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Effects of the slow calcium-channel blocker verapamil on phosphatic metabolism of crystalline lens
Exp. Eye Res. (1988) 45, 139-148
Effects of the Slow C a l c i u m - C h a n n e l Blocker V e r a p a m i l on Phosphatic M e t a b o l i s m of Crystalline Lens JACK V. G R E I N E R * t AND THOMAS GLONEK$
* Howe Laboratory of Ophthalmology, Harvard Medical School, Massachusetts Eye and Ear Infirmary, and the Eye Research Institute of Retina Foundation, Boston, MA, and the ~M R Laboratory and Departments of Physiology and Pathology, "~Chicago College of Osteopathic Medicine, Chicago, I L 60615, U.S.A. (Received 9 March 1987 and accepted 18 July 1987) The effects of calcium-channel blockade on phosphate metabolism in the rabbit lens ex vivo was studied using phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy. Rates-of-change of the intralenticular pH and the following phosphatic metabolites in the lens were determined : ATP, ADP, inorganic orthophosphate, sugar phosphates, nucleoside diphosphosugars, phosphodiesters, dinucleotides, and an uncharacterized resonance peak at 63 in the alp spectrum. Incubation in 20, 200 or 2000/zg/ml verapamil led to the same qualitative changes in the lens's metabolic profile at each different concentration ; only the rates of metabolite change were altered significantly by verapamil concentration. Compared with the control, the entire orthophosphate resonance band increased relative to the signals of ATP, and the a-glycerophosphate and nucleoside monophosphate resonances increased in relation to inorganic orthophosphate, and these two resonance groups shifted relative to each other so that they coalesced, indicating that the two groups of signals arose from compounds in relatively different pH compartments. In addition to altering metabolic rates, verapamil reduced the intralentricular pH of the sugar phosphate and inorganic orthophosphate pools with respect to those containing the nucleoside monophosphates ; the pH-change difference was about one order of magnitude smaller for the mononucleotide pool. Despite the metabolic alterations and changes in pH during verapamil incubation, lens clarity was unchanged. Key words: crystalline lens; intralenticular pH; metabolic rates; phosphatic metabolites; alp NMR; slow calcium-channel blockers; verapamil.
1. I n t r o d u c t i o n Slow c a l c i u m - c h a n n e l blockers have been used to t r e a t a n g i n a pectoris, cardiac a r r h y t h m i a s , h y p e r t r o p h i c c a r d i o m y o p a t h y , systemic hypertension, a n d R a y n a u d ' s p h e n o m e n o n (Brawnwald, 1982) w i t h o u t a p p a r e n t adverse h u m a n o p h t h a l m i c consequences. The calcium a n t a g o n i s t v e r a p a m i l has been d e m o n s t r a t e d to p r e v e n t lens opacification in alloxan-diabetic rats (Fleckenstein, 1983). I n these rats with a blood glucose level of 420 m g / 1 0 0 ml, 62 % of the lenses became opaque w i t h i n 6"5 m o n t h s , b u t 25 m g / k g of v e r a p a m i l a d m i n i s t e r e d b y a stomach t u b e reduced the occurrence of lens c a t a r a c t to 3"9 %. W i t h o u t v e r a p a m i l the calcium c o n t e n t of these diabetic r a t lenses increased from 1"1 to 10-8 mM/kg dry wt, whereas with v e r a p a m i l the m e a n calcium c o n t e n t r e m a i n e d low at 3"5 mM/kg. The influence of v e r a p a m i l on the calcium c o n t e n t of lenses m a y be a m o d u l a t i n g factor of the biochemical processes responsible for p r e v e n t i n g cataractogenesis. W i t h regard to the established correlation between a n elevated calcium level a n d lens opacification, F l e c k e n s t e i n (1983) showed t h a t cataracts develop when the lens calcium c o n t e n t exceeds a threshold value of 7-8 mM/kg dry wt. E l e v a t e d calcium c o n t e n t has been reported in senile c a t a r a c t (Burge, 1909 ; Adams, 1929 ; Salit, 1933 ; :~ To whom correspondence should be addressed. 0014-4835/88/020139+ 10 $03.00/0
9 1988 Academic Press Limited
140
J.V. GREINER AND T. GLONEK
Duncan and Bushell, 1975). Salit (1933) found that the calcium content of cataractous lenses was increased tenfold. Jedziniak, Nicoli, Yates and Benedek (1976) reported that the calcium content of 60 cataractous lenses was 2-13 times higher than the mean calcium concentration of normal lenses. Calcium uptake, even in modest amounts, can result in the loss of lens transparency (Adams, 1929; Jedziniak, Kinoshita, Yates, Hocker and Benedek, 1972 ; Spector and Rothschild, 1973 ; Spector, Adams and Krul, 1974). On the other hand, calcium deficiency also promotes caractogenesis as reviewed previously (Glonek, Kopp, Greiner and Sanders, 1985). Any disturbance of lens calcium homeostasis seems to affect adversely the metabolic processes responsible for maintaining lens transparency (Iwata, 1974). In rabbit lens, disturbances of calcium metabolism were detrimental to phosphate metabolism (Glonek et al., 1985). Ex vivo incubation of the lens in calcium-deficient buffers resulted in a pronounced, time-dependent decrease in lenticular ATP levels, with the half-life of ATP being about 11 hr. A concomitant stoichiometric increase in A D P and inorganic orthophosphate (Pi) was also reported. Increasing with time were the purine nucleotides, inosine monophosphate and adenosine monophosphate. With calcium-deficient incubation, the intralenticular pH became more alkaline, and lens energy metabolism was irreversibly impaired. In view of such findings, we questioned whether the slow calcium-channel blocking agent verapamil would have similar effects on lens phosphate homeostasis and on intralenticular pH in rabbits.
2. M a t e r i a l s a n d M e t h o d s Surgical Twenty-four albino rabbits (2-3 kg) received peritoneal injections containing a lethal dose of sodium pentobarbital. The eyes were enucleated, and frontal and sagittal incisions were made in the posterior pole of each eye using a razor blade. The incisions were extended to the ora serrata with curved blunt scissors. After exposing the crystalline lens by separating the vitreous humour with a glass spatula, the zonules were cut. The lens was removed with the aid of a glass lens loupe and immersed in modified Earle's buffer at 37~ (Greiner, Kopp, Sanders and Glonek, 1981). In vitro incubation Twenty-four eyes were used for the experimental procedure, and 24 eyes served as controls. In all experimental time-course determinations, lenses were first incubated in a control buffer for 2 h to equilibrate the pair of isolated lenses to the in vitro conditions (Greiner et al., 1981). For each of four 22 hr time-course determinations carried out per experimental concentration of verapamil, two isolated lenses were weighed and placed together in a tared 12-mm nuclear magnetic resonance (NMR) sample tube containing 2 ml of modified Earle's buffer. Earle's buffer, which contains 5"6 mM glucose, has an osmolarity of 295 mosM and a pH of 7"4 at 37~ The experimental medium was a modified Earle's buffer containing 20, 200 or 2000#g/ml verapamil. The medium was changed periodically according to previously described procedures (Greiner et al., 1981). Identical procedures were used for the control lenses incubated in Earle's buffer. Lens transparency Following incubation all lenses were analysed by photomacrography (Greiner, Chylack and Pihlaja, 1976). Lens transparency was monitored using grid paper observed through the lens by biomicroscopy. Any distortion of the linear grid lines was considered as loss of lens transparency.
