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Movement of calcium and magnesium across the midgut epithelium of the American cockroach
J. Insect Physiol., 1969, Vol. 15, pp. 789 to 797. Pergamon Press. Printed in Great Britain
:MOVEMENT OF CALCIUM AND MAGNESIUM ACROSS THE MIDGUT EPITHELIUM OF THE AMERICAN COCKROACH JOHN
R. SAUER and RICHARD
R. MILLS
Department of Biology, Tulane University, New Orleans, La. 70118 (Received 22 November 1968)
Abstract-Atomic absorption spectrophotometry has been used to study net movements of calcium and magnesium across the midgut epithelia of the American cockroach. Movements of both ions are affected by the concentration of midgut lumen contents. Generally, influx movements of both ions are dec.reased with increasing lumen osmolality. DNP (2,4_dinitrophenol, 0.001 M) tends to increase the influx of calcium while the inverse is true for magnesium. Only minimal quantities of dinitrophenol are absorbed into the epithelial cells, and only a small fraction of this completely crosses the epithelial cells during the course of the 2 hr experiment. These data and an indication that dinitrophenol placement (lumen or blood side) is correlated with resultant changes in ion movements suggest that the primary effect of dinitrophenol is on or near the epithelial membrane. INTRODUCTION
ALL INSECTS,including those living in terrestrial environments, are faced with the problem of maintaining an internal environment radically different from the surrounding media. The proportions of various haemolymph constituents must be in proper balance to allow the animal to survive and function normally. The insect if; equipped with several systems capable of abetting this problem. For example, an enveloping waxy cuticle imposes a permeability barrier to water evaporation while actively absorbing water from the atmosphere (BEAMENT,1964). The Malpighian tubules and rectum also contribute to the regulation process. This is accomplished by haemolymph fluid being secreted (with but few changes) into the Malpighian tubules (BERRIDGE,1968) and allowed to pass to the rectum for selective reabsorption of ions and water (PHILLIPS, 1964a, b). Substantial evidence has accumulated correlating these functions with overall salt and water balance [reviewed by BARTON-BROWNE(1964); and STOBBARTand SHAW (1964)]. By contrast, little is known about whether other parts of the continuous alimentary tract can perform key r8les in regulating salt and water movements. This is true for the midgut even though many solutes probably enter the haemolymph via this organ (STOBBARTand SHAW, 1964; SAUERet al., 1969). Any selectivity or control over such movements would necessarily imply an important function for the organ in overall maintenance of homeostasis. It is generally conceded 789
790
JOHN R. SAUERAND RICHARD R. MILLS
that two of the most important divalent cations in living organisms are calcium and magnesium. Consistent with this, the present investigation was designed to elucidate net permeability properties of the midgut epithelium to calcium and The results indicate that the organ exhibits varying and differential magnesium. permeability to both ions depending upon the nature of the lumen contents. In addition, each ion is characteristically sensitive to 2,4-dinitrophenol. MATERIALS
AND
METHODS
All studies utilized in vitro midgut preparations dissected from adult male Periplaneta americana (L.). Cockroaches were reared in wire mesh cages and fed Purina dog chow and water ad libitum. Rearing was done at room temperature (275 SC) and daily photoperiod conditions. The midgut was dissected and cannulated as previously described (SAUER et al., 1969). Individual net ion concentration changes were determined by using a Beckman 1301 atomic absorption Ten ~1 samples from the in vitro preparations and controls spectrophotometer. were appropriately diluted with deionized water for linear concentration reading on the instrument. Ion movements were studied over ranges of lumen salt concentration. The effects of dinitrophenol and the changes in osmolarlity and lumen volume were determined by former methods (SAUER et al., 1969). This experimental design is summarized as follows: Saline containing the ions to be studied plus others were placed in the cleansed lumen and on the blood side of the midgut. The initial saline concentrations of calcium and magnesium were kept constant throughout all experiments. Four different amounts of sodium chloride were added to the lumen in separate experiments to vary the osmotic concentration. The initial concentrations of the midgut lumen contents corresponding to the saline plus additional amounts of sodium chloride were 400, 475, 575, or 700 m-osmols. DNP (2,4_dinitrophenol, 0.001 M) was added to either the lumen, blood side, or both sides of the in vitro midgut preparation. All experiments were allowed to proceed for 2 hr at 25°C. W-dinitrophenol was obtained from New England Nuclear, Boston, Mass. Individual isotopic samples were counted with a Beckman LS 200 B liquid scintillation counter and the scintillation cocktail was that of WHARTON et al. (1965). RESULTS
D$erential
absorption of calcium and magnesium by the midgut epithelium
Prior research in this laboratory has established that lumen saline contents decrease in concentration during the course of experiments when the lumen is iso- or hyperosmotic to the blood side bathing solution (SAUER et al., 1969). At the same time, net water movements are minimal precluding any possibility of inward moving solvent diluting the media. One can conclude, therefore, that in these experiments net concentration changes are brought about almost wholly by solute displacements rather than osmosis. Because of this an ideal opportunity was provided to study specific ion movements in the absence of any complicating solvent flow. Taking advantage of this, it was decided to study net divalent
791
CALCIUM AND MAGNESIUM MOVEMENT IN AMERICAN COCKROACH
ion mov’ements from the cockroach midgut. Of particular interest there is selectivity for one or more ions. Table 1 summarizes and compares net midgut permeability to magnesium during the course of isosmotic experiments. More 0.94 m&I/l., was absorbed than calcium, 0.73 mM/l., but since the TABLE I.-ABSORPTION
Ion Calcium Magnesium
No. of animals 13 33
was whether calcium and magnesium, initial saline
OF CALCIUM AND MAGNESIUM FROM THE MIDGUT LUMEN DURING ISOSMOTIC* EXPERIMENTS Initial lumen concentration
Av. net absorption from lumen
Absorption of ion from lumen fluid
(mW1.)
