8-bromocyclic-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

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

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