Bipolar secretion of androgen-binding protein and transferrin by Sertoli cells cultured in a two-compartment culture chamber.

The influence of various medium supplements on Sertoli cell (Sc) monolayer permeability to [3H]inulin and polarized secretion of transferrin (Trf) and androgen-binding protein (ABP) was investigated in a two-compartment culture chamber. Sc from 14- and 18-day-old rats were maintained for 13 days in one of the following media: Modified Eagle's Medium-Ham's F-12 (DFM), DFM plus insulin, epidermal growth factor, progesterone, hydrocortisone, and vitamins A and E (6F), 6F plus testosterone (7F), 7F plus FSH (8F), 6F plus 2% fetal bovine serum (6F2S), 6F plus 5% fetal bovine serum (6F5S). The monolayer permeability to [3H]inulin decreased rapidly during the initial 3-5 days of culture for the Sc isolated from 18- and 14-day old animals, then remained stable in all media except DFM. Morphological examination revealed the presence of tight junctions between neighbouring Sc in both age groups, indicating their de novo formation. Secretion of Trf was lowest in DFM and steadily declined. In all other media, Trf secretion peaked on day 5 and remained relatively constant after day 7. Medium 7F only slightly and inconsistently increased the secretion, whereas 8F was always highly stimulatory compared to 6F. Supplementation of 6F with serum resulted in the greatest Trf secretion. In the case of ABP, three different secretion patterns were noted depending on the medium composition; secretion was greatest in the presence of 5% fetal bovine serum. The medium supplements also differentially affected the polarity of Trf and ABP secretion. The ratio of Trf secreted to the outer and inner compartments (OC/IC) was approximately 2.0 in DFM and was not influenced by supplements in 6F, 7F, and 8F. However, in serum-containing media, the OC/IC ratio gradually increased with time to about 5.0 on day 13. The average OC/IC ratio of ABP was 1.7 in DFM and, in contrast to Trf, declined to 0.7 in other serum-free media. Serum supplementation reversed and increased the ABP ratio to about 6.0 on day 13. These data indicate that Sc grown on permeable supports form confluent monolayers that limit the diffusion of macromolecules, most likely due to the formation of tight junctions. The monolayer permeability as well as the total and polarized secretion of Trf and ABP are differentially regulated by hormones and serum factors.

Regulation of transepithelial electrical resistance in two-compartment Sertoli cell cultures: in vitro model of the blood-testis barrier.

The effects of FSH, testosterone (T), and incubation temperature on the development of inter-Sertoli cell (Sc) tight junctions were investigated in vitro by using repetitive measurements of transepithelial electrical resistance (TER). Control cultures developed stable TER of 100-145 omega cm2 during the initial 3-4 days of incubation at either 33 or 36.5 C, suggesting the formation of simple but continuous tight junctions. The presence of FSH (200 ng/ml) at 33 C delayed the onset of TER development by 3-5 days. The addition of FSH at the time of stable TER (day 5) resulted in a rapid (24 h) decrease of TER to 35-40 omega cm2, which returned to the control level during the subsequent 5-7 days. T alone (0.001-10 microM) caused an early and dose-dependent increase in TER to 165-240 omega cm2. In mono-layers incubated at 36.5 C, the continuous presence of FSH resulted in a dose-dependent increase in TER, which stabilized at 260-380 omega cm2 after 4-6 days. At this temperature, the addition of FSH on day 5 caused a rapid drop of TER similar to that observed at 33 C. This drop could not be prevented by antiproteases (aprotinin, epsilon-aminocaproic acid, or 10% fetal bovine serum) and was followed by an increase in TER up to 260-300-omega cm2. The Sc monolayers developed FSH-induced TER of 230-280 omega cm2 at 33 C, but only after several days of culture at 36.5 C. The effects of T at 36.5 and 33 C were similar, but the maximal TER values were significantly higher (290-380 omega cm2) at 36.5 C. The concomitant presence of T and FSH at 36.5 C resulted in the highest TER levels (580-1200 omega cm2) within 4-6 days, suggesting the synergistic effect of the two hormones on TER development. Dihydrotestosterone was more effective than T when used together with FSH, whereas estradiol had no effect. The different patterns of TER did not result from differences in Sc number or metabolic activity and probably reflected developmental and/or maturational changes in the inter-Sc tight junctions. It is concluded that FSH, T, and temperature play a role in the development of high TER by Sc monolayers (formation of tight junctions) in vitro. FSH and T appear to regulate TER via separate pathways and to cooperate by a yet unknown synergistic mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)

Vectorial secretion of inhibin by immature rat Sertoli cells in vitro: reexamination of previous results.

