GnRH receptor signaling: cross-talk of Ca2+ and protein kinase C.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
Z Naor
Publications and source records attributed to Z Naor.
Arachidonic acid and lipoxygenase products stimulate protein kinase C beta mRNA levels in pituitary alpha T3-1 cell line: role in gonadotropin-releasing hormone action.
The cross-talk of arachidonic acid (AA) and its lipoxygenase products with protein kinase C beta (PKC beta) mRNA levels during the action of gonadotropin-releasing hormone (GnRH) was investigated in the pituitary alpha T3-1 cell line. The addition of AA or its 5-lipoxygenase products 5-hydroxyeicosatetraenoic acid (5-HETE) or leukotriene C4 (LTC4) for 30 or 60 min stimulated PCK beta, but not PKC alpha mRNA levels (3-5-fold); PCK gamma is not expressed by the cells. Other HETEs or leukotrienes tested showed no significant effect. The range of effective concentration for LTC4 and 5-HETE (around 10(-10) M) is the range found in GnRH-stimulated pituitary cells. Although PKC beta mRNA levels were preferentially elevated by LTC4 and 5-HETE at early time points, PKC alpha mRNA levels were elevated at 6-12 h of incubation when PKC beta mRNA levels returned to basal levels. The addition of the phospholipase A2 inhibitor 4-bromophenacyl bromide or the selective 5-lipoxygenase inhibitor L-656,224 abolished [D-Trp6]GnRH (GnRH-A) elevation of PKC beta mRNA levels, whereas PKC alpha mRNA levels were not increased by this neurohormone. The cyclo-oxygenase inhibitor indomethacin elevated basal PKC beta mRNA levels and potentiated the GnRH-A response. Cross-talk exists between AA and some of its lipoxygenase products and PKC beta gene expression during cell signalling. AA, 5-HETE and LTC4 participate in the rapid stimulation of PKC beta mRNA levels by GnRH.
Acetophenones↗
Bilateral comparison of occipital lobe sulci: a sulcus identifying algorithm.
A procedure for automatic identification of sulci of cerebral hemispheres in magnetic resonance images is presented. The algorithm departs from information of the spatial location of a given feature (e.g., sulcal contour) in one hemisphere and automatically defines its course in the other hemisphere. The automatic tracing of sulci was successfully performed in the mesial aspect of both hemispheres in 18 normal individuals. The sulcal location in the target hemisphere was first approximated by assuming an affine transformation between hemispheres, and then refined by local edge analysis. The method produced reliable results in comparing the intersulcal areas (the cuneus), sulcal length, their complexity, and the angle between the pariteooccipital and retrocalcarine fissures.
Adolescent↗
Phosphatidylserine directs differential phosphorylation of actin and glyceraldehyde-3-phosphate dehydrogenase by protein kinase C: possible implications for regulation of actin polymerization.
The phospholipid-dependent protein kinase C is implicated in the regulation of cellular motility and energy metabolism. Phosphatidylserine, a main cofactor of protein kinase C, is involved in the regulation of glyceraldhehyde-3-phosphate dehydrogenase, which as actin, was shown to be phosphorylated by purified protein kinase C. Here, we study the effect of phosphatidylserine on the enzyme-substrate interaction of protein kinase C with glyceraldhehyde-3-phosphate dehydrogenase and actin. The stoichiometry of glyceraldhehyde-3-phosphate dehydrogenase phosphorylation is not affected by varying the level of phosphatidylserine. However, actin phosphorylation is dependent on phosphatidylserine level, peaking at high phosphatidylserine concentration. Moreover, if actin and glyceraldhehyde-3-phosphate dehydrogenase are cophosphorylated at high phosphatidylserine concentration, actin phosphorylation is favored, despite lower affinity for protein kinase C. Hence, phosphatidylserine directs differential phosphorylation of these key proteins of glycolysis and cellular motility and might be capable of recruiting protein kinase C for preferential actin phosphorylation. The sedimentation of phosphorylated actin is increased 3.8 fold and total actin 1.7 fold, suggesting that phosphorylation promotes actin polymerization.
