Suppression of fatty acid ß-oxidation and energy deficiency as a cause of inhibitory effect of E. coli lipopolysaccharide on osmotic water transport in the frog urinary bladder.

Previously we showed that arginine-vasotocin (AVT)-stimulated osmotic water permeability (OWP) of the frog urinary bladder was decreased if the mucosal side of the bladder has been naturally colonized by Gram-negative bacteria, or if bacterial lipopolysaccharide (LPS) was introduced into the lumen of the isolated bladder (J. Exp. Zool., 2013, 319, 487-494). Taking into account that in different tissues and cell types, challenge with LPS causes significant metabolic shift and energy deficiency, we hypothesized that an LPS-induced decrease of AVT-stimulated OWP could depend on the reduction of fatty acid oxidation (FAO), which is important for generation of ATP in epithelia. Using an isolated frog Rana temporaria urinary bladder we showed that the AVT-induced increase of OWP did not depend on the external glucose, but was inhibited by oligomycin, an ATP-synthase inhibitor, and by etomoxir, an inhibitor of carnitine palmitoyltransferase-1. In primary cultured epithelial cells isolated from the bladder mucosa, LPS E. coli (25 µg/ml, 21 h), as well as etomoxir (100 µM), decreased FAO accompanied by triacylglycerol accumulation. Both drugs impaired mitochondrial functions demonstrated by decreased ATP production and a reduced maximal oxygen consumption rate (OCR) and OCR directed at ATP synthesis. Additionally, we found that LPS decreased the expression of peroxisome proliferator-activated receptor alpha, a key player in the regulation of FAO. These data indicate that the impairment of AVT-induced water transport in osmoregulatory epithelium caused by LPS depends at least partly on defects in FAO and FAO-dependent energy production.

Mitochondrial-Targeted Antioxidant MitoQ Prevents E. coli Lipopolysaccharide-Induced Accumulation of Triacylglycerol and Lipid Droplets Biogenesis in Epithelial Cells.

The effect of bacterial lipopolysaccharide (LPS) on eukaryotic cell could be accompanied by a significant metabolic shift that includes accumulation of triacylglycerol (TAG) in lipid droplets (LD), ubiquitous organelles associated with fatty acid storage, energy regulation and demonstrated tight spatial and functional connections with mitochondria. The impairment of mitochondrial activity under pathological stimuli has been shown to provoke TAG storage and LD biogenesis. However the potential mechanisms that link mitochondrial disturbances and TAG accumulation are not completely understood. We hypothesize that mitochondrial ROS (mROS) may play a role of a trigger leading to subsequent accumulation of intracellular TAG and LD in response to a bacterial stimulus. Using isolated epithelial cells from the frog urinary bladder, we showed that LPS decreased fatty acids oxidation, enhanced TAG deposition, and promoted LD formation. LPS treatment did not affect the mitochondrial membrane potential but increased cellular ROS production and led to impairment of mitochondrial function as revealed by decreased ATP production and a reduced maximal oxygen consumption rate (OCR) and OCR directed at ATP turnover. The mitochondrial-targeted antioxidant MitoQ at a dose of 25 nM did not prevent LPS-induced alterations in cellular respiration, but, in contrast to nonmitochondrial antioxidant α-tocopherol, reduced the effect of LPS on the generation of ROS, restored the LPS-induced decline of fatty acids oxidation, and prevented accumulation of TAG and LD biogenesis. The data obtained indicate the key signaling role of mROS in the lipid metabolic shift that occurs under the impact of a bacterial pathogen in epithelial cells.

GM1 and GD1a gangliosides modulate toxic and inflammatory effects of E. coli lipopolysaccharide by preventing TLR4 translocation into lipid rafts.

Exogenous gangliosides are known to inhibit the effects of Escherichia coli lipopolysaccharide (LPS) in different cells exhibiting anti-inflammatory and immunosuppressive activities. The mechanisms underlying ganglioside action are not fully understood. Because LPS recognition and receptor complex formation occur in lipid rafts, and gangliosides play a key role in their maintenance, we hypothesize that protective effects of exogenous gangliosides would depend on inhibition of LPS signaling via prevention of TLR4 translocation into lipid rafts. The effect of GM1 and GD1a gangliosides on LPS-induced toxic and inflammatory reactions in PC12 cells, and in epithelial cells isolated from the frog urinary bladder, was studied. In PC12 cells, GD1a and GM1 significantly reduced the effect of LPS on the decrease of cell survival and on stimulation of reactive oxygen species production. In epithelial cells, gangliosides decreased LPS-stimulated iNOS expression, NO, and PGE2 production. Subcellular fractionation, in combination with immunoblotting, showed that pretreatment of cells with GM1, GD1a, or methyl-ß-cyclodextrin, completely eliminated the effect of LPS on translocation of TLR4 into lipid rafts. The results are consistent with the hypothesis that ganglioside-induced prevention of TLR4 translocation into lipid rafts could be a mechanism of protection against LPS in various cells.

