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Biomedical subjects

M R Hanley

Publications and source records attributed to M R Hanley.

At least 73 records · Page 4Linked to original sources
PubMed1989

Identification of a novel inflammatory stimulant of chondrocytes. Early events in cell activation by bradykinin receptors on pig articular chondrocytes.

The inflammatory peptide bradykinin stimulated a rapid and transient increase in cytoplasmic [Ca2+] in primary pig chondrocytes, as measured by the fluorescent indicator dye Fura-2. This increase occurred in the absence of extracellular Ca2+, indicating a mobilization from intracellular stores. The elevation in intracellular [Ca2+] was mediated by authentic bradykinin receptors, since it was blocked by the specific bradykinin antagonist [beta-(2-thienyl)-L-Ala5,8,D-Phe7]bradykinin. Activation of chondrocytes by bradykinin induced a concentration-dependent [ED50 (dose for half-maximal response) approximately 40 nM] accumulation of inositol monophosphate in the presence of LiCl and a concentration-dependent increase in production of prostaglandin E2. The generation of the secondary mediator prostaglandin E2 was a biologically relevant output response induced by bradykinin, but chondrocyte responses, such as the rate of entry into DNA synthesis, the rate and pattern of new protein synthesis and the rate of synthesis and resorption of cartilage proteoglycan, were unaltered by bradykinin treatment. Chondrocytes were also shown to be activated by two pharmacological mediators of cytosolic [Ca2+] elevation, i.e. the ionophore A23187 and thapsigargin, which both produced alterations in protein synthesis which were mimicked by bradykinin. Thus Ca2+-sensitive pathways exist which are not functionally responsive to a Ca2+-mobilizing and inositol phosphate-generating hormone, potentially indicating other routes of regulation. These results call attention to bradykinin and related peptides as another class of inflammatory mediators which may regulate physiological and pathological chondrocyte metabolism.

Animals↗
PubMed1989

Thapsigargin, a new calcium-dependent epithelial anion secretagogue.

1. Thapsigargin, a sesquiterpene lactone, was shown to cause electrogenic anion secretion in monolayers of human colonic epithelial cells, an effect which was crucially dependent upon calcium and did not involve eicosanoid formation. 2. To measure the secretory effect calcium needed to be present in the external bathing solution. By means of Fura-2 fluorescence measurements thapsigargin was shown to raise Cai by around 250 nM when the bathing solution contained calcium. In the nominal absence of external calcium thapsigargin raised Cai by only 60 nM, but from a lower basal value. This was insufficient to cause secretion. 3. Effects of other calcium-dependent secretagogues (e.g. lysylbradykinin) were inhibited in the presence of thapsigargin, whereas kinin responses were potentiated if the peptide was added following a stimulus which increases cyclic AMP. 4. From the data given here and the known behaviour of colonic epithelia it is concluded that thapsigargin increases Cai by a non-ionophoric mechanism by release from internal stores. Calcium-stimulated calcium influx then follows resulting in the opening of basolateral K channels, increasing the electrochemical gradient for chloride efflux, or alternatively by activating anion channels in the apical membrane. It is concluded that thapsigargin is a potentially important tool for examining epithelial mechanisms.

Adenocarcinoma↗
PubMed1988

The mas oncogene encodes an angiotensin receptor.

The class of receptors coupled to GTP-binding proteins share a conserved structural motif which is described as a 'seven-transmembrane segment' following the prediction that these hydrophobic segments form membrane-spanning alpha-helices. Identified examples include the mammalian opsins, alpha 1-, alpha 2-, beta 1- and beta 2-adrenergic receptors, the muscarinic receptor family, the 5-HT1C-receptor, and the substance-K receptor. In addition, two mammalian genes have been identified that code for predicted gene products with sequence similarity to these receptors, but whose ligand specificity is unknown namely, G21 and the mas oncogene. The mas oncogene shows the greatest sequence similarity to the substance-K receptor, and on this basis it was predicted that it would encode a peptide receptor with mitogenic activity which would act through the inositol lipid signalling pathways. The mas oncogene product was transiently expressed in Xenopus oocytes, and stably expressed in a transfected mammalian cell line. The results demonstrate that the mas gene product is a functional angiotensin receptor.

