The insulin-like growth factor system in mammalian pregnancy--a workshop report.
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Adult↗
Biomedical subjects
C S Watson
Publications and source records attributed to C S Watson.
Extracellular glutamate levels and neuropathology in cerebral white matter following repeated umbilical cord occlusion in the near term fetal sheep.
Umbilical cord occlusion causes fetal hypoxemia which can result in brain injury including damage to cerebral white matter. Excessive glutamate release may be involved in the damage process. This study examined the relation between extracellular glutamate levels in the cerebral white matter of the ovine fetus during and after intermittent umbilical cord occlusion and the degree of resultant fetal brain injury. Fetal sheep underwent surgery for chronic catheterisation and implantation of an intra-cerebral microdialysis probe at 130 days of gestation (term approximately 147 days). Four days after surgery (day 1), seven fetuses were subjected to 5x2 min umbilical cord occlusions, and on the following day (day 2) they were subjected to either 4 or 5x4 min umbilical cord occlusions; seven fetuses served as controls. Microdialysis samples were collected before, during and after the umbilical cord occlusions to determine extracellular glutamate levels in the cerebral white matter. Fetal blood gas status was measured and the fetal electrocorticogram was recorded continuously. During the periods of umbilical cord occlusions on both days 1 and 2, fetal arterial oxygen saturation, arterial partial pressure of oxygen and arterial pH decreased (P<0.05) while arterial partial pressure of carbon dioxide increased (P<0.05). All fetuses showed episodes of isoelectric electrocortical activity during umbilical cord occlusions on both days 1 and 2. In fetuses with patent microdialysis probes there were marked increases of glutamate efflux in the cerebral white matter following umbilical cord occlusion. Fetal brains were removed at autopsy on day 5 and subjected to histological assessment. Brain damage was observed in all fetuses exposed to cord occlusion, particularly in the periventricular white matter, with the most extensive damage occurring in the fetuses with the greatest increases in glutamate levels. We conclude that, in the unanesthetised fetus in utero, glutamatergic processes are associated with umbilical cord occlusion-induced brain damage in the cerebral white matter.
Animals↗
A comparison of membrane vs. intracellular estrogen receptor-alpha in GH(3)/B6 pituitary tumor cells using a quantitative plate immunoassay.
The plasma membrane form of the estrogen receptor-alpha (mER-alpha) is involved in rapid estrogen-induced prolactin release from GH(3)/B6 rat pituitary tumor cells and can be detected immunocytochemically using several estrogen receptor-alpha (ER-alpha) antibodies. We recently described staining of fixed cells via a biotin-avidin-alkaline phosphatase sandwich assay. From this protocol, we have developed a rapid, quantifiable 96-well plate immunoassay for mER-alpha, using a different alkaline phosphatase substrate, para-nitrophenylphosphate, which generates a soluble yellow product, para-nitrophenol. We also permeabilized cells with detergent during fixation to measure intracellular ER-alpha (iER-alpha) with the same assay and then compared intracellular versus membrane ER-alpha levels in two GH(3)/B6 cell subclones originally selected for high and absent mER-alpha expression by immunocytochemistry. While the F10 subclone expresses plentiful amounts of the mER-alpha, the D9 subclone has undetectable levels of mER-alpha using this assay. In addition, there is a seven-fold difference in iER-alpha expression between the high (F10) and no (D9) mER-alpha expressing subclones. In the high mER-alpha expressing cell line, the mER-alpha totals approximately one third of total cellular ER-alpha. Neither membrane or intracellular forms of ER-beta were detected with this assay. The pNp assay allows convenient and quantitative comparison of multiple parameters of mER-alpha and iER-alpha regulation and should be applicable to other antigens that are expressed on the cell surface as well as intracellularly.
Analysis of Variance↗
Membrane estrogen and glucocorticoid receptors--implications for hormonal control of immune function and autoimmunity.