(mM/l.)
(%)
4.5 19.7
* The initial lumen concentration (400 m-osmols) concentration.
0.73 0.94
16.2 4.7
was equal to the initial blood side
ratio of :magnesium to calcium is 5 to 1, higher net midgut permeability to calcium was exhibited. Of the available calcium 16.2 per cent was absorbed as compared to only 4.7 per cent for magnesium (Table 1). On the other hand, as will be shown later, one cannot state generally which of the two is more permeable across the midgut since variations in lumen concentration profoundly affect net fluxes of each ioc.. It is important, however, that under a given set of parameters (lumen concentration and/or nature of ion ratios) selective permeability to one ion over another is possible. Absorption of calcium and magnesium with increasing lumen osmolality Afte:r reviewing the results of these experiments it was decided to test what effect, if’ any, lumen concentration had on relative permeabilities since increasing lumen osmolalities were previously shown to cause greater reductions in total Fig. 1 outlines net absorption of lumen concentrations (SAUER et al., 1969). calcium and magnesium as the lumen osmolalities were increased. Both absolute and relative permeabilities changed with initial lumen concentrations. At 475 and 575 m-osmols, minimal net permeability to calcium was seen but at higher concentrations (700 m-osmols) it increased back to 0.37 mM/l. (Fig. 1A). At 475 and 575 m-osmols, the midgut is relatively more permeable to magnesium than calcium, differing considerably from the values obtained at 400 m-osmols (Fig. 1). For both ions a trend of decreasing net influx with increasing lumen osmolality is apparent. It is strongly indicated that the nature and/or concentration of the lumen contents influence the movement of calcium and magnesium. Moreover, the nature of the influence is not general but is a function of the affected ion. That is to say, net movements of both ions are affected by increasing lumen osmolalities but the effect is not equivalent for a given lumen concentration.
JOHN R. SAUER ANDRICHARDR. MILLS
792
Absorption of calcium and magnesium with increasing lumen osmolality and DNP in the blood side solution Since in previous experiments additions of DNP affected lumen osmolality changes (SAUER et al., 1969) it is desirable to know if such effects also apply to the movements of calcium and magnesium. Both are altered by DNP in the A.
+ 0.4~. 0 b
+0.2-.
s
O -0.2_
I” $ r
:
,, -1:
Magnesium
(IO)
S h
8.
Calcium
(13)
\
-“.4- 0.6.. -0.8-
(11)
/IO)
- 1 -o_. 1
I
r;::1;; ; 400
475 Initial
575 Lumen
; 700 Cont.
In
Milliosmols
FIG. 1. Absorption of calcium and magnesium with increasing lumen milliosmolality. Numbers in parentheses indicate the numbers of animals used. Vertical lines represent 2 x S.E. on either side of the mean.
blood side solution (Fig. 2). DNP increases the influx of calcium at initial lumen concentrations of 400, 475, and 575 m-osmols but not at 700 m-osmols (Fig. 2A). In fact, a net efflux of calcium takes place during these experimental conditions (Fig. 2A). A trend of decreasing calcium and magnesium absorption is again seen with increasing lumen concentration. Net influx movements of magnesium are inhibited by the presence of DNP in the blood side solution (Fig. 2B). It is noteworthy that at 400, 475, and 575 m-osmols, DNP produced opposite effects on the two ions, that is, it enhances net influx of calcium but inhibits the same for magnesium. Absorption of calcium and magnesium with increasing lumen osmolality and DNP in the lumen JEuid Calcium and magnesium movements with DNP in the lumen fluid are outlined in Fig. 3. At 400 and 475 m-osmols calcium changes are little different from those
CALCIUM
793
AND MAGNESIUM MOVEMENT IN AMERICAN COCKROACH
obtained without DNP. However, at 575 and 700 m-osmols the movements closely resemble those achieved with DNP in the blood side solution (Figs. 3A and 1A). Net influx movements of magnesium are inhibited in much the same way as they were when DNP was in the blood side solution (Fig. 3B). At 700 mosmols a net efflux of this ion takes place (Fig. 3B). A. + 0.4
B.
