TNF-alpha modulates the Na+/ K+ ATPase and the Na+K+2Cl- symporter in LLC-PK cells.

BACKGROUND: Tumour necrosis factor alpha (TNF-alpha) has been implicated in the development of diabetic nephropathy and the accompanying increase in sodium retention. Inhibition of renal Na(+)/K(+) ATPase was reported to accompany cell death. As TNF is known to induce both apoptosis and cell survival, this work investigated the effect and mechanism of action of TNF-alpha on the Na(+)/K(+) ATPase and the Na(+)K(+)2Cl(-) symporter using LLC-PK(1) cells, a porcine renal proximal tubules cell line. MATERIALS AND METHODS: Cells were incubated for 2 h with TNF-alpha in presence and absence of pyrrolidinedithiocarbamate, SP600125 and FK009, respective inhibitors of the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB), c-Jun N-terminal kinase (JNK) and caspases. The activity of the pump was assayed by measuring the ouabain-inhibitable release of inorganic phosphate. Changes in its expression and the expression of the symporter were monitored by western blot analysis. RESULTS: TNF-alpha up-regulated both transporters. NF-kappaB, JNK and the caspases were all mediators of the cytokine action. TNF up-regulated the Na(+)/K(+) pump by stimulating JNK which in turn, activated NF-kappaB and inhibited the caspases. TNF effect on the cotransporter was also mediated via activation of JNK which however inhibited NF-kappaB and by so doing prevented activation of caspases. As caspases were demonstrated to down-regulate the two transporters, their inhibition by TNF is responsible for the observed up-regulatory effect. CONCLUSIONS: It was concluded that the Na(+)/K(+) ATPase and Na(+)K(+)2Cl(-) are both targets of TNF-alpha and the effect of the cytokine favours cell survival over cell death.

Larvicidal activity of essential oils extracted from commonly used herbs in Lebanon against the seaside mosquito, Ochlerotatus caspius.

This study investigates the potential of essential oils from commonly used medical and culinary herbs in Lebanon as an environmentally safe measure to control the seaside mosquito, Ochlerotatus caspius. The composition of essential oils extracted from parsley seeds and leaves, alpine thyme inflorescences, anis seeds, and coriander fruits were analyzed by GC-MS, and the major components of these oils were found to be thymol, sabinene, carvacrol, anethole, and linalool, respectively. Mosquito larvicidal assays were conducted to evaluate the LC(50) and LC(90) after 24 and 48h of the essential oils and their major constituents. All of the tested oils proved to have strong larvicidal activity (LC(50): 15-156ppm) against Oc. caspius fourth instars, with the most potent oil being thyme inflorescence extract, followed by parsley seed oil, aniseed oil, and then coriander fruit oil. Toxicity of each oil major constituent was also estimated and compared to a reported larvicidal compound, eugenol.

Comparative impact of live chicken infectious anaemia virus vaccine versus natural exposure in meat chicken breeders on immunity to infectivity by CIA and inclusion body hepatitis viruses in their offspring.

The immunology and histopathology and the distribution of viral antigen in infections with chicken infectious anaemia virus (CIAV) and inclusion body hepatitis virus (IBHV) were compared in the broiler offspring of CIAV-vaccinated meat chicken breeders versus those in the offspring of breeders naturally exposed to field CIAV. No significant difference in the humoral antibody level specific for CIAV was observed between 5 and 33 weeks of age in the two breeder groups (p > 0.05). The maternal humoral immunity to CIAV in the day-old offspring of the groups did not differ significantly (p > 0.05). The humoral immunity to CIAV at 40 days of age indicated an absence of clinical signs of CIAV in the broiler offspring of both groups of breeders and this was associated with mean serum thymulin levels in offspring of both groups not differing significantly at 1 or 40 days of age. Histopathological and immunofluorescence observations did not differ significantly in the offspring of either group by CIAV or IBHV.

