Early-onset Charcot-Marie-Tooth patients with mitofusin 2 mutations and brain involvement.

Mutations of the mitofusin 2 (MFN2) gene have been reported to be the most common cause of the axonal form of Charcot-Marie-Tooth disease (CMT). A prospective brain MRI study was performed on 18 early-onset CMT patients with MFN2 mutations, and a high frequency (39%) of brain abnormalities was found. Early-onset patients showed multiple scattered or confluent brain lesions that involved gray matter as well as white matter. Patterns of brain involvement in early-onset patients differed from those of late-onset patients and other hereditary peripheral neuropathies. In addition, one CMT patient demonstrated a brain lesion before the development of peripheral neuropathy.

Different clinical and magnetic resonance imaging features between Charcot-Marie-Tooth disease type 1A and 2A.

Charcot-Marie-Tooth disease type 1A (CMT1A) is the more frequent cause of demyelinating CMT, and CMT2A is the most common cause of axonal CMT. We conducted a magnetic resonance imaging (MRI) study on 39 CMT1A and 21 CMT2A patients to compare their neuroimaging patterns and correlate with clinical features. CMT1A patients showed selective fatty infiltration with a preference for anterior and lateral compartment muscles, whereas CMT2A patients showed a preference for superficial posterior compartment muscles. Early-onset CMT2A patients showed more severe leg fatty atrophy than late-onset CMT2A patients. In late-onset CMT2A, soleus muscle was the earliest, and most severely affected than the other leg muscles. Selective involvement of intrinsic foot muscles is a characteristic pattern of minimal CMT1A and CMT2A. Our MRI study demonstrates different patterns of fatty infiltration involving superficial posterior compartment muscles in CMT2A (partial T-type), and peroneal nerve innervated muscles in CMT1A (P-type).

Designing a magnesium alloy with high strength and high formability.

Although magnesium alloys, as the lightest structural alloys, offer significant potential for automotive applications, their applications remain limited due to their poor formability at room temperature. Since the strategies used for improving formability usually result in a degradation of strength, there are no high strength magnesium alloys showing good formability. Here we report an alloy design concept that can simultaneously provide high strength and good formability. Such designed alloy when subjected to an appropriate processing technique shows a combination of strength and formability that surpasses those of the existing magnesium alloys reported so far. The alloy design concept used in the present study is based on the utilization of alloying elements that can induce precipitation, as well as maximize the segregation of other texture-controlling alloying elements. Such developed alloy is expected to broaden the application of Mg alloy sheets, which are now starting to gain acceptance by automotive industries.

Inhibition of the EGF-induced activation of phospholipase C-gamma1 by a single chain antibody fragment.

Phospholipase C-gamma1(PLC-gamma1) is known to play an essential role in various cellular responses, such as proliferation and tumorigenesis, and PLC-gamma1-specific inhibitors are commonly employed to investigate the mechanism of the PLC-gamma1-mediated signaling pathway. In this study, we developed a single chain antibody fragment (scFv) as a blocker for PLC-gamma1 mediated signaling. scFv, designated F7-scFv, specifically bound to PLC-gamma1 with high affinity (K(d)=1.9x10(-8) M) in vitro. F7-scFv also bound to PLC-gamma1 in vivo and altered the distribution pattern of PLC-gamma1 from the cytoplasm to the intracellular aggregates, where F7-scFv was localized. Moreover, F7-scFv interrupted the EGF-induced translocation of PLC-gamma1 from the cytosol to the membrane ruffle and attenuated EGF-induced inositol phosphates generation and intracellular calcium mobilization. These results indicate that F7-scFv blocks EGF-induced PLC-gamma1 activation by causing sequestering of PLC-gamma1 into intracellular aggregates, and may therefore be useful in studies of the PLC-gamma1-mediated signaling pathway.

Modulation of H2 histamine receptor-mediated cAMP generation and granulocytic differentiation by extracellular nucleotides via activation of protein kinase C.

