Autoradiography. For autoradiographic localization of [3H]BK receptor binding, guinea pigs were perfused through the heart with 500 ml of a mixture of equal parts of isotonic phosphate-buffered saline (pH 7.4) and 10%o (wt/vol) sucrose. After perfusion, the tissues were removed, embedded in homogenized calf cortex, and frozen on dry ice. Canine tissues were removed without perfusion and immersed in chilled perfusion medium for 20 min, then mounted and frozen as described. Ten-micrometer sections were cut in a cryostat at -12°C, thaw-mounted onto chrome alum/gelatin-coated microscope slides, and stored frozen at -20°C.When ready for use, the slides were warmed to room temperature, incubated for 120 min at 4°C in medium containing 20 mM 2-{[tris(hydroxymethyl)methyl]amino}ethane sulfonic acid (Tes) (pH 6.8), 1 mM 1,10-phenanthroline, 0.2% bovine serum albumin (protease free), 1 mM dithiothreitol, bacitracin (140 ,ug/ml), 0.1 mM SQ20,881, 300 mM sucrose, and 0.5 nM [3H]BK (52 Ci/mmol; 1 Ci = 37 GBq).Blanks were incubated in the same medium with 1 ,uM unlabeled BK or Lys-BK.After incubation, the tissue sections were washed in a solution of 25 mM Tes (pH 6.8) and 10% (vol/vol) sucrose for two 10-min periods at 4°C, dried rapidly under cold dry air, and apposed to either NTB-3 emulsion-coated coverslips or LKB Ultrafilm for 1 month. After exposure, the coverslip/slide or Ultrafilm was developed, and the tissue was stained with cresyl violet.Isolated Tissues. Smooth muscle effects of the BK analogs were determined on the isolated rat uterus and isolated guinea pig ileum by standard methods (10). BK analogs were incubated with the preparation for 30 sec before adding an ED50 dose of BK. BK analogs that decreased BK were then examined in detail to determine pA2 values for antagonism (pA2 is the negative log of the antagonist concentration that causes a 2-fold shift in the agonist dose-response curve) (11). Dose-response curves were determined for analogs that produced contractions at 10 uM.BK-Induced Vascular Pain. Male Sprague-Dawley rats (200-300 g) (Charles River Breeding Laboratories) were housed five per cage with constant access to food and water with a 12-hr light-dark cycle (7 a.m.-7 p.m.). Rats received an indwelling carotid artery cannula routed subcutaneously to exit behind the head. One to 3 days following surgery, the cannula was connected through tubing to a remote syringe allowing intraarterial injections while the rats were conscious and freely moving. In preliminary experiments, BK at 2 nmol/kg caused stereotypic flexion of the right forelimb and dextrorotation of the head in essentially 100% of the rats tested. Rats that displayed this BK response were injected 5 min later, through the same arterial cannula, with BK and a BK antagonist at 2, 20, or 200 nmol/kg.
The purpose of this study was to investigate the effects of angiotensin II and bradykinin on intracellular Ca2+ dynamics in cultured endothelial cells. We used the "second-generation" fluorescent Ca2+ indicator fura-2, in conjunction with dual-wavelength fluorescence spectroscopy, in cultured adherent pulmonary arterial endothelial cells. Angiotensin II (up to 2 microM) had no consistent effect on intracellular Ca2+ levels. In contrast, bradykinin (10 nM) elicited a transient increase of cytosolic free Ca2+, from the resting value of 37 +/- 5 to 647 +/- 123 nM, followed by a decline to a steady-state value of 113 +/- 14 nM, which was significantly higher than the resting Ca2+ levels. Bradykinin's Ca-stimulatory effect was dose dependent, having a half-maximally effective concentration of approximately 1 nM and a maximally effective concentration of 10 nM. A B1-receptor agonist, Des-Arg9-bradykinin, was much less effective than bradykinin as modulator of cytosolic Ca2+. Moreover, a B1-receptor antagonist, Des-Arg9, [Leu8]-bradykinin, did not significantly affect the increase of cytosolic Ca2+ elicited by bradykinin. On the other hand, the bradykinin-elicited increase of Ca2+ was almost completely inhibited by a novel B2-receptor antagonist, D-Arg-[Hyp3, Thi5,8, D-Phe7]-bradykinin. Bradykinin increased cytosolic free Ca2+ levels in cells maintained in Ca2+-deficient extracellular medium, suggesting that the peptide mobilized Ca2+ from intracellular stores. However, the absence of extra-cellular Ca2+ resulted in an 80-90% attenuation of the transient Ca2+ response, whereas the posttransient steady-state response was completely absent. These findings are consistent with the notion that the bradykinin-elicited transient Ca2+ response is dependent on both extra- and intracellular Ca2+ and that the posttransient steady-state response is entirely dependent on extracellular Ca2+. Endothelial cells were responsive to a second dose of bradykinin after a 10-min interim period of incubation in the absence of the peptide hormone. The absence of extracellular Ca2+ during the interim period, or the pretreatment of cells with ionomycin in the absence of extracellular Ca2+, prevented the response of the cells to a second dose of bradykinin. Bradykinin- or ionomycin-desensitized cells could be resensitized by a brief incubation period in Ca2+-replete medium. The results are consistent with the notions that cellular resensitization requires the replenishment of intracellular Ca2+ and that bradykinin, but not angiotensin II, modulates intracellular Ca2+ dynamics in endothelial cells by interacting with a B2-type receptor.
