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Measurement of intracellular Na in the rat salivary gland: a 23Na-NMR study using double quantum filtering
142
Biochimica et Biophysica Acta, 1034(1990) 142-147 Elsevier
BBAGEN 23306
Measurement of intracellular Na in the rat salivary gland: a 23Na-NMR study using double quantum filtering Yoshiteru Seo, Masataka Murakami and Hiroshi Watari Department of Molecular Physiology, National Institute for Physiological Sciences, Okazaki (Japan)
(Received 5 September1989) (Revised manuscript received22 January 1990)
Key words: Sodium,intracellular; Sodium, interstitial; Quadrupole relaxation; Double quantum filter; NMR, 23Na-; (Rat salivarygland)
Z3Na in the prefused rat mandibular salivary gland was measured by spin-echo double quantum filter Z3Na-NMR spectroscopy at 8.45 T. Resonances due to the intracellular 23Na and the interstitial Z3Na were observed in the perfused gland at 25°C. The resonance due to intracellular Z3Na consisted of two Lorentzian signals stemming from the 1 1 / 2 ) ( - 1/21 coherence (sharp resonance) and the 1 - 1 / 2 ) ( 3 / 2 1 and 1 3 / 2 ) ( 1 / 2 1 coherences (broad resonance). The transverse relaxation rate constant corresponding to the 1 1 / 2 ) ( - 1 / 2 1 coherence was 95-1-4 s -I and that corresponding to the I - 1/2)3z( - 3 / 2 [ and ] 3 / 2 ) ( 1 / 2 1 coherences was 1360 + 75 s -~ (mean + S.E., n = 5). The resonance due to the interstitial Na had longer relaxation rate constants, and disappeared upon administration of dysprosium triethylenetetramine-N,N',N ",N "' ,N '"-hexaacetic acid.
Introduction Initial 23Na relaxometry studies in biological systems have suggested a model for the relaxation characteristics of intracellular Na (e.g. Refs. 1, 2). Since 23Na has a spin of 3/2, high-molecular-weight solutes such as proteins would be expected to enhance the quadrupolar relaxation of 23Na. Recently, the method of double quantum filter N M R spectroscopy has been reported [3-6]. This method is selective for the double quantum coherence from the 11/2)( - 3/21 and 13/2)( - 1/21 rank 3 coherences. Preliminary results for intracellular 23Na in erythrocytes have also been reported [7]. In this study, we have detected a resonance corresponding to the double quantum coherence of 23Na in the perfused rat manidbular gland, and we have obtained relaxation parameters for the intracellular 23Na and the interstitial 23Na. We have also used this method to measure changes in intracellular Na content during salivary secretion.
Abbreviations: Dy(TTHA), dysprosium triethylenetetramine-N,N', N ", N " , N "-hexaacetic acid; FID, free induction decay. Correspondence: Y. Seo, Department of Molecular Physiology,National Institute for Physiological Sciences, Myodaiji, Okazaki, 444 Japan.
Experimental procedures Preparation of the isolated salivary gland Mandibular salivary glands were isolated from rats (Wistar-Hamamatsu, 250-350 g) anesthetized with sodium pentobarbital (50 m g / k g body weight i.p.). The glands (wet weight 0.3 g) were placed in an N M R tube (10 mm diameter) and perfused arterially with a modified Krebs solution at a rate of 2 m l / m i n using a peristaltic pump (Cole-Palmer) [8,9]. The composition of the modified Krebs solution (in mM) was: 146 Na, 4.3 K, 1 Ca, 1 Mg, 148.3 C1, 5 glucose and lq Hepes buffer (pH 7.4), saturated with 100% 0 2. The composition of the perfusate containing Dy(TTHA) was (in mM): 10 Dy(TTHA), 146 Na, 4.3 K, 1 Ca, 1 Mg, 118.4 C1, 5 glucose and 10 Hepes buffer (pH 7.4). The space outside the gland was perfused with a 300 mM sucrose solution at a rate of 5 ml/min. Measurement of 23Na-NMR 23Na-NMR spectra were collected using a WM360wb spectrometer (Bruker, 8.45 T) with a broadband probe tuned to 95.24 MHz. The spin-echo double q u a n t u m filter, D-90 o_~./2_180 °-~-/2-90 °-8-90 °acquire, was used with a 32-step phase cycle [4]. ~- was the creation time (6 ms), ~ was the double quantum evolution time (0.01 ms), and D was the relaxation delay (0.4 s). In every experiment, the 180 ° pulse width
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143 ( 6 4 - 6 6 Fs) was measured carefully with an accuracy of + 0 . 1 ~ts. The spectral width was 10 kHz, the receiver dead time after the final 90 o pulse was 25 #s, and 2048 data points were used. The detectable magnetization Mx(z, 8, t) under the on-resonance condition is expected to be given as follows [10-12], Mx (I", 6, t) = 0.75.(e -~:~ - e-~l")-e-Sdq~- (e-~2z --e -~lt)
(1)
where • is the creation time, 8 is the evolution time, t is the time following the detection pulse, Sdq is the double q u a n t u m relaxation rate constant arising from the 1 1 / 2 ) ( - 3 / 2 1 and 1 3 / 2 ) ( - 1 / 2 1 rank 3 coherences, s l and s 2 are the transverse relaxation rate constants (1/T2) of the I - 1 / 2 ) ( - 3 / 2 I and 1 3 / 2 ) ( 1 / 2 I coherences and the 1 1 / 2 ) ( - 1/21 coherence, respectively. In the condition of extreme narrowing (o:% << 1, where ~ is the L a r m o r frequency and % is the correlation time), the double q u a n t u m filter does not evoke a double q u a n t u m coherence. Preliminary observations of 23Na in Krebs solution (7"1 = T2 = 60 ms, 2 5 ° C ) and in a viscous NaC1 solution (150 m m o l / k g , 76% ( w / w ) glycerol, 50 cP, T 1 ~ T2 --~ 2 ms, 25 o C) showed no significant signal from the double q u a n t u m coherence and showed an artifact of dispersion-like line shape with 0.3-0.1% of the signal intensity obtained with the one-pulse sequence. The artifact is p r o b a b l y caused by small errors in setting the 90 ° and 180 ° pulses, and also by errors in the phase shifts of the transmitter and receiver in the spectrometer. The same type of artifact, arising f o r m extracellular 23Na, has been observed with erythrocyte suspensions [7]. W h e n o:'rc is close to or larger than 1 (the slow motion condition), a double q u a n t u m coherence is evoked by the