Biomedical subjects

L A Greene

Publications and source records attributed to L A Greene.

At least 91 records · Page 5Linked to original sources

PubMed1994

A Trk nerve growth factor (NGF) receptor point mutation affecting interaction with phospholipase C-gamma 1 abolishes NGF-promoted peripherin induction but not neurite outgrowth.

We analyzed the function of Trk nerve growth factor (NGF) receptors containing a point mutation (Tyr-->Phe) in a major autophosphorylation site (Tyr-785). Tyr-785 is required for phospholipase C-gamma 1 to interact with Trk and to become tyrosine-phosphorylated in response to NGF. The altered receptors were transfected into a mutant subline of PC12 rat pheochromocytoma cells (designated PC12nnr5) that, unlike wild-type PC12 cells, lack expression of endogenous Trk and responsiveness to NGF. PC12nnr5 cells permanently transfected with Trk Y785F exhibit NGF-dependent autophosphorylation and normal NGF binding and internalization. Moreover, Trk Y785F mediates NGF-stimulated neurite outgrowth as well as a variety of additional responses including induction of immediate-early and late genes. However, in contrast to cells expressing wild-type Trk, cells expressing Trk Y785F lack NGF-promoted elevation of peripherin intermediate filament mRNA and protein. These observations indicate that phospholipase C-gamma 1 activation or other signaling pathways dependent on Tyr-785 autophosphorylation are selectively required for regulation of peripherin expression by NGF, but not for many other functional NGF responses. This supports the presence of multiple and separable signaling pathways in the NGF mechanism of action.

Amino Acid Sequence↗

PubMed1994

Reciprocal regulation of estrogen and NGF receptors by their ligands in PC12 cells.

Recent work has shown that estrogen receptor mRNA and protein co-localize with neurotrophin receptor systems in the developing basal forebrain. In the present study we examined the potential for reciprocal regulation of estrogen and neurotrophin receptor systems by their ligands in a prototypical neurotrophin target, the PC12 cell. Using in situ hybridization histochemistry, RT-PCR and a modified nuclear exchange assay, we found both estrogen receptor mRNA and estrogen binding in PC12 cells. Moreover, while estrogen binding was relatively low in naive PC12 cells, long-term exposure to NGF enhanced estrogen binding in these cells by sixfold. Furthermore, concurrent exposure to estrogen and NGF differentially regulated the expression of the two NGF receptor mRNAs. The expression of trkA mRNA was up-regulated, while p75NGFR mRNA was down-regulated transiently. The present data indicate that NGF may increase neuronal sensitivity to estrogen, and that estrogen, by differentially regulating p75NGFR and trkA mRNA, may alter the ratio of the two NGF receptors, and, consequently, neurotrophin responsivity. In view of the widespread co-localization of estrogen and neurotrophin receptor systems in the developing CNS, the reciprocal regulation of these receptor systems by NGF and estrogen may have important implications for processes governing neural maturation and the maintainance of neural function.

Animals↗

PubMed1994

Trk receptors use redundant signal transduction pathways involving SHC and PLC-gamma 1 to mediate NGF responses.

In response to NGF, the Trk receptor tyrosine kinase forms a complex with SHC, a protein that couples receptor tyrosine kinases to p21ras. Complex formation between Trk and SHC, SHC tyrosine phosphorylation, and association of SHC with Grb2 were mediated by autophosphorylation at Y490 in Trk [sequence: see text]. To determine the role of SHC and other Trk substrates in NGF signaling, Trk receptors with mutations in Y490 and Y785 (the PLC-gamma 1 association site) were introduced into PC12nnr5 cells. NGF treatment of PC12nnr5 cells expressing Trk with mutations in either substrate-binding site resulted in normal neurite outgrowth and Erk1 activity and tyrosine phosphorylation. However, PC12nnr5 cells expressing Trk with mutations at both sites failed to stably extend neurites and efficiently induce Erk1 activity and tyrosine phosphorylation in response to NGF. We postulate that Trk receptors can activate Erk1 by either SHC- or PLC-gamma 1-dependent signaling pathways. These results suggest a model whereby Trk receptors utilize at least partially redundant signal transduction pathways to mediate NGF responses.

