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Cardiovascular Research 2005 65(2):374-386; doi:10.1016/j.cardiores.2004.10.010
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Copyright © 2004, European Society of Cardiology

AT1 receptors and L-type calcium channels: functional coupling in supersensitivity to angiotensin II in diabetic rats

K.H.S. Arun1, C.L. Kaul and P. Ramarao*

Cardiovascular and Receptorology Laboratory, Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Sector-67, S.A.S. Nagar, Punjab-160062, India

* Corresponding author. Tel.: +91 172 2214697, +91 172 2214690; fax: +91 172 2214692. Email address: Kumar.Arun{at}pharma.med.uni-giessen.de ramaraop{at}yahoo.com

Received 5 June 2004; revised 20 September 2004; accepted 7 October 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
Objective: The study was designed to investigate the role of calcium channels in enhanced angiotensin II (Ang II)-induced contraction in thoracic aortic rings from diabetic rats.

Methods: Ang II-induced isometric tension was studied in thoracic aortic rings isolated from control or streptozotocin-induced (8 weeks) diabetic rats. Saturation binding studies at AT1 receptors and L-type calcium channels were performed using [3H] Ang II and [3H] PN200110, respectively. Ang II-induced calcium influx was studied in fura-2-loaded single vascular smooth muscle cells isolated from thoracic aorta of control and diabetic rats.

Results: Ang II did not induce contraction in calcium-free Krebs. In the presence of extracellular calcium, increased Emax (mg/mm2) and pD2 to Ang II was observed in aortic rings from diabetic (795.54 ± 38.19; 8.27 ± 0.12) compared to control (230.09 ± 25.45; 7.68 ± 0.22) rats, respectively. Nimodipine but not verapamil or diltiazem dose-dependently blocked the Ang II-induced contractions in a noncompetitive manner and its –log IC50 was significantly lower in aortic rings from diabetic (8.81 ± 0.10) compared to control (9.34 ± 0.11) rats. The Ang II-induced increase in intracellular calcium levels was significantly enhanced (2.5-fold) in vascular smooth muscle cells from diabetic rats. AT1 receptor saturation binding with [3H] Ang II revealed a significantly higher affinity (nM) and Bmax (pmol/mg protein) in aortic vascular membrane preparation from diabetic (0.31 ± 0.04; 64.18 ± 2.4) compared to control (0.52 ± 0.02; 47.81 ± 2.8) rats, respectively, while L-type calcium channel saturation binding with [3H] PN200110 showed a higher affinity (nM) with no change in the Bmax (fmol/mg protein) in diabetic (0.74 ± 0.08; 4.52 ± 0.40) compared to control (1.49 ± 0.32; 5.43 ± 0.60) aortic membranes, respectively.

Conclusions: Our results suggest that Ang II-induced contraction is dependent on extracellular calcium, and enhanced functional coupling of AT1 receptors and DHP-sensitive L-type calcium channels results in supersensitivity to Ang II in thoracic aorta isolated from diabetic rats.

KEYWORDS Angiotensin II; L-type calcium channel; Thoracic aorta; Diabetes; Streptozotocin

Abbreviations: Ang II, angiotensin II • AT1, angiotensin type I receptor • BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) • Bmax, binding maxima • CCB, calcium channel blockers • CFK, calcium-free Krebs • CRC, concentration–response curve • DCC, dihydropyridine sensitive L-type calcium channels • DHP, dihydropyridine • DMEM, Dulbecco's modified essential medium • KCl, potassium chloride • Kd, dissociation constant • KHB, Krebs-Henseleit buffer • LOE-908, (R, S)-(3,4dihydro-6, 7-dimethoxy-isochinolin-1-yl)-2-phenyl-N, N-di [2-(2,3,4-trimethoxyphenyl) ethyl] acetamid mesylate • PE, Phenylephrine • STZ, streptozotocin • VSMC, vascular smooth muscle cell


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
Diabetes is one among the most costly and burdensome chronic disease of the present day and its incidence is exploding in pandemic proportions. More than the disease, the vascular complications associated with the disease are major cause for the increased morbidity and mortality in diabetic patients [1]. The co-existence of hypertension and diabetes synergistically increases the risk of these vascular complications. The process that are thought to be etiologically important in the development or perpetuation of vascular complications in diabetics are alterations in the balanced production of vasodilator (nitric oxide, prostaglandin's I2 and E2) and vasoconstrictor (angiotensin II {Ang II}, endothelin I, cyclooxygenase and lipoxygenase products of arachidonic acid metabolism) substances from the endothelium and vascular smooth muscle cells [2]. However, alterations in the cellular/extracellular elements that comprise the vascular wall along with other cytokines and growth factors also contribute to the vascular complications in diabetes [3]. Ang II, the principal hormone of the renin–angiotensin system, is implicated as one of the contributing factors in the vascular complications associated with diabetes [4]. We have previously reported enhanced contractile response to Ang II in thoracic aorta from SHR [5]and STZ-induced diabetic rats [6].

