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A study of myocardial muscarinic receptors in streptozotocin-induced diabetic rats using iodine-123 N -methyl-4-iododexetimide

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A study of myocardial muscarinic receptors in streptozotocin-induced diabetic rats using iodine-123 N -methyl-4-iododexetimide
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  Original article  A study of myocardial muscarinic receptors in streptozotocin-induced diabetic rats using iodine-123 N  -methyl-4-iododexetimide Karine Mardon 1 , Michael Kassiou 2 , Andrew Katsifis 1 , Ljubco Najdovski 1 1 Radiopharmaceutical Division R&D, Australian Nuclear Science and Technology Organisation, Sydney NSW, Australia 2 Department of PET and Nuclear Medicine, Royal Prince Alfred Hospital, Sydney 2050 NSW, Australia & misc : Received 16 November and in revised form 16 February 1999 & p.1: Abstract. In previous studies we have shown that io-dine-123  N  -methyl-4-iododexetimide ([ 123 I]MIDEX) is asuitable single-photon emission tomography radiotracerfor the characterisation of myocardial muscarinic acetyl-choline receptors (m-AChR) in the normal state. It hasbeen demonstrated that m-AChR are altered as a conse-quence of diabetes. The aim of the present study was toexamine myocardial m-AChR density using [ 123 I]MI-DEX in streptozotocin (STZ)-induced diabetic rats. Invitro binding experiments were conducted on left andright ventricle and atrium homogenate membranes of 1-week, 5-week and 10-week STZ-induced diabetic andaged-matched normal rats. The m-AChR densities (  B max values), as determined by saturation experiments with[ 123 I]MIDEX, revealed no difference in left and rightventricles or atrium in 1-week and 5-week STZ-diabeticrats when compared with normal rats. However, the 10-week STZ-diabetic group revealed a 39% ( P< 0.001) de-crease in m-AChR density in atrium with no change inleft and right ventricles. The equilibrium dissociationconstant ( K  d values) was similar in all groups. In vitrobinding autoradiography revealed a 40% decrease in m-AChR density in atrium in the same 10-week diabeticrats. No statistically significant difference was found in1-week and 5-week diabetic rats compared with nor-mals. Ex vivo autoradiography showed a 50% decreasein [ 123 I]MIDEX uptake in atrium in 5-week diabetic ratsand a 60% decrease in 10-week diabetic rats. These re-sults demonstrate the ability of the single-photon agent[ 123 I]MIDEX to measure in vitro and ex vivo alterationsin myocardial m-AChR density observed in STZ-in-duced diabetic rats. & kwd: Key words: Muscarinic receptor – Diabetes – Single-photon emission tomography Eur J Nucl Med (1999) 26:743–749 Introduction Muscarinic acetylcholine receptors (m-AChR) play animportant part in the regulation of the rate and force of heart contractions. Changes in m-AChR concentrationand affinity have been reported in various conditions anddiseases such as ageing [1] and congestive heart disease[2]. Previous studies [3] have demonstrated that myocar-dial m-AChR-mediated responses are altered as a conse-quence of diabetes. However, the cellular and molecularbasis of altered performance of the diabetic heart re-mains poorly understood. Positron-emitting radioli-gands, such as carbon-11 labelled methiodidequinuclidinyl benzilate ([ 11 C]MQNB) [4], with high af-finity for myocardial muscarinic receptors have beencharacterised and used to quantify myocardial m-AChRin dogs and humans [5, 6]. Single-photon agents, iodine-123  N  -methyl-4-iododexetimide ([ 123 I]MIDEX) and itspharmacological inactive enantiomer [ 123 I]  N  -methyl-4-iodolevetimide ([ 123 I]MILEV), have been evaluated assingle-photon emission tomography (SPET) radiotracersfor the characterisation of myocardial m-AChR in rats[7], rabbits and dogs [8]. Single-photon agents suitablefor imaging myocardial m-AChR have not previouslybeen used in evaluating m-AChR alterations in diabetes.The aim of the present study was to examine myocardialm-AChR status using [ 123 I]MIDEX (Fig.1) in rats ren-dered diabetic by the injection of streptozotocin (STZ). European Journal of Nuclear MedicineVol. 