Tissue & Cell, 2000 32 (5) 389–398 © 2000 Harcourt Publishers Ltd doi: 10.1054/tice.2000.0127, available online at http://www.idealibrary.com
Tissue&Cell
Integumental amino acid uptake in a carnivorous predator mollusc (Sepia officinalis, Cephalopoda) M. de Eguileor1, M.G. Leonardi2, A. Grimaldi1, G. Tettamanti1, L. Fiandra2, B. Giordana2, R. Valvassori1, G. Lanzavecchia1
Abstract. The epithelial cells of the integument of body, arms and tentacles of Sepia officinalis present on their apical membrane a well-organised brush border and show the morphological and histochemical characteristics of a typical absorptive epithelium. The ability of the integument to absorb amino acids was investigated both in the arms incubated in vitro and in a purified preparation of brush border membrane vesicles (BBMV). Autoradiographic pictures of the integument after incubation of the arms in sea-water with or without sodium, showed that proline intake was Na+-dependent, whereas leucine intake appeared to be a largely cationindependent process. Time course experiments of labelled leucine, proline and lysine uptakes in BBMV evidenced that these amino acids are accumulated within the vesicles in the presence of an inwardly directed sodium gradient. The sodium-driven accumulation proves that cationic and neutral amino acids are taken up by the apical membrane of the epithelium of Sepia integument through a secondary active mechanism. For leucine, a 90% inhibition of the uptake was recorded in the presence of a large excess of the substrate. In agreement with the autoradiography results, an analysis of the cation specificity transport in BBMV showed that leucine uptake had a low cation specificity, whereas lysine and proline uptakes were Na+-dependent. An excess of lysine and proline, which share with alanine two different transport systems in the gill epithelium of marine bivalves, reduced eucine uptake. The possible role of the absorptive ability of the integument in a carnivorous mollusc is discussed. © 2000 Harcourt Publishers Ltd
Keywords: amino acid uptake, brush border membrane vesicles, epidermal cells, arms, cuttlefish.
Introduction The epithelial tissues of marine invertebrates are adapted to absorb dissolved organic material (DOM) across the body surface as shown by the ubiquitous presence of microvilli in
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DBSF University of Insubria Via J.H. Dunant 3,21100 Varese, Italy Department of Biology, University of Milan Via Celoria 26,20133 Milano, Italy
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Received 22 November 1999 Accepted 12 July 2000 Correspondence to: Magda de Eguileor, DBSF-University of Insubria Via J.H. Dunant 3, 21100 Varese, Italy. Tel.: +39 0332 421310; Fax +39 0332 421300; E-mail:
[email protected]
direct contact with sea water (Preston, 1990). Amino acid absorption by the integument has been demonstrated in many phyla and different classes of soft-bodied marine invertebrates (Southward & Southward, 1968, 1980; Wright & Pajor, 1989; Preston, 1993). The transport of amino acids and other small organic compounds by epidermal cells may represent an essential contribution in nutrition (Wright et al., 1987; Stephens, 1988; Rice & Stephens, 1988; Wright & Manahan, 1989; Wright & Pajor, 1989; Preston, 1990;) and play a key role in cell volume regulation. It is well known that most marine invertebrates, which are osmoconformers, maintain the elevate osmolality within cells and body fluids by means of a high level of small organic 389
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compounds instead of inorganic osmolytes, since the former are far less perturbing to the functional activity of proteins (Somero & Bowlus, 1983; Kinne, 1993). Sepia officinalis is an active carnivorous predator as are all other Cephalopods and it lives, buried in the sand when not seeking prey (Boucaud-Camou & Boucher-Rodoni, 1983). The integument of cuttlefish presents a well organised brush border on the body surface (Brocco, 1976; Singley, 1982; Packard, 1988) and it seems feasible, as suggested by Preston (1990) for epithelial tissues with microvilli, that these animals absorb organic compounds directly from the environment, although the physiological role of such ability in animals with an extensive digestive system, a closed circulatory system and a well-developed central nervous system is not immediately evident. In the present work we provide evidence that a well developed brush border is present also in the integumental epithelial cells of arms and tentacles, and, on the basis of a histochemical and immunohistochemical analysis, that both the epithelium and the brush border have the conventional features typical of absorptive epithelia. Autoradiographic pictures of the integument, after incubation of the arms in vitro with labelled leucine or proline in normal or sodiumfree sea water, strongly suggested the ability of the epithelium to absorb the two amino acids with a different sensitivity to the presence of Na+. To substantiate this finding, we characterised leucine and proline uptakes in a purified preparation of vesiculated brush border membranes (BBMV) obtained from the integument. BBMV represent a useful tool to study transport mechanisms, since it makes it possible to separate the transport phenomenon from the metabolic event, and, due to the complete control of the composition of the internal and external compartments, allow a better understanding of transport processes. The results obtained with BBMV demonstrated that the integument of Sepia officinalis is able to accumulate neutral and cationic amino acids via at least two transport systems with different cation-sensitivity.
