Murthy K S, Zhang K M, Jin J G, Grider J R, Makhlouf G M
Department of Physiology, Medical College of Virginia, Richmond 23298.
Am J Physiol. 1993 Oct;265(4 Pt 1):G660-71. doi: 10.1152/ajpgi.1993.265.4.G660.
Vasoactive intestinal peptide (VIP) and peptide histidine-isoleucine (PHI) receptors and the signaling pathways to which they are coupled were characterized in dispersed gastric smooth muscle cells. Radioligand binding using 125I-labeled VIP and PHI identified 4 classes of receptors: VIP-preferring and PHI-preferring receptors recognized by both ligands and readily desensitized by the preferred ligand, and VIP-specific and PHI-specific receptors recognized by only 1 ligand and resistant to desensitization. All except VIP-specific receptors were coupled to adenylate cyclase. VIP-specific receptors mediated a G protein-coupled Ca2+ influx that led to activation of NO synthase (NOS), NO-dependent activation of soluble guanylate cyclase, and activation of guanosine 3',5'-cyclic monophosphate (cGMP) kinase resulting in muscle relaxation. The entire cascade was blocked by Ca2+ channel and/or calmodulin antagonists. The NOS inhibitor NG-nitro-L-arginine abolished L-[3H]citrulline (coproduct of NO synthesis) and cGMP generation and partly inhibited (52 +/- 4%) relaxation. The components of response mediated by VIP-specific receptors (increase in [Ca2+]i, L-[3H]citrulline, and cGMP) were preserved after desensitization. Insertion of guanosine 5'-O-(beta-thio)diphosphate into reversibly permeabilized muscle cells abolished responses mediated by VIP-preferring and VIP-specific receptors. VIP stimulated both adenosine 3',5'-cyclic monophosphate (cAMP)-kinase and cGMP-kinase activities consistent with stimulation of cAMP and cGMP. Both kinases contributed to relaxation that was partly inhibited by cAMP-kinase [H-89 and (R)-p-adenosine 3',5'-cyclic monophosphorothioate] and cGMP-kinase (KT-5823) inhibitors and abolished by a combination of the 2 types of inhibitors. We conclude that VIP-specific receptors mediate a G protein-coupled Ca2+ influx leading to activation of a constitutive Ca2+/calmodulin-dependent NOS and generation of NO, which is partly responsible for relaxation in smooth muscle.
在分散的胃平滑肌细胞中对血管活性肠肽（VIP）和肽组氨酸异亮氨酸（PHI）受体及其偶联的信号通路进行了表征。使用125I标记的VIP和PHI进行放射性配体结合鉴定出4类受体：两种配体均可识别的VIP偏好性和PHI偏好性受体，且易被偏好性配体脱敏；仅被1种配体识别且对脱敏有抗性的VIP特异性和PHI特异性受体。除VIP特异性受体外，所有受体均与腺苷酸环化酶偶联。VIP特异性受体介导一种G蛋白偶联的Ca2+内流，导致一氧化氮合酶（NOS）激活、可溶性鸟苷酸环化酶的NO依赖性激活以及鸟苷3',5'-环磷酸（cGMP）激酶激活，从而导致肌肉松弛。整个级联反应被Ca2+通道和/或钙调蛋白拮抗剂阻断。NOS抑制剂NG-硝基-L-精氨酸消除了L-[3H]瓜氨酸（NO合成的副产物）和cGMP生成，并部分抑制（52±4%）松弛。脱敏后，VIP特异性受体介导的反应成分（[Ca2+]i、L-[3H]瓜氨酸和cGMP增加）得以保留。将鸟苷5'-O-（β-硫代）二磷酸插入可逆透化的肌肉细胞中消除了由VIP偏好性和VIP特异性受体介导的反应。VIP刺激腺苷3',5'-环磷酸（cAMP）激酶和cGMP激酶活性，这与cAMP和cGMP的刺激一致。两种激酶均有助于松弛，cAMP激酶[H-89和（R）-p-腺苷3',5'-环磷酸硫代酯]和cGMP激酶（KT-5823）抑制剂可部分抑制松弛，两种类型抑制剂联合使用则可消除松弛。我们得出结论，VIP特异性受体介导一种G蛋白偶联的Ca2+内流，导致组成型Ca2+/钙调蛋白依赖性NOS激活和NO生成，这部分负责平滑肌的松弛。
