Leiomyoma primary cultures have elevated transcriptional response to estrogen compared with autologous myometrial cultures.

OBJECTIVE: We tested the hypothesis that uterine leiomyomas are hypersensitive to estrogen as compared with autologous human myometrium. METHODS: The estrogen-induced transcriptional responses of uterine leiomyoma and myometrial primary cultures were determined by transient expression assays. The relative levels of estrogen receptor (ER) in myometrial and leiomyoma tissues were determined by immunoblot. RESULTS: Myometrial and leiomyoma primary cultures were transcriptionally responsive to the estrogen ethinyl estradiol (eE2). The partial agonist tamoxifen did not elicit a positive transcriptional response and antagonized estrogen-induced transcription in the cultured cells. The responses of hormone-treated leiomyoma cells averaged 4.5-fold higher than those in controls with no hormone (P = .0001). The myometrial cells from women in the follicular phase exhibited little if any transcriptional response to eE2, whereas myometrial cells from women in the luteal phase had a transcriptional response to eE2 averaging threefold higher than that in no-hormone controls (P = .0083). Differences in response between autologous myometrial and leiomyoma cultures were statistically significant by the two-tailed Wilcoxon paired nonparametric signed-rank test (n = 11; P = .0137). These differences were more pronounced in cultures from women in the follicular or early luteal phase. In addition, the levels of ER increased in follicular and early luteal phase myometrial tissues, which correlated well with the number of days from the last menstrual period (n = 8; r = 0.9046; P = .002). Estrogen-receptor levels in myometrial tissues decreased during the late luteal phase. Levels in leiomyoma tissues did not follow the same pattern as in the myometrium and were elevated in tissues taken from women in the follicular phase. CONCLUSIONS: Autologous leiomyoma cultures have a significantly higher response to estrogen than do matched myometrial cultures, especially if the cultures are derived from the follicular phase. The levels of ER in leiomyoma tissue from women in the follicular phase are significantly elevated.

Acetazolamide inhibits acidification by the turtle bladder independent of cell pH.

Acetazolamide (ACZL) inhibits luminal acidification by the turtle urinary bladder, a process thought to be mediated by the drug's ability to inhibit carbonic anhydrase (CA) and thus elevate cell pH. To test the hypothesis that these transport changes are actually mediated by changes of cell pH, we measured this parameter in single, identifiable mucosal cells using 4-methylumbelliferone and fluorescence microscopy. In control bladders 5 X 10(-4) M ACZL inhibited proton transport by 80 +/- 6%, and alkalinized cell pH, especially in a subpopulation of CA cells. A much larger cell alkalinization was induced by serosal HCO3- but proton transport fell only 30 +/- 7%. When cell pH was clamped at approximately 7.0 using 50 mM dimethyloxazolidinedione, or when cell pH was acidified using 7.5 mM propionate, transport rates still declined by 74 +/- 2, and 100 +/- 12%, respectively, in response to ACZL. In propionate-acidified bladders, 1 mM sodium azide blocked the inhibition of transport seen with 5 X 10(-4) M ACZL and reversed the inhibition with 10(-5) M ACZL. The apical endocytosis rate was increased by ACZL in normal and propionate-acidified bladders, but was not stimulated by alkalinizing the cell with NH4Cl. We conclude that ACZL can induce cellular alkalinization in this tissue, but that this pH change is not required for the inhibition of transport, or the ACZL-associated stimulation of endocytosis. The drug's ability to inhibit acidification appears to be the result of an azide-sensitive mechanism that has yet to be defined.

Constitutive and transport-related endocytotic pathways in turtle bladder epithelium.