Membrane steroid receptors (mSRs) have recently re-emerged as candidates for mediating steroid effects which do not fit the paradigm of nuclear transcription factor mechanisms. We have studied two steroid-binding classes of mSRs, and have noted striking similarities in their characteristics (immunocytochemical appearance, biochemical properties, proteolytic sensitivity, signaling pathways, regulation, and molecular origins). These observations strengthen the conclusion that mSRs can be modified versions of intracellular steroid receptors. The membrane estrogen receptors (mERs) we studied are involved in estrogen-induced release of prolactin. Membrane glucocorticoid receptors (mGRs) in both mouse and human lymphoma cells are necessary for the initiation of glucocorticoid-induced therapeutic apoptosis which is related to the developmental phenomenon of thymic involution. Diseases of autoimmunity such as systemic lupus erythematosus and arthritis are related to estrogen status. Since both of these mSRs have recently been found in both normal and cancerous lymphoid cells, actions of these mSRs may have important consequences for functions and diseases of the immune system. Therefore, the study of these forms of steroid receptors may present novel therapeutic opportunities for the use of steroids and steroid analogs.
Animals↗
Individual differences in the processing of speech and nonspeech sounds by normal-hearing listeners.
While a large portion of the variance among listeners in speech recognition is associated with the audibility of components of the speech waveform, it is not possible to predict individual differences in the accuracy of speech processing strictly from the audiogram. This has suggested that some of the variance may be associated with individual differences in spectral or temporal resolving power, or acuity. Psychoacoustic measures of spectral-temporal acuity with nonspeech stimuli have been shown, however, to correlate only weakly (or not at all) with speech processing. In a replication and extension of an earlier study [Watson et al., J. Acoust. Soc. Am. Suppl. 1 71. S73 (1982)] 93 normal-hearing college students were tested on speech perception tasks (nonsense syllables, words, and sentences in a noise background) and on six spectral-temporal discrimination tasks using simple and complex nonspeech sounds. Factor analysis showed that the abilities that explain performance on the nonspeech tasks are quite distinct from those that account for performance on the speech tasks. Performance was significantly correlated among speech tasks and among nonspeech tasks. Either, (a) auditory spectral-temporal acuity for nonspeech sounds is orthogonal to speech processing abilities, or (b) the appropriate tasks or types of nonspeech stimuli that challenge the abilities required for speech recognition have yet to be identified.
Adolescent↗
Sources of variation in profile analysis. I. Individual differences and extended training.
This study investigated two sources of variance in the ability to discriminate auditory profiles: individual differences and extended training. The goals of the study were (1) to determine the range and origins of individual differences in profile analysis and (2) to determine whether those who initially had poor sensitivity to changes in spectral shape could eventually acquire finer sensitivity. Profile stimuli had 11 components with equal-log spacing from 200-2200 Hz. Thresholds ranged from - 1 to -25 dB (signal level relative to the context level) across 46 listeners. The correlation between spectral-shape discrimination thresholds after 2000 trials and pure-tone intensity-discrimination thresholds was 0.36. The range of individual differences for pure-tone intensity discrimination and spectral-shape discrimination was about the same. Two groups of listeners were given extended practice on the profile task, one group that showed low thresholds after an initial 2000 trials of practice and another that showed much higher initial thresholds. All listeners improved during the course of the first 2000 trials of training. Most of the poor listeners continued to improve during 9000 trials of training. Individual differences in the listeners' sensitivity to changes in spectral shape still existed after the extended practice.
Adult↗
Sources of variation in profile analysis. II. Component spacing, dynamic changes, and roving level.
Profile-analysis experiments have typically employed static profiles with constant frequency components spaced at equal intervals along a logarithmic frequency axis. Most periodic, naturally occurring stimuli, however, have components that are harmonically related and vary dynamically in time. One goal of these studies was to determine whether amplitude-increment detection thresholds are different in dynamic, harmonically spaced profiles compared to those for static-log profiles, and why such differences might exist. A second goal was to determine the impact of roving levels (within-trial variation of level). Thresholds for static-log profiles were, on average, 8.7 dB lower than for static-harmonic profiles. A traditional filter-bank model could not account for this result. No consistent effect of dynamic contour (an exponential rising frequency glide) was observed. Thresholds were consistently poorer by 4 to 7 dB when the level was roved, but the differences in thresholds among the different profiles varied little. It is proposed that the higher thresholds observed in static-harmonic profiles may be accounted for by the more intense pitch strength associated with the harmonic profiles.