double q u a n t u m filter [6]. Preliminary observations of 23Na in an albumin solution (Sigma A6003, 0.5 g in 0.5 ml of 2 M NaC1 solution, 25 ° C) showed a signal from the double q u a n t u m coherence (s 1 = 345 s -1, s 2 = 92 s - l ) . As reported previously [12], the form of Eqn. 1 offers two methods for determining the relaxation rate constants, s I and s2: (1) the transformed signal intensity obtained with various z can be fitted to k - (e -s2" - e - ~ " ) ; (2) the signal intensity of the free induction decay for a given ~" can be fitted to k . (e - s J - e-Slt). The effect of any inhomogeneity in the static magnetic field is relatively small because a typical inhomogeneous decay rate constant (10 s -1) is m u c h smaller than the values of s~ and s 2. Preliminary observations using an albumin solution showed good agreement between the two methods [12]. Four to seven experiments were performed using the same protocol, and the F I D were added. The Fourier transform was then applied with a line-broadening factor of 5 H z after extending the data block to 8192 points. The F I D and spectral data were stored on
magnetic tape, then transferred to a micro c o m p u t e r ( N E C 9801RA, N E C ) [9]. Software routines of a spreadsheet p r o g r a m (Lotus 1-2-3) was used for further analysis. Results
Double quantum filter 23Na-NMR spectra of the salivary gland We f o u n d two c o m p o n e n t s of 23Na in the perfused mandibular salivary gland. Fig. l a shows the F I D of 23Na obtained with the double q u a n t u m filter in the resting gland. The F I D consists of a single c o m p o n e n t with a relative slow relaxation rate. The curve shows the result of fitting k(e -s2t - e -sit) to the data. The values of s t and s 2 for Z3Na in the resting glands were 71 + 3 and 43 + 1 s-1 (mean + S.E., n = 5), respectively. W h e n the content of intracellular N a was raised by application of acetylcholine (1 ttM) with ouabain (1 mM), the 6"
5'
Q)
4"
21" -1' ~-~ - 2 '
5' 4' 3' 2" 1' 8"
time (msec) Fig. 1. The double quantum filtered free induction decays (FID) of interstitial Na and intracellular Na measured by 23Na-NMR at 25 o C. (a) The FID of the unstimulated mandibular gland observed with a creation time (,) of 6 ms and an evolution time (6) of 10 ~ts. The FID derives mainly from the interstitial 23Na. The signal intensity of the free induction decay (M(t)) is shown from 0.025 to 100 ms following the final 90 ° pulse. The curve shows the result of fitting k(e -s'-te -sit) t o the data. Transverse relaxation rate constants of the I1/2)(-3/21 and 13/2)(1/21 coherences (sa) and of the 11/2)(1/21 coherence (s2) were 84 and 40 s-1 (correlation coefficient 0.79, n = 342). (b) The FID of intracellular 23Na in the mandibular gland. The FID was obtained by subtracting the FID of the unstimulated gland from the FID of the gland during the administration of 1 #M acetylcholine and 1 mM ouabain. The curve shows the result of fitting k(e -s:t - e -s~t) to the data over the period from 0.025 ms to 30 ms. Values for s 1 and s 2 were 1095 and 104 s -1 (correlation coefficient 0.81, n = 300).
144 F I D consisted two components. Fig. l b shows the free induction decay ofo the new, faster component which was obtained by subtraction of the slow component. The values of sl and s 2 for the fast component were 1360 + 75 and 95 + 4 s -1 (mean + S.E., n = 5), respectively. Fig. 2a shows the changes in the signal intensity with over the range from 0.25 ms to 40 ms. This should contain the same information on the relaxation rates as the FID. The squares in Fig. 2a show the results obtained from the resting gland. The dotted line shows the function k(e - s : ~ - e -s'~) using sl and s 2 values for the resting gland obtained from the F I D experiment. When the content of intracellular Na was raised using acetylcholine and ouabain, the additional faster component became apparent, that was not detectable in the resting gland (open diamonds in Fig. 2a). Fig. 2b shows the increment in the signal intensity observed during the infusion of acetylcholine and ouabain, and the dotted line shows the function k(e - ~ - e -~,~) using s 1 and s 2 values for the fast component obtained from the F I D experiment. These results confirm the presence of two distinct compartments or pools of N a that evoke double quantum coherence. To confirm the origin of the two components, we
201 o ° ~0
0
a)
!
o .............. [3""
o
8_
b)
201,~ ,. o
""
o
~0
[3
2'0
3"0
40
T. ( msec ) Fig. 2. Changes in the signal intensity of the double q u a n t u m filter 23Na-NMR spectrum of the mandibular gland with the creation time 0") at 2 5 ° C . (a) Signal intensities (MO')) of the unstimulated gland ([3) and of the gland stimulated with 1 /~M acetylcholine and 1 m M ouabain (O) are shown for ~- values over the range from 0.25 ms to 40 ms. The FID of the unstimulated mandibular gland consists mainly of interstitial Na. The curve shows the function k(e -s2"~- e -~1~) using s 1 and s 2 values for the interstitial Na obtained from the F I D experiment. (b) The increment in signal intensity (AM(,r)) due to stimulation with 1 # M acetylcholine and 1 m M ouabain (r-I) obtained by subtraction. The curve shows the function k ( e - S 2 ~ - e -sly) using s I and s 2 values for the intracellular N a obtained from the F I D experiment.
a)
'
4'0
'
2b
'
6
'-~0'-Z0'
Chemical shift ( ppm ) Fig. 3. Double q u a n t u m filter 23Na-NMR spectra of the perfused rat mandibular gland at 25 o C with a 8 value of 10 #s and a ~" value of 6 ms. (a) Intracellular 23Na spectrum without shift reagent. The increased intraceUular 23Na signal due to stimulation with 1 # M acetylcholine and 1 m M ouabain was obtained by subtraction. (b) The 23Na spectrum with 10 m M Dy(q-THA), 1 # M acetylcholine and 1 m M ouabain. The intracellular 23Na resonance (0.6 ppm) and an artifact from the extracellular 23Na (3.69 ppm) are observed.
