William S. Marshall

Transport mechanisms of seawater teleost chloride cells: an inclusive model of a multifunctional cell.

This review assembles recent information on seawater-type chloride cells of marine teleost fish and evaluates the secretion of Na+, Cl-, K+, H+ and NH4+ and the absorption of Ca2+. The evidence for the distribution (apical vs basolateral) and the abundance of the various ion pumps, cotransporters, channels and exchangers is assessed and an inclusive model is constructed. Relationships among the transport systems are presented to suggest that many, if not all, of these systems may be operating simultaneously in individual, multifunctional chloride cells.

Ion balance, acid-base regulation, and chloride cell function in the common killifish, Fundulus heteroclitus-a euryhaline estuarine teleost

The common killifish,Fundulus heteroclitus, is a euryhaline teleost common throughout estuaries of eastern North America. This symposium paper reviews the important contributions of the killifish to our present understanding of ionoregulation in seawater (SW) fish and their mechanisms of euryhalinity, and presents new data developing the killifish as a freshwater (FW) model system. Experiments on killifish have characterized (i) drinking in SW and its reduction in FW; (ii) the adaptive roles of the kidney to SW and FW conditions; (iii) the instantaneous (Phase I) and delayed (Phase II) reductions in Na+ outflux that occur upon transfer from SW to FW; (iv) the importance of prolactin secretion in the Phase II effect; (v) the cortisol-stimulated induction of branchial Na+, K+-ATPase that occurs upon transfer from FW to SW; (vi) the accompanying changes in morphology of the mitochondria-rich (MR) or “chloride cells” on the gills; (vii) the localization of this Na+, K+-ATPase activity to the basolateral membrane of chloride cells; and (viii) the NaCl-secretory function of these cells in SW. The opercular epithelium, which is rich in chloride cells, has been used as an in vitro model to characterize the mechanisms and control of NaCl secretion in SW fish. Much less is known about gill function in fresh water (inward NaCl transport), primarily due to the absence of a comparable freshwater model. Here we show that killifish acclimated to dilute FW ([NaCl] = 1 mmol I−1) possess large numbers of MR cells on the opercular epithelium. When mounted in vitro with FW on the outside, the preparation develops a large inside negative transepithelial potential (TEP) that is a Na+ diffusion potential. By the Ussing flux ratio criterion, Na+ fluxes are passive, but a small active influx of Cl− occurs, an observation that supports the involvement of MR cells in active Cl− uptake. This FW opercular epithelium if bathed with isotonic saline on both sides does not secrete Cl−, indicating that the MR cells indeed are of the FW type. In vivo, the fish exhibits a high rate of Na+ influx and outflux; Cl− outflux is much lower, and there is no detectable Cl− influx. Experimental variation of FW [NaCl] reveals a saturable, low affinity Na+ uptake mechanism, a Cl− influx mechanism that is activated only at much higher concentrations, and no evidence of exchange diffusion. Acid-base disturbance appears to be corrected by differential regulation of the outflux components only. Hence, the FW killifish ionoregulates somewhat differently from the few other FW teleosts that have been examined, and its opercular epithelium will serve as a very useful model system.

NaCl Transport in Gills and Related Structures

This first part of the review will survey our actual knowledge on various aspects of NaCl transport and related mechanisms at work in gills of invertebrates.

Hypotonic shock mediation by p38 MAPK, JNK, PKC, FAK, OSR1 and SPAK in osmosensing chloride secreting cells of killifish opercular epithelium

SUMMARY Hypotonic shock rapidly inhibits Cl- secretion by chloride cells, an effect that is osmotic and not produced by NaCl-depleted isosmotic solutions, yet the mechanism for the inhibition and its recovery are not known. We exposed isolated opercular epithelia, mounted in Ussing chambers, to hypotonic shock in the presence of a variety of chemicals: a general protein kinase C (PKC) inhibitor chelerythrine, Gö6976 that selectively blocks PKCα and β subtypes, H-89 that blocks PKA, SB203580 that blocks p38 mitogen-activated protein kinase (MAPK), as well as serine/threonine protein phosphatase (PP1 and 2A) inhibitor okadaic acid, and finally tamoxifen, a blocker of volume-activated anion channels (VSOAC). Chelerythrine has no effect on hypotonic inhibition but blocked the recovery, indicating PKC involvement in stimulation. Gö6976 had little effect, suggesting that PKCα and PKCβ subtypes are not involved. H-89 did not block hypotonic inhibition but decreased the recovery, indicating PKA may be involved in the recovery and overshoot (after restoration of isotonic conditions). SB203580 significantly enhanced the decrease in current by hypotonic shock, suggesting an inhibitory role of p38 MAPK in the hypotonic inhibition. Okadaic acid increased the steady state current, slowed the hypotonic inhibition but made the decrease in current larger; also the recovery and overshoot were completely blocked. Hypotonic stress rapidly and transiently increased phosphorylated p38 MAPK (pp38) MAPK (measured by western analysis) by eightfold at 5 min, then more slowly again to sevenfold at 60 min. Hypertonic shock slowly increased p38 by sevenfold at 60 min. Phosphorylated JNK kinase was increased by 40-50% by both hypotonic and hypertonic shock and was still elevated at 30 min in hypertonic medium. By immunoblot analysis it was found that the stress protein kinase (SPAK) and oxidation stress response kinase 1 (OSR1) were present in salt and freshwater acclimated fish with higher expression in freshwater. By immunocytochemistry, SPAK, OSR1 and phosphorylated focal adhesion kinase (pFAK) were colocalized with NKCC at the basolateral membrane. The protein tyrosine kinase inhibitor genistein (100 μmol l-1) inhibited Cl- secretion that was high, increased Cl- secretion that was low and reduced immunocytochemical staining for phosphorylated FAK. We present a model for rapid control of CFTR and NKCC in chloride cells that includes: (1) activation of NKCC and CFTR via cAMP/PKA, (2) activation of NKCC by PKC, myosin light chain kinase (MLCK), p38, OSR1 and SPAK, (3) deactivation of NKCC by hypotonic cell swelling, Ca2+ and an as yet unidentified protein phosphatase and (4) involvement of protein tyrosine kinase (PTK) acting on FAK to set levels of NKCC activity.

