Anand-Srivastava M B, Gutkowska J, Cantin M
Clinical Research Institute of Montreal, Quebec, Canada.
Biochem J. 1991 Aug 15;278 ( Pt 1)(Pt 1):211-7. doi: 10.1042/bj2780211.
The effect of atrial natriuretic factor (ANF) on adenylate cyclase activity was studied in rat platelet membranes. ANF-(99-126)-, -(101-126)-, -(103-126)- and -(103-123)-peptide inhibited adenylate cyclase activity in a concentration-dependent manner with an order of potency of ANF-(103-123)-peptide greater than ANF-(99-126)-peptide greater than ANF-(101-126)-peptide greater than ANF-(103-126)-peptide. ANF-(103-123)-peptide and ANF-(99-126)-peptide inhibited the enzyme activity by about 50-55%, with an apparent Ki between 0.1 and 0.5 nM, and ANF-(101-126)-peptide inhibited the enzyme activity by about 35%, with an apparent Ki between 1 and 3 nM. On the other hand, ANF-(103-126)-peptide was the least potent and inhibited the adenylate cyclase activity by about 30% (Ki approximately 10 nM). The inhibitory effect of ANF on adenylate cyclase was also dependent on the presence of guanine nucleotides and was attenuated by amiloride and pertussis toxin. The stimulatory effects of various agonists such as N-ethylcarboxamideadenosine, prostaglandin E1, isoprenaline and forskolin on adenylate cyclase were also inhibited by ANF to various extents; however, the stimulations were not completely abolished. In addition, 125I-labelled ANF-(99-126)-peptide bound specifically to rat platelet membranes. The binding of 125I-ANF was competitively inhibited in a concentration-dependent manner by the unlabelled peptides which were used for adenylate cyclase inhibition. ANF-(103-123)-peptide, ANF-(99-126)-peptide and ANF-(101-126)-peptide were almost equipotent [IC50 (median inhibitory concentration) = 0.1-1 nM], and ANF-(103-126)-peptide was the least potent (IC50 approximately 10 nM). Scatchard analysis of the data revealed the presence of a single class of binding sites of high affinity (Kd approximately 120 pM). Affinity cross-linking of 125I-ANF-(99-126)-peptide to its binding sites in rat platelet membranes and analysis by SDS/PAGE followed by autoradiography showed a predominant labelling of a protein band with an apparent Mr of 66,000. These data indicate the presence of only ANF-R2 (low-Mr) receptors in platelets and suggest that these receptors may be coupled to the adenylate cyclase system.
在大鼠血小板膜中研究了心房利钠因子（ANF）对腺苷酸环化酶活性的影响。ANF-(99 - 126)-、-(101 - 126)-、-(103 - 126)-和-(103 - 123)-肽以浓度依赖性方式抑制腺苷酸环化酶活性，其效力顺序为ANF-(103 - 123)-肽＞ANF-(99 - 126)-肽＞ANF-(101 - 126)-肽＞ANF-(103 - 126)-肽。ANF-(103 - 123)-肽和ANF-(99 - 126)-肽抑制酶活性约50 - 55%，表观Ki在0.1至0.5 nM之间，而ANF-(101 - 126)-肽抑制酶活性约35%，表观Ki在1至3 nM之间。另一方面，ANF-(103 - 126)-肽效力最低，抑制腺苷酸环化酶活性约30%（Ki约为10 nM）。ANF对腺苷酸环化酶的抑制作用还取决于鸟嘌呤核苷酸的存在，并且被氨氯吡咪和百日咳毒素减弱。各种激动剂如N - 乙基羧酰胺腺苷、前列腺素E1、异丙肾上腺素和福斯高林对腺苷酸环化酶的刺激作用也被ANF不同程度地抑制；然而，刺激并未完全消除。此外，125I标记的ANF-(99 - 126)-肽特异性结合大鼠血小板膜。未标记的用于抑制腺苷酸环化酶的肽以浓度依赖性方式竞争性抑制125I - ANF的结合。ANF-(103 - 123)-肽、ANF-(99 - 126)-肽和ANF-(101 - 126)-肽几乎效力相当[IC50（半数抑制浓度）= 0.1 - 1 nM]，而ANF-(103 - 126)-肽效力最低（IC50约为10 nM）。对数据的Scatchard分析显示存在一类高亲和力的单一结合位点（Kd约为120 pM）。125I - ANF-(99 - 126)-肽与大鼠血小板膜中其结合位点的亲和交联以及随后通过SDS/PAGE和放射自显影分析显示，一条表观Mr为66,000的蛋白带出现主要标记。这些数据表明血小板中仅存在ANF - R2（低Mr）受体，并提示这些受体可能与腺苷酸环化酶系统偶联。
