Atrial natriuretic peptide signal pathway upregulated in stomach of streptozotocin-induced diabetic mice

Abstract

Aim: To investigate atrial natriuretic peptide (ANP) secretion from gastric mucosa and the relationship between the ANP/natriuretic peptide receptor type A (NPR-A) pathway and diabetic gastroparesis.

Methods: Male imprinting control region (ICR) mice (4 wk old) were divided into two groups: control mice, and streptozotocin-induced diabetic mice. Eight weeks after injection, spontaneous gastric contraction was recorded by using physiography in control and streptozotocin-induced diabetic mice. The ANP-positive cells in gastric mucosa and among dispersed gastric epithelial cells were detected by using immunohistochemistry and flow cytometry, respectively. ANP and natriuretic peptide receptor type A (NPR-A) gene expression in gastric tissue was observed by using the reverse transcriptase polymerase chain reaction.

Results: The frequency of spontaneous gastric contraction was reduced from 12.9 +/- 0.8 cycles/min in the control group to 8.4 +/- 0.6 cycles/min in the diabetic mice (n = 8, P < 0.05). However, the amplitude of contraction was not significantly affected in the diabetic group. The depletion of interstitial cells of Cajal in the gastric muscle layer was observed in the diabetic mice. ANP-positive cells were distributed in the gastric mucosal layer and the density index of ANP-positive cells was increased from 20.9 +/- 2.2 cells/field in control mice to 51.8 +/- 2.9 cells/field in diabetic mice (n = 8, P < 0.05). The percentage of ANP-positive cells among the dispersed gastric epithelial cells was increased from 10.0% +/- 0.9% in the control mice to 41.2% +/- 1.0% in the diabetic mice (n = 3, P < 0.05). ANP and NPR-A genes were both expressed in mouse stomach, and the expression was significantly increased in the diabetic mice.

Conclusion: These results suggest that the ANP/NPR-A signaling pathway is upregulated in streptozotocin-induced diabetic mice, and contributes to the development of diabetic gastroparesis.

Figure 1

Comparision of gastric contractility and expression of ICCs in normal and STZ-induced diabetic mice. A: Representative traces of spontaneous gastric contraction in control and STZ-induced diabetic mice; B: Summary of amplitudes and frequencies of gastric motility in control and STZ-induced diabetic groups (n = 8; ^aP < 0.05 vs control group); C and D: Expression of c-Kit-positive cells in the muscle layer. Arrow indicates the positive staining area. Expression of c-Kit was more obvious in STZ-induced diabetic mice than in the normal controls. Scale bar = 40 μm.

Figure 2

Immunohistochemical analysis of atrial natriuretic peptide (ANP) in mouse gastric tissue. The photographs show ANP expression in gastric mucosa in the control group (A), STZ-induced diabetic group (B), and negative control group (C); The paranuclear cytoplasmic stained cells that display an intense dark brown color were ANP-positive-staining cells in the mucosa. The red arrows indicate ANP-positive cells; B shows that the number of ANP-positive cells increased in the STZ-induced diabetic group; D shows the density index that referred to the number of ANP-positive cells per field of vision (n = 8, ^aP < 0.05 vs control group). Scale bar = 20 μm.

Figure 3

Flow cytometric analysis of ANP-positive cells among gastric mucosal cells. The percentage of ANP-positive cells was determined by flow cytometry with FITC-conjugated goat anti-rabbit IgG. A: Relative fluorescence intensity of gastric mucosal cells in the control and diabetic groups. M1 indicates the proportions of ANP-positive cells that displayed positive FITC labeling. The inserted fluorescent image is a single ANP-positive cell that had a positive fluorescent signal in its cytoplasm (× 400); B: ANP-positive cells among the gastric mucosal cells (10⁴ cells per sample) in control and STZ-induced diabetic mice (n = 3, ^aP < 0.05 vs control).

Figure 4

RT-PCR of gastric ANP and NPR-A genes in control and STZ-induced diabetic mice. A: Results of agarose gel electrophoresis, including ANP, NPR-A and GAPDH genes. The density signal was normalized to that of GAPDH. The mean value of the density was obtained from three separate experiments; B: Expression of ANP and NPR-A genes was increased in STZ-induced diabetic mice (n = 3; ^aP < 0.05, ^bP < 0.01 vs control).