Regulation of cell shape in Euglena gracilis. V. Time-dependent responses to Ca2+ agonists and antagonists

1988 ◽  
Vol 89 (3) ◽  
pp. 365-371
Author(s):  
T.A. Lonergan ◽  
L.C. Williamson
Keyword(s):  
Euglena Gracilis ◽  
Cell Elongation ◽  
Biological Clock ◽  
Bay K 8644 ◽  
Unexpected Result ◽  
Channel Blockers ◽  
Light Cycle ◽  
Free Medium ◽  
Simultaneous Exposure ◽  
Cell Rounding

The daily changes in cellular shape observed in growth-synchronized cultures of Euglena gracilis Klebs strain Z, were altered by exposure to Ca2+ channel agonists and antagonists. The response of the cells to these pharmacological agents depended, in part, on the time in the growth cycle that the cells were exposed. The Ca2+ channel blockers verapamil and nifedipine and the intracellular Ca2+ antagonist TMB-8 all caused cell rounding when elongated cells from the middle of the light cycle were treated. These results were the same as with other methods used to deprive cells of extracellular Ca2+, such as exposure to EGTA or resuspension in Ca2+-free medium. The cell response in mid light cycle to the channel blockers was reversible by simultaneous exposure to CaCl2, and the nifedipine response was also reversed by simultaneous exposure to the structurally related Ca2+ agonist BAY-K 8644. Exposure of cells in the first hour of the light cycle to verapamil, nifedipine or TMB-8 caused an unexpected result. Instead of preventing the round cells from elongating in the first portion of the light cycle, as do LaCl3, EGTA or resuspension in Ca2+-free medium, a greater than expected percentage of elongated cells was found in the treated population. This represents the first instance in which the biological clock control over the rate and extent of cell elongation was accelerated. The calcium agonist CGP-28392 did not have an effect on cell elongation in the early portion of the light cycle but caused cell rounding in the middle of the light cycle. The calcium agonist BAY-K 8644 did not cause any shape changes alone, but was capable of reversing the effects of nifedipine in the middle of the light cycle.

scholarly journals Regulation of cell shape in Euglena gracilis. IV. Localization of actin, myosin and calmodulin

1985 ◽  
Vol 77 (1) ◽  
pp. 197-208
Author(s):  
T.A. Lonergan
Keyword(s):  
Plasma Membrane ◽  
Euglena Gracilis ◽  
Cell Shape ◽  
Biological Clock ◽  
Free Medium ◽  
Calmodulin Antagonist ◽  
Cell Rounding ◽  
Contractile System ◽  
High Degree ◽  
Shape Changes

The immunofluorescence patterns for actin, myosin, calmodulin and tubulin were observed in Euglena gracilis Klebs strain Z during the biological clock-controlled shape changes observed with division-synchronized cells, and during two shock responses that induce cell rounding. The fluorescence patterns for actin, myosin, calmodulin and tubulin show a high degree of coincidence and are visualized as lines running parallel to, and having the same spacing as, the pellicle strips beneath the plasma membrane. The fluorescence patterns remain intact during the daily shape changes, implying that the shape changes do not result from cycles of polymerization and depolymerization of the microtubules and microfilaments. Resuspension of cells in Ca2+-free medium induces cell rounding of many of the cells. The actin and calmodulin patterns are partially disrupted by the Ca2+-free resuspension, while the myosin pattern is almost totally disrupted. Microtubules are unaffected by this treatment. Prior exposure of cells to the calmodulin antagonist trifluoperazine or to the microfilament-stabilizing peptide phalloidin stabilize the actin, myosin and calmodulin patterns against disruption by the Ca2+-free resuspension and other shock responses. The possibility of an actomyosin contractile system controlled by calmodulin is discussed.

