A micromethod for the determination of the activities of kininases in rat plasma. Kinetics and inhibitory characteristics.

Kininase II (EC 3.4.15.1) (KII) and kininase I (KI) (EC 3.4.12.7) activities of rat plasma were characterized by the hydrolysis of hippuryl-L-histidyl-L-leucine (HHL), hippuryl-L-arginine (HLA) [expressed as carboxypeptidase N1 (CN1) activity] and hippuryl-L-lysine (HLL) [expressed as carboxypeptidase N2 (CN2) activity]. Using a spectrophotometric assay, biochemical characteristics of the three enzymes were investigated. The Michaelis-Menten constants were as follows: KII: Km 2.55 +/- 0.22 mM, Vmax 0.357 +/- 0.017 mumol/min/mL; CN1: Km 6.93 +/- 0.32 mM, Vmax 0.748 +/- 0.019 mumol/min/mL; and CN2: Km 35.8 +/- 1.52 mM, Vmax 13.11 +/- 0.40 mumol/min/mL. EDTA and O-phenanthroline inhibited the three enzyme assays at the same Ki, whereas captopril and 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (MERGETPA), allowed for the demonstration of the specificity of each assay. Furthermore, Ki values of MERGETPA against both CN1 (4.75 microM) and CN2 (2.36 microM) activities do not support the hypothesis that KI activity may be accounted for by the presence of isoenzymes in rat plasma.

[Activity of plasma kininase I and kininase II in hypertensive rats]. / Activité des kininase I et kininase II plasmatiques chez le rat hypertendu.

This study was undertaken to verify the activity of plasma kininases in hypertension. Male Wistar rats (WIS) were used and three models of experimental hypertension were studied: spontaneously hypertensive rats (SHR), renal hypertensive rats, made according to the method of Goldblatt, DOCA-salt hypertensive rats. Normal Wistar rats, nephrectomized rats and sodium-loaded rats were used as control groups. Plasma from these animals was used to evaluate the kininase activities: kininase II activity (KII) was measured by the hydrolysis of hippuryl-L-histidyl-L-leucine (HHL); kininase I activity (KI) was measured by the hydrolysis of hippuryl-L-arginine (HLA) (CN1 activity) and of hippuryl-L-lysine (HLL) (CN2 activity). The three enzyme activities were characterized by their kinetic constants and the inhibitory pattern of various inhibitors. In normal WIS rats, hydrolysis of HHL proceeds with a Km of 2.55 +/- 0.22 mM and at a Vmax of 0.357 +/- 0.017 mumol/min/ml; the enzyme is inhibited by EDTA, 0-phenanthroline and captopril. HLA has a Km of 6.93 +/- 0.32 mM and a Vmax of 0.748 +/- 0.019 mumol/min/ml while the Km and Vmax values of HLL are 35.8 +/- 1.52 mM and 13.11 +/- 0.40 mumol/min/ml. The hydrolysis of both substrates is inhibited by EDTA, 0-phenanthroline and MERGETPA. KII activity is decreased in WKY and SHR rats (Vmax = 0.241 +/- 0.014 and 0.262 +/- 0.011 mumol/min/ml, respectively). In renal hypertensive rats and DOCA-salt hypertensive rats, the KII activity remained unchanged. CN1 activity was increased in 1K, 1C hypertensive animals (Vmax = 0.866 +/- 0.221 mumol/min/ml) and in DOCA-salt hypertensive rats (Vmax = 1.119 +/- 0.049 mumol/min/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

Cellulose sulfate increases blood concentration of kinins in rats.

Intravenous injection of cellulose sulfate (CS) in rats induced a dose-dependent decrease of blood pressure accompanied by a concomitant increase of circulating levels of immunoreactive bradykinin (BK) from a level of 52 pg/mL of blood in control animals to 400 pg/mL in the group injected with 3.0 mg/kg of CS. An increase of desArg9BK was also observed while the blood concentration of BK(1-7) remained constant. Kinins contents in acidic extracts of lungs increased slightly while renal kinins from the same animals showed a slight decrease. The results suggest that plasma kallikrein-kinin system mediates CS-induced hypotension and that glandular kallikreins do not participate to the hypotensive action of CS.

