Hormone-stimulated Increase Research Articles

Abstract P5-14-01: Telapristone acetate abrogates PR-dependent paracrine-mediated mammary cell proliferation

Abstract Background: Blockade of the progesterone (P)-progesterone receptor (PR) axis is a novel but untested strategy for breast cancer prevention. We report preclinical data evaluating telapristone acetate (TPA) compared with mifepristone (MFP), the prototype PR-antagonist. We hypothesize that the progesterone-PR blockade by TPA will inhibit PR-dependent paracrine expression (RANKL, WNT4, ID4, and calcitonin) attenuating cell proliferation and abrogating side branching and alveoli formation of mammary glands induced by hormones similar in extent or superior to MFP. Methods:Adult virgin FVB mice at 12 weeks of age were randomized to four treatment groups: no treatment control, EP (0.3 mg E + 30 mg P), EP + TPA (30mg), and EP + MFP (30mg). Hormone and drug pellets were subcutaneously implanted in flank area between the neck and shoulder. After 28-day treatments, the mice were euthanized to collect mammary glands, and processed as mammary whole mounts and as formalin-fixed paraffin embedding (FFPE) specimens. We evaluated cell proliferation (Ki67) by immunohistochemistry. Total RNA was extracted from FFPE specimens and the Nanostring nCounter assay was utilized to assess paracrine gene expression. The Mann Whitney test was used to calculate statistical significance (p<0.05). Results: We observed a considerable increase in side branches and alveoli in EP treatment group compared to the controls. The growth of the mammary gland stimulated by EP treatment was corroborated by a significant increase in Ki67 compared to control mice (median 41% vs 24%, respectively, p < 0.0001). Both TPA and MFP treatment abrogated side branching and alveoli formation and significantly reduced median Ki67 to 1/3 of that of control mice having endogenous estrus cycle hormone levels (24%, 8%, and 9% for control, EP+TPA and EP+MFP groups, respectively).TPA induced greater Ki67 reduction than MFP (7.5 ± 1.9% vs. 9.4 ± 1.9%, p= 0.04). As expected, EP treatment upregulated the RANKL expression compared to the control group while the administration of TPA and MFP significantly inhibited the RANKL expression stimulated by EP treatment. The mRNA expression measurement revealed that Rankl expression was 18 fold increased by EP treatment but completely inhibited by TPA and MFP treatment (p<0.001 for both drugs). Similarly the Rank expression was increased two-fold by EP treatment (p< 0.05) but down-regulated by TPA and MFP treatment (p<0.01 for both drugs). We observed significant upregulation of Wnt4, and Calca expression by EP treatment compared to controls (p<0.01 for both). Id4 expression showed the same trend but the change was non-significant. The addition of TPA completely opposed the hormone-stimulated increase of three paracrine molecules (Wnt4, Calca, and Id4) to a level below control group (p< 0.001 for all). Conclusions: We have demonstrated that TPA abrogates cell proliferation induced by exogenous EP hormones in an ovary intact mouse model. The blockade of PR-P binding was evident by complete inhibition of paracrine expression (not only RANKL/RANK expression but also WNT4, Calcitonin, and ID4 expression). TPA was efficacious as MFP in opposing paracrine-induced mammary cell proliferation, and warrants further testing in a breast cancer prevention trial. Citation Format: Lee O, Sun L, Karavites LC, Clare SE, Khan SA. Telapristone acetate abrogates PR-dependent paracrine-mediated mammary cell proliferation [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-14-01.

Thyroid hormone-stimulated increases in PGC-1α and UCP2 promote life history-specific endocrine changes and maintain a lipid-based metabolism.

