Anton Hermann

Biochemical Characterization and Histochemical Localization of Nitric Oxide Synthase in the Nervous System of the Snail, Helix pomatia

Abstract: Nitric oxide synthase (NOS) in the snail Helix pomatia was characterized by biochemical and molecular biological techniques and localized by histochemical methods. Central ganglia contained particulate paraformaldehyde‐sensitive and cytosolic paraformaldehyde‐insensitive NADPH‐diaphorase. The cytosolic NADPH‐diaphorase activity coeluted with NOS activity. The activity of NOS was dependent on Ca2+ and NADPH and was inhibited by NG‐nitro‐l‐arginine (l‐NNA). Proteins purified by 2′,5′‐ADP affinity chromatography were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and migrated at 150, 60, 40, and 30 kDa. An antibody to mammalian NOS exclusively labeled the 60‐kDa protein. Characterization of the cDNA of the corresponding 60‐kDa NOS‐immunoreactive protein revealed no sequence homology with any known NOS isoform. The recombinant protein exhibited Ca2+‐ and NADPH‐dependent NOS activity, which was partially inhibited by EGTA and l‐NNA. Histochemistry showed NADPH‐diaphorase activity in discrete regions of the central and peripheral nervous system. About 60% of the NADPH‐diaphorase‐positive neurons colocalize with immunoreactive material detected by antibodies to mammalian NOS. Comparison of organs showed the highest NADPH‐diaphorase activity in the nervous system, whereas moderate activity was present in muscle tissue, digestive tract, and gonads. Our study suggests the presence of NOS and a putative NOS‐associated/regulating protein in mollusk nervous tissue.

Modulation of calcium-activated potassium channels

Potassium currents play a critical role in action potential repolarization, setting of the resting membrane potential, control of neuronal firing rates, and regulation of neurotransmitter release. The diversity of the potassium channels that generate these currents is nothing less than staggering. This diversity is generated by multiple genes (as many as 100 and perhaps more in some creatures) encoding the pore-forming channel α subunits, alternative splicing of channel gene transcripts, formation of heteromultimeric channels, participation of auxiliary (non-pore-forming) β and other subunits, and modulation of channel properties by post-translational modifications and other mechanisms. Prominent among the potassium channels are several families of calcium activated potassium channels, which are highly selective for potassium ions as their charge carrier, and require intracellular calcium for channel gating. The modulation of one of these families, that of the large conductance calcium activated and voltage-dependent potassium channels, has been especially widely studied. In this review we discuss a few selected examples of the modulation of these channels, to illustrate some of the molecular mechanisms and physiological consequences of ion channel modulation.

Hydrogen sulfide increases calcium-activated potassium (BK) channel activity of rat pituitary tumor cells

Hydrogen sulfide (H2S) is the third gasotransmitter found to be produced endogenously in living cells to exert physiological functions. Large conductance (maxi) calcium-activated potassium channels (BK), which play an important role in the regulation of electrical activity in many cells, are targets of gasotransmitters. We examined the modulating action of H2S on BK channels from rat GH3 pituitary tumor cells using patch clamp techniques. Application of sodium hydrogen sulfide as H2S donor to the bath solution in whole cell experiments caused an increase of calcium-activated potassium outward currents. In single channel recordings, H2S increased BK channel activity in a concentration-dependent manner. Hydrogen sulfide induced a reversible increase in channel open probability in a voltage-dependent, but calcium independent manner. The reducing agent, dithiothreitol, prevented the increase of open probability by H2S, whereas, the oxidizing agent thimerosal increased channel open probability in the presence of H2S. Our data show that H2S augments BK channel activity, and this effect can be linked to its reducing action on sulfhydryl groups of the channel protein.

Ethanol actions on the mechanisms of Ca2+ mobilization in rat hippocampal cells are mediated by protein kinase C.

