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M Sugimori

Publications and source records attributed to M Sugimori.

At least 37 records · Page 2Linked to original sources
PubMed1997

Evidence for serotonin sensitivity of adult rat spinal axons: studies using randomized double pulse stimulation.

We have recently shown both inhibitory and excitatory effects of serotonin on neonatal rat dorsal column axons. While neonatal rat dorsal column axons also respond to norepinephrine and GABA, adult rat dorsal columns are insensitive to the actions of both compounds. Therefore, we studied the effects of serotonin agonists on adult rat dorsal column axons using randomized double pulse stimuli at 0.2 Hz with random interpulse intervals of 3, 4, 5, 8, 10, 20, 30, 50 and 80 ms. The serotonin(1A) agonist, 8-hydroxy-dipropylaminotetralin-hydrobromide (8-OH-DPAT), significantly modulated test response amplitudes at 3, 4, 5 and 8 ms interpulse intervals by 29.6+/-4.0%, 17.4+/-2.1%, 9.6+/-2.3%, and 12.4+/-2.2% of conditioning pulse amplitudes, respectively. The mean latencies at 3, 4 and 5 ms interpulse intervals increased by 17.0+/-5.1%, 8.6+/-2.1%, and 5.1+/-1.4%, respectively (P<0.05). However, neither 10 microM 8-OH-DPAT nor 100 microM serotonin hydrochloride affected the compound action potentials evoked by conditioning or test pulses. In contrast, treatment with 100 microM quipazine dimaleate (a serotonin(2A) agonist) decreased the refractory period. While the response amplitudes to a 3-ms double pulse were reduced by 11.0+/-1.5% during the control period, the test response fell to only 2.4+/-1.8% of the conditioning response amplitudes after exposure to 100 microM quipazine. 8-OH-DPAT decreased the amplitude, prolonged the latency and increased the refractory periods of compound action potentials in the adult rat dorsal column, although a high concentration of the agonist (100 microM) was required for these effects. In contrast, the serotonin(2A) agonist, quipazine, decreased refractory periods. These results suggest that both serotonin(1A) and serotonin(2A) receptor subtypes are present on adult spinal dorsal column axons. Further, these receptors have opposing effects on axonal excitability, despite the fact that their sensitivities are relatively low.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1995

Role of the C2A domain of synaptotagmin in transmitter release as determined by specific antibody injection into the squid giant synapse preterminal.

Squid synaptotagmin (Syt) cDNA, including its open reading frame, was cloned and polyclonal antibodies were obtained in rabbits immunized with glutathione S-transferase (GST)-Syt-C2A. Binding assays indicated that the antibody, anti-Syt-C2A, recognized squid Syt and inhibited the Ca(2+)-dependent phospholipid binding to the C2A domain. This antibody, when injected into the preterminal at the squid giant synapse, blocked transmitter release in a manner similar to that previously reported for the presynaptic injection of members of the inositol high-polyphosphate series. The block was not accompanied by any change in the presynaptic action potential or the amplitude or voltage dependence of the presynaptic Ca2+ current. The postsynaptic potential was rather insensitive to repetitive presynaptic stimulation, indicating a direct effect of the antibody on the transmitter release system. Following block of transmitter release, confocal microscopical analysis of the preterminal junction injected with rhodamine-conjugated anti-Syt-C2A demonstrated fluorescent spots at the inner surface of the presynaptic plasmalemma next to the active zones. Structural analysis of the same preparations demonstrated an accumulation of synaptic vesicles corresponding in size and distribution to the fluorescent spots demonstrated confocally. Together with the finding that such antibody prevents Ca2+ binding to a specific receptor in the C2A domain, these results indicate that Ca2+ triggers transmitter release by activating the C2A domain of Syt. We conclude that the C2A domain is directly related to the fusion of synaptic vesicles that results in transmitter release.

Amino Acid Sequence↗
PubMed1995

Role of the C2B domain of synaptotagmin in vesicular release and recycling as determined by specific antibody injection into the squid giant synapse preterminal.