Ang II via its AT1 or AT2 receptors plays an important role in structural and functional integrity of arterial wall and hence is crucial in pathological mechanisms underlying vascular complications in diabetes [7]. In most blood vessels, the angiotensin II responses are predominantly mediated by AT1 receptors [8]. The AT1 receptors are reported to coupled to numerous signal transduction pathways in a variety of vascular cell types, including intracellular Ca2+, phospholipase C, PKC, tyrosine kinases, MAP kinases and JAK/STAT [9]. Ang II-induced Ca2+ influx in vascular smooth muscle cells is reported to involve voltage-dependent calcium channels which are directly or indirectly activated by Ang II, Ca2+-permeable nonspecific dihydropyridine-insensitive cation channels, receptor-gated Ca2+ channels, Ca2+-activated Ca2+ release, and activation of the Na+/Ca2+ exchanger [10]. However, which among these pathways are responsible for the Ang II-induced contraction is unclear. Hence, we planned the present study to investigate which calcium source is involved in the contractile responses to Ang II, identify the calcium channels linked to AT1 receptor mediated contractile response and weather AT1 receptor and ion channel functional link if any is altered under diabetic conditions.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 5. Conclusions
 References
 
2.1. Materials
[3H]Ang II and [3H]PN200110 were procured from Amersham Pharmacia Biotech, UK. Ang II (Bachem, Basel, Switzerland), streptozotocin (STZ; Calbiochem, USA), glucose GOD-POD kit (Glaxo Qualigens, Mumbai, India) was used in the study. Gadolinium, lanthanum, thapsigargin, 1,2-bis(2-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) {BAPTA 2AM}, {omega}-Agatoxin IVA and arachidonic acid were procured from Sigma, St. Louis, MO, USA. (R, S)-(3,4dihydro-6, 7-dimethoxy-isochinolin-1-yl)-2-phenyl-N, N-di [2-(2,3,4-trimethoxyphenyl) ethyl] acetamid mesylate (LOE-908) was kindly gifted by Boehringer Ingelheim, Germany. Valsartan was kindly gifted by Novartis Pharma (Switzerland), respectively. Nimodipine, lercanidipine, nifedipine, verapamil and diltiazem were kindly gifted by Torrent Pharmaceuticals, India. All other chemicals were of reagent grade obtained from Merck, India.

2.2. Animals
All the experiments were approved by the Institutional Animal Ethics Committee (IAEC, NIPER) vide approval #IAEC 02/011 and complied with the NIH guidelines on handling of experimental animals.

Experiments were performed on male Sprague–Dawley rats (Central Animal Facility, NIPER, India) that were 6–8 weeks old and weighed 150 to 180 g at the start of the study. Animals were housed four per cage in a room with controlled ambient temperature (20 ± 1 °C), humidity (50 ± 10%) and 12 h light/dark cycle. Food and water were provided to each rat ad libitum.

2.3. Induction of diabetes
Rats were made diabetic as described previously [6]. Briefly, streptozotocin (STZ, 65 mg/kg., dissolved in ice-cold 10 mM citrate buffer, pH 4.4) was administered once intraperitoneally to rats. Age-matched control rats received an equivalent volume of the vehicle. Blood samples for glucose measurements were collected from the retro-orbital plexus at 48-h post dose. Rats with a blood glucose level >250 mg/dl were selected for the study.

2.4. Blood pressure recording
Blood pressure (Systolic, mean and diastolic) and heart rate were recorded at weekly intervals from pre to 8th week post-STZ administration, using a tail cuff blood pressure recorder (IITC INC, Life Science Instruments, Model no 29,229; California, USA). Rats were acclimatized to heating chamber (24–26 °C) for 20 min before recording the blood pressure (between 9 and 11 AM). Three recordings were measured for each rat and the average was calculated.

2.5. Isometric tension measurement
Animals were sacrificed by cervical dislocation (under xylazine–ketamine anesthesia) 8 weeks post-STZ treatment. The thoracic aorta (from the arch of aorta to the diaphragm) was quickly excised and placed in ice-cold oxygenated (95% O2–5% CO2) Krebs-Henseleit buffer (KHB). A part of the tissue was preserved in buffered formal saline for histological examinations. The aorta was cut into 5-mm segments after it was cleaned of adhering fat and adventitial tissues. Due care was taken to keep the endothelium intact (E+). In some preparations, the endothelium was deliberately removed by passing a steel wire of suitable diameter through the lumen and gently rubbed using the finger (E–). The composition (mM) of KHB was NaCl: 118, KCl: 4.7, MgSO4: 1.2, CaCl2: 2.6, KH2PO4: 1.2, NaHCO3: 25 and glucose: 5.5 (pH7.4; 284 ± 2 mOsmol/l). For the depolarization, KHB with 80 mM KCl was prepared with a corresponding decrease in NaCl.

Each ring was suspended by a pair of stainless steel hooks in a water-jacketed organ bath, filled with 10 ml of oxygenated KHB maintained at 37°C. Isometric tension responses were measured on a physiograph chart recorder (BioDevices, Ambala, India) using an isometric force transducer (T-305, BioDevices). A resting tension of 2 g was applied to the aortic rings, which were then allowed to equilibrate for 2 h during which the buffer was changed every 15 min. After the equilibration, cumulative concentration–response to Ang II (0.1 nM–30 µM) and KCl (20–120 mM) was recorded.

