Biomedical subjects

H Kuromi

Publications and source records attributed to H Kuromi.

At least 19 recordsLinked to original sources

PubMed2004

Analysis of conditional paralytic mutants in Drosophila sarco-endoplasmic reticulum calcium ATPase reveals novel mechanisms for regulating membrane excitability.

Individual contributions made by different calcium release and sequestration mechanisms to various aspects of excitable cell physiology are incompletely understood. SERCA, a sarco-endoplasmic reticulum calcium ATPase, being the main agent for calcium uptake into the ER, plays a central role in this process. By isolation and extensive characterization of conditional mutations in the Drosophila SERCA gene, we describe novel roles of this key protein in neuromuscular physiology and enable a genetic analysis of SERCA function. At motor nerve terminals, SERCA inhibition retards calcium sequestration and reduces the amplitude of evoked excitatory junctional currents. This suggests a direct contribution of store-derived calcium in determining the quantal content of evoked release. Conditional paralysis of SERCA mutants is also marked by prolonged neural activity-driven muscle contraction, thus reflecting the phylogenetically conserved role of SERCA in terminating contraction. Further analysis of ionic currents from mutants uncovers SERCA-dependent mechanisms regulating voltage-gated calcium channels and calcium-activated potassium channels that together control muscle excitability. Finally, our identification of dominant loss-of-function mutations in SERCA indicates novel intra- and intermolecular interactions for SERCA in vivo, overlooked by current structural models.

Animals↗

PubMed2001

Extension of glial processes by activation of Ca2+-permeable AMPA receptor channels.

AMPA type-glutamate receptor channels (AMPARs) assembled without the GluR2 (GluR-B) subunit are characterized by high Ca2+ permeability, and are expressed abundantly in cerebellar Bergmann glial cells. Here we show that the morphology of cultured Bergmann glia-like fusiform cells derived from the rat cerebellum was changed by manipulating expression of Ca2+-permeable AMPARs using adenoviral vector-mediated gene transfer. Converting endogenous Ca2+-permeable AMPARs into Ca2+-impermeable channels by viral-mediated transfer of GluR2 gene induced retraction of glial processes. In contrast, overexpression of Ca2+-permeable AMPARs markedly elongated glial processes. The process extension was blocked by 2,3-Dihydroxy-6-nitro-7-sulfamoylbenzo(F)quinoxaline (NBQX), a specific antagonist of AMPAR. These results indicate that glutamate regulates the morphology of glial processes by activating Ca2+-permeable AMPARs.

Adenoviridae↗

PubMed2000

Tetanic stimulation recruits vesicles from reserve pool via a cAMP-mediated process in Drosophila synapses.

At Drosophila neuromuscular junctions, there are two synaptic vesicle pools, namely the exo/endo cycling pool (ECP) and the reserve pool (RP). We studied the recruitment process from RP using a fluorescent dye, FMI-43. During high-frequency nerve stimulation, vesicles in RP were recruited for release, and endocytosed vesicles were incorporated into both pools, whereas with low-frequency stimulation, vesicles were incorporated into and released from ECP. Release of vesicles from RP was detected electrophysiologically after emptying vesicles in the ECP of transmitter by a H+ pump inhibitor. Recruitment from RP was depressed by inhibitors of steps in the cAMP/PKA cascade and enhanced by their activators. In rutabaga (rut) with low cAMP levels, mobilization of vesicles from RP during tetanic stimulation was depressed, while it was enhanced in dunce (dnc) with high cAMP levels.

Adenylyl Cyclase Inhibitors↗

PubMed2000

Synaptic development is controlled in the periactive zones of Drosophila synapses.

A cell-adhesion molecule fasciclin 2 (FAS2), which is required for synaptic growth and still life (SIF), an activator of RAC, were found to localize in the surrounding region of the active zone, defining the periactive zone in Drosophila neuromuscular synapses. BetaPS integrin and discs large (DLG), both involved in synaptic development, also decorated the zone. However, shibire (SHI), the Drosophila dynamin that regulates endocytosis, was found in the distinct region. Mutant analyses showed that sif genetically interacted with Fas2 in synaptic growth and that the proper localization of SIF required FAS2, suggesting that they are components in related signaling pathways that locally function in the periactive zones. We propose that neurotransmission and synaptic growth are primarily regulated in segregated subcellular spaces, active zones and periactive zones, respectively.

Animals↗

PubMed1999

The optically determined size of exo/endo cycling vesicle pool correlates with the quantal content at the neuromuscular junction of Drosophila larvae.

