The non-peptide neuroprotective agents SR 57746A interacts with neurotrophin 3 to induce differentiation in the PC12 cell-line.

SR 57746A (1-(2beta-naphthylethyl)-4-(3-trifluoromethylphenyl)-1,2,5,6 -tetrahydropyridine hydrochloride) is a neuroprotective compound which potentiates nerve-growth factor (NGF)-induced differentiation in PC12 cells. We have evaluated the interaction of SR 57746A with the other members of the neurotrophin family in this cell-line. In contrast with NGF, neurotrophin-3 did not increase the differentiation of PC12 cells. However, the association of SR 57746A with neurotrophin-3 significantly increased neurite outgrowth. No significant activity on neurite outgrowth was observed with brain-derived neurotrophic factor or neurotrophin-4, either alone or combined with SR 57746A. These results indicate that as well as potentiating the effect of NGF SR 57746A enables neurotrophin-3, which alone is inactive, to increase the differentiation of these cells.

Characterization of a B2-bradykinin receptor in rat renal mesangial cells.

The specific binding of bradykinin (BK) was investigated using membrane fractions from mesangial cells in primary culture, a cloned cell line, and in intact adherent cells with three different radiolabelled BK analogues: 125I-[Tyr0]BK, 125I-[Tyr5]BK and 125I-[Tyr8]BK. The best radioligand was 125I-[Tyr0]BK, and assay conditions were determined to ensure maximal stable binding. Binding appeared to be reversible and not to be inhibited by a wide variety of protease inhibitors including converting enzyme inhibitor and phosphoramidon. The maximum density of binding sites (Bmax) was about 88 +/- 18 fmol/mg protein, which is equivalent to about 6000 sites/cell, and the dissociation constant averaged 2 nM. No significant difference in Bmax was observed between membranes from cells in primary culture and those from cloned cells. Of the BK analogues tested, unmodified BK exhibited the highest inhibition constant (close to 10(-10) M). No displacement of 125I-[Tyr0]BK was observed in the presence of the B1 agonist des-Arg9-BK or several unrelated peptides, including atrial natriuretic factor and angiotensin I and II, whereas 50% inhibition of binding was achieved with the B2 antagonist [D-Arg,Hyp3,D-Phe7]BK (10(-9)M). Addition of BK for 3 min to the incubation medium of cloned mesangial cells induced a dose- and time-dependent increase in PGE2 unlike des-Arg9-BK, which showed no such effect. The secretion was strongly inhibited by prior incubation with the B2 antagonist [D-Arg,Hyp3,D-Phe7]BK. The pharmacological profile of the binding site determined with various BK agonists and antagonists, and the stimulating effect of binding site activation on prostaglandin release strongly suggest that B2-kinin-like receptors are present in rat mesangial cells.

Early acute effects of mercuric chloride on synthesis and release of kallikrein and on distal tubular morphology of rat renal cortical slices.

The effect of mercuric chloride on kallikrein content and secretion of renal cortical slices was studied. Mercuric chloride showed dose-dependent inhibition of the secretion of immunoreactive and active kallikrein in the medium associated with a relative increase in the kallikrein tissue content of slices. However the net de novo biosynthesis was also reduced. Active and inactive kallikrein exhibited the same percentage of inhibition indicating that the activation mechanism of prokallikrein was not affected. These results suggest that mercuric chloride exerts an inhibition on tubular secretion but also on the tissular biosynthesis of kallikrein in these in vitro conditions. Morphological study of slices incubated in the presence of mercury also revealed significant tissular lesions which were located on the proximal tubule, but distal tubular changes were also observed. Distal nephrons were identified by the presence of immunoreactive kallikrein with the peroxidase-antiperoxidase method. These ultrastructural alterations included an increase in number and size of lysozomes, vacuoles and lipid droplets. These lesions were associated with an increased release of the lysozomal enzyme N-acetyl-beta-D-glucosaminidase. Since these distal tubular lesions are associated with inhibition of kallikrein secretion which is specifically located in the distal tubule, these results suggest that acute exposure of kidney cortical slices to mercuric chloride causes rapid and marked ultrastructural changes not only on the proximal tubule but also induced structural and biochemical effects at the distal tubule level. As incubation of mercuric chloride did not induce any direct alterations of immunoreactive and active kallikrein, it is likely that the observed inhibition of kallikrein synthesis and secretion are secondary to the morphological lesions.

Inverse relationship between renal and urinary kallikrein during chromate-induced acute renal failure in rat: urinary kallikrein excretion as a possible recovery index.

Acute renal failure (ARF) was induced in rat following a single injection of sodium chromate. A transient polyuria and a 10-fold decrease in glomerular filtration rate was immediately observed after sodium chromate administration. Urinary sodium and potassium excretion were reduced within 24 h and remained decreased for 8 to 10 days. Progressive recovery of normal renal functions, mainly electrolyte excretion and filtration rate was observed 12 days after sodium chromate administration. Urinary kallikrein excretion (UKE) was decreased only 48 h after sodium chromate administration. However the proportion of the active and inactive form excreted was unchanged. UKE remained also at a reduced level for 8 to 10 days and returned progressively to base-line level. The kallikrein content in the tissue was significantly increased immediately after sodium chromate administration and recovered normal values 12 days later. The increase of kallikrein in the tissue is more likely unspecific due to impaired protein transport than a specific stimulation of renal kallikrein biosynthesis. The decreased UKE may indicate a distal tubular reversible dysfunction in this ARF model. These reductions in electrolyte excretion, glomerular filtration and UKE were associated with selective morphological lesions. Whereas the glomeruli were intact, important damages affected proximal tubule cells which appeared necrotic and showed presence of vacuoles, liquefaction of cytoplasmic material and lost of microvilli. Less marked lesions were however observed in distal tubules, particularly large vacuoles were present at the apical poles of the tubule cells, the sites of kallikrein secretion. These distal damages may be involved in the increase of tissue concentration and in the decrease of UKE.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of changes in sodium or water intake on glomerular B2-kinin-binding sites.

