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MS 8795 Received 30 September 1998; accepted after revision 20 January 1999.
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ABSTRACT |
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 TopAbstract IntroductionMethodsResultsDiscussionReferences |
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cm2. Addition of 50 nM PGE2 caused a stimulation of Isc, Vt and transepithelial conductance, Gt. The increase in Isc was probably due to the elevation in Cl- secretion, since it could be correlated with the stimulation of serosal to mucosal 36Cl- flux. Application of the neurohypophyseal peptide arginine vasotocin (AVT; 50 nM) or the 
-adrenergic agonist isoproterenol (isoprenaline; 0·5 µM) evoked a stimulation in Cl- secretion, as was shown by the increases in Isc and Gt. The excitatory effect of isoproterenol followed by the inhibitory action of propranolol, a -adrenergic antagonist, suggested the presence of -adrenergic receptors. Noradrenaline (0·1 µM) elicited a reduction in Isc, Vt and Gt, which was counterbalanced by the addition of phentolamine, an 
-adrenergic antagonist. This suggested an activation of -adrenergic receptors.
- and -adrenergic receptors.
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INTRODUCTION |
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Teleost fish living in seawater (SW) actively secrete salt through the gill epithelium to maintain their blood osmotic pressure substantially below that of the external medium. Studies on salt transport in gill epithelium have been made in vivo (Forster, 1976), in experiments on isolated perfused heads (Payan & Matty, 1975) and in in vitro studies with another model, the fish opercular membrane (Degnan et al. 1977; Zadunaisky, 1996). Gill primary cell culture has provided a new and complementary approach. The primary cell culture of gills from a fresh water (FW) rainbow trout (Oncorhynchus mykiss) was first introduced by Pärt et al. (1993) and Wood & Pärt (1997). We developed a primary culture of sea bass (Dicentrarchus labrax) gill cells that exhibited morphological (Avella et al. 1994) and functional (Avella & Ehrenfeld, 1997a) characteristics of a Cl- secretory epithelium. When grown on permeable support in the presence of a culture medium supplemented with sea bass serum, the cells form a confluent, highly polarized and tight epithelium. This cultured epithelium is composed of one cell type that exhibits the morphological characteristics of gill respiratory cells, and hence is termed respiratory-like cells (Avella & Ehrenfeld, 1997a). This cell layer of respiratory-like cells exhibited chloride transport and was responsible for the development of a transepithelial potential (Vt, serosal positive), current (Isc) and high resistance (Rt), when placed in an Ussing chamber. In addition, we have identified Cl- channels in gill cells grown on impermeable supports using patch-clamp techniques (Duranton et al. 1997). This preparation therefore provides a new fish gill model for studying the mechanisms and regulation of Cl- secretion.
In the present study, we examined the effects of hormones (prostaglandin (PGE2), arginine vasotocin (AVT), iso-proterenol (isoprenaline) and noradrenaline), which are known to be involved in osmoregulation, on Cl- secretion using the Isc technique and isotopic (36Cl-) fluxes.
Some of our results have already been presented to The Physiological Society (Avella & Ehrenfeld, 1997b).
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METHODS |
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Animals
Sea bass (Dicentrarchus labrax) of average weight 40 g were obtained from a local sea farm (Société 3A, Antibes, France). They were kept at ambient temperature (14-18°C) and with natural lighting in a semi-open circuit (tank water completely renewed every 6 h) in 1 m3 tanks containing Mediterranean SW (36 g l-1 salinity). Fish were fed daily with Aqualim pellets (Aquasarb, France) at 2·5 % of their body weight.
Primary cell culture
Solutions. The solutions and culture conditions used have been previously described by Avella et al. (1994). In brief, the washing medium Leibovitz L15 (ICN Pharmaceuticals, France) was supplemented with 20 mM NaCl, fungizone (0·1 µg ml-1), penicillin (200 i.u. ml-1), streptomycin (200 µg ml-1) and gentamicin (400 µg ml-1). All antibiotics were supplied by Sigma (USA). The culture medium, Leibovitz L15 was supplemented with 10 % sea bass serum (SBS) (see Avella & Ehrenfeld, 1997a, for preparation), 20 mM NaCl, penicillin (100 i.u. ml-1), streptomycin (100 µg ml-1) and gentamicin (200 µg ml-1). The final pH of all solutions was adjusted to 7·8.
