Modulation of guanosine triphosphatase activity of G proteins by arachidonic acid in rat Leydig cell membranes.

Previous results from our group have indicated that arachidonic acid decrease cAMP production through a modification of heterotrimeric G proteins. In the present study, we have characterized the high affinity GTPase activity present in Leydig cell membranes and its regulation by fatty acids. The high-affinity GTPase activity, measured as [gamma32P] GTP hydrolysis rate, was both time and protein concentration dependent. Arachidonic acid elicited a dose-dependent inhibition of enzyme activity with an IC50 = 26.7+/-1.1 microM. The existence of only two double bonds in linoleic acid is reflected by a decrease in its inhibitory activity (IC50 = 34+/-2.3 microM). Saturated fatty acids showed no effect at this level. The kinetic analysis as interpreted by Lineweaver-Burk plots, indicated that 50 microM arachidonic acid had no effect on the apparent affinity for GTP, but resulted in a 40% decreases in the maximal velocity of the reaction. Arachidonic acid modulation of GTPase activity was not attenuated by blocking eicosanoid metabolism with inhibitors of 5'-lipoxygenase, cyclooxygenase, or epoxygenase P-450. The addition of arachidonic acid to pertussis toxin-treated membranes had no effect on the enzyme activity, indicating that arachidonic acid does not modify the GTPase activity present in Galphas protein. However, ADP-ribosylation with cholera toxin followed by arachidonic acid treatment led to a further 40% inhibition when compared with cholera toxin treatment alone. These results allowed us to postulate that arachidonic acid inhibits the GTPase activity of Gi protein family. To further analyze the mechanism of arachidonic acid inhibition of GTPase activity, the effect of arachidonic acid on the [35S]GTPgammaS binding was studied. No effect of this fatty acid on GTP binding was found. Combining our previous results with those found here, we can conclude that arachidonic acid maintains Gi proteins in their active state, which in turn inhibit adenylate cyclase and results in decrease cAMP levels.

Specific effect of arachidonic acid on 17beta-hydroxysteroid dehydrogenase in rat Leydig cells.

It is well known that arachidonic acid (AA) acts as an intratesticular factor regulating luteinizing hormone-mediated testicular steroidogenesis. The present studies were conducted to determine the effect of AA on steroidogenic enzymes in rat Leydig cells. Exogenously added AA significantly inhibited 22(R)-hydroxy-cholesterol-stimulated testosterone production, which is a clear indication that AA is acting at some point after cholesterol transport to the inner mitochondrial membrane. AA failed to block the conversion of 22(R)-hydroxycholesterol to pregnenolone, indicating that the cytochrome P-450 side-chain cleavage enzyme complex is not the site of inhibition. The present results demonstrate that only 17beta-hydroxysteroid dehydrogenase seems to be involved in the AA action, since nearly 60% inhibition of testosterone production was found when the cells were incubated with androstenedione. Furthermore, no effect of AA was found when androstenediol was used as substrate in the testosterone synthesis, which indicates that 3beta-hydroxysteroid dehydrogenase is not affected by AA. The conversion of AA to its metabolites is not required for its action on 17beta-hydroxysteroid dehydrogenase and the activation of protein kinase C is not involved in the inhibitory effect.

Different sites of action of arachidonic acid on steroidogenesis in rat Leydig cells.

