Potentiation of bradykinin effects and inhibition of kininase activity in isolated smooth muscle.

Prolongation of bradykinin half-life following kininase inhibition has been proposed as the reason for the potentiation of kinin effects. We have reassessed this assumption by using three different isolated smooth muscle preparations and simultaneously studying the inhibition of kininase activity and the potentiation of bradykinin effects by enalaprilat and BPP9a. Rat duodenum displayed higher total kininase activity, metabolizing half of the added bradykinin in 6.5 min, while this time for rat uterus was greater than 60 min. Guinea-pig ileum showed the intermediate value of 14.6 min. Enalaprilat and BPP9a slowed the metabolism of bradykinin by 50-100% in rat duodenum and by 50-180% in guinea-pig ileum, showing that a significant fraction of total kininase activity appears to be due to kininase II. In rat duodenum, an almost complete blockade of kininase activity was achieved when bacitracin and mergetpa were used together with enalaprilat. Enalaprilat and BPP9a potentiated bradykinin effects in guinea-pig ileum and rat uterus. In contrast, bradykinin-induced relaxations and contractions in rat duodenum were not potentiated by enalaprilat, BPP9a, or by the enzyme inhibitor mixture (enalaprilat--bacitracin--mergetpa). The results suggest that inhibition of bradykinin enzymatic metabolism by kininases does not necessarily lead to the potentiation of bradykinin effects.     

Potentiation of bradykinin actions by analogues of the bradykinin potentiating nonapeptide BPP9alpha

Synthetic analogues of the bradykinin potentiating nonapeptide BPP9alpha indicate significantly different structural requirements for potentiation of the bradykinin (BK)-induced smooth muscle contraction (GPI) and the inhibition of isolated somatic angiotensin I-converting enzyme (ACE). The results disprove the ACE inhibition as the only single mechanism and also the direct interaction of potentiating peptides with the bradykinin receptors in transfected COS-7 cells as molecular mechanism of potentiation. Our results indicate a stimulation of inositol phosphates (IPn) formation independently from the B2 receptor. Furthermore, the results with La3+ support the role of extracellular Ca2+ and its influx through corresponding channels. The missing effect of calyculin on the GPI disproves the role of phosphatases in the potentiating action. These experimental studies should not only contribute to a better understanding of the potentiating mechanisms but also incorporate a shift in the research towards the immune system, in particular towards the immunocompetent polymorphonuclear leukocytes. The chemotaxis of these cells can be potentiated most likely by exclusive inhibition of the enzymatic degradation of bradykinin. Thus the obtained results give evidence that the potentiation of the bradykinin action can occur by different mechanisms, depending on the system and on the applied potentiating factor.     

Effects of intradermal bradykinin after inhibition of angiotensin converting enzyme.

Inhibitors of angiotensin converting enzyme may cause angio-oedema. To see if this might be due to potentiation of the tissue effects of bradykinin the thickness of weals raised by intradermal injection of saline or 1, 3, or 10 micrograms bradykinin was measured before and three times after single doses of captopril, enalapril, or placebo. The mean thickness increased with increasing doses of bradykinin. It did not change with time after the administration of placebo or captopril but increased from 0.61 mm before enalapril to 1.12 mm two and a half hours and 1.06 mm five hours after enalapril was given. Five subjects flushed when given bradykinin after captopril and four after enalapril, but none flushed when given bradykinin after placebo. It is concluded that angiotensin converting enzyme inhibitors potentiate the effects of intradermal bradykinin in vivo and that this may partially explain why they cause angio-oedema in susceptible patients.     

Serotonin, bradykinin and endothelin signalling in a sheep choroid plexus cell line

The secretion of cerebrospinal fluid by the epithelial cells of choroid plexus is regulated by membrane receptors coupled to adenylyl cyclases or to phospholipase C. These intracellular signalling pathways as their interactions were investigated in a sheep choroid plexus cell line. Endothelin-1, bradykinin and serotonin induced a transient dose-dependent increase in intracellular calcium. EC 50 were 10(-8) M for endothelin-1, 10(-8) M for bradykinin and 10(-6) M for serotonin. Maximal increase in intracellular calcium was comparable for bradykinin and serotonin, but was 3 to 5 fold larger for endothelin-1. Successive stimulations with endothelin-1, serotonin or bradykinin elicited calcium increases similar to single stimulations reflecting absence of heterologous desensitization between these receptors. Forskolin-induced cAMP accumulation was potentiated by bradykinin, but not by serotonin and endothelin-1. This potentiation resulted from an increase in cAMP production rather than to an inhibition of cAMP hydrolysis. These data suggest that serotonin, endothelin-1 and bradykinin each use specific signalling pathways in the sheep choroid plexus cells.     

