Activation of phospholipase C by different effectors in rat placental cells.

The present communication documents the accumulation of inositol phosphates in rat placental cells by fluoride as well as by vanadate. These findings suggest the existence of the phosphoinositide pathway and its modulation by a G-protein. A concomitant action of fluoride on phosphoinositide breakdown was also observed. As is often the case in intact cells from different organs, protein kinase C exerts a feedback regulatory control on this signalling system. Gonadotropin-releasing hormone (GnRH) also stimulated the accumulation of inositol phosphates in cultured cells but no effect could be detected in freshly isolated cells. Therefore, the phosphoinositide pathway seems to be involved in the mechanism of action of GnRH in rat placental cells.

Comparison of inositol phosphates accumulation induced by different effectors in rat thyroid slices.

Rat thyroid slices were submitted to different effectors and hormones in order to investigate their action on the phosphatidylinositol metabolism. Fluoride and vanadate induced a clear increase of the inositol phosphates with half maximal stimulation at 7 mM and 8 mM respectively. The inositol bisphosphate (IP2) and inositol trisphosphate (IP3) accumulation induced by vanadate was relatively higher than that observed in the case of fluoride stimulation. Carbachol stimulated also the generation of inositol phosphates with half maximal activation at 2.5 x 10(-6) M. In the same conditions, no significant effect on inositol phosphates production could be detected by the action of TSH or TRH.

[Isolation of protein kinase C isoforms from human full-term placenta]. / Isolement des isoformes de la protéine kinase C à partir de placenta humain à terme.

Protein kinase C isolated from human term placenta was resolved by hydroxyapatite column chromatography into two major fractions corresponding to type II (beta sequence) and type III (alpha sequence) PKC isolated from rat brain. Both subspecies were stimulated by Ca2+ with a different sensitivity. No effect of arachidonic acid on their activity was observed.

Isolation and characterization of protein kinase C from human placenta.

Protein kinase C (Ca2+ and phospholipid-dependent protein kinase) was detected in human placenta and was partially purified using DEAE cellulose chromatography and Ultrogel filtration. Diolein alone did not act on this enzyme but exerted a strong stimulatory action when associated with phosphatidylserine and Ca2+. Similar results were obtained with the phorbol myristate acetate. The kinetic constants for ATP, histone HI or Mg2+ and the apparent Ka for Ca2+ and phosphatidylserine were determined.

Isolation, characterization and Ca2+ and calmodulin regulation of cyclic nucleotide phosphodiesterases from human placentae of different ages.

Two kinds of phosphodiesterases were isolated from human placenta by DEAE chromatography and characterized: one Ca2+ and calmodulin dependent, the other stimulated by Ca2+ but not by calmodulin. Both hydrolyzed cAMP and cGMP. The first one exhibited a higher affinity for cGMP. Half maximal activation by calmodulin was attained at 10(-8)M of calmodulin concentration independently of the hydrolyzed substrate (cGMP or cAMP). This phosphodiesterase appears to be almost homogeneous by molecular sieve chromatography on Ultragel AcA 34. The second phosphodiesterase exhibited similar affinities for cAMP and cGMP and could be resolved into three active isoforms with different molecular weight on Ultrogel AcA 34. Only minor differences were observed in the characteristics of these enzymes when the phosphodiesterases were prepared from placentae of 7-8 weeks of pregnancy or from normal term placenta.

Growth hormones. II. Structure-function relationships.

Localization of the somatotropic activity of growth hormones from several species and from different organs was attempted using different approaches. Sequences were compared in order to detect one or several regions with a common homology. The technique of peptide recombinants as well as chemical changes affecting some amino acids was applied to these hormones; the biological function in vivo of growth or binding to somatotropic receptors was then estimated. The few data available on biosynthetic molecules and secondary structures of natural growth hormones are reported. This study indicates the somatotropic function of particular sites.

Adenylate cyclase stimulation and luteinizing hormone-receptor interaction in plasma membranes from rat testicular interstitial cells in relation to the chemical structure of the hormone. Role of Mg2+.

