ABSTRACT
AIMS: To evaluate the acute effects of the mitochondrial complex I inhibitor rotenone on rat hippocampal synaptic plasticity. METHODS: Electrophysiological field potential recordings were used to measure basal synaptic transmission and synaptic plasticity in rat coronal hippocampal slices. Synaptic long-term potentiation (LTP) was induced by high-frequency stimulation (100 Hz, 1 second × 3 at an interval of 20 seconds). In addition, mitochondrial complex I function was measured using MitoSOX imaging in mitochondrial preparations. RESULTS: Acute exposure of hippocampal slices to 50 nM rotenone for 1 h did not alter basal CA3-CA1 synaptic transmission though 500 nM rotenone significantly reduced basal synaptic transmission. However, 50 nM rotenone significantly impaired LTP and this rotenone's effect was prevented by co-application of rotenone plus the ketones acetoacetate and ß-hydroxybutyrate (1 mM each). Finally, we measured mitochondrial function using MitoSOX imaging in mitochondrial preparations and found that 50 nM rotenone partially reduced mitochondrial function whereas 500 nM rotenone completely eliminated mitochondrial function. CONCLUSIONS: Our findings suggest that mitochondrial activity driven by complex I is a sensitive modulator of synaptic plasticity in the hippocampus. Acute exposure of the hippocampus to rotenone eliminates complex I function and in turn impairs LTP.
Subject(s)
Electron Transport Complex I/drug effects , Hippocampus/drug effects , Long-Term Potentiation/drug effects , Rotenone/toxicity , Synapses/drug effects , Uncoupling Agents/toxicity , 3-Hydroxybutyric Acid/pharmacology , Acetoacetates/pharmacology , Animals , CA1 Region, Hippocampal/drug effects , Electrophysiological Phenomena , In Vitro Techniques , Ketones/pharmacology , Mitochondria/drug effects , Mitochondria/metabolism , Neuronal Plasticity/drug effects , Oxygen Consumption/drug effects , Rats , Rats, Wistar , Synaptic Transmission/drug effectsABSTRACT
Many patients with type 2 diabetes mellitus (DM2) are at risk for micro and macro vascular complications, which could be observed in heavy smokers. Cigarette smoking increases the risk for type 2 diabetes incidence. Nicotine, acknowledged as the major pharmacologically active chemical in tobacco, is responsible for the association between cigarette smoking and development of diabetes. This minireview summarized recent studies on nicotine effects on insulin action and insulin secretion, indicating the impact of nicotine on type 2 diabetes development.
Subject(s)
Diabetes Mellitus, Type 2/etiology , Nicotine/adverse effects , Smoking/adverse effects , Animals , Diabetes Mellitus, Type 2/complications , Humans , Insulin/metabolism , Insulin Resistance , Insulin Secretion , Microcirculation/drug effects , Nicotinic Agonists/adverse effects , Risk FactorsABSTRACT
In this study, we evaluate the effects of (3beta)-3-[2-(diethylamino)ethoxy]androst-5-en-17-one dihydrochloride (U18666A), a cholesterol synthesis/transporter inhibitor, on selected human neuronal nicotinic acetylcholine receptors (nAChRs) heterologously expressed in the SH-EP1 cell line using whole-cell patch-clamp recordings. The results indicate that with 2-min pretreatment, U18666A inhibited different nAChR subtypes with a rank-order of potency (IC(50) of whole-cell peak current): alpha4beta2 (8.0 +/- 3.0 nM) > alpha3beta2 (1.7 +/- 0.4 microM) > alpha4beta4 (26 +/- 7.2 microM) > alpha7 (> 100 microM), suggesting this compound is more selective to alpha4beta2-nAChRs. Thus, the pharmacological profiles and mechanisms of U18666A acting on alpha4beta2-nAChRs were investigated in detail. U18666A suppresses both peak and steady state components of whole-cell currents mediated by human alpha4beta2-nAChRs in response to nicotine. In nicotine-induced concentration-response curves, U18666A reduces nicotine-induced current at maximally effective agonist concentrations without influencing nicotine's EC(50) value, suggesting a non-competitive inhibition. U18666A-induced inhibition of nAChR function is concentration-, voltage-, and use-dependent, suggesting an open channel block. Taken into consideration of approximately 10 000-fold enhancement of the potency of U18666A after 2-min pre-treatment, this compound also likely inhibits alpha4beta2-nAChRs through a close channel block. In addition, the U18666A-induced inhibition in alpha4beta2-nAChRs is not mediated by either increased receptor endocytosis or altered cell cholesterol. These data indicate that U18666A is a potent antagonist of alpha4beta2-nAChRs and may be useful as a tool in the functional characterization and pharmacological profiling of nAChRs, as well as a potential candidate for smoking cessation.
