European Journal ofPharmacolo6fis202 (1991) 117-120 0 1991 Elsevier Science Publishers B.V. All rights reserved ~1~2999/91/$03,5~ ADONIS 00142999910~1~
117
EJP 20898
Short commuaicat~o~
Laszlo Gaal L* *, Christian Schudt ’ and Peter Illes 1*2 ’ apartment o~phu~aculu~,
Byk Guldm Pha~ace~t~ca~, ok-GLdde~-stray 2, O-7750 ~D~~ta~~ F.R.G. and 2 ~cpa~ment o~Phu~aco~~~, ~nil~e~i~ of Freibtq, Hermann-Herder-Strasse S, O-7800 Freiburg LB., F.R.G.
Received 23 April 1991, revised MS received 20 June 1991, accepted 9 July 1991
~tracel~ular field potentials were evoked in the CA1 pyramidal cell layer of the isolated rat hip~campus by electrical stimulation of the s&ratum radiatum. Of the three phosphodiesterase (PDE) inhibitors, 3-isobutyl-i-methy~anthi~~ (IBMX), zardaverine and rolipram, only the adenosine receptor antagonist, IBMX, increased the amplitudes of the extracellular excitatory postsynaptic potential (EPSP) and population spike (PS). The @-adrecoceptor agonist, isoproterenol, also facilitated these potentials and became more potent in the presence of zardaverine or rolipram. The results suggest that PDE blockade increases the excitability of pyramidal neurones only after preceding stimulation of P-adrenoceptors. Hip~ampal
pyramidal cells; Fiefd potentials; Phosphodiesterase inhibitors; ~-Adrenoceptor Adenosine receptor antagonists
1. Introduction 3-Isobu~l-1-methy~anthine (IBMX~ inhibits various cyclic nucleotide phosphodies:erase (PDE) isozymes (Beavo and Reifsnyder, i,‘90). It was suggested that the alkylxanthine, denbufjllline, increases the excitability of hippocampal CA1 pyramidal cells by blocking a cyclic adenosine 5’-monophosp~ate(CAMP)-specific PDE isozyme (Sutor et al., 1990). IBMX had a similar effect, probably both by inhibition of PDE activity and blockade of the A,-subtype of P,-purinoceptor (Sutor et al., 19901, which mediates tonic inhibition of pyramidal cells in response to endogenous adenosine (Haas and Greene, 1988). However, all xanthine derivatives, including denbufylline (Nicholson et al., 1989), appear to bind to adenosine A,- and A,-receptors (Bruns et al., P986). In order to separate the influence of adenosine receptor antagonism from that of PDE inhibition, we decided to investigate the effect of the non-xanthine PDE in-
Correspondence to: P. files, Department of Pharmacology, Wniversity of Freiburg, He~ann-Herder-Strasse 5, D-7X00 Fteibur~ i.Br., F.R.G. * Dedicated to professor E. Mutschier in honour of his hOth birthday. ** Present address: Chemical Works of Gideon Richter. P.O. Box 27, H-147.5 Budapest, Hungary.
agonists;
hibitors, rolipram and zardaverine, which have no A,or AZ-affinity (Kilian et al., 1989; Nicholson et al., 1991) and therefore are unlikely to interfere with endogenous adenosine. In fact, rolipram and zardaverine increased hippocampal CA1 excitability only when padrenoceptors were activated by isoproterenol.