Synaptotagmin (Syt) is an inositol high-polyphosphate series [IHPS inositol 1,3,4,5-tetrakisphosphate (IP4), inositol 1,3,4,5,6-pentakisphosphate, and inositol 1,2,3,4,5,6-hexakisphosphate] binding synaptic vesicle protein. A polyclonal antibody against the C2B domain (anti-Syt-C2B), an IHPS binding site, was produced. The specificity of this antibody to the C2B domain was determined by comparing its ability to inhibit IP4 binding to the C2B domain with that to inhibit the Ca2+/phospholipid binding to the C2A domain. Injection of the anti-Syt-C2B IgG into the squid giant presynapse did not block synaptic release. Coinjection of IP4 and anti-Syt-C2B IgG failed to block transmitter release, while IP4 itself was a powerful synpatic release blocker. Repetitive stimulation to presynaptic fiber injected with anti-Syt-C2B IgG demonstrated a rapid decline of the postsynaptic response amplitude probably due to its block of synaptic vesicle recycling. Electron microscopy of the anti-Syt-C2B-injected presynapse showed a 90% reduction of the numbers of synaptic vesicles. These results, taken together, indicate that the Syt molecule is central, in synaptic vesicle fusion by Ca2+ and its regulation by IHPS, as well as in the recycling of synaptic vesicles.

Animals↗
PubMed1995

Two types of calcium response limited to single spines in cerebellar Purkinje cells.

Of fundamental importance in understanding neuronal function is the unambiguous determination of the smallest unit of neuronal integration. It was recently suggested that a whole dendritic branchlet, including tens of spines, acts as the fundamental unit in terms of dendritic calcium dynamics in Purkinje cells. By contrast, we demonstrate that the smallest such unit is the single spine. The results show, by two-photon excited fluorescence laser scanning microscopy, that individual spines are capable of independent calcium activation. Moreover, two distinct spine populations were distinguished by their opposite response to membrane hyperpolarization. Indeed, in a subpopulation of spines calcium entry can also occur through a pathway other than voltage-gated channels. These findings challenge the assumption of a unique parallel fiber activation mode and prompt a reevaluation of the level of functional complexity ascribed to single neurons.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1995

Effects of Ca2+ channel blockers on transmitter release and presynaptic currents at the frog neuromuscular junction.

1. The effects of the calcium channel blockers, funnel-web spider toxin (FTX), omega-agatoxin IVA (omega-Aga IVA) and omega-conotoxin GVIA (omega-CgTX), were tested on transmitter release and presynaptic currents in frog motor nerve endings. 2. Evoked transmitter release was blocked by FTX (IC50 = 0.02 microliter ml-1) and omega-CgTX (1 microM) but was not affected by omega-Aga IVA (0.5 microM). When FTX (0.1 microliter ml-1) was assayed on spontaneous release either in normal Ringer solution or in low Ca(2+)-high Mg2+ solution, it was found not to affect miniature endplate potential (MEPP) amplitude but to increase MEPP frequency by approximately 2-fold in both conditions. 3. Presynaptic calcium currents (ICa), measured by the perineurial technique in the presence of 10 mM tetraethylammonium chloride (TEA) and 200 microM BaCl2 to block K+ currents, were blocked by omega-CgTX (5 microM), partially blocked by FTX (1 microliter ml-1) and not affected by omega-Aga IVA (0.5 microM). 4. The presynaptic calcium-activated potassium current (IK(Ca)) measured by the perineurial technique in the presence of 0.5 microM 3,4-aminopyridine (DAP) to block voltage-dependent K+ currents, was strongly affected by charybdotoxin (ChTX) (300 nM) and completely abolished by BaCl2 (200 microM). This current was also blocked by omega-CgTX (5 microM) and by CdCl2 (200 microM) but was not affected by FTX (1 microliter ml-1). The blockade by omega-CgTX could not be reversed by elevating [Ca]o to 10 mM. 5. The results suggest that in frog synaptic terminals two omega-CgTX-sensitive populations might coexist. The transmitter release process seems to be mediated by calcium influx through a omega-CgTX- and FTX-sensitive population.

