A. G. Scicli

A local kallikrein-kinin system is present in rat hearts.

It has been reported that kinins mediate part of the beneficial cardiac effects induced by treatment with angiotensin-converting enzyme inhibitors in situations such as ischemia-reperfusion injury, myocardial infarction, and cardiac hypertrophy. However, it is not known whether the heart contains an independent kallikrein-kinin system. We measured kallikrein in tissue and in the incubation medium of heart slices. Heart slices released active and total (trypsin-activatable) kallikrein into the medium (46 +/- 5 and 380 +/- 18 pg bradykinin/mg, respectively, after 1 hour and 78 +/- 6 and 654 +/- 14 pg bradykinin/mg after 2 hours, n = 7). Release was not due to tissue damage because lactate dehydrogenase, a cytosolic marker, decreased from 8.9 +/- 2.9 to 2.9 +/- 1.0 U/mg per hour. Although kallikrein was released, total tissue kallikrein in the slices did not change (423 +/- 25 pg bradykinin/mg in nonincubated slices and 370 +/- 42 pg bradykinin/mg after 2 hours, P = NS), suggesting pool replenishment. Cardiac kallikrein activity was inhibited by incubation with anti-glandular kallikrein antibodies. Pretreatment with the protein synthesis inhibitor puromycin (10 mg IP) lowered release of active kallikrein from 78 +/- 6 to 22 +/- 4 pg bradykinin/mg and total kallikrein from 654 +/- 14 to 113 +/- 9 pg bradykinin/mg (P < .001). By using reverse transcription polymerase chain reaction with kallikrein family oligonucleotide primers and a specific kallikrein probe, we found that mRNA for tissue kallikrein is present in both atrial and ventricular RNA. Kallikrein activity was also detected in primary cultures of neonatal rat atrial and ventricular cardiocytes and their incubation medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of kinins in the acute antihypertensive effect of the converting enzyme inhibitor, captopril.

SUMMARY The role of kinins in the acute antihypertensive effect of a converting enzyme inhibitor (CEI) was studied in sodium-depleted normotensive and in two-kidney, one clip chronically hypertensive rats (2K- 1C). The 2K-1C were on a normal sodium diet. The acute vasodepressor effect of the CEI was determined in these two groups either after administration of normal rabbit globulins or antikinin globulins. The amount of kinin antibodies administered completely blocked the hypotensive effects of bradykinin, 400 ng/kg, and urinary kallikrein, 4

Measurement of urinary kallikrein activity by kinin radioimmunoassay.

Mg/kg- After administration of CEI in the sodium-depleted rats there was no significant difference (p > 0.05) in the acute changes in mean blood pressure (BP) between the group pretreated with normal rabbit globulins (ABP Δ32.3 ± 3.9 mm Hg) and the group pretreated with antikinin globulins (ABP Δ25 ± 2.5 mm Hg). In the 2K-1C pretreated with normal rabbit globulins, the CEI produced a decrease in BP of −21 ± 4.5 mm Hg. This decrease was almost completely blocked in the group pretreated with the antikinin globulins (ABP Δ4 ± 4.1 mm Hg). These differences in the changes in BP were significant (p < 0.02). These results suggest that the acute antihypertensive effect of the CEI in the sodium-depleted rats is probably due to inhibition of the conversion of angiotensin I to II while in the 2K-1C it is due, in part, to an increase in kinin concentrations secondary to the inhibition of kininase II.

Effect of a kinin antagonist on the acute antihypertensive activity of enalaprilat in severe hypertension.

Abstract A method for measuring urinary kallikrein activity by kinin radioimmunoassay (RIA) is described. Kinins were generated by incubating urine with partially purified dog kininogen in the presence of peptidase inhibitors. Antibodies against kallidin were induced in rabbits by injecting kallidin coupled to ovalbumin. One of the antisera generated was used at a final dilution of 1:18,000 to obtain a 40 per cent binding of approximately 3000 cpm (10 pg) of bradykinin (S-tyrosin)-[ 125 I]triacetate ([ 125 I]bradykinin). Synthetic kallidin (10–500 pg) was used to construct standard curves. When kinins generated by urinary kallikrein were also used, the two displacement curves for [ 125 I]bradykinin were similar. The RIA was sensitive to 10 pg kinins. The antiserum cross-reacted with bradykinin, methionyl- kallidin, and kininogen, but not with angiotensin, oxytocin or SQ 20,881. To increase the specificity of the RIA, the kininogen was removed by ethanol precipitation followed by QAE-Sephadex-A50 chromatography. Kallikrein activity in 81 human urine samples and 8 samples obtained from a dog undergoing stop-flow procedure was measured by RIA and bioassay. Correlations of r = 0.81 and 0.94 were found. This RIA is useful for measuring kallikrein activity in rat, dog and human urine.

