Download - Home | Journal of Cell Science - THE AMPHIBIAN ...J. Cell Sci. 5a, 197-213 (1981) Printed in Great Britain @ Company of Biologists Limited 1981 THE AMPHIBIAN EPIDERMIS: DISTRIBUTION

J. Cell Sci. 5a, 197-213 (1981)Printed in Great Britain @ Company of Biologists Limited 1981

THE AMPHIBIAN EPIDERMIS: DISTRIBUTION

OF MITOCHONDRIA-RICH CELLS AND THE

EFFECT OF OXYTOCIN

D. BROWN1*, A. GROSSOS AND R. C. DE SOUSA8-3

Institute of Histology and Embryology1 and Departments of Physiology1

and Medicine3 University of Geneva Medical School, 1211 Geneva 4, Switzerland

SUMMARYIt is known that the ion-transporting capacity and the permeability to water of amphibian

skins vary greatly both between and within species. Furthermore, the extent to which differentskins respond to hormonal stimulation of these parameters also shows considerable inter- andintra-specific variation. As a first step towards defining a possible morphological basis for thisphysiological heterogeneity, we examined different regions of skins from 3 anurans, Bufo bufo,Rana ridibunda and Xenopus laevis, that are species with widely differing habitats. The mito-chondria-rich cell population of the epidermis was counted and the epidermal thickness wasmeasured. There were large differences in the mitochondria-rich cell content and in the epi-dermal thickness of the skins from different species and from different regions of skin from thesame animal.

In a second set of studies, the same morphological features were examined and, in addition,routine functional parameters were measured to monitor some transport properties of the skinsused. The skins also varied considerably with respect to short-circuit current, potential differ-ence, water permeability and sensitivity to oxytocin. Although no apparent relationship wasnoted between either basal or hormone-stimulated physiological parameters and the morpho-logical features of the individual skins, the striking variation in the density of mitochondria-rich cells in amphibian epidermis merits further studies, including the use of techniques orexperimental designs that allow the movement of individual species of ion across the skin to befollowed.

INTRODUCTION

It is well known that the skin of anurans such as frogs and toads plays a major rolein the adaptation to the environment and homeostasis of these animals, and that theepidermis is a versatile regulatory organ with a wide range of permeabilities to ionsand water in different species (Bentley, 1971; Deyrup, 1964; Lindemann & Voute,1976; Shoemaker & Nagy, 1977; Ussing, i960). Furthermore, various regions of skinfrom the same animal have also been reported to have greatly differing transportproperties (Baldwin, 1979; Bentley & Main, 1972), which can be modified by agentssuch as neurohypophyseal peptides (Fuhrman & Ussing, 1951; Rajerison, Montegut,Jard & Morel, 1972 a; Sawyer, 1951), catecholamines (Fassina, Fiandini, Carpenedo &Santi, 1971; Hillyard, 1979; Rajerison, Montegut, Jard & Morel, 19726), steroids(Cirne & Malnic, 1972; Crabbe", 1964; Maetz, Jard & Morel, 1958; Porter, 1971) and

• Author for correspondence.

198 D. Brown, A. Grosso and R. C. De Sousa

prostaglandins (Fassina, Carpenedo & Santi, 1969; Hall & Martin, 1974; Lote, Rider& Thomas, 1974).

Up to now, however, no systematic effort has been made to correlate the structuraland functional properties of the amphibian epidermis. To this end, we selected3 anuran species with different habitats: Bufo bufo (a mainly terrestrial toad), Ranaridibunda (a truly amphibious frog) and Xenopus laevis (a totally aquatic toad). Pre-liminary observations showed large differences in the number of mitochondria-rich(MR) cells and in the epidermal thickness of the skins. Therefore, as an initial steptowards defining the morphological characteristics of the amphibian skin, a quanti-tative evaluation of these 2 parameters was performed. Furthermore, in order to havesome general information on the transport properties of the skins used, routinefunctional studies were carried out prior to fixation and cell counting in an additionalset of 12 animals. The functional parameters monitored were short-circuit current(s.c.c), transepithelial potential difference (p.d.) and water flux (JH,O)> b o t n in the'resting' state and following exposure to a neurohypophyseal peptide, oxytocin.

