Exisulind, a novel proapoptotic drug, inhibits rat urinary bladder tumorigenesis.

Exisulind (Aptosyn) is a novel antineoplastic drug being developed for the prevention and treatment of precancerous and malignant diseases. In colon tumor cells, the drug induces apoptosis by a mechanism involving cyclic GMP (cGMP) phosphodiesterase inhibition, sustained elevation of cGMP, and protein kinase G activation. We studied the effect of exisulind on bladder tumorigenesis induced in rats by the carcinogen, N-butyl-N-(4-hydroxybutyl) nitrosamine. Exisulind at doses of 800, 1000, and 1200 mg/kg (diet) inhibited tumor multiplicity by 36, 47, and 64% and tumor incidence by 31, 38, and 61%, respectively. Experiments on the human bladder tumor cell line, HT1376, showed that exisulind inhibited growth with a GI(50) of 118 microM, suggesting that the antineoplastic activity of the drug in vivo involved a direct effect on neoplastic urothelium. Exisulind also induced apoptosis as determined by DNA fragmentation, caspase activation, and morphology. Analysis of phosphodiesterase (PDE) isozymes in HT1376 cells showed PDE5 and PDE4 isozymes that were inhibited by exisulind with IC(50)s of 112 and 116 microM, respectively. Inhibition of PDE5 appears to be pharmacologically relevant, because treatment of HT1376 cells increased cGMP and activated protein kinase G at doses that induce apoptosis, whereas cyclic AMP levels were not changed. Immunocytochemistry showed that PDE5 was localized in discrete perinuclear foci in HT1376 cells. Immunohistochemistry showed that PDE5 was overexpressed in human squamous and transitional cell carcinomas compared with normal urothelium. The data lead us to conclude that future clinical trials of exisulind for human bladder cancer treatment and/or prevention should be considered and suggest a mechanism of action involving cGMP-mediated apoptosis induction.

Reinnervation of motor endplates and increased muscle fiber size after human insulin-like growth factor I gene transfer into the paralyzed larynx.

Current surgical strategies for the treatment of laryngeal paralysis are limited by the muscle atrophy associated with denervation. Moreover, attempts at reinnervation have not effected significant change in surgical outcome. To address this clinical problem, we have developed a rat laryngeal paralysis model to study novel gene transfer strategies. Using this model, the human insulin-like growth factor I (hIGF-I) gene was introduced into paralyzed rat laryngeal muscle to assess the benefit of sustained local hIGF-I production. A muscle-specific nonviral vector containing the alpha-actin promoter and hIGF-I gene was used in formulation with a polyvinyl-based delivery system and injected into paralyzed adult rat laryngeal muscle. Twenty-eight days after a single injection, gene transfer efficiency, muscle fiber size, motor endplate length, and nerve-to-motor endplate contact were evaluated. Gene transfer was detected in 100% of injected animals by PCR. Gene transfer with expression, as measured by RT-PCR for hIGF-I mRNA, occurred in 81.3 % of injected animals. When compared with controls, hIGF-I-transfected animals presented a significant increase in muscle fiber diameter [17.56 (+/-0.97 SD) microm versus 14.70 (+/-1.43 SD) microm; p = 0.0002], a significant decrease in motor endplate length [20.88 (+/-1.42 SD) microm versus 25.41 (+/-3.19 SD) microm; p = 0.0025], and a significant increase in percentage of endplates with nerve contact (20.3% (+/-13.9 SD) versus 4.4% (+/-4.2 SD); p = 0.0079). In the context of laryngeal paralysis, gene therapy represents a tremendous opportunity to augment current surgical treatment modalities by preventing or reversing muscle atrophy, and by enhancing nerve sprouting and reinnervation.

A physicochemical approach for predicting the effectiveness of peptide-based gene delivery systems for use in plasmid-based gene therapy.

