Inhibition of cerebrospinal fluid formation by omeprazole

EXPERIMENTAL

NEUROLOGY

115,

3%%-399

(19%)

Inhibition of Cerebrospinal Fluid Formation by Omeprazole MARIALINDVALL-AXELSSON,*,T Departments

CHRISTER NILSSON,*

of *Medical Cell Research, Section of Neurobiology and *Department of Ped~tr~cs, Karoli~~ku institute,

Omeprazole, a specific inhibitor of Ht-K’-activated ATPase, gave a dose-dependent inhibition of CSF production as determined by cerebroventricul~isterual perfusions in the rabbit. The reduction was 35% when the perfusate concentration of omeprazole was lo-‘MT and 25% after an intravenous dose of 0.2 mgfkg of omeprazole, respectively. A similarly substituted benzimidazol (H178142) without II+-Kf-ATPase-inhibiting properties did not affect CSF production at a perfusate concentration of lo-’ M. Omeprazole in a concentration of 2 X lo-* M and more caused a significant but variable reduction in total and Na+-K+-ATPase activity in choroid plexus homogenates. However, in concentrations of 2 X lo-” M and less, no effect on total or NafKf-ATPase activity was obtained. Nor did omeprazole (2 x 10m4 M) influence HCO,-ATPase. Choline uptake in isolated ehoroid plexus was significantly reduced by 86% in the presence of acid-pretreated omeprazole 2 X 10U3 M, but was not affected by 2 X lo-’ M omeprazole (intact or acid-pretreated). Thus, the mechanism for the marked inhibitory influence of omeprazole on CSF production is not yet evident. In doses causing even a 50% reduction of CSF production, no side effects were observed in contrast to Na+-K+-ATPase inhibitors such as ouabain. 0 1992 Academic Press, Inc.

INTRODUCTION The formation of cerebrospinal fluid (CSF) from the choroid plexus takes place by active secretion (2,6, 13, 20, 21, 29, 38). The CSF formation can be inhibited by ouabain suggesting an involvement of transport systems dependent on so~um-potassium-activated adenosine triphosphatase (Nat-K+-ATPase) (2,29,34,38). A high activity of Na+-K’-ATPase has been found in choroid plexus homogenates (I, 17, 18, 23, 24, 29, 34). In rabbit, this enzyme represents about one-third to onefourth of the total ATPase activity in the choroid plexus (17,18,24,29). Surgical removal of the extensive sympathetic innervation of the choroid plexus from the superior cervical ganglia (I5), directly innervating the secretory epithelium (8), increases the rate of CSF formation 0014-4886192 $3.00 Copyright Q 1992 by Academic Press,Inc. Aii rights of reproduction in any form resewed.

CHRISTER OWMAN,”

ANDBIRGERWINBLADH$

and tlnternal Medicine, University of Lund, Lund, Sach’s C~ildre~‘s hospital, Sto~k~lm, Sweden

Sweden;

(15) and the activity of Na+--Kf-ATPase in the rabbit choroid plexus (17). Recently, it has been found that the gastric mucosa of pig, dog, and guinea pig contains another specific ATPase, which is H’-K+-dependent (19, 31). Inhibition of the enzyme with omeprazole, a substituted benzimidazole, markedly and selectively reduces histamineand db-CAMP-stimulated gastric acid secretion (36). This enzyme seems so far to be unique for the gastric mucosa as determined by immunochemical methods for the enzyme protein (31). There are certain similarities between the secretory responses in the gastric and choroid plexus epithelia to drugs affecting the autonomic nervous system. Furthermore, the choroid plexus seems to play a role in the regulation of the pH in the CSF (9). For these reasons it was considered of interest to determine any presence of H+-EC+-ATPase-like activity in the choroid plexus by means of an effect of the selective inhibitor, omeprazole, on choroid plexus ATPase activity, transport functions, and the rate of CSF formation. MATERIAL

AND METHODS

Animals

The experiments were performed on 60 randomly pigmented rabbits of either sex, weighing 2.5-3.5 kg. They were fed standard food and tap water ad lib. ~eter~~~t~o~

of ATE&e

Actiuity

A modification of the spectrophotometric method of Bonting et al. (1) was used. The animals were killed by perfusion through the left ventricle of the heart with 500 ml 0.9% saline under light ethyl ether anesthesia. Plexuses from the four ventricles of the brain were dissected out separately, weighed in 0.2 ml distilled water (5-10 mg each), and placed on dry ice. Each plexus was homogenized in ice-cold distilled water using a Potter-Elvehjem homogenizer. The homogenizer was rinsed twice with 0.1 ml distilled water, and the lot was freeze-dried and stored at -80’ until analysis. Before analysis each homogenate was resuspended in 100 ~1 imidazole buffer 394

