Effect of some anticancer drugs on the surface membrane electrical properties of differentiated murine neuroblastoma cells.

The effect of some anticancer agents that produce toxic effects on electrically excitable cells in vivo was studied in vitro with the use of differentiated N1E-115 murine neuroblastoma cells and single microelectrode electrical recording. In the presence of 10(-7) g/ml tetrodotoxin, following the release of 500-millisecond conditioning hyperpolarization, the cells exhibited Ca2+-dependent action potentials. Local application to N1E-115 neuroblastoma cells of cisplatin (cis-PDD) for 30 seconds from a drug-containing effusion pipette produced a dose-dependent reversible inhibition of the Ca2+-dependent action potential, with a 61% inhibition at 1.7 microM and 67% inhibition at 17 microM cis-PDD. trans-Dichlorodiammineplatinum(II) and platinic(IV) chloride, both of which lacked the growth inhibitory properties of cis-PDD against N1E-115 neuroblastoma cells, at concentrations of 170 and 120 microM produced only an 11 and 19% inhibition of the Ca2+-dependent action potential, respectively. Vincristine at a concentration of 1 microM reversibly inhibited the Ca2+-dependent action potential by 48%. 3'-Deamino-3'-(3''-cyano-4''-morpholinyl)doxorubicin, a more potent experimental antitumor agent than doxorubicin, at 10(-8) M inhibited the Ca2+-dependent action potential by 22%, similar to the inhibition previously reported for doxorubicin. None of the agents affected the cell transmembrane potential, which suggests a lack of an effect on the mechanisms responsible for maintaining the resting cell membrane potential difference. The effects of the agents on the Ca2+-dependent action potential might reflect a direct effect on a plasma membrane Ca2+ channel or on the lipid domain around the channels, or they might be produced by changes in intracellular Ca2+ homeostasis, among other mechanisms. It is not known whether a change in the membrane Ca2+ current is related to the antitumor effects of the agents, but such a change may contribute to the neurotoxicity of cis-PDD and vincristine and the cardiac toxicity of the anthracycline.

Anthracycline-induced inhibition of a calcium action potential in differentiated murine neuroblastoma cells.

The effects of some anthracyclines on a Ca2+ -dependent action potential have been studied in differentiated murine neuroblastoma cells (N1E-115 clone). The differentiated neuroblastoma cell possesses characteristics of an electrically excitable cell and can generate propagated potential spikes in which Ca2+ is the inward charge carrier. This was shown by the fact that action potentials recorded from differentiated neuroblastoma cells in the presence of 10(-7) g of tetrodotoxin per ml, which inhibits active Na+ channels, had a spike amplitude that depended upon the extracellular Ca2+ concentration in a manner close to that predicted by the Nernst equation. The peak potential changed 28.9 mV/decade change in extracellular Ca2+. Local application to a cell of 10(-8) M doxorubicin produced inhibition of this Ca2+ -dependent action potential within 5 s of drug application and a maximum inhibition of 13% 60 s after drug application. There was almost complete recovery to the initial spike amplitude value within 10 min after removing drug. The same concentration of doxorubicin also produced complete inhibition, without recovery, of a Ca2+ -dependent after-discharge which followed the initial action potential in about half the cells studied. Increasing concentrations of doxorubicin produced dose-dependent inhibition of the initial Ca2+ -dependent action potential. Cells exposed to 10(-5) M doxorubicin showed 88% inhibition of the Ca2+ -dependent action potential with no recovery even 10 min after removing the drug. Daunomycin, 10(-6) M, produced 90% inhibition of the Ca2+ -dependent action potential. Daunomycin aglycone (10(-6) M), which lacks antitumor activity, had no significant effect on the Ca2+ -dependent action potential. The rapid onset of the drug-induced response together with the low concentrations of anthracyclines needed to inhibit voltage-dependent Ca2+ channels in the neuroblastoma cells suggest a direct effect of anthracyclines on the cell surface membrane. The findings are discussed in light of the possible role of Ca2+ in cancer cells.

Altered H1 histamine receptor signaling in Balb/3T3 cells transformed by v-K-ras and v-H-ras oncogenes.