In vitro effect of eugenol and cinnamaldehyde on membrane potential and respiratory chain complexes in isolated rat liver mitochondria.

The effect of water extracts of cinnamon and clove on rat mitochondrial F(0)F1ATPase was investigated. Both spices stimulated ATP hydrolysis. Gas chromatography analysis of the water extracts, confirmed the presence of eugenol and cinnamaldehyde as major components in clove and cinnamon, respectively. Both components (1) stimulated ATPase significantly at concentrations equal or greater then 0.3 mM; (2) reduced mitochondrial membrane potential: a 50% decrease in Deltapsi was obtained at 7.56 and 11.6 micromoles/mg protein for eugenol and cinnmaldehyde, respectively; (3) inhibited NADH oxidase or complex I of the respiratory chain with a 50% inhibition at 15 and 20 micromoles/mg protein for eugenol and cinnamaldehyde respectively; (4) had no effect on succinate dehydrogenase activity. The study proposes the mitochondria as a target for the action of the spices resulting in derangement of mitochondrial functions, particularly at proton transferring sites.

The mechanism underlying the laxative properties of parsley extract.

Parsley has been claimed in folk medicine to possess laxative properties attributed to the presence therein of some volatile oils that are more concentrated in seeds than in stems or leaves. The advocated physiological effect of parsley, does not have, however, any proven scientific background and relies mainly on simple observations and empirical information. This work aims at providing the scientific evidence that would confirm or reject the claimed laxative role of parsley, and at determining its mechanism of action if present. A perfusion technique was used to measure the net fluid absorption from the rat colon. The addition of an aqueous extract of parsley seeds to the perfusion buffer, and the omission of sodium, both reduced significantly net water absorption from the colon, as compared to the control. Parsley, added to a sodium-free buffer did not lead to any further significant change in water absorption as compared to parsley alone inferring that with parsley sodium absorption was already inhibited. Since K+ and Cl- secretion depends on the activity of the NaKCl2 transporter, the latter was inhibited with furosemide which increased significantly net water absorption. When parsley and furosemide were added together, net water absorption was significantly higher than with parsley alone and significantly lower than with furosemide alone. In addition, parsley extract was shown to inhibit the in vitro activity of the Na+-K+ATPase in a colon homogenate and the activity of a partially purified dog kidney ATPase. The results suggest that parsley acts by, inhibiting sodium and consequently water absorption through an inhibition of the Na+-K+ pump, and by stimulating the NaKCl, transporter and increasing electrolyte and water secretion.

Epinephrine stimulates the Na+-K+ ATPase in isolated rat jejunal crypt cells.

The effect of epinephrine on the activity of the Na+-K+ ATPase was studied in isolated rat jejunal cells. The activity of the pump was assessed by measuring the ouabain inhibitable K+ accumulation by the enterocytes using 86Rb as a tracer. Epinephrine stimulated significantly the Na+-K+ ATPase in crypt cells but not in villus cells. This effect was still apparent in presence of propranolol and prazocin but disappeared in presence of yohimbine. Amiloride did not affect the epinephrine-induced stimulation. Calcium channel blockers and dibutyryl cAMP enhanced the activity of the pump, and exerted respectively overlapping and additive effects with epinephrine, when added simultaneously. The calcium ionophore A23187 inhibited the basal activity of the ATPase and the stimulatory effect of epinephrine disappeared in its presence. These results suggest that epinephrine stimulates the Na+-K+ ATPase in jejunal crypt cells by activating alpha2 receptors and decreasing intracellular calcium, and not by altering cAMP levels.

Effect of cinnamon, clove and some of their constituents on the Na(+)-K(+)-ATPase activity and alanine absorption in the rat jejunum.