The present study in purified rat Leydig cells shows that arachidonic acid may act as an intratesticular factor regulating LH-mediated testicular steroidogenesis. Arachidonic acid decreased, in a dose-dependent manner, the LH-stimulated cAMP and testosterone levels, over 2 h incubation. Incubation of Leydig cells with arachidonic acid did not modify 125I-hCG binding to the cells as compared to control, showing that the action of arachidonic acid is not related to a decrease of hCG binding to the cells. Forskolin-stimulated cAMP and testosterone production were inhibited by 51.65 and 70.9%, respectively, in the presence of arachidonic acid (100 microM), although the ED50 for the diterpene was not changed. When isobutyl-methyl-xanthine was added to the incubation medium, the same percentage of inhibition was found indicating that arachidonic acid inhibition of cAMP production is not due to stimulation of Leydig cell phosphodiesterase activity. Pretreatment of the cells with pertussis toxin, to inactivate Gi, was also without effect on arachidonic acid inhibition of LH-stimulated cAMP production, but pertussis toxin abolished the inhibitory effects of arachidonic acid when adenylate cyclase was stimulated with forskolin. However, arachidonic acid addition resulted in inhibition of LH- and forskolin-stimulated testosterone production, even if the cells were pretreated with pertussis toxin. It can be concluded that: (1) The inhibitory effect of arachidonic acid is neither due to a decrease of hCG binding to Leydig cells nor to a stimulation of cell phosphodiesterase activity; (2) arachidonic acid modulates cAMP production at two different levels, either by activation of Gi protein and by inhibition of Gs protein or adenylate cyclase; (3) the effect of arachidonic acid on steroidogenesis is also beyond cAMP formation.

Control of steroidogenesis in Leydig cells.

Luteinizing hormone (LH) interacts with its plasma membrane receptor to stimulate steroidogenesis not only via cyclic AMP but also other pathways which include arachidonic acid and leukotrienes and regulation of chloride and calcium channels. The same stimulatory pathways may lead to desensitization and down-regulation of the LH receptor and steroidogenesis. The LH receptor exists in a dynamic state, being truncated, or internalized, degraded or recycled. Desensitization is controlled by protein kinase C (PKC) in the rat and by cyclic AMP dependent protein kinase and PKC in the mouse Leydig cells. Using an adapted anti-sense oligonucleotide strategy we have shown that the cytoplasmic C-terminal sequence of the LH receptor is essential for desensitization to occur. In contrast, these sequences of the LH receptor are not required for the stimulation of cyclic AMP and steroid production. We have also shown that the extracellular domain of the LH receptor is secreted from the Leydig cell and may act as a LH-binding protein.

Somatostatin inhibits VIP- and forskolin-stimulated cyclic AMP accumulation in enterocytes from rat jejunum.

This study demonstrates the dual regulation by somatostatin of vasoactive intestinal peptide (VIP)-stimulated and forskolin-stimulated cyclic AMP accumulation by isolated rat intestinal epithelial cells. Somatostatin non-competitively inhibited (IC50 = 1 microM) the stimulatory effect of VIP on cyclic AMP accumulation, suggesting that the two neuropeptides act through separate receptors. The cyclic AMP accumulation produced by forskolin (a diterpene that stimulates directly the catalytic subunit of adenylate cyclase) was also inhibited by somatostatin in a dose-dependent manner. However, somatostatin did not modify the stimulatory effect of VIP on adenylate cyclase activity in a membrane preparation from the same cells, making it difficult to explain the mechanism of somatostatin action at this level. The data presented here suggest that somatostatin may play a physiological role in the regulation of nutrient absorption and the release of gut hormones or exocrine secretions by intestinal epithelial cells through the modulation of cyclic AMP production.

Direct effect of arachidonic acid on protein kinase C and LH-stimulated steroidogenesis in rat Leydig cells; evidence for tonic inhibitory control of steroidogenesis by protein kinase C.

The role of arachidonic acid in the regulation of steroidogenesis in rat Leydig cells was studied. A dose- and time-dependent biphasic effect on maximal and submaximal LH- and dibutyryl-cAMP-stimulated testosterone production was found. The locus of the inhibition, which occurred during 3 h incubation, was prior to the side chain cleavage of cholesterol and after cAMP production. The same inhibitory effect was found with the protein kinase C (PKC) activators, phorbol-12-myristate, 13-acetate (PMA) and oleic acid, also with no change in LH-stimulated cAMP production. Arachidonic acid, PMA, and diolein, all stimulated PKC activity in a dose-dependent fashion in partially purified Leydig cell homogenates. When the cells were incubated for 5 h, arachidonic acid potentiated LH- and dibutyryl-cAMP-stimulated testosterone production. Similarly, incubation with PMA for 5 h, potentiated subsequent basal and dibutyryl-cAMP-stimulated testosterone production. PKC was down-regulated over 5 h (but not during 3 h) by pretreating Leydig cells with PMA or arachidonic acid in the presence of LH. Lipoxygenase and cyclooxygenase inhibitors did not alter the stimulatory effects of arachidonic acid. We conclude that the short-term inhibitory effect of arachidonic acid (and PMA) is via activation of PKC, but when protein kinase C (PKC) is down-regulated by these ligands, steroidogenesis is enhanced. These results suggest that steroidogenesis is normally under tonic inhibitory control by PKC.

