Rilmenidine Elevates Cytosolic Free Calcium Concentration in Suspended Cerebral Astrocytes
Total Page：16
File Type：pdf, Size：1020Kb
Journal ofNeurochemistry Lippincott—Raven Publishers, Philadelphia © 1998 International Society for Neurochemistry Rilmenidine Elevates Cytosolic Free Calcium Concentration in Suspended Cerebral Astrocytes Mark A. Ozog, *John X. Wilson, *~S.Jeffrey Dixon, and David F. Cechetto Departments of Anatomy and Cell Biology and * Physiology and l’Division of Oral Biology, Faculty of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada Abstract: Rilmenidine, a ligand for imidazoline and a 2- 1990). Imidazoline receptors are divided3H]c!onidine into subtypes adrenergic receptors, is neuroprotective following focal It(Ernsbergerand 12. TheetI~ al.,subtype1995)1 ~!abe!edis present by [ on neurona! cerebral ischemia. We investigated2~concentrationthe effects([Ca2])of rilmeni-in rat plasma membranes (Ernsberger et al., 1995) within dineastrocytes.on cytosolicRilmenidinefree Cacaused concentration-depen- the rostra! ventro!ateral medulla, hippocampus, hypo- dent elevation of [Ca2~], consisting of a transient in- tha!amus, and striatum (Kamisaki et a!., 1990), and crease (1—100 ~iMrilmenidine) or a transient increase followed by sustained elevation above basal levels (1— may be linked to G proteins (Bricca et al., 1994; Ems- 10 mM rilmenidine). A similar elevation in [Ca2~],was berger et al., 1995). In contrast, the ‘2 subtype [labe!ed induced by the imidazoline ligand cirazoline. The transient by [3H]idazoxan (Regunathan et a!., 1993)] is loca!- response to rilmenidine was observed in Ca2~-free me- ized to astrocytic mitochondrial membranes (Tesson dium, indicating that rilmenidine evokes release of Ca2~ and Parini, 1991) and is not linked to G proteins (Re- from intracellular stores. However, the sustained eleva- gunathan et al., 1991). tion of Ca2~was completely dependent on extracellular It has been shown recently that certain imidazoline Ca2~,consistent with rilmenidine activating Ca2 influx. receptorligands are neuroprotective following an isch- Pretreatment with thapsigargin, an inhibitor of the endo- ernie insu!t. The first indication that imidazoline recep- plasmic reticulum Ca2~-ATPase,abolished the response to rilmenidine, confirming the involvement of intracellular tors are invo!ved in neuroprotection was from Gustaf- stores and suggesting that rilmenidine and thapsigargin son et al. (1989, 1990), who showed that idazoxan, activate a common Ca2~influx pathway. The a an imidazo!ine receptorligand that is a!so an a 2-adrener- 2-adren- gic 2~]~antagonistinduced rauwolscineby clonidine attenuated(a selective thea increase in ergic receptor antagonist, reduces the infarct size in [Ca 2 agonist), but rats following occlusion of the middle cerebral artery. not the response to rilmenidine. These2~releaseresultsfromindicateintra- It was later demonstrated that both idazoxan and ri!- thatcellular rilmenidinestores andstimulatesCa2~influx bothbyCaa mechanism indepen- menidine reduce the infarct size by a mechanism dis- dent of a tinct from both a2-adrenoceptor interaction and local 2-adrenergic receptors.2~from theIn vivo,extracellularrilmenidinefluidmayby cerebra! blood flow elevation (Maiese et a!., 1992). enhanceastrocytes,uptakea processof Cathat may contribute to the neuro- protective effects of this agent. Keywords: Astrocytes— In addition, the neuroprotective effect of ri!menidine Rilmenidine—Calcium—Imidazoline receptors—Cloni- has been shown to be dose-dependent (Maiese et al., dine. 1992). J. Neurochem. 71, 1429—1435 (1998). Within the cerebral cortex, neuronal imidazoline re- ceptors are absent (Kamisaki et a!., 1990), whereas cortical astrocytes express the ‘2 subtype on the outer mitochondria! membrane (Wikberg and Uhlén, 1990; Rilmenidine, clonidine, and related compounds bind Tesson and Parini, ! 991; Ma!!ard et al., 1992). There- to both imidazoline receptors and a fore, it is likely that ri!menidine induces neuroprotec- 2-adrenoceptors. tion in cortex via activation of astrocytic imidazoline Imidazoline receptors are distinguished from a2-adre- receptors. Although the signal transduction pathway of noceptors on the basis of anatomical distribution (Co- imidazo!ine receptors remains unclear, it is possible upry et al., 1989; Kamisaki et a!., 1990), signal trans- duction mechanisms (Michel et a!., 1990), binding Received November 27, 1997; revised manuscript received April profiles (Ernsberger et al., 1987, 1988), and insensitiv- 6, 1998; accepted April 28, 1998. ity to catecholamines and GTP analogues (Wikberg Address correspondence and reprint requests to Dr. D. F. Cechetto at Department of Anatomy and Cell Biology, University of Western and Uhlén, 1990; Bricca eta!., 1993, 1994). Cell lines Ontario, London, Ontario, Canada,2~],, cytosolicN6A 