According to the current theory of synaptic transmission, the amplitude of evoked synaptic potentials correlates with the number of synaptic vesicles released at the presynaptic terminals. Synaptic vesicles in presynaptic boutons constitute two distinct pools, namely, exo/endo cycling and reserve pools (). We defined the vesicles that were endocytosed and exocytosed during high K+ stimulation as the exo/endo cycling vesicle pool. To determine the role of exo/endo cycling vesicle pool in synaptic transmission, we estimated the quantal content electrophysiologically, whereas the pool size was determined optically using fluorescent dye FM1-43. We then manipulated the size of the pool with following treatments. First, to change the state of boutons of nerve terminals, motoneuronal axons were severed. With this treatment, the size of exo/endo cycling vesicle pool decreased together with the quantal content. Second, we promoted the FM1-43 uptake using cyclosporin A, which inhibits calcineurin activities and enhances endocytosis. Cyclosporin A increased the total uptake of FM1-43, but neither the size of exo/endo cycling vesicle pool nor the quantal content changed. Third, we increased the size of exo/endo cycling vesicle pool by forskolin, which enhances synaptic transmission. The forskolin treatment increased both the size of exo/endo cycling vesicle pool and the quantal content. Thus, we found that the quantal content was closely correlated with the size of exo/endo cycling vesicle pool but not necessarily with the total uptake of FM1-43 fluorescence by boutons. The results suggest that vesicles in the exo/endo cycling pool primarily participate in evoked exocytosis of vesicles.

Animals↗

PubMed1999

Activation of metabotropic glutamate receptors enhances synaptic transmission at the Drosophila neuromuscular junction.

We examined the effects of activation of metabotropic glutamate receptors (mGluRs) on glutamatergic synaptic transmission at the neuromuscular junction of newly hatched Drosophila larvae. In nominally Ca(2+)-free solutions puff-application of low concentrations of glutamate evoked a transient frequency increase of miniature synaptic currents (mSCs). The mean amplitude of mSCs was unaffected, suggesting that this effect was presynaptic. Similar alterations of the mSC frequency were obtained using the mGluR agonists, (S)-4C3HPG, DCG-IV, or (1S,3S)-ACPD, but not when using agonists for ionotropic glutamate receptors, NMDA, AMPA or kainate. An mGluR antagonist, MCCG-I, blocked the effect of agonists on the mSC frequency. An adenylate cyclase activator, forskolin, and a cAMP analog, CPT-cAMP, mimicked the effects of mGluR activation. Meanwhile, an adenylate cyclase inhibitor, SQ22,536, blocked the mGluR agonist-induced effects, and in rutabaga, an adenylate-cyclase-defective mutant, the effect of the agonist was greatly reduced. In the presence of external Ca2+, (S)-4C3HPG decreased the failure rate and increased the mean amplitude of stimulus-evoked SCs, while MCCG-I decreased the amplitudes. We suggest that at the larval Drosophila neuromuscular junction endogenous glutamate released at the terminal potentiates synaptic transmission via a process involving cAMP.

Adenylyl Cyclases↗

PubMed1998

Two distinct pools of synaptic vesicles in single presynaptic boutons in a temperature-sensitive Drosophila mutant, shibire.

In a temperature-sensitive Drosophila mutant, shibire, synaptic vesicles are completely depleted in nerve terminals after stimulation at 34 degrees C, but upon returning to 22 degrees C, endocytosis resumes. In this study, synaptic vesicles in the boutons of nerve terminals at the mutant neuromuscular junction were loaded with a fluorescent dye, FM1-43, during vesicle reformation at 22 degrees C after complete depletion at 34 degrees C. We found two distinct pools of synaptic vesicles, namely an exo/endo cycling pool, located in the periphery of the bouton, and a reserve pool, located in its center. Cytochalasin D treatment eliminated the reserve pool and reduced synaptic transmission evoked by high frequency stimulation. Thus, the reserve pool may play a crucial role for sustaining high frequency synaptic transmission.

Actins↗

PubMed1998

Altered bcl-2 and bax expression and intracellular Ca2+ signaling in apoptosis of pancreatic cells and the impairment of glucose-induced insulin secretion.

