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Patch clamp reveals powerful blockade of the mitochondrial permeability transition pore by the D2-receptor agonist pramipexole Iqbal Sayeed,1 Suhel Parvez,1 Kirstin Winkler-Stuck, Gordon Seitz, Isabelle Trieu, Claus-Werner Wallesch, Peter Scho¨nfeld,* and Detlef Siemen2 Departments of Neurology and *Biochemistry, University of Magdeburg, D-39120 Magdeburg, Germany To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4748fje; 10.1096/fj.05-4748fje SPECIFIC AIMS The motor scores in Parkinson’s disease decline slower if the patients are treated with dopamine agonists instead of l-dopa. We wanted to see if this protective effect could be explained by the inhibition of the cascade leading to apoptotic cell death with the mitochondrial permeability transition pore (PTP) as a potential target.
PRINCIPAL FINDINGS 1. Proving the identity of the PTP by dose-dependent cyclosporin A (CSA) blockade It could be inhibition of the PTP that causes the neuroprotective effect of the dopamine-D2 agonist pramipexole (PPX). We prepared mitoplasts (swollen and deenergized mitochondria devoid of their outer membrane by hypotonic treatment) from rat liver mitochondria (RLM) and applied patch-clamp techniques in the mitoplast-attached mode for measuring single-channel currents. Almost every second patch with sufficient seal resistance showed a distinct type of Ca2!-induced activity of very large ("1 nS) conductance with several subconductance states and highly variable kinetics that could be activated by depolarization. Activity of this type is characteristic for the PTP. To further ascertain the identity of the PTP, we added its selective inhibitor CSA at a holding potential of !20 mV and found a reversible blockade of the activity within a few minutes. This effect was concentration dependent (8 independent experiments at 4 different concentrations). The corresponding relation was fitted best by Michaelis-Menten (MM) kinetics assuming an IC50 of 26 nM. 2. PPX inhibits PTP
we tried application of the dopamine-D2 agonist PPX at various concentrations to patches from which the PTP was recorded earlier (Fig. 1A). At a holding potential of !20 mV PPX reversibly blocked the PTP. The concentration response relation (n#12) was fitted again by MM kinetics using an IC50 of 500 nM (Fig. 1B). The fact that the curve does not approach zero even at high concentrations owes to the way it was determined. 3. No modulatory effect on PTP by L-dopa and dopamine l-dopa and its metabolite dopamine were not reported to exhibit a pronounced neuroprotective effect when used for treating Parkinson’s disease (PD) patients. Therefore, we checked as a control whether these substances would be able to block the PTP as well. In three experiments with 10 $M l-dopa, the mean Po was 0.59 % 0.11 before, 0.57 % 0.04 in l-dopa, and 0.57 % 0.09 in the control after, respectively. The corresponding values for four experiments with 10 $M dopamine were 0.61 % 0.09 before, 0.66 % 0.05 in dopamine, and 0.64 % 0.08 in the control solution after. These results show that neither substance was able to inhibit the PTP. 4. Inorganic phosphate (Pi) attenuates PTP blockade by PPX It was noticed that phosphate in the buffer solutions can attenuate the inhibitory effect of pramipexole on the PTP. Therefore single-channel experiments with 20 $M PPX (a concentration large enough to show considerable blocking effect) and 10 mM Pi (a concentration covering the intracellular range) were performed. In one set of experiments the phosphate was added to the bath only. In a second set it was also added to the 1
These authors contributed equally to this study. Correspondence: Department of Neurology, University of Magdeburg, Leipziger Str. 44, D-39120 Magdeburg, Germany. E-mail:
[email protected] 2
Bearing in mind the blockade of permeability transition (PT) by CSA with its known anti-apoptotic effect, 556
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6. Acidification-induced PT is decreased by PPX It has been reported that acidification of the incubation medium potentiates PT. As a validative experiment, the tendency of mitochondria to undergo PT was quantified by measuring the time (tPTmax) required for complete PT of RLM suspended in swelling medium at pH values &7.0 with and without 100 $M PPX. We demonstrate that acidification of the medium pH from 7.2 to 6.8 dramatically accelerates PT. Data are means of experiments from 4 – 6 different preparations. In the presence of PPX PT stimulated by acidification was decreased. 7. PPX inhibits lipid peroxidation It is known that PT is promoted by reactive oxygen species (ROS) due to oxidation of critical sulfhydryl groups within the PTP. This might suggest that a secondary activity of PPX could be the protection of PT by suppressing sulfhydryl group oxidation. We demon-
Figure 1. PPX reversibly inhibits current through the PTP. A) Single-channel current traces of the PTP at Eh # !20 mV before, during, and after application of 2 $M PPX by the flow system. B) Concentration response curve for the PPX effect on the PTP (Eh # !20 mV). Data from 12 independent experiments shown as mean of the normalized Po values Po/Po,max (%se), where Po,max is the mean Po of three 1 min segments recorded before adding the test solution. Continuous curve calculated using MM kinetics with IC50 # 500 nM and A # 0.33.
