Short-term functional adaptation of aquaporin-1 surface expression in the proximal tubule, a component of glomerulotubular balance.

Transepithelial water flow across the renal proximal tubule is mediated predominantly by aquaporin-1 (AQP1). Along this nephron segment, luminal delivery and transepithelial reabsorption are directly coupled, a phenomenon called glomerulotubular balance. We hypothesized that the surface expression of AQP1 is regulated by fluid shear stress, contributing to this effect. Consistent with this finding, we found that the abundance of AQP1 in brush border apical and basolateral membranes was augmented >2-fold by increasing luminal perfusion rates in isolated, microperfused proximal tubules for 15 minutes. Mouse kidneys with diminished endocytosis caused by a conditional deletion of megalin or the chloride channel ClC-5 had constitutively enhanced AQP1 abundance in the proximal tubule brush border membrane. In AQP1-transfected, cultured proximal tubule cells, fluid shear stress or the addition of cyclic nucleotides enhanced AQP1 surface expression and concomitantly diminished its ubiquitination. These effects were also associated with an elevated osmotic water permeability. In sum, we have shown that luminal surface expression of AQP1 in the proximal tubule brush border membrane is regulated in response to flow. Cellular trafficking, endocytosis, an intact endosomal compartment, and controlled protein stability are the likely prerequisites for AQP1 activation by enhanced tubular fluid shear stress, serving to maintain glomerulotubular balance.

Calcium regulation of tight junction permeability.

Calcium transport in the kidney is a key element in Ca(2+) homeostasis. Ca(2+) concentration, or more precisely the activity of freely dissociated Ca(2+) ions, is a prerequisite for the appropriate function of virtually every cell. Along the renal tubule, about 85% of the filtered Ca(2+) is transported across tight junctions at the paracellular route of reabsorption. Therefore, claudins, which form the conductive and selective part of the tight junctions, have moved into the focus of interest with respect to regulatory events in the control of Ca(2+) transport. This control is of particular interest for the kidney since it has to defend itself against nephrocalcinosis and kidney stones. Tight junction proteins provide pathways, driving forces, and regulatory targets for Ca(2+) transport. Direct regulation of tight junctions by changing Ca(2+) concentrations allows fast and efficient feedback loops to adapt Ca(2+) transport to the requirements of kidney function and plasma Ca(2+) concentration.

Human beta-defensin-2 increases cholinergic response in colon epithelium.

The human beta-defensin-2 (hBD-2) is expressed in epithelial cells of skin and respiratory and gastrointestinal tracts. Defensins are arginine-rich small cationic peptides with six intramolecular disulfide bonds and are antimicrobially active against a broad spectrum of pathogens. In addition, they have cytokine-like immunomodulatory properties. We hypothesized that hBD-2 also might influence epithelial cells themselves, thereby altering fluid composition in the gastrointestinal tract. We therefore tested its impact on electrogenic ion transport properties of distal colon in Ussing chamber experiments. Application of hBD-2 did not affect transepithelial voltage or resistance in cAMP-stimulated distal colon. However, it increased cholinergic Ca(2+)-dependent Cl(-) secretion. After 20 min of incubation with hBD-2, the effect of carbachol (CCh) on the equivalent short circuit current (I'(sc)) was enhanced twofold compared to vehicle-treated colon. Modulation of Ca(2+) signaling by hBD-2 was validated by Fura-2 measurements in human colon carcinoma HT29 cells. Twenty-minute incubation with hBD-2 increased the CCh-induced Ca(2+) transient by 20-30% compared to either vehicle-treated cells or cells treated with the defensins hBD-1, hBD-3, or HD-5. This effect was concentration-dependent, with an EC(50) of 0.043 microg/ml, and still present in the absence of extracellular Ca(2+). Also, the ionomycin-induced Ca(2+) transient was increased by hBD-2 treatment. We conclude that hBD-2 facilitates cholinergic Ca(2+)-regulated epithelial Cl(-) secretion. These findings contribute to the concept of a specific interaction of antimicrobial peptides with epithelial function.

Targeted deletion of murine Cldn16 identifies extra- and intrarenal compensatory mechanisms of Ca2+ and Mg2+ wasting.

