Biomedical subjects

R L Parsons

Publications and source records attributed to R L Parsons.

At least 37 records · Page 2Linked to original sources

PubMed2000

PACAP peptides modulate guinea pig cardiac neuron membrane excitability and neuropeptide expression.

Morphological studies identified PACAP-immunoreactive nerve fibers in dense pericellular arrangements around virtually every cholinergic parasympathetic neuron of guinea pig cardiac ganglia; all postganglionic cardiac neurons expressed membrane-associated PAC1 receptor protein. Characterization of the alternative splice variants established predominant expression of the PAC1(very short) receptor transcript containing neither HIP nor HOP exons. PACAP depolarized cardiac neurons and increased membrane excitability; the excitability resulted from neither altered action potential properties nor inhibition of IM. Treatment of cardiac ganglia explants with PACAP significantly reduced the numbers of cholinergic neurons coexpressing somatostatin immunoreactivity, which did not appear to be correlated with prosomatostatin mRNA expression. The PACAP-mediated decrease in somatostatin immunoreactive neurons required calcium influx through L-type calcium channels and activation of adenylyl cyclase, whereas activation of phospholipase C or protein kinase A was not required. These observations indicate that PACAP through the PAC1 receptors elicits complex actions on guinea pig parasympathetic cardiac ganglia neurons, including modulation of membrane ion conductances and modulation of neuropeptide expression.

Animals↗

PubMed2000

Differentiated dairy grazing intensity in the Northeast.

As the dairy industry in the Northeast experienced difficult economic conditions in the 1990s, grazing was increasingly viewed as an option for feeding dairy cattle. This analysis used a large sample of dairy farms randomly drawn from three states (Pennsylvania, Vermont, and Virginia) in early 1997 to compare important aspects of the farming operations for four distinct grazing systems: continuous, traditional, moderately intensive, and intensive. Farmers who used intensive grazing tended to be younger, have more cows per acre, and have greater satisfaction with their farming operations. Logit regression results showed that more formal education and a higher debt-to-asset ratio increased the likelihood that a farmer would increase reliance on grazing in the future.

Agriculture↗

PubMed1999

Neuropeptide Y (NPY) expression is increased in explanted guinea pig parasympathetic cardiac ganglia neurons.

While expression of neuropeptides by sympathetic neurons is altered by decentralization and axotomy, it is not known whether similar experimental paradigms also modulate the chemical phenotype of parasympathetic cardiac ganglia neurons. The present study tested whether guinea pig parasympathetic neuron neuropeptide Y (NPY) expression was altered when cardiac ganglia preparations were maintained as organ explants in the presence or absence of colchicine. Two experimental approaches were used to examine NPY expression. First, immunocytochemical techniques were used to quantitate numbers of neurons within the cardiac ganglia exhibiting NPY-immunoreactivity; second, reverse transcription PCR was used to examine proNPY mRNA expression. In control cardiac ganglia preparations, approximately 4% of ganglia neurons exhibited NPY-immunoreactivity. The percentage of NPY-immunopositive neurons in 30- and 72-h explanted cardiac ganglia preparations, maintained in the absence of colchicine, increased to 11 and 16%, respectively. Colchicine treatment of explanted preparations further increased the percentage of NPY-positive ganglia cells 24% (30 h) and 32% (72 h). All NPY-immunoreactive neurons from control ganglia and explanted ganglia were choline acetyltransferase(ChAT)-immunoreactive, indicating retention of the cholinergic phenotype. ProNPY mRNA also was increased following ganglia explantation, consistent with the increase in the numbers of NPY-immunoreactive neurons. NPY transcripts were further increased after 30 h, but not after 72 h in colchicine-treated, explanted cardiac ganglia preparations. These results demonstrate that NPY expression is altered in explanted cardiac ganglia preparations, providing evidence that the chemical phenotype of parasympathetic cardiac neurons can be modulated.

Animals↗

PubMed1999

The maize EmBP-1 orthologue differentially regulates opaque2-dependent gene expression in yeast and cultured maize endosperm cells.

