Laura A. Merriam

Pituitary Adenylate Cyclase 1 Receptor Internalization and Endosomal Signaling Mediate the Pituitary Adenylate Cyclase Activating Polypeptide-Induced Increase in Guinea Pig Cardiac Neuron Excitability

After G-protein-coupled receptor activation and signaling at the plasma membrane, the receptor complex is often rapidly internalized via endocytic vesicles for trafficking into various intracellular compartments and pathways. The formation of signaling endosomes is recognized as a mechanism that produces sustained intracellular signals that may be distinct from those generated at the cell surface for cellular responses including growth, differentiation, and survival. Pituitary adenylate cyclase activating polypeptide (PACAP; Adcyap1) is a potent neurotransmitter/neurotrophic peptide and mediates its diverse cellular functions in part through internalization of its cognate G-protein-coupled PAC1 receptor (PAC1R; Adcyap1r1). In the present study, we examined whether PAC1R endocytosis participates in the regulation of neuronal excitability. Although PACAP increased excitability in 90% of guinea pig cardiac neurons, pretreatment with Pitstop 2 or dynasore to inhibit clathrin and dynamin I/II, respectively, suppressed the PACAP effect. Subsequent addition of inhibitor after the PACAP-induced increase in excitability developed gradually attenuated excitability with no changes in action potential properties. Likewise, the PACAP-induced increase in excitability was markedly decreased at ambient temperature. Receptor trafficking studies with GFP-PAC1 cell lines demonstrated the efficacy of Pitstop 2, dynasore, and low temperatures at suppressing PAC1R endocytosis. In contrast, brefeldin A pretreatments to disrupt Golgi vesicle trafficking did not blunt the PACAP effect, and PACAP/PAC1R signaling still increased neuronal cAMP production even with endocytic blockade. Our results demonstrate that PACAP/PAC1R complex endocytosis is a key step for the PACAP modulation of cardiac neuron excitability.

Pituitary adenylate cyclase-activating polypeptide enhances the hyperpolarization-activated nonselective cationic conductance, Ih, in dissociated guinea pig intracardiac neurons

Pituitary adenylate cyclase-activating polypeptide (PACAP) peptides, which are co-localized with acetylcholine in preganglionic parasympathetic fibers innervating guinea pig intracardiac ganglia, depolarize and increase excitability of intracardiac neurons. Perforated patch whole cell recordings were used to test whether PACAP27-enhanced activation of Ih contributed to the increase in excitability. In current clamp, 100 nM PACAP27 increased rectification during 500-ms hyperpolarizations and increased the number of anodal break action potentials (APs). PACAP27 also increased the number of APs produced by 500-ms depolarizing currents. In voltage clamp, the effects of 100 nM PACAP27 were determined during hyperpolarizing steps from -50 mV to voltages between -60 and -120 mV. PACAP27 increased the amplitude and rate of activation of Ih. PACAP27 shifted the voltage dependence of activation of Ih by 6.6 mV. The effect of PACAP27 was eliminated by pretreatment with the Ih inhibitor ZD7288 (100 microM). The adenylyl cyclase activator forskolin (10 microM) produced a similar shift in the voltage dependence of Ih activation. We conclude that PACAP27 enhances Ih by shifting the voltage dependence of activation and propose that this effect is mediated primarily by PAC1 receptor activation of adenylyl cyclase and generation of cAMP. Furthermore, we propose that the peptide-enhanced Ih contributes to the PACAP27-induced increase in membrane excitability.

Presynaptic function is altered in snake K+-depolarized motor nerve terminals containing compromised mitochondria

1 Presynaptic function was investigated at K+‐stimulated motor nerve terminals in snake costocutaneous nerve muscle preparations exposed to carbonyl cyanide m‐chlorophenylhydrazone (CCCP, 2 μm), oligomycin (8 μg ml−1) or CCCP and oligomycin together. 2 Miniature endplate currents (MEPCs) were recorded at ‐150 mV with two‐electrode voltage clamp. With all three drug treatments, during stimulation by elevated K+ (35 mm), MEPC frequencies initially increased to values > 350 s−1, but then declined. The decline occurred more rapidly in preparations treated with CCCP or CCCP and oligomycin together than in those treated with oligomycin alone. 3 Staining with FM1‐43 indicated that synaptic vesicle membrane endocytosis occurred at some CCCP‐ or oligomycin‐treated nerve terminals after 120 or 180 min of K+ stimulation, respectively. 4 The addition of glucose to stimulate production of ATP by glycolysis during sustained K+ stimulation attenuated the decline in MEPC frequency and increased the percentage of terminals stained by FM1‐43 in preparations exposed to either CCCP or oligomycin. 5 We propose that the decline in K+‐stimulated quantal release in preparations treated with CCCP, oligomycin or CCCP and oligomycin together could result from a progressive elevation of intracellular calcium concentration ([Ca2+]i). For oligomycin‐treated nerve terminals, a progressive elevation of [Ca2+]i could occur as the cytoplasmic ATP/ADP ratio decreases, causing energy‐dependent Ca2+ buffering mechanisms to fail. The decline in MEPC frequency could occur more rapidly in preparations treated with CCCP or CCCP and oligomycin together because mitochondrial Ca2+ buffering and ATP production were both inhibited. Therefore, the proposed sustained elevation of [Ca2+]i could occur more rapidly.

