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It is established that neuropeptide Y (NPY) is a transmitter of parasympathetic secretory impulses in submandibular gland. The neuropeptides substance P, vasoactive intestinal peptide (VIP) and calcitonin gene-related peptide (CGRP) are likely mediators of secretory parasympathetic responses of the gland. Previously, we have shown that substance P, VIP and CGRP modulate voltage-dependent Ca(2+) channels (VDCCs) in hamster submandibular ganglion (SMG) neurons. In this study, we attempt to characterize the effect of NPY on VDCCs current using Ba(2+) (I(Ba)) in SMG neurons. Application of NPY caused both facilitation and inhibition of L-type and N/P/Q-type I(Ba), respectively. Intracellular dialysis of the Gα(s)-protein antibody attenuated the NPY-induced facilitation of I(Ba). The adenylate cyclase (AC) inhibitor, as well as protein kinase A (PKA) inhibitor attenuated the NPY-induced facilitation of I(Ba). Intracellular dialysis of the Gα(i)-protein antibody attenuated the NPY-induced inhibition of I(Ba). Application of a strong depolarizing voltage prepulse attenuated the NPY-induced inhibition of I(Ba). These results indicate that NPY facilitates L-type VDCCs via Gα(s)-protein involving AC and PKA. On the other hand, NPY also inhibits N/P/Q-type VDCCs via Gα(i)-protein βγ subunits in the SMG neurons., (Copyright © 2012 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.)

Objective: The control of saliva secretion is mainly under parasympathetic control. The submandibular ganglion (SMG) is a parasympathetic ganglion which receives inputs from preganglionic cholinergic neurons, and innervates the submandibular salivary gland to control saliva secretion. The aim of this study was to investigate if adrenomedullin (ADM) and/or calcitonin gene-related peptide (CGRP) modulate voltage-dependent calcium channel (VDCCs) current (I(Ca)) in SMG., Design: The profile of CGRP and ADM actions in SMG was studied using the whole-cell configuration of the patch-clamp technique., Results: Both ADM and CGRP facilitated I(Ca). These facilitations were attenuated by intracellular dialysis of the anti-Gα(s)-protein and pretreatment of SQ22536 (an adenylate cyclase inhibitor)., Conclusions: ADM and CGRP facilitates VDCCs mediated by Gα(s)-protein and adenylate cyclase in SMG., (Copyright © 2010. Published by Elsevier Ltd.)

The control of saliva secretion is mainly under parasympathetic control, although there also could be a sympathetic component. Sympathetic nerves are held to have a limited action in secretion in submandibular glands because, on electrical stimulation, only a very small increase to the normal background, basal secretion occurs. Parasympathetic stimulation, on the other hand, caused a good flow of saliva with moderate secretion of acinar mucin, plus an extensive secretion of granules from the granular tubules. The submandibular ganglion (SMG) is a parasympathetic ganglion which receives inputs from preganglionic cholinergic neurons, and innervates the submandibular salivary gland to control saliva secretion. Neurotransmitters and neuropeptides acting via G-protein coupled receptors (GPCRs) change the electrical excitability of neurons. In these neurons, many neurotransmitters and neuropeptides modulate voltage-dependent calcium channels (VDCCs). The modulation is mediated by a family of GPCRs acting either directly through the membrane delimited G-proteins or through second messengers. However, the mechanism of modulation and the signal transduction pathway linked to an individual GPCRs depend on the animal species. This review reports how neurotransmitters and neuropeptides modulate VDCCs and how these modulatory actions are integrated in SMG systems. The action of neurotransmitters and neuropeptides on VDCCs may provide a mechanism for regulating SMG excitability and also provide a cellular mechanism of a variety of neuronal Ca(2+)-dependent processes.

Angiotensin II (Ang II) is one of the most important vasoconstrictive hormones but is also known to act as a neuromodulator and a neurotransmitter in the central and peripheral nervous system. The submandibular ganglion (SMG) neuron is a parasympathetic ganglion which receives inputs from preganglionic cholinergic neurons, and innervates the submandibular salivary gland to control saliva secretion. In this study, the effects of Ang II on SMG neurons were investigated using the whole-cell patch clamp technique. Membrane currents evoked by a ramp pulse from +50 to -100 mV (-150 mV/500 ms) were compared in both the absence and presence of Ang II. In eight neurons tested, 1 microM Ang II increased inward currents by 42.0+/-8.2%. The reversal potentials of the Ang II-induced current were 0.2+/-0.6 mV. These increase of inward currents by Ang II were antagonized by losartan, a selective antagonist of AT(1) receptors. Intracellular dialysis with 0.1mM guanosin 5'-O-(2-thiodiphosphate) (GDP-beta-S), a G-proteins blocker, and anti-G(q/11) antibody attenuated Ang II-induced ionic current. In addition, pretreatment of neurons with 10 microM staurosporine (stauro), a protein kinase C (PKC) inhibitor, 0.5 microM PMA, a PKC activator, and 10 microM KN-93, a Ca2+/calmodulin-dependent protein kinase II (CaM K II) inhibitor, attenuated Ang II-induced ionic current in SMG neurons. The data presented here demonstrated that Ang II-induced ionic current via G(q/11)-proteins involving both PKC and CaM K II pathways in SMG neurons.

