To eat or not to eat: ontogeny of hypothalamic feeding controls and a role for leptin in modulating life-history transition in amphibian tadpoles.

Many animal life histories entail changing feeding ecology, but the molecular bases for these transitions are poorly understood. The amphibian tadpole is typically a growth and dispersal life-history stage. Tadpoles are primarily herbivorous, and they capitalize on growth opportunities to reach a minimum body size to initiate metamorphosis. During metamorphic climax, feeding declines, at which time the gastrointestinal (GI) tract remodels to accommodate the carnivorous diet of the adult frog. Here we show that anorexigenic hypothalamic feeding controls are absent in the tadpole, but develop during metamorphosis concurrent with the production of the satiety signal leptin. Before metamorphosis there is a large increase in leptin mRNA in fat tissue. Leptin receptor mRNA increased during metamorphosis in the preoptic area/hypothalamus, the key brain region involved with the control of food intake and metabolism. This corresponded with an increase in functional leptin receptor, as evidenced by induction of socs3 mRNA and phosphorylated STAT3 immunoreactivity, and suppression of feeding behaviour after injection of recombinant frog leptin. Furthermore, we found that immunoneutralization of leptin in tadpoles at metamorphic climax caused them to resume feeding. The absence of negative regulation of food intake in the tadpole allows the animal to maximize growth prior to metamorphosis. Maturation of leptin-responsive neural circuits suppresses feeding during metamorphosis to facilitate remodelling of the GI tract.

Inverse Drug Screening of Bioelectric Signaling and Neurotransmitter Roles: Illustrated Using a Xenopus Tail Regeneration Assay.

Xenopus embryos and larvae are an ideal model system in which to study the interplay between genetics, physiology, and anatomy in the control of structure and function. An important emerging field is the study of bioelectric signaling, the exchange of ion- and neurotransmitter-mediated messages among all types of cells (not just nerve and muscle cells), in the regulation of growth and form during embryogenesis, regeneration, and cancer. To facilitate the mechanistic investigation of bioelectric events in vivo, it is necessary to identify the endogenous signaling machinery involved in any patterning process of interest. This protocol uses the tail regeneration assay in Xenopus to perform an inverse drug screen; tiers of known compounds are used to probe the involvement of increasingly specific classes of bioelectric and neurotransmitter machinery. By using a hierarchical approach, large classes of targets are ruled out in early rounds, focusing attention on progressively narrower sets of proteins. Such a screen avoids many of the limitations of a molecular-genetic targeting approach and provides a rapid and efficient way to focus on specific targets. Usually, <10 experiments are needed to determine whether bioelectrics and/or neurotransmitter signaling are involved in the process of interest. This protocol describes the strategy in the context of a semiquantitative analysis of tail regeneration but can be applied to any assay in Xenopus or other small aquatic model system (e.g., zebrafish). Given the ever-increasing toolkit of chemical genetics, such screens represent a powerful and versatile methodology for probing the physiological circuits underlying pattern regulation.

A transgenic reporter under control of an es1 promoter/enhancer marks wound epidermis and apical epithelial cap during tail regeneration in Xenopus laevis tadpole.

Rapid wound healing and subsequent formation of the apical epithelial cap (AEC) are believed to be required for successful appendage regeneration in amphibians. Despite the significant role of AEC in limb regeneration, its role in tail regeneration and the mechanisms that regulate the wound healing and AEC formation are not well understood. We previously identified Xenopus laevis es1, which is preferentially expressed in wounded regions, including the AEC after tail regeneration. In this study we established and characterized transgenic Xenopus laevis lines harboring the enhanced green fluorescent protein (EGFP) gene under control of an es1 gene regulatory sequence (es1:egfp). The EGFP reporter expression was clearly seen in several regions of the embryo and then declined to an undetectable level in larvae, recapitulating the endogenous es1 expression. After amputation of the tadpole tail, EGFP expression was re-activated at the edge of the stump epidermis and then increased in the wound epidermis (WE) covering the amputation surface. As the stump started to regenerate, the EGFP expression became restricted to the most distal epidermal region, including the AEC. EGFP was preferentially expressed in the basal or deep cells but not in the superficial cells of the WE and AEC. We performed a small-scale pharmacological screening for chemicals that affected the expression of EGFP in the stump epidermis after tail amputation. The EGFP expression was attenuated by treatment with an inhibitor for ERK, TGF-ß or reactive oxygen species (ROS) signaling. These treatments also impaired wound closure of the amputation surface, suggesting that the three signaling activities are required for es1 expression in the WE and successful wound healing after tail amputation. These findings showed that es1:egfp Xenopus laevis should be a useful tool to analyze molecular mechanisms regulating wound healing and appendage regeneration.