Differences in LH receptor down-regulation between rat and mouse Leydig cells: effects of 3',5'-cyclic AMP and phorbol esters.

The role of cyclic AMP and phorbol esters in luteinizing hormone (LH) receptor down-regulation in Leydig cells has been studied. Dibutyryl cyclic AMP (db-cAMP) (0.01, 0.1 and 1 mM), forskolin (80 microM) and cholera toxin (1.19 nM) caused a 30-50% loss of [125I]hCG binding sites and an inhibition of receptor-[125I]hCG complex internalization in mouse tumour Leydig (MA10, MLTC-1) cells during 2 h. In contrast, db-cAMP had no effect on the level of binding sites or internalization of the hormone receptor complex in rat testis Leydig cells or a rat tumour (R2C) Leydig cell. Phorbol 12-myristate 13-acetate (PMA) at concentrations from 10(-9) to 10(-5) M had no effect on hormone binding or hormone-receptor complex internalization in any of the Leydig cells. In contrast a 2 h preincubation of MLTC-1 cells with 10(-7) M PMA caused a loss of subsequent LH-stimulated cyclic AMP and pregnenolone production. These results indicate that LH receptor down-regulation is mediated by cyclic AMP dependent kinase, but not protein kinase C, in mouse Leydig cells. No down-regulation of rat Leydig cell LH receptor occurs with either kinase.

Somatostatin inhibition of VIP- and isoproterenol-stimulated cyclic AMP accumulation in dissociated cells from rat cerebral cortex.

Freshly dissociated cerebral cortex cells from adult rats have been used in the present study to determine if dual regulation of cyclic AMP levels by inhibitory and stimulatory agents can be expressed in the mature brain. Somatostatin, an inhibitory agent, barely affected the basal cyclic AMP metabolism while vasoactive intestinal peptide (VIP) and isoproterenol, two stimulatory agents enhanced cyclic AMP production. However, this increase was depressed by somatostatin, which decreased the efficiency, but not the potency, of the effects of the two stimulatory agents on cyclic AMP accumulation.

Effect of morphine and acetylcholine on contractile activity and cyclic AMP in guinea-pig ileum.

Neither acute nor prolonged exposure to morphine altered cAMP content or spontaneous movements of longitudinal muscle-myenteric plexus strips of the guinea-pig ileum. By contrast, exogenous acetylcholine or electrical stimulation of the strips elicited both a decrease of cAMP concentration and a twitch response. Atropine blocked the effects of stimulation on these parameters. Addition of morphine to electrically stimulated strips inhibited the twitch response but did not affect cAMP levels. Incubation with morphine led to the development of tolerance to the inhibitory effect on twitch activity and prevented the fall in cAMP normally elicited by electrical stimulation. These results suggest that muscarinic activation is associated with a reduction of cAMP content, an effect which would be impaired in opiate-tolerant tissues.

Effect of haloperidol withdrawal on somatostatin level and binding in rat brain.

The effects of withdrawal on the level and specific binding of somatostatin in the frontoparietal cortex and hippocampus of the rat after chronic haloperidol treatment were examined using 125I-Tyr11 somatostatin as tracer. One week after haloperiodol withdrawal the number of specific somatostatin receptors in both brain areas returned to control values, after having decreased as the result of chronic administration. Neither administration of haloperidol nor withdrawal of it affected the levels of somatostatin-like immunoreactivity (SLI) in the two brain areas studied. The return of the somatostatin receptor number to control values after haloperidol withdrawal may be related to the motor side-effects that are clinically observed when the haloperidol treatment is terminated.

