Inhibitory Effect Of Bradykinin Research Articles

Surface megalin expression is a target to the inhibitory effect of bradykinin on the renal albumin endocytosis

Megalin-mediated albumin endocytosis plays a critical role in albumin reabsorption in proximal tubule (PT) epithelial cells (PTECs). Some studies have pointed out the modulatory effect of bradykinin (BK) on urinary protein excretion, but its role in PT protein endocytosis has not yet been determined. Here, we studied the possible correlation between BK and albumin endocytosis in PT. Using LLC-PK1 cells, a model of PTECs, we showed that BK specifically inhibited megalin-mediated albumin endocytosis. This inhibitory effect of BK was mediated by B2 receptor (B2R) because it was abolished by HOE140, an antagonist of B2R, but it was not affected by Lys-des-Arg9-BK, an antagonist of B1. BK induced the stall of megalin in EEA1+ endosomes, but not in LAMP1+ lysosomes, leading to a decrease in surface megalin expression. In addition, we showed that BK, through B2R, activated calphostin C-sensitive protein kinase C, which mediated its effect on the surface megalin expression and albumin endocytosis. These results reveal an important modulatory mechanism of PT albumin endocytosis by BK, which opens new possibilities to understanding the effect of BK on urinary albumin excretion.

Differential fading of inhibitory and excitatory B2 bradykinin receptor responses in rat sympathetic neurons: a role for protein kinase C

Through inhibitory and excitatory effects on sympathetic neurons, B(2) bradykinin receptors contribute to protective and noxious cardiovascular mechanisms. Presynaptic inhibition of sympathetic transmitter release involves an inhibition of Ca(V)2 channels, neuronal excitation an inhibition of K(V)7 channels. To investigate which of these mechanisms prevail over time, the respective currents were determined. The inhibition of Ca(2+) currents by bradykinin reached a maximum of 50%, started to fade within the first minute, and became attenuated significantly after > or = 4 min. The inhibition of K(+) currents reached a maximum of 85%, started to fade after > 3 min, and became attenuated significantly after > or = 7 min. Blocking Ca(2+)-independent protein kinase C (PKC) enhanced the inhibition of Ca(2+) currents by bradykinin and delayed its fading, left the inhibition of K(+) currents and its fading unaltered, and enhanced the reduction of noradrenaline release and slowed its fading. Conversely, direct activation of PKC abolished the inhibition of noradrenaline release and largely attenuated the inhibition of Ca(2+) currents. These results show that the inhibitory effects of bradykinin in sympathetic neurons are outweighed over time by its excitatory actions because of more rapid, PKC-dependent fading of the inhibitory response.

Rhythmic Expression of Adenylyl Cyclase VI Contributes to the Differential Regulation of Serotonin N-Acetyltransferase by Bradykinin in Rat Pineal Glands

The rhythmic nocturnal production of melatonin in pineal glands is controlled by the periodic release of norepinephrine from the superior cervical ganglion. Norepinephrine binds to the beta-adrenergic receptor and stimulates an increase in intracellular cAMP levels, leading to the transcriptional activation of serotonin N-acetyltransferase, which in turn promotes melatonin production. In the present study, we report that bradykinin inhibits basal- and forskolin-stimulated adenylyl cyclase activity, norepinephrine-induced cAMP generation, and N-acetyltransferase expression in a calcium-dependent manner. These effects were blocked by pretreatment with U73122 (a selective phospholipase C inhibitor), and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (a Ca(2+) chelator), but not pertussis toxin. The calcium ionophore, ionomycin, inhibited isoproterenol-mediated cAMP generation, similar to bradykinin. Interestingly, the inhibitory effect of bradykinin was evident only during the daytime. At midday, bradykinin inhibited the cAMP level by approximately 50% but markedly stimulated cAMP production (by approximately 50%) at night. Northern blotting and immunoblotting data disclosed circadian expression of calcium-inhibitable adenylyl cyclase type 6. Expression of adenylyl cyclase type 6 was maximal at Zeitgeber Time (ZT) 01 and very low at ZT 13. Our results suggest that bradykinin-induced calcium inhibits melatonin synthesis through the mediation of adenylyl cyclase type 6 expression.

