expression of HSP-70 did not block LPS-induced increase in IL-6 synthesis. To further confirm these results, an antisense HSP-70 DNA oligomer was used to block HSP-70 synthesis. The inhibitory effect of HSP-70 on LPS-induced cytokine production in gene- transfected cells was completely reversed after treatment of cells with 5 microM antisense HSP-70. The same concentration of antisense HSP-70 also partially reversed heat-shock-induced inhibition of LPS-stimulated cytokine production. These results suggest that HSP-70 is involved in the regulation of LPS-induced cytokine production and that this family of proteins plays a role in mitigating adverse effects of endotoxin during infection or other pathological stresses.     

The effect of vaccination with the lysate of heat-shocked tumor cells on nitric oxide production in BALB/c mice with fibrosarcoma tumor

The aim of this study was to investigate the effect of heat shock protein-70 (HSP-70) on splenocyte proliferation and nitric oxide (NO) production in the BALB/c mice fibrosarcoma tumor model. To do so, HSP-70 was induced in the lysate of heat-shocked tumor cells and WEHI-164 cells (mouse fibrosarcoma cell line) were injected subcutaneously into the right flank of inbred BALB/c mice to establish a tumor model. Three animal bearing tumor groups were applied: the test group; vaccinated with HSP-70 enriched tumor lysate; control group I, vaccinated with tumor lysate only; and control group II, which received PBS. Using immunoblot analysis, an increase of HSP-70 expression was detected in the lysate of heat-shocked cells in comparison with non-heat-shocked cells. The effect of the test lysate on NO production was measured both in vitro and in vivo in the peritoneal macrophages and splenocytes of tumor bearing mice, respectively. The result showed a significant increase in NO production both in vitro by peritoneal macrophages and in vivo after immunization with HSP-70 enriched tumor lysate. In addition, tumor growth was significantly postponed and the proliferation of splenocytes was increased in the test group. Our results indicate that the lysate of heat-shocked tumor cells was more potent than that of non-heat-shocked tumor cells in inducing anti-tumor immunity. Since production of NO by HSP-activated antigen presenting cells (APCs) is likely to affect innate immunity and tumor growth, the probable mechanism of postponing tumor growth would be NO production by innate immune cells. These findings provide a useful therapeutic model for developing novel approaches to cancer treatments.     

MicroRNA-1 Participates in Nitric Oxide-Induced Apoptotic Insults to MC3T3-E1 Cells by Targeting Heat-Shock Protein-70

Our previous studies showed that nitric oxide (NO) could induce osteoblast apoptosis. MicroRNA-1 (miR-1), a skeletal- and cardiac muscle-specific small non-coding RNA, contributes to the regulation of multiple cell activities. In this study, we evaluated the roles of miR-1 in NO-induced insults to osteoblasts and the possible mechanisms. Exposure of mouse MC3T3-E1 cells to sodium nitroprusside (SNP) increased amounts of cellular NO and intracellular reactive oxygen species. Sequentially, SNP decreased cell survival but induced caspase-3 activation, DNA fragmentation, and cell apoptosis. In parallel, treatment with SNP induced miR-1 expression in a time-dependent manner. Application of miR-1 antisense inhibitors to osteoblasts caused significant inhibition of SNP-induced miR-1 expression. Knocking down miR-1 concurrently attenuated SNP-induced alterations in cell morphology and survival. Consecutively, SNP time-dependently inhibited heat-shock protein (HSP)-70 messenger (m)RNA and protein expressions. A bioinformatic search predicted the existence of miR-1-specific binding elements in the 3''-untranslational region of HSP-70 mRNA. Downregulation of miR-1 expression simultaneously lessened SNP-induced inhibition of HSP-70 mRNA and protein expressions. Consequently, SNP-induced modifications in the mitochondrial membrane potential, caspase-3 activation, DNA fragmentation, and apoptotic insults were significantly alleviated by miR-1 antisense inhibitors. Therefore, this study showed that miR-1 participates in NO-induced apoptotic insults through targeting HSP-70 gene expression.     

