Akt phosphorylation of neuronal nitric oxide synthase regulates gastrointestinal motility in mouse ileum Damian D. Guerraa, Rachael Boka, Vibhuti Vyasa, David J. Orlickyb, Ramón A. Lorcaa, and K. Joseph Hurta,c,1 aDivision of Reproductive Sciences, Department of Obstetrics and Gynecology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045; bDepartment of Pathology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045; and cDivision of Maternal Fetal Medicine, Department of Obstetrics and Gynecology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045 Edited by Solomon H. Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved July 15, 2019 (received for review April 5, 2019) Nitric oxide (NO) is a major inhibitory neurotransmitter that mediates receptor activation (18–20). Some evidence suggests that phosphor- nonadrenergic noncholinergic (NANC) signaling. Neuronal NO syn- ylation of the equivalent Akt/PKA consensus site in nNOS, ser- + thase (nNOS) is activated by Ca2 /calmodulin to produce NO, which ine1412 (S1412), also stimulates neuronal NO synthesis (21). Because + causes smooth muscle relaxation to regulate physiologic tone. nNOS nNOS activity is more sensitive to [Ca2 ](21),andnNOSismore serine1412 (S1412) phosphorylation may reduce the activating rapidly dephosphorylated than eNOS (22), it is technically difficult to 2+ Ca requirement and sustain NO production. We developed and evaluate nNOS S1412 phosphorylation in vivo. Nonetheless, studies S1412A characterized a nonphosphorylatable nNOS knock-in mouse implicate nNOS S1412 phosphorylation in hippocampal excitotoxicity and evaluated its enteric neurotransmission and gastrointestinal (4), penile erection (22), and luteinizing hormone release (23). (GI) motility to understand the physiologic significance of nNOS Enteric neurons express high levels of nNOS, Akt, and PKA S1412 phosphorylation. Electrical field stimulation (EFS) of wild- (24, 25), indicating that nNOS S1412 phosphorylation may reg- type (WT) mouse ileum induced nNOS S1412 phosphorylation ulate GI neurotransmission. To test this, we performed organ that was blocked by tetrodotoxin and by inhibitors of the protein bath experiments using mouse ileum and electrical field stimu- kinase Akt but not by PKA inhibitors. Low-frequency depolariza- lation (EFS) and created a knock-in mouse in which nNOS tion increased nNOS S1412 phosphorylation and relaxed WT S1412 is replaced by nonphosphorylatable alanine (nNOSS1412A). ileum but only partially relaxed nNOSS1412A ileum.Athigherfre- quencies, nNOS S1412 had no effect. nNOSS1412A ileum expressed We found that S1412 phosphorylation facilitates GI relaxation PHYSIOLOGY during minimal neuronal depolarization. Our results suggest less phosphodiesterase-5 and was more sensitive to relaxation by 2+ exogenous NO. Under non-NANC conditions, peristalsis and seg- depolarization activates Akt to promote Ca -independent mentationwerefasterinthenNOSS1412A ileum. Together these find- nNOS activation. These findings offer targets to treat GI motility ings show that neuronal depolarization stimulates enteric nNOS disorders and are consistent with depolarization-dependent Akt phosphorylation by Akt to promote normal GI motility. Thus, phos- stimulation of nNOS as a general NO signaling mechanism in the phorylation of nNOS S1412 is a significant regulatory mechanism for autonomic nervous system and brain. nitrergic neurotransmission in the gut. Results enteric nervous system | gasotransmitter | nitric oxide (NO) | ileum ENS Depolarization Stimulates Akt Phosphorylation of nNOS S1412. Glutamate stimulation of rat cortical neurons induces phos- itric oxide (NO) is a major nonadrenergic noncholinergic phorylation of nNOS S1412 by Akt (4), and direct electrical N(NANC) inhibitory signal in the peripheral nervous system, including the gastrointestinal (GI) tract. The enteric nervous Significance system (ENS) produces NO that relaxes GI smooth muscle to regulate physiologic peristalsis (1–3). Neuronal NO synthase Neuronal nitric oxide synthase (nNOS) is believed to be activated + (nNOS) is the primary source of NO in the gut (4, 5), and altered primarily by Ca2 /calmodulin. Activation of nNOS by phosphor- NO production can disrupt normal GI motility. In mice, NO-cyclic ylation similar to endothelial NOS has been suggested but is guanosine- 3′,5′-monophosphate (NO-cGMP) pathway antagonists technically difficult to confirm in vivo. We developed a knock-in inhibit GI smooth muscle relaxation (2), and genetic deletion of mouse with nNOS serine1412 mutated to alanine and show nNOS delays bowel transit (5, 6). In humans, nNOS inhibition in- using mouse ileum that nNOS phosphoserine1412 regulates gas- creases GI contractility (7), while increased nitrergic neurons are trointestinal smooth muscle relaxation and functional motility. found in idiopathic chronic constipation and other motility disorders nNOS is phosphorylated by the protein kinase Akt during sub- (8). Diabetic patients exhibit defective nitrergic GI relaxation (9), but maximal neuronal depolarization to augment NO-cGMP signal- 2+ enteric nNOS expression appears unchanged (10, 11). This suggests ing in a manner tuned toward resting intracellular Ca levels. that both nNOS protein expression and posttranslational regulation Phosphorylation at nNOS serine1412 represents a previously influence GI motility. unrecognized physiologic mechanism of nNOS regulation linking Depolarization of nitrergic enteric neurons leads to voltage- neuronal depolarization to gastrointestinal motility and clarifies + dependent calcium (Ca2 ) entry, activation of calmodulin (CaM), characteristics of neuronal NO production. and increased NO production by nNOS. NO stimulates soluble Author contributions: D.D.G. and K.J.H. designed research; D.D.G., R.B., V.V., D.J.O., and guanylate cyclase (sGC) to produce cGMP, which activates protein K.J.H. performed research; D.J.O., R.A.L., and K.J.H. contributed new reagents/analytic kinase G (PKG) to promote smooth muscle relaxation (12, 13). El- tools; D.D.G., R.B., V.V., D.J.O., R.A.L., and K.J.H. analyzed data; and D.D.G., R.A.L., and + evated neuronal intracellular [Ca2 ] is therefore a major stimulus for K.J.H. wrote the paper. + nitrergic relaxation (14). Sustained high [Ca2 ]candamagecells(15), The authors declare no conflict of interest. 2+ so generally only brief depolarization transiently activates Ca entry This article is a PNAS Direct Submission. (16). Because GI myocyte relaxation is prolonged (>5s)(17),nNOS Published under the PNAS license. posttranslational modification may sustain NO production in- 1To whom correspondence may be addressed. Email: [email protected]. 2+ 2+ dependently of Ca entry. Ca -inde-pendent activation of endo- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. thelial NOS (eNOS) by Akt or PKA phosphorylation of eNOS 1073/pnas.1905902116/-/DCSupplemental. serine1179 is well established in response to vascular shear stress and www.pnas.org/cgi/doi/10.1073/pnas.1905902116 PNAS Latest Articles | 1of6 Downloaded by guest on September 23, 2021 depolarization of rat pelvic ganglia increases nNOS S1412 can modulate nitrergic signaling via S1412 phosphorylation in phosphorylation by PKA (22). In vitro, the phosphomimetic the gut. + mutant nNOSS1412D is more active than WT nNOS at low [Ca2 ] We next determined the role of Akt and PKA in mediating typical of resting neurons (21). To test if depolarization increases nNOS S1412 phosphorylation in the ileum during EFS stimula- nNOS phosphorylation in the ENS, we applied EFS to rings of tion by adding selective kinase inhibitors. Two Akt inhibitors wild-type (WT) mouse ileum suspended in physiologic organ (Akti and MK-2206 [MK]) significantly attenuated EFS re- C bath under NANC conditions. Both low (0.25 Hz) and medium laxation at 0.25 and 0.5 Hz (Fig. 1 ), suggesting that low- (1 Hz) frequency EFS increased S1412 phosphorylation by at least frequency depolarization promotes relaxation mediated by Akt. 2-fold compared to sham-treated unstimulated rings (Fig. 1A and Activation of Akt by depolarization has been observed in cortical neurons, neuroblastoma cells, and cardiac myocytes (4, 27–29). SI Appendix,Fig.S1) (22). The pan-NOS inhibitor L-NAME blocked In contrast to Akt inhibitors, the PKA inhibitors H89 and myr- EFS-induced ileal relaxation (SI Appendix, Fig. S2), confirming istoylated PKA inhibitor peptide (PKI) had no effect on EFS that NOS was the predominant inhibitory mediator (4, 26). The + responses (Fig. 1D). EFS stimulated Akt S473 phosphorylation voltage-gated Na channel antagonist tetrodotoxin (TTX), which by about 20 to 30% in total ileal lysates, an indication of in- blocks neuronal depolarization, also prevented EFS-stimulated creased Akt kinase activity (Fig. 1E and SI Appendix, Fig. S1). B SI nNOS S1412 phosphorylation (Fig. 1 ) and ileal relaxation ( While Akt inhibitors blocked EFS-induced S1412 phosphoryla- Appendix ,Fig.S2). These data suggest that neuronal depolarization tion (Fig. 1 F and G), PKA inhibition by H89 did not (Fig. 1H). Thus, low-frequency neuronal depolarization stimulates Akt to phosphorylate S1412 and enhance nitrergic GI relaxation. A EFS (Hz) B Veh. + TTX 0 0.25 1.0 Mutating nNOS S1412 to Alanine Attenuates Akt-Dependent Nitrergic EFS 0 0 1.0 1.0 0 0 1.0 1.0 Relaxation. Because Akt inhibition blocks both nNOS S1412 phos- pS1412 pS1412 (Hz) phorylation and ileal relaxation during EFS, we hypothesized