Depletion of substance P from rat primary sensory neurons by ATP, an implication of P2X receptor-mediated release of substance P.

Effects of ATP on substance P immunoreactivity were examined in cultured dorsal root ganglion neurons. We found that treatment of dorsal root ganglion neurons with ATP significantly depleted substance P immunoreactivity on the neurites and somata of the neurons. The effects of ATP were significantly inhibited by the purinergic P2 receptor antagonists suramin (30 microM) and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (10 microM). We also showed that ATP-induced depletion of substance P immunoreactivity from dorsal root ganglion neurons depended on the entry of Ca(2+). In a spinal cord slice preparation, we also found the internalization of neurokinin-1/substance P receptors in many dorsal horn neurons following the application of ATP or alpha,beta-methylene-ATP.Together these results indicate that activation of P2X receptors may result in release of substance P from primary afferent neurons.     

Multiple P2 receptors contribute to a transient increase in intracellular Ca2+ concentration in ATP-stimulated rat brown adipocytes

Extracellular ATP in micromolar concentrations evokes a transient elevation in intracellular free Ca(2+) concentration ([Ca(2+)](i)), which arises primarily from a release of Ca(2+) from intracellular stores in rat brown adipocytes. We investigated the mechanisms underlying this transient nature of [Ca(2+)](i) elevation during exposure to ATP by using fura-2 fluorescence measurements together with the P2 receptor antagonists pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) and suramin. Extracellular ATP (10 microM) almost completely depressed the thapsigargin (100 nM)-evoked [Ca(2+)](i) elevation mediated through store-operated Ca(2+) entry. The inhibitory effect of ATP was antagonized by PPADS with IC(50) of 0.7 microM. In the presence of PPADS at concentrations of more than 5 microM, the ATP-induced [Ca(2+)](i) elevation became sustained during the entire duration of the agonist application, although the magnitude of the sustained [Ca(2+)](i) elevation was reduced in a concentration-dependent manner by PPADS with an IC(50) of 200 microM. In contrast, the ATP-induced [Ca(2+)](i) elevation was blocked by suramin in a concentration range similar to that required to antagonize the inhibitory effect of ATP on the store-operated pathway. These results suggest that the [Ca(2+)](i) responses to extracellular ATP in rat brown adipocytes are mediated through the activation of at least two distinct P2 receptors exhibiting different sensitivities to PPADS but similar sensitivities to suramin. Extracellular ATP stimulates the PPADS-resistant P2 receptor to mobilize intracellular Ca(2+) stores, which is probably followed by the activation of store-operated Ca(2+) entry. Extracellular ATP, however, would inhibit this Ca(2+) entry process through the stimulation of the PPADS-sensitive P2-receptor, which may underlie the transient nature of [Ca(2+)](i) elevation in response to extracellular ATP.     

Receptor-mediated facilitation of cell volume regulation by swelling-induced ATP release in human epithelial cells

Osmotic swelling induces the release of intracellular ATP in a number of cell types. In the immediate vicinity of the cell surface, released ATP has been shown to reach a concentration high enough to stimulate P2-purinergic receptors in a human epithelial cell line, Intestine 407. The role of released ATP in the regulatory volume decrease (RVD) after cell swelling was thus studied in Intestine 407 cells. The RVD was suppressed by an ATP hydrolyzing enzyme, apyrase, or by a purinergic receptor antagonist, suramin. Extracellular application of ATP accelerated the RVD rate in a concentration-dependent manner. An increase in the cytosolic free-Ca(2+) concentration was induced by a hypotonic challenge, and the swelling-induced Ca(2+) response was partially suppressed by apyrase or suramin. A rise in cytosolic Ca(2+) was also induced by extracellular application of ATP or UTP, but not ADP, 2-methylthio-ATP or alpha,beta-methylene ATP. The ATP-induced Ca(2+) response was blocked by suramin. Therefore, it is concluded that RVD is facilitated by ATP, which is released upon cell swelling, by augmenting intracellular Ca(2+) rise via the stimulation of purinergic (P2Y(2)) receptors in the human epithelial cell.     