Cell culture conditions. Before culture preparation, fish were held for 2 h in a 10 l tank of aerated SW containing antibiotics and fungicides: 0·02 % Furaltadone (Sigma) and 0·02 % Temerol (Francodex, France). Fish were killed by a blow to the head and decerebrated. They were then decapitated and the gills excised. The gill arches (cartilage) were removed and the remaining filaments were dipped into washing medium (see above). They were washed under gentle automatic shaking (5 × 10 min, 100 agitations min-1). Single-cell suspensions were prepared by a technique modified from that of Pärt et al. (1993), as described by Avella & Ehrenfeld (1997a). Finally, cells were seeded in Costar-Transwell 0·45 µm pore inserts (25 mm diameter, 4·8 cm2 surface; Costar, MA, USA) in 6-well Costar plates at a high density (2 × 106 cells cm-2). Cells were maintained in a low-temperature incubator (Jouan, France) at 17°C, and in a humidified air atmosphere (i.e. atmospheric PCO2). After 24 h, cells reached confluency. The medium was changed every second day and the cells were used from days 6 to 9.
Morphological studies
Scanning electron microscopy (SEM). Both apical and basal sides were rinsed with serum-free medium and fixed for 45 min at 4°C in 1·5 % glutaraldehyde and 0·8 % paraformaldehyde buffered with 0·05 M sodium cacodylate (pH 7·8). The epithelium was then rinsed three times in phosphate buffer saline and left at 4°C overnight. Cells on filters were dehydrated in serial ethanol concentrations followed by 5 min incubation in 1, 1, 1, 3, 3, 3-Hexamethyldisilazan (Merck, Darmstadt, Germany) at room temperature (21-25°C) and allowed to dry. The cells were mounted on aluminium stubs with conductive double adhesive tape and coated with gold/palladium for 3 min in an argon atmosphere in a vacuum evaporator. Cells were examined with a field transmission electron microscope (Jeol JSM 6300F) at 2 kV.
Fluorescence microscopy. Attempts to identify chloride cells were performed using the mitochondria-rich cell marker DASPEI (dimethylamino-styrylmethylpyridiniumiodide) (Karnaky et al. 1984) used at a concentration of 10 µM (20 min application on the cell layer). The preparation was observed in fluorescence microscopy with fluorescein filters after several washes of the DASPEI-containing solution.
Electrical measurements
The inserts containing the cultured gill cells were placed in a modified Ussing chamber (Avella & Ehrenfeld, 1997a) in order to measure the transepithelial electrical potential (Vt), short-circuit current (Isc) and resistance (Rt) of gill cells in culture. The volumes of the apical and basolateral bathing solutions were 2 and 4 ml, respectively. Spontaneous Vt was measured with agar-KCl salt bridges and was clamped at 0 V, through platinum electrodes, using an automatic voltage clamp (Model VC 600, Physiological Instruments, Houston, TX, USA). The sign of the transepithelial potential was determined by referring the basal side to the apical side (the latter taken as reference). The Isc was continuously recorded on a chart paper recorder (SEFRAM, France) and additional Vt pulses were applied (10 mV, 1 s duration every 60 s) to determine the epithelial resistance (Rt). In all experiments the cell culture medium (antibiotic and serum free) was used on both sides of the cultured epithelium to avoid possible intracellular ion and volume changes due to differing experimental and culture media. Media were not withdrawn, blockers or hormone antagonists being added in the presence of the initial hormone.
Kinetic studies
Unidirectional transepithelial Cl- flux (Jout: outflux from serosal to mucosal side) was performed in a 6-well culture plate in open-circuit conditions. 36Cl- (0·62 µCi ml-1) was added to the basal side of the epithelium. Cells were first equilibrated for 15 min in the presence of serosal isotope. Then a control period of 20 min preceded PGE2 addition (5 × 10-8 M) to the serosal bath (two 5 min periods). In each period the apical solution was replaced by a fresh medium solution. Aliquots of serosal medium (samples of 10 µl taken in duplicate) were collected at the beginning and end of each period in order to determine the specific activity of the isotope containing solution. All samples were placed in counting vials supplemented with 10 ml of Aqueous Counting Scintillant (ACS; Amersham, England) for counting in a liquid scintillation counter (Packard Instruments, USA). Fluxes were expressed as nequiv h-1 cm-2.
Hormones, drugs and cell marker
PGE2, AVT, isoproterenol, propranolol, noradrenaline, phentolamine, ouabain, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), diphenylamine-2-carboxylic acid (DPC), 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), bumetanide, dibutyryl 3', 5'-cyclic monophosphate (dB-cAMP) and DASPEI were purchased from Sigma (USA).
