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Cellular ionic concentrations in the midgut of two larvae of lepidoptera in vivo and in vitro
Comp. Biochem. Physiol., 1978. Vol. 59A, pp. 17 to 20. Pergamon Press. Printed in Great Britain
CELLULAR IONIC CONCENTRATIONS IN THE MIDGUT OF TWO LARVAE OF LEPIDOPTERA I N V I V O and I N V I T R O B. GIORDANA AND F. SACCH] Istituto di Fisiologia Generale e Chimica Biologica dell'Universit/l di Milano, Italy
(Received 24 March 1977) Abstract--1. The concentrations of Na, K, Mg, Ca and C1 across the midgut of Philosamia cynthia and Bombyx mori are reported. Intracellular 4ntestinal concentrations of the same ions both in vivo and in vitro have also been calculated. 2. The haemolymph of both larvae shows a (Na + K)/(Mg + Ca) ratio lower than 0.4; CI concentration is very low. K concentration in the lumen content ranges between 150 and 199 mequivA, while Ca and Mg concentrations are lower than those in the haemolymph. 3. Na, Mg, Ca and CI intracellular concentrations in vivo are low, while K is very high (over 190 mequiv/l cell water). Ion variations in the incubated tissue have been studied.
and 1:39 for Mg determinations. For Na, K, Mg and CI determinations the solutions were diluted 1 : 1 with HCIO4 0.6 N and centrifuged for 5 min to obtain the sedimentation of proteins. Na and K were then assayed by means of a flame photometer (Beckman DU-2). Mg and Ca were assayed colorimetrically after preparation of samples following Magnesium Merckotest (Art. no. 3338) and Calcium Kit (Clinton) indications. Samples were then read by C.E. 343 Single Sample Spectrometer, Cecil Instrument Ltd, Cambridge. Chlorides were assayed by means of mercurimetric tritration according to Chloride Merckotest (Art. no. 3311). To perform ionic determinations in the lumen content, the larvae were cut lengthwise, the midgut was perforated and the intestinal content collected into a test tube and centrifuged for 5 min to obtain a solution free from leaf fragments. The supernatant was then diluted 1:29 with bidistilled water for K and Mg determinations. Determinations were then performed as described for the haemolymph. The intestinal tissue was removed from the larvae as described elsewhere (Giordana and Sacchi, 1977a), lightly blotted with filter paper (Whatman no. 587 E), shattered, placed in a tared tube and weighed. One ml of bidistilled water was added, the suspension thoroughly mixed, frozen, thawed, resuspended and centrifuged for 30min. The supernatant was diluted 1 : 1 with HC104 0.6 N and centrifuged for 5 min for Na, K, Mg and C1 determinations; ions were then assayed as described. Sediments were dried overnight and weighed to obtain total tissue water. Experiments were also performed on incubated midguts, in order to measure extracellular space values and ionic intracellular concentrations. Salines were prepared with an ionic composition as determined in the haemolymph:
INTRODUCTION In a previous paper ( G i o r d a n a & Sacchi, 1977a), the presence of a very low N a concentration (less t h a n 5 mequiv/l) and a relatively high K concentration has been pointed out in haemolymph, intestinal tissue and lumen content o f Philosamia cynthia and Bombyx mori. These larvae belong to the order of Lepidoptera, which is also characterized by a high h a e m o l y m p h a t i c concentration of divalent cations (Florkin & Jeuniaux, 1975). In the midgut of these larvae (Harvey & Nedergaard, 1964; G i o r d a n a & Sacchi, 1977a), a very large transepithelial potential difference (p.d.) with the positive pole in the lumen, due to an active and electrogenic extrusion of K ions, has been observed. The values of these p.d.s, range between 110 and 1 3 0 m V in vivo and between 70 and 8 5 m V in vitro. While cation distribution across the midgut of Hyalophora cecropia in vivo has been well studied (Harvey et al., 1975), very little is k n o w n a b o u t ionic pattern in haemolymph, intestinal tissue and content of Philosamia cynthia and Bombyx mori. Furthermore, i m p o r t a n t suggestions can be supplied by the exact valuation of the intracellular ionic concentration of the intestinal epithelium, which separates two comp a r t m e n t s - - h a e m o l y m p h and l u m e n - - w i t h very different ionic concentrations. In this work, the concentrations of sodium, potassium, magnesium, calcium and chloride across the midgut of Philosamia cynthia and Bombyx mori, and the intracellular intestinal concentrations of the same ions b o t h in vivo and in vitro are reported.
