ABSTRACT
Previous studies have shown that the vascular reactivity of the mouse aorta differs substantially from that of the rat aorta in response to several agonists such as angiotensin II, endothelin-1 and isoproterenol. However, no information is available about the agonists bradykinin (BK) and DesArg9BK (DBK). Our aim was to determine the potential expression of kinin B1 and B2 receptors in the abdominal mouse aorta isolated from C57BL/6 mice. Contraction and relaxation responses to BK and DBK were investigated using isometric recordings. The kinins were unable to induce relaxation but concentration-contraction response curves were obtained by applying increasing concentrations of the agonists BK and DBK. These effects were blocked by the antagonists Icatibant and R-715, respectively. The potency (pD2) calculated from the curves was 7.0 ± 0.1 for BK and 7.3 ± 0.2 for DBK. The efficacy was 51 ± 2 percent for BK and 30 ± 1 percent for DBK when compared to 1 æM norepinephrine. The concentration-dependent responses of BK and DBK were markedly inhibited by the arachidonic acid inhibitor indomethacin (1 æM), suggesting a mediation by the cyclooxygenase pathway. These contractile responses were not potentiated in the presence of the NOS inhibitor L-NAME (1 mM) or endothelium-denuded aorta, indicating that the NO pathway is not involved. We conclude that the mouse aorta constitutively contains B1 and B2 subtypes of kinin receptors and that stimulation with BK and DBK induces contractile effect mediated by endothelium-independent vasoconstrictor prostanoids.
Subject(s)
Animals , Male , Mice , Aorta, Abdominal/drug effects , Bradykinin/agonists , Bradykinin/analogs & derivatives , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/physiology , Receptor, Bradykinin B1/drug effects , /drug effects , Aorta, Abdominal/physiology , Bradykinin/pharmacology , Endothelium, Vascular/drug effects , Endothelium, Vascular/physiology , Indomethacin/pharmacology , Isometric Contraction/drug effects , Isometric Contraction/physiology , Receptor, Bradykinin B1/physiology , /physiology , Vasoconstriction/drug effects , Vasoconstriction/physiologyABSTRACT
Carboxypeptidase M (CPM) is an extracellular glycosylphosphatidyl-inositol-anchored membrane glycoprotein, which removes the C-terminal basic residues, lysine and arginine, from peptides and proteins at neutral pH. CPM plays an important role in the control of peptide hormones and growth factor activity on the cell surface. The present study was carried out to clone and express human CPM in the yeast Pichia pastoris in order to evaluate the importance of this enzyme in physiological and pathological processes. The cDNA for the enzyme was amplified from total placental RNA by RT-PCR and cloned in the vector pPIC9, which uses the methanol oxidase promoter and drives the expression of high levels of heterologous proteins in P. pastoris. The cpm gene, after cloning and transfection, was integrated into the yeast genome, which produced the active protein. The recombinant protein was secreted into the medium and the enzymatic activity was measured using the fluorescent substrate dansyl-Ala-Arg. The enzyme was purified by a two-step protocol including gel filtration and ion-exchange chromatography, resulting in a 1753-fold purified active protein (16474 RFU mg protein-1 min-1). This purification protocol permitted us to obtain 410 mg of the purified protein per liter of fermentation medium. SDS-PAGE showed that recombinant CPM migrated as a single band with a molecular mass similar to that of native placental enzyme (62 kDa), suggesting that the expression of a glycosylated protein had occurred. These results demonstrate for the first time the establishment of a method using P. pastoris to express human CPM necessary to the development of specific antibodies and antagonists, and the analysis of the involvement of this peptidase in different physiological and pathological processes.
Subject(s)
Humans , Metalloendopeptidases/isolation & purification , Pichia/enzymology , Chromatography, Ion Exchange , Metalloendopeptidases/genetics , Pichia/genetics , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The participation of the kallikrein-kinin system, comprising the serine proteases kallikreins, the protein substrates kininogens and the effective peptides kinins, in some pathological processes like hypertension and cardiovascular diseases is still a matter of controversy. The use of different experimental set-ups in concert with the development of potent and specific inhibitors and antagonists for the system has highlighted its importance but the results still lack conclusivity. Over the last few years, transgenic and gene-targeting technologies associated with molecular biology tools have provided specific information about the elusive role of the kallikrein-kinin system in the control of blood pressure and electrolyte homeostasis. cDNA and genomic sequences for kinin receptors B2 and B1 from different species were isolated and shown to encode G-protein-coupled receptors and the structure and pharmacology of the receptors were characterized. Transgenic animals expressing an overactive kallikrein-kinin system were established to study the cardiovascular effects of these alterations and the results of these investigations further corroborate the importance of this system in the maintenance of normal blood pressure. Knockout animals for B2 and B1 receptors are available and their analysis also points to the role of these receptors in cardiovascular regulation and inflammatory processes. In this paper the most recent and relevant genetic animal models developed for the study of the kallikrein-kinin system are reviewed, and the advances they brought to the understanding of the biological role of this system are discussed.