Involvement of the cytoskeleton in oxytocin secretion by cultured bovine luteal cells

A number of substances have been implicated in the regulation of oxytocin (OT) secretion from bovine corpus luteum in vivo. However, isolated bovine luteal cells cultured in a monolayer lose the ability to secrete OT in response to stimulatory substances. The present study investigated how cell-to-cell contact and the cytoskeleton affect OT secretion by isolated bovine luteal cells. In experiment 1, bovine midluteal cells (Days 8-12 of the estrous cycle) were stimulated with prostaglandin F2alpha (PGF2alpha; 1 microM), noradrenaline (NA; 10 microM), or growth hormone (GH; 5 nM) in two culture systems: In one system, cell monolayers were incubated in 24-well culture plates, and in the other system, aggregates of cells were incubated in glass tubes in a shaking water bath. The cells cultured in a monolayer underwent considerable spreading and showed a variety of shapes, whereas the cells cultured in glass tubes remained fully rounded during the experimental period and soon formed aggregates of cells. Although PGF2alpha, NA, and GH did not stimulate OT secretion by the monolayer cells, all tested substances stimulated OT secretion by the aggregated cells (P < 0.01). In experiment 2, the monolayer cells were pre-exposed for 1 h to an antimicrofilament agent (cytochalasin B; 1 microM) or two antimicrotubule agents (colchicine or vinblastine; 1 microM) before stimulation with PGF2alpha, NA, or GH. Although PGF2alpha, NA, and GH did not stimulate OT secretion by the monolayer cells in the presence of colchicine or vinblastine, they all stimulated OT secretion in the presence of cytochalasin B (P < 0.001). The overall results show that OT secretion by bovine luteal cells depends on microfilament function and cell shape. Moreover, the aggregate culture system that allows three-dimensional, cell-to-cell contact seems to be a good model for studying OT secretion by isolated bovine luteal cells.     

Binding of 3H-gossypol in organelles of cultured bovine luteal cells.

Gossypol is an antifertility agent which inhibits steroidogenesis in both sexes. The present study investigated the binding of gossypol in organelles of cultured bovine luteal cell to elucidate its inhibitory site of action in steroid biosynthesis. Cultured bovine luteal cells were incubated with 3H-gossypol (4.3 or 2.15 microM) for 3 hours. At the end of treatment, cultured bovine luteal cells were harvested, homogenized and centrifuged for organelle preparation. The radioactivity of gossypol was measured in each subcellular fraction. The cell membrane fraction has the highest binding capacity for gossypol, and the majority of gossypol was located in the particulate fractions. Results of the present study provide information in understanding the regulatory mechanism of gossypol on antisteroidogenic and/or toxic effects in cultured bovine luteal cells.     

Evidence for coupling of different receptors for gonadotropin-releasing hormone to phospholipases C and A2 in cultured rat luteal cells

Effects of [D-Ala6,Des-Gly10]gonadotropin-releasing hormone (GnRH), ethylamide (GnRHa), and prostaglandin F2 alpha (PGF2 alpha) on inositol phosphate (IPs) formation and arachidonic acid (AA) release were studied in rat luteal cells of primary culture. In the cells obtained from one-day-old corpora lutea, PGF2 alpha (100 nM) and GnRHa (100 nM) significantly increased the IPs formation and the AA release. Antagonists of GnRH added solely or with GnRHa did not stimulate the IPs formation but did stimulate the AA release. In the cells obtained from 5-day-old corpora lutea, GnRHa failed to stimulate the IPs formation but significantly stimulated the AA release. The stimulation of both IPs formation and AA release by PGF2 alpha was consistently found in cells of two different luteal ages. These results suggest that GnRH receptor independently couples to both phospholipases C and A2 through different classes of GnRH receptors.     

Demonstration of oxytocin release by bovine luteal cells utilizing the reverse hemolytic plaque assay.

