Hironori Nakanishi

A novel antagonist, No. 7943, of the Na+/Ca2+ exchange current in guinea-pig cardiac ventricular cells

1 The effects of No. 7943 on the Na+/Ca2+ exchange current and on other membrane currents were investigated in single cardiac ventricular cells of guinea‐pig with the whole‐cell voltage‐clamp technique. 2 No. 7943 at 0.1–10 μm suppressed the outward Na+/Ca2+ exchange current in a concentration‐dependent manner. The suppression was reversible and the IC50 value was approximately 0.32 μm. 3 No. 7943 at 5–50 μm suppressed also the inward Na+/Ca2+ exchange current in a concentration‐dependent manner but with a higher IC50 value of approximately 17 μm. 4 In a concentration‐response curve, No. 7943 raised the KmCa2+ value, but did not affect the Imax value, indicating that No. 7943 is a competitive antagonist with external Ca2+ for the outward Na+/Ca2+ exchange current. 5 The voltage‐gated Na+ current, Ca2+ current and the inward rectifier K+ current were also inhibited by No. 7943 with IC50s of approximately 14, 8 and 7 μm, respectively. 6 In contrast to No. 7943, 3′,4′‐dichlorobenzamil (DCB) at 3–30 μm suppressed the inward Na+/Ca2+ exchange current with IC50 of 17 μm, but did not affect the outward exchange current at these concentrations. 7 We conclude that No. 7943 inhibits the outward Na+/Ca2+ exchange current more potently than any other currents as a competitive inhibitor with external Ca2+. This effect is in contrast to DCB which preferentially inhibits the inward rather than the outward Na+/Ca2+ exchange current.

Differential Expression of Type I, II, and V Adenylyl Cyclase Gene in the Postnatal Developing Rat Brain

Abstract: The developmental changes in the expression of mRNA encoding three major brain adenylyl cyclase (AC; EC 4.6.1.1) subtypes, type I (AC1), II (AC2), and V (AC5), were examined by in situ hybridization in rat brain from neonate to adult. During the early postnatal stage, levels of AC1 transcripts were very high in the cerebral cortex, striatum, thalamus, brainstem, and inferior colliculus. Then, AC1 mRNA levels rapidly decreased to the levels observed in the adult brain. In contrast, AC1 transcripts were very low at the early postnatal stage in the cerebellum and hippocampus and markedly increased during the second postnatal week. AC2 mRNA was widely distributed in rat brain throughout the development, and levels did not vary with different ages of the animal. AC5 mRNA was expressed to a limited extent in the neonatal brain, but levels dramatically increased during the second postnatal week in restricted regions, including the striatum, nucleus accumbens, and olfactory tubercle. The developing profiles of three AC gene transcripts were confirmed by northern blot analyses with mRNA isolated from different brain regions at different postnatal stages. In addition, the basal and forskolin‐, GTPγS‐, or Ca2+/calmodulin‐stimulated AC activity in plasma membrane preparations obtained from different brain regions at different ages were correlated with the age‐dependent changes in the region‐specific AC mRNA levels. These results demonstrate that different AC subtypes are expressed in the developing rat brain in a region‐ and age‐specific manner, suggesting specific roles not only in the synaptic transmission but also in the differentiation and maturation of neuronal cells in the developing brain.

Thromboxane A2 activates phospholipase C in astrocytoma cells via pertussis toxin-insensitive G-protein