Novel synthetic peptides, based on carrier peptide analogs (YKAKnWK) and an amphipathic peptide (GLFEALLELLESLWELLLEA), have been formulated with DNA plasmids to create peptide-based gene delivery systems. The carrier peptides are used to condense plasmids into nanoparticles with a hydrodynamic diameter (DH) ranging from 40 to 200 nm, which are sterically stable for over 100 h. Size and morphology of the carrier peptide/plasmid complex have been determined by photon correlation spectroscopy (PCS) and transmission electron microscopy (TEM), respectively. The amphipathic peptide is used as a pH-sensitive lytic agent to facilitate release of the plasmid from endosomes after endocytosis of the peptide/plasmid complex. Hemolysis assays have shown that the amphipathic peptide destabilizes lipid bilayers at low pH, mimicking the properties of viral fusogenic peptides. However, circular dichroism studies show that unlike the viral fusion peptides, this amphipathic peptide loses some of its alpha-helical structure at low pH in the presence of liposomes. The peptide-based gene delivery systems were tested for transfection efficiency in a variety of cell lines, including 14-day C2C12 mouse myotubes, using gene expression systems containing the beta-galactosidase reporter gene. Transfection data demonstrate a correlation between in vitro transfection efficiency and the combination of several physical properties of the peptide/plasmid complexes, including 1) DNA dose, 2) the zeta potential of the particle, 3) the requirement of both lytic and carrier peptides, and 4) the number of lysine residues associated with the carrier peptide. Transfection data on 14-day C2C12 myotubes utilizing the therapeutic human growth hormone gene formulated in an optimal peptide gene delivery system show an increase in gene expression over time, with a maximum in protein levels at 96 h (approximately 18 ng/ml).

Systemic effect of human growth hormone after intramuscular injection of a single dose of a muscle-specific gene medicine.

A muscle-specific gene medicine is described that provides for long-term secretion of biologically active human growth hormone (hGH) from skeletal muscle into the systemic circulation. The hGH gene medicine is composed of a muscle-specific hGH plasmid expression system complexed with a protective, interactive, non-condensing (PINC) delivery system. The muscle-specific gene expression system, pSK-hGH-GH, was constructed by linking the promoter/enhancer regions of chicken skeletal alpha-actin to hGH gene. C2C12 myoblast transfection with pSK-hGH-GH resulted in the synthesis of hGH in a muscle-specific manner. Direct injection into rat tibialis cranialis muscle of pSK-hGH-GH complexed with a polymeric PINC delivery system, polyvinylpyrrolidone (PVP), produced hGH levels in muscle that were 10- to 15-fold higher compared with plasmid formulated in saline at 14 days post-injection. Intratracheal instillation in rat lung of pSK-hGH-GH did not produce significantly detectable levels of hGH. In hypophysectomized rats, a single intramuscular dose of the pSK-hGH-GH/PVP complex resulted in hGH expression and a subsequent increase in serum levels of rat IGF-I and growth. hGH expression and effects on rat serum IGF-I levels were detectable up to 28 days after injection of formulated plasmid and effects on growth were detectable unto 21 days. Anti-hGH antibodies were detectable in serum at 14 days post-injection, reached a plateau at 21 days, and remained elevated through the study period. Cyclosporin treatment of the pSK-hGH-GH/PVP-injected animals completely inhibited the antibody response and resulted in increased hGH expression.

Expression of biologically active human insulin-like growth factor-I following intramuscular injection of a formulated plasmid in rats.

Recent evidence has shown that insulin-like growth factor-I (IGF-I) plays an important role in the development, maintenance, and regeneration of peripheral nerves and skeletal muscle. IGF-I offers the potential to treat neuromuscular diseases in humans. We have developed a nonviral gene therapy method to express and produce localized and sustained therapeutic levels of IGF-I within target muscles by intramuscular injection of formulated plasmids. The purpose of the present study was to demonstrate that intramuscular injection of a plasmid encoding human IGF-I (hIGF-I) and engineered to restrict expression to skeletal muscle produces sustained local concentrations of biologically active hIGF-I. Normal rats received a single intramuscular injection of plasmids formulated as a complex with polyvinylpyrrolidone (PVP). Results show that hIGF-I mRNA and hIGF-I protein were detectable in the injected muscles for the duration of the study (28 days), whereas the hIGF-I protein was not detected in blood. Biological activity of hIGF-I was determined by immunodetection of a nerve-specific growth-associated protein, GAP-43, an indicator of motor neuron sprouting. Placement of human growth hormone (hGH) 3' untranslated region enhanced GAP-43 staining, probably due to improved secretion of hIGF-I. Enhanced immunoreactivity of GAP-43 was observed in muscles injected with the formulated hIGF-I plasmid when compared to controls. These results demonstrate that intramuscular injection of hIGF-I plasmid formulated as a complex with PVP produces a localized and sustained level of biologically active hIGF-I.

