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Rat renal proximal tubular gluconeogenesis: Possible involvement of nonmitochondrial carbonic anhydrase isozymes
AK(:HIVES
Vol.
OF BIOCHEMISTRY
282, No. 1, October,
APU’D BIOPHYSICS
pp. l-7,
1990
Rat Renal Proximal Tubular Gluconeogenesis: Possible Involvement of Nonmitochondrial Carbonic Anhydrase lsozymes Susanna
J. Dodgson’
Department
Received
of Physiology,
January
and Kay Cherian University
16, 1990, and in revised
of Pennsylvania
form
May
School of Medicine,
Copyright All rights
correspondence
should
(cl 1990 by Academic Press, of reproduction in any form
be addressed.
Inc. reserved.
Pennsylvania
19104-60&G
lo,1990
The carbonic anhydrase (CA) inhibitor ethoxzolamide decreases the rate of glucose synthesis from 10 mM pyruvate by tubules incubating in 25 mM HCO, but not in 50 mM HCO,: this is evidence that rat renal cortical mitochondrial CA (CA V) provides HCO, for pyruvate carboxylation in renal tubular gluconeogenesis at physiological total CO2 (CO2 + HCO;). In renal proximal tubules prepared from 48-h-starved rats and incubating in 10 mM pyruvate in 25 mM HCO, buffered saline (Krebs-Henseleit buffer) the CA inhibitors acetazolamide (AZ) and benzolamide (BZ) decreased the rate of glucose synthesis. Maximal inhibition was reached with 125 pM AZ or with 450 pM BZ. The rate of glucose synthesis increased with increasing pyruvate concentration from 3.33 to 20 mM; including 600 pM BZ or 188 ELM AZ results in glucose synthesis becoming independent of increasing pyruvate concentration. Doubling the physiological concentration of bicarbonate restored the dependence of glucose synthesis on pyruvate concentration and partly, but not completely, alleviated the inhibitory effect of AZ and BZ, leading to the conclusion that AZ and BZ influence gluconeogenesis by affecting enzymes in addition to CA V. Tubules were incubated with substrates which do not require pyruvate carboxylation for synthesis of oxaloacetate. When tubules were incubated in 10 mM malate the rate of glucose synthesis was unaffected by less than 100 pM AZ or 400 pM BZ and was decreased maximally by 40 and 20%, respectively, by 125 ELM AZ, 450 j.tM BZ, and higher concentrations of these drugs. Increasing the malate concentration from 3.33 to 20 mM increased the rate of glucose synthesis; 600 pM BZ inhibited the rate of glucose synthesis only when the malate concentration was greater than 10 mM but 188 MM AZ decreased the rate of glucose synthesis at each concentration of malate. Results were similar when tubules were incu-
’ To whom
Philadelphia,
bated in glutamine with CA inhibitors. The rate of glucose synthesis differed with the substrate metabolized and the substrate concentration except when 600 pM BZ was included. It is concluded that only when pyruvate is carboxylated does BZ inhibition of CA V affect mitochondrial production of oxaloacetate. It is further concluded that the maximum decrease of glucose synthesis observed with BZ results from a block in gluconeogenesis after phosphoenolpyruvate carboxykinase (PEPCK). Greater inhibition of the rate of glucose synthesis was always observed with 188 HAM AZ. This inhibition was independent of substrate concentration, indicating a posterior block; however, the rate of glucose synthesis varied with each substrate, indicating an additional block before PEPCK. It is concluded that AZ and BZ inhibit enzymes involved in gluconeogenesis other than CA V. Since 95% of the CA activity of rat kidney tubules is nonmitochondrial it is suggested that this inhibition may be a secondary effect of inhibition of other CA isozymes. CC 1990 Academic Press, Inc.
It has recently been demonstrated in this laboratory that the rate of glucose synthesis by intact renal tubules is reduced maximally 75% by less than micromolar concentrations of the carbonic anhydrase (CA,’ EC 4.2.1.1) inhibitor ethoxzolamide when pyruvate is the added substrate (1). Glucose synthesis was unaffected by 1.6 yM ethoxzolamide when tubules were incubated in twice physiological total COT, leading to the conclusion that
* Abbreviations used: CA II, the cytoplasmic soluble carbonic anhydrase; CA IV, the brush border bound carbonic anhydrase; CA V, the mitochondrial carbonic anhydrase; PEPCK, phosphoenolpyruvate carboxykinase; PC, pyruvate carboxylase; CPS I, carbamyl phosphate synthetase I; AZ, acetazolamide; BZ, benzolamide; total CO,, CO, + HCO;; EGTA, ethylene glycol bis(&aminoethyl ether)i\i,W-tetraacetic acid; Mops, 4-morpholinepropanesulfonic acid. 1
2
DODGSON
AND
at physiological total COz , the rate of glucose synthesis is controlled by CA V. Carbonic anhydrase activity was first described in the kidney in 1941 (4). The unique mitochondrial carbonic anhydrase, CA V comprises only 5% of the total carbonic anhydrase activity of rat renal proximal tubules (1, 2); the cytosolic CA II and the membrane-bound CA IV together comprise 95%. It is generally believed that either CA II or CA IV or both are needed for 80 to 85% of the bicarbonate resorption (reviewed in (5, 6)); it is likely that CA II is needed for HCO;-Na+ exchange across brush border membranes (7). The major aim of this present study was to determine whether inhibition of the rate of glucose synthesis by CA inhibitors always results entirely from the inhibition of the mitochondrial carbonic anhydrase CA V. The CA inhibitor acetazolamide (AZ) has been used extensively in renal studies (5-8); there is no evidence that it inhibits other enzymes and many conclusions about the functions of the tubular carbonic anhydrases have been based on studies with these inhibitors. The data in this report indicate that acetazolamide (~10~~ M) decreases the rate of renal tubular glucose synthesis solely by inhibiting the mitochondrial CA V, as does another CA inhibitor, benzolamide (BZ) (15 X lop4 M). At higher concentrations of CA inhibitors the rate of glucose synthesis is decreased by the effect of acetazolamide (and to a lesser extent, benzolamide) on other enzymes participating in gluconeogenesis. METHODS Reagents. Collagenase (Worthington Type IV) was bought from Worthington (Freehold, NJ). “O-labeled NaHCO, was prepared from unlabeled NaHCO, and “O-labeled HOH as described (10). All other chemicals and reagents were bought from Sigma Chemical Company (St. Louis, MO). Preparation of biological materials. Intact renal proximal cortical tubule suspensions were prepared from 48-h-starved 200-g male rats according to published techniques (11) exactly as previously described (1). For each preparation tubules were prepared from the perfused kidneys of two animals which had first been anesthetized with sodium barbitol (5 mg/lOO g body weight). The isolated kidney tubules were in a final suspension of 20 to 50 mg dry wt per milliliter. Experiments with intact tubules were finished within 1 h and 10 min after their isolation. Dry weights were determined on each preparation (1). Intact renal cortical mitochondria were prepared from 48.h-starved male rats. Cortices were finely minced and rinsed in 275 mM mannitol, 1 mM EGTA, 2 mM Mops, pH 7.4.The nonmitochondrial fraction was sedimented at 2200g; the mitochondrial fraction sedimented at 72OOg, was resuspended in the mitochondrial buffer, and was pelleted at SSOOg. Renal cortical mitochondria were further purified by centrifugation through a Percoll gradient at 100,OOOg for 30 min (1, 2). Mitochondrial protein concentration was determined by the Lowry technique (12). Production and determination of glucose. Isolated proximal tubule suspensions were incubated in 20 ml scintillation vials with KrebsHenseleit bicarbonate saline incubation buffer (13), pyruvate, malate, or glutamine, and carbonic anhydrase inhibitors. In each vial the 1.2ml fluid volume included between 3.3 and 20 mM pyruvate and 400 ~1 of kidney tubule suspension in incubation buffer. The incubation
