Kaori Inoue

Ca2+ waves in keratinocytes are transmitted to sensory neurons: the involvement of extracellular ATP and P2Y2 receptor activation.

ATP acts as an intercellular messenger in a variety of cells. In the present study, we have characterized the propagation of Ca2+ waves mediated by extracellular ATP in cultured NHEKs (normal human epidermal keratinocytes) that were co-cultured with mouse DRG (dorsal root ganglion) neurons. Pharmacological characterization showed that NHEKs express functional metabotropic P2Y2 receptors. When a cell was gently stimulated with a glass pipette, an increase in [Ca2+]i (intracellular Ca2+ concentration) was observed, followed by the induction of propagating Ca2+ waves in neighbouring cells in an extracellular ATP-dependent manner. Using an ATP-imaging technique, the release and diffusion of ATP in NHEKs were confirmed. DRG neurons are known to terminate in the basal layer of keratinocytes. In a co-culture of NHEKs and DRG neurons, mechanical-stimulation-evoked Ca2+ waves in NHEKs caused an increase in [Ca2+]i in the adjacent DRG neurons, which was also dependent on extracellular ATP and the activation of P2Y2 receptors. Taken together, extracellular ATP is a dominant messenger that forms intercellular Ca2+ waves in NHEKs. In addition, Ca2+ waves in NHEKs could cause an increase in [Ca2+]i in DRG neurons, suggesting a dynamic cross-talk between skin and sensory neurons mediated by extracellular ATP.

An ATP‐activated conductance in pheochromocytoma cells and its suppression by extracellular calcium.

1. ATP‐activated inward current in PC12 pheochromocytoma cells was characterized using the whole‐cell voltage‐clamp technique. 2. ATP (100 microM) applied extracellular elicited an inward rectifying current with a reversal potential of about +7 mV. The current was desensitized in seconds in spite of continued presence of ATP. 3. A comparison was made of the ability of ATP and its analogues. The order of potency in activating the inward current was ATP greater than ATP gamma S greater than ADP; AMP, adenosine and alpha, beta‐methylene ATP were inactive at concentrations up to mM. 4. The ATP‐activated current was also observed when external Na+ and Ca2+ were replaced with K+, TEA, Tris or glucosamine. The order of ion selectivity was Na+ greater than K+ greater than TEA not equal to Tris greater than glucosamine. 5. The ATP‐activated current was also recorded in extracellular solutions containing Ca2+ as the only external cation. The amplitude increased as the concentration of Ca2+ was increased in the range between 1.8 and 16.2 mM. However, the current amplitude decreased at higher Ca2+ concentrations and the current was not recorded in 110 mM‐Ca2+ solution. 6. In the presence of 140 mM‐Na+ in the external solution, the current amplitude also decreased as the external Ca2+ concentration was increased (from 1.8 to 16.2 mM). 7. The results indicate that Ca2+ as well as monovalent cations permeate through the ATP‐sensitive pathway and that Ca2+ blocks ion permeation, including its own permeation through the pathway. This regulation by extracellular Ca2+ is different to the ATP‐activated current in smooth muscle cells.

Effects of nocistatin on nociceptin-induced impairment of learning and memory in mice.

We investigated the effects of nociceptin/orphanin FQ and nocistatin on learning and memory function as measured in a step-down type passive avoidance task and spontaneous alternation of Y-maze with mice. Nociceptin (0.5-5.0 nmol/mouse, i.c.v.) 30 min before the training session or Y-maze test, dose dependently shortened the step-down latency and impaired spontaneous alternation, while there was no significant effect of nocistatin (0.5-5.0 nmol/mouse). Interestingly, nocistatin (5.0 nmol) significantly improved the nociceptin (5.0 nmol)-induced impairment of learning and memory without changing motor activity or response to electric shocks. These results suggest that nocistatin, a new biologically active peptide now found to also counteract the impairment of learning and memory induced by nociceptin, plays an important role in the regulation of learning and memory process in the central nervous system.

Comparison of adenosine triphosphate‐ and nicotine‐activated inward currents in rat phaeochromocytoma cells.

