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Synthetic glucocorticoids (GCs) are widely used to treat inflammatory conditions. However, chronic use of GCs can lead to hypertension. The cause of this undesired side effect remains unclear. Previously, we developed an in vivo rat model to study the mechanisms underlying hypertension induced by the chronic administration of the potent synthetic GC, dexamethasone (DEX) and found that the catecholamine biosynthetic pathway plays an important role. In the current study, we used this model to investigate the role of the adrenal medulla, renal nerves, and other peripheral sympathetic nerves in DEX-induced hypertension. After 5 days of baseline telemetric recording of mean arterial pressure (MAP) and heart rate (HR), rats were subjected to one of the following treatments: renal denervation (RDNX), adrenal medullectomy (ADMX), 6-hydroxydopamine (6-OHDA, 20 mg/kg, i.p.) to induce chemical sympathectomy, or a combination of ADMX and 6-OHDA. On day 11, the animals received vehicle (VEH) or DEX in drinking water for 7 days, with the latter causing an increase in MAP in control animals. ADMX and RDNX by themselves exacerbated the pressor effect of DEX. In the chemical sympathectomy group, DEX still caused a rise in MAP but the response was lower (ΔMAP of 6-OHDA/DEX < VEH/DEX, p = 0.039). However, when ΔMAP was normalized to day 10, 6-OHDA + DEX did not show any difference from VEH + DEX, certainly not an increase as observed in DEX + ADMX or RDNX groups. This indicates that sympathetic nerves do not modulate the pressor effect of DEX. TH mRNA levels increased in the adrenal medulla in both VEH/DEX (p = 0.009) and 6-OHDA/DEX (p = 0.031) groups. In the 6-OHDA group, DEX also increased plasma levels of norepinephrine (NE) (p = 0.016). Our results suggest that the activation of catecholamine synthetic pathway could be involved in the pressor response to DEX in animals even under chemical sympathectomy with 6-OHDA.

Our objective was to study hypertension induced by chronic administration of synthetic glucocorticoid, dexamethasone (DEX), under nonstressful conditions and examine the role of catecholamine biosynthesis. To achieve this, we did the following: 1) used radiotelemetry to record mean arterial pressure (MAP) and heart rate (HR) in freely moving rats, and 2) administered different doses of DEX in drinking water. To evaluate the involvement of tyrosine hydroxylase (TH), the rate-limiting step in catecholamine biosynthesis, we treated rats with the TH inhibitor, α-methyl-para-tyrosine (α-MPT), for 3 days prior to administration of DEX and assessed TH mRNA and protein expression by quantitative real-time polymerase chain reaction and Western blot in the adrenal medulla. We observed a dose-dependent elevation in blood pressure with a DEX dose of 0.3 mg/kg administered for 10 days, significantly increasing MAP by +15.0 ± 1.1 mm Hg, while concomitantly reducing HR. Although this DEX treatment also significantly decreased body weight, pair-fed animals that showed similar decreases in body weight due to lowered food intake were not hypertensive, suggesting that body weight changes may not account for DEX-induced hypertension. Chronic DEX treatment significantly increased the TH mRNA and protein levels in the adrenal medulla, and α-MPT administration not only reduced DEX pressor effects, but also inhibited TH (serine(40)) phosphorylation. Our study thus validates a novel model to study hypertension induced by chronic intake of DEX in freely moving rats not subject to the confounding factors of previous models and establishes its dependence on concomitant activation of peripheral catecholamine biosynthesis.

Exposure to early-life stress is a risk factor for the development of cognitive and emotional disorders later in life. We previously demonstrated that prenatal stress (PNS) in rats results in long-term, stable changes in central stress-response systems and impairs the ability to extinguish conditioned fear responding, a component of post-traumatic stress disorder (PTSD). Maternal corticosterone (CORT), released during prenatal stress, is a possible mediator of these effects. The purpose of the present study was to investigate whether fetal exposure to CORT at levels induced by PNS is sufficient to alter the development of adult stress neurobiology and fear extinction behavior. Pregnant dams were subject to either PNS (60 min immobilization/day from ED 14-21) or a daily injection of CORT (10mg/kg), which approximated both fetal and maternal plasma CORT levels elicited during PNS. Control dams were given injections of oil vehicle. Male offspring were allowed to grow to adulthood undisturbed, at which point they were sacrificed and the medial prefrontal cortex (mPFC), hippocampus, hypothalamus, and a section of the rostral pons containing the locus coeruleus (LC) were dissected. PNS and prenatal CORT treatment decreased glucocorticoid receptor protein levels in the mPFC, hippocampus, and hypothalamus when compared to control offspring. Both treatments also decreased tyrosine hydroxylase levels in the LC. Finally, the effect of prenatal CORT exposure on fear extinction behavior was examined following chronic stress. Prenatal CORT impaired both acquisition and recall of cue-conditioned fear extinction. This effect was additive to the impairment induced by previous chronic stress. Thus, these data suggest that fetal exposure to high levels of maternal CORT is responsible for many of the lasting neurobiological consequences of PNS as they relate to the processes underlying extinction of learned fear. The data further suggest that adverse prenatal environments constitute a risk factor for PTSD-like symptomatology, especially when combined with chronic stressors later in life.

The human tyrosine hydroxylase (hTH) gene has a 42 bp evolutionarily conserved region designated (CR) II at -7.24 kb, which bears 93% homology to the region we earlier identified as containing the glucocorticoid response element, a 7 bp activator protein-1 (AP-1)-like motif in the rat TH gene. We cloned this hTH-CRII region upstream of minimal basal hTH promoter in luciferase (Luc) reporter vector, and tested glucocorticoid responsiveness in human cell lines. Dexamethasone (Dex) stimulated Luc activity of hTH-CRII in HeLa cells, while mifepristone, a glucocorticoid receptor (GR) antagonist, prevented Dex stimulation. Deletion of the 7 bp 5'-TGACTAA at -7243 bp completely abolished the Dex-stimulated Luc activity of hTH-CRII construct. The AP-1 agonist, tetradeconoyl-12,13-phorbol acetate (TPA), also stimulated hTH promoter activity, and Dex and TPA together further accentuated this response. Chromatin immunoprecipitation assays revealed the presence of both GR and AP-1 proteins, especially Jun family members, at this hTH promoter site. Dex did not stimulate hTH promoter activity in a catecholaminergic cell line, which had low endogenous GR levels, but did activate the response when GR was expressed exogenously. Thus, our studies have clearly identified a glucocorticoid-responsive element in a 7 bp AP-1-like motif in the promoter region at -7.24 kb of the human TH gene.

