Pituitary autoantibodies in patients with hypopituitarism and their relatives

In order to investigate the physiological role of calcitonin gene-related peptide (CGRP) in mollusc, both circulating CGRP-related molecules and gill or mantle carbonic anhydrase activity were analysed during the annual growth of Pecten maximus. CGRP like molecules measured by radioreceptor assay increased significantly during the annual cycle. Similarly, gill carbonic anhydrase activity increased and showed a maximum activity when growth is stimulated to the greatest extent. Correlation studies showed a significant relationship between the tissue weight and either the gill carbonic anhydrase activity or the CGRP-related molecules determined by radioreceptorassay. This observation suggests a possible interaction between carbonic anhydrase activity and CGRP. Accordingly, we searched for a direct effect of CGRP on the gill carbonic anhydrase activity. In gill membranes, CGRP stimulated the carbonic anhydrase activity. The maximum effect was obtained at a CGRP concentration of 50 nM.     

A radioreceptor assay for catecholamines. I. A study with 3H-norepinephrine (author's transl)

A radioreceptor assay for catecholamines(CA) has been established utilizing the microsomal fraction of bovine myocardium as CA receptors and 3H-norepinephrine(3H-NA) as a ligand. The bound fraction was separated from the free fraction by filtration through milipore filters. The following results were obtained: 1) The optimal amount of the microsomal fraction was 250 micrograms/tube, when 3H-NA of 12,000 c.p.m was used. The appropriate conditions for incubation were 2 hours at 37 degrees C in a pH 7.4 sucrose solution. 2) By this method, norepinephrine was detectable in the range from 5 to 100ng/tube. 3) Various compounds with catechol nucleus showed a cross-reaction in this radioreceptor assay system. 4) Alpha-adrenergic blocking agents inhibited the binding of 3H-NA, whereas beta-blocking agents were ineffective. These results suggest that the microsomal fraction of bovine myocardium contains alpha-adrenergic receptors which bind the catechol nucleus of CA.     

Alteration of calcitonin gene related peptide and its receptor binding sites during the development of tolerance to mu and delta opioids

Calcitonin gene related peptide (CGRP), one of the most abundant peptides in the spinal cord, is localized in primary afferents and released following nociceptive stimuli. Its colocalization and corelease with substance P, a well-known nociceptive neuropeptide, support the importance of CGRP in pain mechanisms. However, its distinctive function in that regard remains to be fully established. Recently, we reported that increases in CGRP-like immunostaining and decrements in specific 125I-labelled human CGRP alpha ([125I]hCGRP alpha) binding sites in the spinal cord were correlated with the development of tolerance to the spinal antinociceptive action of a mu opioid agonist, morphine. The goal of the present study was to investigate whether the development of tolerance to other classes of opioids, namely, delta and kappa agonists, can also alter CGRP-like immunostaining and receptors in the rat spinal cord. The antinociceptive effects of all opioids were monitored by the tail-immersion test. Tolerance to their antinociceptive properties was induced by the infusion for 7 days of mu (morphine sulfate, 7.5 micrograms/h), delta D([D-Pen2,D-Pen5]enkephalin (DPDPE), 2.0 micrograms/h), and kappa (U-50488H, 10.0 micrograms/h) related agonists at the spinal level (L4), using osmotic minipumps. We confirmed that rats chronically treated with morphine showed significant decreases in [125I]CGRP alpha binding in laminae I, II, and III of the L4 spinal cord, while CGRP-like immunostaining was increased in these same laminae. Similar effects were observed following a treatment with the delta agonist, DPDPE, while the kappa agonist, U-50488H, apparently only slightly decreased [125I]CGRP alpha] binding in lamina II. Binding in other laminae and CGRP-like immunostaining were not affected. These results suggest a specific interaction between spinal CGRP systems and the development of tolerance to the spinal antinociceptive effects of mu- and delta-related agonists.     

