Stephen J. Morris

Amyloid beta-peptide(25-35) changes [Ca2+] in hippocampal neurons.

Insoluble aggregates of the amyloid beta-peptide (A beta) is a major constituent of senile plaques found in brains of Alzheimer disease (AD) patients. The detrimental effects of aggregated A beta is associated with an increased intracellular Ca2+ concentration ([Ca2+]i). We examined the effects of A beta(25-35) on [Ca2+]i and intracellular H+ concentration ([H+]i) in single hippocampal neurons by real time fluorescence imaging using the Ca(2+)- and H(+)-specific ratio dyes, indo-1 and SNARF-1. Incubation of these cultures with A beta(25-35) for 3-12 days in vitro increased [Ca2+]i and [H+]i in large, NMDA-responsive neurons.

The use of cobalt ions as a collisional quencher to probe surface charge and stability of fluorescently labeled bilayer vesicles.

Co2+ quenched the fluorescence of the lipid probes NBD-phosphatidylethanolamine (NBD-PE) and lissamine-rhodamine phosphatidylethanolamine (N-Rh-PE) incorporated into lipid vesicles, according to a collisional quenching mechanism in agreement with the Stern-Vollmer law. The quenching coefficient (Q) for NBD-PE, incorporated into uncharged phosphatidylcholine (PC) vesicles was 13.8 M-1. This value was equal to the quenching coefficient of water-soluble NBD-taurine in aqueous solution, indicating that Co2+ was readily accessible to the outer surface of PC vesicles. In phosphatidylserine-phosphatidylethanolamine (PS-PE) (1:1) vesicles, quenching was also proportional to Co2+ concentration but Q was 114 mM-1, some 8000-fold smaller. Using the Gouy-Chapman-Stern model we demonstrated that the surface density of Co2+ bound to lipid was linear with Co2+ concentration in the medium up to 7%. Co2+-associated phospholipid would in turn quench NBD-PE or N-Rh-PE by collisional quenching with lateral diffusion. We investigated the ability of Co2+ to permeate PS-PE (1:1) vesicles. Co2+ quenched fluorophores on the outer surface of large unilamellar vesicles, formed by reverse-phase evaporation. In small unilamellar vesicles Co2+ quenched probes on both outer and inner surfaces, indicating rapid permeation of the ions into the vesicles. Using stopped-flow rapid mixing, we measured the rate of influx of Co2+, and correcting for surface potential using the Gouy-Chapman-Stern model, we calculated a permeability coefficient of 10(-12) cm/s for Co2+ concentrations below 300 microM. Above this concentration, there was a very steep rise in the permeability coefficient, indicating that binding of Co2+ induces defects in the bilayer of these vesicles. This may be related to the ability of the vesicles to undergo membrane fusion. A method for calculating the membrane surface potential from Co2+ quenching data is presented.

Dopamine D2 Receptor Isoforms Expressed in AtT20 Cells Differentially Couple to G Proteins to Acutely Inhibit High Voltage-Activated Calcium Channels

Abstract : The dopamine D2 receptor belongs to the serpentine superfamily of receptors, which have seven transmembrane segments and activate G proteins. D2 receptors are known to be linked, through Gαo‐ and Gαi‐containing G proteins, to several signaling pathways in neuronal and secretory cells, including inhibition of adenylyl cyclase and high voltage‐activated Ca2+ channels (HVA‐CCs). The dopamine D2 receptor exists in two alternatively spliced isoforms, “long” and “short” (D2L and D2S, respectively), which have identical ligand binding sites but differ by 29 amino acids in the third intracellular loop, the proposed site for G protein interaction. This has led to the speculation that the two isoforms may interact with different G proteins. We have transfected the AtT20 cell line with either D2L (KCL line) or D2S (KCS line) to facilitate experimentation on the individual isoforms. Both lines show dopamine agonist‐dependent inhibition of Q‐type HVA‐CCs. We combined G protein antisense knock‐down studies with multiwavelength fluorescence video microscopy to measure changes in HVA‐CC inhibition to investigate the possibility of differential G protein coupling to this inhibition. The initial, rapid, K+ depolarization‐induced increase in intracellular Ca2+ concentration is due to influx through HVA‐CCs. Our studies reveal that both D2 isoforms couple to Gαo to partially inhibit this influx. However, D2L also couples to Gα13, whereas D2S couples to Gα12. These data support the hypothesis of differential coupling of D2 receptor isoforms to G proteins.

