Functional Analysis Of Antibodies Against Short Peptides Of α1-Adrenergic Receptor

PartⅠFunctional Analysis of Antibodies against Short Peptides of alA-Adrenergic ReceptorAims:α1A-adrenergic receptor (α1A-AR) is the crucial subtype ofα1-adrenergic rec eptors (al-ARs) in maintenance and regulation of blood pressure.α1A-AR maybe the target of lowering high blood pressure. However, the high specific antagonists ofα1A-AR are lack till recently. We aimed to functionally analyze the specific antibodi es against short peptides of rat alA-AR, providing a novel strategy for hypertension therapy.Methods and results:We screened out two short peptides of rat alA-AR:CP-7 (1 ocated in the second extracellular loop) and CPE-8 (located in the third extracellular loop). New Zealand rabbits and spontaneously hypertensive rats (SHR) were immu nized with the conjugation of the above peptides and KLH. Antibodies against CP-7 and CPE-8 were purified from serum of rabbits by 50% saturated ammonium sulfa te precipitating and peptides affinity purification, termed Anti-CP-7 and Anti-CPE-8 respectively. The two antibodies could specifically bind to alA-AR with no cross-re action with the other two subtypes. Their functions were analyzed from different lev els. Only Anti-CP-7 antibody could inhibit the activation of Ca2+ dependent signal t ransduction including protein kinases C (PKC) translocation and extracellular signal-r egulated kinases (ERK1/2) phosphorylation. We further found Anti-CP-7 antibody co uld effectively decrease the beating rates of neonatal rat cardiomyocytes in basal an d phenylephrine (PHE)-induced levels. Compared to KLH immunized group, CP-7-K LH immunizing inhibited the increasing of the systolic blood pressure (SBP) of spo ntaneous hypertensive rats (SHR), which is significantly correlated to the titer of An ti-CP-7. All above effects were not significant in CPE-8-KLH immunized group.Conclusion:Our study demonstrated that Anti-CP-7 antibody could specifically inhi bit the activation of rat alA-AR both in vitro and in vivo. It could become a nove 1 strategy in inhibition of alA-AR or other G protein coupling receptors (GPCRs) which are lack of specific antagonists. CP-7 may be the potential target of hyperten sion therapy. The treatment against CP-7 could become a potential therapeutic meth od for primary hypertension. PartⅡFunctional Analysis of an Antibody against a short peptide ofα1D-Adrenergic ReceptorAims:alD-adrenergic receptor(α1D-AR) is one subtype of al-adrenergic receptors (α1-ARs).It is reported to participate in the maintenance and regulation of systemic bl ood pressure. The elevation of the activity ofα1D-AR can lead to high blood press ure.alD-AR may be the target of treating hypertension. However, the specific inhibi tor ofα1D-AR is lack. In our experiment, we aimed to functionally analyze the spe cific antibody against the short peptide of the second extracellular loop of ratα1D-AR to provide a novel strategy for hypertension therapy.Methods and results:New Zealand rabbits and spontaneous hypertensive rats (SHR) were immunized with a short epitope of ratα1D-AR (CF-8:located in the second extracellular loop) for 10 weeks. Specific antibody to CF-8(Anti-CF-8) was purified from rabbit IgG by 50% saturated ammonium sulfate precipitating and peptide affi nity purification and concentrated. Anti-CF-8 was used in vitro to study its effect o nα1D-AR. We demonstrated that Anti-CF-8 could specifically bindtoα1D-AR expre ssed in thoracic artery and vascular smooth muscle cells (VSMC) of Male Sprague-Dawley rats. Functional analysis in vitro showed it could inhibit protein kinase C (PKC) translocation and extracellular signal-regulated kinase (ERK) phosphorylation stimulated by phenylephrine (PHE).Anti-CF-8 could also inhibit the contraction of th oracic aorta rings stimulated by PHE. Spontaneous hypertensive rats (SHR) were us ed as the model to evaluate the Anti-CF-8 antibody on blood pressure in vivo. How ever, it showed no significant difference between the KLH and CF-8-KLH immuniz ed groups in the whole immunizing process. The peak titer of Anti-CF-8 reached 1: 3200. Conclusion:We concluded that although our purified antibody Anti-CF-8 could inhi bit the activation of alD-AR in vitro specifically, the effect of CF-8-KLH on blood pressure was not significant. We need much higher antibody titer to evaluate the e ffect of Anti-CF-8 in vivo.