VERAPAMIL
E F F E C T S ON L E N S M E T A B O L I S M
141
Lens perchloric acid (PCA ) extraction A t t h e e n d of each time-course, e x p e r i m e n t a l a n d control samples were frozen in liquid nitrogen. PCA e x t r a c t i o n s were t h e n p e r f o r m e d on b o t h t y p e s of samples. These e x t r a c t s were p r e p a r e d according t o s t a n d a r d procedures (Bs a n d Glonek, 1982).
~'P NMR spectroscopic analysis 31p N M R spectroscopic analyses a t 81 M H z h a v e b e e n described p r e v i o u s l y (Greiner et al., 1981). I n t a c t - t i s s u e spectroscopic a n a l y t i c a l conditions were as follows: pulse width, 9 #sec (45 ~ spin-flip angle); a c q u i s i t i o n delay, 2 0 0 # s e c ; n u m b e r of scans, 4400; n u m b e r of d a t a p o i n t s per f r e e - i n d u c t i o n decay, 8192; a c q u i s i t i o n time, 0"819 sec; sweep w i d t h + 2 5 0 0 H z ; t h e filter t i m e - c o n s t a n t used i n t r o d u c e d l 0 H z a d d i t i o n a l line b r o a d e n i n g . Spectroscopic conditions used d u r i n g lens P C A - e x t r a c t analysis were identical to those used d u r i n g t h e i n t a c t - l e n s analysis, e x c e p t for t h e following p a r a m e t e r s : n u m b e r of scans, 30000; n u m b e r
TABLE I
Perchloric acid (PCA )-extract and intact-tissue rabbit lens phosphate profiles after 13-hr verapamil (2000 ~ff / ml) incubation Mole % of total lens profile signal area Phosphorus resonance 5"988 4'658 4'588 4-458 (Hexose 6-P) ~GP 3"868 NMP NADP-2-'P Choline P Pi 0"928 0.858 GPC ATP ADP DN NS
Chemical shift in extract ($)~
PCA extract (% detected phosphorus LS.E.M.)
5"98 4"65 4"58 4"45
1.79_+ 0"17 0"50 ___0' 17 0'68 + 0"21 0-98 • 0"35
4"29 3'86 3"76 3"55 3"32 2"63 0"92 0"85 0-13 w I[ 82 :~
7-45_ 1"03 0-87 + 0'36 5'22 + 1'42 0"27 + 0"85 1-20___0'25 18-79 + 0"78 0.61 ___0"54 0.57 • 0"45 2-03 _+0-71 28-74 • 1"30 6.14• 12"96 + 1-04 11"20 • 2"51
Intact tissue (% detected phosphorus calculated from Table III) 2.53
17"17~
20"80
18"57 3'215
2-97 29-39 5-85 10"01 9"87
* Numbered resonances are unidentified. Following the IUPAC convention with the resonance of 85 % phosphoric acid at 24~ designated 08. $ Summed percentages corresponding to phosphates that contribute respective signal areas to the sugar-phosphate and phosphodiester bands of the intact-tissue spectrum. w ATP gives rise to three P-31 resonance signals corresponding to three phosphate groups in the molecule: a, 10-898;/?, -21-418; ~, -5"768. [I ADP gives rise to two P-31 resonance signals: a, -10"558;/?, -6"138. 82 The principal resonance signals of this band arise from the P, P'-diesterified pyrophosphate residues of NAD and NADP : - 11"373. Resonances from - 1 1 to - 1 3 3 correspond to hexose-phosphate portions of resonance multiplets. Hexose 6-P, hexose 6-phosphate; aGP, a-glycerophosphate; NMP, nucleoside monophosphates; NADP 2-'P, nicotinamide adenine dinucleotide phosphate 2-'P; choline-P, choline phosphate; Pi, inorganic orthophosphate; GPC, glycerol 3-phosphochotine; ATP, adenosine triphosphate; ADP, adenosine diphosphate; DN, dinucleotides; NS, nucleoside diphosphosugars.
142
J. V. G R E I N E R AND T. G L O N E K TABLE II
Time-course data for 200/~g/ml verapamil Coefficients (y = Phosphate resonance band ATP Pi SP NS GPC ADP DN 65
Slope (A) -- 1.205 • @390 • 0.405 • 0"394 _ 0-038 • 0"041 • - 0"064 • 0.000 •
pH
0"058 0.025 0.034 0.053 0.014 0-066 0"013
0"016 • 0.002
Ax+B)*• Intercept (B) 45"05 _ 13.50 • 15"53 • 4-75 • 2.48 • 5-32 • 10"84 • 2"53 •
0"36 0-16 0-21 2.33 0"28 1"20 1.41 0"48
6.81 • 0'03
* For phosphates the ordinate is the mole 5 % phosphorus detected and the abscissa is hours ; for pH the ordinate is pH units. ATP, adenosine triphosphate; Pi, inorganic orthophosphate; SP, sugar phosphates; NS, nucleoside diphosphosugars, GPC, glycerol 3-phosphocholine ; ADP, adenosine diphosphate ; DN, dinucleotides ; 63, unidentified resonance signal. TABLE III
Time-course data for 2000 l~g/ml verapamil Coefficients (y = Phosphate resonance band
Slope (A)
Ax+B)*• Intercept (B)
ATP Pi SP NS GPC ADP DN 65
- 1"248• 0.026 0-372 + 0.027 0"389 • 0"034 0"450 • 0"018 @000 • 0.005 @059 • 0.023 -- 0.004 _ 0.051 - 0"037 • 0"022
45"86 _ 1.20 13.59 _ 0-21 14-84 _ 0"26 5"40 + 2"14 2'56 • 0.40 5'05 • 1.18 10.60 • 1-08 2"30 • 0"77
pH
-- 0"017 + 0"022
6"81 • 0-77
* For phosphates the ordinate is the mole % phosphorus detected and the abscissa is hours ; for pH the ordinate is pH units. ATP, adenosine triphosphate; Pi, inorganic orthophosphate; SP, sugar phosphates; NS, nucleoside diphosphosugars ; GPC, glycerol 3-phosphocholine ; ADP, adenosine diphosphate ; DN, dinucleotides; 65, unidentified resonance signal.
o f d a t a p o i n t s per free-induction decay, 16384; acquisition time, 1"64 sec a n d a filter t i m e c o n s t a n t t h a t i n t r o d u c e d 0"6 H z a d d i t i o n a l line broadening. To c o m p e n s a t e for relative s a t u r a t i o n effects a m o n g the various p h o s p h o r u s signals d e t e c t e d in a single 31p N M R spectroscopic e x t r a c t profile, t h e N M R spectra were s t a n d a r d i z e d a g a i n s t m e a s u r e d a m o u n t s of tissue-profile metabolites. The calibration procedures h a v e been described (Bdrdny a n d Glonek, 1982). I n t a c t - t i s s u e spectra were c o m p a r e d w i t h t h e s e calibrated e x t r a c t s p e c t r a ; t h e q u a n t i t a t i v e c o m p a r i s o n was t h e same, w i t h i n e x p e r i m e n t a l limits, s u p p o r t i n g t h e i n t e r p r e t a t i o n of Greiner et al., (1981) a n d Bdr~ny
VERAPAMIL EFFECTS ON LENS METABOLISM
t43
and Glonek (1982) that using moderately rapid repetition times does not result in measurable discriminatory loss of signal-area fidelity among components contributing to an intact-tissue spectrum. Table I demonstrates that, within the precision of measurement obtainable from intacttissue analysis, the relative metabolite levels detected by the extract spectroscopic analysis are the same as the corresponding values obtained from the intact tissue (Burr, Glonek End Bdr~ny, 1976 ; Bs and Glonek, 1982). The individual resonance peaks that made up the lens extract spectrum are more highly resolved than those from the intact tissue as a result of the optimal physical-chemical conditions present in the extract. Thus, the extract analyses provide more detailed metabolic information regarding levels of certain phosphatic compounds, which are not well resolved in the intact-tissue spectrum.
