Horst Onken

Multiple functions of the crustacean gill: osmotic/ionic regulation, acid-base balance, ammonia excretion, and bioaccumulation of toxic metals

The crustacean gill is a multi-functional organ, and it is the site of a number of physiological processes, including ion transport, which is the basis for hemolymph osmoregulation; acid-base balance; and ammonia excretion. The gill is also the site by which many toxic metals are taken up by aquatic crustaceans, and thus it plays an important role in the toxicology of these species. This review provides a comprehensive overview of the ecology, physiology, biochemistry, and molecular biology of the mechanisms of osmotic and ionic regulation performed by the gill. The current concepts of the mechanisms of ion transport, the structural, biochemical, and molecular bases of systemic physiology, and the history of their development are discussed. The relationship between branchial ion transport and hemolymph acid-base regulation is also treated. In addition, the mechanisms of ammonia transport and excretion across the gill are discussed. And finally, the toxicology of heavy metal accumulation via the gill is reviewed in detail.

Osmoregulation and Excretion

The article discusses advances in osmoregulation and excretion with emphasis on how multicellular animals in different osmotic environments regulate their milieu intérieur. Mechanisms of energy transformations in animal osmoregulation are dealt with in biophysical terms with respect to water and ion exchange across biological membranes and coupling of ion and water fluxes across epithelia. The discussion of functions is based on a comparative approach analyzing mechanisms that have evolved in different taxonomic groups at biochemical, cellular and tissue levels and their integration in maintaining whole body water and ion homeostasis. The focus is on recent studies of adaptations and newly discovered mechanisms of acclimatization during transitions of animals between different osmotic environments. Special attention is paid to hypotheses about the diversity of cellular organization of osmoregulatory and excretory organs such as glomerular kidneys, antennal glands, Malpighian tubules and insect gut, gills, integument and intestine, with accounts on experimental approaches and methods applied in the studies. It is demonstrated how knowledge in these areas of comparative physiology has expanded considerably during the last two decades, bridging seminal classical works with studies based on new approaches at all levels of anatomical and functional organization. A number of as yet partially unanswered questions are emphasized, some of which are about how water and solute exchange mechanisms at lower levels are integrated for regulating whole body extracellular water volume and ion homeostasis of animals in their natural habitats.

Ion-motive ATPases and active, transbranchial NaCl uptake in the red freshwater crab, Dilocarcinus pagei (Decapoda, Trichodactylidae)

SUMMARY The present investigation examined the microanatomy and mRNA expression and activity of ion-motive ATPases, in anterior and posterior gills of a South American, true freshwater crab, Dilocarcinus pagei. Like diadromous crabs, the anterior gills of this hololimnetic trichodactylid exhibit a highly attenuated (2–5 μm), symmetrical epithelium on both lamellar surfaces. In sharp contrast, the posterior gill lamellar epithelia are markedly asymmetrical. Their proximal side consists of thick (18–20μ m) cells, displaying features typical of a transporting epithelium, while the distal epithelium is thin (3–10 μm) and formed entirely by apical pillar cell flanges. Both anterior and posterior gills express Na+/K+- and V-ATPases. Phylogenetic analysis of partial cDNA sequences for the Na+/K+-ATPase α-subunit and V-ATPase B-subunit among various crab species confirmed the previous classification and grouping of D. pagei based on morphological criteria. Semi-quantitative RT-PCR clearly showed that mRNA for both ion pump subunits is more intensely expressed in posterior gills. Na+/K+-ATPase activity in the posterior gills was nearly fourfold that of anterior gills, while V-ATPase and F-ATPase activities did not differ. A negative short-circuit current (Isc) was measured using the distal side of split, posterior gill lamellae, mounted in a modified Ussing chamber and perfused symmetrically with identical hemolymph-like salines. Although hemolymph-side ouabain did not affect this current, concanamycin significantly reduced Isc without altering preparation conductance, suggesting V-ATPase-driven Cl– absorption on the distal side of the posterior gill lamellae, as known to occur in diadromous crabs adapted to freshwater. These findings suggest that active Na+ uptake predominates across the thick proximal epithelium, and Cl– uptake across the thin, distal epithelium of the posterior gill lamellae.

The anterior stomach of larval mosquitoes (Aedes aegypti): effects of neuropeptides on transepithelial ion transport and muscular motility

SUMMARY The present investigation studied the influence of a number of neuropeptides on semi-open preparations of the isolated and perfused anterior stomach of larval Aedes aegypti. Effects of peptides were observed on the lumen negative transepithelial voltage (Vte) that is present with serotonin in the bath; this voltage most likely reflects active HCO3– secretion involved in alkalization of the larval anterior stomach. The five different A. aegypti allatostatins (allatostatin A 1–5) all affected Vte in almost identical ways, causing a 10–15% reduction of the voltage at 10–7 mol l–1. A. aegypti neuropeptide F and proctolin reduced Vte at submicromolar concentrations. At 10–6 mol l–1, neuropeptide F reduced Vte by 30% and proctolin reduced Vte by 50%. In contrast, A. aegypti allatotropin, A. aegypti head peptides I and III and A. aegypti short neuropeptide F were without effect on Vte. During the investigation it was observed that the peristaltic contractions of the preparations caused a dynamic component of Vte. Peristaltic contractions and the correlated voltage fluctuations depended on the presence of serotonin. Peristaltic activity and Vte deflections were progressively inhibited by A. aegypti head peptides I and III by A. aegypti short neuropeptide F and by A. aegypti neuropeptide F when the peptide concentrations were increased from 10–8 to 10–6 mol l–1. These observations show that physiological concentrations of some of the tested neuropeptides affect two processes that require coordination: ion transport and motility of the larval anterior stomach.