Animals↗
PubMed1995

The concept of calcium concentration microdomains in synaptic transmission.

Ever since the initial measurements of presynaptic calcium currents it has been evident that calcium triggers transmitter release quite rapidly. Several models indicate, as did the initial voltage clamp measurements, that the calcium concentration triggering such release could be very high at the entry site and that this concentration should be very short lasting. In order to determine this time course, calcium entry was studied at the squid giant synapse by imaging light emission from n-aequorin-J, intracellularly injected into the presynaptic terminal. The imaging utilized a video system capable of acquiring 4000 frames per sec. The results indicate that the calcium entry, triggered by action potentials, reaches a peak within 200 musec and has an overall duration of close to 800 musec, closely matching the duration of the presynaptic calcium current determined by voltage clamp results under similar conditions.

Animals↗
PubMed1995

Different calcium channels mediate transmitter release evoked by transient or sustained depolarization at mammalian sympathetic ganglia.

We have compared the effect of calcium channel blockers on the potassium-evoked release of tritium-labeled acetylcholine and on preganglionic spike-evoked synaptic transmission in the rat superior cervical ganglion. Transmitter release at the nerve terminals is mediated by the influx of calcium through voltage-gated calcium channels. While four types of voltage-gated calcium channels (T, L, N and P) have been identified in neurons, it is not clear which may actually be involved in excitation-secretion coupling. Release of tritiated acetylcholine evoked by sustained depolarization in high (40 mM) extracellular potassium decreased markedly in the absence of calcium or the presence of cadmium. High potassium-evoked release was substantially inhibited by the P-type channel blockers, purified from funnel-web spider toxin, and omega-agatoxin-IVA, and by the N-type channel blocker omega-conotoxin-GVIA, but was unaffected by the L-type channel blocker nitrendipine. In contrast, postganglionic compound action potentials synaptically triggered by preganglionic stimulation were strongly blocked by funnel-web spider toxin and slightly blocked by a high concentration of omega-agatoxin-IVA, but were unaffected by either omega-conotoxin-GVIA, nitrendipine or a low concentration of omega-agatoxin-IVA. Thus, at the superior cervical ganglion, funnel-web spider toxin-sensitive calcium channels play a dominant role in transmitter release evoked by transient, spike-mediated depolarization, but other types of voltage-gated calcium channels in addition to the funnel-web spider toxin-sensitive channel mediate the transmitter release that is evoked by sustained high potassium depolarization.

Acetylcholine↗
PubMed1995

Time resolved calcium microdomains and synaptic transmission.

The time course for the calcium entry that triggers release was studied at the squid giant synapse by imaging light emission from n-aequorin-J intracellularly injected into the presynaptic terminal. The imaging utilized a video system capable of acquiring 4000 frames per second. The results indicate that the calcium entry triggered by action potentials reaches a peak within 200 microseconds and has an overall duration of close to 800 microseconds, closely matching the duration of the presynaptic calcium current determined by voltage clamp results under similar conditions.

Aequorin↗
PubMed1995

Pharmacological characterization of the voltage-dependent Ca2+ channels present in synaptosomes from rat and chicken central nervous system.