Mathematical analysis The time-course data obtained were reduced by a least-squares regression analysis using the equation, Y = Ax+B. The data are presented in Tables II, I I I and IV. Intralenticular pH was determined by comparing the chemical-shift value of known resonances, e.g., Pi, to standardized pH spectroscopic 31p NMR titration curves (Bdr~ny and Glonek, 1982):
TABLE IV
Time-course data for 20 gg/ml verapamil Coefficients (y = Ax+B)*• Phosphate resonance band ATP Pi SP NS GPC ADP DN 65 pH
Slope (A) - 0"408_ 0-011 @152 + 0"034 @163 _ 0"083 @031 • 0-027 0.011 _ 0"011 @068_ 0"080 - @000_ 0'051 - 0'017 + @024 - 0-003_ @001
Intercept (B) 44.90 _+1.07 13.94 + 0-32 15.85_+0"78 3.45 + 1'55 2'90_ 0.70 4.05 _ 1'32 12"05• 1-48 2.86 • 0.23 6'85 • 0.03
* For phosphates the ordinate is the mole % phosphorus detected and the abscissa is hours; for pH the ordinate is pH units. ATP, adenosine triphosphate; Pi, inorganic orthophosphate; SP, sugar phosphates; NS, nucleoside diphosphosugars ; GPC, glycerol 3-phosphocholine; ADP, adenosine diphosphate; DN, dinucleotides; 63, unidentified resonance signal.
3. R e s u l t s I n control i n c u b a t i o n s (no added verapamil), modified E a r l e ' s buffer supported the m e t a b o l i s m of the r a b b i t lens for the entire 22 hr i n c u b a t i o n , d u r i n g which i n t e r v a l the i n t r a l e n t i c u l a r p H a n d relative c o n c e n t r a t i o n s of all phosphates except A T P r e m a i n e d c o n s t a n t . The relative A T P c o n c e n t r a t i o n increased b y 5 % . Levels of phosphatic metabolites did n o t differ significantly from those reported previously (Greiner et al., 1981). Regardless of the v e r a p a m i l c o n c e n t r a t i o n (20, 200 or 2 0 0 0 # g / m l ) d u r i n g i n c u b a t i o n , the lens' metabolic profile changed similarly ; only the rates of m e t a b o l i t e change were altered m e a s u r a b l y b y v e r a p a m i l c o n c e n t r a t i o n . Figure 1 shows the 31p N M R s p e c t r u m of r a b b i t lens d u r i n g a n initial control i n c u b a t i o n a n d after a 10 hr
144
J.V. GREINER AND T. GLONEK
Control
ATP
p
Pi
,NMP ADP
Experimental
f
8
I
I
I
I
0
I
I
'
'
l
-10
I
'
'
'
I
r
--20
FIG. 1. The alp N-MR spectra of a control and experimental rabbit lens after a 10 hr incubation in 200 pg/ml verapamil in modified Earle's buffer at 37~ 63, unidentified chemical resonance; aGP, resonance band of triose phosphates and hexose 6-phosphates; NMP, nucleoside monophosphates; Pi, inorganic orthophosphate; GPC, glycerol 3-phosphocholine; ATP, a-, fl-, and 9,-phosphate groups; ADPfl, the fl, chain-terminal phosphate of ADP; DN, dinucleotide phosphate resonance band; NS, nucleoside diphosphosugar, hexose-phosphate resonance band.
incubation in 200 # g / m l verapamil buffer. The control spectrum shows 31p resonance bands originating from the following phosphatic substances: 63, a phosphatic compound of undetermined chemical structure, prominent particularly in the rabbit lens; aGP, the resonance band of triose phosphates and hexose 6-phosphates, which contains the signal from a-glycerophosphate in rabbit lens as a principal component (Greiner et al., 1981); NMP, nucleoside monophosphate, the resonance band of the pentose 5-phosphates and other phosophomonoesters, which contains the signals from the two nucleoside monophosphates IMP and AMP in the rabbit lens as principal components (Kopp, Glonek and Greiner, 1983); Pi, the resonance from inorganic orthophosphate ; GPC, the resonance signal from glycerol 3-phosphocholine, which serves as the internal spectroscopic chemical-shift reference; ATP, a, fl, and 7 phosphate signals; A D P /?, the /?, chain-terminal phosphate of ADP; DN, the resonance band of the dinucleotides, principally NAD with some contribution from N A D P and the nucleotide portion of the uridine diphosphosugars; NS, the sugar
VERAPAMIL EFFECTS ON LENS METABOLISM
145
phosphate portion of the nucleoside diphosphosugars, which contains uridine diphosphoglucose as the principal component (Greiner et al., 1981). After a 10 hr incubation in the verapamil, the spectrum was altered (Fig. 1), especially in the orthophosphate region (8~-0~). Relative to the control, the entire orthophosphate band increased in relation to the ATP signals, the a-glycerophosphate (aGP) and NMP resonances increased in relation to Pi and shifted relative to each other so that they coalesced, indicating that the two groups of signals arose from compounds in relatively different pH compartments.
Control
Pi
aGP/~NMP GPC
Pi
i
I
w
8
1
I
5
r
I
I
I
@
F~G.2. Expanded view of the 31p NMR orthophosphate spectral region of a control and experimental rabbit lens after a 10 hr incubation in 200 #g/ml verapami[ in modified Ear|e's buffer, showing the coalescence of the a-glycerophosphate (aGP) and nucleoside monophosphate (NMP) resonance bands.
