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1.
IET Syst Biol ; 5(3): 174-84, 2011 May.
Article in English | MEDLINE | ID: mdl-21639591

ABSTRACT

In this study, the authors explored the utility of a descriptive and predictive bionetwork model for phospholipase C-coupled calcium signalling pathways, built with non-kinetic experimental information. Boolean models generated from these data yield oscillatory activity patterns for both the endoplasmic reticulum resident inositol-1,4,5-trisphosphate receptor (IP(3)R) and the plasma-membrane resident canonical transient receptor potential channel 3 (TRPC3). These results are specific as randomisation of the Boolean operators ablates oscillatory pattern formation. Furthermore, knock-out simulations of the IP(3)R, TRPC3 and multiple other proteins recapitulate experimentally derived results. The potential of this approach can be observed by its ability to predict previously undescribed cellular phenotypes using in vitro experimental data. Indeed, our cellular analysis of the developmental and calcium-regulatory protein, DANGER1a, confirms the counter-intuitive predictions from our Boolean models in two highly relevant cellular models. Based on these results, the authors theorise that with sufficient legacy knowledge and/or computational biology predictions, Boolean networks can provide a robust method for predictive modelling of any biological system. [Includes supplementary material].


Subject(s)
Calcium Signaling , Models, Biological , Type C Phospholipases/metabolism , Animals , GTP-Binding Protein beta Subunits/metabolism , Humans , Inositol 1,4,5-Trisphosphate Receptors/metabolism , Mathematical Concepts , Mice , Mice, Knockout , Serotonin/metabolism , Systems Biology , TRPC Cation Channels/metabolism
2.
Handb Exp Pharmacol ; (179): 575-91, 2007.
Article in English | MEDLINE | ID: mdl-17217080

ABSTRACT

TRPC channels are ubiquitously expressed among cell types and mediate signals in response to phospholipase C (PLC)-coupled receptors. TRPC channels function as integrators of multiple signals resulting from receptor-induced PLC activation, which catalyzes the breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) to produce inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol (DAG). InsP3 depletes Ca2+ stores and TRPC3 channels can be activated by store-depletion. InsP3 also activates the InsP3 receptor, which may undergo direct interactions with the TRPC3 channel, perhaps mediating store-dependence. The other PLC product, DAG, has a direct non-PKC-dependent activating role on TRPC3 channels likely by direct binding. DAG also has profound effects on the TRPC3 channel through PKC. Thus PKC is a powerful inhibitor of most TRPC channels and DAG is a dual regulator of the TRPC3 channel. PLC-mediated DAG results in rapid channel opening followed later by a slower DAG-induced PKC-mediated deactivation of the channel. The decreased level of PIP2 from PLC activation also has an important modifying action on TRPC3 channels. Thus, the TRPC3 channel and PLCgamma form an intermolecular PH domain that has high specificity for binding PIP2. This interaction allows the channel to be retained within the plasma membrane, a further operational control factor for TRPC3. As nonselective cation channels, TRPC channel opening results in the entry of both Na+ and Ca2+ ions. Thus, while they may mediate Ca2+ entry signals, TRPC channels are also powerful modifiers of membrane potential.


Subject(s)
Cell Physiological Phenomena , Signal Transduction/physiology , Transient Receptor Potential Channels/physiology , Animals , Biotransformation/physiology , Humans , Inositol 1,4,5-Trisphosphate/physiology , Protein Kinase C/physiology , Signal Transduction/drug effects , Transient Receptor Potential Channels/drug effects , Type C Phospholipases/physiology
3.
J Biol Chem ; 275(37): 28562-8, 2000 Sep 15.
Article in English | MEDLINE | ID: mdl-10878007

ABSTRACT

The mechanism for coupling between Ca(2+) stores and store-operated channels (SOCs) is an important but unresolved question. SOC-mediated Ca(2+) entry is complex and may reflect more than one type of channel and coupling mechanism. To assess such possible divergence the function and coupling of SOCs was compared with two other distinct yet related Ca(2+) entry mechanisms. SOC coupling in DDT(1)MF-2 smooth muscle cells was prevented by the permeant inositol 1,4,5-trisphosphate (InsP(3)) receptor blockers, 2-aminoethoxydiphenyl borate (2-APB) and xestospongin C. In contrast, Ca(2+) entry induced by S-nitrosylation and potentiated by store depletion (Ma, H-T., Favre, C. J., Patterson, R. L., Stone, M. R., and Gill, D. L. (1999) J. Biol. Chem. 274, 35318-35324) was unaffected by 2-APB, suggesting that this entry mechanism is independent of InsP(3) receptors. The cycloalkyl lactamimide, MDL-12, 330A (MDL), prevented SOC activation (IC(50) 10 micrometer) and similarly completely blocked S-nitrosylation-mediated Ca(2+) entry. Ca(2+) entry mediated by the TRP3 channel stably expressed in HEK293 cells was activated by phospholipase C-coupled receptors but independent of Ca(2+) store depletion (Ma, H.-T., Patterson, R. L., van Rossum, D. B., Birnbaumer, L., Mikoshiba, K., and Gill, D. L. (2000) Science 287, 1647-1651). Receptor-induced TRP3 activation was 2-APB-sensitive and fully blocked by MDL. Direct stimulation of TRP3 channels by the permeant diacylglycerol derivative, 1-oleoyl-2-acetyl-sn-glycerol, was not blocked by 2-APB, but was again prevented by MDL. The results indicate that although the activation and coupling processes for each of the three entry mechanisms are distinct, sensitivity to MDL is a feature shared by all three mechanisms, suggesting there may be a common structural feature in the channels themselves or an associated regulatory component.