To understand more closely the structural requirements of the LH molecule necessary to stimulate adenylate cyclase, we studied the modulation of this enzyme in partially purified plasma membranes prepared from isolated interstitial cells of rat testis submitted to oLH and to some oLH derivatives and natural analogues. The role of Mg2+ was also investigated in relation to the structural modifications of oLH. Some new facts appeared in this study: 1. Methyl oLH, which exhibited the same ability as native oLH to stimulate cAMP accumulation and steroidogenesis in isolated cells, cannot induce the same level of maximal stimulation of adenylate cyclase as native oLH in plasma membranes. This phenomenon is related to the Mg2+ concentration, and the differences between maximal activation induced by methyl oLH and oLH were more apparent at a free Mg2+ concentration of 3.3 mmol/l than at lower concentrations. The maximal activity (in terms of native oLH) of other alkyl derivatives, such as ethyl or isopropyl oLH, on the contrary, was similar in isolated plasma membranes and in intact cells suggesting that the differential behaviour of the membranes specifically concerns the methyl derivative. 2. Guanidyl oLH and guanidyl porcine LH, which were able to induce cAMP accumulation in intact cells, did not exhibit any stimulating activity in plasma membranes. 3. Among the natural analogues, hCG and pLH are distinguished by a lower maximal activity (by comparison with oLH) particularly at high Mg2+ concentration. This work shows that changes in the LH structure have an impact not only on the parameters of the adenylate cyclase complex but also on the transduction of the hormone signal and its modulation by Mg2+.

Activation of a cyclic nucleotide phosphodiesterase and of a protein kinase by chemically modified calmodulin.

1. Several calmodulin derivatives prepared by chemical modification of lysine residues were tested using bovine heart cyclic nucleotide phosphodiesterase and wheat germ calmodulin-dependent protein kinase. 2. The effect of chemical modification on the activation capacity of calmodulin for the two studied enzymes was different. 3. This was particularly noticeable in the case of alkylated derivatives which exhibited a higher affinity than native calmodulin towards phosphodiesterase but a lower affinity towards protein kinase. 4. The efficiency of these derivatives (maximal activation) was higher than that of native calmodulin in relation with the protein kinase.

Action of Mn2+ and vanadium compounds on hormone and forskolin induced stimulation of juvenile rat ovarian adenylate cyclase.

The effect of forskolin on the hormonal (LH, FSH) activation and on the stimulation provided by other effectors (Gpp(NH)p,NaF) of the juvenile rat ovarian adenylate cyclase was investigated. Forskolin exhibited a synergistic action with LH, FSH and Gpp(NH)p but not with NaF. Addition of Ca2+ was inhibitory over a concentration range from 10(-5) to 10(-2) M whereas EGTA enhanced the response at 5.10(-5) M and inhibited it at higher concentration. The cAMP production was increased by addition of Mn2+ at low concentration (up to 5 mM) but markedly decreased at higher concentration (30 mM). FSH induced cAMP production was completely abolished at 30 mM Mn2+. The effect of vanadyl ion was very similar to that of Mn2+ Vanadate anion on the contrary was without effect on FSH stimulation.

A comparative study of adenylate cyclase activity and progesterone synthesis in ovine corpora lutea stimulated by different chemical derivatives or natural analogs of ovine LH.

The adenylate cyclase activation by ovine native LH, natural analogs (porcine LH, hCG) and chemical derivatives of LH (methylated, ethylated, isopropylated, guanidinated) was studied in purified plasma membranes of ovine corpora lutea, including the regulatory effects of guanyl 5'-yl imidophosphate (Gpp(NH)p) and Mg2+. The Ka app. for native LH (about 15 nM) was independent of Gpp(NH)p and Mg2+. Similar maximal activation of the enzyme was obtained by using ovine LH or natural analogs, but differences were remarked concerning the Ka app. values of these hormones. Porcine LH was equipotent with ovine LH; on the contrary, hCG exhibited a lower Ka app. value (3 nM). All chemical derivatives (Me-LH, Et-LH, Iso-LH and Gu-LH) exhibited Ka app. higher than native (about 2- to 4-fold), but similar maximal activation. No modification was observed in the regulatory effects of Gpp(NH)p and Mg2+ on the adenylate cyclase activation as a consequence of structural modifications of the hormone. A comparison of the steroidogenic activity on intact luteal cells and the adenylate cyclase activation ability on purified plasma membranes of the derivatives mentioned above evidenced some interesting discrepancies. The drop in adenylate cyclase activation potency of Me-LH was not reflected in its steroidogenic activity (Me-LH was equipotent with native LH); on the contrary, the capacity of Gu-LH to stimulate adenylate cyclase was not so much decreased as was its steroidogenic potency which was almost abolished.

Modulation of juvenile rat ovarian adenylate cyclase activity by calcium and calmodulin.

