Validation of Salamander Dermal Mucus Swabs as a Novel, Nonlethal Approach for Amphibian Metabolomics and Glutathione Analysis Following Pesticide Exposure.

Evaluating biomarkers of stress in amphibians is critical to conservation, yet current techniques are often destructive and/or time-consuming, which limits ease of use. In the present study, we validate the use of dermal swabs in spotted salamanders (Ambystoma maculatum) for biochemical profiling, as well as glutathione (GSH) stress response following pesticide exposure. Thirty-three purchased spotted salamanders were acclimated to laboratory conditions at Washington College (Chestertown, MD, USA) for 4 weeks. Following acclimation, salamanders were randomly sorted into three groups for an 8-h pesticide exposure on soil: control with no pesticide, 2,4-dichlorophenoxyacetic acid (2,4-D), or chlorpyrifos. Before and after exposure, mucus samples were obtained by gently rubbing a polyester-tipped swab 50 times across the ventral and dorsal surfaces. Salamanders were humanely euthanized and dissected to remove the brain for acetylcholinesterase and liver for GSH and hepatic metabolome analyses, and a whole-body tissue homogenate was used for pesticide quantification. Levels of GSH were present in lower quantities on dermal swabs relative to liver tissues for chlorpyrifos, 2,4-D, and control treatments. However, 2,4-D exposures demonstrated a large effect size increase for GSH levels in livers (Cohen's d = 0.925, p = 0.036). Other GSH increases were statistically insignificant, and effect sizes were characterized as small for 2,4-D mucosal swabs (d = 0.36), medium for chlorpyrifos mucosal swabs (d = 0.713), and negligible for chlorpyrifos liver levels (d = 0.012). The metabolomics analyses indicated that the urea cycle, alanine, and glutamate metabolism biological pathways were perturbed by both sets of pesticide exposures. Obtaining mucus samples through dermal swabbing in amphibians is a viable technique for evaluating health in these imperiled taxa. Environ Toxicol Chem 2024;43:1126-1137. © 2024 SETAC.

Adenosine suppresses exocytosis from cone terminals of the salamander retina.

In the retina, adenosine is released in the dark and has been shown to inhibit Ca2+ influx through voltage-gated Ca2+ channels in cones. Therefore, we tested whether adenosine can inhibit exocytosis from isolated cone photoreceptors. Simultaneous measurements of membrane exocytosis and Ca2+ were made from cones using the activity-dependent dye, Synaptored-C2, and the Ca2+ indicator dye, Fluo-4. Adenosine suppressed exocytosis in cones, indicating that transmitter release is also reduced from cone terminals, and further supports an inhibitory mechanism for modulating transmitter release onto second-order neurons. Furthermore, this raises the possibility that adenosine might be neuroprotective for photoreceptors and second-order neurons by suppressing Ca2+ levels in cones and reducing exocytosis of L-glutamate, respectively.

Goalpha labels ON bipolar cells in the tiger salamander retina.

By using double-label immunocytochemistry and confocal microscopy, we studied rod and cone synaptic contacts, photoreceptor-bipolar cell convergence, and patterns of axon terminal ramification of ON bipolar cells in the tiger salamander retina. An antibody to recoverin, a calcium-binding protein found in photoreceptors and other retinal neurons in various vertebrates, differentially labeled rods and cones by lightly staining rod cell bodies, axons, and synaptic pedicles and heavily staining cone cell bodies and pedicles. An antibody to G(oalpha) labeled most ON bipolar cells, with axon terminals ramified mainly in strata 6-9 and a minor band in stratum 3 of the inner plexiform layer (IPL). Stratum 10 of the IPL was G(oalpha) negative, and previous studies showed that axon terminals of rod-dominated ON bipolar cells are monostratified in that stratum. The axonal morphology of G(oalpha)-positive cells resembled that of the cone-dominated (DBC(C)) or mixed rod and cone ON (DBC(M)) bipolar cells. The G(oalpha)-positive dendritic processes made close contact with all cone pedicles and superficial contact with some rod pedicles, consistent with the idea that G(oalpha) subunits are present in DBC(C)s and DBC(M)s. The size and density of these cells were analyzed, and their spatial distributions were determined. To our knowledge, this is the first study to characterize photoreceptor inputs and axon terminal morphology of a population of ON bipolar cell with the use of a G(oalpha) antibody as an immunomarker in the salamander retina.

