paracellular transport characteristics of aplysia juliana intestine

Camp. Biochem. Physiol. Vol. 72A, No. 4, pp. 721 to 725, 1982 Printed in Great Britain.

0300-9629/82/080721-05$03.00/O 0 1982 Pergamon Press Ltd

PARACELLULAR TRANSPORT CHARACTERISTICS OF APLYSIA JULIANA INTESTINE

GEORGE A. GERENC~ER

Department of Physiology, College of Medicine, University of Florida, Gainesville, FL 32610, U.S.A.

(Received 26 November 1981)

Abstract-l. Current-voltage measurements strongly suggested the existence of a low resistance paracellular pathway in the intestinal epithelium.

2. Na’ and Cl- transport across the intestine involved both a cellular and a paracellular pathway. 3. The paracellular pathway for both Na+ and Cl- constituted the major conductive route for these

ions. 4. Diffusion potentials in the intestinal epithelium were symmetrical.

INTRODUCTION

With recent notable exceptions (White, 1980; You- mans & Armstrong, 1977), intestinal preparations of vertebrates bathed on both mucosal and serosal aspects with a substrate-free, Na+-containing Ringer solution elicit spontaneous transepithelial potential differences ($,,) of the order l-5 mV, the serosal surface being positive relative ti, the mucosal surface (Barry et al., 1965; Clarkson et al., 1961). The SCC in these preparations could be accounted for by means of active Na+ transport from M - S in the absence of, or in excess of, active Cl- transport in the same direction (Barry et al., 1965; Quay & Armstrong, 1969).

Several observations indicate that electrolyte transport by the invertebrate intestine differs somewhat from that of vertebrate intestine. For instance, the JI, across isolated intestine of the Lepidoptera, Hyulo- phora cecropia, was shown to be serosa negative, and this electrical orientation was accounted for by an active electrogenic pump moving potassium, in a net sense, from serosa to mucosa (Harvey et al., 1968). Additionally, the Jlms across both the intestine of the seaborne mollusc, Cryptochiton stelleri, and locust rectum was observed to be serosa negative (Lawrence & Mailman, 1967; Williams et al., 1978). The SCC across the locust rectum, bathed in a Na+-free Ringer medium, was accounted for, in a large measure, by an absorptive flux of Cl- (Williams et al., 1978).

Aplysiu juliana intestinal ion transport also differs substantially from that of vertebrate intestine. For instance, Aplysia juliana intestine bathed in substrate- free Na+-containing seawater medium exhibits a spontaneous ems (0.5-2.0 mV) such that the serosal surface is negative relative to the mucosal surface (Gerencser et al., 1977; Gerencser & Hong, 1977). The SCC is accounted for, wholly or predominantly, by active absorptive mechanisms for both Na+ and Cl-, the Cl- transport mechanism being more vigorous than the Na+. These results were qualitatively no different from those observed with Aplysia californica intestine (Gerencser, 1978a; Gerencser, 1978b) and account for the negative serosal $,, observed in this intestinal preparation.

There is strong evidence that there are two pathways available for ion movement across all epithelia, a transcellular pathway whereby ions traverse at least two cell membranes arranged in series and a parallel, transepithelial, paracellular or shunt pathway which by-passes the transcellular route (Ussing et al., 1974). The relative roles played by each of these pathways is dependent upon the type of epithelial tissue (Friimter & Diamond, 1972). One type of epithelial tissue, exemplified by frog skin (Ussing & Windhager, 1964) and toad urinary bladder (Civan & Frazier, 1968), is characterized by a high IL,,,, and a high transepithelial resistance. The other type of epithelial tissue, such as small intestine (Frizzell & Schultz, 1972), proximal renal tubule (Boulpaep, 1971) and gall bladder (Barry et al., 1971), is characterized by a low, practically neg- ligible, l(lms and a low transepithelial resistance.

The present investigation was therefore undertaken in order to assess:

(1) The ionic and electrical characteristics of the paracellular pathway in Aplysia juliana intestine;

(2) Comparative transport characteristics in Aply- sia juliana intestine relative to other vertebrate and invertebrate epithelial preparations.

MATERIALS AND METHODS

MOllUSC

Seahares, Aplysia juliana, collected off Diamond Head and Kewalo Basin, Oahu, Hawaii were airshipped to Gai- nesville, Florida through the kindness of Drs M. Hadfield and M. Dunlap, Pacific Biomedical Research Center. The molluscs were maintained at 25°C in circulating filtered seawater. In most cases, only animals that had been kept in the laboratory under the above conditions for a week or less were used for experimental purposes.