Materials and methods Fifty specimens of Sepia officinalis, 3 months old, were kept in artificial sea water at 18°C and fed daily with live shrimps. Light microscopy and transmission electron microscopy For routine transmission electron microscopy, anaesthetised cuttlefishes were sectioned and fixed for 2 h in 2% glutaraldehyde in sea water. Specimens were washed in sea water and post fixed for 2 h with 1% osmic acid in cacodylate buffer (pH 7.2). After standard dehydration in an ethanol series, specimens were embedded in an EponAraldite 812 mixture. Sections were cut with Reichert Ultracut E ultrotome (Leica, Nussloch, Germany). Semithin
sections were stained by conventional methods (crystal violet and basic fuchsin; Moore et al., 1960) and observed with an optical microscope (Olympus, Tokyo, Japan). Thin sections were stained by uranyl acetate and lead citrate and were observed with a Jeol 1010 electron microscope (Jeol, Tokyo, Japan). Histochemistry Arms and tentacles from anaesthetised cuttlefishes were dissected in 5 mm blocks. The specimens were rapidly frozen in liquid nitrogen. Cryosections (10 µm) were obtained with a Reichert-Jung Frigocut 2800. The slides were immediately used or stored at –20°C. The sections were stained according to Dubowitz & Brooke (1973) with a histoenzymatic kit (Bio-Optica, Milan, Italy). Various cytoplasmic characteristics were assayed using several histochemical procedures: succinic acid dehydrogenase reaction (SDH); NADH-tetrazolium reductase (NADH-TR) and ATPase attributable to mitochondrial activity (i.e. to the source of energy in cell metabolism); alkaline phosphatase (ALP), whose distribution is commonly close related with absorptive function (Richards & Arme, 1982). For all reactions, controls involving the omission of the substrate were carried out. In all cases, negative results were obtained, thus proving the specific nature of the reactions. Each experiment was repeated three times. Immunocytochemistry Cryosections (10 µm), incubated for 15 min with a solution of 0.05 M NH4Cl to reduce auto-fluorescence, were washed with phosphate-buffered saline solution (PBS) and then incubated with the primary antisera (diluted 1:200) for 30 min. The primary antibodies used were: (mouse) antihuman Alkaline Phosphatase (ALP) (clone 8B6) (Sigma Immunochemicals, St. Louis, MO) and (rabbit) anti-Na+/K+ ATPase α-1 (Upstate biotechnology, Lake Placid, NY). After incubation with the primary antibodies, washed specimens were incubated with the appropriate IgG antisera, tetramethylrhodamine isothiocyanate (TRITC) conjugated (diluted 1:100) (from Jackson, Immuno Research Laboratories, West Grove, PA), as a secondary antibody, for 1 h in a dark moist chamber. The PBS buffer used for washing and dilution of antibodies contained 2% bovine serum albumin (BSA). Coverslips were mounted in Vectashield Mounting Medium for fluorescence (Vector Laboratories, Burlingame, CA) and examined with a confocal laser microscope (laser λ, 568 nm for rhodamine) (MRC 1024, Bio-Rad Laboratories, CA) using X40 and X63 objectives (NA 1.30, 1.25). Confocal images were superimposed using Photoshop 5.0. Incubation of arms and autoradiography Arms were ligated, severed from the animal and incubated at 15°C in an artificial sea water with the following composition: 460 mM NaCl, 10 mM KCl, 10 mM CaCl2, 27 mM MgSO4, 0.5 µM [3H] leucine 1 µCi/ml or 0.5 µM [3H]
