The present study focused on the role of microRNA-139-5p (miRNA-139-5p) in the regulation of basal tone in internal anal sphincter (IAS). Applying genome-wide miRNA microarrays on the phenotypically distinct smooth muscle cells (SMCs) within the rat anorectrum, we identified miRNA-139-5p as differentially expressed RNA repressor with highest expression in the purely phasic smooth muscle of anococcygeus (ASM) vs. the truly tonic smooth muscle of IAS. This pattern of miRNA-139-5p expression, previously shown to target ROCK2, was validated by target prediction using ingenuity pathway (IPA) and by qPCR analyses. Immunoblotting, immunocytochemistry (ICC), and functional assays using IAS tissues and cells subjected to overexpression/knockdown of miRNA-139-5p confirmed the inverse relationship between miRNA-139-5p and ROCK2 expressions/IAS tone. Overexpression of miRNA-139-5p caused a decrease, while knockdown by anti-miRNA-139-5p caused an increase in the IAS tone; these tissue contractile responses were confirmed by single-cell contraction using magnetic twisting cytometry (MTC). These findings suggest miRNA-139-5p is capable of significantly influencing the phenotypic tonicity in smooth muscle via ROCK2: a lack of tone in ASM may be associated with the suppression of ROCK2 by high expression of miRNA-139-5p, whereas basal IAS tone may be associated with the persistence of ROCK2 due to low expression of miRNA-139-5p.

Postnatal maturation of esophageal musculature involves proximal-to-distal replacement of smooth muscle with skeletal muscle by elusive mechanisms. We report that this process is impaired in mice lacking the cell surface receptor Cdo and identify the underlying developmental mechanism. A myogenic transition zone containing proliferative skeletal muscle precursor cells migrated in a proximal-distal direction, leaving differentiated myofibers in its wake. Distal to the transition zone, smooth muscle fascicles underwent a morphogenetic process whereby they changed their orientation relative to each other and to the lumen. Consequently, a path was cleared for the transition zone, and smooth muscle ultimately occupied only the distal-most esophagus; there was no loss of smooth muscle. Cdo(-/-) mice were specifically defective in fascicular reorientation, resulting in an aberrantly proximal skeletal-smooth muscle boundary. Furthermore, Cdo(-/-) mice displayed megaesophagus and achalasia, and their lower esophageal sphincter was resistant to nitric oxide-induced relaxation, suggesting a developmental linkage between patterning and sphincter function. Collectively, these results illuminate mechanisms of esophageal morphogenesis and motility disorders.

Caveolins (CAVs) are structural proteins of caveolae that function as signaling platforms to regulate smooth muscle contraction. Loss of CAV protein expression is associated with impaired contraction in obstruction-induced bladder smooth muscle (BSM) hypertrophy. In this study, microarray analysis of bladder RNA revealed down-regulation of CAV1, CAV2, and CAV3 gene transcription in BSM from models of obstructive bladder disease in mice and humans. We identified and characterized regulatory regions responsible for CAV1, CAV2, and CAV3 gene expression in mice with obstruction-induced BSM hypertrophy, and in men with benign prostatic hyperplasia. DNA affinity chromatography and chromatin immunoprecipitation assays revealed a greater increase in binding of GATA-binding factor 6 (GATA-6) and NF-κB to their cognate binding motifs on CAV1, CAV2, and CAV3 promoters in obstructed BSM relative to that observed in control BSM. Knockout of NF-κB subunits, shRNA-mediated knockdown of GATA-6, or pharmacologic inhibition of GATA-6 and NF-κB in BSM increased CAV1, CAV2, and CAV3 transcription and promoter activity. Conversely, overexpression of GATA-6 decreased CAV2 and CAV3 transcription and promoter activity. Collectively, these data provide new insight into the mechanisms by which CAV gene expression is repressed in hypertrophied BSM in obstructive bladder disease.

Aging-associated decrease in internal anal sphincter (IAS) tone (AADI) is a major contributor in the rectoanal incontinence (RI). To determine the pathogenesis of AADI, we investigated the effect of aging on GPCR activation and related downstream signaling. We particularly investigated two GPCRs that characterize IAS smooth muscle cells (SMCs): thromboxane A2 and angiotensin II type 1. Two groups of Fischer 344 rats (6-month-old [young group] and 26-month-old [old group]) were employed to determine the GPCR function by isometric contraction, the expressions of GPCRs, and their downstream regulatory signaling proteins (regulator of G-protein signaling 2, RGS2; GPCR Kinase 5, GRK5; and β-arrestin, Arrb2) using RT-PCR, qPCR, and western blot analyses. We used reversible biotinylation to monitor the GPCR trafficking using SMCs. Aging selectively attenuated thromboxane A2 and Ang II-induced IAS contraction. RT-PCR, qPCR, and WB data revealed a significant decrease in the expressions of the GPCRs and increase in the expression of RGS2, GRK5, and Arrb2. The increased GPCR internalization and decreased recycling under aging were validated by reversible biotinylation. We conclude that downregulation of GPCR, accompanied by upregulation of regulatory proteins, plays an important role in receptor desensitization and may be important underlying mechanisms of RI in certain aging patients.

Extracellular pH is an important physiological determinant of vascular tone that is normally maintained within 7.35-7.45. Any change outside this range leads to severe pathological repercussions. We investigated the unknown effects of extracellular acidosis on relaxation in the superior mesenteric artery (SMA) of goat. SMA rings were employed to maintain isometric contractions at extracellular pH (pHo) 7.4 and 6.8. We analyzed the effect of acidosis (pHo 6.8) compared to physiological pH (pHo 7.4) on three signaling mediators of endothelium-dependent hyperpolarization: nitric oxide (NO), prostaglandin I2 (PGI2), and myoendothelial gap junctions (MEGJ). NO and cyclic guanosine monophosphate (cGMP) levels were compared between normal and acidic pH. Quantitative real-time PCR (qPCR) studies determined the change in expression of vascular connexin (Cx), Cx37, Cx40, and Cx43. Under acidosis, acetyl choline-induced relaxation was augmented in an endothelium-dependent manner via eNOS-NO-cGMP signaling. Conversely, at normal pH, acetyl choline-induced vasorelaxation was mediated primarily via COX-PGI2 pathway. The functional activity of MEGJ was increased under acidosis as evident from increased sensitivity of connexin blockers and upregulated gene and protein expression of connexins. In conclusion, acetyl choline-induced augmented vasorelaxation under acidosis is mediated by NOS-NO-cGMP, with a partial role of MEGJ as EDH mediators in the SMA. Present data suggest a novel role of connexin as therapeutic targets to attenuate the detrimental effect of acidosis on vascular tone.