Actins↗
Rhythmicity of luteinizing hormone secretion expressed in vitro.
In the present study we explored the possibility that the pituitary functions as an autonomous clock and is capable of generating rhythms of luteinizing hormone (LH) release independently of hypothalamic control. Pituitaries from estrous or diestrous day 1 female mice were perifused separately with Medium-199. Effluent samples were collected at 10-min intervals and assayed for LH levels. Fourier analysis and curve-fit analysis served to elucidate the presence of prominent periods whose significance was then determined by best-fit cosinor. The latter method was used to determine additional parameters for the significant rhythm. All perifused pituitaries exhibited LH release patterns that were composed of significantly long ultradian rhythms (approximately 16 and 8 h, p < 0.001). Continuous stimulation with gonadotropin-releasing hormone (GnRH) or estradiol did not alter the periods of the observed rhythms but affected other rhythm parameters. Gonadotropin-releasing hormone increased the mesor of the rhythm and estradiol increased the amplitude. The results indicate that pituitary gonadotropes are capable of producing rhythms of LH release for a long duration in vitro, in the absence of hypothalamic control. Both GnRH and estradiol affect different rhythm parameters but do not change the periods of these rhythms.
Animals↗
Interaction of purified protein kinase C with key proteins of energy metabolism and cellular motility.
The phospholipid dependent protein kinase C is involved in regulation of cellular motility and energy metabolism. To study a possible direct interaction of protein kinase C with cellular motility and energy metabolism, we used purified rat brain protein kinase C to phosphorylate key proteins of these systems. Protein kinase C phosphorylates with comparable stoichiometry the G- but not the F- form of muscle and brain actin, the key protein of cellular motility. Glyceraldehyde-3-phosphate dehydrogenase, creatine kinase and glutamine synthase, key enzymes of energy metabolism, are also phosphorylated at comparable stoichiometry. The data suggest that protein kinase C might be directly involved in the regulation of cellular motility and energy metabolism.
Actins↗
Mechanism of action of gonadotropin-releasing hormone upon gonadotropin alpha-subunit mRNA levels in the alpha T3-1 cell line: role of Ca2+ and protein kinase C.
Addition of [D-Trp6]gonadotropin-releasing hormone (GnRHa) to alpha T3-1 cells induced a very rapid response upon gonadotropin alpha-subunit mRNA which was detected after 30-60 min and was abolished by pretreatment with actinomycin D. A similar response was obtained with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA), or the Ca2+ ionophore, ionomycin. GnRHa (10 nM) also stimulated a secondary rise in alpha-subunit mRNA levels between 12 and 24 h of incubation. No additivity was obtained (at 60 min) upon the combined addition of GnRHa and PMA, GnRHa and ionomycin, or PMA and ionomycin. The effect of GnRHa upon alpha-subunit mRNA was blocked by the PKC inhibitors staurosporine or GF 109203X. Down-regulation of endogenous PKC activity resulted in inhibition of the stimulatory effect of gonadotropin-releasing hormone (GnRH), PMA and ionomycin. Removal of extra-cellular Ca2+ abolished the effect of GnRHa and PMA upon alpha-subunit mRNA levels. Interestingly PMA and ionomycin had no effect on alpha-subunit mRNA levels at 24 h of incubation; however, the combined addition of the drugs mimicked the late phase of GnRHa (10 nM) action. The data provide evidence that PKC and Ca2+ are involved in mediating the early and the late responses of GnRHa upon alpha-subunit mRNA elevation and that differential cross-talk exists between the messengers.
Calcium↗
Involvement of protein phosphatases in gonadotropin releasing hormone regulated gonadotropin secretion.
