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Biomedical subjects

R L Parsons

Publications and source records attributed to R L Parsons.

At least 19 recordsLinked to original sources

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

Bethanechol-induced responses in mudpuppy parasympathetic neurons.

The effect of bethanechol on membrane potential and excitability was determined in mudpuppy parasympathetic postganglionic neurons. Bethanechol induced a large amplitude hyperpolarization, which was followed by a smaller amplitude depolarization, in 115 out of 135 cells tested. In approximately 20% of these cells, a brief depolarization preceded the hyperpolarization. During the bethanechol-induced hyperpolarization, the membrane input resistance decreased markedly, whereas the input resistance was increased during the subsequent depolarization. The hyperpolarization and depolarization were blocked by atropine and were unaffected by d-tubocurarine, thus, both appeared to be mediated by muscarinic receptors. The bethanechol-induced hyperpolarization was inhibited by the M2 muscarinic receptor antagonist AF-DX 116, whereas the bethanechol-induced depolarization was unaffected. Both a nonselective increase in membrane conductance and a decrease in membrane potassium conductance appeared to be involved in the generation of the bethanechol-induced depolarization. Evidence for the first mechanism was obtained in barium-treated cells in which bethanechol initiated a rapid onset depolarization, which was reversed at membrane potentials near 0 mV. Evidence for the second mechanism was obtained when the hyperpolarization was inhibited by AF-DX 116. In AF-DX 116-treated cells, the membrane input resistance was increased during most of the bethanechol-induced depolarization. Mudpuppy neurons initiate repetitive action potential activity in response to long depolarizing current pulses. Following application of bethanechol, with the hyperpolarization negated electrotonically, the number of action potentials produced by a depolarizing current pulse was greater than that produced prior to application of bethanechol. It is suggested that activation of muscarinic receptors on mudpuppy cardiac neurons influences multiple conductance systems and determines the excitability of these neurons.

Animals

Galanin stimulates phosphatidylinositol turnover in cardiac tissue of the mudpuppy.

A galanin-like peptide has been found in the parasympathetic fibers innervating the mudpuppy myocardium and direct application of galanin produces hyperpolarization of atrial myocytes and a decrease in twitch tension. In the present study, atrial and ventricular strips were incubated with galanin and then evaluated for changes in either phosphatidylinositol turnover or cyclic nucleotide levels. Galanin caused a significant and concentration-dependent increase in phosphatidylinositol turnover in both atrial and ventricular tissue. However, galanin had no effect on cyclic AMP or cyclic GMP levels in either basal or adrenergically-stimulated preparations. These results suggest that the galanin-induced cardio-inhibition in the mudpuppy may be mediated, at least in part, by a change in phosphatidylinositol turnover.

Animals

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

Sexually dimorphic distribution of a galanin-like peptide in the central nervous system of the teleost fish Poecilia latipinna.

Immunohistochemical techniques were used to visualize areas of the brain and spinal cord containing a galanin-like peptide in the teleost fish, the sailfin molly. Galanin-like immunoreactivity (GAL-LI) in both males and females was identified in neurons in the nucleus preopticus periventricularis, nucleus lateralis tuberis, and nucleus commissuralis. GAL-LI fibers had a comparable distribution in the forebrain, preoptic, hypothalamic, and visceral sensory areas of both sexes. In striking contrast to these areas, the optic tectum, torus semicircularis, brainstem tegmentum, and spinal cord of the male contained much higher levels of GAL-LI than the female. GAL-LI in these dimorphic areas in the female was limited to single fiber bundles in the ventromedial tegmentum and in the trigeminal system. Additionally, a population of neurons in the preoptic nucleus was found to contain GAL-LI in the male only. Sexual dimorphism was especially prominent in the spinal cord, where extensive GAL-LI fibers were found in the male only. These fibers were oriented in the longitudinal plane and confined largely to the gray matter. Comparative studies were performed on the goldfish spinal cord, in which GAL-LI was localized solely in the dorsal horn and exhibited no sexual dimorphism. Further, examination of spinal cord material from neonatal mollies revealed a lack of spinal GAL-LI at this developmental stage. As the extent of GAL-LI in the male molly spinal cord differs from both the goldfish and from that reported for the mammalian spinal cord, and a prominent sexual dimorphism in GAL-LI extends from the diencephalon to the caudal spinal cord, it is suggested that a galanin-like peptide may play a unique, sex-specific role in this species.

Animals

Source of sexually dimorphic galanin-like immunoreactive projections in the teleost fish Poecilia latipinna.