used the chemical shift reagent Dy(TTHA). Fig. 3a shows the double quantum filtered spectrum obtained without Dy(TTHA). Previous work has shown that when 10 m M D y ( T T H A ) are applied to the salivary gland, the extracellular N a and the interstitial N a resonances are shifted approx. 3.2 and 2.4 p p m from the intracellular N a resonance, respectively [9]. Fig. 3b shows the double quantum filtered spectrum of the gland perfused with acetylcholine, ouabain and Dy(TTHA). The resonance remaining in its original position corresponds to the intracellular Na. The slight broadening of the line and the small shift (0.6 ppm) might be due to changes in the magnetic susceptibility of the perfusate. The small dispersion signal at 3.1 p p m might be due to an artifact from the extracellular N a as discussed above. The intensity was about 0.2% of the intensity obtained with the one-pulse sequence. The interstitial N a resonance normally seen at 2.4 p p m [9] was not observed in the presence of the shift reagent. Fig. 4a shows the double quantum filtered F I D of the perfused gland without the shift reagent. The slow component was observed as before. Intracellular Na was increased by applying acetylcholine and ouabain. Then, 10 m M D y ( T T H A ) was applied for 1 h and washed out for 20 rain. Thereafter, we obtained the F I D shown in Fig. 4b. Only the fast component, corresponding to the intracellular N a (s 1 = 990 s -1, s2 = 73 s - l ) remained. The acetylcholine and ouabain were then washed out and no significant signal corresponding to the slow component was subsequently observed (Fig. 4c). The vanishing resonance may therefore correspond to the Na previously observed at 2.4 p p m with the single-pulse method during perfusion with 10 m M D y ( T T H A ) [9]. This component may therefore be due
145 TABLE I
0.2'
Relaxation characteristics of intracellular Na and interstitial Na of the rat mandibular gland measured by double quantum filter 23Na-NMR at 25°C
0.1
Values of s I and s 2 are the transverse relaxation rate constants of the I - 1/2) ( - 3/21 and 13/2) (1/21 coherences and the 11/2) ( - 1/2 I coherence, respectively, o~ is the Larmor frequency, ~'c is the correlation time and e2qQ/h is the apparent quadrupolar coupling constant of 23Na. The s I and s 2 values for intracellular 23Na were calculated using FIDs obtained during perfusion with (a) 1 /~M acetylcholine and 1 mM ouabain, and (b) 1 #M acetylcholine alone. Since the increase in Na content obtained with acetylcholine is 1/5 of that obtained with acetylcholine and ouabain, the accumulated FIDs from five glands were used for this calculation• The FIDs of the unstimulated glands were used for calculation of the s I and s 2 values for the interstitial 23Na. Details of the calculation of Tc and eEqQ/h are given in the Discussion. Values are means 5: S.E.
0 -0.1 0.2" 0.1'
~E 0 -0.1 0.2'
c) 0.1 • O'
-0.1
0
20
z,O 60 time ( msec )
80
100
Fig. 4. The double quantum filtered FID of the interstitial Na and the intracellular Na measured by 23Na-NMR with a ~" value of 6 ms and a 8 value of 10 /~s at 25°C. (a) The FID of an unstimulated manidublar gland without shift reagent. The FID derives mainly from the interstitial 23Na. The curve shows the result of fitting k(e - s 2 t e -sit) to the data. Values for s I and s 2 of 69 and 43 s -1 (correlation coefficient 0.46, n = 342) were obtained. (b) The FID of the mandibular gland with 1 mM ouabain and 1 #M acetylcholine without shift reagent after 60 min of 10 mM Dy(TTHA) preperfusion. The curve shows the result of fitting k ( e -s2t --e -sit) to the data over the period from 0.025 ms to 30 ms. Values for s 1 and s 2 of 990 and 73 s-I (correlation coefficient 0.27, n = 342) were obtained. (c) The FID of an unstimulated mandibular gland without shift reagent after 60 min of 10 mM Dy(TTHA) preperfusion. Only a small signal with s 1 and s 2 values of 670 and 55 s -1 (correlation coefficient 0.08, n = 342) were observed.
to N a in t h e i n t e r s t i t i a l s p a c e t h a t i n t e r a c t s w i t h t h e Dy(TTHA).
Relaxation characteristics of 23Na at 25 ° C T h e r e l a x a t i o n c h a r a c t e r i s t i c s o f the i n t r a c e l l u l a r / 3 N a a r e s u m m a r i z e d in T a b l e I. V a l u e s o f s I a n d s 2 for t h e intracellular Na were calculated using FIDs obtained during perfusion with acetylcholine both with and w i t h o u t o u a b a i n . R e l a x a t i o n c h a r a c t e r i s t i c s o f the i n t e r stitial N a are also s u m m a r i z e d in T a b l e I. T h e F I D s o b t a i n e d f r o m t h e r e s t i n g g l a n d s w e r e u s e d to c a l c u l a t e s I a n d s 2 for t h e i n t e r s t i t i a l N a a s s u m i n g t h e i n t r a c e l l u l a r c o m p a r t m e n t to b e negligible. H o w e v e r , t h e s e F I D s m a y c o n t a i n a n a r t i f a c t d u e to t h e e x t r a c e l l u l a r N a w h o s e m a g n i t u d e c o u l d a m o u n t to 1 / 4 to 1 / 2 o f the total i n t e n s i t y o f t h e F I D . T h e i n h o m o g e n e i t y - r e l a t e d d e c a y r a t e c o n s t a n t ( a p p r o x . 10 s - x ) m i g h t also c o n t r i b u t e a little to t h e e r r o r s in t h e v a l u e s o f s 1 a n d
S1
S2
tdT c
Tc
e2qQ/h
(s- 1)
(s- 1)
(tad- s)
(ps)
(MHz)
Intracellular Na (a) 1360 + 75 (b) 610
94.7 + 3.7 130
4.0 _ 0.2 2.0
6.7 _+0.3 3.4
1.95 + 0.02 1.7
Interstitial Na 71.1 +3.1
42.8 +1.0
0.70 +0.07
1.2 +0.1
0.86 +0.01
n
5 (5) 5
s 2. C o n s e q u e n t l y , t h e r e l a x a t i o n p a r a m e t e r s e s t i m a t e d for t h e i n t e r s t i t i a l N a s h o u l d b e r e g a r d e d w i t h c a u t i o n .
Measurement of changes in intracellular N a T h e effects o f a c e t y l c h o l i n e (1 # M ) s t i m u l a t i o n in the p r e s e n c e o f o u a b a i n (1 m M ) o n t h e c o n t e n t o f i n t r a cellular Na were examined using the following protocol. A f t e r 90 m i n o f c o n t r o l p e r f u s i o n , a c e t y l c h o l i n e w a s a d m i n i s t e r e d for 22.5 m i n , t h e n b o t h o u a b a i n a n d a c e t y l c h o l i n e w e r e a d m i n i s t e r e d for 67.5 rain, a n d t h e n the control perfusion without stimulation was continued for a f u r t h e r 45 m i n . 23Na d o u b l e q u a n t u m f i l t e r e d s p e c t r a w e r e o b s e r v e d e v e r y 7.5 m i n d u r i n g t h e e x p e r i m e n t . T h e s p e c t r u m c o l l e c t e d d u r i n g t h e last 60 m i n o f t h e i n i t i a l c o n t r o l p e r i o d was u s e d as t h e c o n t r o l spectrum. Fig. 5 s h o w s c h a n g e s in t h e d i f f e r e n c e s p e c t r a , obtained by subtracting the control spectrum from the s u b s e q u e n t series o f spectra. T h e p e a k h e i g h t is r o u g h l y p r o p o r t i o n a l to t h e i n t r a c e l l u l a r N a c o n t e n t , since the a p p a r e n t l i n e - w i d t h at h a l f - h e i g h t o f t h e p e a k (ul/2 = 33 + 1 H z ( n = 14) -- SE/'h" d- the fine b r o a d e n i n g f a c t o r ) remained constant during the experiment. Upon stimulation with acetylcholine, the intracellular Na content i n c r e a s e d b y 2 8 _ 6 a r b i t r a r y u n i t s ( m e a n _+ S.E. of a v e r a g e d d a t a f r o m 0 to 22.5 m i n , n = 4), a n d r e m a i n e d at this l e v e l d u r i n g s t i m u l a t i o n . T h e i n t r a c e l l u l a r N a content increased again during administration of o u a b a i n w i t h a c e t y l c h o l i n e , a n d s h o w e d a p l a t e a u level
146
I Ouabain [ Acetylcholine
-40
0
4'0
'
da
'
'
Time ( min ) Fig. 5. Changes in the intracellular 2 3 N a r e s o n a n c e induced by 1/~M acetylcholine with 1 mM ouabain at 25 o C. After 22.5 min of preadministration of acetylcholine, ouabain was appfied for 67.5 min. Averaged spectra of the intracellular 23Na measured every 7.5 rain using the double quantum filter with a r value of 6 ms and a 8 value of 10/*s are shown (n = 4). The spectra were obtained by subtraction of the resting gland spectrum from the series of original spectra.
