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PubMed1987

The mechanism of electrodiffusive K+ transport in leaky epithelia and some of its consequences for anion transport.

The ventricular membrane of the epithelium from the choroid plexus of Necturus maculosus was probed with double-barrelled ion-selective microelectrodes while the ventricular bathing solution was changed abruptly. The transient states induced by increasing the external concentration of K+ or by the application of ouabain showed that the passive movements of K+ across the ventricular membrane were electro-diffusive and could be described by the Goldmann equation and one constant permeability (PK) of 24 X 10(-6) cm s-1. The passive efflux was balanced by a ouabain-sensitive influx. PK was different in different steady states; when the cell was acidified by half a pH unit by increasing CO2 in the bathing solutions from 1 to 5%, then PK decreased to 13 X 10(-6) cm s-1. Removal of Cl- from the bathing solution increased PK by 50% and reduction of Cl- transport by furosemide did not alter PK, consequently major movements of K+ were independent of Cl- movements. Depolarizations of the cell caused an increase in the cellular HCO-3 concentration due to an electrodiffusive permeability (PHCO3), the value of which was estimated to 17 X 10(-6) cm s-1.

Animals↗
PubMed2002

Sodium absorption, volume control and potassium channels: in tribute to a great biologist.

It is well established, for all Na-absorbing epithelia, that an increase in the rate of transcellular Na+ absorption is accompanied by an increase in the conductance of the basolateral membrane to K+. For the case of small intestinal epithelial cells from the salamander Necturus maculosus, where the rate of transcellular Na+ absorption can be increased manyfold by the addition of sugars or amino acids to the luminal bathing solution, it appears that this parallelism between Na-K pump rate and basolateral membrane K+ conductance is closely related to volume regulation by the enterocyte. Recent studies have disclosed the presence of stretch-activated K+ channels, in a highly enriched basolateral membrane fraction isolated from these epithelial cells, whose activity is increased by an increase in vesicle volume and inhibited by a decrease in vesicle volume or ATP. The activity of this channel also appears to be regulated by the degree of organization of the cortical actin cytoskeleton; activity is increased by depolymerization of the actin cytoskeleton and decreased by repolymerization of that structure. We postulate that the inhibitory effect of ATP is related to its role in promoting the polymerization of G-actin to form F-actin. We propose that enterocyte swelling that results from the intracellular accumulation of sugars or amino acids in osmotically active forms brings about disorganization of the cortical actin cytoskeleton and activates these channels and is, at least in part, responsible for the "pump-leak parallelism" in this amphibian.

Absorption↗
PubMed1998

Protein kinase C and regulatory volume decrease in mudpuppy red blood cells.

This study examined whether protein kinase C (PKC) stimulates K+ efflux during regulatory volume decrease (RVD) in Necturus maculosus (mudpuppy) red blood cells (RBCs). The limit of osmotic fragility increased with the general protein kinase inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7, 10 micrometer), but not with the cyclic nucleotide-dependent kinase antagonists N-(2'-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004, 10 micrometer) and N-2-(methylamino)ethyl-5-isoquinoline-sulfonamide (H-8, 5 micrometer). Consistent with these results, osmotic fragility also increased with the PKC antagonists bisindolylmaleimide I (GF-109203X or bis I, 100 nm), bisindolylmaleimide II (bis II, 100 nm), and chelerythrine (10 micrometer). The effect of these three antagonists and H-7 was reversed with gramicidin (5 micrometer in a choline Ringer), indicating PKC was linked to K+ efflux (gramicidin is a cationophore that was used to ensure a high K+ permeability). We also measured cell volume recovery from hypotonic shock (0.5x Ringer) with a Coulter counter and estimated cell volume from the hematocrit. The percent RVD compared to control decreased with H-7 (10 micrometer), sphingosine (100 nm), chelerythrine (10 micrometer), bis I (100 nm), and bis II (100 nm), but not with HA-1004 (10 micrometer) nor H-8 (5 micrometer). Inhibition of RVD by H-7, chelerythrine, bis I, and bis II was reversed with gramicidin (5 micrometer). Furthermore, using the patch clamp technique, we found H-7 (10 micrometer) reduced a whole cell conductance that was activated during cell swelling. In addition, a conductance responsible for K+ efflux during cell swelling was inhibited by bis I (100 nm) and bis II (100 nm). These results indicate that a conductive pathway mediating K+ loss during RVD is regulated, at least in part, by protein kinase C.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2000

Chlorinated hydrocarbon concentrations in plasma of the Lake Erie water snake (Nerodia sipedon insularum) and northern water snake (Nerodia sipedon sipedon) from the Great Lakes basin in 1998.