Proton secretion in the urinary bladder of the freshwater turtle is mediated by a proton pump located in the apical membrane of a population of cells characteristically rich in carbonic anhydrase. Earlier studies have demonstrated that these cells exhibit apical-membrane endocytotic and exocytotic processes which are thought to be involved in the regulation of the rate of proton transport via alterations in the number of pumps within the apical membrane. In this study, we sought to characterize these processes using two different methods. Analysis of transepithelial impedance yielded estimates of membrane capacitance which could be related to membrane area, thereby allowing one to monitor net changes in apical-membrane area resulting from changes in the net rates of endo- and exocytosis. Uptake of the fluid-phase marker FITC-dextran provided a measure of net extracellular volume uptake which was related to net rates of endocytosis. Our major conclusions are summarized as follows. The bladder cells exhibit a high baseline rate of endocytosis which appears to be a constitutive process similar to pinocytosis. This process is completely inhibited when ambient temperature is reduced to 15 degrees C. In addition, serosal application of 0.5 mM acetazolamide causes a transient increase in the rate of endocytosis, concomitant with a decrease in the rate of transport. Reduction of ambient temperature to 15 degrees C reduces the rate of acetazolamide-induced endocytosis, but does not abolish it. Addition of 1 mM serosal azide not only prevents the acetazolamide-induced increase in endocytosis, but also prevents the decrease in transport caused by acetazolamide. Azide has no effect on the baseline rate of endocytosis, nor does it prevent inhibition of carbonic anhydrase by acetazolamide. The specificity of azide, coupled with the different temperature sensitivities, demonstrate that the constitutive and transport-dependent endocytotic pathways are distinct processes. The observation that azide prevents both the acetazolamide-induced increase in endocytosis and the decrease in transport strongly supports the notion that endocytosis of proton-pump-containing membrane is requisite for the inhibition of transport by acetazolamide. Finally, the results also demonstrate that acetazolamide does not inhibit proton secretion simply by inhibiting carbonic anhydrase.

Proton transport and membrane shuttling in turtle bladder epithelium.

Proton secretion in the urinary bladder of the fresh-water turtle is mediated by proton pumps located in the apical membrane of carbonic-anhydrase (CA)-rich cells. It has been proposed that the rate of proton transport is regulated by endocytotic and exocytotic fusion processes which alter the apical membrane area, and hence number of exposed pumps. Three techniques were used to study this process. Analyses of transepithelial impedance provided estimates of transport-associated changes in net membrane area, as well as other electrical parameters. Electron microscopy allowed visualization of the endocytotic vesicles thought to be involved in the process. Finally, uptake of a fluorescent fluid-phase marker provided measurements of the rates of endocytosis. We report the following: endocytotic and exocytotic processes occur primarily in the CA-rich cells; inhibition of proton transport resulting from 0.5 mM acetazolamide (AZ) results in a decrease in the apical membrane area of approximately 0.47 cm2/cm2 tissue; the apical membrane specific conductance of the CA-rich cells is approximately 220 microS/microF, and possibly represents a Cl- conductance that may function in counter-ion flow; the decline in transport following AZ is not directly proportional to the decline in apical membrane area, suggesting that changes in pump kinetics are also involved in the regulation of transport; the CA-rich cells exhibit a high rate of constitutive pinocytosis, and hence membrane shuttling, which appears to be independent of the rate of transport; AZ induces a transient increase in the rates of endocytosis and shuttling; and the transport-associated changes in apical membrane area may reflect an effect of AZ on a regulated endocytotic pathway which is distinct from the pinocytotic process.

Fluorescence identifies an alkaline cell in turtle urinary bladder.

Intracellular pH (pHi) of turtle bladder mucosal cells was studied by the trapped fluorescent indicator technique. Bladders efficiently accumulated and converted 4-methylumbelliferyl acetate to its pH-sensitive derivative 4-methylumbelliferone (4MU). Excited at the pH-indifferent wavelength 334 nm, bladders fluoresced a uniform blue. Using pH-sensitive 365-nm excitation, 10-20% of the mucosal cells fluoresced distinctly brighter, suggesting a more alkaline pHi. Using the 365/334 ratio to quantitate pHi, this difference averaged 0.1 pH units. Bright cells were more distinct after SITS or acetazolamide but disappeared after digitonin permeabilization, dinitrophenol, or treatment with propionate, DMO, and NH4Cl. Essentially the same population of bright cells was identified by carboxyfluorescein diacetate. The brighter cell corresponded exactly to a population of cells with distinctive acridine orange staining and bright costaining with the potential-sensing probes Di-O-C5, Di-S-C3, and 4-Di-5-Asp. Two extremes of bright cell shape were seen: an elongate cell, prevalent under conditions stimulating H+ secretion, and a more compact cell, when acidification was inhibited. These observations support the hypothesis that acidification represents H+ secretion via the luminal membrane and that a primary role of carbonic anhydrase in this process is to support the exit of base from the cell. The more alkaline cells appear to be the carbonic anhydrase-rich cells. These cells are chemically isolated from the surrounding granular cells and change their morphology in response to changes in acidification. These special properties indicate a unique role for the carbonic anhydrase cell in H+ secretion.