Transformation of Balb/3T3 cells with the v-K-ras oncogene resulted in the expression of functional Ca(2+)-mobilizing receptors for histamine, whereas v-H-ras-transformed Balb/3T3 cells failed to show a similar response to histamine. Stimulation of histamine receptors in v-K-ras-transformed cells produced a dose-dependent increase in intracellular free calcium ([Ca2+]i), which was inhibited by the H1 histamine antagonist pyrilamine but unaffected by the H2 histamine receptor antagonist cimetidine. Histamine-mediated elevation of [Ca2+]i was partially inhibited by the removal of extracellular Ca2+, which indicates that the H1 histamine receptors mobilize intracellular Ca2+ and also promote Ca2+ influx. H1 histamine receptors were identified in both v-K-ras- and v-H-ras-transformed Balb/3T3 cells, but not in untransformed cells, using the specific H1 antagonist [3H]-pyrilamine. Transformation of Balb/3T3 cells with the viral ras oncogene results in a complex regulation of H1 histamine receptors. K-ras and H-ras transformation results in the expression of H1 histamine receptors; however, H1 receptor expression and Ca2+ mobilization are uncoupled in v-H-ras-transformed cells.

The use of Fura-2 to estimate myoplasmic [Ca2+] in human skeletal muscle.

Fura-2 was used to estimate myoplasmic [Ca2+] in intact fibers and fiber segments from normal and diseased human muscles. Small muscle bundles (20-50 fibers) were loaded with the membrane-permeant form of the dye (Fura-2 AM). High-performance liquid chromatography was utilized to study the ability of these cells to hydrolyze Fura-2 AM. Immediately after the 30 min loading period, Fura-2 (the Ca2+ indicator) was the predominant form of the dye in all preparations and the concentration within these fibers remained stable for over 4 1/2 hours. In addition, the resting myoplasmic [Ca2+] in fiber segments from normal subjects and those susceptible to malignant hyperthermia were the same. However, halothane administration (1.5%) induced correlated increases in myoplasmic [Ca2+] and force only in fibers from the susceptible patients. In contrast, caffeine administration causes correlated increases in myoplasmic [Ca2+] and force in both types of muscle, but lower concentrations were needed to do so in the fibers from the susceptible patients. The effects of halothane and caffeine were reversible. We conclude that Fura-2 can be used successfully to estimate resting levels and changes in myoplasmic [Ca2+] in human skeletal muscle.

Electrophysiological changes of HSV-1-infected dorsal root ganglia neurons in culture.

Dissociated rat dorsal root ganglion cultures were infected with herpes simplex virus type 1 and studied electrophysiologically throughout the course of infection. Alterations in the electrical parameters of the action potentials of these neurons occurred as early as 4 hours post infection. The maximum rate of rise, spike amplitude, and overshoot were decreased, and the full width at half maximum and resting membrane potential were increased. At 6 hours post infection there is a movement back to normal values for these parameters. This reversal is transient, however, and the types of alterations seen at 4 hours post infection are more pronounced at 12 hours post infection. The first morphological alterations occur at 16 hours post infection, at which time less than 50% of the neurons impaled produced action potentials. Application of cyclohexamide to inhibit protein synthesis 1 hour post infection prevented electrophysiological changes normally seen at 4 hours and 6 hours post infection. It is concluded that the sodium conductance is specifically reduced by acute HSV infection, and that this reduction is attributed to viral specific or modified host cell protein synthesis.

Evidence of a neurogenic component during IgE-mediated inflammation in mouse skin.

IgE-mediated inflammation was measured in mouse footpads that lacked sciatic innervation. Mice were passively sensitized with a monoclonal antibody, IgE anti-dinitrophenol, or were immunized for specific IgE production. Antigen-induced swelling in the denervated footpads was reduced 23-39% when compared to sham or untreated controls. Reduced IgE-mediated swelling responses were attributed to the loss of a mast cell-nerve interaction and not to blood vessel sensitivity to vasoamines. Furthermore, electrical stimulation of the distal segment of the sciatic nerve completely restored IgE-mediated inflammation. These data provide in vivo evidence that peripheral nerves participate in cutaneous IgE-mediated swelling reactions with the net effect of increasing inflammation.

