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Serotonin activates Cl− channels in the apical membrane of rat choroid plexus epithelial cells
European Journal of Pharmacology, 239 (1993) 31-37
31
© 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00
EJP 53195
Serotonin activates C1- channels in the apical membrane of rat choroid plexus epithelial cells C a t h e r i n e G a r n e r , Wasyl F e n i u k a a n d P e t e r D. B r o w n Department of Physiological Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK and " Glaxo Institute of Applied Pharmacology, Department of Pharmacology, University of Cambridge, Tetmis Court Road, Cambridge CB2 1QJ, UK
Received 28 January 1993, revised MS received 26 April 1993,accepted 11 May 1993
The effects of serotonin on ion channel activity in epithelial cells from rat choroid plexus were examined. Serotonin (50 nM, 500 nM and 1 /~M) stimulated channel activity in cell-attached patches. The current-voltage (I-V) relationship for the serotonin-activated channel gave a conductance of 26.6 + 1.5 pS and the current reversed at an applied electrode potential (-Vp)--15.3 :i: 3.3 mV with a KCl-rich electrode solution (n = 8). Similar I-V relationships were obtained using electrode solutions in which K + was replaced by other cations (Na + and N-methyl-D-glucamine), suggesting that the serotonin-activated channels are selective to Cl-. The effect of I tzM serotonin on channel activity was inhibited by ritanserin (30 and 100 nM) which has a high affinity for serotonin 5-HTlc receptors and 5-HT2 receptors. Spiperone (30 nM), which binds weakly to 5-HTlc receptors but has a high affinity for 5-HT2 receptors, did not inhibit the actions of serotonin. These data suggest that serotonin increases C1- channel activity by acting on the 5-HTlc receptors on the epithelium. Choroid plexus; 5-HT (5-hydroxytryptamine, serotonin); 5-HTic receptor; Cl- channels
1. Introduction The choroid plexuses are thought to be the principal sites of cerebrospinal fluid (CSF) secretion (Davson et al., 1988; Wright, 1978). It is the epithelial cells of the choroid plexuses which are responsible for the production of CSF. This they achieve by actively transporting ions (Na +, C l - and H C O 3) from the blood to the CSF, in a process which is thought to involve a variety of transport proteins, e.g. Na+-K + ATPase, CI--HCO 3 exchange, Na+-H + exchange, anion and K + channels (Brown and Garner, 1993; Christensen et al., 1989; G a r n e r and Brown, 1992; Wright, 1978). The movement of ions across the epithelium provides the osmotic gradient which drives fluid secretion (Wright, 1978). The whole process of CSF secretion appears to be regulated, since the concentrations of some ions in the CSF are maintainedwithin well defined limits, e.g. K + (Husted and Reed, 1976) and H C O 3 (Husted and Reed, 1977). It has also been suggested that neurotransmitters and hormones may influence the rate of
Correspondence to: P.D. Brown, Department of Physiological Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK. Tel. 44-61 275 5463, fax 44-61 275 5600.
CSF production (Edvinsson et al., 1983; Saito and Wright, 1983). A number of receptors for neurotransmitters have now been identified on choroid plexus epithelial cells, e.g. flE-adrenoceptors (Nathanson, 1983) and serotonin 5-HTlc receptors (Hartig et al., 1990). The serotonin 5-HTlc receptor is expressed in the choroid plexus at densities which are 10-times greater than those found elsewhere in the central nervous system (Pazos and Palacios, 1988; Yagaloff and Hartig, 1985). This receptor has been very well characterised in agonist and antagonist binding studies (Hartig et al., 1990; Hoyer et al., 1989). The molecular structure of the receptor is also known (Julius et al., 1988; Lubbert et al., 1987), and it has been shown to be linked via G-proteins to the mobilisation of intracellular inositol trisphosphate (Conn et al., 1986). The exact role of the receptor in the choroid plexus remains unknown, however it has been suggested that it may be involved in the regulation CSF secretion (Hartig, 1989). Neurotransmitters are thought to regulate fluid secretion in many epithelia by controlling the activity of ion channels in the apical membrane of the epithelial cell (Gogelein, 1988; Petersen and Gallacher, 1988). The purpose of this present study, therefore, was to investigate the effect of serotonin on ion channel activ-
32 ity in the choroid plexus. In patch clamp experiments serotonin was found to increase the activity of the C1channels in the apical membrane of rat choroid plexus epithelial cells. The possible involvement of 5-HT1c receptors was then examined using ritanserin or spiperone, which are respectively antagonists of both 5-HTlc and 5-HT 2 receptors or the 5-HT 2 receptor alone. A preliminary account of some of this work has been presented to the Physiological Society (Garner and Brown, 1993).
2. Materials and methods
2.1. Animals and tissue preparation Adult Sprague-Dawley rats of either sex (150-200 g) were killed by an overdose of diethyl ether. To clear blood from the cerebral vasculature 10 to 15 ml of cold HEPES-buffered saline (4°C) were injected into the left ventricle of the heart. The choroid plexus was then quickly removed from the fourth ventricle of the brain, and a small piece of tissue was placed in a superfusion chamber (volume = 400 /~1). The saline solution used to flush the vasculature and bathe the tissue in all experiments contained (mM): NaC1 140; KCI 2; MgCI 2 1; CaCI 2 1; HEPES 10; glucose 5. This solution was gassed with 100% 0 2 and the pH was adjusted to 7.4 with HCI. The tissue was superfused with this solution at 1 ml • min-1 throughout each experiment. All experiments were performed at room temperature (21-24°C).
2.2. Single channel recording Channel activity was recorded as previously described (Garner and Brown, 1992). Electrodes (tip resistance 20 to 30 M J2) were filled routinely with a K+-rich solution containing (mM): KCI 120; NaC1 1; MgC12 1; HEPES 10; CaCI 2 1; E G T A 10, and the pH was adjusted to 7.4 with KOH (Ca 2÷ activity = 5 nM; Brown et al., 1988). In some experiments a Na+-rich electrode solution was used which had a composition identical to the K+-rich solution in all respects, except that Na + completely replaced K +. In three experiments 60 mM N-methyl-D-glucamine (NMDG ÷) chloride was used to replace 60 mM KCI in the electrode solution. Single channel activity was measured using an Axopatch-lD amplifier (Axon Instruments, Foster City, CA), and current records were stored as a digital signal on video tape (40 kHz). Data were subsequently analyzed by computer using the software package Pclamp 5.5.1 (Axon Instruments). Channel open probability (Po) was estimated for sequential periods of data (20.48 s digitised at a rate of 5 kHz).
