Concentrations and activity coefficients of NA+, K+ and Cl− in Aplysia californica enterocytes

Camp. Biochem.

Phssiol.

Printed in Great Britain

Vol. 77A. No. 4. pp. 717-720,

1984

0300.9629!84

$3.00 + 0.00

,( 1984 Pergamon Press Ltd

CONCENTRATIONS AND ACTIVITY COEFFICIENTS OF Na+, K+ AND Cl - IN APLYSIA CALIlWRNICA ENTEROCYTES GEORGE A. GERENCSER Department

of Physiology,

College

of Medicine, Telephone:

University of Florida, 904-392-3791

(Received

Gainesville,

FL 32610.

USA.

7 July 1983)

Abstract-l. Tissue water averaged 76% of tissue weight in epithelial sheets of Aplysiu &ornica intestine. 2. Extracellular water averaged 19.67; of total intestinal water. 3. Intracellular Cl concentrations averaged 110 mM with most of Cl being bound or sequestered. 4. Intracellular Na+ concentrations were 34.2 mM with approximately 30”/1 bound or sequestered. 5. Intracellular K’ concentrations were not bound and averaged 532 mM. 6. Valinomycin lowered intracellular K A concentrations dramatically.

INTRODUCTION In the absence of muscosally-added organic solutes, small transepithelial potential differences ($,,) can normally be observed across vertebrate intestine, the serosal aspect being positive relative to the mucosal aspect (Baillien and Schoffeniels, 1961; Clarkson et al., 1961). In fact, the short-circuit current (SCC) in these preparations has been shown to consist of either (a) active Nat transport from mucosa to serosa; (b) active Na+ and Cl transport from mucosa to serosa (Barry et al., 1965). Subsequent addition of an actively transported sugar or amino acid to the mucosal bathing medium of vertebrate intestine instantaneously enhanced both $,,,, and SCC (Schultz and Curran, 1970). Enhanced Na+ absorption accounted for these observed electrical increases (Quay and Armstrong, 1969). Several observations indicate that electrolyte transport in invertebrate intestine differs from that in vertebrate intestine. For example, invertebrate intestine bathed in a Na+-containing solution exhibits an electrical polarity such that the serosal aspect is negative relative to the mucosal aspect (Gerencser, 1978b; Lawrence and Mailman, 1967). Furthermore, the negative serosal I/,,,, could be hyperpolarized by metabolizable monosaccharides (Lawrence et ul., 1972). This observation is consistent with greater anion absorption rather than enhanced Na+ absorption Recently, Gerencser (1978b) reported that the $,, across the intestine of the mollusk, Aplysia cahfornica, was serosa negative and the major portion of the SCC was carried by a net active Cl- absorption while the remainder of the SCC was wholly or predominantly a net Na’ absorptive current. Additionally, Gerencser (1978a) reported that addition of actively transported sugars and amino acids (Gerencser, 198 1) to the mucosal bathing solution hyperpolarized and increased SCC across the Aplysia intestine. $ Tre major portion of the enhanced SCC in the presence of D-glucose (Gerencser, 1978a) or glycine (Gerencser. 198 1) was carried by an enhanced net Cl 717

absorption while the minor portion of the SCC was wholly or predominantly an enhanced Na ’ absorption (Gerencser, 1978a). It is not known whether the differences between the intestine of the invertebrate Ap/,vsiu and vertebrate intestine reflect different mechanisms of transepithelial ion transport. To understand better the transfer of ions (Na+, K’, Cl-) across the epithelial cell membranes, a knowledge of the intracellular ionic pools is essential. To the extent that Nat, K’, and Cll are transported through the epithelial cells, such active transport pools must exist. Ideally, a knowledge of the size and location of such pools within the transporting cells as well as the electrochemical potentials of these ions in their respective pools is essential to our understanding of their transepithelial transport. Therefore, in the present study, measurements were made of extracellular water, total water and concentrations of Na+, K+ and Cl- in the enterocytes of Aplysia caljfornica intestine as well as calculations of the activity coefficients for these three ions were made. MATERIALS AND METHODS Mollu.sk Sea hares, adult Aplysia culifornicrr. were obtained from Pacific Bio-Marine, Venice, California, and were maintained at 25’C in circulating filtered seawater. Incubation

