Jeannine Marchetti

Measurement of urinary kallikrein activity species differences in kinin production

A sensitive, specific radioimmunoassay for kinins has been developed, which is able to detect 1.5 pg bradykinin or 3 pg lysyl-bradykinin (Lys-bradykinin). 50% displacement in the standard curve was obtained with 10 pg bradykinin or 15 pg Lys-bradykinin in 0.6-ml incubates. The antisera, raised against bradykinin, recognized well Lys-bradykinin and methionyl-lysyl-bradykinin (Met-lys-Bradykinin), but cross-reacted 0.4% or less with bradykinin fragments. Kininogen cross-reacted only 0.2%. The radioimmunoassay and kininogen from several species were used in the measurement of human and rat urinary kallikrein activity. The peptide generated by hydrolysis of the substrates by rat or human urines was characterized by radioimmunoassay in two different systems: polyacrylamide gel electrophoresis and carboxymethyl cellulose chromatography. Both urines did not produce the same kinin: the kinin produced by human urine migrated like Lys-bradykinin, whereas the kinin produced by rat urine migrated like bradykinin. This gives evidence of differences in the specificity between kinin-forming enzymes in rat and human urines.

NEGATIVE COOPERATIVITY IN THE HUMAN BRADYKININ B2 RECEPTOR

A human kidney bradykinin (BK) B2 receptor cDNA was transfected in CHO-K1 cells to establish cell lines that express stably and at high density a receptor exhibiting B2 receptor properties in terms of coupling to cell signaling effectors, desensitization, and internalization. A cell line with a density of 1.3 × 106 receptors/cell allowed us to carry out a detailed study of BK-receptor interaction over a wide range of BK concentrations. A model assuming that BK binds to two receptor affinity states (depending on guanine nucleotide-sensitive coupling) was not sufficient to account for the kinetics of BK binding. Equilibrium kinetic analysis and studies of the effects of receptor occupancy by agonists or antagonists on the kinetics of BK-receptor complex dissociation revealed features typical of negative cooperative binding. The negative cooperativity phenomenon was also observed in isolated membranes in both the presence and absence of guanine nucleotide. Thus, following the interaction with BK, B2 receptor molecules likely interact with each other, resulting in an acceleration of bound ligand dissociation and a decrease in the apparent affinity of the receptor for BK. This phenomenon can participate in the desensitization process.

Bradykinin attenuates the [Ca2+]i response to angiotensin II of renal juxtamedullary efferent arterioles via an EDHF

Bradykinin (BK) effect on the [Ca2+]i response to 1 nM angiotensin II was examined in muscular juxtamedullary efferent arterioles (EA) of rat kidney. BK (10 nM) applied during the angiotensin II‐stimulated [Ca2+]i increase, induced a [Ca2+]i drop (73±2%). This drop was prevented by de‐endothelialization and suppressed by HOE 140, a B2 receptor antagonist. It was neither affected by L‐NAME or indomethacin, nor mimicked by sodium nitroprusside, 8‐bromo‐cyclic GMP or PGI2. The BK effect did not occur when the [Ca2+]i increase was caused by 100 mM KCl‐induced membrane depolarization and was abolished by 0.1 μM charybdotoxin, a K+ channel blocker. Although proadifen prevented the BK‐caused [Ca2+]i fall, more selective cytochrome P450 inhibitors, 17‐octadecynoic acid (50 μM) and 7‐ethoxyresorufin (10 μM) were without effect. Increasing extracellular potassium from 5 to 15 mM during angiotensin II stimulation caused a [Ca2+]i decrease (26±4%) smaller than BK which was charybdotoxin‐insensitive. Inhibition of inward rectifying K+ channels by 30 μM BaCl2 and/or of Na+/K+ ATPase by 1 mM ouabain abolished the [Ca2+]i decrease elicited by potassium but not by BK. A voltage‐operated calcium channel blocker, nifedipine (1 μM) did not prevent the BK effect but reduced the [Ca2+]i drop. These results indicate that the BK‐induced [Ca2+]i decrease in angiotensin II‐stimulated muscular EA is mediated by an EDHF which activates charybdotoxin‐sensitive K+ channels. In these vessels, EDHF seems to be neither a cytochrome P450‐derived arachidonic acid metabolite nor K+ itself. The closure of voltage‐operated calcium channels is not the only cellular mechanism involved in this EDHF‐mediated [Ca2+]i decrease.

