Nitric oxide inhibits neuroendocrine Ca(V)1 L-channel gating via cGMP-dependent protein kinase in cell-attached patches of bovine chromaffin cells

Abstract

Nitric oxide (NO) regulates the release of catecholamines from the adrenal medulla but the molecular targets of its action are not yet well identified. Here we show that the NO donor sodium nitroprusside (SNP, 200 microM) causes a marked depression of the single Ca(V)1 L-channel activity in cell-attached patches of bovine chromaffin cells. SNP action was complete within 3-5 min of cell superfusion. In multichannel patches the open probability (NP(o)) decreased by approximately 60 % between 0 and +20 mV. Averaged currents over a number of traces were proportionally reduced and showed no drastic changes to their time course. In single-channel patches the open probability (P(o)) at +10 mV decreased by the same amount as that of multichannel patches (approximately 61 %). Such a reduction was mainly associated with an increased probability of null sweeps and a prolongation of mean shut times, while first latency, mean open time and single-channel conductance were not significantly affected. Addition of the NO scavenger carboxy-PTIO or cell treatment with the guanylate cyclase inhibitor ODQ prevented the SNP-induced inhibition. 8-Bromo-cyclicGMP (8-Br-cGMP; 400 microM) mimicked the action of the NO donor and the protein kinase G blocker KT-5823 prevented this effect. The depressive action of SNP was preserved after blocking the cAMP-dependent up-regulatory pathway with the protein kinase A inhibitor H89. Similarly, the inhibitory action of 8-Br-cGMP proceeded regardless of the elevation of cAMP levels, suggesting that cGMP/PKG and cAMP/PKA act independently on L-channel gating. The inhibitory action of 8-Br-cGMP was also independent of the G protein-induced inhibition of L-channels mediated by purinergic and opiodergic autoreceptors. Since Ca(2+) channels contribute critically to both the local production of NO and catecholamine release, the NO/PKG-mediated inhibition of neuroendocrine L-channels described here may represent an important autocrine signalling mechanism for controlling the rate of neurotransmitter release from adrenal glands.

Figure 2. Mean NP_o and null sweeps probability versus time before and during SNP application

Filled bars are data in control conditions obtained by averaging data collected during 1 min of recording from 13 patches. Open bars are values during SNP application obtained at intervals of 30 s (A-C) or by grouping all the values from the third to the sixth minute (D). In A-C, depolarizations were at +10 mV. A shows the mean NP_o calculated including the null sweeps (see Methods) and B shows the probability of null sweeps versus time. Notice the marked decrease of NP_o and the almost threefold increase of null sweeps probability with time. C shows the values of NP_o calculated by excluding the null sweeps from the analysis. NP_o decreased from 0.37 ± 0.05 to 0.21 ± 0.01 with a 43.2 % reduction with respect to controls, P < 0.05. D shows the mean values of NP_o at 0, +10 and +20 mV in control conditions and during SNP application, with the percentage of reduction indicated below (* P < 0.05, ** P < 0.01). The difference between minimal and maximal reduction (57.7 vs. 64.9 %) was not statistically significant (P > 0.05).

Figure 3. Single L-channel parameters in control conditions and during application of SNP

A, histograms showing distribution of single L-channel amplitudes measured at +10 mV before (left) and during exposure to 200 μM SNP (right) collected from 13 patches. The curves are best-fitted Gaussian functions with a mean of −1.24 ± 0.07 pA in controls and −1.27 ± 0.18 pA with SNP. B, open time distribution at +10 mV in control conditions (left) and during SNP application (right). The data were collected from 13 patches: 4 patches with two channel openings and 9 with single channel openings (of these latter, 4 were depolarized with pulses of 200 ms and 5 with pulses of 600 ms). The distributions were fitted with a two-exponential function with the following time constants: t_O1 = 1.9 ms (57 % of channel openings) and t_O2 = 7.1 ms (43 %) in controls, and t_O1 = 1.9 ms (60 %) and t_O2 = 7.5 ms (40 %) with SNP. Mean open times (<t_O>) derived from the fit are given to the top right of each distribution. C, closed time distribution at +10 mV in control conditions (left) and during SNP application (right). The data were collected from 5 patches displaying single channel openings and depolarized with pulses of 600 ms to +10 mV. The distributions were fitted with a three-exponential function with the following time constants: t_C1 = 1.3 ms (65 %), t_C2 = 12.7 ms (32 %) and t_C3 = 127 ms (3 %) in controls, and t_C1 = 1.4 ms (56 %), t_C2 = 12.5 ms (32 %) and t_C3 = 127 ms (12 %) with SNP. Mean closed times (<t_C>) derived from the fit are given to the top right of each distribution. D, mean unitary current amplitudes plotted versus voltage. The linear regressions through data points have mean slope conductances of 21.3 ± 2.6 pS (control) and 21.5 ± 6.8 pS (SNP) (n = 5-13). E and F, average t_O, t_C, P_o and null sweeps probability in controls and with SNP obtained from the arithmetic mean of the values calculated from patches containing a single Bay K-modified L-channel (* P < 0.05, ** P < 0.01).