Constitutive expression of functional GABAB receptors in mIL-tsA58 cells requires both GABAB(1) and GABAB(2) genes

Studies of γ‐aminobutyric acid (GABA)B receptor function in heterologous cell systems have suggested that expression of two distinct seven transmembrane G‐protein coupled receptor subunits is necessary for receptor activation and signal transduction. Some results suggest that both receptor proteins must be inserted into the plasma membrane to create heterodimers; however, it is possible that subunit monomers or homodimers are functional in cells which constitutively express GABAB receptors. A new pituitary intermediate lobe melanotrope cell clone (mIL tsA58) has been isolated which constitutively expresses GABAB, D2 and corticotrophin releasing factor receptors. Here, we report on characterization of the GABAB receptors. Solution hybridization‐nuclease protection assays reveal the presence of GABAB(1) and GABAB(2) transcripts. Western blots show GABAB(1a) and one of two GABAB(2) proteins. Addition of the GABAB agonist baclofen to cultured mIL‐tsA58 (mIL) cells inhibits high voltage activated Ca2+ channels, as measured by agonist‐induced inhibition of the K+‐depolarization‐stimulated increase in Ca2+ influx. CGP55845, a GABAB antagonist, blocks the response to baclofen. Knockdown of either GABAB(1) or GABAB(2) subunits with selective antisense oligodeoxynucleotides reduced GABAB protein levels and completely abolished the GABAB receptor response in the mIL cells. Taken together, these results indicate that functionally active GABAB receptors in mIL cells require the constitutive expression of both GABAB genes. This is a physiologic validation of results from recombinant overexpression in naive cells and shows that the mIL cell line is a useful model for studying GABAB receptor expression, regulation and function.

Transient domains induced by influenza haemagglutinin during membrane fusion

During low pH-induced fusion of influenza virus with erythrocytes we have observed differential dispersion of viral lipid and haemagglutinin (HA) into the erythrocyte membrane, and viral RNA into the erythrocyte using fluorescence video microscopy. The movement of both viral lipid and HA from virus to cell was restricted during the initial stages of fusion relative to free diffusion. This indicates the existence of relatively long-lived barriers to diffusion subsequent to fusion pore formation. Fluorescence anisotropy of phospholipid analogues incorporated into the viral membrane decreased when the pH was lowered to levels required for optimum fusion. This indicates that the restricted motion of viral membrane components was not due to rigidification of membrane lipids. The movement of HA from the fusion site was also assessed by photosensitized labelling by means of a fluorescent substrate (NBD-taurine) passing through the band 3 sialoglycoprotein (the erythrocyte anion transporter). We also examined the flow of lipid and aqueous markers during fusion of HA-expressing cells with labelled erythrocytes. During this cell-cell fusion, movement of lipid between fusing membranes begins before the fusion pore is wide enough to allow diffusion of aqueous molecules (M(r) > 500). The data indicate that HA is capable of creating domains in the membrane and controlling continuity of aqueous compartments which are bounded by such domains.

GABABR1a/R1b‐Type Receptor Antisense Deoxynucleotide Treatment of Melanotropes Blocks Chronic GABAB Receptor Inhibition of High Voltage‐Activated Ca2+ Channels

Abstract: GABAB and dopamine D2 receptors, both of which acutely inhibit adenylyl cyclase and high voltage‐activated Ca2+ channels (HVA‐CCs), are found in high levels in the melanotrope cells of the pituitary intermediate lobe. Chronic D2 receptor agonist application in vitro has been reported to result in inhibition of HVA‐CC activity by down‐regulation. Here we report that chronic GABAB, but not GABAA, agonist treatment also resulted in HVA‐CC inhibition. Two GABAB receptor variants have been cloned and shown to inhibit adenylyl cyclase in HEK‐293 cells. We have constructed an antisense deoxynucleotide knockdown‐type probe that is complementary to 18 bp from the point at which the two sequences first become homologous. Chronic coincubation with baclofen and GABAB antisense nucleotide completely eliminated the inhibition of the channels by baclofen alone but had no reversing effect on HVA‐CC inhibition by the D2 agonist quinpirole. A scrambled, missense nucleotide also had no reversing effect. Incubation with a D2 antisense knockdown probe eliminated the ability of a D2 agonist to inhibit the channels but had no effect on baclofen blockade. These results show the existence an R1a/R1b type of GABAB receptor, which, like the D2 receptor, is coupled to chronic HVA‐CC inhibition in melanotropes.

