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In vitro osmoregulation of taurine in fetal mouse hearts
J Mol Cell Cardiol 16, 311-320 (1984)
In v i t r o O s m o r e g u l a t i o n of Taurine in Fetal Mouse Hearts* Matthew Atlas, Joseph John Bahl, William Roesket, and Rubin Bressler Department of Internal Medicine, Arizona Health Sciences Center, University of Arizona, Tucson, A Z 85724, USA (Received 28 January 1983, accepted in revisedJbrm 3 August 1983) M. ATLAS,J. J. BAHL,W. ROESKE'~AND R. BRESSLER.In vitro Osmoregulation of Taurine in Fetal Mouse Hearts. Journal of Molecularand CellularCardiology(1984) 16, 311-320. Regulation of taurine transport and accumulation in explanted fetal mouse hearts is shown to be under osmotic control. All osmotic agents studied, both ionic (NaC1, LiCl, choline C1) and nonionic (sucrose, glucose) stimulated [~H]-taurine transport during an incubation of 19 h. Hyperosmotic stimulation of transport achieved statistical significance by 3 h in the presence of sucrose (P < 0.05). After 1 h, 40 mM NaC1 engendered a 56% increase in [3H]-taurine transport (P < 0.01). The NaCI stimulation at I h may relate more to the transport system's absolute sodium ion requirement,than hyperosmotic stimulation. Incremental addition of NaC1 or sucrose linearly stimulates [aH]-taurine transport in an incubation of 19 h. Total taurine, measured by HPLC, increased 25% with addition of either 40 mM NaC1 or 80 mM sucrose. Hyperosmotic stimulation of transport was not blocked with propranolol but was additive to ~-adrenergic stimulation of transport. Osmotic stimulation occurred with a large increase in Vmax (0.41 --* 0.81 nmol/mg tissue/h) but only a small change in Km (0.51 --+ 0.43 raM). After 1 h preincubation with a hyperosmotic addition phenylalanine transport was measured, but was not different from control. Phenylalanine accumulation measured during 19 h incubation similarly was not altered. Streptozotocin induced diabetic rats had elevated plasma osmolarities (295 • 2.1 ~ 322 • 1.3 mosmol) and cardiac taurine (24.3 ~- 1.2 --* 36 • 1.0 ~tmol/~ g wet wt.). The data presented demonstrates that mammalian cardiac taurine is regulated by the osmotic environment of the heart, suggesting an osmoregulatory function for intracellular taurine and physiological relevance in disease states such as diabetes. KEy WORDS: Taurine; Osmoregulation; Transport; Diabetes.
Introduction T h e m a m m a l i a n h e a r t strictly r e g u l a t e s t h e level o f c a r d i a c t a u r i n e b y a c a r r i e r m e d i a t e d t r a n s p o r t system w i t h o u t a n y c l e a r d e m o n s t r a t i o n o f significant in situ biosynthesis, b i n d i n g , or s u b c e l l u l a r c o m p a r t m e n t a l i z a t i o n [8, 12]. I n c r e a s e d c o n c e n t r a t i o n s o f c a r d i a c t a u r i n e , h o w e v e r , h a v e b e e n obs e r v e d in h u m a n c o n g e s t i v e h e a r t failure, e x p e r i m e n t a l a n i m a l h e a r t failure a n d in d i a b e t i c rats [10, 12, 20]. As s h o w n previously, s t i m u l a t i o n o f t r a n s p o r t b y ~a d r e n e r g i c agonists c a n be r e a d i l y b l o c k e d b y ~-adrenergic antagonists without alteration o f b a s a l t a u r i n e t r a n s p o r t [2, 11]. D e s p i t e c o n s i d e r a b l e i n t e r e s t in t a u r i n e , n o specific
f u n c t i o n for c a r d i a c t a u r i n e has b e e n c l e a r l y established. T h e w o r k o f T h u r s t o n et al. suggests t h a t t a u r i n e is i n v o l v e d in t h e o s m o r e g u l a t i o n o f the mammalian brain and heart. Using chronic hypernatremic, dehydrated mine 16 a m i n o acids m e a s u r e d in t h e b r a i n w e r e d e t e r m i n e d to be s i g n i f i c a n t l y e l e v a t e d on:'a d r y w e i g h t basis [24]. T a u r i n e a c c o u n t e d f6r o v e r o n e - h a l f o f the t o t a l a m i n o a c i d i n c r e a s e o b s e r v e d . I n a d d i t i o n , T h u r s t o n et al. h a v e d e m o n s t r a t e d a 3 0 % i n c r e a s e in t h e a m o u n t o f t a u r i n e in t h e h e a r t s o f these c h r o n i c a l l y d e h y d r a t e d , h y p e r n a t r e m i c m i c e [25]. S i m i larly, a n o s m o r e g u l a t o r y f u n c t i o n for t a u r i n e has b e e n d e m o n s t r a t e d in the h e a r t s o f fish
* This work was supported by Grants HL-13636, HL-20984 and GM07533. "~ Recipient of United States Public Health Service Research Scientist Development Award HL-00776 from the National Heart, Lung and Blood Institute. 0022-2828/84/040311 + 10 $03.00/0 9 1984 Academic Press Inc. (London) Limited
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and the muscles of invertebrates and amphibians [5, 14, 26]. Previous studies of the effect of osmolarity on cardiac taurine levels have utilized changes in sodium concentration as a model for changes in extracellular fluid osmolarity without consideration of other osmotic particles. It has been demonstrated in several models that taurine transport is sodium dependent [6] and therefore these Studies have not determined whether or not the changes in cardiac taurine are due to a change in sodium concentration, a change in osmolarity or a combination of the two effects. In this study we undertook an investigation of the effect of sodium ion concentration, extracellular hyperosmolarity and ~-adrenergic regulation on taurine transport in the fetal mouse heart. I n addition, the plasma osmolarity and cardiac taurine levels in control and streptozotocin diabetic rats were measured. We chose the intact, beating fetal mouse heart in organ culture as our model of study because: (1) This model provides a spontaneously and rhythmically beating whole heart which can be studied in vitro for periods up to 6 days in a chemically defined culture medium without the addition of hormones and/or serum [13, 27] ; (2) We had previously characterized taurine transport in this model [3, 7, 8]; and (3) The development of the ~-adrenergic receptor has been well characterized in the fetal mouse heart [4 ]. The results demonstrate that taurine transport, but not all amino acid transport, into the m a m m a l i a n myocardium is proportional to and regulated by extracellular fluid osmolarity and that osmotic control is separate and distinct from ~-adrenergic regulation of taurine'transport. An obligatory requirement for sodium ion was demonstrated for taurine transport and appears to be a prerequisite for all taurine transport in m a m m a l i a n myocardium because nonstimulated (basal) ~-adrenergic and osmotically stimulated taurine transport are abolished in the absence of sodium. Materials and Methods
Fetuses, between 16 and 17 days of gestation, were surgically removed from pregnant CFI strain white mice (Charles River Breeding Laboratories, Wilmington, MA) and placed
on ice. The hearts were removed from the fetuses, dissected free from pericardium and vessels and were then placed in ice cold 0.9% sterile saline solution until all of the hearts from the litter had been dissected. Each heart was then placed on individual stainless steel grids in separate organ culture dishes (Falcon Plastics, Div. Bio Quest, Oxnard, CA) containing 0 . 5 0 m l G I B C O (Grand Island Biological Co., Grand Island, NY) medium 199 with Earle's salts (sodium content of 146mu) and allowed 1 h to stabilize. All hearts were maintained in 3.0 1 airtight jars under an atmosphere of 95% 0 2 / 5 % C O 2 at 37~ The preincubation medium was modified accordingly for experiments using an incubation medium other than G I B C O medium 199. With 8 to 16 fetuses per litter, sufficient numbers of hearts were obtained to allow direct comparison between experimentally treated hearts and matched littermate control hearts, thereby minimizing individual variability. The transport of taurine was measuredin hearts from matched littermates in the following manner: After the 1-h preincubation, the preincubation medium was aspirated from the culture dishes and replaced with 0.50 ml of either medium 199 or a modified medium 199 supplemented with 10 ~Ci/ml [3H]-taurine and 1 [zCi/ml [14C]-sorbitol. The experimental and control groups contained appropriate amounts of taurine and/or various solutes (osmotic agents, drugs, etc.). 200 ~ i [3H]-phenylalaline (10 ~Ci/ml, with 1 ~Ci2ml [14C]-sorbitol) in medium 199 supplemented with 200 EZM taurine and osmotic additions as stated in the text was used as a marker of e-amino acid transport. After the specified time of experimental incubation, the hearts, of which typically 90% to 100)o were beating, were removed, blotted and weighed. In order to maximize consistency, only data from hearts weighing between 2 and 4 mg wet wt (fetuses at 16 to 17 days of gestation) at the end of the specified experimental incubation time period were used. The hearts were then dissolved by heating for 1 h at 60~ in 0.2 ml 2N N a O H and then neutralized using 0.2 ml 2N HC1. The radioactivity of the dissolved fetal mouse heart and of the experimental incubation medium (before and after the specified
Taurlne and Osmoregulation
experimental incubation period) was measured using liquid scintillation counting. T h e extracellular space and non-specific uptake for each heart was estimated using the nontransportable carbohydrate [14C]-sorbitol [19]. Within 20 min [14C]-sorbitol has marked the extracellular space of the heart and is thereafter accumulated slowly into intracellular spaces by nonspecific processes. Addition of [3H]-taurine and [14C]-sorbitol to the incubation medium was simultaneous. Net [3H]-taurine transport for each heart was calculated by subtracting the [aH]taurine in the sorbitol space from the total [3H]-taurine measured in each heart. Total taurine was analyzed utilizing reverse phase high performance liquid chromatographic (HPLC) separation of a fluorescent derivative of taurine obtained by the reaction of taurine with an o-phthalaldehyde/ethanethiol reagent [20, 22]. Diabetes was induced in male Sprague Dawley rats by intravenous administration of 75 mg/kg of the diabetogenic agent streptozotocin (Sigma Chemical Co., St Louis, MO) as reported previously [15]. Diabetic animals were hyperphagic, polyuric and had blood glucoses ranging from 375 to 550 mg ~ Plasma osmolarity was measured in both the normal and streptozotocin induced diabetic rats with a vapor pressure osmometer (Model 5100B, Wescor, Inc., Logan, U T ) . Comparisons between single treatment means and control were made by Student's t test. Comparisons between more than one treatment mean and control were made with Dunnett's multiple range test [29]. Best-fit lines were determined by a linear regression method or a nonlinear regression analysis. D a t a is presented as the mean • S.E.M. The taurine used was from Sigma Chemical C o m p a n y (St Louis, M O ) . [G-aH]-taurine, 3.9 Ci/mmol, was custom synthesized by catalytic exchange by New England Nuclear (Boston, MA). I t was purified by ionexchange chromatography as reported previously by H r u s k a et al. [9], and was determined to be greater than 98~ pure by thin layer chromatography (acetone/formic acid/water) and by isotopic dilution and recrystalization to constant specific activity, D[U14-C]-sorbitol, 100mCi/mmol, and L-
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[alanine-2,3-3H]-phenylalanine, 25 Ci/mmol were obtained from New England Nuclear (Boston, MA). Sodium free medium was made from appropriate inorganic salts and the appropriate component solutions supplied by Grand Island Biological Co., Grand Island, NY; Eagle's minimal essential medium (MEM)-vitamin solution ( • 100), antibioticantimycotic mixture ( • 100), M E M - a m i n o acid solution ( • 50).