Metalloendopeptidases EC 3.4.24.15/16 regulate bradykinin activity in the cerebral microvasculature

Bradykinin is a vasoactive peptide that has been shown to increase the permeability of the cerebral microvasculature to blood-borne macromolecules. The two zinc metalloendopeptidases EC (EP 24.15) and EC (EP 24.16) degrade bradykinin in vitro and are highly expressed in the brain. However, the role that these enzymes play in bradykinin metabolism in vivo remains unclear. In the present study, we investigated the role of EP 24.15 and EP 24.16 in the regulation of bradykinin-induced alterations in microvascular permeability. Permeability of the cerebral microvasculature was assessed in anesthetized Sprague-Dawley rats by measuring the clearance of 70-kDa FITC dextran from the brain. Inhibition of EP 24.15 and EP 24.16 by the specific inhibitor N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Aib-Tyr-p-aminobenzoate (JA-2) resulted in the potentiation of bradykinin-induced increases in cerebral microvessel permeability. The level of potentiation was comparable to that achieved by the inhibition of angiotensin-converting enzyme. These findings provide the first evidence of an in vivo role for EP 24.15/EP 24.16 in brain function, specifically in regulating alterations in microvessel permeability induced by exogenous bradykinin.     

Kinins, receptors, kininases and inhibitors--where did they lead us?

Based on studies presented here and other published experiments performed with surviving tissue preparations, with transfected cells and with cells that constitutively express the human angiotensin I converting enzyme ACE and B2 receptors, we concluded the following: ACE inhibitors and other endogenous peptides that react with the active site of ACE potentiate the effect of bradykinin and its ACE resistant peptide congeners on the B2 receptor. They also resensitize receptors which had been desensitized by the agonist. ACE and bradykinin receptors have to be sterically close, possibly forming a heterodimer, for the ACE inhibitors to induce an allosteric modification on the receptor. When ACE inhibitors augment bradykinin effects, they reduce the phosphorylation of the B2 receptor. The primary actions of bradykinin on the receptor are not affected by protein kinase C or phosphatase inhibitors, but the potentiation of bradykinin or the resensitization of the receptor by ACE inhibitors are abolished by the same inhibitors. The results with protein kinase C and phosphatase inhibitors indicate that another intermediate protein may be involved in the processes of signaling induced by ACE inhibitors, and that ACE inhibitors affect the signal transduction pathway triggered by bradykinin on the B2 receptor.     

A potent mercapto bi-product analogue inhibitor for human carboxypeptidase N

The bi-product analogue inhibitor, 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, has been synthesized in high yield and exhibits a K_i of 2.0 nM with human plasma carboxypeptidase N. The ease of synthesis and subsequent availability make it an ideal compound to study potentiation of bradykinin and other vasoactive peptides.     

Effects of bradykinin on PC-12 cell differentiation

PC-12 cells are used as a model for neuronal differentiation because they assume a neuronal phenotype, including the extension of neurites, when exposed to nerve growth factor (NGF). The present results show that bradykinin (BK) also causes PC-12 cells to extend neurites. In addition, BK potentiates the neurite-extending effect of nerve growth factor (NGF), an action which is attenuated by a BK antagonist. The potentiation of neurite extension produced by the combination of BK and NGF may be mediated at the receptor level, as indicated by an NGF-induced alteration of BK binding.     

Cloning of bradykinin precursor cDNAs from skin of Bombina maxima reveals novel bombinakinin M antagonists and a bradykinin potential peptide

Bombinakinin M (DLPKINRKGP-bradykinin) is a bradykinin-related peptide purified from skin secretions of the frog Bombina maxima. As previously reported, its biosynthesis is characterized by a tandem repeats with various copy numbers of the peptide and sometimes co-expressed with other structure-function distinguishable peptides. At present study, two novel cDNAs encoding bombinakinin M and its variants were cloned from a cDNA library from the skin of the frog. The encoded two precursor proteins are common in that each contains three repeats of a novel 16-amino acid peptide unit and one copy of kinestatin at their N- and C-terminal parts, respectively. They differ in that the first precursor contains two copies of bombinakinin M and the second one contains one copy of a novel bombinakinin M variant. Bombinakinin M was found to elicit concentration-dependent contractile effects on guinea pig ileum, with an EC50 value of 4 nM that is four times higher than that of bradykinin (1 nM). Interestingly, the synthetic peptide (DYTIRTRLH-amide), as deduced from the 16-amino acid peptide repeats in the newly cloned cDNAs, possessed weak inhibitory activity on the contractile effects of bombinakinin M, but not on that of bradykinin. Furthermore, the newly identified bombinakinin M variant (DLSKMSFLHG-Ile1-bradykinin), did not show contractile activity on guinea pig ileum, but showed potentiation effect on the myotropic activity of bradykinin. In a molar ratio of 1:58, it augmented the activity of bradykinin up to two-fold.     

