Effects of bradykinin and angiotensin II on intracellular Ca2+ dynamics in endothelial cells.
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.
Morgan-Boyd
R
R
Department of Pharmacology, University of Tennessee, Memphis 38163.
Stewart
J M
JM
Vavrek
R J
RJ
Hassid
A
A
D000804:Angiotensin II / Q000494:pharmacology*; D000818:Animals; D001572:Benzofurans; D001920:Bradykinin / Q000494:pharmacology*; D002118:Calcium / Q000493:pharmacokinetics*; D002417:Cattle; D004727:Endothelium / Q000187:drug effects / Q000378:metabolism*; D004987:Ethers / Q000494:pharmacology; D005029:Ethylenediamines / Q000494:pharmacology; D016257:Fura-2; D015759:Ionomycin; D007700:Kinetics; D008433:Mathematics; D009543:Nifedipine / Q000494:pharmacology
D001572:Benzofurans; D004987:Ethers; D005029:Ethylenediamines; D000804:Angiotensin II; D015759:Ionomycin; D009543:Nifedipine; C044387:N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine; D001920:Bradykinin; D002118:Calcium; D016257:Fura-2