The noradrenergic locus coeruleus (LC) has been implicated in the promotion of arousal, in focused attention and learning, and in the regulation of the sleep/waking cycle. The complex biological functions of the central noradrenergic system have been investigated largely through electrophysiological recordings and neurotoxic lesions of LC neurons. Activation of LC neurons through electrical or chemical stimulation has also led to important insights, although these techniques have limited cellular specificity and short-term effects. Here, we describe a novel method aimed at stimulating the central noradrenergic system in a highly selective manner for prolonged periods of time. This was achieved through the conditional expression of a transgene for chlorotoxin (Cltx) in the LC of adult mice. Chlorotoxin is a component of scorpion venom that partially blocks small conductance chloride channels. In this manner, the influence of GABAergic and glycinergic inhibitory inputs on LC cells is greatly reduced, while their ability to respond to excitatory inputs is unaffected. We demonstrate that the unilateral induction of Cltx expression in the LC is associated with a concomitant ipsilateral increase in the expression of markers of noradrenergic activity in LC neurons. Moreover, LC disinhibition is associated with the ipsilateral induction of the immediate early gene NGFI-A in cortical and subcortical target areas. Unlike previous gain of function approaches, transgenic disinhibition of LC cells is highly selective and persists for at least several weeks. This method represents a powerful new tool to assess the long-term effects of LC activation and is potentially applicable to other neuronal systems.

Interaction of the neural cell adhesion molecule (N-CAM) with astrocytes activates a transcription factor, NF-kappaB, that mediates inflammatory responses after neural injury. Here we describe intracellular signaling events that link N-CAM binding to NF-kappaB-mediated transcription. Addition of the third immunoglobulin domain of N-CAM (Ig III), which mimics the activity of intact N-CAM, or of cytokines (interleukin-1beta or tumor necrosis factor-alpha), increased transcription from an NF-kappaB-responsive luciferase reporter gene construct that had been transiently transfected into neonatal rat forebrain astrocytes. NF-kappaB activity induced by Ig III or cytokines was decreased by inhibition of nonreceptor protein tyrosine kinases (PTKs), phospholipase C, protein kinase C (PKC), calcium/calmodulin-dependent protein kinase II (CaMKII), or oxidative stress. Inhibition of PKC blocked nuclear translocation of NF-kappaB protein while binding of NF-kappaB to DNA was decreased by modulation of redox homeostasis. In contrast, inhibition of CaMKII and nonreceptor PTKs altered neither nuclear translocation nor DNA binding, suggesting that these kinases affect NF-kappaB transactivation. A number of agents that inhibit NF-kappaB activation in other cell types did not affect activation in astrocytes. These findings suggest that activation of NF-kappaB by N-CAM and cytokines in astrocytes involves multiple signals that differentially affect NF-kappaB nuclear translocation, DNA binding, and transactivation.

In neurons, translation of dendritically localized mRNAs is thought to play a role in affecting synaptic efficacy. Inasmuch as components of the translation machinery may be limiting in dendrites, we investigated the mechanisms by which translation of five dendritically localized mRNAs is initiated. The 5′ leader sequences of mRNAs encoding the activity-regulated cytoskeletal protein, the α subunit of calcium–calmodulin-dependent kinase II, dendrin, the microtubule-associated protein 2, and neurogranin (RC3) were evaluated for their ability to affect translation in the 5′ untranslated region of a monocistronic reporter mRNA. In both neural and nonneural cell lines, the activity-regulated cytoskeletal protein, microtubule-associated protein 2, and α-CaM Kinase II leader sequences enhanced translation, whereas the dendrin and RC3 5′ untranslated regions slightly inhibited translation as compared with controls. When cap-dependent translation of these constructs was suppressed by overexpression of a protein that binds the cap-binding protein eIF4E, it was revealed that translation of these mRNAs had both cap-dependent and cap-independent components. The cap-independent component was further analyzed by inserting the 5′ leader sequences into the intercistronic region of dicistronic mRNAs. All five leader sequences mediated internal initiation via internal ribosome entry sites (IRESes). The RC3 IRES was most active and was further characterized after transfection in primary neurons. Although translation mediated by this IRES occurred throughout the cell, it was relatively more efficient in dendrites. These data suggest that IRESes may increase translation efficiency at postsynaptic sites after synaptic activation.