Cyclic GMP Regulates Nicotine-Induced Secretion from Cultured Bovine Adrenal Chromaffin Cells: Effects of 8–Bromo–Cyclic GMP, Atrial Natriuretic Peptide, and Nitroprusside (Nitric Oxide)

Methacholine, atrial natriuretic peptide (ANP), nitroprusside (nitric oxide), angiotensin II, and bradykinin raised cyclic GMP (cGMP) levels in cultured bovine adrenal chromaffin cells. The role of cGMP in secretion from chromaffin cells was examined using 8-bromo-cGMP. This analogue had no effect on basal secretion or secretion due to angiotensin II, bradykinin, or a high K+ level but potentiated secretion due to low doses of nicotine. At supramaximal doses of nicotine, 8-bromo-cGMP inhibited secretion. These effects of 8-bromo-cGMP were not due to changes in the nicotine-induced rise in cytosolic calcium concentration. A potentiation of secretion due to low doses of nicotine was also found following simultaneous addition of ANP or nitroprusside, a result suggesting that ANP and nitric oxide (endothelium-derived relaxing factor) could be important regulators of secretion from adrenal chromaffin cells.     

A possible alternative mechanism of kinin generation in vivo by cathepsin L

We investigated the ability of cathepsin L to induce a hypotensive effect after intravenous injection in rats and correlated this decrease in blood pressure with kinin generation. Simultaneously with blood pressure decrease, we detected plasma kininogen depletion in the treated rats. The effect observed in vivo was abolished by pre-incubation of cathepsin L with the cysteine peptidase-specific inhibitor E-64 (1 microM) or by previous administration of the bradykinin B2 receptor antagonist JE049 (4 mg/kg). A potentiation of the hypotensive effect caused by cathepsin L was observed by previous administration of the angiotensin I-converting enzyme inhibitor captopril (5 mg/kg). In vitro studies indicated that cathepsin L excised bradykinin from the synthetic fluorogenic peptide Abz-MTSVIRRPPGFSPFRAPRV-NH2, based on the Met375-Val393 sequence of rat kininogen (Abz = o-aminobenzoic acid). In conclusion, our data indicate that in vivo cathepsin L releases a kinin-related peptide, and in vitro experiments suggest that the kinin generated is bradykinin. Although it is well known that cysteine proteases are strongly inhibited by kininogen, cathepsin L could represent an alternative pathway for kinin production in pathological processes.     

Bradykinin,angiotensin-(1-7), and ACE inhibitors: how do they interact?

The beneficial effect of ACE inhibitors in hypertension and heart failure may relate, at least in part, to their capacity to interfere with bradykinin metabolism. In addition, recent studies have provided evidence for bradykinin-potentiating effects of ACE inhibitors that are independent of bradykinin hydrolysis, i.e. ACE-bradykinin type 2 (B(2)) receptor 'cross-talk', resulting in B(2) receptor upregulation and/or more efficient activation of signal transduction pathways, as well as direct activation of bradykinin type 1 receptors by ACE inhibitors. This review critically reviews the current evidence for hydrolysis-independent bradykinin potentiation by ACE inhibitors, evaluating not only the many studies that have been performed with ACE-resistant bradykinin analogues, but also paying attention to angiotensin-(1-7), a metabolite of both angiotensin I and II, that could act as an endogenous ACE inhibitor. The levels of angiotensin-(1-7) are increased during ACE inhibition, and most studies suggest that its hypotensive effects are mediated in a bradykinin-dependent manner.     

