Effects of composite films of silk fibroin and graphene oxide on the proliferation, cell viability and mesenchymal phenotype of periodontal ligament stem cells.

In regenerative dentistry, stem cell-based therapy often requires a scaffold to deliver cells and/or growth factors to the injured site. Graphene oxide (GO) and silk fibroin (SF) are promising biomaterials for tissue engineering as they are both non toxic and promote cell proliferation. On the other hand, periodontal ligament stem cells (PDLSCs) are mesenchymal stem cells readily accessible with a promising use in cell therapy. The purpose of this study was to investigate the effects of composite films of GO, SF and GO combined with fibroin in the mesenchymal phenotype, viability, adhesion and proliferation rate of PDLSCs. PDLSCs obtained from healthy extracted teeth were cultured on GO, SF or combination of GO and SF films up to 10 days. Adhesion level of PDSCs on the different biomaterials were evaluated after 12 h of culture, whereas proliferation rate of cells was assessed using the MTT assay. Level of apoptosis was determined using Annexin-V and 7-AAD and mesenchymal markers expression of PDLSCs were analyzed by flow cytometry. At day 7 of culture, MTT experiments showed a high rate of proliferation of PDLSCs growing on GO films compared to the other tested biomaterials, although it was slightly lower than in plastic (control). However PDLSCs growing in fibroin or GO plus fibroin films showed a discrete proliferation. Importantly, at day 10 of culture it was observed a significant increase in PDLSCs proliferation rate in GO films compared to plastic (P < 0.05), as well as in GO plus fibroin compared to fibroin alone (P < 0.001). Flow cytometry analysis showed that culture of PDLSCs in fibroin, GO or GO plus fibroin films did not significantly alter the level of expression of the mesenchymal markers CD73, CD90 or CD105 up to 168 h, being the cell viability in GO even better than obtained in plastic. Our findings suggest that the combination of human dental stem cells/fibroin/GO based-bioengineered constructs have strong potential for their therapeutic use in regenerative dentistry.

Naloxone-precipitated morphine withdrawal evokes phosphorylation of heat shock protein 27 in rat heart through extracellular signal-regulated kinase.

Heat shock protein 27 (Hsp27) is a well-known stress response protein that becomes phosphorylated through extracellular signal-regulated kinase (ERK). Different drugs of abuse, such as morphine and/or its withdrawal, induce severe stress situations. In this study, we investigated Hsp27 and phospho-Hsp27 expression during morphine dependence and withdrawal and evaluated the involvement of ERK in the phosphorylation of Hsp27 in the rat right ventricle. Dependence on morphine was induced by a 7-day s.c. implantation of morphine pellets. Morphine withdrawal was precipitated on day 8 by injection of naloxone (2 mg/kg, s.c.). ERK1/2, Hsp27 and phospho-Hsp27 at Ser15 were determined by quantitative blot immunolabeling using specific antibodies. Hsp27 expression was increased 30, 60, 90 and 120 min (144.5±14.2%, P<0.0001; 128.9±4.6%, P=0.04; 177.4±12.7, P<0.0001; and 136.2±11.0%, P=0.042, respectively) after saline injection to rats dependent on morphine. Naloxone-precipitated morphine withdrawal also increased the phosphorylation of Hsp27 at Ser15 at those time points (146.8±19.8%, P=0.034; 143.9±17.9%, P=0.032; 161.2±33.3%, P=0.029; and 152.2±25.5%, P=0.008, respectively). However, there were no changes in Hsp27 phosphorylation in the morphine dependent group injected with saline. In addition, there was an increase in the phosphorylation of ERK 60 min after naloxone injection in morphine dependent rats (pERK1: 116.3±4.2%, P=0.015 and pERK2: 117.2±1.5%, P=0.05). Pretreatment with SL327, an inhibitor of ERK phosphorylation, decreased activation (phosphorylation) of both ERK and Hsp27 (pERK1: 4.5±3.6%, P<0.0001; pERK2: 42.3±3.3%, P<0.0001; and pHsp27: 97.6±1.5%, P=0.008), suggesting that ERK activation triggers Hsp27 phosphorylation. The present findings demonstrate that morphine withdrawal is capable of inducing the activation of Hsp27 in the heart and suggest that phosphorylation of Hsp27 is closely linked to and also dependent on the ERK pathway.

