Vitamin D receptor gene, biochemical bone markers and bone mineral density in Mexican women on dialysis.

BACKGROUND: The influence of the Bsm1 polymorphism of the vitamin D receptor (VDR) gene on mineral and bone disorders in chronic kidney disease (CKD) is still under discussion. The aim of this study was to analyse the relationship between VDR polymorphism, bone mineral density (BMD), biochemical bone markers and clinical factors in women on peritoneal dialysis (PD) and haemodialysis (HD). METHODS: In a cross-sectional study, 197 women (42 +/- 10 years; 25% with diabetes mellitus (DM); body mass index (BMI) 25.26 +/- 4.77 kg/m(2)) treated by PD (72%) or HD (28%) underwent measurements of BMD (measured at the calcaneus by quantitative ultrasound; expressed as T- and Z-scores) and plasma total calcium (tCa), intact parathyroid hormone 1-84 (iPTH), phosphorus, albumin, glucose, osteoprotegerin (OPG), fetuin-A, intact osteocalcin-49 and N-MID fragment 1-43 aa (N-MID osteocalcin) N-terminal propeptide of type 1 procollagen (PINP) and C-terminal telopeptide-beta aspartic acid (BCL). DNA was extracted from peripheral blood. PCR products were digested with Bsm1 to analyse VDR polymorphism. RESULTS: The Z-score of BMD was -1.1 +/- 1.03. According to the values of osteopenia (T-score = -1.0), patients with higher BMD were younger, had lower frequency of amenorrhoea and diabetes and had higher serum creatinine and fetuin levels as well as lower levels of PINP. In a stepwise multivariate logistics analysis, osteopenia was associated with presence of genotype BB+Bb (OR = 3.26, P < or = 0.003) and age (OR = 0.95, P = 0.050). According to the B allele, bb: n = 126 (64%) and BB+Bb: n = 71(36%), group bb had significantly higher mean Z-scores (-0.97 +/- 1.0 vs -1.3+/-0.92; P < or = 0.021). CONCLUSIONS: The high frequency of osteopenia observed in female CKD patients on dialysis is associated with age and genetic predisposition as revealed by its association to the Bsm1 VDR polymorphism.

Age-related differences in the beta-adrenergic vasodilator response in rat aortic rings.

Mechanisms underlying age-dependent changes in vasodilator responses to beta-adrenergic drugs are poorly understood. The aim of the current study was to compare responses to isoproterenol (a non-selective beta-adrenergic receptor agonist) in phenylephrine or KCl precontracted aortic rings from 3 week and 3 month old male Wistar rats. Both the mechanism and the subtype of beta-adrenergic receptor underlying the response to isoproterenol in the both age groups were examined. Endothelial removal, pre-contraction with KCl (40 mM), pre-treatment with tetraethylammonium or with N(omega)-Nitro-L-arginine methyl ester inhibited the vasodilator response to isoproterenol only in aortic rings from older rats. The inhibition was total when TEA and L-NAME were administered together. In both age groups the response to isoproterenol was unaffected by the beta1-adrenergic antagonist CGP20712A, but was significantly inhibited by ICI 118551 (a beta2-adrenergic-antagonist) and to a greater extent by SR 59230A (a non-selective beta 3-adrenergic antagonist), the inhibition being more evident in the older rats. Unlike younger rats, in older animals the response to isoproterenol was partially dependent on endothelial nitric oxide and on K+ channels. In both age groups, beta2- and beta3-, but not beta1-adrenergic receptors were involved. The degree of relative participation of beta2 and beta3 adrenergic receptors may change with age and explain the differences in response to isoproterenol.

Influence of triiodothyronine on the contractile effect of D-nor-pseudoephedrine.

Here we contrast the vascular smooth muscle contractility produced by D-nor-pseudoephedrine, alone or combined with triiodothyronine, on the aorta and coronary vasculature of the rat. At high concentrations (greater than those found in normal therapeutic dosing) D-nor-pseudoephedrine contracted the aorta. In contrast, it produced vasodilation on pre-contracted aorta independently of the vasoconstrictor employed or the presence of vascular endothelium. D-nor-pseudoephedrine increased coronary perfusion pressure, but the effect was smaller than the vasoconstriction produced by phenylephrine (a full alpha 1 adrenergic agonist), and was blocked by the pre-treatment with triiodothyronine. These results suggest cardiovascular risks in the use of D-nor-pseudoephedrine for weight loss.