Subject(s)
Androstenes/pharmacology , Anticholesteremic Agents/pharmacology , Gene Expression/drug effects , Receptors, Nicotinic/drug effects , Receptors, Nicotinic/metabolism , Androstenes/chemistry , Biophysical Phenomena/physiology , Cell Line, Transformed , Dose-Response Relationship, Drug , Electric Stimulation/methods , Epithelial Cells/drug effects , Epithelial Cells/physiology , Guanosine Diphosphate/analogs & derivatives , Guanosine Diphosphate/pharmacology , Humans , Membrane Potentials/drug effects , Nicotine/pharmacology , Patch-Clamp Techniques , Receptors, Nicotinic/genetics , Thionucleotides/pharmacology , trans-1,4-Bis(2-chlorobenzaminomethyl)cyclohexane Dihydrochloride/chemistry , trans-1,4-Bis(2-chlorobenzaminomethyl)cyclohexane Dihydrochloride/pharmacologyABSTRACT
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.
Subject(s)
Calcium/metabolism , Hepatocytes/drug effects , Hepatocytes/metabolism , Hydrogen Peroxide/pharmacology , Oxidants/pharmacology , Adenosine Triphosphate/metabolism , Amiloride/pharmacology , Animals , Enzyme Inhibitors/pharmacology , Hepatocytes/cytology , Male , Membrane Potential, Mitochondrial/drug effects , Membrane Potential, Mitochondrial/physiology , Membrane Potentials/physiology , Mitochondria/drug effects , Mitochondria/metabolism , Oxidation-Reduction , Patch-Clamp Techniques , Rats , Rats, Wistar , Reperfusion Injury/metabolism , Signal Transduction/physiology , Sodium Channel Blockers/pharmacology , Sodium Cyanide/pharmacology , Type C Phospholipases/antagonists & inhibitors , Type C Phospholipases/metabolismABSTRACT
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.
Subject(s)
Diabetes Mellitus, Experimental/pathology , Diabetes Mellitus, Type 2/pathology , Dietary Sucrose/pharmacology , Insulin-Secreting Cells/pathology , Mitochondria/pathology , Animals , Blood Glucose/metabolism , Body Weight/drug effects , Cell Death/drug effects , Dietary Sucrose/adverse effects , Disease Models, Animal , Insulin/blood , Insulin-Secreting Cells/drug effects , Membrane Potential, Mitochondrial/drug effects , Mitochondria/drug effects , Mitochondria/ultrastructure , Rats , Rats, WistarABSTRACT
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.
Subject(s)
Insulin-Secreting Cells/drug effects , Insulin/metabolism , Potassium Channel Blockers/pharmacology , Potassium Channels, Inwardly Rectifying/physiology , Propylamines/pharmacology , Animals , Calcium/metabolism , Cell Line , Cells, Cultured , Diazoxide/pharmacology , Dose-Response Relationship, Drug , Female , Glucose/pharmacology , Humans , Insulin-Secreting Cells/metabolism , Insulin-Secreting Cells/physiology , Ion Channel Gating/drug effects , KATP Channels , Membrane Potentials/drug effects , Nifedipine/pharmacology , Oocytes/drug effects , Oocytes/metabolism , Oocytes/physiology , Potassium Channels, Inwardly Rectifying/antagonists & inhibitors , Potassium Channels, Inwardly Rectifying/genetics , Rats , Tolbutamide/pharmacology , Xenopus laevisABSTRACT
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.