Pharmacological characterization of P2Y receptor subtypes on isolated tiger salamander Müller cells.

Müller cells express a variety of neurotransmitter receptors that permit them to "sense" the extracellular environment within the retina. We have used a battery of agonists and antagonists to characterize the purinergic receptor subtypes expressed on isolated tiger salamander Müller cells. Changes in intracellular calcium ion concentration ([Ca(2+)](i)) in Müller cells were measured using the Ca(2+) indicator dye Fura-2 and digital imaging microscopy. ATP, 2-methylthio-ATP, 2-methylthio-ADP, ADP, UTP, UDP, deoxyATP, and 3'-O-(4-benzoyl)benzoyl ATP evoked increases in [Ca(2+)](i) in both the presence and absence of extracellular Ca(2+). Therefore, the increases we observed were likely due to intracellular Ca(2+) release mediated by G-protein-coupled P2Y receptor activation, rather than Ca(2+) influx via P2X receptor channels. The P2Y(1) receptor agonists 2-methylthio-ATP, 2-methylthio-ADP, and ADP evoked increases in [Ca(2+)](i) that were inhibited by the P2Y(1) receptor antagonists adenosine 3'-phosphate 5'-phosphosulfate and 2'-deoxy-N(6)-methyleneadenosine-3',5'-bisphosphate. Responses to ADP were not completely inhibited by the P2Y(1) receptor antagonists. The residual response to ADP could be mediated by P2Y(13) receptors. UTP evoked an increase in [Ca(2+)](i) that was partially inhibited by suramin, suggesting that Müller cells express P2Y(2) and P2Y(4) receptors. The P2Y(6) receptor agonist UDP, and the P2Y(11) receptor agonists deoxyATP, and 3'-O-(4-benzoyl)benzoyl ATP, evoked increases in [Ca(2+)](i) in Müller cells. We conclude that isolated tiger salamander Müller cells express P2Y(1), P2Y(2), P2Y(6), P2Y(11), and possibly P2Y(4) and P2Y(13) receptors. Therefore, the physiological release of ATP, ADP, UTP, and UDP and/or their accumulation in the retina under pathological conditions could stimulate increases in [Ca(2+)](i) in Müller cells.

Expression of fibroblast growth factors 4, 8, and 10 in limbs, flanks, and blastemas of Ambystoma.

Members of the fibroblast growth factor (FGF) family of molecules are critical to limb outgrowth. Here, we examine the expression of Fgfs in three types of limbs-embryonic (developing), mature (differentiated), and regenerating-as well as in the surrounding non-limb tissues in the Mexican axolotl, Ambystoma mexicanum. We have previously cloned partial cDNAs of Fgf4, 8, and 10 from the axolotl (Christensen et al., 2001); the complete Fgf10 cDNA sequence is presented here. Axolotl Fgf10 showed deduced amino acid sequence identity with all other vertebrate Fgf10 coding sequences of >62%, and also included conserved 5' and 3' untranslated regions in nucleotide sequence comparisons. Semiquantitative reverse transcriptase-polymerase chain reaction showed that fibroblast growth factors are differentially expressed in axolotl limbs. Only Fgf8 and 10 were highly expressed during axolotl limb development, although Fgf4, 8, and 10 are all highly expressed during limb development of other vertebrates. Fgf4 expression, however, was highly expressed in the differentiated salamander limb, whereas expression levels of Fgf8 and 10 decreased. Expression levels of Fgf8 and 10 then increased during limb regeneration, whereas Fgf4 expression was completely absent. In addition, axolotl limb regeneration contrasted to limb development of other vertebrates in that Fgf8 did not seem to be as highly expressed in the distal epithelium; rather, its highest expression was found in the blastema mesenchyme. Finally, we investigated the expression of these Fgfs in non-limb tissues. The Fgfs were clearly expressed in developing flank tissue and then severely downregulated in mature flank tissue. Differential Fgf expression levels in the limb and shoulder (limb field) versus in the flank (non-limb field) suggest that FGFs may be instrumental during limb field specification as well as instrumental in maintaining the salamander limb in a state of preparation for regeneration.

