Electrocatalytic synthesis of ammonia by surface proton hopping.

Highly efficient ammonia synthesis at a low temperature is desirable for future energy and material sources. We accomplished efficient electrocatalytic low-temperature ammonia synthesis with the highest yield ever reported. The maximum ammonia synthesis rate was 30 099 µmol gcat-1 h-1 over a 9.9 wt% Cs/5.0 wt% Ru/SrZrO3 catalyst, which is a very high rate. Proton hopping on the surface of the heterogeneous catalyst played an important role in the reaction, revealed by in situ IR measurements. Hopping protons activate N2 even at low temperatures, and they moderate the harsh reaction condition requirements. Application of an electric field to the catalyst resulted in a drastic decrease in the apparent activation energy from 121 kJ mol-1 to 37 kJ mol-1. N2 dissociative adsorption is markedly promoted by the application of the electric field, as evidenced by DFT calculations. The process described herein opens the door for small-scale, on-demand ammonia synthesis.

SH2-containing inositol 5'-phosphatase 2 selectively impairs hypothalamic insulin signalling and regulation of food intake in mice.

SH2-containing inositol 5'-phosphatase 2 (SHIP2) is a lipid phosphatase that negatively regulates the metabolic signalling of insulin in peripheral tissues; however, the expression of SHIP2 in the hypothalamus and its functional roles are largely unknown. In the present study, immunohistochemical analysis demonstrated that SHIP2 protein exists in neuronal cells expressing neuropeptide Y and pro-opiomelanocortin in the arcuate nucleus of the hypothalamus in C57BL/6J mice. Interestingly, the expression levels of SHIP2 in the hypothalamus were elevated in aged C57BL/6J mice and diabetic db/db mice. To clarify the significance of the increased expression of SHIP2 in the hypothalamus, we examined the central effects of insulin and leptin in transgenic mice overexpressing SHIP2 (SHIP2-Tg). Accumulation of phosphatidylinositol (3,4,5)-trisphosphate and phosphorylation of Akt in the hypothalamus, induced by i.c.v. injection of insulin, were attenuated in SHIP2-Tg compared to wild-type mice, whereas leptin-induced phosphorylation of signal transducer and activator of transcription 3 in the hypothalamus was comparable between them. The suppression of food intake after i.c.v. administration of insulin (but not leptin) was attenuated consistently in SHIP2-Tg. In addition, SHIP2-Tg showed increased food consumption after starvation and become heavier with visceral fat accumulation than wild-type mice, despite normal levels of oxygen consumption and spontaneous movement. These results suggest that SHIP2 contributes to the regulation of food intake mainly via the attenuation of insulin signalling in the hypothalamus of mice.

Role of orexin in the regulation of glucose homeostasis.

Orexin-A (hypocretin-1) and orexin-B (hypocretin-2) are hypothalamic neuropeptides that play key roles in the regulation of wakefulness, feeding, reward, autonomic functions and energy homeostasis. To control these functions indispensable for survival, orexin-expressing neurones integrate peripheral metabolic signals, interact with many types of neurones in the brain and modulate their activities via the activation of orexin-1 receptor or orexin-2 receptor. In addition, a new functional role of orexin is emerging in the regulation of insulin and leptin sensitivities responsible for whole-body glucose metabolism. Recent evidence indicates that orexin efficiently protects against the development of peripheral insulin resistance induced by ageing or high-fat feeding in mice. In particular, the orexin receptor-2 signalling appears to confer resistance to diet-induced obesity and insulin insensitivity by improving leptin sensitivity. In fact, the expression of orexin gene is known to be down-regulated by hyperglycaemia in the rodent model of diabetes, such as ob/ob and db/db mice. Moreover, the levels of orexin receptor-2 mRNA have been shown to decline in the brain of mice along with ageing. These suggest that hyperglycaemia due to insulin insensitivity during ageing or by habitual consumption of a high-fat diet leads to the reduction in orexin expression in the hypothalamus, thereby further exacerbating peripheral insulin resistance. Therefore, orexin receptor controlling hypothalamic insulin/leptin actions may be a new target for possible future treatment of hyperglycaemia in patients with type 2 diabetes.