The involvement of calcium and calmodulin in the regulation of juvenile rat ovarian adenylate cyclase activity was investigated. Both basal and LH-stimulated cAMP production were inhibited by adding Ca2+ to the incubation medium at concentrations higher than 10(-5) M. Conversely, up to 10(-3) M concentrations of EGTA increased cAMP production (basal, stimulated by LH, FSH, NaF and Gpp(NH)p); higher concentrations of the chelator led to an inhibition of cAMP formation. However, when the homogenates were previously deprived of Ca2+ by treatment with buffer containing EGTA, a biphasic response to LH and Gpp(NH)p stimulation was obtained in the presence of increasing concentrations of added Ca2+:cAMP production was first enhanced at low concentrations and then inhibited at higher concentrations. These observations suggest that the optimal concentration of Ca2+ needed to obtained maximal stimulation of the enzyme was much lower than the Ca2+ content in the homogenates and that a minimal concentration of Ca2+ was required to activate it. In the presence of micromolar concentrations of trifluoperazine and pimozide, two potent inactivators of calmodulin, LH-stimulated cAMP production was markedly decreased. Reactivation was obtained by adding exogenous calmodulin to the assay medium. The addition of Ca2+-free exogenous calmodulin (10(-6) M) caused a specific and significant enhancement of cAMP accumulation induced by an optimal dose of LH. These results suggest that calcium ions regulated the adenylate cyclase activity in the rat ovaries and had a dual effect that was first stimulatory at low concentration and mediated by calmodulin and then inhibitory at high (non-physiological) concentration.

Relationships between structure and function of lactogenic hormones.

Lactogenic activity of several hormone derivatives obtained by chemical modifications of lysine residues was studied by radioreceptor assay. The relationships between structure and binding to lactogenic receptors are discussed taking into account lysine residue positions liable to be involved in the location of lactogenic function.

The liver is the main organ to clear plasma and tissue kallikreins from rat plasma, in vivo.

We report observations regarding the in vivo distribution of labelled kallikreins in plasma, liver and some other organs, twenty minutes following their intravenous injection in the rat. The kallikreins used were: tritiated homogeneous human plasma (HuPK) and horse urinary (HoUK) as well as highly purified iodinated rat plasma kallikrein (RPK). The main findings were: the liver cleared 15% of HuPK, 38% of RPK and 69% HoUK; with both types (plasma and tissue) of native kallikreins the liver was the main clearing organ.

[Steroidogenic action of 2 analogs of ovine LH on cells isolated from ovine corpus luteum]. / Activité stéroïdogénique de deux analogues de la LH ovine sur cellules isolées de corps jaunes ovins.

Lysine residues appear to play an important role in the biological activity of luteinizing hormone or lutropin (LH). Some derivatives obtained by chemical modification such as N-methylated LH exhibit the same hormonal activity than LH in the different steps of the mechanism of steroidogenesis. Some others, on the contrary, preserve the hormonal activity only at some steps. In this work was investigated the action of ovine LH and some derivatives on isolated cells prepared from ovine corpora lutea. Guanidinated LH (which is able to bind to LH receptors in Leydig cells but whose steroidogenic potency is very low) exhibits no binding or steroidogenic activity in the female (sheep or rat). As a consequence guanidyl LH can act as an inhibitor of LH action in the male (Leydig cells).

Involvement of lysine residues in the binding of ovine prolactin and human growth hormone to lactogenic receptors.

The lactogenic activity (L.A.) of oPRL and hGH derivatives obtained by chemical modifications of lysine residues was studied by radioreceptor assay. Control treatment with borohydride had a slight effect on the L.A. of hGH but drastically reduced the oPRL activity; this latter was preserved in the presence of iodoacetamide. Methylation, ethylation, guanidination and acetimidination affected the L.A. of both hormones as a function of the degree of modification. The structure-binding relationships to the lactogenic receptors are discussed, suggesting that the lysine or arginine residues in homologous positions 42, 51, 73, 128, 146 of oPRL and 47, 50, 73, 128, 147 of hGH might be particularly involved.

Involvement of lysine residues in the binding of hGH and bGH to somatotropic receptors.

The biological activities of human (hGH) and bovine (bGH) growth hormone derivatives obtained by chemical modification of the lysine residues were studied by radioreceptor assays using rabbit liver homogenates for somatotropic activity (SA). Control treatment with BH4 had a very slight effect on the SA, whereas the methylation and ethylation drastically reduced the activity of both hormones. Guanidination of these hormones and even acetimidination at a lower rate are accompanied by a considerable loss of biological activity. These results show the involvement of lysine residues in the interaction of hGH and bGH with somatotropic receptors. The structure-function relationship of these molecules is discussed, suggesting that the lysine or arginine residues in positions 41, 64, 70 and 115 might be particularly implicated.