Dopamine D4 receptor-mediated regulation of rod opsin mRNA expression in tiger salamander.

Light stimulates dopamine release in the retina and has been shown to rapidly up-regulate rod opsin mRNA. In the present study, we tested the effect of dopamine on rod opsin mRNA expression and examined the hypothesis that dopamine can mediate a light-evoked increase in opsin gene expression. Northern blots showed that a 30-min light-exposure increased rod opsin mRNA expression 27%. In situ hybridization on isolated rods showed that 500 nM dopamine and 1 microM quinpirole (dopamine D2/D3/D4 agonist) increased opsin mRNA 45% and 26%, respectively. The effect of quinpirole was selectively blocked by the D4 antagonist, L750,667 (20 microM). In very low density cultures, quinpirole increased opsin expression 46%, suggesting a direct effect on rod photoreceptors. Consistent with a dopamine D4 receptor mechanism, 1 microM H-89 (protein kinase A inhibitor) increased opsin mRNA 39%. Finally, intravitreal injection of quinpirole increased opsin mRNA 21% whereas injection of L750,667 (10 microM) blocked the light-evoked increase in opsin expression. These data show that rod opsin mRNA is up-regulated by dopamine binding a D4-like receptor on rods, possibly through inhibition of protein kinase A, and that endogenous dopamine can mediate the light-evoked increase in opsin mRNA expression.

Ontogeny of neurohormonal peptides, serotonin, and nitric oxide synthase in the gastrointestinal neuroendocrine system of the axolotl (Ambystoma mexicanum): an immunohistochemical analysis.

The ontogeny of the neurohormonal peptides vasoactive intestinal polypeptide (VIP), neurotensin (NT), substance P (SP), calcitonin gene-related peptide (CGRP), gastrin/cholecystokinin (GAS/CCK), and somatostatin (SOM) as well as serotonin (SER) and nitric oxide synthase (NOS) was investigated in the gastrointestinal tract of the urodele Ambystoma mexicanum, the axolotl, using immunohistochemical techniques. The first regulatory substances to appear were SP, SOM, and SER that could be immunohistochemically detected up from stage 1. At early stage 2, VIP immunoreactivity was observed infrequently in enteric nerve fibers. With the onset of external feeding at late stage 2, SP-immunoreactive (IR) and SER-IR endocrine cells and VIP-IR nerve fibers were present throughout the gastrointestinal tract. Furthermore, in the small intestine NT-IR and GAS/CCK-IR endocrine cells appeared. At stage 3, SER immunoreactivity was observed not only in endocrine cells but also in nerve fibers. CGRP-IR and SP-IR nerve fibers were detectable at stage 4 and stage 5, respectively. From stage 5 on, a minority of the CGRP immunoreactivity occurred in SP-IR nerve fibers. NOS immunoreactivity did not appear before stage 6 when it was found infrequently in nerve fibers. Thus, several phases of development can be distinguished: (1) at the yolk sac stages only few regulatory substances are present. (2) At the onset of external feeding, all endocrine cell types investigated were readily detectable. Thus, the onset of external feeding seems to trigger the development of the gastrointestinal endocrine system. (3) The endocrine cells are first found in the proximal part of the gastrointestinal tract and later in higher numbers in the distal parts. (4) The dually distributed neurohormonal peptides and SER first appear in endocrine cells and later additionally in nerve fibers. Thus, the nerve fibers likely set up the fine regulation of gastrointestinal blood flow and motility.

Differential modulation of rod and cone calcium currents in tiger salamander retina by D2 dopamine receptors and cAMP.