Age-related insulin resistance in hypothalamus and peripheral tissues of orexin knockout mice.

AIMS/HYPOTHESIS: Orexin/hypocretin is a hypothalamic neuropeptide that regulates motivated behaviours, such as feeding and arousal, and, importantly, is also involved in energy homeostasis. The aim of this study was to reveal the role of orexin in the regulation of insulin sensitivity for glucose metabolism. METHODS: Orexin knockout mice fasted overnight underwent oral glucose tolerance testing and insulin tolerance testing. The impact of orexin deficiency on insulin signalling was studied by Western blotting to measure levels of Akt phosphorylation and its upstream and downstream molecules in the hypothalamus, muscle and liver in orexin knockout mice. RESULTS: We found that orexin deficiency caused the age-related development of impaired glucose tolerance and insulin resistance in both male mice without obesity and female mice with mild obesity, fed a normal chow diet. When maintained on a high-fat diet, these abnormalities became more pronounced exclusively in female orexin knockout mice that developed severe obesity. Insulin signalling through Akt was disrupted in peripheral tissues of middle-aged (9-month-old) but not young adult (2-to-3-month-old) orexin knockout mice fed a normal chow diet. Moreover, basal levels of hypothalamic Akt phosphorylation were abnormally elevated in orexin knockout mice at every age studied, and insulin stimulation failed to increase the level of phosphorylation. Similar abnormalities were observed with respect to GSK3beta phosphorylation in the hypothalamus and peripheral tissues of middle-aged orexin knockout mice. CONCLUSIONS/INTERPRETATION: Our results demonstrate a novel role for orexin in hypothalamic insulin signalling, which is likely to be responsible for preventing the development of peripheral insulin resistance with age.

Calcium mobilization elicited by two types of nicotinic acetylcholine receptors in mouse substantia nigra pars compacta.

Nicotinic acetylcholine receptors (nAChRs) are expressed in the midbrain ascending dopaminergic system, a target of many addictive drugs. Here we assessed the intracellular Ca2+ level by imaging fura-2-loaded cells in substantia nigra pars compacta in mouse brain slices, and we examined the influence on this level of prolonged exposures to nicotine using mice lacking the nAChR beta2-subunit. In control cells, superfusion with nicotine (10-100 microM) caused a long-lasting rise of intracellular Ca2+ level which depended on extracellular Ca2+. This nicotinic response was almost completely absent in beta2-/- mutant mice, leaving a small residual response to a high concentration (100 microM) of nicotine which was inhibited by the alpha7-subunit-selective antagonist, methyllycaconitine. Conversely, the alpha7-subunit-selective agonist choline (10 mM) caused a methyllycaconitine-sensitive increase in intracellular Ca2+ level both in wild-type and beta2-/- mutant mice. Nicotine-elicited Ca2+ mobilization was reduced by the Na+ channel blocker tetrodotoxin (TTX) and by T-type Ca2+ channel blocking agents, whereas the choline-elicited Ca2+ increase was insensitive to TTX. Neither nicotine nor choline produced Ca2+ increase following inhibition of the release of Ca2+ from intracellular stores by dantrolene. These results demonstrate that in nigral dopaminergic neurons, nicotine can elicit Ca2+ mobilization via activation of two distinct nAChR subtypes: that of beta2-subunit-containing nAChR followed by activation of Na+ channel and T-type Ca2+ channels, and/or activation of alpha7-subunit-containing nAChR. The Ca2+ influx due to nAChR activation is subsequently amplified by the recruitment of intracellular Ca2+ stores. This Ca2+ mobilization may possibly contribute to the long-term effects of nicotine on the dopaminergic system.