The effect of a water extract of some spices on the in vitro activity of the rat jejunal Na(+)-K(+)-ATPase was investigated. Extracts of nutmeg, cinnamon, clove, cumin, coriander, turmeric and caraway all inhibited the ATPase, while anise seed and white pepper exerted no significant effects. The extracts of clove and cinnamon had the most potent inhibitory effect on the intestinal ATPase as compared to extracts of other spices. They also inhibited the in vitro Na(+)-K(+)-ATPase activity in a crude kidney homogenate and the activity of an isolated dog kidney Na(+)-K(+)-ATPase. The alcoholic extract of cinnamon, compared to the aqueous extract, had a stronger inhibitory action on the jejunal enzyme and a lower IC(50) value, which was not significantly different from the one observed with cinnamaldehyde, the major volatile oil present cinnamon, suggesting that in alcoholic extracts cinnamaldehyde is the major inhibitory component. The IC(50) values of eugenol, aqueous clove extract and ethanolic clove extract all fell within the same range and were not significantly different from each other, suggesting that eugenol is the major inhibitory component in both alcoholic and aqueous extracts. Based on the IC(50) values, the order of sensitivity of the enzyme to the spices extracts is as follows: isolated dog kidney ATPase>rat kidney ATPase>rat intestine ATPase. The aqueous extracts of clove and cinnamon also significantly lowered the absorption of alanine from the rat intestine. It was concluded that the active principle(s) in clove and cinnamon can permeate the membrane of the enterocytes and inhibit the Na(+)-K(+)-ATPase that provides the driving force for many transport processes.

Cyclic AMP and furosemide stimulate the Na(+)-K(+) ATPase in isolated rat jejunal cells.

The effect of cAMP on the activity of Na(+)-K(+) ATPase in the rat jejunum was studied by measuring the ouabain-sensitive (86)Rb accumulation by isolated villus cells. The cyclic nucleotide increased significantly (86)Rb accumulation by the enterocytes. The involvement of the NaKCl(2) symporter, the H(+)-K(+) exchanger and the Na(+)-K(+) pump was tested by measuring, respectively the furosemide-sensitive, the omeprazole sensitive, and the ouabain-sensitive (86)Rb accumulation. Furosemide significantly increased the basal level of intracellular (86)Rb and exerted overlapping effects with cAMP. Inhibiting the Na(+)-H(+) exchanger with amiloride, decreased the basal level of intracellular (86)Rb, and in its presence the stimulatory effect of cAMP was still observed but not that of furosemide. cAMP and furosemide did not exert any significant effect on (86)Rb accumulation by the enterocytes in presence of ouabain or in the absence of sodium but their stimulatory effect still appeared in the absence of chloride or in the presence of omeprazole or K(+)-channel blockers. These data suggest that cAMP and furosemide stimulate the Na(+)-K(+) ATPase in rat jejunal cells, and that this effect is, in the case of furosemide, mediated by an activation of the Na(+)-H(+) exchanger.

Interleukin-1 beta inhibits the intestinal transport of [14C] 3-O-methylglucose in the rat.

Interleukin-1 (IL-1) is known to be a hypoglycemic cytokine, but its mechanism of action is still unknown. Since the blood glucose levels depend on the amount of glucose entering and leaving the circulation, this work was conducted to test the hypothesis that the hypoglycemia observed with IL-1beta might result, at least partially, from a reduced intestinal glucose absorption. Male Sprague Dawley rats were injected intraperitoneally (i.p.) with IL-1beta, and a jejunal segment was perfused with [14C] 3-O-methylglucose for 5, 15, 25 and 40 min. Our results showed that IL-1beta significantly inhibited the mucosal uptake of this hexose and reduced its intestinal retention. The time course and the dose response effect for this cytokine were also determined. Studies on the effect of IL-1beta on the activity of the intestinal Na+-K+ ATPase demonstrated a significant inhibition of the pump. The effect of IL-1beta on the hexose transport across the brush border membrane may thus be attributed to its inhibitory effect on the Na+-K+ ATPase.