Tyrosine Kinase and Mitogen-Activated Protein Kinase/Extracellularly Regulated Kinase Differentially Regulate Intracellular Calcium Concentration Responses to Angiotensin II/III and Bradykinin in Rat Cortical Thick Ascending Limb

The cortical thick ascending limb (CTAL) coexpresses angiotensin (Ang) II/Ang III receptor type 1A (AT(1A)-R) and bradykinin (BK) receptor type 2 (B2-R). In several cell types, these two receptors share the same signaling pathways, although their physiological functions are often opposite. In CTAL, little is known about the intracellular transduction events leading to the final physiological response induced by these two peptides. We investigated and compared in this segment the action of Ang II/III and BK on intracellular calcium concentration ([Ca2+]i) response and metabolic CO2 production, an index of Na+ transport, by using inhibitors of protein kinase C (bisindolylmaleimide), Src tyrosine kinase (herbimycin A and PP2), and MAPK/ERK (PD98059 and UO126). Ang II/III and BK (10(-7) mol/liter) released Ca2+ from the same intracellular pools but activated different Ca2+ entry pathways. Ang II/III- or BK-induced [Ca2+]i increases were similarly potentiated by bisindolylmaleimide. Herbimycin A and PP2 decreased similarly the [Ca2+]i responses induced by Ang II/III and BK. In contrast, PD98059 and UO126 affected the effects of BK to a larger extent than those of Ang II/III. Especially, the Ca2+ influx induced by BK was more strongly inhibited than that induced by Ang II/III in the presence of both compounds. The Na+ transport was inhibited by BK and stimulated by Ang II/III. The inhibitory action of BK on Na+ transport was blocked by UO126, whereas the stimulatory response of Ang II/III was potentiated by UO126 but blocked by bisindolylmaleimide. These data suggest that the inhibitory effect of BK on Na+ transport seems to be directly mediated by an increase in Ca2+ influx dependent on MAPK/ERK pathway activation. In contrast, the stimulatory effect of Ang II/III on Na+ transport is more complex and involves PKC and MAPK/ERK pathways.

PLA 2/PGE 2 are involved in the inhibitory effect of bradykinin on the angiotensin-(1–7)-stimulated Na +-ATPase activity of the proximal tubule

Recently, we demonstrated that bradykinin (BK) counteracts the stimulatory effect of Ang-(1–7) on the Na +-ATPase activity from basolateral membrane of the proximal tubule through B 2 receptor. In the present paper, the signaling pathway involved in the inhibitory response of the Na +-ATPase activity to BK was investigated. The following results indicate that the phospholipase A 2 (PLA 2)/COX/prostaglandin E (PGE 2) pathway is implicated in this process: (1) The inhibitory effect of BK on Ang-(1–7)-stimulated enzyme is abolished in a dose-dependent manner by quinacrine (10 −9;-10 −6M), a nonspecific PLA 2 inhibitor, and by PACOCF 3 (10 −7M), an inhibitor of a Ca 2+-independent PLA 2. However, AACOCF 3 (2×10 −4 M), an inhibitor of the cytosolic PLA 2, does not modify the inhibitory effect of BK. (2) The inhibitory effect of BK on the Ang-(1–7)-stimulated enzyme is reversed by cyclooxygenase (COX) inhibitors diclofenac (10 −12 M) and indomethacin (10 −12 M). (3) PGE 2 (10 −12–10 −5 M) inhibits the Na +-ATPase activity in a dose dependent manner. (4)The inhibitory effects of PGE 2 and BK on the Na +-ATPase activity are not cumulative. (5) PGE 2 (10 −12–10 −8 M) counteracts the stimulatory effect of Ang-(1–7) on the enzyme activity in a dose-dependent manner.