Calcium
Magnesium
4
+ 0.2_ C)
2)
O_ -0.2_ -C&I_ - 0.6_ -0.8_ -1 .o_
;: <
-1.2_ -1.44bo
I 475 initial
I 575 Lumen
I 700 Cont.
I 400
1 475
I 575
I 700
In Milliosmols
FIG. 2. Calcium and magnesium absorption with 10m3 M DNP in the blood side solution ( x ) compared to absorption without DNP ( 0). Both reactions were allowed to proceed with increasing lumen concentrations. Numbers in parentheses indicate the numbers of animals used. Calcium results are statistically difYerent (P-CO-OS) at 475, 57.5, and 700 m-osmols. Magnesium results are significa.ntly different (P-C 0.001) at 400 m-osmols, (P
Absorp;!ion of calcium and magnesium with increasing lumen osmolality and DNP on both sides of the midgut In this series of experiments, calcium movements closely resembled those achieved when DNP was in only the lumen solution (Fig. 4A). However, there was a somewhat greater tendency to increase net calcium influx, especially at 400 and 475 m-osmols (Fig. 4A). At 700 m-osmols only slight differences between these results and those without DNP were observed (Fig. 4A). Net movements of magnesium were reduced (Fig. 4B) and closely resembled those obtained with DNP on the blood side. The generalization appears true again that DNP tends to increasle calcium influx but inhibits the same for magnesium.
JOHN R. SAUER AND RICHARD R. MILLS
794
A.
4b
4f5 h-dial
B.
Calcium
5% Lumen
700 Cont.
4bo
495
Magnesium
575 I
700
In Milliosmols
FIG. 3. Calcium and magnesium absorption with DNP in the lumen ( x ) compared to absorption without DNP (0). Calcium differences are significantly different (P
The preceding information points up several interesting phenomena: (1) The concentration and/or composition of the lumen contents markedly affects absorption of calcium and magnesium. (2) The movements of both ions are DNP sensitive. (3) At all but the highest lumen osmolality, DNP tends to increase net ’ fl ux movements of magnesium are influx movements of calcium. (4) N e t in definitely inhibited by DNP. The idea that fluid concentrations can inversely affect rates of absorption is not new to insects. Water absorption rates in the locust and cockroach recta are apparently thus affected by rectal lumen concentrations (PHILLIPS, 1964a; WALL, 1967). Rates of rectal ion absorption are apparently determined by the ionic concentration of the haemolymph (PHILLIPS, 1964b). Indeed, the concept of osmoregulation is one which rests on the assumption that concentration ultimately It is not surprising, therefore, controls the various regulatory mechanisms. that epithelial permeability in the midgut is affected by osmotic concentrations of the lumen contents. It is difficult to assess, however, the exact mechanisms
CALCIUM ANDMAGNESIUM MOVEMENT IN AMRRICAN COCKROACH
795
involved in such occurrences. One such mechanism might be active transport. With this in mind, DNP was added in the various experiments since this metabolic poison inhibits active potassium transport in the midgut of Cecropia (HASKELL et al., 1965), water transport in the cockroach rectum (WALL, 1967) and the B. Magnesium
A. Calcium + 0.4 ‘0.2 0
“l-l
1
O_.-
s\
-0.2_
3
- 04.
>,
-0.6.
%
$.
- 0.8_
-1.0.. -1.2_
z Q
-14
--
.
lx I & ! (9)
\t
(s)
1
(12)
400
475
575
Initial
Lumen
7bo Cone.