Methyllycaconitine-sensitive neuronal nicotinic receptor-operated slow Ca2+ signal by local application or perfusion of ACh at the mouse neuromuscular junction.

Local application of acetylcholine (ACh; 0.3 mM, 20 microl) elicited bi-phasic elevation of intracellular Ca2+ concentrations (contractile fast and non-contractile slow Ca2- signal measured as aequorin luminescence) in diaphragm muscle preparation. A neuronal nicotinic antagonist methyllycaconitine (MLA; 0.01-1 microM), which did not affect the fast Ca2+ transients and twitch tension, concentration-dependently depressed only the slow Ca2+ component. Ca2+ channel blockers, Cd2+ (200 microM), nitrendipine (1 microM), verapamil (1 microM) and diltiazem (1 microM), or a Na+ channel blocker tetrodotoxin (TTX; 0.1 microM) failed to prevent the generation of slow Ca2+ response. Perfusion of ACh (1 microM) to isolated single skeletal (flexor digitorum brevis) muscle cells pretreated with TTX (0.1 microM) also elicited a slow Ca2+ signal measured as confocal imaging with a fluorescent dye, fluo-3, at the endplate region. MLA (1 microM) antagonized against the ACh perfusion-elicited slow Ca2+ signal. Perfusion of choline (1 mM), a neuronal nicotinic agonist, also elicited the MLA-sensitive slow Ca2+ signal. These results strongly suggest that the ACh-induced slow Ca2+ signal reflects Ca2+ entry through a postsynaptic MLA-sensitive neuronal nicotinic ACh receptor subtype at the neuromuscular junction.

[Desensitizing function of calcium mobilized by the postsynaptic neuronal-type nicotinic acetylcholine receptors at the neuromuscular junction].

Neuronal-type nicotinic acetylcholine receptors (N-nAChR) are co-localized with muscle-type (M-)nAChR in the postjunctional endplate membrane of adult skeletal muscle fibers. The postsynaptic desensitizing functions of the N-nAChR at the neuromuscular junction and at single skeletal muscle cells have been investigated using aequorin luminescence and fluorescence confocal imaging. A biphasic elevation of local intracellular Ca2+ is elicited by prolonged nicotinic action at the mouse muscle endplates. The contractile fast and non-contractile slow Ca2+ components are operated by postsynaptic M- and colocalized N-type nAChR, respectively. We have named the latter slow one RAMIC (receptor-activity modulating intracellular Ca2+). The N-nAChR are activated by nicotine and choline, and RAMIC are antagonized by methyllycaconitine and dihydro-beta-erythroidine. Neuromuscular functions may be regulated by a dual nAChR system to maintain the normal postsynaptic excitability. Certain N-nAChR may be also endowed with the same functional role in the central nervous system.

Calcitonin gene-related peptide potentiates nicotinic acetylcholine receptor-operated slow Ca2+ mobilization at mouse muscle endplates.

1. The involvement of calcitonin gene-related peptide (CGRP) in the non-contractile slow Ca2+ mobilization induced by prolonged nicotinic stimulation was investigated by measurement of [Ca2+], levels in mouse single muscle cells (flexor digitorum brevis; FDB) loaded with a Ca2+ indicator fluo-3 using confocal laser scanning microscopy. 2. CGRP (3-30 nM) potentiated acetylcholine (ACh, 1 microM)-elicited slow Ca2+ mobilization in a concentration-dependent manner. 3. The potentiation by CGRP of the slow Ca2+ component was greatly depressed by a competitive nicotinic antagonist (+)-tubocurarine (5 microM). The Ca2+ channel blocker nitrendipine (1 microM) affected neither ACh responses nor the CGRP potentiation. 4. The slow Ca2+ component was completely abolished by reducing [Ca2+]0 from 2.5 to 0.25 mM whereas the fast component was not affected. The CGRP-induced potentiation of slow Ca2+ signal was also depressed by decreasing [Ca2+]0. 5. Isoproterenol (30 microM) and 8-bromo-adenosine 3',5'-cyclic monophosphate (1 mM) potentiated the ACh-elicited slow Ca2+ response. The potentiation by CGRP of the slow Ca2+ component was completely abolished by a protein kinase-A inhibitor H-89 (1 microM). 6. These findings indicate that CGRP potentiates the nicotinic ACh receptor-operated slow Ca2+ signal via the activation of protein kinase-A system at the skeletal muscle endplates.