Inhibition of IGF-I–induced Erk 1 and 2 activation and mitogenesis in mesangial cells by bradykinin

The beneficial effects of therapeutic angiotensin-converting enzyme (ACE) inhibitor treatment against the worsening of glomerulosclerosis during the course of diabetic nephropathy have been widely documented. ACE inhibitors inhibit both angiotensin II formation and bradykinin (BK) degradation, thereby reducing angiotensin II type 1 (AT1) receptor activity and favoring B2-kinin receptor (B2 receptor) activation. Since the involvement of growth factors such as insulin-like growth factor (IGF-I) has been implicated in the early steps of diabetic nephropathy, we investigated the effect of BK on Erk 1 and 2 activation and cell proliferation by IGF-I. The activation of Erk 1 and 2 in mesangial cells (MCs) and isolated glomeruli (IG) was investigated by immunoprecipitation and Western blotting during activation of the IGF-I receptor in the presence or absence of BK and of protein kinase C (PKC), tyrosine-kinase and phosphatase selective inhibitors. Mesangial cell proliferation was assessed in vitro by cell counting. In untreated MCs and IG, when added separately, BK and IGF-I both activated Erk 1 and 2. In contrast, in MCs and IG pretreated with BK, the IGF-I-induced Erk 1 and 2 activation was dose-dependently reduced. The inhibitory effect of BK on IGF-I-induced activation of Erk 1 and 2 was completely abolished by addition of a B2 antagonist, by chelation of intracellular calcium and by tyrosine phosphatase inhibition. Additionally, BK reduced MC proliferation induced by IGF-I. A new inhibitory pathway of the early steps of IGF-I signaling by the B2 receptor is found both in cultured MCs and in IG, which involves a calcium-dependent tyrosine phosphatase activity. Recruitment of this mechanism may account for the beneficial effects of ACE inhibitor treatment on glomerulosclerosis associated with diabetic nephropathies.

Effect of bradykinin on membrane properties of guinea pig bronchial parasympathetic ganglion neurons.

The effect of bradykinin on membrane properties of parasympathetic ganglion neurons in isolated guinea pig bronchial tissue was studied using intracellular recording techniques. Bradykinin (1-100 nM) caused a reversible membrane potential depolarization of ganglion neurons that was not associated with a change in input resistance. The selective bradykinin B(2) receptor antagonist HOE-140 inhibited bradykinin-induced membrane depolarizations. Furthermore, the cyclooxygenase inhibitor indomethacin attenuated bradykinin-induced membrane depolarizations to a similar magnitude ( approximately 70%) as HOE-140. However, neurokinin-1 and -3 receptor antagonists did not have similar inhibitory effects. The ability of bradykinin to directly alter active properties of parasympathetic ganglion neurons was also examined. Bradykinin (100 nM) significantly reduced the duration of the afterhyperpolarization (AHP) that followed four consecutive action potentials. The inhibitory effect of bradykinin on the AHP response was reversed by HOE-140 but not by indomethacin. These results indicate that bradykinin can stimulate airway parasympathetic ganglion neurons independent of sensory nerve activation and provide an alternative mechanism for regulating airway parasympathetic tone.

Bradykinin prevents postischemic leukocyte adhesion and emigration and attenuates microvascular barrier disruption.

Although a number of recent reports indicate that bradykinin attenuates ischemia- reperfusion (I/R)-induced tissue injury, the mechanisms underlying its protective actions are not fully understood. However, because bradykinin induces endothelial nitric oxide (NO) production and NO donors have been shown to attenuate postischemic leukocyte adhesion, endothelial barrier disruption, and tissue injury, we hypothesized that bradykinin may act to reduce I/R-induced tissue injury by preventing leukocyte recruitment and preserving microvascular barrier function. To address this postulate, we used intravital videomicroscopic approaches to quantify leukocyte-endothelial cell interactions and microvascular barrier function in single postcapillary venules in the rat mesentery. Reperfusion after 20 min of ischemia significantly decreased wall shear rate and leukocyte rolling velocity, increased the number of rolling, adherent, and emigrated leukocytes, and disrupted the microvascular barrier as evidenced by enhanced venular albumin leakage. Superfusion of the mesentery with bradykinin (10 nM) during I/R significantly reduced these deleterious effects of I/R. Although these inhibitory effects of bradykinin were not affected by cyclooxygenase blockade with indomethacin (10 microM), coadministration with NO synthase (N(omega)-nitro-L-arginine methyl ester, 10 microM) or bradykinin B(2)-receptor (HOE-140, 1 microM) antagonists abolished the protective actions of bradykinin. Plasma NO concentration was measured in the mesenteric vein and was significantly decreased after I/R, an effect that was prevented by bradykinin treatment. These results indicate that bradykinin attenuates I/R-induced leukocyte recruitment and microvascular dysfunction by a mechanism that involves bradykinin B(2)-receptor-dependent NO production.