MATERIALS AND METHODS

X. laevis were obtained from the South African Snake Farm, Fish Hoek, South Africa,B. bufo from Barilli and Biaggi, Bologna, Italy and R. ridibunda from a local dealer. All experi-ments were carried out between April and July. Xenopus were used within 1 month of theirarrival in the laboratory.

Morphological studies

Pieces of dorsal and ventral skin were fixed with 2% glutaraldehyde in 0-05 M-phosphatebuffer, before being dehydrated in alcohols and embedded in Epon 812. Thin sections wereexamined in a Philips EM 300 electron microscope and semithin (approx. 1 fim) sections werecut from the same blocks and examined either with phase-contrast or following staining withtoluidine blue.

For freeze-fracture, skins were briefly fixed (5-10 min) in 2% glutaraldehyde and werestored in 0-05 M-phosphate buffer prior to use. The tissue was infiltrated with 30% glycerolin phosphate buffer for at least 1 h prior to quenching in Freon 22 (cooled with liquid nitrogen)and fracturing (Moor, Muhlethaler, Waldner & Frey-Wyssling, 1961) at — n o °C in a Balzers301 freeze-etch device (Balzers, Liechtenstein). Platinum/carbon replicas were cleaned bysequential treatment with sodium hypochlorite, chloroform/methanol (2:1) and distilled waterbefore being picked up on Parlodion-coated copper grids and examined in a Philips EM 300microscope.

Estimation of mitochondria-rich (MR) cell number

After dehydration, the tissue was orientated during the embedding process to permit thesubsequent cutting of sections that were either tangential or perpendicular to the surface ofthe epithelium. With tangential sections, photographs at a final magnification of x 425 wereused for all counts, and the number of MR cells in a square of area 10 000 fim1 (equivalent to18-1 cm1 on the photograph) was counted. With perpendicular sections, the number of MRcells was expressed per cm length of epidermis.

For both tangential and perpendicular sections, 4 random samples were taken from eachepidermis. Each sample consisted of a 2 mm x 2 mm square of skin, and 3 photographs weretaken from different regions of each sample. The MR cell numbers for the epidermis of eachanimal are, therefore, based on counts from 12 photographs.

Mitochondria-rich cells 199

Epithelial thickness

Twelve photographs of perpendicular sections from each epidermis were used, and thethickness was measured at 3-cm intervals (110/tm) on each photograph, giving 4 values perphotograph. The values given are, therefore, the average of 48 measurements for each epidermis.

Carbonic anhydrase localization

Carbonic anhydrase was localized in mounted cryostat sections of glutaraldehyde-fixed/skinsby a modification (see Brown, 1980) of the cobalt/phosphate method of Hansson (1967).Specificity was demonstrated by the incubation of some sections in medium containingio~6 M-acetazolamide (Sigma), a specific inhibitor of carbonic anhydrase activity.

Physiological studies

Potential difference (p.d.) and short-circuit current (s.c.c.) were monitored with techniquesdescribed elsewhere (De Sousa & Grosso, 1973), and water-flow measurements were performedwith an automatic, volumetric technique (Grosso & De Sousa, 1978; Ruphi, De Sousa,Favrod-Coune & Posternak, 1972). The transepithelial osmotic gradient was establishedby exposing the internal side of the skin to normal Ringer solution and the external side to10-fold diluted Ringer. Oxytocin (Syntocinon, Sandoz S.A.) was used at a final concentrationof 50 milliunits/ml. Student's Mest for paired data was used for all statistical analysis.

RESULTS

Mitochondria-rich cells of amphibian epidermis have been described previously inseveral ultrastructural studies (Farquhar & Palade, 1965; Lavker, 1971, 1972;Whitear, 1975). In perpendicular sections of the epidermis, they have a long, flask-likeshape and their narrower, apical pole, lies just beneath the stratum corneum (Figs. 1-3).In tangential sections, however, they appear as round profiles of various sizes,depending on the level of sectioning (Figs. 4-6), which are scattered between theother, much larger epithelial cells.

