Dopamine D1 Receptor Immunoreactivity on Fine Processes of GFAP-Positive Astrocytes in the Substantia Nigra Pars Reticulata of Adult Mouse.

Substantia nigra pars reticulata (SNr), the major output nucleus of the basal ganglia, receives dopamine from dendrites extending from dopaminergic neurons of the adjacent nucleus pars compacta (SNc), which is known for its selective degeneration in Parkinson's disease. As a recipient for dendritically released dopamine, the dopamine D1 receptor (D1R) is a primary candidate due to its very dense immunoreactivity in the SNr. However, the precise location of D1R remains unclear at the cellular level in the SNr except for that reported on axons/axon terminals of presumably striatal GABAergic neurons. To address this, we used D1R promotor-controlled, mVenus-expressing transgenic mice. When cells were acutely dissociated from SNr of mouse brain, prominent mVenus fluorescence was detected in fine processes of glia-like cells, but no such fluorescence was detected from neurons in the same preparation, except for the synaptic bouton-like structure on the neurons. Double immunolabeling of SNr cells dissociated from adult wild-type mice brain further revealed marked D1R immunoreactivity in the processes of glial fibrillary acidic protein (GFAP)-positive astrocytes. Such D1R imunoreactivity was significantly stronger in the SNr astrocytes than that in those of the visual cortex in the same preparation. Interestingly, GFAP-positive astrocytes dissociated from the striatum demonstrated D1R immunoreactivity, either remarkable or minimal, similarly to that shown in neurons in this nucleus. In contrast, in the SNr and visual cortex, only weak D1R immunoreactivity was detected in the neurons tested. These results suggest that the SNr astrocyte may be a candidate recipient for dendritically released dopamine. Further study is required to fully elucidate the physiological roles of divergent dopamine receptor immunoreactivity profiles in GFAP-positive astrocytes.

Syntheses of 2-NBDG analogues for monitoring stereoselective uptake of D-glucose.

2-NBDG is a widely used fluorescent tracer for monitoring d-glucose uptake into single living cells. However, 2-NBDG alone is not sufficient for monitoring the net stereoselective uptake of d-glucose, unless its possible non-stereoselective uptake is properly evaluated. l-Glucose derivatives, which emit fluorescence distinct from that of 2-NBDG, should provide valuable information on the stereoselective uptake, when used with 2-NBDG in combination. In the present study, we synthesized Texas Red (sulforhodamine 101 acid)-coupled and [2-(benz-2-oxa-1,3-diazol-4-yl)amino]-coupled 2-deoxy-D-glucose, referred to as [2-TRG] and [2-BDG], respectively. These derivatives showed emission wavelength longer and shorter than that of 2-NBDG, respectively. 2-TRLG, an antipode of 2-TRG, proved to be an effective tracer for evaluating the extent of non-stereoselective uptake of 2-NBDG when used simultaneously with 2-NBDG. On the other hand, 2-BDG exhibited very weak fluorescence, but the application of a novel cross coupling in the presence of a benzoxadiazole group may be useful for the future development of effective glucose tracers.

Hydrogen peroxide mobilizes Ca2+ through two distinct mechanisms in rat hepatocytes.

AIM: Hydrogen peroxide (H2O2) is produced during liver transplantation. Ischemia/reperfusion induces oxidation and causes intracellular Ca2+ overload, which harms liver cells. Our goal was to determine the precise mechanisms of these processes. METHODS: Hepatocytes were extracted from rats. Intracellular Ca2+ concentrations ([Ca2+](i)), inner mitochondrial membrane potentials and NAD(P)H levels were measured using fluorescence imaging. Phospholipase C (PLC) activity was detected using exogenous PIP2. ATP concentrations were measured using the luciferin-luciferase method. Patch-clamp recordings were performed to evaluate membrane currents. RESULTS: H2O2 increased intracellular Ca2+ concentrations ([Ca2+](i)) across two kinetic phases. A low concentration (400 micromol/L) of H2O2 induced a sustained elevation of [Ca2+](i) that was reversed by removing extracellular Ca2+. H2O2 increased membrane currents consistent with intracellular ATP concentrations. The non-selective ATP-sensitive cation channel blocker amiloride inhibited H2O2-induced membrane current increases and [Ca2+](i) elevation. A high concentration (1 mmol/L)of H2O2 induced an additional transient elevation of [Ca2+](i), which was abolished by the specific PLC blocker U73122 but was not eliminated by removal of extracellular Ca2+. PLC activity was increased by 1 mmol/L H2O2 but not by 400 micromol/L H2O2. CONCLUSIONS: H2O2 mobilizes Ca2+ through two distinct mechanisms. In one, 400 micromol/L H2O2-induced sustained [Ca2+](i) elevation is mediated via a Ca2+ influx mechanism, under which H2O2 impairs mitochondrial function via oxidative stress,reduces intracellular ATP production, and in turn opens ATP-sensitive, non-specific cation channels, leading to Ca2+ influx.In contrast, 1 mmol/L H2O2-induced transient elevation of [Ca2+](i) is mediated via activation of the PLC signaling pathway and subsequently, by mobilization of Ca2+ from intracellular Ca2+ stores.

