inositol-1-4-5-trisphosphate and Disease-Models--Animal

Huntington's disease (HD) and spinocerebellar ataxias (SCAs) are autosomal-dominant neurodegenerative disorders. HD is caused by polyglutamine (polyQ) expansion in the amino-terminal region of a protein huntingtin (Htt) and primarily affects medium spiny striatal neurons (MSN). Many SCAs are caused by polyQ-expansion in ataxin proteins and primarily affect cerebellar Purkinje cells. The reasons for neuronal dysfunction and death in HD and SCAs remain poorly understood and no cure is available for the patients. Our laboratory discovered that mutant huntingtin, ataxin-2 and ataxin-3 proteins specifically bind to the carboxy-terminal region of the type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R1), an intracellular Ca(2+) release channel. Moreover, we found that association of mutant huntingtin or ataxins with IP(3)R1 causes sensitization of IP(3)R1 to activation by IP(3) in planar lipid bilayers and in neuronal cells. These results suggested that deranged neuronal Ca(2+) signaling might play an important role in pathogenesis of HD, SCA2 and SCA3. In support of this idea, we demonstrated a connection between abnormal Ca(2+) signaling and neuronal cell death in experiments with HD, SCA2 and SCA3 transgenic mouse models. Additional data in the literature indicate that abnormal neuronal Ca(2+) signaling may also play an important role in pathogenesis of SCAl, SCA5, SCA6, SCA14 and SCA15/16. Based on these results I propose that IP(3)R and other Ca(2+) signaling proteins should be considered as potential therapeutic targets for treatment of HD and SCAs.

Topics: Animals; Ataxins; Calcium Signaling; Disease Models, Animal; Humans; Huntingtin Protein; Huntington Disease; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Mice; Mice, Transgenic; Nerve Tissue Proteins; Neurons; Nuclear Proteins; Receptors, N-Methyl-D-Aspartate; Spinocerebellar Ataxias

Topics: Active Transport, Cell Nucleus; Animals; Autoimmune Diseases; Calcium Signaling; Chromosomes, Human, Pair 7; Cyclic AMP Response Element Modulator; Cyclic AMP-Dependent Protein Kinases; Disease Models, Animal; DNA-Binding Proteins; Genetic Predisposition to Disease; Humans; Inositol 1,4,5-Trisphosphate; Interleukin-2; Isoenzymes; Leukocyte Common Antigens; Lupus Erythematosus, Systemic; MAP Kinase Signaling System; Membrane Proteins; Mice; Mice, Inbred MRL lpr; Models, Immunological; NF-kappa B; Phosphorylation; Protein Processing, Post-Translational; Receptors, Antigen, T-Cell; Repressor Proteins; Signal Transduction; T-Lymphocyte Subsets; Transcription Factor RelA

Pulmonary arterial hypertension (PAH) is a pathophysiological syndrome associated with pulmonary/systemic inflammation. Melatonin relieves PAH, but the molecular mode of action remains unclear. Here, we investigated the role of melatonin in normalizing vascular homeostasis.. Light-time mean serum melatonin concentration was lower in patients with PAH than in normal controls [11.06 ± 3.44 (7.13-15.6) vs. 14.55 ± 1.28 (8.0-19.4) pg/mL], which was negatively correlated with increased serum levels of interleukin-1β (IL-1β) in patients with PAH. We showed that inflammasomes were activated in the PAH mice model and that melatonin attenuated IL-1β secretion. On one hand, melatonin reduced the number of macrophages in lung by inhibiting the endothelial chemokines and adhesion factors. Moreover, use of Il1r-/- mice, Caspase1/11-/- mice, and melatonin-treated mice revealed that melatonin reduced hypoxia-induced vascular endothelial leakage in the lung. On the other hand, we verified that melatonin reduced the formation of inflammasome multiprotein complexes by modulating calcium ions in macrophages using a live cell station, and melatonin decreased inositol triphosphate and increased cAMP. Furthermore, knockdown of melatonin membrane receptors blocked melatonin function, and a melatonin membrane receptors agonist inactivated inflammasomes in macrophages.. Melatonin attenuated inflammasome-associated vascular disorders by directly improving endothelial leakage and decreasing the formation of inflammasome multiprotein complexes in macrophages. Taken together, our data provide a theoretical basis for applying melatonin clinically, and inflammasomes may be a possible target of PAH treatment.

Topics: Adult; Animals; Anti-Inflammatory Agents; Biomarkers; Calcium; Case-Control Studies; Caspase 1; Caspases, Initiator; Cells, Cultured; Cyclic AMP; Disease Models, Animal; Endothelial Cells; Female; Human Umbilical Vein Endothelial Cells; Humans; Inflammasomes; Inositol 1,4,5-Trisphosphate; Interleukin-1beta; Macrophage Activation; Macrophages, Peritoneal; Male; Melatonin; Mice, Inbred C57BL; Mice, Knockout; Middle Aged; Permeability; Pulmonary Arterial Hypertension; Receptors, Interleukin-1 Type I; Receptors, Melatonin; Signal Transduction

Spinocerebellar ataxias 17 (SCA17) is caused by polyglutamine (polyQ) expansion in the TATA box-binding protein (TBP). The selective neurodegeneration in the cerebellum in SCA17 raises the question of why ubiquitously expressed polyQ proteins can cause neurodegeneration in distinct brain regions in different polyQ diseases. By expressing mutant TBP in different brain regions in adult wild-type mice via stereotaxic injection of adeno-associated virus, we found that adult cerebellar neurons are particularly vulnerable to mutant TBP. In SCA17 knock-in mice, mutant TBP inhibits SP1-mediated gene transcription to down-regulate INPP5A, a protein that is highly abundant in the cerebellum. CRISPR/Cas9-mediated deletion of Inpp5a in the cerebellum of wild-type mice leads to Purkinje cell degeneration, and Inpp5a overexpression decreases inositol 1,4,5-trisphosphate (IP

Topics: Animals; Disease Models, Animal; Down-Regulation; Gene Knock-In Techniques; HEK293 Cells; Humans; Inositol 1,4,5-Trisphosphate; Inositol Polyphosphate 5-Phosphatases; Mice; Mice, Transgenic; Peptides; Purkinje Cells; Sp1 Transcription Factor; Spinocerebellar Ataxias; TATA-Box Binding Protein; Trinucleotide Repeat Expansion

Hypertension is a cardiovascular disease that severely impairs human health; however, its specific etiology and pathogenesis are complex. The present study investigated the effects of the calcium sensing receptor (CaSR) on vascular tone in spontaneously hypertensive rats (SHRs), and clarified the role and mechanism of CaSR in regulating this property with respect to the phospholipase C (PLC)‑inositol 1,4,5‑triphosphate (IP3)/adenylate cyclase‑V(AC‑V)/cyclic adenosine monophosphate (cAMP)/renin‑angiotensin system (RAS) pathway in these animals. CaSR protein expression in the mesenteric artery (MA) of rats and CaSR protein expression in SHRs were significantly reduced. Based on wire myography studies, vasoconstriction was significantly augmented and vasodilatation was attenuated in SHRs, and this effect was endothelium‑independent. The CaSR calcimimetic NPSR568 and inhibitor NPS2143 reduced vasoconstriction and enhanced vasodilation in SHRs. Furthermore, pretreatment with PLC‑IP3/AC‑V/cAMP/RAS pathway blockers significantly reduced the vasoconstriction response and enhanced the vasodilator response in SHRs and Wistar‑Kyoto rats (WKY), and these effects were partially dependent on the endothelium. Additionally, pretreatment with CaSR inhibitors were determined to cooperate with the PLC‑IP3/AC‑V/cAMP/RAS pathway inhibitors to significantly reduce vasoconstriction and enhance vasodilation in SHRs and WKY. Our results demonstrated that CaSR is functionally expressed in the MA of SHRs, and that CaSR expression is decreased in SHRs. Additionally, vasoconstriction was enhanced while vasodilatation was attenuated in SHRs; these processes were determined to be endothelium‑independent. CaSR is involved in the regulation of blood pressure and vascular tension in SHRs and WKYs. In association with mechanistic differences, this effect was proposed to be partially endothelium‑dependent and mediated by the PLC‑IP3/AC‑V/cAMP/RAS pathway.

Topics: Adenylyl Cyclases; Animals; Blood Pressure; Cyclic AMP; Disease Models, Animal; Inositol 1,4,5-Trisphosphate; Male; Mesenteric Arteries; Rats; Rats, Inbred SHR; Receptors, Calcium-Sensing; Renin-Angiotensin System; Type C Phospholipases; Vasoconstriction; Vasodilation

Fine-tuning of the activity of inositol 1,4,5-trisphosphate receptors (IP

Topics: Acute Disease; Animals; Calcium Signaling; Disease Models, Animal; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Ion Channel Gating; Male; Mice; Mice, Inbred C57BL; Pancreatitis; Proteolysis; Rats; Rats, Wistar

Hyperalgesic priming, a model of pain chronification in the rat, is mediated by ryanodine receptor-dependent calcium release. Although ryanodine induces priming in both sexes, females are 5 orders of magnitude more sensitive, by an estrogen receptor α (EsRα)-dependent mechanism. An inositol 1,4,5-triphosphate (IP

Topics: Animals; Cells, Cultured; Dinoprostone; Disease Models, Animal; Enzyme Inhibitors; Female; Ganglia, Spinal; Hyperalgesia; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Macrocyclic Compounds; Male; Oligodeoxyribonucleotides, Antisense; Oxazoles; Pain Measurement; Pain Threshold; Rats; Rats, Sprague-Dawley; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sensory Receptor Cells; Sex Characteristics; Thapsigargin

Activation of platelets by subendothelial collagen results in an increase of cytosolic Ca(2+) concentration ([Ca(2+)]i) and is followed by platelet activation and thrombus formation that may lead to vascular occlusion. The present study determined the role of phosphoinositide-dependent protein kinase 1 (PDK1) in collagen-dependent platelet Ca(2+) signaling and ischemic stroke in vivo.. Platelet activation with collagen receptor glycoprotein VI agonists collagen-related peptide or convulxin resulted in a significant increase in PDK1 activity independent of second-wave signaling. PDK1 deficiency was associated with reduced platelet phospholipase Cγ2-dependent inositol-1,4,5-trisphosphate production and intracellular [Ca(2+)]i in response to stimulation with collagen-related peptide or convulxin. The defective increase of [Ca(2+)]i resulted in a substantial defect in activation-dependent platelet secretion and aggregation on collagen-related peptide stimulation. Furthermore, Rac1 activation and spreading, adhesion to collagen, and thrombus formation under high arterial shear rates were significantly diminished in PDK1-deficient platelets. Mice with PDK1-deficient platelets were protected against arterial thrombotic occlusion after FeCl3-induced mesenteric arterioles injury and ischemic stroke in vivo. These mice had significantly reduced brain infarct volumes, with a significantly increased survival of 7 days after transient middle cerebral artery occlusion without increase of intracerebral hemorrhage. Tail bleeding time was prolonged in pdk1(-/-) mice, reflecting an important role of PDK1 in primary hemostasis.. PDK1 is required for Ca(2+)-dependent platelet activation on stimulation of collagen receptor glycoprotein VI, arterial thrombotic occlusion, and ischemic stroke in vivo.