1 A new synthetic bradykinin analogue was found to be an antagonist of bradykinin-induced vascular permeability in rabbit skin. It was effective in equimolar concentrations. 2 These analogues also antagonized the action of bradykinin in contracting the guinea-pig isolated ileum. The mean pA2 values of five different antagonists ranged from 5.3-6.4 respectively, on this preparation. 3 Our observations, together with those of others suggest that these antagonists act on the same receptor types, viz., B2, in rabbit blood vessels and in smooth muscle of guinea-pig ileum. 4 Our results support the view that the way is now promising for the synthesis of potent specific antagonists of bradykinin for experimental and therapeutic use.
In this paper we examine the effect of the vasodilator peptide bradykinin on endothelial cell regulation of phosphoinositide (PI) turnover. The data show that the activation of PI turnover by bradykinin in bovine pulmonary artery endothelial cells is insensitive to pertussis toxin, which ADP ribosylates a membrane protein of mol wt 40,000. However, this effect of bradykinin can be potentiated by guanosine 5'-O-(3-thio)triphosphate (GTP gamma S), an activator of G proteins, and depressed by guanosine 5'-O-(2-thio)diphosphate (GDP beta S), an inhibitor of G proteins. After endothelial cells were preincubated for 1 h with GTP gamma S, there was a three- to fourfold increase in PI turnover. Preincubation of cells with GDP beta S did not affect the basal level of PI turnover, but completely prevented activation of PI turnover by bradykinin. 4 beta-Phorbol-12 beta-myristate-13 alpha-acetate can block the bradykinin-stimulated inositol monophosphate formation in cultured endothelial cells. The effects of bradykinin on PI turnover were blocked by B2 antagonists but not by B1 antagonists. Taken together, these results indicate that in endothelial cells the bradykinin B2 receptor is coupled to phospholipase C via a G protein (or proteins) that is not a substrate for pertussis toxin (neither Gi nor Go).
SUMMARY This study was designed to examine the contribution of bradykinin to the depressor effect of different antihypertensive drugs in two-kidney renovascular hypertensive rats, using a new specific antagonist of bradykinin. First, the inhibitory capacity of this peptide for exogenously injected bradykinin (75-200 ng) was tested. An inhibition of the vasodepressor action of bradykinin by over 50% was found when the bradykinin inhibitor was infused at a rate of 40 /xg/min, with little difference at higher rates of infusion. This inhibitor then was infused in three groups of renovascular hypertensive rats after their blood pressure had been decreased by pretreatment with the converting enzyme inhibitor enalapril (MK 421), saralasin, or sodium nitroprusside, respectively. Infusion of the inhibitor produced an immediate 30% increase in blood pressure only in the enalapril-treated group. These results indicate that bradykinin is involved in the decrease of blood pressure produced by converting enzyme inhibition in experimental renovascular hypertension. (Hypertension 8: 000-000, 1986) KEY WORDS • experimental renovascular hypertension • converting enzyme inhibitor saralasin • sodium nitroprusside A LTHOUGH bradykinin (BK) is considered to be / \ a powerful vasodilator, 1 its contribution to A. \ -blood pressure regulation remains obscure. Exogenous BK injected systemically causes a decrease in blood pressure, 2 but it is rapidly inactivated after passage through the pulmonary vascular bed.3 Following the discovery of a BK-potentiating factor by Ferreira, 4 it was demonstrated that the kininase II degrading BK in the lung and the angiotensin converting enzyme (ACE) transforming the inactive angiotensin I into the biologically active angiotensin II are in fact identical 5 and that both effects are inhibited by the same factor.6 Theoretically, both actions should contribute to the hypotensive effect of various ACE inhibitors, yet only the elimination of angiotensin II has been proved beyond doubt. The actual accumulation of BK remains controversial, 7 " 9 and its role is mostly based on indirect evidence.10 " 12In several instances, the action of a vasoactive hormone has been demonstrated by the use of a specific Received March 8, 1986; accepted May 20, 1986. competitive antagonist of that hormone at the vascular receptor level; examples include saralasin to determine the pressor effect of angiotensin II 13 and V, antagonists to prove the pressor effect of vasopressin.14 In this study, we used a competitive antagonist of BK, the compound B4146 (synthesized by J.M.S. and R.J.V.), to prove that the depressor effect of ACE inhibition is indeed partly due to BK. The inhibitory capacity of this agent has been demonstrated in vitro on vascular, uterine, and ileum smooth muscle.13 - 16Materials and Methods The first part of the study consisted of a series of experiments designed to test the inhibitory capacity of the BK inhibitor B4146 (Arg-Pro-Hyp-Gly-Thi-SerDPhe-Thi-Arg.TFA; Hyp = L^-hydroxyproline; Thi = /3-(2-thienyl)...
1. The effect of bradykinin (BK) and some analogues of BK on the human blister base was studied. 2. BK produced reproducible dose-related increases in pain responses. A characteristic delay, which was not dose-related occurred between application of BK and the resultant response. 3. The rank order of potency of several kinin analogues on the pain response was BK much much greater than sigma-cyclo-(Lys1-Gly6)-BK = sigma-cyclo-kallidin greater than des-Arg9-BK. 4. No increase in pain response was seen with repeated application of the selective B1 receptor agonist des-Arg9-BK to the same blister base at 4 h intervals. The B1 receptor antagonist des-Arg9-Leu8-BK was without effect against BK-induced responses. 5. The B2 receptor antagonists, D-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-D-Phe-Thi-Arg-TEA and D-Pro-Phe-Arg-heptylamide produced significant antagonism of the bradykinin-induced pain responses at doses which had no effect against 5-hydroxytryptamine or potassium chloride. 6. It is concluded that the kinin receptor mediating pain on the human blister base is of the B2 type.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.