Corpora lutea (CL) of a number of species produce oxytocin (OXT). In the present experiments we studied basal, prostaglandin (PG) F2 alpha-stimulated and ascorbate-stimulated OXT release from individual bovine luteal cells utilizing the reverse hemolytic plaque assay (RHPA). Using a mixture of C- and N-terminus-specific antisera against OXT, we were able to demonstrate OXT plaque formation by individual luteal cells. CL consist of two steroidogenic cell types: large luteal cells (LLC), believed to derive from granulosa cells and to produce and secrete OXT, and small luteal cells (SLC), thought to derive from theca cells. To distinguish between these two cell types, we designated cells greater than 20 microns as LLC and those less than 20 microns as SLC. On the basis of this morphological parameter, OXT release from both LLC and SLC was demonstrable. After an incubation period of 15 h, 7% of both cell types formed OXT plaques. PGF 2 alpha and ascorbate increased the size of plaques surrounding both LLC and SLC to more than 200% and 240%, respectively (basal plaque size = 100%). The number of plaque-forming cells increased only slightly in the presence of either PGF 2 alpha or ascorbate in comparison to basal conditions. We suggest that the RHPA can be used to demonstrate peptide release from luteal cells. It is concluded that LLC may be subdivided into functional subclasses because less than 10% of bovine luteal cells release OXT. Known OXT secretagogues increased the amount of OXT released. It appears that not only LLC but also SLC secrete this peptide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fas-mediated apoptosis is suppressed by calf serum in cultured bovine luteal cells

Calf serum (CS) is a common supplement used in cell culture. It has been suggested that CS contains substances protecting cells against apoptosis. To examine whether a culture system including CS is appropriate for studying apoptosis in bovine luteal cells, we examined the influence of CS on the expression of Fas, bcl-2 and bax gene. Since progesterone (P(4)) is known to be an anti-apoptotic factor in bovine luteal cells, the present study was carried out to examine the P(4) effect on apoptosis. Bovine mid-luteal cells were exposed to Fas ligand (Fas L) in the presence or in the absence of P(4) antagonist (onapristone, OP) in a basal medium (BM) containing 5% CS (BM-CS) or BM containing 0.1% BSA (BM-BSA). Although Fas L alone, OP alone or Fas L plus OP did not show any cytotoxic effect on the cells cultured in BM-CS, administration of OP or OP in combination with Fas L resulted in the killing of 30% and 55% of the cells cultured in BM-BSA medium, respectively (p<0.05). Concomitantly, CS inhibited bax mRNA expression and stimulated bcl-2 expression in the cells (p<0.05). Moreover, in the cells cultured with BM-CS, Fas mRNA expression was smaller than that of cells incubated in BM-BSA medium (p<0.05). The overall results suggest that CS suppressed Fas-mediated cell death in cultured bovine luteal cells by promoting the ratio of bcl-2 to bax expression and by inhibiting Fas expression. Therefore, it may be suggested that CS contains such anti-apoptotic substances (growth factors) amplifying the cell survival pathways in the bovine corpus luteum (CL) in vitro.     

Evidence for a systemic role for ovarian oxytocin in luteal regression in sheep

Jugular venous concentrations of oxytocin and progesterone changed in parallel during the oestrous cycle in the ewe, falling at luteal regression and rising with formation of the new corpus luteum. These fluctuations in the circulating concentration of oxytocin were not caused by changes in its metabolic clearance rate. On Days 6-9 of the cycle circulating oxytocin concentrations exhibited a diurnal rhythm, peaking at 09:00 h; this rhythm was absent on Days 11-14. Although there was no evidence for increased production of oxytocin at or preceding luteal regression in samples taken daily, more frequent sampling revealed that two thirds of detected surges of uterine secretion of prostaglandin (PG) F-2 alpha were accompanied by raised levels of oxytocin. This oxytocin was not of pituitary origin. Luteal regression induced with cloprostenol on Day 8 after oestrus caused a decrease in circulating progesterone level followed after 24 h by a fall in oxytocin. Measurements of oxytocin in the ovary and other organs before and after treatment with cloprostenol identified the corpora lutea as a major potential source of oxytocin, and suggested that 98% of luteal oxytocin was available for secretion in response to prostaglandin stimulation. The data are consistent with a role for ovarian secretion of oxytocin in response to uterine release of PGF-2 alpha in the control of luteal regression.     

