Electrotonic coupling between stratum oriens interneurones in the intact in vitro mouse juvenile hippocampus.
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Using the isolated juvenile (7-14 days) mouse whole hippocampus preparation, which contains intact complex local circuitry, 145 dual whole cell recordings were made from stratum oriens (s.o.) interneurones under infrared microscopy. In 11.7% of paired recordings, evidence for direct electrotonic coupling between the s.o. interneurones was obtained from the response of one interneurone to a long (400-600 ms) constant current pulse passed into the coupled interneurone. When specifically orienting the dual recordings in the transectional plane of the hippocampus, 18.5% of paired recordings showed electrotonic coupling. The coupling coefficient, estimated from averaged data, was 6.9 +/- 4.7%, ranging from 1.3 to 17.6%. The time constant of the electrotonically transmitted hyperpolarization was inversely related to the coupling coefficient between the two neurones. The electrotonic responses of one neurone to constant current pulses injected into the other coupled neurone were intermittent. Spikes in one of the coupled neurones were associated with small electrotonic EPSPs (spikelets) in the other coupled neurone, in those neuronal pairs with coupling coefficients greater than 10%. Failure of spikelet production following a spike in the coupled cell occurred 5-10% of the time. Electrotonic coupling and spikelets persisted in the presence of chemical synaptic transmission blockade by CNQX, APV and bicuculline, or in zero Ca(2+) perfusate, but were abolished by carbenoxolone (100 microm), a gap junctional blocker. These data confirm the existence of electrotonic coupling between s.o. interneurones, presumably via gap junctions located in dendrites. (+info)
Coexistence of gamma and high-frequency oscillations in rat medial entorhinal cortex in vitro.
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High frequency oscillations (> 80-90 Hz) occur in neocortex and hippocampus in vivo where they are associated with specific behavioural states and more classical EEG frequency bands. In the hippocampus in vitro these oscillations can occur in the absence of pyramidal neuronal somatodendritic compartments and are temporally correlated with on-going, persistent gamma frequency oscillations. Their occurrence in the hippocampus is dependent on gap-junctional communication and it has been suggested that these high frequency oscillations originate as collective behaviour in populations of electrically coupled principal cell axonal compartments. Here we demonstrate that the superficial layers of medial entorhinal cortex can also generate high frequency oscillations associated with gamma rhythms. During persistent gamma frequency oscillations high frequency oscillations occur with a high bispectral coherence with the field gamma activity. Bursts of high frequency oscillations are temporally correlated with both the onset of compound excitatory postsynaptic potentials in fast-spiking interneurones and spikelet potentials in both pyramidal and stellate principal neurones. Both the gamma frequency and high frequency oscillations were attenuated by the gap junction blocker carbenoxolone. These data suggest that high frequency oscillations may represent the substrate for phasic drive to interneurones during persistent gamma oscillations in the medial entorhinal cortex. (+info)
Identification of small molecule synthetic inhibitors of DNA polymerase beta by NMR chemical shift mapping.
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DNA polymerase beta (beta-pol) plays a central role in repair of damaged DNA bases by base excision repair (BER) pathways. A predominant phenotype of beta-pol null mouse fibroblasts is hypersensitivity to the DNA-methylating agent methyl methanesulfonate. Residues in the 8-kDa domain of beta-pol that seem to interact with a known natural product beta-pol inhibitor, koetjapic acid, were identified by NMR chemical shift mapping. The data implicate the binding pocket as the hydrophobic cleft between helix-2 and helix-4, which provides the DNA binding and deoxyribose phosphate lyase activities of the enzyme. Nine structurally related synthetic compounds, containing aromatic or other hydrophobic groups in combination with two carboxylate groups, were then tested. They were found to bind to the same or a very similar region on the surface of the enzyme. The ability of these compounds to potentiate methyl methanesulfonate cytotoxicity, an indicator of cellular BER capacity, in wild-type and beta-pol null mouse fibroblasts, was next ascertained. The most active and beta-pol-specific of these agents, pamoic acid, was further characterized and found to be an inhibitor of the deoxyribose phosphate lyase and DNA polymerase activities of purified beta-pol on a BER substrate. Our results illustrate that NMR-based mapping techniques can be used in the design of small molecule enzyme inhibitors including those with potential use in a clinical setting. (+info)
Increased in vivo regeneration of cortisol in adipose tissue in human obesity and effects of the 11beta-hydroxysteroid dehydrogenase type 1 inhibitor carbenoxolone.