INHIBITION

OF

CSF

FORMATION

solution (imidazole 25 mM, EDTA 1 mM) per mg plexus wet weight. After vigorous shaking, 50 ~1 of the homogenate was mixed with 0.2 ml of one of three different solutions. All had the same basic composition (in mM): MgCl,, 3.75; KCl, 25; NaCl, 163; EDTA, 0.2; Tris, 41.3; and histidine, 5 mg/ml. Two of the solutions, in addition, contained either 1 mM ouabain (Sigma), an inhibitor of Na+-K+-ATPase, and/or variable concentrations of omeprazole (H168/68; 5-methoxy-2-[[(4methoxy-3,5-dimethyl-2-pyridinyl)methyl]sulfinyl]lH-benzimidazole; Hgssle, Sweden), which is an inhibitor of H+-K+-ATPase. Both inhibitors were dissolved in 99.5% ethanol. For the determination of HCO,-ATPase, NaSCN in water solution was added to a final concentration of 2 X lop2 M. Ten microliters of ATP (Aldrich) solution, final concentration 3 X 10V3 M, was added after 10 min of incubation at 37°C in an oscillating bath, and the incubation was then continued for 1 h. The reaction was stopped by the addition of 0.1 ml of ice-cold trichloracetic acid (TCA; 20% w/v) followed after 5 min by centrifugation for 5 min at 2000 rpm. A 0.3-ml aliquot of the supernatant was mixed with freshly prepared reagent (1.09 M H,SO, with 1% ammonium molybdate, to which FeSO, had been added immediately before use to a final concentration of 40 mg/ml) and allowed to stand for 30 min before reading at 700 nm in a spectrophotometer. TCA- or heat-denatured homogenates processed as above served as blanks. Phosphate standards were incorporated in each set of analysis. At omeprazole concentrations of 3 X lop4 M or more the addition of TCA caused turbidity which could be eliminated by centrifugation. This lowered the phosphate readings and compensation was done in the calculations with the aid of the blanks and phosphate standards. The different ATPase activities were calculated from the difference in phosphate concentration between the solution containing no inhibitors and that containing ouabain, omeprazole, and NaSCN, respectively. Uptake

of Choline in Choroid

Plexus in Vitro

The method used has been described in detail elsewhere (37). In short, animals were sacrificed and the tissue dissected out as described above. The tissues were preincubated for 10 min in a Krebs-Henseleit buffer (pH 7.4) under continuous gassing with 95% 0, and 5% CO, at 37°C in a shaking bath. Thereafter [‘4C]choline ([methyl-‘4C]choline chloride; Radiochemical Centre, Amersham) was added (final concentration 10V5 M) and incubation was continued for 20 min. After incubation the tissues were removed, rinsed in cold Krebs-Henseleit buffer, gently blotted on filter paper, wrapped in plastic film to avoid drying, weighed on a microbalance, and dissolved in Soluene 350 (Packard). Radioactivity was measured by liquid scintillation counting using conventional techniques. Since choline is not metabolized