Apoptosis is the process of cellular self-destruction, and genes such as bcl-2 and bax are known to inhibit and promote apoptosis, respectively. In this study, we show that apoptosis can be induced in pancreatic beta-cell lines, and we investigate the apoptotic pathways through the bcl-2 and bax genes and intracellular Ca2+. Serum deprivation induces apoptosis in the MIN6 and RINm5F pancreatic beta-cell lines, and alters the bcl-2 messenger RNA (mRNA) and protein. KCl, BayK, A23187, and ionomycin elicit an elevation of cytosolic/nuclear Ca2+, which, however, is insufficient to evoke apoptosis or to alter bcl-2 or bax mRNA expression in MIN6 cells. The extracellular Ca2+ chelators, EGTA and 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetrapotassium salt, hydrate, evoke apoptosis and also alter the ratio of bcl-2 to bax mRNA and protein concomitantly with the depletion of cytosolic/nuclear Ca2+. This indicates that there are at least two apoptotic pathways in pancreatic beta-cells: through serum deprivation and through a decrease in cytosolic/nuclear Ca2+. MIN6 cells exhibit reduced insulin secretion induced by glucose regardless of the molecular pathway of apoptosis. Apoptosis in pancreatic beta-cells, therefore, may be closely related to the impairment of insulin secretion in certain pathological conditions such as diabetes mellitus.

Animals↗

PubMed1997

An inhibitory role of calcineurin in endocytosis of synaptic vesicles at nerve terminals of Drosophila larvae.

In this study, we tested a hypothesis that activation of calcineurin, Ca2+/calmodulin-dependent protein phosphatase 2B, is an initiating signal for synaptic vesicle endocytosis. We examined effects of calcineurin inhibitors, cyclosporin A or FK506 and calmodulin inhibitors on stimulus-induced FM1-43 uptake into nerve terminals of Drosophila larvae. Fluorescent FM1-43 labels recycling synaptic vesicles in nerve terminals. Pretreatment with cyclosporin A (5-40 microM) or with FK506 (5-10 microM) enhanced FM1-43 uptake induced by high (60 mM) K+ in a dose-dependent manner. The effect required some preincubation time of about 10 min. The nerve terminals loaded with FM1-43 were destained by electrical nerve stimulation in the cyclosporin A-pretreated preparations, confirming that FM1-43 was taken up into synaptic vesicles. Pretreatment with rapamycin (2 or 20 microM), a structural analog of FK506 which has no effect on calcineurin, or calyculin A (0.3-50 nM), an inhibitor of protein phosphatase 1 and 2A, had no detectable effect on FM1-43 uptake. On the other hand, pretreatment with trifluoperazine (1-50 microM) or with phenoxybenzamine (100 microM), inhibitors of calmodulin, enhanced FM1-43 uptake. Since endocytosis is coupled with exocytosis, it is possible that the enhancement of FM1-43 uptake results from facilitation of exocytosis. However, the frequency of spontaneous junctional potentials and the mean amplitude of evoked potentials did not change after the cyclosporin A treatment, suggesting that the exocytosis process was not significantly affected by the drug. Furthermore, we can temporally separate synaptic vesicle exocytosis and endocytosis in a Drosophila mutant, shibire (shi(ts1)). By taking advantage of this mutation, we showed that cyclosporin A and trifluoperazine enhanced synaptic vesicle recycling by directly acting on the endocytotic process. Present results are not compatible with the hypothesis, but suggest that calcineurin inhibits synaptic vesicle recycling.

Animals↗

PubMed1997

Suramin inhibits glucose-induced Ca2+ response in single rat pancreatic beta cells.

It is reported that glucose releases ATP with insulin from pancreatic beta cells. Suramin (50-200 microM), a purinoceptor blocker, reversibly and dose-dependently inhibited the rise in cytoplasmic calcium concentration ([Ca2+]i) induced by glucose and by ATP in single beta cells. Suramin did not inhibit the glucose-induced increase in NAD(P)H fluorescence. The rise in [Ca2+]i induced by tolbutamide (an inhibitor of ATP-sensitive K+ channels), arginine (a cationic amino acid) and acetylcholine was unaffected by suramin (50-200 microM). This suggests that suramin inhibits the glucose-induced Ca2+ response without an inhibitory effect on glucose metabolism, K+ channels, voltage-dependent Ca2+ influx or Ca2+ release from internal Ca2+ stores and that a purinergic mechanism is involved in the glucose response in beta cells.

