The Rectal Gland of the Shark: The Road to Understanding the Mechanism and Regulation of Transepithelial Chloride Transport.

Pictured, described, and speculated on, for close to 400 years, the function of the rectal gland of elasmobranchs remained unknown. In the late 1950s, Burger discovered that the rectal gland of Squalus acanthias secreted an almost pure solution of sodium chloride, isosmotic with blood, which could be stimulated by volume expansion of the fish. Twenty five years later, Stoff discovered that the secretion of the gland was mediated by adenyl cyclase. Studies since then have shown that vasoactive intestinal peptide (VIP) is the neurotransmitter responsible for activating adenyl cyclase; however, the amount of circulating VIP does not change in response to volume expansion. The humoral factor involved in activating the secretion of the gland is C-type natriuretic peptide, secreted from the heart in response to volume expansion. C-type natriuretic peptide circulates to the gland where it stimulates the release of VIP from nerves within the gland, but it also has a direct effect, independent of VIP. Sodium, potassium, and chloride are required for the gland to secrete, and the secretion of the gland is inhibited by ouabain or furosemide. The current model for the secretion of chloride was developed from this information. Basolateral NaKATPase maintains a low intracellular concentration of sodium, which establishes the large electrochemical gradient for sodium directed into the cell. Sodium moves from the blood into the cell (together with potassium and chloride) down this electrochemical gradient, through a coupled sodium, potassium, and two chloride cotransporter (NKCC1). On activation, chloride moves from the cell into the gland lumen, down its electrical gradient through apical cystic fibrosis transmembrane regulator. The fall in intracellular chloride leads to the phosphorylation and activation of NKCC1 that allows more chloride into the cell. Transepithelial sodium secretion into the lumen is driven by an electrical gradient through a paracellular pathway. The aim of this review was to examine the history of the origin of this model for the transport of chloride and suggest that it is applicable to many epithelia that transport chloride, both in resorptive and secretory directions.

Using 15N to determine the metabolic fate of dietary nitrogen in North Pacific spiny dogfish (Squalus acanthias suckleyi).

Marine elasmobranchs are ureosmotic, retaining large concentrations of urea to balance their internal osmotic pressure with that of the external marine environment. The synthesis of urea requires the intake of exogenous nitrogen to maintain whole-body nitrogen balance and satisfy obligatory osmoregulatory and somatic processes. We hypothesized that dietary nitrogen may be directed toward the synthesis of specific nitrogenous molecules in post-fed animals; specifically, we predicted the preferential accumulation and retention of labelled nitrogen would be directed towards the synthesis of urea necessary for osmoregulatory purposes. North Pacific spiny dogfish (Squalus acanthias suckleyi) were fed a single meal of 7 mmol l-1 15NH4Cl in a 2% ration by body mass of herring slurry via gavage. Dietary labelled nitrogen was tracked from ingestion to tissue incorporation and the subsequent synthesis of nitrogenous compounds (urea, glutamine, bulk amino acids, protein) in the intestinal spiral valve, plasma, liver and muscle. Within 20 h post-feeding, we found labelled nitrogen was incorporated into all tissues examined. The highest δ15N values were seen in the anterior region of the spiral valve at 20 h post-feeding, suggesting this region was particularly important in assimilating the dietary labelled nitrogen. In all tissues examined, enrichment of the nitrogenous compounds was sustained throughout the 168 h experimental period, highlighting the ability of these animals to retain and use dietary nitrogen for both osmoregulatory and somatic processes.

Osmorespiratory compromise in an elasmobranch: oxygen consumption, ventilation and nitrogen metabolism during recovery from exhaustive exercise in dogfish sharks (Squalus suckleyi).