CHERIAN buffer was either Krebs-Henseleit bicarbonate saline buffer, which was continuously bubbled with 5% CO,/95% 0, or 50 mM bicarbonate buffer (Krebs-Henseleit but with 25 mM NaCl replaced with an additional 25 mM NaHC03) continuously bubbled with 10% C02/90% OZ. The former buffer has physiological concentrations of total CO* and the latter has doubled physiological concentrations. Before and after addition of 0.4 ml of kidney tubules (8 to 20 mg dry wt) the vials, on ice, were flushed with gas and then tightly closed. At 30-s intervals vials were removed from ice and added to the 37°C shaking water bath; 0.250-ml aliquots were removed from each vial after 20, 40, and 60 min, and added to 0.1 ml of HClO,. Each vial was flushed with gas and tightly capped, then returned to the incubating water bath. The pH of the acidified aliquot was neutralized with KHCOB, the solution centrifuged at 13,000g for 5 min, and glucose estimated in the supernatant (14). The rate of glucose production was calculated in terms of micromoles.Gram dry weight-’ 60 minss’ by constructing curves such as are shown in Fig. 2 and calculating the concentration of glucose at 60 min from the line of best fit with Cricket software. Statistical analyses of rates of glucose synthesis from different substrates were made with Student’s two-tailed t tests for paired samples, by Statworks software. Mass spectrometric analyses. The carbonic anhydrase activity of renal cortical mitochondria was assayed by the “O-exchange technique (10) as previously described for intact hepatic mitochondria (15, 16), and intact cells (17). The mass spectrometer recorded the mass 46 (C’60180), the mass 44 (C160,) and the mass 32 (i602) peaks alternately every 15 s. Mass 46 peak heights reach a final equilibrium value, 46cc,, which is subtracted from the peak height at time t, 46,. The addition of a carbonic anhydrase containing sample accelerates the decrease; conversely total carbonic anhydrase inhibition returns the decrease to the uncatalyzed rates. The experiments with intact renal cortical mitochondria were conducted in 50 mM Mops, 35 mM KCl, 5 mM KH,PO,, 1 mM EGTA, 1 mM MgC&, 25 mM KHCOB (1% labeled with “0) at pH 7.4, 37°C. The CA V activity is presented in terms of the ratio of mass 46 to mass 44 peak heights (Fig. 1). The mass 44 peak height does not decay, it increases with increased acidity and decreases with increased alkalinity; thus the ratio of peak heights reflects the true decay of C’sOi60, independent of pH changes. Experiments with disrupted renal cortical mitochondria were conducted in 25 mM NaHCO; (1% labeled with “0). The mass 46 peak Ci601’0 decreases as ‘so exchanges with the 55 M I60 pool in water as outlined by Mills and Urey (18): ~160180
+ ~~160
:-“;
~~~180160~
E;%EO=”
=k\\ k\\
1135 \\
C=02 + H2”O H’ + HC’801602The plot of log 10(46,/44, - 46a;/44co) vs time is linear with slope n,, from which the uncatalyzed CO, hydration rate is calculated. On addition of a vesicular carbonic anhydrase containing sample there is a rapid “step” decrease in the plot, followed by a slower linear portion with slope neat. When the permeability barrier to HCO; has been removed by disruption of the membrane, the disappearance of mass 46 is monophasic (10). The constant k.,, (in ml sec~i mg dry weight-‘) was calculated for disrupted mitochondria from equations given previously (15).
RESULTS
Carbonic anhydrase activity of disrupted renal cortical mitochondria at 37°C was determined by mass spectrometry with concentrations of acetazolamide and ben-
RENAL
1
Starved IO mM
5
g
Rat Pyruvate
TUBULE
GLUCONEOGENESIS
IN
I
'E
Control
0
Pyruvate Rats
200
8 i
100 t
3
RAT
10 mM Starved
z
200
-z Z E 2
PROXIMAL
s
100
A, ? 0 0
>ul
"0
20
40
Time
(minutes)
60
= 0.98).
The rate of glucose synthesis at 60 min was determined by incubating renal proximal tubules in an isotonic bicarbonate buffer for 60 min and measuring the concentration of glucose in aliquots at 20,40, and 60 min as described (see Methods). Figure 1 represents two typical experiments in which rat proximal tubules were incubated with 10 mM pyruvate with or without the carbonic anhydrase inhibitor benzolamide. The rate of glucose synthesis was linear over 60 min in the control experiment as well as the one in which 781 ELM benzolamide was included; this was also observed for each experiment described in this report. The rate of glucose synthesis was decreased incrementally by including in the incubation solutions increasing concentrations of acetazolamide and benzolamide (Fig. 2). The concentrations of CA inhibitors needed for maximal inhibition were 125 PM for acetazolamide and 400 PM for benzolamide. These data contrast with the concentration of ethoxzolamide needed for maximal inhibition, 1 PM (1). In subsequent experiments the rates of glucose synthesis were maximally inhibited by including either 600 pM benzolamide or 188 PM acetazolamide. At the concentrations of acetazolamide and benzolamide needed to maximally decrease glucose synthesis, CA V of intact mitochondria is fully inhibited; Fig. 3 indicates that 42 yM acetazolamide inhibits CA V completely as does 335 pM benzolamide. These concentra-
200
300
400
[Acetazolamide]
FIG. 1. Glucose synthesis over 60 min by proximal tubules prepared from 46h-fasted rats. Incubation vials contained a total volume of 1.2 ml (of which 0.4 ml was the final addition of suspended proximal tubules) with tubules and all solutes in Krebs-Henseleit bicarbonate saline buffer gassed with 5% CO,/95% 0, with 10 mM pyruvate. At t = 0 the closed vials were added to the 37°C shaking water bath. At t = 20, 40, and 60 min, 250-~1 aliquots were removed and added to HCIO,, the vials regassed, tightly recapped, and replaced in the water bath.
zolamide between 0 and lop6 M. A concentration of lop7 M of each drug was sufficient to completely inhibit all the carbonic anhydrase activity of 1 mg mitochondrial protein/ml reaction solution. Ki’S CA V were determined with disrupted renal cortical mitochondria at 37°C from Easson-Stedman plots (19) for acetazolamide, 0.04 pM (n = 4, r = 0.89), and for benzolamide, 0.08 PM (n = 4, r
100
0
z
0
pM
500
1000
[Benzolamide]
FM
FIG. 2. Glucose synthesis by intact rat kidney cortical proximal tubules in 60 min as a function of either benzolamide or acetazolamide in bicarbonate buffer at pH 7.4,37”C with 10 mM pyruvate. Each curve is a composite of 15 to 30 incubations conducted on between 2 and 4 different days exactly according to the legend in Fig. 1.
tions are several orders of magnitude higher than their Ki, and lower than the concentrations needed for maximal inhibition of glucose synthesis. AZ and BZ are considerably less lipophilic than ethoxzolamide (with which total inhibition of CA V in intact mitochondria was observed with 0.15 pM (1)) and it could not be assumed that they cross mitochondrial membranes even at these high
335 FM B2
i
”
0
2
4
Time
6
a
(minutes)
FIG. 3. Plots of log,, ((mass 46 peak height at time = t/mass 44 peak height at time = t), minus (mass 46 peak height at time = z/ mass 44 peak height at time = x,)} against time before and after rat renal cortical mitochondrial suspensions were added to the reaction chamber of the mass spectrometer. Mitochondria (0.5 mg/ml) were added at the first arrow to the Mops buffer described under Methods with 25 mM KHCO, (1% labeled with “0) at pH 7.4,37”C and either 81 pM acetazolamide (AZ) or 335 pM benzolamide (BZ) added at the second arrow.