Figure 2 shows the orthophosphate spectral region on an expanded scale. The three greatest changes resulting from verapamil incubation were the upfield shift (to lower 3 values) of the a-GP band and Pi and the increased NMP concentration. The Pi upfield shift of 0-283 corresponded to a decrease in intralenticular pH over 10 hr of 0-20 from the initial value of 6"83 (BKrs and Glonek, 1982). The corresponding shift in the a G P signal was 0"433, which corresponded over 10 hr to a decrease in pH of 0"31 from an initial value of 6"83. The nucleoside monophosphate shift was less ( 0"123, S.E.M. • 0"03) and corresponded to a pH decrease of 0"07, S.E.M. • 0"006 from an initial value of 6"80. Under the influence of verapamil, the lenticular ATP concentration and
146
J.V. GREINER AND T. GLONEK
intralenticular pH declined with time. Pi, the combined sugar phosphates, and the nucleoside diphosphosugars increased. The remaining signals were unaffected by the verapamil incubation. Equations corresponding to changes in spectral signals with time are presented in Tables II, III and IV. All changes with time for all three verapamil concentrations tested are expressed as linear processes. Significant concentration changes {slope in column A in Tables II-IV) were observed only for ATP, Pi, the sugar phosphates (4"293-3"763) and the nucleoside diphosphosugars. GPC, ADP, the uncharacterized substance at 63 and the dinucleotides remained generally constant throughout the incubation regardless of the verapamil concentration. (The small apparent slopes in these data are rendered insignificant by a large degree of scatter.) The intercepts (column B in Tables II-IV) correspond to control rabbit lens mole-percentages previously reported (Greiner et al., 1981). The loss of ATP phosphorus corresponded to nearly equivalent rates of increase in Pi, sugar phosphates and nucleoside diphosphosugars. From the PCA-extract data, the nucleoside monophosphate component, not a glycolytic or shunt-pathway intermediate, increased within the sugar-phosphate band. This component ordinarily accounts for 2 % of the total detectable phosphorus in control spectra. At 200/~g/ml, verapamil incubation nearly doubled this value. Despite the metabolic alterations and changes in pH observed during the 22 hr verapamil incubations, lens clarity was unchanged. 4. D i s c u s s i o n
The most noticeable effect of verapamil incubation on mammalian lens was the reduction of the intralenticular pH in pools containing Pi and aGP. Both these substances reside within the same hydrogen-ion pool, i.e. they exhibit the same spectroscopic pH. This pool, however, differs from that containing the mononucleotides AMP and IMP in which the spectroscopic pH rate of change with verapamil incubation is less than that of Pi and aGP. Gradients of pH within a tissue have been observed spectroscopically using other systems, the most well-documented example being the acid and alkaline Pi pools in skeletal muscles (Burr et al., 1976; Busby, Gadian, Radda, Richards and Seely, 1978) and heart (Garlick, Brown, Sullivan and Ugurbil, 1983). Chemical-shift data have been interpreted to indicate the existence of multiple pH pools within lens (Greiner, Kopp and Glonek, 1982; Kopp et al., 1983), although challenges to the lens system, such as incubation with ouabain, have always resulted in parallel changes in the lenticular pH pools (Greiner, Kopp and Glonek, 1985a). Our study provides the first example of a pH change in one pool not paralleled by a corresponding change in another. According to the pH effect, the metabolite pool containing the nucleoside monophosphate must be distinct from that containing Pi and aGP. How a calcium-channel blocking agent can selectively affect these two metabolite pools is unknown. However, one broad interpretation can be applied. In a study on the distribution of phosphatic metabolites within the crystalline lens (Greiner, Kopp and Glonek, 1985b), inosine monophosphate was more prominent in the lens nucleus (mole %, 3"95) than in the cortex (mole %, 0"90). The remaining orthophosphate esters were distributed more uniformly, although the entire sugar phosphate band was more prominent in the cortex than in the nucleus. Consistent with these data, verapamil may act primarily on the cortex or may be prevented from reaching nuclear cells, which contain the bulk of the nucleoside monophosphate
VERAPAMIL EFFECTS ON LENS METABOLISM
147
component. The spectroscopic pH effects of the intact lens may occur because the cortex is becoming more acidic in response to verapamil, but the deeper and apparently less metabolically active nucleus is unaffected. To explain the verapamil-induced acidosis, it has been hypothesized that the calcium channel and slow sodium channel use the same enzymatic components (Nayler and Krikler, 1974 ; Rattke and Glasser, 1978), and that a blocking agent, such as verapamil, inhibits both enzymatic activities. Inhibiting sodium translocation promotes acidosis by inhibiting the charge-transfer process. Without sodium translocation, a charge imbalance occurs with hydronium-ion pumping mlless anions are translocated simultaneously. Since cells cannot tolerate a charge imbalance or excessive salt pumping, hydrogen ions generated from lenticular glycolysis accumulate in the absence of sodium translocation. The excess of hydrogen ions is apparently not distributed uniformly throughout the lenticular cells but is restricted to the specific low-pH pools. The ATP depletion during incubation is not great in relation to the stress placed upon the lens system by the great reduction of intralenticular pH. For this mason, we interpret the loss of lenticular ATP as secondary to hydrogen ion accumulation and resulting from an increasing energy demand produced by increasing acidosis. Pi and the nueleoside monophosphates are the products of ATP hydrolysis and accumulate if ATP levels cannot be restored at the required rate. Elevation of the nucleoside diphosphosugar levels suggests a stimulation of fl-Dglucuronide or glycogen biosynthesis, or both. Since the pathway for glycuronide biosynthesis also generates hydrogen ions, its activation can exacerbate the intralenticular pH decrease. The most probable need for the increased uridine diphosphosugars, therefore, may be activated glucose for glycogen biosynthesis. Limiting our study is the lack of a supporting medium to provide the lens with metabolic stability for more than 26 hr at 37~ (Glonek et al., 1985). Thus, assessing long-term effects on the metabolic and pH changes of the lens must await development of such a medium. In summary, this study demonstrates that verapamil significantly alters the rates of phosphorus metabolite change in the lens and lowers intralenticular pH. Despite these metabolic changes, however, verapamil may protect against cataractogenesis as reported in a study of alloxan-diabetic rats undergoing long-term treatment with verapamil (Fleckenstein, Witzleben, Frey and Milner, 1981). ACKNOWLEDGMENTS We thank Donna G. Peace, M.S., and Manuel G. Flores, B.S., for technical assistance. This study was supported by grant EY-03988 from the National Eye Institute, National Institutes of Health, Bethesda, MD, U.S.A. REFERENCES Adams, D. R. (1929). Role of calcium in senile cataract. Biochem. J. 23, 902-12. BgrAny, M. and Glonek, T. (1982). Phosphorus-31 nuclear magnetic resonance of contractile systems. In Methods in Enzymoloyy, Vol. 85B, (eds Frederiksen, D. L. and Cunningham, L. W.) Pp. 624-76.Academic Press: N e w York. Brawnwald, E. (1982). Mechanism of action of calcium-channel-blocking agents. N. Engl. J. Med. 307, 1618-27. Burge, W.E. (1909). Analyses of the ash of the normal and cataractous lens. Arch. Ophthalmol. 38, 435-50.