A galanin-like peptide has a sexually dimorphic distribution in the teleost fish, the sailfin molly. An extensive system of galanin-like immunoreactive (GAL-LI) fibers has been described in the brainstem and spinal cord of the male molly, which is absent in the female (Cornbrooks and Parsons, companion paper). As GAL-LI in the mammalian spinal cord has been localized to neurons of origin in the dorsal root ganglia and dorsal and ventral horns, the present study was undertaken to determine whether the sexually dimorphic GAL-LI in the male molly may originate in part from corresponding sources in this species. Colchicine treatments of the spinal cord and dorsal root ganglia did not result in GAL-LI staining in neuronal somata in these regions. Following complete transection of the spinal cord and at any level of the spinal cord, there was a complete absence of GAL-LI caudal to the lesion site. In fish that received unilateral spinal transection, there was a loss of GAL-LI ipsilateral and caudal to the lesion. Finally, in fish that received lesions in the rostral hypothalamus, there was a complete loss of GAL-LI in the sexually dimorphic fiber system in the brainstem and spinal cord, but not in non-dimorphic GAL-LI regions of the brainstem. Thus the sexually dimorphic fiber system in the male molly may originate in neurons of the preoptic nucleus that are sexually dimorphic for a GAL-LI peptide. This preoptico-spinal pathway may mediate sex-specific behaviors in this species.

Animals

Microtubule-associated protein-2 and neurofilament immunoreactivity in neurons and small, intensely fluorescent cells of an amphibian cardiac ganglion.

The localization of two cytoskeletal proteins was analysed in the cell bodies and processes of ganglionic neurons and small, intensely fluorescent cells of the parasympathetic cardiac ganglion of Necturus maculosus (mudpuppy). Antibodies against microtubule-associated protein-2 and against the highly phosphorylated isoforms of high and middle molecular weight neurofilament subunits were used as somatodendritic and axonal markers, respectively. The ganglionic neurons, which usually have only one major process, and small, intensely fluorescent cells, which have several processes, showed distinctly different staining patterns with the two antibodies. In control and denervated ganglia, the ganglionic cell bodies and several hundred micrometers of the proximal processes were labeled with the antibody against microtubule-associated protein-2, whereas small, intensely fluorescent cells and processes showed a paucity of immunoreactivity. The neurofilament antibody labeled numerous axons in the ganglion but did not label the proximal part of the postganglionic process or small, intensely fluorescent cell processes. Denervation resulted in the presence of phosphorylated neurofilament subunit immunoreactivity in the soma and proximal process of the ganglionic neuron. These data suggest that (i) small, intensely fluorescent cells and ganglionic neurons in the mudpuppy cardiac ganglion contain distinctly different cytoskeletal proteins, (ii) the proximal part of postganglionic "axons" contains dendrite-like and not axon-like cytoskeletal proteins, and (iii) deafferentation promotes the localization of phosphorylated forms of neurofilament subunits in the soma and proximal process of parasympathetic ganglionic neurons.

Animals

Analysis of the galanin-induced decrease in membrane excitability in mudpuppy parasympathetic neurons.

Previously, we showed that the neuropeptide galanin hyperpolarizes and decreases membrane excitability of mudpuppy parasympathetic neurons [Konopka L. M., McKeon T. W. and Parsons R. L. (1989) J. Physiol. 410, 107-122]. We also demonstrated that membrane excitability remains depressed when the agonist-induced potential change is negated electrotonically. We hypothesized that galanin inhibits the membrane conductances associated with spike generation. However, we cannot rule out the possibility that the decreased excitability is due to a galanin-induced increase in membrane potassium conductance which reduces the effectiveness of subsequent depolarizing stimuli. Therefore, in the present study we tested, with the galanin-induced hyperpolarization negated, whether the galanin-induced increased membrane potassium conductance was responsible for the decreased excitability. The results showed that the galanin-induced decreased excitability was not dependent on the peak amplitude of the galanin-induced hyperpolarization. Furthermore, the decreased excitability occurred in cells in which there was no measurable galanin-induced hyperpolarization. Moreover, in most cells the galanin-induced decrease in input resistance, measured at the peak of the hyperpolarization (3-25 mV), was less than 15% and when the hyperpolarization was negated electronically, the decrease was even less (approximately 2%). These results indicated that when the hyperpolarization was negated, the galanin-induced increase in potassium conductance was not responsible for the decreased excitability. In preparations pretreated with 5 mM tetraethylammonium, galanin decreased excitability which indicated that a galanin-induced decrease in the calcium-dependent potassium current was not necessary for the decreased excitability. Galanin also decreased excitability in preparations exposed to either 1-3 microM tetrodotoxin or 100-200 microM cadmium. Following galanin application, the threshold for initiation of tetrodotoxin-insensitive spikes was shifted to more positive membrane potentials. Galanin also decreased the amplitude and hyperpolarizing afterpotential of barium spikes in the absence of any agonist-induced hyperpolarization. These observations confirmed that galanin decreased the voltage-dependent calcium conductance. In the present study, we showed that when the hyperpolarization was negated, galanin decreased excitability by shifting the threshold for spike generation regardless of whether voltage-dependent sodium or calcium currents were primarily responsible for the depolarizing component of the action potential.