Extracellular ATP exerts a variety of biological actions through several kinds of P2 receptor in HL-60 promyelocytes. We show that stimulation of P2Y2 receptors with ATP and analogs resulted in the inhibition of a subsequently histamine-induced cAMP production and functional differentiation. Treatment of the cells with phorbol 12-myristate 13-acetate also blocked the histamine-mediated cAMP generation just as ATP did. Incubation of the cells with the protein kinase C inhibitor bisindolylmaleimide (GF109203X) abolished the inhibitory effects of extracellular nucleotides, suggesting that protein kinase C may act as an inter-regulator between two receptors. However, ATP did not affect the binding affinity or total binding of [3H]histamine to membrane receptors; it also did not heterologously desensitize H2 receptors. The ATP treatment synergistically elevated the cAMP levels induced directly by forskolin or indirectly by G protein activation after cholera toxin treatment. This indicates that the site of the protein kinase C action is not the G protein or effector enzyme. Co-stimulation of the cells with nucleotides and histamine inhibited histamine-mediated granulocytic differentiation, which was evaluated by looking at the extent of N-formyl-methionyl-leucyl-phenylalanine responses. Taken together, the results demonstrate that extracellular nucleotides are negatively involved in the modulation of histamine signaling via activation of protein kinase C, probably by inhibiting coupling between receptor and G protein.

Differential regulation of P2Y(11) receptor-mediated signalling to phospholipase C and adenylyl cyclase by protein kinase C in HL-60 promyelocytes.

The regulatory mode of the P2Y(11) purinoceptor-mediated signalling cascades towards phospholipase C and adenylyl cyclase was studied in HL-60 promyelocytes. Treatment with the potent P2Y(11) receptor activator dATP evoked an elevated intracellular Ca(2+) concentration ([Ca(2+)](i)) and inositol 1,4,5-trisphosphate (IP(3)) production that was sustained for longer than 30 min. However, the dATP-induced responses were significantly inhibited by the activation of protein kinase C after a short exposure to phorbol 12-myristate 13-acetate (PMA). dATP also potently stimulated cyclic AMP production with half maximum effect seen at 23+/-7 microM dATP. In addition, a 5-min pretreatment with PMA enhanced the dATP-stimulated cyclic AMP accumulation. PMA potentiated the cyclic AMP production when adenylyl cyclase was activated directly by forskolin or indirectly by G protein activation after cholera toxin treatment. dATP also enhanced the forskolin-mediated cyclic AMP generation. Treatment of the cells with 10 microM U-73122, which almost completely blocked the dATP-stimulated IP(3) production and [Ca(2+)](i) rise, had no effect on cyclic AMP accumulation, while 10 microM 9-(tetrahydro-2-furyl)adenine (SQ 22536), which inhibited the adenylyl cyclase activation, did not effect the dATP-stimulated phosphoinositide turnover. Taken together, the results indicate that P2Y(11) receptor-mediated activation of phospholipase C and adenylyl cyclase occurs through independent pathways and is differentially regulated by protein kinase C in HL-60 cells.

Pharmacological characterization of beta2-adrenoceptor in PGT-beta mouse pineal gland tumour cells.

1. The adrenoceptor in a mouse pineal gland tumour cell line (PGT-beta) was identified and characterized using pharmacological and physiological approaches. 2. Adrenaline and noradrenaline, adrenoceptor agonists, stimulated cyclic AMP generation in a concentration-dependent manner, but had no effect on inositol 1,4,5-trisphosphate production. Adrenaline was a more potent activator of cyclic AMP generation than noradrenaline, with half maximal-effective concentrations (EC50) seen at 175+/-22 nM and 18+/-2 microM for adrenaline and noradrenaline, respectively. 3. The addition of forskolin synergistically stimulated the adrenaline-mediated cyclic AMP generation in a concentration-dependent manner. 4. The pA2 value for the specific beta2-adrenoceptor antagonist ICI-118,551 (8.7+/-0.4) as an antagonist of the adrenaline-stimulated cyclic AMP generation were 3 units higher than the value for the betaI-adrenoceptor antagonist atenolol (5.6+/-0.3). 5. Treatment of the cells with adrenaline and forskolin evoked a 3 fold increase in the activity of serotonin N-acetyltransferase with the peak occurring 6 h after stimulation. 6. These results suggest the presence of beta2-adrenoceptors in mouse pineal cells and a functional relationship between the adenylyl cyclase system and the regulation of N-acetyltransferase expression.

