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PubMedDate not supplied

Regulation of gap junction coupling through the neuronal connexin Cx35 by nitric oxide and cGMP.

Gap-junctional coupling among neurons is subject to regulation by a number of neurotransmitters including nitric oxide. We studied the mechanisms by which NO regulates coupling in cells expressing Cx35, a connexin expressed in neurons throughout the central nervous system. NO donors caused potent uncoupling of HeLa cells stably transfected with Cx35. This effect was mimicked by Bay 21-4272, an activator of guanylyl cyclase. A pharmacological analysis indicated that NO-induced uncoupling involved both PKG-dependent and PKG-independent pathways. PKA was involved in both pathways, suggesting that PKG-dependent uncoupling may be indirect. In vitro, PKG phosphorylated Cx35 at three sites: Ser110, Ser276, and Ser289. A mutational analysis indicated that phosphorylation on Ser110 and Ser276, sites previously shown also to be phosphorylated by PKA, had a significant influence on regulation. Ser289 phosphorylation had very limited effects. We conclude that NO can regulate coupling through Cx35 and that regulation is indirect in HeLa cells.

Cell Communication↗
PubMed1999

Longitudinal spread of second messenger signals in isolated rod outer segments of lizards.

1. In vertebrate rods activation of the phototransduction cascade by light triggers changes in the concentrations of at least two diffusible intracellular second messengers (cGMP and Ca2+) whose actions depend on how far they spread from their site of production or entry. To address questions about their spatial spread, cell-attached patch current recording and fluorescence imaging of Calcium Green-dextran were used to measure the longitudinal spread of cGMP and Ca2+, respectively, in functionally intact isolated Gecko gecko lizard rod outer segments under whole-cell voltage clamp. 2. The light-evoked changes in cGMP and Ca2+ concentrations decayed with distance from a site of steady focal activation by two-photon absorption of 1064 nm light with similar decay lengths of approximately 3.5 microm. 3. These results can be understood on the basis of a quantitative model of coupled diffusible intracellular messengers, which is likely to have broad relevance for second messenger signalling pathways in general. 4. The decay length for the spread of adaptation from a site of steady local illumination was about 8 microm, i.e. substantially longer than the decay lengths measured for the spread of cGMP and Ca2+. There are a number of factors, however, that could broaden the apparent relationship between functional changes in the light response and the concentration of a diffusible messenger. For these reasons the measured decay length is an upper limit estimate of the spread of adaptation and does not rule out the possibility that Ca2+ and/or cGMP carry the adaptation signal.

Adaptation, Physiological↗
PubMed2005

A direct relationship between plasma aldosterone and cardiac L-type Ca2+ current in mice.

Aldosterone is involved in a variety of pathophysiological processes that ultimately cause cardiovascular diseases. Despite this, the physiological role of aldosterone in heart function remains elusive. We took advantage of transgenic mouse models characterized by a renal salt-losing (SL) or salt-retaining (SR) phenotype, thus exhibiting chronically high or low plasma aldosterone levels, respectively, to investigate the chronic effects of aldosterone in cardiomyocytes devoid of pathology. On a diet containing normal levels of salt, these animals do not develop any evidence of cardiovascular disease. Using the whole cell patch-clamp technique on freshly isolated adult ventricular cardiomyocytes, we observed that the amplitude of L-type Ca(2)(+) currents (I(Ca)) correlates with plasma aldosterone levels. Larger values of I(Ca) are associated with high aldosterone concentrations in SL models, whereas smaller values of I(Ca) were observed in the SR model. Neither the time- nor the voltage-dependent properties of I(Ca) varied measurably. In parallel, we determined whether modulation of I(Ca) by blood concentration of aldosterone has a major physiological impact on the excitation-contraction coupling of the cardiomyocytes. Action potential duration, [Ca(2)(+)](i) transient amplitude and contraction are increased in the SL model and decreased in the SR model. In conclusion, we demonstrate that the blood concentration of aldosterone exerts chronic regulation of I(Ca) in mouse cardiomyocytes. This regulation has important consequences for excitation-contraction coupling and, potentially, for other Ca(2)(+)-regulated functions in cardiomyocytes.

