Greg G. Goss

Evidence for a morphological component in acid-base regulation during environmental hypercapnia in the brown bullhead (Ictalurus nebulosus)

SummaryExposure of adult brown bullheads Ictalurus nebulosus (120–450 g) to environmental hypercapnia (2% carbon dioxide in air) and subsequent recovery caused transient changes in whole body net sodium flux (JnetNa+) and net chloride flux (JnetCl-) resulting largely from changes in whole body sodium influx (JinNa+) and chloride influx (JinCl-). Scanning electron microscopy (SEM) revealed that the fractional area of chloride cells (CCs) on the interlamellar regions was reduced by 95% during environmental hypercapnia. During post-hypercapnic recovery, gill filament CC fractional area increased. The changes in JinCl-during and after environmental hypercapnia were closely associated with the changes in CC fractional area while the changes in JinNa+did not correspond to the changes in CC fractional area. Transmission electron microscopy (TEM) supported the SEM observations of CC surface area changes and demonstrated that these changes were caused by covering/uncovering by adjacent pavement cells (PVCs). Lamellar and filament PVC microvilli density increased during hypercapnia while there was a subsequent reduction in the post-hypercapnic period. These data suggest that an important mechanism of acid-base regulation during hypercapnic acidosis is modification of the chloride cell-associated Cl-/HCO3-exchange mechanism. We suggest that bullheads vary availability, and thus functional activity, of this transporter via reversible morphological alterations of the gill epithelium. The increase in density of PVC microvilli may be associated with sodium uptake and/or acidic equivalent excretion during acidosis.

Interrelationships between gill chloride cell morphology and calcium uptake in freshwater teleosts

The involvement of the freshwater fish gill chloride cells (CCs) in trans-branchial calcium uptake (JinCa2+) was investigated. This was accomplished by assessing the interspecific relationships between the apical surface area of CCs exposed to the external environment and JinCa2+. Three species of freshwater teleosts, the rainbow trout (Oncorhynchus mykiss), the American eel (Anguilla rostrata) and the brown bullhead catfish (Ictalurus nebulosus), were used. Chronic (ten-day) treatment with cortisol in each species was used as a tool to evoke variations in both JinCa2+ and gill CC morphology in order to assess intraspecific relationships between CC surface area and JinCa2+. The results of quantitative morphometry, based on analysis of scanning electron micrographs, demonstrated that catfish possessed the lowest fractional area of exposed CC (CCFA) on the gill filament epithelium (12,744 ± 2248 μm2/mm2) and was followed, in increasing order, by American eel (21,355 ± 981 μm2/mm2) and rainbow trout (149,928 ± 26,545 μm2/mm2). With the exception of catfish, chronic treatment with cortisol caused significant increases in CCFA owing to proliferation of CCs and/or enlargement of individual CCs (eel only). The rates of JinCa2+ closely reflected the CC fractional area in each species. The results of correlation analysis revealed significant correlations between CC fractional area and JinCa2+ in trout and eel. Owing to the absence of an effect of cortisol treatment, there was no significant correlation in catfish because of insufficient variation in CC fractional area in this species. CC fractional area was significantly correlated with JinCa2+ among the three species examined. These results suggest that CC is involved in calcium uptake in freshwater teleosts and that both intra- and interspecific differences in the rates of calcium uptake can be accounted for by variability in the surface area of exposed CCs on the gill epithelia.

Mannitol at Clinical Concentrations Activates Multiple Signaling Pathways and Induces Apoptosis in Endothelial Cells