Pharmacological characterization of adenosine receptors in PGT-beta mouse pineal gland tumour cells.

1. The adenosine receptor in mouse pinealocytes was identified and characterized using pharmacological and physiological approaches. 2. Expression of the two adenosine receptor subtypes A2B and A3 was detected in mouse pineal glands and PGT-beta cells by polymerase chain reaction and nucleotide sequencing. 3. Adenosine and 5'-N-ethylcarboxamidoadenosine (NECA) evoked cyclic AMP generation but the A2)-selective agonist 2-(4-(2-carboxyethyl)phenylethylamino)adenosine-5'-N-ethylcarboxamideadenosine (CGS 21680) and the A1-specific agonists R-N(6)-(2-phenylisopropyl)adenosine (R-PIA) and N(6)-cyclopentyladenosine (CPA) had little effect on intracellular cyclic AMP levels. The A2B receptor selective antagonists alloxazine and enprofylline completely blocked NECA-mediated cyclic AMP accumulation. 4. Treatment of cells with the A3-selective agonist N(6)-(3-iodobenzyl)-5'-(N-methylcarbamoyl)adenosine (IB-MECA) inhibited the elevation of the cyclic AMP level induced by NECA or isoproterenol in a concentration-dependent manner with maximal inhibition of 40 - 50%. These responses were blocked by the specific A3 adenosine receptor antagonist MRS 1191. Pretreatment of the cells with pertussis toxin attenuated the IB-MECA-induced responses, suggesting that this effect occurred via the pertussis toxin-sensitive inhibitory G proteins. 5. IB-MECA also caused a concentration-dependent elevation in [Ca(2+)]i and IP3 content. Both the responses induced by IB-MECA were attenuated by treatment with U73122 or phorbol 12-myristate 13-acetate. 6. These data suggest the presence of both A2B and A3 adenosine receptors in mouse pineal tumour cells and that the A2B receptor is positively coupled to adenylyl cyclase whereas the A3 receptor is negatively coupled to adenylyl cyclase and also coupled to phospholipase C.

Inhibition by ethaverine of catecholamine secretion through blocking L-type Ca2+ channels in PC12 cells.

Ethaverine, a derivative of papaverine, is used as a vasodilator and antispasmodic drug. We have investigated the effects of ethaverine on the secretion of [3H]norepinephrine from PC12 cells, of neuroendocrine origin. Treatment with ethaverine reduced catecholamine secretion in a concentration-dependent manner. The maximal inhibitory effect (90%) was achieved with 10 microM ethaverine, and the IC50 for secretion was approximately 2 microM. Ethaverine pretreatment for 1 min prior to stimulation by 70 mM K+ also decreased the level of intracellular Ca2+ in a concentration-dependent manner, as measured by fura-2 fluorescence. The IC50 for the inhibition of the increase in intracellular Ca2+ was approximately 2 microM. Nifedipine, a dihydropyridine L-type Ca2+ channel blocker, did not enhance the inhibitory effect of ethaverine on the 70 mM K(+)-induced increase in [Ca2+]i or catecholamine secretion. In contrast, the addition of the N-type voltage-sensitive Ca2+ channel antagonist omega-conotoxin with ethaverine resulted in further reductions in the increase in [Ca2+]i and catecholamine release. Maximally effective concentrations of ethaverine and nifedipine showed the same inhibitory effect on the 70 mM K(+)-evoked responses. However, ethaverine pretreatment did not inhibit the bradykinin-induced secretion and [Ca2+]i rise, which are known to be produced through the receptor-operated Ca2+ channels. We conclude that ethaverine reduces catecholamine secretion by blocking the L-type voltage-sensitive Ca2+ channel.

Chlorpromazine-induced inhibition of catecholamine secretion by a differential blockade of nicotinic receptors and L-type Ca2+ channels in rat pheochromocytoma cells.