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proline 1 µCi/ml. In the experiments without Na+, NaCl was omitted and replaced by 920 mM sucrose. The incubation medium was aerated and stirred by bubbling 100% O2. After an incubation of 30 min, the arms were washed twice in unlabelled artificial sea water with or without Na+, lightly blotted and immediately frozen in liquid nitrogen. Frozen sections of all specimens were cut at 12 µm thickness and collected on slides. These slides were coated with Kodak fine-grain Autoradiography Stripping Plate AR 10 emulsion, air-dried overnight in the dark and then stored at 4°C for 3–5 weeks in an airtight lightproof box with silica gel dessicator. Slides were developed in a Kodak D-19 developer, stained with hematoxylin or eosin and mounted in Permount. Control slides containing specimens that had not been exposed to radioactivity were treated in the same manner. Isolation of integument, preservation and preparation of BBMV The arm integument of anaesthetised cuttlefishes was scraped, lightly blotted on filter paper, weighed, placed in cryotubes, frozen in liquid nitrogen and then stored. At the moment of use, the cryotubes were kept in a 37°C water bath until the tissue began to melt. BBMV were prepared by Ca++-precipitation and differential centrifugation according to Giordana et al., 1982. The tissue was homogenised in 100 mM mannitol, 10 mM Hepes-Tris at pH 7.6 and the final pellet was resuspended by 10 passes through a 22 gauge needle in 600 mM mannitol, 10 mM Hepes-Tris at pH 7.6. The membrane protein concentration was assessed with the Coomassie Brilliant Blue G-250 protein assay (Pierce), with bovine serum albumin as standard, and adjusted to a final concentration of 2 mg/ml. The protein yield in homogenate and BBMV from 1 g of fresh tissue was respectively 21.00 ± 1.35 mg and 0.34 ± 0.04 mg (3 determinations). The purity of the vesicle preparation was assessed by measuring the activity of the basolateral and brush border membrane marker enzymes in the crude homogenate and in the final pellet: Na+/K+-ATPase (EC 3.6.1.3) was assayed according to Quigley and Gotterer (1969) and alkaline phosphatase (EC 3.1.3.1) was determined by measuring the release of pnitrophenol from p-nitrophenylphosphate. Incubations were carried out in a spectrophotometer (Ultrospec 3000 Pharmacia Biotech, Cambridge, UK) with a thermostatised (25°C) cuvette holder, under conditions in which the activity was proportional to protein concentrations and time. Transport experiments Transport experiments were performed in triplicate at 20 °C by mixing 10 µl of the vesicle suspension with 10 µl of the radiolabeled incubation mixture, whose final composition is reported in the legends to the figures. At selected times, 20 µl samples were withdrawn from the incubation mixture, diluted with 2 ml of ice-cold stop solution (80 mM mannitol, 400 mM NaCl, 10 mM Hepes-Tris at pH 7.6), filtered through a 0.45 µm pore size cellulose-nitrate filter
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(MFS Micro Filtration System) and rapidly washed with 3 ml of ice-cold stop solution. The filters were then dissolved in a scintillation mixture (Ultima Gold, Packard) and the radioactivity counted in a liquid scintillation spectrometer (Packard, Tri-Carb model 300C). To measure the initial rates of amino acid transport, incubations lasting 10 s were performed in quadruplicate with an automated device. All the figures report a typical experiment, but each experiment was repeated at least three times with different BBMV preparations.
