Plasma from patients with seronegative myasthenia gravis inhibit nAChR responses in the TE671/RD cell line

Myasthenia gravis (MG) is an autoimmune disorder in which anti-acetylcholine receptor (AChR) antibodies cause muscle weakness. In 10–15% of MG patients anti-AChR antibodies are undetectable (seronegative MG, SMG), though clinical and experimental evidence points to causative circulating factors. Using whole-cell patch-clamp techniques, we investigated the effects of heat-inactivated plasma from SMG patients (n=7) on voltage-gated sodium [I _Na(V)] and ACh-induced nicotinic AChR (nAChR) currents in the human rhabdomyosarcoma cell line TE671/RD, comparing the results to those obtained with plasma from healthy individuals (HC, n=6), patients with Guillain-Barré syndrome (GBS, n=3) or those with other neurological diseases (OND, n=3). None of the plasma samples inhibited I _Na(V). nAChR currents were rapidly (<1 min) and significantly (P<0.01) reduced by a 110 dilution of plasma from SMG patients compared with plasma from healthy controls and were not restored by washing. The inhibition appeared in some cases to be calcium dependent since for one of three plasmas it was prevented by 10 mM EGTA in the patch pipette. Currents were also reduced by two of three plasmas obtained from GBS patients at 13 dilution, but not by the three plasmas from patients with ONDs. The rapid action of plasma from SMG patients argues against an antibodyinduced reduction in nAChR numbers; its calcium dependence in one case suggests action by a second messenger that might involve nAChR phosphorylation.     

Cytoplasmic Ca2+ determines the rate of Ca2+ entry into Mardin-Darby canine kidney-focus (MDCK-F) cells

Transformed Mardin-Darby canine kidney-focus (MDCK-F) cells exhibit spontaneous Ca²⁺ oscillations from an inositol 1,4,5-trisphosphate-sensitive cytoplasmic Ca²⁺ store. In this study, Ca²⁺ entry from the extracellular space and its role in generation of oscillations were investigated by means of Ca²⁺ video imaging and the Fura-2/Mn²⁺ quenching technique. Oscillations were dependent on extracellular Ca²⁺ concentration and were inhibited by extracellularly applied La³⁺, Co²⁺ and Ni²⁺. Depolarization of the cell membrane with high K⁺ concentrations and the L-type Ca²⁺ channel blocker nifedipine had no effect on oscillations, indicating the lack of involvement of voltage-gated Ca²⁺ channels. Mn²⁺ quenching experiments disclosed significant Ca²⁺ influx into MDCK-F cells. The rate of this influx was constant between Ca²⁺ spikes, but markedly increased during the spontaneous Ca²⁺ spikes. Similar transient increases in Ca²⁺ entry could be mimicked by agents triggering intracellular Ca²⁺ release such as bradykinin and thapsigargin. We conclude that the plasma membrane of MDCK-F cells exhibits a marked voltage-independent Ca²⁺ permeability permitting Ca²⁺ entry into the cytoplasm. The rate of Ca²⁺ entry which determines the frequency of oscillations is most likely to be regulated by the cytoplasmic Ca²⁺ concentration.     

Inositol 1,4,5-trisphosphate formation and ryanodine-sensitive oscillations of cytosolic free Ca2+ concentrations in neuroblastoma×fibroblast hybrid NL308 cells expressing m2 and m4 muscarinic acetylcholine receptor subtypes