Involvement of lysine residues in the binding of ovine chorionic somatomammotropin to lactogenic and somatotropic receptors.

The biological activities of several ovine chorionic somatomammotropin (oCS) derivatives obtained by chemical modification of the lysine residues were studied by radioreceptor assays using rabbit mammary homogenates (lactogenic activity, L.A.) and liver homogenates (somatotropic activity, S.A.). Even if the control treatment with BH-4 markedly decreased the L.A., it was clear that methylation mainly affected the S.A. and that ethylation reduced both activities. Guanidination inactivated almost completely both activities and acetimidination at a very low degree (3 of 14 lysines) led to less than 50% of both activities. These results show the involvement of lysine residues in the interaction of oCS with lactogenic and somatotropic receptors.

Action of nitroguanidinated luteinizing hormone on different rat gonadal tissues.

The biological activities of nitroguanidinated derivatives prepared from ovine or porcine luteinizing hormone were investigated using rat Leydig cells and pseudopregnant rat ovaries. In these tissues nitroguanidyl ovine luteinizing hormone (NGoLH) or nitroguanidyl porcine luteinizing hormone (NGpLH) were unable to stimulate adenylate cyclase or steroidogenesis but were able to inhibit the binding of ovine or porcine native LH to their specific receptors. When added to incubations of isolated Leydig cells or pseudopregnant ovary slices, NGoLH as well as NGpLH inhibited the stimulating action of native LH on adenylate cyclase or steroidogenesis. However, these derivatives had no inhibiting action on the stimulation of adenylate cyclase and steroidogenesis induced in the Leydig cells by choleratoxin or on the stimulation of testosterone production induced by 8-bromo-cyclic AMP. Since NGpLH (which does not contain lysine residues or free alpha-amino groups in the beta-subunit) exhibits the same antagonist action as NGoLH, we conclude that the nitroguanidination of the alpha-subunit is sufficient to endow the derivative with antihormone properties.

Action of some luteinizing hormone derivatives in ovaries from pseudopregnant rats: dissimilarities between their activities on this organ and on Leydig cells.

Biological activities of several derivatives of ovine LH obtained by chemical modification of the amino groups were investigated using ovaries from pseudopregnant rats. Binding-inhibition activities and steroidogenic potencies of ethylated, isopropylated and guanidinated LH were in good agreement, whereas adenylate cyclase activities were relatively greater. When compared with previous results on binding-inhibition activities and steroidogenic potencies using isolated rat Leydig cells, the ovaries from pseudopregnant rats appeared to be more discriminating. Ethylated and isopropylated derivatives exhibited lower binding-inhibition activities and steroidogenic potencies in female gonads. This difference was particularly evident in the case of guanidinated LH which exhibited a very low binding-inhibition activity and consequently was unable to act as an inhibitor of the action of LH on the ovaries. Guanidinated porcine LH (in which all the lysine residues of the alpha-subunit were transformed into homoarginine, without modification of the beta-subunit which does not contain lysine) showed similar biological activities to guanidinated ovine LH in the isolated Leydig cells as well as in pseudopregnant ovaries. It can, consequently, act as an inhibitor of LH action on Leydig cells but not on the ovary of the pseudopregnant rat. Thus, the inhibitory properties of this derivative can be ascribed to the modification introduced in the alpha-subunit.

LH receptors in ovine corpora lutea in relation to various physiological states and effects PGF-2 alpha on LH-induced steroidogenesis in vitro.

The LH binding properties (determined using tritiated methylated LH) and the in-vitro steroidogenic activity of CL from ewes in the oestrous cycle or early pregnancy (Day 18) were compared. No significant alteration in the Kd values was observed. However, the number of sites was maximal at Day 10 of the cycle and in early pregnant animals which had not been pregnant for at least 3 months (dry ewes). Non-lactating or suckling ewes had half the numbers of binding sites. The increase of the number of receptor sites was accompanied by a steroidogenic response at lower LH concentration. During incubation or superfusion for 5 h, a refractoriness to LH stimulation appeared after 1 h with high LH concentrations and after 3 h with low concentrations. The opposite effect of the addition of indomethacin or PGF-2 alpha suggests the intervention of PGs in this phenomenon.