Electrophysiologic effects of acute ethanol exposure. I. Alterations in the action potentials of dorsal root ganglia neurons in dissociated culture.

Dissociated dorsal root ganglion neurons from embryonic rats were exposed to 0.05, 0.15 and 0.30 g% ethanol for 1 h. Action potentials were recorded and statistically compared for differences from the control values. Exposure to ethanol produced no significant alterations in the resting membrane potential, spike amplitude, or maximum rate of rise in any of the experimental conditions. Significant differences were noted in parameters associated with the repolarization phase including decreases in the maximum rate of fall and one-half time to convergence and increases in the full width of half maximum and full width of base parameters. These alterations were reversed within 30 min after ethanol removal. In order to determine the specific ionic conductances affected by ethanol, action potentials were recorded from neurons exposed to specific Na+, Ca2+, and K+ current blockers. Analysis of the data indicates that K+ conductance is decreased as a result of a decreased Ca2+ current. Further, maximum Na+ conductance is not significantly affected by physiologic concentrations of ethanol.

Electrophysiologic effects of acute ethanol exposure. II. Alterations in the calcium component of action potentials from sensory neurons in dissociated culture.

Dissociated dorsal root ganglion neurons were exposed to 0.05-0.50 g% ethanol. The recording medium was altered in order to produce an action potential with an increased duration which was Ca2+-dependent. Ethanol was observed to decrease the duration of sensory neuron action potentials indicating a decreased inward Ca2+ current. The ethanol induced decrease in duration was reversible and dose-dependent. No alterations in resting membrane potential, spike amplitude, or maximum rate of rise were observed during ethanol exposure. An increased threshold was also observed with application of ethanol. These findings are discussed in regard to the possible effects of ethanol on Ca2+ movement at the presynaptic terminal.

The effects of ethanol on the electrophysiology of calcium channels.

Acute ethanol intoxication affects many systems in the body, especially the central nervous system. Because early experiments using axonal preparations required very high concentrations of ethanol to produce ionic current alterations, researchers turned their attention away from specific effects on electrogenesis and looked for effects at the synapse. The role of Ca2+ in the release of neurotransmitters was well known and was considered a possible site of action for ethanol. Indeed, several studies demonstrated that ethanol alters Ca2+ binding or transport in synaptosomes and neural tissue. The purpose of this chapter is to present electrophysiological evidence for the acute effects of ethanol on calcium channels. It is necessary first to define the relevant ethanol concentrations and to describe the characteristics of tissue preparations that may best help to determine the effects of ethanol. A discussion of these two points along with a brief synopsis of the role of Ca2+ in excitable tissues is presented. This is followed by a discussion of the effects of ethanol on Ca2+ and Ca2+-activated conductances in both nonmammalian and mammalian cells, and a model is presented in an attempt to unify the experimental evidence of the acute effects of ethanol.

Morphological destruction of cultured cells by the attachment of Treponema pallidum.

The incubation of Treponema pallidum with rabbit testicular cells, HEP-2 cells, human foreskin cells, rat cardiac cells, and rat skeletal muscle cells caused morphological disruption of these cultured cells. Control preparations of heat-inactivated treponemes, a high-speed supernatant in which treponemes had been pelleted, and culture medium failed to damage the tissue cells, as did viable treponemes when the cells were incubated in inverted Sykes-Moore chambers. Thus, cellular disruption is not associated with soluble treponemal, soluble inflammatory, or soluble testicular constituents but is mediated by the specific attachment of T pallidum. This organism apparently elaborates some type of toxic activity that lyses membranes: this may explain some of the histopathology of syphilitic disease.

Histamine-induced intracellular free Ca++, inositol phosphates and electrical changes in murine N1E-115 neuroblastoma cells.

