Signaling mechanisms during the response of pituitary gonadotropes to GnRH.
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Animals↗
Biomedical subjects
A Tse
Publications and source records attributed to A Tse.
Immunomodulatory and antiviral activities of 2',3'-dideoxy-beta-L-cytidine and 2',3'-dideoxy-beta-L-5-fluorocytidine.
Two dideoxynucleosides, 2',3'-dideoxy-beta-L-cytidine and 2',3'-dideoxy-beta-L-5-flurocytidine, containing unnatural L-configuration in their sugar moieties, were synthesized and assayed for antiviral activities. Both compounds were shown to possess potent anti-human immunodeficiency virus type 1 and antihepatitis B virus activities, while demonstrating no anti-herpes simplex viruses 1 and 2 activity. These two compounds exhibited in vitro cellular toxicities for several leukocytic cell lines and were shown to inhibit phytohemagglutinin-stimulated human peripheral blood mononuclear leukocyte proliferations. At inhibitory concentrations, both compounds caused accumulations of cells in the S phase. While demonstrating no obvious morphological toxicity in vivo in mice at concentrations of 75 and 150 mg/kg, 2',3'-dideoxy-beta-L-5-fluorocytidine- treated animals were shown to have considerable increases in CD4/CD8 double positive T lymphocyte population in their blood circulation.
Animals↗
Cyclic Ca2+ changes in intracellular stores of gonadotropes during gonadotropin-releasing hormone-stimulated Ca2+ oscillations.
Gonadotropin-releasing hormone induces oscillatory release of Ca2+ from inositol trisphosphate-sensitive stores of gonadotropes. Simultaneously with electrophysiological measures of cytoplasmic [Ca2+], corresponding changes in [Ca2+] within intracellular stores were monitored with a fluorescent dye, mag-indo-1. Each cycle of oscillation released only 10% of the detectable stored Ca2+. Some Ca2+ was recovered by the stores using a mechanism sensitive to inhibitors of intracellular Ca2+ ATPases, and much of the remainder was temporarily and rapidly pumped into other intracellular compartments or out of the cell. The dynamics of Ca2+ oscillations are thus more complex than a repeated emptying and refilling of a single compartment. The free concentrations measured show that intracellular Ca2+ store compartments contain strong Ca2+ buffers.
Animals↗
Calcium homeostasis in identified rat gonadotrophs.
1. Whole-cell voltage clamp was used in conjunction with the fluorescent Ca2+ indicator indo-1 to measure extracellular Ca2+ entry and intracellular Ca2+ concentrations ([Ca2+]i) in rat gonadotrophs identified with the reverse haemolytic plaque assay. 2. Depolarizations to potentials more positive than -40 mV elicited inward Ca2+ current (ICa) and transient elevations of [Ca2+]i. 3. The relationship between [Ca2+]i elevations and Ca2+ entry with different Ca2+ buffer concentrations in the pipette showed that endogenous Ca2+ buffers normally bind approximately 99% of the Ca2+ entering the cell. 4. With [Ca2+]i elevations less than 500 nM, decay of [Ca2+]i could be approximated by an exponential whose time constant increased with the concentration of exogenous Ca2+ buffers. 5. Inhibitors of intracellular Ca(2+)-ATPases, thapsigargin, cyclopiazonic acid (CPA) and 2,5-di-(tert-butyl)-1,4-benzohydroquinone (BHQ), caused [Ca2+]i to rise. Application of BHQ during [Ca2+]i oscillations induced by gonadotrophin-releasing hormone (GnRH) terminated the oscillation in a slowly decaying elevation. BHQ slowed the decay of depolarization-induced [Ca2+]i elevations about 3-fold. 6. Taking into account the Ca2+ buffering properties of the cytoplasm permitted estimation of the fluxes and rate constants for Ca2+ movements in gonadotrophs. The intracellular store is a major determinant of Ca2+ homeostasis in gonadotrophs.
Animals↗
Calcium oscillations and exocytosis in pituitary gonadotropes.
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Animals↗
Effect of antibiotic treatment on inflammatory markers and lung function in cystic fibrosis patients with Pseudomonas cepacia.