MR cells can be distinguished from other epidermal cells on the basis of 2 othercharacteristic features. Firstly, they contain large amounts of the enzyme, carbonicanhydrase, and in skins incubated to reveal this enzyme activity, only the MR cells,which can be easily recognized by their shape and position, are positive (Fig. 7)1Secondly, in freeze-fracture replicas, the plasma membranes of these cells containa population of elongated (rod-shaped) intramembrane particles. Such particles havebeen described previously in MR cells of R. ridibunda and X. laevis epidermis (Brown,Ilic & Orci, 1978) and they have now been found also on the plasma membrane ofMR cells from B. bufo epidermis (Fig. 9). This figure shows such particles on theapical microvilli of an MR cell, although in common with the other species, elongatedparticles were also found on the basolateral plasma membrane as well as on themembranes of some cytoplasmic vesicles.

MR cell number

In order to quantify the MR cell density of these skins, 8 specimens for each specieswere used in the initial counting studies. The physiological parameters of these skinswere not measured. Tangential sections were considered to be at an appropriate depth

2OO

X. laevis

D. Brown, A. Grosso and R. C. De Sousa

\


for counting when MR cells were distributed throughout most of the section. Insections from deeper into the epidermis MR cells were fewer, and in the deepestlayers they were no longer found. The 4-25 cm x 4-25 cm square used for counting wasplaced over the region that, by eye, appeared to contain the largest number of MRcells. Results obtained in this way were much more consistent than earlier resultsbased on counting the number of cells per photograph. This was due to the difficultyin cutting truly tangential sections, so that some areas of most photographs hadregions of low MR cell density, which probably represented deeper regions of theepidermis where MR cells are scarce.

Table 1 shows the distribution of MR cells in dorsal, abdominal and thoracic skinof R. ridibunda and X. laevis. In R. ridibunda, the abdominal skin was much richer inMR cells than the dorsal region, whereas the reverse was true for X. laevis. In bothspecies, however, the thoracic skin contained about the same number of MR cells asthe abdominal skin.

Although the technique of tangential sectioning worked well for R. ridibunda andX. laevis, the skin of B. bufo was so uneven and undulating that it was impossible tocut a large area tangentially. To circumvent this problem, the counting of MR cellswas done in perpendicular sections of B. bufo skin, and the results are presented inTable 2. As in R. ridibunda, in B. bufo the abdominal skin was richer in MR cellsthan the dorsal skin. In addition, when different regions of the ventral skin werecompared, the thoracic region had the lowest density (comparable to that of the dorsalskin), whereas the pelvic region consistently showed the highest counts. The relation-ships between the abdominal, thoracic and dorsal skins of these 3 species are sum-marized in Table 3.

A second series of cell counts was performed in animals whose skins were alsocharacterized from the functional viewpoint. Only perpendicular sections were used,thus allowing the results obtained in the 3 species to be compared directly. In theseanimals, the relationship between dorsal and abdominal skin was the same as de-scribed above, but it could now clearly be seen that the dorsal skin of X. laevis con-tained more MR cells than any of the others examined, whereas the dorsal skin fromR. ridibunda contained by far the fewest (Tables 4-6).

Epidermal thickness

As shown in Figs. 1-3, the thickness of the epidermis varied considerably amongthe 3 species, the average values for the ventral skin being (in /tm) 56-913-4 forR. ridibunda, 129-5 ± 6-3 for X. laevis and 40-7 ± 2-4 for B. bufo. The correspondingvalues for dorsal skin were 54-5 ± 2-7, 73-2 ± 3-3 and 43-5 ± 2-6. Only in X. laevis was

Figs. 1-3. Semi-thin sections of ventral skin from the 3 species used in this study, seenin the phase-contrast microscope. Sections were cut perpendicular to the surface toshow the position of MR cells in the epidermis (arrows) and the thickness of theepidermis. The epidermis of X. laevis (Fig. 1) is by far the thickest, that of R. ridi-bunda is intermediate (Fig. 2) and that of B. bufo (Fig. 3) is the thinnest, x 300. Bar,100 /tin.