Immunocytochemical localization of salivary peptide P-C in human submandibular gland.

Human saliva contains a proline-rich polypeptide, salivary peptide P-C, which potentiates insulin release and reduces glucagon release from perfused rat pancreas to decrease blood glucose level. To elucidate the process of secretion into humoral fluid of this peptide morphologically, we investigated ultrastructural localization of P-C in human submandibular gland by immunogold technique with anti-peptide P-C whose specificity to P-C was confirmed by immunoblotting. The labeling with gold particles which represents the distribution of P-C-like-immunoreactivity (P-C-LI) was detected in the secretory granules and rough endoplasmic reticula of the acinar serous cells and in few mucosa cells. P-C-LI was also observed in the lumen of striated duct but not intracellularly in the ductal cells themselves, indicating that P-C is not probably reabsorbed there. These results suggest that salivary peptide P-C is present in acinar serous cells, is secreted into the oral cavity, and may be reabsorbed through the digestive tract to modulate the blood glucose level after feeding.

Platelet-derived growth factor blocks the cell-cycle transition from the G0 to G1 phase in subcultured angiogenic endothelial cells in rat thoracic aorta.

Platelet-derived growth factor (PDGF)-BB induces tube formation by the differentiating (tube-forming) endothelial cells (EC) of rat thoracic aorta, although PDGF-BB does not affect the proliferative EC (increasing the cell numbers) at the progression phase. These changes in the responses to PDGF-BB were due to the phenotype-dependent expression of PDGF beta-receptor (PDGFR-beta) on EC because PDGFR-beta-like immunoreactivity was observed in the angiogenic EC forming a tube-like structure in 35-day culture with 10% fetal bovine serum, but not in the proliferative EC in 5-day culture. To elucidate the functional role of PDGFR-beta in the alteration of EC phenotype, the influence of PDGF-BB on the cell cycle of EC was investigated by flow cytometry. This analysis demonstrates that PDGF-BB blocks the transition from the G0 to G1 phase in the 35-day cultured EC, although no effect was observed on any phases of the cell cycle in 5-day culture. We conclude that 1) PDGFR-beta is expressed in mature angiogenic EC of rat aorta, and 2) PDGF-BB may contribute to promotion of the EC differentiation with tubular morphogenesis by inhibiting cell growth.

Neuronal nicotinic receptor operates slow Ca2+ mobilization at mouse muscle endplate.

The contribution to neuromuscular functions by neuronal nicotinic acetylcholine receptor (nAChR) expressed at skeletal muscle endplate was investigated using intracellular Ca2+ measurements. A neuronal nAChR blocker, methyllycaconitine (MLA), depressed non-contractile Ca2+ mobilization without affecting muscle nAChR activity in nerve-stimulated mouse diaphragm muscle, after cholinesterase inhibition. Confocal imaging demonstrates that the MLA-sensitive Ca2+ mobilization also occurred at the endplate in single flexor digitorum brevis muscle cells as the slow component of two-phasic Ca2+ elevation after the prolonged nicotinic stimulation. A monoclonal antibody to alpha 1 subunit of muscle nAChR depressed the fast but not the slow component. Thus, muscle neuronal-nAChR can induce the localized rise of Ca2+ at the postjunctional sites.

[Slow Ca2+ (RAMIC) mobilization operated by postsynaptic neuronal nicotinic receptor regulates synaptic function at the mouse neuromuscular junction].

