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Adrenaline (Adr) is known to directly or indirectly modulate bone cell activity under physiological and pathological conditions. Osteoblasts play a major role in bone formation, employing intracellular Ca(2+) as a second messenger to modulate hormonal responses and as a cofactor for mineralization. Voltage-dependent Ca(2+) channels (VDCCs) mediate the influx of Ca(2+) in response to membrane depolarization. The purpose of this study was to investigate the effects of Adr on VDCC currents in osteoblasts using a patch-clamp recording method. Application of 1 mM Adr facilitated VDCC currents in a concentration-dependent manner. Pre-treatment with b receptor antagonist propranolol blocked Adr-induced facilitation of VDCC currents carried by Ba(2+) (IBa). These results indicate that Adr-induced facilitation of IBa was mediated by b receptors in MC3T3-E1 osteoblast-like cells. To our knowledge, the data presented here demonstrate for the first time that Adr facilitates VDCCs in MC3T3-E1 osteoblast-like cells.

The hypothalamic nonapeptide and neurohypophyseal hormone arg-vasopressin (AVP), also known as antidiuretic hormone, is best known for its effects on water reabsorption in kidney. Osteoblasts play a major role in bone formation, employing intracellular Ca(2+) as a second messenger to modulate hormonal responses and as a cofactor for mineralization. Voltage-dependent Ca(2+) channels (VDCCs) mediate the influx of Ca(2+) in response to membrane depolarization. The purpose of this study was to investigate the effects of AVP on VDCC currents in osteoblasts using a patch-clamp recording method. An application of 1μM AVP facilitated VDCC currents in osteoblasts. To our knowledge, the data presented here demonstrate for the first time that AVP facilitates VDCCs in osteoblasts.

Angiotensin II (Ang II) plays a major role in the maintenance of extracellular fluid volume and blood pressure. In addition to its well-established role in circulatory homeostasis, it has been implicated in the process of bone formation. Osteoblasts play a major role in bone formation, employing intracellular Ca(2+) as a second messenger to modulate hormonal responses and as a cofactor for mineralization. Voltage-dependent Ca(2+) channels (VDCCs) mediate the influx of Ca(2+) in response to membrane depolarization. The purpose of this study was to investigate the effects of Ang II on VDCC currents in osteoblasts using a patch-clamp recording method. To our knowledge, the data presented here demonstrate for the first time that Ang II facilitates VDCCs in osteoblasts.

Osteoblasts play a major role in bone formation. Osteoblasts employ intracellular Ca(2+) as a second messenger to modulate hormonal responses and a cofactor for bone mineralization. Adrenomedullin (ADM) promotes osteoblast growth and proliferation, inducing an increase in bone mass. Voltage-dependent Ca(2+) channels (VDCCs) mediate the influx of Ca(2+) in response to membrane depolarization. Voltage-dependent Ca(2+) channels serve as crucial mediators of many Ca(2+)-dependent functions, including growth of bone and regulation of proliferation. The purpose of this study was to investigate the effects of ADM on VDCC currents in osteoblasts using a patch-clamp recording method. To our knowledge, the data presented here demonstrate for the first time that ADM facilitates VDCCs in osteoblasts.

Osteoblasts play a major role in bone formation. Osteoblasts employ intracellular Ca(2+) as a second messenger modulating hormonal responses and a cofactor for bone mineralization. Voltage-dependent Ca(2+) channels (VDCCs) are most commonly present in excitable cell membranes. They are also present at lower levels even in most nonexcitable cells too. In both types of cell, they mediate the influx of Ca(2+) in response to membrane depolarization. Prepulse facilitation is a phenomenon in which a long and strong depolarizing pulse induces a form of VDCC that exhibits an increased opening probability. We believe this to be the first study to demonstrate that strong depolarization prepulses both increase and decrease VDCCs in osteoblasts.

The biologically active form of vitamin D, 1α,25-dihydroxy vitamin D₃ (VD), regulates the synthesis of the bone Ca-binding proteins osteocalcin and osteopontin. The actions of VD are mediated through the vitamin D receptor (VDR). Liganded VDR heterodimerizes with the retinoid X receptor and interacts with a vitamin D response element (VDRE). Recently, it has been demonstrated that vitamin D responses elicited in osteoblasts can be rapid as well as long-term. The purpose of this study was to elucidate the mechanism of Ca(2+) signaling of VD in osteoblasts using intracellular Ca(2+) ([Ca(2+)]i) measurements. A rapid VD (10 nM)-induced increase in [Ca(2+)]i was observed within 40 sec. This increase, however, was negated with application of Ca(2+)-free Krebs' solution. These results indicate that VD induces an increase in [Ca(2+)]i from extracellular Ca(2+) in osteoblasts.

The postsynaptic actions of exogenously applied adenosine 5'-triphosphate (ATP) were investigated in nucleus tractus solitarius (NTS) of the rat. Whole cell patch-clamp recordings were used to examine the regulation of voltage-dependent Ca2+ channels (VDCCs) currents (I(Ca)) by ATP in freshly dissociated NTS. Application of ATP inhibited I(Ca) from -905 pA to -741 pA. In addition to this inhibition, application of ATP facilitated I(Ca) from -941 pA to -1,094 pA in other neurons. The data presented here demonstrate for the first time that ATP has both inhibitory and facilitative effects on I(Ca) in NTS. It can be considered that ATP acts as a neurotransmitter in the NTS by having multiple regulatory effects on VDCCs.

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.

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.

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.

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.

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.

In the majority of autonomic ganglia, the responses to tachykinins such as substance P, neurokinin A and neurokinin B are primarily mediated by neurokinin-3 (NK-3) receptors. Neurokinin B (NK-B) and senktide are known as an endogenous tachykinin and the related peptide selective for NK-3 receptor. In this study, the generation mechanism of NK-3 receptor agonist-induced response in the hamster submandibular ganglion (SMG) cells was investigated using the current-clamp technique. The SMG cells responded to the NK-3 receptor agonists with two types of depolarizations accompanied by either a decrease or an increase in membrane input resistance. The results showed that K+ channels alone or the combination of K+ and nonselective cation channels coupled with NK-3 receptors on the SMG cells were involved in generation of the NKB- and senktide-depolarizations.

We described in a previous paper (Bull Tokyo dent Coll 33: 33-35, 1992) that the submandibular ganglion cells (SMG) of hamster responded to substance P (SP) with a depolarization, which could be divided into three subtypes according to the change in membrane input resistance (Rm) during depolarization: depolarizations accompanied by either an increase or a decrease in Rm, and without change in Rm. In this study, the generation mechanism of the SP-induced depolarization accompanied by a decrease in Rm was investigated using the current-clamp technique. The result showed that cation channels coupled with NK-1 receptors on SMG cells were involved in generation of the SP-depolarization, and that the main charge carrier was the sodium ion.

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).