Nerve Growth Factor Potentiates Bradykinin-Induced Calcium Influx and Release in PC12 Cells

To investigate how the response to agonists changes during neuronal differentiation, we examined the effect of nerve growth factor (NGF) on bradykinin-induced calcium increases in PC12 cells. Short-term (1 h) treatment with NGF increased the potency of bradykinin to raise intracellular calcium by about 10-fold, whereas long-term (1 week) treatment, which was associated with the expression of the differentiated phenotype, increased the potency about 100-fold. Neither treatment affected the maximal response to bradykinin. NGF alone had no acute effect on calcium levels. Short-term potentiation appeared to be mainly a result of greater release of calcium from intracellular stores, whereas the effect of long-term treatment apparently was due to increases in both release from intracellular stores and calcium influx. [3H]Bradykinin binding to intact PC12 cells was unaltered by short-term NGF treatment, whereas differentiated cells displayed a 50% increase in receptor number and about a twofold increase in affinity as compared with cells not treated with NGF. The production of inositol phosphates in response to bradykinin correlated poorly with the calcium transients, in that large calcium responses were associated with small increases in inositol phosphates. Neither NGF treatment had a significant effect on the appearance of inositol phosphates in response to bradykinin. Experiments with permeabilized cells revealed that differentiated cells did not display a heightened response to exogenously added inositol 1,4,5-trisphosphate. Our results demonstrate that NGF modulates the bradykinin signaling pathway without acutely activating this pathway itself.     

Neuroligand-Evoked Calcium-Dependent Release of Excitatory Amino Acids from Cultured Astrocytes

Abstract: The release of excitatory amino acids (EAAs) from neuron-free cultures of neocortical astrocytes was monitored using HPLC. The neuroligand bradykinin caused a dose-dependent receptor-mediated increase in release of the EAAs glutamate and aspartate from type 1 astrocyte cell cultures obtained from rat cerebral cortex. Removal of calcium from the extracellular fluid prevented the bradykinin-induced release of EAAs from astrocytes. The addition of the calcium ionophore ionomycin caused a calcium-dependent release of EAAs. Inhibitors of the glutamate transporters p -chloromercuriphenylsulfonic acid, l - trans -pyrrolidine-2,4-dicarboxylate, and dihydrokainate failed to impair the ability of bradykinin to stimulate glutamate release from astrocytes. α-Latrotoxin, an active compound of black widow spider venom, caused a significant increase of the release of glutamate in calcium-containing saline. In calcium-depleted saline, α-latrotoxin produced an initial increase in the concentration of glutamate followed by a decline in the concentration of glutamate indicating stimulation of exocytosis coupled with low calcium-induced inhibition of endocytosis. Taken together, these data suggest that astrocytes may release neurotransmitter through a mechanism that is similar to the neuronal secretory process. Given the important role of glutamate in the induction of long-term potentiation, learning, memory, and excitotoxicity, it will be important to determine external signals that control both the uptake and release of glutamate by astrocytes.     

Neuropeptide tyrosine (NPY)-induced potentiation of the pressor activity of catecholamines in conscious rats

IV bolus administration of 2.5-50 micrograms NPY (0.6-12.5 nmol) to conscious rats produced a dose- and time-dependent increase in systolic and diastolic blood pressure. Following priming with 2.5 micrograms NPY, or larger doses, the subsequent administrations of noradrenaline produced pressor responses that were potentiated both in magnitude and duration. The NPY-induced potentiation of the pressor response to noradrenaline was dose-dependent and extended to the pressor action of adrenaline and angiotensin II but not to the hypotensions produced by bradykinin or isoproterenol. The potentiation was not related to the fact that multiple doses of catecholamines were repeated. Reserpine did not substantially modify the NPY-induced potentiation of the pressor activity of the catecholamines. Chemical sympathectomy following 6-hydroxydopamine caused a marked supersensitivity to the catecholamines and NPY but obliterated the NPY-induced potentiation of the pressor effect of adrenaline. Nifedipine reduced the pressor action of the catecholamines and NPY but did not attenuate the NPY-induced potentiation of the pressor action of catecholamines. It is concluded that the acute pressor effect of NPY and of the potentiation of the catecholamine pressor effects involve different mechanisms.     

Different effect of the ''recombinant'' bradykinin on the contractile activity of cultured atrial and ventricular cardiomyocytes

The physiological activity of the "recombinant" bradykinin expressed by retrovirus recombinant pPS-3-neo (brd) was tested on cultural atrial (aCMC) and ventricular (vCMC) cardiomyocytes in newborn rats. The "recombinant" bradykinin was shown to have a chronotropic effect on aCMC and an inotropic effect on vCMC. The effects are in line with the action of the synthetic bradykinin preparation at a concentration of around 10(-15) M. A pretreatment of CMC by parmidine, i.e. a bradykinin antagonist, blocked the effect of bradykinin. The contractive CMC activity in the cultural cell medium, transferred by pPS-3-neo without the bradykinin gene, was not different from the control value.     