Tectal corticotropin-releasing factor (CRF) neurons respond to fasting and a reactive stressor in the African Clawed Frog, Xenopus laevis.

It is well established that hypothalamic neurons producing the peptide corticotropin-releasing factor (CRF) play a key role in stress adaptation, including reduction of food intake when a threat or stressor is present. We have previously reported on the presence of an intrinsic CRF signaling system within the optic tectum (OT), a brain area that plays a key role in visually guided prey capture/predator avoidance decisions. To better understand the potential role of tectal CRF neurons in regulating adaptive behavior and energy balance during stress we examined evidence for modulation of tectal CRF neuronal activity after stressor exposure and food deprivation in the African clawed frog Xenopus laevis. We tested two predictions, 1) that exposure to categorically distinct stressors (ether vapors and shaking) will reduce food intake and modulate the activity of tectal CRF cells, and 2) that food deprivation will modulate the activity of tectal CRF cells. Exposure to ether increased tectal content of CRF and CRF transcript, but lowed CRFR1 transcript abundance. Two weeks of food deprivation reduced total fat stores in frogs and decreased tectal content of CRF content while having no effect on CRF and CRFR1 transcript abundance. Our data are consistent with a role for tectal CRF neurons in modulating food intake in response to certain stressors.

Diclofenac can exhibit estrogenic modes of action in male Xenopus laevis, and affects the hypothalamus-pituitary-gonad axis and mating vocalizations.

Diclofenac (DCF) is a non-steroidal analgesic and antiphlogistic. Due to its tremendous use, DCF can be found in the environment, especially in sewage, but also surface waters, ground and drinking water. Previous studies indicated that DCF can modulate the reproductive physiology of fish by altering the expression of important key enzymes of the hypothalamus-pituitary-gonad-axis (HPG-axis) and might act as an estrogenic endocrine disrupting chemical (EDC). Other studies, however, demonstrated that DCF does not exhibit any estrogenicity. Thus, in the present study we investigated whether an exposure to DCF can affect reproductive behavior and physiology of adult male X. laevis by analyzing DCF effects on the mate calling behavior of the frogs and on gene expression patterns of key biomarkers of the HPG-axis. In addition, plasma sex steroid levels were determined to gain detailed insights into the mechanisms of DCF action. We could demonstrate that DCF can act as EDC by exhibiting slight estrogenic modes of action. In addition, pharmacological impacts on gonadal steroidogenesis could be revealed leading to imbalances in sex steroid levels and ratios. DCF furthermore altered the calling behavior of exposed males, potentially reducing the mating and reproductive success of the frogs, possibly leading to severe population effects.

Thiol-reactive compounds from garlic inhibit the epithelial sodium channel (ENaC).

The epithelial sodium channel (ENaC) is a key factor in the transepithelial movement of sodium, and consequently salt and water homeostasis in various organs. Dysregulated activity of ENaC is associated with human diseases such as hypertension, the salt-wasting syndrome pseudohypoaldosteronism type 1, cystic fibrosis, pulmonary oedema or intestinal disorders. Therefore it is important to identify novel compounds that affect ENaC activity. This study investigated if garlic (Allium sativum) and its characteristic organosulfur compounds have impact on ENaCs. Human ENaCs were heterologously expressed in Xenopus oocytes and their activity was measured as transmembrane currents by the two-electrode voltage-clamp technique. The application of freshly prepared extract from 5g of fresh garlic (1% final concentration) decreased transmembrane currents of ENaC-expressing oocytes within 10 min. This effect was dose-dependent and irreversible. It was fully sensitive to the ENaC-inhibitor amiloride and was not apparent on native control oocytes. The effect of garlic was blocked by dithiothreitol and l-cysteine indicating involvement of thiol-reactive compounds. The garlic organosulsur compounds S-allylcysteine, alliin and diallyl sulfides had no effect on ENaC. By contrast, the thiol-reactive garlic compound allicin significantly inhibited ENaC to a similar extent as garlic extract. These data indicate that thiol-reactive compounds which are present in garlic inhibit ENaC.