The voltage-dependent calcium channels present in mammalian and chicken brain synaptosomes were characterized pharmacologically using specific blockers of L-type channels (1,4-dihydropyridines), N-type channels (omega-conotoxin GVIA), and P-type channels [funnel web toxin (FTX) and omega-agatoxin IVA]. K(+)-induced Ca2+ uptake by chicken synaptosomes was blocked by omega-conotoxin GVIA (IC50 = 250 nM). This toxin at 5 microM did not block Ca2+ entry into rat frontal cortex synaptosomes. FTX and omega-agatoxin IVA blocked Ca2+ uptake by rat synaptosomes (IC50 = 0.17 microliter/ml and 40 nM, respectively). Likewise, in chicken synaptosomes, FTX and omega-agatoxin IVA affected Ca2+ uptake, FTX (3 microliters/ml) exerted a maximal inhibition of 40% with an IC50 similar to the one obtained in rat preparations, whereas with omega-agatoxin IVA saturation was not reached even at 5 microM. In chicken preparations, the combined effect of saturating concentrations of FTX (1 microliter/ml) and different concentrations of omega-conotoxin GVIA showed no additive effects. However, the effect of saturating concentrations of FTX and omega-conotoxin GVIA was never greater than the one observed with omega-conotoxin GVIA. We also found that 60% of the Ca2+ uptake by rat and chicken synaptosomes was inhibited by omega-conotoxin MVIID (1 microM), a toxin that has a high index of discrimination against N-type channels. Conversely, nitrendipine (10 microM) had no significant effect on Ca2+ uptake in either the rat or the chicken. In conclusion, Ca2+ uptake by rat synaptosomes is potently inhibited by different P-type Ca2+ channel blockers, thus indicating that P-type channels are predominant in this preparation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

A new water-soluble camptothecin derivative, DX-8951f, exhibits potent antitumor activity against human tumors in vitro and in vivo.

CPT-11, a semisynthetic derivative of camptothecin, exhibited strong antitumor activity against lymphoma, lung cancer, colorectal cancer, gastric cancer, ovarian cancer, and cervical cancer. CPT-11 is a pro-drug that is converted to an active metabolite, SN-38, in vivo by enzymes such as carboxylesterase. We synthesized a water-soluble and non-pro-drug analog of camptothecin, DX-8951f. It showed both high in vitro potency against a series of 32 malignant cell lines and significant topoisomerase I inhibition. The anti-proliferative activity of DX-8951f, as indicated by the mean GI50 value, was about 6 and 28 times greater than that of SN-38 or SK&F 10486-A (Topotecan), respectively. These three derivatives of camptothecin showed similar patterns of differential response among 32 cell lines, that is, their spectra of in vitro cytotoxicity were almost the same. The antitumor activity of three doses of DX-8951f administered i.v. at 4-day intervals against human gastric adenocarcinoma SC-6 xenografts was greater than that of CPT-11 or SK&F 10486-A. Moreover, it overcame P-glycoprotein-mediated multi-drug resistance. These data suggest that DX-8951f has a high antitumor activity and is a potential therapeutic agent.

Animals↗
PubMed1994

The inositol high-polyphosphate series blocks synaptic transmission by preventing vesicular fusion: a squid giant synapse study.

Presynaptic injection of inositol 1,3,4,5-tetraphosphate, inositol 1,3,4,5,6-pentakisphosphate, or inositol 1,2,3,4,5,6-hexakisphosphate--which we denote here the inositol high-polyphosphate series (IHPS)--is shown to block synaptic transmission when injected into the preterminal of the squid giant synapse. This effect is not produced by injection of inositol 1,4,5-trisphosphate. The synaptic block is characterized by a time course in the order of 15-45 min, depending on the injection site in the preterminal fiber; the fastest block occurs when the injection is made at the terminal release site. Presynaptic voltage clamp during transmitter release demonstrates that IHPS block did not modify the presynaptic inward, calcium current. Analysis of synaptic noise at the postsynaptic axon shows that both the evoked and spontaneous transmitter release are blocked by the IHPS. Tetanic stimulation of the presynaptic fiber at frequencies of 100 Hz indicates that block is accompanied by gradual reduction of the postsynaptic response, demonstrating that the block interferes with vesicular fusion rather than with vesicular docking. These results, in combination with the recently demonstrated observation that the IHPS bind the C2B domain in synaptotagmin [Fukada, M., Aruga, J., Niinobe, M., Aimoto, S. & Mikoshiba, K. (1994) J. Biol. Chem. 269, 29206-29211], suggest that IHPS elements are involved in vesicle fusion and exocytosis. In addition, a scheme is proposed in which synaptotagmin triggers transmitter release directly by promoting the fusion of synaptic vesicles with the presynaptic plasmalemma, in agreement with the very rapid nature of transmitter release in chemical synapses.