The influence of constitutive COX-2 in smooth muscle tissue on the contractile effect of phenylephrine in the rat abdominal aorta.

Prostanoids are involved in the phenylephrine-induced contraction of the aorta. Here, we examined whether or not constitutive cyclooxygenase-2 (phosholipases C and A2) is the source of prostanoids in the smooth muscle of the arterial wall of the thoracic and abdominal aorta. Both cyclooxygenase isoforms (COX-1 and COX-2) were expressed in the two aortic segments, but their expression was not altered by phenylephrine, the protein synthesis inhibitor cycloheximide, or the phospholipase A2 inhibitors arachidonyl trifluoromethyl ketone and methyl arachidonyl fluorophosponate. Indomethacin and NS398, which are a non-selective and selective COX-2 inhibitor, respectively, but not SC-560, which is a COX-1-selective inhibitor, inhibited the effect of phenylephrine on the abdominal, but not the thoracic, aorta. Similarly, U73122, which is a phospholipase C inhibitor, and RHC80267, which is a diacylglycerol lipase inhibitor, inhibited the effect of phenylephrine. These findings suggest that prostanoids, which are produced by constitutively active COX-2, influence the contractile response of the abdominal aorta and that the production of arachidonic acid relies on phospholipase C and diacylglycerol lipase.

Bioassay-guided isolation of an anti-ulcer chromene from Eupatorium aschenbornianum: role of nitric oxide, prostaglandins and sulfydryls.

Eupatorium aschenbornianum is considered useful in the treatment of gastric ulcer. In the current study the validity of this practice was tested by using the experimental model of an ethanol induced gastric ulcer in rats. The results show that E. aschenbornianum had gastroprotective activity, that the hexane extract had the highest protective activity (85.65+/-4.76%), and that encecanescin isolated from this extract was the main active gastroprotective agent. The effect elicited by encecanescin was attenuated by N(G)-nitro-L-arginine methyl ester, N-ethylmaleimide and indomethacin, which suggests that NO, prostaglandins and sulfydryl groups are involved in the mechanisms of gastroprotective action.

Enhancement of embryonic stem cell differentiation promoted by avian chorioallantoic membranes.

Avian chorioallantoic membrane (CAM) has been used as a model to explore angiogenesis and to study the microvasculature of transplanted tissues. Because CAM provides a vascular bed, cells can be implanted, and their development can be monitored and modified. We used the CAM model to study the differentiation process of embryoid bodies (EBs) derived from mouse embryonic stem cells (ESCs) influenced by the CAM vascular bed. After EBs were incubated in CAM for 5 days, they underwent further differentiation and became tissue masses (TMs) of different morphologies from those that grew outside CAM. Immunohistochemical analysis of TMs demonstrated tissue-specific markers such as neurofilament light, CD34, collagen IV, cardiac myosin heavy chain (MHC), and cardiotin. Differentiated mouse blood vessels stained with anti-CD31 were found within the TMs, as well as blood vessels stained positive for QH1 and QCPN, markers for quail endothelial cells and perinuclear quail antigen, respectively. Quail erythrocytes inside mouse blood vessels suggested a connection between existing quail vessels and blood vessels growing inside the TMs as a result of EB differentiation. Therefore, CAM could be a suitable model to trigger and study the differentiation of EBs in close interaction with surrogate quail blood vessels.

Bioassay-guided isolation of an anti-ulcer diterpenoid from Croton reflexifolius: role of nitric oxide, prostaglandins and sulfhydryls.

