Interactions of ANG II AT2 receptor signaling with ANF

心钠素（ANF）和血管紧张素Ⅱ（Ang Ⅱ）在调节电解质平衡和血压方面发挥重要作用。ANF和Ang II通常相互拮抗。然而，血管紧张素II和心钠素的相互作用的分子基础的事件仍有待建立。已显示Ang II AT 2受体通过未知蛋白酪氨酸磷酸酶抑制ANF刺激的鸟苷酸环化酶活性。已显示PC 12和兔肾小管上皮细胞具有AT 2受体亚型。使用这些细胞，我们发现，血管紧张素II抑制心钠素刺激的鸟苷酸环化酶的活性和刺激蛋白酪氨酸磷酸酶的活动，并转染促分裂原活化的蛋白激酶磷酸酶（MKP- 1）和蛋白酪氨酸磷酸酶，SHP-1，抑制心钠素刺激的鸟苷酸环化酶的活性。因此，MKP-1或SHP-1可能是介导Ang II抑制ANF信号的蛋白酪氨酸磷酸酶。我们的研究表明，ANF的结合诱导GCRP/GCRP-2（鸟苷酸环化酶调节蛋白）与鸟苷酸环化酶的关联，导致酶激活。最近，道格拉斯博士的实验室发现，AT 2受体通过G蛋白β γ亚基释放花生四烯酸，然后激活EGF受体/ERK（丝裂原活化蛋白激酶）级联反应。我们已经发现花生四烯酸诱导MKP-1的表达。因此，G蛋白β γ亚单位/花生四烯酸或EGF受体/ERK级联可能介导Ang II对MKP-1诱导或MKP-1/SHP-1激活的作用。基于这些发现，我们假设：1）Ang II通过G蛋白β γ亚基/花生四烯酸或EGF受体/ERK级联激活或诱导MKP-1/SHP-1; 2）MKP-1或SHP-1然后与鸟苷酸环化酶或GCRP/GCRP-2缔合并使其去磷酸化，从而抑制ANF刺激的鸟苷酸环化酶活性。为了验证这些假设，我们用PC 12和AT 1和AT 2受体基因敲除小鼠的肾小管上皮细胞，提出：1）检测MKP-1/SHP-1是否位于Ang Ⅱ激活后G蛋白β γ亚基/花生四烯酸或EGF受体/ERK级联反应的下游;以及3）检测MKP- 1/SHP-1是否与鸟苷酸环化酶或GCRP（或GCRP-2）结合并使其去磷酸化，从而导致抑制ANF刺激的鸟苷酸环化酶活性。从这些研究中产生的信息将有助于了解在分子水平上的血管紧张素II和心钠素信号的相互作用。

Atrial natriuretic factor (ANF) and angiotensin (Ang II) exert potent effects on the regulation of electrolyte balance and blood pressure. ANF and Ang II normally antagonize each other. However, the molecular event underlying the interplay of Ang II and ANF remains to be established. The Ang II AT2 receptor has been shown to inhibit ANF- stimulated guanylate cyclase activity through an unknown protein tyrosine phosphatase. PC12 and rabbit renal tubular epithelial cells have been shown to possess an AT2 receptor subtype. Using these cells, we have found that Ang II inhibits ANF-stimulated guanylate cyclase activity and stimulates protein tyrosine phosphatase activities, and that transfection of the mitogen-activated protein kinase phosphatase (MKP- 1) and protein tyrosine phosphatase, SHP-1, inhibits ANF-stimulated guanylate cyclase activity. Therefore, MKP-1 or SHP-1 may be the protein tyrosine phosphatase that mediates the inhibitory effect of Ang II on ANF signaling. Our studiers indicate that binding of ANF induces the association of GCRP/GCRP-2 (guanylate cyclase regulatory proteins) with guanylate cyclase leading to enzyme activation. Recently, Dr. Douglas's laboratory has found that the AT2 receptor, through G protein betagamma subunits, releases arachidonic acid which then activates EGF receptor/ERK (mitogen-activate protein kinase) cascade. We have found that arachidonic acid induces the expression of MKP-1. Therefore, G protein betagamma subunits/arachidonic acid or EGF receptor/ERK cascade may mediate the effect of Ang II on MKP-1 induction or MKP-1/SHP-1 activation. Based on these findings, we hypothesize that: 1) Ang II activates or induces MKP-1/SHP-1 through G protein betagamma subunits/arachidonic acid or EGF receptor/ERK cascade; and 2) MKP-1 or SHP-1 then associates with and dephosphorylates either guanylate cyclase or GCRP/GCRP-2 leading to the inhibition of ANF-stimulated guanylate cyclase activity. To evaluate these hypotheses, using PC12 and renal tubular epithelial cells from rabbit angiotensin II AT1 and AT2 receptor knock out mice, we propose: 1) to examine whether MKP-1/SHP-1 is downstream of G protein betagamma subunits/arachidonic acid or EGF receptor/ERK cascade following Ang II activation; and 3) to examine whether MKP- 1/SHP-1 associate with and dephosphorylates either guanylate cyclase or GCRP (or GCRP-2) leading to the inhibition of ANF-stimulated guanylate cyclase activity. The information generated from these studies will help to understand the interplay of Ang II and ANF signaling in the molecular level.