2.3. Addition of serotonin and antagonists Channel activity was recorded in cell-attached patches. A hyperpolarising potential was applied to the patch ( - V p = - 4 0 mV), so that C1- channel currents could be easily distinguished above the noise level. A control period of activity was recorded at the beginning of each experiment (90 s in the absence of serotonin). Patches which exhibited a low level of control activity (Po < 0.5) were selected for the study (approximately 80% of patches). After the control period, the tissue was superfused with bath solution containing serotonin (50 nM, 500 nM and 1 /zM) for 2 min. This was followed by a further 4 min control period. Experiments were also performed in the presence of serotonin and 5-HT receptor antagonists (30 and 100 nM ritanserin and 30 nM spiperone). The bath was perfused with the control bath solution for at least 10 min following the exposure of tissue to any of the above substances. This was to allow time for the complete removal of the drug from the bath, and to prevent desensitisation of the 5-HTlc receptor (Walter et al., 1991). Current-voltage (I-V) relationships for channels activated by serotonin were determined by recording currents at -V p = - 8 0 to 40 mV, using the KCl-rich, NaCl-rich or N M D G + / K + electrode solution. When possible, patches were excised into the inside-out configuration at the end of the experiment. The number of ion channels present in the patch could then be assessed by applying depolarising potentials (the Po of the channel is known to increase with depolarisation; see Garner and Brown, 1992). Data presented in the Results are from patches which contained not more than one channel. Data from patches which could be shown in the inside-out configuration not to contain any channels have been omitted.
2.4. Statistics Channel activity (Po) in control conditions is given as the average value for the whole of the control period. For test conditions (e.g. the presence of serotonin) activity is quoted either as a maximum value, or as a 'pooled' value which is the average activity for the whole of the test period. Differences in channel activity between control and pooled test conditions were tested for significance using Student's t-test for paired data. Data for I-V relationships were subjected to linear regression analysis.
2.5. Chemicals Serotonin (Sigma) was dissolved directly in HEPES-buffered NaCI solution. Ritanserin (Janssen Pharmaceutical), and spiperone (synthesised by the
33
Chemistry Research Department at Glaxo Group Research) were dissolved as 1 mM stock solutions in 10 mM tartaric acid. The stocks were then diluted with HEPES-buffered NaCI to give a final concentration of 30 or 100 nM ritanserin and 30 nM spiperone.
l(pA] -80
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Single-channel current records from a cell-attached patch (held at - V p = - 4 0 mV) in the presence and absence of serotonin are shown in fig. 1A. The patch exhibited very little channel activity during the control period, i.e. before the addition of serotonin (upper trace). Frequent channel openings (inward current steps) were observed in the presence of serotonin (middle trace: 90 s from the start of the superfusion with serotonin). Channel activity returned towards control levels once serotonin was removed from the bathing solution (lower trace). The time course for the changes in channel activity (open probability; Po) is illustrated in fig. lB. Channel activity increased almost immediately (within 30 s) after the addition of serotonin. The maximum activation of the channel (Po = 0.98, comA. Control . . . . . . ; . . . . it~ ; ~_-:-
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Time (s| Fig. 1. Effect of serotonin on channel activity in the apical membrane of rat choroid plexus. (A) Current records are shown for a cell-attached patch held at - V p = - 4 0 mV (solid line indicates the closed state of the channel; filtered at 500 Hz). Upper trace: channel activity in control conditions (before addition of serotonin). Middle trace: the increase in channel activity (inward current) 90 s after the addition of 1 /zM serotonin. Bottom trace: activity 4 min after removal of serotonin. (B) Time course for changes in channel open state probability (Po) during the superfusion with serotonin. Po was estimated for sequential 20 s periods of channel activity. The data are taken from the single experiment shown in fig. 1A, although similar data were obtained with 1 /zM serotonin in 8 out of 11 patches.
-3 Fig. 2. Current-voltage relationship for the serotonin-activated channel. Currents (mean + S.E.M.) are plotted for experiments in which the electrode contained: 120 mM KCI (n = 8; D) or 120 mM NaCI (n = 5; • ) , the bath contained the HEPES-buffered NaC1 solution and serotonin. The line was fitted by linear regression to the data obtained with the KCI electrode solution. The slope of this line gives a conductance of 26.6+ 1.5 pS and a reversal potential at - V p = 15.3 :t:3.3 mV. The line fitted through the data for NaCI electrode solution (not shown) gave similar values (conductance = 27.5_+ 1.8 pS; reversal potential - Vp = 19.4 + 4.2 mV).
pared to the control Po = 0.01) was observed after only 90 s of the superfusion period. Channel activity returned towards the control level following the removal of the serotonin (Po = 0.02 after 3 min). Similar results were obtained in eight out of eleven experiments with 1 /xM serotonin. The pooled values of Po from all eleven experiments were: control = 0.026 + 0.016; serotonin = 0.262 _+ 0.095 (significantly different by paired t-test at P < 0.05). An increase in channel activity was also observed in three out of four patches with 500 nM serotonin (control = 0.011 + 0.02; serotonin = 0.120 + 0.090), and two out of four patches with 50 nM serotonin (control = 0.008 + 0.005; serotonin = 0.065 + 0.040). The current-voltage (l-V) relationship for the serotonin-activated channel is shown in fig. 2. Data (means + S.E.M.) obtained using either a KCl-rich or a NaCIrich electrode solution are plotted. The line was fitted by linear regression through the data obtained with the KCl-rich solution. The conductance of the channel estimated from the slope of this line was 26.6 + 1.5 pS and current reversal occurred at - V p --- 15.3 __+3.3 mV (n = 8). Similar values were obtained with the NaCI electrode solution: c o n d u c t a n c e - 27.5 + 1.8 pS and - Vp = 19.4 + 4.2 mV (n = 5). These data indicate that the channel activated by serotonin is either selective to CI- or is a nonselective channel which is permeable to Na ÷ or K ÷. The selectivity of the channel was further examined by replacing 60 mM K ÷ in the electrode solution with the impermeant cation N M D G ÷ (60 mM). The I-V relationships obtained in three experiments with N M D G ÷ ( - V o = 17.4 + 0.4 mV and G = 21.5 + 1.8 pS), were similar to those obtained with the KC1 or NaC1 electrode solution (not significantly different by unpaired t-test, P > 0.1). This suggests that the channel is C1--selective rather than a non-selective cation channel.
