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, 302 (1), G66-76

Altered Calcium Signaling in Colonic Smooth Muscle of Type 1 Diabetic Mice

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Altered Calcium Signaling in Colonic Smooth Muscle of Type 1 Diabetic Mice

Ketrija Touw et al. Am J Physiol Gastrointest Liver Physiol.

Abstract

Seventy-six percent of diabetic patients develop gastrointestinal symptoms, such as constipation. However, the direct effects of diabetes on intestinal smooth muscle are poorly described. This study aimed to identify the role played by smooth muscle in mediating diabetes-induced colonic dysmotility. To induce type 1 diabetes, mice were injected intraperitoneally with low-dose streptozotocin once a day for 5 days. Animals developed hyperglycemia (>200 mg/dl) 1 wk after the last injection and were euthanized 7-8 wk after the last treatment. Computed tomography demonstrated decreased overall gastrointestinal motility in the diabetic mice. In vitro contractility of colonic smooth muscle rings from diabetic mice was also decreased. Fura-2 ratiometric Ca(2+) imaging showed attenuated Ca(2+) increases in response to KCl stimulation that were associated with decreased light chain phosphorylation in diabetic mice. The diabetic mice also exhibited elevated basal Ca(2+) levels, increased myosin phosphatase targeting subunit 1 expression, and significant changes in expression of Ca(2+) handling proteins, as determined by quantitative RT-PCR and Western blotting. Mice that were hyperglycemic for <1 wk also showed decreased colonic contractile responses that were associated with decreased Ca(2+) increases in response to KCl stimulation, although without an elevation in basal Ca(2+) levels or a significant change in the expression of Ca(2+) signaling molecules. These data demonstrate that type 1 diabetes is associated with decreased depolarization-induced Ca(2+) influx in colonic smooth muscle that leads to attenuated myosin light chain phosphorylation and impaired colonic contractility.