Results
The fetal mouse hearts used in these experiments contained 18.99 ~ 5.7 nmol (n = 19) of endogenous taurine/mg of tissue. Unless stated otherwise, all fetal mouse hearts were incubated in the presence of 200 ~R [3H]taurine. Because Thurston et al. [24] had reported increased plasma sodium from 146 meq/1 to 186 meq/l in chronically dehydrated, hypernatremic mice for an estimated total of 410mosmol/1 we chose an addition of 80 mosmol/1 as our maximal hyperosmotic condition.
Effect of hyperosmolarity on [3H]-taurine transport Hearts were incubated individually for 1 or 19h in medium 199 with and without 80 mosg additions of either NaC1, sucrose, LiC1, choline chloride or glucose (see Table 1). During 1 h, supplementation of medium t99 with a 40 mM addition of NaC1 stimulated [3H]-taurine transport 56}/o (P < 0.01) as compared to control hearts incubated in unsupplemented medium 199. The mean value of [3H]-taurine transport at 1 h was greater in the presence of an 80 mosR addition of either sucrose, LiC1 or choline chloride but not glucose, however, the increases were not statistically significant as compared to the control hearts. This sug, gested the possibility that a statistically significant increase might be observed after a more chronic exposure. After an incubation of 3 h, addition of 80 mR sucrose or 40 mR sodium resulted in 313 4- 22.5 (n = 4) and 345 :~ 7.4 :(n = 3) pmol taurine transported per mg wet wt tissue, both values of which were statistically different from the
314
M. Atlas e t al.
TABLE I. [SH]-taurine transport during 1 and 19 h hyperosmolarity [~H]-taurine transport (prnol/mg tissue wet wt) Additions None (control) NaC1 (+40 mM) Sucrose ( + 8 0 raM) LiC1 (+40 mM) Choline chloride (+40 mM) Glucose (+80 raM) Values represent mean • S.E.M. 1 h incubation: 85 hearts from 13 litters. 19 h incubation: 39 hearts from 3 litters.
1h 123.8 191.5 146.2 134.8 144.0 120.0
4444i 4-
5.7 14.8 a 7.2 7.8 14.8 4.3
n
19 h
28 21 19 6 6 5
2215i84 4157 -4- 297 a 4053-4- 158a 30844-89 a 2880 4- 224 b 31944-249 a
a p < 0.01 compared to control. b p < 0.05 compared to control.
control value of 256 4- 7.6 (n = 4) pmol taurine transported per m g wet wt (P < 0.05). A n incubation of 19 h allowed for one day's experiments to end and the next day's to begin in a 24-h period. [3H]-taurine transport f o r ' 1 9 h was significantly stimulated (P < 0.05) in the presence of each of the osmotic agents by at least 30% to as m u c h as 90o/0 above the value observed in the control hearts. Although not included in T a b l e 1, data from hearts of a single litter incubated for both t and 19 h show the s a m e result as the litters incubated for either 1 or 19 h with the osmotic agents listed in T a b l e 1.
Effect of a range of osmolarity on 19 h of [SH]-taurine transport The a m o u n t of [~H]-taurine transported over 19 h was observed to be dependent upon the concentration of either the ionic (NaC1) or the nonionic (sucrose) osmotic agent added to the incubation m e d i u m as shown in Figure 1. Incremental increases in the concentration of either NaC1 or sucrose resulted in a corresponding linear increase in [SH]taurine transl6ort. Addition of as little as 5 r a m NaC1 o r 10mM sucrose resulted in t h e m e a n value of [~H]-taurine tra.nsport being 12% above c o n t r o l while an 80 mosM addition of either osmotic agent stimulated [SH]-taUi"ine transport as m u c h as 97%
240 o,.~
210
180 = ~ .,90,
150
I
% ~
120i 9O
I I0
1
I
20 30
[ 40
Additional
I
I
I
I
50 60 70 80 mosmoles
F I G U R E 1. Incremental additions of NaC1 ( O ) or sucrose ( A ) during 1 9 h [aH]-taurine transport. Values represent mean 4- S.E.M. (99 hearts, 8 litters).
above control. A n analysis of covariance performed by a stepwise, d u m m y variable, multiple regression procedure showed no statistical difference between the slopes of the NaC1 and sucrose addition curves.
Total taurine after 19 h hyperosmolarity As shown in Table 2, no significant change of cardiac taurine (measured by H P L C ) was observed after 19 h incubation in the presence of 200 ~M taurine (control osmolarity) c o m p a r e d to the a m o u n t observed after 1 h incubation ~n m e d i u m 199 containing no taurine (control). I n c u b a t i o n of fetal mouse h e a r t s in the presence of an addition of
315
Taurine and Osmoregulation TABLE 2. Total cardiac taurine measured after 19 h hyperosmolarity Total taurine (nmol/mg tissue wet wt)
Treatment t h, no murine 19 h, 200 aM taurine 19 h, 200 aM taurine, + 4 0 mM NaC1 19 h, 200 pM taurine, + 8 0 mM sucrose
19.1 20.5 24.9 25.8
~ 0.3 :k 1.2 st= 1.3a ~ 1.6a
HPLC analysis presented as the mean -1- S.E.M. (n -----3). The data from one litter is presented. The results were consistent in three experiments, but the data was not combined because of the litter to litter variation in endogenous taurine. a p < 0.05 compared to 19 h, 200 p~mtaurine.
4 0 r a m NaC1 or 80 mM sucrose for 19 h resulted in a statistically significant increase ( N a C 1 2 1 % , sucrose 25%) (P < 0.05) in total cardiac t a u r i n e as c o m p a r e d to control hearts.