Acetylcholine↗

PubMed1996

PACAP/VIP receptors in pancreatic beta-cells: their roles in insulin secretion.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide belonging to the vasoactive intestinal polypeptide (VIP)/glucagon/secretin family. We have isolated a third PACAP receptor subtype, designated PACAPR-3, by molecular cloning. The cDNA encoding PACAPR-3 has been isolated from a mouse insulin-secreting beta-cell line MIN6 cDNA library. Mouse PACAPR-3 is a protein of 437 amino acids that has 50% and 51% identity with rat PACAP type I and type II receptors, respectively. We have expressed PACAPR-3 in mammalian cells and Xenopus oocytes. PACAPR-3 binds to VIP as well as PACAP-38 and -27, with a slightly higher affinity for PACAP-38, and is positively coupled to adenylate cyclase. PACAP-38, -27, and VIP evoked Ca2+ activated-Cl- currents in Xenopus oocytes. RNA blotting studies reveal that PACAPR-3 mRNA is expressed widely in tissues and cell lines, including pancreatic islets, insulin-secreting cell lines (MIN6, HIT-T15, and RINm5F), lung, brain, stomach, colon, and heart. Furthermore, insulin secretion from the MIN6 cells is stimulated significantly by PACAP-38 and VIP. The possible mechanisms of insulin secretion by PACAP and VIP are also discussed.

Amino Acid Sequence↗

PubMed1995

A monitor of secretion from single pancreatic beta-cells.

ATP is known to be coreleased with insulin from pancreatic beta-cells. To monitor insulin secretion from single beta-cells, a single beta-cell was surrounded in culture by Fura 2-loaded calf pulmonary artery endothelium (CPAE) cells, which can detect the ATP. CPAE cells did not respond with an elevation in cytoplasmic calcium concentration ([Ca2+]i) to either tolbutamide (100 mumol/l) or kainate (1 mmol/l) but did respond with an elevation in [Ca2+]i to ATP (0.1-10 mumol/l) without desensitization and in a dose-dependent manner. A brief application of tolbutamide (10 mumol/l) increased [Ca2+]i in both the beta-cell and the adjacent CPAE cells in co-culture. Suramin (100 mumol/l), an ATP-receptor blocker, inhibited the tolbutamide-induced elevation in [Ca2+]i in the CPAE cells but did not inhibit the elevation in [Ca2+]i in the beta-cell, confirming that the insulin secretagogue-induced Ca2+ response in CPAE cells in co-culture is mediated by ATP released from the beta-cell. When co-culture of the beta-cell and CPAE cells was stimulated by kainate (1 mmol/l) and then tolbutamide (10 mumol/l), the CPAE cells showed elevations in [Ca2+]i in response to kainate and tolbutamide during elevation in [Ca2+]i in the beta-cell. This strongly suggests that insulin secretion as well as an increase in [Ca2+]i in response to different agents, i.e., kainate and tolbutamide, occurs in a single beta-cell. A long exposure of tolbutamide (100 mumol/l, 4 min) resulted in a long-lasting elevation in [Ca2+]i in the beta-cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed1995

Expression and role of ionotropic glutamate receptors in pancreatic islet cells.

Although the excitatory amino acid glutamate and its receptors play crucial roles in many functions of the central nervous system (CNS), their presence in the peripheral tissues has remained unclear. In the present study, we have identified kainate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), and N-methyl-D-aspartate (NMDA) receptor subtype mRNAs in pancreatic islets, using reverse transcriptase polymerase chain reaction (RT-PCR). Intracellular calcium ([Ca2+]i) measurements and electrophysiological recordings indicate that kainate, AMPA, and NMDA all elicit increases of [Ca2+]i in single pancreatic beta-cells and depolarize them. In addition, kainate and AMPA stimulate insulin secretion from isolated pancreatic islets, whereas NMDA does not. Also, immunocytochemical study shows the presence of intense glutaminase immunoreactivity in pancreatic alpha-cells and intrapancreatic ganglia, a finding compatible with the possibility that glutamate is released from alpha-cells as well as from neurons. Because the inhibitory amino acid gamma-amino butyric acid (GABA) is present in beta-cells as well as in neurons and inhibits glucagon secretion from alpha-cells, the present study suggests that glutamate and GABA are coordinated in the regulation of hormone secretion in pancreatic islets.

Animals↗

PubMed1994

Functional neuronal ionotropic glutamate receptors are expressed in the non-neuronal cell line MIN6.