The vectorial secretion of immunoactive and bioactive inhibin by immature rat Sertoli cells (Sc) cultured in a two-compartment system was investigated using various culture supports. When Sc were cultured on Millipore-HA filters (used in all previous studies on vectorial secretion of inhibin), both immuno- and bioactive inhibin were found almost exclusively in the apical compartment, suggesting predominantly apical secretion of the glycoprotein. However, the cell-free Millipore-HA filters completely blocked the passage of Sc-conditioned medium (SCCM) inhibin, even after pretreatment with BSA and SCCM to saturate the protein-binding sites. On the other hand, polycarbonate Nucleopore filters or Millicell-CM membranes, both exhibiting extremely low protein-binding capacity, did not significantly block the passage of SCCM inhibin. When Sc were cultured on Nucleopore filters, the immunoactive inhibin was detected in both culture compartments; the basal compartment/apical compartment (BC/AC) ratio was about 1.5 (range, 1.2-1.9). The maximal effective dose of FSH or (Bu)2cAMP caused a 6- to 9-fold increase in the total (BC plus AC) secretion of immunoactive inhibin, but only a 60% increase in the secretion of bioactive inhibin, as evaluated by RIA and pituitary cell bioassay, respectively. The latter phenomenon was not accompanied by any significant change in the basal/apical distribution of either bioactive nor immunoactive inhibin. The presence of testosterone alone (10(-6) M) did not affect either total immunoactive inhibin secretion or its BC/AC ratio. The effects of the concomitant presence of FSH and testosterone did not differ significantly from those of FSH alone. Similarly to testosterone, the lack of any significant effect was observed for 17 beta-estradiol, dihydrotestosterone, androstenediol, and androstenedione regardless of the presence or absence of FSH. The striking dissimilarity of BC/AC ratios of inhibin noted in cultures maintained on Millipore-HA and Nucleopore filters was not due to differences in permeability barrier or Sc functional polarity. When cultured on either support, Sc monolayers developed comparable permeability barriers, as evaluated by measuring the passage of [3H]inulin and development of electrical resistance. The maximal electrical resistance (130-150 omega cm2) developed after 6-8 days of culture on either support. Also, total transferrin secretion and transferrin BC/AC ratio were similar on both supports, suggesting comparable cell numbers and functional polarities. These findings demonstrate that immature Sc in vitro secrete inhibin bidirectionally (BC/AC ratio, approximately 1.5); the polarity of secretion is unaffected by either FSH or various naturally occurring steroids, including testosterone.(ABSTRACT TRUNCATED AT 400 WORDS)

Action kinetics of inhibin in superfused pituitary cells depend on gonadotropin-releasing hormone treatment.

We examined the effects of partly purified inhibin from porcine follicular fluid on FSH and LH release in superfused rat pituitary cell cultures exposed to different GnRH stimuli. Pituitary cells from immature male rats were cultured in chemically defined medium. After 4 days of static culture in the absence of inhibin preparation and GnRH, the cell monolayers were superfused for approximately 10 h at a constant speed (0.15 or 0.25 ml/min) with medium with or without inhibin preparation (1 micrograms/ml). During the superfusion, some cultures were stimulated with GnRH (10 nM) continuously or intermittently (1 min/0.5 h or 6 min/1 h). In the basal condition (no GnRH), inhibin suppressed FSH release after 5 h of exposure (P less than 0.01), whereas LH secretion was not affected. In cultures treated with GnRH pulses (of either frequency), the inhibitory effects on the GnRH-stimulated FSH and LH release were statistically significant (P less than 0.01) after 2 h of exposure, became more pronounced in the next several hours, then remained stable until the end of the experiment. In cultures exposed to GnRH continuously, the suppressing effects of inhibin preparation became significant (P less than 0.01) after 3 h of exposure and were maximal at 4 h (52% and 61% of control values for FSH and LH, respectively). Later, the suppressing effect became less pronounce due to the decreasing rate of gonadotropin secretion in control (no inhibin) cultures exposed continuously to GnRH. The magnitude of FSH and LH suppression after 9 h of exposure to the inhibin preparation was statistically different (P less than 0.05) for different GnRH treatments and was more pronounced with GnRH pulses (24-27% and 54-57% of control values for FSH and LH, respectively) than with cultures exposed to GnRH continuously (77% and 89% of control values for FSH and LH, respectively) or in the absence of GnRH (50% and 92% of control values for FSH and LH, respectively). We conclude that both the kinetics and magnitude of action of the inhibin preparation on FSH and LH release can differ significantly depending on the presence or absence of GnRH as well as on the mode of GnRH stimulation. Of particular importance is the observation that suppressive effects of inhibin preparation decline in cultures that have been desensitized to GnRH after prolonged continuous GnRH exposures. These differences stress the role of GnRH-inhibin interactions in the regulation of gonadotropin secretion and emphasize the importance of the mode of GnRH stimulation in studies concerning inhibin action on pituitary cells in vitro.

