Does gill boundary layer carbonic anhydrase contribute to carbon dioxide excretion: a comparison between dogfish (Squalus acanthias) and rainbow trout (Oncorhynchus mykiss).
In vivo experiments were conducted on spiny dogfish (Squalus acanthias) and rainbow trout (Oncorhynchus mykiss) in sea water to determine the potential role of externally oriented or gill boundary layer carbonic anhydrase in carbon dioxide excretion. This was accomplished by assessing pH changes in expired water using a stopped-flow apparatus. In dogfish, expired water was in acid-base disequilibrium as indicated by a pronounced acidification (delta pH=-0.11+/-0.01; N=22; mean +/- s.e.m.) during the period of stopped flow; inspired water, however, was in acid-base equilibrium (delta pH=-0.002+/-0.01; N=22). The acid-base disequilibrium in expired water was abolished (delta pH=-0.005+/-0.01; N=6) by the addition of bovine carbonic anhydrase (5 mg l-1) to the external medium. Addition of the carbonic anhydrase inhibitor acetazolamide (1 mmol l-1) to the water significantly reduced the magnitude of the pH disequilibrium (from -0.133+/-0.03 to -0.063+/-0.02; N=4). However, after correcting for the increased buffering capacity of the water caused by acetazolamide, the acid-base disequilibrium during stopped flow was unaffected by this treatment (control delta [H+]=99.8+/-22.8 micromol l-1; acetazolamide delta [H+]=81.3+/-21.5 micromol l-1). In rainbow trout, expired water displayed an acid-base disequilibrium (delta pH=0.09+/-0.01; N=6) that also was abolished by the application of external carbonic anhydrase (delta pH=0.02+/-0.01). The origin of the expired water acid-base disequilibrium was investigated further in dogfish. Intravascular injection of acetazolamide (40 mg kg-1) to inhibit internal carbonic anhydrase activity non-specifically and thus CO2 excretion significantly diminished the extent of the expired water disequilibrium pH after 30 min (from -0.123+/-0.01 to -0.065+/-0.01; N=6). Selective inhibition of extracellular carbonic anhydrase activity using a low intravascular dose (1.3 mg kg-1) of the inhibitor benzolamide caused a significant reduction in the acid-base disequilibrium after 5 min (from -0.11+/-0.01 to -0.07+/-0. 01; N=14). These results demonstrate that the expired water acid-base disequilibrium originates, at least in part, from excretory CO2 and that extracellular carbonic anhydrase in dogfish may have a significant role in carbon dioxide excretion. However, externally oriented carbonic anhydrase (if present in dogfish) plays no role in catalysing the hydration of the excretory CO2 in water flowing over the gills and thus is unlikely to facilitate CO2 excretion. (+info)
Characterization of an amphioxus paired box gene, AmphiPax2/5/8: developmental expression patterns in optic support cells, nephridium, thyroid-like structures and pharyngeal gill slits, but not in the midbrain-hindbrain boundary region.
On the basis of developmental gene expression, the vertebrate central nervous system comprises: a forebrain plus anterior midbrain, a midbrain-hindbrain boundary region (MHB) having organizer properties, and a rhombospinal domain. The vertebrate MHB is characterized by position, by organizer properties and by being the early site of action of Wnt1 and engrailed genes, and of genes of the Pax2/5/8 subfamily. Wada and others (Wada, H., Saiga, H., Satoh, N. and Holland, P. W. H. (1998) Development 125, 1113-1122) suggested that ascidian tunicates have a vertebrate-like MHB on the basis of ascidian Pax258 expression there. In another invertebrate chordate, amphioxus, comparable gene expression evidence for a vertebrate-like MHB is lacking. We, therefore, isolated and characterized AmphiPax2/5/8, the sole member of this subfamily in amphioxus. AmphiPax2/5/8 is initially expressed well back in the rhombospinal domain and not where a MHB would be expected. In contrast, most of the other expression domains of AmphiPax2/5/8 correspond to expression domains of vertebrate Pax2, Pax5 and Pax8 in structures that are probably homologous - support cells of the eye, nephridium, thyroid-like structures and pharyngeal gill slits; although AmphiPax2/5/8 is not transcribed in any structures that could be interpreted as homologues of vertebrate otic placodes or otic vesicles. In sum, the developmental expression of AmphiPax2/5/8 indicates that the amphioxus central nervous system lacks a MHB resembling the vertebrate isthmic region. Additional gene expression data for the developing ascidian and amphioxus nervous systems would help determine whether a MHB is a basal chordate character secondarily lost in amphioxus. The alternative is that the MHB is a vertebrate innovation. (+info)
Regulation of Cl- secretion in seawater fish (Dicentrarchus labrax) gill respiratory cells in primary culture.