The functional trade-off between respiratory gas exchange versus osmolyte and water balance that occurs at the thin, highly vascularized gills of fishes has been termed the osmorespiratory compromise. Increases in gas exchange capacity for meeting elevated oxygen demands can end up favoring the passive movement of osmolytes and water, potentially causing a disturbance in osmotic balance. This phenomenon has been studied only sparsely in marine elasmobranchs. Our goal was to evaluate the effects of exhaustive exercise (as a modulator of oxygen demand) on oxygen consumption (MO2), branchial losses of nitrogenous products (ammonia and urea-N), diffusive water exchange rates, and gill ventilation (frequency and amplitude), in the Pacific spiny dogfish (Squalus suckleyi). To that end, MO2, osmolyte fluxes, diffusive water exchange rate, and ventilation dynamics were first measured under resting control conditions, then sharks were exercised until exhaustion (20 min), and the same parameters were monitored for the subsequent 4 h of recovery. While MO2 nearly doubled immediately after exercise and remained elevated for 2 h, ventilation dynamics did not change, suggesting that fish were increasing oxygen extraction efficiency at the gills. Diffusive water flux rates (measured over 0-2 h of recovery) were not affected. Ammonia losses were elevated by 7.6-fold immediately after exercise and remained elevated for 3 h into recovery, while urea-N losses were elevated only 1.75-fold and returned to control levels after 1 h. These results are consistent with previous investigations using different challenges (hypoxia, high temperature) and point to a tighter regulation of urea-N conservation mechanisms at the gills, likely due to the use of urea as a prized osmolyte in elasmobranchs. Environmental hyperoxia offered no relief from the osmorespiratory compromise, as there were no effects on any of the parameters measured during recovery from exhaustive exercise.

The aquaporin 8 (AQP8) membrane channel gene is present in the elasmobranch dogfish (Squalus acanthias) genome and is expressed in brain but not in gill, kidney or intestine.

The transport mechanisms for water, ammonia and urea in elasmobranch gill, kidney and gastrointestinal tract remain to be fully elucidated. Aquaporin 8 (AQP8) is a known water, ammonia and urea channel that is expressed in the kidney and respiratory and gastrointestinal tracts of mammals and teleost fish. However, at the initiation of this study in late 2019, there was no copy of an elasmobranch aquaporin 8 gene identified in the genebank even for closely related holocephalon species such as elephant fish (Callorhinchus milii) or for the elasmobranch little skate (Leucoraja erinacea). A transcriptomic study in spiny dogfish (Squalus acanthias) also failed to identify a copy. Hence this study has remedied this and identified the AQP8 cDNA sequence using degenerate PCR. Agarose electrophoresis of degenerate PCR reactions from dogfish tissues showed a strong band from brain cDNA and faint bands of a similar size in gill and liver. 5' and 3' RACE was used to complete the AQP8 cDNA sequence. Primers were then designed for further PCR reactions to determine the distribution of AQP8 mRNA expression in dogfish tissues. This showed that AQP8 is only expressed in dogfish brain and AQP8 therefore clearly can play no role in water, ammonia and urea transport in the gill, kidney or gastrointestinal tract. The role of AQP8 in dogfish brain remains to be determined.

Microstructure and characteristic properties of dogfish skin gelatin gels prepared by freeze/spray-drying methods.

The effects of two pretreatments (microwaves or oven-drying) on the dogfish (Squalus acanthias) skin as well as two drying processes (freeze-drying or spray-drying) on the extracted gelatins were studied. Thus six types of gelatins were obtained, three of which were freeze-dried (FG) and the others were spray-dried (SG), from the untreated skin (US), microwaves-pretreated skin (MS) and oven-pretreated skin (OS). The highest yield (8.67%) was obtained for the OSFG, while the lowest one (3.06%) was measured for the OSSG. Interestingly, all gelatins exhibited relatively high protein (84.02-89.53%), and low lipid (0.50-1.71%) and ash (3.05-7.17%) contents. In addition, gelatins were analyzed by the Fourier transform infrared and the spectra displayed important differences in some specific peaks, particularly in the amide I, amide II and amide III. The gelatins extracted from the untreated skin, regardless the drying method, presented the highest foaming capacity. The textural profile analysis showed that USSG was the hardest (213.6 g) and the chewier (23.8 N × mm) gelatin. Moreover, analysis of thermal properties showed that USSG also has the highest glass-transition temperature. The interesting properties of gelatin extracted from dogfish skin encourage their future use as a functional ingredient in industrial food formulations.

