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The endothelial cell should be regarded as a highly active metabolic and endocrine organ interacting with the blood streak and the interstitium. Kinins are vasodepressor hormones that may participate in local blood flow regulation as part of an autocrine-paracrine system. We have previously reported that tissue kallikrein, its mRNA and kininogen are present in vascular tissue. The present study was undertaken to examine the release of the components of this system from isolated perfused rat vessels. These vessels were perfused at 4 ml/min with physiological saline solution containing 3% Ficoll and 0.1% BSA. Kallikrein was released into the perfusate at a rate of 75 +/- 5 ng Bk/100 g bw/30 min (n = 10). Kininogen was released at a rate of 55 +/- 5 pg Bk/100 g bw/30 min. Pre-treatment with puromycin, a protein synthesis inhibitor, significantly reduces kallikrein and kininogen release. Vascular derived kinins were released at a constant rate of 38 +/- 6 pg Bk/100 g bw/30 min for at least 120 min. This basal kinin release was increased 3-fold when perfused with the angiotensin converting enzyme inhibitor ramiprilat (p < 0.05). When purified kininogen was added to the physiological saline solution, immunoreactive kinins in the perfusate increased from 42 +/- 7 to 3140 +/- 210 pg Bk/100 g bw/30 min (n = 6; p < 0.002). Increase in flow rate (from 2 ml/min up to 4 ml/min and 8 ml/min) causes a parallel increase in the release of kinins (from 32 +/- 4 up to 48 +/- 6 and 62 +/- 8 pg Bk/100 g bw/30 min, respectively; p < 0.01); the increase may be due to the effect of shear stress upon the endothelial cells. The present data confirm that vascular tissue synthesizes and releases continuously kallikrein, kininogen and kinins. Vascular kinins induce potent vasodilatation through the release of prostacyclin, nitric oxide and endothelium-derived hyperpolarization factor, and some of the converting enzyme inhibitors effect may be explained by potentiation of vascular-derived kinins.

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)

We performed this study to examine the presence of a kallikrein-kinin system in rat fetal and maternal tissues. Uteri and placenta from Wistar pregnant and nonpregnant rats were perfused to eliminate blood, and fetal membranes were washed several times with saline. Amniotic fluids were obtained without blood contamination by amniocentesis from eight rats. The different samples were homogenized and centrifuged (2000g during 20 minutes), and the supernatant was incubated with dog kininogen and 0.1 mol/L Tris-HCl buffer (pH 8.5) in the presence of peptidase inhibitors. Kinins released were measured by radioimmunoassay. Kininogenase activity was found in rat uteri, placental vessels, amniotic fluids, and fetal membranes. The enzymes were present in active but mostly in inactive forms. The kallikrein-like enzymes found in the different preparations and rat urinary kallikrein used as control had similar molecular weights, immunologic characteristics, and inhibition profiles with protease inhibitors. We conclude that kallikrein-like enzymes are present in rat organs of reproduction. These data suggest that kinins released locally may act as paracrine hormones in the regulation of blood pressure during pregnancy.

We determined the status of vascular kallikrein in rats with severe hypertension caused by treatment with deoxycorticosterone acetate (DOCA) and drinking of 1% NaCl for 6 weeks. We assayed active and total kininogenase (kallikrein) activity in the perfusate and in arterial and venous tissue. DOCA-salt rats had higher systolic blood pressure at 6 weeks (214 +/- 5 mm Hg) than rats drinking tap water (135 +/- 4 mm Hg) or saline (145 +/- 8 mm Hg). Kininogenase in the perfusate (nanograms bradykinin per minute per kilogram body weight) increased significantly at 2 weeks, from 5.8 +/- 2.1 to 8.9 +/- 1.4 for active kallikrein and from 28.7 +/- 0.4 to 48.7 +/- 2.9 for total kallikrein. Total kallikrein returned to control values at 4 weeks, whereas it was significantly reduced at 6 weeks (20.9 +/- 0.7). Active kallikrein was significantly depressed at 4 and 6 weeks (1.08 +/- 0.1 and 0.85 +/- 0.1, respectively [P < .05]). Active kallikrein in arterial tissue (picograms bradykinin per milligram per minute) showed a small but significant increase at 2 weeks, from 156 +/- 7 to 201 +/- 10 (P < .05), finally decreasing significantly by 6 weeks to 64 +/- 3; however, total kallikrein showed a significant decrease only at 6 weeks, from 844 +/- 17 to 427 +/- 27. Both active and total kallikrein in the veins were higher than control values at 2 weeks, changing from 437 +/- 7 to 541 +/- 19 and from 1619 +/- 17 to 2062 +/- 86, respectively. Venous kallikrein remained elevated until the end of the experiment.(ABSTRACT TRUNCATED AT 250 WORDS)