Adult male bonnet monkeys maintained under regulated light: dark conditions exhibit a nycthemeral surge of testosterone. The present study attempts to determine the effect of administration of drugs that modulate prolactin levels like ergobromocriptine (EBC) and chlorpromazine (CPZ) on testosterone production. The injection of EBC, a known inhibitor of prolactin secretion, could abolish nocturnal testosterone surge irrespective of the drug being given at 8.00 or 17.00 h. Testosterone surge could likewise be inhibited by treating animals with CPZ, a potent stimulator of prolactin secretion. This suggests that alteration in endogenous prolactin level from the normal effects nycthemeral surges of testosterone. The in vivo responsiveness of the testes of monkeys injected either CPZ oder EBC to exogenous LH or LHRH stimulation was tested. While LH could completely override the CPZ induced inhibition in testosterone production it could only partially reverse the EBC effect.

Glucocorticoids (GCs) generally stimulate gene transcription via consensus glucocorticoid response elements (GREs) located in the promoter region. To identify the GRE in the rat tyrosine hydroxylase (TH) gene promoter, we transiently transfected PC12 cells with a 9-kilobase (kb) TH promoter-luciferase (Luc) construct. Dexamethasone (Dex) stimulated Luc activity, which was abolished by mifepristone (RU486). Serial deletion mutations revealed a Dex-responsive 7-base pair (bp) sequence, TGACTAA, located at -5734 to -5728. Deletion of just these seven nucleotides from the 9-kb promoter completely abolished the Dex response and partially reduced the response to phorbol ester but not to forskolin. The Dex response was fully retained in a construct in which most of the 9-kb promoter was deleted, except for 100 bp around the -5.7-kb region, clearly identifying this 7-bp sequence as solely responsible for GC responsiveness. Conversely, deletion of the proximal cAMP-response element (-45/-38) or activator protein-1 (AP-1) (-207/-201) sites in the 9-kb promoter did not affect Dex and phorbol ester responses. A radiolabeled 25-bp promoter fragment bearing the 7-bp TH-GRE/AP-1 showed specific binding to PC12 nuclear proteins. Using antibodies against the glucocorticoid receptors and AP-1 family of proteins and primers for the TH-GRE/AP-1 region, we detected a specific DNA amplicon in a chromatin immunoprecipitation assay. This 7-bp TH-GRE/AP-1 sequence (TGACTAA) does not bear similarity to any known GRE but closely resembles the consensus AP-1 binding site, TGACTCA. Our studies describe for the first time a novel GRE/AP-1 site present in the TH gene promoter that is critical for glucocorticoid regulation of the TH gene.

We examined the mRNA and protein levels of GABA(A) and NMDA receptor (NMDAR) subunits in cultured mouse cortical neurons following exposure to chronic ethanol (CE) or chronic intermittent ethanol (CIE), and after 5 days of withdrawal. With respect to GABA(A) receptor mRNA, both treatments decreased the levels of alpha1 and alpha2 subunits, and increased the level of alpha4. However, only CE treatment caused parallel changes in the protein levels; alpha2 and alpha4 protein levels did not change after CIE. Both treatments did not alter beta2 and beta3 mRNA levels, but they increased beta2/3 protein levels. The gamma2 subunit mRNA levels decreased with both treatments, but protein levels did not change. Most of the changes returned to control levels after withdrawal, except for the gamma2 subunit protein, which was lower than controls. In the case of NMDAR subunit, both treatments greatly increased the levels of NR2B mRNA, but barely altered NR1 mRNA and polypeptide levels. CIE treatment caused a relatively higher increase in NR2B protein, and this was the only sustained increase after long-term withdrawal. Taken together, our results show that CIE regimen has less pronounced effects on GABA(A) receptor expression, but increases NR2B expression more dramatically than CE treatment in cultured cortical neurons. These differential effects on subunit expression may result in altered receptor structure and function as a result of ethanol exposure.

Neuron-restrictive silencer factor (NRSF) is a transcriptional repressor of multiple neuronal genes. This study addressed the role of NRSF in N-methyl-D-aspartate (NMDA) receptor NR2B promoter activity and the molecular mechanisms of ethanol-induced NR2B up-regulation in fetal cortical neurons. The 5'-flanking region of the NR2B gene contains five NRSE-like elements. Functional analysis of the upstream regions of the NR2B gene by transient transfection of neurons revealed that neuron-restrictive silencer element (NRSE) motifs located between base pair -1407 and -2741 represses transcription of the gene. Analysis by electrophoretic mobility shift assay and reporter gene assay identified NRSE2 and 3 as responsible for repressing NR2B gene transcription. The identity of NRSF as the functional binding factor is suggested by the specific binding of in vitro synthesized NRSF or cell lysate to the labeled probes and the specific antibody-induced supershift. Furthermore, whereas mutations of NRSE2 and 3 motifs increased the promoter activity, overexpression of NRSF reduced it significantly. The pattern of NRSF expression during development was investigated and demonstrated that the highest expression is on embryonic day 14 with moderate expression on postnatal day 0, reflecting a possible role of NRSF as a regulator during development. Treatment of cultured cortical neurons with 100 mM ethanol for 5 days caused a significant decrease in the NRSF mRNA and protein levels, NRSF/NRSE binding activity, and an increase in the promoter activity. Therefore, our studies suggest that NRSF is a negative regulator of NR2B expression and may contribute to the ethanol-induced up-regulation of the NR2B gene in fetal cortical neurons.