From the Great Lakes basin, concentrations of 59 congener-specific polychlorinated biphenyls (PCBs) and 14 organochlorine pesticides were measured in blood plasma of northern water snake (Nerodia sipedon sipedon) and Lake Erie water snake (Nerodia sipedon insularum), which is endangered in Canada. In 1998, four male adult Lake Erie water snakes were sampled from Pelee Island, western Lake Erie; four male northern water snakes were sampled at Little Lake, about 20 km north of Parry Sound in central Ontario; and four adult gravid female northern water snakes were sampled from Garden Island, eastern Lake Ontario. The blood plasma was pooled by site for a total of three samples analyzed. The Pelee Island sample from male Lake Erie water snakes contained less than half the lipid concentration (0.349%) than samples from the other sites, but it was the most contaminated with PCBs, even on a wet weight basis. Summed concentration of individual PCBs in the Pelee Island sample was 167 ng/g (wet weight), which was 14-fold higher than the next most contaminated sample, which was from Little Lake. The plasma sample from Little Lake contained 12 ng/g (WW) and was four times more contaminated with PCBs than the sample from female snakes from Garden Island, Lake Ontario. Organochlorine pesticide concentrations in plasma were relatively similar among sites. None of the pesticides was found above trace concentrations (0.1-0.9 ng/g) except pp'-DDE, which occurred at 2-5 ng/g among sites. PCB congener patterns in the Lake Erie water snakes were compared to PCB patterns in plasma of common snapping turtle (Chelydra serpentina serpentina) from Lake Ontario, herring gull eggs (Larus argentatus) from western Lake Erie, and mudpuppy eggs (Necturus maculosus) from the Detroit River. The PCB patterns in water snake and herring gull sample were most similar, followed by the pattern in snapping turtle plasma. The presence of more lower-chlorinated chlorobiphenyls in the mudpuppy eggs relative to the other species made this sample distinct from the water snake, gull, and turtle.

Animals↗
PubMed1991

Role of Na+/K+ exchange and organic acids in base induced hyperpolarization of renal proximal amphibian tubule.

Fast peritubular alkaline perturbations in Necturus renal proximal tubule evoke hyperpolarizations of the basolateral membrane. These voltage changes are partly due to an increase in basolateral K(+)-permeability. Additional role of the Na(+)/K(+)-ATPase and organic acids in generating these base induced hyperpolarizations (BIH) can be deduced from the reduction in BIH during low K+, high amiloride or omission of organic acids.

Amino Acids↗
PubMed1985

Structure and function of urodele myelin lacking alpha-hydroxy fatty acid-containing galactosphingolipids: slow nerve conduction and unusual myelin thickness.

Myelin of several Caudata (Urodela) species appears to be unique in the fact that it lacks hydroxycerebrosides and hydroxysulfatides although it contains their non-hydroxy counterparts. Comparison of the nerve conduction velocities in the Urodeles Necturus (salamander) and Notophthalmus (newt) with that in a reptile, Anolis (chameleon) which contains hydroxycerebrosides and -sulfatides indicated that the values were significantly reduced in the urodeles. Furthermore, urodele myelin thickness remained uniformly the same regardless of the size of the nerve fiber. Despite these differences the myelins appeared structurally similar. Electron microscopic and X-ray diffraction studies did not disclose any structural difference between the two orders. A teased fiber technique established that the ratio of internodal distance and fiber diameter in urodele nerves was essentially similar to that in Anolis. These findings suggest that the absence of hydroxycerebroside and -sulfatide may be related to the reduction in nerve conduction velocity and unusual myelin thickness in the urodele nervous system.

Animals↗
PubMed1992

An in vitro preparation of the mudpuppy for simultaneous intracellular and electromyographic recording during locomotion.

In this report we describe the development of an in vitro preparation of the mudpuppy (Necturus maculatus) used to investigate locomotion in walking vertebrates. The preparation consists of the first 5 segments of the cervical spinal cord and the attached forelimb. The preparation is bathed in a cooled (15 degrees C) and oxygenated spinal cord Ringers solution and remains viable for 36-100 h. Locomotion can be elicited during the first 36-48 h by applying the excitatory amino acid N-methyl D-aspartate (NMDA) to the bath. Cutaneous and dorsal root reflexes remain unchanged for much longer periods of time (72-100 h). During locomotion, intracellular recordings can be made from interneurons and motoneurons while simultaneous electromyographic (EMG) recordings are made from forelimb muscles. Rhythmically active interneurons can be classified according to their phase of activity during the step cycle. Further classification of interneurons involves both monitoring the afferent input to these cells from dorsal root and cutaneous afferents as well as using their action potentials as a trigger for averaging the ongoing locomotor EMG activity. In this way some of the input and output characteristics of the interneurons can be monitored. The ability to record simultaneously from interneurons and muscles offers distinct advantages over current in vitro preparations.