In a previous study, we characterized a B2-kinin-binding site in rat glomerular membranes. We have now investigated the density and affinity of this binding site for bradykinin (BK) in rat glomerular membranes, in relation to the renal and urinary levels of kallikrein during changes in sodium and water intake. The density of BK-binding sites was reduced after 28 days of low-sodium diet (12.5 +/- 1.3 vs. control: 32.1 +/- 5.2 fmol/mg protein, P less than 0.05), whereas the concentration and activity of kallikrein was increased in the renal tissue and in the urine. Water deprivation for 4 days also resulted in a decreased density of binding sites (13.2 +/- 1.2 vs. control 32.1 +/- 5.2 fmol/mg protein, P less than 0.05) and, interestingly, renal kallikrein levels increased. During high salt consumption, urinary kallikrein activity increased but the renal concentration and activity of kallikrein was unchanged. In this high-sodium-loaded group, BK-binding site density was similar to controls receiving a normal-sodium diet (41.6 +/- 8.4 vs. control 32.1 +/- 5.2 fmol/mg protein). On the other hand, the binding affinity of BK did not change significantly in all experimental groups, and the binding specificity was not altered as BK remained the most potent agonist. Sodium depletion and water deprivation led to the same effects on BK-binding sites and on renal kallikrein. The renal glomerular BK-binding site density decreased in both conditions, whereas the renal level of kallikrein was enhanced. An inverse relationship between renal kinin levels and the density of glomerular BK-binding sites is therefore suggested.

[Evidence for a glomerular receptor for bradykinin in rats. Effect of sodium intake on density and affinity of the receptor]. / Mise en évidence d'un récepteur glomérulaire à la bradykinine chez le rat. Effet de l'apport sodé sur la densité et l'affinité de ce récepteur.

Bradykinin (BK) have been involved in a lot of pharmacological and biological effects including natriuresis, vasodilatation, inflammation and pain mediation. All these potent effects of BK are presumably mediated via one or more specific receptors which have been classified in two types named B1 and B2 receptors. In the kidney, specific binding have been reported successively in cortical epithelial membranes, in renomedullary interstitial cells and in cortical collecting tubules. Furthermore, since a large number of vasoactive compounds have been shown to regulate renal glomerular hemodynamics, we examined the binding of BK in rat glomerular membranes and the effect of variable salt diet on the density (Bmax) and the dissociation constant (KD) of this binding. Incubation of a radiolabeled bradykinin analog, [125I]-Tyr8-BK with a crude membrane preparation obtained from isolated rat glomeruli revealed a time dependent binding. The binding was saturable, reversible and was a linear function of protein membrane concentration. The radiolabeled Tyr8-BK bound to a single class of binding sites with an equilibrium dissociation constant (KD) of 2.75 +/- 0.7 nM and a density (Bmax) of 32.1 +/- 5.2 fmol/mg protein. [125I]-Tyr8-BK binding was reversed by bradykinin (Ki = 0.4 10(-9) M) and by other kinin analogs. However, Des-Arg9-BK had no effect on binding of the radiolabelled BK. These results are consistent with the presence of a B2-kinin like receptor in rat glomeruli. Low salt diet (during one month) and water deprivation (during 4 days) induced a decrease in the density of glomerular BK like receptors respectively (Bmax = 12.45 +/- 1.3 and 13.25 +/- 1.17 fmol/mg protein).(ABSTRACT TRUNCATED AT 250 WORDS)

Direct radioimmunoassay of active and inactive human glandular kallikrein: some physiological and pathological variabilities.

We have developed a sensitive and specific radioimmunoassay which allows the detection of human glandular kallikrein in biologic fluids at a level of 40 pg/ml. The antisera did not recognize human plasma kallikrein and glandular kallikrein from other species including marmoset. Furthermore the antibody did not bind pro-kallikrein but was specific for the trypsin activated kallikrein. The antibody inhibited the kininogenase activity of standard kallikrein incubated with human kininogen. However active kallikrein inhibited by inhibitors bound at the active site is still detectable, indicating that the antibody is specific for the structure of the active form but not for the active site. In normotensive subjects, daily urinary kallikrein excretion increased with age until 30, then a decrease was observed. In renal transplanted recipients a progressive increase of the active form was found. A low concentration of immunoreactive active kallikrein was detected in lymphatic fluids of patients suffering from acute pancreatitis treated by lymphatic drainage; although this kallikrein is the active immunoreactive form, a very weak kininogenase activity was measured, suggesting a partial inhibition by anti-proteases. These data provide complementary evidence for the physiological and pathological role of glandular kallikrein.