5Cl.free Ca2~concentration; transfected with a2-adrenoceptor genes do not demon- I~,Abbreviations‘2, and 12, subtypeused:1, [Ca2, and 3 imidazoline receptor, respectively. strate imidazoline receptor binding (Kamisaki et al., 1429 1430 M. A. OZOG ET AL. equation [Ca2~]~ = K that rilmenidine stimulates astrocytes to buffer poten- 2~.In this 0 [(R — Rmin)/(Rmax — R)]13,2~complexwhere regard,tia!!y neurotoxic ri!rnenidinelevels has beenof extracellularshown to induceCa uptake K0havingis theadissociationvalue of 250constantnM, Rforis thethe indo-lfluorescence—Ca intensity of 45Ca2~in a concentration-dependent manner in mm- at 405 nm divided by the intensity at 485 nm, R,,,~,and Rmin islices of cerebra! cortex (Regunathan et a!., 1995). are the fluorescence ratio values under saturation and low Ca2~conditions, respectively, and /3 is the ratio of fluores- In this study, we have investigated the action of cence values for low and saturating Ca2~concentrations rilmenidine on the cytosolic free Ca2~concentration measured at 485 nm. To obtain saturating [Ca2~]~, the fluo- ([Ca2~I rescence of indo- 1 pentapotassium salt in Na + buffer (1 mM 1) in cerebra! astrocytes. In addition, we have Ca2~)was measured. Low [Ca2~]~was obtained by using examined whether the effects of ri!menidine are depen- Ca2~-free Na~buffer containing EGTA (10mM) and alka- dent on a2-adrenoceptor activation. linizing the buffer with Tris. Data analysis EXPERIMENTAL PROCEDURES The change in [Ca2~I, was calculated by measuring either Materials the peak height of the response above basal level or the Minimum essential medium containing ! .8 mM CaC12 was average height during the first 100 softhe response. The latter means ofmeasurement wasaccomplished using Mocha prepared according to the method outlined by Dixon and Wi!son (1995). Horse serum and Hanks’ balanced salt solu- image analysis software (Jandel Scientific) to determine the tion without CaC12 were obtained from GIBCO Laboratories area between the response curve and the predicted basal level for the first 100 s following injection of drug. All (Burlington, ON, Canada). Pluronic F-127 and both the experiments were done on three or more astrocyte prepara- acetoxymethy! ester and pentapotassium salt of indo-1 were purchased from Molecular Probes (Eugene, OR, U.S.A.). tions from individual litters of rats (except for cirazoline ATP, EGTA, trypsin, phentolamine, c!onidine, and thapsi- experiments, in which two litters were used). The n values gargin were obtained from Sigma Chemical Co. (St. Louis, (stated in parentheses) represent measures from astrocyte MO, U.S.A.). Rauwoiscine was acquired from Research Bio- cultures grown in separate dishes. Data are presented as chemica!s Internationa! (Natick, MA, U.S.A.). Cirazo!ine means ±SEM. Comparisons between mean values based on asingle level of treatmentwere evaluated using the paired was obtained from Tocris (Ballwin, MO, U.S.A.). Ri!meni- test (two-tailed). A p value of <0.05 was considered sig- dine was a gift from Servier Laboratories (Paris, France). nificant. No significant changes in [Ca2~]~ were caused by Falcon brand cu!ture dishes were purchased from VWRCan- the administration of the solvents for all substances tested. ada (Mississauga, ON, Canada).2~-freeTrypsinbuffer containingsolution consistedtrypsin of(0.05%)nomina!!yand CaEDTA2+ - and(0.5MgmM). Na~buffer contained !35 mM NaC1, 5 mM KC1, 1 mM MgC1 RESULTS 2, 1 mM CaC!2, !0 mM glucose, and 20 mM HEPES,2~-freepHNa~7.30buffer±0.02,hadosmolarityadditional [Ca2~ 1. response to rilmenidine 290NaC!±substituted5 mosmol/L.for CaClCa To examine the effect of rilmenidine on [Ca2~I~in 2. astrocytes, 0.1 mM (final concentration) was added to indo-l-!oaded astrocytes suspended in Na~buffer. Cells and cell culture Primary cultures of type 1 astrocytes were prepared from Rilmenidine consistently produced significant in- 1-day-old Wistarrats according to the method of Dixon and creases in [Ca2~°]~immediately following its addition Wilson (!995). Astrocytic cultures were maintained for 14— to the buffer (Fig. 1A). For comparison, we also illus- 18 days before being used for experiments. Immunohisto- trate the increase in [Ca2~°]~induced by 0.1 mM ATP, chemical analysis indicated that 95% of the cultured cells which activates astrocytic purinergic receptors, re- expressed theastrocytic marker, glia! fibri!lary acidic protein sulting in the release of Ca2~from intracellular stores (data not shown). and Ca2~influx (Peuchen et a!., 1996). Rilmenidine 2~], and ATP induced an increase in the fluorescence inten- MeasurementPrimary culturesofof[Caastrocytes were incubatedwith Hanks’ sity at 405 nm concurrently with a decrease in fluores- balanced salt solution containing Pluronic F- 127 (0.03%) and indo- 1 acetoxymethyl ester (5 cence intensity at 485