Angiotensins↗
PubMed1988

Neural function: metabolism and actions of inositol metabolites in mammalian brain.

In the nervous system, a variety of cell types respond to external stimuli through the inositol lipid signalling pathways. The stimulus-coupled sequence of intracellular events has been investigated in a homogeneous model system, the cloned mammalian neural cell line NG115-401L. The neural peptide bradykinin stimulates a rapid production of identified inositol phosphate isomers and an intracellular Ca2+ discharge followed by a persistent plasma membrane influx. The temporal sequence suggests that Ins(1,4,5)P3 or Ins(1,3,4,5)P4 or both may coordinate these events in a neuronal cell, as has been suggested in other cell types. Thapsigargin, an irritant and tumour-promoting plant product, produces calcium transients in the absence of inositol phosphate production, and may provide a new tool for investigating the interactions between inositol phosphates and changes in cellular calcium homeostasis. In the 401L line, high levels of radiolabelled InsP5 and InsP6 have been detected, which has led to the evaluation of their possible occurrence and actions in normal brain. Both InsP5 and InsP6 are produced from a radiolabelled myo-inositol precursor in intact mature brain in a region-specific manner. This suggests that both inositol polyphosphates may be end products of regionally regulated biosynthetic pathways. When microinjected into a nucleus of the brainstem, or iontophoretically applied to the dorsal horn of the spinal cord, both InsP5 and InsP6, but not Ins(1,3,4,5)P4 isomers, appear to be potent neural stimulants. These results suggest that the inositol lipid signalling pathways may generate both intracellular and extracellular signals in brain.

Animals↗
PubMed1988

A novel tumour promoter, thapsigargin, transiently increases cytoplasmic free Ca2+ without generation of inositol phosphates in NG115-401L neuronal cells.

Thapsigargin, a sesquiterpene lactone with potent irritant and tumour-promoting activities, stimulates a rapid (within 15 s) transient increase in intracellular [Ca2+] in the NG115-401L neural cell line, as measured by the fluorescent indicator dye fura-2. This increase in cytoplasmic free [Ca2+] is concentration-dependent (ED50 around 20 nM) and occurs in the absence of extracellular Ca2+. Activation of NG115-401L cells by the inflammatory peptide bradykinin generates inositol phosphates, which parallel increases in intracellular [Ca2+]. However, the rise in cytoplasmic [Ca2+] stimulated by thapsigargin occurs in the absence of detectable production of inositol phosphates. Thapsigargin is unlike phorboid tumour promoters in that it has no action on two non-invasive indicators of phorbol stimulation of these cells, i.e. [3H]choline metabolite production and rise in intracellular pH. These data suggest that thapsigargin releases Ca2+ from an intracellular store by a novel mechanism, independent of the hydrolysis of phosphoinositides and concomitant activation of protein kinase C. Thus thapsigargin may provide a valuable tool for the analysis of intracellular signalling mechanisms.

Bradykinin↗
PubMed1988

Solubilization and characterization of substance P-binding sites from chick brain membranes.

Sites binding monoiodinated-Bolton-Hunter-reagent-labelled substance P were solubilized from 1-day-old-chick brain membrane by using non-ionic detergents (1% digitonin/1% n-octyl glucoside) and a high concentration of NaCl (0.5 M). The solubilized preparation retained the pharmacological properties of the high-affinity binding sites found in the native membrane. The high density of specific binding sites (approximately 2 pmol of binding sites/mg of protein) suggests that the chick brain membranes may be a useful source for the purification of the substance P-binding sites.

Adenosine Triphosphate↗
PubMed1988

A phosphoinositide-linked peptide response in astrocytes: evidence for regional heterogeneity.