Acute effects of glucocorticoids on ATP-induced Ca2+ mobilization and nitric oxide production in cochlear spiral ganglion neurons

Rapid, non-genomic effects of glucocorticoids on extracellular adenosine 5'-triphosphate (ATP)-induced intracellular Ca(2+) concentration ([Ca(2+)](i)) changes and nitric oxide (NO) production were investigated in type I spiral ganglion neurons (SGNs) of the guinea-pig cochlea using the Ca(2+)-sensitive dye fura-2 and the NO-sensitive dye 4,5-diaminofluorescein (DAF-2). Pretreatment of SGNs with 1 microM dexamethasone for 10 min, a synthetic glucocorticoid hormone, enhanced the ATP-induced [Ca(2+)](i) increase in SGNs. RU 38486, a competitive glucocorticoid receptor antagonist eliminated the effects of dexamethasone on the ATP-induced [Ca(2+)](i) increase in SGNs. These acute effects of dexamethasone were dependent on the presence of extracellular Ca(2+), thereby suggesting that dexamethasone may rapidly enhance the Ca(2+) influx through the activation of ionotropic P2X receptors which may interact with glucocorticoid-mediated membrane receptors. Extracellular ATP increased the intensity of DAF-2 fluorescence, indicating NO production in SGNs. The ATP-induced NO production was mainly due to the Ca(2+) influx through the activation of P2 receptors. S-nitroso-N-acetylpenicillamine, a NO donor, enhanced the ATP-induced [Ca(2+)](i) increase in SGNs while L-N(G)-nitroarginine methyl ester (L-NAME), a NO synthesis inhibitor, inhibited it. Dexamethasone enhanced the ATP-induced NO production in SGNs. The augmentation of dexamethasone on ATP-induced NO production was abolished in the presence of l-NAME. It is concluded that the ATP-induced [Ca(2+)](i) increase induces NO production which enhances a [Ca(2+)](i) increase in SGNs by a positive-feedback mechanism. Dexamethasone enhances the ATP-induced [Ca(2+)](i) increase in SGNs which results in the augmentation of NO production. The present study suggests that NO may play an important role in auditory signal transduction. Our results also indicate that glucocorticoids may rapidly affect auditory neurotransmission due to a novel non-genomic mechanism.     

Signaling mechanisms of down-regulation of voltage-activated Ca2+ channels by transient receptor potential vanilloid type 1 stimulation with olvanil in primary sensory neurons

Olvanil ((N-vanillyl)-9-oleamide), a non-pungent transient receptor potential vanilloid type 1 agonist, desensitizes nociceptors and alleviates pain. But its molecular targets and signaling mechanisms are little known. Calcium influx through voltage-activated Ca(2+) channels plays an important role in neurotransmitter release and synaptic transmission. Here we determined the effect of olvanil on voltage-activated Ca(2+) channel currents and the signaling pathways in primary sensory neurons. Whole-cell voltage-clamp recordings were performed in acutely isolated rat dorsal root ganglion neurons. Olvanil (1 microM) elicited a delayed but sustained inward current, and caused a profound inhibition (approximately 60%) of N-, P/Q-, L-, and R-type voltage-activated Ca(2+) channel current. Pretreatment with a specific transient receptor potential vanilloid type 1 antagonist or intracellular application of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid abolished the inhibitory effect of olvanil on voltage-activated Ca(2+) channel current. Calmodulin antagonists (ophiobolin-A and calmodulin inhibitory peptide) largely blocked the effect of olvanil and capsaicin on voltage-activated Ca(2+) channel current. Furthermore, calcineurin (protein phosphatase 2B) inhibitors (deltamethrin and FK-506) eliminated the effect of olvanil on voltage-activated Ca(2+) channel current. Notably, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, calmodulin antagonists, and calcineurin inhibitors each alone significantly increased the amplitude of voltage-activated Ca(2+) channel current. In addition, double immunofluorescence labeling revealed that olvanil induced a rapid internalization of Ca(V)2.2 immunoreactivity from the membrane surface of dorsal root ganglion neurons. Collectively, this study suggests that stimulation of non-pungent transient receptor potential vanilloid type 1 inhibits voltage-activated Ca(2+) channels through a biochemical pathway involving intracellular Ca(2+)-calmodulin and calcineurin in nociceptive neurons. This new information is important for our understanding of the signaling mechanisms of desensitization of nociceptors by transient receptor potential vanilloid type 1 analogues and the feedback regulation of intracellular Ca(2+) and voltage-activated Ca(2+) channels in nociceptive sensory neurons.     

