To investigate the effect of endothelin on intracellular free calcium ([Ca(2+)]i) mobilization and the existence of the endothelin receptor in cultured bovine corneal endothelial cells(BCEC), endothelin-1(ET-1) -induced [Ca(2+)]i increase was measuredby using calcium sensitive indicator, fura-2/AM, and the studies on its desensitizaton and receptor antagonists were also performed. ET-1 increased [Ca(2+)]i in a concentration-dependent manner(10(-9)M-10(-5)M) and ET-1 -unduced [Ca(2+)]i transient increase was significantly ingibited (~50%) by the pretreatment with EGTA for 1 min. Similarly, neomycin also attenuated ET-1 -unduced [Ca(2+)]i transient increase in a concentration-dependent mannet. Desensitizatin study with successive treatment of ET-1 and ET-3, and the experiments of antagonists(BQ-610 for the ET(A) receptor and BQ-788 for the ET(B) receptor) showed the presence of ET(B) receptor in BCEC. In addition, ET-1(10(-6)M) accumulated inositol trisphosphate about two folds (310+/-6.8 cpm) comparing to control(154+/-11.6 cpm) and this accumulation was significantly inhibited by the treatment with neomycin (187+/-28 cpm). These results suggest that endothelin-induced calcium mobilization is receptor-mediated response and TE(B) receptor exists in BCEC.
Calcium* ; Egtazic Acid ; Endothelins ; Endothelium, Corneal* ; Inositol ; Neomycin ; Receptors, Endothelin

PURPOSE: In a previous study increasing the extracellular clacium concentration enhanced the phasic contractile response to low frequency stimulation(2Hz) to a significantly greater degree than the enhancement of high frequency stimulation(16Hz). To investigate the sensitivity of the detrusor contractile responses to field stimulation, bethanechol and ATP in calcium free buffer, the current study was designed. MATERIALS AND METHODS: Each rabbit bladder strip of 5x10mm in size was incubated for 30 minutes in the Tyrode's solution. Individual strips were utilized to generate the response to field stimulation(2, 8 and 32Hz), bethanechol(1.0-250microM) or ATP(0.25-2mM). Upon completion of the first stimulation in Tyrode's solution, each tissue was washed 3 1.tomes at 15 minute intervals with fresh Tyrodes. At 15 minutes following the last wash, the Tyrode's solution was replaced with solution containing no calcium+1.0mM EGTA and incubated for ditional 5, 15 or 30 minutes. At the end of 5, 15 or 30-min period of tulibration a second round of field stimulation or dose-response curves to bethanechol or ATP were generated. The contractile responses were monitored via an FT03 force transducer and recorded on a Grass 7D polygraph and expressed as the g tension per 100mg of tissue. RESULTS: (1) Progressive decrease in both basal tension and spontaneous contractile activity (2) more rapid decrease in the contractile response to 2 and 8Hz field stimulation than to 32Hz stimulation (3) more rapid decrease in the contractile response to lower concentrations of bethanechol and ATP than to high concentrations (4) greater maximal inhibition of the contractile response to low concentrations of bethanechol and ATP than to high concentrations. CONCLUSIONS: These results indicated that detrusor contractility to a sub-maximal stimulation rather than maximal stimulation is more sensitive to extracellular calcium depletion.
Adenosine Triphosphate* ; Bethanechol* ; Calcium* ; Egtazic Acid ; Poaceae ; Transducers ; Urinary Bladder*

Firing patterns of injured nerve fibers were recorded using the single-fiber firing recording technique. Under the same background firing pattern, three types of bursting were induced separately by EGTA, veratridine or high [Ca(2+)](o) in the same type of nerve fibers. The results suggest that different firing patterns are related to different stimuli, which means that each firing pattern carries corresponding neural information.
Action Potentials ; Animals ; Calcium ; pharmacology ; Egtazic Acid ; pharmacology ; Nerve Fibers ; drug effects ; pathology ; Rats ; Veratridine ; pharmacology

The outward currents elicited in hamster eggs by depolarizing pulses were studied. The currents appeared to comprise at least two components, a transient outward component (Ito) and a steady-state outward component (Iinfin). Ito was transiently followed by the cessation of inward Ca2+ current (ICa), and its current-voltage (I-V) relation was a mirror image of that of ICa. Either blockade of ICa by Co2+ or replacement of Ca2+ with Sr2+ abolished Ito without change in Iinfin. Intracellular EGTA (10 mM) inhibited Ito but not Iinfin. suggesting strongly that generation of Ito requires intracellular Ca2+. Apamin (1 nM) abolished selectively Ito, indicating that Ito is Ca2+-dependent K+ current. On the other hand, Iinfin was Ca2+-independent. Both Ito and Iinfin were completely inhibited by internal Cs+ and external TEA. The estimated reversal potential of Ito was close to the theoretical EK. Taken together, both outward currents were carried by K+ channels. From these results, Ito is likely to be a current responsible for the hyperpolarizing responses seen in hamster eggs at fertilization.
Animals ; Apamin ; Cricetinae* ; Eggs ; Egtazic Acid ; Fertilization ; Hand ; Oocytes* ; Ovum ; Tea