A phosphoinositide-linked peptide response in cultured rat astrocytes was studied by measuring the accumulation of [3H]inositol phosphates in the presence of lithium. Cultures derived from cortex, cerebellum and spinal cord each showed a unique pattern or degree of stimulation to a panel of neuropeptides. Cortical and cerebellar astrocytes were similar, responding to bradykinin, oxytocin, vasopressin, eledoisin and neurokinin beta, whereas spinal cord astrocytes were stimulated by substance P, bradykinin, eledoisin, and neurokinins alpha and beta. These observations are evidence in favour of regional specialisations of astrocytes which may respond uniquely to peptides released by particular populations of neurons.

Animals↗
PubMed1988

Development of macroglial cells in rat cerebellum. I. Use of antibodies to follow early in vivo development and migration of oligodendrocytes.

The origin of oligodendrocytes and astrocytes in the CNS is still a focus of much experimentation and controversy. We have used antibodies against ganglioside GD3 and galactocerebroside (GC) to follow the origin and development of rat cerebellar oligodendrocytes both in vitro and in vivo. The immunofluorescent identification of GC+ cells in the rat neonatal cerebellum in vivo, revealed that cells initially GD3+/GC- appeared to make the transition via GD3+/GC+ cells to GD3-/GC+ oligodendrocytes. This sequence of events closely paralleled the maturation of cerebellar oligodendrocyte precursors found in serum-free dissociated culture. In contrast, whereas both GD3+ and glial fibrillary acidic protein-positive cells were seen in serum-containing dissociated culture and also in freshly dissociated suspensions of cerebellum at postnatal days 0 to 6, such cells could not be identified in situ. Putative GD3+/GC- oligodendrocyte precursor cells arose from the deeper regions of the cerebellum at birth, perhaps initially from the superior medullary velum adjacent to the fourth ventricle, and appeared to migrate into the developing folia just prior to myelination and the acquisition of GC.

Animals↗
PubMed1988

Evidence for multiple tachykinin receptor subtypes on the rabbit iris sphincter muscle.

The actions of mammalian tachykinins (substance P, substance K/neurokinin a, neuromedin K/neurokinin b) and non-mammalian tachykinins (eledoisin, kassinin, physalaemin) were compared on the rabbit pupillary sphincter. All acted as direct spasmogens with potencies in the order: eledoisin greater than physalaemin = neurokinin b = substance P greater than kassinin greater than neurokinin a. However, their actions could be divided into at least two categories on the basis of similar kinetics of contractions, differential sensitivity to the tachykinin antagonist (D-Arg1, D-Pro2, D-Trp7,9, Leu11) substance P and specific cross-protection against phenoxybenzamine inactivation by structurally related tachykinins. The relationship between these observations and the suggested "P" and "E" subtypes of tachykinin receptors is discussed.

Animals↗
PubMed1987

Down-regulation of phorbol diester binding to NG115-401L neuronal cells is dependent on structure, concentration and time.

The down-regulation of [3H]PDBu binding to a neural cell line, NG115-401L, has been examined in response to two biologically active phorbol diesters, PDBu and PMA. Chronic treatment with PDBu or PMA causes a concentration- and time-dependent loss of specific [3H]PDBu binding. The action of PMA is biphasic with respect to both concentration and time dependence.

Caenorhabditis elegans Proteins↗
PubMed1987

Bradykinin stimulation of inositol phosphate and calcium responses in insensitive to pertussis toxin in NG115-401L neuronal cells.

Stimulation of NG115-401L neuronal cells with bradykinin produces a dose-dependent increase in inositol phosphate production which is not blocked, rather slightly increased, after treatment with pertussis toxin. Nevertheless, pertussis toxin stimulates ADP-ribosylation of a 41K membrane protein, and blocks opioid receptor-mediated inhibition of stimulated cAMP production in these cells. These results suggest that bradykinin responses in the NG115-401L cells are pertussis-insensitive, unlike bradykinin responses reported in other neuronal cell lines.

Adenosine Diphosphate Ribose↗