Angiotensin-(1-7) Induces Bradykinin-Mediated Hypotensive Responses in Anesthetized Rats

The purpose of this study was to assess the role of kinins in the acute antihypertensive effect of the converting enzyme inhibitor (CEI) enalaprilat in rats with severe hypertension induced by aortic ligation between both renal arteries. For this study, we used a bradykinin analogue, D-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-DPhe-Thi-Arg-TFA, with in vivo antagonistic properties. Hypertensive rats were infused intra-aortically for 15 minutes with either saline (30 microliters/min) or the kinin antagonist (40 micrograms/kg/min). Five minutes after the infusion was begun, a bolus injection of enalaprilat (60 micrograms/kg) was given. The blood pressure of the saline-infused animals decreased 48 +/- 6 mm Hg (from 180 +/- 7 to 132 +/- 7 mm Hg), while that of the rats treated with the antagonist decreased only 21 +/- 4 mm Hg (from 175 +/- 3 to 154 +/- 3 mm Hg). The difference between both decrements was significant (p less than 0.01). In another group of hypertensive animals (n = 9), we measured kinin concentration in plasma from arterial blood before and after administration of CEI (41 +/- 10 vs 68 +/- 20 pg/ml, respectively; NS). These results are consistent with the hypothesis that kinins play a role in the acute antihypertensive effect of CEIs in rats with severe hypertension. However, since arterial blood kinin concentrations were not increased significantly after CEI administration, the effect of the CEI may be due to an increase in tissue kinins, which could act as autacoids regulating vascular resistance.

Kallikrein messenger RNA in rat arteries and veins.

Angiotensin-(1-7) [Ang-(1-7)] reportedly potentiates hypotensive responses to bradykinin. We studied whether increases in circulating bradykinin would alter responses to Ang-(1-7). In rats anesthetized with thiobutabarbital, bradykinin infusion (5 microg/kg per minute I.A.) resulted in a rapid decrease in mean arterial pressure (MAP) of about 20 mm Hg (P<.01, n=9), although MAP slowly increased by 10 mm Hg after 15 minutes. When Ang-(1-7) (20, 80, and 380 nmol per rat I.A.) was given during bradykinin infusion, it elicited hypotension at 80 and 380 nmol (deltaMAP: -15+/-2.7 and -21+/-3.3 mmHg, respectively; P<.001); this hypotension was not affected by the angiotensin type 1 antagonist L-158,809 (200 microg/kg I.A.), the angiotensin type 2 antagonist PD 123319 (10 mg/kg I.A.), saralasin, or sarthran (10 microg/kg per minute). The bradykinin type 2 receptor antagonist icatibant (30 microg per rat) eliminated the hypotensive responses to Ang-(1-7), which now increased MAP at all doses tested (P<.005). Thus in the presence of bradykinin, Ang-(1-7) induces hypotensive responses that are blocked by icatibant and unaffected by angiotensin receptor antagonists. Ang-(1-7) given to saline-infused rats elicited hypertensive responses at all doses (deltaMAP: 6.4+/-1.5, 12+/-1.6, and 16.3+/-2.7 mmHg, respectively; P<.01); these responses were abolished by L-158,809 and sarthran. In rats pretreated with saralasin, Ang-(1-7) induced hypotension at 80 and 380 nmol (deltaMAP: -7.7+/-2.3 and -9.5+/-2.7, respectively; P<.05), whereas icatibant abolished this response. Thus in the rat, Ang-(1-7) can decrease blood pressure by a mechanism involving the bradykinin type 2 receptor and participates with bradykinin in a vasodepressor pathway that may serve a counterregulatory role, modulating the vasoconstrictor effects of Ang II.

Characterization of a kininogenase from rat vascular tissue resembling tissue kallikrein.