Topics: 3-Phosphoinositide-Dependent Protein Kinases; Animals; Blood Platelets; Calcium Signaling; Collagen; Disease Models, Animal; Genetic Predisposition to Disease; Infarction, Middle Cerebral Artery; Inositol 1,4,5-Trisphosphate; Mice, Knockout; Neuropeptides; Phenotype; Phospholipase C gamma; Platelet Activation; Platelet Adhesiveness; Platelet Aggregation; Platelet Membrane Glycoproteins; rac1 GTP-Binding Protein; Thrombosis; Time Factors

It is important to increase our understanding of gustatory detection of dietary fat and its contribution to fat preference. We studied the roles of the fat taste receptors CD36 and GPR120 and their interactions via Ca(2+) signaling in fungiform taste bud cells (TBC).. We measured Ca(2+) signaling in human TBC, transfected with small interfering RNAs against messenger RNAs encoding CD36 and GPR120 (or control small interfering RNAs). We also studied Ca(2+) signaling in TBC from CD36(-/-) mice and from wild-type lean and obese mice. Additional studies were conducted with mouse enteroendocrine cell line STC-1 that express GPR120 and stably transfected with human CD36. We measured release of serotonin and glucagon-like peptide-1 from human and mice TBC in response to CD36 and GPR120 activation.. High concentrations of linoleic acid induced Ca(2+) signaling via CD36 and GPR120 in human and mice TBC, as well as in STC-1 cells, and low concentrations induced Ca(2+) signaling via only CD36. Incubation of human and mice fungiform TBC with lineoleic acid down-regulated CD36 and up-regulated GPR120 in membrane lipid rafts. Obese mice had decreased spontaneous preference for fat. Fungiform TBC from obese mice had reduced Ca(2+) and serotonin responses, but increased release of glucagon-like peptide-1, along with reduced levels of CD36 and increased levels of GPR120 in lipid rafts.. CD36 and GPR120 have nonoverlapping roles in TBC signaling during orogustatory perception of dietary lipids; these are differentially regulated by obesity.

Topics: Animals; Behavior, Animal; Calcium Signaling; CD36 Antigens; Cell Line; Diet, High-Fat; Disease Models, Animal; Food Preferences; Glucagon-Like Peptide 1; Humans; Inositol 1,4,5-Trisphosphate; Linoleic Acid; Membrane Microdomains; Mice; Mice, Inbred C57BL; Mice, Knockout; Obesity; Receptors, G-Protein-Coupled; RNA Interference; Serotonin; Taste; Taste Buds; Taste Perception; Transfection

Sleep apnea is associated with cardiovascular disease, and patients with sleep apnea have elevated plasma endothelin (ET)-1 concentrations. Rats exposed to intermittent hypoxia (IH), a model of sleep apnea, also have increased plasma ET-1 concentrations and heightened constriction to ET-1 in mesenteric arteries without an increase in global vascular smooth muscle cell Ca(2+) concentration ([Ca(2+)]). Because ET-1 has been shown to increase the occurrence of propagating Ca(2+) waves, we hypothesized that ET-1 increases Ca(2+) wave activity in mesenteric arteries, rather than global [Ca(2+)], to mediate enhanced vasoconstriction after IH exposure. Male Sprague-Dawley rats were exposed to sham or IH conditions for 7 h/day for 2 wk. Mesenteric arteries from sham- and IH-exposed rats were isolated, cannulated, and pressurized to 75 mmHg to measure ET-1-induced constriction as well as changes in global [Ca(2+)] and Ca(2+) wave activity. A low concentration of ET-1 (1 nM) elicited similar vasoconstriction and global Ca(2+) responses in the two groups. Conversely, ET-1 had no effect on Ca(2+) wave activity in arteries from sham rats but significantly increased wave frequency in arteries from IH-exposed rats. The ET-1-induced increase in Ca(2+) wave frequency in arteries from IH rats was dependent on phospholipase C and inositol 1,4,5-trisphosphate receptor activation, yet inhibition of phospholipase C and the inositol 1,4,5-trisphosphate receptor did not prevent ET-1-mediated vasoconstriction. These results suggest that although ET-1 elevates Ca(2+) wave activity after IH exposure, increases in wave activity are not associated with increased vasoconstriction.

Topics: Animals; Arterial Pressure; Calcium Signaling; Disease Models, Animal; Endothelin-1; Hypoxia; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Male; Mesenteric Arteries; Phosphodiesterase Inhibitors; Rats; Rats, Sprague-Dawley; Second Messenger Systems; Time Factors; Type C Phospholipases; Vasoconstriction; Vasoconstrictor Agents

The mechanisms of autonomic imbalance and subsequent cardiovascular manifestations in HIV-1-infected patients are poorly understood. We report here that HIV-1 transactivator of transcription (Tat, fragment 1-86) produced a concentration-dependent increase in cytosolic Ca(2+) in cardiac-projecting parasympathetic neurons of nucleus ambiguus retrogradely labeled with rhodamine. Using store-specific pharmacological agents, we identified several mechanisms of the Tat-induced Ca(2+) elevation: 1) lysosomal Ca(2+) mobilization, 2) Ca(2+) release via inositol 1,4,5-trisphosphate-sensitive endoplasmic reticulum pools, and 3) Ca(2+) influx via transient receptor potential vanilloid type 2 (TRPV2) channels. Activation of TRPV2, nonselective cation channels, induced a robust and prolonged neuronal membrane depolarization, thus triggering an additional P/Q-mediated Ca(2+) entry. In vivo microinjection studies indicate a dose-dependent, prolonged bradycardic effect of Tat administration into the nucleus ambiguus of conscious rats, in which neuronal TRPV2 played a major role. Our results support previous studies, indicating that Tat promotes bradycardia and, consequently, may be involved in the QT interval prolongation reported in HIV-infected patients. In the context of an overall HIV-dependent autonomic dysfunction, these Tat-mediated mechanisms may account for the higher prevalence of sudden cardiac death in HIV-1-infected patients compared with general population with similar risk factors. Our results may be particularly relevant in view of the recent findings that significant Tat levels can still be identified in the cerebrospinal fluid of HIV-infected patients with viral load suppression due to efficient antiretroviral therapy.

Topics: Animals; Bradycardia; Calcium; Consciousness; Disease Models, Animal; Endoplasmic Reticulum; Female; Inositol 1,4,5-Trisphosphate; Male; Medulla Oblongata; Microinjections; Parasympathetic Nervous System; Peptide Fragments; Rats; Rats, Sprague-Dawley; tat Gene Products, Human Immunodeficiency Virus; TRPV Cation Channels

SKF83959 stimulates the phospholipase Cβ/inositol phosphate 3 pathway, resulting in the activation of Ca(2+)/calmodulin-dependent kinase IIα, which affects the synthesis of brain-derived neurotrophic factor, a neurotrophic factor critical for the pathophysiology of depression. Previous reports showed that SKF83959 elicited antidepressant activity in the forced swim test and tail suspension test as a novel triple reuptake inhibitor. However, there are no studies showing the effects of SKF83959 in a chronic stress model of depression and the role of phospholipase C/inositol phosphate 3/calmodulin-dependent kinase IIα/brain-derived neurotrophic factor pathway in SKF83959-mediated antidepressant effects.. In this study, SKF83959 was firstly investigated in the chronic social defeat stress model of depression. The changes in hippocampal neurogenesis, dendrite spine density, and brain-derived neurotrophic factor signaling pathway after chronic social defeat stress and SKF83959 treatment were then investigated. Pharmacological inhibitors and small interfering RNA/short hairpin RNA methods were further used to explore the antidepressive mechanisms of SKF83959.. We found that SKF83959 produced antidepressant effects in the chronic social defeat stress model and also restored the chronic social defeat stress-induced decrease in hippocampal brain-derived neurotrophic factor signaling pathway, dendritic spine density, and neurogenesis. By using various inhibitors and siRNA/shRNA methods, we further demonstrated that the hippocampal dopamine D5 receptor, phospholipase C/inositol phosphate 3/ calmodulin-dependent kinase IIα pathway, and brain-derived neurotrophic factor system are all necessary for the SKF83959 effects.. These results suggest that SKF83959 can be developed as a novel antidepressant and produces antidepressant effects via the hippocampal D5/ phospholipase C/inositol phosphate 3/calmodulin-dependent kinase IIα/brain-derived neurotrophic factor pathway.

