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K R Pennypacker

Publications and source records attributed to K R Pennypacker.

At least 19 recordsLinked to original sources
PubMed1995

DNA binding activity of CREB transcription factors during ontogeny of the central nervous system.

During the early postnatal period, the rat brain contains high basal levels of AP-1 DNA binding activity which declines to the low levels found in the adult by the third postnatal week. Although the individual transcription factors that comprise this AP-1 DNA binding complex had not been identified, we discovered that these proteins were immunoreactive to the cAMP responsive element binding protein (CREB) and also recognized the CRE element. The 45 kDa CREB-immunoreactive protein was detected at high levels only during the first postnatal week. CRE and AP-1 DNA binding activities were studied in the olfactory bulb, striatum, hindbrain, hippocampus, hypothalamus and cerebellum. In general, the DNA binding activity correlated with the stage of maturation of the particular brain region. However, basal AP-1 DNA binding in the olfactory bulb from adults remained slightly elevated relative to other brain regions. Interestingly, the DNA binding complex in the olfactory bulb began to include fos-related antigen as well as CREB by the third postnatal week. The fra-containing complex only recognizes the AP-1 element, while the CREB complex can bind to either CRE or AP-1 sequences. Thus, there is crosstalk between the signal transduction systems that activate CREB and AP-1 transcription factors. This elevated CREB DNA binding activity may be a sensitive index for studying the development of the brain and could be involved in modulating the genomic program in differentiating cells.

Aging↗
PubMed1995

Acute repeated nicotine injections increase enkephalin and decrease AP-1 DNA binding activity in rat adrenal medulla.

Previously we reported that a single injection of nicotine decreased AP-1 DNA binding activity in adrenal medullae, although chronic bidaily nicotine (and saline) injections increased this binding activity [15]. Repeated acute nicotine injections (3 mg/kg i.p., 7 injections equi-spaced over a 3 h period) effectively increased adrenal tyrosine hydroxylase [3] and [Met5]enkephalin levels and also profoundly decreased adrenal medulla AP-1 DNA binding activity for over 8 h.

Adrenal Medulla↗
PubMed1995

Implications of prolonged expression of Fos-related antigens.

The AP-1 transcription factors are composed of the Fos and Fos-related antigens as well as Jun and related proteins. These factors have been extensively studied in many diverse paradigms using acute stimuli. Recent attention has focussed on long-term elevation of Fos-related antigens in the CNS, and this is discussed by Keith Pennypacker, Jau-S. Hong and Michael McMillian. Repeated or chronic treatment elevates Fos-related antigen levels for days in many different brain regions. Both direct and indirect stimulation are responsible for the protracted increase in Fos-related antigen-immunoreactive proteins, which may modulate late onset genes involved in neuroplasticity. Understanding the role of these factors in long-lasting or permanent disease states may provide insight into potential therapeutic strategies to treat chronic CNS disorders.

Animals↗
PubMed1995

Pharmacological regulation of transcription factor binding.

Organisms respond to extracellular stimuli by changing the expression of genes. Stimulation of the cell often induces a cascade of intracellular events that leads to activation of transcription factor DNA-binding complexes which modulate the transcription rate. Many cellular processes including development of the organism are dependent on these proteins to maintain proper levels of mRNA. A diversity of mechanisms has evolved to coordinate transcription factor binding to the specific DNA element which affects mRNA synthesis. Precise regulatory processes are present at the level of transcription, translation and posttranslation. Often, posttranslational processes alter affinities of factors to DNA-binding sites. In this review, the molecular controls of transcription factor binding to DNA will be examined, with specific examples of the pharmacologic regulation of transcription factor binding to DNA.

Base Sequence↗
PubMed1995

AP-1 transcription factor complexes in CNS disorders and development.

