Reciprocity in the developmental regulation of aquaporins 1, 3 and 5 during pregnancy and lactation in the rat.

Milk secretion involves significant flux of water, driven largely by synthesis of lactose within the Golgi apparatus. It has not been determined whether this flux is simply a passive consequence of the osmotic potential between cytosol and Golgi, or whether it involves regulated flow. Aquaporins (AQPs) are membrane water channels that regulate water flux. AQP1, AQP3 and AQP5 have previously been detected in mammary tissue, but evidence of developmental regulation (altered expression according to the developmental and physiological state of the mammary gland) is lacking and their cellular/subcellular location is not well understood. In this paper we present evidence of developmental regulation of all three of these AQPs. Further, there was evidence of reciprocity since expression of the rather abundant AQP3 and less abundant AQP1 increased significantly from pregnancy into lactation, whereas expression of the least abundant AQP5 decreased. It would be tempting to suggest that AQP3 and AQP1 are involved in the secretion of water into milk. Paradoxically, however, it was AQP5 that demonstrated most evidence of expression located at the apical (secretory) membrane. The possibility is discussed that AQP5 is synthesized during pregnancy as a stable protein that functions to regulate water secretion during lactation. AQP3 was identified primarily at the basal and lateral membranes of the secretory cells, suggesting a possible involvement in regulated uptake of water and glycerol. AQP1 was identified primarily at the capillary and secretory cell cytoplasmic level and may again be more concerned with uptake and hence milk synthesis, rather than secretion. The fact that expression was developmentally regulated supports, but does not prove, a regulatory involvement of AQPs in water flux through the milk secretory cell.

Expression of the small conductance Ca²âº-activated potassium channel subtype 3 (SK3) in rat uterus after stimulation with 17ß-estradiol.

Preterm births accounts for roughly 9% of all births worldwide and can have detrimental or even lethal consequences for the infant. However to develop new treatment that will lower the rate of preterm births, more knowledge is required on the factors contributing to the contraction and relaxation of the myometrium. The small conductance Ca²âº-activated potassium channel subtype 3 (SK3) has been identified in the myometrium of several species including humans, mice and rats, but with great inter species variation of the expression pattern and regulation. The aim of this study was to investigate the expression of SK3 in the uterus of rats stimulated with 17ß-estradiol and progesterone in order to get an in depth understanding of the rat uterine SK3. Using immunohistochemistry SK3 was localized to the glandular and luminal endometrial lamina epitheliali. Furthermore, a weak signal was observed in the myometrium. Using Western blot the protein level of SK3 was found to increase in uteri from animals treated with 17ß-estradiol, an effect that was not reflected at the mRNA level. The levels of mRNA for SK3 were significantly lower in the uterus of 17ß-estradiol-treated animals than in the uterus of ovariectomized animals. We conclude that the SK channels are present in the endometrial epithelium, and possibly also in the myometrium of the rat uterus. Furthermore, the hormonal effect on SK3 caused by 17ß-estradiol includes divergent regulation at mRNA and protein levels.

Effect of the SK/IK channel modulator 4,5-dichloro-1,3-diethyl-1,3-dihydro-benzoimidazol-2-one (NS4591) on contractile force in rat, pig and human detrusor smooth muscle.

OBJECTIVE: • To investigate the importance of small (SK)- and intermediate (IK)-conductance Ca2(+) -activated K(+) channels on bladder function, by studying the effects of 4,5-dichloro-1,3-diethyl-1,3-dihydro-benzoimidazol-2-one (NS4591), a new modulator of SK/IK channels, on contractions induced by electrical field stimulation (EFS) and carbachol in rat, pig and human detrusor. PATIENTS AND METHODS: • Detrusor biopsies were obtained from rats, pigs and male patients undergoing cystectomy because of bladder cancer. • Force was recorded using myographs. • Intracellular free Ca(2+) was measured in myocytes using microfluorimetry. RESULTS: • In rat bladder rings subjected to EFS, cumulative addition of NS4591 (0.1-30 µM) decreased force by 82 ± 2.9% (n = 6).This effect was reduced by 64 ± 5.2% in the presence of 0.3 µM apamin, a specific inhibitor of SK channels. Apamin increased the force evoked by EFS significantly: force was increased by 14.2 ± 3.4% (n = 5) and 10.1 ± 2.6% (n = 7) in pig and human detrusor strips, respectively (P = 0.04 and P = 0.02). • The cumulative addition of NS4591 (0.3-30 µM) significantly reduced the amplitude of carbachol-induced rhythmic oscillations by 62.0 ± 12.0% (n = 12) and the minimum force between oscillations by 30 ± 5% (n = 9) in pig detrusor strips (P < 0.005). In the presence of 10 µM NS4591, carbachol (1 µM) induced rhythmic contractions with an amplitude and normalized mean power frequency (nmeanPF) of 8.4 ± 5.1% and 0.11 ± 0.06 mN root mean square (rms) Hz (n = 12), respectively, vs. 21 ± 3.4% and 0.17 ± 0.04 mN rms Hz in control strips (n = 13). Apamin induced 6- and 11-fold increases in amplitude and nmeanPF vs. 1.3- and 2-fold increases in control strips. • In human detrusor strips (n = 15), the cumulative addition of NS4591 (1-30 µM) significantly reduced the amplitude by 69 ± 11%, the nmeanPF by 78 ± 6% and the minimum force between carbachol-induced oscillations by 59 ± 5% (P < 0.008). The addition of apamin (0.3 µM) before application of 1 µM carbachol abolished the effects of NS4591 on amplitude and partially abolished its effect on nmeanPF by 41 ± 7%, vs. a 78 ± 6% reduction in the absence of apamin (n = 8). • In spontaneously active detrusor preparations, NS4591 reduced or abolished contractions. • Furthermore, NS4591 (10 µM) decreased the carbachol-induced increase in the fura-2 ratio by 43 ± 3% compared with control (n = 12) (P < 0.03). CONCLUSIONS: • The SK/IK channel modulator NS4591 inhibits EFS- and carbachol-induced contractions in rat, pig and human detrusor muscle. • NS4591 may have therapeutic potential for treatment of detrusor overactivity.