Action Potentials

Biogenic amine localization in cardiac ganglion intrinsic neurons: electron microscopic histochemistry of SIF cells.

The parasympathetic cardiac ganglion in the mudpuppy, N. maculosus, contains postganglionic nerve cells and intrinsic neurons, many of which are small intensely fluorescent (SIF) cells. Several bioactive substances have been localized in the intrinsic nerve cells which may have integrative effects at synapses within the ganglion. Ganglionic intrinsic neurons can be identified electron microscopically by the presence of numerous cytoplasmic granular vesicles 80-120 nm in diameter. Throughout the ganglion there are bundles of unmyelinated fibers some of which are filled with granular and agranular vesicles and axosomatic terminals with similar vesicles synapsing on principal parasympathetic nerve cells. To understand the aminergic contribution to ganglionic synaptic circuitry the chromaffin reaction was used. The intrinsic neurons (i.e., SIF cells) were readily identified by their characteristic intracellular granule population. All intrinsic nerve cells identified showed granules which were positively labelled by the chromaffin reaction. Granular vesicles in synaptic profiles on principal cells (P cells) were also labelled indicating a direct aminergic synaptic innervation to these cells. The cell bodies of intrinsic neurons, ensheathed with supportive glial-like cellular processes, rarely received synapses. Elemental microanalysis was used to verify the chromium content of the electron dense product within the granular vesicles. These studies demonstrated direct aminergic synaptic input to at least a subpopulation of principal parasympathetic cells in the cardiac ganglion of mudpuppy.

Animals

Staurosporine inhibits the extent of acetylcholine receptor recovery from carbachol-induced desensitization in snake twitch fibres.

1. The effect of the protein kinase inhibitor, staurosporine, on the extent and time course of recovery following carbachol-induced desensitization was studied in snake twitch-muscle fibres maintained in an isotonic potassium propionate solution and voltage-clamped to +30 mV. 2. Pretreatment with staurosporine (0.5 microM) decreased the extent of recovery of spontaneous miniature endplate current (m.e.p.c.) amplitudes following desensitization by a sustained application of 540 microM carbachol. Recovery was inhibited by approximately 50% without altering the time course of m.e.p.c. recovery. 3. Staurosporine also produced a concentration-dependent (10 nM to 0.5 microM) decrease in the amplitude of a second carbachol-induced current, following a wash period, as compared to the amplitude of the current produced by the initial carbachol application. Pretreatment with 0.5 microM K252a, another wide spectrum protein kinase inhibitor, also decreased the extent of recovery of the response to a second carbachol application following desensitization. 4. Staurosporine pretreatment (0.5 microM) had no effect on either the kinetics of receptor-channel gating or the initial endplate sensitivity to agonist. This was determined by comparing the amplitude of the carbachol (540 microM)-induced currents and the amplitude and decay rate of m.e.p.cs in control and staurosporine-treated fibres. 5. Staurosporine had no effect on the time course of desensitization onset produced during the initial application of 540 microM carbachol or the depth of desensitization produced by the end of a 2-3 min exposure to 540 microM carbachol.6. Elevation of the external calcium concentration from 1 to 10mM during the 540 microM carbachol application completely antagonized the decreased extent of recovery of m.e.p.c. amplitude produced by pretreatment with 0.5 microM staurosporine.7. We suggest that phosphorylation of a population of acetylcholine receptors is required for complete recovery from desensitization, and that staurosporine inhibits the protein kinases responsible for this phosphorylation.8. We further propose that a transient increase in intracellular calcium, produced by an increase in calcium influx through agonist-activated endplate channels, stimulates additional protein kinase activity, which in turn, antagonizes the effect of staurosporine-treatment on recovery.

Alkaloids

Identification of the active site tyrosine of Flp recombinase. Possible relevance of its location to the mechanism of recombination.