Identification of G protein-coupled endothelin receptors in cultured bovine endothelial cells.

Autocrine and paracrine regulation of vascular endothelial cells by endothelins has been postulated and has been the target of many recent investigations. In the present study, we demonstrated, by affinity labeling, the presence of endothelin-1- and endothelin-3-specific receptors on cultured bovine endothelial cells that secrete endothelin; the endothelin-3 receptor was the major form. Analysis using the GTP analogue guanosine 5'-O- (thiotriphosphate) indicated that these receptors are coupled to G proteins.     

Interferon-gamma induction of major histocompatibility complex antigens on cultured bovine luteal cells

To investigate immunological mechanisms that may be involved in luteal function, the presence of Class I and Class II major histocompatibility complex (MHC) antigens on cultured bovine luteal cells was examined. After 72 h in serum-free culture, Class I antigens were markedly expressed on luteal cells, as determined by indirect immunofluorescence, whereas expression of Class II antigens was limited. The expression of MHC antigens on luteal cells was increased by treatment with the T-lymphocyte factor, interferon-gamma (IFN-gamma). Class I and II antigens were elevated 25% and 370% above controls, respectively, after IFN-gamma exposure. Since the corpus luteum is regulated by luteinizing hormone (LH), luteal cells were treated with either hormone alone or hormone in addition to IFN-gamma, and antigen expression was determined. LH treatment attenuated IFN-gamma-induction of Class II antigens on bovine luteal cells. These observations are the first to demonstrate the presence of MHC antigens on bovine luteal cells and the modulation of antigen expression by the lymphokine IFN-gamma and by LH.     

3H] prostaglandins binding to dispersed bovine luteal cells: evidence for discrete prostaglandin receptors.

Suspensions of dispersed bovine luteal cells prepared by collagenase digestion of luteal tissue specifically bound [³H]Prostaglandin (PG) E₁ and [³H]PGF_2α. While the number of sites per cell (～ 1.8 × 10⁵) were about the same for both [³H]PGs, the apparent Kds were different: [³H]PGE₁ ? 2.4 nM; [³H]PGF_2α ? 11 nM. The [³H]PGs binding was inhibited in a dose-dependent manner in the presence of increasing concentrations of unlabeled PGs. Potency order for inhibition of [³H]PGE₁ binding was: PGE₂ > PGE₁ > PGF_2α > PGF_1α. The corresponding data for [³H]PGF_2α was: PGF_2α > PGF_1α > PGE₂ > PGE₁. While [³H]PGE₁ and [³H]PGF_2α bind to their own receptors with high affinity, their affinities for each other's binding were extremely low. Thus, these results demonstrate that luteal cells, like plasma membranes isolated from luteal tissue, contain receptors for PGEs and PGF_2α which are discrete with respect to specificity and affinity.     

Regulation of low-density lipoprotein receptors in cultured bovine adrenocortical cells

