Abstract
The intracellular motor protein myosin Va is involved in nitrergic neurotransmission possibly by trafficking of neuronal nitric oxide synthase (nNOS) within the nerve terminals. In this study, we examined the role of myosin Va in the stomach and penis, proto-typical smooth muscle organs in which nitric oxide (NO) mediated relaxation is critical for function. We used confocal microscopy and co-immunoprecipitation of tissue from the gastric fundus (GF) and penile corpus cavernosum (CCP) to localize myosin Va with nNOS and demonstrate their molecular interaction. We utilized in vitro mechanical studies to test whether smooth muscle relaxations during nitrergic neuromuscular neurotransmission is altered in DBA (dilute, brown, non-agouti) mice which lack functional myosin Va. Myosin Va was localized in nNOS-positive nerve terminals and was co-immunoprecipitated with nNOS in both GF and CCP. In comparison to C57BL/6J wild type (WT) mice, electrical field stimulation (EFS) of precontracted smooth muscles of GF and CCP from DBA animals showed significant impairment of nitrergic relaxation. An NO donor, Sodium nitroprusside (SNP), caused comparable levels of relaxation in smooth muscles of WT and DBA mice. These normal postjunctional responses to SNP in DBA tissues suggest that impairment of smooth muscle relaxation resulted from inhibition of NO synthesis in prejunctional nerve terminals. Our results suggest that normal physiological processes of relaxation of gastric and cavernosal smooth muscles that facilitate food accommodation and penile erection, respectively, may be disrupted under conditions of myosin Va deficiency, resulting in complications like gastroparesis and erectile dysfunction.
Highlights
Nitric oxide (NO) produced by splice variants of the enzyme neuronal nitric oxide synthase has been shown to be a major inhibitory neurotransmitter at smooth muscle neuromuscular junctions
The present study demonstrates an important role for myosin Va in nitrergic neurotransmission
We showed, using disparate smooth muscle neuromuscular junctions, that: 1) myosin Va localizes in nitrergic nerve fibers in gastric fundus (GF) and corpus cavernosum of the penis (CCP); 2) neuronal nitric oxide synthase (nNOS) interacts with myosin Va in both GF and CCP; 3) smooth muscle relaxation in response to nitrergic nerve stimulation is suppressed in DBA mice in both the GF and CCP due to impaired synthesis of NO rather than defective post-junctional NO signaling
Summary
Nitric oxide (NO) produced by splice variants of the enzyme neuronal nitric oxide synthase (nNOS) has been shown to be a major inhibitory neurotransmitter at smooth muscle neuromuscular junctions. Smooth muscle relaxation facilitated by NO released from nerve varicosities subserves essential physiological functions in many organ systems that are as diverse as gastric motility and penile erection [1,2,3,4]. In these prototypical examples of nitrergic neurotransmission, impaired relaxation has been shown to cause, respectively, a variety of gastrointestinal motility disorders including loss of gastric accommodation [5] and erectile dysfunction [6]. Failed nitrergic neurotransmission may result from an absence or critical reduction in the amount of nNOS or impairment of its catalytic function. The subcellular localization of nNOS regulates its catalytic activity [13,14,15]
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