MATERIALS
AND
METHODS
Philosamia Bombyx
4 25 - 74 18 156 mequiv/l 1.7 25 21 88 18 110 mequiv/l Sucrose was added to both solutions to reach the osmolarity as determined in the haemolymph (Philosamia: 284.6 + 9.3 mOsm/l, three experiments; Bombyx: 290 + 2.9 mOsm/l, three experiments). These salines were aerated and stirred by bubbling with 95% 0 2 and 5~o CO2, pH 7.4. The midgut was excised from the larvae, the peritrophic membrane and the enclosed intestinal content gently
The experiments were performed during June, July and September on mature larvae of Philosamia cynthia reared on Ailanthus #landulosa leaves, and of Bombyx mori fed on Morus alba leaves. In order to measure cations and CI concentrations in the haemolymph, the larvae were bled by cutting a proleg and the haemolymph was collected in a test tube, diluted with bidistilled water 1:9 for Na, K and Ca determinations 17 { BI' St} IA
I~
18
B. G[ORDANAAND F. SACCHI
removed and the tissue mounted as a tube on the perfusion Bombyx mori not only of a steep K gradient between apparatus described in a previous paper (Giordana & the lumen and the haemolymph, but also of a relevant Sacchi, 1976). The tissues were incubated for different Mg gradient with the higher concentration in the haeperiods (10, 60 and 120rain). In order to measure total molymph. The~ potassium distribution is in these larextracellular space (ECS), the salin~-ix~r~sing both sides vae opposite to the high, lumen positive, transepiof the epithelium contained sucrose ~4C or sulphate 3sS 1 #Ci/ml (from Radioehemical Center, Amersham). Sul- thelial p.d., and consistent with an active transport phate was used for short incubations (up to 40 min) and of this cation from the haemolymph to the lumen, as sucrose for longer ones (60 and 120 rain). After the incuba-. has been well demonstrated in the isolated midgut tion, the midguts were removed, blotted with filter paper 6f~H~alophora cecropia (Harvey et al., 1968). In fact, (Whatman 587 E), shattered, placed in a tared tube and the lumen positive p.d. in these two larvae depends rapidly weighed. One ml of bidistilled water was added in vitro entirely on the availability of K in the haemoand the suspensions thoroughly mixed, frozen, thawed and lymphatic side of the epithelium (Giordana & Sacchi, resuspended. After centrifuging for 30min, supernatants 1977a). Differently from K, the Mg and Ca distribuwere assayed for radioactivity by a liquid scintillation spection across the midgut could be justified by the p.d.; trometer (Tri-Carb Packard 3003 series), and for Na, K, Mg, Ca and C1. For Na, K, Mg and CI determinations, on the other hand, an active transport of both Mg supernatants were previously diluted 1:1 with HCIO4 and Ca from the lumen to the haemolymph have been 0.6 N and centrifuged. The sediments were dried overnight found in Hyalophora cecropia (Wood et al., 1975; and weighed to have dry tissue weight and total tissue W o o d and Harvey, 1976). The low concentration of water. Total ECS was calculated, and the intracellular con- CI in the haemolymph of these iarvae confirms that centrations then calculated by the following formula: cations are neutralized in this order mainly by organic acids and free aminoacids (Florkin & c,v, - c,v, ci Jeuniaux, 1975). In Table 1, the ionic concentrations v, - v, determined in the fresh tissue are also reported. The where: Ci is intracellular concentration (mequiv/I), C~ is tissue K concentration is in Philosamia and, to a concentration in tissue water (mequiv/i), C, is concen- smaller extent, in Bombyx, higher than those pretration in salines (mequiv/l), Vf is volume tissue water (ml), viously reported (Giordana & Sacchi, 1977a): in fact, V~ is volume of ECS (ml). in these experiments, to avoid K dilution in the In order to calculate the intracellular concentrations in luminal extracellular space, the tissues were not rinsed the midguts immediately after excision, experiments were in physiological saline before blotting with filter performed on midguts of Philosamia to measure separately the luminal and the haemolymphatic ECS: midguts were paper. In Bombyx mori, K tissue determinations were incubated for 10 min, adding labelled sulphate 2 #Ci/ml in also performed rinsing rapidly (about 15 sec) the midthe saline bathing either the luminal or the haemolympha- guts in physiological saline: under these conditions, tic side of the tissue. the tissue K concentration was 121.5 + 5.2mequiv/l tissue water (five experiments), i.e. significantly lower than in those not washed. K midgut values reported RESULTS AND DISCUSSION in Table 1 are considerably higher than those referred Ionic distribution in intestinal content, intestinal tissue by Harvey et al. (1975) for the midgut of Hyalophora and haemolymph cecropia; these authors washed the tissues three times The results reported in Table 1 confirm the presin cold sucrose solution. Their values are in good ence in the mature larvae of Philosamia cynthia and agreement with the K concentrations in the rinsed Table 1. Ionic concentrations in the lumen content, fresh tissue and haemolymph of Philosamia cynthia and Bombyx mori Ion K Na
Philosaraia cynthia
Mg Ca CI K Na
Bombyx mori
Mg Ca
Cl
Lumen content (mequiv/l) 196.8 _+ 7.I (6) 1.0 + 0.2 (6) 8.6 ± 0.4 (5) 11.0 + 1.0 (5) 14.7 + 1.5 (4) 149.5 + 2.9 (9) 1.3 + 0.1 (9) 29.4 ___3.4 (4) 19.6 _+ 2.0 (4) 17.5 ___ 1.0 (4)
Mean -4- &E--number of experiments in parentheses.