Bioassay of thyrotropin receptor antibodies with Chinese hamster ovary cells transfected with recombinant human thyrotropin receptor: clinical utility in children and adolescents with Graves disease

OBJECTIVE: The objective of this study was to compare the clinical utility of a new bioassay for thyrotropin (TSH) receptor antibodies (Abs) with the conventional radioreceptor assay and with measurement of thyroid peroxidase Abs in the diagnosis of Graves disease in childhood. STUDY DESIGN: Serum samples obtained from 22 children and adolescents with Graves disease (19 hyperthyroid, 3 in remission), 13 children and adolescents with chronic lymphocytic thyroiditis, and 17 normal children in a control group were evaluated. RESULTS: TSH receptor Abs were detected by bioassay in 10 (91%) of 11 patients with active Graves disease but in 0 of 2 patients in remission, 0 of 13 normal members of the control group, and 0 of 11 patients with chronic lymphocytic thyroiditis including 1 with thyrotoxicosis. The sensitivity and specificity of TSH receptor Abs detected by radioreceptor assay studied in the same 11 patients and in an additional 11 patients was similar to bioassay. In contrast, thyroid peroxidase Abs were detected in only 12 (71%) of 17 patients with Graves disease but in 11 of 11 patients with chronic lymphocytic thyroiditis and in 0 of 17 members of the control group. CONCLUSION: Bioassay of TSH receptor Abs is both sensitive and specific for the diagnosis of active Graves disease in the young. When cost and simplicity are considered, however, bioassay offers no advantage over radioreceptor assay for initial diagnostic screening. Rather, bioassay for TSH receptor Abs may be useful in thyrotoxic patients who are negative initially in the radioreceptor assay or in treated patients whose clinical picture is discordant with results in the radioreceptor assay.     

Desensitization of CGRP and adrenomedullin receptors in SK-N-MC cells: implications for the RAMP hypothesis

Recent evidence (1) suggests that the related peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) bind to the same heptahelical transmembrane receptor, with receptor specificity being determined by a receptor associated modifying protein (RAMP). If correct, this hypothesis would predict that each peptide should desensitize the cellular response to subsequent stimulation by itself or the other peptide. We have therefore studied the patterns of desensitization of these receptors in SK-N-MC cells. SK-N-MC cells were stimulated for 20 minutes in either serum free medium alone (control) or SFM containing AM 10(-8) M or CGRP 10(-7) M. Cells were then incubated for a further 20 minutes in SFM containing a second agonist and 1 mM isobutyryl methylxanthine (IBMX), before harvesting and assay for cAMP. Pre-exposure of cells to CGRP or AM decreased cAMP generation in response to subsequent stimulation with CGRP by 58% (+/-14) and 42% (+/-14) (SD) respectively. Pre-incubation of cells with 100 nM H-89 abolished this effect, indicating that desensitization was mediated through PKA. In contrast, there was no attenuation of the cAMP response to stimulation with AM by pre-exposure to AM or CGRP. These results suggest that CGRP and AM receptors exhibit different patterns of desensitization in SK-N-MC cells: a finding with significant implications for the RAMP hypothesis.     

Tissue distribution of liver regulating protein. Evidence for a cell recognition signal common to liver, pancreas, gonads, and hemopoietic tissues

Liver regulating protein (LRP) is an integral plasma membrane protein that plays a critical role in maintaining the differentiated phenotype of adult rat hepatocytes by mediating cell-cell interactions with rat liver epithelial cells. Using a specific monoclonal antibody (MAb L8) capable of inhibiting the interactions between these two cell types, the cellular distribution of LRP was analyzed in the liver. Various cell types, including hepatocytes and several sinusoidal cells, were found to be positive, whereas vascular endothelial cells and bile duct cells were consistently negative. This observation led us to question whether cells of nonhepatic origin would also express LRP. We show that MAb L8 immunoreactive material was detected in only three groups of tissues and corresponded to molecules similar to LRP but with different molecular weights. LRP-like molecules were demonstrated on acinar cells of the exocrine pancreas and on all hemopoietic cells regardless of their localization in the organism. LRP-like molecules were also expressed by germ cells and surrounding feeder cells in the testis and ovary in a stage-dependent manner. These results demonstrate the existence of a family of LRP proteins and strongly suggest a critical role for these molecules in regulating cell-cell communication in specific tissues.     