BACKGROUND: The acquisition of Pseudomonas cepacia in patients with cystic fibrosis is associated with increasing deterioration in lung function and more frequent hospital admissions. Pseudomonas cepacia is usually resistant to several antibiotics in vitro, but the response of patients colonised with the organism has not been extensively studied in vivo. METHODS: A three month prospective study was performed to investigate the response of 14 Ps cepacia positive patients and 10 Ps cepacia negative patients to a two week course of intravenous antibiotics. All those who were Ps cepacia negative and six of the 14 Ps cepacia positive patients had Ps aeruginosa in their sputum which was sensitive to the prescribed therapy. The inflammatory markers C-reactive protein, white blood cell count, serum lactoferrin, neutrophil elastase/alpha 1-antitrypsin complex, and tumour necrosis factor alpha were measured at the start and end of each antibiotic course. RESULTS: The median (range) % improvement in baseline FEV1 and FVC following treatment in the group as a whole was 15.2% (-23.5% to 156.3%) and 23.9% (-36.8% to 232.7%) respectively. There was no statistical difference in improvement in lung function, body weight, or inflammatory markers between individuals who were Ps cepacia positive and those who were Ps cepacia negative. CONCLUSIONS: Patients who are Ps cepacia positive appear to respond as well to intravenous antibiotics as those who are Ps cepacia negative, despite having lower lung function and a bacterium in their sputum which is resistant in vitro to the antibiotics used.
Adult↗
Rhythmic exocytosis stimulated by GnRH-induced calcium oscillations in rat gonadotropes.
In pituitary gonadotropes, gonadotropin-releasing hormone (GnRH) induces the rhythmic release of Ca2+ from an inositol 1,4,5-trisphosphate (IP3)-sensitive store. Simultaneous measurement of the concentration of cytosolic free Ca2+ ([Ca2+]i) and exocytosis in single identified gonadotropes showed that each elevation of [Ca2+]i induced a burst of exocytosis. These phenomena were largely suppressed by buffering of [Ca2+]i but persisted in the absence of extracellular Ca2+. Activation of voltage-gated Ca2+ channels by brief depolarizations seldom supplied enough Ca2+ for exocytosis, but [Ca2+]i elevations induced by photolysis of caged IP3 did trigger exocytosis, confirming that GnRH-stimulated gonadotropic hormone secretion is closely coupled to intracellular Ca2+ release. Agonist-induced oscillations of [Ca2+]i in secretory cells may be a mechanism to optimize the secretory output while avoiding the toxic effects of sustained elevation of [Ca2+]i.
Action Potentials↗
Modulation of ion channels underlying excitation-secretion coupling in identified lactotrophs and gonadotrophs.
The role of ion channel activity in the response of rat pituitary lactotrophs and gonadotrophs to dopamine (DA) and GnRH, respectively, was investigated. Single lactotrophs and gonadotrophs were unambiguously identified with the reverse hemolytic plaque assay, and recordings of membrane potential and current were obtained using whole-cell and single-channel patch-clamp techniques. In lactotrophs, DA inhibited spontaneous electrical activity by activating a K+ conductance that hyperpolarized the cells. A 50 pS K+ channel underlies this response and was activated following agonist binding to a D2 type receptor via a "direct" interaction with a pertussis toxin-sensitive G-protein. In gonadotrophs, GnRH triggers rhythmic hyperpolarizations due also to a K+ conductance increase. The K+ channel underlying the GnRH response is an apamin-sensitive, Ca(2+)-activated channel. Although both agonists produce hyperpolarizations in their respective target cells via K+ channel activation, differences in intracellular calcium response probably discriminate the stimulatory (GnRH) and inhibitory (DA) actions on hormone secretion. Each K+ channel type plays a different role in modulating the intracellular Ca2+ levels to yield these actions.
Action Potentials↗
ZB51: a monoclonal antibody reactive with human plasma cells.
Several monoclonal antibodies have been raised in the past which react with human plasma cells but they have all shown additional reactions with other cell types. In this paper we describe a new monoclonal antibody, ZB51, which recognizes an intracellular antigen in normal plasma cells in cryostat tissue sections and cell smears, and which also reacts with neoplastic cells in most cases of myeloma and with plasma cell lines. The antibody shows minimal reactivity with a few eosinophils and myelocytes in bone marrow and stains a myeloid cell line. Whilst normal epithelium is not labelled, antibody ZB51 stains two carcinoma cell lines. Although it was not possible to characterize the target antigen in terms of molecular weight, the reactivity of antibody ZB51 with normal and neoplastic plasma cells makes it a useful new immunocytochemical reagent.
Antibodies, Monoclonal↗
Role of voltage-gated Na+ and Ca2+ channels in gonadotropin-releasing hormone-induced membrane potential changes in identified rat gonadotropes.