Metabolites of sphingomyelin, ceramide and sphingosine, have previously been implicated in cell growth regulation. Here we show that cell-permeable ceramide analogs and treatment with sphingomyelinase, which hydrolyzes sphingomyelin located on the outer leaflet of the bilayer, increase the progression of quiescent Swiss 3T3 fibroblasts through the S phase of the cell cycle leading to an increase in cell division. Although both potentiate the mitogenic effects of several growth factors [14], sphingomyelinase treatment antagonized the mitogenic effect of the tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA), while ceramide analogs had no effect, and sphingosine, a further metabolite of ceramide, potentiated the mitogenic effect of TPA. Concomitantly, sphingomyelinase, but not ceramide analogs, blunted the rapid increase in membrane-associated protein kinase C (PKC) activity induced by TPA without affecting the translocation of PKC alpha, delta, epsilon or zeta isoforms. Moreover, in contrast to sphingosine which activates phospholipase D (PLD) leading to an increase in phosphatidic acid levels, sphingomyelinase, but not ceramide analogs, reduced TPA-stimulated PLD activity. Our results suggest that the signaling pathways utilized by sphingomyelinase differ from those of cell-permeable ceramide analogs, and both act differently than sphingosine. The differential effects of exogenous short-chain ceramide analogs and sphingomyelinase call for caution in using these analogs as tools to study the role of ceramide in diverse cellular functions.

Platelet-derived growth factor (PDGF) and serum, but not epidermal growth factor (EGF), stimulated sphingosine kinase activity in Swiss 3T3 fibroblasts and increased intracellular concentrations of sphingosine 1-phosphate (SPP), a sphingolipid second messenger (Olivera, A., and Spiegel, S. (1993) Nature 365, 557–560). We report herein thatdl-threo-dihydrosphingosine (DHS), a competitive inhibitor of sphingosine kinase that prevents PDGF-induced SPP formation, specifically inhibited the activation of two cyclin-dependent kinases (p34cdc2 kinase and Cdk2 kinase) induced by PDGF, but not by EGF. SPP reversed the inhibitory effects of DHS on PDGF-stimulated cyclin-dependent kinases and DNA synthesis, demonstrating that the DHS effects were mediated via inhibition of sphingosine kinase. DHS also markedly reduced PDGF-stimulated but not EGF-stimulated mitogen-activated protein kinase activity and DNA binding activity of activator protein-1. Examination of the early signaling events of PDGF action revealed that DHS did not affect PDGF-induced autophosphorylation of the growth factor receptor or phosphorylation of the SH2/SH3 adaptor protein Shc and its association with Grb2. This sphingosine kinase inhibitor did not abrogate activation of phosphatidylinositol 3-kinase by PDGF. In agreement, treatment with SPP had no effect on these responses but did, however, potently stimulate phosphorylation of Crk, another SH2/SH3 adaptor protein. Moreover, DHS inhibited PDGF-stimulated, but not EGF-stimulated, Crk phosphorylation. Thus, regulation of sphingosine kinase activity defines divergence in signal transduction pathways of PDGF and EGF receptors leading to mitogen-activated protein kinase activation.

In an attempt to define the basis for sphingolipid regulation of cell proliferation, we studied the effects of glucosylceramide (GlcCer) synthase inhibition by threo1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) on NIH 3T3 cells overexpressing insulin-like growth factor-1 (IGF-1) receptors. PDMP treatment resulted in a time-dependent decrease in GlcCer levels and an increase in cellular ceramide levels. PDMP abolished serum and IGF-1-stimulated cell proliferation, as measured by a reduction in [3H]thymidine incorporation, protein, and DNA levels. However it did not affect IGF-1-mediated early signaling events, including receptor tyrosine kinase, MAP kinase, and phosphatidylinositol 3-kinase activities. Two-color flow cytometry with propidium iodide and 5-bromo-2′-deoxyuridine monophosphate labeling revealed an arrest of the cell cycle at G1/S and G2/M transitions in an asynchronous population of cells. These changes were time dependent, with maximal effects seen by 12-24 h. Removal of PDMP from the cell medium resulted in reversal of the cell cycle changes, with cells re-entering the S phase. The cell cycle arrest at the G1/S and G2/M transitions was confirmed in cells synchronized by pretreatment with nocodazole, aphidicolin, or hydroxyurea, and released from blockade in the presence of PDMP. A decrease in the activities of two cyclin-dependent kinases, p34cdc2 kinase and cdk2 kinase, was observed with PDMP treatment. When cell ceramide levels were increased by N-acetylsphingosine, comparable changes in the cell cycle distribution were seen. However, sphingomyelinase treatment was without effect. Therefore, it appears that ceramide mediates in part the inhibitory effect of GlcCer synthase inhibition on IGF-1-induced cell proliferation in 3T3 cells. The rapid production of decreased cyclin-dependent kinase activities by PDMP suggests that one of the crucial sites of action of the inhibitor lies in this area.

Amiodarone is an antiarrhythmic drug that often induces thyroid disorders. Its effects on several aspects of thyroid function were studied using cultured dog thyroid cells. Within 5-60 min of incubation of cell membranes with amiodarone, there were profound changes in adenylate cyclase activity and TSH receptor binding. Amiodarone specifically decreased TSH-stimulated adenylate cyclase activity, but not the basal or forskolin-stimulated activities, while it increased the binding of 125I-labeled TSH to its receptors. Significant effects were seen with 5-10 microM amiodarone, with maximal effects at 50-100 microM, when TSH-stimulated adenylate cyclase activity was completely blocked and the labeled TSH binding increased 4- to 5-fold over control. These effects of amiodarone were reversible, since membranes exposed to 50 microM amiodarone for 1 h exhibited normal binding and cyclase activities, when amiodarone was removed by washing before the assay. The above effects of amiodarone were also observed when cells, instead of membranes, were treated with the drug, although the magnitude of changes was less than in membranes. Lower concentrations of amiodarone (10-25 microM) caused significant inhibition of iodide organification, without affecting iodide uptake, while higher concentrations (50-100 microM) inhibited organification by nearly 75% and uptake by about 20%. Amiodarone (10-100 microM) also inhibited [3H]2-deoxy-glucose uptake and the increase in intracellular calcium concentration in response to TSH and carbachol. In contrast to membranes, treatment of cells with amiodarone caused persistent inhibition of TSH-stimulated cAMP formation and iodide organification even 24-48 h after removal of the drug. However, amiodarone had no effect on cell viability, as judged by trypan blue exclusion and ability to remain attached to the culture dishes. These results suggest that amiodarone has specific inhibitory effects on agonist-stimulated functions in thyroid cells, possibly by interfering with TSH-receptor interactions and also at the level of cholinergic receptors.

