Role of prostaglandins in lipopolysaccharide effects on K+-induced contractions in rabbit small intestine

AIM: The mediators of the pathophysiologcal symptoms of septic shock are not completely understood. The aim of this work was to investigate the effect of lipopolysaccharide (LPS) on the K+-induced response of longitudinal segments of rabbit small intestine in vitro and the possible role of prostaglandins. METHODS AND RESULTS: Rabbits were treated with intravenously injected LPS. After 90 min animals were killed and intestinal segments were mounted in an organ bath. Lipopolysaccharide (0.2 microg kg-1) inhibited K+-induced contractions (60 mm) by 68% in duodenum, 58% in jejunum and 52% in ileum. Indomethacin antagonized LPS actions when injected 15 min before LPS. PGE2 reduced K+-induced contractions, imitating LPS effects. In contrast, contractions induced by K+ increased when intestinal segments were incubated in vitro with LPS for 90 min. The LPS (0.3 microg mL-1) increased K+-induced contractions (60 mm) by 46% in duodenum, 63% in jejunum and 85% in ileum. The LPS effect was antagonized by indomethacin at 10-6 m in duodenum and jejunum and at 10-8 m in ileum. PGE2 evoked dose-dependent contractions when added to the bath in duodenum, jejunum and ileum. CONCLUSION: These results suggest that effect of LPS on K+-induced contractions in the rabbit small bowel may be mediated by prostaglandin E2.     

Elastic properties in the circumferential direction in isolated rat small intestine

The aim was to study the passive biomechanical wall properties in the isolated duodenum, jejunum and ileum of the rat. The organ bath contained a Krebs–Ringer solution with 10^-2m MgCl₂ to abolish smooth muscle contractile activity. Stepwise inflation of an intraluminal balloon, in which the cross-sectional area (CSA) was measured, provided the distension stimulus. The circumferential wall stress–strain distributions were computed from steady-state values of these measurements and the unstressed wall thickness was measured in order to evaluate the passive elastic properties. The CSAs measured increased from duodenum to jejunum and to ileum (P<0.001). At the highest applied pressure load the CSA was 16.64±0.46 mm² for the duodenum, 18.70±0.53 mm² for the jejunum and 21.73±0.49 mm² for the ileum. The wall thickness was 0.19±0.01, 0.25±0.01 and 0.28±0.02 mm, in duodenum, jejunum and ileum, respectively (P<0.01). All segments showed stress–strain distributions that were exponential by nature. Applying the function Y=exp(a+bX) gave a median determination coefficient of 0.98 (quartiles 0.96–0.99). The duodenum was significantly stiffer than the two other segments. The values of a and b were 2.03±0.11 and 14.82±1.81, 1.39±0.20 and 13.15±1.42, and 1.13±0.13 and 10.69±0.65 for the duodenum, jejunum and ileum, respectively. The a differed between the duodenum and the other segments (P<0.001), whereas the b differed only between the duodenum and ileum (P<0.005). In conclusion, differences in the luminal CSA, wall thickness and elastic properties were found between the duodenum, jejunum and ileum.     

Characterization of the prostanoid receptors and of the contractile effects of prostaglandin F2a in human pial arteries

The contractile and relaxant effects of various prostanoids were studied on isolated human pial arteries. Contractions were elicited with the following order of potency: U46619?U44069>PGB₂>PGF_2a>PGE₂?PGD₂>PGF_1a≥TXB₂, indicating that prostanoid-induced contractions probably are mediated by a thromboxane-sensitive receptor. Relaxation of PGF_2a-contracted arteries was induced with the order of potency: PGE₂> PGE₁>PGD₂?PGD₁. Vessels contrated by K⁺ were relaxed only by PGE,. Since PGI₂ was previously found to be more potent than all the prostanoids tested in the present study, relaxant responses are probably mediated via a PGI₂-sensitive receptor. The roles of free extracellular and cellularly bound calcium for the contractile effects of PGF_2a and K⁺ were estimated by incubating the arteries for various times in calcium-free medium containing 10^-5 M EGTA. Incubation for 5–10 min abolished K⁺-induced contractions, whereas after 40 min of incubation PGF_2a still induced contractions that reached 70% of control. The PGF_2a-induced contraction was biphasic in 8 out of 10 preparations. The second phase could be eliminated by increasing the EGTA-concentration to 10^-4 M, as well as by nifedipine pretreatment. In calcium-free, high K⁺ medium calcium-induced contractions were elicited at lower concentrations in the presence of PGF_2a. The results suggest that PGF_2a-induced contractions in human pial arteries are relatively independent of free extracellular calcium. PGF_2a may promote trans-membrane influx of calcium, as well as release calcium from seemingly superficially located cellular stores.     

