The effects of cyclic nucleotides and some related agents on 32Pi labeling of renal polyphosphoinositides in vitro.

When rabbit kidney cortex slices were incubated in the presence of ³²P_i and dibutyrylcyclic AMP (dbcAMP)⁴ a significant decrease in the labeling of phosphatidyl inositol phosphate (DPI) but not phosphatidyl inositol bisphosphate (TPI) was observed. In the presence of 0.3 mm caffeine cyclic AMP (cAMP) produced a similar effect. Caffeine potentiated the inhibitory effect of dbcAMP. At high concentrations (3 mm) caffeine alone decreased the ³²P_i labeling of both DPI and TPI. These decreases in ³²P_i labeling were not mediated by decreases in the labeling of intracellular P_i or ATP as measured by 10-min acid-labile nucleotide phosphate (10′-ALNP). Addition of cyclic GMP (cGMP) to the incubation medium decreased the labeling of DPI and to a lesser extent that of TPI also. Addition of parathyroid hormone (PTH) to the incubation medium (in the absence of exogenous cyclic nucleotides) also decreased the ³²P_i labeling of DPI but not that of TPI. In contrast to the effects of cAMP, dbcAMP, cGMP, PTH, and caffeine, the addition of insulin to the incubation medium resulted in increased ³²P_i labeling of DPI with no effect on TPI labeling. DPI isolated from kidney cortex slices prelabeled with ³²P_i and subsequently incubated with cAMP or dbcAMP contained less label than DPI isolated from slices similarly prelabeled but subsequently incubated in the absence of either cAMP or dbcAMP. These data suggest an increased rate of DPI breakdown in the presence of elevated cAMP or dbcAMP concentrations. This hypothesis was supported by the fact that cAMP stimulated the hydrolysis of DPI but not of TPI by a polyphosphoinositide phosphodiesterase present in the supernatant fraction of rabbit kidney cortex.     

Polyphosphoinositide metabolism in rat brain: effects of neuropeptides, neurotransmitters and cyclic nucleotides

This study describes effects of various peptides, neurotransmitters and cyclic nucleotides on brain polyphosphoinositide metabolism in vitro. The interconversion of the polyanionic inositol phospholipids was studied by incubation of a lysed crude mitochondrial/synaptosomal fraction with [gamma-32P]-ATP. The reference peptide ACTH1-24 stimulated the formation of radiolabelled phosphatidylinositol 4,5-diphosphate (TPI) and inhibited that of phosphatidic acid (PA). Substance P inhibited both TPI and PA labelling, whereas beta-endorphin inhibited that of PA without any effect on TPI. Morphine had no effect at any concentration tested, whereas high concentrations of naloxone inhibited the labelling of both PA and TPI. Naloxone did not counteract the effects of ACTH1-24. The other peptides tested (lysine 8-vasopressin and angiotensin II) were without any effect. Under the conditions used, adrenaline, noradrenaline and acetylcholine did not affect the labelling of the (poly)phosphoinositides. Both dopamine and serotonin, however, dose-dependently inhibited the formation of radiolabelled TPI and PA. Low concentrations of cAMP stimulated TPI, but higher concentrations had an overall inhibitory effect on the labelling of TPI, PA and especially phosphatidylinositol 4-phosphate (DPI). The cyclic nucleotide did not mediate or counteract the effects of ACTH, and cGMP was without any effect. These results are discussed in the light of current ideas on the mechanism of action of neuropeptides.     

Glucose inhibits insulin release when not promoting the entry of calcium into the beta-cells

The effect of antigen on the metabolism of polyphosphoinositides was investigated in sensitized rat peritoneal mast cells. Addition of antigen to rat peritoneal mast cells prelabelled with [3H]arachidonic acid resulted in a very rapid decrease in the level of phosphatidylinositol 4-phosphate (DPI) within 5 sec, which appeared to precede the breakdown of phosphatidylinositol (PI), while there was no significant decline of PI 4,5-bisphosphate (TPI). The reduced levels of these phosphoinositides returned almost to control or even slightly higher values by 300 sec in parallel with the antigen-stimulated [32P]phosphate incorporation into these lipids. This early and transient disappearance in DPI prior to that in PI was also observed in [3H]glycerol-prelabelled cells. These data suggest that DPI degradation upon stimulation by antigen in mast cells may be an initial step in the histamine release process.     

