scholarly journals Activation of MEK/ERK signaling contributes to the PACAP-induced increase in guinea pig cardiac neuron excitability

2016 ◽  
Vol 311 (4) ◽  
pp. C643-C651 ◽  
Author(s):  
John D. Tompkins ◽  
Todd A. Clason ◽  
Jean C. Hardwick ◽  
Beatrice M. Girard ◽  
Laura A. Merriam ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Neuronal Excitability ◽  
Low Voltage ◽  
Ionic Currents ◽  
Erk Signaling ◽  
Repetitive Firing ◽  
Neuronal System ◽  
Cellular Mechanisms ◽  
Pac1 Receptor ◽  
Neuron Excitability

Pituitary adenylate cyclase (PAC)-activating polypeptide (PACAP) peptides ( Adcyap1) signaling at the selective PAC1 receptor ( Adcyap1r1) participate in multiple homeostatic and stress-related responses, yet the cellular mechanisms underlying PACAP actions remain to be completely elucidated. PACAP/PAC1 receptor signaling increases excitability of neurons within the guinea pig cardiac ganglia, and as these neurons are readily accessible, this neuronal system is particularly amenable to study of PACAP modulation of ionic conductances. The present study investigated how PACAP activation of MEK/ERK signaling contributed to the peptide-induced increase in cardiac neuron excitability. Treatment with the MEK inhibitor PD 98059 blocked PACAP-stimulated phosphorylated ERK and, in parallel, suppressed the increase in cardiac neuron excitability. However, PD 98059 did not blunt the ability of PACAP to enhance two inward ionic currents, one flowing through hyperpolarization-activated nonselective cationic channels ( Ih) and another flowing through low-voltage-activated calcium channels ( IT), which support the peptide-induced increase in excitability. Thus a PACAP - and MEK/ERK-sensitive, voltage-dependent conductance(s), in addition to Ih and IT, modulates neuronal excitability. Despite prior work implicating PACAP downregulation of the KV4.2 potassium channel in modulation of excitability in other cells, treatment with the KV4.2 current blocker 4-aminopyridine did not replicate the PACAP-induced increase in excitability in cardiac neurons. However, cardiac neurons express the ERK target, the NaV1.7 sodium channel, and treatment with the selective NaV1.7 channel inhibitor PF-04856264 decreased the PACAP modulation of excitability. From these results, PACAP/PAC1 activation of MEK/ERK signaling may phosphorylate the NaV1.7 channel, enhancing sodium currents near the threshold, an action contributing to repetitive firing of the cardiac neurons exposed to PACAP.

scholarly journals Recruitment of endosomal signaling mediates the forskolin modulation of guinea pig cardiac neuron excitability

2017 ◽  
Vol 313 (2) ◽  
pp. C219-C227 ◽  
Author(s):  
Jean C. Hardwick ◽  
Todd A. Clason ◽  
John D. Tompkins ◽  
Beatrice M. Girard ◽  
Caitlin N. Baran ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Neuronal Excitability ◽  
Receptor Activation ◽  
Erk Signaling ◽  
Pac1 Receptor ◽  
Signaling Mechanism ◽  
Erk Activation ◽  
Neuron Excitability ◽  
Endosomal Signaling ◽  
Gated Channel

Forskolin, a selective activator of adenylyl cyclase (AC), commonly is used to establish actions of G protein-coupled receptors (GPCRs) that are initiated primarily through activation of AC/cAMP signaling pathways. In the present study, forskolin was used to evaluate the potential role of AC/cAMP, which is a major signaling mechanism for the pituitary adenylate cyclase-activating polypeptide (PACAP)-selective PAC1 receptor, in the regulation of guinea pig cardiac neuronal excitability. Forskolin (5–10 µM) increases excitability in ~60% of the cardiac neurons. The forskolin-mediated increase in excitability was considered related to cAMP regulation of a cyclic nucleotide gated channel or via protein kinase A (PKA)/ERK signaling, mechanisms that have been linked to PAC1 receptor activation. However, unlike PACAP mechanisms, forskolin enhancement of excitability was not significantly reduced by treatment with cesium to block currents through hyperpolarization-activated nonselective cation channels ( Ih) or by treatment with PD98059 to block MEK/ERK signaling. In contrast, treatment with the clathrin inhibitor Pitstop2 or the dynamin inhibitor dynasore eliminated the forskolin-induced increase in excitability; treatments with the inactive Pitstop analog or PP2 treatment to inhibit Src-mediated endocytosis mechanisms were ineffective. The PKA inhibitor KT5702 significantly suppressed the forskolin-induced change in excitability; further, KT5702 and Pitstop2 reduced the forskolin-stimulated MEK/ERK activation in cardiac neurons. Collectively, the present results suggest that forskolin activation of AC/cAMP/PKA signaling leads to the recruitment of clathrin/dynamin-dependent endosomal transduction cascades, including MEK/ERK signaling, and that endosomal signaling is the critical mechanism underlying the forskolin-induced increase in cardiac neuron excitability.

