A calcium requirement for electric field-induced cell shape changes and preferential orientation

C3H/10T1/2 mouse embryo fibroblasts stimulated by a steady electric field (10 V/cm) for 30 min exhibited lamellar retraction on the sides facing the electrodes. Some cells elongated and preferentially oriented with their long axis perpendicular to the field direction. Depletion of external calcium or blockage of calcium influx with lanthanum or the calcium channel blocker D-600 resulted in a reduction of the field-induced response. When external calcium was elevated stepwise from 0 to 10 mM, the field-induced response increased correspondingly. Electric stimulation in the presence of the calcium ionophore A23187 resulted in an increase of spindle-shaped cells with no preferential orientation. This response was blocked by calcium depletion and lanthanum, but not by D-600. The anticalmodulin drug W-13 inhibited the field-induced responses observed in normal buffer as well as in the presence of A23187. Some cell death resulted from prolonged electric field exposure, and the mortality was reduced by calcium depletion, lanthanum or D-600, but was not affected by W-13. We postulate that local calcium influx through channels opened by the electric field produces areas of high intracellular calcium which stimulate the cytoskeletal network to induce lamellar retraction. Prolonged field-induced calcium influx may eventually overcome the cell's mitochondrial calcium-buffer system, leading to necrotic calcification.     

Calcium,calmodulin, and the production of prostacyclin by cultured vascular endothelial cells

The production of prostacyclin (PGI2) by cultured porcine aortic endothelial cells, in response to serum and the calcium ionophore A23187, was inhibited by TMB-8, an antagonist of intracellular calcium mobilization. The calcium-channel blocker methoxyverapamil (D600) inhibited serum-induced PGI2 production in but had little effect on A23187-induced PGI2 production. Calmodulin activity was detected in endothelial-cell lysates and was inhibited by the calmodulin antagonist W7, which also inhibited PGI2 production in response to both agonists. Calcium and calmodulin appear to play an important role in mediating PGI2 production by the vascular endothelium.     

Trifluoperazine Stimulates Acetylcholine Receptor Synthesis in Cultured Chick Myotubes

Acetylcholine receptor appearance rate in the presence of the phenothiazines trifluoperazine and chlorpromazine was measured in cultured embryonic chick myotubes by means of 125I-alpha-bungarotoxin. At drug concentrations of 5 to 10 X 10(-6) M, receptor appearance rate was significantly enhanced while receptor half-life, cellular protein, net protein synthesis rate, and acetylcholinesterase levels were not similarly affected. The sulfoxide derivatives were without effect. At concentrations of 3 X 10(-5) M and above, both trifluoperazine and chlorpromazine caused myotube contracture and cell loss. Drug combination experiments revealed that receptor stimulation caused by phenothiazines is overcome by low concentrations of veratridine and ryanodine, but not by membrane depolarization with 20 mM KCl. These results lend support to the role of calcium as an intracellular messenger in acetylcholine receptor synthesis regulation, but are difficult to reconcile with the notion that cytosolic calmodulin serves as the calcium receptor in this signaling pathway. Since the trifluoperazine effect resembles that caused by the calcium antagonist D-600, phenothiazines may stimulate receptor synthesis by blocking a voltage-gated calcium channel.     

Trifluoperazine Inhibits 45Ca2+ Uptake and Catecholamine Secretion and Synthesis in Adrenal Medullary Cells