We investigated the effect of chlorpromazine (CPZ), a phenothiazine neuroleptic, on catecholamine secretion in rat pheochromocytoma (PC12) cells. CPZ inhibited [3H]norepinephrine ([3H]NE) secretion induced by 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), an agonist of nicotinic acetylcholine receptors (nAChRs) with an IC50 value of 1.0 +/- 0.2 microM. The DMPP-induced rise in cytosolic free Ca2+ concentration [Ca2+]i was inhibited by CPZ with an IC50 of 1.9 +/- 0.1 microM. The DMPP-induced increase in cytosolic free Na+ concentration [Na+]i was also inhibited by CPZ with a similar potency. Furthermore, the binding of [3H]nicotine to PC12 cells was inhibited by CPZ with an IC50 value of 2.7 +/- 0.6 microM, suggesting that the nAChRs themselves are inhibited by CPZ. In addition, both 70 mM K+-induced [3H]NE secretion and [Ca2+]i increase were inhibited by CPZ with IC50 of 7.9 +/- 1.1 and 6.2 +/- 0.3 microM, respectively. Experiments with Ca2+ channel antagonists suggest that L-type Ca2+ channels are mainly responsible for the inhibition. We conclude that CPZ inhibits catecholamine secretion by blocking nAChRs and L-type Ca2+ channels, with the former being more sensitive to CPZ.

Differential stereoselectivity of D- and L-myo-inositol 1,2,4, 5-tetrakisphosphate binding to the inositol 1,4,5-trisphosphate receptor and 3-kinase.

D- and L-myo-inositol 1,2,4,5-tetrakisphosphate (Ins(1,2,4,5)P(4)) were investigated for their ability to bind to the D-myo-inositol 1, 4,5-trisphosphate (Ins(1,4,5)P(3)) receptor in a bovine adrenal cortical membrane fraction, to mobilize intracellular Ca(2+) stores in Xenopus oocytes, and to bind to the rat brain Ins(1,4,5)P(3) 3-kinase overexpressed and purified in E. coli. In competitive binding experiments with the Ins(1,4,5)P(3) receptor, D-Ins(1,2,4, 5)P(4) effectively displaced [(3)H]Ins(1,4,5)P(3) in a concentration-dependent manner with a potency comparable to that of D-Ins(1,4,5)P(3), while L-Ins(1,2,4,5)P(4) was approximately 50-fold less effective than D-Ins(1,4,5)P(3) and D-Ins(1,2,4,5)P(4). The DL-Ins(1,2,4,5)P(4) racemate bound to the Ins(1,4,5)P(3) receptor with an apparent intermediate efficiency. Injection of D-Ins(1,2,4, 5)P(4) into oocytes evoked a chloride current dependent on intracellular Ca(2+) mobilization in which the agonists ranked in a similar order of potency as in the Ins(1,4,5)P(3) receptor binding. On the other hand, D-Ins(1,2,4,5)P(4) only inhibited the binding of [(3)H]Ins(1,4,5)P(3) to 3-kinase very weakly with a markedly reduced potency compared to D-Ins(1,4,5)P(3), indicating that D-Ins(1,2,4, 5)P(4) is not an effective competitor in the phosphorylation of [(3)H]-Ins(1,4,5)P(3) by 3-kinase. The results, therefore, clearly indicate that D-Ins(1,2,4,5)P(4) is as effective as D-Ins(1,4,5)P(3) in the binding to the receptor but not 3-kinase, and access of Ins(1, 2,4,5)P(4) over the Ins(1,4,5)P(3) receptor calls for stringent stereospecificity with D-Ins(1,2,4,5)P(4) being the active form in DL-Ins(1,2,4,5)P(4)-mediated Ca(2+) mobilization.

Characterization of Mas-7-induced pore formation in SK-N-BE(2)C human neuroblastoma cells.

Mastoparan, a peptide toxin from wasp venome, mimics receptors by stimulating the GTPase activity of guanine nucleotide binding proteins and the G-protein-coupled phospholipase C (PLC). By using Mas-7, the active analog of mastoparan, we showed that it makes pores in the plasma membrane. Treatment with Mas-7 but not Mas-17, the inactive analog, produced a concentration-dependent rise in intracellular Ca2+ concentration ([Ca2+]i) and facilitated the uptake of ethidium bromide (EtBr) (314 Da) to a sustained level during the stimulation. In addition, Mas-7 triggered the influx of lucifer yellow (457 Da), while it did not induce the influx of fura-2 (831 Da) and Evans blue (961 Da). However, the Mas-7-induced permeability was selectively prevented by the addition of La3+, Ni2+, and Co2+, but not Cd2+. This blocking activity was concentration-dependent. While the stimulatory effect of Mas-7 on PLC activity was dependent on extracellular Ca2+, the pore forming activity of Mas-7 was not effected by removal of extracellular Ca2+. These results, therefore, suggest that the mastoparan's action in pore formation is independent from its action in PLC stimulation and is negatively effected by inorganic cations.