Croton reflexifolius H. B. K (Euphorbiaceae) is a very common medicinal plant in the Huastecan region of Mexico that, according to local folk medicine, is considered useful in the treatment of gastritis and gastric ulcer. We have aimed to test the validity of this practice by using the experimental model of an ethanol-induced gastric ulcer in male Wistar rats. The results showed that C. reflexifolius had gastroprotector activity, that the hexane extract had the highest protective activity (64.38+/-7.72%), and that polyalthic acid isolated from this extract was the main active gastroprotector agent. Rats treated orally with polyalthic acid showed a gastroprotective effect similar to that elicited by carbenoxolone. As with carbenoxolone, the effect elicited by polyalthic acid was attenuated by pretreatment with either N(G)-nitro-L-arginine methyl ester (70 mgkg(-1), i.p.), a nitric oxide (NO) synthase inhibitor, or N-ethylmaleimide (10 mgkg(-1), s.c.), a blocker of sulfhydryl groups. This suggested that the gastroprotective mechanism of this diterpenoid involved the participation of both NO and endogenous sulfhydryl groups. Contrary to carbenoxolone, the gastroprotective effect of polyalthic acid was not affected by the inhibition of prostaglandin synthesis with indometacin (10 mgkg(-1), s. c.). In conclusion, Croton reflexifolius contains compounds with gastroprotector activity. Polyalthic acid, which was isolated from this plant, was the main compound with gastroprotector activity, having effectiveness similar to that found with the use of carbenoxolone. Whereas NO and sulfhydryl groups were involved in the mechanisms of gastroprotective action of polyalthic acid, prostaglandins were not.

Kidney damage after renal ablation is worsened in endothelial nitric oxide synthase -/- mice and improved by combined administration of L-arginine and antioxidants.

AIM: Reduction in nitric oxide (NO) levels during kidney failure has been related to the reaction of NO with superoxide anions to yield peroxynitrite which possesses the biological activity responsible for renal damage. However, stimulation of the NO pathway ameliorates the progression of kidney failure. Thus, it is unclear whether NO prevents or acts as the compound responsible for the cytotoxicity observed during kidney failure. METHODS: We evaluated the development of kidney failure in animals that were wild type and deficient in endothelial NO synthase (eNOS -/-) and tested the effects of an antioxidant treatment and NO precursors on the generation of superoxide anion and kidney failure parameters. RESULTS: In wild-type mice, five-sixths nephrectomy increased proteinuria from 3.0 +/- 0.35 to 14.5 +/- 0.76 mg protein/24 h (P < 0.05), blood pressure from 83.1 +/- 1.8 to 126.6 +/- 1.7 mmHg (P < 0.05), and superoxide production from 1.4 +/- 0.6% to 74.3 +/- 0.8% (P < 0.05). The effects of five-sixths nephrectomy on the eNOS -/- mice were greater compared with wild-type mice. Proteinuria increased from 6.7 +/- 0.5 to 22.7 +/- 2.0 mg protein/24 h (P < 0.05), blood pressure increased from 93.3 +/- 0.9 to 151.2 +/- 3.4 mmHg (P < 0.05), and superoxide production increased from 12.9 +/- 0.5% to 99.8 +/- 1.3% (P < 0.05). The nitrotyrosine levels were lower in eNOS -/- mice as compared to wild-type mice. A combination of L-arginine and antioxidant treatment ameliorated renal damage. The effect was improved in wild-type animals. CONCLUSION: Our data support the relevance of NO as an antagonist to superoxide in renal tissues and suggest that the loss of this mechanism promotes the progression of kidney failure.

Relaxant action mechanism of berberine identified as the active principle of Argemone ochroleuca Sweet in guinea-pig tracheal smooth muscle.

In this study we investigated the relaxant effect of the aerial parts of Argemone ochroleuca (Papaveraceae), which is used in Mexican traditional medicine for the treatment of various respiratory diseases such as cough, bronchitis and asthma. The alkaloid berberine was identified as one of the active relaxant principles (EC50 = 118.50 +/-3.91 microM) in the dichloromethane extract of A. ochroleuca (EC50 = 78.03 +/- 2.15 microg mL(-1) with 95.12 +/- 3.56% of relaxation). Berberine concentration-dependently relaxed the carbachol-induced precontractions but not histamine- or KCl-induced precontraction. The relaxant effect of berberine was unaffected by the presence of propranolol (3 microM), glibenclamide (10 microM) or ODQ (10microM). However, 2', 5'-dideoxyadenosine (10 microM) blocked the log concentration-response curves of berberine. On the other hand, berberine produced a leftward shift of the log concentration-response curves of isoproterenol, forskolin and nitroprusside. Additionally, berberine produced a parallel rightward shift of the concentration-response curve of carbachol in a competitive manner with a pA2 of 3.87 +/- 0.045. The above results suggest that the relaxant effect of berberine on tracheal muscle is due to its antagonistic effect on muscarinic acetylcholine receptors.