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Burt, C.T., Glonek, T. and Bdr~ny, M. (1976). Analysis of phosphate metabolites, the intracellular pH, and the state of adenosine triphosphate in intact muscle by phosphorus nuclear magnetic resonance. J. Biol. Chem. 251, 2584-91. Busby, S.J., Gadian, D.G., Radda, G.K., Richards, R.E. and Seeley, P.J. (1978). Phosphorus nuclear-magnetic-resonance studies of compartmentation in muscle. Biochem. J. 170, 103-14. Duncan, G. and Bushell, A. R. (1975). Ion analyses of human eataractous lenses. Exp. Eye Res. 20, 223-30. Fleckenstein, A. (1983). Prevention by verapamil of arterial calcium overload and concomitant lenticular calcification causing cataracts in alloxan-diabetic rats. In Calcium Antaqonism in Heart and Smooth Muscle, Experimental Facts and Therapeutic Prospects, (ed. Fleckenstein, A.) Pp. 280-5. John Wiley and Sons, New York. Fleckenstein, A., Witzleben, H.V., Frey, M. and Milner, T.G. (1981). Prevention of cataracts of alloxan-diabetic rats by long-term treatment with verapamil. Pfluegers Arch. Eur. J. Physiol. 391 (Suppl 1), R12. Garlick, P. B., Brown, T. R., Sullivan, R. H. and Ugurbil, K. (1983). Observation of a second phosphate pool in the perfused heart by alp NRM; is this the mitochochondrial phosphate ? J. Mol. Cell. Cardiol. 15, 855-8. Glonek, T., Kopp, S.J., Greiner, J.V. and Sanders, D.R. (1985). Lenticular energy metabolism during exogenous calcium deprivation and during recovery: Effects of Dextran-40. Exp. Eye Res. 40, 169-78. Greiner, J.V., Chylack, L.T., Jr. and Pihlaja, D.J. (1976). Photomacrography of the crystalline normal and cataractous lens. Exp. Eye Res. 32, 281-4. Greiner, J. V., Kopp, S. J. and Glonek, T. (1982). Dynamic changes in the organophosphate profile upon treatment of the crystalline lens with dexamethasone. Invest. Ophthalmol. Vis. Sei.23, 14-22. Greiner, J. V., Kopp, S. J. and Glonek, T. (1985a). Phosphorus-31 NMR analysis of dynamic energy metabolism in intact crystalline lens treated with ouabain: Phosphorylated metabolites. Ophthalmic Res. 17, 269-78. Greiner, J. V., Kopp, S. J. and Glonek, T. (1985b). Distribution of phosphatic metabolites in the crystalline lens. Invest. Ophthalmol. Vis. Sci. 25, 537-44. Greiner, J. V., Kopp, S. J., Sanders, D. R. and Glonek, T. (1981). Organophosphates of the crystalline lens: a nuclear magnetic resonance spectroscopic study. Invest. Ophthalmol. Vis. Sci. 21,700-13. Iwata, S. (1974). Process of lens opacification and membrane function : A review. Ophthalmic Res. 6,138-54. Jedziniak, J. A., Kinoshita, J. H., Yates, E. M., Hocker, L. O. and Benedek, G. B. (1972). Calcium-induced aggregation of bovine lens alpha erystallins. Invest. Ophthalmol. Vis. Sci. 11,905-15. Jedziniak, J.A., Nicoli, D.F., Yates, E.M. and Benedek, G.B. (1976). On the calcium concentration of cataractous and normal human lenses and protein fractions of cataractous lenses. Exp. Eye Res. 23, 325-32. Kopp, S. J., Glonek, T. and Greiner, J. V. (1983). Dynamic changes in intact crystalline lens metabolism modulated by alkaline earth metals : I. Effects of magnesium. Exp. Eye Res. 36, 327-35. Nayler, W. G. and Krikler, D. (1974). Verapamil and the myocardium. Postgrad. Med. J. 50, 441-6. Rattke, W. and Glasser, D. (1978). Effects of verapamil on bovine lens epithelium in primary culture. Ophthalmic Res. 10, 162-7. Salit, P.W. (1933). Calcium content and weight of human cataractous lenses. Arch. Ophthalmol. 9, 571-8. Spector, A., Adams, D. and Krul, K. (1974). Calcium and high molecular weight protein aggregates inbovine and human lens. Invest. Ophthalmol. Vis. Sei. 13, 982-90. Spector, A. and Rothschild, C. (1973). The effect of calcium upon the reaggregation of bovine alpha-crystallin. Invest. Ophthalmol. Vis. Sci. 12, 225-31.
Effects of the Slow C a l c i u m - C h a n n e l Blocker V e r a p a m i l on Phosphatic M e t a b o l i s m of Crystalline Lens JACK V. G R E I N E R * t AND THOMAS GLONEK$
* Howe Laboratory of Ophthalmology, Harvard Medical School, Massachusetts Eye and Ear Infirmary, and the Eye Research Institute of Retina Foundation, Boston, MA, and the ~M R Laboratory and Departments of Physiology and Pathology, "~Chicago College of Osteopathic Medicine, Chicago, I L 60615, U.S.A. (Received 9 March 1987 and accepted 18 July 1987) The effects of calcium-channel blockade on phosphate metabolism in the rabbit lens ex vivo was studied using phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy. Rates-of-change of the intralenticular pH and the following phosphatic metabolites in the lens were determined : ATP, ADP, inorganic orthophosphate, sugar phosphates, nucleoside diphosphosugars, phosphodiesters, dinucleotides, and an uncharacterized resonance peak at 63 in the alp spectrum. Incubation in 20, 200 or 2000/zg/ml verapamil led to the same qualitative changes in the lens's metabolic profile at each different concentration ; only the rates of metabolite change were altered significantly by verapamil concentration. Compared with the control, the entire orthophosphate resonance band increased relative to the signals of ATP, and the a-glycerophosphate and nucleoside monophosphate resonances increased in relation to inorganic orthophosphate, and these two resonance groups shifted relative to each other so that they coalesced, indicating that the two groups of signals arose from compounds in relatively different pH compartments. In addition to altering metabolic rates, verapamil reduced the intralentricular pH of the sugar phosphate and inorganic orthophosphate pools with respect to those containing the nucleoside monophosphates ; the pH-change difference was about one order of magnitude smaller for the mononucleotide pool. Despite the metabolic alterations and changes in pH during verapamil incubation, lens clarity was unchanged. Key words: crystalline lens; intralenticular pH; metabolic rates; phosphatic metabolites; alp NMR; slow calcium-channel blockers; verapamil.
1. I n t r o d u c t i o n Slow c a l c i u m - c h a n n e l blockers have been used to t r e a t a n g i n a pectoris, cardiac a r r h y t h m i a s , h y p e r t r o p h i c c a r d i o m y o p a t h y , systemic hypertension, a n d R a y n a u d ' s p h e n o m e n o n (Brawnwald, 1982) w i t h o u t a p p a r e n t adverse h u m a n o p h t h a l m i c consequences. The calcium a n t a g o n i s t v e r a p a m i l has been d e m o n s t r a t e d to p r e v e n t lens opacification in alloxan-diabetic rats (Fleckenstein, 1983). I n these rats with a blood glucose level of 420 m g / 1 0 0 ml, 62 % of the lenses became opaque w i t h i n 6"5 m o n t h s , b u t 25 m g / k g of v e r a p a m i l a d m i n i s t e r e d b y a stomach t u b e reduced the occurrence of lens c a t a r a c t to 3"9 %. W i t h o u t v e r a p a m i l the calcium c o n t e n t of these diabetic r a t lenses increased from 1"1 to 10-8 mM/kg dry wt, whereas with v e r a p a m i l the m e a n calcium c o n t e n t r e m a i n e d low at 3"5 mM/kg. The influence of v e r a p a m i l on the calcium c o n t e n t of lenses m a y be a m o d u l a t i n g factor of the biochemical processes responsible for p r e v e n t i n g cataractogenesis. W i t h regard to the established correlation between a n elevated calcium level a n d lens opacification, F l e c k e n s t e i n (1983) showed t h a t cataracts develop when the lens calcium c o n t e n t exceeds a threshold value of 7-8 mM/kg dry wt. E l e v a t e d calcium c o n t e n t has been reported in senile c a t a r a c t (Burge, 1909 ; Adams, 1929 ; Salit, 1933 ; :~ To whom correspondence should be addressed. 0014-4835/88/020139+ 10 $03.00/0
9 1988 Academic Press Limited
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J.V. GREINER AND T. GLONEK
Duncan and Bushell, 1975). Salit (1933) found that the calcium content of cataractous lenses was increased tenfold. Jedziniak, Nicoli, Yates and Benedek (1976) reported that the calcium content of 60 cataractous lenses was 2-13 times higher than the mean calcium concentration of normal lenses. Calcium uptake, even in modest amounts, can result in the loss of lens transparency (Adams, 1929; Jedziniak, Kinoshita, Yates, Hocker and Benedek, 1972 ; Spector and Rothschild, 1973 ; Spector, Adams and Krul, 1974). On the other hand, calcium deficiency also promotes caractogenesis as reviewed previously (Glonek, Kopp, Greiner and Sanders, 1985). Any disturbance of lens calcium homeostasis seems to affect adversely the metabolic processes responsible for maintaining lens transparency (Iwata, 1974). In rabbit lens, disturbances of calcium metabolism were detrimental to phosphate metabolism (Glonek et al., 1985). Ex vivo incubation of the lens in calcium-deficient buffers resulted in a pronounced, time-dependent decrease in lenticular ATP levels, with the half-life of ATP being about 11 hr. A concomitant stoichiometric increase in A D P and inorganic orthophosphate (Pi) was also reported. Increasing with time were the purine nucleotides, inosine monophosphate and adenosine monophosphate. With calcium-deficient incubation, the intralenticular pH became more alkaline, and lens energy metabolism was irreversibly impaired. In view of such findings, we questioned whether the slow calcium-channel blocking agent verapamil would have similar effects on lens phosphate homeostasis and on intralenticular pH in rabbits.