mediu ,for intestinal tissue

The formula for the standard seawater Ringer’s solution used was: NaCl, 0.462 M; MgSO,, 7H,O. 0.00243 M; KCI, 0.0121 M; NaHCO,. 0.00238 M; MgCl,, 6H,O, 0.00983 M; CaCI,, 0.01135 M. The total osmolality of the bathing medium was approximately 1000 mOsm/l and the final pH was 7.8 at 25’C. The activity coefficient of the seawater medium was calculated as 0.66 (Conway, 1952). were deterIntracellular Na* and K + concentrations mined specifically in cells lining the villi of Ap/ysia intestine bv the followine method (White. 1976). Segments of intestine were cut open and pinned to parakin in the bottom of a Lucite chamber containing continuously oxygenated sea-

718

GEORGE A. GERENCSER Table

Before 9 FM valinomycin After 9 GM valinomycin Probability

I.

Total tissue water (S$)

Extracellular water (:;)

intracellular water (“6)

76.2 k 4.3 (5)

19.6 f 3.1 (5)

84.8 5 7.3 (5)

75.8 + 5.6 (5) NS

21.3 k 2.6(5) NS

85.6 IX.1 (5) NS

Values are means k SEM. Numbers in parentheses are numbers of observations. Total tissue water is relative to tissue weight while extracellular water is relative to total tissue water. Intracellular water is calculated as the difference between total tissue water and extracellular water.

water. With a dissecting microscope and fine scissors, strips of villous tissue were cut from the intestine. These were incubated for 90min in oxygenated seawater containing 1 mM unlabeled inulin. Some of the segments were exposed to “C-inulin (New England Nuclear) for the last 60 min to assess extracellular space. All segments were filtered by suction onto filter paper, blotted, and weighed in aluminum boats previously boiled in nitric acid. Villous segments exposed to “C-inulin were digested in 3 ml of 0.1 N HNO, for 48 hr with continuous shaking. Duplicate 1 ml aliquots were counted for radioactivity along with duplicate 0.1 ml aliquots of a I:40 dilution (0.1 N HNO,) of incubation solution. The samples were counted in a three-channel Beckman LS-330 liquid scintillation counter. Two additional groups of villous segments were oven-dried at 100°C for 40 hr to obtain dry tissue weight. One group was solubilized in 25 ml 0.1 N HNO, and, after 48 hr of continuous shaking, analyzed for Na+, K+ and Cl- with a Perkin-Elmer model 303 atomic absorption spectrophotometer. All data 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

As seen in Table 1, in a normal NaCl seawater medium, the mean total water to epithelial tissue wet weight ratio averaged 76.2%. The tissue wet weight was comprised of a scraped epithelium devoid of muscle tissue. Extracellular water averaged 15.2% of the tissue wet weight whereas extracellular water averaged 19.6% of the total water of the epithelial tissue. By difference, therefore, intracellular water averaged 84.8% of tissue wet weight and intracellular water averaged 80.4:Jj of total tissue water. Valinomycin (9pM) had no significant effect on epithelial tissue wet weight, total water, intracellular water or extracellular water. In order to determine the physico-chemical state of Na + , K + and Cl - in the Aplysia enterocyte, villi were surgically removed from the remaining mucosa and analyzed for Na+, KC and Cl-. Also, in the present study, the effect of 9 PM valinomycin on intracellular Table 2. Intracellular

Before 9 FM valinomycin After 9 PM valinomycin

Ck (meq/kg cell water) 532 + 47 (IO)

Nat,

K’

K+ concentrations (Cl,) was studied. As shown in Table 2 intracellular concentrations of Nat (CQ, CL and intracellular concentrations of Cll (C&) averaged 34.2 + 3.5, 532 _+47 and 109.6 f 4.3 meq/kg cell water, respectively. After valinomycin addition to the bathing medium there was a significant decrease in CL (P < 0.05). That a significant amount of intracellular Na+ and Cl- is bound or compartmentalized is evident from the ratios u;U,/Ch, and a&/C’\.,, which are equivalent to the activity coefficients (yka, ;s;.,) of these ions. However, virtually no K+ was found to be bound or compartmentalized (Table 2). The activity values used for calculating the activity coefficients of from previous Na+, K+ and Cl- were obtained studies done under identical conditions (Gerencser and White, 1980; Gerencser, 1983). DISCUSSION