DODGSON
AND
CHERIAN
TABLE
I
Rates of Glucose Synthesis by Intact Rat Renal Proximal Tubules Incubating in Different Pyruvate and Bicarbonate Concentrations in the Presence and Absence of Carbonic Anhydrase Inhibitors Rate 25 [Pyruvate] (mM)
Control
+BZ
(6) 3.3 6.7 10 13.3 16.7 20
124 162 179 184 202 214
f 27 +- 39 f 43 + 79 +I 44 f 68
107 129 106 108 136 117
(4) f f zk + + 2
39 33 62 45 49 48
mM
of glucose
synthesized
(rmol/g
dry wt 60 min)
HCO;
50
1 (%I
+AZ
14 21 41 41 33 45
(3) 86 t 96+81 f 98 + 94 IL 91+
I(%)
Control
+BZ
(6) 18 9 30 18 25 27
31 41 51 41 53 57
86 123 158 182 187 215
f + + + + +
18 36 52 50 66 30
Note. Tubules were incubated for 60 min in either Krebs-Henseleit bicarbonate saline buffer sphere of 5% C02/95% 0, or in the 50 mM NaHCOs buffer (see Methods) under an atmosphere refer to the number of times the series of experiments was conducted with a fresh preparation. benzolamide (BZ) were 188 and 600 pM, respectively.
concentrations. It is concluded from Figs. 2 and 3 that these drugs cross the mitochondrial membranes. In the preceding report from this laboratory (1) it was demonstrated that glucose synthesis from 10 mM pyruvate was maximally inhibited by 1.0 PM ethoxzolamide. In subsequent experiments the pyruvate concentrations in the incubation solutions were varied between 5 and 20 mM pyruvate and the rates of glucose synthesis determined with and without 1.6 ~.LM ethoxzolamide. At each pyruvate concentration, doubling the total COz removed the inhibitory effect of ethoxzolamide entirely, leading to the conclusion that the inhibitory effect of ethoxzolamide at physiological total CO2 was entirely due to inhibition of CA V. The next series of experiments was designed to test whether this same conclusion could be made about the effects of BZ and AZ. Tubules were incubated in either 25 mM total bicarbonate (physiological total CO,) or 50 mM bicarbonate (twice physiological total CO,; Table I). At physiological pC0, the rate of glucose increased with increasing pyruvate concentration (2) only in the absence of CA inhibitors. Doubled physiological total COz had two effects on the experiments in which AZ and BZ were included. First, inhibition of the rate of glucose synthesis was only partly alleviated, this alleviation was greater for BZ than for AZ. Second, the rate of glucose synthesis became dependent on pyruvate concentration, as in the control experiments conducted at physiological pH. It is concluded that AZ and BZ inhibition of the rate of glucose synthesis is not just a simple effect from CA V inhibition and suggested that other enzymes involved in gluconeogenesis are also affected. The remaining experiments in this report were all aimed at determining possible enzymes involved in glucose synthesis which AZ and BZ inhibit either directly or indirectly. Direct effects would be expected for cytosolic and
mM
85 93 126 130 154 176
(4) AZ 18 f 17 + 34 * 13 + 46 k 22
(containing 25 of 10% C02/90% Concentrations
HCO, 1 (%I
+AZ
0 24 20 29 18 18
(4) 90f 92 f 100 f 111 It 116+ 135 +
mM
1 (So)
9 13 22 22 8 36
0 25 37 39 38 37
NaHCO;) under an atmo0,. Numbers in parentheses of acetazolamide (AZ) and
membrane-bound CA isozymes, although these other isozymes have never been implicated in gluconeogenesis. Indirect effects would be predicted for any gluconeogenic enzyme requiring any of the products of CA-mediated catalysis, i.e., H+ or HCO,. Enzymes which are possible candidates are those needed for transmitochondrial transport of substrates and products; the mitochondrial enzymes involved in producing oxaloacetate, the gluconeogenic enzymes after phosphoenolpyruvate carboxykinase, or those needed for glucose 6-phosphate transport into the endoplasmic reticulum and glucose transport out of the endoplasmic reticulum. Figure 4 is a metabolic scheme in which pathways to glucose from pyruvate, malate, and glutamine are depicted. When gluconeogenesis proceeds from malate or glutamine, pyruvate carboxylation is not an intermediate reaction and thus the requirement for CA V does not exist, Fig. 4. When tubules were incubated in 10 mM malate or 10 mM glutamine, concentrations of acetazolamide less than 10e4 M neither increased nor decreased the rate of glucose synthesis, Fig. 5. When 10 mM pyruvate is the substrate 125 pM acetazolamide reduces glucose synthesis to a minimum rate; similarly, acetazolamide reduces the rate of glucose synthesis when either 10 mM malate or 10 mM glutamine are substrates to minimum rates. When 10 mM malate is the substrate, the rate of glucose synthesis was not decreased by 400 pM benzolamide, Fig. 5; higher concentrations of this resulted in an abrupt decrease in glucose synthesis down to a minimum rate as also observed when 10 mM glutamine is the substrate. The data in Fig. 5 also indicate that low concentrations of benzolamide actually may increase the rate of glucose synthesis when 10 mM malate is the substrate. The rates of glucose synthesis increase with increasing malate concentration, Table II. Inclusion of 188 yM
RENAL
PROXIMAL
TUBULE
GLUCONEOGENESIS
IN
5
RAT
It is concluded that >125 PM acetazolamide and >450 benzolamide decrease the rates of glucose synthesis. The first set of evidence for this conclusion is that doubled total CO;! does not completely override AZ and BZ inhibition when pyruvate is the sole metabolic substrate, see Fig. 2 and Table I. The second set of evidence is that these concentrations of AZ and BZ also decrease the rates of glucose synthesis when glutamine and malate are the added substrates, Fig. 5, Table II. If acetazolamide, benzolamide, and ethoxzolamide have their effects only on CA isozymes, these data indicate that the CA isozymes throughout the proximal tubules have either different affinities or accessibilities to the CA inhibitors, or both. The carbonic anhydrase isozymes known to be present in rat kidney are the mitochondrial CA V (a), the cytosolic CA II, and the membrane-bound CA IV (20-22). All isozymes are sensitive to the specific sulfonamide CA inhibitors (2, 22). Carbonic anhydrase activity in the membrane of the endoplasmic reticulum has been detected by histochemical studies of the peripheral nervous system (23), and may also be present in the renal endoplasmic reticulum, although there have been no reports published confirming this. Ca2+ stored within the endoplasmic reticulum is released by IPB in response to P-adrenergic stimulation (24). While there is no evidence in the present report for interference of the CA inhibitors with Ca2+ release, this has been reported in PM
CYTOSOI ,\
c
:
:
:
:
phosphoenolpyruvate
t’
+ GDP f P. I
I
FIG. 4. Enzymes involved in glucose synthesis from pyruvate, malate, and glutamine in rat renal cortical proximal tubules. Also indicated are the carbonic anhydrase isozymes CA II, CA IV, and CA V. Enzymes of glucose synthesis are pyruvate carboxylase, PC, phosphoenolpyruvate carboxykinase, PEPCK. The gluconeogenic enzymes after PEPCK are also glycolytic enzymes, all located in the cytosol; the single exception is the irreversible reaction catalyzed by glucose 6 phosphatase, a soluble enzyme located inside the endoplasmic reticulum. The only other irreversible reaction in the cytosol after PEPCK is catalyzed by fructose-1,6-biphosphatase.
acetazolamide decreased the rate of glucose synthesis at each malate concentration; these rates are similar to the rates when glutamine is the substrate but 30% less than the rates when pyruvate is the substrate (Table I). The minimum rate of glucose synthesis when acetazolamide (188 PM) was included was significantly lower (by paired t tests, P < 0.05) than the minimum rate when benzolamide (600 FM) was included in these experiments. When 600 PM benzolamide is included, the rates of glucose synthesis from malate, glutamine (Table II), and pyruvate (Table I) are similar. Benzolamide does not decrease glucose synthesis at low substrate concentrations no matter which substrate is included; these data are contrary to those obtained after acetazolamide inclusion.
i
Oo1,
1001.
2001.
[Acetazolamide]
a
k
3001,
4001
I
00
pM
100
200
[Acetazolamide]
300
400
pM
DISCUSSION
It is concluded that carbonic anhydrase inhibitors decrease renal proximal tubular glucose synthesis by their effect on the mitochondrial carbonic anhydrase at low concentrations but at high concentrations the inhibitors have an additional effect on glucose synthesis. Low concentrations of the CA inhibitors decreased the rate of glucose synthesis only when pyruvate was the added substrate (Figs. 1 and 2); these data support the hypothesis that mitochondrial carbonic anhydrase is needed to provide the HCO; substrate for pyruvate carboxylase (see scheme in Fig. 4) and also indicate that these CA inhibitors have no other effect on glucose synthesis at these concentrations.
