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J. MembraneBiol. 146, 47-57 (1995)
Mechanisms of Cell Volume Regulation in the Proximal Segment of the Malpighian
Tubule of Rhodnius neglectus
I.R. Arenstein 1, C. Caruso-Neves 2, L.F. Onuchic 1, A.G. Lopes 2
1Departmentof Physiology, Instituto de Ci~ncias Biom6dicas, Universidadede Sao Paulo, SP, Brazil
eInstituto de BioffsicaCarlos Chagas Filho, UniversidadeFederal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
Received: 20 September 1994/Revised: 7 March 1995
Abstract. The cell volume regulation of the lower seg-
ment cells of the Malpighian tubule of Rhodnius neglec-
tus in anisosmotic media was evaluated by using video-
optic techniques. When the medium osmolality was in-
creased with addition of 100 mM mannitol the cells
shrank to a minimum of 16.84 + 2.62% and subsequently
swelled towards their initial volume undergoing a typical
regulatory volume increase (RVI). Replacement of ei-
ther K + or Cl-or HCO 3 by Na +, gluconate and phos-
phate, respectively, abolished the RVI response. Fur-
thermore, the substitution of Na + by tetrarnethylammo-
nium (TMA +) in isosmotic conditions led to cellular
swelling and death. Addition of either amiloride 10 -4 M,
anthracene-9-COOH 5 x 10.4 M, furosemide 5 x 10-4 M
or ethacrynic acid 5 x 10-5 M, also abolished RVI. On
the other hand, addition of either Ba 2+ 10-3 M, SITS 5 x
10-4 M, ouabain 10-3 M or vanadate 10-3 M, did not
change the RVI response. When the tubules were incu-
bated in hyperosmotic media with EGTA 2 rr~ or vera-
pamil 10-6 M, the RVI response was abolished. In con-
trast, a decrease of NaC1 concentration from 129 to 79
rnM induced a cell swelling to a maximum of 33.11 +
1.73%, but the cells maintained swollen, only partially
regulating their volume. These results show that the
proximal cells of Malpighian tubule of R. neglectus are
able to regulate their volume in hyperosmotic but only
partially regulating in hyposmotic solutions. The mech-
anisms in RVI involve Na +, K +, CI-, Ca 2+ and HCO 3
transport pathways and a ouabain-insensitive ATPase
stimulated by Na §
Key words: RVD - - RVI - - Cell volume regulation - -
Malpighian tubule - - Furosemide - - Ethacrynic acid
Correspondence to: A.G. Lopes
The Journal of
Membrane
Biolo9Y
9 Springer-Verlag New York Inc. 1995
Introduction
Most cells are able to regulate their volume in hypos-
motic medium with a typical regulatory volume decrease
(RVD). Only few cell types regulate volume in hyper-
osmotic conditions by regulatory volume increase (RVI)
(Montrose-Rafizadeh & Guggino, 1990; Hoffmann &
Ussing, 1992; MacCarty & O'Neil, 1992; Onuchic et al.,
1992). Some cells, however, recover their volume when
exposed to hyperosmotic medium, with a typical RV1
after a pretreatment in hyposmotic medium (Corasanti et
al., 1990; Hoffmann & Ussing, 1992; Onuchic et al.,
1992). In mouse medullary thick ascending limb, it was
shown that the RVI depends on the antidiuretic hormone
(ADH) (Hebert, 1986). It has been suggested that this
difference between the cell's capacity to regulate it vol-
ume in hyposmotic or hyperosmotic media developed
early in evolution and allowed animals to live in fresh
water (Hoffmann & Ussing, 1992).
In isosmotic conditions, cell volume regulation has
been explained by a "pump-leak" hypothesis, in which
the (Na + + K+)ATPase is the crucial mechanism to main-
tain Na + and K + gradients (Leaf, 1959; Tosteson &
Hoffmann, 1960). On the other hand, several groups
have found that in some cells volume regulation is re-
sistant to ouabain (Russo et al., 1985; Proverbio et al.,
1989). These observations led some investigators to pro-
pose that a cardiac glycoside-insensitive sodium pump
plays an important role in cell volume regulation (Del
Castillo et al., 1982; Marfn et al., 1985; Proverbio et al.,
1986; 1989).
Cell volume regulation involves several steps before
the activation of its final effectors (Lewis & Donaldson,
1990; MacCarty & O'Neil, 1992; Hoffmann & Ussing,
1992). After the detection of the volume change, the cell
effectors have to be stimulated to start the regulatory
response. The activation of effectors proceeds via sec-
48
o n d m e s s e n g e r s i n c l u d i n g C a 2+, cyclic A M P and others
( M a c C a r t y & O ' N e i l , 1992). It h a s b e e n r e p o r t e d that
C a 2+ is i n v o l v e d in the R V D r e s p o n s e o f s o m e cells.
I n s o m e others, C a ~+ does n o t s e e m to b e i n v o l v e d i n the
R V I r e s p o n s e ( C a l a et al., 1983; F o s k e t t & Spring, 1985;
B e a r , 1990; P i e r c e & Politis, 1990; L e w i s & D o n a l d s o n ,
1990; M o n t r o s e - R a f i z a d e h & G u g g i n o , 1991; M a c C a r t y
& O ' N e i l , 1991; 1992; H o f f m a n n & Ussing, 1992). Thus,
the n a t u r e o f t h e s e c o n d m e s s e n g e r s c o n t r o l l i n g v o l u m e
r e g u l a t i o n varies a m o n g d i f f e r e n t cells.
T h e first step in i n s e c t u r i n e f o r m a t i o n is t h e secre-
tion o f an i s o s m o t i c fluid i n distal s e g m e n t o f the M a l -
pighian tubule. In following segments including the
p r o x i m a l s e g m e n t o f M a l p i g h i a n tubule, h i n d g u t a n d rec-
tum, solutes are r e a b s o r b e d selectively. D u r i n g diuresis,
t r a n s c e l l u l a r fluid t r a n s p o r t across t h e s e i n s e c t e p i t h e l i a l
