Cane toad (Rhinella marina) vitamin A, vitamin E, and carotenoid kinetics.

Many amphibian species are threatened with extinction. Understanding their vitamin A (retinol), E (alpha-tocopherol), and carotenoid requirements is vital, as normal levels of these nutrients have a known connection to breeding success with abnormal levels leading to disease. This research examined vitamins A, E, and carotenoids (apocarotenoid, beta-carotene; beta-cryptoxanthin, lutein, zeaxanthin, and esters) concentration kinetics in the liver and plasma of 65 (57.8) cane toads (Rhinella marina) over 4 months supplemented with commercially available invertebrates in human care. Cane toads were opportunistically collected as part of a population control program for use as an amphibian model species. Toads were randomly assigned to one of two diets: treatment 1 was brown house crickets (Acheta domesticus) consuming Mazuri® Hi Calcium Gut Loading Diet without vitamin A or E supplement, plus fresh raw vegetables (carrot/sweet potato); Treatment 2 was the same diet except no vegetables. Ten toads were euthanized on Day 0 to analyze baseline free-ranging liver and plasma metabolites. Six toads consuming each treatment were euthanized on Days 22, 50, and 81, and n = 7 on Day 119 for analysis. Regardless of dietary treatment, most liver and blood metabolites were substantially higher at time 0 than all time points thereafter (p < .05); Ex: liver vitamin A at time 0 was 87.7 ± 16.12 µg/g while Day 119 for treatments 1 and 2 were 11.6 ± 1.19 and 8.2 ± 0.74, respectively. Few statistically significant differences between diets at the same time point were noted (p < .05). The results from this study indicate that additional or alternative diet supplementation may be needed for cane toads (and potentially other amphibians) to mimic their free-ranging diets.

Monitoring the ecotoxicity of γ-Al2O3 and Ni/γ-Al2O3 nanomaterials by means of a battery of bioassays.

The increasing application of nanoparticles (NPs) to a variety of new technologies has become a matter of concern due to the potential toxicity of these materials. Many questions about the fate of NPs in the environment and the subsequent impact on ecosystems need to be answered. The aim of this work was to evaluate the ecotoxicity of two alumina-based nanoceramics, γ-Al2O3 (NC) and Ni/ γ-Al2O3 (NiNC) by means of three different standardized tests: Biochemical Oxygen Demand (BOD5), bioassay with luminescent bacteria (Vibrio fischeri; Microtox), and bioassay on amphibian larvae (Rhinella arenarum) (AMPHITOX). BOD5 values of a very biodegradable mixture (glucose/glutamic acid) decreased with the addition of NiNC(43.8%) and NC (31.6%) with respect to control samples (52.9%). Microtox test results indicated that NiNC presents higher toxicity than NC, with EC50s values of 16.1% and 29.9% respectively; a reduced toxicity was observed, however, in presence of organic matter, thus obtaining EC50s of 37.8% and 19.4%. The results of AMPHITOX test showed a significant increase in the toxicity of both substances over time, the NiNC toxicity being greater than that of NC. The values of 96h-LC50 and 504h-LC50 determined for NiNC were 1.58 and 0.83mg/L, respectively, and 14.5 and 10.5mg/L for NC samples. Amphibian larvae exhibited collapsed cavities, edema, axial flexures, and behavioral alterations as hyperkinesia and reduced movements. These results evidence the vulnerability of wildlife to xenobiotics and the need to develop specific standardized ecotoxicity tests in order to help environmental sustainability and natural species conservation.

