Epidermal growth factor, transforming growth factor-alpha, and epidermal growth factor receptor expression and localization in the canine endometrium during the estrous cycle and in bitches with pyometra.

Gene expression and immunohistochemical localization of epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), and epidermal growth factor receptor (EGF-R) were compared between the endometrium of bitches (Canis familiaris) with pyometra accompanied by cystic endometrial hyperplasia (CEH) and that of healthy bitches at similar stages of the estrous cycle. In normal bitches, endometrial TGF-alpha mRNA levels were highest at proestrus and gradually decreased as the cycle progressed to anestrus. Epidermal growth factor receptor mRNA levels were not significantly affected by the stage of the estrous cycle. Epidermal growth factor mRNA levels were higher at Day 35 of diestrus than at other stages of the estrous cycle (P<0.05). In bitches with pyometra, endometrial TGF-alpha and EGF-R mRNA levels did not differ significantly from those at diestrus in normal bitches, but EGF mRNA levels were lower than those at Day 35 of diestrus in normal bitches (P<0.05). In normal bitches, positive immunohistochemical staining for TGF-alpha, EGF, and EGF-R was mainly present in the glandular and luminal epithelial cells of the endometrium. In contrast, in bitches with pyometra, immunoreactivity for EGF was clearly present in endometrial stromal cells. Inflammatory cells that had infiltrated the endometrial stroma stained strongly for TGF-alpha and EGF-R. Luminal and glandular epithelial cells also stained positive for EGF-R. In conclusion, expression of TGF-alpha by inflammatory cells and a low level of expression and differential localization of EGF may be involved in aberrant growth of endometrial glands and development of CEH.

The mechanisms of alpha(2)-adrenoceptor agonist-induced contraction in longitudinal muscle of the porcine uterus.

The aim of the present study was to clarify the cellular mechanisms underlying the alpha(2)-adrenoceptor-mediated contraction of porcine myometrium (nonvascular smooth muscle). Acetylcholine (3 nM-1 microM), clonidine (1 nM-10 microM) and 5-bromo-N-[2-imidazolin-2-yl]-6-quinoxalinamine (UK14304) (1 nM-10 microM) in Krebs solution caused a concentration-dependent contraction in the longitudinal muscles of the porcine uterus with similar EC(50) values and maximum responses. A lowered external Ca(2+) concentration and verapamil (10 nM-10 microM) decreased the contractile response to clonidine and UK14304 more markedly than the response to acetylcholine. However, in Kumagai solution, neither clonidine nor UK14304 caused contractile responses, but acetylcholine remained effective. The effects of alpha(2)-adrenoceptor agonists on intracellular Ca(2+) concentration ([Ca(2+)](i)) and smooth muscle force were measured simultaneously using fura-PE3-loaded muscle preparations. Clonidine and UK14304 caused increases in [Ca(2+)](i) and force of the longitudinal muscle. The increases in [Ca(2+)](i) and muscle force were markedly inhibited by verapamil and in Ca(2+)-free solution (EGTA, 1 mM). In the absence of external Ca(2+), clonidine caused only a small increase in [Ca(2+)](i) in Ca(2+)-loaded preparations compared with those increases caused by carbachol, histamine, and oxytocin. Ca(2+) (2.5 mM) caused increases in [Ca(2+)](i) and force of the longitudinal muscles in a Ca(2+)-free high K(+) solution. Clonidine concentration dependently potentiated the Ca(2+)-induced contraction without significantly changing the increase in [Ca(2+)](i), and this potentiation was inhibited by yohimbine. These results suggested that clonidine increases the Ca(2+) sensitivity of the contractile elements through activation of alpha(2)-adrenoceptors. During the development of the contractile response to clonidine (1 microM, 0-5 min), tissue cyclic AMP levels did not change significantly. In vitro treatment with pertussis toxin (1 microg/ml for 2 h) significantly decreased the contraction induced by clonidine without affecting the responses to carbachol and high K(+). The present results indicate that in porcine myometrium, alpha(2)-adrenoceptor stimulation caused contraction of the longitudinal muscles by mechanisms largely dependent on the influx of extracellular Ca(2+), probably through voltage-dependent Ca(2+) channels (VDCCs), and that the potentiation of the Ca(2+) sensitivity of the contractile elements is another mechanism of the contractile responses. These actions involve a pertussis-toxin-sensitive G protein (probably G(i) type) in the signal transduction pathway.