Lys-Bradykinin

Aminopeptidase A (APA) is a type II membrane-bound protein implicated in the regulation of blood pressure in the brain renin-angiotensin system. In this study, a recombinant soluble form of APA was expressed in a baculovirus system, purified to homogeneity, and characterized. By using synthetic substrates, it was shown that although the enzyme has a rather broad substrate specificity in the absence of Ca2+, the preferential release of acidic amino acid residues was observed in the presence of Ca2+. Moreover, Ca2+ up- or down-regulated the enzymatic activity depending on the substrate. By searching for natural substrates of APA, we found that peptides having acidic amino acids at their N terminus (angiotensin II, neurokinin B, cholecystokinin-8, and chromogranin A) were cleaved by the enzyme efficiently in the presence but not in the absence of Ca2+. Moreover kallidin (Lys-bradykinin) was converted to bradykinin effectively only in the absence of Ca2+. These results suggest that Ca2+ increases the preference of the enzyme for the peptide substrates having N-terminal acidic amino acids. In addition, we found that angiotensin IV could bind to APA both in the presence and absence of Ca2+ and inhibited the enzymatic activity of APA competitively, suggesting that angiotensin IV acts as a negative regulator of the enzyme once generated from angiotensin II by the serial actions of aminopeptidases.

Using Indo-1 as a fluorescent probe, we studied the dynamics and the underlying mechanisms of the response of cytosolic free calcium ([Ca2+]i) to different concentrations of four prolactin secretagogues, thyrotropin-releasing hormone, angiotensin II, bradykinin, and lys-bradykinin in rat anterior pituitary cells. Low concentrations (1-100 pM) of these peptides caused a sustained increase in [Ca2+]i, whereas high concentrations (up to 100 nM) caused a large transient elevation of [Ca2+]i that was followed by a lower sustained plateau. Experiments with protein kinase C-depleted cells suggested that phorbol diester-sensitive protein kinase C was not involved in the transition of [Ca2+]i from spike to plateau seen with high concentrations of secretagogue. Specific concentrations of secretagogue mobilized different pools of [Ca2+]i, as indicated by experiments with Ca2(+)-depleted medium. Low concentrations of secretagogue induced a Ca2+ response that was abolished by Ca2(+)-depleted medium, whereas high concentrations generated a [Ca2+]i response that was refractory to Ca2(+)-depleted medium. Dopamine (100 nM) abolished the [Ca2+]i plateau response to all four agents at low concentrations and selectively reduced the plateau component of the responses elicited at high concentrations of secretagogue.

The tritiated bradykinin B1 receptor agonist [3H]des-Arg(10)-kallidin bound to a single class of high-affinity binding sites (K(d) = 0.5 +/- 0.16 nM; B(max) = 15,000 +/- 8,000 sites/cell) on cultured rat aortic smooth muscle cells. [3H]Des-Arg(10)-kallidin association and dissociation kinetics were monoexponential, making it possible to determine the association and dissociation rate constants (k(+1) = 1.5 10(5) M(-1) sec(-1); k(-1) = 4.2 10(-5) sec(-1)). [3H]Des-Arg(10)-kallidin binding was inhibited by specific ligands of bradykinin B1 and B2 receptors with a rank order of potency consistent with that known for bradykinin B1 receptors in other species (des-Arg(9)-[Leu(8)]bradykinin = des-Arg(10)-kallidin = des-Arg(9)-bradykinin = des-Arg(10)-[Leu(9)]kallidin > des-Arg(10)-HOE-140 >> bradykinin >> HOE-140). Bradykinin B1 receptor mRNA was also detected in these cells. Des-Arg(10)-kallidin increased cytosolic free Ca2+ levels, phosphoinositide turnover, and arachidonic acid release at nanomolar concentrations (respective EC(50) values: 16 +/- 2, 4 +/- 2.7, 6 +/- 2 nM). These functional effects of des-Arg(10)-kallidin could be blocked by the bradykinin B1 receptor antagonist des-Arg(9)-[Leu(8)]bradykinin, but were not sensitive to bradykinin B2 receptor antagonists.