A new diabetes model induced by neonatal alloxan treatment in rats

Anti-leishmanial activity of chloroform and methanol extracts of Vernonia amygdalina, a plant widely used in Ethiopia for the treatment of parasitic infections, has been assessed in vitro on Leishmania aethiopica. Amastigotes were more sensitive to V. amygdalina than promastigotes. The chloroform extract had a stronger parasiticidal activity, with median effective doses (ED50) of 18.5 micrograms/ml and 13.3 micrograms/ml for promastigotes and amastigotes, than the methanol extract with ED50 of 74.4 micrograms/ml and 45.8 micrograms/ml respectively. Cytotoxicity caused by V. amygdalina to host cells, the human leukaemia monocyte THP-1 cell line, as determined by the methyl tetrazolium assay, resulted in a median lethal dose (LD50) of 19.6 micrograms/ml for the chloroform extract and 243.4 micrograms/ml for the methanol extract. In comparison, the ED50 and LD50 of pentamidine, a standard anti-leishmanial drug, were 0.5 micrograms/ml and 1.4 micrograms/ml respectively. These results indicate that V. amygdalina displays potent anti-leishmanial activities and warrants further investigation.     

Radioiodinated estrogen derivatives

Monoiodohexestrol exhibits 10 to 15% specific binding to the 8S estrogen receptor while the remainder binds to nonreceptor 4S proteins. Reduction of nonreceptor binding with either thyroxine or 8-anilino-1-naphthalene sulfonic acid was not quantitative. Thus no accurate determination of the concentration of receptor sites in the radioreceptor assay was possible by graphical analysis. Two additional estrogens--17 alpha [125I]iodoethynylestradiol and 17 alpha-[125I]iodoethynyl-11 beta-methoxy estradiol--were synthesized at high specific activity. Although the iodoethynyl derivatives were stable under synthetic conditions, deiodination in the presence of proteins is too fast to allow either in vivo or in vitro use. To make these compounds clinically useful, therefore, chemical modification to reduce nonreceptor binding and the rate of dehalogenation must be undertaken.     

Functional coupling of endogenous serotonin (5-HT1B) and calcitonin (C1a) receptors in CHO cells to a cyclic AMP-responsive luciferase reporter gene

A cyclic AMP-responsive reporter cell line has been established through the stable expression of a luciferase reporter plasmid in Chinese hamster ovary (CHO) cells. Reporter cells showed a dose-dependent expression of luciferase in response to incubation with forskolin. These CHO cells were screened for endogenous G protein-coupled receptors capable of stimulating or inhibiting adenylyl cyclase, by monitoring changes in luciferase expression. Serotonin (5-HT) receptor agonist ligands caused an inhibition of forskolin-stimulated luciferase expression in the rank order 5-carboxamidotryptamine > 5-HT > sumatriptan > 8-hydroxy-2-(di-n-propylamino)tetralin. The response to 5-HT was reversed by the 5-HT1 receptor antagonists cyanopindolol and pindolol, but not the 5-HT2 receptor antagonist ketanserin. Calcitonin was more potent than calcitonin gene-related peptide (CGRP) at stimulating luciferase expression in this cell line, and these responses were insensitive to the CGRP receptor antagonist, CGRP (8-37). These results were consistent with the presence of 5-HT(1B-like) and calcitonin (C1a-like) receptors in CHO cells, with the responses to 5-HT and CGRP being pertussis and cholera toxin-sensitive, respectively. This reporter gene assay gave the expected pharmacological profile for these receptors when compared with cyclic AMP accumulation assays, confirming its value as a functional assay for G protein-coupled receptors linked to adenylyl cyclase.     

Rapid nitric oxide- and prostaglandin-dependent release of calcitonin gene-related peptide (CGRP) triggered by endotoxin in rat mesenteric arterial bed

1. Our objective was to determine whether endotoxin (ETX) could directly trigger the release of calcitonin gene-related peptide (CGRP) from perivascular sensory nerves in the isolated mesenteric arterial bed (MAB) of the rat and to determine whether nitric oxide (NO) and prostaglandins (PGs) are involved. 2. ETX caused time- and concentration-dependent release of CGRP, and as much as a 17 fold increase in CGRP levels in the perfusate at 10-15 min after the administration of ETX (50 micrograms ml-1). 3. CGRP-like immunoreactivity in the perfusate was shown to co-elute with synthetic rat CGRP by reverse-phase h.p.l.c. 4. Pretreatment of MAB with capsaicin or ruthenium red inhibited ETX-induced CGRP release by 90% and 71%, respectively. ETX-evoked CGRP release was decreased by 84% during Ca2(+)-free perfusion. 5. The release of CGRP evoked by ETX was enhanced by L-arginine by 43% and inhibited by N omega-nitro-L-arginine (L-NOARG) and methylene blue by 37% and 38%, respectively. L-Arginine reversed the effect of L-NOARG. 6. Indomethacin and ibuprofen also inhibited the ETX-induced CGRP release by 34% and 44%, respectively. No additive inhibition could be found when L-NOARG and indomethacin were concomitantly incubated. 7. The data suggest that ETX triggers the release of CGRP from capsaicin-sensitive sensory nerves innervating blood vessels. The ETX-induced CGRP release is dependent on extracellular Ca2+ influx and involves a ruthenium red-sensitive mechanism. Both NO and PGs appear to be involved in the ETX-induced release of CGRP in the rat mesenteric arterial bed.     