We have previously reported that GnRH induces rhythmic hyperpolarizations in male rat (35- to 45-day-old) gonadotropes by periodically opening apamin-sensitive Ca(2+)-activated K+ channels. Using the whole cell recording technique, we now show that these gonadotropes, identified with the reverse hemolytic plaque assay, express tetrodotoxin-sensitive Na+ channels and omega-conotoxin-insensitive, high voltage-activated Ca2+ channels that are partially sensitive to dihydropyridines. We found no low voltage-activated Ca2+ channels in these cells. At the normal resting potential, about 93% of the Na+ channels and 50% of the Ca2+ channels are inactivated. The GnRH-induced hyperpolarizations transiently remove the resting inactivation of Na+ and Ca2+ channels, enabling them to initiate action potentials at the termination of each hyperpolarization. Opening of Na+ channels accounts for the high rate of rise and the positive peak of the action potential. In addition, a significant fraction of Ca2+ channels should be activated during the action potentials, allowing a voltage-gated entry of extracellular Ca2+ that can enhance the frequency and amplitude of GnRH-induced intracellular Ca2+ oscillations. Therefore, we envision the following role for action potentials in GnRH-stimulated Ca2+ responses: action potentials will open voltage-gated Ca2+ channels that allow entry of extracellular Ca2+, which can help to replenish the intracellular Ca2+ store and act as a coactivator in the stimulation of intracellular Ca2+ release from the inositol 1,4,5-trisphosphate-sensitive store.
Action Potentials↗
Perinatal grief: response to the loss of an infant.
The grief response of parents following loss of an infant from a multiple gestation pregnancy was explored to describe the experience, compare and contrast mother's and father's responses, and analyze the properties of the Perinatal Grief Scale. Twenty-seven parents participated. Parental responses were complex and variable. Most parents disagreed with experiencing difficulty coping and despair. Confrontive, self reliant, and supportant coping strategies were used most frequently, while supportant, confrontive and optimistic strategies were reported the most effective. Anxiety, depression and alterations in activities of daily living were reported. The total impact of life events was slightly positive for this group. Although the instruments were reliable in this population of parents, sensitivity was reduced. A model of grief associated with multiple gestation perinatal loss was generated. Selection and effectiveness of coping strategies appear to be not only interrelated, but dependent on the time since the infants death and the length of the infants life. The length of an infant's life and time since death were affirmed as related to both the strength and type of relationship between variables associated with the grief response and to use and effectiveness of coping strategies. Responses of mothers and fathers appear to be similar.
Adult↗
GnRH-induced Ca2+ oscillations and rhythmic hyperpolarizations of pituitary gonadotropes.
Secretion of gonadotropic hormones from pituitary gonadotropes in response to gonadotropin-releasing hormone (GnRH) is essential for regulation of reproductive potential. Gonadotropes from male rats exhibited an unusual form of cellular excitation that resulted from periodic membrane hyperpolarization. GnRH induced an oscillatory release of intracellular Ca2+ via a guanosine triphosphate (GTP) binding protein-coupled phosphoinositide pathway and hyperpolarized the gonadotrope periodically by opening apamin-sensitive Ca(2+)-activated K+ (SK) channels. Each hyperpolarization was terminated by firing of a few action potentials that may result from removal of inactivation from voltage-gated Na+ and Ca2+ channels.
Animals↗
Structural features of 5,10-dideaza-5,6,7,8-tetrahydrofolate that determine inhibition of mammalian glycinamide ribonucleotide formyltransferase.
We have investigated the structural features of 5,10-dideaza-5,6,7,8-tetrahydrofolate (DDATHF) that determine the activity of this compound as an inhibitor of glycinamide ribonucleotide formyltransferase (GARFT) purified from mouse L1210 cells. 5-Deazatetrahydrofolate was as good an inhibitor of GARFT as DDATHF, indicating that isosteric replacement of nitrogen by carbon at the 5-position of tetrahydrofolate is sufficient for inhibition of GARFT. 5,10-Dideazafolic acid, 5,8,10-trideazatetrahydrofolate, and 2-desamino-5,10-dideazatetrahydrofolate were poor inhibitors of GARFT, indicating that a reduced pyridopyrimidine ring, N-8, and the 2-amino group of DDATHF, respectively, play an important role in the binding of tetrahydrofolate analogues to this enzyme. DDATHF analogues in which the phenyl ring was replaced either by a cyclohexyl ring or by methylene groups retained activity as inhibitors. 5,10-Dideazatetrahydrohomofolate was about 6 times more potent as an inhibitor of GARFT than DDATHF, but 5,10-dideazatetrahydronorfolate had about one-fifth of the activity of DDATHF. An analogue of DDATHF in which the glutamic acid side chain was replaced by aspartic acid (which was not a substrate for polyglutamation and was only weakly cytotoxic) was equiactive with DDATHF as an inhibitor of purified GARFT. Surprisingly, 5,10-dideazatetrahydropteroic acid was about as active as DDATHF as an inhibitor of GARFT, an indication that the glutamic acid in the side chain of DDATHF does not play a role in this ligand-enzyme interaction. The polyglutamate derivatives of DDATHF bound up to 100 times tighter to GARFT than DDATHF itself; longer chain polyglutamates conformed to Goldstein's zone B behavior under experimental conditions and were projected to be in zone C, i.e., stoichiometric inhibition, in vivo. We conclude that the presence of carbon at the 5-position of tetrahydrofolate analogues is sufficient for inhibition of GARFT, that N-8 and the 2-amino group are involved in binding of DDATHF to GARFT, probably through hydrogen bonds, and that the structures of the phenyl ring and amino acid side chain of DDATHF analogues are not primary determinants of GARFT inhibition by monoglutamate forms of these compounds. We also conclude that polyglutamation plays a major role in the potent cytotoxicity of DDATHF.