Regulation of bradykinin-induced phosphoinositide turnover in cultured cerebellar astrocytes: possible role of protein kinase C

Phosphoinositide hydrolysis was studied in primary cultures of rat cerebellar astrocytes pre-labeled with [³H]myo-inositol. Among the agonists examined, the rank order of efficacies in causing phosphoinositide hydrolysis was bradykinin > endothelin-1 > ATP > norepinephrine. The bradykinin response was robust (24-fold increase) with EC₅₀ value of 30 nM and saturating concentration of 1 μM. Preincubation of cells with pertussis toxin did not affect the activation of phosphoinositide turnover by bradykinin. Although short-term (within 90 min) treatment of cells with phorbol dibutyrate attenuated bradykinin-induced phosphoinositide breakdown, the inhibitory effect was lost after 3–6 h of phorbol dibutyrate treatment. Extended (24 h) preincubation resulted in a potentiation of bradykinin response. Homologous desensitization of bradykinin response was observed in cells prestimulated with bradykinin for up to 6 h. However, similar to the effect of phorbol dibutyrate. 24-h pretreatment with bradykinin selectively sensitized the response to bradykinin. Up-regulation of the bradykinin response was also observed in cells prestimulated with endothelin-1 or norepinephrine for 24 h, although these treatments resulted in only homologous desensitization to their own response. Our results suggest that cultured cerebellar astrocytes express bradykinin receptors coupled to phospholipase C and in these cells protein kinase C plays a more prominent role in the negative-feedback regulation of bradykinin-evoked phosphoinositide response.     

Dose-dependent effect of bradykinin on muscular blood flow and glucose uptake in man

Using the forearm technique, the effect of bradykinin on muscular blood flow and glucose uptake in healthy man in the postabsorptive state (n = 8) was studied at different doses of an intra-arterial infusion of bradykinin (2.5-150 ng/min). The blood flow of the forearm was increased dose-dependently from basal 2.8 +/- 0.3 up to 14.7 +/- 2.8 ml/(100 g X min). At lower bradykinin concentrations (2.5-25 ng/min), muscular glucose uptake was raised parallel to the increased blood flow from basal 0.71 +/- 0.30 to 2.93 +/- 0.50 mumol/(100 g X min). However, at higher doses (50-150 ng/min) glucose uptake was decreased again. Thus, the greatest metabolic effect of bradykinin was seen at a calculated bradykinin concentration of approximately 1 X 10(-9)M in the blood.     

Stimulation by Bradykinin, Angiotensin II, and Carbachol of the Accumulation of Inositol Phosphates in PC-12 Pheochromocytoma Cells: Differential Effects of Lithium Ions on Inositol Mono-and Polyphosphates

Rat PC-12 pheochromocytoma cells respond to stimulation with bradykinin, angiotensin II, and carbachol with an increased formation of labeled inositol phosphates after preincubation of the cells with [3H]inositol. Li+ potentiates greatly the agonist-induced increase in amount of inositol mono-, bis-, and trisphosphate but not the increase in amount of inositol tetrakisphosphate. Separation of the isomers of inositol trisphosphate shows that the lithium-induced increase in amount of inositol trisphosphate is due to potentiation evoked by lithium of the accumulation of inositol-1,3,4-trisphosphate.     

Nerve Growth Factor Potentiates the Hormone-Stimulated Intracellular Accumulation of Inositol Phosphates and Ca2+ in Rat PC12 Pheochromocytoma Cells: Comparison with the Effect of Epidermal Growth Factor

The effects of nerve growth factor (NGF) and epidermal growth factor (EGF) on the intracellular accumulation of inositol phosphates and on cytosolic free Ca2+ concentrations were studied in rat PC12 pheochromocytoma cells. Both NGF and EGF potentiate in these cells the increase in the accumulation of inositol phosphates that is elicited by bradykinin and carbachol. A corresponding potentiation was also found for the agonist-induced increase of cytosolic Ca2+ concentrations. The effect of NGF, but not that of EGF, is abolished when the cells are preincubated with 5'-deoxy-5'-methylthioadenosine, an inhibitor of S-adenosylhomocysteine hydrolase. These results suggest that an increased response to hormones, which act via phosphoinositide-derived second messengers, may be important in the mechanism of action of NGF and EGF.