At the end of experiment, the aortic ring was blotted dry, its length measured and weighed to calculate tension as normalized for cross-sectional area using the following formula:



Formula

(The density of vascular smooth muscle=1.05 mg/mm3).

The Ang II and KCl responses are expressed as mg/mm2.

To evaluate the role of extracellular calcium in the agonist-mediated contraction, cumulative concentration–response curve (CRC) to Ang II and KCl was recorded in calcium-free Krebs (CFK) with the following composition: (mM) NaCl: 121, KCl: 4.7, MgCl2: 1.2, KH2PO4: 1.2, NaHCO3: 25, glucose: 5.5 and EGTA: 0.1. In yet other experiments, aortic rings were loaded with BAPTA, using the BAPTA 2AM and cumulative CRC to Ang II, and KCl were recorded. To identify the type of calcium channels involved in the contractile process, the CRC to Ang II and KCl were constructed in the presence of various calcium channel blockers.

To evaluate the functional coupling of AT1 receptors and L-type calcium channels, CRC to Ang II was constructed in the presence of various concentrations of nimodipine and the –log IC50 of nimodipine was estimated by nonlinear regression analysis of the percent Ang II contraction inhibition vs. log nimodipine concentration plot.

2.6. Vascular smooth muscle cell isolation
VSMC were isolated from rat thoracic aorta by a modified method of Smith and Brock [11]. Briefly, thoracic aorta was isolated from control or diabetic rats as described above. The artery was cleaned of connective tissue and cut open longitudinally and endothelial layer was denuded by gentle rubbing with a sterile cotton swab. The tissue was minced with a fine scissor and then transferred to an enzyme mixture (prepared in serum free Dulbecco's modified essential medium {DMEM}) at 37 °C containing collagenase (1 mg/ml), 0.1 mg/ml elastase and bovine serum albumin (1 mg/ml) for 30–45 min. After which the tissue was triturated with a pasture pipette and transferred to 35-mm petri dish consisting of DMEM with penicillin (10,000 U/ml), streptomycin (100 µg/ml), glutamine (2 mM), and glucose (5 mM) and incubated at 37 °C for 6–8 h before being loaded with fura-2 for measurement of intracellular calcium concentration.

2.7. Measurement of intracellular calcium concentration
Fura-2, a calcium sensitive dye, was used to assess changes in [Ca2+]i in single VSMC using dual excitation digital Ca2+ imaging system (TILLvisION 4.0., TILL Photonics, Germany) equipped with IMAGO camera. The imaging system was mounted on an inverted microscope (Nikon eclipse TE2000U) outfitted with a 40 x oil immersion objective. Fura-2 AM was dissolved in DMSO and added from a 1-mM stock to the cell suspension (5 x 106 cells/ml) at a final concentration of 5 µM. Cells were incubated with fura-2 AM for 30 min at 37 °C. After which, cells were washed twice with KHB (pH 7.4 and 285–295 mOsmol/l) and placed in a perfusion chamber (Warner Instruments, USA) at room temperature in the dark for 20 min. Cells were illuminated with a xenon arc lamp (TILL Photonics) at 340 and 380 nm and emitted light was collected from regions that encompassed single cells with a CCD camera (IMAGO, TILL Photonics) at 510 nm. If cells moved, the experiment was paused and the regions of interest resized. The images acquired were stored on compact disks for later analysis. Although it is difficult to accurately measure intracellular calcium ([Ca2+]i) [12], estimates were made from the following equation:



Formula

The values for the maximum 380 fluorescence (Sf2), minimum 380 fluorescence (Sb2), minimum ratio (Rmin) and maximum ratio (Rmax) were determined from calibrations of fura-2 for each cell. The Kd for fura-2 was assumed to be 224 nM [13]. At the end of each experiment, cells were treated with 0.1% triton X-100 to determine Rmax, while Rmin was determined in CFK containing 10 mM EGTA [14].

2.8. Vascular smooth muscle membrane preparation
Vascular smooth muscle membrane was prepared as per the method of Maillard et al. [15]. Briefly the rat thoracic aorta (from aortic arch to diaphragm) was isolated as described above and was cleaned of all the adhering tissues and fat. The aorta was slit longitudinally and homogenized in 20 volumes of ice-cold Tris–hydrochloride buffer (Tris–HCl: 20 mM; EDTA: 1 mM; dithiothreitol: 1 mM; pH 8.0) using a Polytron homogenizer at 49,000 x g for 1 min. The homogenate was incubated with equal volume of 1 M KCl for 10 min under ice-cold temperature following which the homogenate was centrifuged (SORVALL 5B RC plus, high-speed centrifuge floor model) at 49,000 x g for 20 min at 4 °C and the pellet obtained was resuspended in the incubation Tris–HCl buffer (Tris–HCl: 50 mM; NaH2PO4: 50 mM; MgCl2: 10 mM; pH 7.1). The homogenate was incubated at 37 °C for 30 min in order to remove the endogenous ligands from their binding sites. The suspension was centrifuged a second time, at 49,000 x g for 20 min (4 °C), and the pellet obtained was resuspended in incubation Tris–HCl buffer and used for binding studies. The protein content was determined by the method of Lowry [16].