The properties of thromboxane A2 (TXA2) receptors were examined in 1321N1 human astrocytoma cells. 9,11-Epithio-11,12-methanothromboxane A2 (STA2), a stable analogue of TXA2, stimulated the accumulation of inositol phosphates (IPs) with an EC50 of about 50 nM. The STA2-induced accumulation of IPs was inhibited concentration dependently by ONO3708, a TXA2 receptor antagonist, with an inhibition constant (Ki) of about 10 nM. Inositol trisphosphate (IP3) was accumulated more rapidly than inositol bisphosphate (IP2) in response to STA2. HPLC analysis indicated that inositol 1,4,5-trisphosphate accumulated in the presence of STA2. STA2 alone had no effect on the accumulation of IPs in membrane preparations but it potentiated the accumulation induced by GTP gamma S. [3H]SQ29548, a TXA2 receptor antagonist, bound specifically to TXA2 receptors, expressing a single binding site with a dissociation constant (Kd) of 10.9 nM. The competition curve for STA2 inhibition of [3H]SQ29548 binding was shifted to the right and was steeper in the presence of GTP gamma S. Pertussis toxin (IAP) elicited ADP-ribosylation of 41KD protein but had no effect on the sensitivity to GTP of the STA2 inhibition of SQ29548 binding or of STA2-induced accumulation of IPs. It is concluded from these results that the stimulation of TXA2 receptors results in activation of phospholipase C via a GTP binding protein and that the protein is not a substrate for IAP.

The presence of thromboxane A2 receptors in cultured astrocytes from rabbit brain

We have previously shown that human astrocytoma cells (1321N1) express thromboxane A2 (TXA2) receptors, of which stimulation activates phosphoinositide hydrolysis (Nakahata et al., Eur. J. Pharmacol. 162 (1989) 407). In order to examine whether TXA2 receptors exist in native astrocytes or not, rabbit cultured astrocytes were used. Glial fibrillary acidic protein (GFAP)-positive astrocytes were obtained three weeks after culture of brain. [3H]ONO NT-126, a TXA2 antagonist, bound to the membranes derived from cultured rabbit astrocytes with the dissociation constant (Kd) of 0.23 nM and the maximum binding site (Bmax) of 69.5 fmol/mg protein. STA2, a stable TXA2 receptor agonist, activates phosphoinositide hydrolysis in a concentration-dependent manner, and S-145, a TXA2 antagonist, inhibited STA2-induced phosphoinositide hydrolysis. The results indicate that TXA2 receptors exist in cultured rabbit astrocytes and the activation of TXA2 receptors results in phosphoinositide hydrolysis.

Mastoparan inhibits phosphoinositide hydrolysis via pertussis toxin-intensive G-protein in human astrocytoma cells

Mastoparan inhibited [3H]inositol phosphate accumulation induced by carbachol as well as cyclic AMP accumulation induced by isoproterenol in 1321N1 human astrocytoma cells. Mastoparan inhibited GTPγS‐induced, but not Ca2+‐induced, [3H]inositol phosphate accumulation in membrane preparations with an IC 50 of approximately 10 μM. The inhibitory effect of mastoparan on carbachol‐induced [3pH]inositol phosphate accumulation was resistant to pertussis toxin (IAP) treatment in intact cells. These results suggest that mastoparan inhibits phospholipase C in human astrocytoma cells via a GTP binding protein, which is not a substrate for IAP.

Inhibitory effect of 8-bromo cyclic GMP on an extracellular Ca2+-dependent arachidonic acid liberation in collagen-stimulated rabbit platelets.

The inhibitory effect of cyclic GMP on collagen-induced platelet activation was studied using 8-bromo cyclic GMP (8brcGMP) in washed rabbit platelets. Addition of collagen (1 micrograms/ml) to platelet suspension caused shape change and aggregation associated with thromboxane (TX) A2 formation. 8brcGMP (10-1000 microM) inhibited collagen-induced platelet aggregation and TXA2 formation in a concentration-dependent manner. 8brcGMP did not affect platelet cyclooxygenase pathways, but markedly inhibited collagen-induced arachidonic acid (AA) liberation from membrane phospholipids in [3H]AA-prelabeled platelets, indicating that the inhibitory effect of 8brcGMP on collagen-induced aggregation is due to an inhibition of AA liberation. In [32P]orthophosphate-labeled platelets, collagen stimulated phosphorylation of a 20,000 dalton (20-kD) and 40-kD proteins. 8BrcGMP stimulated phosphorylation of a specific protein having molecular weight of 46-kD and inhibited collagen-induced both 20- and 40-kD protein phosphorylation. Collagen could stimulate the AA liberation without activation of phospholipase C or Na+-H+ exchange, but could not in the absence of extracellular Ca2+. These findings suggest that cyclic GMP inhibits collagen-induced AA liberation which is mediated by an extracellular Ca2+-dependent phospholipase A2. However, cyclic GMP seems to inhibit the Ca2+-activated phospholipase A2 indirectly, since 8brcGMP had no effect on Ca2+ ionophore A23187-induced platelet aggregation or AA liberation. It is therefore suggested that cyclic GMP may regulate collagen-induced increase in an availability of extracellular Ca2+ which is responsible for phospholipase A2 activation in rabbit platelets.