Statistics
Maximal or minimal values of Isc are reported in Tables 1-4. Data variability is expressed as the standard error of the mean (S.E.M.). Student's unpaired t test was used first for estimating the significance of differences between means.
Considering the relatively great variability found in control levels of Isc, Vt and Gt, the effects of hormones, blockers and antagonists were analysed using Student's paired t test.
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RESULTS |
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Morphological studies
SEM observations revealed a regular epithelia-like structure composed of a mosaic of polygonal epithelial cells (longest axis about 10-30 µm) presenting well-defined intercellular junctions (Fig. 1). The apical cell surface was formed of a complex system of convoluted microridges closely resembling those of respiratory (pavement) cells often described in teleost gills (Hughes, 1979; Laurent & Dunel, 1980).
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The confluent cell culture is composed of contiguous epithelial polygonal cells. Cells cultured in the presence of sea bass serum reveal a regular arrangement of convoluted microridges and well-defined intercellular junctions. The culture therefore consists of a mosaic of respiratory-like cells with surface microridges that are similar to the original epithelium. mr, microridges; pm, plasma membrane. Scale bar, 10 µm. | ||
Observations of gill cultured epithelium treated with DASPEI (10 µM) revealed no particular fluorescent cell staining (data not shown), confirming our SEM data showing that chloride cells were absent from this preparation.
Ion transport studies
Due to the high seeding density, primary cultures of sea bass gill cells reached confluence within 24 h. However, cells were maintained in a culture medium supplemented with sea bass serum for 6-9 days before electrical measurements were performed. The short-circuit current (Isc) of cultured cells grown on permeable filters decreased slightly in the first 5-10 min but was then stable (Isc = 8·8 ± 0·4 µA cm-2, n = 30) for longer periods of time (hours). The transepithelial electrical potential (Vt) after the stabilization period was 28·6 ± 0·6 mV (serosal side positive) and the transepithelial resistance (Rt) was high (5026 ± 127 cm2), characteristic of a very tight epithelium. The stability of the cell electrophysiological parameters facilitated assessment of hormonal effects on Cl- secretion.
Effects of PGE2
We have previously found that Isc was correlated with the net excretion of chloride measured by 36Cl- fluxes (Avella & Ehrenfeld, 1997a). Addition of 50 nM PGE2 to the serosal side stimulated Isc, Vt and transepithelial conductance Gt (Fig. 2A and Table 1). We confirmed that the current activated by PGE2 application was a chloride current with 36Cl- efflux experiments upon PGE2 application (cf. Fig 2A and Fig 3). As evident from Fig 2A and Fig 3, the Isc and 36Cl- outfluxes were nearly doubled after PGE2 addition within the first 5 min.
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A representative trace is shown. PGE2 and AVT were added to the serosal side to a final concentration of 50 nM. The transient deflections in the current result from periodic Vt pulses (10 mV). | ||
Table 1. Effect of PGE2 addition (5 × 10-8 M) to the serosal side on Isc, Vt and Gt: inhibition by bumetanide and DIDS (10-4 M, serosal side)
| Isc (µA cm-2) |
Vt (mV) |
Gt (mS cm-2) |
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| Effect of PGE2 (n = 6) |
a | Control | 4·2 ± 1·3 | 21·0 ± 6·2 | 0·29 ± 0·08 |
| b | PGE2 | 5·6 ± 1·4 | 25·5 ± 7·1 | 0·32 ± 0·08 | |
| Difference (b - a) | 1·4 ± 0·4  |
4·5 ± 1·7 |
0·03 ± 0·01 |
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| Effect of bumetanide in PGE2-treated cells (n = 5) |
a | PGE2 | 5·6 ± 1·3 | 27·9 ± 7·6 | 0·30 ± 0·09 |
| b | Bumetanide | 3·0 ± 0·9 | 12·6 ± 3·0 | 0·28 ± 0·09 | |
| Difference (a - b) | 2·6 ± 0·5 |
15·2 ± 5·6 |
0·02 ± 0·01 * | ||
| Effect of DIDS in PGE2 + bumetanide- treated cells (n = 4) |
a | PGE2 | 5·9 ± 1·6 | 31·1 ± 8·8 | 0·28 ± 0·12 |
| b | Bumetanide | 3·2 ± 1·2 | 13·8 ± 3·6 | 0·26 ± 0·12 | |
| c | DIDS | 1·4 ± 0·6 | 8·9 ± 1·8 | 0·21 ± 0·09 | |
| Difference (a - c) | 4·5 ± 1·2 |
22·2 ± 7·3 |
0·07 ± 0·02 * | ||
| Difference (b - c) | 1·8 ± 0·7 * | 4·9 ± 2·8 | 0·05 ± 0·03 | ||
P < 0·05 (two-tailed). DIDS was added in the presence of PGE2 + bumetanide.