Thyroid hormones (THs) regulate metabolism, but are typically suppressed during times of stressful physiological conditions, including fasting. Interestingly, prolonged fasting in northern elephant seal pups is associated with reliance on a lipid-based metabolism and increased levels of circulating THs that are partially attributed to active secretion as opposed to reduced clearance. This apparent paradox is coupled with complementary increases in cellular TH-mediated activity, suggesting that in mammals naturally adapted to prolonged fasting, THs are necessary to support metabolism. However, the functional relevance of this physiological paradox has remained largely unexplored, especially as it relates to the regulation of lipids. To address the hypothesis that TSH-mediated increase in THs contributes to lipid metabolism, we infused early and late-fasted pups with TSH and measured several key genes in adipose and muscle, and plasma hormones associated with regulation of lipid metabolism. TSH infusion increased the mRNA expressions of peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) more than 6.5-fold at 60 min in muscle, and expression of uncoupling protein 2 (UCP2) more than 27-fold during the early fast at 60 min, in adipose. Additionally, during the late fast period, the protein content of adipose CD36 increased 1.1-fold, and plasma nonesterified fatty acid (NEFA) concentrations increased 25% at 120 min, with NEFA levels returning to baseline after 24 h. We show that the TSH-induced increases in THs in fasting pups are functional and likely contribute to the maintenance of a lipid-based metabolism.

Nmp4/CIZ Suppresses the Response of Bone to Anabolic Parathyroid Hormone by Regulating Both Osteoblasts and Osteoclasts

How parathyroid hormone (PTH) increases bone mass is unclear, but understanding this phenomenon is significant to the improvement of osteoporosis therapy. Nmp4/CIZ is a nucleocytoplasmic shuttling transcriptional repressor that suppresses PTH-induced osteoblast gene expression and hormone-stimulated gains in murine femoral trabecular bone. To further characterize Nmp4/CIZ suppression of hormone-mediated bone growth, we treated 10-week-old Nmp4-knockout (KO) and wild-type (WT) mice with intermittent human PTH(1-34) at 30μg/kg daily or vehicle, 7days/week, for 2, 3, or 7weeks. Null mice treated with hormone (7weeks) gained more vertebral and tibial cancellous bone than WT animals, paralleling the exaggerated response in the femur. Interestingly, Nmp4/CIZ suppression of this hormone-stimulated bone formation was not apparent during the first 2weeks of treatment. Consistent with the null mice enhanced PTH-stimulated addition of trabecular bone, these animals exhibited an augmented hormone-induced increase in serum osteocalcin 3weeks into treatment. Unexpectedly, the Nmp4-KO mice displayed an osteoclast phenotype. Serum C-terminal telopeptide, a marker for bone resorption, was elevated in the null mice, irrespective of treatment. Nmp4-KO bone marrow cultures produced more osteoclasts, which exhibited elevated resorbing activity, compared to WT cultures. The expression of several genes critical to the development of both osteoblasts and osteoclasts was elevated in Nmp4-KO mice at 2weeks, but not 3weeks, of hormone exposure. We propose that Nmp4/CIZ dampens PTH-induced improvement of trabecular bone throughout the skeleton by transiently suppressing hormone-stimulated increases in the expression of proteins key to the required enhanced activity and number of both osteoblasts and osteoclasts.

Involvement of JAK2 and Src kinase tyrosine phosphorylation in human growth hormone-stimulated increases in cytosolic free Ca2+ and insulin secretion

We previously reported that human growth hormone (hGH) increases cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) and proliferation in pancreatic beta-cells (Sjöholm A, Zhang Q, Welsh N, Hansson A, Larsson O, Tally M, and Berggren PO. J Biol Chem 275: 21033-21040, 2000) and that the hGH-induced rise in [Ca(2+)](i) involves Ca(2+)-induced Ca(2+) release facilitated by tyrosine phosphorylation of ryanodine receptors (Zhang Q, Kohler M, Yang SN, Zhang F, Larsson O, and Berggren PO. Mol Endocrinol 18: 1658-1669, 2004). Here we investigated the tyrosine kinases that convey the hGH-induced rise in [Ca(2+)](i) and insulin release in BRIN-BD11 beta-cells. hGH caused tyrosine phosphorylation of Janus kinase (JAK)2 and c-Src, events inhibited by the JAK2 inhibitor AG490 or the Src kinase inhibitor PP2. Although hGH-stimulated rises in [Ca(2+)](i) and insulin secretion were completely abolished by AG490 and JAK2 inhibitor II, the inhibitors had no effect on insulin secretion stimulated by a high K(+) concentration. Similarly, Src kinase inhibitor-1 and PP2, but not its inactive analog PP3, suppressed [Ca(2+)](i) elevation and completely abolished insulin secretion stimulated by hGH but did not affect responses to K(+). Ovine prolactin increased [Ca(2+)](i) and insulin secretion to a similar extent as hGH, effects prevented by the JAK2 and Src kinase inhibitors. In contrast, bovine GH evoked a rise in [Ca(2+)](i) but did not stimulate insulin secretion. Neither JAK2 nor Src kinase inhibitors influenced the effect of bovine GH on [Ca(2+)](i). Our study indicates that hGH stimulates rise in [Ca(2+)](i) and insulin secretion mainly through activation of the prolactin receptor and JAK2 and Src kinases in rat insulin-secreting cells.