I
4bo
4%
575
7&J
In Milliosmols
FIG. 4. Absorption with DNP on both sides of the midgut ( x) compared to absorption without DNP (0). Calcium differences are significantly different (I’ < 0.05) at 575 m-osmols. Magnesium differences are significant (P< 0.001) at 400 m-osmols, (P< 0.01) at 575 m-osmols, and (P
JOHNR. SALTER ANDRICHARDR. MILLS
796
resemble those seen for calcium thus making this argument untenable (SAUER and MILLS, 1969). It may be that variations in the lumen sodium chloride cause considerable changes in an actively maintained transcellular potential difference across the epithelial cells. This seems credible in the light of what is known about potentials across the midgut of Cecropia (HARVEY and NEDERGAARD,1964). It is interesting that for each of the ions, two DNP experimental conditions yield very similar results. This may suggest that structures on the blood or lumen surface are most important in controlling movements of a particular ion. That is to say, there may be some significance in the fact that calcium movements are alike with DNP in the lumen fluid and with DNP on both sides of the cells (Figs. 3A and 4A). In the same way, magnesium movements are similar when DNP is in the blood side solution and when it is on both sides of the midgut (Figs. 2B and 4B). Tests with 14C-dinitrophenol indicate that less than 10 per cent of the available DNP is absorbed into the cells and very little moves entirely across when applied to only one side (Table 2). This indicates that the highest concentration TABLE2--‘PC-orNrrRoPHENoLABSORPTION ANDMOVEMENT ACROSS THEEPITHELIAL
Exp. W-DNP (inside only) W-DNP (blood side only) 14C-DNP (inside and out) 14C-DNP (outside) W-DNP (lumen)
No. of animals
Experimental sample taken from
Mean % of Mean counts/min 14C-DNP absorbed absorbed
4
Lumen
1515
6.9
4
Blood side
2055
9.7
4
Lumen
1090
4.9
3
Lumen
-
-
3
Blood side
-
-
Mean counts/min appearing in experimental sample
CELLS
Mean % of W-DNP crossing epithelial cells
-
54.5 (background = 40) 276 (background = 40)
<1 5
of DNP remains near the epithelial limiting surfaces, i.e. lumen and/or blood side. It suggests that most of its effects may occur along these surfaces. This concept would concur with the inhibiting effect of DNP on active potassium transport in the midgut of Cecropia, where placement of it on the blood side inhibits much more effectively than with it in the lumen (HASKELL et al., 1965). In summary, it appears the midgut may exert control over divalent ion movements. This concept is supported by data on variable movements as the lumen
CALCIUMAND MAGNESIUM MOVEMENT IN AMERICAN COCKROACH
797
osmolality is changed. Energy processes could be implicated as shown by changes brought about by adding DNP. Since only small amounts of DNP are actually absorbed into the cells and almost none moves entirely across the cells it appears probable that the primary target of DNP is on the epithelial membrane. Acknowledgements-The authors would like to thank C. A. BAYS, H. APPLEBAUM,and M. MICHELSONfor technical assistance and MARGIE M. SAUERfor drawing the figures. One of us (J. R. S.) was the recipient of a NASA predoctoral fellowship. This work was supported in part by grants from NSF (GB-4773, GB-6026, GB-7428), a cell biology training grant (NIH-GM-669) from USPHS NIH and a Sigma Xi grant-in-aid of research to J. R. S. REFERENCES BARTON-BROWNE L. B. (1964) Water regulation in insects. A. Rev. Ent. 9, 63-82. BEAMENTJ. W. L. (1964) The active and passive movement of water in insects. Adv. Insect Physiol. 2, 67-129. BERRIDGEM. J. (1966) Metabolic pathways of isolated Malpighian tubules of the blowfly functioning in an artificial medium. J. Insect Physiol. 12, 1523-1533. BERRIDGEM. J. (1968) Ion and water transport across epithelia. In Insects and Physiology (Ed. by BEAMENTJ. W. L. and TREHERNE J. E. ) pp. 329-347. Elsevier, New York. BIELAWSX.IJ., THOMPSONT. E., and LEHNINGERA. L. (1967) The effect of 2,4-dinitrophencl on the electrical resistance of phospholipid bilayer membranes. In Mitochondriul Structure and Compartmentation (Ed. by QUAGLIARIELLO E., PAPA S., SLATERE. C. and TAGEKJ. M.) pp. 181-184. Adriatica Editrice, Bari. HARVEYIV. R. and NEDERGAAIUI S. (1964) Sodium-independent active transport of potassium in the isolated midgut of the cecropia silkworm (Hyalophora cecropia). Proc. nut. Acad. Sci. LTSA 51, 757-765. HASKELLJ. A., CLEMONSR. D. and HARVEYW. R. (1965) Active transport by the cecropiu midgut-I. Inhibitors, stimulants and potassium transport. r. cell camp. Physiol. 65, 45-56. PHILLIPSJ. E. (1964a) Rectal absorption in the desert locust, Schistocerca gregariu ForskalI. Water. J. exp. Biol., 41, 15-38. PHILLIPSJ. E. (1964b) Rectal absorption in the desert locust, Schistocercagregariu ForskalII. Sodium, potassium and chloride. J. exp. Biol. 41, 39-61. SAUERJ. :R. and MILLS R. R. (1969) Movement of potassium and sodium across the midgut epithelium of the American cockroach. In preparation. SAUERJ. R., SCHLENZ-TRUER., and MILLS R. R. (1969) Salt and water transport by the in vitro cockroach midgut. J. Insect Physiol. 15, 483-493. STOBBARTR. H. and SHAW J. (1964) Salt and water balance: Excretion. In The Physiology of Insecta (Ed. by ROCKSTEINM.) 3, 189-258. Academic Press, New York. WALL B. J. (1967) Evidence for antidiuretic control of rectal water absorption in the cockroach Periplaneta americana (L.). J. Insect Physiol. 13, 565-578. WHARTOND. R. A., WHARTONM. L., and LOLA J. (1965) Blood volume and water content of the American cockroach, Periplaneta americana (L.). Methods and the influence of age ard starvation. J. Insect Physiol. 11,391404.