The neural cell adhesion molecule, N-CAM, is expressed on the surface of astrocytes and neurons, and N-CAM homophilic binding has been shown to alter gene expression in both of these cell types. To determine mechanisms by which N-CAM regulates gene expression, we have analyzed DNA binding of and transcriptional activation by NF-kappa B after N-CAM binding to the cell surface. Addition of purified N-CAM, the recombinant third immunoglobulin domain of N-CAM, or N-CAM antibodies either to neonatal rat forebrain astrocytes or to cerebellar granule neurons increased NF-kappa B/DNA binding activity in nuclear extracts as measured by electrophoretic mobility shift assays. Analysis using supershifting antibodies indicated that, in both cell types, p50 and p65 but not p52, c-Rel, or Rel B were contained in the NF-kappa B-binding complex. NF-kappa B-mediated transcription was increased after N-CAM binding to astrocytes and neurons as demonstrated by the activation of two different luciferase reporter constructs containing NF-kappa B-binding sites. N-CAM binding also resulted in degradation of I kappa B-alpha protein followed by an increase in the levels of I kappa B-alpha mRNA and protein. These results indicate that N-CAM homophilic binding at the cell membrane leads to increased NF-kappa B binding to DNA and transcriptional activation in both neurons and astrocytes. Activation of NF-kappa B, however, did not influence the previously reported ability of N-CAM to inhibit astrocyte proliferation. These observations together support the hypothesis that N-CAM binding activates multiple pathways leading to changes in gene expression in both astrocytes and neurons.

Neural cell adhesion molecules (CAMs) of the immunoglobulin superfamily nucleate and maintain groups of cells at key sites during early development and in the adult. In addition to their adhesive properties, binding of CAMs can affect intracellular signaling. Their ability to influence developmental events, including cell migration, proliferation, and differentiation can therefore result both from their adhesive as well as their signaling properties. This review focuses on the two CAMs for which the most information is known, the neural CAM, N-CAM, and L1. N-CAM was the first CAM to be characterized and, therefore, has been studied extensively. The binding of N-CAM to cells leads to a number of signaling events, some of which result in changes in gene expression. Interest in L1 derives from the fact that mutations in its gene lead to human genetic diseases including mental retardation. Much is known about modifications of the L1 cytoplasmic domain and its interaction with cytoskeletal molecules. The study of CAM signaling mechanisms has been assay-dependent rather than molecule-dependent, with particular emphasis on assays of neurite outgrowth and gene expression, an emphasis that is maintained throughout the review. The signals generated following CAM binding that lead to alterations in cell morphology and gene expression have been linked directly in only a few cases. We also review information on other CAMs, giving special consideration to those that are anchored in the membrane by a phospholipid anchor. These proteins, including a form of N-CAM, are presumed to be localized in lipid rafts, membrane substructures that include distinctive subsets of cytoplasmic signaling molecules such as members of the src-family of nonreceptor protein tyrosine kinases. In the end, these studies may reveal that what CAMs do after they bind cells together may have as profound consequences for the cells as the adhesive interactions themselves. This area will therefore remain a rich ground for future studies.

Excitatory synaptic activity can evoke transient and substantial elevations of postsynaptic calcium. Downstream effects of elevated calcium include the activation of the calcium-dependent protease calpain. We have developed a reagent that identifies dendritic spines in which calpain has been activated. A fusion protein was expressed that contained enhanced yellow and enhanced cyan fluorescent protein (EYFP and ECFP, respectively) linked by a peptide that included the μ-calpain cleavage site from α-spectrin. A PDZ-binding site fused to ECFP anchored this protein to postsynaptic densities. The fusion protein exhibited fluorescence resonance energy transfer (FRET), and diminution of FRET by proteolysis was used to localize calpain activity in situ by fluorescence microscopy. Incubation of the fusion protein with calpain in the presence of calcium resulted in the separation of EYFP and ECFP into monomeric fluorophores. In transiently transfected cell lines and dissociated hippocampal neurons, FRET was diminished by raising intracellular calcium levels with an ionophore or with glutamatergic agonists. Calpain inhibitors blocked these changes. Under control conditions, FRET levels in different dendritic spines of cultured neurons and in hippocampal slices were heterogeneous but showed robust decreases upon treatment with glutamatergic agonists. Immunostaining of cultured neurons with antibodies to a spectrin epitope produced by calpain-mediated digestion revealed an inverse correlation between the amount of FRET present at postsynaptic elements and the concentration of spectrin breakdown products. These results suggest that the FRET methodology identifies sites of synaptically induced calpain activity and that it may be useful in analyzing synapses undergoing changes in efficacy.