A Dominant Negative Gαs Mutant That Prevents Thyroid-stimulating Hormone Receptor Activation of cAMP Production and Inositol 1,4,5-Trisphosphate Turnover: COMPETITION BY DIFFERENT G PROTEINS FOR ACTIVATION BY A COMMON RECEPTOR

A Ser to Asn mutation at position 54 of the alpha subunit of G(s) (designated N54-alpha(s)) was characterized after transient expression of it with various components of the receptor-adenylyl cyclase pathway in COS-1, COS-7, and HEK 293 cells. Previous studies of the N54-alpha(s) mutant revealed that it has a conditional dominant negative phenotype that prevents hormone-stimulated increases in cAMP without interfering with the regulation of basal cAMP levels (Cleator, J. H., Mehta, N. D., Kurtz, D. K., Hildebrandt, J. D. (1999) FEBS Lett. 243, 205-208). Experiments reported here were conducted to localize the mechanism of the dominant negative effect of the mutant. Competition studies conducted with activated alpha(s)* (Q212L) showed that the N54 mutant did not work down-stream by blocking the interaction of endogenous alpha(s) with adenylyl cyclase. The co-expression of wild type or N54-alpha(s) along with the thyroid-stimulating hormone (TSH) receptor and adenylyl cyclase isotypes differing with respect to betagamma stimulation (AC II or AC III) revealed that the phenotype of the mutant is not dependent upon the presence of adenylyl cyclase isoforms regulated by betagamma. These studies ruled out a downstream site of action of the mutant. To investigate an upstream site of action, N54-alpha(s) was co-expressed with either the TSH receptor that activates both alpha(s) and alpha(q) or with the alpha(1B)-adrenergic receptor that activates only alpha(q). N54-alpha(s) failed to inhibit alpha(1B)-adrenergic receptor stimulation of inositol 1,4,5-trisphosphate production but did inhibit TSH stimulation of inositol 1,4,5-trisphosphate. These results show that G(s) and G(q) compete for activation by the TSH receptor. They also indicate that the N54 protein has a dominant negative phenotype by blocking upstream receptor interactions with normal G proteins. This phenotype is different from that seen in analogous mutants of other G protein alpha subunits and suggests that either regulation or protein-protein interactions differ among G protein alpha subunits.

Inhibitory effect of lysophosphatidylcholine and phospholipase A2 - treated low density lipoproteins on receptor-dependent regulation of {Ca2+}i in platelets

Treatment of LDL with phospholipase A from bee venom resulted in formation of lipid-protein particles (pl2 LDL) with increased content of lysophosphatidylcholine (LPC). At the same time, composition of other lipids and protein structure were unaffected. Both pl-LDL and LPC abolished PAF-, ADP- and thrombin-induced Ca2+ elevation in platelets and platelet aggregation, while LDL had no effect on hormone-stimulated increase in the intracellular Ca2+ content ({Ca2+}i). The effect persisted in Ca2+-free medium, indicating that pl-LDL and LPC also abolish Ca2+ mobilisation from intracellular stores. Neither LPC nor pl-LDL changed platelet Ca2+ levels and inhibited platelet aggregation induced by thapsigargin, a specific inhibitor of the endoplasmic reticulum Ca2+ ATPase. The inhibitory effect depended on LPC concentration, incubation time and the structure of LPC: lysophosphatidylethanolamine and phosphatidylcholine produced no inhibitory effect. The half-maximum-effective concentrations were the same for LPC and pl-LDL (2-4 muM). The results obtained indicate that LPC and pl-LDL inhibit the receptor-dependent increase in Ca2+. It can be suggested that the effect of LPC is mediated by redistribution of the plasma membrane integral proteins, which leads to disintegration of the intracellular signalling systems. phorbolmyristate acetate, a PKC activator, and

Eclosion hormone-stimulated cGMP levels in the central nervous system of Manduca sexta: inhibition by lipid metabolism blockers, increase in inositol(1,4,5)trisphosphate and further evidence against the involvement of nitric oxide.