Vascular tissue contains kallikrein and kallikrein mRNA, suggesting a vascular kallikrein-kinin system. We questioned whether 1) kallikrein concentration varies among large and small vessels; 2) kallikrein is released by vascular tissue; and 3) blocking protein synthesis inhibits release, suggesting de novo synthesis. Using rat vascular rings and isolated-perfused hindquarters, we examined kallikrein in the bath and perfusate. Active kallikrein was higher in tail arteries than the aorta (P < 0.001); tail veins had six times more kininogenase than the vena cava (P < 0.001). Total kallikrein showed a similar pattern, being highest in tail vessels. Arterial rings released active and total kallikrein. After 1, 2, and 3 h incubation, cumulative release was as follows: active, 90 +/- 13, 201 +/- 25, and 311 +/- 41 pg.h-1 x mg tissue-1; total, 170 +/- 14, 366 +/- 24, and 537 +/- 40 pg.h-1 x mg tissue-1, indicating constant release up to > or = 3 h. In contrast, lactic dehydrogenase fell from 6.7 +/- 2.5 to 2.5 +/- 0.4 U.h-1 x mg tissue-1. Total kallikrein in the rings was 302 +/- 51 pg bradykinin/mg wt tissue before 3 h and 298 +/- 68 afterward. Kallikrein released by the hindquarters after 3 h was as follows: active, 6.2 +/- 2.8 ng bradykinin.min-1 x kg.body wt-1; total, 85.2 +/- 17 ng bradykinin.min-1 x kg body wt-1. Puromycin pretreatment (10 mg ip) reduced total perfusate kallikrein from 105 +/- 19 to 8.5 +/- 3.6 (P < 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)

Kallikrein was identified in the adrenal glands of the rat. The enzyme was present in active and inactive forms (n = 9), since preincubation with trypsin increased kininogenase activity from 54.8 +/- 11.8 to 230 +/- 23 pg bradykinin per milligram protein per minute. Adrenal kininogenase activity was inhibited by 91% by phenylmethylsulfonyl fluoride (2 mM), 81% by D-Phe-Phe-Arg-chloromethyl ketone (1 microM), 88% by aprotinin (1,000 KIU), and only 16% by soybean trypsin inhibitor (50 microM). Preincubation with antibodies against rat urinary kallikrein resulted in over 90% inhibition of kininogenase activity. Immunoreactive glandular kallikrein was 30.7 +/- 4.8 ng/mg protein (n = 11). The apparent molecular weight of the adrenal kininogenase on gel filtration chromatography was 33,000 +/- 500 D. Both the adrenal enzyme and the purified submandibular gland kallikrein used as a control had the same mobility on alkaline polyacrylamide gel electrophoresis. To determine whether messenger RNA (mRNA) for glandular kallikrein is present in adrenal gland RNA, we used the polymerase chain reaction employing oligonucleotide primers and glandular kallikrein 32P complementary DNA (cDNA) as a probe, which should give a cDNA fragment of 370 bp. Southern blots of the amplified products revealed a fragment of the predicted size. In conclusion, glandular kallikrein has been identified in the adrenal glands. The presence of mRNA for glandular kallikrein suggests that kallikrein is synthesized locally in this tissue. This provides an anatomic basis for possible participation of a local kallikrein-kinin pathway in the regulation of adrenal function.

Kallikrein and minute amounts of kininogen have been found in rat cardiac tissue. The mRNA for kallikrein was also determined by the polymerase chain reaction using KK-specific probe. The existence of an intrinsic kallikrein-kinin system in the heart raises the possibility that the enzyme-peptide system is involved in local regulation of cardiac function and metabolism.

We have previously reported that vascular tissue contains kallikrein and kallikrein mRNA. We can now show that kallikrein is present throughout the vascular tree and is released from arterial and venous rings incubated "in vitro". Using the isolated perfused rat hindquarters as a model, we found that kallikrein appeared in the perfusate in concentrations that increased linearly with time. Treatment with puromycin inhibited kallikrein release by 87% (p < 0.01), these data suggest that kallikrein is synthesized and released by the vascular wall. Local generation of kinins (autocrine/paracrine system) may contribute to the regulation of vascular homeostasis.

SBTI-resistant kininogenase activity was found in nonpregnant and pregnant rat uterus, placenta, amniotic fluid and fetal membranes. After trypsin treatment the kininogenase activity increased 2-3 fold. Total kininogenase (active plus inactive) was completely blocked by kallikrein antibodies. The physiological role of these kallikrein-like enzymes is unknown. It is speculated that these enzymes play a local role, perhaps in the processing of polypeptide hormones of through the release of kinins in the regulation of uterine blood flow.

Active and inactive kallikrein or a kallikrein-like enzyme are found in the aorta, vena cava, and tail artery and veins of the rat. We studied the concentration of vascular kininogenase in rats with one-kidney, one clip renovascular hypertension and in unilaterally nephrectomized normotensive rats. Six weeks after surgery, active and total vascular kininogenase activity (active plus trypsin-activated) was measured. Blood pressure was 212 +/- 4 mm Hg in the hypertensive rats (n = 33) and 120 +/- 1 mm Hg in the normotensive rats (n = 32) (p less than 0.001). Active kininogenase was lower in the hypertensive rats; although the difference was not significant in the thoracic aorta (56 +/- 8 versus 77 +/- 15), it was highly significant in the abdominal aorta (63 +/- 13 versus 167 +/- 17, p less than 0.001) and tail artery (48 +/- 8 versus 197 +/- 31, p less than 0.003). Total vascular kininogenase activity (active plus trypsin-activated) was lower in the hypertensive rats in all arteries examined: thoracic aorta (183 +/- 16 versus 380 +/- 38, p less than 0.003), abdominal aorta (565 +/- 61 versus 1,093 +/- 74, p less than 0.001), and tail artery (532 +/- 112 versus 1,243 +/- 135, p less than 0.003). Active kininogenase in the vena cava was higher in the hypertensive rats (213 +/- 56 versus 131 +/- 31); however, this difference was not statistically significant, whereas in the tail veins it was highly significant (1,803 +/- 221 versus 771 +/- 79, p less than 0.003).(ABSTRACT TRUNCATED AT 250 WORDS)