Intracellular free Ca²⁺ concentrations ([Ca²⁺]_i) were measured in subclones of NL308 neuroblastoma x fibroblast hybrid cells expressing each of the individual muscarinic acetylcholine receptor (mAChR) subtypes m1, m2, m3 and m4. Application of 100 M acetylcholine (ACh) increased [Ca²⁺]_i in all four subclones. The increased [Ca²⁺]_i levels were significantly higher in m1- and m3-transformed cells than those in m2- and m4-transformed cells. In more than 95% of m2- and m4-transformed cells, [Ca²⁺]_i showed sinusoidal oscillations. ACh-induced increases in [Ca²⁺]_i were not observed in cells treated with an intracellular Ca²⁺ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N,N'-tetraacetic acid (BAPTA). Removal of extracellular Ca²⁺ with ethyleneglycol-bis-(-aminoethyl)-N,N,N,N'-tetraacetate (EGTA) did not affect the initial [Ca²⁺]_i increases, but reduced the late phases of [Ca²⁺]_i in m1- and m3-transformed cells by 20–30%. Oscillations in m2- and m4-transformed cells persisted in EGTA solution (though sometimes slowed in frequency), suggesting that they were of intracellular origin. ACh-induced [Ca²⁺]_i and inositol 1,4,5-trisphosphate formation was completely suppressed by pre-treatment with 50–100 ng ml^–1 Pertussis toxin (PTX) for 12 h in m2- and m4-transformed cells, but not in m1 and m3-transformed cells. In all cells, extracellular application of caffeine and ryanodine, or intracellular application of cyclic adenosine diphosphate ribose (cADPR) produced a rise in [Ca²⁺]_i. ACh-induced [Ca²⁺]_i oscillations were not observed in ryanodine-treated m2-transformed cells. These results show that, while all four mAChRs utilize Ca²⁺ as a common second messenger, m2 and m4 receptors use a different signalling pathway to that used by m1 and m3 receptors.     

Effect of cytosolic Mg2+ on mitochondrial Ca2+ signaling

Cytosolic Ca²⁺ signals are followed by mitochondrial Ca²⁺ uptake, which, in turn, modifies several biological processes. Mg²⁺ is known to inhibit Ca²⁺ uptake by isolated mitochondria, but its significance in intact cells has not been elucidated. In HEK293T cells, activation of purinergic receptors with extracellular ATP caused cytosolic Ca²⁺ signals associated with parallel changes in cytosolic [Mg²⁺]. Neither signals were affected by omitting bivalent cations from the extracellular medium. The effect of store-operated Ca²⁺ influx on cytosolic Mg²⁺ concentration ([Mg²⁺]_c) was negligible. Uncaged Ca²⁺ displaced Mg²⁺ from cytosolic binding sites, but for an equivalent Ca²⁺ signal, the change in [Mg²⁺] was significantly smaller than that measured after adding extracellular ATP. Inositol 1,4,5-trisphosphate mobilized Ca²⁺ and Mg²⁺ from internal stores in permeabilized cells. The increase of [Mg²⁺] in the range that occurred in ATP-stimulated cells inhibited mitochondrial Ca²⁺ uptake in permeabilized cells without affecting mitochondrial Ca²⁺ efflux. Therefore, the Mg²⁺ signal generated by Ca²⁺ mobilizing agonists may attenuate mitochondrial Ca²⁺ uptake.     

Vasopressin responses in electrically coupled A7r5 cells

Changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and in membrane potential were monitored in single A7r5 smooth-muscle cells during spontaneous spiking and after arginine vasopressin stimulation. Spontaneous Ca²⁺ oscillations, which were associated with the occurrence of action potentials, occurred in about 90% of the confluent monolayers investigated. This spontaneous activity was synchronized amongst all the cells of the monolayer, indicating that the cells were electrically coupled. Arginine vasopressin stimulation produced a [Ca²⁺]_i rise that was about 5 times higher than the amplitude of the spontaneous Ca²⁺ oscillations and resulted in a subsequent cessation of spontaneous electrical activity and associated Ca²⁺ spiking, which persisted after [Ca²⁺]_i returned to baseline. Individual cells in the monolayer responded to arginine vasopressin with a different latency. Agonist-induced Ca²⁺ waves within one cell propagated much more slowly than spontaneous [Ca²⁺]_i rises. We conclude that agonist-induced [Ca²⁺]_i increases in an electrically coupled cell monolayer can be asynchronous.     

Cholinergic agonists increase cell calcium in rat medullary collecting tubules

The intracellular free calcium concentration [Ca²⁺]_i of rat medullary collecting tubules was calculated from microscope fluorescence measurements in single pieces of fura-2-loaded tubules superfused at 37 °C. When carbachol (10^–4–10^–3M) was added in the superfusate, a biphasic increase in [Ca²⁺]_i was generally obtained, which included an early peak phase and a sustained plateau thereafter; sometimes, the peak phase was not apparent; the plateau was maintained as long as the agonist was applied. Several responses could be induced successively without a fall in responsiveness. From dose/response curves, K_1/2 values of about 10^–5M for carbachol and 10^–6M for acetylcholine were obtained. The effects of the agonists were suppressed with 10^–4M of atropine or pirenzepine, indicating the presence of muscarinic receptors of the M₁ type. In the absence of external calcium, the peak phase of the response was preserved while the plateau phase was suppressed; thus, the peak involves the release of calcium stored in organelles, whereas the plateau involves the entry of external calcium through calcium channels which were voltage independent and insensitive to the usual calcium blockers.     