Subject(s)
Calcium Channels/physiology , Calcium/metabolism , Animals , Cricetinae , Diglycerides/pharmacology , Imines/pharmacology , Inositol 1,4,5-Trisphosphate Receptors , Male , Receptors, Cytoplasmic and Nuclear/physiology , TRPC Cation Channels , Triazoles/pharmacology
4.
Science ; 287(5458): 1647-51, 2000 Mar 03.
Article in English | MEDLINE | ID: mdl-10698739

ABSTRACT

The coupling mechanism between endoplasmic reticulum (ER) calcium ion (Ca2+) stores and plasma membrane (PM) store-operated channels (SOCs) is crucial to Ca2+ signaling but has eluded detection. SOCs may be functionally related to the TRP family of receptor-operated channels. Direct comparison of endogenous SOCs with stably expressed TRP3 channels in human embryonic kidney (HEK293) cells revealed that TRP3 channels differ in being store independent. However, condensed cortical F-actin prevented activation of both SOC and TRP3 channels, which suggests that ER-PM interactions underlie coupling of both channels. A cell-permeant inhibitor of inositol trisphosphate receptor (InsP3R) function, 2-aminoethoxydiphenyl borate, prevented both receptor-induced TRP3 activation and store-induced SOC activation. It is concluded that InsP3Rs mediate both SOC and TRP channel opening and that the InsP3R is essential for maintaining coupling between store emptying and physiological activation of SOCs.


Subject(s)
Calcium Channels/metabolism , Calcium Signaling , Calcium/metabolism , Endoplasmic Reticulum/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Actins/metabolism , Boron Compounds/pharmacology , Calcium Channels/chemistry , Carbachol/pharmacology , Cell Line , Cell Membrane/metabolism , Diglycerides/metabolism , Diglycerides/pharmacology , Enzyme Inhibitors/pharmacology , Humans , Inositol 1,4,5-Trisphosphate Receptors , Ionomycin/pharmacology , Macrocyclic Compounds , Marine Toxins , Oxazoles/pharmacology , Phosphoprotein Phosphatases/antagonists & inhibitors , Receptors, Cytoplasmic and Nuclear/chemistry , Strontium/metabolism , TRPC Cation Channels , Thapsigargin/pharmacology , Transfection , Type C Phospholipases/metabolism
5.
Cell ; 98(4): 487-99, 1999 Aug 20.
Article in English | MEDLINE | ID: mdl-10481913

ABSTRACT

The elusive coupling between endoplasmic reticulum (ER) Ca2+ stores and plasma membrane (PM) "store-operated" Ca2+ entry channels was probed through a novel combination of cytoskeletal modifications. Whereas coupling was unaffected by disassembly of the actin cytoskeleton, in situ redistribution of F-actin into a tight cortical layer subjacent to the PM displaced cortical ER and prevented coupling between ER and PM Ca2+ entry channels, while not affecting inositol 1,4,5-trisphosphate-mediated store release. Importantly, disassembly of the induced cortical actin layer allowed ER to regain access to the PM and reestablish coupling of Ca2+ entry channels to Ca2+ store depletion. Coupling is concluded to be mediated by a physical "secretion-like" mechanism involving close but reversible interactions between the ER and the PM.


Subject(s)
Calcium Channels/metabolism , Calcium Signaling/physiology , Calcium/metabolism , Cell Membrane/metabolism , Endoplasmic Reticulum/metabolism , Ion Channel Gating/physiology , Actins/physiology , Animals , Cell Line, Transformed , Cell Membrane/ultrastructure , Cells, Cultured , Cytochalasin D/pharmacology , Cytoskeleton/drug effects , Cytoskeleton/physiology , Cytoskeleton/ultrastructure , Inositol 1,4,5-Trisphosphate/metabolism , Ion Transport , Marine Toxins , Microscopy, Fluorescence , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/ultrastructure , Okadaic Acid/pharmacology , Oxazoles/pharmacology , Phosphoprotein Phosphatases/antagonists & inhibitors , Phosphoprotein Phosphatases/physiology , Rats
6.
Trends Neurosci ; 22(7): 290-5, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10370249

ABSTRACT

A wide heterogeneity in dendritic-spine morphology is observed and ultrastructural changes can be induced following experimental stimulation of neurons. Morphological adaptation of a given spine might, thus, reflect its history or the current state of synaptic activity. These changes could conceivably result from rearrangements of the cytoskeleton that is subjacent to excitatory synapses. This article dicusses the direct and indirect interactions, between glutamate receptors and the cytoskeletal proteins, which include PDZ-containing proteins, actin and tubulin, as well as associated proteins. In fact, the synaptic-activity-controlled balancing of monomeric, dimeric and polymeric forms of actin and tubulin might underlie the changes in spine shape. These continuous adaptations could be relevant for physiological events, such as learning and the formation of memory.