cells is v e r y fast (Phillips, 1981, M a d d r e l l & O ' D o n n e l l ,
1992). T h u s , b e c a u s e t h e c o n t e n t o f t h e s e cell e x c h a n g e s
e v e r y f e w m i n u t e s , it is i m p o r t a n t t h a t t h e s e cells regu-
late t h e i r cell v o l u m e accurately.
In this paper, w e s t u d y w h e t h e r t h e p r o x i m a l seg-
m e n t cells o f t h e M a l p i g h i a n t u b u l e o f R. neglectus reg-
ulate t h e i r v o l u m e in a n i s o s m o t i c s o l u t i o n s a n d i n v e s t i -
gate the m e c h a n i s m s i n v o l v e d in this r e g u l a t i o n . M o s t o f
the p r e v i o u s studies c o n c e r n i n g t r a n s p o r t m e c h a n i s m s i n
the M a l p i g h i a n t u b u l e h a v e f o c u s e d o n distal cells ( M a d -
drell, 1969, Phillips, 1981, M a d d r e l l & O ' D o n n e l l , 1992,
N i c o l s o n , 1993). T h u s , i n this paper, w e c o m p a r e the
results o b t a i n e d in p r o x i m a l cells w i t h t h o s e o b t a i n e d in
distal s e g m e n t .
Materials and Methods
PREPARATION OF MALPIGHIAN TUBULES
Male Rhodnius neglectus, fasted for 5-8 days, were used in the exper-
iments. After decapitation, the animals were kept in isosmotic solution
and the tubules were dissected out with thin tweezers (Dumont #5)
under a stereoscopic microscope. For the microperfusion experiments,
1 mm long lower segments were cut with microsurgical needles (Cir-
con, Micro Surgical, USA) and transferred to the microperfusion cham-
ber. The tubule was viewed through an inverted microscope (Dia-
phot--TMD, Nikon, Tokyo, Japan). The condenser lens was removed
and replaced by a revolving nosepiece containing a 40x water immer-
sion objective (40/0,75 W, 160/-, Carl Zeiss, Germany). This lens was
important both to allow a larger amount of light and to help to maintain
a laminar flow in the perfusion chamber, which is essential to avoid
vibration and to keep the tubule in focus. The condenser was changed
to a 10x lens by rotating the nosepiece when a large light beam was
required in order to position the pipettes and perfuse the tubules under
a lower magnification. The tubules were cannulated and perfused ac-
cording to techniques used in a previous work adapted from the original
ones described by Burg et al., 1966) to permit exchange of luminal and
peritubular solutions (Lopes et al., 1988). These modifications made it
possible to exchange the solutions of the bath or the lumen perfusate in
less than 5 sec.
The holding pipettes were drawn in a microforge in order to
obtain a 800 p,m long parallel section with diameter of 75 gm at the
I.R. Arenstein et al.: Cell Volume Regulation in Malpighian Tubule
tip and a constriction of 45 pm. The perfusion pipettes were 1,200 gm
long with a external diameter of 35 gm.
To allow the best exchange of luminal solution and to avoid
vibration, cell volume was always analyzed in cells placed close to the
tip of the perfusion pipette.
SOLUTIONS
The control solution contained (in mg): 129 NaC1, 8.6 KC1, 8.5 MgC12,
2 CaC12, 4.3 Na2HPO4/NaH2PO4, 10.2 HCO3, 34 glucose and 3 ala-
nine, with final osmolality of 320 _+ 5 mOsm/Kg, The solution was
gassed with a mixture of 95% 02-5% CQ, and the pH was adjusted to
7.0. The hyposmotic solution had the same ionic composition as the
control, except that NaC1 concentration was decreased to 79 mM, lead-
ing to a final osmolality of 220 _+5 mOsm/Kg. Hyperosmotic solutions
were prepared by adding 100 mM mannitol to the control solution, to
obtain a final osmolality of 420 + 5 mOsm/Kg. Solutions free of Na+,
K+, C1- and HCO~ were prepared by replacement of Na§ by with
tetramethylammonium (TMA+), K+ by Na+, C1- by gluconate and
HCO3 by HEPES. Ion substitutions were always made in both perfus-
ate and bath, simultaneously after the tubules had been equilibrated for
20 min in the chosen isosmotic ion-depleted solution prior to the hy-
perosmotic shock. To block the role of K+ and of C1- channels, 10.3 M
Ba2+ and 5 x 10.4 M anthracene-9-COOH were used, respectively.
To determine if the Na+/I-I+, CI-/HCO~ and Na+/K+/2CF cotransporters
were involved in the RVI response the follows specific inhibitors were
added: 10 - 4 M amiloride, 5 x 10.4 u SITS and 5 x 10~4 M furosemide,
respectively. Ouabain and vanadate were added to the bath at concen-
trations of 10 -3 M and used to detect a possible role of basolateral
(Na+ + K+)ATPase in RVI. Ethacrynic acid 5 x 10.4 M was used to
determine if the ouabain-insensitive Na+-ATPase was involved in RVI
response. In the experiments where these drugs were added, tubules
were incubated for 20 min in isosmotic solutions containing the drug
prior to the hyperosmotic shock. In all experimefits, each condition
was studied during a period of 20 min.
CELL VOLUME MEASUREMENTS
Cell volume measurements were carried out by video-optical tech-
niques similar to those used in previous publications (Guggino et al.,
1985; Lopes et al., 1988). Briefly, to evaluate cell volume, the level of
focus of a 32x objective was adjusted near the center of the tubule
lumen to get a longitudinal side view, which was observed in a tele~
vision monitor. Besides file use of an air table to hold the microper-
fusion setup, two basic procedures were employed to avoid undesirable
movements of the tubule during the experiments: (1) the tubule length