We have shown previously that long-term ethanol treatment causes an up-regulation of N-methyl-D-aspartate (NMDA) receptor 2B subunit (NR2B) number and function in cultured fetal mouse cortical neurons. To examine the intracellular signaling pathways involved in this NR2B gene transcription, we have subjected fetal cortical neurons to long-term treatment with ethanol and studied its effect on cAMP response element-binding protein (CREB) and extracellular signal-regulated kinase (ERK) levels by Western blot and enzyme-linked immunosorbent assay. We find a significant increase in phosphorylated CREB, without change in total CREB protein, in cells treated with ethanol for 5 days. Long-term ethanol treatment did not increase levels of both total and phospho-ERK in serum-free medium, whereas it did increase ERK phosphorylation in medium containing serum, without affecting total ERK levels. CREB phosphorylation was increased by ethanol treatment in both media, irrespective of the presence of serum. Electrophoretic mobility shift assay, using a 25-base pair (bp) double-stranded DNA fragment containing the cyclic AMP response element (CRE)-like sequence of the NR2B promoter as (32)P-labeled probe, showed an increase in specific CRE binding to nuclear proteins isolated from cells undergoing long-term ethanol treatment. A 467-bp DNA fragment of the NR2B promoter containing the CRE sequence cloned into the luciferase vector exhibited high reporter activity in transient cotransfection assay of mouse cortical neurons, and ethanol treatment increased this activity. Introducing site-directed mutation in the CRE sequence significantly reduced the reporter activity relative to the wild-type construct, and it also abolished the stimulatory effect by ethanol. Our results indicate that CREB is probably involved in mediating ethanol-induced up-regulation of NR2B gene.

Odontoblasts and osteoblasts produce similar highly mineralized extracellular matrices. In bone, osteoblasts/stromal cells regulate osteoclast (ocl) formation and bone resorption by producing factors like osteoprotegerin (OPG), osteoclast differentiating factor (ODF/RANKL), and macrophage colony-stimulating factor (M-CSF) that interact with hematopoietic ocl precursor cells. Using odontoblast and pulp cell lines, we detected a constitutive expression of OPG, RANKL, and M-CSF mRNA in both cell types. OPG and RANKL proteins were also detectable. In vivo, RANKL and OPG were localized to odontoblasts, ameloblasts, and pulp cells in developing mouse teeth by immunohistochemistry. In a coculture system, we found the dental cells to be inhibitory to ocl formation from spleen and bone marrow precursors, despite their production of osteoclast stimulatory factors. Our data indicate for the first time that dental cells express factors important in regulation of osteoclastogenesis and bone resorption. Since both stimulatory (RANKL, M-CSF) and inhibitory (OPG) factors are expressed, a balance between positive and negative factors may contribute to regulation of bone resorption.

The mechanism and site(s) of the defect responsible for desensitization to hormone stimulation of adenylyl cyclase (AC) vary with cell type. Plasma membrane preparations were assayed after treatment of primary cultured dog thyroid cells to determine the role of the TSH receptor, stimulatory and inhibitory guanine nucleotide binding proteins (Gs and Gi), and catalytic unit in AC desensitization. Exposure of cells to TSH or the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), caused time dependent decreases in TSH-stimulated AC and [125I]TSH binding with approximately 50% decreases seen after 18 h; Bt2cAMP was unable to reproduce the TSH effect. Whereas TSH treatment caused concomitant decreases (approximately 25%) in both cyclase activity and [125I]TSH binding after 2 h, TPA treatment decreased AC activity after 6 h and binding only after 18 h. The protein kinase C inhibitor, H-7, prevented TPA-induced but not TSH-induced effects on AC and hormone binding. Membrane AC activation by cholera toxin or forskolin was not altered by 18 h pretreatment of cells with TSH or TPA, indicating that these agents had no apparent effect on intrinsic functionality of either Gs or the catalytic unit. TSH or TPA pretreatment of cells reduced subsequent toxin-mediated AD[32P]-ribosylation of Gs and Gi in isolated membranes. However, the TSH- and TPA-induced decreases in AD[32P]-ribosylation and desensitization do not appear to be due to endogenous ribosylation of G proteins, since treatment of cells with pertussis toxin, for example, to endogenously ribosylate Gi, both increased TSH-stimulated AC activity and failed to affect the ability of TSH or TPA to desensitize. Thus, in this system, although specific hormone-induced AC desensitization and receptor down-regulation conform to several aspects of classic homologous processes, similar effects are also induced by a nonreceptor (phorbol ester) pathway; desensitization, however, can precede down-regulation, possibly due to receptor-Gs uncoupling.

Publisher Summary This chapter discusses acute stimulatory effects of luteinizing hormone-releasing hormone (LHRH) and LHRH analogues on the preovulatory rat follicle. In a study described in the chapter, immature Sprague–Dawley rats, 26 days old, were treated with 10 IU PMSG. This treatment resulted in the ovulation of 12–18 ova early on the morning of day 29. Ovarian follicles or granulosa cells were isolated on the morning of day 28. The follicles were incubated in Krebs–Ringer bicarbonate buffer with glucose and 1% bovine serum albumin. Granulosa cells were cultured in Eagle's minimal essential medium with Hepes and 10% fetal calf serum. For the in vivo experiments, the rats were hypophysectomized on the morning of day 28. It was found that mammalian oocytes remain arrested in the prophase stage of the first meiotic division until meiosis is resumed in the preovulatory follicle following the LH surge. Oocyte meiosis can also be initiated in vitro by the addition of gonadotrophin to isolated preovulatory rat follicles.

The effect of TSH, carbachol (CC), and ATP on intracellular calcium concentration ([Ca2+]i) in primary cultures of dog thyroid cells was examined using the fluorescent Ca2+ indicator fura-2. TSH caused an increase in [Ca2+]i at 37 C, but not 22 C, while it increased cAMP formation in these cells at both 22 and 37 C. CC and ATP increased [Ca2+]i at both 22 and 37 C. The CC-induced increase in [Ca2+]i was under muscarinic receptor control, and it was biphasic, with an initial spike followed by a sustained increase at a lower level. TSH and ATP were weaker agonists compared to CC, since maximal doses of TSH (100-500 mU/ml) and ATP (100-500 microM) increased [Ca2+]i by 40-70% over basal levels, compared to a 2- to 4-fold increase in [Ca2+] induced by maximal doses of CC (10-50 microM). The TSH-induced increase in [Ca2+]i was transient, returning to basal levels within 1-2 min after application of the agonist. All three agents were able to transiently increase [Ca2+]i to be internal stores. In the presence of the inorganic Ca2+ channel blockers La3+, Ni2+, and Co2+, the peak [Ca2+]i change was little affected, while the persistent response to CC and ATP was blocked, indicating dependence of this phase on influx of Ca2+. Paradoxically, these channel blockers abolished the effect of TSH on [Ca2+]i. TSH stimulation of cAMP formation was also inhibited 80-90% by these blockers, but not in Ca2+-free/EGTA buffer. These results suggest that the Ca2+ channel blockers may have actions in addition to inhibition of Ca2+ entry in these cells. TMB-8 [8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate HCl] specifically blocked both the initial and sustained increase induced by CC, while having no effect on ATP or TSH-induced [Ca2+]i, suggesting that TMB-8 may not be a general antagonist of Ca2+ mobilization. Activators of protein kinase-C, such as phorbol esters or an analog of diacylglycerol, inhibited the [Ca2+]i rise induced by all the three agonists used, indicating a regulatory role of protein kinase-C activation on [Ca2+]i in these cells. In FRTL-5 cells, [Ca2+]i was also increased by TSH and ATP, but not by CC. ATP, however, was a more effective agonist than in dog thyroid cells, while TSH increased [Ca2+]i by a similar magnitude in both cell types. The results of the present study demonstrate that TSH, albeit of lesser potency than CC, increases [Ca2+]i by causing intracellular Ca2+ mobilization in cultured dog thyroid cells.(ABSTRACT TRUNCATED AT 400 WORDS)

The effect of neutralizing FSH or LH on ovarian lipids in the cycling hamster was studied. In the normal cycling hamster on the day of proestrus, histochemical examination revealed the presence of sudanophilic lipids in the granulosa cells of the follicles and in the interstitium. A clear reduction in the intensity of lipid staining was observed on proestrus in the ovary of hamsters treated with FSH antiserum on the previous proestrus. Similar treatment with antiserum to LH, on the other hand, caused an accumulation of lipids in these structures. Estimation of the free and esterified fractions of cholesterol and triglycerides in the nonluteal tissue of the ovary of hamsters on proestrus following treatment with FSH antiserum on the previous proestrus revealed a significant reduction in all 3 lipid components. Even a short term deprivation of FSH caused a similar reduction in these lipids in the ovary. In contrast, treatment with LH antiserum either on the previous proestrus or on the previous day (diestrus-2) resulted in an enhancement in esterified cholesterol and triglycerides, while it caused a reduction in the free cholesterol fraction of the ovary on proestrus.It is suggested that though treatment with antisera to either FSH or LH causes a disruption in follicular maturation, their effect on lipid metabolism is different. A positive role for FSH and LH in maintaining normal sterol and triglyceride levels in the nonluteal ovarian tissue of cycling hamster is indicated.

Initiation of follicular growth by specific hormonal stimuli in ovaries of immature rats and hamsters was studied by determining the rate of incorporation of3H-thymidine into ovarian DNAin vitro. Incorporation was considered as an index of DNA synthesis and cell multiplication.

The effects of prior exposure of normal rat adrenocortical cells to ACTH on the responsiveness of the cells to subsequent stimulation with the hormone have been studied. ACTH induces a time- and concentration-dependent refractoriness of both cAMP formation and steroidogenesis. Desensitization of either response was observed only upon activation of the response. Thus, both ACTH and 8-Br-cAMP caused desensitization of the steroidogenic response. The ACTH-induced desensitization of steroidogenesis, however, was completely prevented by blocking the steroidogenic action of ACTH with aminoglutethimide during exposure of cells to the hormone. Aminoglutethimide had no effect on ACTH-induced desensitization of the cAMP response. Studies with analogs of the hormone also confirmed that induction of desensitization of the steroidogenic response is independent of the desensitization of the cAMP response. Binding studies showed that the insignificant decrease in ACTH receptors could not account for the large changes in the responsiveness induced by prior exposure of cells to ACTH. Desensitization of the steroidogenic response appears to result from a defect in the rate-limiting first step of the steroidogenic pathway, namely conversion of cholesterol to pregnenolone.

The changes that occur during follicular growth and atresia in the lysosomal enzyme activities and in gonadotropin receptors of isolated granulosa cells were studied. At different intervals after an injection of pregnant mare serum gonadotropin (PMSG) to 21-day-old female rats, the granulosa cells (GC) were isolated and the total activity of cathepsin-D [9025-26-7], a representative lysosomal enzyme, and FSH [9002-68-0] receptor activity in terms of binding of $[^{125}I]oFSH$ to GC and the steroidogenic response of GC to FSH in vitro were detd. During the period of both follicular growth and atresia there was an inverse correlation between the lysosomal enzyme activity and FSH receptor activity of granulosa cells, the former being low during follicular growth or tropic phase when the levels of FSH receptors were increasing. During the atretic phase, beyond 48 h of PMSG treatment (perhaps due to the normal metabolic clearance of the injected PMSG), the cathepsin-D activity showed an increase while the FSH receptors were on the decline. That lack of the hormone was responsible for atresia was confirmed when an antiserum to PMSG was injected during the tropic phase (24 h after PMSG); this resulted in a sharp increase in cathepsin-D activity. Injection of xcess amts. of either FSH or LH [9002-67-9], along with or soon after PMSG antiserum, prevented the increase in cathepsin-D activity, and also resulted in the maintenance of gonadotropin receptors. This suggested that gonadotropins can rescue follicles from undergoing atresia. In the unprimed immature rat FSH, but not LH or hCG, mimicked the effects of PMSG, in terms of bringing about a redn. in cathepsin-D activity of GC. Also in such rats, neutralization of endogenous FSH with an antiserum to FSH resulted in an increase in the cathepsin-D activity of GC, suggesting that even the prepubertal rat ovary is dependent on the tropic support of FSH for prevention of atresia.