2. Materials and methods 2.1. Preparation and recording Male Wistar rats ilSO-220 g) were anaesthetized with isoflurane (Forene @,Abbott, Wiesbaden, F.R.G.) and decapitated. Hippocampal slices (400 pm thick) prepared with a McIlwain chopper were placed in a recording chamber (Illes and NBrenberg, 1990) and superfused with medium at a rate of 2 ml/min. The medium (mM): NaCl 124; KCI 3; NaH2P0, 1.3; MgSG, 2; CaCI, 2; NaHCO, 26, and glucose 10; pH 7.4 was saturated with 95% 0*-s% CO, and maintained at 33°C. A bipolar stimulation electrode was placed in the stratum radiatum, Pulses of O.l-ms duration were supplied by the isolation unit of a HI-MED stimulator at a frequency of 0.1 Hz. The voltage was adjusted to evoke 60-70% of the maximal amplitude of the population spike (PS). Experiments were started only if the extra-
~~~f~~~r~~c~tato~ postsy~apt~c potentials (EPSP) and pS had been stable for at feast 30 min. Responses were recorded with glass micro-electrodes (resistances 2-8 ‘Ml)) fifled with 2 M NaCI. The electrode was placed in the CA1 pyramidal cell layer at a depth of 100-200 em and was connected to an AC amplifier (WPI DAM SO): the EPSP and PS were sampled, stored and anafyzed by means of a computer (IBM XT-2861 fitted with an AD converter ~~etrabyt~, DASH f6F). The amplitude of the EPSP was measured from the second maximal negativity to the isoelectric fine, while the amplitude of the PS was determined from the positive peak to the line connecting the first and second maximal negativities.
The drugs used were: 6-(4-difluoromethoxy-3metho~phenyf-3(2H)pyridazinone tzardaverine; Byk Gufden. Konstanz, F.R.G.); 4-(3-cyc!o~entyfo~-4m-thoxyphenyll-2-pyrrofidone (rofipram; Schering, Berlin, F.R.G.); 3-isobutyl-1-methyfxanthine (IBMXI, ( - Iisoproterenol (Sigma, Miinchen, F.R.G.). Stock solutions (IO mMf of IBMX and isoproterenof were prepared with distilled water; zardaverine was dissolved in I N NaOH and rolipram in 1% ethanol. Further dilutions were made with medium. Equivalent quantities of the solvents had no effect. 2.3. Sintistics The results are expressed as means + S.E.M. The paired, two-tailed Student’s r-test was used for comparison of means, and for comparison of the means with zero. A probability level of 0.05 or fess was considered to be statistically significant.
3. Results
In two preliminary experiments IBMX 10 FM was applied for 30 min. IBMX increased both the EPSP and I’S; the peak enhancement was reached IO-15 min after addition. Slow recovery of responses occurred when the compound was washed out and became complete after about 60 min. In the subsequent six experiments increasing concentrations (0.01-10 PM) of IBMX were applied CUmufatively for 10 min each. IBMX (O.l-f0 FM) produced a concentrat~on~dep~ndent increase of the EPSP and PS ffig. 1; 6 slices). IBMX 10 FM evoked a second
i
,IBMX
p----i----
B
\.
--
i
-I 5
ms
. IIIII
0. I
Concenrratmn
5 mV
1 (pM1
---+a-RL 10
Fig. I. Effects of IBMX, zardaverine and rolipram on field potentials in hippocampal CA1 pyramidal cells of the rat. (A) Percent changes in the amplitude of the excitatory postsynaptic potential (EPSP). 0, IBMX; 0, zardaverine (ZRD); 11, rolipram (RL). (B) Percent changes in the ampIitude of the populatjon spike (PSI. e, IBMX, zardaverine; A. rolipram. Inset: average of 4 field potentials before (continuous line) and after (broken line) the application of zardaverine IO WM. Means* S.E.M. of 6 (IBMX, ZRDI and 4 (RL) experimcnts both in A and B. * P < 0.05-0.01; significant differences from zero.