ATP-inhibition of M current in frog sympathetic neurons involves phospholipase C but not Ins P(3), Ca(2+), PKC,or Ras

Suppression of the voltage-activated, noninactivating K(+) conductance (M conductance; g(M)) by muscarinic agonists, P(2Y) agonists or bradykinin increases neuronal excitability. All agonist effects are mediated, at least in part, via the Gq/(11) class of G protein. We found, using whole cell or perforated patch recording from bullfrog sympathetic B neurons that ATP-induced suppression of g(M) was attenuated by the phospholipase C (PLC) inhibitor, U73122 (IC(50) approximately 0.14 microM) but not by the inactive isomer, U73343. The ability of extracellularly applied U73122 to inhibit PLC was confirmed by its antagonism of ATP-induced elevation of intracellular Ca(2+) as measured by fura-2 photometry. ATP-induced g(M) suppression was not antagonized by the protein kinase C (PKC) inhibitor, chelerythrine (5 microM extracellular +10 microM intracellular), by the Ca(2+)-ATPase inhibitor, thapsigargin (5 microM), or by inositol trisphosphate (InsP(3)) receptor antagonists, heparin (approximaterly 300 microM) or xestospongin C (1.8 microM). The effect of ATP on g(M) was thus dependent on PLC yet independent of PKC and of InsP(3)-induced release of intracellular Ca(2+). We therefore tested the involvement of a PKC-independent action of diacylglycerol (DAG) that could occur via activation of Ras. This low-molecular-weight G protein is activated following DAG binding to Ras-GRP, a neuronal Ras-GTP exchange factor. However, impairment of Ras function by culturing neurons with isoprenylation inhibitors (perillic acid, 0.1 mM, or alpha-hydroxyfarnesyl-phosphonic acid, 10 microM) failed to affect ATP-induced g(M) suppression. Inhibition of MEK (mitogen-activated protein kinase), a downstream target of Ras, by using PD 98059 (10 microM) was also ineffective. The transduction mechanism used by ATP to suppress g(M) in frog sympathetic neurons therefore differs from the PLC-independent mechanism used by muscarine and from the PLC and Ca(2+)-dependent mechanism used by bradykinin and UTP in mammalian ganglia. The possibility remains that "lipid-signaling" mechanisms, perhaps involving PLC-induced depletion of phosphatidylinositol bisphosphate, are involved in PLC-mediated inhibition of g(M) by ATP in amphibian sympathetic neurons.     

Effects of extracellular atp on axonal transport in cultured mouse dorsal root ganglion neurons

In primary sensory neurons, extracellular ATP plays important roles in nociception and afferent neurotransmission. Here we investigated the effects of ATP on axonal transport in cultured adult mouse dorsal root ganglion neurons using video-enhanced microscopy. Continuous application (26 min) of ATP (100 microM) significantly increased axonal transport of membrane-bound organelles in anterograde and retrograde directions. All neurons tested (n=5) responded to ATP. The number of transported organelles per min began to increase within 2 min and peaked at 11-14 min after the start of ATP application, and thereafter gradually declined. The peak values in both directions were approximately 140% of the initial values before application. The P2 receptor antagonist suramin (1 mM) completely blocked the effect of ATP. Uridine 5'-triphosphate (UTP; 100 microM) produced a similar effect to ATP, with peak values at 11 min reaching 140% in both directions (n=6). ADP (100 microM; n=5), alpha,beta-methylene ATP (100 microM; n=6), or 2-methylthio ATP (100 microM; n=5) had no effect on axonal transport.Our findings indicate that extracellular ATP is able to increase axonal transport in primary sensory neurons. The equal potency of ATP and UTP with no detectable response to ADP, alpha,beta-methylene ATP, or 2-methylthio ATP suggests the possible involvement of P2Y(2) receptors. Extracellular ATP may play an important role in the modulation of axonal transport in sensory neurons.     

