PubMed Health⌕ Search

Biomedical subjects

H Kuromi

Publications and source records attributed to H Kuromi.

At least 37 records · Page 2Linked to original sources
PubMed1991

An in vitro method assaying recognition ability of nerve terminals for special substances.

The present study provides a simple in vitro method for detecting the ability of nerve terminals to recognize special molecules. Test molecules were applied to a restricted narrow region on the surface of culture dishes, and dissociated sympathetic neurons which were embedded in a collagen gel were placed on the surface next to the region. Sympathetic fibers distributed densely on the laminin-applied region, whereas they were missing on the heparin- or hyaluronic acid-applied regions. The neurites showed no response to the fibronectin- or heparan sulfate-applied region. These results indicate that sympathetic fibers have an affinity for laminin, whereas they recognize a repellent property of heparin or hyaluronic acid.

Adrenergic Fibers↗
PubMed1991

Selective promotion of neuronal adhesion by target tissue-derived materials.

Chick sympathetic nerves densely innervate expansor secundariorum muscle, but not skeletal muscle. By contrast, parasympathetic ciliary nerves innervate striated muscle, but no parasympathetic innervation occurs in expansor secundariorum muscle. The present study revealed accordingly that sympathetic ganglionic neurons from chick embryos adhered firmly to a dish precoated with expansor secundariorum muscle-conditioned medium (ECM), but not to one precoated with skeletal muscle-conditioned medium (SCM), while parasympathetic ciliary ganglionic neurons adhered to a dish precoated with SCM, but not to one precoated with ECM. These results indicate that there are certain materials which mediate specific adhesion between neurons and their target cells.

Animals↗
PubMed1990

Sympathetic innervation-mediating protein.

The present study was performed to elucidate the mechanisms and molecular basis of specific innervation of the peripheral tissues. Chick sympathetic nerve fibers densely innervate expansor secundariorum muscle, but not skeletal muscle. When a sympathetic ganglion was cultured in collagen gel with muscle explants, the ganglion extended neurites towards two types of muscles to the same extent. Dissociated sympathetic ganglion neurons adhered firmly to the dish precoated with materials from expansor secundariorum but not to the dish precoated with those from skeletal muscle. Sympathetic nerve fibers were found densely distributed on the substrate from expansor secundariorum but not on the substrate from skeletal muscle. These results suggest that neuronal recognition mechanisms are involved in the process of selective sympathetic innervation of the expansor secundariorum muscle of the chick. The protein which caused the dense distribution of sympathetic fibers as the substrate has been purified from heart-cell-conditioned medium. The most purified fraction showed a single band with an apparent molecular weight of 370,000 daltons on SDS-PAGE under non-reducing and reducing conditions. The biological activity of the protein was over 100 times higher than that of laminin. Antiserum was raised against the factor in heart-cell-conditioned medium that induced the dense distribution of sympathetic fibers in culture. Addition of the antiserum to the culture medium inhibited the dense distribution of sympathetic fibers on the purified protein-substrate without affecting the general growth of the fibers. Subcutaneous injection of the antiserum into the chicks inhibited the regeneration of adrenergic fibers following 6-hydroxydopamine-induced axotomy in peripheral tissues (heart, spleen, kidney and blood vessel).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

[Mechanism for acetylcholine receptor localization at nerve-muscle synapse].

This paper summarizes the hypothesis proposed to explain the mechanism for AChR localization at the neuromuscular synapse. Two theories have been proposed to explain the neuronal control of extrajunctional AChR. One theory claimed that motoneurons decreased the ACh sensitivity of the extrajunctional membrane through neurotrophic influence. However, direct electrical stimulation of denervated muscles resulted in a decrease of extrajunctional ACh sensitivity, supporting the other hypothesis that loss of extrajunctional AChR of the innervated muscle is directly related to muscle activity per se. AChR clusters (high density of AChR) at the neuro-muscular junction were supposed to result from the association of nerves with preexisting AChR clusters. However, Xenopus nerve-muscle cocultures clearly demonstrated that AChR clusters at the neuromuscular junction were formed after the nerve came in contact with the muscle membrane. Two hypothesis are proposed for nerve-induced formation of AChR clusters. Preferential insertion of AChR into the end-plate was suggested by the finding that AChR messenger RNA was more abundant near to than far from the end-plate in adult muscle fibers. On the other hand, in cultured and embryonic muscles, AChR clusters were formed at nerve-muscle junctions through receptor redistribution which was mediated by the passive diffusion-trap mechanism.

Animals↗
PubMed1986

Concanavalin A prevents acetylcholine receptor redistribution in Xenopus nerve-muscle cultures.

