Characterization of receptor-mediated actions of T-kinin.

T-Kinin (Ile-Ser-bradykinin) is unique to the rat. This study characterizes the receptors involved in T-kinin activity on both the intact isolated rat uterus and membrane receptor preparations of the rat uterus. The results show that T-kinin acts through kinin B2 receptors in the rat uterus as demonstrated by B2 receptor-antagonist inhibition. While the potency of T-kinin on rat uterus contraction was similar to that of bradykinin, binding studies showed that the affinity of T-kinin to the receptor was 10-fold lower than that of bradykinin. On the other hand, the D isomer of T-kinin, D-Ile-Ser-bradykinin, had an affinity for the receptor greater than that of T-kinin and was more potent in causing contraction. Comparing this finding with our previously published report that D-Ile-Ser-bradykinin is not active on the kinin receptor for vascular permeability indicates that the kinin receptors in the rat uterus are not the same as those previously reported in the smooth muscle of the vasculature, i.e. there exists subclasses of kinin B2 receptors. The data from binding studies on a variety of T-kinin analogues show that the substitution of hydroxyproline (Hyp) for Pro5, together with the D-configuration at Ile1 and/or Ser2 may be useful for the development of selective T-kinin antagonists. Studies involving pretreatment of the tissue with indomethacin demonstrated that prostaglandin release was more of a component of T-kinin's activity on the rat uterus than that of bradykinin.

Characterization of the kinin system in the ovary during ovulation in the rat.

Ovulation has been noted for some time to bear a remarkable similarity to an inflammatory response. One of the principal components that is activated and helps mediate the events during an inflammatory response is the kinin system. Therefore, the purpose of the present study was to examine whether this system could be similarly activated and involved in the cascade of events that leads to ovulation. To answer this question, immature 23-day-old female rats were primed with eCG (10 IU) and ovulation was induced by administration of hCG (10 IU) 48 h later. Groups of rats were killed at 0 h, 10 h, 20 h, and 30 h after hCG for determination of ovulation, ovarian steroid levels, and changes in the levels of kinin system components. Plasma total kininogen levels did not change during the entire period studied. In contrast, ovarian total kininogen levels rose from 0 h to reach a peak at 10 h--a time immediately preceding the beginning of ovulation--after which the levels fell at 20 h, only to rise again at 30 h. Three species of kininogens, high molecular weight (HMW), low molecular weight (LMW), and T-kininogen, were shown to be present in the ovary. T-kininogen was the major kininogen present in the ovary, accounting for 60-92% of the total kininogen at any given time point during the ovulatory process. HMW kininogen levels accounted for only 1.2% of the total ovarian kininogen.(ABSTRACT TRUNCATED AT 250 WORDS)

T-kininogen, processing and functions.

Studies are presented which indicate that T-kininogen, the acute phase kininogen of the rat, could be a healing protein because of its properties as a cysteine protease inhibitor. Evidence is also presented that mRNA of T-kininogen synthesis may be a function of interleukin 6 production. A regulatory mechanism is postulated by which SH cofactors could determine if T-kinin is released or whether the T-kininogen molecule would remain intact. Evidence is also presented that T-kinin acts through kinin B2 receptors. No specific binding of bradykinin or T-kinin could be detected in rat heart preparations.

Lack of specific binding of bradykinin and D-Arg [Hyp3, Thi5, D-Tic, Oic8] bradykinin in membranes from rat heart.

Recent data demonstrated effects of bradykinin (BK) in the isolated perfused rat heart which could be blocked by BK antagonists. However, so far BK receptors in heart tissue have not been characterized. To search for BK receptors in rat hearts iodinated D-Arg[Hyp3,Thi5,D-Tic,Oic8]BK (Hoe 140) was used as a ligand for binding due to its high affinity and to its resistance to degradation. The labeled antagonist bound well to membranes prepared from rat ileum and could be displaced by the unlabeled antagonist as well as by BK and T-kinin in a concentration dependent manner. However, there was no specific binding detectable when membranes from rat left cardiac ventricle were used. To assure integrity of at least one other receptor in these membranes, binding of 3H-methylscopolamine and its displacement by the respective cold ligand was demonstrated. To rule out an occupation of the BK receptors by endogenous formed BK, the tissue was treated with an acidic buffer with high ionic strength to remove surface bound BK. However, this treatment did not unmask any specific binding for the BK antagonist. In view of the possibility that the structure of the labeled antagonist prevented its binding, experiments were carried out using tritiated BK itself. These experiments also failed to demonstrate specific binding sites which indicate that the structural aspects of Hoe 140 are probably not interfering in binding of the antagonist, especially since it binds to ileum. It is concluded, that there are too few binding sites for BK in the rat heart homogenate for ordinary binding studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduction of T-kininogen messenger RNA levels by dexamethasone in the adjuvant-treated rat.