Bovine adrenocortical cells in monolayer culture produce cortisol and respond to corticotropin (ACTH) by an increase in cortisol secretion. Several lines of evidence are indicative that much of the cholesterol that serves as precursor for steroid hormone biosynthesis by these cells is derived from low-density lipoprotein (LDL) cholesterol that is taken up endocytotically by means of specific receptors localized in bovine adrenocortical plasma membranes. ACTH stimulated this process concomitant with an increase in steroid production. In the absence of LDL, ACTH had no effect on steroid biosynthesis. ACTH action in bovine adrenocortical cells resulted in an increase in the number of LDL receptor sites in the membrane fractions, whereas the dissociation constant for LDL binding was not changed. Chloroquine and NH₄Cl, considered to be inhibitors of lysosomal degradative activity, caused an increase in the number of [¹²⁵I]iodoLDL binding sites in the plasma membrane but the effect of ACTH was still apparent in the presence of these agents. These results are suggestive that the lifetime of the LDL receptor is increased when lysosomal activity is inhibited. When aminoglutethimide was added to block cholesterol side-chain cleavage activity and inhibit steroid production, the number of [¹²⁵I]iodoLDL binding sites in the membrane fractions prepared from bovine adrenocortical cells cultured in the presence of ACTH was reduced to 50% of that in cells maintained in aminoglutethimide-free medium. However, under these conditions the number of binding sites was still significantly greater than in cells maintained in the absence of ACTH. The effects of aminoglutethimide on uptake and degradation of [¹²⁵I]iodoLDL were similar to the effects on the number of [¹²⁵I]iodoLDL binding sites. Based on these results, we conclude that the action of ACTH to stimulate LDL metabolism in bovine adrenocortical cells results from an increase in the number of LDL binding sites in the plasma membranes. This action of ACTH appears to be, at least in part, independent of cholesterol utilization for cortisol biosynthesis. However, the effect of aminoglutethimide is indicative that changes in the intracellular cholesterol concentration might modulate the action of ACTH to increase the number of LDL binding sites and therefore to stimulate LDL degradation.     

Hormonal regulation of oestrogen and progesterone receptors in cultured bovine endometrial cells.

Changes in the number of progesterone and oestradiol receptors in the endometrium are thought to play a role in the induction of luteolysis. The effect of oestradiol and progesterone on the regulation of their receptors in cultured bovine uterine epithelial and stromal cells was examined to determine the mechanisms involved in this process. Cells were obtained from cows at days 1-3 of the oestrous cycle and were cultured for 4 or 8 days in medium alone (RPMI medium + 5% (v/v) charcoal-dextran stripped newborn calf serum) or with oestradiol, progesterone or oestradiol and progesterone. At the end of culture, receptor binding was measured by saturation analysis. Specific binding of both [3H]ORG 2058 (16 alpha-ethyl-21-hydroxy-19-nor (6,7-3H) pregn-4-ene-3,20-dione) and [3H]oestradiol to epithelial and stromal cells showed high affinities (Kd = 1.1 x 10(-9) and 6 x 10(-10) mol l-1, respectively, for progesterone receptors; Kd = 5.5 x 10(-9) and 7 x 10(-10) mol l-1, respectively, for oestradiol receptors). In the stromal cells, oestradiol (0.1-10 nmol l-1) increased the number of oestradiol receptors from 0.21 +/- 0.06 to 0.70 +/- 0.058 fmol microgram-1 DNA and the number of progesterone receptors from 1.4 +/- 0.83 to 6.6 +/- 0.70 fmol microgram-1 DNA in a dose-dependent manner after 4 days of culture (P < 0.01). After culture for 8 days, the stimulatory effect of oestradiol increased. Progesterone (50 nmol l-1) had no effect on the number of oestradiol or progesterone receptors (P > 0.05). However, progesterone inhibited the stimulatory effect of oestradiol. In epithelial cells, the lower concentrations of oestradiol (0.1 and 1 nmol l-1) stimulated the number of progesterone receptors (P = 0.05) after 4 days culture, whereas the highest concentration of oestradiol (10 nmol l-1), progesterone (50 nmol l-1) and progesterone (50 nmol l-1) plus oestradiol (1 nmol l-1) had no effect. After culture for 8 days, the stimulatory effect of oestradiol decreased. In contrast to progesterone receptors, the number of oestradiol receptors increased with oestradiol concentration (P < 0.01). These data show that the number of progesterone receptors was higher in the stromal cells than in epithelial cells, whereas the number of oestradiol receptors was higher in the epithelial cells than in stromal cells. Oestradiol upregulates its own receptor and increases the number of progesterone receptors in both cell types in vitro, whereas progesterone has little effect, but inhibits the effects of oestradiol on progesterone receptors.     