Influence of dietary background on the response of pigs to the beta-adrenergic agonist BRL 47672.

The level of protein in the diet both before and during treatment with beta-adrenergic agonist (BAA) was investigated with regard to its effect on growth performance and carcass characteristics of pigs. A total of 76 pigs were divided into two groups and given ad libitum access to diets containing either 12 or 18% crude protein (CP) during growth from 15 to 60 kg. At 60 kg, six pigs from each group were slaughtered and carcass composition was determined. For the test period (growth from 60 to 90 kg) the remainder of the pigs were divided into eight groups of eight pigs each. Half of the pigs from each of the two dietary pretreatment groups were switched to the other diet and half remained on the same diet. Of the four dietary groups, half were fed 0 mg/kg (controls) or 1 mg/kg of BRL 47672. During the test period, all pigs were fed at 90% of their calculated ad libitum DE intake. All pigs were slaughtered at 90 kg and carcass composition was determined. Addition of BRL 47672 to the diet resulted in an overall improvement (P < .05) in daily gain (.72 vs .64 kg) and gain/feed (.28 vs .26); the greatest improvement occurred with pigs fed the 18% CP diet (24 and 14% for daily gain and gain/feed, respectively). Pigs that had received BRL 47672 also had 12% less backfat, 10% less carcass lipid, 16% larger longissimus muscle area, and 5% more carcass protein (P < .05 for each), and the greatest changes occurred in pigs fed the 18% CP diet (-26, -16, +21, and +10%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Bovine corpus luteum function after removal of granulosa cells from the preovulatory follicle.

Experiments were conducted to determine the effects of removing granulosa cells from bovine preovulatory follicles on subsequent corpus luteum (CL) function. Holstein heifers were assigned to three groups: untreated controls (n = 6), removal of granulosa cells (n = 9) and removal and return of granulosa cells (n = 7). Surgery was performed 18-24 hr after the onset of estrus and in all cases after the preovulatory luteinizing hormone (LH) surge. Jugular venous blood was collected and estrous activity monitored twice daily. Corpora lutea were formed in six heifers in each group. Concentrations of plasma progesterone were reduced (P less than 0.05) on Days 7-17 in heifers from which granulosa cells were removed when compared to the other two groups. There were no differences in the lengths of the estrous cycles nor concentrations of LH in the three groups. In summary, these experiments support the concept that granulosa cells make a substantial contribution to the output of progesterone by the cyclic CL but may have a limited role in determining the functional lifespan of the CL. These experiments also establish the fact that granulosa cells develop into functional luteal cells after their removal and return to the preovulatory follicle.

Differential origin and control mechanisms in small and large bovine luteal cells.

Studies of the calcium requirement and the relationship of intracellular calcium to progesterone synthesis in highly purified preparations of bovine luteal cells reveal a remarkably close relationship between intracellular calcium levels and steroidogenesis. The differential responses of the two cell types, summarized in Table 2, are beginning to reveal how the two cell types may co-operate to produce both luteotrophic and luteolytic responses at different stages of the oestrous cycle and early pregnancy. The luteotrophic mechanisms in the small cells are fairly clear; in addition to the luteotrophic effects of LH and cAMP, activation of protein kinase C leads to increased progesterone synthesis. Accordingly, PGF-2 alpha and several other prostanoids are luteotrophic in these cells. PGF-2 alpha stimulates phospholipase C activity in the small cells but does not reduce LH-stimulated cAMP or progesterone accumulation (Davis et al., 1989). This acute stimulus of protein kinase C activation to progesterone production in bovine small luteal cells is rapidly desensitized, although its stimulus to prostanoid production continues for at least 24 h. Large cells respond to LH, but only at relatively high levels. In addition, we have no good evidence for a role for protein kinase C in the control of progesterone synthesis in the large bovine luteal cells from mid-cycle corpora lutea. Phorbol esters have no effect on steroidogenesis and it is not yet established that protein kinase C provides the same high affinity receptor for phorbol esters that is found in the small cells. Experiments with inhibitors of protein kinase C, such as staurosporine, in large cells have been inconclusive. Evidence for several species suggests that both cell types co-operate, in ways not yet fully understood, to bring about maximal progesterone production at mid-cycle. Some evidence suggests that they may also co-operate to bring about luteolysis. The concept that PGF-2 alpha initiates luteolysis by inhibiting LH stimulated progesterone production in the large cells must be revised in light of the relative insensitivity of these cells to LH and the fact that they probably constitutively express the cholesterol side-chain cleavage enzymes (P-450scc) that represent the rate-limiting step in progesterone production. Oonk et al. (1989) have reported that, once P-450scc mRNA is induced in rat granulosa cells by the LH surge, it is constitutively maintained by the luteinized cells in the absence of gonadotrophins and is no longer regulated by cAMP.(ABSTRACT TRUNCATED AT 400 WORDS)