2.9. Saturation binding of [3H]Ang II at AT1 receptors
2.9.1. Binding assay
The saturation binding of [3H]Ang II (0.05–2.5 nM) to thoracic aortic membranes from control and diabetic rats was carried out in a total volume of 0.25 ml which consisted of Tris–HCl buffer (50 mM, pH 7.4) with or without valsartan (10 µM). Binding was initiated by adding tissue protein equivalent to 180–200 µg per tube. The binding assay was carried out in triplicate at 24 °C for 60 min. Non-specific binding was determined as the difference in binding in the absence and presence of 10 µM unlabelled valsartan. Binding was terminated by rapidly washing with ice-cold Tris–HCl buffer and filtering the contents of the incubation tubes through Whatman GF/B filter paper under reduced pressure using a Brandel cell harvester (Biomedical Research and Development Laboratories, Gaithersberg, MD, USA). The filter disc was washed thrice with 5 ml of ice-cold buffer and then transferred to liquid scintillation vials and 5 ml of scintillation cocktail, containing 3 g PPO (2,5-diphenyloxazole) and 100 mg of POPOP (2,2'-phenylene-bis (5-phenyloxazole) in 1000 ml of sulphur-free xylene, was dispensed using a Brandel cocktail dispenser. After a 6-h equilibration period, the radioactivity in the samples was determined using a Wallac (model 1409) liquid scintillation beta counter.

2.10. Saturation binding of [3H]PN200110 at L-type calcium channels
2.10.1. Binding assay
The saturation binding of [3H] PN200110 (0.05–2.5 nM) to thoracic aortic membranes from control and diabetic rats (prepared as mentioned above) was carried out in a total volume of 0.25 ml, which consisted of Tris–HCl buffer (50 mM, pH 7.1) with or without unlabeled nimodipine (10 µM). Binding was initiated by adding tissue protein equivalent to 180–200 µg per tube. The binding assay was carried out in triplicate at 27 °C for 60 min. Non-specific binding was determined as the difference in binding in the absence and presence of 10 µM unlabelled nimodipine. The termination of the binding and rest of the procedure was similar to as mentioned above for saturation binding of [3H] Ang II at AT1 receptors.

2.11. Statistical analysis
Cumulative concentration–response curves were analyzed for pD2 value (the negative log concentration required to produce 50% of the maximal response). Experimental values are expressed as mean ± S.E.M., and Student's t-test at 5% level of significance (p<0.05) was used to assess the differences between any two groups. Comparison of mean values between various groups was performed by analysis of variance followed by multiple comparisons by Scheffe's F-test. Data analysis was done using GraphPad Prism version 3.00 for Windows (GraphPad Software, San Diego, CA, USA).


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 5. Conclusions
 References
 
3.1. General observations
STZ (65-mg/kg, i.p)-administered rats developed diabetes as evident from the sustained elevation of blood glucose levels and at 8th week post-STZ administration had significantly lower body weight (Table 1). These rats progressively developed hypertension as evident from the elevated systolic, diastolic and mean blood pressure with no change in the heart rate (Table 1, Fig. 1). Elevated systolic and diastolic pressure was observed from 1st and 4th weeks post-STZ administration, respectively (Fig. 1). In the histological sections of thoracic aorta from 8-week diabetic rats, we observed endothelial damage and increased connective tissue mass in the medial layer suggesting the development of diabetic associated vasculopathy in conduit vessels.


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  		Table 1 Body weight, plasma glucose concentration, systolic, mean and diastolic pressure and heart rate of control and 8-week STZ-induced diabetic rats

 

Figure 1
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  		Fig. 1 Weekly changes in systolic, diastolic and mean blood pressure in control and streptozotocin injected rats. Each value is represented as mean ± S.E.M., n=20. **p<0.01, ***p<0.001 vs. respective control group.

 
3.2. Characteristics of vascular responses
Ang II- and KCl-induced contraction in the endothelium-intact (E+) and -denuded (E–) aortic rings in a concentration-dependent manner (Figs. 2 and 3Go). Only sustained contractions to Ang II were observed, while there was no transient phase.


Figure 2
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  		Fig. 2 Cumulative concentration–response curves to angiotensin II (Ang II) in presence or absence {calcium-free Krebs (CFK)} of extracellular calcium in endothelium intact (E+, upper panel) and denuded (E–, lower panel) aortic rings isolated from control (Con, left panel) or 8-week diabetic (Dia, right panel) rats. Each point is represented as mean ± S.E.M., n=10.

 

Figure 3
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  		Fig. 3 Cumulative concentration–response curves to potassium chloride (KCl) in presence or absence {calcium-free Krebs (CFK)} of extracellular calcium in endothelium intact (E+, upper panel) and denuded (E–, lower panel) aortic rings isolated from control (Con, left panel) or 8-week diabetic (Dia, right panel) rats. Each point is represented as mean ± S.E.M., n=10.

