Biomedical subjects

L A Merriam

Publications and source records attributed to L A Merriam.

11 recordsLinked to original sources

PubMed2004

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.

Action Potentials↗

PubMed2001

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

Presynaptic function was investigated at K+-stimulated motor nerve terminals in snake costocutaneous nerve muscle preparations exposed to carbonyl cyanide m-chlorophenylhydrazone (CCCP, 2 M), oligomycin (8 g x ml(-1)) or CCCP and oligomycin together. 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. 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. 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. 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.

Action Potentials↗

PubMed2001

Number of K(Ca) channels underlying spontaneous miniature outward currents (SMOCs) in mudpuppy cardiac neurons.

Spontaneous miniature outward currents (SMOCs) in parasympathetic neurons from mudpuppy cardiac ganglia are caused by activation of TEA- and iberiotoxin-sensitive, Ca(2+)-dependent K(+) (BK) channels. Previously we reported that SMOCs are activated by Ca(2+)-induced Ca(2+) release (CICR) from caffeine- and ryanodine-sensitive intracellular Ca(2+) stores. In the present study, we analyzed the single channel currents that contribute to SMOC generation in mudpuppy cardiac neurons. The slope conductance of BK channels, determined from the I-V relationship of single-channel currents recorded with cell-attached patches in physiological K(+) concentrations, was 84 pS. The evidence supporting the identity of this channel as the channel involved in SMOC generation was its sensitivity to internal Ca(2+), external TEA, and caffeine. In cell-attached patch recordings, 166 microM TEA applied in the pipette reduced single-channel current amplitude by 32%, and bath-applied caffeine increased BK channel activity. The ratio between the averaged SMOC amplitude and the single-channel current amplitude was used to estimate the average number of channels involved in SMOC generation. The estimated number of channels involved in generation of an averaged SMOC ranged from 18 to 23 channels. We also determined that the Po of the BK channels at the peak of a SMOC remains constant at voltages more positive than -20 mV, suggesting that the transient rise in intracellular Ca(2+) from ryanodine-sensitive intracellular stores in the vicinity of the BK channel reached concentrations most likely exceeding 40 microM.

Animals↗

PubMed1999

Empty synaptic vesicles recycle and undergo exocytosis at vesamicol-treated motor nerve terminals.

We investigated whether recycled cholinergic synaptic vesicles, which were not refilled with ACh, would join other synaptic vesicles in the readily releasable store near active zones, dock, and continue to undergo exocytosis during prolonged stimulation. Snake nerve-muscle preparations were treated with 5 microM vesamicol to inhibit the vesicular ACh transporter and then were exposed to an elevated potassium solution, 35 mM potassium propionate (35 KP), to release all preformed quanta of ACh. At vesamicol-treated endplates, miniature endplate current (MEPC) frequency increased initially from 0.4 to >300 s-1 in 35 KP but then declined to <1 s-1 by 90 min. The decrease in frequency was not accompanied by a decrease in MEPC average amplitude. Nerve terminals accumulated the activity-dependent dye FM1-43 when exposed to the dye for the final 6 min of a 120-min exposure to 35 KP. Thus synaptic membrane endocytosis continued at a high rate, although MEPCs occurred infrequently. After a 120-min exposure in 35 KP, nerve terminals accumulated FM1-43 and then destained, confirming that exocytosis also still occurred at a high rate. These results demonstrate that recycled cholinergic synaptic vesicles that were not refilled with ACh continued to dock and undergo exocytosis after membrane retrieval. Thus transport of ACh into recycled cholinergic vesicles is not a requirement for repeated cycles of exocytosis and retrieval of synaptic vesicle membrane during prolonged stimulation of motor nerve terminals.

Acetylcholine↗

PubMed1999

Ca(2+)-induced Ca(2+) release activates spontaneous miniature outward currents (SMOCs) in parasympathetic cardiac neurons.