Apparent intracellular free Ca++ concentration [(Ca++]i) was measured in differentiated N1E-115 neuroblastoma by microinjecting cells with aequorin (estimated intracellular concentration, 4 microM) and measuring light emission. Histamine produced a transient, dose-dependent increase in [Ca++]i. Pyrilamine blocked completely the response to histamine whereas cimetidine had no effect. Omitting Ca++ from the external medium reversibly blocked the response. As well as a rise in [Ca++]i, histamine caused a concomitant cell hyperpolarization that was not blocked by ouabain, low Cl-, tetraethylammonium chloride/tetradotoxin or metiamide but was blocked by apamin and pyrilamine. A secondary small depolarization caused by histamine was also blocked by apamin but not by ouabain, low Cl- or tetraethylammonium chloride/tetrodotoxin. Direct iontophoretic injection of Ca++ into cells caused only hyperpolarization. Injection of inositol 1,4,5-trisphosphate [IP3(1,4,5)] caused an increase in [Ca++]i and rapid hyperpolarization. Inositol 1,3,4-trisphosphate [IP3(1,3,4)] caused an increase in [Ca++]i, rapid hyperpolarization and a slower depolarization. Repeated injections of IP3(1,3,4) led to a diminished [Ca++]i response and decreased hyperpolarization but had no effect on depolarization. Inositol 1,3,4,5-tetrakisphosphate was without effect on [Ca++]i or on cellular membrane potential. The results suggest that histamine causes an H1 receptor-dependent increase in [Ca++]i, probably by the increased entry of extracellular Ca++, although there may be a contribution from intracellular Ca++ released by IP3(1,4,5). The increase in [Ca++]i activates K+ channels leading to cell hyperpolarization. IP3(1,3,4) formed from inositol 1,3,4,5-tetrakisphosphate, which is itself a product of IP3(1,4,5), causes a slower depolarization by a mechanism that does not involve Na+ channels or an increase in [Ca++]i.

An increase in intracellular free Ca2+ associated with serum-free growth stimulation of Swiss 3T3 fibroblasts by epidermal growth factor in the presence of bradykinin.

Bradykinin gave a biphasic increase in intracellular free Ca2+ concentration ([Ca2+]i) in serum-deprived Swiss 3T3 fibroblasts loaded with the photoprotein aequorin. Epidermal growth factor (EGF) alone did not increase [Ca2+]i, but when added after bradykinin there was an increase in [Ca2+]i. The EGF-dependent increase in [Ca2+]i was maximal at 3 min and disappeared with a half-life of 6 min after bradykinin. Removing Ca2+ from the external medium did not abolish either the bradykinin or the EGF-induced [Ca2+]i responses. Although prostaglandins E2 and F2 alpha also gave [Ca2+]i responses and permitted an EGF-dependent [Ca2+]i response, the effect of bradykinin did not appear to be mediated by prostaglandins since it was not blocked by indomethacin. Vasopressin and phorbol 12-myristate 13-acetate both gave a [Ca2+]i response but did not facilitate a [Ca2+]i response by EGF. Bradykinin or EGF alone did not increase DNA synthesis in growth-arrested Swiss 3T3 fibroblasts, but EGF added together with, or after, bradykinin increased DNA synthesis. The effect disappeared with a half-life of 180 min after the addition of bradykinin. It is concluded that stimulation of receptor protein tyrosine kinase is unlikely, by itself, to explain the increase in DNA synthesis produced by EGF. The observed increase in [Ca2+]i caused by EGF after bradykinin probably reflects the interaction of intracellular second messenger pathways leading to facilitation of DNA synthesis.

Incomplete hydrolysis of the calcium indicator precursor fura-2 pentaacetoxymethyl ester (fura-2 AM) by cells.