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11beta-Hydroxysteroid dehydrogenase type 1 (11HSD1) regenerates cortisol from cortisone within adipose tissue and liver. 11HSD1 inhibitors may enhance insulin sensitivity in type 2 diabetes and be most efficacious in obesity when 11HSD1 is increased in subcutaneous adipose biopsies. We examined the regeneration of cortisol in vivo in obesity, and the effects of the 11HSD1 inhibitor carbenoxolone. We compared six lean and six obese men and performed a randomized, placebo-controlled crossover study of carbenoxolone in obese men. The obese men had no difference in their whole-body rate of regenerating cortisol (measured with 9,11,12,12-[(2)H(4)]cortisol tracer), but had more rapid conversion of [(3)H]cortisone to [(3)H]cortisol in abdominal subcutaneous adipose tissue (measured with microdialysis). During insulin infusion, adipose 11HSD1 activity fell markedly in lean but not in obese men. Carbenoxolone inhibited whole-body cortisol regeneration, but did not significantly inhibit adipose 11HSD1 and had no effects on insulin sensitivity (measured by [(2)H(2)]glucose infusion with or without hyperinsulinemia). Thus, in vivo cortisol generation is increased selectively within adipose tissue in obesity, perhaps reflecting resistance to insulin-mediated downregulation of 11HSD1. However, obese men are less susceptible than lean men to the insulin-sensitizing effects of carbenoxolone. To be useful in obese patients, 11HSD1 inhibitors will need to inhibit the enzyme more effectively in adipose tissue. (+info)
Inner and outer retinal pathways both contribute to surround inhibition of salamander ganglion cells.
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Illumination of the receptive-field surround reduces the sensitivity of a retinal ganglion cell to centre illumination. The steady, antagonistic receptive-field surround of retinal ganglion cells is classically attributed to the signalling of horizontal cells in the outer plexiform layer (OPL). However, amacrine cell signalling in the inner plexiform layer (IPL) also contributes to the steady receptive-field surround of the ganglion cell. We examined the contributions of these two forms of presynaptic lateral inhibition to ganglion cell light sensitivity by measuring the effects of surround illumination on EPSCs evoked by centre illumination. GABA(C) receptor antagonists reduced inhibition attributed to dim surround illumination, suggesting that this inhibition was mediated by signalling to bipolar cell axon terminals. Brighter surround illumination further reduced the light sensitivity of the ganglion cell. The bright surround effects on the EPSCs were insensitive to GABA receptor blockers. Perturbing outer retinal signalling with either carbenoxolone or cobalt blocked the effects of the bright surround illumination, but not the effects of dim surround illumination. We found that the light sensitivities of presynaptic, inhibitory pathways in the IPL and OPL were different. GABA(C) receptor blockers reduced dim surround inhibition, suggesting it was mediated in the IPL. By contrast, carbenoxolone and cobalt reduced bright surround, suggesting it was mediated by horizontal cells in the OPL. Direct amacrine cell input to ganglion cells, mediated by GABA(A) receptors, comprised another surround pathway that was most effectively activated by bright illumination. Our results suggest that surround activation of lateral pathways in the IPL and OPL differently modulate the sensitivity of the ganglion cell to centre illumination. (+info)
Signal propagation via gap junctions, a key step in the regulation of liver metabolism by the sympathetic hepatic nerves.