Binding of 125I-insulin on capillary endothelial and myofiber cell membranes in normal and streptozotocin-induced diabetic perfused rat hearts.

A heart-perfusion technique was employed to measure 125I-insulin binding on capillary endothelial and myocyte cell membranes in Sprague-Dawley rats. Animals were anesthetized, and the anterior chest wall excised to expose the mediastinal contents. The right and left superior and inferior venae cavae were dissected and tied, and another tie was passed around the aorta. A polyethylene catheter was introduced into the aortic lumen from cephalad to caudad to sit with its tip above the aortic valve. Another catheter was introduced into the cavity of the right atrium and both were anchored by sutures. Oxygenated Ringer-Lock buffer containing 20 mM/L K+ and 125I-insulin was perfused at a rate of 1 mL/min via the aortic catheter. Concomitantly, the distal ascending aorta and venae cavae were ligated. The effluent was collected from the right atrial catheter at the same infusion rate. Animals were divided into two groups, the normal group and streptozotocin-induced diabetic group. Heart perfusion was done on both groups either without or after treatment with detergent (CHAPS) to remove the capillary endothelial lining. A physical model for 125I-insulin sequestration as a ligand to its receptors on endothelial and/or myocyte plasma membranes was proposed. The model described a reversible binding of ligand on cellular surface receptor concentration to fit a conservation equation and a first order Bessel function. The binding constants (kn), reversal constants (k-n), dissociation constants kd = k-n/kn, and residency time constants tau = 1/k-n of 125I-insulin in normal untreated, normal CHAPS-treated, diabetic untreated, and diabetic CHAPS-treated hearts were estimated using a theoretically generated curve-fit to the data. Since insulin receptor binding on the capillary endothelial cell surfaces may serve to transport insulin from the intravascular to the subendothelial space, and since streptozotocin-induced diabetes was shown to diminish receptor autophosphorylation and kinase activity and hence internalization of insulin, then one can conclude the following from the data. In the normal heart, removal of the capillary endothelial lining with CHAPS did not alter kn, k-n, kd, and tau of insulin binding as compared to the normal untreated, whereas in the diabetic untreated heart these constants were altered, compared to the diabetic treated. Furthermore, the kn and k-n values in the diabetic CHAPS-treated hearts were the same as for the normals untreated and CHAPS-treated, respectively. In conclusion, the dissociation constants and residency time constants of all groups indicated the possible existence of two types of insulin receptors: the capillary endothelial cell surface insulin receptors with lower residency time (low affinity receptor or combination of insulin and IGF-1 receptors) and the myocyte plasma membrane insulin receptors with higher residency times (high affinity).

Binding and distribution of three prototype calcium channel blockers in perfused rat liver.

This work represents a study of the binding and distribution of three different calcium channel blockers in the Sprague-Dawley rat liver, using an in situ perfusion technique. For this purpose, [3H] desmethoxyverapamil, [3H] PN200-110 (isradipine) and [3H] azidopine were used as binding probes interacting with calcium channels. The perfusion steps of the liver involved both portal vein and thoracic inferior vena cava cannulations as inlet and outlet respectively. The subhepatic inferior vena cava was ligated to prevent leakage of the perfusate. Buffer, containing the tracer drug, was administered via the portal vein at a rate of 1 mL/min and perfusate collected at the same rate within specified time intervals during 50 min. The concentration of the tracer solutes in the perfusate's outlet increased with time, and steady state was observed for all tracers at > or = 40 min. The effect of adding cold isradipine to tracer desmethoxyverapamil, or cold verapamil to tracer PN200-110 were also assessed. First order rate constants for hepatocellular influx, efflux and calcium channel binding of the tracer substances were obtained using a simplified model from Goresky et al. These constants were mathematically manipulated and changed into permeability constants, second order binding constants, and residency times. Tracer solute influx across hepatocellular membranes is solubility-diffusion controlled, is inversely related to the molecular weights and is different in value from the efflux constants. Cold isradipine reduced the binding constant of desmethoxyverapamil by 36%, while cold verapamil reduced the binding constant of PN200-110 by 23%. Azidopine cellular distribution was low, however, binding to its receptor was analogous to desmethoxyverapamil and PN200-110. Moreover, PN200-110 had the highest residency time with no effect of cold verapamil on its receptor binding, while desmethoxyverapamil had the lowest residency time which significantly increased in the presence of cold isradipine.