In addition to the bZIP protein Opaque2 (O2), there are other maize endosperm nuclear proteins that recognize the O2 box in 22 kDa zein gene promoters. In an effort to understand the effect of these factors on 22 kDa zein expression, we have cloned one of these and identified it as the putative maize (Zea mays L.) orthologue of the wheat bZIP protein EmBP-1 (mEmBP-1). The mEmBP-1 protein exhibits 52% sequence identity and 68% similarity with the wheat protein and recognizes a similar spectrum of DNA sequences, albeit with slightly altered specificity. The mEmBP-1 gene exists as duplicate loci in maize on chromosomes 7S (mEmBP-1a) and 2L (mEmBP-1b). The mEmBP-1 genes are expressed in endosperm, embryo, immature ears, tassel, roots, and seedling shoots at low levels. Although mEmBP-1 binds to the O2 box from the 22 kDa zein gene promoter as a homodimer, it is unable to heterodimerize with O2. The mEmBP-1 protein can activate transcription from a truncated promoter containing a pentamer of the O2 site in yeast cells; however, it inhibited regulated transcription of a 22 kDa zein promoter in a transient expression assay using cultured maize endosperm cells.

Amino Acid Sequence↗

PubMed1999

Role of mitochondrial dysfunction in the Ca2+-induced decline of transmitter release at K+-depolarized motor neuron terminals.

The present study tested whether a Ca2+-induced disruption of mitochondrial function was responsible for the decline in miniature endplate current (MEPC) frequency that occurs with nerve-muscle preparations maintained in a 35 mM potassium propionate (35 mM KP) solution containing elevated calcium. When the 35 mM KP contained control Ca2+ (1 mM), the MEPC frequency increased and remained elevated for many hours, and the mitochondria within twitch motor neuron terminals were similar in appearance to those in unstimulated terminals. All nerve terminals accumulated FM1-43 when the dye was present for the final 6 min of a 300-min exposure to 35 mM KP with control Ca2+. In contrast, when Ca2+ was increased to 3.6 mM in the 35 mM KP solution, the MEPC frequency initially reached frequencies >350 s-1 but then gradually fell approaching frequencies <50 s-1. A progressive swelling and eventual distortion of mitochondria within the twitch motor neuron terminals occurred during prolonged exposure to 35 mM KP with elevated Ca2+. After approximately 300 min in 35 mM KP with elevated Ca2+, only 58% of the twitch terminals accumulated FM1-43. The decline in MEPC frequency in 35 mM KP with elevated Ca2+ was less when 15 mM glucose was present or when preparations were pretreated with 10 microM oligomycin and then bathed in the 35 mM KP with glucose. When glucose was present, with or without oligomycin pretreatment, a greater percentage of twitch terminals accumulated FM1-43. However, the mitochondria in these preparations were still greatly swollen and distorted. We propose that prolonged depolarization of twitch motor neuron terminals by 35 mM KP with elevated Ca2+ produced a Ca2+-induced decrease in mitochondrial ATP production. Under these conditions, the cytosolic ATP/ADP ratio was decreased thereby compromising both transmitter release and refilling of recycled synaptic vesicles. The addition of glucose stimulated glycolysis which contributed to the maintenance of required ATP levels.

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↗

PubMed1998

Mechanisms of pituitary adenylate cyclase activating polypeptide (PACAP)-induced depolarization of sympathetic superior cervical ganglion (SCG) neurons.

