Rabito S F, Minshall R D, Nakamura F, Wang L X
Department of Anesthesiology, Cook County Hospital, Chicago, IL 60612, USA.
Diabetes. 1996 Jan;45 Suppl 1:S29-33. doi: 10.2337/diab.45.1.s29.
To determine the presence of bradykinin receptors in skeletal muscle, we examined in both displacement and saturation studies the binding of [125I-Tyr8]bradykinin or [3H]bradykinin in three types of skeletal muscle preparations: membrane fractions from guinea pig hindlimb quadriceps, dog semimembranosus and semitendinosus muscles, and L8 rat skeletal muscle myoblasts. Scatchard analysis of [125I-Tyr8]bradykinin x bradykinin competition binding demonstrated specific bradykinin binding of 4.9 and 3.2 fmol/mg protein in dog and guinea pig skeletal muscle preparations, respectively. Unlabeled bradykinin specifically displaced [125I-Tyr8]bradykinin with IC50 values of 36.5 +/- 6 and 118.0 +/- 16.0 pmol/l from dog and guinea pig muscle membranes, respectively. The B2 bradykinin receptor antagonist HOE 140 and the B1 bradykinin receptor antagonist des-Arg9[Leu8]bradykinin displaced the binding of [3H]bradykinin from dog membranes with IC50 values of 0.38 and 217.3 nmol/l, respectively, suggesting that bradykinin binds to a B2-type receptor. In addition, unlabeled bradykinin competed with [3H]bradykinin for binding to dog skeletal muscle membrane preparations in a biphasic manner. To assess whether this represents multiple bradykinin receptor subtypes present in skeletal muscle homogenates or several affinity states of a single binding site, we examined bradykinin receptors on a pure skeletal muscle system, the L8 neonatal rat skeletal muscle myoblast cell line. These myoblasts also contain specific [3H]bradykinin-binding sites with a Bmax of 271 fmol/mg protein and a Kd of 0.83 nmol/l. Competitive agonist binding curves were biphasic (high-affinity IC50 = 3.9 pmol/l, low-affinity IC50 = 22.6 nmol/l) in the absence of guanosine 5'-O-(3-thio-trisphosphate) (GTP gamma S); they shifted to a model of one affinity (8.1 nmol/l) in the presence of GTP gamma S. Because the enzyme neutral endopeptidase 24.11 is an important kininase in skeletal muscle, we examined the effect of the neutral endopeptidase inhibitor phosphoramidon on the binding of bradykinin to dog skeletal muscle membranes. We found that phosphoramidon decreased the apparent Bmax from 7.3 to 5.8 fmol/mg protein. In addition, in this cell line we investigated the action of bradykinin on phosphoinositide hydrolysis. Inositol 1,4,5-trisphosphate (IP3) was measured with a radioreceptor assay. Bradykinin (0.1 nmol/l to 1 mumol/l) induced IP3 formation in a dose-dependent manner (EC50 = 1.42 nmol/l) from a basal level of 72.8 +/- 16 pmol/mg protein to 433 +/- 35.5 at the highest (1 mumol/l) concentration. We conclude that bradykinin B2 receptors are expressed in skeletal muscle. Phosphoinositide hydrolysis upon stimulation of this receptor is an indicator of intracellular signal transduction. Part of the bradykinin binding in skeletal muscle is due to interaction with the enzyme neutral endopeptidase.
为了确定骨骼肌中缓激肽受体的存在，我们在置换和饱和研究中检测了[125I-Tyr8]缓激肽或[3H]缓激肽在三种骨骼肌制剂中的结合情况：豚鼠后肢股四头肌、犬半膜肌和半腱肌的膜组分，以及L8大鼠骨骼肌成肌细胞。对[125I-Tyr8]缓激肽×缓激肽竞争结合进行Scatchard分析表明，在犬和豚鼠骨骼肌制剂中，缓激肽的特异性结合分别为4.9和3.2 fmol/mg蛋白质。未标记的缓激肽能特异性置换[125I-Tyr8]缓激肽，在犬和豚鼠肌膜中的IC50值分别为36.5±6和118.0±16.0 pmol/l。缓激肽B2受体拮抗剂HOE 140和缓激肽B1受体拮抗剂去-Arg9[Leu8]缓激肽从犬膜中置换[3H]缓激肽结合的IC50值分别为0.38和217.3 nmol/l，这表明缓激肽与B2型受体结合。此外，未标记的缓激肽与[3H]缓激肽竞争结合犬骨骼肌膜制剂呈双相性。为了评估这是代表骨骼肌匀浆中存在多种缓激肽受体亚型还是单一结合位点的几种亲和状态，我们在一个纯骨骼肌系统——L8新生大鼠骨骼肌成肌细胞系上检测了缓激肽受体。这些成肌细胞也含有特异性的[3H]缓激肽结合位点，Bmax为271 fmol/mg蛋白质，Kd为0.83 nmol/l。在不存在鸟苷5'-O-(3-硫代三磷酸)(GTPγS)的情况下，竞争性激动剂结合曲线呈双相性(高亲和力IC50 = 3.9 pmol/l，低亲和力IC50 = 22.6 nmol/l)；在存在GTPγS的情况下，它们转变为单一亲和力(8.1 nmol/l)的模式。由于中性内肽酶24.11是骨骼肌中一种重要的激肽酶，我们检测了中性内肽酶抑制剂磷酰胺素对缓激肽与犬骨骼肌膜结合的影响。我们发现磷酰胺素使表观Bmax从7.3降至5.8 fmol/mg蛋白质。此外，在这个细胞系中，我们研究了缓激肽对磷脂酰肌醇水解的作用。用放射受体分析法测量肌醇1,4,5-三磷酸(IP3)。缓激肽(0.1 nmol/l至1 μmol/l)以剂量依赖性方式诱导IP3形成(EC50 = 1.42 nmol/l)，从基础水平72.8±16 pmol/mg蛋白质在最高(1 μmol/l)浓度时升至433±35.5。我们得出结论，缓激肽B2受体在骨骼肌中表达。该受体受刺激后磷脂酰肌醇水解是细胞内信号转导的一个指标。骨骼肌中部分缓激肽结合是由于与中性内肽酶的相互作用。