During neuromuscular junction formation ACh receptors accumulate at the nerve-contact region. It has been shown that this is at least partly due to lateral migration of existing receptors in the membrane (Anderson et al., 1977). Randomly diffusing ACh receptor molecules in the membrane may be trapped at the nerve-contact region to form a high receptor density area. If this were the major mechanism, cross-linking ACh receptors by tetravalent concanavalin A (Con A) should immobilize receptors and prevent nerve-induced receptor accumulation. We examined the effect of Con A on nerve-induced receptor accumulation and on the mobility of ACh receptors in cultured Xenopus muscle cells. ACh receptors were stained with tetramethyl rhodamine conjugated alpha-bungarotoxin. The cells were then treated briefly with Con A, and neural tube cells were added to these cultures. The mobility of ACh receptors was measured by the fluorescence photobleaching recovery method. The Con A treatment prevented rapid diffusion of ACh receptors as well as nerve-induced receptor accumulation. Functional synapse formation was not inhibited by this treatment. In contrast, divalent succinyl Con A did not affect the mobility of ACh receptors nor prevent nerve-induced ACh receptor accumulation. When the Con A concentration was varied, the blocking effect on the nerve-induced receptor accumulation changed in parallel with the mobile fraction of receptors. Newly inserted ACh receptors after the Con A treatment were found to be mobile and to accumulate at the nerve-contact region. In these cultures, new receptors accumulated around old, immobilized receptors in some areas along the nerve contact. This observation suggests that new receptors were inserted elsewhere and migrated to the nerve-contact region surrounding immobilized old ones. In addition to the accumulation of receptors, the nerve disperses preexisting receptor clusters prior to induction of high-density regions along the contact area, and, at this early stage, denervation disperses nerve-induced receptor clusters in Xenopus cultures (Kuromi and Kidokoro, 1984a, b). When cultures were treated with Con A, neither of these events occurred, suggesting that these are also diffusion-mediated.

Animals↗
PubMed1985

Formation of acetylcholine receptor clusters at neuromuscular junction in Xenopus cultures.

The formation of acetylcholine receptor (AChR) clusters at the neuromuscular junction was investigated by observing the sequential changes in AChR cluster distribution on cultured Xenopus muscle cells. AChRs were labeled with tetramethylrhodamine-conjugated alpha-bungarotoxin (TMR-alpha BT). Before innervation AChRs were distributed over the entire surface of muscle cells with occasional spots of high density (hot spots). When the nerve contacted the muscle cell, the large existing hot spots disappeared and small AChR clusters (less than 1 micron in diameter) initially emerged from the background along the area of nerve contact. They grew in size, increased in number, and fused to form larger clusters over a period of 1 or 2 days. Receptor clusters did not migrate as a whole as observed during "cap" formation in B lymphocytes. The rate of recruitment of AChRs at the nerve-muscle junction varied from less than 50 binding sites to 1000 sites/hr for alpha BT. In this study the diffusion-trap mechanism was tested for the nerve-induced receptor accumulation. The diffusion coefficient of diffusely distributed AChRs was measured using the fluorescence photobleaching recovery method and found to be 2.45 X 10(-10) cm2/sec at 22 degrees C. There was no significant difference in these values among the muscle cells cultured without nerve, the non-nerve-contacted muscle cells in nerve-muscle cultures, and the nerve-contacted muscle cells. It was found that the diffusion of receptors in the membrane is not rate-limiting for AChR accumulation.

Animals↗
PubMed1984

Nerve disperses preexisting acetylcholine receptor clusters prior to induction of receptor accumulation in Xenopus muscle cultures.

We have investigated the sequential changes of acetylcholine receptor (AChR) distribution on identified Xenopus laevis muscle cells in culture before and after innervation. AChRs on muscle cells were stained with tetramethylrhodamine-conjugated alpha-bungarotoxin and the distribution of AChR clusters was examined on a fluorescence microscope using an image intensifier. Large receptor clusters were identified on muscle cells and their fate was followed afterward. In muscle cells cultured without neural tube cells, about one-half of the identified AChR clusters survived for 2 days. In nerve-muscle cocultures, preexisting AChR clusters survived longer on non-nerve-contacted muscle cells than on muscle cells cultured without nerve. However, in nerve-contacted muscle cells the great majority of preexisting AChR clusters dispersed within 2 days. The dispersal of preexisting AChR clusters preceded receptor accumulation along the path of nerve contact by about 12-16 hr. Therefore, an accelerated dispersal of receptor clusters in innervated muscle cells is not a consequence of receptor accumulation along the nerve. The preexisting AChR clusters located near and far from the nerve contact sites dispersed along a similar time course. Protease inhibitors, trasylol and leupeptin, reduced the nerve-induced dispersal of the preexisting AChR clusters in the period before AChR accumulation at the nerve contact sites but did not do so during the period when AChRs began to accumulate at nerve-muscle contact. The significance of the dispersal of preexisting receptor clusters is discussed with regard to neuromuscular junction formation.

