Safety and Effectiveness of Conversion From Cyclosporine to Once-Daily Prolonged-Release Tacrolimus in Stable Kidney Transplant Patients: A Multicenter Observational Study in Japan.

This study investigated the safety and effectiveness of conversion from cyclosporine- to prolonged-release tacrolimus (PR-T)-based immunosuppression in kidney transplant recipients (KTRs) in Japanese routine clinical practice. MATERIALS AND METHODS: This was a prospective observational study of stable KTRs who were converted from cyclosporine to PR-T according to local clinical practice. Clinical data were collected up to 12 months postconversion. Study outcomes included conversion dosing ratios, PR-T dose and trough levels, change in estimated glomerular filtration rate between conversion and month 12, graft/patient survival, and rejection rate (Kaplan-Meier). Outcomes of ongoing preconversion hypertrichosis, gingival hypertrophy, and cyclosporine-related renal toxicity were detailed. Data for adverse drug reactions were collected. RESULTS: Overall, 266 patients (mean ± SD age 51.9 ± 13.5 years) were included. The mean ± SD conversion ratio (PR-T:cyclosporine, mg:mg) was 0.029 ± 0.017. After an initial decrease between conversion and month 3, mean ± SD PR-T daily dose remained stable up to month 12 (2.4 ± 1.5 mg at months 3 and 12), as did tacrolimus trough blood levels (3.5 ± 1.8 vs 3.6 ± 1.7 ng/mL, respectively). Estimated glomerular filtration rate was stable over 12 months (mean ± SD change from conversion to month 12 was 0.3 ± 7.8 mL/min/1.73m2). Month 12 Kaplan-Meier patient and graft survival rates were 99.6% and 95.5%, respectively. Eight patients reported 9 rejection episodes. PR-T demonstrated potential to improve cyclosporine-related renal toxicity, hypertrichosis, and gingival hypertrophy. Postconversion, 46 adverse drug reactions were reported in 39 patients (14.7%); there was 1 death. CONCLUSIONS: Conversion from cyclosporine to PR-T in Japanese stable KTRs was effective and tolerable over 12 months, with low rates of rejection reported.

Safety and efficacy of once-daily modified-release tacrolimus in kidney transplant recipients: interim analysis of multicenter postmarketing surveillance in Japan.

INTRODUCTION: Modified-release formulation of tacrolimus (TAC-MR) has been developed with the intent of improving patient adherence and quality of life. A number of studies have indicated that the efficacy and safety of once-daily TAC-MR were comparable with those of the original formulation, twice-daily TAC. However, its dosage, trough level, safety, and efficacy in the multicenter clinical experience of Japanese kidney transplant recipients have not been reported. METHODS: This post-marketing surveillance designed as an open-label, prospective, noncomparative, noninterventional observational study was performed. The 256 patients were enrolled for de novo transplantation, and the 106 patients were enrolled for conversion to TAC-MR from 52 medical institutions in Japan. The follow-up period in de novo transplantation was 5 years, but here we report the results of the 24-week interim analysis. The observation period in conversion was 24 weeks. RESULTS: Regarding de novo transplantation, the median daily TAC-MR dose was 0.150 mg/kg/d at the initial administration and the median TAC trough level was 12.1 ng/mL at 3 days. The common adverse drug reactions were infections, renal disorders, and glucose tolerance disorders at incidence rates of 23.6%, 6.8%, and 5.6%, respectively. Both patient and graft survival rates at 24 weeks were 98.2% and the rejection rate was 16.1%. Regarding conversion to TAC-MR, the median conventional TAC dose before conversion was 3.2 mg/d, and the median TAC-MR dose at the converted day was 3.2 mg/d. The median TAC trough level was 5.4 ng/mL before conversion, and it was 5.2 ng/mL after conversion. The most common adverse drug reactions were infections at an incidence rate of 4.9%. There was 1 graft loss and death, and there was 1 episode of rejection. CONCLUSION: This interim analysis shows that a TAC-MR-based immunosuppressive regimen is safe and effective as used in Japanese clinical practice.

