PubMed Health⌕ Search

Biomedical subjects

E L Wee

Publications and source records attributed to E L Wee.

16 recordsLinked to original sources
PubMed1986

Caffeine effects on cyclic AMP levels in the mouse embryonic limb and palate in vitro.

Caffeine is a teratogen that causes limb and palate malformations in rodents. Since the ability to raise cyclic nucleotide levels is a known biological action of caffeine, cyclic AMP levels were measured in CD-1 mouse embryonic forelimb from whole embryo culture and embryonic limb and palate cells grown in primary culture following treatment with various concentrations of caffeine (0, 1, 3, or 10 mM). In forelimb buds from whole embryo culture, a dose-dependent response was observed. Caffeine at 1 mM concentration stimulated cyclic AMP levels to 151% of control value at 60 min. Even greater stimulation of cyclic AMP occurred at higher caffeine concentrations. A dose-dependent response was seen in both limb and palate cell culture. In limb cell culture, all caffeine concentrations significantly stimulated cyclic AMP after 10 min compared to control. In palate cell culture, there was a twofold increase in cyclic AMP at the 1-mM caffeine concentration. At higher caffeine concentrations, cyclic AMP was significantly increased after 60 min. In addition, stimulation of cyclic AMP in cultured limb and palate cells by isoproterenol, a beta-adrenergic agonist, was used as a positive control. Isoproterenol stimulated a 2.5-fold greater response in the palate cells than in the limb bud cells at isoproterenol levels of 10(-5) or 10(-4) M. The increase of cyclic AMP may be influential in the process of abnormal limb or palate development.

Animals↗
PubMed1986

Presence of gamma-aminobutyric acid in embryonic palates of AJ and SWV mouse strains.

The presence of gamma-aminobutyric acid (GABA) in the embryonic palate was sought as a criterion for its role in regulating palate development. GABA was measured by a gas chromatographic-mass spectrometric (GC-MS) method using the heptafluorobutyryl (HFB)-cyclohexyl-GABA derivative, which gave the necessary sensitivity and specificity to measure low levels of GABA in the presence of contaminating substances. GABA was measured in dissected embryonic palates at various times of development in the AJ mouse strain. GABA levels were lower in day 14 AJ palates (0.19 +/- 0.01 nmol/mg protein) than at days 13 (0.28 +/- 0.03) and 15 (0.30 +/- 0.04). Comparable levels were observed in fore- and hindlimbs at day 14, whereas levels were lower in embryonic tongue and higher, as was expected, in embryonic brain. To confirm the presence of GABA in the palate, it was analyzed in growing palate mesenchymal cells in primary and secondary cultures as well as in serum-free medium. In addition, GABA levels were compared in the SWV mouse strain; this strain exhibits a more efficient active uptake mechanism and diazepam produces a higher frequency of cleft palate in this strain than in AJ. SWV contained one and one-half to three times higher concentrations of GABA in excised palates and cultured palate cells than the AJ strain. Furthermore, when GABA levels in skin fibroblasts of the two strains were measured, SWV cells contained 2.7-fold greater GABA than AJ cells. The present results provide additional evidence for the role of GABA in palate development.

Amniotic Fluid↗
PubMed1985

Rapid gas chromatographic--mass spectrometric quantitation of gamma-aminobutyric acid in biological specimens.

A mass fragmentographic method for gamma-aminobutyric acid (GABA) quantitation using the heptafluorobutyryl-cyclohexyl-GABA derivative is described. Both capillary and packed column gas chromatography were used. This procedure employs 2,2[2H2]GABA as an internal standard and allows the rapid, sensitive, and specific measurement of GABA with a minimum of sample clean-up. Application of the method is demonstrated in mouse embryonic brain, body, and palate and human platelets, plasma, cerebrospinal fluid, and urine.

Animals↗
PubMed1985

GABA uptake in embryonic palate mesenchymal cells of two mouse strains.