scholarly journals Nickel suppresses the PACAP-induced increase in guinea pig cardiac neuron excitability

2015 ◽  
Vol 308 (11) ◽  
pp. C857-C866 ◽  
Author(s):  
John D. Tompkins ◽  
Laura A. Merriam ◽  
Beatrice M. Girard ◽  
Victor May ◽  
Rodney L. Parsons
Keyword(s):  
Guinea Pig ◽  
Neuronal Excitability ◽  
Low Voltage ◽  
Calcium Influx ◽  
Quantitative Polymerase Chain Reaction ◽  
Intercellular Signaling ◽  
Neuron Excitability ◽  
Unique System ◽  
Cell Recording ◽  
Recording Conditions

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a potent intercellular signaling molecule involved in multiple homeostatic functions. PACAP/PAC1 receptor signaling increases excitability of neurons within the guinea pig cardiac ganglia, making them a unique system to establish mechanisms underlying PACAP modulation of neuronal function. Calcium influx is required for the PACAP-increased cardiac neuron excitability, although the pathway is unknown. This study tested whether PACAP enhancement of calcium influx through either T-type or R-type channels contributed to the modulation of excitability. Real-time quantitative polymerase chain reaction analyses indicated transcripts for Cav3.1, Cav3.2, and Cav3.3 T-type isoforms and R-type Cav2.3 in cardiac neurons. These neurons often exhibit a hyperpolarization-induced rebound depolarization that remains when cesium is present to block hyperpolarization-activated nonselective cationic currents ( Ih). The T-type calcium channel inhibitors, nickel (Ni2+) or mibefradil, suppressed the rebound depolarization, and treatment with both drugs hyperpolarized cardiac neurons by 2–4 mV. Together, these results are consistent with the presence of functional T-type channels, potentially along with R-type channels, in these cardiac neurons. Fifty micromolar Ni2+, a concentration that suppresses currents in both T-type and R-type channels, blunted the PACAP-initiated increase in excitability. Ni2+ also blunted PACAP enhancement of the hyperpolarization-induced rebound depolarization and reversed the PACAP-mediated increase in excitability, after being initiated, in a subset of cells. Lastly, low voltage-activated currents, measured under perforated patch whole cell recording conditions and potentially flowing through T-type or R-type channels, were enhanced by PACAP. Together, our results suggest that a PACAP-enhanced, Ni2+-sensitive current contributes to PACAP-induced modulation of neuronal excitability.

scholarly journals Src family kinase inhibitors blunt PACAP-induced PAC1 receptor endocytosis, phosphorylation of ERK, and the increase in cardiac neuron excitability

2018 ◽  
Vol 314 (2) ◽  
pp. C233-C241 ◽  
Author(s):  
John D. Tompkins ◽  
Todd A. Clason ◽  
Thomas R. Buttolph ◽  
Beatrice M. Girard ◽  
Anne K. Linden ◽  
...  
Keyword(s):  
G Protein ◽  
Neuronal Excitability ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Src Family Kinases ◽  
Receptor Internalization ◽  
Src Family Kinase ◽  
Pac1 Receptor ◽  
Neuron Excitability ◽  
Endosomal Signaling

Pituitary adenylate cyclase activating polypeptide (PACAP, Adcyap1) activation of PAC1 receptors ( Adcyap1r1) significantly increases excitability of guinea pig cardiac neurons. This modulation of excitability is mediated in part by plasma membrane G protein-dependent activation of adenylyl cyclase and downstream signaling cascades. However, additional mechanisms responsible for the enhanced excitability are activated following internalization of the PAC1 receptor and endosomal signaling. Src family kinases play critical roles mediating endocytosis of many trophic factor and G protein-coupled receptors. The present study investigated whether Src family kinases also support the PACAP-induced PAC1 receptor internalization, phosphorylation of ERK, and enhanced neuronal excitability. Using human embryonic kidney cells stably expressing a green fluorescent protein-tagged PAC1 receptor, treatment with the Src family kinase inhibitor PP2 (10 µM) markedly reduced the PACAP-induced PAC1 receptor internalization, and in parallel, both PP2 and Src inhibitor 1 (Src-1, 2 µM) reduced ERK activation determined by Western blot analysis. In contrast, Src family kinase inhibitors did not eliminate a PACAP-induced rise in global calcium generated by inositol (1,4,5)-trisphosphate-induced release of calcium from endoplasmic reticulum stores. From confocal analysis of phosphorylated ERK immunostaining, PP2 treatment significantly attenuated PACAP activation of ERK in neurons within cardiac ganglia whole mount preparations. Intracellular recordings demonstrated that PP2 also significantly blunted a PACAP-induced increase in cardiac neuron excitability. These studies demonstrate Src-related kinase activity in PAC1 receptor internalization, activation of MEK/ERK signaling, and regulation of neuronal excitability. The present results provide further support for the importance of PAC1 receptor endosomal signaling as a key mechanism regulating cellular function.