:MOVEMENT OF CALCIUM AND MAGNESIUM ACROSS THE MIDGUT EPITHELIUM OF THE AMERICAN COCKROACH JOHN
R. SAUER and RICHARD
R. MILLS
Department of Biology, Tulane University, New Orleans, La. 70118 (Received 22 November 1968)
Abstract-Atomic absorption spectrophotometry has been used to study net movements of calcium and magnesium across the midgut epithelia of the American cockroach. Movements of both ions are affected by the concentration of midgut lumen contents. Generally, influx movements of both ions are dec.reased with increasing lumen osmolality. DNP (2,4_dinitrophenol, 0.001 M) tends to increase the influx of calcium while the inverse is true for magnesium. Only minimal quantities of dinitrophenol are absorbed into the epithelial cells, and only a small fraction of this completely crosses the epithelial cells during the course of the 2 hr experiment. These data and an indication that dinitrophenol placement (lumen or blood side) is correlated with resultant changes in ion movements suggest that the primary effect of dinitrophenol is on or near the epithelial membrane. INTRODUCTION
ALL INSECTS,including those living in terrestrial environments, are faced with the problem of maintaining an internal environment radically different from the surrounding media. The proportions of various haemolymph constituents must be in proper balance to allow the animal to survive and function normally. The insect if; equipped with several systems capable of abetting this problem. For example, an enveloping waxy cuticle imposes a permeability barrier to water evaporation while actively absorbing water from the atmosphere (BEAMENT,1964). The Malpighian tubules and rectum also contribute to the regulation process. This is accomplished by haemolymph fluid being secreted (with but few changes) into the Malpighian tubules (BERRIDGE,1968) and allowed to pass to the rectum for selective reabsorption of ions and water (PHILLIPS, 1964a, b). Substantial evidence has accumulated correlating these functions with overall salt and water balance [reviewed by BARTON-BROWNE(1964); and STOBBARTand SHAW (1964)]. By contrast, little is known about whether other parts of the continuous alimentary tract can perform key r8les in regulating salt and water movements. This is true for the midgut even though many solutes probably enter the haemolymph via this organ (STOBBARTand SHAW, 1964; SAUERet al., 1969). Any selectivity or control over such movements would necessarily imply an important function for the organ in overall maintenance of homeostasis. It is generally conceded 789
790
JOHN R. SAUERAND RICHARD R. MILLS
that two of the most important divalent cations in living organisms are calcium and magnesium. Consistent with this, the present investigation was designed to elucidate net permeability properties of the midgut epithelium to calcium and The results indicate that the organ exhibits varying and differential magnesium. permeability to both ions depending upon the nature of the lumen contents. In addition, each ion is characteristically sensitive to 2,4-dinitrophenol. MATERIALS
AND
METHODS
All studies utilized in vitro midgut preparations dissected from adult male Periplaneta americana (L.). Cockroaches were reared in wire mesh cages and fed Purina dog chow and water ad libitum. Rearing was done at room temperature (275 SC) and daily photoperiod conditions. The midgut was dissected and cannulated as previously described (SAUER et al., 1969). Individual net ion concentration changes were determined by using a Beckman 1301 atomic absorption Ten ~1 samples from the in vitro preparations and controls spectrophotometer. were appropriately diluted with deionized water for linear concentration reading on the instrument. Ion movements were studied over ranges of lumen salt concentration. The effects of dinitrophenol and the changes in osmolarlity and lumen volume were determined by former methods (SAUER et al., 1969). This experimental design is summarized as follows: Saline containing the ions to be studied plus others were placed in the cleansed lumen and on the blood side of the midgut. The initial saline concentrations of calcium and magnesium were kept constant throughout all experiments. Four different amounts of sodium chloride were added to the lumen in separate experiments to vary the osmotic concentration. The initial concentrations of the midgut lumen contents corresponding to the saline plus additional amounts of sodium chloride were 400, 475, 575, or 700 m-osmols. DNP (2,4_dinitrophenol, 0.001 M) was added to either the lumen, blood side, or both sides of the in vitro midgut preparation. All experiments were allowed to proceed for 2 hr at 25°C. W-dinitrophenol was obtained from New England Nuclear, Boston, Mass. Individual isotopic samples were counted with a Beckman LS 200 B liquid scintillation counter and the scintillation cocktail was that of WHARTON et al. (1965). RESULTS
D$erential
absorption of calcium and magnesium by the midgut epithelium
Prior research in this laboratory has established that lumen saline contents decrease in concentration during the course of experiments when the lumen is iso- or hyperosmotic to the blood side bathing solution (SAUER et al., 1969). At the same time, net water movements are minimal precluding any possibility of inward moving solvent diluting the media. One can conclude, therefore, that in these experiments net concentration changes are brought about almost wholly by solute displacements rather than osmosis. Because of this an ideal opportunity was provided to study specific ion movements in the absence of any complicating solvent flow. Taking advantage of this, it was decided to study net divalent
791
CALCIUM AND MAGNESIUM MOVEMENT IN AMERICAN COCKROACH
ion mov’ements from the cockroach midgut. Of particular interest there is selectivity for one or more ions. Table 1 summarizes and compares net midgut permeability to magnesium during the course of isosmotic experiments. More 0.94 m&I/l., was absorbed than calcium, 0.73 mM/l., but since the TABLE I.-ABSORPTION
Ion Calcium Magnesium
No. of animals 13 33
was whether calcium and magnesium, initial saline
OF CALCIUM AND MAGNESIUM FROM THE MIDGUT LUMEN DURING ISOSMOTIC* EXPERIMENTS Initial lumen concentration
Av. net absorption from lumen
Absorption of ion from lumen fluid
(mW1.)