Glandular kallikrein (EC 3.4.21.8) belongs to a subgroup of serine proteases coded by a multigene family. A kininogenase resembling glandular kallikrein has been identified in vascular tissue; however, it is not clear whether it is synthesized by vascular tissue or taken up from plasma. To determine the potential for kallikrein synthesis in vascular tissues, we tested whether messenger RNA (mRNA) for glandular kallikrein is present in rat arteries and veins. Poly(A+) RNA was isolated from pools of arteries or veins (n = 3, 30 rats each). Poly(A+) RNA from the kidney and liver was used as a positive and negative control, respectively. As a probe, we used rat pancreatic kallikrein 32P-labeled complementary DNA, which recognizes mRNA of the entire rat kallikrein family. Slot-blot analysis indicated that kallikrein mRNA was present in mRNA from the arteries, veins, and kidney but not from the liver. Poly(A+) RNA from arteries and veins contained approximately 1% as much kallikrein mRNA as that from the kidney. To confirm the slot-blot results and determine whether the mRNA for true glandular kallikrein was present in vascular tissue, we employed a polymerase chain reaction assay, first using primers specific for the entire kallikrein family (which amplify a 430-bp fragment) and then using primers specific for true glandular kallikrein mRNA (which amplify a 370-bp fragment). After the polymerase chain reaction assay, both arteries and veins showed fragments of these sizes when tested with rat kallikrein complementary DNA probe, thus confirming the presence of glandular kallikrein mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Urinary kallikrein in rats bred for their susceptibility and resistance to the hypertensive effect of salt. A new radioimmunoassay for its direct determination.

A kininogenase resembling glandular kallikrein was partially purified from vascular tissue and characterized. Saline-perfused rat tail arteries and veins were homogenized in 0.25 m sucrose containing 10 mM Tris-HCl (pH 7.4). The homogenate was centrifuged at 105,000 g for 60 minutes, and a vascular kininogenase was purified from the supernatant by chromatofocusing, affinity chromatography on immobilized antibodies against rat urinary kallikrein, and gel filtration on Sephadex G-100. The inhibitory effects of antibodies against rat urinary kallikrein were tested with equivalent kinin-forming concentrations of rat urinary kallikrein and vascular kininogenase. Kininogenase activities of both enzymes were similarly inhibited by both polydonal and monoclonal antibodies. Aprotinin (1,000 KIU) completely inhibited vascular kininogenase activity, while soybean trypsin inhibitor (100 μg) did not modify its kinin-forming activity. Vascular kininogenase and rat urinary kallikrein had the same elution volume when chromatographed on a Sephadex G-100 column, and had similar mobilities in 10% polyacrylamide gel electrophoresis. Kinins released by vascular kininogenase were identified as bradykinin by reverse-phase high performance liquid chromatography. Rat vascular kininogenase appears to be similar to glandular kallikrein. Kinins released locally by vascular kininogenase may contribute to the regulation of vascular tone.

Evidence against a role of vasopressin in the maintenance of high blood pressure in mineralocorticoid and renovascular hypertension.

Urinary kallikrein was measured in rats bred to be susceptible (S) or resistant (R) to the hypertensive effect of salt. To determine kallikrein, three different methods were used: (1) a new direct radioimmunoassay (RIA) for the enzymic protein; (2) a method based on the capability of kallikrein, when incubated with kininogen, to generate kinins which were then measured by RIA (kininogenase activity); and (3) a method based on the capability of kallikrein to break the ester bond of p-tosyl-L- arginine methyl ester HCI (TAMe). A significant correlation (r = 0.90) was found between the direct RIA and the kininogenase method. It was found that urinary kallikrein was significantly decreased in the S as compared to the R rats by the use of these three methods. Urinary kallikrein in the S rats was much lower when measured by the kininogenase method than by direct RIA or esterolytic assay. This difference could be due to excretion of pre-kallikrein and/or kallikrein bound to an inhibitor (inactive kallikrein). It is suggested that the decrease of urinary kallikrein excretion (active and inactive) in the S rats could be a consequence of a genetic defect that may affect the development of hypertension perhaps through the alteration of sodium and water excretion by the kidney.

Role of angiotensin converting enzyme and other peptidases in in vivo metabolism of kinins.

SUMMARY To determine the role of vasopressin in the maintenance of high blood pressure, the antihypertensive effect of the antagonists of the vasopressor effect of vasopressin, [l-deaminopenicillamine, 4- valine, 8-D-arginine] vasopressin (dPVDAVP), and [l-(β-mercapto-β, β-cyclopentamethylenepropionic acid), 4-valine, 8-D-arginine] vasopressin (cyclo dVDAVP), was studied in unanestfaetized, nonsurgically stressed rats with adrenal regeneration hypertension, malignant DOCA-salt hypertension, and malignant twokidney, one clip Goldblatt hypertension. The doses of vasopressin antagonist used blocked the blood pressure (BP) response to vasopressin almost completely, with no changes in the pressor response to norepinephrine and angiotensln II. Administration of the vasopressin antagonists did not induce significant changes in the mean BP in any of the three experimental groups studied. It is suggested that in unanesthetized, nonsurgically stressed rats with adrenal regeneration hypertension, malignant DOCA-salt hypertension, and malignant two-kidney, one clip Goldblatt hypertension, vasopressin does not have a role in the maintenance of high BP.