 
Ang II and KCl did not induce contraction in calcium-free Krebs (CFK) while the contraction was observed on replacing the calcium in the organ bath. Ang II- (Fig. 2) and KCl-induced (Fig. 3) contraction was also observed when extracellular calcium was replaced with non-physiological divalent cation, barium (Ba2+, 6 mM) or strontium (Sr2+, 10 mM).

Ang II-induced contraction was blocked by valsartan (10 µM, AT1 receptor blocker), dihydropyridine (DHP) calcium channel blockers (CCB, 10 µM, nimodipine, lercanidipine or nifedipine) but not by verapamil (100 µM) or diltiazem (100 µM) (Fig. 4). Gadolinium (10 µM, store operated calcium channel blocker) or lanthanum (10 µM, capacitative calcium entry blocker) or LOE 908 (100 µM, receptor operated calcium channel blocker) did not affect Ang II-induced contraction (Fig. 4). Lignocaine (10 µM, sodium channel blocker) did not block/reduce Ang II-induced response suggesting that the contraction is independent of the membrane depolarization process. {omega}-Agatoxin IVA did not affect the Ang II contraction thus excluding the role of P/Q-type Ca2+ channels. In the presence of thapsigargin (100 nM, sarcoplasmic reticulum calcium ATPase pump inhibitor, i.e., releases intracellular calcium) or in aortic rings loaded with BAPTA (10 µM, selective chelator of Ca2+) (Fig. 4) Ang II-induced contraction was unaffected.


Figure 4
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  		Fig. 4 Effect of various modulators on angiotensin II (Ang II, 100 nM)-induced contraction in thoracic aortic rings isolated from control rats. Panel A: Ang II-induced maximal contraction in presence or extracellular barium (Ba2+, 6 mM), calcium (Ca2+, 2.4 mM), strontium (Sr2+, 10 mM) or valsartan (Val, 10 µM). Panel B: Effect of nimodipine (Nim, 10 µM), lercanidipine (Ler, 10 µM), nifedipine (10 µM) and verapamil (Ver, 100 µM) on Ang II-induced contraction in presence of extracellular calcium. Panel C: Effect of diltiazem (Dil, 100 µM), lanthanum (Lan, 10 µM), LOE 908 (LOE, 100 µM) and gadolinium (Gad, 10 µM) on Ang II-induced contraction in presence of extracellular calcium. Panel D: Effect of lignocaine (Lig, 10 µM), thapsigargin (TG, 100 nM) and BAPTA (10 µM) loading on Ang II-induced contraction in the presence of extracellular calcium. Each point is represented as mean ± S.E.M., n=6–8.

 
Supersensitivity (->pD2) to Ang II but not KCl was observed in thoracic aorta isolated from diabetic as compared to control rats (Table 2). While enhanced response (->Emax) to both Ang II and KCl was observed (Fig. 5). Further, in diabetic rats, the increased and comparable response to Ang II in the absence and presence of endothelium indicates that both endothelium and smooth muscle components are involved in the vascular pathology in diabetes.


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  		Table 2 Characteristics of cumulative concentration–response curves to angiotensin II (Ang II), and potassium chloride (KCl) in endothelium intact and denuded aortic rings isolated from control or 8-week diabetic rats

 

Figure 5
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  		Fig. 5 Cumulative concentration–response curves to angiotensin II (Ang II, upper panel) and potassium chloride (KCl, lower panel) in endothelium intact (E+, left panel) and denuded (E–, right panel) aortic rings isolated from control (Con) or 8-week diabetic (Dia) rats. Each point is represented as mean ± S.E.M., n=10. **p<0.01, ***p<0.001 vs. respective control group.

 
3.3. Effect of nimodipine on Ang II- and KCl-induced contraction
Since DHP CCB blocked the responses to Ang II and KCl, we selected nimodipine (a prototype of the group) for our further studies. Nimodipine dose-dependently blocked the Ang II- and KCl-induced contraction in a noncompetitive manner (Fig. 6) in thoracic aortic rings from diabetic and control rats. –log IC50 of nimodipine to Ang II, and KCl-induced contraction in intact as well as denuded thoracic aortic rings was significantly lower in diabetic as compared to control rats (Table 2).


Figure 6
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  		Fig. 6 Cumulative concentration–response curves to angiotensin II (Ang II, upper panel) and potassium chloride (KCl, lower panel) in the presence of various concentration of nimodipine (Nim) in endothelium intact (E+) aortic rings isolated from control (Con, Left panel) or 8-week diabetic (Dia, right panel) rats. Each point is represented as mean ± S.E.M., n=3–10.