The effects of ethanol on intracellular free Ca(2+) concentration, [Ca](i), were studied in cultured rat hippocampal neurons using fluo-3 and confocal microscopy. Ethanol application transiently elevAted [Ca](i) due to Ca(2+)-induced Ca(2+) release from internal stores since the effect was observed also in solutions containing zero Ca(2+) or 0.3 mM La(3+) and restoration of external Ca(2+) content led to secondary response in presence of ethanol. The sites of highest [Ca]i increases correlated well with those obtained after Ca(2+) release from caffeine-and IP3-sensitive internal stores. After single ethanol exposure the caffeine-evoked [Ca](i) transients were potentiated whereas Ca(2+) release induced by IP(3)-mobilizing agonists was suppressed. Similar effects were observed by activation of protein kinase C (PKC) by phorbol esters which also occluded ethanol actions. Ethanol increased fluorescence of Rim-1, a PKC indicator dye. The data obtained are consistent with ethanol activation of PKC whereby Ca(2+) release via ryanodine receptors is potentiated and IP(3) receptors are down-modulated. Since the effects of both ethanol and phorbol esters were mimicked by cytochalasins B and D, PKC-induced cytoskeleton phosphorylation and its subsequent rearrangements can be responsible for observed effects.

Sarcoplasmic calcium-binding protein

Sarcoplasmic calcium-binding proteins (SCPs) are members of the EF-hand calcium-binding protein family which are characterized by the presence of helix-loop-helix motifs in their amino acid sequence. SCPs have an M(r) of approximately 20,000, a pI of approximately 5 and interact with two to three calcium ions (Ca2+) with a KD of 10(-7) to 10(-8) M. Mg2+ ions antagonize Ca2+ ion binding in a complex manner so that these proteins are exquisitely fine-tuned to interfere with the Ca2+ signal. SCPs apparently fulfil no specific activatory function. They exhibit strong polymorphism, show a marked homology to coelenterate photoproteins (aequorin, luciferin) and have been found only in invertebrates, predominantly in muscle and neurons. In mollusks, SCPs are distributed in a tissue-specific manner, with immunoreactivity to SCP I-like isoforms localized in electrically silent neurons colocalized with serotonin, and immunoreactivity to SCP II-like isoforms exclusively present in muscle.

Pharmacological and functional characterization of galanin-like peptide fragments as potent galanin receptor agonists

The hypothalamic galanin-like peptide (GALP) was isolated by its ability to activate galanin receptors. The mature porcine GALP is a 60-amino acid neuropeptide proteolytically processed from a 120-amino acid precursor protein. It contains a region identical to the N-terminal 13-amino acids of the neuropeptide galanin. Within the sequence of human GALP (1-60) a potential proteolytic cleavage site between two basic amino acids is present at position 33, which might lead to a shorter C-terminally amidated peptide. In addition, the first two amino acids could be potentially removed via the action of dipeptidase IV. Ligand binding assays using the human neuroblastoma cell line SH-SY5Y transfected with the respective galanin receptors revealed that human GALP (1-60) displayed the highest affinity for the galanin receptor subtype GalR3 (IC50 = 10 nM) followed by GalR2 (IC50 = 28 nM) and GalR1 (IC50 = 77 nM). Ligand binding assays and functional studies showed that the human GALP (3-32) fragment was at least as potent as full length GALP (1-60). Other studies have shown that shorter fragments like human GALP (1-21) and GALP (22-60) were not effective on feeding responses in mice as compared to the full length peptide. Taken together these data suggest that the putative fragment GALP (3-32) might represent the strongest mediator of biological GALP activity. Furthermore it might be a useful tool to study the affinity of GALP to galanin receptors and to search for specific GALP receptors.

Role of calcium in photodynamically induced cell damage of human fibroblasts.

Photodynamically induced changes in the cytoplasmic free calcium concentration ([Ca2+]i) and its role in cell damage were investigated in human skin fibroblasts using confocal laser microscopy. Fluorescence and absorbance spectrophotometry measurements indicate that the photosensitizer aluminum phthalocyanine tetrasulfonate (AlPcS4) binds to the plasma membrane and only after irradiation is able to enter the cells, causing massive morphologic alterations. Upon irradiation of sensitizer‐treated cells, the increase in [Ca2+]i is related to the amount of light and extracellular [Ca2+]e. The increase in [Ca2+]i was substantially reduced in the absence of [Ca2+]e. Cell damage or death after photodynamic treatment was prevented and shifted toward higher fluence by increasing [Ca2+]i at high [Ca2+]e and was greater at low [Ca2+]e. Application of Ca2+ channel blockers, such as Co2+, Cd2+ or verapamil, could not prevent the increase of [Ca2+]i. Our results indicate that activation of the photosensitizer, AlPcS4, causes an influx of Ca2+, which protects cells from photodamage. At low [Ca2+]e and high fluence values, release of Ca2+ from internal stores probably as a protective measure occurs in order to increase the [Ca2+]i.

