cyclic-gmp and lucifer-yellow

The effect of several second messengers on the functional expression of gap junctions was investigated in primary cultures of newborn rat microglia. As previously reported, microglia cultured under resting conditions expressed low levels of the gap junction protein connexin 43, and exhibited little dye coupling. After treatment with 4bromo-A23187, a Ca(2+) ionophore, the incidence of dye coupling between microglia increased progressively over a 12-h period. Dye coupling was markedly reduced by gap junction blockers. Induction of dye coupling by 4bromo-A23187 was prevented by the addition of a synthetic peptide with the same sequence as a region of the extracellular loop 1 of connexin 43 (residues 53-66). The increase in dye coupling induced by 4bromo-A23187 was associated with increased connexin 43 mRNA and protein levels. Treatment of microglia with phorbol 12-myristate 13-acetate, an activator of protein kinase C, did not promote gap junctional communication in untreated microglia and reversed 4bromo-A23187-induced dye coupling. Thus, gap junctional communication between microglia can be regulated oppositely by calcium- and protein kinase C-dependent pathways. Activators of cGMP-dependent protein kinase (8bromo-cGMP) or protein kinase A (8bromo-cAMP) had no effect on untreated microglia or on 4bromo-A23187-induced dye coupling. Differential regulation of gap junctions by intracellular calcium concentration and protein kinase C activity may help to explain how various stimuli evoke differences in microglia responses, such as synthesis and secretion of cytokines and proteases.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Animals, Newborn; Calcimycin; Calcium; Calcium Signaling; Cells, Cultured; Central Nervous System; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Fluorescent Dyes; Gap Junctions; GAP-43 Protein; Gliosis; Ionophores; Isoquinolines; Microglia; Protein Kinase C; Rats; RNA, Messenger; Second Messenger Systems; Tetradecanoylphorbol Acetate; Up-Regulation

The stomatogastric ganglion (STG) of the crab Cancer productus contains approximately 30 neurons arrayed into two different networks (gastric mill and pyloric), each of which produces a distinct motor pattern in vitro. Here we show that the functional division of the STG into these two networks requires intact NO-cGMP signaling. Multiple nitric oxide synthase (NOS)-like proteins are expressed in the stomatogastric nervous system, and NO appears to be released as an orthograde transmitter from descending inputs to the STG. The receptor of NO, a soluble guanylate cyclase (sGC), is expressed in a subset of neurons in both motor networks. When NO diffusion or sGC activation are blocked within the ganglion, the two networks combine into a single conjoint circuit. The gastric mill motor rhythm breaks down, and several gastric neurons pattern switch and begin firing in pyloric time. The functional reorganization of the STG is both rapid and reversible, and the gastric mill motor rhythm is restored when the ganglion is returned to normal saline. Finally, pharmacological manipulations of the NO-cGMP pathway are ineffective when descending modulatory inputs to the STG are blocked. This suggests that the NO-cGMP pathway may interact with other biochemical cascades to partition rhythmic motor output from the ganglion.

Topics: Animals; Arginine; Brachyura; Citrulline; Cyclic GMP; Digestive System; Enzyme Inhibitors; Fluorescent Dyes; Ganglia, Invertebrate; Guanylate Cyclase; Immunohistochemistry; In Vitro Techniques; Isoquinolines; Male; Nerve Net; Neurons; Neurotransmitter Agents; Nitric Oxide; Nitric Oxide Donors; Periodicity; Signal Transduction

Unlike many neuron populations, supraoptic nucleus (SON) neurons are rich in both nitric oxide synthase (NOS) and the NO receptor-soluble guanylyl cyclase (GC), the activation of which leads to cGMP accumulation. Elevations in cGMP result in increased coupling among SON neurons. We investigated the effect of NO on dye coupling in SONs from male, proestrus virgin female, and lactating rats. In 167 slices 263 SON neurons were recorded; 210 of these neurons were injected intracellularly (one neuron per SON) with Lucifer yellow (LY). The typically minimal coupling seen in virgin females was increased nearly fourfold by the NO precursor, L-arginine, or the NO donor, sodium nitroprusside (SNP). L-Arginine-induced coupling was abolished by a NOS inhibitor. In slices from male and lactating rats who have a higher basal incidence of coupling, SNP increased coupling by approximately twofold over control (p < 0.03). SNP effects were prevented by the NO scavenger hemoglobin (20 microM) and by the selective blocker of NO-activated GC, ODQ (10 microM). These results suggest that NO released from cells within the SON can expand the coupled network of neurons and that this action occurs via cGMP-dependent processes. Because increased coupling is associated with elevated SON neuronal excitability, we also studied the effects of 8-bromo-cGMP on excitability. In both phasically and continuously firing neurons 8-bromo-cGMP (1-2 mM), but not cGMP, produced membrane depolarizations accompanied by membrane conductance increases. Conductance increases remained when depolarizations were eliminated by current-clamping the membrane potential. Thus, NO-induced cGMP increases SON neuronal coupling and excitability.