Subject(s)
Cytoskeletal Proteins/metabolism , Cytoskeleton/metabolism , Dendrites/metabolism , Nerve Tissue Proteins/metabolism , Receptors, Glutamate/metabolism , Actin Cytoskeleton/metabolism , Actins/metabolism , Animals , Cytoskeleton/ultrastructure , Dendrites/ultrastructure , Humans , Tubulin/metabolism
7.
J Neurochem ; 72(3): 962-73, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10037467

ABSTRACT

The cytoplasmic C-terminal domains (CTs) of the NR1 and NR2 subunits of the NMDA receptor have been implicated in its anchoring to the subsynaptic cytoskeleton. Here, we used affinity chromatography with glutathione S-transferase-NR1-CT and -NR2B-CT fusion proteins to identify novel binding partner(s) of these NMDA receptor subunits. Upon incubation with rat brain cytosolic protein fraction, both NR1-CT and NR2B-CT, but not glutathione S-transferase, specifically bound tubulin. The respective fusion proteins also bound tubulin purified from brain, suggesting a direct interaction between the two binding partners. In tubulin polymerization assays, NR1-CT and NR2B-CT significantly decreased the rate of microtubule formation without destabilizing preformed microtubules. Moreover, only minor fractions of either fusion protein coprecipitated with the newly formed microtubules. Consistent with these findings, ultrastructural analysis of the newly formed microtubules revealed a limited association only with the CTs of the NR1 and NR2B. These data suggest a direct interaction of the NMDA receptor channel subunit CTs and tubulin dimers or soluble forms of tubulin. The efficient modulation of microtubule dynamics by the NR1 and NR2 cytoplasmic domains suggests a functional interaction of the receptor and the subsynaptic cytoskeletal network that may play a role during morphological adaptations, as observed during synaptogenesis and in adult CNS plasticity.


Subject(s)
Glutathione Transferase/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Recombinant Fusion Proteins/metabolism , Tubulin/metabolism , Animals , Chromatography, Affinity , Cytoplasm/metabolism , Cytoplasm/ultrastructure , Glutathione Transferase/genetics , In Vitro Techniques , Microscopy, Electron , Microtubules/metabolism , Microtubules/ultrastructure , Rats , Receptors, N-Methyl-D-Aspartate/genetics , Solubility , Tubulin/biosynthesis
8.
Prog Neurobiol ; 55(6): 611-40, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9670221

ABSTRACT

The synaptic plasticity that is addressed in this review follows neurodegeneration in the brain and thus has both structural as well as functional components. The model of neurodegeneration that has been selected is the kainic acid lesioned hippocampus. Degeneration of the CA3 pyramidal cells results in a loss of the Schaffer collateral afferents innervating the CA1 pyramidal cells. This is followed by a period of structural plasticity where new synapses are formed. These are associated with changes in the numbers and shapes of spines as well as changes in the morphometry of the dendrites. It is suggested that this synaptogenesis is responsible for an increase in the ratio of NMDA to AMPA receptors mediating excitatory synaptic transmission at these synapses. Changes in the temporal and spatial properties of these synapses resulted in an altered balance between LTP and LTD. These properties together with a reduction in the inhibitory drive increased the excitability of the surviving CA1 pyramidal cells which in turn triggered epileptiform bursting activity. In this review we discuss the insights that may be gained from studies of the underlying molecular machinery. Developments in one of the collections of the cogs in this machinery has been summarized through recent studies characterizing the roles of neural recognition molecules in synaptic plasticity in the adult nervous systems of vertebrates and invertebrates. Such investigations of neural cell adhesion molecules, cadherins and amyloid precursor protein have shown the involvement of these molecules on the morphogenetic level of synaptic changes, on the one hand, and signal transduction effects, on the other. Further complex cogs are found in the forms of the low-density lipoprotein receptor (LDL-R) family of genes and their ligands play pivotal roles in the brain development and in regulating the growth and remodelling of neurones. Evidence is discussed for their role in the maintenance of cognitive function as well as Alzheimer's. The molecular mechanisms responsible for the clustering and maintenance of transmitter receptors at postsynaptic sites are the final cogs in the machinery that we have reviewed. Postsynaptic densities (PSD) from excitatory synapses have yielded many cytoskeletal proteins including actin, spectrin, tubulin, microtubule-associated proteins and calcium/calmodulin-dependent protein kinase II. Isolated PSDs have also been shown to be enriched in AMPA, kainate and NMDA receptors. However, recently, a new family of proteins, the MAGUKs (for membrane-associated guanylate kinase) has emerged. The role of these proteins in clustering different NMDA receptor subunits is discussed. The MAGUK proteins are also thought to play a role in synaptic plasticity mediated by nitric oxide (NO). Both NMDA and non-NMDA receptors are highly clustered at excitatory postsynaptic sites in cortical and hippocampal neurones but have revealed differences in their choice of molecular components. Both GABAA and glycine (Gly) receptors mediate synaptic inhibition in the brain and spinal cord. Whilst little is known about how GABAA receptors are localized in the postsynaptic membrane, considerable progress has been made towards the elucidation of the molecular mechanisms underlying the formation of Gly receptors. It has been shown that the peripheral membrane protein gephyrin plays a pivotal role in the formation of Gly receptor clusters most likely by anchoring the receptor to the subsynaptic cytoskeleton. Evidence for the distribution as well as function of gephyrin and Gly receptors is discussed. Postsynaptic membrane specializations are complex molecular machinery subserving a multitude of functions in the proper communication between neurones. Despite the fact that only a few key players have been identified it will be a fascinating to watch the story as to how they contribute to structural and functional plasticity unfold.