To study regulation in vivo of the promoter for the neural cell adhesion molecule, N-CAM, we have used homologous recombination to insert the bacterial lacZ gene between the transcription and translation initiation sites of the N-CAM gene. This insertion disrupts the gene and places the expression of beta-galactosidase under the control of the N-CAM promoter, Animals homozygous for the disrupted allele did not express N-CAM mRNA or protein, but the pattern of beta-galactosidase expression in heterozygous and homozygous embryos was similar to that of N-CAM mRNA in wild-type animals, The homozygotes exhibited many of the morphological abnormalities observed in previously reported N-CAM knockout mice, with the exception that hippocampal long-term potentiation in the Schaffer collaterals was identical in homozygous, heterozygous, and wild-type animals. Heterozygous mice were used to examine the regulation of the N-CAM promoter in response to enhanced synaptic transmission, Treatment of the mice with an ampakine, an allosteric modulator of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors that enhances normal glutamate-mediated synaptic transmission, increased the expression of beta-galactosidase in vivo as well as in tissue slices in vitro. Similar treatments also increased the expression of N-CAM mRNA in the heterozygotes. The effects of ampakine in slices were strongly reduced in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an AMPA receptor antagonist, Taken together, these results indicate that facilitation of AMPA receptor-mediated transmission leads to activation of the N-CAM promoter and provide support for the hypothesis that N-CAM synthesis is regulated in part by synaptic activity.

Tenascin (TN) is an extracellular matrix protein found in areas of cell migration during development and expressed at high levels in migratory tumor cells. TN was previously shown to support the attachment and migration of glioma cells in culture. To determine the domains responsible for glioma migration and attachment, we produced recombinant fusion proteins that collectively span the majority of the molecule including its epidermal growth factor-like repeats, fibronectin type III repeats and fibrinogen domain. These domains were tested for their ability to support migration of C6 glioma cells in an aggregate migration assay. A recombinant fusion protein including fibronectin type III (FNIII) repeats 2-6 (TNfn2-6) was the only fragment found to promote migration of C6 glioma cells at levels similar to that promoted by intact TN. Evaluation of smaller segments and individual FNIII repeats revealed that TNfn3 promoted migration and attachment of glioma cells and TNfn6 promoted migration but not attachment. While TNfn3 and TNfn6 promoted migration individually, the presence of both TNfn3 and TNfn6 was required for migration on segments of the FNIII region that included TNfn5. TNfn5 inhibited migration in a dose dependent manner when mixed with TNfn3 and also promoted strong attachment and spreading of C6 glioma cells. TNfn3 and TNfn6 promote cell migration and may function cooperatively to overcome the inhibitory activity of TNfn5. Additional cell attachment studies suggested that both beta1 integrins and heparin may differentially influence the attachment of glioma cells to TN fragments. Together, these findings show that C6 glioma cells integrate their response upon binding to at least three domains within TN.

The neural cell adhesion molecule (N-CAM) inhibits astrocyte proliferation in vitro and in vivo , and this effect is partially reversed by the glucocorticoid antagonist RU-486. The present studies have tested the hypothesis that N-CAM-mediated inhibition of astrocyte proliferation is caused by homophilic binding and involves the activation of glucocorticoid receptors. It was observed that all N-CAM Ig domains inhibited astrocyte proliferation in parallel with their ability to influence N-CAM binding. The proliferation of other N-CAM-expressing cells also was inhibited by the addition of N-CAM. In contrast, the proliferation of astrocytes from knockout mice lacking N-CAM was not inhibited by added N-CAM. These findings support the hypothesis that it is binding of soluble N-CAM to N-CAM on the astrocyte surface that leads to decreased proliferation. Signaling pathways stimulated by growth factors include activation of mitogen-activated protein (MAP) kinase. Addition of N-CAM inhibited MAP kinase activity induced by basic fibroblast growth factor in astrocytes. In accord with previous findings that RU-486 could partially prevent the proliferative effects of N-CAM, inhibition of MAP kinase activity by N-CAM was reversed by RU-486. The ability of N-CAM to inhibit astrocyte proliferation was unaffected, however, by agents that block the ability of N-CAM to promote neurite outgrowth. Together, these findings indicate that homophilic N-CAM binding leads to inhibition of astrocyte proliferation via a pathway involving the glucocorticoid receptor and that the ability of N-CAM to influence astrocyte proliferation and neurite outgrowth involves different signal pathways.