Aldosterone↗
PubMed1999

C-Linker of cyclic nucleotide-gated channels controls coupling of ligand binding to channel gating.

Cyclic nucleotide-gated channels are composed of a core transmembrane domain, structurally homologous to the voltage-gated K+ channels, and a cytoplasmic ligand-binding domain. These two modules are joined by approximately 90 conserved amino acids, the C-linker, whose precise role in the mechanism of channel activation by cyclic nucleotides is poorly understood. We examined cyclic nucleotide-gated channels from bovine photoreceptors and Caenorhabditis elegans sensory neurons that show marked differences in cyclic nucleotide efficacy and sensitivity. By constructing chimeras from these two channels, we identified a region of 30 amino acids in the C-linker (the L2 region) as an important determinant of activation properties. An increase in both the efficacy of gating and apparent affinity for cGMP and cAMP can be conferred onto the photoreceptor channel by the replacement of its L2 region with that of the C. elegans channel. Three residues within this region largely account for this effect. Despite the profound effect of the C-linker region on ligand gating, the identity of the C-linker does not affect the spontaneous, ligand-independent open probability. Based on a cyclic allosteric model of activation, we propose that the C-linker couples the opening reaction in the transmembrane core region to the enhancement of the affinity of the open channel for agonist, which underlies ligand gating.

Amino Acid Sequence↗
PubMed2002

Spontaneous rhythmicity in cultured cell clusters isolated from mouse small intestine.

To investigate spontaneous rhythmicity in smooth muscle tissue, we have developed a cell cluster preparation. Cell clusters were enzymatically isolated from the muscle layer of mouse small intestine and cultured for several days. They included smooth muscle, neurones, and c-Kit-immunopositive interstitial cells. c-Kit-immunopositive cells in myenteric plexus, showing a networklike structure, are putative pacemaker cells. The cultured cell clusters routinely show spontaneous contraction and preserve characteristic features in this tissue: (1) high temperature dependency of contractile frequency; (2) spontaneous electrical activities measured with patch clamp techniques are insensitive to tetradotoxin (TTX) and dihydropyridine Ca(2+) antagonists. This preparation could therefore be used as a good model system to investigate the underlying mechanisms of intestinal motility and pacemaker function. The relationship between the frequency of electrical activity and cluster size suggests that the minimum unit of small intestine tissue to yield normal pacemaker activity is approximately 100 microm in diameter, or less. The applications of 100-120 microM Cd(2+) and Ni(2+) significantly suppressed the spontaneous activity. Ca(2+) influx pathways other than L-type and "classical" T-type voltage-sensitive Ca(2+) channels seem very likely to play an important role, such as nonselective cation channels and capacitative Ca(2+) entry. Furthermore, applications of heptanol reduced the amplitude and the frequency of the oscillating inward currents and eventually terminated them, suggesting that electrical cell-to-cell coupling may also make some contribution to the generation of spontaneous activity.

Anesthetics, Local↗
PubMed2004

Experimental evidence and modeling studies support a synchronizing role for electrical coupling in the cat thalamic reticular neurons in vivo.

Thalamic reticular (RE) neurons are crucially implicated in brain rhythms. Here, we report that RE neurons of adult cats, recorded and stained intracellularly in vivo, displayed spontaneously occurring spikelets, which are characteristic of central neurons that are coupled electrotonically via gap junctions. Spikelets occurred spontaneously during spindles, an oscillation in which RE neurons play a leading role, as well as during interspindle lulls. They were significantly different from excitatory postsynaptic potentials and also distinct from fast prepotentials that are presumably dendritic spikes generated synaptically. Spikelets were strongly reduced by halothane, a blocker of gap junctions. Multi-site extracellular recordings performed before, during and after administration of halothane demonstrated a role for electrical coupling in the synchronization of spindling activity within the RE nucleus. Finally, computational models of RE neurons predicted that gap junctions between these neurons could mediate the spread of low-frequency activity at great distances. These experimental and modeling data suggest that electrotonic coupling within the RE nucleus plays an important role in the generation and synchronization of low-frequency (spindling) activities in the thalamus.