Cross-talk between protein kinase A and mitogen-activated protein kinases signalling in the adaptive changes observed during morphine withdrawal in the heart.

Our previous studies have shown that morphine withdrawal induced an increase in the expression of protein kinase (PK) A and mitogen-activated extracellular kinase (MAPK) pathways in the heart during morphine withdrawal. The purpose of the present study was to evaluate the interaction between PKA and extracellular signal-regulated kinase (ERK) signaling pathways mediating the cardiac adaptive changes observed after naloxone administration to morphine-dependent rats. Dependence on morphine was induced by a 7-day subcutaneous implantation of morphine pellets. Morphine withdrawal was precipitated on day 8 by an injection of naloxone (2 mg/kg). ERK1/2 and tyrosine hydroxylase (TH) phosphorylation was determined by quantitative blot immunolabeling using phosphorylation state-specific antibodies. Naloxone-induced morphine withdrawal activates ERK1/2 and phosphorylates TH at Ser31 in the right and left ventricle, with an increase in the mean arterial blood pressure and heart rate. When N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004), a PKA inhibitor, was infused, concomitantly with morphine, it diminished the expression of ERK1/2. In contrast, the infusion of calphostin C (a PKC inhibitor) did not modify the morphine withdrawal-induced activation of ERK1/2. The ability of morphine withdrawal to activate ERK that phosphorylates TH at Ser31 was reduced by HA-1004. The present findings demonstrate that the enhancement of ERK1/2 expression and the phosphorylation state of TH at Ser31 during morphine withdrawal are dependent on PKA and suggest cross-talk between PKA and ERK1/2 transduction pathway mediating morphine withdrawal-induced activation (phosphorylation) of TH.

Vitamin E supplementation reverses renal altered vascular reactivity in chronic bile duct-ligated rats.

An altered vascular reactivity is an important manifestation of the hemodynamic and renal dysfunction during liver cirrhosis. Oxidative stress-derived substances and nitric oxide (NO) have been shown to be involved in those alterations. In fact, both can affect vascular contractile function, directly or by influencing intracellular signaling pathways. Nevertheless, it is unknown whether oxidative stress contributes to the impaired systemic and renal vascular reactivity observed in cirrhosis. To test this, we evaluated the effect of vitamin E supplementation (5,000 IU/kg diet) on the vasoconstrictor and vasodilator responses of isolated perfused kidneys and aortic rings of rats with cirrhosis induced by bile duct ligation (BDL), and on the expression of renal and aortic phospho-extracellular regulated kinase 1/2 (p-ERK1/2). BDL induced a blunted renal vascular response to phenylephrine and ACh, while BDL aortic rings responded less to phenylephrine but normally to ACh. Cirrhotic rats had higher levels of oxidative stress-derived substances [measured as thiobarbituric acid-reactive substances (TBARS)] and NO (measured as urinary nitrite excretion) than controls. Vitamin E supplementation normalized the renal hyporesponse to phenylephrine and ACh in BDL, although failed to modify it in aortic rings. Furthermore, vitamin E decreased levels of TBARS, increased levels of NO, and normalized the increased kidney expression of p-ERK1/2 of the BDL rats. In conclusion, BDL rats showed a blunted vascular reactivity to phenylephrine and ACh, more pronounced in the kidney and reversed by vitamin E pretreatment, suggesting a role for oxidative stress in those abnormalities.

Omapatrilat normalizes renal function curve in spontaneously hypertensive rats.

BACKGROUND: The present study was designed to analyze the chronic renal response to omapatrilat, a new vasopeptidase inhibitor, in spontaneously hypertensive rats (SHR). To that end, the renal and blood pressure response to a 4-day salt loading protocol was analyzed and the respective chronic renal curves constructed. RESULTS: In non treated animals, and under normal sodium intake (around 2 mEq/day), mean arterial pressure (MAP), was significantly higher in the SHR as compared with the controls (WKY). After increasing salt intake (8 times normal), MAP did not change significantly in any group and the animals reached a normal sodium balance in four days. In a second group of animals, omapatrilat was given orally for 15 days at the dose of 40 mg/kg/day in the drinking water. In these omapatrilat-treated animals, and under normal sodium intake, MAP was significantly lower in both groups, although the antihypertensive effect was much greater in the SHR, so that the MAP of the SHR group was completely normalized and similar to the WKY-treated group. The subsequent elevation of sodium intake did not significantly elevate MAP in any group and the animals could manage the sodium excess as well as the non treated groups. CONCLUSIONS: These results indicate that chronic treatment with omapatrilat normalizes blood pressure in SHR without affecting adversely the renal ability to eliminate a sodium load. Chronic treatment with omapatrilat resets the chronic pressure natriuresis relationship of the SHR to a normal level, thus without altering the normal salt-independence of this arterial hypertension model.