Intestinal tissue (mequiv/l tiss. water) 174.1 _ (6) 3.0 + (6) 35.6 + (6) 11.6 + (6) --
3.8 0.4 2.0 1.0
142.0 + 1.0 (4) 2.2 + 0.2 (6) 39.6 __ 1.4 (6) 24.2 + 2.8 (4) 30.6 + 1.7 (7)
Haemolymph (mequiv/l) 23.8 + 0.4 (6) 4.6 + 0.8 (6) 74.6 _+ 2.0 (8) 25.2 + 1.8 (4) 6.3 ___0.7 (4) 46.2 + 0.9 (6) 1.7 ± 0.1 (6) 89.0 ± 2.4 (4) 43.4 + 1.0 (9) 19.5 + 1.2 (4)
Cellular ionic concentrations in the midgut of two larvae
19
Table 2. Total, luminal and haemolymphatic extraceiiu|ar space (ECS) values determined in Philosamia cynthia and Bombyx mori isolated midguts by means of Sulphate and sucrose
Philosamia cynthia
ECS
Sulphate
Sucrose
Total
40.2 + 1,0 (11) 10.9 4- 3.0
42,8 4- 1,8 (8)
Luminal
(4) Haemolymphatic
Bombyx mori
Total
29.4 _ 2.1 (4) 45.7 _+ 1.5 (14)
44.3 ± 0.8 (13)
ECS expressed as percentage of tissue water. Means ± S.E.--number of experiments in parentheses. midguts of Philosamia cynthia (107.2 _ 3.0 mequivfl tissue water). In order to have the exact intracellular ionic concentrations, tissue values must be corrected for the ion concentrations in the extracellular luminal and haemolymphatic volumes.
Extracellular space determinations The ECS has been measured in incubated midguts by means of two different markers: sulphate asS and sucrose ~4C. These molecules reach the total extracellular volume in different times but give a same final ECS (Table 2). An ample discussion of these ECS determinations has been reported elsewhere (Giordana & Sacchi, 1977b). Luminal and haemolymphatic ECS determined with sulphate in Philosamia are also reported in Table 2. The luminal to haemolymphatic ECS ratio is 0.37 and a similar ratio has been found for Bombyx. Harvey & Wood (1973) reported for cecropia midgut a ratio equal to 4.7: it should be considered that a different technique had been used
by these authors, who also determined a very small total ECS.
Intracellular ionic concentrations Table 3 reports the ionic intracellular concentrations in the midgut of both larvae. Tissue values have been corrected for total ECS value determined experimentally in incubated tissues, while in fresh tissues corrections have been made using the mean values of luminal and haemolymphatic ECS. In this case, it has been assumed that the ECS in vivo is not different from that determined in vitro, since in vivo determinations have not yet been carried out. In these midguts it is impossible to scrape mucosal cells, so that it should be noted that ion intracellular concentrations include not only epithelial cells but also muscle cells and tracheae: any way, columnar and goblet cells are the major fraction of the tissue (Anderson & Harvey, 1966; histological preparations of Bombyx ~ r i midgut made in this laboratory and observed by light microscopy confirm these findings).
Table 3. lntracellular ionic concentrations in the midgut of Philosamia cynthia and Bombyx mori determined in fresh tissue immediately after excision and in incubated tissues
Ion K Na
Philosamia cynthia
Mg Ca CI f K Na
Bombyx mori
Mg Ca CI
Experimental period Incubation Incubation Incubation Fresh tissue 10 rain 60 rain 120 min 242.5 ± (6) 2.6 ± (6) 23.8 ± (6) 5.6 ± (6) --
6.5 0.5 4.0 1.8
197.5 ±_ 1.8 (4) ,2.9 ± 0.4 (6) 9.0 ± 2.2 (6) 13.4 ± 5.2 (4) 41.2 ± 2.7 (7)
188.3 _ 9.1 (4) 3.7 ± 0.2 (4) 12.0 4- 3.2 (4) 7.2 (2) 33.9 ± 1.9 (4) 185.3 ± 11.0 (11) 14.2 ± 3.0 (6) 15.6 + 3.8 (6) 16.2 (2) 41.9 ± 5.3 (4)
190.3 4- 8.4 163.1 ± 4.9 (4) (4) 5.9 4- 1.6 4.1 ± 0.7 (4) (4) 10.0 4- 3.8 14.6 + 2.0 (4) (5) 4.2 4- 1.2 5.2 (4) (2) "39.2 4- 5.1 41.5 ± 4.6 (4) (4) 180.9 4- 6.8 141.1 4- 8.4 (8) (9) 9.8 ± 1.5 14.9 ± 1.5 (6) (4) 14.8 4- 3.8 -(8) 18.4 ± 1.8 23.6 ± 6.6 (8) (4) 42.8 _ 3.4 34.6 + 2.3 (4) (5)
Values expressed as mequiv/l cell water. Mean ± S.E.--number of experiments in parentheses.