Novel anti-idiotype antibody therapy for lipooligosaccharide-induced experimental autoimmune neuritis: use relevant to Guillain-Barré syndrome.

Campylobacteriosis is a frequent antecedent event in Guillain-Barré syndrome (GBS), inducing high-titer serum antibodies for ganglioside antigens in the peripheral nervous system (PNS). Molecular mimicry between the lipooligosaccharide (LOS) component of Campylobacter jejuni and human peripheral nerve gangliosides is believed to play an important role in the pathogenesis of GBS. Conventional treatment strategies for patients with GBS include plasmapheresis, intravenous immunoglobulin (IVIG), and immunosuppression, which are invasive or relatively ineffective. In this study, we used our animal model of GBS, in which Lewis rats were immunized with GD3-like LOS isolated from C.jejuni. The animals developed anti-GD3 ganglioside antibodies and manifested neuromuscular dysfunction. To develop novel therapeutic strategies, we treated the animals by intraperitoneal administration of an anti-GD3 antiidiotype monoclonal antibody (BEC2) that specifically interacts with the pathogenic antibody. The treated animals had a remarkable reduction of anti-GD3 antibody titers and improvement of motor nerve functions. The results suggest that ganglioside mimics, such as antiidiotype antibodies, may be powerful reagents for therapeutic intervention in GBS by neutralizing specific pathogenic antiganglioside antibodies.

Inhibition of adenylylcyclase activity in mouse cerebellum membranes upon hydrolysis of triacylglycerols by triacylglycerol lipase, but not phospholipids by phospholipase A(2).

We previously showed that arachidonic acid and related unsaturated free fatty acids (U-FFAs) inhibit the activity of adenylylcyclase in brain membranes of mice. The level of U-FFAs elevates when the hydrolysis of triacylglycerols (TAGs) and phospholipids is promoted. In this study, we examined whether activation of triacylglycerol lipase (TAG lipase) and phospholipase A(2) (PLA(2)) results in the inhibition of adenylylcyclase activity in cerebellum membranes of mice. Incubation of Intralipos with TAG lipase in the presence of membranes mainly released oleic acid and linoleic acid and caused > or =95% inhibition of adenylylcyclase activity. In contrast, PLA(2), though releasing substantial amounts of U-FFAs, increased the enzymatic activity. To account for this difference, we examined how by-products formed in U-FFA release by TAG lipase and PLA(2) operated on the arachidonic acid-induced inhibition. Lysophosphatidylcholne and some other lysophospholipids, produced by PLA(2), enhanced the adenylylcyclase activity and attenuated the inhibitory effect of arachidonic acid. On the other hand, no such effects were found with by-products of TAG lipase-mediated lipolysis. Rather, monoacylglycerols having U-FFAs, possibly formed by TAG lipase, potentiated the arachidonic acid-induced inhibition of adenylylcyclase. Bovine serum albumin, added into the mixture for the pretreatment of membranes with TAG lipase, prevented the inhibition of adenylylcyclase. These results indicate that by-products formed in U-FFA release have a crucial role for the U-FFA's action on adenylylcyclase and that U-FFAs released from TAG are an inhibitor of adenylylcyclase. It may be that albumin in plasma, and thus FFA-binding proteins within cells, are of importance in protecting adenylylcyclase upon U-FFA release.

GM2 ganglioside regulates the function of ciliary neurotrophic factor receptor in murine immortalized motor neuron-like cells (NSC-34).