1. The adenosine triphosphate (ATP)‐activated inward current was compared to the nicotine‐activated inward current in nerve growth factor (NGF)‐treated rat phaeochromocytoma PC12 cells. 2. Both ATP and nicotine activated an inward current at negative holding potentials. The concentration of ATP necessary to activate the inward current was about 10‐fold higher than that of nicotine; the EC50 was 20.5 microM for ATP and 2.4 microM for nicotine. The maximal responses induced by ATP and nicotine were almost identical in the same cells. The current‐voltage relationship for the ATP‐activated current was very similar to that for the nicotine‐activated current, and both currents reversed around 0 mV in a physiological saline. 3. The ATP‐activated current and the nicotine‐activated current were not additive; the current activated by a combined administration of ATP (100 microM) and nicotine (10 microM) was only about 20% larger than the current activated by either ATP or nicotine alone. Nicotine (100 microM) did not increase the current activated by 1 microM‐ATP. 4. ATP could activate an inward current in the cells even after desensitization to nicotine had developed. 5. Hexamethonium (100 microM) selectively blocked the nicotine‐activated current whereas suramin (100 microM), a purinoceptor antagonist, selectively blocked the ATP‐activated current. 6. Ionic selectivity was studied by changing compositions of extracellular solutions. When external Na+ was replaced with Cs+, both ATP and nicotine activated inward currents. However, with an extracellular solution containing Tris or glucosamine as a major cation, only ATP, not nicotine, activated an inward current. 7. ATP‐ and nicotine‐activated currents were also recorded from cells bathed in a solution containing 1.8 mM‐Ca2+ as the only external cation, suggesting that both pathways are Ca2+ permeable. 8. The results suggest that the ATP‐sensitive ionic pathway is not independent of the nicotine‐sensitive pathway in these cells. Our working hypothesis is that ATP and nicotine activate the same channels but the binding sites and the open‐states of the channels are different between these two agonists.

Glutamate-evoked release of adenosine 5'-triphosphate causing an increase in intracellular calcium in hippocampal neurones

ATP evoked an increase in intracellular Ca2+ concentration ([Ca]i) in hippocampal neurons in the presence of tetrodotoxin (TTX, 3 microM), hexamethonium (C6, 100 microM), D-2-amino-phosphonovalerate (APV, 100 microM), 6-cyano-7-nitro quinoxalline-2,3-dione (CNQX, 30 microM), bicuculline (10 microM) and cadmium (Cd2+, 300 microM). The increase was blocked by suramin (100 microM), a P2-purinoceptor blocker. These results suggest that ATP evokes an increase in [Ca]i through P2-purinoceptor. As a result of measuring [Ca]i in multiple cells simultaneously, it was suggested that glutamatergic neurones connect with purinergic neurones and glutamate (10 microM) stimulated the release of ATP causing an increase in [Ca]i in postsynaptic neurones.

Improvement by low doses of nociceptin on scopolamine-induced impairment of learning and/or memory.

The effects of fmol doses of nociceptin/orphanin FQ on scopolamine-induced impairment of learning and/or memory were examined using spontaneous alternation of Y-maze and step-down type passive avoidance tasks. While fmol doses of nociceptin alone had no effect on spontaneous alternation or passive avoidance behavior in normal mice, administration of nociceptin (10 and/or 100 fmol/mouse) 30 min before spontaneous alternation performance or the training session of the passive avoidance task, significantly improved the scopolamine-induced impairment of spontaneous alternation and passive avoidance behavior. This ameliorating effect was not antagonized by nocistatin (0.5 and 5.0 nmol/mouse, i.c.v.), naloxone benzoylhydrazone (2.3, 11.2, and 56.1 micromol/kg, s.c.) or nor-binaltorphimine (4.9 nmol/mouse, i.c.v.). These results indicated that very low doses of nociceptin ameliorate impairments of spontaneous alternation and passive avoidance induced by scopolamine, and suggested that this peptide has bidirectional modulatory effects on learning and memory; impairment at high doses and amelioration at low doses.

Mechanical-stimulation-evoked calcium waves in proliferating and differentiated human keratinocytes

Calcium dynamics in the epidermis play a crucial role in barrier homeostasis and keratinocyte differentiation. We have recently suggested that the electro-physiological responses of the keratinocyte represent the frontier of the skin sensory system for environmental stimuli. In the present study, we have evaluated the responses of proliferating and differentiated human keratinocytes to mechanical stress by measuring the intracellular calcium level. Before differentiation, mechanical stress induces a calcium wave over a limited area; this is completely blocked by apyrase, which degrades ATP. In the case of differentiated keratinocytes, the calcium wave propagates over a larger area. Application of apyrase does not completely inhibit this wave. Thus, in differentiated cells, the induction of calcium waves might involve not only ATP, but also another factor. Immunohistochemical studies indicate that connexins 26 and 43, both components of gap junctions, are expressed in the cell membrane of differentiated keratinocytes. Application of octanol or carbenxolone, which block gap junctions, significantly reduces calcium wave propagation in differentiated keratinocytes. Thus, signaling via gap junctions might be involved in the induction of calcium waves in response to mechanical stress at the upper layer of the epidermis.

Nociceptin/orphanin FQ and nocistatin on learning and memory impairment induced by scopolamine in mice.