Light-mediated breakdown of phosphatidylinositol-4,5-bisphosphate in isolated rod outer segments of frog photoreceptor

When isolated frog (Rana catesbeiana) rod outer segment (ROS) fragments were incubated with [gamma-32P]ATP in the dark, only two of phospholipids, i.e., phosphatidylinositol-4-phosphate (DPI) and phosphatidic acid (PA) incorporated 32P. Upon addition of DPI (100 microM), considerable amount of 32P was incorporated into phosphatidylinositol-4,5-bisphosphate (TPI) as well as DPI and PA. Exposure of the ROS membranes to 5 sec flash of light resulted in approx. 20% decrease in the labeled TPI, while no significant effect was observed on DPI and PA. It was also observed that Ca2+ markedly accelerated the production of PA in the dark, while it reduced the 32P-incorporation into TPI. These results suggest that there is light- and/or Ca2+-dependent TPI-specific phospholipase C in ROS of vertebrate photoreceptors.     

Calcium regulation of phosphatidyl inositol turnover in macrophage activation by formyl peptides

Stimulation by the tripeptide N-formyl norleucyl leucyl phenylalanine (FNLLP) of the guinea pig alveolar macrophage gives rise to transient production of superoxide anion (O2-). Components of the phosphatidyl inositol (PI) cycle (phosphatidic acid (PA), phosphatidyl inositol-4,5-bisphosphate (TPI) and phosphatidyl inositol-4-phosphate (DPI) were monitored using 32P in order to examine the possible association of this cycle with the FNLLP-stimulated production of O2-. Macrophage stimulation by FNLLP led to an increased flux of metabolites through the PI cycle. The level of 32P label in both TPI and DPI rapidly decreased upon exposure to FNLLP, followed by a 5-min period during which the 32P label in TPI and DPI approached prestimulated levels. During this period, there was a fivefold increase in 32P-PA. It is suggested that diacylglycerol (DAG) is the O2- -activating intermediate in the stimulated mechanism, as evidenced by the buildup of PA (for which DAG is the precursor) in parallel with the time course of O2- production. The importance of continued cycling of PI in the stimulated mechanism is demonstrated by the inhibition by LiCl of the extent, but not the initial rate, of both O2- production and the formation of 32P-PA upon peptide stimulation after 1-h preincubation with 10 mM LiCl. The influence of calcium on this mechanism was also examined. It has previously been demonstrated that intracellular availability of calcium can influence the rate and extent of O2- production. In cells preloaded with quin-2, which acts as a high-affinity sink for calcium in the cytosol, the initial rate of FNLLP-stimulated O2- production is inhibited in low (10 microM) extracellular calcium medium. High extracellular calcium (1 mM) completely reverses this inhibition and also significantly extends the time course of O2- production in both quin-2 and control cells (Stickle et al., 1984). In parallel with these effects on O2- production, varying calcium conditions is demonstrated to influence the rate and extent of PA formation. These same calcium conditions were found to have little or no effect on the initial unstimulated levels of TPI, DPI, and PA. These results indicate that the influence of an intracellular pool of calcium on O2- production may be via its influence on stimulated PI turnover.     