26, No. 7, July 1999 – © Springer-Verlag 1999 Correspondence to: K. Mardon, Radiopharmaceutical DivisionR&D, Ansto, Private Mail Bag 1, Menai NSW 2234, Australia &  /fn-bloc k: Fig.1. Chemical structure of [ 123 I]MIDEX &  /fig.c :  744European Journal of Nuclear Medicine Vol. 26, No. 7, July 1999 Materials and methods The study protocol was approved by ANSTO Animal Care andEthics Committee and all animal procedures were carried out incompliance with Australian laws governing animal experimenta-tion.  Radiosynthesis of [ 123  I]MIDEX. & p.2: The preparation of [ 123 I]MIDEXinvolves the preparation of 4-[ 123 I]iododexetimide followed by N-methylation as previously described [7]. Briefly, a freshly pre-pared solution of 4-(trimethylsilyl)dexetimide HCl (0.5mg) andchloramine-T (0.7mg) in trifluoroacetic acid (100  µ l) was addedto a small vial containing aqueous sodium [ 123 I]iodide. The reac-tion mixture was allowed to stand at room temperature for 15minfollowed by addition of high-performance liquid chromatography(HPLC) mobile phase (200  µ l) and concentrated NH 4 OH (120  µ l).The mixture was injected onto a µ -Bondapak C-18 (10  µ m,300mm×7.8mm) semi-preparative column eluted at a flow rateof 2.5ml/min. The mobile phase consisted of 45:55 acetoni-trile:water (0.1  M  ammonium acetate). The effluent from the col-umn was monitored with a UV detector (254nm) and in line ra-dioactivity detector (Berthold, LB 507). The radioactivity corre-sponding to 4-[ 123 I]iododexetimide ( t  R =38min) was collected andevaporated to dryness. To the residue was added tributyl phos-phate (50  µ l) followed by methyl iodide (300  µ l). The reactionmixture was sealed, heated at 90°C for 15min then injected ontothe same HPLC column as above under the same HPLC condi-tions. The radioactivity peak corresponding to [ 123 I]MIDEX( t  R =26min) was collected and evaporated to dryness. The residuewas dissolved in sterile saline and filtered through a sterile 0.22- µ m filter into a sterile pyrogen-free evacuated vial. For determina-tion of radiochemical purity, chemical purity and specific activity,an aliquot of the final product was applied to an analytical Gold-pak Exsil C-18 reverse-phase HPLC column (10  µ m,250mm×4.6mm) with a mobile phase of 50:50 ethanol:water(0.1  M  ammonium acetate). With a flow rate of 1ml/min,[ 123 I]MIDEX eluted at 6.5min. For all pharmacological work car-ried out in this study, cold mass of MIDEX was added to lowerthe specific activity to 100Ci/mmol.  Animal treatments. & p.2: Male Wistar rats, 12 weeks old and weighing250–300g, were used. Half of the rats received a single intrave-nous injection of STZ (45mg/kg in citrate buffer, 50m  M  ,pH4.5), via the tail vein, and the other half were used as age-matched controls and received intravenous injection of the samevolume of citrate buffer without streptozotocin. The rats were ob-served daily and weighed weekly throughout the experiment. Allrats were given standard food and water ad libitum. Glucosuriawas routinely checked with urinalysis strips (Clinistix, Bayer Di-agnostics, Australia). On the day of the experiment, a blood sam-ple was collected and glucose levels determined by Rapid BloodAnalyser (Reflolux S, Boehringer, Germany). Between 1 and 10weeks after the single injection of STZ or citrate buffer, the ratswere sacrificed by CO 2 administration followed by cervical frac-ture. Hearts were rapidly removed from each animal and weighed.  In vitro binding assays. & p.2: Membrane preparations for binding as-says were prepared as previously described with some minormodifications [9, 10]. Control and diabetic hearts were dissectedto obtain right ventricle, left ventricle and atrium. The tissueswere stored at –80°C. Tissue samples were thawed, placed in 50volumes of ice-cold buffer (50m  M  Tris-HCl, 10m  M  MgCl 2 ,pH7.4) and minced finely with scissors. After homogenising thepreparation with a polytron (PCU Kinematica, Bioblock, Switzer-land) with one 5-s burst at a setting of 7, 1ml of ice-cold 2.5  M  KCl was added to extract contractile proteins, followed by stirringat 4°C for 15min. The suspension was then centrifuged at40,000 g for 20min at 4°C. The pellets were resuspended