Claudin-16 (CLDN16) is critical for renal paracellular epithelial transport of Ca(2+) and Mg(2+) in the thick ascending loop of Henle. To gain novel insights into the role of CLDN16 in renal Ca(2+) and Mg(2+) homeostasis and the pathological mechanisms underlying a human disease associated with CLDN16 dysfunction [familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC), OMIM 248250], we generated a mouse model of CLDN16 deficiency. Similar to patients, CLDN16-deficient mice displayed hypercalciuria and hypomagnesemia. Contrary to FHHNC patients, nephrocalcinosis was absent in our model, indicating the existence of compensatory pathways in ion handling in this model. In line with the renal loss of Ca(2+), compensatory mechanisms like parathyroid hormone and 1,25(OH)(2)D(3) were significantly elevated. Also, gene expression profiling revealed transcriptional upregulation of several Ca(2+) and Mg(2+) transport systems including Trpv5, Trpm6, and calbindin-D9k. Induced gene expression was also seen for the transcripts of two putative Mg(2+) transport proteins, Cnnm2 and Atp13a4. Moreover, urinary pH was significantly lower when compared with wild-type mice. Taken together, our findings demonstrate that loss of CLDN16 activity leads to specific alterations in Ca(2+) and Mg(2+) homeostasis and that CLDN16-deficient mice represent a useful model to further elucidate pathways involved in renal Ca(2+) and Mg(2+) handling.

Insights into driving forces and paracellular permeability from claudin-16 knockdown mouse.

Tight junction (TJ) properties are determined by membrane protein complexes of neighboring cells that form both a barrier and a selective pathway for paracellular substrate transport. Our previous work supports the view that paracellular permeability changes in the thick ascending limb (TAL) may underlie the mechanism for familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC), a rare autosomal recessive disease linked to mutations in claudin-16 (CLDN16) and claudin-19 (CLDN19). CLDN16 knockdown (KD) mice are lacking CLDN16 expression by transgenic RNA interference. We observed that the transport defect for Mg(2+) and Ca(2+) in this animal model is caused by a loss of paracellular cation selectivity. The permeability ratio for Na(+) over Cl(-) in KD mice was lower by a factor of two without a change in paracellular conductance, compared to wild type (WT). This resulted in a collapse of the transepithelial voltage, which is the driving force for Mg(2+) and Ca(2+) absorption in TAL. Since CLDN16 KD mice revealed lower blood pressure and an increased aldosterone plasma concentration, we hypothesize that the reduction in paracellular selectivity could allow backflow of Na(+) and Cl(-) into the lumen of the TAL, thus enhancing the distal NaCl load and challenging the organism with a latent NaCl loss.

[Endothelin-1 and nitric oxide mediated the lipopolysaccharide-induced cardiac negative inotropic role].

AIM: To investigate the effects of endothelin-1 (ET-1) and nitric oxide (NO) on lipopolysaccharide(LPS)-induced myocardial dysfunction, and explore the related underlying mechanisms. METHODS: Experimental septic model was established by intraperitoneal injection of LPS (10 mg x kg(-1)). The study was carried out on the isolated rat hearts to determine the roles of ET-1 and NO in the effect of LPS on the cardiac contractility and on the isolated rat ventricular myocytes model to observe the [Ca2+]i homeostasis in cardiac myocytes. RESULTS: (1) The levels of serum NO2-/NO3- and plasma ET-1 were markedly increased by LPS treatment for 4 hours. (2) LPS induced the decrease in rate-pressure product (RPP), and increase in left ventricular end-diastolic pressure (LVEDP) in the isolated perfused rat hearts. Pretreatment with either aminoguanidine (AMG) (100 mg x kg(-1), i.p.) or BQ-123 (1 mg x kg(-1), i.p.) partially attenuated LPS-induced myocardial depression. When these two drugs were simultaneously given, myocardial depression elicited by LPS was almost abolished. (3) LPS significantly decreased the amplitude of caffeine induced [Ca2+]i transients compared to the control cells. The activity of SR Ca22+ -ATPase was significantly decreased in the cardiac myocytes from LPS-treated rats. Single pretreatment with either AMG or BQ-123 did not attenuate the impairment of SR Ca2+ -ATPase induced by LPS. CONCLUSION: ET-1 and NO mediate myocardial dysfunction in hearts isolated and decrease [Ca2+]i transients in cardiac myocytes from LPS-treated rats. But neither ET-1 nor NO participates in the impairment of SR Ca2+ -ATPase induced by LPS.