Complete recovery by nerve growth factor of neuropeptide content and function in capsaicin-impaired sensory neurons

In the present study the ability of nerve growth factor (NGF) to facilitate the recovery of peptidergic primary sensory C-fibers after an acute capsaicin treatment (50 mg/kg s.c.) was investigated in adult rats. NGF (4 micrograms 1/day for 3 days) was injected into the plantar of one hind paw starting 24 h after the capsaicin treatment. Without NGF, there was a significant reduction of calcitonin gene-related peptide (CGRP) and substance P content of the paw skin and the sciatic nerve. CGRP and substance P levels were completely replenished in the NGF-treated paw skin and in the innervating sciatic nerve they even increased over control levels as determined 40 h after the last injection of NGF. CGRP levels also recovered in the contralateral paw and sciatic nerve, but no recovery was observed in other tissues such as the front paw, the auricle, or the urinary bladder. Mustard oil-induced neurogenic plasma extravasation, taken as a functional parameter for peptidergic primary sensory C-fibers, was significantly decreased after the capsaicin treatment and showed a complete recovery by NGF in the injected paw as well as in the contralateral paw skin. These results show that NGF not only was able to reverse the decrease of transmitter content caused by capsaicin but also restored the peripheral function of primary afferent neurons.     

Characterization of placentation-specific binucleate cell glycoproteins possessing a novel carbohydrate. Evidence for a new family of pregnancy-associated molecules

The ovine binucleate cell-specific glycoproteins recognized by the monoclonal antibody SBU-3 first appear at the initiation of placentation, and their expression continues throughout gestation. These placenta-specific proteins have not been detected in any other adult or fetal sheep tissues and are specific to the materno-fetal interface. The SBU-3 monoclonal antibody recognizes the carbohydrate epitope common to a group of proteins ranging in molecular mass from 30 to 200 kDa whose function during pregnancy remains undefined. The biochemical properties of these uniquely expressed glycoproteins were investigated by analyzing both the carbohydrate and protein portion of the molecules. Analysis of phytohemagglutinin and concanavalin A binding to electrophoretically separated SBU-3 proteins revealed that the major proteins between 40 and 70 kDa bind phytohemagglutinin. In contrast, concanavalin A bound only to minor proteins in the SBU-3 glycoprotein preparation. Analysis of the carbohydrate conjugated to the SBU-3 glycoproteins revealed that the major chains are sialylated O-linked and complex partially sialylated multiple antennary N-linked chains. The presence of N-glycolylneuraminic acid in an N-linked structure indicates the unique nature of this carbohydrate epitope. The differential binding to phytohemagglutinin and concanavalin A provided a method for further purification and characterization of the major protein components with monoclonal antibody immunoaffinity-purified SBU-3 proteins being further separated by concanavalin A-Sepharose chromatography. Microsequence analysis of the major non-concanavalin A-binding proteins (69, 62, and 57 kDa) revealed partial homology to ovine and bovine pregnancy-associated glycoprotein and rabbit pepsinogen F. Immunoblot analysis of the SBU-3 proteins showed cross-reactivity with polyclonal antisera directed against ovine placental-associated glycoprotein and pregnancy-specific glycoprotein B. These results suggest that together these glycoproteins represent members of a binucleate cell-derived family of pregnancy-associated molecules in the ruminant placenta.     

Cardiac expression of genes encoding putative adrenomedullin/calcitonin gene-related peptide receptors

Recent studies have suggested that adrenomedullin (AM) may play a role in the pathophysiology of heart disease, though the specific cardiac receptors involved have not been defined. RT-PCR cloned fragments of three putative AM/calcitonin gene-related peptide (CGRP) receptors were used to established a quantitative RNase protection assay to identify and quantitate expression of receptor mRNAs in heart and in cardiac myocytes. Intact rat heart expressed mRNA encoding the putative AM/CGRP receptors RDC1 and CRLR at 37- and 15-fold higher levels, respectively, than the AM-selective receptor L1, with a qualitatively similar profile in cultured neonatal cardiac myocytes. The high level of expression of RDC1 and CRLR suggests that both AM and CGRP may have direct actions on the cardiac myocyte via common receptors that can interact with either ligand.     