We have found that non-contractile slow Ca2+ mobilization (RAMIC; Receptor-Activity Modulating Intracellular Ca2+) is generated by motor nerve stimulation with anti-cholinesterase at the skeletal muscle, and desensitizes muscle nicotinic receptor (nAChR). To confirm this Ca2+ mobilization without anti-cholinesterase, acetylcholine (ACh) was locally applied by N2-gas pressure onto endplate region at the mouse phrenic nerve-diaphragm muscle preparation. ACh (0.1-3 mM, 20 microliters) elicited bi-phasic elevation of [Ca2+]i (fast and slow Ca2+ mobilization measured as Ca(2+)-aequorin luminescence) in muscle cells. The peak amplitude of slow Ca2+ mobilization (not accompanied by contraction) was increased by ACh concentration-dependently, whereas that of fast component (accompanied by contraction) reached a maximum response at a lower concentration of ACh. The slow Ca2+ mobilization was blocked by lower concentrations of competitive nAChR antagonists which did not affect the fast Ca2+ transients. Moreover, the slow Ca2+ signal was selectively depressed by a neuronal nAChR antagonist methyllycaconitine. Neither Ca2+ channel blockers nor a Na+ channel blocker tetrodotoxin prevented the generation of the slow Ca2+ mobilization. These results suggest that RAMIC is mobilized through postsynaptic neuronal nAChR subtype to desensitize muscle nAChR at the neuromuscular junction.

Enhancement by calcitonin gene-related peptide of non-contractile Ca2(+)-induced nicotinic receptor desensitization at the mouse neuromuscular junction.

1. Nicotinic acetylcholine receptor (AChR)-operated non-contractile Ca2+ mobilization (unaccompanied by muscle contraction) depressed contractile Ca2+ mobilization (accompanied by muscle contraction) in mouse diaphragm muscles. In the process of nicotinic AChR desensitization, the enhancing role of calcitonin gene-related peptide (CGRP) on the non-contractile Ca2(+)-induced depression of contractile Ca2+ mobilization was investigated by measurement of Ca2(+)-aequorin luminescence in the presence of neostigmine (0.1 microM). 2. When the phrenic nerve was stimulated with paired pulses at intervals of 150, 300, 600, 1000 and 2000 ms, contractile Ca2+ transients were elicited during the generation of non-contractile Ca2+ mobilization. The amplitude of the contractile Ca2 transients elicited by the second pulse (S2) was depressed at the shorter pulse intervals, but not at the longer pulse intervals. 3. The extent of depression of S2 was enhanced when the duration of non-contractile Ca2+ mobilization was prolonged by CGRP (10 nM). However, CGRP failed to enhance the depression of S2 when non-contractile Ca2+ mobilization was not observed at the low external Ca2+ concentration (1.3 mM). 4. The enhancing effect by CGRP on the depression of S2 was counteracted by staurosporine (3 nM), a protein kinase-C inhibitor, despite prolongation of the duration of non-contractile Ca2+ mobilization. 5. When H-89 (1 microM), a protein kinase-A inhibitor, completely blocked non-contractile Ca2+ mobilization, the depression of S2 was diminished. The prolongation of the duration of non-contractile Ca2+ mobilization by AA373 (300 microM), a protein kinase-A activator, enhanced the depression of S2. The enhancing effect was observed neither with CGRP nor with AA373, in the presence of H-89 (0.1 microM). 6. These findings suggest that the CGRP mobilizes non-contractile Ca2+ through activation of protein kinase-A, which in turn may activate protein kinase-C, then enhance the desensitization of postsynaptic nicotinic AChRs at the neuromuscular junction.

Diabetic state-induced rapid inactivation of noncontractile Ca2+ mobilization operated by nicotinic acetylcholine receptor in mouse diaphragm muscle.