The Paraguayan Rhinella toad venom: Implications in the traditional medicine and proliferation of breast cancer cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Toads belonging to genus Rhinella are used in Paraguayan traditional medicine to treat cancer and skin infections. AIM OF THE STUDY: The objective of the study was to determine the composition of venoms obtained from three different Paraguayan Rhinella species, to establish the constituents of a preparation sold in the capital city of Paraguay to treat cancer as containing the toad as ingredient, to establish the effect of the most active Rhinella schneideri venom on the cell cycle using human breast cancer cells and to assess the antiprotozoal activity of the venoms. METHODS: The venom obtained from the toads parotid glands was analyzed by HPLC-MS-MS. The preparation sold in the capital city of Paraguay to treat cancer that is advertised as made using the toad was analyzed by HPLC-MS-MS. The effect of the R. schneideri venom and the preparation was investigated on human breast cancer cells. The antiprotozoal activity was evaluated on Leishmania braziliensis, L. infantum and murine macrophages. RESULTS: From the venoms of R. ornata, R. schneideri and R. scitula, some 40 compounds were identified by spectroscopic and spectrometric means. Several minor constituents are reported for the first time. The preparation sold as made from the toad did not contained bufadienolides or compounds that can be associated with the toad but plant compounds, mainly phenolics and flavonoids. The venom showed activity on human breast cancer cells and modified the cell cycle proliferation. The antiprotozoal effect was higher for the R. schneideri venom and can be related to the composition and relative ratio of constituents compared with R. ornata and R. scitula. CONCLUSIONS: The preparation sold in the capital city of Paraguay as containing the toad venom, used popularly to treat cancer did not contain the toad venom constituents. Consistent with this, this preparation was inactive on proliferation of human breast cancer cells. In contrast, the toad venoms of Rhinella species altered the cell cycle progression, affecting the proliferation of malignant cells. The findings suggest that care should be taken with the providers of the preparation and that the crude drug present a strong activity towards human breast cancer cell lines. The antiprotozoal effect of the R. schneideri venom was moderate while the venom of R. ornata was devoid of activity and that of R. scitula was active at very high concentration.

Nerve-muscle activation by rotating permanent magnet configurations.

KEY POINTS: The standard method of magnetic nerve activation using pulses of high current in coils has drawbacks of high cost, high electrical power (of order 1 kW), and limited repetition rate without liquid cooling. Here we report a new technique for nerve activation using high speed rotation of permanent magnet configurations, generating a sustained sinusoidal electric field using very low power (of order 10 W). A high ratio of the electric field gradient divided by frequency is shown to be the key indicator for nerve activation at high frequencies. Activation of the cane toad sciatic nerve and attached gastrocnemius muscle was observed at frequencies as low as 180 Hz for activation of the muscle directly and 230 Hz for curved nerves, but probably not in straight sections of nerve. These results, employing the first prototype device, suggest the opportunity for a new class of small low-cost magnetic nerve and/or muscle stimulators. ABSTRACT: Conventional pulsed current systems for magnetic neurostimulation are large and expensive and have limited repetition rate because of overheating. Here we report a new technique for nerve activation, namely high-speed rotation of a configuration of permanent magnets. Analytical solutions of the cable equation are derived for the oscillating electric field generated, which has amplitude proportional to the rotation speed. The prototype device built comprised a configuration of two cylindrical magnets with antiparallel magnetisations, made to rotate by interaction between the magnets' own magnetic field and three-phase currents in coils mounted on one side of the device. The electric field in a rectangular bath placed on top of the device was both numerically evaluated and measured. The ratio of the electric field gradient on frequency was approximately 1 V m(-2) Hz(-1) near the device. An exploratory series of physiological tests was conducted on the sciatic nerve and attached gastrocnemius muscle of the cane toad (Bufo marinus). Activation was readily observed of the muscle directly, at frequencies as low as 180 Hz, and of nerves bent around insulators, at frequencies as low as 230 Hz. Nerve-muscles, with the muscle elevated to avoid its direct activation, were occasionally activated, possibly in the straight section of the nerve, but more likely in the nerve where it curved up to the muscle, at radius of curvature 10 mm or more, or at the nerve end. These positive first results suggest the opportunity for a new class of small, low-cost devices for magnetic stimulation of nerves and/or muscles.

Serum and hepatic vitamin A levels in captive and wild marine toads (Bufo marinus).