Fura-2 AM is an esterified cell-permeant form of the Ca2+ indicator fura-2 (1-[2-(5-carboxyoxal-2-yl)-6-aminobenzofuran-5-oxyl]-2-(2'-a mino-5'- methylphenoxy)-ethane-N,N,N',N'-tetraacetic acid). Fura-2 AM has been reported to be completely cleaved by cellular esterases to fura-2 which is trapped within cells and is used to measure intracellular free Ca2+ concentration by a fluorescence ratio method. Successful application of the method requires that fura-2 be the major cellular fluorescent metabolite of fura-2 AM. We have used high-performance liquid chromatography to study fura-2 AM hydrolysis by cells. Murine N1E-115 neuroblastoma cells incubated with 10 microM fura-2 AM formed fura-2 at a rate of 9.7 pmol/min/10(6) cells. The concentration of fura-2 in the cells after 60 min, assuming uniform distribution, was 137 microM. Smaller amounts of at least four other metabolites were present, as well as large amounts of unhydrolyzed fura-2 AM. Washing the cells with medium containing 2% bovine serum albumin decreased the concentration of fura-2 to 40 microM and that of fura-2 AM to 90 microM. The half-time for loss of fura-2 from neuroblastoma cells after washing was 34 min. Human pulmonary artery endothelial (HPAE) cells formed fura-2 at a rate of 2.6 nmol/min/10(6) cells and the concentration of fura-2 after 60 min of incubation and washing with albumin containing medium was 130 microM, and the concentration of fura-2 AM was 58 microM. The half-time for loss of fura-2 from washed HPAE cells was 74 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Scanning electron microscopy of the attachment of Treponema pallidum to nerve cells in vitro.

Treponema pallidum (Nichols strain) was incubated with cultured nerve cells derived from rat embryos. Primary cultures were established from dorsal root ganglia, superior cervical ganglia, and spinal cord. Using phase contrast microscopy treponemes were seen to interact with the nerve cells in a similar manner to other cultured mammalian cells. Organisms began to attach within minutes after inoculation, actively motile organisms attached at the tip of one end, higher numbers of organisms attached with continued incubation, and attached organisms survived longer than unattached organisms. T pallidum attached both to nerve cell bodies and to neuronal processes of each of the three nerve cell cultures. As shown by scanning electron microscopy the mechanism of attachment was identical to that of cultured cells derived from rabbits testis, rat skeletal muscle, and human cervical carcinoma. There was no indentation or swelling of the cultured cell surface at the point of attachment, just a close physical proximity of organisms and cells. These techniques provide a biological means of studying the in-vitro detrimental influences of micro-organisms on nerve tissue.

Electrophysiological dysfunction and cellular disruption of sensory neurones during incubation with Treponema pallidum.

Treponema pallidum (Nichols strain) was incubated with cultured nerve cells derived from dorsal root ganglia of rat embryos. The electrophysiological response of these neuronal cells was then investigated. Cells exposed to 2 X 10(8) treponemes/ml responded abnormally after 13 hours and failed to respond after 18 hours. In contrast, control preparations exposed to heat-inactivated treponemes or to culture medium responded normally after 72 hours. Extended incubation with viable treponemes resulted in various degrees of nerve cell disruption as shown by scanning electron microscopy. With some cells holes in the cytoplasmic membrane were detected; with others a coagulated matrix of apparent nuclear material and remnants of cytoskeletal elements indicated more severe destruction. These findings may explain the painless nature of many of the clinical manifestations of syphilis as well as the severe damage to central nervous system tissue in tertiary and congenital syphilis.

Doxorubicin blocks the increase in intracellular Ca++, part of a second messenger system in N1E-115 murine neuroblastoma cells.

Exposure of differentiated N1E-115 murine neuroblastoma cells, microinjected with the Ca(++)-sensitive photoprotein aequorin, to doxorubicin for 1 hr, but not for 2 min, produced a reversible block of the rise in intracellular free Ca++ [( Ca++]i) produced by histamine. The resting level of [Ca++]i was increased from 0.23 to 1.22 microM (P less than 0.05) by 10(-4) M histamine. After exposure to 10(-6) M doxorubicin for 1 hr, histamine increased [Ca++]i to only 0.34 microM (P less than 0.05 compared to the histamine alone value). Doxorubicin exposure for 1 hr completely blocked the increase in inositol trisphosphate caused by histamine. There was no block by doxorubicin of the release of intracellular Ca++ after microinjection of the cells with inositol 1,4,5-trisphosphate. Based on the results from studies with differentiated N1E-115 neuroblastoma cells doxorubicin may: 1) block the histamine-induced rise in [Ca++]i by decreasing synthesis of inositol polyphosphates, 2) block plasma membrane Ca++ channels that allow entry of extracellular Ca++ in response to histamine and/or 3) prevent recovery of histamine receptors after desensitization.