Ghrelin signalling in guinea-pig femoral artery smooth muscle cells

Aim: Our aim was to study the new signalling pathway of ghrelin in the guinea‐pig femoral artery using the outward I_K as a sensor. Methods: Whole‐cell patch‐clamp experiments were performed on single smooth muscle cells, freshly isolated from the guinea‐pig femoral artery. The contractile force of isometric preparations of the same artery was measured using a wire‐myograph. Results: In a Ca²⁺‐ and nicardipine‐containing external solution, 1 mmol L^?1 tetraethylammonium reduced the net I_K by 49 ± 7%. This effect was similar and not additive to the effect of the specific BK_Ca channel inhibitor iberiotoxin. Ghrelin (10^?7 mol L^?1) quickly and significantly reduced the amplitudes of tetraethylammonium‐ and iberiotoxin‐sensitive currents through BK_Ca channels. The application of 5 × 10^?6 mol L^?1 desacyl ghrelin did not affect the amplitude of the control I_K but it successfully prevented the ghrelin‐induced I_K decrease. The effect of ghrelin on I_K was insensitive to selective inhibitors of cAMP‐dependent protein kinase, soluble guanylyl cyclase, cGMP‐dependent protein kinase or a calmodulin antagonist, but was effectively antagonized by blockers of BK_Ca channels, phosphatidylinositol‐phospholipase C, phosphatidylcholine‐phospholipase C, protein kinase C, SERCA, IP₃‐induced Ca²⁺ release and by pertussis toxin. The ghrelin‐induced increase in the force of contractions was blocked when iberiotoxin (10^?7 mol L^?1) was present in the bath solution. Conclusions: Ghrelin reduces I_K(Ca) in femoral artery myocytes by a mechanism that requires activation of Gα_i/o‐proteins, phosphatidylinositol phospholipase C, phosphatidylcholine phospholipase C, protein kinase C and IP₃‐induced Ca²⁺ release.     

Segmental heterogeneity of swelling-induced Cl transport in rat small intestine

The effect of cell swelling induced by hypotonic media was studied in segments of rat small intestine. In the Ussing chamber, exposure to a hypotonic medium caused a decrease in short-circuit current (I _sc) and potential difference (V ^ms) in the jejunum, whereas the ileum responded with an increase in I _sc and V ^ms. The transition from one pattern to the other was located about in the middle of the small intestine. Tissue conductance decreased in both segments, probably due to a reduction of paracellular shunt conductance induced by the cell swelling. Voltage scanning experiments revealed that the observed decrease in total tissue conductance in the ileum was caused solely by a decrease in local conductance in the villus region while the crypt conductance did not change, suggesting that the decrease in paracellular conductance of the crypts is compensated by an increase in cellular conductance. The response in both segments was dependent on the presence of Cl⁻ and was blocked by the Cl⁻ channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB). It was not affected by the neurotoxin tetrodotoxin. In the jejunum the swelling-induced decrease in I _sc was reduced in the presence of the cyclooxygenase inhibitor, indomethacin, or the lipoxygenase inhibitor, nordihydroguaiaretic acid. In the ileum the Cl⁻ secretion induced by hypotonicity was blocked by the K⁺ channel blocker quinine and was reversed into a decrease in I _sc when serosal Ca²⁺ was zero. We conclude that the observed volume regulatory changes are initiated in the jejunum by an eicosanoid-mediated opening of basolateral Cl⁻ channels and in the ileum by a Ca²⁺-mediated opening of K⁺ channels which enhances apical Cl⁻ efflux. Received: 27 June 1995/Received after revision: 8 December 1995/Accepted: 28 December 1995     

Effects of prostanoids on isolated feline cerebral arteries

The effects of various prostanoids on isolated feline basilar arteries (BA) were studied. Contractions were induced with the following order of potency: U 46619 ? U44069 > PGB₂ > PGF_2a? PGA, ? PGB, ≥ PGA, ? PGE₂= PGD₂ > PGF_1a? TXB₂? PGE₁? PGD₁. Distinct bimodal responses with a relaxation at low concentrations followed by a contraction at high concentrations, were induced by PGA₁, PGA₂, PGD, and PGE₂. None of the tested prostanoids relaxed K⁺-contracted arteries, and a sizable relaxant effect in PGF_2a-contracted arteries could be induced only by PGE₁. As judged by the relative order of potency, PG-induced contractions of the feline BA seem to be mediated by a thromboxane sensitive receptor. PGF_2a-induced contractions apparently do not involve the release of noradrenaline from perivascular nerves since phentolamine failed to affect contractions induced by this agent.     