1. Diabetic modifications of nicotinic receptor-operated noncontractile Ca2- mobilization observed in the presence of anticholinesterase were investigated by measuring Ca(2+)-aequorin luminescence in diaphragm muscles of mice with diabetes induced by injections of streptozotocin (150 mg kg-1, bolus i.v.) and alloxan (85 mg kg-1, bolus i.v.). 2. The diabetic state accelerated the decline of noncontractile Ca2+ transients without affecting their peak amplitude. Insulin treatment reversed this alteration. 3. The increase in contractile Ca2+ transients by cholinesterase inhibition was attenuated 0.6 fold and became resistant to changes in [Ca2+]o in the diabetic state. 4. Changes in extracellular pH from 7.6 to 5.6 depressed the peak amplitude of noncontractile Ca2+ transients without affecting their duration, and enhanced the peak amplitude of contractile Ca2+ transients. 5. These results suggest that the inactivation process of noncontractile Ca2+ mobilization is promoted in diabetic muscles, presumably by desensitization of the nicotinic acetylcholine receptor.

Accelerated desensitization of nicotinic receptor channels and its dependence on extracellular calcium in isolated skeletal muscles of streptozotocin-diabetic mice.

1. To elucidate the influence of the diabetic state on desensitization of nicotinic acetylcholine (ACh) receptor channels, we investigated the time course of the decrease in amplitude of ACh potentials elicited by iontophoretic application to isolated diaphragm muscle of streptozotocin-diabetic mice. We also investigated time- and extracellular Ca(2+)-dependent changes in the channel opening frequency of ACh-activated channel currents and the involvement of protein kinases by use of the cell-attached patch clamp technique in single skeletal muscle cells. 2. When ACh potentials were evoked at 10 Hz, the decline in trains of ACh potentials was accelerated in the diabetic state. 3. The time-dependent decrease in the channel opening frequency of diabetic muscle cells was greatly accelerated compared with normal cells in 2.5 mM Ca2+ medium. 4. This accelerated decrease in channel opening frequency was restored by pretreatment with a protein kinase C inhibitor, staurosporine (10 nM) but neither a protein kinase A inhibitor, H-89 (3 microM) nor a calmodulin kinase II inhibitor, KN-62 (5 microM) were able to restore the fall in opening frequency. 5. These results demonstrate that in the diabetic state the desensitization of nicotinic ACh receptor channels may be greatly accelerated by activating protein kinase C, which is caused by an increase in the amount of available intracellular Ca2+.

Immunohistochemical localization of neuronal nicotinic receptor subtypes at the pre- and postjunctional sites in mouse diaphragm muscle.

The existence of neuronal nicotinic acetylcholine receptor (nAChR) subunits was investigated in the cryostat sections of mouse diaphragm muscles using the indirect immunofluorescence technique. The specific immunolabelings with monoclonal antibodies (mAbs) to beta 2 and to alpha 8 subunits of neuronal nAChR were observed at the endplate determined by labeling with a fluorescent dye (BODIPY)-conjugated alpha-bungarotoxin. The immunoreactivity of mAb to the alpha 3 subunit of neuronal nAChR was detected on the motor nerve fibers including the nerve terminals. These results provide evidence that the subtypes of postsynaptic nAChR, recognized by the anti-beta 2 and/or anti-alpha 8 mAbs, and the presynaptic nAChR recognized by the anti-alpha 3 mAb, are present at the neuromuscular junction, in addition to the classical muscle nAChR.

Postsynaptic nicotinic receptor desensitized by non-contractile Ca2+ mobilization via protein kinase-C activation at the mouse neuromuscular junction.