Previous studies have shown that the neuropeptide, eclosion hormone, stimulates a nitric oxide-independent increase in the levels of cGMP in the nervous system of Manduca sexta. By contrast, recent results in Bombyx mori suggest that eclosion hormone increases cGMP via the production of nitric oxide. In view of these conflicting results we have carried out additional studies to test whether nitric oxide is involved in this process in Manduca. Evidence presented here supports our earlier observations that in Manduca the eclosion hormone-stimulated increase in cGMP is nitric oxide- and carbon monoxide-independent. In addition, we show that a wide variety of inhibitors of lipid metabolism block the eclosion hormone-stimulated cGMP increase. This supports the hypothesis that the activation of the guanylate cyclase is mediated by a lipid messenger. We also show that eclosion hormone stimulates an increase in the levels of inositol(1,4,5)trisphosphate. The time-course of this increase is consistent with the hypothesis that eclosion hormone stimulation of a phospholipase C is an early event in the cascade that results in an increase in cGMP. Receptor-mediated lipid hydrolysis is often mediated by G protein-coupled receptors. Experiments using pertussis toxin show that the eclosion hormone-stimulated increase in cGMP is not mediated by a pertussis toxin-sensitive G protein.

Endothelin Receptors, Second Messengers, and Actions in Bone

Endothelins are a class of peptides that are produced by and elicit responses in many tissues. A growing literature documents the presence and effects of endothelins in bone. Both endothelinA and endothelinB receptors have been demonstrated in osteoblastic cells by ligand binding. Major signal transduction pathways for endothelin in bone cells appear to be stimulation of phospholipid turnover, by activation of A, C and D phospholipases, stimulation of calcium flux from intracellular and extracellular stores and activation of tyrosine kinases. Endothelins also modulate calcium signaling elicited by other agents in osteoblastic cells. The parathyroid hormone-stimulated calcium transient in UMR-106 cells is enhanced by endothelins, acting through an endothelinB receptor, whereas the parathyroid hormone-stimulated increase in cyclic AMP is inhibited by endothelins. Phenotypic responses to endothelin-1 include changes in alkaline phosphatase activity, stimulation of osteocalcin and osteopontin message, stimulation of collagen and noncollagenous protein synthesis, inhibition of osteoclast motility and stimulation of prostaglandin-dependent resorption. Endothelin-1 also enhances the interleukin-1-induced increase in interleukin-6. Endothelins can also potentially affect calcium metabolism through their actions to inhibit the secretion of parathyroid hormone.

Eclosion hormone stimulates cyclic GMP levels in Manduca sexta nervous tissue via arachidonic acid metabolism with little or no contribution from the production of nitric oxide.

The neuropeptide eclosion hormone acts directly on the nervous system of the tobacco hornworm, Manduca sexta, to trigger ecdysis behavior at the end of each molt. Previous studies have shown that the action of eclosion hormone is mediated via the intracellular messenger cyclic GMP. In the present study we have investigated the mechanisms involved in the eclosion hormone-stimulated increases in cyclic GMP. No stimulation of guanylate cyclase was seen in homogenized nervous tissue, suggesting that eclosion hormone does not directly stimulate a membrane-bound form of guanylate cyclase. Nitric oxide synthase inhibitors, N-methylarginine and nitroarginine, had no effect on eclosion hormone-stimulated cyclic GMP levels. By contrast, 4-bromophenacyl bromide, an inhibitor of arachidonic acid release, and nordihydroguaiaretic acid, an inhibitor of arachidonic acid metabolism, almost completely abolished the eclosion hormone-stimulated cyclic GMP increase. We hypothesize that eclosion hormone receptors are coupled to a lipase, activation of which causes the release of arachidonic acid. Either the arachidonic acid directly stimulates the soluble guanylate cyclase or further metabolism of arachidonic acid yields compounds that activate guanylate cyclase.

