ON-LINE SUPPLEMENT

NEURONAL PROSTACYCLIN IS AN AUTOCRINE REGULATOR OF ARTERIAL BARORECEPTOR ACTIVITY.

Vladislav Snitsarev1, Carol A. Whiteis1, Mark W. Chapleau1,2,3, and
Francois M. Abboud1,2

Cultured nodose neurons

Experiments were performed at 37°C in PDMI–2 heated bath (Harvard Apparatus, Holliston, MA) from 1 to 3 days after dissociation. The extracellular buffer contained (in mmol/L): NaCl (116), KCl (5.4), NaH2PO4 (1), MgSO4 (0.8), MgCl2 (1), CaCl2 (1.8), Dglucose (5.6), NaHCO3 (26), and pH 7.4 (5%CO2).

Microelectrodes and membrane potential (MP) measurements

MP was recorded with sharp microelectrodes made of 1.0 mm O.D/0.5 mm I.D. borosilicate glass tubing (catalog #BF100-50-10, Sutter, Novato, CA). For microelectrode fabrication, a preset program #10 for P–2000 laser puller (Sutter, Novato, CA) was used. The outer tip of such microelectrodes is ~100 nm. These microelectrodes were filled with KCl (1 mol/L) solution and had a resistance between 100 and 250 MΩ 1. Brown (1986) has recommended the use of 1 mol/L KCl instead of the usual 3 mol/L KCl, which would have substantially decreased the microelectrode resistance. However, the lower K+ concentration we used and the resulting higher resistance diminished the artifact of K+ flow to the cytoplasm.

In measuring electrophysiological responses to repeated mechanical stimulations, sharp microelectrodes have advantages over patch clamp pipettes. First, the area of contact between the sharp microelectrode and the cellular membrane is much smaller (100 nm) than in the case of a patch pipette (outer tip diameter is about
2 µm). This provides a better chance of cell survival after impalement for a longer time. We have been able to record reliably resting MPs and APs for more than one hour from a neuron in vitro. Second, after impaling and obtaining a seal, the sharp microelectrode can be retracted or moved several micrometers without disrupting the seal and affecting resting MP 2. Hence, it is more likely to remain in the cell without dislodgement and allow continuous electrophysiologic recording during repeated mechanical deformations.

Mechanical Stimulation

We have developed models of mechanical stimulation of BRNs over several years. We have empirically found that pressure puffs of 10 psi give more consistent and reversible depolarizations of mechanosensitive cells ranging from 3 to 30 mV and in some neurons induce a burst of APs.

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The 1 sec duration of depolarizing currents was chosen because first, we had to do repeated injections over a period of 15 minutes to test the temporal pattern of recovery, and second, the depolarizing current of 0.5 nA usually produced an early rapid burst of APs which slowed significantly within 1 sec. This feature was useful as the enhanced recovery following NA was often evident by a more rapid and more sustained generation of APs.

Generation of PGI2 by nodose neurons in culture

Neurons from 16 nodose ganglia of eight rats were dissociated. Eight ganglia from each set of four rats were pooled and evenly distributed to eight wells each in a 48 well Costar cell culture plate (Corning Inc., Corning, NY) precoated with poly-D-lysine (Sigma, St. Louis, MO) and laminin (Sigma, St. Louis, MO), and cultured for 24 hours in 0.5 ml of culture medium. On the next day the culture media were washed twice with 0.5 ml Earle’s buffer (Sigma, St. Louis, MO). Indomethacin (Sigma, St. Louis, MO) (10 µmol/L) was added to four wells in each set, and cells were incubated for another 30 min. Arachidonic acid (Cayman Chemical, Ann Arbor, MI) (10 µmol/L) was then added to two wells with indomethacin and two wells without indomethacin in each set, and cells were incubated for another 30 min. Electric field stimulation (20 Hz pulses of 10 mA amplitude for two min.) was applied with platinum electrodes placed in four of the eight wells, in each set: one containing indomethacin, one arachidonic acid, one indomethacin and arachidonic acid, and one without chemicals. The remaining four wells with similar chemical compositions were not stimulated. All wells were sonicated with Microson XL2000 ultrasonic cell disruptor (Misonix, Formingdale, NY) and their contents centrifuged at 1500 rpm for 10 min. at 4ºC (VSMC-13, PRO Scientific Inc., Oxford, CT). The supernatant was analyzed in duplicate aliquots of 100 μl of the total of 500 μl from each well with a 6-keto-Prostaglandin F1a Enzyme Immunoassay Kit (Assay Designs Inc., Ann Arbor, MI). The quantity of 6-keto-Prostaglandin F1a is expressed as pg/100 μl of supernatant from each well.

Reagents

cPGI (5-[hexahydro-5hydroxy-4-(3-hydroxy-1-octenyl)-2(1H)-pentalenylidene]-,[3aS-[2E,3α,4α(1E,3R*),5β,6α]]) was purchased from Cayman Chemical (Ann Arbor, MI). DiI was purchased from Molecular Probes (Eugene, OR). Indomethacin was purchased from Sigma (St. Louis, MO). All other reagents were from either Sigma (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA).

References

1 Brown Kt. Advanced Micropipette Techniques for Cell Physiology. Chichester: John Wiley & Sons; 1986.

2 Brown KT, Flaming DG. Glass-membrane seals during intracellular recording with penetrating micropipettes. In: Brown KT, Flaming DG, editors. Advanced Micropipette Techniques for Cell Physiology. Chichester: John Wiley & Sons; 1986. p. 251.

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