The role of persistent protein phosphorylation upon gonadotropin releasing hormone (GnRH) stimulated luteinizing hormone (LH) release was investigated by the use of the selective inhibitors of protein phosphatase type 1 (PP1) and 2A (PP2A), okadaic acid (OA) and calyculin A. Pre-incubation of cultured rat pituitary cells with OA (24 h) or calyculin A (30 min) resulted in inhibition of GnRH-stimulated LH release with significant inhibition being detected at 10 nM and 30 nM for OA and calyculin A, respectively. The inactive OA analog norokadone and the protein tyrosine phosphatase inhibitor vanadyl hydroperoxide had no significant effect on GnRH-induced LH release. The stimulatory effects of the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol 13-acetate (TPA, 50 ng/ml) or the Ca2+ ionophore, ionomycin (1 micron), upon LH release were also abolished by pretreatment with OA (10-20 nM) or calyculin A (30 nM). Stimulation of LH release by high K+ (28 mM) or residual LH release stimulated by GnRH in Ca(2+)-free medium were also blocked by OA. These observations indicate that protein dephosphorylation is involved positively in GnRH-stimulated LH release. The site of action of the protein phosphatases PP1 and PP2A is most likely downstream to Ca2+ elevation and PKC activation by GnRH.
Animals↗
Hypothalamic and thyroidal regulation of growth hormone in tilapia.
A radioimmunoassay (RIA) for recombinant tilapia growth hormone (GH) was established and validated. The ability of various hypothalamic factors to regulate GH secretion in the tilapia hybrid (Oreochromis niloticus x Oreochromis aureus) was studied. Somatostatin1-14 (SRIF1-14; 10-100 micrograms/kg) was found to reduce circulating GH levels in a dose-dependent manner. SRIF1-14 (0.1-1000 nM) inhibited GH release from perifused pituitary fragments (ED50 0.83 nM). Human growth hormone-releasing hormone fragment 1-29 (hGHRH1-29; 100 micrograms/kg) doubled circulating GH levels and modestly stimulated GH secretion in vitro. Carp growth hormone-releasing hormone (cGHRH) stimulated GH secretion in vitro to a similar degree at the same dose (1 microM). Injection of salmon gonadotropin-releasing hormone (sGnRH) superactive analog (10-100 micrograms/kg) increased plasma GH levels sixfold. sGnRH also stimulated GH release in vitro (ED50 142.56 nM). Dopamine (0.1-10 microM) and the D1 DA receptor agonist SKF 38393 increased GH secretion from perifused pituitary fragments dose-relatedly. Thyrotropin-releasing hormone (TRH) had no effect on GH secretion from perifused pituitary fragments, but increased plasma GH levels, as did bovine thyroid stimulating hormone (bTSH). The increased plasma GH in the bTSH-treated fish coincided with a dramatic increase in T4; however, TRH increased GH without changing T4 levels. T3 increased the synthesis of GH by isolated pituitaries (incorporation of [3H]leucine). SRIF1-14 seems to be a most potent hypothalamic regulator of GH secretion in tilapia; sGnRH and DA both increased GH secretion, although sGnRH elicited considerably greater responses at lower doses. Two forms of GHRH increased GH levels, although the unavailability of the homologous peptide prevented an accurate evaluation of its importance in regulating GH secretion. The thyroid axis (TRH, TSH, and T3) stimulates both synthesis and release of GH, although TRH did not appear to have a direct effect on the level of the pituitary.
Animals↗
Signal transduction of the gonadotropin releasing hormone (GnRH) receptor: cross-talk of calcium, protein kinase C (PKC), and arachidonic acid.
1. The decapeptide neurohormone gonadotropin releasing hormone (GnRH) is the first key hormone of the reproductive system. Produced in the hypothalamus, GnRH is released in a pulsatile manner into the hypophysial portal system to reach the anterior pituitary and stimulates the release and synthesis of the gonadotropin hormones LH and FSH. GnRH, a Ca2+ mobilizing ligand, binds to its respective binding protein, which is a member of the seven transmembrane domain receptor family and activates a G-protein (Gq). 2. The alpha subunit of Gq triggers enhanced phosphoinositide turnover and the elevation of multiple second messengers required for gonadotropin release and biosynthesis. 3. The messenger molecules IP3, diacylglycerol, Ca2+, protein kinase C, arachidonic acid and leukotriene C4 cross-talk in a complex networks of signaling, culminating in gonadotropin release and gene expression.