Augmented beta cell loss and mitochondrial abnormalities in sucrose-fed GK rats.

Progressive decline of islet beta cell mass is a hallmark of type 2 diabetes, where nutritional insults are invoked in the pathologic process. Its detailed mechanisms are, however, incompletely understood. We explored the effect of sucrose diet on mitochondria in Goto Kakizaki (GK) rats, a spontaneously diabetic model. Six-week-old male GK rats were given 30% sucrose orally for 2 weeks. Normal Wistar rats fed with sucrose served as controls. Compared to untreated GK rats, sucrose-fed GK rats showed severe degeneration and death of beta cells with disrupted and swollen mitochondria and a greater beta cell loss. Submicroscopic analysis disclosed a smaller mean volume and a greater number of mitochondria in beta cells in GK rats compared to those in Wistar rats. Mitochondria in sucrose-fed GK rats were 2.4-fold greater in mean volume than those in untreated state. Without sucrose feeding, there was no significant difference in mitochondrial membrane potentials (MmPs) of isolated islets between Wistar and GK rats. MmPs were reduced by 44% in sucrose-fed GK rats but not influenced in sucrose-fed Wistar rats. Current results suggest that nutritional insults like sucrose feeding may exert deleterious effects on mitochondria, resulting in augmented beta cell loss in type 2 diabetes.

Iptakalim, a vascular ATP-sensitive potassium (KATP) channel opener, closes rat pancreatic beta-cell KATP channels and increases insulin release.

Sulfonylureas have been the leading oral antihyperglycemic agents, and they presently continue to be the most popular antidiabetic drugs prescribed for treatment of type 2 diabetes. However, concern has arisen over the side effects of sulfonylureas on the cardiovascular system. Here, we tested the hypothesis that iptakalim, a novel vascular ATP-sensitive potassium (K(ATP)) channel opener, closes rat pancreatic beta-cell K(ATP) channels and increases insulin release. Rat pancreatic beta-cell K(ATP) channels and heterologously expressed K(ATP) channels in both human embryonic kidney (HEK) 293 cells and Xenopus oocytes were used to test the pharmacological effects of iptakalim. Patch-clamp recordings, Ca(2+) imaging, and measurements of insulin release were applied. Patch-clamp whole-cell recordings revealed that iptakalim depolarized beta-cells, induced action potential firing, and reduced K(ATP) channel-mediated currents. Single-channel recordings revealed that iptakalim reduced the open probability of K(ATP) channels without changing channel sensitivity to ATP. By closing beta-cell K(ATP) channels, iptakalim elevated intracellular Ca(2+) concentrations and increased insulin release. In addition, iptakalim decreased the open probability of recombinant Kir6.2FL4A (a trafficking mutant of the Kir6.2) K(ATP) channels heterologously expressed in HEK 293 cells, suggesting that iptakalim suppressed the function of beta-cell K(ATP) channels by directly inhibiting the Kir6.2 subunit. Finally, iptakalim inhibited Kir6.2/SUR1, but it activated Kir6.1/SUR2B (vascular-type), K(ATP) channels heterologously expressed in Xenopus oocytes. Iptakalim bidirectionally regulated pancreatic-type and vascular-type K(ATP) channels, and this unique pharmacological property suggests the potential use of iptakalim as a new therapeutic strategy for treating type 2 diabetes with the additional benefit of alleviating vascular disorders.

Iptakalim modulates ATP-sensitive K(+) channels in dopamine neurons from rat substantia nigra pars compacta.