Abstract: In isolated adrenal medullary cells, carbamyl-choline and high K⁺ cause the calcium-dependent secretion of catecholamines with a simultaneous increase in the synthesis of ¹⁴C-catecholamines from [¹⁴C]tyrosine. In these cells, trifluoperazine, a selective antagonist of calmodulin, inhibited both the secretion and synthesis of catecholamines. The stimulatory effect of carbamyl-choline was inhibited to a greater extent than that of high K⁺. The inhibitory effect of trifluoperazine on carbamylcholine-evoked secretion of catecholamines was not overcome by an increase in either carbamylcholine or calcium concentration, showing that inhibition by trifluoperazine occurs by a mechanism distinct from competitive antagonism at the cholinergic receptor and from direct inactivation of calcium channels. Doses of trifluoperazine that inhibited catecholamine secretion and synthesis also inhibited the uptake of radioactive calcium by the cells. These results suggest that trifluoperazine inhibits the secretion and synthesis of catecholamines mainly due to its inhibition of calcium uptake. Trifluoperazine seems to inhibit calcium uptake by uncoupling the linkage between cholinergic receptor stimulation and calcium channel activation.     

Cytoprotection and intracellular calcium.

It seems that prostacyclin has an increasing effect on gastric mucosal (antral and fundic) calmodulin level in rats. Using either the calcium channel blocker verapamil or anti-calmodulin drugs (diazepam, trifluoperazine,) the cytoprotective effect of prostacyclin can be inhibited. Therefore, it is probable that calcium ions and calcium-activated calmodulin play a role in the effect of prostacyclin.     

The role of calcium ions in the mechanism of ACTH stimulation of cortisol synthesis

Removal of free calcium ions from the incubation medium of isolated bovine adrenocortical cells with EGTA reduced basal cortisol synthesis and blocked the effects of ACTH; additional calcium restored normal steroid synthesis. Calcium channel blockers, verapamil and nitrendipine and the calmodulin antagonist, trifluoperazine inhibited ACTH-stimulated cortisol synthesis in a dose-dependent manner (IC50s of 6.2, 10 and 5.2 microM, respectively). Steroidogenic effects of dibutyryl cyclic AMP were prevented with 50 microM verapamil or trifluoperazine. Calcium ionophore A23187 at 1 microM increased cortisol synthesis 2-3 fold which was less than the normal response to ACTH. Stimulatory effects of ionophore and cyclic AMP or ACTH were not additive. ACTH-stimulation of cortisol synthesis appears to involve cyclic AMP-dependent uptake of extracellular calcium ions, possibly by a mechanism requiring calmodulin. Increases in intracellular calcium ions cannot wholly mimic ACTH actions.     

钙信使系统对机械刺激诱导的烟草悬浮培养细胞中H_2O_2爆发的调控

机械刺激可诱导烟草悬浮培养细胞H2O2的爆发,外源Ca2＋有强化作用,而Ca2＋螯合剂EGTA、质膜Ca2＋通道阻塞剂La3＋、胞内Ca2＋通道阻断剂钌红,以及钙调素拮抗剂氯丙嗪和三氟拉嗪则削弱机械刺激诱导的氧化爆发。这暗示以钙和钙调素为核心的钙信使系统对机械刺激诱发的烟草悬浮培养细胞中H2O2的爆发有调控作用。     

Effect of trifluoperazine on renal epithelioid Madin-Darby canine kidney cells

Following exposure to a number of hormones, the cell membrane in Madin-Darby Canine Kidney (MDCK) cells is hyperpolarized by increase of intracellular calcium activity. The present study has been performed to elucidate the possible role of calmodulin in the regulation of intracellular calcium activity and cell membrane potential. To this end trifluoperazine has been added during continuous recording of cell membrane potential or intracellular calcium. Trifluoperazine leads to a transient increase of intracellular calcium as well as a sustained hyperpolarization of the cell membrane by activation of calcium sensitive K+ channels. Half-maximal effects are observed between 1 and 10 mumol/L trifluoperazine. A further calmodulin antagonist, chlorpromazine, (50 mumol/L), similarly hyperpolarizes the cell membrane. The effects of trifluoperazine are virtually abolished in the absence of extracellular calcium. Pretreatment of the cells with either pertussis toxin or phorbol-ester TPA does not interfere with the hyperpolarizing effect of trifluoperazine. In conclusion, calmodulin is apparently involved in the regulation of calcium transfer across the cell membrane but not in the stimulation of K+ channels by intracellular calcium.     

