Limitations on the use of phenothiazines and local anaesthetics as indicators of calmodulin function in intact cells

Corps, Anthony N.; Hesketh, T R; Metcalfe, James C.

doi:10.1016/0014-5793(82)80461-4

Cited by 59 publications

(17 citation statements)

References 29 publications

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“…2) was similar to comparable findings for other calmodulin-dependent reaction. These aspects render it likely that the actions were mediated through calmodulin and not via one of the reported nonspecific actions of phenothiazines on cells (23,24).…”

Section: Discussionmentioning

confidence: 99%

Infection of B lymphocytes by a human herpesvirus, Epstein-Barr virus, is blocked by calmodulin antagonists.

Nemerow¹,

Cooper²

1984

Proc. Natl. Acad. Sci. U.S.A.

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Epstein-Barr virus (EBV) is a human herpesvirus that selectively binds to and infects human B lymphocytes (B cells). In the studies presented here, we found that several phenothiazines, including trifluoperazine, chlorpromazine, prochlorpromazine, and promethazine, blocked EBV infectivity of isolated adult human B cells as measured either by outgrowth of transformed cell colonies-or by [3ll~thymidine incorporation. Trifluoperazine, chlorpromazine, and prochlorpromazine were equally effective with 20 ,uM fully inhibiting infectivity, whereas 100 ,AM promethazine was required for a comparable effect. Inhibition by trifluoperazine was partially reversible. Studies with radiolabeled EBV demonstrated that the inhibitors did not impair virus binding to B cells. Electron microscopic examination of B lymphocytes revealed that trifluoperazine reduced the number of large uncoated cell vacuoles and the number of membrane microvilli, indicating that this agent interfered with cell pinocytosis. This process was accompanied by inhibition of EBV endoeytosis iQto B cells.Phenothiazines bind to and inhibit calmodulin, an intracellular calcium-binding protein that regulates several key enzymes, some of which directly affect cytoskeletal elements, although they alsQ may interact nonspecifically with other cellular constituents. In this regard, haloperidol, a non-phenothiazine calmodulin antagonist, and R24571, a derivative of the antimycotic miconazole, which is a potent and highly specific calmodulin inhibitor, also blocked EBV infection. These studies suggest that calmodulin or a calmodulin-regulated cellular enzyme(s) is involved in normal cellular endocytic processes in B lymphocytes and thereby in the early stages of EBV infection.Recent studies in our laboratory have shown that EpsteinBarr virus (EBV) infects normal human B cells by an endocytic pathway that is somewhat different (1) than the clathrin-receptosome-lysosome pathway used by many other protein ligands (2) as well as several viruses (3) to enter cells. One difference in the endocytic pathway of EBV in normal B cells is the lack of participation of clathrin-coated pits and vesicles in the infectious process. The studies presented here were undertaken to further examine the early events in EBV infection and particularly the' mechanisms involved in EBV endocytosis into normal B lymphocytes. One element that provides an early signal for me'mbrane protein redistribution (capping) and cell activation processes in lymphocytes is intracellular calcium (4-8).. Caltcium ions markedly influence many biological processes in lymphocytes, including capping but not patching, cytoplasjnic streaming, endocytosis, and exocytosis (4, 9-11). The effects of calcium on several important cell enzymes such as mefhbrane-associated Ca2+, Mg2+-ATPase, phosphodiesterase, and myosin light chain kinase implicated in the above noted cellular activities are regulated by the intracellular calcium-binding protein calmodulin (7,9,(12)(13)(14). In pursuing our studies of the mechanisms invo...

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Section: Discussionmentioning

confidence: 99%

Infection of B lymphocytes by a human herpesvirus, Epstein-Barr virus, is blocked by calmodulin antagonists.

Nemerow¹,

Cooper²

1984

Proc. Natl. Acad. Sci. U.S.A.

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“…In our sys tem of fetal neurons, neither calmidazolium nor W-7 (two inhibitors of calmodulin) could modify A 23187-induced SRIF release; another antagonist, chlorpromazine, decreased it by 45%, but only at high concentrations. It should be kept in mind that chlorpromazine, a phenothiazine type of cal modulin-inhibiting agent, can affect a wide variety of en zymes and/or cellular processes [4,9,17,19], including Ca2 +-influx [10,II,13,30]. These effects could explain the chlorpromazine inhibition of ionophore-stimulated SRIF re lease observed in our work.…”

Section: Discussionmentioning

confidence: 47%

Calcium Dependence of Somatostatin (SRIF) Release and Cyclic AMP Levels in Cultured Diencephalic Neurons

