DIABETES, VOLUME 45, SUPPLEMETN 1, JANUARY 1996, PAGES S29-SS33

Bradykinin B2 Receptors on Skeletal Muscle Are Coupled to Inositol 1,4,5-Trisphosphate Formation

Sara F. Rabito, Richard D. Minshall, Fumiaki Nakamura, and Li-Xiu Wang

To determine the presence of bradykinin receptors in skeletal muscle, we examined in both displacement and saturation studies the binding of [^l25I-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 [^l25I-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/1 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/1, 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 myeblast cell line. These myoblasts also contain specific [³H]bradykinin-binding sites with a B_max of 271 fmol/mg protein and a K^d of 0.83 nmol/1. Competitive agonist binding curves were biphasic (high-affinity IC₅₀ = 3.9 pmol/1, low-affinity IC₅₀ = 22.6 nmol/l) in the absence of guanosine 5'-0-(3-thiotrisphosphate) (GTPYS); they shifted to a model of one affinity (8.1 nmol/1) in the presence of GTPYS. 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 D_max 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/1 to 1 ?mol/l) induced IP3 formation in a dose-dependent manner (EC₅₀ = 1.42 nmol/1) from a basal level of 72.8 + 16 pmol/mg protein to 433 + 35.5 at the highest (1 µmol/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. Diabetes 45 (Suppl. 1):S29-S33, 1996

Copyright © 1996 American Diabetes Association

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