Animals↗
PubMed1987

Distribution of calcitonin gene-related peptide immunoreactive nerve fibers in the mudpuppy cardiac septum.

An immunohistochemical study was undertaken to determine the distribution of calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers in the cardiac septum of the mudpuppy, Necturus maculosus. Numerous long, CGRP-immunoreactive nerve fibers course across the septum, run in the nerve trunks connecting clusters of postganglionic parasympathetic cells, form complexes over groups of ganglion cells and make pericellular networks around individual ganglion cells. The postganglionic parasympathetic neurons and small intensely fluorescent (SIF)-like cells did not exhibit CGRP immunoreactivity. Most of the CGRP-immunoreactive nerve fibers also are labeled for substance P. In freshly dissected preparations, the staining pattern for CGRP was not similar to that obtained using an antiserum against synaptic vesicle membrane, which appears to preferentially label cholinergic preganglionic terminals on all postganglionic parasympathetic cells in the mudpuppy preparation. Further, in explanted ganglia (maintained 10 days in culture) almost no reactivity was obtained with the antivesicle antiserum whereas numerous nerve fibers still exhibited CGRP-immunoreactivity. These observations demonstrate that the CGRP-immunoreactive nerve fibers are not parasympathetic preganglionic axons. Rather we suggest that the CGRP-immunoreactive nerve fibers are processes of primary sensory fibers.

Animals↗
PubMed1990

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↗
PubMed1990

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↗
PubMed1989

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↗
PubMed1991

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↗
PubMed1985

Capacitative transients in voltage-clamped epithelia.

In voltage-clamped epithelia the cell membrane potential transient during a + 10-mV transepithelial pulse conforms to the expected behavior for a series combination of two linear resistance-capacitance (RC) circuits. The evolution of the cell potential is characterized by a single time constant with values of 30-130 ms in frog skin and Necturus gallbladder. These observations have important consequences for the measurement of cell membrane resistance ratios and the interpretation of current-voltage relations.

Amiloride↗
PubMed1991

Ba2+ release from soda glass modifies single maxi K+ channel activity in patch clamp experiments.

Glasses used to fabricate patch pipettes may release components which affect ion channels (Cota, G., and C.M. Armstrong. 1988. Biophys. J. 53:107-109; Furman, R.E., and J.C. Tanaka. 1988. Biophys. J. 53:287-292; Rojas, L., and C. Zuazaga. 1988. Neurosci. Lett. 88:39-44). The gating properties of maxi K+ channels from Necturus gallbladder epithelium depend on whether borosilicate glass (BG) or blue tip hematocrit glass (SG) is used to construct the patch pipettes. The data are consistent with solubilization from SG of a component which exerts voltage-dependent, cytosolic-side specific block, closely resembling "slow block" by Ba2+ ions. Ringer's solution preincubated with SG, but not with BG, blocked inside-out maxi K+ channels when used as bathing solution. Mass spectrometry revealed that Ba2+ is released by the glass from fast and slow-release compartments (SG contains 3% wt/wt BaO), and is the only ion found in the solution at concentrations consistent with the observed channel block. Additionally, SG released O2-, Na+, Ca2+, and Mg2+, all to micromolar concentrations. These elements do not interfere with maxi K+ channels but they could in principle alter the properties of other ion channels. Thus, screening for channel-modifying substances released by the glass may be necessary for the adequate interpretation of patch-clamp results.

Animals↗
PubMed2000

Pituitary adenylate cyclase-activating polypeptide innervation of the mudpuppy cardiac ganglion.