A Potential Antineoplastic Peptide of Human Prostate Cancer Cells Derived from the Lesser Spotted Dogfish (Scyliorhinus canicula L.).

The purpose of the present paper is to investigate the mechanism of action of a pyroglutamate-modified peptide (pE-K092D) on in vitro growth inhibition of MDA-Pca-2b prostate cancer cells. This peptide was derived from a peptide previously isolated from the testis of the lesser spotted dogfish and identified as QLTPEALADEEEMNALAAR (K092D). The effect of the peptide on cell proliferation and cell death mechanisms was studied by flow cytometry. Cellular morphology and cytoskeleton integrity of peptide-treated cells were observed by immunofluorescence microscopy. Results showed the onset of peptide induced early cytoskeleton perturbation, inhibition of autophagy, inhibition of cell proliferation and, at the end, non-apoptotic cell death mechanisms (membrane destabilization and necrosis). All those mechanisms seem to contribute to MDA-Pca-2b growth inhibition by a main cytostatic fate.

Climate change and overfishing increase neurotoxicant in marine predators.

More than three billion people rely on seafood for nutrition. However, fish are the predominant source of human exposure to methylmercury (MeHg), a potent neurotoxic substance. In the United States, 82% of population-wide exposure to MeHg is from the consumption of marine seafood and almost 40% is from fresh and canned tuna alone1. Around 80% of the inorganic mercury (Hg) that is emitted to the atmosphere from natural and human sources is deposited in the ocean2, where some is converted by microorganisms to MeHg. In predatory fish, environmental MeHg concentrations are amplified by a million times or more. Human exposure to MeHg has been associated with long-term neurocognitive deficits in children that persist into adulthood, with global costs to society that exceed US$20 billion3. The first global treaty on reductions in anthropogenic Hg emissions (the Minamata Convention on Mercury) entered into force in 2017. However, effects of ongoing changes in marine ecosystems on bioaccumulation of MeHg in marine predators that are frequently consumed by humans (for example, tuna, cod and swordfish) have not been considered when setting global policy targets. Here we use more than 30 years of data and ecosystem modelling to show that MeHg concentrations in Atlantic cod (Gadus morhua) increased by up to 23% between the 1970s and 2000s as a result of dietary shifts initiated by overfishing. Our model also predicts an estimated 56% increase in tissue MeHg concentrations in Atlantic bluefin tuna (Thunnus thynnus) due to increases in seawater temperature between a low point in 1969 and recent peak levels-which is consistent with 2017 observations. This estimated increase in tissue MeHg exceeds the modelled 22% reduction that was achieved in the late 1990s and 2000s as a result of decreased seawater MeHg concentrations. The recently reported plateau in global anthropogenic Hg emissions4 suggests that ocean warming and fisheries management programmes will be major drivers of future MeHg concentrations in marine predators.

Growth-related changes in non-essential and essential metals in the liver of star-spotted smooth-hounds (dogfish) Mustelus manazo from the northern region of Japan.

We analyzed Cd, Hg, Zn, Cu, Mn and Fe concentrations in liver samples from male and female star-spotted smooth-hounds at various life stages. Male sharks of this species are known to reach their maximum body length (BL) more quickly than females, while females are known to mature later and live longer than males. Hepatic Cd and Hg concentrations in males and females markedly increased after maturation, but these increases proceeded earlier in males than in females. Hepatic Zn and Cu concentrations decreased during the growth stage of males and females, and thereafter increased concomitantly with increases of Cd and Hg burdens, forming a U-shaped curve over their lifespan, and the BL at which the lowest concentrations of Zn and Cu were observed was smaller in males than in females. These gender-related differences in those metals could reflect the faster growth and earlier cessation of growth in males.

A Developmental Study of the Cerebellar Nucleus in the Catshark, a Basal Gnathostome.

