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The effect of furosemide and bumetanide on cerebrospinal fluid formation
Brain Research, 221 (1981) 171-183 Elsevier/North-Holland Biomedical Press
171
THE EFFECT OF FUROSEMIDE AND BUMETANIDE ON CEREBROSPINAL FLUID FORMATION
BETTY P. VOGH and MAX R. LANGHAM, Jr. University of Florida College of Medicine, Department of Pharmacology and Therapeutics, Gainesville, Fla. 32610 (U.S.A.) (Accepted December 11th, 1980) Key words: cerebrospinal fluid - CSF formation - furosemide - bumetanide - acetazolamidecarbonic anhydrase - choroid plexus
SUMMARY
Complete inhibition of carbonic anhydrase maximally reduces CSF flow 40-60 %34. We have confirmed that CSF flow in rabbits may be maximally decreased in this way by acetazolamide 30 mg/kg i.v., but have found only about 20 %reduction from control after furosemide at 50 mg/kg i.v. This effect of furosemide is consistent with specific but partial inhibition of carbonic anhydrase of choroid plexus (and perhaps other sites of CSF secretion) based on the affinity of furosemide for carbonic anhydrase. Bumetanide, with 7 times lower affinity for carbonic anhydrase and 14-40 times higher inhibition of renal electrolyte transport processes than furosemide 2 ,18,3o, did not decrease CSF flow.
INTRODUCTION
The physiology of the cerebrospinal fluid (CSF) secretion has been a subject of interest to investigators for many years. The effects of several drugs on CSF production, CSF composition, and rate constants for isotopic Na+ movement into CSF have been studied to elucidate basic mechanisms of the system and to find rational methods for treating hydrocephalus and other conditions involving high CSF pressure 12 ,23,34. Earlier reports of the effect of furosemide upon CSF production or influx of 22Na i to CSF are inconsistent but mostly indicate CSF reduction 6 ,14,19,28,32,33. Questions remained whether the effects seen were due to volume depletion through diuresis, to direct interference with CI- transport at the choroid plexus (as in the kidney7) or to inhibition of carbonic anhydrase (CA) at sites of CSF formation. Furosemide is a weak inhibitor of CA (see ref. 19 and data below).
172 In this study, in which volume depletion is ruled out by preventing renal action of the drugs studied, we show that the measured mean effect of furosemide on CSF flow is small and cannot be differentiated from control. The lack of effect of bumetanide, a congener of furosemide 40 times more potent as a diuretic in man 2 and animals 30 than furosemide, and 14 times more potent as an inhibitor of renal C 1 transport in isolated renal tubules of rabbit than furosemide 18, strongly supports the likelihood that any action of furosemide at sites of CSF formation is by CA inhibition.
MATERIALS AND METHODS New Zealand white rabbits weighing 1,9-3.0 kg (2.3 ± 0.1) of either sex were anesthetized by injecting isotonic sodium pentobarbital into an ear vein. Anesthesia to depress the corneal reflex was maintained with supplemental i.v. doses. After tracheotomy, a cannula was inserted for maintenance on a Harvard respirator, using room air enriched to 3 0 - 4 0 ~ oxygen, and expired CO2 was monitored continuously. A femoral artery was cannulated for arterial pH measurements (Instrumentation Laboratories, Model 113). Respiration was adjusted to maintain arterial pH at 7.35-7.45. In some animals NaHCO3 was given i.v. to assist in pH control. Arterial blood pressure was also monitored continuously. From a :flank approach, the renal arteries, veins and ureters were ligated. Body temperature was kept at 38 ~ 1 °C using a heating pad responsive, by relay switches, to changes in the deep colonic temperature of the animals. For perfusion of the ventricles, artificial CSF was prepared ~6. This buffer solution, dye-labeled with 0.5 mg/ml Blue Dextran 2000 (Sigma or Pharmacia), was gassed with 5 ~ CO2 to pH 7.4 at room temperature before filling the perfusion system. Rabbits were placed in a sphinx-like position, their heads held rigidly. An 18-gauge spinal needle filled with artificial CSF and connected to a Harvard infusion pump was then placed stereotactically into the right lateral ventricle. CSF pressure was continuously recorded by means of a Statham transducer in line with the infusion tubing. Pressures recorded as the needle penetrated a lateral ventricle were between 0 and 10 cmH20 with respect to the auditory meatus. A 19-gauge hubless needle was manually placed into the cisterna magna after incising the skin and spreading the muscles of the neck. True CSF was collected at - - 1 0 cmH20 for 5 min. This was done to facilitate complete replacement of CSF by the artificial CSF containing the dye. To further wash out residual CSF, the ventricular system was then perfused for 5 min at 200 #l/min, then 5 min at 100/~l/min, and finally 15 min at 54 #l/min before starting to collect the perfusate for analysis. The steady rate of perfusion for the experiment was 54 #l/min, and the perfusate was collected at a pressure which did not vary by more than :!: 2 c m H 2 0 from the measured physiological pressure. Each sample of perfusate was collected over a period of 30 min. Samples were spun in a clinical centrifuge for 20 min at maximum speed to remove any possible material which might interfere with light transmittance. The measure of concentration of Blue Dextran was its optical density at 640 rim.
173 Formation of new CSF was calculated for each sample according to Heisey et al. l6 as: optical density of inflow - optical density of outflOW) (rate of perfusion) ( . optical density of outflow In the main study, the rabbits served as control animals or were treated, 30-40 min before collecting the first sample of perfusate, with furosemide (50 mg/kg) or acetazolamide (30 mg/kg) i.v. Furosemide, supplied by Hoescht-Roussel Pharmaceuticals, was freshly dissolved for each experiment in 0.1 M phosphate buffer, pH 7.4, at 20 mg/ml, by heating to 60 DC. Acetazolamide, supplied by American Cyanamid, was prepared as the sodium salt, at 50 mg/ml, pH 8.9. When acetazolamide was given, an additional volume of pH 7.4 phosphate buffer was administered equivalent to the volume of the furosemide dose. Furosemide dose was followed by pH 8.9 buffer equivalent to the dose of acetazolamide. Controls were given phosphate buffer equivalent in volume and pH to the furosemide and acetazolamide doses. In a group of 3 rabbits, bumetanide, supplied by Hoffman LaRoche, was given i.p., 50 mg/kg, prepared as 30 mg/ml in 95 % ethanol. This route and solvent were chosen due to the low solubility of bumetanide in aqueous buffer. The treatment of these rabbits was identical in other respects to that of the other groups, except that 30-50 min extra were allowed for drug absorption. For inclusion of an experiment in the group for statistical analysis, the animal was required to survive 2.5 h of perfusion with mean arterial blood pressure above 50 mmHg at all times. Most in the main groups were in the range 70-90 mmHg, but the animals treated with bumetanide averaged 53 mmHg. CSF data were discarded for any animal for which autopsy failed to confirm successful bilateral occlusion of the renal pedicle. In 6 rabbits given furosemide and sUbjected to the above procedures, blood samples were drawn at 10, 30, 60 and 120 min after injection of drug for determination of furosemide in plasma. Plasma, diluted I: 10, was prepared for assay by heating to 100 DC for 5 min to denature CA in any red cell contents which might be present. (Such treatment did not destroy furosemide in standards treated identically.) The assay for furosemide levels was based on inhibition of dog red cell CA24 after the drug sample was incubated with CA and CO 2 5 min. Plasma drawn prior to drug treatment was subjected to equilibrium dialysis to determine the per cent binding of furosemide to plasma protein. Two ml of plasma from each of two rabbits was dialyzed in 500 ml of 25 pM furosemide overnight during refrigeration. Aliquots of plasma and dialysate were then analyzed for furosemide concentration. The dissociation constants (K,) or drug concentrations giving 50 %inhibition of CA (ho) were determined 26 for furosemide, bumetanide and acetazolamide, using a range of drug concentrations with rabbit choroid plexus CA, pure human CA-C (isozyme C, CA II) and dog red cell preparations. The data for CSF production were submitted to multiple regression analysis as detailed in Results. Computations were made at the Northeast Regional Data Center, Gainesville, Fla. using the Statistical Analysis System (SAS)3.
t74 RESULTS I n Fig. 1, after washing o u t o f residual C S F , successive 30-min samples of perfused artificial C S F c o n t a i n i n g the dilution m a r k e r are c o m p a r e d for effect of t r e a t m e n t o n C S F rate o f p r o d u c t i o n . I t can be readily seen t h a t the m e a n rate for the b u m e t a n i d e g r o u p is like t h a t o f the controls. R a t e after f u r o s e m i d e is also like control, with the suggestion t h a t g r o u p s larger t h a n 6 or 7 a n i m a l s m i g h t verify the t e n d e n c y for f u r o s e m i d e to reduce flow slightly. There is a consistent p a t t e r n o f l o w e r m e a n value, 9 - 2 0 % lower for furosemide. A c e t a z o l a m i d e clearly differs f r o m controls. r a n g i n g 41-78 % less over 5 s a m p l e periods. Because the plots for individual animals, t r e a t e d a n d untreated, showed a significant decline in C S F flow with time (as previously observed, see Discussion) and because the times f r o m d r u g a d m i n i s t r a t i o n to start o f s a m p l e I v a r i e d somewhat. linear m o d e l s were used to fit the d a t a to e x a m i n e the effect o f time a n d the effect o f d r u g t r e a t m e n t a p a r t f r o m time.