3T3 Cells↗

PubMed1994

Neurotrophic factors in the rat penis.

An intact nerve supply is essential for normal erectile function. We have undertaken a study to examine the presence and synthesis of growth factors of the penis that support neural function. Extracts were obtained from deskinned penises of Sprague-Dawley rats, aged 3, 6 and 10 weeks, representing prepubertal, pubertal and postpubertal states. Penile extracts were subjected to Northern blot analysis to evaluate expression of nerve growth factor-beta (beta-NGF)-mRNA, PC-12 bioassay to quantitate the nerve growth promoting activity and immunoassay to detect the amount of beta-NGF protein. These initial experiments showed a disproportionately abundant level of nerve growth promoting activity as compared with the levels detected with the immunoassay. The PC-12 bioassay is sensitive to both beta-NGF and fibroblast growth factors (FGFs). To further investigate these findings, the bioassay was conducted again after heparin chromatography, with beta-NGF receptor blockade, or with the addition of anti-beta-NGF, anti-basic-FGF, or anti-acidic-FGF. These studies confirmed that the abundant nerve growth promoting activity in the rat penis is due largely to basic FGF. In conclusion, the neurotrophin NGF is expressed in the rat penis at levels consistent with its expression in other peripheral tissues. Basic-FGF, on the other hand, has been detected at levels far in excess of NGF. Since erectile function is dependent on the integrity of the vascular structure and its intact innervation and since basic FGF presents as an abundant penile growth factor with both angiogenic and neurotrophic activities, basic FGF might play a significant role in erectile physiology.

Animals↗

PubMed1994

KT5926 selectively inhibits nerve growth factor-dependent neurite elongation.

We have examined the effects of the protein kinase inhibitor KT5926 on NGF-promoted responses in PC12 and PC12-C41 cells (a subclone of the parental cell line). Our findings reveal that this compound specifically and reversibly prevents the NGF-induced outgrowth and regeneration of neurites. In addition, neurites of NGF-pretreated cells cease further elongation upon exposure to KT5926. However, preexisting neurite networks in the cultures remain intact in the presence of the drug. The inhibition of neuritic growth appears to occur at least in part at the level of growth cones since KT5926 also causes these structures to collapse and inhibits NGF-promoted reactivation of NGF-deprived growth cones. Although KT5926 is an analogue of K-252a, which blocks all responses to NGF, it does not affect other NGF-elicited cellular responses examined, including NGF-dependent priming of cells, gp140prototrk autophosphorylation, immediate-early gene induction, and phosphorylation of several known cytoskeletal proteins (MAP 1.2/1B, chartin MAPs, and beta-tubulin). However, phosphate incorporation into a cytoskeletally localized 58 kDa phosphoprotein, designated pp58, is selectively reduced in KT5926-treated cultures (+/- NGF). Although KT5926 is an in vitro inhibitor of myosin light chain kinase and calmodulin-dependent protein kinase II, inhibition of these two kinase activities by ML-9 and KN-62, respectively, applied alone or together, does not mimic the effects of KT5926 on neurite growth and on pp58 phosphorylation. Taken together, our findings suggest that KT5926, via a previously unidentified protein kinase inhibitory activity, differentially interferes with NGF-promoted growth cone function and consequently affects neuritic outgrowth. This compound should therefore be a useful tool for dissecting the mechanism of NGF actions and affords a means to identify phosphoproteins that play specific roles in neurite growth/elongation.

Alkaloids↗

PubMed1993

Association of protein kinases ERK1 and ERK2 with p75 nerve growth factor receptors.