Our understanding of PACAP expression and regulation of sympathetic neuronal function has been augmented considerably over the last few years. Among the three major VIP/PACAP receptor subtypes, the SCG appears to express preferentially one particular variant of the PACAP-selective PACAP1 receptor coupled to multiple intracellular signaling cascades. The in situ histochemical hybridization and immunocytochemical studies of PACAP1 receptor mRNA and protein are in good agreement; nearly all of the SCG neurons express the PACAP-selective receptor, suggesting that most of the sympathetic neurons are under PACAP neuromodulation. In accord with that possibility, several independent studies have now demonstrated PACAP peptide expression in the IML sympathetic preganglionic neurons and fibers, including those projecting to the SCG, further emphasizing the significance of PACAP peptides as a preganglionic noncholinergic mediator of sympathetic function. Given the high potency of PACAP on any of a number of cellular responses, the functional relevance of PACAP peptides on SCG neurons is considerable. We have previously demonstrated the potency and efficacy of both PACAP27 and PACAP38 on sympathetic neuron neurotransmitter/neuropeptide production and secretion; the ability of these peptides to stimulate neuronal second messenger activation was also in the nanomolar range. These results are congruous with our current electrophysiological studies, which were driven to further define the dynamic sympathetic responses to PACAP. In line with the morphological studies, for example, more than 90% of the sympathetic neurons responded to PACAP. In agreement with previous neuropharmacological data, the PACAP-induced depolarizations were elicited at physiologically relevant peptide concentrations at high affinity PACAP-selective receptors. The effects were direct and the alterations in postganglionic neuronal membrane properties appeared to be mediated by several ionic mechanisms. If these studies were analogous to pieces in a puzzle to understand the effects of PACAP in sympathetic development and function, the picture of late has been more completely assembled. But several important challenges still remain. What are the signal transduction mechanisms that mediate the PACAP-induced changes in sympathetic membrane properties? How do the resulting alterations impact the acute and more long-term responses of sympathetic neurons? Does the coupling of PACAP1 receptors to intracellular signaling pathways differ during development, resulting in a transition from the neurotrophic properties of PACAP in neuroblasts to neuromodulatory roles of the peptides in postmitotic neurons? By looking at these issues in one distinct neuronal system, we enlarge our understanding and appreciation of peptides, and PACAP in particular, in the molecular and cellular events guiding neuronal development, function, and plasticity.

Animals↗

PubMed1998

Pituitary adenylate cyclase-activating polypeptide expression and modulation of neuronal excitability in guinea pig cardiac ganglia.

Cardiac output is regulated by the coordinate interactions of stimulatory sympathetic and inhibitory parasympathetic signals. Intracardiac parasympathetic ganglia are integrative centers of cardiac regulation, and modulation of the parasympathetic drive on the heart is accomplished by altering intrinsic cardiac ganglion neuron excitability. The pituitary adenylate cyclase-activating polypeptide (PACAP)/vasoactive intestinal peptide (VIP) family of peptides modulates cardiac function, and in guinea pig heart, PACAP appears to act directly on intrinsic parasympathetic cardiac ganglia neurons through PACAP-selective receptors. A multidisciplinary project tested whether cardiac PACAP peptides act through PACAP-selective receptors as excitatory neuromodulators amplifying the parasympathetic inhibition from guinea pig cardiac ganglia. The in vivo sources of regulatory PACAP peptides were localized immunocytochemically to neuronal fibers and a subpopulation of intrinsic postganglionic cardiac neurons. RT-PCR confirmed that cardiac ganglia expressed proPACAP transcripts and have PACAP peptide biosynthetic capabilities. Messenger RNA encoding PACAP-selective PAC1 receptor isoforms were also present in cardiac ganglia. Alternative splicing of PAC1 receptor transcripts produced predominant expression of the very short variant with neither HIP nor HOP cassettes; lower levels of the PAC1HOP2 receptor mRNA were present. Almost all of the parasympathetic neurons expressed membrane-associated PAC1 receptor proteins, localized immunocytochemically, which correlated with the population of cells that responded physiologically to PACAP peptides. PACAP depolarized cardiac ganglia neurons and increased neuronal membrane excitability. The rank order of peptide potency on membrane excitability in response to depolarizing currents was PACAP27>PACAP38>VIP. The PACAP-induced increase in excitability was not a function of membrane depolarization nor was it caused by alterations in action potential configuration. These results support roles for PACAP peptides as integrative modulators amplifying, through PACAP-selective receptors, the parasympathetic cardiac ganglia inhibition of cardiac output.

Adenylyl Cyclases↗

PubMed1998

Expression and physiological actions of neuropeptide Y in guinea pig parasympathetic cardiac ganglia.

