Tissue kallikrein and kinin infusion promotes neovascularization in limb ischemia.

Adenovirus-mediated kallikrein delivery has been shown to promote blood vessel growth in the limb under both ischemic and normoperfused conditions. Here we investigated whether a continuous supply of kallikrein and kinin peptide can induce neovascularization in a rat model of hindlimb ischemia. Rats underwent femoral artery ligation and localized injection of tissue kallikrein, bradykinin or B1 receptor agonist, followed by infusion of proteins by osmotic minipump. Regional blood flow was monitored weekly by laser Doppler perfusion imaging. Three weeks after surgery, rats receiving kallikrein and kinins showed a significant increase in the perfusion ratio of ischemic vs. normoperfused limb compared to control rats. Similarly, a microsphere assay showed that kallikrein and kinins significantly increased regional blood flow without altering blood pressure. Moreover, kallikrein and kinins significantly augmented capillary and arteriole densities, as quantified by immunostaining with CD-31 and smooth muscle alpha-actin. Both tissue kallikrein and bradykinin increased hemoglobin content in Matrigel implants in mice, providing further evidence of the angiogenic properties. Kinins, when delivered subcutaneously via Matrigel in rats, also increased regional perfusion. This is the first demonstration that local application of tissue kallikrein protein or kinin peptide has therapeutic value in the treatment of ischemic disease by promoting neovascularization.

Tissue kallikrein and kinin infusion rescues failing myocardium after myocardial infarction.

BACKGROUND: Tissue kallikrein is a serine proteinase that generates the vasoactive kinin peptide, which produces vasodilatory, angiogenic, and antiapoptotic effects. In this study, we investigated the effect of a stable supply of kallikrein and kinin on ventricular remodeling and blood vessel growth in rats after myocardial infarction. METHODS AND RESULTS: At 1 week after coronary artery ligation, tissue kallikrein or kinin was infused through a minipump for 4 weeks. At 5 weeks after myocardial infarction, kallikrein and kinin infusion significantly improved cardiac contractility and reduced diastolic dysfunction without affecting systolic blood pressure. Kallikrein and kinin infusion significantly increased capillary density in the noninfarcted region. Kallikrein and kinin infusion also reduced heart weight/body weight ratio, cardiomyocyte size, and atrial natriuretic peptide and brain natriuretic peptide expression in the noninfarcted area. Moreover, kallikrein and kinin infusion inhibited interstitial collagen deposition, collagen fraction volume, and collagen I and collagen III mRNA levels, transforming growth factor (TGF)-beta1 and plasminogen activator inhibitor-1 expression, and Smad2 phosphorylation. The effects of kallikrein and kinin on cardiac remodeling were associated with increased nitric oxide levels and reduced NADPH oxidase expression and activity, superoxide formation, and malondialdehyde levels. Furthermore, in cultured cardiac fibroblasts, kinin inhibited angiotensin II-stimulated TGF-beta1 production, and the effect was blocked by icatibant. CONCLUSION: These results indicate that a subdepressor dose of kallikrein or kinin can restore impaired cardiac function in rats with postinfarction heart failure by inhibiting hypertrophy and fibrosis and promoting angiogenesis through increased nitric oxide formation and suppression of oxidative stress and TGF-beta1 expression.

Modulation of hypotensive effects of kinins by cathepsin K.

Kinins are pro-inflammatory peptides, which participate in the maintenance of cardiovascular homeostasis, and play a key role in numerous diseases, including lung fibrosis and hypertension. Evidence has been provided recently for the presence of alternative mechanisms of bradykinin generation and/or degradation. Here we showed that cathepsin K may act as a potent kinin-degrading enzyme in bloodstream. Contrary to cathepsin L, cathepsin K attenuates kallikrein-induced decrease of rat blood pressure, and reduces the hypotensive effect of bradykinin in a dose-dependent manner. Moreover, we identified, by engineering the S2 subsite of both recombinant enzymes, two critical residues involved respectively in the kininase activity of cathepsin K, i.e. Tyr67/Leu205, versus kininogenase activity of cathepsin L, i.e. Leu67/Ala205. In conclusion, according to its ability to modulate hypotensive effects of kinins, we propose that cathepsin K is a kininase of biological relevance, in complement of well-documented neutral endopeptidase or angiotensin-converting enzyme.

Role of synergistic action of PAF and kinin in bacterial endotoxin-induced hypotension in rats.