Mechanisms of relaxant action of a crude hexane extract of Gnaphalium liebmannii in guinea pig tracheal smooth muscle.

We investigated the mechanisms of action of Gnaphalium liebmannii which is used as a folk medicine in México for treating various respiratory diseases such as gripe, fever, asthma, cough, cold, bronchitis, expectorating, and bronchial affections. The tension changes of guinea pig tracheal segments were isometrically recorder on a polygraph. Hexane extract of Gnaphalium liebmannii was the most active relaxant extract (IC(30)=54.23+/-19.79 microg/mL with 99.5+/-3.2 % of relaxation), followed by dichloromethane extract (IC(30)=120.22+/-5.27 microg/mL) and methanol extract (IC(30)=190.25+/-30.02 microg/mL). Hexane extract produced a parallel rightward shift of the concentration-response curve of carbachol in a competitive manner (pA(2)=-2.4), but did not modify the concentration-response curves for histamine. The relaxant effect of hexane extract of Gnaphalium liebmannii was unaffected by the presence of propranolol (3x10(-6)M) or glibenclamide (10 microM). However hexane extract produced a leftward shifts of the concentration-response curve of forskolin (10(-8) to 10(-3)M), nitroprusside (10(-10) to 10(-6)M), isoproterenol (3x10(-10) to 3x10(-5)M) and aminophylline (10(-11) to 10(-2)M). The above results suggest that Gnaphalium liebmannii induce relaxation of the tracheal muscle, probably via phosphodiesterase inhibition. The bronchodilator effect of Gnaphalium liebmannii might explain in part their traditional use as anti-asthmatic remedy.

Nerve root degeneration and regeneration by intrathecal phenol in rats: a morphologic approach.

Intrathecal injection of phenol (ITP) has been used to control intractable pain and spasticity. Direct caustic nerve damage has been postulated as the mechanism of analgesia. Sensation is commonly recovered, suggesting that a spontaneous regeneration process takes place. There is, however, a lack of mechanistic information on ITP therapy. To define morphologically the neurolysis and regeneration phenomena produced by ITP, anesthetized rats were subjected to laminectomy at L5; 5 microl of 22% phenol in saline solution or vehicle (control) was injected. Light and electron microscopy studies of nerve roots were performed at 2, 14, and 60 days after injection. Rats given ITP showed at the early stage a variable amount of roots with signs of infarction characterized by loss of axon-myelin units and thrombosis of intra-root vessels. At 14 days, abundance of macrophages removing debris, open vessels, and nerve sprouts was identified in damaged roots. At this time, non-myelinating glial fibrillary acidic protein-positive Schwann cells were observed in both damaged and apparently undamaged roots. At 60 days, abundance of 2',3'-cyclic nucleotide 3'-phosphodiesterase-positive Schwann cells myelinating newly formed axons was observed in damaged roots. Control rats did not show signs of neural or vascular pathology. Attempting to prevent thrombosis, another group of rats received heparin before ITP; these anti-coagulated rats developed radicular thrombosis, neurolysis, and hemorrhage. In conclusion, neurolysis produced by ITP is associated with acute ischemia (not prevented by heparin) and is followed by vascular, nerve, and myelin regeneration. Our results help understand the lack of efficacy of and some complications by ITP clinical therapy.

[Intracellular calcium-dependent contractile effects of serotonin in the human umbilical artery]. / Efectos contráctiles dependientes del calcio intracelular inducidos por la serotonina en la arteria umbilical humana.