We previously reported that ciliary neurotrophic factor (CNTF) increased the serum-free cell survival of immortalized motor neuron-like cells (NSC-34), and addition of the exogenous ganglioside GalNAc beta4(Neu5Ac alpha3)Gal beta4GlcCer (GM2) facilitated cell survival together with CNTF. Moreover beta 1,4 N-acetylgalactosaminyltransferase (GM2 synthase) activity increased in NSC-34 cells cultured with CNTF. We now have examined whether CNTF-induced cell survival is associated with the collaboration between GM2 and the CNTF receptor (CNTF-R). Despite the presence of CNTF (50 ng/ml), anti-CNTF-R antibody caused cell death and prevented the up-regulation of GM2 synthase expression. The addition of GM2 (1 to 20 microM) abrogated the anti-CNTF-R antibody effect which shortened cell survival and blocked GM2 synthase activation. Use of [125I]CNTF showed the specificity of CNTF binding in NSC-34 cells in situ. GM2 produced a 5-fold increase in the CNTF binding affinity per cell but did not change the binding site number. The study by metabolic labeling with [1-(14)C]N-acetyl-D-galactosamine ([14C]GalNAc) showed that biosynthesized GM2 was involved in the immunoprecipitation of CNTF-R. These findings indicate that up-regulated GM2 synthesis induces functional conversion of CNTF-R to the activated state, in which it has affinity for CNTF. We conclude that GM2 is a bio-regulating molecule of CNTF-R in motor neurons.

Effect of pituitary adenylate cyclase-activating polypeptide (PACAP) on progestin biosynthesis in cultured luteal cells from rat ovary.

We examined the effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on progestin biosynthesis in cultured luteal cells from rat ovary. Luteal cells from immature rats treated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) were cultured in the absence or presence of ovine luteinizing hormone (LH) (100 ng/ml) and PACAP-38 (10, 100 and 1000 ng/ml). Following 48 hours of incubation, the levels of progesterone, 20 alpha-hydroxy-4-pregnene-3-one (20 alpha-OH-P) and adenosine 3',5'-monophosphate (cAMP) were measured in the culture media. PACAP alone significantly stimulated the production of progesterone and 20 alpha-OH-P in a dose-dependent manner (p < 0.01 and 0.05, respectively, ANOVA). LH-induced production of progesterone and accumulation of cAMP were significantly decreased by increasing concentrations of PACAP (p < 0.05 for each, ANOVA). Conversely, LH-stimulated 20 alpha-OH-P production was enhanced by PACAP in a dose-dependent manner (p < 0.05). Since PACAP decreased the ratio of progesterone to 20 alpha-OH-P production in LH-stimulated cells, PACAP-mediated inhibition of the stimulatory action of LH on progesterone production may be involved in the initiation of luteolysis. PACAP-38 also suppressed increases in LH receptor content in cultured luteal cells. These results suggest that PACAP regulates the effects of LH on luteal cell function and that PACAP might be closely linked to reproduction.

Metastasis suppressor gene KiSS-1 encodes peptide ligand of a G-protein-coupled receptor.

Metastasis is a major cause of death in cancer patients and involves a multistep process including detachment of cancer cells from a primary cancer, invasion of surrounding tissue, spread through circulation, re-invasion and proliferation in distant organs. KiSS-1 is a human metastasis suppressor gene, that suppresses metastases of human melanomas and breast carcinomas without affecting tumorigenicity. However, its gene product and functional mechanisms have not been elucidated. Here we show that KiSS-1 (refs 1, 4) encodes a carboxy-terminally amidated peptide with 54 amino-acid residues, which we have isolated from human placenta as the endogenous ligand of an orphan G-protein-coupled receptor (hOT7T175) and have named 'metastin'. Metastin inhibits chemotaxis and invasion of hOT7T175-transfected CHO cells in vitro and attenuates pulmonary metastasis of hOT7T175-transfected B16-BL6 melanomas in vivo. The results suggest possible mechanisms of action for KiSS-1 and a potential new therapeutic approach.

Inhibition of adenylyl cyclase activity in brain membrane fractions by arachidonic acid and related unsaturated fatty acids.

