Calcium-Dependent Increases in Protein Kinase-A Activity in Mouse Retinal Ganglion Cells Are Mediated by Multiple Adenylate Cyclases

Neurons undergo long term, activity dependent changes that are mediated by activation of second messenger cascades. In particular, calcium-dependent activation of the cyclic-AMP/Protein kinase A signaling cascade has been implicated in several developmental processes including cell survival, axonal outgrowth, and axonal refinement. The biochemical link between calcium influx and the activation of the cAMP/PKA pathway is primarily mediated through adenylate cyclases. Here, dual imaging of intracellular calcium concentration and PKA activity was used to assay the role of different classes of calcium-dependent adenylate cyclases (ACs) in the activation of the cAMP/PKA pathway in retinal ganglion cells (RGCs). Surprisingly, depolarization-induced calcium-dependent PKA transients persist in barrelless mice lacking AC1, the predominant calcium-dependent adenylate cyclase in RGCs, as well as in double knockout mice lacking both AC1 and AC8. Furthermore, in a subset of RGCs, depolarization-induced PKA transients persist during the inhibition of all transmembrane adenylate cyclases. These results are consistent with the existence of a soluble adenylate cyclase that plays a role in calcium-dependent activation of the cAMP/PKA cascade in neurons.     

The GAR-3 muscarinic receptor cooperates with calcium signals to regulate muscle contraction in the Caenorhabditis elegans pharynx

Muscarinic acetylcholine receptors regulate the activity of neurons and muscle cells through G-protein-coupled cascades. Here, we identify a pathway through which the GAR-3 muscarinic receptor regulates both membrane potential and excitation-contraction coupling in the Caenorhabditis elegans pharyngeal muscle. GAR-3 signaling is enhanced in worms overexpressing gar-3 or lacking GPB-2, a G-protein beta-subunit involved in RGS-mediated inhibition of G(o)alpha- and G(q)alpha-linked pathways. High levels of signaling through GAR-3 inhibit pharyngeal muscle relaxation and impair feeding--but do not block muscle repolarization--when worms are exposed to arecoline, a muscarinic agonist. Loss of gar-3 function results in shortened action potentials and brief muscle contractions in the pharyngeal terminal bulb. High levels of calcium entry through voltage-gated channels also impair terminal bulb relaxation and sensitize worms to the toxic effects of arecoline. Mutation of gar-3 reverses this sensitivity, suggesting that GAR-3 regulates calcium influx or calcium-dependent processes. Because the effects of GAR-3 signaling on membrane depolarization and muscle contraction can be separated, we conclude that GAR-3 regulates multiple calcium-dependent processes in the C. elegans pharyngeal muscle.     

A putative cation channel, NCA-1, and a novel protein, UNC-80, transmit neuronal activity in C. elegans

Voltage-gated cation channels regulate neuronal excitability through selective ion flux. NALCN, a member of a protein family that is structurally related to the α1 subunits of voltage-gated sodium/calcium channels, was recently shown to regulate the resting membrane potentials by mediating sodium leak and the firing of mouse neurons. We identified a role for the Caenorhabditis elegans NALCN homologues NCA-1 and NCA-2 in the propagation of neuronal activity from cell bodies to synapses. Loss of NCA activities leads to reduced synaptic transmission at neuromuscular junctions and frequent halting in locomotion. In vivo calcium imaging experiments further indicate that while calcium influx in the cell bodies of egg-laying motorneurons is unaffected by altered NCA activity, synaptic calcium transients are significantly reduced in nca loss-of-function mutants and increased in nca gain-of-function mutants. NCA-1 localizes along axons and is enriched at nonsynaptic regions. Its localization and function depend on UNC-79, and UNC-80, a novel conserved protein that is also enriched at nonsynaptic regions. We propose that NCA-1 and UNC-80 regulate neuronal activity at least in part by transmitting depolarization signals to synapses in C. elegans neurons.     

