INTRODUCTION

Two general mechanisms of intestinal transcellular CI⁻ absorption have been well established (Gerencser, 1996). One of these is an electrically silent symport which drives CI⁻ uphill into enterocytes by means of an inward flux of Na⁺ moving down a favorable electrochemical gradient as is exemplified in intestinal epithelia of prawn (Ahearn et al., 1977), flounder (Field et al., 1978), bullfrog(Quay and Armstrong, 1969) and human (Turnberg et al., 1970). The other transcellular CI⁻ absorptive mechanism is a CI⁻ /anion antiport as is found in intestinal epithelia of Amphiuma (Gunter-Smith and White, 1979) and rabbit (Frizzell et al., 1976).

However, It has been hypothesized that Cl⁻ absorption across the Aplysia gut is mediated by a primary active transport process (Gerencser and White, 1980; Gerencser, 1983) located in the basolateral membrane (BLM) Gerencser, 1983]. Lending credence to this idea were the observations that both BLM-localized Cl- stimulated ATPase and ATP-dependent Cl⁻ transport activities were reconstituted into proteoliposomes (Gerencser, 1990) and were shown to be properties of the same transporter protein (Gerencser and Zelezna, 1993). Serosal N-ethylmalemide (NEM) inhibited the active transepithelial Cl⁻ absorptive flux across the Aplysia gut (Gerencser, 1990) while it had been demonstrated in other tissues that NEM specifically inhibited vacuolar H⁺-ATPases (V-ATPases) (Pederson and Carafoli, 1987). Therefore, the present study was undertaken to further clarify whether the nature of the Aplysia Cl⁻ pump was P, V or F (Pederson and Carafoli, 1987).

METHODS

Seahares (Aplysia californica) were obtained from Marinus, Inc. (Long Beach, CA) and were maintained at 25°C in circulating filtered seawater. Adult Aplysia (600–1000g) were used in these experiments. Tris (hydroxymethyl) aminomethane (Tris)-ATP, phenylmethylsulfonylfluoride (PMSF) and EDTA were purchased from Sigma Chemical. (St.Louis MO). All other reagent grade purity chemicals, including sodium orthovanadate, were purchased from Fisher Scientific (Norcross, GA). ³⁶Cl⁻,[carboxyl¹⁴-C]inulin and [methyl⁻³H] triphenylphosphonium bromide ([³H] TPMP⁺) were purchased from New England Nuclear (Boston, MA).

The inside-out BLM vesicles were prepared from Aplysia foregut epithelial cells by homogenization and differential and discontinuous sucrose density-gradient centrifugation techniques as described previously (Gerencser, 1990). Marker enzymes of the BLM were determined as previously described (Gerencser, 1990; Gerencser and Zelezna, 1993).

Both Cl⁻-stimulated ATPase and ATP-dependent transport activities were measured as described previously (Gerencser and Lee, 1985; Gerencser, 1988). The BLMV electrical potential (ΔΨ) was estimated from the distribution of the lipophilic cation triphenylmethylphosphonium (TPMP⁺) between the extra- and intravesicular space by ultrafiltration as described by Gerencser (1988) utilizing a double labeling method. When used, inhibitors were preincubated with the BLMV for 10 min., as was valinomycin, the K⁺ ionophore (Gerencser, 1988).

All date are reported as means ±SEM. Differences between means were analyzed statistically using Student's t-test with a P<0.05 used as the statistical significant difference criterion.

RESULTS AND DISCUSSION

The present series of experiments probed the effects of various reactants and inhibitors on ATP-dependent ΔΨ, ATP-dependent Cl⁻ transport, and Cl⁻ -ATPase activity in the BLMV preparation. As seen in Table 1, the only inhibitors that significantly reduced ATP-driven ΔΨ, ATP-dependent Cl⁻ accumulation, and Cl⁻ -stimulated ATPase activity in the BLMV were orthovanadate, PCMBS and NEM. The PCMBS-induced inhibition of ATP-driven ΔΨ, ATP-dependent Cl⁻ accumulation, and Cl⁻-stimulated ATPase activity was reversed by the subsequent addition of dithiothreitol (DTT).

Table 1

Effect of Inhibitors and/or Reactants on ATP-dependent ΔΨ, ATP-dependent Cl⁻ Transport and Cl⁻ ATPase Activity^a

The present finding that ATP, in the presence of Cl⁻, can stimulate both ΔΨ and its associated intravesicular negativity, and Cl⁻ accumulation in the BLMV (Table 1), containing Cl⁻ ATPase extracted from Aplysia gut BLM (Gerencser and Lee, 1985), strongly suggests that the mechanism responsible for this phenomenon is electrogenic. Electrogenicity is defined as generation of a potential difference and that is what was observed directly by the ATP-generated ΔΨ in Table 1.