Mechanisms of the increased pressor response to vasopressors in the mesenteric bed of nitric oxide-deficient hypertensive rats.

In the present study we analyzed mesenteric vascular reactivity of chronic nitric oxide (NO)-deficient hypertensive rats (NW-nitro-L-Arginine Methyl Ester, L-NAME, 50 mg/kg/day, oral, 3 weeks). Perfusion pressure changes in response to cumulative additions of methoxamine and KCl were significantly increased in the mesenteric vessels of the L-NAME-treated as compared with vessels of the controls. Verapamil reduced the responses to methoxamine, but those of the hypertensive rats were still enhanced. In contrast, responses to KCl were almost completely abolished by verapamil. In mesenteric vessels perfused with zero calcium and high-potassium Krebs, pressor responses to the re-addition of calcium were also significantly enhanced in the hypertensive rats compared to the controls. Vasodilator responses to acetylcholine in KCl-preconstricted vessels, while still significant, were reduced in the L-NAME-treated rats. In this case, acute inhibition of NO blocked the vasodilator responses to acetylcholine and abolished the differences between the two groups. In methoxamine-preconstricted vessels and in the presence of acute inhibition of NO and prostaglandins, vasodilator responses to acetylcholine were significantly greater in the hypertensive vessels than in controls. In conclusion, the mesenteric vessels of L-NAME hypertensive rats show an enhanced response to vasopressors which is related to calcium entry. These data also reveal the existence of an enhanced role of a NO and prostaglandin-independent vasodilator factor, probably endothelium-derived hyperpolarizing factor that may play a compensatory role in the deficiency of NO.

[Renal effects of the chronic inhibition of nitric oxide synthesis in cirrhotic rats with ascites]. / Efectos renales de la inhibición crónica de la síntesis de óxido nítrico en ratas cirróticas con ascitis.

Previous studies have shown that acute inhibition of nitric oxide (NO) synthesis improves sodium and water excretion and increases blood pressure in cirrhotic rats with ascites, thus suggesting that NO is an important factor contributing to the arterial hypotension and sodium retention of liver cirrhosis. In the present work we have analyzed the renal effects derived from the chronic oral treatment (10 days) with aminoguanidine (AG, 100 mg/kg/day), a preferential inhibitor of inducible NO synthase (iNOS), or Nw-Nitro-L-Arginine Methyl Ester (L-NAME, 0.5 mg/kg/day), a nonselective inhibitor of NOS, in an experimental model of liver cirrhosis with ascites (carbon tetrachloride inhalation). Untreated cirrhotic rats showed lower mean arterial pressure (MAP), diuresis, natriuresis and glomerular filtration rate (GFR) and similar renal blood flow (RBF) compared with the untreated control rats. Chronic administration of AG did not modify significantly any parameter in cirrhotic and control animals. Conversely, long-term L-NAME administration to cirrhotic rats normalized MAP and significantly increased water and sodium excretion, whereas in control animals these parameters were not significantly modified. These results show that chronic NO synthesis inhibition with L-NAME, but not with aminoguanidine, improves renal perfusion pressure and increases the lower sodium and water excretion of cirrhotic rats with ascites. Thus, an enhanced production of NO is an important factor contributing to the renal sodium and water retention characteristic of liver cirrhosis.

Mesenteric hyporesponsiveness in cirrhotic rats with ascites: role of cGMP and K+ channels.