Iptakalim, a novel cardiovascular ATP-sensitive K(+) (K(ATP)) channel opener, exerts neuroprotective effects on dopaminergic (DA) neurons against metabolic stress-induced neurotoxicity, but the mechanisms are largely unknown. Here, we examined the effects of iptakalim on functional K(ATP) channels in the plasma membrane (pm) and mitochondrial membrane using patch-clamp and fluorescence-imaging techniques. In identified DA neurons acutely dissociated from rat substantia nigra pars compacta (SNc), both the mitochondrial metabolic inhibitor rotenone and the sulfonylurea receptor subtype (SUR) 1-selective K(ATP) channel opener (KCO) diazoxide induced neuronal hyperpolarization and abolished action potential firing, but the SUR2B-selective KCO cromakalim exerted little effect, suggesting that functional K(ATP) channels in rat SNc DA neurons are mainly composed of SUR1. Immunocytochemical staining showed a SUR1-rather than a SUR2B-positive reaction in most dissociated DA neurons. At concentrations between 3 and 300 microM, iptakalim failed to hyperpolarize DA neurons; however, 300 microM iptakalim increased neuronal firing. In addition, iptakalim restored DA neuronal firing during rotenone-induced hyperpolarization and suppressed rotenone-induced outward current, suggesting that high concentrations of iptakalim close neuronal K(ATP) channels. Furthermore, in human embryonic kidney 293 cells, iptakalim (300-500 microM) closed diazoxide-induced Kir6.2/SUR1 K(ATP) channels, which were heterologously expressed. In rhodamine-123-preloaded DA neurons, iptakalim neither depolarized mitochondrial membrane nor prevented rotenone-induced mitochondrial depolarization. These data indicate that iptakalim is not a K(ATP) channel opener in rat SNc DA neurons; instead, iptakalim is a pm-K(ATP) channel closer at high concentrations. These effects of iptakalim stimulate further pharmacological investigation and the development of possible therapeutic applications.

Novel mechanism of chronic exposure of oleic acid-induced insulin release impairment in rat pancreatic beta-cells.

A sustained, high circulating level of free fatty acids (FFAs) is an important risk factor for the development of insulin resistance, islet beta-cell dysfunction, and pathogenesis of type 2 diabetes. Here, we report a novel mechanism of chronic exposure of oleic acid (OA)-induced rat insulin release impairment. Following a 4-day exposure to 0.1 mM OA, there was no significant difference in basal insulin release when comparing OA-treated and untreated islets in the presence of 2.8 mM glucose, whereas 16.7 mM glucose-stimulated insulin release increased 2-fold in control, but not in OA-treated, islets. Perforated patch-clamp recordings showed that untreated beta-cells exhibited a resting potential of -62.1 +/- 0.9 mV and were electrically silent, whereas OA-treated beta-cells showed more positive resting potentials and spontaneous action potential firing. Cell-attached single-channel recordings revealed spontaneous opening of ATP-sensitive potassium (K(ATP)) channels in control, but not in OA-treated, beta-cells. Inside-out excised patch recordings showed similar activity in both OA-treated and untreated beta-cells in the absence of ATP on the inside of the cellular membrane, whereas in the presence of ATP, K(ATP) channel activity was significantly reduced in OA-treated beta-cells. Electron microscopy demonstrated that chronic exposure to OA resulted in the accumulation of triglycerides in beta-cell cytoplasm and reduced both the number of insulin-containing granules and insulin content. Collectively, chronic exposure to OA closed K(ATP) channels by increasing the sensitivity of K(ATP) channels to ATP, which in turn led to the continuous excitation of beta-cells, depletion of insulin storage, and impairment of glucose-stimulated insulin release.

Modulation of Ca2+ influx by corticotropin-releasing factor (CRF) family of peptides via CRF receptors in rat pancreatic beta-cells.

The actions of the corticotropin-releasing factor (CRF) family of peptides are mediated by the seven transmembrane-domain G-protein-coupled receptors, the CRF receptors type 1 (CRF1 receptor) and type 2 (CRF2 receptor). In a previous study, we reported that CRF, an endogenous ligand for CRF1 receptor, modulated Ca2+ influx in rat pancreatic beta-cells. In addition to CRF, other additional members of the family, urocortins, have been identified in mammals. Urocortin 1 (UCN 1), a peptide of the CRF family, binds both CRF1 receptor and CRF2 receptor with equal affinities. Urocortin 3 (UCN 3), a highly selective ligand for CRF2 receptor with little affinity for CRF1 receptor, has been shown in rat pancreatic beta-cells. The present study focused on the effects of the CRF family peptides on intracellular Ca2+ ([Ca2+]i) concentration via CRF receptors in rat pancreatic beta-cells. Microfluorimetric experiments showed that CRF (0.2 nM) and UCN 1 (0.2 nM) elevated [Ca2+]i levels. Both CRF and UCN 1 effects were attenuated by astressin, a non-selective CRF receptor antagonist. Antisauvagine-30, a selective CRF2 receptor antagonist, appeared to enhance the UCN 1 effect on the elevation of [Ca2+]i. The CRF effect on the elevation of [Ca2+]i was inhibited by the addition of UCN 3. Taken together, the activation of CRF2 receptor antagonizes the CRF1 receptor-stimulated Ca2+ influx.

