Burgess N. Christensen

Multiple protein kinase pathways are involved in gastrin-releasing peptide receptor-regulated secretion.

Gastrin-releasing peptide (GRP) and its amphibian homolog, bombesin, are potent secretogogues in mammals. We determined the roles of intracellular free Ca2+([Ca2+] i ), protein kinase C (PKC), and mitogen-activated protein kinases (MAPK) in GRP receptor (GRP-R)-regulated secretion. Bombesin induced either [Ca2+] i oscillations or a biphasic elevation in [Ca2+] i . The biphasic response was associated with peptide secretion. Receptor-activated secretion was blocked by removal of extracellular Ca2+, by chelation of [Ca2+] i , and by treatment with inhibitors of phospholipase C, conventional PKC isozymes, and MAPK kinase (MEK). Agonist-induced increases in [Ca2+] i were also inhibited by dominant negative MEK-1 and the MEK inhibitor, PD89059, but not by an inhibitor of PKC. Direct activation of PKC by a phorbol ester activated MAPK and stimulated peptide secretion without a concomitant increase in [Ca2+] i . Inhibition of MEK blocked both bombesin- and phorbol 12-myristate 13-acetate-induced secretion. GRP-R-regulated secretion is initiated by an increase in [Ca2+] i ; however, elevated [Ca2+] i is insufficient to stimulate secretion in the absence of activation of PKC and the downstream MEK/MAPK pathways. We demonstrated that the activity of MEK is important for maintaining elevated [Ca2+] i levels induced by GRP-R activation, suggesting that MEK may affect receptor-regulated secretion by modulating the activity of Ca2+-sensitive PKC.

N-methyl-D-aspartate receptors coexist with kainate and quisqualate receptors on single isolated catfish horizontal cells

Horizontal cells enzymatically isolated from catfish retina were exposed to the putative neurotransmitters aspartate (Asp) or N-methyl-D-aspartate (NMDA). Under voltage clamp conditions, inward currents were recorded when the holding potential was more negative than zero and outward currents were recorded when the membrane potential was more positive than zero. The current voltage curve was highly non-linear in the range of membrane potential between -30 and -100 mV. This non-linearity was largely removed in zero magnesium solution. 2-Amino-phosphonovaleric acid selectively blocked Asp and NMDA responses. These response characteristics are consistent with the presence of NMDA receptors in these cells.

Protonation of histidine groups inhibits gating of the of the quisqualate/kainate channel protein in isolated catfish cone horizontal cells

Increases in the extracellular hydrogen ion concentration ([H+]o) but not the intracellular concentration ([H+]i) antagonized the inward going membrane currents recorded from isolated cone horizontal cells during application of quisqualate, alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid, and kainate. The pK determined from a titration curve was 6.5 with a slope greater than 1, indicating protonation of several histidines. The reduction in membrane current was voltage-independent. The affinity of the agonist for the receptor, the single-channel conductance, and the open time were unaffected by [H+]o. [H+]o antagonism was not the result of charge neutralization such as screening surface charge. Diethylpyrocarbonate, a histidine-modifying reagent, reduced the agonist-induced current, but disulfide- and sulfhydryl-modifying reagents were ineffective. These results suggest that histidine groups on the external face of the channel protein provide a functional site regulating channel gating.

Connexin43 immunoreactivity in the catfish retina

We used antibodies directed against rat heart connexin43 (Cx43) to perform immunoblot and immunohistochemical (IHC) analyses of the catfish retina. The antibodies recognized a retinal protein which co-migrated with mouse brain Cx43. IHC staining resulted in punctate labeling of the external limiting membrane and the outer nuclear layer. Although infrequent, labeling was also observed in the inner nuclear layer. These results suggest that a Cx43 isoform may be present in Muller glial cells and neurons of the distal catfish retina.

A quantitative analysis of ultrastructural changes induced by electrical stimulation of identified spinal cord axons in the larval lamprey.