between the pipette tips was kept very short, and (2) the tubule was
positioned between the bottom of the chamber and the water-
immersion lens used as a condenser. With this care, it was possible to
obtain a laminar flow and a very stable preparation. By this means, the
cells were kept stable at the same focal plane during the whole proce-
dure, which was essential for the reliability of the results. Images of
the tubule were recorded during all the experiments. The frozen dis-
play of the video image on the television screen was used for the direct
measurement of tubule diameter and cell height. The volume of a
constant tubule segment was calculated by means of a mathematical
model based on the difference between its external and internal cylin-
ders. The temporal resolution for cell volume measurements was lim-
ited by the system to a frame-by-frame sampling rate. However, due to
the slow rate of phenomenon, we took one measurement every minute.
The total cell volume obtained under each experimental condition
was normalized to the total cell volume of the same segment under
I.R. Arenstein et al.: Cell Volume Regulation in Malpighian Tubule
Table 1. Cell volume regulation during hyposmotic and hyperosmotic
shocks, expressed in % of control volume
Hyperosmotic Hyposmotic
V1 -16.84 _+2.62 33.11 _+ 1.73
V2 -2.05 _+ 1.53 16.11 + 3.18
V4 19.14 + 2.74 -7.06 -+ 1.10
n 6 6
P values
V 2 vs. W0 NS <~0,05
V2 vs. V 1 <0.05 <0.05
V4 v s . V o <0.05 <0.05
Values are means _+SE expressed as % change of cell volume obtained
under each experimental condition normalized to the total cell volume
of the same segment under control conditions, n = number of experi-
ments. Ns = not statistically significant. V 0 = control volume in isos-
motic solution; V 1 = maximal % change during hyperosmotic or hy-
posmotic stress; V 2 = final % change during hyposmotic or hyperos-
motic stress; V 4 = % change after reintroduction of isosmotic solution.
Positive and negative values represent swelling and shrinking, respec-
tively.
control conditions, and the results are shown as percent cell volume
variation calculated by the following equation:
% cell volume change = (100V/Vo) - 100,
where Vo = control cell volume and V = cell volume under a given
experimental condition, as indicated in the table legends.
The data were analyzed by two-way analysis of variance
(ANOVA), considering as factors the treatments. The magnitude of the
differences were verified " a posteriori" by the least significant differ-
ence statistical contrast test (Montgomery, 1976). The considered level
of significance was 0.05. The statistical comparisons for each experi-
mental group are shown in the tables.
Results
VOLUME REGULATION DURING OSMOTIC SHOCK
To verify if the cells of Malpighian tubule were able to
regulate their volume in hyposmotic or in hyperosmotic
media, the osmolality of both intraluminal and bath flu-
ids was decreased or increased, respectively. Table 1
shows that the cells' shrinkage occurred when the osmo-
lality was increased by the addition of 100 mM mannitol.
Following shrinkage, the cell volume returned to the ini-
tial value. This pattern is a typical response to cells dur-
ing RVI. In contrast, when the osmolality was decreased
by reduction of NaC1 from 129 to 79 mM the cells
swelled, but only partially recovered. They remained
partially swollen after 15 rain of the osmotic shock. In
addition, upon return to normal osmolality, after a hy-
perosmotic or hyposmotic shock, the cells swelled or
shrank. This observed "undershoot" or "overshoot"
indicates that during the RVI or the partial RVD, there
occurs either an increase or a decrease of intracellular
49
~-@ I - - Hyposmotie I Isosmotic_
0-0 [ Hyperosmotic [ Isosmotic-
30
20
215
r,.)
-10 Time (mln)
-20
Fig. 1. Cell volume regulation during hyposmotic and hyperosmotic
shocks expressed in % of initial volume. Experimental conditions are
shown at the top.
solutes, respectively. One representative experiment is
illustrated in Fig. 1. These results indicate that the prox-
imal cells of Malpighian tubule of R. neglectus are able
to fully regulate their volume during hyperosmotic
shock. On the other hand, the regulation of cell volume
in hyposmotic is incomplete.
IONIC DEPENDENCE OF R V I RESPONSE
The dependence of RVI on K +, C1- or HCO~ is shown in
Table 2. In separate experiments, tubules were first in-
cubated for 20 rain in an isosmotic medium in which K +
was replaced by Na +, C1- by gluconate, or HCO 3- by
HEPES. Substitution of these ions in both luminal and
bath solutions did not result in a significant change in
cellular volume during this period. When these tubules
were exposed to hyperosmolality, the cells showed no
RVI. Re-addition of K +, CI-- or HCO 3, in the hyperos-
motic solution, restored RVI. Upon return to the isos-
motic solution the cell swelled, in a way similar to that
observed in Fig. 1. One of these experiments is shown in
Fig. 2. When Na + was replaced by TMA +, even in isos-
motic solution, the cell swelled and died (data not
shown). Based on these results, we may conclude that
the RVI depends upon Na +, K +, C1- and HCO~.
IONS TRANSPORT MECHANISMS INVOLVED IN RVI RESPONSE
To determine the ion transport mechanisms involved in
RVI, we utilized inhibitors of several transport process,
50 I.R. Arenstein et al.: Cell Volume Regulation in Malpighian Tubule