Dependence of CSF production upon perfusion time A general linear m o d e l o f the form, Piit = fi0
/31t × fi2t2 +
Y, 6i~z Ri -- e r r o r : j -~ 1. . . . . 4.
(Eqn. 1)
i
was used to fit the data, to e x a m i n e the effect o f time. In this f o r m u l a t i o n PHt =: C S F p r o d u c t i o n in # l / m i n at time t for the iTM r a b b i t receiving the jth t r e a t m e n t : N0 intercept for a reference r a b b i t ; t ~ min after starting ventriculocisternal perfusion; t2 = t, squared;/31,/32 ~ coefficients o f the m o d e l variables t a n d t 2. related to slope; Ri = a variable used to eliminate the effect o f inherent differences in flow a m o n g r a b b i t s : i,e. Ri = 1 for the ith r a b b i t a n d Ri ~= zero for all others: ~i~ 2 :=- coefficient for Rl to
4
z C
3
~ ~o 5 g s £)
-
u©-
A(5)
_
_
4
a.l
0
- -
2
'
~
z-
5
S A M P L E NUMBER
Fig. 1. Rates of CSF formation during ventriculocisternal perfusion in nephrectomized rabbits are shown as mean and S.E.M. for successive 30 rain periods following drugtreatment and the washing out of residual CSF. Control (C); burnetanide, 50 mg/kg (B); furosemide, 50 mg/kg (F): and acetazolamide. 30 mg/kg (A). See text for discussion of decline in flow in control experiments. It is clear that the effects of furosemide or bumetanide 50 mg/kg do not approximate that of acetazolamide 30 mg/kg.
175 TABLE l Effect of time on formation of CSF* The values are given with S.E.M.
/31 (pl/min e) [;2(/~l/min3)
Control (n 7)
Acetazolamide Furosemide Bumetanide Pooled data (30mg/kgi.v.) (50mg/kgi.v.) (50mg/kgi.p.) (n 19) (n 3) (n -- 6) (n - 3)
- - 0.0500 ± 0.0216 0.0001 ± 0.0001
--0.0804 ± 0.0418 0.0001 ± 0.0002
--0.0841 ± 0.0170 0.0002 :~ 0.0001
--0.1153 ± 0.0406 0.0004 ± 0.0001
- - 0.0680 i- 0.0126 0.0001 ± 0.00005
* See Eqn. 1 in text. The fact that fll values are negative shows that CSF flow falls with time; positive values for/32indicate a slower rate of decline as time passes. take into a c c o u n t the shift in intercept for the i t~ r a b b i t f r o m fl0; a n d e r r o r = error for the i th r a b b i t at t. A q u a d r a t i c with respect to time was used because it a p p e a r e d t h a t p r o d u c t i o n decreased at a decreasing rate with time. The d u m m y variable, Ri, allowed e s t i m a t i o n o f the effect o f time a p a r t from the average level o f p r o d u c t i o n o f individual rabbits. T h e resulting coefficients for the time variables are given in Table I. F o r the c o n t r o l group, /31 is - - 0 . 0 5 # l / m i n each min a n d f12 is + 0 2 0 0 1 #l/rain 2 each rain. The fie coefficient shows t h a t the rate o f decrease diminishes with time. A n F-test indicated t h a t the effect o f time on furosemide, acetazolamide, b u m e t a n i d e and c o n t r o l groups did n o t differ. Therefore, the d a t a for all d r u g treatments a n d controls were p o o l e d to estimate a general coefficient o f decline with time, n o t due to d r u g (Table I). The p r i m a r y coefficient from the p o o l e d d a t a is - - 0 . 0 6 8 0 / ~ l / m i n per minute o f perfusion time or - - 4 . 0 8 / ~ l / m i n / h . This slope differs f r o m zero at P <~ 0.001. Dependence o f C S F production upon drug treatment apart f r o m decline due to time T h e effects o f the d r u g treatments a p a r t f r o m the effect o f time were then examined. A general linear m o d e l o f the form, Pijt ~ fl0 + tilt .t- fl2t ~ +
Z flj+2Tj + error,
(Eqn. 2)
J
was used to fit the data. I n this f o r m u l a t i o n : Pair, ill, f12, t, t 2 a n d error are defined as in Eqn. 1 ; t30 ~ intercept for the c o n t r o l g r o u p ; Tj = a variable used to g r o u p rabbits a c c o r d i n g to treatment, i.e. Tj - - 1 for rabbits in g r o u p being examined, a n d Tj = zero for all others; a n d f l j , 2 ~ coefficient o f Tj to t a k e into a c c o u n t the shift in intercept for the jth g r o u p from/30. The e r r o r was specified to follow a s e c o n d - o r d e r auto-regressive scheme to allow for the systematic deviation o f a p a r t i c u l a r r a b b i t * * . ** The AUTOREG procedure of SAS was used ~. This procedure first estimates the model with ordinary least-squares, then estimates the first- and second-order autocorrelation inherent among all points for a particular rabbit from the ordinary least-squares residuals. These estimates of autocorrelation are used to transform the original data to correct for this. Coefficients for the terms of the model are then regenerated from the transformed data.
176 TABLE II Estimated CSF produetion based on Eqn. 2 applied at mean time o f all observations* Drug group and dose
CSF production ( itl min)
Change Ji'om control ~ % decrease)
Control Furosemide (50 mg/kg i.v.) Acetazolamide (30 mg/kg i.v.) Bumetanide (50 mg/kg i.p.)
9.4 7.2** 4. I *** 8.4§
0 23 56 l1
* Time when 9 5 ~ confidence interval is closest to mean, i.e. at ~ 120 rain of perfusion (most samples were collected between 30 and 210 rain after starting perfusion). ** Different from control at P --~ 0.05. *** Different from control at P < 0.005. Different from furosemide at P - 0.01. § Not statistically different from control. The model was fitted for furosemide, acetazolamide, b u m e t a n i d e a n d control groups. U s i n g the coefficients for the model, curves giving m e a n C S F p r o d u c t i o n values for each t r e a t m e n t were generated along with 95 ~ confidence intervals. C S F flow-rates at the m e a n observation time, where the 9 5 ~ confidence interval is narrowest, are shown in T a b l e II. The m e a n c o n t r o l rate is 9.4 #l/rain, a n d the residual m e a n flows after furosemide, acetazotamide a n d b u m e t a n i d e treatments represent 23, 56 a n d 11 ~ decreases f r o m the m e a n c o n t r o l rate. U s i n g this approach, the effects of furosemide a n d acetazolamide (but not b u m e t a n i d e ) differed from control, a n d furosemide differed from acetazolamide (Table II). These results agree with those of Fig. 1 except t h a t furosemide is n o w shown to differ from control. The c o n c e n t r a t i o n of C A in homogenized r a b b i t choroid plexus (corrected for CA in blood), f o u n d to be 5.4 :~: 1.4 # M (S.E.M.) (n -----3) in a recent study ~5. was confirmed by new d e t e r m i n a t i o n s giving 5.1, 5.6 a n d 7.6 # M . The ability of furosemide, acetazolamide and b e n z o l a m i d e to i n h i b i t rabbit TABLE 1II Ability o f druga' used in rabbits to inhibit carbonic anhydrase in vitro (15o or Ki)
I50 is the concentration of inhibitor necessary to reduce enzyme activity to 50 ~ Of original activity in the assay. K, is the dissociation constant for enzyme-inhibitorcomplex. !5o ~ K~in these assays where enzyme concentration is kept low, < 5 x 10-9 M (March et al.2n), in every case drug was incubated with CA during constant COZ gas flow for 2 or 5 rain as shown in parentheses in the table:Such in, cubation enhances the inhibitionof dog red ceil CA by these drugs and does not destroy enzyme activity in the uninhibited control. To reach maximal inhibition, 5 min are required for furosernide and 2 min for acetazolamide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -7
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Source o f CA
Furosemide
Aeetazolamide
Bumetahide
Rabbit choroid plexus Dog red Cell CA Human red cell, isozyme CA-C
2.5 × 10 7 M (5rain) 2.9 x IO 7 M(5min)
1.5 ~ 10 ~ M(5min) 1 × 10 8 M(2min)
---
4.0 × 10:v M(2min)
1 × 10-SM(2min)
2.7 :~ 10 6 M(2min)
177 TABLE IV Concentration (pM) offurosemide in plasma, (after 50 mg/kg i.v. dose, mean and S.E.M.for 6 rabbits) and fractional inhibition of carbonic anhydrase
Total drug was determined by CA inhibitory activity compared to that of a set of furosemide standards, by the method of Maren et a1. 26 , using 5 min incubation of dog red cell CA with drug and C02 substrate before addition of bicarbonate bufferfor assay of pH change. The value of total drug for 90 min is taken from a plot of the other measured values. Binding of furosemide to plasma protein (determined by equilibrium dialysis of plasma taken prior to drug treatment) in 2 rabbits was 91 and 92.4 %for 25 pM furosemide in the dialyzing bath. The fractional inhibition was calculated as Itree/(Irree + Kl) (ref. 20); Itree is the unbound furosemide in plasma, taken to be the same as unbound furosemide in the secretory tissue. Minimum value for maximal inhibition of CSF flow appears to be 0.9995. Sample time 10 min