Differential effects of calcium on progesterone production in small and large bovine luteal cells.

We studied the effects of calcium (Ca2+) ions in progesterone (P) production by separated small and large luteal cells. Corpora lutea were collected from 31 heifers between days 10 and 12 of the estrous cycle. Purified small and large cells were obtained by unit gravity sedimentation and flow cytometry. P accumulation in cells plus media was determined after incubating 1 x 10(5) small and 5 x 10(3) large cells for 2 and 4 h respectively. Removal of Ca2+ from the medium did not influence basal P production in the small cells (P greater than 0.05). However, stimulation of P by luteinizing hormone (LH), prostaglandin E2 (PGE2), 8-bromo-cyclic 3',5' adenosine monophosphate (8-Br-cAMP) and prostaglandin F2 alpha (PGF2 alpha) was impaired (P less than 0.05) by low Ca2+ concentrations. LH and PGE2-stimulated cAMP production was not altered by low extracellular Ca2+ concentrations, and PGF2 alpha had no effect on cAMP. In contrast, basal as well as LH and forskolin-stimulated P production were attenuated (P less than 0.05) in Ca2(+)-deficient medium in the large cells. However, P production stimulated by 8-Br-cAMP was not altered in Ca2(+)-deficient medium. Steroidogenesis in large cells was also dependent on intracellular Ca2+, since 8-N, N-diethylamineocytyl-3,4,5-trimethoxybenzoate (TMB-8), an inhibitor of intracellular Ca2+ release and/or action, suppressed (P less than 0.05) basal, LH and 8-Br-cAMP stimulated P. In contrast, basal P in small cells was not altered by TMB-8; whereas LH-stimulated P was reduced 2-fold (P less than 0.05). The calcium ionophore, A23187, inhibited LH-stimulated P in small cells and both basal and agonist-stimulated P in large cells. These studies show that basal P production in small cells does not require Ca2+ ions, while hormone-stimulated P production in small cells and both basal and hormone-stimulated P in large cells do require Ca2+. The inhibitory effect of Ca2+ ion removal was exerted prior to the generation of cAMP in the large cells, but distal to cAMP generation in hormone-stimulated small cells. The calmodulin/protein kinase C antagonist, W-7, also inhibited both basal and hormone-stimulated P production in both small and large luteal cells, indicating that P production in luteal cells also involves Ca2(+)-calmodulin/protein kinase C-dependent mechanisms.

Arachidonic acid and its metabolites increase cytosolic free calcium in bovine luteal cells.