The mechanisms that mediate hyporesponsiveness to vasoconstrictors in liver cirrhosis are not completely established. In the present study we have explored the role of NO and potassium channels by studying the pressor response to methoxamine in rats with carbon tetrachloride-induced cirrhosis with ascites. Experiments were performed in the isolated and perfused mesenteric arterial bed of control rats and of cirrhotic rats with ascites. Pressor responses to methoxamine, an alpha-adrenergic agonist, were analysed under basal conditions, after inhibition of guanylate cyclase with Methylene Blue (MB; 10 microM), after inhibition of NO synthesis with N(G)-nitro-L-arginine (L-NNA; 100 microM) and after blockade of potassium channels with tetraethylammonium (TEA; 3 mM). Compared with those from controls, preparations from cirrhotic rats showed a lower pressor response to methoxamine (maximum: controls, 114.4+/-6.8 mmHg; cirrhotic rats, 74.7+/-7.3 mmHg). Pretreatment with MB or L-NNA increased the responses in both groups, but without correcting the lower than normal response of the cirrhotic rats. Pretreatment with TEA alone did not modify the responses as compared with the untreated groups. Pretreatment with TEA plus MB or TEA plus L-NNA also potentiated the responses, and the responses of the cirrhotic animals were greater than those of the groups treated with MB or L-NNA alone. However, no treatment completely normalized the lower response of the mesenteries from cirrhotic animals, suggesting that factors other than NO and potassium channels also participate, although to a lesser degree, in the lower pressor response of the mesenteric arterial bed of animals with cirrhosis. These results confirm that NO and potassium channels are important mediators of the lower vascular pressor response of the mesenteric bed of cirrhotic rats with ascites. This effect seems to be mediated by the NO-dependent formation of cGMP and by the NO-dependent and -independent activation of potassium channels.

Haemodynamic and renal evolution of the bile duct-ligated rat.

In the present study we have characterized the evolution of changes in systemic haemodynamics (thermodilution in conscious animals) and sodium balance (metabolic cages) in a model of liver cirrhosis induced by chronic bile duct ligation (BDL). Mean arterial pressure (BDL, 111.5+/-4.7 mmHg; sham-operated, 122.9+/-3.0 mmHg) and peripheral vascular resistance (BDL, 2.63+/-0.08 units; sham-operated, 2.93+/-0.09 units) were lower in BDL rats from day 12 after surgery and decreased progressively throughout the following days. Portal hypertension was evident earlier in BDL rats and was maintained throughout the study period. Cardiac index (BDL, 58.8+/-3. 9 ml.min(-1).100 g(-1); sham-operated, 43.9+/-1.5 ml.min(-1).100 g(-1)) and stroke volume (BDL, 147.2+/-12.7 ml.beat(-1).100 g(-1); sham-operated, 109.0+/-4.2 ml.beat(-1).100 g(-1)) were significantly elevated in the BDL rats only from day 18 after surgery. There were no significant differences in sodium balance between the groups until day 16 after surgery, at which time BDL animals started to retain significantly more sodium than the controls. Sodium retention increased progressively, and at day 20 BDL rats had retained 0.7 mmol/100 g more than the control animals (accumulated retention: BDL, 2.2+/-0.2 mmol/100 g; sham-operated, 1.5+/-0.2 mmol/100 g). Plasma renin activity and aldosterone concentration were not elevated in the BDL animals at days 12, 16 or 20 after surgery. These data indicate that the BDL rat model shows early portal hypertension, peripheral vasodilation and arterial hypotension, several days before sodium retention is detectable, and in the absence of changes in plasma levels of renin and aldosterone. Overall, these data suggest that, in the BDL rat model, sodium retention is secondary to portal hypertension and peripheral vasodilation.

Pressor and renal effects of cross-linked hemoglobin in anesthetized cirrhotic rats.

