Hiroshi Kuromi

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, α‐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 β‐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 α‐cells and intrapancreatic ganglia, a finding compatible with the possibility that glutamate is released from α‐cells as well as from neurons. Because the inhibitory amino acid γ‐amino butyric acid (GABA) is present in β‐cells as well as in neurons and inhibits glucagon secretion from α‐cells, the present study suggests that glutamate and GABA are coordinated in the regulation of hormone secretion in pancreatic islets.—Inagaki, N., Kuromi, H., Gonoi, T., Okamoto, Y., Ishida, H., Seino, Y., Kaneko, T., Iwanaga, T., Seino, S. Expression and role of ionotropic glutamate receptors in pancreatic islet cells. FASEB J. 9, 686‐691 (1995)

PACAP/VIP Receptors in Pancreatic β-Cells: Their Roles in Insulin Secretiona

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.

Changes in acetylcholine and noradrenaline sensitivity of chick smooth muscle wholly innervated by sympathetic nerve during development

Developmental changes in sensitivity of the isolated expansor sedumdariorum muscle of posthatching chicks to noradrenaline (NA), Acetylcholine (ACh) and some other drugs were investigated. This muscle responded to both NA and ACh in early life. The sensitivity to ACh decreased progressively with increasing age and disappeared on day 40 after hatching, however, a corresponding elevation of cholinesterase activity was not observed. The sensitivity to NA remained at the same level during the period of 2-60 days after hatching. The Contractile action of ACh on this muscle was not affected by d-tubocurarine, hexamethonium or phentolamine, but was completely abolished by atropine. These results suggest that there are at least two kinds of responsive sites on the expansor secumdariorum muscle in the new-born chick and that the sites sensitive to ACh degenerate progressively during the developmental processes. The cholinergic sensitive sites of this muscle in the new-born chick may be muscarinic.

Partial purification and characterization of neurotrophic 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.

Effects of spinal cord and other tissue extracts on resting and action potentials of organ-cultured mouse skeletal muscle.

Summary The trophic effects of spinal cord extract on resting potential, action potential and sensitivity of action potential to tetrodotoxin (TTX) in organ-cultured mouse skeletal muscle were examined. Spinal cord extract blocked decrease in the maximal rates of rise and fall of the action potential caused by cultivation and also partially prevented decrease in its TTX-sensitivity and in the resting potential. In another experiment, spinal cord extract was added to organ cultures of skeletal muscle that had been previously denervated for 3 days in vivo. Spinal cord extract partially reversed post-denervation fall in the maximal rate of rise of action potential and its TTX-sensitivity. Extracts of non-nervous tissues (kidney, spleen, pancreas and adrenal gland) were ineffective. These results indicate that mouse spinal cord appears to possess neurotrophic substance(s) regulating membrane properties of mouse skeletal muscle.

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.

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.

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.

A Monitor of Secretion From Single Pancreatic β-Cells

ATP is known to be coreleased with insulin from pancreatic β-cells. To monitor insulin secretion from single β-cells, a single β-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 μmol/l) without desensitization and in a dose-dependent manner. A brief application of tolbutamide (10 μmol/l) increased [Ca2+]i in both the β-cell and the adjacent CPAE cells in co-culture. Suramin (100 μmol/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 β-cell, confirming that the insulin secretagogue-induced Ca2+ response in CPAE cells in co-culture is mediated by ATP released from the β-cell. When co-culture of the β-cell and CPAE cells was stimulated by kainate (1 μmol/l) and then tolbutamide (10 μmol/l), the CPAE cells showed elevations in [Ca2+]i in response to kainate and tolbutamide during elevation in [Ca2+]i in the β-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 β-cell. A long exposure of tolbutamide (100 μmol/l, 4 min) resulted in a long-lasting elevation in [Ca2+]i in the β-cell. During the elevation in [Ca2+]i, induced by tolbutamide in the β-cell, secretion was observed immediately after its application and again after some resting time. This temporal analysis shows that the secretory response of a single intact β-cell rapidly desensitizes despite the [Ca2+]i remaining elevated during the secretagogue stimulation and that it can return, after some desensitized period, in an intact cell. The present co-culture system using CPAE cells as reporter cells is, therefore, useful for monitoring insulin secretion from single intact β-cells.

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.