BACKGROUND: Absence of innervation is a hallmark of human umbilical vessels. Intervillous space blood flow is regulated by vasoactive substances and calcium dependent contractility, both in normal and pathological conditions such as preeclampsia-eclampsia. OBJECTIVE: To obtain additional information on the intracellular calcium contractile effects of serotonin in human umbilical arteries. MATERIALS AND METHODS: Umbilical arteries from normal pregnancies were dissected, cut in 5 mm rings and mounted in a temperature-controlled isolated organ chamber, using calcium-free Krebs solution. The contractile effects of serotonin, lantane, verapamil and cyclopiazonic acid were evaluated at different concentrations using a computer coupled biopac polygraph. RESULTS: No differences in response were observed in the presence and absence of intracellular calcium. The positive contractile effects observed with serotonin were significantly decreased with repeated stimulation. An increase in the basal tone of the vessel was observed after calcium supplementation was added to the solution. This effect was minimized in the presence of verapamil and lantane. The contractile effects of serotonin in the calcium-free solution were not affected by the presence of cyclopiazonic acid. CONCLUSIONS: Serotonin contractile effects in the human umbilical artery depend mainly on intracellular calcium levels which favor the gradual entrance of this ion over time. Calcium influx induced by serotonin is possible through L and Non-L channels apparently insensitive to ciclopiozonic acid.

Efectos contráctiles dependientes del calcio intracelular inducidos por la serotonina en la arteria umbilical humana / Intracellular calcium-dependent contractile effects of serotonin in the human umbilical artery

Antecedentes: Los vasos umbilicales humanos se caracterizan por la ausencia de inervación y por consecuencia, las diferentes sustancias vasoactivas y la respuesta contráctil a través de los iones de calcio son los factores que determinan el flujo sanguíneo al espacio intervelloso en condiciones normales y patológicas, como es el caso de la preeclampsia eclampsia. Objetivo: Aportar información adicional de la respuesta contráctil de la serotonina en relación al calcio intracelular en la arteria umbilical humana. Material y métodos: Se utilizaron vasos umbilicales procedentes de mujeres con embarazo normoevolutivo, los cuales una vez disecados se cortaron en anillos de 5 mm y se montaron en cámara de órgano aislado, utilizando solución de Krebs con y sin calcio, burbujeada con carbógeno y la temperatura controlada. Se evaluó el efecto contráctil inducido por serotonina a diferentes concentraciones molares y se contrastó con verapamil, lantano y ácido ciclopiazónico, cuantificando la respuesta contráctil mediante un polígrafo biopac acoplado a un sistema computacional. Resultados: No se observaron diferencias significativas en la magnitud de la respuesta obtenida en presencia y ausencia de calcio extracelular. Se apreció el efecto contráctil a la serotonina que disminuyó significati vamente como respuesta a la estimulación repetida a la misma; así mismo, se incrementó el tono basal posterior a la adición del calcio al medio de incubación, lo que dependió del tiempo de exposición. También se observó la inhibición parcial del incremento en el tono basal con vera pamil y lantano. Finalmente, el pretratamiento con ácido ciclopiazónico no modificó la respuesta contráctil a la serotonina en un medio sin calcio. Conclusiones: La contracción inducida por serotonina en la arteria umbilical humana, depende principalmente de calcio intracelular y favorece el ingreso capacitativo de este ion, el cual se incrementa gradualmente a través del tiempo. El ingreso capacitativo del calcio secundario al vaciamiento de los depósitos intracelulares de este ion con serotonina se efectúa a través de canales tipo L y no-L, y no parecen ser sensibles al ácido ciclopiazónico.

Background: Absence of innervation is a hallmark of human umbilical vessels. Intervillous space blood flow is regulated by vasoactive substances and calcium dependent contractility, both in normal and pathological conditions such as preeclampsia eclampsia. Objective: To obtain additional information on the intracellular calcium contractile effects of serotonin in human umbilical arteries. Materials and Methods: Umbilical arteries from normal pregnancies were dissected, cut in 5 mm rings and mounted in a temperature controlled isolated organ chamber, using calciumfree Krebs solution. The contractile effects of serotonin, lantane, verapamil and cyclopiazonic acid were evaluated at different concentrations using a computer coupled biopac polygraph. Results: No differences in response were observed in the presence and absence of intracellular calcium. The positive contractile effects observed with serotonin were significantly decreased with repeated stimulation. An increase in the basal tone of the vessel was observed after calcium supplementation was added to the solution. This effect was minimized in the presence of verapamil and lantane. The contractile effects of serotonin in the calcium free solution were not affected by the presence of cyclopiazonic acid. Conclusions: Serotonin contractile effects in the human umbilical artery depend mainly on intracellular calcium levels which favor the gradual entrance of this ion over time. Calcium influx induced by serotonin is possible through L and Non-L channels apparently insensitive to cyclopiozonic acid.