BACKGROUND/AIMS: Cross-linked hemoglobin (XL-Hb), a hemoglobin-based oxygen carrier, is currently under investigation as a blood substitute. In the present study we have evaluated its pressor and renal effects in a rat model of liver cirrhosis by bile duct ligation. METHODS: Experiments were performed 3 weeks after surgery in anesthetized rats In the first protocol, the ability of XL-Hb to recover blood pressure after a hypotensive hemorrhage (0.5 ml/min, 10 min) was analyzed. In the second protocol, the pressor and renal effects produced by the administration of XL-Hb were evaluated during a period of 3 h. RESULTS: After a hypotensive hemorrhage (0.5 ml/min, 10 min), resuscitation with XL-Hb resulted in greater and faster recovery of blood pressure than with the administration of blood. In non-hemorrhaged rats, administration of XL-Hb (5% of blood volume) reversibly increased blood pressure in bile duct ligation and in control rats, but this effect was of longer duration in the control animals. XL-Hb also induced brisk increases in water and sodium excretion in both groups of animals, but the response of the control animals was more intense and sustained than that of the bile duct ligation rats. Glomerular filtration rate and renal blood flow showed slight decreases, but they were well maintained around the baseline levels. All the parameters studied were normalized 3 h later. In additional experiments, the effect of a bolus of L-NAME (10 mg/kg), an inhibitor of nitric oxide synthase, 1 h after the administration of XL-Hb was partially reduced, suggesting that the effect of XL-Hb may be secondary to the disappearance of circulating nitric oxide. CONCLUSIONS: XL-Hb seems to be effective as a resuscitative solution in case of hemorrhage in cirrhotic rats Moreover, this blood substitute only moderately and reversibly elevates blood pressure and does not adversely affects renal function.

Effect of endothelin blockade on pressure natriuresis in nitric oxide-deficient hypertensive rats.

OBJECTIVE: Chronic inhibition of nitric oxide synthesis has been shown to cause arterial hypertension and an important blunting of the pressure diuresis and natriuresis response. The mechanisms mediating these abnormalities are not completely established. We therefore studied the effects of endothelin on these alterations. MATERIALS AND METHODS: Pressure diuretic and natriuretic relationships were evaluated in rats treated chronically (3 weeks) with the nitric oxide synthesis inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME; 40 mg/kg per day), alone or in combination with bosentan sodium salt (acute treatment: 10 mg/kg, intravenously; chronic treatment: 10 mg/kg per day). RESULTS: Chronic treatment with L-NAME significantly elevated mean arterial pressure (143.7 +/- 2.8 mmHg versus 102.8 +/- 1.6 in controls), reduced the glomerular filtration rate and renal blood flow and shifted the pressure diuretic and natriuretic responses to the right. Treatment with bosentan, either acute or chronically, did not attenuate the arterial hypertension of the L-NAME-treated rats but normalized the glomerular filtration rate and renal blood flow. In spite of the normalization of renal hemodynamics, the pressure diuretic and natriuretic responses of the bosentan-treated groups were not normalized, although chronic bosentan significantly improved the pressure natriuretic response. CONCLUSIONS: These results indicate that endothelin participates in the renal hemodynamic and excretory alterations that follow chronic inhibition of nitric oxide synthesis. However, the arterial hypertension is not mediated by endothelin activation.

Pressure natriuresis in nitric oxide-deficient hypertensive rats: effect of antihypertensive treatments.

Chronic inhibition of nitric oxide (NO) synthesis has been shown to result in arterial hypertension and an important blunting of the pressure diuresis and natriuresis response (PDN). The mechanisms mediating these abnormalities are not completely understood. In the present study, the role of several antihypertensive drugs to ameliorate these alterations was evaluated. The PDN relationships have been evaluated in rats chronically (8 wk) treated with the NO synthesis inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME; 40 mg/kg per d in the drinking water). Appropriate groups of rats were simultaneously treated with the angiotensin II receptor blocker candesartan at a low (1.5 mg/kg per d) and high (2.5 mg/kg per d) dose, with the converting enzyme inhibitor captopril (60 mg/kg per d) and with the calcium channel blocker verapamil (100 mg/kg per d). Chronic treatment with L-NAME significantly elevated mean BP (163.6 +/- 6.5 mmHg versus 105.1 +/- 3.6 in controls), reduced GFR and renal blood flow (RBF), and shifted to the right the PDN responses. Chronic administration of low-dose candesartan, captopril, or verapamil prevented the arterial hypertension and improved renal hemodynamics, but these levels were not completely normalized. High-dose administration also improved renal hemodynamics but induced reduced BP below the levels of control animals. Despite the normalization of the elevated BP, the PDN responses of these hypertensive treated groups were not normalized, and the slopes of the respective diuretic or natriuretic responses were very similar to those of the hypertensive untreated rats. The results indicate that interruption or blockade of the renin-angiotensin system and calcium channel blockade are effective treatments for the NO-deficient arterial hypertension and renal vasoconstriction. However, the PDN responses are not normalized, and this finding suggests that the antihypertensive treatment is not enough to overcome the renal alterations associated with the chronic deficiency of NO.