Indoleamine 2,3-dioxygenase participates in the interferon-gamma-induced cell death process in cultured bovine luteal cells

Interferon-gamma (IFNG) induces apoptotic cell death in bovine luteal cells, but the pathway(s) involved in this process are not well defined. Evidence supporting the involvement of an IFNG-inducible enzymatic pathway that degrades tryptophan in IFNG-induced death of bovine luteal cells is presented in this study. The IFNG-inducible enzyme indoleamine 2,3-dioxygenase (INDO) catalyzes the first step in a metabolic pathway that degrades tryptophan. In the first experiment, RT-PCR revealed the presence of INDO mRNA in luteal cells treated with IFNG, but not in untreated cells. To determine whether INDO participates in IFNG-induced death of bovine luteal cells, an experiment was performed to test the effect of 1-methyl-D-tryptophan (1-MT), an inhibitor of INDO, on IFNG-induced DNA fragmentation in luteal cells. Single-cell gel electrophoresis and microscopic image analysis revealed that 1-MT inhibited DNA fragmentation induced by IFNG. To determine whether supplementation of cell cultures with additional tryptophan could also protect luteal cells from IFNG-induced DNA fragmentation, luteal cells were cultured in the presence of IFNG, and L-tryptophan was added to cultures to achieve final concentrations that were 5-, 10-, or 25-fold higher than the concentration of L-tryptophan found in nonsupplemented culture medium. Supplementation of IFNG-treated luteal cell cultures with elevated concentrations of tryptophan also prevented IFNG-induced DNA fragmentation. We conclude that INDO participates in IFNG-induced death of bovine luteal cells, through a mechanism that involves degradation of tryptophan, thereby reducing tryptophan concentrations to a point insufficient to meet luteal cells needs.     

Characterization of beta-adrenergic receptors in cultured human and bovine endothelial cells

We used radioligand binding methods to characterize beta-adrenergic receptors on endothelial cells cultured from adult human iliac vein (HIVE) and bovine fetal aorta (BFAE). For comparison, we also studied the well-characterized C6 glioma cell line (C6). Both human and bovine endothelial cells showed specific saturable binding of [125I]iodopindolol. There was no difference in the binding affinity (KD) of iodopindolol to membranes from the three cell types. However, the beta-receptor density (Bmax) was greater on HIVE cells and BFAE cells than on C6 cells. Displacement of ligand from HIVE and BFAE cells by zinterol or from BFAE cells by ICI 89,406 was consistent with binding to the beta 2-subtype. In contrast, displacement of ligand from C6 cells by zinterol or ICI 89,406 was consistent with binding to both beta 1- and beta 2-subtypes. Exposing BFAE cells in culture to 10 microM isoproterenol for 6 h resulted in a 55% decrease in Bmax without a change in KD. We conclude that 1) human and bovine endothelial cells in culture contain a substantial population of beta-adrenergic receptors, which are predominantly of the beta 2-subtype, and 2) endothelial beta-receptors exhibit downregulation by beta-agonists in culture.     

Effects of prostaglandin F2 alpha on agonist-induced progesterone production in cultured bovine luteal cells

The present study examines the effects of prostaglandin F2 alpha (PGF2 alpha) on basal and agonist-stimulated progesterone (P4) production utilizing long-term, serum-free cultures of bovine luteal cells. During the first 24 h of culture, PGF2 alpha had no significant effect on P4 production, and was unable to inhibit either luteinizing hormone (LH)- or dibutyryl cAMP (dbcAMP)-stimulated increases in P4. Treatment with PGF2 alpha on Day 1 produced a moderate, nonsignificant (P greater than 0.05) inhibition of cholera toxin (CT)- and forskolin (FKN)-stimulated P4 synthesis. Beyond Day 1 of culture (Days 3-11), PGF2 alpha continued to have no significant effect on basal P4 production, but suppressed all stimulatory effects of LH, dbcAMP, CT and FKN. Treatment with indomethacin inhibited prostaglandin synthesis by the cultured cells and also elevated levels of P4 from Days 3 to 11 of culture. Concurrent treatment with PGF2 alpha suppressed the steroidogenic effect of indomethacin. From these studies it was concluded that in cultured bovine luteal cells, PGF2 alpha does not affect basal P4 production, but is able to inhibit agonist-stimulated P4 production at a site beyond the accumulation of cAMP. This inhibitory effect is not apparent during the first 24 h of culture, but appears after Day 1 and persists throughout the remaining 10 days of the culture period.     