Nociceptin, also known as orphanin FQ, is an endogenous ligand for the orphan opioid receptor‐like receptor 1 (ORL1) and involves in various functions in the central nervous system (CNS). On the other hand, nocistatin is recently isolated from the same precursor as nociceptin and blocks nociceptin‐induced allodynia and hyperalgesia. Although ORL1 receptors which display a high degree of sequence homology with classical opioid receptors are abundant in the hippocampus, little is known regarding their role in learning and memory. The present study was designed to investigate whether nociceptin/orphanin FQ and nocistatin could modulate impairment of learning and memory induced by scopolamine, a muscarinic cholinergic receptor antagonist, using spontaneous alternation of Y‐maze and step‐down type passive avoidance tasks in mice. While nocistatin (0.5–5.0 nmol mouse−1, i.c.v.) administered 30 min before spontaneous alternation performance or the training session of the passive avoidance task, had no effect on spontaneous alternation or passive avoidance behaviours, a lower per cent alternation and shorter median step‐down latency in the retention test were obtained in nociceptin (1.5 and/or 5.0 nmol mouse−1, i.c.v.)‐treated normal mice. Administration of nocistatin (1.5 and/or 5.0 nmol mouse−1, i.c.v.) 30 min before spontaneous alternation performance or the training session of the passive avoidance task, attenuated the scopolamine‐induced impairment of spontaneous alternation and passive avoidance behaviours. These results indicated that nocistatin, a new biologically active peptide, ameliorates impairments of spontaneous alternation and passive avoidance induced by scopolamine, and suggested that these peptides play opposite roles in learning and memory.

Enhancement by zinc of ATP-evoked dopamine release from rat pheochromocytoma PC12 cells

The effects of zinc (Zn2+) on ATP-evoked dopamine release was investigated in rat pheochromocytoma PC12 cells. Zn2+ potentiated the dopamine release evoked by 30 microM ATP in a concentration-dependent manner over a concentration range from 3 to 300 microM. High concentration of Zn2+ (> 1 mM) inhibited the release. Zn2+ (10 microM) shifted the concentration-response curve of the ATP-evoked dopamine release to the left without affecting the maximal response. The dopamine release evoked by 40 mM KCl was not affected by Zn2+ (1-100 microM), whereas high concentration of Zn2+ ( > 300 microM) attenuated the response. The dopamine release evoked by 30 microM ATP in the presence of 10 microM Zn2+ were suppressed by suramin (30 microM), an antagonist to P2-purinoceptors, to an extent similar to that in the absence of Zn2+. Zn2+ (1-100 microM) enhanced the ATP-evoked increase in intracellular Ca2+ concentration ([Ca]i) in the cells. The Ca2+ responses to ATP in the presence and absence of Zn2+ were abolished by external Ca(2+)-depletion. Under whole-cell voltage-clamp, Zn2+ (10 microM) augmented by two-fold the peak amplitude of an inward current evoked by 30 microM ATP. Taken together, it is suggested that Zn2+ enhances the ATP-evoked dopamine release by increasing sensitivity to ATP. The enhancement may be due to the augmentation of ATP-gated Ca(2+)-influx, but not due to modulation of cellular machinery downstream to [Ca]i rise. The enhancement of the ATP-mediated responses may underlie modulation by Zn2+ of physiological functions in various types of neuronal cells.

Inhibitory effects of capsaicin on acetylcholine‐evoked responses in rat phaeochromocytoma cells

1 The effects of capsaicin on cellular responses evoked by acetylcholine (ACh) and those by adenosine 5′‐triphosphate (ATP) were investigated in rat phaeochromocytoma PC12 cells. 2 Capsaicin (1 to 30 μm) suppressed dopamine release and the intracellular Ca2+ increase evoked by 100 μm ACh. The concentration‐dependence of the ACh‐evoked release of dopamine was not shifted but the maximal response was reduced by capsaicin. Dopamine release evoked by 80 mm KC1 was also suppressed by capsaicin (3 and 30 μm), but the extent of suppression was smaller than that of the ACh‐evoked release. 3 Under whole‐cell voltage‐clamp, capsaicin (1 to 30 μm) reversibly inhibited the inward current activated by ACh (30 to 300 μm). The inhibition exhibited dependence on the concentration of ACh, and the current activated by a higher concentration of ACh was less inhibited. Voltage‐dependence of block by capsaicin was not observed when it was tested either by applying a ramp pulse during the current activation by ACh or by eliciting the current in cells held at various potentials. 4 High concentrations of capsaicin (30 to 100 μm) enhanced the inward current as well as dopamine release evoked by 30 μm ATP. 5 The results suggest that low concentrations of capsaicin selectively antagonize responses mediated by nicotinic receptor‐channels without affecting those mediated by purinoceptor‐coupled channels. As the antagonism by capsaicin of the ACh‐evoked responses was observed at concentrations as low as 1 μm, the influence on nicotinic receptors should be taken into account when this compound is used as a pharmacological tool to deplete neuropeptides.