Total drug Estimated unbound drug Fractional inhibition of choroid plexus CA
30 min
60 min
90 min
120 min
1017 ± 158 619 ± 120 81 49
376 ± 99 30
310 25
281 ± 91 22
0.9963
0.9900
0.9881
0.9865
0.9939
choroid plexus CA and other CA sources is shown in Table III. Because the enzyme concentration is low in these assays « 5 X 10-9 M), the Iso approximately equals K, the dissociation constant for the inhibitor 26 • The general conclusion from Table III is that the inhibitory potency (affinity for CA) for acetazolamide is 17-40 times greater than for furosemide, and that of furosemide is 7 times greater than for bumetanide. Binding offurosemide to plasma protein averaged 92 %. Physiological inhibition of tissue CA depends upon the unbound (free) level of inhibitor in plasma and the access of this free drug to the enzyme site. Mean levels of total furosemide measured in plasma of kidney-ligated rabbits given 50 mg/kg are in Table IV, along with the estimated free drug concentrations and the degree of inhibition of CA obtained when free drug in plasma is in equilibrium with free drug in secretory tissues, as expected here. This dose gave a theoretical concentration at zero time of 1400 flM and a half-life of 30-40 min over the first hour; loss is much slower thereafter. The plasma drug concentration at 90 min probably forecasts secretory function at about 120 min (near the mean observation time in the regression analysis). At 90 min, total furosemide was 310 flM, from a plot of the data in Table IV; free furosemide was 25 flM, down from 81 flM at 10 min. DISCUSSION
Control flow-rates for our rabbits were 12.3 fll/min in the first 30 min sample, 11.1 in the second sample (in Fig. I) and 9.4 fll/min at the mean observation time in the regression (Table II), in good agreement with CSF flow for rabbits given by Davson and Pollay13.
178 Choroid plexus is responsible for a large fraction of CSF production (reviewed by Segal and Pollay 34 and by Rapoport3t). CA of plexus, by catalyzing the formation of HCO3 from CO2 and hydroxyl ion. serves the secretory function importantly22,a~,36. Inhibition of CA in choroid plexus to the level of at least 99.95 % was required to achieve maximal flow inhibition (40-60 % decrease) through the CA mechanism m cats aS, in which plexus CA averaged 22/~M. In rabbits with about one-third as much plexus CA as cat (see ref. 19 and data above), but with a higher fraction of acetazolamide bound to plasma protein (ref. 21, p. 642), maximal flow reduction also appeared to require 99.95 ~o inh ibition19. Such inhibition was provided by acetazolamide, 30 mg/kg i.v. in the cats a5 and 15-30 mg/kg i.v. in the rabbits tg. These animals were not nephrectomized. Acetazolamide at 30 mg/kg should, therefore, provide maximal flow reduction m the nephrectomized rabbits of this study. The data show that this is indeed the case: flow was reduced 41-78% from control rates in the data of Fig. 1. or 56% in the regression data of Table II. The 50 mg/kg i.v. dose of furosemide gave a 9--20 % flow reduction in Fig. ! and 23 % flow reduction in the regression in which the effect of time on flow was minimized. The 50 mg/kg dose was chosen because it (or less) has been reported to give maximal or near maximal CSF flow reduction in rabbits t9.32 and cats 33. It is widely accepted that CA inhibitors such as acetazolamide decrease flow through specific inhibition of CA. In the case of furosemide (and its congener, bumetanide) 3 mechanisms for decreasing flow must be considered: (1) decrease due to loss of body fluid through diuresis; (2) inhibition of CA; and (3) inhibition of a primary transport system for CI-. The first is removed from the present study by nephrectomy, The second is mandated by the structural feature, R-SO~NH~ (Fig. 2), which is specific for inhibition of CA (ref. 21, p. 630). The third is brought to be the fore by data suggesting that a C1- transporting system may exist in choroid plexus 14 and the facts that furosemide inhibits CI- exchange in red cells 5, furosemide decreases
N--N
°
H2NSO2
S
ii NHC CH3
CI
~
NHCH2/~-~
HzNSOz ~ ' / ~ C O O H
H2~N
Acetazotamide
Furosemide
NH CHzCHzCH2CH~
0 0 ~
COOH
Bumefanide
Fig. 2. Structures of the three drugs used in this study. The structure R-SO~N/-I~ is the necessary
feature for CA inhibition.
179
the electrogenic transport of Cl- across frog isolated cornea8, and the diuretic effects of furosemide and bumetanide are related to inhibition of Cl- transport in the ascending limb of the nephron 7.18 . Deciding whether observed effects of furosemide could be due to CA inhibition rests heavily upon the Iso or Kt values. Published values of Iso or Ki for inhibition of CA in vitro by furosemide range from 3 X 10-3 M to 2.4 X 10-7 M19.29.37. Our own determinations, using several sources of CA, averaged to 3.0 X 10-7 M (Table III). These wide discrepancies in Kt values most probably are related to incubation times (see footnote to Table III). McCarthy and Reed 19 reported maximal CSF flow reduction ('" 50 %decrease in flow) in non-nephrectomized rabbits for furosemide at 50-100 mg/kg, a result quite different from our results in nephrectomized rabbits. For these experiments they measured fractional inhibition in vitro just as we did, with the exception that they apparently did not allow incubation time necessary for full interaction of furosemide with CA. Their values are, therefore, low. (In our experiments the CA inhibitory activity of furosemide increased IS-fold with 2 min preexposure of drug, enzyme and C02, 40-fold with 5 min pre-exposure, and 50-fold with 10 min. The 5-min time was selected for standard assay.) Because their values for fractional inhibition by furosemide in vitro are low, their Kt is low and their calculated adjustment of fractional inhibition to the in vivo condition is also low. We do not know why McCarthy and Reed 19 found maximal decrease in CSF flow after furosemide, but it may be the result of a profound diuretic effect on circulating fluid volume. They assumed their maximal reduction in flow (equivalent to acetazolamide effect) to be due entirely to a direct physiological effect on the CSF secretory mechanisms. The combination of underestimating the inhibition and overestimating the direct effect on choroid plexus led them to conclude that CA was not involved in the action of furosemide. In the case of acetazolamide, for which the reaction between drug and enzyme in vitro is much speedier, the value for KI given by McCarthy and Reed 19 agrees with the widely accepted Kt of 1-2 x 10-8 M26, and their finding that the full effect on CSF flow by acetazolamide is seen only at 99.95 % inhibition (or more) in vivo is reasonable. Such an effect occurred with 15 mg/kg of acetazolamide 19 . When it is appreciated that furosemide is 17--40 times less active than acetazolamide (Table III), it is not surprising to get a partial effect at 50 mg/kg (Fig. 1). We have shown that inhibition of cat and rabbit choroid plexus CA is analagous to that of dog red cells or purified human red cell CA-C, all of which are in the family of 'fast' CA (Table III and ref. 35). Based on our Kt values from Table III, if 15 mg/kg acetazolamide is the lowest dose for maximal CSF flow inhibition in rabbits 19, then 17--40 times this or 250--600 mg/kg furosemide would be the theoretical dose to given maximal decrease in flow by the CA mechanism, and a partial effect is all that could be expected from 50 mg/kg. Using the Kt for furosemide as 3 X 10-7 M and taking the maximum concentration offree furosemide in choroid plexus to be the same as plasma unbound furosemide, we calculated fractional inhibition (i) in vivo for inhibitor (I) as: 1=
Itree I'rce
+ Kt
(ref. 20)