BACKGROUND AND PURPOSE Hyperosmotic mannitol therapy is widely used in the clinical setting for acute and subacute reduction in brain edema, to decrease muscle damage in compartment syndrome, and to improve renal perfusion. Though beneficial rheological effects commonly are attributed to mannitol, its direct effects on endothelial cells are poorly understood. METHODS We studied the effect of hypertonic and hypotonic stress on bovine aortic endothelial (BAE) cells, using mannitol, urea, and sodium chloride and medium dilution in vitro. RESULTS Exposure to incremental osmolar concentrations of 300 mOsm of each osmotic agent increased apoptosis in BAE cells (mannitol congruent withNaCl>urea). Induced programmed cell death was detected by DAPI staining of intact cell nuclei, and by TUNEL and DNA fragmentation ladder assays. Mannitol-induced apoptosis exhibited dose dependence (42% of cells at 300 mOsm [P<0.0001] compared with 1.2% of control cells) and was also observed in bovine smooth muscle cells. Mannitol-induced apoptosis was attenuated approximately 50% in the presence of cycloheximide or actinomycin D. Hypertonic mannitol and NaCl, but not urea, increased tyrosine phosphorylation of the focal adhesion contact-associated proteins paxillin and FAK. Hypotonic medium, which did not lead to apoptosis, increased protein tyrosine phosphorylation of FAK but not of paxillin. Addition of mannitol or NaCl also produced sustained increases in c-Jun NH2-terminal kinase (JNK) activity. In addition, hypertonic mannitol increased intracellular free [Ca2+] in a dose-dependent manner. Chelation of intracellular Ca2+ with quin2-AM (10 micromol/L) inhibited mannitol-induced apoptosis approximately 50%, as to a lesser extent did inhibition of tyrosine kinase activity with herbimycin (1 micromol/L). CONCLUSIONS We have shown that hypertonic mannitol exposure induces endothelial cell apoptosis, accompanied by activation of tyrosine and stress kinases, phosphorylation of FAK and paxillin, and elevation of intracellular free [Ca2+]. The apoptosis is attenuated by inhibition of transcription or translation, by inhibition of tyrosine kinases, or by intracellular Ca2+ buffering. These data suggest that clinical use of the osmotic diuretic mannitol may exert direct deleterious effects on vascular endothelium.

Morphological responses of the rainbow trout (Oncorhynchus mykiss) gill to hyperoxia, base (NaHCO3) and acid (HCl) infusions

Marked morphological responses occur in the gills of freshwater rainbow trout in response to experimental acid-base disturbance and these responses play an important role in acid-base correction. Compensated respiratory acidosis induced by 70h exposure to environmental hyperoxia (elevated water PO2) caused a 33% decrease in branchial chloride cell fractional surface area (CCFA). Metabolic alkalosis induced by normoxic recovery (6h) from hyperoxia (72h) caused a 50% increase in CCFA, whereas metabolic alkalosis induced by infusion (19h) of NaHCO3 caused a 70% rise. However, the largest increase (135%) in CCFA was seen in response to infusion (19h) of HCl. NaCl infusion had no effect. A particular goal was to assess the relative importance of changes in CCFA vs. changes in internal substrate (HCO3−) availability in regulating the activity of the branchial Cl−/HCO3− exchange system. For each of the experimental treatments, the accompanying blood acid-base status and branchial transport kinetics (Km, Jmax) for Cl− uptake had been determined in earlier studies. In the present study, a positive linear relationship was established between CCFA and JCl−max in individual control fish in the absence of an acid-base disturbance. By reference to this relationship, observed changes in JCl−max during metabolic acid-base disturbances were clearly due to changes in both CCFA and internal substrate levels (plasma [HCO3−]) with the two factors having approximately equal influence.

Two-substrate kinetic analysis : a novel approach linking ion and acid-base transport at the gills of freshwater trout, Oncorhynchus mykiss

SummaryThe novel application of a two-substrate model (Florini and Vestling 1957) from enzymology to transport kinetics at the gills of freshwater trout indicated that Na+/acidic equivalent and Cl-/basic equivalent flux rates are normally limited by the availability of the internal acidic and basic counterions, as well as by external Na+ and Cl- levels. Adult rainbow trout fitted with dorsal aortic and bladder catheters were chronically infused (10–16 h) with isosmotic HCl to induce a persistent metabolic acidosis. Acid-base neutral infusions of isosmotic NaCl and non-infused controls were also performed. Results were compared to previous data on metabolic alkalosis in trout induced by either isosmotic NaHCO3 infusion or recovery from environmental hyperoxia (Goss and Wood 1990a, b). Metabolic acidosis resulted in a marked stimulation of Na+ influx, no change in Cl- influx, positive Na+ balance, negative Cl- balance, and net H+ excretion at the gills. Metabolic alkalosis caused a marked inhibition of Na+ influx and stimulation of Cl- influx, negative Na+ balance, positive Cl- balance, and net H+ uptake (=base excretion). Mean gill intracellular pH qualitatively followed extracellular pH. Classical one-substrate Michaelis-Menten analysis of kinetic data indicated that changes in Na+ and Cl- transport during acid-base disturbance are achieved by large increases and decreases in Jmax, and by increases in Km. However, one-substrate analysis considers only external substrate concentration and cannot account for transport limitations by the internal substrate. The kinetic data were fitted successfully to a two-substrate model, using extracellular acid-base data as a measure of internal HCO3-and H+ availability. This analysis indicated that true Jmax values for Na+/acidic equivalent and Cl-/basic equivalent transport are 4–5 times higher than apparent Jmax values by one-substrate analysis. Flux rates are limited by the availability of the internal counterions; transport Km values for HCO3-and H+ are far above their normal internal concentrations. Therefore, small changes in acid-base status will have large effects on transport rates, and on apparent Jmax values, without alterations in the number of transport sites. This system provides an automatic, negative feedback control for clearance or retention of acidic/basic equivalents when acid-base status is changing.