202 D. Brovm, A. Grosso and R. C. De Sousa


there a significant difference between the thickness of dorsal and ventral epidermis.Both regions were much thicker in X. laevis than in other species, whereas the epi-dermis of B. bufo was the thinnest. The differences in thickness were due mainly tovariation in the number of layers of living cells, and not to the depth of the cornifiedlayer.

Potential difference and short-circuit current measurements

Tables 4-6 show the p.d. and s.c.c. values measured in dorsal and abdominal skinsof the same animal. In each instance, pieces of skin were mounted in double chambersof the Ussing type so that changes in p.d. and s.c.c. induced by oxytocin (natrifericeffect) could be compared with spontaneous changes in basal p.d. and s.c.c. valuesin 2 adjacent areas of the same skin. These areas are referred to as 'experimental' and'control' tissues, respectively. In general, s.c.c. and p.d. were measured sequentiallywith a voltage clamp having a 2-min cycle for automatic switching between open-circuit and short-circuit modes (De Sousa & Grosso, 1973); in some instances,however, only the s.c.c. was measured. Comparable steady-state values were achievedwith either technique, as shown by the A and B values of the control tissues (seeTables 4—6). The p.d. and s.c.c. varied somewhat from one specimen to the next but,with few exceptions, there was excellent symmetry between adjacent pieces of thesame skin. This can be seen by comparing the A values in the tables, which representp.d. or s.c.c. recorded in both skin segments prior to addition of oxytocin to theexperimental tissue.

The lowest values for p.d. and s.c.c. were recorded in X. laevis, particularly in thedorsal skin. Higher readings were obtained from both R. ridibunda and B. bufo, andvalues from dorsal and abdominal skin were in the same range. Concerning the re-sponse to oxytocin, the stimulation of p.d. and s.c.c. was minor in X. laevis, variablein B. bufo and most evident in the ventral skin of R. ridibunda (see Tables 4-6).

Hydrosmotic flow measurements

The second functional parameter to be studied was the change in water perme-ability of the skin following exposure to oxytocin (hydrosmotic effect). The resultsare shown in Fig. 10. In the presence of the standard osmotic gradient (see Materialsand Methods), basal flows were very small in X. laevis, intermediate in R. ridibunda

Fig. 4. Semi-thin, toluidine blue-stained section of R. ridibunda dorsal epidermis cuttangentially to the surface. There are only a few MR cells (circled) scattered betweenthe other, polyhedric epithelial cells, x 600. Bar, 50 fim.Fig. 5. Semi-thin, toluidine blue-stained tangential section of R. ridibunda ventralepidermis, showing large numbers of MR cells (circles) amongst the other epithelialcells. Depending on the level of sectioning, the MR cell nucleus is not always visiblein such sections, x 600. Bar, 50 /tm.Fig. 6. Electron micrograph of an MR cell from R. ridibunda sectioned transversely atthe level of the nucleus. Note the large number of mitochondria arranged around thenucleus and the relative scarcity of large desmosomes between the MR cell and thesurrounding epithelial cells, x 9000. Bar, 5 fim.



Table i. Quantitative evaluation of MR cell density in tangential sections o/X. laevisand R. ridibunda epidermis

Animal

i

2

345678

Mean ± s.E.

X. laevis

Dorsal

19272 2

2 1

2923242 2

23"2± 1-2a

(cells per

Thoracic

14141 0

161 1

14IS18

14010-9b

io4 fim1)

Abdominal

1 1

151 1

1 1

1816

1413

13-410-9c

R. ridibunda (cells per

Dorsal

22

93422

53-7±o-8

d

Thoracic

IS2 0

18

15IS141517

161 ±o-8e

10* fim*)

Abdominal

27272 0

1816

13172 1

i9-8±i-8f

Statistical significance ((-test): a-b: o-oi > P > o-ooia-c: P < 0001b-c: Not significantd-e: P < 0001d-f: P < 0001e-f: 0-05 > P > 0-02

and largest in B. bufo. Exposure to oxytocin induced no measurable stimulation ofwater flow in X. laevis, a moderate increase in R. ridibunda and a marked increase inB. bufo.