Dual effects of mastoparan on intracellular free Ca2+ concentrations in human astrocytoma cells

1 The effect of mastoparan, a wasp venom toxin, on intracellular free Ca2+ concentration ([Ca2+]i) was examined in human astrocytoma cells. Mastoparan inhibited [Ca2+]i induced by carbachol (100 μm) in a concentration‐dependent manner in the absence of extracellular Ca2+, consistent with our previous results showing that mastoparan inhibits phosphoinositide hydrolysis in human astrocytoma cells. 2 In contrast, mastoparan itself increased [Ca2+]i and augmented carbachol‐induced increase in the [Ca2+]i in the presence of extracellular Ca2+, suggesting that mastoparan elicited Ca2+ influx from the extracellular medium. The increase appeared to be maximum at extracellular Ca2+ concentrations of 0.1–0.2 mm. The higher concentrations of extracellular Ca2+ depressed the influx. 3 Pertussis toxin did not affect mastoparan‐induced inhibition of [Ca2+]i in the absence of extracellular Ca2+, consistent with the previous results that pertussis toxin did not affect mastoparan‐induced inhibition of phosphoinositide hydrolysis. 4 Pertussis toxin augmented mastoparan‐induced increase in [Ca2+]i in the presence of extracellular Ca2+, suggesting that pertussis toxin substrate(s) seems to be inhibitory for Ca2+ influx induced by mastoparan. 5 Verapamil, nifedipine and diltiazem (each 10 μm), L‐type Ca2+ antagonists, did not affect mastoparan‐induced Ca2+ influx. However, verapamil (10 μm) slightly inhibited the increase in [Ca2+]i induced by carbachol in the presence of mastoparan. 6 The results obtained in the present study indicate that mastoparan has two opposite effects on [Ca2+]i in human astrocytoma cells and possibly has at least two sites of action.

Identification of P2X7 (P2Z) receptor in N18TG-2 cells and NG108-15 cells

Abstract: ATP activates a nonselective cation current by stimulating the P2Z receptor in NG108‐15 cells—a hybrid cell line of the mouse neuroblastoma N18TG‐2 cells and the rat glioma C6Bu‐1 cells. Recently, the P2X7 receptor was cloned from the rat brain and was found to have electrophysiological properties similar to those of the P2Z receptor. We examined the expression of P2X7 receptor mRNA in NG108‐15 cells as well as in their parent cell lines, N18TG‐2 and C6Bu‐1 cells, by reverse transcription‐polymerase chain reaction (RT‐PCR). The cDNA templates from these cell lines were amplified with primers specific to the P2X7 receptor sequence. Positive signals were detected in the RT‐PCR products from NG108‐15 and N18TG‐2 cells but not from C6Bu‐1 cells. We next examined the effect of ATP on the membrane current in N18TG‐2 cells and C6Bu‐1 cells by whole‐cell voltage clamp. In N18TG‐2 cells, ATP induced a sustained current with a reversal potential of 9.3 ± 1.2 mV (n = 22) in a concentration‐dependent manner with an EC50 of 1.76 ± 0.18 mM (n = 36). In contrast, ATP (1 mM) did not induce any current in C6Bu‐1 cells. The ATP‐induced current in N18TG‐2 cells resembled that in NG108‐15 cells in the following points: (a) The currents did not desensitize significantly. (b) EC50 values of ATP are of millimolar order. (c) Benzoylbenzoyl‐ATP was a more potent agonist than ATP. (d) The current was larger in methanesulfonate than in Cl− external solution. (e) The current was larger at lower external Mg2+ concentrations. These results suggest that the hybrid NG108‐15 cells possess a P2X7 receptor like the P2Z receptor and that the ability of expressing this channel originates from N18TG‐2 cells but not from C6Bu‐1 cells.