(mM/l.)
(%)
4.5 19.7
* The initial lumen concentration (400 m-osmols) concentration.
0.73 0.94
16.2 4.7
was equal to the initial blood side
ratio of :magnesium to calcium is 5 to 1, higher net midgut permeability to calcium was exhibited. Of the available calcium 16.2 per cent was absorbed as compared to only 4.7 per cent for magnesium (Table 1). On the other hand, as will be shown later, one cannot state generally which of the two is more permeable across the midgut since variations in lumen concentration profoundly affect net fluxes of each ioc.. It is important, however, that under a given set of parameters (lumen concentration and/or nature of ion ratios) selective permeability to one ion over another is possible. Absorption of calcium and magnesium with increasing lumen osmolality Afte:r reviewing the results of these experiments it was decided to test what effect, if’ any, lumen concentration had on relative permeabilities since increasing lumen osmolalities were previously shown to cause greater reductions in total Fig. 1 outlines net absorption of lumen concentrations (SAUER et al., 1969). calcium and magnesium as the lumen osmolalities were increased. Both absolute and relative permeabilities changed with initial lumen concentrations. At 475 and 575 m-osmols, minimal net permeability to calcium was seen but at higher concentrations (700 m-osmols) it increased back to 0.37 mM/l. (Fig. 1A). At 475 and 575 m-osmols, the midgut is relatively more permeable to magnesium than calcium, differing considerably from the values obtained at 400 m-osmols (Fig. 1). For both ions a trend of decreasing net influx with increasing lumen osmolality is apparent. It is strongly indicated that the nature and/or concentration of the lumen contents influence the movement of calcium and magnesium. Moreover, the nature of the influence is not general but is a function of the affected ion. That is to say, net movements of both ions are affected by increasing lumen osmolalities but the effect is not equivalent for a given lumen concentration.
JOHN R. SAUER ANDRICHARDR. MILLS
792
Absorption of calcium and magnesium with increasing lumen osmolality and DNP in the blood side solution Since in previous experiments additions of DNP affected lumen osmolality changes (SAUER et al., 1969) it is desirable to know if such effects also apply to the movements of calcium and magnesium. Both are altered by DNP in the A.
+ 0.4~. 0 b
+0.2-.
s
O -0.2_
I” $ r
:
,, -1:
Magnesium
(IO)
S h
8.
Calcium
(13)
\
-“.4- 0.6.. -0.8-
(11)
/IO)
- 1 -o_. 1
I
r;::1;; ; 400
475 Initial
575 Lumen
; 700 Cont.
In
Milliosmols
FIG. 1. Absorption of calcium and magnesium with increasing lumen milliosmolality. Numbers in parentheses indicate the numbers of animals used. Vertical lines represent 2 x S.E. on either side of the mean.
blood side solution (Fig. 2). DNP increases the influx of calcium at initial lumen concentrations of 400, 475, and 575 m-osmols but not at 700 m-osmols (Fig. 2A). In fact, a net efflux of calcium takes place during these experimental conditions (Fig. 2A). A trend of decreasing calcium and magnesium absorption is again seen with increasing lumen concentration. Net influx movements of magnesium are inhibited by the presence of DNP in the blood side solution (Fig. 2B). It is noteworthy that at 400, 475, and 575 m-osmols, DNP produced opposite effects on the two ions, that is, it enhances net influx of calcium but inhibits the same for magnesium. Absorption of calcium and magnesium with increasing lumen osmolality and DNP in the lumen JEuid Calcium and magnesium movements with DNP in the lumen fluid are outlined in Fig. 3. At 400 and 475 m-osmols calcium changes are little different from those
CALCIUM
793
AND MAGNESIUM MOVEMENT IN AMERICAN COCKROACH
obtained without DNP. However, at 575 and 700 m-osmols the movements closely resemble those achieved with DNP in the blood side solution (Figs. 3A and 1A). Net influx movements of magnesium are inhibited in much the same way as they were when DNP was in the blood side solution (Fig. 3B). At 700 mosmols a net efflux of this ion takes place (Fig. 3B). A. + 0.4
B.