Subject(s)
Nerve Degeneration/physiopathology , Neuronal Plasticity/physiology , Synaptic Membranes/physiology , Animals , Hippocampus/drug effects , Hippocampus/pathology , Humans , Kainic Acid/toxicity , Multigene Family , Neural Cell Adhesion Molecules/physiology , Receptors, LDL/genetics
9.
Neurosci Biobehav Rev ; 21(5): 649-78, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9353797

ABSTRACT

Calcitonin generelated peptide (CGRP) is a neuropeptide discovered by a molecular approach over 10 years ago. More recently, islet amyloid polypeptide or amylin, and adrenomedullin were isolated from human insulinoma and pheochromocytoma respectively, and revealed between 25 and 50% sequence homology with CGRP. This review discusses findings on the anatomical distributions of CGRP mRNA, CGRP-like immunoreactivity and receptors in the central nervous system, as well as the potential physiological roles for CGRP. The anatomical distribution and biological activities of amylin and adrenomedullin are also presented. Based upon the differential biological activity of various CGRP analogs, the CGRP receptors have been classified in two major classes, namely the CGRP1 and CGRP2 subtypes. A third subtype has also been proposed (e.g. in the nucleus accumbens) as it does not share the pharmacological properties of the other two classes. The anatomical distribution and the pharmacological characteristics of amylin binding sites in the rat brain are different from those reported for CGRP but share several similarities with the salmon calcitonin receptors. The receptors identified thus far for CGRP and related peptides belong to the G protein-coupled receptor superfamily. Indeed, modulation of adenylate cyclase activity following receptor activation has been reported for CGRP, amylin and adrenomedullin. Furthermore, the binding affinity of CGRP and related peptides is modulated by nucleotides such as GTP. The cloning of various calcitonin and most recently of CGRP1 and adrenomedullin receptors was reported and revealed structural similarities but also significant differences to other members of the G protein-coupled receptors. They may thus form a new subfamily. The cloning of the amylin receptor(s) as well as of the other putative CGRP receptor subtype(s) are still awaited. Finally, a broad variety of biological activities has been described for CGRP-like peptides. These include vasodilation, nociception, glucose uptake and the stimulation of glycolysis in skeletal muscles. These effects may thus suggest their potential role and therapeutic applications in migraine, subarachnoid haemorrhage, diabetes and pain-related mechanisms, among other disorders.


Subject(s)
Calcitonin Gene-Related Peptide/physiology , Nervous System Physiological Phenomena , Nervous System/anatomy & histology , Receptors, Calcitonin Gene-Related Peptide/physiology , Amino Acid Sequence , Animals , Humans , Molecular Sequence Data , Rats , Receptors, Calcitonin Gene-Related Peptide/drug effects
10.
Neuroscience ; 79(3): 799-818, 1997 Aug.
Article in English | MEDLINE | ID: mdl-9219943