Plasticity of melanotrope cell regulations in Xenopus laevis.

This review focuses on the plasticity of the regulation of a particular neuroendocrine transducer cell, the melanotrope cell in the pituitary pars intermedia of the amphibian Xenopus laevis. This cell type is a suitable model to study the relationship between various external regulatory inputs and the secretion of an adaptive endocrine message, in this case the release of α-melanophore-stimulating hormone, which activates skin melanophores to darken when the animal is placed on a dark background. Information about the environmental conditions is processed by various brain centres, in the hypothalamus and elsewhere, that eventually control the activity of the melanotrope cell regarding hormone production and secretion. The review discusses the roles of these hypothalamic and extrahypothalamic nuclei, their neurochemical messengers acting on the melanotrope, and the external stimuli they mediate to control melanotrope cell functioning.

Electric field effects on human spinal injury: Is there a basis in the in vitro studies?

An important basis for the clinical application of small DC electric current to mammalian spinal injury is the responses of neurons in culture to applied electric fields. Our recent finding that zebrafish neurons were unresponsive to applied fields prompted us to critically examine previous results. We conclude that compelling evidence for neuronal guidance and directional stimulation of growth toward either the cathode or anode in an electric field exists only for cultured Xenopus neurons, and not for any mammalian neurons. No basis for the reported success in treating spinal injury exists in the in vitro studies, and considerable research will be required if the conditions of field application in mammalian spinal injury are to be optimized.

Tone and call responses of units in the auditory nerve and dorsal medullary nucleus of Xenopus laevis.

The clawed frog Xenopus laevis produces vocalizations consisting of distinct patterns of clicks. This study provides the first description of spontaneous, pure-tone and communication-signal evoked discharge properties of auditory nerve (n.VIII) fibers and dorsal medullary nucleus (DMN) cells in an obligatorily aquatic anuran. Responses of 297 n.VIII and 253 DMN units are analyzed for spontaneous rates (SR), frequency tuning, rate-intensity functions, and firing rate adaptation, with a view to how these basic characteristics shape responses to recorded call stimuli. Response properties generally resemble those in partially terrestrial anurans. Broad tuning exists across characteristic frequencies (CFs). Threshold minima are -101 dB re 1 mm/s at 675 Hz; -87 dB at 1,600 Hz; and -61 dB at 3,000 Hz (-90, -77, and -44 dB re 1 Pa, respectively), paralleling the peak frequency of vocalizations at 1.2-1.6 kHz with approximately 500 Hz in 3 dB bandwidth. SRs range from 0 to 80 (n.VIII) and 0 to 73 spikes/s (DMN). Nerve and DMN units of all CFs follow click rates in natural calls, < or =67 clicks/s and faster. Units encode clicks with a single spike, double spikes, or bursts. Spike times correlate closely with click envelopes. No temporal filtering for communicative click rates occurs in either n.VIII or the DMN.

Vitellogenin-inducing activities of natural, synthetic, and environmental estrogens in primary cultured Xenopus laevis hepatocytes.