34
To explore the possibility that the effect of serotonin on channel activity was a receptor-mediated event, 1 /zM serotonin was added to the tissue in the presence of 30 and 100 nM ritanserin in the bath solution. At these concentrations ritanserin is an effective antagonist of both 5-HTlc and 5-HT 2 receptors (Hoyer et al., 1989; Sahin-Erdemli et al., 1991). Figure 3 shows the effect of serotonin and 100 nM ritanserin on channel activity in a single cell-attached patch ( - V p =-40 mV). Channel openings were not observed during the control period before the addition of ritanserin and 5-HT (fig. 3A, upper trace). Serotonin failed to cause an increase in channel activity in the presence of ritanserin (fig. 3A, middle trace). However, serotonin alone was able to elicit channel activity when ritanserin was removed from the bath solution (fig. 3A, lower trace). The time course of this experiment is summarised in fig. 3B, channel activity was observed within 60 s of the removal of ritanserin. Serotonin (1 /zM) failed to cause a significant increase in Po in the presence of 100 nM ritanserin in four experiments. However, in each of these experiments serotonin alone caused a significant increase in Po, from 0.010 + 0.005 in the presence of ritanserin to 0.062 + 0.008 (P < 0.05, paired t-test, n = 4). The effects of 1 /~M serotonin on channel activity were also
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Fig. 4. The effects of 5-HT receptor antagonists on the serotonininduced channel activity (Po)- Data (means _+S.E.M.) were calculated for cell-attached patches whilst the bath was peffused with solutions containing: 5-HT: 1 /zM serotonin; Rit: ] /zM serotonin and 30 nM ritanserin (5-HT1c and 5-HT 2 receptor antagonist); Spip: 1 p.M serotonin and 30 nM spiperonc (5-HT 2 receptor antagonist); Con: is the pooled control value from all 20 experiments. The figures in parentheses are the number of individual experiments performed. Data for each experimental condition were compared with the appropriate control values by Student's t-test for paired data, * indicates data which are significantly different form controls (P < 0.05).
inhibited by 30 nM ritanserin in three experiments (see fig. 4), but activity was observed in each case with serotonin alone. The inhibitory actions of the ritanserin did not appear to be due to a direct inhibition of the ion channels, since channel activity was not affected by 100 nM ritanserin in three inside-out patches (data not shown). Ritanserin may have been acting at either 5-HTlc or 5-HT 2 receptors. To investigate the type of receptor involved more precisely, and since there are no specific 5-HTlc receptor antagonists, the effects of spiperone (which has a low affinity for the 5-HT~c receptor, but a high affinity for the 5-HT 2 receptor; Barker et al., 1991; Canton et al., 1990) were examined. In six experiments 30 nM spiperone in the bath solution failed to inhibit the serotonin-induced increase in channel activity (fig. 4). In control conditions (the absence of serotonin and spiperone) there was little channel activity (Po = 0.02 + 0.011). Addition of 1 / z M serotonin and 30 nM spiperone caused a significant increase in channel activity (Po = 0.140 + 0.050; P < 0.05).
I
4. Discussion
0.1
0
120
240
360
480
600
Time (s) Fig. 3. Ritanserin inhibits the serotonin-induced increase in channel activity. T h e tissue was superfused with 1 p,M serotonin in the presence of 100 n M ritanserin (an antagonist at serotonin 5-HT]c and 5-HT e receptors). (A) Single channel current records ( - Vp = 40 mV; filter 500 Hz): U p p e r trace: before the addition of serotonin and ritanserin; middle trace: in the presence of ritanserin and serotonin; bottom trace: in the presence of serotonin alone. (B) Time course for changes in open probability for the channel in fig. 3A. Similar results were obtained in four experiments with 100 n M ritanserin.
4.1. Channel activation by serotonin is mediated by the 5-HTzc receptor In this study serotonin was shown to activate ion channels in the apical membrane of the rat choroid plexus. The data suggest that the activated channel is identical to a C1--selective channel which has previously been identified in the rat choroid plexus (Garner and Brown, 1992). The main evidence for this is the I-V relationship for the channel, which is virtually
35 identical in the presence of the KCl-rich, the NaCl-rich or the N M D G + / K ÷ electrode solution (fig. 2). The reversal potential and the conductance of the channel were not significantly different under any of these conditions, suggesting that the channel is selective to CI- and not a nonselective cation channel. Experiments on this channel in inside-out patches have also shown that it is selective to CI- (Garner and Brown, 1992), and other anions e.g. HCO3, Br- and I - (Lynch et al., 1993). A similar study of the mouse choroid plexus has also shown that CI- channels are activated by serotonin (Hung et al., 1990). The channel identified in the mouse, however, had a much lower conductance (12 pS). The reason for the difference between the data from the rat and mouse is not clear, although it may simply be a species difference. Future studies of choroid plexus tissue from other mammalian species may help explain this difference. The effects of serotonin on ion channel activity in the rat choroid plexus were further characterised by investigating the nature of the receptor involved. In the absence of a good specific antagonist for the 5-HTlc receptor, the differential effects of ritanserin (an antagonist of 5-HTxc and 5-HT 2 receptors; Hoyer et al., 1989; Sahin-Erdemli., 1991) and spiperone (an antagonist of 5-HT 2 receptors; Barker et al., 1991; Canton et al., 1990) were examined. In seven experiments ritanserin (30 and 100 nM) inhibited the effect of 1 /~M serotonin on CI- channel activity (figs. 3 and 4). In each of these experiments channels could be activated by serotonin alone (e.g. fig. 3). The effect of ritanserin did not appear to be caused by a direct block of the C1- channels, because channel activity could be observed in inside-out patches in the presence of both concentrations of ritanserin. The experiments with ritanserin suggest that the activation of the C1- channels by serotonin is a receptor-mediated event. However, the ritanserin could be acting either on the 5-HT~c receptors on the epithelial cells, or possibly indirectly at 5-HT 2 receptors on cells adjacent to the epithelium, e.g. fibroblasts (Barker et al., 1991) or nerves (Aghajanian et al., 1990). The experiments in which 30 nM spiperone, at this concentration an effective antagonist of 5-HT 2 receptors, failed to inhibit the effects of serotonin (fig. 4) suggest that serotonin is acting upon the 5-HTIc receptors. These data support the findings of Hung et al. (1990), who used 1 mM mianserin, which like ritanserin is an antagonist of both 5-HTlc and 5-HT 2 receptors, to inhibit the effect of serotonin on mouse choroid plexus.