Transcription factors are regulatory proteins that modify gene expression. Any cellular function requiring alterations in mRNA levels depends upon these factors. The CNS, AP-1 (activator protein-1; c-fos and fos-related antigens plus jun-related factors) and CREB (cAMP responsive element binding protein) families of transcription factors have been extensively studied. The DNA binding complex is composed of dimers formed between the AP-1 and CREB factors and binding specificity is dictated by which proteins comprise the complex. Whereas the AP-1 factors are inducible, CREB and related proteins are constitutive and regulate gene transcription through phosphorylation. Due to seizure activity, many AP-1 factors are induced, but rapidly return to basal levels. However, if neuronal death occurs, fos-related antigens of 35 kDa persist for an extended period and may be involved in regulating genes related to neuronal plasticity. Similar factors are expressed after chronic drug treatment indicating a role in drug tolerance. However, during early CNS development, elevated AP-1 DNA binding consisting of c-jun and CREB occurs in every brain region and is inversely related to the degree of maturation of a particular brain area. These transcription factors are important for gene regulation during CNS dysfunction and development and those present specify which genes are activated.

Activating Transcription Factor 2↗
PubMed1994

Regulation of tyrosine hydroxylase in olfactory bulb cultures: selective inhibition of depolarization-induced increase by endogenous opioids.

Regulation of tyrosine hydroxylase (TH) by second messenger pathway activators was examined in rat olfactory bulb cell cultures. The number of TH-immunoreactive neurons was increased 2-3-fold by 36 h treatments with forskolin (Fsk, 10(-6) M) or phorbol myristate acetate (PMA, 10(-7) M), but was not significantly increased by a depolarizing concentration of KCl (45 mM). In contrast, KCl increased media [Met5]enkephalin (ME) immunoreactivity 2-fold in these cultures, equivalent to stimulation with Fsk or PMA. The possibility was examined that ME or another opioid produced by the cultures selectively inhibited the TH response to KCl. Pretreatment with the opioid receptor antagonist naloxone (10(-6) M) greatly increased the number of TH-immunoreactive neurons observed in response to KCl treatment, but had no effect on basal or Fsk-stimulated TH immunostaining, nor on basal or stimulated ME release. The increase in TH-immunoreactivity observed with combined KCl plus naloxone treatment was prevented by pretreating the cultures with the calcium channel blocker nimodipine (10(-6) M), which had no effect on Fsk stimulation or basal TH immunostaining. These data suggest that endogenous opioids selectively inhibit KCl-stimulated Ca2+ entry and thus TH induction in olfactory bulb cell cultures. These cultures offer a simple model system for further study of TH regulation in dopaminergic neurons.

Animals↗
PubMed1994

Brain injury in a dish: a model for reactive gliosis.

Reactive gliosis is a powerful response to brain injury and subsequent neuronal damage in vivo. Neuronal cell cultures are now well established as assays to study this process in vitro. However, equivalent studies of purified glial cell populations have only recently been achieved, following the realization that glial cells produce many of the neuropeptides, transmitters and growth factors that are produced also by neurons. There is now scope for studies in vitro that use mixed, identified populations of glial and neuronal cells to dissect the interactions between the two. Such cultures also lend themselves to assays for potential therapeutic strategies for brain injury that take account of all the different cell types found in the brain.

Animals↗
PubMed1994

Role of a 35 kDa fos-related antigen (FRA) in the long-term induction of striatal dynorphin expression in the 6-hydroxydopamine lesioned rat.

D1 dopamine (DA) receptor agonists induce the expression of the opioid peptide dynorphin (DYN) in the striatum, an effect accentuated several fold by removing the dopaminergic innervation to the striatum (e.g., by lesioning the DA cell bodies in the substantia nigra [SN]). D1 receptor-mediated effects are thought to involve cAMP and/or phosphoinositides as second messengers. However, it is unclear what third messengers are involved in the regulation of DYN expression. The present experiments evaluated the possible role of two families of immediate-early gene (IEG) proteins, Fos and Jun, in the induction of DYN biosynthesis following repeated treatment with DA agonists. In addition, the role of N-methyl-D-aspartate (NMDA) receptors in modulating DA-induced changes in DYN and IEG protein expression was assessed. Adult male rats received unilateral 6-hydroxydopamine (6-OHDA) or sham lesions of the SN. Following a recovery period, animals were injected twice daily with the DA agonist, apomorphine (APO; 5 mg/kg), for 4 or 7 days. As expected, APO induced DYN biosynthesis, at both the peptide and mRNA level, several fold more in the striatum ipsilateral to the 6-OHDA lesion than in the contralateral control side (or a sham lesioned striatum). These effects appeared to be mediated by D1 receptors since the D1 agonist, SKF 38393 (5 mg/kg), caused the same changes in DYN expression as APO whereas a D2 agonist, quinpirole (1 mg/kg), had no effect. Paralleling the increase in DYN expression, APO also induced the expression of c-Fos and Fos-related antigens (FRA's), in particular a 35 kDa FRA, but had no effect on the expression of various Jun-related IEG proteins (i.e., c-Jun, Jun B, Jun D). Consistent with the notion that Fos and FRA proteins alter transcriptional activity by binding to AP-1 (or AP-1-like) DNA sequences in the promoter regions of target genes, we found that repeated APO treatment caused large increases in AP-1 binding activity in striata ipsilateral to 6-OHDA lesions. These data indicate that repeated activation of D1 receptors increases both the expression of a 35 kDa FRA and AP-1 binding, events which may mediate the large increases in DYN expression in the DA denervated striatum. While co-administration of the NMDA receptor antagonist, MK-801, inhibited APO-induced increases in DYN and Fos/FRA expression in the intact striatum, its only effect in the DA-denervated striatum was a partial (35%) inhibition of the APO-induced increase in DYN-ir concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed1992

