Differential regulation of protein kinase C isoforms in human uterine leiomyoma.

The protein kinase C (PKC) isoenzyme expression pattern in human uterine leiomyoma was compared with that obtained in homologous myometrium distal from the tumor. The six PKC isoforms (PKCalpha, PKCbeta1, PKCbeta2, PKCdelta, PKCepsilon and PKCzeta) evidenced in the myometrium were found to be similarly expressed in leiomyoma. Quantitative immunoblotting revealed that all PKC isoforms were preferentially localized in the particulate fraction. To gain insight into the possible functional consequences of PKC expression patterns, subcellular redistribution in response to the mitogenic peptide endothelin-1 (ET-1) was studied. After stimulation with ET-1, differential redistribution occurred in leiomyoma and myometrium, suggesting a selective role of PKC isoforms in the myometrial growth process.

Contribution of protein kinase C to ET-1-induced proliferation in human myometrial cells.

The role of protein kinase C (PKC) in endothelin-1 (ET-1)-induced proliferation of human myometrial cells was investigated. ET-1 dose dependently stimulated DNA synthesis and the number of cultured myometrial cells. Inhibition of PKC by calphostin C or Ro-31-8220 or downregulation of PKC eliminated the proliferative effects of ET-1. The failure of two protein tyrosine kinase (PTK) inhibitors (tyrphostin 51 and tyrphostin 23) to affect ET-1-induced proliferation supports the hypothesis of noninvolvement of the tyrosine kinase signaling pathway in this process. The expression and distribution of PKC isoforms were examined by Western blot analysis. The five PKC isoforms (PKC-alpha, -beta1, -beta2, -zeta, -epsilon) evidenced in human myometrial tissue were found to be differentially expressed in myometrial cells, with a predominant expression of PKC-alpha and PKC-zeta. Treatment with phorbol 12, 13-dibutyrate (PDBu) resulted in the translocation of all five isoforms to the particulate fraction, whereas ET-1 induced a selective increase in particulate PKC-beta1, PKC-beta2, and PKC-epsilon. Our findings that multiple PKC isoforms are differentially responsive to ET-1 or PDBu suggest that they play distinct roles in the myometrial growth process.

Role of protein kinase C in endothelin-1-induced contraction of human myometrium.

The role of protein kinase C (PKC) in the contraction of human myometrium induced by endothelin-1 was investigated. The PKC inhibitor, calphostin C, reduced the sustained phase of endothelin-1-induced contraction. The expression and subcellular distribution of PKC isoforms were determined in unstimulated myometrium by Western blotting using isoform-specific antisera. At least five PKC isoforms (PKCalpha, PKCbeta1, PKCbeta2, PKCzeta, and trace amounts of PKCepsilon) were detected. Quantitative immunoblotting revealed that all these isoforms were diversely distributed between the cytosolic and particulate fractions. After stimulation with phorbol 12,13-dibutyrate (PDB) and endothelin-1, differential redistribution occurred, suggesting a selective role of these isoforms in the physiological function of the myometrium. Biochemical assay confirmed that PDB as well as endothelin-1 evoked a decrease in cytosolic PKC activity. Taken together, these results suggest that PKC may play a role in endothelin-1-induced contraction of human uterine smooth muscle.

Regulation of protein kinase C in the muscular layer of human placental stem villi vessels.

Protein kinase C (PKC) activity in the muscular layer of stem villi vessels from the human term placenta was studied. Resting state PKC activity was distributed evenly between the cytosol and the particulate fractions. Upon stimulation by three different activators, phorbol 12-myristate 13-acetate, fluoride and endothelin-1, a translocation of PKC activity from the cytosolic to the particulate fraction was observed. The expression and distribution of PKC isoforms were then examined by Western blot analysis using specific antibodies to PKC isoforms. At least four PKC isoforms, PKCalpha, PKCbeta1, PKCbeta2, PKCzeta, and trace amounts of PKCepsilon were detected in both fractions. Their relative responses to the different agonists were examined by quantifying their subcellular redistribution. No significant differential activation of the four mainly expressed PKC isoforms were observed in response to stimulation with any of the stimuli. Moreover, our results show that endothelin-1 induced translocation/activation of PKC in this vascular smooth muscle.