Histamine inhibits ATP-induced [Ca2+]i rise through the activation of protein kinase A in HL-60 cells.

We investigated the cross-talk between the histamine and ATP receptors in HL-60 human promyelocytes. While both histamine and extracellular ATP increase intracellular Ca2+ concentration ([Ca2+]i) we found that histamine treatment causes a decrease in the subsequent ATP-induced Ca2+ release from intracellular stores and Ca2+ influx from extracellular space. In addition, histamine also inhibited the subsequent ATP-induced inositol 1.4,5-trisphosphate (IP3) generation in a manner comparable to the Ca2+ release. However, histamine did not inhibit thapsigargin-induced Ca2+ release and influx, thus indicating that histamine does not directly inhibit the Ca2+ release-activated channel (CRAC). Ca2+ elevation induced by 2'- and 3'-O-(4-benzoylbenzoyl) ATP (BzATP), which does not produce IP3, was also inhibited by treatment with histamine, suggesting the presence of ATP-gated channels that are regulated by histamine. Treatment with dibutyryl cAMP or 8-bromo-cAMP inhibited the subsequent ATP-induced response similar to histamine. Moreover, the incubation of cells with N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89), a protein kinase A inhibitor abolished histamine's inhibitory effect on the ATP-induced [Ca2+]i rise and IP3 formation. These results suggest that histamine inhibits both ATP-induced IP3 production and ATP-activated channel opening, through protein kinase A activation.

Synergistic activation of adenylyl cyclase is dependent upon phospholipase C-mediated processes in human neuroblastoma SK-N-BE(2)C cells.

1-[6-[17 beta-3-Methoxyestra-1,3.5(10)-trien-17-yl]amino]hexyl]-1 H-pyrrole-2,5-dione (U-73122), an inhibitor of processes involved in the activation of phospholipase C, was used to assess the role of phospholipase C activation in the synergistic elevation of cAMP induced by carbachol and prostaglandin E2 in human neuroblastoma (SK-N-BE(2)C cells. Pre-treatment of the cells with U-73122 resulted in inhibition of carbachol-induced intracellular Ca2+ ([Ca2+]i) rise and inositol 1,4,5-trisphosphate (InsP3) generation, with maximal and half maximal inhibition (IC50) occurring at approximately 15 microM and 3.2 microM, respectively. U-731222 also inhibited the synergistic enhancement of cAMP accumulation induced by carbachol and prostaglandin E2 in a concentration-dependent manner with maximum and IC50 at 12 +/- 4 microM and 3.4 +/- 0.3 microM, respectively. However, U-73122 did significantly inhibit prostaglandin E2-induced production. While 1,2-bis(o-aminophenoxy)ethane-N,N,N,'N'-tetraacetic acid (BAPTA/AM) treatment decreased the synergistic cAMP accumulation by 28%m addition U-73122 further decreased it down to complete inhibition. Furthermore, GTP gamma S- and A1F4(-)-induced InsP3 generation in digitonin-mediated permeabilized cells was also inhibited by U-73122 treatment. Pre-treatment of the cells with neomycin, another blocker of the phospholipase C pathway, also resulted in inhibition of the carbachol-induced [Ca2+]i rise, InsP3 generation, and the enhancing effect on cAMP accumulation, to a comparable extent. But, Ca2+ chelation by BAPTA/AM in addition to neomycin treatment further decreased the cAMP accumulation. These results suggest that the increase in cytosolic Ca2+ and the coupling process between muscarinic receptor-like G-protein and phospholipase C are important for the synergistic activation of adenylyl cyclase in SK-N-BE(2)C cells.