Purification and characterization of the zeta isoform of protein kinase C from bovine kidney.

The zeta isoform of protein kinase C (PKC zeta) was purified to near homogeneity from the cytosolic fraction of bovine kidney by successive chromatography on DEAE-Sephacel, heparin-Sepharose, phenyl-5PW, hydroxyapatite, and Mono Q. The purified enzyme had a molecular mass of 78 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein was recognized by an antibody raised against a synthetic oligopeptide corresponding to the deduced amino acid sequence of rat PKC zeta. The enzymatic properties of PKC zeta were examined and compared with conventional protein kinase C purified from rat brain. The activity of PKC zeta was stimulated by phospholipid but was unaffected by phorbol ester, diacylglycerol, or Ca2+. PKC zeta did not bind phorbol ester, and autophosphorylation was not affected by phorbol ester. Unsaturated fatty acid activated PKC zeta, but this activation was neither additive nor synergistic with phospholipid. These results indicate that regulation of PKC zeta is distinct from that of other isoforms and suggest that hormone-stimulated increases in diacylglycerol and Ca2+ do not activate this isoform in cells. It is possible that PKC zeta belongs to another enzyme family, in which regulation is by a different mechanism from that for other isoforms of protein kinase C.

Protein kinase C does not participate in carbachol's secretory action in T84 cells.

We investigated the role of protein kinase C (PKC) in mediating carbachol's stimulation of transepithelial Cl- secretion in T84 cells. Direct PKC activation with phorbol 12-myristate 13-acetate (PMA) stimulated transepithelial Cl- transport (measured as the short-circuit current), demonstrating that PKC could interact with the secretory apparatus. Carbachol stimulated PKC activity, suggesting that the enzyme might participate in the hormone's action. Diacylglycerol metabolism inhibitors (DMIs), known to augment hormone-stimulated increases in diacylglycerol levels, potentiated the short-circuit current response to carbachol. The effect of DMIs was not due to amplification of carbachol-induced increases in PKC activity, however; PKC activity during carbachol stimulation was no higher in the presence of DMIs than in their absence. Augmentation of carbachol's action by DMIs appeared to be due to the direct activation of PKC which, like PMA, stimulated the Cl- conductance of the apical membrane (GCl). The effects of DMIs and carbachol on GCl were additive. Carbachol itself stimulated GCl but not by activating PKC; staurosporine did not blunt the effect of carbachol on GCl. Nor did staurosporine reduce the effect of carbachol on transepithelial Cl- secretion. These observations demonstrate that PKC does not participate in the secretory action of carbachol in T84 cells and suggest that direct PKC activation with DMIs and PMA stimulates an apical pool of PKC that is not accessible to carbachol applied to the basolateral membrane.

Participation of protein kinase C in desensitization to bradykinin and to carbachol in MDCK cells.

To explore the possibility that protein kinase C (PKC) participates in desensitization to Ca(2+)-mobilizing hormones in MDCK cells, we measured intracellular free Ca2+ concentration ([Ca2+]i) using fura-2 and video microscopy. We first examined the response of MDCK cells grown on plastic dishes. Exposure of cells to bradykinin (BK) or to carbachol, followed by reexposure after washing off the hormone, revealed two features of hormone desensitization. First, the initial hormone-induced peak response of [Ca2+]i was transitory; [Ca2+]i returned to control levels despite continued presence of hormone. Second, cells remained refractory to hormone rechallenge for 5 min after washing off hormone; [Ca2+]i response on re-exposure was reduced 70% compared with initial hormone-stimulated peak. Subsequent experiments demonstrated involvement of PKC in both desensitization processes. Pretreatment with the phorbol ester, phorbol 12-myristate 13-acetate, significantly blunted initial response to BK and to carbachol by 70 and 86%, respectively. When hormone-stimulated C kinase activity was enhanced with the diglyceride lipase inhibitor, RG 80267, BK- and carbachol-induced increases in [Ca2+]i were blunted 50%. Pretreatment with sphingosine, an inhibitor of PKC, resulted in an amplification of initial hormone-stimulated increase in [Ca2+]i and restored the response to rechallenge. To examine the possible interaction between BK and carbachol,both of which use PKC to induce desensitization, we measured [Ca2+]i in cells grown as monolayers on permeable, collagen-coated supports. Both carbachol and BK induced desensitization to the other hormone (heterologous desensitization)provided that the two hormones were applied to the same side of the polarized monolayer (apical).(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of rat uterine glycerylphosphorylcholine diesterase and its tissue-specific induction by 17 beta-estradiol.

