Claude Jacquemin

Control by TSH of a phospholipase A2 activity, a limiting factor in the biosynthesis of prostaglandins in the thyroid

Tbyreostimuhn QTSH) stimulates the thyroid sdenyi-cyclase and increases the level of cyclic 3*-S’adcnosine monophosphate (c-AMP)_ The model of Sutherland, in which c-AMP is the second messenger, explains many effects of T§H [ t 1. On the creher hand, the prostagtandins (EYE2 and PGFZa) which are abondane in the ehyroid [2], seimulate the thyroid adenyl-cyciase f3--51. Recently Burke 161 has shown an increase in the ineracellu%ar concentration of thy&d proseaglandins under the infh.rence of TSH, The hypothesis of Kuehl [7-S], according to which the action of the luteinizin~ hormone @JI) on the ovary adenyl-cyclase svould necessarily involve a stimulation of prostaglandins biosynthesis, has been applied to the ehyroid by Burke [4-g] _ Qr; the other side, several authors have reached the conclusion that the release of prostaglandins from a tissue was due to a neo-synthesis and not to a depletion of storage material [lo, I 9 1: The efficiency of the prostagiandin synthetase seems to depend only on the amount of suhstrate available, as indeed the addition of arachidonic acid causes a rapid formation of ‘prostaglandins [ 12,13] . The polyunsaturated fatty acids, precursors of the prostaghmdins, exist at a low concentration as free acids, but their concentration is not negligible in the membrane phosphohpids. It has been suggested that the a&hey ofendogenous phospholipases A could be the factor regulating the iriosynthesis of prostagEandins [ 14, IS] _ It has been demonstrated that phospholipids can play the role of precursors in the biosynthesis of prostaglandins [ 16, 171 and that prior treatment with a venom phosphoIipase A increases the conversion yield [ 15,17f . fn this work we have studied 3;~ one hand whether the prostaglandins are obligatory intermediates in ihe action of TSH on the adenyl-cycLse, as suggested by Burke, and on the other hand if there is a TSH-depenclent control mechanism of the production of E, and F2ar prostaglandins from thyrrjid phosphohpids. ‘We have obtained the following results: i) TSH increases the biosynthesis of prostagiandins in the thyroid in vifro; we thus confirm the results of Burke et al. [6] using a different methodology. This effect of TCSH is abolished by indomethacin and aspirin, which are inhibitors of proseaglandin synthetaso

The Expression of Thyrotropin Receptor in the Brain1

The regulation of the thyroid gland by TSH is mediated by a heterotrimeric G protein-coupled receptor. Nonthyroid effects of TSH have been reported, and expression of its receptor has been described in adipocytes and lymphocytes. We have previously reported the existence of specific and saturable binding sites of TSH and specific TSH effects in primary cultured rat brain astroglial cells. We now report expression of the TSH receptor gene in these cells; the coding sequence of the corresponding complementary DNA is identical to that previously established in thyroid. Using specific antisense RNA probe, expression of this gene was detected in some isolated or clustered glial fibrillary acidic protein-positive primary cultured cells by in situ hybridization. With this technique, we further detected TSH receptor messenger RNA (mRNA) expression in rat brain cryoslices in both neuronal cells and astrocytes. Its presence predominated in neuron-rich areas (pyriform and postcingulate cortex, hippocampus, and hypot...The regulation of the thyroid gland by TSH is mediated by a heterotrimeric G protein-coupled receptor. Nonthyroid effects of TSH have been reported, and expression of its receptor has been described in adipocytes and lymphocytes. We have previously reported the existence of specific and saturable binding sites of TSH and specific TSH effects in primary cultured rat brain astroglial cells. We now report expression of the TSH receptor gene in these cells; the coding sequence of the corresponding complementary DNA is identical to that previously established in thyroid. Using specific antisense RNA probe, expression of this gene was detected in some isolated or clustered glial fibrillary acidic protein-positive primary cultured cells by in situ hybridization. With this technique, we further detected TSH receptor messenger RNA (mRNA) expression in rat brain cryoslices in both neuronal cells and astrocytes. Its presence predominated in neuron-rich areas (pyriform and postcingulate cortex, hippocampus, and hypothalamic nuclei) and was mostly colocalized with neuron-specific enolase. In astrocytes, this mRNA was detected in the ependymal cell layer and the subependymal zone, and several isolated cells were also found in the brain parenchyma. We also detected TSH receptor mRNA and protein in primary cultured human astrocytes. The protein was detected as well in both rat and human brain cryoslices. Together, these findings clearly demonstrate the expression of the TSH receptor gene in the brain in both neuronal cells and astrocytes.