BY

OMEPRAZOLE

395

in the tissue (37), tissue/medium (TIM) ratios were calculated as the quotient of radioactivity between the tissue wet weight and the incubation medium. Rate of CSF Formation A modification of the intraventricular [14C]inulin dilution technique (10, 26) was used during ventriculocisternal perfusion. The animals were sedated with pentobarbital, tracheotomized, and then anesthetized with halothane and a mixt,ure of N,O and 0, in an open system during artificial ventilation. The rabbits were placed in prone position with the head fixed in a metal frame. Artificial CSF containing the labeled inulin (inulin [14C]carboxylic acid, 1 pCi/lOO ml; Radiochemical Centre, Amersham) was infused at a constant rate of 33 yllmin through an inflow probe into the right lateral ventricle during continuous recording of infusion pressure by a Statham model P23 AC transducer. The fluid was drained at the same rate by means of a needle placed in the cisterna magna connected with a roller pump. The outflow was collected in 5-min aliquots (150 ~1). Blood gases were frequently measured throughout the experiment in an Astrup system (Radiometer, Copenhagen) and systemic blood pressure was monitored continuously. Blood gases were maintained within narrow limits throughout the experiments by small adjustments of the ventilatory rate. For further methodological details, see Lindvall et al. (16). Radioactivity was measured by liquid scintillation counting, quench corrections being obtained according to the conventional principles. Omeprazole was given in varying concentrations in the perfusate from 10m4 to lo-‘M, corresponding to 0.2 X 10-l to 0.2 X lob6 mg X kg-’ h-l. In another set of experiments the drug was given as an intravenous (iv) injection in an ear vein in doses of 2 X 10-l to 2 x lop3 mg/kg. The effect of omeprazole was compared with a similarly substituted benzimidazole (H178142; 5-methoxy-2-[[(3,4,5,6-tetrametyl-2-pyridynyl)metyl]sulfinyl]-lH-benzimidazole), given at a concentration of 10e5 M in the CSF perfusate. H178/42 has a considerably lower effect on H+-K+-ATPase than omeprazole, i.e., Ki > lop4 for H178/42 vs. 0.3 X 10efi for omeprazole (Karlsson, 1990, personal communication). Because of their low water solubility, the stock solutions were made up in 99.5% ethanol. Addition of equivalent amounts of ethanol to the incubation and the perfusion solutions as those used in the experiments did not influence in vitro choline uptake, ATPase activities or CSF production. The exact mechanism of action of omeprazole on the H+-K+-activated ATPase has not been clarified. Omeprazole is not in itself an inhibitor of H+-K+-ATPase but is decomposed in acid milieu to its active components. This activation is very rapid (19, 35). For this reason the in vitro experiments were also performed

396

LINDVALL-AXELSSON Omeprazole

i.c.v.

lo-g I

10.’ I

1o-B I

L

concentration 1o-6 I

1o-5 I

(MI lo-‘ 1

1 (Ll

a” 60

.*

FIG. 1.

Changes in the rate of cerebrospinal fluid (CSF) production during intracerebroventricular (icv) perfusion of the selective H+-Kf-ATPase inhibitor, omeprazole, at various concentrations in the CSF infusate. The values are related to the control values of CSF production obtained in each animal prior to administration of omeprazole without compensating for any methodological errors, amounting to a mean of 8% (see Lindvall et al. (16)) related to, e.g., change of syringe and/or the long total infusion period. Values are mean percentage changes f SEM; number of animals indicated within parentheses. Significant differences from the control values were determined according to the paired t test (based on the absolute values for the CSF formation): *O.Ol < P < 0.05, **O.OOl < P < 0.01. (The intraventricular perfusion of 10m4 M omeprazole corresponds to a dose rate to the rabbit of around 20 gg X kg-’ X h-i.)

with omeprazole treated with acid as follows: pH in an omeprazole solution in water was lowered to pH 2.5 with HCl. The solution was kept in room temperature and darkness for 10 min and then neutralized to pH 7.5 with THAM buffer. Assay for ATPase activity was further performed at low pH (4-6) in additional experiments (by lowering the pH during the preincubation procedure).

ET

AL.

Because of the long equilibration time of the perfusion method (i.e., 0.5-l h) no more precise conclusions about the time course of the onset of the effect can be drawn. The physiological variables recorded during the control and experimental periods showed no statistically significant differences, except at the omeprazole concentration of lo-’ M, where mean pH showed a slight decrease from pH 7.45 to 7.40 (P < 0.05). No other side effects were seen. In contrast, when CSF production is lowered to a similar extent with ouabain, severe side effects are noted in the animals (2,29). Addition of alcohol without omeprazole to the infusate did not influence the CSF production. Injection (iv) of omeprazole in 10 animals significantly decreased CSF production (but to a smaller extent than the highest concentrations used intraventricularly) by around 25% at the highest doses of 2 X lop2 and 2 X 10-l mg/kg (Fig. 2). Higher doses were not tested. The benzimidazole H178/42 did not affect CSF production at a concentration of 10e5M in the perfusate (n = 4). The activity of ouabain-sensitive Na+-K+-ATPase comprised on average 32% of total ATPase activity in the (blood-free) choroid plexuses from the various brain ventricles. Since there were no significant differences between the plexuses from the different ventricles, the data are not reported separately in the following text. Omeprazole in low concentrations, 2 X 10e5and 2 X lOA M, did not affect total or Na+-K+-ATPase activity. At 2 X 10e4M concentration, a variable influence was seen. At 2 X 10e3M omeprazole concentration a considerable decrease (46%) in total ATPase activity was seen. Addition of ouabain further decreased the activity, but less than ouabain alone, showing that both Na+-K+-ATPase and Na+-K+-independent ATPase activities were affected (Table 1). Pretreatment of omeprazole with