To obtain further evidence that the inhibitory neurotransmitter GABA functions in palate development, the presence of an active GABA uptake mechanism was sought using primary cultures of embryonic palate mesenchymal cells. Uptake was compared from cells of two inbred mouse strains in which the SWV strain shows greater sensitivity than the AJ strain to effects of GABA on palate morphogenesis and of diazepam in producing cleft palate. Palate cells were capable of accumulating [3H]GABA by saturable uptake mechanisms characteristic of a high and low affinity active transport as indicated by temperature, Na+ ion and carrier dependence as well as Km and Vmax values that were comparable to other biological systems. The Vmax of the high-affinity uptake system from cells of the SWV strain was 1.8 fold higher than that of the AJ. GABA uptake was also observed in fibroblasts from various sources including embryonic mouse limb cells, human skin fibroblasts and 3T3 cells. When active GABA uptake was measured in skin fibroblasts from the mouse SWV and AJ strains, the rate of uptake from SWV cells under high affinity conditions was also 1.8 fold greater than in AJ cells. Thus active GABA uptake appears to be genetically regulated in non-neural cells which may contribute to differential responses to GABA.

Animals↗
PubMed1985

Role of neurotransmitters and teratogens on palate development.

Neurotransmitters regulate palate shelf reorientation. Acetylcholine and serotonin stimulate, whereas GABA inhibits reorientation. Serotonin stimulates cell movement in an in vitro chemotactic system. Diazepam may cause cleft palate by mimicking GABA. Diazepam sensitivity may be caused by genotypic differences in a GABA-ergic system in the embryo.

Abnormalities, Drug-Induced↗
PubMed1983

Involvement of GABA in palate morphogenesis and its relation to diazepam teratogenesis in two mouse strains.

Previous studies have indicated that serotonin and acetylcholine stimulate palate shelf reorientation. The present studies were undertaken to determine whether gamma-aminobutyric acid (GABA) functions as an inhibitory neurotransmitter in the palate and whether diazepam mimics GABA to inhibit shelf reorientation and cause cleft palate. First, it was shown that 10(-4) M GABA inhibits palate shelf reorientation in day 14.5 AJ embryos cultured for 2 hours. Anterior palate reorientation stimulated by 10(-5) M serotonin was decreased by GABA; 10(-5) M picrotoxin (GABA antagonist) stimulated anterior shelf reorientation and reversed the effect of GABA. Diazepam (10(-4) M) partially inhibited palate shelf reorientation and that stimulated by 10(-5) M serotonin. Diazepam (400 mg/kg) was administered to AJ mice at day 13.5 of gestation and embryos were cultured at day 14.5. The inhibition produced by diazepam was significantly reduced by 10(-5) M picrotoxin. The teratogenic effect of diazepam was compared with AJ and Swiss-Webster Vancouver (SWV) inbred strains. Diazepam produced greater clefting in SWV mice (57% net) than in the AJ (18% net) when compared to their water- and food-starved controls. The greater sensitivity of the SWV strain than the AJ strain to diazepam, as well as to GABA, was also observed in embryo culture. GABA (10(-5) M) markedly inhibited posterior palate reorientation and reversed the stimulation produced by bethanechol in SWV mice. The inhibitory effects of GABA on the posterior palate were partially reversed by picrotoxin. Furthermore, diazepam inhibited palate reorientation either when administered to the pregnant dam or added in embryo culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1981

Palate morphogenesis. VI. Identification of stellate cells in culture.

Mesenchymal cells from the palate of mouse embryos at day 14.5 of gestation produce a minor population of stellate cells in culture. These cells are often bipolar and spindle-shaped with long cytoplasmic processes similar to neural-crest cells. Culturing of explants of palatal mesenchyme enriched for this type of cell. Stellate cells were the first to migrate from explants, followed by fibroblast-like cells and then by squamous cells. The majority of the cells in the explant were fibroblast-like. Squamous cells were present mostly in the anterior and mid-palate and least frequently in those from the posterior palate. They may represent tooth-germ epithelium. When pieces of palate were dissected out and cultured for enrichment of non-muscle contractile systems, most of the migrating cells were stellate. These may represent the highly migratory cells that are, in part, responsible for elevation of the palate shelf. Serotonin was measured in cultured mesenchymal cells from the palate. Its occurrence is consistent with regulation of movement of palate cells.