The captive breeding program for the endangered Puerto Rican crested toad (Peltophryne [Bufo] lemur) has been hampered by an undiagnosed condition called "Brown Skin Disease" (BSD). Toads develop widespread skin darkening, skin thickening and abnormal shedding and eventually succumb to a chronic loss of viability. This project evaluated the marine toad (Bufo marinus) as a model for the PRCT, examining vitamin A deficiency as a potential cause of BSD. Wild caught marine toads had significantly higher liver vitamin A concentrations (61.89 ± 63.49 µg/g) than captive born marine toads (0.58 ± 0.59 µg/g); P<0.001). A significant difference in serum vitamin A concentration was found between the captive and wild caught toads (P=0.013) and between the low vitamin A-fed and wild caught toads (P=0.004), when controlling for liver vitamin A concentrations. After captive toads were treated with topical and/or oral vitamin A, their hepatic vitamin A concentrations were similar to those of the wild toads, averaging 48.41 ± 37.03 µg/g. However, plasma vitamin A concentrations pre- and post-vitamin A supplementation did not differ statistically. We concluded that plasma vitamin A concentrations do not provide a linear indication of liver/body vitamin A status, and that both topical and oral supplementation with an oil-based vitamin A formulation can increase liver stores in amphibians. No evidence of BSD or other signs of deficiency were noted in the marine toads, although this feeding trial was relatively short (127 days). To date, clinical, pathological and research findings do not support vitamin A deficiency as a primary factor underlying BSD.

[Study on display of acupuncture meridian based on electric signal transfer characteristics].

OBJECTIVE: To analyze the real-time effects of electroacupuncture (EA) stimulation by means of multiport network theory and measurement of the electric signals transfer coefficient, so as to explore a way for determining the running course of acupuncture meridian and for characterizing its physiological activities in the living body. METHODS: The body was modeled as a complex inhomogeneous 3-dimensions multi-port network, and the meridians were assumed to be "the most smoothly channels for signal transmission". Experiments were performed in 12 beheaded toads whose forelimbs and hindlimbs were divided into proportional coordinates. A concentric electrode with a conical tip was inserted into the toad leg for electrical stimulation of the local muscle, and another electrode alike was inserted into different spots of the limbs to detect the spreading signals in a 3-dimensional scanning mode. Following detecting the response electric voltage values of various spots and calculating their transfer coefficients, the spots which acquired a maximum signal value were considered to be the "acupoints". The imaginary connective lines passing the "acupoints" were regarded as the running courses of acupuncture meridians. RESULTS: A total of twelve 3-dimensional curves were detected based on the connected lines of electric signal transfer function extremum spots 2 mm beneath the skin of the ipsilateral fore- and hind-limbs of 12 spinal toads. CONCLUSION: The present study initially validates the feasibility of electric signal transfer coefficient measurement for displaying the running course of acupuncture meridian in the toad fore- and hindlimbs.

Effects of multiple chemical, physical, and biological stressors on the incidence and types of abnormalities observed in Bermuda's cane toads (Rhinella marina).

The interactive effects of contaminants and ultraviolet light (UV)-exposure on the incidence and types of abnormalities observed were measured in newly metamorphosed cane toads (Rhinella marina) from four Bermuda ponds contaminated with petrochemicals and metals. Abnormalities were compared in toadlets that were field-collected, reared in predator exclusion cages, reared in laboratory microcosms exposed to control media or corresponding pond media, and reared in laboratory microcosms exposed to UV-light and control media or media from two ponds. Percent abnormal for field-collected, cage-reared, and microcosm-reared toadlets were equivalent per site and ranged between 14% and 63%. All treatments produced similar limb abnormalities but the percentage of hind versus forelimb defects was statistically greater only in field-collected toadlets. UV-exposed control media did not induce abnormalities in larvae exhibiting no maternal effect, and did not alter the types of abnormalities observed in larvae exhibiting a maternal or latent effect. Site media treatments without UV exposure induced significant cephalic and limb abnormalities, proved additive to the observed maternal/latent effect, and produced limb defects predominantly in forelimbs. Concurrent exposure to site media and UV-light induced similar types of abnormalities but a significantly higher percentage of hind limb abnormalities (68-89%) than exposure to site media alone (7-13%). Our results suggest that the types of abnormalities expressed were principally determined by direct and/or transgenerational contaminant exposure, but that UV-light exposure caused limb abnormalities to occur primarily in the hind limbs, mirroring field observations. Our field observations also suggest that ectromelia and brachydactyly in some field-collected specimens may be predator-induced.

The toxicological effects of petroleum spray oils on insects - evidence for an alternative mode of action and possible new control options.