Calcium-induced calcium release regulates action potential generation in guinea-pig sympathetic neurones.

Experiments were done using guinea‐pig sympathetic neurones dissociated from the stellate ganglia to establish whether calcium‐induced calcium release (CICR) modulated action potential (AP) generation in mammalian neurones. Using measurements of intracellular calcium ([Ca2+]i) with the Ca2+‐sensitive dye fluo‐3, we demonstrated that 10 mm caffeine activated ryanodine receptors and caused a rise in [Ca2+]i in both Ca2+‐containing and Ca2+‐deficient solutions. We also demonstrated that combined treatment with caffeine and 1 μm thapsigargin or caffeine and 20 μm ryanodine blocked subsequent caffeine‐induced elevations of [Ca2+]i. Treatment with thapsigargin, ryanodine or 200 μm Cd2+ to disrupt CICR decreased the latency to AP generation during 400 ms depolarizing current ramps using the perforated patch whole cell patch clamp in current clamp mode. Treatment with 500 μm tetraethylammonium also decreased the latency to AP generation during depolarizing current ramps in control cells, but not in cells pretreated with thapsigargin to deplete internal Ca2+ stores. In summary, we propose that an outward current, carried at least in part through BK channels, is activated by CICR at membrane voltages approaching the threshold for AP initiation and that this current opposed depolarizing current ramps applied to guinea‐pig sympathetic stellate neurones.

Galanin Activates an Inwardly Rectifying Potassium Conductance and Inhibits a Voltage-Dependent Calcium Conductance in Mudpuppy Parasympathetic Neurons a

Abstract: Galanin‐induced activation of an inwardly rectifying membrane potassium (K+) current and inhibition of barium current (IBa) were studied using whole cell voltage clamp recording techniques in parasympathetic neurons dissociated from the mudpuppy cardiac ganglion. Both activation of the K+ current and inhibition of IBa were concentration‐dependent with an EC50 (or IC50) of ˜35 nM and ˜0.4 nM, respectively. Both actions of galanin were eliminated by pretreatment with pertussis toxin, which suggested involvement of Gi/Go protein activation. Galantide antagonized the galanin‐induced activation of K+ current with an IC50 equal to 4 nM. By contrast, galantide, by itself, inhibited IBa with an EC50 equal to 16 nM. Another galanin analog, M40, primarily antagonized the galanin‐induced activation of K+ current, but in some cells, M40 also acted as a weak agonist. M40, like galantide, inhibited IBa. The NH2‐terminal fragment galanin‐(1–16) activated the K+ current and inhibited IBa, indicating that the first 16 amino acids of the galanin peptide were sufficient for both actions. In summary, it is postulated that the effects of galanin on mudpuppy parasympathetic neurons might be mediated by activation of two different subtypes of galanin receptor, one that regulates membrane K+ conductance and a second that modulates calcium conductance.

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

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 (Ni(2+)) 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 Ni(2+), a concentration that suppresses currents in both T-type and R-type channels, blunted the PACAP-initiated increase in excitability. Ni(2+) 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, Ni(2+)-sensitive current contributes to PACAP-induced modulation of neuronal excitability.

The modulation of action potential generation by calcium-induced calcium release is enhanced by mitochondrial inhibitors in mudpuppy parasympathetic neurons.