Glucose-induced, calcium-mediated protein phosphorylation in intact pancreatic islets

Conditions for studying protein phosphorylation in intact pancreatic islets were developed in order to study the effects of glucose and other effectors. Islets were incubated in Krebs-Ringer bicarbonate buffer containing 5 mM malate and 5 mM pyruvate (metabolic fuels that are not insulin secretagogues) for 150 min to permit incorporation of 32Pi into islet phosphate pools. Glucose or other effectors were then added, and the incubation was terminated after 10 to 30 min. Glucose increased phosphorylation of four islet peptides with molecular weights of 20,000, 33,000, 43,000 and 57,000. The calcium channel blockers, verapamil and D-600, inhibited phosphorylation of each of the four proteins, and trifluoperazine inhibited phosphorylation of the proteins with molecular weights of 20,000 and 57,000. The results indicate that glucose-induced insulin release may be mediated in part by protein phosphorylation, and that calcium may act as an intracellular messenger in coupling the glucose stimulus to the secretory process.     

Neurotensin regulation of TSH secretion in the rat

The ionophore A23187 (6.7 microM) increased the rates of formation of prostaglandins and cyclic AMP in suspensions of thioglycollate-elicited rat peritoneal macrophages. Both effects were inhibited by the calmodulin blocker trifluoperazine (50 microM) and the calcium channel blocker verapamil (500 microM). Inhibitors of phospholipase A2 and cyclo-oxygenase also blocked both actions of A23187. The stimulated prostaglandin formation was markedly reduced when the cells were preincubated with 8-bromo-cyclic AMP (1mM), dibutyryl cyclic AMP (1mM) or cholera toxin (500ng/ml). Addition of exogenous arachidonic acid (30 microM) alleviated this inhibition. We propose that the effect of A23187 on macrophages includes a 'self-limiting' mechanism whereby newly-synthesized prostaglandins can inhibit, via cyclic AMP, a step(s) prior to the transformation of arachidonic acid and thus modulate their own production.     

Involvement of calmodulin in phagocytotic respiratory burst of leukocytes

The superoxide release of guinea pig exudate leukocytes induced by phagocytosis or by stimulation with cytochalasin D, digitonin or calcium ionophore A23187 was completely inhibited by the inhibitors of calmodulin-stimulated processes such as trifluoperazine at 10 μM. A particulate NADPH-dependent superoxide-forming enzyme from the cytochalasin D-stimulated cells was also inhibited by the inhibitors and by EGTA. The activation of heart phosphodiesterase by a boiled extract of the cells which was dependent on calcium ions and abolished by trifluoperazine was observed. These results suggest the presence of calmodulin in leukocytes and its possible role in the stimulation of the superoxide formation.     

Phorbol myristate acetate stimulates formation of phosphatidyl inositol 4-phosphate and phosphatidyl inositol 4,5-bisphosphate in human platelets

There are conflicting data in the literature as to whether or not the Ca2+ activation of phospholipase A2 is mediated by the calcium binding protein calmodulin. In the present study the membrane-bound phospholipase A2 enzymes in rat and human platelets were shown to be absolutely Ca2+ dependent but were not stimulated by the addition of calmodulin. A partially purified phospholipase A2 from rat platelet membrane, which contained little endogenous calmodulin, also was not stimulated by calmodulin addition. Both isolated and membrane-bound phospholipase A2 were inhibited by the non-specific calmodulin antagonist trifluoperazine but the inhibition was not overcome by adding calmodulin. There was thus no evidence from these studies that phospholipase A2 is calmodulin regulated.     