180 and found that even if equilibrium between plasma and plexus free drug had existed 10 rain after dose, inhibition of CA would not have been sufficient to give maximal flow reduction (Table IV). However. all the calculated fractional inhibition values during 120 min after drug (Table IV) are consistent with a partial effect on CSF formation (rather than no effect as assumed by McCarthy and Reed19). Our previous studies with acetazolamide 35 suggest that doses of 1-3 mg/kg acetazolamide give the same mini maI flow decrease seen here after 50 mg/kg furosemide. Following this line, bumetanide, which is < 1 ~o as active as acetazolamide, should show no effect at 50 mg/kg. In the nephron, furosemide and bumetanide inhibit C1- transport from lumen to blood 7,18. Secretion of furosemidel~,15, ~7 and of bumetanide 3° helps to concentrate these drugs at the C1- transporting site. l f a transport system for CI-- exists for choroid plexus elaboration of CSF. with characteristics similar to the nephron, then furosemide might decrease CSF flow by acting from the blood side to decrease C1 transport into CSF when the plasma concentration of free furosemide is similar to that which inhibits in the nephron. To examine this possibility, we consider the data of Burg et al. 7 and lmai t8 in which luminal furosemide at 10-4-10 -6 M had a marked effect on transepithetial potential through inhibition of CI- transport from the lumen in the thick ascending limb of isolated rabbit nephron. From Cutler et al. ~t it may be calculated from renal clearance and serum data that, after the 1 mg/kg oral dose which causes maximal diuresis, final urine contains 30-220 ~. 10-~ M furosemide during the first 2 h. This final urine concentration would, of course, be greater than at the C1- site. In our data. free plasma furosemide was 22-81 × 10--6 M over 2 h. We might expect, therefore, a large decrease m CSF flow if this could be achieved through inhibition of C1transport. This was not the case. Most convincing is the finding that bumetanide, a drug with 40 times more dose potencyZ,10, a0 for diuresis and 14 times more concentration potency at the CI- site in the tubule t8 than furosemide, gave no effect on CSF flow. It appears that inhibition of flow by furosemide is not via direct Ct- inhibition. The half-time for furosemide in the nephrectomized rabbits of Table 1V averages 30-40 rain over the first hour and 130 min over the second hour: Cutler et al.21 give the half-time in anephric man as 80.7 rain. These rates must be presumed to be rates o f loss by metabolism or biliary excretion. In dog both furosemide 1° and bumetanide 1°,3° are excreted unchanged. The plasma binding data we have for furosemide in rabbit (92~o) may be compared with data for other species: dog 83-9I ~10,15,30, and man 95 o/~H Acetazolamide is 90-95 o; bound in man, 75-85 o//,, bound in rabbit (ref. 21, p. 642). F r o m the data of Wistrand z8 for 20 and 40 mg/kg acetazolamide in rabbit, it can be estimated that 30 mg/kg i.v. gives ,-~ 50 # M free acetazolamide at zero time. Because this drug is normally excreted by kidney 25 ,38 unmetabolized and the kidneys of our rabbits were ligated, a level near 50 ~M should have been present throughout. K~ t.5 ~ t0 ~8 M, for acetazolamide in choroid plexus (Table III), and 50 # M free acetazolamide yield i -~ 99.97 ~o. This means only 3 parts CA are not bound to drug in 10,000 parts compared to 37-I 35 parts not drug-bound in the calculation ofi for furosemide during 2 h after dose.
181
The steepness of the decline in CSF flow with time exceeded that in cats 35 or monkeys27. At present we can offer no explanation for this decline. McCarthy and Reed 19 , who also anesthetized their rabbits with sodium pentobarbital, have reported reasonably stable CSF flows in perfused rabbits over several hours, but data showing flow with time were not presented. Of the variables which Martins et al. (who first reported falling CSF flow-rate during perfusion)27 regard as possible contributors, we have controlled intracranial pressure and body temperature. Also pC02 was presumably held within physiological limits by the respiratory adjustments made to regulate arterial pH; however, compensatory respiratory alkalosis may have been present. Blood pressure was inclined to be low throughout our experiments. This, if anything, would have the effect of lowering the entire curve describing CSF f1ow 9. But, since blood pressure was lowest in the bumetanide experiments, in which flow was unchanged from control, blood pressure does not appear to be a primary factor. By pretreating the animals in these experiments rather than having each serve as its own control, we have avoided problems of comparing pre- and post-drug flow-rates that differ both due to unavoidable slope and by drug action. Our statistical treatment has shown that the effect of time is about the same for all the groups and that procedures to eliminate the effect of time on the results did not generate conclusions unlike those taken from raw data. Our results differ from previous findings showing either a slight increase or maximal decrease in CSF flow by furosemide1 4 ,19,28,32,33. It is our conclusion that furosemide has a very weak direct effect to decrease CSF flow, and that effect is mediated by CA inhibition. ACKNOWLEDGEMENTS
We gratefully acknowledge the guidance and critical review of Dr. T. H. Maren; advice and personal assistance with analysis of the data given by Dr. Max R. Langham, of University of Florida Institute of Food and Agricultural Sciences; the technical assistance of Mr. David R. Godman; and drugs supplied by manufacturers for this study (see Methods). This study was supported by NIH Grant EY 02227.
REFERENCES 1 Abbott, J., Davson, H., Glen, I. and Grant, N., Chloride transport and potential across the blood-CSF barrier, Brain Research, 29 (1971) 185-193. 2 Ashbury, M. J., Gatenby, P. B. B., O'Sullivan, S. and Bourke, E., Bumetanide: potent new 'loop' diuretic, Brit. Med. J., 1 (1972) 211-213. 3 Barr, A., Goodnight, J., Sail, J. and Helwig, J., A User's Guide to SAS '76, SAS Institute, Raleigh 1976. 4 Bourke, R. S., Gabelnick, H. L. and Young,O., Mediated transport of chloride from blood into cerebrospinal fluid, Exp. Brain Res., 10 (1970) 17-38. 5 Brazy, P. C. and Gunn, R. B., Furosemide inhibition of chloride transport in human red blood cells, J. gen. Physiol., 68 (1976) 583-599. 6 Buhrley, L. E. and Reed, D. J., The effect of furosemide on sodium-22 uptake into cerebrospinal fluid and brain, Exp. Brain Res., 14 (1972) 503-510.
182 7 Burg, M., Stoner, L., Cardinal, J. and Green, N., Furosemide effect on isolated perfused tubules, Amer. J. Physiol., 225 (1973) 119-124. 8 Candia, O. A., Short-circuit current related to active transport of chloride in frog cornea: effects of furosemide and ethaerynic acid, Biochim. biophys. Acta (Arnst.), 298 (1973) 1011-1014. 9 Carey, M. E. and Vela, A. R., Effect of systemic arterial hypotension on the rate ofcerebrospinal fluid formation in dogs, J. Neurosurg., 41 (1974) 350-355. 10 Cohen, M. R., Hinsch, E., Vergona, R., Ryan, J., Kolis, S. J. and Schwartz, M. A., A comparauve diuretic and tissue distribution study of bumetanide and furosemide in the dog, J. PharmacoL exp. Ther., 197 (1976) 697-702. 11 Cutler, R. E., Forrey, A. W., Christopher, T. G. and Kimpel, B. M.. Pharmacokinetics of furosemide in normal subjects and functionally anephric patients, Clin. PharmacoL Ther.. 15 (1974) 588-596. 12 Davson, H., Physiology of the Cerebrospinal Fluid, Little-Brown, Boston, 1967. 13 Davson, H. and Pollay, M., Influence of various drugs on the transport of ~31I and PAH across the cerebrospinal fluid-blood barrier., J. Physiol. (Lond.), 167 (1963) 239-246. 14 Domer, F. R., Effects of diuretics on cerebrospinal fluid formation and potassium movement, Exp. Neurol., 24 (1969) 54-64. 15 Gayer, J., Die Ausscheidung von Furosemid durch die Niere des Hundes. Verh. deutseh Ges. inn Med., 70 (1964) 702-704. 16 Heisey, S. R., Held, D. and Pappenheimer, J. R., Bulk flow and diffusion in the cerebrospinal fluid system of the goat, Amer. J. Physiol.. 203 (1962) 775-781. 17 Hook, J. B. and Williamson, H. E,, Influence of probenecid and alterations in acid-base balance on the saluretic activity of furosemide, J. Pharmaeol. exp. Ther., 149 (1965) 404-408. 18 Imai. M., Effect of bumetanide a n d furosemide on the thick ascending limb of Henie's loop of rabbits and rats perfused in vitro, Europ. J. Pharmacol., 41 (1977) 409-416. 19 McCarthy, K. D. and Reed, D. J., The effect of acetazolamide and furosemide oil cerebrospinat fluid production and choroid plexus carbonic anhydrase activity, J. Pharmacol. exp. Ther., 189 (1974) 194-201. 20 Maren, T. H., The relation between enzyme inhibition and physiological response ~rt the carbonic anhydrase system, J. Pharmacol. exp. Ther.. 139 (1963) 14ff-153. 