The output of the cerebellar cortex is mainly released via cerebellar nuclei which vary in number and complexity among gnathostomes, extant vertebrates with a cerebellum. Cartilaginous fishes, a basal gnathostome lineage, show a conspicuous, well-organized cerebellar nucleus, unlike ray-finned fishes. To gain insight into the evolution and development of the cerebellar nucleus, we analyzed in the shark Scyliorhinus canicula (a chondrichthyan model species) the developmental expression of several genes coding for transcription factors (ScLhx5,ScLhx9,ScTbr1, and ScEn2) and the distribution of the protein calbindin, since all appear to be involved in cerebellar nuclei patterning in other gnathostomes. Three regions (subventricular, medial or central, and lateral or superficial) became recognizable in the cerebellar nucleus of this shark during development. Present genoarchitectonic and neurochemical data in embryos provide insight into the origin of the cerebellar nucleus in chondrichthyans and support a tripartite mediolateral organization of the cerebellar nucleus, as previously described in adult sharks. Furthermore, the expression pattern of ScLhx5,ScLhx9, and ScTbr1 in this shark, together with that of markers of proliferation, migration, and early differentiation of neurons, is compatible with the hypothesis that, as in mammals, different subsets of cerebellar nucleus neurons are originated from progenitors of 2 different sources: the ventricular zone of the cerebellar plate and the rhombic lip. We also present suggestive evidence that Lhx9 expression is involved in cerebellar nuclei patterning early on in gnathostome evolution, rather than representing an evolutionary innovation of the dentate nucleus in mammals, as previously hypothesized.

Functional and molecular identification of a TASK-1 potassium channel regulating chloride secretion through CFTR channels in the shark rectal gland: implications for cystic fibrosis.

In the shark rectal gland (SRG), apical chloride secretion through CFTR channels is electrically coupled to a basolateral K+ conductance whose type and molecular identity are unknown. We performed studies in the perfused SRG with 17 K+ channel inhibitors to begin this search. Maximal chloride secretion was markedly inhibited by low-perfusate pH, bupivicaine, anandamide, zinc, quinidine, and quinine, consistent with the properties of an acid-sensitive, four-transmembrane, two-pore-domain K+ channel (4TM-K2P). Using PCR with degenerate primers to this family, we identified a TASK-1 fragment in shark rectal gland, brain, gill, and kidney. Using 5' and 3' rapid amplification of cDNA ends PCR and genomic walking, we cloned the full-length shark gene (1,282 bp), whose open reading frame encodes a protein of 375 amino acids that was 80% identical to the human TASK-1 protein. We expressed shark and human TASK-1 cRNA in Xenopus oocytes and characterized these channels using two-electrode voltage clamping. Both channels had identical current-voltage relationships (outward rectifying) and a reversal potential of -90 mV. Both were inhibited by quinine, bupivicaine, and acidic pH. The pKa for current inhibition was 7.75 for shark TASK-1 vs. 7.37 for human TASK-1, values similar to the arterial pH for each species. We identified this protein in SRG by Western blot and confocal immunofluorescent microscopy and detected the protein in SRG and human airway cells. Shark TASK-1 is the major K+ channel coupled to chloride secretion in the SRG, is the oldest 4TM 2P family member identified, and is the first TASK-1 channel identified to play a role in setting the driving force for chloride secretion in epithelia. The detection of this potassium channel in mammalian lung tissue has implications for human biology and disease.

Dogfish glucagon analogues counter hyperglycaemia and enhance both insulin secretion and action in diet-induced obese diabetic mice.