0
500
[Benzolamide]
1000
pM
0
500
[Benzolamide]
1000
pM
FIG. 5. Glucose synthesis by intact rat kidney cortical proximal tubules in 60 min as a function of benzolamide and acetazolamide in bicarbonate buffer at pH 7.4,37”C with 10 mM malate or 10 mM glutamine. Each curve is a composite of 8 to 30 incubations conducted on between 2 and 4 different days exactly according to the legend in Fig. 1.
6
DODGSON TABLE
AND
II
Rates of Glucose Synthesis by Renal Proximal Tubules Incubating in Different Substrate Concentrations in the Presence and Absence of Carbonic Anhydrase Inhibitors Rate of glucose synthesized (wmol/g dry wt 60 min) [Substrate] mM
Malate
Control
$600
(2)
(2)
3.3 6.7 10 13.3 16.7 20
go+31 121* 121k 136 f 141 k 151*
21 6 11 6 19
101k 107 113 121 120 98
Glutamine
(4) 92 + 106 k 122 f 144 f 146 2 146 2
37 44 51 32 26 17
81 113 112 100 93 90
3.3 6.7 10 13.3 16.7 20
BZ
/LM
f f k + +
3 17 32 59 41 14
(2) k +++ 2 +
34 34 19 29 25 32
+188
MM
AZ
(2) 66 + 16 67+ 8 51* 11 59 3125 69 t 11 82 t 15 (1) 64 56 53 71 76 64
Note. Tubules, prepared from 48-h-starved male rats, were incubated for 60 min in Krebs-Henseleit bicarbonate saline buffer under an atmosphere of 5% CO,/95% 02. Numbers in parentheses refer to the number of times the series of experiments was conducted with a fresh preparation.
experiments with the sarcoplasmic reticulum, gous organelle of the skeletal muscle, which
the analoare known
have carbonic anhydrase activity (25). The possibility that acetazolamide and benzolamide inhibit enzymes other than the carbonic anhydrases cannot be ignored. Intracellular inhibition of other enzymes has never been demonstrated; the renal y-glutamy1 transpeptidase (26) was found to be 35% inhibited by 5 mM acetazolamide, a concentration far higher than that in the tubules in the experiments of this report. Benzolamide decreasesthe rate of glucose synthesis to a minimum rate which is not significantly different for pyruvate or malate, malate or glutamine, pyruvate or glutamine. This contrasts with the very different rates of glucose synthesis in the absence of benzolamide. These data indicate there is a common block for glucose synthesis which is independent of substrate, or substrate concentration, and point to this block being after phosphoenolpyruvate carboxykinase, PEPCK. From examination of the metabolic scheme in Fig. 4, it is suggested that a possible site of this block could be the irreversible ultimate gluconeogenic enzyme, glucose-6-phosphatase. This enzyme is located inside the endoplasmic reticulum, and the final step in the synthesis of glucose is its release from the endoplasmic reticulum; thus there could be a block in the transport of either glucose B-phosphate or glucose across the membranes of the endoplasmic re-
CHERIAN
ticulum or even a direct inhibition of glucose-6-phosphatase. If cytosolic CA II is needed for providing H’ or HCO, for transport of glucose 6-phosphate into or glucose out of the endoplasmic reticulum then CA inhibition would alter the rate at which glucose is synthesized by the renal proximal tubule. It is noted that other substrates for glucose-6-phosphatase are H+ and OH-, as for the carbonic anhydrases and the other irreversible cytosolic gluconeogenic enzyme fructose-1,6-biphosphatase. Acetazolamide decreasesthe rates of glucose synthesis to lower minimum rates than does benzolamide; these mimima are significantly different for pyruvate and malate (by unpaired Student’s t tests, P < 0.002) and pyruvate and glutamine (P < 0.002) but not malate and glutamine. The rates of glucose synthesis by tubules incubating in 125 PM acetazolamide were the same when malate and glutamine were substrates. This rate was different when pyruvate was the substrate. It is thus concluded that acetazolamide has an additional effect on gluconeogenesis before PEPCK which is more effective at reducing the rate of glucose synthesis than the effect after PEPCK. Twice physiological total CO2 overrides inhibition of glucose synthesis by ethoxzolamide (1) but not by acetazolamide or by benzolamide. The concentrations of ethoxzolamide needed for decreased rates are lop2 to lo-” times lower than for the other CA inhibitors. A complete cessation of glucose synthesis by kidney tubules has been observed in this laboratory when greater than 4 pM ethoxzolamide is included (Dodgson, unpublished results). It has been stated that when a concentration of greater than lop4 M CA inhibitor is needed for an effect to be ascribed to the carbonic anhydrases, this effect is possibly nonspecific (28, 29), resulting from the uptake of other ions with the CA inhibitors. In this present report the concentrations of the inhibitors needed for decreasing glucose synthesis to minimal rates are exactly in this range. The data in this report do not indicate whether the effects of acetazolamide or benzolamide result
from
ion influx
or inhibition
of cytosolic
or mem-
brane-bound carbonic anhydrases which in some way decrease the activity of other gluconeogenic enzymes. The rate of glucose synthesis is decreased to a minimum with 1 pM ethoxzolamide, 125 pM acetazolamide, or 450 PM benzolamide (1:125:450); this ratio compares well with the relative permeances of these CA inhibitors across cellular and organellular membranes (30). Benzolamide has been used in studies with intact cells because it is relatively impermeant across cell membranes. The data in this report indicate clearly that benzolamide is permeant across membranes of isolated mitochondria (Fig. 3). The need for higher concentrations of benzolamide to decrease glucose synthesis than the other CA inhibitors agrees with previous observations that ben-
RENAL
PROXIMAL
TUBULE
zolamide is less freely permeable across cellular membranes (5,8, 25). In a limited study, it has previously been reported that 1 mM acetazolamide decreased rat renal tubular glucose synthesis 70% when 10 mM malate was the substrate and 11% when 2 mM glutamine was the substrate (31). These data are not greatly different from the present data, indicating that the same effect is seen even with extremely high concentrations of the CA inhibitor acetazolamide. Thus, whatever the reason for the observed abrupt decrease in the rate of glucose synthesis at 100 PM acetazolamide or 450 PM benzolamide, increasing the concentration of either drug does not decrease the rate still further. It has previously been reported from this laboratory that hepatocytes prepared from 48-h-starved male guinea pigs synthesized glucose from pyruvate dependent of CA V but from glutamine this rate was independent of carbonic anhydrase inhibitors (32). Rat kidney CA V differs from guinea pig liver CA V in its K, and its lower pH sensitivity (2); it is clear from this report that they both provide HCO, for pyruvate carboxylation in the same manner. ACKNOWLEDGMENTS The IXXISS spectrometer facility is supported by HL P019737, Program Director Dr. Robert E. Forster II. Dr. Thomas Maren provided benzolamide; Lederle, Pearl River, NY provided acetazolamide. Thanks always to Mrs. Lydia Lin, who maintains the mass spectrometer and prepares KHC’“O”0, and NaHCi80i602, and to Dr. Forster for constant encouragement. This research was supported by NIH DK 38041 (to S.J.D.).
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0. H., Rosebrough, N. J., Farr, J. Biol. Cham. 193,265-275.
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H. A., and Henseleit, A 210,33-66.
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A. I,., and Randall, Hoppe-S&r’s
P. J.
2. Physiol.
14. Bergmeyer, H. U., Bernt, E., Schmidt, F., and Stork, H. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U., Ed.), Vol. 4, 2nd ed. Verlag Chemie, Weinham, Federal Republic of Germany. 15. Dodgson, Proc. Nat. 16. Dodgson, (1983) J. 17. Dodgson, 18. Mills, 1026.
S. J., Forster, R. E., II, Storey, B. T., and Mela, L. (1980) Acad. Sci. USA 77,5562%5566. S. J., Forster, R. E., II, Schwed, D. A., and Storey, B. T. Biol. Chem. 258, 7696-7701. S. J. J. Appl. Physiol. 63,2134-2141, 1987.
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E. (1936-1937)
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Proc.
R. Sot. London
P/lugers
Arch.