Quantitation of plasma membrane expression of a fusion protein of Na/H exchanger NHE3 and green fluorescence protein (GFP) in living PS120 fibroblasts.

We developed a confocal morphometric analysis to quantitate the relative plasma membrane (PM) expression of the Na/H exchanger NHE3 in living PS120 fibroblasts. NHE3 is a membrane transport protein that is acutely regulated by changes in the number of molecules expressed at the PM. To quantitate the PM expression of NHE3 under various experimental conditions, we stably expressed a chimera of rabbit NHE3 and green fluorescent protein (NHE3-GFP) in PS120 fibroblasts. A three-dimensional (3D) map of the intracellular distribution of NHE3-GFP was obtained by confocal laser scanning microscopy (CLSM) of cells superfused with a styryl dye, FM 4-64. This fluorophore rapidly and reversibly labeled the outer lipid layer of the PM, which allowed generation of a digital mask of the PM and calculation of the fraction of a total cellular NHE3-GFP expressed at the PM. This analysis was successfully used to quantitate the relative PM expression of NHE3-GFP in control cells (25%) and a decrease in the expression caused by subsequent exposure of cells to wortmannin (5.1%). Reliability of the method was confirmed by cell surface biotinylation, which yielded very similar results. Confocal morphometric analysis is fast and reproducible and could potentially be used for investigations on regulation of expression of other membrane proteins.

Vectorial secretion of transferrin and androgen binding protein in Sertoli cell cultures: effect of extracellular matrix, peritubular myoid cells and medium composition.

We examined the effect of various culture conditions on the polarized secretion of androgen binding protein (ABP) and transferrin (Trf) by Sertoli cells (Sc) in vitro. Sc from 18-day-old rats were cultured as confluent monolayers on permeable membranes in two-compartment chambers for up to 11 days. Coating of the membranes with extracellular matrix (ECM) components: collagen IV + laminin (CL) or reconstituted basement membrane (RBM) enhanced ABP and Trf secretion (200% and 150%, respectively), with RBM being more effective than CL in stimulating ABP but not Trf secretion. Neither CL nor RBM significantly influenced the relative amounts of ABP and Trf secreted into the outer (OC) and inner (IC) compartments of the culture chamber (OC/IC ratio). All of these effects were not significantly influenced by the presence of testosterone and serum. Co-culture of Sc with peritubular myoid cells (Pc) significantly increased the secretion of both ABP and Trf, although the magnitude of stimulation and the time-response patterns were different for each protein. Co-culture with Pc also dramatically increased the OC/IC ratios for ABP and Trf. Testosterone (10(-6) M) enhanced the Pc effects. In cultures of Sc alone, presence of 2% fetal bovine serum increased the OC/IC ratios, whereas testosterone had no effect. Based on these results, we suggest a possible role of Pc in the regulation of Sc polarized secretions.

Effects of cyclic AMP and phorbol ester on transepithelial electrical resistance of Sertoli cell monolayers in two-compartment culture.