PS in all 8 slices tested, and led to epifeptiform discharges in 2 slices.
after-
3.2. Effects of zardu~~erine and roiipram A cumulative concentration-response cutve was determined for zardavcrine (0.01-10 PM) according to the protocol described for IBMX. Even the highest concentration (10 PM) of zardaverine failed to alter the field potentials (fig. 1; 6 slices). When the contact time with zardaverine 10 PM was increased from 10 to 30 min, the amplitude of EPSP or PS did not change either (3 slices). In order to obtain information about the effect of zardaverine at various stimulation voltages, the inputoutput curves were determined both before and IO min after the application of zardaverine 10 FM. The curves obtained were practically identical in the absence and presence of the PDE inhibitor (2 slices). Zardaverine 10 FM did not evoke a second PS in any of the 11 slices tested. When rofipram Uf.Ol-10 PM) was applied cumulatively, there was no change in the field potentials either (fig. I; 4 slices). Rolipram 10 FM failed to evoke a second PS.
119 EPSP
PS
IPR IPR ZROclPR
IPR IPR ZRD+IPR
EPSP
PS
IPR IPR RL+IPR
IPR IPR RL+lPR
Fig. 2. Effects of zardaverine and rolipram on the enhancement of field potentials by isoproterenol. (A) Percent increase of the excitatory postsynaptic potential (EPSPf and population spike (PSI by isoprotereno1 I /LM before (IPR), during CZRDi-IPR) and after (IPR) the application of zardaverine 0.1 &M. (Bl Percent increase of the EPSP and PS by isoproterenol 1 FM before (IPRl, during (RLi-IPRl and after (IPR) the application of rolipram I PM. MeansfS.E.M. obtained from 5 (A3 and h (Elf experiments. * P < O&5--0.01: significant differences from the effects of isoproterenol alone.
3.3. Effect of ,isoproterenol and its interaction with zardaserine or rolipram
Since isoproterenol was expected to induce desensitization, increasing non-cumulative concentrations (0.01-l @Ml of the P-agonist were applied for 5 min every 20-30 min. The enhancement of the EPSP was 4.8rtO.9% at 0.01 FM, 12.6+1.1% at 0.1 FM and 16.5 rt 1.4% at 1 PM isoprotercnol, while the corresponding increases of the PS were 4.3 + 0.8, 7.7 & 1.3 and IO.0 + 2.1% (P < 0.01 in each case; 5 slices). When the incubation with isoproterenof 1 PM was for 10 min, the maximum effect was reached after about 5 min, and thereafter the responses decreased in spite of the presence of the drug (2 slices). isoproterenol 1 FM evoked a second PS in only 1 out of 18 slices. Although zardaverine 0.1 PM and rolipram 1 PM did not change the field potentials on their own (see fig. l), they markedly increased the effect of isoproterenol 1 PM (5 and 6 slices, respectively). The PDE inhibitors were added for 10 min alone and for 5 min in combination with isoproterenol. An interval of 30-50 min was allowed between subsequent applications of isoproterenol. The enhancement of the EPSP and PS
by zardaverine (fig. 2AI and rolipram (fig. 2B) was significant, and reversible on washout.