Alpha and beta adrenoceptors mediate the inhibitory effect of epinephrine on the mucosal uptake of phenylalanine in the rat jejunum.

Although adrenergic agonists and antagonists have extensive clinical use, their effects on the intestinal absorption of amino acids have rarely been investigated. This work attempts to study the effect of epinephrine on the mucosal uptake of phenylalanine in the rat jejunum, using an in situ double perfusion technique that allows the intestine and its vasculature to be perfused simultaneously and respectively with the amino acid and the hormone and its antagonists. Epinephrine reduced, by about 30%, the mucosal transport of phenylalanine by activating alpha 1, alpha 2, and beta adrenergic receptors. The effect of the latter was not apparent however, until the alpha 2 receptors were blocked.

Asymmetric signal transduction in polarized ileal Na(+)-absorbing cells: carbachol activates brush-border but not basolateral-membrane PIP2-PLC and translocates PLC-gamma 1 only to the brush border.

In ileal Na+ absorptive cells, carbachol inhibits NaCl absorption and its component brush-border Na+/H+ exchanger, acting via basolateral membrane (BLM) receptors. This carbachol effect involves brush-border but not BLM protein kinase C. In the present work we describe another asymmetric aspect of signal transduction in these epithelial cells, this time involving phosphatidylinositol 4,5-bisphosphate (PIP2)-specific phospholipase C (PLC). Thirty seconds and 1 min after carbachol treatment, brush-border PIP2-specific PLC activity increased, returning to control levels by 2.5 min. Involvement of brush-border tyrosine kinase(s) in this effect was suggested by inhibition of the carbachol effect on NaCl absorption by the tyrosine kinase inhibitor genistein, added to the mucosal but not the serosal surface. Luminal genistein pretreatment also prevented the carbachol-induced increase in brush-border PLC activity. In contrast, carbachol exposure did not change the BLM PIP2-specific PLC activity. Western analysis and immunoprecipitation demonstrated that PLC-gamma 1 is present in the brush border and that carbachol increases the PLC-gamma 1 amount in the brush border. Both the brush border and BLM contain PLC-beta 3 and a small amount of PLC-delta 1 but no PLC-beta 1, whereas BLM lacks detectable PLC-gamma 1. No change in PLC-beta 3 or PLC-delta 1 amount in the brush border occurred with carbachol exposure. No change in tyrosine phosphorylation of brush-border PLC-gamma 1 occurred with carbachol treatment. The Ca2+ ionophore A23187 did not alter PIP2-specific PLC activity in either the brush border or the BLM. These studies demonstrate that carbachol but not Ca2+ ionophore effects on brush-border NaCl absorption are associated with increases in brush-border but not BLM PIP2-specific PLC activity and in the amount of brush-border PLC-gamma 1, and involve tyrosine phosphorylation. This asymmetric aspect of epithelial signal transduction, together with the previous demonstration of localization of high-sensitivity IP3 stores to the apical membrane area in intestinal epithelial cells, shows that different aspects of signal transduction occur at the apical and basolateral membranes in epithelial and requires studies in both domains to define mechanisms of intracellular signalling.

Inhibitors in tea of intestinal absorption of phenylalanine in rats.