Pretreatment of mouse brain membranes with arachidonic acid (AA) and related unsaturated fatty acids at 30 degrees C for 10 min decreased basal activity and isoproterenol/guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)- and forskolin-stimulated activities of adenylyl cyclase to a level less than 5% of control. The presence of the carboxyl group on the fatty acids was essential for the inhibition, because no such inhibition was found with ethyl arachidonate or AA attached to diacylglycerols and phospholipids. The AA-mediated inhibition was observed when the activity was measured in the presence of Mn2+ or forskolin and was insensitive to pertussis toxin or guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS), indicating a mechanism independent of GTP-binding proteins. In addition, the fact that stimulators of the adenylyl cyclase catalytic unit, ATP, GTP gamma S and forskolin, when present during pretreatment, attenuate the inhibitory effect of AA may suggest that the catalytic unit is a target of AA. Bovine serum albumin suppressed the inhibition when present in the mixtures for pretreatment, but could not restore the adenylyl cyclase activity that had been reduced by AA, indicating an irreversible inhibition by AA. The effect of AA was found to be additive to P-site-mediated inhibition. The present study suggests the existence of another mechanism of regulation of adenylyl cyclase by unsaturated fatty acids.

Plasma endothelin and LH-RH, LH, FSH, prolactin, progesterone, 17alpha-hydroxyprogesterone, estrone, 17beta-estradiol, delta4-androstenedione, testosterone, active renin, angiotensin-II and ANP levels in blood and LH, estrone and 17beta-estradiol and pregnanediol levels in urine of normal cycling women.

In order to investigate the endothelin (ET) levels and their relationship to various hormones during the menstrual cycle, we measured endothelin-1, -2 and -3 (ET-1, -2 and -3), big ET-1 and big ET-3 levels in 27 normally cycling women (mean age 27 years). Simultaneous determination of luteinizing hormone-releasing hormone (LH-RH), luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin, progesterone, 17alpha-hydroxyprogesterone (17-OHP), estrone, 17beta-estradiol, delta4-androstenedione, testosterone, active renin, angiotensin-II (A-II) and atrial natriuretic peptide (ANP) in blood, and LH, estrone and 17beta-estradiol and pregnanediol levels in urine was made in the same 27 women. The levels of ET-2 in plasma were found to be 20% of those of ET-1. In addition, ET-1 levels were measured in the endometrium of the normal uterus. Plasma ET-1 and ET-3 levels fluctuated during the menstrual cycle, with a peak at the luteal phase, but showed only a negative relationship (p < 0.01) to each other at the menstrual phase, whereas big ET-1 and big ET-3 levels showed no significant changes. Plasma ET-1 and ET-3 levels showed no significant relationship to the big ETs. As for the relationship to other hormones, plasma ET-3 had a negative relationship (p < 0.01) to prolactin and a positive correlation (p < 0.01) with ANP during the entire menstrual cycle. Plasma ET-1 and ET-3 showed a partial positive or negative correlation to LH, FSH, prolactin and ANP levels, depending upon the cycle phase, whereas plasma ETs and big ETs were unrelated to other hormones in the blood, and LH, estrone, 17beta-estradiol and pregnanediol in the urine throughout the menstrual cycle. At each menstrual stage, plasma ET-3 levels were more significantly related to LH, FSH, prolactin and ANP than ET-1, indicating a closer relationship between ET-3 and these circulating hormones during the menstrual cycle. The ET-1 level showed no significant change in the endometrium during the menstrual cycle.

Serum testosterone responses to continuous and intermittent exercise training in male rats.