Calcium
Magnesium
4
+ 0.2_ C)
2)
O_ -0.2_ -C&I_ - 0.6_ -0.8_ -1 .o_
;: <
-1.2_ -1.44bo
I 475 initial
I 575 Lumen
I 700 Cont.
I 400
1 475
I 575
I 700
In Milliosmols
FIG. 2. Calcium and magnesium absorption with 10m3 M DNP in the blood side solution ( x ) compared to absorption without DNP ( 0). Both reactions were allowed to proceed with increasing lumen concentrations. Numbers in parentheses indicate the numbers of animals used. Calcium results are statistically difYerent (P-CO-OS) at 475, 57.5, and 700 m-osmols. Magnesium results are significa.ntly different (P-C 0.001) at 400 m-osmols, (P
Absorp;!ion of calcium and magnesium with increasing lumen osmolality and DNP on both sides of the midgut In this series of experiments, calcium movements closely resembled those achieved when DNP was in only the lumen solution (Fig. 4A). However, there was a somewhat greater tendency to increase net calcium influx, especially at 400 and 475 m-osmols (Fig. 4A). At 700 m-osmols only slight differences between these results and those without DNP were observed (Fig. 4A). Net movements of magnesium were reduced (Fig. 4B) and closely resembled those obtained with DNP on the blood side. The generalization appears true again that DNP tends to increasle calcium influx but inhibits the same for magnesium.
JOHN R. SAUER AND RICHARD R. MILLS
794
A.
4b
4f5 h-dial
B.
Calcium
5% Lumen
700 Cont.
4bo
495
Magnesium
575 I
700
In Milliosmols
FIG. 3. Calcium and magnesium absorption with DNP in the lumen ( x ) compared to absorption without DNP (0). Calcium differences are significantly different (P
The preceding information points up several interesting phenomena: (1) The concentration and/or composition of the lumen contents markedly affects absorption of calcium and magnesium. (2) The movements of both ions are DNP sensitive. (3) At all but the highest lumen osmolality, DNP tends to increase net ’ fl ux movements of magnesium are influx movements of calcium. (4) N e t in definitely inhibited by DNP. The idea that fluid concentrations can inversely affect rates of absorption is not new to insects. Water absorption rates in the locust and cockroach recta are apparently thus affected by rectal lumen concentrations (PHILLIPS, 1964a; WALL, 1967). Rates of rectal ion absorption are apparently determined by the ionic concentration of the haemolymph (PHILLIPS, 1964b). Indeed, the concept of osmoregulation is one which rests on the assumption that concentration ultimately It is not surprising, therefore, controls the various regulatory mechanisms. that epithelial permeability in the midgut is affected by osmotic concentrations of the lumen contents. It is difficult to assess, however, the exact mechanisms
CALCIUM ANDMAGNESIUM MOVEMENT IN AMRRICAN COCKROACH
795
involved in such occurrences. One such mechanism might be active transport. With this in mind, DNP was added in the various experiments since this metabolic poison inhibits active potassium transport in the midgut of Cecropia (HASKELL et al., 1965), water transport in the cockroach rectum (WALL, 1967) and the B. Magnesium
A. Calcium + 0.4 ‘0.2 0
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-0.2_
3
- 04.
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-0.6.
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- 0.8_
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z Q
-14
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.
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\t
(s)
1
(12)
400
475
575
Initial
Lumen
7bo Cone.