Topics: 2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; Animals; Antidepressive Agents; Behavior, Animal; Brain-Derived Neurotrophic Factor; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Chronic Disease; Depression; Disease Models, Animal; Dopamine Agonists; Dose-Response Relationship, Drug; Enzyme Activation; Hippocampus; Inositol 1,4,5-Trisphosphate; Male; Membrane Glycoproteins; Mice, Inbred C57BL; Protein-Tyrosine Kinases; Receptors, Dopamine D5; RNA Interference; RNA, Small Interfering; Signal Transduction; Social Behavior; Stress, Psychological; Type C Phospholipases

The aim of this study was to investigate the effect of Withania somnifera (WS) extract, withanolide A (WA), and carbamazepine (CBZ) on cerebellar AMPA receptor function in pilocarpine-induced temporal lobe epilepsy (TLE). In the present study, motor learning deficit was studied by rotarod test, grid walk test, and narrow beam test. Motor learning was significantly impaired in rats with epilepsy. The treatment with WS and WA significantly reversed the motor learning deficit in rats with epilepsy when compared with control rats. There was an increase in glutamate content and IP3 content observed in rats with epilepsy which was reversed in WS- and WA-treated rats with epilepsy. alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor dysfunction was analyzed using radiolabeled AMPA receptor binding assay, AMPA receptor mRNA expression, and immunohistochemistry using anti-AMPA receptor antibody. Our results suggest that there was a decrease in Bmax, mRNA expression, and AMPA receptor expression indicating AMPA receptor dysfunction, which is suggested to have contributed to the motor learning deficit observed in rats with epilepsy. Moreover, treatment with WS and WA resulted in physiological expression of AMPA receptors. There was also alteration in GAD and GLAST expression which supplemented the increase in extracellular glutamate. The treatment with WS and WA reversed the GAD and GLAST expression. These findings suggest that WS and WA regulate AMPA receptor function in the cerebellum of rats with TLE, which has therapeutic application in epilepsy.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Anticonvulsants; Carbamazepine; Cerebellum; Disease Models, Animal; Epilepsy, Temporal Lobe; Excitatory Amino Acid Transporter 1; Gene Expression Regulation; Glutamate Decarboxylase; Glutamic Acid; Inositol 1,4,5-Trisphosphate; Learning Disabilities; Linear Models; Locomotion; Male; Motor Activity; Phytotherapy; Pilocarpine; Protein Binding; Psychomotor Performance; Rats; Rats, Wistar; Receptors, AMPA; RNA, Messenger; Time Factors; Tritium; Withania; Withanolides

AβpE3-42 (N-terminal truncated amyloid-β peptide starting with pyroglutamate at the third position) is abundant in Alzheimer's disease (AD) brain and has high aggregation propensity and cellular toxicity. Transgenic TBA42 mice expressing AβpE3-42 exhibit a neurological phenotype evident at the age of 12 months. As AD has a long presymptomatic period, early detection of imminent neurodegeneration is highly desirable. In the present work we used four-month-old presymptomatic TBA42 mice and performed a whole-brain proteome analysis in order to elucidate early AD-related pathological changes and the molecular networks involved. At least three proteins were found to be moderately (by 17% to 28%) but statistically significantly upregulated, including: nectin-like molecule 1 involved in cell-cell adhesion; Homer proteins involved in scaffolding, organizing proteins at synapse and regulating intracellular calcium within neurons; and inositol-trisphosphate 3-kinase A, which is important for InsP3 induced calcium signaling in the brain. Analysis of key nodes (regulatory molecules found on pathway intersections) identified Rho-kinase (ROCK), a serine/threonine kinase and one of the major downstream effectors of the small GTPase Rho, as well as three key nodes of the mTOR/p70S6K signaling pathway previously implicated in multiple fundamental biological processes including synaptic plasticity, and upregulated in AD. These data confirm that AD-typical molecular pathways can be detected by whole-brain shotgun proteomics in young presymptomatic mice long before the onset of behavioral changes.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Brain; Cell Adhesion Molecules, Neuronal; Disease Models, Animal; Gene Expression Regulation; Humans; Inositol 1,4,5-Trisphosphate; Mice; Mice, Inbred C57BL; Mice, Transgenic; Peptide Fragments; Principal Component Analysis; Protein Interaction Mapping; Proteomics; rho-Associated Kinases; Signal Transduction; Tandem Mass Spectrometry; Thyrotropin-Releasing Hormone

We investigated the contribution of inositol(1,4,5)-trisphosphate (Ins(1,4,5)P3 [IP3]) receptors (IP3-R) to disease progression in mouse models of dilated cardiomyopathy (DCM) and pressure overload hypertrophy. Mice expressing mammalian sterile 20-like kinase and dominant-negative phosphatidylinositol-3-kinase in heart (Mst1×dn-PI3K-2Tg; DCM-2Tg) develop severe DCM and conduction block, associated with increased expression of type 2 IP3-R (IP3-R(2)) and heightened generation of Ins(1,4,5)P3. Similar increases in Ins(1,4,5)P3 and IP3-R(2) are caused by transverse aortic constriction.. To evaluate the contribution of IP3-R(2) to disease progression, the DCM-2Tg mice were further crossed with mice in which the type 2 IP3-R (IP3-R(2)-/-) had been deleted (DCM-2Tg×IP3-R(2)-/-) and transverse aortic constriction was performed on IP3-R(2)-/- mice. Hearts from DCM-2Tg mice and DCM-2Tg×IP3-R(2)-/- were similar in terms of chamber dilatation, atrial enlargement, and ventricular wall thinning. Electrophysiological changes were also similar in the DCM-2Tg mice, with and without IP3-R(2). Deletion of IP3-R(2) did not alter the progression of heart failure, because DCM-2Tg mice with and without IP3-R(2) had similarly reduced contractility, increased lung congestion, and atrial thrombus, and both strains died between 10 and 12 weeks of age. Loss of IP3-R(2) did not alter the progression of hypertrophy after transverse aortic constriction.. We conclude that IP3-R(2) do not contribute to the progression of DCM or pressure overload hypertrophy, despite increased expression and heightened generation of the ligand, Ins(1,4,5)P3.

Topics: Animals; Cardiomyopathy, Dilated; Disease Models, Animal; Disease Progression; Electrocardiography; Gene Expression Profiling; Gene Expression Regulation; Genotype; Heart Block; Heart Failure; Hypertrophy, Left Ventricular; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Myocardium; Phenotype; Phosphatidylinositol 3-Kinase; Protein Serine-Threonine Kinases; Time Factors; Ventricular Function, Left; Ventricular Pressure; Ventricular Remodeling

In animals, inositol 1,4,5-trisphosphate receptors (IP3 Rs) are ion channels that play a pivotal role in many biological processes by mediating Ca(2+) release from the endoplasmic reticulum. Here, we report the identification and characterization of a novel IP3 R in the parasitic protist, Trypanosoma cruzi, the pathogen responsible for Chagas disease. DT40 cells lacking endogenous IP3 R genes expressing T. cruzi IP3 R (TcIP3 R) exhibited IP3 -mediated Ca(2+) release from the ER, and demonstrated receptor binding to IP3 . TcIP3 R was expressed throughout the parasite life cycle but the expression level was much lower in bloodstream trypomastigotes than in intracellular amastigotes or epimastigotes. Disruption of two of the three TcIP3 R gene loci led to the death of the parasite, suggesting that IP3 R is essential for T. cruzi. Parasites expressing reduced or increased levels of TcIP3 R displayed defects in growth, transformation and infectivity, indicating that TcIP3 R is an important regulator of the parasite's life cycle. Furthermore, mice infected with T. cruzi expressing reduced levels of TcIP3 R exhibited a reduction of disease symptoms, indicating that TcIP3 R is an important virulence factor. Combined with the fact that the primary structure of TcIP3 R has low similarity to that of mammalian IP3 Rs, TcIP3 R is a promising drug target for Chagas disease.

Topics: Animals; Chagas Disease; Disease Models, Animal; DNA, Protozoan; Gene Expression Regulation; Gene Knockout Techniques; Genes, Essential; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Mice; Molecular Sequence Data; Sequence Analysis, DNA; Trypanosoma cruzi; Virulence; Virulence Factors

This study examines the extent to which the antiapoptotic Bcl-2 proteins Bcl-2 and Bcl-x(L) contribute to diabetic Ca(2+) dysregulation and vessel contractility in vascular smooth muscle cells (VSMCs) through their interaction with inositol 1,4,5-trisphosphate receptor (InsP(3)R) intracellular Ca(2+) release channels. Measurements of intracellular ([Ca(2+)](i)) and sarcoplasmic reticulum ([Ca(2+)](SR)) calcium concentrations were made in primary cells isolated from diabetic (db/db) and nondiabetic (db/m) mice. In addition, [Ca(2+)](i) and constriction were recorded simultaneously in isolated intact arteries. Protein expression levels of Bcl-x(L) but not Bcl-2 were elevated in VSMCs isolated from db/db compared with db/m age-matched controls. In single cells, InsP(3)-evoked [Ca(2+)](i) signaling was enhanced in VSMCs from db/db mice compared with db/m. This was attributed to alterations in the intrinsic properties of the InsP(3)R itself because there were no differences between db/db and db/m in the steady-state [Ca(2+)](SR) or InsP(3)R expression levels. Moreover, in permeabilized cells the rate of InsP(3)R-dependent SR Ca(2+) release was increased in db/db compared with db/m VSMCs. The enhanced InsP(3)-dependent SR Ca(2+) release was attenuated by the Bcl-2 protein inhibitor ABT-737 only in diabetic cells. Application of ABT-737 similarly attenuated enhanced agonist-induced [Ca(2+)](i) signaling only in intact aortic and mesenteric db/db vessels. In contrast, ABT-737 had no effect on agonist-evoked contractility in either db/db or db/m vessels. Taken together, the data suggest that in type 2 diabetes the mechanism for [Ca(2+)](i) dysregulation in VSMCs involves Bcl-2 protein-dependent increases in InsP(3)R excitability and that dysregulated [Ca(2+)](i) signaling does not appear to contribute to increased vessel reactivity.