The submandibular ganglion (SMG) neuron has been well established as the parasympathetic ganglion that innervates the submandibular and sublingual salivary glands. Thus this neuron plays a key role in salivary secretion. In a previous study, we reported that SMG possessed T-, L-, N-, P/Q- and R-type voltage-dependent calcium channels (VDCCs). In this study, we analyzed the contribution of the distinct subtypes of VDCCs currents (ICa) using the whole-cell configuration of the patch clamp technique in SMG neurons. In addition, we also investigated the effects of a strong voltage prepulse on the contributions of the subtypes of VDCCs. In SMG neuronal ICa without a prepulse, the mean percentages of L-, N-, P-, Q- and R-type were 39.7, 31.5, 10.6, 7.1 and 7.9%. In SMG neuronal ICa with prepulse, the mean percentages of L-, N-, P-, Q- and R-type were 37.2, 34.0, 14.0, 7.6 and 7.0%. Thus, these results showed that SMG possess multiple types of VDCCs and that N- and P-type VDCCs are facilitated by a prepulse in SMG neurons.

Angiotensin II (AngII) is one of the most important vasoconstrictive hormones but is also known to act as a neuromodulator and a neurotransmitter in the central and peripheral nervous systems. In a previous study, we have shown that AngII, mediated by AT(1) receptors, inhibits voltage-dependent calcium channel (VDCC) currents (I(Ca)) via G-proteins in submandibular ganglion (SMG) neurons. In this study, we further characterized the signal transduction of AngII-induced inhibition of I(Ca). Application of 1 microM AngII inhibited I(Ca) by 32.1+/-2.7% (mean+/-S.E.M., n=9). Intracellular dialysis of anti-G(q/11) antibodies attenuated these inhibition (8.8+/-1.3%, n=6). In addition, treatment of protein kinase C (PKC) activator and inhibitor also attenuated these inhibition (8.0+/-0.9 and 9.8+/-0.9%, n=6 and 9, respectively). We therefore conclude that AngII inhibits VDCC via G(q/11)-proteins involving in SMG neurons. In addition, such PKC-dependent pathways mediated mainly L-type VDCC inhibition.

In a previous study, we demonstrated that angiotensin II (AngII) inhibited voltage-dependent calcium channels (VDCCs) currents (ICa) in hamster submandibular ganglion (SMG) neurons. In sinoatrial node cells, it has been reported that AngII inhibits ICa by suppressing cyclic AMP production. In this study, to investigate the possible involvement of a cyclic AMP-cyclic AMP dependent protein kinase (PKA) pathway in the AngII-induced inhibition of ICa, effects of AngII were examined in SMG neurons after treatment with an activator and inhibitor of PKA. Neither pretreatment of neurons with membrane permeable cyclic AMP nor intracellular dialysis of PKA blocker attenuated the AngII-induced inhibition of ICa. These results indicate that AngII inhibited ICa via a cyclic AMP-PKA-independent mechanism in SMG neurons.

In this study, we investigated the effects of vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide 1-38 (PACAP) on the voltage-gated calcium currents in hamster submandibular ganglion neurons. VIP and PACAP inhibited the high threshold voltage-gated calcium current in a voltage-independent and a concentration-dependent manner via the G protein-mediated pathway. L-, N- and P/Q-type components of the total maximum voltage-gated calcium current accounted for 48.0 +/- 3.1% (n = 4), 35.1 +/- 4.7% (n = 4), and 13.5 +/- 2.3% (n = 3) of the total peak amplitude, respectively. VIP at a concentration of 1 microM inhibited the L-type calcium current by 33.2% +/- 1.4% (n = 4), the N-type current by 31.0 +/- 3.6%, and the P/Q-type current by 3.2 +/- 1.1% (n = 3). PACAP at a concentration of 1 microM inhibited the L-type current by 35.6 +/- 5.7%, the N-type current by 34.4 +/- 3.1% (n = 4), and the P/Q-type current by 6.4 +/- 2.1% (n = 2). However, VIP and PACAP did not inhibit the low threshold voltage-gated (T-type) calcium current. The rank order of potency was PACAP > VIP. In experiments replacing GTP with GDP-beta-S, the inhibitory effects of VIP and PACAP were prevented. In experiments of double-pulse protocol, depolarizing conditioning pulses could not relieve the inhibition of total high threshold voltage-gated calcium currents produced by VIP and PACAP. Therefore, the inhibition of the high threshold voltage-gated calcium channels produced by VIP and PACAP in hamster parasympathetic neurons differed in its mechanisms from that of N-type calcium channels in rat sympathetic neurons.