Effect of fusaric acid on phosphoinositide metabolism in the erythrocyte membranes of rats with spontaneous hypertension

2- and 4-month-old male spontaneously hypertensive rats (SHR) were injected fusaric acid at a dose of 50 mg/kg body weight. Fusaric acid increased diphosphoinositide (DPI) and triphosphoinositide (TPI) levels in erythrocyte membranes of 4-month-old SHR by 41% and 20%, respectively. 32P incorporation into TPI decreased by 24% in 2- and by 20% in 4-month-old SHR. Phosphatidylinositol metabolism remained unchanged. The results also suggest that fusaric acid normalized DPI and TPI metabolism in erythrocyte membranes of SHR.     

Reversible,long-term inhibition of ovulation with a gonadotropin-releasing hormone antagonist in the marmoset monkey (Callithrix jacchus)

The effects of weekly injections of a gonadotropin-releasing hormone (GnRH) antagonist (GnRHa) ([N-acetyl-DβNal1-D-pCl-Phe2-D-Phe3-D-Arg6-Phe7-Arg8D-Ala10] NH2 GnRH) on pituitary and ovarian function were examined in the marmoset monkey, Callithrix jacchus. In experiment 1, five cyclic females were given weekly injections of vehicle (50% propylene glycol in saline) for 6 weeks followed by GnRHa for 20 weeks, animals receiving either 200 μg GnRHa/injection (n = 2) or 67 μg GnRHa/injection (n = 3) for 10 weeks, after which the treatment was reversed. Bioactive luteinizing hormone (LH) and progesterone (Po) were measured in blood samples (0.2–0.4 ml) collected twice weekly until at least 8 weeks after the last GnRHa injection. GnRHa treatment, timed to begin in the midluteal phase, caused a rapid decline in LH and Po and luteal regression after a single injection (both doses). Po levels were consistently low (<10 ng/ml), and ovulation was inhibited throughout 200 μg treatment in all animals. Short periods of elevated Po (>10 ng/ml) were, however, occasionally seen during 67 μg treatment, indicating incomplete ovarian suppression. Mean LH levels were significantly lower during GnRHa treatment compared with the period of vehicle injection (all animals 200 μg; three animals 67 μg), and there were significant differences in LH levels between GnRHa treatments (200 μg vs. 67 μg) in four animals. Four animals resumed normal ovarian cycles after the end of GnRHa treatment (15/16 days, three animals; 59 days, one animal); the fifth animal died of unknown causes 32 days after the last GnRHa injection. In a second experiment, pituitary responsiveness to exogenous GnRH was tested 1 day after a single injection of vehicle or antagonist (200 or 67 μg). Measurement of bioactive LH indicated that pituitary response to 200 ng native GnRH was significantly suppressed in animals receiving the antagonist, the degree of suppression being dose related. A third experiment examined the effect of four weekly injections of 200 μg GnRHa on follicular size and granulosa cell responsiveness to human follicle-stimulating hormone (hFSH) in vitro. Follicular development beyond 1 mm was inhibited by GnRHa treatment (preovulatory follicles normally 2-4 mm) although granulosa cell responsiveness to FSH during 48 hr of culture was not impaired. These results suggest that the GnRHa-induced suppression of follicular development and ovulation was mediated primarily by an inhibition of pituitary gonadotropin secretion and not by a direct action at the level of the ovary.     

Effects of Corticotropin-(1–24)-Tetracosapeptide on Polyphosphoinositide Metabolism and Protein Phosphorylation in Rabbit Iris Subcellular Fractions