Topics: Animals; Aorta, Thoracic; bcl-X Protein; Biphenyl Compounds; Blood Glucose; Calcium; Calcium Signaling; Cells, Cultured; Diabetes Mellitus, Type 2; Diabetic Angiopathies; Disease Models, Animal; Dose-Response Relationship, Drug; Homeostasis; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Male; Mice; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Sarcoplasmic Reticulum; Sulfonamides; Time Factors; Up-Regulation; Vasoconstriction

Mutations in amyloid precursor protein (APP), and presenilin-1 and presenilin-2 (PS1 and PS2) have causally been implicated in Familial Alzheimer's Disease (FAD), but the mechanistic link between the mutations and the early onset of neurodegeneration is still debated. Although no consensus has yet been reached, most data suggest that both FAD-linked PS mutants and endogenous PSs are involved in cellular Ca2+ homeostasis. We here investigated subcellular Ca2+ handling in primary neuronal cultures and acute brain slices from wild type and transgenic mice carrying the FAD-linked PS2-N141I mutation, either alone or in the presence of the APP Swedish mutation. Compared with wild type, both types of transgenic neurons show a similar reduction in endoplasmic reticulum (ER) Ca2+ content and decreased response to metabotropic agonists, albeit increased Ca2+ release induced by caffeine. In both transgenic neurons, we also observed a higher ER-mitochondria juxtaposition that favors increased mitochondrial Ca2+ uptake upon ER Ca2+ release. A model is described that integrates into a unifying hypothesis the contradictory effects on Ca2+ homeostasis of different PS mutations and points to the relevance of these findings in neurodegeneration and aging.

Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Animals; Calcium; Disease Models, Animal; Endoplasmic Reticulum; Inositol 1,4,5-Trisphosphate; Mice; Mice, Transgenic; Mitochondria; Neurons; Presenilin-2; Ryanodine

To compare the effects of instant electroacupuncture (EA) at the different acupoints on IP3 in the uterus tissue of dysmenorrhea model rats so as to investigate the specificity of acupoints.. Fifty female SD rats were randomly divided into a saline group, a model group, a Sanyinjiao (SF 6) group, a Xuehai (SP 10) group and a Hegu (LI 4) group, 10 rats in each group. The rats were given subcutaneous injection of Estradiol Beozoate injection for 10 consecutive days except those in the saline group, and intraperitoneal injection of 2U Oxytocin at 1 h after the last administration to create the dysmenorrhea rats model, and the saline group was given the same dose of saline every day. On the 10th day the rats in each EA group were given EA 20 min, and the rats in the saline group and model group were bound 20 min, and the writhing response was observed at the same time. The uterine IP3 contents were detected with enzyme-linked immunosorbent assay method.. (1) Compared with (0.311+/- 0.253) in the saline group, the writhing scores per minute of (5.867 +/- 3.442) in the model group and (2.311 +/- 0.957) in the Xuehai (SP 10) group were both increased significantly (P < 0.01, P < 0.05), and (1.833 +/- 1.355) in the Sanyinjiao (SP 6) group and (0.743 +/- 0.306) in the Hegu (LI 4) group showed no significant differences (P > 0.05). Compared with that in the model group, the writhing scores per minute decreased significantly (all P < 0.01) in all the EA groups, with no significant differences among all the EA groups (all P > 0.05). (2) Compared with (2.698 +/- 1.491) ng/mg in the saline group, IP3 contents of the uterus of (0.813 +/- 0.899) ng/mg in the model group, (1.740 +/- 0.375) ng/mg in the Sanyinjiao (SP 6) group and (0.692 +/- 0.212) ng/mg in the Hegu (LI 4) group were all lower significantly (P < 0.05, P < 0.01), and (0.743+/- 0.306) ng/mg in the Xuehai (SP 10) group showed no significant differences (P > 0.05). Compared with that in the model group, IP3 content of the uterus in the Hegu (LI 4) group showed no significant difference (P > 0.05), and those in the Sanyinjiao (SP 6) group and in the Xuehai (SP 10) group increased significantly (both P < 0 05), which were significantly higher than that in the Hegu (II 4) group (P < 0.05, P < 0.01).. There are no significant differences among the instant EA groups in improving the dysmenorrhea symptoms, but there is obvious specificity of acupoint effects in the regulation of IP3. Electroacupuncture at "Sanyinjiao (SP 6) " Xuehai (SP 10)" has more marked effect in dysmenorrhea model rats.

Topics: Acupuncture Points; Animals; Disease Models, Animal; Dysmenorrhea; Electroacupuncture; Female; Humans; Inositol 1,4,5-Trisphosphate; Random Allocation; Rats; Rats, Sprague-Dawley; Uterus

Working memory (WM) is a process of actively maintaining information in the mind for a relatively short period of time, and prefrontal cortex (PFC) has been thought to play a central role in its function. However, our understanding of underlying molecular events that translate into WM behavior remains elusive. To shed light on this issue, we have used three distinct nonhuman primate models of WM where each model represents three WM conditions: normal control, WM-deficient, and recuperated to normal from WM deficiency. Based on the hypothesis that there is a common molecular substrate for the coding of WM behavior, we have studied the relationship of these animals' performance on a WM task with their PFC levels of molecular components associated with Gq-phospholipase C and cAMP pathways, with the idea of identifying the footprints of such biomolecules. We observed that in all of the primate models WM deficiency was strongly related to the reduced concentration of IP(3) in PFC, whereas recuperation of WM-deficient animals to normal condition was associated with the normalization in IP(3) level. However, this correlation was absent or weak for cAMP, active protein kinase A, dopamine D(1) receptor, and Gq protein. In addition, WM deficiency related not only to pharmacological conditions but also to aging. Thus, it is suggested that optimal IP(3) activity is essential for normal WM function and the maintenance of intracellular IP(3)-mediated Ca(2+) level in PFC may serve as biochemical substrate for the expression of WM behavior.

Topics: Amphetamine; Animals; Antipsychotic Agents; Calcium; Calcium Signaling; Central Nervous System Stimulants; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Disease Models, Animal; GTP-Binding Protein alpha Subunits, Gq-G11; Haloperidol; Inositol 1,4,5-Trisphosphate; Macaca mulatta; Memory Disorders; Memory, Short-Term; Phospholipases; Prefrontal Cortex; Receptors, Dopamine D1; Recovery of Function

Inositol 1,4,5-trisphosphate (IP(3)) is a second messenger that regulates intracellular Ca(2+) release through IP(3) receptors located in the sarco(endo)plasmic reticulum of cardiac myocytes. Many prohypertrophic G protein-coupled receptor (GPCR) signaling events lead to IP(3) liberation, although its importance in transducing the hypertrophic response has not been established in vivo.. Here, we generated conditional, heart-specific transgenic mice with both gain- and loss-of-function for IP(3) receptor signaling to examine its hypertrophic growth effects following pathological and physiological stimulation.. Overexpression of the mouse type-2 IP(3) receptor (IP(3)R2) in the heart generated mild baseline cardiac hypertrophy at 3 months of age. Isolated myocytes from overexpressing lines showed increased Ca(2+) transients and arrhythmias in response to endothelin-1 stimulation. Although low levels of IP(3)R2 overexpression failed to augment/synergize cardiac hypertrophy following 2 weeks of pressure-overload stimulation, such levels did enhance hypertrophy following 2 weeks of isoproterenol infusion, in response to Galphaq overexpression, and/or in response to exercise stimulation. To inhibit IP(3) signaling in vivo, we generated transgenic mice expressing an IP(3) chelating protein (IP(3)-sponge). IP(3)-sponge transgenic mice abrogated cardiac hypertrophy in response to isoproterenol and angiotensin II infusion but not pressure-overload stimulation. Mechanistically, IP(3)R2-enhanced cardiac hypertrophy following isoproterenol infusion was significantly reduced in the calcineurin-Abeta-null background.. These results indicate that IP(3)-mediated Ca(2+) release plays a central role in regulating cardiac hypertrophy downstream of GPCR signaling, in part, through a calcineurin-dependent mechanism.

Topics: Age Factors; Angiotensin II; Animals; Arrhythmias, Cardiac; Calcineurin; Calcium Signaling; Cardiomegaly; Disease Models, Animal; Endothelin-1; GTP-Binding Protein alpha Subunits, Gq-G11; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Isoproterenol; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Myocytes, Cardiac; Phenotype; Physical Exertion

We have developed a caged IP(3) analogue for two-photon photolysis in living animals. This probe is a cell permeable version and was coloaded with a fluorescent Ca(2+) dye into astrocytes in layer 1 of the somatosensory cortex of anesthetized mice. Two-photon irradiation of single cells at 720 nm produced rapid and robust increases in intracellular Ca(2+) concentrations monitored using two-photon microscopy at 950 nm. The photoevoked intracellular Ca(2+) waves were similar in magnitude to intrinsic signals in wild type mice. These waves did not propagate to other cells beyond the targeted astrocyte. In contrast, we observed intercellular astrocytic Ca(2+) waves in two mouse models of familial Alzheimer's disease. These data suggest that Alzheimer's might perturb gliotransmission but not IP(3) signaling per se in mouse models of the disease.

Topics: Alzheimer Disease; Animals; Astrocytes; Calcium Signaling; Disease Models, Animal; Esterases; Fluorescent Dyes; Green Fluorescent Proteins; Humans; Inositol 1,4,5-Trisphosphate; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Fluorescence, Multiphoton; Photolysis; Photons; Pia Mater; Presenilin-1; Presenilin-2; Transgenes

Sex hormones regulate cholangiocyte hyperplasia in bile duct-ligated (BDL) rats. We studied whether follicle-stimulating hormone (FSH) regulates cholangiocyte proliferation. FSH receptor (FSHR) and FSH expression was evaluated in liver sections, purified cholangiocytes, and cholangiocyte cultures (NRICC). In vivo, normal female and male rats were treated with FSH or immediately after BDL with antide (a gonadotropin-releasing hormone antagonist blocking FSH secretion) or a neutralizing FSH antibody for 1 wk. We evaluated 1) cholangiocyte proliferation in sections and cholangiocytes and 2) changes in secretin-stimulated cAMP (functional index of cholangiocyte growth) levels, and ERK1/2 and Elk-1 phosphorylation. NRICC were stimulated with FSH before evaluation of proliferation, cAMP/IP(3) levels, and ERK1/2 and Elk-1 phosphorylation. To determine whether FSH regulates cholangiocyte proliferation by an autocrine mechanism, we evaluated the effects of 1) cholangiocyte supernatant (containing FSH) on NRICC proliferation and 2) FSH silencing in NRICC before measuring proliferation and ERK1/2 and Elk-1 phosphorylation. Cholangiocytes and NRICC express FSHR and FSH and secrete FSH. In vivo administration of FSH to normal rats increased, whereas administration of antide and anti-FSH antibody to BDL rats decreased 1) ductal mass and 2) secretin-stimulated cAMP levels, proliferation, and ERK1/2 and Elk-1 phosphorylation in cholangiocytes compared with controls. In NRICC, FSH increased cholangiocyte proliferation, cAMP levels, and ERK1/2 and Elk-1 phosphorylation. The supernatant of cholangiocytes increased NRICC proliferation, inhibited by preincubation with anti-FSH antibody. Silencing of FSH gene decreases cholangiocyte proliferation and ERK1/2 and Elk-1 phosphorylation. Modulation of cholangiocyte FSH expression may be important for the management of cholangiopathies.