Adolescent↗
5'UTR sequences of the glucocorticoid receptor 1A transcript encode a peptide associated with translational regulation of the glucocorticoid receptor.
We have recently reported that glucocorticoid receptor (GR) transcript 1A, one of the five mouse GR splice variants (1A-1E), encodes membrane GR (mGR), which subsequently participates in mediating the apoptotic effects of glucocorticoids (GCs); all transcripts vary at their 5'UTR. Computer analysis of the entire1026 bp comprising the 5'UTR of transcript 1A identified five putative translation start sites at positions 85, 217, 478, 628, and 892 with the potential to encode peptides of 33, 93, 6, 18, and 41 amino acids, respectively. We then separately generated point mutations at these five upstream AUG codons of the GR 1A cDNA and performed in vitro transcription/translation experiments to investigate the regulatory effects of these sites on GR synthesis. GR translation products were immuno-captured with BUGR-2 antibody (Ab), then subjected to Western blot analysis. Mutation of the uAUG codon-2 completely inhibited GR synthesis, while mutations at the other four uAUG codons had no significant effect on the translation of transcript 1A. Antibodies (Abs) against the uORF-2 and uORF-5 protein products were used to perform Western blot analysis on cytosolic proteins from S-49 cells (which express GR transcript 1A), U937 cells transfected with GR 1A cDNA, or in vitro translation products from this cDNA. This assay identified an intense immunoreactive band of approximately 8.5 kDa recognized only with Ab to the uORF-2 peptide; this size is consistent with the computer-predicted size of the uORF-2 product, suggesting that the uORF-2 product is indeed synthesized in cells. No peptide was identified with Ab to uORF-5 peptide. Indirect fluorescent Ab staining, confocal microscopy and FACS analysis all showed that the ORF-2 peptide is localized both in the interior of the cell and at the plasma membrane. Using Ab to ORF-2 peptide for immunoadsorption we then asked whether cellular factors interact with the product of uORF-2. Immuno-captured uORF-2 peptide levels correlated with the concentrations of several salt-wash-sensitive cellular proteins, suggesting that protein-protein interactions occur between this upstream open reading frame (uORF) product and other factors. The uORF-2 product, however, does not appear to directly interact with GR, since there was no reciprocal immuno-capture between these two proteins. In summary, our results show that cells can synthesize the uORF-2 peptide, blocking of the synthesis of the uORF-2 peptide product abolishes translation of GR from the GR 1A transcript, and the peptide product of uORF-2 interacts with other cellular factors which might be involved in translation of GR.
5' Untranslated Regions↗
Antibodies to the estrogen receptor-alpha modulate rapid prolactin release from rat pituitary tumor cells through plasma membrane estrogen receptors.
Antibodies (Abs) raised against the estrogen receptor-alpha (ERalpha) were used to investigate the role of ERalpha proteins located at the plasma membrane in mediating the rapid, estrogen-stimulated secretion of prolactin (PRL) from rat pituitary GH(3)/B6/F10 cells. Exposure of the cells to 1 nM 17beta-estradiol (E(2)) significantly increased PRL release after 3 or 6 min. When ERalpha Abs that bind specifically to ERalpha but are too large to diffuse into cells were tested for activity at the cell membrane, Ab R4, targeted to an ERalpha hinge region sequence, increased PRL release in a time- and concentration-dependent fashion. Ab H151, directed against a different hinge region epitope, decreased PRL release and blocked the stimulatory action of E(2). Abs raised against the DNA binding domain (H226) or the carboxyl terminus (C542) were not biologically active. When each Ab was examined for recognition of ERalpha on the cell surface by immunocytochemistry, all except H151 generated immunostaining in aldehyde-fixed cells. In live cells, however, Ab H151 but not Ab R4 blocked the membrane binding of fluorescently tagged E(2)-BSA. Overall, the data indicate that plasma membrane ERalpha proteins mediate estrogen-stimulated PRL release from GH(3)/B6/F10 cells. These results may also convey information about conformationally sensitive areas of the membrane form of ERalpha involved in rapid, nongenomic responses to estrogens.
