H-ras inhibits RhoA/ROCK leading to a decrease in the basal tone in the internal anal sphincter.
OBJECTIVE
The present studies evaluated the role of H-ras and its implications in the RhoA/Rho kinase (ROCK) pathway in regulating basal tone in the internal anal sphincter (IAS).


METHODS
Studies were performed in the IAS from the wild-type (H-ras(+/+)) and knock-out (H-ras(-/-)) mice. The basal tone of smooth muscle strips was measured by isometric force transducers. Length of smooth muscle cells (SMC) isolated from the IAS in the basal state was determined by phase contrast microscopy. Experiments were repeated in the presence of Y 27632, a ROCK inhibitor. Involvement of the RhoA/ROCK machinery was analyzed by reverse-transcription polymerase chain reaction, Western blot, and immunocytochemistry. Reversal of H-ras knock-out effect was evaluated by transfection of SMCs with the constitutively activated (G12V) mutant.


RESULTS
Basal tone of the H-ras(-/-) IAS was significantly higher and resistant to relaxation by Y 27632, compared with the H-ras(+/+) IAS. Similarly, the length of SMCs from H-ras(-/-) IAS was significantly shorter. Y 27632 eliminated this difference. RhoA immunoreactivity shifted from cytoplasm to plasma membrane in H-ras(-/-) SMCs, a change typically associated with contraction. Further, SMCs from H-ras(-/-) mice exhibited higher levels of the contractile proteins ROCK II, phosphorylated-MYPT(1) and phosphorylated-MLC(20). Transfection with the G12V mutant increased the length of H-ras(-/-) cells. Conversely, the dominant negative H-ras (S17N) mutant decreased the length of H-ras(+/+) cells.


CONCLUSIONS
H-ras negatively regulates basal tone in the IAS by inhibiting RhoA/Rho-kinase machinery. Studies may have significant relevance in the pathophysiology and therapy of certain anorectal motility disorders associated with the IAS dysfunction.
de Godoy
Márcio A F
MA
Departments of Medicine, Division of Gastroenterology & Hepatology, Pharmacology and Experimental Therapeutics, and Emergency Medicine, Jefferson Medical College of Thomas Jefferson University, Philadelphia, Pennyslvania 19107, USA.
Patel
Chirag A
CA
Waldman
Scott A
SA
Katsuki
Motoya
M
Regan
Raymond F
RF
Rattan
Satish
S
D000577:Amides / Q000494:pharmacology; D001003:Anal Canal / Q000166:cytology / Q000502:physiology*; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics*; D004587:Electrophoresis, Agar Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D011905:Genes, ras / Q000502:physiology*; D005838:Genotype; D007150:Immunohistochemistry; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000235:genetics*; D051379:Mice; D018345:Mice, Knockout; D008858:Microscopy, Phase-Contrast; D009119:Muscle Contraction / Q000187:drug effects / Q000235:genetics; D009129:Muscle Tonus / Q000235:genetics*; D009130:Muscle, Smooth / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics*; D011725:Pyridines / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D054460:rho-Associated Kinases
D000577:Amides; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D011725:Pyridines; C108830:Y 27632; D004247:DNA; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases