Physiologic regulation of heart rate and blood pressure involves connexin 36-containing gap junctions.

Chronically elevated sympathetic nervous activity underlies many cardiovascular diseases. Elucidating the mechanisms contributing to sympathetic nervous system output may reveal new avenues of treatment. The contribution of the gap junctional protein connexin 36 (Cx36) to the regulation of sympathetic activity and thus blood pressure and heart rate was determined using a mouse with specific genetic deletion of Cx36. Ablation of the Cx36 protein was confirmed in sympathetic preganglionic neurons of Cx36-knockout (KO) mice. Telemetric analysis from conscious Cx36 KO mice revealed higher variance in heart rate and blood pressure during rest and activity compared to wild-type (WT) mice, and smaller responses to chemoreceptor activation when anesthetized. In the working heart-brain stem preparation of the Cx36-KO mouse, respiratory-coupled sympathetic nerve discharge was attenuated and responses to chemoreceptor stimulation and noxious stimulation were blunted compared to WT mice. Using whole cell patch recordings, sympathetic preganglionic neurons in spinal cord slices of Cx36-KO mice displayed lower levels of spikelet activity compared to WT mice, indicating reduced gap junction coupling between neurons. Cx36 deletion therefore disrupts normal regulation of sympathetic outflow with effects on cardiovascular parameters.-Lall, V. K., Bruce, G., Voytenko, L., Drinkhill, M., Wellershaus, K., Willecke, K., Deuchars, J., Deuchars, S. A. Physiologic regulation of heart rate and blood pressure involves connexin 36-containing gap junctions.

Na+/K+ ATPase α1 and α3 isoforms are differentially expressed in α- and γ-motoneurons.

The Na(+)/K(+) ATPase (NKA) is an essential membrane protein underlying the membrane potential in excitable cells. Transmembrane ion transport is performed by the catalytic α subunits (α1-4). The predominant subunits in neurons are α1 and α3, which have different affinities for Na(+) and K(+), impacting on transport kinetics. The exchange rate of Na(+)/K(+) markedly influences the activity of the neurons expressing them. We have investigated the distribution and function of the main isoforms of the α subunit expressed in the mouse spinal cord. NKAα1 immunoreactivity (IR) displayed restricted labeling, mainly confined to large ventral horn neurons and ependymal cells. NKAα3 IR was more widespread in the spinal cord, again being observed in large ventral horn neurons, but also in smaller interneurons throughout the dorsal and ventral horns. Within the ventral horn, the α1 and α3 isoforms were mutually exclusive, with the α3 isoform in smaller neurons displaying markers of γ-motoneurons and α1 in α-motoneurons. The α3 isoform was also observed within muscle spindle afferent neurons in dorsal root ganglia with a higher proportion at cervical versus lumbar regions. We confirmed the differential expression of α subunits in motoneurons electrophysiologically in neonatal slices of mouse spinal cord. γ-Motoneurons were excited by bath application of low concentrations of ouabain that selectively inhibit NKAα3 while α-motoneurons were insensitive to these low concentrations. The selective expression of NKAα3 in γ-motoneurons and muscle spindle afferents, which may affect excitability of these neurons, has implications in motor control and disease states associated with NKAα3 dysfunction.

Influence of retinoblastoma-related gene silencing on the initiation of DNA replication by African cassava mosaic virus Rep in cells of mature leaves in Nicotiana benthamiana plants.

BACKGROUND: Geminiviruses mainly infect terminally differentiated tissues and cells in plants. They need to reprogramme host cellular machinery for DNA replication. This process is thought to be mediated by inactivation of cell-cycle repressor proteins and by induction of host DNA synthesis protein expression through actions of the geminviral replication initiator protein (Rep). FINDINGS: Exploiting a Nicotiana benthamiana pOri2 line, which is transformed with a transgene consisting of a direct repeat of the African cassava mosaic virus (ACMV)-replication origin (Ori) flanking a non-viral DNA region, and virus-induced RNA silencing (VIGS), the impact of host gene expression on replication of the ACMV-derived replicon was investigated. The ACMV Rep trans-replicated the viral episomal replicon in leaves of young but not older pOri2 plants. Upon VIGS-mediated down-regulation of N. benthamiana NbRBR1, the retinoblastoma-related protein gene coding for a negative cell-cycle suppressor, recovered the ability of ACMV Rep for trans DNA replication, whereas the silencing of NbPCNA coding for the sliding clamp of DNA polymerase had no effect. CONCLUSIONS: These results suggest that the cellular machinery for DNA replication in differentiated tissues of older leaves cannot be reprogrammed by Rep alone but may need other uncharacterised viral and plant factors.

The role of the membrane potential in chondrocyte volume regulation.