Amino Acid Sequence↗
Perimembrane localization of the estrogen receptor alpha protein in neuronal processes of cultured hippocampal neurons.
There is clear evidence of rapid, nongenomic responses to estrogen in a variety of neuronal model systems. To address the question of whether some of these rapid estrogen signals might be transduced by the classical estrogen receptor (ER) alpha or a closely related protein in nontransformed neurons, we undertook the present study using isolated fetal rat hippocampal neurons. Several antibodies developed to detect ERalpha were tested in this system and showed positive membrane staining in nonpermeabilized neurons. MC-20, an affinity purified anti-ERalpha, rabbit polyclonal IgG antibody which does not recognize ERbeta was selected to carry out the majority of the experiments. When permeabilized, the hippocampal neurons exhibited low levels of nuclear staining for ERalpha, but abundant labeling for ERalpha throughout the entire cell including the neurites. In addition to traditional immunocytochemistry controls, incubation of neurons for 24 h in the presence of 10 microM antisense oligonucleotide directed against the translation start site of ERalpha reduced ERalpha immunoreactivity throughout the neurons providing further evidence that the immunostaining was specific for ERalpha. Confocal and conventional microscopy demonstrated that the antigen was predominately extranuclear and localization of ERalpha in the neurites suggests that the receptor is in close proximity to the plasma membrane. This localization is consistent with a role for ERalpha as a transducer of rapid, nongenomic estrogen responses in hippocampal neurons.
Amino Acid Sequence↗
Signaling themes shared between peptide and steroid hormones at the plasma membrane.
Steroid hormone receptors are primarily thought of as transcriptional regulators that are activated by ligand binding. However, there is also evidence for "nongenomic" actions of steroid hormones. Cheryl Watson summarizes a recent meeting that brought together researchers exploring these rapid effects of steroid hormones that occur in the absence of new protein synthesis. Current evidence for the existence of membrane steroid receptors, which may or may not be related to the well-known nuclear receptors, is discussed.
Animals↗
Multiple glucocorticoid receptor transcripts in membrane glucocorticoid receptor-enriched S-49 mouse lymphoma cells.
A cDNA library from plasma membrane glucocorticoid receptor-enriched (mGR(++)) S- 49 mouse T lymphoma cells was screened with full-length rat intracellular GR (iGR) cDNA, BUGR-2 antibody, and PCR amplimers to portions of the mouse GR cDNA. One or two single-base substitutions resulting in amino acid changes (which do not incapacitate the receptor) were found in all but one clone: Val437 --> Gly (located in the first zinc finger), and Glu546 --> Gly (in the steroid-binding domain). Two previously unidentified exon 1 variants (1D and 1E), and two of three previously reported variants (1A, 1B) were found to be spliced onto the common exon 2. Exon 1D- and 1E-containing transcripts were confirmed by direct sequencing of amplimers from reverse transcriptase-coupled PCR. RNase protection studies revealed that one of these transcripts was expressed in mGR(++) cells only, but not in two mGR-less (mGR(--) S-49, and AtT-20 mouse pituitary) cell lines. These studies suggest that at least four promoters may be responsible for the control of GR (iGR and mGR) types in mouse lymphoma cells.
5' Untranslated Regions↗
Association of the glucocorticoid receptor alternatively-spliced transcript 1A with the presence of the high molecular weight membrane glucocorticoid receptor in mouse lymphoma cells.
