Insulin enhances the bradykinin response in L8 rat skeletal myoblasts

Inhibitors of ACE/kininase II enhance insulin sensitivity, an action that i s mediated in part by bradykinin (BK). We investigated whether insulin inte racts with the BK receptor signaling to modulate the inositol 1,4,5-trispho sphate (IP3) response to BK in 18 rat skeletal myoblasts. Stimulation of th e cultures with BR (10 nmol/l) for 15 s increased IP3 from a basal level of 75.2 +/- 7.6 to 200.2 +/- 15.7 pmol/mg protein. Treatment of the cultures with 1, 2, and 20 nmol/l of insulin for 90 min before adding BK increased I P3 formation by the same BK dose to 328.2 +/- 19, 434.5 +/- 18, and 460.8 /- 21.3 pmol/mg protein, respectively. When wortmannin was administered to inhibit phosphatidylinositol (PI) 3-kinases at lower concentration (1 nmol/ l), it increased IP3 formation stimulated by BK only when insulin was prese nt. At a higher concentration (100 nmol/l), wortmannin significantly enhanc ed BK-induced IP3 formation in the absence of insulin. Genistein and tyrpho stin A-23, tyrosine kinase inhibitors, completely reversed the elevated IP3 formation by BK and insulin. The IP3 response to 10 nmol/l BK was 223.3 +/ - 11.8 pmol/mg protein in the absence of insulin and 402.2 +/- 12.0 pmol/mg protein in the presence of 2 nmol/l insulin. However, when exposing the cu ltures to 1 nmol/l genistein or tyrphostin A-23, the IP3 response to BK in the presence of insulin decreased to 211.8 +/- 46.7 and 187.7 +/- 19.9 pmol /mg protein. Tyrphostin A-1, the inactive analog, was ineffective. Exposing the cells to 1 mu mol/l 3,4,5-trimethoxybenzoic acid 8-[diethylamino]octyl ester, an intracellular Ca2+ antagonist, did not change the potentiation b y insulin. But, exposing them to 0.1 mu mol/l n-[6-aminohexyl]-5- chloro-1- naphthalene-sulfonamide, a calmodulin antagonist, resulted in enhanced IP3 response to BK alone to 292.2 +/- 18.5 pmol/mg protein and to BK in the pre sence of 1, 2, and 20 nmol/l insulin to 488 +/- 22.2, 625.5 +/- 11.61 and 6 85.2 +/- 15.9 pmol/mg protein, respectively. In conclusion, insulin potenti ates BK-induced IP3 production in 18 rat skeletal myoblasts, and this actio n of insulin involves a tyrosine kinase. Inhibition of PI Q-kinases potenti ated BK-induced IP3 formation in the presence of insulin. Calmodulin blocke d the action of insulin. These results support a modulatory effect of insul in on the BK signaling system via a tyrosine kinase in 18 rat skeletal myob lasts that results in increased IP3 formation. Because BK release from skel etal muscle increases during contractions, this action of insulin is likely to play a role in the modulation of the excitation-contraction coupling pr ocess of the skeletal muscle.