Phylogenetic Analysis of the MS4A and TMEM176 Gene Families

Background The MS4A gene family in humans includes CD20 (MS4A1), FcRβ (MS4A2), Htm4 (MS4A3), and at least 13 other syntenic genes encoding membrane proteins, most having characteristic tetraspanning topology. Expression of MS4A genes is variable in tissues throughout the body; however, several are limited to cells in the hematopoietic system where they have known roles in immune cell functions. Genes in the small TMEM176 group share significant sequence similarity with MS4A genes and there is evidence of immune function of at least one of the encoded proteins. In this study, we examined the evolutionary history of the MS4A/TMEM176 families as well as tissue expression of the phylogenetically earliest members, in order to investigate their possible origins in immune cells. Principal Findings Orthologs of human MS4A genes were found only in mammals; however, MS4A gene homologs were found in most jawed vertebrates. TMEM176 genes were found only in mammals and bony fish. Several unusual MS4A genes having 2 or more tandem MS4A sequences were identified in the chicken (Gallus gallus) and early mammals (opossum, Monodelphis domestica and platypus, Ornithorhyncus anatinus). A large number of highly conserved MS4A and TMEM176 genes was found in zebrafish (Danio rerio). The most primitive organism identified to have MS4A genes was spiny dogfish (Squalus acanthus). Tissue expression of MS4A genes in S. acanthias and D. rerio showed no evidence of expression restricted to the hematopoietic system. Conclusions/Significance Our findings suggest that MS4A genes first appeared in cartilaginous fish with expression outside of the immune system, and have since diversified in many species into their modern forms with expression and function in both immune and nonimmune cells.

The effects of experimentally altered gill chloride cell surface area on acid-base regulation in rainbow trout during metabolic alkalosis

To evaluate the role of the gill chloride cells in regulating metabolic alkalosis in rainbow trout (Oncorhynchus mykiss), the surface area of branchial chloride cells was altered experimentally using combined cortisol/ovine growth hormone injections. Long-term (10-day) treatment of fish with cortisol/ovine growth hormone caused an increase in the two-dimensional chloride cell fractional surface area when compared to uninjected fish (from 8.4 to 29.7%). This was the combined result of an increase in the size of individual cells (from 34.6 to 59.2 μm2) and increased numbers of cells (from 2368 to 5006 cells · mm-2). Metabolic alkalosis was induced by intra-arterial infusion of 140 mmol · l-1 NaHCO3; control fish were infused with 140 mmol · l-1 NaCl. Blood pH and plasma [HCO3-] increased in both the untreated and the cortisol/ovine growth hormone-treated fish. However, the increases in pH (from 8.05 to 8.53) and [HCO3-] (from 5.9 to 22.2 mmol · l-1) in the untreated fish were significantly greater than in the cortisol/ovine growth hormone-treated fish (pH increased from 7.78 to 8.11; [HCO3-] increased from 5.5 to 13.9 mmol · l-1). In all fish, NaHCO3 infusion elicited an increase in the rate of branchial basic equivalent excretion (acidic equivalent uptake) which, in turn, was caused by decreases and increases in branchial Na+ uptake and Cl- uptake, respectively. In the untreated fish, there was a pronounced increase (75%) in chloride cell surface area during NaHCO3 infusion. The attenuation of the metabolic alkalosis during HCO3- infusion in the cortical/ovine growth hormone-treated fish was caused, at least in part, by an enhancement of branchial basic equivalent excretion. In these fish that already displayed a proliferation of chloride cells, there was no further increase in chloride cell surface area. The changes in Na+ influx and Cl- influx were quantitatively similar during NaHCO3 infusion in both groups. This suggests that the greater rate of base excretion in the cortisol/ovine growth hormone-treated fish was caused by a greater percentage of Cl- uptake being coupled to HCO3- excretion and less to Cl- excretion (Cl- exchange diffusion).