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MS ID#: HYPERTENSION/2006/081166
MS TITLE: INCREASED CHRONOTROPIC ACTION OF ANGIOTENSIN II IN SHR
NEURONS: ROLE OF MACROPHAGE MIGRATION INHIBITORY FACTOR
Materials
Rabbit anti-rat MIF antibody was purchased from Torrey Pines Biolabs (Houston, TX). Ang II and all other chemicals were obtained from Sigma-Aldrich Chemical (St. Louis, MO). 125I-(Sar1-Ile8)-Ang II (~2150 Ci/mmol) was obtained from the University of Mississippi (Oxford, MS). Mouse recombinant MIF (rMIF) and C60S-MIF mutant were prepared from an Escherichia coli expression system and purified free of endotoxin by C8 chromatography as described previously1,2. Mouse MIF differs from rat MIF by a single amino acid substitution (mMIF:asn54, rMIF:ser54) that has not been found to affect the influence of the bioactivity or immunoreactivity of the protein in different murine assays1,2. Rat/Mouse Macrophage Inhibitory Factor ELISA kits were obtained from Chemicon International (Temecula, CA). RNeasy kits and OneStep RT-PCR Kits were obtained from Qiagen (Valencia, CA). TA Cloning® Kits were from Invitrogen (Carlsbad, CA). Adeno-X™ Rapid Titer Kits were from BD Biosciences (Palo Alto, CA).
Electrophysiological Recordings
Spontaneous and depolarizing pulse-elicited APs were recorded with the whole-cell voltage clamp configuration in current clamp mode3,4. Experiments were performed at room temperature (23-24°C) with an Axopatch 200B amplifier and a Digidata 1200B interface (Axon Instruments, Burlingame, CA). Data acquisition and analyses were performed using pClamp 8.0. Cells were bathed in Tyrode's solution containing (in mM) 140NaCl, 5.4KCl, 2.0CaCl2, 2.0MgCl2, 0.3NaH2PO4, 10HEPES, and 10dextrose, pH adjusted to 7.4with NaOH. Neurons in the culture dish (volume 1.5ml) were superfused at a rate of 2-4 ml/min. The patch electrodes (Kimax-5.1, Kimble Glass, Toledo, OH) had resistances of 3-4 MOhms when filled with an internal pipette solution containing (in mM) 140KCl, 4MgCl2, 4ATP, 0.1guanosine 5'-triphosphate, 10dextrose, and 10HEPES, pH adjusted to 7.2with KOH. The whole-cell configuration was formed by applying negative pressure to the patch electrode. A junction potential of -8 mV was corrected for all membrane potentials. The resting membrane potential (RMP) was defined as the potential within a 1s time period during which there was no spontaneously firing AP. The neuronal firing rate was measured as the number of fully developed APs (depolarization beyond 0 mV) per second (Hz).
Intracellular application of rMIF, C60S-MIF and MIF-neutralizing antibodies during the electrophysiological recordings were achieved by injection through the patch pipette5. In brief, a sidearm pipette holder is attached to the head stage of the Axopatch. One side arm is used to apply suction for seal formation, and the second side arm is used to advance a very fine polyethylene catheter (PE-50) down the inside of the patch pipette. Control measurements of are made 5min after the whole cell configuration is established in a given neuron. After this, the protein or antibody solution (1µl) is injected into the tip of the recording electrode via the PE-50 tube. From the pipette tip, the MIF, etc. are allowed to diffuse into the neuron, and measurements of firing rate are made 4min later, at which time a stable peak response is obtained. Care is taken not to overperfuse the neuron, and this is monitored electrically via the Axopatch and on the TV monitor. Thus the concentrations of rMIF, C60S-MIF and MIF-neutralizing antibodies that are given in the Results indicate the amounts that were injected at the pipette tip and are likely higher than the amounts that reach the site of action.
AT1R mRNA and AT1R binding
The effects of Ad5-SYN-MIF and Ad5-SYN-EGFP on AT1R mRNA levels was examined using quantitative real-time PCR. Neuronal cultures from WKY rats or SHR were incubated with Ad5-SYN-MIF, Ad5-SYN-EGFP, or phosphate buffered saline (PBS) as a control for 3 days at 37oC in a CO2 incubator. Total RNA was isolated from these neurons using an RNeasy Kit. For each reverse transcription-PCR reaction, 2 µg of total RNA was converted into cDNA using reverse transcriptase (Qiagen). Two microliters of reverse transcription reaction was then quantified by quantitative real-time PCR using an ABI PRISM 7900 Sequence Detector and ABI TaqMan ® PCR Master Mix (Applied Biosystems, CA). The quantitative results of AT1R mRNA were normalized to18S RNA from the same sample. Primers and probes for AT1R and 18S were purchased from Applied Biosystems, and the experimental protocol utilized was exactly as described in the instructions provided by the company.
References
1. Bernhagen J, Mitchell RA, Calandra T, Voelter W, Cerami A, Bucala R. Purification, bioactivity, and secondary structure analysis of mouse and human macrophage migration inhibitory factor (MIF). Biochemistry. 1994; 33:14144-14155.
2. Kleemann R, Kapurniotu A, Mischke R, Held J, and Bernhagen J. Characterization of catalytic centre mutants of macrophage migration inhibitory factor (MIF) and comparison to Cys81Ser MIF. Eur J Biochem. 1999; 261:753-766.
3. Zhu M, Sumners C, Gelband CH, Posner P. Chronotropic effect of angiotensin II via type 2 receptors in rat brain neurons. J Neurophysiol. 2001; 85:2177-2183.
4. Sun C, Li H, Leng L, Raizada MK, Bucala R, Sumners C. Macrophage migration inhibitory factor: an intracellular inhibitor of angiotensin II-induced increases in neuronal activity. J Neuroscience 2004; 24: 9944-9952.
5. Zhu M, Gelband CH, Posner P, Sumners C. Angiotensin II decreases neuronal delayed rectifier potassium current: role of calcium/calmodulin-dependent protein kinase II. J Neurophysiol. 1999; 82:1560-1568.