Subject(s)
Adenosine Triphosphate/pharmacology , Potassium Channels/drug effects , Propylamines/pharmacology , Substantia Nigra/drug effects , ATP-Binding Cassette Transporters/drug effects , Animals , Humans , Membrane Potentials/drug effects , Multidrug Resistance-Associated Proteins/drug effects , Potassium Channels, Inwardly Rectifying/drug effects , Rats , Rats, Wistar , Receptors, Drug , Rotenone/pharmacology , Sulfonylurea Receptors , Tolbutamide/pharmacologyABSTRACT
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.
Subject(s)
Insulin-Secreting Cells/drug effects , Insulin/metabolism , Oleic Acid/toxicity , Adenosine Triphosphate/pharmacology , Animals , Glucose/pharmacology , Insulin/analysis , Insulin Secretion , Insulin-Secreting Cells/chemistry , Insulin-Secreting Cells/metabolism , Male , Membrane Potentials/drug effects , Potassium Channels, Inwardly Rectifying/drug effects , Potassium Channels, Inwardly Rectifying/physiology , Rats , Rats, Sprague-DawleyABSTRACT
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.
Subject(s)
Calcium/metabolism , Corticotropin-Releasing Hormone/metabolism , Insulin-Secreting Cells/metabolism , Animals , Corticotropin-Releasing Hormone/pharmacology , Dose-Response Relationship, Drug , Ligands , Peptide Fragments/pharmacology , Peptides/chemistry , Rats , Rats, Wistar , Receptors, Corticotropin-Releasing Hormone/metabolism , UrocortinsABSTRACT
We designed and fabricated an array of sugar micro needles of the length ranging from 150 micro m to 2 mm for transdermic delivery of drugs. Micro needles were molded out of maltose mixed with pharmaceutical material, being expected bio-degradable in the human skin. To test basic tolerance to the healthy human skin, a clinical experiment was carried out for 10 healthy adult volunteers. 500 microm-needles containing 5 wt% of ascorbate-2-glycoside were inserted into the skin of the forearm and snapped off to be left in the skin. They spontaneously dissolved by hydrolysis to release ascorbate in the epidermis and the dermis. No dermatological problems were observed in terms of the International Contact Dermatitis Research Group criteria. These observations indicate that the present system is a novel approach to achieve transdermic drug delivery.
Subject(s)
Drug Delivery Systems/instrumentation , Glycosides/administration & dosage , Injections, Subcutaneous/instrumentation , Microinjections/instrumentation , Needles/adverse effects , Adult , Biocompatible Materials/adverse effects , Dermatitis, Contact/etiology , Dermatitis, Contact/pathology , Drug Delivery Systems/adverse effects , Drug Delivery Systems/methods , Equipment Design , Equipment Failure Analysis , Female , Humans , Injections, Subcutaneous/adverse effects , Injections, Subcutaneous/methods , Male , Materials Testing , Microinjections/methods , MiniaturizationABSTRACT
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.