I
4bo
4%
575
7&J
In Milliosmols
FIG. 4. Absorption with DNP on both sides of the midgut ( x) compared to absorption without DNP (0). Calcium differences are significantly different (I’ < 0.05) at 575 m-osmols. Magnesium differences are significant (P< 0.001) at 400 m-osmols, (P< 0.01) at 575 m-osmols, and (P
JOHNR. SALTER ANDRICHARDR. MILLS
796
resemble those seen for calcium thus making this argument untenable (SAUER and MILLS, 1969). It may be that variations in the lumen sodium chloride cause considerable changes in an actively maintained transcellular potential difference across the epithelial cells. This seems credible in the light of what is known about potentials across the midgut of Cecropia (HARVEY and NEDERGAARD,1964). It is interesting that for each of the ions, two DNP experimental conditions yield very similar results. This may suggest that structures on the blood or lumen surface are most important in controlling movements of a particular ion. That is to say, there may be some significance in the fact that calcium movements are alike with DNP in the lumen fluid and with DNP on both sides of the cells (Figs. 3A and 4A). In the same way, magnesium movements are similar when DNP is in the blood side solution and when it is on both sides of the midgut (Figs. 2B and 4B). Tests with 14C-dinitrophenol indicate that less than 10 per cent of the available DNP is absorbed into the cells and very little moves entirely across when applied to only one side (Table 2). This indicates that the highest concentration TABLE2--‘PC-orNrrRoPHENoLABSORPTION ANDMOVEMENT ACROSS THEEPITHELIAL
Exp. W-DNP (inside only) W-DNP (blood side only) 14C-DNP (inside and out) 14C-DNP (outside) W-DNP (lumen)
No. of animals
Experimental sample taken from
Mean % of Mean counts/min 14C-DNP absorbed absorbed
4
Lumen
1515
6.9
4
Blood side
2055
9.7
4
Lumen
1090
4.9
3
Lumen
-
-
3
Blood side
-
-
Mean counts/min appearing in experimental sample
CELLS
Mean % of W-DNP crossing epithelial cells
-
54.5 (background = 40) 276 (background = 40)
<1 5
of DNP remains near the epithelial limiting surfaces, i.e. lumen and/or blood side. It suggests that most of its effects may occur along these surfaces. This concept would concur with the inhibiting effect of DNP on active potassium transport in the midgut of Cecropia, where placement of it on the blood side inhibits much more effectively than with it in the lumen (HASKELL et al., 1965). In summary, it appears the midgut may exert control over divalent ion movements. This concept is supported by data on variable movements as the lumen
CALCIUMAND MAGNESIUM MOVEMENT IN AMERICAN COCKROACH
797
osmolality is changed. Energy processes could be implicated as shown by changes brought about by adding DNP. Since only small amounts of DNP are actually absorbed into the cells and almost none moves entirely across the cells it appears probable that the primary target of DNP is on the epithelial membrane. Acknowledgements-The authors would like to thank C. A. BAYS, H. APPLEBAUM,and M. MICHELSONfor technical assistance and MARGIE M. SAUERfor drawing the figures. One of us (J. R. S.) was the recipient of a NASA predoctoral fellowship. This work was supported in part by grants from NSF (GB-4773, GB-6026, GB-7428), a cell biology training grant (NIH-GM-669) from USPHS NIH and a Sigma Xi grant-in-aid of research to J. R. S. REFERENCES BARTON-BROWNE L. B. (1964) Water regulation in insects. A. Rev. Ent. 9, 63-82. BEAMENTJ. W. L. (1964) The active and passive movement of water in insects. Adv. Insect Physiol. 2, 67-129. BERRIDGEM. J. (1966) Metabolic pathways of isolated Malpighian tubules of the blowfly functioning in an artificial medium. J. Insect Physiol. 12, 1523-1533. BERRIDGEM. J. (1968) Ion and water transport across epithelia. In Insects and Physiology (Ed. by BEAMENTJ. W. L. and TREHERNE J. E. ) pp. 329-347. Elsevier, New York. BIELAWSX.IJ., THOMPSONT. E., and LEHNINGERA. L. (1967) The effect of 2,4-dinitrophencl on the electrical resistance of phospholipid bilayer membranes. In Mitochondriul Structure and Compartmentation (Ed. by QUAGLIARIELLO E., PAPA S., SLATERE. C. and TAGEKJ. M.) pp. 181-184. Adriatica Editrice, Bari. HARVEYIV. R. and NEDERGAAIUI S. (1964) Sodium-independent active transport of potassium in the isolated midgut of the cecropia silkworm (Hyalophora cecropia). Proc. nut. Acad. Sci. LTSA 51, 757-765. HASKELLJ. A., CLEMONSR. D. and HARVEYW. R. (1965) Active transport by the cecropiu midgut-I. Inhibitors, stimulants and potassium transport. r. cell camp. Physiol. 65, 45-56. PHILLIPSJ. E. (1964a) Rectal absorption in the desert locust, Schistocerca gregariu ForskalI. Water. J. exp. Biol., 41, 15-38. PHILLIPSJ. E. (1964b) Rectal absorption in the desert locust, Schistocercagregariu ForskalII. Sodium, potassium and chloride. J. exp. Biol. 41, 39-61. SAUERJ. :R. and MILLS R. R. (1969) Movement of potassium and sodium across the midgut epithelium of the American cockroach. In preparation. SAUERJ. R., SCHLENZ-TRUER., and MILLS R. R. (1969) Salt and water transport by the in vitro cockroach midgut. J. Insect Physiol. 15, 483-493. STOBBARTR. H. and SHAW J. (1964) Salt and water balance: Excretion. In The Physiology of Insecta (Ed. by ROCKSTEINM.) 3, 189-258. Academic Press, New York. WALL B. J. (1967) Evidence for antidiuretic control of rectal water absorption in the cockroach Periplaneta americana (L.). J. Insect Physiol. 13, 565-578. WHARTOND. R. A., WHARTONM. L., and LOLA J. (1965) Blood volume and water content of the American cockroach, Periplaneta americana (L.). Methods and the influence of age ard starvation. J. Insect Physiol. 11,391404.