Influence of sodium balance on urinary excretion of immunoreactive kinins in rats.

Antibodies against bradykinin (BK) and its metabolites, namely des-Arg9-BK and des-Phe8,Arg9-BK were raised in rabbits, and specific radioimmunoassays (RIA) for these peptides were developed. Specificity studies showed that each RIA was specific for its antigen, since the cross-reactivities of various kinin-related peptides were less than 1.5%. The lowest concentration of peptide that could be measured in these assays was approximately 60 pg/ml. The antibodies were used to measure concentrations of BK and its metabolites in urine and kidneys of rats maintained on different sodium balance for 5 wk. The results showed that normal rats excrete low quantities of BK (63.78 +/- 2.98 ng/day, 88 determinations). The urinary excretion of des-Arg9-BK averaged 77.69 +/- 5.53 ng/day, whereas the amount of des-Phe8,Arg9-BK is equal to 7.13 +/- 0.42 ng/day. Sodium loading brings about a small decrease in the concentration of BK (45.57 +/- 2.36 ng/day, 76 determinations), whereas sodium depletion significantly increased the excretion of BK (94.23 +/- 5.50, 102 determinations, P less than 0.01) accompanied by no modification of the excretion of metabolites. Regression analysis of the results showed a positive correlation between urinary volume and BK in control and sodium-loaded animals and urinary BK and sodium in the sodium-loaded group. In kidney homogenates, sodium depletion increased not only the concentration of BK (10-fold) but also that of des-Arg9-BK and des-Phe8,Arg9-BK by a factor of four and two, respectively, when compared with normal and sodium-loaded animals. These results support the hypothesis that the renal kallikrein-kinin system may be regulated by corticosteroids.

Alternative excision products originating from a single integration of polyomavirus DNA.

The Cyp cell line consists of mouse cells transformed by a thermosensitive polyomavirus (Py) genome and routinely propagated at 39 degrees C. Cyp cells are readily induced to synthesize free Py DNA by being transferred to 33 degrees C. In one subclone (C12/a1/S48, or S48) of this line, such induction resulted in the intracellular accumulation of three discrete species of cyclic DNA, i.e., genomic Py DNA, RmI, and RmII. RmI and RmII are Py-mouse chimeras, each of which contains a distinct set of sequences originating from the site of integration. Conceivably, genomic Py DNA, RmI, and RmII could persist at 39 degrees C as free replicating plasmids or originate from distinct populations of cells in S48 cultures. The data indicated that all three species arise at 33 degrees C from a genetically homogeneous cell population in which neither RmI nor RmII replicates at 39 degrees C. Examination of the sequence at the viral-cellular junction unique to RmII indicated that this chimera is excised from the host chromosome through a recombination event involving a complex viral sequence and a simple cellular sequence. Therefore, RmII provides another example of precise recombination occurring between nonhomologous sequences in a mammalian cell, as already observed for RmI (B. S. Sylla, D. Huberdeau, D. Bourgaux-Ramoisy, and P. Bourgaux, Cell 37:661-667, 1984).

Site-specific excision of integrated polyoma DNA.

Cyp cells are permissive murine cells carrying a thermosensitive polyoma virus genome that remains integrated at 39 degrees C, but is effectively excised and replicated after transfer to 33 degrees C. In rare subclones of the Cyp line, temperature shift-down yields predominantly homogeneous populations of chimeric molecules that appear to reflect the circularization of defined segments of DNA spanning one of the junctions between the integrated viral genome and the adjacent cellular DNA. Such accurate and frequent excision requires a specific recombination mechanism. We examined both the cellular and the viral sequences that cross-over to generate one of these chimeric molecules, Rm I. The homology at the cross-over site is one of 1 or 2 base pairs at most; patches of homology, amounting in total to 19 or 20 base pairs, are found in perfect register on both sides of this site; and the two stretches of DNA that are joined to form RM I contain similar 12-14 base pair sequences (5'- CTCCTTTACAGAGG -3' and 5'- CTCCTTTCAAGG -3') in opposite orientations.