When inflammation is induced in rats following injection of Freund's complete adjuvant, steady state levels of T-I and T-II kininogen mRNAs increase markedly as do plasma levels of T-I and T-II kininogens. When rats are additionally treated with dexamethasone, T-I and T-II steady state mRNA levels and plasma levels of T-kininogens are reduced. The results suggest that dexamethasone may affect the magnitude of T-kininogen gene induction caused by inflammation.

Identification of thiol-activated T-kininogenases in the rat and mouse submandibular glands.

T-kininogen is a unique protein(s) which is directed for synthesis following inflammation such as that caused by adjuvant arthritis and carrageenin. The properties of the protein include thiol-protease inhibition and the potential to act as a substrate to release Ile-Ser-bradykinin (T-kinin). During the inflammatory response, free T-kinin is found in the blood and inflammatory fluids indicating that T-kininogenase (T-kgnase) enzymes exist that release T-kinin and perhaps related T-kinins involved in inflammation. We report here that T-kininogenases have been found in the rat and mouse submandibular glands and in rat peritoneal white cells. The pH optimum is about 8.0. The enzymes must be thiol-activated. Thus, the rat has the complete T-kgnase-T-kinin system. Since T-kininogen is a member of the superfamily of cysteine protease inhibitors, members of the superfamily may be directed for synthesis in inflammatory diseases including ascites in other species including humans.

Increase in vascular permeability of rat and guinea pig skin by T-kinin.

The vascular permeability-increasing response to T-kinin in rat and guinea pig skin was investigated. The vascular permeability was measured with 125I-labeled bovine serum albumin [( 125I]BSA) as a tracer. Plasma exudation rapidly occurred 0-15 min after the intradermal injection of T-kinin. T-kinin in doses of 0.3, 1, and 3 nM/spot significantly increased the vascular permeability in a dose-dependent manner. The vascular response of T-kinin is similar to that of bradykinin. On the other hand, T-kinin analogs, D-Ile-Ser-bradykinin and Ile-D-Ser-bradykinin only weakly enhanced the vascular permeability. Prostaglandin E1, forskolin, and the angiotensin-converting enzyme inhibitor, SQ-14225, potentiated the T-kinin-induced plasma exudation. Cyproheptadine and indomethacin did not affect the T-kinin-induced response. The results suggest that T-kinin will play an important role in increasing vascular permeability associated with inflammation.

Isolation of a thiol-activated T-kininogenase from the rat submandibular gland.

T-kininogenase (T-kgnase) activity has been investigated in tissues of the rat and submandibular glands of the rat, mouse and guinea pig. Both rat and mouse submandibular homogenates showed high T-kgnase activity. The enzyme has been purified 360-fold from rat submandibular gland homogenate supernatant fluid. The enzyme has an apparent molecular mass of 28 kDa and a pH optimum of 8.0 toward T-kininogen. It cleaved T-kininogen in catalytic quantities to release T-kinin (Ile-Ser-bradykinin) and small quantities of bradykinin and an unknown kinin. The activity of the enzyme was increased 10-fold in the presence of thiol groups (dithiothreitol) and inhibited by leupeptin (90%) and to a lesser extent by aprotinin (49%), TLCK (46%) and soybean trypsin inhibitor (27%). Pepstatin and PMSF did not inhibit the enzyme. Studies on substrate specificity, pH optimum and agents which inhibit T-kgnase activity demonstrate that this enzyme is different from plasma and tissue kallikreins, cathepsin D, esterase A and esterase B (other known kininogenases). It is the first thiol-activated kininogenase to be reported.