In earlier studies, the neural cell adhesion molecule, N-CAM, was found to inhibit the proliferation of rat astrocytes bothin vitroandin vivo. To identify the gene targets involved, we used subtractive hybridization to examine changes in gene expression that occur after astrocytes are exposed to N-CAMin vitro. While the mRNA levels for N-CAM decreased after such treatment, the levels of mRNAs for glutamine synthetase and calreticulin increased. Since glutamine synthetase and calreticulin are known to be involved in glucocorticoid receptor pathways, we tested a number of steroids for their effects on astrocyte proliferation. Dexamethasone, corticosterone, and aldosterone were all found to inhibit rat cortical astrocyte proliferation in culture in a dose-dependent manner. RU-486, a potent glucocorticoid antagonist, reversed the inhibitory effects of dexamethasone. These observations prompted the hypothesis that the inhibition of proliferation by N-CAM might be mediated through the glucocorticoid receptor pathway. Consistent with this hypothesis, the inhibition of astrocyte proliferation by N-CAM was reversed in part by a number of glucocorticoid antagonists, including RU-486, dehydroepiandrosterone, and progesterone. In transfection experiments with cultured astrocytes, N-CAM treatment increased the expression of a luciferase reporter gene under the control of a minimal promoter linked to a glucocorticoid response element. The enhanced activity of this construct stimulated by N-CAM was abolished in the presence of RU-486. The combined data suggest that astrocyte proliferation is in part regulated by alterations in glucocorticoid receptor pathways.

Polysialylation of the neural cell adhesion molecule (N-CAM) reduces the efficacy of N-CAM-mediated homophilic binding and is regulated both during development and in regions undergoing neurogenesis or remodeling in the adult. Hamster PST-1 (PST) and rat STX are two related sialytransferases that catalyze the polysialylation of N-CAM. We have isolated a cDNA clone for the rat homologue of PST and compared its amino acid and nucleotide sequence to that of rat STX. This analysis revealed regions of high sequence similarity corresponding to the enzymatic domains of the two molecules. Other regions of lower similarity were used to generate specific probes for in situ hybridization. The distribution of PST and STX mRNAs, polysialic acid, and N-CAM were analyzed at three developmental stages. PST and STX mRNAs were expressed abundantly throughout the nervous system at embryonic day 15 and postnatal day 4 and were coexpressed in most tissues examined. In the adult brain, STX expression was reduced relative to PST and expression of both mRNAs was restricted to subsets of cells in areas undergoing constant synaptic rearrangement including hippocampus and olfactory system. The results suggest that both PST and STX participate in the polysialylation of N-CAM in vivo and that their expression levels are dynamically controlled during development and regeneration.

Conditional gene expression and gene deletion are important experimental approaches for examining the functions of particular gene products in development and disease. The cre-loxP system from bacteriophage P1 has been used in transgenic animals to induce site-specific DNA recombination leading to gene activation or deletion. To regulate the recombination in a spatiotemporally controlled manner, we constructed a recombinant adenoviral vector, Adv/cre, that contained the cre recombinase gene under regulation of the herpes simplex virus thymidine kinase promoter. The efficacy and target specificity of this vector in mediating loxP-dependent recombination were analyzed in mice that had been genetically engineered to contain loxP sites in their genome. After intravenous injection of the Adv/cre vector into adult animals, the liver and spleen showed the highest infectivity of the adenovirus as well as the highest levels of recombination, whereas other tissues such as kidney, lung, and heart had lower levels of infection and recombination. Only trace levels of recombination were detected in the brain. However, when the Adv/cre vector was injected directly into specific regions of the adult brain, including the cerebral cortex, hippocampus, and cerebellum, recombination was detectable at the injection site. Furthermore, when the Adv/cre vector was injected into the forebrains of neonatal mice, the rearranged toxP locus from recombination could be detected in the injected regions for at least 8 weeks. Taken together, these results demonstrate that the Adv/cre vector expressing a functional cre protein is capable of mediating loxP-dependent recombination in various tissues and the recombined gene locus may in some cases be maintained for an extended period. The use of the adenovirus vector expressing cre combined with localized delivery to specific tissues may provide an efficient means to achieve conditional gene expression or knockout with precise spatiotemporal control.