Subject(s)
Calcium Signaling/drug effects , Enterotoxins/pharmacology , Intestinal Mucosa/metabolism , Nitric Oxide Synthase/metabolism , Calcium/metabolism , Cations, Divalent/metabolism , Guanidine/pharmacology , Humans , Intestinal Mucosa/drug effects , Intestinal Mucosa/enzymology , Ion Transport , Nitric Oxide Synthase/antagonists & inhibitors , Tumor Necrosis Factor-alpha/pharmacology , omega-N-Methylarginine/pharmacologyABSTRACT
Hippocampal gamma oscillations, as a form of neuronal network synchronization, are speculated to be associated with learning, memory and attention. Nicotinic acetylcholine receptor alpha7 subtypes (alpha7-nAChRs) are highly expressed in hippocampal neurons and play important roles in modulating neuronal function, synaptic plasticity, learning and memory. However, little is known about the role of alpha7-nAChRs in hippocampal gamma oscillations. Here, we examined the effects of selective alpha7- and non-alpha7-nAChR antagonists on tetanic gamma oscillations in rat hippocampal slices. We found that brief tetanic stimulation-induced gamma oscillations (30-80 Hz) and pharmacological blockade of alpha7-nAChRs using the relatively selective alpha7-nAChR antagonists, methyllycaconitine (10 or 100 nM) or alpha-bungarotoxin (10 nM), significantly reduced the frequency spectrum power, the number of spikes, and burst duration of evoked gamma oscillations. Neither mecamylamine nor dihydro-beta-erythroidine, which are selective antagonists of non-alpha7-nAChRs, demonstrated significant effects on tetanic gamma oscillations. Nicotine exposure promotes hippocampal gamma oscillations in a methyllycaconitine-sensitive manner. It is concluded that alpha7-nAChRs in hippocampal slices play important roles in regulation of gamma oscillations, thus potentially helping to explain roles of nAChRs in cognitive functions such as learning, memory and attention.
Subject(s)
Aconitine/analogs & derivatives , Biological Clocks/radiation effects , Electric Stimulation/methods , Hippocampus/physiology , Hippocampus/radiation effects , Receptors, Nicotinic/physiology , Aconitine/pharmacology , Animals , Animals, Newborn , Bicuculline/pharmacology , Biological Clocks/drug effects , Biological Clocks/physiology , Bungarotoxins/pharmacology , Dose-Response Relationship, Drug , Dose-Response Relationship, Radiation , Electrophysiology/methods , GABA Antagonists/pharmacology , Hippocampus/cytology , Hippocampus/drug effects , In Vitro Techniques , Nicotine/pharmacology , Nicotinic Agonists/pharmacology , Nicotinic Antagonists/pharmacology , Rats , Rats, Wistar , Spectrum Analysis , Time Factors , alpha7 Nicotinic Acetylcholine ReceptorABSTRACT
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.
Subject(s)
Calcium Channels, L-Type/metabolism , Calcium Signaling/physiology , Glucose/metabolism , Islets of Langerhans/metabolism , Pertussis Toxin/pharmacology , Potassium Channels, Voltage-Gated/metabolism , Animals , Calcium Signaling/drug effects , Cells, Cultured , Clonidine/pharmacology , Dose-Response Relationship, Drug , Male , Membrane Potentials/drug effects , Membrane Potentials/physiology , Rats , Rats, WistarABSTRACT
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.
Subject(s)
Boron Compounds/pharmacology , Calcium Signaling/drug effects , Calcium/metabolism , Pancreas/drug effects , Acetylcholine/antagonists & inhibitors , Animals , Calcium Signaling/physiology , Cells, Cultured , Inositol 1,4,5-Trisphosphate/antagonists & inhibitors , Mice , Mice, Inbred ICR , Pancreas/metabolism , Type C Phospholipases/metabolismABSTRACT
BACKGROUND: To better understand the pathogenesis of photodynamic therapy (PDT)-induced apoptosis cytosolic calcium [Ca2+]i was measured using cultured fetal rat keratinocytes (FRSKs). Moreover, the influence of 1,25 dihydroxy vitamin D3 (1,25(OH)2D3) with the action of increasing [Ca2+]i on the PDT effect was studied. METHODS: FRSKs were treated with a medium containing the photosensitizer, aluminum phthalocyanine tetrasulfonate (AlPcTs), and were then exposed to selective visible light derived from a halogen lamp. Electrophoresis of DNA extracted from the PDT-treated cells revealed DNA fragmentation, a sign of apoptosis in cultured FRSKs under the condition with or without 1,25(OH)2D3. RESULTS: PDT-treated FRSKs exhibited increased levels of [Ca2+]i; these levels were significantly elevated further by the treatment of cells with 1,25(OH)2D3. However, cells treated with ethylene glycol bis (b-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), a chelator of extracellular calcium, prior to PDT did not show any DNA fragmentation either in the presence or absence of 1,25(OH)2D3. CONCLUSION: PDT-induced apoptosis in FRSKs may be caused by the influx of extracellular calcium. Addition of 1,25(OH)2D3 clearly enhanced the DNA fragmentation in the cultured FRSKs, indicating the effect of increased [Ca2+]i. The combination therapy of AlPcTs-PDT with the administration of 1,25(OH)2D3 may contribute to the enhancement of the AlPcTs-PTD effect.