Guinea pig atrial whole mount preparations containing the parasympathetic cardiac ganglia were used to establish the expression, distribution and actions of neuropeptide Y (NPY) in atrial tissues. NPY-immunoreactive fibers densely innervated the atrial myocardium and blood vessels. Fibers containing NPY also innervated intrinsic parasympathetic cardiac neurons. Four percent of the cardiac neurons, identified using microtubule associated protein-2 antiserum, were NPY-positive. An endogenous source of NPY was confirmed with reverse transcription PCR which demonstrated the presence of proNPY mRNA. Sixty percent of the parasympathetic cardiac neurons were hyperpolarized by local application of NPY. NPY also decreased the amplitude and duration of the action potential after hyperpolarization in 60% of the neurons and decreased the fast excitatory postsynaptic potential in about 50% of the cells. These observations indicate that NPY is anatomically positioned to directly alter the output of the parasympathetic cardiac ganglia either by hyperpolarizing the cardiac neurons or by decreasing the fast synaptic input which drives individual neurons.

Animals↗

PubMed1998

Profitability of moderate intensive grazing of diary cows in the northeast.

Empirical analysis of the profitability of moderate grazing are presented based on sample dairy farms in Pennsylvania and New York. Net income per cow was higher for dairy farms that employed moderate intensive grazing than for dairy farms that employed extensive grazing. Income appeared to be adequate for family living expenses, but moderate intensive grazing could not be considered a high profit system. Detailed analyses of dairy farms that employed moderate and extensive grazing in northern Pennsylvania indicated that returns to management and owner equity were higher for pasture enterprises than for corn silage or hay enterprises. Positive dairy profits were related to lower feed costs. Milk production was lower on farms that employed moderate grazing than on farms that employed extensive grazing. Logit regression analysis characterized farms that employed moderate intensive grazing as oriented toward dairy rather than toward crop production; these farms had lower culling rates and a greater dependence on milk sales as a share of total sales. The reduced use of fertilizers and chemicals suggests that moderate grazing had environmentally sustainable features.

Animal Feed↗

PubMed1997

Tachykinin-induced activation of non-specific cation conductance via NK3 neurokinin receptors in guinea-pig intracardiac neurones.

1. Whole mount preparations from guinea-pig hearts were used to characterize the receptors and ionic mechanisms mediating the substance P (SP)-induced depolarization of parasympathetic postganglionic neurones of the cardiac ganglion. 2. Measurement of the amplitude of depolarization in response to superfusion of different tachykinin agonists (neurokinins A (NKA) and B (NKB), SP, and senktide) gave a rank-order potency of NKB = senktide > NKA > SP, indicating involvement of an NK3 receptor. The use of the selective tachykinin receptor antagonists SR 140333, SR 48986, and SR 142801 demonstrated that only the NK3 receptor antagonist SR 142801 inhibited the SP-induced depolarization. 3. The SP-induced depolarization was not inhibited by Ba2+, TEA, or niflumic acid, or altered by reduced Cl- solutions, but was attenuated in reduced Na+ solutions. Single electrode voltage clamp studies demonstrated that the SP-induced inward current increased in amplitude at more negative potentials, had a reversal potential of approximately 0 mV, and was reduced in amplitude in reduced Na+ solutions. 4. We conclude that the SP-induced depolarization in guinea-pig postganglionic parasympathetic neurones of the cardiac ganglion is due to NK3-mediated activation of a non-selective cation conductance.

Animals↗

PubMed1997

A maize zinc-finger protein binds the prolamin box in zein gene promoters and interacts with the basic leucine zipper transcriptional activator Opaque2.

The prolamin box (P-box) is a highly conserved 7-bp sequence element (5'-TGTAAAG-3') found in the promoters of many cereal seed storage protein genes. Nuclear factors from maize endosperm specifically interact with the P-box present in maize prolamin genes (zeins). The presence of the P-box in all zein gene promoters suggests that interactions between endosperm DNA binding proteins and the P-box may play an important role in the coordinate activation of zein gene expression during endosperm development. We have cloned an endosperm-specific maize cDNA, named prolamin-box binding factor (PBF), that encodes a member of the recently described Dof class of plant Cys2-Cys2 zinc-finger DNA binding proteins. When tested in gel shift assays, PBF exhibits the same sequence-specific binding to the P-box as factors present in maize endosperm nuclei. Additionally, PBF interacts in vitro with the basic leucine zipper protein Opaque2, a known transcriptional activator of zein gene expression whose target site lies 20 bp downstream of the P-box in the 22-kDa zein gene promoter. The isolation of the PBF gene provides an essential tool to further investigate the functional role of the highly conserved P-box in regulating cereal storage protein gene expression.

