Proteolytic activation of a putative receptor leading to vasoconstriction and platelet aggregation.

Submandibular enzymatic vasoconstrictor (SEV), a member of the kallikrein family of enzymes, elicits biological effects by a proteolytically mediated mechanism. We studied 1) whether SEV is able to aggregate platelets and 2) whether SEV may activate a receptor other than the cloned thrombin receptor. SEV (10(-8)M) aggregated platelets, released ATP and increased intracellular Ca2+. Elastase treatment rendered human platelets unresponsive to SEV and thrombin (TH), but not to cathepsin G. In desensitization experiments performed with gamma-TH, after two successive additions of approximately 50 nM gamma-TH, a third dose elicited 15.8 +/- 3.4% of the initial response (n = 4), but platelets responded to approximately 20 nM SEV by 33.8 +/- 7.2% of control (p < 0.03 vs last response to gamma-TH). After desensitization to SEV (n = 4), the response to a third dose was 4 +/- 1.3% of control, but gamma-TH still induced 37.7 +/- 12.4% aggregation (p < 0.02 vs last response to SEV). Incubation of washed rabbit platelets with alpha-TH digested with elastase (10(-10) M TH added to 7 micrograms/ml elastase for 1 min) rendered them unresponsive to additional challenges with TH, but they still responded to an equipotent dose of SEV (2.7 x 10(-9) M) by 86 +/- 48% of control. In isolated rabbit aortic rings contracted with 10(-6) M norepinephrine (NE) to 42 +/- 3% of maximum. SEV (2.8 x 10(-8) M) caused further contraction to 87 +/- 4%. In contrast, alpha-TH (1.6 x 10(-7) M) tended to relax both NE- and SEV-contracted rings by 14 +/- 2 and 16.2 +/- 2%, respectively (n = 3 each). We concluded that part of the platelet-aggregating effect of SEV may be mediated by activation of a receptor(s) different from that of TH.

T-kininogenase activity of the rat submandibular gland is predominantly due to the kallikrein-like serine protease antigen gamma.

T-kininogen, the major kininogen in rat plasma, releases Ile-Ser-bradykinin (T-kinin) when incubated with trypsin, but is not a substrate for tissue kallikrein. Enzymes able to release T-kinins from T-kininogen have been found in the rat submandibular gland, but precise identification of these enzymes and their possible relationship to kallikrein-like enzymes has not been established. We studied T-kininogenase activity in fractionated submandibular gland homogenate. The main T-kininogen catalytic enzyme was purified and characterized, and found to be identical to antigen gamma, a kallikrein-like enzyme which we have previously characterized. Of other identified kallikrein-like enzymes only tonin showed weak T-kininogenase activity, which was about 0.25% of that of antigen gamma. No other T-kininogen catalytic enzymes were observed. Antigen gamma released a kinin which was identified as T-kinin by reverse-phase h.p.l.c. The T-kininogenase activity of antigen gamma had a Km of 29 +/- 4 microM and a kcat/Km of 140 M-1.s-1, and was comparable with its high and low molecular mass-kininogenase activity (7.4 and 10 micrograms of kinin/h per mg respectively). In contrast, tissue kallikrein released 0.2 and 42,200 micrograms of kinin/h per mg respectively. Thus antigen gamma is a weak kininogenase. The isoelectric point of antigen gamma, but not its molecular mass, differed from that of other kallikrein-like enzymes. Isoelectrofocusing in flat-bed gels combined with immunostaining was therefore a convenient method for identification. The kallikrein-like nature of antigen gamma was demonstrated by its immunological similarity to tissue kallikrein and tonin and by 91% and 87% amino acid sequence similarity with tonin and kallikrein respectively (67 amino acids sequenced). Complete identity was also not observed with other sequenced kallikrein genes, mRNAs or proteins.

Tissue kallikrein processes small proenkephalin peptides.

Tissue kallikrein may play a role in processing precursor polypeptide hormones. We investigated whether hydrolysis of natural enkephalin precursors, peptide F and bovine adrenal medulla docosapeptide (BAM-22P), by hog pancreatic kallikrein is consistent with this concept. Incubation of peptide F with this tissue kallikrein resulted in the release of Met5-enkephalin and Met5-Lys6-enkephalin. Met5-Lys6-enkephalin was the main peptide released, indicating that the major cleavage site was between two lysine residues. At 37 degrees C and pH 8.5, the KM values for formation of Met5-enkephalin and Met5-Lys6-enkephalin were 129 and 191 microM, respectively. Corresponding kcat values were 0.001 and 0.03 s-1 and kcat/KM ratios were 8 and 1.6.10(2) M-1.s-1, respectively. Cleavage of peptide F at acidic pH (5.5) was negligible. When BAM-22P was used as a substrate, Met5-Arg6-enkephalin was released, thus indicating cleavage between two arginine residues. At pH 8.5, KM was 64 microM, kcat was 4.5 s-1, and the kcat/KM ratio was 7.10(4) M-1.s-1. At 5.5, the pH of the secretory granules, KM, kcat and kcat/KM were 184 microM, 1.9 s-1 and 10(4) M-1.s-1, respectively. It is unlikely that peptide F could be a substrate for kallikrein in vivo; however, tissue kallikrein could aid in processing proenkephalin precursors such as BAM-22P by cleaving Arg-Arg peptide bonds.