1. Primary cultures of sea bass (Dicentrarchus labrax) gill cells grown on permeable membranes form a highly differentiated tight epithelium composed of respiratory-like cells. This preparation was also found to provide a functional model for investigating the hormonal regulation of Cl- secretion. 2. In control conditions, i.e. in the absence of hormones or other stimuli, the cultured epithelium showed a short-circuit current (Isc) of 8.8 +/- 0.4 microA cm-2, a transepithelial potential (Vt) of 28.6 +/- 0.6 mV (serosal side positive), and a transepithelial resistance (Rt) of 5026 +/- 127 Omega cm2. Addition of 50 nM PGE2 caused a stimulation of Isc, Vt and transepithelial conductance, Gt. The increase in Isc was probably due to the elevation in Cl- secretion, since it could be correlated with the stimulation of serosal to mucosal 36Cl- flux. Application of the neurohypophyseal peptide arginine vasotocin (AVT; 50 nM) or the beta-adrenergic agonist isoproterenol (isoprenaline; 0. 5 microM) evoked a stimulation in Cl- secretion, as was shown by the increases in Isc and Gt. The excitatory effect of isoproterenol followed by the inhibitory action of propranolol, a beta-adrenergic antagonist, suggested the presence of beta-adrenergic receptors. Noradrenaline (0.1 microM) elicited a reduction in Isc, Vt and Gt, which was counterbalanced by the addition of phentolamine, an alpha-adrenergic antagonist. This suggested an activation of alpha-adrenergic receptors. 3. This study provides evidence for hormonal control of the Cl- secretion in sea bass gill respiratory cells in culture, involving AVT, prostaglandin (PGE2), and beta- and alpha-adrenergic receptors. (+info)
Thermal compensation in protein and RNA synthesis during the intermolt cycle of the American lobster, Homarus americanus.
1. The in vitro rates of incorporation of precursors into protein and RNA and the concentration of RNA were measured in tissues of intermolt and premolt lobsters acclimated to 5 degrees C and 20 degrees C. Midgut gland, abdominal muscle and gill of intermolt lobsters respond to temperature acclimation by a compensatory translation of the rate-temperature (R-T) curves with respect to the rates of incorporation of 3H-leucine and 3H-uridine into the acid-insoluble fraction. Midgut gland and muscle of premolt animals exhibit either no compensation or inverse compensation; gill tissue exhibits a rotation of the R-T curve. 2. The existence of the complete de novo pathway of pyrimidine biosynthesis is demonstrated in the class Crustacea. NaH14 CO2 is incorporated into orotic acid and orotic-14 C-acid is incorporated into the acid-insoluble fraction. 3. Both the concentration of RNA and the rates of incorporation of precursors of both the salvage and de novo pyrimidine pathways are enhanced in the midgut gland of premolt lobsters, relative to intermolt tissue, under conditions of warm-acclimation. (+info)
Identification by differential display of a hypertonicity-inducible inward rectifier potassium channel highly expressed in chloride cells.
By using differential mRNA display to monitor the molecular alterations associated with adaptation of euryhaline eels to different salinities, we identified a cDNA fragment strongly induced in seawater eel gills. Cloning of a full-length cDNA and its expression in COS-7 cells indicated that the clone codes for an inward rectifier K+ channel (eKir) of 372 amino acid residues, which has two transmembrane segments and a typical pore-forming region (H5). Only low sequence similarities are present, except the H5 region, compared with other members of the inward rectifier K+ channel family (Kir). Consistent with this divergence in the amino acid sequence, a phylogenetic analysis indicated early divergence and independent evolution of eKir from other members; it is only distantly related to the Kir5.0 subfamily members. RNase protection analysis showed that eKir is highly expressed in the seawater eel gill, kidney, and posterior intestine but very weakly in freshwater eels. Immunohistochemistry of gill sections revealed dense localization of eKir in the chloride cells. Immunoelectron microscopy indicated that eKir is mainly present in the microtubular system in the chloride cell. This location and its salt-inducible nature suggest that the eKir channel cloned here is a novel member of the Kir5.0 subfamily of the Kir family and is implicated in osmoregulation. (+info)
Developmental expression of Pax1/9 genes in urochordate and hemichordate gills: insight into function and evolution of the pharyngeal epithelium.