Animals↗
PubMed1981

Presence of serotonin in the palate just prior to shelf elevation.

Since serotonin and its antagonists affect shelf rotation in mouse embryo culture, experiments were carried out to determine whether a serotonergic system is present in the palate. Employing [3H]5-HT, day-14.5 embryos incorporated the monoamine into palates. Active uptake of [3H]5-HT was shown since excised palates incorporated 9-fold more radioactivity at 37 degrees C than at 4 degrees C. Synthesis of palatal serotonin was measured. Embryos were cultured in the presence of the serotonin precursor, [3H]5-HTP, and radioactive 5-HT was monitored in the palate by thin-layer chromatography. Furthermore, excised palates were incubated with [3H]5-HTP and radioactive 5-HT was measured. Incorporation was linear for about 6 h. In addition, another radioactive compound was detected which had the same Rf as the methylated derivative, 5-methoxytryptamine. Synthesis of this compound was appreciable, about 30% of that of serotonin. Levels of serotonin in the palate were measured by high pressure liquid chromatography. Palates at day 14.5 of gestation contained 0.40 ng serotonin/mg protein, which was greater than that of tongue (0.33), body (0.14) but less than that of brain (3.09). Serotonin in palate and other embryonic tissues increased with time of development. Dopamine levels in the palate and other tissues were also determined. The distribution of serotonin in the palate was analyzed by culturing day-14.5 embryos in the presence of [3H]5-HTP, and after aldehyde fixation, paraffin embedment and sectioning, autoradiography was performed. Grains were observed throughout the palate in cells of regions 2 and 3, internal mesenchyme, tooth germ, and epithelium. Surprisingly, the pterygopalatine nerve, maxillary nerve and pterygopalatine ganglion contained an appreciable concentration of grains. Thus, the presence of serotonin in the palate is consistent with the neurotransmitter playing a role in shelf elevation.

Animals↗
PubMed1980

Palate morphogenesis: II. Contraction of cytoplasmic processes in ATP-induced palate rotation in glycerinated mouse heads.

It has been previously shown that non-muscle contractile system(s) exist in mouse palate mesenchyme underlying the palatal epithelium before shelf rotation. In order to obtain evidence that the non-muscle contractile system(s) function to elevate the palate, glycerinated heads have been incubated with ATP. It was shown that 5 mM ATP and a 30 min incubation at 25 degrees C stimulated palate rotation optimally. Elevation of the anterior end of the palate was nearly complete (PSI = 3.90, p less than 10(-6)). Although rotation of the posterior end was significant (p less than 0.02), movement was limited (PSI = 1.70). Light microscopy of the palate revealed that ATP caused a marked condensation of the cytoplasmic processes of the mesenchymal cells. The contraction of the processes of the mesenchymal cells induced by ATP increased roughly with increased palate shelf rotation and was greater at the peripheral than at the internal mesenchyme. Cytochalasin B pretreatment at 40 microM completely blocked the ATP-induced rotation at the anterior end. The effect of other nucleotides on palate rotation was tested. GTP caused a significant stimulation of anterior shelf rotation (p less than 0.005), which was less than ATP, while ADP and CTP were ineffective. Low temperature (6 degrees C) prevented the ATP-induced shelf rotation. These results suggest that the non-muscle contractile cells in the mesenchyme play a role in palate elevation and that contraction of the actomyosin containing microfilaments supplies the motive force.

Actomyosin↗
PubMed1980

Palate morphogenesis. V. Effects of cholinergic agonists and antagonists on rotation in embryo culture.