We tested the most widely held theory about the mode of action of petroleum spray oils (PSOs) on insects (i.e. anoxia). An nC24 petroleum oil was applied topically to cotton aphids (Aphis gossypii) and cluster caterpillars (Spodoptera litura), which then showed signs of mortality that are inconsistent with anoxia. The insects died soon after treatment, with most of the mortality occurring within the first 10min. Toxicity symptoms included loss of locomotory ability, unusual abdominal contractions associated with spiracular fluttering, and ultimately dehydration and necrosis within 24h. We therefore investigated the main mechanism(s) by which the nC24 petroleum oil interacts with the insects' cells and organs, and ultimately kills the insects. The results suggest a mode of action that relates to the liphophylic properties of the oil. This includes rapid penetration through the insect cuticle followed by accumulation in the lipid-containing tissues, mainly those of the CNS, and finally penetration into the nerve cells themselves. In vitro tests with isolated insect cells further revealed that the oil penetrates the cytoplasm and induces 100% mortality of these cells within 2min of application. No signs of oil accumulation within the tracheae were observed, so it is unlikely that anoxia is taking place at any stage of the intoxication process. Electrophysiological studies confirm that oil accumulation in the nerve ganglia has the direct effect of suppressing synaptic transmission in insect ganglia as well as in the neuromuscular junctions of vertebrates (toads and rats). These results demonstrate conclusively that at least some modern PSOs do not kill insects by anoxia, but by a range of cellular disruptions that lead to rapid insect death. The implications of our findings for the development of oil-based integrated pest management strategies are discussed.

On the anticonvulsant activity of kaurenic acid.

Kaurenic acid [(-)-kaur-16-en-19-oic acid] is a diterpene isolated from the aerial parts of Espeletia semiglobulata, one of 85 species of Espeletiinae found in Venezuela. Its anticonvulsive activity was studied using two different models of experimental seizures: spinal seizures induced by sudden cooling (SSSC) in amphibians and seizures induced by pentylenetetrazol (PTZ) in mice. In SSSC, kaurenic acid (KA) inhibited the tonic hind-limb extension with an ED50 of 2.5 mg/kg. It was 4-fold more potent than known anticonvulsant drugs such as carbamazepine and phenytoin and 100-fold more potent than valproic acid. However, KA as well as valproic acid were ineffective against the clonic phase of SSSC. In the PTZ-induced seizures, KA at doses of 0.625 and 1.25 mg/kg increased the latency of seizure onset and protected against generalized clonic-tonic seizures by 45% and 65%, respectively. The sedative effects of KA had an ED50 of 8.5 mg/kg in mice and 75 mg/kg in amphibians. This work provides experimental evidence supporting the potential value of kaurenic acid as an anticonvulsive drug.

Characterization of a major secretory protein in the cane toad (Bufo marinus) choroid plexus as an amphibian lipocalin-type prostaglandin D synthase.

Here we report the enzymatic and ligand-binding properties of a major secretory protein in the choroid plexus of cane toad, Bufo marinus, whose protein is homologous with lipocalin-type prostaglandin (PG) D synthase (L-PGDS) and is recombinantly expressed in Xenopus A6 cells and Escherichia coli. The toad protein bound all-trans retinal, bile pigment, and thyroid hormones with high affinities (K(d)=0.17 to 2.00 microM). The toad protein also catalysed the L-PGDS activity, which was accelerated in the presence of GSH or DTT, similar to the mammalian enzyme. The K(m) value for PGH(2) (17 microM) of the toad protein was almost the same as that of rat L-PGDS (14 microM), whereas the turnover number (6 min(-1)) was approximately 28 fold lower than that of rat L-PGDS. Site-directed mutagenesis based on a modeled structure of the toad protein revealed that Cys(59) and Thr(61) residues were crucial for the PGDS activity. The quadruple Gly(39)Ser/Ala(75)Ser/Ser(140)Thr/Phe(142)Tyr mutant of the toad protein, resembling mouse L-PGDS, showed a 1.6 fold increase in the turnover number and a shift in the optimum pH for the PGDS activity from 9.0 to 8.5. Our results suggest that the toad protein is a prototype of L-PGDS with a highly functional ligand-binding pocket and yet with a primitive catalytic pocket.

Interphotoreceptor retinoid-binding protein (IRBP) promotes the release of all-trans retinol from the isolated retina following rhodopsin bleaching illumination.