Staphylococcal enterotoxin A modulates intracellular Ca2+ signal pathway in human intestinal epithelial cells.

We demonstrate here that staphylococcal enterotoxin A (SEA) induces an increase in intracellular calcium ([Ca2+]i) in human intestinal epithelial cells and the [Ca2+]i is released from intracellular stores. SEA-induced increase of [Ca2+]i was clearly inhibited by treatment with a nitric oxide synthase (NOS) inhibitors, N(G)-monomethyl-L-arginine and guanidine. Intestinal epithelial cells express endothelial NOS in resting cell condition, and express inducible NOS after stimulating with tumor necrosis factor (TNF)-alpha. TNF-alpha-pretreated cells showed a significant increase in [Ca2+]i that was also inhibited by the NOS inhibitor. These results suggest that SEA modulated [Ca2+]i signal is dependent on NOS expression in human intestinal epithelial cells.

Pertussis toxin-sensitive pathway inhibits glucose-stimulated Ca2+ signals of rat islet beta-cells by affecting L-type Ca2+ channels and voltage-dependent K+ channels.

A role of pertussis toxin (PTX)-sensitive pathway in regulation of glucose-stimulated Ca2+ signaling in rat islet beta-cells was investigated by using clonidine as a selective agonist to alpha2-adrenoceptors which link to the pathway. An elevation of extracellular glucose concentration from 5.5 to 22.2 mM (glucose stimulation) increased the levels of [Ca2+]i of beta-cells, and clonidine reversibly reduced the elevated levels of [Ca2+]i. This clonidine effect was antagonized by yohimbine, and abolished in beta-cells pre-treated with PTX. Clonidine showed little effect on membrane currents including those through ATP-sensitive K+ channels induced by voltage ramps from -90 to -50 mV. Clonidine showed little effect on the magnitude of whole-cell currents through L-type Ca2+ channels (ICa(L)), but increased the inactivation process of the currents. Clonidine increased the magnitude of the voltage-dependent K+ currents (IVK). These clonidine effects on ICa(L) and IVK were abolished in beta-cells treated with PTX or GDP-betaS. These results suggest that the PTX-sensitive pathway increases IVK activity and decreases ICa(L) activity of islet beta-cells, resulting in a decrease in the levels of [Ca2+]i elevated by depolarization-induced Ca2+ entry. This mechanism seems responsible at least in part for well-known inhibitory action of PTX-sensitive pathway on glucose-stimulated insulin secretion from islet beta-cells.

2-aminoethoxydiphenyl borate inhibits agonist-induced Ca2+ signals by blocking inositol trisphosphate formation in acutely dissociated mouse pancreatic acinar cells.

Evidence suggests that 2-aminoethoxydiphenyl borate (2-APB) modulates intracellular Ca(2+) signals in a complex manner. 2-APB inhibits or potentiates intracellular Ca(2+) signals in different cell types, perhaps through different mechanisms. Here, we report a novel mechanism underlying 2-APB-induced inhibition of agonist-activated Ca(2+) oscillations in mouse pancreatic acinar cells, using patch-clamp and biochemical techniques. Pre-treatment of the cells with 100 microM 2-APB completely abolished ACh- but not inositol trisphosphate (InsP(3))-induced Ca(2+) oscillations, suggesting that the mechanism of inhibition occurs between cytoplasmic receptors and InsP(3) receptor activation. In addition, 100 microM 2-APB significantly inhibited ACh-induced phospholipase C (PLC) activation. These findings indicate that, in mouse pancreatic acinar cells, in addition to modulating InsP(3) receptors and blocking the store-operated Ca(2+) pathway, high concentrations of 2-APB also inhibit agonist-induced Ca(2+) signals by reducing InsP(3) formation.

Masked excitatory action of noradrenaline on rat islet beta-cells via activation of phospholipase C.