Transient ischemia inhibits nonexocytotic release of norepinephrine following sustained ischemia in rat heart: is bradykinin involved?

Previous studies have demonstrated that transient ischemia inhibits the release of norepinephrine (NE) following a sustained ischemia. However, the mechanism underlying this inhibition is unknown. Therefore, this study was designed to investigate whether bradykinin (BK) may be involved in the inhibition of NE release following ischemic preconditioning. The effects of transient ischemia, exogenous BK, and kinin receptor blockers on NE release after a prolonged ischemia were tested in the isolated rat heart preparation. Three cycles of 5-min ischemia and reperfusion resulted in the reduction of NE release from 115.3 +/- 14.5 to 51.6 +/- 9.3 pmol.g-1 (p < 0.05) after 30 min of subtotal global ischemia. This effect was not prevented by the administration of either Lys-[Leu8]-des-Arg9-BK (1 mumol.L-1), a B1 antagonist, or HOE-140 (1 mumol.L-1), a B2 antagonist. Three cycles of 5-min BK or des-Arg9-BK infusion also resulted in a dose-dependent inhibition of NE release after 30 min of ischemia. The inhibitory effects of BK (1 mumol.L-1) or des-Arg9-BK (0.5 mumol.L-1) were blocked by Lys-[Leu8]-des-Arg9-BK (1 mumol.L-1), but not by HOE-140 (1 mumol.L-1). The results show that transient ischemia and BK protect sympathetic nerve endings in the isolated rat heart. The inhibition of NE release by pretreatment with BK is mediated by the activation of B1 receptors, whereas preconditioning provided by transient ischemia may be mediated by a different, yet unknown, mechanism in the rat heart.

Transient ischemia inhibits nonexocytotic release of norepinephrine following sustained ischemia in rat heart: is bradykinin involved?

Previous studies have demonstrated that transient ischemia inhibits the release of norepinephrine (NE) following a sustained ischemia. However, the mechanism underlying this inhibition is unknown. Therefore, this study was designed to investigate whether bradykinin (BK) may be involved in the inhibition of NE release following ischemic preconditioning. The effects of transient ischemia, exogenous BK, and kinin receptor blockers on NE release after a prolonged ischemia were tested in the isolated rat heart preparation. Three cycles of 5-min ischemia and reperfusion resulted in the reduction of NE release from 115.3 +/- 14.5 to 51.6 +/- 9.3 pmol.g-1 (p < 0.05) after 30 min of subtotal global ischemia. This effect was not prevented by the administration of either Lys-[Leu8]-des-Arg9-BK (1 mumol.L-1), a B1 antagonist, or HOE-140 (1 mumol.L-1), a B2 antagonist. Three cycles of 5-min BK or des-Arg9-BK infusion also resulted in a dose-dependent inhibition of NE release after 30 min of ischemia. The inhibitory effects of BK (1 mumol.L-1) or des-Arg9-BK (0.5 mumol.L-1) were blocked by Lys-[Leu8]-des-Arg9-BK (1 mumol.L-1), but not by HOE-140 (1 mumol.L-1). The results show that transient ischemia and BK protect sympathetic nerve endings in the isolated rat heart. The inhibition of NE release by pretreatment with BK is mediated by the activation of B1 receptors, whereas preconditioning provided by transient ischemia may be mediated by a different, yet unknown, mechanism in the rat heart.

Lysophosphatidic acid and bradykinin have opposite effects on phenotypic transformation of normal rat kidney cells

The bioactive lipid lysophosphatidic acid is besides a strong mitogen for quiescent fibroblasts, a potent inducer of phenotypic transformation of normal rat kidney cells. The lysophosphatidic acid induced loss of density-arrest is strongly inhibited by bradykinin. Although their effects on normal rat kidney cell proliferation are opposite, bradykinin mimics many of the intracellular effects induced upon lysophosphatidic acid receptor activation, including phosphoinositide turnover, Ca(2+)-mobilization and arachidonic acid release. Bradykinin does not counteract the lysophosphatidic acid induced reduction of cAMP levels in normal rat kidney cells. However, bradykinin inhibits the lysophosphatidic acid and other growth factor induced phenotypic transformation through the induction of a so far uncharacterized prostaglandin G/H synthase product. The growth inhibitory effect of bradykinin is limited to density-arrested cells, while upon prolonged treatment bradykinin itself is capable to induce the loss of density-dependent growth control. It is concluded that bradykinin is a bifunctional regulator of normal rat kidney cell proliferation and that its inhibitory effects are mediated via the induction of a prostaglandin derivative.