Acyltransferases↗
The refractory phase of experimental allergic encephalomyelitis in the Lewis rat is antigen specific in its induction but not in its effect.
Rats that have recovered from experimental allergic encephalomyelitis (EAE) are refractory to attempts to induce further episodes of the disease. The specificity of this refractoriness was examined by determining the susceptibility of such animals to the related disease experimental allergic neuritis (EAN). As EAE and EAN are induced respectively by central nervous system myelin basic protein (MBP) and the P2 peptide of peripheral nerve myelin, it was possible to test whether the refractory phase of EAE represented a generalized hyporeactivity to antigenic challenge or was solely a failure to respond to the antigen with which disease was induced. For some experiments the roles of the two antigens were reversed, i.e. the effects of preimmunizing with P2 peptide on the susceptibility to EAE was examined. Animals preimmunized with MBP or P2 peptide became profoundly refractory to EAE and EAN respectively and rats preimmunized with one antigen but challenged with the other were susceptible to the challenge. It is concluded that refractoriness to EAE and EAN is largely antigen specific. Preimmunization with one antigen followed by challenge with a mixture of both suggested that the immunosuppression responsible for refractoriness, although specific in its induction, was non-specific in its effect.
Amino Acid Sequence↗
Prevalence of illicit drug use among prenatal patients and predictive validity of nurses' judgments.
The purposes of this investigation were to: (1) estimate the prevalence of illicit drug use within the prenatal population of a midwestern suburban ambulatory care center; (2) explore the relationship between nurses' judgments of patient illicit drug use and urine toxicology results; and (3) determine if a difference exists between nurses' judgments of illicit drug use by private physicians' patients (PPP) and nurses' judgments of illicit drug use by resident physicians' patients (RPP). For a 16-week period, all new prenatal patients (N = 189) were included in this study; 78 (41%) were in the RPP group and 111 (59%) were in the PPP group. Urine remaining from routine urinalysis was assayed for seven drugs or drug classes. Fourteen (7.5%) subjects tested positive for illicit drugs, 8 (10.3%) in the RPP group, and 6 (5.4%) in the PPP group (chi 2, P greater than .33). Using a Likert-type scale, nurses rated the likelihood of a positive result of each patient's urine test. Nurses' judgments of patient illicit drug use and positive results were related, r = .28. Nurses were more likely (P less than .0001) to suspect residents' patients of illicit drug use than private physicians' patients.
Adult↗
Assembly of MHC class I molecules analyzed in vitro.
Recent evidence suggests that peptide ligands take part in the assembly of class I molecules in living cells. We now describe a simple system for studying class I assembly in vitro. Detergent extracts of the mutant cells RMA-S and .174, in which class I assembly does not occur spontaneously, will support assembly in vitro when specific peptides are added. Peptides stabilize a conformational change in the class I heavy chain and association with beta 2-microglobulin, at concentrations approximately 100-fold lower than required in "peptide feeding" experiments with whole cells. We show that peptides bind class I molecules during assembly and demonstrate that the conformational change induced in the heavy chain is influenced by the concentrations of both peptide and beta 2-microglobulin.
Amino Acid Sequence↗
Electrophysiology of parasympathetic neurones isolated from the interatrial septum of bull-frog heart.