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Statistical comparison was by Student's unpaired t test (n = 3): the difference is significantly different from control levels with P < 0·05 (two-tailed). | ||
Basal Isc correlated with Cl- secretion via the bumetanide-sensitive Na+-K+-2Cl- cotransporter, the DIDS-sensitive Cl--HCO3- exchanger, both located on the basolateral membranes, and a Cl- channel located on the apical membranes (Avella & Ehrenfeld, 1997a). Application of bumetanide (100 µM) to the serosal solution of PGE2-treated cells was found to rapidly (< 1 min) reduce Isc and Vt, and slightly reduce Gt (Table 1). An example of the bumetanide effect when added after PGE2 stimulation of Isc is given in Fig. 4. Serosal application of 100 µM DIDS to bumetanide plus PGE2-treated cells induced an additional decrease in Cl- transport (Fig. 4 and Table 1).
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A representative trace is shown. The medium was not changed and all agents were added successively to the serosal side to final concentrations of 50 nM (PGE2) and 100 µM (bumetanide and DIDS). The transient deflections in the current result from periodic Vt pulses (10 mV). | ||
Effects of AVT
AVT has been shown to be involved in fish osmoregulation (Takei, 1993). We therefore tested its possible effect on Cl- transport in our primary cell culture. Addition of 50 nM AVT caused an immediate (within 1 min) enhancement of Isc (22·9 ± 8·3 %, n = 9) and Gt (14·2 ± 4·6 %, n = 8) (Fig. 2B and Table 2). Application of DPC, a commonly used Cl- channel blocker, has been shown previously to block basal Cl- secretion (Avella & Ehrenfeld, 1997a). When added to the mucosal solution on AVT-treated cells, this agent also caused an immediate fall in Isc and Vt (Fig. 5 and Table 2). Isc was even reduced to levels (1·6 ± 0·4 µA cm-2, n = 4, Table 2) well below that of the experimental control group (15·5 ± 6·0 µA cm-2).
Table 2. Effect of AVT addition (5 × 10-8 M) to the serosal side on Isc, Vt and Gt: inhibition by DPC (1 mM, mucosal side)
| Isc (µA cm-2) |
Vt (mV) |
Gt (mS cm-2) |
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| Effect of AVT (n = 9) |
a | Control | 14·0 ± 5·1 | 35·7 ± 7·0 | 0·40 ± 0·20 |
| b | AVT | 15·8 ± 5·3 | 37·3 ± 6·6 | 0·43 ± 0·20 | |
| Difference (b - a) | 1·8 ± 0·5 |
1·6 ± 1·1 | 0·03 ± 0·005 |
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| Effect of DPC in AVT-treated cells (n = 5) |
a | AVT (baseline) | 15·5 ± 6·0 | 35·3 ± 11·6 | 0·59 ± 0·25 |
| b | DPC | 1·6 ± 0·4 | 10·2 ± 3·6 | 0·20 ± 0·05 | |
| Difference (a - b) | 13·9 ± 6·1* | 25·1 ± 11·5 * | 0·39 ± 0·22 | ||
P < 0·05 (two-tailed).
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A representative trace is shown. AVT was added to the serosal side to a final concentration of 50 nM and DPC to the mucosal side to a final concentration of 1 mM. The transient deflections in the current result from periodic Vt pulses (10 mV). | ||
cAMP-dependent Cl- channels have been implicated in most epithelia that secrete chloride and exhibit transport stimulation upon AVT application (Verrey, 1994). We have shown previously that cAMP itself was able to stimulate Isc in cultured gill cells (Avella & Ehrenfeld, 1997a). An unexpected feature occurred when AVT was added after cAMP application. Serosal addition of 1 mM dibutyryl cAMP (a membrane-permeant analogue of cAMP) resulted in an increase in Isc and Gt (Fig. 6). Subsequent addition of AVT to the cAMP-treated cells initially evoked an increase in Isc and Gt, which rapidly (2-3 min) declined to lower levels of Isc. When compared with the maximal cAMP effect, Isc and Gt were reduced by 42·6 ± 16·9 and 26·3 ± 9·5 %, respectively (n = 3) (Fig. 6).