HETEROGENEOUS EFFECTS OF BRADYKININ AND A23187 ON GLIOMA GROWTH, MIGRATION AND INVASION

To evaluate the role of receptor interaction and calcium mobilization in growth of gliomas, we examined the effects of bradykinin (BK) and the calcium ionophore A23187 on growth, migration and invasion of the continuous human glioma cell lines GaMG, D37-MG and D54-MG. BK induced a transient increase in intracellular calcium concentration only in D54-MG. A23187 but not BK inhibited glioma proliferation both during monolayer and spheroid growth. Migration of cells from glioma spheroids was slightly inhibited in GaMG both in response to BK and A23187. No effects were seen in D37-MG or D54-MG. Both BK and A23187 inhibited the glioma invasion as assessed with the glioma/brain aggregate co-culture technique in all three cell lines studied. It is concluded that an increase in the intracellular calcium concentration inhibits both growth and invasion of gliomas. The inhibitory effect of BK on glioma invasion may be mediated by a calcium-independent mechanism.

Bradykinin inhibits cyclic AMP accumulation in D384-human astrocytoma cells via a calcium-dependent inhibition of adenylyl cyclase

Bradykinin causes a concentration-dependent, transient rise in intracellular Ca 2+ and a sustained inhibition of forskolin-, dopamine- and 5′- N-ethyl-carboxamidoadenosine (NECA)-stimulated cAMP accumulation in D384 astrocytoma cells. Chelation of intracellular calcium abolished bradykinin's inhibitory effect on cAMP accumulation. Chelating extracellular Ca 2+ did not block the initial, but eliminated the sustained inhibition of cAMP accumulation. Increasing Ca 2+ influx by calcium ionophore A23187 caused a concentration-dependent inhibition of stimulated cAMP accumulation. A hydroquinone derivative 2,5-di(tert-butyl)-1,4-benzohydroquinone (tBuBHQ), which inhibits microsomal Ca 2+ sequestration, did not mimic the effect of bradykinin, although it increased [Ca 2+] i even more than A23187 did. The inhibitory effect of bradykinin was not mediated by Ca 2+/CaM-dependent stimulation of phosphodiesterase (PDE). Forskolin-stimulated adenylyl cyclase activity was inhibited by Ca 2+ (10 −7 to 10 −3 M), both in ethyleneglycol-bis-( β-aminoethyl ether)- N, N, N′, N′-tetraacetic acid (EGTA) washed and native D384 plasma membranes. This effect was not altered by calmodulin (CaM) or CaM-antagonists. Bradykinin treatment, which attenuates cAMP accumulation in intact cells, did not do so in plasma membranes. These findings suggest that bradykinin-induced inhibition of cAMP formation in D384 cells requires mobilization of [Ca 2+] i and subsequent entry of Ca 2+ which directly interacts with a component of the adenylyl cyclase system.

Bradykinin inhibition of cyclic AMP accumulation in D384 astrocytoma cells. Evidence against a role of cyclic GMP.

The present studies were performed in order to examine the possible role of cyclic GMP-stimulated phosphodiesterase (cGMP-PDE) activity in the inhibitory action of the inflammatory peptide bradykinin on cyclic AMP (cAMP) accumulation in D384 cells. Bradykinin decreased the forskolin-stimulated cAMP accumulation in the presence of the phosphodiesterase inhibitor rolipram, and caused a transient 50% rise in cellular cGMP in the presence of the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX). Both basal and bradykinin-stimulated cGMP accumulation were about 8 times higher in the presence of IBMX than in the presence of rolipram. Sodium nitroprusside, which caused a 20-70-fold increase in cGMP levels reduced forskolin stimulated cAMP accumulation, whereas hydroxylamine, which maximally caused a 16-fold increase in cGMP, did not. 8-bromo-cGMP or dibutyryl cGMP had no effect on cAMP accumulation induced by forskolin. The inhibitory effect of nitroprusside was totally reversed by blocking the soluble guanylate cyclase activity by methylene blue treatment; however, the inhibitory action of bradykinin on cAMP accumulation was not changed by this treatment. Additionally, inhibition of nitric oxide synthesis, which is known to be regulated by Ca2+ and in turn stimulates cGMP production, by N omega-nitro-L-arginine (L-NAME) treatment did not alter the inhibitory effect of bradykinin on forskolin-induced cAMP accumulation. These results indicate that large increases in cGMP may regulate cAMP via cGMP-PDE whereas the small increase induced by bradykinin is insufficient and that cGMP is not involved in the inhibitory action of bradykinin on cAMP levels in D384 cells.

