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We show that BDNF regulates the timing of neurodevelopment via a novel mechanism of extranuclear sequestration of NFATc4 in Golgi. This leads to accelerated derepression of an NFI temporal occupancy gene program in cerebellar granule cells that includes Bdnf itself, revealing an autoregulatory loop within the program driven by BDNF and NFATc4.

The development of the mammalian cerebellum is orchestrated by both cell-autonomous programs and inductive environmental influences. Here, we describe the main processes of cerebellar ontogenesis, highlighting the neurogenic strategies used by developing progenitors, the genetic programs involved in cell fate specification, the progressive changes of structural organization, and some of the better-known abnormalities associated with developmental disorders of the cerebellum., Competing Interests: The authors declare that they have no competing interests.

Nuclear Factor One (NFI) transcription factors regulate temporal gene expression required for dendritogenesis and synaptogenesis via delayed occupancy of target promoters in developing cerebellar granule neurons (CGNs). Mechanisms that promote NFI temporal occupancy have not been previously defined. We show here that the transcription factor ETV1 directly binds to and is required for expression and NFI occupancy of a cohort of NFI-dependent genes in CGNs maturing in vivo. Expression of ETV1 is low in early postnatal cerebellum and increases with maturation, mirroring NFI temporal occupancy of coregulated target genes. Precocious expression of ETV1 in mouse CGNs accelerated onset of expression and NFI temporal occupancy of late target genes and enhanced Map2(+) neurite outgrowth. ETV1 also activated expression and NFI occupancy of the Etv1 gene itself, and this autoregulatory loop preceded ETV1 binding and activation of other coregulated target genes in vivo. These findings suggest a potential model in which ETV1 activates NFI temporal binding to a subset of late-expressed genes in a stepwise manner by initial positive feedback regulation of the Etv1 gene itself followed by activation of downstream coregulated targets as ETV1 expression increases. Sequential transcription factor autoregulation and subsequent binding to downstream promoters may provide an intrinsic developmental timer for dendrite/synapse gene expression., (© 2016 Ding et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Dendrite and synapse development are critical for establishing appropriate neuronal circuits, and disrupted timing of these events can alter neural connectivity. Using microarrays, we have identified a nuclear factor I (NFI)-regulated temporal switch program linked to dendrite formation in developing mouse cerebellar granule neurons (CGNs). NFI function was required for upregulation of many synapse-related genes as well as downregulation of genes expressed in immature CGNs. Chromatin immunoprecipitation analysis revealed that a central feature of this program was temporally regulated NFI occupancy of late-expressed gene promoters. Developing CGNs undergo a hyperpolarizing shift in membrane potential, and depolarization inhibits their dendritic and synaptic maturation via activation of calcineurin (CaN) (Okazawa et al., 2009). Maintaining immature CGNs in a depolarized state blocked NFI temporal occupancy of late-expressed genes and the NFI switch program via activation of the CaN/nuclear factor of activated T-cells, cytoplasmic (NFATc) pathway and promotion of late-gene occupancy by NFATc4, and these mechanisms inhibited dendritogenesis. Conversely, inhibition of the CaN/NFATc pathway in CGNs maturing under physiological nondepolarizing conditions upregulated the NFI switch program, NFI temporal occupancy, and dendrite formation. NFATc4 occupied the promoters of late-expressed NFI program genes in immature mouse cerebellum, and its binding was temporally downregulated with development. Further, NFI temporal binding and switch gene expression were upregulated in the developing cerebellum of Nfatc4 (-/-) mice. These findings define a novel NFI switch and temporal occupancy program that forms a critical link between membrane potential/CaN and dendritic maturation in CGNs. CaN inhibits the program and NFI occupancy in immature CGNs by promoting NFATc4 binding to late-expressed genes. As maturing CGNs become more hyperpolarized, NFATc4 binding declines leading to onset of NFI temporal binding and the NFI switch program.

The cerebellum plays an important role in motor control, motor skill acquisition, memory and learning among other brain functions. In rodents, cerebellar development continues after birth, characterized by the maturation of granule neurons. Cerebellar granule neurons (CGNs) are the most abundant neuronal type in the central nervous system, and they provide an excellent model for investigating molecular, -cellular, and physiological mechanisms underlying neuronal development as well as neural circuitry linked to behavior. Here we describe a procedure to isolate and culture CGNs from postnatal day 6 mice. These cultures can be used to examine numerous aspects of CGN differentiation, electrophysiology, and function.