The effect of long-term passive immunization with a specific homologous antiserum to ovine FSH on testicular function in the adult bonnet monkey was studied. Testicular function was assessed by studying biochemical parameters such as hyaluronidase activity and the rate of incorporation of [3H]thymidine into DNA of testis in vitro. The above parameters were determined in one group of monkeys at 210 days and in another group at 130 days after initiation of antiserum treatment. The testis of both groups of monkeys showed a marked reduction in the rate of [3H]thymidine incorporation into testicular DNA as well as in hyaluronidase activity compared to the corresponding control animals. Further, on withdrawal of antiserum treatment in one set of monkeys, a clear recovery was observed in these parameters, thus demonstrating that the earlier observed effects were specifically due to lack of FSH. The results clearly indicate that chronic lack of FSH affects testicular function in the adult monkey and it is suggested that FSH could have a positive role in spermatogenesis in the adult sub-human primate.

Since 12-O-tetradecanoyl-phorbol-13-acetate (TPA) reproduced some of the effects of TSH on phosphorylation of polypeptides in the thyroid, its effects on several thyroid metabolic variables were investigated. Like TSH, TPA stimulated glucose oxidation, iodide organification, and 32P incorporation into phospholipids in thyroid slices. However, in contrast to TSH, it did not augment cAMP accumulation. An inactive phorbol ester, 4 alpha-phorbol, did not reproduce any of the effects of TPA. An initial incubation of thyroid slices with TPA decreased the stimulation of cAMP, glucose oxidation, and colloid droplet formation induced by TSH. However, an initial incubation with TPA did not modify the subsequent stimulation of glucose oxidation induced by (Bu)2 cAMP. TPA potentiated the ability of TSH to desensitize the adenylate cyclase system. Although both TPA and TSH increased 32P incorporation into phospholipids, the patterns were different when individual phospholipids were examined. These results indicate another regulatory mechanism for thyroid cell functions independent of cAMP.

The role of the proestrous surge of FSH and LH in the initiation of follicular maturation in the cyclic hamster was studied. Blockade of gonadotropin action was confined to the period of the surge by injecting specific antisera to FSH or LH at the beginning of the surge, followed by injection of the respective hormone at the end of the surge to neutralize any persisting excess antibody. The criteria used to assess the effect of such an acute neutralization of FSH or LH surge on follicular development were: 1) the occurrence of ovulation on the expected day of estrus at the end of the next cycle, considering that ovulation is the physiological endpoint of normal follicular development, and 2) the rate of [3H]thymidine incorporation into DNA in vitro by the non-luteal tissue of ovaries on the day after the surge (i.e., estrus). The latter was based on our observation that during the normal estrous cycle, the highest rate of incorporation of [3H]thymidine into ovarian DNA occurred at 1200 h of estrus, about ...

The induction of LH receptors by FSH in cultured rat granulosa cells and the effects of ovarian steroids on this process were examined. Granulosa cells were isolated from the ovaries of untreated immature rats (25 days old) and cultured with highly purified FSH (Sairam; 250 ng/ml). After culture (48-96 h in chemically defined media), both the binding of [125I]hCG and the responsiveness (cAMP and progesterone production) to an acute LH stimulus (100 ng/ml; NIH B8) were measured. The appearance of LH/hCG-binding sites and LH responsiveness indicates the presence of functional LH receptors. The induction of LH receptors by FSH requires a lag period of 24-48 h. After 48 h, the concentration of LH receptors in cultured granulosa cells continues to increase with time in culture with FSH; the continuous presence of FSH is required to maintain the induction process. If granulosa cells are cultured without hormone for 24-48 h before FSH is added, no induction of LH receptors occurs. However, if 17 beta-estradiol (5 X 10(-7) M) is added during this initial period, then the cells are responsive to the later addition of FSH. This maintenance of FSH responsiveness is not observed when dihydrotestosterone or progesterone is substituted for 17 beta-estradiol in the initial culture period. The FSH-dependent induction of LH receptors in cultured rat granulosa cells can be blocked if an inhibitor of steroidogenesis, such as aminoglutethimide phosphate (AGP; 1 mM), is added along with FSH at the initiation of the culture. The inclusion of progesterone, dihydrotestosterone, or 17 beta-estradiol (but not 20 alpha-dihydroprogesterone) in the culture together with FSH and AGP will overcome the inhibition by AGP and restore the induction of LH receptors. The results suggest that steroids produced by the developing follicle can modulate the FSH-dependent induction of LH receptors, and this may play a role in the development of follicular responsiveness to LH.

We recently reported that treatment of mouse Leydig cell cultures for 5 days with LH or cAMP caused an induction of the microsomal cytochrome P-450 activities 17 alpha-hydroxylase and C17-20 lyase. We also have shown that the microsomal P-450s are very sensitive to oxygen-mediated loss of these activities and that this decrease is enhanced by steroids produced during acute cAMP stimulation of Leydig cells. In the present study, we investigated whether steroids produced during chronic cAMP treatment of Leydig cells limit the extent of induction of 17 alpha-hydroxylase and C17-20 lyase. Treatment of Leydig cell cultures with 8-bromo-cAMP in the presence of aminoglutethimide, an inhibitor of cholesterol side-chain cleavage, resulted in a 4- to 7-fold enhancement of cAMP-mediated induction of the 17 alpha-hydroxylase and C17-20 lyase activities and, after 11 days of treatment, completely restored the activities to those found in freshly isolated Leydig cells. Treatment with aminoglutethimide in the absence of cAMP had no effect on these enzyme activities. Addition of the steroid products androstenedione and/or testosterone (5 microM) to cAMP-plus amino-glutethimide-treated cultures caused a significant reduction in cAMP-mediated induction of microsomal P-450, while addition of estradiol (50 nM) had no significant effect. 3 beta-Hydroxysteroid dehydrogenase-isomerase, another microsomal enzyme that is not a P-450 enzyme, was not induced by cAMP in either the presence or absence of aminoglutethimide. The data suggest that Leydig cell microsomal P-450 activities are maintained in vivo by a balance between two processes: cAMP-mediated induction and steroid product-induced degradation.