Topics: Animals; Arginine; Cyclic GMP; Female; Fluorescent Dyes; Guanylate Cyclase; In Vitro Techniques; Isoquinolines; Male; Membrane Potentials; Neurons; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitroprusside; Rats; Rats, Sprague-Dawley; Supraoptic Nucleus

In the teleost retina the intercellular messenger nitric oxide can be synthesized by several cell types including cone photoreceptors and H1 horizontal cells, indicating a modulatory role within the outer plexiform layer, the first stage of the visual information processing. Therefore, the aim of this study was to elucidate the effects of nitric oxide on the physiology of cone horizontal cells in the intact retina. The nitric oxide donor sodium nitroprusside (0.5-2.5 mM) enhanced the light responsiveness of cone horizontal cells and reduced the degree of electrical coupling in the network. Furthermore, the spread of intracellularly injected Lucifer Yellow was restricted. The effects on light responsiveness and electrical coupling were qualitatively mimicked by 8-bromo-cGMP (0.5 mM) and could not be achieved by ferrocyanide (1 mM), the byproduct of nitric oxide liberation from nitroprusside. The effects of NO on the responsiveness of horizontal cells may be due to an action on green- and red-sensitive cones. Nitroprusside (0.1 mM) diminished the K(+)-stimulated release of endogenous dopamine by 50%, whereas the basal dopamine release was not affected, indicating that the effects on electrotonic horizontal cell coupling were not elicited by an NO-induced release of dopamine. With respect to the morphologic plasticity of the cone-horizontal cell synapse the inhibitor of endogenous nitric oxide synthesis L-nitroarginine (0.1 mM) had no influence on the formation or retraction of spinules. These results show that NO affects the responsiveness and coupling of the horizontal cell network in a dopamine-independent way.

Topics: Animals; Carps; Cyclic GMP; Dark Adaptation; Dopamine; Evoked Potentials, Visual; Fluorescent Dyes; Isoquinolines; Membrane Potentials; Nitric Oxide; Nitroprusside; Photic Stimulation; Retina; Time Factors; Visual Perception

Primary cultures of osteoblast-like cells (OB) derived from calvarial fragments of newborn rats and juvenile guinea pigs formed numerous gap junctions between neighboring cells in vitro. Intracellular injection of Lucifer yellow led to a staining of up to 30 adjacent cells. Parallel intracellular recordings showed that amplitudes of stimulated membrane potential changes (4-5 mV) were closely related between coupled cells. The coupling factor, which was derived from the ratio of these amplitudes, ranged between 0.1 and 0.8. The coupling factor (1) was not dependent on the membrane potential or the injected current strength; (2) strong acidosis (pH < 6.6) and hypercapnia (pCO2 > 80 mm Hg) did not affect electric or dye coupling; (3) elevation of intracellular cAMP level was ineffective; (4) rise of the extra- and intracellular Ca2+ concentration did not effect the electric coupling; (5) the anticonvulsant drugs carbamazepine and phenytoin impaired the coupling factor up to 59%. The findings show that cell-cell communication between OB via gap junctions proved stable under various conditions which, in other tissues, were found to reduce the coupling strength of gap junctions.

Topics: 1-Octanol; Animals; Anticonvulsants; Calcium; Cell Communication; Cells, Cultured; Cyclic AMP; Cyclic GMP; Electrophysiology; Guinea Pigs; Hydrogen-Ion Concentration; Intercellular Junctions; Isoquinolines; Membrane Potentials; Octanols; Osteoblasts; Rats; Staining and Labeling

Topics: Animals; Arginine; Carps; Cyclic AMP; Cyclic GMP; In Vitro Techniques; Isoquinolines; Nitric Oxide; Photoreceptor Cells; Rana catesbeiana; Retina; Spectrometry, Fluorescence; Turtles

The influence of cAMP on the cell-to-cell diffusion of Lucifer Yellow CH in heart muscle was further investigated. It was found that isoproterenol (10(-5) M) enhanced the diffusion coefficient (D) from 4 +/- 0.63 X 10(-7) cm2/s (control) to 2.4 +/- 0.66 X 10(-6) cm2/s. Dibutyryl-cAMP (5 X 10(-4) M) plus theophylline (0.4 mM) also increased the cell-to-cell diffusion of Lucifer Yellow CH (D = 3.2 +/- 0.69 X 10(-6) cm2/s). The effect of dBcAMP was increased by theophylline. Since the permeability of the surface cell membrane to the dye is negligible in presence of these drugs and the binding of Lucifer Yellow CH to cytoplasmic proteins was not altered, it is thus concluded that the enhanced longitudinal diffusion of the dye was due to an increase in junctional permeability. As the enhanced cell-to-cell diffusion of the dye caused by isoproterenol was quickly reversed the hypothesis of a greater synthesis of intercellular channels seems unlikely. Acetylcholine (10(-5) M) that increases the intercellular concentration of cGMP had no effect on cell-to-cell diffusion of the dye. The present results support the view that cAMP is a modulator of junctional permeability in heart muscle.

Topics: Acetylcholine; Animals; Bucladesine; Cell Membrane Permeability; Cyclic AMP; Cyclic GMP; Dogs; Heart; In Vitro Techniques; Intercellular Junctions; Isoquinolines; Mathematics; Theophylline

The influence of dB-cAMP (5 X 10(-4) M) and 8-Br-cGMP (5 X 10(-5) M; 5 X 10(-9) M) on the longitudinal diffusion of Lucifer Yellow CH along dog trabeculae was investigated. It was found that dB-cAMP enhances the diffusion coefficient of the dye from 4 +/- 0.63 X 10(-7) cm2/s (control) to 2 +/- 0.53 X 10(-6) cm2/s. Efflux studies showed that the permeability of the surface cell membrane to Lucifer Yellow is negligible ruling out the possibility that the dye moved from cell-to-cell through the extracellular space. The permeability of the nexal membrane (Pnexus = 3 X 4 cm/s) was appreciably enhanced in fibers exposed to dB-cAMP (9.1 X 10(-4) cm/s). No change in the longitudinal redistribution of Lucifer Yellow CH was found with 8-Br-cGMP. The results support the hypothesis that cAMP is a modulator of junctional permeability in the normal heart.

Topics: Animals; Bucladesine; Cell Membrane Permeability; Cyclic GMP; Diffusion; Dogs; Intercellular Junctions; Isoquinolines; Myocardium