Ca2+-Activated K+-Current Density Is Correlated With Soma Size in Rat Vestibular-Afferent Neurons in Culture

2005 ◽  
Vol 94 (6) ◽  
pp. 3751-3761 ◽  
Author(s):  
Agenor Limón ◽  
Cristina Pérez ◽  
Rosario Vega ◽  
Enrique Soto
Keyword(s):  
Low Voltage ◽  
Ionic Currents ◽  
Vestibular Nuclei ◽  
Head Movements ◽  
Small Cells ◽  
Repetitive Firing ◽  
Afferent Neurons ◽  
Patch Clamp Technique ◽  
Soma Size ◽  
Discharge Patterns

Vestibular-afferent neurons (VANs) transmit information about linear and angular accelerations during head movements from vestibular end organs to vestibular nuclei. In situ, these neurons show heterogeneous discharge patterns that may be produced by differences in their intrinsic properties. However, little is known about the ionic currents underlying their different firing patterns. Using the whole cell patch-clamp technique, we analyzed the expression of Ca2+ and Ca2+-activated K+ currents ( IKCa) in primary cultured neurons isolated from young rats (p7–p10). We found two overlapping subpopulations of VANs classified according to low-threshold Ca2+-current [low-voltage–activated (LVA)] expression; LVA (−) neurons, formed by small cells, and LVA (+) neurons composed of medium to large cells. The IKCa in both cell-groups was carried through channels of high (BK), intermediate (IK), and low conductance (SK), besides a resistant channel to classical blockers (IR). BK was expressed preferentially in LVA (+) cells, whereas IR expression was preferentially in LVA (−) cells. No correlation between SK and IK expression with the soma size was found. Current-clamp experiments showed that BK participates in the adaptation of discharge and in the duration of the action potential, whereas SK and IK did not show a significant contribution to electrical discharge of cultured VANs. However, because of the low number of VANs in culture with repetitive firing it is difficult to interpret our results in terms of discharge patterns. Our results demonstrate that vestibular-afferent neurons possess different Ca2+-activated K+ (KCa) channels and that their expression, heterogeneous among the cells, would contribute to explain some of the differences in the electrical-firing properties of these neurons.

scholarly journals Novel neuronal and astrocytic mechanisms in thalamocortical loop dynamics

2002 ◽  
Vol 357 (1428) ◽  
pp. 1675-1693 ◽  
Author(s):  
Vincenzo Crunelli ◽  
Kate L. Blethyn ◽  
David W. Cope ◽  
Stuart W. Hughes ◽  
H. Rheinallt Parri ◽  
...  
Keyword(s):  
Low Voltage ◽  
Electrical Coupling ◽  
Low Frequency ◽  
Network Activity ◽  
Thalamic Neuron ◽  
Cellular Mechanisms ◽  
Brain Areas ◽  
Sensory Stimuli ◽  
Loop Dynamics ◽  
Intracellular Ca

In this review, we summarize three sets of findings that have recently been observed in thalamic astrocytes and neurons, and discuss their significance for thalamocortical loop dynamics. (i) A physiologically relevant ‘window’ component of the low–voltage–activated, T–type Ca 2+ current ( I Twindow ) plays an essential part in the slow (less than 1 Hz) sleep oscillation in adult thalamocortical (TC) neurons, indicating that the expression of this fundamental sleep rhythm in these neurons is not a simple reflection of cortical network activity. It is also likely that I Twindow underlies one of the cellular mechanisms enabling TC neurons to produce burst firing in response to novel sensory stimuli. (ii) Both electrophysiological and dye–injection experiments support the existence of gap junction–mediated coupling among young and adult TC neurons. This finding indicates that electrical coupling–mediated synchronization might be implicated in the high and low frequency oscillatory activities expressed by this type of thalamic neuron. (iii) Spontaneous intracellular Ca 2+ ([Ca 2+ ] i ) waves propagating among thalamic astrocytes are able to elicit large and long–lasting N –methyl–D–aspartate–mediated currents in TC neurons. The peculiar developmental profile within the first two postnatal weeks of these astrocytic [Ca 2+ ] i transients and the selective activation of these glutamate receptors point to a role for this astrocyte–to–neuron signalling mechanism in the topographic wiring of the thalamocortical loop. As some of these novel cellular and intracellular properties are not restricted to thalamic astrocytes and neurons, their significance may well apply to (patho)physiological functions of glial and neuronal elements in other brain areas.

