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A H Sharp

Publications and source records attributed to A H Sharp.

At least 55 records · Page 3Linked to original sources
PubMed1993

Inositol 1,4,5-trisphosphate receptors: immunohistochemical localization to discrete areas of rat central nervous system.

The second messenger inositol 1,4,5-trisphosphate triggers the release of intracellular Ca2+ stores upon binding to the inositol 1,4,5-trisphosphate receptor protein, a calcium channel that has been purified and molecularly cloned. To clarify the roles of inositol 1,4,5-trisphosphate receptor in the central nervous system, we have examined in detail the distribution of inositol 1,4,5-trisphosphate receptors in the rat brain and spinal cord using immunohistochemical methods. Inositol 1,4,5-trisphosphate receptors are present in neuronal cells, fibers and terminals in a wide distribution of areas throughout the central nervous system. These include a number of areas not previously reported, such as the olfactory bulb, thalamic nuclei and dorsal horn of the spinal cord. In addition, we have noted a strikingly high density of inositol 1,4,5-trisphosphate receptors in circumventricular organs and neuroendocrine structures such as the area postrema, choroid plexus, subcommisural organ, pineal gland and pituitary. The distribution of inositol 1,4,5-trisphosphate receptors in discrete structures throughout the central nervous system, including interconnected neuronal systems and neuroendocrine and circumventricular organ structures, presumably reflects the importance of Ca2+ release mediated by the phosphoinositide second messenger system in control of diverse physiological processes.

Animals↗
PubMed1993

Differential localization of phosphoinositide-linked metabotropic glutamate receptor (mGluR1) and the inositol 1,4,5-trisphosphate receptor in rat brain.

The type 1 metabotropic glutamate receptor (mGluR1) is through to act via the phosphoinositide (PI) system with the associated formation of inositol 1,4,5-trisphosphate (IP3) and Ca2+ release. Utilizing immunohistochemistry and in situ hybridization, we have localized protein and mRNA, respectively, for the mGluR1 and the IP3 receptor (IP3R). We have also localized glutamate-linked PI turnover by autoradiography with 3H-cytidine. We observe a striking contrast in localizations of mGluR1 and IP3R both for protein and mRNA. For instance, mGluR1 occurs in the apparent absence of IP3R in neurons of the stratum oriens of the CA1 hippocampus, islands of Calleja, anterodorsal nucleus of thalamus, lateral nucleus of hypothalamus, and the granular cell layer and the deep nuclei of cerebellum. mGluR1 actions in these brain regions may primarily be mediated through the protein kinase C limb of the PI system, as they contain moderate amounts of 3H-phorbol ester binding. The subthalamic nucleus, red nucleus, and Darkshevich's nucleus, which possess high levels of mGluR1, are devoid of both IP3R immunoreactivity and 3H-phorbol ester binding. These reciprocal localizations suggest that mGluR1 actions in many brain areas may not primarily involve IP3, reflecting instead influences on protein kinase C or other second messengers.

Animals↗
PubMed1993

Differential immunohistochemical localization of inositol 1,4,5-trisphosphate- and ryanodine-sensitive Ca2+ release channels in rat brain.

Ca2+ release from inositol 1,4,5-trisphosphate (IP3)-sensitive and ryanodine-sensitive intracellular Ca2+ stores is mediated by distinct proteins identified as IP3 receptors (IP3R) and ryanodine receptors (RyR), respectively. We have compared the immunohistochemical localizations of IP3R and RyR in the brain at the light and electron microscopic levels and have also evaluated the distribution of the major brain intracellular Ca(2+)-pumping ATPase. IP3R and RyR occur in overlapping populations of neurons in widespread areas of the brain, but labeling is distinct in a number of areas. For example, IP3R is enriched in cerebellar Purkinje cells and hippocampal CA1 pyramidal cells, while RyR is present at relatively low levels in these cells. RyR is most enriched in the dentate gyrus and CA3/4 areas of the hippocampus, where IP3R levels are low. In the cortex, IP3R is found in pyramidal cell bodies and proximal dendrites, whereas RyR is located predominantly in long, thin apical dendrites of pyramidal cells. In deep cerebellar nuclei, RyR is located in cell bodies that appear devoid of IP3R, whereas IP3R is enriched in terminals surrounding cell bodies. Electron microscopy in the hippocampus reveals RyR in axons, dendritic spines, and dendritic shafts near dendritic spines while IP3R is primarily identified in dendritic shafts and cell bodies. These results suggest that the IP3- and ryanodine-sensitive Ca2+ pools have largely distinct roles in controlling intracellular Ca2+ levels, though in some sites they may interact to varying degrees.