2. M a t e r i a l s a n d M e t h o d s Surgical Twenty-four albino rabbits (2-3 kg) received peritoneal injections containing a lethal dose of sodium pentobarbital. The eyes were enucleated, and frontal and sagittal incisions were made in the posterior pole of each eye using a razor blade. The incisions were extended to the ora serrata with curved blunt scissors. After exposing the crystalline lens by separating the vitreous humour with a glass spatula, the zonules were cut. The lens was removed with the aid of a glass lens loupe and immersed in modified Earle's buffer at 37~ (Greiner, Kopp, Sanders and Glonek, 1981). In vitro incubation Twenty-four eyes were used for the experimental procedure, and 24 eyes served as controls. In all experimental time-course determinations, lenses were first incubated in a control buffer for 2 h to equilibrate the pair of isolated lenses to the in vitro conditions (Greiner et al., 1981). For each of four 22 hr time-course determinations carried out per experimental concentration of verapamil, two isolated lenses were weighed and placed together in a tared 12-mm nuclear magnetic resonance (NMR) sample tube containing 2 ml of modified Earle's buffer. Earle's buffer, which contains 5"6 mM glucose, has an osmolarity of 295 mosM and a pH of 7"4 at 37~ The experimental medium was a modified Earle's buffer containing 20, 200 or 2000#g/ml verapamil. The medium was changed periodically according to previously described procedures (Greiner et al., 1981). Identical procedures were used for the control lenses incubated in Earle's buffer. Lens transparency Following incubation all lenses were analysed by photomacrography (Greiner, Chylack and Pihlaja, 1976). Lens transparency was monitored using grid paper observed through the lens by biomicroscopy. Any distortion of the linear grid lines was considered as loss of lens transparency.
VERAPAMIL
E F F E C T S ON L E N S M E T A B O L I S M
141
Lens perchloric acid (PCA ) extraction A t t h e e n d of each time-course, e x p e r i m e n t a l a n d control samples were frozen in liquid nitrogen. PCA e x t r a c t i o n s were t h e n p e r f o r m e d on b o t h t y p e s of samples. These e x t r a c t s were p r e p a r e d according t o s t a n d a r d procedures (Bs a n d Glonek, 1982).
~'P NMR spectroscopic analysis 31p N M R spectroscopic analyses a t 81 M H z h a v e b e e n described p r e v i o u s l y (Greiner et al., 1981). I n t a c t - t i s s u e spectroscopic a n a l y t i c a l conditions were as follows: pulse width, 9 #sec (45 ~ spin-flip angle); a c q u i s i t i o n delay, 2 0 0 # s e c ; n u m b e r of scans, 4400; n u m b e r of d a t a p o i n t s per f r e e - i n d u c t i o n decay, 8192; a c q u i s i t i o n time, 0"819 sec; sweep w i d t h + 2 5 0 0 H z ; t h e filter t i m e - c o n s t a n t used i n t r o d u c e d l 0 H z a d d i t i o n a l line b r o a d e n i n g . Spectroscopic conditions used d u r i n g lens P C A - e x t r a c t analysis were identical to those used d u r i n g t h e i n t a c t - l e n s analysis, e x c e p t for t h e following p a r a m e t e r s : n u m b e r of scans, 30000; n u m b e r
TABLE I
Perchloric acid (PCA )-extract and intact-tissue rabbit lens phosphate profiles after 13-hr verapamil (2000 ~ff / ml) incubation Mole % of total lens profile signal area Phosphorus resonance 5"988 4'658 4'588 4-458 (Hexose 6-P) ~GP 3"868 NMP NADP-2-'P Choline P Pi 0"928 0.858 GPC ATP ADP DN NS
Chemical shift in extract ($)~
PCA extract (% detected phosphorus LS.E.M.)
5"98 4"65 4"58 4"45
1.79_+ 0"17 0"50 ___0' 17 0'68 + 0"21 0-98 • 0"35
4"29 3'86 3"76 3"55 3"32 2"63 0"92 0"85 0-13 w I[ 82 :~
7-45_ 1"03 0-87 + 0'36 5'22 + 1'42 0"27 + 0"85 1-20___0'25 18-79 + 0"78 0.61 ___0"54 0.57 • 0"45 2-03 _+0-71 28-74 • 1"30 6.14• 12"96 + 1-04 11"20 • 2"51
Intact tissue (% detected phosphorus calculated from Table III) 2.53
17"17~
20"80
18"57 3'215
2-97 29-39 5-85 10"01 9"87
* Numbered resonances are unidentified. Following the IUPAC convention with the resonance of 85 % phosphoric acid at 24~ designated 08. $ Summed percentages corresponding to phosphates that contribute respective signal areas to the sugar-phosphate and phosphodiester bands of the intact-tissue spectrum. w ATP gives rise to three P-31 resonance signals corresponding to three phosphate groups in the molecule: a, 10-898;/?, -21-418; ~, -5"768. [I ADP gives rise to two P-31 resonance signals: a, -10"558;/?, -6"138. 82 The principal resonance signals of this band arise from the P, P'-diesterified pyrophosphate residues of NAD and NADP : - 11"373. Resonances from - 1 1 to - 1 3 3 correspond to hexose-phosphate portions of resonance multiplets. Hexose 6-P, hexose 6-phosphate; aGP, a-glycerophosphate; NMP, nucleoside monophosphates; NADP 2-'P, nicotinamide adenine dinucleotide phosphate 2-'P; choline-P, choline phosphate; Pi, inorganic orthophosphate; GPC, glycerol 3-phosphochotine; ATP, adenosine triphosphate; ADP, adenosine diphosphate; DN, dinucleotides; NS, nucleoside diphosphosugars.
142
J. V. G R E I N E R AND T. G L O N E K TABLE II
Time-course data for 200/~g/ml verapamil Coefficients (y = Phosphate resonance band ATP Pi SP NS GPC ADP DN 65
Slope (A) -- 1.205 • @390 • 0.405 • 0"394 _ 0-038 • 0"041 • - 0"064 • 0.000 •
pH
0"058 0.025 0.034 0.053 0.014 0-066 0"013
0"016 • 0.002
Ax+B)*• Intercept (B) 45"05 _ 13.50 • 15"53 • 4-75 • 2.48 • 5-32 • 10"84 • 2"53 •
0"36 0-16 0-21 2.33 0"28 1"20 1.41 0"48
6.81 • 0'03
* For phosphates the ordinate is the mole 5 % phosphorus detected and the abscissa is hours ; for pH the ordinate is pH units. ATP, adenosine triphosphate; Pi, inorganic orthophosphate; SP, sugar phosphates; NS, nucleoside diphosphosugars, GPC, glycerol 3-phosphocholine ; ADP, adenosine diphosphate ; DN, dinucleotides ; 63, unidentified resonance signal. TABLE III
Time-course data for 2000 l~g/ml verapamil Coefficients (y = Phosphate resonance band
Slope (A)
Ax+B)*• Intercept (B)
ATP Pi SP NS GPC ADP DN 65
- 1"248• 0.026 0-372 + 0.027 0"389 • 0"034 0"450 • 0"018 @000 • 0.005 @059 • 0.023 -- 0.004 _ 0.051 - 0"037 • 0"022
45"86 _ 1.20 13.59 _ 0-21 14-84 _ 0"26 5"40 + 2"14 2'56 • 0.40 5'05 • 1.18 10.60 • 1-08 2"30 • 0"77
pH
-- 0"017 + 0"022
6"81 • 0-77
* For phosphates the ordinate is the mole % phosphorus detected and the abscissa is hours ; for pH the ordinate is pH units. ATP, adenosine triphosphate; Pi, inorganic orthophosphate; SP, sugar phosphates; NS, nucleoside diphosphosugars ; GPC, glycerol 3-phosphocholine ; ADP, adenosine diphosphate ; DN, dinucleotides; 65, unidentified resonance signal.