Metabolic studies on rabbit bladder smooth muscle and mucosa

Recent studies indicate that the mucosa of the urinary bladder may play a major role in the maintenance of normal bladder function. The mucosal surface of the urinary bladder serves as a protective layer against the irritative solutes found in the urine. The integrity of this barrier can be broken by overdistension, anoxia, detergents, alcohols, bacterial infection and by contact with agents to which the mucosa has been sensitized.In view that both anoxia and ischemia can mediate a breakdown in the role of the mucosal layer as a permeability barrier, it is reasonable to assume that this function is dependent on cellular metabolism. As an initial investigation we have compared a variety of biochemical and metabolic parameters between the mucosal layer (consisting of the lamina propria, urothelium, and any connective tissue and vascular tissue within this layer); and the muscularis layer.The results of these studies demonstrated that the rate of glucose metabolism to lactic acid (LA) of the mucosa was more than three-fold greater than that of the smooth muscle. The rate of CO₂ production of the mucosa was 60% greater than that of the unstimulated smooth muscle. The maximal activity of the mitochondrial enzyme citrate synthase was significantly greater in the mucosa than in the smooth muscle, however, the activity of malate dehydrogenase was similar for both tissues. The maximal activity of the cytosolic enzyme creatine kinase was more than two-fold greater in the bladder smooth muscle than in the mucosa; although the affinities of the creatine kinase isoforms of the mucosa were sigificantly greater than those of the muscle.Although the concentrations of ATP and ADP were similar in both muscle and mucosa, the level of creatine phosphate (CP) was over four-fold greater in the bladder muscle while the level of AMP in the muscle was only 58% of that in the mucosal epithelium.In summary, the rate of glucose metabolism was greater in the mucosa than in the smooth muscle although the concentrations of high energy phosphates (ATP+CP) was significantly greater in the smooth muscle. Future studies will be directed at identifying the specific cellular processes within the mucosal layer that relate to the function of the urothelium as a permeability barrier.     

Unique properties of muscularis mucosae smooth muscle in guinea pig urinary bladder

The muscularis mucosae, a type of smooth muscle located between the urothelium and the urinary bladder detrusor, has been described, although its properties and role in bladder function have not been characterized. Here, using mucosal tissue strips isolated from guinea pig urinary bladders, we identified spontaneous phasic contractions (SPCs) that appear to originate in the muscularis mucosae. This smooth muscle layer exhibited Ca(2+) waves and flashes, but localized Ca(2+) events (Ca(2+) sparks, purinergic receptor-mediated transients) were not detected. Ca(2+) flashes, often in bursts, occurred with a frequency (～5.7/min) similar to that of SPCs (～4/min), suggesting that SPCs are triggered by bursts of Ca(2+) flashes. The force generated by a single mucosal SPC represented the maximal force of the strip, whereas a single detrusor SPC was ～3% of maximal force of the detrusor strip. Electrical field stimulation (0.5-50 Hz) evoked force transients in isolated detrusor and mucosal strips. Inhibition of cholinergic receptors significantly decreased force in detrusor and mucosal strips (at higher frequencies). Concurrent inhibition of purinergic and cholinergic receptors nearly abolished evoked responses in detrusor and mucosae. Mucosal SPCs were unaffected by blocking small-conductance Ca(2+)-activated K(+) (SK) channels with apamin and were unchanged by blocking large-conductance Ca(2+)-activated K(+) (BK) channels with iberiotoxin (IbTX), indicating that SK and BK channels play a much smaller role in regulating muscularis mucosae SPCs than they do in regulating detrusor SPCs. Consistent with this, BK channel current density in myocytes from muscularis mucosae was ～20% of that in detrusor myocytes. These findings indicate that the muscularis mucosae in guinea pig represents a second smooth muscle compartment that is physiologically and pharmacologically distinct from the detrusor and may contribute to the overall contractile properties of the urinary bladder.     

Localisation and measurement of VIP in the genitourinary system of man and animals

VIP is present in the genitourinary system of man and animals. In man the highest concentrations are found in the penis, the uterus and vagina and in the urinary bladder. VIP nerves heavily innervate the erectile tissue of the male external genitalia, the uterine smooth muscle and blood vessels, the seromucous glands of the cervix, and the lamina propria and vaginal epithelium. In the urinary bladder, VIP nerves are located beneath the transitional epithelium, in the lamina propria and in the smooth muscle. Other areas well innervated by VIP nerves include the prostate, seminal vesicles and vasa deferentia. Chemical (phenol- and 6-OHDA) or surgical (hypogastric or pelvic nerve section) extrinsic denervation fail to deplete the genitourinary system of its VIP content, supporting the view that VIP-containing nerves originate from local ganglion cells. Indeed, neuronal cell bodies containing VIP are seen in the paracervical ganglia of the female genitalia, the para- or intramural bladder ganglia and scattered through the base of the cavernosum body, the neck of the bladder and the prostate. The finding of elevated levels of VIP in the local circulation after induced penile erection in man and mammals and the ability of VIP to relax the detrusor muscle of the bladder suggests that the peptide may be involved in penile erection and bladder relaxation, as does the marked VIP depletion in the penis or bladder in patients suffering from diabetic impotence or bladder instability.     

