Kallikrein stimulates prostacyclin production in bovine vascular endothelial cells

Cultured endothelial cells isolated from bovine carotid aorta produce prostacyclin (prostaglandin I2) and a small amount of prostaglandin E2. The effects of kallikrein (EC 3.4.21.8) on the release of prostacyclin from the cells were studied with the radioimmunoassay technique. Kallikrein stimulated the release of prostacyclin in a dose-dependent manner. The maximal stimulation reached up to 9.2-fold at 0.1 micrograms/ml of kallikrein. The effect was not associated with the activation of the fatty acid cyclooxygenase, but with the stimulation of arachidonic acid release. But kallikrein itself did not have phospholipase activity. On the other hand, at the same doses, kallikrein failed to induce platelet aggregation or enhance platelet aggregation induced by collagen. Our findings suggest that the vasodilator effect of kallikrein is mediated in part by prostacyclin production. Furthermore, we investigated the possibility that the stimulatory effect of kallikrein on prostacyclin production in endothelial cells is associated with kinin formation. Bradykinin and lysylbradykinin (kallidin) also stimulated the release of prostacyclin, but the effects were far less than that of kallikrein. And the stimulation due to the addition of both kallikrein and bradykinin on prostacyclin and arachidonic acid release was not competitive or additive, but synergistic. Moreover, even if fetal calf serum was incubated with kallikrein, bradykinin was not detected at all. When kallikrein was pre-incubated with aporotinin, which is an inactivator of kallikrein, the effect of kallikrein was completely abolished. These findings suggest that the stimulatory effect of kallikrein on the release of prostacyclin from vascular cells is possibly not due to kinin formation, but to other substance(s) produced by this serine proteinase.     

De-aggregatory action of prostacyclin in vivo and its enhancement by theophylline.

In the mixed venous blood of anaesthetized, heparinized cats prostacyclin de-aggregated platelet thrombi, which were formed on the surface of blood-superfused collagen strips or on the surface of blood-superfused aortic strips from atherosclerotic rabbits. The reversal of platelet aggregation by prostacyclin was still achieved 3 hrs after the formation of platelet clumps. After an intravenous injection of prostacyclin the ID50 for its de-aggregatory action was 7.5 microgram/kg. Theophylline ethyl-diamine (aminophylline), at a dose of 3 mg/kg i.v., did not reverse platelet aggregation but it enhanced the duration of the de-aggregatory action of prostacyclin; it had little effect on the hypotensive action of prostacyclin. It is concluded that prostacyclin disintegrates platelet clumps long after they are formed in heparinized blood in vivo and that its anti-platelet action, but not hypotensive action, is selectively potentiated by a phosphodiesterase inhibitor. The above experimental data indicate the possibility of the combined use of theophylline and prostacyclin in arterial thrombosis.     

De-aggregatory action of prostacyclin and its enhancement by theophylline

In the mixed venous blood of anaesthetized, heparinized cats prostacyclin de-aggregated platelet thrombi, which were formed on the surface of blood-superfused collagen strips or on the surface of blood-superfused aortic strips from atherosclerotic rabbits. The reversal of platelet aggregation by prostacyclin was still achieved 3 hrs after the formation of platelet clumps. After an intravenous injection of prostacyclin the ID₅₀ for its de-aggregatory action was 7.5 μg/kg. Theophylline ethyldiamine (aminophylline), at a dose of 3 mg/kg i.v., did not reverse platelet aggregation but it enhanced the duration of the de-aggregatory action of prostacyclin; it had little effect on the hypotensive action of prostacyclin. It is concluded that prostacyclin disintegrates platelet clumps long after they are formed in heparinized blood and that its anti-platelet action, but not hypotensive action, is selectively potentiated by a phosphodiesterase inhibitor. The above experimental data indicate the possibility of the combined use of theophylline and prostacyclin in arterial thrombosis.     