Table 2. Cell volume variations during hyperosmotic shock in the Table 3. Cell volume variations during hyperosmotic shock in the
absence of K§ C1- or HCO~, expressed in % of control volume presence of amiloride 10-~ M, expressed in % of control volume

0 K+ 0 C1- 0 HCO~ Amiloride

V1 -24.60 + 1.26 -20.94 5:1.39 -22.26 + 1.25 VI -17.04 + 3.30

V2 -18.89 - 2.90 -20.94 _+_1.39 -22.26 + 1.25 V2 -17.04 + 3.30
V3 -1.09 + 0.40 0 -1.26 5:0.69 V3 -17.04 + 3.30
V4 17.93 + 6.38 23.80 -+ 1.56 17.89 ---3.84 V4 -7.19 + 2.98
n 6 6 6 n 4
P value P value
V 2 vs. V o <0.05 <0.05 <0.05 V 2 vs. Vo <0.05
g 2 VS. V 1 NS NS NS V 2 VS. V 1 NS
V s vs. V 2 <0.05 <0.05 <0.05 V 3 vs. V 2 NS
V4 vs. V o <0.05 <0.05 <0.05 NS
V 4 vs. Vo
Values are means + SE expressed as % change of cell volume obtained
under each experimental condition normalized to the total cell volume Values are means + SE expressed as % change of celi volume obtained
of the same segment under control conditions, n = number of experi- under each experimental condition normalized to the total cell volume
ments. NS = not statistically significant. V o = control volume in isos- of the same segment under control conditions, n = number of experi-
motic solution; V 1 = maximal % change during hyperosmotic stress in ments. NS = not statistically significant. V o = control volume in isos-
the absence of the ions shown at the top of each column; V 2 = final % motic solution; V 1 = maximal % change during hyperosmotic stress in
change during hyperosmotic stress in the absence of the ions shown at the presence of amiloride; V 2 = final % change during hyperosmotic
the top of each column; V 3 = % change after re-addition of the ions stress in the presence of amiloride; V 3 = % change after amiloride
shown at the top of each column, still in hyperosmolality; V4 = % removal, still in hyperosmolality; V4 = % change after re-introduction
change after re-introduction of isosmotic solution containing all the of isosmotic solution, in the absence of amiloride. Positive and negative
ions. Positive and negative values represent swelling and shrinking, values represent swelling and shrinking, respectively.
respectively.
a m i l o r i d e o n RVI. T h e p r e i n c u b a t i o n o f t h e t u b u l e for
2 0 rain in the i s o s m o t i c m e d i u m w i t h amiloride, d i d not
cr e - - e l - - 0mM I 148.1mM alter cell v o l u m e significantly. T h e t u b u l e w a s t h e n ex-
He03- ~--0 ] 0 mM [ 10.2 mIVl p o s e d to h y p e r o s m o l a l i t y in the p r e s e n c e o f this drug.
K§ ~--01 OmM I 8,6raM T h e cells s h r a n k w i t h o u t a n y R V I , e v e n after the d r u g
Hyperosmotie ] lsosmotic_ had been washed out under hyperosmotic medium. The
30- r e t u r n to i s o s m o t i c s o l u t i o n led to s o m e R V I , b u t to a
v a l u e t h a t w a s still l o w e r t h a n the c o n t r o l v o l u m e . O n e
o f t h e s e e x p e r i m e n t s is r e p r e s e n t e d i n Fig. 3. T h e s e data
20-
are c o n s i s t e n t w i t h the c o n c l u s i o n t h a t a n a m i l o r i d e -
s e n s i t i v e p a t h w a y i n v o l v i n g e i t h e r N a + c h a n n e l s or Na§
r) lo
H § c o t r a n s p o r t e r s or b o t h is i n v o l v e d in RVI.
In the f o l l o w i n g g r o u p o f e x p e r i m e n t s , cells w e r e

.•
0 initially i n c u b a t e d in i s o s m o t i c s o l u t i o n s i n the p r e s e n c e
30 40 50
o f 10 -3 M B a 2§ or 5 x 10 -4 U f u r o s e m i d e in b o t h l u m i n a l
Q n.)
-10 - a n d b a t h fluids ( T a b l e 4). B o t h d r u g s h a d n o e f f e c t o n
cell v o l u m e u n d e r i s o s m o t i c c o n d i t i o n s . W h e n the os-
Q,) -20 - m o l a l i t y w a s i n c r e a s e d in the p r e s e n c e o f B a 2+, the cells
shrank and recovered their volume completely. Thus,
R V I w a s n o t i n h i b i t e d b y B a 2+. O n t h e o t h e r h a n d , w h e n
-30
the o s m o l a l i t y w a s i n c r e a s e d in t h e p r e s e n c e o f furose-
Fig. 2. Cell volume variations during hyperosmotic shock in the ab- m i d e the cells s h r a n k w i t h o u t R V I . A f t e r w a s h o u t o f t h e
sence of K§ C1- or HCO3 expressed in % of initial volume. Experi- cells w i t h a f u r o s e m i d e - f r e e h y p e r o s m o t i c solution, t h e y
mental conditions are shown at the top. s w e l l e d to t h e i r initial v o l u m e . T h e r e t u r n to i s o s m o t i c
solution, in b o t h cases, p r o m o t e d cell s w e l l i n g a b o v e
t h e i r initial v o l u m e , as s h o w n in T a b l e 4. O n e r e p r e s e n -
as d e s c r i b e d in M a t e r i a l a n d M e t h o d s . T o s t u d y t h e pos- t a t i v e e x p e r i m e n t is s h o w n in Fig. 4. T h e s e data d e m -
sible i n v o l v e m e n t o f N a § volume regulatory o n s t r a t e t h a t K § p a r t i c i p a t e s in R V I p r o b a b l y t h r o u g h
m e c h a n i s m s (either N a c h a n n e l s or N a § + e x c h a n g e or Na+/K+/2C1 - c o t r a n s p o r t e r s .
b o t h ) , a n o t h e r g r o u p o f t u b u l e s w a s e x p o s e d to h y p e r o s - T o i n v e s t i g a t e C1- p a t h w a y s i n v o l v e d in the R V I
m o t i c s h o c k in t h e p r e s e n c e o f 10 -4 M a m i l o r i d e in b o t h response, a group of experiments were conducted with
l u m i n a l a n d b a t h fluids. T a b l e 3 s h o w s the e f f e c t o f addition of 5 x 10 ~ M a n t h r a c e n e - 9 - C O O H or 5 x 10 .4 M
I.R. Arenstein et al.: Cell Volume Regulation in Malpighian Tubule 51

Amiloride L - - IO'4M [ - - OM Fur~3~de ] 5x104M I - - 0 M

Hyperosmolie j Isosmotic__ Barium ~ _ _ 10"3M [ 0M

Hyperosmotic i
I -
Isosmotie --
~0 10 1 40-
r..) 12OO
0.[ i i i , i 30-
! 10 20 30 40 50
m
Time (min.) ,.~ 2o-
~10

> I0-
O
~
o 7- 0,
10 20 .r ,0
i

,o

Fig. 3. Cell volume variations during hyperosmotic shock in the pres- -10 - C~=g~ Time (mm)
ence of amiloride expressed in % of intial volume. Experimental con-
ditions are shown at the top. ~) -20 -

-30
Table 4. Cell volume variations during hyperosmotic shock in the
presence of furosemide 5 x 10-4 M or Ba 2+ 10-3 M, expressed in % of Fig. 4. Cell volume variations during hyperosmotic shock in the pres-
control volume ence of furosemide or barium expressed in % of initial volume. Ex-
perimental conditions are shown at the top.
Furosemide Ba 2+