Sodium dependence of [SH]-taurine transport atlh [ZH]-taurine transport was found to be d e p e n d e n t u p o n the presence of sodium in the i n c u b a t i o n m e d i u m as shown in T a b l e 3. I o n i c strength, chloride c o n c e n t r a t i o n a n d osmolarity were m a i n t a i n e d by r e p l a c e m e n t of the sodium chloride with l i t h i u m chloride. I s o p r o t e r e n o l si~gnificantly stimulated (P < 0.01) t a u r i n e transport i n the presence of 145 m g NaC1 (control). However, the total substitution of sodium chloride with l i t h i u m
chloride completely abolished b o t h basal a n d isoproterenol stimulated transport of labeled taurine. I n data not shown, p r o p r a n o l o l (2 • 10-v g) was found to block isoproterenol stimulated [ZH]-taurine transport a n d to have no effect on n o n - s t i m u l a t e d (basal) t a u r i n e transport. I n a second group of experiments (data not shown) using three litters (n = 32), complete r e p l a c e m e n t of sodium chloride with either l i t h i u m chloride, choline chloride, or sucrose resulted in no transport of taurine. W e were u n a b l e to study hyperosmolarity i n the absence of NaC1 at 19 h because the hearts did n o t survive. I n contrast to sodium d e p e n d e n c e experiments performed by Grosso et al. [7, 8], we p r e i n c u b a t e d the fetal mouse hearts in the same m e d i u m as the e x p e r i m e n t a l i n c u b a t i o n
TABLE 3. Sodium dependence of [ZH]-taurine transport at 1 h
Treatment 145 mM NaC1a Control Isoproterenol 2 • 10-n M NaC1b Control (145 mM LiC1) Isoproterenol 2 • 10-e M (145 mM LiC1)
[SH]-taurine transport (pmol/mg tissue wet wt)
125.1 =E 5.3 184.6 =L 9.1 e
0 mM
--4.3 • 0.3 c --6.3 ~z 1.3c
Values represent mean -k S.E.M. (43 hearts from four litters). pre and experimental incubation in medium 199 (145 mM NaCI). b pre and experimental incubation in modified sodium-free medium (145 mM LiC1). c p < 0.01 compared to 145 mM NaC1 control.
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M. Atlas e t al.
medium with respect to the concentrations of sodium chloride and the osmotic agent; lithium chloride, choline chloride or sucrose. Grosso et al. preincubated all fetal mouse hearts in medium 199 and then briefly washed the hearts with a modified medium 199 containing no sodium before placing the hearts in the experimental incubation medium. Our conditions, which ensured a more complete removal of the sodium from the fetal mouse hearts, resulted in no sodium independent taurine transport. We found that [aH]-taurine transport for 1 h in medium 199 was not affected by 1 h preincubation in modified (NaC1 free) medium 199 (control medium 199 preincubation 123.8 ~ 5.7 pmol/ mg wet wt; NaC1 free modified medium 199 preincubation 126.4 _+_0.8 pmol/mg wet wt).
taurine transport observed after 19 h is diminished by about 25% from linear extrapolation of the accumulation during 1 to 9 h. This is due at least in part to efflux of [3H]-taurine which becomes more significant as the label mixes with the intracellular pool of unlabeled taurine.
8
2.4z'8!
===2.o.
.='_
Time course of [aH]-taurine transport Hearts were incubated individually for selected times between 1 and 19 h in medium 199 with either 200 ~xM or 2 mM [3H]-taurine (Fig. 2). [3H]-taurine transport is shown to be linear over a 9~ time period. [3H]-
1:6
6
4
E E
-~ 0 . 4 0
7-5 5
0.8
Osmotic and ~-adrenergic regulation of [aH]-taurine transport over 19 h Propranolol, a ~-adrenergic antagonist, has been demonstrated to block isoproterenol stimulated taurine transport but to have no effect on non-stlmulated (basal) taurine transport [2, 12]. Osmotically stimulated [nH]-taurine transport at 19 h is also not blocked in the presence of 2 • 10-TM propranolol as shown in Table 4.
............................
2
4
6
8
I0 12 14 16 1819
Time ( h )
F I G U R E 2. T i m e course of [aH]-taurine transport. Hearts were incubated for varying times u p to 19 h with 200 ~,M ( O ) or 2 mM ( 9 taurine (means =k S.Z.M. 79 hearts, 7 litters).
Concentration dependence of [3H]-taurine transport Taurine transport was measured over the concentration range of 0.05 to 2 raM. We chose the time period between the 8th and 9th hour in the presence and in the absence of an 80 mM sucrose addition because the transport of 2 mM taurine was determined to be linear at that time (see Fig. 2). The
T A B L E 4. H y p e r o s m o t i c s t i m u l a t i o n o f [ 3 H ] - t a u r i n e t r a n s p o r t o v e r 19 h a n d lack o f i n h i b i t i o n b y ~-adrenergic blockade
Additions
[aH]-taurine transport ( p m o l / m g tissue w e t wt)
Control
2098•
Propranolol 2 • 10-7 M
2385 -~ 114 4389• a 4841 • 213b
S u c r o s e 80 mM Propranolol 2 •
10 -7 M, s u c r o s e 80 mM
[3H]-taurine transport m e a n • S.E.M. (33 hearts from three litters). a p < 0.01 compared to control. b p < 0.01 compared to control b u t not different from 80 mM sucrose.
Taurine and Osmoregulation ,~
0.8
,$
.~
317
__----t--
f
0.6
24
-6 E "C 0.4
-I>
8 12
O
0.2
_g
6
T
m ua
I
0.2
I
I
0.6
i
I
I
i
i
1.0 ETaurinelmM
t
[ 2.0
i0
I I0
I 20
I
[sJ
F I G U R E 3. Concentration dependence of the rate of [3H]-taurine transport between the 8th and 9th hour after 8 h in the presence ( A ) and absence (@) of 80 mM sucrose additional. Each point represents net [SH]taurine transport into a single heart.
[3H]-labeled taurine and [l*C]-sorbitol was added to the incubation medium after 8 h and [3H]-taurine transport was measured at 9 h. [3H]-taurine transport in the presence and in the absence of 80 mM sucrose as shown in Figure 3 is a saturable process indicative of a carrier mediated transport system. A linear regression analysis of a double reciprocal plot of the data was performed in order to determine the kinetic parameters (Kin and Vmax)' In the control fetal mouse hearts (no osmotic agent) the Kra and Vm.x were determined to be 0.51 mM and 0.41 nmol/ mg tissue/h respectively. In the presence of 80 mosM sucrose the Km and "Vmax were determined to be 0.43 mM and 0.81 nmol/mg tissue/h respectively. Thus the increased osmolarity appeared to engender a significant increase in the Vma* for taurine transport.
Specificity of hyperosmotic stimulated transport Unlike taurine transport that does not saturate for many hours, phenylalanine transport saturates rapidly [17]. To determine if phenylalanine transport was affected by hyperosmolarity hearts were preincubated for I h with and without addition of 80 mM
sucrose prior to being transferred on their stainless steel grid to a culture dish containing [3H]-phenylalanine and [ar in medium 199, with and without 80mM sucrose as in the preincubation. [3H]-phenylalanine transport was not saturated by 1 or 3 min and no difference was found with or without hyperosmotic addition at either of these time points. By 5 rain the pool of phenylalanine was saturated but the 12.9 • 1.0 pmol/mg wet wt (n = 20) observed with 80 mM sucrose was not significantly different from the 12.0 • 0.8 pmol/mg wet wt (n = 21) of the control. No statistically significant difference was observed between groups in the same litter and neither the presence of 200 ~XMtaurine nor extending the preincubation time with the osmotic addition to 4 h changed this observation. Phenylalanine accumulation continues over time as phenylalanine is incorporated into protein. After 19 h [3H]-phenylalanine accumulation (3 litters, 21 hearts) in the presence of 80 mM added sucrose (1.43 40.05 nmol/mg wet wt) was not significantly different from control (1.45 4- 0.05 nmol/mg wet wt). Sucrose was used so as not to alter the sodium gradient which is known to affect amino acid transport.