DISCUSSION

We examined the skin of 3 anurans with very different habitats and found 2 majormorphological differences: the epithelial thickness and the density of MR cells in theepidermis. From the functional view-point, the skins also varied considerably withrespect to p.d., s.c.c, permeability to water and sensitivity to a neurohypophysealhormone, oxytocin.

Fig. 7. Cryostat section of R. ridibunda ventral skin incubated to reveal carbonicanhydrase activity. The blackened, positive cells (arrows) in the outer epidermis arethe MR cells. Some cells in the cutaneous glands are also positive, x 120. Bar, 200 fim.Fig. 8. Cryostat section of R. ridibunda ventral skin incubated in the medium forcarbonic anhydrase detection, but with the addition of io"5 M-acetazolamide, an in-hibitor of carbonic anhydrase activity. The specific reaction in the MR cells has beencompletely abolished, but the non-specific deposits found in the dermis are stillpresent, x 95. Bar, 200 fim.

Fig. 9. Freeze-fracture replica of the apical region of an MR (mr) cell from B. bufoepidermis. The apical microvilli have a population of rod-shaped particles, character-istic of this cell type, on their P-face (inset). The cytoplasm of adjacent cells from thereplacement cell layer (rcl) contains mainly filamentous material, x 12600. Bar, 2 fim.Inset: x 74500. Bar, 025 fim.

2o6 D. Brovm, A. Grosso and R. C. De Sousa

Table 2. Quantitative evaluation of the number of MR cells in perpendicular sectionsof B. bufo epidermis

Animal

1

2

345678

Mean±s.E.

Dorsal

81

65787465768880

75-9 ±2-8a

Number of MR

Thoracic

8868866188649582

79'° ±4-5b

cells/cm epidermis

Abdominal

107

94I O I

632 0 8125

153126

I22-I ± 15-4C

Pelvic

1 4 0

1251821 4 0

1851362 3 0

138

I59-5 ± 1 2 7d

Statistical significance (t-test):a—b: Not significanta-c: 0-05 > P > O'O2a-d: P < o-ooib-c: 0-02 > P > o-oib-d: P < 0001c-d: 0-05 > P > 0-02

Table 3. Ratio of MR cells in abdominal and thoracic skin compared to the dorsal skinin X. laevis, R. ridibunda and B. bufo (summary of data from Tables 1 and 2)

Species Abdominal/dorsal Thoracic/dorsal

X. laevis 0-58 o-6oR. ridibunda 5-35 4-35B. bufo 1 63 1 05

2-12 (pelvic)

X. laevis, the species with a permanent aquatic habitat, had a very low resting waterflux, which was not increased by oxytocin (Fig. 10). This does not imply the absenceof hormone receptors, since a small natriferic effect of oxytocin was detectable in thesame tissue, as also described by other authors (Civan & Di Bona, 1974; Yorio &Bentley, 1978). The basal permeability to water and the hydrosmotic response tooxytocin (Fig. 10) appeared to be inversely related to the epidermal thickness in theventral skin, although this relationship did not hold for the dorsal skins, which wereall relatively impermeable (data not shown). In addition, our data, using a very precisetechnique to measure water flux, do not suggest a relationship between MR celldensity and the magnitude of the transepithelial water fluxes.

The conspicuous differences in MR cell density and their possible relation withion-transport properties of the skin deserve a few comments. In X. laevis the dorsalepidermis contained the most MR cells, whereas in the other 2 species the ventralepidermis had the most. The relative abundance of MR cells in our Xenopus speci-mens is intriguing; the skin of this animal consistently showed much lower values for


Table 4. Potential difference (p.d.) and short-circuit current (s.c.c.) of'resting' (control)and oxytocin-treated (experimental) X. laevis skin: comparison with MR cell density ofthe epidermis*

Animal no.No. MR cells(per cm)

Dorsal skin

p.d. (mV)ControlExperimental

s.c.c. (jiA)ControlExperimental

No. MR cells(per cm)

Ventral skin


8.C.C. (JlA)