Salt and acid-base metabolism in claudin-16 knockdown mice: impact for the pathophysiology of FHHNC patients.

Claudin-16 is defective in familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC). Claudin-16 knockdown (CLDN16 KD) mice show reduced cation selectivity in the thick ascending limb. The defect leads to a collapse of the lumen-positive diffusion voltage, which drives Ca(2+) and Mg(2+) absorption. Because of the reduced tight junction permeability ratio for Na(+) over Cl(-), we proposed a backleak of NaCl into the lumen. Systemic analysis had revealed lower blood pressure and a moderately increased plasma aldosterone concentration. In this study, we measured the amiloride-sensitive equivalent short-circuit current in isolated, perfused collecting ducts and found it increased by fivefold in CLDN16 KD mice compared with wild-type (WT) mice. Amiloride treatment unmasked renal Na(+) loss in the thick ascending limb of the nephron. Under amiloride treatment, CLDN16 KD mice developed hyponatremia and the renal fractional excretion of Na(+) was twofold higher in CLDN16 KD compared with WT mice. The loss of claudin-16 also resulted in increased urinary flow, reduced HCO(3)(-) excretion, and lower urine pH. We conclude that perturbation in salt and acid-base metabolism in CLDN16 KD mice has its origin in the defective cation permselectivity of the thick ascending limb of the nephron. This study has contributed to the still incomplete understanding of the symptoms of FHHNC patients.

Epithelial K⁺ channels: driving force generation and K⁺ recycling for epithelial transport with physiological and clinical implications.

K(+) channels form a large family of membrane proteins that are expressed in a polarized fashion in any epithelial cell. Based on the transmembrane gradient for K(+) that is maintained by the Na(+)-K(+)-ATPase, these channels serve two principal functions for transepithelial transport: generation of membrane voltage and recycling of K(+). In this brief review, we will outline the importance of this ancient principle by examples of epithelial transport in the renal proximal tubule and gastric parietal cells. In both tissues, K(+) channel activity is rate-limiting for transport processes across the epithelial cells and essential for cell volume regulation. Recent experimental data using pharmacological tools and genetically modified animals have confirmed the original physiological concepts and specified the knowledge down to the molecular level. The development of highly active and tissue selective small molecule therapeutics has been impeded by two typical features of K(+) channels: their molecular architecture challenges the design of molecules with high affinity binding and they are expressed in a variety of tissues at the same time. Nevertheless, new insights into pathophysiology, e.g. that K(+) channel inhibition can block gastric acid secretion, render the clinical use of K(+) channel drugs in gastric disease and as kidney transport inhibitors highly attractive.

Transgenic RNAi depletion of claudin-16 and the renal handling of magnesium.

Tight junctions play a key role in mediating paracellular ion reabsorption in the kidney. Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is a human disorder caused by mutations in the tight junction protein claudin-16. However, the molecular mechanisms underlining the renal handling of magnesium and its dysfunction causing FHHNC are unknown. Here we show that claudin-16 plays a key role in maintaining the paracellular cation selectivity of the thick ascending limbs of the nephron. Using RNA interference, we have generated claudin-16-deficient mouse models. Claudin-16 knock-down (KD) mice exhibit chronic renal wasting of magnesium and calcium and develop renal nephrocalcinosis. Our data suggest that claudin-16 forms a non-selective paracellular cation channel, rather than a selective Mg(2+)/Ca(2+) channel as previously proposed. Our study highlights the pivotal importance of the tight junction in renal control of ion homeostasis and provides answer to the pathogenesis of FHHNC. We anticipate our study to be a starting point for more sophisticated in vivo analysis of tight junction proteins in renal functions. Furthermore, tight junction proteins could be major targets of drug development for electrolyte disorders.

[The alterations of nitric oxide synthase activity of ventricular cardiac muscle of rats in two septic shock models].

