Characterization of a mammalian smooth muscle cell line that has retained transcriptional and posttranscriptional potencies.

Unlike skeletal and cardiac muscle cells that differentiate irreversibly, smooth muscle cells (SMCs) retain a high degree of plasticity. During the so-called phenotypic modulation, SMCs can undergo transition between a contractile phenotype and a highly proliferative synthetic phenotype, as apparent from the extinction of numerous smooth muscle (SM) markers when they are passaged in culture. It would be very useful to have an SMC line that can be indefinitely propagated for the cellular and molecular analysis of the mechanisms that underlie the control of SM differentiation. This report describes an immortalized rabbit aorta SMC-derived cell line (U8A4) that has conserved differentiated properties through multiple subcultures. U8A4 cells can grow in the absence of serum and express the SMC markers studied, including SM alpha-actin, SM calponin, SM22alpha, SM alpha-tropomyosin (alpha-TM), SM myosin heavy chain (SM-MHC), and myocardin. U8A4 cells can activate SMC-restricted promoters like those of SM22alpha, SM calponin, and SM-MHC genes as efficiently as described previously for rat SMC lines (PAC1, A7r5, and A10). These cells can also process exogenous alpha-TM transcripts according to an SM-specific pattern. These results demonstrate that the U8A4 cell line constitutes a good alternative model to existing SMC lines that could facilitate the study of the transcriptional and posttranscriptional regulatory mechanisms underlying SMC differentiation.

Acute effects of adenosine triphosphates, cyclic 3',5'-adenosine monophosphates, and follicle-stimulating hormone on cytosolic calcium level in cultured immature rat Ssertoli cells.

The ability of ATP and FSH to induce intracellular calcium [Ca(2+)](i) changes in Sertoli cells is imperfectly understood and reports are conflicting. We have applied the single-cell microfluorometry technique with the calcium probe indo-1 to investigate [Ca(2+)](i) in individual cultured Sertoli cells. When cells were exposed to ATP, cAMP, and FSH, a fast and biphasic increase in [Ca(2+)](i) was obtained in 100%, 70%, and 56% of cells, respectively. Caffeine did not activate Ca(2+) mobilization, while thapsigargin suppressed the peak response. External calcium free-EGTA buffer suppressed the plateau phase, while blockers of voltage-operated Ca(2+) channels did not abolish the response to cAMP and ATP. We conclude that the three messengers mobilized Ca(2+) from intracellular thapsigargin-sensitive stores, which induced a subsequent Ca(2+) influx from the extracellular medium by a voltage-independent Ca(2+) entry. The well-documented mechanisms by which these messengers act on cells support the idea that they release Ca(2+) from smooth endoplasmic reticulum by two different pathways, or that FSH and cAMP first release ATP, which then acts on cells. Among the cells, 77% and 80% responded, respectively, to FSH and cAMP by a delayed long-lasting decrease in [Ca(2+)](i) that was never recorded in the presence of ATP. This suggests that FSH and cAMP also promote a slow redistribution of [Ca(2+)](i) from the exchangeable pool to the bound nonexchangeable pools. Involvement of voltage-operated and voltage-independent calcium channels in the response of Sertoli cells to ATP, FSH, and cAMP is discussed.

Inhibition by cAMP of calcium-activated chloride currents in cultured Sertoli cells from immature testis.

We have characterized a Ca2+-dependent Cl- current (ClCa) in cultured Sertoli cells from immature rat testis by using the whole cell recording patch-clamp technique. Cells dialyzed with pipette solutions containing 3 mm adenoside-triphosphate (ATP) and 1 microM free Ca2+, exhibited outward currents which were inhibited by 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and anthracene-9-carboxylic acid (9-AC) but insensitive to tetraethylammonium (TEA). Dialysis of cells with pipette solutions containing less than 1 nm free Ca2+ strongly reduced the currents indicating that they were Ca2+ dependent. With cells dialyzed with Cs+ glutamate-rich pipette solutions containing 0.2 mm EGTA, 10 microM ionomycin induced outward currents having properties of Ca2+-activated Cl- currents. With ATP-free pipette solution, the magnitude of currents was not modified suggesting the direct control by Ca2+. By contrast, addition of 0.1 mm cAMP in the pipette solution or the superfusion of cells by a permeant analogue of cAMP strongly reduced the currents. These results may suggest that ClCa is inhibited by cAMP-dependent protein kinase. Finally, our results do not agree with the model of primary fluid secretion by exocrine cells, but are in agreement with a hyperpolarizing effect of cAMP in primary culture of Sertoli cells and the release of a low Cl- and bicarbonate-rich primary fluid by these cells.

Voltage-dependent calcium current with properties of T-type current in Sertoli cells from immature rat testis in primary cultures.

We have applied the whole-cell configuration of the patch-clamp technique to investigate calcium currents in Sertoli cells from immature rat testis in primary culture. Cesium-filled pipettes were used to block potassium currents. In the presence of 10 mM extracellular calcium (Ca+), depolarizations elicited transient inward currents in the range -70 to +10 mV with a maximal amplitude of -1.3 pA/pF at -30 mV. This component activated in the range -70 to -20 mV (V0.5 = -42 mV) and inactivated in the range -75 to -45 mV (V0.5 = 61 mV). Currents were not modified when barium (Ba2+) was substituted for Ca2+. They were suppressed by Ca(2+)-free solutions, insensitive to Bay K 8644 (an L-type channel opener), and inhibited by 1 mM cobalt, 10 microM nickel, 10 microM isradipine, and 1-10 microM omega conotoxine GVIA (four calcium-channel blockers). We conclude that calcium channels with the properties of T-type calcium channels of excitable cells are located in the membrane of immature Sertoli cells in primary cultures. Because these channels do not appear to be directly sensitive to FSH, their involvement in calcium movements remains to be determined.