Animals↗
PubMed1993

Phosphoinositide second messenger system is enriched in striosomes: immunohistochemical demonstration of inositol 1,4,5-trisphosphate receptors and phospholipase C beta and gamma in primate basal ganglia.

The neurochemical organization of the basal ganglia has been studied extensively with respect to neurotransmitters, neuropeptides, and their receptors. The chemoarchitecture of the striatum has been found particularly striking, because it distinguishes many substances by their relative distributions within the striosome and matrix compartments of the striatum. Very little is yet known about the differential distribution of second messenger systems in the basal ganglia, however, and no information is available about whether the distribution of second messenger systems is related to the prominent neurochemical compartmentalization of the striatum. We have examined the distribution of the phosphoinositide second messenger system in the primate basal ganglia and substantia nigra, as detected with polyclonal antisera against the inositol 1,4,5-trisphosphate receptor (IP3R), and monoclonal antisera against phospholipase C beta (PLC beta) and phospholipase C gamma (PLC gamma). In the striatum, immunostaining for each of the three proteins was present predominantly in medium-sized neuronal perikarya and in the neuropil. Circumscribed zones of enhanced IP3R, PLC beta, and PLC gamma immunoreactivity appeared in a background of generally weaker staining, and these zones corresponded to striosomes as identified by calbinidin D28k and substance P immunostaining in adjacent sections. Thus, the richest representation of the phosphoinositide system in the primate striatum appears to be in striosomes. In the substantia nigra pars compacta, neurons and neuropil were immunopositive, but in the substantia nigra pars reticulata and in each segment of the globus pallidus, immunostaining was mainly confined to the neuropil. Perikaryal PCL gamma immunoreactivity in the absence of detectable PLC beta or IP3R immunolabeling was found in the magnocellular neurons embedded in the medullary layer between the putamen and the globus pallidus. These observations demonstrate that the phosphoinositide second messenger system is selectively enhanced in neuronal subsystems of the basal ganglia, including striosomes, and suggest that signaling by phosphoinositide pathways elicits discrete effects on input-output processing by the basal ganglia.

Animals↗
PubMed1992

Inositol 1,4,5-trisphosphate receptors. Localization in epithelial tissue.

Using a polyclonal antiserum raised against the inositol 1,4,5-trisphosphate receptor (IP3R) purified from rat cerebellum, we examined the subcellular distribution of IP3R in canine pancreatic homogenates. IP3R was present primarily in a smooth microsomal fraction (low density), a (high density) rough microsomal (RM) fraction previously shown to consist of highly purified rough endoplasmic reticulum (RER) vesicles, and, to a much lesser extent, in an intermediate density microsomal fraction which did not contain markers for RER or plasma membrane. When the RM fraction was subjected to isopycnic centrifugation on sucrose gradients, IP3R equilibrated at high sucrose densities. When ribosomes were extracted from the RM fraction by treatment with puromycin/high salt, IP3R equilibrated at considerably lighter sucrose densities. This shift in density indicated that IP3R which was present in the RM fraction is associated with the RER. Because of a significant amount of IP3R fractionating into the smooth microsomal fraction (which contains plasma membrane, among other "smooth" membranes) and a considerable amount of IP3R present in the nuclear pellet which is also enriched in plasma membrane, we examined the possibility that IP3R may be present in plasma membrane. Further subfractionation of a crude plasma membrane pellet from rat liver revealed that IP3R coenriched with a plasma membrane marker and strongly suggested an association of IP3R with plasma membrane. The issue of why the same receptor is found in multiple biochemically and morphologically distinct membrane fractions is discussed in terms of the possibility of RER subcompartmentalization and IP3R subtypes. The fractionation pattern of IP3R in pancreas is significantly different from that previously reported for calcium (Ca2+)-binding proteins and an intracellular Ca-ATPase (Nigam, S. K. and Towers, T. (1990) J. Cell Biol. 111, 197-200), raising questions as to links between these latter proteins and IP3 sensitive Ca2+ pools. Nevertheless, although the fractionation patterns are different, all of these proteins are clearly associated with the RER.

