David L. Dyer

Lysophosphatidic Acid‐Induced Neurite Retraction in PC12 Cells: Control by Phosphoinositide‐Ca2+ Signaling and Rho

Abstract: The endogenous phospholipid mediator lysophosphatidic acid (LPA) caused growth cone collapse, neurite retraction, and cell flattening in differentiated PC12 cells. Neurite retraction was blocked by cytochalasin B and ADP‐ribosylation of the small‐molecular‐weight G protein Rho by the Clostridium botulinum C‐3 toxin. LPA induced a transient rise in the level of inositol 1,4,5‐trisphosphate, and retraction was blocked by inhibitors of phospholipase β. Repeated application of LPA elicited homologous desensitization of the Ca2+ mobilization response. The activation of the phosphoinositide (PIP)‐Ca2+ second messenger system played a permissive role in the morphoregulatory response. Blockers of protein kinase C—chelerythrine, a myristoylated pseudosubstrate peptide, staurosporine, and depletion of protein kinase C from the cells by long‐term phorbol ester treatment—all diminished neurite retraction by interfering with LPA‐induced Ca2+ mobilization, which was required for the withdrawal of neurites. A brief 15‐min treatment with 4β‐phorbol 12‐myristate 13‐acetate also blocked retraction and Ca2+ mobilization, by inactivating the LPA receptor. Inhibition of protein tyrosine phosphorylation by herbimycin diminished retraction. Although activation of the PIP‐Ca2+ second messenger system appears necessary for the Rho‐mediated rearrangements of the actin cytoskeleton, bradykinin, which activates similar signaling events, failed to cause retraction, indicating that a yet unidentified novel mechanism is also involved in the LPA‐induced morphoregulatory response.

Lysophosphatidic acid-induced neurite retraction in PC12 cells: neurite-protective effects of cyclic AMP signaling.

Abstract: Effects of the cyclic AMP second messenger system were studied on the retraction of neurites elicited by the phospholipid mediator lysophosphatidic acid (LPA) in PC12 cells. LPA stimulation inhibited adenylyl cyclase, indicating that the LPA receptor couples to the heterotrimeric Gi proteins. However, pertussis toxin or expression of dominant negative Ras did not prevent neurite retraction. In contrast, cholera toxin, forskolin, and application of dibutyryl‐cyclic AMP prevented neurite retraction. The neurite‐protective effect of forskolin was blocked by Rp‐adenosine 3′,5′‐phosphorothioate. Forskolin and dibutyryl‐cyclic AMP both failed to protect neurites in A126‐1B2 and 123.7 cells, which lack cyclic AMP‐activated protein kinase. Data indicate that elevation of cyclic AMP levels triggers a cyclic AMP‐activated protein kinase‐dependent mechanism that opposes the functioning of the morphoregulatory signaling activated by LPA. ADP‐ribosylation of Rho by the Clostridium botulinum C‐3 toxin in 123.7 cells caused neuronal differentiation, indicated by neurite extension, and blocked LPA‐induced neurite retraction. LPA activates Gq‐ and Gi‐linked signaling in parallel; therefore, a morphoregulatory signaling network hypothesis is proposed versus the simplistic approach of a signaling pathway. The signaling network integrates the receptor‐activated individual, sequential, and parallel signaling events into an interactive network whose individual components may fulfill required and permissive functions encoding the cellular response.

Biotin uptake by human colonic epithelial NCM460 cells: a carrier-mediated process shared with pantothenic acid

Previous studies showed that the normal microflora of the large intestine synthesizes biotin and that the colon is capable of absorbing intraluminally introduced free biotin. Nothing, however, is known about the mechanism of biotin absorption in the large intestine and its regulation. To address these issues, we used the human-derived, nontransformed colonic epithelial cell line NCM460. The initial rate of biotin uptake was found to be 1) temperature and energy dependent, 2) Na+ dependent (coupling ratio of 1:1), 3) saturable as a function of concentration [apparent Michaelis constant ( K m) of 19.7 μM], 4) inhibited by structural analogs with a free carboxyl group at the valeric acid moiety, and 5) competitively inhibited by the vitamin pantothenic acid (inhibition constant of 14.4 μM). Pretreatment with the protein kinase C (PKC) activators phorbol 12-myristate 13-acetate (PMA) and 1,2-dioctanoyl- sn-glycerol significantly inhibited biotin uptake. In contrast, pretreatment with the PKC inhibitors staurosporine and chelerythrine led to a slight, but significant, increase in biotin uptake. The effect of PMA was mediated via a marked decrease in maximal uptake velocity and a slight increase in apparent K m. Pretreatment of cells with modulators of the protein kinase A-mediated pathway, on the other hand, showed no significant effect on biotin uptake. These results demonstrate, for the first time, the functional existence of a Na+-dependent, specialized carrier-mediated system for biotin uptake in colonic epithelial cells. This system is shared with pantothenic acid and appears to be under the regulation of an intracellular PKC-mediated pathway.