V1 -19.45 + 2.12 -16.54 + 2.16 Table 5. Cell volume variations during hyperosmotic shock in the
V2 -19.45 + 2.12 -1.42 + 1.34 presence of anthracene-9-COOH 5 x 10-4 M or SITS 5 x 10-4 M,
V3 -1.10 + 0.50 -0.41 _+0.20 expressed in % of control volume
V4 25.15 + 4.03 19.79 _+4.20
n 6 6 Anthracene-9-COOH SITS
P values
V 2 vs. Vo <0.05 NS VI -21.01 + 2.45 -21.79 +_ 1.51
V 2 vs. V 1 NS <0.05 V2 -19.63 _+3.00 -3.58 _+ 1.64
V 3 Vs. V 2 <0.05 NS V3 -12.98 _+4.92 -2.59 _+ 1.43
V 4 VS. V o <0.05 <0.05 V4 11.75 + 6.75 12.17 + 2.86
n 6 6
Values are means + sE expressed as % change of cell volume obtained P values
under each experimental condition normalized to the total cell volume V 2 vs. Vo <0.05 Ns
of the same segment under control conditions, n = number of experi- V 2 vs. V1 NS <0,05
ments. NS = not statistically significant. V o = control volume in isos- V 3 vs. V2 NS NS
motic solution; V 1 = maximal % change during hyperosmotic stress in V 4 vs. Vo <0.05 <0.05
the presence of furosemide or barium; V z = final % change during
hyperosmotic stress in the presence of furosemide or barium; V 3 = % Values are means + sz expressed as % change of cell volume obtained
change after furosemide or barium removal, still in hyperosmolality; V 4 under each experimental condition normalized to the total cell volume
= % change after re-introduction or isosmotic solution, in the absence of the same segment under control conditions, n = number of experi-
of furosemide or barium. Positive and negative values represent swell- ments. NS = not statistically significant. V o = control volume in isos-
ing and shrinking, respectively. motic solution; V 1 = maximal % change during hyperosmotic stress in
the presence of anthracene-9-COOH or SITS; V 2 = final % change
during hyperosmotic stress in the presence of anthracene-9-COOH or
S I T S ( T a b l e 5). T h e s e a g e n t s d i d n o t c a u s e a n y s i g n i f -
SITS; V 3 = % change after anthracene-9-COOH or SITS removal, still
i c a n t c h a n g e s in v o l u m e d u r i n g t h e i s o s m o t i c i n c u b a t i o n in hyperosmolality; V 4 = % change after re-introduction of isosmotic
period. Hyperosmotic shock in the presence of an- solution, in the absence of anthracene-9-COOH or SITS. Positive and
thracene-9-COOH or SITS, however, was followed by a negative values represent swelling and shrinking, respectively.
s i g n i f i c a n t cell s h r i n k a g e ( s e e T a b l e 5). T h e s e c e l l s w e r e
a b l e to r e c o v e r t h e i r v o l u m e in t h e p r e s e n c e o f S I T S
(3.58 + 1 . 6 4 % ) , b u t w e r e u n a b l e to r e c o v e r t h e i r v o l u m e m o s t l y l i k e l y i n v o l v i n g C1- c h a n n e l s . O n e r e p r e s e n t a -
with anthracene-9-COOH even after a washout with a t i v e e x p e r i m e n t is i l l u s t r a t e d in F i g . 5. It is i m p o r t a n t to
blocker-free, hyperosmotic solution. Re-addition of the stress that some C1-/HCO~ cotransporters are insensitive
i s o s m o t i c s o l u t i o n l e d to a c e l l u l a r s w e l l i n g o n l y in t h e to S I T S , as in t u r t l e b l a d d e r ( K o h n et al., 1990) a n d
presence of SITS, indicating that the participation of CI- c o l l e c t i n g d u c t i n t e r c a l a t e d c e l l s ( F u r u y a et al., 1991;
in t h e R V I r e s p o n s e is m e d i a t e d b y a n a n t h r a c e n e - 9 - S c h u s t e r , 1993). T h e r e f o r e , it c a n n o t b e i n f e r r e d f r o m
COOH-sensitive and not a SITS-sensitive pathway the SITS insensitivity of RVI that the anion cotransport-
52 I.R. Arenstein et al.: Cell Volume Regulation in Malpighian Tubule

Anthracene- T a b l e 6. Cell volume variations during hyperosmotic shock in the

9-COOH } - - 5x104M- [ _ 0M_
presence of ouabain 10 -3 M, vanadate 10-3 M or ethacrynic acid 5 x
SITS t-- 5 x 104M [ 0M 10-5 M, expressed in % of control volume
0--43
_ _ Hyperosmotic _ _ i ]sosmotic- -
Ouabain Vanadate Ethacrynic
30
acid

20 V1 -16.75_+2.93 -19.18_+0.50 -15.43-+3.09

V2 - 2 . 1 2 + 0.92 - 2 . 6 0 +__2.05 -15.43 + 3.09
,s= V3 -1.16+0.60 -2.45_+2.11 -0.37_+0.30
Q) 10
V4 22.06 -+ 2.97 12.70 _+9.25 20.70 + 3.67
n 3 6 6
0 -- I P value
E 20 30 so V 2 vs. Vo NS NS <0.05
O Time (min.) V 2 vs. V~ <0.05 <0.05 NS
~;~ -10
V 3 vs. V 2 NS NS <0.05
V 4 vs. V o <0.05 <0.05 <0.05
Q,) -20
Values are means _+ SE expressed as % change of cell volume obtained
under each experimental condition normalized to the total cell volume
-30
of the same segment under control conditions, n = number of experi-
ments. NS = not statistically significant. V o - control volume in isos-
Fig. 5. Cell volume variations during hyperosmotic shock in the pres-
motic solution; V 1 = maximal % change during hyperosmotic stress in
ence of anthracene-9-COOH or SITS expressed in % of initial volume.
the presence of ouabain, vanadate or ethacrynic acid; V 2 = final %
Experimental conditions are shown at the top.
change during hyperosmotic stress in the presence of ouabain, vanadate
or ethacrynic acid; V 3 = % change after ouabain, vanadate or ethacrynic
acid removal, still in hyperosmolality; V 4 = % change after re-
introduction or isosmotic solution, in the absence of ouabain, vanadate
ers do not play a role in this process. This is especially
or ethacrynic acid. Positive and negative values represent swelling and
true because the dependence of RVI on HCO~ in the shrinking, respectively.
medium is consistent with the participation of a HCO 3-
dependent anion cotransporter. Ethacrynic
To determine whether primary active transport Acid ~ - - 5x 104M 0 M -- 1