of 136 + 15 arbitrary units (mean + S.E. of averaged data from 67.5 to 90 min, n = 4).
Discussion
This is to our knowledge the first study in which the • 23parameters of quadrupolar relaxation of N a have been determined in the mammalian salivary gland. The 23Na-NMR spectrum of intracellular N a in living cells has been interpreted as an inhomogeneous powder pattern spectrum, a homogeneous biexponential spectrum, and a spectrum of the intermediate of a rapid exchange process [2,11]. In this study, we have observed two Lorentzian resonances corresponding to the 1 1 / 2 ) ( 1 / 2 I coherence and the i - 1 / 2 ) ( - 3 / 2 I and J 3 / 2 ) { 1 / 2 i coherences of the intracellular Na. The difference in the resonance frequency between the two resonances was negligibly small. We therefore exclude the classical inhomogeneous powder pattern model. Consequently, the apparent reduction in the N M R visibility of intracellular N a (e.g., Refs. 1, 2, 13) might be due to the relatively rapid relaxation of the 1 - 1 / 2 ) ( 3 / 2 1 and 1 3 / 2 ) ( 1 / 2 1 coherences and also due to the relatively long dead time following the sampling pulse.
The homogeneous biexponential spectrum is expected from a homogeneous population of 23Na under the condition that ~ 1 >> rc >> ~-1, where ~Q is the time-averaged values of the quadrupolar coupling constant in frequency units. The resonance is a superposition of a narrow Lorentzian resonance corresponding to the 1 1 / 2 ) ( - 1 / 2 J coherence and a broad Lorentzian resonance corresponding to J - 1 / 2 ) ( 3 / 2 I and 1 3 / 2 ) ( 1 / 2 J coherences at the same frequency [11]. If we assume that there is a single, homogeneous ~opulation of intracellular Na, the correlation time of N a and the quadrupolar coupling constant (assuming the quadrupolar asymmetry parameter to be zero), is calculated to be approx. 6.7.10 - 9 S and 1.9 MHz, respectively, from s 1 and s 2 using equations (57), (58) and (5) from the paper by Jaccard et al. [6]. The value for the intracellular N a elevated by acetylcholine stimulation alone was 1.7 MHz, in good agreement in spite of the low signal-to-noise ratio. These values for the quadrupolar coupling constant are similar to those estimated for N a in glycerol (1.6 M H z [14]), associated with ionophores (0.5-2 M H z [15]) and in sodium compounds (0.3-3.7 M H z [16,17]). A model consisting of rapid exchange between immobilized and free 23Na within an isotropic medium also gives rise to a spectrum consisting of two Lorentzian resonances. In the rapid exchange model, the apparent value for the quadrupolar coupling constant might be e x p e c t e d to be smaller (approx. 0.1 M H z for sodium laurate [14]). Furthermore, when the fraction of free N a was increased significantly (approx. 5-times) by administration of acetylcholine with ouabain, the apparent quadrupolar coupling constant would be expected to decrease• However, the value observed during administration of ouabain with acetylcholine tended to be higher than that obtained during administration of acetylcholine alone (Table I). At this stage, however, we do not have strong evidence for eliminating the rapid exchange process• Studies of the field and temperature dependencies of the relaxation parameters will be necessary to confirm this conclusion [14]. We also demonstrated that the double quantum filter allows us to measure the changes in intracelhilar Na content without the use of a chemical shift reagent. The following factors might contribute to the errors in the measurement: (1) changes in the size of the double quantum coherence deriving from the interstitial Na; (2) changes in the size of the artifact due to the extracellular Na. In the isolated, perfused preparation at constant perfusion rate, small changes in the size of the interstitial and extracellular spaces could possibly cause these errors. However, the size of the error due to changes in the extent of the extracellular oedema is likely to be no more than 10-20% of the increment due to 1 ~tM acetylcholine, which is not unreasonable in this type of experiment.
147
Acknowledgements We thank H. Hattori, O, Ichikawa, A. Ikeda, K. Suzuki and H. Ohkawara for their technical assistance, and Dr. K. Nagayama and Dr. M.C. Steward for helpful discussion.
References 1 Civan, M.M. and Shporer, M. (1978) in Biological Magnetic Resonance. I (Berliner, L.J. and Reuben, J., eds.), pp. 1-32, Plenum Press, New York. 2 Civan, M.M. and Shporer, M. (1989) in Current Topics in Membranes and Transport, Vol. 34, pp. 1-19, Academic Press, New York, in press. 3 Bax, A., Freeman, R. and KempseU, S.P. (1980) J. Am. Chem. Soc. 102, 4849-4851. 4 Piantini, U., Sorensen, O.W. and Ernst, R.R. (1982) J. Am. Chem. Soc. 104, 6800-6801. 5 Shaka, A.J. and Freeman, R. (1983) J. Magn. Reson. 51, 169-173. 6 Jaccard, G., Wimperis, S. and Bodenhausen, G. (1986) J. Chem. Phys. 85, 6282-6293.
7 Pekar, J., Renshaw, P.F. and Leigh, Jr.S. (1987) J. Magn. Reson. 72, 159-161. 8 Murakami, M., Seo, Y., Watari, H., Ueda, H., Hashimoto, T. and Tagawa, K. (1987) Jpn. J. Physiol. 37, 411-423. 9 Seo, Y., Murakami, M., Matsumoto, T., Nishikawa, H. and Watari, H. (1987) Pfliigers Arch. 409, 343-348. 10 Pekar, J. and Leigh, Jr., S. (1986) J. Magn. Reson. 69, 582-584. 11 Rooney, W.D., Barbara, T.M., and Springer, C.S. (1988) J. Am. Chem. Soc. 110, 674-681. 12 Seo, Y., Murakami, M., Suzuki, E., Kuki, S., Nagayama, K. and Watari, H. (1990) Biochemistry 29, 599-603. 13 Springer, Jr., C.S. (1987) Annu. Rev. Biophys. Chem. 16, 375-399. 14 Lerner, L. and Torchia, D.A. (1986) J. Am. Chem. Soe. 108, 4264-4268. 15 Haynes, D.H., Pressman, B.C. and Kowalsky, A. (1971) Biochemistry 10, 853-860. 16 Das, T.P. and Hahn, E.L. (1958) in Solid State Physics Suppl. 1 (Seitz, F. and Turnbull, D., eds.), pp. 119-163, Academic Press, New York. 17 Edmounds, D.T. and Mailer, J.P.G. (1979) J. Magn. Reson. 36, 411-418.