Protein-DNA interactions are critical to maintain genome stability, DNA replication, chromosome -segregation and to regulate gene expression. Chromatin immunoprecipitation (ChIP) is a powerful technique to study these interactions within living neurons and nervous tissue. In particular, ChIP analysis of chromatin in which protein-DNA interactions are first fixed in situ provides a valuable approach to identify specific transcription factor-DNA interactions and their regulation in the developing nervous system. Here we describe a procedure utilizing Percoll gradient purification of nuclei from fresh brain tissue pre-fixed with formaldehyde for ChIP analysis. This purification protocol provides an enrichment of neuronal nuclei in high yield. We also illustrate the suitability of chromatin prepared from Percoll-purified brain nuclei for ChIP analysis of regulated transcription factor interactions with neuronal gene promoters.

Lentiviral vectors have become very useful tools for transgene delivery. Based on their ability to transduce both dividing and nondividing cells and to produce long-term transgene expression, lentiviruses have found numerous applications in the biomedical sciences, including developmental neuroscience. This protocol describes how to prepare lentiviral vectors by calcium phosphate transfection and to concentrate viral particles by ultracentrifugation. Functional vector titers can then be determined by methods such as fluorescence-activated cell sorting or immunostaining. Effective titers in the range of 10(8)-10(9) infectious units/ml can be routinely obtained using these protocols. Finally, we describe the infection of primary neuronal cultures with lentiviral vectors resulting in 85-90 % cell transduction using appropriate multiplicities of infection.

Granule neurons have a central role in cerebellar function via their synaptic interactions with other neuronal cell types both within and outside this structure. Establishment of these synaptic connections and its control is therefore essential to their function. Both intrinsic as well as environmental mechanisms are required for neuronal development and formation of neuronal circuits, and a key but poorly understood question is how these various events are coordinated and integrated in maturing neurons. In this review, we summarize recent work on the role of the Nuclear Factor I family in the transcriptional programming of cerebellar granule neuron maturation and synapse formation. In particular, we describe (1) the involvement of this family of factors in key developmental steps occurring throughout postmitotic granule neuron development, including dendrite and synapse formation and synaptic receptor expression, and (2) the mediation of these actions by critical downstream gene targets that control cell-cell interactions. These findings illustrate how Nuclear Factor I proteins and their regulons function as a “bridge” between cell-intrinsic and cell-extrinsic interactions to control multiple phases of granule neuron development.

The cell adhesion molecule Tag-1 is highly expressed in immature cerebellar granule neurons (CGNs) during axonogenesis and is down-regulated prior to onset of radial migration. However, its precise role(s) during development of mammalian CGNs has been unclear. Here we studied the effects of anti-Tag-1 function blocking antibodies on the development of mouse CGNs in primary cell culture and in situ. Interfering antibodies inhibited axon formation by mouse CGNs in both cell cultures and in cerebellar slices. Effects on axon extension in cell cultures were observed under conditions of homotypic cell-cell contact, consistent with inhibition of cell adhesion activity. Further, when used as a substratum Tag-1 protein strongly stimulated neurite outgrowth by CGNs. Antagonism of Tag-1 also enhanced CGN migration in modified Boyden chamber assays. Radial migration was inhibited by Tag-1 antibodies in cerebellar slices, possibly reflecting a block in early CGN maturation in situ. These findings are consistent with a regulatory role for Tag-1 in axon emergence as well as migratory behavior by developing mouse CGNs.

Developing neurons undergo a series of maturational stages, and the timing of these events is critical for formation of synaptic circuitry. Here we addressed temporal regulation of the Gabra6 gene, which is expressed in a delayed manner during dendritogenesis in maturing cerebellar granule neurons (CGNs). Developmental up-regulation of Gabra6 transcription required a binding site for nuclear factor I (NFI) proteins. The amounts and DNA binding activities of NFI proteins were similar in immature and mature CGNs; however, NFI occupancy of the Gabra6 promoter in native chromatin was temporally delayed in parallel with Gabra6 gene expression, both in vivo and in culture. The trans-repressor RE1 silencing transcription factor (REST) occupied the Gabra6 proximal promoter in CGN progenitors and early postmitotic CGNs, and its departure mirrored the initial onset of NFI binding as CGNs differentiated. Furthermore constitutive REST expression blocked both Gabra6 expression and NFI occupancy in mature CGNs, whereas REST knockdown in immature CGNs accelerated the initiation of both events. These studies identify a novel mechanism for controlling the timing of dendritogenesis-associated gene expression in maturing neurons through delayed binding of NFI proteins to chromatin. They also establish a temporal function for REST in preventing premature promoter occupancy by NFI proteins in early-stage postmitotic neurons.