Effects of voltage‐gated calcium channel subunit genes on calcium influx in cultured C. elegans mechanosensory neurons

Voltage‐gated calcium channels (VGCCs) serve as a critical link between electrical signaling and diverse cellular processes in neurons. We have exploited recent advances in genetically encoded calcium sensors and in culture techniques to investigate how the VGCC α₁ subunit EGL‐19 and α₂/δ subunit UNC‐36 affect the functional properties of C. elegans mechanosensory neurons. Using the protein‐based optical indicator cameleon, we recorded calcium transients from cultured mechanosensory neurons in response to transient depolarization. We observed that in these cultured cells, calcium transients induced by extracellular potassium were significantly reduced by a reduction‐of‐function mutation in egl‐19 and significantly reduced by L‐type calcium channel inhibitors; thus, a main source of touch neuron calcium transients appeared to be influx of extracellular calcium through L‐type channels. Transients did not depend directly on intracellular calcium stores, although a store‐independent 2‐APB and gadolinium‐sensitive calcium flux was detected. The transients were also significantly reduced by mutations in unc‐36, which encodes the main neuronal α₂/δ subunit in C. elegans. Interestingly, while egl‐19 mutations resulted in similar reductions in calcium influx at all stimulus strengths, unc‐36 mutations preferentially affected responses to smaller depolarizations. These experiments suggest a central role for EGL‐19 and UNC‐36 in excitability and functional activity of the mechanosensory neurons. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Minimal Change in the Cytoplasmic Calcium Dynamics in Striatal GABAergic Neurons of a DYT1 Dystonia Knock-In Mouse Model

DYT1 dystonia is the most common hereditary form of primary torsion dystonia. This autosomal-dominant disorder is characterized by involuntary muscle contractions that cause sustained twisting and repetitive movements. It is caused by an in-frame deletion in the TOR1A gene, leading to the deletion of a glutamic acid residue in the torsinA protein. Heterozygous knock-in mice, which reproduce the genetic mutation in human patients, have abnormalities in synaptic transmission at the principal GABAergic neurons in the striatum, a brain structure that is involved in the execution and modulation of motor activity. However, whether this mutation affects the excitability of striatal GABAergic neurons has not been investigated in this animal model. Here, we examined the excitability of cultured striatal neurons obtained from heterozygous knock-in mice, using calcium imaging as indirect readout. Immunofluorescence revealed that more than 97% of these neurons are positive for a marker of GABAergic neurons, and that more than 92% are also positive for a marker of medium spiny neurons, indicating that these are mixed cultures of mostly medium spiny neurons and a few (~5%) GABAergic interneurons. When these neurons were depolarized by field stimulation, the calcium concentration in the dendrites increased rapidly and then decayed slowly. The amplitudes of calcium transients were larger in heterozygous neurons than in wild-type neurons, resulting in ~15% increase in cumulative calcium transients during a train of stimuli. However, there was no change in other parameters of calcium dynamics. Given that calcium dynamics reflect neuronal excitability, these results suggest that the mutation only slightly increases the excitability of striatal GABAergic neurons in DYT1 dystonia.     

The mAKAP signaling complex: integration of cAMP, calcium, and MAP kinase signaling pathways

Following its production by adenylyl cyclases, the second messenger cAMP is in involved in pleiotrophic signal transduction. The effectors of cAMP include the cAMP-dependent protein kinase (PKA), the guanine nucleotide exchange factor Epac (exchange protein activated by cAMP), and cAMP-dependent ion channels. In turn, cAMP signaling is attenuated by phosphodiesterase-catalyzed degradation. The association of cAMP effectors and the enzymes that regulate cAMP concentration into signaling complexes helps to explain the differential signaling initiated by members of the G(s)-protein coupled receptor family. The signal transduction complex formed by the scaffold protein mAKAP (muscle A kinase-anchoring protein) at the nuclear envelope of both striated myocytes and neurons contains three cAMP-binding proteins, PKA, Epac1, and the phosphodiesterase PDE4D3. In addition, the mAKAP complex also contains components of the ERK5 MAP kinase signaling pathway, the calcium release channel ryanodine receptor and the phosphatases PP2A as well as calcineurin. Analysis of the mAKAP complex illustrates how a macromolecular complex can serve as a node in the intracellular signaling network of cardiac myocytes to integrate multiple cAMP signals with those of calcium and MAP kinases to regulate the hypertrophic actions of several hormones.     