AIM: To observe the differences of hemodynamics and nitric oxide synthase(NOS) activity of ventricular cardiac muscle in two septic shock models and explore the possible mechanism. METHODS: Two rat models of septic shock[lipopolysaccharide(LPS)-induced and cecal ligation and puncture (CLP)-induced septic shock] were used. The hemodynamic parameters and nitric oxide synthase activity of ventricular cardiac muscle were measured. RESULTS: The hemodynamic parameters in CLP-induced model were increased in the early stage and decreased in the late stage while in LPS-induced model the parameters showed the same change of the CLP late stage. Both LPS model and CLP model (late stage) showed significant increase in NOS activity, but there was no difference between the two models. After treatment of the NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME), the parameters of CLP-late stage and LPS model increased significantly. The NOS activity reached the highest level in the CLP-middle stage. The production of nitrite/nitrate decreased significantly in LPS model and CLP model(late stage) after treatment of L-NAME, but the nitrite/nitrate produced by constitutive NOS in LPS model was higher than CLP model(late stage). CONCLUSION: The increase of the NOS activity may be the main reason to lead to the depression of the hemodynamic parameters. Inducible NOS may play the leading role in the LPS model while cNOS and iNOS have the same effect in the CLP model.

[Effect of cinobufacini on vascular contractile of rat thoracic aorta].

OBJECTIVE: To examine the effect of cinobufacini on rat thoracic aorta and its mechanism. METHODS: Isolated rat thoracic aorta was perfused and isometric tension was recorded by organ bath technique before and after cinobufacini treatment. RESULT: Cinobufacini induced contraction of isolated thoracic aorta with or without endothelium in a concentration-dependent manner (at concentration of 2.5,5.0,7.5,10.0 g/L). The vasoconstriction effect of cinobufacini was more potent in endothelium-denuded aorta ring [(16.3+/-3.39)%, (52.5+/-7.70)%, (60.9+/-8.84)%, (69.2+/-11.34)%] than in endothelium-intact aorta ring [(6.2+/-2.07)%, (14.7+/-4.91), (17.6+/-5.86)%, (20.3+/-6.78)% (P<0.01)]. Its contractile effect was attenuated in Ca(2+)-free solution (about 1/10 of that in buffer with 1.25 mmol/L CaCl(2)) or by the treatment with verapamil (10(-7)mol/L), an L-type calcium channel antagonist. Cinobufacini induced contraction on the endothelium-intact rat aorta was augmented by pretreatment with L-NAME (10(-4)mol/L), a nitric oxide synthase inhibitor. CONCLUSION: Cinobufacini contracts rat thoracic aorta by opening the voltage-dependent Ca(2+) channel and increasing Ca(2+) influx into vascular smooth muscle. Cinobufacini can also stimulate the release of vascular relaxant factor, nitric oxide, from the endothelium and thus antagonize cinobufacini-induced contraction.

Diphasic effects of Astragalus membranaceus BUNGE (Leguminosae) on vascular tone in rat thoracic aorta.

This study was designed to investigate the effects of the aqueous ethanol extract of Astragalus membranaceus BUNGE (Leguminosae) on rat thoracic aorta. Isometric tension was recorded in response to drugs in organ bath. In endothelium-intact aortic rings, A. membranaceus extract induced a significant dose-dependent relaxation of the rings precontracted by phenylephrine, which could be inhibited by preincubation with L-N(omega)-nitro-arginine methyl ester or methylthioninium chloride. In endothelium-denuded ones, the extract could dose-dependently relax the rings contracted by phenylephrine, not by KCl; and it could also attenuate contractile response to phenylephrine, not to caffeine or phorbol-12,13-diacetate in Ca(2+)-free medium; but it failed to affect the CaCl(2)-induced enhancement of contractile response to phenylephrine in Ca(2+)-free medium. These results indicate that nitric oxide signaling and Ca(2+)-handling pathway are involved in the A. membranaceus extract-induced vasodilatation.

[Relaxant effect of Astragalus membranaceus on smooth muscle cells of rat thoracic aorta].