Long-term haloperidol treatment decreases somatostatin binding in rat brain.

The effects of short and long-term haloperidol treatment on somatostatin concentration and specific binding in rat cerebral cortex and hippocampus were examined using the binding ligand 125I-Tyr1-somatostatin. Haloperidol treatment did not affect the concentration of somatostatin-like immunoreactivity in the two brain areas. Nevertheless, long-term, and not short-term, haloperidol treatment decreased the number of somatostatin receptors in the cerebral cortex and hippocampus. No significant differences in the apparent binding affinity values were seen after haloperidol treatment. When added at the time of the binding assay haloperidol 34.2 microM produced a 42% and 27% decrease in cerebrocortical and hippocampal membrane somatostatin receptors respectively.

Cysteamine normalizes cerebral somatostatin level and binding in pentylenetetrazol-kindled rats.

Rats were kindled by intraperitoneal injection of pentylenetetrazol (PTZ) (30 mg/Kg) every 48 h. Once kindled, some of the animals received a single injection of cysteamine (200 mg/Kg). Somatostatin-like immunoreactivity (SLI) and 125 I-Tyr11-somatostatin binding were measured in the frontoparietal cortex and hippocampus of the two experimental groups and the control rats. After PTZ kindling the following was observed: 1) SLI content was increased in the two areas; 2) Somatostatin receptor affinity decreased in the frontoparietal cortex and was unaltered in the hippocampus; 3) The number of somatostatin receptors decreased in the hippocampus and was unaltered in the frontoparietal cortex. Cysteamine, an agent which depletes brain somatostatin and suppresses kindled seizures in PTZ-treated rats, reversed the altered SLI levels and binding in these rats.

Effects of alloxan-induced diabetes on somatostatin binding to cytosolic components of rabbit gastric fundic mucosa.

Diabetes was induced by administration of alloxan (150 mg/Kg) to 24 h-fasted rabbits. Somatostatin-like immunoreactivity (SLI) and cytosolic binding sites for somatostatin in gastric fundic mucosa were studied using radiolabelled Tyr11-somatostatin. Three months after the onset of the disease, the specific binding of somatostatin to these sites was seen to be significantly lower, due to a reduction in the number (but not the affinity) of specific somatostatin binding sites of high-affinity and a disappearance of the specific somatostatin binding sites of low-affinity. These changes were associated with an increase in the SLI concentration in both gastric fundic mucosa and plasma.

Species variations of somatostatin concentrations and binding sites in jejunal mucosa.

1. Somatostatin-like immunoreactivity (SLI) and specific binding of 125I-Tyr11-somatostatin were measured in jejunal mucosa of the mouse, rat, hamster, rabbit and guinea-pig. 2. The SLI concentrations in guinea-pig and rabbit were much greater than those in other rodents considered. 3. Somatostatin binding varied greatly with the species examined, the highest values being observed in cytosolic fraction of guinea-pig and rabbit jejunal mucosa, but the lowest ones in mouse. 4. The observed differences in somatostatin binding were not related to varying extents of degradation by the diverse cytosolic preparations studied. 5. The binding sites were highly specific for somatostatin in all rodent species studied. 6. There appears to be a direct relationship between somatostatin levels and somatostatin binding sites in jejunal mucosa when considering a variety of rodent species usually employed as laboratory animals.

Postnatal development of somatostatin levels and its binding sites in rabbit gastric mucosa.

Using a specific radioimmunoassay technique, we have determined somatostatin-like immunoreactivity (SLI) in acid extracts of gastric (fundic and antral) mucosa as well as the specific binding of 125I-Tyr11-somatostatin to cytosol of the stomach of 0 to 150 days postnatal rabbits. The levels of somatostatin in both fundus and antrum decreased from birth up to day 5 followed by a sharp increase from 5 to 10 days, then decreased progressively until day 35. After this age, the somatostatin concentration remained relatively stable. The number of specific somatostatin binding sites of both high- and low-affinity increased gradually (without changes in the affinity values) with the development of rabbits, reaching the adult level by 35 days. However, there was an apparent lack of high-affinity sites immediately after birth (day 0). The somatostatin binding sites had characteristics identical with those found in adult animals with regard to their respective specific ligands.