A Putative Cation Channel, NCA-1, and a Novel Protein, UNC-80, Transmit Neuronal Activity in C. elegans

Voltage-gated cation channels regulate neuronal excitability through selective ion flux. NALCN, a member of a protein family that is structurally related to the α1 subunits of voltage-gated sodium/calcium channels, was recently shown to regulate the resting membrane potentials by mediating sodium leak and the firing of mouse neurons. We identified a role for the Caenorhabditis elegans NALCN homologues NCA-1 and NCA-2 in the propagation of neuronal activity from cell bodies to synapses. Loss of NCA activities leads to reduced synaptic transmission at neuromuscular junctions and frequent halting in locomotion. In vivo calcium imaging experiments further indicate that while calcium influx in the cell bodies of egg-laying motorneurons is unaffected by altered NCA activity, synaptic calcium transients are significantly reduced in nca loss-of-function mutants and increased in nca gain-of-function mutants. NCA-1 localizes along axons and is enriched at nonsynaptic regions. Its localization and function depend on UNC-79, and UNC-80, a novel conserved protein that is also enriched at nonsynaptic regions. We propose that NCA-1 and UNC-80 regulate neuronal activity at least in part by transmitting depolarization signals to synapses in C. elegans neurons.     

VIP and PACAP regulate localized Ca2+ transients via cAMP-dependent mechanism

Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) have been suggested as participants in enteric inhibitory neural regulation of gastrointestinal motility. These peptides cause a variety of postjunctional responses including membrane hyperpolarization and inhibition of contraction. Neuropeptides released from enteric motor neurons can elicit responses by direct stimulation of smooth muscle cells as opposed to other transmitters that rely on synapses between motor nerve terminals and interstitial cells of Cajal. Therefore, we studied the responses of murine colonic smooth muscle cells to VIP and PACAP(1–38) with confocal microscopy and patch-clamp technique. Localized Ca²⁺ transients (Ca²⁺ puffs) were observed in colonic myocytes, and these events coupled to spontaneous transient outward currents (STOCs). VIP and PACAP increased Ca²⁺ transients and STOC frequency and amplitude. Application of dibutyryl cAMP had similar effects. The adenylyl cyclase blocker MDL-12,330A alone did not affect spontaneous Ca²⁺ puffs and STOCs but prevented responses to VIP. Disruption of A-kinase-anchoring protein (AKAP) associations by application of AKAP St-Ht31 inhibitory peptide had effects similar to those of MDL-12,330A. Inhibition of ryanodine receptor channels did not block spontaneous Ca²⁺ puffs and STOCs but prevented the effects of dibutyryl cAMP. These findings suggest that regulation of Ca²⁺ transients (which couple to activation of STOCs) may contribute to the inhibitory effects of VIP and PACAP. Regulation of Ca²⁺ transients by VIP and PACAP occurs via adenylyl cyclase, increased synthesis of cAMP, and PKA-dependent regulation of ryanodine receptor channels. calcium puffs; ryanodine receptor channels; enteric nervous system; gastrointestinal motility     

Functional alterations in gut contractility after connexin36 ablation and evidence for gap junctions forming electrical synapses between nitrergic enteric neurons

Neurons in the enteric nervous system utilize numerous neurotransmitters to orchestrate rhythmic gut smooth muscle contractions. We examined whether electrical synapses formed by gap junctions containing connexin36 also contribute to communication between enteric neurons in mouse colon. Spontaneous contractility properties and responses to electrical field stimulation and cholinergic agonist were altered in gut from connexin36 knockout vs. wild-type mice. Immunofluorescence revealed punctate labelling of connexin36 that was localized at appositions between somata of enteric neurons immunopositive for the enzyme nitric oxide synthase. There is indication for a possible functional role of gap junctions between inhibitory nitrergic enteric neurons.     

Molecular mechanisms of pituitary endocrine cell calcium handling

Endocrine pituitary cells express numerous voltage-gated Na(+), Ca(2+), K(+), and Cl(-) channels and several ligand-gated channels, and they fire action potentials spontaneously. Depending on the cell type, this electrical activity can generate localized or global Ca(2+) signals, the latter reaching the threshold for stimulus-secretion coupling. These cells also express numerous G-protein-coupled receptors, which can stimulate or silence electrical activity and Ca(2+) influx through voltage-gated Ca(2+) channels and hormone release. Receptors positively coupled to the adenylyl cyclase signaling pathway stimulate electrical activity with cAMP, which activates hyperpolarization-activated cyclic nucleotide-regulated channels directly, or by cAMP-dependent kinase-mediated phosphorylation of K(+), Na(+), Ca(2+), and/or non-selective cation-conducting channels. Receptors that are negatively coupled to adenylyl cyclase signaling pathways inhibit spontaneous electrical activity and accompanied Ca(2+) transients predominantly through the activation of inwardly rectifying K(+) channels and the inhibition of voltage-gated Ca(2+) channels. The Ca(2+)-mobilizing receptors activate inositol trisphosphate-gated Ca(2+) channels in the endoplasmic reticulum, leading to Ca(2+) release in an oscillatory or non-oscillatory manner, depending on the cell type. This Ca(2+) release causes a cell type-specific modulation of electrical activity and intracellular Ca(2+) handling.     