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A representative trace is shown. All agents were added to the serosal side to final concentrations of 1 mM (dB-cAMP) and 50 nM (AVT). The transient deflections in the current result from periodic Vt pulses (10 mV). | ||
Effects of catecholamines
Application of isoproterenol (a -adrenergic agonist) to the serosal side (0·5 µM) elicited a marked increase in Isc and Vt, and a small increase in Gt (Fig 7 and Fig 8 and Table 3). As illustrated in Fig. 7, apical addition of 1 mM DPC to isoproterenol-treated cells elicited an immediate decline in Isc to very low levels (1·3 ± 0·3 µA cm-2; n = 4). A reduction of Vt and Gt was also observed (Table 3 and Fig. 7). Following basal addition of propranolol (10 µM), a specific -adrenergic antagonist, to isoproterenol-treated cells, Isc, Vt and Gt returned to control levels (Fig. 8 and Table 3).
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A representative trace is shown. Isoproterenol was added to the serosal side to a final concentration of 0·5 µM and DPC to the mucosal side to a final concentration of 1 mM. The transient deflections in the current result from periodic Vt pulses (10 mV). | ||
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A continuing trace is shown in three sections. The medium was not changed and all agents were added successively to the serosal side to final concentrations of 0·5 µM (isoproterenol), 10 µM (propranolol and phentolamine), 0·1 µM (noradrenaline) and 100 µM (ouabain). The transient deflections in the current result from periodic Vt pulses (10 mV). | ||
Table 3. Effect of isoproterenol addition (0·5 µM) to the serosal side on Isc, Vt and Gt: inhibition by propranolol (10 µM, serosal side) or DPC (1 mM, mucosal side)
| Isc (µA cm-2) |
Vt (mV) |
Gt (mS cm-2) |
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| Effect of isoproterenol (n = 11) |
a | Control | 11·3 ± 5·4 | 24·5 ± 3·6 | 0·65 ± 0·26 |
| b | Isoproterenol | 14·8 ± 6·8 | 27·6 ± 3·9 | 0·68 ± 0·26 | |
| Difference (b - a) | 3·5 ± 1·5 |
3·1 ± 0·6 |
0·03 ± 0·01 |
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| Effect of DPC in isoproterenol- treated cells (n = 4) |
a | Isoproterenol | 7·7 ± 2·2 | 34·1 ± 7·1 | 0·23 ± 0·07 |
| b | DPC | 1·3 ± 0·3 | 9·7 ± 1·8 | 0·11 ± 0·02 | |
| Difference (a - b) | 6·4 ± 2·1 * | 24·4 ± 8·2 * | 0·11 ± 0·05 * | ||
| Effect of propranolol in isoproterenol- treated cells (n = 4) |
a | Control | 20·8 ± 14·7 | 16·5 ± 5·6 | 1·38 ± 0·57 |
| b | Isoproterenol | 25·1 ± 15·6 | 20·5 ± 6·6 | 1·43 ± 0·57 | |
| c | Propranolol | 18·3 ± 14·0 | 13·0 ± 4·8 | 1·38 ± 0·57 | |
| Difference (b - c) | 6·8 ± 2·3 * | 7·6 ± 3·0 * | 0·04 ± 0·02 * | ||
P < 0·05 (two-tailed).
Addition of noradrenaline (0·1 µM) to the serosal side caused an initial decrease in Isc, Vt and Gt, which stabilized after 2-4 min (Fig. 8 and Table 4). To investigate the involvement of -adrenergic receptors, phentolamine (an -antagonist), was applied to the basal side of propranolol (-antagonist)- plus noradrenaline-treated cells. Phentolamine application (10 µM) reversed the noradrenaline effect (Fig. 8 and Table 4). Finally, as described previously (Avella & Ehrenfeld, 1997a), ouabain completely blocked the Isc, indicating that Na+-K+-ATPase may be indirectly involved in Cl- secretion (Fig. 8).
