Protein kinase A facilitates relaxation of mouse ileum via phosphorylation of neuronal nitric oxide synthase

Abstract

Background and purpose: The enteric neurotransmitter nitric oxide (NO) regulates gastrointestinal motility by relaxing smooth muscle. Pharmacological cAMP induction also relaxes gastrointestinal smooth muscle, but it is uncertain whether cAMP augments or suppresses enteric NO signalling. In other organ systems, cAMP can increase neuronal NO production by stimulating protein kinase A (PKA) to phosphorylate neuronal NOS (nNOS) Serine-1412 (S1412). We hypothesized that cAMP also increases nNOS S1412 phosphorylation by PKA in enteric neurons to augment nitrergic relaxation of mouse ileum.

Experimental approach: We measured contractile force and nNOS S1412 phosphorylation in ileal rings suspended in an organ bath. We used forskolin to induce cAMP-dependent relaxation of wild type, nNOS^S1412A knock-in and nNOSα-null ileal rings in the presence or absence of PKA, protein kinase B (Akt) and NOS inhibitors.

Key results: Forskolin stimulated phosphorylation of nNOS S1412 in mouse ileum. Forskolin relaxed nNOSα-null and nNOS^S1412A ileal rings less than wild-type ileal rings. PKA inhibition blocked forskolin-induced nNOS phosphorylation and attenuated relaxation of wild type but not nNOS^S1412A ileum. Akt inhibition did not alter nNOS phosphorylation with forskolin but did attenuate relaxation of wild type and nNOS^S1412A . NOS inhibition with L-NAME eliminated the effects of PKA and Akt inhibitors on relaxation.

Conclusion and implications: PKA phosphorylation of nNOS S1412 augments forskolin-induced nitrergic ileal relaxation. The relationship between cAMP/PKA and NO is therefore synergistic in enteric nitrergic neurons. Because NO regulates gut motility, selective modulation of enteric neuronal cAMP synthesis may be useful for the treatment of gastrointestinal motility disorders.

Figure 1

PKA and Akt inhibitors attenuate nitrergic forskolin (FSK) relaxation of ileum. (a) The NO signalling inhibitors cPTIO (300 μM), ODQ (5 μM), and Rp‐cGMPs (5 μM) attenuate FSK relaxation. (b) PKA inhibitors (H‐89, 1 μM; Myr‐PKI, 10 μM; and Rp‐cAMPs, 25 μM) and the Akt inhibitor MK‐2206 (MK; 10 μM) attenuate FSK relaxation. (c) Akt and PKA inhibitors do not further attenuate FSK relaxation under NOS blockade with l‐NAME (1 mM), but the NOS substrate l‐Arginine (l‐Arg; 2 mM) partially restores FSK relaxation. (d) The EPAC inhibitor CE3F4 (10 μM) attenuates FSK relaxation independently of PKA, and adenylate cyclase blockade with dideoxyadenosine (DDA; 20 μM) attenuates relaxation as much as combined PKA and EPAC inhibition. FSK IC₅₀ values are bold, and 95% confidence intervals are in parentheses. ^* P < .05 versus Veh IC₅₀. Veh: DMSO = control. Error bars: SEM. n, number of ileal rings

Figure 2

Forskolin (FSK) promotes nNOS S1412 phosphorylation by PKA in the ileum. (a) FSK enhances phosphorylation of nNOS at S1412 (pS1412). (b) PKA (H‐89 and Rp‐cAMPs), but not Akt (MK), inhibition blocks FSK‐enhanced phosphorylation of nNOS S1412. Representative immunoblots and quantifications are shown on the top and bottom panels respectively. Quantification of pS1412/total nNOS is normalized to Veh (a) or Veh for each treatment (b). Symbols represent individual ileal samples, and bars are means ± SEM. FSK: 300 nM. ^* P < .05 versus Veh by Kruskal–Wallis and Dunn's post hoc tests (a) or versus Veh for each treatment by Mann–Whitney tests (b). n.s., not significant

Figure 3

nNOS Serine‐1412 potentiates forskolin (FSK) ileal relaxation. (a) FSK relaxation is reduced in both nNOS^S1412A and nNOSα‐null (nNOSα KO) compared to wild‐type (WT) mice. (b) WT, nNOS^S1412A and nNOSα KO ilea exhibit the same sensitivity to FSK under NOS blockade with l‐NAME. (c) Akt (MK), but not PKA (H‐89), inhibition further attenuates nNOS^S1412A ileal relaxation by FSK. D. Under NOS blockade with l‐NAME, neither PKA (H‐89) nor Akt (MK) inhibitors further attenuate FSK relaxation. ^* P < .05 versus WT IC₅₀ (a) or Veh IC₅₀ (c)

Figure 4

NO‐dependent forskolin (FSK) relaxation primarily requires nNOS. (a) FSK relaxation of endothelial NOS‐null (eNOS KO) ilea is sensitive to PKA (H‐89) and NOS (l‐NAME) inhibition, but not Akt inhibition (MK). (b) FSK relaxation of the eNOS KO nNOS^S1412A double homozygote (Dob. Hom.) is indistinguishable from the eNOS KO and nNOS^S1412A single mutants. (c) Inhibition of neuronal depolarization (TTX; 10 μM) or nNOS (100 μM NANT or 50 μM 1400W) blocks most NOS‐dependent FSK relaxation. ^* P < .05 versus Veh IC₅₀ (a,c). ^§Datasets from Figure 3a repeated to illustrate non‐additivity of eNOS KO and nNOS^S1412A mutations

Figure 5

nNOS S1412 phosphorylation augments cAMP‐dependent enteric relaxation. Forskolin (FSK) relaxes GI smooth muscle via nitrergic (NO‐dependent) and non‐nitrergic mechanisms. Left. A model for nitrergic FSK relaxation. FSK stimulates AC to synthesize cAMP, which activates PKA phosphorylation of nNOS S1412 in myenteric inhibitory neurons. Phosphorylation enhances nNOS activity, thereby increasing NO‐cGMP‐dependent relaxation in myocytes. Akt phosphorylation of eNOS facilitates basal NO release, but eNOS stimulation plays a small role in nitrergic FSK relaxation. Inset shows gut layers. Right. Contribution of nNOS, eNOS and other factors in FSK relaxation. Top. At low to moderate [FSK], nNOS activation is the primary relaxation mechanism in WT animals with a small contribution from eNOS. At high [FSK], relaxation is primarily non‐nitrergic and may involve AC‐independent effects of FSK. Bottom. nNOS^S1412A mutation abolishes most nitrergic FSK relaxation, but non‐phosphorylation dependent nNOS activation and possibly eNOS can still mediate nitrergic relaxation at low to moderate [FSK]