B. GIORDANA AND F. SACCHI
20 I-IK
Philosamia cyntio m
[] Mg
Bombyx mori a Lumen content
Potassium and magnesium concentrations in the lumen content, midgut cells and the haemolymph of both larvae are summarized in Fig. 1.
b Cells
Acknowledgements--We are indebted to Professor V. Capraro for his helpful criticism and advice.
c Haemotymph
200
I i ,
REFERENCES
.>
~: IOC
iiiii:!:~:i:!: a
b
c
a
b
c
Fig. 1. K and Mg distribution in the lumen content, intestinal cells and haemolymph of Philosamia cynthia and Bombyx mori. In both animals, intestinal cells are poor not only in Na but also in divalent cations, while K concentration is, expecially in Philosamia, extremely high, higher than those found in the epithelial cells of vertebrates (Esposito et al., 1973; Cremaschi & Henin, 1975), and also than that reported by Zerahn (1975) for the incubated midgut of Hyalophora. This author found a total ECS value very similar to those reported in this paper, and referred to a K concentration of about 140 mequiv/I cell water. The potassium concentration alone would give an intracellular osmolarity higher than the haemolymphatic one: on the other hand, K ions are not only presumably in part osmotically inactive, but they can also be neutralized by polyvalent anions. In fact, cellular chloride is quite low. Na, Mg, Ca and C1 cellular concentrations do not vary appreciably even after 2 hr of incubation (Table 3). The cells lose a certain amount of K in the first minutes of incubation, reaching a concentration which remains constant for 1 hr. It should be emphasized that, in the experimental conditions considered, the luminal K concentration is equal to the haemolymphatic one and much lower than that of lumen content in vivo (Table 1). The slight influence on cellular K concentration of the gradient between the cell and the lumen in vitro can be explained by a low passive permeability to K of the luminal membrane (Harvey & Wood, 1973). The transepithelial potential difference also is, in Philosamia, scarcely influenced by luminal K absence (Giordana & Sacchi, 1977a).
ANDERSON E. & HARVEY W. R. (1966) Active transport by the cecropia midgut. II. Fine structure of the midgut epithelium. J. Cell Biol. 31, 107-134. CREMASCHID. & HENINS. (1975) Extracellular space determination in gallbladder mucosa. Biochim. biophys. Acta 411, 291-294. EsPOSITO G., FAELLIA. & CAPRAROV. (1973) Sugar and electrolyte absorption in the rat intestine perfused in vivo. Pfliigers Arch. ges. Physiol. 340, 335-348. FLORKI~M. & JEUNIAUXC. (1975) Haemolymph composition. In The Physiology of Insecta (Edited by ROCKSTEIN M.), p. 255. Academic Press, New York. GIORDA~AB. & SACCm F. (1976) Transepithelial electrical parameters in the midgut of three different larvae of Lepidoptera. Rc. Acc. naz. Lincei 60, 151-155. GIOROANAB. & SACCHIF. (1977a) Some ionic and electrical parameters of the intestinal epithelium in three mature larvae of Lepidoptera. Comp. Biochem. Physiol. 56A, 95-99. GIORDANA B. & SACCHI F. (1977b) Extracellular space values and intracellular ionic concentrations in the isolated midgut of Philosamia cynthia and Bombyx mori. Experientia In press. HARVEYW. R. & NEDERGAARDS. (1964) Sodium independent active transport of potassium in the cecropia silkworm. Proc. natn. Acad. Sei. U.S.A. 51, 757-762. HARVEY W. R. & WOOD J. L. (1973) The route of cation transport across the silkworm midgut. In Transport Mechanisms in Epithelia (Edited USSINGH. H. & THORN N. A.), p. 342. Munskgaard, Copenhagen. HARVEY W. R., HASKELLJ. A. & NEDERGAARDS. (1968) Active transport by the cecropia midgut. III Midgut potential generated directly by active K transport. J. exp. Biol. 48, 1-12. HARVEY W. R., WOOD J. L., QUATRALER. P. & JUNGREIS A. M. (1975) Cation distributions across the larval and pupal midgut of the Lepidopteran, Hyalophora cecropia, in vivo. J. exp. Biol. 63, 321-330. WOOD J. L. & HARVEYW. R. (1976) Active transport of calcium alcross the isolated midgut of Hyalophora cecropia. J. exp. Biol. 65, 347-360. WOODJ. L., JUNGREISA. M. & HARVEYW. R. (1975) Active transport of magnesium across the isolated migut of Hyalophora cecropia. J. exp. Biol. 63, 313-320. ZERAHN K. (1975) Potassium exchange between bathing solution and midgut of Hyalophora cecropia and time delay for potassium flux through the midgut. J. exp. Biol. 63, 295-300.