pipette, i.e., to either side of the membrane. Although there was no change of the pronounced PPX effect by Pi when given from the bath side only, it nearly abolished the PPX inhibition when given from both sides (Fig. 2A). The corresponding Po values were 0.58 % 0.02 before PPX, 0.20 % 0.04 (n#3) in PPX alone, and 0.19 % 0.02 (n#4) in PPX plus Pi at one side. In a different set of 4 experiments Po was reduced from 0.52 % 0.03 to 0.42 % 0.03 when Pi as added from both sides. In other words, the Po -reduction by PPX of 66% was attenuated to a 19% reduction in the presence of Pi on both sides (Fig. 2B). 5. PPX inhibits Ca2!-triggered swelling of mitochondria A pronounced inhibition of the PTP by PPX seen with mitoplasts raises the question of to which extent PPX prevents Ca2!-triggered PT in intact mitochondria. Absorbance measurements on RLM and on rat brain mitochondria (RBM) indicated that 100 $M PPX inhibited the CSA-sensitive (1 $M), Ca2!-triggered large amplitude swelling even in the presence of Pi. PRAMIPEXOLE BLOCKS PERMEABILITY TRANSITION PORE
Figure 2. Phosphate attenuates the PPX inhibition of the PTP if given from both sides of the patch. A) Upper trace shows inhibition by 20 $M PPX in the presence of 10 mM Pi given only from the flow system at the time marked by arrow. Lower trace is from another experiment with Pi both in the flow system and inside the measuring pipette. Record was taken after "3 min to be sure that a PPX effect had not been overlooked. Eh # !20 mV for both traces. B) Mean Po values (%se) of experiments with PPX alone (n#3) or with PPX plus Pi in flow system only (n#4) vs. mean Po before adding test substances. Reduction was significant on the 5% and 1% level. Pi on either side of the pipette compared with and without PPX showed no significant difference (n#4). 557
strate that lipid peroxidation of RLM exposed to oxidative stress is partly suppressed by 0.2 mM and even more at 1 mM PPX, as indicated by the decline of formed thiobarbituric acid reactive. Consequently, PPX exerts anti-oxidative protection on mitochondria. CONCLUSIONS AND SIGNIFICANCE PPX is the most frequently used dopamine agonist, acting on D2/D3 receptor subtypes. Other effects of PPX are reported such as 1) an anti-oxidative property on cells, and 2) an anti-apoptotic action. PPX exists as (!)- and as (–)-enantiomer. In contrast to stereoselective D2 receptor binding of the PPX (–)-enantiomer, the anti-apoptotic effect is exerted by both enantiomers. Finally, PPX may induce 3) down-regulation of the dopamine transport, which in turn may downregulate MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) uptake. The clinically established neuroprotective effect of PPX is likely caused by one or more of these activities. This is further supported by reports demonstrating in a human neuroblastoma cell line that PPX-induced neuroprotection against MPP! and rotenone acts via a non-dopaminergic mechanism. We demonstrate here by application of PPX in singlechannels measurements its direct inhibition of the PTP in the inner mitochondrial membrane. Characterization of the PT, such as quantification of the time course of PTP opening, is best done with liver mitochondria. Most of our experiments were performed on RLM and mitoplasts. Despite minor differences in the protein composition of various tissues, it is well established that the PT is modulated by the same effectors (such as pH, Pi , Ca2!, depolarization, and others). Only the sensitivity to CSA may be reduced in RBM, as seen in our experiments. For this reason, we are convinced that the protective action of PPX described here is not specific for liver mitochondria. The PTP seems to be an extraordinary large pore (or several cooperating pores) characterized by large conductance ("1 nS), a variety of substates, activation by membrane depolarization, and its biochemistry. Strong modulation of the PTP was demonstrated with CSA, melatonin, and pH (inhibition), and with Ca2!, Pi , and PAO (activation). For this study, the identity of the PTP-related current events was proved by blockade with CSA. Since the concentration response of single PTPs to CSA had not been reported, we measured the Po over a broad range of CSA concentrations. The IC50 of 26 nM obtained is in line with the crude estimate of 10 to 100 nM for inhibition by Szabo´ and Zoratti. The high Po at rest seen in our experiments is explained by the presence of Ca2! (needed to obtain and keep reliable patches). l-dopa, still considered the gold standard for treating PD, is metabolized to dopamine acting on the receptors of the striatal neurons, where it modulates the postsynaptic response. We explored whether dopa-
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Figure 3. Under normal conditions, S. nigra neurons project on striatum neurons by means of the transmitter dopamine. In Parkinson’s disease, apoptosis of S. nigra neurons leads to a reduced availability of dopamine. l-dopa, being metabolized to dopamine, may compensate for the loss of transmitter partly. Besides its agonistic effect on the D2 receptor, the dopamine agonist pramipexole can interrupt the apoptosis signaling chain by blocking the PTP. It could thus protect the neurons from programmed cell death.
mine and PPX had a common effect on the PTP as well, and found there was no direct effect either by dopamine or its precursor l-dopa on the PTP. Nor does dopamine affect Ca2!-induced PT in intact mitochondria. It has to be asked if the measured concentration response curve for PPX is relevant for the patient. A fluctuation of the intracellular Pi concentration, e.g. is likely to interfere with the blockade by PPX (Fig. 2). It is not known what the precise concentration of PPX in the cytosol of the treated patients really is. However, from volunteers treated with the usual doses of PPX, mean plasma concentrations of PPX were measured to be in the range of 4 – 6 ng/mL. When it is assumed that PPX can permeate the plasma membrane of neuronal cells as shown by earlier experiments, a 100-fold enrichment inside the cell can be estimated from the plasma membrane potential (& – 60 mV) and the doublecharged nature of PPX. This resulted in a total mean intracellular concentration of 1.6 $M PPX, a concentration that corresponds to half-maximal blockade of PTP (Fig. 1B). It was reported that the binding of PPX to plasma proteins is ' 20%. Thus, inhibition of the PTP by free PPX seems to be sufficient for considerable reduction of mitochondria-linked apoptosis without suppressing it completely. A crucial test for the assumed reduction of apoptosis by PPX would be the presence of a beneficial neuroprotective effect in patients with neurodegenerative diseases other than PD like Alzheimer’s disease or amyotrophic lateral sclerosis. This paper summarizes strong arguments that PPX could prevent PT. Therefore, it could help suppress the mitochondria-linked pathway of apoptosis.
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