Action Potentials↗
PubMed2000

Cortical synchronization suggests neural principles of visual feature grouping.

Compositions of visual scenes are related here to neural signals in visual cortex and to cortical circuit models to understand neural mechanisms of perceptual feature grouping. Starting from the hypothesis that synchronization and decoupling of cortical gamma-activities (35-90 Hz) define the relations among visual objects, we concentrate on synchronization related to (1) static retinal stimulation during ocular fixation, and (2) transient stimulation by sudden shifts in object position. The synchronization hypothesis has been tested by analyzing signal correlations in visual cortex of monkeys with the following results: Static retinal stimuli induce loosely phase-coupled gamma-activities among neurons of an object's cortical representation. Patches of gamma-synchronization become decoupled across the representation of an object's contour, and thereby can code figure-ground segregation. Transient stimuli evoke synchronized volleys of stimulus-locked activities that are typically non-rhythmic and include low frequency components in addition to those in the gamma-range. It is argued that stimulus-induced and stimulus-locked synchronizations may play different roles in perceptual feature grouping.

Animals↗
PubMed2005

An arginine residue in the pore region is a key determinant of chloride dependence in cardiac pacemaker channels.

The modulation of ion channel activity by extracellular ions plays a central role in the control of heart function. Here, we show that the sinoatrial pacemaker current I(f) is strongly affected by the extracellular Cl- concentration. We investigated the molecular basis of the Cl- dependence in heterologously expressed hyperpolarization-activated cyclic nucleotide-gated (HCN) channels that represent the molecular correlate of I(f). Currents carried by the two cardiac HCN channel isoforms (HCN2 and HCN4) showed the same strong Cl- dependence as the sinoatrial I(f) and decreased to about 10% in the absence of external Cl-. In contrast, the neuronal HCN1 current was reduced to only 50% under the same conditions. Depletion of Cl- did not affect the voltage dependence of activation or the ion selectivity of the channels, indicating that the reduction of I(f) was caused by a decrease of channel conductance. A series of chimeras between HCN1 and HCN2 was constructed to identify the structural determinants underlying the different Cl- dependence of HCN1 and HCN2. Exchange of the ion-conducting pore region was sufficient to switch the Cl- dependence from HCN1- to HCN2-type and vice versa. Replacement of a single alanine residue in the pore of HCN1 (Ala-352) by an arginine residue present in HCN2 at equivalent position (Arg-405) induced HCN2-type chloride sensitivity in HCN1. Our data indicate that Arg-405 is a key component of a domain that allosterically couples Cl- binding with channel activation.

Allosteric Regulation↗
PubMed2006

Genetic elimination of Suppressor of fused reveals an essential repressor function in the mammalian Hedgehog signaling pathway.

The Hedgehog (Hh) pathway plays important roles during embryogenesis and carcinogenesis. Here, we show that ablation of the mouse Suppressor of fused (Sufu), an intracellular pathway component, leads to embryonic lethality at approximately E9.5 with cephalic and neural tube defects. Fibroblasts derived from Sufu(-/-) embryos showed high Gli-mediated Hh pathway activity that could not be modulated at the level of Smoothened and could only partially be blocked by PKA activation. Despite the robust constitutive pathway activation in the Sufu(-/-) fibroblasts, the GLI1 steady-state localization remained largely cytoplasmic, implying the presence of an effective nuclear export mechanism. Sufu(+/-) mice develop a skin phenotype with basaloid changes and jaw keratocysts, characteristic features of Gorlin syndrome, a human genetic disease linked to enhanced Hh signaling. Our data demonstrate that, in striking contrast to Drosophila, in mammals, Sufu has a central role, and its loss of function leads to potent ligand-independent activation of the Hh pathway.

Animals↗
PubMed2001

Potentiation and inhibition of Ca(2+) release-activated Ca(2+) channels by 2-aminoethyldiphenyl borate (2-APB) occurs independently of IP(3) receptors.