Mudpuppy parasympathetic cardiac neurons exhibit spontaneous miniature outward currents (SMOCs) that are thought to be due to the activation of clusters of large conductance Ca(2+)-activated K(+) channels (BK channels) by localized release of Ca(2+) from internal stores close to the plasma membrane. Perforated-patch whole cell recordings were used to determine whether Ca(2+)-induced Ca(2+) release (CICR) is involved in SMOC generation. We confirmed that BK channels are involved by showing that SMOCs are inhibited by 100 nM iberiotoxin or 500 microM tetraethylammonium (TEA), but not by 100 nM apamin. SMOC frequency is decreased in solutions that contain 0 Ca(2+)/3.6 mM Mg(2+), and also in the presence of 1 microM nifedipine and 3 microM omega-conotoxin GVIA, suggesting that SMOC activation is dependent on calcium influx. However, Ca(2+) influx alone is not sufficient; SMOC activation is also dependent on Ca(2+) release from the caffeine- and ryanodine-sensitive Ca(2+) store, because exposure to 2 mM caffeine consistently caused an increase in SMOC frequency, and 10-100 microM ryanodine altered the configuration of SMOCs and eventually inhibited SMOC activity. Depletion of intracellular Ca(2+) stores by the Ca-ATPase inhibitor cyclopiazonic acid (10 microM) inhibited SMOC activity, even when Ca(2+) influx was not compromised. We also tested the effects of the membrane-permeable Ca(2+) chelators, bis-(o-aminophenoxy)-N,N,N', N'-tetraacetic acid-AM (BAPTA-AM) and EGTA-AM. EGTA-AM (10 microM) caused no inhibition of SMOC activation, whereas 10 microM BAPTA-AM consistently inhibited SMOCs. After SMOCs were completely inhibited by BAPTA, 3 mM caffeine caused SMOC activity to resume. This effect was reversible on removal of caffeine and suggests that the source of Ca(2+) that triggers the internal Ca(2+) release channel is different from the source of Ca(2+) that activates clusters of BK channels. We propose that influx of Ca(2+) through voltage-dependent Ca(2+) channels is required for SMOC generation, but that the influx of Ca(2+) triggers CICR from intracellular stores, which then activates the BK channels responsible for SMOC generation.

Animals↗

PubMed1998

Galanin activates an inwardly rectifying potassium conductance and inhibits a voltage-dependent calcium conductance in mudpuppy parasympathetic neurons.

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 approximately 35 nM and approximately 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.

Animals↗

PubMed1995

Galantide distinguishes putative subtypes of galanin receptors in mudpuppy parasympathetic neurons.

The effect of the chimeric ligand galantide on the galanin-induced activation of membrane K+ conductance and inhibition of voltage-dependent Ca2+ conductance has been studied using voltage-clamped dissociated mudpuppy parasympathetic neurons. Galantide did not activate the K+ conductance but produced a concentration-dependent antagonism (IC50 = 4 nM) of the galanin-induced increase in K+ conductance. Galantide acted like galanin and inhibited the voltage-dependent Ba2+ current (IBa). The inhibition of IBa also was concentration dependent (IC50 = 16 nM) and the maximum inhibition produced by galantide was approximately 40%. We also demonstrate that the galanin-(1-16) fragment increased the membrane K+ conductance and decreased IBa, suggesting that the NH2 portion of the galanin molecule is sufficient to mediate both actions. One interpretation of these observations is that different galanin receptors mediate the different effects of galanin on the mudpuppy parasympathetic neurons.

Animals↗

PubMed1995

Neuropeptide galanin inhibits omega-conotoxin GVIA-sensitive calcium channels in parasympathetic neurons.

1. We determined the effect of the neuropeptide galanin on barium currents (IBa) flowing through voltage-gated calcium channels. We voltage clamped parasympathetic neurons dissociated from mudpuppy cardiac ganglia using both the standard whole cell and the perforated-patch variations of the patch-clamp technique. 2. Galanin produced a concentration-dependent inhibition of IBa. The maximal inhibition was 50-60% and the concentration that produced half-maximal inhibition (IC50) was 0.42 nM. In mud-puppy parasympathetic neurons, omega-conotoxin-GVIA (CTX)-sensitive channels are the predominant type of calcium channels, and only a small portion of IBa is contributed by dihydropyridine-sensitive channels. Galanin preferentially inhibited a portion of the CTX-sensitive current. 3. In currents recorded with the standard whole cell technique, activation of IBa was slowed in the presence of galanin. In contrast, in the majority of neurons studied with the perforated-patch technique, galanin decreased IBa without altering the kinetics of current activation. With both recording methods, the decrease in IBa was greatest with voltage steps to 0 mV and persisted with steps to +50 mV. For control currents, large depolarizing voltage steps (+70 to +120 mV) did not markedly facilitate IBa when either recording technique was used. However, the degree of facilitation in galanin was significantly greater with the standard whole cell recording technique. 4. IBa exhibited inactivation under the conditions of these experiments. Inactivation of IBa recorded during a 900-ms depolarizing voltage step was fitted to a double exponential. Galanin decreased the amplitude of IBa but did not alter the time constants of inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Galanin activates an inwardly rectifying potassium conductance in mudpuppy atrial myocytes.