Animals↗
PubMed1992

The endoplasmic reticulum of Purkinje neuron body and dendrites: molecular identity and specializations for Ca2+ transport.

Immunofluorescence and immunogold labeling, together with sucrose gradient separation and Western blot analysis of microsomal subfractions, were employed in parallel to probe the endoplasmic reticulum in the cell body and dendrites of rat cerebellar Purkinje neurons. Two markers, previously investigated in non-nerve cells, the membrane protein p91 (calnexin) and the lumenal protein BiP, were found to be highly expressed and widely distributed to the various endoplasmic reticulum sections of Purkinje neurons, from the cell body to dendrites and dendritic spines. An antibody (denominated anti-rough-surfaced endoplasmic reticulum), which recognized two membrane proteins, p14 and p40, revealed a similar immunogold labeling pattern. However, centrifugation results consistent with a widespread distribution were obtained for p14 only, while p40 was concentrated in the rough microsome-enriched subfractions. The areas enriched in the inositol 1,4,5-triphosphate receptor and thus presumably specialized in Ca2+ transport (stacks of multiple smooth-surfaced cisternae; the dendritic spine apparatus) also exhibited labeling for BiP and p91, and were positive for the anti-rough-surfaced endoplasmic reticulum antibody (presumably via the p14 antigen). Additional antibodies, that yielded inadequate immunocytochemical signals, were employed only by Western blotting of the microsomal subfractions, while the ryanodine receptor was studied by specific binding. The latter receptor and the Ca2+ ATPase, known in other species to be concentrated in Purkinje neurons, exhibited bimodal distributions with a peak in the light and another in the heavy subfractions. A similar distribution was also observed with another lumenal protein, protein disulfide isomerase. Taken as a whole, the results that we have obtained suggest the existence in the endoplasmic reticulum of Purkinje neurons of two levels of organization; the first identified by widespread, probably general markers (BiP, p91, possibly p14 and others), the second by specialization markers, such as the inositol 1,4,5-triphosphate receptor and, possibly, p40, which appear restricted to areas where specific functions appear to be localized.

Animals↗
PubMed1991

Purified omega-conotoxin GVIA receptor of rat brain resembles a dihydropyridine-sensitive L-type calcium channel.

The omega-conotoxin GVIA (CTX) receptor has been purified 1900-fold to apparent homogeneity by monitoring both reversible binding of 125I-labeled CTX (125I-CTX) and photoincorporation of N-hydroxysuccinimidyl-4-azidobenzoate-125I-CTX (HSA-125I-CTX). Photoincorporation of HSA-125I-CTX into a 230-kDa protein exhibits a pharmacologic and chromatographic profile indicating that the 230-kDa protein is the CTX-binding subunit of the receptor. The pharmacologic specificity of 125I-CTX binding to the purified CTX receptor closely resembles that of the native membrane-bound form with respect to sensitivity towards CTX (Kd = 32 pM) and other peptide toxin antagonists. The purified CTX receptor comprises the 230-kDa protein (alpha 1) and four additional proteins with apparent molecular masses of 140 (alpha 2), 110, 70 (beta 2), and 60 (beta 1) kDa. This subunit structure closely resembles that of the 1,4-dihydropyridine-sensitive L-type calcium channel.

Affinity Labels↗
PubMed1991

Structural characterization of the dihydropyridine-sensitive calcium channel alpha 2-subunit and the associated delta peptides.

Upon disulfide bond reduction, the alpha 2-subunit of the dihydropyridine-sensitive Ca2+ channel undergoes a characteristic mobility shift on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis with the concurrent appearance of the three delta peptides delta 1 (25,000 Da), delta 2 (22,000 Da), and delta 3 (17,000 Da). Densitometric scanning of Coomassie Blue-stained gels shows a stoichiometric ration of 1.0:0.31.47:0.08 for the alpha 2-subunit and the delta peptides 1, 2, and 3, respectively. Characterization of the delta peptides using antibodies, photoincorporation of a hydrophobic probe, and lectin staining shows tham to be antigenically similar hydrophobic glycoproteins. Amino-terminal sequence analysis of the delta peptides reveals three identical sequences that match the predicted amino acid sequence of the alpha 2-subunit starting at Ala935. Enzymatic deglycosylation of the reduced alpha 2.delta complex produces individual core peptides of 105,000 and 17,000 Da, respectively. Treatment of skeletal muscle membranes with high pH in the presence of reducing agents is able to extract the larger amino-terminal peptide but not the smaller carboxyl (delta) peptide, consistent with a single transmembrane domain in the carboxyl (delta) region. The data support a model of the alpha 2-subunit in which the propeptide is processed into two chains that remain attached through disulfide linkages.