Regional intestinal adaptations in Na+,K+‐ATPase in experimental colitis and the contrasting effects of interferon‐γ

Aims: This study evaluated Na⁺,K⁺‐ATPase activity and the abundance of α₁ subunit Na⁺,K⁺‐ATPase in experimental colitis and gathered evidence on the effects of interferon‐γ (IFN‐γ) on intestinal Na⁺,K⁺‐ATPase. Methods: Colitis was induced by the intrarectal administration of 2,4,6‐trinitrobenzene sulphonic acid (TNBS, 30 mg/250 μL). Na⁺,K⁺‐ATPase activity was determined as the difference between total and ouabain‐insensitive ATPase. The abundance of Na⁺,K⁺‐ATPase was analysed by immunoblotting. Results: Na⁺,K⁺‐ATPase activity was markedly reduced in the proximal colonic mucosa of TNBS‐treated rats, whereas upstream in the terminal ileal mucosa a marked increase in sodium pump activity was observed. At the jejunal level no significant changes in Na⁺,K⁺‐ATPase activity were observed between TNBS‐treated rats and corresponding controls (ethanol‐treated rats). No changes were observed in the abundance of α₁ subunit Na⁺,K⁺‐ATPase in the proximal colon, terminal ileum and jejunum. The administration of IFN‐γ (50 000 U) 48 h before sacrifice reduced both Na⁺,K⁺‐ATPase activity and the abundance of α₁ subunit Na⁺,K⁺‐ATPase in the proximal colon. Dexamethasone prevented colonic inflammation and decreases in proximal colonic Na⁺,K⁺‐ATPase activity in TNBS‐treated rats, but did not affect the INF‐γ‐induced decrease in colonic Na⁺,K⁺‐ATPase activity. Conclusions: The increase in ileal Na⁺,K⁺‐ATPase activity upstream to the lesioned colonic mucosa, where Na⁺,K⁺‐ATPase activity was markedly reduced, might indicate a compensatory process to counteract the decrease in water and electrolyte absorption at the colonic level. This decrease in colonic Na⁺,K⁺‐ATPase activity is likely not related to INF‐γ‐induced downregulation of Na⁺,K⁺‐ATPase.     

Oxidative stress‐induced glucocorticoid resistance is prevented by dual PDE3/PDE4 inhibition in human alveolar macrophages

Background Oxidative stress is present in airway diseases such as severe asthma or Chronic Obstructive Pulmonary Disease and contributes to the low response to glucocorticoids through the down‐regulation of histone deacetylase (HDAC) activity. Objective To study the effects of the phosphodiesterase (PDE)‐3 and 4 inhibitors and their combination vs. glucocorticoids in a model of lipopolysaccharide (LPS)‐induced cytokine release in alveolar macrophages under oxidative stress conditions. Methods Differentiated U937 or human alveolar macrophages were stimulated with H₂O₂ (10–1000 μm ) or cigarette smoke extract (CSE, 0–15%) for 4 h before LPS (0.5 μg/mL, 24 h) addition. In other experiments, cells were pre‐treated with dexamethasone or budesonide (10^?9–10^?6 m ), with the PDE4 inhibitor rolipram (10^?9–10^?5 m ), PDE3 inhibitor motapizone (10 μm ), 3′,5′‐cyclic monophosphate enhancer PGE₂ (10 nm ), or with the combination of rolipram (10^?6 m )+PGE₂ (10 nm )+motapizone (10 μm ) 15 min before oxidants. IL‐8 and TNF‐α were measured by ELISA and HDAC activity by a colorimetric assay. Results Budesonide and dexamethasone produced a concentration‐dependent inhibition of the LPS‐induced IL‐8 and TNF‐α secretion with an E_max about 90% of inhibition, which was reduced by approximately 30% in the presence of H₂O₂ or CSE. Pre‐treatment with rolipram, motapizone or PGE2 only reached about 20% of inhibition but was not affected by oxidative stress. In contrast, PDE4/PDE3 combination in presence of PGE2 effectively inhibited the LPS‐induced cytokine secretion by about 90% and was not affected by oxidative stress. Combined PDE4 and PDE3 inhibition reversed glucocorticoid resistance under oxidative stress conditions. HDAC activity was reduced in the presence of oxidative stress, and in contrast to glucocorticoids, pre‐treatment with PDE4/PDE3 combination was able to prevent HDAC inactivity. Conclusions & Clinical Relevance This study shows that the combination of the PDE3/PDE4 inhibitors prevents alveolar macrophage activation in those situations of glucocorticoid resistance, which may be of potential interest to develop new effective anti‐inflammatory drugs in airway diseases. Cite this as: J. Milara, A. Navarro, P. Almudéver, J. Lluch, E. J. Morcillo and J. Cortijo, Clinical & Experimental Allergy, 2011 (41) 535–546.     