Amino Acid Sequence↗

PubMed1997

Transmitter release differs at snake twitch and tonic endplates during potassium-induced nerve terminal depolarization.

Twitch and tonic muscle fibers of snake skeletal muscle differ in their synpatic as well as mechanical properties. These experiments were aimed at detemining the basis of the difference in vesicular release properties of nerve terminals at twitch and tonic endplates. Miniature endplate currents (MEPCs) were recorded from voltage-clamped garter snake muscle fibers depolarized by high K+ in either a control Ca2+ or high-Ca2+ solution. MEPC frequency increased at twitch and tonic endplates and remained elevated for 8 h during depolarization in control Ca2+. At twitch endplates depolarized in the presence of high Ca2+, an increase in MEPC frequency was followed by a progressive decline. In contrast, MEPC frequency remained elevated in high Ca2+ at tonic endplates. The observed decrease in MEPC frequency at depolarized twitch endplates in high Ca2+ was not a function of the level of depolarization or initial MEPC frequency, nor was it due to a reduction in MEPC amplitude and loss of MEPCs in baseline noise. An optical assay of presynaptic function in which the activity-dependent dye FM1-43 was used confirmed that quantal releases differs at twitch and tonic endplates. Most twitch nerve terminals were labeled by FM1-43 during prolonged depolarization with control Ca2+ or after brief depolarization with high Ca2+. In contrast, the number of twitch nerve terminals and the degree to which they were stained was greatly reduced after prolonged exposure to high K+ and high Ca2+, whereas depolarized tonic endplates were well stained by FM1-43 during brief and prolonged exposure to high Ca2+. FM1-43 staining also revealed variable levels of quantal release between individual boutons at twitch endplates after prolonged depolarization in high-Ca2+ solution. The observed reduction in presynaptic function at twitch nerve terminals after prolonged depolarization in high-Ca2+ solution was reversible and therefore not due to irreversible damage to terminal boutons. MEPC frequency increased at both twitch and tonic endplates when either Sr2+ or Ba2+ was substituted for high Ca2+ during K(+)-induced depolarization. Over time, in Sr2+ or Ba2+ solutions, MEPC frequency remained elevated at tonic endplates but declined at twitch endplates with a time course similar to that observed in high Ca2+. MEPC amplitudes at both endplates remained constant. We conclude that the regulation of quantal release differs in nerve terminals innervating twitch and tonic endplates and postulate that differential intraterminal accumulation of Ca2+ may underlie the observed difference in presynaptic function.

Animals↗

PubMed1996

Expression of choline acetyltransferase immunoreactivity in guinea pig cardiac ganglia.

Recent reports indicate that a considerable amount of heterogeneity exists amongst cardiac postganglionic neurons in their chemical coding patterns and electrical properties, and that some of these cells may serve in roles as sensory and interganglionic neurons as well as motor neurons. This study was undertaken to ascertain whether or not all of these neurons are cholinergic by immunostaining whole-mount preparations of the guinea pig heart for choline acetyltransferase (ChAT). Counts of neurons that were immunostained for microtubule-associated protein-2 revealed that about 1000 neurons exist in about 100 ganglia on the posterior atrial surface. ChAT immunoreactivity was expressed by all of the postganglionic neurons in the cardiac ganglia, including the 5% of neurons that also expressed immunoreactivity for nitric oxide synthase. Varicose nerve fibers that were immunoreactive for ChAT were abundant in ganglia, with every cardiac neuron lying in close apposition to one or more labelled varicosities. ChAT-immunoreactive nerve fibers were also observed in large vagosympathetic fiber bundles, in interganglionic fiber bundles, and passing individually within the myocardium. Immunoreactivity for ChAT was also observed in a large proportion of the small tyrosine hydroxylase-immunoreactive neurons that exist in guinea pig cardiac ganglia. These results indicate that all postganglionic neurons in guinea pig cardiac ganglia are likely to utilize acetylcholine as a neurotransmitter, regardless of their functional role in circuitry of cardiac innervation, and each of these neurons is likely to receive cholinergic input.