Biochimica et Biophysica Acta, 1034(1990) 142-147 Elsevier
BBAGEN 23306
Measurement of intracellular Na in the rat salivary gland: a 23Na-NMR study using double quantum filtering Yoshiteru Seo, Masataka Murakami and Hiroshi Watari Department of Molecular Physiology, National Institute for Physiological Sciences, Okazaki (Japan)
(Received 5 September1989) (Revised manuscript received22 January 1990)
Key words: Sodium,intracellular; Sodium, interstitial; Quadrupole relaxation; Double quantum filter; NMR, 23Na-; (Rat salivarygland)
Z3Na in the prefused rat mandibular salivary gland was measured by spin-echo double quantum filter Z3Na-NMR spectroscopy at 8.45 T. Resonances due to the intracellular 23Na and the interstitial Z3Na were observed in the perfused gland at 25°C. The resonance due to intracellular Z3Na consisted of two Lorentzian signals stemming from the 1 1 / 2 ) ( - 1/21 coherence (sharp resonance) and the 1 - 1 / 2 ) ( 3 / 2 1 and 1 3 / 2 ) ( 1 / 2 1 coherences (broad resonance). The transverse relaxation rate constant corresponding to the 1 1 / 2 ) ( - 1 / 2 1 coherence was 95-1-4 s -I and that corresponding to the I - 1/2)3z( - 3 / 2 [ and ] 3 / 2 ) ( 1 / 2 1 coherences was 1360 + 75 s -~ (mean + S.E., n = 5). The resonance due to the interstitial Na had longer relaxation rate constants, and disappeared upon administration of dysprosium triethylenetetramine-N,N',N ",N "' ,N '"-hexaacetic acid.
Introduction Initial 23Na relaxometry studies in biological systems have suggested a model for the relaxation characteristics of intracellular Na (e.g. Refs. 1, 2). Since 23Na has a spin of 3/2, high-molecular-weight solutes such as proteins would be expected to enhance the quadrupolar relaxation of 23Na. Recently, the method of double quantum filter N M R spectroscopy has been reported [3-6]. This method is selective for the double quantum coherence from the 11/2)( - 3/21 and 13/2)( - 1/21 rank 3 coherences. Preliminary results for intracellular 23Na in erythrocytes have also been reported [7]. In this study, we have detected a resonance corresponding to the double quantum coherence of 23Na in the perfused rat manidbular gland, and we have obtained relaxation parameters for the intracellular 23Na and the interstitial 23Na. We have also used this method to measure changes in intracellular Na content during salivary secretion.
Abbreviations: Dy(TTHA), dysprosium triethylenetetramine-N,N', N ", N " , N "-hexaacetic acid; FID, free induction decay. Correspondence: Y. Seo, Department of Molecular Physiology,National Institute for Physiological Sciences, Myodaiji, Okazaki, 444 Japan.
Experimental procedures Preparation of the isolated salivary gland Mandibular salivary glands were isolated from rats (Wistar-Hamamatsu, 250-350 g) anesthetized with sodium pentobarbital (50 m g / k g body weight i.p.). The glands (wet weight 0.3 g) were placed in an N M R tube (10 mm diameter) and perfused arterially with a modified Krebs solution at a rate of 2 m l / m i n using a peristaltic pump (Cole-Palmer) [8,9]. The composition of the modified Krebs solution (in mM) was: 146 Na, 4.3 K, 1 Ca, 1 Mg, 148.3 C1, 5 glucose and lq Hepes buffer (pH 7.4), saturated with 100% 0 2. The composition of the perfusate containing Dy(TTHA) was (in mM): 10 Dy(TTHA), 146 Na, 4.3 K, 1 Ca, 1 Mg, 118.4 C1, 5 glucose and 10 Hepes buffer (pH 7.4). The space outside the gland was perfused with a 300 mM sucrose solution at a rate of 5 ml/min. Measurement of 23Na-NMR 23Na-NMR spectra were collected using a WM360wb spectrometer (Bruker, 8.45 T) with a broadband probe tuned to 95.24 MHz. The spin-echo double q u a n t u m filter, D-90 o_~./2_180 °-~-/2-90 °-8-90 °acquire, was used with a 32-step phase cycle [4]. ~- was the creation time (6 ms), ~ was the double quantum evolution time (0.01 ms), and D was the relaxation delay (0.4 s). In every experiment, the 180 ° pulse width
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143 ( 6 4 - 6 6 Fs) was measured carefully with an accuracy of + 0 . 1 ~ts. The spectral width was 10 kHz, the receiver dead time after the final 90 o pulse was 25 #s, and 2048 data points were used. The detectable magnetization Mx(z, 8, t) under the on-resonance condition is expected to be given as follows [10-12], Mx (I", 6, t) = 0.75.(e -~:~ - e-~l")-e-Sdq~- (e-~2z --e -~lt)
(1)
where • is the creation time, 8 is the evolution time, t is the time following the detection pulse, Sdq is the double q u a n t u m relaxation rate constant arising from the 1 1 / 2 ) ( - 3 / 2 1 and 1 3 / 2 ) ( - 1 / 2 1 rank 3 coherences, s l and s 2 are the transverse relaxation rate constants (1/T2) of the I - 1 / 2 ) ( - 3 / 2 I and 1 3 / 2 ) ( 1 / 2 I coherences and the 1 1 / 2 ) ( - 1/21 coherence, respectively. In the condition of extreme narrowing (o:% << 1, where ~ is the L a r m o r frequency and % is the correlation time), the double q u a n t u m filter does not evoke a double q u a n t u m coherence. Preliminary observations of 23Na in Krebs solution (7"1 = T2 = 60 ms, 2 5 ° C ) and in a viscous NaC1 solution (150 m m o l / k g , 76% ( w / w ) glycerol, 50 cP, T 1 ~ T2 --~ 2 ms, 25 o C) showed no significant signal from the double q u a n t u m coherence and showed an artifact of dispersion-like line shape with 0.3-0.1% of the signal intensity obtained with the one-pulse sequence. The artifact is p r o b a b l y caused by small errors in setting the 90 ° and 180 ° pulses, and also by errors in the phase shifts of the transmitter and receiver in the spectrometer. The same type of artifact, arising f o r m extracellular 23Na, has been observed with erythrocyte suspensions [7]. W h e n o:'rc is close to or larger than 1 (the slow motion condition), a double q u a n t u m coherence is evoked by the double q u a n t u m filter [6]. Preliminary observations of 23Na in an albumin