OBJECTIVE: To investigate the effect of Astragalus membranaceus(AM) on vascular circles and the underlying mechanisms. METHODS: The study was performed with the model of isolate rat thoracic aorta rings in organ bath. When the endothelium of rat thoracic aorta was removed,the effect of accumulated AM on aorta rings in resting tension, or pre-constricted with KCl, or pre-constricted with phenylephrine (PE) was observed. And to explove the mechanism, the aorta rings were incubated with Ca(2+)-free medium alone, or Ca(2+)-free medium plus heparin, or propranolol alone before pre-contraction with PE. RESULTS: AM had no significant effects on aorta rings in resting tension or pre-constricted with KCl. When the concentration of AM was cumulated to 10(-1), 3 x 10(-1),10(0), 3 x 10(0) g/L, it caused concentration-dependent relaxation while aorta rings were pre-constricted with PE(3 x 10(-7)mol/L), compared with the control [(90.4 +/-4.2)% compared with (94.7 +/-2.4)%,(86.1 +/-5.0)% compared with (92.6 +/-3.2)%, (82.3 +/-5.9)% compared with (90.4 +/-3.6) %, (78.3 +/-6.0)% compared with (88.1 +/-4.0)%]. This effect was not inhibited by Ca(2+)-free medium or propranolol alone. However, the effect was attenuated by the co-incubation with heparin and Ca(2+)-free medium [without heparin:(76.2+/-4.3)% compared with (92.3 +/-5.9)%, with heparin: (95.3+/-0.5)% compared with (95.1+/-0.6)%]. CONCLUSION: The results indicate that AM can relax the rat thoracic aorta rings without endothelium. The mechanism may include the inhibition of intracellular calcium ions release by the 1,4,5-triphosphate inositol-receptor-dependent pathway in vascular smooth muscle cells.

The relationship of ventricular dynamics and mitochondrial nitric oxide synthase activity in septic shock models.

The aim of the present study was to investigate the role of mitochondrial nitric oxide synthase (mtNOS) in the septic shock and analyze its relationship to ventricular contractility. Two models of septic shock [lipopolysaccharide (LPS)-induced and cecal ligation and puncture (CLP)-induced] were used. There was a significant depression of ventricular contractile parameters recorded in the late stage of the septic shock. After measurement of ventricular-dynamic parameters, mitochondrial and cytoplasmic fractions were isolated and their nitric oxide synthase (NOS) activity was assessed using a NOS activity assay kit. Both models showed a larger increase in mitochondrial NOS activity than that in cytosol. However, the increase in mtNOS activity in the LPS-induced shock model was less pronounced than in the CLP-induced model. Regression analysis shows that mitochondrial nitric oxide synthase (mtNOS) activity is negatively correlative to the left ventricular developed pressure in CLP model. The results suggest that mitochondrial NOS may mainly contribute to the ventricular depression in the septic shock.

Endothelin-1-mediated coronary vasoconstriction deteriorates myocardial depression in hearts isolated from lipopolysaccharide-treated rats: interaction with nitric oxide.

1. The aim of the present study was to evaluate the contribution of disturbance of coronary perfusion to myocardial depression in hearts isolated from lipopolysaccharide (LPS)-treated rats and to investigate the involvement of endothelin (ET)-1 and nitric oxide (NO). 2. Rats were treated with LPS (10 mg/kg, i.p.) and, 4 h later, plasma ET-1 concentrations were measured by radioimmunoassay and hearts were excised for perfusion at a constant perfusion flow. The selective ETA receptor antagonist BQ-123, in the absence or presence of aminoguanidine, a specific inhibitor of inducible NO synthase, was given 15 min before LPS challenge. Coronary perfusion pressure (CPP) and measures of myocardial contractile function were recorded. 3. In hearts isolated from LPS-treated rats, there was a marked increase in CPP that was abolished by pretreatment with BQ-123. In parallel, an increase in plasma ET-1 concentrations was seen in these rats. Lipopolysaccharide also induced decreases in left ventricular developed pressure (LVDP), the product of LVDP and heart rate and maximal rate of rise/fall of left ventricular pressure (+/- dP/dtmax). Single treatment with BQ-123 or aminoguanidine attenuated LPS-induced myocardial depression. However, when these two drugs were given simultaneously, myocardial depression elicited by LPS was blocked significantly. 4. Endothelin-1-mediated coronary vasoconstriction, together with NO, contributes to myocardial depression in hearts isolated from LPS-treated rats.

Role of cyclooxygenase in ventricular effects of adrenomedullin: is adrenomedullin a double-edged sword in sepsis?