Influence of nitric oxide and noradrenaline on prostaglandin F(2)(alpha)-induced oxytocin secretion and intracellular calcium mobilization in cultured bovine luteal cells

Although prostaglandin (PG) F(2alpha) released from the uterus has been shown to cause regression of the bovine corpus luteum (CL), the neuroendocrine, paracrine, and autocrine mechanisms regulating luteolysis and PGF(2alpha) action in the CL are not fully understood. A number of substances produced locally in the CL may be involved in maintaining the equilibrium between luteal development and its regression. The present study was carried out to determine whether noradrenaline (NA) and nitric oxide (NO) regulate the sensitivity of the bovine CL to PGF(2alpha) in vitro and modulate a positive feedback cascade between PGF(2alpha) and luteal oxytocin (OT) in cows. Bovine luteal cells (Days 8-12 of the estrous cycle) cultured in glass tubes were pre-exposed to NA (10(-5) M) or an NO donor (S-nitroso-N:-acetylpenicillamine [S-NAP]; 10(-4) M) before stimulation with PGF(2alpha) (10(-6) M). Noradrenaline significantly stimulated the release of progesterone (P(4)), OT, PGF(2alpha), and PGE(2) (P: < 0.01); however, S-NAP inhibited P(4) and OT secretion (P: < 0.05). Oxytocin secretion and the intracellular level of free Ca(2+) ([Ca(2+)](i)) were measured as indicators of CL sensitivity to PGF(2alpha). Prostaglandin F(2alpha) increased both the amount of OT secretion and [Ca(2+)](i) by approximately two times the amount before (both P: < 0.05). The S-NAP amplified the effect of PGF(2alpha) on [Ca(2+)](i) and OT secretion (both P: < 0.001), whereas NA diminished the stimulatory effects of PGF(2alpha) on [Ca(2+)](i) (P: < 0.05). Moreover, PGF(2alpha) did not exert any additionally effects on OT secretion in NA-pretreated cells. The overall results suggest that adrenergic and nitrergic agents play opposite roles in the regulation of bovine CL function. While NA stimulates P(4) and OT secretion, NO may inhibit it in bovine CL. Both NA and NO are likely to stimulate the synthesis of luteal PGs and to modulate the action of PGF(2alpha). Noradrenaline may be the factor that is responsible for the limited action of PGF(2alpha) on CL and may be involved in the protection of the CL against premature luteolysis. In contrast, NO augments PGF(2alpha) action on CL and it may be involved in the course of luteolysis.     

Evidence for oxytocin receptors in the urinary bladder of the rabbit

Experiments were performed on isolated detrusor smooth muscle from New Zealand White rabbits. Oxytocin was shown to exhibit high intrinsic contractile activity on isolated strips of detrusor muscle, where the maximum contractile amplitude was 12% greater than control responses to 1 microM carbachol. Repeated applications of 1 microM oxytocin were associated with tachyphylaxis representing a 49% decrease in the amplitude which became reproducible after several applications without further decay of contractile strength. Dose-response experiments indicated that threshold contractions to oxytocin occur at 3 nM and were maximum at 10 microM with mean effective concentration of 125 nM. The contractile responses to 1 microM oxytocin were not antagonized by phentolamine, atropine, methysergide, saralasin, or naloxone, but were partially inhibited by 1 microM of indomethacin. Ligand binding studies on partially purified membrane preparations from detrusor smooth muscle were performed over a range of 78 pM to 10 nM with 125I-labelled oxytocin. Scatchard analysis of specific bound receptors indicated a KD of 2.5 nM and Bmax of 187 fmol/mg protein and a second compartment that was unsaturable at the concentrations of ligand employed. Nonspecific binding ranged from 36 to 77% of the total binding.     