De-aggregatory action of prostacyclin in vivo and its enhancement by theophylline

In the mixed venous blood of anaesthetized, heparinized cats prostacyclin de-aggregated platelet thrombi, which were formed on the surface of blood-superfused collagen strips or on the surface of blood-superfused aortic strips from atherosclerotic rabbits. The reversal of platelet aggregation by prostacyclin was still achieved 3 hrs after the formation of platelet clumps. After an intravenous injection of prostacyclin the ID₅₀ for its de-aggregatory action was 7.5 μg/kg. Theophylline ethyldiamine (aminophylline), at a dose of 3 mg/kg i.v., did not reverse platelet aggregation but it enhanced the duration of the de-aggregatory action of prostacyclin; it had little effect on the hypotensive action of prostacyclin. It is concluded that prostacyclin disintegrates platelet clumps long after they are formed in heparinized blood $\text{in vivo}$ and that its anti-platelet action, but not hypotensive action, is selectively potentiated by a phosphodiesterase inhibitor. The above experimental data indicate the possibility of the combined use of theophylline and prostacyclin in arterial thrombosis.     

The effect of aspirin and two nitric oxide donors on the infarcted heart in situ

Nitric oxide (NO) donors are heterogeneous substances which release NO, a biologically active compound. NO released by nitric oxide donors has important effects on the circulation by causing vasodilation, diminishing myocardial contractile force, inhibiting platelet aggregation, and counteracting the effects of thromboxane A2. In the infarcted heart, activation of the inducible form of nitric oxide synthase (iNOS) and the formation of prostacyclin and thromboxane A2 by cyclooxygenase (COX) were increased. Myocardial infarction also resulted in increased myocardial NO production. Aspirin (acetylsalicylic acid. ASA) at low concentration (35 mg/kg/day) fails to change iNOS production, in contrast to higher dose (150 mg/kg/day) which, as previously shown, inhibits iNOS activity. ASA at all doses also suppresses myocardial prostanoid formation because of inhibition of COX. Recently, two NO donors have been synthesized: NCX 4016 and Diethylenetriamine/NO (DETA/NO). NCX 4016 combines an NO-releasing moiety with a carboxylic residue via an esteric bond. We describe here that NCX 4016 (65 mg/kg/day) increased prostacyclin and thromboxane A2 production in the infarcted heart muscle, overcoming the inhibitory effects of ASA. As a result of nitric oxide release, oxidation products of NO (NO2- and NO3-; NOx) in arterial blood rose following administration of NCX 4016. On oral administration, NCX 4016 did not change systemic arterial pressure. The effects of a single NO donor, DETA/NO (1.0 mg/kg/day) on the infarcted heart were also investigated On intravenous administration, the compound increased NO concentration in arterial blood slightly but to a lesser degree than NCX 4016. Like NCX 4016, it raised myocardial production of prostacyclin and thromboxane A2 in the infarcted heart. However, it caused a severe fall in blood pressure. These findings demonstrate that newly-synthesized NO donors release nitric oxide in situ and increase myocardial production of prostanoids. NCX 4016 has therapeutic potential because it can be orally administered, lacks hypotensive effects, increases blood levels of nitric oxide and myocardial prostacyclin production.     

Kallikrein-kinin system in hepatic experimental models

The purpose of this brief review is to describe some characteristics of the kallikrein-kinin system (KKS) in the liver. The liver synthesizes kininogens and prekallikrein and the synthesis of both proteins is increased in rats during the acute phase reaction. It is also the main organ to clear tissue as well as plasma kallikrein from the circulation in normal and pathological conditions. Bradykinin (BK), yielded by the kallikrein-kinin system, is a potent arterial hypotensive peptide, but in the liver it induces a portal hypertensive response. The portal hypertensive action of bradykinin is mediated by B2 receptors located on sinusoidal cells of the periportal region and is followed by its hydrolysis by angiotensin-converting enzyme, which is primarily present in the perivenous (centrolobular) region.     

A stable prostacyclin-like substance produced by the medicinal leech Hirudo medicinalis.

The medicinal leech Hirudo medicinalis produces a low-molecular mass compound with properties similar to those of prostacyclin. It extracted with organic solvent, had affinity to 6-keto-PGF1alpha antibodies, inhibited human platelet aggregation induced in vitro by thrombin (by 50% at 4 pg/ml), and caused hypotension and secretion of plasminogen (t-PA) into the blood stream of rats. A main distinction from prostacyclin is stability of the substance due to covalent binding with the polypeptide chain of destabilase. Because of the high aggregability of destabilase, the molecules of the protein-lipid complex are organized into micelles that can change their spatial orientation depending on the nature of the solvent. Incorporation of hirudin and blood plasma kallikrein inhibitor into the micelle structure causes the formation of liposomes (with a molecular mass of the structural monomer 25 kDa). This complex with polypeptides provides not only stability but also rapid transmembrane penetration. The pure prostacyclin-like substance has a molecular mass of 391 Da and can be produced on destruction of the destabilase polypeptide chain.     

