Kaliuretic response to potassium loading in amiloride-treated dogs.

To assess whether an intact mechanism of sodium transport in the distal nephron is a prerequisite for the development of a kaliuresis in response to an acute potassium load (0.4 M KCl, 1 ml/min), the effects of a simultaneous infusion of KCl and amiloride (1 mg/kg/h) were evaluated in anesthetized dogs. A major reduction in potassium excretion mainly due to a sharp decrease in urine K+ concentration to one tenth of control levels was found after amiloride. The simultaneous infusion of KCl and amiloride resulted in a rapid and major increase in kaliuresis that was accounted for mostly by the rise in urine K+ concentration. The increased kaliuresis after the acute potassium infusion was of similar magnitude when expressed as percent value of control to that previously reported in dogs not receiving amiloride; the absolute rates of K+ excretion, however, were only 2.7 and 7.3% (before and after KCl infusion, respectively) of the values in dogs not receiving amiloride. Our observations suggest that potassium infusion in the intact dog increases kaliuresis primarily as a result of a more favorable chemical gradient of this cation between blood and/or distal tubular cells and urine. Yet, when a chemical gradient is the only driving force of potassium secretion, as was the case in our amiloride-treated dogs, the absolute rate of kaliuresis is very modest. The presence of an unimpaired electrical profile and sodium transport mechanisms in the distal nephron, although not critical for the development of kaliuresis in response to a K+ load, accounts for a severalfold rise in renal potassium excretion above basal levels.     

Vanadium compounds

The direct effect of different vanadium compounds upon alkaline phosphatase (ALP) activity was investigated. Vanadate and vanadyl inhibited both the soluble and particulate ALP activity from UMR.106 cells and from bovine intestinal ALP. We have also shown the inhibition of ALP activity in the soluble fraction of osteoblasts by peroxo and hydroperoxo vanadium compounds. ALP activity in the particulate fraction was not inhibited by these species; nor was the bovine intestinal ALP. Using inhibitors of Tyr-phosphatase (PTPases), the soluble ALP was partially characterized as a PTPase. The major activity in the particulate fraction represents the bone-specific ALP-activity. This study demonstrates that different forms of vanadium are direct inhibitors of ALP activity. This effect is dependent on the enzymatic activity investigated and on the origin of the ALP.     

Vanadium derivatives act as growth factor — mimetic compounds upon differentiation and proliferation of osteoblast-like UMR106 cells

The effect of different vanadium compounds on proliferation and differentiation was examined in osteoblast-like UMR106 cells. Vanadate increased the cell growth in a biphasic manner, the higher doses inhibiting cell progression. Vanadyl stimulated cell proliferation in a dose-responsive manner. Similar to vanadate, pervanadate increased osteoblast-like cell proliferation in a biphasic manner but no inhibition of growth was observed. Vanadyl and pervanadate were stronger stimulators of cell growth than vanadate. Only vanadate was able to regulate the cell differentiation as measured by cell alkaline phosphatase activity. These results suggest that vanadium derivatives behave like growth factors on osteoblast-like cells and are potential pharmacological tools in the control of cell growth.     

Revue rapide: 381 sujets impuissants,explores en 2 ans,dont 207 avec Prostaglandine E1

During the last two years, 381 impotent patients have been tested, 174 with papaverine and 207 with prostaglandin E1 (PGE1: Alprostadil). More positive responses were obtained using PGE1 (76.8%) than papaverine (44.8%), and some patients whose response to an initial PGE1 test was negative demonstrated a positive effect after a second injection. Two non-responders to PGE1 showed a positive response to papaverine+phentolamine. Tolerance of PGE1 was extremely good, provoking fewer prolonged erections than papaverine. Pain following intracavernosal injection of PGE1 was distressing in 10% of cases, all young men with psychogenic impotence. A therapeutic trial of repeated injections, carried out in 49 selected patients with mild organic dysfunction but strong psychogenic inhibition, typically showed a rapid clinical improvement. Moreover, 16 patients with cavernosal sclerosis and vascular impotence who were treated by injections two or three months apart (instead of having a prosthetic implant) showed a recovery of sexual activity. Consequently, during these two years only 35 patients (9.1%) were treated surgically, 21 by the implantation of hydraulic prostheses. The significant benefits of PGE1, especially the ease of self-administration, demonstrate the need for new therapeutic trials.     