ABSTRACT

Interleukin-2 is an immunoregulatory cytokine with several recently established CNS activities. Central effects of interleukin-2 include growth promotion for neuronal and glial cells as well as modulatory influences on neurotransmission and hormone release. However, little is known about the consequences in the CNS of chronically elevated levels of interleukin-2. Alterations in the interleukin-2/interleukin-2 receptor system are not only associated with CNS trauma, inflammation and certain neuropathologies; elevated interleukin-2 concentrations are especially induced during the therapeutic use of interleukin-2 in cancer treatments. In the present study, intracerebroventricular (i.c.v.) interleukin-2 infusions (5 15 U/h) were performed in Sprague Dawley rats for up to 14 days. Interleukin-2-treated animals showed significantly increased plasma levels of corticosterone indicating an hyperfunctioning of the hypothalamic-pituitary-adrenocortical axis that lasted over the 14 day infusion period. Moreover, the performance of interleukin-2-treated animals in the Morris swim maze task was transiently impaired. Quantitative receptor autoradiographic analyses revealed changes in the binding levels of cholinergic M1 and M2 as well as dopaminergic D1 and D2 receptors in selected brain areas in which interleukin-2 was shown to modulate neurotransmission and which are enriched with interleukin-2 receptor expression. Decreased receptor binding levels were observed in the frontoparietal cortex (M2, D1, D2), hippocampal CA1 region (M1, M2) and the nucleus accumbens (D2). Histological and immunohistochemical examination of the brains of interleukin-2-treated animals revealed multiple alterations. Interleukin-2 treatment resulted in an intracranial accumulation of non-neural, MHC class II-positive cells as well as T and B lymphocytes within the infused brain hemisphere. Cellular infiltrates were associated with angiogenesis and the deposition of extracellular matrix material, such as fibronectin. Adjacent brain regions that were partly invaded and dislodged by the cellular masses were characterized by reactive astrogliosis, microglial activation, endothelial upregulation of adhesion molecules, myelin damage and neuronal loss. Together the data suggest that persistently elevated central levels of interleukin-2 can interfere with several CNS functions and may lead to nervous tissue injury. These findings could be relevant to CNS pathologies characterized by abnormal interleukin-2 production and to central responses to interleukin-2 treatments.


Subject(s)
Brain/drug effects , Interleukin-2/toxicity , Animals , Autoradiography , Brain/ultrastructure , Injections, Intraventricular , Male , Microscopy, Confocal , Rats , Rats, Sprague-Dawley , Time Factors
11.
Neuroreport ; 7(18): 2883-8, 1996 Nov 25.
Article in English | MEDLINE | ID: mdl-9116202

ABSTRACT

Intracerebroventricular infusion of interleukin-2 (IL-2, 15 U h-1 for 14 days) chronically activated the hypothalamic-pituitary-adrenocortical (HPA) axis in rats. IL-2 induced increases in plasma levels of adrenocorticotropic hormone (ACTH, up to two-fold) and corticosterone (up to four-fold) compared with controls. Continuously elevated brain levels of IL-2 did not lead to a persistent HPA activation, but resulted in (two) periods of hormonal hypersecretion. ACTH and corticosterone levels were elevated between days 3 and 5, with changes in corticosterone preceding those of ACTH. Concentrations of corticosterone, but not of ACTH, increased again on day 11. Underscoring its importance as a neuroendocrine regulator, this study reveals that, in addition to its immediate effects, IL-2 induces a complex pattern of chronic HPA stimulation. These findings may functionally relate to several CNS disorders and certain endocrine dysfunctions observed during IL-2 immunotherapy.


Subject(s)
Hypothalamo-Hypophyseal System/physiology , Interleukin-2/pharmacology , Adrenocorticotropic Hormone/blood , Animals , Corticosterone/blood , Histocytochemistry , Hypothalamo-Hypophyseal System/drug effects , Injections, Intraventricular , Interleukin-2/administration & dosage , Male , Radioimmunoassay , Rats , Rats, Sprague-Dawley
12.
J Neurosci ; 16(7): 2342-51, 1996 Apr 01.
Article in English | MEDLINE | ID: mdl-8601814

ABSTRACT

Tolerance to morphine analgesia is believed to result from a neuronal adaptation produced by continuous drug administration, although the precise mechanisms involved have yet to be established. Recently, we reported selective alterations in rat spinal calcitonin gene-related peptide (CGRP) markers in morphine-tolerant animals. In fact, increases in CGRP-like immunostaining and decrements in specific [125]hCGRP binding in the superficial laminae of the dorsal horn were correlated with the development of tolerance to the spinal antinociceptive action of morphine. Other spinally located peptides such as substance P, galanin, and neuropeptide Y were unaffected. Thus, the major goal of the present study was to investigate whether the development of tolerance to spinally infused morphine could be modulated by the blockade of dorsal horn CGRP receptors using the potent CGRP antagonist hCGRP(8-37). Indeed, cotreatments with hCGRP(8-37) prevented, in a dose-dependent manner, the development of tolerance to morphine-induced analgesia in both the rat tail-flick/tail-immersion and paw-pressure tests. Moreover, alterations in spinal CGRP markers seen in morphine-tolerant animals were not observed after a coadministration of morphine and hCGRP(8-37). These results demonstrate the existence of specific interaction between CGRP and the development of tolerance to the spinal antinociceptive effects of morphine. They also suggest that CGRP receptor antagonists could become useful adjuncts in the treatment of pain and tolerance to the antinociceptive effects of morphine.