Role of Heat Shock Proteins in the Effect of NMDA and KCl on Cerebellar Granule Cells Survival

Cerebellar granule cells (CGC) die apoptotically after five days in culture (DIV) at physiological concentrations of potassium (5 mM; K5). When CGC are depolarized (K25) or treated with NMDA (150 M) cell survival is increased. CGC changed from K25 to K5 die after 24–48 h. It is known that heat shock protein (HSP) may protect from cell death. Here, we found that cells in K5 showed an increase in HSP-70 levels after 3 DIV. Similarly, in cells changed from K25 to K5, HSP-70 levels were increased after 6 h. Neither NMDA nor K25 treatment affected HSP-70 levels from 2–7 DIV. Ethanol or thermal stress induced HSP-70, but cell survival was not affected in K5 medium. These results suggest that HSP, particularly HSP-70, are not involved in the mechanisms by which NMDA and KCl promote cell survival.     

Localization of urokinase-type plasminogen activator messenger RNA in the normal mouse by in situ hybridization

The histological distribution of urokinase-type plasminogen activator (u-PA) mRNA was analyzed in normal mouse tissue by in situ hybridization with anti-sense RNA transcribed from three different subclones of a mouse u-PA cDNA. Hybridization signal was found over a distinct fibroblast-like cell in the lamina propria of the gastrointestinal tract, over proximal, distal, and collecting tubules of the kidney, and over the epithelium of the bladder, ductus deferens, and epididymis. No hybridization signal was found over cells of the lung, pancreas, liver, adrenal, pituitary, cerebrum, hypothalamus, cerebellum, sciatic nerve, and striated muscle, nor over endothelial cells in any tissue investigated. The lack of u-PA mRNA in lung tissue was confirmed by Northern analysis and is in contrast to the high amounts of u-PA protein found in this tissue.     

Characterization of HSP-70 cognate proteins from wheat

Summary Animal and plant cells contain a family of constitutively expressed HSP-70 cognate proteins that are localized in different subcellular locations and are presumed to play a role in protein folding and transport. Utilizing antibodies raised against the yeast endoplasmicreticulum-localized HSP-70 cognate termed BiP/GRP-78, as well as antibodies raised against the Escherichia coli HSP-70 protein DnaK, we have identified and characterized a large family of closely related proteins in wheat. One protein band of 78 kDa that is apparently closely related to yeast BiP was localized in the endoplasmic reticulum. This band cross-reacted with the yeast BiP but not with the DnaK-specific antibodies. The yeast BiP antibodies also recognized a cytoplasmic protein of 70 kDa that is probably related to the HSC-70 cognate proteins. These two proteins were further confirmed as HSP-70 cognates by their ability to bind to an ATP-agarose column. Probing of proteins from purified wheat mitochondrial preparations with the yeast BiP and DnaK-specific antibodies showed that this organelle contained a family of HSP-70-related proteins. The yeast BiP antibodies recognized two mitochondrial proteins of 60 and 58 kDa, but failed to detect any protein in the size rang of 70 to 80 kDa. However, the presence of immunologically distinct proteins of 90 and 78 kDa, as well as of lower molecular weight from this family in the mitochondria, was shown by probing with the DnaK-specific antibodies. A new protein of 30 kDa, cross-reacting with anti-yeast BiP antibodies, was detected only in developing seeds, close to their maturity. The evolution of HSP-70 cognate proteins in wheat as shown in this study is discussed.     