Roles of inositol trisphosphate and protein kinase C in the spontaneous outward current modulated by calcium release in rabbit portal vein

We examined the effects of heparin, guanosine nucleotides, protein kinase C (PKC) modulators, such as phorbol 12,13-dibutylate (PDBu) and H-7 on Ca²⁺-dependent K⁺ currents in smooth muscle cells of the rabbit portal vein using the whole-cell patch-clamp technique, to explore the effects of PKC on the oscillatory outward current (I _oo). Neomycin (30 M), an inhibitor of phospholipase C, and intracellular applications of heparin (10 g/ml) and guanosine 5-O-(2-thiodiphosphate) (GDP[S]; 1 mM) partly but consistently inhibited the generation of I _oo, whereas a higher concentration of heparin (100 g/ml) transiently enhanced then suppressed the generation of I _oo. Inhibition of I _oo generation by heparin was more powerful at the holding potential of + 20 mV than at –20 mV. Inositol 1,4,5-trisphosphate (InsP ₃; 30 M) continuously generated I _oo at holding potentials more positive than –60 mV. Noradrenaline (10 M) and caffeine (3–20 mM) transiently augmented, then reduced the generation of I _oo. Heparin (10 g/ml) completely inhibited responses induced by InsP ₃ and noradrenaline, but not those induced by caffeine. Intracellular application of guanosine 5-triphosphate (GTP; 200 M) or low concentrations of guanosine 5-O-(3-thiotriphosphate) (GTP[S]; 3 M) continuously augmented the generation of I _oo. High concentrations of GTP[S] (10 M) transiently augmented, then inhibited I _oo. Neither GTP[S] nor noradrenaline induced the transient augmentation or the subsequent inhibition of I _oo when applied in the presence of GDP[S] (1 mM), neomycin (30 M) or heparin (10 g/ml). PDBu (0.1 M) reduced the generation of I _oo but failed to produce an outward current following application of caffeine (3–5 mM). This action of PDBu was inhibited by pretreatment with H-7 (20 M). In the presence of H-7, GTP[S] continuously enhanced the generation of I _oo. The suppression of the generation of I _oo during application of noradrenaline (10 M) was reduced by pretreatment with H-7. Thus both InsP₃ and protein kinase C contribute to the generation of I _oo in smooth muscle cells of the rabbit portal vein and heparin is not a specific InsP ₃ antagonist on the InsP ₃-induced Ca²⁺-release channel (PIRC). InsP ₃ opens PIRC and protein kinase C may deplete the stored Ca²⁺ by either inhibiting the reuptake of Ca²⁺ or by enhancement of the releasing actions of InsP ₃.     

The pathway for refilling intracellular Ca2+ stores passes through the cytosol in human leukaemia cells

The pathway for refilling the intracellular Ca²⁺ stores of HL60 and U937 human leukaemia cells loaded with fura-2 has been investigated. On addition of external Ca²⁺ to cells with empty stores there was an increase in the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) which preceded the refilling of the stores. The increase in [Ca²⁺]_i was faster than the refilling, by 3-to 15-fold, depending on the cell type. In measurements in single HL60 cells we found that the refilling of the stores correlated with the extent of the [Ca²⁺]_i increase on addition of external Ca²⁺. The cells showing no [Ca²⁺]_i increase were unable to refill their stores. The addition of Ni²⁺ to the extracellular medium prevented both the [Ca²⁺]_i increase and the refilling of the stores. These results indicate that the limiting step for store refilling is the entry of Ca²⁺ from the extracellular medium to the cytosol. Hence, we conclude that extracellular Ca²⁺ cannot gain access directly to the intracellular Ca²⁺ stores in these cells, but must first enter the cytosol and be taken up from there into the stores.     