Abstract: Effects of the neuropeptide corticotropin-(1–24) -tetracosapeptide (ACTH) on the endogenous and exogenous phosphorylation of lipids and endogenous phosphorylation of proteins were investigated in microsomes and a 110,000 ×g supernatant fraction [30–50% (NH₄)₂SO₄ precipitate; ASP_30–50] obtained from rabbit iris smooth muscle. Subcellular distribution studies revealed that both of these fractions are enriched in diphosphoinositide (DPI) kinase. The ³²P labeling of lipids and proteins was measured by incubation of the subcellular fractions with [γ-³²P]ATP. The labeled lipids, which consisted of triphosphoinositide (TPI), DPI, and phosphatidic acid (PA) were isolated by TLC. The microsomal and ASP_30–50 fractions were resolved into six and nine labeled phosphoprotein bands, respectively, by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The basal labeling of both lipids and proteins was rapid (30–60 s), and it was dependent on the presence of Mg²⁺ in the incubation medium; in general it was inhibited by high concentrations (>0.2 mM) of Ca²⁺. ACTH stimulated the labeling of TPI and inhibited that of PA in a dose-dependent manner, with maximal effect observed at 50–100 μ of the peptide. ACTH appears to increase TPI labeling by stimulating the DPI kinase. Under the same experimental conditions ACTH (100 μM) inhibited significantly the endogenous phosphorylation of six microsomal phosphoproteins (100K, 84K, 65K, 53K, 48K, and 17K). In the ASP_30–50 fraction, ACTH inhibited the phosphorylation of three phosphoproteins (53K, 48K, and 17K) and stimulated the labeling of six phosphoprotein bands (117K, 100K, 84K, 65K, 42K, and 35K). The effects of ACTH on lipid and protein phosphorylation are probably Ca²⁺-independent; thus the neuropeptide effects were not influenced by either 1 μM EGTA or low concentrations of Ca²⁺ (50 μ.M). We conclude that a relationship may exist between polyphosphoinositide metabolism and protein phosphorylation in the rabbit iris smooth muscle.     

Calcium- and Calmodulin-Dependent Phosphorylation of Diphosphoinositide in Acetylcholine Receptor-Rich Membranes from Electroplax of Narke japonica

The phosphorylation of phosphoinositides in the acetylcholine receptor (AChR)-rich membranes from the electroplax of the electric fish Narke japonica has been examined. When the AChR-rich membranes were incubated with [gamma-32P]ATP, 32P was incorporated into only two inositol phospholipids, i.e., tri- and diphosphoinositide (TPI and DPI). Even after the alkali treatment of the membrane, AChR-rich membranes still showed a considerable DPI kinase activity upon addition of exogenous DPI. It is likely that the 32P-incorporation into these lipids was realized by the membrane-bound DPI kinase and phosphatidyl inositol (PI) kinase. Such a membrane-bound DPI kinase was activated by Ca2+ (greater than 10(-6) M), whereas the PI kinase appeared to be inhibited by Ca2+. The effect of Ca2+ on the DPI phosphorylation was further enhanced by the addition of ubiquitous Ca2+-dependent regulator protein calmodulin. Calmodulin antagonists such as chlorpromazine (CPZ), trifluoperazine (TFP), and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibited the phosphorylation of DPI in the AChR-rich membranes. It is suggested that the small pool of TPI in the plasma membrane is replenished by such Ca2+- and calmodulin-dependent DPI kinase responding to the change in the intracellular Ca2+ level.     

Subcellular incorporation of 32P into phosphoinositides and other phospholipids in isolated hepatocytes

Isolated rat hepatocytes were incubated with 32Pi for various times and then fractionated into plasma membranes, mitochondria, nuclei, lysosomes, and microsomes by differential centrifugation and Percoll density gradient centrifugation. The phospholipids were isolated and deacylated by mild alkaline treatment. The glycerophosphate esters were separated by anion exchange high pressure liquid chromatography and assayed for radioactivity. It was found that plasma membranes, mitochondria, nuclei, lysosomes, and microsomes displayed similar rates of 32P incorporation into the major phospholipids, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid. This suggests that the phospholipids of these organelles are undergoing rapid turnover and replacement with newly synthesized phospholipids from the endoplasmic reticulum. However, the plasma membrane fraction incorporated 32P into phosphatidylinositol 4-phosphate (DPI) and phosphatidylinositol 4,5-bisphosphate (TPI) at rates 5-10 and 25-50 times, respectively, faster than any of the other subcellular fractions. Although the plasma membrane is the primary site of 32P incorporation into DPI and TPI, this study also demonstrates that significant incorporation of 32P into DPI occurs in other subcellular sites, especially lysosomes.     