Estradiol inhibits atp-induced intracellular calcium concentration increase in dorsal root ganglia neurons

Estrogen has been implicated in modulation of pain processing. Although this modulation occurs within the CNS, estrogen may also act on primary afferent neurons whose cell bodies are located within the dorsal root ganglia (DRG). Primary cultures of rat DRG neurons were loaded with Fura-2 and tested for ATP-induced changes in intracellular calcium concentration ([Ca(2+)](i)) by fluorescent ratio imaging. ATP, an algesic agent, induces [Ca(2+)](i) changes via activation of purinergic 2X (P2X) type receptors and voltage-gated Ca(2+) channels (VGCC). ATP (10 microM) caused increased [Ca(2+)](i) transients (226.6+/-16.7 nM, n = 42) in 53% of small to medium DRG neurons. A 5-min incubation with 17 beta-estradiol (100 nM) inhibited ATP-induced [Ca(2+)](i) (164+/-14.6 nM, P<0.05) in 85% of the ATP-responsive DRG neurons, whereas the inactive isomer 17 alpha-estradiol had no effect. Both the mixed agonist/antagonist tamoxifen (1 microM) and specific estrogen receptor antagonist ICI 182780 (1 microM) blocked the estradiol inhibition of ATP-induced [Ca(2+)](i) transients. Estradiol coupled to bovine serum albumin, which does not diffuse through the plasma membrane, blocked ATP-induced [Ca(2+)](i), suggesting that estradiol acts at a membrane-associated estrogen receptor. Attenuation of [Ca(2+)](i) transients was mediated by estrogen action on VGCC. Nifedipine (10 microM), an L-type VGCC antagonist mimicked the effect of estrogen and when co-administered did not increase the estradiol inhibition of ATP-induced [Ca(2+)](i) transients. N- and P-type VGCC antagonists omega-conotoxin GVIA (1 microM) and omega-agatoxin IVA (100 nM), attenuated the ATP-induced [Ca(2+)](i) transients. Co-administration of these blockers with estrogen induced a further decrease of the ATP-induced [Ca(2+)](i) flux. Together, these results suggest that although ATP stimulation of P2X receptors activates L-, N-, and P-type VGCC, estradiol primarily blocks L-type VGCC. The estradiol regulation of this ATP-induced [Ca(2+)](i) transients suggests a mechanism through which estradiol may modulate nociceptive signaling in the peripheral nervous system.     

Stimulation of mouse cultured sympathetic neurons by uracil but not adenine nucleotides

Cultured neurons from the paravertebral sympathetic chain of rats possess excitatory P2X as well as excitatory uracil nucleotide-sensitive P2Y receptors. Preliminary observations had indicated that the analogous neurons of mice lacked P2X receptors. This difference was now investigated. Thoracolumbar sympathetic neurons from one- to three-day-old mice were cultured for seven days. When the neurons were preincubated with [3H]noradrenaline and then superfused, ATP failed to cause any change in tritium outflow. UTP (3-300 microM) and UDP (30-100 microM), in contrast, caused marked increases, and so did nicotine (3-100 microM). The effect of UTP was not changed by suramin but abolished by tetrodotoxin and in the absence of calcium. The effect of nicotine was antagonized by hexamethonium and also abolished by tetrodotoxin and in the absence of calcium. Pre-exposure to UDP prevented the effect of UTP. In neurons studied by means of whole-cell patch-clamp techniques under current clamp, ATP lacked any effect. UTP (100 microM), UDP (100 microM) and nicotine (10 microM) caused depolarization accompanied by action potentials. Pre-exposure to UDP prevented the effect of UTP. In neurons studied under voltage clamp, ATP, UTP and UDP failed to cause any detectable current. Nicotine (10 microM), in contrast, elicited inward currents. Neither UTP nor UDP reduced the M-type potassium outward current. These results demonstrate a pronounced difference between cultured sympathetic neurons from the mouse and the rat paravertebral chain. Neurons from both species possess the nicotinic acetylcholine receptor. Neurons from both species also possess uracil nucleotide-sensitive P2Y receptors which, when activated, mediate depolarization, action potential firing and noradrenaline release; these effects are not due to inhibition of M-type potassium channels. Only the rat but not the mouse neurons, however, possess P2X receptors which, when activated, mediate cation entry, depolarization, action potential generation and transmitter release. The absence of functional P2X receptors makes the mouse neurons suitable for further study of the uracil nucleotide-sensitive P2Y receptors.     