in50m  M  Tris/HCl, 10m  M  MgCl 2 , pH7.4 ice-cold buffer with adounce (manual homogenizer) to yield an appropriate protein con-centration. Protein measurements were made by Lowry’s method[11] using bovine serum albumin as standard.The incubation buffer contained 0.1ml Tris/HCl 50m  M  ,MgCl 2 10m  M  , pH7.4 or 0.1ml of atropine 1m  M  (non-specificbinding), 0.1ml of [ 123 I]MIDEX (specific activity 100Ci/mmol,concentration of 0.1–25n  M  ) and 0.3ml of membrane preparation.The assay was incubated for 30min at 37°C and filtered throughWhatman GF/B filter papers using a cell harvester (Inotech, Swit-zerland). Each filter was washed twice with 5ml of the ice-coldbuffer to remove free [ 123 I]MIDEX. The filters were counted withan automated gamma counter and corrected for decay. The maxi-mum number of specific binding sites (  B max ) and [ 123 I]MIDEXequilibrium dissociation constant ( K  d ) were calculated using acomputer-assisted program (EBDA, LIGAND) [12]. Calculatedvariables as well as parameter estimates from radioligand bindingdata are given as mean ±SD of three experiments.  Ex vivo and in vitro autoradiographic distribution of [ 123  I]MIDEX in the rat heart. & p.2: In vitro autoradiography was performed on ratheart from 1 to 10 weeks after the single injection of STZ or ci-trate buffer. Following sacrifice of the rats as outlined above,hearts were rapidly removed from each animal, weighed and rap-idly frozen in isopentane chilled at –80°C. Horizontal sections20  µ m thick were cut with a cryostat (Leica, Germany), mountedonto gelatin-coated slides and stored in a freezer (–20°C) untiluse. Myocardial m-AChR were labelled with concentrations rang-ing from 0.1n  M  to 50n  M  of [ 123 I]MIDEX (100Ci/mmol) inTris/HCl 50m  M  , MgCl 2 10m  M  , pH7.4 at 37°C for 30min. Non-specific binding was defined by incubating adjacent tissue sec-tions with 1m  M  of atropine. The incubation was terminated byrinsing sections twice for 2min in the cold incubation buffer. Sec-tions were then dipped briefly in cold distilled water and driedrapidly under a stream of cold air. Half of the slices were affixed(together with heart paste calibration standards) to radiographicfilms (Amersham Hyperfilm- β max) overnight and the optical den-sity of the autoradiograms was quantified by using a microcom-puter imaging device (MCID, Imaging research, Ontario, Cana-da). Half of the slices were wiped off from the microscope slideand the radioactivity counted with an automated gamma counterfor determination of saturation parameters (  B max and K  d ).Ex vivo autoradiography was performed on control and diabet-ic rats from 1 to 10 weeks after the injection of STZ or citratebuffer. Rats received an intravenous injection via the tail vein of [ 123 I]MIDEX (200  µ Ci/100  µ l of saline) or atropine (1mg/kg,non-specific binding) 5min prior to [ 123 I]MIDEX injection. Ratswere sacrificed 10min after the injection of [ 123 I]MIDEX. Theheart was rapidly removed, frozen in isopentane chilled at –80°Cand mounted onto microtome chucks. Horizontal sections (20  µ min thickness) were then cut with a cryostat, thaw-mounted ontoglass slides, dried and affixed (together with heart paste calibra-tion standards) to radiographic films (Amersham Hyperfilm-ßmax) overnight. The optical density of the autoradiograms wasquantified by using a microcomputer imaging device (MCID, Im-aging research, Ontario, Canada). Statistics. & p.2: Data are presented as means ±SD. Differences betweengroups were analysed using an unpaired t  test or, where appropri-ate, analysis of variance testing (ANOVA). Probability levels of 0.05 or smaller were considered significant.  745European Journal of Nuclear Medicine Vol. 26, No. 7, July 1999 Results Urine glucose, blood glucose and heart weight  Diabetes was confirmed by the finding of glucosuria 2days after the injection of STZ. Blood glucose and bodyweight findings are summarised in Table 1. No changesin body weight or heart weight were found in 1-week di-abetic rats when compared with the age-matched controlrats. STZ-treated animals were found to have significant-ly lower body, ventricle and atrium weights when com-pared with the age-matched controls in either the 5-week or the 10-week studies. Ten weeks after STZ injection,diabetic animals exhibited significantly higher ventricleweight to body weight ratios and atrium weight to bodyweight ratios in comparison to control rats.  