The effects of dibutyryl cyclic AMP [Bu)2cAMP) and phorbol ester (TPA), in the absence or presence of follicle-stimulating hormone (FSH) and/or testosterone, on the development of tight junctions by immature rat Sertoli cells (Sc) were investigated in vitro using the two-compartment culture system. The tight junction status was evaluated by repeated measurements of transepithelial electrical resistance (TER). Untreated cell monolayers developed stable TER of approximately 120 omega cm2 during 3 days of culture. Continuous presence of FSH (200 ng/ml) from day 1 onward significantly increased the TER up to approximately 300 omega cm2 after a transient (24-36 h) delay. The initial delay was prolonged to 3-4 days by the addition of 1-methyl-3-isobutylxanthine (MIX) (0.2 mM), whereas the subsequent increase of TER was significantly potentiated by the concomitant presence of testosterone (10 microM). Cholera toxin (CHT; 10 ng/ml) and forskolin (FR; 50 microM) mimicked these FSH effects. (Bu)2cAMP, at concentrations which maximally stimulated immunoactive inhibin secretion (100-500 microM), inhibited the initial TER increase and significantly decreased the TER level when added on days 1 and 5 of culture, respectively. In contrast, low concentrations of (Bu)2cAMP (4-20 microM) consistently stimulated the TER development, mimicking the stimulatory phase of FSH action. TPA (100 nM) alone had no effect on TER development, but potentiated the stimulatory effect of testosterone in a manner similar to FSH, CHT, FR or low concentrations of (Bu)2cAMP. These results demonstrate, for the first time, a concentration-dependent, dual effect of exogenous cAMP on the Sc function.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of germ cells on Sertoli cell secretory activity in direct and indirect co-culture with Sertoli cells from rats of different ages.

We examined the effect of direct and indirect Sertoli-germ cell co-culture on androgen binding protein (ABP) and transferrin (TRF) secretion by Sertoli cells (Sc) from 10-, 18-, and 26-day-old rats. Addition of germ cells (Gc), mainly (greater than 80%) pachytene spermatocytes, directly to Sc monolayers enhanced basal and follicle-stimulating hormone (FSH) + testosterone-stimulated ABP and TRF secretion at all three ages. When the Gc were co-cultured indirectly with Sc (separated by a Nucleopore filter), only 50% of the direct stimulatory effect was found at 18- and 26-day-old groups, whereas no difference between direct and indirect co-culture was noted with Sc from 10-day-old rats. With 18- and 26-day-old rat Sc, the Gc effect on ABP and TRF secretion declined after 6 days of Sc culture, reaching the level of Sc-only cultures after 10 days, whereas the direct effect was maintained throughout the entire culture period. With Sc from 10-day-old animals, both direct and indirect effect of Gc decreased after 6 days but the levels of ABP and TRF secretion remained above those of Sc-only cultures. The viability and number of Gc in indirect co-cultures were maintained significantly higher than in Gc-only control cultures. The direct and indirect Gc effect was completely reversed 48 h after the Gc were removed from Sc cultures of 18- and 26-day-old rats, whereas in Sc cultures from 10-day-old rats 40% of the stimulatory effect remained after 48 h of Gc removal. We conclude that Gc can influence Sc secretory activity through both direct contact and some released factor(s). These two pathways may have different relevance at different ages during sexual maturation.

Stimulatory effect of Sertoli cell secretory products on testosterone secretion by purified Leydig cells in primary culture.

We investigated the influence of media from nonstimulated (SCCM) and FSH-stimulated (F-SCCM) cultured rat Sertoli cells on testosterone secretion by purified rat Leydig cells maintained in culture for 4 days. Both SCCM and F-SCCM stimulated Leydig cell secretory activity to a level 2-6 times that of the control, the effect being always maximal on day 3 of culture. On day 3, concentrated SCCM had a greater stimulatory effect on testosterone secretion than the original (i.e. non-concentrated) one, the effect being dose-related and similar to that exerted by concentrated F-SCCM. On the other hand, original as well as concentrated F-SCCM stimulated the basal testosterone secretion in a dose-dependent manner on day 1 to about 200% and 400% of the control level, respectively, whereas SCCM exerted the 'early' effect only as a concentrated preparation. Preincubation of Leydig cells with F-SCCM enhanced both basal (190% control) and LH-stimulated (274% control) testosterone secretion when the LH (10 ng/ml) was added for 3 h on day 1. The enhanced influence of SCCM was noted only with the LH-stimulated cells (140% control). It is concluded that, in culture, Sertoli cells release at least 2 factors which enhance testosterone secretion by Leydig cells in vitro. One of them seems to be FSH-dependent and increases both basal and LH-stimulated testosterone secretion. This factor (MW greater than 1 kDa) is heat-labile and exerts its maximal effect between 12 and 18 h of culture. The second factor(s) acts predominantly on day 3 of culture, is apparently FSH-independent, and its influence on Leydig cell testosterone may be, at least in part, nonspecific.