Inositol phosphates in the heart: controversy and consensus

Summary and Conclustions Numerous studies have addressed various aspects of inositol phosphate release and metabolism in myocardial preparations, and many different viewpoints have been expressed. The various results and interpretations presented often appear confusing and extracting a consensus view can be difficult. The differences often derive from the differing cardiac preparations used, especially isolated cells versus intact tissue. Despite these problems there are aspects where consensus prevails. Both the metabolism and the functional activity of inositol phosphates in heart appear to differ from those previously described in non-excitable cells. Inositol phosphates do not appear to be of major importance in the control of cardiac function under physiological conditions but may well have greater influence under pathological conditions such as myocardial ischaemia and reperfusion. Hopefully, the near future will see remaining controversies resolved.Abbreviations ANP Atrial natriuretic peptide - DAG sn-1,2-diacylglycerol - Ins Inositol - PCA Perchloric acid - PKC Protein kinase C - PLC Phospholipase C - PtdIns Phosphatidylinositol - TCA Trichloroacetic acid     

Initiation sites for Ca2+ signals in endothelial cells

Intracellular Ca²⁺ signals in response to inositol 1,4,5-trisphosphate-producing agents often present themselves as Ca²⁺ oscillations and propagating Ca²⁺ waves originating at discrete initiation sites. We studied the spatial organization of the Ca²⁺ signal in single CPAE endothelial cells stimulated with adenosine triphosphate. The long, thin processes presented a higher agonist sensitivity and, for the same agonist concentration, a faster rise in cytoplasmic Ca²⁺ concentration and rate of wave propagation than the cell body. Ca²⁺ waves originated preferentially in one of these processes and then invaded the cell body. Removal of external Ca²⁺ induced a progressive inhibition up to blockade of the response in the process but not in the cell body. These findings suggest that CPAE cells contain many individual store units, each of which has the inherent ability to set the stage for Ca²⁺ release. A diffusing messenger originating from the initiation zone then coordinates the events leading to Ca²⁺ release in the individual store units to produce a Ca²⁺ wave.     

An α3β4 subunit combination acts as a major functional nicotinic acetylcholine receptor in male rat pelvic ganglion neurons

We identified major subunits of the nicotinic acetylcholine receptor (nAChR) involved in excitatory postsynaptic potential and intracellular Ca²⁺ ([Ca²⁺] _i ) increase in the major pelvic ganglion (MPG) neurons of the male rat. ACh elicited fast inward currents in both sympathetic and parasympathetic MPG neurons. Mecamylamine, a selective antagonist for α3β4 nAChR, potently inhibited the ACh-induced currents in sympathetic and parasympathetic neurons (IC₅₀; 0.53 and 0.22 μM, respectively). Furthermore, α-conotoxin AuIB (10 μM), a new selective antagonist for α3β4 nAChR, blocked more than 80% of the ACh-induced currents in MPG neurons. Conversely, α-bungarotoxin, α-methyllycaconitine, and dihydro-β-erythroidine, known as blockers of the α7 or α4β2, did not show selective blocking effects on MPG neurons. ACh transiently increased [Ca²⁺] _i which was subsequently abolished in the extracellular Ca²⁺-free environment. Simultaneous recording of [Ca²⁺] _i and ionic currents revealed that ACh increased [Ca²⁺] _i under the conditions of the voltage-clamped (at −80 mV) state, and this resulted from the influx through nAChR itself. ACh-induced [Ca²⁺] _i increase was blocked by mecamylamine (10 μM), but was not affected by atropine (1 μM). RT-PCR analysis showed that, among subunits of nAChR, α3 and β4 were predominantly expressed in MPG. We suggest that activation of α3 and β4 nAChR subunits in MPG neurons induce fast inward currents and [Ca²⁺] _i increase, possibly mediating a major role in pelvic autonomic synaptic transmission.     

The acid test: the discovery of two-pore channels (TPCs) as NAADP-gated endolysosomal Ca2+ release channels

In this review, we describe the background and implications of our recent discovery that two-pore channels (TPCs) comprise a novel class of calcium release channels gated by the intracellular messenger nicotinic acid adenine dinucleotide phosphate (NAADP). Their localisation to the endolysosomal system highlights a new function for these organelles as targets for NAADP-mediated Ca²⁺ mobilisation. In addition, we describe how TPCs may also trigger further Ca²⁺ release by coupling to the endoplasmic reticular stores through activation of IP₃ receptors and ryanodine receptors.     