Active NaCl absorption across posterior gills of hyperosmoregulating Chasmagnathus granulatus.

SUMMARY Split lamellae of posterior gills of Chasmagnathus granulatus adapted to 2.5‰ salinity were mounted in a modified Ussing chamber. With NaCl-saline on both sides of the preparation a transepithelial voltage (Vte) of 4.1±0.5 mV (outside positive) was measured. After voltage-clamping, the negative short-circuit current (Isc) amounted to -142±21 μA cm-2 at a conductance (Gte) of 44±5 mS cm-2. Substitution of either chloride (by nitrate) or sodium (by choline) on both sides of split gill lamellae significantly reduced Isc (by 70-80%) and Gte (by 30-50%). External CsCl (but not BaCl2 or furosemide) inhibited the negative Isc without affecting Gte. Addition of ouabain, BaCl2 or diphenylamine-2-carboxylate to the internal bath inhibited Isc at unchanged Gte. Internal acetazolamide did not affect Isc or Gte across split gill lamellae. Unidirectional Na+ influx across isolated and perfused posterior gills, however, was reduced by internal acetazolamide by approximately 20% at constant Vte. The results suggest that posterior gills of hyperosmoregulating C. granulatus display a high conductance epithelium that actively absorbs NaCl in a coupled way by an electrogenic mechanism similar to that seen in the thick ascending limb of Henles loop and, to a minor degree, by an electroneutral mechanism, presumably via apical Na+/H+- and Cl-/HCO3--antiports.

Alkalinization in the isolated and perfused anterior midgut of the larval mosquito, Aedes aegypti.

Abstract In the present study, isolated midguts of larval Aedes aegypti L. (Diptera: Culicidae) were mounted on perfusion pipettes and bathed in high buffer mosquito saline. With low buffer perfusion saline, containing m-cresol purple, transepithelial voltage was monitored and luminal alkalinization became visible through color changes of m-cresol purple after perfusion stop. Lumen negative voltage and alkalinization depended on metabolic energy and were stimulated in the presence of serotonin (0.2 µmol l-1). In some experiments a pH microelectrode in the lumen recorded pH values up to 10 within minutes after perfusion stop. The V-ATPase inhibitor concanamycin (50 µmol l-1) on the hemolymph side almost abolished Vte and inhibited luminal alkalinization. The carbonic anhydrase inhibitor, methazolamide (50 µmol l-1), on either the luminal or hemolymph-side, or the inhibitor of anion transport, DIDS (1 mmol l-1) on the luminal side, had no effect on Vte or alkalinization. Cl- substitution in the lumen or on both sides of the tissue affected Vte, but the color change of m-cresol purple was unchanged from control conditions. Hemolymph-side Na+ substitution or addition of the Na+/H+ exchange inhibitor, amiloride (200 µmol l-1), reduced Vte and luminal alkalinization. Luminal amiloride (200 µmol l-1) was without effects on Vte or alkalinization. High K+ (60 mmol l-1) in the lumen reduced Vte without affecting alkalinization. These results indicate that strong luminal alkalinization in isolated and perfused anterior midgut of larval A. aegypti depends on basolateral V-ATPase, but is apparently independent of carbonic anhydrase, apical Cl-/HCO3- exchange or apical K+/2H+ antiport.

Revisiting the cellular mechanisms of strong luminal alkalinization in the anterior midgut of larval mosquitoes

SUMMARY Here we critically review two recent hypotheses about the mechanism of strong alkalinization by the anterior midgut of mosquito larvae and our tests of these hypotheses. We present experimental evidence against the major components of transport models proposed in these hypotheses. Measurements of the transapical and transbasal proton electrochemical gradients provide an indication of driving forces faced by and generated by the transport mechanisms of the tissue. These measurements confirmed that basal V-ATPase energizes alkalinization. Serotonin stimulates the V-ATPase, as indicated by the ensuing increase in proton-motive force across the basal membrane. Moreover, the neurohormone resulted in a surprisingly large increase in the intracellular pH. The results of inhibitor studies indicate that, contrary to previous proposals, carbonic anhydrase is apparently not involved in supplying acid–base equivalents to the respective transporters. Furthermore, any apical processes proposed to be involved in alkali secretion or acid absorption must be Cl– independent and insensitive to DIDS, amiloride, Zn2+ and ouabain. These results argue against the involvement of putative apical Cl–/HCO –3 exchangers, apical H+ channels, apical cation/proton exchangers and the importance of the apical Na+/K+ pump. The studies analyzed here thus provide both a limitation and direction for further studies of the mechanism of strong alkalinization in this system.