NPC-14686, a novel anti-inflammatory agent, increased intracellular Ca(2+) concentrations in MDCK renal tubular cells

The effect of NPC-14686 (Fmoc-L-homophenylalanine), a novel anti-inflammatory agent on intracellular free Ca(2+) concentrations ([Ca(2+)](i)) in Madin Darby canine kidney (MDCK) renal tubular cells, was investigated, using fura-2 as a Ca(2+) dye. At concentrations between 10 and 200 microM NPC-14686 increased [Ca(2+)](i) concentration dependently. The [Ca(2+)](i) signal comprised an initial rise and a sustained phase. Ca(2+) removal inhibited the Ca(2+) signals by 90%. In Ca(2+)-free medium, pretreatment with 100 microM NPC-14686 nearly abolished the [Ca(2+)](i) increase induced by 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor) and abolished the [Ca(2+)](i) increase induced by 2 microM carbonylcyanide m-chlorophenylhydrazone (CCCP) (a mitochondrial uncoupler). NPC-14686 (100 microM) induced a slight [Ca(2+)](i) increase after pretreatment with 2 microM CCCP and 1 microM thapsigargin. Addition of 3 mM Ca(2+) elicited a [Ca(2+)](i) increase in cells pretreated with 100 microM NPC-14686 in Ca(2+)-free medium. Inhibition of inositol-1,4,5-trisphosphate (IP(3)) production by suppressing phospholipase C with 2 microM U73122 did not alter NPC-14686-induced Ca(2+) release. Trypan blue exclusion revealed that incubation with 10 or 200 microM NPC-14686 for 1-30 min decreased cell viability by 10-20% concentration dependently. Collectively, the results demonstrate that, in MDCK tubular cells, NPC-14686 induced Ca(2+) release followed by Ca(2+) entry, with the latter playing a major role. NPC-14686 appears to release intracellular Ca(2+) in an IP(3)-uncoupled manner. NPC-14686 may be of mild cytotoxicity.     

ATP acting on P2Y receptors triggers calcium mobilization in primary cultures of rat neurohypophysial astrocytes (pituicytes)

 The effect of adenosine triphosphate (ATP) on the intracellular Ca²⁺ concentration ([Ca²⁺]_i) of cultured neurohypophysial astrocytes (pituicytes) was studied by fluorescence videomicroscopy. ATP evoked a [Ca²⁺]_i increase, which was dose dependent in the 2.5–50 μM range (EC₅₀=4.3 μM). The ATP-evoked [Ca²⁺]_i rise was not modified during the first minute following the removal of external Ca²⁺. Application of 500 nM thapsigargin inhibited the ATP-dependent [Ca²⁺]_i increase. Caffeine (10 mM) and ryanodine (1 μM) did not affect the ATP-induced [Ca²⁺]_i rise. The pituicytes responded to various P₂ purinoceptor agonists with the following order of potency: ATP=ATP[γ-S]=2-MeSATP≥ADP, where ATP[γ-S] is adenosine 5′-O-(3-thiotriphosphate) and 2-MeSATP is 2-methylthio-adenosine-5′-triphosphate. Adenosine, AMP, α,β-methylene adenosine-5′-triphosphate (α,β-MeATP), β,γ methylene adenosine-5′-triphosphate (β,γ-MeATP) and uridine 5′-triphosphate (UTP) were ineffective. The P₂ purinoceptor antagonists blocked the ATP-evoked [Ca²⁺]_i increase with the following selectivity: RB-2>suramin>PPADS, where RB-2 is Reactive Blue 2 and PPADS is pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid. The ATP-evoked [Ca²⁺]_i increase was substantially blocked by pertussis toxin treatment, suggesting that it might be mediated by a pertussis-toxin-sensitive G protein. The phospholipase C (PLC) inhibitor U-73122 (0.5 μM) abolished the ATP-evoked [Ca²⁺]_i rise, whereas its inactive stereoisomer U-73343 (0.5 μM) remained ineffective. Our results indicate that, in rat cultured pituicytes, ATP stimulation induces an increase in [Ca²⁺]_i due to PLC-mediated release from intracellular stores through activation of a pertussis-toxin-sensitive, G-protein-linked P_2Y receptor. Received: 24 September 1998 / Received after revision: 10 December 1998 / Accepted: 18 December 1998     

Melatonin inhibits high voltage activated calcium currents in cultured rat dorsal root ganglion neurones.