370,
121-
21. Wistrand, P. J., and Knuutila. (1989) Kidney Int. 35, 851-859. 22. Wistrand, P. J., Ann. N. Y. Acad. Sci. 429, 195-206, 1984. 23. Riley, D. A., Ellis, S., Lang, D. H., and Rain, cJ. L. W. (1984) Ann. N. Y. Acad. Sci. 429,408-412. 24. Streb, H., Irvine, R. F., Berridge, M., and Schulz, I. (1983) Nature (London) 306,67-69. 25. Geers, C., and Gras, G. in The Carbonic Anhydrases: Cellular Physiology and Molecular Genetics (Dodgson, S. J., Tashian, R. E., Gras, G., and Carter, N. D., Eds.), Plenum, New York, in press. 26. Thornley-Brown, D., Dass, P. D., and B&hem. Pharmacol. 31,3347-X%3. 27. Nordlie, 2160.
R. C., and Arion,
W. J. (1965)
Welbourne, J. Biol.
T. C. (1984)
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240,
2155-
28. Maren, T. H. (1977) Amer. J. Physiol. 232, F291lF297. 29. Swenson, E. R. in The Carbonic Anhydrases: Cellular Physiology and Molecular Genetics (Dodgson, S. J., Tashian, R. E., Gros, G., and Carter, M. D., Eds.), Plenum, New York, in press. 30. Maren, T. H. (1967) Physiol. Reu. 47,595&781. 31. Tannen, 536-542.
50,
IN
32
R. L., and Ross,
Dodgson, S. J., and Forster, phvs. 251, 198-204.
B. D. (1983)
J. Lab.
R. E., II. (1986)
Clin.
Arch.
Med.
Biochem.
102, Bio-
Vol.
OF BIOCHEMISTRY
282, No. 1, October,
APU’D BIOPHYSICS
pp. l-7,
1990
Rat Renal Proximal Tubular Gluconeogenesis: Possible Involvement of Nonmitochondrial Carbonic Anhydrase lsozymes Susanna
J. Dodgson’
Department
Received
of Physiology,
January
and Kay Cherian University
16, 1990, and in revised
of Pennsylvania
form
May
School of Medicine,
Copyright All rights
correspondence
should
(cl 1990 by Academic Press, of reproduction in any form
be addressed.
Inc. reserved.
Pennsylvania
19104-60&G
lo,1990
The carbonic anhydrase (CA) inhibitor ethoxzolamide decreases the rate of glucose synthesis from 10 mM pyruvate by tubules incubating in 25 mM HCO, but not in 50 mM HCO,: this is evidence that rat renal cortical mitochondrial CA (CA V) provides HCO, for pyruvate carboxylation in renal tubular gluconeogenesis at physiological total CO2 (CO2 + HCO;). In renal proximal tubules prepared from 48-h-starved rats and incubating in 10 mM pyruvate in 25 mM HCO, buffered saline (Krebs-Henseleit buffer) the CA inhibitors acetazolamide (AZ) and benzolamide (BZ) decreased the rate of glucose synthesis. Maximal inhibition was reached with 125 pM AZ or with 450 pM BZ. The rate of glucose synthesis increased with increasing pyruvate concentration from 3.33 to 20 mM; including 600 pM BZ or 188 ELM AZ results in glucose synthesis becoming independent of increasing pyruvate concentration. Doubling the physiological concentration of bicarbonate restored the dependence of glucose synthesis on pyruvate concentration and partly, but not completely, alleviated the inhibitory effect of AZ and BZ, leading to the conclusion that AZ and BZ influence gluconeogenesis by affecting enzymes in addition to CA V. Tubules were incubated with substrates which do not require pyruvate carboxylation for synthesis of oxaloacetate. When tubules were incubated in 10 mM malate the rate of glucose synthesis was unaffected by less than 100 pM AZ or 400 pM BZ and was decreased maximally by 40 and 20%, respectively, by 125 ELM AZ, 450 j.tM BZ, and higher concentrations of these drugs. Increasing the malate concentration from 3.33 to 20 mM increased the rate of glucose synthesis; 600 pM BZ inhibited the rate of glucose synthesis only when the malate concentration was greater than 10 mM but 188 MM AZ decreased the rate of glucose synthesis at each concentration of malate. Results were similar when tubules were incu-
’ To whom
Philadelphia,
bated in glutamine with CA inhibitors. The rate of glucose synthesis differed with the substrate metabolized and the substrate concentration except when 600 pM BZ was included. It is concluded that only when pyruvate is carboxylated does BZ inhibition of CA V affect mitochondrial production of oxaloacetate. It is further concluded that the maximum decrease of glucose synthesis observed with BZ results from a block in gluconeogenesis after phosphoenolpyruvate carboxykinase (PEPCK). Greater inhibition of the rate of glucose synthesis was always observed with 188 HAM AZ. This inhibition was independent of substrate concentration, indicating a posterior block; however, the rate of glucose synthesis varied with each substrate, indicating an additional block before PEPCK. It is concluded that AZ and BZ inhibit enzymes involved in gluconeogenesis other than CA V. Since 95% of the CA activity of rat kidney tubules is nonmitochondrial it is suggested that this inhibition may be a secondary effect of inhibition of other CA isozymes. CC 1990 Academic Press, Inc.
It has recently been demonstrated in this laboratory that the rate of glucose synthesis by intact renal tubules is reduced maximally 75% by less than micromolar concentrations of the carbonic anhydrase (CA,’ EC 4.2.1.1) inhibitor ethoxzolamide when pyruvate is the added substrate (1). Glucose synthesis was unaffected by 1.6 yM ethoxzolamide when tubules were incubated in twice physiological total COT, leading to the conclusion that
* Abbreviations used: CA II, the cytoplasmic soluble carbonic anhydrase; CA IV, the brush border bound carbonic anhydrase; CA V, the mitochondrial carbonic anhydrase; PEPCK, phosphoenolpyruvate carboxykinase; PC, pyruvate carboxylase; CPS I, carbamyl phosphate synthetase I; AZ, acetazolamide; BZ, benzolamide; total CO,, CO, + HCO;; EGTA, ethylene glycol bis(&aminoethyl ether)i\i,W-tetraacetic acid; Mops, 4-morpholinepropanesulfonic acid. 1
2
DODGSON
AND
at physiological total COz , the rate of glucose synthesis is controlled by CA V. Carbonic anhydrase activity was first described in the kidney in 1941 (4). The unique mitochondrial carbonic anhydrase, CA V comprises only 5% of the total carbonic anhydrase activity of rat renal proximal tubules (1, 2); the cytosolic CA II and the membrane-bound CA IV together comprise 95%. It is generally believed that either CA II or CA IV or both are needed for 80 to 85% of the bicarbonate resorption (reviewed in (5, 6)); it is likely that CA II is needed for HCO;-Na+ exchange across brush border membranes (7). The major aim of this present study was to determine whether inhibition of the rate of glucose synthesis by CA inhibitors always results entirely from the inhibition of the mitochondrial carbonic anhydrase CA V. The CA inhibitor acetazolamide (AZ) has been used extensively in renal studies (5-8); there is no evidence that it inhibits other enzymes and many conclusions about the functions of the tubular carbonic anhydrases have been based on studies with these inhibitors. The data in this report indicate that acetazolamide (~10~~ M) decreases the rate of renal tubular glucose synthesis solely by inhibiting the mitochondrial CA V, as does another CA inhibitor, benzolamide (BZ) (15 X lop4 M). At higher concentrations of CA inhibitors the rate of glucose synthesis is decreased by the effect of acetazolamide (and to a lesser extent, benzolamide) on other enzymes participating in gluconeogenesis. METHODS Reagents. Collagenase (Worthington Type IV) was bought from Worthington (Freehold, NJ). “O-labeled NaHCO, was prepared from unlabeled NaHCO, and “O-labeled HOH as described (10). All other chemicals and reagents were bought from Sigma Chemical Company (St. Louis, MO). Preparation of biological materials. Intact renal proximal cortical tubule suspensions were prepared from 48-h-starved 200-g male rats according to published techniques (11) exactly as previously described (1). For each preparation tubules were prepared from the perfused kidneys of two animals which had first been anesthetized with sodium barbitol (5 mg/lOO g body weight). The isolated kidney tubules were in a final suspension of 20 to 50 mg dry wt per milliliter. Experiments with intact tubules were finished within 1 h and 10 min after their isolation. Dry weights were determined on each preparation (1). Intact renal cortical mitochondria were prepared from 48.h-starved male rats. Cortices were finely minced and rinsed in 275 mM mannitol, 1 mM EGTA, 2 mM Mops, pH 7.4.The nonmitochondrial fraction was sedimented at 2200g; the mitochondrial fraction sedimented at 72OOg, was resuspended in the mitochondrial buffer, and was pelleted at SSOOg. Renal cortical mitochondria were further purified by centrifugation through a Percoll gradient at 100,OOOg for 30 min (1, 2). Mitochondrial protein concentration was determined by the Lowry technique (12). Production and determination of glucose. Isolated proximal tubule suspensions were incubated in 20 ml scintillation vials with KrebsHenseleit bicarbonate saline incubation buffer (13), pyruvate, malate, or glutamine, and carbonic anhydrase inhibitors. In each vial the 1.2ml fluid volume included between 3.3 and 20 mM pyruvate and 400 ~1 of kidney tubule suspension in incubation buffer. The incubation