A combination of site-directed mutagenesis and amino acid sequence analysis identifies Tyr-343 of Flp recombinase as the residue that covalently attaches to DNA during the strand-cleavage step of recombination. This residue is part of the invariant His-Arg-Tyr triad of the Int family of recombinases. Tyr-343 is located in a highly protease-accessible (and hence "open") region of Flp. This placement may provide the conformational flexibility required for the dual role of Tyr-343 in recombination: nicking of the DNA strands to initiate recombination and joining of the nicked strands across partner substrates to complete recombination. In-frame insertion of a few amino acids close to Tyr-343 (and to its amino-terminal side) does not affect substrate recognition by Flp but abolishes its catalytic function.

Amino Acid Sequence

Galanin-induced hyperpolarization of mudpuppy neurons is calcium dependent.

The calcium dependence of the hyperpolarization produced by galanin has been studied in parasympathetic postganglionic neurons of the mudpuppy. Necturus maculosus. The galanin-induced hyperpolarization, but not the bethanechol-induced hyperpolarization, was reduced when manganese or magnesium were substituted for external calcium. Also, the galanin-induced, but not the bethanechol-induced hyperpolarization, was decreased in the presence of cadmium or nitrendipine. Because the inhibition of the galanin-induced hyperpolarization developed gradually and reversed slowly in the absence of extracellular calcium or presence of the voltage-gated calcium channel blockers, it is suggested that an intracellular pool rather than an extracellular pool of calcium is involved in the generation of the galanin-induced hyperpolarization.

Animals

Functional analysis of Arg-308 mutants of Flp recombinase. Possible role of Arg-308 in coupling substrate binding to catalysis.

The arginine residue at position 308 in the Flp recombinase corresponds to the only invariant arginine within the Int family of recombinases. Alterations of this residue result in Flp variants that retain substrate recognition, but form weaker protein-DNA complexes than wild type Flp. Furthermore, their DNA cleavage activity is significantly diminished. A conservative change of R308K results in a functional Flp variant; however, this protein has a lowered temperature optimum for recombination. The Arg-308 mutants can be stabilized on the DNA substrate through cooperativity with a partner Flp mutant that is tight binding. Thus, interactions between Flp monomers must be a relevant feature of the normal recombination reaction.

Arginine

Galanin immunoreactivity in the mudpuppy cardiac ganglion.

The source of galanin-immunoreactive fibers in the cardiac ganglion and on cardiac muscle in mudpuppy (Necturus maculosus) has been determined utilizing immunohistochemical techniques. The galanin-immunoreactive fibers are not processes of afferent fibers originating in either the rostral four dorsal root ganglia or vagal sensory ganglia. Following colchicine treatment, all of the postganglionic parasympathetic neurons and a subpopulation of the small intrinsic neurons in the cardiac ganglion exhibit galanin immunoreactivity. The majority of the galanin-immunoreactive fibers that form complexes on the parasympathetic postganglionic neurons are derived from galanin-immunoreactive small intrinsic neurons, although some of these connections may represent collateral processes from other parasympathetic postganglionic neurons. All of the galanin-immunoreactive processes that innervate cardiac muscle are derived from postganglionic parasympathetic neurons in the cardiac ganglion.

Animals

End-plate ion channel block produced by lincosamide antibiotics and their chemical analogs.

Five lincosamide compounds were studied for their effects on end-plate currents (epcs), miniature end-plate currents and acetylcholine-induced current fluctuations in the garter snake costocutaneous nerve-muscle preparation. At high concentrations, lincomycin and clindamycin reduced epc amplitude, but analysis of driving functions showed that only with clindamycin was this due solely to changes in epc quantal content. The effect of lincomycin on epc amplitude was exaggerated by rapid channel block during the rising phase of the epc. Clindamycin produced currents with a single exponential decay and single Lorentzian noise spectra. All the other compounds produced currents which decayed as the sum of two exponential components. For lincomycin and epilincomycin, noise spectra consisted of two Lorentzian components. For epiclindamycin and deoxylincomycin, although epcs and miniature end-plate currents decayed with two components, it was not possible to separate two components in the noise spectra. A kinetic analysis of ion channel blocking actions showed only small differences between the two pairs of stereoisomers studied. End-plate ion channel blocking and unblocking rate constants did not vary greatly among the compounds but the end-plate ion channel unblocking rate constant values for the two lincomycin stereoisomers were larger than those for the two clindamycin stereoisomers. Deoxylincomycin exhibited properties similar to those of the clindamycins. It was concluded that lipid solubility, not stereochemical conformation, plays the greater role in determining the ion channel blocking properties within the series, particularly that of the rate of dissociation of the compound from end-plate ion channels.