Psychopathological disorders and coronary diseases

BACKGROUND: Endotoxin is an inflammatory made by gram negative bacteria that can irritate the skin, induce respiratory problems, fever, and shock. It is an adjuvant for both delayed hypersensitivity and IgE production and has been shown to magnify antigen specific mediator release. Since many of the clinical problems associated with natural latex products involve similar clinical sequelae, we investigated the possibility that latex gloves might be contaminated with endotoxin. OBJECTIVE: To measure the endotoxin content of a variety of natural latex gloves, investigate the its distribution and origin, associated with latex proteins, and determine the particle sizes associated with its release. METHODS: Endotoxin, protein, and allergen were measured using a quantitative kinetic Limulus assay, modified Lowry, and RAST inhibition, respectively. Particle size and density were determined using an Anderson multistage air sampler and CsCl2 gradient. RESULTS: Endotoxin was found to be a highly significant contaminant of some latex gloves. Levels ranged from 0.09 ng to 2.8 micrograms/g of glove. Protein levels ranged from < 25 to 1150 micrograms/g of glove while allergen levels ranged from < 1 to 837 micrograms/g of glove. Endotoxin and protein eluted rapidly from the interior of the gloves tested. Greater than 70% of the endotoxin was found to be associated with particles in the < 7 microns aerodynamic diameter range. The highest levels of endotoxin were found in nonsterile examination gloves with a tendency towards powdered gloves containing more endotoxin and protein. A slurry containing cross-linked dextran through which gloves were dipped revealed very high endotoxin contamination (64 micrograms/mL) while unused cross-linked dextran has very little associated endotoxin. CONCLUSIONS: These data demonstrate that some natural rubber latex gloves, particularly nonsterile examination gloves, are contaminated with high amounts of endotoxin and proteins. These were found mostly on the inside of gloves and were released as very small respirable particles that were not physically associated with the powder. These findings support the hypothesis that endotoxin may be responsible for some of the tissue irritation associated with latex glove use. In addition, this material may be responsible for the enhancement of delayed and immediate hypersensitivity reactions to chemicals and proteins found in these products and offers a possible explanation for the disproportionate severity of these reactions.     

Importance of the intracellular domain of NR2 subunits for NMDA receptor function in vivo

The definitive function of pancreatic polypeptide in mammalian physiology remains unknown. The identification of specific PP target tissues should be helpful to further investigations into the possible regulatory actions of this peptide. An in vivo radioreceptor assay was used in the rat to locate potential binding sites of I(125) bovine PP. In vitro, high concentrations of unlabeled hormone competitively inhibit binding to receptors by low concentrations of labeled hormone. In vivo studies showed that, in the presence of concentrated unlabeled pancreatic polypeptide, labeled PP distributes between the plasma and interstitial fluid. When excess unlabeled PP is replaced with saline in the companion animals, the labeled peptide appears to distribute in a volume that exceeds the combined plasma volume and interstitial fluid volume of the tissue. Using this in vivo receptor assay, the distribution volume that exceeds the anatomic extracellular volume has been identified as the receptor compartment. With this assay we demonstrated in the rat specific and displaceable PP binding to the ductus choledochus, duodenum, ileum, and adrenal gland. The NVV determined in the adrenal gland of experimental animals was 3.9 times greater than that found in the control group. Binding was rapid and was displaced only by excess unlabeled pancreatic polypeptide. Neither excess insulin nor excess neuropeptide Y significantly reduced this binding.     

Determinants of tPA antigen and associations with coronary artery disease and acute cerebrovascular disease

A serum-free organ culture model for chondrocyte maturation, using the Avian sternum, was developed. Day-14 chick embryo sterna were placed in organ culture in the presence of defined medium. The optimal medium for chondrocyte terminal differentiation contained specific concentrations of dexamethasone, insulin, thyroid hormone and ascorbic acid. Three parameters, including sternal growth, cell diameter and type X collagen production, were analyzed as indicators of chondrocyte terminal differentiation. These parameters were analyzed in cephalic, middle and caudal regions of the organ-cultured chick sterna and compared to sterna grown in ovo. This study demonstrates that the organ-cultured tissue maintains normal morphological characteristics and terminal differentiation in the cephalic region only, similar to in ovo development, while maintaining normal cell-matrix relationships.     