Role of cyclic guanosine monophosphate and K+ channels as mediators of the mesenteric vascular hyporesponsiveness in portal hypertensive rats.

The mechanisms mediating the hyporesponsiveness to vasoconstrictors in portal hypertension are not completely established. In the present study, we evaluated the role of cyclic guanosine monophosphate (cGMP) and potassium channels as contributors to the pressor hyporesponsiveness to methoxamine (MTX) of the mesenteric vascular bed of portal vein-ligated (PVL) hypertensive rats. In basal conditions, and compared with sham-operated control rat (SHAM) vessels, PVL preparations showed a blunted pressor response (maximum: 39.3 +/- 6.1 vs. 94.5 +/- 8.9 mm Hg), which increased by pretreatment with methylene blue (MB), a guanylate cyclase inhibitor (118.7 +/- 8.9 vs. 152.0 +/- 10.0, respectively), and even more with the nitric oxide (NO) synthesis inhibitor N(omega)-nitro-L-arginine (NNA) (159.9 +/- 7.4 vs. 194.1 +/- 5.7, respectively), suggesting that NO acts through cGMP-dependent and independent mechanisms. In all cases, however, the pressor responses of PVL vessels were lower than those of SHAM. Pretreatment of the vessels with the potassium channel inhibitors, tetraethylammonium (TEA), glibenclamide (GLB), or charybdotoxin (CHX), did not improve the reduced pressor responses of the PVL rats. However, when the preparations were simultaneously pretreated with MB and TEA or with NNA and TEA, the pressor responses were potentiated with respect to groups treated with MB or NNA alone, and the differences between PVL and SHAM vessels were completely corrected. These data suggest that both NO and potassium channels mediate the vascular hyporesponsiveness to methoxamine of the PVL mesenteric vasculature. Our results also disclose that NO blunts the pressor response of the PVL vessels by a dual mechanism of action, through activation of potassium channels and through the formation of cGMP. Finally, the NO-independent component mediated by potassium channels can be only seen when the main cGMP-NO component is inactivated. In conclusion, both cGMP and potassium channels mediate the vascular hyporesponsiveness to MTX of the mesenteric bed of portal hypertensive rats.

Role of AT1 receptors in the renal papillary effects of acute and chronic nitric oxide inhibition.

Nitric oxide (NO) is a vasodilator substance controlling renal papillary blood flow (PBF) in the rat. In this study we have evaluated the role of AT1 angiotensin II receptors as modulators of the whole kidney and papillary vasoconstrictor effects induced by the acute or chronic inhibition of NO synthesis. Experiments have been performed in anesthetized, euvolemic Munich-Wistar rats prepared for the study of renal blood flow (RBF) and PBF. In normal rats, acute administration of the NO synthesis inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) increased mean arterial pressure (MAP) and decreased RBF and PBF. Either acute or chronic treatment with the AT1 receptor blocker losartan did not modify the decreases in RBF or PBF secondary to L-NAME. In animals made hypertensive by chronic inhibition of NO, basal MAP was higher, whereas RBF and PBF were lower than in the controls. In these animals, acute or chronic administration of losartan decreased MAP and increased both RBF and PBF significantly. These results indicate that, under normal conditions, the decreases in RBF or PBF induced by the acute inhibition of NO synthesis are not modulated by AT1-receptor stimulation. However, the arterial hypertension, renal vasoconstriction, and reduced PBF present in chronic NO-deficient hypertensive rats is partially due to the effects of angiotensin II, via stimulation of AT1-receptors.

Elevated glomerular and blood mononuclear lymphocyte nitric oxide production in rats with chronic bile duct ligation: role of inducible nitric oxide synthase activation.