Extracellular signal-regulated protein kinases (ERKs) constitute a family of protein serine-threonine kinases implicated in a variety of cell-signaling pathways. In cultured rat pheochromocytoma PC12 cells, ERK1 and ERK2 are activated by nerve growth factor (NGF), which also induces rapid association between ERK1 and the high affinity gp140prototrk tyrosine kinase NGF receptor. In the present work, we investigated the possible association between ERKs and the low affinity NGF receptor, p75. Extracts of PC12 cells (before and after NGF treatment) were subjected to immunoprecipitation with anti-p75 antibodies or antiserum; the immune complexes were then assessed for the presence of ERK proteins and tyrosine phosphorylation or for ERK activity using a specific substrate peptide. ERK1 and, to a lesser extent, ERK2 were found to be constitutively associated with p75. NGF did not modulate the total amount of ERK proteins coimmunoprecipitated with p75 but did markedly stimulate the level of p75-associated ERK catalytic activity. NGF treatment also enhanced the tyrosine phosphorylation of a p75-associated species that co-migrates with ERK1 in Western blots. Finally, K-252a, a compound that specifically inhibits activation by NGF of gp140prototrk, abolished the latter effect. These findings indicate that NGF, via activation of gp140prototrk, leads to association of enzymatically active ERKs with p75 and raise the possibility that this interaction may play a role in the NGF mechanism of action.

Animals↗

PubMed1993

Mammalian myosin I alpha, I beta, and I gamma: new widely expressed genes of the myosin I family.

A polymerase chain reaction strategy was devised to identify new members of the mammalian myosin I family of actin-based motors. Using cellular RNA from mouse granular neurons and PC12 cells, we have cloned and sequenced three 1.2-kb polymerase chain reaction products that correspond to novel mammalian myosin I genes designated MMI alpha, MMI beta, MMI gamma. The pattern of expression for each of the myosin I's is unique: messages are detected in diverse tissues including the brain, lung, kidney, liver, intestine, and adrenal gland. Overlapping clones representing full-length cDNAs for MMI alpha were obtained from mouse brain. These encode a 1,079 amino acid protein containing a myosin head, a domain with five calmodulin binding sites, and a positively charged COOH-terminal tail. In situ hybridization reveals that MMI alpha is highly expressed in virtually all neurons (but not glia) in the postnatal and adult mouse brain and in neuroblasts of the cerebellar external granular layer. Expression varies in different brain regions and undergoes developmental regulation. Myosin I's are present in diverse organisms from protozoa to vertebrates. This and the expression of three novel members of this family in brain and other mammalian tissues suggests that they may participate in critical and fundamental cellular processes.

Aging↗

PubMed1993

Internucleosomal DNA cleavage and neuronal cell survival/death.

Serum-free PC12 cell cultures have been used to study the mechanisms of neuronal death after neurotrophic factor deprivation. We previously reported that PC12 cells undergo "apoptotic" internucleosomal DNA cleavage after withdrawal of trophic support. Here, we have used a sensitive method to detect PC12 cell DNA fragmentation within three hrs of serum removal and have exploited this assay to examine several aspects regarding the mechanisms of neuronal survival/death. Major advantages of this assay are that it permits acute experiments to be performed well before other manifest signs of cell death and under conditions that cannot be applied chronically. We find that this apopotic DNA fragmentation is distinct from the random DNA degradation that occurs during necrotic death. Major observations include the following: (a) There is a good correlation between the ability of trophic substances to promote PC12 cell survival and to inhibit early DNA fragmentation. (b) Phorbol ester, an activator of PKC, acutely suppresses DNA fragmentation, but does not promote long-term survival or inhibition of endonuclease activity when applied chronically due to its downregulation of PKC. (c) Cells undergoing apoptosis within 3 h of serum withdrawal have a "commitment point" of only 1.0-1.5 h beyond which they can no longer be rescued by NGF. (d) Aurin, a non-carboxylic analog of the endonuclease inhibitor ATA, also inhibits DNA fragmentation and promotes short-term survival of PC12 cells. (e) Macromolecular synthesis is not required for DNA fragmentation or for NGF to prevent this event. (f) Extracellular Ca2+ is not required for internucleosomal DNA cleavage caused by serum withdrawal or for suppression of this by NGF. (g) DNA fragmentation can also be detected in cultures of rat sympathetic neurons as early as 10 h after removal of NGF. As in PC12 cell cultures, this precedes morphological signs of cell death.