CELLULAR IONIC CONCENTRATIONS IN THE MIDGUT OF TWO LARVAE OF LEPIDOPTERA I N V I V O and I N V I T R O B. GIORDANA AND F. SACCH] Istituto di Fisiologia Generale e Chimica Biologica dell'Universit/l di Milano, Italy
(Received 24 March 1977) Abstract--1. The concentrations of Na, K, Mg, Ca and C1 across the midgut of Philosamia cynthia and Bombyx mori are reported. Intracellular 4ntestinal concentrations of the same ions both in vivo and in vitro have also been calculated. 2. The haemolymph of both larvae shows a (Na + K)/(Mg + Ca) ratio lower than 0.4; CI concentration is very low. K concentration in the lumen content ranges between 150 and 199 mequivA, while Ca and Mg concentrations are lower than those in the haemolymph. 3. Na, Mg, Ca and CI intracellular concentrations in vivo are low, while K is very high (over 190 mequiv/l cell water). Ion variations in the incubated tissue have been studied.
and 1:39 for Mg determinations. For Na, K, Mg and CI determinations the solutions were diluted 1 : 1 with HCIO4 0.6 N and centrifuged for 5 min to obtain the sedimentation of proteins. Na and K were then assayed by means of a flame photometer (Beckman DU-2). Mg and Ca were assayed colorimetrically after preparation of samples following Magnesium Merckotest (Art. no. 3338) and Calcium Kit (Clinton) indications. Samples were then read by C.E. 343 Single Sample Spectrometer, Cecil Instrument Ltd, Cambridge. Chlorides were assayed by means of mercurimetric tritration according to Chloride Merckotest (Art. no. 3311). To perform ionic determinations in the lumen content, the larvae were cut lengthwise, the midgut was perforated and the intestinal content collected into a test tube and centrifuged for 5 min to obtain a solution free from leaf fragments. The supernatant was then diluted 1:29 with bidistilled water for K and Mg determinations. Determinations were then performed as described for the haemolymph. The intestinal tissue was removed from the larvae as described elsewhere (Giordana and Sacchi, 1977a), lightly blotted with filter paper (Whatman no. 587 E), shattered, placed in a tared tube and weighed. One ml of bidistilled water was added, the suspension thoroughly mixed, frozen, thawed, resuspended and centrifuged for 30min. The supernatant was diluted 1 : 1 with HC104 0.6 N and centrifuged for 5 min for Na, K, Mg and C1 determinations; ions were then assayed as described. Sediments were dried overnight and weighed to obtain total tissue water. Experiments were also performed on incubated midguts, in order to measure extracellular space values and ionic intracellular concentrations. Salines were prepared with an ionic composition as determined in the haemolymph:
INTRODUCTION In a previous paper ( G i o r d a n a & Sacchi, 1977a), the presence of a very low N a concentration (less t h a n 5 mequiv/l) and a relatively high K concentration has been pointed out in haemolymph, intestinal tissue and lumen content o f Philosamia cynthia and Bombyx mori. These larvae belong to the order of Lepidoptera, which is also characterized by a high h a e m o l y m p h a t i c concentration of divalent cations (Florkin & Jeuniaux, 1975). In the midgut of these larvae (Harvey & Nedergaard, 1964; G i o r d a n a & Sacchi, 1977a), a very large transepithelial potential difference (p.d.) with the positive pole in the lumen, due to an active and electrogenic extrusion of K ions, has been observed. The values of these p.d.s, range between 110 and 1 3 0 m V in vivo and between 70 and 8 5 m V in vitro. While cation distribution across the midgut of Hyalophora cecropia in vivo has been well studied (Harvey et al., 1975), very little is k n o w n a b o u t ionic pattern in haemolymph, intestinal tissue and content of Philosamia cynthia and Bombyx mori. Furthermore, i m p o r t a n t suggestions can be supplied by the exact valuation of the intracellular ionic concentration of the intestinal epithelium, which separates two comp a r t m e n t s - - h a e m o l y m p h and l u m e n - - w i t h very different ionic concentrations. In this work, the concentrations of sodium, potassium, magnesium, calcium and chloride across the midgut of Philosamia cynthia and Bombyx mori, and the intracellular intestinal concentrations of the same ions b o t h in vivo and in vitro are reported.