Subject(s)
Analgesics, Opioid/pharmacology , Calcitonin Gene-Related Peptide Receptor Antagonists , Morphine/pharmacology , Spinal Cord/drug effects , Animals , Dose-Response Relationship, Drug , Drug Tolerance , Immunohistochemistry , Male , Motor Activity/drug effects , Motor Neurons/drug effects , Motor Neurons/ultrastructure , Rats , Rats, Sprague-Dawley
13.
Can J Physiol Pharmacol ; 73(7): 1084-8, 1995 Jul.
Article in English | MEDLINE | ID: mdl-8846406

ABSTRACT

Adrenomedullin (ADM) is a recently identified peptide that shows some homology (approximately 25%) with calcitonin gene related peptide (CGRP) and is now considered to be a new member of this peptide family. Because it shares biological effects with CGRP, we evaluated the possible affinity of human adrenomedullin (hADM) for 125I-labelled human CGRP alpha ([125I]hCGRP alpha) binding sites in the rat brain. Moreover, we evaluated the potential existence of cross-reactivity for 125I-labelled Bolton-Hunter rat amylin ([125I]BHrAMY), another member of this peptide family. In all brain areas investigated, hADM only competed with relatively low affinities for both [125I]hCGRP alpha and [125I]BHrAMY binding sites, with IC50 values generally in the high nanomolar-low micromolar range, the lowest affinity being observed for [125I]BHrAMY binding sites. Interestingly, the lowest affinities of hADM against both radioligands were detected in the nucleus accumbens and ventral striatum. These areas are known to be enriched with atypical CGRP - salmon calcitonin - amylin sensitive sites. It thus appears that hADM is unlikely to bind to this atypical site. Moreover, hADM demonstrated limited affinity for either [125I]hCGRP alpha or [125I]BHrAMY binding sites in the rat brain. This suggests that the potential biological effects of ADM in the brain could be mediated through a different class of receptors with higher affinity for this newly isolated peptide.


Subject(s)
Antihypertensive Agents/metabolism , Brain/metabolism , Peptides/metabolism , Receptors, Calcitonin Gene-Related Peptide/metabolism , Receptors, Peptide/metabolism , Adrenomedullin , Animals , Binding Sites , Humans , Male , Rats , Rats, Sprague-Dawley , Receptors, Islet Amyloid Polypeptide
14.
Can J Physiol Pharmacol ; 73(7): 1089-95, 1995 Jul.
Article in English | MEDLINE | ID: mdl-8846407

ABSTRACT

Calcitonin gene related peptide (CGRP), one of the most abundant peptides in the spinal cord, is localized in primary afferents and released following nociceptive stimuli. Its colocalization and corelease with substance P, a well-known nociceptive neuropeptide, support the importance of CGRP in pain mechanisms. However, its distinctive function in that regard remains to be fully established. Recently, we reported that increases in CGRP-like immunostaining and decrements in specific 125I-labelled human CGRP alpha ([125I]hCGRP alpha) binding sites in the spinal cord were correlated with the development of tolerance to the spinal antinociceptive action of a mu opioid agonist, morphine. The goal of the present study was to investigate whether the development of tolerance to other classes of opioids, namely, delta and kappa agonists, can also alter CGRP-like immunostaining and receptors in the rat spinal cord. The antinociceptive effects of all opioids were monitored by the tail-immersion test. Tolerance to their antinociceptive properties was induced by the infusion for 7 days of mu (morphine sulfate, 7.5 micrograms/h), delta D([D-Pen2,D-Pen5]enkephalin (DPDPE), 2.0 micrograms/h), and kappa (U-50488H, 10.0 micrograms/h) related agonists at the spinal level (L4), using osmotic minipumps. We confirmed that rats chronically treated with morphine showed significant decreases in [125I]CGRP alpha binding in laminae I, II, and III of the L4 spinal cord, while CGRP-like immunostaining was increased in these same laminae. Similar effects were observed following a treatment with the delta agonist, DPDPE, while the kappa agonist, U-50488H, apparently only slightly decreased [125I]CGRP alpha] binding in lamina II. Binding in other laminae and CGRP-like immunostaining were not affected. These results suggest a specific interaction between spinal CGRP systems and the development of tolerance to the spinal antinociceptive effects of mu- and delta-related agonists.


Subject(s)
Calcitonin Gene-Related Peptide/analysis , Narcotics/pharmacology , Receptors, Calcitonin Gene-Related Peptide/analysis , Receptors, Opioid, delta/agonists , Receptors, Opioid, mu/agonists , Spinal Cord/drug effects , 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer , Animals , Autoradiography , Drug Tolerance , Enkephalin, D-Penicillamine (2,5)- , Enkephalins/pharmacology , Immunohistochemistry , Male , Morphine/pharmacology , Pyrrolidines/pharmacology , Rats , Rats, Sprague-Dawley , Spinal Cord/chemistry
15.
J Pharmacol Exp Ther ; 273(2): 887-94, 1995 May.
Article in English | MEDLINE | ID: mdl-7752094