The calcium ion influx through voltage-dependent calcium channels (VDCCs) has a vital role in the control of neurotransmitter release and membrane excitability. Prepulse facilitation is a phenomenon in which a strong depolarizing pulse induces a form of the VDCCs that exhibits an increased opening probability in response to a given test potential; this persists for several seconds after repolarization. It has been reported that prepulse facilitation occurs via dissociation of the guanosine triphosphate (GTP)-binding proteins (G-proteins) from the VDCCs and that recovery from facilitation involves rebinding of the G-proteins. The heterotrimeric G-proteins act as switches that regulate information processing circuits connecting cell surface G-protein-coupled-receptors to a variety of effectors. In this study, we have studied the characterization of G-protein subtypes in prepulse facilitation of VDCCs currents (Ica) in hamster submandibular ganglion (SMG) neurons, using whole-cell patch clamp recordings. Under control conditions, with GTP (0.1 mM) in the recording pipette, the rate of prepulse facilitation was 19.0 +/- 1.9% (n = 13). Intracellular dialysis with GDP-beta-S (0.1 mM), G-protein blocker, and pretreatment of neurons with N-ethylmaleimide (NEM) (100 microM for 2 min), Gi/o blocker, attenuated the rate of prepulse facilitation. Intracellular dialysis of anti-Gq/11-antibody did not alter it. These results suggest that prepulse facilitation of VDCCs is due to Gi/o-types of G-protein, but not to the Gq/11-type, in SMG neurons.

Angiotensin II (Ang II) is one of the most important vasoconstrictive hormones but is also known to act as a neuromodulator and a neurotransmitter in the central and peripheral nervous systems. In a previous study, we have shown that Ang II, via AT1 receptors, induced depolarization by inhibition of M-type K(+) channels and SK channels in submandibular ganglion (SMG) neurons. In this study, we investigated the effects of Ang II on calcium channel current (I(Ca)) in acutely dissociated SMG neurons by the patch-clamp technique using the whole-cell configuration. Ang II inhibited total I(Ca) by 32.1+/-2.7%. The half-maximum inhibitory concentration (IC(50)) of Ang II for inhibiting I(Ca) was 0.8 microM. In the presence of 1 microM losartan, which is a selective antagonist of AT1 receptors, the effect of Ang II was attenuated (7.6+/-1.5%). Application of a strong depolarizing voltage prepulse did not affect the Ang II-induced inhibition of I(Ca) (32.8+/-2.8%). Intracellular dialysis of GDP-beta-S attenuated the inhibition of I(Ca) (6.8+/-2.1%). The mean percentage inhibitions of L-, N- and P/Q-type VDCCs by Ang II were 29.1+/-1.7, 16.3+/-6.0 and 1.2+/-0.8%, respectively, of the total I(Ca).

The calcium ion influx through voltage-dependent calcium channels (VDCCs) has a vital role in the control of neurotransmitter release and membrane excitability. The modulation of VDCCs controls the extent of calcium entry and thus provides a way of regulating neuronal function. Prepulse facilitation is a phenomenon in which a strong depolarizing pulse induces a form of the VDCCs that exhibits an increased opening probability in response to a given test potential that persists for several seconds after repolarization. In this study, we have studied the characterization of prepulse facilitation of VDCCs currents (Ica) in hamster submandibular ganglion (SMG) neurons, using whole-cell patch clamp recordings. In SMG neurons, application of a strong depolarizing prepulse caused a Ica. In 8 SMG neurons, rate of facilitation was 1.1 +/- 0.1. The greatest value of prepulse facilitation was obtained with prepulse to +100 mV, 10 ms duration in this neuron. The magnitude of facilitation was dependent on changing the interval between the -prepulse and the +prepulse and reached a maximum at a interval of 500 ms.

Both substance P and neurokinin A are known as neurotransmitters of the submandibular ganglion cell. In this study, the effects of neurokinin (NK) receptor-subtype agonists on hamster submandibular ganglion cells were investigated using the whole-cell patch-clamp technique. Membrane currents evoked by a ramp pulse from +50 to -100 mV (-150 mV/1000 msec) were compared in both the absence and presence of NK receptor agonist. The NK-1 receptor agonist [Sar9, Met (O2)11]-substance P, the NK-2 receptor agonist [Ala5, beta-Ala8]-alpha-neurokinin fragment 4-10, and the NK-3 receptor agonist senktide were used. The three agonists dose-dependently increased the amplitude of the inward current with a reversal potential near 0 mV. Their rank order was NK-1 = NK-3 > NK-2. Even when the external solution was replaced with Cs+ or N-methyl-D-glucamine+ instead of Na+, the NK receptor agonists also increased the amplitude of the inward current. Thus, NK-1 and NK-3 receptors are apparently coupled with non-selective cation channels in submandibular ganglion cells.