 
3.4. Effect of Ang II on interaction of calcium concentration in single VSMC
To understand the role of calcium in vascular response to Ang II, we studied the effect of Ang II on intracellular calcium concentration ([Ca2+]i) in single VSMCs isolated from the thoracic aorta of control and diabetic rats. We observed a biphasic pattern wherein a transient phase was followed by a sustained phase (Fig. 7A). In calcium-free Krebs, Ang II did induce the increase in [Ca2+]i (as evident from the increase in fura ratio340/380), however, this release did not differ in VSMCs isolated from control and diabetic rats (Fig. 7C). In the presence of extracellular calcium, we observed a 2.5-fold increase in the maximal [Ca2+]i in Ang II exposed VSMC isolated from diabetic than control rats (Fig. 7B). pD2 value of Ang II was significantly higher in diabetic than control rat VSMCs (Fig. 7B). Nimodipine (1 µM) reduced the Ang II-induced rise in [Ca2+]i (Fig 7D) while in BAPTA-loaded VSMCs, nimodipine blocked the Ang II-induced rise in [Ca2+]i. Thus, these results support our in vitro observations of enhanced Ang II response in thoracic aortic rings from diabetic rats.


Figure 7
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  		Fig. 7 Effect of angiotensin II (Ang II) on intracellular calcium concentration ([Ca2+]i) of a single vascular smooth muscle cell (VSMC). Panel A: Changes in fura ratio340/380 following Ang II (100 nM) exposure in presence of extracellular calcium in single VSMC isolated from thoracic aorta of control or 8-week diabetic rats. Panel B: Cumulative concentration–response curve of Ang II (1 pM–3 µM) effect on [Ca2+]i in single VSMC isolated from thoracic aorta of control or 8-week diabetic rats. Panel C: Changes in fura ratio340/380 following Ang II (100 nM) exposure in calcium-free Krebs (CFK). Panel D: Response to Ang II (100 nM) in presence of nimodipine (1 µM). A representative of mean time courses derived from 6–10 individual experiments is shown in panels A, C and D. Each point in panel B is represented as mean ± S.E.M., of four to five independent experiments. **p<0.01, ***p<0.001 vs. respective control group.

 
3.5. Saturation binding of [3H] Ang II at AT1 receptors
To assess whether the enhanced Ang II response was due to altered AT1 receptor status, we studied the binding characteristics of [3H] Ang II at AT1 receptors and observed significantly higher affinity and Bmax in VSMC membranes from diabetic than control rats (Fig. 8).


Figure 8
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  		Fig. 8 Binding characteristics of [3H]angiotensin II (Ang II) at AT1 receptors in vascular smooth muscle membrane preparation from thoracic aorta of age match control and 8-week diabetic rats. AT1 receptor antagonist valsartan (10 µM) was used to define nonspecific binding. The panel shows a representative scatchard plot from a single experiment. Kd and Bmax values reported were derived from nonlinear regression of specific bound vs. free [3H]Ang II concentration. Each value is represented as mean ± S.E.M., of three independent experiments. **p<0.01 vs. respective control group.

 
3.6. Saturation binding of [3H]PN200110 at L-type calcium channels
Since the Ang II-induced vasocontractile effect was mediated by extracellular calcium influx via DHP sensitive L-type calcium channels, we studied its binding characteristics in VSMC membranes prepared from thoracic aorta isolated from control and diabetic rats. As the nimodipine –log IC50 value to Ang II-induced contraction was significantly lower in diabetic tissue, we expected the binding affinity to be lower in VSMC membranes from diabetic rats. Contrary to this, we observed a higher affinity with no change in the Bmax in VSMC membranes from diabetic than control rats (Fig. 9).


Figure 9
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  		Fig. 9 Binding characteristics of [3H]PN200110 at L-type calcium channels in vascular smooth muscle membrane preparation from thoracic aorta of age match control and 8-week diabetic rats. Nimodipine (10 µM, dihydropyridine L-type calcium channel blocker) was used to define nonspecific binding. The panel shows a representative scatchard plot from a single experiment. Kd and Bmax values reported were derived from nonlinear regression of specific bound vs. free [3H]PN200110 concentration. Each value is represented as mean ± S.E.M., of three independent experiments. **p<0.01 vs. respective control group.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
5. Conclusions
 References
 
4.1. General observations
STZ (65 mg/kg, i.p) administered rats developed diabetes as evident from the sustained elevation of blood glucose levels and at 8th week post-STZ administration had significantly lower body weight. These rats progressively developed hypertension as evident from the elevated systolic, diastolic and mean blood pressure with no change in the heart rate. Although several studies report elevated systolic pressure in STZ injected rats [17–19], change in diastolic pressure is not widely reported or studied. However, numbers of published reports provide diverse and conflicting results with respect to the change in blood pressure in STZ-injected rats, which we believe is largely due to the difference in the weight range of the rats used for the study. When the rats selected have higher weight range (250–350 g) at the beginning of the study, they do not show increase in BP following STZ administration (unpublished observation). Another factor contributing to the variability is the way STZ is administered. STZ being highly unstable once diluted (which according to some of our unpublished data is about less than 2 min). It is very important that STZ is diluted in citrate buffer just before administration. Generally when i.v. administration is adopted, it is not possible to be very quick with it, hence we have used the i.p route of administration. Also there is large variation in the dose of STZ used in different labs, which further supports the less stable nature of STZ and thus contributing to the diversified observations to the change in blood pressure. In the histological sections of thoracic aorta from 8-week diabetic rats, we observed endothelial damage and increased connective tissue mass in the medial layer suggesting the development of diabetic associated vasculopathy in conduit vessels. Our observations are consistent with several other reports on STZ-induced structural changes in aortic endothelium and medial layer [20–22].