We studied the effects of arachidonic acid and its metabolites on intracellular free calcium concentrations ([Ca2+]i) in highly purified bovine luteal cell preparations. Corpora lutea were collected from Holstein heifers between days 10 and 12 of the estrous cycle. The cells were dispersed and small and large cells were separated by unit gravity sedimentation and flow cytometry. The [Ca2+]i was determined by spectrofluorometry in luteal cells loaded with the fluorescent Ca2+ probe, Fura-2. Arachidonic acid elicited a dose-dependent increase in [Ca2+]i in both small and large luteal cells, having an effect at concentrations as low as 5 microM; and was maximally effective at 50 microM. Several other fatty acids failed to exert a similar response. Addition of nordihydroguaiaretic acid (NDGA) or indomethacin failed to suppress the effects of arachidonic acid. In fact, the presence of both inhibitors resulted in increases of [Ca2+]i, with NDGA exerting a greater stimulation of [Ca2+]i than indomethacin. Prostaglandin F2 alpha (PGF2 alpha) as well as prostaglandin E2 (PGE2) increased [Ca2+]i in the small luteal cells. These results support the idea that arachidonic acid exerts a direct action in mobilizing [Ca2+]i, in the luteal cells. Furthermore, they demonstrate that the cyclooxygenase (PGF2 alpha and PGE2) and lipoxygenase products of arachidonic acid metabolism also play a role in increasing [Ca2+]i in bovine luteal cells. Since the bovine corpus luteum contains large quantities of arachidonic acid, these findings suggest that this compound may regulate calcium-dependent functions of the corpus luteum, including steroid and peptide hormone production and secretion.

Phorbol ester receptors in bovine luteal cells: relationship to protein kinase C.

We investigated the binding kinetics of the tumor-promoting phorbol ester, phorbol-12,13-dibutyrate (PBt2) to dispersed total bovine luteal cells, purified small luteal cells, and purified luteal protein kinase C (PKC). Saturation analysis and competitive displacement techniques were used. Binding of [3H]PBt2 to total luteal cell preparations resulted in two distinct affinities. The high affinity component was characterized by a Kd of 4.5 +/- 1.5 nM. Analysis of [3H]PBt2 binding to total cells using competitive displacement demonstrated that the low affinity binding was specific and displaceable but dependent on concentrations of [3H]PBt2 far above the Kd for the high affinity binding. In contrast to the total cell preparations, only high affinity binding was observed in intact purified small luteal cells (Kd = 0.96 +/- 0.04 nM). Partial purification of luteal cytosolic PKC by DEAE-Sephadex chromatography resulted in co-elution of PKC enzyme activity and the [3H]PBt2 binding activity. Under conditions of saturating calcium (0.1 mM) and phosphatidylserine (PS) (100 micrograms/tube) concentrations, binding to the partially purified PKC preparation was found to be of a single high affinity and exhibited a Kd (1.3 +/- 0.2 nM) similar to the high affinity binding observed in intact cells. These results suggest that the primary phorbol ester receptor in luteal cells is PKC. However, a low affinity, high capacity [3H]PBt2 binding site also exists within the corpus luteum, either in the large cells or in the accessory cell fraction which consists mainly of endothelial cells.

Second messenger systems and progesterone secretion in the small cells of the bovine corpus luteum: effects of gonadotropins and prostaglandin F2a.

The present studies were conducted to determine the effects of gonadotropins (LH and hCG) and prostaglandin F2a (PGF2a) on the production of "second messengers" and progesterone synthesis in purified preparations of bovine small luteal cells. Corpora lutea were removed from heifers during the luteal phase of the normal estrous cycle. Small luteal cells were isolated by unit-gravity sedimentation and were 95-99% pure. LH provoked rapid and sustained increases in the levels of [3H]inositol mono-, bis-, and trisphosphates (IP, IP2, IP3, respectively), cAMP and progesterone in small luteal cells. LiCl (10 mM) enhanced inositol phosphate accumulation in response to LH but had no effect on LH-stimulated cAMP or progesterone accumulation. Time course studies revealed that LH-induced increases in IP3 and cAMP occurred simultaneously and preceded the increases in progesterone secretion. Similar dose-response relationships were observed for inositol phosphate and cAMP accumulation with maximal increases observed with 1-10 micrograms/ml of LH. Progesterone accumulation was maximal at 1-10 ng/ml of LH. LH (1 microgram/ml) and hCG (20 IU/ml) provoked similar increases in inositol phosphate, cAMP and progesterone accumulation in small luteal cells. 8-Bromo-cAMP (2.5 mM) and forskolin (1 microM) increased progesterone synthesis but did not increase inositol phosphate accumulation in 30 min incubations. PGF2a (1 microM) was more effective than LH (1 microgram/ml) at stimulating increases in inositol phosphate accumulation (4.4-fold vs 2.2-fold increase for PGF2a and LH, respectively). The combined effects of LH and PGF2a on accumulation of inositol phosphates were slightly greater than the effects of PGF2a alone. In 30 min incubations, PGF2a had no effect on cAMP accumulation and provoked small increases in progesterone secretion. Additionally, PGF2a treatment had no significant effect on LH-induced cAMP or progesterone accumulation in 30 min incubations of small luteal cells. These findings provide the first evidence that gonadotropins stimulate the cAMP and IP3-diacylglycerol transmembrane signalling systems in bovine small luteal cells. PGF2a stimulated phospholipase C activity in small cells but did not reduce LH-stimulated cAMP or progesterone accumulation. These results also demonstrate that induction of functional luteolysis in vitro requires more than the activation of the phospholipase C-IP3/calcium and -diacylglycerol/protein kinase C transmembrane signalling system.