Animals↗

PubMed1993

Association of a purine-analogue-sensitive protein kinase activity with p75 nerve growth factor receptors.

Purine analogues are protein kinase inhibitors, and they block with varying potency and specificity certain of the biological actions of nerve growth factor (NGF). The analogue 6-thioguanine (6-TG) has been shown to inhibit with high specificity protein kinase N (PKN), a serine/threonine protein kinase activated by NGF in several cellular systems. In the present work, immunoprecipitates of p75 NGF receptors from PC12 cells (+/-NGF treatment) were assayed for protein kinase activity using the substrate myelin basic protein under phosphorylating conditions optimal for PKN and in the presence or absence of purine analogues. An NGF-inducible activity was detected, and approximately 80% was inhibited by purine analogues. This activity was maximally stimulated by NGF within 5-10 min, partially decreased by 60 min, and returned to basal levels after 15 h of NGF treatment. The analogue 6-TG inhibited the NGF-inducible p75-associated kinase activity with an IC50 in the range of 15-35 microM. In mutant PC12 nnr-5 cells that lack the Trk NGF receptor, the purine-analogue-sensitive p75-associated kinase activity was not inducible by NFG. In normal PC12 cells, cyclic AMP analogues and epidermal growth factor failed to induce the same activity. Application of either 2-aminopurine or 6-TG to intact cells only slightly inhibit the NGF-dependent induction of the purine-analogue-inhibited p75-associated kinase activity. This activity shares many similarities but also displays some significant differences with cytosolic PKN. Our findings therefore indicate the association of a purine-analogue-sensitive protein kinase with p75 NGF receptors.

2-Aminopurine↗

PubMed1993

A purine analog-sensitive protein kinase activity associates with Trk nerve growth factor receptors.

Previous studies showed that purine analogs block with varying efficiency and specificity certain effects of nerve growth factor (NGF) on PC12 cells. These compounds also inhibit protein kinase activities. The analog 6-thioguanine has thus far been shown to inhibit only protein kinase N, an NGF-activated protein kinase, whereas 2-aminopurine also blocks other kinases. In the present study, immunoprecipitates of Trk NGF receptors from PC12 cells (+/- NGF treatment) were assayed for protein kinase activity by using the substrates myelin basic protein and histone HF1 under phosphorylating conditions optimal for protein kinase N and in the presence or absence of purine analogs. Activity was detected and approximately 50-80% was inhibited by these compounds. The purine analog-sensitive activity was maximally stimulated by NGF within 5 min, was partially decreased by 10 min, and still remained over basal levels after 15 h of NGF treatment. Analysis of myelin basic protein phosphorylated by anti-Trk immunoprecipitates revealed an NGF-stimulated increase in phosphothreonine and phosphotyrosine. Phosphorylation of threonine, but not of tyrosine residues, was inhibited by 6-thioguanine, which therefore inhibits a serine/threonine kinase associated with NGF receptor rather than the receptor kinase itself. Neither 2-aminopurine nor 6-thioguanine inhibited the NGF-dependent induction of Trk-associated kinase activity. Our findings thus indicate association of a purine analog-sensitive serine/threonine protein kinase activity with Trk NGF receptors.

2-Aminopurine↗

PubMed1993

Characterization of a PC12 cell sub-clone (PC12-C41) with enhanced neurite outgrowth capacity: implications for a modulatory role of high molecular weight tau in neuritogenesis.