4.2. Characteristics of vascular responses
Ang II- and KCl-induced contraction in concentration-dependent manner. Only sustained contractions to Ang II were observed, while there was no transient phase. Sustained phase of contraction is reported to be extracellular calcium dependent [23], thus suggesting that extracellular calcium could be the major contributor to Ang II-induced contraction in thoracic aortic rings, which was further explored in the present study.

Ang II- and KCl-induced contraction was not observed in calcium-free krebs (CFK) while the contraction was observed on replacing the calcium in the organ bath, suggesting that extracellular calcium is the only source for Ang II- and KCl-induced contraction. Further, Ang II- and KCl-induced contraction was also observed when extracellular calcium was replaced with non-physiological divalent cation, barium (Ba2+, 6 mM) or strontium (Sr2+, 10 mM). Several other studies have reported extracellular calcium dependence for KCl-induced contraction [24,25], hence our results are in concurrence to these findings. Ang II couples with calcium influx, efflux or release events [10] by various pathways and our results suggest that only the pathways involved in extracellular calcium influx are responsible for the Ang II-induced contraction, hence we attempted to identify this/these pathway/s in the present study.

Ang II-induced contraction was blocked by valsartan (10 µM, AT1 receptor blocker), dihydropyridine (DHP) calcium channel blockers (CCB, 10 µM, nimodipine, lercanidipine or nifedipine) but not by verapamil (100 µM) or diltiazem (100 µM). Thus suggesting that Ang II activated calcium channels are DHP sensitive and DHP insensitive channels are not involved in the process. Gadolinium (10 µM, store operated calcium channel blocker) or lanthanum (10 µM, capacitative calcium entry blocker) or LOE 908 (100 µM, receptor operated calcium channel blocker) did not affect Ang II-induced contraction. Lignocaine (10 µM, sodium channel blocker) did not block/reduce Ang II-induced response suggesting the contraction is independent of the membrane depolarization process. Supporting our observation, a previous study showed that Ang II activates L-type Ca2+ channels by direct binding of the G-protein beta/gamma subunits, thus independent of changes in membrane voltage [26]. {omega}-Agatoxin IVA did not affect the Ang II contraction thus excluding the role of P/Q-type Ca2+ channels. P/Q-type Ca2+ channels are expressed in rat aorta and renal arteries where in their activation is depolarization dependent [27], since Ang II-induced contraction was independent of the depolarization process, this further excludes the involvement of these channels in Ang II-induced contraction in rat thoracic aorta. In the presence of thapsigargin (100 nM, sarcoplasmic reticulum calcium ATPase pump inhibitor i.e., releases intracellular calcium) or in aortic rings loaded with BAPTA (10 µM, selective chelator of Ca2+), Ang II-induced contraction was unaffected, thus excluding the involvement of the intracellularly released calcium in the Ang II-induced contraction in rat thoracic aorta. Hence, our results suggest that Ang II-induced contraction is mediated via AT1 receptors by the activation of plasma membrane DHP sensitive L-type calcium channels. Using apoE null mouse model, a recent study demonstrates that vascular complications (atherosclerosis) encountered in STZ-induced diabetic mouse were ameliorated by AT1 selective antagonist but not Ca2+ channel antagonist, thus excluding the role of L-type calcium channels in the formation of atherosclerotic lesions [28]. However, this does not exclude their role in Ang II-induced contraction, since Ang II may regulate the structural and functional parameters in the vasculature differentially. Hence, L-type calcium channels although not involved in Ang II-mediated structural events in vasculature may be involved in the functional events such as contraction.

Supersensitivity (->pD2) to Ang II but not KCl was observed in thoracic aorta isolated from diabetic as compared to control rats. While enhanced response (->Emax) to both Ang II and KCl was observed, several studies have reported enhanced vasocontractile response to PE [29,30], Ang II [6,31], KCl [32,33], noradrenaline [34,35] and 5-hydroxytryptamine [35], in diabetic vasculature, thus suggesting that the enhanced response is not agonist specific. Our results are in concurrence with these findings. Since Ang II and KCl did not induce contraction in CFK, we could reasonably conclude that enhanced response to Ang II and KCl in thoracic aorta of diabetic rats is extracellular calcium dependent. Further, in diabetic rats, the increased and comparable response to Ang II in the absence and presence of endothelium indicates that both endothelium and smooth muscle components are involved in the vascular pathology in diabetes, unlike most studies claiming only the role of endothelial component [36,37].