Previously, we demonstrated that outward currents activated by calcium-induced calcium release (CICR) opposed depolarization-induced action potential (AP) generation in dissociated mudpuppy parasympathetic neurons [J Neurophysiol 88 (2002) 1119]. In the present study, we tested whether AP generation by depolarizing current ramps could be altered by dissipating the mitochondrial membrane potential and thus interrupting mitochondrial Ca2+ buffering. Exposure to the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP; 2 microM) alone or in combination with the mitochondrial ATP synthase inhibitor oligomycin (8 microg/ml), increased the latency to AP generation. Exposure to the electron transport chain inhibitor rotenone (10 microM) alone or in combination with oligomycin (8 microg/ml) similarly increased the latency to AP generation. CCCP and oligomycin or rotenone and oligomycin treatment caused rhodamine 123 loss from mitochondria within a few minutes, confirming that the mitochondrial membrane potential was dissipated during drug exposure. Oligomycin alone had no effect on the latency to AP generation and did not cause loss of rhodamine 123 from mitochondria. The increase in latency induced by CCCP and oligomycin was similar when recordings were made with either the perforated patch or standard whole cell patch recording configuration. Exposure to the endoplasmic reticulum Ca-ATPase inhibitor thapsigargin (1 microM), decreased the latency to AP generation. In cells pretreated with thapsigargin to eliminate CICR, CCCP and oligomycin had no effect on AP latency. Pretreatment with iberiotoxin (IBX; 100 nM), an inhibitor of large conductance, calcium- and voltage-activated potassium channels, reduced the extent of the CCCP- and oligomycin-induced increase in latency to AP generation. These results indicate that treatment with CCCP or rotenone to dissipate the mitochondrial membrane potential, a condition which should minimize sequestration of Ca2+ by mitochondria, facilitated the Ca(2+)-induced Ca2+ release activation of IBX-sensitive and IBX-insensitive conductances that regulate AP generation.

Activation of MEK/ERK signaling contributes to the PACAP-induced increase in guinea pig cardiac 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.

Decrease in neuronal nicotinic acetylcholine receptor subunit and PSD-93 transcript levels in the male mouse MPG after cavernous nerve injury or explant culture

Quantitative real-time PCR was used to test whether cavernous nerve injury leads to a decrease in major pelvic ganglia (MPG) neuronal nicotinic ACh receptor (nAChR) subunit and postsynaptic density (PSD)-93 transcript levels. Subunits α3, β4, and α7, commonly expressed in the MPG, were selected for analysis. After 72 h in explant culture, MPG transcript levels for α3, β4, α7, and PSD-93 were significantly depressed. Three days after cavernous nerve axotomy or crush in vivo, transcript levels for α3, β4, and PSD-93, but not for α7, were significantly depressed. Three days after dissection of the cavernous nerve free of underlying tissue and application of a 5-mm lateral stretch (manipulation), transcript levels for α3 and PSD-93 were also significantly decreased. Seven days after all three surgical procedures, α3 transcript levels remained depressed, but PSD-93 transcript levels were still decreased only after axotomy or nerve crush. At 30 days postsurgery, transcript levels for the nAChR subunits and PSD-93 had recovered. ACh-induced currents were significantly smaller in MPG neurons dissociated from 3-day explant cultured ganglia than from those recorded in neurons dissociated from acutely isolated ganglia; this observation provides direct evidence showing that a decrease in nAChR function was coincident with a decrease in nAChR subunit transcript levels. We conclude that a downregulation of nAChR subunit and PSD-93 expression after cavernous nerve injury, or even manipulation, could interrupt synaptic transmission within the MPG and thus contribute to the loss of neural control of urogenital organs after pelvic surgeries.

Pretreatment with Nonselective Cationic Channel Inhibitors Blunts the PACAP-Induced Increase in Guinea Pig Cardiac Neuron Excitability

Calcium influx is required for the pituitary adenylyl cyclase activating polypeptide (PACAP)-induced increase in guinea pig cardiac neuron excitability, noted as a change from a phasic to multiple action potential firing pattern. Intracellular recordings indicated that pretreatment with the nonselective cationic channel inhibitors, 2-aminoethoxydiphenylborate (2-APB), 1-[β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole HCl (SKF 96365), and flufenamic acid (FFA) reduced the 20-nM PACAP-induced excitability increase. Additional experiments tested whether 2-APB, FFA, and SKF 96365 could suppress the increase in excitability by PACAP once it had developed. The increased action potential firing remained following application of 2-APB but was diminished by FFA. SKF 96365 transiently depressed the PACAP-induced excitability increase. A decrease and recovery of action potential amplitude paralleled the excitability shift. Since semiquantitative PCR indicated that cardiac neurons express TRPC subunit transcripts, we hypothesize that PACAP activates calcium-permeable, nonselective cationic channels, which possibly are members of the TRPC family. Our results are consistent with calcium influx being required for the initiation of the PACAP-induced increase in excitability, but suggest that it may not be required to sustain the peptide effect. The present results also demonstrate that nonselective cationic channel inhibitors could have other actions, which might contribute to the inhibition of the PACAP-induced excitability increase.