Thyrotropin-releasing hormone activates a Ca2+-dependent polyphosphoinositide phosphodiesterase in permeable GH3 cells. GTP gamma S potentiation by a cholera and pertussis toxin-insensitive mechanism

Numerous hormones are known to rapidly activate polyphosphoinositide turnover in target cells by promoting phosphodiesteratic cleavage of the phospholipids; however, little is known about the enzymology of receptor-mediated phosphoinositide breakdown. In the present study, thyrotropin-releasing hormone (TRH) stimulation of polyphosphoinositide turnover has been characterized in electrically permeabilized, [3H]myoinositol-labeled GH3 cells. The permeable cells allow the influence of small molecular weight (Mr less than or equal to 1000) cofactors to be determined. We present evidence for the following: 1) TRH stimulates inositol phosphate generation in permeable cells; 2) optimal hormone-stimulated inositol phosphate generation requires Mg2+, ATP, and Ca2+; 3) Mg2+ and ATP requirements reflect polyphosphoinositide kinase reactions; 4) in the absence of MgATP, TRH stimulates the phosphodiesteratic breakdown of pre-existing polyphosphoinositides in a reaction which requires only low Ca2+ (10(-7) M); 5) hormone activation is potentiated in the presence of the stable guanine nucleotide, GTP gamma S; neither TRH-stimulated nor GTP gamma S-potentiated hydrolysis is inhibited by cholera or pertussis toxin treatment. These results demonstrate that hormone-induced phospholipid hydrolysis involves activation of a phosphoinositide phosphodiesterase; activation results in lowering the Ca2+ requirement of the phosphodiesterase such that maximal activity is observed at Ca2+ levels characteristic of a resting cell (10(-7) M). Furthermore, TRH regulation of polyphosphoinositide hydrolysis is modulated by guanine nucleotides; however, nucleotide regulation appears to involve a GTP-binding factor (Np) other than Ns or Ni.     

Evidence for the presence of muscarinic acetylcholine receptors in bovine brain coated vesicles

Evidence obtained from quinuclidinylbenzilate binding determinations suggested that muscarinic acetylcholine receptor molecules are present in purified bovine brain coated vesicles. Immunoprecipitates formed from coated vesicles with polyclonal antibodies to clathrin bound on the surface of fixed Staphylococcus aureus cells also showed quinuclidinylbenzilate binding activity. The high purity of coated vesicles was established by assays for biochemical markers and by electron microscopy. Muscarinic receptor sites for quinuclidinylbenzilate binding to coated vesicles displayed a Kd of 25 pM and a Bmax of about 191 fmol/mg of protein. Binding competition experiments using atropine, N-methylscopolamine, oxotremorine, and carbamylcholine confirmed the typical muscarinic nature of the binding site. Ranking order of potency for the receptors was: atropine greater than N-methylscopolamine greater than oxotremorine greater than carbachol Analysis of data using a two-site model revealed 13% high-affinity sites for oxotremorine, 66% high-affinity sites for carbachol, and 62% for the antagonist N-methylscopolamine. Heterogeneity of binding affinities for muscarinic drugs detected in the coated vesicles may be related to the presence of coated vesicle subpopulations in brain tissue, (Kohtz, D. S., Kohtz, J. D., Schook, W. J., and Puszkin, S. (1985) J. Cell Biol. 101, 48a; Pfeffer, S. R., and Kelly, R. B. (1985) Cell 40, 949-957).     