Involvement of PAF and kinin in the endotoxin (LPS)-induced hypotension was examined in the rat strains, Brown Norway (B/N);Kitasato (Normal) and B/N-Kitasato rats, intravenous injection of 10 mg/kg LPS caused a hypotension composed on two phases (15 min and 70 min after LPS injection). However in the kininogen-deficient B/N-Katholiek rats. LPS induced only the second phase. Pretreatment with bradykinin (BK) antagonist. Hoe 140 suppressed LPS-induced hypotension of normal rats to the level of Katholiek rats. TCV 309, a PAF-antagonist, suppressed the LPS-hypotension almost completely in the both strains. The reason why the PAF-antagonist showed almost complete inhibition, could be explained by a synergism. Because concomitant injection of a small amount of BK with PAF caused potentiation of the effect of the PAF action. These results suggest that formation of endogenous BK contributes mainly to the 1st phase of LPS-hypotension, and PAF contributes to both phases, by either direct and synergistic actions.

Mediation by B1 and B2 receptors of vasodepressor responses to intravenously administered kinins in anaesthetized dogs.

1. Vasodepressor responses to intravenous (i.v.) injection of bradykinin (BK) and des-Arg9-BK, a selective B1 kinin receptor agonist, were characterized following i.v. pretreatment with selective B1 ([Leu8]-des-Arg9-BK) and B2 (Hoe 140) kinin receptor antagonists in anaesthetized dogs. 2. Des-Arg9-BK (0.05-3.3 nmol kg-1) produced dose-dependent decreases in mean arterial blood pressure with a ED50 0.4 nmol kg-1. The vasodepressor effects evoked by des-Arg9-BK (0.6 nmol kg-1) and BK (0.2 nmol kg-1) were greater after i.v. and i.a. injections, respectively. 3. The vasodepressor response to BK (0.6 nmol kg-1) but not to des-Arg9-BK (0.6 nmol kg-1) was significantly (P < 0.001) blocked by pretreatment with the B2 receptor antagonist, Hoe 140. 4. The vasodepressor response to des-Arg9-BK (0.6 nmol kg-1) but not to BK (0.6 nmol kg-1) was significantly (P < 0.001) reduced by pretreatment with the selective B1 receptor antagonist, [Leu8]-des-Arg9-BK. Although both B1 and B2 receptor antagonists caused a transient fall in blood pressure, their inhibitory action was unlikely to be related to a desensitization mechanism. 5. Inhibition of prostaglandin synthesis with indomethacin prevented the vasodepressor response induced by arachidonic acid (1 mg kg-1, i.v.) but not that to BK or des-Arg9-BK (0.6 nmol kg-1). 6. These results suggest, firstly, that the vasodepressor responses to i.v. BK and des-Arg9-BK are mediated by the activation of B2 and B1 receptors, respectively; secondly, that prostaglandins are not involved in the vasodepressor responses to kinins.These findings provide pharmacological evidence for the existence of functionally active B1 receptors in canine cardiovascular homeostasis.

[The response of the airways in asthmatic patients to kinin inhalations define a type-B2 receptor profile]. / La risposta delle vie aeree di pazienti asmatici ad inalazioni di chinine definisce un profilo recettoriale di tipo B2.

Kinins (i.e. bradykinin, kallidin and [desArg9]-bradykinin) are vasoactive oligopeptides which may contribute as mediators in the pathogenesis of asthma by interacting with specific receptors designated as B1 and B2. The aim of the present study was to investigate the airway response to inhaled histamine, bradykinin, kallidin and [desArg9]-bradykinin in normal and asthmatic subjects. Changes in airway caliber were followed as FEV1 (forced expiratory volume in 1 sec) and as Vp30 (maximum expiratory flow at 30% of the vital capacity). Neither histamine, bradykinin, kallidin nor [desArg9]-bradykinin had any measurable bronchoconstrictor effect in the normal subjects. However, in the asthmatic subjects, histamine, bradykinin and kallidin, but not [desArg9]-bradykinin, produced prompt concentration-related bronchoconstriction. The geometric mean PC20FEV1 (provocation-concentration of inhaled agonist reducing the FEV1 by 20% from baseline) values were 0.027, 0.082 and 0.44 mg/mL for bradykinin, kallidin and histamine respectively. Because bradykinin and kallidin are agonists of B2 receptors and [desArg9]-bradykinin is an agonist for B1 receptors, our data suggest that asthmatic, but not normal, airways are hyperresponsive to kinins and that this potent bronchoconstrictor response is due to a specific pharmacologic effect compatible with the stimulation of B2 receptors.

Role of NK-2 receptors in the antidipsogenic activity of neurokinins in the mouse.

1. The receptors involved in the anti-dipsogenic activity of neurokinin (NK) agonists were investigated in water-deprived mice. 2. Intracerebral administration of agonists selective at all three NK receptors (NK-1, NK-2, NK-3) caused inhibition of drinking in this model. However, only the NK-2 receptor agonist, GR64349, inhibited drinking without producing other behavioural effects. Both NK-1 (GR73632) and NK-3 (senktide) agonists induced a variety of behavioural effects which appeared to compete with the drinking response. 3. The inhibitory effect on drinking observed after central injection of the NK-2 agonist, GR64349, was attenuated by co-administration of the NK-2 antagonist, L-659,877, but not by the NK-1 antagonist, GR82334. 4. These results illustrate that the antidipsogenic activity of the NKs, in mice, is mediated via NK-2 receptors.