318
M. Atlas e t al.
Correlation of cardiac taurine and plasma osmolarity in streptozotocin-diabetic rats and age-matched controls Total cardiac taurine and plasma osmolarity increased in rats (n = 15) 2 weeks after streptozotocin induced diabetes as compared to age matched controls (n = 10) (plasma osmolarity: 295 4- 2.1 ~ 322 ~ 1.3 mosmol P < 0.001; endogenous cardiac taurine: 24,3 =~ 1.2 -+ 36 • 1.0 ~tM/gram wet wt tissue, P < 0.001). Discussion
The physiological basis for the close regulation of the high taurine concentrations found in the m a m m a l i a n heart has remained obscure. In this study it was found that during 19h all of the osmotic agents tested both ionic and nonionic stimulated taurine transport (Table 1). The magnitude of the stimulation was observed to be proportional to the concentration of the osmotic agent indicating the sensitivity of taurine transport to changes in osmolarity. This suggested that in vivo alterations of plasma osmolarity might affect changes in the levels of cardiac taurine. The physiological significance of the osmotic effect on taurine transport and accumulation is suggested by the increase in plasma osmolarity and cardiac taurine levels observed in streptozotocin-induced diabetic rats as compared to nondiabetic controls. Whether the increased accumulation of cardiac taurine observed in diabetes plays ar~y role in the natural history of diabetic complications is unknown. The fact that the exact magnitude of stimulation was different for' each of the osmotic agents during 19 h suggests that the fetal mouse heart responds differently to various osmotic agents. Because NaC1 directly increases taurine transport at l h and different osmotic agents result in different amounts of taurine transport over 19 h, it is most likely to be coincidental that the 19 h sodium and sucrose stimulations are equivalent. The effect of NaC1 is most likely to be the sum of direct NaC1 stimulation and osmotic stimulation. Stimulation of [SH]-taurine transport by increased osmolarity was shown to be separate from the ~-adrenergic mediated taurine transport during 19h (Table 4). Whereas chronic
~-adrenergic stimulation m a y be the cause of the elevated taurine observed in congestive heart failure and perhaps hypertension, osmotic stimulation of taurine transport appears to be the cause of the reported increased cardiac taurine observed in diabetes [20]. In the absence of an osmotic stress maintenance of the steady state level of cardiac taurine allows only for the exchange o f extracellular labeled taurine for an equivalent amount of unlabeled intracellular taurine (Tables 1 and 2). However, in the presence of an osmotic stress, we observed an increase in the amount of [3H]-taurine transported over a range of concentrations and a 98% increase in the maximal velocity of transport without any meaningful change in the Km of transport (Fig. 3). Alterations in efflux which would contribute to defining steady state levels has not yet been studied. All taurine transport in the fetal mouse heart was demonstrated to have an absolute requirement for the presence of sodium (Table 3). With incremental increases in the sodium ion concentration, above 145 mM (control), taurine transport is linearly stimulated (Fig. 1). The stimulation of taurine transport by sodium is a phenomenon separate from ~-adrenergic stimulation of taurine transport and at 1 h is greater than the response to an equivalent non-sodium increase in osmolarity. The dependence of the taurine transport system on sodium has been demonstrated in various tissues from rat and fish [1, 5, 6, 18, 22] and would appear to be a universal characteristic of taurine transport. Studies by Schaffer and Kulakowski using purified taurine binding proteins solubilized from swine sarcolemma and reconstituted into proteoliposomes demonstrate a sodium requirement for taurine transport and suggest that taurine transport is an electrogenic process driven by the inward gradient of sodium ions [22]. In our model we show a significant (P < 0.01) increase in taurine transport at I h with the addition of 40 mM NaC1 while addition of sucrose, LiCI, choline chloride or glucose did not result in statistically significant stimulation of taurine transport (Table 1). The use of free amino acids in the osmoregulation of m a m m a l i a n tissue was first
Taurine and Osmoregulation
suggested by the work of McDowell et al. [16]. Their work demonstrated the ability of dogs and cats, injected with hypertonic solutions, to generate osmotically active particles in what they explained as an attempt to limit cellular dehydration. The work by Thurston et al. showing the elevation of taurine levels in the brains and hearts of hypernatremic, dehydrated mice further demonstrates the possible significance of osmolarity in the regulation of taurine transport in mammalian tissues [24, 25]. Our studies establish that taurine transport, more than just being dependent on sodium ion concentration, is under general osmotic regulation. Phenylalanine is transported by a separate transport system that was not stimulated by hyperosmolarity, thus the response of taurine transport and accumulation to increased osmolarity is selective. The osmoregulation of taurine would enable the cell to alter its osmotic content without involving the various ions required to main-
319
tain necessary membrane potentials and gains additional advantage by sparing amino acids required for protein synthesis. A role for taurine in the osmoregulatory system of the mammalian myocardium is suggested by ou'r work in the fetal mouse heart and previous work in the adult fish hearts of the Platichthys flesus {flounder) [26] and the Raja erinacea (little skate) [6] indicating that this role for cardiac taurine may be general. The data presented has demonstrated that cardiac taurine transport is regulated by the osmotic environment of the myocardium and suggests that taurine is an osmoregutator in the mammalian heart.
Acknowledgement The authors wish to thank Dr Murray Korc for his generous supply of streptozotocindiabetic rats and age-matched controls and many helpful discussions.
References 1 AWAPARA,J., BERG, M. Uptake of taurine by slices of rat heart and kidney. In: Huxtable AND Barbeau, (Eds) Taurine, pp. 135-143. New York: Raven Press (1976). 2 AZARI,J., ttUXTABL% R. J. The mechanism of the adrenergic stimulation of taurine influx in the heart. E u r J Pharmacol 51, 217-223 (1980). 3 BRESSLER,R., GROSSO, D. S., ROESKE, W. R. Carrier-mediated taurine uptake in the fetal mouse heart. Trans Assoc Am Phys 90, 257-269 (1977). 4 CHEN,F. M., YAMAMURA,H. I., ROESKE,W. R. Ontogeny of mammalian myocardial [3-adrenergic receptors. Eur J Pharmacol 58, 255-264 (1979). 5 FORSTER,R. P., GOLDSTm~,L. Amino acids and cell volume regulation. YaleJ Biol Med 52, 497-515 (1979). 6 FORSTER,R. P., HANNAFIN,J. A. Taurine uptake in atrial myocardium by a sodium-dependent p-amino acid system in the elasmobranch, Raja erinacea. Comp Biochem Physiol 67, 107-113 (1980). 7 GRosso, D. S., ROESKE,W. R., BRESSLER,R. Taurine transport in the fetal mouse heart. Proc West Pharmacol Soc 20, 239-243 (1977). 8 GRosso, D. S., ROESKF.,W. R., BRESSLER,R. Characterization of a carrier-mediated transport system for taurine in the fetal mouse heart in vitro..J Clin Invest 61, 944-952 (1978). 9 HRosr~, R. E., HOXTA~LE, R. J., YAMAMO~, H. I. Purification of [3H]-taurine of high specific activity. Anal Biochem 79, 568-570 (1977). !0 HUXTABLE,R., BRESSLER, R. Taurine concentration in congestive heart failure. Science 184, 1187-I188 (1974). 11 HUXTABLE,R., CHUBB,,J. Adrenergic stimulation of taurine transport by the heart. Science 198, 409-411 (1977). 12 HUXTAnLE, R. J. Regulation of taurine in the heart. In: Barbeau AND Huxtable, (Eds) Taurine and Neurological Disorders, pp. 5-17. New York: Raven Press (1978). 13 INGWALL,J. S., ROESKE,W. R., WXLDENTHAL,K. The fetal mouse heart in organ culture: Maintenance of the differentiated state. Methods Cell Biol 21A, 167-187 (1980). 14 JACOBSEN,J. G., SMITH, L. H. Biochemistry and physiology of taurine derivatives. Physiol Rev 48, 424-511 (1968). 15 KORC, M., IWAMOTO, Y., SANKARAN,H., WILLIAMS,J. A., GOLDFINE, I. D. Insulin action in pancreatic acini from streptozotocin-treated rats. I. Stimulation of protein synthesis. Am J Physio1240, G56-G62 (1981). 16 McDOWELC, M. E., WOLF, A. V., STEER, A. Osmotic volumes of distribution: Idiogenic changes inosmotic pressure associated with administration of hypertonic solutions. Am J Physiol 180, 545-558 (1955).
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17 McKEE, E. E., CHEUNG,J. Y., RANNELS,D. E., MORGAN,H. E. Measurement of the rate of protein symthesis and compartmentation of heart phenylalanine. J Biol Chem 253, 1030-1040 (1978). 18 MEINERS,B. A., SPETH, R. C., BR~SOLIN,N., HUXTABLE,R.J., YAUAUURA,H. I. Sodium-dependent, highaffinity taurine transport into rat brain synaptosomes. Fed Proc 39, 2695-2700 (1981). 19 MORGAN,H. E., HENDERSON,H. J., REGAN, D. M., PARK, C. R. Regulation of glucose uptake in muscle. J Biol Chem 236, 253-261 (1961). 20 REmEL, D. K., SI~AFPER,J. E., Kocsis, J. J., NEELV,J. R. Changes in taurine content in heart and other organs of diabetic rats. J Mol Cell Cardiol 11,827-830 (1979). 21 ROTH, M. Fluorescence reaction for amino acids. Anal Chem 43, 880-882 (1971). 22 SCnAFFSR,S., KULA~OWSKI,E. Reconstitution of taurine transport in membrane vesicles. Fed Proc 40, 891 (1981). 23 STUART,J. D., WILSON,T. D., HILL, D. W., WALTERS,F. H., FENO, S. Y. High performance liquid chromatographic separation and fluorescent measurement of taurine, a key amino acid. J Liq Chromatogr 2, 809-821 (1979). 24 TnURSTON,J. H., HAUnAR% H. E., DinGo, D. A. Taurine: A role in osmotic regulation of mammalian brain and possible clinical significance. Life Sci 26, 1561-1568 (1980). 25 TnURSTON,J. H., HAUHART, H. E., NACCARATO,E. F. Taurine: Possible role in osmotic regulation of mammalian hearts. Science 214, 1373-1374 (1981). 26 VIsLm, T., FUOELLI,K. Cell volume regulation in flounder (Platichthysflesus) heart muscle accompanying an alteration in plasma osmolarity. Comp Biochem Physiol 52A, 415-418 (1975). 27 WILDENTHAL,K. Long term maintenance of spontaneously beating mouse hearts in organ culture. J Appl Physiol 30, 153-157 (1971). 28 WILDENTnAL,K. Studies of foetal mouse hearts in organ culture: Metabolic requirements for prolonged function in vitro and the influence of cardiac maturation on substrate utilization. J Mol Cell Cardiol 5, 87-99 (1973). 29 ZAR,J. H. Multiple comparison. In: Biostatistical Methods, pp. 151-162. Englewood Cliffs, N.J.: PrenticeHall, Inc. (1974).
