Effects of ageing and streptozotocin-induced diabetes on connexin43 and P2 purinoceptor expression in the rat corpora cavernosa and urinary bladder

Abstract

Objective: To investigate whether ageing and diabetes alter the expression of the gap junction protein connexin43 (Cx43) and of particular purinoceptor (P2R) subtypes in the corpus cavernosum and urinary bladder, and determine whether changes in expression of these proteins correlate with development of erectile and bladder dysfunction in diabetic and ageing rats.

Materials and methods: Erectile and bladder function of streptozotocin (STZ)-induced diabetic, insulin-treated and age-matched control Fischer-344 rats were evaluated 2, 4 and 8 months after diabetes induction by in vivo cystometry and cavernosometry. Corporal and bladder tissue were then isolated at each of these sample times and protein expression levels of Cx43 and of various P2R subtypes were determined by Western blotting.

Results: In the corpora of control rats ageing was accompanied by a significant decrease in Cx43 and P2X(1)R, and increase in P2X(7)R expression. There was decreased Cx43 and increased P2Y(4)R expression in the ageing control rat bladder. There was a significant negative correlation between erectile capacity and P2X(1)R expression levels, and a positive correlation between bladder spontaneous activity and P2Y(4)R expression levels. There was already development of erectile dysfunction and bladder overactivity at 2 months after inducing diabetes, the earliest sample measured in the study. The development of these urogenital complications was accompanied by significant decreases in Cx43, P2Y(2)R, P2X(4)R and increase in P2X(1)R expression in the corpora, and by a doubling in Cx43 and P2Y(2)R, and significant increase in P2Y(4)R expression in the bladder. Changes in Cx43 and P2R expression were largely prevented by insulin therapy.

Conclusion: Ageing and diabetes mellitus markedly altered the expression of the gap junction protein Cx43 and of particular P2R subtypes in the rat penile corpora and urinary bladder. These changes in Cx43 and P2R expression provide the molecular substrate for altered gap junction and purinergic signalling in these tissues, and thus probably contribute to the early development of erectile dysfunction and higher detrusor activity in ageing and in diabetic rats.

FIG. 1

The effect of ageing on Cx43 expression in healthy rat penile corporal and urinary bladder tissues. Expression levels of Cx43 in the corporal (A) and bladder (B) tissue of 2-, 4- and 8-month control rat groups were determined by Western blot and normalized by GAPDH. Note the significant decrease in Cx43 expression as the corpora ages and the earlier halving in Cx43 expression in bladders from younger (2-month group) to more mature rats (4-month group), which persisted with further ageing (8-month group). Values are the mean (sem) of tissues from at least three rats in each group. *P < 0.05 vs the 2-month group.

FIG. 2

The effect ageing on P2 receptor expression in healthy rat penile corporal and urinary bladder tissues. Expression levels of metabotropic P2Y₂R and P2Y₄R, and ionotropic P2X₁R, P2X₃R, P2X₄R and P2X₇R subtypes in the corporal (A) and bladder (B) tissue of 2-, 4- and 8-month control rat groups were determined by Western blot and normalized by GAPDH. Note the significant change in P2R expression profile in the corpora, characterized by a decrease in P2X₁R as the corpora ages and significant increase in P2X₇R in younger 4-month adult rats that is followed by a significant decrease in older rats. Ageing did not significantly alter P2R expression profile in the bladder but expression of P2Y₄Rs markedly increased in ageing bladders. Values are the mean (sem) of tissues from at least 3 three rats in each group. Significant differences between the 2-, 4- and 8-month groups for each P2R subtype were determined by one-way anova followed by Tukey's multiple comparison test.

FIG. 3

The effect of diabetes on P2R expression in rat penile corporal and urinary bladder tissues. Expression levels of metabotropic P2Y₂R and P2Y₄R, and ionotropic P2X₁R, P2X₃R, P2X₄R and P2X₇R subtypes in age-matched control, in IRT-treated and in 2-, 4- and 8-month STZ-rat corporal (A) and bladder (B) tissues were determined by Western blot and normalized by GAPDH. Note the significant decrease in P2Y₂R, P2X₄R and increase in P2X₁R expression in 2-month diabetic corpora. Different from what was observed in the corpora, P2X₁R expression was not altered but P2Y₂R and P2Y₄R expression is up-regulated in the 2-month diabetic bladders. Values are the mean (sem) of tissues from at least three rats in each group. Significant differences between control, diabetic and IRT-treated groups for each P2R subtype were determined by one-way anova followed by Tukey's multiple comparison test.

FIG. 4

The effect of diabetes on Cx43 expression in rat penile corporal and urinary bladder tissues. Expression levels of Cx43 in 2-, 4- and 8-month STZ-diabetic, IRT-treated and age-matched control corporal (A) and bladder (B) tissue were determined by (C) Western blot and normalized by GAPDH. Note the significant decrease in Cx43 expression as the corpora ages and the `ageing effect' of diabetes in the corpora of 2-month STZ-rats. In diabetic bladders Cx43 was markedly up-regulated and in older diabetic bladders remained almost double that in age-matched controls. Note also the changes in Cx43 phosphorylation, with a marked increase in the slower migrating P1 and P2 phosphorylated forms of Cx43 in diabetic bladders. Values are the mean (sem) of tissues from at least three rats in each group. *P < 0.05 vs age-matched control; ^@P < 0.05 vs 2-month control corpora. Significant differences were determined by one-way anova followed by Tukey's multiple comparison test.