Evidence for cAMP-dependent Platelet Ectoprotein Kinase Activity That Phosphorylates Platelet Glycoprotein IV (CD36)

The dephosphorylating enzyme alkaline phosphatase, by removing phosphate groups from the external platelet membrane proteins, modulates platelet activation (Hatmi, M., Haye, B., Gavaret, J. M., Vargaftig, B. B., and Jacquemin, C. (1991) Br. J. Pharmacol. 104, 554-558). This observation, together with findings reported by others (Ehrlich, Y. H., Davis, T. B., Bock, E., Kornecki, E., and Lenox, R. H. (1986) Nature 320, 67-70; Dusenbery, K. E., Mendiola, J. R., and Skubitz, K. M. (1988) Biochem. Biophys. Res. Commun. 153, 7-13), indicate the existence of ectoprotein kinase activity on the blood platelet surface. In this study, we demonstrate that washed human platelets phosphorylate the synthetic heptapeptide kemptide in a cAMP-dependent mode. The intensity of the phosphorylation was concentration-dependent for kemptide. In addition, incubation of platelets with [γ-32P]ATP resulted in a rapid incorporation of [32P] phosphate into proteins at the outer membrane surface that was sensitive to alkaline phosphatase treatment. When cAMP was added to the medium, major phosphorylation of an 88-kDa ectoprotein occurred. Its isoelectric point determined by isoelectric focusing SDS-polyacrylamide gel electrophoresis was around pH 6.2. Phosphorylations of this 88-kDa polypeptide and of the exogenous kemptide substrate were both prevented by the specific protein kinase A inhibitor peptide. When platelets were preincubated with [32P]inorganic phosphate to label intracellular proteins, the protein phosphorylation pattern was different from that obtained with [γ-32P]ATP, indicating that the latter occurred at the outer surface of the cells. Prostacyclin, which induces the increase of intracellular cAMP levels and, consequently, its liberation into the extracellular medium, increased phosphorylation of both kemptide and platelet 88-kDa polypeptide. The major protein of 88-kDa, which was phosphorylated in the presence of cAMP and external [γ-32P]ATP, was identified by immunoprecipitation to GPIV (CD36), one of thrombospondin and collagen binding sites on platelets. The phosphorylation of CD36 also occurred in platelet-rich plasma, suggesting a physiological role for this ectoenzyme. In the present study, we clearly demonstrate the presence of an ectoprotein kinase A activity at the surface of intact human platelets, and we revealed its principal endogenous substrate as being CD36.

Existence of two pools of prostaglandins during stimulation of the thyroid by TSH

In a previous work [l] we have shown that Kuehl’s hypothesis [2,3], taken over by Burke [4,5], according to which the prostaglandins would represent an obligatory intermediate in the hormonal stimulation of adenyl cyclase, could not be applied to the stimulation of the thyroid by TSH. Our results have been simultaneously confirmed by those of Wolff [6]. Finally, Burke himself was puzzled by the observation [7] that dibutyryl adenosine monophosphate cyclic (DBC) was able to increase the level of prostaglandins in rat or mouse thyroid. We decided to study the relations which could exist between the process of prostaglandin biosynthesis that we have described, where TSH stimulates a phospholipase A2 which preferentially liberates arachidonate from phosphatidyl-inositol, and the effect of DBC. The results that we have obtained show the independence of the two pathways. DBC or CAMP have no effect on phospholipase AZ, but they activate a lipase which liberates arachidonate from neutral lipids. It is therefore possible to distinguish two pools of prostaglandins, one which is pre-CAMP and not compulsory, the other which is post-CAMP and is a consequence of the activation of adenyl-cyclase.

Control by thyrotropin of the production by thyroid cells of an inositol phosphate-glycan

The increased turnover of phosphatidylinositol promoted by thyrotropin (TSH) in pig thyroid tissue does not seem to be caused by an increased production of inositol tris-phosphate. We have explored another possibility, the synthesis of an inositol phosphate-glycan (IP-gly). Our results show that thyroid cells in culture produced this substance from a precursor phosphatidylinositol-glycan (Gly-PI). The obtained IP-gly seemed, by its analytical and biological properties, to be identical, or similar, to the previously described insulin mediator.

Tetradecanoyl phorbol-13-acetate counteracts the responsiveness of cultured thyroid cells to thyrotropin

We have studied the effects of TPA on the metabolism of porcine thyroid cells cultured for 1-4 days in the absence (control cells) and in the presence of 0.1 mU/ml TSH (TSH cells). The phospholipid turnover, evaluated after a 2 hr incorporation of 32P-phosphate into phospholipids, is markedly modified by the presence of TPA (1.5 microM, 2 hr) in the incubation medium of control and TSH treated cells. The total incorporation is 3-4 times higher than untreated cells, the labelling of phosphatidylinositol (PI) is slightly decreased or unchanged whereas that of phosphatidylcholine (PC) is strongly increased. The increased labelling of PI, promoted by an acute TSH treatment is counteracted by TPA. This TPA effect is not observed when prelabelled cells are challenged for 5 min with the drug. A similar effect is observed when 10 nM TPA is added in the culture medium for 20 hr. The addition of TPA does not affect significantly the protein iodine content in 3 or 4 days control cells incubated for 45 min or 2 hr with 125I-iodine, but dramatically decreases the very high iodination rate of TSH cells. We have tested the TPA effect on the cyclic AMP accumulation for the last 5 min of a 2 hr incubation. TPA inhibits by about 50-80% the stimulation evoked by TSH and only by 10% that evoked by forskolin (0.1 mM). These results suggest a possible link between the PC turnover and the adenylate cyclase responsiveness to TSH and the iodination rate.