The main finding of this work is that, in contrast to the xanthine derivative IBMX, zardavcrine and roIipram increase the excitabili~ of hip~campaI pyramidal CA1 neurones only in the presence of isoproterenol. IBMX is a non-selective inhibitor of all known PDE isozymes (Beavo and Reifsnyder, 1990). However, this compound is also an antagonist at the A,-type of P,-purinoceptor (Bruns et al., 1986) and thereby may counteract a tonic inhibition of pyramidal cells by endogenous adenosine; adenosine antagonistic methylxanthines were shown to increase the EPSP and PS of CA1 cells (Haas and Green, 1988). Rofipram and denbufylfine are selective blockers of the CAMP-specific PDE isozyme; zardaverine also blocks the PDE inhibited by cyclic guanosine S-monophosphate icGMPI (Nicholson et al., 1991). It was suggested that the excitability of pyramidal cells is regulated by the CAMP-specjfic PDE, since denbufylline increased the field potentials (Sutor et al., 1990). It was an unexpected finding that, in our experiments, neither rolipram nor zardaverine enhanced the EPSP or the PS. The explanation for this result may be that denbufylline is a weak antagonist at adenosine A ,- and A,-receptors (Nicholson et al., 19891, while rolipram and zardaverine are inactive in this respect (Kifian et at., 1989; Nicholson et al., 1991). In the rat hippocampus isoproterenol activates adrenoceptors of the /3,-subtype and thereby stimulates the enzyme, adenyfate cyclase (Fowler and O’Donnell, 19881. In consequence, the level of intracellular CAMP increases and so does the amplitude of the PS (Fowfer and O’Donnell, 1988). It is striking that rofipram (and zardaverine) did not increase the excitability of CA1 neurones, although rolipram enhanced the level of CAMP in cortical (Challis and Nicholson, 1990) and hippocampal slices of rats (W. Bohnenkamp and C. Schudt, personal communications to a larger extent than did isoproterenol. However, rolipram potentiated the effects of isoproterenol on both the field potentials and the concentration of CAMP. There are two alternative explanations for the discrepancy between these efectrophysiological and biochemical findings. Firstly, there are a number of various cells in hippocampa1 slices and, tberefore, changes of CAMP levels in the whole slice may not reflect correctly the changes of CAMP concentrations in PYramidaf neurones. Under in vitro conditions adenylate cyclase probably has to be stimulated by exogenous catecholamines before an effect of PDE inhibition on
the e~e~tr~~bysio~o~i~al properties of CA1 ueurones can be demonstrated. The situation may be different in in viva experiments since. in contrast to the slice preparation, the noradrenergic input to the pyramidal ~e~~o~es is intact and therefore tonically active. Secondly, isopr~te~no~ may increase the exc~tab~I~ty of CA1 neurones, not only as a consequence of its adenylate cyciase stimulatory properties, but also by some additional uukno~ mechanism which is potent~ated by PDE inhibition.
Challis. R.A.J. and CD. Nicholson, 1990. Effects of selective phosphodiesterase ~ub~bitionon cyclic AMP hydrol~is in rat cerebral cortical slices. Br. J. Pharmacol. 99. 47. Fowler. J.C. and J.M. O’Donnell, 1988. Antagonism of the responses to isoproterenol in the rat hippocampal slice with subtype-selective antagonists. Eur. J. Fharmacol. t.53, JO.% Haas, H.L. and R.W+ Greene, 1988, ~Ie~troph~ioIogi~~ anafysis of the effects of exogenous and endogenous adenosine in hippocampal slices, in: Neurotransmitters and Cortical Function, eds. M. Avoli, T.A. Reader, R.W. Dykes and P. Gloor (Plenum Publishing Corporation. New York1 p. 483. fllcs. P. and W. Nurenberg, 1990. Blockade of u,-adrenoceptors increases opioid e-receptor-mediated inhibition of the firing rate of rat locus coeruleu- neurones, Naunyn-~hmiedeb. Arch. PharIElkSCO!.
We are grateful to Mrs. E. Forster and A. BOB fur technizal assistance.
Bravo. J.A. and D.H. Reifsnyder, 19nt). Primary sequence of cyclic nucieotide phosphodiesterasr isozymes and the design of selective inhihitors. Trends Pharntacol. Sci. 11. 150. Bruns. R-F.. G.H. Lu and T.A. Pugsley. 1986. Characterization of the A, adenosine receptor fabeied by [-‘HfNECA in rat striatat membranes. Mot, Pharmaeoi. 29. 33f.
342. 490.