4.2. Physiological role of serotonin-activated CI- channels Most of the available evidence suggests that 5-HTlc receptors are found on the apical membrane of choroid
plexus epithelial cells, so that they are exposed to the CSF (Hartig, 1989). The concentration of serotonin in the CSF is in the range of 1 to 30 nM, and has been reported to vary over a 24 h cycle (Anderson et al., 1987; Garrick et al., 1983). These concentrations are similar to the K D for the 5-HT~c receptor (6.5 to 30 nM, Hartig, 1989), but it is difficult to predict whether or not these concentrations are sufficient to stimulate channel activity in the choroid plexus. Of the three concentrations of serotonin used in this study (50 nM, 500 nM and 1 ~M), only 1 /~M caused a significant increase in channel activity. However, the single-channel patch clamp method may lack resolution when dealing with means of open probabilities, since there was a great deal of variation in channel activity in the presence of each concentration of serotonin (e.g. see the values of the S.E.M.). It is likely that whole-cell patch clamp studies, in which the activity of all the C1channels in a cell can be monitored, will be needed to examine the effects of lower concentrations ( < 1/xM) of serotonin. If the concentration of serotonin in the CSF is sufficient to stimulate C1- activity in the choroid plexus, then serotonin may have a role in regulating the rate of CSF secretion, (e.g. in many other secretory epithelia the activation of anion channels causes an increase in the rate of CI- and fluid secretion; see Gogelein, 1988; Petersen and Gallacher, 1988). This prediction, however, is not supported by experiments which have directly measured CSF production. Lindvall-Axelsson et al. (1988, 1989), found that high concentrations of serotonin (10/xM) caused a 30% decrease in CSF production in in vivo studies on rabbits. While these data should be interpreted with caution, because serotonin may have some nonspecific effects at this concentration, they clearly do not support the hypothesis that serotonin increases CSF production. One possible explanation for this discrepancy is that the CI- channels have only a minor role in transporting C1- from the choroid plexus into the CSF. Christensen et al. (1989), working on the amphibian choroid plexus, suggested that the majority of CI- is transported into the CSF by K+-CI - cotransport or by low conductance channels which cannot be identified in single channel studies. The increase in C1- conductance evoked by serotonin may therefore have very little effect on the overall rate of CSF production. The channels, however, may be a major route for HCO~- transport into the CSF. Saito and Wright (1983, 1984), suggested that much of the electrogenic transport of ions across the bull frog choroid plexus was HCO~--dependent. Serotonin did not affect the short-circuit current across bull-frog choroid plexus (Saito and Wright, 1983), but this may be because of a lack of serotonin receptors in the amphibian choroid plexus. Recent experiments in this laboratory have
36
shown that the Cl--selective channels in inside-out patches from rat choroid plexus have a finite HCO 3
permeability (Pbicarbonate ; P C I - =
0.53;
Lynch et al., 1993).
The opening of these channels by serotonin may therefore increase HCO 3 transport into the CSF. This may help explain how HCO 3 transport is regulated, so that HCO 3 activity in the CSF is maintained within welldefined limits (Husted and Reed, 1977). However, this will need to be confirmed by investigating the effects of serotonin on the ionic composition of the CSF. In summary, the results presented show that serotonin activates anion channels in the apical membrane of the rat choroid plexus. The serotonin appears to exert this effect by acting on 5-HTIc receptors. The precise importance of the change in channel activity is not yet known, but it is probably related to the transport of ions into the cerebrospinal fluid.
Acknowledgements We would like to thank Drs. Susan Greenwood and Tohru Kotera for their constructive comments on the manuscript. C.G. is the recipient of a S.E.R.C. Case award studentship with Glaxo Research Ltd.
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intracranial pressure, in: Neurobiology of Cerebrospinal Fluid 2, ed. J.H. Wood (Plenum, New York) p. 671. Garner, C. and P.D. Brown, 1992, Two types of chloride channel in the apical membrane of rat choroid plexus epithelial cells, Brain Res. 591, 137. Garner, C. and P.D. Brown, 1993, Serotonin activates CI- channels in the apical membrane of epithelial cell in isolated rat choroid plexus, J. Physiol. (London) 459, 38P. Garrick, N.A., L. Tamarkin, P.L. Taylor, S.P. Markey and D.L. Murphy, 1983, Light and propranolol suppress the nocturnal elevation of serotonin in the cerebrospinal fluid of rhesus monkeys, Science 221, 474. Gogelein, H., 1988, Chloride channels in epithelia, Biochim. Biophys. Acta 947, 521. Hartig, P.R., 1989, Serotonin 5-HTlc receptors: what do they do?, in: Serotonin: Actions, Receptors, Pathophysiology, eds. E.J. Mylecharone, J.A. Angus, I.S. De la Londe and P.P.A. Hmphrey (Macmillan Press, London) p. 180. Hartig, P., B.J. Hoffman, M.J. Kaufman and F. Hirata, 1990, The 5-HTIc receptor, Ann. N.Y. Acad. Sci. 60, 149. Hoyer, D., C. Waeber, P. Schoeffter, J.M. Palacios and A. Dravid, 1989, 5-HTlc receptor-mediated stimulation of inositol phosphate production in pig choroid plexus, Naunyn-Schmiedeb. Arch. Pharmacol. 339, 252. Hung, B.C.P, D.D.F. LOo and E.M. Wright, 1990, Serotonin regulates CI channels in mouse choroid plexus, Biophys. J. 57, 83a. Husted, R.F. and D.J. Reed, 1976, Regulation of cerebrospinal fluid potassium by the cat choroid plexus, J. Physiol. (London) 259, 213. Husted, R.F. and D.J. Reed, 1977, Regulation of cerebrospinal fluid bicarbonate by the cat choroid plexus, J. Physiol. (London) 267, 411. Julius, D., A.B. MacDermott, R. Axel and T.M. Jessel, 1988, Molecular characterisation of a functional cDNA encoding the serotonin 1C receptor, Science 241,558. Lindvall-Axelsson, M., C. Matthew, C. Nilsson and C. Owman, 1988, Effect of 5-hydroxytryptamine on the rate of cerebrospinal fluid production in rabbit, Exp. Neurol. 99, 362. Lindvail-Axelsson, M., C. Nilsson, C. Owman and P. Svensson, 1989, Involvement of 5-HTlc receptors in the production of c.s.f, from the choroid plexus, J. Cereb. Blood Flow Metab. 9 (Suppl. 