The effect of noradrenaline (NE) on rat islet beta-cells was examined. NE reduced insulin secretion from rat islets exposed to extracellular solutions containing glucose at 5.5 or 16.6 mM. In islets treated with pertussis toxin (PTX), however, NE increased insulin secretion. The NE-induced augmentation of insulin secretion was inhibited by prazosin. In intact islets, NE increased phospholipase C (PLC) activity, an effect that was prevented by treatment of islets with U-73122. NE elevated intracellular [Ca2+] ([Ca2+]i) in isolated beta-cells independently of PTX. Although this NE effect was inhibited by prazosin, phenylephrine did not mimic it. The [Ca2+]i response to NE was also prevented by the treatment of cells with U-73122. NE produced depolarization of beta-cells followed by nifedipine-sensitive action potentials. NE reduced the whole-cell membrane currents through ATP-sensitive K+ channels (KATP), responsible for the depolarization. This NE effect was prevented by treatment of beta-cells with U-73122 or BAPTA/AM. Although at least some of our results imply the presence of alpha1-adrenoceptors, beta-cells were not stained by a polyclonal IgG antibody recognizing all adrenergic alpha1-receptor subtypes so far identified. These results suggest that an interaction of NE with an unknown type of receptor activates rat islet beta-cells via a PLC-dependent signal pathway. This effect is, however, masked by the inhibitory action via a PTX-sensitive pathway also activated by NE.

Intracellular Ca(2+) modulation of ATP-sensitive K(+) channel activity in acetylcholine-induced activation of rat pancreatic beta-cells.

We investigated the mechanism by which acetylcholine (ACh) regulates insulin secretion from rat pancreatic beta-cells. In an extracellular solution with 5.5 mM glucose, ACh increased the rate of insulin secretion from rat islets. In islets treated with bisindolylmaleimide (BIM), a PKC inhibitor, ACh still increased insulin secretion, but the increment was lower than that without BIM. In the presence of nifedipine, an L-type Ca(2+) channel blocker, on the other hand, ACh did not increase insulin secretion. In isolated rat pancreatic beta-cells, ACh caused depolarization followed by action potentials. This ACh effect was observed even in cells treated with BIM. In the presence of nifedipine, ACh caused only depolarization. These ACh effects were prevented by atropine. In the perforated whole-cell configuration, ramp pulses from -90 to -50 mV induced membrane currents mostly through ATP-sensitive K(+) channels (K(ATP)). These currents were reduced in size by ACh in cells either treated or untreated with BIM; whereas the loading of cells with U-73122 (a phospholipase C inhibitor) or BAPTA/AM (a Ca(2+) chelator) abolished the ACh effect. In the standard whole-cell configuration, ACh reduced the currents through K(ATP) with 0.5 mM EGTA, but not with 10 mM EGTA, in the pipette solution. Intracellular application of GDPbetaS or heparin also inhibited the ACh effect. In the inside-out single-channel recordings, elevation of the Ca(2+) concentration inside the membrane from 10 nM-10 microM decreased K(ATP) activity only in the presence of ATP. The affinity of ATP to K(ATP) became 4.5 times higher with the higher concentration of Ca(2+). These results suggest that Ca(2+) from ACh receptor signaling modulates the sensitivity of K(ATP) to ATP. A positive-feedback mechanism of intracellular Ca(2+)-dependent Ca(2+) influx was also demonstrated.

Role of mitochondrial NADH shuttle system in acute amylase secretion by acetylcholine from mouse pancreatic acinar cells.

Using the mice that lack mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH), a rate limiting enzyme of the glycerol-phosphate NADH shuttle, we investigated the role of the NADH shuttle system in amylase secretion in response to acetylcholine (ACh) in pancreatic acinar cells. The pancreatic acinar cells of mGPDH-deficient mice were not different in histology and immunohistochemistry from those of wild-type mice. In both types of pancreatic acinar cells from wild-type and mGPDH-deficient mice, ACh similarly potentiated amylase secretion, measured in 30 minutes after the ACh stimulation. A 30 minutes pre-treatment of wild-type cells with aminooxyacetate (AOA), an inhibitor of aspartate aminotransferases of the malate-aspartate NADH shuttle, did not change the rate of ACh-induced amylase secretion, measured in the following 30 minutes. In also mGPDH-deficient cells treated with AOA, thus in this situation all mitochondrial NADH shuttles being dysfunctioning, ACh induced amylase release in a similar amount to that in AOA-untreated cells. The basal levels of intracellular Ca2+ concentration ([Ca2+]i), the ACh-stimulated levels of [Ca2+]i and Ca2+ oscillation patterns in response to ACh were similar in wild-type and mGPDH-deficient cells, and the AOA-treatment did not affect these [Ca2+]i responses. The levels of intracellular concentration of ATP before and during stimulation with ACh were similar in wild-type and mGPDH-defficient cells. In only AOA-treated mGPDH-deficient cells, the level of ATP decreased after the ACh stimulation. These results suggest that acute response of amylase secretion to ACh from mouse pancreatic acinar cells does not require simultaneous functioning of the mitochondrial NADH shuttle system, although the supply of intracellular ATP decreases during the ACh stimulation.