Blue light-promoted stomatal opening in abaxial epidermis of Commelina benghalensis is maximal at low calcium

The requirement for calcium in blue light-promoted stomatal opening, in comparison with that in red light, was studied in epidermal strips of Commelina benghalensis L. Blue light promoted stomatal opening in the presence of a low level of calcium, whereas in red light opening was relatively tolerant to calcium. Stomatal opening under blue light was restricted by external calcium (above 5 μ M ) or abscisic acid. When present in the incubation medium, EGTA increased the extent of stomatal opening under blue light. Verapamil (a calcium-channel blocker) and trifluoperazine (TFP, a calmodulin antagonist) reduced the stimulation of stomatal opening by blue light. Lanthanum, an external calcium-channel antagonist, had no significant effect on stomatal opening under either blue or red light. These observations indicate that blue light-promoted stomatal opening preferentially occurs at low levels of calcium, and modulation by calmodulin is strongly suggested. We conclude that a fine-tuning of the calcium level within guard cells is essential during the transduction of the blue light signal.     

Kinetics of acetylcholine-activated cation channel blockade by the calcium antagonist D-600 inAplysia neurons

The effects of the calcium channel blocker D-600 on the cation channels activated by acetylcholine (ACh) was studied in voltage-clamped Aplysia neurons by voltage-jump relaxation analysis. D-600 blocked the steady-state ACh current in a highly voltage-dependent manner, the degree of antagonism increasing with membrane hyperpolarization. In the presence of D-600 the current relaxations following hyperpolarizing command steps became biphasic. The time constants of ACh-induced current relaxations (tau f), which approximate the mean channel lifetime, were reduced in a voltage-dependent manner, the degree of reduction of tau f increasing with increasing membrane potential. In addition to the acceleration of tau f, a slow, inverse kinetic component (tau s) of the relaxation appeared in the presence of D-600. The rate of this inverse kinetic component was accelerated either by increasing the agonist or antagonist dose or by increasing the membrane potential. These results suggest that D-600 acts to antagonize the acetylcholine response through a blockade of the open state of the transmitter-activated cation channel. Possible kinetic schemes for this interaction are discussed.     

Modulation of elastase-like activity in fibroblasts stimulated with elastin peptides

Elastin-derived peptides, kappa-elastin, prepared by chemical degradation of insoluble elastin from bovine ligamentum nuchae, were shown to increase the elastase-like activity in the culture medium and cell fractions in fibroblasts. Preincubation of cells with nifedipine (calcium channel blocker) and trifluoperazine (calmodulin antagonist) induced a decrease in the activities of the enzyme under study. These data suggest the possibility of pharmacological modulation of the biological effects induced by elastin-derived peptides.     

Modulation of calcium mobilization in aortic rings of pregnant rats: Contribution of extracellular calcium and of voltage-operated calcium channels

Pregnancy is associated with decreased vascular responsiveness to vasopressor stimuli. We have tested the involvement of Ca2+ mobilization in myotropic responses of aortic rings obtained from pregnant and virgin rats. Contractions of the rings to phenylephrine, in the absence of calcium in the bathing medium, were lower in tissues from virgin than from pregnant rats. Concentration-response curves to CaCl2 that were measured after stimulation by phenylephrine in the absence of Ca2+ were shifted to higher levels of contraction. This was not observed when KCl was used to prestimulate the aorta. D-600, a phenylalkylamine calcium channel blocker, similarly inhibited these responses to CaCl2 in tissues from both pregnant and virgin animals. D-600 exerted a concentration-dependent inhibition of responses to phenylephrine and KCl. However, the calcium antagonist was less effective in aortic rings of pregnant than of virgin rats. Basal 45Ca2+ uptake was lower in aortic rings from pregnant than from virgin rats, and Bay K 8644 was unable to reverse this difference. The time course of basal and stimulated (KCl) 45Ca2+ influx was lower in aorta of pregnant rats at all times studied. Moreover, when the intracellular calcium pools were emptied with phenylephrine, the refilling of these pools was delayed in aortic rings of pregnant rats. These results indicate an altered extracellular calcium mobilization of aortic rings from pregnant rats. These changes may be due to a functional alteration of the voltage-operated calcium channels during pregnancy.     