Incubation media for intestinal tissue The formula for the standard seawater Ringer’s solution

used was: NaCl, 462mM; MgS0,.7H20, 2.4mM; KCI, 12.2mM; NaHCOs, 2.4mM; MgCI,, 9.8 mM; Cat&, 11.4 mM. A Na+-free medium was prepared by totally replacing Na+ with choline+, using the chloride and bicarbonate salts. A Cl--free medium was prepared by totally replacing Cl- with SO:, using sodium salts. Mannitol was

721

722 GEORGE A. GEKtUCStR

used to adjust the osmolality of the SO, ‘-based medium. The total osmolality of the standard bathing media was 1000 mOsm/l and their final pH was 7.8.

Experimental procedures

Adult Aplysia juliana were used in these experiments. The animals were sacrificed and the guts were removed and positioned as a flat sheet between two halves of a lucite chamber as described by Gerencser (1978b), which allowed measurement of $I~,, clamped transmural PDs or diffusion PDs. The intestine was oxygenated between either identical seawater media or asymmetric media as will be described in the diffusion potential experiments.

Electrical measurements

The methods used to measure $,, clamped transmural PDs and diffusion PDs were essentially similar to those employed for rabbit ileum by Schultz & Zalusky (1964), except that agar bridges from calomel half-cells, instead of silver-silver chloride electrodes, were used to apply external current to the system. The electrolyte content of these bridges was identical to that of the bathing solution in each experiment. The agar bridges from the potential-sensing electrodes contained saturated potassium chloride. To minimize potential offset between these electrodes, the ends of these bridges, which were immersed in the bathing fluid, were pre-equilibrated with the appropriate bathing solution for several hours before the experiment. Offset between the potential-sensing electrodes was measured at the beginning of the experiment and again at the end of the run, following removal of the tissue and replacement of the bathing fluid. The potential drop between the potential- sensing electrodes due to the resistance of the bathing solution was compensated automatically by the voltage clamp device (VCD), as described by Rothe et al. (1969). The VCD had the capacity to voltage clamp the tissue over a wide range of transmural PDs (k 50 mV).

Voltage clamp serosal to mucosal jhxes

The technique used to measure the unidirectional serosal to mucosal (S + M) flux of ion from the mucosal bathing solution into the intestinal tissue has been previously described by Desjeux et a[. (1974). Briefly, isotopes were added to the bathing solutions immediately after mounting the tissue. Following a 30 min equilibration period under short-circuit conditions, samples for flux determinations were taken every 10 min for 50min. The transmural PD was clamped at zero for 30 min, at either + I5 or - 15 mV from 3&60min, at the opposite polarity from 6s90min and finally at zero from 90-120 min. The flux determinations under each condition were averaged to give a single value for flux in that tissue at the particular PD.

DifSusion potential measurements

Flat sheets of intestine were clamped between the halves of the chamber described above. The tissue was initially bathed on both sides by the standard NaCl seawater medium containing I mM ouabain and $,, was recorded as described above, using 3% agar bridges containing the standard NaCl seawater medium. After approximately 1 hr. the spontaneous t/j,,,, had declined to zero and the compo-

sition of the mucosal or serosal solutton was then sertally diluted with seawater solutions in which substitute seawater media replaced the standard NaCl seawater medium. The absence of an initial transepithelial PD ($,,) was taken to indicate that active ion transport by the tissue had ceased (Schultz & Zalusky, 1964; Gerencser. 1981) so that the transmural PDs resulting from changing the composition of the mucosal or serosal solution reflected only diffusion potentials across the intestine. The values reported are steady-state values that were achieved within I min after changing the composition of the mucosal or serosal solution; these values remained constant for the 2 min interval between serial dilutions.

The experimental data are complicated by asymmetric junction potentials, arising at the tips of the potential-mea- suring electrodes, which can neither be measured directly nor calculated satisfactorily. However, compensation for these PDs in the diffusion potential measurements was done as demonstrated by Caldwell (1968) and Picknett (1968); these authors have shown that the corrected PDs are not likely to be in error by more than I mV.

The application of the radiotracer technique for the de- termination of unidirectional fluxes of “Cl- and “Na+ under voltage-clamped conditions was as described by Quay & Armstrong (1969).

All data are reported as means +SEM. Difference between means were analyzed statistically using Student’s t-test.