Topics: Animals; Antibodies; Apoptosis; Autocrine Communication; Bile Ducts; Cell Proliferation; Cells, Cultured; Cholestasis; Culture Media, Conditioned; Cyclic AMP; Disease Models, Animal; ets-Domain Protein Elk-1; Female; Follicle Stimulating Hormone; Hepatocytes; Hormone Antagonists; Infusion Pumps, Implantable; Inositol 1,4,5-Trisphosphate; Ligation; Liver; Male; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Oligopeptides; Phosphorylation; Rats; Rats, Inbred F344; Receptors, FSH; RNA Interference

Emotional disturbances, depressive mood, anxiety, aggressive behavior, and memory impairment are the common psychiatric features associated with temporal lobe epilepsy (TLE). The present study was carried out to investigate the role of Bacopa monnieri extract in hippocampus of pilocarpine-induced temporal lobe epileptic rats through the 5-HT(2C) receptor in relation to depression. Our results showed upregulation of 5-HT(2C) receptors with a decreased affinity in hippocampus of pilocarpine-induced epileptic rats. Also, there was an increase in 5-HT(2C) gene expression and inositol triphosphate content in epileptic hippocampus. Carbamazepine and B. monnieri treatments reversed the alterations in 5-HT(2C) receptor binding, gene expression, and inositol triphosphate content in treated epileptic rats as compared to untreated epileptic rats. The forced swim test confirmed the depressive behavior pattern during epilepsy that was nearly completely reversed by B. monnieri treatment.

Topics: Animals; Anticonvulsants; Bacopa; Carbamazepine; Disease Models, Animal; Epilepsy; Ergolines; Hippocampus; Inositol 1,4,5-Trisphosphate; Male; Pilocarpine; Plant Preparations; Protein Binding; Radioligand Assay; Rats; Rats, Wistar; Receptor, Serotonin, 5-HT2C; Serotonin Antagonists; Statistics, Nonparametric; Swimming; Tritium; Up-Regulation

Lipopolysaccharide (LPS) induces tumor necrosis factor-alpha (TNF-alpha) expression in cardiomyocytes, which contributes to myocardial dysfunction during sepsis. The purpose of this study was to investigate the role of phosphatidylinositol (PI) phospholipase Cgamma1 (PLCgamma1) in cardiac TNF-alpha expression, and myocardial dysfunction during endotoxemia.. In cultured mouse neonatal cardiomyocytes, LPS increased PLCgamma1 phosphorylation. Knockdown of PLCgamma1 with specific siRNA or inhibition of PLCgamma1 with U73122 attenuated TNF-alpha expression induced by LPS. This action of PLCgamma1 was mediated through inositol-1,4,5-trisphosphate (IP3)/IP3 receptor (IP3R) pathways since blocking either IP3 or IP3R decreased LPS-induced TNF-alpha expression. In contrast, neither diacylglycerol agonist nor antagonist had any evident effect on LPS-induced TNF-alpha expression in cardiomyocytes. To investigate the role of PLCgamma1 in endotoxemia in vivo, wild-type and heterozygous PLCgamma1 knockout (PLCgamma1(+/-)) mice were pre-treated with either U73122, or its inactive analog U73343, or vehicle for 15 min, followed by LPS for 4 h. Inhibition of PLCgamma1 by U73122 or by heterozygous deletion of the PLCgamma1 gene decreased cardiac TNF-alpha expression. More importantly, LPS-induced myocardial dysfunction was also attenuated in PLCgamma1(+/-) mice or by U73122 treatment.. PLCgamma1 signalling induces cardiac TNF-alpha expression and myocardial dysfunction during LPS stimulation. The action of PLCgamma1 on TNF-alpha expression is mediated through IP3/IP3R pathways. The present results suggest that PLCgamma1 may be a potential therapeutic target for myocardial dysfunction in sepsis.

Topics: Animals; Animals, Newborn; Cardiomyopathies; Cells, Cultured; Diglycerides; Disease Models, Animal; Endotoxemia; Estrenes; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Mice, Knockout; Myocardial Contraction; Myocytes, Cardiac; Phosphodiesterase Inhibitors; Phospholipase C gamma; Phosphorylation; Pyrrolidinones; RNA Interference; Signal Transduction; Transfection; Tumor Necrosis Factor-alpha

Protease-activated receptor-2 (PAR-2) is activated when trypsin cleaves its NH(2) terminus to expose a tethered ligand. We previously demonstrated that PAR-2 activates ion channels in pancreatic duct epithelial cells (PDEC). Using real-time optical fluorescent probes, cyan fluorescence protein-Epac1-yellow fluorescence protein for cAMP, PH(PLC-delta1)-enhanced green fluorescent protein for phosphatidylinositol 4,5-bisphosphate, and protein kinase Cgamma (PKCgamma)-C1-yellow fluorescence protein for diacylglycerol, we now define the signaling pathways mediating PAR-2 effect in dog PDEC. Although PAR-2 activation does not stimulate a cAMP increase, it induces phospholipase C to hydrolyze phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-trisphosphate and diacylglycerol. Intracellular Ca(2+) mobilization from inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores and a subsequent Ca(2+) influx through store-operated Ca(2+) channels cause a biphasic increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), measured with Indo-1 dye. Single-cell amperometry demonstrated that this increase in [Ca(2+)](i) in turn causes a biphasic increase in exocytosis. A protein kinase assay revealed that trypsin also activates PKC isozymes to stimulate additional exocytosis. Paralleling the increased exocytosis, mucin secretion from PDEC was also induced by trypsin or the PAR-2 activating peptide. Consistent with the serosal localization of PAR-2, 1 microm luminal trypsin did not induce exocytosis in polarized PDEC monolayers; on the other hand, 10 microm trypsin at 37 degrees C damaged the epithelial barrier sufficiently so that it could reach and activate the serosal PAR-2 to stimulate exocytosis. Thus, in PDEC, PAR-2 activation increases [Ca(2+)](i) and activates PKC to stimulate exocytosis and mucin secretion. These functions may mediate the reported protective role of PAR-2 in different models of pancreatitis.

Topics: Animals; Calcium; Calcium Channels; Calcium Signaling; Cells, Cultured; Cyclic AMP; Disease Models, Animal; Dogs; Epithelial Cells; Exocytosis; Inositol 1,4,5-Trisphosphate; Ion Channels; Mucins; Pancreatic Ducts; Pancreatitis; Phosphatidylinositol 4,5-Diphosphate; Protein Kinase C; Receptor, PAR-2; Trypsin; Type C Phospholipases

Ca2+ signaling regulation plays an important role in triggering and/or maintaining atrial fibrillation (AF). Little is known about the relationship of the inositol-1,4,5-triphosphate receptors (InsP3Rs) and ryanodine receptors (RyRs) in left atrium to chronic AF. In this study, we investigated the expression and function of InsP3R1, InsP3R2 and RyR2 in a chronic dog model of AF. AF was induced in 6 dogs by rapid right atrial pacing for 24 weeks, and a sham procedure was performed in 5 dogs (control group). The intact left atrial myocytes were used to examine the expression and function of InsP3Rs, RyRs by BODIPY(O,R) TR-X ryanodine, heparin-fluorescein conjugate, and were stimulated by caffeine, ATP to release Ca2+ through RyRs, InsP3Rs separately. We also assessed the molecular components of left atrial tissue underlying the amount of RyR2, InsP3R1 and InsP3R2 determined by RT-PCR, immunohistochemistry and Western blot analysis. In the chronic AF group, the Ca2+ released through RyRs is not altered, but the Ca2+ released through InsP3Rs increased significantly. RyR2 distributed in cytosol of myocytes, cellular membrane; its expression significantly decreased in AF group compared to controls. InsP3R1 distributed in cytosol, InsP3R2 distributed not only in cytosol, cellular membrane, but also in nuclear envelope and intercalated discs. The InsP3R1 and InsP3R2 expression significantly increased in chronic AF group compared to controls. These results indicated that in a chronic dog model of AF, the expression and function of RyR2 down-regulated; on the contrary, the expression and function of InsP3R1, InsP3R2 up-regulated, and InsP3R2 may be the major InsP3Rs, which regulate intracellular or even intercellular Ca2+ signal transmission.

Topics: Animals; Atrial Fibrillation; Calcium; Chronic Disease; Disease Models, Animal; Dogs; Heart Atria; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Myocytes, Cardiac; Ryanodine Receptor Calcium Release Channel; Signal Transduction

Medicinal drugs do not always have clearly understood mechanisms of action, especially as regards psychiatric treatment. Identification of genes involved in drug resistance is an important step toward elucidating the genetic basis of disease and the molecular mechanism of drug action. However, this approach is impractical in higher animals, as ablation and screening of every gene in an animal is not currently possible. Dictyostelium has proven a good model system for molecular pharmacological research as a result of its genetic tractability, ease of gene knockout, and creation of isogenic lines. In this system, we have identified genes that confer resistance to bipolar disorder drugs. This work has implicated inositol (1,4,5) trisphosphate (InsP3) signaling as a common mechanism of action for these drugs in patients.

