3h camp

Methylphenidate hydrochloride (MPD; Ritalin®) was produced by Novartis AG (Basel, Switzerland). Cocaine hydrochloride, R(+)-SCH 23390 hydrochloride (SCH 23390) and Dopamine Hydrochloride were purchased from Sigma Aldrich Co. (St. Louis. MO, USA). [³H]-Dopamine (specific activity: 56.8 Ci/mmol) and [³H]-cAMP (specific activity: 28.1 Ci/mmol) were acquired from PerkinElmer (Massachusetts, USA). [³H]-SCH23390 (specific activity: 70.3 Ci/mmol) was acquired from Amersham (USA). All other chemicals were of the highest purity obtainable from regular commercial sources. Each experiment was performed at least 3 times.

The cell lines, culture, and cAMP assay are all described in our previous work (Apostol et al., 2021 (link)). Briefly, human PAC1, VPAC1, and VPAC2 receptors expressed in CHO cells were used for all experiments. Cells were grown in 50:50 DMEM-F12 medium with 10% heat-inactivated fetal bovine serum, 1X penicillin-streptomycin supplement, and 500 μg/ml G418 (neomycin) to maintain selection (all from ThermoFisher) at 37°C with 5% CO₂ atmosphere. For the cAMP assay, cells were seeded at 20,000 cells/well in a 96 well plate, recovered overnight, then serum starved for 4 h. The cells were then incubated with 500 μM IBMX for 20 min, followed by agonist concentration curves in 500 μM IBMX media for 10 min. The reaction was terminated, boiled, then collected, followed by competition with 7 μg of bovine protein kinase A (Sigma-Aldrich) and ∼1 pmol of ³H-cAMP (PerkinElmer). The assay was incubated at room temp for 1 h, then collected onto GF/B filter plates and the data read using a MicroBeta2 scintillation counter (PerkinElmer). The resulting data was fit to a 3 variable non-linear regression curve using GraphPad Prism, which resulted in potency (EC₅₀) and efficacy (E_MAX) measurements. Native PACAP_1–27 was used as a positive control, and also used to define an E_MAX level of 100%.

(-)-Propranolol, (-)-isoproterenol, epidermal growth factor (EGF), hydrocortisone, bovine serum albumin (BSA), phenylarsine oxide (PAO) and poly-L-lysine, IV collagen and laminin were from Sigma-Aldrich (Saint Louis, MO, USA). Culture medium, fetal bovine serum (FBS) and other products for cell culture were from Invitrogen-Thermo Fisher Scientific (Waltham, MA, USA). Fibronectin was from Santa Cruz Biotechnology (Dallas, TX, USA). FuGENE^® was purchased from Promega (Madison, WI, USA). Human recombinant insulin was kindly donated by Denver Farma (Buenos Aires, Argentina). Methyl-[³H]-thymidine and [³H]-cAMP were purchased from PerkinElmer Life Sciences (Waltham, MA, USA). BMS-3 was from SynKinase and PF3758309 was from Medkoo Biosciences (Morrisville, NC, USA).

Angiotensin-(1-7), cell culture medium, bovine serum albumin (BSA), isobutyl methylxanthine (IBMX), cAMP, forskolin, Ang-1-7, FURA-2AM, histamine dihydrochloride, triton X-100, pertussis toxin and protease inhibitors were obtained from Sigma Chemical Company (St. Louis, MO, United States). Fetal calf serum was from Natocor (Argentina). [³H] cAMP, was purchased from PerkinElmer Life Sciences (Boston, MA, United States). AVE 0991: 5-formyl-4-methoxy-2-phenyl-1-[[4-[2-ethylaminocarbonylsulfonamido-5-isobutil-3-thienyl] phenyl]-methyl]imidazole was obtained from ApexBio Technology LLC (Houston, TX, United States). Other chemicals used were of analytical grade and obtained from standard sources.

Cell culture medium, antibiotics, isobutylmethyl xanthine (IBMX), cAMP, bovine serum albumin (BSA), cycloheximide, amthamine, famotidine, forskolin, and PD98059 were obtained from Sigma Chemical Company (St. Louis, MO, USA). Tiotidine were from Tocris Cookson Inc. (Ballwin, MO, USA). [³H]cAMP, and [³H]tiotidine were purchased from Perkin Elmer Life Sciences (Boston, MA, USA). Fetal calf serum was from Natocor (Argentina). Other chemicals used were of analytical grade and obtained from standard sources.

Prazosin hydrochloride, (-)isoprenaline hydrochloride, timolol maleate, atropine sulphate, lidocaine (2-diethylamino-N-[2,6-dimethylphenyl]-acetamide) hydrochloride, L-ascorbic acid and 3-isobutyl-1-methylxanthine (IBMX) were purchased from Sigma-Aldrich (St. Louis, MO, USA). CGP20712 dihydrochloride, CGS 15943, cilostamide and rolipram were purchased from Tocris Bioscience (Bristol, UK). Forskolin was from LC Laboratories (Woburn, MA, USA). (R,R)- and (R,S)-fenoterol were a kind gift from J. Kozocas (SRI International, Menlo Park, CA, USA). Isoflurane (1-chloro-2,2,2-trifluoroethyl difluoromethyl ether; Forene) was from Abbot Scandinavia (Solna, Sweden). Pertussis toxin was from Merck chemicals (Nottingham, UK). [³H]cAMP was from PerkinElmer (Boston, MA, USA).