Orthovanadate is a specific competitive inhibitor of phosphate-binding on P-type ATPase catalytic (sub)units, and all P-type ATPases have a catalytic (sub)unit whose molecular weight approximates 100 kDa (Pederson and Carafoli, 1987). In the present study orthovanadate inhibited the ATP -dependent ΔΨ and ATP-dependent Cl⁻ transport and Cl⁻-ATPase activity in the BLM (Table 1) suggesting that the 110 kDa Cl⁻ATPase catalytic (sub)unit previously detected in the proteoliposome (Gerencser and Zelezna, 1993) was responsible for generating the negative intravesicular potential difference in the BLM preparation through transport of Cl⁻ and its associated negative charge. (Slayman and Zuckier, 1989; Pederson and Carafoli, 1987). In contrast, DCCD, an inhibitor of all known H⁺ -ATPases, whether they belong to P, V, or F groups (Pederson and Carafoli, 1987) had no effect on ATP driven ΔΨ, ATP-dependent Cl⁻ accumulation or Cl⁻-ATPase activity (Table 1) strongly suggesting that contaminant H⁺ - ATPase (Gerencser, 1996) could not express the observed Cl⁻ pump activity.

As demonstrated in the present study (Table 1), the addition of PCMBS or NEM to BLM vesicles containing Cl⁻-ATPase evoked an inhibition of ATP-dependent ΔΨ, ATP-dependent Cl⁻ transport and Cl⁻-ATPase activity significantly lower from that of control. Although NEM nor PCMBS are not absolutely specific for sulfhydryl ligands and have been shown to inhibit other ligands such as carboxyl, amino, phosphoryl, and tyrosyl (Rothstein, 1970) it is strongly suggested that, at least, PCMBS inhibition was, in a major part if not totally, through sulfhydryl ligand binding, since DTT, a specific thiol-reducing agent (Rothstein, 1970), almost totally reversed the inhibition by PCMBS (Table 1). Since NEM isan inhibitor of V-ATPases (Pederson and Carafoli, 1987) and has been shown to inhibit Cl⁻ pump characteristics (Table 1) and Cl⁻ transport (Gerencser, 1990), it is not necessarily true that the Cl⁻ pump is a V-ATPase because NEM is relatively non-specific inhibitor of all types of ligands on molecules (Rothestein, 1990). The fact that the PCMBS inhibition of the Cl- pump is reversed by DTT and NEM inhibition is not reversed by DTT attests to the non-specificity of this molecule (Table 1). Further proofs of the non V-ATPase nature of the Cl⁻ pump rests with the insensitivity of the Cl⁻ pump characteristics to DCCD, bafilomycin and NBD- Cl⁻, all of which are V-ATPase inhibitors (Pedersen and Carafoli, 1987).

Additionally, Cl⁻ -ATPase activity ATP-dependent ΔΨ and ATP-dependent Cl⁻ transport in the Aplysia gut BLMV were insensitive to the F-ATPase inhibitors, efrapeptin and azide (Table 1) (Pederson and Carafoli, 1987). All of these negative results suggest that neither proton pump, nor F-ATPase, nor V-ATPase activity is involved in ATP-dependent ΔΨ, ATP-dependent Cl⁻ transport or Cl⁻-ATPase activity. The present results warrant strong speculation that the active Cl⁻ absorptive mechanism in the Aplysia gut BLM is electrogenic and is driven by a Cl⁻ stimulated ATPase which is a P-type ATPase. Since all F-and V-ATPases are proton pumps (Pederson and Carafoli, 1987), the finding that the CI⁻-ATPase is a P-type ATPase attests to a biological efficiency of this unimolecular mechansism (Gerencser and Zelezna, 1993). Otherwise, a primary proton pump(i.e.a V-ATPase) would need a second transporter in order to translocate CI⁻ up an electrochemical potential grandient (i.e. a H⁺-CI⁻ symporter). This event would be after the proton pump had set up a favorable H⁺ gradient that would drive CI⁻ energetically uphill by the symporter as has been described by Wiezorek (1992) in his “proton recycling process” hypothesis.

This study concurs with several others that postulate a CI⁻-ATPase as a mechanism that translocates CI⁻, in a net sense, across a zoological membrane system. These include an electrogenic CI⁻ transporter found in locust rectal epithelium (Hanrahan and Phillips, 1983) rat brain (Shiroya et al., 1989), blue crab gill (Lee, 1982) and human placenta (Boyd and Chipperfield, 1982).