In this study, we investigated the modulation of calcium channels by vasoactive intestinal polypeptide (VIP) in hamster submandibular ganglion (SMG) cells using the whole-cell patch clamp technique. VIP (1 microM) inhibited the high voltage-gated calcium channels in the SMG neurons via activation of VIP receptors and G-protein coupled to these receptors, but did not affect the low voltage-gated calcium channels. VIP at 1 microM reduced the peak amplitude of the maximum high voltage-gated calcium current by 26.0 +/- 6.2% (mean +/- S.E., n = 5) and slowed the rising phase of the calcium current. The time to peak of the maximum high voltage-gated calcium current was prolonged from 16.8 msec to 22.4 msec. In a representative cell, 1 microM VIP reduced the peak amplitude of maximum high voltage-gated calcium current by 50.5% and the amplitude at the end of voltage step after 300 msec by 51.9%. The VIP-induced inhibition of the calcium current was produced in a voltage-dependent manner. Inhibition was maximal (50.5%) at the level of -20 mV. Therefore, VIP inhibits both transient and sustained types of high voltage-gated calcium channels in the hamster SMG neuron.

Both substance P (SP) and neurokinin A (NKA) are known as neurotransmitters of the submandibular ganglion (SMG) neurons. SP released from collaterals of the sensory nerves also regulates the excitability of SMG neurons. It has recently been shown that neurokinins (NK) inhibit calcium channels in various neurons. In this study, the effects of NK on voltage-dependent calcium channel current (I(Ca)) in SMG cells were investigated using the whole-cell patch-clamp recording method. NK-1 receptor agonist and SP caused inhibition of I(Ca) in SMG cells in a dose-dependent manner. NK-1 receptor agonist inhibited L-, N- and P/Q-type I(Ca) components. GDP-beta-S included in the pipette solution reduced the NK-1 receptor agonist-induced inhibition of I(Ca). In addition, NK-1 receptor agonist-induced inhibition of I(Ca) was reduced by stimulation of protein kinase C (PKC) but not cyclic AMP-dependent protein kinase (PKA). The results provided evidence for a signal transduction pathway in which calcium channel inhibition by NK receptors required activation of G-protein and PKC-affected step phosphorylation in SMG neurons.

In this study, we analyzed the effect of alpha-calcitonin gene-related peptide (CGRP) on the neurons of hamster submandibular ganglion (SMG). CGRP induced a small slow-depolarization accompanied with an increase in input resistance in about 60% of tested cells. CGRP enhanced durations of their Ca2+ spikes. The Ca2+ spikes were subdivided into two subgroups according to duration short duration within 1 sec and long duration of more than 1 sec. Forskolin, an activator of adenylate cyclase, mimicked the effects of CGRP. These data indicated that the effects of CGRP on SMG cells were mediated via the intracellular cyclic AMP signalling system. The effect of CGRP on the voltage-activated Ca2+ currents was characterized by enhancement of both transient and sustained components. The enhancement was greater in the transient component than in the sustained component. The subgroups of the Ca2+ spike may indicate differences in mechanism accelerating transmitter release and may reflect distinct functional roles of two different populations of SMG cells.

The effects of agonists for delta-opioid receptors on synaptic transmission in hamster submandibular ganglion were studied in vitro, using an intracellular recording technique. [Met5]enkephalinamide (17 microM), [D-Ser2¿Leu-enkephalin-Thr (14 microM) and [D-Pen2, D-Pen5]enkephalin (10 microM), delta selective agonists caused reversible depression of the fast EPSP amplitude in 10 of 30 cells tested. The inhibitory actions of [D-Pen2, D-Pen5]enkephalin were antagonized by ICI 174,864, a delta selective antagonist. Naloxon a non-selective antagonist and ICI 174,864 both also potentiated the fast EPSP amplitude in 5 of 11 cells tested.

In this study, effects of exogenously applied Met-enkephalin on hamster submandibular ganglion cells were investigated using an intracellular recording technique. Met-enkephalin (10 microM) was applied to cells by pressure ejection (2 kg/cm2, 50 msec) from a micropipette. More than 30% of the cells caused a hyperpolarization (amplitude: about 3 mV) accompanied by an increase in potassium conductance. After 30-40 ejections, the Ca2+ spike was shortened by 18.1%, and the nicotinic transmission was depressed. The inhibitory effect on presynaptic terminals was blocked by naloxone (10 microM).