Bernhard F.X. Reber

Differential cytoskeletal changes during growth cone collapse in response to hSema III and thrombin.

Growth cones are known as the site of action of many factors that influence neurite growth behavior. To assess how different collapsing agents influence the growth cone cytoskeleton, we used recombinant human Semaphorin III (hSema III) and the serine protease thrombin. Embryonic chick dorsal root ganglion neurons showed a dramatic depolymerization of actin filaments within 5 min upon hSema III exposure and virtually no influence on microtubules (MT). Only at later time points (20-30 min) was the polymerization/depolymerization rate of MT significantly affected. Thrombin induced a morphologically and kinetically similar growth cone collapse. Moreover, thrombin induced an early and selective depolymerization of dynamic MT, accompanied by the formation of loops of stable MT bundles. Selective changes in the phosphorylation pattern of tau were associated with microtubule assembly in thrombin-induced responses. Our data provide evidence that different signal transduction pathways lead to distinct changes of the growth cone cytoskeleton.

Detection of a trigger zone of bradykinin-induced fast calcium waves in PC12 neurites

Bradykinin and caffeine were used as two different agonists to study inositol 1,4,5-trisphosphate (IP3)-sensitive and caffeine/ryanodine-sensitive intracellular Ca2+ release in the outgrowing neurites of nerve-growth-factor (NGF)-treated rat phaeochromocytoma cells (PC12). Changes in neuritic intracellular free Ca2+ ([Ca2+]i) in single cells were measured after loading with a 1:1 mixture of the acetoxymethyl (AM) ester of the Ca2+-sensitive dyes Fura-red and Fluo-3, in combination with confocal microscopy. Bradykinin-induced Ca2+ release was blocked by U73211, a specific phospholipase C inhibitor. Caffeine-induced Ca2+ release was very low in neurites at rest. It increased after the cells were pre-loaded with Ca2+. The Ca2+ signal evoked at high concentrations of bradykinin (> 500 nM) arose from a trigger zone in the proximal part of the neurite, as a bi-direction-al wave towards the growth cone and cell body. The speed of neuritic Ca2+ waves was reduced in cells loaded with the Ca2+ chelator l,2-bis(2-aminophenoxy)ethane-tetraacetic acid/AM. Preloading of Ca2+ stores led to increased bradykinin-induced Ca2+ release, as seen for caffeine, and faster Ca2+ wave speeds. Caffeine evoked a simultaneous [Ca2+]i rise along the neurites of Ca2+ pre-loaded cells. Higher Ca2+ signal amplitudes and faster Ca2+ wave speeds, but no longer-lasting IP3-induced [Ca2+]i signals, correlated with increased caffeine-induced Ca2+ release in the neurites. At low concentrations of bradykinin (< 1.0 nM), the Ca2+ signals ceased to propagate as complete Ca2+ waves. Instead, repetitive stochastic Ca2+ release events (neuritic Ca2+ puffs) were observed. Neuritic Ca2+ puffs spread across only a few microns, at a slower speed than neuritic Ca2+ waves. These Ca2+ puffs represent elementary Ca2+ release units, whereby the released Ca2+ ions form these elementary events into the shape of a Ca2+ wave.

Activation of different pathways for calcium elevation by bradykinin and ATP in rat pheochromocytoma (PC 12) cells.

We have studied the pathways by which extra-cellular bradykinin and adenosine 5′-triphosphate (ATP) elicit changes in intracellular free calcium ([Ca2+]i) in nerve-growth-factor(NGF)- treated rat pheochromocytoma (PC 12) cells. Both substances caused a significant rise in [Ca2+]i as assessed by fura-2 based micro-fluorimetry. The bradykinin-induced response consisted of an initial Ca2+ mobilization from an internal pool followed by a sustained increase in [Ca2+]i, which was due to activation of a small inward current. The initial response always started at a localized site opposite to the cell nucleus. The inward current was partially carried by Ca2+ and began with a time lag of about 4 s after the start of the initial transient signal. Stepwise hyperpolarization of the plasma membrane, after activation of the inward current by bradykinin, caused a simultaneous increase in current amplitude and in [Ca2+]i, due to an increase in the driving force for Ca2+ influx. With ATP as an agonist the onset of inward current coincided with an increase in [Ca2+]i. Inward current and [Ca2+]i were enhanced during hyperpolarizing steps indicating a substantial Ca2+ influx through ATP-activated channels. No release of Ca2+ from internal stores, but a large Na+ inward current, was observed in Ca2+-free external solution after addition of ATP. While the bradykinin-induced responses were much more pronounced in cell bodies than in growth cones, the ATP effects were somewhat variable in cell bodies and more homogeneous in growth cones.