Active and passive respiratory mechanics in anesthetized dogs

In six spontaneously breathing anesthetized dogs (pentobarbital sodium, 30 mg/kg) airflow, volume, and tracheal and esophageal pressures were measured. The active and passive mechanical properties of the total respiratory system, lung, and chest wall were calculated. The average passive values of respiratory system, lung, and chest wall elastances amounted to, respectively, 50.1, 32.3, and 17.7 cmH2O X l-1. Resistive pressure-vs.-flow relationships for the relaxed respiratory system, lung, and chest wall were also determined; a linear relationship was found for the former (the total passive intrinsic resistance averaged 4.1 cmH2O X l-1 X s), whereas power functions best described the others: the pulmonary pressure-flow relationship exhibited an upward concavity, which for the chest wall presented an upward convexity. The average active elastance and resistance of the respiratory system were, respectively, 64.0 cmH2O X l-1 and 5.4 cmH2O X l-1 X s. The greater active impedance reflects pressure losses due to force-length and force-velocity properties of the inspiratory muscles and those due to distortion of the respiratory system from its relaxed configuration.     

Gastrointestinal peptides and the adaptation to extrauterine nutrition

The adaptation to extrauterine nutrition involves complex physiological changes at birth which may be regulated by genetic endowment; enteral nutrients, secretions, and bacteria; and endogenous hormones and exogenous hormones in breast milk. The hypothesis is explored that enteral feeding after birth may trigger key adaptations in the gut and in metabolism partly through the mediation of gastrointestinal hormone secretion. Gut peptides are found in the early human fetal gut and by the second trimester some are found in high concentrations in the fetal circulation and amniotic fluid. Major plasma hormonal surges occur during the neonatal period in term and preterm infants: notably in enteroglucagon, gastrin, motilin, neurotensin, gastrointestinal peptide, and pancreatic polypeptide. These events do not occur in neonates deprived of enteral feeding. A progressive development of dynamic gut hormonal responses to feeding is observed. The pattern of gut endocrine changes after birth is influenced by the type and route of feeding. Potential pathophysiological effects of depriving high risk neonates of enteral feeding are considered. It is speculated that infants committed to prolonged total parenteral nutrition from birth may benefit from the biological effects of intraluminal nutrients used in subnutritional quantities.     

Stimulation by calcium and carbamoylcholine of the ouabain-sensitive uptake of 86Rb+ in isolated rat pancreatic acinar cells

The uptake of 86Rb+ was assayed in isolated rat pancreatic acinar cells to determine the effect of calcium and carbamoylcholine on the ouabain-sensitive and ouabain-insensitive components. The presence of calcium in the medium bathing the cells during the preincubation and the main incubation periods was needed to preserve in optimum conditions the uptake of 86Rb+, the stimulation by carbamoylcholine and the sensitivity to ouabain. In the presence of calcium, the ouabain-sensitive component of 86Rb+ uptake was higher than the ouabain-insensitive. The ouabain-sensitive component was 3-times lower in cells incubated in a medium lacking calcium and containing 1 mM EGTA, as compared to cells incubated in the presence of calcium. Carbamoylcholine, at 5 X 10(-4) M, stimulated the uptake of 86Rb+ and this effect depended on the presence of calcium in the bathing medium. Maximal stimulation by carbamoylcholine was reached at 0.2 mM calcium. The nett stimulation by carbamoylcholine was inhibited up to 85% by 1 mM ouabain. As judged by digitonin-disruption of plasma membrane, the above-indicated effects were limited to a cytoplasmic pool of 86Rb+ and a leaky plasma membrane could be ruled out. The results suggest that in rat pancreatic acinar cells, carbamoylcholine stimulated the ouabain-sensitive uptake of 86Rb+ and required the presence of calcium in the bathing medium.     

The biological activity of cyclopropyl analogs of all-trans- and 13-cis-retinoic acid in the rat vaginal smear assay

The biological activity of a series of cyclopropyl analogs of all-trans- and 13-cis-retinoic acid has been evaluated in the vaginal smear assay carried out in vitamin A-deficient rats. These analogs were designed to probe the role of the 13-cis isomer in the actions of the parent all-trans-retinoic acid by blocking the interconversion of these two compounds. Although relatively less active, the potency of some of the cyclopropyl analogs suggests that 13-cis-retinoic acid is a fully active metabolite of all-trans-retinoic acid. Since 13-cis-retinoic acid represents a small percentage of the retinoic acid metabolites, the physiological significance of this activity is still unclear. Possible reasons for the reduced activity of the cyclopropyl analogs, as well as an aromatic analog of retinoic acid, are discussed.     