RESULTS

It is assumed that the transmural unidirectional flux of ion from serosa to mucosa can, in principle, be described as the sum of a flux through a cellular path-

way, J1,,, and a diffusional flux through the paracel- Mar shunt, Jt,,,. Thus.

Jf, = J’r,, + Jis,,,. (1)

It is further assumed, in accordance with the consider- ations of Frizzell & Schultz (1972) that Jf,, can be characterized as a simple diffusion process. On this basis, their analysis indicates that with identical ion concentrations in the two bathing solutions, J;,y, should be simply a function of transmural potential difference. For relatively small values of PD, this relationship can be approximated by the following ex- pression,

J;,,,, = oJ6,,[exp(F$,$RT)]“* = oJ~_,,<~‘*, (2)

in which oJLsm is flux through the shunt pathway under short-circuit conditions, I/I,,,, is potential difference across the tissue with the mucosal side taken as reference and F, R and T have their usual physio-

chemical meanings. Thus, equation I can be written

J:, = Ji,,, + oJ,&~~~. (3)

In principle, Ji,,, could also depend on potential difference. However, Frizzell & Schultz (1972) have

Table I. Sodium and chloride fluxes from serosa to mucosa

Na+ cl-

(peq$;ihr)

21.0 f I.1 (5) 23.1 & 1.2(5)

J:Sm (fleq/cm’/hr)

JL, (peq/cm2/hr)

0.9 * 0.2 (5) 20.0 f I.3 (5) 0.9 * 0.1 (5) 22.2 * 1.4(S)

see

Wq/cm’/hr)

4.5 + 0.3 (5) 4.3 + 0.3 (5)

Average values k SEM are given for the number of experiments shown in parentheses. oJ~, is the observed flux under short-circuit conditions while Jf,, and J;,,,, are calculated from equation 3.

Intestinal ion transport 723

Fig. 1.

9 ms’ m”

Current-voltage relationship of Aplysia juliana intestine clamped at voltages +15mV.

shown that SS-90% of the current passed across rabbit ileum is carried via the shunt pathway. Thus, for reasonable values of external current, there should be little change in &,, because there is little current flow via the cellular pathway.

The Jdsm (paracellular component) for both Na+ and Cl- were linear functions of the clamped voltages varied from + 15 to - 15 mV (N = 3).

Table 1 shows that for both Na* and Cl- the mean oJ,, greatly exceeds the corresponding average SCC. Also, the mean J,, for both Na+ and Cl- is significantly less (P < 0.001) than the mean Jdam of NaC and Cl- , respectively. That is, the cellular component for both Na+ and Cl- (oJ,,,) fluxes are very small frac- tions of the total oJ, flux for the tissue. Conversely, the paracellular component of oJ,, transfer for both Na+ and Cl- predominates in conductance of these two ions across the tissue.

Resistance

The slope of the current-voltage relation indicates that the transepithelial resistance is only 2@-30 R cm’ (Fig. 1). In contrast, the resistance of most single cell membranes, including those of gall-bladder epithelial

Table 2. NaCl diffusion potentials

Seawater solutions Mucosal Serosal W(mV)

A. NaCl NaCl 0.0 f O.O(l8) B. Choline Cl NaCl -2.1 f 0.8 (10) C. NaCl Choline Cl + 1.8 L- 0.5 (10) D. Na2S04 NaCl -1.3 f 0.4(19) E. NaCl Na2S04 - 3.0 * 0.5 (19)

(L mr values are means f SEM. Numbers in parentheses are numbers of observations. Polarity of $,,,, is relative to mucosal solution. All mean experimental +,,,, (lines, B, C, D and E) are significantly different at P < 0.05 from the con- trol mean II/,,,, (line A).

from - 15 to

cells (Fromter & Diamond, 1972) and proximal tubule (Boulpaep, 1971), is several thousand ohm cm*. Hence, the principal pathway of transepithelial ion permeation in Aplysia juliana intestine must be a shunt that by-passes the high-resistance epithelial cells.