L-type Ca2+ channels and purinergic P2X2 cation channels participate in calcium-tyrosine kinase-mediated PC12 growth cone arrest.

During development and regeneration of the nervous system, growth cones of the various nerve cells navigate and direct neurite elongation by detecting and responding to cues in the environment. To investigate changes in growth cone behaviour due to calcium influx we used nerve growth factor (NGF)‐induced growth cones of PC12 (rat pheochromocytoma cells) cells as a model. High external concentrations of potassium and ATP depress growth cone motility, induce club‐shaped growth cones and reduce filopodia length and the number and relative F‐actin contents of single growth cones (r.a.c.), respectively. The cellular responses are mediated by a sustained increase in the intracellular free Ca2+ concentrations ([Ca2+]i) as monitored by calcium‐sensitive fluorescent dyes and confocal microfluorimetry. The responses are not detectable in the presence of the protein tyrosine kinase inhibitor genistein. Immunocytochemistry revealed an increased level of tyrosine‐phosphorylated proteins in cell bodies and growth cones but not in cell nuclei. Paxillin, a cytoskeleton‐associated protein located in neurites and growth cones, was detected among the phosphotyrosine proteins. The sustained (> 30 s) Ca2+ influx through voltage‐gated L‐type but not N‐ or P‐type Ca2+ channels induced the F‐actin loss and tyrosine phosphorylation. Ca2+ entry through P2X2 ligand‐gated channels caused the same effects. Our data suggest the following mechanism: increased [Ca2+]i levels activate tyrosine kinases located close to the ion channels which then leads to changes in morphology due to tyrosine phosphorylation of proteins, e.g. paxillin.

Unidirectional interaction between two intracellular calcium stores in rat phaeochromocytoma (PC12) cells.

1. A clone of the rat phaeochromocytoma cell line (PC12) was treated with nerve growth factor (NGF) for 4‐6 days and used to study caffeine‐ and bradykinin‐induced Ca2+ release from intracellular Ca2+ stores. The caffeine‐sensitive store can be depleted by Ca(2+)‐induced Ca2+ release (CICR), while the bradykinin‐induced release is mediated by inositol 1,4,5‐trisphosphate (IP3). The effect of Ca2+ release from these Ca2+ stores on cytosolic free Ca2+ ([Ca2+]i) was measured by means of fura‐2 single cell microfluorimetry. 2. Caffeine application caused no or only a small Ca2+ release in untreated cells in normal culture medium. The caffeine‐sensitive pool could be filled by Ca2+ entry into cells through either voltage‐activated Ca2+ channels or ligand‐gated cation channels. 3. Bradykinin application produced substantial Ca2+ release in untreated cells in normal culture medium. The response was enhanced after K(+)‐depolarization of the cells. The bradykinin‐induced release of Ca2+ also caused depletion of the caffeine‐sensitive pool by CICR. However, Ca2+ released from the IP3‐sensitive store was not sequestered into the caffeine‐sensitive Ca2+ store. 4. The caffeine‐induced rise in [Ca2+]i was blocked by ryanodine in a use‐dependent manner. In addition, a substantial use‐dependent ryanodine block resulted from the bradykinin‐induced rise of [Ca2+]i and subsequent CICR. By contrast, the K(+)‐induced rise of [Ca2+]i caused only a marginal use‐dependent ryanodine inhibition of Ca2+ release. 5. Our results suggest an enhancement of the IP3‐induced [Ca2+]i rise in the cytoplasm by CICR from the caffeine‐sensitive pool. 6. A mathematical model adequately simulates our experimental data.

Inhibition of protein kinases in rat pheochromocytoma (PC12) cells promotes morphological differentiation and down-regulates ion channel expression

We have studied morphological differentiation and ion channel expression in PC12 cells under different culture conditions. Differentiation mediated by nerve growth factor (NGF) was compared with that induced by depletion and inhibition of protein kinases (phorbol ester β-PMA plus staurosporine). Morphological differentiation was similar under both conditions. However, ion channel densities, studied by means of the patch-clamp technique, were enhanced by NGF and reduced by β-PMA + staurosporine. Similar changes were also observed for ω-conotoxin-sensitive Ca2+ channels by measuring radioligand binding. The decrease in Ca2+ channel density, after treatment of the cells with β-PMA + staurosporine, resulted in a reduced increase in the intracellular Ca2+ concentration during K+ depolarization. We conclude that morphological differentiation, but not ion channel expression, can occur during depression of protein kinase activities in PC12 cells.

