Sympathetic Voltage-Independent Regulation of Voltage-Gated Calcium Channels in Pancreatic β-Cells

Voltage-gated calcium (CaV) channels are regulated by G proteins via voltage-dependent and independent pathways. Voltage-independent regulation of calcium channels is important for intracellular calcium concentration and insulin secretion. Voltage dependence of each pathway can be elucidated by a prepulse facilitation protocol. Using this experimental approach, we compared CaV regulation by GTPγS and noradrenaline (NA) in rat pancreatic β-cells and rat superior cervical ganglion (SCG) neurons. The SCG neuron is a model in which the bases of CaV channel regulation by G proteins have been established. CaV channel regulation through activation of the sympathetic nervous system has been poorly studied in native insulin-secreting cells. We recorded CaV channel currents by means of the patch-clamp technique in the whole-cell configuration. We found that application of both GTPγS (a nonspecific activator of G proteins) by cell dialysis and noradrenaline (NA)-exposure reduced CaV current amplitude in pancreatic β-cells and in SCG neurons. However, the inhibition of CaV channel currents in GTPγS-dialyzed SCG neurons was relieved by a strong depolarizing pulse. By contrast, in pancreatic β-cells, the inhibition was maintained after a strong depolarizing pulse. In SCG neurons, the CaV channel inhibition by NA is predominantly voltage-dependent, whereas in pancreatic β-cells it is only 40%. Thus, it appears that CaV channels in rat pancreatic β-cells are regulated mainly through a voltage-independent pathway. The signaling pathway for CaV channel regulation by NA in pancreatic β-cells appears to differ from the classic signaling pathway described in SCG neurons. Therefore, voltage-independent regulation of Ca2+ entry through CaV channels is a critical step in understanding the pathophysiology of type 2 diabetes.


Introduction
The islets of Langerhans are innervated by the sympathetic nervous system [1,16].Secretion of insulin by cells in these islets is inhibited by the neurotransmitter noradrenaline (NA) through activation of α2-adrenergic receptors coupled to G proteins [1,19].NA has been shown to regulate voltage-gated calcium (Ca V ) channels in insulin-secreting cell lines, however, this has not been observed in native pancreatic β-cells [3,13,19].
G protein regulation of Ca V channels has been studied extensively in neurons of the superior cervical ganglion (SCG), and the biophysical properties of Ca V channels are differentially expressed via voltage-dependent and -independent pathways [11,20].Both pathways reduce the Ca V channel current amplitude and regulate Ca 2+ -dependent processes, such as neurotransmitter release.NA-induced inhibition of Ca V channels in neurons is a fast voltage-dependent mechanism [6].The voltage-dependent pathway is thought to be delimited to the plasma membrane by G protein βγ subunits; the voltageindependent pathway is less understood.There is evidence that the membrane lipid PIP 2 is the molecule responsible for the voltage-independent inhibition of Ca V channels triggered by M 1 R activation [9,10,15].Furthermore, some other voltageindependent pathways have been documented [20].
Voltage-independent regulation of calcium channels governs intracellular calcium concentration and insulin secretion.
The voltage-dependent and -independent pathways can be elucidated by a prepulse protocol that consists of two identical voltage pulses separated by a strong depolarizing pulse [8].The strong depolarizing pulse is commonly used as a tool to differentiate the two mechanisms.It can be mimicked by action potential burst firing and thus it is physiologically relevant to study the voltage-dependence of Ca V channel regulation [4,17].
Pancreatic β-cells and SCG neurons express α2-adrenergic receptors, therefore, we examined NA regulation of Ca V channels.We used rat SCG neurons, a model in which signaling pathways have been elucidated, so that we could compare NA regulation of Ca V channels in pancreatic β-cells against NA regulation of Ca V channels in SCG neurons.In order to compare G protein regulation in both cell types, endogenous G proteins were activated with GTPγS.A prepulse protocol was used to isolate the voltage-dependent and -independent pathways.

Cell Culture
Wistar rats were provided by the Universidad Nacional Autonóma de México School of Medicine's animal breeding facility and were handled according to the Mexican Official Norm for Use, Care and Reproduction of Laboratory Animals (NOM-062-ZOO-1999).
Neurons were dissociated from the SCG by mechanical and enzymatic methods, as previously described [14].Pancreatic βcells were also obtained as previously described [5].SCG neurons and pancreatic β-cells were plated and incubated at 37°C in a humidified atmosphere of 95% air and 5% CO 2 for 16-24 hours before electrophysiological recordings.