Because synaptic refinement of medial nucleus of trapezoid body (MNTB) - lateral superior olive (LSO) synapses is most active during the first postnatal week and the long term depression (LTD) has been suggested as one of its mechanisms, LTD of MNTB-LSO synapses was investigated in neonatal rat brain stem slices with the whole cell voltage clamp technique. In Mg2+ free condition, tetanus (10 stimuli at 10 Hz for 2 min) in the current clamp mode induced a robust LTD of isolated D, L-APV-sensitive postsynaptic currents (PSCs) for more than 30 min (n=6, 2.4+/-0.4% of the control), while isolated CNQX-sensitive PSCs were not suppressed (n=6, 95.3+/-1.6%). Tetanus also elicited similar LTD in the isolated GABAergic/glycinergic PSCs (n=5, 3.6+/-0.5%) and mixed PSCs (GABAergic/glycinergic/glutamatergic) (n=4, 2.2+/-0.7%). However, such a strong LTD was not observed in the mixed PSCs when 10 mM EGTA was added in the internal solution (n=10), indicating that postsynaptic Ca2+ rise is needed for the strong LTD. This robust LTD might contribute to the active synaptic refinement occurring during the first postnatal week.
Animals ; Brain Stem ; Depression ; Egtazic Acid ; Olea ; Rats* ; Synapses* ; Synaptic Potentials ; Tetanus

The thioureylene drugs, propylthiouracil and methylmercaptoimidazol(MMI), exert their antithyroid effect primarily through inhibition of thyroid peroxidase-catalyzed iodination of thyroglobulin. Recently the interest about the effect to the thyroglobulin synthesis of these drugs have been increasing. So we studied the MMI effect to the thyroglobulin synthesis in cultured porcine thyroid cells. Porcine thyroid cells were isolated by sequential trypsinization in the presence of EGTA, seeded at high density(1X10^6 cells/cm^2) and cultured. One week later, MMI was added in different concentrations(0, 0.2, 1, 5mM) with TSH only or with 4H(b-TSH, Insulin, Transferrin, Hydrocortisone) or without hormone. Medias were collected after 24 hours and compared the amount of thyroglobulin secreted. And also pulse-labeling were performed with S^35 cysteine/methionine(1-2uCi/well) for 30, 60, 90min at the same conditions.There was no significant change in the amount of the secreted thyroglobulin by MMI, and there was no significant change in the pulse-labeled interacellular thyroglobulin by MMI. And also there was no significant change in the secretion of TSH-stimulated thyroglobulin by MMI. So we conclude that MMI has no effect on the thyroglobulin synthesis in cultured porcine thyroid cells and also MMI has no effect on the TSH-stimulated thyroglobulin synthesis in cultured porcine thyroid cell.
Antithyroid Agents ; Egtazic Acid ; Halogenation ; Insulin ; Methimazole ; Propylthiouracil ; Thyroglobulin ; Thyroid Gland ; Transferrin ; Trypsin

The amount of thyroglobulin synthesized from thyroid cells and stored in colloid space in very important in thyroid hormone synthesis. The thyroglobulin synthesis is mainly regulated by TSH secreted from the pituitary gland. But recently there were some reports about the possibility that iodine regulated the thyroid protein synthesis. So our studied were conducted to determined whether iodine could have inhibitory effect on thyroglobulin synthesis and methimazole could abolish the inhibitory effect of idoine.Porcine thyroid cells were isolated by sequential trypsinization in the presence of EGTA, seeded at high density(1X10^6 cells/cm^2) and cultured. One week later, Nal was added in different concentrations(10^-7, 10^-6, 10^-5, 10^-4M). 24hour medias were collected and checked the amount of thyroglobulin secreted. And also pulse-labeling were performed with[^35S] cysteine/methionine(1-2 uCi/well) for 1 hour at the same conditions. We used 3mM methimazole and 10^-4M NaI to observe the blocking effect of methimazol in iodine.The extracellular thyroglobulin secretion was significantly decreased by iodine in dose dependent manner(82.4%, 80.7%, 76.8% and 73.1% of control). And also intracellular thyroglobulin synthesis was significantly decreased by iodide in dose dependent manner(100.5%, 83.4%, 82.3% and 79.4% of control). The inhibitory effect of iodide was abolished by methimazole(74.7% to 101.3% of control). These data indicate that high iodide inhibit the thyroglobulin synthesis and secretion from the thyroid cells, and furthermore autoregulation by iodide may include thyroglobulin synthesis. And also this effect is dependent on the generation of an organic form of iodine because methimazole abolish the inhibitory effect of iodide.
Colloids ; Egtazic Acid ; Homeostasis ; Iodine ; Methimazole ; Pituitary Gland ; Thyroglobulin ; Thyroid Gland ; Trypsin