Many cell types have significant negative resting membrane potentials (RMPs) resulting from the activity of potassium-selective and chloride-selective ion channels. In excitable cells, such as neurones, rapid changes in membrane permeability underlie the generation of action potentials. Chondrocytes have less negative RMPs and the role of the RMP is not clear. Here we examine the basis of the chondrocyte RMP and possible physiological benefits. We demonstrate that maintenance of the chondrocyte RMP involves gadolinium-sensitive cation channels. Pharmacological inhibition of these channels causes the RMP to become more negative (100 µM gadolinium: ΔV(m) = -30 ± 4 mV). Analysis of the gadolinium-sensitive conductance reveals a high permeability to calcium ions (PCa/PNa ≈80) with little selectivity between monovalent ions; similar to that reported elsewhere for TRPV5. Detection of TRPV5 by PCR and immunohistochemistry and the sensitivity of the RMP to the TRPV5 inhibitor econazole (ΔV(m) = -18 ± 3 mV) suggests that the RMP may be, in part, controlled by TRPV5. We investigated the physiological advantage of the relatively positive RMP using a mathematical model in which membrane stretch activates potassium channels allowing potassium efflux to oppose osmotic water uptake. At very negative RMP potassium efflux is negligible, but at more positive RMP it is sufficient to limit volume increase. In support of our model, cells clamped at -80 mV and challenged with a reduced osmotic potential swelled approximately twice as much as cells at +10 mV. The positive RMP may be a protective adaptation that allows chondrocytes to respond to the dramatic osmotic changes, with minimal changes in cell volume.

Gap junction-mediated spontaneous Ca(2+) waves in differentiated cholinergic SN56 cells.

Neuronal gap junctions are receiving increasing attention as a physiological means of intercellular communication, yet our understanding of them is poorly developed when compared to synaptic communication. Using microfluorimetry, we demonstrate that differentiation of SN56 cells (hybridoma cells derived from murine septal neurones) leads to the spontaneous generation of Ca(2+) waves. These waves were unaffected by tetrodotoxin (1microM), but blocked by removal of extracellular Ca(2+), or addition of non-specific Ca(2+) channel inhibitors (Cd(2+) (0.1mM) or Ni(2+) (1mM)). Combined application of antagonists of NMDA receptors (AP5; 100microM), AMPA/kainate receptors (NBQX; 20microM), nicotinic AChR receptors (hexamethonium; 100microM) or inotropic purinoceptors (brilliant blue; 100nM) was also without effect. However, Ca(2+) waves were fully prevented by carbenoxolone (200microM), halothane (3mM) or niflumic acid (100microM), three structurally diverse inhibitors of gap junctions, and mRNA for connexin 36 was detected by PCR. Whole-cell patch-clamp recordings revealed spontaneous inward currents in voltage-clamped cells which we inhibited by Cd(2+), Ni(2+) or niflumic acid. Our data suggest that differentiated SN56 cells generated spontaneous Ca(2+) waves which are propagated by intercellular gap junctions. We propose that this system can be exploited conveniently for the development of neuronal gap junction modulators.

GABA(B) Mediated Regulation of Sympathetic Preganglionic Neurons: Pre- and Postsynaptic Sites of Action.

Modulatory influences on sympathetic nervous system activity are diverse and far reaching, acting at select points in the complex pathways controlling sympathetic outflow to enable subtle changes or more global effects. Changes in the degree of sympathetic neuromodulation can have serious consequences on homeostatic variables such as heart rate, blood pressure and gut motility. At the level of the spinal cord, the sympathetic preganglionic neurons (SPNs) can be modulated by activation of presynaptic GABA(B) heteroreceptors on glutamatergic terminals and by postsynaptic GABA(B) receptors. Here we show that a low concentration of the GABA(B) agonist baclofen (1 µM) attenuated GABAergic inhibitory postsynaptic potentials in SPNs elicited from stimulation of either the central autonomic area or descending fibers in the lateral funiculus. This low baclofen concentration also elicited three categories of postsynaptic response: a large hyperpolarization with a decrease in input resistance, a moderate hyperpolarization with no change in input resistance and no response. Using cesium-loaded, tetraethylammonium chloride containing electrodes (to block potassium conductance), baclofen elicited moderate hyperpolarizations with no change in input resistance in 50% of SPNs; the remainder were unaffected. These modest hyperpolarizations were reduced in Ca(2+) free solution or cadmium. Hyperpolarizing responses were also observed in interneurons in the vicinity of SPNs. These studies provide the first evidence for GABA(B) autoreceptors involved in inhibitory GABAergic transmission onto SPNs and for postsynaptic GABA(B) receptors on interneurons. The data also indicate that there is heterogeneity in the postsynaptic responses of SPNs.