Kilian. U.. R. Beume. M. Ettze. C. Schudt, 1989. Is phosphodiesterase inhibition a relevant bronchospasmolytic principle? Agents Actions Suppl. 28. 331. Nicholson. CD., S.A. Jackman and R. Wilke, 1989, The ability of denbu~~Ijne to inhibit cyclic nucieotide p~sph~iesterase a..d its affinity for adenosine receptors and the adenosine re-uptake site, Br. J. Pharmacol. 97, 889. Nicholson, CD.. R.A.J. Challiss and M. Shahid, 1991, Differential modulation of tissue function and therapeutic potential of selective inhibitors of cyclic nucleotide phosphodiesterase isaenqmes, Trends Pharmacol. Sci. 11, 19. Sutor. B., C. Alzheimer, A. Ameri and G. Ten Bruggencate, 1990. The tow K~-pbosphod~esteras~ inhibjtor denbu~~line enhances neuronal excitability in guinea-pig hip~ampus in vitro, Naunyn-Schmiedeb. Arch. Pharmacol. 342,349.
117
EJP 20898
Short commuaicat~o~
Laszlo Gaal L* *, Christian Schudt ’ and Peter Illes 1*2 ’ apartment o~phu~aculu~,
Byk Guldm Pha~ace~t~ca~, ok-GLdde~-stray 2, O-7750 ~D~~ta~~ F.R.G. and 2 ~cpa~ment o~Phu~aco~~~, ~nil~e~i~ of Freibtq, Hermann-Herder-Strasse S, O-7800 Freiburg LB., F.R.G.
Received 23 April 1991, revised MS received 20 June 1991, accepted 9 July 1991
~tracel~ular field potentials were evoked in the CA1 pyramidal cell layer of the isolated rat hip~campus by electrical stimulation of the s&ratum radiatum. Of the three phosphodiesterase (PDE) inhibitors, 3-isobutyl-i-methy~anthi~~ (IBMX), zardaverine and rolipram, only the adenosine receptor antagonist, IBMX, increased the amplitudes of the extracellular excitatory postsynaptic potential (EPSP) and population spike (PS). The @-adrecoceptor agonist, isoproterenol, also facilitated these potentials and became more potent in the presence of zardaverine or rolipram. The results suggest that PDE blockade increases the excitability of pyramidal neurones only after preceding stimulation of P-adrenoceptors. Hip~ampal
pyramidal cells; Fiefd potentials; Phosphodiesterase inhibitors; ~-Adrenoceptor Adenosine receptor antagonists
1. Introduction 3-Isobu~l-1-methy~anthine (IBMX~ inhibits various cyclic nucleotide phosphodies:erase (PDE) isozymes (Beavo and Reifsnyder, i,‘90). It was suggested that the alkylxanthine, denbufjllline, increases the excitability of hippocampal CA1 pyramidal cells by blocking a cyclic adenosine 5’-monophosp~ate(CAMP)-specific PDE isozyme (Sutor et al., 1990). IBMX had a similar effect, probably both by inhibition of PDE activity and blockade of the A,-subtype of P,-purinoceptor (Sutor et al., 19901, which mediates tonic inhibition of pyramidal cells in response to endogenous adenosine (Haas and Greene, 1988). However, all xanthine derivatives, including denbufylline (Nicholson et al., 1989), appear to bind to adenosine A,- and A,-receptors (Bruns et al., P986). In order to separate the influence of adenosine receptor antagonism from that of PDE inhibition, we decided to investigate the effect of the non-xanthine PDE in-
Correspondence to: P. files, Department of Pharmacology, Wniversity of Freiburg, He~ann-Herder-Strasse 5, D-7X00 Fteibur~ i.Br., F.R.G. * Dedicated to professor E. Mutschier in honour of his hOth birthday. ** Present address: Chemical Works of Gideon Richter. P.O. Box 27, H-147.5 Budapest, Hungary.
agonists;
hibitors, rolipram and zardaverine, which have no A,or AZ-affinity (Kilian et al., 1989; Nicholson et al., 1991) and therefore are unlikely to interfere with endogenous adenosine. In fact, rolipram and zardaverine increased hippocampal CA1 excitability only when padrenoceptors were activated by isoproterenol.