Inactivation of kallikrein and kininases and stabilization of whole rat brain kinin levels following focused microwave irradiation.

Focused microwave irradiation was employed to stabilize endogenous whole rat brain bradykinin levels prior to a simple extraction procedure. Skull microwave exposure (2450 MHz, 3.8 kW., 2.45 sec) resulted in inactivation to less than 5% of control of whole brain kallikrein and kininase activity. Using this adequate exposure duration whole rat brain kinin levels as measured by a sensitive radioimmunoassay were approximately 0.6 pmol/g (wet weight). Further purification of irradiated brain extracts using HPLC revealed that immunoreactive kinin eluted as a single peak that co-chromatographed with authentic bradykinin. Microwave fixation duration of 1.25 sec yielded greatly increased levels of immunoreactive kinin which following HPLC purification eluted in two peaks, corresponding to authentic bradykinin and T-kinin, respectively. The tissue injury resulting from incomplete microwave fixation resulted in the release of kinins. This excess immunoreactive kinin may be derived from cerebral blood, since the predominant form of kinin-generating protein in plasma is T-kininogen.

Isolation and properties of two rat plasma T-kininogens.

Two species of T-kininogen which release T-kinin (Ile-Ser-bradykinin) have been purified from plasma of rats treated with Freund's complete adjuvant. The molecular weight was estimated to be 69,000 for either T-kininogen I and II by SDS-polyacrylamide gel electrophoresis. Trypsin released one mole of T-kinin from one mole of either T-kininogen, but glandular kallikrein, including rat urinary and rat submandibular gland kallikreins and human urinary kallikrein, did not release any kinin from T-kininogens. Cathepsin D, which was purified from rat liver, released T-kinin from T-kininogens at pH 4.0. These results indicate that rat plasma contains two types of T-kininogen which differ from high molecular weight and low molecular weight kininogens.

Increased plasma level of T-kininogen in rats treated with Freund's adjuvant.

Studies have been carried out to clarify which component of plasma kininogen in rats increased in the inflammatory condition induced by an injection of Freund's complete adjuvant. Plasma T-kininogen, which was measured by assaying the amount of T-kinin liberated by trypsin treatment, remarkably increased in parallel with the severity of paw swelling following the intradermal injection of adjuvant into the rat hindpaw. Treatment with indomethacin or dexamethasone following an injection of adjuvant suppressed the increase in T-kininogen level as well as the development of paw swelling in rats. These results indicate that T-kininogen, the newly found precursor of T-kinin, is the main component of plasma kininogen which responds to the inflammatory stimulus in adjuvant arthritis.

Release of T-kinin and bradykinin in carrageenin induced inflammation in the rat.

Plasma and inflammatory fluid kininogen levels, and blood and inflammatory fluid free kinin levels were determined in rats 24 h after the injection of carrageenin into an air pouch. Plasma T-kininogen levels increased 7-fold. In the inflammatory fluid levels reached 8 micrograms/ml. Blood levels of free kinin showed a 5-fold increase. The kinins were identified on HPLC as T-kinin (Ile-Ser-bradykinin) and bradykinin, 63 and 37%, respectively. These results indicate for the first time that free T-kinin as well as bradykinin is released during an inflammatory response in rat and confirms our previous finding that T-kininogen may be a major acute-phase protein in inflammation.

T-kininogen--the major plasma kininogen in rat adjuvant arthritis.

Total kininogen in plasma of Freund's adjuvant treated rats increased 20-fold 7 days following the injection. Analysis of the kininogens demonstrated that increases in T-kininogen was the major reason for the rise in kininogen. High molecular weight and low molecular weight kininogens showed little or no change. The increase in T-kininogen paralleled the inflammatory condition. Anti-inflammatory agents which reduced paw swelling also reduced plasma T-kininogen levels. Unidentified peaks on HPLC of kinin following plasma treatment by trypsin were shown to be oligopeptides containing T-kinin (Ile-serbradykinin). The relationship of T-kininogen to the inflammatory response is discussed.