Recent work indicates that nitric oxide (NO) plays an important role in the systemic and renal alterations of cirrhosis. In the present study, we have evaluated whether the inducible NO synthase (iNOS) isoform participates in the enhanced renal and systemic NO production of a rat model of cirrhosis. In vitro and in vivo experiments were performed in rats subjected to chronic bile duct ligation (BDL) and in sham-operated (SO) animals. Plasma nitrite (3.1 +/- 0.1 micromol/L in SO and 6.6 +/- 0.2 micromol/L in BDL), glomerular nitrite production (6.4 +/- 0.1 vs. 9.8 +/- 0.1 nmol/24h/7,000 glomeruli, respectively), and mononuclear lymphocyte cells nitrite production (0.3 +/- 0.04 vs. 0.6 +/- 0.12 nmol/10(6) cells, respectively) were all significantly higher in BDL than in SO. Moreover, mononuclear lymphocytes and glomeruli from BDL rats showed an increased expression of macrophage-type iNOS, detected by Western blot. Kidneys from BDL animals also showed an increased calcium-independent NO synthase activity, compared with those from SO rats. Constitutive endothelial-type NO synthase expression in glomeruli or the activity of calcium-dependent NO synthase in whole kidney did not show differences between BDL and SO rats. In cultured mesangial cells from normal rats, the addition of plasma from BDL but not of plasma from SO significantly stimulated (35%) nitrite production and increased the expression of macrophage-type iNOS. In addition, administration of aminoguanidine (AG), a preferential iNOS inhibitor, elevated dose-dependently mean arterial pressure in both groups, but this increase was greater in BDL (26.5 +/- 4.4 mm hg) than in SO (13.6 +/- 2.6). In BDL, AG also increased sodium and water excretion and glomerular filtration rate. In contrast, there were only small nonsignificant changes in SO animals. Therefore, these results indicate that the expression, activity and production of NO in kidneys, glomeruli, and mononuclear lymphocyte cells is elevated in BDL rats, and this is partly because of a plasma-derived substance(s), which stimulates iNOS formation. The amelioration of the arterial hypotension and the associated reduced excretory levels of these cirrhotic animals by aminoguanidine further support the involvement of the inducible NO synthase isoform in the renal alterations observed in BDL animals.

Age-related changes in the pressure diuresis and natriuresis response.

The renal-excretory responses to changes in renal perfusion pressure (RPP) were studied in anesthetized young (3 mo old), adult (12 mo old), and senescent (24 mo old) rats to evaluate whether the pressure diuresis and natriuresis mechanism is altered as a function of age. Experiments were performed in anesthetized animals in which nervous and systemic hormonal influences to the kidney were fixed. Mean arterial pressure was similar in all three groups: 97.6 +/- 2.6, 102.1 +/- 3.7, and 95.2 +/- 5.2 mmHg in young, adult, and senescent rats, respectively. The relationships between RPP and diuresis/natriuresis or fractional excretions of water and sodium were similar in young and adult rats. However, in senescent rats the pressure-diuretic and pressure-natriuretic responses were slightly shifted to the right, so that diuresis and natriuresis were significantly lower at higher levels of RPP. Glomerular filtration rate was well autoregulated, and there were no differences between young and adult rats at each level of RPP. However, a significantly lower glomerular filtration rate was observed in senescent rats. These results indicate an age-related decline in the pressure-dependent sodium and water excretion that appears to be due to a decrease in glomerular filtration and an increase in tubular sodium reabsorption.

Vascular hyporesponsiveness in aortic rings from cirrhotic rats: role of nitric oxide and endothelium.

1. The role of nitric oxide as mediator of the vascular alterations present in different models of experimental liver cirrhosis is controversial. In the present study, we evaluated the role of nitric oxide and that of the endothelium in the response to phenylephrine and acetylcholine of isolated aortic rings from chronic bile duct-ligated (29 days) rats and their corresponding controls. Experiments were performed in rings with or without endothelium, in rings pretreated with N-omega-nitro-L-arginine methyl ester (10(-4) mol/l) to inhibit nitric oxide synthesis and in rings pretreated with aminoguanidine (10(-4) mol/l) to inhibit inducible nitric oxide synthesis. 2. Under basal conditions, the maximum absolute tension developed in response to cumulative addition of phenylephrine was significantly decreased in rings from bile duct-ligated animals (1.62 +/- 0.06 g) compared with the control rings (2.15 +/- 0.099). This hyporesponsiveness to phenylephrine of rings from bile duct-ligated animals was corrected after treatment with N-omega-nitro-L-arginine methyl ester and reduced, but not completely eliminated, in rings without endothelium. In contrast, aminoguanidine did not modify the lower response to phenylephrine rings from bile duct-ligated animals. ED50 values were not different between groups under any experimental conditions. 3. The endothelium-dependent vasodilatation to acetylcholine in phenylephrine-constricted rings was similar in both groups of animals, control and bile duct ligated, under all experimental conditions. N-omega-nitro-L-arginine methyl ester pretreatment and removal of the endothelium completely abolished the response to acetylcholine in cirrhotic and control rings. 4. These results demonstrate that in aortic rings from cirrhotic, bile duct-ligated rats, increased production of nitric oxide, mainly of endothelial origin, is responsible for the lower contractile response to phenylephrine. Our data, however, do not support the involvement of the inducible nitric oxide synthase isoform in this alteration. In contrast, endothelial vasodilatory response to acetylcholine is not altered in this model of cirrhosis, which indicates that not all mechanisms of nitric oxide release are abnormal.