Differential effects of luteinizing hormone on intracellular free Ca2+ in small and large bovine luteal cells.

The effect of LH on the intracellular free Ca2+ concentration ([Ca2+]i) was investigated in highly purified small and large bovine luteal cell populations. Luteal cells were obtained from midcycle corpora lutea dispersed with collagenase and separated by flow cytometry into large and small cells. Resting levels of Ca2+ were higher (P less than 0.05) in the large than small cells [314 +/- 25 nM (n = 5) vs. 186 +/- 13 nM (mean +/- SE; n = 13) for large and small cells, respectively]. LH rapidly increased [Ca2+]i in both small and large cells loaded with the fluorescent Ca2+ probe fura-2. In the small cells, [Ca2+]i was immediately increased 2- to 6-fold (from 176 +/- 8 to 468 +/- 8 nM; n = 5) after adding LH. The LH induced [Ca2+]i rise occurred in two phases: an initial peak due to intracellular Ca2+ mobilization and a secondary rise due to Ca2+ influx from extracellular sources. Preincubation of the small cells with EGTA reduced the initial phase and abolished the secondary rise in [Ca2+]. Both forskolin and 8-bromo-cAMP increased [Ca2+]i in the small cells. In contrast, only a single phase of [Ca2+]i rise was observed in LH-treated large cells, and the response was 1.5- to 2-fold greater than the resting Ca2+ levels [314 +/- 25 vs. 435 +/- 60 nM (n = 4), for resting vs. LH-treated values, respectively]. The addition of both LH and prostaglandin F2 alpha (PGF2 alpha) to the large cells resulted in increases in [Ca2+]i that were greater than those induced by each hormone separately (2.0-fold for LH and 2.7-fold for PGF2 alpha vs. 7- to 9-fold in the presence of both hormones). These findings demonstrate that LH induces rapid increases in intracellular [Ca2+]i that differ in magnitude and profile between the small and large bovine luteal cells. Furthermore, LH and PGF2 alpha interacted to promote increases in [Ca2+]i in the large cells, that were higher than the sum of [Ca2+]i induced by each hormone separately.

The effects of platelet-activating factor and platelet-derived compounds on bovine luteal cell progesterone production.

This study was conducted to characterize bovine platelets with respect to serotonin (5-HT) concentration and platelet-activating factor (PAF)-activation and to examine the in vitro effects of PAF and platelet-derived compounds on bovine luteal progesterone (P4) production. The concentration of 5-HT in platelets, as determined by high-performance liquid chromatography, was 538.8 +/- 40.83 ng/1 x 10(8) platelets. Based on a circulating platelet concentration range of 2.3 x 10(8) 5.8 x 10(8) platelets/ml, the circulating concentration of 5-HT would be approximately 1239-3125 ng/ml of blood. Bovine platelets were found to aggregate in response to PAF (1-40 ng/0.5 ml), with maximal aggregation occurring at 20-40 ng/0.5 ml. Coincubation of luteal cells with platelets (1 x 10(7)-4 x 10(8] enhanced luteal P4 production (p less than 0.05). Addition of the 5-HT receptor antagonist mianserin blocked the platelet-induced increases in P4 (p less than 0.05). Preincubation of platelets with indomethacin did not alter the production of P4 (p greater than 0.05), nor did the addition of propranolol (p greater than 0.05). Platelet-derived growth factor at 8 and 16 ng/ml enhanced basal P4 production (p less than 0.05) but had no effect on the responsiveness of luteal cells to luteinizing hormone (LH) (p greater than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

A comparison of the effects of cyclooxygenase prostanoids on progesterone production by small and large bovine luteal cells.