Electrophysiological study
Recordings of Ca V currents in SCG neurons and pancreatic βcells were obtained at room temperature (22-24°C) by the patch-clamp technique in the whole-cell configuration and using an EPC-9 amplifier (HEKA Electronic, Lambrecht, Germany).Voltage protocols were generated, and current responses were digitized and stored by means of the Patchmaster software (HEKA Electronic).Pipettes were pulled from borosilicate glass capillaries with a horizontal patch electrode puller (Sutter Instrument, Novato, CA, USA), and SCG neurons were filled with internal solution consisting of (in mM) 140 CsCl, 20 TEA-Cl, 10 HEPES, 0.1 BAPTA-tetra cesium, 5 MgCl 2 , 5 Na 2 ATP, 0.3 Na 2 GTP, and 0.1 leupeptin; the solution was adjusted to pH 7.2 with CsOH.Resistance of the pipettes was 1.8-2.0MΩ.Pancreatic βcells were filled with internal solution consisting of (in mM) 140 CsCl, 32 TEA-Cl, 10 HEPES, 0.1 BAPTA-4 Cs, 1 MgCl 2 , 3 Na 2 ATP, 3 Na 2 GTP, and 0.1 leupetine; the solution was adjusted to pH 7.4 with CsOH.Resistance of the pipettes was 2.5-3.5 MΩ.

Data Analysis
Inhibition of the current induced by GTPγS or NA was calculated as the amplitude of the steady-state current under GTPγS dialysis or during NA exposure minus the current amplitude under control conditions.Thereafter, it was divided by the current amplitude under control conditions (thus reported as a percentage).The facilitation index was calculated by measuring the peak current amplitude at P2 divided by the peak current amplitude at P1.Time constants (τ) were calculated from an exponential equation.Data are shown as mean ± SEM.
Statistical differences were analyzed by t-test.P< 0.05 was considered significant.

Inhibition of Ca V Current by GTPγS
We first examined the fraction of voltage-dependent andindependent regulation by activation of endogenous G proteins with GTPγS.Although both types of regulation were seen in SCG neurons, the voltage-dependent pathway was predominant under G protein activation [7].Representative Ca V currents evoked by the prepulse protocol in control and in GTPγS-dialyzed SCG neurons and in pancreatic β-cells are shown in Figure 1A and B, respectively.The Ca V current amplitude was reduced by GTPγS in both cell types, however, this inhibition was released after a strong depolarizing pulse only in SCG neurons (Figure 1A and B).In SCG neurons the time course of the reduction in Ca V current amplitude by GTPγS was biphasic (Figure 1C; τ1 = 17.2 ± 8.8 seconds and τ2 = 29.9 ± 6.09 seconds), whereas in pancreatic βcells it was monophasic (Figure 1D; τ= 31.2 ± 2.6 seconds).Ca V current inhibition was greater in SCG neurons (P1 = 88 ± 2.3% and P2 = 28 ± 4.3%) than in pancreatic β-cells (P1 = 53 ± 4.6% and P2 = 50 ± 5.6%) (Figure 1E).The facilitation index was used to evaluate the fraction of voltage-dependent and voltageindependent regulation.Facilitation indices in SCG cells were 1.08 ± 0.06 (control) and 2.82 ± 0.026 (GTPγS), whereas in pancreatic β-cells, they were 1.04 ± 0.01 and 0.99 ± 0.042, respectively.In summary, these data support that Ca V currents are reduced by endogenous G protein activation.However, voltage-dependent regulation is predominant in rat SCG neurons, whereas voltageindependent regulation does the same in rat pancreatic β-cells

Voltage-Independent Inhibition of Ca V Current by NA in Pancreatic β-Cells
Ca V current in sympathetic neurons has been shown to be reduced by NA mainly through a voltage-dependent pathway [6].Thus, we hypothesized that Ca V currents in rat pancreatic β-cells are regulated by NA in a similar manner.We showed recently that Ca V channels are regulated in SCG neurons and pancreatic β-cells via similar pathways when M 1 R is activated.We used a prepulse protocol and characterized a specific time course as a hallmark of a voltage-dependent pathway.Representative P1 and P2 Ca V current traces elicited in control and NA-treated sympathetic neurons and pancreatic β-cells are shown in Figure 2A and B, respectively.Ca V current was reduced by NA in both cells types, however, the current inhibition was released by the prepulse only in sympathetic neurons.The time course of I Ba amplitude in NA-treated sympathetic neurons and pancreatic βcells is shown in Figure 2C and D, respectively.Notably, there was a reduction in Ca V current amplitude in treated SCG neurons (87 ± 8.8% in P1 and 25 ± 3.5% in P2) and treated pancreatic β-cells (44.7 ± 4% in P1 and 34.5 ± 4.4% in P2) (Figure 2).However, the reduced current was scarcely restored by a depolarizing prepulse in pancreatic β-cells compared to SCG neurons (Figure 2F: SCG control= 1.1 ± 0.06, SCG NA= 2.6 ± 0.42 and pancreatic β control= 1.02 ± 0.02, pancreatic β NA= 1.4 ± 0.03 cells).Taking together, these data show that NA reduces the Ca V current amplitude in rat pancreatic β-cells through a voltage-independent pathway and suggest that this Ca V current regulation by NA is significantly different from that observed in SCG neurons.