Effect of cysteamine on cytosolic somatostatin binding sites in rabbit duodenal mucosa.

Administration of cysteamine in rabbits elicited a rapid depletion of both duodenal mucosa and plasma somatostatin. A significant reduction was observed within 5 min, returning toward control values by 150 min. The depletion of somatostatin was associated with an increase in the binding capacity and a decrease in the affinity of both high- and low-affinity binding sites present in cytosol of duodenal mucosa. Incubation of cytosolic fraction from control rabbits with 1 mM cysteamine did not modify somatostatin binding. Furthermore, addition of cysteamine at the time of binding assay did not affect the integrity of 125I-Tyr11-somatostatin. It is concluded that in vivo administration of cysteamine to rabbits depletes both duodenal mucosa and plasma somatostatin and leads to up-regulation of duodenal somatostatin binding sites.

Ontogeny of somatostatin binding sites in the rabbit small intestinal epithelial tract.

The ontogenic patterns of somatostatin content and its binding sites were studied in small intestine in the developing rabbit from birth until the adult stage. A peak of somatostatin concentration was observed immediately after birth (day 0) and at day 10 in duodenum and jejunum, followed by a decrease at day 15 and a new and gradual augment thereafter. Ileal somatostatin concentrations decreased after birth up to day 15 and then increased progressively with age until the adult period. The somatostatin binding capacity in cytosol of the mucosal layer of duodenum, jejunum and ileum decreased from birth until 10 days and increased thereafter up to the adult period. However, no age differences in dissociation constants were observed. Interestingly, there was an apparent lack of high-affinity sites immediately after birth (day 0) whereas two classes of binding sites were seen thereafter. These results suggest that somatostatin may be involved in the functional and anatomical development of the small intestine during the neonatal period.

Effect of small intestinal resection on somatostatin binding to cytosol of rabbit gastric mucosa.

Small bowel resection in the rabbit increased gastric (fundus and antrum) somatostatin content and decreased the number of somatostatin binding sites (but not their corresponding affinity values) in gastric (fundus and antrum) cytosol three weeks after surgery. Five weeks after resection the number of somatostatin binding sites at both fundic and antral levels as well as antral somatostatin content returned towards control values whereas the fundic concentration of the peptide remained increased. Present results together with the known inhibitory role of somatostatin on various gastric functions suggest that the gastric alterations showed by animals subjected to small bowel resection may be due, at least in part, to the observed decrease of the number of gastric somatostatin binding sites.

Specific binding of Leu-enkephalin to small and large intestinal epithelial cells from guinea-pig.

Leu-enkephalin receptors were identified in guinea-pig intestinal mucosa in small as well as in large epithelial cells. Binding studies at apparent equilibrium could be interpreted in terms of two populations of receptors in every intestinal segment. Leu-enkephalin receptors were unequally distributed along the intestinal mucosa, with the lowest density but the highest affinity values in the caecum and colon. Duodenal epithelial cells exhibited the highest binding values due to a great number of low-affinity receptors. Receptors exhibited a high degree of specificity for Leu-enkephalin as evidenced by the poor competition shown by a variety of enkephalin analogues and naloxone and the lack of effect of other unrelated peptides present in the intestinal tract.

Modulation of somatostatin binding sites in cytosol of rabbit gastric fundic mucosa by cysteamine administration.

Cysteamine, when given in vivo to rabbits, depleted immunoreactive somatostatin in rabbit gastric fundic mucosa. Depletion of immunoreactive somatostatin was associated with both an increase in the number and a decrease in the affinity of the low-affinity somatostatin binding sites. The in vitro incubation of cysteamine (0.1 mM) with the cytosolic fraction did not result in any modification of somatostatin binding. These results suggest that a decrease in the endogenous immunoreactive somatostatin might lead to up-regulation of somatostatin binding sites in the gastric fundic mucosa.