Tolbutamide and diazoxide modulate phospholipase C-linked Ca2+ signaling and insulin secretion in β-cells

2000 ◽  
Vol 278 (4) ◽  
pp. E639-E647 ◽  
Author(s):  
Christof Schöfl ◽  
Julia Börger ◽  
Thilo Mader ◽  
Mark Waring ◽  
Alexander von zur Mühlen ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Phospholipase C ◽  
Insulin Release ◽  
Arginine Vasopressin ◽  
Bay K 8644 ◽  
Free Medium ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Activating Receptors

Arginine vasopressin (AVP), bombesin, and ACh increase cytosolic free Ca2+ and potentiate glucose-induced insulin release by activating receptors linked to phospholipase C (PLC). We examined whether tolbutamide and diazoxide, which close or open ATP-sensitive K+ channels (KATP channels), respectively, interact with PLC-linked Ca2+ signals in HIT-T15 and mouse β-cells and with PLC-linked insulin secretion from HIT-T15 cells. In the presence of glucose, the PLC-linked Ca2+ signals were enhanced by tolbutamide (3–300 μM) and inhibited by diazoxide (10–100 μM). The effects of tolbutamide and diazoxide on PLC-linked Ca2+ signaling were mimicked by BAY K 8644 and nifedipine, an activator and inhibitor of L-type voltage-sensitive Ca2+channels, respectively. Neither tolbutamide nor diazoxide affected PLC-linked mobilization of internal Ca2+ or store-operated Ca2+ influx through non-L-type Ca2+ channels. In the absence of glucose, PLC-linked Ca2+ signals were diminished or abolished; this effect could be partly antagonized by tolbutamide. In the presence of glucose, tolbutamide potentiated and diazoxide inhibited AVP- or bombesin-induced insulin secretion from HIT-T15 cells. Nifedipine (10 μM) blocked both the potentiating and inhibitory actions of tolbutamide and diazoxide on AVP-induced insulin release, respectively. In glucose-free medium, AVP-induced insulin release was reduced but was again potentiated by tolbutamide, whereas diazoxide caused no further inhibition. Thus tolbutamide and diazoxide regulate both PLC-linked Ca2+signaling and insulin secretion from pancreatic β-cells by modulating KATP channels, thereby determining voltage-sensitive Ca2+ influx.

Evidence for two types of internal Ca2+ stores in canine mesenteric artery with different refilling mechanisms

1992 ◽  
Vol 262 (1) ◽  
pp. H31-H37 ◽  
Author(s):  
A. M. Low ◽  
C. Y. Kwan ◽  
E. E. Daniel
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Mesenteric Artery ◽  
Cyclopiazonic Acid ◽  
Bay K 8644 ◽  
Krebs Solution ◽  
Tetraacetic Acid ◽  
Free Medium ◽  
Phenylephrine Hydrochloride ◽  
Dog Mesenteric Artery ◽  
Biphasic Responses

Novel transient biphasic responses of the dog mesenteric artery to phenylephrine hydrochloride (PE, 10 microM) in Ca(2+)-free medium containing 50 microM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) have been analyzed. The initial component was significantly inhibited by ryanodine (30-100 microM), an agonist enhancing Ca2+ release from the sarcoplasmic reticulum, whereas the second was significantly inhibited by nifedipine (1 microM), and L-type Ca2+ channel antagonist, or EGTA, to chelate Ca2+, and was potentiated by BAY K 8644 (1 microM), an L-type Ca2+ channel agonist. After repletion of Ca2+ stores in normal Krebs solution or in high KCl (60 mM) Krebs, the first component was inhibited by cyclopiazonic acid (CPA, 30 microM), a putative, reversible, and selective microsomal Ca2+ pump adenosinetriphosphatase inhibitor. BAY K 8644 potentiated the second component in the presence of CPA. The inhibition of the first component by CPA suggests that the refilling ultimately requires the CPA-sensitive Ca2+ pump for Ca2+ resequestration. However, the second component may refill by a CPA-independent route opened by BAY K 8644. These results, taken as a whole, indicate that the biphasic PE response in Ca(2+)-free medium may reflect compartmentalization of Ca2+ storage related to the different routes of refilling.

scholarly journals Photoaffinity labelling of the cardiac calcium channel. (−)-[3H]azidopine labels a 165 kDa polypeptide, and evidence against a [3H]-1,4-dihydropyridine-isothiocyanate being a calcium-channel-specific affinity ligand