Discussion
We showed in rat pancreatic β-cells that Ca V channels are inhibited by G proteins mainly via voltage-independent pathways.Voltage-independent regulation of calcium channels determines intracellular calcium concentration and insulin secretion.The macroscopic Ca V channel current was reduced to 55% by GTPγS dialysis.Similarly, in mouse pancreatic β-cells, GTPγS dialysis has been shown to reduce Ca 2+ currents by 30% [2].Inhibition in both mouse and rat pancreatic β-cells is minimal compared to that in SCG cells.The Ca V channel inhibition in the mouse model is partially released by a strong depolarizing pulse, suggesting a predominant voltage-dependent pathway under endogenous G protein activation [2].On the contrary, Ca V channel inhibition was not relieved despite the application of a strong depolarizing pulse in GTPγS-dialysed rat pancreatic β-cells.According to Elmslie and colleagues [8], the voltage-dependent regulation by G proteins in SCG neurons is released by a strong depolarizing pulse, and this relief is the hallmark of G protein regulation in neurons.Our data suggest that regulation of Ca V channels by G protein-coupled receptors in insulin-secreting pancreatic cells could be mediated by pathways that have no relationship to the classic regulatory pathways described in SCG neurons [8].
In SCG neurons, the NA signal is transmitted mainly through α 2 -adrenergic receptors coupled to Gi proteins.This signaling pathway is voltage-dependent and requires the action of Gβγ subunits [9,10].Similarly, α 2 -adrenergic receptor activation by NA reduces insulin release in rat pancreatic β-cells [18,19].
Ca V channels are thought to be similarly inhibited by activation of α 2 -adrenergic receptors in human β-, RINm5F, and HIT cells [13,19].The regulation in RINm5F is predominantly voltagedependent, whereas in human β-cells the inhibition is voltageindependent [13].Our data suggest that NA inhibits Ca V currents via a voltage-independent pathway, in a similar way to human β-cells.Interestingly, α 2 -adrenoreceptor stimulation does not inhibit L-type calcium channels in mouse pancreatic β-cells [3].Our study in primary cultured cells is the very first providing evidence that NA inhibits Ca V channels in pancreatic β-cells.The classic understanding is that voltage-independent regulation is mediated by G q/11 proteins involving PIP 2 hydrolysis, however, there is no evidence that activation of α 2 -adrenergic receptors hydrolyzes PIP 2 .The signaling cascade and specific

Sympathetic Voltage-Independent Regulation of Voltage-Gated Calcium Channels in Pancreatic β-Cells
activities of G protein subunits and their link to α 2 -adrenergic receptors in the modulation of β-cell Ca V channels remain to be elucidated.
Inhibition of the insulin secreting process by activation of the sympathetic nervous system has been reported [18,19].Our findings are consistent with this notion.We showed that Ca V current diminishes in rat pancreatic β-cells treated with NA, resulting in a decreased Ca 2+ entry.Although calcium influx through Ca V channels is a known part of this process, little is known about Ca V channel regulation that occurs via G-protein activation of the voltage-independent pathway.In conclusion, regulation of Ca V channels by G proteins in pancreatic β-cells via a voltage-independent pathway appears to be an additional mechanism by which the Ca 2+ concentration is controlled and a key step in such an important physiological process.Whatever the mechanism turns out to be, a voltage-independent mechanism is involved in pancreatic β-cells.However, an important question for future studies is whether or not a specific voltage-independent mechanism may account for the Ca V current inhibition in pancreatic β-cells.

Figure 1 :
Figure 1: Inhibition of Ca V current amplitude by GTPγS via a voltage-independent pathway in rat pancreatic β-cells.(A) and (B) Representative current traces elicited by a prepulse protocol, which consisted of a pair of 10-ms depolarizing pulses to -5 mV (P1, P2) from a holding potential of -80 mV and separation of P1 and P1 by a prepulse (PP) of +125 mV during a 25-ms period.Left panel shows overlapping P1 and P2 current traces from rat superior cervical ganglion (SCG) neurons under control and GTPγS conditions.Right panel shows overlapping P1 and P2 current traces from rat pancreatic β-cells under control and noradrenaline (NA)-treatment.Time course of I Ba relative (P1) in a GTPγS-dialyzed neuron (C) and a pancreatic β-cell (D).Summary of the percentage inhibition of P1 and P2 (E) and facilitation index (F) in rat SCG neurons and rat pancreatic β-cells.

Figure 2 :
Figure 2: Inhibition of Ca V current amplitude by(NA)-treatment via a voltage-independent pathway in rat pancreatic β-cells.(A) and (B) Representative current traces elicited by the prepulse protocol, which consisted of a pair of 10-ms depolarizing pulses to -5 mV (P1, P2) from a holding potential of -80 mV and separation of P1 and P1 by a prepulse (PP) of +125 mV during a 25-ms period.(A) and (B), overlapping P1 and P2 current traces from (A) rat superior cervical ganglion (SCG) neurons and (B) rat pancreatic β-cells under control and NA-treatment.Time course of I Ba relative (P1) in NA-treated neurons (C) and in pancreatic β-cells (D).Summary of percentage inhibition of P1 and P2 (E) and facilitation index (F) in rat SCG neurons and rat pancreatic β-cells.