All-trans retinol generated in rod photoreceptors upon the bleaching of rhodopsin is known to move from the rods to the retinal pigment epithelium (RPE), where it is enzymatically converted to 11-cis retinal in the retinoid visual cycle. Interphotoreceptor retinoid-binding protein (IRBP) contained in the extracellular compartment (interphotoreceptor matrix) that separates the retina and RPE has been hypothesized to facilitate this movement of all-trans retinol, but the precise role of IRBP in this process remains unclear. To examine the activity of IRBP in the release of all-trans retinol from the rods, initially dark-adapted isolated retinas obtained from toad (Bufo marinus) eyes were bleached and then incubated in darkness for defined periods (5-180 min) in physiological saline (Ringer solution) supplemented with IRBP (here termed 'IRBP I') at defined concentrations (2-90 microm). Retinoids present in the retina and extracellular medium were then determined by extraction and HPLC analysis. Preparations incubated with > or =10 microm IRBP I showed a pronounced release of all-trans retinol with increasing period of incubation. As determined with 25 microm IRBP I, the increase of all-trans retinol in the extracellular medium was accompanied by a significant decrease in the combined amount of all-trans retinal and all-trans retinol contained in the retina. This effect was not mimicked by unsupplemented Ringer solution or by Ringer solution containing 25 or 90 microm bovine serum albumin. However, incubation with 'IRBP II', a previously described variant of IRBP with altered lectin-binding properties, led to the appearance of substantial all-trans retinol in the extracellular medium. The results suggest that in vivo, IRBP plays a direct role in the release of all-trans retinol from the rods during operation of the visual cycle.

Cannibalism in a population of the medicinal leech (Hirudo medicinalis L.).

Medicinal leeches (Hirudo medicinalis L.) were maintained in large ponds in a commercial leech farm at Biebertal, Germany. The feeding of hungry adult leeches was performed on representative individuals that were placed on cloth soaked with mammalian blood obtained from a local butchery (pig, Sus scrofa). In a second set of experiments, cane toads (Bufo marinus) were used as host organisms. The leeches rapidly attached to the toads, explored the body and sucked blood. After feeding, the fully engorged leeches were placed into the pond or an aquarium. In this artificial habitat, the satiated leeches were attacked by hungry conspecifics, sucked off and killed. This observation demonstrates that H. medicinalis must be classified as a cannibalistic annelid.

Bufo marinus oocytes as a model for ion channel protein expression and functional characterization for electrophysiological studies.

Oocytes from Xenopus laevis are commonly used as an expression system for ion channel proteins. The aim of this study was to determine whether oocytes from the Colombian native toad, Bufo marinus, could be used as an alternative expression system for ion channel protein expression and functional characterization using the two-microelectrode voltage clamp method. B. marinus oocytes and X. laevis were isolated and cultured in similar conditions. The mean resting membrane potential of B. marinus oocytes was similar to that of X. laevis oocytes as well as the whole-cell basal currents. The potassium ion channel Kv1.1 was successfully expressed in B. marinus oocytes and showed a typical outward rectifying current. Potassium channel blockers reduced these currents. The similarities on electrical properties and expression of ion channel proteins show that B. marinus oocytes can be used effectively to express these proteins, making these cells a viable heterologous system for the expression of ion channel proteins and their electrophysiological characterization.

Effects of low intensity infrared laser radiation on the water transport in the isolated toad urinary bladder.

BACKGROUND AND OBJECTIVES: The aim of this work was to study the effects of low intensity laser radiation on water transport in the toad bladder in vitro. STUDY DESIGN/MATERIALS AND METHODS: The water flow through the membrane was measured gravimetrically in bag preparations of the membrane. RESULTS: Laser radiation did not alter the water transport in the presence nor in the absence of vasopressin. In contrast, when the hemibladders were previously treated with vasopressin, the laser decreased by approximately 33.70% arginine-vasopressin (AVP)-mediated water transport. Laser radiation increased 3'5'-cyclic adenosine monophosphate (3'5'-cAMP) mediated water transport by approximately 23%. The association of laser radiation with indomethacin (IND) did not affect AVP-mediated water transport. CONCLUSIONS: This data suggests that the laser may have two effects on AVP-mediated water transport: one inhibitory effect on 3'5'-cAMP synthesis by inhibiting the adenylate cyclase complex and another stimulatory effect by inhibiting nucleotide-phosphodiesterase activity. Our results also indicate that the laser does not interfere in the prostaglandins biosynthesis induced by AVP.