1987 ◽  
Vol 243 (1) ◽  
pp. 127-135 ◽  
Author(s):  
D R Ferry ◽  
A Goll ◽  
H Glossmann
Keyword(s):  
Guinea Pig ◽  
Calcium Channel ◽  
Polyacrylamide Gel Electrophoresis ◽  
Dimethyl Ester ◽  
High Capacity ◽  
Bay K 8644 ◽  
Channel Blockers ◽  
Guinea Pig Heart ◽  
Affinity Ligand ◽  
Heart Membranes

The arylazide 1,4-dihydropyridine (-)-[3H]azidopine binds to a saturable population of sites in guinea-pig heart membranes with a dissociation constant (KD) of 30 +/- 7 pM and a density (Bmax.) of 670 +/- 97 fmol/mg of protein. This high-affinity binding site is assumed to reside on voltage-operated calcium channels because reversible binding is blocked stereoselectively by 1,4-dihydropyridine channel blockers and by the enantiomers of Bay K 8644. A low-affinity (KD 25 +/- 7 nM) high-capacity (Bmax. 21.6 +/- 9 pmol/mg of protein) site does not bind (-)- or (+)-Bay K 8644, but is blocked by high concentrations (greater than 500 nM) of dihydro-2,6-dimethyl-4-(2-isothiocyanatophenyl)-3,5-pyridinedicarboxy lic acid dimethyl ester (1,4-DHP-isothiocyanate) or, e.g., (+/-)-nicardipine. (-)-[3H]Azidopine was photoincorporated covalently into bands of 165 +/- 8, 39 +/- 2 and 35 +/- 3 kDa, as determined by SDS/polyacrylamide-gel electrophoresis. Labelling of the 165 kDa band is protected stereoselectively by 1,4-dihydropyridine enantiomers at low (nM) concentrations and by (-)- and (+)-Bay K 8644, whereas the lower-Mr bands are not. Thus, only the 165 kDa band is the calcium-channel-linked 1,4-dihydropyridine receptor. Photolabelling of the 39 or 35 kDa bands was only blocked by 10 microM-1,4-DHP-isothiocyanate or 50 microM-(+/-)-nicardipine but not by 10 microM-(-)-Bay K 8644. [3H]-1,4-DHP-isothiocyanate binds to guinea-pig heart membranes with a KD of 0.35 nM and dissociates with a k-1 of 0.2 min-1 at 30 degrees C. [3H]-1,4 DHP-isothiocyanate irreversibly labels bands of 39 and 35 kDa which are protected by greater than 10 microM-(+/-)-nicardipine or unlabelled ligand but not by 10 microM-(-)-Bay K 8644. Thus, [3H]-1,4-DHP-isothiocyanate is not an affinity probe for the calcium channel.

scholarly journals SUN-LB121 Nifedipine Worsens Glucose Tolerance in C57BL/6J Mice Exposed to Intermittent Hypoxia

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Stanley M Chen Cardenas ◽  
Larissa A Shimoda ◽  
Naresh M Punjabi
Keyword(s):  
Glucose Tolerance ◽  
Murine Model ◽  
Fasting Glucose ◽  
Tolerance Test ◽  
Automated System ◽  
Channel Blockers ◽  
Light Cycle ◽  
Glucose Levels ◽  
Obstructive Sleep ◽  
Tolerance Curve