In vitro binding assay [ 123 I]MIDEX bound to m-AChR in right and left ventri-cles and atrium in STZ-diabetic and age-matched controlrats with the same affinity ( K  d =5.5–8.5n  M  ) (Table 2).The number of binding sites was also estimated in rightand left ventricles and atrium from STZ-diabetic rats andage-matched control rats (Table 2). No change in myo-cardial m-AChR density was found in 1-week and 5-week STZ-diabetic rats when compared with the age-matched controls. A 39% decrease in m-AChR densitywas found in the atrium of 10-week STZ-diabetic ratswhen compared with control rats (  B max =2.65±0.40pmol/mg protein versus  B max =4.34±0.48 pmol/mg pro-tein, P =0.002, respectively). m-AChR density did notdiffer in the right and left ventricles between 10-week STZ-diabetic rats and control rats. The binding of [ 123 I]MIDEX to homogenates of rat atrium in 10-week  Table1. Urine glucose, blood glucose and body and heart weights in control rats and rats with STZ-induced diabetes a &  /t bl.c :& t bl.b: 1-week rats5-week rats10-week ratsCDCDCDBW (g)323±11297±25410±9290±16**471±5284±10**LV (mg)634±22567±24754±46567±24*807±12536±13*LV/BW (mg/g)1.96±0.011.94±0.081.82±0.131.98±0.071.73±0.031.93±0.05*RV (mg)184±20162±20196±24158±23*231±20155±22*RV/BW (mg/g)0.57±0.080.54±0.050.47±0.050.55±0.060.50±0.050.56±0.09A (mg)47±1050±569±550±1369±1060±6*A/BW (mg/g)0.14±0.030.17±0.020.17±0.020.17±0.040.15±0.020.21±0.02*Blood glucose (mmol/l)4.9±0.2517±0.55**4.75±0.6523.5±2**5±0.4026±2**Urine glucose  − +  − +  − + a All values are means ±SD ( n= 3–5). The values in diabetic animals (D) are compared with the values in corresponding controls (C) andthe level of significant difference is indicated by * P< 0.05 and ** P< 0.001BW, Body weight; LV, left ventricle; RV, right ventricle; A, atrium &  /t bl.b: Table2. m-AChR density measured with [ 123 I]MIDEX and ligand affinity in atrium and ventricles from STZ-diabetic and age-matchedcontrol rats a &  /t bl.c :& t bl.b: 1-week rats5-week rats10-week ratsCDCDCD  B max (pmol/mg protein)Left ventricle2.16±0.271.92±0.362.26±0.362.22±0.552.45±0.302.33±0.28Right ventricle2.51±0.502.28±0.532.68±0.432.50±0.342.50±0.342.16±0.39Atrium4.55±0.604.13±0.634.31±0.384.34±0.484.34±0.482.65±0.40*Kd (n  M  )Left ventricle7.50±2.136.75±2.056.71±1.85.00±1.415.00±1.415.57±0.98Right ventricle7.62±2.935.50±1.707.43±1.526.50±2.386.50±2.386.57±1.90Atrium8.5±1.385.50±1.526.60±2.085.75±1.705.75±1.706.00±2.36 a All values are means ±SD ( n= 3–5 for each group). The values in diabetic animals are compared with the values in correspondingcontrols and the level of significant difference is indicated by * P <0.05C, Control; D, Diabetic &  /t bl.b:  746European Journal of Nuclear Medicine Vol. 26, No. 7, July 1999 STZ-diabetic rats and control rats was saturable with thespecific binding displaying typical hyperbolic curves(Fig.2). At a concentration of 15n  M  of [ 123 I]MIDEX,the non-specific binding in the atrium of control rats and10-week STZ-diabetic rats represented 26% and 25%,respectively of the total binding. Protein yield data on membrane homogenate  preparations of rat hearts No changes in protein yield on membrane homogenatepreparations were found in 1-week, 5-week and 10-week diabetic rats when compared with the age-matched con-trol rats (Table 3).  In vitro and ex vivo autoradiographic distribution of [ 123  I]MIDEX binding sites in STZ-diabetic rats and age-matched control rats In vitro autoradiography. & p.1: Scatchard analysis of satura-tion isotherms of [ 123 I]MIDEX binding to horizontalheart sections (measured by gamma counter) revealedthat the binding of [ 123 I]MIDEX was specific, saturableand of high affinity. A typical equilibrium saturation iso-therm of [ 123 I]MIDEX binding to whole heart sections of a 10-week STZ-diabetic rat and a control rat is shown inFig.3. Scatchard analysis of saturation data revealed that[ 123 I]MIDEX bound to m-AChR with the same affinityfor