Animals↗

PubMed1996

Activation of the protein phosphatase calcineurin during carbachol exposure decreases the extent of recovery from end-plate desensitization.

1. Our previous studies demonstrated that protein kinase C (PKC) activity is required for acetylcholine (ACh) sensitivity to recover fully at snake twitch fiber end plates after prolonged exposure to carbachol. In the present studies, we have investigated whether protein phosphatase(s), activated during carbachol exposure, dephosphorylated critical membrane proteins, which required rephosphorylation by PKC to maintain end-plate sensitivity. End-plate sensitivity was assessed from measurements of miniature end-plate currents (MEPCs) and carbachol-activated currents (EPCCARBS). Conductance of ACh-activated channels was determined from patch-clamp recordings of single-channel currents. 2. Pretreatment of snake muscle preparations with the protein kinase inhibitor staurosporine (0.5 microM), followed by a 10-min exposure to 540 microM carbachol, reduced mean MEPC amplitudes to values 30-40% less than those recorded before carbachol exposure. Conversely, at control end plates exposed to carbachol, the mean MEPC amplitude was reduced by only approximately 5% compared with precarbachol values. This staurosporine-induced decrease in ACh sensitivity could be prevented by pretreatment with the protein phosphatase 2B (calcineurin) inhibitor deltamethrin (0.5 microM), whereas okadaic acid (5 microM) and calyculin A (0.5 microM), inhibitors of protein phosphatases 1 and 2A, had no effect. 3. After a 10-min exposure to 540 microM carbachol, EPCCARB amplitudes (produced by local superfusion with 20 microM carbachol) were significantly smaller at staurosporine-treated end plates than at control end plates. In contrast, the EPCCARB amplitude recorded from end plates pretreated with both deltamethrin and staurosporine was not significantly different from that recorded at control end plates. 4. Substitution of 10 mM Mn2+ for external Ca2+ during the exposure to 540 microM carbachol prevented the decrease in MEPC amplitude recovery at staurosporine-treated end plates. These results suggested that the alteration in sensitivity at staurosporine-treated end plates was calcium dependent. 5. At control end plates, a single population of ACh-activated channels (45-50 pS) is observed both before and after a 10-min exposure to 540 microM carbachol. Conversely, at staurosporine-treated end plates, after exposure to carbachol, a second population of small-conductance (25-30 pS) ACh-activated channels is present in addition to the predominant 45- to 50-pS ACh-activated channels. In preparations pretreated with both deltamethrin and staurosporine, after carbachol exposure, there was a significant decrease in the frequency of small-conductance ACh-activated channels. Deltamethrin treatment alone produced no small-conductance channels before or after a 10-min exposure to 540 microM carbachol. Also, no small-conductance ACh-gated channels were recorded at PKC-inhibited end plates after carbachol exposure either with pretreatment with 10 microM cyclosporin A (another inhibitor of calcineurin) or with the substitution of 10 mM Mn2+ for Ca2+ during the 10-min agonist exposure. 6. We propose that during prolonged exposure to the nicotinic agonist carbachol, calcium influx through ACh-gated channels elevates the level of ionized calcium at the inner surface of the post-junctional membrane and that this local rise in intracellular calcium activates the calcium-dependent phosphatase calcineurin. Dephosphorylation of some key membrane protein by calcineurin leads to a decrease in the extent of recovery from desensitization. Under normal conditions, this process is effectively reversed by PKC activity and end-plate sensitivity recovers fully. However, when PKC is inhibited, the extent of recovery of end-plate sensitivity is decreased, and associated with this decrease is the presence of small-conductance ACh-activated channels not normally recorded at snake twitch fiber end plates.

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↗