Expression of a Rho guanine nucleotide exchange factor, Ect2, in the developing mouse pituitary.

The pituitary gland is a highly mitotically active tissue after birth. Various cell types are known to undergo proliferation in the anterior pituitary. However, little is known about the mechanisms regulating mitotic activity in this tissue. When searching for genes specifically expressed in the pituitary gland among those that we previously screened in Drosophila, we found epithelial cell-transforming gene 2 (Ect2). Ect2 is a guanine nucleotide exchange factor for Rho GTPases, which is known to play an essential role in cytokinesis. Although there have been many cellular studies regarding the function of Ect2, the temporal and spatial expression patterns of Ect2 in vivo have not been determined. In the present study, we examined the postnatal developmental expression of Ect2 in the mouse pituitary. Enhanced Ect2 expression was detected in the mouse pituitary gland during the first 3 weeks after birth, which coincided well with the period of rapid pituitary expansion associated with increased growth rate. Immunostaining analysis showed that Ect2-expressing cells were distributed in the anterior and intermediate lobes, but not the posterior lobe, of the pituitary. These Ect2-expressing cells frequently incorporated the thymidine analogue, EdU (5-ethynyl-2'-deoxyuridine), indicating that these cells were mitotically active. Taken together, the results demonstrate the functional role of Ect2 in postnatal proliferating cells in the two lobes of the pituitary, thereby suggesting roles in developmental growth of the mammalian pituitary.

Angiotensin II stimulates cyclic ADP-ribose formation in neonatal rat cardiac myocytes.

To examine the role of cyclic ADP-ribose (cADP-ribose) as a second messenger downstream of angiotensin II (Ang II) receptor activation in the heart, ADP-ribosyl cyclase activity was measured in a crude membrane fraction of ventricular myocytes. Ang II at 10-100 nM increased ADP-ribosyl cyclase activity by 40-90% in the ventricular muscle of neonatal (2-4-day-old) rats, but not in fetal or adult hearts. This increase was inhibited by the Ang II antipeptide. Stimulation of ADP-ribosyl cyclase was reproduced by GTP and guanosine 5'-[gamma-thio]triphosphate, and prevented by guanosine 5'-[beta-thio]diphosphate. Prior treatment of the rats with cholera toxin A and B subunits also blocked the Ang II-induced activation. The density of Ang II receptors detected as [(3)H]Ang II binding was higher in neonatal than adult rats. These results demonstrate the existence of a signalling pathway from Ang II receptors to membrane-bound ADP-ribosyl cyclase in the ventricular muscle cell and suggest that the Ang II-induced increase in cADP-ribose synthesis is involved in the regulation of cardiac function and development.

Sympathetic potentiation of cyclic ADP-ribose formation in rat cardiac myocytes.

We examined the role of cyclic ADP-ribose (cADP-ribose) as a second messenger downstream of adrenergic receptors in the heart after excitation of sympathetic neurons. To address this question, ADP-ribosyl cyclase activity was measured as the rate of [(3)H]cADP-ribose formation from [(3)H]NAD(+) in a crude membrane fraction of rat ventricular myocytes. Isoproterenol at 1 microM increased ADP-ribosyl cyclase activity by 1.7-fold in ventricular muscle; this increase was inhibited by propranolol. The stimulatory effect on the cyclase was mimicked by 10 nM GTP and 10 microM guanosine 5'-3-O-(thio)triphosphate, whereas 10 microM GTP inhibited the cyclase. Cholera toxin blocked the activation of the cyclase by isoproterenol and GTP. The above effects of isoproterenol and GTP in ventricular membranes were confirmed by cyclic GDP-ribose formation fluorometrically. These results demonstrate the existence of a signal pathway from beta-adrenergic receptors to membrane-bound ADP-ribosyl cyclase via G protein in the ventricular muscle cells and suggest that increased cADP-ribose synthesis is involved in up-regulation of cardiac function by sympathetic stimulation.