Serum testosterone (T) were investigated at rest and following exercise during 6 weeks of continuous and intermittent swimming training in male rats, and the regulatory mechanisms of the changes were discussed by evaluating serum luteinizing hormone (LH), and conducting GnRH (gonadotropin releasing hormone, 1.5 microg/kg body weight) or hCG (human chorionic gonadotropin, 25 IU/kg body weight) challenge tests. Relative to the resting level, serum T increased after intermittent exercise (6.47 +/- 1.58 vs 3.08 +/- 2.85 nmol/l), which was followed with the same changes in LH (12.81 +/- 4.21 vs 5.70 +/- 1.56 nmol/l). Serum T was lower after continuous exercise compared to the resting level (2.02 +/- 0.53 vs 10.96 +/- 3.11 nmol/l), while LH level was higher than that in sedentary group (11.23 +/- 5.61 vs 5.00 +/- 1.61 nmol/l). No significant changes were observed in resting T during and after intermittent training. A lower resting T level was shown at the end of 3 weeks of continuous training as compared to the sedentary group (1.88 +/- 0.69 vs 12.36 +/- 2.10 nmol/l), but it increased after 6 weeks of training. Serum T increased significantly in the intermittent training group after hCG treatment as compared to the saline treatment (52.42 +/- 12.10 vs 6.81 +/- 6.22 nmol/l), but insignificantly in the continuous training group. The similar increases in serum LH were observed in all the groups after GnRH treatment.

GM2 promotes ciliary neurotrophic factor-dependent rescue of immortalized motor neuron-like cell (NSC-34).

We have examined whether ciliary neurotrophic factor (CNTF) can alter serum-free cell survival of immortalized motor neuron-like cells, which were established by fusing mouse neuroblastoma N18TG2 with mouse motor neurons. One of the cell lines, NSC-34 exhibited cell survival in the presence of CNTF. NSC-34 preserves the most characteristics of motor neurons, such as the formation of neuromuscular junctions on co-cultured myotube. GM2 ganglioside is characteristic of motor neurons, and expressed highly in NSC-34. When NSC-34 was cultured with exogenous GM2 ganglioside and CNTF, GM2 facilitated the cell survival effect of CNTF. In the addition, beta 1,4 N-acetylgalactosaminyltransferase (GM2 synthase) activity was enhanced up to 3.9-fold by culture in the presence of CNTF. GM2 might be a functional modulator of CNTF in motor neurons. It might be presented to cell surface by its enzyme activation, and become a signal of early stage, when CNTF rescues motor neurons.

Biological roles of angiotensin II via its type 2 receptor during rat follicle atresia.

Type 1 angiotensin II (ANG II) receptors play crucial roles in the regulation of blood pressure and fluid osmolarity, whereas the physiological roles of type 2 ANG II receptors (AT2) remain unclear. Because AT2 is expressed in atretic follicles where granulosa cells undergo apoptosis, we examined the space and time relationship between AT2 expression and follicle atresia in vivo and the effect of AT2 on follicle-stimulating hormone (FSH) actions in vitro. Binding studies, autoradiography, and RT-PCR of AT2 revealed that the AT2 content in granulosa cells was time dependently increased at both protein and mRNA levels in equine chorionic gonadotropin-treated immature female rats. This increase paralleled the progression of atresia. ANG II suppressed FSH-caused prevention of DNA fragmentation, increases in luteinizing hormone receptor content, and estrogen production through AT2 in cultured granulosa cells. Moreover, FSH-induced stimulation of extracellular signal-regulated kinase activity, critical for cell survival, was inhibited by AT2 stimulation. These results suggest that AT2 mediates the progression of follicle atresia through granulosa cell apoptosis by inhibiting FSH actions.

Serum insulin-like growth factor-I, insulin-like growth factor binding protein-3, sex steroids, osteocalcin and bone mineral density in male and female rats.