mechanisms generate the ionic gradients responsible for Vanadate 10-3M 0M I

(>-O I - -
volume regulation, hyperosmotic shock was induced in
Ouabain _ _ 10-3M 0M I
the presence of either 10 -3 M ouabain, 10-3 M vanadate or
5 x 10-5 M ethacrynic acid (Table 6). The incubation of Hyperosmotic _ _ I Isosmotic __

tubules in an isosmotic solution with these drugs did not

significantly change cell volume. When the hyperos-
ID
motic shock was induced in the presence of ouabain, 20

vanadate or ethacrynic acid the cells shrank. RVI was

,.~
abolished by ethacrynic acid but not by ouabain or van- Q) 10
adate. When the cells were washed with hyperosmotic
solution in absence of ethacrynic acid, full RVI was ob-
served. In three experimental conditions, return to isos-
~,1 0 , L ~ I
T ~_ _ _ .2_~ .~ e ~ 3 0. 4~ ime ( r a i n ' ;
motic solutions led to cellular swelling. One representa- O -10
tive experiment is shown in Fig. 6.
ID
~) -20
SIGNALING MECHANISMS IN RVI
-30
It was reported that, in several cells, RVI response is
independent of the intracellular Ca 2+ concentration (Ho- Fig. 6. Cell volume variations during hyperosmotic shock in the pres-
ffmann & Ussing, 1992; MacCarty & O'Neil, 1992). ence of ouabain, vanadate or ethacrynic acid expressed in % of initial
Therefore, to verify if the RVI response is dependent on volume. Experimental conditions are shown at the top.
extracellular Ca 2§ the Malpighian tubule was exposed to
hyperosmotic medium in the presence of 2 mM EGTA or
10.6 M verapamil (blocker of voltage dependent Ca 2§ added or verapamil was removed the cells showed a
channels). Initially, the cells shrank to a minimum in the typical RVI response. The return to isosmotic solution
presence of EGTA and verapamil, respectively. In both promoted the increase of cell volume (Table 7). A typ-
cases, RVI response was abolished. When Ca 2+ was re- ical experiment is shown in Fig. 7.
I.R. Arenstein et al.: Ceil Volume Regulation in Malpighian Tubule
Table 7. Cell volume variations during hyperosmotic shock in the
presence of EGTA 2 mM or verapamil 10-3 M
EGTA Ver~amil
V1 -22.00 _+ 1.70 -15.39 + 1.84
V2 -19.57 + 1.23 -15.39 • 1.84
V3 -1.79 + 0.45 -3.14 • 1.20
V4 11.66 • 4.05 16.00 • 6 . 6 0
n 6 6 [,,)
P values
V2 vs. Vo <0.05 >0.05
V 2 vs. V 1 NS NS
V 3 v s . V2 <0.05 <0.05
V 4 vs. Vo <0.05 <0.05
Values are means _+SE expressed as % change of cell volume obtained
under each experimental condition normalized to the total cell volume
of the same segment under control conditions, n = number of experi-
ments. NS = not statistically significant. V o = control volume in isos-
motic solution; V 1 = maximal % change during hyperosmotic stress in
the presence of EGTA or verapamil; V 2 = final % change during
hyperosmotic stress in the presence of EGTA or verapamil; V 3 = %
change after EGTA or verapamil removal, still in hyperosmolality; V 4
= % change after the re-introduction of isosmotic solution, in the ab-
sence of EGTA or verapamil. Positive and negative values represent
swelling and shrinking, respectively.
Discussion
In the present work, we have used video-optical tech-
niques similar to those used previously (Guggino et al.,
1985; Lopes et al., 1988; Onuchic et al., 1992) to study
the cell volume regulation in proximal cells of Mal-
pighian tubule of R. negIectus exposed to hyperosmotic
or hyposmotic solutions. These cells regulate their vol-
ume in hyperosmotic medium with a typical RVI, but
they are not able to regulate completely their volume in
hyposmotic medium (i.e., they do not show a full RVD).
These results are in contrast with the data obtained in
previous studies performed on amphibian and mamma-
lian cells (Ussing, 1982; Hoffmann et al., 1984; Davis &
Finn, 1985). Several studies have shown that: (1) many
mammalian renal epithelial cells regulate their volume in
hyposmolality but fail to completely regulate cell volume
when submitted to hyperosmotic stress (Montrose-
Rafizadeh & Guggino, 1990; Onuchic et al., 1992); (2)
organic osmolyte accumulation may contribute to RVI in
renal medullary cells, although no direct evidence has
been shown so far. This process may involve uptake
from extracellular compartment (Nakanishi et al., 1988)
or intracellular synthesis of organic solutes (Bagnasco et
al., 1988). While the incorporation of extracellular ions
constitutes an acute response (Hebert, 1986), the organic
osmolyte mechanisms are slow and progressive, consti-
tuting probably a second line of cell volume defense.
These processes are more stable and may refine the abil-
ity to regulate cell volume, and (3) the electrolyte uptake
53
Ve<~-~ t 10-6M I OM
EGTAI 2x 10"3M I 0M
Hypcrosmolie ] lsosmotic __
0 30 ]
2O
,xZ
10
-10 - ~ Time (rain.)
~.) -20 -
-30
Fig. 7. Cell volume variations during hyperosmotic shock in the pres-
ence of EGTA or verapamil expressed in % of initial volume. Exper-
imental conditions are shown at the top.
mechanisms involved in the short term response of RVI
of some of these cells require the presence of specific
hormones in solution (Hebert, 1986) or specific pre-
osmotic exposure to be activated (Montrose-Rafizadeh &
Guggino, 1990). In contrast, in proximal cells of the
Malpighian tubule: (1) volume regulation in hyperos-
motic stress is acutely established by ionic uptake, with-
out the need of pre-activation processes; (2) the RVI
response observed was complete, whereas RVD was in-
complete; and (3) several ionic mechanisms seem to be
involved in the RVI response, giving the cell a handful of
possibilities to regulate volume. Therefore, we postulate
that, in general, insect Malpighian tubule cells may reg-
ulate their volume in hyperosmolality using ionic trans-
porters independent of pre-activation mechanisms, while
renal mammalian cells might require organic osmolyte
dependent or pre-activated processes to regulate cell vol-