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.

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.

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.

Effects of antimicrotubular agents in cAMP production and in steroidogenic response of isolated rat Leydig cells.

In dispersed rat Leydig cells, colchicine was found to stimulate basal cAMP production and testosterone secretion in a dose and time-dependent manner, but to a lesser extent than LH. However, these drugs are unable to stimulate adenylate cyclase activity in plasma membranes isolated from these cells. The amount of testosterone secreted at 150 min under the influence of colchicine and LH added simultaneously was not different from the amount produced during stimulation by LH alone. It is only after exposure of the cells for 1 hr to colchicine that the accumulation of cAMP in response to LH was inhibited; furthermore, both intracellular and medium testosterone accumulation in response to the hormone were reduced. Similar effects were observed with two other alkaloids, vinblastine and podophyllotoxin. The three drugs also inhibited the stimulation of testosterone secretion by 8-Br-cAMP or choleratoxin. These studies suggest that the state of microtubule polymerization and/or tubulin can influence the process of steroidogenesis in rat Leydig cells.

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.

A comparative study of the action of several lutropin derivatives on rat Leydig cells.

Rat intestinal cells prepared from testes were incubated in the presence of different lutropin derivatives obtained by chemical modification of the amino groups. The cAMP accumulation and the testosterone biosynthesis were determined in the cell homogenates. Binding determinations were carried out by a radioligand receptor assay using tritiated methylated lutropin. The binding activities--relative to native LH--of three different derivatives obtained by reductive alkylation (methylated, ethylated and isopropylated LH) were in good agreement with the relative potencies assessed by their capacity to stimulate cAMP and testosterone production. Guanidinated LH (11-NH2 groups modified) exhibited a binding activity and a relative potency relatively high with regard to cAMP accumulation (as compared with that of native LH). Its steroidogenic potency, however, was very low. When Leydig cells were incubated in the presence of native and guanidinated LH, the testosterone production was similar to that induced by the derivative alone, indicating that the derivative exerted a competitive inhibitory action preventing the stimulation of steroidogenesis by native LH. These results suggest that a guanidinated derivative is able to bind to the LH receptor and the complex so formed is able to be coupled with an adenylate cyclase pool (or cAMP compartment) which is not connected with the steroidogenic pathway.

[Stimulation of rat ovarian adenyl cyclase by methylated luteinizing hormone (LH). The effect of ethylenediaminetetraacetic acid]. / Stimulation de l'adényl-cyclase ovarienne de ratte par l'hormone lutéinisante (LH) méthylée. Influence de l'acide éthylène-diamino-têtra-acétique

A comparative study was made of the stimulation of rat ovary adenyl cyclase by ovine luteinizing hormone (LH) and its methylated drrivative (obtained by reductive alkylation of the lysine residues). The apparent Km (half maximal activity) of native LH and methylated LH was similar but the maximal activity obtained with the methylated derivative was significantly higher (1.2-1.8 times). When EDTA was added to the incubation medium the maximal activity obtained with the methylated LH diminished and became even lower than that obtained with native LH.

Characterization of the mannosyl and fucosyl transferases in the ovine anterior pituitary glands.

Ovine anterior pituitary glands contain mannosyl- and fucosyl-transferases localized in the microsomes and able to incorporate mannose or fucose as such from GDP-mannose or GDP-fucose into endogenous glycoproteins. The requirements and conditions necessary for maximum activity were investigated. The value of the Km is very similar for the two enzyme systems, 3 X 10(-7) M in the case of mannosyl-transferases and 5 X 10(-7) M in the case of fucosyl-transferases.