Bradykinin release from contracting skeletal muscle of the cat.

Results of previous studies from our laboratory suggest that bradykinin has a role in the exercise pressor reflex elicited by static muscle contraction. The purpose of this study was to quantify the release of bradykinin from contracting skeletal muscle. In 18 cats, blood samples were withdrawn directly from the venous effluent of the triceps surae muscles immediately before and after 30 s of static contraction producing peak muscle tensions of 33, 50, and 100% of maximum electrically stimulated contraction. Contractions producing muscle tensions of 50 and 100% of maximum increased muscle venous bradykinin levels by 27 +/- 9 and 19 +/- 10 pg/ml, respectively. Conversely, 33% maximum contraction did not alter muscle venous bradykinin concentrations. However, when captopril was administered to slow the degradation of bradykinin, muscle venous bradykinin increased from 68 +/- 15 pg/ml at rest to 106 +/- 18 after contractions of 33% of maximum. When muscle ischemia was induced by 2 min of arterial occlusion before and during 30 s of 33% of maximum contraction, muscle venous bradykinin increased by 15 +/- 5 pg/ml. In addition, contraction-induced changes in muscle venous pH and lactate strongly correlated with bradykinin concentrations (r = 0.80 and 0.83, respectively). These data demonstrate that static contraction of relatively high intensity evokes the release of bradykinin from skeletal muscle and that ischemia, decreased pH, and increased lactate are strongly correlated with this release.

A new kinin moiety in human plasma kininogens.

Recently, we isolated a new kinin from human urine and tentatively identified it as [Ala3]-Lys-bradykinin. However, there were inconsistencies between the properties of the naturally occurring new kinin and synthetic [Ala3]-Lys-bradykinin. In the present work, we determined whether the new kinin was released from human plasma kininogen, and further investigated the structure of the new kinin. After incubation of plasma (n = 6) with human urinary kallikrein, kinins were separated by HPLC and measured by RIA. The new kinin and Lys-bradykinin were found representing 23 +/- 3 and 76 +/- 6%, respectively, of total kinins released (2.0 +/- 0.4 micrograms/ml). The new kinin was also released from both purified low- and high-molecular-weight kininogens, representing 40-42% of total kinins released. Amino acid sequencing and composition analysis indicated that the structure of the new kinin was [Hyp3]-Lys-bradykinin (Lys-Arg-Pro-Hyp-Gly-Phe-Ser-Pro-Phe-Arg) and not [Ala3]-Lys-bradykinin. We conclude that an important proportion of human kininogens contain hydroxyproline instead of proline in position three of the bradykinin moiety.

T-kininogen in rat plasma after nephrectomy and other surgical traumas.

Plasma kininogen is known to increase after nephrectomy in the rat, and we studied the type of kininogen responsible for the increase. Total kininogen in plasma was estimated by measuring the kinins released after incubation with an excess of trypsin, and expressed as micrograms bradykinin equivalents per ml of plasma (bk eq/ml). Plasma Kininogen in 24-hournephrectomized rats increased five-fold over the control values (16.4 +/- 0.8 vs 3.0 +/- 0.3 micrograms bk eq/ml; p less than 0.001). However, we found that the increase in total kininogen was not due to nephrectomy per se, but rather to surgery, since similar increases were found after 24 hours in shamnephrectomized rats (15.5 +/- 0.5 micrograms bk eq/ml), and in rats with a catheter implanted into the carotid artery (14.2 +/- 1.7 micrograms bk eq/ml). The increase in kininogen was proportional to the extent of the surgery since simple exposure of the carotid artery increased kininogen to 5.3 +/- 0.5 micrograms bk eq/ml, and sham carotid cannulation to 8.3 +/- 0.4 micrograms bk eq/ml. Anesthesia without surgery did not affect plasma kininogen concentration. To determine whether the adrenals or the pituitary glands are involved in mediating the changes, plasma kininogen was measured after adrenalectomy and hypophysectomy. Total kininogen in plasma of 24 hour-adrenalectomized rats was sevenfold higher than that of intact rats, also higher than the total kininogen in plasma of sham-adrenalectomized rats (21.6 +/- 1.7 vs. 16.5 +/- 1.1 micrograms bk eq/ml; p less than 0.05). The role of the hypophysis was studied by nephrectomizing rats 12 days after they had been hypophysectomized. Total plasma kininogen in the control group of rats was still four-fold higher than in intact rats. Twenty-four hours after nephrectomy, a further two-fold increase was obtained (12.7 +/- 1.6 vs. 25.6 +/- 2.0 micrograms bk eq/ml; p less than 0.001). Kinins released by trypsin from plasma of the various groups of rats were separated by reserve-phase (C18) high pressure liquid chromatography (HPLC), and then quantitated. Only T-kinin was found to be increased. Unknown kinins, which were partially converted to T-kinin by increasing trypsin concentration from 10 to 80 mg/ml plasma, were also found by HPLC. The results indicate that surgical trauma induces a marked increase in the concentration of T-kininogen in rat plasma. Neither the hypophysis, nor the adrenals seem to be involved in mediating the increase in T-kininogen.

