Aquatic Toxicology 106–107 (2012) 95–103 Contents lists available at SciVerse ScienceDirect Aquatic Toxicology jou rn al h om epa ge: www.elsevier.com/locate/aquatox Lead hampers gill cell volume regulation in marine crabs: Stronger effect in a weak osmoregulator than in an osmoconformer Enelise M. Amado b , Carolina A. Freire b , Marco T. Grassi c , Marta M. Souza a,∗ a Departamento de Ciências Fisiológicas, Universidade Estadual de Londrina, Londrina, Paraná, Brazil b Departamento de Fisiologia, Setor de Ciências Biológicas, Universidade Federal do Paraná, Curitiba, Paraná, Brazil c Departamento de Química, Setor de Ciências Exatas, Universidade Federal do Paraná, Curitiba, Paraná, Brazil a r t i c l e i n f o Article history: Received 30 June 2011 Received in revised form 14 October 2011 Accepted 23 October 2011 Keywords: Callinectes ornatus Crab Gill cells Hepatus pudibundus In vitro Pb 2+ a b s t r a c t Hepatus pudibundus is a strictly marine osmoconformer crab, while Callinectes ornatus inhabits estuar- ine areas, behaving as a weak hyper-osmoregulator in diluted seawater. Osmoconformers are expected to have higher capacity for cell volume regulation, but gill cells of a regulator are expected to display ion transporters to a higher degree. The influence of lead nitrate (10 M) on the ability of isolated gill cells from both species to volume regulate under isosmotic and hyposmotic conditions were here eval- uated. Without lead, under a 25% hyposmotic shock, the gill cells of both species were quite capable of cell volume maintenance. Cells of C. ornatus, however, had a little swelling (5%) during the hyposmotic shock of greater intensity (50%), while cells of H. pudibundus were still capable of volume regulation. In the presence of lead, even under isosmoticity, the gill cells of both species showed about 10% volume reduction, indicating that lead promotes the loss of water by the cells. When lead was associated with 25% and 50% hyposmotic shock, C. ornatus cells lost more volume (15%), when compared to isosmotic conditions, while H. pudibundus cells showed volume regulation. We then analyzed the possible ways of action of lead on the mechanisms of cell volume regulation in the two species. Verapamil (100 M) was used to inhibit Ca 2+ channels, ouabain (100 M) to inhibit Na + /K + -ATPase, and HgCl 2 (100 M) to inhibit aquaporins. Our results suggest that: (1) Ca 2+ channels are candidates for lead entry into gill cells of H. pudibundus and C. ornatus, being the target of lead action in these cells; (2) aquaporins are much more relevant for water flux in H. pudibundus; and (3) the Na + /K + -ATPase is much more relevant for volume regulation in C. ornatus. Osmoregulators may be more susceptible to metal contamination than osmoconformers, especially in situations of reduced salinity, for two basic reasons: (1) lower capacity of volume regulation and (2) putative higher uptake of Pb 2+ through ionic pathways that operate in salt absorption, such as, for example, the Na + /K + -ATPase. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Cell volume regulation is an essential process for maintain- ing cellular homeostasis. The maintenance of a constant volume in face of osmotic disturbances, both extra- and intracellular, is a critical problem faced by all animal cells. Volume changes are usu- ally grouped into two broad categories: anisosmotic and isosmotic. Anisosmotic volume changes are induced by changes in extra- cellular osmolality and isosmotic volume changes occur through changes in intracellular solute content (Strange, 2004; Hoffmann et al., 2009). ∗ Corresponding author at: Instituto de Ciências Biológicas, Universidade Federal do Rio Grande – FURG, Rio Grande, RS, Brazil, Tel.: +55 53 3233 6852; fax: +55 53 3233 6848. E-mail address: mmsouza@furg.br (M.M. Souza). The function and location of gills leads to the fact that their cells are exposed to changes in the external environment, such as changes in salinity and the presence of contaminants (which most often do not affect the osmolality of the water). Furthermore, besides constitutive metabolic processes common to all cells, the function of transepithelial salt transport will also contribute to vol- ume disturbances of gill cells, even in the absence of changes in the external environment. The volume of gill cells can thus be chal- lenged by these two types of osmotic disturbances. Moreover, the external medium of gills cells, given their polarity and condition of interface epithelium, is represented by two possibly different environments, especially in osmoregulators that keep a gradient between their extracellular medium and the water (Freire et al., 2008a,b). To maintain their vital functions, gill cells must have ability to volume regulate after such changes. Under normal conditions, the osmolality of the cytoplasm is equal to the osmolality of the extracellular fluid. Changes in solute 0166-445X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2011.10.012