Intracellular Na+, K+ and Cll concentrations were obtained from the villous epithelial cells of Aplysia californica intestine, as were the intracellular Na + , K + and Cl _ activity measurements (Gerencser and White, 1980; Gerencser, 1983). This insures reasonably valid activity coefficients (y; = u;/C:) for the three ions when considering the heterogeneity of the intestine relative to villous, intervillous and crypt cell regions. The data in Table 2 indicate that a large fraction of the Nat associated with the absorptive cells is not available to the Na+-microelectrode since the experimental activity coefficients, ah,/Ch,, is much lower than the activity coefficient for Na+ in the bath (0.66). This is in conformity with the results previously reported for Aplysia intestine (Gerencser, 1983) bullfrog intestine maintained in sodium sulfate media (Armstrong et al., 1975) or in sodium chloride media (Armstrong et al., 1979a) and with the data reported for other species (Lev and Armstrong, 1975; Walker and Brown, 1977). The C& found in this investigation gave an r&, of 16.7%. This is considerably lower than the value (0.66) predicted, on the basis of a free solution model

and Cl- concentrations

;IK

Ck (meq/kg cell water)

0.72

34.2 f 3.5 (IO)

and activity

coefficients

Y’k,

Cti (meq/kg cell water)

;‘[I

N

0.50

109.6 k 4.3 (IO)

0 II

5

14.4 * 1.2(10)

Values are means k SEM. Numbers

in parentheses

are numbers

of observations;

N is number

of animals.

Intestinal

ion concentrations

for cytoplasmic Cl- and could be interpreted as suggestive of some intracellular binding or compartmentation of Cl-. This conclusion is in concert with those findings of y& in Amphiuma small intestine (White, 1977), rabbit ileum (Frizzell et al., 1973) and bullfrog small intestine (Armstrong et al., 1979b). It appears that most or all intracellular K+ is in free solution in the cytosol. This agrees with the activity coefficient for KC found in Aplysia intestine (Gerencser, 1983), bullfrog intestinal epithelial cells (Lee and Armstrong, 1972) and a great many more species for which aK has been directly measured (Lev and Armstrong, 1975; Walker and Brown, 1977). On the other hand a K+ activity coefficient of 0.28 was reported for Amphiuma intestine (White, 1976) and, similarly, low values for K+ activity coefficient were described (Khuri, 1974) for Necrurus (0.57) and rat proximal tubule cells (0.40) and rat distal tubule cells (0.34). It, therefore, appears that C; reflects a true transport pool for K+; however, CL, severely overestimates the transport pool for Na+ because of the significant fraction of intracellular Na+ that is bound. The Cl- transport pool is approximately 17% of the C;, which creates steep electrochemical gradients for this anion across both mucosal and basolateral membranes of the Aplysia enterocytes. This provides both a large downhill electrochemical driving force for Clfrom the mucosal solution, across the mucosal membrane, into the cytoplasmic aspect of the enterocyte and a large uphill electrochemical driving force for Cl- from the intracellular aspect of the enterocyte across the basolateral membrane into the serosal solution. The finding that valinomycin decreases CL to an extremely low value relative to control suggests that K+ is virtually the only ion sensed by the K+-sensitive microelectrode for valinomycin is a specific ionophore for K+ (Krasne et al., 1971). The possibility does exist, however, that interfering cations can contribute to the K+-electrode potential. However, in light of the valinomycin effect on CL the maximum contribution to the K+-electrode potential by interfering cations would constitute only a minute portion (negligible) of the total potential. Also, this observation indirectly confirms the notion that K+ is the major ion contributing to intracellular osmolality in the Aplysia intestinal epithelial cells. Assuming that the intracellular activity coefficient for K + is the same as that in the external medium (Lev and Armstrong, 1975), the intracellular K+ concentration found in other intestinal preparations (Armstrong et al., 1979a; Garcia-Diaz er al., 1980; Lee and Armstrong, 1972) accounts for some 45 to 60’%,of the total intracellular osmolality. The same calculation shows that my estimate of ak is equivalent to an intracellular K+ concentration of ca. 532 mM; that is, K + accounts for 53% of the total intracellular osmolality, in good agreement with earlier reports by others (Armstrong et al., 1979a; Garcia-Diaz et al., 1980; Lee and Armstrong, 1975).

Acknowledgement-These studies were supported hall Foundation Grant (No. 78-156 ck-1).

by White-

719 REFERENCES

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