Frog urinary bladder epithelial cells express TLR4 and respond to bacterial LPS by increase of iNOS expression and L-arginine uptake.

As in mammals, epithelium of the amphibian urinary bladder forms a barrier to pathogen entry and is a first line of defense against penetrating microorganisms. We investigated the effect of Escherichia coli LPS on generation of nitric oxide (NO), a critically important mediator during infectious processes, by primary cultured frog (Rana temporaria) urinary bladder epithelial cells (FUBEC). It was found that FUBEC constitutively express Toll-like receptor 4 (TLR4), a receptor of LPS, and respond to LPS (10 µg/ml) by stimulation of inducible nitric oxide synthase (iNOS) mRNA/protein expression and NOS activity measured by nitrite produced in the culture medium and by citrulline assay. We characterized uptake of l-arginine, a precursor in NO synthesis, by FUBEC and showed that it is mediated mainly by the y+ cationic amino acid transport system. LPS stimulated l-arginine uptake, and this effect was blocked by the iNOS inhibitor 1400W. Arginase II was found to be expressed in FUBEC. Inhibition of arginase activity by (S)-(boronoethyl)-l-cysteine increased generation of NO, suggesting contribution of arginase to NO production via competing with NOS for the substrate. LPS altered neither total arginase activity nor arginase II expression. Among epithelial cells, phagocytic macrophage-like cells were observed, but they did not contribute to LPS-induced NO production. These data demonstrate that amphibian urinary bladder epithelial cells recognize LPS and respond to it by increased generation of NO via stimulation of iNOS expression and l-arginine uptake, which appears to be essential for the regulation of the innate immune response and the inflammation in bladder epithelium.

Prostaglandin E2 inhibits vasotocin-induced osmotic water permeability in the frog urinary bladder by EP1-receptor-mediated activation of NO/cGMP pathway.

PGE(2) is a well-known inhibitor of the antidiuretic hormone-induced increase of osmotic water permeability (OWP) in different osmoregulatory epithelia; however, the mechanisms underlying this effect of PGE(2) are not completely understood. Here, we report that, in the frog Rana temporaria urinary bladder, EP(1)-receptor-mediated inhibition of arginine-vasotocin (AVT)-induced OWP by PGE(2) is attributed to increased generation of nitric oxide (NO) in epithelial cells. It was shown that the inhibitory effect of 17-phenyl-trinor-PGE(2) (17-ph-PGE(2)), an EP(1) agonist, on AVT-induced OWP was significantly reduced in the presence of 7-nitroindazole (7-NI), a neuronal NO synthase (nNOS) inhibitor. NO synthase (NOS) activity in both lysed and intact epithelial cells measured as a rate of conversion of l-[(3)H]arginine to l-[(3)H]citrulline was Ca(2+) dependent and inhibited by 7-NI. PGE(2) and 17-ph-PGE(2), but not M&B-28767 (EP(3) agonist) or butaprost (EP(2) agonist), stimulated NOS activity in epithelial cells. The above effect of PGE(2) was abolished in the presence of SC-19220, an EP(1) antagonist. 7-NI reduced the stimulatory effect of 17-ph-PGE(2) on NOS activity. 17-ph-PGE(2) increased intracellular Ca(2+) concentration and cGMP in epithelial cells. Western blot analysis revealed an nNOS expression in epithelial cells. These results show that the inhibitory effect of PGE(2) on AVT-induced OWP in the frog urinary bladder is based at least partly on EP(1)-receptor-mediated activation of the NO/cGMP pathway, suggesting a novel cross talk between AVT, PGE(2), and nNOS that may be important in the regulation of water transport.

Nitric oxide inhibits arginine-vasotocin-induced increase of water osmotic permeability in frog urinary bladder.