AIMS: To investigate the antidiabetic actions of three dogfish glucagon peptide analogues [known glucagon-like peptide-1 and glucagon receptor co-agonists] after chronic administration in diet-induced high-fat-diet-fed diabetic mice. MATERIALS AND METHODS: National Institutes of Health Swiss mice were pre-conditioned to a high-fat diet (45% fat) for 100 days, and control mice were fed a normal diet (10% fat). Normal diet control and high-fat-fed control mice received twice-daily intraperitoneal (i.p.) saline injections, while the high-fat-fed treatment groups (n = 8) received twice-daily injections of exendin-4(1-39), [S2a]dogfish glucagon, [S2a]dogfish glucagon exendin-4(31-39) or [S2a]dogfish glucagon-Lys(30) -γ-glutamyl-PAL (25 nmol/kg body weight) for 51 days. RESULTS: After dogfish glucagon analogue treatment, there was a rapid and sustained decrease in non-fasting blood glucose and an associated insulinotropic effect (analysis of variance, p < .05 to <.001) compared with saline-treated high-fat-fed controls. All peptide treatments significantly improved i.p. and oral glucose tolerance with concomitant increased insulin secretion compared with saline-treated high-fat-fed controls (p <.05 to <.001). After chronic treatment, no receptor desensitization was observed but insulin sensitivity was enhanced for all peptide-treated groups (p < .01 to <.001) except [S2a]dogfish glucagon. Both exendin-4 and [S2a]dogfish glucagon exendin-4(31-39) significantly reduced plasma triglyceride concentrations compared with those found in lean controls (p = .0105 and p = .0048, respectively). Pancreatic insulin content was not affected by peptide treatments but [S2a]dogfish glucagon and [S2a]dogfish glucagon exendin-4(31-39) decreased pancreatic glucagon by 28%-34% (p = .0221 and p = .0075, respectively). The percentage of ß-cell area within islets was increased by exendin-4 and peptide analogue treatment groups compared with high-fat-fed controls and the ß-cell area decreased (p < .05 to <.01). CONCLUSIONS: Overall, dogfish glucagon co-agonist analogues had several beneficial metabolic effects, showing therapeutic potential for type 2 diabetes.

Novel dual agonist peptide analogues derived from dogfish glucagon show promising in vitro insulin releasing actions and antihyperglycaemic activity in mice.

The antidiabetic potential of thirteen novel dogfish glucagon derived analogues were assessed in vitro and in acute in vivo studies. Stable peptide analogues enhanced insulin secretion from BRIN-BD11 ß-cells (p < 0.001) and reduced acute glycaemic responses following intraperitoneal glucose (25 nmol/kg) in healthy NIH Swiss mice (p < 0.05-p<0.001). The in vitro insulinotropic actions of [S2a]dogfish glucagon, [S2a]dogfish glucagon-exendin-4(31-39) and [S2a]dogfish glucagon-Lys(30)-γ-glutamyl-PAL, were blocked (p < 0.05-p<0.001) by the specific GLP-1 and glucagon receptor antagonists, exendin-4(9-39) and (desHis(1)Pro(4)Glu(9))glucagon amide but not by (Pro(3))GIP, indicating lack of GIP receptor involvement. These analogues dose-dependently stimulated cAMP production in GLP-1 and glucagon (p < 0.05-p<0.001) but not GIP-receptor transfected cells. They improved acute glycaemic and insulinotropic responses in high-fat fed diabetic mice and in wild-type C57BL/6J and GIPR-KO mice (p < 0.05-p<0.001), but not GLP-1R-KO mice, confirming action on GLP-1 but not GIP receptors. Overall, dogfish glucagon analogues have potential for diabetes therapy, exerting beneficial metabolic effects via GLP-1 and glucagon receptors.

Morphogenesis of the cerebellum and cerebellum-related structures in the shark Scyliorhinus canicula: insights on the ground pattern of the cerebellar ontogeny.