The rapid polyphosphoinositide metabolism may be a triggering event for thrombin-mediated stimulation of human platelets

The metabolism of polyphosphoinositides was examined in human platelets activated by thrombin. The addition of thrombin to [3H]glycerol-labeled platelets induced an initial loss and a subsequent increase of the radioactivity in phosphatidylinositol-4,5-bisphosphate (TPI) without any significant change in phosphatidylinositol-4-phosphate (DPI). A marked enhancement of [32P]Pi incorporation into TPI occurred in parallel with an increase in this lipid content, which was accompanied with a conccurent decrease in phosphatidylinositol (PI). The rate of this subsequent increase in TPI was smaller than that observed in [3H]arachidonic acid-labeled platelets, suggesting that formed TPI in activated platelets may contain much greater amount of arachidonate than preexisting TPI in resting platelets. These data indicate that thrombin causes a rapid change in TPI metabolism (initial degradation of preexisting TPI and subsequent production of arachidonate-rich TPI), which might be a primary candidate to modulate thrombin-induced function in human platelets.     

Possible regulation of phospholipase C activity in human platelets by phosphatidylinositol 4',5'-bisphosphate

Phospholipase C from human platelets was found to catalyze the Ca2+-dependent degradation of phosphatidylinositol (PI), phosphatidylinositol 4'-phosphate (DPI), and phosphatidylinositol 4',5'-bisphosphate (TPI) at Ca2+ concentrations from 150 microM to 5 mM. Both DPI and TPI inhibited the hydrolysis of [2-3H]inositol-labeled PI (250 microM) in a concentration-dependent manner. The use of DPI and TPI from beef brain, both of which have fatty acid compositions different from that of soybean PI, permitted an assessment of the inhibitory effect of polyphosphoinositides on the hydrolysis of PI by phospholipase C. Fatty acid analysis of the diacylglycerols formed demonstrated that DPI and TPI, when incubated in mixture with PI, were competitive substrates for PI hydrolysis. Increasing the DPI/PI ratio from 0 to 0.3 caused a shift in the degradation of PI to DPI without greatly affecting the formation of 1,2-diacylglycerol. TPI alone, or in mixture with PI, was a poor substrate for phospholipase C. Increasing the TPI/PI ratio from 0 to 0.21, on the other hand, inhibited both PI degradation (greater than or equal to 95%) and overall formation of 1,2-diacylglycerol (greater than or equal to 82%). Kinetic analysis revealed that TPI acts as a mixed-type inhibitor with a Ki of about 10 microM. The Ka for Ca2+ in PI hydrolysis was profoundly increased from 5 to 180 microM when TPI (36 microM) was included with PI (250 microM). Optimum PI degradation under these conditions was only attained when the calcium concentration approached 4 mM. Analysis of phospholipids from unstimulated human platelets from five different donors revealed DPI/PI and TPI/PI ratios of 0.42 and 0.16, respectively. These findings, combined with the observed inhibition of PI hydrolysis by TPI at a TPI/PI ratio of 0.16, would suggest that in unstimulated platelets phospholipase C activity may be inhibited by greater than or equal to 75%. Changes in 33P-prelabeled phospholipids of intact platelets upon stimulation with thrombin indicated a transient decline in 33P label of both TPI and DPI (15 s) followed by an increase in [33P]phosphatidic acid but no change in [33P]PI. The finding that DPI is selectively degraded by phospholipase C in mixture with PI at DPI/PI ratios determined to be present in unstimulated platelets indicates that DPI may be more important than PI in the formation of 1,2-diacylglycerol which is believed to serve as precursor of arachidonic acid for thromboxane biosynthesis. Furthermore, the results suggest that in human platelets TPI may serve as modulator for the formation of 1,2-diacylglycerol from inositol phospholipids.     