1), $680. Lubbert, H., B.J. Hoffman, T.P. Snutch, T. Van Dyke, A.J. Levine, P.R. Hartig, H.A. Lester and N. Davidson, 1987, eDNA cloning of a serotonin 5-HT~c receptor by electrophysiological assays of mRNA injected Xenopus oocytes, Proc. Natl. Acad. Sci. U.S.A. 84, 4332. Lynch, P.S., P.G. Smith, C. Garner and P.D. Brown, 1993, Bicarbonate permeable channels in the apical membrane of epithelial ceils in isolated rat choroid plexus, J. Physiol. (London) 459, 426P. Nathanson, J.A., 1983, Adrenergic-receptor mechanisms in mammalian choroid plexus, in: Neurobiology of Cerebrospinal Fluid 2, ed. J.H. Wood (Plenum, New York) p. 677. Pazos, A. and J.M. Palacios, 1985, Quantitative autoradiographic mapping of serotonin receptors in the rat brain. 1. Serotonin-1 receptors, Brain Res. 346, 205. Petersen, O.H. and D.V. Gallacher, 1988, Electrophysiology of pancreatic and salivary acinar cells, Ann. Rev. Physiol. 50, 65. Sahin-Erdemli, I., P. Schoeffter and D. Hoyer, 1991, Competitive antagonism by recognised 5-HT 2 antagonists at 5-HTlc receptors in pig choroid plexus, Naunyn-Schmiedeb. Arch. Pharmacol. 344, 137. Saito, Y. and E.M. Wright, 1983, Bicarbonate transport across the frog choroid plexus and its control by cyclic nuclectides, J. Physiol. (London) 336, 635. Saito, Y. and E.M. Wright, 1984, Regulation of bicarbonate trans-
37 port across the brush border membrane of the bull-frog choroid plexus, J. Physiol. (London) 350, 327. Walter, A.E., J.H. Hoger, C. Labarca, L. Yu, N. Davidson and H.A. Lester, 1991, Low molecular weight mRNA encodes a protein that controls serotonin 5-HT1c and acetylcholine M l receptor sensitivity in Xenopus oocytes, J. Gen. Physiol. 98, 399.
Wright, E.M., 1978, Transport processes in the formation of cerebrospinal fluid, Rev. Physiol. Biochem. Pharmacol. 83, 1. Yagaloff, K.A. and P.R. Hartig, 1985, 125I-Lysergic acid diethylamide binds to a novel serotonergic site on rat choroid plexus epithelial cells, J. Neurosci. 5, 3178.
31
© 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00
EJP 53195
Serotonin activates C1- channels in the apical membrane of rat choroid plexus epithelial cells C a t h e r i n e G a r n e r , Wasyl F e n i u k a a n d P e t e r D. B r o w n Department of Physiological Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK and " Glaxo Institute of Applied Pharmacology, Department of Pharmacology, University of Cambridge, Tetmis Court Road, Cambridge CB2 1QJ, UK
Received 28 January 1993, revised MS received 26 April 1993,accepted 11 May 1993
The effects of serotonin on ion channel activity in epithelial cells from rat choroid plexus were examined. Serotonin (50 nM, 500 nM and 1 /~M) stimulated channel activity in cell-attached patches. The current-voltage (I-V) relationship for the serotonin-activated channel gave a conductance of 26.6 + 1.5 pS and the current reversed at an applied electrode potential (-Vp)--15.3 :i: 3.3 mV with a KCl-rich electrode solution (n = 8). Similar I-V relationships were obtained using electrode solutions in which K + was replaced by other cations (Na + and N-methyl-D-glucamine), suggesting that the serotonin-activated channels are selective to Cl-. The effect of I tzM serotonin on channel activity was inhibited by ritanserin (30 and 100 nM) which has a high affinity for serotonin 5-HTlc receptors and 5-HT2 receptors. Spiperone (30 nM), which binds weakly to 5-HTlc receptors but has a high affinity for 5-HT2 receptors, did not inhibit the actions of serotonin. These data suggest that serotonin increases C1- channel activity by acting on the 5-HTlc receptors on the epithelium. Choroid plexus; 5-HT (5-hydroxytryptamine, serotonin); 5-HTic receptor; Cl- channels
1. Introduction The choroid plexuses are thought to be the principal sites of cerebrospinal fluid (CSF) secretion (Davson et al., 1988; Wright, 1978). It is the epithelial cells of the choroid plexuses which are responsible for the production of CSF. This they achieve by actively transporting ions (Na +, C l - and H C O 3) from the blood to the CSF, in a process which is thought to involve a variety of transport proteins, e.g. Na+-K + ATPase, CI--HCO 3 exchange, Na+-H + exchange, anion and K + channels (Brown and Garner, 1993; Christensen et al., 1989; G a r n e r and Brown, 1992; Wright, 1978). The movement of ions across the epithelium provides the osmotic gradient which drives fluid secretion (Wright, 1978). The whole process of CSF secretion appears to be regulated, since the concentrations of some ions in the CSF are maintainedwithin well defined limits, e.g. K + (Husted and Reed, 1976) and H C O 3 (Husted and Reed, 1977). It has also been suggested that neurotransmitters and hormones may influence the rate of
Correspondence to: P.D. Brown, Department of Physiological Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK. Tel. 44-61 275 5463, fax 44-61 275 5600.
CSF production (Edvinsson et al., 1983; Saito and Wright, 1983). A number of receptors for neurotransmitters have now been identified on choroid plexus epithelial cells, e.g. flE-adrenoceptors (Nathanson, 1983) and serotonin 5-HTlc receptors (Hartig et al., 1990). The serotonin 5-HTlc receptor is expressed in the choroid plexus at densities which are 10-times greater than those found elsewhere in the central nervous system (Pazos and Palacios, 1988; Yagaloff and Hartig, 1985). This receptor has been very well characterised in agonist and antagonist binding studies (Hartig et al., 1990; Hoyer et al., 1989). The molecular structure of the receptor is also known (Julius et al., 1988; Lubbert et al., 1987), and it has been shown to be linked via G-proteins to the mobilisation of intracellular inositol trisphosphate (Conn et al., 1986). The exact role of the receptor in the choroid plexus remains unknown, however it has been suggested that it may be involved in the regulation CSF secretion (Hartig, 1989). Neurotransmitters are thought to regulate fluid secretion in many epithelia by controlling the activity of ion channels in the apical membrane of the epithelial cell (Gogelein, 1988; Petersen and Gallacher, 1988). The purpose of this present study, therefore, was to investigate the effect of serotonin on ion channel activ-
32 ity in the choroid plexus. In patch clamp experiments serotonin was found to increase the activity of the C1channels in the apical membrane of rat choroid plexus epithelial cells. The possible involvement of 5-HT1c receptors was then examined using ritanserin or spiperone, which are respectively antagonists of both 5-HTlc and 5-HT 2 receptors or the 5-HT 2 receptor alone. A preliminary account of some of this work has been presented to the Physiological Society (Garner and Brown, 1993).