The epithelium of the pharynx contributes to the formation of gills in hemichordates, urochordates, cephalochordates and primitive vertebrates, and is therefore a key structure for understanding developmental mechanisms underlying the establishment of chordate body plans. Pax1- and Pax9-related genes encode transcription factors which are expressed in the pharyngeal region of cephalochordates as well as in the vertebrate pharyngeal pouch epithelium that forms the thymus and parathyroid glands. To explore the molecular basis underlying the occurrence and modifications of the pharyngeal epithelium during evolution, we isolated cDNA clones for Pax1- and Pax9-related genes of urochordates (HrPax1/9 of Halocynthia roretzi and CiPax1/9 of Ciona intestinalis) and a hemichordate (PfPax1/9 of Ptychodera flava) from gill cDNA libraries. Each gene is present as a single copy per haploid genome. All of the cDNAs encode typical paired domains and octapeptides but not a homeodomain, as is also true of other Pax1- and Pax9-related genes. Molecular phylogenetic analysis based on comparison of the paired domain amino-acid sequences suggests that HrPax1/9, CiPax1/9 and PfPax1/9 belong to the Pax1/9 subfamily, and that they are descendants of a single precursor of Pax1/Pax9. Screening of HrPax1/9 cDNA clones yielded six different types of transcripts which were generated by alternative splicing. Northern blot, RT-PCR/Southern and in situ hybridization analyses revealed that HrPax1/9, CiPax1/9 and PfPax1/9 are not expressed during early embryogenesis but are expressed in the epithelia of differentiating gills, suggesting that these genes encode gill-specific transcription factors. The Pax1/9 genes therefore might provide the first developmental genetic corroboration of hypotheses of organ-level homology that unifies hemichordates, urochordates and cephalochordates. (+info)
Extent of gill pathology in the toadfish Tetractenos hamiltoni caused by Naobranchia variabilis (Copepoda: Naobranchiidae).
Sanguinivorous Naobranchia variabilis prefer the first gill arch, external hemibranch and anterior end of the gill arch. The smallest N. variabilis observed attached to fish by a thin filament which connects fused tips of second maxillae to a 'plug' inserted into the gill tissue. Second maxillae enlarge to encircle and increasingly compress the gill filament, which results in a thin layer of epithelium and connective tissue overlying the cartilaginous supporting bar. Early juveniles cause little tissue proliferation, but the extent of proliferated epithelial and connective tissue (PR) adjacent to the maxillae increases from late juveniles to subadult and adult copepods. Most variation in length of gill filament damage (PL, proliferated and compressed tissue) among age classes is explained by maxilla length (ML, length of compressed gill filament); adult trunk width (TRW) explains an extra, small amount of variation, but not trunk length (TRL) or total fish length (TL). Most variation in ML is explained by TRW of adults, subadults and late juveniles, and TRL of early juveniles. PR is explained by TRW for adults, but by ML for other ages. These patterns are due to elongation of the juvenile trunk during growth and lateral expansion of adult egg pouches during maturation. Up to 38 N. variabilis, average (avg.) = 9.3, infected individual Tetractenos hamiltoni and damaged up to 3.4% (avg. 0.72%) of total filament length and 8.6% (avg. 2.1%) of gill filaments per fish. (+info)
Mechanics of ventilation in swellsharks, Cephaloscyllium ventriosum (Scyliorhinidae).
A simple two-pump model has served to describe the mechanics of ventilation in cartilaginous and bony fishes since the pioneering work of G. M. Hughes. A hallmark of this model is that water flow over the gills is continuous. Studies of feeding kinematics in the swellshark Cephaloscyllium ventriosum, however, suggested that a flow reversal occurred during prey capture and transport. Given that feeding is often considered to be simply an exaggeration of the kinematic events performed during respiration, I investigated whether flow reversals are potentially present during respiration. Pressure and impedance data were coupled with kinematic data from high-speed video footage and dye studies and used to infer patterns of water flow through the heads of respiring swellsharks. Swellsharks were implanted with pressure transducers to determine the pattern and magnitude of pressures generated within the buccal and parabranchial (gill) cavities during respiration. Pressure traces revealed extended periods of pressure reversal during the respiratory cycle. Further, impedance data suggested that pressures within the buccal and parabranchial cavities were not generated by the cyclic opening and closing of the jaws and gills in the manner previously suggested by Hughes. Thus, the classic model needs to be re-evaluated to determine its general applicability. Two alternative models for pressure patterns and their mechanism of generation during respiration are provided. The first depicts a double-reversal scenario common in the swellshark whereby pressures are reversed following both of the pump stages (the suction pump and the pressure pump) rather than after the pressure-pump stage only. The second model describes a scenario in which the suction pump is insufficient for generating a positive pressure differential across the gills; thus, a pressure reversal persists throughout this phase of respiration. Kinematic analysis based on high-speed video footage and dye studies, however, suggested that during respiration, as opposed to feeding, distinct flow reversals do not result from the pressure reversals. Thus, water is probably pooling around the gill filaments during the long periods of pressure reversal. (+info)