Liver glycogen phosphorylase is upregulated in glioblastoma and provides a metabolic vulnerability to high dose radiation

Channelling of glucose via glycogen, known as the glycogen shunt, may play an important role in the metabolism of brain tumours, especially in hypoxic conditions. We aimed to dissect the role of glycogen degradation in glioblastoma (GBM) response to ionising radiation (IR). Knockdown of the glycogen phosphorylase liver isoform (PYGL), but not the brain isoform (PYGB), decreased clonogenic growth and survival of GBM cell lines and sensitised them to IR doses of 10–12 Gy. Two to five days after IR exposure of PYGL knockdown GBM cells, mitotic catastrophy and a giant multinucleated cell morphology with senescence-like phenotype developed. The basal levels of the lysosomal enzyme alpha-acid glucosidase (GAA), essential for autolysosomal glycogen degradation, and the lipidated forms of gamma-aminobutyric acid receptor-associated protein-like (GABARAPL1 and GABARAPL2) increased in shPYGL U87MG cells, suggesting a compensatory mechanism of glycogen degradation. In response to IR, dysregulation of autophagy was shown by accumulation of the p62 and the lipidated form of GABARAPL1 and GABARAPL2 in shPYGL U87MG cells. IR increased the mitochondrial mass and the colocalisation of mitochondria with lysosomes in shPYGL cells, thereby indicating reduced mitophagy. These changes coincided with increased phosphorylation of AMP-activated protein kinase and acetyl-CoA carboxylase 2, slower ATP generation in response to glucose loading and progressive loss of oxidative phosphorylation. The resulting metabolic deficiencies affected the availability of ATP required for mitosis, resulting in the mitotic catastrophy observed in shPYGL cells following IR. PYGL mRNA and protein levels were higher in human GBM than in normal human brain tissues and high PYGL mRNA expression in GBM correlated with poor patient survival. In conclusion, we show a major new role for glycogen metabolism in GBM cancer. Inhibition of glycogen degradation sensitises GBM cells to high-dose IR indicating that PYGL is a potential novel target for the treatment of GBMs.Subject terms: Cancer metabolism, CNS cancer     

NARC-1/PCSK9 and its natural mutants: zymogen cleavage and effects on the low density lipoprotein (LDL) receptor and LDL cholesterol

The discovery of autosomal dominant hypercholesterolemic patients with mutations in the PCSK9 gene, encoding the proprotein convertase NARC-1, resulting in the missense mutations suggested a role in low density lipoprotein (LDL) metabolism. We show that the endoplasmic reticulum-localized proNARC-1 to NARC-1 zymogen conversion is Ca2+-independent and that within the zymogen autocatalytic processing site SSVFAQ [downward arrow]SIP Val at P4 and Pro at P3' are critical. The S127R and D374Y mutations result in approximately 50-60% and > or =98% decrease in zymogen processing, respectively. In contrast, the double [D374Y + N157K], F216L, and R218S natural mutants resulted in normal zymogen processing. The cell surface LDL receptor (LDLR) levels are reduced by 35% in lymphoblasts of S127R patients. The LDLR levels are also reduced in stable HepG2 cells overexpressing NARC-1 or its natural mutant S127R, and this reduction is abrogated in the presence of 5 mm ammonium chloride, suggesting that overexpression of NARC-1 increases the turnover rate of the LDLR. Adenoviral expression of wild type human NARC-1 in mice resulted in a maximal approximately 9-fold increase in circulating LDL cholesterol, while in LDLR-/- mice a delayed approximately 2-fold increase in LDL cholesterol was observed. In conclusion, NARC-1 seems to affect both the level of LDLR and that of circulating apoB-containing lipoproteins in an LDLR-dependent and -independent fashion.     

Rab35: GEFs,GAPs and Effectors

Rabs are the largest family of small GTPases and are master regulators of membrane trafficking. Following activation by guanine‐nucleotide exchange factors (GEFs), each Rab binds a specific set of effector proteins that mediate the various downstream functions of that Rab. Then, with the help of GTPase‐activating proteins, the Rab converts GTP to GDP, terminating its function. There are over 60 Rabs in humans and only a subset has been analyzed in any detail. Recently, Rab35 has emerged as a key regulator of cargo recycling at endosomes, with an additional role in regulation of the actin cytoskeleton. Here, we will focus on the regulation of Rab35 activity by the connecdenn/DENND1 family of GEFs and the TBC1D10/EPI64 family of GTPase‐activating proteins. We will describe how analysis of these proteins, as well as a plethora of Rab35 effectors has provided insights into Rab35 function. Finally, we will describe how Rab35 provides a novel link between the Rab and Arf family of GTPases with implications for tumor formation and invasiveness .