The rat uterine enzyme glycerylphosphorylcholine (GPC) diesterase found in the proestrous secretions was purified and characterized biochemically with respect to its subunit mol wt, native size, pI, and amino acid and carbohydrate composition. The 30-kDa protein was assessed to have a native mol wt of 105 kDa, as determined by analytical gel filtration. It had a basic pI with an unusually high carbohydrate/protein ratio (0.83:1). The estrogen inducibility of the protein was identified by its de novo synthesis in vitro after in vivo 17 beta-estradiol administration. For this experiment, uteri from estradiol-treated immature rats were incubated in the presence of [14C]glucosamine, and the 14C-labeled total glycoconjugates were then subjected to the enzyme purification procedure. The peak enzyme fraction was observed to correspond to one of the 14C-labeled protein peaks in the elution profile of the glycoconjugates. Assay of GPC diesterase activity showed significant enhancement only in the uterus, not in other organ systems, after in vivo estradiol treatment, and this hormone-stimulated increase was inhibited by nafoxidine, a potent estrogen antagonist. The enzyme activity profile of the three main separated uterine cellular types showed that the enzyme was secreted by epithelial cells. No antigenic homology of the enzyme was observed with GPC diesterase from nonmammalian sources. The results suggested that GPC diesterase was one of the estrogen-regulated proteins of the preovulatory uterine fluid of rats, and the availability of a purified preparation of the enzyme could serve as a useful tool to study the mechanism of estrogen action.

Increases in Intracellular Ca2+ Regulate the Binding of [3H]Phorbol 12,13-Dibutyrate to Intact 1321N1 Astrocytoma Cells

The redistribution of protein kinase C (Ca2+/phospholipid-dependent protein kinase) from a cytosolic or a loosely associated membrane compartment to a more integral membrane compartment is stimulated by Ca2+ in vitro. This event is thought to be necessary for activation of the enzyme. To determine whether such a redistribution of protein kinase C occurs following hormonally stimulated increases in cytoplasmic Ca2+, we measured [3H]phorbol 12,13-dibutyrate ([3H]PDB) binding to protein kinase C in intact 1321N1 astrocytoma cells. The muscarinic agonist carbachol causes a 2-fold increase in [3H]PDB binding. This increase is transient, peaking at 1 min and returning toward control levels by 5 min. Scatchard analysis of [3H]PDB binding in the presence of carbachol reveals a 2-fold increase in the Bmax and no change in the KD compared to control values. This increase in Bmax likely represents a redistribution of protein kinase C to the membrane because [3H]PDB binding in intact cells is predominantly to membrane-associated enzyme. The Ca2+ ionophore ionomycin, and two other Ca2+-mobilizing hormones, bradykinin and histamine, mimic the effects of carbachol. Furthermore, when hormone-sensitive Ca2+ stores are depleted by prior agonist treatment, the carbachol-induced increases in intracellular [Ca2+] and [3H]PDB binding are completely blocked. Under these conditions, phosphoinositide hydrolysis and diacylglycerol (DAG) formation are not inhibited. We also examined the time course of DAG accumulation in response to carbachol. DAG is not yet significantly elevated when the increase in [3H]PDB binding is maximal. Furthermore, [3H]PDB binding has returned to control levels when DAG concentrations are maximally elevated. These data suggest that hormone-stimulated increases in cytoplasmic Ca2+ cause a marked and rapid redistribution of protein kinase C which precedes any significant increase in DAG. Our findings also demonstrate that [3H]PDB binding to intact cells may be a useful measure of the ability of Ca2+-mobilizing hormones to affect protein kinase C.

