Inhibitory Effect Of Neomycin Research Articles

Extracellular Ca2+ induces desensitized cytosolic Ca2+ rise sensitive to phospholipase C inhibitor which suppresses root growth with Ca2+ dependence

Calcium (Ca) is an essential element for all organisms. In animal cells, the plasma membrane-localized Ca receptor CaSR coupled to a phospholipase C (PLC)-dependent signaling cascade monitors extracellular Ca2+ concentrations ([Ca2+]ext) and responds with increases in cytosolic calcium concentrations ([Ca2+]cyt). Plant roots encounter variable soil conditions, but how they sense changes in [Ca2+]ext is largely unknown. In this study, we demonstrate that increasing [Ca2+]ext evokes a transient increase in [Ca2+] in the cytosol, mitochondria, and nuclei of Arabidopsis thaliana root cells. These increases were strongly desensitized to repeat applications of [Ca2+]ext, a typical feature of receptor-mediated cellular signaling in animal and plant cells. Treatment with gadolinium (Gd3+), a CaSR activator in animal cells, induced concentration-dependent increases in [Ca2+]cyt in roots, which showed self-desensitization and cross-desensitization to [Ca2+]ext-induced increases in [Ca2+]cyt (EICC). EICC was sensitive to extracellular H+, K+, Na+, and Mg2+ levels. Treatment with the PLC inhibitor neomycin suppressed EICC and Ca accumulation in roots. The inhibitory effect of neomycin on root elongation was fully rescued by increasing [Ca2+]ext but not [Mg2+] or [K+] in the growth medium. These results suggest that [Ca2+]ext and the movement of Ca2+ into the cytosol of plant roots are regulated by a receptor-mediated signaling pathway involving PLC.

Osteoblasts detect pericellular calcium concentration increase via neomycin-sensitive voltage gated calcium channels

The mechanisms underlying the detection of critically loaded or micro-damaged regions of bone by bone cells are still a matter of debate. Our previous studies showed that calcium efflux originates from pre-failure regions of bone matrix and MC3T3-E1 osteoblasts respond to such efflux by an increase in the intracellular calcium concentration. The mechanisms by which the intracellular calcium concentration increases in response to an increase in the pericellular calcium concentration are unknown. Elevation of the intracellular calcium may occur via release from the internal calcium stores of the cell and/or via the membrane bound channels. The current study applied a wide range of pharmaceutical inhibitors to identify the calcium entry pathways involved in the process: internal calcium release from endoplasmic reticulum (ER, inhibited by thapsigargin and TMB-8), calcium receptor (CaSR, inhibited by calhex), stretch-activated calcium channel (SACC, inhibited by gadolinium), voltage-gated calcium channels (VGCC, inhibited by nifedipine, verapamil, neomycin, and ω-conotoxin), and calcium-induced-calcium-release channel (CICRC, inhibited by ryanodine and dantrolene). These inhibitors were screened for their effectiveness to block intracellular calcium increase by using a concentration gradient induced calcium efflux model which mimics calcium diffusion from the basal aspect of cells. The inhibitor(s) which reduced the intracellular calcium response was further tested on osteoblasts seeded on mechanically loaded notched cortical bone wafers undergoing damage. The results showed that only neomycin reduced the intracellular calcium response in osteoblasts, by 27%, upon extracellular calcium stimulus induced by concentration gradient. The inhibitory effect of neomycin was more pronounced (75% reduction in maximum fluorescence) for osteoblasts seeded on notched cortical bone wafers loaded mechanically to damaging load levels. These results imply that the increase in intracellular calcium occurs by the entry of extracellular calcium ions through VGCCs which are sensitive to neomycin. N-type and P-type VGCCs are potential candidates because they are observed in osteoblasts and they are sensitive to neomycin. The calcium channels identified in this study provide new insight into mechanisms underlying the targeted repair process which is essential to bone adaptation.