Hexose transport across the apical and basolateral membrane of enterocytes from different regions of the chicken intestine

The properties of hexose transport across the apical and basolateral membranes of chicken enterocytes have been studied in the small and large intestine. Results show that (a) isolated epithelial cells from all segments except the coprodeum can accumulate 3-O-methylglucose (Glc3Me) against a concentration gradient, by a Na⁺-dependent and phloridzin-sensitive mechanism, (b) The cell cumulative capacity for Glc3Me (control/phloridzin-incubated cells) is lower in the small intestine than in the large intestine (rectum = proximal caecum = ileum > jejunum > duodenum). (c) Theophylline enhances the cell Glc3Me cumulative capacity 2.9-fold in the duodenum and 2.4-fold in the jejunum but has no effect in the other segments studied. (d) Analysis of sugar uptake indicates that net hexose influx rates decrease from proximal to distal regions: jejunum > duodenum > ileum = proximal caecum = rectum for the apical transport system (-methyl glucoside as substrate and phloridzin as inhibitor) and duodenum > jejunum > ileum = proximal caecum = rectum for the basolateral system (2-deoxyglucose; theophylline). (e) The duodenum and the jejunum show high apical and basolateral hexose transport rates, which confer a significant capacity for sugar absorption on the proximal intestine. More distal regions, including the ileum, the proximal caecum and the rectum, have transport systems analogous to those of the proximal intestine that keep a considerable potential capability to recover hexoses from the lumen.     

Exercise-induced regulation of phospholemman (FXYD1) in rat skeletal muscle: implications for Na+/K+-ATPase activity

Background: Na⁺/K⁺‐ATPase activity is upregulated during muscle exercise to maintain ionic homeostasis. One mechanism may involve movement of α‐subunits to the outer membrane (translocation). Aim: We investigated the existence of exercise‐induced translocation and phosphorylation of phospholemman (PLM, FXYD1) protein in rat skeletal muscle and exercise‐induced changes in V_max and K_m for Na⁺ of the Na⁺/K⁺‐ATPase. Methods: Two membrane fractionation methods and immunoprecipitation were used. Results: Both fractionation methods revealed a 200–350% increase in PLM in the sarcolemma after 30 min of treadmill running, while the phosphorylation of Ser‐68 of PLM appeared to be unchanged. Exercise did not change V_max or K_m for Na⁺ of the Na⁺/K⁺‐ATPase in muscle homogenate, but induced a 67% increase in V_max in the sarcolemmal giant vesicle preparation; K_m for Na⁺ remained constant. The main part of the increase in V_max is related to a 36–53% increase in the level of α‐subunits; the remainder may be related to increased PLM content. Similar results were obtained with another membrane purification method. In resting muscle, 29% and 32% of α₁‐ and α₂‐subunits, respectively, were co‐immunoprecipitated by PLM antibodies. In muscle homogenate prepared after exercise, immunoprecipitation of α₁‐subunits was increased to 227%, whereas the fraction of precipitated α₂ remained constant. Conclusion: Exercise translocates PLM to the muscle outer membrane and increases its association with mainly the α₁‐subunit, which may contribute to the increased V_max of the Na⁺/K⁺‐ATPase.     