In v i t r o O s m o r e g u l a t i o n of Taurine in Fetal Mouse Hearts* Matthew Atlas, Joseph John Bahl, William Roesket, and Rubin Bressler Department of Internal Medicine, Arizona Health Sciences Center, University of Arizona, Tucson, A Z 85724, USA (Received 28 January 1983, accepted in revisedJbrm 3 August 1983) M. ATLAS,J. J. BAHL,W. ROESKE'~AND R. BRESSLER.In vitro Osmoregulation of Taurine in Fetal Mouse Hearts. Journal of Molecularand CellularCardiology(1984) 16, 311-320. Regulation of taurine transport and accumulation in explanted fetal mouse hearts is shown to be under osmotic control. All osmotic agents studied, both ionic (NaC1, LiCl, choline C1) and nonionic (sucrose, glucose) stimulated [~H]-taurine transport during an incubation of 19 h. Hyperosmotic stimulation of transport achieved statistical significance by 3 h in the presence of sucrose (P < 0.05). After 1 h, 40 mM NaC1 engendered a 56% increase in [3H]-taurine transport (P < 0.01). The NaCI stimulation at I h may relate more to the transport system's absolute sodium ion requirement,than hyperosmotic stimulation. Incremental addition of NaC1 or sucrose linearly stimulates [aH]-taurine transport in an incubation of 19 h. Total taurine, measured by HPLC, increased 25% with addition of either 40 mM NaC1 or 80 mM sucrose. Hyperosmotic stimulation of transport was not blocked with propranolol but was additive to ~-adrenergic stimulation of transport. Osmotic stimulation occurred with a large increase in Vmax (0.41 --* 0.81 nmol/mg tissue/h) but only a small change in Km (0.51 --+ 0.43 raM). After 1 h preincubation with a hyperosmotic addition phenylalanine transport was measured, but was not different from control. Phenylalanine accumulation measured during 19 h incubation similarly was not altered. Streptozotocin induced diabetic rats had elevated plasma osmolarities (295 • 2.1 ~ 322 • 1.3 mosmol) and cardiac taurine (24.3 ~- 1.2 --* 36 • 1.0 ~tmol/~ g wet wt.). The data presented demonstrates that mammalian cardiac taurine is regulated by the osmotic environment of the heart, suggesting an osmoregulatory function for intracellular taurine and physiological relevance in disease states such as diabetes. KEy WORDS: Taurine; Osmoregulation; Transport; Diabetes.
Introduction T h e m a m m a l i a n h e a r t strictly r e g u l a t e s t h e level o f c a r d i a c t a u r i n e b y a c a r r i e r m e d i a t e d t r a n s p o r t system w i t h o u t a n y c l e a r d e m o n s t r a t i o n o f significant in situ biosynthesis, b i n d i n g , or s u b c e l l u l a r c o m p a r t m e n t a l i z a t i o n [8, 12]. I n c r e a s e d c o n c e n t r a t i o n s o f c a r d i a c t a u r i n e , h o w e v e r , h a v e b e e n obs e r v e d in h u m a n c o n g e s t i v e h e a r t failure, e x p e r i m e n t a l a n i m a l h e a r t failure a n d in d i a b e t i c rats [10, 12, 20]. As s h o w n previously, s t i m u l a t i o n o f t r a n s p o r t b y ~a d r e n e r g i c agonists c a n be r e a d i l y b l o c k e d b y ~-adrenergic antagonists without alteration o f b a s a l t a u r i n e t r a n s p o r t [2, 11]. D e s p i t e c o n s i d e r a b l e i n t e r e s t in t a u r i n e , n o specific
f u n c t i o n for c a r d i a c t a u r i n e has b e e n c l e a r l y established. T h e w o r k o f T h u r s t o n et al. suggests t h a t t a u r i n e is i n v o l v e d in t h e o s m o r e g u l a t i o n o f the mammalian brain and heart. Using chronic hypernatremic, dehydrated mine 16 a m i n o acids m e a s u r e d in t h e b r a i n w e r e d e t e r m i n e d to be s i g n i f i c a n t l y e l e v a t e d on:'a d r y w e i g h t basis [24]. T a u r i n e a c c o u n t e d f6r o v e r o n e - h a l f o f the t o t a l a m i n o a c i d i n c r e a s e o b s e r v e d . I n a d d i t i o n , T h u r s t o n et al. h a v e d e m o n s t r a t e d a 3 0 % i n c r e a s e in t h e a m o u n t o f t a u r i n e in t h e h e a r t s o f these c h r o n i c a l l y d e h y d r a t e d , h y p e r n a t r e m i c m i c e [25]. S i m i larly, a n o s m o r e g u l a t o r y f u n c t i o n for t a u r i n e has b e e n d e m o n s t r a t e d in the h e a r t s o f fish
* This work was supported by Grants HL-13636, HL-20984 and GM07533. "~ Recipient of United States Public Health Service Research Scientist Development Award HL-00776 from the National Heart, Lung and Blood Institute. 0022-2828/84/040311 + 10 $03.00/0 9 1984 Academic Press Inc. (London) Limited
312
M. Atlas e t al.
and the muscles of invertebrates and amphibians [5, 14, 26]. Previous studies of the effect of osmolarity on cardiac taurine levels have utilized changes in sodium concentration as a model for changes in extracellular fluid osmolarity without consideration of other osmotic particles. It has been demonstrated in several models that taurine transport is sodium dependent [6] and therefore these Studies have not determined whether or not the changes in cardiac taurine are due to a change in sodium concentration, a change in osmolarity or a combination of the two effects. In this study we undertook an investigation of the effect of sodium ion concentration, extracellular hyperosmolarity and ~-adrenergic regulation on taurine transport in the fetal mouse heart. I n addition, the plasma osmolarity and cardiac taurine levels in control and streptozotocin diabetic rats were measured. We chose the intact, beating fetal mouse heart in organ culture as our model of study because: (1) This model provides a spontaneously and rhythmically beating whole heart which can be studied in vitro for periods up to 6 days in a chemically defined culture medium without the addition of hormones and/or serum [13, 27] ; (2) We had previously characterized taurine transport in this model [3, 7, 8]; and (3) The development of the ~-adrenergic receptor has been well characterized in the fetal mouse heart [4 ]. The results demonstrate that taurine transport, but not all amino acid transport, into the m a m m a l i a n myocardium is proportional to and regulated by extracellular fluid osmolarity and that osmotic control is separate and distinct from ~-adrenergic regulation of taurine'transport. An obligatory requirement for sodium ion was demonstrated for taurine transport and appears to be a prerequisite for all taurine transport in m a m m a l i a n myocardium because nonstimulated (basal) ~-adrenergic and osmotically stimulated taurine transport are abolished in the absence of sodium. Materials and Methods
Fetuses, between 16 and 17 days of gestation, were surgically removed from pregnant CFI strain white mice (Charles River Breeding Laboratories, Wilmington, MA) and placed
on ice. The hearts were removed from the fetuses, dissected free from pericardium and vessels and were then placed in ice cold 0.9% sterile saline solution until all of the hearts from the litter had been dissected. Each heart was then placed on individual stainless steel grids in separate organ culture dishes (Falcon Plastics, Div. Bio Quest, Oxnard, CA) containing 0 . 5 0 m l G I B C O (Grand Island Biological Co., Grand Island, NY) medium 199 with Earle's salts (sodium content of 146mu) and allowed 1 h to stabilize. All hearts were maintained in 3.0 1 airtight jars under an atmosphere of 95% 0 2 / 5 % C O 2 at 37~ The preincubation medium was modified accordingly for experiments using an incubation medium other than G I B C O medium 199. With 8 to 16 fetuses per litter, sufficient numbers of hearts were obtained to allow direct comparison between experimentally treated hearts and matched littermate control hearts, thereby minimizing individual variability. The transport of taurine was measuredin hearts from matched littermates in the following manner: After the 1-h preincubation, the preincubation medium was aspirated from the culture dishes and replaced with 0.50 ml of either medium 199 or a modified medium 199 supplemented with 10 ~Ci/ml [3H]-taurine and 1 [zCi/ml [14C]-sorbitol. The experimental and control groups contained appropriate amounts of taurine and/or various solutes (osmotic agents, drugs, etc.). 200 ~ i [3H]-phenylalaline (10 ~Ci/ml, with 1 ~Ci2ml [14C]-sorbitol) in medium 199 supplemented with 200 EZM taurine and osmotic additions as stated in the text was used as a marker of e-amino acid transport. After the specified time of experimental incubation, the hearts, of which typically 90% to 100)o were beating, were removed, blotted and weighed. In order to maximize consistency, only data from hearts weighing between 2 and 4 mg wet wt (fetuses at 16 to 17 days of gestation) at the end of the specified experimental incubation time period were used. The hearts were then dissolved by heating for 1 h at 60~ in 0.2 ml 2N N a O H and then neutralized using 0.2 ml 2N HC1. The radioactivity of the dissolved fetal mouse heart and of the experimental incubation medium (before and after the specified
Taurlne and Osmoregulation
experimental incubation period) was measured using liquid scintillation counting. T h e extracellular space and non-specific uptake for each heart was estimated using the nontransportable carbohydrate [14C]-sorbitol [19]. Within 20 min [14C]-sorbitol has marked the extracellular space of the heart and is thereafter accumulated slowly into intracellular spaces by nonspecific processes. Addition of [3H]-taurine and [14C]-sorbitol to the incubation medium was simultaneous. Net [3H]-taurine transport for each heart was calculated by subtracting the [aH]taurine in the sorbitol space from the total [3H]-taurine measured in each heart. Total taurine was analyzed utilizing reverse phase high performance liquid chromatographic (HPLC) separation of a fluorescent derivative of taurine obtained by the reaction of taurine with an o-phthalaldehyde/ethanethiol reagent [20, 22]. Diabetes was induced in male Sprague Dawley rats by intravenous administration of 75 mg/kg of the diabetogenic agent streptozotocin (Sigma Chemical Co., St Louis, MO) as reported previously [15]. Diabetic animals were hyperphagic, polyuric and had blood glucoses ranging from 375 to 550 mg ~ Plasma osmolarity was measured in both the normal and streptozotocin induced diabetic rats with a vapor pressure osmometer (Model 5100B, Wescor, Inc., Logan, U T ) . Comparisons between single treatment means and control were made by Student's t test. Comparisons between more than one treatment mean and control were made with Dunnett's multiple range test [29]. Best-fit lines were determined by a linear regression method or a nonlinear regression analysis. D a t a is presented as the mean • S.E.M. The taurine used was from Sigma Chemical C o m p a n y (St Louis, M O ) . [G-aH]-taurine, 3.9 Ci/mmol, was custom synthesized by catalytic exchange by New England Nuclear (Boston, MA). I t was purified by ionexchange chromatography as reported previously by H r u s k a et al. [9], and was determined to be greater than 98~ pure by thin layer chromatography (acetone/formic acid/water) and by isotopic dilution and recrystalization to constant specific activity, D[U14-C]-sorbitol, 100mCi/mmol, and L-
313
[alanine-2,3-3H]-phenylalanine, 25 Ci/mmol were obtained from New England Nuclear (Boston, MA). Sodium free medium was made from appropriate inorganic salts and the appropriate component solutions supplied by Grand Island Biological Co., Grand Island, NY; Eagle's minimal essential medium (MEM)-vitamin solution ( • 100), antibioticantimycotic mixture ( • 100), M E M - a m i n o acid solution ( • 50).