Rapid Incorporation In Vivo of Intracerebrally Injected 32Pi into Polyphosphoinositides of Three Subfractions of Rat Brain Myelin

Abstract: At intervals ranging from 1 to 10 min after injection of ³²Pi into rat brain, myelin was prepared and separated into three subfractions: heavy, medium, and light. The radioactivity of total phospholipids and polyphospho-inositides (PPI) was then determined. There was rapid incorporation of ³²Pi into PPI, which contained 50–70% of the radioactivity among total brain lipids and more than 70% among myelin lipids. The myelin fraction had incorporated ³²Pi into total recovered PPI in the order of medium > heavy > light fraction: however, the order of relative specific radioactivities was heavy > light > medium. Labeling of the PPI precursors, phosphatidic acid (PA) and phos-phatidylinositol (PI), was considerably lower in the purified myelin than in total brain. The di- (DPI) and triphosphoinositides (TPI) in heavy myelin exchanged ³²Pi at rates 2 to 3 times faster than those in medium and light myelin. DPI of all subfractions of myelin exchanged much faster than TPI. The results show that the most active phosphate turnover of myelin PPI occurs in the heavy myelin fraction (probably largely consisting of myelin appurtenant regions). However, medium and light myelin (most probably representing the closely packed layers of myelin sheaths) also showed rapid turnover of PPI.     

Disruption of neuroendocrine control of luteinizing hormone secretion by aroclor 1254 involves inhibition of hypothalamic tryptophan hydroxylase activity

Mechanisms governing the effect of polychlorinated biphenyl (PCB) toxicity on hypothalamic serotonergic function and the neuroendocrine system controlling LH secretion were investigated in Atlantic croaker (Micropogonias unulatus) exposed to the PCB mixture Aroclor 1254 (1 microg x g body weight(-1) x day(-1)) in the diet for 30 days. PCB treatment caused a decrease in hypothalamic 5-hydroxytryptamine (5-HT) concentrations and significant inhibition of hypothalamic tryptophan hydroxylase (TPH), the rate-limiting enzyme in 5-HT synthesis, but did not alter the activity of monoamine oxidase, the catabolic enzyme. Further, PCB treatment caused significant decreases in GnRH content in the preoptic-anterior hypothalamic area. Significant decreases in pituitary GnRH receptor concentrations and the LH response to the GnRH analogue (GnRHa) were also observed in PCB-exposed fish, possibly as a consequence of a decline in GnRH release. The possible association between impaired serotonergic and neuroendocrine functions after PCB treatment was explored using serotonergic drugs. Treatment of croaker with p-chlorophenylalanine, an irreversible TPH inhibitor, mimicked the effects of PCB on the GnRH system and the LH response to GnRHa. Bypassing the TPH-dependent hydroxylation step with the administration of 5-hydroxytryptophan restored 5-HT to control levels and prevented the deleterious effects of PCB on the neuroendocrine parameters. Moreover, slow-release GnRH implants prevented the PCB-induced decline in GnRH receptors and restored the LH response to GnRHa, suggesting that GnRH therapy can reverse PCB-induced disruption of LH secretion. These results demonstrate that TPH is one of the targets of PCB neurotoxicity and indicate that a decrease in 5-HT availability in PCB-exposed croaker results in disruption of the stimulatory 5-HT/GnRH pathway controlling LH secretion.     