The levels of cholesterol in the fore, mid and hind brain regions were determined in normal and Pars distalis ablatedRana cyanophlictis, at various time intervals after the operation. The time course of the effects of removal of Pars distalis showed an initial overshoot (shock effect) and a recovery phase leading to a new stabilized stage. In general ablation of Pars distalis resulted in the elevation of the levels of cholesterol during the first week after the operation (P>0·05, 0·001 and 0·001 for the fore, mid and hind brain on the first day after the operation). Regional differences in the magnitude of response were significant. A possible relationship between adrenal steroids and thyroid hormones and cholesterol synthesis is suggested.

The effect of TSH, phorbol ester, norepinephrine (NE), and carbachol, agents known to influence thyroid metabolism, was compared on iodide organification in dog thyroid slices, freshly isolated follicles, and cultured cells. TSH stimulated iodide organification in all three types of preparations, and this effect was mimicked by (Bu)2cAMP. In contrast, the phorbol ester, tetradecanoyl phorbol acetate (TPA) stimulated iodide organification in slices and follicles but inhibited it in cells. The dose and time required for these divergent effects were similar. Other stimulators of protein kinase C such as aplysiatoxin and teleocidin mimicked the effects of TPA, and these effects were partially reversed by H-7, an inhibitor of protein kinase C. Pretreatment of cells with TPA for 4 h did not affect TSH-stimulated cAMP production, but TPA inhibited iodide organification in cells even in the presence of TSH or (Bu)2cAMP. Similarly NE and carbachol stimulated iodide organification in follicles but inhibited it in cells under basal as well as TSH-stimulated conditions. These effects of NE and carbachol were via alpha 2-adrenergic and muscarinic cholinergic receptors, respectively. However, NE and carbachol inhibited TSH-stimulated cAMP production in both follicles and cells. In thyroid cells, carbachol inhibited uptake and increased efflux of iodide within minutes. TPA also produced similar effects after longer periods of incubation, where an inhibition of uptake was seen by 1 h and an increase in efflux by 2 h. NE had a marginal inhibitory effect on uptake and had no effect on efflux of iodide. In contrast to these agents, TSH increased uptake but not efflux of iodide. The present results suggest that the response of the freshly isolated tissue to phorbol esters, NE and carbachol differs from that of cells in culture with respect to an important metabolic function of the thyroid gland. These agents seem to have direct effects on iodide transport and organification unrelated to their effects on cAMP production.

The effect of an agonistic gonadotropin releasing hormone (GnRH) -analog (D -Ala’ ,desGly’#{176} -NH3 -GnRH -ethylamide, GnRHa) on granulosa cell steroidogenesis in the presence or absence of follicle-stimulating hormone (FSH) or luteinizing hormone (LH) was studied. Granulosa cells, isolated from preovulatory follicles of pregnant mare’s serum gonadotropin (PMSG) -treated immature rats or from the less mature follicles of untreated immature rats, were cultured for a period of 72 h with daily changes of medium, and progesterone and its metabolite, 20a -dihydro progesterone (20o-OHP), were assayed in the medium. In granulosa cells from preovulatory follicles, LH and FSH caused a much greater stimulation of steroidogenesis than did GnRHa. There appeared to be no interaction between GnRHa and FSH during the first 10 h, but at 24 h and later the presence of GnRHa clearly inhibited the steroidogenic response to LH and FSH. Steroidogenesis in granulosa cells from immature rats was considerably lower and the effects of GnRHa and FSH alone less pronounced. In these cells, FSH #{149}stimulated progesterone secretion was inhibited by GnRHa only at 72 h. In contrast, 20o-OHP secretion in the same cultures was potentiated by the combined presence of FSH and GnRHa. In conclusion, it seems as though the effects of GnRHa on granulosa cell steroidogenesis varies with exposure time, the initial response being stimulatory and the later inhibitory. Further more, the response is also to some extent determined by the maturational stage of the granulosa cells.

The ability of FSH to stimulate the activity of ornithine decarboxylase (ODC) in the ovary of the immature rat and cycling hamster has been examined using specific antisera to gonadotropins. The stimulatory effect of FSH on ODC activity in the ovary of the immature rat was abolished when LH antiserum was administered along with FSH, while similar administration of FSH antiserum had no effect on LH action in stimulating ODC activity, thereby demonstrating the specificity of the LH effect. During the estrus cycle of the hamster, ODC activity in the ovary could be detected only on the evening of proestrus, the maximal activity seen at 1700 h being associated with both the Graafian follicles and the rest of the ovarian tissue. Neutralization of the proestrous FSH surge had no effect on the activity of ODC in either of these tissues, while similar administration of LH antiserum at 1300 h of proestrus completely inhibited the ODC activity in both large follicles and the rest of the ovarian tissue. Thus, the surge of LH, but not of FSH, appears to be responsible for regulating the ODC activity in the ovary of the cycling hamster.