Results
The fetal mouse hearts used in these experiments contained 18.99 ~ 5.7 nmol (n = 19) of endogenous taurine/mg of tissue. Unless stated otherwise, all fetal mouse hearts were incubated in the presence of 200 ~R [3H]taurine. Because Thurston et al. [24] had reported increased plasma sodium from 146 meq/1 to 186 meq/l in chronically dehydrated, hypernatremic mice for an estimated total of 410mosmol/1 we chose an addition of 80 mosmol/1 as our maximal hyperosmotic condition.
Effect of hyperosmolarity on [3H]-taurine transport Hearts were incubated individually for 1 or 19h in medium 199 with and without 80 mosg additions of either NaC1, sucrose, LiC1, choline chloride or glucose (see Table 1). During 1 h, supplementation of medium t99 with a 40 mM addition of NaC1 stimulated [3H]-taurine transport 56}/o (P < 0.01) as compared to control hearts incubated in unsupplemented medium 199. The mean value of [3H]-taurine transport at 1 h was greater in the presence of an 80 mosR addition of either sucrose, LiC1 or choline chloride but not glucose, however, the increases were not statistically significant as compared to the control hearts. This sug, gested the possibility that a statistically significant increase might be observed after a more chronic exposure. After an incubation of 3 h, addition of 80 mR sucrose or 40 mR sodium resulted in 313 4- 22.5 (n = 4) and 345 :~ 7.4 :(n = 3) pmol taurine transported per mg wet wt tissue, both values of which were statistically different from the
314
M. Atlas e t al.
TABLE I. [SH]-taurine transport during 1 and 19 h hyperosmolarity [~H]-taurine transport (prnol/mg tissue wet wt) Additions None (control) NaC1 (+40 mM) Sucrose ( + 8 0 raM) LiC1 (+40 mM) Choline chloride (+40 mM) Glucose (+80 raM) Values represent mean • S.E.M. 1 h incubation: 85 hearts from 13 litters. 19 h incubation: 39 hearts from 3 litters.
1h 123.8 191.5 146.2 134.8 144.0 120.0
4444i 4-
5.7 14.8 a 7.2 7.8 14.8 4.3
n
19 h
28 21 19 6 6 5
2215i84 4157 -4- 297 a 4053-4- 158a 30844-89 a 2880 4- 224 b 31944-249 a
a p < 0.01 compared to control. b p < 0.05 compared to control.
control value of 256 4- 7.6 (n = 4) pmol taurine transported per m g wet wt (P < 0.05). A n incubation of 19 h allowed for one day's experiments to end and the next day's to begin in a 24-h period. [3H]-taurine transport f o r ' 1 9 h was significantly stimulated (P < 0.05) in the presence of each of the osmotic agents by at least 30% to as m u c h as 90o/0 above the value observed in the control hearts. Although not included in T a b l e 1, data from hearts of a single litter incubated for both t and 19 h show the s a m e result as the litters incubated for either 1 or 19 h with the osmotic agents listed in T a b l e 1.
Effect of a range of osmolarity on 19 h of [SH]-taurine transport The a m o u n t of [~H]-taurine transported over 19 h was observed to be dependent upon the concentration of either the ionic (NaC1) or the nonionic (sucrose) osmotic agent added to the incubation m e d i u m as shown in Figure 1. Incremental increases in the concentration of either NaC1 or sucrose resulted in a corresponding linear increase in [SH]taurine transl6ort. Addition of as little as 5 r a m NaC1 o r 10mM sucrose resulted in t h e m e a n value of [~H]-taurine tra.nsport being 12% above c o n t r o l while an 80 mosM addition of either osmotic agent stimulated [SH]-taUi"ine transport as m u c h as 97%
240 o,.~
210
180 = ~ .,90,
150
I
% ~
120i 9O
I I0
1
I
20 30
[ 40
Additional
I
I
I
I
50 60 70 80 mosmoles
F I G U R E 1. Incremental additions of NaC1 ( O ) or sucrose ( A ) during 1 9 h [aH]-taurine transport. Values represent mean 4- S.E.M. (99 hearts, 8 litters).
above control. A n analysis of covariance performed by a stepwise, d u m m y variable, multiple regression procedure showed no statistical difference between the slopes of the NaC1 and sucrose addition curves.
Total taurine after 19 h hyperosmolarity As shown in Table 2, no significant change of cardiac taurine (measured by H P L C ) was observed after 19 h incubation in the presence of 200 ~M taurine (control osmolarity) c o m p a r e d to the a m o u n t observed after 1 h incubation ~n m e d i u m 199 containing no taurine (control). I n c u b a t i o n of fetal mouse h e a r t s in the presence of an addition of
315
Taurine and Osmoregulation TABLE 2. Total cardiac taurine measured after 19 h hyperosmolarity Total taurine (nmol/mg tissue wet wt)
Treatment t h, no murine 19 h, 200 aM taurine 19 h, 200 aM taurine, + 4 0 mM NaC1 19 h, 200 pM taurine, + 8 0 mM sucrose
19.1 20.5 24.9 25.8
~ 0.3 :k 1.2 st= 1.3a ~ 1.6a
HPLC analysis presented as the mean -1- S.E.M. (n -----3). The data from one litter is presented. The results were consistent in three experiments, but the data was not combined because of the litter to litter variation in endogenous taurine. a p < 0.05 compared to 19 h, 200 p~mtaurine.
4 0 r a m NaC1 or 80 mM sucrose for 19 h resulted in a statistically significant increase ( N a C 1 2 1 % , sucrose 25%) (P < 0.05) in total cardiac t a u r i n e as c o m p a r e d to control hearts.