Adrenomedullin (ADM) is upregulated in cardiac tissue under various pathophysiological conditions. However, the direct inotropic effect of ADM on normal and compromised cardiomyocytes is not clear. In rat ventricular myocytes, ADM produced an initial (<30 min) increase in cell shortening and Ca(2+) transient and, on prolonged incubation (>1 h), a marked decrease in cell shortening and Ca(2+) transient. Both effects were sensitive to inhibition by the ADM antagonist ADM-(22-52). The increase and decrease in cell shortening and Ca(2+) transient were attenuated by pretreatment with indomethacin [a nonspecific cyclooxygenase (COX) inhibitor], nimesulide and SC-236 (specific COX-2 inhibitors), and tranylcypromine (a prostacyclin synthase inhibitor); SQ-29548 (a thromboxane receptor antagonist) was without effect. Cells isolated from LPS-treated rats that were in the late, hypodynamic phase of septic shock also showed a marked decrease in cell shortening and Ca(2+) transient. Because ADM is overexpressed in sepsis, we repeated the above protocol in cells isolated from LPS-treated rats. At 4 h after LPS injection, ADM levels markedly increased in plasma, ventricles, and freshly isolated ventricular myocytes. Decreases in cell shortening and Ca(2+) transient in LPS-treated cells were reversed by pretreatment with ADM-(22-52). Anti-ADM (rat) IgG also reversed the decrease in cell shortening and other parameters of cell kinetics. Indomethacin, SC-236, and tranylcypromine restored cell contractility and the decrease in Ca(2+) transient, whereas SQ-29548 had no effect, implying that prostacyclin played a role in both effects. However, with regard to cell-shortening kinetics, indomethacin and SQ-29548 decreased the amount of time taken by the cells to return to baseline, whereas SC-236 and tranylcypromine did not, implying that not only prostacyclin, but also thromboxane, is involved. The results indicate that ADM interacts with COX to yield prostanoids, which mediate its negative inotropic effect in LPS-treated rat ventricular myocytes.

[Endothelium-independent vasorelaxant effect of puerarin on rat thoracic aorta].

OBJECTIVE: To investigate the vasorelaxant effect of puerarin in rat aortic rings and the mechanism. METHOD: The isolated thoracic aortic rings of male Sprague-Dawley rats were mounted on the organ bath and the contractile responses of the vessel were recorded. RESULT: Puerarin completely relaxed the contractions induced by phenylephrine in a concentration-dependent manner in endothelium-intact and endothelium-denuded rat aorta, but it had no effect on those preconstricted by a high concentration of potassium chloride (KCl, 60 mmol x L(-1)). The relaxant effect of puerarin was significantly inhibited by pretreatment of endothelium-denuded aorta with potassium channel antagonists tetraethylammonium, 4-aminopyridine but not glibenclamide. CONCLUSION: Puerarin induces an endothelium-independent relaxation in rat aortic rings. The mechanisms may involve the reduction in Ca2+ influx through the calcium channels operated by alpha-adrenergic receptor and the activation of the potassium channels (Kv and BKca, but not KATP).

[Modulation of iron on the vasodilating effect of interleukin-2 in the isolated aortic ring].

OBJECTIVE: To explore the effect and mechanism of iron on the vasodilating effect of interleukin-2 (IL-2) in the isolated aortic ring. METHODS: Isometric tension was recorded in response to drugs in organ bath. Ferric ammonium citrate (FAC) was added to the bath 30 min before phenylephrine (1 micromol/L), which was followed by IL-2 in a cumulative fashion. Spectrophotometry was used to determine the activity of nitric oxide synthase (NOS) of the thoracic aorta. RESULTS: FAC (0.1 - 10 micromol/L) alone did not affect the tension of rings,but inhibited the vasodilating effect of IL-2 (1 - 1,000 U/ml) in a dose dependent manner. IL-2(1, 10, 100, 1000 U/ml) decreased the aortic tension to (78.47+/-4.31)%, (66.86+/-5.55)%, (52.62+/-4.51)% and (42.39+/-4.27)% of pre-drug control, respectively. However, after incubation with 10 micromol/L FAC in the presence of IL-2, the aortic tension was reduced to (89.81+/-1.94)%, (86.13+/-3.11)%, (77.16+/-5.66)% and (68.76+/-5.69)% of pre-drug control, respectively. Pretreatment with L-arginine (1 mmol/L) abolished the inhibitory effect of FAC. Pretreatment with FAC attenuated the increased activity of NOS induced by IL-2 from (22.10+/-1.87)U/mg prot to (15.71+/-0.89)U/mg prot. High Ca(2+) (2.5 mmol/L) incubation did not change the inhibitory effect of FAC. Pretreatment with FAC attenuated the increased caffeine-releasable pool of Ca(2+) by IL-2. High K(+) (10 mmol/L) incubation abolished the inhibitory effect of FAC. CONCLUSION: FAC inhibits the vasodilating effect of IL-2 in the isolated aortic ring,which may be mediated by decreasing the activity of NOS. Intracellular calcium release and inward rectifier potassium channel are involved in the inhibitory effect of FAC.