CHERIAN buffer was either Krebs-Henseleit bicarbonate saline buffer, which was continuously bubbled with 5% CO,/95% 0, or 50 mM bicarbonate buffer (Krebs-Henseleit but with 25 mM NaCl replaced with an additional 25 mM NaHC03) continuously bubbled with 10% C02/90% OZ. The former buffer has physiological concentrations of total CO* and the latter has doubled physiological concentrations. Before and after addition of 0.4 ml of kidney tubules (8 to 20 mg dry wt) the vials, on ice, were flushed with gas and then tightly closed. At 30-s intervals vials were removed from ice and added to the 37°C shaking water bath; 0.250-ml aliquots were removed from each vial after 20, 40, and 60 min, and added to 0.1 ml of HClO,. Each vial was flushed with gas and tightly capped, then returned to the incubating water bath. The pH of the acidified aliquot was neutralized with KHCOB, the solution centrifuged at 13,000g for 5 min, and glucose estimated in the supernatant (14). The rate of glucose production was calculated in terms of micromoles.Gram dry weight-’ 60 minss’ by constructing curves such as are shown in Fig. 2 and calculating the concentration of glucose at 60 min from the line of best fit with Cricket software. Statistical analyses of rates of glucose synthesis from different substrates were made with Student’s two-tailed t tests for paired samples, by Statworks software. Mass spectrometric analyses. The carbonic anhydrase activity of renal cortical mitochondria was assayed by the “O-exchange technique (10) as previously described for intact hepatic mitochondria (15, 16), and intact cells (17). The mass spectrometer recorded the mass 46 (C’60180), the mass 44 (C160,) and the mass 32 (i602) peaks alternately every 15 s. Mass 46 peak heights reach a final equilibrium value, 46cc,, which is subtracted from the peak height at time t, 46,. The addition of a carbonic anhydrase containing sample accelerates the decrease; conversely total carbonic anhydrase inhibition returns the decrease to the uncatalyzed rates. The experiments with intact renal cortical mitochondria were conducted in 50 mM Mops, 35 mM KCl, 5 mM KH,PO,, 1 mM EGTA, 1 mM MgC&, 25 mM KHCOB (1% labeled with “0) at pH 7.4, 37°C. The CA V activity is presented in terms of the ratio of mass 46 to mass 44 peak heights (Fig. 1). The mass 44 peak height does not decay, it increases with increased acidity and decreases with increased alkalinity; thus the ratio of peak heights reflects the true decay of C’sOi60, independent of pH changes. Experiments with disrupted renal cortical mitochondria were conducted in 25 mM NaHCO; (1% labeled with “0). The mass 46 peak Ci601’0 decreases as ‘so exchanges with the 55 M I60 pool in water as outlined by Mills and Urey (18): ~160180
+ ~~160
:-“;
~~~180160~
E;%EO=”
=k\\ k\\
1135 \\
C=02 + H2”O H’ + HC’801602The plot of log 10(46,/44, - 46a;/44co) vs time is linear with slope n,, from which the uncatalyzed CO, hydration rate is calculated. On addition of a vesicular carbonic anhydrase containing sample there is a rapid “step” decrease in the plot, followed by a slower linear portion with slope neat. When the permeability barrier to HCO; has been removed by disruption of the membrane, the disappearance of mass 46 is monophasic (10). The constant k.,, (in ml sec~i mg dry weight-‘) was calculated for disrupted mitochondria from equations given previously (15).
RESULTS
Carbonic anhydrase activity of disrupted renal cortical mitochondria at 37°C was determined by mass spectrometry with concentrations of acetazolamide and ben-
RENAL
1
Starved IO mM
5
g
Rat Pyruvate
TUBULE
GLUCONEOGENESIS
IN
I
'E
Control
0
Pyruvate Rats
200
8 i
100 t
3
RAT
10 mM Starved
z
200
-z Z E 2
PROXIMAL
s
100
A, ? 0 0
>ul
"0
20
40
Time
(minutes)
60
= 0.98).
The rate of glucose synthesis at 60 min was determined by incubating renal proximal tubules in an isotonic bicarbonate buffer for 60 min and measuring the concentration of glucose in aliquots at 20,40, and 60 min as described (see Methods). Figure 1 represents two typical experiments in which rat proximal tubules were incubated with 10 mM pyruvate with or without the carbonic anhydrase inhibitor benzolamide. The rate of glucose synthesis was linear over 60 min in the control experiment as well as the one in which 781 ELM benzolamide was included; this was also observed for each experiment described in this report. The rate of glucose synthesis was decreased incrementally by including in the incubation solutions increasing concentrations of acetazolamide and benzolamide (Fig. 2). The concentrations of CA inhibitors needed for maximal inhibition were 125 PM for acetazolamide and 400 PM for benzolamide. These data contrast with the concentration of ethoxzolamide needed for maximal inhibition, 1 PM (1). In subsequent experiments the rates of glucose synthesis were maximally inhibited by including either 600 pM benzolamide or 188 PM acetazolamide. At the concentrations of acetazolamide and benzolamide needed to maximally decrease glucose synthesis, CA V of intact mitochondria is fully inhibited; Fig. 3 indicates that 42 yM acetazolamide inhibits CA V completely as does 335 pM benzolamide. These concentra-
200
300
400
[Acetazolamide]
FIG. 1. Glucose synthesis over 60 min by proximal tubules prepared from 46h-fasted rats. Incubation vials contained a total volume of 1.2 ml (of which 0.4 ml was the final addition of suspended proximal tubules) with tubules and all solutes in Krebs-Henseleit bicarbonate saline buffer gassed with 5% CO,/95% 0, with 10 mM pyruvate. At t = 0 the closed vials were added to the 37°C shaking water bath. At t = 20, 40, and 60 min, 250-~1 aliquots were removed and added to HCIO,, the vials regassed, tightly recapped, and replaced in the water bath.
zolamide between 0 and lop6 M. A concentration of lop7 M of each drug was sufficient to completely inhibit all the carbonic anhydrase activity of 1 mg mitochondrial protein/ml reaction solution. Ki’S CA V were determined with disrupted renal cortical mitochondria at 37°C from Easson-Stedman plots (19) for acetazolamide, 0.04 pM (n = 4, r = 0.89), and for benzolamide, 0.08 PM (n = 4, r
100
0
z
0
pM
500
1000
[Benzolamide]
FM
FIG. 2. Glucose synthesis by intact rat kidney cortical proximal tubules in 60 min as a function of either benzolamide or acetazolamide in bicarbonate buffer at pH 7.4,37”C with 10 mM pyruvate. Each curve is a composite of 15 to 30 incubations conducted on between 2 and 4 different days exactly according to the legend in Fig. 1.
tions are several orders of magnitude higher than their Ki, and lower than the concentrations needed for maximal inhibition of glucose synthesis. AZ and BZ are considerably less lipophilic than ethoxzolamide (with which total inhibition of CA V in intact mitochondria was observed with 0.15 pM (1)) and it could not be assumed that they cross mitochondrial membranes even at these high
335 FM B2
i
”
0
2
4
Time
6
a
(minutes)
FIG. 3. Plots of log,, ((mass 46 peak height at time = t/mass 44 peak height at time = t), minus (mass 46 peak height at time = z/ mass 44 peak height at time = x,)} against time before and after rat renal cortical mitochondrial suspensions were added to the reaction chamber of the mass spectrometer. Mitochondria (0.5 mg/ml) were added at the first arrow to the Mops buffer described under Methods with 25 mM KHCO, (1% labeled with “0) at pH 7.4,37”C and either 81 pM acetazolamide (AZ) or 335 pM benzolamide (BZ) added at the second arrow.