Mechanism of the ATP-induced rise in cytosolic Ca2+ in freshly isolated smooth muscle cells from human saphenous vein

Effects of exogenous adenosine 5-triphosphate (ATP) were studied by measurements of intracellular Ca²⁺ concentration ([Ca²⁺]_i) and membrane currents in myocytes freshly isolated from the human saphenous vein. At a holding potential of –60 mV, ATP (10 M) elicited a transient inward current and increased [Ca²⁺]_i. These effects of ATP were inhibited by ,-methylene adenosine 5-triphosphate (AMPCPP, 10 M). The ATP-gated current corresponded to a non-selective cation conductance allowing Ca²⁺ entry. The ATP-induced [Ca²⁺]_i rise was abolished in Ca²⁺-free solution and was reduced to 30.1±5.5% (n=14) of the control response when ATP was applied immediately after caffeine, and to 23.7±3.8% (n=11) in the presence of thapsigargin. The Ca²⁺-induced Ca²⁺ release blocker tetracaine inhibited the rise in [Ca²⁺]_i induced by both caffeine and ATP, with apparent inhibitory constants of 70 M and 100 M, respectively. Of the ATP-induced increase in [Ca²⁺]_i 29.3±3.9% (n=8) was tetracaine resistant. It is concluded that the effects of ATP in human saphenous vein myocytes are only mediated by activation of P_2x receptor channels. The ATP-induced [Ca²⁺]_i rise is due to both Ca²⁺ entry and Ca²⁺ release activated by Ca²⁺ ions that enter the cell through P_2x receptor channels.     

Visualization and manipulation of phosphoinositide dynamics in live cells using engineered protein domains

There is hardly a membrane-associated molecular event that is not regulated by phosphoinositides, a minor but critically important class of phospholipids of cellular membranes. The rapid formation, elimination, and conversion of these lipids in specific membrane compartments are ensured by a wealthy number of inositol lipid kinases and phosphatases with unique localization and regulatory properties. The existence of multiple inositol lipid pools have been indicated by metabolic labeling studies, but the level of functional compartmentalization revealed by the identification of numerous protein effectors acted upon by phosphoinositides could not have been foreseen. The changing perception of inositides from just serving as lipid precursors of second messengers to becoming highly dynamic local membrane-bound regulators poses new challenges concerning the detection of their rapid localized changes. Moreover, it is increasingly evident that manipulation of lipids in highly defined compartments would be a highly superior approach to soaking the cells with a particular phosphoinositide when studying the local regulation of the lipid on any effectors. In this review, we will summarize our efforts to improve our tools in studying phosphoinositide dynamics and discuss our views on the values of these methods compared to other options currently used or being explored.     

Neuropeptide Y2-type receptor-mediated activation of large-conductance Ca2+-sensitive K+ channels in a human neuroblastoma cell line

We have proposed recently that a pertussistoxin-insensitive Ca²⁺ influx stimulated by Y₂-type receptor activation in CHP-234 human neuroblastoma cells underlies increases in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) induced by neuropeptide Y (NPY), which were strictly dependent on extracellular Ca²⁺ and independent of internal Ca²⁺ stores. We describe here the actions of NPY in these same cells, using the activity of Ca²⁺-activated K⁺ channels as an indicator of [Ca²⁺]_i. The elementary slope conductance of these channels was 110±3 pS (with an asymmetrical K⁺gradient), their activity was greatly increased by application of ionomycin, and they were reversibly blocked by 1 mM tetraethylammonium (TEA) and 100 nM charybdotoxin. Application of 100 nM NPY, in the presence but not in the absence of extracellular Ca²⁺, increased the channel open probability. ATP applied in the absence of external Ca²⁺ caused rises both in channel open probability and [Ca²⁺]_i. Inositol trisphosphate production was stimulated by ATP but not by NPY. In outside-out patches, NPY increased channel open probability, indicating that NPY-associated Ca²⁺ influx does not require all the intracellular machinery present in intact cells. Channel activation by NPY was unaffected by the replacement of guanosine 5-triphosphate (GTP) by (guanosine 5-O-(2-thiodiphosphate) (GDP[S]), a non-hydrolysable GDP analogue, in the pipette internal solution, consistent with the lack of involvement of G-proteins in the coupling of Y₂-type receptors to Ca²⁺ influx in CHP-234 cells.     