The actions of melatonin on high-voltage activated calcium channels (HVACC) and intracellular free Ca(2+) concentration in cultured dorsal root ganglion (DRG) neurones from neonatal rats were investigated using the whole-cell patch clamp and the fura-2 fluorescence ratio Ca(2+)-imaging techniques. HVACC were pharmacologically and biophysically isolated and the effects of melatonin were investigated. Extracellular application of melatonin inhibited HVACC in a dose dependent manner. In calcium imaging experiments, application of extracellular recording medium containing 30 mM KCl evoked increases in intracellular free Ca(2+) that were dependent upon external Ca(2+) ions. This increase was prevented by both low (10 microM) and high dose (100 microM) of melatonin pre-treatment. The results of this study indicate that the pineal hormone melatonin has inhibitory actions on voltage dependent calcium entry in cultured rat DRG neurones.     

Localized IP3-evoked Ca2+ release activates a K+ current in primary vagal sensory neurons

Electrophysiological and microfluorimetric techniques were used to determine whether intracellular photorelease of caged IP(3), and the consequent release of Ca(2+), could trigger a Ca(2+)-activated K(+) current (I(IP3)). Photorelease of caged IP(3) evoked an I(IP3) that averaged 2.36 +/- 0.35 (SE) pA/pF in 24 of 28 rabbit primary vagal sensory neurons (nodose ganglion neurons, NGNs) voltage-clamped at -50 mV. I(IP3) was abolished by intracellular BAPTA (2 mM), a Ca(2+) chelator. Changing the K(+) equilibrium potential by increasing extracellular K(+) ion concentration caused a predicted Nernstian shift in the reversal potential of I(IP3). These results indicated that I(IP3) was a Ca(2+)-dependent K(+) current. I(IP3) was unaffected by three common antagonists of Ca(2+)-activated K(+) currents: bath-applied iberiotoxin (50 nM) or apamin (100 nM), and intracellular 8-Br-cAMP (100 microM) included in the patch pipette. We have previously demonstrated that both IP(3)-evoked Ca(2+) release and Ca(2+)-induced Ca(2+) release (CICR) are co-expressed in NGNs and that CICR can trigger a Ca(2+)-activated K(+) current. In the present study, using caffeine, a CICR agonist, to selectively attenuate intracellular Ca(2+) stores, we showed that IP(3)-evoked Ca(2+) release occurs independently of CICR, but interestingly, that a component of I(IP3) requires CICR. These data suggest that IP(3)-evoked Ca(2+) release activates a K(+) current that is pharmacologically distinct from other Ca(2+)-activated K(+) currents in NGNs. We describe several models that explain our results based on Ca(2+) signaling microdomains in NGNs.     

Calcium release from internal stores is required for the generation of spontaneous hyperpolarizations in dopaminergic neurons of neonatal rats

We recently have demonstrated the existence of spontaneous hyperpolarizations in midbrain dopaminergic neurons of neonatal but not adult rats. These events are mediated by the opening of apamin-sensitive K(+) channels after a rise in the intracellular concentration of Ca(2+). They are resistant to tetrodotoxin in most cases and are probably endogenous (i.e., not synaptically activated). Here their mechanism was investigated. Cyclopiazonic acid (10 microM), a specific inhibitor of endoplasmic reticulum Ca(2+) ATPases, reversibly abolished the events. Caffeine, which promotes Ca(2+) release from intracellular stores, had concentration-dependent effects. At 1 mM, it markedly and steadily increased the frequency and the amplitude of the hyperpolarizations. At 10 mM, it induced a transient increase in their frequency followed by their cessation. All these effects were quickly reversible. Ryanodine (10 microM), which decreases the conductance of Ca(2+) release channels, irreversibly blocked the spontaneous hyperpolarizations. Dantrolene (100 microM), a blocker of Ca(2+) release from sarcoplasmic reticulum of striated muscle, did not affect the events. On the other hand, Cd(2+) (100-300 microM), a broad antagonist of membrane voltage-gated Ca(2+) channels, significantly reduced the amplitude and the frequency of the hyperpolarizations. However, when the frequency of the events was increased by 1 mM caffeine, Cd(2+) affected them to a smaller extent, whereas cyclopiazonic acid still abolished them. We conclude that internal stores are the major source of Ca(2+) ions that induce the K(+) channel openings underlying the spontaneous hyperpolarizations of these neurons.     