Effects of recombinant human inhibin and testosterone on gonadotropin secretion and subunit mRNA in superfused male rat pituitary cell cultures stimulated with pulsatile gonadotropin-releasing hormone.

Effects of recombinant human inhibin (rh inhibin) and testosterone on follicle-stimulating hormone (FSH) and luteinizing hormone (LH) secretion and mRNA levels of gonadotropin subunits were investigated in superfused male rat pituitary cell cultures. During superfusion, the cells were stimulated with gonadotropin-releasing hormone (GnRH) pulses (10 nM, 6 min/h) and exposed to rh inhibin (2 ng/ml) and/or testosterone (10 nM) for up to 20 h. The concentrations of FSH and LH were measured in effluent media by radioimmunoassay (RIA), and subunit mRNAs were determined by Northern blot hybridizations using rat FSH beta, LH beta and alpha genomic and cDNA probes. Rh inhibin suppressed the secretion of FSH (30-40% of control) and the secretion of LH to 50-60% of control, but inhibited only FSH beta mRNA (to non-detectable levels). Testosterone alone suppressed the release of LH to 50% of control, whereas FSH release was increased to 130-160% (P less than 0.05) of control. This increase was due to higher interpulse values without significant changes in the pulse amplitude. Also FSH beta mRNA level was increased (1.5-fold, P less than 0.05) but only after 17-20 h of treatment. On the other hand, testosterone had no effect on LH beta and alpha subunit mRNA levels. Testosterone in combination with rh inhibin showed an inhibitory effect on LH beta mRNA; however, the pattern of LH release was not significantly different from that observed with rh inhibin or testosterone alone. Combined effects of testosterone and rh inhibin on FSH secretion and FSH beta mRNA were similar to those observed with rh inhibin alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Short-term regulation of NHE3 by EGF and protein kinase C but not protein kinase A involves vesicle trafficking in epithelial cells and fibroblasts.

NHE3 is an intestinal epithelial isoform Na+/H+ exchanger that is present in the brush border of small intestinal, colonic, and gallbladder Na(+)-absorbing epithelial cells. NHE3 is acutely up- and downregulated in response to some G protein-linked receptors, tyrosine kinase receptors, and protein kinases when studied in intact ileum, when stably expressed in PS120 fibroblasts, and in the few studies reported in the human colon cancer cell line Caco-2. In most cases this is due to changes in Vmax of NHE3, although in response to cAMP and squalamine there are also changes in the K'(H+)i of the exchanger. The mechanism of the Vmax regulation as shown by cell surface biotinylation and confocal microscopy in Caco-2 cells and biotinylation in PS120 cells involves changes in the amount of NHE3 on the plasma membrane. In addition, in some cases there are also changes in turnover number of the exchanger. In some cases, the change in amount of NHE3 in the plasma membrane is associated with a change in the amount of plasma membrane. A combination of biochemical studies and transport/inhibitor studies in intact ileum and Caco-2 cells demonstrated that the increase in brush border Na+/H+ exchange caused by acute exposure to EGF was mediated by PI 3-kinase. PI 3-kinase was also involved in FGF stimulation of NHE3 expressed in fibroblasts. Thus, NHE3 is another example of a transport protein that is acutely regulated in part by changing the amount of the transporter on the plasma membrane by a process that appears to involve vesicle trafficking and also to involve changes in turnover number.

Polarized Sertoli cell functions in a new two-compartment culture system.

Sertoli cells in vivo are highly polarized and interact with the inner (tubular) and outer (interstitial) fluids. To simulate this condition in vitro we developed a two-compartment culture system in which confluent Sertoli cell monolayers were grown on permeable supports (Millipore filters) separating the inner and outer fluid compartments. Monolayer permeability to (3H)-inulin decreased by 90% after 5 to 7 days of culture, presumably due to formation of tight junctions. This process was influenced by cell plating density. The cells were highly polarized morphologically, resembling their appearance in vivo, and secreted transferrin bidirectionally into both fluid compartments. The amount of transferrin secreted was 166% to 250% of that secreted by the same number of Sertoli cells cultured in plastic dishes. Testosterone (5 X 10(-8) M) doubled and testosterone + FSH (0.1 microgram/ml) increased transferrin secretion 3.6-fold. These results demonstrate that under suitable culture conditions the Sertoli cells remain both morphologically and functionally polarized, reflecting a more physiologic state.