Characterization of dynorphin-containing neurons on dissociated dentate gyrus cell cultures.

In the dentate gyrus, the synthesis of the opioid peptide, dynorphin, is modulated by a variety of stimuli. In order to elucidate the cellular and molecular mechanisms regulating the synthesis of dynorphin in the hippocampus, we have established a routine primary cell culture of dentate granule neurons and identified granule-like neurons by a characteristic marker, dynorphin, in these cultures. Cultures were prepared from 7-day-old rat pups and maintained in medium with 2% fetal bovine serum. These cultures contained approximately 20% neurons and survived for over 4 weeks. After 2 weeks in culture, neurons expressing dynorphin-A and its messenger RNA were detected using immunocytochemistry and in situ hybridization, respectively. In dentate cultures, enkephalin-, cholecystokinin-, neuropeptide Y- and substance P-positive cells were observed in addition to dynorphin-positive cells with immunocytochemistry. The results suggest that dentate gyrus cell cultures provide a valid in vitro model for studying molecular mechanisms regulating prodynorphin gene expression.

Animals↗
PubMed1992

Preferential activation of [3H]phorbol-12,13-dibutyrate binding by AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) in neonatal striatal cell cultures.

Activation of excitatory amino acid receptors increased [3H]phorbol-12,13-dibutyrate ([3H]PdBu) binding in four week cultures of striatal cells from postnatal day 7 rat pups (PN7), and in PN7 cells co-cultured the fourth week with striatal cells from postnatal day 1 rat pups. Kainate (KA), trans-1-amino-cyclopentyl-1,3-dicarboxylate (ACPD), and N-methyl-D-aspartate (NMDA) increased [3H]PdBu binding equally in both types of cultures, but alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) increased binding by 3-fold in the co-cultures. Thus, [3H]PdBu binding in these two types of striatal cultures offers a simple model system for studying the regulation of AMPA/KA receptor responses.

Animals↗
PubMed1992

Constitutive expression of AP-1 transcription factors in the rat adrenal. Effects of nicotine.

The AP-1 proteins (c-fos and fos-related antigens and jun proteins) are transcription factors that are induced in most cells by various stimuli, but the expression of these proteins is low or undetectable in the basal state. Our data indicate that the rat adrenal gland contains a basally expressed elevated level of AP-1 proteins and mRNAs (40- and 35-kDa fos-related antigens, a 39-kDa c-jun protein, c-jun, junB, and junD mRNA), as well as high basal AP-1 DNA binding activity. Both the medulla and cortex contained equivalent levels of fos-related antigens and c-jun protein; however, the majority of AP-1 DNA binding was detected in the medulla. Handling of rats for several days prior to sacrifice failed to affect protein expression or binding; on the other hand, repeated injections of nicotine increased AP-1 DNA binding. A single nicotine injection 60 min prior to removing the adrenals caused a significant reduction in AP-1 DNA binding without any detectable changes in AP-1 protein expression. Pretreatment of adrenal nuclear extracts with alkaline phosphatase prior to incubation with the AP-1 oligomer decreased binding activity. Thus, unlike most tissues where AP-1 DNA binding activity is controlled by increased expression of AP-1 proteins, DNA binding in the rat adrenal gland appears to be controlled at the post-translational level, possibly through dephosphorylation.

