The effect of cicloxolone sodium on the replication in cultured cells of adenovirus type 5, reovirus type 3, poliovirus type 1, two bunyaviruses and Semliki Forest virus.
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The effect of cicloxolone sodium (CCX) on the replication of typical representatives of different virus families [adenovirus type 5 (Ad-5), reovirus type 3 (Reo-3), Bunyamwera and Germiston viruses, poliovirus type 1 (Polio-1) and Semliki Forest virus (SFV)] in tissue culture was investigated. The Golgi apparatus inhibitor monensin (Mon) and CCX were shown to have analogous effects on some aspects of virus replication. Although the Mon-like effect of CCX played no role in the antiviral activity against Ad-5, Reo-3 or Polio-1, it could entirely account for the antiviral activity against the Bunyamwera and Germiston viruses, for which inhibition of glycoprotein processing was responsible for the antiviral activity. In the case of SFV, the Mon-like activity of CCX caused cytoplasmic assembly of fully infectious SFV within vacuoles and thus impaired virus release without altering total infectious virus yield. Fewer Ad-5 and Reo-3 progeny were produced in the presence of the drug. CCX had a dose-dependent biphasic effect on the particle:p.f.u. ratio of the Reo-3 yield. At low CCX concentration (less than 50 microM) the virus yield contained poor quality, non-infectious virus, but at higher CCX concentration (greater than or equal to 100 microM) low quality virus could no longer be successfully assembled. We conclude that the antiviral effect can be manifested in three ways: (i) by a reduction in the virus particle yield produced; (ii) by a loss of quality (relative infectivity); (iii) by a virucidal effect of the drug. We have previously defined three CCX sensitivity classes. Mechanisms (i), (ii) and (iii) operate against viruses belonging to class CCXs-1 [herpes simplex virus (HSV) type 1, HSV-2 and vesicular stomatitis virus], but essentially only (i) and (ii) affect Reo-3 (CCXs-2), whereas (i) and possibly (iii) affect Ad-5 (CCXs-2). In the case of SFV (CCXs-3) none of these mechanisms operate, but relocation of assembled virus is found. (+info)
Carbenoxolone blockade of neuronal network activity in culture is not mediated by an action on gap junctions.
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Spontaneous activity in the central nervous system is strongly suppressed by blockers of gap junctions (GJs), suggesting that GJs contribute to network activity. However, the lack of selective GJ blockers prohibits the determination of their site of action, i.e. neuronal versus glial. Astrocytes are strongly coupled through GJs and have recently been shown to modulate synaptic transmission, yet their role in neuronal network activity was not analysed. The present study investigated the effects and site of action of the GJ blocker, carbenoxolone (CBX), on neuronal network activity. To this end, we used cultures of hippocampal or cortical neurons, plated on astrocytes. In these cultures neurons display spontaneous synchronous activity and GJs are found only in astrocytes. CBX induced in these neurons a reversible suppression of spontaneous action potential discharges, synaptic currents and synchronised calcium oscillations. Moreover, CBX inhibited oscillatory activity induced by the GABAA antagonist, bicuculline. These effects were not due to blockade of astrocytic GJs, since they were not mimicked nor occluded by endothelin-1 (ET-1), a peptide known to block astrocytic GJs. Also, these effects were still present in co-cultures of wild-type neurons plated on astrocytes originating from connexin-43 (Cx43) knockout mice, and in neuronal cultures which contain few isolated astrocytes. CBX was not likely to exert its effect through neuronal GJs either, as immunostaining for major neuronal connexins (Cx) as well as dye or electrical coupling, were not detected in the different models of cultured neurons examined. Finally while CBX (at 100 microM) did not modify presynaptic transmitter release and postsynaptic responses to glutamate, it did cause an increase in the action potential threshold and strongly decreased the firing rate in response to a sustained depolarising current. These data demonstrate that CBX does not exert its action on network activity of cultured neurons through astrocytic GJs and suggest that it has direct effects on neurons, not involving GJs. (+info)
Different mechanisms promote astrocyte Ca2+ waves and spreading depression in the mouse neocortex.