Sevoflurane reduces severity of acute lung injury possibly by impairing formation of alveolar oedema

Pulmonary oedema is a hallmark of acute lung injury (ALI), consisting of various degrees of water and proteins. Physiologically, sodium enters through apical sodium channels (ENaC) and is extruded basolaterally by a sodium–potassium–adenosine–triphosphatase pump (Na⁺/K⁺‐ATPase). Water follows to maintain iso‐osmolar conditions and to keep alveoli dry. We postulated that the volatile anaesthetic sevoflurane would impact oedema resolution positively in an in‐vitro and in‐vivo model of ALI. Alveolar epithelial type II cells (AECII) and mixed alveolar epithelial cells (mAEC) were stimulated with 20 µg/ml lipopolysaccharide (LPS) and co‐exposed to sevoflurane for 8 h. In‐vitro active sodium transport via ENaC and Na⁺/K⁺‐ATPase was determined, assessing ²²sodium and ⁸⁶rubidium influx, respectively. Intratracheally applied LPS (150 µg) was used for the ALI in rats under sevoflurane or propofol anaesthesia (8 h). Oxygenation index (PaO₂/FiO₂) was calculated and lung oedema assessed determining lung wet/dry ratio. In AECII LPS decreased activity of ENaC and Na⁺/K⁺‐ATPase by 17·4% ± 13·3% standard deviation and 16·2% ± 13·1%, respectively. These effects were reversible in the presence of sevoflurane. Significant better oxygenation was observed with an increase of PaO₂/FiO₂ from 189 ± 142 mmHg to 454 ± 25 mmHg after 8 h in the sevoflurane/LPS compared to the propofol/LPS group. The wet/dry ratio in sevoflurane/LPS was reduced by 21·6% ± 2·3% in comparison to propofol/LPS‐treated animals. Sevoflurane has a stimulating effect on ENaC and Na⁺/K⁺‐ATPase in vitro in LPS‐injured AECII. In‐vivo experiments, however, give strong evidence that sevoflurane does not affect water reabsorption and oedema resolution, but possibly oedema formation.     

Effects of prostanoids on isolated feline cerebral arteries

The roles of extra-and intracellular calcium for the contractile effects of PGF_2α in the feline basilar artery (BA) were investigated. Comparisons were made with contractions induced by K⁺ and noradrenaline (NA). Addition of nifedipine to PGF_2α-or K⁺ (124 mM)-contracted arteries resulted in an incomplete relaxation, whereas NA-contracted vessels were completely relaxed. Incubation of the preparations in a calcium-free medium containing 10^-5 M EGTA for 5–10 min almost abolished contractions induced by K⁺ and NA. In contrast, 63 % of the response to PGF_2α remained after pretreatment of the arteries in a calcium-free solution for 40 min; PGF_2α produced a biphasic contraction in 17 out of 20 preparations consisting of a rapidly developing initial phase followed by a second increase in tension after 1–6 min. The second phase was absent if the EGTA-concentration was increased to 10^-4 M, or if the arteries were pre-treated with nifedipine. After incubation of the arteries in a calcium-free medium for 40–120 min and K⁺-depolarization, re-addition of calcium elicited contractions at lower concentrations in the presence of PGF_2α than in controls. The results suggest that PGF_2α-induced contractions in the feline BA are considerably less dependent on extracellular calcium than contractions evoked by K⁺ or NA. PGF_2α appears to be able to release calcium from two cellular stores, and may also promote calcium influx through the cell membrane.     

Activation of nonselective cation channels in the basolateral membrane of rat distal colon crypt cells by prostaglandin E2

Ion channels in the basolateral membrane of colonic crypts were investigated with the patch-clamp technique during stimulation of secretion. Intact crypts were isolated from rat distal colon and the cell potential was recorded by addition of nystatin to the pipette solution. The cell resting potential in the base of the crypt was –74±1 mV (n=90). Addition of 100 M carbachol to the bath resulted in a transient hyperpolarization by 9 mV, which was probably due to the opening of basolateral K⁺ channels. In contrast, application of prostaglandin E₂ (PGE₂, 1 nM–1 M) caused a dose-dependent depolarization in the base of the crypt. With 1 M PGE₂ cells depolarized from -74±1 to –27±2 mV (n=26). Cell potential recordings in the midcrypt showed only a slight and transient depolarization after application of PGE₂, whereas cells close to the surface of the crypt had no response. In the base of the crypt the PGE₂-induced depolarization could be completely inhibited by addition of 50 M flufenamic acid, a known blocker of nonselective cation channels. After substitution of all monovalent cations by N-methyl-D-glucamine in the bath, PGE₂ had no significant effect on the cell potential. Cell-attached experiments with no nystatin in the patch pipette revealed the activation of ion channels in the basolateral membrane after application of PGE₂. After excision of the membrane patch, these channels could be identified as nonselective cation channels. Experiments involving substitution of the bath solution showed that the channel is impermeable for Cl^– and scarcely permeable for Ca²⁺ ions. The permeability sequence for monovalent cations, as calculated from reversal potentials, is NH ₄ ⁺ >Na⁺=K⁺>Rb⁺=Li⁺TRIS⁺=NMDG⁺. Single channels are completely inhibited by flufenamic acid (50 M), mefenamic acid (200 M), as well as by 3, 5-dichlorodiphenylamine-2-carboxylate. In conclusion, PGE₂ activates nonselective cation channels in the basolateral membrane of cells in the base of colonic crypts. It is suggested that this mechanism initiates the secretion of K⁺ ions. Na⁺ influx through the nonselective cation channel will stimulate the Na⁺/K⁺ pump and active uptake of K⁺ at the basolateral side. K⁺ can leave the cell at the luminal side through K⁺-selective channels.     