5-FU concentrations in the blood and tumor tissue after 5'-DFUR or UFT administration in the patients with uterine cervical cancer

In order to verify the antitumor activity of fluorinated pyrimidine drugs, we conducted an investigation of the clinical pharmacology with two drugs, 5'-DFUR and UFT. Total 21 cases of cervical cancer were alloted randomly into 5'-DFUR group (daily dose 800 mg for 3 days) consisting of 11 patients and UFT group (daily dose 600 mg for 3 days) consisting of 10 patients, the unchanged substances (5'-DFUR in the 5'-DFUR group and tegafur concentrations in the UFT group) and 5-FU concentrations in serum and tissues were measured 6 hours after administration of the drugs. The 5'-DFUR concentration in the 5'-DFUR group was not detected in serum and less than a detectable limit for all of cancerous tissues, normal cervical tissues, and lymph nodes. The tegafur concentrations in the UFT treated group was 13.8 +/- 7.0 micrograms/ml for serum, 10.4 +/- 6.4 micrograms/g for cancerous tissues, 14.1 +/- 7.3 micrograms/g for normal cervical tissues, and 4.5 +/- 4.2 micrograms/g for lymph nodes. The 5-FU concentrations in the 5'-DFUR treated group were 0.018 +/- 0.046 micrograms/g for cancerous tissues, but less than a detectable limit for serum and normal cervical tissues. On the other hand, in the UFT group, 0.271 +/- 0.247 micrograms/g for a cancerous tissue, 0.035 +/- 0.018 micrograms/ml for serum, 0.125 +/- 0.073 micrograms/g for normal cervical tissues, showing significantly high values (p < 0.01, p < 0.001, and p < 0.01, respectively) compared to those in the 5'-DFUR treated group. These results suggest that UFT is a promising drug for the treatment of cancer of the uterine cervix.     

The involvement of nitric oxide (NO) in the CGRP-induced behavior of rats

The possible role of nitric oxide (NO) in CGRP-induced passive avoidance, active avoidance, and open field behavior was tested in rats. A specific NO synthase inhibitor, N omega-nitro-L-arginine (L-NA), was used to disrupt NO synthesis. ICV administration of 5 micrograms of L-NA reversed the action of CGRP in passive and active avoidance tests. In an open field, L-NA prevented the action of CGRP on locomotion and grooming. The inactive isomer D-NA had no effect on behavior of animals. The data suggest that NO might contribute to CGRP-induced behavior in rats.     

An aminopeptidase inhibitor, bestatin, enhances progesterone and oestradiol secretion by porcine granulosa cells stimulated with follicle stimulating hormone in vitro

We examined the presence of cell surface aminopeptidase on cultured porcine granulosa cells by employing the aminopeptidase assay using alanine-p-nitroanilide and histochemical staining using L-leucyl-beta-naphthylamide. Porcine granulosa cells obtained from follicles 4-5 mm in diameter were cultured for 7 days. The aminopeptidase assay showed that the porcine granulosa cell culture had aminopeptidase activity and that this activity was inhibited in a dose-dependent manner by bestatin which binds to cell surfaces and inhibits cell surface aminopeptidases. Histochemical staining also indicated that cultured granulosa cells had aminopeptidase activity. Porcine granulosa cells were cultured in the presence or absence of porcine follicle stimulating hormone (FSH, 3.125 nmol/l) and/or bestatin (0.4, 4.0 and 40.0 micrograms/ml) for 7 days, and the production of progesterone and oestradiol was measured. In the presence of porcine FSH, the production of progesterone and oestradiol by granulosa cells was increased significantly by approximately 5- and 2-fold respectively. These increases were enhanced further by bestatin (40.0 micrograms/ml). In the absence of porcine FSH, progesterone production was enhanced by bestatin (40.0 micrograms/ml), whereas no significant effect of bestatin on oestradiol secretion was observed. These findings indicate that the inhibition of membrane-bound aminopeptidase(s) on the cell surfaces affects the steroidogenesis of granulosa cells, and that these aminopeptidase(s) are important regulators of granulosa cell differentiation.