Synaptic transmission from vertebrate photoreceptors involves activation of L-type calcium currents (ICa). Dopamine is an important circadian neuromodulator in the retina and photoreceptors possess D2 dopamine receptors. We examined modulation of ICa by dopamine and cAMP in retinal slices and isolated cells of larval tiger salamander. Results show that dopamine and a D2 agonist, quinpirole, enhanced ICa in rods and red-, blue- and UV-sensitive small single cones but inhibited ICa in red-sensitive large single cones. A D1 agonist, SKF-38393, was without effect. Quinpirole effects were blocked by pertussis toxin (PTx) pretreatment indicating involvement of PTx-sensitive G-proteins. Like dopamine, inhibition of cAMP-dependent protein kinase (PKA) by Rp-cAMPS enhanced ICa in rods and small single cones, but inhibited ICa in large single cones. In contrast, forskolin and Sp-cAMPS, which stimulate PKA, inhibited ICa in rods and small single cones but enhanced ICa in large single cones. Sp-cAMPS also occluded effects of quinpirole. These results suggest that D2 receptors modulate ICa via inhibition of cAMP. Differences among the responses of photoreceptors to cAMP are consistent with the possibility that small single cones and rods may possess different Ca2+ channel subtypes than large single cones. The results with dopamine and quinpirole showing inhibition of ICa in large single cones and enhancement of rod ICa were unexpected because previous studies have shown that dopamine suppresses rod inputs and enhances cone inputs into second-order neurons. The present results therefore indicate that the dopaminergic enhancement of cone inputs does not arise from modulation of photoreceptor ICa.

Fate and the biochemical effects of 2,4,6-trinitrotoluene exposure to tiger salamanders (Ambystoma tigrinum).

Biotransformation, metabolic enzyme profiles, and the glutathione antioxidant system in tiger salamanders (Ambystoma tigrinum) from a 14-day exposure to 2,4,6-trinitrotoluene (TNT) in situ were examined. Concentrations of parent compound and metabolites were measured in skin, kidney, and liver tissue. In addition, cytochrome P450 (P450) and cytochrome b5 content and their dependent isozyme activities, ethoxyresorufin O-dealkylation, pentoxyresorufin O-dealkylation, and the glutathione antioxidant system in the skin, liver, lung, kidneys, and blood were evaluated. Considerable differences were found in relative concentrations of TNT and its metabolites in the skin, relative to the liver and kidney. Trace amounts of TNT were detected only in the skin and liver of exposed animals while one of the secondary reduction metabolites, 2,6-diaminonitrotoluene, was found only in liver and kidney. Differences in the metabolite concentrations between systemic organs (liver, kidneys) and the skin suggest that the skin may be important in the primary reduction of TNT. In addition, measurable levels of these basal enzyme indicators were detected; yet of those evaluated only hepatic P450 content was affected by TNT exposure. The qualitative and quantitative differences in TNT and its metabolites in tissues suggest the fate and metabolism of the TNT in salamanders. Furthermore, results indicate that tiger salamanders possess considerable levels of xenobiotic metabolizing and antioxidant enzymes in these tissues but are not sensitive indicators of TNT exposure.

Immunocytochemical localization of gamma-aminobutyric acid plasma membrane transporters in the tiger salamander retina.

Gamma-aminobutyric acid (GABA) plasma membrane transporters (GATs) play an important role in regulating GABA neurotransmission in the nervous system. The distribution of two GATs, GAT 1 and GAT 3, in salamander retina was investigated by using affinity-purified polyclonal antisera directed to the predicted C-terminals of rat GAT 1 and rat GAT 3. GAT 1-immunoreactivity (-IR) was found in type IB and IIB orthotopic bipolar cells (BCs) located in the distal and middle of the inner nuclear layer (INL), respectively; in type IIA and IA amacrine cells (ACs) located in the middle and proximal INL, respectively; and in interplexiform cells and cells in the ganglion cell layer (GCL). No detectable staining was found in horizontal cells (HCs) or in structures resembling Müller cells. GAT 1-immunoreactive fibers were present in the outer plexiform layer (OPL) and inner plexiform layer (IPL) in three bands corresponding to the three bands previously reported to be GABA-IR. GAT 3 antibodies labeled fewer cells and cell types than GAT 1 antibodies. GAT 3-IR was localized to type IIA and IA ACs and cells in the GCL, but not to BCs, HCs, or Müller cell-like structures. There was weak labeling of the OPL and stronger labeling of the IPL, with three distinct bands at the same depth as observed with GAT 1-IR. Double-labeling showed that the majority of GAT 1-IR BCs (88%), ACs (88%), and cells in the GCL (78%) colocalized with GABA-IR. The present study provides the first direct evidence of the expression of two GAT subtypes in neurons of nonmammalian retinas. These transporters could regulate GABA neurotransmission by reuptake and termination of GABA's action and, perhaps, by GABA release mechanisms. The presence of GAT 1-IR/GABA-IR bipolar cells further supports our earlier observations that a subgroup of orthotopic bipolar cells are likely to be GABAergic.