Figures

Fig. 1.
Fig. 1.
Computed tomography (CT) scans reveal decreased gastrointestinal (GI) motility in long-term diabetic mice. At 7–8 wk after becoming hyperglycemic, streptozotocin (STZ)-treated mice (n = 3) or paired saline injected control mice (control; n = 2) were fasted overnight and subjected to CT scan. CT images were taken 3, 5, 9, and 15 h after contrast agent administration. ST, stomach; SI, small intestine; LI, large intestine.
Fig. 2.
Fig. 2.
Diabetic mice show decreased colon contractility. At 7–8 wk after becoming hyperglycemic, mice were killed, and colons were harvested for contractility measurements, as described in materials and methods. A: schematic diagram showing the location of the colonic segments used in B–E. B: representative tension recordings from colon rings contracted by 60 mM KCl stimulation. C: quantification of changes (Δ) in peak force produced by colonic rings from control and STZ-induced mice. Data shown are the relative Δforce of rings from STZ-treated mice expressed as a percentage of the Δforce observed in a parallel paired ring obtained from a control mouse (set to 100%). P1–P4, proximal parts of the colon; D1–D4, distal parts of the colon. Each bar represents the mean ± SE of 6–12 different mice: P1 (n = 10), P2 (n = 11), P3 (n = 10), P4 (n = 12), D4 (n = 6), D3 (n = 9), D2 (n = 11), D1 (n = 10). ΔForce that were significantly less than 100% are indicated: *P < 0.05, **P < 0.005. D: colon rings from the central portion of the colon of control and 7-wk diabetic mice (STZ) were equilibrated at optimal resting tension in Krebs buffer for 1 h and contracted using 60 mM KCl. Following washing, rings were then incubated with tetrodotoxin (1 μM) for 5 min and then stimulated with 60 mM KCl. Values are expressed as percentage of KCl-induced contraction obtained from colon of control mice (set to 100%) (n = 7). ΔForce that were significantly different from 100% are indicated: *P < 0.05. E: average Δforce of all of the colonic segments combined (n = 79, segments from 6–12 different mice). ΔContractility of control mice were set to 100%. ***P < 0.0005.
Fig. 3.
Fig. 3.
Basal levels of intracellular Ca2+ are increased, whereas the Ca2+ response to 60 mM KCl is decreased in diabetic mice. A: representative averaged recordings of intracellular Ca2+ measured using fura-2 in a middle part of colonic smooth muscle strip isolated from an STZ-treated mouse (7 wk) or a control saline-injected mouse. B: Δbasal levels of intracellular Ca2+ in STZ-treated mice compared with control mice. C: Δintracellular Ca2+ in response to 60 mM KCl in STZ-treated mice compared with control mice. For A–C, each tracing/bar represents the mean ± SE obtained from 6–8 different mice. For B and C, intracellular Ca2+ levels of control mice are set to 1. *P < 0.05. D: parallel colonic smooth muscle samples were used to measure myosin light chain (MLC) phosphorylation levels (n = 4 mice). MLC phosphorylation is expressed as MLCphosphorylated/MLCtotal. Values are means ± SE. E: CPI-17 phosphorylation levels were measured in colonic smooth muscle tissue samples (n = 9). CPI-17 phosphorylation is expressed as CPI-17phosphorylated/CPI-17total. Values are means ± SE. F: myosin phosphatase targeting subunit 1 (MYPT1) phosphorylation levels were measured in colonic smooth muscle tissue samples (n = 8). MYPT1 T696 phosphorylation is expressed as MYPTphosphorylated/MYPTtotal ± SE. G: CPI-17, MYPT1, and MLC kinase (MLCK) expression levels were quantitated by Western blotting and compared between control mice and mice that were hyperglycemic for 7–8 wk. Means ± SE (n = 8) following normalization to meta-vinculin or β-actin as an internal control are shown. *P < 0.05.
Fig. 4.
Fig. 4.
Changes in mRNA and protein levels of calcium-handling proteins in diabetic mice. The middle part (P4–D2) of the colon was dissected and cleaned of the epithelial cell layer. A: transcripts were quantitated by real-time RT-PCR. Transcript levels were first normalized to a hypoxanthine phosphoribosyltransferase (HPRT) internal loading control, and then samples from STZ-treated mice were expressed relative to samples obtained from control-treated mice. Relative expression = 2−ΔΔCt, where ΔΔCt = (CtSTZ − CtHPRT) − (Ctcontrol − CtHPRT). Each bar represents the mean ± SE of samples obtained from 4–10 different mice. *P < 0.05. B, left: immunoblots of protein extracts obtained from control and STZ-treated mice (7 wk). Molecular mass markers (kDa) are indicated at the left of the blots. Right: quantification of protein expression levels following normalizing to vinculin as an internal control. Values are means ± SE of 4 different mice. *P < 0.05, **P < 0.005. PMCA, plasma membrane Ca2+-ATPase; TRPC, canonical transient receptor potential; SERCA2b, sarco(endo)plasmic reticulum Ca2+-ATPase 2b; RyR, ryanodine receptor; IP3R, inositol-trisphosphate receptor; NCX2, sodium-calcium exchanger 2.
Fig. 5.
Fig. 5.
Contractility in mice that were hyperglycemic for <1 wk is decreased due to attenuated intracellular Ca2+ responses and decreased MLC phosphorylation. One week after becoming hyperglycemic, mice were euthanized, and a middle part of colons was isolated for contractility measurements, Ca2+ imaging, mRNA and protein analysis, and MLC phosphorylation measurements. A: colon rings from the central portion of the colon of control (open bars) and 1-wk diabetic mice (STZ; solid bars) were equilibrated at optimal resting tension in Krebs buffer for 1 h and contracted using 60 mM KCl. Following washing, rings were then incubated with tetrodotoxin (1 μM) for 5 min and then stimulated with 60 mM KCl. Values are expressed as percentage of KCl-induced contraction obtained from colon of control mice. ΔForce that were significantly different from 100% are indicated: *P < 0.05 (n = 6–13). B: basal levels of intracellular Ca2+ measured in the middle part of the colon obtained from 5–6 control and STZ-treated mice. C: mean ± SE relative change in intracellular Ca2+ levels in response to 60 mM KCl stimulation in control and STZ-treated mice (n = 5–6 mice). *P < 0.05. D: RNA was isolated from the middle to distal part of colon (P4–D2) obtained from control or STZ-treated (1 wk) mice, and transcripts were quantitated as in Fig. 4. Each bar represents the mean ± SE values obtained from 4–5 different mice. *P < 0.05. E, left: immunoblots of proteins obtained from parallel samples to those shown in D. Molecular mass markers (kDa) are indicated at the left of the blots. Right: quantification of immunoblots following normalizing to vinculin. Each bar represents the mean ± SE of 8 different mice. *P < 0.05, **P < 0.005. F: colonic rings obtained from control and STZ-treated (1 wk) mice were hung in a muscle bath, and MLC phosphorylation levels were determined under resting conditions (n = 7 mice) and at the peak of a 60 mM KCl-induced contraction (n = 8 mice). MLC phosphorylation is expressed as MLCphosphorylated/MLCtotal. Values are means ± SE. *P < 0.05.
Fig. 6.
Fig. 6.
Diabetic mice do not exhibit altered smooth muscle structure, neural density, or inflammation. A: hematoxylin- and eosin-stained sections of the middle part of the colon obtained from 7-wk diabetic and control mice. B: immunohistochemical staining of neurons (visualized by antibodies to neurofilament 200) from 1-wk diabetic mice. C: quantitative RT-PCR analysis of RNA expression in colons obtained from 1-wk diabetic and control mice. D: quantitative RT-PCR analysis of RNA expression in 7-wk diabetic and control mice. For C and D, transcripts were quantitated as described in Fig. 4. Each bar represents the mean ± SE of 4–7 different mice. *P < 0.05, **P < 0.005. iNOS, inducible nitric oxide synthase; IL-1β, interleukin-1β; TNF-α, tumor necrosis factor-α.

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