8

Statistics

I

Omeprazale

i.v. dose (pg/kgl

2

20

200

“0 15 E 1

Differences between mean values were evaluated with the paired t test. RESULTS

The mean rate of CSF formation during the 2-h steady-state period before administration of the enzyme inhibitor omeprazole was 9.9 f 0.5 (SEM) pl/min. Various concentrations of omeprazole were then added to the inulin-containing perfusion solution in 18 animals during a subsequent 2-h experimental period. There was a dose-related pronounced reduction in CSF formation (Fig. l), being about 50% at the highest concentration of the inhibitor tested (lo-* M), while it was not significantly altered at the concentration of lo-’ M.

b

I

2 20 .c .s 25 5 i ;30 0, u P

(3) *

13) ***

(6) **

2 FIG. 2. Changes in the rate of CSF formation after intravenous (iv) injection of various doses of omeprazole. The values are obtained as in Fig. 1 and show mean percentage values + SEM; number of animals indicated within parentheses. Significant differences from the control value before omeprazole injection as in Fig. 1: *P -c 0.05, **0.001 < P < 0.01, ***p < 0.001.

INHIBITION

TABLE

OF

CSF

FORMATION

1

Inhibition of ATPase Activity by Different Concentrations of Ouabain, Omeprazole, Ouabain and Omeprazole, or Omeprazole Pretreated with Acid” Percentage Inhibitor concentration of) 2 2 2 2

x x x x

1om3 1o-4 1om5 1o-6

Ouabain 32 + 9 (19) 32 + 4 (4) 25 f 3 (3) 11 Zk 7 (2)

a Mean f standard within parentheses.

Omeprazole 46 f

8 (18)

11 + 6 (4) 7 -c 10 (2) 0 f 0 (3)

deviation; For details,

inhibition Omeprazole + ouabain

Omeprazole pretreated with acid

67 f 3 (3) (3) (3) (3)

39 + 7 25 + 3 7 t 2

number of determinations see Materials and Methods.

62

k 8 (16)

11f 4 2 -c4 -

(16) (13)

indicated

acid significantly increased its inhibitory activity in the concentration 2 X 1O-3 M but not in lower concentrations. Acidification of the incubation medium to pH 6.5 and 4, respectively, during the preincubation gave a small decrease in total ATPase activity with decreasing pH. However, the pattern and magnitude of inhibition of omeprazole was the same as at pH 7.5. Addition of NaHCO, (40 mM) to ouabain-inhibited incubate increased ATPase activity significantly by 28 +- 9% (SD; n = 8), suggesting the presence of a bicarbonate-activated ATPase (HCO;-ATPase). Consequently, this increase in activity was partly inhibited by NaSCN 10e2 M, a known HCO;-ATPase inhibitor. However, omeprazole 2 X lop4 M did not influence the increase in ATPase activity caused by NaHCO,, 34 -t 8% (SD; n = 4). The choroid plexus uptake in vitro of choline (organic base; 10e5 M) into the choroid plexus showed a T/M ratio of 82 -t 13 (n = 12) in controls and was significantly reduced by 68 and 86%, respectively, with intact and acid-pretreated omeprazole 2 X 10e3 M in the incubation medium (Table 2). On the other hand, the plexus uptake of choline 10e5M was not influenced by omeprazole 2 X 10e5M, and pretreatment of the inhibitor with acid did not change the results (Table 2). DISCUSSION Omeprazole decreased CSF production significantly already in low concentrations when infusedintraventricularly, and in low doses when given iv. The highest dose given iv was about the same as that recommended for clinical use in man (calculated per kilogram body weight). In the dog 0.5 mg/kg gives a plasma concentration of 4 X 10e6Mat 5 min and 10e6Mat 60 min (Mikulski et al., personal communication). In our experiments 0.2 mg/ kg intravenously gave a 25% reduction, while 10F7and low6 M in the ventricular perfusions gave a 15 and 35%