Animals↗
Atrial natriuretic peptide induces acrosomal exocytosis in bovine spermatozoa.
The induction of acrosomal exocytosis in capacitated bull spermatozoa by atrial natriuretic peptide (ANP) was studied in vitro. ANP markedly stimulated acrosomal exocytosis in a calcium-dependent manner. Typically, ANP exerts its action via activation of the ANP receptor (ANPR-A), a particulate guanylyl cyclase-linked receptor, and subsequent formation of guanosine 3',5'-cyclic monophosphate (cGMP). We found that the ANP-induced acrosome reaction was inhibited by the competitive ANPR-A receptor antagonist-anantin, indicating a receptor-mediated effect. We could mimic the effect of ANP on the acrosome reaction by using 8-bromo-cGMP, suggesting that cGMP may serve as a signal transducer mediating the acrosome reaction. Indeed, the ANP-induced acrosome reaction was associated with elevation of cGMP levels. cGMP can also be formed by activation of the soluble form of guanylyl cyclase. Sodium nitroprusside (SNP) stimulated cGMP accumulation and acrosome reaction of capacitated spermatozoa. Thus ANP and the nitric oxide-releasing compound SNP, via activation of guanylyl cyclase (the former activating the particulate and the latter activating the soluble form of the enzyme), may play a significant role in the induction of the acrosome reaction.
Acrosome↗
Ultrastructural localization of protein kinase C in human sperm.
We localized protein kinase C (PKC) in human sperm cells at the ultrastructural level by the immunogold technique. The sperm head PKC was localized in the acrosome, equatorial segment, and post-acrosomal region. In the flagellum, PKC was associated with the segmented column of the neck and was distributed along the mid, principal, and end pieces. Immunoreactive sites were observed in patches along the axoneme and outer dense fibers and were evenly distributed between these regions. Pre-absorption of the antibody used with rat brain PKC (alpha and beta) eliminated gold labeling of the sperm head but only reduced labeling of the sperm tail. The co-localization of PKC with various cytoskeletal and other structural elements suggests that the proteins involved are potential substrates for sperm PKC subspecies. The localization of PKC in distinct structures of the human sperm (head, neck, and tail) strongly suggests a role for this enzyme in various aspects of sperm physiology.
Adult↗
Activation of protein kinase C beta gene expression by gonadotropin-releasing hormone in alpha T3-1 cell line. Role of Ca2+ and autoregulation by protein kinase C.
The gonadotroph-derived alpha T3-1 cell line was used to investigate the effect of gonadotropin-releasing hormone (GnRH) upon conventional protein kinase C sub-types (cPKCs) gene expression. Addition of the stable analog [D-Trp6]GnRH (GnRH-A, 0.1 nM) resulted in a rapid increase (30 min) of the steady state levels of PKC beta, but not PKC alpha, mRNA levels, while PKC gamma is not expressed in the cells. The rapid stimulatory effect of GnRH-A was blocked by pretreatment with actinomycin D or with the GnRH antagonist (D-pGlu1, pC1Phe2,D-Trp3,6)GnRH and was not mimicked by thyrotropin-releasing hormone. Addition of the PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted also in a rapid (30 min) and selective increase in PKC beta, but not PKC alpha, mRNA levels. In contrast, the calcium ionophore, ionomycin, increased rapidly (30 min) both PKC alpha and PKC beta mRNA levels, and its stimulatory effect on PKC beta was not additive with that of TPA. The rapid stimulatory effect of GnRH-A was blocked by the PKC inhibitor bisindolylmaleimide (GF 109203X) or by down-regulation of endogenous PKC. Similarly, the rapid effect of GnRH-A was abolished by the intracellular Ca2+ chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) or by removal of extracellular Ca2+. Stimulation of PKC beta mRNA levels by ionomycin was only reduced by GF 109203X and was not affected by down-regulation of PKC. In contrast the effect of TPA on PKC beta mRNA levels was reduced by BAPTA and abolished by removal of Ca2+. We conclude that Ca2+ and PKC act sequentially during GnRH-A-induced PKC beta gene expression and that PKC beta gene expression induced by GnRH-A is autoregulated by PKC.