Vitellogenin (VTG)-inducing activities of natural estrogens (E1: estrone, E2:17beta-estradiol, E3: estriol, alpha-E2: 17alpha-estradiol), synthetic estrogens (EE2: 17alpha-ethynyl estradiol, DES: diethylstilbestrol,), phytoestrogen (GEN: genistein), and xeno-estrogens (BPA: bisphenol A, NP: nonylphenol, OP: octylphenol) were investigated by an assay system using primary-cultured hepatocytes of Xenopus laevis. An enzyme-linked immunoabsorbent assay (ELISA) was able to detect VTG at a minimum detection limit of 0.06 ng/mL. Relative estrogenic activities of the compounds were determined from their dose-response curves. The activities relative to E2 activity were 138% for DES, 121% for EE2, 6.1% for E3, 0.33% for E1, 0.29% for alpha-E2, 0.037% for GEN, 0.008% for BPA, 0.005% for NP, and 0.002% for OP. Comparison with data reported for other bioassay systems revealed that there were significant interspecies-and cell-type-differences in the activities of DES, E3, E1 and alpha-E2. BPA was found to have a substantial antagonistic activity (approximately 0.8% of tamoxifen activity) under the influence of physiological concentrations of E2. Complex-effects of endocrine disrupters on aquatic animals will be discussed.

Xenopus laevis is a potential alternative model animal species to study reproductive toxicity of phytoestrogens.

This study investigated effects of phytoestrogen quercetin on the gonadal development in Xenopus laevis. X. laevis at Nieuwkoop and Faber stage 46/47 were exposed to 50, 100 and 200 microg/L quercetin till 1 month postmetamorphosis. Gonads from frogs at 1 and 3 months postmetamorphosis were examined in gross morphology and histology. The highest dose of quercetin as well as estradiol (E2) significantly increased the percentages of phenotypic females. Exposure to quercetin at all doses induced abnormal testes with certain ovarian characteristics to some degree in gross morphology, including ovotestes. The abnormality rate exceeded 10% in each quercetin treatment. Histologic examination revealed that some abnormal testes exhibited intersexuality with testicular structure and ovarian structure or oocytes interspersed in testicular structure at 1 month postmetamorphosis. At 3 months postmetamorphosis, testicular abnormalities were more obvious, such as necrosis or apoptosis of spermatogonia, occurrence of developed or undeveloped oocytes, delay of the development of seminiferous tubes without or less late stage spermatocytes. The results have shown that quercetin cannot only feminize but also impair testicular development of X. laevis, i.e. X. laevis is sensitive to phytoestrogen. It is suggested that X. laevis might be an alternative model species to study reproductive toxicity of phytoestrogens.

Impairment of hyperpolarization-activated, cyclic nucleotide-gated channel function by the intravenous general anesthetic propofol.

Propofol (2,6-diisopropylphenol) is a widely used intravenous general anesthetic, which has been reported to produce bradycardia in patients at concentrations associated with profound sedation and loss of consciousness. Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels conduct a monovalent cationic current I(h) (also known as I(q) or I(f)) that contributes to autorhythmicity in both the brain and heart. Here we studied the effects of propofol on recombinant HCN1, HCN2, and HCN4 channels and found that the drug inhibits and slows activation of all three channels at clinically relevant concentrations. In oocyte expression studies, HCN1 channel activation was most sensitive to slowing by propofol (EC(50) values of 5.6 +/- 1.0 microM for fast component and 31.5 +/- 7.5 microM for slow component). HCN1 channels also showed a marked propofol-induced hyperpolarizing shift in the voltage dependence of activation (EC(50) of 6.7 +/- 1.0 microM) and accelerated deactivation (EC(50) of 4.5 +/- 0.9 microM). Furthermore, propofol reduced heart rate in an isolated guinea pig heart preparation over the same range of concentrations. These data suggest that propofol modulation of HCN channel gating is an important molecular mechanism that can contribute to the depression of central nervous system function and also lead to bradyarrhythmias in patients receiving propofol during surgical anesthesia.

Auditory evoked potentials from medulla and midbrain in the clawed frog, Xenopus laevis laevis.

Auditory evoked potentials (AEPs) to clicks and tonal pulses were recorded from medulla and midbrain in Xenopus laevis laevis. They comprise three components: an initial peak (I) at 2.2-3 ms latency, a fast series of peaks (F) at 5-15 ms latency, and a slow negative wave (S) at 20-40 ms latency. In medullary recordings, the initial peak was largest, whereas in midbrain recordings typically the two other components prevailed. For all components and animals, response threshold at 4 clicks/s was approximately 69 dB SPL. In response to tonal stimuli, AEP amplitudes were maximal at 1.3-2.0 and 3.5 kHz. Raising the click rate to 100/s gradually reduced the amplitude of the I and the first F peaks, whereas later F peaks and the S wave virtually disappeared at 20-40 clicks/s. On the other hand, extending the plateau duration of tonal stimuli from 4 to 10 ms hardly affected the I and F peaks but doubled the S amplitude. This suggests two systems for stimulus processing, a fast system capable to follow clicks up to high repetition rates and a slow system with longer integration time.