Transferrin secretion in response to different modes of FSH stimulation and cycloheximide in superfused Sertoli cell cultures.

The influence of different modes of FSH stimulation and cycloheximide on transferrin secretion by rat Sertoli cells was investigated using a superfusion culture system. Sertoli cells from 18-day-old rats were cultured in serum-free medium on Matrigel-covered slides first in static conditions for 19 hours, and then superfused at a flow rate of 2.5 ml/hour. After an equilibration period of 48 hours to establish the basal rate of transferrin secretion, the cultures were exposed to various modes of FSH stimulation. Sertoli cells stimulated intermittently (20 min/2 hours) up to 22 hours responded to each consecutive FSH pulse with a rapid increase of transferrin secretion followed by a decline toward basal values. Continuous 22-hour exposure to FSH elicited an immediate increase followed by irregular fluctuations and a transient decline towards the baseline. With either mode of FSH stimulation, there was a secondary prolonged increase in transferrin secretion. Although cultures stimulated intermittently or continuously during the entire experimental period (22 hours) secreted similar cumulative amounts of transferrin (10.8 +/- 0.5 micrograms and 11.1 +/- 0.8 micrograms, respectively), there was a direct correlation between the secreted amount of transferrin and the duration of FSH exposure up to 8 hours. Addition of cycloheximide decreased both basal and FSH-stimulated transferrin secretion. However, even when cycloheximide was added 1 hour before FSH, an early secretory peak in response to FSH was still observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of germ cells on vectorial secretion of androgen binding protein and transferrin by immature rat Sertoli cells in vitro.

The influence of germ cells (greater than 85% pachytene spermatocytes) on vectorial secretion of androgen binding protein (ABP) and transferrin by immature rat Sertoli cells was investigated using two-compartment culture chambers. The ratio of ABP secreted into the outer and inner compartment in control cultures of Sertoli cells alone was 1.9, and was not influenced by either FSH or testosterone. Co-culture of Sertoli cells in direct contact with germ cells in the presence of FSH decreased this ratio, the decrease being most pronounced (0.7) after 2 days of co-culture. This effect was not observed if the germ cells were not in direct contact with Sertoli cell monolayers. The outer to inner compartment ratio of transferrin in Sertoli cell-alone cultures was 1.6 and, in contrast to ABP, was not significantly influenced by the addition of germ cells, even in the presence of FSH. It is concluded that in immature rat Sertoli cells the polarity of ABP secretion, but not that of transferrin, may be regulated by pachytene spermatocytes (and possibly other germ cells), and that this process is FSH-dependent.

The modified method for isolation and culture of highly homogeneous Leydig cell population from rat testes.

A modified method for isolation and culture of a pure population of rat Leydig cells is described. For obtaining crude interstitial cell suspension, decapsulated testes were dispersed in 0.02% collagenase solution in Ca2+, Mg2+--free Hanks medium for 1 hour. Then, approx. 5 X 10(7) cells were centrifuged in 10-90% discontinuous, isoosmotic Percoll gradient at 3000 g for 20 min. The cells from eight fractions obtained were collected and cultured in Eagle's MEM for 4 days. Using morphological methods, 1.059-1.070 g/ml density fraction contained 97% and 1.070-1.080 g/ml fraction contained 90% viable Leydig cells. The cells secreted testosterone to the culture medium and responded to LH stimulation with over four-fold increase in hormone secretion.

Why should a clinician care about the molecular biology of transport?

This review outlines the progress made over the last few years in three chosen areas of intestinal ion transport. In the field of intestinal secretion, research on the secretion of bicarbonate by pancreatic ducts and duodenal epithelia in cystic fibrosis revealed the crucial role of chloride channel (CFTR) in the control of activity of other transporters involved in bicarbonate secretion. In the area of intestinal absorption, studies on the regulation and physiologic roles of epithelial Na(+)/H(+) exchangers confirmed the suspected involvement of recycling in the acute regulation of NHE3 activity and resulted in formulation of new concepts for the roles of NHE3 and NHE2 in the gastrointestinal tract. Finally, the recent discovery of the first known viral enterotoxin revolutionized our understanding of pathomechanisms of secretory diarrhea during viral infections in humans. All of these findings are discussed in the context of their utility to the practicing gastroenterologist.