The effect of active serum albumin on PC12 cells: I. Neurite retraction and activation of the phosphoinositide second messenger system

Vertebrate blood sera contain a factor that triggers oscillatory chloride currents in Xenopus oocytes through activation of the phosphoinositide/Ca2+ second system. The active serum component consists of lipids bound to an isoform of serum albumin that we have named active serum albumin (ASA). In undifferentiated PC12 cells, micromolar concentrations of ASA inhibit the early morphological changes induced by NGF, whereas in differentiated PC12 cells ASA caused a rapid withdrawal of neurites, which was reversible and dependent upon culture age. In contrast to normal serum, plasma and thrombin did not cause neurite retraction. Preincubation of ASA with monospecific antibodies to serum albumin suppressed its ability to induce neurite retraction in a dose dependent fashion. As in the oocyte, ASA activated the phosphatidylinositol second messenger system of PC12 cells, causing a several fold increase in Ins1,4,5P3 levels within minutes of application. The Ins1,4,5P3 increase was also blocked, in a titratable fashion, when ASA was preincubated with monospecific antibodies to serum albumin. This suggests that ASA-induced neurite retraction in PC12 cells may depend, at least in part, on activation of the phosphatidylinositol second messenger system. Results involving albumin-depleted sera show that ASA is the main factor responsible for serum vulnerability of neurites in PC12 cells. These findings point to some limitations in the use of serum in culture media, and raise the possibility that the serum factor may impair neuronal plasticity in disorders that are accompanied by the activation of blood coagulation together with a breakdown of the blood-brain barrier.

Shrink film patterning by craft cutter: complete plastic chips with high resolution/high-aspect ratio channel

This paper presents a rapid, ultra-low-cost approach to fabricate microfluidic devices using a polyolefin shrink film and a digital craft cutter. The shrinking process (with a 95% reduction in area) results in relatively uniform and consistent microfluidic channels with smooth surfaces, vertical sidewalls, and high aspect ratio channels with lateral resolutions well beyond the tool used to cut them. The thermal bonding of the layers results in strongly bonded devices. Complex microfluidic designs are easily designed on the fly and protein assays are also readily integrated into the device. Full device characterization including channel consistency, optical properties, and bonding strength are assessed in this technical note.

The effect of active serum albumin on PC12 cells: II. Intracellular Ca2+ transients and their role in neurite retraction

In the preceding paper it was shown that an isoform of serum albumin (ASA; active serum albumin) causes a rapid retraction of neurites and increases intracellular content of Ins1,4,5P3 in PC12 cells. Here we examined whether ASAs effects in nerve growth factor-differentiated PC12 cells were mediated through the Ins1,4,5P3/Ca2+ second messenger pathway by monitoring intracellular Ca2+ (Ca2+i) with Fura2. It was found that ASA caused a dose-dependent increase in Ca2+i. In Ca(2+)-free medium, the increase in Ca2+i elicited by ASA was smaller, but the rise in Ins1,4,5P3 content was not appreciably changed. The small Ca2+i increase seen in Ca(2+)-free medium was probably due to the release of Ca2+ from Ins1,4,5P3-sensitive intracellular stores. In Ca(2+)-containing medium the Ca2+ transient induced by ASA was not affected by organic Ca2+ channel blockers, but decreased when Co2+, Mn2+ or Zn2+ were present in the extracellular medium. The effect of other ligands, such as carbachol and bradykinin, whose receptors are coupled to the phosphoinositide system was also investigated. Carbachol at concentrations from 2 to 200 microM, and bradykinin at a concentration of 2 microM did not cause neurite retraction, whereas 200 microM bradykinin caused an approximately 40% decrease in neurite length. Thapsigargin, a Ca(2+)-ATPase inhibitor, caused a sustained elevation of Ca2+i and retraction of neurites, whereas depolarization of the cells by high K+ gave only a transient elevation of Ca2+i, and no neurite retraction. Therefore, a sustained elevation in Ca2+i might be a sufficient trigger to induce neurite retraction in differentiated PC12 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Sequential shrink photolithography for plastic microlens arrays

Endeavoring to push the boundaries of microfabrication with shrinkable polymers, we have developed a sequential shrink photolithography process. We demonstrate the utility of this approach by rapidly fabricating plastic microlens arrays. First, we create a mask out of the childrens toy Shrinky Dinks by simply printing dots using a standard desktop printer. Upon retraction of this pre-stressed thermoplastic sheet, the dots shrink to a fraction of their original size, which we then lithographically transfer onto photoresist-coated commodity shrink wrap film. This shrink film reduces in area by 95% when briefly heated, creating smooth convex photoresist bumps down to 30 µm. Taken together, this sequential shrink process provides a complete process to create microlenses, with an almost 99% reduction in area from the original pattern size. Finally, with a lithography molding step, we emboss these bumps into optical grade plastics such as cyclic olefin copolymer for functional microlens arrays.

Biotin uptake in cultured cell lines.

Publisher Summary This chapter investigates biotin uptake in different tissues (intestine, liver, kidney, and placenta) using cell culture systems. The cell line Hep G2 is used as a model of biotin uptake in the hepatocyte. Hep G2 is a human-derived hepatoma cell line that possesses many of the normal hepatocyte functions, including similar membrane transport processes, and more general aspects of liver physiology. Biotin transport in Hep G2 cells is shown to be similar to biotin transport in the native hepatocyte, being Na + , temperature, and energy dependent, and saturable as a function of concentration. As in the intestine, the uptake process in the liver is also specific for biotin; moreover, biotin analogs with a free carboxyl group on the valeric acid side-chain moiety, such as thioctic acid and dethiobiotin, inhibit biotin uptake, while analogs with a blocked carboxyl group, such as biocytin and biotin methyl ester, do not. Studies on biotin transport in the intact central nervous system are quite difficult to evaluate because of cellular heterogeneity, and therefore in vitro studies using pure cultures of astrocytes have been quite useful and are extensively employed.