21 Maren T. H., Carbonic anhydrase: chemistry, physiology, inhibition, Physiol. Rev.. 47 (1967) 595-781. 22 Maren T. H., Bicarbonate formation in cerebrospinal fluid: role in sodium transport and pH regulation, Amer. J. Physiol., 222 (1972) 885-899. 23 Maren T . H . , Effect of varying CO2equilibriaonratesofHCO3-formationincerebrospinalfluid, J. appl. Physiol. : Respirat. Environ. Exercise Physiol., 47 (1979) 47t-477. 24 Maren T. H., Ash, V. I. and Bailey, E. M,, Jr.. A method for determination of carbonic anhydrase inhibitors, particularly of Diamox®, Bull. Johns Hopk. Hosp., 95 11954) 244-255. 25 Maren T. H., Mayer, E. and Wadsworth, B. C., The pharmacology of Diamox®, 2-acetylamino1,3.4-thiadiazote-5-sulfonamide, Bull. Johns Hopk. Hosp., 95 (1954) 199-243. 26 Maren, T. H., Parcell, A. L. and Malik, M.N., A kinetic analysis of carbonic anhydrase inhibition. J. Pharmacol. exp. Ther., 130 (1960~ 389-400, 27 Martins, A. N., Newby, N. and Doyle, T. F.. Sources of error in measuring cerebrospinal fluid formation by ventriculocisternaI perfusion, J. Neurol. Neurosurg. Psychiat., 40 (1977) 645-650. 28 Melby, J. M. and Reed, D. J., Effect of acetazolamide and furosemide on CSF production by cat choroid plexus in situ, Abstract in Proceedings for Society for Neuroscience. 8th Ann. Meet., 1978. p. 246. 29 Muscbaweck, R. and Hajdu, P., Die Salidiuretische Wirksamkeit der Chlor-N-(2 furylmethylF5sulfamylanthranilsaure, Arzneim. Forsch., 14 (1964) 44-47. 30 Ostergaard, E. H., Magnussen, M. P.. Nielsen, C. K., Eilertsen, E. and Frey, lq. H.. Pharmacological properties of 3-n-butylamino-4-phenoxy-5-sulfamylbenzote acid (bumetanide), a new potent diuretic, Arzneirn. Forsch., 22 (1972) 66-72. 31 Rapoport, S. I., The Blood-Brain Barrier in Physiology and Medicine, Raven Press, New York. 1976, pp. 85-86. 32 Reed, D. J., The effect of furosemide on cerebrospinal fluid flow in rabbits. Arch. htt. Pharmaeodyn., 178 (1969) 324-330. 33 Sahar, A. and Tsipstein, E., Effects of mannitol and furosemide on the rate of formation of cerebrospinal fluid, Exp. Neurol., 60 (1978) 584-591.
183 34 Segal, M, B. and Pollay, M., The secretion of cerebrospinal fluid. In L. Z. Bito, H. Davson and J. D. Fenstermacher (Eds.), The Ocular and Cerebrospinal Fluids, Proceedings of a Fogarty International Symposium, Exp. Eye Res. (Suppl,) 25 (1977) 127-148. 35 Vogh, B. P., The relation of ehoroid plexus carbonic anhydrase activity to cerebrospinal fluid formation: study of three inhibitors in cat, with extrapolation to man, J. PharmacoL exp. Ther., 213 (1980) 321-331. 36 Vogh, B. P. and Maren, T. H., Sodium, chloride, and bicarbonate movement fi'om plasma to cerebrospinal fluid in cats, Amer. J. Physiol,, 228 (1975) 673 683. 37 Wilson, C. B., Stein, J. H. and Kirkendall, W. M., Differences in the effect of ethacrynic acid and furosemide on urinary acidification in man (abstract), Clin. Res., 15 (1967) 374. 38 Wistrand, P. J., The effect of carbonic anhydrase inhibitor on intra-ocular pressure with observations on the pharmacology of acetazolamide in the rabbit, Acta Pharmacol. Toxicol., 16 (1959) 171-193.
171
THE EFFECT OF FUROSEMIDE AND BUMETANIDE ON CEREBROSPINAL FLUID FORMATION
BETTY P. VOGH and MAX R. LANGHAM, Jr. University of Florida College of Medicine, Department of Pharmacology and Therapeutics, Gainesville, Fla. 32610 (U.S.A.) (Accepted December 11th, 1980) Key words: cerebrospinal fluid - CSF formation - furosemide - bumetanide - acetazolamidecarbonic anhydrase - choroid plexus
SUMMARY
Complete inhibition of carbonic anhydrase maximally reduces CSF flow 40-60 %34. We have confirmed that CSF flow in rabbits may be maximally decreased in this way by acetazolamide 30 mg/kg i.v., but have found only about 20 %reduction from control after furosemide at 50 mg/kg i.v. This effect of furosemide is consistent with specific but partial inhibition of carbonic anhydrase of choroid plexus (and perhaps other sites of CSF secretion) based on the affinity of furosemide for carbonic anhydrase. Bumetanide, with 7 times lower affinity for carbonic anhydrase and 14-40 times higher inhibition of renal electrolyte transport processes than furosemide 2 ,18,3o, did not decrease CSF flow.
INTRODUCTION
The physiology of the cerebrospinal fluid (CSF) secretion has been a subject of interest to investigators for many years. The effects of several drugs on CSF production, CSF composition, and rate constants for isotopic Na+ movement into CSF have been studied to elucidate basic mechanisms of the system and to find rational methods for treating hydrocephalus and other conditions involving high CSF pressure 12 ,23,34. Earlier reports of the effect of furosemide upon CSF production or influx of 22Na i to CSF are inconsistent but mostly indicate CSF reduction 6 ,14,19,28,32,33. Questions remained whether the effects seen were due to volume depletion through diuresis, to direct interference with CI- transport at the choroid plexus (as in the kidney7) or to inhibition of carbonic anhydrase (CA) at sites of CSF formation. Furosemide is a weak inhibitor of CA (see ref. 19 and data below).
172 In this study, in which volume depletion is ruled out by preventing renal action of the drugs studied, we show that the measured mean effect of furosemide on CSF flow is small and cannot be differentiated from control. The lack of effect of bumetanide, a congener of furosemide 40 times more potent as a diuretic in man 2 and animals 30 than furosemide, and 14 times more potent as an inhibitor of renal C 1 transport in isolated renal tubules of rabbit than furosemide 18, strongly supports the likelihood that any action of furosemide at sites of CSF formation is by CA inhibition.
MATERIALS AND METHODS New Zealand white rabbits weighing 1,9-3.0 kg (2.3 ± 0.1) of either sex were anesthetized by injecting isotonic sodium pentobarbital into an ear vein. Anesthesia to depress the corneal reflex was maintained with supplemental i.v. doses. After tracheotomy, a cannula was inserted for maintenance on a Harvard respirator, using room air enriched to 3 0 - 4 0 ~ oxygen, and expired CO2 was monitored continuously. A femoral artery was cannulated for arterial pH measurements (Instrumentation Laboratories, Model 113). Respiration was adjusted to maintain arterial pH at 7.35-7.45. In some animals NaHCO3 was given i.v. to assist in pH control. Arterial blood pressure was also monitored continuously. From a :flank approach, the renal arteries, veins and ureters were ligated. Body temperature was kept at 38 ~ 1 °C using a heating pad responsive, by relay switches, to changes in the deep colonic temperature of the animals. For perfusion of the ventricles, artificial CSF was prepared ~6. This buffer solution, dye-labeled with 0.5 mg/ml Blue Dextran 2000 (Sigma or Pharmacia), was gassed with 5 ~ CO2 to pH 7.4 at room temperature before filling the perfusion system. Rabbits were placed in a sphinx-like position, their heads held rigidly. An 18-gauge spinal needle filled with artificial CSF and connected to a Harvard infusion pump was then placed stereotactically into the right lateral ventricle. CSF pressure was continuously recorded by means of a Statham transducer in line with the infusion tubing. Pressures recorded as the needle penetrated a lateral ventricle were between 0 and 10 cmH20 with respect to the auditory meatus. A 19-gauge hubless needle was manually placed into the cisterna magna after incising the skin and spreading the muscles of the neck. True CSF was collected at - - 1 0 cmH20 for 5 min. This was done to facilitate complete replacement of CSF by the artificial CSF containing the dye. To further wash out residual CSF, the ventricular system was then perfused for 5 min at 200 #l/min, then 5 min at 100/~l/min, and finally 15 min at 54 #l/min before starting to collect the perfusate for analysis. The steady rate of perfusion for the experiment was 54 #l/min, and the perfusate was collected at a pressure which did not vary by more than :!: 2 c m H 2 0 from the measured physiological pressure. Each sample of perfusate was collected over a period of 30 min. Samples were spun in a clinical centrifuge for 20 min at maximum speed to remove any possible material which might interfere with light transmittance. The measure of concentration of Blue Dextran was its optical density at 640 rim.