Comparison of intracellular calcium signals evoked by heat and capsaicin in cultured rat dorsal root ganglion neurons and in a cell line expressing the rat vanilloid receptor, VR1

The cloning of the receptor for capsaicin, vanilloid receptor 1, has shown it to be non-selective cation channel with a high calcium permeability which can be opened by noxious heat as well as capsaicin. Here we compare the calcium signals produced by native and recombinant capsaicin receptors when activated by either heat or capsaicin by imaging intracellular calcium levels ([Ca2+](i)) in rat dorsal root ganglion neurons and Chinese hamster ovary cells transfected with the rat vanilloid receptor, vanilloid receptor 1. Vanilloid receptor 1 transfected cells and a subset of dorsal root ganglion neurons responded to both capsaicin and to heating to 50 degrees C with rapid, substantial and reversible rises in [Ca2+](i). All except one of the dorsal root ganglion neurons responsive to capsaicin also showed sensitivity to heat, and most, but not all, heat-sensitive neurons also responded to capsaicin. Both capsaicin and heat responses were dependent on the presence of extracellular Ca2+. Non-transfected Chinese hamster ovary cells and non-responsive dorsal root ganglion neurons showed only small rises in [Ca2+](i) in response to heat which did not depend on the presence of external Ca2+. Responsive dorsal root ganglion neurons and vanilloid receptor 1 transfected cells showed a clear temperature threshold, above which [Ca2+](i) increased rapidly. This was estimated to be 42.6+/-0.3 degrees C for vanilloid receptor 1 transfected cells and 42.0+/-0.6 degrees C for dorsal root ganglion neurons. The competitive capsaicin antagonist capsazepine (10microM) abolished [Ca2+](i) increases stimulated by capsaicin in both dorsal root ganglion neurons and vanilloid receptor 1 transfected cells. However, responses to heat of a similar magnitude in the same cells were inhibited by only 37% by capsazepine (10microM). In vanilloid receptor 1 transfected cells, Ruthenium Red (10microM) blocked responses to both capsaicin and heat. These results demonstrate that imaging of [Ca2+](i) can identify dorsal root ganglion neurons which are responsive to both heat and capsaicin. They show that heat and capsaicin responses mediated by native and recombinant capsaicin receptors are similar with respect to the characteristics and pharmacology examined, suggesting that expression of recombinant vanilloid receptor 1 in cell lines accurately reproduces the properties of the native receptor.     

Ca(2+)-independent but voltage-dependent secretion in mammalian dorsal root ganglion neurons

We have investigated the Ca(2+) dependence of vesicular secretion from the soma of dorsal root ganglion (DRG) neurons, which secrete neuropeptides by exocytosis of dense-core vesicles. In patch-clamped somata of rat DRG neurons, we found a depolarization-induced membrane capacitance increase (DeltaC(m)) in the absence of extracellular Ca(2+) and in the presence of a Ca(2+) chelator (BAPTA) in the intracellular solution. Depletion of internal Ca(2+) stores by thapsigargin in the Ca(2+)-free bath also did not block the DeltaC(m), indicating that Ca(2+) release from internal Ca(2+) stores may not have been involved. Furthermore, the Ca(2+)-independent DeltaC(m) was blocked by whole-cell dialysis with tetanus toxin and was accompanied by pulsatile secretion of false transmitters, as detected by amperometric measurements. These results indicate the existence of Ca(2+)-independent but voltage-dependent vesicular secretion (CIVDS) in a mammalian sensory neuron.     

Ca2+ influx, but not Ca2+ release from internal stores, is required for the PACAP-induced increase in excitability in guinea pig intracardiac neurons

