Atenolol

Autonomic regulation of the heart was analyzed through recording changes in HR after selective pharmacological blockade of the parasympathetic and sympathetic nervous system using anti-cholinergic and beta-blocker drugs, respectively 26 (link). The bradycardic response obtained after β-adrenergic receptor blockade with atenolol (1 mg/kg, i.v.; Sigma-Aldrich Co, St Louis, MO, USA) was used to estimate sympathetic tone (atenolol Δ). The tachycardia response after muscarinic cholinergic receptor blockade with methyl atropine (3 mg/kg, i.v.; Sigma-Aldrich Co, St Louis, MO, USA) was used to estimate vagal tone (Atropine Δ). At the end of the experiment, hexamethonium bromide, a ganglion blocker that also inhibits the effects of noradrenalin on vessels, was slowly administered (1 mg/kg, iv; Sigma-Aldrich Co, St Louis, MO, USA). The sympathetic tone to the vessels was considered the difference between the minimum MAP obtained after hexamethonium blockade and the basal MAP (Hexamethonium Δ).

After the baroreflex sensitivity assessment, the AP and PI were continuously recorded at a basal state and after intravenous injection of methylatropine (4 mg/kg, Sigma, Kawasaki, Japan; 0.2 mL). Because the HR response to these drugs reaches its peak within 3 to 5 min, this time interval was allowed to elapse before the heart rate measurement. Atenolol (8 mg/kg, Sigma; 0.2 mL) was intravenously injected 10 min after methylatropine, and again the response was evaluated after a simultaneous blockade with Atenolol and methylatropine. On the following day, the sequence of injections was inverted (first Atenolol and then methylatropine). The intrinsic heart rate (IHR) was evaluated after a simultaneous blockade with Atenolol and methylatropine. The sympathetic tonus was determined as the difference between the maximum heart rate after the methylatropine injection and IHR. The vagal tonus was obtained by the difference between the lowest heart rate after the Atenolol injection and IHR [37 (link)].

A total of 134 B6D2F1 (C57BL/6 female × DBA/2 male) male mice were maintained under SPF conditions receiving ad libitum a standard rodent chow (Panlab, Spain) in individual mouse cages during their whole lifespan. Eighty-six of these animals (43 Old control and 43 Old-AT-treated) were used only to obtain the survival curves. The Atenolol treatment started at 2 months of age. Forty-eight separated ‘pilot’ animals were used to measure the different physiological and biochemical parameters at 16 months of age after sacrifice by cervical dislocation. A young control group (6 months of age) was also included. Atenolol (Sigma, A7655) was given in drinking water at 0.1 g L⁻¹, which resulted in a mean Atenolol intake of 0.559 mg per mouse per day. Just after cervical dislocation, tissues were immediately processed to isolate functional mitochondria for respiration and mitROS generation measurements. Tissue and excess mitochondrial samples were stored at −80 °C for the posterior analyses. Detailed methods of the physiological parameters and the measurements in functional mitochondria referred to below are described in the online Supporting Information.

DNJ (purity >98.0%) and SZ–A (lot number: 20130717-2), containing 37.5% of DNJ, 6.4% of FGM, and 4.8% of DAB, was kindly provided by the Department of Pharmaceutics (Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing, China). Miglitol (internal standard, IS) was purchased from J and K Scientific Ltd., (Beijing, China). FGM (purity >98.0%) was obtained from Medchem Express Co., Ltd., (Princeton, NJ, USA). DAB∙HCl (purity >98.0%), propranolol, atenolol, β-nicotinamide adenine dinucleotide phosphate (β-NADP) hydrate, d-glucose 6-phosphate (G-6-P) disodium salt hydrate, glucose-6-phosphate dehydrogenase (G-6-PDH), and ammonium hydroxide solution were products of Sigma-Aldrich Co., Ltd., (St. Louis, MO, USA). Tris(hydroxymethyl)aminomethane (Tris) was obtained from Sinopharm Chemical Reagent Co., Ltd., (Beijing, China). Anaerobic medium was purchased from Beijing Land Bridge Technology Co., Ltd., (Beijing, China). Acetonitrile and methanol were of HPLC grade (Merck KGaA, Darmstadt, Germany). Ultrapure water was prepared by a Milli-Q Reagent water system (Millipore, Billerica, MA, USA). All other chemicals were of analytical reagent grade and commercially available.

Clear cell renal cell carcinoma primary tumor cultures (ccRCC) were obtained from surplus of resected surgery tumor samples from VHL patients following the procedure previously described (16), and cultured in RPMI medium supplemented with 20% fetal bovine serum (FBS), 2 mM L-glutamine, and 100 U/mL penicillin/streptomycin (all from GIBCO, Grand Island, NY, USA). All patients provided written informed consent to use their tissue samples for this study.
HUVECs (ATCC-CRL-1730) were cultured in EGM-2 (Lonza, Walkersville, MD, USA) supplemented with 10% FBS, 2 mM L-glutamine, and 100 U/mL penicillin/streptomycin (GIBCO, Grand Island, NY, USA).
The human renal cancer cell line 786-O (ATCC CRL-1932) was cultured in RPMI supplemented with 20% FBS, 2 mM L-glutamine, and 100 U/mL penicillin/streptomycin.
ccRCC primary tumors, 786-O cells, and HUVECs were incubated with different doses of ADRB antagonists for the time and dose indicated in each experiment. Atenolol (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in DMSO (Merck, Darmstadt, Germany), while propranolol and ICI (Sigma-Aldrich, St. Louis, MO, USA) were dissolved in distilled water.
All the cellular assays were performed at 37 °C, 5% CO₂ and humidity conditions.