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Aim: These studies test the hypothesis that norepinephrine (NE) impairs renal NCC regulation to evoke salt-sensitive hypertension in Sprague-Dawley (SD) and Dahl Salt-Sensitive (DSS) rats.Methods: SD rats receiving a s.c. saline vehicle or NE (600ng/min) infusion were fed a 0.4% (NS) or high 8% NaCl (HS) diet for 14 days. Naïve DSS rats were placed on a NS or HS diet for 21 days. On day 14 or 21, MAP and peak natriuresis to i.v. hydrochlorothiazide (HCTZ; 2mg/kg) infusion was assessed. Plasma NE was measured and NCC immunoblotting was performed on kidney cortex tissue (N=5/6).Results: Excess NE significantly increased MAP and plasma NE levels in NE infused SD rats. A HS diet further increased plasma NE levels and MAP in both NE infused SD and DSS rats. HS intake failed to suppress NCC activity in NE infused SD rats and naïve DSS rats. Impaired NCC expression was shown in NE and HS treated SD rats.Conclusion: These findings reveal that NE infusion combined with a high salt diet in a salt-resistant phenotype evokes the physiological responses found in a genetic model of salt-sensitive hypertension. These data from both animal models support a direct role of NE on NCC function and the pathophysiology of salt-sensitivity. [ABSTRACT FROM AUTHOR]

Aim: Recent controversial studies have proposed that excess norepinephrine (NE) evokes impaired NCC regulation to drive salt-sensitive hypertension. The following studies examine the impact of excess NE on salt-sensitivity and sodium homeostasis in conscious Sprague-Dawley (SD) rats.Methods: Naïve male SD rats, rats receiving a s.c. vehicle infusion (DMSO/Saline, 50:50), or rats receiving a s.c. NE infusion (600ng/min) were fed a 0.4% (NS) or 8% NaCl (HS) diet for 14 days. Additional rats received s.c. hydrochlorothiazide (HCTZ, 4mg/kg/d) in combination with NE (600ng/min) for 14 days on HS. On day 14, MAP, FENa, MAP response to i.v. hexamethonium (30mg/kg), and peak natriuresis to i.v. HCTZ (2mg/kg) infusion were assessed (N=4/gp). A PCR array examining NCC associated genes was performed on kidney cortex samples from each group.Results: NE increased MAP, FENa and vascular sympathetic tone (MAP [mmHg] NS 127±2, NE+NS 151±3, p<0.05). We observed no difference between the naïve and vehicle rats. A HS diet exacerbated NE induced hypertension (MAP [mmHg] HS 129±2, NE+HS 172±4, p<0.05), reduced FENa and prevented a salt stimulated reduction in HCTZ evoked natriuresis. Co-infusion of HCTZ with NE abolished the salt-sensitive component of NE-induced hypertension (MAP [mmHg] NE+HCTZ+HS: 152±3, p<0.05). PCR analysis revealed a significant increase in serine/threonine kinase 39 (0.83-fold increase vs. Naïve SD on NS) mRNA in NE+HS rats. We did not see NE or HS evoked changes in OSR-1, WNK4 or NCC mRNA in any group.Conclusion: The results support previous studies in mice and highlight an opposing interaction between excess NE and high salt intake on sodium homeostasis which exacerbated NE-induced hypertension via a mechanism independent of NE-mediated vascular constriction. Physiologically, our results show impaired NCC function, supporting previous data. In contrast, we failed to detect elevated NCC or WNK4 mRNA in response to NE infusion contradicting data generated in mice and suggesting a key role of altered NCC phosphorylation versus expression in NE treated rats. [ABSTRACT FROM AUTHOR]

In this study, mechanisms for the hydrolysis of the Gly-Pro bond in Gly-Pro-Met and Gly-Pro-His, the Gly-Sar bond in Gly-Sar-Met, and the Gly-Gly bond in the Gly-Gly-Met peptide catalyzed by [Pd(H2O)4]2 (I) have been investigated at the DFT level. In all cases, the optimized structure of the active bidentate complex, formed by the reaction of I with the substrate [Pd(H2O)2((Gly)-(Pro)-(Met-KS,KN))]1+ complex for the Gly-Pro-Met peptide, was found to exist in the trans conformation. This structure is in agreement with the experimentally measured TOCSY and POESY 1H NMR spectra, After the formation of this complex, the following two mechanisms have been proposed experimentally: (1) external attack mechanism and (2) internal delivery mechanism. The DFT calculations suggest that in the external attack mechanism the calculated barriers are prohibitively high (i,e., 50-70 kcal/mol) for the cleavage of all the peptide bonds, and therefore, this mechanism is ruled out. However, in the internal delivery mechanism, the bidentate complex is first transformed from the trans to the cix conformation. Here, the overall barriers for the hydrolysis of the Gly-ProMet, Gly-Pro-His, Gly-Sar-Met, and Gly-Gly-Met peptide bonds are 38.3,41.4,39.8, and 39.2 kcal/mol, respectively. These barriers are in much better agreement with the experimentally measured rate constants at pH 2.0 and at 60 5C. The substitution of Pd(ll) with Pt(ll) was found to make a negligibly small difference (0.53 kcal/mol) on the barrier for the cleavage of the Gly-Pro-His bond. These calculations indicate that after the creation of the active bidentate complex in the trans conformation the internal delivery mechanism is the most energetically feasible. [ABSTRACT FROM AUTHOR]

Background: CD133 (AC133) is a surface antigen that defines a broad population of stem cells, including myogenic and endothelial progenitors. CD133+ cells are rare in adult tissues, and the factors that support their differentiation into mature angiomyogenic cells are not known. These hurdles have hampered the use of CD133+ cells for therapeutic purposes. Because human fetal liver is a rich source of CD133+ cells, we sought to identify the growth factors that promote codifferentiation of these cells into angiogenic and myogenic cells., Methods and Results: Human fetal liver CD133+ and CD133- cell subpopulations were cultured with 5'-azacytidine or vascular endothelial growth factor (VEGF165) and/or brain-derived nerve growth factor (BDNF). CD133+ but not CD133- cells from human fetal liver codifferentiated into spindle-shaped cells, as well as flat adherent multinucleated cells capable of spontaneous contractions in culture. The resulting spindle-shaped cells were confirmed to be endothelial cells by immunohistochemistry analysis for von Willebrand factor and by acetylated LDL uptake. Multinucleated cells were characterized as striated muscles by electron microscopy and immunohistochemistry analysis for myosin heavy chain. Presence of VEGF165 and BDNF significantly enhanced angiomyogenesis in vitro. Inoculation of cells derived from CD133+ cells, but not CD133- cells, into the ear pinna of NOD/SCID mice resulted in the formation of cardiomyocytes, as identified by immunostaining with cardiac troponin-T antibody. These cells generated electrical action potentials, detectable by ECG tracing., Conclusions: CD133 defines a population of human fetal liver cells capable of differentiating into both angiogenic and myogenic cells. Preconditioning of these CD133+ cells with VEGF165 and BDNF enhances the angiomyogenesis. CD133+ fetal liver cells ultimately may be used for therapeutic angiomyogenesis.