MATERIALS
AND
METHODS
Philosamia Bombyx
4 25 - 74 18 156 mequiv/l 1.7 25 21 88 18 110 mequiv/l Sucrose was added to both solutions to reach the osmolarity as determined in the haemolymph (Philosamia: 284.6 + 9.3 mOsm/l, three experiments; Bombyx: 290 + 2.9 mOsm/l, three experiments). These salines were aerated and stirred by bubbling with 95% 0 2 and 5~o CO2, pH 7.4. The midgut was excised from the larvae, the peritrophic membrane and the enclosed intestinal content gently
The experiments were performed during June, July and September on mature larvae of Philosamia cynthia reared on Ailanthus #landulosa leaves, and of Bombyx mori fed on Morus alba leaves. In order to measure cations and CI concentrations in the haemolymph, the larvae were bled by cutting a proleg and the haemolymph was collected in a test tube, diluted with bidistilled water 1:9 for Na, K and Ca determinations 17 { BI' St} IA
I~
18
B. G[ORDANAAND F. SACCHI
removed and the tissue mounted as a tube on the perfusion Bombyx mori not only of a steep K gradient between apparatus described in a previous paper (Giordana & the lumen and the haemolymph, but also of a relevant Sacchi, 1976). The tissues were incubated for different Mg gradient with the higher concentration in the haeperiods (10, 60 and 120rain). In order to measure total molymph. The~ potassium distribution is in these larextracellular space (ECS), the salin~-ix~r~sing both sides vae opposite to the high, lumen positive, transepiof the epithelium contained sucrose ~4C or sulphate 3sS 1 #Ci/ml (from Radioehemical Center, Amersham). Sul- thelial p.d., and consistent with an active transport phate was used for short incubations (up to 40 min) and of this cation from the haemolymph to the lumen, as sucrose for longer ones (60 and 120 rain). After the incuba-. has been well demonstrated in the isolated midgut tion, the midguts were removed, blotted with filter paper 6f~H~alophora cecropia (Harvey et al., 1968). In fact, (Whatman 587 E), shattered, placed in a tared tube and the lumen positive p.d. in these two larvae depends rapidly weighed. One ml of bidistilled water was added in vitro entirely on the availability of K in the haemoand the suspensions thoroughly mixed, frozen, thawed and lymphatic side of the epithelium (Giordana & Sacchi, resuspended. After centrifuging for 30min, supernatants 1977a). Differently from K, the Mg and Ca distribuwere assayed for radioactivity by a liquid scintillation spection across the midgut could be justified by the p.d.; trometer (Tri-Carb Packard 3003 series), and for Na, K, Mg, Ca and C1. For Na, K, Mg and CI determinations, on the other hand, an active transport of both Mg supernatants were previously diluted 1:1 with HCIO4 and Ca from the lumen to the haemolymph have been 0.6 N and centrifuged. The sediments were dried overnight found in Hyalophora cecropia (Wood et al., 1975; and weighed to have dry tissue weight and total tissue W o o d and Harvey, 1976). The low concentration of water. Total ECS was calculated, and the intracellular con- CI in the haemolymph of these iarvae confirms that centrations then calculated by the following formula: cations are neutralized in this order mainly by organic acids and free aminoacids (Florkin & c,v, - c,v, ci Jeuniaux, 1975). In Table 1, the ionic concentrations v, - v, determined in the fresh tissue are also reported. The where: Ci is intracellular concentration (mequiv/I), C~ is tissue K concentration is in Philosamia and, to a concentration in tissue water (mequiv/i), C, is concen- smaller extent, in Bombyx, higher than those pretration in salines (mequiv/l), Vf is volume tissue water (ml), viously reported (Giordana & Sacchi, 1977a): in fact, V~ is volume of ECS (ml). in these experiments, to avoid K dilution in the In order to calculate the intracellular concentrations in luminal extracellular space, the tissues were not rinsed the midguts immediately after excision, experiments were in physiological saline before blotting with filter performed on midguts of Philosamia to measure separately the luminal and the haemolymphatic ECS: midguts were paper. In Bombyx mori, K tissue determinations were incubated for 10 min, adding labelled sulphate 2 #Ci/ml in also performed rinsing rapidly (about 15 sec) the midthe saline bathing either the luminal or the haemolympha- guts in physiological saline: under these conditions, tic side of the tissue. the tissue K concentration was 121.5 + 5.2mequiv/l tissue water (five experiments), i.e. significantly lower than in those not washed. K midgut values reported RESULTS AND DISCUSSION in Table 1 are considerably higher than those referred Ionic distribution in intestinal content, intestinal tissue by Harvey et al. (1975) for the midgut of Hyalophora and haemolymph cecropia; these authors washed the tissues three times The results reported in Table 1 confirm the presin cold sucrose solution. Their values are in good ence in the mature larvae of Philosamia cynthia and agreement with the K concentrations in the rinsed Table 1. Ionic concentrations in the lumen content, fresh tissue and haemolymph of Philosamia cynthia and Bombyx mori Ion K Na
Philosaraia cynthia
Mg Ca CI K Na
Bombyx mori
Mg Ca
Cl
Lumen content (mequiv/l) 196.8 _+ 7.I (6) 1.0 + 0.2 (6) 8.6 ± 0.4 (5) 11.0 + 1.0 (5) 14.7 + 1.5 (4) 149.5 + 2.9 (9) 1.3 + 0.1 (9) 29.4 ___3.4 (4) 19.6 _+ 2.0 (4) 17.5 ___ 1.0 (4)
Mean -4- &E--number of experiments in parentheses.