1. The effects of the IP(3)-receptor antagonist 2-aminoethyldiphenyl borate (2-APB) on the Ca(2+) release-activated Ca(2+) current (I(CRAC)) in Jurkat human T cells, DT40 chicken B cells and rat basophilic leukaemia (RBL) cells were examined. 2. 2-APB elicited both stimulatory and inhibitory effects on Ca(2+) influx through CRAC channels. At concentrations of 1-5 microM, 2-APB enhanced Ca(2+) entry in intact cells and increased I(CRAC) amplitude by up to fivefold. At levels > or = 10 microM, 2-APB caused a transient enhancement of I(CRAC) followed by inhibition. 3. 2-APB altered the kinetics of fast Ca(2+)-dependent inactivation of I(CRAC). At concentrations of 1-5 microM, 2-APB increased the rate of fast inactivation. In contrast, 2-APB at higher concentrations (> or = 10 microM) reduced or completely blocked inactivation. 4. 2-APB inhibited Ca(2+) efflux from mitochondria. 5. 2-APB inhibited I(CRAC) more potently when applied extracellularly than intracellularly. Furthermore, increased protonation of 2-APB at low pH did not affect potentiation or inhibition. Thus, 2-APB may have an extracellular site of action. 6. Neither I(CRAC) activation by passive store depletion nor the effects of 2-APB were altered by intracellular dialysis with 500 microg ml(-1) heparin. 7. I(CRAC) is present in wild-type as well as mutant DT40 B cells lacking all three IP(3) receptor isoforms. 2-APB also potentiates and inhibits I(CRAC) in both cell types, indicating that 2-APB exerts its effects independently of IP(3) receptors. 8. Our results show that CRAC channel activation does not require physical interaction with IP(3) receptors as proposed in the conformational coupling model. Potentiation of I(CRAC) by 2-APB may be a useful diagnostic feature for positive identification of putative CRAC channel genes, and provides a novel tool for exploring the physiological functions of store-operated channels.

Animals↗
PubMedDate not supplied

Regulation of trabecular meshwork contractility.

Ample evidence supports the theory that trabecular meshwork possesses smooth-muscle-like properties. Trabecular meshwork cells express a large number of transporters, channels and receptors, many of which are known to regulate smooth-muscle contractility. It has been shown that trabecular meshwork can be induced to contract and relax in response to pharmacological agents. In the model of the bovine eye, confirmed in some cases by experiments on primates, agents that contract trabecular meshwork reduce outflow. On the cellular level, this is coupled with depolarization and a rise in intracellular calcium. Relaxation of trabecular meshwork, on the other hand, appears to be coupled to a stimulation of the maxi-K channel, inducing hyperpolarization and a closure of L-type calcium channels. No significant differences between cells from a human and a bovine source emerged, either in classical measurements of membrane voltage, in measurements of intracellular calcium or patch-clamp experiments. Thus, pharmacological agents that relax trabecular meshwork seem promising candidates for further research - the ultimate goal being an improvement of glaucoma therapy in humans.

Adrenergic Agonists↗
PubMed2001

NADPH oxidase does not account fully for O2-sensing in model airway chemoreceptor cells.

A key feature of O2 sensing by chemoreceptor tissues is the hypoxic inhibition of K+ channels. However, mechanisms coupling a fall of pO2 to channel closure differ between tissues: O2 regulation of K+ channels in chemoreceptive neuroepithelial bodies and their immortal counterparts, H146 cells, involves altered reactive oxygen species generation by NADPH oxidase. In contrast, this enzyme complex is not involved in O2 sensing by the carotid body and pulmonary vasculature. Here, we provide pharmacological evidence to support a role for NADPH oxidase in hypoxic inhibition of K+ currents in H146 cells. Two structurally unrelated NADPH oxidase inhibitors, diphenylene iodonium and phenylarsine oxide, suppressed hypoxic inhibition of K+ currents recorded using the patch-clamp technique. Most importantly, however, neither inhibitor fully blocked this response. Our findings provide the first evidence that multiple mechanisms may coexist within a specific cell type to account for hypoxic suppression of K+ channel activity.