Cyclic monkeys were deprived of FSH for specific periods on different days of the follicular phase by injecting them with minimal doses of an FSH antiserum characterized for specificity and bioneutralizing ability. The effect of the antiserum on follicular maturation was assessed by determining (a) serum oestrogen concentrations through the midcycle period, (b) serum progesterone concentrations as an index of ovulation and luteal function, (c) laparoscopic examination of the surface of the ovary when necessary, and (d) overall cycle length. While antiserum injection on Day 5 of the cycle caused delay in the oestrogen surge from Days 9 to 11, injection on Day 6 led to the occurrence of two oestrogen surges, on Days 9 and 14. Laparoscopic examination showed that the earlier follicle had disappeared and a new follicle had appeared by Day 14. Antiserum injection on Day 7 of the cycle arrested further growth of the maturing follicle, but a new follicle appeared 9 days later, as indicated by a surge of oestrogen on Day 16. Injection of antiserum beyond Day 7 had no effect on follicular development, ovulation and luteal function. These observations suggest that the mature follicle becomes relatively independent of FSH support about 48 h before ovulation and this event could be a marker for follicular dominance.

Previous studies have shown that gonadotrophin-releasing hormone (GnRH) can induce resumption of meiosis in follicle-enclosed rat oocytes. In the present study a GnRH antagonistic analogue ([d-pGlul, d-Phe2,-d-Trp3,6]LRF) was found to effectively abolish the stimulatory effect of a GnRH agonist upon resumption of meiosis and lactate accumulation in isolated pre-ovulatory rat follicles but the have no effect on LH stimulation of these parameters. It is concluded that although LH and GnRH can evoke a similar response they act through separate receptor sites and that it is unlikely that GnRH mediates the effect of LH on meiosis or glycolysis.

Publisher Summary This chapter discusses the preparation and characterization of gonadotropin antibodies. Antibodies to gonadotropic hormones by virtue of their ability to react with gonadotropins across species barriers are utilized in wide applications. Immunoassays of heterologous nature have provided information on the similar secretory patterns of gonadotropins in a wide variety of animals in different physiological states and under different stresses. The chapter discusses the causal relationship of gonadotropin levels to physiological function. Hormone antibodies are used as specific antagonists in studies on the physiological functions of hormones. An analysis of events occurring at midcycle or proestrus shows that the threshold of luteinizing hormone (LH) for induction of ovulation and perhaps initiation of luteinization is high, and this appears to be reflected by the surge. FSH surge occurring at the same time is involved in triggering a fresh wave of folliculogenesis and this has no direct association with the immediate ovulatory process. The need for FSH appears to be much more stringent at this phase than at any other time during follicular maturation. The role of LH during the follicular phase appears to be one of synergism with FSH, its need perhaps increasing as the follicle matures.

A method is described for monitoring the concentration of endogenous receptor-bound gonadotropin in the ovarian tissue. This involved development of a radioimmunoassay procedure, the validity of which for measuring all of the tissue-bound hormone has been established. The specificity of the method of measurement was indicated by the fact that high levels of FSH could be measured only in target tissue such as follicles, while non-target organs showed little FSH. Using this method, the amount of FSH in the non-luteal ovarian tissue of the hamster at different stages of the estrous cycle was quantitated and compared with serum FSH levels found at these times. No correlation could be found between serum and tissue FSH levels at all times. On the morning of estrus, for example, when the serum level of FSH was high, the ovarian concentration was low, and on the evening of diestrus-2 the ovary exhibited high concentration of FSH, despite the serum FSH concentration being low at this time. The highest concentration of FSH in the ovary during the cycle was found on the evening of proestrus. Although a large amount of this was found in the Graafian follicles, a considerable amount could still be found in the ‘growing’ follicles. Ovarian FSH concentration could be considered to be a reflection of FSH receptor content, since preventing the development of FSH receptors by blocking initiation of follicular development during the cycle resulted in a decrease in the concentration of FSH in the ovary. The high concentration of FSH in the ovary seen on the evening of diestrus-2 was not influenced either by varying the concentration of estrogen or by neutralization of LH. Neutralization of FSH on diestrus-2, on the other hand, caused a drastic reduction in the ovarian LH concentration on the next day (i.e. at proestrus), thus suggesting the importance of FSH in the induction of LH receptors.

The intracellular free calcium concentration, [Ca2+]i, has been measured in dog thyroid cells using the fluorescent Ca2+-indicator, quin2. Acetylcholine or its non-hydrolyzable analog, carbamylcholine rapidly increased [Ca2+]i by 40 +/- 4% (mean +/- SE) over the basal level of 81 +/- 2 nM. This increase was totally abolished by atropine, a muscarinic cholinergic receptor blocker, but was not influenced by verapamil, a voltage dependent-calcium channel blocker. Depletion of extracellular Ca2+ by the addition of EGTA, diminished but did not abolish the response to carbamylcholine. These data suggest that cholinergic effectors increase [Ca2+]i by mobilization of Ca2+ from intracellular stores rather than from an influx of Ca2+. Addition of TSH, isoproterenol, phorbol ester, dibutyryl cyclic GMP or cyclic AMP did not elicit any change in [Ca2+]i suggesting that their action may not involve any mobilization of intracellular Ca2+. These data provide direct evidence that in the thyroid cell, cholinergic agents act via their receptors to cause a rapid increase in [Ca2+]i, which may mediate their metabolic effects.

The effect of a GnRH analogue ((D-Ala6, des-Gly10-NH2)-GnRH-ethylamide,GnRHa) on granulosa and cumulus cell glycolysis in presence or absence of FSH was studied. Cumulus complexes and granulosa cells from PMSG-treated rats were cultured in Eagle's minimal essential medium (MEM) for a period of 72 h. Media were changed at 24 and 48 h and lactate content was assayed by fluorimetry. GnRHa alone stimulated lactate production in granulosa cells. GnRH combined with FSH increased lactate production in granulosa cells during the 0–24 h period and decreased it during the 48–72 h period as compared to FSH alone. GnRHa did not stimulate lactate production in cumulus complexes during 72 h culture in MEM, while FSH did. In a less complex culture medium, BMOC, GnRHa caused a small increase in lactate production and slightly enhanced the FSH effect. In conclusion, GnRHa has a direct stimulatory effect on granulosa cell glycolysis. GnRHa also modulates the FSH stimulation of granulosa cells biphasically, i.e. early enhancement (0–24 h) and late inhibition (48–72 h). GnRHa has no consistent direct effects on cumulus cell glycolysis.