Animals↗
PubMed1984

Denervation disperses acetylcholine receptor clusters at the neuromuscular junction in Xenopus cultures.

The effect of denervation on acetylcholine receptor (AChR) cluster distribution on cultured Xenopus muscle cells has been examined in order to study the role of intact nerve in the maintenance of clusters at the nerve-muscle junction during development. AChRs on the muscle cell were labeled with tetramethyl rhodamine-conjugated alpha-bungarotoxin and sequential changes in AChR cluster distribution were examined with a fluorescence microscope using an image intensifier. Denervation was carried out by exposing the nerve cell body to a focused laser light of a high intensity. After this procedure the neurites originating from the cell quickly disintegrated and large AChR clusters associated with nerve divided into smaller clusters. Individual clusters subsequently decreased in size and finally disappeared. In about 30% of the cases new AChR clusters appeared at the extrajunctional region after denervation. These observations indicate that intact nerves are necessary for the maintenance of receptor localization at the nerve-muscle junction and that nerve-induced accumulation is seemingly reversible during the early period of synapse formation. We tested the idea that receptor clusters were lost due to diffusion of receptors in the muscle membrane after denervation. However, the rate of receptor cluster dispersal after denervation was much slower than that predicted by the diffusion model, suggesting that diffusion of receptors is not a rate-limiting step. Furthermore, we found that receptor clusters at the junction stabilize during days in culture. Thus, 80-90% of receptor clusters at the nerve-muscle junction disappeared at 7 hr after denervation in 1-day cocultures, while about 50% of receptor clusters remained after denervation in 3-day cocultures.

Animals↗
PubMed1981

Papaverine-insensitive and papaverine-sensitive drug-induced contractions of chick smooth muscle.

The mechanisms by which acetylcholine (ACh) and noradrenaline (NA) induce muscle contraction were examined in the chick expansor secundariorum muscle in early life, because responsiveness to ACh disappeared and responsiveness to NA persisted during development. ACh-induced contractions decreased with lowered temperature, while NA-induced contractions increased between 30 and 20 degrees C. Papaverine depressed and antagonized NA-induced contractions, but did not depress ACh-induced contractions. The second application of ACh in Ca2+-free solution produced a negligible contraction. In contrast, even the third application of NA in Ca2+-free solution still evoked a contraction of more than 50% of the response in normal solution. These results suggest that the pathway from cholinergic receptor activation to contraction is mainly mediated through a papaverine-insensitive and external Ca2+-dependent process, whereas the pathway from adrenergic receptor activation to contraction is mainly mediated through a papaverine-sensitive and external Ca2+-independent process.

Animals↗
PubMed1981

Neurotrophic substance develops tetrodotoxin-sensitive action potential and increases curare-sensitivity of acetylcholine response in cultured rat myotubes.

We examined the trophic effects of a partially purified trophic substance from mouse spinal cord extract on the tetrodotoxin (TTX)-sensitivity of action potentials and on acetylcholine-sensitivity of rat skeletal myotubes in 7- and 8-day-old cultures. Many myotubes grown in control medium generate action potentials in the presence of TTX (10(-6) M). The addition of fraction E (Fr.E) from a Biogel P2 column, which exhibited trophic activity on adult denervated muscle in organ culture, decreased TTX-resistivity of action potentials of myotubes in cell culture. The trophic substance was also effective when further purified by paper chromatography and electrophoresis. The response to iontophoretically applied acetylcholine of Fr.E-treated myotubes was much more reduced by D-tubocurarine (10(-7) g/ml) than those of control cultured myotubes. No difference in morphological differentiation, protein synthesis, creatine phosphokinase activity or specific binding of [125I]alpha-bungarotoxin was observed between control and Fr.E-treated cultures. These results suggest that the trophic substance in Fr.E may be involved in the normal development of TTX-sensitive sodium channels and of acetylcholine receptor properties.

Acetylcholine↗
PubMed1979

Partial purification and characterization of neutrophic substance affecting tetrodotoxin sensitivity of organ-cultured mouse muscle.

From mouse spinal cord homogenate, we isolated a trophic substance which reverses the post-denervation decrease in tetrodotoxin sensitivity of action potential in organ-cultured extensor digitorum longus muscle of mouse and characterized its physicochemical properties. The trophic substance was separated from macromolecules in homogenate by gel filtration on Biogel P2 column. The partially purified trophic substance was heat-stable, acid-stable and alkaline-labile. The trophic activity was destroyed by lyophilization at neutral pH but not at acidic pH. The trophic activity was abolished by incubation with pronase or leucine aminopeptidase, but not by trypsin, chymotrypsin, thermolysin or carboxypeptidase A. The trophic substance passed through an ultrafiltration membrane UM10 freely. A small part of the trophic activity passed through a UM2 or UM05, and the rest was retained on the membranes. The trophic substance adsorbed on CM-Sephadex at pH 7.2 but passed through DEAE-Sephadex at pH 8.4. These results suggest that the trophic substance regulating tetrodotoxin sensitivity of action potential in mouse skeletal muscle is a peptide with a rather low molecular weight of less than 10,000 and that while the N-terminus of the peptide is free, the C-terminus is probably blocked. This peptide differs from other trophic substances reported previously by other investigators.