The LCROSS Cratering Experiment

Watering the Moon About a year ago, a spent upper stage of an Atlas rocket was deliberately crashed into a crater at the south pole of the Moon, ejecting a plume of debris, dust, and vapor. The goal of this event, the Lunar Crater Observation and Sensing Satellite (LCROSS) experiment, was to search for water and other volatiles in the soil of one of the coldest places on the Moon: the permanently shadowed region within the Cabeus crater. Using ultraviolet, visible, and near-infrared spectroscopy data from accompanying craft, Colaprete et al. (p. 463; see the news story by Kerr; see the cover) found evidence for the presence of water and other volatiles within the ejecta cloud. Schultz et al. (p. 468) monitored the different stages of the impact and the resulting plume. Gladstone et al. (p. 472), using an ultraviolet spectrograph onboard the Lunar Reconnaissance Orbiter (LRO), detected H2, CO, Ca, Hg, and Mg in the impact plume, and Hayne et al. (p. 477) measured the thermal signature of the impact and discovered that it had heated a 30 to 200 square-meter region from ∼40 kelvin to at least 950 kelvin. Paige et al. (p. 479) mapped cryogenic zones predictive of volatile entrapment, and Mitrofanov et al. (p. 483) used LRO instruments to confirm that surface temperatures in the south polar region persist even in sunlight. In all, about 155 kilograms of water vapor was emitted during the impact; meanwhile, the LRO continues to orbit the Moon, sending back a stream of data to help us understand the evolution of its complex surface structures. A controlled spacecraft impact into a crater in the lunar south pole plunged through the lunar soil, revealing water and other volatiles. As its detached upper-stage launch vehicle collided with the surface, instruments on the trailing Lunar Crater Observation and Sensing Satellite (LCROSS) Shepherding Spacecraft monitored the impact and ejecta. The faint impact flash in visible wavelengths and thermal signature imaged in the mid-infrared together indicate a low-density surface layer. The evolving spectra reveal not only OH within sunlit ejecta but also other volatile species. As the Shepherding Spacecraft approached the surface, it imaged a 25- to-30-meter–diameter crater and evidence of a high-angle ballistic ejecta plume still in the process of returning to the surface—an evolution attributed to the nature of the impactor.

Transepithelial potential and short-circuit current across the isolated skin ofGillichthys mirabilis (Teleostei: Gobiidae), acclimated to 5% and 100% seawater

SummarySkin samples taken from the scaleless and well-vascularized area of the lower jaw were used in a modified Ussing chamber to test for electrogenic ion transport. In the absence of electrochemical gradients the skin developed a transepithelial potential (TEP) of 10–30 mV, serosa-positive, with a short-circuit current (SCC) of 13.7±3 or 20.8±7 μamp/cm2 for fish acclimated to 5% or 100% sea water, respectively. Iodoacetamide +2,4-dinitrophenol, ouabain, or acetazolamide rapidly inhibited the TEP and SCC when perfused on the serosal side, but had little effect when added to the mucosal side. Sodium-, potassium-, or chloride-free Ringer, on both sides, reversibly reduced the TEP and SCC to near zero. The results indicate active ion transport acrossGillichthys skin and suggest a functional chloride excreting pump in the skin of seawater-adapted fish.

