Bradykinin elevates cytosolic Ca2+ concentration in smooth muscle cells isolated from rat duodenum.

The effect of bradykinin on the cytosolic Ca2+ concentration were measured in single, Fura-2 loaded, smooth muscle cells isolated from rat duodenum. All cells responded with a Ca2+ signal when exposed to bradykinin. The bradykinin response consisted of an initial Ca2+ spike followed by a plateau. Pre-treatment of single muscle cells with either the phospholipase C blocker U-73122 or thapsigargin, which is a potent inhibitor of the endoplasmic reticulum Ca2+-ATPase, inhibited the response to bradykinin. Pre-treatment of the cells with EGTA or La3+ to inhibit the Ca2+ influx, abolished the response induced by bradykinin. We conclude that bradykinin applied to single smooth muscle cells from rat duodenum, increases cytosolic Ca2+ by emptying intracellular Ca2+ stores, and by contribution from extracellular Ca2+. In contrast to bradykinin-induced response in isolated rat duodenum (a relaxation followed by a contraction), we did not observe a biphasic effect of bradykinin on cytosolic Ca2+ in single muscle cells. Bradykinin may thus cause relaxation of duodenal smooth muscle indirectly through an effect on neighbouring cells as dilatation is brought about by this agent in blood vessels.

Mechanisms of the relaxant and contractile responses to bradykinin in rat duodenum.

The signal pathway for bradykinin-induced relaxation followed by contraction in the isolated rat duodenum was investigated by comparing the effect of blocking agents on the response to bradykinin and acetylcholine. The phospholipase C inhibitor U-73122 inhibited the relaxation induced by bradykinin, but had no effect on the contraction to either bradykinin or acetylcholine. The same response pattern was observed when the tissues were pre-treated with thapsigargin, a selective inhibitor of microsomal Ca2+ pumps. An inhibitor of non-voltage-dependent Ca2+ influx, SK&F 96365, inhibited the relaxant response to bradykinin and the contraction induced by acetylcholine, but not the contraction induced by bradykinin. In Ca2+-free Krebs-Henseleit buffer, the tissues failed to respond when they were exposed to either bradykinin or acetylcholine. When the tissues were partly depolarized (30 mM KCI), both bradykinin and acetylcholine induced contraction, while the relaxant response to bradykinin was almost completely abolished. Apamin (an antagonist of low-conductance calcium-activated K+ channel) together with charybdotoxin (CTX, an antagonist of large-conductance calcium-activated K+ channel) and CTX alone inhibited the relaxant but not the contractile response to bradykinin. We conclude that the biphasic response in isolated rat duodenum to bradykinin involves two distinct pathways. We propose that the relaxant component is induced indirectly via inositol-mediated increase in cytosolic Ca2+ in non-muscle cells with subsequent signals to the smooth muscle cells, whereas the contractile response is induced by direct effect on the smooth muscle cells.

Bradykinin causes contraction in rat uterus through the same signal pathway as oxytocin.

The signal pathway for bradykinin-induced contraction of the uterine smooth muscle was investigated by comparing the effect of blocking agents on bradykinin and oxytocin induced contractions of the isolated rat uterus in organ bath. The phospholipase C inhibitor U-73,122 abolished the effect of both bradykinin and oxytocin. Inhibition of non-voltage-dependent Ca2+ influx by SK & F 96,365 reduced the contraction induced by both agonists to about 20% of control. The tissues failed to contract when they were exposed to bradykinin or oxytocin in Ca(2+)-free Krebs-Henseleit buffer with 2 mM EDTA. Both bradykinin and oxytocin induced further contraction when the tissues were partially depolarized and partially contracted by 30 mM KCl. These observations suggest that bradykinin, like oxytocin, activates phospholipase C which generates IP3 with a subsequent release of Ca2+ from intracellular stores followed by store-operated Ca2+ influx. Thus, membrane potential independent steps appear to be important in bradykinin-induced contraction in the rat uterus.

Kallikrein rK10-induced kinin-independent, direct activation of NO-formation and relaxation of rat isolated aortic rings.