ABSTRACT

Tolerance to the spinal antinociceptive effects of morphine develops rapidly after its chronic administration. The mechanism involved in this phenomenon is unclear, but it is unlikely due to a direct regulation of spinal opioid peptides and their receptor binding sites. A variety of neuropeptides, especially the neurokinins and calcitonin gene-related peptide (CGRP) are concentrated in primary sensory afferents and have thus been proposed to play significant roles in spinal nociceptive mechanisms. However, their functions in the development of tolerance to the antinociceptive properties of morphine have not been explored fully. We therefore investigated the possible involvement of various sensory neuropeptides including CGRP, substance P, galanin, neurotensin and neuropeptide Y and their receptors in the dorsal horn of the spinal cord during the development of tolerance to the antinociceptive action of intrathecal morphine. Morphine sulfate (7.5 micrograms/microliters/hr) was administered continuously at lumbar level L4 using mini-osmotic pumps for 3, 5, 7 and 14 days. Tolerance to the antinociceptive effect of morphine was verified with the tail-immersion test and became evident on the 5th day of treatment. In tolerant animals, there was a marked increase in CGRP-like immunostaining and a decrease (30-45%) in [125I]human CGRP alpha binding in laminae I, II and III of the dorsal horn of the spinal cord. These changes coincided with the onset of morphine tolerance and persisted for the 14-day period during which tolerance was present. Similar changes were not observed in the immunostaining or binding of the other neuropeptides studied.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Calcitonin Gene-Related Peptide/metabolism , Morphine/pharmacology , Receptors, Neuropeptide/metabolism , Spinal Cord/drug effects , Animals , Autoradiography , Drug Tolerance , Humans , Male , Neuropeptides/metabolism , Protein Binding , Rats , Rats, Sprague-Dawley , Spinal Cord/metabolism
16.
Appl Environ Microbiol ; 61(4): 1599-609, 1995 Apr.
Article in English | MEDLINE | ID: mdl-7538280

ABSTRACT

Seventeen Bradyrhizobium sp. strains and one Azorhizobium strain were compared on the basis of five genetic and phenetic features: (i) partial sequence analyses of the 16S rRNA gene (rDNA), (ii) randomly amplified DNA polymorphisms (RAPD) using three oligonucleotide primers, (iii) total cellular protein profiles, (iv) utilization of 21 aliphatic and 22 aromatic substrates, and (v) intrinsic resistances to seven antibiotics. Partial 16S rDNA analysis revealed the presence of only two rDNA homology (i.e., identity) groups among the 17 Bradyrhizobium strains. The partial 16S rDNA sequences of Bradyrhizobium sp. strains form a tight similarity (> 95%) cluster with Rhodopseudomonas palustris, Nitrobacter species, Afipia species, and Blastobacter denitrificans but were less similar to other members of the alpha-Proteobacteria, including other members of the Rhizobiaceae family. Clustering the Bradyrhizobium sp. strains for their RAPD profiles, protein profiles, and substrate utilization data revealed more diversity than rDNA analysis. Intrinsic antibiotic resistance yielded strain-specific patterns that could not be clustered. High rDNA similarity appeared to be a prerequisite, but it did not necessarily lead to high similarity values between RAPD profiles, protein profiles, and substrate utilization. The various relationship structures, coming forth from each of the studied features, had low compatibilities, casting doubt on the usefulness of a polyphasic approach in rhizobial taxonomy.


Subject(s)
Arachis/microbiology , Rhizobiaceae/genetics , Bacterial Proteins/isolation & purification , Bacterial Typing Techniques , Base Sequence , DNA Fingerprinting , DNA Primers/genetics , DNA, Bacterial/genetics , DNA, Ribosomal/genetics , Drug Resistance, Microbial/genetics , Genotype , Molecular Sequence Data , Phenotype , RNA, Bacterial/genetics , RNA, Ribosomal, 16S/genetics , Rhizobiaceae/classification , Rhizobiaceae/metabolism
17.
J Pharmacol Exp Ther ; 270(2): 779-87, 1994 Aug.
Article in English | MEDLINE | ID: mdl-8071870

ABSTRACT

Amylin is a recently isolated peptide from amyloid plaques in noninsulin-dependent diabetic patients and showed high sequence homology with calcitonin gene-related peptide. We investigated the distribution and the binding profile of [125I]Bolton Hunter-rat amylin ([125I]BH-rat amylin) binding sites in the rat brain, as well as the affinity of rat amylin for [125I]hCGRP alpha binding sites in the brain, atrium (CGRP1 receptor-enriched tissue) and vas deferens (CGRP2 receptor-enriched tissue). High amounts of high affinity [125I]BH-rat amylin binding sites were observed in the nucleus accumbens, various hypothalamic nuclei, amygdaloid body, dorsal raphe, tegmental and parabrachial nuclei and the locus ceruleus. Interestingly, both rat amylin and salmon calcitonin revealed low nanomolar affinities (2-19 nM) for [125I] BH-rat amylin binding sites in the various brain areas, whereas human calcitonin gene-related peptide-alpha (hCGRP alpha) showed lower affinities ranging between 13 to 150 nM. Moreover, the affinity of rat amylin was much lower than that of hCGRP alpha for [125I]hCGRP alpha binding in the brain, atrium and vas deferens, except for very few areas such as the nucleus accumbens and ventral striatum. Similarly, rat amylin was much weaker (100- to 400-fold) than hCGRP alpha to induce a biological effect in the atrium and vas deferens. These results thus suggest the existence of unique [125I]BH-rat amylin binding sites in the rat brain as well as limited cross-reactivity between rat amylin and [125I]hCGRP alpha receptors present in the brain, atrium and vas deferens.