Topics: Animals; Antimanic Agents; Bipolar Disorder; Dictyostelium; Disease Models, Animal; DNA Transposable Elements; Drug Evaluation, Preclinical; Drug Resistance; Gene Deletion; Gene Targeting; Humans; Inositol 1,4,5-Trisphosphate; Pharmacogenetics; Restriction Mapping

Platelet-activating factor (PAF) is a pro-inflammatory lipid mediator that increases vascular permeability by simultaneous activation of two pathways, one dependent on the cyclooxygenase metabolite prostaglandin E2 and the other on the sphingomyelinase metabolite ceramide. The hypothesis that part of the PAF-induced oedema is mediated via the inositol 1,4,5-trisphosphate (IP3) pathway or Rho kinase pathway was investigated. Oedema formation was induced in isolated perfused rat lungs by injection of 5 nmol PAF into the pulmonary artery. Lungs were pre-treated with specific inhibitors: edelfosine (L108) to block phosphatidyl-inositol-specific phospholipase C, xestospongin to block the IP3 receptor, 5-iodonaphthalene-1-sulphonyl-homopiperazine (ML-7) to block myosin light chain kinase, and (+)-R-trans-4-(aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide (Y27632) to block Rho-associated protein kinase. Pre-treatment with L108 or xestospongin reduced PAF-induced oedema formation by 58 and 56%, respectively. The effect of L108 was additive to that of the cyclooxygenase inhibitor acetyl salicylic acid (88% oedema reduction). PAF-induced oedema formation was also reduced if extracellular calcium concentrations were lowered. Furthermore, treatment with ML-7 reduced oedema formation by 54%, whereas Y27632 was without effect. It is concluded that platelet-activating-factor-triggered oedema is mediated by activation of the inositol 1,4,5-trisphosphate pathway, influx of extracellular calcium and subsequent activation of a myosin light chain kinase-dependent and Rho-associated-protein-kinase-independent mechanism.

Topics: Animals; Calcium; Disease Models, Animal; Endothelium; Female; Inositol 1,4,5-Trisphosphate; Intracellular Signaling Peptides and Proteins; Lung; Myosin-Light-Chain Kinase; Organ Size; Platelet Activating Factor; Protein Serine-Threonine Kinases; Pulmonary Edema; Rats; Rats, Wistar; Reference Values; rho-Associated Kinases

Vascular smooth muscle cells (VSMCs) that accumulate in neointima after angioplastic injury show different phenotypic characteristics from those of medial layer and an impaired reactivity to contracting agents. The aim of the study was to correlate the vascular hyporesponsiveness to the changes in intracellular calcium concentration [Ca(2+)](i) and the expression of proteins necessary for its utilization in mechanically injured rat carotid arteries (IC) at 14 and 28 days after angioplastic balloon. IC showed a significant reduction (P<0.01) to PE- or KCl-induced contraction as compared to uninjured carotid (UC). Fura-2AM-loaded VSMCs isolated from IC revealed that this hyporeactivity to PE or KCl was accompanied by the impairment of the increase in [Ca(2+)](i) induced by contracting agents in both Ca(2+)-free or -containing medium. Similar results were observed following the ryanodine challenge in VSMC. Western blot analysis showed a significant (P<0.05) reduction in myosin heavy chain (MHC) and IP(3)-type III receptor expression in IC isolated at 14 days from injury compared to UC, while an improvement of these proteins expression was observed at 28 days after damage. On the other hand, in IC tissue, SERCA2 and alpha-actin expression, compared to UC was significantly higher at 14 days than at 28 days. These data indicate that vascular hyporeactivity induced by mechanical injury may be due to alterations of either [Ca(2+)](i) or contractile proteins. These modifications could be related to the changes of VSMC phenotypic characteristics, as supported by the observed modifications in MHC, SERCA2 and alpha-actin expression, proteins considered as biological markers of cellular differentiation.

Topics: Actins; Angioplasty, Balloon; Animals; Blotting, Western; Calcium; Calcium Channels; Calcium-Transporting ATPases; Carotid Artery Injuries; Carotid Artery, External; Cells, Cultured; Disease Models, Animal; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Male; Muscle, Smooth, Vascular; Myosin Heavy Chains; Phenotype; Phenylephrine; Potassium Chloride; Rats; Rats, Wistar; Receptors, Cytoplasmic and Nuclear; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Spectrometry, Fluorescence; Vasoconstriction; Vasoconstrictor Agents

Huntington's disease (HD) is caused by polyglutamine expansion (exp) in huntingtin (Htt). The type 1 inositol (1,4,5)-triphosphate receptor (InsP3R1) is an intracellular calcium (Ca2+) release channel that plays an important role in neuronal function. In a yeast two-hybrid screen with the InsP3R1 carboxy terminus, we isolated Htt-associated protein-1A (HAP1A). We show that an InsP3R1-HAP1A-Htt ternary complex is formed in vitro and in vivo. In planar lipid bilayer reconstitution experiments, InsP3R1 activation by InsP3 is sensitized by Httexp, but not by normal Htt. Transfection of full-length Httexp or caspase-resistant Httexp, but not normal Htt, into medium spiny striatal neurons faciliates Ca2+ release in response to threshold concentrations of the selective mGluR1/5 agonist 3,5-DHPG. Our findings identify a novel molecular link between Htt and InsP3R1-mediated neuronal Ca2+ signaling and provide an explanation for the derangement of cytosolic Ca2+ signaling in HD patients and mouse models.

Topics: Action Potentials; Animals; Blotting, Western; Calcium; Calcium Channels; Calcium Signaling; Cells, Cultured; Cerebellum; Cerebral Cortex; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Fura-2; Green Fluorescent Proteins; Humans; Huntingtin Protein; Huntington Disease; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Lipid Bilayers; Luminescent Proteins; Methoxyhydroxyphenylglycol; Nerve Tissue Proteins; Neurons; Nuclear Proteins; Patch-Clamp Techniques; Peptide Fragments; Plasmids; Protein Binding; Receptors, Cytoplasmic and Nuclear; Recombinant Proteins; Time Factors; Two-Hybrid System Techniques

Decreased platelet aggregation to collagen is a cause for bleeding diathesis of Chediak-Higashi syndrome (CHS). We investigated whether the collagen receptor-Ca2+ signaling system was impaired in platelets from cattle affected with CHS. A collagen-induced increase in cytosolic Ca2+ ([Ca2+]i) was depressed in CHS platelets, which was accompanied by a decrease in the production of inositol 1,4,5-trisphosphate. When the influences of endogenous arachidonic acid metabolites and ADP were excluded, convulxin or collagen-related peptide, which are specific agonists for the collagen receptor GPVI, increased [Ca2+]i in both normal and CHS platelets. In contrast, rhodocytin, which was thought to activate another collagen receptor GPIa/IIa, increased [Ca2+]i in CHS platelets to a lesser extent than in normal ones. Cytochalasin D, an actin polymerization inhibitor, depressed the response to collagen or rhodocytin but not the response to convulxin. Adhesion of CHS platelets to acid soluble type I collagen, which was mediated by GPIa/IIa, was similar to that of normal platelets. These results suggest that a defect in the rhodocytin-sensitive pathway is responsible for decreasing the response to collagen in CHS platelets. It remains to be determined which receptor is associated with the mechanism.

Topics: Animals; Calcium Signaling; Carrier Proteins; Cattle; Cattle Diseases; Chediak-Higashi Syndrome; Collagen; Crotalid Venoms; Cytochalasin D; Disease Models, Animal; Inositol 1,4,5-Trisphosphate; Integrins; Lectins; Lectins, C-Type; Peptides; Platelet Adhesiveness; Receptors, Collagen; Viper Venoms

Topics: Animals; Anti-Arrhythmia Agents; Arrhythmias, Cardiac; Disease Models, Animal; Humans; Inositol 1,4,5-Trisphosphate; Myocardial Reperfusion; Rats; Sodium-Calcium Exchanger; Thiourea

Nuclear magnetic resonance (NMR) spectroscopy was used to evaluate cytosolic compounds and membrane phospholipids simultaneously in trisomy 16 (Ts16) and euploid (control) murine brain at fetal day 15 in order to examine the cellular biochemistry that underlies the neurodevelopmental consequences of chromosome triplication in this model of Down syndrome (DS). Proton NMR spectroscopic analysis of brain tissue extracts demonstrated decreased levels of choline and increased levels of myo-inositol (MI) in Ts16 brains compared with control. These data are consistent with the cholinergic deficits and elevated MI levels previously described in Ts16. Compared with euploid brains. Ts16 brains also possess higher levels of creatine, adenosine, and tyrosine. Increased levels of MI and creatine, compounds that are localized to glia, imply abnormalities in the trophic environment of Ts16 brain. Phosphorus NMR spectroscopic analysis of extracts further revealed elevated levels of anionic phospholipid membrane components, such as phosphatidylinositol (PtdIno) and phosphatidylethanolamine, in Ts16 brains. Since these compounds are confined to the inner leaflet of the membrane, the findings suggest that membrane composition is altered specifically in the cytosolic bilayer at this stage. Together our proton and phosphorus NMR spectroscopic results indicate that multiple biochemical pathways are affected in Ts16 brain development. Understanding the effects of these aberrations may elucidate the processes that lead to neural dysfunction and Alzheimer's disease (AD) neuropathology in DS individuals.

Topics: Alanine; Animals; Aspartic Acid; Biomarkers; Brain Chemistry; Choline; Creatine; Disease Models, Animal; Down Syndrome; Female; gamma-Aminobutyric Acid; Glutamic Acid; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Lactic Acid; Magnetic Resonance Spectroscopy; Male; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Phosphorus Isotopes; Protons; Taurine; Threonine

In this study, the dynamic interrelationships between myocardial functional state and changes in the second messenger content in pressure-overloaded hypertrophied hearts were investigated. Forty-three rat hearts were used after partial clamping of the abdominal aorta. The isolated hearts were perfused with Krebs-Henseleit buffer and allocated to perfusion for 20 s or 40 min as controls (n = 12); or with noradrenaline (10(-6) mol l-1, n = 11); carbachol (3 x 10(-7) mol l-1, n = 9); or noradrenaline plus carbachol (10(-6) mol l-1 + 3 x 10(-7) mol l-1, respectively, n = 11). maxdP/dt increased more than 2-fold already after 20 s on noradrenaline stimulation, followed by a significant increase in cAMP. After 40 min, maxdP/dt was lower than the maximal value, although higher than controls. cAMP was also decreased, but still significantly higher than controls. Perfusion with noradrenaline plus carbachol produced the same changes in maxdP/dt as those seen after noradrenaline stimulation alone, but failed to increase cAMP content after both 20 s and 40 min. The inositol trisphosphate (IP3) content was increased 40 min of control perfusion (p < 0.05). Noradrenaline and carbachol, separately, produced an increase in IP3 content already after 20 s (p < 0.05). The combination of noradrenaline plus carbachol also produced an increase of IP3 (p < 0.05; compared to controls), but to a lesser extent when compared either to noradrenaline or carbachol (p < 0.05). After 40 min of perfusion, IP3 was in the same range regardless of added agonist(s) and still slightly above control level (p < 0.05). The early increase in maxdP/dt induced by noradrenaline or the combination of noradrenaline plus carbachol was not paralleled by a decrease in ATP content. This was also the case upon addition of carbachol alone. However, after 40 min of agonistic perfusion, ATP levels were substantially decreased. In conclusion, myocardial IP3 content in pressure-overloaded hypertrophied hearts was not different from that of sham-operated hearts. After agonistic stimulation, an early increase in IP3 formation was seen. Attenuation of the IP3 response by combined stimulation with noradrenaline and carbachol was initially present in pressure-overloaded hypertrophied hearts. After 40 min no attenuation was found for either IP3 or for cAMP content, suggestive of induction of a desensitization.