Identification of a new kinin in human urine.

The types of kinins excreted in fresh urine of dogs, rats, and humans were compared. Urinary kinins were separated by reverse-phase (C18) high performance liquid chromatography and quantitated by radioimmunoassay using an antibody directed against the COOH-terminal region of the peptide. Kinins were found in the following proportions: 53 +/- 3% bradykinin, 23 +/- 4% Lys-bradykinin, and 13 +/- 7% des-Arg1-bradykinin in dog urine; 67 +/- 6% bradykinin, 6 +/- 3% Lys-bradykinin, and 10 +/- 3% des-Arg1-bradykinin in rat urine; and 12 +/- 4% bradykinin, 30 +/- 3% Lys-bradykinin, 2 +/- 1% des-Arg1-bradykinin, and 41 +/- 3% unknown kinin in human urine. The unknown kinin was purified from a pool of human urine. Amino acid sequencing revealed a structure similar to Lys-bradykinin except that proline in position 4 was replaced by alanine ([Ala3]Lys-bradykinin). Synthetic and endogenous [Ala3]Lys-bradykinins had similar high performance liquid chromotography elution volumes and both had vasodilator activity and contracted the rat uterus. Human urinary kallikrein incubated with semipurified human low molecular weight kininogen released 76% of the total kinins as Lys-bradykinin, 7% as bradykinin, and 17% as [Ala3]Lys-bradykinin. In contrast, rat urinary kallikrein released 86% bradykinin, 18% Lys-bradykinin, and negligible amounts of [Ala3]Lys-bradykinin. The study revealed the presence of a new kinin, [Ala3]Lys-bradykinin, in human urine and it also proves that the types of kinins generated intrarenally are species-dependent.

Blood kinins, their concentration in normal subjects and in patients with congenital deficiency in plasma prekallikrein and kininogen.

Kinins are potent vasodilator peptides that may participate in the regulation of local blood flow and blood pressure. Here we report a new method to measure kinins in blood. For this, 6 ml of blood are collected in less than 10 sec directly into 25 ml of ethanol. Kinins are further purified by extracting lipids with ether and by removing kininogen and other interfering substances by chromatography on QAE Sephadex and BioRex 70; then they are measured by a sensitive RIA. In 22 normal subjects, after correction for recovery (50%), the kinin concentration in peripheral venous blood was 25.2 +/- 2.6 pg/ml (mean +/- S.E.M.). To determine whether the circulating kinins are formed by plasma kallikrein or other kininogenases, the concentration of blood kinins was measured in the venous blood of three patients with congenital deficiency in plasma prekallikrein (Fletcher trait) and in one patient with congenital deficiency in the substrate of plasma kallikrein, high-molecular-weight kininogen (Fitzgerald trait). In the three subjects with Fletcher trait, blood kinins were 16, 21, and 31 pg/ml, whereas in the subject with Fitzgerald trait they were 26 pg/ml. Normal subjects had concentrations in the same range (9 to 55 pg/ml), indicating that the concentration of blood kinins in normal subjects is much lower than previously reported (70 to 5000 pg/ml). These results also suggest that kininogenases other than plasma kallikrein may generate circulating kinins.

Quantitative study of extrathyroid enzymatic conversion of T4 to T3 in man.

The authors present a quantitative study of the extrathyroid conversion process of T4 to T3 in 3 athyroid patients, on the basis of an original biochemical model and a rigorous mathematical treatment, utilizing the kinetic isotope method. The values of thor the constant of the reaction rate of conversion of T4 to T3 and of the T4 fraction that undergoes this process--serum concentrations of T4 and T2--were determined in two completely different ways: 1) the radiochromatographic method, and 2) radioimmunoassay of T3 and competitive protein binding of T4. The results thus obtained supported each other and revealed that more than 1/3 of the body total thyroxine is turned into triiodothyronine by the extrathyroid conversion process. The maximum concentration of the triiodothyronine thus formed is reached after 5-6 days, if the reference level of this hormone is zero. However, the higher the T3 reference concentration in the body, up to the normal physiologic level, the shorter the time interval for reaching the T3 maximum concentration following a single moderate dose of T4.