During development of the vertebrate nervous system, the neural cell adhesion molecule (N-CAM) is expressed in a defined spatiotemporal pattern. We have proposed that the expression of N-CAM is controlled, in part, by proteins encoded by homeobox genes. This hypothesis has been supported by previous in vitro experiments showing that products of homeobox genes can both bind to and transactivate the N-CAM promoter via two homeodomain binding sites, HBS-I and HBS-II. We have now tested the hypothesis that the N-CAM gene is a target of homeodomain proteins in vivo by using transgenic mice containing native and mutated N-CAM promoter constructs linked to a beta-galactosidase reporter gene. Segments of the 5' flanking region of the mouse N-CAM gene were sufficient to direct expression of the reporter gene in the central nervous system in a pattern consistent with that of the endogenous N-CAM gene. For example, at embryonic day (E) 11, beta-galactosidase staining was found in postmitotic neurons in dorsolateral and ventrolateral regions of the spinal cord; at E14.5, staining was seen in these neurons throughout the spinal cord. In contrast, mice carrying an N-CAM promoter-reporter construct with mutations in both homeodomain binding sites (HBS-I and HBS-II) showed altered expression patterns in the spinal cord. At E11, beta-galactosidase expression was seen in the ventrolateral spinal cord, but was absent in the dorsolateral areas, and at E 14.5, beta-galactosidase expression was no longer detected in any cells of the cord. Homeodomain binding sites found in the N-CAM promoter thus appear to be important in determining specific expression patterns of N-CAM along the dorsoventral axis in the developing spinal cord. These experiments suggest that the N-CAM gene is an in vivo target of homeobox gene products in vertebrates.

After a penetrating lesion in the central nervous system, astrocytes enlarge, divide, and participate in creating an environment that adversely affects neuronal regeneration. We have recently shown that the neural cell adhesion molecule (N-CAM) partially inhibits the division of early postnatal rat astrocytes in vitro. In the present study, we demonstrate that addition of N-CAM, the third immunoglobulin-like domain of N-CAM, or a synthetic decapeptide corresponding to a putative homophilic binding site in N-CAM partially inhibits astrocyte proliferation after a stab lesion in the adult rat brain. Animals were lesioned in the cerebral cortex, hippocampus, or striatum with a Hamilton syringe and needle at defined stereotaxic positions. On one side, the lesions were concomitantly infused with N-CAM or with one of the N-CAM-related molecules. As a control, a peptide of the same composition as the N-CAM decapeptide but of random sequence was infused on the contralateral side of the brain. We consistently found that the population of dividing astrocytes was significantly smaller on the side in which N-CAM or one of the N-CAM-related molecules was infused than on the opposite side. The inhibition was greatest in the cortical lesion sites (approximately 50%) and was less pronounced in the hippocampus (approximately 25%) and striatum (approximately 20%). Two weeks after the lesion, the cerebral cortical sites infused with N-CAM continued to exhibit a significantly smaller population of dividing astrocytes than the sites on the opposite side. When N-CAM and basic fibroblast growth factor, which is known to stimulate astrocyte division in vitro, were coinfused into cortical lesion sites, astrocyte proliferation was still inhibited. These results suggest the hypothesis that, by reducing glial proliferation, N-CAM or its peptides may help create an environment that is more suitable for neuronal regeneration.

Early postmitotic cortical neurons are mostly corticofugal projection neurons that take up positions in deep cortical laminae. Later postmitotic neurons are preferentially localized to superficial cortical laminae. In reeler mutant mice it appears that cortical laminar positions with respect to birthdate are reversed (Caviness, 1982). In a reanalysis of reeler lamination we found that early postmitotic cortical neurons labeled by embryonic day (E) 11–13 injections of a birthdate marker, or by early postnatal day (PND) 2 retrograde labeling through their output projections, appear to take up positions both in the superficial and deep cortex. Neurons born on E11 and E12 are more likely to be situated superficially in the reeler cortex and neurons born on E13 are more likely to be situated in the deep reeler cortex. Many corticofugal projection neurons in the deep (but not superficial) reeler cortex either die or retract their axons before PND 21. We hypothesize that the earliest postmitotic (E11-E12) of the early postmitotic reeler cortical neurons are overly adhesive and act as a barrier to later postmitotic migrating neurons. In vitro cortical aggregation cultures confirmed that early postmitotic (E12) reeler neurons are more adhesive than early postmitotic (E12) wild-type neurons or late postmitotic (E16) reeler or wild-type cortical neurons. We suggest that the moderate wild-type preferential adhesion of early postmitotic cortical neurons to each other helps deep and superficially fated lineages to form cortical laminae.