Effect of neomycin B on rotavirus plus- and minus-strand RNA synthesis.

Rotavirus multiplication was inhibited by neomycin B by affecting the synthesis of the viral genome. Genome replication involves a two-step mechanism, one is plus-strand synthesis or transcription, and the other is minus-strand synthesis or replication. The results indicate that both activities are inhibited by aminoglycoside. The effect on the minus-strand was determined by an in vitro assay using a template-dependant open core preparation. The effect on transcription was explored using transcriptionally active virus particles. In the case of transcription, the inhibitory effect of neomycin B was studied in both initiation and the elongation of the mRNA. Initiation was defined by the synthesis of short transcripts of less than 25 nucleotides in length and elongation as an extension of those molecules into full-length transcripts. The inhibitory effect of neomycin was also mimicked by other aminoglycosides such as lividomycin, paromomycin, and tobramycin. The results may be explained based on the ability of the drug to interact with the stem and loop regions, which, in the case of rotavirus, have been identified at the end of the templates required for both transcription and minus-strand synthesis.

Decrease in acetylcholine-induced current by neomycin in PC12 cells

The effects of neomycin, one of the aminoglycoside antibiotics, on the acetylcholine (ACh)-induced current ( I ACh) were studied in pheochromocytoma cells by using the whole-cell clamp technique. The I ACh proved to be generated through neuronal nicotinic receptor. ACh (30 μM) induced an inward current at a holding potential of −80 mV. When cells were treated with neomycin (0.01–1 mM) and ACh (30 μM) simultaneously, an inhibitory effect of neomycin on the peak of I ACh was found. This effect was fast, reversible, and concentration dependent. Pretreatment with neomycin for 3–8 min had no effect on the inhibition of I ACh induced by neomycin. External application of 0.1 mM neomycin neither shifted the dose–response curve of the peak I ACh to the right (dissociation constant ( K d) = 16.5 μM ) nor affected its coefficient (1.8) but inhibited the curve amplitudes by ∼33%. Stimulated protein kinase C activation by using an exogenous activator produced inhibition of I ACh, while using protein kinase C inhibitor (PKCI 19-31) had no effect on the inhibition of I ACh induced by neomycin. These results suggest that neomycin has an inhibitory effect on I ACh without the involvement of phospholipase C. It indicates that neomycin binds to a specific site on the cell membrane, probably on the neuronal nicotinic receptor-coupled channel, and inhibits the I ACh in a noncompetitive manner, thus controlling the immediate catecholamine release from the sympathetic cells.

Involvement of PKC-alpha in regulatory volume decrease responses and activation of volume-sensitive chloride channels in human cervical cancer HT-3 cells.

1. The present study was carried out to identify the specific protein kinase C (PKC) isoform involved in regulatory volume decrease (RVD) responses, and to investigate the signal transduction pathways underlying the activation of volume-sensitive chloride channels in human cervical cancer HT-3 cells. The role of Ca2+ in RVD and in the activation of chloride currents was also studied. 2. The time course of RVDs was prolonged by microinjection of PKC-alpha antibody but not by PKC-beta or PKC-gamma antibody, and also by exposure to Ca2+-free medium, in particular when combined with microinjection of EDTA. Immunofluorescence staining showed that hypotonic superfusion evoked the translocation of PKC-alpha to the cell membrane, whereas PKC-beta or PKC-gamma remained unaffected. The translocation of PKC-alpha was observed a few minutes after hypotonic stress, reaching peak intensity at 30 min, and returned to the cytoplasm 60 min after hypotonic exposure. Western blot analyses showed an increased PKC-alpha level in terms of intensity and phosphorylation in the cell membrane, while neither PKC-beta nor PKC-gamma was activated upon hyposmotic challenge. 3. Whole-cell patch-clamp studies demonstrated that neomycin and PKC blockers such as staurosporine and H7 inhibited volume-sensitive chloride currents. The inhibitory effect of neomycin on chloride currents can be reversed by the PKC activator phorbol 12-myristate, 13-acetate (PMA). Moreover, the PKC inhibitor and PKC-alpha antibody, but not PKC-beta or PKC-gamma antibody, significantly attenuated the chloride currents. The activation of volume-sensitive chloride currents were insensitive to the changes of intracellular Ca2+ but required the presence of extracellular Ca2+. 4. Our results suggest the involvement of PKC-alpha and extracellular Ca2+ in RVD responses and the activation of volume-sensitive chloride channels in HT-3 cells.