o f d a t a p o i n t s per free-induction decay, 16384; acquisition time, 1"64 sec a n d a filter t i m e c o n s t a n t t h a t i n t r o d u c e d 0"6 H z a d d i t i o n a l line broadening. To c o m p e n s a t e for relative s a t u r a t i o n effects a m o n g the various p h o s p h o r u s signals d e t e c t e d in a single 31p N M R spectroscopic e x t r a c t profile, t h e N M R spectra were s t a n d a r d i z e d a g a i n s t m e a s u r e d a m o u n t s of tissue-profile metabolites. The calibration procedures h a v e been described (Bdrdny a n d Glonek, 1982). I n t a c t - t i s s u e spectra were c o m p a r e d w i t h t h e s e calibrated e x t r a c t s p e c t r a ; t h e q u a n t i t a t i v e c o m p a r i s o n was t h e same, w i t h i n e x p e r i m e n t a l limits, s u p p o r t i n g t h e i n t e r p r e t a t i o n of Greiner et al., (1981) a n d Bdr~ny
VERAPAMIL EFFECTS ON LENS METABOLISM
t43
and Glonek (1982) that using moderately rapid repetition times does not result in measurable discriminatory loss of signal-area fidelity among components contributing to an intact-tissue spectrum. Table I demonstrates that, within the precision of measurement obtainable from intacttissue analysis, the relative metabolite levels detected by the extract spectroscopic analysis are the same as the corresponding values obtained from the intact tissue (Burr, Glonek End Bdr~ny, 1976 ; Bs and Glonek, 1982). The individual resonance peaks that made up the lens extract spectrum are more highly resolved than those from the intact tissue as a result of the optimal physical-chemical conditions present in the extract. Thus, the extract analyses provide more detailed metabolic information regarding levels of certain phosphatic compounds, which are not well resolved in the intact-tissue spectrum.
Mathematical analysis The time-course data obtained were reduced by a least-squares regression analysis using the equation, Y = Ax+B. The data are presented in Tables II, I I I and IV. Intralenticular pH was determined by comparing the chemical-shift value of known resonances, e.g., Pi, to standardized pH spectroscopic 31p NMR titration curves (Bdr~ny and Glonek, 1982):
TABLE IV
Time-course data for 20 gg/ml verapamil Coefficients (y = Ax+B)*• Phosphate resonance band ATP Pi SP NS GPC ADP DN 65 pH
Slope (A) - 0"408_ 0-011 @152 + 0"034 @163 _ 0"083 @031 • 0-027 0.011 _ 0"011 @068_ 0"080 - @000_ 0'051 - 0'017 + @024 - 0-003_ @001
Intercept (B) 44.90 _+1.07 13.94 + 0-32 15.85_+0"78 3.45 + 1'55 2'90_ 0.70 4.05 _ 1'32 12"05• 1-48 2.86 • 0.23 6'85 • 0.03
* For phosphates the ordinate is the mole % phosphorus detected and the abscissa is hours; for pH the ordinate is pH units. ATP, adenosine triphosphate; Pi, inorganic orthophosphate; SP, sugar phosphates; NS, nucleoside diphosphosugars ; GPC, glycerol 3-phosphocholine; ADP, adenosine diphosphate; DN, dinucleotides; 63, unidentified resonance signal.
3. R e s u l t s I n control i n c u b a t i o n s (no added verapamil), modified E a r l e ' s buffer supported the m e t a b o l i s m of the r a b b i t lens for the entire 22 hr i n c u b a t i o n , d u r i n g which i n t e r v a l the i n t r a l e n t i c u l a r p H a n d relative c o n c e n t r a t i o n s of all phosphates except A T P r e m a i n e d c o n s t a n t . The relative A T P c o n c e n t r a t i o n increased b y 5 % . Levels of phosphatic metabolites did n o t differ significantly from those reported previously (Greiner et al., 1981). Regardless of the v e r a p a m i l c o n c e n t r a t i o n (20, 200 or 2 0 0 0 # g / m l ) d u r i n g i n c u b a t i o n , the lens' metabolic profile changed similarly ; only the rates of m e t a b o l i t e change were altered m e a s u r a b l y b y v e r a p a m i l c o n c e n t r a t i o n . Figure 1 shows the 31p N M R s p e c t r u m of r a b b i t lens d u r i n g a n initial control i n c u b a t i o n a n d after a 10 hr
144
J.V. GREINER AND T. GLONEK
Control
ATP
p
Pi
,NMP ADP
Experimental
f
8
I
I
I
I
0
I
I
'
'
l
-10
I
'
'
'
I
r
--20
FIG. 1. The alp N-MR spectra of a control and experimental rabbit lens after a 10 hr incubation in 200 pg/ml verapamil in modified Earle's buffer at 37~ 63, unidentified chemical resonance; aGP, resonance band of triose phosphates and hexose 6-phosphates; NMP, nucleoside monophosphates; Pi, inorganic orthophosphate; GPC, glycerol 3-phosphocholine; ATP, a-, fl-, and 9,-phosphate groups; ADPfl, the fl, chain-terminal phosphate of ADP; DN, dinucleotide phosphate resonance band; NS, nucleoside diphosphosugar, hexose-phosphate resonance band.
incubation in 200 # g / m l verapamil buffer. The control spectrum shows 31p resonance bands originating from the following phosphatic substances: 63, a phosphatic compound of undetermined chemical structure, prominent particularly in the rabbit lens; aGP, the resonance band of triose phosphates and hexose 6-phosphates, which contains the signal from a-glycerophosphate in rabbit lens as a principal component (Greiner et al., 1981); NMP, nucleoside monophosphate, the resonance band of the pentose 5-phosphates and other phosophomonoesters, which contains the signals from the two nucleoside monophosphates IMP and AMP in the rabbit lens as principal components (Kopp, Glonek and Greiner, 1983); Pi, the resonance from inorganic orthophosphate ; GPC, the resonance signal from glycerol 3-phosphocholine, which serves as the internal spectroscopic chemical-shift reference; ATP, a, fl, and 7 phosphate signals; A D P /?, the /?, chain-terminal phosphate of ADP; DN, the resonance band of the dinucleotides, principally NAD with some contribution from N A D P and the nucleotide portion of the uridine diphosphosugars; NS, the sugar
VERAPAMIL EFFECTS ON LENS METABOLISM
145
phosphate portion of the nucleoside diphosphosugars, which contains uridine diphosphoglucose as the principal component (Greiner et al., 1981). After a 10 hr incubation in the verapamil, the spectrum was altered (Fig. 1), especially in the orthophosphate region (8~-0~). Relative to the control, the entire orthophosphate band increased in relation to the ATP signals, the a-glycerophosphate (aGP) and NMP resonances increased in relation to Pi and shifted relative to each other so that they coalesced, indicating that the two groups of signals arose from compounds in relatively different pH compartments.