Recent studies have shown that the nuclear factor I (NFI) family controls multiple stages of the postmitotic differentiation of cerebellar granule neurons (CGNs). Regulation of cell-cell signaling is an integral part of this NFI program, which involves expression of the cell adhesion molecules N cadherin and ephrin B1 throughout postmitotic CGN development. Here, we identify two additional downstream targets of NFI that are involved in extracellular CGN interactions. The cell adhesion molecule Tag-1 is highly enriched in CGNs undergoing parallel fiber formation and is down-regulated prior to onset of radial migration. We found that Tag-1 expression was strongly reduced by NFI dominant repression in immature primary CGNs and in the cerebella of E18 Nfib-null mice. Transient transfection and chromatin immunoprecipitation suggested that the Tag-1 gene is directly regulated by NFI. Furthermore, functional, Nfi knockout and chromatin immunoprecipitation studies implicated Wnt7a as a direct target of NFI in maturing CGNs. Wnt7a is secreted by developing CGNs and is required for maturation of mossy fiber-CGN synaptic rosettes. Consistent with this, synapsin I was greatly reduced within the internal granule cell layer of P17 Nfia-null mice. These findings indicated that NFI controls CGN postmitotic maturation through a combination of extracellular signaling molecules that operate either continuously to regulate multiple stages of development (N cadherin and ephrin B1) or primarily at early (Tag-1) or late (Wnt7a) maturation steps. They also illustrate the importance of NFI as a critical link between cell-intrinsic mechanisms and cell-cell interactions in the development of the mouse cerebellum., (2009 Wiley-Liss, Inc.)

A hallmark of male germ cell gene expression is the generation by alternative polyadenylation of cell-specific mRNAs, many of which utilize noncanonical A(A/U)UAAA-independent polyadenylation signals. Cleavage factor I (CFIm), a component of the pre-mRNA cleavage and polyadenylation protein complex, can direct A(A/U)UAAA-independent polyadenylation site selection of somatic cell mRNAs. Here we report that the CFIm subunits NUDT21/CPSF5 and CPSF6 are highly enriched in mouse male germ cells relative to somatic cells. Both subunits are expressed from spermatogenic cell mRNAs that are shorter than the corresponding somatic transcripts. Complementary DNA sequencing and Northern blotting revealed that the shorter Nudt21 and Cpsf6 mRNAs are generated by alternative polyadenylation in male germ cells using proximal poly(A) signals. Both sets of transcripts contain CFIm binding sites within their 3'-untranslated regions, suggesting autoregulation of CFIm subunit formation in male germ cells. CFIm subunit mRNA and protein levels exhibit distinct developmental variation during spermatogenesis, indicating stage-dependent translational and/or posttranslational regulation. CFIm binding sites were identified near the 3' ends of numerous male germ cell transcripts utilizing A(A/U)UAAA-independent sites. Together these findings suggest that CFIm complexes participate in alternative polyadenylation directed by noncanonical poly(A) signals during spermatogenesis.

A central question is how various stages of neuronal development are integrated as a differentiation program. Here we show that the nuclear factor I (NFI) family of transcriptional regulators is expressed and functions throughout the postmitotic development of cerebellar granule neurons (CGNs). Expression of an NFI dominant repressor in CGN cultures blocked axon outgrowth and dendrite formation and decreased CGN migration. Inhibition of NFI transactivation also disrupted extension and fasciculation of parallel fibers as well as CGN migration to the internal granule cell layer in cerebellar slices. In postnatal day 17 Nfia-deficient mice, parallel fibers were greatly diminished and disoriented, CGN dendrite formation was dramatically impaired, and migration from the external germinal layer (EGL) was retarded. Axonal marker expression also was disrupted within the EGL of embryonic day 18 Nfib-null mice. NFI regulation of axon extension was observed under conditions of homotypic cell contact, implicating cell surface proteins as downstream mediators of its actions in CGNs. Consistent with this, the cell adhesion molecules ephrin B1 and N-cadherin were identified as NFI gene targets in CGNs using inhibitor and Nfi mutant analysis as well as chromatin immunoprecipitation. Functional inhibition of ephrin B1 or N-cadherin interfered with CGN axon extension and guidance, migration, and dendritogenesis in cell culture as well as in situ. These studies define NFI as a key regulator of postmitotic CGN development, in particular of axon formation, dendritogenesis, and migratory behavior. Furthermore, they reveal how a single transcription factor family can control and integrate multiple aspects of neuronal differentiation through the regulation of cell adhesion molecules.