Pharmacological comparison of bPTH-(1–34) and other hypotensive peptides in the dog

The purpose of the present study was to compare the potency, effectiveness and duration of action of synthetic bPTH-(1–34) with those of other known hypotensive peptides in the anesthetized dog. Of sixteen peptides tested in the present study only 8 were demonstrated to possess hypotensive activity. While bPTH-(1–34) was one of the least potent of the hypotensive peptides, it was equal to or greater than the other peptides in terms of effectiveness and duration of action. Of all the peptides studied, substance P and eledoisin were the most potent in terms of their hypotensive action. It is suggested that perhaps substance P and eledoisin might act at a different site or through different mechanisms than do vasoactive intestinal peptide (V.I.P.), corticotropin inhibiting peptide (C.I.P.), neurotensin, xenopsin, bradykinin and bPTH-(1–34).     

Pharmacological comparison of bPTH-(1–34) and other hypotensive peptides in the dog

The purpose of the present study was to compare the potency, effectiveness and duration of action of synthetic bPTH-(1–34) with those of other known hypotensive peptides in the anesthetized dog. Of sixteen peptides tested in the present study only 8 were demonstrated to possess hypotensive activity. While bPTH-(1–34) was one of the least potent of the hypotensive peptides, it was equal to or greater than the other peptides in terms of effectiveness and duration of action. Of all the peptides studied, substance P and eledoisin were the most potent in terms of their hypotensive action. It is suggested that perhaps substance P and eledoisin might act at a different site or through different mechanisms than do vasoactive intestinal peptide (V.I.P.), corticotropin inhibiting peptide (C.I.P.), neurotensin, xenopsin, bradykinin and bPTH-(1–34).     

Effect of prostacyclin infusion on active and inactive renin release in the isolated perfused kidney

The effect of prostacyclin infusion into the renal artery of the isolated perfused hog kidney on the release of active and inactive renin was investigated. Infusion of prostacyclin at a rate of 0.1 μg/min resulted in a significant increase (p<0.01) in active renin and a significant fall (p<0.01) in inactive renin. Prostacyclin also increased urinary kallikrein excretion (p<0.05). The results indicate that the kidney secretes not only active renin but also inactive renin, and suggest that prostacyclin stimulates the conversion of inactive renin to the active form through the activation of the renal kallikrein system.     

Hypotensive activity of novokinin, a potent analogue of ovokinin(2-7), is mediated by angiotensin AT(2) receptor and prostaglandin IP receptor

Novokinin (Arg-Pro-Leu-Lys-Pro-Trp) is a potent hypotensive peptide previously designed based on the structure of ovokinin(2-7) (Arg-Ala-Asp-His-Pro-Phe), a vasorelaxing and hypotensive peptide derived from ovalbumin. Novokinin exhibited an affinity for the angiotensin AT(2) receptor (Ki=7.35 microM). Novokinin significantly lowered systolic blood pressure at a dose of 0.03 and 0.1 mg/kg after intravenous and oral administration, respectively, in spontaneously hypertensive rats (SHRs), and the hypotensive activity was blocked by PD123319, an antagonist of the AT(2) receptor. Novokinin lowered blood pressure in C57BL/6J mice after oral administration at a dose of 50 mg/kg. However, in AT(2) receptor-deficient mice, novokinin did not reduce blood pressure. These results demonstrate that the hypotensive activity of novokinin is mediated by the AT(2) receptor. The hypotensive activity of novokinin in SHRs was completely blocked by indomethacin and CAY10441, an inhibitor of cyclooxygenase and an antagonist of the prostaglandin IP receptor, respectively. These suggest that the hypotensive activity is mediated by prostacyclin and the IP receptor downstream of the AT(2) receptor.     

