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Inhibition of ACh on the delayed rectifier--like potassium current in acutely isolated cerebral cortical neurons of rats

Cui Lanwei, Li Yurong, Yang Lei, Jia Shuwei, Qu Lihui, Yao Kun, Jin Hongbo

Department of Physiology, Harbin Medical University. Harbin 150086, China

Abstract

The modulation of ACh on delayed rectifier-like potassium currents (I_(K)) was studied in freshly dissociated cerebral cortical neurons using the whole-cell patch-clamp technique. Wistar rats between 10- and 14-day old of both sexes were used. After rats were decapitated, their brains were quickly removed, iced, and then manually cut into 400 µm slices. Slices were then incubated for 0.5 h at 32℃ in a buffered artificial cerebrospinal fluid (ACSF) bubbled with 95% O_(2), 5% CO_(2). Slices were then removed into buffered ACSF containing protease (0.5 mg/ml) at 32℃. After 30 min of enzyme digestion, tissue was rinsed three times in the buffered saline. Then the enzyme-treated slices were mechanically dissociated with a graded series of fire-polished Pasteur pipettes. The cell suspension was then plated into a 35 mm dish and placed on the stage of a Olympus inverted microscope. For whole-cell recordings of currents, standard voltage-clamp techniques were used. Neurons were held at -80 mV, and the I_(K) was evoked by 2 000 ms depolarizing voltage commands to potential between -40 mV and +60 mV in 10 mV steps applied at a frequency of 0.5 Hz. It was found that the inhibitory effect of ACh (0.1, 1, 10, 100 µmol/L) on I_(K) was dose-dependent. It was also found that ACh affected the activation process of I_(K) significantly, i.e., the activation curve of I_(K) was characterized by half-activation potential of (-41.8±9.7) mV and a slope factor of (30.7±7.2) mV in the cortical neurons and they were changed to (-122.4±38.6) mV and (42.4±7.0) mV, respectively, after giving ACh (10µmol/L). Tubocurarine (100 µmol/L) antagonized the inhibitory effect of ACh on I_(K), and the drop of currents varied from the control value of (36.5±7.8)% to (16.9±13.8)% (n=8, P<0.01). 4-DAMP (10 µmol/L) blocked the inhibitory effect of ACh on I_(K), and the currents reduced from the control value of (36.5±7.8)% to (26.8±4.7)% (n=6, P<0.05). Pirenzepin did not antagonize the inhibition of ACh on I_(K) (n=7, P>0.05). Chelerythrine (20 #mu#mol/L) blocked the inhibitory effect of ACh on I_(K) and the currents reduced from the control value of (36.5±7.8)% to (11.7±17.3)% (n=6, P<0.05). On the contrary, PDBu (10 #mu#mol/L) strengthened the inhibition of ACh on I_(K) and the drop of currents changed from the control value of (36.5±7.8)% to (59.2±14.0)% (n=5, P<0.05). PDBu abolished the antagonism of chelerythrine on ACh in cortical neurons. It is suggested that the ACh-induced depolarization of neurons in the cortex is attributed to the inhibition of I_(K) that is most likely evoked by the activation of nicotinic ACh receptors and muscarinic M_(3) receptor via protein kinase C (PKC) signal transduction pathway.

Key words: Acetylcholine receptors;Cortexin;Delayed rectifier-like potassium currents;Whole-cell recording;Protein kinase C

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Citing This Article：

Cui Lanwei, Li Yurong, Yang Lei, Jia Shuwei, Qu Lihui, Yao Kun, Jin Hongbo. Inhibition of ACh on the delayed rectifier--like potassium current in acutely isolated cerebral cortical neurons of rats . Acta Physiol Sin 2006; 58 (1): (in Chinese with English abstract).