Results The epithelium of the integument in cuttlefish body, as just described by Brocco (1976), Singley (1982) and Packard (1988), is characterised on the free margin by a brush border of closely pressed microvilli. We found this specialisation also on arms and tentacles (Figs. 1–4). It is important to remember that both arms and tentacles, used in prey capture, are structured in analogous manner with the difference concerning the presence or not of a circular muscle layer (Kier, 1982, 1991). In these two structures, the microvillar distribution and organisation were rather different: in the former case microvilli were closely resembling those present on the body and the intestinal wall (Figs. 4–5) and in the latter case, microvilli were short and sparse (Fig. 2). Moreover the epidermal cells showed, in addition to the apical microvillar surface, the lateral side infoldings closely associated with mitochondria (Fig. 3). In all cases (arm and tentacle surfaces) the epithelial cells showed the same histochemical picture with a strong positivity for SDH (Fig. 6), NADH-TR (Fig. 7) and ATPase (Fig. 8) suggesting a high mitochondrial activity. In addition a strong positivity was evident for alkaline phosphatase marking the apical portion of the epithelia (as typically observed in many absorptive epithelia) but not the arm/tentacle sucker epithelia (Figs. 9–10). To establish if the integument of Sepia officinalis can absorb amino acids from the environment, we performed experiments with arms ligated, severed and isolated in vitro. The arms were incubated with 0.5 µM labelled leucine or proline in normal or Na+ free sea water. The autoradiography shows that leucine is taken up by the integument and crosses the epithelial layer both in the presence (Fig. 11) and in the absence of sodium (Fig. 12), whereas for proline a heavy accumulation of silver particles in the tissue occurred only in specimens incubated in the presence of sodium (Figs. 13, 14). To extend the study on the ability of the epithelium to absorb amino acids by means of Na+-dependent and Na+independent processes, as observed in the intact integument, we prepared brush border membrane vesicles from this tissue by Ca++-precipitation. The final preparation is shown in Fig. 15. The purity of this preparation was evaluated from the enrichment factor (ratio of the enzyme specific activity
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Fig. 1.
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Semi-thin longitudinal section of elongated cuttlefish tentacle. Under the epithelium (E), thick muscular fields are visible (M). Scale bar, 50 µm.
Figs. 2–5. Thin sections of cuttlefish epithelial cells. The brush border (arrowheads) of tentacles 2 (Scale bar, 0.5 µm) and arms 4 (Scale bar 0.4 µm) showing well structured microvilli, can be compared with the intestinal brush border 5 (Scale bar, 0.5 µm). The epithelial cells show the nucleus (N) and the mithocondria (m) associated with the infoldings (V) of lateral membrane 3 (Scale bar, 2 µm). Figs. 6–10. Cryosections of arm or tentacle show the same histochemical responses. The epithelial cells (E), on muscle fields (M), react strongly (arrowheads) for SDH 6 (Scale bar, 40 µm), NADH-TR 7 (Scale bar, 40 µm), ATPase 8 (Scale bar, 80 µm) and for ALP 9 (Scale bar, 40 µm). The positivity (arrowheads) of the last enzime, typical of absorptive epithelia, is localised on the surface but not on the suckers surface (S).
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Figs 11–12. Cryosections. [3H]-leucine autoradiography of the arms incubated in normal 11 or sodium-free sea water 12. There is a high density of silver grains in the epithelium of both preparations (▲). Figs 13–14. Cryosections. [3H]-proline autoradiography of the arms incubated in normal 13 or sodium-free sea water 14. When the arms are incubated in the presence of sodium there is a heavy accumulation of silver particles (▲) in the arms tissues, while with the incubation in the sodium-free medium, the silver grains are especially located outwardly (▲). Fig. 15. Thin section of the brush border membrane vesicle preparation. Scale bar, 1 µm.