Characterization of ion and acid‐base transport in the fresh water adapted mummichog (Fundulus heteroclitus)

We examined whether ionoregulatory mechanisms of fresh water Fundulus heteroclitus in vivo are similar to those of typical freshwater species (e.g., rainbow trout, goldfish, and catfish). Under control conditions ([NaCl]ext ~1 mmol/l), the mummichog exhibits very large Na+ influx and efflux rates but virtually no Cl– influx and a small Cl– efflux component. External NaCl levels were varied to reveal a saturable, low affinity (Km = 1,723 ± 223 μmol/l), high capacity (Jmax = 2,258 ± 288 nEq/g/h) Na+ uptake system that was independent of both Na+ efflux and ammonia excretion. A measurable Cl– influx did not occur until NaCl levels surpassed 2 mmol/l and did not saturate within the freshwater range, suggesting a completely different uptake mechanism. Cl– efflux was also independent of Cl– influx. A systemic acidosis (intraperitoneal HCl injection) was induced in order to investigate the connection between ionoregulation and acid-base balance. The acidosis did not affect influx rates but induced an elevated Cl– efflux and an attenuated Na+ efflux. This resulted in an excess of net Cl– loss over Na+ loss which effected a net acid excretion by strong ion difference theory. These results concur with the measured acid-base fluxes which indicate that over 50% of the acid load was excreted within 4 h by differential efflux modulation. Therefore an ion/acid-base link does exist in the mummichog but differs in nature from that of other freshwater fish. Indeed, virtually all of these findings differ from the current model for most other teleosts, indicating that alternate models of ionoregulation in fresh water exist. J. Exp. Zool. 279:208–219, 1997.

NaCl transport and ultrastructure of opercular epithelium from a freshwater-adapted euryhaline teleost, Fundulus heteroclitus

We adapted killifish to defined freshwater (FW: 1.0 mM Na, 1.0 mM Cl, + 0.1 mM Ca) and by fluorescence light microscopy and scanning and transmission electron microscopy found that the opercular epithelium retained mitochondria rich (MR) cells that were significantly larger but less numerous than in SW-acclimated tissues. Opercular epithelia mounted in vitro with FW bathing the mucosal surface take up Cl against a large negative inside transepithelial potential (Vt, grand mean –64.1 mV) and concentration gradient; the observed flux ratio was significantly different (P < 0.001) from that predicted for passive ion distribution but the net flux was consistently negative. The Na flux ratio suggested that Na was passively distributed. Vt was largely a Na diffusion potential, based on unilateral manipulations of [Na]. Cl unidirectional uptake was unaffected by mucosally added SITS (0.1 mM) but was inhibited by SCN (1.0 mM) and by anaerobiosis. Killifish transferred from SW to FW for 48 h had reduced Cl secretion by the opercular epithelium compared to SW controls but had not yet developed Cl uptake, indicating a slow adaptive process for development of Cl absorptive transport. Opercular epithelia of FW adapted fish, if bathed with isotonic saline on both sides, has a modest net Na and Cl uptake, unlike SW opercular epithelium that strongly secretes Cl under similar conditions. FW killifish opercular epithelium may provide a model to study ion regulation by euryhaline fish. J. Exp. Zool. 277:23–37, 1997.

Cystic fibrosis transmembrane conductance regulator in teleost fish.

The gills and intestinal epithelia of teleost fish express cystic fibrosis transmembrane conductance regulator (CFTR), and utilize this low conductance anion channel in the apical membrane for ion secretion in seawater gill and in the basolateral membrane for ion absorption in freshwater gill. Similarly, in the intestine CFTR is present in the basolateral membrane for intestinal absorption and also in the apical membrane of secreting intestine. The expression of CFTR and the directed trafficking of the protein to the apical or basolateral membrane is salinity-dependent. The CFTR gene has been cloned and sequenced from several teleost species and although all the major elements in the human gene are present, including two nucleotide binding domains that are common to all ATP binding cassette (ABC) transporters, the sequences are divergent compared to shark or human. In euryhaline fish adapting to seawater, CFTR, localized immunocytochemically, redistributes slowly from a basolateral location to the apical membrane while ion secretory capacity increases. The facility with which teleosts regulate CFTR expression and activation during salinity adaptation make this system an appealing model for the expression and trafficking operation of this labile gene product.

Cellular and paracellular pathway resistances in the "tight" Cl- -secreting epithelium of rabbit cornea.

SummaryThe high transverse resistance of the isolated rabbit cornea (6–12 kΩ·cm2) is associated with the corneal epithelium, a Cl−-secreting tissue which is modulated by β-adrenergic and serotonergic receptors. Three methods were employed to determine the resistances for the apical membrane, basolateral membrane, and paracellular conductive pathways in the epithelium. In the first method, the specific resistance of the apical membrane was selectively and reversibly changed. Epinephrine was used to increase apical Cl− conductance and Ag+ was used to increase apical cation permeability. The second method utilized a direct measure of the spontaneous cellular ionic current. The third method obtained estimates of shunt resistance using transepithelial electrophysiological responses to changes in apical membrane resistance. The results of the first method were largely independent of the agent used. In addition, the three methods were in general agreement, and the ranges of mean values for apical membrane, basolateral membrane, and shunt resistances were 23–33, 3–4, and 12–16 kΩ·cm2, respectively, for the normal cornea. The apical membrane was the major, physiologically-modulated barrier to ion permeation. The shunt resistance of the corneal epithelium was comparable to that found previously for other “tight” epithelia. Experiments using Ag+ in tissues that were bathed in Cl− and HCO3-free solutions indicated that under resting conditions the apical membrane is anion-selective.