solution (Sigma A6003, 0.5 g in 0.5 ml of 2 M NaC1 solution, 25 ° C) showed a signal from the double q u a n t u m coherence (s 1 = 345 s -1, s 2 = 92 s - l ) . As reported previously [12], the form of Eqn. 1 offers two methods for determining the relaxation rate constants, s I and s2: (1) the transformed signal intensity obtained with various z can be fitted to k - (e -s2" - e - ~ " ) ; (2) the signal intensity of the free induction decay for a given ~" can be fitted to k . (e - s J - e-Slt). The effect of any inhomogeneity in the static magnetic field is relatively small because a typical inhomogeneous decay rate constant (10 s -1) is m u c h smaller than the values of s~ and s 2. Preliminary observations using an albumin solution showed good agreement between the two methods [12]. Four to seven experiments were performed using the same protocol, and the F I D were added. The Fourier transform was then applied with a line-broadening factor of 5 H z after extending the data block to 8192 points. The F I D and spectral data were stored on
magnetic tape, then transferred to a micro c o m p u t e r ( N E C 9801RA, N E C ) [9]. Software routines of a spreadsheet p r o g r a m (Lotus 1-2-3) was used for further analysis. Results
Double quantum filter 23Na-NMR spectra of the salivary gland We f o u n d two c o m p o n e n t s of 23Na in the perfused mandibular salivary gland. Fig. l a shows the F I D of 23Na obtained with the double q u a n t u m filter in the resting gland. The F I D consists of a single c o m p o n e n t with a relative slow relaxation rate. The curve shows the result of fitting k(e -s2t - e -sit) to the data. The values of s t and s 2 for Z3Na in the resting glands were 71 + 3 and 43 + 1 s-1 (mean + S.E., n = 5), respectively. W h e n the content of intracellular N a was raised by application of acetylcholine (1 ttM) with ouabain (1 mM), the 6"
5'
Q)
4"
21" -1' ~-~ - 2 '
5' 4' 3' 2" 1' 8"
time (msec) Fig. 1. The double quantum filtered free induction decays (FID) of interstitial Na and intracellular Na measured by 23Na-NMR at 25 o C. (a) The FID of the unstimulated mandibular gland observed with a creation time (,) of 6 ms and an evolution time (6) of 10 ~ts. The FID derives mainly from the interstitial 23Na. The signal intensity of the free induction decay (M(t)) is shown from 0.025 to 100 ms following the final 90 ° pulse. The curve shows the result of fitting k(e -s'-te -sit) t o the data. Transverse relaxation rate constants of the I1/2)(-3/21 and 13/2)(1/21 coherences (sa) and of the 11/2)(1/21 coherence (s2) were 84 and 40 s-1 (correlation coefficient 0.79, n = 342). (b) The FID of intracellular 23Na in the mandibular gland. The FID was obtained by subtracting the FID of the unstimulated gland from the FID of the gland during the administration of 1 #M acetylcholine and 1 mM ouabain. The curve shows the result of fitting k(e -s:t - e -s~t) to the data over the period from 0.025 ms to 30 ms. Values for s 1 and s 2 were 1095 and 104 s -1 (correlation coefficient 0.81, n = 300).
144 F I D consisted two components. Fig. l b shows the free induction decay ofo the new, faster component which was obtained by subtraction of the slow component. The values of sl and s 2 for the fast component were 1360 + 75 and 95 + 4 s -1 (mean + S.E., n = 5), respectively. Fig. 2a shows the changes in the signal intensity with over the range from 0.25 ms to 40 ms. This should contain the same information on the relaxation rates as the FID. The squares in Fig. 2a show the results obtained from the resting gland. The dotted line shows the function k(e - s : ~ - e -s'~) using sl and s 2 values for the resting gland obtained from the F I D experiment. When the content of intracellular Na was raised using acetylcholine and ouabain, the additional faster component became apparent, that was not detectable in the resting gland (open diamonds in Fig. 2a). Fig. 2b shows the increment in the signal intensity observed during the infusion of acetylcholine and ouabain, and the dotted line shows the function k(e - ~ - e -~,~) using s 1 and s 2 values for the fast component obtained from the F I D experiment. These results confirm the presence of two distinct compartments or pools of N a that evoke double quantum coherence. To confirm the origin of the two components, we
201 o ° ~0
0
a)
!
o .............. [3""
o
8_
b)
201,~ ,. o
""
o
~0
[3
2'0
3"0
40
T. ( msec ) Fig. 2. Changes in the signal intensity of the double q u a n t u m filter 23Na-NMR spectrum of the mandibular gland with the creation time 0") at 2 5 ° C . (a) Signal intensities (MO')) of the unstimulated gland ([3) and of the gland stimulated with 1 /~M acetylcholine and 1 m M ouabain (O) are shown for ~- values over the range from 0.25 ms to 40 ms. The FID of the unstimulated mandibular gland consists mainly of interstitial Na. The curve shows the function k(e -s2"~- e -~1~) using s 1 and s 2 values for the interstitial Na obtained from the F I D experiment. (b) The increment in signal intensity (AM(,r)) due to stimulation with 1 # M acetylcholine and 1 m M ouabain (r-I) obtained by subtraction. The curve shows the function k ( e - S 2 ~ - e -sly) using s I and s 2 values for the intracellular N a obtained from the F I D experiment.
a)
'
4'0
'
2b
'
6
'-~0'-Z0'
Chemical shift ( ppm ) Fig. 3. Double q u a n t u m filter 23Na-NMR spectra of the perfused rat mandibular gland at 25 o C with a 8 value of 10 #s and a ~" value of 6 ms. (a) Intracellular 23Na spectrum without shift reagent. The increased intraceUular 23Na signal due to stimulation with 1 # M acetylcholine and 1 m M ouabain was obtained by subtraction. (b) The 23Na spectrum with 10 m M Dy(q-THA), 1 # M acetylcholine and 1 m M ouabain. The intracellular 23Na resonance (0.6 ppm) and an artifact from the extracellular 23Na (3.69 ppm) are observed.