Intestinal tissue (mequiv/l tiss. water) 174.1 _ (6) 3.0 + (6) 35.6 + (6) 11.6 + (6) --
3.8 0.4 2.0 1.0
142.0 + 1.0 (4) 2.2 + 0.2 (6) 39.6 __ 1.4 (6) 24.2 + 2.8 (4) 30.6 + 1.7 (7)
Haemolymph (mequiv/l) 23.8 + 0.4 (6) 4.6 + 0.8 (6) 74.6 _+ 2.0 (8) 25.2 + 1.8 (4) 6.3 ___0.7 (4) 46.2 + 0.9 (6) 1.7 ± 0.1 (6) 89.0 ± 2.4 (4) 43.4 + 1.0 (9) 19.5 + 1.2 (4)
Cellular ionic concentrations in the midgut of two larvae
19
Table 2. Total, luminal and haemolymphatic extraceiiu|ar space (ECS) values determined in Philosamia cynthia and Bombyx mori isolated midguts by means of Sulphate and sucrose
Philosamia cynthia
ECS
Sulphate
Sucrose
Total
40.2 + 1,0 (11) 10.9 4- 3.0
42,8 4- 1,8 (8)
Luminal
(4) Haemolymphatic
Bombyx mori
Total
29.4 _ 2.1 (4) 45.7 _+ 1.5 (14)
44.3 ± 0.8 (13)
ECS expressed as percentage of tissue water. Means ± S.E.--number of experiments in parentheses. midguts of Philosamia cynthia (107.2 _ 3.0 mequivfl tissue water). In order to have the exact intracellular ionic concentrations, tissue values must be corrected for the ion concentrations in the extracellular luminal and haemolymphatic volumes.
Extracellular space determinations The ECS has been measured in incubated midguts by means of two different markers: sulphate asS and sucrose ~4C. These molecules reach the total extracellular volume in different times but give a same final ECS (Table 2). An ample discussion of these ECS determinations has been reported elsewhere (Giordana & Sacchi, 1977b). Luminal and haemolymphatic ECS determined with sulphate in Philosamia are also reported in Table 2. The luminal to haemolymphatic ECS ratio is 0.37 and a similar ratio has been found for Bombyx. Harvey & Wood (1973) reported for cecropia midgut a ratio equal to 4.7: it should be considered that a different technique had been used
by these authors, who also determined a very small total ECS.
Intracellular ionic concentrations Table 3 reports the ionic intracellular concentrations in the midgut of both larvae. Tissue values have been corrected for total ECS value determined experimentally in incubated tissues, while in fresh tissues corrections have been made using the mean values of luminal and haemolymphatic ECS. In this case, it has been assumed that the ECS in vivo is not different from that determined in vitro, since in vivo determinations have not yet been carried out. In these midguts it is impossible to scrape mucosal cells, so that it should be noted that ion intracellular concentrations include not only epithelial cells but also muscle cells and tracheae: any way, columnar and goblet cells are the major fraction of the tissue (Anderson & Harvey, 1966; histological preparations of Bombyx ~ r i midgut made in this laboratory and observed by light microscopy confirm these findings).
Table 3. lntracellular ionic concentrations in the midgut of Philosamia cynthia and Bombyx mori determined in fresh tissue immediately after excision and in incubated tissues
Ion K Na
Philosamia cynthia
Mg Ca CI f K Na
Bombyx mori
Mg Ca CI
Experimental period Incubation Incubation Incubation Fresh tissue 10 rain 60 rain 120 min 242.5 ± (6) 2.6 ± (6) 23.8 ± (6) 5.6 ± (6) --
6.5 0.5 4.0 1.8
197.5 ±_ 1.8 (4) ,2.9 ± 0.4 (6) 9.0 ± 2.2 (6) 13.4 ± 5.2 (4) 41.2 ± 2.7 (7)
188.3 _ 9.1 (4) 3.7 ± 0.2 (4) 12.0 4- 3.2 (4) 7.2 (2) 33.9 ± 1.9 (4) 185.3 ± 11.0 (11) 14.2 ± 3.0 (6) 15.6 + 3.8 (6) 16.2 (2) 41.9 ± 5.3 (4)
190.3 4- 8.4 163.1 ± 4.9 (4) (4) 5.9 4- 1.6 4.1 ± 0.7 (4) (4) 10.0 4- 3.8 14.6 + 2.0 (4) (5) 4.2 4- 1.2 5.2 (4) (2) "39.2 4- 5.1 41.5 ± 4.6 (4) (4) 180.9 4- 6.8 141.1 4- 8.4 (8) (9) 9.8 ± 1.5 14.9 ± 1.5 (6) (4) 14.8 4- 3.8 -(8) 18.4 ± 1.8 23.6 ± 6.6 (8) (4) 42.8 _ 3.4 34.6 + 2.3 (4) (5)
Values expressed as mequiv/l cell water. Mean ± S.E.--number of experiments in parentheses.