2. Materials and methods
2.1. Animals and tissue preparation Adult Sprague-Dawley rats of either sex (150-200 g) were killed by an overdose of diethyl ether. To clear blood from the cerebral vasculature 10 to 15 ml of cold HEPES-buffered saline (4°C) were injected into the left ventricle of the heart. The choroid plexus was then quickly removed from the fourth ventricle of the brain, and a small piece of tissue was placed in a superfusion chamber (volume = 400 /~1). The saline solution used to flush the vasculature and bathe the tissue in all experiments contained (mM): NaC1 140; KCI 2; MgCI 2 1; CaCI 2 1; HEPES 10; glucose 5. This solution was gassed with 100% 0 2 and the pH was adjusted to 7.4 with HCI. The tissue was superfused with this solution at 1 ml • min-1 throughout each experiment. All experiments were performed at room temperature (21-24°C).
2.2. Single channel recording Channel activity was recorded as previously described (Garner and Brown, 1992). Electrodes (tip resistance 20 to 30 M J2) were filled routinely with a K+-rich solution containing (mM): KCI 120; NaC1 1; MgC12 1; HEPES 10; CaCI 2 1; E G T A 10, and the pH was adjusted to 7.4 with KOH (Ca 2÷ activity = 5 nM; Brown et al., 1988). In some experiments a Na+-rich electrode solution was used which had a composition identical to the K+-rich solution in all respects, except that Na + completely replaced K +. In three experiments 60 mM N-methyl-D-glucamine (NMDG ÷) chloride was used to replace 60 mM KCI in the electrode solution. Single channel activity was measured using an Axopatch-lD amplifier (Axon Instruments, Foster City, CA), and current records were stored as a digital signal on video tape (40 kHz). Data were subsequently analyzed by computer using the software package Pclamp 5.5.1 (Axon Instruments). Channel open probability (Po) was estimated for sequential periods of data (20.48 s digitised at a rate of 5 kHz).
2.3. Addition of serotonin and antagonists Channel activity was recorded in cell-attached patches. A hyperpolarising potential was applied to the patch ( - V p = - 4 0 mV), so that C1- channel currents could be easily distinguished above the noise level. A control period of activity was recorded at the beginning of each experiment (90 s in the absence of serotonin). Patches which exhibited a low level of control activity (Po < 0.5) were selected for the study (approximately 80% of patches). After the control period, the tissue was superfused with bath solution containing serotonin (50 nM, 500 nM and 1 /zM) for 2 min. This was followed by a further 4 min control period. Experiments were also performed in the presence of serotonin and 5-HT receptor antagonists (30 and 100 nM ritanserin and 30 nM spiperone). The bath was perfused with the control bath solution for at least 10 min following the exposure of tissue to any of the above substances. This was to allow time for the complete removal of the drug from the bath, and to prevent desensitisation of the 5-HTlc receptor (Walter et al., 1991). Current-voltage (I-V) relationships for channels activated by serotonin were determined by recording currents at -V p = - 8 0 to 40 mV, using the KCl-rich, NaCl-rich or N M D G + / K + electrode solution. When possible, patches were excised into the inside-out configuration at the end of the experiment. The number of ion channels present in the patch could then be assessed by applying depolarising potentials (the Po of the channel is known to increase with depolarisation; see Garner and Brown, 1992). Data presented in the Results are from patches which contained not more than one channel. Data from patches which could be shown in the inside-out configuration not to contain any channels have been omitted.
2.4. Statistics Channel activity (Po) in control conditions is given as the average value for the whole of the control period. For test conditions (e.g. the presence of serotonin) activity is quoted either as a maximum value, or as a 'pooled' value which is the average activity for the whole of the test period. Differences in channel activity between control and pooled test conditions were tested for significance using Student's t-test for paired data. Data for I-V relationships were subjected to linear regression analysis.
2.5. Chemicals Serotonin (Sigma) was dissolved directly in HEPES-buffered NaCI solution. Ritanserin (Janssen Pharmaceutical), and spiperone (synthesised by the
33
Chemistry Research Department at Glaxo Group Research) were dissolved as 1 mM stock solutions in 10 mM tartaric acid. The stocks were then diluted with HEPES-buffered NaCI to give a final concentration of 30 or 100 nM ritanserin and 30 nM spiperone.
l(pA] -80
i
-Vp(mV)
1
-40
J
i /
40
"--2
3. R e s u l t s
Single-channel current records from a cell-attached patch (held at - V p = - 4 0 mV) in the presence and absence of serotonin are shown in fig. 1A. The patch exhibited very little channel activity during the control period, i.e. before the addition of serotonin (upper trace). Frequent channel openings (inward current steps) were observed in the presence of serotonin (middle trace: 90 s from the start of the superfusion with serotonin). Channel activity returned towards control levels once serotonin was removed from the bathing solution (lower trace). The time course for the changes in channel activity (open probability; Po) is illustrated in fig. lB. Channel activity increased almost immediately (within 30 s) after the addition of serotonin. The maximum activation of the channel (Po = 0.98, comA. Control . . . . . . ; . . . . it~ ; ~_-:-
_7:'-L"'~~::..~..,?~,t!~::~l=,3a,_~
5-HT
Control
......... r. . . .
' qV 2pA [
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1.0~
t
lpM 5-HT
~
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I
,
0.5 0
0
,
120
, 240
,
--
360
Time (s| Fig. 1. Effect of serotonin on channel activity in the apical membrane of rat choroid plexus. (A) Current records are shown for a cell-attached patch held at - V p = - 4 0 mV (solid line indicates the closed state of the channel; filtered at 500 Hz). Upper trace: channel activity in control conditions (before addition of serotonin). Middle trace: the increase in channel activity (inward current) 90 s after the addition of 1 /zM serotonin. Bottom trace: activity 4 min after removal of serotonin. (B) Time course for changes in channel open state probability (Po) during the superfusion with serotonin. Po was estimated for sequential 20 s periods of channel activity. The data are taken from the single experiment shown in fig. 1A, although similar data were obtained with 1 /zM serotonin in 8 out of 11 patches.