Abstract Background: Intermittent hypoxemia (IH), a pathognomonic component of obstructive sleep apnea (OSA), has been independently associated with development of glucose intolerance, insulin resistance, and type 2 diabetes. L-type calcium channel blockers (CCB) influence glucose homeostasis including insulin sensitivity and secretion. To date, the potential impact of the combined effects of L-type CCB and IH on fasting glycemia and glucose tolerance have not been examined. The objective of this study was to determine whether CCB alters glucose metabolism in a murine model of IH. Methods: Adult male C57BL6/J mice (age 19-week-old) were exposed to IH using an automated system with specially-modified cages that oscillated FiO2from 21% to 5.5% at a target rate of 60 events/h during a 12 h (7am – 7pm) light cycle to simulate severe OSA for 5 days. The L-type CCB, nifedipine, or vehicle (polyethleneglycol-400) were administered at a dose of 20mg/kg/day via subcutaneous osmotic pumps (Alzet model 2001). Mice were exposed to IH or intermittent air (IA) with four resulting groups: IA-vehicle (n=12), IH-vehicle (n=16), IA–nifedipine (n=10), and IH–nifedipine (n=13). Fasting glucose, intraperitoneal glucose tolerance test, and insulin levels were obtained after exposures. Results: In the absence of a L-type CCB, IH increased fasting (105.1 vs. 71.2 mg/dL; p<0.001) and 2-hour glucose levels (104.8 vs. 82.0 mg/dL; p=0.003). The area under the glucose tolerance curve (AUC) was also higher with IH than IA in mice treated with vehicle (17896.3 vs.13965.8 mg-min/dL; p<0.001). Although the effects of IH on fasting glucose levels were comparable with and without L-type CCB treatment, the 2-hour glucose levels and the AUCs were substantially different. A statistically significant interaction was noted for the 2-hr glucose levels between IH and treatment with a L-type CCB (IH-CCB: 193.7; IH-V: 122.6; IA-CCB: 103.5; and IA-V: 82.0 mg/dL; p<0.05 for interaction between IH and CCB). Finally, the AUC for IH-CCB treated mice was significantly higher than the AUC for IH-V treated mice (IH-CCB: 30223.1; IH-V: 17896.3. mg-min/dL; p=0.0001) Conclusions: In a murine model of IH, treatment with an L-type CCB exacerbates the deleterious effects of IH on glucose tolerance. Thus, use of CCB in patients with OSA should take into consideration these unfavorable effects particularly in those who are metabolically compromised.

The Effect of a Biological Clock on the Developmental Rate of Drosophila Pupae

1965 ◽  
Vol 42 (2) ◽  
pp. 323-337
Author(s):  
JANET E. HARKER
Keyword(s):  
Biological Clock ◽  
Bright Light ◽  
Developmental Rate ◽  
Time Of Day ◽  
Time Interval ◽  
Light Cycle ◽  
Continuous Darkness ◽  
Pupal Development ◽  
Wide Range ◽  
Three Stages

1. A study of the course of pupal development in two strains of Drosophila melanogaster has been made in an attempt to establish the factors affecting the time of adult eclosion. 2. The time taken to complete three stages of pupal development has been measured for pupae entering each stage at each particular hour of the day when insects were kept in 12 hr. light:12 hr. darkness, 12 hr. bright light: 12 hr. dim light, or in continuous darkness. 3. The duration of each stage, in both strains, is affected by the time of day, relative to the light cycle, at which the stage is entered. The duration of each stage for pupae kept in continuous darkness is affected by the time of day at which the stage is entered, relative to the light cycle to which they had been exposed as larvae. 4. The time-interval curves for all three stages of any one strain take the same form. 5. Because of the very wide range of developmental rates, dependent upon the time within the light cycle at which each stage begins, a population in which the larvae all pupate within a 24 hr. period will continue to produce adult flies over several days. 6. The eclosion rhythm is a population effect and does not reflect the phasing of individuals to a dawn eclosion; the majority of adults emerge at dawn because of the summation effect of circadian rhythms of development at earlier stages.

A role for Ca(2+)-conducting ion channels in mechanically-induced signal transduction of airway epithelial cells

1994 ◽  
Vol 107 (11) ◽  
pp. 3037-3044 ◽  
Author(s):  
S. Boitano ◽  
M.J. Sanderson ◽  
E.R. Dirksen
Keyword(s):  
Epithelial Cells ◽  
Mechanical Stimulation ◽  
Airway Epithelial Cells ◽  
Effective Concentration ◽  
Channel Blockers ◽  
Airway Epithelial ◽  
Free Medium ◽  
Half Maximal Effective Concentration ◽  
Induced Signal ◽  
Stimulation Of

Mechanical stimulation of a single cell in a cultured monolayer of airway epithelial cells initiates an intercellularly communicated increase in intracellular Ca2+ concentration ([Ca2+]i) that propagates radically through adjacent cells via gap junctions, forming an intercellular Ca2+ wave. Mechanically-induced intercellular Ca2+ waves also occur in the absence of extracellular Ca2+. However, in Ca(2+)-free medium an increase in [Ca2+]i of the stimulated cell does not occur. Thus, mechanically-induced [Ca2+]i changes in the stimulated cell are influenced by the extracellular Ca2+ concentration. To investigate if a channel-mediated Ca2+ flux across the plasma membrane contributes to the elevation of [Ca2+]i in the stimulated cell we used digital image microscopy to measure mechanically-induced [Ca2+]i changes in the presence of Ca2+ channel blockers. In Ca(2+)-free medium containing Gd3+ (20 microM) mechanical stimulation resulted in an [Ca2+]i increase in the stimulated cell. The delay time between mechanical stimulation and increase in [Ca2+]i of the stimulated cell was dependent on extracellular [Gd3+], with a half-maximal effective concentration of approximately 40 microM. Mechanical stimulation in Ca(2+)-free medium containing La3+ (10 microM) or Ni2+ (100 microM) gave similar results. Mechanical stimulation in Ca(2+)-free medium containing the dihydropyridine Ca2+ channel blockers nifedipine (10 microM) and nimodipine (10 microM) also resulted in an increase of [Ca2+]i of the stimulated cell. Mechanical stimulation of cells treated with thapsigargin to deplete intracellular Ca2+ stores, in the presence of 1.3 mM extracellular Ca2+, results in an increase in [Ca2+]i of the stimulated cell without the propagation of an intercellular Ca2+ wave.(ABSTRACT TRUNCATED AT 250 WORDS)

Ca2+ channels in chick neural retina cells characterized by 1,4-dihydropyridine antagonists and activators

1989 ◽  
Vol 67 (5) ◽  
pp. 506-514 ◽  
Author(s):  
X. Y. Wei ◽  
A. Rutledge ◽  
Q. Zhong ◽  
J. Ferrante ◽  
D. J. Triggle
Keyword(s):  
Calcium Channel ◽  
Intact Cell ◽  
Neural Retina ◽  
Bay K 8644 ◽  
Single Type ◽  
Channel Blockers ◽  
Binding Affinities ◽  
High Affinity ◽  
Voltage Dependent ◽  
High Affinity Binding Site

The voltage-sensitive calcium channel in cultured chick neural retina cells was characterized by the actions of the enantiomers of Bay K 8644 and 202-791 and other 1,4-dihydropyridines. These cells showed time- and voltage-dependent Ca2+ uptake that was stimulated by K+ depolarization and blocked by the inorganic calcium channel blockers Cd2+ and Co2+. A small fraction only (15% maximum) of the uptake was inactivated by predepolarization of the cells with 80 mM K+. Ca2+ uptake was sensitive to the 1,4-dihydropyridine calcium channel antagonists and activators. (S)-Bay K 8644 and (S)-202-791 stimulated the Ca2+ uptake, and (R)-Bay K 8644 and (R)-202-791 as well as nitrendipine and PN 200-110 inhibited Ca2+ uptake stimulated by K+ depolarization or channel activators. The K+ depolarization-stimulated uptake was inhibited by 90%, but the activator-stimulated uptake was completely blocked by the 1,4-dihydropyridine antagonists. The potencies of these agents as inhibitors of Ca2+ uptake were significantly lower than the binding affinities in membrane preparations from the same cells or their binding and pharmacologic affinities in vascular smooth muscle. K+ depolarization or (S)-Bay K 8644 induced 45Ca2+ uptake was not observed in a glial cell culture. [3H]Nitrendipine and [3H]PN 200-110 bound to membrane preparations of the cells consistent with the presence of a single type of high affinity binding site. [3H]PN 200-110 bound with higher affinity (KD = 7.09 ± 0.90 × 10−11 M) than did [3H]nitrendipine (KD = 4.10 ± 0.92 × 10−10 M), but the Bmax values were similar for the two ligands (98.9 ± 4.1 and 99.4 ± 6.58 fmol/mg protein, respectively). The discrepancy between binding and pharmacologic activities of the antagonist ligands does not appear to be due to the presence of 1,4-dihydropyridine-insensitive Ca2+ channels, but may relate to the inability of these agents to access a high affinity inactivated state in the intact cell or to the presence of discrete categories of binding sites. This marked discrepancy between affinities does not exist for the activator ligands studied. This study confirms the presence of voltage-dependent 1,4-dihydropyridine sensitive Ca2+ channels in chick neural retina cells.Key words: Ca2+ channels, 1,4-dihydropyridines, chick neural retina, retinal neurons.

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