Evidence for contribution of Ca2+ storage sites on unitary K+ channel currents in inside-out membrane of rabbit portal vein

While making use of the inside-out membrane patch, we examined the effects of caffeine and heparin on unitary currents of the large conductance Ca²⁺ -dependent K⁺ (maxi-K⁺) channel in the rabbit portal vein. About half of the inside-out membranes we used contained a functional Ca²⁺ -store site which facilitated modification of the maxi-K⁺ channel.When high-K⁺ solution containing 0.05mM EGTA was superfused in the bath, simultaneous openings of more than 20 maxi-K⁺ channels were observed in 39 of 83 patch membranes, and multi-channel opening appeared periodically or continuously at the holding potential of – 10mV. Most channel activities of these patch membranes were inhibited by caffeine or heparin, and some heparin-insensitive channel activities were inhibited by caffeine. The remaining patch membranes (44 out of 83) showed low activity of the maxi-K⁺ channel, and neither caffeine nor heparin modified channel activity.Therefore, in our experimental set-up, half the number of excised patch membranes contained a Ca²⁺ store site. Most Ca²⁺ store sites have inositol 1,4,5-trisphosphate (InsP₃)-activated Ca²⁺ release (IACR) and caffeine-activated Ca²⁺ release (CACR) channels and few lack the IACR channel. The mechanisms of activation of the maxi-K⁺ channel in relation to release of Ca²⁺ from the store sites can be examined in detail using the approaches we have described.     

Evidence that Ca/calmodulin-dependent protein kinase mediates the modulation of the Ca2+-dependent K+ current,I AHP,by acetylcholine,but not by glutamate,in hippocampal neurons

Muscarinic and metabotropic glutamate receptor agonists increase the excitability of hippocampal and other cortical neurons by suppressing the Ca²⁺-activated K⁺current,I _AHP, which underlies the slow afterhyperpolarization (AHP) and spike frequency adaptation. We have examined the mechanism of action of a muscarinic agonist (carbachol) and a metabotropic glutamate receptor agonist (1-Aminocyclopentane-trans-1,3-dicarboxylic acid; t-ACPD) onI _AHP in hippocampal CA1 neurons in slices, by using highly specific protein kinase inhibitors. We found that inhibition of protein kinase A (PKA) with the adenosine 3,5-cyclic monophosphate (cAMP) analogue Rp-adenosine-3,5-cyclic phosphorothioate Rp-cAMPS, did not prevent the muscarinic and glutamatergic suppression ofI _AHP. In contrast, two specific peptide inhibitors of Ca²⁺/calmodulin-dependent protein kinase II (CaM-K II), each partially blocked the effect of carbachol, but not the effect of t-ACPD onI _AHP. We conclude that CaM-K II, but not PKA, is involved in mediating the muscarinic suppression ofI _AHP, although other pathways may also contribute. In contrast, neither CaM-K II nor PKA seems to mediate the metabotropic glutamate receptor action onI _AHP.     

Inhibitory M2 muscarinic receptors are upregulated in both axotomized and intact small diameter dorsal root ganglion cells after peripheral nerve injury

Acetylcholine reduces nociceptive input in part by activating inhibitory M2 muscarinic receptors on primary sensory neurons, and acetylcholinesterase inhibitors and muscarinic agonists produce analgesia in humans and animals. M2 muscarinic receptors are upregulated in animals with diabetic neuropathy, but their level of expression and function after peripheral nerve injury has not been previously examined. This study tested, using intracellular Ca(2+) response to membrane depolarization, the effect of the M2 muscarinic receptor agonist bethanechol on individual dorsal root ganglion cells from normal and L5-6 spinal nerve-ligated rats, followed by M2 muscarinic receptor immunostaining. We also examined functional transient receptor potential for vanilloids-1 activity by determining intracellular Ca(2+) response evoked by capsaicin in M2 muscarinic receptor immunoreactive cells. In normal dorsal root ganglion cells, bethanechol inhibited the Ca(2+) response in a concentration-related fashion, and this inhibition was blocked by the M2 muscarinic receptor antagonist gallamine. Cells expressing M2 muscarinic receptors by immunostaining were significantly inhibited by bethanechol, whereas those lacking positive staining were not. The proportion of studied dorsal root ganglion neurons with positive M2 muscarinic receptor staining increased significantly in the injured ipsilateral L5-6 and the uninjured ipsilateral L4 ganglia, but not in the contralateral dorsal root ganglion neurons compared with normals. In contrast, the proportion of neurons responding to capsaicin significantly decreased in the injured ipsilateral L5-6 dorsal root ganglion cells. These results suggest that inhibitory M2 muscarinic receptors are upregulated in small- and medium-sized axotomized dorsal root ganglion neurons and their uninjured neighbors following nerve injury, and may represent an appropriate target for analgesia in this setting.     