Role of endothelium in the abnormal response of mesenteric vessels in rats with portal hypertension and liver cirrhosis.

BACKGROUND & AIMS: Previous studies have shown that nitric oxide synthesis inhibition corrects the hyporesponsiveness to vasoconstrictors present in the mesenteric vascular bed of portal-hypertensive rats. The origin of this elevated NO production, whether endothelial or muscular, is unknown. The aim of this study was to evaluate the role of vascular endothelium in the hyporesponsiveness to methoxamine (MTX) in the mesenteric vascular bed of portal vein-ligated (PVL) and cirrhotic rats. METHODS: Endothelial denudation was achieved using a combined treatment of cholic acid and distilled water. RESULTS: Compared with the respective control groups, PVL rats showed a reduced vascular response to MTX. Similar results were obtained in cirrhotic animals. The presence of ascites was associated with a more severe reduction in the response to MTX. Removal of the endothelium completely corrected the vascular hyporesponsiveness of PVL, cirrhotic nonascitic, and ascitic animals. In these experiments, acetylcholine-mediated vasodilation was practically absent whereas that of sodium nitroprusside was potentiated, which indicates a successful elimination of the endothelium and the preservation of smooth muscle function. Immunostaining for NO synthase isoforms revealed the presence of endothelial NO synthase protein in healthy and PVL rats exclusively in the endothelium. CONCLUSIONS: The mesenteric vascular hyporesponsiveness to MTX present in these models of liver diseases and portal hypertension is solely due to endothelium-dependent factors.

Renal and pressor effects of aminoguanidine in cirrhotic rats with ascites.

Recent work indicates that nitric oxide (NO) plays an important role in the systemic and renal alterations of liver cirrhosis. This study used aminoguanidine (AG), a preferential inhibitor of inducible nitric oxide synthase (iNOS), to evaluate the role of this NOS isoform in the systemic and renal alterations of an experimental model of liver cirrhosis with ascites (carbon tetrachloride/ phenobarbital). Experiments have been performed in anesthetized cirrhotic rats and their respective control rats prepared for clearance studies. Administration of AG (10 to 100 mg/kg, iv) elevated dose-dependent mean arterial pressure (MAP, in mm Hg) in the cirrhotic rats from a basal level of 79.3 +/- 3.6 to 115.0 +/- 4.7, whereas in the control animals, MAP increased only with the highest dose of the inhibitor (from 121.8 +/- 3.6 to 133.3 +/- 1.4). In the cirrhotic group, AG also significantly increased sodium and water excretion, whereas these effects were very modest in the control group. Plasma concentration of nitrates+nitrites, measured as an index of NO production, were significantly increased in the cirrhotic animals in the basal period and decreased with AG to levels not significantly different from the control animals. Similar experiments performed with the nonspecific NOS inhibitor N omega-nitro-L-arginine (NNA) also demonstrated an increased pressor sensitivity of the cirrhotic rats, but the arterial hypotension was completely corrected. These results, in an experimental model of liver cirrhosis with ascites, show that AG exerts a beneficial effect as a result of inhibition of NO production, increasing blood pressure and improving the reduced excretory function. Because NNA, but not AG, completely normalized the arterial hypotension, it is suggested that the constitutive NOS isoform is also contributing in an important degree. It is concluded that the activation of both inducible and constitutive NOS isoforms plays an important role in the lower systemic blood pressure and associated abnormalities that characterize liver cirrhosis.