Endothelium-Derived and Intraneuronal Nitric Oxide-Dependent Inhibition of Norepinephrine Efflux From Sympathetic Nerves by Bradykinin

Evidence is presented that bradykinin inhibits norepinephrine efflux from sympathetic nerves innervating canine mesenteric and pulmonary arteries, in part, by releasing endothelium-derived relaxing factor (EDRF) from the vascular endothelium. Moreover, in the absence of vascular endothelium, bradykinin also inhibits norepinephrine efflux from the sympathetic nerve terminals innervating these blood vessels. This inhibition is attenuated by canavanine and LNMMA, which inhibit the conversion of arginine to nitric oxide, and is enhanced after overnight incubation of blood vessels with arginine. In endothelium-rubbed blood vessels the inhibitory effect of bradykinin on norepinephrine efflux is enhanced by increasing extracellular calcium ion ([Ca2+]o) and attenuated by nitrendipine. We propose that bradykinin inhibits norepinephrine efflux by stimulating intraneuronal nitric oxide from arginine.

Bradykinin activates protein kinase C in cultured cortical collecting tubular cells

Bradykinin inhibits vasopressin-stimulated water transport in cortical collecting tubular cells. The biochemical mechanism of this effect was explored by means of primary cultures of rabbit cortical collecting tubular cells. Bradykinin was found to produce a rapid release of calcium from intracellular stores, an increase in sn-1,2-diacylglycerol levels, and a fivefold increase in membrane-bound protein kinase C activity, consistent with stimulation of phospholipase C and activation of protein kinase C in rabbit cortical collecting tubular cells. In addition, bradykinin produced a dose-dependent 46% inhibition of vasopressin-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) formation. Pretreatment with the protein kinase C inhibitors, H-7 and staurosporine, reversed the bradykinin-mediated inhibition of vasopressin-stimulated cAMP accumulation. In contrast, pretreatment with either the phospholipase A2 inhibitor, mepacrine, or pertussis toxin did not prevent the inhibitory effect of bradykinin on vasopressin-stimulated cAMP production, suggesting that the effects are not mediated by prostaglandin E2 or activation of a pertussis-toxin sensitive guanine nucleotide regulatory protein (e.g., Gi). Because bradykinin also inhibits isoproterenol-stimulated cAMP formation but does not inhibit either basal-, forskolin-, or cholera toxin-stimulated cAMP accumulation, the site of this inhibition appears to involve the hormone receptor or coupling of the receptor to the stimulatory guanine nucleotide regulatory subunit (Gs). The results demonstrate that bradykinin stimulates phospholipase C leading to activation of protein kinase C, which then inhibits vasopressin-stimulated cAMP production at the level of the hormone receptor or coupling of the receptor to Gs in cultured cortical collecting tubular cells.

Conversion of kinins and their antagonists into B 1 receptor activators and blockers in isolated vessels