Tapia‐Arancibia¹,

Parés‐Herbuté²,

Astier³

1989

Neuroendocrinology

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Calmodulin has been reported to be involved in the Ca²⁺-dependent hypothalamus in vitro. The present experiments were undertaken to determine whether at an early stage of development (in diencephalic primary cultures secreting SRIF, on the 11^th day) the activation of a Ca^2+-calmodulin kinase system is also involved in the release of the peptide. Since a calmodulin-dependent adenylate cyclase activity has been detected in the brain, we measured intracellular cyclic AMP accumulation as an additional parameter of calmodulin activity. SRIF release and cyclic AMP accumulation were stimulated by K⁺ (56 mM) and by the Ca²⁺ ionophores ionomycin (0.5 µM) and A 23187 (in a dose-dependent manner). Incubation of cells in Ca²⁺-free Locke medium or in the presence of Co²⁺ (1 mM) completely blocked ionophore-induced SRIF release and cyclic AMP accumulation. Three calmodulin antagonists (calmidazolium, W-7, and chlorpromazine) and two blockers of calmodulin-dependent kinase (phenytoin and diazepam) were tested on evoked-SRIF release and cyclic AMP formation. Neither W-7 nor calmidazolium modified A 23187-induced SRIF release at any dose tested, although they inhibited, in a dose-dependent manner, the stimulatory effect of the Ca²⁺ ionophore on cyclic AMP accumulation. High concentrations of chlorpromazine (100 µM) were needed to decrease A 23187-induced SRIF release by 45% whereas the same concentration completely blocked the cyclic AMP accumulation evoked by the ionophore (1 µM). K⁺-induced SRIF release was not modified by 0.5 µM calmidazolium but was inhibited by rather high concentrations of W-7 (100 µM); both antagonists significantly decreased K⁺-evoked cyclic AMP accumulation. Phenytoin (1 µM to 100 µM) and diazepam (20 µM) failed to block the stimulatory effect of A 23187 (0.5 µM) on either parameter. The present results: (1) confirm the Ca²⁺-dependence of SRIF release in fetal diencephalic cells but without the participation of calmodulin-dependent mechanisms; (2) show that agents which increase cytosolic calcium also stimulate cyclic AMP formation, probably by stimulating a calmodulin-dependent adenylate cyclase, and (3) suggest that a relationship between these two events is unlikely in the model used here.

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“…1-3). While the specificity of these calmodulin antagonists is established under appropriate experimental conditions using soluble proteins (Levin & Weiss, 1979), all the calmodulin antagonists are also known to have nonspecific side effects, including hydrophobic actions, causing perturbation of cell membranes (Seeman, 1972;Landry, Amellal & Ruckstuhl, 1981;Douglas & Nemeth, 1982) and ATP-depletion within the cells (Montecucco et al, 1981;Ruben & Rasmussen, 1981;Corps, Hesketh & Metcalfe, 1982). Under present experimental conditions, several observations argue against the possibility of nonspecific side effects.…”

Section: Discussionmentioning

confidence: 90%

Evidence for the involvement of calmodulin in the operation of Ca-activated K channels in mouse fibroblasts

et al. 1987

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The oscillation of membrane potential in fibroblastic L cells is known to result from periodic stimulation of Ca2+-activated K+ channels due to the oscillatory increase in the intracellular Ca2+ concentration. These repeated hyperpolarizations were inhibited by putative calmodulin antagonists, trifluoperazine (TFP), N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and promethazine (PMZ), and the concentrations required for half-maximal inhibition were 25, 30 and 300 microM, respectively. These doses were lower than those for reducing the membrane resistance due to nonspecific cell damages. Another calmodulin antagonist, chlorpromazine (CPZ), was also effective, but CPZ-sulfoxide was not. Intracellular pressure injections of calmodulin-interacting divalent cations, Ca2+, Sr2+, Mn2+ and Ni2+, elicited slow hyperpolarizations, whereas Mg2+ and Ba2+, which are known to be essentially inert for calmodulin, failed to evoke any responses. The injection of purified calmodulin also brought about a similar hyperpolarization. Quinine, an inhibitor of Ca2+-activated K+ channels, abolished both Ca2+- and calmodulin-induced hyperpolarizations. TFP prevented Ca2+-induced hyperpolarizations. The TFP effect was partially reversed by the calmodulin injection. It is concluded that calmodulin is involved in the operation of Ca2+-activated K+ channels in fibroblasts.

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Limitations on the use of phenothiazines and local anaesthetics as indicators of calmodulin function in intact cells

Cited by 59 publications

References 29 publications

Infection of B lymphocytes by a human herpesvirus, Epstein-Barr virus, is blocked by calmodulin antagonists.

Infection of B lymphocytes by a human herpesvirus, Epstein-Barr virus, is blocked by calmodulin antagonists.

Calcium Dependence of Somatostatin (SRIF) Release and Cyclic AMP Levels in Cultured Diencephalic Neurons

Evidence for the involvement of calmodulin in the operation of Ca-activated K channels in mouse fibroblasts

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