used the chemical shift reagent Dy(TTHA). Fig. 3a shows the double quantum filtered spectrum obtained without Dy(TTHA). Previous work has shown that when 10 m M D y ( T T H A ) are applied to the salivary gland, the extracellular N a and the interstitial N a resonances are shifted approx. 3.2 and 2.4 p p m from the intracellular N a resonance, respectively [9]. Fig. 3b shows the double quantum filtered spectrum of the gland perfused with acetylcholine, ouabain and Dy(TTHA). The resonance remaining in its original position corresponds to the intracellular Na. The slight broadening of the line and the small shift (0.6 ppm) might be due to changes in the magnetic susceptibility of the perfusate. The small dispersion signal at 3.1 p p m might be due to an artifact from the extracellular N a as discussed above. The intensity was about 0.2% of the intensity obtained with the one-pulse sequence. The interstitial N a resonance normally seen at 2.4 p p m [9] was not observed in the presence of the shift reagent. Fig. 4a shows the double quantum filtered F I D of the perfused gland without the shift reagent. The slow component was observed as before. Intracellular Na was increased by applying acetylcholine and ouabain. Then, 10 m M D y ( T T H A ) was applied for 1 h and washed out for 20 rain. Thereafter, we obtained the F I D shown in Fig. 4b. Only the fast component, corresponding to the intracellular N a (s 1 = 990 s -1, s2 = 73 s - l ) remained. The acetylcholine and ouabain were then washed out and no significant signal corresponding to the slow component was subsequently observed (Fig. 4c). The vanishing resonance may therefore correspond to the Na previously observed at 2.4 p p m with the single-pulse method during perfusion with 10 m M D y ( T T H A ) [9]. This component may therefore be due
145 TABLE I
0.2'
Relaxation characteristics of intracellular Na and interstitial Na of the rat mandibular gland measured by double quantum filter 23Na-NMR at 25°C
0.1
Values of s I and s 2 are the transverse relaxation rate constants of the I - 1/2) ( - 3/21 and 13/2) (1/21 coherences and the 11/2) ( - 1/2 I coherence, respectively, o~ is the Larmor frequency, ~'c is the correlation time and e2qQ/h is the apparent quadrupolar coupling constant of 23Na. The s I and s 2 values for intracellular 23Na were calculated using FIDs obtained during perfusion with (a) 1 /~M acetylcholine and 1 mM ouabain, and (b) 1 #M acetylcholine alone. Since the increase in Na content obtained with acetylcholine is 1/5 of that obtained with acetylcholine and ouabain, the accumulated FIDs from five glands were used for this calculation• The FIDs of the unstimulated glands were used for calculation of the s I and s 2 values for the interstitial 23Na. Details of the calculation of Tc and eEqQ/h are given in the Discussion. Values are means 5: S.E.
0 -0.1 0.2" 0.1'
~E 0 -0.1 0.2'
c) 0.1 • O'
-0.1
0
20
z,O 60 time ( msec )
80
100
Fig. 4. The double quantum filtered FID of the interstitial Na and the intracellular Na measured by 23Na-NMR with a ~" value of 6 ms and a 8 value of 10 /~s at 25°C. (a) The FID of an unstimulated manidublar gland without shift reagent. The FID derives mainly from the interstitial 23Na. The curve shows the result of fitting k(e - s 2 t e -sit) to the data. Values for s I and s 2 of 69 and 43 s -1 (correlation coefficient 0.46, n = 342) were obtained. (b) The FID of the mandibular gland with 1 mM ouabain and 1 #M acetylcholine without shift reagent after 60 min of 10 mM Dy(TTHA) preperfusion. The curve shows the result of fitting k ( e -s2t --e -sit) to the data over the period from 0.025 ms to 30 ms. Values for s 1 and s 2 of 990 and 73 s-I (correlation coefficient 0.27, n = 342) were obtained. (c) The FID of an unstimulated mandibular gland without shift reagent after 60 min of 10 mM Dy(TTHA) preperfusion. Only a small signal with s 1 and s 2 values of 670 and 55 s -1 (correlation coefficient 0.08, n = 342) were observed.
to N a in t h e i n t e r s t i t i a l s p a c e t h a t i n t e r a c t s w i t h t h e Dy(TTHA).
Relaxation characteristics of 23Na at 25 ° C T h e r e l a x a t i o n c h a r a c t e r i s t i c s o f the i n t r a c e l l u l a r / 3 N a a r e s u m m a r i z e d in T a b l e I. V a l u e s o f s I a n d s 2 for t h e intracellular Na were calculated using FIDs obtained during perfusion with acetylcholine both with and w i t h o u t o u a b a i n . R e l a x a t i o n c h a r a c t e r i s t i c s o f the i n t e r stitial N a are also s u m m a r i z e d in T a b l e I. T h e F I D s o b t a i n e d f r o m t h e r e s t i n g g l a n d s w e r e u s e d to c a l c u l a t e s I a n d s 2 for t h e i n t e r s t i t i a l N a a s s u m i n g t h e i n t r a c e l l u l a r c o m p a r t m e n t to b e negligible. H o w e v e r , t h e s e F I D s m a y c o n t a i n a n a r t i f a c t d u e to t h e e x t r a c e l l u l a r N a w h o s e m a g n i t u d e c o u l d a m o u n t to 1 / 4 to 1 / 2 o f the total i n t e n s i t y o f t h e F I D . T h e i n h o m o g e n e i t y - r e l a t e d d e c a y r a t e c o n s t a n t ( a p p r o x . 10 s - x ) m i g h t also c o n t r i b u t e a little to t h e e r r o r s in t h e v a l u e s o f s 1 a n d
S1
S2
tdT c
Tc
e2qQ/h
(s- 1)
(s- 1)
(tad- s)
(ps)
(MHz)
Intracellular Na (a) 1360 + 75 (b) 610
94.7 + 3.7 130
4.0 _ 0.2 2.0
6.7 _+0.3 3.4
1.95 + 0.02 1.7
Interstitial Na 71.1 +3.1
42.8 +1.0
0.70 +0.07
1.2 +0.1
0.86 +0.01
n
5 (5) 5
s 2. C o n s e q u e n t l y , t h e r e l a x a t i o n p a r a m e t e r s e s t i m a t e d for t h e i n t e r s t i t i a l N a s h o u l d b e r e g a r d e d w i t h c a u t i o n .
Measurement of changes in intracellular N a T h e effects o f a c e t y l c h o l i n e (1 # M ) s t i m u l a t i o n in the p r e s e n c e o f o u a b a i n (1 m M ) o n t h e c o n t e n t o f i n t r a cellular Na were examined using the following protocol. A f t e r 90 m i n o f c o n t r o l p e r f u s i o n , a c e t y l c h o l i n e w a s a d m i n i s t e r e d for 22.5 m i n , t h e n b o t h o u a b a i n a n d a c e t y l c h o l i n e w e r e a d m i n i s t e r e d for 67.5 rain, a n d t h e n the control perfusion without stimulation was continued for a f u r t h e r 45 m i n . 23Na d o u b l e q u a n t u m f i l t e r e d s p e c t r a w e r e o b s e r v e d e v e r y 7.5 m i n d u r i n g t h e e x p e r i m e n t . T h e s p e c t r u m c o l l e c t e d d u r i n g t h e last 60 m i n o f t h e i n i t i a l c o n t r o l p e r i o d was u s e d as t h e c o n t r o l spectrum. Fig. 5 s h o w s c h a n g e s in t h e d i f f e r e n c e s p e c t r a , obtained by subtracting the control spectrum from the s u b s e q u e n t series o f spectra. T h e p e a k h e i g h t is r o u g h l y p r o p o r t i o n a l to t h e i n t r a c e l l u l a r N a c o n t e n t , since the a p p a r e n t l i n e - w i d t h at h a l f - h e i g h t o f t h e p e a k (ul/2 = 33 + 1 H z ( n = 14) -- SE/'h" d- the fine b r o a d e n i n g f a c t o r ) remained constant during the experiment. Upon stimulation with acetylcholine, the intracellular Na content i n c r e a s e d b y 2 8 _ 6 a r b i t r a r y u n i t s ( m e a n _+ S.E. of a v e r a g e d d a t a f r o m 0 to 22.5 m i n , n = 4), a n d r e m a i n e d at this l e v e l d u r i n g s t i m u l a t i o n . T h e i n t r a c e l l u l a r N a content increased again during administration of o u a b a i n w i t h a c e t y l c h o l i n e , a n d s h o w e d a p l a t e a u level
146
I Ouabain [ Acetylcholine
-40
0
4'0
'
da
'
'
Time ( min ) Fig. 5. Changes in the intracellular 2 3 N a r e s o n a n c e induced by 1/~M acetylcholine with 1 mM ouabain at 25 o C. After 22.5 min of preadministration of acetylcholine, ouabain was appfied for 67.5 min. Averaged spectra of the intracellular 23Na measured every 7.5 rain using the double quantum filter with a r value of 6 ms and a 8 value of 10/*s are shown (n = 4). The spectra were obtained by subtraction of the resting gland spectrum from the series of original spectra.