173 Formation of new CSF was calculated for each sample according to Heisey et al. l6 as: optical density of inflow - optical density of outflOW) (rate of perfusion) ( . optical density of outflow In the main study, the rabbits served as control animals or were treated, 30-40 min before collecting the first sample of perfusate, with furosemide (50 mg/kg) or acetazolamide (30 mg/kg) i.v. Furosemide, supplied by Hoescht-Roussel Pharmaceuticals, was freshly dissolved for each experiment in 0.1 M phosphate buffer, pH 7.4, at 20 mg/ml, by heating to 60 DC. Acetazolamide, supplied by American Cyanamid, was prepared as the sodium salt, at 50 mg/ml, pH 8.9. When acetazolamide was given, an additional volume of pH 7.4 phosphate buffer was administered equivalent to the volume of the furosemide dose. Furosemide dose was followed by pH 8.9 buffer equivalent to the dose of acetazolamide. Controls were given phosphate buffer equivalent in volume and pH to the furosemide and acetazolamide doses. In a group of 3 rabbits, bumetanide, supplied by Hoffman LaRoche, was given i.p., 50 mg/kg, prepared as 30 mg/ml in 95 % ethanol. This route and solvent were chosen due to the low solubility of bumetanide in aqueous buffer. The treatment of these rabbits was identical in other respects to that of the other groups, except that 30-50 min extra were allowed for drug absorption. For inclusion of an experiment in the group for statistical analysis, the animal was required to survive 2.5 h of perfusion with mean arterial blood pressure above 50 mmHg at all times. Most in the main groups were in the range 70-90 mmHg, but the animals treated with bumetanide averaged 53 mmHg. CSF data were discarded for any animal for which autopsy failed to confirm successful bilateral occlusion of the renal pedicle. In 6 rabbits given furosemide and sUbjected to the above procedures, blood samples were drawn at 10, 30, 60 and 120 min after injection of drug for determination of furosemide in plasma. Plasma, diluted I: 10, was prepared for assay by heating to 100 DC for 5 min to denature CA in any red cell contents which might be present. (Such treatment did not destroy furosemide in standards treated identically.) The assay for furosemide levels was based on inhibition of dog red cell CA24 after the drug sample was incubated with CA and CO 2 5 min. Plasma drawn prior to drug treatment was subjected to equilibrium dialysis to determine the per cent binding of furosemide to plasma protein. Two ml of plasma from each of two rabbits was dialyzed in 500 ml of 25 pM furosemide overnight during refrigeration. Aliquots of plasma and dialysate were then analyzed for furosemide concentration. The dissociation constants (K,) or drug concentrations giving 50 %inhibition of CA (ho) were determined 26 for furosemide, bumetanide and acetazolamide, using a range of drug concentrations with rabbit choroid plexus CA, pure human CA-C (isozyme C, CA II) and dog red cell preparations. The data for CSF production were submitted to multiple regression analysis as detailed in Results. Computations were made at the Northeast Regional Data Center, Gainesville, Fla. using the Statistical Analysis System (SAS)3.
t74 RESULTS I n Fig. 1, after washing o u t o f residual C S F , successive 30-min samples of perfused artificial C S F c o n t a i n i n g the dilution m a r k e r are c o m p a r e d for effect of t r e a t m e n t o n C S F rate o f p r o d u c t i o n . I t can be readily seen t h a t the m e a n rate for the b u m e t a n i d e g r o u p is like t h a t o f the controls. R a t e after f u r o s e m i d e is also like control, with the suggestion t h a t g r o u p s larger t h a n 6 or 7 a n i m a l s m i g h t verify the t e n d e n c y for f u r o s e m i d e to reduce flow slightly. There is a consistent p a t t e r n o f l o w e r m e a n value, 9 - 2 0 % lower for furosemide. A c e t a z o l a m i d e clearly differs f r o m controls. r a n g i n g 41-78 % less over 5 s a m p l e periods. Because the plots for individual animals, t r e a t e d a n d untreated, showed a significant decline in C S F flow with time (as previously observed, see Discussion) and because the times f r o m d r u g a d m i n i s t r a t i o n to start o f s a m p l e I v a r i e d somewhat. linear m o d e l s were used to fit the d a t a to e x a m i n e the effect o f time a n d the effect o f d r u g t r e a t m e n t a p a r t f r o m time.
Dependence of CSF production upon perfusion time A general linear m o d e l o f the form, Piit = fi0
/31t × fi2t2 +
Y, 6i~z Ri -- e r r o r : j -~ 1. . . . . 4.
(Eqn. 1)
i
was used to fit the data, to e x a m i n e the effect o f time. In this f o r m u l a t i o n PHt =: C S F p r o d u c t i o n in # l / m i n at time t for the iTM r a b b i t receiving the jth t r e a t m e n t : N0 intercept for a reference r a b b i t ; t ~ min after starting ventriculocisternal perfusion; t2 = t, squared;/31,/32 ~ coefficients o f the m o d e l variables t a n d t 2. related to slope; Ri = a variable used to eliminate the effect o f inherent differences in flow a m o n g r a b b i t s : i,e. Ri = 1 for the ith r a b b i t a n d Ri ~= zero for all others: ~i~ 2 :=- coefficient for Rl to
4
z C
3
~ ~o 5 g s £)
-
u©-
A(5)
_
_
4
a.l
0
- -
2
'
~
z-
5
S A M P L E NUMBER
Fig. 1. Rates of CSF formation during ventriculocisternal perfusion in nephrectomized rabbits are shown as mean and S.E.M. for successive 30 rain periods following drugtreatment and the washing out of residual CSF. Control (C); burnetanide, 50 mg/kg (B); furosemide, 50 mg/kg (F): and acetazolamide. 30 mg/kg (A). See text for discussion of decline in flow in control experiments. It is clear that the effects of furosemide or bumetanide 50 mg/kg do not approximate that of acetazolamide 30 mg/kg.
175 TABLE l Effect of time on formation of CSF* The values are given with S.E.M.
/31 (pl/min e) [;2(/~l/min3)
Control (n 7)
Acetazolamide Furosemide Bumetanide Pooled data (30mg/kgi.v.) (50mg/kgi.v.) (50mg/kgi.p.) (n 19) (n 3) (n -- 6) (n - 3)
- - 0.0500 ± 0.0216 0.0001 ± 0.0001
--0.0804 ± 0.0418 0.0001 ± 0.0002
--0.0841 ± 0.0170 0.0002 :~ 0.0001
--0.1153 ± 0.0406 0.0004 ± 0.0001
- - 0.0680 i- 0.0126 0.0001 ± 0.00005
* See Eqn. 1 in text. The fact that fll values are negative shows that CSF flow falls with time; positive values for/32indicate a slower rate of decline as time passes. take into a c c o u n t the shift in intercept for the i t~ r a b b i t f r o m fl0; a n d e r r o r = error for the i th r a b b i t at t. A q u a d r a t i c with respect to time was used because it a p p e a r e d t h a t p r o d u c t i o n decreased at a decreasing rate with time. The d u m m y variable, Ri, allowed e s t i m a t i o n o f the effect o f time a p a r t from the average level o f p r o d u c t i o n o f individual rabbits. T h e resulting coefficients for the time variables are given in Table I. F o r the c o n t r o l group, /31 is - - 0 . 0 5 # l / m i n each min a n d f12 is + 0 2 0 0 1 #l/rain 2 each rain. The fie coefficient shows t h a t the rate o f decrease diminishes with time. A n F-test indicated t h a t the effect o f time on furosemide, acetazolamide, b u m e t a n i d e and c o n t r o l groups did n o t differ. Therefore, the d a t a for all d r u g treatments a n d controls were p o o l e d to estimate a general coefficient o f decline with time, n o t due to d r u g (Table I). The p r i m a r y coefficient from the p o o l e d d a t a is - - 0 . 0 6 8 0 / ~ l / m i n per minute o f perfusion time or - - 4 . 0 8 / ~ l / m i n / h . This slope differs f r o m zero at P <~ 0.001. Dependence o f C S F production upon drug treatment apart f r o m decline due to time T h e effects o f the d r u g treatments a p a r t f r o m the effect o f time were then examined. A general linear m o d e l o f the form, Pijt ~ fl0 + tilt .t- fl2t ~ +
Z flj+2Tj + error,
(Eqn. 2)
J
was used to fit the data. I n this f o r m u l a t i o n : Pair, ill, f12, t, t 2 a n d error are defined as in Eqn. 1 ; t30 ~ intercept for the c o n t r o l g r o u p ; Tj = a variable used to g r o u p rabbits a c c o r d i n g to treatment, i.e. Tj - - 1 for rabbits in g r o u p being examined, a n d Tj = zero for all others; a n d f l j , 2 ~ coefficient o f Tj to t a k e into a c c o u n t the shift in intercept for the jth g r o u p from/30. The e r r o r was specified to follow a s e c o n d - o r d e r auto-regressive scheme to allow for the systematic deviation o f a p a r t i c u l a r r a b b i t * * . ** The AUTOREG procedure of SAS was used ~. This procedure first estimates the model with ordinary least-squares, then estimates the first- and second-order autocorrelation inherent among all points for a particular rabbit from the ordinary least-squares residuals. These estimates of autocorrelation are used to transform the original data to correct for this. Coefficients for the terms of the model are then regenerated from the transformed data.