Morphological studies have shown that the pterygopalatine ganglion in the day-14.5 mouse palatal shelf lies adjacent to the putative contractile system of region-2 cells in the posterior palate. It is of interest to learn whether the ganglion could influence rotation of the palate. Results of embryo culture experiments showed that acetylcholine appeared to play a role in posterior shelf rotation since cholinergic agents (pyridostigmine, bethanechol and carbachol) stimulated elevation of that end of the palate. To characterize the putative receptors mediating the effects of the cholinergic agonists on palate shelf elevation, muscarinic or nicotinic antagonists were introduced into the emrbyo culture system. Atropine, a muscarinic blocking agent, did not produce any significant effect on palate shelf rotation when tested in overnight and 2 h embryo cultures at 3 X 10(-5) M and 10(-4) M, respectively. Neither did atropine inhibit significantly the stimulation produced by 10(-8) M bethanechol, Thus, the cholinergic effect was probably not on muscarinic receptors of the contractile system. However, hexamethonium, a nicotinic antagonist, at 10(-6) M and 10(-4) M profoundly inhibited posterior shelf rotation to about 35% of the control value in a 2 h incubation. In addition, 10(-4) M hexamethonium inhibited posterior palate rotation to 11% of the control value after overnight culture. Furthermore, hexamethonium was able to reverse the stimulation of posterior rotation produced by carbachol. Partial inhibition of palate rotation by hexamethonium was also demonstrated when pregnant dams were injected with drug at doses approximately corresponding to 10(-6) and 10(-4) M. Hexamethonium treatment resulted in approximately 30% of the palates not completely rotated at day 15.5 compared to only 9.3% in the control. Hexamethoniuim also produced a significant increase in the palate gap and a comparable decrease in palate fusion. These effects were slightly greater at the posterior end of the palate. Thus, the cholinergic ganglion in the posterior palate may play a role in regulating shelf rotation at that end through a nicotinic pathway.

Acetylcholinesterase↗
PubMed1979

Palate morphogenesis. III. Changes in cell shape and orientation during shelf elevation.

The process of palate shelf elevation has been analyzed by light microscopy in mouse embryos cultured in vitro. The observations presented correlate changes in cell shape and orientation in the palate with the morphogenetic movement of the shelf. These studies suggest that in addition to any physical-chemical force elevating the shelf an active contraction of specific palate cells could also aid the process. Contribution to elevation could be derived from masses of contracting cells from the previously described non-muscle contractile systems in posterior (region 2) and mid-anterior (region 3) palate as well as other peripheral mesenchymal cells. Finally, elongation and contraction of the tongue side epithelial cells may also play a role in palate elevation.

Animals↗
PubMed1979

Palate morphogenesis. IV. Effects of serotonin and its antagonists on rotation in embryo culture.

Previous studies have localized non-muscle contractile systems in the posterior (region 2) and the anterior (region 3) ends of mouse palates at the time of shelf movement. In order to determine whether these contractile systems function in shelf rotation, effects of pharmacologic agents have been analyzed in embryo culture. First, it was shown that the posterior end of the palate rotates before the anterior end, and its rotation in culture was proportionally greater as development of the embryo progressed. Generally, the posterior end of the palate was more easily inhibited in embryo culture than the anterior end. Serotonin at 10(-8) M to 10(-5) M was shown to significantly stimulate rotation at the anterior end of the palate after 2 h in embryo culture. The effect on the posterior palate was less pronounced. To investigate further the role of this neurotransmitter on palate shelf rotation, serotonin antagonists were employed. Methysergide (10(-4) M) inhibited anterior shelf rotation to 12% of control values (P less than 0.005), while not significantly affecting the posterior end. Ergotamine (10(-6) M) significantly inhibited the stimulation induced by 10(-5) M serotonin (P less than 0.025). Cyproheptadine (10(-9) M) partially inhibited anterior and posterior shelf rotation in embryo culture. When injected into the pregnant dam, cyproheptadine partially inhibited shelf rotation and fusion. The palate was examined histologically after embryo culture. In the presence of 10(-4) M methysergide, the elongated contractile cells in region 3 at the anterior and midpalatal mesenchyme were prevented from rounding. Thus, serotonin may be regulating rotation of the anterior end of the palate by an effect on cell-mediated process.

Animals↗