of 136 + 15 arbitrary units (mean + S.E. of averaged data from 67.5 to 90 min, n = 4).
Discussion
This is to our knowledge the first study in which the • 23parameters of quadrupolar relaxation of N a have been determined in the mammalian salivary gland. The 23Na-NMR spectrum of intracellular N a in living cells has been interpreted as an inhomogeneous powder pattern spectrum, a homogeneous biexponential spectrum, and a spectrum of the intermediate of a rapid exchange process [2,11]. In this study, we have observed two Lorentzian resonances corresponding to the 1 1 / 2 ) ( 1 / 2 I coherence and the i - 1 / 2 ) ( - 3 / 2 I and J 3 / 2 ) { 1 / 2 i coherences of the intracellular Na. The difference in the resonance frequency between the two resonances was negligibly small. We therefore exclude the classical inhomogeneous powder pattern model. Consequently, the apparent reduction in the N M R visibility of intracellular N a (e.g., Refs. 1, 2, 13) might be due to the relatively rapid relaxation of the 1 - 1 / 2 ) ( 3 / 2 1 and 1 3 / 2 ) ( 1 / 2 1 coherences and also due to the relatively long dead time following the sampling pulse.
The homogeneous biexponential spectrum is expected from a homogeneous population of 23Na under the condition that ~ 1 >> rc >> ~-1, where ~Q is the time-averaged values of the quadrupolar coupling constant in frequency units. The resonance is a superposition of a narrow Lorentzian resonance corresponding to the 1 1 / 2 ) ( - 1 / 2 J coherence and a broad Lorentzian resonance corresponding to J - 1 / 2 ) ( 3 / 2 I and 1 3 / 2 ) ( 1 / 2 J coherences at the same frequency [11]. If we assume that there is a single, homogeneous ~opulation of intracellular Na, the correlation time of N a and the quadrupolar coupling constant (assuming the quadrupolar asymmetry parameter to be zero), is calculated to be approx. 6.7.10 - 9 S and 1.9 MHz, respectively, from s 1 and s 2 using equations (57), (58) and (5) from the paper by Jaccard et al. [6]. The value for the intracellular N a elevated by acetylcholine stimulation alone was 1.7 MHz, in good agreement in spite of the low signal-to-noise ratio. These values for the quadrupolar coupling constant are similar to those estimated for N a in glycerol (1.6 M H z [14]), associated with ionophores (0.5-2 M H z [15]) and in sodium compounds (0.3-3.7 M H z [16,17]). A model consisting of rapid exchange between immobilized and free 23Na within an isotropic medium also gives rise to a spectrum consisting of two Lorentzian resonances. In the rapid exchange model, the apparent value for the quadrupolar coupling constant might be e x p e c t e d to be smaller (approx. 0.1 M H z for sodium laurate [14]). Furthermore, when the fraction of free N a was increased significantly (approx. 5-times) by administration of acetylcholine with ouabain, the apparent quadrupolar coupling constant would be expected to decrease• However, the value observed during administration of ouabain with acetylcholine tended to be higher than that obtained during administration of acetylcholine alone (Table I). At this stage, however, we do not have strong evidence for eliminating the rapid exchange process• Studies of the field and temperature dependencies of the relaxation parameters will be necessary to confirm this conclusion [14]. We also demonstrated that the double quantum filter allows us to measure the changes in intracelhilar Na content without the use of a chemical shift reagent. The following factors might contribute to the errors in the measurement: (1) changes in the size of the double quantum coherence deriving from the interstitial Na; (2) changes in the size of the artifact due to the extracellular Na. In the isolated, perfused preparation at constant perfusion rate, small changes in the size of the interstitial and extracellular spaces could possibly cause these errors. However, the size of the error due to changes in the extent of the extracellular oedema is likely to be no more than 10-20% of the increment due to 1 ~tM acetylcholine, which is not unreasonable in this type of experiment.
147
Acknowledgements We thank H. Hattori, O, Ichikawa, A. Ikeda, K. Suzuki and H. Ohkawara for their technical assistance, and Dr. K. Nagayama and Dr. M.C. Steward for helpful discussion.
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7 Pekar, J., Renshaw, P.F. and Leigh, Jr.S. (1987) J. Magn. Reson. 72, 159-161. 8 Murakami, M., Seo, Y., Watari, H., Ueda, H., Hashimoto, T. and Tagawa, K. (1987) Jpn. J. Physiol. 37, 411-423. 9 Seo, Y., Murakami, M., Matsumoto, T., Nishikawa, H. and Watari, H. (1987) Pfliigers Arch. 409, 343-348. 10 Pekar, J. and Leigh, Jr., S. (1986) J. Magn. Reson. 69, 582-584. 11 Rooney, W.D., Barbara, T.M., and Springer, C.S. (1988) J. Am. Chem. Soc. 110, 674-681. 12 Seo, Y., Murakami, M., Suzuki, E., Kuki, S., Nagayama, K. and Watari, H. (1990) Biochemistry 29, 599-603. 13 Springer, Jr., C.S. (1987) Annu. Rev. Biophys. Chem. 16, 375-399. 14 Lerner, L. and Torchia, D.A. (1986) J. Am. Chem. Soe. 108, 4264-4268. 15 Haynes, D.H., Pressman, B.C. and Kowalsky, A. (1971) Biochemistry 10, 853-860. 16 Das, T.P. and Hahn, E.L. (1958) in Solid State Physics Suppl. 1 (Seitz, F. and Turnbull, D., eds.), pp. 119-163, Academic Press, New York. 17 Edmounds, D.T. and Mailer, J.P.G. (1979) J. Magn. Reson. 36, 411-418.