SummaryMicroelectrodes filled with horseradish peroxidase (HRP) were used to label single identified giant axons in the isolated lamprey spinal cord. Subsequent to the iontophoretic injection of HRP, the spinal cord was stimulated at repetition rates of 20–30/s and the activity in labelled axons monitored. Immediately following failure of the action potential, the spinal cord was fixed by immersion and processed for light and electron microscopy. Electron micrographs were taken of synaptic contacts made by the labelled axons. Several quantitative measures were made from each synapse using a digitizing tablet interfaced with a digital computer. These measures included vesicle number (VN), vesicle area (VA), length of differentiated membrane (DM), vesicle density (VD=VN/VA), vesicle frequency (VF = VN/DM), and a relative measure of the amount of vesicle membrane added to the axolemma during the stimulation period, the curvature ratio (CR). Measures from 106 stimulated synapses were compared with 134 synapses from injected but unstimulated giant axons. The results from these experiments suggest that measurable ultrastructural changes occur during transmitter release at identified C.N.S. synapses, which are consistent with the hypothesis of synaptic vesicle recycling.

Distribution of the inositol trisphosphate receptor in the catfish retina

Inositol 1,4,5-trisphosphate (InsP3) mobilizes intracellular stored Ca2+ by binding to specific receptors that are similar to the ryanodine receptor of skeletal and cardiac muscle. We have immunolocalized the InsP3 receptor to the inner nuclear layer and limiting membranes of the catfish retina. Immunocytochemistry on dissociated retinal cells further localized the receptor in the horizontal, bipolar and Müller glial cells. Immunostaining of the rat retina localized the InsP3 receptor to the plexiform layers. These data show a different distribution of InsP3 receptor in the catfish retina compared to that of other vertebrates, that may be suggestive of a different functional role for this receptor in different species.

Proton inhibition of the NMDA-gated channel in isolated catfish cone horizontal cells.

The effect of H+ on the N-methyl-D-aspartate-induced (NMDA) membrane current in enzymatically isolated catfish cone horizontal cells was investigated. Extracellular acidification to pH 5.5 blocked nearly completely the NMDA-induced current and reduced desensitization. The pK for the H+ effect was 6.5, near that for the free amino acid histidine. Protons did not alter the receptor affinity for NMDA and the inhibition was insensitive to the membrane potential and surface charge screening. However, extracellular H+ increased the IC50 for Zn2+. These results indicate that protons can modulate the NMDA-induced membrane current by a mechanism that may include interaction with histidine residues.

The relationship between some measures of synaptic ultrastructure as a function of distance from the soma on lamprey reticulospinal neurons

SummarySynaptic junctions located on the dendrites of lamprey (Petromyzon marinus) reticulospinal neurons labelled with intracellularly-injected horseradish peroxidase were studied. The normal ultrastructure of the synaptic junctions was defined and several quantitative measures made from each junction in order to test the hypothesis that distally-located synapses are ultrastructurally different from those located at proximal dendritic sites. A total of 820 contacts from one neuron and 279 from a second neuron ranging from 20 to 340 μm from the soma were quantified. The vast majority of the presynaptic endings contained round, clear-cored vesicles and formed an asymmetrical membrane differentiation with the postsynaptic dendrite. A small fraction of the population contained flattened or pleomorphic vesicles and these synapses were equally distributed with respect to distance from the soma. Many of the terminals contained a few large dark- and clear-cored vesicles. Four quantitative measures of each synaptic contact were made. These included vesicle number, length of differentiated membrane, vesicle area and terminal area. Four ratios relating the different quantitative measures were also calculated. Each ratio or measurement from the synaptic junctions was plotted as a function of distance from the soma to determine if differences existed at any distance. It was found that synaptic junctions are uniformly similar and that distal junctions did not differ significantly (P > 0.05) from those at proximal dendritic sites. It is concluded that if distal synapses do compensate for their remote location they do this in some other way, possibly by increasing the number of synaptic contacts made by each presynaptic axon.

A Mathematical Model of Retinal Photoreceptors

Retinal photoreceptors transmit light stimuli via electrical signals generated by a change in the photocurrent. This voltage-sensitive current is gated by cGMP and allows the entry of both Na+ and Ca2+ into the cell. cGMP levels are controlled by the opposing actions of guanylate cyclase and phosphodiesterase, which are in turn controlled by buffered Ca2+ and photon absorption, respectively. We have constructed a model that reproduces photocurrent responses to light stimuli. In addition to the processes noted above. the actions of the Na+/K+ and Na+/Ca2+-K+ exchangers present in the photoreceptor inner and outer segments are considered. Previously published models have not incorporated all of these processes in a biologically relevant manner. Model light responses are compared to those described in the literature, and the role of Ca2+ buffering in light adaptation is explored.