Because the cerebellum emerged at the agnathan-gnathostome transition and cartilaginous fishes are at the base of the gnathostome lineage, this group is crucial to determine the basic developmental pattern of the cerebellum and to gain insights into its origin. We have systematically analyzed key events in the development of cerebellum and cerebellum-related structures of the shark Scyliorhinus canicula. Three developmental periods are distinguished based on anatomical observations combined with molecular analysis. We present neurochemical and genoarchitectonic evidence on the onset of cerebellar development, the rostral and caudal cerebellar boundaries, the compartmentalization of the cerebellum, and correspondence of cerebellar domains to rhombomeric segmentation of the rostral hindbrain. Our observations, mainly based on the expression pattern of ScHoxA2, support the origin of both the upper and lower auricular leaves from r1 and exclude any cerebellar origin from r2. Correlation between subrhombomeres r1a/r1b and cerebellar domains is proposed based on the ScEn2 expression. The ScEn2 and ScOtx2 expression patterns revealed an antero-posterior cerebellar compartmentalization similar to that of mammals, and supported certain fissures (commonly used to define cerebellar domains) as reliable anatomical landmarks. At difference from mammals, the expression of ScEn2 along the cerebellar median-lateral axis does not reveal a multiple-banded pattern. The present study provides an atlas of cerebellar development in one of the most basal extant gnathostome lineages and emphasizes the importance of combining classic descriptive with modern molecular studies to gain knowledge on the ancestral condition of cerebellar developmental processes and the origins and evolution of the cerebellum.

Production of Hyaluronic Acid by Streptococcus zooepidemicus on Protein Substrates Obtained from Scyliorhinus canicula Discards.

This work investigates the production of hyaluronic acid (H) by Streptococcus equi subsp. zooepidemicus in complex media formulated with peptones obtained from Scyliorhinus canicula viscera by-products. Initially, in batch cultures, the greatest productions were achieved using commercial media (3.03 g/L) followed by peptones from alcalase hydrolyzed viscera (2.32 g/L) and peptones from non-hydrolyzed viscera (2.26 g/L). An increase of between 12% and 15% was found in subsequent fed-batch cultures performed on waste peptones. Such organic nitrogen sources were shown to be an excellent low-cost substrate for microbial H, saving more than 50% of the nutrient costs.

Examining urea flux across the intestine of the spiny dogfish, Squalus acanthias.

Recent examination of urea flux in the intestine of the spiny dogfish shark, Squalus acanthias, has shown that feeding significantly enhances urea uptake across the intestine, and this was significantly inhibited following mucosal addition of phloretin. The present study examined potential mechanisms of urea uptake across the dogfish intestine in starved and fed dogfish. Unidirectional flux chambers were used to examine the kinetics of urea uptake, and to determine the influence of sodium, ouabain, competitive urea analogues, and phloretin on urea uptake across the gut of fed dogfish. Intestinal epithelial preparations from starved and fed dogfish were mounted in Ussing chambers to examine the effect of phloretin on bidirectional solute transport across the intestine. In the unidirectional studies, the maximum uptake rate of urea was found to be 35.3±6.9 µmol.cm(-2).h(-1) and Km was found to be 291.8±9.6 mM in fed fish, and there was a mild inhibition of urea uptake following mucosal addition of competitive agonists. Addition of phloretin, Na-free Ringers and ouabain to the mucosal side of intestinal epithelia also led to a significant reduction in urea uptake in fed fish. In the Ussing chamber studies there was a net influx of urea in fed fish and a small insignificant efflux in starved fish. Addition of phloretin blocked urea uptake in fed fish when added to the mucosal side. Furthermore, phloretin had no effect on ion transport across the intestinal epithelia with the exception of the divalent cations, magnesium and calcium.

Mercury bioaccumulation in cartilaginous fishes from Southern New England coastal waters: contamination from a trophic ecology and human health perspective.