Animals

Characteristics of the galanin-induced depolarization of mudpuppy parasympathetic postganglionic neurons.

The depolarization of mudpuppy parasympathetic cardiac neurons, produced by pressure application of the neuropeptide galanin, has been characterized. The amplitude and duration of the depolarization were dependent on the duration of the galanin application. The amplitude of the depolarization increased with hyperpolarization and the reversal potential determined by extrapolation was approximately +10 mV. The amplitude and time course of the galanin-induced depolarization were not changed by substitution of either manganese or magnesium for extracellular calcium, but were decreased by exposure to 50-100 microM d-tubocurarine. It is proposed that the galanin-induced depolarization results from a receptor-activated, nonselective, cation channel; and further, that the receptor initiating depolarization differs from the galanin receptor mediating hyperpolarizing responses in these parasympathetic neurons.

Action Potentials

The presence and possible role of a galanin-like peptide in the mudpuppy heart.

A correlated histochemical and pharmacological study was undertaken to establish the presence, origin, and possible function of nerve fibers containing a galanin-like peptide in the mudpuppy (Necturus maculosus) heart. Whole mount preparations of septum-sinus venosus or atria and sections of ventricular muscle were prepared for immunocytochemistry. Galanin-immunoreactive fibers were found coursing diffusely across the septum-sinus venosus to form complex networks over cardiac muscle strands. Individual atrial muscle strands were densely innervated by galanin-immunoreactive fibers and galanin-immunoreactive fibers were also observed in the epicardial and myocardial layers of the ventricle. Most of the parasympathetic postganglionic neurons in the cardiac ganglion and many of the small intensely fluorescent-like cells exhibited galanin immunoreactivity. Galanin-immunoreactive fibers were present in the nerve trunks connecting clusters of parasympathetic postganglionic neurons. Close associations between galanin-positive fibers and individual parasympathetic postganglionic neurons were also observed. The presence of the galanin-immunoreactive fibers was similar in preparations taken from animals pretreated with 6-hydroxydopamine to that seen in preparations taken from control animals, indicating that the galanin-positive fibers were not sympathetic postganglionic axons. Moreover, the galanin-immunoreactive nerve fibers were separate from fibers containing substance P and/or calcitonin gene-related peptide that have previously been shown to be processes of afferent fibers. In twitch-tension experiments, galanin in the range 1 x 10(-7) to 1 x 10(-6) M caused cardioinhibition of spontaneously beating isolated septal-sinus venosus preparations. Galanin also produced a concentration-dependent (1 x 10(-7) to 1 x 10(-6) M) decrease in the twitch-tension development of electrically stimulated atrial or ventricular preparations. Local application of galanin produced hyperpolarization of cardiac muscle fibers in both isolated septal-sinus venosus preparations and atrial preparations. The response of individual parasympathetic ganglion cells to local application of galanin varied between neurons; some neurons were depolarized whereas others were hyperpolarized. We conclude that a galanin-like peptide is contained in both the parasympathetic postganglionic neurons and small intensely fluorescent-like cells and their processes. Further, we hypothesize that in the case of the parasympathetic postganglionic neurons, the galanin-like peptide may work in conjunction with acetylcholine to regulate cardiac activity.

Animals

Galanin-induced hyperpolarization and decreased membrane excitability of neurones in mudpuppy cardiac ganglia.

1. Membrane hyperpolarization and decreased excitability produced by galanin were investigated in vitro on parasympathetic postganglionic neurones in the cardiac ganglion of the mudpuppy, Necturus maculosus. 2. Galanin produced a slowly developing hyperpolarization which, in 2.5 mM-KCl, reversed at -105.4 +/- 2.7 mV. The reversal potential was shifted by 38.7 +/- 4.9 mV following a fourfold elevation of the extracellular potassium concentration. 3. Galanin inhibited action potential firing in spontaneously active neurones and decreased the number of spikes in a train produced by long (500-680 ms) depolarizing current pulses. Both effects were independent of the galanin-induced hyperpolarization. 4. Galanin increased the threshold for spike generation, prolonged the spike hyperpolarizing after-potential and decreased the maximum rate of rise, amplitude and maximum rate of fall of the sodium spike. These effects occurred independently of the galanin-induced hyperpolarization. 5. Galanin decreased the amplitude and duration of TTX-insensitive spikes initiated in cells maintained in a solution containing 9 mM-calcium, 20 mM-TEA and 1.5 microM-TTX. 6. These results suggest that a galanin-like peptide may act as an inhibitory transmitter in the mudpuppy cardiac ganglion.

Action Potentials