Control of gonadotropin secretion by follicle-stimulating hormone-releasing factor, luteinizing hormone-releasing hormone, and leptin.

Fractionation of hypothalamic extracts on a Sephadex G-25 column separates follicle-stimulating hormone-releasing factor (FSHRF) from luteinizing hormone-releasing hormone (LHRH). The FSH-releasing peak contained immunoreactive lamprey gonadotropin-releasing hormone (lGnRH) by radioimmunoassay, and its activity was inactivated by an antiserum specific to lGnRH. The identity of lGnRH-III with FSHRF is supported by studies with over 40 GnRH analogs that revealed that this is the sole analog with preferential FSH-releasing activity. Selective activity appears to require amino acids 5-8 of lGnRH-III. Chicken GnRH-II has slight selective FSH-releasing activity. Using a specific lGnRH-III antiserum, a population of lGnRH-III neurons was visualized in the dorsal and ventral preoptic area with axons projecting to the median eminence in areas shown previously to control FSH secretion based on lesion and stimulation studies. Some lGnRH-III neurons contained only this peptide, others also contained LHRH, and still others contained only LHRH. The differential pulsatile release of FSH and LH and their differential secretion at different times of the estrous cycle may be caused by differential secretion of FSHRF and LHRH. Both FSH and LHRH act by nitric oxide (NO) that generates cyclic guanosine monophosphate. lGnRH-III has very low affinity to the LHRH receptor. Biotinylated lGnRH-III (10(-9) M) labels 80% of FSH gonadotropes and is not displaced by LHRH, providing evidence for the existence of an FSHRF receptor. Leptin has equal potency as LHRH to release gonadotropins by NO. lGnRH-III specifically releases FSH, not only in rats but also in cows.

Fourth module of Xenopus interphotoreceptor retinoid-binding protein: activity in retinoid transfer between the retinal pigment epithelium and rod photoreceptors.

PURPOSE: Interphotoreceptor retinoid-binding protein (IRBP), an extracellular protein believed to support the exchange of retinoids between the neural retina and retinal pigment epithelium (RPE) in the vertebrate eye, exhibits a modular, i.e., repeat, structure. The present study was undertaken to determine whether an individual module of IRBP has activity in retinoid transfer between the RPE and rod photoreceptors. METHODS: The retinoid transfer activity of a recombinant protein corresponding to the fourth module of Xenopus laevis IRBP (X4IRBP) was examined in two ways. First, X4IRBP was tested for its ability to support the regeneration of porphyropsin in detached/reattached Xenopus retina/RPE-eyecups. Following illumination and removal of native IRBP, Xenopus eyecups supplemented with 42 microM X4IRBP or (as a control) Ringer's solution were incubated in darkness and then analyzed for regenerated porphyropsin. Second, toad (Bufo marinus) RPE-eyecup preparations were used to evaluate X4IRBP's ability to promote the release of 11-cis retinal from the RPE. RESULTS: The regeneration of porphyropsin in X4IRBP-supplemented Xenopus retina/RPE-eyecups (0.45 +/- 0.04 nmol; mean +/- SEM, n = 11) exceeded that in controls (0.13 +/- 0.02 nmol, n = 11). For promoting the release of 11-cis retinal from the toad RPE, 42 microM X4IRBP was more effective than equimolar bovine serum albumin although considerably less than that of 26 microM native bovine IRBP. CONCLUSIONS: The results indicate a low but significant activity of IRBP's fourth module in reactions relevant to retinoid exchange.

Cloning and functional characterization of the amphibian mesotocin receptor, a member of the oxytocin/vasopressin receptor superfamily.