Mastoparan elicits prostaglandin E2 generation and inhibits inositol phosphate accumulation via different mechanisms in rabbit astrocytes.

The effects of mastoparan on phosphoinositide hydrolysis and prostaglandin E2 (PGE2) generation were investigated in astrocytes cultured from rabbit brain. Mastoparan inhibited the accumulations of [3H]inositol phosphates induced by bradykinin (1 microM) in a time- and concentration-dependent manner. Mastoparan (3-30 microM) also released PGE2 in a time- and concentration-dependent manner. Mastoparan-induced release of PGE2 was inhibited by indomethacin, a cyclooxygenase inhibitor, by dexamethasone, a steroidal anti-inflammatory drug, and by pertussis toxin, an inactivator of some G proteins, such as Gi and Go. Mastoparan also caused [3H]arachidonic acid liberation, which was inhibited by dexamethasone or pertussis toxin. In contrast, indomethacin, dexamethasone and pertussis toxin failed to attenuate mastoparan-induced inhibition of [3H]inositol phosphate accumulation induced by bradykinin. Thus, mastoparan-induced inhibition of phosphoinositide hydrolysis does not involve pertussis toxin-sensitive G protein nor arachidonic acid metabolites. In addition to the inhibition of phospholipase C, mastoparan activates phospholipase A2 through pertussis toxin-sensitive G protein.

ATP-activated nonselective cation current in NG108-15 cells

Abstract: ATP (1 mM) induced a biphasic increase in intracellular Ca2+ concentration ([Ca2+]i), i.e., an initial transient increase decayed to a level of sustained increase, in NG108‐15 cells. The transient increase was inhibited by a phospholipase C inhibitor, 1‐[6‐[[17β‐3‐methoxyestra‐1,3,5(10)‐trien‐17‐yl]amino]hexyl]‐1H‐pyrrole‐2,5‐dione (U73122), whereas the sustained increase was abolished by removal of external Ca2+. We examined the mechanism of the ATP‐elicited sustained [Ca2+]i increase using the fura‐2 fluorescent method and the whole‐cell patch clamp technique. ATP (1 mM) induced a membrane current with the reversal potential of 12.5 ± 0.8 mV (n = 10) in Tyrode external solution. The EC50 of ATP was ∼0.75 mM. The permeability ratio of various cations carrying this current was Na+ (defined as 1) > Li+ (0.92 ± 0.01; n = 5) > K+ (0.89 ± 0.03; n = 6) > Rb+ (0.55 ± 0.02; n = 6) > Cs+ (0.51 ± 0.01; n = 5) > Ca2+ (0.22 ± 0.03; n = 3) > N‐methyl‐d‐glucamine (0.13 ± 0.01; n = 5), suggesting that ATP activated a nonselective cation current. The ATP‐induced current was larger at lower concentrations of external Mg2+. ATP analogues that induced the current were 2‐methylthio‐ATP (2MeSATP), benzoylbenzoic‐ATP, adenosine 5′‐thiotriphosphate (ATPγS), and adenosine 5′‐O‐(2‐thiodiphosphate), but not adenosine, ADP, α,β‐methylene‐ATP (AMPCPP), β,γ‐methylene‐ATP (AMPPCP), or UTP. Concomitant with the current data, 2MeSATP and ATPγS, but not AMPCPP or AMPPCP, increased the sustained [Ca2+]i increase. We conclude that ATP activates a class of Ca2+‐permeable nonselective cation channels via the P2z receptor in NG108‐15 cells.