Animals↗
PubMed1991

Heterogeneity of microsomal Ca2+ stores in chicken Purkinje neurons.

Chicken cerebellum microsomes were subfractionated on isopycnic, linear sucrose (15-50%) density gradients. The distribution of four markers of intracellular, rapidly-exchanging Ca2+ stores, i.e. the Ca2+ pump, the receptors for inositol 1,4,5-trisphosphate (IP3) and ryanodine (Ry), and calsequestrin (CS, an intralumenal, high capacity Ca2+ binding protein) was investigated biochemically and immunologically. In the cerebellum, high levels of these markers are expressed by one of the cell types, the Purkinje neuron. Heavy subfractions were enriched in both CS and Ry receptor, intermediate subfractions in the IP3 receptor, while the Ca2+ pump was present in both intermediate and heavy subfractions. Intact cells and pelleted subfractions were examined by conventional and immuno-electron microscopy (immunogold labeling of ultrathin cryosections with anti-CS and anti-IP3 receptor antibodies). Of the strongly CS-labeled, moderately dense-cored vacuoles (calciosomes) recently described in chicken Purkinje neurons only partly exhibited labeling for the IP3 receptor as well, and the rest appeared negative. The latter were enriched in a heavy subfraction of the gradient where Ry receptors were also concentrated, whereas the CS-rich vacuoles in an intermediate subfraction were almost always IP3 receptor-positive. The population of CS-rich calciosomes of chicken Purkinje neurons appears therefore to be molecularly heterogeneous, with a part responsive to IP3 and the rest possibly sensitive to Ry.

Animals↗
PubMed1991

Localization of the inositol 1,4,5-trisphosphate receptor in synaptic terminals in the vertebrate retina.

Inositol 1,4,5-trisphosphate (InsP3) mobilizes internal Ca2+ in cells by binding to a receptor protein, which has recently been purified and molecularly cloned. To clarify those neuronal functions that are regulated by InsP3, we have localized this InsP3 receptor protein immunocytochemically in the retina, a neural tissue of well-defined structure and function. Positive staining in neurons is confined almost exclusively to the synaptic layers. Using dissociated retinal neurons, we have further localized the receptor to presynaptic terminals of photoreceptors and bipolar cells, as well as the synaptic processes of amacrine cells. The specific association of InsP3 receptors with synaptic terminals suggests a role for InsP3 in synaptic modulation, especially with respect to transmitter release.

Ambystoma↗
PubMed1989

Characterization of the 1,4-dihydropyridine receptor using subunit-specific polyclonal antibodies. Evidence for a 32,000-Da subunit.

The purified receptor for the 1,4-dihydropyridine Ca2+ channel blockers from rabbit skeletal muscle contains protein components of 170,000 Da (alpha 1), 175,000 Da (alpha 2), 52,000 Da (beta), and 32,000 Da (gamma) when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. Subunit-specific polyclonal antibodies have now been prepared and used to characterize the association of the 32,000-Da polypeptide (gamma subunit) with other subunits of the dihydropyridine receptor. Immunoblot analysis of fractions collected during purification of the dihydropyridine receptor shows that the 32,000-Da polypeptide copurified with alpha 1 and alpha 2 subunits at each step of the purification. In addition, monoclonal antibodies against the alpha 1 and beta subunits immunoprecipitate the digitonin-solubilized dihydropyridine receptor as a multisubunit complex which includes the 32,000-Da polypeptide. Polyclonal antibodies generated against both the nonreduced and reduced forms of the alpha 2 subunit and the gamma subunit have been used to show that the 32,000-Da polypeptide is not a proteolytic fragment of a larger component of the dihydropyridine receptor and not disulfide linked to the alpha 2 subunit. In addition, polyclonal antibodies against the rabbit skeletal muscle 32,000-Da polypeptide specifically react with similar proteins in skeletal muscle of other species including avian and amphibian species. Thus, our results demonstrate that the 32,000-Da polypeptide (gamma subunit) is an integral and distinct component of the dihydropyridine receptor.