ControlExperimental

i

154

A

—

—

i"52'O

196A

A

8 0

8 0

S o5 0

B

—

—

i-s2'O

B

6-S13-0

S'O8 0

2

179

A

4-o

SoIO'O

139

t

A

S OIO'O

5-0

9 5

B

4 0IO-5

S'°14-0

B

4-S8-5

4 5I2-O

3

303A

A

4-52 'O

5-0

2 5

104A

1

A

7'S5 0

7-S9 5

B

4-S2-0

4'52 O

B

7-57-S

7 0

11 5

4

35OA

A

- i - s— i - o

0

o-5

231A

/

A

3-o5-0

2-S4 0

B

O

3-

0

3-

a

3'8'

2

5

0

5

\

• 0

0

•5S

• The A values in the tables represent the p.d. or s.c.c. of both control and experimentalskins immediately prior to addition of oxytocin to the experimental tissue. The B values arethe p.d. and 8.c.c. of the experimental tissue at the peak of the response to the hormone, atwhich time the p.d. and s.c.c. values of the control skins were also recorded.

s.c.c, p.d. and water permeability than the other 2 species, and it was only poorlyresponsive to oxytocin. On the other hand, the dorsal skin of R. ridibunda containedvery few MR cells but s.c.c. could be increased by oxytocin (see Table 5). It has beensuggested that the higher sensitivity of amphibian ventral epidermis to neurohypo-physeal hormones may result from a higher MR cell density in this region (Ehrenfeldet al. 1976), but there are conflicting reports in the literature concerning the sensitivityof MR cells to such hormones (Goodman, Bloom, Battenberg, Rasmussen & Davis,1975; Handler & Preston, 1976; Rossier, Rossier, Pfeiffer & Krahenbuhl, 1979; Scott,Sapirstein & Yoder, 1974). Indeed, we were unable to find a consistent patternbetween M R cell content of the epidermis and the electrical parameters measured eitherbefore or after exposure to oxytocin. This observation should, however, be interpretedwith caution for 2 main reasons. Firstly, despite the fact that s.c.c. is essentiallydetermined by sodium transport in all 3 species used here (Larsen & Kristensen,1978; Li & De Sousa, 1979; Yorio & Bentley, 1978), subtle but significant differencesin the transport of other ions could be undetectable by the s.c.c. technique. Secondly,


Table 5. Potential difference (p.d.) and short-circuit current (s.c.c.) of resting (control)and oxytocin-treated (experimental) R. ridibunda skin. Comparison with MR celldensity of the epidermis*

Animal no. ...No. MR cells(per cm) ...

Dorsal skin



No. MR cells(per cm) ...

Ventral skin



A

23-01 7 0

17-52 5 0

f

A

—

—

20-SI 7 S

I

3

B

22-S2 5 0

I7-S3 2 5

169A

B

—

—

19-028's

•

2

11A

r

A

45-546-0

57-04 2 5

B

46-05 4 0

57-545-o

130A

t

A

41-044-0

5 i - 546-0

See legend

B

41-053-o

50-067-0

to Table

3

10A

I

A

—

—

25-03O'O

139

A

26-032-5

15-5175

4-

B

—

—

24-S

35'°

B

24-047-0

14-53 2 5

4

2 2A

A

38-029'O

3 6 0

37-5

i nA

r

A

45-045-5

5 2 0

55°

B

3 8 0

37'0

37°50-5

B

46-059-°

50-075-o

MR cells may exist in 'active' and 'inactive' states, based on morphological criteria(Frazier, 1978; Stetson, Wade & Giebisch, 1980; Voute, Hanni & Amman, 1972;Wade, 1976). In this case, the number of active cells in an epithelium may be moreimportant to overall function than the total number of MR cells, as counted in ourexperiments.