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in the final vesicle suspension and in the homogenate) of the two enzymes alkaline phosphatase and Na+/K+-ATPase specific markers for the apical plasma membrane and for basolateral membrane, respectively, in absorptive epithelia. Their presence in these specific areas of the epithelial membrane in the integument of Sepia officinalis is demonstrated by their immunochemical localisation (Figs. 16–18). Table 1 shows that the activity of alkaline phosphatase, but not of Na+/K+-ATPase, is increased in BBMV in comparison with that in homogenate, and confirms that the final fraction is enriched in brush border membranes. A typical time course of the uptake of 0.5 µM leucine into BBMV in the presence of an inwardly directed Na+gradient is reported in Fig. 19. Leucine was translocated and transiently accumulated within the vesicles. The amino acid accumulation value (ratio between the maximal and the final uptake values) was only fourfold: possibly this value is underestimated, if the equilibrium value was not yet reached after 120 min. Longer incubation times could not be performed because the transport activity, measured as initial rates, was appreciably decreased in BBMV maintained at room temperature for longer times (data not shown). Lysine and proline uptakes with time were also measured in the presence of a sodium gradient. Fig. 20 shows that both lysine and proline were accumulated within the vesicles, even if in both cases the occurrence of the maximal peak was delayed compared to that of leucine and the equilibrium value was clearly not yet reached after 120 min. Even if the height and the shape of the time course curves are strictly linked to the kinetic characteristics of the transport system (Leonardi et al., 1993), other membrane properties such as a low passive permeability for the cation, solute and/or water, as observed in bivalves (Wright et al., 1987; Pajor & Wright, 1989), may cause a delay in the attainment of the maximum value. Sepia officinalis, as several marine species (Preston, 1990; Kinne, 1993), presents a high cellular amino acid concentration (Robertson, 1965), therefore amino acid uptake in vivo has to be performed against a steep chemical gradient (Stephens, 1988; Wright & Pajor, 1989; Preston, 1990, 1993) so that amino acid accumulation has to be energised by the downhill gradient of a driver cation. The specificity for the driver cation was tested by measuring leucine, lysine and proline initial uptake rates in the presence of a Li+-, Na+- or K+-gradient. The cation specificity of the transport agency involved in leucine uptake is poor (Fig. 21), since both lithium and potassium can efficiently substitute for sodium in catalysing leucine uptake. On the contrary, proline initial rate was significantly higher in the presence of Na+ compared to that in the presence of Li+ or K+ (Fig. 21) and lysine uptake appears to require Na+, even if lithium and potassium can partially support the amino acid uptake (Fig. 21). In the gills of Mytilus edulis the uptake of neutral amino acids occurs via two distinct transport systems: an alaninelysine (AK) pathway with a poor cation selectivity and an alanine-proline (AP) pathway with a high cation selectivity
(Wright & Pajor, 1989). Therefore, the inhibition induced by a large excess (1 mM) of lysine and proline on 0.5 µM leucine uptake at 10 s was tested (Fig. 22). Lysine caused a 42% and proline a 22% inhibition of leucine uptake (percent inhibitions were referred to the control uptake, 8.32 ± 0.10 pmol/10 s/mg protein, after subtraction of the residual uptake in the presence of 1 mM leucine, 0.64 ± 0.01 pmol/10 s/mg protein). Therefore these two amino acids seem to share with leucine the same transport system, even if the inhibition of leucine transport by the two amino acids in Sepia officinalis is far less remarkable than that exerted on alanine in M. edulis (Pajor & Wright, 1989; Wright & Pajor, 1989).
