Alzet osmotic minipumps model 1002

PTH(1-34) or [Cys25]PTH(1-34) (40 or 120 μg/kg/d) were infused, as described,^{(52 (link))} for 4 days by Alzet osmotic minipumps (Model 1002, DURECT Corporation, Cupertino, CA, USA) into 7-week-old male C57BL6 mice; control animals received vehicle. Both peptides were diluted in a solution of 150 mM NaCl, 1 mM HCl, and 2% heat-inactivated mouse serum. Blood ionized calcium and plasma phosphate levels were determined before and daily after starting the infusion.
To determine the biological activity of circulating PTH(1-34) or [Cys25]PTH(1-34), pooled plasma collected from mice infused with either ligand was incubated with GP2 cells (stable hPTH1R expression) and cAMP-dependent increases in luminescence were measured, as described.^{(55 (link))}For some experiments, heparinized plasma was collected before and daily after minipump implantation. Furthermore, aliquots of the peptide solution in each minipump were recovered before implantation and stored at −80°C. Four days after implantation into an interscapular subcutaneous pocket, aliquots were obtained from each pump and stored at −80°C; aliquots from pumps loaded with vehicle served as controls. All samples were evaluated in the same assays.

All animals were used in accordance with the guidelines of the National Institutes of Health Institute of Laboratory Animal Resources, 1996) and American Heart Association for the care and use of laboratory animals. The procedures were performed in accordance with experimental protocols that were approved by the University Committee on Animal Resources at the University of Rochester. C57/BL6 male mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Animals were housed under a 12-h light-dark regimen. Cardiac remodeling was induced in vivo by subcutaneous infusion of Ang II (800 ng/min/kg) for 2 weeks or corresponding vehicle using Alzet osmotic mini-pumps (model 1002, Durect Corp, Cupertino, CA) as described previously [12 (link), 13 (link)]. Animals at age of 10 weeks were anesthetized with inhaled isoflurane and osmotic mini-pumps were implanted subcutaneously on the back slightly posterior to the scapulae. For systemic vinpocetine treatment, mice were intraperitoneally injected with 5 mg/kg vinpocetine or vehicle every day as described previously [10 (link), 11 (link)]. Blood pressure was recorded by tail artery blood pressure measurement. Mouse hearts were excised, excised hearts were washed with saline to remove the blood, and the whole hearts were weighed. Hearts were used for histological and immunoblotting analyses.