Animals↗
PubMed1994

Antitumor agents. 7. Synthesis and antitumor activity of novel hexacyclic camptothecin analogues.

Eleven novel hexacyclic and three 7,9-disubstituted pentacyclic derivatives of camptothecin were synthesized and evaluated for in vitro cytotoxic activity against P388, HOC-21, and QG-56 and in vivo antileukemic activity against P388 in mice. Hexacyclic compounds which have an additional 5-, 6-, or 7-membered ring cyclized at positions 7 and 9 of camptothecin were prepared by intramolecular cyclization of pentacyclic camptothecin derivatives or Friedländer condensation of the appropriate bicyclic amino ketone and tricyclic ketone. All of the hexacyclic compounds exhibited compatible or superior activity of 7-ethyl-10-hydroxycamptothecin (SN-38) in in vitro assays without regard to the size or type of the additional ring, and three of six compounds showed more than 300% T/C on in vivo assay. These results suggest that the potency of the hexacyclic ring system is higher than that of the original pentacyclic ring system of camptothecin and that the conformational rigidity of substituents at positions 7 and 9 is favorable for antitumor activity.

Animals↗
PubMed1994

Transmission at the squid giant synapse was blocked by tetanus toxin by affecting synaptobrevin, a vesicle-bound protein.

1. The effect of whole tetanus toxin (TeTX) and of its light chain (TeTX L-chain) on transmitter release was determined by presynaptic pressure-injection in the squid giant synapse. 2. The results indicate that whole TeTX does not modify transmission while the L-chain blocks transmission within 20-30 min. This block does not involve changes in the sodium or potassium conductances responsible for spike generation or the voltage-dependent presynaptic calcium current responsible for transmitter release. 3. Western blotting of protein fractions from the squid optic lobe demonstrated the presence of a protein which reacted with specific antibodies against mammalian synaptobrevin, a vesicular protein. In addition, this protein was enzymatically cleaved by the L-chain component of the toxin in a similar fashion to its mammalian counterpart. 4. These results demonstrate that TeTX L-chain toxin acts directly on a squid synaptobrevin and prevents synaptic release probably by interfering with the docking-fusion synaptic vesicles at the active zone.

Animals↗
PubMed1994

Progressive bronchial obstruction during the acute stage of respiratory tract infection in asthmatic children.

To study the time-course of infectious asthma, we retrospectively examined FEV1 from 5 days before to 10 days after the onset of illness in 31 asthmatic children (20 boys and 11 girls), aged 8 to 12 years. Infections were confirmed by a rise of at least fourfold in serum compliment fixation titers (respiratory syncytial virus, adenovirus, and Mycoplasma pneumoniae) and hemoagglutination inhibition titers (parainfluenza virus types 1, 2, and 3). All the patients had 20 percent or more fall in FEV1 from baseline value during acute phase, but were clinically tolerable and required minimum or no bronchodilators. Regardless of infectious agent, FEV1 began to fall on the first disease day or the previous day, and deteriorate for the first few days. Mean(SD) maximum fall in FEV1 ranged from 39(12) percent to 45(20) percent. Thereafter, FEV1 began to improve and returned to the preillness level by the seventh to tenth day. These results suggest that progressive bronchial obstruction may be inevitable during the acute stage of any infectious asthma.

Acute Disease↗
PubMed1994

Time-resolved imaging of Ca(2+)-dependent aequorin luminescence of microdomains and QEDs in synaptic preterminals.

