Leg intravenous pressure during head-up tilt

Leg vascular resistance is calculated as the arterial-venous pressure gradient divided by blood flow. During orthostatic challenges it is assumed that the hydrostatic pressure contributes equally to leg arterial, as well as to leg venous pressure. Because of venous valves, one may question whether, during orthostatic challenges, a continuous hydrostatic column is formed and if leg venous pressure is equal to the hydrostatic pressure. The purpose of this study was, therefore, to measure intravenous pressure in the great saphenous vein of 12 healthy individuals during 30 degrees and 70 degrees head-up tilt and compare this with the calculated hydrostatic pressure. The height difference between the heart and the right medial malleolus level represented the hydrostatic column. The results demonstrate that there were no differences between the measured intravenous pressure and the calculated hydrostatic pressure during 30 degrees (47.2 +/- 1.0 and 46.9 +/- 1.5 mmHg, respectively) and 70 degrees head-up tilt (83.9 +/- 0.9 and 85.1 +/- 1.2 mmHg, respectively). Steady-state levels of intravenous pressure were reached after 95 +/- 12 s during 30 degrees and 161 +/- 15 s during 70 degrees head-up tilt. In conclusion, the measured leg venous pressure is similar to the calculated hydrostatic pressure during orthostatic challenges. Therefore, the assumption that hydrostatic pressure contributes equally to leg arterial as well as to leg venous pressure during orthostatic challenges can be made.     

A MBP-FAAH fusion protein as a tool to produce human and rat fatty acid amide hydrolase: expression and pharmacological comparison

Summary. Fatty acid amide hydrolase (FAAH), a membrane-anchored enzyme responsible for the termination of endocannabinoid signalling, is an attractive target for treating conditions such as pain and anxiety. Inhibitors of the enzyme, optimized using rodent FAAH, are known but their pharmacology and medicinal chemistry properties on the human FAAH are missing. Therefore recombinant human enzyme would represent a powerful tool to evaluate new drug candidates. However, the production of high amounts of enzyme is hampered by the known refractiveness of FAAH to overexpression. Here, we report the successful overexpression of rat and human FAAH as a fusion to the E. coli maltose-binding protein, retaining catalytic properties of native FAAH. Several known FAAH inhibitors were tested and differences in their potencies toward the human and rat FAAH were found, underscoring the importance of using a human FAAH in the development of inhibitors. Authors’ address: Didier M. Lambert, Unité de Chimie pharmaceutique et de Radiopharmacie, Université catholique de Louvain, Avenue E. Mounier 73.40, 1200 Bruxelles, Belgique     

Coordinate induction of PPAR alpha and SREBP2 in multifunctional protein 2 deficient mice

Mice with inactivation of the D-specific multifunctional protein 2 (MFP2), a crucial enzyme of peroxisomal beta-oxidation, develop multiple pathologies in diverse tissues already starting in the postnatal period. Gene expression profiling performed on liver of 2-day-old pups revealed up-regulation of PPAR alpha responsive genes in knockout mice. Surprisingly, also genes involved in cholesterol biosynthesis were markedly induced. Real-time PCR confirmed the induction of PPAR alpha target genes and of HMGCR and SREBP2, both involved in cholesterol synthesis, in lactating and in adult MFP2 knockout mice. In accordance, the rate of cholesterol biosynthesis was significantly increased in liver of knockout mice but the hepatic cholesterol concentration was unaltered. In MFP2/PPAR alpha double knockout mice, up-regulations of SREBP2 and HMGCR were markedly attenuated. These data demonstrate a tight interrelationship between induction of PPAR alpha by endogenous ligands and up-regulation of genes of cholesterol biosynthesis through increased expression of SREBP2.     

Na-P(i) cotransporter type I activity causes a transient intracellular alkalinization during ATP depletion in rabbit medullary thick ascending limb cells

The cellular pathophysiology of renal ischemia-reperfusion injury was investigated in primary cell cultures from rabbit medullary thick ascending limb (MTAL). Metabolic inhibition (MI) was achieved with cyanide and 2-deoxyglucose. Sixty minutes of MI caused a profound but reversible decrease in intracellular concentration of ATP ([ATP]i). Intracellular pH (pHi) first decreased after initiation of MI, followed by a transient alkalinization. When [ATP]i reached its lowest value (<1% of control), the cells slowly acidified to reach a stable pHi of 6.92 after 50 min of MI. In the presence of EIPA (10 micromol/L), the pattern of changes in pHi was unchanged and acidification was not increased, indicating that the Na+/H+ exchangers were inactive during ATP depletion. When inorganic phosphate (P(i)) or Na+ was omitted from the apical solutions during MI, the transient alkalinization was no longer observed and the cytosol slowly acidified. Experiments on Na+-dependent alkalinizations revealed the presence of a Na-P(i) cotransporter in the apical cell membrane. With indirect immunofluorescence, the Na-P(i) cotransporter expressed in these primary cell cultures could be identified as Na-P(i) type I. Although the exact physiological role of Na-P(i) type I still is unresolved, these experiments demonstrate that apical Na-P(i) type I activity is increased at the onset of ATP depletion in MTAL cells.     

Arg(1098) is critical for the chloride dependence of human angiotensin I-converting enzyme C-domain catalytic activity.

Angiotensin (Ang) I-converting enzyme (ACE) is a Zn(2+) metalloprotease with two homologous catalytic domains. Both the N- and C-terminal domains are peptidyl dipeptidases. Hydrolysis by ACE of its decapeptide substrate Ang I is increased by Cl(-), but the molecular mechanism of this regulation is unclear. A search for single substitutions to Gln among all conserved basic residues (Lys/Arg) in human ACE C-domain identified R1098Q as the sole mutant that lacked Cl(-) dependence. Cl(-) dependence is also lost when the equivalent Arg in the N-domain, Arg(500), is substituted with Gln. The Arg(1098) to Lys substitution reduced Cl(-) binding affinity by approximately 100-fold. In the absence of Cl(-), substrate binding affinity (1/K(m)) of and catalytic efficiency (k(cat)/K(m)) for Ang I hydrolysis are increased 6.9- and 32-fold, respectively, by the Arg(1098) to Gln substitution, and are similar (<2-fold difference) to the respective wild-type C-domain catalytic constants in the presence of optimal [Cl(-)]. The Arg(1098) to Gln substitution also eliminates Cl(-) dependence for hydrolysis of tetrapeptide substrates, but activity toward these substrates is similar to that of the wild-type C-domain in the absence of Cl(-). These findings indicate that: 1) Arg(1098) is a critical residue of the C-domain Cl(-)-binding site and 2) a basic side chain is necessary for Cl(-) dependence. For tetrapeptide substrates, the inability of R1098Q to recreate the high affinity state generated by the Cl(-)-C-domain interaction suggests that substrate interactions with the enzyme-bound Cl(-) are much more important for the hydrolysis of short substrates than for Ang I. Since Cl(-) concentrations are saturating under physiological conditions and Arg(1098) is not critical for Ang I hydrolysis, we speculate that the evolutionary pressure for the maintenance of the Cl(-)-binding site is its ability to allow cleavage of short cognate peptide substrates at high catalytic efficiencies.