Epc 8 patch clamp amplifier

Catecholamine secretion from glomus cells or chromaffin cells in CB or AM slices, respectively, was performed following a procedure developed in our laboratory^{54 (link),69 (link)}. Secretory events elicited by different stimuli were detected with a 10 μm carbon-fiber electrode. Amperometric currents were recorded with an EPC-8 patch clamp amplifier (HEKA Electronics), filtered at 100 Hz and digitized at 250 Hz before storage on computer. Data acquisition and analysis were performed with an ITC-16 interface (Instrutech Corporation) and PULSE/PULSEFIT software (HEKA Electronics). The secretion rate (femtocoulombs (fC)/min) was calculated as the amount of charge transferred to the recording electrode during a given period of time.

To perform the experiments, single slices were transferred to the chamber placed on the stage of an upright microscope (Axioscope, Zeiss, Oberkochen, Germany) and continuously perfused by gravity (flow 1–2 ml min^-1) with a solution containing (in mM): 117 NaCl, 4.5 KCl, 23 NaHCO₃, 1 MgCl₂, 2.5 CaCl₂, 5 glucose and five sucrose. The ‘normoxic’ solution was bubbled with 5% CO₂, 20% O₂ and 75 % N₂ (PO₂ ≈ 150 mmHg). The ‘hypoxic’ solution was bubbled with 5% CO₂ and 95% N₂ (PO₂ ≈ 10–20 mmHg). The ‘hypercapnic’ solution was bubbled with 20% CO₂, 20% O₂ and 60 % N₂ (PO₂ ≈ 150 mmHg). The osmolality of solutions was ≈ 300 mosmol kg⁻¹ and pH = 7.4. In the 20 mM K⁺ solution, NaCl was replaced by KCl equimolarly (20 mM). All experiments were carried out at a chamber temperature of 36°C. Amperometric currents were recorded with an EPC-8 patch-clamp amplifier (HEKA Electronics), filtered at 100 Hz and digitized at 250 Hz for storage in a computer. Data acquisition and analysis were carried out with an ITC-16 interface and PULSE/PULSEFIT software (HEKA Electronics). The secretion rate (fC min⁻¹) was calculated as the amount of charge transferred to the recording electrode during a given period of time.