Table 4. Effect of noradrenaline (NA) addition (0·1 µM) to the serosal side on Isc, Vt and Gt: stimulation by phentolamine (10 µM, serosal side)
| Isc (µA cm-2) |
Vt (mV) |
Gt (mS cm-2) |
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| Effect of noradrenaline (n = 8) |
a | Control | 5·0 ± 1·2 | 16·6 ± 3·6 | 0·33 ± 0·06 |
| b | NA decrease | 2·7 ± 0·7 | 11·5 ± 3·9 | 0·28 ± 0·05 | |
| Difference (a - b) | 2·3 ± 0·8 |
5·2 ± 1·7 |
0·05 ± 0·02 * | ||
| Effect of phentolamine in NA-treated cells (n = 4) |
a | Control | 2·7 ± 0·7 | 9·3 ± 2·2 | 0·31 ± 0·07 |
| b | NA | 0·9 ± 1·7 | 3·6 ± 0·3 | 0·27 ± 0·05 | |
| c | Phentolamine | 3·4 ± 0·6 | 12·8 ± 1·8 | 0·29 ± 0·05 | |
| Difference (c - b) | 2·5 ± 0·5 |
9·2 ± 2·0 |
0·02 ± 0·01 | ||
P < 0·05 (two-tailed) and * P < 0·05 (one-tailed).
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DISCUSSION |
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The structural heterogeneity of the gill epithelium with both chloride and respiratory cells, together with the complex branchial circulatory system, made it difficult to assess the contributions of each cell type to the various gill functions, particularly that of ion transport.
Sea bass gill cells in primary culture were found to exhibit only one cell type, which presents the characteristics of the gill respiratory cell, revealed by scanning and transmission electron microscopy studies (Avella et al. 1994; Avella & Ehrenfeld, 1997a; present study). In addition, the cultured epithelium showed functional characteristics of a Cl- secretory epithelium. Cl- excretion is mediated by the Na+-K+-2Cl- cotransporter and the Cl--HCO3- exchanger, both located on the basolateral membranes, and by the presence of Cl- channels on the apical membranes (Avella & Ehrenfeld, 1997a). Patch-clamp studies have confirmed the presence of Cl- channels on apical membranes of gill cells (plated onto Petri dishes) (Duranton et al. 1997). We found that these channels were of low conductance (8 pS), were voltage dependent and could be inhibited by NPPB, DPC and I-, but were insensitive to DIDS. Furthermore, channel activity could be stimulated by PKA plus ATP in the inside-out excised configuration. The sea bass gill cells in culture composed of respiratory-like cells therefore represent a more simple model than the natural gill tissue for investigation of gill function. Whilst our preparation is highly polarized and functional, we are aware that primary cell cultures may also present incomplete differentiation.
In this study, we investigated the effects of hormones involved in fish osmoregulation on chloride secretion in sea bass gill cells in culture. Our results show that Cl- secretion is stimulated by the neurohypophyseal hormone AVT, by prostaglandin (PGE2) and by isoproterenol, and can be inhibited by noradrenaline.
Effect of PGE2
The role of eicosanoids in mammals is well documented, but little information is available concerning their synthesis and effects in lower vertebrates. Eicosanoid production has been demonstrated in renal tissue of goldfish during seawater adaptation (Lowenstein, 1991) and in gill tissue in the killifish (Van Praag et al. 1987). However, to our knowledge, no study has examined the effect of prostaglandins on ion transport in fish gills.
In the present study, PGE2 was found to stimulate chloride secretion, as indicated by the correlated increase of Isc and 36Cl- outfluxes. Bumetanide and DIDS blocked the PGE2-elicited current, suggesting that Cl- entry through basolateral membranes occurs via the anionic exchanger Cl--HCO3- and the Na+-K+-2Cl- cotransporter. This result is consistent with the PGE2-stimulated inward short-circuit current resulting from basal to apical Cl- secretion described previously in unstimulated preparations (Avella & Ehrenfeld, 1997a).
The results are different from those obtained using fish opercular membrane, since the addition of PGE2 or PGE1 decreased Cl- secretion through this epithelium (Eriksson et al. 1985; Van Praag et al. 1987). No comparison is available in the literature regarding the effect of eicosanoids in gill tissue. However, it is relevant to note that our primary culture is composed of respiratory-like cells, whereas the operculum transport is expected to be mediated by the dominant cell type in this epithelium, the chloride cells (Karnaky & Kinter, 1977). Moreover, the stimulatory effect of prostaglandin on Cl- secretion in our fish gill cell preparation may be compared with prostaglandin action on frog skin (Ziyadeh et al. 1986), frog cornea (Schaeffer & Zadunaisky, 1986) and various cell lines in culture (Keeler & Wong, 1986; Simmons, 1991; Armstrong et al. 1992).