The Effect of Neomycin on PDGF-Induced Mitogenic Response and Actin Organization in Cultured Human Fibroblasts

Actin organization and DNA synthesis were studied in cultured, serum-starved, subconfluent human fibroblasts in response to human recombinant platelet-derived growth factor (PDGF)-BB and neomycin, an inhibitor of phosphoinositide (PI) turnover. The labeling of F-actin with TRITC-phalloidin showed that PDGF (10 ng/ml) and neomycin (3.5 mM) induced, within minutes, similar changes in the actin cytoskeleton and when used together potentiated each other's effects. PDGF stimulated a twofold increase in DNA synthesis, as shown by [3H]thymidine incorporation, 48 h after the beginning of incubation. Neomycin did not induce DNA synthesis but reduced the mitogenic response to PDGF to control levels. Stimulation of DNA synthesis was correlated with an intense increase in lamellipodia formation and ruffling, whereas inhibition of the DNA synthesis was correlated with the development of elongated cell shape with few ruffles and lamellipodia, similarly to untreated control cells. Our findings support the conclusion that early changes in the actin cytoskeleton and induction of DNA synthesis are separable biological processes that are mediated by different pathways. The inhibitory effect of neomycin on PDGF-induced DNA synthesis was dose-dependent during the first 24 h of PDGF incubation, after which the cells were irreversibly and maximally stimulated to synthesize DNA. Results of experimental treatments with neomycin at various times during the incubation with PDGF indicate that the critical period during which PDGF acts to stimulate DNA synthesis is after the 1st h but before the 12th h of incubation.

Neurochemical evidence that [Ca2+]o antagonizes the effect of neomycin on acetylcholine release from mouse hemidiaphragm preparation: an attempt to assess the margin to safety.

Although the neuromuscular junction is the most thoroughly studied synapse of any type and has become the model of our understanding of synaptic transmission, some questions remain unanswered; e.g. there has been no direct assessment of the size of margin of safety. In this study the [Ca2+]o-dependent, quantally released acetylcholine measured by a neurochemical method, and the contraction of the mouse hemidiaphragm in response to phrenic nerve stimulation were recorded, and the effect of neomycin was studied. It was found that a much higher concentration of neomycin was needed to depress contractions, than to reduce acetylcholine release to the same extent, and that there was an inverse correlation between [Ca2+]o and the inhibitory effect of neomycin on acetylcholine release. Ninety percent of the release of acetylcholine had to be reduced by neomycin before any reduction in muscle responses could be seen. This indicates that the margin of safety is about 10. In conclusion, at the neuromuscular junction any reduction in ACh release, whatever the mechanism, first produces a reduction in the margin of safety. The nondepolarizing neuromuscular blocking agents block primarily the nicotinic receptors located on the postjunctional site. Many receptors have to be blocked before a reduction of muscle response is observed. This is probably the reason why unexpected clinical problems (e.g. recurarization) have been described when a patients has been treated with antibiotics, even though the dose of muscle relaxant injected was relatively low.

Decreased cellular toxicity of neomycin in a clonal cell line isolated from LLC-PK1.