Folliculogenesis Is Not Fully Inhibited during GnRH Analogues Treatment in Mice Challenging Their Efficiency to Preserve the Ovarian Reserve during Chemotherapy in This Model

As many chemotherapy regimens induce follicular depletion, fertility preservation became a major concern in young cancer patients. By maintaining follicles at the resting stage, gonadotropin-releasing hormone analogues (GnRHa) were proposed as an ovarian-protective option during chemotherapy. However, their efficacy and mechanisms of action remain to be elucidated. Mice were dosed with cyclophosphamide (Cy, 100–500mg/kg i.p) to quantify follicular depletion and evaluate apoptosis at different times. We observed a dose-dependent depletion of the follicular reserve within 24 hours after Cy injection with a mean follicular loss of 45% at the dose of 200mg/kg. Apoptosis occurs in the granulosa cells of growing follicles within 12 hours after Cy treatment, while no apoptosis was detected in resting follicles suggesting that chemotherapy acutely affects both resting and growing follicles through different mechanisms. We further tested the ability of both GnRH agonist and antagonist to inhibit oestrus cycles, follicular growth and FSH secretion in mice and to protect ovarian reserve against chemotherapy. Although GnRHa were efficient to disrupt oestrus cycles, they failed to inhibit follicular development, irrespective of the doses and injection sites (sc or im). Around 20% of healthy growing follicles were still observed during GnRHa treatment and serum FSH levels were not reduced either by antagonist or agonist. GnRHa had no effect on Cy-induced follicular damages. Thus, we showed that GnRHa were not as efficient at inhibiting the pituitary-gonadal axis in mice as in human. Furthermore, the acute depletion of primordial follicles observed after chemotherapy does not support the hypothesis that the ovary may be protected by gonadotropin suppression.     

Microdetermination of phosphoinositides in a single extract

A method that allows the quantification of phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (DPI), and phosphatidylinositol 4,5-biphosphate (TPI) on a nanomolar scale is presented. The method is based on the simultaneous separation of lipids on high-performance thin-layer chromatography plates, followed by a microassay for phosphorus of PI spots and a densitometric assay of DPI and TPI. The new procedure allows the determination of the phospholipids in small amounts (100 micrograms protein) of synaptosomes and synaptic plasma membranes, and in homogenates of microwave-fixed brain tissue (1 mg wet wt). The usefulness of the method is illustrated by showing the effect of Ca2+ on the breakdown of DPI and TPI in synaptosomal plasma membranes.     

Onset of neurophysin self-association upon neurophysin/neuropeptide hormone precursor biosynthesis

32P-Labelled washed rabbit platelets were incubated with 0.6 nM platelet activating factor (PAF-acether), giving a full aggregation and release response within 30-60 s. The major phospholipid changes observed under these conditions were: (1) An increased labelling of phosphatidic acid (PA) within 10 s and of phosphatidylinositol (MPI) at 30 s, reflecting the activation of the MPI cycle via the cytosolic phospholipase C; (2) an enhancement of phosphatidylinositol-4-phosphate (DPI) and phosphatidylinositol-4,5-bisphosphate (TPI) labelling at later incubation times; (3) an early degradation of TPI with a counterbalancing formation of DPI. The latter changes suggest a receptor-mediated stimulation of TPI-phosphomonoesterase, the role of which in the mechanism of platelet activation is discussed.     

Phorbol ester-induced LH release in pituitary gonadotrophs: effects of antagonists of calmodulin and GnRH.

The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent activator of Ca(2+)- and phospholipid-dependent protein kinase (C kinase), stimulates luteinizing hormone (LH) release from rat pituitary cells. The actions of TPA upon LH release were compared with those of the GnRH superagonist [D-Ala6] des-Gly10-GnRH N-ethylamide (GnRHa) in cultured pituitary cells. LH release was stimulated by 0.1 nM TPA and the maximum response at 10 nM TPA was 50% of the LH response to GnRHa. The ED50 values for TPA and GnRHa were 1.2 and 0.037 nM, respectively, and the maximum stimulatory effects of TPA and GnRHa on LH release were not additive. GnRHa-stimulated LH release was decreased by calmodulin (CaM) antagonists including pimozide, trifluoperazine, W5 and W7, being most effectively reduced (by 70%) by 10 microM pimozide. In contrast to their inhibition of GnRH action, these antagonists enhanced TPA-stimulated LH release, so that 10 microM pimozide and W7 doubled the maximum LH response. The potent GnRH antagonist [Ac-D-p-Cl-Phe1.2, D-Trp3, D-Lys6, D-Ala10]GnRH, which completely inhibited GnRHa-stimulated LH release with ID50 of 6.8 nM, also reduced maximum TPA-stimulated LH release by about 50%. These results suggest that both Ca2+/CaM and C kinase pathways are involved in the LH release mechanism, and indicate that C kinase plays a major role in the action of GnRH upon gonadotropin secretion. The synergism between CaM antagonists and TPA suggests that blockade of CaM-mediated processes leads to enhanced activation of the C kinase pathway, possibly by removal of an inhibitory influence. Furthermore, the partial inhibition of TPA-stimulated LH release by a GnRH antagonist suggests that the pathway(s), specifically connected with LH release in the diverse effects of C kinase, might be locked by the continuous receptor inactivation by antagonist and indicates the complicated pathways which diverge from the receptor and converge into specific cellular response.     