Sodium dependence of [SH]-taurine transport atlh [ZH]-taurine transport was found to be d e p e n d e n t u p o n the presence of sodium in the i n c u b a t i o n m e d i u m as shown in T a b l e 3. I o n i c strength, chloride c o n c e n t r a t i o n a n d osmolarity were m a i n t a i n e d by r e p l a c e m e n t of the sodium chloride with l i t h i u m chloride. I s o p r o t e r e n o l si~gnificantly stimulated (P < 0.01) t a u r i n e transport i n the presence of 145 m g NaC1 (control). However, the total substitution of sodium chloride with l i t h i u m
chloride completely abolished b o t h basal a n d isoproterenol stimulated transport of labeled taurine. I n data not shown, p r o p r a n o l o l (2 • 10-v g) was found to block isoproterenol stimulated [ZH]-taurine transport a n d to have no effect on n o n - s t i m u l a t e d (basal) t a u r i n e transport. I n a second group of experiments (data not shown) using three litters (n = 32), complete r e p l a c e m e n t of sodium chloride with either l i t h i u m chloride, choline chloride, or sucrose resulted in no transport of taurine. W e were u n a b l e to study hyperosmolarity i n the absence of NaC1 at 19 h because the hearts did n o t survive. I n contrast to sodium d e p e n d e n c e experiments performed by Grosso et al. [7, 8], we p r e i n c u b a t e d the fetal mouse hearts in the same m e d i u m as the e x p e r i m e n t a l i n c u b a t i o n
TABLE 3. Sodium dependence of [ZH]-taurine transport at 1 h
Treatment 145 mM NaC1a Control Isoproterenol 2 • 10-n M NaC1b Control (145 mM LiC1) Isoproterenol 2 • 10-e M (145 mM LiC1)
[SH]-taurine transport (pmol/mg tissue wet wt)
125.1 =E 5.3 184.6 =L 9.1 e
0 mM
--4.3 • 0.3 c --6.3 ~z 1.3c
Values represent mean -k S.E.M. (43 hearts from four litters). pre and experimental incubation in medium 199 (145 mM NaCI). b pre and experimental incubation in modified sodium-free medium (145 mM LiC1). c p < 0.01 compared to 145 mM NaC1 control.
316
M. Atlas e t al.
medium with respect to the concentrations of sodium chloride and the osmotic agent; lithium chloride, choline chloride or sucrose. Grosso et al. preincubated all fetal mouse hearts in medium 199 and then briefly washed the hearts with a modified medium 199 containing no sodium before placing the hearts in the experimental incubation medium. Our conditions, which ensured a more complete removal of the sodium from the fetal mouse hearts, resulted in no sodium independent taurine transport. We found that [aH]-taurine transport for 1 h in medium 199 was not affected by 1 h preincubation in modified (NaC1 free) medium 199 (control medium 199 preincubation 123.8 ~ 5.7 pmol/ mg wet wt; NaC1 free modified medium 199 preincubation 126.4 _+_0.8 pmol/mg wet wt).
taurine transport observed after 19 h is diminished by about 25% from linear extrapolation of the accumulation during 1 to 9 h. This is due at least in part to efflux of [3H]-taurine which becomes more significant as the label mixes with the intracellular pool of unlabeled taurine.
8
2.4z'8!
===2.o.
.='_
Time course of [aH]-taurine transport Hearts were incubated individually for selected times between 1 and 19 h in medium 199 with either 200 ~xM or 2 mM [3H]-taurine (Fig. 2). [3H]-taurine transport is shown to be linear over a 9~ time period. [3H]-
1:6
6
4
E E
-~ 0 . 4 0
7-5 5
0.8
Osmotic and ~-adrenergic regulation of [aH]-taurine transport over 19 h Propranolol, a ~-adrenergic antagonist, has been demonstrated to block isoproterenol stimulated taurine transport but to have no effect on non-stlmulated (basal) taurine transport [2, 12]. Osmotically stimulated [nH]-taurine transport at 19 h is also not blocked in the presence of 2 • 10-TM propranolol as shown in Table 4.
............................
2
4
6
8
I0 12 14 16 1819
Time ( h )
F I G U R E 2. T i m e course of [aH]-taurine transport. Hearts were incubated for varying times u p to 19 h with 200 ~,M ( O ) or 2 mM ( 9 taurine (means =k S.Z.M. 79 hearts, 7 litters).
Concentration dependence of [3H]-taurine transport Taurine transport was measured over the concentration range of 0.05 to 2 raM. We chose the time period between the 8th and 9th hour in the presence and in the absence of an 80 mM sucrose addition because the transport of 2 mM taurine was determined to be linear at that time (see Fig. 2). The
T A B L E 4. H y p e r o s m o t i c s t i m u l a t i o n o f [ 3 H ] - t a u r i n e t r a n s p o r t o v e r 19 h a n d lack o f i n h i b i t i o n b y ~-adrenergic blockade
Additions
[aH]-taurine transport ( p m o l / m g tissue w e t wt)
Control
2098•
Propranolol 2 • 10-7 M
2385 -~ 114 4389• a 4841 • 213b
S u c r o s e 80 mM Propranolol 2 •
10 -7 M, s u c r o s e 80 mM
[3H]-taurine transport m e a n • S.E.M. (33 hearts from three litters). a p < 0.01 compared to control. b p < 0.01 compared to control b u t not different from 80 mM sucrose.
Taurine and Osmoregulation ,~
0.8
,$
.~
317
__----t--
f
0.6
24
-6 E "C 0.4
-I>
8 12
O
0.2
_g
6
T
m ua
I
0.2
I
I
0.6
i
I
I
i
i
1.0 ETaurinelmM
t
[ 2.0
i0
I I0
I 20
I
[sJ
F I G U R E 3. Concentration dependence of the rate of [3H]-taurine transport between the 8th and 9th hour after 8 h in the presence ( A ) and absence (@) of 80 mM sucrose additional. Each point represents net [SH]taurine transport into a single heart.
[3H]-labeled taurine and [l*C]-sorbitol was added to the incubation medium after 8 h and [3H]-taurine transport was measured at 9 h. [3H]-taurine transport in the presence and in the absence of 80 mM sucrose as shown in Figure 3 is a saturable process indicative of a carrier mediated transport system. A linear regression analysis of a double reciprocal plot of the data was performed in order to determine the kinetic parameters (Kin and Vmax)' In the control fetal mouse hearts (no osmotic agent) the Kra and Vm.x were determined to be 0.51 mM and 0.41 nmol/ mg tissue/h respectively. In the presence of 80 mosM sucrose the Km and "Vmax were determined to be 0.43 mM and 0.81 nmol/mg tissue/h respectively. Thus the increased osmolarity appeared to engender a significant increase in the Vma* for taurine transport.
Specificity of hyperosmotic stimulated transport Unlike taurine transport that does not saturate for many hours, phenylalanine transport saturates rapidly [17]. To determine if phenylalanine transport was affected by hyperosmolarity hearts were preincubated for I h with and without addition of 80 mM
sucrose prior to being transferred on their stainless steel grid to a culture dish containing [3H]-phenylalanine and [ar in medium 199, with and without 80mM sucrose as in the preincubation. [3H]-phenylalanine transport was not saturated by 1 or 3 min and no difference was found with or without hyperosmotic addition at either of these time points. By 5 rain the pool of phenylalanine was saturated but the 12.9 • 1.0 pmol/mg wet wt (n = 20) observed with 80 mM sucrose was not significantly different from the 12.0 • 0.8 pmol/mg wet wt (n = 21) of the control. No statistically significant difference was observed between groups in the same litter and neither the presence of 200 ~XMtaurine nor extending the preincubation time with the osmotic addition to 4 h changed this observation. Phenylalanine accumulation continues over time as phenylalanine is incorporated into protein. After 19 h [3H]-phenylalanine accumulation (3 litters, 21 hearts) in the presence of 80 mM added sucrose (1.43 40.05 nmol/mg wet wt) was not significantly different from control (1.45 4- 0.05 nmol/mg wet wt). Sucrose was used so as not to alter the sodium gradient which is known to affect amino acid transport.