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Cell-to-cell communication via gap junctions has been proposed to be involved in the metabolic actions of sympathetic liver nerves in the rat. The effects of hepatic nerve stimulation and noradrenaline-, PGF2 alpha- and glucagon infusion on glucose metabolism and perfusion flow were studied in perfused rat liver in the absence and presence of the gap junctional inhibitors, heptanol, carbenoxolone and (4 beta)phorbol 12-myristate 13-acetate (4 beta PMA). (i) Stimulation of the hepatic nerve plexus increased glucose output, decreased flow and caused an overflow of noradrenaline into the hepatic vein. (ii) Heptanol completely inhibited not only the nerve stimulation-dependent metabolic and hemodynamic alterations but also the noradrenaline overflow. Thus the heptanol-dependent inhibitions were caused primarily by a strong impairment of transmitter release. (iii) Carbenoxolone inhibited the effects of neurostimulation on glucose metabolism partially by about 50%, whereas it left perfusion flow and noradrenaline overflow essentially unaltered. (iv) 4 beta PMA reduced the nerve stimulation-dependent enhancement of glucose release by about 80% but the noradrenaline-dependent increase in glucose output only by about 30%; the increase in glucose release by PGF2 alpha and by glucagon remained essentially unaltered. 4 beta PMA reduced the nerve stimulation-dependent decrease in portal flow by about 35% but did not affect the noradrenaline-and PGF2 alpha-elicited alterations, nor did it alter noradrenaline overflow. The results allow the conclusion that gap junctional communication plays a major role in the regulation of hepatic carbohydrate metabolism by sympathetic liver nerves, but not by circulating noradrenaline, PGF2 alpha or glucagon. (+info)
Electrical coupling in sustentacular cells of the mouse olfactory epithelium.
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Sustentacular cells (SCs) line the apical surface of the olfactory epithelium (OE) and provide trophic, metabolic, and mechanical support for olfactory receptor neurons. Morphological studies have suggested that SCs possess gap junctions, although physiological evidence for gap junctional communication in mammalian SCs is lacking. In the present study we investigated whether coupling exists between SCs situated in tissue slices of OE from neonatal (P0-P4) mice. Using whole cell and cell-attached patch recordings from SCs, we demonstrate that SCs are electrically coupled by junctional resistances on the order of 300 M(omega). Under whole cell recording conditions, Alexa 488 added to the pipette solution failed to reveal dye coupling between SCs. Electrical coupling was deduced from the biexponential decay of capacitive currents recorded from SCs and from the bell-shaped voltage dependency of a P2Y-receptor-activated current, both of which were abolished by 18beta-glycyrrhetinic acid (20-50 microM), a blocker of gap junctions. These data provide strong evidence for functional coupling between SCs, the physiological importance of which is discussed. (+info)
Mechanical strain opens connexin 43 hemichannels in osteocytes: a novel mechanism for the release of prostaglandin.
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Mechanosensing bone osteocytes express large amounts of connexin (Cx)43, the component of gap junctions; yet, gap junctions are only active at the small tips of their dendritic processes, suggesting another function for Cx43. Both primary osteocytes and the osteocyte-like MLO-Y4 cells respond to fluid flow shear stress by releasing intracellular prostaglandin E2 (PGE2). Cells plated at lower densities release more PGE2 than cells plated at higher densities. This response was significantly reduced by antisense to Cx43 and by the gap junction and hemichannel inhibitors 18 beta-glycyrrhetinic acid and carbenoxolone, even in cells without physical contact, suggesting the involvement of Cx43-hemichannels. Inhibitors of other channels, such as the purinergic receptor P2X7 and the prostaglandin transporter PGT, had no effect on PGE2 release. Cell surface biotinylation analysis showed that surface expression of Cx43 was increased by shear stress. Together, these results suggest fluid flow shear stress induces the translocation of Cx43 to the membrane surface and that unapposed hemichannels formed by Cx43 serve as a novel portal for the release of PGE2 in response to mechanical strain. (+info)