To address the means by which diversity of neuronal morphology is generated, we have isolated and characterized naturally occurring variants of rat PC12 pheochromocytoma cells that exhibit altered neurite outgrowth properties in response to nerve growth factor (NGF). We describe here a PC12 cell sub-clone, designated PC12-clone 41 (PC12-C41), that displays significant increases in neurite abundance and stability when compared with the parental line. This difference does not appear to be due to an altered sensitivity or responsiveness to NGF or to a more rapid rate of neurite extension. Because of the role of the cytoskeleton in neuritogenesis, we examined a panel of the major cytoskeletal proteins (MAP 1.2/1B, beta-tubulin, chartins, peripherin, and high and low molecular weight (HMW and LMW) taus) whose levels and/or extent of phosphorylation are regulated by NGF in PC12 cultures. Although most cytoskeletal proteins showed little difference between PC12 and PC12-C41 cells (+/- NGF treatment), there was a significant contrast between the two lines with respect to tau expression. In particular, while NGF increases the total specific levels of tau in both cell types to similar extents (by about twofold), the proportion comprising HMW tau is threefold higher in the PC12-C41 clone than in PC12 cells. A comparable difference was observed under substratum conditions that were non-permissive for neurite outgrowth and so this effect was not merely a consequence of the differential neuritogenic capacities of the two lines. The distinction between the expression of HMW and LMW taus in PC12 and PC12-C41 cells (+/- NGF) was also observed at the level of the messages encoding these proteins. Such findings indicate that initiation of neurite outgrowth in PC12 cultures does not require a massive induction of tau expression and raise the possibility that HMW and LMW taus may have differential capacities for modulating neuronal morphology.

Animals↗

PubMed1993

Bcl-2 affects survival but not neuronal differentiation of PC12 cells.

Past studies have shown that serum-free cultures of PC12 cells are a useful model system for studying the mechanisms of neuronal death after neurotrophic factor deprivation. These cultures, as well as NGF-deprived cultures of sympathetic neurons, manifest and endonuclease activity that leads to "apoptotic" internucleosomal DNA cleavage. Overexpression of the proto-oncogene bcl-2 blocks apoptotic death in various cell types. To study the actions of this protein in neuronal cells, we derived PC12 cell lines stably transfected with a cDNA encoding human bcl-2. It is reported here that lines expressing high levels of the exogenous bcl-2 protein are protected from both death and apoptotic DNA fragmentation caused by removal of trophic support. However, expression of high levels of exogenous bcl-2 neither mimics nor interferes with promotion of neurite outgrowth by NGF.

Animals↗

PubMed1993

Transfection with trk restores "slow" NGF binding, efficient NGF uptake, and multiple NGF responses to NGF-nonresponsive PC12 cell mutants.

NGF binds to and activates the protein tyrosine kinase gp 140prototrk. Expression of this receptor is required for at least some responses to NGF. Three outstanding issues are addressed in the present work. First, we determined whether expression of gp 140prototrk is required for all neuronal NGF responses. Second, we examined the role of gp 140prototrk in NGF binding and internalization. Third, we addressed the utility of NGF-nonresponsive PC12nnr5 cells for study of the NGF mechanism. In contrast to wild-type PC12 cells, PC12nnr5 cells do not express endogenous gp 140prototrk. We therefore asked whether they possess other defects that compromise NGF signaling pathways. To answer these questions, we transfected PC12nnr5 cells with a cDNA encoding full-length human gp 140prototrk and isolated cell lines permanently expressing the receptor. Introduction of trk rescued all of the many and varied NGF responses assessed, including enhanced protein tyrosine phosphorylation, induction of immediate-early and neural-specific genes, neurite outgrowth and regeneration, maintenance of survival in serum-free medium, and stimulation of AChE activity. In contrast to PC12nnr5 cells, the trk-transfected lines also bind and internalize NGF with wild-type PC12 cell characteristics. These findings indicate that gp 140prototrk is required for many, if not all, responses of neuronal cells to NGF and is necessary for proper NGF binding and internalization. Additionally, as no signaling defect other than the absence of trk expression was revealed in PC12nnr5 cells, this work supports the utility of this line for genetic dissection of the NGF mechanism of action.