Ambient UV-B radiation causes deformities in amphibian embryos.

There has been a great deal of recent attention on the suspected increase in amphibian deformities. However, most reports of amphibian deformities have been anecdotal, and no experiments in the field under natural conditions have been performed to investigate this phenomenon. Under laboratory conditions, a variety of agents can induce deformities in amphibians. We investigated one of these agents, UV-B radiation, in field experiments, as a cause for amphibian deformities. We monitored hatching success and development in long-toed salamanders under UV-B shields and in regimes that allowed UV-B radiation. Embryos under UV-B shields had a significantly higher hatching rate and fewer deformities, and developed more quickly than those exposed to UV-B. Deformities may contribute directly to embryo mortality, and they may affect an individual's subsequent survival after hatching.

Enkephalin immunoreactivity in the paraneurons of the tiger salamander (Ambystoma tigrinum) tongue.

Immunohistochemical tests have demonstrated the presence of leu-5-enkephalin and other bioactive compounds (serotonin and neuron-specific enolase) in the basal cells of lingual taste buds in Ambystoma tigrinum; there was also a weak reaction for met-5-enkephalin. Similar reactions were obtained from particular cells dispersed within the lingual epithelium, which are provisionally identified as Merkel cells.

Immunofluorescent studies on titin and myosin in developing hearts of normal and cardiac mutant axolotls.

Homozygous recessive cardiac mutant gene c in the axolotl, Ambystoma mexicanum, results in a failure of the embryonic heart to initiate beating. Previous studies show that mutant axolotl hearts fail to form sarcomeric myofibrils even though hearts from their normal siblings exhibit organized myofibrils beginning at stage 34-35. In the present study, the proteins titin and myosin are studied using normal (+/+) axolotl embryonic hearts at stages 26-35. Additionally, titin is examined in normal (+/c) and cardiac mutant (c/c) embryonic axolotl hearts using immunofluorescent microscopy at stages 35-42. At tailbud stage 26, the ventromedially migrating sheets of precardiac mesoderm appear as two-cell-layers. Myosin shows periodic staining at the cell peripheries of the presumptive heart cells at this stage, whereas titin is not yet detectable by immunofluorescent microscopy. At preheartbeat stages 32-33, a myocardial tube begins to form around the endocardial tube. In some areas, periodic myosin staining is found to be separated from the titin staining; other areas in the heart at this stage show a co-localization of the two proteins. Both titin and myosin begin to incorporate into myofibrils at stage 35, when normal hearts initiate beating. Additionally, areas with amorphous staining for both proteins are observed at this stage. These observations indicate that titin and myosin accumulate independently at very early premyofibril stages; the two proteins then appear to associate closely just before assembly into myofibrils. Staining for titin in freshly frozen and paraffin-embedded tissues of normal embryonic hearts at stages 35, 39, and 41 reveals an increased organization of the protein into sarcomeres as development progresses. The mutant siblings, however, first show titin staining only limited to the peripheries of yolk platelets. Although substantial quantities of titin accumulate in mutant hearts at later stages of development (39 and 41), it does not become organized into myofibrils as in normal cells at these stages.

Phototaxic behavior and the retinotectal transport of horseradish peroxidase (HRP) in surgically created cyclopean salamander larvae (Ambystoma).