Inflammatory cytokines in paraventricular nucleus modulate sympathetic activity and cardiac sympathetic afferent reflex in rats

Aims: G protein‐coupled receptors such as the AT_1aR are frequently subject to desensitization, extensively studied in cell culture but to small extent in hypertensive models. Recently, angiotensin II (ANG II)‐induced desensitization was shown to last 10 min in isolated afferent arterioles (AAs), suggesting impact on ANG II vasoactivity. In the present study, we explored ANG II desensitization and effects of adenosine (Ado) in AAs from two‐kidney, one‐clip (2K1C) hypertensive rats. Our main hypothesis was that Ado affects ANG II contractility differently in 2K1C, because of persistently elevated levels of ANG II. Methods: Afferent arterioles were isolated with the agarose‐infusion/enzyme‐treatment technique from normotensive and 2K1C hypertensive rats, and stimulated with ANG II (10^?7 m ) at baseline and re‐stimulated after 20 or 40 min, with or without Ado (2.5 × 10^?5 m ) in the vessel bath. Results: Afferent arterioles from normotensive rats re‐stimulated with ANG II after 20 min displayed a blunted contraction (Δ12.8 ± 4.3%, P < 0.05), which disappeared when AAs were stimulated after 40 min (Δ2.7 ± 2.3%, NS), indicating that desensitization lasted for 30 ± 10 min. Ado augmented ANG II contractions after 20 min, but not after 40 min, suggesting that only de‐sensitized vessels were affected. Similar experiments in AAs from the clipped and non‐clipped kidneys revealed no desensitization when re‐stimulated with ANG II after 20 and 40 min, and contractions were unaffected by Ado. Conclusions: Reduced duration of desensitization in AAs from 2K1C may cause vessels to be sensitized longer and increase vasoconstriction. The present study demonstrates that Ado does not augment ANG II‐induced contractions in AAs from 2K1C as in normotensive rats, possibly because of a reduced period of desensitization.     

Angiotensin II induced contraction of rat and human small intestinal wall musculature in vitro

Background: Angiotensin II (Ang II) is a well‐known activator of smooth muscle in the vasculature but has been little explored with regard to intestinal wall muscular activity. This study investigates pharmacological properties of Ang II and expression of its receptors in small‐intestinal smooth muscle from rats and humans. Methods: Isometric recordings were performed in vitro on small intestinal longitudinal muscle strips. Protein expressions of Ang II typ 1 (AT1R) and typ 2 (AT2R) receptors were assessed by Western blot. Results: Ang II elicited concentration‐dependent contractions of rat jejunal and ileal muscle preparations. The concentration–response curve (rat ileum, EC₅₀: 1.5 ± 0.9 × 10^?8 m ) was shifted to the right by the AT1R receptor antagonist losartan (10^?7 m ) but was unaffected by the AT2R antagonist PD123319 (10^?7 m ) as well as by the adrenolytic guanethidine (3 × 10^?6 m ) and the anticholinergic atropine (10^?6 m ). Human duodenal, jejunal and ileal longitudinal muscle preparations all contracted concentration‐dependently in response to Ang II. The concentration–response curve (human jejunum, EC₅₀: 1.5 ± 0.8 × 10^?8 m ) was shifted to the right by losartan (10^?7 m ) but was unaffected by PD123319 (10^?7 m ). Both AT1R and AT2R were detected in all segments of the rat small intestinal wall musculature, whereas only AT1R was readily detectable in the human samples. Conclusion: Ang II elicits contractions of small‐intestinal longitudinal muscle preparations from the small intestine of rats and man. The pharmacological pattern and protein expression analyses indicate mediation via the AT1R.     