Cardiovascular responses elicited by intrathecal kinins in the conscious rat.

In the conscious, unrestrained rat, intrathecal (i.t.) injection of 0.81 pmol-81 nmol bradykinin (BK), kallidin (KD) and T-kinin at the T-9 spinal cord level produced transient (less than 10 min) increases in mean arterial pressure (MAP) and longer lasting decreases in heart rate (HR). These effects were dose-dependent and similar with respect to intensity and time course for the three kinins. The des-Arg9-BK fragment, a selective agonist for B1 receptors, was active only at 81 nmol. The pressor response induced by BK was enhanced by propranolol and by transection of the cervical spinal cord but was converted to a vasodepressor effect by prazosin. The bradycardia was converted to tachycardia by prazosin, atropine, pentolinium, capsaicin and in spinal transected rats. However, the cardiovascular responses to BK remained unaffected by diphenhydramine plus cimetidine, morphine, indomethacin, adrenal medullectomy, i.t. idazoxan and after bulbospinal noradrenaline deafferentation with 6-hydroxydopamine. These results suggest that the increase in MAP induced by i.t. BK is mediated by the sympathoadrenal system while the decrease in HR is ascribable to a vagal reflex involving sensory C-fibers and a spinobulbar pathway. This pharmacological evidence therefore supports a role for kinins in cardiovascular regulation in the spinal cord.

Capillary permeability induced by intravenous neurokinins. Receptor characterization and mechanism of action.

The effects on plasma extravasation of three increasing doses from 6.5 pmol to 650 nmol/kg of substance P (SP), SP fragments, neurokinin A (NKA), neurokinin B (NKB) and selective agonists for neurokinin receptors were assessed in three cutaneous tissues (skin of hind paws, dorsal skin and ears) by intravenous (i.v.) administration in the pentobarbitone anaesthetized rat. Dose-dependent increases in plasma extravasation were observed with the following rank orders of potency (SP greater than NKA greater than NKB) for neurokinins and (SP greater than [p-Glu6]SP(6-11) greater than SP(4-11) greater than [p-Glu5]SP(5-11) greater than SP(7-11] for C-terminal SP fragments. The metabolically stable SP analogue [p-Glu5, MePhe8, Sar9]SP(5-11) was slightly more potent than [p-Glu5]SP(5-11). The N-terminal fragments SP(1-4), SP(1-7) and SP(1-9) were inactive up to 650 nmol/kg. The NK-1 receptor selective agonists [Sar9, Met(O2)11]SP and [beta-Ala4, Sar9, Met (O2)11]SP(4-11) were more potent than the NK-2 [( Nle10]NKA(4-10] and NK-3 [( MePhe7]NKB and [beta-Asp4, MePhe7]NKB(4-10] receptor selective agonists. Plasma extravasation induced by SP (6.5 nmol/kg) was unchanged in the presence of atropine, methysergide, diphenhydramine or during the i.v. and intra-arterial (i.a.) infusion of D-Arg0[Hyp3.D-Phe7]BK, an antagonist of bradykinin. Plasma extravasation induced by SP and [Sar9, Met(O2)11]SP was significantly reduced by indomethacin while that induced by NKA, NKB, [beta-Ala4, Sar9, Met(O2)11]SP(4-11), SP(4-11) and [p-Glu6]SP(6-11) was unaffected by the cyclooxygenase inhibitor. Compound 48/80 (0.75 mg/kg), histamine (10 mg/kg) and 5-HT (10 mg/kg) caused an increase in plasma extravasation, only the effect of compound 48/80 was abolished by indomethacin. Pretreatment with compound 48/80 prevented its own action on plasma extravasation and significantly reduced that induced by 6.5 nmol/kg of SP. These results rule out the involvement of acetylcholine (muscarinic receptors), 5-HT (5-HT1 and 5-HT2 receptors), histamine (H1 receptors) and kinins (B2 receptors) in the response to SP and indicate that the two positively charged amino acids (Arg, Lys) at the N-terminal end of the SP molecule are essential to trigger the release of prostaglandins from mast cells. This mechanism is responsible for the indirect effect of SP and related peptides on capillary permeability and does not appear to be mediated by a selective SP receptor. In addition, neurokinins may increase capillary permeability by direct activation of a NK-1 receptor type on the vascular endothelium.

Changes in rectal temperature of the rabbit by intracerebroventricular injection of bradykinin and related kinins.

