Presynaptic cyclic nucleotide-gated ion channels modulate neurotransmission in the mammalian olfactory bulb.
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Cyclic nucleotide-gated channels (CNGCs) on the dendritic cilia of olfactory receptor neurons (ORNs) are critical for sensory transduction in the olfactory system. Do CNGCs also play a role in the axons and/or nerve terminals of ORNs? We find that the cyclic nucleotides cAMP and cGMP can both facilitate and depress synaptic transmission between olfactory nerve fibers and their targets in olfactory bulb glomeruli. Cyclic nucleotides increase intracellular Ca(2+) in ORN terminals and enhance spontaneous transmitter release; at higher concentrations, cyclic nucleotides depress evoked transmission by altering olfactory nerve excitability. Cyclic nucleotides have no effect on transmission or nerve excitability, however, in mice lacking olfactory CNGCs. Taken together, our results identify a novel role for presynaptic CNGCs in modulating neurotransmission. (+info)
Regulation of intracellular cyclic AMP in skeletal muscle cells involves the efflux of cyclic nucleotide to the extracellular compartment.
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(1) This report analyses the intracellular and extracellular accumulation of cyclic AMP in primary rat skeletal muscle cultures, after direct and receptor-dependent stimulation of adenylyl cyclase (AC). (2) Isoprenaline, calcitonin gene-related peptide (CGRP) and forskolin induced a transient increase in the intracellular cyclic AMP that peaked 5 min after onset stimulation. (3) Under stimulation with isoprenaline or CGRP, the intracellular cyclic AMP initial rise was followed by an exponential decline, reaching 46 and 52% of peak levels in 10 min, respectively. (4) Conversely, the forskolin-dependent accumulation of intracellular cyclic AMP decreased slowly and linearly, reaching 49% of the peak level in 30 min. (5) The loss of intracellular cyclic AMP from peak levels, induced by direct or receptor-induced activation of AC, was followed by an increase in the extracellular cyclic AMP. (6) This effect was independent on PDEs, since it was obtained in the presence of 3-isobutyl-1-methylxanthine (IBMX). (7) Besides, in isoprenaline treated cells, the beta-adrenoceptor antagonist propranolol reduced both intra- and extracellular accumulation of cyclic AMP, whereas the organic anion transporter inhibitor probenecid reduced exclusively the extracellular accumulation. (8) Together our data show that direct or receptor-dependent activation of skeletal muscle AC results in a transient increase in the intracellular cyclic AMP, despite the continuous presence of the stimulus. The temporal declining of intracellular cyclic AMP was not dependent on the cyclic AMP breakdown but associated to the efflux of cyclic nucleotide to the extracellular compartment, by an active transport since it was prevented by probenecid. (+info)
Phosphorylation of the heat shock-related protein, HSP20, mediates cyclic nucleotide-dependent relaxation.
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Cyclic nucleotide-dependent relaxation of vascular smooth muscle is associated with increases in the phosphorylation of the small heat shock-related protein, HSP20. To determine whether phosphorylated HSP20 directly mediates relaxation, we used gene transfection and protein transduction of HSP20 analogues. Rat mesangial cells were transfected with constructs containing wild-type HSP20-enhanced green fluorescent protein (EGFP), phosphorylation site mutated HSP20 (S16A-HSP20-EGFP), or EGFP alone. Contractile properties were determined on a silicone polymer substrata. In the presence of serum, EGFP-vector transfected control cells and S16A-HSP20 transfected cells formed wrinkles on the polymer (contracted). Activation of cyclic nucleotide signaling pathways in the EGFP-vector transfected control cells led to a time-dependent decrease in the wrinkles (relaxation). The S16A-HSP20 transfected cells were refractory to cyclic nucleotide-dependent relaxation. Cells overexpressing the wild-type HSP20 did not form wrinkles on the polymer in response to serum (refractory to contraction). Treatment of precontracted strips of intact bovine carotid artery smooth muscle with synthetic peptides containing HIV-trans-activating transcriptional activator and a phosphopeptide motif of HSP20 led to dose-dependent relaxation. These data provide evidence that phosphorylated HSP20 has a direct role in smooth muscle relaxation and that small phosphopeptide motifs of HSP20 can mimic the effects of the entire molecule. (+info)
Connecting the eye to the brain: the molecular basis of ganglion cell axon guidance.
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In the past several years, a great deal has been learnt about the molecular basis through which specific neural pathways in the visual system are established during embryonic development. This review provides a framework for understanding the principles of retinal ganglion cell axon guidance, and introduces some of the families of axon guidance molecules involved. In addition, the potential relevance of retinal axon guidance to human visual developmental disorders, and to retinal axon regeneration, is discussed. (+info)
Heat induced HSP20 phosphorylation without increased cyclic nucleotide levels in swine carotid media.