Synthesis and biological studies of catechol ether type derivatives as potential phosphodiesterase (PDE) IV inhibitors.

New series of catechol ether type derivatives 5, 6 have been synthesized and applied to biological tests. Even though it is a preliminary data, some of our target molecules show the promising result against PDE IV inhibition. SAR and biological studies with synthetic compounds will be discussed in detail.

Management and clinical outcome of acute basilar artery dissection.

BACKGROUND AND PURPOSE: There have been inconsistencies on the prognosis and controversies as to the proper management of acute basilar artery dissection. The aim of this study was to evaluate acute basilar artery dissection and its outcome after management. MATERIALS AND METHODS: A total of 21 patients (mean age, 53 years; range, 24-78 years) with acute basilar artery dissection were identified between January 2001 and October 2007. Clinical presentation, management, and outcomes were retrospectively evaluated. RESULTS: The patients presented with subarachnoid hemorrhage (n = 10), brain stem ischemia (n = 10), or stem compression sign (n = 1). Ruptured basilar artery dissections were treated by stent placement with coiling (n = 4), single stent placement (n = 3), or conservatively (n = 3). Of the patients treated with endovascular technique, 6 had favorable outcome (modified Rankin scale [mRS], 0-2) and the remaining patient, who was treated by single stent placement, died from rebleeding. All 3 conservatively managed patients experienced rebleeding, of whom 2 died and the other was moderately disabled. Unruptured basilar artery dissections were treated conservatively (n = 7) or by stent placement (n = 4). Of the patients with unruptured basilar artery dissection, 9 had favorable outcome and the remaining 2 patients, both of whom were conservatively managed, had poor outcome because of infarct progression. The group with the ruptured basilar artery dissection revealed a higher mortality rate than the group with the unruptured dissection (30% vs 0%). The group treated with endovascular means revealed more favorable outcome than the group that was treated with conservative measures (90.9% vs 50%). CONCLUSION: The ruptured basilar artery dissections were at high risk for rebleeding, resulting in a grave outcome. Stent placement with or without coiling may be considered to prevent rebleeding in ruptured basilar dissections and judiciously considered in unruptured dissections with signs of progressive brain stem ischemia.

Stimulation of adenylyl cyclase mediated by phospholipase C-linked M3 muscarinic receptor in human neuroblastoma SK-N-BE (2) C cells.

Muscarinic receptor in human neuroblastoma SK-N-BE(2)C cells was identified and characterized. Treatment of the cells with carbachol evoked the generation of inositol 1,4,5-trisphosphate (IP3) with a peak level reached at 1 min after stimulation. Carbachol increased intracellular Ca2+ ([Ca2+]i) with an EC50 value of 35 microM. In addition, carbachol produced a 1.3-3-fold increase in the cyclic AMP (cAMP) level compared with untreated control and elevated synergistically the cAMP level in the treatment with prostaglandin E2 (PGE2). The M3 antagonist p-fluorohexahydrosiladifenidol (IC50 = 0.5-0.8 microM) inhibited the increases in [Ca2+]i, IP3, and cAMP more effectively than the M1 antagonist pirenzepine (IC50 = 5-9 microM) and the M2 antagonist methoctramine (IC50 = 20-30 microM). The involvements of [Ca2+]i elevation and protein kinase C activation induced by phospholipase C activation were tested in the carbachol-induced cAMP production. The calcium chelator BAPTA/AM (75 microM) inhibited significantly the synergistic effects of carbachol and PGE2 on the production of cAMP, whereas the Ca2+ ionophore ionomycin (1 microM) clearly enhanced PGE2-induced cAMP production. However, phorbol 12-myristate 13-acetate did not enhance PGE2-stimulated cAMP production. These data suggest that phospholipase C-linked M3 receptors are present and that stimulation of the receptors activates adenylyl cyclase, at least in part, by the Ca(2+)-dependent system in the neuronal cells.

Inhibition of bradykinin-induced cytosolic Ca2+ elevation by muscarinic stimulation without attenuation of inositol 1,4,5-trisphosphate production in human neuroblastoma SK-N-BE(2)C cells.