UNLABELLED: Negative phototaxis (NP) was used to evaluate the recovery of vision in albino axolotl larvae with one eye discarded and the other transplanted either to the orbit (orthoclops) or to the top of the head (cyclops). NP was assessed at approximately 1, 2 and 3 months postoperatively, using an automated, infrared monitor. Some 88% of the orthoclopes and 64% of the cyclopes recovered NP. However, among the cyclopes that did recover, the quantitative aspects of NP were virtually the same as those of the orthoclopes. That the cyclopean eye can regenerate retinotectal pathways was established by anterograde tracing of horseradish peroxidase (HRP). But where previously uninjured animals transported HRP to the contralateral tectum, both the cyclopes and the orthoclopes distributed the enzyme to the left and right tectal halves. Heavy deposits of HRP were found in the tecta of some animals that lacked NP. To find out if an optic tectum is actually required for NP, a series of ablation experiments were performed, using Ambystoma punctatum larvae. Tectectomy had the same effect on NP as bilaterally extirpating the eyes or intracranially severing both optic nerves, i.e. removing the tectum abolished NP. THE RESULTS: (1) confirm the efficacy of the ectopic eye in the cyclops preparation; (2) show that the ectopic eye can regenerate retinotectal pathways; (3) indicate that retinotectal contact is a necessary but insufficient condition for NP.

Peripheral projections of nervus terminalis LHRH-containing neurons in the tiger salamander, Ambystoma tigrinum.

The peripheral projections of the nervus terminalis (NT) have been difficult to examine due to the weak immunoreactivity of the processes to various antibodies. We performed two experimental manipulations in the tiger salamander in an attempt to increase the luteinizing hormone-releasing hormone-immunoreactive (LHRH-ir) labelling in the peripheral processes of the NT:1) the NT was sectioned centrally, or 2) a 100 mg melatonin pellet was embedded subcutaneously for 3 days prior to sacrifice. Following these manipulations, animals were sacrificed and tissue was processed with standard immunocytochemical techniques for the analysis of the distribution of LHRH-ir processes. In the nasal cavity, LHRH-ir fibers were observed projecting 1) into the rostral olfactory epithelium, 2) to Bowman's glands in the lamina propria of the rostromedial olfactory mucosa and ventrolateral mucosa between the main nasal cavity and Jacobson's organ, 3) into the naris constrictor muscle, and 4) along the palatine nerves and ganglia. These lesion and hormone manipulations have enabled the detection of peripheral projections of the NT not observed previously with immunocytochemical procedures alone. The wide distribution of LHRH-ir NT processes in the nasal cavity and cranium suggests that this nerve may influence many different cranial structures during appropriate pheromonal or neuroendocrine events.

Characterization of the glutamate transporter in retinal cones of the tiger salamander.

L-Glutamate elicits an inwardly rectifying current at hyperpolarized potentials in isolated retinal cones of the tiger salamander, as measured under whole-cell patch clamp. Evidence presented in this article supports the notion that cones possess a high-affinity glutamate transporter. This glutamate-elicited current shows no desensitization over a period of several minutes, and has an affinity (Km) of 10 microM. The inward current is mimicked by the amino acids L-aspartate, D-aspartate, L-cysteate, and to a lesser extent D-glutamate. It is neither blocked by the glutamate receptor antagonists kynurenic acid (1 mM), 6-cyano-7-nitroquinoxaline-2,3-dione (100 microM), or 2-amino-5-phosphonovalerate (100 microM), nor elicited by the glutamate receptor agonists (100 microM each) kainate, quisqualate, NMDA, or 2-amino-4-phosphonobutyrate. The glutamate-elicited current was reduced by the glutamate transport blockers dihydrokainate (DHKA), DL-threo-beta-hydroxyaspartate (beta HA), and L-trans-pyrrolidine-2,4-dicarboxylic acid. When glutamate was present on both sides of the membrane, the blockers reduced both uptake and release; the blocker-sensitive current as a function of membrane potential represents the transport current-voltage relation (I-V), and the reversal potential of the I-V represents the transporter equilibrium potential. This potential was a function of the equilibrium potential for glutamate. DHKA and beta HA depolarized horizontal cells in a retinal slice, and abolished their light responses, suggesting that in the absence of glutamate transport, glutamate concentrations in the cleft rise to a level that saturates the postsynaptic receptors. The high capacity of the cone glutamate transporter is well suited for the rapid removal of glutamate from the synaptic cleft required for the signaling of a light onset to postsynaptic cells.