1. rK10, a weak T-kininogenase isolated from the rat submandibular gland, is a protein belonging to the rat kallikrein family. In the present work, we have studied the biological effects of rK10 with respect to its ability to alter vascular resistance, either directly like rK9, i.e. another kallikrein-like protein, trypsin and thrombin, or through the release of kinins like tissue kallikrein (rK1). The direct effect was studied by its vasomotor activity on rat isolated aortic rings since this preparation was insensitive to the action of kinins. Its ability to induce altered vascular resistance through kinin-generation was investigated by blood pressure studies in whole animals. The studies were performed in comparison to rK1. 2. Unlike rK1, which induces hypotension when administered intravenously to rats (delta BP = -56 +/- 5 mmHg, 5 micrograms kg-1), rK10 did not have any effect on systemic blood pressure (delta BP = -3 +/- 1, 5 micrograms kg-1, i.v.). 3. rK10 was without effect on uncontracted aortic rings, but showed a concentration-dependent (10(-8)-10(-6) M) relaxant effect on tissue precontracted with phenylephrine (10(-6) M). After removal of endothelial cells, no relaxation was observed. The relaxant response to rK10 was transient. rK1 (with and without endothelium), bradykinin and T-kinin (with endothelium) had no effect on contracted or uncontracted aortic rings. 4. The relaxant effect of rK10 was dependent on its enzymatic activity since preincubation with aprotinin (1.02 mM) significantly reduced vasorelaxation from 74 +/- 4% to 24 +/- 3%. 5. The relaxant effect was not inhibited by the kinin antagonist Hoe 140 (10-7 M; 34 +/- 4% without,versus 30 +/- 2% with Hoe 140), but was totally inhibited by the NO-synthase inhibitor N omega.nitro-L-arginine methyl ester (L-NAME) (2.5 x 10-4 M; 27 +/- 3% without and 2 +/- 1% with L-NAME).6. These results show that rKlO has the ability to induce vascular relaxation by a specific, direct effect on endothelial cell NO-synthesis, dependent on rK1O proteolytic activity, but independent of its ability to generate kinin. This effect, or its T-kininogenase activity in blood, was not sufficient for rK1O to have an effect on peripheral vascular resistance since intravenous injections of rK1O, unlike rKl, did not induce hypotension. Thus, rKlO does not seem to play a role in blood pressure homeostasis but may have a local effect on vascular resistance.

Purification of enzymes of the kallikrein gene family (rK8 and rK9) from the rat prostate.

The rat kallikrein family consists of multiple closely related proteins. A method for demonstration and identification of kallikrein-like proteins has been developed based on their differences in isoelectric point and their immunological similarity. The method, which involved separation in flat-bed isoelectro-focusing gels (pH range 3-9) and detection by immunoblotting using polyclonal antiserum against one of the family members, has been used in the present study to detect kallikrein-like proteins in the rat prostate. Nine immunoreactive kallikrein-like protein bands were detected with pI ranging from 5.30 to 8.35. Of these, six were completely purified and three were partially purified. Two proteins (pI 5.30 and 6.75-6.90) corresponded to protein bands in gels of rat submandibular-gland extracts, and were identified by partial amino acid sequence analysis as rK8 and rK9 respectively. In addition, sequence analysis revealed complete sequence similarity between rK9 and the immunoreactive prostate proteins with pI 7.15, 7.25, 7.50 and 8.27. On the basis of this finding and immunological and biochemical characterization, we concluded that all the kallikrein-like proteins detected, except for rK8, represented isoenzymes of rK9. The molecular masses of the prostate rK9 isoenzymes (24,600-29,300 Da) were close to that of submandibular-gland rK9 (24,600 Da), although differences were observed after reduction with mercaptoethanol. The prostate rK9 isoenzymes were, like submandibular-gland rK9, inhibited by soya-bean trypsin inhibitor but not by aprotinin, and were classified as serine proteases as they were inhibited by phenylmethanesulphonyl fluoride. rK8 (28,700 Da) showed no activity with any of the substrates tested, and its inhibitory profile could therefore not be studied. No other enzymes of the kallikrein family were found in the rat prostate.

Characterization of a new kallikrein-like enzyme (KLP-S3) of the rat submandibular gland.

The submandibular gland of the rat contains several enzymes belonging to the kallikrein family. These include tissue kallikrein, antigen gamma (T-kininogenase), esterase B and tonin. In the present study, a new member of this family, which we have named KLP-S3, was identified and purified from the submandibular gland. KLP-S3 was classified as a kallikrein-like enzyme on the basis of its immunological similarity to other kallikrein-like enzymes and its showing 70% and 73% identity in partial amino acid sequence with tissue kallikrein and tonin respectively. Furthermore, the 44 sequenced amino acid residues showed complete correspondence to the mRNA S3 of the kallikrein gene family, which was the rationale for the name kallikrein-like protein (KLP) S3. KLP-S3 consisted of three isoenzymes with pI 6.75, 6.90 and 6.95, which significantly differed from those of other kallikrein-like enzymes. In conjunction with its immunological relationship to kallikrein, this parameter (pI) was considered robust enough to identify the enzyme during purification, since a specific physiological substrate for KLP-S3 has yet to be identified. In SDS/PAGE the three isoenzymes ran as one band with a molecular mass of 25,800 Da, which after reduction with 2-mercaptoethanol was split into two chains with molecular masses of 16,500 and 13,300 Da. In common with other kallikrein-like enzymes, KLP-S3 was inhibited by phenylmethanesulphonyl fluoride, and was thus classified as a serine protease. It was also inhibited by soya-bean trypsin inhibitor but not by aprotinin. It showed weak reactivity against the chromogenic substrates S2288, S2266, S2366 and S2302 (D-Ile-Pro-Arg 4-nitroanilide, D-Val-Leu-Arg 4-nitroanilide, Glu-Pro-Arg 4-nitroanilide and D-Pro-Phe-Arg 4-nitroanilide respectively) and did not cleave rat T-kininogen or dog high-molecular-mass/low-molecular-mass kininogen. Its specific angiotensin II-generating activity (angiotensin I as substrate) was 0.04% of that of rat tonin. KLP-S3 (1-100 nM) induced a statistically significant angiotensin-independent contraction of isolated rat aorta rings. The maximum contraction was 15% of the response to the alpha-adrenoceptor agonist phenylephrine (1 microM). The concentration of KLP-S3 in the rat submandibular gland was by single radial immunodiffusion estimated to be 47 +/- 3 micrograms/mg of protein.