Localized elevation of intracellular free calcium [Ca2+]i concentration serves as the trigger for a wide variety of physiological processes, e.g., neurotransmitter release at most chemical synapses (1-3). The details of the mechanisms that regulate these processes are still unresolved (3-6), but they must involve precise temporal sequences of molecular events initiated by a transient localized elevation of Ca2+ concentration (i.e., a Ca2+ microdomain [3,7-15]). A microdomain is defined as an autonomous compartment of minimal spatio-temporal volume within which a signaled process can occur (8, 10, 12). A quantum emission domain (QED) is a quantal signal element (3, 16, 17). The concept of a QED was first applied to Ca2+ signaling at the synaptic preterminal (3, 4) and for large-diameter mitotic cells (16, 17). The concept of Ca2+ microdomains was tested by labeling preterminals of squid giant synapses with low-sensitivity aequorin (a photoprotein that emits a photon upon binding Ca2+ [18, 19]). That work confirmed earlier modeling efforts (10, 16) and showed that, upon depolarization, the [Ca2+]i profile reaches 200-300 microM within the microdomains, and that these [Ca2+]i profiles are composed of groups of short-lived 0.5 microns diameter QEDs. In those records, obtained with 2:1 interlacing devices operating at the RS-170 standard, QEDs appeared as striped dots or chevrons rather than as solid dots, indicating that a QED lasted less than 16.6 ms (one video field), and thus establishing the need for higher sampling rates to better characterize the QED.(ABSTRACT TRUNCATED AT 250 WORDS)

Aequorin↗
PubMed1994

High-resolution measurement of the time course of calcium-concentration microdomains at squid presynaptic terminals.

Transmitter release is considered to be a secretory event triggered by localized calcium influx which, by binding to a low-affinity Ca2+ site at the presynaptic active zone, initiates vesicular exocytosis (1-7). In previous experiments with aequorin-loaded presynaptic terminals we visualized, upon tetanic presynaptic stimulation, small points of light produced by calcium concentration microdomains of about 300 microM (5). These microdomains had a diameter of about 0.5 microns (5) and covered 5-10% of the total presynaptic membrane with an average density of 8.4 microns2 per 100 microns2, corresponding closely to the size and distribution of the active zones in that junction (6, 7). To understand in more detail the nature of these concentration microdomains, we obtained rapid video images (400/s) after injecting the photoprotein n-aequorin-J into the presynaptic terminals of squid giant synapses. Using that experimental approach, we determined that microdomains evoked by presynaptic spike activation had a duration of about 800 microseconds. Spontaneous quantum emission domains (QEDs) observed at about the same locations as the microdomains were smaller in amplitude, shorter in duration, and less frequent. These results illustrate the time course of the calcium concentration profiles responsible for transmitter release. Their extremely short duration compares closely with that of calcium current flow during a presynaptic action potential and indicates that, as theorized in the past (6-8), intracellular calcium concentration at the active zone remains high only for the duration of transmembrane calcium flow.

Action Potentials↗
PubMed1993

IgG from amyotrophic lateral sclerosis patients increases current through P-type calcium channels in mammalian cerebellar Purkinje cells and in isolated channel protein in lipid bilayer.

The effect of the IgG from amyotrophic lateral sclerosis (ALS) patients was tested on the voltage-dependent barium currents (IBa) in mammalian dissociated Purkinje cells and in isolated P-type calcium channels in lipid bilayers. Whole cell clamp of Purkinje cells demonstrates that ALS IgG increases the amplitude of IBa without modifying their voltage kinetics. This increased IBa could be blocked by a purified nonpeptide toxin from Agelenopsis aperta venom (purified funnel-web spider toxin) or by a synthetic polyamine analog (synthetic funnel-web spider toxin) and by a peptide toxin from the same spider venom, omega-Aga-IVA. Similar results were obtained on single-channel recordings from purified P channel protein. The addition of ALS IgG increased single-channel IBa open time without affecting slope conductance. The results described above were not seen with normal human IgG nor with boiled ALS IgG. It is concluded that ALS IgG enhances inward current through P-type calcium channels. Since P-type Ca2+ channels are present in motoneuron axon terminals, we propose that the enhanced calcium current triggered by ALS IgG may contribute to neuronal damage in ALS.

Amyotrophic Lateral Sclerosis↗