The aim of the present study is to investigate the contribution of Ca2+-activated K+ (KCa) channels and delayed rectifier K+ (KV) channels to the resting membrane potential (RMP) in rabbit middle cerebral arterial smooth muscle cells. The RMP and membrane currents were recorded using the whole-cell patch configuration and single KCa channel was recorded using the outside-out patch configuration. Using the pipette solution containing 0.05 mM EGTA, the RMP was -25.76+/-5.08 mV (n=12) and showed spontaneous transient hyperpolarizations (STHPs). The membrane currents showed time- and voltage-dependent outward currents with spontaneous transient outward currents (STOCs). When we recorded the membrane potential using the pipette solution containing 10 mM EGTA, the RMP was depolarized and did not show STHPs. The membrane currents showed no STOCs but only showed slowly inactivating outward currents. External TEA (1 mM) reversibly inhibited the STHPs, depolarized the RMP, reduced the membrane currents, abolished STOCs, and decreased the open probability of single KCa channel. When KV currents were isolated, the application of 4-AP (5 mM) depolarized the RMP. The important aspect of our results is that KCa channel is responsible for the generation of the STHPs in the membrane potential and plays an important role in the regulation of the RMP and KV channel is also responsible for the regulation of the RMP in rabbit middle cerebral arterial smooth muscle cells.
Egtazic Acid ; Membrane Potentials* ; Membranes ; Muscle, Smooth* ; Myocytes, Smooth Muscle* ; Tea

Ghrelin is a newly discovered peptide, which is released from the stomach and neurons in the hypothalamic arcuate nucleus (ARC), and potently stimulates growth hormone release and food intake. In the present study, we investigated the effect of ghrelin on [Ca2+]i in thyroid FRTL-5 cells. Ghrelin (5 nM) increased [Ca2+]i and TSH (1 unit/l) had an additive effect on [Ca2+]i when extracellular normal solution was 1.1mM Ca2+ containing Coon's modified Ham's F12 medium. When Ca2+-free medium containing 2 mM EGTA replaced the above normal solution, ghrelin also induced a similar rise in [Ca2+]i. In the middle of [Ca2+]i increment by ghrelin, nifedipine (1 micrometer), nickel (100micrometer) and La3+ (100micrometer) had no effect on [Ca2+]i. After endoplasmic reticulum was depleted by cyclopiazonic acid (CPA; 10micrometer), ghrelin caused no visible change on [Ca2+]i in Ca2+-free/2 mM EGTA solution. These results suggest that ghrelin can increase [Ca2+]i through endoplasmic reticulum in thyroid FRTL-5 cells.
Arcuate Nucleus ; Eating ; Egtazic Acid ; Endoplasmic Reticulum ; Ghrelin* ; Growth Hormone ; Neurons ; Nickel ; Nifedipine ; Stomach ; Thyroid Gland*

OBJECTIVE: This study was to determine the effect of different concentration of calcium in medium on the preimplantational development of zygotes and early 2-cell embryos. METHODS: Female mice of ICR strain (5~8 weeks old) were superovulated and mated with fertile males. Zygotes or early 2-cell embryos were collected by flushing the oviducts 31~32 hours after hCG injection. The embryos were cultured in various concentrations of Ca2+ in medium or with EDTA, EGTA and Ni2+. RESULT AND CONCLUSION: Treatment of high concentration of Ca2+ (3.42 mM (2X)~17.1 mM (10X) in medium didn't develop well compared to the control Low concentrations of Ca2+ (0.214 mM (1/8X)~0.855 mM (1/2X)) were deterimental to development beyond 2-cell stage. EDTA, Ca2+ chelating agent was treated with ranged concentrations of eDTA (0.014 mM~0.107 mM) to medium contaning 1.71 mM Ca2+ showed beneficial effect to development to blastocyst compared to the control. EGTA, extracellular Ca2+ chelator, was treated with ranged concentrations of EGTA (0.014~0.107 mM) to the medium contaning 1.71 mM Ca2+. There is no significant difference with the control. Ni2+ (50 micrometer), T-type Ca2+-channel blocker was treated to medium contaning low concentration of Ca2+. It overcame 2-cell block significantly. Rate of degenerated embryos decreased and developmental rate to morula and blastocyst increased more than low Ca2+ concentration alone. Further studies are needed for the overcoming effect of 2-cell block by Ni2+.
Animals ; Blastocyst ; Calcium ; Edetic Acid ; Egtazic Acid ; Embryonic Structures* ; Female ; Flushing ; Humans ; Male ; Mice* ; Morula ; Oviducts ; Zygote