Ionic selectivity of Ih channels of rod photoreceptors in tiger salamanders.

Ionic selectivity of Ih channels of tiger salamander rod photoreceptors was investigated using whole-cell voltage clamp. Measured reversal potentials and the Goldman-Hodgkin-Katz voltage equation were used to calculate permeability ratios with 20 mM K+ as a reference. In the absence of external K+, Ih is small and hard to discern. Hence, we defined Ih as the current blocked by 2 mM external Cs+. Some small amines permeate Ih channels, with the following permeability ratios (PX/PK):NH4+, 0.17; methylammonium, 0.06; and hydrazine, 0.04. Other amines are tially impermeant: dimethylammonium (< 0.02), ethylammonium (< 0.01), and tetramethylammonium (< 0.01). When K+ is the only external permeant ion and its concentration is varied, the reversal potential of Ih follows the Nernst potential for a K+ electrode. Ih channels are also permeable to other alkali metal cations (PX/PK): T1+, > 1.55; K+, 1; Rb+, > 0.55; Na+, 0.33; Li+, 0.02. Except for Na+, the relative slope conductance had a similar sequence (GX/GK): T1+, 1.07; K+, 1; Rb+, 0.37; NH4+, 0.07; Na+, 0.02. Based on permeabilities to organic cations, the narrowest part of the pore has a diameter between 4.0 and 4.6 A. Some permeant cations have large effects on the gating kinetics of Ih channels; however, permeant cations appear to have little effect on the steady-state activation curve of Ih channels. Lowering K+ or replacing K+ with Na+ reduces the maximal conductance of Ih but does not shift or change the steepness of its voltage dependence. With ammonium or methylammonium replacing K+ a similar pattern is seen, except that there is a small positive shift of approximately 10 mV in the voltage dependence.

Leydig cells in the lingual epithelium of the axolotl, Ambystoma mexicanum, are immunoreactive for serotonin.

The Leydig cells in the lingual epithelium of the axolotl were investigated by immunohistochemistry using serotonin antiserum. Serotonin-immunoreactivity was found in their secretory granules. The physiological role of serotonin in the Leydig cell, a type of exocrine cell, is unknown.

Isolation of lactose-binding lectins from axolotl (Ambystoma mexicanum).

1. Lactose-inhibitable hemagglutination activity was identified in extracts of axolotl (Ambystoma mexicanum) larvae. 2. Two types of lectin were isolated from extracts by affinity chromatography on lactose-Sepharose. 3. A thiol-independent lectin of subunit mol. wt 15 kDa and a thiol-dependent lectin of subunit mol. wt 18 kDa were identified. 4. The 15 kDa and a 18 kDa polypeptides were weakly reactive with polyclonal anti-human galaptin serum.

Reinvestigation of DNA ligase I in axolotl and Pleurodeles development.

We have recently shown that the exclusion process causing the replacement of DNA ligases II by DNA ligase I in amphibian eggs after fertilization does not occur in the case of Xenopus laevis [Hardy, S., Aoufouchi, S., Thiebaud, P., and Prigent, C., (1991) Nucleic Acids Res. 19, 701-705]. Since this result is in contradiction with the situation reported in axolotl and Pleurodeles we decided to reinvestigate such results in both species. Three different approaches have been used: (1) the substrate specificity of DNA ligase I; (2) the DNA ligase-AMP adduct reaction and (3) the immunological detection using antibodies raised against the X.laevis DNA ligase I. Our results clearly demonstrate that DNA ligase I activity is associated with a single polypeptide which is present in oocyte, unfertilized egg and embryo of both amphibians. Therefore, the hypothesis of a change in DNA ligase forms, resulting from an expression of the DNA ligase I gene in axolotl and Pleurodeles early development must be rejected. We also show that, in contradiction with published data, the unfertilized sea urchin egg contains a DNA ligase activity able to join blunt ended DNA molecules.