2. Materials and methods 2.1. Preparation and recording Male Wistar rats ilSO-220 g) were anaesthetized with isoflurane (Forene @,Abbott, Wiesbaden, F.R.G.) and decapitated. Hippocampal slices (400 pm thick) prepared with a McIlwain chopper were placed in a recording chamber (Illes and NBrenberg, 1990) and superfused with medium at a rate of 2 ml/min. The medium (mM): NaCl 124; KCI 3; NaH2P0, 1.3; MgSG, 2; CaCI, 2; NaHCO, 26, and glucose 10; pH 7.4 was saturated with 95% 0*-s% CO, and maintained at 33°C. A bipolar stimulation electrode was placed in the stratum radiatum, Pulses of O.l-ms duration were supplied by the isolation unit of a HI-MED stimulator at a frequency of 0.1 Hz. The voltage was adjusted to evoke 60-70% of the maximal amplitude of the population spike (PS). Experiments were started only if the extra-
~~~f~~~r~~c~tato~ postsy~apt~c potentials (EPSP) and pS had been stable for at feast 30 min. Responses were recorded with glass micro-electrodes (resistances 2-8 ‘Ml)) fifled with 2 M NaCI. The electrode was placed in the CA1 pyramidal cell layer at a depth of 100-200 em and was connected to an AC amplifier (WPI DAM SO): the EPSP and PS were sampled, stored and anafyzed by means of a computer (IBM XT-2861 fitted with an AD converter ~~etrabyt~, DASH f6F). The amplitude of the EPSP was measured from the second maximal negativity to the isoelectric fine, while the amplitude of the PS was determined from the positive peak to the line connecting the first and second maximal negativities.
The drugs used were: 6-(4-difluoromethoxy-3metho~phenyf-3(2H)pyridazinone tzardaverine; Byk Gufden. Konstanz, F.R.G.); 4-(3-cyc!o~entyfo~-4m-thoxyphenyll-2-pyrrofidone (rofipram; Schering, Berlin, F.R.G.); 3-isobutyl-1-methyfxanthine (IBMXI, ( - Iisoproterenol (Sigma, Miinchen, F.R.G.). Stock solutions (IO mMf of IBMX and isoproterenof were prepared with distilled water; zardaverine was dissolved in I N NaOH and rolipram in 1% ethanol. Further dilutions were made with medium. Equivalent quantities of the solvents had no effect. 2.3. Sintistics The results are expressed as means + S.E.M. The paired, two-tailed Student’s r-test was used for comparison of means, and for comparison of the means with zero. A probability level of 0.05 or fess was considered to be statistically significant.
3. Results
In two preliminary experiments IBMX 10 FM was applied for 30 min. IBMX increased both the EPSP and I’S; the peak enhancement was reached IO-15 min after addition. Slow recovery of responses occurred when the compound was washed out and became complete after about 60 min. In the subsequent six experiments increasing concentrations (0.01-10 PM) of IBMX were applied CUmufatively for 10 min each. IBMX (O.l-f0 FM) produced a concentrat~on~dep~ndent increase of the EPSP and PS ffig. 1; 6 slices). IBMX 10 FM evoked a second
i
,IBMX
p----i----
B
\.
--
i
-I 5
ms
. IIIII
0. I
Concenrratmn
5 mV
1 (pM1
---+a-RL 10
Fig. I. Effects of IBMX, zardaverine and rolipram on field potentials in hippocampal CA1 pyramidal cells of the rat. (A) Percent changes in the amplitude of the excitatory postsynaptic potential (EPSP). 0, IBMX; 0, zardaverine (ZRD); 11, rolipram (RL). (B) Percent changes in the ampIitude of the populatjon spike (PSI. e, IBMX, zardaverine; A. rolipram. Inset: average of 4 field potentials before (continuous line) and after (broken line) the application of zardaverine IO WM. Means* S.E.M. of 6 (IBMX, ZRDI and 4 (RL) experimcnts both in A and B. * P < 0.05-0.01; significant differences from zero.
PS in all 8 slices tested, and led to epifeptiform discharges in 2 slices.
after-
3.2. Effects of zardu~~erine and roiipram A cumulative concentration-response cutve was determined for zardavcrine (0.01-10 PM) according to the protocol described for IBMX. Even the highest concentration (10 PM) of zardaverine failed to alter the field potentials (fig. 1; 6 slices). When the contact time with zardaverine 10 PM was increased from 10 to 30 min, the amplitude of EPSP or PS did not change either (3 slices). In order to obtain information about the effect of zardaverine at various stimulation voltages, the inputoutput curves were determined both before and IO min after the application of zardaverine 10 FM. The curves obtained were practically identical in the absence and presence of the PDE inhibitor (2 slices). Zardaverine 10 FM did not evoke a second PS in any of the 11 slices tested. When rofipram Uf.Ol-10 PM) was applied cumulatively, there was no change in the field potentials either (fig. I; 4 slices). Rolipram 10 FM failed to evoke a second PS.
119 EPSP
PS
IPR IPR ZROclPR
IPR IPR ZRD+IPR
EPSP
PS
IPR IPR RL+IPR
IPR IPR RL+lPR
Fig. 2. Effects of zardaverine and rolipram on the enhancement of field potentials by isoproterenol. (A) Percent increase of the excitatory postsynaptic potential (EPSPf and population spike (PSI by isoprotereno1 I /LM before (IPR), during CZRDi-IPR) and after (IPR) the application of zardaverine 0.1 &M. (Bl Percent increase of the EPSP and PS by isoproterenol 1 FM before (IPRl, during (RLi-IPRl and after (IPR) the application of rolipram I PM. MeansfS.E.M. obtained from 5 (A3 and h (Elf experiments. * P < O&5--0.01: significant differences from the effects of isoproterenol alone.
3.3. Effect of ,isoproterenol and its interaction with zardaserine or rolipram
Since isoproterenol was expected to induce desensitization, increasing non-cumulative concentrations (0.01-l @Ml of the P-agonist were applied for 5 min every 20-30 min. The enhancement of the EPSP was 4.8rtO.9% at 0.01 FM, 12.6+1.1% at 0.1 FM and 16.5 rt 1.4% at 1 PM isoprotercnol, while the corresponding increases of the PS were 4.3 + 0.8, 7.7 & 1.3 and IO.0 + 2.1% (P < 0.01 in each case; 5 slices). When the incubation with isoproterenof 1 PM was for 10 min, the maximum effect was reached after about 5 min, and thereafter the responses decreased in spite of the presence of the drug (2 slices). isoproterenol 1 FM evoked a second PS in only 1 out of 18 slices. Although zardaverine 0.1 PM and rolipram 1 PM did not change the field potentials on their own (see fig. l), they markedly increased the effect of isoproterenol 1 PM (5 and 6 slices, respectively). The PDE inhibitors were added for 10 min alone and for 5 min in combination with isoproterenol. An interval of 30-50 min was allowed between subsequent applications of isoproterenol. The enhancement of the EPSP and PS
by zardaverine (fig. 2AI and rolipram (fig. 2B) was significant, and reversible on washout.
The main finding of this work is that, in contrast to the xanthine derivative IBMX, zardavcrine and roIipram increase the excitabili~ of hip~campaI pyramidal CA1 neurones only in the presence of isoproterenol. IBMX is a non-selective inhibitor of all known PDE isozymes (Beavo and Reifsnyder, 1990). However, this compound is also an antagonist at the A,-type of P,-purinoceptor (Bruns et al., 1986) and thereby may counteract a tonic inhibition of pyramidal cells by endogenous adenosine; adenosine antagonistic methylxanthines were shown to increase the EPSP and PS of CA1 cells (Haas and Green, 1988). Rofipram and denbufylfine are selective blockers of the CAMP-specific PDE isozyme; zardaverine also blocks the PDE inhibited by cyclic guanosine S-monophosphate icGMPI (Nicholson et al., 1991). It was suggested that the excitability of pyramidal cells is regulated by the CAMP-specjfic PDE, since denbufylline increased the field potentials (Sutor et al., 1990). It was an unexpected finding that, in our experiments, neither rolipram nor zardaverine enhanced the EPSP or the PS. The explanation for this result may be that denbufylline is a weak antagonist at adenosine A ,- and A,-receptors (Nicholson et al., 19891, while rolipram and zardaverine are inactive in this respect (Kifian et at., 1989; Nicholson et al., 1991). In the rat hippocampus isoproterenol activates adrenoceptors of the /3,-subtype and thereby stimulates the enzyme, adenyfate cyclase (Fowler and O’Donnell, 19881. In consequence, the level of intracellular CAMP increases and so does the amplitude of the PS (Fowfer and O’Donnell, 1988). It is striking that rofipram (and zardaverine) did not increase the excitability of CA1 neurones, although rolipram enhanced the level of CAMP in cortical (Challis and Nicholson, 1990) and hippocampal slices of rats (W. Bohnenkamp and C. Schudt, personal communications to a larger extent than did isoproterenol. However, rolipram potentiated the effects of isoproterenol on both the field potentials and the concentration of CAMP. There are two alternative explanations for the discrepancy between these efectrophysiological and biochemical findings. Firstly, there are a number of various cells in hippocampa1 slices and, tberefore, changes of CAMP levels in the whole slice may not reflect correctly the changes of CAMP concentrations in PYramidaf neurones. Under in vitro conditions adenylate cyclase probably has to be stimulated by exogenous catecholamines before an effect of PDE inhibition on
the e~e~tr~~bysio~o~i~al properties of CA1 ueurones can be demonstrated. The situation may be different in in viva experiments since. in contrast to the slice preparation, the noradrenergic input to the pyramidal ~e~~o~es is intact and therefore tonically active. Secondly, isopr~te~no~ may increase the exc~tab~I~ty of CA1 neurones, not only as a consequence of its adenylate cyciase stimulatory properties, but also by some additional uukno~ mechanism which is potent~ated by PDE inhibition.
Challis. R.A.J. and CD. Nicholson, 1990. Effects of selective phosphodiesterase ~ub~bitionon cyclic AMP hydrol~is in rat cerebral cortical slices. Br. J. Pharmacol. 99. 47. Fowler. J.C. and J.M. O’Donnell, 1988. Antagonism of the responses to isoproterenol in the rat hippocampal slice with subtype-selective antagonists. Eur. J. Fharmacol. t.53, JO.% Haas, H.L. and R.W+ Greene, 1988, ~Ie~troph~ioIogi~~ anafysis of the effects of exogenous and endogenous adenosine in hippocampal slices, in: Neurotransmitters and Cortical Function, eds. M. Avoli, T.A. Reader, R.W. Dykes and P. Gloor (Plenum Publishing Corporation. New York1 p. 483. fllcs. P. and W. Nurenberg, 1990. Blockade of u,-adrenoceptors increases opioid e-receptor-mediated inhibition of the firing rate of rat locus coeruleu- neurones, Naunyn-~hmiedeb. Arch. PharIElkSCO!.
We are grateful to Mrs. E. Forster and A. BOB fur technizal assistance.
Bravo. J.A. and D.H. Reifsnyder, 19nt). Primary sequence of cyclic nucieotide phosphodiesterasr isozymes and the design of selective inhihitors. Trends Pharntacol. Sci. 11. 150. Bruns. R-F.. G.H. Lu and T.A. Pugsley. 1986. Characterization of the A, adenosine receptor fabeied by [-‘HfNECA in rat striatat membranes. Mot, Pharmaeoi. 29. 33f.
342. 490.
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