Topics: Adrenergic Agonists; Animals; Aorta, Thoracic; Cyclic AMP; Cyclic GMP; Disease Models, Animal; Hypertrophy, Left Ventricular; Inositol 1,4,5-Trisphosphate; Ligation; Male; Muscarinic Agonists; Myocardium; Rats; Rats, Sprague-Dawley; Second Messenger Systems; Ventricular Pressure

Although the signaling pathway involving polyphosphoinositide (poly-PI) hydrolysis and release of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] is an important mechanism for regulation of neuronal calcium homeostasis, the effect of cerebral ischemia-reperfusion on this calcium signaling pathway is not well understood. Because activity of this pathway is dependent on availability of ATP, this study is aimed at examining the poly-PI signaling pathway and high-energy metabolites in a rat stroke model.. Focal cerebral ischemia in rats was induced by temporary occlusion of the right middle cerebral artery and both common carotid arteries. Levels of Ins(1,4,5)P3 were determined by use of the radioreceptor binding assay. Poly-PI turnover in rat cortex was assessed with an in vivo protocol involving intracerebral injection of [3H] inositol and systemic administration of lithium. High-energy metabolites (ATP, ADP, and AMP) were analyzed by high-performance liquid chromatography.. Ischemia induced an increase in poly-PI turnover in the right middle cerebral artery cortex, but reperfusion led to a decline in this signaling activity. However, Ins(1,4,5)P3 levels decreased during ischemia, and these levels were not restored if ischemic insults were longer than 30 minutes. ATP levels decreased to 26% of control during ischemia and recovered to 80% of control during the initial 4 hours of reperfusion; these changes were followed by a second phase of decline.. Results show an important relationship between ischemia-induced depletion of high-energy metabolites and poly-PI signaling activity. However, the uncoupling between Ins(1,4,5)P3 and ATP during reperfusion after severe ischemia suggests that metabolism of Ins(1,4,5)P3 is more stringently regulated than ATP.

Topics: Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Animals; Brain; Calcium; Carotid Artery, Common; Cerebral Arteries; Cerebral Cortex; Chromatography, High Pressure Liquid; Disease Models, Animal; Homeostasis; Hydrolysis; Inositol 1,4,5-Trisphosphate; Ischemic Attack, Transient; Neurons; Phosphatidylinositol Phosphates; Rats; Reperfusion; Signal Transduction; Tritium

The origin of Ca2+ contributing to the enhancement of acetylcholine-induced bronchial smooth muscle constriction in airway hyperresponsiveness induced by antigen challenge was investigated. Under Ca(2+)-free (concomitant with 10(-6) M nicardipine) conditions, the contractile responses of bronchial rings to 1 mM acetylcholine were significantly greater in rings from rats with hyperresponsive airways (0.15 +/- 0.04 g) than those of rings from normal rats (0.02 +/- 0.004 g; P < 0.05). The cumulatively administered Ca2+ induced a markedly greater bronchoconstriction in rings from rats with hyperresponsive airways in Ca(2+)-free solution when muscles were pretreated with 1 mM acetylcholine (in the presence of 10(-6) M nicardipine) than in rings from normal rats, whereas no significant difference in Ca(2+)-induced bronchoconstriction was observed between the two groups when muscles were pretreated with 60 mM K+ (in the presence of 10(-6) M atropine). These findings suggest that enhancement of the availability of Ca2+ released from intracellular stores and/or influxed through receptor-operated Ca2+ channels in airway smooth muscles might be involved in the airway hyperresponsiveness to acetylcholine in rats.

Topics: Acetylcholine; Animals; Antigens, Helminth; Ascaris suum; Bronchial Hyperreactivity; Bronchoconstriction; Calcium; Calcium Channels; Disease Models, Animal; Dose-Response Relationship, Drug; Inositol 1,4,5-Trisphosphate; Male; Muscle Contraction; Muscle, Smooth; Nicardipine; Rats; Rats, Wistar

Because the phosphatidylinositol pathway may be part of the signaling system associated with stretch-induced release of atrial natriuretic peptide (ANP), we tested the hypothesis that formation of the intermediate inositol-1,4,5-trisphosphate (IP3) is impaired when ANP release is decreased in response to atrial stretch in hearts from aging genetically hypertensive (GH) rats. Immunoreactive ANP release into the coronary effluent and IP3 levels were studied in cardiac tissues of isolated perfused hearts from normotensive control (WAG) or GH rats aged 4, 11, or 16 months. Left atria were repeatedly distended and released with a latex balloon. ANP was measured in coronary effluent, and IP3 was measured in cardiac tissues. In all age groups, stretch and relief of stretch evoked considerably less ANP release in spontaneously beating hearts from GH than from WAG rats. Hearts from GH rats aged 16 months released no ANP, but electrical pacing restored some stretch-induced ANP secretion. With repeated stretch and release of stretch of the left atrium for 2 min, IP3 levels increased in left atrial tissue in WAG but not in GH hearts of all age groups. IP3 levels in (unstretched) left ventricles were much lower than in left atria and were unaltered by atrial stretch. In aging GH rats, the capacity to release ANP on atrial stretch is largely lost, in association with complete suppression of stimulus-induced increase in IP3 levels. These data support a role for IP3 in stretch-mediated atrial ANP secretion and suggest a progressive uncoupling of this signaling pathway in aging hypertensive rats.

Topics: Aging; Analysis of Variance; Angioplasty, Balloon; Animals; Atrial Function; Atrial Natriuretic Factor; Blood Pressure; Body Weight; Cardiac Pacing, Artificial; Compliance; Disease Models, Animal; Hypertension; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Male; Myocardium; Organ Size; Rats; Rats, Wistar

While it is probable that the cerebrovascular spasm which occurs after subarachnoid haemorrhage results from the action of haemoglobin, the mechanism of that process remains unclear. These studies were thus designed to test the hypothesis that the action of oxyhaemoglobin results from the iron-catalyzed formation of free radicals and subsequent lipid peroxidation resulting in intracellular changes in the second messengers for contraction.. Levels of intracellular calcium and of inositol (1,4,5)-trisphosphate were measured in cultured vascular smooth muscle cells derived from primate cerebral arteries. Contractility of rings of canine cerebral vessels were examined in vitro using standard pharmacological techniques. Vessels in spasm were obtained from the "two haemorrhage" canine model and the presence of vasospasm was confirmed angiographically. In each case, the effects of oxyhaemoglobin and sometimes of free radicals generated from iron salts were examined in the presence and in the absence of free-radical scavenging agents or the iron chelating agent, deferoxamine.. Oxyhaemoglobin produces a slowly-developing sustained contraction of arterial rings which is accompanied by a sustained elevation of intracellular calcium. It also produces a transient but significant elevation of inositol (1,4,5)-trisphosphate, but this is not correlated with the development of sustained constriction. Deferoxamine and the lazaroid compounds U-74389G and U-83836E were effective in preventing the effects of oxyhaemoglobin and free radicals in the models tested, although in vessels in spasm, all effects were smaller.. The present study provides results which are consistent with the hypothesis that the actions of haemoglobin on vascular smooth muscle are mediated by the formation of free radicals which subsequently affect intracellular calcium concentrations. This also implies that agents which impair free radical production or other processes leading to iron-catalyzed lipid peroxidation, are of potential value in cerebrovascular spasm.

Topics: Animals; Antioxidants; Calcium; Cell Line; Cell Survival; Chelating Agents; Chromans; Deferoxamine; Disease Models, Animal; Dogs; Female; Free Radical Scavengers; Hydrogen Peroxide; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Iron; Ischemic Attack, Transient; Male; Muscle Contraction; Muscle, Smooth, Vascular; Oxyhemoglobins; Piperazines; Pregnatrienes; Subarachnoid Hemorrhage

Stimulation of T lymphocytes through the T cell receptor in the absence of costimulatory signal(s) induces a state of unresponsiveness to subsequent antigen presentation. We have employed solubilized complexes consisting of rat class II MHC molecules containing an immunodominant peptide of the acetylcholine receptor (AChR alpha 100-116) to induce unresponsiveness in the autoreactive T lymphocytes mediating an animal model of myasthenia gravis. In vitro incubation of rat T cell lines specific for peptide AChR alpha 100-116 with solubilized complexes of MHC II and AChR alpha 100-116 (MHC II:AChR alpha 100-116) rendered the T cells unresponsive to subsequent stimulation by antigen presenting cells and the peptide. T cell lines with a broader specificity to the entire AChR protein pentamer had an 81% reduction in proliferation to AChR following a preincubation with solubilized MHC II:AChR alpha 100-116. Treatment with the solubilized MHC II:AChR alpha 100-116 induced phosphatidylinositol 4,5-bisphosphate hydrolysis, an early signalling event associated with binding to the TCR. Rats primed with AChR and injected intravenously with MHC II:AChR alpha 100-116 had reduced in vitro T cell proliferation to the AChR alpha 100-116 peptide and to whole AChR. Solubilized MHC II:AChR alpha 100-116 injected i.v. into rats exhibiting serological clinical symptoms of experimental autoimmune myasthenia gravis (EAMG) prevented death in 67% of the treated animals, compared to a 0-20% survival rate in all other control groups. These results demonstrate that solubilized MHC II complexed with an immunodominant autoantigenic peptide is tolerogenic and improves the survival rate of rats with EAMG, suggesting the basis for an antigen-specific therapy in autoimmune diseases such as MG.