176 TABLE II Estimated CSF produetion based on Eqn. 2 applied at mean time o f all observations* Drug group and dose
CSF production ( itl min)
Change Ji'om control ~ % decrease)
Control Furosemide (50 mg/kg i.v.) Acetazolamide (30 mg/kg i.v.) Bumetanide (50 mg/kg i.p.)
9.4 7.2** 4. I *** 8.4§
0 23 56 l1
* Time when 9 5 ~ confidence interval is closest to mean, i.e. at ~ 120 rain of perfusion (most samples were collected between 30 and 210 rain after starting perfusion). ** Different from control at P --~ 0.05. *** Different from control at P < 0.005. Different from furosemide at P - 0.01. § Not statistically different from control. The model was fitted for furosemide, acetazolamide, b u m e t a n i d e a n d control groups. U s i n g the coefficients for the model, curves giving m e a n C S F p r o d u c t i o n values for each t r e a t m e n t were generated along with 95 ~ confidence intervals. C S F flow-rates at the m e a n observation time, where the 9 5 ~ confidence interval is narrowest, are shown in T a b l e II. The m e a n c o n t r o l rate is 9.4 #l/rain, a n d the residual m e a n flows after furosemide, acetazotamide a n d b u m e t a n i d e treatments represent 23, 56 a n d 11 ~ decreases f r o m the m e a n c o n t r o l rate. U s i n g this approach, the effects of furosemide a n d acetazolamide (but not b u m e t a n i d e ) differed from control, a n d furosemide differed from acetazolamide (Table II). These results agree with those of Fig. 1 except t h a t furosemide is n o w shown to differ from control. The c o n c e n t r a t i o n of C A in homogenized r a b b i t choroid plexus (corrected for CA in blood), f o u n d to be 5.4 :~: 1.4 # M (S.E.M.) (n -----3) in a recent study ~5. was confirmed by new d e t e r m i n a t i o n s giving 5.1, 5.6 a n d 7.6 # M . The ability of furosemide, acetazolamide and b e n z o l a m i d e to i n h i b i t rabbit TABLE 1II Ability o f druga' used in rabbits to inhibit carbonic anhydrase in vitro (15o or Ki)
I50 is the concentration of inhibitor necessary to reduce enzyme activity to 50 ~ Of original activity in the assay. K, is the dissociation constant for enzyme-inhibitorcomplex. !5o ~ K~in these assays where enzyme concentration is kept low, < 5 x 10-9 M (March et al.2n), in every case drug was incubated with CA during constant COZ gas flow for 2 or 5 rain as shown in parentheses in the table:Such in, cubation enhances the inhibitionof dog red ceil CA by these drugs and does not destroy enzyme activity in the uninhibited control. To reach maximal inhibition, 5 min are required for furosernide and 2 min for acetazolamide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -7
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
Source o f CA
Furosemide
Aeetazolamide
Bumetahide
Rabbit choroid plexus Dog red Cell CA Human red cell, isozyme CA-C
2.5 × 10 7 M (5rain) 2.9 x IO 7 M(5min)
1.5 ~ 10 ~ M(5min) 1 × 10 8 M(2min)
---
4.0 × 10:v M(2min)
1 × 10-SM(2min)
2.7 :~ 10 6 M(2min)
177 TABLE IV Concentration (pM) offurosemide in plasma, (after 50 mg/kg i.v. dose, mean and S.E.M.for 6 rabbits) and fractional inhibition of carbonic anhydrase
Total drug was determined by CA inhibitory activity compared to that of a set of furosemide standards, by the method of Maren et a1. 26 , using 5 min incubation of dog red cell CA with drug and C02 substrate before addition of bicarbonate bufferfor assay of pH change. The value of total drug for 90 min is taken from a plot of the other measured values. Binding of furosemide to plasma protein (determined by equilibrium dialysis of plasma taken prior to drug treatment) in 2 rabbits was 91 and 92.4 %for 25 pM furosemide in the dialyzing bath. The fractional inhibition was calculated as Itree/(Irree + Kl) (ref. 20); Itree is the unbound furosemide in plasma, taken to be the same as unbound furosemide in the secretory tissue. Minimum value for maximal inhibition of CSF flow appears to be 0.9995. Sample time 10 min
Total drug Estimated unbound drug Fractional inhibition of choroid plexus CA
30 min
60 min
90 min
120 min
1017 ± 158 619 ± 120 81 49
376 ± 99 30
310 25
281 ± 91 22
0.9963
0.9900
0.9881
0.9865
0.9939
choroid plexus CA and other CA sources is shown in Table III. Because the enzyme concentration is low in these assays « 5 X 10-9 M), the Iso approximately equals K, the dissociation constant for the inhibitor 26 • The general conclusion from Table III is that the inhibitory potency (affinity for CA) for acetazolamide is 17-40 times greater than for furosemide, and that of furosemide is 7 times greater than for bumetanide. Binding offurosemide to plasma protein averaged 92 %. Physiological inhibition of tissue CA depends upon the unbound (free) level of inhibitor in plasma and the access of this free drug to the enzyme site. Mean levels of total furosemide measured in plasma of kidney-ligated rabbits given 50 mg/kg are in Table IV, along with the estimated free drug concentrations and the degree of inhibition of CA obtained when free drug in plasma is in equilibrium with free drug in secretory tissues, as expected here. This dose gave a theoretical concentration at zero time of 1400 flM and a half-life of 30-40 min over the first hour; loss is much slower thereafter. The plasma drug concentration at 90 min probably forecasts secretory function at about 120 min (near the mean observation time in the regression analysis). At 90 min, total furosemide was 310 flM, from a plot of the data in Table IV; free furosemide was 25 flM, down from 81 flM at 10 min. DISCUSSION
Control flow-rates for our rabbits were 12.3 fll/min in the first 30 min sample, 11.1 in the second sample (in Fig. I) and 9.4 fll/min at the mean observation time in the regression (Table II), in good agreement with CSF flow for rabbits given by Davson and Pollay13.
178 Choroid plexus is responsible for a large fraction of CSF production (reviewed by Segal and Pollay 34 and by Rapoport3t). CA of plexus, by catalyzing the formation of HCO3 from CO2 and hydroxyl ion. serves the secretory function importantly22,a~,36. Inhibition of CA in choroid plexus to the level of at least 99.95 % was required to achieve maximal flow inhibition (40-60 % decrease) through the CA mechanism m cats aS, in which plexus CA averaged 22/~M. In rabbits with about one-third as much plexus CA as cat (see ref. 19 and data above), but with a higher fraction of acetazolamide bound to plasma protein (ref. 21, p. 642), maximal flow reduction also appeared to require 99.95 ~o inh ibition19. Such inhibition was provided by acetazolamide, 30 mg/kg i.v. in the cats a5 and 15-30 mg/kg i.v. in the rabbits tg. These animals were not nephrectomized. Acetazolamide at 30 mg/kg should, therefore, provide maximal flow reduction m the nephrectomized rabbits of this study. The data show that this is indeed the case: flow was reduced 41-78% from control rates in the data of Fig. 1. or 56% in the regression data of Table II. The 50 mg/kg i.v. dose of furosemide gave a 9--20 % flow reduction in Fig. ! and 23 % flow reduction in the regression in which the effect of time on flow was minimized. The 50 mg/kg dose was chosen because it (or less) has been reported to give maximal or near maximal CSF flow reduction in rabbits t9.32 and cats 33. It is widely accepted that CA inhibitors such as acetazolamide decrease flow through specific inhibition of CA. In the case of furosemide (and its congener, bumetanide) 3 mechanisms for decreasing flow must be considered: (1) decrease due to loss of body fluid through diuresis; (2) inhibition of CA; and (3) inhibition of a primary transport system for CI-. The first is removed from the present study by nephrectomy, The second is mandated by the structural feature, R-SO~NH~ (Fig. 2), which is specific for inhibition of CA (ref. 21, p. 630). The third is brought to be the fore by data suggesting that a C1- transporting system may exist in choroid plexus 14 and the facts that furosemide inhibits CI- exchange in red cells 5, furosemide decreases
N--N
°
H2NSO2
S
ii NHC CH3
CI
~
NHCH2/~-~
HzNSOz ~ ' / ~ C O O H
H2~N
Acetazotamide
Furosemide
NH CHzCHzCH2CH~
0 0 ~
COOH
Bumefanide
Fig. 2. Structures of the three drugs used in this study. The structure R-SO~N/-I~ is the necessary
feature for CA inhibition.