This study examined total mercury (Hg) concentrations in cartilaginous fishes from Southern New England coastal waters, including smooth dogfish (Mustelus canis), spiny dogfish (Squalus acanthias), little skate (Leucoraja erinacea), and winter skate (Leucoraja ocellata). Total Hg in dogfish and skates were positively related to their respective body size and age, indicating Hg bioaccumulation in muscle tissue. There were also significant inter-species differences in Hg levels (mean ± 1 SD, mg Hg/kg dry weight, ppm): smooth dogfish (3.3 ± 2.1 ppm; n = 54) > spiny dogfish (1.1 ± 0.7 ppm; n = 124) > little skate (0.4 ± 0.3 ppm; n = 173) âˆ¼ winter skate (0.3 ± 0.2 ppm; n = 148). The increased Hg content of smooth dogfish was attributed to its upper trophic level status, determined by stable nitrogen (δ(15)N) isotope analysis (mean δ(15)N = 13.2 ± 0.7‰), and the consumption of high Hg prey, most notably cancer crabs (0.10 ppm). Spiny dogfish had depleted δ(15)N signatures (11.6 ± 0.8‰), yet demonstrated a moderate level of contamination by foraging on pelagic prey with a range of Hg concentrations, e.g., in order of dietary importance, butterfish (Hg = 0.06 ppm), longfin squid (0.17 ppm), and scup (0.11 ppm). Skates were low trophic level consumers (δ(15)N = 11.9-12.0‰) and fed mainly on amphipods, small decapods, and polychaetes with low Hg concentrations (0.05-0.09 ppm). Intra-specific Hg concentrations were directly related to δ(15)N and carbon (δ(13)C) isotope signatures, suggesting that Hg biomagnifies across successive trophic levels and foraging in the benthic trophic pathway increases Hg exposure. From a human health perspective, 87% of smooth dogfish, 32% of spiny dogfish, and <2% of skates had Hg concentrations exceeding the US Environmental Protection Agency threshold level (0.3 ppm wet weight). These results indicate that frequent consumption of smooth dogfish and spiny dogfish may adversely affect human health, whereas skates present minimal risk.

cGMP inhibition of type 3 phosphodiesterase is the major mechanism by which C-type natriuretic peptide activates CFTR in the shark rectal gland.

The in vitro perfused rectal gland of the dogfish shark (Squalus acanthias) and filter-grown monolayers of primary cultures of shark rectal gland (SRG) epithelial cells were used to analyze the signal transduction pathway by which C-type natriuretic peptide (CNP) stimulates chloride secretion. CNP binds to natriuretic receptors in the basolateral membrane, elevates cellular cGMP, and opens cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels in the apical membrane. CNP-provoked chloride secretion was completely inhibitable by the nonspecific protein kinase inhibitor staurosporine and the PKA inhibitor H89 but insensitive to H8, an inhibitor of type I and II isoforms of cGMP-dependent protein kinase (cGKI and cGKII). CNP-induced secretion could not be mimicked by nonhydrolyzable cGMP analogs added alone or in combination with the protein kinase C activator phorbolester, arguing against a role for cGK or for cGMP-induced PKC signaling. We failed to detect a dogfish ortholog of cGKII by molecular cloning and affinity chromatography. However, inhibitors of the cGMP-inhibitable isoform of phosphodiesterase (PDE3) including milrinone, amrinone, and cilostamide but not inhibitors of other PDE isoenzymes mimicked the effect of CNP on chloride secretion in perfused glands and monolayers. CNP raised cGMP and cAMP levels in the SRG epithelial cells. This rise in cAMP as well as the CNP and amrinone-provoked chloride secretion, but not the rise in cGMP, was almost completely blocked by the Gαi-coupled adenylyl cyclase inhibitor somatostatin, arguing against a role for cGMP cross-activation of PKA in CNP action. These data provide molecular, functional, and pharmacological evidence for a CNP/cGMP/PDE3/cAMP/PKA signaling cascade coupled to CFTR in the SRG.

Characterization of spermatogonial markers in the mature testis of the dogfish (Scyliorhinus canicula L.).