Subject(s)
Calcitriol/pharmacology , Calcium/pharmacology , Keratinocytes/drug effects , Keratinocytes/radiation effects , Ultraviolet Rays , Animals , Apoptosis/drug effects , Apoptosis/radiation effects , Calcitriol/administration & dosage , Calcium/administration & dosage , DNA/analysis , DNA Fragmentation/drug effects , DNA Fragmentation/radiation effects , Embryo, Mammalian , Keratinocytes/metabolism , Photochemotherapy , RatsABSTRACT
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.
Subject(s)
Action Potentials/drug effects , Islets of Langerhans/drug effects , Norepinephrine/pharmacology , Type C Phospholipases/metabolism , Action Potentials/physiology , Animals , Enzyme Activation/drug effects , Enzyme Activation/physiology , Islets of Langerhans/enzymology , Male , Membrane Potentials/drug effects , Membrane Potentials/physiology , Rats , Rats, WistarABSTRACT
Hyperthermic spreading depression (HSD) in immature rat hippocampal slices is mediated by Na+/K(+)-ATPase failure. Here, we test whether depleting intracellular ATP serves as a possible mechanism for HSD genesis. Results indicate that (1) pre-incubation with 3 mM creatine for 3 h failed to prevent hyperthermic spreading depression occurrence; and (2) intracellular ATP concentration doubled during experimental hyperthermia. This study suggests that HSD is not be mediated by depletion of intracellular ATP during hyperthermia.
Subject(s)
Cortical Spreading Depression/physiology , Energy Metabolism/physiology , Fever/metabolism , Hippocampus/metabolism , Intracellular Fluid/metabolism , Animals , Animals, Newborn , Cortical Spreading Depression/drug effects , Creatine/pharmacology , Energy Metabolism/drug effects , Hippocampus/drug effects , In Vitro Techniques , Intracellular Fluid/drug effects , Rats , Rats, Sprague-DawleyABSTRACT
Recently, mutations of KCNQ2 or KCNQ3, members of the KCNQ-related K(+)-channel (KCNQ-channel) family, were identified as cause of benign familial neonatal convulsions (BFNC). However, the exact pathogenic mechanisms of age-dependent development and spontaneous remission of BFNC remain to be elucidated. To clarify the age-dependent etiology of BFNC, we determined age-dependent functional switching of KCNQ-channels, GABAergic- and glutamatergic-transmission in rat hippocampus. The effects of inhibitors of KCNQ-channel, GABA- and glutamate-receptors on propagation of neuronal-excitability and neurotransmitter release were determined by 64-channel multielectrode-dish (MED64), whole-cell recording, in vitro release technique and in vivo microdialysis biosensor, using rat hippocampus from day of birth (P0) to postnatal-day 56 (P56). Inhibition of KCNQ-channels enhanced depolarization-induced glutamate and GABA releases during P0-P7, but not during P14-P28. Inhibition of KCNQ-channels magnified neuronal-excitability propagation from P0 to P14: maximal at P3, but this effect disappeared by P28. GABA(A)-receptor inhibition surprisingly reduced neuronal-excitability propagation during P0-P3, but not at P7. AMPA/glutamate-receptors inhibition reduced propagation of neuronal-excitability throughout the study period. KCNQ-channels inhibition shortened spike-frequency adaptation, but this stimulation was more predominant during P<7 than P>14. During the first week of life, KCNQ-channels performed as a predominant inhibitory system, whereas after this period GABAergic-transmission switched from excitatory to inhibitory function. Contrary, glutamatergic-transmission has acquired as excitatory function from P0. These findings suggest that the pathogenic mechanisms of age-dependent development and spontaneous remission of BFNC are, at least partially, associated with the interaction between age-dependent reduction of inhibitory KCNQ-channel activity and age-dependent functional switching of the GABAergic-system from excitatory to inhibitory action in neonatal CNS.