Subject(s)
Amyloid/metabolism , Brain/metabolism , Receptors, Peptide/metabolism , Animals , Autoradiography , Binding, Competitive , Calcitonin Gene-Related Peptide/metabolism , Guinea Pigs , In Vitro Techniques , Islet Amyloid Polypeptide , Male , Rats , Rats, Sprague-Dawley , Receptors, Islet Amyloid Polypeptide
18.
J Pharmacol Exp Ther ; 269(2): 846-53, 1994 May.
Article in English | MEDLINE | ID: mdl-8182554

ABSTRACT

The calcitonin gene-related peptide (CGRP) C-terminal fragment human CGRP8-37 acts as a potent antagonist of various in vitro and in vivo effects of CGRP. Its iodinated counterpart, [125I-Tyr] hCGRP8-37, binds with high affinity (KD values between 7.5 x 10(-11)-2.1 x 10(-10) M) to what is apparently a single class of CGRP receptors in brain, atrium and vas deferens membrane preparations. The relative potencies of various CGRP-related fragments and analogs in competing for [125I-Tyr]hCGRP8-37 binding sites were similar in these three preparations, with hCGRP alpha being the most potent competitor, followed by unlabeled hCGRP8-37, the linear agonist [acetamidomethyl-cysteine2,7] hCGRP alpha, and finally by rat amylin-amide and salmon calcitonin. Competition profiles suggested the existence of a single affinity site (except in the case of hCGRP alpha, for which competition binding data were best fitted to two apparent affinity constants in all three tissues), whereas rat amylin-amide revealed two affinity constants in the rat brain. Guanylylimidodiphosphate (100 microM) failed to alter specific [125I-Tyr]hCGRP8-37 binding in the various tissues studied here. Quantitative receptor autoradiography in the rat brain revealed [125I-Tyr]hCGRP8-37 binding sites mostly concentrated in the nucleus accumbens (shell), caudate putamen (tail), amygdaloid body, pontine nuclei, cerebellum and inferior olive, whereas lower quantities of sites were present in the olfactory tubercle, nucleus accumbens (core), medial geniculate, superior colliculus, temporal cortex, inferior colliculus, lateral lemniscus, vestibular nuclei and principal sensory trigeminal nucleus.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Brain/metabolism , Calcitonin Gene-Related Peptide Receptor Antagonists , Calcitonin Gene-Related Peptide/metabolism , Peptide Fragments/metabolism , Animals , Autoradiography , Binding, Competitive , Guinea Pigs , In Vitro Techniques , Male , Radioligand Assay , Rats , Receptors, Calcitonin Gene-Related Peptide/metabolism
19.
Brain Res ; 617(2): 249-57, 1993 Jul 23.
Article in English | MEDLINE | ID: mdl-8402153

ABSTRACT

Recent data have suggested the existence of at least two major classes of calcitonin gene-related peptide (CGRP) receptors in brain and peripheral tissues [Henke et al., Brain Res., 410 (1987) 404-408; Dennis et al., J. Pharmacol. Exp. Ther., 251 (1989) 718-725; ibid, 254 (1990) 123-128; Quirion et al., Ann. NY Acad. Sci., 657 (1992) 88-105]. However, little is currently known in the structure characteristics of CGRP receptors as cloning as yet to be reported. In the present study, the sensitivity of [125I]humanCGRP alpha binding to guanine nucleotides and temperature was investigated in guinea pig atria (prototypical CGRP1 tissue) guinea pig vas deferens (prototypical CGRP2 tissue) and in the rat brain and cerebellum (mixed assay). Binding isotherms of [125I]hCGRP alpha in those four tissue preparations were curvilinear and best fitted to a two-site model under most assay conditions. The high affinity binding component was highly temperature-sensitive and accounted, under experimental conditions, for up to 18% of the total population of receptors. Moreover, these high affinity sites were also highly sensitive to guanine nucleotides (Gpp(NH)p, 100 microM) in all preparations although to a different extend depending upon assay temperatures. Taken together, this suggests that the different CGRP receptor subtypes present in these tissue all belong to a G-protein coupled receptor family.


Subject(s)
Brain/drug effects , Guanine Nucleotides/pharmacology , Receptors, Calcitonin Gene-Related Peptide/drug effects , Temperature , Animals , Brain/metabolism , Cerebellum/drug effects , Cerebellum/metabolism , GTP-Binding Proteins/metabolism , Guanylyl Imidodiphosphate/physiology , Guinea Pigs , Heart Atria/drug effects , Heart Atria/metabolism , Iodine Radioisotopes , Male , Radioligand Assay , Rats , Rats, Sprague-Dawley , Vas Deferens/drug effects , Vas Deferens/metabolism
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