Low temperature stimulates alpha-melanophore-stimulating hormone secretion and inhibits background adaptation in Xenopus laevis.

It is well-known that alpha-melanophore-stimulating hormone (alpha-MSH) release from the amphibian pars intermedia (PI) depends on the light condition of the animal's background, permitting the animal to adapt the colour of its skin to background light intensity. In the present study, we carried out nine experiments on the effect of low temperature on this skin adaptation process in the toad Xenopus laevis, using the skin melanophore index (MI) bioassay and a radioimmunoassay to measure skin colour adaptation and alpha-MSH secretion, respectively. We show that temperatures below 8 degrees C stimulate alpha-MSH secretion and skin darkening, with a maximum at 5 degrees C, independent of the illumination state of the background. No significant stimulatory effect of low temperature on the MI and alpha-MSH plasma contents was noted when the experiment was repeated with toads from which the neurointermediate lobe (NIL) had been surgically extirpated. This indicates that low temperature stimulates alpha-MSH release from melanotrope cells located in the PI. An in vitro superfusion study with the NIL demonstrated that low temperature does not act directly on the PI. A possible role of the central nervous system in cold-induced alpha-MSH release from the PI was tested by studying the hypothalamic expression of c-Fos (as an indicator for neuronal activity) and the coexistence of c-Fos with the regulators of melanotrope cell activity, neuropeptide Y (NPY) and thyrotrophin-releasing hormone (TRH), using double fluorescence immunocytochemistry. Upon lowering temperature from 22 degrees C to 5 degrees C, in white-adapted animals c-Fos expression decreased in NPY-producing suprachiasmatic-melanotrope-inhibiting neurones (SMIN) in the ventrolateral area of the suprachiasmatic nucleus (SC) but increased in TRH-containing neurones of the magnocellular nucleus. TRH is known to stimulate melanotrope alpha-MSH release. We conclude that temperatures around 5 degrees C inactivate the SMIN in the SC and activate TRH-neurones in the magnocellular nucleus, resulting in enhanced alpha-MSH secretion from the PI, darkening the skin of white-adapted X. laevis.

The dopamine precursor L-dihydroxyphenylalanine is transported by the amino acid transporters rBAT and LAT2 in renal cortex.

The intrarenal autocrine-paracrine dopamine (DA) system is critical for Na(+) homeostasis. l-Dihydroxyphenylalanine (l-DOPA) uptake from the glomerular filtrate and plasma provides the substrate for DA generation by the renal proximal tubule. The transporter(s) responsible for proximal tubule l-DOPA uptake has not been characterized. Renal cortical poly-A(+) RNA injected into Xenopus laevis oocytes induced l-DOPA uptake in a time- and dose-dependent fashion with biphasic K(m)s in the millimolar and micromolar range and independent of inward Na(+), K(+), or H(+) gradients, suggesting the presence of low- and high-affinity l-DOPA carriers. Complementary RNA from two amino acid transporters yielded l-DOPA uptake significantly above water-injected controls the rBAT/b(0,+)AT dimer (rBAT) and the LAT2/4F2 dimer (LAT2). In contradistinction to renal cortical poly-A(+), l-DOPA kinetics of rBAT and LAT2 showed classic Michaelis-Menton kinetics with K(m)s in the micromolar and millimolar range, respectively. Sequence-specific antisense oligonucleotides to rBAT or LAT2 (AS) caused inhibition of rBAT and LAT2 cRNA-induced l-DOPA transport and cortical poly-A(+)-induced arginine and phenylalanine transport. However, the same ASs only partially blocked poly-A(+)-induced l-DOPA transport. In cultured kidney cells, silencing inhibitory RNA (siRNA) to rBAT significantly inhibited l-DOPA uptake. We conclude that rBAT and LAT2 can mediate apical and basolateral l-DOPA uptake into the proximal tubule, respectively. Additional l-DOPA transport mechanisms exist in the renal cortex that remain to be identified.