DISCUSSION The morphological and immunohistochemical characterisation of Sepia officinalis integument carried out in the present study has evidenced that the epithelial cells possess the classical features of absorptive epithelia (Figs. 1–10). As stressed by Preston (1990), the epithelial tissues specifically adapted for absorption invariably have microvilli, and the positive histochemical responses obtained in cuttlefish arms and tentacles are comparable to those observed in those epithelia in which passage of nutritive substances regularly occurs, i.e. the gut epithelium of invertebrate and vertebrate (Barnet & Munday, 1972; Adibi & Mecer, 1973), the external integument of parasitic helminths (Mettrick & Podesta, 1974), adult/young adhering zones of brooding leeches (de Eguileor et al., 1994), the larvae of Strongylocentrotus (Davis et al., 1985), the gutless organisms such as the pogonophora (Southward & Southward, 1968, 1980) (Figs. 6–10). The presence of an extensive array of microvilli on the body surface is a rather surprising finding in an active strictly carnivorous animal. In addition, it seems peculiar that arms and tentacles, capturing and attacking structures exposed to physical stress, have a surface with microvilli (Figs. 2–4, 16). We investigated the ability of Sepia officinalis integument to absorb amino acids by uptake experiments in the isolated arms. The autoradiographic pictures clearly show that the labelled amino acids are taken up from the external milieu and suggest that proline transport might be dependent on the sodium gradient, whereas leucine uptake would not (Figs. 11–14). To determine if concentrative transport systems are involved in this amino acid transport, brush border membrane vesicles were purified from Sepia officinalis arm integument. Electron microscopy (Fig. 15) and the enrichment of the marker enzyme alkaline phosphatase (Table 1) provide evidence that the final preparation contained membrane vesicles enriched in brush border. The transient intravesicular accumulation of leucine which occurred in the presence of an inwardly directed sodium gradient (Fig. 19) indicates that the uptake is
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Fig. 16. Thin section of arm epithelial cells (E) at low magnification to easily identify the next immunofluorescence staining. Scale bar, 3 µm. Fig. 17. Cryosection. Superimposed confocal image. The immunofluorescence staining using anti-ALP antibody showed a localization of the enzyme at the level of the brush border membrane. Fig. 18. Cryosection. Superimposed confocal image. The immunofluorescence staining using anti-Na+/K+ ATPase, an integral membrane protein complex responsible of electrochemical gradients of Na+ and K+ across the plasma membrane, was localized in the baso-lateral portion of epithelial cell membranes.
performed by a carrier-mediated mechanism, further proved by the 90% inhibition of leucine uptake by an excess of the substrate (Fig. 22). As suggested by the experiments with the arms incubated in vitro (Figs. 11–12), the transport agency involved in leucine uptake appears to have a low specificity for Na+ (Fig. 21) and recognises also cationic amino acids, since an excess of lysine caused a 42% inhibition of leucine uptake (Fig. 22). Lysine, but also proline, is indeed absorbed in Sepia integument by a cotransport mechanism, as shown by the presence of the peak (Fig. 20) and of an increased uptake (Fig. 21) in the presence of the sodium gradient. The results here presented suggest that neutral and cationic amino acids are absorbed by Sepia integument through a secondary active mechanism, in agreement with what found in other molluscs (Wright et al., 1987; Rice & Stephens, 1988; Stephens, 1988; Wright & Manahan, 1989;
Wright & Pajor, 1989; Preston, 1990). However, the neutral amino acid leucine crosses the apical membrane through pathways with different features as compared to those observed in Mytilus, since only 40% or less of leucine uptake was inhibited by either lysine or proline, while an inhibition of 84% was observed in marine bivalves. The ability of cuttlefish to absorb amino acids by the integument via a secondary active mechanism, in order to accumulate them in cells and body fluids, is shared by many other molluscs (Wright et al., 1987; Rice & Stephens; 1988, Stephens, 1988; Wright & Manahan, 1989; Wright & Pajor, 1989; Preston, 1990) and may represent a phylogenetical remnant. The integument could, alternatively, be the largest absorptive surface in S. officinalis, since in a specimen 10 cm long it has an extension of tenfold the intestinal one. In this rough estimate we have a value by defect of the entire
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Table 1. Enzyme activities in homogenate and BBMV. Enzyme Na+-K+ ATPase Alkaline phosphatase
Homogenate mU/mg protein
BBMV mU/mg protein
R.E.