Induction of heme oxygenase in guinea‐pig stomach: roles in contraction and in single muscle cell ionic currents

The role of heme oxygenase reaction products in modulation of stomach fundus excitability was studied. The presence of constitutive heme oxygenase 2 was verified in myenteric ganglia by immunohistochemistry. The role of inducible heme oxygenase isoenzyme was investigated after invivo treatment of animals with CoCl₂ (80 mg kg^?1 b.w) injected subcutaneously 24 h before they were killed. This treatment resulted in increased production of bilirubin and positive staining for the inducible isoform in stomach smooth muscle and vast induction in the liver. In both control and treated animals haemin, applied to the bath as a substrate of heme oxygenase caused significant decrease of prostaglandin F_2α‐induced tone, and ameliorated the relaxatory response of the fundic strips to electrical field stimulation. Both effects were antagonized by Sn‐protoporphyrin IX, competitive heme oxygenase inhibitor, and were found to be neuronally dependent. In single freshly isolated smooth muscle cells from control animals haemin caused a concentration‐dependent increase of the whole cell K⁺ currents, which was not affected by Sn‐protoporphyrin IX, cyclic guanosine monophosphate (cGMP)‐dependent protein kinase or guanylyl cyclase antagonists, but was reversed by various antioxidants and abolished by an NO scavenger. In cells from treated animals the K⁺ current increasing effect of haemin did not depend on the presence of antioxidants, but was abolished by protein kinase G and guanylyl cyclase inhibitors, depletors of intracellular Ca²⁺ pools or Sn‐protoporphyrin IX. Biliverdin did not affect contraction or ionic currents. Thus, this is the first study demonstrating that heme oxygenase is an inducible enzyme in guinea‐pigs, which exerts a modulatory role on gastric smooth muscle excitability via carbon monoxide production.     

Enhancement of depolarization-induced contractions after endothelium denudation is not related to an impaired production of nitric oxide or prostacyclin in the rabbit basilar artery

Enhancement of the extracellular potassium ion (K⁺) concentration combined with endothelial injury have been suggested to occur during cerebral ischaemia-reperfusion and vasospasm after subarachnoid hemorrhage. The effect of potassium (K⁺) depolarization was therefore investigated in isolated segments of the rabbit basilar artery with and without an intact endothelial cell layer. Addition of potassium chloride to the organ bath induced a concentration-dependent contraction. Endothelial denudation of the artery resulted in an unstable baseline tension and a leftward shift of the K⁺ concentration-response curve. The K⁺ concentration eliciting half maximum contraction decreased from 26 mmol l^-1 in the presence to 12 mmol l“¹ in the absence of an intact endothelium. Nimodipine (3 × 10^-7 mol l^-1) or exposure to a calcium-free medium abolished the spontaneous as well as K⁺-induced contractions. A^-nitro-L-arginine (10^-4 mol l^-1), indomethacin (3 × 10^-6 mol l^-1) and glibenclamide (10^-5 mol I^-1) did not affect the contractile response to K⁺ in intact arteries. However, N_w-nitro-D-arginine increased the baseline tension, and this effect could not be reproduced with N_w-nitro-D-arginine. Pinacidil (10^-6mol l^-l) abolished the spontaneous contractile activity in endothelium-denuded arteries and reduce the K⁺ sensitivity to the same level as in intact arteries. Tetraethylammonium (3 mmol l^-1) and ouabain (10^-5 mol I^-l) increased the basal tension and shifted the K⁺ concentration-response curve to the left. Calcium-induced contractions in preparations exposed to a calcium-free, 124 mmol l^-l K.⁺ solution did not differ between endothelium-denuded and intact arteries. It is suggested that the endothelium of the rabbit basilar artery releases a hyperpolarizing factor, distinct from nitric oxide or a cyclooxygenase product, which attenuates the vasoconstrictor effect of K⁺ depolarization.     

Effect of aflatoxin B1 on IgA+ cell number and immunoglobulin mRNA expression in the intestine of broilers