External action of di- and polyamines on maxi calcium-activated potassium channels: an electrophysiological and molecular modeling study.

In this study we compared polyamines to various diamines, and we modeled flexibility as well as hydrophobicity properties of these molecules to examine possible structural differences that could explain their external effects on the channels. The natural polyamines (putrescine, cadaverine, spermidine, spermine) and diamines increasing in CH2 chain length from C2 to C12 were used to probe maxi calcium-activated potassium (BK) channels in GH3 pituitary tumor cells when applied extracellularly. In single-channel recordings we found polyamines as well as diamines up to 1,10-diaminodecane to be ineffective in altering channel current amplitudes or kinetics. In contrast, 1,12-diamino dodecane (1,12-DD) was found to be a reversible blocker, with a blocking site at an electrical distance (z delta) of 0.72 within the channel. It reduced single-channel current amplitude, mean channel open time, and channel open probability. In computer simulations structural data, such as flexibility, hydration, and log D values, were calculated. 1,12-DD showed the largest flexibility of all diamines (minimum N-N distance 9.9 A) combined with a marked hydrophobicity due to a 4-5 A hydrophobic intersegment between hydrophilic ends in the molecule, as confirmed by GRID water probe maps and a log D value of -1.82 at pH 7.2. We propose that the amount of hydration of the molecule, more than its flexibility, constitutes an essential parameter for its ability to act as a channel blocker.

Nitric oxide-mediated cGMP synthesis in Helix neural ganglia

The central nervous system of the mollusc Helix pomatia was stimulated with NO donors sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP) or hydroxylamine, in the presence of a phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine (IBMX). Radioimmunoassay revealed that all of the three NO donors significantly increased cGMP levels by 22-27-fold above basal levels. Compared with controls, strong cGMP immunoreactivity was observed in axons and cytoplasm of the stimulated neurons. About 80% of cGMP-immunoreactive neurons colocalized with NADPH-diaphorase activity. Some glial cells and giant neurons were not stained by NADPH-diaphorase histochemistry but were cGMP-immunoreactive. The results suggest the existence of a NO/cGMP pathway and indicate NO as an intra- and intercellular signaling molecule in the Helix central nervous system.

Intracellular Action of Spermine on Neuronal Ca2+ and K+ Currents

Intra‐and extracellular effects of the polyamine spermine on electrical activity and membrane currents of identified neurons in the abdominal ganglion of Aplysia californica were studied under current‐and voltage‐clamp conditions. Lonophoretic injection of spermine reduced the amplitude of action potentials and altered their time course as well as spontaneous discharge activity. Investigation of membrane currents showed that intracellular spermine suppressed the total outward current but increased the inward rectifier current. After separation of ion currents it was found that the voltage‐activated, delayed K+ outward current and the Ca2+ inward current were reduced by intracellular spermine in a dose‐ and voltage‐dependent manner. The block of the K+ current can be described by a voltage‐dependent reaction, where one spermine molecule binds to one channel. The binding constant Kb, at zero voltage, and the effective valency, zδ, had values of 176/M and 0.41 for cell R‐15, 223/M and 0.64 for cell L‐11, and 137/M and 0.42 for cell L‐3. Apparently, more than one spermine cation is needed to block one Ca2+ channel, since the coefficient n, which absorbs the molecularity and cooperativity of the reaction, had non‐integral values between 1.34 and 2.22. The binding constant Kb and the effective valency zδ had values of 265/M and 0.64 for cell R‐15, 821M and 0.56 for cell L‐4, and 263/M and 0.51 for cell L‐6. Intracellular spermine also blocked the Ca2+‐activated K+ current induced by ionophoretic Ca2+‐injections, but increased the current at prolonged times after spermine injection. Extracellular spermine had no effect on electrical activity or on membrane currents. The results indicate that intracellular spermine affects the electrical discharge activity of neurons by acting as a blocker and/or modulator at voltage‐dependent membrane conductances.