BY

397

OMEPRAZOLE

reduction in CSF production, respectively. The CSF concentrations are about 10% of the plasma concentrations after iv administration (Mikulski et al., personal communication). Because of the good lipid solubility of omeprazole, there is no reason to believe that the drug exerts its action after penetration into the CSF. If the disposition data from the dog roughly can be applied to the rabbit, there is a reasonable agreement between the effect on CSF production of the intravenous and intraventricular administration routes. The mechanism for the influence of omeprazole on the CSF production is obscure. In concentrations causing a pronounced reduction of the CSF production, i.e., 2 X 1O-5 M, no influence on the ATPase activity was obtained. The inhibitory action on ATPase activity seen with 2 X 10d4 M and higher concentrations might be rather unspecific and has been observed also by others (Wallmark, personal communication). In this case it is of doubtful physiological significance. The same holds for the effect of omeprazole on the active uptake of choline in choroid plexus in vitro where a significant inhibition was observed only at an omeprazole concentration of 2 X 10V3M but none at 2 X 10d5M. An influence on CSF production via the ATPase system could be possible if the choroid plexuses accumulated omeprazole to a high concentration. However, at 2 X lop5 M omeprazole no reduction in choline uptake was obtained in choroid plexuses in vitro. As mentioned earlier, omeprazole needs transformation in acid milieu to obtain specific H+-K+-ATPase-inhibiting properties. No such compartment is known to exist in the choroid plexus. Altogether, from these experiments it seemsunlikely that omeprazole reduces CSF production by interfering with the ATPase system in the choroid plexus. An intact sodium pump seems to be necessary for at least part of the CSF production from the choroid plexus (5, 13, 14, 29, 32-34). High concentrations of

TABLE

2

Percentage Change in Mean Plexus Tissue Medium Ratio (TIM) for Choline 1O-5 M in Vitro with Omeprazole (Pretreated or Not Pretreated with Acid) Present in the Incubation

Medium”~*

Compared

to Controls Choline (W5 M)

Omeprazole Omeprazole Omeprazole Omeprazole

2 2 2 2

X X X X

10d3 M 10W3 M 10m5 M 10e5 M

not pretreated pretreated not pretreated pretreated

-68.3 -85.6 +7.3 +19.6

D Significant differences from the control values (without zole) according to Student’s t test; number of determinations parentheses. *P < 0.05. * T/M in controls for choline low5 M 82 k 13. Reduction indicated by -; increase in T/M by +.

(12)* (12)* (12)“.“’ (12)“.“’ omeprawithin in T/M

398

LINDVALL-AXELSSON

ouabain do not completely inhibit CSF production when the latter is determined by cerebroventricular perfusions (29). It has been claimed that up to around half of the total CSF might be derived from extrachoroidal sources in the brain (3,4,22,28-30). Omeprazole might hypothetically interfere with these less well known mechanisms. The maximal inhibition of the CSF production obtained with omeprazole was around 50% which is less than the maximal effect obtained with ouabain (29). This further supports the theory that CSF is produced by several different mechanisms. Other possible mechanisms for the effect of omeprazole would be an influence of the drug on choroid plexus blood flow or a more general toxic metabolic influence. However, no influence on the general circulation or general toxic effects were seen in the intact animals even at the highest dose intravenously or highest intraventricular perfusate concentration of omeprazole. This is in sharp contrast with the profound general effects seen on animals receiving high concentrations of ouabain in ventriculocisternal perfusions (2,11,34) with comparable influence on CSF production rates. However, recently Pollay et al. (29) minimized the strong ouabain effects on blood pressure and heart by avoiding exposure of the 4th ventricle in a ventriculoaqueductal perfusion system. It is intriguing that the influence of omeprazole on CSF production seems coupled to the specific structure inhibiting H+-K+-ATPase, since the much less potent analogue H178/42 was without effect on CSF production. It has recently been reported that omeprazole, besides its effect on H+-K*-ATPase, also influences steroid synthesis (7, 12). These reports supplied no data, however, as to the specificity of this influence of omeprazole on steroid synthesis. We are now investigating if omeprazol has an influence on human CSF production. If that is the case it might be useful in acute situations with increased intracranial pressure due to obstruction of CSF drainage or CSF overproduction. ACKNOWLEDGMENTS Supported by grants from the Swedish Medical Research Council (grant No 04X-732) and the Medical Faculty of Lund. The skillful technical assistance of MS Ann Brun and Mrs Ulla-Britt Andersson is gratefully acknowledged.

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