This work studies the effect of tea extract on the mucosal and serosal transport of phenylalanine, and attempts to identify the active ingredient(s) therein by studying the effect of known tea constituents like theophylline, caffeine and tannic acid. Tea and all the constituents tested inhibited the mucosal uptake of phenylalanine. The serosal transport was unaffected by caffeine and tannic acid, but inhibited by theophylline and high concentrations of tea. The in vitro activity of the intestinal Na+-K+ATPase was also assayed from a jejunal homogenate in presence of theophylline, caffeine, tannic acid and cAMP. All were found to inhibit significantly the enzyme. The in vitro activity of a purified Na+-K+ATPase was however stimulated by theophylline and caffeine, and inhibited only by tannic acid. It was concluded that the inhibitory effect of tea is exerted mainly through its constituents which inhibit the Na+-K+ pump directly (tannic acid) or indirectly (theophylline and caffeine), possible by elevating cAMP levels, dissipating thus the sodium gradient needed for the mucosal uptake of the amino acid.

Tea extract inhibits intestinal absorption of glucose and sodium in rats.

The effect of tea extract on the intestinal absorption of glucose and sodium was studied in rats. Tea extract reduced the mucosal uptake of glucose and its portal plasma concentration, but was without any effect on its serosal transport. The mucosal uptake of sodium was similarly inhibited. The tea extract reduced also the in vitro activity of the Na(+)-K+ ATPase in an intestinal mucosal homogenate. These data are consistent with the hypothesis that an active ingredient in tea reduced sodium extrusion from the enterocytes by inhibiting the Na(+)-K+ pump, thus destroying the gradient needed for the mucosal transport of glucose.

Effects of cytochalasin, colchicine, and ethylenediaminetetraacetic acid on linoleic acid transport across rat jejunal enterocytes.

The involvement and the site of interference of the cytoskeleton in the transport of linoleic acid across the rat jejunum was investigated by administration of microfilamentous and microtubular altering agents such as cytochalasin, colchicine, and ethylenediaminetetraacetic acid (EDTA). An isolated jejunal segment was perfused with a buffer containing labeled linoleic acid, and portal blood and perfusate samples were collected concomitantly at 5-min intervals and assayed for their radioactivity. At the end of the perfusion, the amount of radioactivity retained in the intestine was also determined. The results were analyzed by using a three-compartment physical model that allows the determination of mucosal and serosal permeability coefficients, from which changes in the permeability of the mucosal and serosal membranes were assessed. Cytochalasin decreased the permeability of the mucosal membrane to linoleic acid, but not that of the serosal membrane. The administration of colchicine, EDTA, or cytochalasin + colchicine increased the permeability of the serosal membrane but did not affect the mucosal membrane.

System approach to the transepithelial transport across rat jejunal enterocytes: effect of cytochalasin B, colchicine, and ethylenediaminetetraacetic acid on butyric acid transport.

A three-compartment physical model is devised for transepithelial passive transport across intestinal cells. The mathematical equations derived from the model allow the simultaneous and quantitative measurements, in the form of permeability coefficients, of solute transport across both the luminal-serosal and serosal-blood barriers. The proposed model is used to study the involvement of the cytoskeleton in butyric acid absorption by the rat jejunum. Alterations in cytoskeletal functions are introduced by the administration of microfilamentous and microtubular altering agents such as cytochalasin, colchicine, or EDTA. An isolated jejunal segment perfused with a buffer containing labeled butyric acid was homogenized at the end of the experiment and assayed for its butyric acid content. During the perfusion, portal blood samples, as well as perfusate samples collected 10 cm distal to the perfusion site were drawn at 5-min intervals and assayed for their radioactivity. Cytochalasin was found to decrease the permeability of the mucosal membrane to butyric acid and to increase that of the serosal membrane. Colchicine did not have any effect either on the mucosal or on the serosal side. Cytochalasin and colchicine, when given in the same experiment, increased the permeability of the serosal membrane to butyric acid, but were without any effect on the mucosal barrier. Also, EDTA had no effect on the mucosal side, but decreased significantly the permeability of the serosal membrane to the fatty acid.