Objectives: The purpose of this study was to investigate whether stent-based delivery of an inhibitor of mammalian target of rapamycin (mTOR) can selectively clear macrophages in rabbit atherosclerotic plaques., Background: Current pharmacologic approaches to stabilize atherosclerotic plaques have only partially reduced the incidence of acute coronary syndromes and sudden death. Macrophages play a pivotal role in plaque destabilization, whereas smooth muscle cells (SMC) promote plaque stability., Methods: Stents eluting the mTOR inhibitor everolimus were implanted in atherosclerotic arteries of cholesterol-fed rabbits. In addition, in vitro experiments using explanted atherosclerotic segments and cultured macrophages as well as SMC were performed., Results: Stents eluting everolimus led to a marked reduction in macrophage content without altering the amount of SMC compared with polymer control stents. In vitro studies showed that everolimus treatment induced inhibition of translation in both cultured macrophages and SMC. However, cell death occurred only in macrophages and was characterized by bulk degradation of long-lived proteins, processing of microtubule-associated protein light chain 3, and cytoplasmic vacuolization, which are all markers of autophagy. Everolimus-induced autophagy was mediated by mTOR inhibition, because cell viability was not affected using tacrolimus, an mTOR-independent everolimus analog. Moreover, mTOR gene silencing was associated with selective induction of macrophage cell death. Autophagic macrophage cell death was confirmed by transmission electron microscopy both in cultured cells and in atherosclerotic explants., Conclusions: Stent-based delivery of everolimus selectively cleared macrophages in rabbit atherosclerotic plaques by autophagy, an mTOR inhibition-dependent and novel mechanism to induce cell death in mammalian cells.

Spermatogeniccells elaborate a highly specialized differentiation program that is mediated in part by germ cell-enriched transcription factors. This includes a novel member of the sterol response element-binding factor family, SREBF2_v1/SREBP2gc. Somatic SREBFs are predominantly synthesized as precursor proteins and are critical regulators of cholesterol and fatty acid synthesis. In contrast, SREBF2_v1 bypasses the precursor pathway and has been directly implicated in spermatogenic cell-specific gene expression. During spermatogenesis, SREBF2 precursor transcripts predominate in premeiotic stages, while SREBF2_v1 is highly upregulated specifically in pachytene spermatocytes and round spermatids. In the present study, we demonstrate thatSrebf2_v1mRNAs are present in the testis of several mammalian species, including humans. The basis for the stage-dependent transition in SREBF2 isoforms was also investigated. A 3' rapid amplification of cDNA ends (RACE)-PCR analysis of the rat and human revealed thatSrebf2_v1transcripts are generated by alternative pre-mRNA cleavage/polyadenylation. This involves the use of an intronic, A(A/U)UAAA-independent poly(A) signal within intron 7 of theSrebf2gene. Developmentally regulated competition between germ cell factors that control RNA splicing and pre-mRNA cleavage/polyadenylation may underlie this process. These results define an important role for alternative polyadenylation in male germ cell gene expression and development by controlling a stage-dependent switch in transcription factor structure and function during spermatogenesis. TheSrebf2gene thus provides a useful model to explore the role of alternative polyadenylation in regulating stage-dependent functions of important protein regulators in spermatogenic cells.

Cerebellar granule neurons (CGNs) undergo a well-defined, intrinsic differentiation program that is recapitulated in vitro. Thus, homogeneous cultures of CGNs provide an excellent opportunity to define the mechanisms underlying their development. The ability to alter endogenous gene expression in CGNs on a population-wide basis would greatly facilitate the elucidation of these events. In the present study, we show that self-inactivating lentiviruses efficiently infect both dividing progenitors and post-mitotic CGN cultures in a quantitative manner without altering their cellular properties. The time course for protein expression was biphasic for both types of cultures, with the first peak occurring during the initial infection period. Thus, lentiviruses can express proteins in CGNs both acutely and on a long-term basis to study developmental and other processes continuously over an extended time period. These vectors also infected CGNs in cerebellar slice preparations. In addition, lentiviruses harboring a transgene for the mouse GABA(A) receptor alpha6 subunit promoter recapitulated the differentiation-dependent expression of this gene in CGN cultures. Self-inactivating lentiviruses are extremely versatile vectors that offer important advantages for studies of protein function and gene regulation. The ability to alter protein function on a global scale in CGN cultures permits biochemical assessment of its impact on mRNA and protein populations, as well as on protein--protein and protein--DNA interactions. Further, integrated lentiviruses can be used to study chromatin-dependent promoter regulation and transcription factor interactions in CGNs over time in a facile manner.