Calcium↗
Arachidonic acid and lipoxygenase products stimulate gonadotropin alpha-subunit mRNA levels in pituitary alpha T3-1 cell line: role in gonadotropin releasing hormone action.
The role of arachidonic acid (AA) and its lipoxygenase metabolites in gonadotropin releasing hormone (GnRH) induced alpha-subunit gene expression was investigated in the transformed gonadotroph cell line alpha T3-1. The stable analog [D-Trp6]GnRH (GnRHa) stimulated [3H]AA release from prelabeled cells after a lag of 1-2 min. Addition of AA stimulated alpha-subunit mRNA levels in a dose-dependent manner, a significant effect being detected at 5 microM AA. Among various lipoxygenase metabolites of AA, only the 5-lipoxygenase products 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene C4 (LTC4) stimulated alpha-subunit mRNA levels. However, while 5-HETE and LTC4 (0.1 nM each) were active already after 30 min of incubation, similar to GnRHa, AA (20 microM) stimulated alpha-mRNA levels after 1 h of incubation. Addition of the phospholipase A2 inhibitor 4-bromophenacyl bromide (BPB) or the selective 5-lipoxygenase inhibitor L-656,224 inhibited GnRHa elevation of alpha-subunit mRNA by 65%, while the cyclooxygenase inhibitor indomethacin had no effect. Addition of AA (20 microM) or LTC4 (0.1 nM) to normal cultured rat pituitary cells mimicked the rapid (30 min) stimulatory effect of GnRH (1 nM) upon alpha-subunit, LH beta, and FSH beta mRNA levels, while 5-HETE (0.1 nM) stimulated only FSH beta mRNA levels at this time point. Thus AA and selected 5-lipoxygenase products, in particular LTC4, participate in GnRHa-induced alpha-subunit mRNA elevation.
Animals↗
Regulation of gonadotropin mRNA levels in cultured rat pituitary cells by gonadotropin-releasing hormone (GnRH): role for Ca2+ and protein kinase C.
Incubation of cultured rat pituitary cells with gonadotropin-releasing hormone (GnRH, 1 nM) resulted in a rapid elevation of gonadotropin subunit steady-state mRNA levels(alpha, 2.2-fold, LH beta, 2.1-fold, and FSH beta 2.2-fold increases at 30 min). Addition of actinomycin D abolished the stimulatory effect of GnRH upon alpha and LH beta and reduced the effect upon FSH beta mRNA levels. The effect of GnRH is biphasic, where the early phase is being observed at 30-60 min, while the late phase is noticed between 12-24 h. A significant decrease in FSH beta mRNA levels was found after 6 h of incubation when using a stable GnRH analog. The unique profile of the time response enabled us to attempt to dissect the signal transduction cascade involved in the neurohormone action. Addition of the protein kinase C (PKC) activator, 12-O-tetradecanoylphorbol 13-acetate (TPA), or the Ca2+ ionophore, ionomycin, mimicked the profile of GnRH-induced alpha and LH beta mRNA elevation. The two phases of FSH beta mRNA elevation induced by GnRH could be mimicked by TPA, while the decrease at 6 h was mimicked by ionomycin. The rapid stimulatory effect of GnRH on gonadotropin subunit mRNA levels was abolished by the PKC inhibitors, staurosporine and GF 109203X. Similarly, the rapid stimulatory effect of GnRH on alpha and LH beta, but not FSH beta, was abolished in Ca(2+)-free medium. While additivity in LH release is obtained upon the combined addition of TPA and ionomycin for 30 min of incubation, LH beta and FSH beta gene expression is inhibited.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Developmental expression of protein kinase C subspecies in rat brain-pituitary axis.
