Store operated calcium channels are associated with diabetic cystopathy in streptozotocin‑induced diabetic rats

Abstract

Store operated calcium channels (SOCCs) have been suggested to play a critical role in many diabetic complications. Diabetic cystopathy (DCP) is common in patients with diabetes, but the role of SOCCs in DCP is still unclear. The aim of the present study was to investigate the role of SOCCs in DCP with streptozocin (STZ)‑induced diabetic rats. Specifically, the authors investigated whether SOCCs were altered in streptozocin (STZ)‑induced diabetic rats and, if so, how this may contribute to the contraction of bladder detrusor strips and the intracellular Ca2+ concentration of bladder smooth muscle cells in diabetic rats. Cyclopiazonic acid (CPA, 10 µM) and SKF‑96365 (10 µM) were used to activate and inhibit SOCCs respectively, to research the effects of SOCCs on the contraction of the bladder detrusor strips in normal and STZ‑induced diabetic rats at the 4th, 8th and 12th week after the diabetic rat model was established. The changes of intracellular Ca2+ were also evaluated under confocal microscopy with pretreated Fluo‑4AM. In addition, the expressions of Orai1 and STIM1 were detected by reverse transcription‑quantitative polymerase chain reaction and western blotting at different time points. According to the results, the contractive frequency of diabetic bladder muscle strips was higher than that of controls in the 4th and 8th week. The increased fluorescence intensity was detected after using CPA and SKF‑96365 in diabetic groups. The expressions of Orai1 and STIM1 changed in a time‑dependent manner.

Keywords: store-operated calcium channels; diabetic cystopathy; Ca2+; bladder dysfunction; Orai1; STIM1.

Figure 1.

Changes of contractions of bladder detrusor strips after activation and inhibition store operated calcium channels. (A) Changes of contractions of bladder detrusor strips in control groups. (B) Changes of contractions of bladder detrusor strips in DM groups; (C) Changes of contractive frequency of bladder detrusor strips. *P<0.05 vs. control groups after using CPA. (D) Changes of contractive frequency of bladder detrusor strips *P<0.05 vs. control groups after using SFK-96365. Among diabetic groups, ^#P<0.05 vs. DM4W and DM8W groups. DM, diabetes mellitus; CPA, cyclopiazonic acid; SKF, SKF-96365.

Figure 2.

Changes of intracellular Ca²⁺ in BSMCs after activation (CPA, 10 µM) and inhibition (SKF-96365, 10 µM) SOCCs. (A) shown as green fluorescence intensity of Fluo-4 AM at 516 nm. CPA, after using CPA; SKF, after using SKF-96365. (A-d, A-e and A-f) represent DM4W, DM8W and DM12W group, respectively. (A-a, A-b and A-c) represent the control groups. Magnification, ×200. (B) The fluorescent line graphs of real-time changes of intracellular Ca²⁺ in BSMCs. (B-d, B-e and B-f) represent DM4W, DM8W and DM12W group respectively. (B-a, B-b and B-c) represent the control groups. (C) *P<0.05, vs. control groups after using CPA. (D) *P<0.05 vs. control groups after using SKF-96365. ^#P<0.05, DM12W group compared with other two DM groups. BSMCs, bladder smooth muscle cells; CPA, cyclopiazonic acid; F1, the measured values of fluorescence intensity in BSMCs; F0, the background values of fluorescence intensity; DM, diabetes mellitus; SKF, SKF-96365.

Figure 3.

The relative mRNA expressions of Orai1 and STIM1 in all experimental groups. (A) *P<0.05 vs. control groups; (B) *P<0.05 vs. control groups. ^#P<0.05 vs. other DM groups (n=6 for every group).

Figure 4.

The expression expressions of Orai1 and STIM1 protein in all experimental groups. (A) The typical blots of Orai1, STIM1 and β-actin proteins in different groups. (B) *P<0.05 vs. control groups, and ^#P<0.05 vs. DM4W and DM8W groups. (C) *P<0.05 vs. control groups, and ^#P<0.05 among three DM groups (n=6 for every group).