Chronic and acute effects of forskolin on isolated thyroid cell metabolism

The chronic treatment (2 days or more) of cultured thyroid cells with 1-10 microM forskolin (forskolin-treated cells) sensitizes the response of adenylate cyclase to further acute stimulation by 100 microM forskolin or 10 mU/ml thyrotropin (TSH). This positive regulation, similar to that produced by 0.1 mU/ml TSH (TSH-treated cells), is obtained between 2 and 3 days of culture. The acute response to TSH or forskolin of cells treated for 4 days with forskolin increases with the concentration of forskolin present during the chronic treatment. This result is different from that obtained after a chronic treatment with TSH which induces refractoriness beyond 0.1 mU/ml. These cells are then desensitized to TSH but not to forskolin. When both agonists are mixed together, their acute effect is additive on control, TSH- and forskolin-treated cells. The chronic treatment of cultured thyroid cells with 1-10 microM forskolin produces, just like 0.1 mU/ml TSH, a chronic phospholipid effect characterized by enhanced incorporation of 32Pi into phosphatidylinositol (PI) and phosphatidic acid. The acute challenge of these cells with 100 microM forskolin evokes a reverse phospholipid effect, i.e. a decreased incorporation of 32Pi into PI. The acute stimulation of TSH-treated cells with TSH produces a reverse phospholipid effect whereas the acute stimulation of forskolin-treated cells with TSH gives a normal phospholipid effect as it does on control cells. These results show that the observed effects of TSH on cAMP accumulation and phospholipid turnover are not independent and are regulated in an inverse reciprocal pattern.

Insulin and insulin-like growth factor 1 receptors during postnatal development of rat brain

The binding of insulin and insulin-like growth factor 1 (IGF1) to high-affinity sites in the brain of rats aged 2-37 days was studied. Specific binding of insulin and IGF1 was assessed using tracer concentrations of 125I-insulin or 125I-IGF1. Sites for insulin and IGF1 were distinguished in these conditions as shown by competition experiments. The Kd were 3.6 nM (insulin) and 2.0 nM (IGF1). These values did not change significantly over the age range studied. The numbers of high-affinity binding sites for insulin and IGF1 were similar in adult animals. IGF1 binding was higher than the insulin binding in 2-day-old animals. The binding capacity for both insulin and IGF1 decreased from birth to age 15 and days remained stable thereafter. Tyrosine kinase activity, which is associated with these receptors, was measured using the artificial substrate poly (Glu, Tyr). It decreased over the first 15 days of life and remained stable thereafter. Autophosphorylation of the receptors confirmed this result. This decrease appears to be due to changes in the numbers of the two types of receptors, and is probably a reflection mainly of the variation in the number of IGF1 receptors. Similar results for insulin and IGF1 binding as well as tyrosine kinase activity were obtained with hypothyroid rats.

Activation of S6 kinase activity in astrocytes by insulin, somatomedin C and TPA

Treatment of cultured astrocytes from 2‐day‐old rat cerebral hemispheres with insulin or somatomedin C (IGF1) promoted a rapid activation of a cytosolic protein kinase which phosphorylates ribosomal protein S6. Phosphorylation of substrates currently used for protein kinase assays (histone H2B and phosvitin) was not stimulated. Neither the cyclic AMP‐dependent protein kinase activity nor that of protein kinase C was modified. Treatment of these astrocytes with TPA also promoted a rapid increase in S6 kinase activity in the cytosolic fraction. Simultaneously, protein kinase C disappeared from the cytosol. Neither cyclic AMP‐dependent protein kinase activity nor phosvitin kinase activity was modified. The effects of insulin, IGF1 and TPA were also observed in the presence of cycloheximide. Cycloheximide also potentiated their effects. These data indicate that S6 kinase activity in astrocytes is promoted from a pre‐existing molecule via the tyrosine kinase‐insulin receptor and suggest that protein kinase C is implicated in the process.

Cholinergic control by endogenous prostaglandins of cAMP accumulation under TSH stimulation in the thyroid

Recently, Goldberg et al. [l] have studied the relation of cyclic GMP with regulatory processes in various systems. According to the concept of Yin-Yang, they hypothesized that the intracellular concentration of cyclic AMP and of cyclic GMP are correlated and that these concentrations vary inversely in numerous regulatory events. This hypothesis implies that the system under study is modulated by two antagonistic effecters. Many of the biological effects of thyroid stimulating hormone (TSH) on the thyroid appear to be mediated by an increase of the synthesis of cyclic AMP (CAMP) [2] Qn the contrary cyclic AMP is not implicated in the 32P incorporation into phospholipids evoked either by TSH [3,4] or by acetylcholine. This latter result is supported by the fact that acetylcholine does not increase cyclic AMP concentrations in thyroid slices [SJ . Moreover, cyclic GMP levels are significantly increased by acetylcholine [6] .The present studies demonstrate that acetylcholine antagonizes the increase of cyclic AMP promoted by TSH and that prostaglandins Fa are implicated in this process.