1. Non-contractile Ca2+ mobilization (unaccompanied by muscle contraction) was initiated by nerve stimulation in the presence of neostigmine (more than 0.03 microM) at the endplate region of mouse diaphragm muscles. In the process of nicotinic receptor desensitization, the depressant effect of non-contractile Ca2+ on contractile Ca2+ mobilization was investigated by measurement of Ca(2+)-aequorin luminescence. 2. When the phrenic nerve was stimulated with paired pulses having intervals of 150, 300, 600, 1000 and 2000 ms, contractile Ca2+ transients were elicited during the generation of non-contractile Ca2+ mobilization. The amplitude of the contractile Ca2+ transients elicited by the second pulse (S2) was depressed at the shorter pulse intervals, but recovered to the initial contractile response (S1) at longer pulse intervals. 3. The extent of depression of S2 was enhanced by increasing the concentration of neostigmine (0.03 to 0.3 microM). When a low concentration (0.05 microM) of pancuronium, a competitive nicotinic antagonist, completely blocked non-contractile Ca2+ mobilization, the depression of S2 was diminished. 4. The depression of S2 was enhanced when the peak amplitude of non-contractile Ca2+ mobilization was raised by increasing the external Ca2+ concentration from 1.3 to 5 mM. 5. Staurosporine (10 nM), a protein kinase-C inhibitor, diminished the depression of S2 despite large amounts of non-contractile Ca2+ mobilization. The diminishing effect of staurosporine was counteracted by TPA (0.1 microM), a protein kinase-C activator. 6. These findings suggest that non-contractile Ca2+ mobilization may enhance the desensitization of the postsynaptic nicotinic receptor via activation of protein kinase-C at the neuromuscular junction.

Monoclonal antibody to beta 2 subunit of neuronal nicotinic receptor depresses the postjunctional non-contractile Ca2+ mobilization in the mouse diaphragm muscle.

The involvement of subtypes of nicotinic acetylcholine receptor (nAChR) in the postjunctional non-contractile Ca2+ mobilization was investigated in mouse diaphragm muscles treated with an anticholinesterase, using monoclonal antibodies (mAbs) to nAChR subunits. mAb 210 (specific for alpha 1 subunit of muscle nAChR) depressed contractile Ca2+ transients without affecting non-contractile Ca2+ transients. mAb 270 (specific for beta 2 subunit of neuronal nAChR) depressed only non-contractile Ca2+ transients. mAb 210 did not completely block the ACh-activated channel currents in flexor digitorum brevis muscle cells. The present findings indicate that the anti-beta 2 mAb 270-related subtype of nAChR may postsynaptically operate the non-contractile Ca2+ mobilization at the neuromuscular junction, suggesting the involvement of a subtype different from the usual muscle-type nAChR.

Independent regulation of activation and inactivation phases in non-contractile Ca2+ transients by nicotinic receptor at the mouse neuromuscular junction.

Non-contractile Ca2+ mobilization (not accompanied by muscle contraction) occurs by the prolonged activation of nicotinic acetylcholine receptor in mouse diaphragm muscles treated with anticholinesterase. To elucidate the regulation properties of non-contractile Ca2+ mobilization by nicotinic receptor, the modes of action of competitive and depolarizing neuromuscular blockers were investigated. (+)-Tubocurarine (0.07-0.1 microM), pancuronium (0.05 microM) and alpha-bungarotoxin (0.03-0.06 microM) decreased decay time (T2, duration of inactivation phase) without changes in rise time (T1, duration of activation phase) of non-contractile Ca2+ transients. These competitive antagonists also suppressed their peak amplitude at higher concentrations than those affecting T2. Contractile Ca2+ transients were not inhibited by these antagonists at the concentrations used. Decamethonium (1 microM), a depolarizing blocker, suppressed the peak amplitude of non-contractile Ca2+ transients without affecting their duration. In contrast, succinylcholine (0.3 microM) suppressed both peak amplitude and T1 without changing T2, presumably via the receptor desensitization. Succinylcholine but not decamethonium inhibited contractile Ca2+ transients at the concentrations used. These results demonstrate that the activation and inactivation phases in non-contractile Ca2+ transients are independently regulated by nicotinic acetylcholine receptor.