-3 Fig. 2. Current-voltage relationship for the serotonin-activated channel. Currents (mean + S.E.M.) are plotted for experiments in which the electrode contained: 120 mM KCI (n = 8; D) or 120 mM NaCI (n = 5; • ) , the bath contained the HEPES-buffered NaC1 solution and serotonin. The line was fitted by linear regression to the data obtained with the KCI electrode solution. The slope of this line gives a conductance of 26.6+ 1.5 pS and a reversal potential at - V p = 15.3 :t:3.3 mV. The line fitted through the data for NaCI electrode solution (not shown) gave similar values (conductance = 27.5_+ 1.8 pS; reversal potential - Vp = 19.4 + 4.2 mV).
pared to the control Po = 0.01) was observed after only 90 s of the superfusion period. Channel activity returned towards the control level following the removal of the serotonin (Po = 0.02 after 3 min). Similar results were obtained in eight out of eleven experiments with 1 /xM serotonin. The pooled values of Po from all eleven experiments were: control = 0.026 + 0.016; serotonin = 0.262 _+ 0.095 (significantly different by paired t-test at P < 0.05). An increase in channel activity was also observed in three out of four patches with 500 nM serotonin (control = 0.011 + 0.02; serotonin = 0.120 + 0.090), and two out of four patches with 50 nM serotonin (control = 0.008 + 0.005; serotonin = 0.065 + 0.040). The current-voltage (l-V) relationship for the serotonin-activated channel is shown in fig. 2. Data (means + S.E.M.) obtained using either a KCl-rich or a NaCIrich electrode solution are plotted. The line was fitted by linear regression through the data obtained with the KCl-rich solution. The conductance of the channel estimated from the slope of this line was 26.6 + 1.5 pS and current reversal occurred at - V p --- 15.3 __+3.3 mV (n = 8). Similar values were obtained with the NaCI electrode solution: c o n d u c t a n c e - 27.5 + 1.8 pS and - Vp = 19.4 + 4.2 mV (n = 5). These data indicate that the channel activated by serotonin is either selective to CI- or is a nonselective channel which is permeable to Na ÷ or K ÷. The selectivity of the channel was further examined by replacing 60 mM K ÷ in the electrode solution with the impermeant cation N M D G ÷ (60 mM). The I-V relationships obtained in three experiments with N M D G ÷ ( - V o = 17.4 + 0.4 mV and G = 21.5 + 1.8 pS), were similar to those obtained with the KC1 or NaC1 electrode solution (not significantly different by unpaired t-test, P > 0.1). This suggests that the channel is C1--selective rather than a non-selective cation channel.
34
To explore the possibility that the effect of serotonin on channel activity was a receptor-mediated event, 1 /zM serotonin was added to the tissue in the presence of 30 and 100 nM ritanserin in the bath solution. At these concentrations ritanserin is an effective antagonist of both 5-HTlc and 5-HT 2 receptors (Hoyer et al., 1989; Sahin-Erdemli et al., 1991). Figure 3 shows the effect of serotonin and 100 nM ritanserin on channel activity in a single cell-attached patch ( - V p =-40 mV). Channel openings were not observed during the control period before the addition of ritanserin and 5-HT (fig. 3A, upper trace). Serotonin failed to cause an increase in channel activity in the presence of ritanserin (fig. 3A, middle trace). However, serotonin alone was able to elicit channel activity when ritanserin was removed from the bath solution (fig. 3A, lower trace). The time course of this experiment is summarised in fig. 3B, channel activity was observed within 60 s of the removal of ritanserin. Serotonin (1 /zM) failed to cause a significant increase in Po in the presence of 100 nM ritanserin in four experiments. However, in each of these experiments serotonin alone caused a significant increase in Po, from 0.010 + 0.005 in the presence of ritanserin to 0.062 + 0.008 (P < 0.05, paired t-test, n = 4). The effects of 1 /~M serotonin on channel activity were also
A.
Control
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Po
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Fig. 4. The effects of 5-HT receptor antagonists on the serotonininduced channel activity (Po)- Data (means _+S.E.M.) were calculated for cell-attached patches whilst the bath was peffused with solutions containing: 5-HT: 1 /zM serotonin; Rit: ] /zM serotonin and 30 nM ritanserin (5-HT1c and 5-HT 2 receptor antagonist); Spip: 1 p.M serotonin and 30 nM spiperonc (5-HT 2 receptor antagonist); Con: is the pooled control value from all 20 experiments. The figures in parentheses are the number of individual experiments performed. Data for each experimental condition were compared with the appropriate control values by Student's t-test for paired data, * indicates data which are significantly different form controls (P < 0.05).
inhibited by 30 nM ritanserin in three experiments (see fig. 4), but activity was observed in each case with serotonin alone. The inhibitory actions of the ritanserin did not appear to be due to a direct inhibition of the ion channels, since channel activity was not affected by 100 nM ritanserin in three inside-out patches (data not shown). Ritanserin may have been acting at either 5-HTlc or 5-HT 2 receptors. To investigate the type of receptor involved more precisely, and since there are no specific 5-HTlc receptor antagonists, the effects of spiperone (which has a low affinity for the 5-HT~c receptor, but a high affinity for the 5-HT 2 receptor; Barker et al., 1991; Canton et al., 1990) were examined. In six experiments 30 nM spiperone in the bath solution failed to inhibit the serotonin-induced increase in channel activity (fig. 4). In control conditions (the absence of serotonin and spiperone) there was little channel activity (Po = 0.02 + 0.011). Addition of 1 / z M serotonin and 30 nM spiperone caused a significant increase in channel activity (Po = 0.140 + 0.050; P < 0.05).
I
4. Discussion
0.1
0
120
240
360
480
600
Time (s) Fig. 3. Ritanserin inhibits the serotonin-induced increase in channel activity. T h e tissue was superfused with 1 p,M serotonin in the presence of 100 n M ritanserin (an antagonist at serotonin 5-HT]c and 5-HT e receptors). (A) Single channel current records ( - Vp = 40 mV; filter 500 Hz): U p p e r trace: before the addition of serotonin and ritanserin; middle trace: in the presence of ritanserin and serotonin; bottom trace: in the presence of serotonin alone. (B) Time course for changes in open probability for the channel in fig. 3A. Similar results were obtained in four experiments with 100 n M ritanserin.