Arsenicals↗
PubMed2005

Morphological, electrophysiological and coupling characteristics of bone marrow-derived mononuclear cells-an in vitro-model.

OBJECTIVE: Transplantation of bone marrow-derived mononuclear cells (BMC) may be suitable to prevent myocardial remodeling and improve left ventricular function after myocardial infarction. However, it is unknown whether or not cardiomyocytes and BMCs can form functioning cell-to-cell coupling and develop adequate electrophysiological properties. METHODS: BMCs were isolated from minipig leg bones, treated with 5-azacytidine (10 microM) for 24 h, cultured for 7 days and labeled with a fluoroscopic dye (DIL). BMCs were cocultured with spontaneously beating cardiomyocytes of neonatal rats. On days 4, 7 and 14 cocultured cells were analyzed. Immunhistochemistry (Connexin 43, alpha-actinin) was used to assess cardiomyogenic differentiation. Action potential characteristics were recorded in whole cell patch clamp mode and to investigate intercellular communication a second gap junction permeable fluoroscopic dye was brought into BMCs by microinjection (Lucifer yellow, LY). RESULTS: From day 7 in coculture BMCs beated synchronously with neonatal rat cardiomyocytes. On day 14, 55.9% of BMCs expressed alpha-actinin and 98.3% were positive for gap junction protein connexin 43. BMC action potential duration (APD90) was mean 11.1 ms with dV/dt(max) of 26.8 V/s and similar to atrial cardiac type. However, microinjection of LY revealed only little dye transfer into adjacent rat cardiomyocytes. CONCLUSIONS: Cocultured BMCs have the potential for early expression of muscle specific proteins in about 60% after 14 days and for cardiac gap junction proteins. Synchronous beating indicates an effective electromechanical coupling. In this heterologous setting we could prove only weak metabolic coupling.

Actinin↗
PubMed2005

Effect of electrical coupling on ionic current and synaptic potential measurements.

Recent studies have found electrical coupling to be more ubiquitous than previously thought, and coupling through gap junctions is known to play a crucial role in neuronal function and network output. In particular, current spread through gap junctions may affect the activation of voltage-dependent conductances as well as chemical synaptic release. Using voltage-clamp recordings of two strongly electrically coupled neurons of the lobster stomatogastric ganglion and conductance-based models of these neurons, we identified effects of electrical coupling on the measurement of leak and voltage-gated outward currents, as well as synaptic potentials. Experimental measurements showed that both leak and voltage-gated outward currents are recruited by gap junctions from neurons coupled to the clamped cell. Nevertheless, in spite of the strong coupling between these neurons, the errors made in estimating voltage-gated conductance parameters were relatively minor (<10%). Thus in many cases isolation of coupled neurons may not be required if a small degree of measurement error of the voltage-gated currents or the synaptic potentials is acceptable. Modeling results show, however, that such errors may be as high as 20% if the gap-junction position is near the recording site or as high as 90% when measuring smaller voltage-gated ionic currents. Paradoxically, improved space clamp increases the errors arising from electrical coupling because voltage control across gap junctions is poor for even the highest realistic coupling conductances. Furthermore, the common procedure of leak subtraction can add an extra error to the conductance measurement, the sign of which depends on the maximal conductance.

Analysis of Variance↗
PubMed1997

Modulation of stimulus-secretion coupling in single rat gonadotrophs.

