Vasopressin

Vasopressin and SR49059 were obtained from Sigma-Aldrich and dissolved in ultrapure distilled water on the day of the study.

Working heart-brainstem preparations (Paton, 1996 (link)) were surgically prepared one week after the microinjections in the A5, as previously described (Zoccal et al., 2008 (link)). The animals were initially deeply anesthetized with halothane (AstraZeneca, Cotia, SP, Brazil) until the loss of the paw withdrawal reflex, transected caudal to the diaphragm, submerged in a chilled Ringer solution (in mM: NaCl, 125; NaHCO₃, 24; KCl, 3; CaCl₂, 2.5; MgSO₄, 1.25; KH₂PO₄, 1.25; dextrose, 10) and decerebrated at the precollicular level. Lungs were removed the preparations were then transferred to a recording chamber. The descending aorta was cannulated and perfused retrogradely using a roller pump (Watson-Marlow 502 s, Falmouth, Cornwall, UK) via a double-lumen cannula. The perfusate consisted of Ringer solution containing 1.25% Polyethylene glycol (an oncotic agent, Sigma, St Louis, USA) and a neuromuscular blocker (vecuronium bromide, 3–4 μg mL⁻¹, Cristália Produtos Químicos Farmacêuticos Ltda., São Paulo, Brazil). This solution was gassed continuously with 5% CO₂–95% O₂, warmed to 31–32 °C and filtered using a nylon mesh (pore size: 25 μm, Millipore, Billirica, MA, USA). The perfusion pressure was maintained in the range of 50–70 mmHg by adjusting the rate flow to 21–25 mL min⁻¹ and by adding vasopressin to the perfusate (0.6–1.2 nM, Sigma, St. Louis, MO, USA).

Working heart-brainstem preparations (Paton, 1996 (link)) were surgically prepared, as previously described (Zoccal et al., 2008 (link)). Juvenile Holtzman rats were deeply anesthetized with halothane until the paw and tail pinch reflexes were abolished. The animals were then transected below the diaphragm and submerged in a cold Ringer solution (in mm: NaCl, 125; NaHCO₃, 24; KCl, 3; CaCl₂, 2.5; MgSO₄, 1.25; KH₂PO₄, 1.25; dextrose, 10). Animals were then decerebrated and their skin removed. The lungs were removed with care to not damage the descending aorta or the phrenic nerve at the attachment to the diaphragm. The left cervical vagus nerve was isolated. Preparations were then transferred to a recording chamber; the descending aorta was cannulated and perfused retrogradely (21–25 mL·min⁻¹; Watson-Marlow 502s, Falmouth, Cornwall, UK), via a double-lumen cannula, with Ringer solution containing 1.25% Polyethylene glycol (an oncotic agent, Sigma, St Louis, USA) and vecuronium bromide (a neuromuscular blocker, 3–4 μg·mL⁻¹). The perfusion pressure was held within 50–70 mmHg by adding vasopressin (0.6–1.2 nM, Sigma, St. Louis, MO, USA) to the perfusate. The perfusate was continuously gassed with 5% CO₂ and 95% O₂, warmed to 31–32 °C and filtered using a nylon mesh (25 μm).

Ubiquitin (sc-8017), LAMP1 (sc-20011), phospho-PERK (sc-32577), PERK (sc-13073), phospho-c-Jun (sc-822), c-Jun (sc-376488), SERCA2 (sc-376235, sc-8095), ATF6 (sc-22799), eIF2α (sc-11386), CaMKII (sc-5306), α1_C/Ca_v1.2 (sc-16229-R), α1_D/Ca_v1.3 (sc-25687), LC3 (sc-271625) and p62 (sc-28359) antibodies were obtained from Santa Cruz Biotechnology, Inc. IRE1α (3294), phospho-CaMKII (12716), phospho-eIF2α (3398), PERK (5683), LC3 (2775), p62 (5114) and Calnexin (2679) antibodies were purchased from Cell Signaling Technology, Inc. Pan-keratin antibody was obtained from Dr. Omary (University of Michigan)^{65 (link)}. Actin (JLA20) and tubulin (T5168) antibodies were purchased from Developmental Studies Hybridoma Bank (DSHB) and Sigma, respectively. Fatty acid free and low endotoxin bovine serum albumin (BSA), palmitic acid (PA), stearic acid (SA), oleic acid (OA), docosahexaenoic acid (DHA), tunicamycin (Tm), thapsigargin (Tg), butylated hydroxyanisole (BHA), N-acetylcysteine (NAC), chlorocresol, L-cycloserine, glucagon, phenylephrine, vasopressin, verapamil and nicardipine were purchased from Sigma. SP600125, bafilomycin and rapamycin were from LC Labs, and fumonisin B1 and suramin were from Cayman Chemical.