1. Whole-cell voltage-clamp techniques were used to study the voltage-dependent membrane conductances in parasympathetic neurones enzymatically isolated from the interatrial septum of bull-frog heart and maintained in short-term (1-10 day) tissue culture. 2. The resting potential of the isolated neurones averaged -55.4 +/- 1.1 mV (+/- S.E.M., n = 11). Action potentials evoked in the isolated cells by brief (1-2 ms) current injections were similar to those recorded from neurones in the 'intact' septum. The amplitude of action potentials of isolated neurones averaged about 113 mV, with a peak depolarization of +32.8 +/- 2.8 mV and after-hyperpolarization of -80.0 +/- 2.8 mV. 3. The pattern of membrane currents recorded using voltage clamp with 'normal' external (containing 110 mM-Na+) and internal (110 mM-K+) solutions consisted of a rapidly activating and inactivating inward current followed by a slower, sustained outward current. 4. The inward components of current were isolated by using an internal solution in which Cs+ and TEA+ (tetrathylammonium) ions replaced K+. Depolarizations from holding potentials of -50 to -70 mV produced inward currents which had an initial transient phase followed by a maintained, or very slowly inactivating, component. The current-voltage relation for the initial transient phase reached a peak at membrane potentials near 0 mV, while the maintained phase, measured, for example, at the end of 50 ms voltage-clamp steps, had its peak near +10 mV. 5. The transient component of inward current was carried primarily by Na+ ions, as replacement of Na+ by TEA+ in the external solution abolished the transient. This current was thus identified as a voltage-dependent Na+ current, INa. The maintained component was greatly attenuated by removing 80-90% of the external Ca2+ ions, and it was abolished by divalent cations such as Cd2+ (0.2-0.4 mM), Ni2+ (0.5 mM) and La3+ (10-100 microM). This maintained component was thus a voltage-dependent calcium current, ICa. 6. About 80% of INa recorded in the presence of low (0.2-0.5 mM) external Ca2+ and 2 microM-LaCl3 was blocked by tetrodotoxin (TTX) with an apparent Kd of about 8 nM. The remaining 20% of INa was resistant to block by 2-10 microM-TTX. However, the 'TTX-resistant' component of INa was blocked by Cd2+ (0.2-0.4 mM). 7. The voltage-dependent calcium current, ICa, measured in saline in which Na+ was replaced by N-methyl-D-glucamine, activated near -40 mV and reached a peak near +10 to +15 mV.(ABSTRACT TRUNCATED AT 400 WORDS)
Action Potentials↗
Muscarinic modulation of calcium current in neurones from the interatrial septum of bull-frog heart.
1. The effects of activation of muscarinic receptors on the voltage-dependent calcium current, ICa, in parasympathetic neurones were examined. 2. Neurones were enzymatically isolated from the interatrial septum of bull-frog (Rana catesbeiana) heart, and were maintained in short-term (1-6 day) tissue culture. ICa was recorded from the cells using whole-cell patch-clamp methods (Clark, Tse & Giles, 1990). 3. External application of 2 nM to 10 microM acetylcholine (ACh) reduced the amplitude and slowed the time course of activation of ICa. These effects were dependent on membrane potential; they were most pronounced at potentials near the peak of the current-voltage relation for ICa (i.e. +10 to +15 mV), whereas at more-negative potentials (i.e. -15 to -25 mV) the effects on both amplitude and time course were relatively small. 4. Atropine (1 microM) completely blocked the action of 1 microM-ACh, indicating that the effects of ACh on ICa were mediated by activation of muscarinic receptors. 5. Other muscarinic agonists, such as carbamylcholine (0.1-10 microM), DL-muscarine (0.1-2.5 microM) and oxotremorine (5 microM), had similar effects on ICa to ACh. 6. A guanine nucleotide-binding protein (G-protein) is involved in this muscarinic inhibition of ICa. Inclusion of the non-hydrolysable guanosine triphosphate analogue guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S; 200 microM) in the intracellular solutions mimicked the effects of ACh, and application of external ACh in the presence of internal GTP-gamma-S produced smaller changes in ICa than in control conditions. Inclusion of another non-hydrolysable analogue, guanosine 5'-O-(2-thiodiphosphate) (GDP-beta-S; 0.5-5 mM), blocked the inhibitory effect of ACh on ICa. 7. The G-protein involved in the inhibition of ICa was sensitive to pertussis toxin (islet-activating protein; IAP). The inhibition of ICa by carbamylcholine (5 microM) was reduced by about 90% after incubating cells for 12-15 h in culture medium containing 200 ng/ml IAP. 8. The possible roles of cyclic AMP or cyclic GMP-dependent protein kinases, or protein kinase C, in the muscarinic inhibition of ICa were tested, but these enzymes appear not to be directly involved.
Acetylcholine↗