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Ca²⁺-SENSITIZATION OF COLONIC SMOOTH MUSCLE IS ALTERED IN A MURINE EXPERIMENTAL COLITIS MODEL

Eikichi Ihara¹, Paul Beck², Justin MacDonald¹
Smooth Muscle and Gastrointestinal Research Groups, Departments of ¹Biochemistry and Molecular Biology; ²Medicine, University of Calgary, Calgary, Alberta

BACKGROUNDS: Colonic smooth muscle contractility is altered in patients with inflammatory bowel disease (IBD) and its underlying mechanisms remain to be investigated. Intracellular Ca²⁺ ([Ca²⁺]_i) is the primary determinant of smooth muscle contraction; however, agonist stimulation can lead to increased contractile force without any changes in [Ca²⁺]_i. This enhancement of the contractile response to Ca²⁺ is referred to as ‘Ca²⁺-sensitization’. Pathological alterations in Ca²⁺-sensitization underlie many diseases associated with smooth muscle dysfunction. We investigated whether the Ca²⁺-sensitization were altered during acute and recovery states of IBD episodes.
METHODS: Colonic circular smooth muscle strips were prepared from normal and DSS-treated mice, where contractile responses were examined. DSS-treated mice were administered 5% DSS in the drinking water for 7 days after which time a portion of the animals were sacrificed (acute inflammation state). The remaining animals were provided normal drinking water for the next 35 days and were then sacrificed (recovered state).
RESULTS: The muscarinic receptor agonist carbachol (CCh, 10 µM) generated contractile force (43.7±8.0 % of the 118 mM K⁺-induced contraction) in colon isolated from control mice. Interestingly, this contraction was potentiated (3.5-fold) in acute inflammation during DSS-treatment but not in recovery. CCh-induced Ca²⁺-sensitization in alpha-toxin permeabilized colonic smooth muscle was potentiated (2.2-fold) with acute inflammation in DSS-treated mice. However, Ca²⁺-sensitization was significantly reduced following the 35 days recovery from DSS treatment. The application of microcystin unmasks basal activated protein kinases and induces Ca²⁺-independent contraction. Microcystin (1 µM)-induced force development was 42.2±0.8 % in beta-escin permeabilized colonic smooth muscle strips from control mice. This contraction was potentiated with acute inflammation in DSS-treated mice (60.6±4.7 %) but not in recovery. The Ca²⁺-sensitized contraction was inhibited with GF109203x, PD98059 and SB203580, but not with Y27632. Immunohistochemistry showed that expressions levels both of ERK1/2 and p38MAPK were increased in the muscularis propria at acute state of DSS-induced colitis.
CONCLUSIONS: Carbachol and microcystin-induced Ca²⁺-sensitization in colonic smooth muscle were increased with acute inflammation in DSS-treated mice but not in recovery. PKC, ERK1/2 and p38 MAPK play an important role in smooth muscle contractile dysfunction observed in IBD.

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