Role of calcium channel in postvagal potentiation of contraction as evident from the effects of Mn2+, La3+, D-600, and deoxycholate on isolated guinea pig atria-vagus nerve preparation

The role of the calcium channel in the first large contraction (postvagal potentiation, PVP) of the atria at the end of the inhibitory phase of its response (IPR) to vagal stimulation has been investigated by studying the effects of agents acting on the calcium channel (e.g., Ca2+, Mn2+, La3+, and D-600) or sarcoplasmic reticulum (SR) (e.g., deoxycholate (DOC)). IPR was potentiated by high [Ca2+]o (3-16 mM) and also by the calcium channel blockers, Mn2+ (1 microM-0.5 mM), La3+ (0.1 microM-0.5 mM), D-600 (1.0-10 microM), and DOC (1 microM-0.5 mM). PVP was also potentiated by enhanced [Ca2+]o, but the PVP ratio, which employs a correction for the simultaneous changes in the force of spontaneous contraction was inhibited. This indicated greater potentiation of contractility during spontaneous activity by Ca2+ than during PVP. Mn2+, La3+, and D-600 and even DOC in the above concentrations inhibited PVP but increased the PVP ratio. High concentrations of DOC (greater than 1 mM), which disrupt SR, strongly inhibited PVP. It is concluded that the calcium channel plays a more prominent role in spontaneous contractions than in PVP in guinea pig atria. PVP is suggested to be generated by excessive triggered release of Ca2+ from SR leading to a marked increase in [Ca2+]i. The calcium channel and the calcium trapped in the glycocalyx also play significant roles in PVP.     

Effect of compound D-600 (methoxyverapamil) on gluconeogenesis and on acceleration of the process by alpha-adrenergic stimuli in rat kidney tubules.

1. Tubule fragments were isolated from renal cortex of fed rats and glucose formation was measured after incubation with 5 mM-sodium lactate. 20 Compound D-600 (10-100 microM) decreased gluconeogenesis from lactate. This inhibition of the process by compound D-600 increased with increasing extracellular Ca2+ concentration, was overridden by noradrenaline and diminished by starvation for 24 h. 3. Inhibition of lactate-supported gluconeogenesis by compound D-600 was not prevented by the alpha 1-adrenoceptor antagonist thymoxamine. 4. Compound D-600 had little effect on gluconeogenesis from 2-oxoglutarate and increased gluconeogenesis from succinate. 5. Compound D-600 opposed stimulation of gluconeogenesis by noradrenaline or oxymetazoline (a selective alpha-adrenoceptor agonist) in a manner suggesting that compound D-600 is an alpha-adrenoceptor blocker. Oxymetazoline was more sensitive than noradrenaline to blockade by both compound D-600 and by the conventional alpha-adrenoceptor antagonist phentolamine. Noradrenaline became more sensitive to blockade by compound D-600 when extracellular Ca2+ was decreased. 6. Compound D-600 did not block stimulation of gluconeogenesis by angiotensin or cyclic AMP.     

Effect of calcium and calmodulin modulators on the development of Erysiphe pisi on pea leaves

The effect of calcium and calmodulin modulators, viz., ethylene glycol bis (beta-amino ethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), a calcium chelator; verapamil, a plasma membrane Ca2+ channel blocker; ruthenium red, an organelle Ca2+ channel blocker; and chlorpromazine, a calmodulin antagonist; on the development of Erysiphe pisi was studied by floating the inoculated leaves on the respective solutions of chemicals. All the modulators affected the development of E. pisi by inhibiting the colony diameter, number of secondary branches, number of hyphal cells per colony and number of haustoria. The calmodulin antagonist was more effective in reducing E. pisi development. The results suggest the possible involvement of calcium and calmodulin in the development of E. pisi on pea leaves.