Animals↗
PubMed1989

Specific absence of the alpha 1 subunit of the dihydropyridine receptor in mice with muscular dysgenesis.

Muscular dysgenesis is a lethal mutation in mice that results in a complete absence of skeletal muscle contraction due to the failure of depolarization of the transverse tubular membrane to trigger calcium release from the sarcoplasmic reticulum. In order to determine whether the defect in muscular dysgenesis leads to a specific loss of one of the components of excitation-contraction coupling or to a generalized loss of all components of excitation-contraction coupling, we have analyzed skeletal muscle from control and dysgenic mice for the sarcoplasmic reticulum and transverse tubular proteins which are believe to function in excitation-contraction coupling. We report that the proteins involved in sarcoplasmic reticulum calcium transport, storage, and release [Ca2+ + Mg2+)-ATPase, calsequestrin, and calcium release channel) are present in dysgenic muscle. Also present in dysgenic muscle is the 175/150-kDa glycoprotein subunit (alpha 2) of the dihydropyridine receptor. However, the 170-kDa dihydropyridine binding subunit (alpha 1) of the dihydropyridine receptor is absent in dysgenic muscle. These results suggest that the specific absence of the alpha 1 subunit of the dihydropyridine receptor is responsible for the defects in muscular dysgenesis and that the alpha 1 subunit of the dihydropyridine receptor is essential for excitation-contraction coupling in skeletal muscle.

Animals↗
PubMed1989

32,000-Dalton subunit of the 1,4-dihydropyridine receptor.

Polyclonal antibodies to the 32,000-Da polypeptide of the 1,4-dihydropyridine receptor of the voltage-dependent Ca2+ channel have been produced and used to characterize the association of the 32,000-Da polypeptide (gamma subunit) with other subunits of the dihydropyridine receptor. The 32,000-Da polypeptide was found to copurify with alpha 1 and alpha 2 subunits at each step of the purification of the dihydropyridine receptor. Monoclonal antibodies against the alpha 1 and beta subunits immunoprecipitate the digitonin-solubilized dihydropyridine receptor as a multisubunit complex that includes the 32,000-Da polypeptide. Polyclonal antibodies generated against both the nonreduced and reduced forms of the alpha 2 subunit and the gamma subunit have been used to show that the 32,000-Da polypeptide is not a proteolytic fragment of a larger component of the dihydropyridine receptor and not disulfide linked to the alpha 2 subunit. Our results demonstrate that the 32,000-Da polypeptide (gamma subunit) is an integral and distinct component of the dihydropyridine receptor.

Animals↗
PubMed1988

Sequence and expression of mRNAs encoding the alpha 1 and alpha 2 subunits of a DHP-sensitive calcium channel.

Complementary DNAs were isolated and used to deduce the primary structures of the alpha 1 and alpha 2 subunits of the dihydropyridine-sensitive, voltage-dependent calcium channel from rabbit skeletal muscle. The alpha 1 subunit, which contains putative binding sites for calcium antagonists, is a hydrophobic protein with a sequence that is consistent with multiple transmembrane domains and shows structural and sequence homology with other voltage-dependent ion channels. In contrast, the alpha 2 subunit is a hydrophilic protein without homology to other known protein sequences. Nucleic acid hybridization studies suggest that the alpha 1 and alpha 2 subunit mRNAs are expressed differentially in a tissue-specific manner and that there is a family of genes encoding additional calcium channel subtypes.

Amino Acid Sequence↗
PubMed1987

Identification and characterization of the dihydropyridine-binding subunit of the skeletal muscle dihydropyridine receptor.

