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| Claudio Grassi |
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Institute of Human Physiology, UCSC, Largo F. Vito 1, Rome
e-mail: grassi@rm.unicatt.it |
Poster Presentation: |
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| INHIBITION OF N-TYPE Ca2+-CHANNEL GATING INDUCED BY NITRIC OXIDE VIA cGMP-DEPENDENT PROTEIN KINASE, IN HUMAN NEUROBLASTOMA IMR32 CELLS. |
| G. Martinotti, M. D'Ascenzo, G.B. Azzena, and C. Grassi | |
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Institute of Human Physiology, Medical School, Catholic University "S. Cuore", Rome, Italy |
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| Nitric oxide (NO), which is produced in both neurons and glial cells under different physiological and patho-physiological conditions, acts as a non-conventional intercellular messenger in the central and peripheral nervous systems. The functional role played by NO in the nervous system is partly related to its action on various ion channels, including the voltage-gated Ca2+ channels. In previous studies, we have found that NO-donors and cGMP analogues markedly inhibit both L- and P/Q-type Ca2+ channels (Grassi et al., Pflügers Arch. 437:241-247, 1999; Carabelli et al., J. Physiol. 541:351-366, 2002). Several investigators have suggested that Ca2+ influx through N-type channels is instead to some degree enhanced by NO donors (Chen and Schofield, 1995; Hirooka et al., 2000). The present study was conducted at the levels of both macroscopic currents and single channels in an attempt to clarify the nature of NO’s modulatory effects on the N channels of human neuronal cells.
The experiments were performed by using the patch-clamp technique in the whole-cell and cell-attached configurations (10 and 100 mM Ba2+ as charge carrier, respectively). In order to isolate the current flowing through N-type Ca2+ channels, the occasionally found T-type current was eliminated by means of pre-pulse depolarization at -40 mV and L-channels were blocked by 5 uM nifedipine.
During depolarization at +10 mV, the NO-donor, sodium nitroprusside (SNP, 200 uM), reduced macroscopic N-currents by 34.1+/-1.5% (n=21; P<0.01). No significant inhibition was found when SNP was applied with the NO-scavenger carboxy-PTIO (300 uM) or in the presence of the guanylate cyclase inhibitor ODQ (10 uM). The membrane permeable analogue of cGMP, 8-Br-cGMP (400 uM) mimicked SNP effects, reducing Ba2+-currents by 37.2+/-3.7% (n=7; P<0.001). These effects were virtually abolished by the protein kinase G (PKG) inhibitors KT5823 (1 uM) or Rp-8-pCPT-cGMPS (20 uM). At the single-channel level, 8-Br-cGMP reduced the open probability by 54.5% (0.05+/-0.01 vs. 0.11+/-0.01, P<0.001) and increased the null sweep probability from 0.13+/-0.06 to 0.39+/-0.02. Channel conductance was instead not significantly affected by 8-Br-cGMP (20.2+/-0.6 vs. 19.4+/-2.4 pS). Plots of the open time distribution were fitted according to one exponential with to= 1.12 ms in controls and 1.19 ms during 8-Br-cGMP application. The closed time distribution was fitted with a three exponential function, all time constants being significantly prolonged by the cGMP analogue. The mean tc values were 8.53 ms in controls and 14.47 ms with 8-Br-cGMP.
In conclusion, our data suggest that the N-channels of neuroblastoma IMR32 cells are inhibited by NO. This effect is mediated by cGMP and PKG and it consists of a reduction in the channel-open probability and an increase in closed times, which are not associated with significant changes in either channel conductance or mean open time. The consequent decrease in Ca2+ influx through these channels may affect numerous neuronal functions, including neurotransmitter release from nerve endings and synaptic transmission.
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