The present study addressed the question of whether nitric oxide (NO) participates in regulation of osmotic water permeability in the urinary bladder of the frog Rana temporaria L. Experiments were carried out on isolated, paired hemi-bladders filled with amphibian Ringer solution diluted 1:10 with distilled water. Sodium nitroprusside (SNP, 125-250 micro M), an NO donor, markedly attenuated the increase of osmotic water flow elicited by arginine-vasotocin (AVT) (AVT 10(-10) M: 2.20+/-0.26; AVT plus 200 micro M SNP: 1.21+/-0.15 micro l/min cm(2), n=20, P<0.001). This effect of SNP was apparent only in the presence of 50 micro M zaprinast, an inhibitor of the cGMP-specific phosphodiesterase-5 (PDE5). In the presence of zaprinast, SNP elevated cGMP production significantly both in control and AVT-stimulated urinary bladders, but had no effect on the level of cAMP (AVT 5 x 10(-10) M: 7.6+/-0.6; AVT plus SNP 200 micro M: 7.5+/-0.4 pmol/mg protein, n=8, N.S.). 1 H-[1,2,4]-oxadiazole-[4,3-a]-quinoxalin-1-one (ODQ, 25-100 micro M), an inhibitor of soluble guanylate cyclase, enhanced the AVT-induced water flow, decreased the SNP-stimulated increase of cGMP in the bladder tissue and almost abolished the inhibitory effect of SNP on the AVT-induced hydroosmotic response. 8-( p-Chlorophenylthio)-cGMP (8-pCPT-cGMP, 25 or 50 micro M), a membrane-permeable cGMP analogue specific for cGMP-dependent protein kinase (PKG), inhibited, whereas 2 micro M KT-5823, an inhibitor of PKG, significantly stimulated the increase of water flow induced by AVT. The inhibitory effect of SNP on AVT-induced water flow was almost completely reversed by KT-5823, but not by 50-100 micro M erythro-9-[2-hydroxy-3-nonyl]adenine (EHNA), an inhibitor of cGMP-activated PDE2. Immunohistochemistry of urinary bladder slices with antibodies against different types of NO synthase (NOS) revealed a positive immunostaining for neuronal NOS (nNOS) in the mucosal epithelium. These results suggest that in the frog urinary bladder endogenous NO is involved in regulation of water osmotic permeability. NO inhibits the AVT-induced increase of water flow at least partly by activation of PKG, which interferes with the hydroosmotic effect of AVT probably at (a) post-cAMP step(s).

Regulation of urea permeability in frog urinary bladder by prostaglandin E(2).

The present study was performed to investigate the role of prostaglandin E(2) (PGE(2)) in the regulation of urea transport in the frog urinary bladder, which is known to occur via a specialized arginine-vasotocin- (AVT-) regulated urea transporter. The bladders isolated from Rana temporaria L. were filled with amphibian Ringer solution containing 370 Bq/ml (0.01 microCi/ml) of [14C]urea, and urea permeability ( P(urea)) was determined by sampling the serosal and mucosal bathing medium at 30-min intervals for measurement of radioactivity. It was found that, from the serosal side, PGE(2) (10 nM to 1 microM) caused a dose-dependent increase in P(urea) [(7.2+/-1.8)x10(-6) cm/s in the presence of 0.5 microM PGE(2)versus (1.0+/-0.2)x10(-6) cm/s in control, n=9, P<0.001]. As in response to AVT, the PGE(2)-induced P(urea)reached a maximum in 1-1.5 h after the agonist was added. The stimulatory effects of PGE(2) and AVT applied together were not additive. PGE(2)-induced urea transport was strongly inhibited by nearly 75% in the presence of mucosal or serosal phloretin (10(-4) M). P(urea) was enhanced up to (4.7+/-0.8)x10(-6) cm/s (n=12, P<0.001) by butaprost (5 x 10(-6) M), a selective EP(2) receptor agonist, while sulprostone (EP(1)/EP(3) agonist, 10(-6) M) caused no changes in P(urea). PGE(2)dose-dependently increased the content of cAMP in mucosal epithelial cells (control: 18.0+/-1.8; 10(-6) M PGE(2): 74.2+/-9.3 pmol cAMP/mg protein per 30 min, n=7, P<0.001). Phorbol esters did not alter PGE(2)-induced P(urea), whereas H-89 (20 microM), a protein kinase A inhibitor, reduced it by 45.1+/-9.9% ( n=5, P<0.05). PGE(2)did not change the AVT-stimulated P(urea) measured in isoosmotic conditions, but inhibited the last one in the presence of a serosa-to-mucosa osmotic gradient. The data obtained show that, in the frog urinary bladder, PGE(2)is a stimulator of phloretin-inhibitable urea transport. Its effect seems to be mediated by EP(2) receptor-coupled generation of intracellular cAMP.