Effects of hyperthermia on DNA interstrand crosslinking after treatment with BCNU in 9L rat brain tumor cells

The effects of hyperthermia (42 degrees C) on 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-mediated DNA interstrand crosslink formation were investigated in 9L rat brain tumor cells using the technique of alkaline elution. When cells were treated with 60 microM BCNU for 1 hr at 37 degrees C and incubated for 6 hr in drug-free medium at 42 degrees C, there was a 50% increase in crosslinking; and when cells were treated at 42 degrees C and incubated at 37 degrees C, there was a 45% increase in crosslinking compared with the results for cells treated and incubated at 37 degrees C. When cells were treated and incubated at 42 degrees C, there was a 129% increase in DNA crosslinking. The same relative order of results was found for cell survival. These results suggest that hyperthermia can increase DNA interstrand crosslink formation and the consequent cell death through two independent mechanisms: an increase in the amount of initial alkylation because of the increased rate of hydrolysis of BCNU at higher temperatures, and the effect of heat on DNA structure that leads to an increase in the number of crosslinks formed.     

Nitrogenase activity in the non-heterocystous cyanobacterium Oscillatoria sp. grown under alternating light-dark cycles

The non-heterocystous cyanobacterium Oscillatoria sp. strain 23 fixes nitrogen under aerobic conditions. If nitrate-grown cultures were transferred to a medium free of combined nitrogen, nitrogenase was induced within about 1 day. The acetylene reduction showed a diurnal variation under conditions of continuous light. Maximum rates of acetylene reduction steadily increased during 8 successive days. When grown under alternating light-dark cycles, Oscillatoria sp. fixes nitrogen preferably in the dark period. For dark periods longer than 8 h, nitrogenase activity is only present during the dark period. For dark periods of 8 h and less, however, nitrogenase activity appears before the beginning of the dark period. This is most pronounced in cultures grown in a 20 h light – 4 h dark cycle. In that case, nitrogenase activity appears 3–4 h before the beginning of the dark period. According to the light-dark regime applied, nitrogenase activity was observed during 8–11 h. Oscillatoria sp. grown under 16 h light and 8 h dark cycle, also induced nitrogenase at the usual point of time, when suddenly transferred to conditions of continuous light. The activity appeared exactly at the point of time where the dark period used to begin. No nitrogenase activity was observed when chloramphenicol was added to the cultures 3 h before the onset of the dark period. This observation indicated that for each cycle, de novo nitrogenase synthesis is necessary.     

Morbidity in Chagas' disease. IV. Longitudinal study of 10 years in Pains and Iguatama, Minas Gerais, Brazil

An evolutive study of the "case-control" type was carried out in an endemic area of Chagas' disease in Minas Gerais State, Brazil, using two cross-section evaluations with an interval of ten years between them (1974-1984). Patients were paired for sex and age. In the first cross-section study 264 pairs one with a positive serology and the other with a negative serology for T. cruzi antibodies were included. In the second evaluation, ten years later, 235 patients among those with previous positive serology and 216 with negative serology were located, but only 110 pairs could be recomposed and reexamined (clinical examination, ECG and Rx of the heart and esophagus). The incidence of chagasic cardiopathy in the cases with positive serology but previously assymptomatic was 38.3% during the ten year period. On the other hand there was a deterioration in 24% of the patients with chagasic cardiopathy since the first examination. Considering all clinical forms of the disease in 34.5% of the patients the clinical situation deteriorated, in 57.3% there was no change and in 8.2% the situation improved. The general mortality in the period was 23% in the chagasic group and 10.6% in the control group, but the lethality by cardiopathy was 17% in chagasic group and only 23.3% in the control group. The mortality was twice as high in males than in females, mainly in the age group from 30 to 59 years.