Ozone exposure induces iNOS expression and tyrosine nitration in rat aorta.

The aim was to study whether ozone affects vascular endothelium by causing inducible nitric oxide synthase (iNOS) expression and tyrosine nitration. We also studied biomarkers of endothelial function. Male Wistar rats were exposed to ozone (0.25ppm, 4h/day) or filtered air (control, ozone <0.05ppm). After ozone exposure, blood samples were taken to measure 6-keto prostaglandin F1α (6-keto PGF1α), dehydro-thromboxane B(2) (DH-TxB(2)), endothelin-1 and NO(2)(-)/NO(3)(-) (NO(x)(-)). iNOS and nitrotyrosine were detected in aorta by immunohistochemistry. Nitrotyrosine was also detected by immunoelectromicroscopy. Control aortae failed to show either iNOS or nitrotyrosine. Time-dependent positive iNOS and nitrotyrosine cells were observed in exposed animals. Except for NO(x)(-), endothelial markers decreased after 14 days of ozone exposure (P<0.05). After 28 days of ozone, 6-keto PGF1α remained low (P<0.05) while DH-TxB(2) increased (P<0.05). It is concluded that ozone causes endothelial dysfunction manifested early with peroxynitrite formation and lately with changes in endothelial markers.

Nitric oxide synthase in Entamoeba histolytica: its effect on rat aortic rings.

NADPH-diaphorase activity has been considered as a nitric oxide synthase (NOS) marker. Therefore, the presence of NADPH-d activity in Entamoeba histolytica suggests that they have NOS activity. The aim of this work was to provide support for this contention. The amebic culture medium or amebic purified proteins induced relaxation of endothelium-denuded rat aortic rings pre-contracted with phenylephrine (10(-6) M), which was inhibited when the amebas were incubated with NG-monomethyl-L-arginine or aminoguanidine (NOS inhibitors), or by pretreatment of the aortic rings with methylene blue. L-Arginine reverted the L-NAME inhibitory effect. In addition, trophozoites produce NO in culture and they have proteins which were recognized by antibodies specific to NOS and show activity of NO synthase. In conclusion, our results provide evidence about the production of NO by trophozoites. This molecule may be responsible for the relaxation elicited by the amebic culture medium and may participate in the pathogenesis of the invasive amebiasis. Index Descriptors and Abbreviations: Entamoeba histolytica; NO, nitric oxide; NOS, nitric oxide synthase; iNOS, inducible nitric oxide synthase; ecNOS, endothelial nitric oxide synthase; NADPH-d, NADPH-diaphorase enzyme; beta-NADPH, beta-nicotinamide-adenine dinucleotide; L-NAME, N-omega-nitro-L-arginine methyl ester hydrochloride; NBT, nitobluetetrazolium; PBS, phosphate-buffered saline; EDTA, ethylenediaminetetraacetic acid; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Synthesis and evaluation of the cardiovascular effects of two, membrane impermeant, macromolecular complexes of dextran-testosterone.

The incidence of cardiovascular disease is greater in men than in premenopausal women. Testosterone has been considered a significant risk factor for cardiovascular disease, but testosterone's mechanism of action and its cellular site of action are still not clear. However, it is likely that non-genomic extracellular effects of the hormone are involved. With the aim of providing further information about this phenomenon, two membrane impermeant, macromolecular complexes of testosterone were synthesized and their cardiovascular effects were evaluated. We covalently bound testosterone (through carbon 3 or C-17 functional groups) to dextran (2 MDa) and evaluated its effects on isolated and perfused rat hearts (Langerdorff model). Our results showed that the macromolecular complexes increased vascular resistance similarly to free testosterone and blocked adenosine-induced vasodilatation. These effects were exerted rapidly and possibly through a non-genomic mechanism. Blockade of C-3 or C-17 functional groups by binding to macromolecular dextran induced no qualitative and/or quantitative changes in testosterone-induced effects.