318
M. Atlas e t al.
Correlation of cardiac taurine and plasma osmolarity in streptozotocin-diabetic rats and age-matched controls Total cardiac taurine and plasma osmolarity increased in rats (n = 15) 2 weeks after streptozotocin induced diabetes as compared to age matched controls (n = 10) (plasma osmolarity: 295 4- 2.1 ~ 322 ~ 1.3 mosmol P < 0.001; endogenous cardiac taurine: 24,3 =~ 1.2 -+ 36 • 1.0 ~tM/gram wet wt tissue, P < 0.001). Discussion
The physiological basis for the close regulation of the high taurine concentrations found in the m a m m a l i a n heart has remained obscure. In this study it was found that during 19h all of the osmotic agents tested both ionic and nonionic stimulated taurine transport (Table 1). The magnitude of the stimulation was observed to be proportional to the concentration of the osmotic agent indicating the sensitivity of taurine transport to changes in osmolarity. This suggested that in vivo alterations of plasma osmolarity might affect changes in the levels of cardiac taurine. The physiological significance of the osmotic effect on taurine transport and accumulation is suggested by the increase in plasma osmolarity and cardiac taurine levels observed in streptozotocin-induced diabetic rats as compared to nondiabetic controls. Whether the increased accumulation of cardiac taurine observed in diabetes plays ar~y role in the natural history of diabetic complications is unknown. The fact that the exact magnitude of stimulation was different for' each of the osmotic agents during 19 h suggests that the fetal mouse heart responds differently to various osmotic agents. Because NaC1 directly increases taurine transport at l h and different osmotic agents result in different amounts of taurine transport over 19 h, it is most likely to be coincidental that the 19 h sodium and sucrose stimulations are equivalent. The effect of NaC1 is most likely to be the sum of direct NaC1 stimulation and osmotic stimulation. Stimulation of [SH]-taurine transport by increased osmolarity was shown to be separate from the ~-adrenergic mediated taurine transport during 19h (Table 4). Whereas chronic
~-adrenergic stimulation m a y be the cause of the elevated taurine observed in congestive heart failure and perhaps hypertension, osmotic stimulation of taurine transport appears to be the cause of the reported increased cardiac taurine observed in diabetes [20]. In the absence of an osmotic stress maintenance of the steady state level of cardiac taurine allows only for the exchange o f extracellular labeled taurine for an equivalent amount of unlabeled intracellular taurine (Tables 1 and 2). However, in the presence of an osmotic stress, we observed an increase in the amount of [3H]-taurine transported over a range of concentrations and a 98% increase in the maximal velocity of transport without any meaningful change in the Km of transport (Fig. 3). Alterations in efflux which would contribute to defining steady state levels has not yet been studied. All taurine transport in the fetal mouse heart was demonstrated to have an absolute requirement for the presence of sodium (Table 3). With incremental increases in the sodium ion concentration, above 145 mM (control), taurine transport is linearly stimulated (Fig. 1). The stimulation of taurine transport by sodium is a phenomenon separate from ~-adrenergic stimulation of taurine transport and at 1 h is greater than the response to an equivalent non-sodium increase in osmolarity. The dependence of the taurine transport system on sodium has been demonstrated in various tissues from rat and fish [1, 5, 6, 18, 22] and would appear to be a universal characteristic of taurine transport. Studies by Schaffer and Kulakowski using purified taurine binding proteins solubilized from swine sarcolemma and reconstituted into proteoliposomes demonstrate a sodium requirement for taurine transport and suggest that taurine transport is an electrogenic process driven by the inward gradient of sodium ions [22]. In our model we show a significant (P < 0.01) increase in taurine transport at I h with the addition of 40 mM NaC1 while addition of sucrose, LiCI, choline chloride or glucose did not result in statistically significant stimulation of taurine transport (Table 1). The use of free amino acids in the osmoregulation of m a m m a l i a n tissue was first
Taurine and Osmoregulation
suggested by the work of McDowell et al. [16]. Their work demonstrated the ability of dogs and cats, injected with hypertonic solutions, to generate osmotically active particles in what they explained as an attempt to limit cellular dehydration. The work by Thurston et al. showing the elevation of taurine levels in the brains and hearts of hypernatremic, dehydrated mice further demonstrates the possible significance of osmolarity in the regulation of taurine transport in mammalian tissues [24, 25]. Our studies establish that taurine transport, more than just being dependent on sodium ion concentration, is under general osmotic regulation. Phenylalanine is transported by a separate transport system that was not stimulated by hyperosmolarity, thus the response of taurine transport and accumulation to increased osmolarity is selective. The osmoregulation of taurine would enable the cell to alter its osmotic content without involving the various ions required to main-
319
tain necessary membrane potentials and gains additional advantage by sparing amino acids required for protein synthesis. A role for taurine in the osmoregulatory system of the mammalian myocardium is suggested by ou'r work in the fetal mouse heart and previous work in the adult fish hearts of the Platichthys flesus {flounder) [26] and the Raja erinacea (little skate) [6] indicating that this role for cardiac taurine may be general. The data presented has demonstrated that cardiac taurine transport is regulated by the osmotic environment of the myocardium and suggests that taurine is an osmoregutator in the mammalian heart.
Acknowledgement The authors wish to thank Dr Murray Korc for his generous supply of streptozotocindiabetic rats and age-matched controls and many helpful discussions.
References 1 AWAPARA,J., BERG, M. Uptake of taurine by slices of rat heart and kidney. In: Huxtable AND Barbeau, (Eds) Taurine, pp. 135-143. New York: Raven Press (1976). 2 AZARI,J., ttUXTABL% R. J. The mechanism of the adrenergic stimulation of taurine influx in the heart. E u r J Pharmacol 51, 217-223 (1980). 3 BRESSLER,R., GROSSO, D. S., ROESKE, W. R. Carrier-mediated taurine uptake in the fetal mouse heart. Trans Assoc Am Phys 90, 257-269 (1977). 4 CHEN,F. M., YAMAMURA,H. I., ROESKE,W. R. Ontogeny of mammalian myocardial [3-adrenergic receptors. Eur J Pharmacol 58, 255-264 (1979). 5 FORSTER,R. P., GOLDSTm~,L. Amino acids and cell volume regulation. YaleJ Biol Med 52, 497-515 (1979). 6 FORSTER,R. P., HANNAFIN,J. A. Taurine uptake in atrial myocardium by a sodium-dependent p-amino acid system in the elasmobranch, Raja erinacea. Comp Biochem Physiol 67, 107-113 (1980). 7 GRosso, D. S., ROESKE,W. R., BRESSLER,R. Taurine transport in the fetal mouse heart. Proc West Pharmacol Soc 20, 239-243 (1977). 8 GRosso, D. S., ROESKF.,W. R., BRESSLER,R. Characterization of a carrier-mediated transport system for taurine in the fetal mouse heart in vitro..J Clin Invest 61, 944-952 (1978). 9 HRosr~, R. E., HOXTA~LE, R. J., YAMAMO~, H. I. Purification of [3H]-taurine of high specific activity. Anal Biochem 79, 568-570 (1977). !0 HUXTABLE,R., BRESSLER, R. Taurine concentration in congestive heart failure. Science 184, 1187-I188 (1974). 11 HUXTABLE,R., CHUBB,,J. Adrenergic stimulation of taurine transport by the heart. Science 198, 409-411 (1977). 12 HUXTAnLE, R. J. Regulation of taurine in the heart. In: Barbeau AND Huxtable, (Eds) Taurine and Neurological Disorders, pp. 5-17. New York: Raven Press (1978). 13 INGWALL,J. S., ROESKE,W. R., WXLDENTHAL,K. The fetal mouse heart in organ culture: Maintenance of the differentiated state. Methods Cell Biol 21A, 167-187 (1980). 14 JACOBSEN,J. G., SMITH, L. H. Biochemistry and physiology of taurine derivatives. Physiol Rev 48, 424-511 (1968). 15 KORC, M., IWAMOTO, Y., SANKARAN,H., WILLIAMS,J. A., GOLDFINE, I. D. Insulin action in pancreatic acini from streptozotocin-treated rats. I. Stimulation of protein synthesis. Am J Physio1240, G56-G62 (1981). 16 McDOWELC, M. E., WOLF, A. V., STEER, A. Osmotic volumes of distribution: Idiogenic changes inosmotic pressure associated with administration of hypertonic solutions. Am J Physiol 180, 545-558 (1955).
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17 McKEE, E. E., CHEUNG,J. Y., RANNELS,D. E., MORGAN,H. E. Measurement of the rate of protein symthesis and compartmentation of heart phenylalanine. J Biol Chem 253, 1030-1040 (1978). 18 MEINERS,B. A., SPETH, R. C., BR~SOLIN,N., HUXTABLE,R.J., YAUAUURA,H. I. Sodium-dependent, highaffinity taurine transport into rat brain synaptosomes. Fed Proc 39, 2695-2700 (1981). 19 MORGAN,H. E., HENDERSON,H. J., REGAN, D. M., PARK, C. R. Regulation of glucose uptake in muscle. J Biol Chem 236, 253-261 (1961). 20 REmEL, D. K., SI~AFPER,J. E., Kocsis, J. J., NEELV,J. R. Changes in taurine content in heart and other organs of diabetic rats. J Mol Cell Cardiol 11,827-830 (1979). 21 ROTH, M. Fluorescence reaction for amino acids. Anal Chem 43, 880-882 (1971). 22 SCnAFFSR,S., KULA~OWSKI,E. Reconstitution of taurine transport in membrane vesicles. Fed Proc 40, 891 (1981). 23 STUART,J. D., WILSON,T. D., HILL, D. W., WALTERS,F. H., FENO, S. Y. High performance liquid chromatographic separation and fluorescent measurement of taurine, a key amino acid. J Liq Chromatogr 2, 809-821 (1979). 24 TnURSTON,J. H., HAUnAR% H. E., DinGo, D. A. Taurine: A role in osmotic regulation of mammalian brain and possible clinical significance. Life Sci 26, 1561-1568 (1980). 25 TnURSTON,J. H., HAUHART, H. E., NACCARATO,E. F. Taurine: Possible role in osmotic regulation of mammalian hearts. Science 214, 1373-1374 (1981). 26 VIsLm, T., FUOELLI,K. Cell volume regulation in flounder (Platichthysflesus) heart muscle accompanying an alteration in plasma osmolarity. Comp Biochem Physiol 52A, 415-418 (1975). 27 WILDENTHAL,K. Long term maintenance of spontaneously beating mouse hearts in organ culture. J Appl Physiol 30, 153-157 (1971). 28 WILDENTnAL,K. Studies of foetal mouse hearts in organ culture: Metabolic requirements for prolonged function in vitro and the influence of cardiac maturation on substrate utilization. J Mol Cell Cardiol 5, 87-99 (1973). 29 ZAR,J. H. Multiple comparison. In: Biostatistical Methods, pp. 151-162. Englewood Cliffs, N.J.: PrenticeHall, Inc. (1974).