The effects of intracerebrovascular injections of bradykinin (BK), lysyl-bradykinin (L-BK), methionyl-lysyl-bradykinin (ML-BK) and des-arginyl-bradykinin (Des-Arg9-BK) on the rectal temperature of rabbits were investigated. A linear dose-response curve could be obtained with bradykinin. Equimolar doses to BK of L-BK and ML-BK gave responses of the rectal temperature undistinguishable from those of BK. In contrast, the smaller polypeptide Des-Arg9-BK did not show any hyperthermic properties. Since prostaglandins (PG) are known to potentiate (facilitate) the peripheral effects of BK and display a hyperthermic action, their synthesis being inhibited by antipyretics, we studied the effect of indomethacin and paracetamol on the BK-induced rise in rectal temperature in rabbits. It could be shown that these drugs inhibited partially the response to BK. The possible participation of BK in thermoregulation will be discussed.

[Special indications for the use of soft contact lenses as a drug-release-system (author's transl)]. / Besondere Indikationen für die Anwendung weicher Kontaktlinsen als Augentropfenreservoir (Drug Release System)

Research has been performed, both experimentally and clinically, to establish the value of the association of soft contact lenses and some types of eye drops. The use of soft contact lenses with eye drops may be useful in some special cases: a) more prolonged and more sustained effect compared with the usual way of administration of eye drops (especially antiglaucomatous substances, antimetabolites, mydriatics); b) possibility of reducing the concentration to avoid local discomfort or systemic side-effects, without loss of their effectiveness on the eye conditions to be treated. The combined use of soft lenses (12.5-15 mm in diameter) with eye drops may be obtained either by presoaking the lens in the liquid or by regular instillation of eye drops after insertion of the lens; the two techniques may of course be associated. In the present research the advantages of utilizing hydrophylic lenses with osmotically active substances, to obtain a better and more protracted dehydration of the cornea, were first examined, in vitro and in vivo. The following substances were tested: 10% propylenglycol, 10% glycerol, 10% glucose and 5% natrium chloride. The clearing effect of the different types of treatment was evaluated in 45 patients with edematous bullous keratopathy with an instrument which measured the infrared light emitted by an optic fiber and reflected by the cornea. The effects were more marked for the epithelial than for the stromal oedema. Another group of investigations was performed with two polypeptides with high molecular weight: Eledoisin, extracted from a mediterranean octopus, Eledone moschata, and Physalaemin, extracted from the skin of a south american batrachian, Physalaemus fuscomaculatus, both of these stimulate the lacrimal secretion and were previously successfully employed topically by the authors against keratoconjunctivitis sicca. The increase of the amount of fluid was however short-lived. Eledoisin at a concentration of 200 mug/ml, was examined in its effects both in vitro and in vivo, whereas physalaemin, at a concentration of 20 mug/ml, only in vitro, owing to the present shortage of the product. The clinical tests in 23 eyes of 14 patients with keratoconjunctivitis sicca proved satisfactory, since the lacrymal stimulating effect is not only greater, but lasts three times longer by combining the instillation of eledoisin with a presoaked soft lens. Some antiglaucomatous products (propranolol, clonidine, prostigmine) were, finally, used in association with a soft lens to reduce the concentration of the eye drops for a better tolerance locally (propranolol: a beta-adrenergic blocking agent) or generally (clonidine: alpha-adrenergic agent), also with the advantage of protracted release. With propranolol the concentration could be reduced to 0.01-0,10% (instead of 0.125 to 0.25%) and to 1.5% (instead of 3%) with prostigmine, when lenses were presoaked or instillations took place at regular time intervals, after insertion of the lenses.

Antinociceptive action of bradykinin and related kinins of larger molecular weights by the intraventricular route.

1. It was shown previously that bradykinin (Bk) when given intraventricularly increases the threshold of electrical stimulation of dental pulp in the rabbit.2. Bradykinin derivatives with increasing molecular weights (lysyl-Bk (L-Bk), methionyl-lysyl-Bk (ML-Bk) and glycyl-arginyl-methionyl-lysyl-Bk (GAML-Bk)) were found to produce effects of decreasing intensity on the threshold of electrical stimulation according to the following scale: Bk (1.00)>L-Bk (0.28)>ML-Bk (0.060)>GAML-Bk (0.047).3. The four peptides had similar relative activities on the guinea-pig ileum but an inverse relationship in their effects on vascular permeability and rat blood pressure.4. The discrimination index, increase in vascular permeability/antinociceptive effect rose to values of the order of 170 to 550, taking Bk as the reference peptide (potency=1.00).5. We conclude that the increase in threshold of electrical stimulation could not be due to an increase in vascular permeability or decrease of blood pressure.