Evidence for coupled transport of bicarbonate and sodium in cultured bovine corneal endothelial cells

Summary Usin gintracellular microelectrode technique, the response of the voltageV across the plasma membrane of cultured bovine corneal endothelial cells to changes in sodium and bicarbonate concentrations was investigated. (1) The electrical response to changes in [HCO ₃ ^– ] _o (depolarization upon lowering and hyperpolarization upon raising [HCO ₃ ^– ] _o ) was dependent on sodium. Lithium could fairly well be substituted for sodium, whereas potassium or choline were much less effective. (2) Removal of external sodium caused a depolarization, while a readdition led to a hyperpolarization, which increased with time of preincubation in the sodium-depleted medium. (3) The response to changes in [Na⁺] _o was dependent on bicarbonate. In a nominally bicarbonate-free medium, its amplitude was decreased or even reversed in sign. (4) Application of SITS or DIDS (10^–3 m) had a similar effect on the response to sodium as bicarbonate-depleted medium. (5) At [Na⁺] _o =151mm and [HCO ₃ ^– ] _o =46mm, the transients ofV depended, with 39.0±9.0 (sd) mV/decade, on bicarbonate and, with 15.3±5.8 (sd) mV/decade, on sodium. (6) After the preincubation of cells with lithium, replacement of Li by choline led to similar effects as the replacement of sodium by choline, though the response ofV was smaller with Li. This response could be reduced or reversed by the removal of bicarbonate or by the application of SITS. (7) Amiloride (10^–3 m) caused a reversible hyperpolarization of the steady-state potential by 8.5±2.6 mV (sd). It did not affect the immediate response to changes in [Na⁺] _o or [HCO ₃ ^– ] _o , but reduced the speed of regaining the steady-state potential after a change in [HCO ₃ ^– ] _o . (8) Ouabain (10^–4 m) caused a fast depolarization of –6.8±1.1 (sd) mV, which was followed by a continuing slower depolarization. The effect was almost identical at 10^–5 m. (9) It is suggested, that corneal endothelial cells possess a cotransport for sodium and bicarbonate, which transports net negative charage with these ions. It is inhibitable by stilbenes, but not directly affected by amiloride or ouabain. Lithium is a good substitute for sodium with respect to bicarbonate transport and is transported itself. In addition, the effect of amiloride provides indirect evidence for the existence of a Na⁺/H⁺-antiport. A model for the transepithelial transport of bicarbonate across the corneal endothelium is proposed.     

Gossypolone suppresses progesterone synthesis in bovine luteal cells

Gossypolone, a proposed major metabolite of gossypol, was synthesized and investigated for its effect on progesterone synthesis in cultured bovine luteal cells. Gossypolone inhibited human chorionic gonadotropin(hCG)-stimulated progesterone secretion, reduced substrate-enhanced conversions of 25-hydroxycholesterol to pregnenolone and of pregnenolone to progesterone in a dose-dependent fashion. These findings indicate that gossypolone inhibits not only 3β-hydroxysteroid dehydrogenase (3β-HSD) activity, as gossypol does, but also side-chain cleavage enzyme complex (cytochrome P450scc activity. However, the two compounds appear to have a similar potency in inhibiting progesterone secretion. Both gossypolone and gossypol (8.5 μM) induced morphological changes in cellular organelles.     

Sensitivity of cultured chick embryo heart cells to acetylcholine. Evidence for nicotinic and muscarinic receptors

Sensitivity of cultured chick embryo heart cells to acetylcholine changes with time in culture. In 24 h cultures, about 25% of the cells exhibit a positive chronotropic response to acetylcholine. This effect is no longer observed after 48 h in culture. Positive and negative chronotropic effects of acetylcholine can be related to the presence of nicotinic and muscarinic receptors evidenced by autoradiography. Some data suggest a possible relationship between the type of sensitivity to acetylcholine and the changes in cell membrane properties occurring in culture.