Highly purified preparations of small and large bovine luteal cells were utilized to examine the effects of prostaglandins F2 alpha (PGF2 alpha), E2 (PGE2) and I2 (PGI2) analog on progesterone production. Corpora lutea were obtained from Holstein heifers between days 10 and 12 of the estrous cycle. Purified small and large cells were obtained by unit gravity sedimentation and flow cytometry. Progesterone accumulation was determined in 1 x 10(5) small and 5 x 10(3) large cells after 2 and 4 h incubations respectively. Progesterone synthesis was increased (p less than 0.05) in the small cells by the increasing levels of PGF2 alpha, PGE2, carba-PGI2 and LH. PGF2 alpha, but not PGE2 or carba-PGI2 increased (p less than 0.05) LH-stimulated progesterone production. There was no interaction of various combinations of prostaglandins on progesterone production in the small cells. In the large cells, PGF2 alpha had no effect on basal progesterone production. However, it inhibited LH-stimulated progesterone synthesis. In contrast, PGE2 and carba-PGI2 stimulated (p less than 0.05) basal progesterone production in the large cells. In the presence of LH, high levels of carba-PGI2 inhibited (p less than 0.05) progesterone synthesis. The PGE2 and PGI2-stimulated progesterone production in the large luteal cells was also inhibited in the presence of PGF2 alpha. These data suggest all of the prostaglandins used exert a luteotropic action in the small cells. In the large cells only PGE2 and carba-PGI2 are luteotropic, while PGF2 alpha exerts a luteolytic action. The effects of the prostaglandins in the small and large luteal cells suggest that their receptors are present in both cell types.

Acute effects of prostaglandin F2 alpha on inositol phospholipid hydrolysis in the large and small cells of the bovine corpus luteum.

The present studies were conducted to determine whether the large or small bovine luteal cell was the site for the stimulatory effect of prostaglandin F2 alpha (PGF) on phospholipase C-catalyzed inositol phospholipid hydrolysis. Corpora lutea were removed from heifers during the luteal phase of the normal estrous cycle. Small luteal cells were isolated by unit-gravity sedimentation and large luteal cells were isolated by flow cytometry using a Becton Dickson FACS 440 cell sorter. PGF provoked rapid (5-30 s) and sustained (up to 30 min) increases in the levels of inositol mono-, bis-, and trisphosphates (IP, IP2, IP3, respectively) in small luteal cells. IP3 was formed more rapidly than IP2 or IP following PGF treatment. The PGF-stimulated increase in IP3 was accompanied by a transient reduction in the levels of 3H-labeled phosphatidylinositol 4,5-bisphosphate. LiCl (10 mM) enhanced inositol phosphate accumulation in response to PGF. Maximal increases in inositol phosphate accumulation were observed with 1-10 microM PGF and half-maximal increases were observed with 60 nM PGF. PGF (1-10 microM) had no effect on cAMP levels but stimulated small increases in progesterone accumulation in 30 min incubations of small luteal cells. PGF also increased the accumulation of inositol phosphates in large luteal cells. The increases were apparent within 5 min of incubation (the earliest time examined) and further increases were observed in incubations lasting 30 min. PGF had no significant effect on cAMP or progesterone in 30 min incubations of large cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Control of progesterone production in small and large bovine luteal cells separated by flow cytometry.