Dissimilar mechanisms of Ca2+response to bradykinin in different types of juxtamedullary glomerular arterioles

Bradykinin (BK)-induced changes in intracellular calcium level ([Ca2+]i) were studied on fura 2-loaded afferent (AA) and efferent glomerular arterioles (EA) microdissected from juxtamedullary renal cortex. A distinction was made between thin and muscular EA. In AA and both types of EA, BK increased [Ca2+]ithrough activation of B2 receptors located only on the endothelium. The responses were not affected by nifedipine (10-6 M) and were smaller in a Ca2+-free medium, providing evidence that BK opens voltage-independent Ca2+ channels and mobilizes intracellular Ca2+. Thin EA differed from AA and muscular EA by a lower sensitivity to BK (EC50 = 6.95 ± 3.81 vs. 0.21 ± 0.08 and 0.18 ± 0.13 nM, respectively; P < 0.05), a higher maximal response (89 ± 5 vs. 57 ± 5 and 44 ± 7 nM; P < 0.001), and a spontaneous return to basal Ca2+ level, even in the presence of BK. Genistein (10-4 M) and herbimycin A (25 × 10-6M), specific inhibitors of tyrosine kinases, inhibited the [Ca2+]iresponses exclusively in AA. Genistein reduced the peak and plateau phases of responses by 69 ± 9 and 82 ± 6%, respectively, in a medium with Ca2+ and the peak by 48 ± 9% in a Ca2+-free medium. Similar reductions were observed with herbimycin A. These results show that dissimilar signal transduction pathways are involved in BK effects on juxtamedullary arterioles and that a tyrosine kinase activity could participate in the regulation of BK effect on AA but not on EA.

Abnormal regulation of antidiuretic hormone in idiopathic edema

Idiopathic edema is characterized by impaired water excretion, particularly in the upright posture. Indirect evidence has shown that antidiuretic hormone is involved in this disease. For this reason, we measured urinary arginine vasopressin by radioimmunoassay before and during water loading (15 ml/kg) in 10 normal women and in 10 subjects with idiopathic edema in both the supine and upright postures. Daily sodium intake was 100 meq. Renin and aldosterone were concomitantly investigated, and abnormally high values were observed both in the recumbent and upright postures. Basal values for urinary arginine vasopressin were identical in control subjects and in patients with idiopathic edema. The water load significantly reduced urinary arginine vasopressin in normal women in both positions, but in those with idiopathic edema only in the supine position. In those with idiopathic edema, assumption of the upright posture was accompanied by a transient decrease in glomerular filtration, a major decrease in osmolar clearance and no decrease in urinary arginine vasopressin after water loading. Significant correlations were established between urinary arginine vasopressin and osmolar or volemic parameters in normal women, but these correlations were not found in those with idiopathic edema in either position. Arginine vasopressin regulation was abnormal in idiopathic edema, and this hormone was believed to play a part in the pathogenesis of this disease.

Influence of previous diuretic intake on the humoral and hormonal profile of idiopathic oedema

Abstract. Three groups of women underwent water loading tests: normal subjects, idiopathic oedema patients who had taken no medication for at least 3 months, and a second oedema group with recent diuretic intake.

Prostaglandin E2 enhances type 2-bradykinin receptor expression in human glomerular podocytes

We examined the effect of prostaglandin E2 (PGE2) on bradykinin (BK) binding, BK-dependent intracellular calcium and inositol phosphate (i.p.) concentrations and BK mRNA in human glomerular visceral epithelial cells (hGVEC). PGE2 (10 nM) produced a concentration-dependent increase in [3H]-BK specific binding after a lag time of 24 h with a threshold at 0.1 nM. This increase appeared to be mediated exclusively by an increase in BK receptor (BKR)-2 expression. Scatchard analysis of [3H]-BK saturation binding showed that PGE2 produced an increase in the receptor site density without a change in the apparent dissociation constant. PGE2 also markedly stimulated cAMP production. This effect was thought to mediate the increase in expression of BKR-2 as 8-bromo cAMP and various cAMP-stimulating agents acted similarly. PGE2 did not change the BK-dependent intracellular IP3 and cytosolic calcium increases. The overexpression of BKR-2 in the presence of PGE2 was associated with an increase in mRNA as shown by the nuclease protection assay without any change in mRNA half-life. Cycloheximide, an inhibitor of protein synthesis, enhanced BKR-2 mRNA expression. In conclusion, treatment with PGE2 stimulates the synthesis of BKR-2 in hGVEC, possibly by interfering with an inhibitory protein involved in BKR-2 transcription.