We have examined the neonatal developmental expression of protein kinase C subspecies (PKCs) in rat brain, pituitary glands and cells by enzymatic activity assays, immunohistochemistry and Western blot analysis with type-specific antibodies. A very large increase (455%) was noticed in brain PKC activity during the first week of life with the particulate fraction (22% of total enzyme activity on day 1) increasing dramatically (900%) during the first week to 50% of enzyme activity. In contrast, the pituitary gland showed high activity on day 1 that decreased progressively to reach the lowest levels at 1 year of age. Paradoxically, the number of pituitary cells immunolabeled for PKC increases as a function of age. Western blot analysis showed only small changes in PKC alpha, PKC beta and PKC epsilon when brains from 6-day-old and 3-month-old female rats were compared, whereas PKC tau and PKC delta increased markedly during this period. On the other hand, brain PKC zeta decreased between 6 days and 3 months of age. Western blot analysis showed no major changes in pituitary PKC alpha, PKC beta and PKC zeta when 6-day-old and 3-month-old female rats were compared, while PKC tau was not detected. The major band of pituitary PKC delta (76 kDa) decreased markedly between 6 days and 3 months of age whereas the minor band (68 kDa) did not change.(ABSTRACT TRUNCATED AT 250 WORDS)
Aging↗
Dissociation between release and gene expression of gonadotropin alpha-subunit in gonadotropin-releasing hormone-stimulated alpha T3-1 cell line.
The alpha T3-1 cell line which was derived by targeted tumorigenesis in transgenic mice [Windle et al. (1990) Mol. Endocrinol. 4, 597-603] possesses high-affinity binding sites for GnRH analogs coupled to enhanced phosphoinositide turnover and phospholipase D activity. Incubation of alpha T3-1 cells with [D-Trp6]-GnRH analog (GnRH-A) resulted in a rapid increase in gonadotropin alpha-subunit mRNA levels which was detected already at 30 min of incubation (0.1 nM GnRH-A, 3-fold, p < 0.01). The effect diminished with time to reach basal levels at about 12 h of incubation, with a secondary rise in alpha mRNA levels between 12 and 24 h of incubation. Addition of the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA, 100 ng/mL) or the Ca2+ ionophore ionomycin (1 microM) to alpha T3-1 cells also resulted in a rapid increase in alpha-subunit mRNA levels. Surprisingly, GnRH-induced alpha-subunit release was detected only after a lag of 4 h of incubation. Thus, dissociation between exocytosis and gene expression can be demonstrated in GnRH-stimulated alpha T3-1 cell line.
Animals↗
Gonadotropin releasing hormone activates the lipoxygenase pathway in cultured pituitary cells: role in gonadotropin secretion and evidence for a novel autocrine/paracrine loop.
The formation and role of arachidonic acid (AA) and its metabolites during gonadotropin releasing hormone- (GnRH-) induced gonadotropin secretion were investigated in primary cultures of rat pituitary cells. Prelabeled cells ([3H]AA) responded to GnRH challenge with increased formation (about 2-fold) of the leukotrienes LTC4, LTD4, and LTE4 as well as 5- and 15-eicosatetraenoic acids (5- and 15-HETE) as identified by HPLC. Formation of leukotrienes and 15-HETE was further verified by specific radioimmunoassays. No significant increase in the formation of 12-HETE or of the cyclooxygenase products prostaglandin E (PGE) and thromboxane A2 by GnRH was noticed. Addition of physiological concentrations of LTC4 enhanced basal LH release, while subphysiological concentrations of LTC4 (10(-15)-10(-12) M) inhibited GnRH-induced LH release by about 35% (p less than 0.02). Using specific lipoxygenase inhibitors L-656,224 and MK 886, we found inhibition of GnRH-induced LH release by about 40% at concentrations known to specifically inhibit the 5-lipoxygenase pathway. The peptidoleukotriene receptor antagonist ICI 198,615 inhibited LTC4- and LTE4-induced LH release and surprisingly also the effect of GnRH on LH release by 40%. The data strongly suggest a role for AA and its lipoxygenase metabolites in the on/off reactions of GnRH upon LH release. The data also present a novel amplification cycle in which newly formed leukotrienes become first messengers and establish an autocrine/paracrine loop.
Animals↗