Stimulation and inhibition of Leydig cell steroidogenesis by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate: similarity to the effects of gonadotropin-releasing hormone

To explore the mechanism of gonadotropin-releasing hormone (GnRH) action on Leydig cell steroidogenesis the effects of a GnRH analog (GnRHa) were compared to those of 12-O-tetradecanoylphorbol 13-acetate (TPA). Both compounds acutely stimulated androgen production 2-4 fold with EC50's of 9 nM (TPA) and 0.2 nM (GnRHa). The effects of TPA and GnRHa were not additive and neither compound acutely altered the luteinizing hormone (LH) concentration-response relationship. After 24 h of exposure to TPA or GnRHa the ability of LH to stimulate androgen production was impaired. The parallel effects of TPA and GnRHa on Leydig cell steroidogenesis suggest that they are acting via similar mechanisms; presumably the activation protein kinase C.     

Cytoplasmic versus nuclear localization of Fos-related proteins in the frog,Rana esculenta,testis: in vivo and direct in vitro effect of a gonadotropin-releasing hormone agonist

Evidence has been accumulated indicating that GnRH-like peptides are present in a variety of extrabrain areas of mammalian and nonmammalian vertebrates. A pioneer study carried out in the frog, Rana esculenta, demonstrated that testicular GnRH induced spermatogonial proliferation. Recently, we have shown that in proliferating spermatogonia (SPG) of frogs, a change of localization of the oncoprotein Fos, from the cytoplasm to the nucleus, occurs. This leads to the hypothesis that one or more testicular GnRH peptides may regulate SPG proliferation through Fos family proteins. Therefore, in vivo experiments in intact R. esculenta and in vitro incubations of testis fragments have been carried out using GnRH agonist (GnRHa; buserelin) and GnRH antagonist (D-pGlu(1),D-Phe(2),D-Trp(3,6)-GnRH). Cytoplasmic and nuclear Fos-like protein localization has been found by Western blot analysis in testicular extracts. Immunocytochemistry confirmed that cytoplasmic immunostaining was restricted to SPG; change of localization into the nuclear compartment was observed after GnRHa treatment. Northern blot analysis showed that treatments of testis fragments with GnRHa did not modify testicular c-fos mRNA expression. On the contrary, a Fos-like protein of 52 kDa, while not affected in vivo, disappeared from testicular cytosolic extracts after in vitro treatment with GnRHa. Contemporaneously, a 55-kDa Fos-related signal appeared in nuclear extracts. The GnRH antagonist counteracted the effects of GnRHa. Furthermore, in vivo treatments showed that GnRHa acted negatively on a 43-kDa nuclear Fos-related signal and that gonadotropins caused the decrease of 52-kDa cytoplasmic signal. In conclusion, we show, to our knowledge for the first time, that Fos is regulated by GnRHa directly (not through the pituitary) at the testicular level. The main effect appears to be related to Fos translocation from cytoplasmic to nuclear compartments of SPG.