ume in hyperosmotic stress. On the other hand, insect
Malpighian tubule cells may require prestimulation ei-
ther by hormones or previous activation of transport sys-
tems for complete RVD. This might be attributed to ev-
olutionary adaptation of animal cells, such as different
basal or variations of environmental osmolality to which
the cells are submitted, as well as by different functions.
However, further experiments are still required to test
this hypothesis.
The observation that replacement of Na § by TMA §
promoted cell swelling and death in isosmotic medium
indicates that cell volume regulation in basal state re-
quires Na § The addition of amiloride abolished RVI,
suggesting that Na § participates in RVI either through a
Na+/I-I+ exchanger or/and Na + channels. It has been well
known that Na § transport pathways inhibited by
54
amiloride play a central role in fluid secretion in the
distal portions of the Rhodnius Malpighian tubule (Mad-
drell, 1969; Gee, 1976; Phillips, 1981; Baldrick et al.,
1988; Maddrell & O'Donnell, 1992; Nicolson; 1993).
Phillips (1981), proposed that the amiloride-sensitive
Na § transport is energized by a "second Na § pump,"
located in the basolateral membrane. More recent obser-
vations are compatible with the presence of an amiloride-
sensitive Na+/I-I+ cotransport in the luminal membrane of
the distal portion of the Malpighian tubule, energized by
H+-ATPase located in same membrane (Maddrell &
O'Donnell, 1992; Nicolson, 1993). In addition, it has
been shown that the basal Na § permeability of this seg-
ment is low. On the other hand, it has been proposed that
there is a Na § channel stimulated by cAMP that is
present in the same portion of Aedes and Glossima Mal-
pighian tubules (Nicolson, 1993). Sawyer & Beyenbach
(1985) also working with fluid secretion in the distal
portion of Aedes Malpighian tubules showed that addi-
tion of cAMP depolarizes Vb (basolateral voltage) but
has no effect no Va (apical voltage), indicating that the
Na + channel is located in the basolateral membrane. The
electrochemical gradient for Na + favors the influx across
the basolateral membrane (Maddrell, 1969; Phillips,
1981).
Because 5-8 day-fasting animals were used in the
present experiments, we expect that the level of diuretic
hormone is very low (Beyenbach & Petzel, 1987).
Therefore, we postulate that in the antidiuretic state that
the Na+/H+ antiporters and/or amiloride-sensitive Na +
channels, which are involved in fluid secretion in the
diuretic state, may participate in the RVI response during
periods of antidiuresis. Our observation that replace-
ment of Na + by TMA § in isosmotic solutions induces cell
swelling and death suggests that Na+-dependent transport
pathways may also be important in maintaining cell ho-
meostasis in resting conditions. On the other hand, if we
consider that the Malpighian tubule plasma membrane is
permeable to TMA +, the favorable gradient could ex-
plain the cell swelling caused by TMA + influx.
In our experiments, SITS did not change the RVI
response, suggesting the independence of this mecha-
nism on a SITS-sensitive C1-/HCO5 antiport. The par-
ticipation of this transporter in fluid secretion also had
been ruled out (Phillips, 1981, Nicolson, 1993). On the
other hand, most of the animal cells studied so far that
regulate volume using a Na+/H+ antiporter also utilize a
C1-/HCO~ antiporter in this process (Ericson & Spring,
1982; Grinstein et al., 1984; Cala, 1985; Hoffmann et al.,
1992). In these cells, both antiporter and exchanger
work together to support net CF and Na + influx in hy-
perosmotic solutions. This operates because the ex-
change of Na + for I-V increases intracellular pH and
HCO~ concentration. Higher HCO~ concentrations en-
hance the C1-/HCO~ exchange, resulting in the net
movement of C1- into the cell and removing excess
I.R. Arensteinet al.: Cell VolumeRegulationin MalpighianTubule
HCO 3. As a consequence, the parallel function of these
transporters results in the exchange of osmotically active
for osmotically inactive particles, represented by the in-
flux of Na + and C1- and the outflow of CO 2 and H20
(Cala, 1980; Hoffmann & Ussing, 1992). Despite the
SITS-insensitivity, we did observe that the RVI response
requires the presence of HCO 3 in medium. For these
reasons, we postulate that there may be a SITS-
insensitive CI-/HCO~ antiporter involved in RVI re-
sponse of Malpighian tubule of R. neglectus.
The present data clearly indicate that RVI is inhib-
ited by anthracene-9-COOH, a well known C1--channel
blocker. It has been reported in previous studies the par-
ticipation of C1- channels in RVD response in several
cell types, but not in RVI response (Knoblauch et al.,
1989; Hoffmann & Ussing, 1992; McCarty & O'Neil,
1992; Onuchic et al., 1992). The presence of CI- chan-
nels in the apical membrane of the Malpighian tubule
cells involved in fluid secretion has been described
(Maddrell, 1969; Phillips, 1981; O'Donnell & Maddrell,
1984; Wright & Beyenbach, 1987; Nicolson, 1993).
Moreover, the CI- electrochemical gradient in apical
membrane favors C1- secretion. It becomes difficult then
to postulate that apical CI- channels are involved in RVI
response. However, because the CI- electrochemical
gradient in the basolateral membrane favors the C1- up-
take, it is possible that there are CI- channels in the
basolateral membrane that activate only during RVI re-
sponse (Maddrell, 1969; Phillips, 1981; Hevert, 1984).
In agreement with this hypothesis, there is evidence that
Cu2+-sensitive anionic channels are indeed present in the
basolateral membrane (Phillips, 1981). Following the
electroneutrality principle, the participation of CI- chan-
nel in RVI response may indicate that the mechanism of
Na + transport during RVI could be through Na + channels