Adrenal Glands↗
PubMed1992

A 35 kDa Fos-related antigen is co-localized with substance P and dynorphin in striatal neurons.

The rat striatum after dopamine denervation followed by repeated apomorphine treatment was examined for the co-expression of c-fos and Fos-related antigens with dynorphin, substance P and [Met5]enkephalin using Western blot and immunohistochemical techniques. Administration of apomorphine, a dopamine agonist, elevated the level of 35 kDa Fos-related antigen which co-localized with dynorphin and substance P, but not enkephalin, in striatal neurons.

Animals↗
PubMed1992

Apomorphine induction of AP-1 DNA binding in the rat striatum after dopamine depletion.

The expression of AP-1 transcription factors was assessed in the dopamine-depleted rat striatum over a 1 week period of repeated apomorphine injections. A single injection of apomorphine increased the expression of a 35 kDa Fos-related antigen and Jun proteins and their expression continued to increase until day 3 of repeated apomorphine treatment in dopamine-depleted striata. Apomorphine induces AP-1 transcription factors which may be involved in modulating gene expression in the striatum.

Animals↗
PubMed1992

Dopamine stimulates [3H]phorbol 12,13-dibutyrate binding in cultured striatal cells.

The effect of dopamine (DA) on the binding of [3H]phorbol 12,13-dibutyrate ([3H]PdBu) in cultured rat striatal cells was examined. DA maximally increased specific [3H]PdBu binding by 70 +/- 10%, an increase comparable to that observed with norepinephrine (NE). This finding suggests that DA activates protein kinase C in cultured striatal cells, because increases in [3H]PdBu binding reflect translocation of protein kinase C. Half-maximal stimulation was observed with 10(-6) M DA. The peak response was observed at 2-3 min after addition of 10(-4) M DA, but [3H]PdBu binding was still increased above basal at 30 min. DA was not acting via an adrenergic receptor. Prazosin (10(-6) M) blocked the response to NE, suggesting mediation by an alpha 1-adrenergic receptor, but had little effect on the response to DA. Conversely, the D1 receptor antagonist SCH-23390 (10(-6) M) blocked the response to DA, but only partially inhibited the response to NE. Morphine (10(-6) M) inhibited the response to DA by 46 +/- 14%, but did not affect significantly the response to NE. The DA effect on [3H]PdBu binding is apparently independent of the increase in cyclic AMP seen on D1 receptor activation. Forskolin, apomorphine, and the D1 agonist SKF-38393 all increased cyclic AMP in striatal cells, but were less effective than DA in stimulating [3H]PdBu binding. The D2 agonist quinpirole was ineffective in stimulating either cyclic AMP or [3H]PdBu binding.

Animals↗
PubMed1990

Identification of calmodulin-binding proteins.

We have outlined and partially characterized a series of biotinylated calmodulin derivatives that may be useful in the study of calmodulin-binding protein expression, physical points of calmodulin-target interaction, and proteolytic mapping of related calmodulin-binding proteins. Biotinylated calmodulins offer several advantages as probes of protein-protein interactions. First, biotinylation can be directed to different amino acid residues. Second, biotinylation can be carried out under mild, near-physiological conditions, reducing the likelihood that conditions of protein modification would destroy biological function. Third, biotinylated proteins are stable, and reagents needed for their preparation and detection are relatively inexpensive. Fourth, the sensitivity of avidin-chromogenic enzyme systems is approaching that of radioactivity, with the added advantage that chromogens can be visualized in a relatively short time with respect to autoradiography. However, as with any protein modification procedure, one must be cautious when interpreting the results obtained with biotinylated proteins. For calmodulin-binding proteins, some interactions are impaired by modification of specific lysyl residues. On the other hand, interaction of biotinylated calmodulin with phosphodiesterase occurs, but this interaction may obscure recognition of the biotin residue by avidin. One approach to circumvent this problem is to have a series of site-directed biotinylated proteins available for use as outlined in this chapter. The choice of which agent to use is determined by the primary sequence of the protein of interest and whether any information is available concerning the effects of chemical modification on structure (i.e., acetylation experiments, modification of free sulfhydryls). In the absence of such information, an empirical approach can be taken. Photobiotin affords an easy means for biotinylation of proteins; however, the sites of modification are not always predictable. NHS-biotin derivatives are readily available and are relatively easy to use. Finally, one may wish to biotinylate the protein while liganded to its normal interacting molecule, in the case of calmodulin, calcium ion is the obvious choice. However, calmodulin could also be biotinylated while bound to a specific binding protein such as calcineurin. The latter method may be of use in determination of changes in reactivities of specific amino acid residues subsequent to binding. Finally, it may prove advantageous to biotinylate genetically engineered calmodulin, yeast calmodulin, or plant calmodulin to further define calmodulin-target protein interactions. Thus, the use of biotinylated calmodulin derivatives may offer insights into a range of structural and functional questions relevant to regulation of specific calmodulin-binding proteins.