A carboxypeptidase inhibitor (DL-2-mercaptomethyl-3-guanidoethylthiopropranoic acid) (mergetpa) was used to block the conversion of kinins and B 2 receptor antagonists into metabolites devoid of the C-terminal Arg. Experiments were carried out on rabbit isolated aortae (a B 1 receptor system) or rabbit jugular veins and dog carotid arteries (two B 2 receptor systems). The contractile effect of bradykinin in the rabbit aorta was significantly reduced by mergetpa while that of desArg 9-BK was not modified. pA 2 values of B 2 receptor antagonists, [Thi 5,8,D-Phe 7]bradykinin and [Thi 6,9,D-Phe 8]kallidin were markedly reduced by mergetpa. The apparent affinity (pA 2) of a B 1 receptor antagonist, [Leu 9]desArg 10-kallidin was not affected. Carboxypeptidases inhibition did not modify the activities of bradykinin or the affinities of B 2 receptor antagonists in the rabbit jugular vein and the dog carotid artery. An inhibitor of kininase II (D-3-mercapto-2-methylpropranoyl-L-proline (S,S)) (captopril) reduced the contractile effects of angiotensin I in the three preparations and potentiated the stimulatory or inhibitory effects of bradykinin: captopril did not have effect on the affinities of B 2 receptor antagonists and did not modify the effects of angiotensin II. Comparative experiments performed in tissues with or without endothelium gave the same results with both mergetpa and captopril. The present findings suggest that bradykinin and B 2 receptor antagonists are converted by carboxypeptidases into biologically active B 1 receptor agonist or antagonists. This is the reason why B 2 receptor antagonists are not selective.

The actions of some vasoactive polypeptides and their antagonists on the anococcygeus muscle.

1 The action of three polypeptides, bradykinin, substance P and eledoisin known to inhibit vascular smooth muscle has been examined on the anococcygeus muscle of the rat, cat and rabbit.2 In the atonic rat muscle, bradykinin and substance P had little or no effect on tone but eledoisin produced a sustained dose-related contraction which could be abolished by phentolamine (1 muM) and is, therefore, probably an indirect sympathomimetic effect. On the motor response to field stimulation of adrenergic nerves, bradykinin had no effect whereas both substance P and eledoisin reduced this response. The mechanism of action was further analysed with eledoisin by examining its effect on the response to noradrenaline. Eledoisin did not alter the dose-response curve to noradrenaline and its inhibitory action is likely, therefore, to be presynaptic.3 In the rat anococcygeus muscle in which the tone was raised by guanethidine or carbachol, bradykinin and substance P reduced this tone whereas eledoisin continued to exert a motor action. Compared with substance P the inhibitory effect of bradykinin appeared at lower concentrations (threshold 0.01 mug/ml), developed more rapidly and the size of the response was greater.4 The effect of bradykinin on the tonically contracted cat and rabbit anococcygeus muscles was examined in addition to that of the rat. In all three species bradykinin caused inhibition and the magnitude of the response was equal to the maximum effect of inhibitory nerve stimulation. None of the peptides affected the inhibitory response to nerve stimulation itself.5 The effects of three substances, hesperitin, khellin and apiin, reported in other tissues to antagonize the action of bradykinin were examined both on the inhibitory response to bradykinin and to field stimulation. None of them was able to inhibit either response, although they reduced tone when given by themselves. During these experiments it was found that ethanol antagonized the inhibitory response to field stimulation.6 The possibility that bradykinin or some related peptide might play a part in the inhibitory response to nerve stimulation in the anococcygeus is discussed.

Effect of bradykinin on transepithelial transfer of sodium and water in vitro.

Mucosal sodium and water transfer were measured in everted sacs of rat jejunum. 2. Bradykinin (7.86 X 10(-12)M), when present in both mucosal and serosal solutions, produced a biphasic effect on mucosal sodium and water transfer. When basal transfer was low a stimulation was observed whereas an inhibition of transfer was observed when basal transfer was high. Bradykinin at concentrations of 7-86 X 10(-11) and 7-86 X 10(-13) M produced qualitatively similar effects. 3. Inhibition of transfer was observed whether bradykinin was present in the mucosal, serosal or both solutions. Stimulation of transfer was observed only when bradykinin was present in the serosal solution. 4. Theophylline (1 mM), alone inhibited water transfer at high and intermediate levels of basal transfer, and significantly potentiated the inhibitory effect of bradykinin (7-86 X 10(-12)M) on water transfer at intermediate levels of control transfer. 5. Cyclic AMP (1 mM) inhibited water transfer when basal transfer was high. Dibutyryl cyclic AMP (1 mM) inhibited water transfer at all levels of basal transfer. Dibutyryl cyclic AMP (1 mM) and bradykinin (7-86 X 10(-12)M) together produced a significantly greater inhibition of water transfer than either agent alone, at intermediate basal transfer. 6. It was observed that the action of bradykinin upon sodium and water transfer consists of two different and opposing effects. It is possible that the inhibitory effect of bradykinin upon water transfer is related to increased cyclic AMP activity.