Cross talk between two phospholipase C (PLC)-linked receptor signalings was investigated in SK-N-BE(2)C human neuroblastoma cells. Sequential stimulation with two agonists at 5-min intervals was performed to examine the interaction between muscarinic and bradykinin (BK) receptors. Pretreatment of cells with a maximal effective concentration (5 microM) of BK did not affect the subsequent carbachol (CCh)-induced [Ca2+]i rise, but CCh (1 mM) pretreatment completely abolished the BK-induced [Ca2+]i rise without inhibition of BK-induced inositol 1,4,5-trisphosphate (IP3) generation. Thapsigargin (1 microM) pretreatment abolished the subsequent BK- and CCh-induced [Ca2+]i rise, though it did not affect agonist-induced IP3 generation. However, the addition of atropine at plateau phases of CCh-induced [Ca2+]i rise and IP3 production caused a rapid decline to the basal levels and then restored the [Ca2+]i rise by BK. Treatment of cells with both CCh and BK at the same time showed additive effects in IP3 production. However, the [Ca2+]i rise induced by both agonists in the presence or absence of extracellular Ca2+ was the same as the responses triggered by CCh alone. The results suggest that each receptor or receptor-linked PLC activity is not influenced by pretreatment with the other agonist but IP3-sensitive Ca2+ stores are shared by signal pathways from both receptors.

Signal flows from two phospholipase C-linked receptors are independent in PC12 cells.

Bradykinin (BK) receptor and P2-purinergic receptor are known to be coupled to phospholipase C (PLC) in PC12 cells. To study the interaction between these two PLC-linked receptors, the presence of both receptors on individual cells was demonstrated by sequential Ca2+ spikes caused by BK and ATP in a single fura-2-loaded cell. BK- and ATP-induced catecholamine (CA) secretions were desensitized within 5 min. However, in the sequential experiment, the BK-induced homologous desensitization of CA secretion did not block the ATP-induced secretion, and vice versa. Each agonist-induced an increase in inositol 1,4,5-trisphosphate (IP3) production and intracellular free Ca2+ concentration also led to homologous desensitization. However, there was no heterologous desensitization between the two agonists. When the cells were treated with both BK and ATP simultaneously, the amounts of CA secretion, IP3 production, internal Ca2+ mobilization, and Ca2+ influx were all additive. We also found that both IP3-induced Ca2+ release from intracellular Ca2+ stores and Ca2+ influx from extracellular space were able to release [3H]norepinephrine, and the secretion induced by both agonists was exactly additive in the absence or presence of extracellular Ca2+. The data suggest that the CA secretions caused by BK or ATP may have separate secretory pathways even though they activate identical second messenger pathways.

Feedback regulation of ATP-induced Ca2+ signaling in HL-60 cells is mediated by protein kinase A- and C-mediated changes in capacitative Ca2+ entry.

Extracellular ATP increases intracellular Ca2+ ([Ca2+]i) in HL-60 cells. When cells are stimulated with supramaximal concentrations of ATP, although the initial [Ca2+]i increase is similar over a range of 30, 100, and 300 microM ATP, the rate of the return to basal [Ca2+]i level is faster in cells treated with higher concentrations of ATP. This probably results from differences in Ca2+ influx rather than Ca2+ release, since the influx of the unidirectional Ca2+ surrogates Ba2+ and Mn2+ also exhibit similar responses. Furthermore, while 300 microM ATP had an inhibitory effect on the thapsigargin-induced capacitative Ca2+ entry, 30 microM ATP potentiated the response. However, the inhibitory action of 300 microM ATP was blocked by protein kinase C (PKC) inhibitors, such as GF 109203X and chelerythrine, and the potentiating action of 30 microM ATP was blocked by protein kinase A (PKA) inhibitors H89 and Rp-cAMPS. The PKC inhibitors also slowed the decay rate of the Ca2+ response induced by 300 microM ATP, and the PKA inhibitors increased it when induced by 30 microM ATP. In the measurements of PKA and PKC activity, 30 microM ATP activates only PKA, while 300 microM ATP activates both kinases. Taken together, these data suggest that the changes in the ATP-induced Ca2+ response result from differential modulation of ATP-induced capacitative Ca2+ entry by PKC and PKA in HL-60 cells.