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Cortical spreading depression (CSD) is thought to play an important role in different pathological conditions of the human brain. Here we investigated the interaction between CSD and Ca2+ waves within the astrocyte population in slices from mouse neocortex (postnatal days 10-14). After local KCl ejection as a trigger for CSD, we recorded the propagation of Ca2+ increases within a large population of identified astrocytes in synchrony with CSD measured as intrinsic optical signal (IOS) or negative DC-potential shift. The two events spread with 39.2 +/- 3.3 mum/sec until the IOS and negative DC-potential shift decayed after approximately 1 mm. However, the astrocyte Ca2+ wave continued to propagate for up to another 500 microm but with a reduced speed of 18.3 +/- 2.5 microm/sec that is also typical for glial Ca2+ waves in white matter or culture. While blocking CSD using MK-801 (40 microm), an NMDA-receptor antagonist, the astrocyte Ca2+ wave persisted with a reduced speed (13.2 +/- 1.5 microm/sec). The specific gap junction blocker carbenoxolon (100 microm) did not prevent CSD but decelerated the speed (2.9 +/- 0.9 microm/sec) of the astrocyte Ca2+ wave in the periphery of CSD. We also found that interfering with intracellular astrocytic Ca2+ signaling by depletion of internal Ca2+ stores does not affect the spread of the IOS. We conclude that CSD determines the velocity of an accompanying astrocytic Ca2+ response, but the astrocyte Ca2+ wave penetrates a larger territory and by this represents a self-reliant phenomenon with a different mechanism of propagation. (+info)
Conduction slowing by the gap junctional uncoupler carbenoxolone.
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BACKGROUND: Cellular electrical coupling is essential for normal propagation of the cardiac action potential, whereas reduced electrical coupling is associated with arrhythmias. Known cellular uncoupling agents have severe side effects on membrane ionic currents. We investigated the effect of carbenoxolone on cellular electrical coupling, membrane ionic currents, and atrial and ventricular conduction. METHODS AND RESULTS: In isolated rabbit left ventricular and right atrial myocytes, carbenoxolone (50 micromol/l) had no effect on action potential characteristics. Calcium, potassium, and sodium currents remained unchanged. Dual current clamp experiments on poorly coupled cell pairs revealed a 21+/-3% decrease in coupling conductance by carbenoxolone (mean+/-S.E.M., n=4, p<0.05). High-density activation mapping was performed in intact rabbit atrium and ventricle during Langendorff perfusion of the heart. The amplitude of the Laplacian of the electrograms, a measure of coupling current in intact hearts, decreased from 1.45+/-0.66 to 0.75+/-0.51 microA/mm(3) (mean+/-SD, n=32, p<0.05) after 15 min of carbenoxolone. Carbenoxolone reversibly decreased longitudinal and transversal conduction velocity from 66+/-15 to 49+/-16 cm/s and from 50+/-14 to 35+/-15 cm/s in ventricle, respectively (mean+/-SD, n=5, both p<0.05). In atrium, longitudinal and transversal conduction velocity decreased from 80+/-29 to 60+/-16 cm/s and from 49+/-10 to 38+/-10 cm/s (mean+/-SD, n=8, both p<0.05). CONCLUSIONS: Carbenoxolone-induced uncoupling causes atrial and ventricular conduction slowing without affecting cardiac membrane currents. Activation delay is larger in poorly coupled cells. (+info)
The hippocampal intrinsic network oscillator.