Kinins mediate kallikrein-induced endothelium-dependent relaxations in isolated canine coronary arteries.

ACE inhibitors elicit the release of endothelium-derived relaxing factors in perfused isolated canine arteries (Mombouli and Vanhoutte, J. Cardiovasc. Pharmacol. 1991, 18: 926-927); this action is antagonized by bradykinin-receptor antagonists suggesting that it is mediated by local kinin generation. The effects of exogenous tissular kallikrein (porcine) were examined in vitro in the isolated canine coronary artery. Isometric tension was measured in blood vessel rings (with and without endothelium) contracted with prostaglandin F2 alpha. The kallikrein elicited relaxations in rings with, but not in those without, endothelium. This response was augmented by the angiotensin converting enzyme inhibitor perindoprilat, and it was antagonized by the selective B2-kinin receptor antagonist HOE 140 and aprotinin, an inhibitor of tissular kallikrein. These data suggest that in the canine coronary artery, kallikrein causes relaxations that may be mediated by kinins generated from endogenous kininogens present in the vascular wall.     

Responses of the rat cardiovascular system to substance P,neurotensin and bombesin

The carotid arterial blood pressure and heart rate responses to intravenous injections of substance P, neurotensin and bombesin were compared in anaesthetized rats. In rats anaesthetized with urethane neurotensin produced only a fall in blood pressure but in rats anaesthetized with sodium thiobutabarbitone, the fall was preceded by a transient rise in blood pressure. The reason for the different responses to neurotensin with the two anaesthetics was not investigated. The hypotensive effect of neurotensin observed with both anaesthetics was abolished by mepyramine and therefore appeared to be mediated by action on H₁ receptors either of neurotensin directly or of histamine released. On the other hand, catecholamines might be implicated in the pressor response to neurotensin observed in rats anaesthetized with sodium thiobutabarbitone since it was reduced by phentolamine and hexamethonium. Low doses of substance P produced a depressor response which was not inhibited by the antagonists tested. At higher doses marked tachycardia occurred and the depressor response was less and was often followed by a pressor response. The tachycardia was abolished by propranolol but not by cervical cord section or by hexamethonium. Bombesin produced a pressor response which was unaffected by hexamethonium but was reversed to depressor by phentolamine. This depressor response to bombesin was abolished by propranolol. It was concluded that substance P produced a depressor response by action on its own specific receptors and tachycardia by catecholamine release whereas neurotensin and bombesin produced cardiovascular actions which were mediated entirely by amine release.     

Arachidonic acid is preferentially metabolized by cyclooxygenase-2 to prostacyclin and prostaglandin E2

The two cyclooxygenase isoforms, cyclooxygenase-1 and cyclooxygenase-2, both metabolize arachidonic acid to prostaglandin H2, which is subsequently processed by downstream enzymes to the various prostanoids. In the present study, we asked if the two isoforms differ in the profile of prostanoids that ultimately arise from their action on arachidonic acid. Resident peritoneal macrophages contained only cyclooxygenase-1 and synthesized (from either endogenous or exogenous arachidonic acid) a balance of four major prostanoids: prostacyclin, thromboxane A2, prostaglandin D2, and 12-hydroxyheptadecatrienoic acid. Prostaglandin E2 was a minor fifth product, although these cells efficiently converted exogenous prostaglandin H2 to prostaglandin E2. By contrast, induction of cyclooxygenase-2 with lipopol- ysaccharide resulted in the preferential production of prostacyclin and prostaglandin E2. This shift in product profile was accentuated if cyclooxygenase-1 was permanently inactivated with aspirin before cyclooxygenase-2 induction. The conversion of exogenous prostaglandin H2 to prostaglandin E2 was only modestly increased by lipopolysaccharide treatment. Thus, cyclooxygenase-2 induction leads to a shift in arachidonic acid metabolism from the production of several prostanoids with diverse effects as mediated by cyclooxygenase-1 to the preferential synthesis of two prostanoids, prostacyclin and prostaglandin E2, which evoke common effects at the cellular level.     