Roles of corticotropin-releasing factor, neuropeptide Y and corticosterone in the regulation of food intake in Xenopus laevis.

In mammals, hypothalamic control of food intake involves counterregulation of appetite by an orexigenic peptides such as corticotropin-releasing factor (CRF), and orexigenic peptides such as neuropeptide Y (NPY). Glucocorticoids also stimulate food intake by inhibiting CRF while facilitating NPY actions. To gain a better understanding of the diversity and evolution of neuroendocrine feeding controls in vertebrates, we analysed the effects of CRF, NPY and glucocorticoids on food intake in juvenile Xenopus laevis. We also analysed brain CRF and NPY mRNA content and plasma corticosterone concentrations in relation to nutritional state. Intracerebroventricular (i.c.v.) injection of ovine CRF suppressed food intake while CRF receptor antagonist alpha helical CRF(9-41) significantly increased food intake relative to uninjected and placebo controls. By contrast, i.c.v. injection of frog NPY and short-term corticosterone treatment increased food intake. Semi-quantitative reverse transcription-polymerase chain reaction analyses showed that CRF and NPY mRNA fluctuated with food intake in the brain region containing the mid-posterior hypothalamus, pretectum, and optic tectum: CRF mRNA decreased 6 h after a meal and remained low through 31 days of food deprivation; NPY mRNA content also decreased 6 h after a meal, but increased to prefeeding levels by 24 h. Plasma corticosterone concentration increased 6 h after a meal, returned to prefeeding levels by 24 h, and did not change with prolonged food deprivation. This postprandial increase in plasma corticosterone may be related to the subsequent increase in plasma glucose and body water content that occurs 24 h postfeeding. Overall, our data support the conclusion that, similar to other vertebrates, CRF is anorexigenic while NPY is orexigenic in X. laevis, and CRF secretion modulates food intake in the absence of stress by exerting an inhibitory tone on appetite. Furthermore, the stress axis is activated in response to food intake, but in contrast to mammals and birds is not activated during periods of food deprivation.

Environmental estrogens and reproductive biology in amphibians.

In this paper, the effects of an estrogenic compound, 4-nonyl-phenol (NP), on the amphibians Rana esculenta and Triturus carnifex are described together with those on sexual differentiation in Xenopus laevis. NP increased plasma vitellogenin in male frogs and newts in a dose-related manner; moreover, inhibitory effects on gonadotropin and prolactin (PRL) secretion by pituitary were found together with an elevation of plasma androgens. NP treatment also caused a remarkable increase in number of prolactin-immunolabeled cells, suggesting that xenoestrogen might induce, at least in the newt pituitary, a PRL accumulation possibly due to a reduction of the hormone release. In addition, both NP and bisphenol A caused feminization by increasing the percentage of female phenotypes in X. laevis, and the in vivo effects were more pronounced than those of estradiol-17beta.

XCL-2 is a novel m-type calpain and disrupts morphogenetic movements during embryogenesis in Xenopus laevis.

We identified a novel cDNA, XCL-2, encoding an m-type calpain, a calcium-dependent intracellular protease. This protein has all characteristic structures and active sites of canonical calpains. Zygotic transcription of the gene was first detected at stage 10. It is expressed exclusively in the ventral circumblastoporal collar and the mesoderm-free zone at the most anterior tip of neural fold in late gastrulae and neurulae. In later stages, expression is only found in cement gland and proctodeum. It is also expressed in a tissue-specific manner. In adult tissues, various levels of expression were detected in brain, eye, heart, intestine, kidney, lung, stomach and testis, but not in liver, muscle, nerve, ovary, skin and spleen. Overexpression of wild-type XCL-2 suggests that this gene is involved in gastrulation movement and convergent extension during gastrulation and neurulation. Overexpression of a dominant-negative mutant caused a phenotype morphologically similar to, but histologically different from, that caused by overexpression of wild-type XCL-2. The mutant phenotype can be rescued by injection of wild-type XCL-2. These data suggest that XCL-2 plays an important role in convergent extension movements during embryogenesis in Xenopus laevis.