We report that a non-neuronal cell line, MIN6, derived from insulin-secreting pancreatic beta-cells, naturally expresses functional ionotropic glutamate receptors. Electrophysiological recordings show that kainate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), and N-methyl-D-aspartate (NMDA) depolarize single MIN6 cells and evoke inward ionic currents. These agents also increase the intracellular calcium concentration in MIN6 cells. Furthermore, insulin secretion from MIN6 cells is stimulated by kainate, AMPA, and NMDA. The presence of AMPA/kainate and NMDA receptor subtypes is confirmed by reverse transcriptase-polymerase chain reaction. These results demonstrate that ionotropic glutamate receptors with properties similar to those in neuronal cells are expressed in a non-neuronal cell line, MIN6. Thus, MIN6 provides a useful and valuable model system for biochemical, pharmacological, and physiological studies of ionotropic glutamate receptors.

Animals↗

PubMed1993

Isolation of sympathonectin; a substrate-bound protein which induces preferential growth of sympathetic fibers in vitro.

A protein named 'sympathonectin', was purified from chick heart cell-conditioned medium (HCM), on the basis of its ability to direct the neurite outgrowth of cultured sympathetic neurons. The most purified fraction showed a doublet band on SDS-PAGE with an apparent molecular weight of 370 kDa. The biological activity of sympathonectin was over 100 times higher than that of laminin. Immunoblotting with anti-sympathonectin of the 100,000 x g pellet (particulate) fraction of HCM identified a distinct 370 kDa band; this molecule did not react to the anti-laminin serum. The immunohistochemical analysis showed that the antibody against sympathonectin stained heart tissue but not skeletal muscle tissue, whereas anti-laminin serum stained both tissues. These results suggest that sympathonectin may play a role during sympathetic innervation of target organs.

Animals↗

PubMed1993

A variant form of laminin is responsible for the neurite outgrowth-promoting activity in conditioned medium from a squamous carcinoma cell line.

We examined the effects of conditioned medium (CM) obtained from a squamous carcinoma cell line, termed SAS, on chick sympathetic neuritic outgrowth. Neurons grown on a substratum coated with CM extended their neurites. Antisera raised against human laminin and mouse EHS laminin immunoprecipitated the neurite outgrowth-promoting factor in CM. The most purified fraction of CM contained a 740-kDa protein which reacted with the anti-laminin sera and was composed of three polypeptides of 330 kDa, 215 kDa and 195 kDa. The 215-kDa and 195-kDa polypeptides, but not the 330-kDa polypeptide were shown to be antigenically related to mouse laminin and human laminin by immunoblotting. The presence of merosin M chain in SAS cells was ruled out by using the method of reverse transcription polymerase chain reaction (RT-PCR). These results suggest that the 740-kDa protein is a laminin variant having a novel heavy chain of 330 kDa and is responsible for neurite outgrowth-promoting activity in CM.

Animals↗

PubMed1992

Characterization of substances which promote or repel sympathetic fiber growth in vitro.

To determine whether a recognition mechanism is involved in determination of sympathetic innervation patterns of various tissues, tissue-derived substances were applied to a restricted test surface region of dishes and the responses of cultured sympathetic neurites were examined. Sympathetic fibers exhibited a turning or ramifying response, resulting in a dense fiber growth on test regions coated with particulate (adheron) fractions of a conditioned-medium (CM) from expansor secundariorum, heart, peripheral blood vessel or abdominal aorta, whereas on test regions coated with those from lung, skeletal muscle or dorsal aorta the neurite growth was repelled and sparse fiber growth was observed. Particulate fractions of brain- or gizzard-CM had no effect. These patterns in vitro were in parallel with the dense sympathetic innervation in expansor secundariorum, heart, peripheral blood vessel and abdominal aorta, but little or no sympathetic innervation in lung, skeletal muscle and dorsal aorta in vivo. These results suggest that adheron particles may participate in determination of sympathetic innervation patterns. Activity which repels or promotes the sympathetic fiber growth was inactivated by pronase E or trypsin but not by DNase or neuraminidase. Repelling activity was lost after treatment with heparinase or heparitinase but not with chondroitinase ABC or hyaluronidase. Promoting activity was retained after treatment with these glycosidases. These results suggest that the factor(s) possessing a repellent effect is a heparan sulfate proteoglycan and one(s) possessing a promoting effect is a protein.

Adrenergic Fibers↗

PubMed1991

Molecules secreted from target and non-target tissues promote and repel sympathetic fiber distribution in vitro.

By using an in vitro assay system detecting the ability of nerve terminals to recognize the test molecules, I found that sympathetic fibers distributed densely on substrates coated with the particulate (adheron) fractions of growth conditioned medium from expansor secundariorum (target tissue), but did not on substrates coated with those from skeletal muscle (non-target). This result suggests that adheron particles are involved in the haptotactic process of specific sympathetic innervation on the target tissue.

Animals↗