Synergistic effect of D-003 and aspirin on experimental thrombosis models

D-003 is a mixture of higher primary aliphatic saturated acids purified from sugarcane wax, with antiplatelet and antithrombotic effects experimentally demonstrated. Octacosanoic acid is the main component of D-003, followed by triacontanoic, dotriacontanoic, and tetracontanoic acids, while other acids are minor components. This work investigates the effects of combination therapy D-003+aspirin (ASA) on arachidonic acid (AA)-induced sudden death in mice and bleeding time in rats. In addition, the effects of D-003 on serum levels of two metabolites of AA: thromboxane A(2) and prostacyclin, assessed through the measurement of their stable metabolites: thromboxane B(2) (TxB(2)) and 6 keto PgF1alpha by radioimmunoassay kits, were also investigated. Combination therapy of D-003 (50mg/kg) and ASA (3mg/kg) significantly increased bleeding time in rats in a synergistic manner compared with D-003 or ASA alone. Moreover, the combined treatment of D-003 (200mg/kg) and ASA (5mg/kg) in mice protected against AA-induced sudden death (83% survivors) in a synergistic manner which was compared with each treatment alone (33% survivors). These results indicate that antiplatelet effects of D-003 are not mediated by a cyclooxygenase inhibition. D-003 and ASA monotherapies reduced serum TxB(2) levels, whereas D-003, but not ASA, significantly increased 6 keto PgF1alpha levels.     

Taprostene, a stable prostacyclin analogue, enhances the thrombolytic efficacy of saruplase (recombinant single-chain urokinase-type plasminogen activator) in rabbits with pulmonary embolized thrombi

Cooperative effects of the prostacyclin analogue taprostene and the thrombolytic agent saruplase (r-scu-PA) were studied in anesthetized rabbits with pulmonary thromboembolism. Thrombolysis was evaluated as decrease of the total weight and of the incorporated 125J-fibrin-radioactivity of the embolized thrombi. Saruplase (10.0-46.4 micrograms/kg.min, i.v.) produced dose-dependent lytic effects. Taprostene, infused in a dose (0.1 microgram/kg.min, i.v.) that inhibited ADP-induced decrease of circulating platelets by 56%, reduced the total thrombus weight (p less than 0.05 vs control) and in combination it further augmented the saruplase (21.5 micrograms/kg.min)-induced thrombolysis (p less than 0.05 vs saruplase alone). Taprostene did not increase the spontaneous lysis rate of the incorporated 125J-fibrin (7.3 +/- 1.4% vs 8.1 +/- 1.4%), but further enhanced the fibrinolytic effect of saruplase (37.2 +/- 5.6% saruplase vs 53.6 +/- 2.3% saruplase + taprostene; p less than 0.05). This overadditive synergism is tentatively ascribed to the platelet inhibition by the prostacyclin analogue that may facilitate the action of the thrombolytic agent. Taprostene lowered mean arterial blood pressure by 22% in anesthetized rabbits; it did not significantly modify the slight decrease of the plasma fibrinogen level (20-30%) by 21.5 micrograms/kg.min saruplase. The results show that the prostacyclin analogue taprostene reduces the total thrombus weight and enhances the efficacy of the thrombolytic agent saruplase in pulmonary thromboembolism in rabbits.     

Suppression of the voltage-gated K+ current of human megakaryocytes by thrombin and prostacyclin

We examined the effects of platelet activators and inhibitors of platelet function on the voltage-gated delayed rectifier K⁺ current of human megakaryocytes. We found that both the activators such as thrombin, the thrombin receptor peptide (TRP^42–47) and ADP and the inhibitors such as prostacyclin suppressed the delayed rectifier current through two different mechanisms. The cAMP dependent protein kinase (A-kinase) inhibitor IP₂₀ blocked the suppression of the delayed rectifier current by prostacyclin and failed to block the suppression by thrombin, TRP^42–47 and ADP. The effects of IP₂₀ suggest that the action of prostacyclin is mediated by A-kinase and the action of the three activators is not mediated by A-kinase. Pertussis toxin (PTX) an inhibitor of the inhibitory GTP-binding proteins (G_i) blocked the suppression of the delayed rectifier current by thrombin, TRP^42–47 and ADP and failed to block the suppression by prostacyclin. The effects of PTX suggests that the action of the three activators is mediated by G_i or some other PTX-sensitive GTP-binding protein. We speculate that thrombin and other platelet activators that activate G_i may be suppressing the delayed rectifier current via a direct interaction of G_i or a subunit of it with the delayed rectifier potassium channel itself.     