Figure 1. The NO donor SNP markedly inhibits the single L-channel activity in bovine chromaffin cells

A, representative traces of L-channel activity, recorded in a cell-attached patch containing more than one channel under control conditions (left) and during exposure to 200 μM SNP (right). ω-CTx-MVIIC (10 μM), Bay K 8644 (5 μM) and a mixture of purinergic and opioidergic receptor antagonists (100 μM suramin, 10 μM naloxone) were present in the pipette solution. Bottom traces are averaged currents calculated over 10 (control) and 40 sweeps (SNP) from the same patch. B, NP_oversus time before (▪) and during SNP exposure (□). Horizontal segments indicate the selected traces shown in A. C, averaged currents obtained from 13 patches, taking 10 traces in control and 40 traces from the third to the sixth minute of SNP application from each individual patch. D, NP_oversus time for a representative control cell. Channel activity was tested for rundown for 18 min.

Figure 4. The NO scavenger carboxy-PTIO prevents the inhibitory effects of SNP

A, representative traces of L-channel activity recorded in control conditions (left) and during the simultaneous application of 200 μM SNP and 300 μM carboxy-PTIO (right). Bottom traces are averaged currents calculated from the same patch over 10 and 40 sweeps, respectively. B, time course of NP_o during cell exposure to both solutions. Filled bars are NP_o values in control conditions and open bars are NP_o values during drug application. C, mean NP_o values obtained by averaging data collected from 7 patches over 1 min (control, ▪) and 30 s periods (drug application, □). D, normalized mean NP_o with respect to control, for cells exposed to SNP + carboxy PTIO (n = 7; □) or carboxy PTIO alone (n = 4; ). All the patches contained multichannel openings.

Figure 5. The guanylate cyclase inhibitor ODQ prevents SNP action

A, during simultaneous application of SNP (200 μM) and ODQ (10 μM; right), L-channel activity is not significantly different from that in the presence of ODQ alone (left). Bottom traces are averaged currents from the same patch over 10 and 40 sweeps, respectively. Notice the close similarities between the two traces. B, NP_o values versus time calculated from the same patch shown in A. Horizontal segments indicate the representative traces shown in A. C, mean NP_o obtained by averaging data from 5 patches over 1 min (control + ODQ, ▪) and 30 s periods (SNP + ODQ, □), respectively. All the patches contained multichannel openings.

Figure 6. 8-Br-cGMP mimics the effects of SNP by reducing L-channel activity through a PKG-mediated mechanism

A, cell exposure to 400 μM 8-Br-cGMP (right) reduces the probability of channel opening with respect to controls (left). B, after incubation with the specific PKG inhibitor, KT-5823 (1 μM), 8-Br-cGMP fails to evoke the marked reduction in NP_o shown in A. Bottom traces in A and B are averaged currents obtained from 10 (control) and 40 traces (drug) in both cases. C and D show the mean NP_o values versus time obtained from 5 patches. Filled bars in C and D are mean control values collected over 1 min; open bars are mean values obtained by averaging data over 30 s periods during application of 8-Br-cGMP and 8-Br-cGMP + KT-5823, respectively. The inset in C shows normalized mean NP_o with respect to controls exposed to KT-5823 for about 10 min (n = 4), pretreated with KT-5823 and then exposed to 8-Br-cGMP (n = 5), or exposed to 8-Br-cGMP alone (n = 5). All the patches contained multichannel openings.

Figure 7. SNP preserves its action even when the PKA inhibitor H89 prevents cAMP-mediated up-regulation of L-channel

A, L-channel activity in a chromaffin cell incubated for 20 min with 1 μM H89 (left) is effectively inhibited by exposure to SNP (200 μM; right). Bottom traces are averaged currents obtained from 10 (control + H89) and 40 traces (SNP + H89) of the same patch. Notice the strong depression induced by SNP, which is comparable to that of Fig. 1. B and C show the time course of NP_o and mean NP_o derived from 6 patches following the same protocols as in Fig. 5. Horizontal segments in B indicate the representative traces shown in A. All the patches contained multichannel openings.

Figure 8. 8-Br-cGMP inhibits L-channel activity regardless of the down- and up-modulation induced by G_i/G_o proteins and cAMP

A, L-channel activity recorded in the presence of purinergic and opioidergic receptor agonists (100 μM ATP, 10 μM DAMGO and 1 μM DPDPE) in the patch pipette (left) is inhibited by cell exposure to 400 μM 8-Br-cGMP (right). Averaged currents from 10 traces in the presence of agonists alone and 40 traces in the presence of agonists + 8-Br-cGMP are shown at the bottom. B shows the averaged currents and the NP_o values derived from the data of 6 patches. NP_o with the agonists was 0.25 ± 0.04 (▪) and decreased to 0.10 ± 0.03 (* P < 0.02) with 8-Br-cGMP (□). C, L-channel activity recorded from a chromaffin cell incubated for 30 min with 8-CPT-cAMP (1 mm) (left) in which a single L-channel displayed high-P_o activity. Addition of 400 μM 8-Br-cGMP (right) produced a marked inhibition, which started to become significant from the third minute of application. The selected traces to the right were recorded between the third and sixth minute of drug application. Bottom traces are averaged currents obtained from 10 (cAMP) and 40 traces (cAMP + 8-Br-cGMP; right). Notice their larger amplitude with respect to those in A. D shows the averaged currents and the NP_o values derived from 4 patches. NP_o with 8-CPT-cAMP was 0.45 ± 0.08 (▪) and decreased to 0.19 ± 0.04 (* P < 0.05) with 8-Br-cGMP (□). All the patches contained multichannel openings.