The influenza haemagglutinin-induced fusion cascade: effects of target membrane permeability changes

To define the stages in influenza haemagglutinin (HA)-mediated fusion the kinetics of fusion between cell pairs consisting of single influenza HA-expressing cells and single erythrocytes (RBC) which had been labelled with both a fluorescent lipid (Dil) in the membrane and a fluorescent solute (calcein) in the aqueous space have been monitored. It is shown that release of solute from the target cell occurs, following the formation of the hemi-fusion diaphragm. These results are discussed in terms of a model in which fusion peptide insertion into the target membrane induces lipid stalks, which results in the formation of a hemifusion diaphragm and a fusion pore. Bilayer expansion due to overproduction of these stalks can give rise to collateral damage of target membranes.

POMC mRNA levels in individual melanotropes and GFAP in glial-like cells in rat pituitary

In this study we demonstrated that individual melanotropes expressed differing levels of POMC mRNA. Interspersed among the melanotropes was a small population of process-bearing cells expressing GFAP. We compared cellular resolution and feasibility for quantitation of hybrids formed by oligonucleotide probes labeled with two different markers using in situ hybridization histochemistry. In hybridizations using [35S]-labeled probes the signal could not easily be assigned to individual melanotropes, although digoxigenin-labeled probes provided good cellular resolution. Heterogeneous staining intensities of individual melanotropes for POMC mRNA were measured, and relative quantitation of changes in POMC mRNA levels following interactions with the D2 dopamine receptor was performed. We combined in situ hybridization histochemistry with immunohistochemistry to demonstrate that cells within the lobe parenchyma not expressing POMC mRNA were immunoreactive for GFAP.

Differential innervation of individual melanotropes suggests a role for nonsynaptic inhibitory regulation of the developing and adult rat pituitary intermediate lobe

Dopamine and GABA were detected in intermediate lobe axons around birth, and early axons were closely apposed to glial cells and processes, possibly using them for guidance. In the adult, axons containing colocalized dopamine and GABA were distributed in a distinct pattern within the lobe, with plexuses located dorsally and ventrally. Axons preferentially followed glial processes in interlobular septa, yet were also interspersed between melanotropes. Individual melanotropes were contacted by varying numbers of axon terminals, with some devoid of contacts. Boutons contained both small clear vesicles and large dense‐cored vesicles; membrane specializations were not well‐developed. From these findings we concluded that in addition to direct synaptic inhibition, dopamine and GABA could stimulate their receptors by mechanisms similar to “parasynaptic” [Schmitt (1984) Neuroscience, 13:991–1001] or “volume” [Agnati et al. (1995) Neuroscience, 69:711–726] transmission as proposed for the CNS. Humoral agents passing into the intermediate lobe from portal vessels, thus acting as classical hormones, further regulate the melanotropes. Moreover, approximately 50% of the axonal elements were closely apposed to glia, suggesting that glia could have regulatory roles.

Types and activities of voltage-operated calcium channels change during development of rat pituitary neurointermediate lobe

Cultures of pituitary neurointermediate lobe cells were established from rats aged 1, 12, and 42 days to identify the types and assess the activities of Ca2+ channels present in melanotropes, glial‐like cells, and fibroblasts during development. Day 12 represents the time at which dopaminergic axons have become distributed throughout the lobe, glial cells begin to lose their radial orientation, and melanotropes robustly express the short isoform of the dopamine D2 receptor. Thus, we studied Ca2+ channels in relation to the event of innervation of melanotropes. Real‐time fluorescence video microscopy, in the presence of pharmacological agents, which block L‐, N‐, P‐, and T‐type channels, was used as an indirect measurement of channel activity. Assessment of cell type was verified by triple‐label fluorescence immunohistochemistry. In melanotropes, extracellular Ca2+ addition caused Ca2+ influx through ω‐conotoxin GVIA‐sensitive, N‐type channels on days 1 and 12 but not on day 42. The K+ depolarization induced an increase in intracellular Ca2+ concentration in all age‐groups. This effect was decreased by nifedipinc, an L‐type channel blocker, at all ages, and by ω‐agatoxin IVa, a P‐type blocker, only on day 42. These results demonstrate that the predominance of N‐ or P‐type channels on melanotropes is age‐dependent and can be correlated with other developmental changes. The T‐type blocker, NiSO4, had no effect. In glial‐like cells of all ages, extracellular Ca2+ addition resulted in an increase in intracellular Ca2+ concentration, which was inhibited only by NiSO4. The percentage of responsive glial‐like cells was equally high in days 1 and 12 cultures, then declined by day 42. The K+ depolarization had no effect on glial‐like cells. Fibroblasts did not respond significantly to extracellular Ca2+ or K+ depolarization, indicating little detectable activity by this methodology from functional voltage‐operated Ca2+ channels.