Effects of voltage-gated calcium channel subunit genes on calcium influx in cultured C. elegans mechanosensory neurons

Voltage-gated calcium channels (VGCCs) serve as a critical link between electrical signaling and diverse cellular processes in neurons. We have exploited recent advances in genetically encoded calcium sensors and in culture techniques to investigate how the VGCC alpha1 subunit EGL-19 and alpha2/delta subunit UNC-36 affect the functional properties of C. elegans mechanosensory neurons. Using the protein-based optical indicator cameleon, we recorded calcium transients from cultured mechanosensory neurons in response to transient depolarization. We observed that in these cultured cells, calcium transients induced by extracellular potassium were significantly reduced by a reduction-of-function mutation in egl-19 and significantly reduced by L-type calcium channel inhibitors; thus, a main source of touch neuron calcium transients appeared to be influx of extracellular calcium through L-type channels. Transients did not depend directly on intracellular calcium stores, although a store-independent 2-APB and gadolinium-sensitive calcium flux was detected. The transients were also significantly reduced by mutations in unc-36, which encodes the main neuronal alpha2/delta subunit in C. elegans. Interestingly, while egl-19 mutations resulted in similar reductions in calcium influx at all stimulus strengths, unc-36 mutations preferentially affected responses to smaller depolarizations. These experiments suggest a central role for EGL-19 and UNC-36 in excitability and functional activity of the mechanosensory neurons.     

Increase of cytosolic calcium induced by trichosanthin suppresses cAMP/PKC levels through the inhibition of adenylyl cyclase activity in HeLa cells

Increase of cytosolic free calcium played a pivotal role in apoptotic cells induced by trichosanthin. However, little is known about the influence of cytosolic calcium increase on adenylyl cyclase activity and intracellular cAMP signaling pathway in HeLa cells. The present study showed that an influx of extracellular Ca²⁺ initiated by trichosanthin was required for the suppression of adenylyl cyclase activity and decrease of intracellular cAMP level. Furthermore, this inhibition was abolished by activation of PKC rather than PKA. Therefore, our results suggested that increase of cytosolic calcium induced by trichosanthin inhibits cAMP levels via suppression of adenylyl cyclase activity.     

Signaling pathway underlying the octopaminergic modulation of myogenic contraction in the cricket lateral oviduct

Octopamine (OA), a biogenic monoamine, is a neurotransmitter and neuromodulator in invertebrates. Here, we report the effect of OA on the spontaneous rhythmic contractions (SRCs) of the lateral oviduct of the cricket Gryllus bimaculatus and the possible signaling pathway involved. Application of OA increased both the frequency and amplitude of SRCs in a dose-dependent manner. The effect of OA was inhibited by subsequent application of the OA receptor antagonist epinastine, indicating that the action of OA is mediated by OA receptor. To investigate the predominant signaling pathway underlying the action of OA, we first examined a possible involvement of the cAMP/cAMP-dependent protein kinase A (PKA) signaling pathway. Application of the membrane-permeable cAMP analog 8-Br-cAMP had little effect on SRCs and the effect of OA was not influenced by subsequent application of the PKA inhibitor H89, indicating that the cAMP/PKA signaling pathway is not the predominant pathway in the action of OA. Next, we examined a possible involvement of the second messenger inositol 1,4,5-trisphosphate in the action of OA. The effect of OA on SRCs was inhibited by subsequent application of the phosphoinositide-specific phospholipase C (PLC) inhibitor U73122, indicating that the PLC pathway is involved in the action of OA. The OA-induced increase in the frequency of SRCs was inhibited by pretreatment of the cell with the ryanodine receptor antagonist tetracaine but was not significantly affected by the IP₃ receptor antagonist 2-aminoethoxydiphenyl borate (2-APB). On the other hand, the OA-induced increase in the amplitude of SRCs was inhibited by pretreatment of the cells with 2-APB but was not significantly affected by tetracaine. Taken together, these results suggest that the OA-induced excitatory effect on SRCs is mediated by the PLC signaling pathway: Ca²⁺ release from IP₃ receptors may contribute to the modulation of the amplitude of SRCs, whereas Ca²⁺ release from ryanodine receptors may contribute to the modulation of the frequency of SRCs.     