Although it has been reported that the rate of weight gain and linear growth increases markedly during puberty in rats, little is known about the relationship between endocrine changes and bone mineral density (BMD) changes upon sexual maturation in these animals. The aim of this study was to examine the levels of serum insulin-like growth factor-I (IGF-I), IGF binding protein (IGFBP)-3, sex steroids and osteocalcin, and the changes in BMD in normal aging male and female rats. Male rats exhibited increases in serum IGF-I and IGFBP-3 concentrations before increases in serum testosterone levels. IGF-I and testosterone peaked at 9 weeks of age, and thereafter remained in a steady state, whereas IGFBP-3 reached a peak at 7 weeks of age, and then gradually declined. A strong correlation between serum IGF-I and IGFBP-3 levels was found in subjects 3-9 weeks old. A highly significant correlation between serum IGF-I and testosterone levels was also found. In females, serum 17 beta-estradiol, IGF-I and IGFBP-3 levels increased gradually from 3 to 5 weeks old, peaked at 9 weeks, and then decreased slowly thereafter. The correlation coefficient between serum IGF-I and IGFBP-3 was highly significant. The correlation coefficient between serum IGF-I and 17 beta-estradiol levels was weak, although it was strongest when the subjects were 3-9 weeks old. Serum osteocalcin is a marker of bone formation; its level remained relatively high from 3 to 9 and from 3 to 7 weeks of age in males and females, respectively, although osteocalcin in both sexes declined gradually with age. As for bone mass, sharp increases in BMD in the tibia, femur and lumbar vertebrae appeared earlier in female than in male rats, and the BMD in females tended to be higher than in males between 5 and 9 weeks old. After 9 weeks of age, BMD in males was higher than that in females, as BMD in males continued to increase whereas females tended to remain in a steady state after this stage. The correlation coefficients between tibial BMD and serum IGF-I or IGFBP-3 levels were highly significant when the subjects were from 3 to 9 weeks old. Taken together, these results suggest that BMD development occurs earlier in female than in male rats. This sex-related difference in changes in the BMD pattern may result from the earlier onset of puberty in females, and from sex-specific differences in concentrations of IGF-I, IGFBP-3 and sex steroids during maturation.

Effect of growth hormone releasing hormone on luteinizing hormone stimulated progestin biosynthesis in cultured rat ovarian granulosa cells.

In the present study, we examined the effects of growth hormone releasing hormone (GHRH) on luteinizing hormone (LH)-stimulated progestin biosynthesis. Granulosa cells from pregnant mare serum gonadotropin (PMSG)-treated immature rats were cultured in vitro in the absence or presence of ovine. National Institute of Health LH (100 ng/ml) with various doses of GHRH (10(-9), 10(-8) and 10(-7) mol/l) for 24 h. At the end of the incubation period, the incubation media were collected and levels of progesterone, 20 alpha-hydroxypregn-4-en-3-one and cyclic adenosine 3',5'-monophosphate (cAMP) were measured. GHRH significantly stimulated progesterone production and cAMP accumulation in media in a dose-dependent manner in the basal state (p < 0.01 and p < 0.001, respectively). In the presence of LH, GHRH significantly inhibited LH-stimulated progesterone production in a dose-dependent manner (p < 0.01), whereas increasing concentrations of GHRH produced progressive increases in cAMP accumulation (p < 0.05). Since increasing concentrations of GHRH produced progressive decreases in the ratio of progesterone to 20 alpha-hydroxypregn-4-en-3-one production in the LH-stimulated state (p < 0.01), GHRH may be involved in modulation of key steroidogenic steps concerned with progesterone degradation rather than formation in LH-stimulated rat granulosa cells. These results suggest that GHRH may regulate the effects of LH in granulosa cells and play an important role in the reproductive process.

Comparisons of serum testosterone and corticosterone between exercise training during normoxia and hypobaric hypoxia in rats.