In dogfish, spermatogenesis progresses from a restricted germinative zone, which lines the dorsal testicular vessel. Single spermatogonia (A(s)), including the spermatogonial stem cells (SSCs), produce successively paired (A(p)), undifferentiated (A(u4) to A(u512)), and differentiated (A(d1) to A(d8)) spermatogonia and preleptotene (PL) spermatocytes through 13 mitoses. Dogfish spermatogonial subpopulations present classical morphological characteristics but cannot be distinguished on the basis of molecular markers. This characterization has been initiated in mammals despite the difficulty to separate each spermatogonial subpopulation. For instance, both glial cell-derived neurotrophic factor family receptor alpha 1 (GFRα1) and promyelocytic leukemia zinc finger protein (PLZF) are markers of undifferentiated spermatogonia, whereas receptor tyrosine kinase C-kit is a marker of differentiated spermatogonia. The aim of this study is to characterize spermatogonial markers and to differentiate several spermatogonial subpopulations. Dogfish cDNA sequences have been identified and validated by phylogenetic analyses for gfrα1, plzf, pou2, as well as for high-mobility group box proteins 2 and 3 (hmgb2 and 3) and for mini-chromosome maintenance protein 6 (mcm6). We have used the anatomical advantage of the polarized dogfish testis to analyze the expression of those markers by RT-PCR and in situ hybridization. gfrα1, pou2, and plzf have been detected in the testicular germinative zone, suggesting that spermatogonial markers are relatively well conserved among vertebrates but with a less restricted expression for plzf. Moreover, hmgb3 and mcm6 have been identified as new markers of differentiated spermatogonia. Finally, this first molecular characterization of spermatogonial subpopulations in a chondrichthyan model will be useful for further studies on the SSC niche evolution.

Determination of total mercury in biological tissue by isotope dilution ICPMS after UV photochemical vapor generation.

A method is developed for the determination of trace mercury in biological samples using photo chemical vapor generation (PVG) and isotope dilution inductively coupled plasma mass spectrometry (ID ICPMS) detection. Biological tissues were solubilized in formic acid. Subsequently, the sample solutions were exposed to an ultraviolet (UV) source for the reduction of mercury into vapor species prior to ICPMS measurements. The formic acid served not only as a tissue solubilizer in the sample preparation procedure, but also as a photochemical reductant for mercury in the PVG process. The problem arising from the opaque formic acid digested solution was efficiently solved by using ID method. The optimum conditions for sample treatment and PVG were investigated. A limit of detection (LOD) of 0.5 pg g(-1), based on an external calibration, provided 350-fold improvement over that obtained by utilizing conventional pneumatic nebulization sample introduction. Method validation was demonstrated by the determination of total mercury in several biological tissue certified reference materials (CRMs). The results were in good agreement with the certified values.

Cyanovanadate(III) complexes as novel additives for efficient generation of volatile cadmium species in complex samples prior to determinations by inductively coupled plasma mass spectrometry (ICP-MS).

A new method has been described for generation of volatile species of Cd using vanadium(III) cyanide complex. Aqueous solutions of 0.04 mol L(-1) vanadium chloride (VCl3) and 0.12 mol L(-1) potassium cyanide (KCN) were reacted on-line yielding a suspension of vanadium hydroxide, V(OH)3. This suspension was dissolved along the stream of sample solution in dilute HCl to form heptacyanovanadate(III) complex, [V(CN)7]4-. Volatile Cd species were generated by reacting the stream of sample solution and cyanovanadate(III) complex with sodium borohydride (NaBH4). Feasibility of off-line and on-online approaches was investigated for quantitative determinations. Better precision and daily stability were achieved with on-line settings. Optimum signals were obtained from sample solutions within a range of 3 to 5% v/v HCl. A concentration of 2% m/v NaBH4 was adequate to achieve an enhancement of 20-fold in the presence of cyanovanadate(III) complex. The limits of detection were 5.0 and 4.5 ng L(-1) for 110Cd and 111Cd isotopes, respectively. Precision (%RSD) was better than 4.7% for six replicate measurements. The interferences of Cu(II) and Ni(II) were marginal (<10%) at 1.0 µg mL(-1). Depressive effects from Bi, Se and Sn were not significant below 0.1 µg mL(-1). The method was validated by determination of Cd using ICP-MS in certified reference materials of Nearshore seawater (CASS-4), Bone ash (SRM 1400), Dogfish liver (DOLT-4) and Mussel tissue (SRM 2976).