179
the electrogenic transport of Cl- across frog isolated cornea8, and the diuretic effects of furosemide and bumetanide are related to inhibition of Cl- transport in the ascending limb of the nephron 7.18 . Deciding whether observed effects of furosemide could be due to CA inhibition rests heavily upon the Iso or Kt values. Published values of Iso or Ki for inhibition of CA in vitro by furosemide range from 3 X 10-3 M to 2.4 X 10-7 M19.29.37. Our own determinations, using several sources of CA, averaged to 3.0 X 10-7 M (Table III). These wide discrepancies in Kt values most probably are related to incubation times (see footnote to Table III). McCarthy and Reed 19 reported maximal CSF flow reduction ('" 50 %decrease in flow) in non-nephrectomized rabbits for furosemide at 50-100 mg/kg, a result quite different from our results in nephrectomized rabbits. For these experiments they measured fractional inhibition in vitro just as we did, with the exception that they apparently did not allow incubation time necessary for full interaction of furosemide with CA. Their values are, therefore, low. (In our experiments the CA inhibitory activity of furosemide increased IS-fold with 2 min preexposure of drug, enzyme and C02, 40-fold with 5 min pre-exposure, and 50-fold with 10 min. The 5-min time was selected for standard assay.) Because their values for fractional inhibition by furosemide in vitro are low, their Kt is low and their calculated adjustment of fractional inhibition to the in vivo condition is also low. We do not know why McCarthy and Reed 19 found maximal decrease in CSF flow after furosemide, but it may be the result of a profound diuretic effect on circulating fluid volume. They assumed their maximal reduction in flow (equivalent to acetazolamide effect) to be due entirely to a direct physiological effect on the CSF secretory mechanisms. The combination of underestimating the inhibition and overestimating the direct effect on choroid plexus led them to conclude that CA was not involved in the action of furosemide. In the case of acetazolamide, for which the reaction between drug and enzyme in vitro is much speedier, the value for KI given by McCarthy and Reed 19 agrees with the widely accepted Kt of 1-2 x 10-8 M26, and their finding that the full effect on CSF flow by acetazolamide is seen only at 99.95 % inhibition (or more) in vivo is reasonable. Such an effect occurred with 15 mg/kg of acetazolamide 19 . When it is appreciated that furosemide is 17--40 times less active than acetazolamide (Table III), it is not surprising to get a partial effect at 50 mg/kg (Fig. 1). We have shown that inhibition of cat and rabbit choroid plexus CA is analagous to that of dog red cells or purified human red cell CA-C, all of which are in the family of 'fast' CA (Table III and ref. 35). Based on our Kt values from Table III, if 15 mg/kg acetazolamide is the lowest dose for maximal CSF flow inhibition in rabbits 19, then 17--40 times this or 250--600 mg/kg furosemide would be the theoretical dose to given maximal decrease in flow by the CA mechanism, and a partial effect is all that could be expected from 50 mg/kg. Using the Kt for furosemide as 3 X 10-7 M and taking the maximum concentration offree furosemide in choroid plexus to be the same as plasma unbound furosemide, we calculated fractional inhibition (i) in vivo for inhibitor (I) as: 1=
Itree I'rce
+ Kt
(ref. 20)
180 and found that even if equilibrium between plasma and plexus free drug had existed 10 rain after dose, inhibition of CA would not have been sufficient to give maximal flow reduction (Table IV). However. all the calculated fractional inhibition values during 120 min after drug (Table IV) are consistent with a partial effect on CSF formation (rather than no effect as assumed by McCarthy and Reed19). Our previous studies with acetazolamide 35 suggest that doses of 1-3 mg/kg acetazolamide give the same mini maI flow decrease seen here after 50 mg/kg furosemide. Following this line, bumetanide, which is < 1 ~o as active as acetazolamide, should show no effect at 50 mg/kg. In the nephron, furosemide and bumetanide inhibit C1- transport from lumen to blood 7,18. Secretion of furosemidel~,15, ~7 and of bumetanide 3° helps to concentrate these drugs at the C1- transporting site. l f a transport system for CI-- exists for choroid plexus elaboration of CSF. with characteristics similar to the nephron, then furosemide might decrease CSF flow by acting from the blood side to decrease C1 transport into CSF when the plasma concentration of free furosemide is similar to that which inhibits in the nephron. To examine this possibility, we consider the data of Burg et al. 7 and lmai t8 in which luminal furosemide at 10-4-10 -6 M had a marked effect on transepithetial potential through inhibition of CI- transport from the lumen in the thick ascending limb of isolated rabbit nephron. From Cutler et al. ~t it may be calculated from renal clearance and serum data that, after the 1 mg/kg oral dose which causes maximal diuresis, final urine contains 30-220 ~. 10-~ M furosemide during the first 2 h. This final urine concentration would, of course, be greater than at the C1- site. In our data. free plasma furosemide was 22-81 × 10--6 M over 2 h. We might expect, therefore, a large decrease m CSF flow if this could be achieved through inhibition of C1transport. This was not the case. Most convincing is the finding that bumetanide, a drug with 40 times more dose potencyZ,10, a0 for diuresis and 14 times more concentration potency at the CI- site in the tubule t8 than furosemide, gave no effect on CSF flow. It appears that inhibition of flow by furosemide is not via direct Ct- inhibition. The half-time for furosemide in the nephrectomized rabbits of Table 1V averages 30-40 rain over the first hour and 130 min over the second hour: Cutler et al.21 give the half-time in anephric man as 80.7 rain. These rates must be presumed to be rates o f loss by metabolism or biliary excretion. In dog both furosemide 1° and bumetanide 1°,3° are excreted unchanged. The plasma binding data we have for furosemide in rabbit (92~o) may be compared with data for other species: dog 83-9I ~10,15,30, and man 95 o/~H Acetazolamide is 90-95 o; bound in man, 75-85 o//,, bound in rabbit (ref. 21, p. 642). F r o m the data of Wistrand z8 for 20 and 40 mg/kg acetazolamide in rabbit, it can be estimated that 30 mg/kg i.v. gives ,-~ 50 # M free acetazolamide at zero time. Because this drug is normally excreted by kidney 25 ,38 unmetabolized and the kidneys of our rabbits were ligated, a level near 50 ~M should have been present throughout. K~ t.5 ~ t0 ~8 M, for acetazolamide in choroid plexus (Table III), and 50 # M free acetazolamide yield i -~ 99.97 ~o. This means only 3 parts CA are not bound to drug in 10,000 parts compared to 37-I 35 parts not drug-bound in the calculation ofi for furosemide during 2 h after dose.
181
The steepness of the decline in CSF flow with time exceeded that in cats 35 or monkeys27. At present we can offer no explanation for this decline. McCarthy and Reed 19 , who also anesthetized their rabbits with sodium pentobarbital, have reported reasonably stable CSF flows in perfused rabbits over several hours, but data showing flow with time were not presented. Of the variables which Martins et al. (who first reported falling CSF flow-rate during perfusion)27 regard as possible contributors, we have controlled intracranial pressure and body temperature. Also pC02 was presumably held within physiological limits by the respiratory adjustments made to regulate arterial pH; however, compensatory respiratory alkalosis may have been present. Blood pressure was inclined to be low throughout our experiments. This, if anything, would have the effect of lowering the entire curve describing CSF f1ow 9. But, since blood pressure was lowest in the bumetanide experiments, in which flow was unchanged from control, blood pressure does not appear to be a primary factor. By pretreating the animals in these experiments rather than having each serve as its own control, we have avoided problems of comparing pre- and post-drug flow-rates that differ both due to unavoidable slope and by drug action. Our statistical treatment has shown that the effect of time is about the same for all the groups and that procedures to eliminate the effect of time on the results did not generate conclusions unlike those taken from raw data. Our results differ from previous findings showing either a slight increase or maximal decrease in CSF flow by furosemide1 4 ,19,28,32,33. It is our conclusion that furosemide has a very weak direct effect to decrease CSF flow, and that effect is mediated by CA inhibition. ACKNOWLEDGEMENTS
We gratefully acknowledge the guidance and critical review of Dr. T. H. Maren; advice and personal assistance with analysis of the data given by Dr. Max R. Langham, of University of Florida Institute of Food and Agricultural Sciences; the technical assistance of Mr. David R. Godman; and drugs supplied by manufacturers for this study (see Methods). This study was supported by NIH Grant EY 02227.
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