in addition to a Na+/H+ antiporter. However, in this pa-
per, whether the amiloride effect was through the Na+/H+
antiporter or through the Na § channel was not further
investigated.
Since the effects of amiloride on the Na+/I-I+ an-
tiporter or Na § channel and anthracene-9-COOH on the
CI- channel are readily reversible, the present observa-
tion of an irreversible action could also indicate that
these drugs disrupt cell volume regulation through dif-
ferent amiloride- and anthracene-9-COOH-sensitive
pathways.
The inhibition of the RVI promoted by substitution
of K + by Na + could be explained by the K + depletion
established during the 20-min removal of K + during the
isosmotic equilibrium period. The intracellular K § is
necessary to sustain the (Na§ + K+)ATPase and in its
absence would inhibit the enzyme activity. However,
this possibility can be ruled out because addition of oua-
bain and vanadate, blockers of the (Na§ + K+)ATPase,
did not inhibit the RVI response. Therefore, we con-
clude the involvement of K+ in RVI could be through
I.R. Arenstein et al.: Cell Volume Regulation in Malpighian Tubule
Na+/K+/2C1- cotransporter, as it has been shown in other
cells (Gargus & Slayman, 1980; Mercer & Hoffman,
1985; Montrose-Rafizadeh & Guggino, 1990; Hoffmann
& Ussing, 1992). It has been proposed that this trans-
porter is involved in fluid secretion of the Malpighian
tubule, and that it is located in the basolateral membrane
(Maddrell, 1969; Gee, 1976; Phillips, 1981; O'Donnell
& Mad&ell, 1984; Nicolson, 1993). This possibility is
supported further by the observation that the addition of
furosemide, an inhibitor of Na+/K+/2C1- cotransporter,
blocked the RVI response.
Some cells exhibit a volume regulatory response
which is insensitive to ouabain (Russo et al., 1985, Pro-
verbio et al., 1989). In our experiments, we observed
that even with the addition of very high concentrations of
ouabain or vanadate the cells shrank after hyperosmotic
shock followed by a typical RVI. On the other hand, the
addition of ethacrynic acid abolished RVI. Ethacrynic
acid can be associated with the possible inhibition of the
Na+/K+/2C1- cotransporter, as described in other ceils
(O'Grady et al., 1987). But, it is also known that this
drug blocks a ouabain-insensitive pump (Proverbio et al.,
1989). These observations could be interpreted to indi-
cate that the primary active transport involved in RVI in
the Malpighian proximal tubules after hyperosmotic
stress is via a ouabain-insensitive, ethacrynic acid-
sensitive Na + pump (Proverbio et al., 1989). Similar
pumps have been proposed to be present in other cells,
where after swelling, they regulate volume by a ouabain-
insensitive mechanism (Whittembury & Proverbio,
1970; Del Castillo et al., 1982; Marfn et al., 1985; Pro-
verbio et al., 1986; Proverbio et al., 1989). It has also
been proposed that that the "primary" energy source for
fluid secretion in the Malpighian tubule is a Na + stimu-
lated, ouabain-insensitive ATPase, located in the apical
membrane (Maddrel, 1969; Gee, 1976; Phillips, 1981;
O'Donnell & Maddrell, 1984; Pannabecker et al., 1992).
Thus, we postulate that the primary active transport in-
volved in RVI could be this ouabain-insensitive, Na +-
stimulated pump. However, it has also been proposed
that the "prime mover" for fluid secretion in Malpighian
tubule is a H+-ATPase in parallel with K+/H+ or Na+/H+
exchangers (Wieczorek et al., 1991; Bertram et al., 1991;
Nicolson, 1993). Thus, further experiments will be
needed to prove our hypothesis.
Finally, this paper shows that RVI of the cells in the
Malpighian tubule is dependent on extracellular Ca 2+ and
is mediated by verapamil-sensitive transport pathways.
Several papers have shown that Ca 2+ is involved in RVD
in some cells. Whereas, the role of the Ca 2+ in RVI
response has been ruled out in some others (Bear, 1990;
Pierce & Politis, 1990; Montrose-Rafizadeh & Guggino,
1991; Hoffmann & Ussing, 1992; Lewis & Donaldson,
1990; MacCarty & O'Neil, 1991, 1992). MacCarty &
O'Neil (1990, 1991, 1992) showed that verapamil-
sensitive Ca 2+ channels play a role in RVD in rabbit
55
Basolateral lntracellular
Na+@
( Furosemide~ "[
~thacrynic Aeid~ ~ ~ Na+
Z ~2C1-
K+
[ Amiloride
~'C ~ Na+
aco,- L. Cl-
--C
[ .ceno z cl-
[ Verapamil y Zll~Ca++
Fig. 8. Working model showing postulated transporters involved in
cell volume regulation in the Malpighian tubule during hyperosmotic
shock. Inhibitors of the different pathways shown in boxes.
proximal tubule cells. It seems that these channels are
inactive in isosmotic medium and would activate in hy-
perosmotic solutions increasing the intracellular concen-
tration of Ca >. Thus, increases in intracellular Ca 2+
concentration would stimulate the volume regulatory
mechanisms either directly or indirectly (MacCarty &
O'Neil, 1992). Because RVI is Ca2+-and verapamil-
sensitive, it is possible that a similar mechanism partic-
ipates in RVI in our insect tubules. Figure 8 illustrates
the possible membrane ion transport systems involved in
RVI as discussed above: (1) Na+/H+ antiporter and/or
Na + channel; (2) CI- channels; (3) CI-/HCO 3 antiporter;
(4) Na+/K+/2C1- cotransporter; (5) ouabain-insensitive,
Na+-stimulated ATPase, and (6) verapamil-sensitive
Ca 2+ channels.
Thus, we conclude that proximal segment cells of
Malpighian tubules of R. neglectus regulate their volume
effectively in hyperosmotic solutions but only partially
in hyposmotic solutions.
We thank Drs. Gerhard Malnic, Guillermo Whittembury and William
B. Guggino for critical reading of the manuscript.
This work was supported by grants from the Funda~o de Amparo
h Pesquisa do Estado de S~o Paulo FAPESP; Conselho Nacional de
Desenvolvimento Cientffico e Tecnoldgico--CNPq e Financiadora de
Projetos e Pesquisas-FINEP.
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