1. Exocytosis and intracellular [Ca2+] were determined simultaneously in single anterior pituitary gonadotrophs from ovariectomized female rats. Dispersed cells were cultured for 2-4 days with or without 0.2 nM oestradiol-17 beta (E2) before use. Cells were stimulated with either gonadotrophin releasing hormone (GnRH) or by membrane depolarization. Exocytosis was determined from the change in membrane capacitance (Cm) using the perforated-patch whole-cell recording technique. Intracellular [Ca2+] was measured using fura-2 fluorescence. 2. The exocytotic response to 1 nM GnRH was characterized by a wide spectrum of responses, ranging from exocytotic bursts to relatively slow, graded increases that were dependent on the evoked intracellular Ca2+ pattern. A kinetic model is presented that incorporates the observed steep dependence of exocytosis on measured intracellular [Ca2+]; simulated exocytosis reasonably approximated observed exocytotic responses, both kinetically and quantitatively. The model also suggests that the modulatory effects of E2 are brought about either by a change in the Ca2+ sensitivity of exocytosis or by a preferential clustering of docked-secretory granules close to sites of Ca2+ release. The results suggest that in gonadotrophs an oscillatory Ca2+ signal is sensed by the exocytotic apparatus in a modified form of digital encoding. 3. Exocytosis in E2-treated cells was 3-fold greater than in non-treated cells for GnRH-evoked secretion, and 38% greater for depolarization; however, there was no effect of E2 on the intracellular Ca2+ response to either stimulus. The results show that maximum expression of the effect of E2 on exocytosis requires activation of GnRH-dependent pathways.

Animals↗
PubMed2006

Lectin anchored stabilized biodegradable nanoparticles for oral immunization 1. Development and in vitro evaluation.

The investigation comprises development of a stable and targeted formulation of HBsAg for the oral immunization against Hepatitis B. PLGA nanoparticles bearing HBsAg was prepared by double emulsion method. The antigen was protected from organic/aqueous interface by using protein stabilizer, trehalose. The acidic environment generated within PLGA nanoparticles was neutralized by co-encapsulation of a basic additive, Mg(OH)(2) which provides an additional stabilization to the antigen especially against acid induced antigen inactivation. Furthermore, lectin from Arachis hypogaea (PNA) was anchored on to the surface of the HBsAg loaded nanoparticles in order to enhance their affinity towards the antigen presenting cells of the Peyer's patches. The developed system was characterized for shape, size and loading efficiency. The antigen integrity was assessed by using SDS-PAGE followed by isoelectric focusing analysis. Bovine submaxillary mucin (BSM) was used as a biological model for in vitro ligand affinity determination and activity studies. The lectin anchored nanoparticles exhibited 52.18+/-4.73% loading while ligand density was estimated to be of 17.90+/-1.14 microg/mg. The results suggest that HBsAg can be successfully stabilized by co-encapsulation of an appropriate protein stabilizer, i.e. trehalose and a basic additive, Mg(OH)(2). The ligand-coupled nanoparticles demonstrated approximately four folds increase in degree interaction with the BSM as compared to plain nanoparticles. Additionally, the nanoparticles maintained their intrinsic sugar specificity as associated due to lectin (PNA).

Administration, Oral↗
PubMed2002

Ischemia-induced brain damage depends on specific gap-junctional coupling.

Ischemic brain injury results in neuronal loss and associated neurologic deficits. Although there is some evidence that intercellular communication via gap junctions can spread oxidative cell injury, the possible role of gap-junctional communication in ischemia-induced cell death is the object of debate. Because gap junctions directly connect the cytoplasms of coupled cells, they offer a way to propagate stress signals from cell to cell. The authors investigated the contribution of gap-junctional communication to cell death using an in vitro ischemia model, which was reproduced by submersion of organotypic hippocampal slices into glucose-free deoxygenated medium. The gap-junctional blocker carbenoxolone significantly decreased the spread of cell death, as measured by propidium iodide staining, over a 48-hour period after the ischemic episode. Carbenoxolone ameliorated the hypoxia-induced impairment of the intrinsic neuronal electrophysiologic characteristics, as measured by whole-cell patch clamp recordings. To determine whether specific connexins were involved in the spread of postischemic cell death, the authors partially reduced the synthesis of specific connexins using antisense oligodeoxynucleotides. Simultaneous knockdown of two connexins localized mostly in neurons, connexins 32 and 26, resulted in significant neuroprotection 48 hours after the hypoxic-hypoglycemic episode. Similarly, partial reduction of the predominant glial connexin 43 significantly decreased cell death. These results indicate that gap-junctional communication contributes to the propagation of hypoxic injury and that specific gap junctions could be a novel target to reduce brain damage.

Animals↗