D&ion potential measurements

The effects on $ms of replacing NaCl in the mucosal or serosal bathing medium with either Na,SO, or choline Cl has shown symmetrical electrical properties, i.e., reversing the mucosal and serosal bathing solutions gives a $,, of opposite polarity but approximately the same magnitude (Table 2). In these experiments 1 mM serosal ouabain was included in the seawater media; the I,!J,,,~ measurements were taken after the spontaneous $,, (l-2mV) had declined to zero. This procedure assures that the effects of Na+ or Cl- replacement can be attributed entirely to diffusion potentials, uncomplicated by the effects of a reduced mucosal or serosal Na+ or Cl- concentration on the spontaneous $,, (Schultz & Zalusky, 1964). Replace- ment of Na+ in the mucosal solution with choline+ produced a significant increase in the spontaneous serosal negativity of *,,. In contrast, replacing Na+ in the serosal solution with choline+ depolarized the spontaneous serosal negative $,,,s to a steady-state value in which the polarity of $ms reversed, i.e., the serosal aspect was positive relative to the mucosal solution. Replacing Cl- with SO;* in the mucosal bathing solutions depolarized the spontaneous serosal negative $,,,.s and sometimes, reversed the polarity of the $,,,, Replacing Cl- with SO;’ in the serosal bathing solution hyperpolarized the normal spontaneous serosal negative Ic/,,

DISCUSSION

The very low l(/,,,s of Aplysia juliana intestine (Ger- encser et al., 1977; Gerencser & Hong, 1977) and the

124 GEORGE A. GERENCSER

low tissue resistance of approximately 20 R cm’ (Fig. I) strongly suggests that a low resistance paracellular shunt pathway exists in Aplysia julianu intestine and that it plays a major role in ionic conductance across the tissue. Strengthening this idea was the previous finding (Gerencser & Hong, 1977), which demonstrated that the J,, fluxes for both Na’ and Cl- were much greater than the net active mucosal to serosal fluxes (Ji:‘) of these ions. The finding that for both Na+ and Cl- the paracellular shunt component (Jdsm) was greater than 900/:, of the total serosal to mucosal (oJ,,) movement of these ions across the intestinal tissue (Table 1) buttressed the notion that the paracellular pathway, and not the cellular pathway, was the predominent route for ionic conductance across this tissue.

Schultz & Zalusky (1964) demonstrated that the unidirectional transmural flux of Nat from the serosal solution to the mucosal solution was unaffected by ouabain, and that this appeared to be entirely attribu- table to simple ionic diffusion. This alone suggested (Schultz et al., 1967) that most, if not all, of the serosal to mucosal Na+ flux takes place through a shunt pathway; a major transcellular contribution to the serosal to mucosal flux of Na+ would be influenced by intracellular Na* (which is affected by ouabain). Empirically they demonstrated that there is little or no entry of Na+ from the serosal solution across the lateral-serosal membranes, which is similar to the finding in Aplysin juliana intestine (Table 1) for Na+ and Cl-. They implied complete or near-complete rectification of Na+ transport across the membrane that appears to be responsible for active Na’ transport from mucosa to serosa, and this is consistent with the model presented by Clarkson (1967) for rat intestine and the conclusions of Civan (1970) for toad urinary bladder.

Thus, the $,,,, resulting from ion concentration gradients across the Aplysia juliana intestine may be interpreted as simple diffusion potentials. The previous finding that the serosal to mucosal flux of Cl- is greater than the serosal to mucosal flux of Na’ under short-circuited conditions indicates that the paracellular pathway in Aplysia juliana intestine is more per- meable to anions than cations (Gerencser & Hong, 1977). In concert with the results for rabbit gall- bladder (Diamond, 1962), diffusion potentials in the intestine are symmetrical; that is reversing the mucosal and serosal bathing solutions gives a $,,,, of opposite polarity but approximately the same magnitude (Table 2). This result is the immediate expectation for a single membrane, such as the tight junction, but it is not generally expected for a series system of two membranes separated by a reservoir such as a cell (Sandblom & Eisenman, 1967), unless the two membranes happen to have the same relative permeability coefficients. However, since the J,,, for both Na+ and Cl- is less than 5% of the .I,,,, for these ions (Table 1) and, coupled with the previous observation (Ger- encser & Hong, 1977) that the net mucosal to serosal fluxes for both Na+ and Cl- are greater than 15% and 25%, respectively, than the J,, fluxes of these ions, dispels this hypothesis that both the mucosal and lateral-serosal membranes of the Apll~~ia juliana intestinal epithelial cells have the same relative permeability coefficients.

Acknoa,/edyrments~~~The technical assistance of Mr. Scott Sircus in some of these experiments is gratefully ac- knowledged. This mvestigation was supported by White- hall Foundation Grant 78-156 ck-1 DSR.

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paracellular transport characteristics of aplysia juliana intestine

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