Dogfish, Scyliorhinus canicula, rectal gland epithelial cells were successfully cultured using two different techniques: 1) a perfusion based technique and 2) a modified Valentich's technique. The morphology of the primary rectal gland epithelial cell cultures was investigated using light, fluorescence and electron microscopy. These studies demonstrated that the cell cultures express most of the structural features of native shark rectal gland cells, including numerous mitochondria, complex tight junctions and extensive membrane folding. The cultured cells using the perfusion technique adopted an extremely flattened morphology when grown on collagen. These cells whether grown in suspension or on collagen, displayed a striking level of vacuole formation, these vacuoles were not associated with transport epithelia. The rectal gland cell cultures were then used to investigate the effect increasing extracellular sodium chloride concentration has on rectal gland cells Na, K-ATPase activity. Increasing sodium chloride concentration in the growth medium by 50% (240 mM to 360 mM) resulted in a transient 3-4 fold increase in Na, K-ATPase activity in cell homogenates approximately 12 hours after the medium change. The response was dependent upon both sodium and chloride ions and was also inhibited by the loop diuretic bumetanide (0.1 mM within 30 minutes), indicating that entry of the ions into the cell is via the Na, K, C1 cotransporter. Incubation of cells in normal medium in the presence of the sodium ionophore monensin also resulted in a dose dependant sustained increase in Na, K-ATPase activity following a 12 hour incubation. The increase in Na, K-ATPase activity associated with increased extracellular sodium chloride concentration was only seen in cells grown on collagen and not in cells grown in suspension. Increases in activity are sensitive to the protein synthesis inhibitor cycloheximide (10 mug/ml), but not the transcriptional inhibitor actinomycin D suggesting that up-regulation of the Na, K-ATPase occurs at the level of translational regulation. Unfortunately this result could not be confirmed using Northern analysis due to unforeseen difficulties in extracting sufficient RNA from the cell cultures. Addition of bumetanide (0.1 mM) to cells grown in normal medium caused a rapid but reversible down-regulation (by 70%) of basal Na, K-ATPase activity within 30 minutes. The anti-microtubular agent colchicine (0.1 mug/ml) inhibited the bumetanide induced down-regulation of Na, K-ATPase and also the recovery of activity following bumetanide removal. The rectal gland cell cultures were used to investigate potential hormonal regulators of the shark rectal gland. The effect of the putative regulators of sodium chloride secretion scyliorhinin II and sCNP on intracellular concentrations of cAMP and cGMP was investigated. The cell cultures were shown be hormonally active as they responded with an increase in intracelllular cAMP concentration to forskolin, PGE1 and PGE2. When scyliorhinin II (10 muM) and IBMX (1 mM) was perfused through the isolated rectal gland a 2 fold increase in cAMP concentration was found in the perfusate after 8 minutes, however no increase was seen in cAMP levels when cell cultures were treated with scyliorhinin II. Shark CNP increased cGMP concentrations in the perfusates of the perfused rectal gland by up to four fold after seven minutes but there was no consistent effect on cGMP concentrations in the cultured cell monolayer. In conclusion it is believed that sCNP and scyliorhinin II mediate their actions on the regulation of sodium chloride secretion by the rectal gland at the vascular level, controlling the extent of perfusion of the gland. This study showed that high salt levels in the medium of shark rectal gland cell monolayers increased the measurable Na, K-ATPase activity and that this response was dependent on protein synthesis but not transcription. It also showed that the response is inhibited by the loop diuretic bumetanide, indicating that entry of the ions into the cell is via the Na, K, Cl cotransporter and that the increase in Na, K-ATPase activity is presumably due to an increase in intracellular sodium concentration. The hormones sCNP and scyliorhinin II appear to mediate their actions on the regulation of sodium chloride secretion by the rectal gland at the vascular level controlling the extent of perfusion of the gland. In conclusion although sodium chloride transport in the dogfish rectal gland requires much more investigation, this study has hopefully proved that dogfish epithelial cell cultures provide a good model for further investigations involving the regulation of activity and expression of the sodium pump.