Regulation of distinct muscle behaviors controls the C. elegans male's copulatory spicules during mating.

We demonstrate through cell ablation, molecular genetic, and pharmacological approaches that during C. elegans male mating behavior, the male inserts his copulatory spicules into the hermaphrodite by regulating periodic and prolonged spicule muscle contractions. Distinct cholinergic neurons use different ACh receptors and calcium channels in the spicule muscles to mediate these contractile behaviors. The PCB and PCC sensory neurons facilitate periodic contraction through muscle-encoded UNC-68 ryanodine receptor calcium channels. The SPC motor neurons trigger prolonged contraction through EGL-19 L-type voltage-gated calcium channels. The male gonad then lengthens the duration of EGL-19-mediated prolonged muscle contraction. This regulation of muscle contraction provides a paradigm to explain how animals initiate, monitor, and maintain a behavioral motor program.     

Ca2+ channel-sarcoplasmic reticulum coupling: a mechanism of arterial myocyte contraction without Ca2+ influx

Contraction of vascular smooth muscle cells (VSMCs) depends on the rise of cytosolic [Ca2+] owing to either Ca2+ influx through voltage-gated Ca2+ channels of the plasmalemma or receptor-mediated Ca2+ release from the sarcoplasmic reticulum (SR). We show that voltage-gated Ca2+ channels in arterial myocytes mediate fast Ca2+ release from the SR and contraction without the need of Ca2+ influx. After sensing membrane depolarization, Ca2+ channels activate G proteins and the phospholipase C-inositol 1,4,5-trisphosphate (InsP3) pathway. Ca2+ released through InsP3-dependent channels of the SR activates ryanodine receptors to amplify the cytosolic Ca2+ signal. These observations demonstrate a new mechanism of signaling SR Ca(2+)-release channels and reveal an unexpected function of voltage-gated Ca2+ channels in arterial myocytes. Our findings may have therapeutic implications as the calcium-channel-induced Ca2+ release from the SR can be suppressed by Ca(2+)-channel antagonists.     

ZD7288 inhibits exocytosis in an HCN-independent manner and downstream of voltage-gated calcium influx in pituitary lactotrophs

Pituitary lactotrophs fire action potentials spontaneously and the associated voltage-gated calcium influx is sufficient to maintain high prolactin release. Here we studied the role of hyperpolarization-activated cation channels in pacemaking activity, calcium signaling, and prolactin secretion in these cells. A slowly developing and hyperpolarization-activated inward current was identified but only in a fraction of lactotrophs. The current was blocked by ZD7288, a relatively specific blocker of these channels. However, the pacemaking activity increased in ZD7288-treated cells independently of the presence of this current. This in turn facilitated voltage-gated calcium influx and transiently stimulated prolactin secretion. Sustained ZD7288 application in concentrations that are commonly used to block the hyperpolarization-activated cation channels inhibited hormone release at elevated intracellular calcium concentrations. Agonist and Bay K 8644-stimulated prolactin release was also inhibited by ZD7288, indicating that this compound attenuates the exocytotic pathway downstream of calcium influx.     

Acetylcholine-induced contractions in a holothurian (Isostichopus badionotus) smooth muscle are blocked by the calcium antagonists,diltiazem and verapamil

1. The longitudinal muscle of the body wall (LMBW) of the holothurian, Isostichopus badionotus contracted when treated with acetylcholine (ACh). The threshold concentration for initiating a contraction was 10⁻⁸M ACh.2. Inward calcium (Ca²⁺) current blockers, diltiazem and verapamil, blocked contractions induced by ACh suggesting that Ca²⁺ channels are involved. Verapamil caused small rhythmic contractions to occur in some muscle preparations.3. Caffeine initiated contractions only at the high concentration of 10 mM and caused rhythmic contractions in otherwise non-spontaneously beating muscle. The caffeine-contractions were partially blocked by verapamil.