Animals↗

PubMed1993

Depolarization maintains neurites and priming of PC12 cells after nerve growth factor withdrawal.

In contrast to its actions on certain neural populations, membrane depolarization by elevated K+ promotes neither the survival nor the differentiation of PC12 cells. We therefore employed this model system to examine directly the actions of elevated K+ on neurites. Here we report that elevated K+ prevents the degeneration of neurites that occurs when NGF is withdrawn from PC12 cell cultures. This effect is inhibited by the L-type Ca2+ channel blockers verapamil and nitrendipine. Although depolarization preserves preexisting neurites, unlike NGF, it does not promote neurite elongation. In addition to neurite stabilization, elevated K+ also maintains NGF-deprived cells in a "primed" state in which they can rapidly regenerate neurites when re-treated with NGF. Elevated K+ alone has no priming effect, nor is it neuritogenic on either naive or NGF-pretreated cells. To probe the molecular basis for these actions of depolarization, we examined several cytoskeletal proteins whose phosphorylations (beta-tubulin, MAP 1.2/1B, and 64, 72 and 80 kDa chartins) or levels (MAP 1.2/1B and peripherin) are regulated by NGF in parallel with neurite outgrowth. Elevated K+ alone does not mimic these effects of NGF. In all cases, NGF withdrawal leads to the return of these proteins to levels characteristic of naive cells; in contrast, with the exception of the 80 kDa chartins, depolarization of NGF-deprived cultures maintained these proteins at or near their NGF-stimulated states. Similar observations were obtained with the NILE/L1 glycoprotein. These findings suggest that elevated K+ preserves priming and preexisting neurites by maintaining NGF-induced changes in cell composition. Our experiments invoke the possibility that elevation of intraneuronal Ca2+ may lead to selective stabilization of preexisting axons or dendrites in the intact nervous system, especially under circumstances in which the supply of neurotrophic factors is absent or limiting.

Animals↗

PubMed1993

Gangliosides rescue neuronal cells from death after trophic factor deprivation.

Serum-free cultures of PC 12 cells have been used as a model system for studying neuronal death occurring after neurotrophic factor deprivation. In this system, NGF rescues cells from death and prevents apoptotic DNA fragmentation. We report here that GM1 also promotes long-term survival of naive and NGF-pretreated PC 12 cells in serum-free medium and prevents internucleosomal cleavage of PC 12 cell DNA. In contrast to NGF, GM1 does not promote neurite outgrowth or somatic hypertrophy. The survival effects of GM1 are concentration dependent, with maximal activity at 30-50 microM. Optimal promotion of survival is obtained with multiple additions of GM1. Asialo-GM1 and sialic acid do not mimic these actions, indicating a requirement for the intact GM1 molecule. Prevention of serum-free PC 12 cell death is also obtained with di-, tri-, and tetrasialogangliosides. The ganglioside effects on survival and DNA fragmentation appear to be independent of macromolecular synthesis. GM1 is also effective under conditions in which cellular protein kinase C activity is downregulated by preexposure to high concentration of 12-O-tetradecanoylphorbol-13-acetate. Furthermore, GM1 promotes long-term survival of cultured rat sympathetic neurons after withdrawal of NGF. These findings complement prior observations that gangliosides protect cerebellar granule neurons from neurotoxicity caused by exposure to excitatory amino acids and extend the actions of gangliosides to rescue of neuronal cells deprived of neurotrophic factor support.

Animals↗

PubMed1992

Nerve growth factor-activated protein kinase N. Characterization and rapid near homogeneity purification by nucleotide affinity-exchange chromatography.