both control and 10-week STZ-diabetic rats( K  d =14±3.4n  M  versus K  d =12.9±4.2n  M  , respectively).A 36% decrease in m-AChR density was found in wholeheart horizontal sections of 10-week STZ-diabetic ratswhen compared with control rats (  B max =0.92±0.14n  M  Fig.2A,B. Equilibrium bindingdata from a typical experiment.Total, non-specific and specificbinding of [ 123 I]MIDEX in thehomogenate of the rat atrium of a control rat ( A ) and a 10-week STZ-diabetic rat ( B ). The top panel is a representative Scat-chard plot of specific bindingdata; ratio of bound to free(  BS/F  ) [ 123 I]MIDEX is plottedas a function of specificallybound [ 123 I]MIDEX. Datashown are the mean of three dif-ferent experiments &  /fig.c : Table3. Protein yield obtained on membrane homogenate preparations of control and diabetic rat hearts a &  /t bl.c :& t bl.b: 1-week rats5-week rats10-week ratsCDCDCDProtein yield (mg/g organ)Left ventricle87.4±7.292.2±6.790.5±2.991.7±6.089.2±2.587±7.2Right ventricle86.2±3.589.6±5.282.5±1.782.7±6.481.5±5.081.8±6.3Atrium47.8±1.946±4.441.2±4.238.2±2.239.1±3.640±2.9 a All values are means ±SD ( n= 3–5 for each group). The values in diabetic animals are compared with the values in correspondingcontrolsC, Control; D, diabetic &  /t bl.b:  747European Journal of Nuclear Medicine Vol. 26, No. 7, July 1999 versus  B max =1.45±0.17n  M  , P =0.015, respectively). At aconcentration of 20n  M  of [ 123 I]MIDEX, the non-specif-ic binding in the whole heart horizontal sections of con-trol rats and 10-week STZ-diabetic rats represented 27%and 29%, respectively of the total binding. No statistical-ly significant difference in m-AChR density or ligand af-finity was found in the 5-week STZ-diabetic rats whencompared with age-matched control rats.The computer-assisted image analysis of autoradio-grams prepared from whole heart horizontal sections of STZ-diabetic rats and age-matched control rats revealeda heterogeneous distribution of [ 123 I]MIDEX bindingsites. The highest levels of specific [ 123 I]MIDEX bindingwere found in atrium, followed by the right ventricle andleft ventricle. For each region a ratio was calculated be-tween control rats and diabetic rats. The control/diabeticratio for the 5-week rats was calculated for each region:1.04–1.10 for atrium, 1.10 for right ventricle and 1.05for left ventricle. The levels of [ 123 I]MIDEX bindingwere decreased by 40% in the atrium of the 10-week STZ-diabetic rat when compared with control rats. Thecontrol/diabetic ratio for the 10-week rats was calculatedfor each region: 1.66–1.76 for atrium, 1.10 for right ven-tricle and 1.15 for left ventricle.  Ex vivo autoradiography. & p.1: The non-specific binding of [ 123 I]MIDEX (determined with pre-injection of atropine)was subtracted from the total binding of [ 123 I]MIDEXusing the MCID computer analysing system. Figure 4represents the specific binding of [ 123 I]MIDEX in wholeheart horizontal sections of STZ-diabetic rats and age-matched control rats. A 50% decrease in [ 123 I]MIDEXuptake was found in atrium of 5-week STZ-diabetic ratswhen compared with control rats. The control/diabeticratio was calculated for each region: 1.89–1.95 for atri-um, 1.09 for right ventricle and 0.98 for left ventricle. A60% decrease in [ 123 I]MIDEX uptake was found in atri-um of 10-week STZ-diabetic rats when compared withcontrol rats. The control/diabetic ratio was calculated foreach region: 2.38–2.68 for atrium, 1.14 for right ventri-cle and 1.10 for left ventricle. No significant change in[ 123 I]MIDEX uptake was found in the right and left ven- Fig.3A,B. Saturation of [ 123 I]MIDEX binding in hori-zontal sections of rat heart. A Control rat; B 10-week STZ-diabetic rat. The top panel is arepresentative Scatchard plot of specific binding data; ratio of bound to free (  BS/F  ) [ 123 I]MI-DEX is plotted as a function of specifically bound [ 123 I]MI-DEX. Each  point  represents themean of triplicate determina-tions &  /fig.c : ABC Fig.4A–C. Ex vivo autoradiograms of the specific binding of [ 123 I]MIDEX in whole heart horizontal sections of STZ-diabetic rats (  D )and age-matched control rats ( C  )
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