Mesotocin is the oxytocin-like hormone found in most terrestrial vertebrates from lungfishes to marsupials, which includes all non-mammalian tetrapods (amphibians, reptiles, and birds). It has the largest distribution in vertebrates after vasotocin found in all non-mammalian vertebrates and isotocin identified in bony fishes. In this study, we report the cloning and functional characterization of the cDNA for the mesotocin receptor (MTR) from the urinary bladder of the toad Bufo marinus. The cloned cDNA encodes a polypeptide of 389 amino acids that shows the greatest similarity to the teleost fish isotocin receptor and to mammalian oxytocin receptors with mutations in extracellular loops which are involved in ligand binding. When expressed in COSM6 cells, MTR exhibits the following relative order of ligand affinity: mesotocin > vasotocin = oxytocin > vasopressin > hydrin 1, isotocin, hydrin 2. Injection of MTR cRNA into Xenopus laevis oocytes induces membrane chloride currents in response to mesotocin, which indicates the coupling of the mesotocin receptor to the inositol phosphate/calcium pathway. This response is inhibited by an oxytocin antagonist, but not by a vasopressin antagonist specific for V2 vasopressin receptors. MTR mRNA is not only found in toad urinary bladder, but also in kidney, muscle, and brain tissue of the toad as revealed by northern blot analysis and reverse-transcriptase PCR. The results suggest a variety of function for mesotocin and its receptor including, in particular, an involvement in the regulation of water and salt transport.

The rat distal colon P-ATPase alpha subunit encodes a ouabain-sensitive H+, K+-ATPase.

The functional properties and the pharmacological profile of the recently cloned cDNA colonic P-ATPase alpha subunit (Crowson, M.S., and Shull, G.E. (1992) J. Biol. Chem. 267, 13740-13748) were investigated by using the Xenopus oocyte expression system. Xenopus oocytes were injected with alpha subunit cRNAs from Bufo marinus bladder or rat distal colon and/or with beta subunit cRNA from B. marinus bladder. Two days after injection, K+ uptake was measured by using 86 Rb+ as a K+ surrogate, and pH measurements were performed by means of ion-selective microelectrodes. Co-injection of alpha and beta subunit cRNAs led to a large increase in 86Rb+ uptake, an intracellular alkalinization, and an extracellular medium acidification, as compared to alpha or beta injection alone. These results indicate that the colonic P-ATPase alpha subunit, like the bladder alpha subunit, acts as a functional H+,K+-ATPase, and that co-expression of alpha and beta subunits is required for the function. External K+ activation of the 86Rb+ uptake had a K1/2 of approximately 440 microM for the bladder isoform (consistent with the previously reported value (Jaisser, F., Horisberger, J.D., Geering, K., and Rossier, B.C. (1993) J. Cell. Biol. 123, 1421-1431) and a K1/2 of approximately 730 microM for the colonic isoform. Sch28080 was ineffective to reduce 86Rb+ uptake whereas ouabain inhibited the activity expressed from rat colon alpha subunit with a Ki of 970 microM when measured at the Vmax of the enzyme. We conclude that, when expressed in Xenopus oocytes, the rat colon P-ATPase alpha subunit encodes a ouabain-sensitive H+,K+-ATPase.

Influence of pretectal lesions on tectal responses to visual stimulation in anurans: field potential, single neuron and behavior analyses.

Investigating the fine structure of the anuran's optic tectum (OT), Székely and Lázár suggested that a focal tectal excitation would spread across the tectal network if there were no control by thalamic pretectal (TP) inhibitory influences. Disconnecting OT from TP by lancet lesions in toads, we show that visual prey-catching is hyperexcited and stimulus discrimination nearly abolished. Functional recovery exists. Micro-administration of the axon sparing excitotoxins kainic acid (KA) or ibotenic acid (IBO) confirm that the TP region is actually involved. Recordings from prey-selective tectal neurons in immobilized frogs reveal a KA-induced impairment of stimulus discrimination and an increase in spontaneous firing. Following TP-lesions, in freely moving toads a correlation is observed between any moving stimulus, enhanced neuron firing, and pray-catching. Tectal field potentials evoked by diffuse light on- and off-stimuli before and after administration of the conduction-blocking drug procaine (PRC) suggest that populations of tectal neurons are affected by pretectal inhibitory influences. Considering previous work on physiologically identified pretecto-tectal projection cells, three different kinds of pretecto-tectal influences are discussed. A working hypothesis suggests a loop by which striatal influences are controlling TP, thus gating and tuning the visual information processing in OT.