The presence and potential origin of the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) was determined in cardiac ganglia of the mudpuppy, Necturus maculosus. Although PACAP has been implicated in the regulation of cardiac function in several mammalian species, the presence of this peptide in the autonomic nervous system (ANS) of other species is unclear. Thus, this study is the first to characterize this highly conserved peptide in the ANS of a non-mammalian species. PACAP-immunoreactivity was observed in nerve fibers throughout the mudpuppy cardiac ganglia and often was co-localized with the sensory neuropeptides substance P and calcitonin gene-related peptide. Removal of all extrinsic inputs to the ganglia by organ culture eliminated PACAP-immunoreactivity in the cardiac ganglia, whereas bilateral vagotomies only partially reduced PACAP-labeling. PACAP-immunoreactive neurons were observed in both high thoracic dorsal root ganglia and in vagal sensory ganglia. While no PACAP-positive neurons were observed in caudal medulla brainstem regions, PACAP-containing nerve fibers were found in the region of the nucleus solitarius. These results suggest that, in the mudpuppy, PACAP is found primarily in visceral afferent fibers, originating from cells in either the dorsal root ganglia or vagal sensory ganglia. Based on their anatomic localization, these afferent fibers may function to transmit important sensory information to cardiovascular centers in the brain as well as serving as local reflex inputs to modulate postganglionic parasympathetic output within the cardiac ganglion itself.

Afferent Pathways↗
PubMedDate not supplied

Towards molecular understanding of species differences in dioxin sensitivity: initial characterization of Ah receptor cDNAs in birds and an amphibian.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related planar halogenated aromatic hydrocarbons (PHAHs) are highly toxic to most vertebrate animals, but there are dramatic species differences in sensitivity, both within and among vertebrate classes. For example, studies in cultured avian hepatocytes have revealed differential sensitivity of birds to PHAHs [Kennedy et al. (1996). Toxicol. Appl. Pharmacol., 141, 214-230]. Differences in the characteristics or expression of the aryl hydrocarbon receptor (AHR) could contribute to these species differences in PHAH responsiveness. To investigate the molecular mechanism of differential PHAH sensitivity, we have begun to characterize the AHR in white leghorn chicken (Gallus gallus), Pekin duck (Anas platyrhynchos), and common tern (Sterna hirundo), as well as an amphibian, mudpuppy (Necturus maculosus). Partial AHR cDNAs encompassing the helix-loop-helix and PAS domains were cloned and sequenced. Comparison of amino acid sequences in this region indicated a high degree of sequence conservation among the bird species (97% amino acid identity). The percent identity between bird sequences and either mouse or mudpuppy was lower (79%); the mudpuppy AHR was 74% identical to the mouse AHR. Phylogenetic analysis of these and other AHR amino acid sequences showed that the bird and mudpuppy AHRs were more closely related to mammalian and fish AHR1 forms than to fish AHR2. Future studies include the in vitro expression and functional characterization of AHRs from these and other non-mammalian vertebrates.

Amino Acid Sequence↗
PubMed2000

Intrafascicular electrodes for stimulation and recording from mudpuppy spinal roots.

This paper presents a technique for stimulating and recording from multiple intact spinal roots in the in vitro mudpuppy (Necturus maculatus) spinal cord-forearm preparation using fine wire electrodes, a modified intrafascicular electrode. We found that multiple spinal roots of the preparation could be implanted with these modified electrodes for independent stimulation or recording of the roots without inducing mechanical vibrations, disrupting conduction, or obscuring the view of or access to the spinal cord. Recording and stimulation performance using these electrodes was compared with results obtained using conventional hook electrodes. We found that intrafascicular electrodes were more efficient than hook electrodes for stimulating nerve fibers, being able to produce equivalent levels of activation using stimulation levels that were an order of magnitude smaller. Compound action potential signals recorded from electrodes implanted in the spinal roots were found to be larger than those from hook electrodes placed around the corresponding spinal nerve, showing that intrafascicular electrodes are more efficient at recording activity in the nerve. Moreover, it was possible to record evoked activity from cutaneous mechanoreceptors, even though the signal to noise ratio was low. Rough estimates of the conduction velocities for the fastest components in the compound action potentials were calculated and found to be around 17.5 m/s for both dorsal and ventral roots.

Action Potentials↗
PubMed1985

Coupling and uncoupling of amphibian neuroglia.

Glial cells in the optic nerve of Necturus are coupled to each other by low resistance pathways which also permit the diffusion of the fluorescent dye Lucifer Yellow CH among the cells. The spread of dye is readily observed as nuclei of cells distant from the site of intracellular injection are stained. By contrast, horseradish peroxidase does not traverse the intercellular pathways. This protein remains in the injected cell. The addition of weak acids (carbonic or propionic) to the bathing medium reversibly uncouples the glia; it blocks the spread of ionic current and Lucifer Yellow among the cells. A block of ionic coupling will block the spatial buffering of potassium by the glial syncytium.

Animals↗