76.5 ± 8.9 4.8 ± 0.6
93.3 ± 9.8 27.6 ± 2.4
1.2 ± 0.1 5.9 ± 0.2
R.E.: relative enrichment factor, is the ratio between BBMV and homogenate specific activities (mU/mg of protein). Means ± S.E. of three different preparations.
Fig. 19. Time course of leucine uptake in BBMV in the presence of a Na+-gradient. BBMV were incubated in a medium of the following final composition: 300 mM mannitol, 10 mM Hepes-Tris at pH 7.6, 260 mM NaCl, 0.5 µM [3H] leucine 30 µCi/ml. Each point is the mean ± S.E. of an experiment in triplicate. When not given, S.E. bars were smaller than symbols.
body surface. In fact we assumed that the body and arms could be compared to a frustum of cones and tentacles and gut to cilinders. The effective absorptive surface per unit area could be comparable, if we consider that the length and number of microvilli appear to be similar in body/arm integumental and intestinal epithelial cells (Figs. 2–5). Although no information is available on the amino acid transport capability of the intestinal brush border, it seems
feasible that the remarkable absorptive properties of arms and body surface may play a significant role in nutrition and in other aspects of S. officinalis physiology. For example, even if cuttlefishes rely primarily on prey capture as a feeding source, they could exploit the absorptive ability of their integument to gain amino acids from DOM as osmolytes for the regulation of their internal environment. In fact, S. officinalis, like other marine molluscs which utilise nitrogenous solutes as major intracellular osmotic effectors (Somero & Bowlus, 1983), has a high intracellular amino acid concentration (Preston, 1993). Concurrently, amino acids could represent a supplement to traditional food and an additional energy source when the animals remain buried under the sand, where the dissolved organic matter is very high. The superficial absorption could be also of direct advantage to arm and tentacle muscle metabolism, particularly active because of the non-stop use of these appendages to catch the prey, to dig in the sand or to perceive, like a search periscope, any presence in the environment. The intriguing problems of the functional properties and of the physiological role of integumental amino acid transport in cuttlefish is under current investigation.
Fig. 20. Time course of lysine and proline uptake in BBMV in the presence of a Na+-gradient. BBMV were incubated in a medium of the following final composition: 300 mM mannitol, 10 mM Hepes-Tris at pH 7.6, 260 mM NaCl, 0.5 µM [3H] lysine or [3H] proline 30 µCi/ml. Each point is the mean ± S.E. of an experiment in triplicate. When not given, S.E. bars were smaller than symbols.
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Fig. 21. Effect of different monovalent cations on the initial uptake rates of leucine, lysine and proline. BBMV were incubated in a medium of the following final composition: 300 mM mannitol, 10 mM Hepes-Tris at pH 7.6, 260 mM LiCl or NaCl or KCl, 0.5 µM [3H] leucine or [3H] lysine or [3H] proline 30 µCi/ml. Means ± S.E. of an experiment in triplicate. Difference with the uptake in the presence of sodium was tested by Student’s t test. *p < 0.05; **p < 0.01; ***p< = 0.001.
Fig. 22. Inhibition of leucine uptake by different amino acids. BBMV were incubated for 10 s in a buffer of the following final composition: 300 mM mannitol, 10 mM Hepes-Tris at pH 7.6, 0.5 µM [3H] leucine 30 µCi/ml, 1 mM leucine, lysine or proline. Means ± S.E. of an experiment in triplicate. Difference with the uptake in the absence of inhibitor was tested by Student’s t test: *p
