Impaired metabolic modulation of alpha-adrenergic vasoconstriction in dystrophin-deficient skeletal muscle

Abstract

The neuronal isoform of nitric oxide synthase (nNOS) is highly expressed in mammalian skeletal muscle, but its functional role has not been defined. NO has been implicated in the local metabolic regulation of blood flow in contracting skeletal muscle in part by antagonizing sympathetic vasoconstriction. We therefore hypothesized that nNOS in skeletal muscle is the source of the NO mediating the inhibition of sympathetic vasoconstriction in contracting muscle. In the mdx mouse, a model of Duchenne muscular dystrophy in which dystrophin deficiency results in greatly reduced expression of nNOS in skeletal muscle, we found that the normal ability of skeletal muscle contraction to attenuate alpha-adrenergic vasoconstriction is defective. Similar results were obtained in mutant mice that lack the gene encoding nNOS. Together these data suggest a specific role for nNOS in the local metabolic inhibition of alpha-adrenergic vasoconstriction in active skeletal muscle.

Figure 1

Expression of NOS isoforms in skeletal muscle and in vivo responses to endogenous and exogenous NO in C57 and mdx mice. (a) Western blots showing nNOS and eNOS immunoreactivity in supernatant (S) and pellet (P) fractions of gastrocnemius muscle homogenates. In mdx muscle, eNOS expression was normal, whereas nNOS expression was greatly reduced. Note that NOS immunoreactivity in the supernatant fractions is caused by the presence of the detergent CHAPS in the homogenizing buffer. Data shown are representative of results obtained in 2 C57 and 3 mdx mice. (b and c) Hypotensive responses to i.v. injection of the endogenous NO donor acetylcholine (3 μg/kg) or the exogenous NO donor nitroprusside (0.8 μg/kg) were similar in C57 (n = 8) and mdx (n = 8) mice (solid bars). In both groups of mice, NOS inhibition with l-NAME (open bars) attenuated the acetylcholine-mediated hypotension and enhanced the nitroprusside-mediated hypotension. AUC, area under the curve in arbitrary units; ∗, significantly different from response before l-NAME treatment.

Figure 2

Metabolic inhibition of α-adrenergic vasoconstriction in contracting skeletal muscle is prevented by NOS inhibition in wild type C57 mice. (a and c) Segments of an original record from an experiment in one C57 mouse showing the arterial blood pressure (BP) and femoral blood flow velocity (FBF) responses to intraarterial injection of norepinephrine (NE) in resting and contracting hindlimbs before and after NOS inhibition with l-NAME. Before l-NAME treatment, NE injection in resting hindlimbs increased arterial pressure and decreased femoral blood flow velocity (Left in a). In contrast, NE-mediated vasoconstriction was attenuated in contracting hindlimbs, as demonstrated by an increase, rather than decrease, in femoral blood flow velocity in response to NE (Right in a). In this same mouse, l-NAME treatment prevented the contraction-induced inhibition of adrenergic vasoconstriction as indicated by the decrease in femoral blood flow velocity in response to NE during hindlimb contraction (c). (b and d) Summary data showing the NE-mediated decreases in calculated femoral vascular conductance (FVC) in resting and contracting (Contr) hindlimb before and after l-NAME treatment. Hindlimb contraction greatly attenuated the NE-mediated decrease in vascular conductance before (b; n = 10), but not after (d; n = 8), l-NAME treatment.

Figure 3

Metabolic inhibition of α-adrenergic vasoconstriction is defective in contracting nNOS-deficient skeletal muscle of mdx and nNOS knockout mice. (a and c) Segments of original records from experiments in an mdx mouse (a) and in an nNOS knockout mouse (c) showing the arterial blood pressure (BP) and femoral blood flow velocity (FBF) responses to intraarterial injection of norepinephrine (NE) in resting and contracting hindlimb. In both mice, NE injection in resting hindlimb increased arterial pressure and decreased femoral blood flow velocity (Left in a and c). This NE-mediated vasoconstriction was not attenuated in contracting hindlimbs, indicating that metabolic modulation of α-adrenergic vasoconstriction was impaired in nNOS-deficient muscle (Right in a and c). (b and d) Summary data showing the NE-mediated decreases in calculated femoral vascular conductance (FVC) in resting and contracting (Contr) hindlimb. The NE-mediated decreases in vascular conductance were not attenuated by hindlimb contraction in mdx mice (b; n = 10) or in nNOS knockout mice (d; n = 6).