Using the combination of a cDNA library prepared from membrane glucocorticoid (mGR)-enriched S-49 cells and a mouse leukocyte genomic library, we have cloned a 7.3 kb full-length glucocorticoid receptor 1A cDNA. Primer extension, 5'RACE, and long distance PCR identified the transcription start site as being located at 1026 bp from the ATG codon. The first 1,013 nucleotides (nts) of the full length sequence constitute 5' UTR sequence (exon 1), the next 2349 bp, the coding region, and the last 3,907 bp, the 3'UTR. The entire 5'UTR sequence is unique to transcript 1A. The 3'UTR sequence is approximately 88.5 % conserved with the rat 3'UTR. Western blot analysis compared the molecular weight of in vitro translation products from the cloned 1A cDNA with partially purified cellular mGR. Both preparations contained the novel 150 KD and the 94 KD classical GR peptides, suggesting that transcript 1A encodes both receptor forms. Transfection of mGR-less and glucocorticoid lysis-resistant AtT-20 and HL-60 cells with full-length GR 1A cDNA imparted both mGR expression and glucocorticoid lysis-sensitivity to these cells.
3' Untranslated Regions↗
Immunoaffinity isolation of native membrane glucocorticoid receptor from S-49++ lymphoma cells: biochemical characterization and interaction with Hsp 70 and Hsp 90.
The membrane glucocorticoid receptor (mGR), previously correlated with glucocorticoid-induced lymphocytolytic competency, was purified under nondenaturing conditions from mGR-enriched mouse S-49 T lymphoma cells. Proteins were immunoaffinity batch adsorbed to BUGR-2 monoclonal antibody-coupled protein A Sepharose 4B beads, and elution by epitope competition was compared with standard denaturation procedures. Elution with BUGR-2 epitope peptides released multiple mGRs (42-150 kDa) and heat shock proteins 70 and 90, suggesting that mGR interacts with these protein chaperones under physiological conditions. The mGR-heat shock protein 90 interaction was inhibited by 1 microM geldanamycin. Several other mGR binding partners were captured and most were dissociated from mGR by 0.6 M salt. Peptide maps of purified mGR displayed immunoreactive bands unique to mGR. Scatchard analysis estimated a k(d) value of 239 nM and a Bmax of 384 fmol/mg protein for mGR, compared to a k(d) of 19.5 nM and a Bmax of 90.3 fmol/mg protein for the intracellular GR (iGR). The rank order of affinities for mGR were RU-486 > dexamethasone > triamcinolone acetonide = aldosterone. Other steroids had no significant binding affinity. These results show that epitope-purified mGR on the plasma membrane of mouse lymphoma cells is similar but not identical to iGR.
Aldosterone↗
Membrane-initiated steroid actions and the proteins that mediate them.
Membrane-initiated or nongenomic activities of steroids contribute to the effects of steroids on a wide variety of target organs, including those with low receptor numbers, recently discovered due to the increased sensitivity of new measurement techniques. Membrane-initiated responses are the cell's rapid and first response to steroids. The responses that emanate from the membrane can have direct functional consequences, such as secretion of other peptide hormones or rapid behavioral changes. Other rapid responses are prerequisites for subsequent genomic responses. The wide variety of signal transduction schemes employed by various tissues and hormones are summarized and discussed in terms of the identity of proteins that mediate these responses. Probable mixed binding systems for steroids in plasma membranes are compared to similar multiple hormone-binding protein systems in extracellular fluids and inside cells. These issues are related to steroid-dependent tumor growth, developmental and therapeutic apoptosis, and the actions of endocrine disrupters. The integration of membrane-initiated effects with genomic mechanisms results in the complete cellular response to steroids.
Animals↗
Membrane oestrogen receptors on rat pituitary tumour cells: immuno-identification and responses to oestradiol and xenoestrogens.
Our laboratory has identified plasma membrane oestrogen receptors on a GH3/B6 rat pituitary tumour cell line and several sublines which produce rapid (within minutes), non-genomic responses to oestrogens. Oestrogen receptors have been identified by their binding to nine different antibodies (Abs) which together recognize at least seven epitopes on the oestrogen receptor-alpha. GH3/B6/F10 cells, a membrane oestrogen receptor-enriched subline, elevate intracellular calcium levels in response to 10 nM oestradiol. Prolactin release in these cells is triggered by both 1 pM and 1 nM oestradiol and diethylstilbestrol (DES). A membrane oestrogen receptor-alpha immunocytochemical signal rapidly disappears (at 3 min) and reappears (at 12-15 min) when 1 nM oestradiol, 10 nM diethylstilbestrol, or 10 nM nonylphenol is applied to the cells. This suggests that both oestrogens and xenoestrogens can utilize this alternative pathway for oestrogenic action. Xenoestrogens, which have so far shown weak effects in genomic assay systems, should now be retested for activity in eliciting membrane-initiated oestrogenic responses.