Plasma sodium, chloride and urea concentrations and plasma osmolalities were determined after dogfish were dietary adapted to both acute and chronic feeding regimes. Following single feeding events (20g squid / kg body wt) plasma sodium and chloride concentrations dropped transiently by a maximum of 27% and 18% respectively, two days after feeding. There was however no significant change in the plasma urea concentration or plasma osmolality. Determination of blood vessel calibre by histological examination of rectal glands before, and 12 hours after, a single feeding event suggested an increase in the blood supply to the gland. Repeated feeding events (2.5g pellets / kg body wt) over a period of 1 month were conducted with pelleted diets containing either 1% or 6% w / w NaCl and blood samples were taken weekly for analysis. Plasma sodium concentrations were significantly reduced by approximately 10% after 1 and 3 weeks of feeding and plasma osmolalities fell again by approximately 10% at 1, 2 and 3 weeks when fish were fed on the 1% w / w NaCl pellet diet. However both parameters returned to normal values by the end of the study period. Likewise when fish were fed the high salt (6% w / w NaCl) pellet diet, plasma sodium concentrations and plasma osmolalities again fell by 10-15% after 1, 2 and 3 weeks of repeated feeding. Plasma sodium recovered to normal values within 28 days however the plasma osmolality remained significantly lower than controls. Total RNA was extracted from the rectal gland and homologous and heterologous cDNA probes for the alpha1, beta1 subunits of the Na, K-ATPase and the sCFTR and Na-K-Cl cotransporter were used in Northern and dot blot analyses to identify and quantify the levels of mRNA expression of these major ion transporter proteins during dietary adaptation. Maximal Na, K-ATPase activities in rectal gland homogenates increased transiently by over 40-fold, 9 hours after a single feeding episode (20g squid / kg body wt). There was no concomitant increase in either alpha1 or beta1 subunit of the Na, K-ATPase mRNAs over the same time period however the abundance of both al and (31 Na, K-ATPase subunit mRNAs slowly increased by 75% and 39% respectively two days after feeding and were maintained at that level for either 10 days or 5 days respectively. In chronic dietary adaptations to a squid diet, a 40% increase in beta1 Na, K-ATPase subunit mRNA was found after 4 weeks of repeated feeding however there was no significant increase in al subunit mRNA expression or Na, K-ATPase activity. In contrast chronic dietary adaptation to the 6% w / w NaCl pellet diet resulted in a 3-fold increase in Na, K-ATPase activity however there was no concomitant increase in either the alpha1 or beta1 subunit of the Na, K-ATPase mRNAs. After a single feeding event with the squid diet, mRNAs for both the sCFTR and Na-K-Cl cotransporter proteins were significantly increased, by 55% and 65% respectively, five days after the feeding episode compared to controls.

1. Dogfish, Scyliorhinus canicula, rectal gland epithelial cells were successfully cultured using two different techniques: 1) a perfusion based technique and 2) a modified Valentich's technique (Valentich, 1991). 2. Growth stages of cultures were monitored, showing attachment of tubules at approximately two days with a complete monolayer formed between seven and ten days. Cultures were able to be maintained for up to twenty days. Photos were taken illustrating epithelial cell migration and cell viability. 3. Administration of Ca+2 and Mg+2 free Ringer + 2 mM ethylenediamine tetra-acetic acid (EDTA) + 1% trypsin successfully reduced cultures growing in 96-well plates to single cells after a time course of 20 min to allow for accurate cell counts of approximately 22,000 cells per well. 4. 10-6 M shark C-type natriuretic peptide (sCNP) induced stimulation of cGMP in cultured rectal gland epithelial cells over a time course of 240 sec with maximal stimulation occurring at 180 sec. Limited experiments with scyliorhinin II and rectin showed little effects in stimulating cGMP.

1. Groups of dogfish, Scyliorhinus canicula, were adapted to either a high or a low protein diet over a period of 30 days. The fish readily ate both diets and maintained body weights. 2. Dietary adapted fish were acclimated to 130%, 100% and 50% seawater and the metabolic clearance rates (MCR) and blood production rates (BPR) for urea and 1a-hydroxycorticosterone (1a-OH-B) determined. 3. In 100% seawater low protein diet (LPD) fish had significantly lower urea BPR and MCR than the high protein diet (HPD) fish. 4. In 50% seawater plasma osmolality, sodium, chloride and urea concentrations decreased in both dietary groups. Plasma 1a-OH-B concentration and BPR were significantly increased and in HPD fish this was accompanied by an increase in 1a-OH-B MCR. The increase in plasma 1a-OH-B concentration was significantly greater in the HPD fish than in the LPD fish. 5. In 130% seawater plasma osmolality was increased in both dietary groups. In HPD fish this was achieved by increasing plasma urea, sodium and chloride concentrations. In LPD fish this was achieved by significantly elevating plasma sodium concentration only. Plasma 1a-OH-B concentration and BPR were significantly increased and MCR significantly decreased in LPD fish, but remained unaltered in HPD fish. 6. An isolated perifused interrenal gland preparation was developed for Scyliorhinus canicula, which produced a constant 1a-OH-B basal secretion rate after two hours and remained viable for more than 22 hours. 7. ACTH, All, ANP, AVT, forskolin, dibutyryl cAMP and dibutyryl cGMP stimulated interrenal steroidogenesis. 8. ACTH-induced steroidogenesis was unaffected by the presence of dantrolene and verapamil but significantly increased in the presence of cholera toxin and significantly decreased in the absence of extracellular calcium. 9. Ile5-All-induced steroidogenesis was unaffected by the presence of verapamil but significantly decreased in the presence of dantrolene and in the absence of extracellular calcium. 10. Increases in potassium concentration were significantly steroidogenic only at 28mM and alterations in sodiimi concentration had no effect.