Comparative length-tension relationship of urinary bladder strips from hamsters, rats, guinea-pigs, rabbits and cats

1. Comparative passive tension-active tension curves were constructed for urinary bladder body strips from hamster, rat, guinea-pig, rabbit and cat. 2. Equally sized strips from rabbit and cat bladders had a significantly greater mass and cross-sectional area than strips from other species. 3. There was a greater change in cross-sectional area of strips from rabbit and cat bladders with increasing length than in strips from other species. 4. Cat bladder strips developed a significantly greater absolute active tension than did strips from all other species at passive tensions greater than 5 g. 5. The length-tension relationships of the urinary bladder differs from skeletal or vascular smooth muscle in that there is no significant decrease in active tension at strip lengths greater than L0. 6. The ability of the urinary bladder to generate active tension at tissue lengths considerably greater than L0 is a prime importance in the physiological role of the urinary bladder to accommodate and store urine.     

Pathways for movement of ions and water across toad urinary bladder

Hypertonicity of the mucosal bathing medium increases the electrical conductance of toad urinary bladder by osmotic distension of the epithelial "tight" or limiting junctions. However, toad urine is not normally hypertonic to plasma. In this study, the transmural osmotic gradient was varied strictly within the physiologic range; initially hypotonic mucosal bathing media were made isotonic by addition of a variety of solutes. Mucosal NaCl increased tissue conductance substantially. This phenomenon could not have reflected soley an altered conductance of the transcellular active transport pathway since mucosal KCl also increased tissue conductance, whether or not Na+ was present in the bathing media. The effect of mucosal NaCl could not have been mediated solely by a parallel transepithelial pathway formed by damaged tissue since mucosal addition of certain nonelectrolytes also increased tissue conductance. Finally, the osmotically-induced increase in conductance could not have occurred soley in transcellular transepithelial channels in parallel with the active pathway for Na+, since the permeability to 22Na from serosa to mucosa (s to m) was also increased by mucosal addition of NaCl; a number of lines of evidence suggest that s-to-m movement of Na+ proceeds largely through paracellular transepithelial pathways. The results thus establish that the permeability of the limiting junctions is physiologically dependent on the magnitude of the transmural osmotic gradient. A major role is proposed for this mechanism, serving to conserve the body stores of NaCl from excessive urinary excretion.     

Helicobacter pylori Induces Inflammation in Mouse Urinary Bladder and Pelvis

Helicobacter pylori was transurethrally inoculated into the mouse urinary tract. The organism established infection and induced inflammation in the urinary bladder and pelvis. During the infection, urinary pH was elevated, probably due to the production of NH3 by bacterial urease. H. pylori was recovered from the urinary bladder, kidney and urine of the infected mice. Histopathologically, severe neutrophil infiltration was observed in the mucosal layer of both organs. H. pylori was detected on the surface of the epithelial cells. These results indicate that low pH and bacterial flora were not essential factors in establishing the mucosal infection with H. pylori. This experimental system is useful to investigate the pathogenicity of H. pylori in mucosal organs.     

HSP-70 mitigates LPS/SKI-induced cell damage by increasing sphingosine kinase 1 (SK1)

Heat shock proteins (HSPs) are potent protectors of cellular integrity against environmental stresses, including toxic microbial products. To investigate the mechanism of HSP-70 cell protection against bacterial lipopolysaccharide (LPS), we established a stable HSP-70 gene-transfected RAW 264.7 murine macrophage model of LPS-induced cell death. Bacterial LPS increases the activity of sphingosine kinase 1 (SK1), which catalyzes formation of sphingosine-1-phosphate (S1P). S1P functions as a critical signal for initiation and maintenance of diverse aspects of immune cell activation and function. When mouse macrophages were incubated with Escherichia coli LPS (1 μg/ml) and sphingosine kinase inhibitor (SKI, 5 μM), 90% of cells died. Neither LPS nor SKI alone at these doses damaged the cells. The LPS/SKI-induced cell death was partially reversed by overexpression of HSP-70 in gene-transfected macrophages. The specificity of HSP-70 in this reversal was demonstrated by transfection of HSP-70-specific siRNA. Down-regulation of HSP-70 expression after transfection of siRNA specific for HSP-70 was associated with increased LPS/SKI-induced cell damage. Overexpression of human or murine HSP-70 (HSPA1A and Hspa1a, respectively) increased both cellular SK1 mRNA and protein levels. Cellular heat shock also increased SK1 protein. These studies confirm the importance of SK1 as a protective moiety in LPS-induced cell injury and demonstrate that HSP-70-mediated protection from cells treated with LPS/SKI is accompanied by upregulating expression of SK1. HSP-70-mediated increases in SK1 and consequent increased levels of S1P may also play a role in protection of cells from other processes that lead to programmed cell death.