The purpose of this study was to compare the effects of continuous and intermittent exercise training on serum testosterone [T] and corticosterone concentrations [Cort] during normoxia and hypobaric hypoxia. Male rats swam with loads of 3% (normoxia) or 2.25% (462 mmHg) body mass for 60 min in the continuous training groups, and 15 min separated by a 7-min rest x 4, with 60-min total exercise duration in the intermittent training groups, 5 days week(-1) for 6 weeks. Serum [T] were measured at rest and following exercise after 6 weeks of training. Serum [Cort] were measured immediately after an acute period of exercise or after 6 weeks of training at rest and following exercise. Continuous exercise induced decreases in [T] under both conditions. Intermittent exercise showed a tendency to increase [T] during normoxia, but caused a suppression during hypobaric hypoxia. The [Cort] was elevated by a similar margin after an acute period of exercise during both conditions. After 6 weeks of training, however, [Cort] increased slightly after exercise during normoxia. A lower resting [Cort], which was increased after exercise, was found in the training groups during hypoxia. No relevant relationship was found between the behaviours of [T] and [Cort] after exercise during either conditions.

A synthetic ceramide analog (L-PDMP) up-regulates neuronal function.

To address the role of brain gangliosides in synaptic activity, the ceramide analogs, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) and its enantiomer, L-PDMP, were used to inhibit and stimulate ganglioside biosynthesis in cultured cortical neurons. Prolonged treatment with both PDMP isomers exhibited opposite effects on functional synapse formation measured by spontaneous synchronized oscillatory activity of intracellular Ca2+ between the neurons: suppression by D-PDMP and facilitation by L-PDMP. Up-regulation of synaptic activity by L-PDMP could be correlated with the slow but robust activation of p42 mitogen-activated protein kinase. Treatment with L-PDMP after transient forebrain ischemia in rats ameliorated the deficit of a well-learned spatial memory by an 8-arm maze task, suggesting a new potential therapeutic approach for neurodegenerative disorders.

Effects of follicle-stimulating hormone on endothelin receptors in cultured rat ovarian granulosa cells.

Two subtypes of the endothelin (ET) receptor (ETA and ETB) were studied in cultured ovarian granulosa cells. Immature 21-day-old female Wistar-Imamichi rats were implanted with diethylstilbestrol (DES) pellets for 5 days and granulosa cells were collected by repeated puncturing. Viable cells (2.5 or 5 x 10(5)) were cultured with 50--400 ng/ml of ovine NIH follicle-stimulating hormone (FSH) in the presence or absence of [125I-Tyr13]ET-1 (50 pM) in 1 ml McCoy's 5a medium for 72 h. FSH gradually increased the [125I-Tyr13]ET-1 binding to granulosa cells, whereas FSH-untreated granulosa cells had no significant changes. The dose of 200 ng/ml of FSH was most effective for [125I-Tyr13]ET-1 binding for 48-h culture, thereafter revealing a plateau. After 48 h of culture with 200 ng/ml of FSH, granulosa cells were further incubated with [125I-Tyr13]ET-1 (10 pM-1 nM) and/or [125I]IRL1620, the selective ETB receptor agonist (10 pM-1 nM) for 2 h for equilibrium study, and then the dissociation constant and the maximal binding capacity between receptors and ligands were determined by saturation curve and Scatchard plot analysis. ETA + ETB, ETB, and ETA (sites/cells) showed a 4.4-, 2.6-, and 7.5-fold increase, respectively. As for steroidogenesis, ET-1 (100 nM) or ET-3 (100 nM) suppressed FSH-induced progesterone and 17 beta-estradiol production. These results indicate that FSH upregulates both ETA and ETB receptors in DES-treated immature rat granulosa cells, with no significant differences between ET-1 and ET-3, and that ET-1 or ET-3 suppresses FSH-induced steroidogenesis. ETs may affect the granulosa cell function through the ETA and ETB receptors, and the increase in amount of ET binding does not reflect ET effects on granulosa cell function. The ET receptor plays an important role in the development of the ovary.

Changes with age and sex in levels of plasma and ovarian or testicular endothelin-1 in rats.