Different mechanisms of lysophosphatidylcholine-induced Ca mobilization in N2a mouse and SH-SY5Y human neuroblastoma cells

In mice, lysophosphatidylcholine (LPC) was found to be a physiological substrate of neuropathy target esterase, which is also bound by organophosphates that cause a delayed neuropathy in human and some animals. However, the mechanism responsible for causing the different symptoms in mice and humans that are exposed to neuropathic organophosphates still remains unknown. In the present study, we examined and compared the effect of exogenous LPC on intracellular Ca²⁺ overload in mouse N2a and human SH-SY5Y neuroblastoma cells. LPC caused an intracellular Ca²⁺ level ([Ca²⁺]_i) increase in both N2a and SH-SY5Y cells; moreover, the amplitude was higher in N2a cells than that in SH-SY5Y cells. Preincubation of the cells with verapamil, an L-type Ca²⁺ channel blocker, did not affect the LPC-induced Ca²⁺ increase in N2a cells, verapamil inhibited the response by 23% in SH-SY5Y cells. In Ca²⁺-free medium, LPC produced a significant [Ca²⁺]_i decrease in N2a cells, while it caused 64% of total [Ca²⁺]_i increase in SH-SY5Y cells. The results of a cell viability test suggest that N2a cells were more sensitive to LPC than were SH-SY5Y cells. These data suggested that the LPC-induced [Ca²⁺]_i increase was produced in each cell line through different mechanisms. In particular, the [Ca²⁺]_i increase occurred via entry through a permeabilized membrane in N2a cells, but through L-type Ca²⁺ channels as well as by Ca²⁺ release from intracellular Ca²⁺ stores in SH-SY5Y cells. Thus, the symptomatic differences of organophosphate-induced neurotoxicity between mice and humans are probably not related to the diverse amplitudes of intracellular Ca²⁺ overload produced by LPC. Moreover, the demyelination effect induced by LPC in mice may be a consequence of its detergent effect on membranes.     

G-Protein- and capacitatively regulated Ca2+ entry pathways are activated by muscarinic receptor stimulation in a human submandibular ductal cell line

In the human submandibular ductal cell line (HSG) thapsigargin and carbachol stimulated Ca²⁺ release from the internal Ca²⁺ pool, resulting in the activation of capacitatively regulated Ca²⁺ entry (CRCE). This entry pathway was permeant to both Ca²⁺ and Mn²⁺, blocked by Ni²⁺ and insensitive to the muscarinic antagonist, atropine. Carbachol also stimulated an increase in cytosolic [Ca²⁺] in internal Ca²⁺-pool-depleted (i.e.thapsigargin-treated) cells which was dependent on the presence of external Ca²⁺ and blocked by Ni²⁺, demonstrating that it was due to Ca²⁺ entry. However, under the same experimental conditions, carbachol was unable to stimulate Mn²⁺ entry. Additionally, this latter carbachol-stimulated Ca²⁺ entry pathway was blocked by atropine. Pretreatment of HSG cells with AlF₄-increased basal rates of Mn²⁺ entry due to CRCE activation, but attenuated carbachol-stimulated Ca²⁺ entry into thapsigargin-treated cells. The data suggest that two distinct divalent cation entry pathways are activated in muscarinic-receptor-stimulated HSG cells; a CRCE mechanism, permeable to both Mn²⁺ and Ca²⁺, and a second entry mechanism, permeable only to Ca²⁺. The latter does not depend on internal pool depletion, but appears to be regulated via G-protein activation.We thank Dr. Bruce Baum for his encouragement and support during the course of this work. We also thank our colleagues for their cooperation and assistance.