Hormone-induced intracellular calcium oscillations and mitochondrial energy supply in single hepatocytes

Abstract Mitochondria in liver cells behave as a regulated stable power supply — optimized with respect to efficiency and stability against fluctuations of ATP-utilizing reactions in the case of starvation — even in the presence of intracellular Ca 2+ oscillations.

Differential modulation of pharmacologically distinct components of Ca2+ currents by protein kinase C activators and phosphatase inhibitors in nerve-growth-factor-differentiated rat pheochromocytoma (PC12) cells.

We have studied the effects of protein kinase C (PKC) activators 4β-phorbol 12-myristate 13-acetate (4β-PMA) and 1-oleoyl-2-acetylglycerol (OAG) and of phosphatase inhibitors (okadaic acid and calyculin A) on voltage-activated Ca2+ and K+ channels in nerve growth factor-(NGF)-differentiated pheochromocytoma (PC12) cells. Whole-cell Ba2+ and K+ currents were recorded at room temperature with the patch-clamp technique. By using ω-conotoxin (CgTX) and isradipine, two specific Ca2+ channel blockers, we found three types of Ba2+ currents (IBa): (1) a ω-CgTX-sensitive IBa; (2) an isradipine-sensitive IBa; and (3) a ω-CgTX plus isradipine-resistant IBa. The external application of 4β -PMA or OAG down-modulated the isradipine-sensitive IBa whereas the two other IBa were not affected. 4β-PMA-induced inhibition of IBa was prevented by staurosporine (a protein kinase inhibitor) and PKC (19–31) (a specific PKC inhibitor). The delayed rectifier K+ current (IK) was unaffected by PKC activators. Both okadaic acid and calyculin A affected the components of the IBa in different manners. The presence of okadaic acid decreased the isradipine-sensitive IBa more than the ω-CgTX-sensitive IBa. The ω-CgTX plus isradipine-resistant IBa was not affected. Calyculin A down-modulated all three components of IBa to a similar degree. Our results suggest a differential modulation of voltage-activated Ca2+ and K+ channels by the PKC signalling pathway in NGF-differentiated PC12 cells.

Calyculin-A-induced fast neurite retraction in nerve growth factor-differentiated rat pheochromocytoma (PC12) cells

Rat pheochromocytoma (PC12) cells were treated with nerve growth factor (NGF) for 3-4 days. They formed growth cones and extended neurites. Addition of the phosphatase inhibitor calyculin A (CL-A) caused a concentration-dependent complete retraction of neurites within 15 min. Retraction of growth cones started with the filopodia still present. The cell bodies acquired a grape-like shape opposite to the cell nucleus. These morphological changes were reversible. After washout of the inhibitor, the cell bodies recovered to normal shape within about 30-60 min while neurites started to grow again within 1 day. Okadaic acid (OA) which, compared to CL-A, is less potent as a PP-1 and equally potent as a PP-2A class inhibitor, caused neurite retraction only when added at more than a thousand-fold higher concentration than CL-A. Ca2+ levels within neurites and cell bodies remained stable and low during neurite retraction as measured with fura-2. However, cells treated with CL-A showed reduced activity of voltage-gated Ca2+ channels. The results suggest that the observed reversible changes in cell morphology occur at a constant low Ca2+ level and are most likely due to the inhibition of PP-1 class phosphatases.

Inhibition of membrane currents and rises of intracellular Ca2+ in PC12 cells by CGS 9343B, a calmodulin inhibitor

The calmodulin inhibitor 1,3-dihydro-1-[1-((4-methyl-4H,6H-pyrrolo[1,2-a]-[4,1]benzoxazepin - 4-yl)methyl)-4-piperidinyl]-2H-benzimidazol-2-one maleate (CGS 9343B) caused a reversible block of voltage-activated Ca2+, Na+, and K+ currents in differentiated rat pheochromocytoma (PC12) cells. The drug also inhibited nicotinic acetylcholine receptor (nAChR) channel currents but not inward currents evoked by extracellular ATP. Depolarization-induced intracellular Ca2+ transients were almost completely inhibited in growth cones and cell bodies by CGS 9343B. Our results suggest actions of CGS 9343B on ion fluxes unrelated to calmodulin inhibition.