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Oscillatory activity characterizes the activity of the hippocampus in vivo; however, the underlying mechanism remains unknown. It is also known that during oscillations the number of action potentials provided by the principal cells is surprisingly low, and it is still an open question how oscillations can emerge under such constraints. One suggestion is that the discharge activity of inhibitory cells takes this function; however, this has been found, in my previous studies, not to be the case for cholinergically mediated and intrinsically generated hippocampal oscillations. This study identifies the hippocampal intrinsic network oscillator and the interactions which underlie the concurrent expression of cholinergically mediated theta (4-15 Hz) and gamma (20-80 Hz) oscillations. A particular axonal network that involves the hippocampal associative pathway, shown to consist of axonal collaterals of CA2 and some CA3 pyramidal cells, forms the oscillator core element. It is functionally activated via two cholinergically mediated reactions. First, direct activation of CA2 and CA3 pyramidal cells to discharge. Second, enhancement of gap junction-mediated axo-axonic interactions among axons of the core element and associated axons of interneurones, which together form the full oscillator. With these two reactions it is possible to explain the rhythmicities and patterns of activity, under the condition of a low number of action potentials. The discharge of CA3 pyramidals serves mainly as a trigger, while firing by CA2 pyramidals, and to a lesser degree by CA3 pyramidals, maintains the oscillatory activity. The cholinergically mediated 2-fold increase in axonal gap junction communication between cells serves two functions: (a) creation of specific activation pathways to produce the rhythmicities and patterns, and (b) formation of a reverberatory system that extends the time during which the sparsely generated action potentials can interact in the network, thereby providing a new source of action potentials, critical for the expression of oscillatory activity. (+info)
Ultrastructure of junction areas between neurons and astrocytes in rat supraoptic nuclei.
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AIM: To determine the ultrastructure of junction areas between neurons and astrocytes of supraoptic nuclei in rats orally administered 30 g/L NaCl solution for 5 days. METHODS: The anti-connexin (CX) 43 and anti-CX32 double immunoelectromicroscopic labeled method, and anti-Fos or anti-glial fibrillary acidic protein (GFAP) immunohistochemistry were used to detect changes in the junctional area between neurons and astrocytes in supraoptic nuclei of 5 rats after 30 g/L NaCL solution was given for 5days. RESULTS: A heterotypic connexin32/connexin43 gap junction (HGJ) between neurons and astrocytes (AS) in rat supraoptic nuclei was observed, which was characterized by the thickening and dark staining of cytomembranes with a narrow cleft between them. The number of HGJs and Fos like immunoreactive (-LI) cells was significantly increased following hyperosmotic stimuli, that is, the rats were administered 30 g/L NaCl solution orally or 90 g/L NaCl solution intravenously. HGJs could be blocked with carbenoxolone (CBX), a gap junction blocker, and the number of Fos-LI neurons was significantly decreased compared with that in rats without CBX injection, while Fos-LI ASs were not affected. CONCLUSION: HGJ may be a rapid adaptive signal structure between neurons and ASs in response to stimulation. (+info)
Carbenoxolone inhibition of voltage-gated Ca channels and synaptic transmission in the retina.
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We show that carbenoxolone, a drug used to block hemichannels in the retina to test the ephaptic model of horizontal cell inhibitory feedback, has strong inhibitory effects on voltage-gated Ca channels. Carbenoxolone (100 microM) reduced photoreceptor-to-horizontal cell synaptic transmission by 92%. Applied to patch-clamped, isolated cone photoreceptors, carbenoxolone inhibited Ca channels with an EC(50) of 48 microM. At 100 microM, it reduced cone Ca channel current by 37%, reduced depolarization-evoked [Ca(2+)] signals in fluo-4 loaded retinal slices by 57% and inhibited Ca channels in Muller cells by 52%. A synaptic transfer model suggests that the degree of block of Ca channels accounts for the reduction in synaptic transmission. These results suggest broad inhibitory actions for carbenoxolone in the retina that must be considered when interpreting its effects on inhibitory feedback. (+info)
The classical receptive field surround of primate parasol ganglion cells is mediated primarily by a non-GABAergic pathway.
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Although the center-surround receptive field is a fundamental property of retinal ganglion cells, the circuitry that mediates surround inhibition remains controversial. We examined the contribution of horizontal cells and amacrine cells to the surround of parasol ganglion cells of macaque and baboon retina by measuring receptive field structure before and during the application of drugs that have been shown previously to affect surrounds in a range of mammalian and nonmammalian species. Carbenoxolone and cobalt, thought to attenuate feedback from horizontal cells to cones, severely reduced the surround. Tetrodotoxin, which blocks sodium spiking in amacrine cells, and picrotoxin, which blocks the inhibitory action of GABA, only slightly reduced the surround. These data are consistent with the hypothesis that the surrounds of light-adapted parasol ganglion cells are generated primarily by non-GABAergic horizontal cell feedback in the outer retina, with a small contribution from GABAergic amacrine cells of the inner retina. (+info)