It has been shown that prostaglandin E1 stimulates cAMP formation via adenylate cyclase in isolated trout gill cells composed of different cell types (Guibbolini & Lahlou, 1987a, b). It is therefore likely that PGE2 also increases cAMP levels, which in turn activates chloride channels in the apical membranes to secrete Cl-. This assumption is consistent with our previous studies where dB-cAMP stimulated chloride secretion in cultured epithelium composed of fish gill cells in culture (Avella & Ehrenfeld, 1997a). Furthermore, we also found that the activity of patched chloride channels of cultured gill cells was stimulated by protein kinase A plus ATP (Duranton et al. 1997).
The presence of PGE2 receptors in fish gill respiratory cells in culture is a new finding. Circulating or locally produced PGE2 is therefore expected to play an important role in controlling ion homeostasis of SW fish.
Effects of AVT
AVT appears to be the ancestral peptide of the vertebrate neurohypophyseal hormone family, and is the teleost equivalent of the mammalian antidiuretic hormone AVP. However, the exact involvement of AVT in fish osmoregulation is not clear (Takei, 1993).
In our study, AVT was used at 50 nM, which is twice the highest concentration of circulating levels reported (between 26 nM and 0·001 nM). The disparity in the endogenous concentrations found by various authors may be explained by the different techniques used or the salinity of the environmental conditions (Warne et al. 1994; Pierson et al. 1995). We now report for the first time a stimulatory effect of AVT on chloride secretion through fish gill respiratory cells in culture from a marine fish. Under these conditions of elevated Isc (AVT-treated cells), Isc was almost completely inhibited when adding DPC to block the putative apical exit pathway for Cl- (Cl-channel). This result is consistent with a vasotocin-stimulated inward Isc resulting from basal to apical Cl- secretion described previously in unstimulated preparations that were also sensitive to DPC (Avella & Ehrenfeld, 1997a). This effect is comparable to that found in two kidney cell lines (A6 and mIMCD-K2), which have served as models of chloride-secreting epithelia. In these cells, AVT was reported to elicit an initial increase in cAMP levels followed by an increase in apical Cl- permeability (Verrey 1994; Kizer et al. 1995).
The effect of AVT may be compared with previous ion transport studies in gill epithelium in vivo. In SW, AVT increased sodium efflux in the gill of the euryhaline flounder (Maetz & Lahlou, 1974). The physiological significance of the AVT-stimulated Cl- secretion found in this study is consistent with the necessity for fish living in SW to excrete ions through the gills.
The cellular signalling pathways involved in the AVT effect should also be considered. In comparison with mammalian studies, the stimulatory effect of AVT on Isc in sea bass gill cells in culture may be either by V2 receptors, via cAMP-dependent protein kinase A, or V1 receptors via IP3/Ca2+-dependent protein kinase C pathways (Vallotton, 1991). Activation of either pathway may lead to the phosphorylation and activation of the apical chloride channels. However, it has been suggested that AVT inhibits adenylate cyclase activity in trout and eel gill cells in suspension (Guibbolini & Lahlou, 1987b; Guibbolini et al. 1988; Sainsbury & Balment, 1991) via the Gi protein (Guibbolini & Lahlou, 1992) and in lamprey kidney tubules and trout hepatocytes (Lahlou et al. 1988; Guibbolini et al. 1994). Therefore, the present AVT stimulatory effect on Isc is unlikely to be mediated by an AVT-dependent cAMP increase via V2 receptors. Moreover, V1-type vasopressin receptors have recently been cloned and characterized in teleosts (Mahlmann et al. 1994). It is therefore more likely that AVT stimulates the IP3-Ca2+ pathway (V1-type receptor), which in turn may increase the chloride permeability of sea bass gill cells in culture.
When AVT was added to cAMP-treated cells, a transient stimulation of Isc was observed, which was followed by a large decline in Isc (Fig. 6). The fact that cAMP stimulates Isc (see also Avella & Ehrenfeld, 1997a) suggests that the apical Cl- channels are activated by a phosphorylation of the channel itself (or of an associated regulatory protein). The additional stimulation of Isc by AVT is likely to be mediated by V1-receptors and the IP3-Ca2+ pathway. The following Isc inhibition may reflect complex interactions between second messengers. It may be linked to possible opposite effects between the intracellular messengers involved (cAMP and Ca2+), as described with large concentrations of AVP in mammalian kidney tubules (Breyer & Ando, 1994). More experiments will be needed to elucidate this point.
In conclusion, this study shows (1) the presence of AVT receptors in gill respiratory cells in culture, and for the first time (2) that AVT is involved in ion transport through the gill epithelium from a marine fish by stimulating Cl- secretion.