We have previously shown in LLC-PK1 cells, that apical membrane enzyme activity was inhibited by aminoglycoside antibiotics (Am. J. Physiol. 254, C251-C257, 1988). In the present study, the relationship between the lethal cytotoxic effect of aminoglycoside and its effect on apical membrane enzyme was examined by establishing aminoglycoside resistant cells. A clonal cell line, LLC-PK1/NRa3, was isolated from parent LLC-PK1 cells in the presence of neomycin. Neomycin inhibited colony formation and increased the number of floating dead cells in parent LLC-PK1 cultures. In contrast, these cytotoxic effects of neomycin were negligible or less pronounced in NRa3 cells, indicating that NRa3 cells were more resistant to neomycin compared with the parent cells. The inhibitory effect of neomycin on apical enzyme activity was significantly weaker in NRa3 cells than in the parent cells. These results suggest that a common mechanism is involved in the aminoglycoside-induced reductions in the apical enzyme activity and in cell viability of LLC-PK1 cells.

Effect of Calcium and Calcium Antagonists on Phospholipid Secretion Induced by Lung Inflation in Newborn Rabbits

The effect of calcium and selected calcium antagonists on the lung phospholipid secretion associated with lung distension was examined in newborn rabbits. Lung distension was produced by saline lavage or air inflation in freshly killed pups. Calcium or calcium antagonists were added to the lavage solution. The fractional stimulation of secretion with 1.0 mM calcium was 1.94 +/- 0.28 (p less than 0.01), but ethylene diamine tetraacetic acid (EDTA) did not have the expected inhibitory effect. There was dose-related inhibition of phospholipid secretion induced by the calcium antagonist, lanthanum, the fractional recovery decreasing from 1.0 +/- 0.23 at 10(-5) molar, to 0.43 +/- 0.19 at 0.5 x 10(-4) molar, to 0.22 +/- 0.03 at 10(-4) molar and 0.19 +/- 0.05 at 10(-3) molar (p less than 0.001). This inhibition was significantly reversed by the addition of 10 mM calcium. Although nifedipine and verapamil had no effect, nickel and cadmium also inhibited secretion. Therefore, it is likely that lanthanum acted as a calcium channel blocker, suggesting the importance of extracellular calcium. The observation that intense calcium chelation with ethylene glycol bis-aminoethylether tetraacetic acid-bis-aminophenoxyethane tetraacetic acid (EGTA-BAPTA) had a significant inhibitory effect, supports the conclusion that a superficial calcium pool in the plasma membrane may be an important stimulus for phospholipid secretion during lung distension. The inhibitory effect of neomycin, a drug known to displace superficial calcium, was consistent with this conclusion.

The pre- and postjunctional components of the neuromuscular effect of antibiotics.

The relative contributions of the pre- and postsynaptic components of the myoneural blocking effect of different antibiotics were studied using: (a) a radio-active method that measures selectively the Ca(2+)-dependent, stimulation evoked, quantally released, (3)H-acetylcholine ((3)H-ACh) from the mouse in vitro phrenic nerve-hemidiaphragm preparation without cholinesterase inhibition; (b) measurement of the force of contraction of the indirectly or directly stimulated muscle. The antibiotics studied (neomycin, polymyxin B and lincomycin), reduced the release of (3)H-ACh evoked by stimulation (18 trains of 40 shocks at 50 Hz) in a concentration dependent manner. While the inhibitory effect of neomycin was inversely related to [Ca(2+)](o), that of lincomycin was moderately and that of polymyxin B was not affected by increasing [Ca(2+)](o) from 0.75 to 5.0 mM. Similarly, the d-tubocurarine (d-Tc)-induced inhibition of the release of (3)H-ACh was independent of [Ca(2+)](o). The K-channel blocking agent, 4-aminopyridine (4-AP), enhanced the release of ACh in a concentration dependent manner and prevented the neuromuscular effect of neomycin. However, the neuromuscular effect of polymyxin B and of lincomycin was not affected by 4-AP. Atropine, enhanced the release of (3)H-ACh. Antibiotics, however, were still able to reduce the release of ACh when the negative muscarinic feedback mechanism of ACh release was eliminated by atropine. Our findings indicate that the antibiotics studied possess both pre- and postsynaptic effects. Presynaptically they reduce the evoked release of ACh; postsynaptically they inhibit muscle contractility. The rank order of presynaptic action is neomycin >polymyxin B >lincomycin.