The present study was undertaken to examine whether there is a kallikrein-like enzyme in vascular tissue. Isolated saline-perfused rat mesenteric arteries were used. A kallikrein-like enzyme and acid protease from saline-perfused mesenteric arteries were separated by CM-cellulose chromatography with a linear NaCl gradient. The separation made it possible to study the optimum pH of the kallikrein-like enzyme and acid protease. The kallikrein-like enzyme showed optimal activity in the range of pH 7-9 and the acid protease in the range of pH 4-5. Both enzymes when acting on a protein plasma substrate release an active peptide that has a similar chemical and pharmacological properties to bradykinin. Using antibodies that specifically inhibit kinins, the biological action was completely abolished. These results indicate that arterial tissue contains two enzymes which generate kinins, the characteristics of one of the enzymes are quite similar to those of a lysosomal protease of the cathepsin-like type of enzyme and the other differs clearly from the acid protease and plasma kallikreins and has physicochemical characteristics quite similar to those of the kallikreins of glandular origin.

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 X g for 60 min 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 using equivalent kinin-forming concentrations of rat urinary kallikrein and vascular kininogenase. Kininogenase activities of both enzymes were similarly inhibited by urinary kallikrein antibodies. Aprotinin (1,000 KIU) completely inhibited vascular kininogenase activity while soybean trypsin inhibitor (100 micrograms) 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.

A kallikrein-like kininogenase was identified in the rat adrenal gland. Most of the enzyme was present in an inactive form, since pre-incubation with trypsin markedly increased kininogenase activity from 54.8 +/- 11.8 to 230 +/- 23.0 pg bradykinin/mg protein/min. Adrenal kininogenase was inhibited 90% by phenyl methyl sulfonyl fluoride, 92% by D-Phe-Phe-Arg-chloromethylketone, 91% by aprotinin, and only 15% by soybean trypsin inhibitor. Pre-incubation with antibodies against rat urinary kallikrein resulted in 85% inhibition. The apparent molecular weight of adrenal kininogenase on gel filtration chromatography was 33 Kd. The enzyme was strongly adsorbed to immobilized rat urinary kallikrein antibodies and required drastic conditions for elution. In canine adrenal glands, we found that there was no difference in the cortical and medullary distribution of active and inactive SBTI resistant kininogenase activity. We conclude that an enzyme which closely resembles glandular kallikrein is present in adrenal glands.

We studied whether the components of the kallikrein-kinin system are present in the central nervous system. We found that human cerebrospinal fluid (CSF) contains free kinins: 53 +/- 15 pg/ml; kininogen: 10.9 +/- 2.1 ng kinin equivalent/ml, and kininogenase activity: 5.0 +/- 2.1 ng kinins/ml/minute. Kininogenase activity was 2-3 fold augmented by preincubation with trypsin. Soybean trypsin inhibitor completely inhibited untreated CSF and partially inhibited trypsin activated kininogenase. Kininogenase activity and immunoreactive glandular kallikrein were present in rat brain, and their concentrations in hypothalamus is several-fold higher than in cortex, pons-medulla, basal ganglia and cerebellum. In the hypophysis, activity in pars-intermedia was between 6- and 20-fold higher than in posterior and anterior hypophysis, respectively. High activity was also found in the pineal gland. The kallikrein-kinin system is present in the central nervous system where it may participate in modulation of nervous and neuroendocrine functions.

The relative rates of release of active and inactive kallikrein were investigated in the rat kidney by studying in vitro kidney slices. The slices were kept in Warburg flasks in a Krebs-Ringer solution for 15 up to 60 minutes, and their kallikrein content and the kallikrein-like activity released by them were measured at different times. Renal kallikrein concentration from two-kidney, one clip hypertensive rats was less than in the sham-operated controls (p less than 0.01). Active kallikrein released into the bathing medium was 4.7 +/- 0.3 ng/mg/hr in normotensive rats (n = 10) and 3.3 +/- 0.3 in two-kidney hypertensive animals (n = 12); the difference between both groups was statistically significant (p less than 0.01). Inactive kallikrein is between 60% and 70% of the total kallikrein released by kidneys of normal and hypertensive rats. Thus, under the conditions of these experiments, the kidney slices were able to release a kallikrein-like enzyme in an active and an inactive form. The rate of active kallikrein released from slices of hypertensive animals was lower than in the normotensive controls.