Transport of (22)Na(+) and( 45)Ca(2+) by Xenopus laevis oocytes expressing mRNA from lobster hepatopancreas.

This paper describes the development of a functional assay system to express crustacean epithelial electrogenic 2Na(+)/1H(+) antiporters in Xenopus laevis oocytes. Subsequent publications will use this assay method to establish nucleotide and amino acid sequence information about this transporter by functionally screening an hepatopancreatic cDNA library. In this method, oocytes were injected with hepatopancreatic mRNA (50 ng) isolated from Homarus americanus, while control oocytes received injections of an equivalent volume of distilled water. Three to five days post-injection, oocytes were incubated in media containing either (22)Na(+) or (45)Ca(2+) for specific time intervals and the rates of ion transfer into the oocytes were monitored under a variety of experimental conditions. Uptakes of both radiolabelled cations were stimulated by mRNA injection. mRNA-stimulated (22)Na(+) uptake was significantly (P < 0.05) inhibited by addition of calcium, amiloride, or by an antiporter-specific monoclonal antibody to the external medium. mRNA-stimulated (45)Ca(2+) uptake was significantly (P < 0.05) inhibited by addition of sodium, amiloride, cadmium, zinc, or by the antiporter-specific monoclonal antibody (also inhibitory for (22)Na(+) transport) to the external medium. The kinetics of (22)Na(+) influx in mRNA-injected oocytes were sigmoidal functions of external sodium concentration, exhibiting a Hill Coefficient (n) of approximately 3.0. Both calcium and amiloride significantly (P < 0.05) reduced sigmoidal sodium influx kinetics by alterations in the J(max) (amiloride) or K(Na) (calcium) of the transporter. Size fractionation of hepatopancreatic mRNA resulted in a single fraction that was most stimulatory for sodium and calcium transport and which likely contains the antiporter transcript. The results of this study provide the basis for using (22)Na(+) and (45)Ca(2+) transport assays of lobster mRNA-injected oocytes to functionally screen an hepatopancreatic cDNA library for clones that will provide full length nucleotide and amino acid sequences of the invertebrate electrogenic 2Na(+)/1H(+) antiporter protein.

Chronic feeding of a low boron diet adversely affects reproduction and development in Xenopus laevis.

The aims of this work were as follows: 1) to determine whether a purified diet currently used for studies with rats was acceptable for reproductive studies in frogs; and 2) to determine whether frogs are sensitive to a deficit of boron (B) in the diet. Adult Xenopus laevis were fed a nonpurified beef liver and lung (BLL) diet (310 microg B/kg), a purified diet supplemented with boron (+B; 1850 microg B/kg), or a purified diet low in boron (-B; 45 microg B/kg) for 120 d. Frogs fed the BLL and +B diets produced 11.3 and 12.2% necrotic eggs, respectively. Abnormal gastrulation occurred in <4% of the fertilized eggs in both groups, and 96-h larval survival exceeded 75% in both groups. In contrast, frogs fed the -B diet for 120 d produced a high proportion of necrotic eggs (54%). Fertilized embryos from the -B diet-fed frogs showed a high frequency of abnormal gastrulation (26.8%), and >80% of the embryos died before 96 h of development. Mean embryo cell counts at X. laevis developmental stage 7.5 (mid-blastula) were significantly lower in the -B embryos than in the BLL or +B embryos. BLL and -B embryos grown in low boron culture media had a high frequency of malformations compared with embryos grown in boron-supplemented media. These studies show that a purified diet that has been used in rodent studies was acceptable for reproduction studies in X. laevis. This work also demonstrates that a diet low in boron markedly impairs normal reproductive function in adult X. laevis, and that administration of the low boron diet results in an increase in both incidence and severity of adverse effects. In addition, these studies demonstrate the usefulness of the X. laevis model in nutrition studies.