The striatum of the mammalian forebrain is divided into two compartments: the patches and the matrix. Neurons of the patch compartment in the rat striatum become postmitotic earlier in neurogenesis than neurons of the matrix compartment. The selective adhesion of patch neurons to one another has been suggested previously to be an important developmental mechanism of striatal compartmentation. We asked if the selective adhesion of patch neurons is expressed before or after the migration of the majority of the matrix neurons into the striatum. Patch neurons were labelled in vivo by a fluorescent retrograde tracer injected into the substantia nigra on embryonic day 19, which almost exclusively labelled patch neurons. Matrix neurons were labelled with a maternal injection of bromodeoxyuridine at embryonic day 18. When animals were killed at embryonic day 20, the majority of the retrogradely labelled patch neurons were intermixed with the bromodeoxyuridine‐labelled matrix neurons, although there appeared to be clustering of some of the patch neurons. However, by postnatal day 2 there was a complete segregation of the clusters of the retrogradely labelled patch neurons from the bromodeoxyuridine‐labelled matrix neurons in the striatum. This process was modelled in vitro. The patch and matrix compartments were labelled in vivo at embryonic day 13 and 18 respectively, with different birthdate markers ([3H]thymidine or bromodeoxyuridine). At embryonic day 20 the striatal tissue was removed, dissociated and reaggregated in suspension cultures. After 1 day in vitro, labelled patch and matrix neurons were randomly intermixed within the reaggregates. Examination of the cultures at 2.5 and 4 days in vitro revealed clumping of the labelled patch neurons towards the centres of the reaggregates. Over this same period, the labelled matrix neurons did not clump and were dispersed towards the periphery of the reaggregates. The results suggest that patch neuron adhesiveness may appear relatively soon after these neurons become postmitotic, but that this adhesiveness is unable to overcome the initial force produced by the massive migration of matrix neurons into the striatum. We hypothesize that a migratory phase of embryonic striatal development exists, when fated patch and matrix neurons intermix. After this migratory phase, patch neuron adhesiveness can produce the mature segregation of the striatal compartments.

Cytotactin/tenascin is a multidomain extracellular matrix protein that inhibits both cell spreading and intracellular alkalinization. The protein has multiple different domains which are homologous to regions in epidermal growth factor, fibronectin, and fibrinogen. In previous studies, we produced nonoverlapping fusion proteins corresponding to these domains and examined their effects on cell attachment and spreading. Based on their ability either to promote or to inhibit cell attachment, two of these fusion proteins were shown to be adhesive and two were shown to be counteradhesive. To determine how the adhesive and counteradhesive activities of different cytotactin/tenascin domains alter intracellular pH(i) (designated pH(i)), we have measured pH(i) in NIH3T3 and U251MC cells in the presence of the cytotactin/tenascin fusion proteins and intact cytototactin/tenascin, as well as fibronectin. Cells incubated in the presence of intact cytotactin/tenascin or of the counteradhesive fusion proteins had a pH(i) lower than control cells. In contrast, the presence of the adhesive fusion proteins or of fibronectin caused cells to have higher pH(i) values than control cells. When two fragments were simultaneously presented, one of which alone increased pH(i) and the other of wh ich alone decreased pH(i), the predominant effect was that of lowered pH,. incubation with an RGD-containing peptide derived from the cytotactin/tenascin sequence inhibited alkalinization promoted by the adhesive fragment containing the second through sixth fibronectin type III repeats that was known to bind to integrins. Incubation of the cells with heparinase I or III inhibited the intracellular alkalinization of cells plated in the presence of the other adhesive fusion protein containing the fibrinogen domain, suggesting that heparan sulfate proteoglycans were involved in these pH(i) changes. The activity of protein kinase C appeared to be important for the changes in pH(i) mediated by all of the proteins. The protein kinase C inhibitor Calphostin C blocked the rise in pH(i) elicited by the adhesive fusion protei ns and by fibronectin. Moreover, activation of protein kinase C by the addition of phorbol esters increased the pH(i), in cells plated on cytotactin/tenascin or counteradhesive fusion proteins and reversed their effects. The results of this study support the hypothesis that cytotactin/tenascin can bind to multiple cell surface receptors and thereby elicit different physiological responses. Decreases in pH(i), are correlated with the phenomenon of counteradhesion whereas the ability to increase pH, is associated with cell attachment via at least two different types of cell surface receptors. The data raise the possibility that binding of cytotactin/tenascin may influence primary cellular processes such as migration and proliferation through the differential regulation of pH(i). (C) 1994 Wiley-Liss, Inc.