Alkaline Phosphatase↗
PubMed1989

Changes in expression of tyrosine hydroxylase immunoreactivity in human SMS-KCNR neuroblastoma following retinoic acid or phorbol ester-induced differentiation.

Human SMS-KCNR cells differentiated in response to either retinoic acid or phorbol esters; differentiated cells extended numerous, complex neurites and showed reduced proliferation. Tyrosine hydroxylase (TH) immunoreactivity was measured in this cell line following treatment with retinoic acid (1-10 microM), 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 16-160 nM), or combinations of these agents. After 21 days of treatment with either TPA or retinoic acid (RA), TH immunoreactivity was measured in this using densitometric scans of Western blots, was doubled relative to untreated or serum-deprived SMS-KCNR cultures. Increases in TH immunoreactivity could be detected after 6 days of treatment. Treatment with RA for 3 days followed by phorbol esters for an additional 3 days resulted in a 3-fold increase in TH immunoreactivity at day 6; reversing the order of drug treatment did not have this effect. Treatment of cultures with the divalent cationophore A23187 caused treated cells to retract neurites; expression of TH immunoreactivity was decreased relative to drug-treated and control cultures. These results suggest that retinoic acid treatment may 'prime' SMS-KCNR cells for the subsequent effects of phorbol esters, and indicate that the patterns of biochemical differentiation induced by TPA or RA are different.

Cell Differentiation↗
PubMed1989

Expression of calmodulin-dependent phosphodiesterase, calmodulin-dependent protein phosphatase, and other calmodulin-binding proteins in human SMS-KCNR neuroblastoma cells.

Calmodulin (CaM)-dependent enzymes, such as CaM-dependent phosphodiesterase (CaM-PDE), CaM-dependent protein phosphatase (CN), and CaM-dependent protein kinase II (CaM kinase II), are found in high concentrations in differentiated mammalian neurons. In order to determine whether neuroblastoma cells express these CaM-dependent enzymes as a consequence of cellular differentiation, a series of experiments was performed on human SMS-KCNR neuroblastoma cells; these cells morphologically differentiate in response to retinoic acid and phorbol esters [12-O-tetradecanoylphorbol 13-acetate (TPA)]. Using biotinylated CaM overlay procedures, immunoblotting, and protein phosphorylation assays, we found that SMS-KCNR cells expressed CN and CaM-PDE, but did not appear to have other neuronal CaM-binding proteins. Exposure to retinoic acid, TPA, or conditioned media from human HTB-14 glioma cells did not markedly alter the expression of CaM-binding proteins; 21-day treatment with retinoic acid, however, did induce expression of novel CaM-binding proteins of 74 and 76 kilodaltons. Using affinity-purified polyclonal antibodies, CaM-PDE immunoreactivity was detected as a 75-kilodalton peptide in undifferentiated cells, but as a 61-kilodalton peptide in differentiated cells. CaM kinase II activity and subunit autophosphorylation was not evident in either undifferentiated or neurite-bearing cells; however, CaM-dependent phosphatase activity was seen. Immunoblot analysis with affinity-purified antibodies against CN indicated that this enzyme was present in SMS-KCNR cells regardless of their state of differentiation. Although SMS-KCNR cells did not show a complete pattern of neuronal CaM-binding proteins, particularly because CaM kinase II activity was lacking, they may be useful models for examination of CaM-PDE and CN expression. It is possible that CaM-dependent enzymes can be used as sensitive markers for terminal neuronal differentiation.

Avidin↗