Experimental pitfalls in evaluating vectorial protein secretion in vitro; Sertoli cell secretion of androgen-binding protein and transferrin in two-compartment culture chambers.

We examined the influence of various Millipore filter pretreatments on the amounts of androgen-binding protein (ABP) and transferrin (Trf) found in the outer (OC) and inner (IC) compartment of two-compartment Sertoli cell (Sc) cultures. When Sc were cultured on untreated Millipore filters, less than 10% of ABP was found in OC during 3 initial culture days compared to similar cultures on pretreated filters. Most of the glycoprotein was shown to be bound by the filter. Pretreatment of Millipore filters with 5% bovine serum albumin (BSA) or 2% fetal bovine serum (FBS) maximally saturated the nonspecific protein-binding sites resulting in OC:IC ratio of ABP similar to that found in cultures on polycarbonate membranes, which exhibit very low protein-binding capacity. In contrast to ABP, about 40% of Trf was bound by the Millipore filter on Day 1, with only trace amounts bound thereafter. These differences were due to much higher secretion rate of Trf than ABP, resulting in a relatively smaller fraction of Trf bound to the filter. Again, the nonspecific binding of Trf was greatly reduced by filter pretreatment with 5% BSA or 2% FBS. It is concluded that complete saturation of protein-binding sites of cellulose ester supports is necessary for reliable evaluation of vectorial protein secretion by Sc and other polarized epithelial cells maintained in this type of culture. The implications of partial saturation of protein-binding sites of culture support in interpreting experimental results are discussed.

Study of the dynamics of Sertoli cell secretions in a new superfusion, two-compartment culture system.

A new superfusion, two-compartment culture system recently developed in our laboratory was used to investigate the dynamic changes in bidirectional secretion of transferrin (Trf) and androgen binding protein (ABP) by rat Sertoli cells (Sc) cultured for up to 12 d under various experimental conditions. The system is unique in that the cells are grown on porous substrate and can be superfused independently at the apical (A) and basal (B) surfaces. The Sc formed confluent monolayers with tight junctions and were highly polarized, morphologically resembling their normal appearance in vivo. The bidirectional secretion patterns (total amount and A:B ratio) of both Trf and ABP were affected by the addition of hormones (testosterone, 10(-6) M; follicle stimulating hormone, 0.1 microgram/ml; and fetal bovine serum 2%), but not by changes in the medium flow rate (0.8 to 3.2 ml/h). The superfusion, two-compartment culture system provides a very useful model for culture of polarized cell monolayers and for the study of bidirectional secretions under more "physiologic" conditions than those provided by static cultures.

Age-dependent Sertoli cell responsiveness to germ cells in vitro.

This study investigated the effect of germ cells (greater than 80% mid- and late-pachytene spermatocytes) on the secretion of androgen binding protein (ABP) and transferrin by monolayer cultures of Sertoli cells isolated from rats aged 10, 18 or 26 days. There was an age-dependent increase in secretion of ABP and transferrin. Treatment of the Sertoli cell monolayers with hypotonic buffer to remove residual germ cells reduced this increase significantly. On the other hand, addition of germ cells to hypotonic-treated Sertoli cell monolayers increased both basal and FSH + testosterone-stimulated ABP and transferrin secretion at all three ages, although Sertoli cells from 10-day-old animals showed the greatest response. Moreover, addition of germ cells reduced responsiveness to FSH + testosterone in Sertoli cell monolayers obtained from rats aged 18 or 26 days. In monolayers obtained from 10-day-old rats, the opposite effect was noted in the case of ABP secretion. The stimulatory effect of germ cells on ABP and transferrin secretion was proportional to their number, and was reversed 48 h after the germ cells added previously were removed by hypotonic treatment. Whereas the reversal was complete with cultures of Sertoli cells isolated from 18- and 26-day-old rats, approximately 40% of the stimulatory effect remained after removal of germ cells from cultures from the 10-day-old age group. Adhesion of germ cells to Sertoli cell monolayers was also found to be age-dependent, with the largest proportion of added germ cells adhering to Sertoli cells isolated at 18 and 26 days of age. It is concluded that germ cells can significantly and differentially modulate the basal and hormone-stimulated secretory activity of Sertoli cells in vitro and that Sertoli cell responsiveness to germ cells (pachytene spermatocytes) is age-dependent and seems to appear early during the maturation process, before these germ cells appear in the testis.