Corpora lutea were collected from Holstein heifers on Days 10 and 12 of the oestrous cycle and the cells were dispersed with collagenase. The dispersed cells were separated into preparations of highly purified (90-99%) small (less than 20 microns) and large (greater than 25 microns) luteal cells by unit gravity sedimentation and fluorescence-activated cell sorting. Net progesterone accumulation by 1 x 10(5) small cells and 1 x 10(3) large cells during 2 and 4 h incubations, respectively, were measured after additions of LH, PGF-2 alpha, and phorbol esters, alone and in combination. Progesterone synthesis was increased (P less than 0.05) by phorbol dibutyrate (PBt2) or PGF-2 alpha (P less than 0.05) in small, but not in large, luteal cells (10.1 +/- 3.0 and 18.1 +/- 5.0 ng/10(5) cells for 0 and 50 nM-PBt2, and 19.9 +/- 3.2 and 44.2 +/- 9.3 ng/10(5) cells for 0 and 1 microgram PGF-2 alpha/ml). The previously reported stimulatory effects of PKC activation and PGF-2 alpha addition to total dispersed cell preparations are therefore entirely attributable to the small, theca-derived cells. Small cells responded to low levels of LH (9.1 +/- 1.1, 69.0 +/- 5.4 and 154.7 +/- 41.4 ng/10(5) cells for 0, 1 and 5 ng LH/ml, respectively, P less than 0.05), while large cells responded only to high levels of LH (1635 +/- 318, 2662 +/- 459 and 3386 +/- 335 pg/10(3) cells for 0, 100 and 1000 ng LH/ml, respectively, P less than 0.05). PGF-2 alpha inhibited LH-, 8-Br-cAMP- and forskolin-stimulated progesterone synthesis in the large cells (3052 +/- 380, 3498 +/- 418, 3202 +/- 391 pg/10(3) cells for 1 microgram LH/ml, and 0.5 mM-8-Br-cAMP, and 1 microM-forskolin respectively and 1750 +/- 487, 2255 +/- 468, 2165 +/- 442 pg/10(3) cells for PGF-2 alpha + LH, PGF-2 alpha + 8-Br-cAMP and PGF-2 alpha + forskolin, respectively), indicating that the inhibitory effect of PGF-2 alpha on progesterone synthesis in large cells occurs at a site distal to cAMP generation. These results suggest that the large cells are the targets of the luteolytic effects of PGF-2 alpha, while the small cells are responsible for the previously reported luteotrophic effect of PGF-2 alpha in vitro.

Effects of prolactin on steroidogenesis by human luteal cells in culture.

We have examined the effects of prolactin (PRL) on progesterone (P) and estradiol (E2) synthesis by cells obtained from human corpora lutea of early and midluteal phases. The cells were cultured in the presence or absence of low (200 mIU/l) or high levels (1000 mIU/l) of PRL. Basal P, but not E2 was significantly (P less than 0.05) enhanced by the low level of PRL, whereas the high level of PRL increased P only in midcycle cells during the initial 48 hours of culture. The high PRL dose significantly (P less than 0.05) reduced E2 production in both early and midcycle cells during the initial 48 hours of culture. Both levels of PRL significantly (P less than 0.05) inhibited human chorionic gonadotropin (hCG)-stimulated P and E2 production in early luteal cells. In the midcycle corpora lutea cells, the low dose of PRL enhanced hCG-stimulated P but not E2, whereas the high dose of PRL was inhibitory to both P and E2. The high dose of PRL also inhibited prostaglandin E2 but not 8-bromoadenosine 3':5'-cyclic monophosphate-stimulated P and E2 synthesis. These results demonstrate that PRL has a direct effect on luteal cell steroidogenesis.

Control of steroidogenesis in small and large bovine luteal cells.

Evidence was cited to show that: (1) prostacyclin (PGI2) plays a luteotrophic role in the bovine corpus luteum and that products of the lipoxygenase pathway of arachidonic acid metabolism, especially 5-hydroxyeicosatetraenoic acid play luteolytic roles; (2) oxytocin of luteal cell origin plays a role in development, and possibly in regression, of the bovine corpus luteum; and (3) luteal cells arise from two sources; the characteristic small luteal cells at all stages of the oestrous cycle and pregnancy are of theca cell origin; the large cells are of granulosa cell origin early in the cycle, but a population of theca-derived large cells appears later in the cycle. Results of in vitro studies with total dispersed cells and essentially pure preparations of large and small luteal cells indicate that: (1) the recently described Ca2+-polyphosphoinositol-protein kinase C second messenger system is involved in progesterone synthesis in the bovine corpus luteum; (2) activation of protein kinase C is stimulatory to progesterone synthesis in the small luteal cells; (3) activation of protein kinase C has no effect on progesterone synthesis in the large luteal cells; and (4) protein kinase C exerts its luteotrophic effect in total cell preparations, in part at least, by stimulating the production of prostacyclin. The protein kinase C system may cause down regulation of LH receptors in the large cells.