scholarly journals Metabotropic glutamate receptor 5 regulates excitability and Kv4.2-containing K+ channels primarily in excitatory neurons of the spinal dorsal horn

2011 ◽  
Vol 105 (6) ◽  
pp. 3010-3021 ◽  
Author(s):  
Hui-Juan Hu ◽  
Robert W. Gereau
Keyword(s):  
Spinal Cord ◽  
Dorsal Horn ◽  
Spinal Dorsal Horn ◽  
Neuronal Excitability ◽  
Gabaergic Neurons ◽  
Erk Signaling ◽  
Metabotropic Glutamate ◽  
Dorsal Horn Neurons ◽  
Spinal Dorsal ◽  
Excitatory Neurons

Metabotropic glutamate (mGlu) receptors play important roles in the modulation of nociception. Previous studies demonstrated that mGlu5 modulates nociceptive plasticity via activation of ERK signaling. We have reported recently that the Kv4.2 K+ channel subunit underlies A-type currents in spinal cord dorsal horn neurons and that this channel is modulated by mGlu5-ERK signaling. In the present study, we tested the hypothesis that modulation of Kv4.2 by mGlu5 occurs in excitatory spinal dorsal horn neurons. With the use of a transgenic mouse strain expressing enhanced green fluorescent protein (GFP) under control of the promoter for the γ-amino butyric acid (GABA)-synthesizing enzyme, glutamic acid decarboxylase 67 (GAD67), we found that these GABAergic neurons express less Kv4.2-mediated A-type current than non-GAD67-GFP neurons. Furthermore, the mGlu1/5 agonist, (R,S)-3,5-dihydroxyphenylglycine, had no modulatory effects on A-type currents or neuronal excitability in this subgroup of GABAergic neurons but robustly modulated A-type currents and neuronal excitability in non-GFP-expressing neurons. Immunofluorescence studies revealed that Kv4.2 was highly colocalized with markers of excitatory neurons, such as vesicular glutamate transporter 1/2, PKCγ, and neurokinin 1, in cultured dorsal horn neurons. These results indicate that mGlu5-Kv4.2 signaling is associated with excitatory dorsal horn neurons and suggest that the pronociceptive effects of mGlu5 activation in the spinal cord likely involve enhanced excitability of excitatory neurons.

Low-Voltage Activated T-Type Calcium Currents Are Differently Expressed in Superficial and Deep Layers of Guinea PigPiriform Cortex

1998 ◽  
Vol 79 (2) ◽  
pp. 808-816 ◽  
Author(s):  
Jacopo Magistretti ◽  
Marco de Curtis
Keyword(s):  
Guinea Pig ◽  
Calcium Current ◽  
Low Voltage ◽  
Piriform Cortex ◽  
Neuronal Firing ◽  
Calcium Currents ◽  
Patch Clamp Technique ◽  
Voltage Range ◽  
Deep Layers ◽  
Group 2

Magistretti, Jacopo and Marco de Curtis. Low-voltage activated T-type calcium currents are differently expressed in superficial and deep layers of guinea pig piriform cortex. J. Neurophysiol. 79: 808–816, 1998. A variety of voltage-dependent calcium conductances are known to control neuronal excitability by boosting peripheral synaptic potentials and by shaping neuronal firing patterns. The existence and functional significance of a differential expression of low- and high-voltage activated (LVA and HVA, respectively) calcium currents in subpopulations of neurons, acutely isolated from different layers of the guinea pig piriform cortex, were investigated with the whole cell variant of the patch-clamp technique. Calcium currents were recorded from pyramidal and multipolar neurons dissociated from layers II, III, and IV. Average membrane capacitance was larger in layer IV cells [13.1 ± 6.2 (SD) pF] than in neurons from layers II and III (8.6 ± 2.8 and 7.9 ± 3.1 pF, respectively). Neurons from all layers showed HVA calcium currents with an activation voltage range positive to −40 mV. Neurons dissociated from layers III and IV showed an LVA calcium current with the biophysical properties of a T-type conductance. Such a current displayed the following characteristics: 1) showed maximal amplitude of 11–16 pA/pF at −30 mV, 2) inactivated rapidly with a time constant of ∼22 ms at −30 mV, and 3) was completely steady-state inactivated at −60 mV. Only a subpopulation of layer II neurons (group 2 cells; circa 18%) displayed an LVA calcium current similar to that observed in deep layers. The general properties of layer II-group 2 cells were otherwise identical to those of group 1 neurons. The present study demonstrates that LVA calcium currents are differentially expressed in neurons acutely dissociated from distinct layers of the guinea pig piriform cortex.

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