Role of the sympathetic nervous system in the alcohol-guanabenz hemodynamic interaction.

The present study evaluated the contribution of the sympathetic nervous system to the adverse hemodynamic action of ethanol on hypotensive responses in conscious unrestrained spontaneously hypertensive rats. Ethanol caused a dose-related attenuation of the hypotensive effect of guanabenz. An equivalent hypotensive response to sodium nitroprusside was not influenced by ethanol, which indicates a potential specific interaction between ethanol and guanabenz. Alternatively, it is possible that a preexisting high sympathetic nervous system activity, which occurred during nitroprusside infusion, may mask a sympathoexcitatory action of ethanol. Further, ethanol (1 g/kg) failed to reverse the hypotensive effect of the ganglionic blocker hexamethonium. This suggests that a centrally mediated sympathoexcitatory action of ethanol is involved, at least partly, in the reversal of hypotension. In addition, the antagonistic interaction between ethanol and guanabenz seems to take place within the central nervous system and involves opposite effects on central sympathetic tone. Finally, changes in plasma catecholamines provide supportive evidence for the involvement of the sympathetic nervous system in this interaction. In a separate group of conscious spontaneously hypertensive rats, ethanol (1 g/kg) reversed the guanabenz-evoked decreases in blood pressure and plasma catecholamine levels. It is concluded that (i) ethanol adversely interacts with centrally acting antihypertensive drugs through a mechanism that involves a directionally opposite effect on sympathetic activity, and (ii) a sympathetically mediated pressor effect of ethanol is enhanced in the presence of an inhibited central sympathetic tone.     

A potent hypotensive peptide, novokinin, induces relaxation by AT2- and IP-receptor-dependent mechanism in the mesenteric artery from SHRs

In this study, we found that novokinin (Arg-Pro-Leu-Lys-Pro-Trp), a potent hypotensive peptide acting through the AT(2) receptor, has vasorelaxing activity in the mesenteric artery isolated from spontaneously hypertensive rats. The vasorelaxing activity was significantly blocked by PD123319, indomethacin, and CAY10441, which are an AT(2) receptor antagonist, a cyclooxygenase inhibitor, and an IP receptor antagonist, respectively. N(G)-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthase, did not block the vasorelaxing activity. These results suggest that the vasorelaxing activity of novokinin, which contributes to the hypotensive effect, is mainly mediated by prostaglandin I(2) (prostacyclin) and the IP receptor downstream of the AT(2) receptor.     

Tissue kallikrein and bradykinin do not have direct insulin-like actions on skeletal muscle glucose utilization

Studies suggest that the actions of insulin on glucose metabolism may be mediated through activation of a membrane-bound serine protease with properties similar to a kallikrein-like enzyme. Also, bradykinin, a vasoactive product of kallikrein's action upon kininogen substrates, increases glucose uptake when infused into the human forearm. To determine whether a kallikrein or a kinin directly affects cellular glucose metabolism or participates in mediating insulin's actions, we studied their effects on isolated rat soleus muscle. Although trypsin (1.34 microM) increased incorporation of glucose into muscle glycogen to the same extent as insulin (200 mu units/ml), a purified rat tissue (urinary) kallikrein (0.4-1.34 microM) produced no such effect. Furthermore, the tissue kallikrein inhibitor, aprotinin, or a polyclonal kallikrein antiserum did not inhibit the action of insulin on incorporation of glucose into muscle glycogen. Treatment of the muscle preparation with bradykinin (1nM - 10 microM) did not result in any change in basal or insulin-stimulated (20 - 2000 mu units/ml) entry of glucose into glycogen or the glycolytic pathway. Bradykinin (1nM - 10 microM) also did not influence basal or insulin-stimulated (1000 mu units/ml) initial rates of glucose transport. These studies suggest that the previously observed in vivo effects of bradykinin on peripheral glucose uptake are probably mediated by changes in tissue perfusion rather than direct kinin effects on skeletal muscle, and that the putative membrane serine protease involved in the insulin-effector system is not tissue kallikrein.