Identification of proteins of the kallikrein family by isoelectrofocusing and immunoblotting.

We have found that kallikrein-like proteins differ in their isoelectric point but share antigenic determinants. For identification of kallikrein-like proteins an initial separation was carried out in flat-bed isoelectrofocusing gels. The kallikrein-like nature was demonstrated by an immunological similarity to kallikrein-like proteins by immunoblotting using antiserum against a kallikrein family member for staining. We used this system to identify different kallikrein-like proteins during purification of both known as well as new enzymes.

Immunohistochemical localization of rat submandibular gland esterase B (homologous to the RSKG-7 kallikrein gene) in relation to other serine proteases of the kallikrein family.

The rat submandibular gland contains several members of the kallikrein family. In the present study we purified and raised an antiserum against one of these enzymes, i.e., esterase B, which was first described by Khullar et al. in 1986. N-terminal amino acid analysis revealed complete homology between esterase B and the kallikrein family gene RSKG-7. For characterization of the antiserum, flat-bed isoelectrofocusing with immunoblotting was superior to immunoelectrophoresis and double immunodiffusion in detecting and identifying crossreacting proteins. This was due to the fact that kallikrein-like enzymes were readily separated by isoelectrofocusing, and immunoreactivity was easily detected by the sensitive peroxidase-anti-peroxidase staining after blotting onto nitrocellulose membrane. Immunohistochemical controls were carried out accordingly, including homologous as well as crossreacting antigens. In the submandibular gland, esterase B was detected exclusively in all granular convoluted tubular cells, co-localized with tissue kallikrein and tonin. Some staining was also observed in striated duct cells; however, this staining reaction was induced by cross-reactivity with kallikrein, since staining was abolished by addition of kallikrein as well as esterase B to the primary antiserum. It was therefore concluded that like tonin and antigen gamma, but unlike kallikrein, esterase B was not detected in the striated ducts of the submandibular, parotid, or sublingual glands. This separation in anatomic distribution between esterase B and kallikrein may indicate that prokallikrein activation is not the only biological function of esterase B.

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.

Investigation of a possible correlation between rates of secretion and microsomal membrane association of plasma proteins synthesized by rat liver.

The rates of secretion of complement C3, haptoglobin and plasminogen have been determined after pulse labelling with [3H]leucine, and compared to the secretion of prothrombin, albumin and transferrin investigated previously (Kvalvaag, A.H., Tollersrud, O.K. and Helgeland, L. (1988) Biochim. Biophys. Acta 937, 319-327). To study membrane association, rough microsomes were treated with increasing concentrations of saponin, sodium deoxycholate or Triton X-100. All six proteins were quantitated in the soluble and membrane fraction by enzyme immunoassays. At concentrations of saponin from 0.08% to 0.32%, each secretory protein showed a characteristic distribution, almost identical to that obtained with 0.05% sodium deoxycholate or 0.08% Triton X-100. Albumin and transferrin with half-times for secretion (t1/2) 30 and 75 min, respectively, are both almost exclusively found in the luminal fraction (greater than 95%). Prothrombin and plasminogen, which both show an intermediate t1/2 (approx. 55 min), are partially associated with the membranes, as only about 60% was released. Haptoglobin and complement C3 also show some association with the membranes (80-85% released). C3 is secreted at the same rate as prothrombin and plasminogen (t1/2 = 55 min), whereas haptoglobin is secreted more rapidly (t1/2 = 40 min). Accordingly, no correlation between kinetics of secretion and membrane association was demonstrated.

A simple and rapid method for purification of rat haptoglobin for production of antiserum.

Plasma from rats with acute inflammatory response was fractionated on Blue Sepharose CL 6B, to separate haptoglobin from albumin and lipoproteins. Affinity chromatography on Blue Sepharose proved to be a convenient method for crude fractionation of plasma. Pure haptoglobin was obtained by the subsequent affinity chromatography on a rabbit-haemoglobin Sepharose column. Minor amounts of rabbit haemoglobin co-eluted from the haemoglobin Sepharose column but did not influence the monospecificity of the antiserum raised in rabbits. By use of the antiserum in single radial immunodiffusion, the concentration of haptoglobin in plasma from normal rats was measured to be 0.5 g/l.