Cholinergic-induced [ 3H] noradrenaline release in rat brain cortical slices is mediated via a pertussis toxin sensitive GTP binding protein and involves activation of protein kinase C

The involvement of a GTP-binding protein (G-protein) in the process of neurotransmitter release was examined using pertussis toxin and cholerra toxin. Cholinergic agonists are shown to mediate [ 3H]noradrenaline release in rat brain slices via a pertussis toxin (1.2 μg/ml) sensitive, and cholera toxin (0.5 μg/ml) insenstive G. protein. An indication for the involvement of a G-protein and phospholipase C activation in the release process was implied from the inhibitory effect of neomycin on K +-, veratridine, and carbachol-induced-norepinephrine release. Depolarizing agents mediate a neomycin-senstive release, which is not affected either by pertussis toxin or cholera toxin, suggesting a different mode of phospholipase C activation, unlike carbachol-induced release, which is both neomycin and pertussis toxin senstive. Similarly, a hormone-sensitive carrier activated by phenylephrine not via α 1-adrenergic receptors, mediates a non-exocytosis efflux which is not affected by neomycin and is shown to be pertussis toxin-insensitive. The inhibitory action of protein kinase C inhibitors polymyxin B, K252 a and H-7 [(1-(1-(5-isoquinolinesulphonyl)-2-methyl-piperazine] on release, strongly suggests its participation in the process. Polymyxin B, a relatively selective protein kinase C inhibitor, inhibted carbachol-induced release ( ic 50 = 0.53 μ M) as well as the K + and the veratridine induced [ 3H] noradrenaline release. K252 a, an inhibitor of various protein kinases at the ATP site, and H-7, another protein kinase C inhibitor, inhibited carbachol-induced noradrenaline released with ic = 35 nM and 3 μ M respectively. Consistent with its inability to activate phospholipase C, phenylephrine-induced noradrenaline efflux was unaffected by polymyxin B (> 70 μM). The results offer more supportive evidence for a major role played by the dual messengers inositol triphosphate and diacylglycerol (IP 3/DG) in the mechanisms of neuronal release.

Role of bacteria in gastric ulceration produced by indomethacin in the rat: Cytoprotective action of antibiotics

Indomethacin produces mucosal lesions in both the gastric antrum and small intestine in rats refed for 1 h after a 24-h fast. This study was designed to determine the role of bacteria in the formation of the antral lesions. A mixture of antibiotics (bacitracin, neomycin, and polymyxin B) prevented the antral lesions as well as intestinal lesions. The antibiotics also decreased the gastric corpus lesions induced by indomethacin in the fasted rat. Under a germ-free condition, indomethacin did not produce severe lesions in the small intestine of the refed rat but provoked many lesions in the antrum of the refed rat and in the corpus of the fasted rat. Corpus lesions induced by indomethacin in the fasted rat were decreased markedly by neomycin and slightly by polymyxin B, but not by bacitracin. Corpus lesions produced by an absolute ethanol, however, were prevented by each of the antibiotics. The inhibitory effect of neomycin on the corpus lesions was not blocked by pretreatment with indomethacin. In pylorus-ligated rats, neomycin did not decrease gastric acid secretion. The concentration of nonprotein sulfhydryls in the gastric mucosa was not altered by the treatment with neomycin. The antibiotic solutions were hypotonie. It is concluded that (a) bacteria are not important in the formation of antral and corpus lesions induced by indomethacin, and (b) antibiotics prevent gastric ulceration not by its antibacterial action, but by a “cytoprotective” action. The mechanism is unknown, but it may be different from that of antisecretory drugs, prostaglandins, mild irritants, hypertonic solutions, and sulfhydryl compounds.