DODGSON
AND
CHERIAN
TABLE
I
Rates of Glucose Synthesis by Intact Rat Renal Proximal Tubules Incubating in Different Pyruvate and Bicarbonate Concentrations in the Presence and Absence of Carbonic Anhydrase Inhibitors Rate 25 [Pyruvate] (mM)
Control
+BZ
(6) 3.3 6.7 10 13.3 16.7 20
124 162 179 184 202 214
f 27 +- 39 f 43 + 79 +I 44 f 68
107 129 106 108 136 117
(4) f f zk + + 2
39 33 62 45 49 48
mM
of glucose
synthesized
(rmol/g
dry wt 60 min)
HCO;
50
1 (%I
+AZ
14 21 41 41 33 45
(3) 86 t 96+81 f 98 + 94 IL 91+
I(%)
Control
+BZ
(6) 18 9 30 18 25 27
31 41 51 41 53 57
86 123 158 182 187 215
f + + + + +
18 36 52 50 66 30
Note. Tubules were incubated for 60 min in either Krebs-Henseleit bicarbonate saline buffer sphere of 5% C02/95% 0, or in the 50 mM NaHCOs buffer (see Methods) under an atmosphere refer to the number of times the series of experiments was conducted with a fresh preparation. benzolamide (BZ) were 188 and 600 pM, respectively.
concentrations. It is concluded from Figs. 2 and 3 that these drugs cross the mitochondrial membranes. In the preceding report from this laboratory (1) it was demonstrated that glucose synthesis from 10 mM pyruvate was maximally inhibited by 1.0 PM ethoxzolamide. In subsequent experiments the pyruvate concentrations in the incubation solutions were varied between 5 and 20 mM pyruvate and the rates of glucose synthesis determined with and without 1.6 ~.LM ethoxzolamide. At each pyruvate concentration, doubling the total COz removed the inhibitory effect of ethoxzolamide entirely, leading to the conclusion that the inhibitory effect of ethoxzolamide at physiological total CO2 was entirely due to inhibition of CA V. The next series of experiments was designed to test whether this same conclusion could be made about the effects of BZ and AZ. Tubules were incubated in either 25 mM total bicarbonate (physiological total CO,) or 50 mM bicarbonate (twice physiological total CO,; Table I). At physiological pC0, the rate of glucose increased with increasing pyruvate concentration (2) only in the absence of CA inhibitors. Doubled physiological total COz had two effects on the experiments in which AZ and BZ were included. First, inhibition of the rate of glucose synthesis was only partly alleviated, this alleviation was greater for BZ than for AZ. Second, the rate of glucose synthesis became dependent on pyruvate concentration, as in the control experiments conducted at physiological pH. It is concluded that AZ and BZ inhibition of the rate of glucose synthesis is not just a simple effect from CA V inhibition and suggested that other enzymes involved in gluconeogenesis are also affected. The remaining experiments in this report were all aimed at determining possible enzymes involved in glucose synthesis which AZ and BZ inhibit either directly or indirectly. Direct effects would be expected for cytosolic and
mM
85 93 126 130 154 176
(4) AZ 18 f 17 + 34 * 13 + 46 k 22
(containing 25 of 10% C02/90% Concentrations
HCO, 1 (%I
+AZ
0 24 20 29 18 18
(4) 90f 92 f 100 f 111 It 116+ 135 +
mM
1 (So)
9 13 22 22 8 36
0 25 37 39 38 37
NaHCO;) under an atmo0,. Numbers in parentheses of acetazolamide (AZ) and
membrane-bound CA isozymes, although these other isozymes have never been implicated in gluconeogenesis. Indirect effects would be predicted for any gluconeogenic enzyme requiring any of the products of CA-mediated catalysis, i.e., H+ or HCO,. Enzymes which are possible candidates are those needed for transmitochondrial transport of substrates and products; the mitochondrial enzymes involved in producing oxaloacetate, the gluconeogenic enzymes after phosphoenolpyruvate carboxykinase, or those needed for glucose 6-phosphate transport into the endoplasmic reticulum and glucose transport out of the endoplasmic reticulum. Figure 4 is a metabolic scheme in which pathways to glucose from pyruvate, malate, and glutamine are depicted. When gluconeogenesis proceeds from malate or glutamine, pyruvate carboxylation is not an intermediate reaction and thus the requirement for CA V does not exist, Fig. 4. When tubules were incubated in 10 mM malate or 10 mM glutamine, concentrations of acetazolamide less than 10e4 M neither increased nor decreased the rate of glucose synthesis, Fig. 5. When 10 mM pyruvate is the substrate 125 pM acetazolamide reduces glucose synthesis to a minimum rate; similarly, acetazolamide reduces the rate of glucose synthesis when either 10 mM malate or 10 mM glutamine are substrates to minimum rates. When 10 mM malate is the substrate, the rate of glucose synthesis was not decreased by 400 pM benzolamide, Fig. 5; higher concentrations of this resulted in an abrupt decrease in glucose synthesis down to a minimum rate as also observed when 10 mM glutamine is the substrate. The data in Fig. 5 also indicate that low concentrations of benzolamide actually may increase the rate of glucose synthesis when 10 mM malate is the substrate. The rates of glucose synthesis increase with increasing malate concentration, Table II. Inclusion of 188 yM
RENAL
PROXIMAL
TUBULE
GLUCONEOGENESIS
IN
5
RAT
It is concluded that >125 PM acetazolamide and >450 benzolamide decrease the rates of glucose synthesis. The first set of evidence for this conclusion is that doubled total CO;! does not completely override AZ and BZ inhibition when pyruvate is the sole metabolic substrate, see Fig. 2 and Table I. The second set of evidence is that these concentrations of AZ and BZ also decrease the rates of glucose synthesis when glutamine and malate are the added substrates, Fig. 5, Table II. If acetazolamide, benzolamide, and ethoxzolamide have their effects only on CA isozymes, these data indicate that the CA isozymes throughout the proximal tubules have either different affinities or accessibilities to the CA inhibitors, or both. The carbonic anhydrase isozymes known to be present in rat kidney are the mitochondrial CA V (a), the cytosolic CA II, and the membrane-bound CA IV (20-22). All isozymes are sensitive to the specific sulfonamide CA inhibitors (2, 22). Carbonic anhydrase activity in the membrane of the endoplasmic reticulum has been detected by histochemical studies of the peripheral nervous system (23), and may also be present in the renal endoplasmic reticulum, although there have been no reports published confirming this. Ca2+ stored within the endoplasmic reticulum is released by IPB in response to P-adrenergic stimulation (24). While there is no evidence in the present report for interference of the CA inhibitors with Ca2+ release, this has been reported in PM
CYTOSOI ,\
c
:
:
:
:
phosphoenolpyruvate
t’
+ GDP f P. I
I
FIG. 4. Enzymes involved in glucose synthesis from pyruvate, malate, and glutamine in rat renal cortical proximal tubules. Also indicated are the carbonic anhydrase isozymes CA II, CA IV, and CA V. Enzymes of glucose synthesis are pyruvate carboxylase, PC, phosphoenolpyruvate carboxykinase, PEPCK. The gluconeogenic enzymes after PEPCK are also glycolytic enzymes, all located in the cytosol; the single exception is the irreversible reaction catalyzed by glucose 6 phosphatase, a soluble enzyme located inside the endoplasmic reticulum. The only other irreversible reaction in the cytosol after PEPCK is catalyzed by fructose-1,6-biphosphatase.
acetazolamide decreased the rate of glucose synthesis at each malate concentration; these rates are similar to the rates when glutamine is the substrate but 30% less than the rates when pyruvate is the substrate (Table I). The minimum rate of glucose synthesis when acetazolamide (188 PM) was included was significantly lower (by paired t tests, P < 0.05) than the minimum rate when benzolamide (600 FM) was included in these experiments. When 600 PM benzolamide is included, the rates of glucose synthesis from malate, glutamine (Table II), and pyruvate (Table I) are similar. Benzolamide does not decrease glucose synthesis at low substrate concentrations no matter which substrate is included; these data are contrary to those obtained after acetazolamide inclusion.
i
Oo1,
1001.