Electrophysiology of posterior, NaCl-absorbing gills of Chasmagnathus granulatus: rapid responses to osmotic variations.

SUMMARY In the present study, the influence of short-term osmotic variations on some electrophysiological properties related to NaCl absorption across posterior gills of Chasmagnathus granulatus was investigated. The transepithelial potential difference (Vte) of isolated and perfused gills increased significantly when hyposmotic saline (699 mosmol l-1) was used instead of isosmotic solution (1045 mosmol l-1). A reduction of the concentration of Na+ or Cl- at constant osmolarity did not produce any change in Vte. Transepithelial short-circuit current (Isc) and conductance (Gte), measured with split gill lamellae mounted in a modified Ussing chamber, also increased after changing to hyposmotic salines (Isc: from -89.0±40.8 μA cm-2 to -179.3±37.0 μA cm-2; Gte: from 40.5±16.9 mS cm-2 to 47.3±15.8 mS cm-2). The observed effects of reduced osmolarity were fast, reversible and gradually dependent on the magnitude of the osmotic variation. The activity of the Na+/K+-ATPase increased significantly after perfusion with hyposmotic saline, from 18.73±6.35 μmol Pi h-1 mg-1 to 41.84±14.54 μmol Pi h-1 mg-1. Theophylline maintained part of the elevated Vte induced by hyposmotic saline, suggesting that an increased cellular cyclic AMP level is involved in the response to reduced osmolarity. In summary, the results indicate that the hemolymph osmolarity regulates active transbranchial NaCl absorption by modulating the activity of the basolateral Na+/K+-ATPase and by changing a conductive pathway, probably at the apical membrane.

Cellular mechanisms of acid secretion in the posterior midgut of the larval mosquito (Aedes aegypti).

SUMMARY The gut contents of larval mosquitoes are alkalinized by the anterior midgut and reacidified by the posterior midgut. In the present study the cellular mechanisms of reacidification were studied in isolated, perfused posterior midgut by measuring the transepithelial voltage (Vte) and the rate of acid secretion as indicated by the color change of m-cresol purple during intervals of perfusion stop. The lumen-positive Vte and reacidification were significantly increased by serotonin (0.2 μmol l−1). The V-type H+-ATPase inhibitor concanamycin A (10 μmol l−1) on the luminal side inhibited acidification and decreased Vte. On the hemolymph side the carbonic anhydrase (CA) inhibitor acetazolamide (1 mmol l−1) almost abolished Vte, but had no effect on acidification. Similarly, hemolymph-side DIDS (0.1 mmol l−1), DPC (0.5 mmol l−1), amiloride (1 mmol l−1) and ouabain (2.5 mmol l−1) significantly reduced Vte, whereas Ba2+ (5 mmol l−1) was without effect. DPC and amiloride also reduced Vte when applied to the luminal side of the epithelium. Unilateral substitution of gluconate for Cl− affected Vte in a way consistent with a greater permeability for Cl− versus Na+. Cl− replacement in the lumen decreased Vte, whereas replacement on the hemolymph side increased it. Bilateral replacement left the control voltage unaffected. Na+ replacement on either side of the tissue reduced Vte to different degrees. Omission of luminal amino acids was followed by a significant decrease in Vte. Except for concanamycin A, none of the above manipulations impaired acidification, indicating that acidification requires only the apical proton pump. However, the chemical source of secreted H+ is still unknown and needs to be investigated.

Serotonin-induced high intracellular pH aids in alkali secretion in the anterior midgut of larval yellow fever mosquito Aedes aegypti L.

SUMMARY The anterior midgut of the larval yellow fever mosquito Aedes aegypti generates a luminal pH in excess of 10 in vivo and similar values are attained by isolated and perfused anterior midgut segments after stimulation with submicromolar serotonin. In the present study we investigated the mechanisms of strong luminal alkalinization using the intracellular fluorescent indicator BCECF-AM. Following stimulation with serotonin, we observed that intracellular pH (pHi) of the anterior midgut increased from a mean of 6.89 to a mean of 7.62, whereas pHi of the posterior midgut did not change in response to serotonin. Moreover, a further increase of pHi to 8.58 occurred when the pH of the luminal perfusate was raised to an in vivo-like value of 10.0. Luminal Zn2+ (10 μmol l–1), an inhibitor of conductive proton pathways, did not inhibit the increase in pHi, the transepithelial voltage, or the capacity of the isolated tissue to alkalinize the lumen. Finally, the transapical voltage did not significantly respond to luminal pH changes induced either by perfusion with pH 10 or by stopping the luminal perfusion with unbuffered solution which results in spontaneous luminal alkalinization. Together, these results seem to rule out the involvement of conductive pathways for proton absorption across the apical membrane and suggest that a serotonin-induced alkaline pHi plays an important role in the generation of an alkaline lumen.