Galanin- and bethanechol-activated K+ currents have been studied in mudpuppy atrial myocytes. The galanin and bethanechol K+ currents were time-dependent and inwardly rectifying. In GTP gamma S, the galanin and bethanechol currents were reduced progressively as G-protein gated K+ channels became activated. GDP beta S inhibited agonist-induced outward currents. We conclude that galanin and bethanechol activate the same or a very similar inwardly rectifying K+ conductance and that activation of a G protein is required.

Animals↗

PubMed1992

Galanin and bethanechol appear to activate the same inwardly rectifying potassium current in mudpuppy parasympathetic neurons.

Galanin- and bethanechol-activated whole cell currents were studied in dissociated mudpuppy parasympathetic neurons kept in 12.5 mM KCl. The bethanechol-induced current was reduced when generated during a galanin-induced current. The galanin and bethanechol currents reversed at -45 mV and exhibited inward rectification at more negative potentials. Both the galanin- and bethanechol-induced current relaxations recorded during negative voltage steps were fitted best by two exponentials with the averaged time constant values not significantly different for the galanin and bethanechol currents. We conclude that galanin and bethanechol activate a similar membrane potassium conductance which is different from the background potassium conductance.

Animals↗

PubMed1992

Aminergic and peptidergic elements and actions in a cardiac parasympathetic ganglion.

Correlated histochemical, immunocytochemical, and electrophysiological experiments have been undertaken to identify putative neurotransmitter-neuromodulator substances in cells and fibers in the parasympathetic cardiac ganglion of the mudpuppy, Necturus maculosus, and to determine the action of these agents on the properties of the parasympathetic postganglionic neurons. The mudpuppy cardiac ganglion contains two neuron types: large parasympathetic postganglionic neurons and smaller intrinsic neurons initially identified as small intensely fluorescent cells. We have shown that the postganglionic neurons contain both acetylcholine and a galanin-like neuropeptide. Also, we have demonstrated that the intrinsic neurons contain a number of different biogenic amines such as dopamine and serotonin, as well as neuropeptides including a substance P-like peptide and a galanin-like peptide. The results of these studies indicate that the anatomical and histochemical organization of the mudpuppy cardiac ganglion is more complex than that seen in other amphibians and is very similar to that found in most mammalian species. Previously, we showed that galanin has actions that make it of interest as a potential inhibitory neurotransmitter in the mudpuppy cardiac ganglion. Galanin hyperpolarizes and decreases membrane excitability in most parasympathetic neurons. Here we show that galanin initiates membrane hyperpolarization by activating a voltage- and time-dependent potassium conductance. We also present the initial results of ongoing studies which indicate that calcitonin gene-related peptide can depolarize some of the parasympathetic neurons as well as evidence that serotonin initiates depolarization in many parasympathetic neurons. This serotonin-induced depolarization consists of an initial transient depolarization followed by a longer, more slowly developing depolarization. Action potential activity is stimulated during the initial period of depolarization, but depressed during the later, slow depolarization. The results of these electrophysiological experiments suggest that many of the bioactive substances that have been identified in the different cells and nerve fibers within the cardiac ganglion affect the excitability of the postganglionic neurons. In conclusion, we suggest that the results of the studies summarized in this review demonstrate that the cardiac ganglion in the mudpuppy is not simply a relay station. Rather, the cardiac ganglion has a complex organization and exhibits a diversity of physiological responses, indicating that it very likely is another site of integration for control of cardiac function.

Animals↗