Abstract

Aflatoxin B₁ (AFB₁) is the most toxic group of mycotoxins produced by two species of the Aspergillus, common contaminants of food and animal feed. The purpose of our study was to determine the effect of AFB₁ on the number of IgA⁺ cell and immunoglobulin mRNA expression in the intestine of broilers. One hundred and fifty six one-day-old healthy Cobb broilers were randomly divided into the control group (the dosage of 0?mg/kg AFB₁) and AFB₁ group (the dosage of 0.6?mg/kg AFB₁) with three replicates per group and 26 birds per replicate for 21 days, respectively. After necropsy at 7, 14 and 21 days of age, duodenum, jejunum and ileum samples were taken for analyzing IgA⁺ cell by immunohistochemistry and IgA, pIgR, IgM and IgG mRNA expression by qRT-PCR. IgA⁺ cells were mainly distributed in the lamina propria of small intestinal mucosa in both groups at 14 and 21 days of age. A significant decrease in the number of IgA⁺ cells in the duodenum, jejunum and ileum was revealed in the AFB₁ group compared with that of the control group. The expression levels of IgA, pIgR, IgM and IgG mRNA in the intestinal mucosa were lower in the AFB₁ group than those in the control group at 14 and 21 days of age. Our data demonstrated that the dosage of 0.6?mg/kg AFB₁ in broiler diet reduced the number of IgA⁺ cell and the expression of IgA, pIgR, IgM and IgG mRNA in the small intestine.     

Regulation of K+ transport in the rat distal colon via angiotensin II subtype receptors and K+‐pathways

The role of angiotensin subtype‐1 (AT₁) and ‐2 (AT₂) receptors in mediating the effects of angiotensin II (ANG II) on several K⁺ transporters was studied in rat distal colon using an Ussing chamber. Angiotensin II induced K⁺ secretion at two different doses. Secretion occurred at 10^–8 and 10^–4 M , as a result of an increase in serosal‐to‐mucosal flux (J_s–m). The ANG II‐induced stimulation of J_s–m at a low dose (10^–8 M ) was abolished by PD123319 while losartan did not alter the low‐dose ANG II‐dependent increase in J_s–m. In contrast, the increase in J_s–m induced by a high‐dose of ANG II (10^–4 M ) was blocked by losartan, whereas PD123319 partially reduced the stimulatory effect. In the presence of both blockers, high‐dose ANG II induced an inhibition of basal J_s–m. Low‐dose ANG II activated the barium‐sensitive K⁺ channels, whereas the Na⁺, K⁺, 2Cl^– cotransporter and the Na⁺, K⁺‐ATPase pump were unchanged. At the high dose, ANG II activated the barium‐sensitive K⁺ channels and the Na⁺, K⁺, 2Cl^– cotransporter and inhibited the Na⁺, K⁺‐ATPase pump. These data indicate that ANG II stimulates serosal‐to‐mucosal K⁺ flux in the rat distal colon at high and low doses via different receptors and K⁺ transporters.     

Role of Na+,K+ pumps in restoring contractility following loss of cell membrane integrity in rat skeletal muscle

Background and aim: In skeletal muscles, electrical shocks may elicit acute loss of force, possibly related to increased plasma membrane permeability, induced by electroporation (EP). We explore the role of the Na⁺,K⁺ pumps in force recovery after EP. Methods: Isolated rat soleus or extensor digitorum longus (EDL) muscles were exposed to EP paradigms in the range 100–800 V cm^?1, and changes in tetanic force, Na⁺,K⁺ contents, membrane potential, ¹⁴C‐sucrose space and the release of the intracellular enzyme lactic acid dehydrogenase (LDH) were characterized. The effects of Na⁺,K⁺ pump stimulation or inhibition were followed. Results: Electroporation caused voltage‐dependent loss of force, followed by varying rates and degrees of recovery. EP induced a reversible loss of K⁺ and gain of Na⁺, which was not suppressed by tetrodotoxin, but associated with increased ¹⁴C‐sucrose space and release of LDH. In soleus, EP at 500 V cm^?1 induced complete loss of force, followed by a spontaneous, partial recovery. Stimulation of active Na⁺,K⁺ transport by adrenaline, the β₂‐agonist salbutamol, calcitonin gene‐related peptide (CGRP) and dibutyryl cyclic AMP increased initial rate of force recovery by 183–433% and steady‐state force level by 104–143%. These effects were blocked by ouabain (10^?3 m ), which also completely suppressed spontaneous force recovery. EP caused rapid and marked depolarization, followed by a repolarization, which was accelerated by salbutamol. Also in EDL, EP caused complete loss of force, followed by a spontaneous partial recovery, which was markedly stimulated by salbutamol. Conclusion: Electroporation induces reversible depolarization, partial rundown of Na⁺,K⁺ gradients, cell membrane leakage and loss of force. This may explain the paralysis elicited by electrical shocks. Na⁺,K⁺ pump stimulation promotes restoration of contractility, possibly via its electrogenic action. The major new information is that the Na⁺,K⁺ pumps are sufficient to compensate a simple mechanical leakage. This may be important for force recovery in leaky muscle fibres.