The steroid hormone 17β-oestradiol (E2) rapidly affects K⁺ transport in human colon via signal transduction involving Na⁺/H+ exchange and the activation of protein kinase C (PKC) isoforms (McNamara et al. 2000). In rat distal colonic crypts E2 is known to inhibit Cl^– secretion under basal conditions and when secretion has been activated by cAMP or Ca²⁺ agonists (Condliffe et al. 2001). The anti-secretory effect of E2 is female gender-specific and is dependent on the activation of PKCα and PKCδ (Harvey et al. 2002). Downstream targets of PKC maybe ion transporters or other kinases. For example, the protein kinase D (PKD/PKCµ) family of serine-threonine kinases is a downstream target for classical (PKCα) and novel (PKCδ, and h{special}) PKC isoforms. In this study we investigated rapid protein kinase signalling events stimulated by E2 in rat distal colonic crypts and, in particular, the rapid activation of PKD. We also sought to identify any gender differences in the basal expression levels in non-activated crypts of the PKC isoforms known to be activators of PKD.

Sprague-Dawley rats (3 months old) were killed humanely by cervical dislocation and distal colonic crypts were isolated as described previously (Harvey et al. 2002). To ensure heterogeneity, crypt samples were pooled for basal expression studies (n = 3) and activation studies (n = 7). PKC basal expression levels were determined by Western blotting. PKD activation was determined by western blotting.

Gender-specific differential expression of PKC isoforms was observed in non-activated distal colonic crypts. We observed higher levels of basal expression of PKCδ (3-5 fold) and PKC (5-10 fold) in crypts isolated from female rats as compared to males. We have, for the first time, demonstrated the expression of PKD in rat distal colonic crypts with females showing a greater level (2-fold) of basal expression of PKD. No difference was observed in the basal expression levels of PKCα in crypts isolated from males and females. Also, no difference was observed in PKC isoform expression in crypts from female rats at different stages of the oestrus cycle. Treatment with E2 (10 nM) produced a rapid (< 2 min) activation of PKD in rat colonic crypts.

In conclusion, we provide the first evidence for differential female gender-specific expression of PKC isoforms and expression of PKD in the rat colon. The physiological significance of PKD activation in the rapid responses to steroid hormones remains to be elucidated, however the known PKD effectors; NHE-1, ERK1/2 and cJun maybe important in the anti-secretory and proliferative effects of oestrogen.

This work was supported by the Higher Education Authority.