Action Potentials↗
PubMed1979

Isolation and culture of motoneurons from embryonic chicken spinal cords.

A method is described for isolating cholinergic alpha motoneurons from the spinal cord of chicken embryos at stage 17-18 (Hamburger and Hamilton numbering), at the time when it has been shown that motoneurons withdraw from the mitotic cycle but neurons of other types and glia are still mitotic. Fragments of the ventral half of the spinal cord are incubated for 24 hr in the presence of 10 microM 1-beta-D-arabinofuranosylcytosine in order to eliminate dividing cells and are subsequently dissociated into a suspension of single cells. The following evidence has been obtained and suggests that these cells are neuronal and appear to be alpha motoneurons: (i) they are resistant to the lethal effect of arabinofuranosylcytosine, and thus are postmitotic at stage 17-18; (ii) when grown in vitro, they exhibit morphological characteristics similar to those of ventral spinal neurons, which include the ability to be stained with silver, Nissl, methylene blue vital stain at pH 6.5-7.0, and choline acetyltransferase histochemistry; (iii) they have high choline acetyltransferase activity; (iv) they are capable of forming functional synapses with muscle.

Animals↗
PubMed1978

Development of sympathetic nerve--muscle transmission in chick.

The development of sympathetic nerve--muscle transmission was investigated pharmacologically by using the nerve-expansor secundariorum muscle preparation of 2- to 35-day-old chicks. The nerve-induced muscle contraction was not affected by atropine and neostigmine after hatch but was slightly depressed by phentolamine and completely depressed by guanethidine. Since the responsiveness of the muscle to some adrenomimetic drugs remained constant after hatch, adrenergic receptor function does not change during development. The nerve-induced muscle response was scarcely influenced by cocaine or reserpine in neonatal chick while it was potentiated by cocaine and changed by reserpine in mature chicks. These results suggest that cocaine- and reserpine-sensitive mechanisms gradually develop in sympathetic nerve function after hatch. The presence of a potentiating effect of cocaine on the nerve-induced muscle response in dimethylphenylpiperazinium-treated neonatal chick suggests that the development of sympathetic nerve function is regulated by nerve activity itself.

Aging↗
PubMed1976

Influence of sympathetic nerves on development of responsiveness of the chick smooth muscle to drugs.

The role of sympathetic nerves in the development of responsiveness of smooth muscle to drugs was investigated using the expansor secondariorum muscle of 2- to 40-day-old chicks. The normal developmental decrease in the responsiveness of the muscle to acetylcholine was prevented by surgical transection of the nerve or chronic treatment with guanethidine, while it was facilitated by chronic treatment with dimethylphenylpiperazinium. Sensitivity of the muscle to noradrenaline, remaining constant during normal development, was increased by nerve section or guanethidine treatment, while it was slightly decreased by dimethylphenylpipe-azinium treatment. These results suggest that sympathetic nerves regulate the development of the responsiveness of the chick expansor secundariorum muscle to drugs, at least the developmental decrease in responsiveness to acetylcholine.

Acetylcholine↗
PubMed1976

Inhibition of thalamic and hypothalamic somatosensory evoked potentials by stimulation of substantia nigra and its modification by morphine and methotrimeprazine (levomepromazine).

A brief electrical stimulation of the substantia nigra induced a marked and long lasting inhibition of the somatosensory evoked potentials recorded from the centrum medianum of the thalamus (CM) and posterior hypothalamic area (PHA) following sciatic stimulation in unanesthetized rabbits. The nigral inhibitory effect on CM was prolonged by the administration of morphine (4 mg/kg i.v.) but not influenced by that of methotrimeprazine (2-4 mg/kg i.v.). In contrast, the nigral inhibitory effect on PHA was enhanced by the injection of methotrimeprazine (2 mg/kg i.v.), but not changed by that of morphine (4 mg/kg i.v.). These results indicate that the inhibitory system originating from the substantia nigra operates on the somatosensory transmissions from the peripheral nerve to the thalamus and hypothalamus, and that morphine or methotrimeprazine in small doses induced a selective potentiation of the nigral inhibitory influence on the thalamus or hypothalamus, respectively.

Animals↗