Control
Pi
aGP/~NMP GPC
Pi
i
I
w
8
1
I
5
r
I
I
I
@
F~G.2. Expanded view of the 31p NMR orthophosphate spectral region of a control and experimental rabbit lens after a 10 hr incubation in 200 #g/ml verapami[ in modified Ear|e's buffer, showing the coalescence of the a-glycerophosphate (aGP) and nucleoside monophosphate (NMP) resonance bands.
Figure 2 shows the orthophosphate spectral region on an expanded scale. The three greatest changes resulting from verapamil incubation were the upfield shift (to lower 3 values) of the a-GP band and Pi and the increased NMP concentration. The Pi upfield shift of 0-283 corresponded to a decrease in intralenticular pH over 10 hr of 0-20 from the initial value of 6"83 (BKrs and Glonek, 1982). The corresponding shift in the a G P signal was 0"433, which corresponded over 10 hr to a decrease in pH of 0"31 from an initial value of 6"83. The nucleoside monophosphate shift was less ( 0"123, S.E.M. • 0"03) and corresponded to a pH decrease of 0"07, S.E.M. • 0"006 from an initial value of 6"80. Under the influence of verapamil, the lenticular ATP concentration and
146
J.V. GREINER AND T. GLONEK
intralenticular pH declined with time. Pi, the combined sugar phosphates, and the nucleoside diphosphosugars increased. The remaining signals were unaffected by the verapamil incubation. Equations corresponding to changes in spectral signals with time are presented in Tables II, III and IV. All changes with time for all three verapamil concentrations tested are expressed as linear processes. Significant concentration changes {slope in column A in Tables II-IV) were observed only for ATP, Pi, the sugar phosphates (4"293-3"763) and the nucleoside diphosphosugars. GPC, ADP, the uncharacterized substance at 63 and the dinucleotides remained generally constant throughout the incubation regardless of the verapamil concentration. (The small apparent slopes in these data are rendered insignificant by a large degree of scatter.) The intercepts (column B in Tables II-IV) correspond to control rabbit lens mole-percentages previously reported (Greiner et al., 1981). The loss of ATP phosphorus corresponded to nearly equivalent rates of increase in Pi, sugar phosphates and nucleoside diphosphosugars. From the PCA-extract data, the nucleoside monophosphate component, not a glycolytic or shunt-pathway intermediate, increased within the sugar-phosphate band. This component ordinarily accounts for 2 % of the total detectable phosphorus in control spectra. At 200/~g/ml, verapamil incubation nearly doubled this value. Despite the metabolic alterations and changes in pH observed during the 22 hr verapamil incubations, lens clarity was unchanged. 4. D i s c u s s i o n
The most noticeable effect of verapamil incubation on mammalian lens was the reduction of the intralenticular pH in pools containing Pi and aGP. Both these substances reside within the same hydrogen-ion pool, i.e. they exhibit the same spectroscopic pH. This pool, however, differs from that containing the mononucleotides AMP and IMP in which the spectroscopic pH rate of change with verapamil incubation is less than that of Pi and aGP. Gradients of pH within a tissue have been observed spectroscopically using other systems, the most well-documented example being the acid and alkaline Pi pools in skeletal muscles (Burr et al., 1976; Busby, Gadian, Radda, Richards and Seely, 1978) and heart (Garlick, Brown, Sullivan and Ugurbil, 1983). Chemical-shift data have been interpreted to indicate the existence of multiple pH pools within lens (Greiner, Kopp and Glonek, 1982; Kopp et al., 1983), although challenges to the lens system, such as incubation with ouabain, have always resulted in parallel changes in the lenticular pH pools (Greiner, Kopp and Glonek, 1985a). Our study provides the first example of a pH change in one pool not paralleled by a corresponding change in another. According to the pH effect, the metabolite pool containing the nucleoside monophosphate must be distinct from that containing Pi and aGP. How a calcium-channel blocking agent can selectively affect these two metabolite pools is unknown. However, one broad interpretation can be applied. In a study on the distribution of phosphatic metabolites within the crystalline lens (Greiner, Kopp and Glonek, 1985b), inosine monophosphate was more prominent in the lens nucleus (mole %, 3"95) than in the cortex (mole %, 0"90). The remaining orthophosphate esters were distributed more uniformly, although the entire sugar phosphate band was more prominent in the cortex than in the nucleus. Consistent with these data, verapamil may act primarily on the cortex or may be prevented from reaching nuclear cells, which contain the bulk of the nucleoside monophosphate
VERAPAMIL EFFECTS ON LENS METABOLISM
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component. The spectroscopic pH effects of the intact lens may occur because the cortex is becoming more acidic in response to verapamil, but the deeper and apparently less metabolically active nucleus is unaffected. To explain the verapamil-induced acidosis, it has been hypothesized that the calcium channel and slow sodium channel use the same enzymatic components (Nayler and Krikler, 1974 ; Rattke and Glasser, 1978), and that a blocking agent, such as verapamil, inhibits both enzymatic activities. Inhibiting sodium translocation promotes acidosis by inhibiting the charge-transfer process. Without sodium translocation, a charge imbalance occurs with hydronium-ion pumping mlless anions are translocated simultaneously. Since cells cannot tolerate a charge imbalance or excessive salt pumping, hydrogen ions generated from lenticular glycolysis accumulate in the absence of sodium translocation. The excess of hydrogen ions is apparently not distributed uniformly throughout the lenticular cells but is restricted to the specific low-pH pools. The ATP depletion during incubation is not great in relation to the stress placed upon the lens system by the great reduction of intralenticular pH. For this mason, we interpret the loss of lenticular ATP as secondary to hydrogen ion accumulation and resulting from an increasing energy demand produced by increasing acidosis. Pi and the nueleoside monophosphates are the products of ATP hydrolysis and accumulate if ATP levels cannot be restored at the required rate. Elevation of the nucleoside diphosphosugar levels suggests a stimulation of fl-Dglucuronide or glycogen biosynthesis, or both. Since the pathway for glycuronide biosynthesis also generates hydrogen ions, its activation can exacerbate the intralenticular pH decrease. The most probable need for the increased uridine diphosphosugars, therefore, may be activated glucose for glycogen biosynthesis. Limiting our study is the lack of a supporting medium to provide the lens with metabolic stability for more than 26 hr at 37~ (Glonek et al., 1985). Thus, assessing long-term effects on the metabolic and pH changes of the lens must await development of such a medium. In summary, this study demonstrates that verapamil significantly alters the rates of phosphorus metabolite change in the lens and lowers intralenticular pH. Despite these metabolic changes, however, verapamil may protect against cataractogenesis as reported in a study of alloxan-diabetic rats undergoing long-term treatment with verapamil (Fleckenstein, Witzleben, Frey and Milner, 1981). ACKNOWLEDGMENTS We thank Donna G. Peace, M.S., and Manuel G. Flores, B.S., for technical assistance. This study was supported by grant EY-03988 from the National Eye Institute, National Institutes of Health, Bethesda, MD, U.S.A. REFERENCES Adams, D. R. (1929). Role of calcium in senile cataract. Biochem. J. 23, 902-12. BgrAny, M. and Glonek, T. (1982). Phosphorus-31 nuclear magnetic resonance of contractile systems. In Methods in Enzymoloyy, Vol. 85B, (eds Frederiksen, D. L. and Cunningham, L. W.) Pp. 624-76.Academic Press: N e w York. Brawnwald, E. (1982). Mechanism of action of calcium-channel-blocking agents. N. Engl. J. Med. 307, 1618-27. Burge, W.E. (1909). Analyses of the ash of the normal and cataractous lens. Arch. Ophthalmol. 38, 435-50.
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