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BACKGROUND: Heat pretreatment of swine carotid artery has been shown to increase ser16-heat shock protein 20 (HSP20) phosphorylation and suppress force, i.e., reduce force with only minimal reduction in ser19-myosin regulatory light chain (MRLC) phosphorylation. RESULTS: We further investigated this response in intact histamine stimulated swine carotid artery rings. There was a heat threshold such that increased ser16-HSP20 phosphorylation and force suppression were observed between 43 degrees C and 46 degrees C. The increased ser16-HSP20 phosphorylation persisted up to 16 hours after 44.5 degrees C heat treatment. Pretreatment of swine carotid media at 44.5 degrees C increased ser16-HSP20 phosphorylation without increases in [cAMP] or [cGMP], suggesting an alternate mechanism, perhaps phosphatase inhibition, for the increase in ser16-HSP20 phosphorylation. Heat pretreatment at 47.5 degrees C reduced force by decreasing MRLC phosphorylation rather than by large increases in ser16-HSP20 phosphorylation. HSP20 phosphorylation at the putative PKC site did not change with any treatment. CONCLUSION: These results demonstrate that multiple mechanisms can induce force suppression that is correlated with ser16-HSP20 phosphorylation: 1) nitrovasodilators via cGMP, 2) forskolin via cAMP, and 2) thermal stress in a cyclic nucleotide independent manner. (+info)
Transduction of biologically active motifs of the small heat shock-related protein HSP20 leads to relaxation of vascular smooth muscle.
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Activation of cyclic nucleotide-dependent signaling pathways leads to phosphorylation of the small heat shock-related protein, HSP20, on serine 16, and relaxation of vascular smooth muscle. In this study, we used an enhanced protein transduction domain (PTD) sequence to deliver HSP20 phosphopeptide analogs into porcine coronary artery. The transduction of phosphoHSP20 analogs led to dose-dependent relaxation of coronary artery smooth muscle. Peptides containing the protein transduction domain coupled to a random orientation of the same amino acids did not. Direct fluorescence microscopy of arterial rings incubated with fluorescein isothiocyanate (FITC)-PTD or FITC-PTD-HSP20 peptides showed a diffuse peptide uptake. Mass spectrometric immunoassays (MSIAs) of smooth muscle homogenates were used to determine whether the phosphopeptide analogs affected the phosphorylation of endogenous HSP20. Treatment with the phosphodiesterase inhibitor papaverine led to a mass shift of 80 Da. However, there was no mass shift of HSP20 in muscles treated with phosphoHSP20 analogs. This suggests that the PTD-phosphoHSP20 peptide alone is sufficient to inhibit force maintenance and likely has a direct effect on the target of phosphorylated HSP20. These results suggest that transduction of phosphopeptide analogs of HSP20 directly alters physiological responses of intact muscles. The data also support a direct role for phosphorylated HSP20 in mediating vasorelaxation. (+info)
Nitric oxide and cyclic nucleotides are regulators of neuronal migration in an insect embryo.
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The dynamic regulation of nitric oxide synthase (NOS) activity and cGMP levels suggests a functional role in the development of nervous systems. We report evidence for a key role of the NO/cGMP signalling cascade on migration of postmitotic neurons in the enteric nervous system of the embryonic grasshopper. During embryonic development, a population of enteric neurons migrates several hundred micrometers on the surface of the midgut. These midgut neurons (MG neurons) exhibit nitric oxide-induced cGMP-immunoreactivity coinciding with the migratory phase. Using a histochemical marker for NOS, we identified potential sources of NO in subsets of the midgut cells below the migrating MG neurons. Pharmacological inhibition of endogenous NOS, soluble guanylyl cyclase (sGC) and protein kinase G (PKG) activity in whole embryo culture significantly blocks MG neuron migration. This pharmacological inhibition can be rescued by supplementing with protoporphyrin IX free acid, an activator of sGC, and membrane-permeant cGMP, indicating that NO/cGMP signalling is essential for MG neuron migration. Conversely, the stimulation of the cAMP/protein kinase A signalling cascade results in an inhibition of cell migration. Activation of either the cGMP or the cAMP cascade influences the cellular distribution of F-actin in neuronal somata in a complementary fashion. The cytochemical stainings and experimental manipulations of cyclic nucleotide levels provide clear evidence that NO/cGMP/PKG signalling is permissive for MG neuron migration, whereas the cAMP/PKA cascade may be a negative regulator. These findings reveal an accessible invertebrate model in which the role of the NO and cyclic nucleotide signalling in neuronal migration can be analyzed in a natural setting. (+info)
Disruption of an intersubunit interaction underlies Ca2+-calmodulin modulation of cyclic nucleotide-gated channels.
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Cyclic nucleotide-gated channels are key molecular elements for olfactory transduction. Olfactory adaptation caused by repeated exposure to an odorant has been proposed to be mediated by the binding of Ca2+-calmodulin to the NH2-terminal domain of the channel, breaking its interaction with the COOH-terminal domain and downregulating the channel. We used a fluorescence resonance energy transfer (FRET) approach to study the structural aspects of this domain-domain interaction under physiological conditions in real time. Fluorescent proteins enhanced cyan fluorescent protein and enhanced yellow fluorescent protein were genetically attached at sites adjacent to the NH2- and COOH-terminal interacting domains, respectively, allowing direct observation of molecular rearrangements in intact channels. FRET signals caused by the specific interdomain interaction were observed in both intact cells and excised patches. Comparison of the effective FRET efficiencies demonstrated that the interaction occurs specifically between subunits but not within the same subunit. Binding of Ca2+-calmodulin caused a reversible decrease in FRET with the same time course as channel downregulation. These results suggest that a separation or reorientation of the interacting domains between subunits by Ca2+-calmodulin leads to channel downregulation. The quaternary arrangement presents a structural framework for understanding the molecular mechanism of olfactory adaptation. (+info)