4.3. Effect of nimodipine on Ang II- and KCl-induced contraction
Since DHP CCB blocked the responses to Ang II and KCl, we selected nimodipine (a prototype of the group) for our further studies. Nimodipine dose dependently blocked the Ang II- and KCl-induced contraction in a noncompetitive manner in thoracic aortic rings from diabetic and control rats. The –log IC50 of nimodipine to Ang II- and KCl-induced contraction in intact as well as denuded thoracic aortic rings was significantly lower in diabetic as compared to control rats. In other words, higher concentration of nimodipine was needed to block the Ang II- and KCl-induced contraction in thoracic aortic rings from diabetic rats. The pA2 of nifedipine to KCl-induced contraction in the aorta of diabetic rats was decreased by one order of magnitude [38], suggesting that diabetes reduces the sensitivity of aortic tissue to nifedipine due to higher stimulation–contraction coupling of vascular smooth muscles, resulting in increased calcium influx [39,40]. Such mechanisms may be responsible for diabetes-induced vascular complications [38]. Hence, our results support the hypothesis that increased influx of calcium may play an important role in the vascular complications associated with diabetes. We did cross check our data with another DHP calcium channel blocker (lercanidipine) (data not shown) as few reports suggest nimodipine being nonselective. We also observed similar pattern of change in the –log IC50 of nimodipine to phenylephrine-induced contraction in aortic rings from diabetic rats as compared to control, since our objective in the present study was to elucidate the Ang II-induced contraction mechanisms, we have not shown or discussed this data.

4.4. Effect of Ang II on interaction of calcium concentration in single VSMC
To understand the role of calcium in vascular response to Ang II, we studied the effect of Ang II on intracellular calcium concentration ([Ca2+]i) in single VSMCs isolated from the thoracic aorta of control and diabetic rats. We observed a biphasic pattern wherein a transient phase was followed by a sustained phase, which is consistent with other reports [23]. In calcium-free Krebs, Ang II did induce increase in [Ca2+]i (as evident from the increase in fura ratio340/380), however, this release did not differ in VSMCs isolated from control and diabetic rats. Thus, from these results, we can conclude that although Ang II induces intracellular calcium release, it is unaltered in diabetic VSMCs, and moreover, this calcium is not involved in activating the contractile response. We studied the CRC to Ang II (0.1 nM–1 µM)-induced [Ca2+]i in single VSMC. In the presence of extracellular calcium, we observed a 2.5-fold increase in the maximal [Ca2+]i in Ang II exposed VSMC isolated from diabetic than control rats. pD2 value of Ang II was significantly higher in diabetic than control rat VSMCs. Nimodipine (1 µM) reduced the Ang II-induced rise in [Ca2+]i while in BAPTA-loaded VSMCs, nimodipine blocked the Ang II-induced rise in [Ca2+]i. Thus, these results support our in vitro observations of enhanced Ang II response in thoracic aortic rings from diabetic rats being due to enhanced extracellular calcium influx via DHP sensitive L-type calcium channels.

4.5. Saturation binding of [3H]Ang II at AT1 receptors
To assess whether the enhanced Ang II response was due to altered AT1 receptor status, we studied the binding characteristics of [3H]Ang II at AT1 receptors and observed significantly higher affinity and Bmax in VSMC membranes from diabetic than control rats. Hence, in the present study, supersensitivity to Ang II observed in diabetic tissue could be due to increased affinity of Ang II to its AT1 receptor and increase in AT1 receptor density. This enhanced AT1 receptor density and affinity may significantly contribute to the diabetic vascular pathophysiology.

4.6. Saturation binding of [3H]PN200110 at L-type calcium channels
Since the Ang II-induced vasocontractile effect was mediated by extracellular calcium influx via DHP sensitive L-type calcium channels, we studied its binding characteristics in VSMC membranes prepared from thoracic aorta isolated from control and diabetic rats. As the nimodipine –log IC50 value to Ang II-induced contraction was significantly lower in diabetic tissue, we expected the binding affinity to be lower in VSMC membranes from diabetic rats. Contrary to this, we observed a higher affinity with no change in the Bmax in VSMC membranes from diabetic than control rats. With the increase in affinity of [3H]PN200110, we needed higher nimodipine concentration to reduce the Ang II-induced contraction by 50%. Moreover, with no change in the calcium channel density, we observed a significant increase in the [Ca2+]i by Ang II in single VSMCs from diabetic compared to control rats. Hence, our results prompt us to propose that there could be enhanced functional coupling between AT1 receptors and DHP sensitive L-type calcium channels, which could be responsible for the supersensitivity to Ang II observed in thoracic aorta isolated from diabetic rats. Such an increase in the AT1 receptor and DHP sensitive L-type calcium channels functional coupling may significantly contribute to the diabetic vascular complications.


   5. Conclusions
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
References
 
From our results, we conclude that Ang II-induced contraction mediated by AT1 receptors involves extracellular calcium influx by functional coupling with the plasma membrane DHP sensitive L-type calcium channels in rat thoracic aorta. The functional coupling is enhanced in the aortic rings from diabetic rats, which could be responsible for the supersensitivity to Ang II and may significantly contribute to the vascular pathophysiology in diabetes.


   Acknowledgements
 
Arun KHS acknowledges the Council of Scientific and Industrial Research (CSIR, New Delhi, India) for a Senior Research Fellowship (9/727(18)/2002-EMR-I).


   Notes
 
1 Present address: Rudolf Buchheim Institut Für Pharmakologie, Universitätsklinikum Giessen, Frankfurter Strasse 107, D-35392, Giessen, Germany. Back

Time for primary review 29 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 

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