Effects of catecholamines
Catecholamines have been shown to control gill physiology, in particular by their action on ion transport or circulatory processes. Catecholamines affect gill ion transport in in vivo and 'in vitro' (isolated perfused gills or head) studies (Pic et al. 1975; Girard & Payan, 1977) and also affect gill transepithelial potential and resistance (Shuttleworth, 1978). A distinction between vascular and epithelial effects was difficult to evaluate in these studies. We therefore tested on the gill cell epithelium in culture the effects of two catecholamines, isoproterenol (a -adrenergic agonist) and noradrenaline, on the regulation of Isc (Cl- secretion).
We found that isoproterenol stimulated Isc. In isoproterenol-treated cells, the current is also blocked to a large extent by DPC. This finding is therefore interpreted as the stimulation by isoproterenol of the basal Cl- secretion measured previously in the absence of stimuli (Avella & Ehrenfeld, 1997a). This effect was blocked by propranolol, a -adrenergic antagonist, confirming the involvement of -adrenergic receptors. Similar results on Isc have been found in the opercular membrane (Mendelsohn et al. 1981; Eriksson et al. 1985; Marshall et al. 1993) and in fish skin (Marshall & Bern, 1980). In our study, addition of isoproterenol also caused a stimulation of Vt, a result found in the operculum (Zadunaisky et al. 1988). The present investigation establishes the existence of -adrenergic receptors in gill respiratory-like cells of a marine fish. These receptors, when activated, elicit the stimulation of Cl- secretion. This effect is most likely to be mediated by an increase in adenylate cyclase activity and elevated cellular cAMP levels similar to -adrenoreceptor activation in gill tissues (Djabali & Pic, 1982; Guibbolini & Lahlou, 1987a).
Noradrenaline addition under control conditions or after propranolol (-antagonist) application inhibited Isc, Vt and Gt. This effect was completely counterbalanced by phentolamine, an -antagonist, since Isc returned to control values after phentolamine application. The phentolamine result confirms that noradrenaline decreased Cl- secretion through activation of -adrenergic receptors. Similar observations revealed an -adrenergic inhibition of NaCl efflux through the gill epithelium in SW-adapted mullet in vivo (Pic et al. 1975). The addition of noradrenaline or an -adrenergic agonist also produced a decrease in Isc and Cl- secretion through the activation of -adrenergic receptors in the fish operculum (Degnan et al. 1977; Mayer-Gostan & Maetz, 1980; Foskett et al. 1982; Marshall et al. 1993) and in skin (Marshall & Bern, 1980). The present study shows the existence of -adrenergic receptors in gill cells, which, when activated, lead to a decrease in Cl- secretion. This inhibition is probably mediated by a decrease in cellular cAMP levels, as has been measured in gill tissues with -adrenoreceptor activation (Djabali & Pic, 1982). However, an alternative would be that -adrenergic agonists may produce their inhibitory action via 1-receptor and IP3- Ca2+ signalling mechanisms. Determination of intracellular messengers levels as -adrenergic receptor subtypes could help to clarify the nature of the mechanism involved.
This study establishes the presence of both - and -adrenergic receptors in gill respiratory-like cells of a marine fish, their activation leading to stimulation and inhibition of Cl- excretion, respectively.
Conclusion
The present investigation in sea bass gill cells in culture confirms the presence of receptors for AVT and for - and -adrenergic agonists (isoproterenol and noradrenaline), and shows for the first time the presence of PGE2 receptors in these cells. A basolateral localization for these receptors is proposed in the respiratory-like cells. In addition, our study demonstrates for the first time, a stimulatory effect of AVT and PGE2 on Cl- secretion through the SW gill epithelium studied in culture. Finally, this study, in conjunction with our earlier results (Avella & Ehrenfeld, 1997a), emphasizes the importance of gill respiratory cells as a site for regulated ion transport. This has previously been attributed to gill chloride cells only. More studies are needed to elucidate the precise mechanism of intracellular signalling following hormonal regulation.
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Acknowledgements
The authors express their gratitude to Professor B. Lahlou (LPCM, Nice University, France) for his scientific advice throughout this study, to Dr P. Poujeol (LPCM, Nice University) for his comments and to Corinne Raschi (Laboratoire Jean Maetz, Nice University) for her technical assistance.
Corresponding author
M. Avella: Laboratoire de Physiologie Cellulaire et Moléculaire, UMR CNRS 6548, Université de Nice Sophia-Antipolis, Faculté des Sciences, Parc Valrose, 06108 Nice Cedex 2, France.
Email: avella{at}unice.fr
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