Animals↗
Increased cerebral extracellular adenosine and decreased PGE2 during ethanol-induced inhibition of FBM.
Adenosine and PGE2 are neuromodulators, both of which inhibit fetal breathing movements (FBM). Although circulating PGE2 has been implicated as a mediator of ethanol-induced inhibition of FBM in the late-gestation ovine fetus, a role for adenosine has not been examined. The objective of this study was to determine the effect of maternal ethanol infusion on ovine fetal cerebral extracellular fluid adenosine and PGE2 concentrations by using in utero microdialysis and to relate any changes to ethanol-induced inhibition of FBM. Dialysate samples were obtained from the fetal parietal cortex over 70 h after surgery to determine steady-state extracellular fluid adenosine and PGE2 concentrations. On each of postoperative days 3 and 4, after a 2-h baseline period, ewes received a 1-h infusion of ethanol (1 g/kg maternal body wt) or an equivalent volume of saline, and the fetus was monitored for a further 11 h with 30-min dialysate samples collected throughout. Immediately after surgery, dialysate PGE2 and adenosine concentrations were 3.7 +/- 0.7 and 296 +/- 127 nM, respectively. PGE2 did not change over the 70 h, whereas adenosine decreased to 59 +/- 14 nM (P < 0.05) at 4 h and then remained unchanged. Ethanol decreased dialysate PGE2 concentration for 2 h (3.3 +/- 0.3 to 1.9 +/- 0.4 nM; P < 0.05) and increased adenosine concentration for 6 h (87 +/- 13 to a maximum of 252 +/- 59 nM, P < 0.05). Ethanol decreased FBM incidence from 47 +/- 7 to 16 +/- 5% (P < 0.01) for 8 h. Saline infusion did not change dialysate adenosine or PGE2 concentrations or FBM incidence. These data are consistent with the hypothesis that fetal cerebral adenosine, and not PGE2, is the primary mediator of ethanol-induced inhibition of FBM at 123 days of gestation in sheep.
Adenosine↗
The adenosine A(1)-receptor antagonist 8-CPT reverses ethanol-induced inhibition of fetal breathing movements.
Administration of either ethanol or adenosine inhibits fetal breathing movements (FBM), eye movements, and low-voltage electrocortical activity (LV ECoG). The concentration of adenosine in ovine fetal cerebral extracellular fluid increases during ethanol-induced inhibition of FBM. The purpose of this study was to determine the effect of a selective adenosine A(1)-receptor antagonist, 8-cyclopentyltheophylline (8-CPT) on the incidence of FBM during ethanol exposure. After a 2-h control period, seven pregnant ewes received a 1-h intravenous infusion of ethanol (1 g/kg maternal body wt), followed 1 h later by a 2-h fetal intravenous infusion of either 8-CPT (3.78 +/- 0.08 microg. kg(-1). min(-1)) or vehicle. Ethanol reduced the incidence of FBM from 44.0 +/- 10.4 to 2.7 +/- 1.3% (P < 0.05) and 51.2 +/- 7.6 to 11.9 +/- 5.0% (P < 0.05) in fetuses destined to receive 8-CPT or vehicle, respectively. In the vehicle group, FBM remained suppressed for 7 h. In contrast, during the first hour of 8-CPT infusion, FBM returned to baseline (31 +/- 11%) and was not different from control throughout the rest of the experiment. Ethanol also decreased the incidence of both low-voltage electrocortical activity and eye movements, but there were no differences in the incidences of these behavioral parameters between the 8-CPT and vehicle groups throughout the experiment. These data are consistent with the hypothesis that adenosine, acting via A(1) receptors, may play a role in the mechanism of ethanol-induced inhibition of FBM.
Animals↗