2001.
[Acetazolamide]
a
k
3001,
4001
I
00
pM
100
200
[Acetazolamide]
300
400
pM
DISCUSSION
It is concluded that carbonic anhydrase inhibitors decrease renal proximal tubular glucose synthesis by their effect on the mitochondrial carbonic anhydrase at low concentrations but at high concentrations the inhibitors have an additional effect on glucose synthesis. Low concentrations of the CA inhibitors decreased the rate of glucose synthesis only when pyruvate was the added substrate (Figs. 1 and 2); these data support the hypothesis that mitochondrial carbonic anhydrase is needed to provide the HCO; substrate for pyruvate carboxylase (see scheme in Fig. 4) and also indicate that these CA inhibitors have no other effect on glucose synthesis at these concentrations.
0
500
[Benzolamide]
1000
pM
0
500
[Benzolamide]
1000
pM
FIG. 5. Glucose synthesis by intact rat kidney cortical proximal tubules in 60 min as a function of benzolamide and acetazolamide in bicarbonate buffer at pH 7.4,37”C with 10 mM malate or 10 mM glutamine. Each curve is a composite of 8 to 30 incubations conducted on between 2 and 4 different days exactly according to the legend in Fig. 1.
6
DODGSON TABLE
AND
II
Rates of Glucose Synthesis by Renal Proximal Tubules Incubating in Different Substrate Concentrations in the Presence and Absence of Carbonic Anhydrase Inhibitors Rate of glucose synthesized (wmol/g dry wt 60 min) [Substrate] mM
Malate
Control
$600
(2)
(2)
3.3 6.7 10 13.3 16.7 20
go+31 121* 121k 136 f 141 k 151*
21 6 11 6 19
101k 107 113 121 120 98
Glutamine
(4) 92 + 106 k 122 f 144 f 146 2 146 2
37 44 51 32 26 17
81 113 112 100 93 90
3.3 6.7 10 13.3 16.7 20
BZ
/LM
f f k + +
3 17 32 59 41 14
(2) k +++ 2 +
34 34 19 29 25 32
+188
MM
AZ
(2) 66 + 16 67+ 8 51* 11 59 3125 69 t 11 82 t 15 (1) 64 56 53 71 76 64
Note. Tubules, prepared from 48-h-starved male rats, were incubated for 60 min in Krebs-Henseleit bicarbonate saline buffer under an atmosphere of 5% CO,/95% 02. Numbers in parentheses refer to the number of times the series of experiments was conducted with a fresh preparation.
experiments with the sarcoplasmic reticulum, gous organelle of the skeletal muscle, which
the analoare known
have carbonic anhydrase activity (25). The possibility that acetazolamide and benzolamide inhibit enzymes other than the carbonic anhydrases cannot be ignored. Intracellular inhibition of other enzymes has never been demonstrated; the renal y-glutamy1 transpeptidase (26) was found to be 35% inhibited by 5 mM acetazolamide, a concentration far higher than that in the tubules in the experiments of this report. Benzolamide decreasesthe rate of glucose synthesis to a minimum rate which is not significantly different for pyruvate or malate, malate or glutamine, pyruvate or glutamine. This contrasts with the very different rates of glucose synthesis in the absence of benzolamide. These data indicate there is a common block for glucose synthesis which is independent of substrate, or substrate concentration, and point to this block being after phosphoenolpyruvate carboxykinase, PEPCK. From examination of the metabolic scheme in Fig. 4, it is suggested that a possible site of this block could be the irreversible ultimate gluconeogenic enzyme, glucose-6-phosphatase. This enzyme is located inside the endoplasmic reticulum, and the final step in the synthesis of glucose is its release from the endoplasmic reticulum; thus there could be a block in the transport of either glucose B-phosphate or glucose across the membranes of the endoplasmic re-
CHERIAN
ticulum or even a direct inhibition of glucose-6-phosphatase. If cytosolic CA II is needed for providing H’ or HCO, for transport of glucose 6-phosphate into or glucose out of the endoplasmic reticulum then CA inhibition would alter the rate at which glucose is synthesized by the renal proximal tubule. It is noted that other substrates for glucose-6-phosphatase are H+ and OH-, as for the carbonic anhydrases and the other irreversible cytosolic gluconeogenic enzyme fructose-1,6-biphosphatase. Acetazolamide decreasesthe rates of glucose synthesis to lower minimum rates than does benzolamide; these mimima are significantly different for pyruvate and malate (by unpaired Student’s t tests, P < 0.002) and pyruvate and glutamine (P < 0.002) but not malate and glutamine. The rates of glucose synthesis by tubules incubating in 125 PM acetazolamide were the same when malate and glutamine were substrates. This rate was different when pyruvate was the substrate. It is thus concluded that acetazolamide has an additional effect on gluconeogenesis before PEPCK which is more effective at reducing the rate of glucose synthesis than the effect after PEPCK. Twice physiological total CO2 overrides inhibition of glucose synthesis by ethoxzolamide (1) but not by acetazolamide or by benzolamide. The concentrations of ethoxzolamide needed for decreased rates are lop2 to lo-” times lower than for the other CA inhibitors. A complete cessation of glucose synthesis by kidney tubules has been observed in this laboratory when greater than 4 pM ethoxzolamide is included (Dodgson, unpublished results). It has been stated that when a concentration of greater than lop4 M CA inhibitor is needed for an effect to be ascribed to the carbonic anhydrases, this effect is possibly nonspecific (28, 29), resulting from the uptake of other ions with the CA inhibitors. In this present report the concentrations of the inhibitors needed for decreasing glucose synthesis to minimal rates are exactly in this range. The data in this report do not indicate whether the effects of acetazolamide or benzolamide result
from
ion influx
or inhibition
of cytosolic
or mem-
brane-bound carbonic anhydrases which in some way decrease the activity of other gluconeogenic enzymes. The rate of glucose synthesis is decreased to a minimum with 1 pM ethoxzolamide, 125 pM acetazolamide, or 450 PM benzolamide (1:125:450); this ratio compares well with the relative permeances of these CA inhibitors across cellular and organellular membranes (30). Benzolamide has been used in studies with intact cells because it is relatively impermeant across cell membranes. The data in this report indicate clearly that benzolamide is permeant across membranes of isolated mitochondria (Fig. 3). The need for higher concentrations of benzolamide to decrease glucose synthesis than the other CA inhibitors agrees with previous observations that ben-
RENAL
PROXIMAL
TUBULE
zolamide is less freely permeable across cellular membranes (5,8, 25). In a limited study, it has previously been reported that 1 mM acetazolamide decreased rat renal tubular glucose synthesis 70% when 10 mM malate was the substrate and 11% when 2 mM glutamine was the substrate (31). These data are not greatly different from the present data, indicating that the same effect is seen even with extremely high concentrations of the CA inhibitor acetazolamide. Thus, whatever the reason for the observed abrupt decrease in the rate of glucose synthesis at 100 PM acetazolamide or 450 PM benzolamide, increasing the concentration of either drug does not decrease the rate still further. It has previously been reported from this laboratory that hepatocytes prepared from 48-h-starved male guinea pigs synthesized glucose from pyruvate dependent of CA V but from glutamine this rate was independent of carbonic anhydrase inhibitors (32). Rat kidney CA V differs from guinea pig liver CA V in its K, and its lower pH sensitivity (2); it is clear from this report that they both provide HCO, for pyruvate carboxylation in the same manner. ACKNOWLEDGMENTS The IXXISS spectrometer facility is supported by HL P019737, Program Director Dr. Robert E. Forster II. Dr. Thomas Maren provided benzolamide; Lederle, Pearl River, NY provided acetazolamide. Thanks always to Mrs. Lydia Lin, who maintains the mass spectrometer and prepares KHC’“O”0, and NaHCi80i602, and to Dr. Forster for constant encouragement. This research was supported by NIH DK 38041 (to S.J.D.).
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