B. GIORDANA AND F. SACCHI
20 I-IK
Philosamia cyntio m
[] Mg
Bombyx mori a Lumen content
Potassium and magnesium concentrations in the lumen content, midgut cells and the haemolymph of both larvae are summarized in Fig. 1.
b Cells
Acknowledgements--We are indebted to Professor V. Capraro for his helpful criticism and advice.
c Haemotymph
200
I i ,
REFERENCES
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Fig. 1. K and Mg distribution in the lumen content, intestinal cells and haemolymph of Philosamia cynthia and Bombyx mori. In both animals, intestinal cells are poor not only in Na but also in divalent cations, while K concentration is, expecially in Philosamia, extremely high, higher than those found in the epithelial cells of vertebrates (Esposito et al., 1973; Cremaschi & Henin, 1975), and also than that reported by Zerahn (1975) for the incubated midgut of Hyalophora. This author found a total ECS value very similar to those reported in this paper, and referred to a K concentration of about 140 mequiv/I cell water. The potassium concentration alone would give an intracellular osmolarity higher than the haemolymphatic one: on the other hand, K ions are not only presumably in part osmotically inactive, but they can also be neutralized by polyvalent anions. In fact, cellular chloride is quite low. Na, Mg, Ca and C1 cellular concentrations do not vary appreciably even after 2 hr of incubation (Table 3). The cells lose a certain amount of K in the first minutes of incubation, reaching a concentration which remains constant for 1 hr. It should be emphasized that, in the experimental conditions considered, the luminal K concentration is equal to the haemolymphatic one and much lower than that of lumen content in vivo (Table 1). The slight influence on cellular K concentration of the gradient between the cell and the lumen in vitro can be explained by a low passive permeability to K of the luminal membrane (Harvey & Wood, 1973). The transepithelial potential difference also is, in Philosamia, scarcely influenced by luminal K absence (Giordana & Sacchi, 1977a).
ANDERSON E. & HARVEY W. R. (1966) Active transport by the cecropia midgut. II. Fine structure of the midgut epithelium. J. Cell Biol. 31, 107-134. CREMASCHID. & HENINS. (1975) Extracellular space determination in gallbladder mucosa. Biochim. biophys. Acta 411, 291-294. EsPOSITO G., FAELLIA. & CAPRAROV. (1973) Sugar and electrolyte absorption in the rat intestine perfused in vivo. Pfliigers Arch. ges. Physiol. 340, 335-348. FLORKI~M. & JEUNIAUXC. (1975) Haemolymph composition. In The Physiology of Insecta (Edited by ROCKSTEIN M.), p. 255. Academic Press, New York. GIORDA~AB. & SACCm F. (1976) Transepithelial electrical parameters in the midgut of three different larvae of Lepidoptera. Rc. Acc. naz. Lincei 60, 151-155. GIOROANAB. & SACCHIF. (1977a) Some ionic and electrical parameters of the intestinal epithelium in three mature larvae of Lepidoptera. Comp. Biochem. Physiol. 56A, 95-99. GIORDANA B. & SACCHI F. (1977b) Extracellular space values and intracellular ionic concentrations in the isolated midgut of Philosamia cynthia and Bombyx mori. Experientia In press. HARVEYW. R. & NEDERGAARDS. (1964) Sodium independent active transport of potassium in the cecropia silkworm. Proc. natn. Acad. Sei. U.S.A. 51, 757-762. HARVEY W. R. & WOOD J. L. (1973) The route of cation transport across the silkworm midgut. In Transport Mechanisms in Epithelia (Edited USSINGH. H. & THORN N. A.), p. 342. Munskgaard, Copenhagen. HARVEY W. R., HASKELLJ. A. & NEDERGAARDS. (1968) Active transport by the cecropia midgut. III Midgut potential generated directly by active K transport. J. exp. Biol. 48, 1-12. HARVEY W. R., WOOD J. L., QUATRALER. P. & JUNGREIS A. M. (1975) Cation distributions across the larval and pupal midgut of the Lepidopteran, Hyalophora cecropia, in vivo. J. exp. Biol. 63, 321-330. WOOD J. L. & HARVEYW. R. (1976) Active transport of calcium alcross the isolated midgut of Hyalophora cecropia. J. exp. Biol. 65, 347-360. WOODJ. L., JUNGREISA. M. & HARVEYW. R. (1975) Active transport of magnesium across the isolated migut of Hyalophora cecropia. J. exp. Biol. 63, 313-320. ZERAHN K. (1975) Potassium exchange between bathing solution and midgut of Hyalophora cecropia and time delay for potassium flux through the midgut. J. exp. Biol. 63, 295-300.