Topics: Amino Acid Sequence; Animals; Cell Division; Cell Line; Disease Models, Animal; Histocompatibility Antigens Class II; Immune Tolerance; Inositol 1,4,5-Trisphosphate; Male; Molecular Sequence Data; Myasthenia Gravis; Rats; Rats, Inbred Lew; Receptors, Cholinergic; T-Lymphocytes

In this report, we have used a multiple stepwise regression analysis to determine the best derived multivariate equation between a dependent and multiple independent parameters. All parameters were derived from harvested gastrocnemius muscle subjected to chronic burn trauma. Correlations between seven different parameters were tested. Physiologic tension, calcium, adenosine 3':5' cyclic monophosphate (cAMP), inositol 1,4,5-triphosphate (IP3) and its catabolites comprised one large data matrix. From it, the various parameters were substituted as the dependent variable. The remaining parameters all comprised a multiple independent group. From such a format, the stepwise regression was run. The simultaneous co-governing of independent parameters over one dependent parameter provides a means of mathematically proving multiple correlations. Such findings provide a new perspective in interpreting chemical signalling.

Topics: Animals; Burns; Calcium; Cyclic AMP; Disease Models, Animal; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Male; Mice; Muscle, Smooth; Regression Analysis; Second Messenger Systems

Myo-inositol-1,4,5-triphosphoric acid (IP3) formation stimulated by (+-)-1-amino-1,3-cyclopentane-trans-dicarboxylic acid (trans-ACPD) was examined in the cortex, hippocampus and cerebellum of iron-induced epileptic rats and epileptic El mice. Increased IP3 formation by trans-ACPD was observed in the cortex, hippocampus and cerebellum of iron-injected rats while it was found in the hippocampus and cerebellum of the saline-injected control rats. Increased IP3 formation by trans-ACPD was remarkably higher in the hippocampus of iron-injected rats than the other regions. Increased IP3 formation by trans-ACPD was observed in the cortex, hippocampus and cerebellum of ddY mice, while such an increase was found only in the cerebral cortex and not in the hippocampus and cerebellum of El mice. These findings suggest that the inositol response may be involved in the seizure mechanisms of iron-induced epileptic rats and epileptic El mice in some different forms.

Topics: Animals; Brain; Chlorides; Cycloleucine; Disease Models, Animal; Epilepsy; Ferric Compounds; Inositol 1,4,5-Trisphosphate; Male; Mice; Neurotoxins; Rats; Rats, Sprague-Dawley

It has been presumed that alteration in the concentrations of second messengers leads to alterations in the function of the ryanodine receptor. Consequently, we have determined the basal content of cyclic AMP and inositol phosphates in skeletal and cardiac muscle of malignant hyperthermia (MH) susceptible (MHS) and healthy normal control (MHN) swine. Since alpha 1- and beta-adrenoceptors are linked to these second messenger systems, the densities of alpha 1- and beta-adrenoceptors were also determined. In skeletal as well as cardiac muscle, a higher basal concentration of almost all of the inositol phosphates was found. Of all inositol phosphates measured, the presumed second messenger inositol 1,4,5-trisphosphate (1,4,5-IP3) was mostly concentrated in both tissues. Each MHS sample contained more 1,4,5-IP3 than the highest value observed in MHN muscle, indicating that a threshold of 1,4,5-IP3 concentration for determination of MHS or MHN status can be defined. In addition, MHS skeletal muscle contained more cAMP than MHN, whereas there was no difference between MHS and MHN in cardiac muscle. The changes observed in the different inositol phosphate and cAMP contents were not accompanied by an altered alpha 1- or beta-adrenoceptor density in skeletal or cardiac muscle between MHS and MHN. However, the total number of beta-adrenoceptors of MHN and MHS was significantly higher in cardiac (about 80 fmol/mg protein) than skeletal muscles (about 30 fmol/mg protein). The cardiac muscles revealed about 80% beta 1- and beta 2- and 20% beta 2-adrenoceptors, whereas skeletal muscles were characterized by over 95% beta 2-adrenoceptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Cyclic AMP; Disease Models, Animal; Heart Ventricles; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Malignant Hyperthermia; Muscles; Myocardium; Receptors, Adrenergic, alpha-1; Receptors, Adrenergic, beta-1; Receptors, Adrenergic, beta-2; Second Messenger Systems; Swine

In this report, we have used multivariate and vectorial analyses to aid in understanding perturbations in diaphragmatic signal transduction in consequence to burn trauma. This trauma results from shock emanating from skin scald burn. Its respiratory complications are well-known. Relational matrices derived from experimental animal groups of varying body surface area (% BSA) burn size were subjected to individual multiple linear regression and vectorial analyses. Such individual matrices tested the simultaneous dependency of inositol 1,4,5-triphosphate (IP3) on inositol 1,4-biphosphate (I1,4P2), inositol 1-phosphate (I1P) and inositol. Understanding the contribution of multiple independent parameters simultaneously co-governing the dependency of IP3 offers new insight in regulating signal transduction mechanisms under the duress of burn trauma.

Topics: Analysis of Variance; Animals; Body Surface Area; Burns; Diaphragm; Disease Models, Animal; Inositol; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Male; Mice; Multivariate Analysis; Ointments; Polymers; Regression Analysis; Signal Transduction; Silver Sulfadiazine

Skeletal muscle weakness associated with burn trauma prolongs the time of rehabilitation of burn patients. Understanding the underlying chemical changes that impact on physiological tension may provide new therapeutic options for the treatment of burn patients. This report demonstrates the novelty of applying 3-dimensional graphic capabilities, involving area and vector changes to understand variations in inositol derivatives and their co-modulating influence on physiological tension in skeletal muscle. This muscle was distant from the primary anatomical burn site. It was subjected to circulatory shock emanating from burn trauma. Burn injury was achieved by scalding of predefined areas (0, 20% and 50%) on the dorsal and ventral surfaces of mice. At day 21, tension studies via muscle twitch analyses were performed. Through multiple regression, the dependency of physiologic tension was determined with respect to three poly-inositol forms each representing independent parameters simultaneously. The contribution of each of these parameters was assigned to a three-dimensional axis. Relationships of tension on three fixed independent parameters were found only for the 20% and 50% burn groups. Vector analysis on a plane in three-dimensional space determined the relationship of tension to each of the independent parameters in 20% and 50% burn groups. No significant relationship of tension dependency on three fixed poly-inositol variables was found in the control group. Such vector analysis, using solid and differential analytical geometry, allowed for a clear visualization of the interrelationships that existed between secondary messenger systems (viz, IP3) and a resulting physiologic manifestation (viz, tension). This clear visualization allows for a greater understanding of messenger systems that may lead to more effective treatment of skeletal muscle weakness associated with the systemic effects of severe burn trauma.

Topics: Animals; Burns; Disease Models, Animal; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Male; Mice; Mice, Inbred Strains; Multivariate Analysis; Muscle Contraction; Muscles; Regression Analysis; Signal Transduction

In this study incorporation of [3H]inositol into inositol phosphates and phosphoinositides as well as tissue Ins(1,4,5)P3 levels of the atria and ventricles of isolated, perfused rat hearts were compared. Although the incorporation of [3H]inositol into the phosphoinositides of atria and ventricles was similar, significantly higher (2-3 fold) incorporation rates into inositol phosphates were observed in atrial tissue. Using a D-myo-[3H]Ins(1,4,5)P3 assay system, the Ins(1,4,5)P3 levels observed in atria from perfused rat hearts were also significantly higher than those obtained under the same experimental circumstances in the ventricles. Since previous studies on whole hearts showed inhibition of the phosphatidylinositol (PI) pathway during ischaemia with an immediate significant stimulation upon reperfusion [12, 20], the effects of ischaemia and 1 min postischaemic reperfusion were also examined separately in atria and ventricles. The results showed that 20 min of global ischaemia significantly depressed Ins(1,4,5)P3 levels as well as incorporation of [3H]inositol into ventricular InsP2 and InsP3. Reperfusion caused an immediate (within 1 min) increase in Ins(1,4,5)P3 levels and also [3H]inositol incorporation into all three cytosolic inositol phosphates in the ventricles. However, the effect of ischaemia and reperfusion on Ins(1,4,5)P3 levels as well as the incorporation of [3H]inositol into the inositol phosphates were less prominent in the atria. It therefore appears that the differential responses of the atria and the ventricles to an oxygen deficiency [41] are also reflected in the differences in PI metabolism during ischaemia-reperfusion.

Topics: Animals; Disease Models, Animal; Heart Atria; Heart Ventricles; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Male; Myocardial Ischemia; Myocardial Reperfusion; Rats; Rats, Wistar; Tritium

Slice preparations were made from the hippocampus of gerbils after 5 min of ischemia by carotid artery occlusion and the membrane properties of pyramidal neurons were examined. A majority of CA1 neurons lost the capacity for long-term potentiation following tetanic stimulation of the input fibers. CA3 pyramidal neurons, in contrast, preserved responses similar to those in the normal gerbil. Following ischemia, CA1 pyramidal neurons showed increased spontaneous firing that was highly voltage dependent and was blocked by intracellular injection of the Ca2+ chelator, EGTA. Thirty-five percent of CA1 neurons showed an abnormal slow oscillation of the membrane potential after 24 h following ischemia. Intracellular injection of GTP gamma S or IP3 produced facilitation of the oscillations followed by irreversible depolarization. Our results indicate that ischemia-damaged CA1 neurons suffer from abnormal Ca2+ homeostasis, involving IP3-induced liberation of Ca2+ from internal stores.

Topics: Animals; Calcium; Cell Death; Disease Models, Animal; Egtazic Acid; Gerbillinae; Guanosine 5'-O-(3-Thiotriphosphate); Hippocampus; Inositol 1,4,5-Trisphosphate; Ischemic Attack, Transient; Male; Membrane Potentials; Pyramidal Tracts; Reperfusion