Mammalian ovaries and testes contain various intragonadal peptides necessary to control gonadal function. Effects of aging and sex on total plasma and ovarian or testicular endothelin-1 (ET-1) levels were studied in female and male rats aged 4-140 days. At 2 weeks, plasma ET-1 levels were 2.69 +/- 0.09 and 2.41 +/- 0.03 pg/ml (mean +/- SEM; n = 10) in the female and male, respectively. After a temporary decrease, mean ET-1 concentrations increased at 10 weeks and again decreased at 20 weeks in the female, thereafter revealing a plateau, whereas the ET-1 concentrations gradually decreased in the male. In adult females, plasma ET-1 levels were 3.7-fold higher than male levels. Ovarian ET-1 levels gradually increased after birth to 2 weeks, then decreased, and again increased at 3 weeks. Thereafter they gradually decreased, showing a plateau after 10 weeks. In contrast, testicular ET-1 levels gradually decreased after birth. This difference is parallel to but much larger than the sex difference in ovarian or testicular ET-1. Gonadal ET-1 showed a very different ontogeny.

Effect of pituitary adenylate cyclase-activating polypeptide (PACAP) on progestin biosynthesis in cultured granulosa cells from rat ovary and expression of mRNA encoding PACAP type IA receptor.

The purpose of this study was to detect the presence of mRNA encoding pituitary adenylate cyclase-activating polypeptide (PACAP) type I receptor in granulosa cells from rat ovary and to examine the effect of PACAP on progestin biosynthesis. mRNA was isolated from granulosa cells from the ovaries of immature rats treated with pregnant mares' serum gonadotrophin. The technique of reverse transcription and polymerase chain reaction with primers specific to PACAP type I receptor were used to demonstrate the expression of mRNA encoding PACAP type IA receptor in these cells. Granulosa cells were also cultured in the absence or presence of 100 ng LH ml-1 with various doses of PACAP-38 (10, 100 and 1000 ng ml-1). At the end of the incubation period, the incubation media were collected and concentrations of progesterone, 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P) and cAMP were measured. Increasing concentrations of PACAP-38 significantly stimulated the production of progestins (progesterone and 20 alpha-OH-P) and cAMP accumulation in a dose-dependent manner (P < 0.01; ANOVA). This effect was observed in media cultured for 24 and 48 h in both basal and LH-stimulated states. PACAP-38 did not significantly affect the ratio of progesterone: 20 alpha-OH-P produced by granulosa cells cultured for 24 h in the LH-stimulated state. However, at 1000 ng ml-1, PACAP-38 significantly decreased the ratio of progesterone to 20 alpha-OH-P production in granulosa cells cultured for 48 h (P < 0.01). These results suggest that granulosa cells from rat ovary express mRNA encoding PACAP type IA receptor and that PACAP may regulate granulosa cell differentiation and play an important role in the reproductive process.

Up-regulation of ganglioside biosynthesis, functional synapse formation, and memory retention by a synthetic ceramide analog (L-PDMP).

To address the role of brain gangliosides in synaptic activity, the ceramide analogs, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) and its enantiomer, L-PDMP, were used to inhibit and stimulate ganglioside biosynthesis in cultured cortical neurons. Prolonged treatment with both PDMP isomers exhibited opposite effects on functional synapse formation measured by spontaneous synchronized oscillatory activity of intracellular Ca2+ between the neurons: suppression by D-PDMP and facilitation by L-PDMP. Up-regulation of synaptic activity by L-PDMP could be correlated with the slow but robust stimulation of ganglioside biosynthesis through activating GM3, GD3 and GQ1b synthases. In a similar time course, the activity of p42 mitogen-activated protein kinase was also enhanced by L-PDMP. To evaluate the efficacy of this drug in long-term memory, rats were trained for 2 weeks using an 8-arm radial maze task, and then forebrain ischemia was induced by 4-vessel occlusion. Treatment with L-PDMP starting 24 hours after the transient ischemia ameliorated the deficit of a well-learned spatial memory, demonstrating the potential therapeutic intervention of the ceramide analog for neurodegenerative disorders.