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Nitrergic neuromuscular transmission in the mouse internal anal sphincter is accomplished by multiple pathways and postjunctional effector cells.

ABSTRACT: The effector cells and second messengers participating in nitrergic neuromuscular transmission (NMT) were investigated in the mouse internal anal sphincter (IAS). Protein expression of guanylate cyclase (GC?, GC?) and cyclic GMP-dependent protein kinase I (cGKI) were examined in cryostat sections with dual-labeling immunohistochemical techniques in PDGFR?(+) cells, interstitial cells of Cajal (ICC), and smooth muscle cells (SMC). Gene expression levels were determined with quantitative PCR of dispersed cells from Pdgfr?(egfp/+), Kit(copGFP/+), and smMHC(Cre-egfp) mice sorted with FACS. The relative gene and protein expression levels of GC? and GC? were PDGFR?(+) cells > ICC ? SMC. In contrast, cGKI gene expression sequence was SMC = ICC > PDGFR?(+) cells whereas cGKI protein expression sequence was neurons > SMC ? ICC = PDGFR?(+) cells. The functional role of cGKI was investigated in cGKI(-/-) mice. Relaxation with 8-bromo (8-Br)-cGMP was greatly reduced in cGKI(-/-) mice whereas responses to sodium nitroprusside (SNP) were partially reduced and forskolin responses were unchanged. A nitrergic relaxation occurred with nerve stimulation (NS, 5 Hz, 60 s) in cGKI(+/+) and cGKI(-/-) mice although there was a small reduction in the cGKI(-/-) mouse. N(?)-nitro-l-arginine (l-NNA) abolished responses during the first 20-30 s of NS in both animals. The GC inhibitor ODQ greatly reduced or abolished SNP and nitrergic NS responses in both animals. These data confirm an essential role for GC in NO-induced relaxation in the IAS. However, the expression of GC and cGKI by all three cell types suggests that each may participate in coordinating muscular responses to NO. The persistence of nitrergic NMT in the cGKI(-/-) mouse suggests the presence of a significant GC-dependent, cGKI-independent pathway.

SUBMITTER: Cobine CA

PROVIDER: S-EPMC4254957 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Nitrergic neuromuscular transmission in the mouse internal anal sphincter is accomplished by multiple pathways and postjunctional effector cells.

Cobine C A CA Sotherton A G AG Peri L E LE Sanders K M KM Ward S M SM Keef K D KD

American journal of physiology. Gastrointestinal and liver physiology 20141009 11

The effector cells and second messengers participating in nitrergic neuromuscular transmission (NMT) were investigated in the mouse internal anal sphincter (IAS). Protein expression of guanylate cyclase (GCα, GCβ) and cyclic GMP-dependent protein kinase I (cGKI) were examined in cryostat sections with dual-labeling immunohistochemical techniques in PDGFRα(+) cells, interstitial cells of Cajal (ICC), and smooth muscle cells (SMC). Gene expression levels were determined with quantitative PCR of di ...[more]

PMID: 25301187

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Project description:BACKGROUND AND PURPOSE: The internal anal sphincter has been shown to contract in response to alpha1-adrenoceptor stimulation and therefore alpha1-adrenoceptor agonists may be useful in treating faecal incontinence. This study characterizes the alpha1-adrenoceptor subtype responsible for mediating contraction of the internal anal sphincter of the pig. EXPERIMENTAL APPROACH: The potency of agonists and the affinities of several receptor subtype selective antagonists were determined on smooth muscle strips for the pig internal anal sphincter. Cumulative concentration-response curves were performed using phenylephrine and noradrenaline. KEY RESULTS: The potency of the alpha1A-adrenoceptor selective agonist A61603 (pEC50=7.79+/-0.04) was 158-fold greater than that for noradrenaline (pEC50=5.59+/-0.02). Phenylephrine (pEC50=5.99+/-0.05) was 2.5-fold more potent than noradrenaline. The alpha1D-adrenoceptor selective antagonist BMY7378 caused rightward shifts of the concentration-response curves to phenylephrine and noradrenaline, yielding low affinity estimates of 6.59+/-0.15 and 6.33+/-0.13, respectively. Relatively high affinity estimates were obtained for the alpha1A-adrenoceptor selective antagonists, RS100329 (9.01+/-0.14 and 9.06+/-0.22 with phenylephrine and noradrenaline, respectively) and 5-methylurapidil (8.51+/-0.10 and 8.31+/-0.10, respectively). Prazosin antagonized responses of the sphincter to phenylephrine and noradrenaline, yielding mean affinity estimates of 8.58+/-0.10 and 8.15+/-0.08, respectively. The Schild slope for prazosin with phenylephrine was equal to unity (1.01+/-0.24), however the Schild slope using noradrenaline was significantly less than unity (0.50+/-0.11, P<0.05). CONCLUSION AND IMPLICATIONS: The results suggest that contraction of circular smooth muscle from the pig internal anal sphincter is mediated via a population of adrenoceptors with the pharmacological characteristics of the alpha1A/L-adrenoceptor, most probably the alpha1L-adrenoceptor form of this receptor.

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Nitrergic neuromuscular transmission in the mouse internal anal sphincter is accomplished by multiple pathways and postjunctional effector cells.

Publications

Nitrergic neuromuscular transmission in the mouse internal anal sphincter is accomplished by multiple pathways and postjunctional effector cells.

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