[Treatment of interferon-alpha in reducing the endothelium-dependent relaxation of rat thoracic aorta].

OBJECTIVE: To investigate the vascular effect of acute and chronic treatment of interferon-alpha (IFN-alpha) in rat aortic rings. METHODS: Isolated thoracic aortic rings were mounted on the organ bath and the tension of the vessel was recorded. RESULTS: IFN-alpha(10, 100, 1,000 and 10,000 U/ml) caused concentration -dependent relaxation of endothelium-intact aorta rings preconstricted with phenylephrine (PE,10(-6)mol/L), to(90.1+/-0.91)%, (65.1+/-5.21)%, (39.5+/-8.22)% and (35.3+/-8.27)% of pre-drug control, respectively. Removal of the endothelium inhibited the relaxation by IFN-alpha. The vasorelaxant effect of IFN-alpha (100 U/ml ) was attenuated by pretreatment with L-NAME (10(-4)mol/L), methylene blue (10(-5)mol/L) or AMG (10(-4)mol/L), to (97.2+/-5.34)%, (95.1+/-6.25)% and (93.7+/-8.82)% of the control, respectively. Pretreatment with IFN-alpha (1,000,000 U/d, i.p.) for five days markedly inhibited the endothelium-dependent relaxation of the aortic rings to acetylcholine. But the endothelium-dependent relaxation to acetylcholine was not changed by pretreatment of IFN-alpha (10,000 U/ml) with the isolated aorta rings for 2 h. CONCLUSION: The vasorelaxation induced by IFN-alpha in rat aorta rings is endothelium-dependent and is possibly mediated by inducible nitric oxide synthase. Chronic treatment of IFN-alpha may impair the endothelium or NO-sGC pathway.

[NO/cGMP signal pathway involved in the disturbance of calcium homeostasis in vascular smooth muscle during the late phase of sepsis].

OBJECTIVE: To evaluate the alterations in calcium metabolism of the vascular smooth muscle of rat thoracic aorta in the late phase of sepsis and to investigate the involvement of nitric oxide (NO)/cyclic-GMP(cGMP) signal transduction pathway in the sepsis-induced vascular hyporeactivity. METHODS: Male Sprague-Dawley rats were subjected to sepsis by cecal ligation and puncture (CLP). Eighteen hours post CLP, rat aortic rings were removed for measurement of contractile responses to vasoconstrictors by using organ bath technique. RESULT: In endothelium intact aortic rings from CLP rats, concentration-contraction curves to phenylephrine (PE) and high KCl were significantly decreased when compared with those from control rats. The transient contraction induced by PE in calcium-free Krebs solution and the concentration-dependent contraction to CaCl(2)in KCl-depolarized medium were also markedly reduced. The hyporeactivity to vasoconstrictors was completely reversed by pretreatment either with aminoguanidine (AMG), a selective inducible nitric oxide synthase inhibitor, or with 1H [1,2,4] oxadiazolo[4,3-a] quininoxalin-1-one(ODQ), an inhibitor of NO-sensitive guanylyl cyclase. CONCLUSION: A generalized impairment in calcium handling in vascular smooth muscle,including the calcium influx through the voltage-operated and receptor-operated channels and calcium release from intracellular calcium stores, is involved in vascular hyporeactivity during the late phase of sepsis. The NO/cGMP signal transduction pathway might be involved in this defect in vascular smooth muscle.