Protein kinase N (PKN) is a protein kinase rapidly activated by nerve growth factor (NGF) and other agents in PC12 pheochromocytoma and additional cell types. PKN is selectively inhibited by purine analogs, and this property has served both as a diagnostic for PKN activity and to establish its apparent involvement in certain pathways of the NGF mechanism of action. The present work has focused on further characterization, identification, and purification of NGF-activated PKN. We show here that PKN can be substantially enriched by elution from ion exchange resins with ATP. We exploited this novel technique (nucleotide affinity exchange chromatography) to devise two alternative isolation schemes for PKN. One utilizes sequential chromatographic steps and provides a preparation that is apparently 60% homogeneous for PKN and represents a total enrichment of approximately 10,000-fold. The other is a single column procedure and includes prewashes with NAD. This method yields material that is about 5-10% homogeneous for PKN, requires about 1 h, and can be applied to multiple samples in parallel. The ATP elution technique furthermore distinguishes NGF-regulated from basal PKN activity and thereby suggests the presence of distinct PKN isoforms. The applications of sucrose gradient centrifugation, gel filtration chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/silver staining, affinity labeling with 8-azido-ATP/SDS-PAGE, and autophosphorylation (after SDS-PAGE, blotting and renaturation) all indicate that PKN has an apparent molecular mass of 45-47 kDa and is mainly monomeric in solution. These and additional properties appear to distinguish PKN from many previously described protein kinases.

Adenosine Triphosphate↗

PubMed1992

Growth of tumour cell lines in polymer capsules: ultrastructure of encapsulated PC12 cells.

Recent studies indicate that polymer-encapsulated PC12 cells release sufficient amounts of dopamine to significantly alter behavioural paradigms in animals with unilateral lesions of dopaminergic midbrain neurons. Because cell fine structure provides a useful measure for assessment of storage function, exocytosis, metabolism, cell activity and cell viability, we examined the ultrastructure of PC12 cells grown in semi-permeable polymer capsules maintained in vitro or implanted into the forebrain of rats or guinea pigs. Encapsulated PC12 cells remained viable and continued to divide for the entire evaluation period of six months. Overall morphologies of encapsulated PC12 cells were similar in both environments and they resembled PC12 cells grown in monolayer cultures. In short-term cultures, encapsulated PC12 cells typically contained abundant quantities of chromaffin cell-like granules. The encapsulated cells had initially abundant microvilli on their surfaces which decline in frequency over time. After long-term enclosure for ten weeks or more, fewer secretory granules were detected in the cytoplasm of cells in capsules cultured in vitro and in brain-implanted capsules. Some cells in implanted capsules had long slender filipodia that were not present on PC12 cells in cultured capsules. The morphological changes of PC12 cells may correlate with altered growth conditions such as serum and oxygen concentrations, the presence or absence of growth factors in different environments, and with changes of cell interactions related to cell densities and build up of debris within the capsules over time. Since dopaminergic PC12 pheochromocytoma cells remain viable in semi-permeable polymer capsules for at least six months, such 'cell-capsules' could provide an alternative to dopamine-secreting embryonic neural grafts in dopamine replacement therapies.

Animals↗

PubMed1992

NGF and other growth factors induce an association between ERK1 and the NGF receptor, gp140prototrk.

As detected by coimmunoprecipitation from PC12 cells, NGF induces rapid association between ERK1 (a growth factor-activated serine/threonine protein kinase) and gp140prototrk NGF receptors. In contrast, no such association is found with the closely related ERK2. Anti-trk immunocomplexes generated from NGF-treated cells also contain protein kinase activity that shares many properties with soluble ERK1. The association of both ERK1 protein and ERK-like kinase activity with gp140prototrk is maximal by 5 min of NGF treatment, persists for approximately 1 hr, and subsequently declines by 18 hr. Treatment with either basic fibroblast growth factor, epidermal growth factor, or orthovanadate also leads to association of ERK1 with gp140prototrk without tyrosine phosphorylation of the latter. The interaction between ERK1 and gp140prototrk may prove relevant to the NGF mechanism.

Animals↗