4.1. Channel activation by serotonin is mediated by the 5-HTzc receptor In this study serotonin was shown to activate ion channels in the apical membrane of the rat choroid plexus. The data suggest that the activated channel is identical to a C1--selective channel which has previously been identified in the rat choroid plexus (Garner and Brown, 1992). The main evidence for this is the I-V relationship for the channel, which is virtually
35 identical in the presence of the KCl-rich, the NaCl-rich or the N M D G + / K ÷ electrode solution (fig. 2). The reversal potential and the conductance of the channel were not significantly different under any of these conditions, suggesting that the channel is selective to CI- and not a nonselective cation channel. Experiments on this channel in inside-out patches have also shown that it is selective to CI- (Garner and Brown, 1992), and other anions e.g. HCO3, Br- and I - (Lynch et al., 1993). A similar study of the mouse choroid plexus has also shown that CI- channels are activated by serotonin (Hung et al., 1990). The channel identified in the mouse, however, had a much lower conductance (12 pS). The reason for the difference between the data from the rat and mouse is not clear, although it may simply be a species difference. Future studies of choroid plexus tissue from other mammalian species may help explain this difference. The effects of serotonin on ion channel activity in the rat choroid plexus were further characterised by investigating the nature of the receptor involved. In the absence of a good specific antagonist for the 5-HTlc receptor, the differential effects of ritanserin (an antagonist of 5-HTxc and 5-HT 2 receptors; Hoyer et al., 1989; Sahin-Erdemli., 1991) and spiperone (an antagonist of 5-HT 2 receptors; Barker et al., 1991; Canton et al., 1990) were examined. In seven experiments ritanserin (30 and 100 nM) inhibited the effect of 1 /~M serotonin on CI- channel activity (figs. 3 and 4). In each of these experiments channels could be activated by serotonin alone (e.g. fig. 3). The effect of ritanserin did not appear to be caused by a direct block of the C1- channels, because channel activity could be observed in inside-out patches in the presence of both concentrations of ritanserin. The experiments with ritanserin suggest that the activation of the C1- channels by serotonin is a receptor-mediated event. However, the ritanserin could be acting either on the 5-HT~c receptors on the epithelial cells, or possibly indirectly at 5-HT 2 receptors on cells adjacent to the epithelium, e.g. fibroblasts (Barker et al., 1991) or nerves (Aghajanian et al., 1990). The experiments in which 30 nM spiperone, at this concentration an effective antagonist of 5-HT 2 receptors, failed to inhibit the effects of serotonin (fig. 4) suggest that serotonin is acting upon the 5-HTIc receptors. These data support the findings of Hung et al. (1990), who used 1 mM mianserin, which like ritanserin is an antagonist of both 5-HTlc and 5-HT 2 receptors, to inhibit the effect of serotonin on mouse choroid plexus.
4.2. Physiological role of serotonin-activated CI- channels Most of the available evidence suggests that 5-HTlc receptors are found on the apical membrane of choroid
plexus epithelial cells, so that they are exposed to the CSF (Hartig, 1989). The concentration of serotonin in the CSF is in the range of 1 to 30 nM, and has been reported to vary over a 24 h cycle (Anderson et al., 1987; Garrick et al., 1983). These concentrations are similar to the K D for the 5-HT~c receptor (6.5 to 30 nM, Hartig, 1989), but it is difficult to predict whether or not these concentrations are sufficient to stimulate channel activity in the choroid plexus. Of the three concentrations of serotonin used in this study (50 nM, 500 nM and 1 ~M), only 1 /~M caused a significant increase in channel activity. However, the single-channel patch clamp method may lack resolution when dealing with means of open probabilities, since there was a great deal of variation in channel activity in the presence of each concentration of serotonin (e.g. see the values of the S.E.M.). It is likely that whole-cell patch clamp studies, in which the activity of all the C1channels in a cell can be monitored, will be needed to examine the effects of lower concentrations ( < 1/xM) of serotonin. If the concentration of serotonin in the CSF is sufficient to stimulate C1- activity in the choroid plexus, then serotonin may have a role in regulating the rate of CSF secretion, (e.g. in many other secretory epithelia the activation of anion channels causes an increase in the rate of CI- and fluid secretion; see Gogelein, 1988; Petersen and Gallacher, 1988). This prediction, however, is not supported by experiments which have directly measured CSF production. Lindvall-Axelsson et al. (1988, 1989), found that high concentrations of serotonin (10/xM) caused a 30% decrease in CSF production in in vivo studies on rabbits. While these data should be interpreted with caution, because serotonin may have some nonspecific effects at this concentration, they clearly do not support the hypothesis that serotonin increases CSF production. One possible explanation for this discrepancy is that the CI- channels have only a minor role in transporting C1- from the choroid plexus into the CSF. Christensen et al. (1989), working on the amphibian choroid plexus, suggested that the majority of CI- is transported into the CSF by K+-CI - cotransport or by low conductance channels which cannot be identified in single channel studies. The increase in C1- conductance evoked by serotonin may therefore have very little effect on the overall rate of CSF production. The channels, however, may be a major route for HCO~- transport into the CSF. Saito and Wright (1983, 1984), suggested that much of the electrogenic transport of ions across the bull frog choroid plexus was HCO~--dependent. Serotonin did not affect the short-circuit current across bull-frog choroid plexus (Saito and Wright, 1983), but this may be because of a lack of serotonin receptors in the amphibian choroid plexus. Recent experiments in this laboratory have
36
shown that the Cl--selective channels in inside-out patches from rat choroid plexus have a finite HCO 3
permeability (Pbicarbonate ; P C I - =
0.53;
Lynch et al., 1993).
The opening of these channels by serotonin may therefore increase HCO 3 transport into the CSF. This may help explain how HCO 3 transport is regulated, so that HCO 3 activity in the CSF is maintained within welldefined limits (Husted and Reed, 1977). However, this will need to be confirmed by investigating the effects of serotonin on the ionic composition of the CSF. In summary, the results presented show that serotonin activates anion channels in the apical membrane of the rat choroid plexus. The serotonin appears to exert this effect by acting on 5-HTIc receptors. The precise importance of the change in channel activity is not yet known, but it is probably related to the transport of ions into the cerebrospinal fluid.
Acknowledgements We would like to thank Drs. Susan Greenwood and Tohru Kotera for their constructive comments on the manuscript. C.G. is the recipient of a S.E.R.C. Case award studentship with Glaxo Research Ltd.
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