Subject(s)
Aging/physiology , Epilepsy, Benign Neonatal/genetics , Hippocampus/physiology , Potassium Channels/physiology , Animals , Biosensing Techniques , Cortical Spreading Depression/physiology , Glutamic Acid/physiology , Male , Membrane Potentials/physiology , Microdialysis , Neurons/drug effects , Neurons/physiology , Neurotransmitter Agents/metabolism , Potassium/pharmacology , Potassium Channels/deficiency , Potassium Channels/genetics , Rats , Rats, Wistar , Remission, Spontaneous , Synaptic Transmission , gamma-Aminobutyric Acid/physiologyABSTRACT
Phosphodiesterase (PDE) was shown to be downregulated in the failing hearts of transplant recipients, while it was upregulated in hypertrophied hearts induced by isoproterenol and calsequesterin overexpression. We examined the time course of gene expression and the activity of PDE3 and PDE4 in an animal model of salt-induced hypertension, left ventricular hypertrophy, and congestive heart failure (CHF). Dahl salt-sensitive (DS, n = 25) and salt-resistant rats (DR, n = 25) were fed with an 8% NaCl diet after the age of 6 weeks. At 11 weeks (hypertension and hypertrophy stage in DS), PDE4 activity in the heart was higher in DS than in DR. At 18 weeks (hypertension and CHF stage in DS), both PDE3 and PDE4 activity in both the heart and aorta was approximately twofold higher in DS than in DR. The ratios of PDE3 and PDE4 mRNA to GAPDH mRNA in the heart were both approximately twofold higher in DS than in DR at 11 and 18 weeks. The cardiac cyclic adenosine monophosphate content and plasma nitric oxide concentration were higher in DS than in DR at 11 weeks but both of them were lower in DS than in DR at 18 weeks of age. In this animal model, gene expressions of PDE3 and PDE4 were augmented from the hypertrophic stage. PDE3 and PDE4 activities were subsequently enhanced in the CHF stage and seemed to contribute to the development and exacerbation of CHF.
Subject(s)
3',5'-Cyclic-AMP Phosphodiesterases/genetics , Gene Expression Regulation, Enzymologic , Heart Failure/enzymology , Animals , Catecholamines/blood , Cyclic Nucleotide Phosphodiesterases, Type 3 , Cyclic Nucleotide Phosphodiesterases, Type 4 , Disease Models, Animal , Heart Failure/etiology , Hypertension/complications , Hypertrophy, Left Ventricular/enzymology , Rats , Rats, Inbred Dahl , Ventricular Function, LeftABSTRACT
We review a new method to explore the cellular functions in multicellular system by application of the perforated patch-clamp technique to intact pancreatic islet of Langerhans. Using this approach, the integrity of the islet is preserved and intercellular communication via gap junctions and paracrine processes are maintained. By using low-resistance patch electrodes, rapid current responses can be monitored under voltage-clamp control. We have applied this methodology to answer questions not resolved by patch-clamp experiments on isolated single insulin-secreting beta-cells. First, the role of a K(+)-current dependent on Ca(2+)-influx for the termination of burst of action potentials in beta-cells could be documented. Neither the current, nor the bursting pattern of electrical activity is preserved in isolated beta-cells. Second, the conductance of gap junctions (approximately 1 nS) between beta-cells was determined. Third, electrical properties of glucagon-producing alpha- and somatostatin-secreting delta-cells and the different mechanisms for glucose-sensing in these cells could be explored. The findings emanating from these experiments may have implications for neuroscience research such as the mechanism of oscillatory electrical activity in general and processes involved in the glucose-sensing in some neurons, which response to changes of blood glucose concentration.