In the past, MR cells have been considered to be involved in moulting (Masoni &Garcia-Romeu, 1979; Whitear, 1975), keratinization, sensory perception (see Whitear,1975) and some secretory processes (Ehrenfeld et al. 1976; Voute et al. 1975). How-ever, similar cells occur in a number of other transporting epithelia including the toadurinary bladder (Choi, 1963), the amphibian and mammalian kidney collecting tubule(Bargmann & Welsch, 1972; Rhodin, 1958), and the rat epididymis (Sun & Flickinger,1980). All these cells contain large amounts of the enzyme carbonic anhydrase (Cohen,Hoffer & Rosen, 1976; Lonnerholm, 1971; Lonnerholm & Ridderstrale, 1974; Rosen& Friedley, 1973; Rosen, 1972), and their plasma membranes possess characteristicrod-shaped intramembrane particles when examined by freeze-fracture methods(Brown, 1978; Brown et al. 1978; Brown & Montesano, 1980; Humbert, Pricam,Perrelet & Orci, 1975; Orci et al. 1975; Wade, 1976). It is likely, therefore, that all


Table 6. Potential difference (p.d.) and short-circuit current (s.c.c.) of resting (control)and oxytocin-treated (experimental) B. bufo skin. Comparison with MR cell density ofthe epidermis*

Animal no.No. MR cells

(per cm) ...

Dorsal skin


s.c.c. QiA)ControlExperimental

No. MR cells(per cm)

Ventral skin



i

24A

A

—

21-028-5

52A

A

7-0

15-015-0

*B

—

20-5

27-5

B

7O

I5-O

I5-O

• See

2

72A

A

26-O44-0

15-5

B

27-0

49-5

15-037-5

100A

A

4O-547-o

31-030-0

legend 1

B

42-053-5

35-0

to Table

3

103

A

—

15015-0

162A

A

I2'OI4-O

io-o90

4-

B

—

I5-O2S-O

B

150500

10530-0

A

25-O

31-5

to

toO

to

ui u

i

A

I2-Oio-o

14-5io-o

4

69

B

27035O

22523-5

129

B

953 5 0

12525-0

of these cells share a specialized transport function in their respective epithelia. Inaddition to H+ transport (Frazier, 1978; Rosen et al. 1974) other physiological roleshave also been proposed for these cells, including anion transport (Voute & Meier,1978; Whitear, 1972) and K+ transport (Richet & Hagege, 1975; Stetson et al. 1980).In fact, recent work from our laboratory has revealed marked and apparently selectivechanges in the morphology of MR cells from skins exposed to high potassium con-centrations in the internal bathing medium (unpublished data). In addition, MR cellsfrom X. laevis maintained for up to 1 month in 1-25% NaCl solutions decrease innumber, lose their slender flask shape and develop extensive glycogen deposits (Ilic &Brown, 1980).

In summary, we have revealed that the MR cell population varies both betweenthe epidermis of different species and between different regions of skin from the sameanimal, and we have emphasized the specialized nature of these cells, when examinedby freeze-fracture electron microscopy and histochemical techniques to detectcarbonic anhydrase. Our preliminary physiological data did not reveal any obviouscorrelation with the transport functions examined, i.e. water and sodium transport(as estimated by the s.c.c. technique), but more work is obviously needed in this area.

2 1 0 D. Brown, A. Grosso and R. C. De Sousa

1-6 r-

1-2

u

E 0-8

o

0-4

00 L

MR cell counts

Bufo

Rana

Xenopus

, 1

153

172

190

_ 2 _

99

159

69

89

169

97

Bufo Rana Xenopus

Fig. io. Transepithelial water flow C7H,O) under basal conditions and following oxytocintreatment of the ventral skin from 3 animals of each species. The dotted, broken andcomplete lines represent separate animals and the point on the left of each slope is thebasal water flow, while the extreme right of each line is the peak hormone-inducedflow. The inset table shows the number of mitochondria-rich cells per cm in perpen-dicular sections of the same skins (dotted, broken or complete lines) used for the water-flow measurements.

It is hoped that techniques allowing individual species of ions to be followed acrossthe skin will provide a physiological correlation with this cellular heterogeneity.

We thank Professor L. Orci for helpful advice and criticism, Mme M. Sidler-Ansermet forphotographic work, Mr P. Sors for technical help and Mme N. Dupont for secretarial assistance.This work was supported by grants 3.120.77 and 3.043-0.76 from the Swiss National ScienceFoundation.

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(Received 5 January 1981)