Photoaffinity labeling of isolated triads and purified dihydropyridine receptor with [3H]azidopine and (+)-[3H]PN200-110 has been used to identify and characterize the dihydropyridine-binding subunit of the 1,4-dihydropyridine receptor of rabbit skeletal muscle. The 1,4-dihydropyridine receptor purified from rabbit skeletal muscle triads contains four protein subunits of 175,000, 170,000, 52,000, and 32,000 Da (Leung, A., Imagawa, T., and Campbell, K. P. (1987) J. Biol. Chem. 262, 7943-7946). Photoaffinity labeling of isolated triads with [3H]azidopine resulted in specific and covalent incorporation of [3H]azidopine into only the 170,000-Da subunit of the dihydropyridine receptor and not into the 175,000-Da glycoprotein subunit of the receptor. The [3H]azidopine-labeled 170,000-Da subunit was separated from the 175,000-Da glycoprotein subunit by sequential elution from a wheat germ agglutinin-Sepharose column with 1% sodium dodecyl sulfate followed by 200 mM N-acetylglucosamine. Photoaffinity labeling of purified dihydropyridine receptor with [3H]azidopine or (+)-[3H]PN200-110 also resulted in the specific and covalent incorporation of either ligand into only the 170,000-Da subunit. Therefore, our results show that the dihydropyridine-binding subunit of the skeletal muscle 1,4-dihydropyridine receptor is the 170,000-Da subunit and not the 175,000-Da glycoprotein subunit.

Affinity Labels↗
PubMed1987

Affinity purification of antibodies specific for 1,4-dihydropyridine Ca2+ channel blockers.

High-affinity antibodies specific for the 1,4-dihydropyridine Ca2+ channel blockers have been produced in sheep and affinity purified using a dihydropyridine-Sepharose affinity column. Dihydropyridine-Sepharose affinity matrix was synthesized by reaction of aminohexyl-Sepharose with an affinity analogue of nifedipine, dimethyl 1,4-dihydro-2,6-dimethyl-4-(2-isothiocyanatophenyl)-3,5-pyridine-dicarbo xylate. Residual amine groups were then blocked by carbodiimide-catalyzed acetylation. [3H]Nitrendipine-binding activity in serum was specifically absorbed by the dihydropyridine-Sepharose affinity column. The bound antibody was eluted with diethylamine (pH 11.5) in 10% dioxane or with a low-affinity dihydropyridine ligand (diethyl 1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate), pH 7.4. Thirty-six milligrams of highly pure IgG antibody, as demonstrated by sodium dodecyl sulfate-gel electrophoresis, was isolated from 50 ml hyperimmune sheep serum. The affinity-purified anti-dihydropyridine antibodies have been shown to have high affinity (Kd approximately 0.1 nM) and specificity for the 1,4-dihydropyridine Ca2+ channel blockers and, therefore, exhibit dihydropyridine-binding properties similar to the membrane receptor for the 1,4-dihydropyridine Ca2+ channel blockers. Immunoblot staining of an azidopine-bovine serum albumin conjugate with affinity-purified antidihydropyridine antibodies demonstrated that the anti-dihydropyridine antibodies recognize the 1,4-dihydropyridine Ca2+ channel blockers when covalently coupled to protein and, therefore, should be useful in the identification and purification of receptors covalently labelled with 1,4-dihydropyridine Ca2+ channel blockers.

Animals↗
PubMed1980

Standardization of allergen extracts by inhibition of RAST, skin test, and chemical composition.

Five allergen extracts of Dermatophagoides pteronyssinus, Lolium perenne, Alternaria tenuis, Aspergillus fumigatus and Cladosporium herbarum, obtained from four different manufacturers, were examined by inhibition of RAST, content of protein and carbohydrate, contents of phosphorylcholine (Pc) and tridacnin reactive components, and by skin test. Inhibition of RAST was used as a primary method for establishing allergenic potency and demonstrated wide variations for each preparation supplied by the different manufacturers. The extracts also varied widely in protein and carbohydrate content and in the ratio of these parameters, indicating internal heterogeneity. Pc content was significantly related to RAST potency for extracts of A. fumigatus and A. tenuis, suggesting that Pc content may be used as a primary standarization procedure for these extracts. Skin test reactions undertaken at a single concentration did not show any significant variation in weal size between preparations of a given allergen extract. However, of particular importance to practising clinicians is the finding that varying numbers of patients showed negative skin reactions to one preparation of a particular allergen yet were positive to the corresponding preparations supplied by the other companies.

Allergens↗