Effect of parathyroid hormone on amino acid transport by cultured neonatal mouse calvarial bone cells.

The effect of synthetic bovine parathyroid hormone [bPTH-(1-34)] on amino acid uptake by confluent primary cultures of osteoblast-like cells isolated from neonatal mouse calvaria was studied. The uptake of proline and leucine by membrane transport Systems A, ASC, and L was discriminated on the basis of their sodium dependency and sensitivity to the system-specific amino acid analogs 2-(methylamino)-isobutyric acid (MeAIB) for System A and 2-amino-(2,2,1)-heptane-2-carboxylic acid (BCH) for System L. Treatment with 24 nM bPTH-(1-34) in serum-free EBSS for 4 hr increased the initial uptake rate of proline by 50-80% but had no effect on the uptake of leucine. Temporally, the increase in proline uptake was preceded by a 2-hr lag period and plateaued after 5-6 hr. A 5-min exposure to the hormone was sufficient to cause a significant increase in proline uptake measured 4 hr later. The magnitude of the increase was dose-related from 0.24 to 240 nM bPTH-(1-34), with the half-maximal effect occurring at 2.4 nM. Only the sodium-dependent, MeAIB-inhibitable component of proline uptake was elevated. Eadie-Hofstee analysis indicated that bPTH-(1-34) increased Vmax without changing the Km. Actinomycin D and cycloheximide prevented the hormone-stimulated increase, suggesting that RNA and protein synthesis were required. Treatment with either inhibitor alone caused a 30-35% decrease in proline transport that was not observed in the presence of bPTH-(1-34), indicating an effect not dependent on macromolecular synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Substrate phosphoprotein availability regulates eclosion hormone sensitivity in an insect CNS.

The final step in the moulting of all insects is ecdysis, the shedding of the cuticle of the previous instar, which is triggered in Lepidoptera by the neurosecretory peptide eclosion hormone. This hormone acts directly on the nervous system to release the appropriate motor patterns for larval, pupal and adult ecdysis, but there are only brief periods near the end of each moult when the nervous system is competent to respond to the hormone. Previous experiments have shown that the action of eclosion hormone on the nervous system at pupal ecdysis in the tobacco hornworm, Manduca sexta, is mediated by the second messenger cyclic GMP. Here we report that the hormone-stimulated increase in cGMP results in the phosphorylation of two proteins, each with an apparent relative molecular mass (Mr) of 54,000. Moreover, the brief periods during which the central nervous system (CNS) is responsive to eclosion hormone seem to result from the transient presence of these substrate proteins within the nervous system. This provides a novel mechanism by which hormonal responsiveness can be regulated.

Regulation of Uterine Nucleolar RNA Synthesis by Estrogens

Administration of estradiol to ovariectomized mature rats results in a biphasic early (4 h) and late (24 h) increase in transcriptional activity of isolated uterine nucleoli. The increased rate of nucleolar RNA synthesis is dependent upon the dose of estradiol over the range of 0.1 to 1 micrograms/animal, and exhibits hormone specificity. Administration of cycloheximide, an inhibitor of protein synthesis, prior to the administration of hormone or during the early phase of estrogen action (less than 4 h) blocks the estrogen-induced increase in uterine nucleolar transcriptional activity. Administration of cycloheximide during the later phase (greater than 8 h) of estrogen action is without effect on the estrogen-induced increase in transcriptional activity of isolated uterine nucleoli. This suggests that the longer term maintenance of the hormone-stimulated increase in nucleolar RNA synthesis is independent of continuous protein synthesis. Results indicate that the estrogen-induced accumulation and subsequent decline in uterine nuclear estradiol receptor levels is unaffected by cycloheximide treatment. Together, these results indicate the presence of the receptor-hormone complex in the nucleus is not solely responsible for the increased transcriptional activity of uterine nucleoli following in vivo hormone treatment. The early activation of uterine nucleolar RNA synthesis by estrogen seems to result from the synthesis of a short-lived protein(s) that modified RNA polymerase I and/or the nucleolar chromatin template.

Effect of androgens on plasma somatomedin-C/insulin-like growth factor I responses to growth hormone.