Mechanisms modulating the pituitary adenylate cyclase activating polypeptide (PACAP)-induced increase in excitability have been studied using dissociated guinea pig intrinsic cardiac neurons and intact ganglion preparations. Measurements of intracellular calcium (Ca2+) with the fluorescent Ca2+ indicator dye fluo-3 indicated that neither PACAP nor vasoactive intestinal polypeptide (VIP) at either 100 nM or 1 microM produced a discernible elevation of intracellular Ca2+ in dissociated intracardiac neurons. For neurons in ganglion whole mount preparations kept in control bath solution, local application of PACAP significantly increased excitability, as indicated by the number of action potentials generated by long depolarizing current pulses. However, in a Ca2+ -deficient solution in which external Ca2+ was replaced by Mg2+ or when cells were bathed in control solution containing 200 microM Cd2+, PACAP did not enhance action potential firing. In contrast, in a Ca2+ -deficient solution with Ca2+ replaced by strontium (Sr2+), PACAP increased excitability. PACAP increased excitability in cells treated with a combination of 20 microM ryanodine and 10 mM caffeine to interrupt release of Ca2+ from internal stores. Experiments using fluo-3 showed that ryanodine/caffeine pretreatment eliminated subsequent caffeine-induced Ca2+ release from intracellular stores, whereas exposure to the Ca2+ -deficient solution did not. In dissociated intracardiac neurons voltage clamped with the perforated patch recording technique, 100 nM PACAP decreased the voltage-dependent barium current (IBa). These results show that, in the guinea pig intracardiac neurons, the PACAP-induced increase in excitability apparently requires Ca2+ influx through Cd2+ -sensitive calcium permeable channels other than voltage-dependent Ca2+ channels, but not Ca2+ release from internal stores.     

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.     

Cooperativity between extracellular adenosine 5'-triphosphate and activation of N-methyl-D-aspartate receptors in long-term potentiation induction in hippocampal CA1 neurons

The mechanism of ATP-induced long-term potentiation (LTP) was studied pharmacologically using guinea-pig hippocampal slices. LTP, induced in CA1 neurons by 10 min application of 10 microM ATP, was blocked by co-application of the N-methyl-D-aspartate (NMDA) receptor antagonist, D,L-2-amino-5-phosphonovalerate (5 or 50 microM). In ATP-induced LTP, the delivery of test synaptic inputs (once every 20 s) to CA1 neurons could be replaced by co-application of NMDA (100 nM) during ATP perfusion. These results suggest that, in CA1 neurons, a co-operative effect between extracellular ATP and activation of NMDA receptors is required to trigger the process involved in ATP-induced LTP. In addition, ATP-induced LTP was blocked by co-application of an ecto-protein kinase inhibitor, K-252b (40 or 200 nM), whereas a P2X purinoceptor antagonist, pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid 4-sodium (50 microM), or a P2Y purinoceptor antagonist, basilen blue (10 microM), had no effect.The results of the present study, therefore, indicate that the mechanisms of ATP-induced LTP involve the modulation of NMDA receptors/Ca(2+) channels and the phosphorylation of extracellular domains of synaptic membrane proteins, one of which could be the NMDA receptor/Ca(2+) channel.     

ATP-induced Ca2+ response mediated by P2U and P2Y purinoceptors in human macrophages: signalling from dying cells to macrophages

The activation of macrophages by various stimuli leading to chemotactic migration and phagocytosis is known to be mediated by an increase in intracellular Ca2+ concentration ([Ca2+]i). We measured changes in [Ca2+]i using a Ca2+ imaging method in individual human macrophages differentiated from freshly prepared peripheral blood monocytes during culture of 1-2 days. A transient rise in [Ca2+]i (duration 3-4 min) occurred in 10-15 macrophages in the vicinity of a single tumour cell that was attacked and permeabilized by a natural killer cell in a dish. Similar Ca2+ transients were produced in 90% of macrophages by application of supernatant obtained after inducing the lysis of tumour cells with hypo-osmotic treatment. Ca2+ transients were also evoked by ATP in a dose-dependent manner between 0.1 and 100 microm. The ATP-induced [Ca2+]i rise was reduced to less than one-quarter in Ca2+-free medium, indicating that it is mainly due to Ca2+ entry and partly due to intracellular Ca2+ release. UTP (P2U purinoceptor agonist) was more potent than ATP or 2-chloro-ATP (P2Y agonist). Oxidized ATP (P2Z antagonist) had no inhibitory effect. Both cell lysate- and ATP-induced Ca2+ responses were inhibited by Reactive Blue 2 (P2Y and P2U antagonist) to the same extent, but were not affected by PPADS (P2X antagonist). Sequential stimuli by cell lysate and ATP underwent long-lasting desensitization in the Ca2+ response to the second stimulation. The present study supports the view that macrophages respond to signal messengers discharged from damaged or dying cells to be ingested, and ATP is at least one of the messengers and causes a [Ca2+]i rise via P2U and P2Y receptors.