Schroederichthys saurisqualus Soto 2001 são tubarões ovíparos, de pequeno porte (alcançando até 692 mm de comprimento total), geralmente associados a substratos consolidados por organismos como corais, gorgônias e esponjas tubo, podendo ser encontrados em profundidades de até 250 m. No presente estudo, a distribuição geográfica da espécie foi ampliada até São Paulo. A espécie apresentou padrão de coloração com máculas brancas e pretas e selas dispostas por todo corpo, sendo proposta uma nova nomenclatura para este padrão, que poderá ser utilizada para outras espécies da família, descritas somente com base na coloração. Fígado grande ocupando quase toda a cavidade visceral mostrou ser importante taxonomicamente. Uma abordagem inédita relativa à contagem de cúspides dentárias laterais foi efetuada, sendo encontradas de três a cinco nos machos e de três a nove nas fêmeas. Uma nova série dentária foi observada, que variou do tipo alternada dependente até alternada independente, mostrando ter grande importância para a taxonomia e dimorfismo sexual. O condrocrânio mostrou-se de extrema importância taxonômica quando comparado às outras espécies do gênero. O clásper apresentou estruturas anatômicas não conhecidas para a espécie, tais como, a marginal dorsal acessória 4, que está ligada à marginal dorsal acessória 2 e situada abaixo da marginal acessória 3. Com relação aos arcos branquiais, foi verificada uma fusão entre o quarto e o quinto faringobranquiais. Os dados aqui obtidos são potencialmente úteis em futuros estudos filogenéticos e taxonômicos do gênero. / Schroederichthys saurisqualus Soto, 2001 are represented by small oviparous sharks (reaching up to 692 mm in total length), usually associated with consolidated substrates such as corals, tube sponges and gorgonians, and can be found at depths up to 250 m. In this study the geographical distribution of the species was extended to São Paulo. It shows a color pattern with black and white spots and saddles arranged throughout the body, which allowed to purpose a new nomenclature for this pattern, which could be used for other species of the family, described only on the basis of color. Large liver occupying almost the entire visceral cavity showed to be important taxonomically. A new approach on the counting of lateral theeth cusplets was proposed indicating the presence of three to five in males and females from three to nine. A new tooth series was found, which varied from alternated type to alternated independent type, showing to have a major relevance for taxonomy and sexual dimorphism. The neurocranial morphology proved to be extremely important when compared to other species of the genus. The clásper revealed anatomical structures unknown to the species, such as the accessory dorsal marginal 4, which is linked to the accessory dorsal marginal 2 and below the accessory dorsal marginal 3. Regarding the branchial arches, a fusion between the fourth and fifth pharingobranchials was observed. The data obtained here are potentially useful in future taxonomic and phylogenetic studies of the genus

Schroederichthys saurisqualus Soto 2001 são tubarões ovíparos, de pequeno porte (alcançando até 692 mm de comprimento total), geralmente associados a substratos consolidados por organismos como corais, gorgônias e esponjas tubo, podendo ser encontrados em profundidades de até 250 m. No presente estudo, a distribuição geográfica da espécie foi ampliada até São Paulo. A espécie apresentou padrão de coloração com máculas brancas e pretas e selas dispostas por todo corpo, sendo proposta uma nova nomenclatura para este padrão, que poderá ser utilizada para outras espécies da família, descritas somente com base na coloração. Fígado grande ocupando quase toda a cavidade visceral mostrou ser importante taxonomicamente. Uma abordagem inédita relativa à contagem de cúspides dentárias laterais foi efetuada, sendo encontradas de três a cinco nos machos e de três a nove nas fêmeas. Uma nova série dentária foi observada, que variou do tipo alternada dependente até alternada independente, mostrando ter grande importância para a taxonomia e dimorfismo sexual. O condrocrânio mostrou-se de extrema importância taxonômica quando comparado às outras espécies do gênero. O clásper apresentou estruturas anatômicas não conhecidas para a espécie, tais como, a marginal dorsal acessória 4, que está ligada à marginal dorsal acessória 2 e situada abaixo da marginal acessória 3. Com relação aos arcos branquiais, foi verificada uma fusão entre o quarto e o quinto faringobranquiais. Os dados aqui obtidos são potencialmente úteis em futuros estudos filogenéticos e taxonômicos do gênero. / Schroederichthys saurisqualus Soto, 2001 are represented by small oviparous sharks (reaching up to 692 mm in total length), usually associated with consolidated substrates such as corals, tube sponges and gorgonians, and can be found at depths up to 250 m. In this study the geographical distribution of the species was extended to São Paulo. It shows a color pattern with black and white spots and saddles arranged throughout the body, which allowed to purpose a new nomenclature for this pattern, which could be used for other species of the family, described only on the basis of color. Large liver occupying almost the entire visceral cavity showed to be important taxonomically. A new approach on the counting of lateral theeth cusplets was proposed indicating the presence of three to five in males and females from three to nine. A new tooth series was found, which varied from alternated type to alternated independent type, showing to have a major relevance for taxonomy and sexual dimorphism. The neurocranial morphology proved to be extremely important when compared to other species of the genus. The clásper revealed anatomical structures unknown to the species, such as the accessory dorsal marginal 4, which is linked to the accessory dorsal marginal 2 and below the accessory dorsal marginal 3. Regarding the branchial arches, a fusion between the fourth and fifth pharingobranchials was observed. The data obtained here are potentially useful in future taxonomic and phylogenetic studies of the genus