In search of the mechanism for the relatively high plasma somatomedin-C (Sm-C) concentrations in pubertal boys and girls, we have measured the plasma Sm-C responses to exogenous growth hormone (GH) in prepubertal hypopituitary boys before and after testosterone administration. Sm-C responses were determined in 5 hypopituitary boys (9-14 years of age) who were given two successive injections of GH (0.1 U/kg) 48 h apart. Eight days later, after administering 200 mg testosterone IM, their Sm-C responses to the same GH challenge were reassessed. There were no significant differences between the pre-testosterone Sm-C response to GH and the post-testosterone response, despite evidence for reductions in 24 h urinary nitrogen excretion and serum urea nitrogen concentrations in response to testosterone. The results provide no evidence that androgen augments the effect of GH to raise plasma Sm-C during puberty, or that androgen has a direct stimulatory effect on Sm-C production. By inference and from published reports, it appears more likely that a sex hormone-stimulated increase in GH secretion is responsible for the increased Sm-C observed during puberty.

Peptide-induced prostaglandin biosynthesis in the renal-vein-constricted kidney.

The ipsilateral kidney was removed from a rabbit 48h after unilateral partial renal-vein-constriction and was perfused with Krebs-Henseleit media at 37 degrees C. Hourly administration of a fixed dose of bradykinin to the renal-vein-constricted kidney demonstrated a marked time-dependent increase in the release of bioassayable prostaglandin E(2) and thromboxane A(2) into the venous effluent as compared with the response of the contralateral control kidney. The renal-vein-constricted kidney produced up to 60 times more prostaglandin E(2) in response to bradykinin after 6h of perfusion as compared with the contralateral kidney; thromboxane A(2) was not demonstratable in the contralateral kidney. Inhibition of protein synthesis de novo in the perfused renal-vein-constricted kidney with cycloheximide lessened the hormone-stimulated increase in prostaglandin E(2) by 94% and in thromboxane A(2) by 90% at 6h of perfusion. Covalent acetylation of the renal cyclo-oxygenase by prior oral administration of aspirin to the rabbit inhibited initial bradykinin-stimulated prostaglandin E(2) biosynthesis 71% at 1h of perfusion. However, there was total recovery from aspirin in the renal-vein-constricted kidney by 2h of perfusion after bradykinin stimulation. Total cyclo-oxygenase activity as measured by [(14)C]arachidonate metabolism to labelled prostaglandins by renal cortical and renal medullary microsomal fractions prepared from 6h-perfused kidneys demonstrated that renal-vein-constricted kidney-cortical cyclo-oxygenase activity was significantly greater than the contralateral-kidney-cortical conversion, whereas medullary arachidonate metabolism was comparable in both the renal-vein-constricted kidney and contralateral kidney. These data suggest that perfusion of a renal-vein-constricted kidney initiates a time-dependent induction of synthesis of prostaglandin-producing enzymes, which appear to be primarily localized in the renal cortex. The presence of the synthetic capacity to generate very potent vasodilator and vasoconstrictor prostaglandins in the renal cortex suggests that these substances could mediate or modulate changes in renal vascular resistance in pathological states.

Granulosa cell desensitization: effects of gonadotropin on antral and preantral follicles.

The present studies examine the time course, hormonal specificity, and reversibility of gonadotropin-induced desensitization in rat granulosa cells in vivo and relate these events to changes in FSH and LH/hCG receptor availability. To these ends, desensitization to gonadotropins was compared in granulosa cells of preantral and antral follicles. Granulosa cells from antral follicles of estradiol- and FSH-primed hypophysectomized rats possess receptors for both FSH and LH/hCG. These cells appeared to exhibit heterologous desensitization of gonadotropin-stimulated cAMP accumulation in vivo; refractoriness initiated by either gonadotropin (5 μg human FSH or 5–50 IU hCG, administered iv) was communicated to both gonadotropin/adenylate cyclase response systems and prevented a secondary hormone-stimulated increase in cAMP accumulation. Heterologous desensitization was demonstrable within 120 min and occurred in the absence of detectable changes in the binding of 125I-labeled heterologous hormone to isolated gran...