Despite their advantages, percutaneous coronary interventional procedures are less effective in diabetic patients. Changes in the mechanical properties of vascular walls secondary to long-term hyperglycemia as well as other factors such as age may influence coronary distensibility. This investigation is aimed at deciphering the extent of these effects on distensibility of postmortem human coronary arteries in a controlled manner. Excised human left anterior descending (LAD) coronary arteries were obtained within 24 h postmortem. With the use of intravascular ultrasound, vascular deformation was analyzed at midregions of 51 moderate lesions. Intraluminal pressure was systematically altered using a computerized pressure pump system and monitored by a pressure-sensing guidewire. Distensibility, a normalized compliance term, was defined as the change in lumen area normalized by the initial reference area over a given pressure interval. With the use of multivariate analysis and repeated-measures ANOVA, coronary distensibility was independently influenced by hyperglycemia and age (P < 0.05) through the entire pressure range. Within physiological pressure range, distensibility was significantly reduced with age in nonhyperglycemic coronary specimens (10.55 +/- 4.41 vs. 6.99 +/- 2.45, x10(3) kPa(-1), P = 0.01), whereas the hyperglycemic vessels were stiff even in the younger group (7.90 +/- 5.82 vs. 7.20 +/- 3.36, x10(3) kPa(-1), P = 0.79). Similar results were observed with stiffness index and elastic modulus of the arteries. Hyperglycemia and age independently influenced the distensibility of moderately atherosclerotic LAD coronary arteries. The stiffening with age was overshadowed in the hyperglycemic group by as-yet-undetermined factors.

Approximately 12 million Americans have coronary artery disease, and almost one in five deaths in the United States can be attributed to this disease. In addition, 1.2 million Americans undergo cardiac catheterization and over one-half million receive a percutaneous coronary intervention such as balloon angioplasty, atherectomy, or stent implantation annually. This article will provide an overview of (1) atherosclerosis, the progressive disease which can lead to thrombotic events and/or the development of hemodynamically significant coronary artery lesions; (2) restenosis, the reappearance of significant lesions after coronary interventions such as stent placement; and (3) drug-eluting stents, the devices which, by using appropriate polymers to elute the appropriate drug with the appropriate pharmacokinetics, have almost completely eliminated restenosis.

Nervous system formation requires the elaboration of a complex series of differentiation events in both a spatially and maturation-regulated manner. A fundamental question is how neuronal subtype specification and developmental gene expression are controlled within maturing neurons. The alpha6 subunit of the gamma-aminobutyric acid type A (GABA(A)) receptor (GABRA6) is preferentially expressed in cerebellar granule neurons and is part of an intrinsic program directing their differentiation. We have employed a lentiviral approach to examine the transcriptional mechanisms controlling neuronal subtype-selective expression of this gene. These studies demonstrated that nuclear factor I (NFI) proteins are required for both transgenic GABRA6 promoter activity as well as endogenous expression of this gene in cerebellar granule neurons. Chromatin immunoprecipitation also showed that NFI proteins are bound to the GABRA6 promoter in these cells in vivo. Furthermore, analyses of gene knockout mice revealed that Nfia is specifically required for normal expression of the GABRA6 gene in cerebellar granule neurons. NFI expression and DNA binding activity are highly enriched in granule neurons, implicating this transcription factor family in the neuronal subtype-selective expression of the GABRA6 gene. These studies define a new role for NFI proteins as neuronal subtype-enriched transcriptional regulators that participate in an intrinsic transcriptional program directing the differentiation of cerebellar granule neurons.

Sperm are highly specialized cells, and their formation requires the synthesis of a large number of unique mRNAs. However, little is known about the transcriptional mechanisms that direct male germ cell differentiation. Sterol response element binding protein 2gc (SREBP2gc) is a spermatogenic cell-enriched isoform of the ubiquitous transcription factor SREBP2, which in somatic cells is required for homeostatic regulation of cholesterol. SREBP2gc is selectively enriched in spermatocytes and spermatids, and, due to its novel structure, its synthesis is not subject to cholesterol feedback control. This suggested that SREBP2gc has unique cell- and stage-specific functions during spermatogenesis. Here, we demonstrate that this factor activates the promoter for the spermatogenesis-related gene proacrosin in a cell-specific manner. Multiple SREBP2gc response elements were identified within the 5'-flanking and proximal promoter regions of the proacrosin promoter. Mutating these elements greatly diminished in vivo expression of this promoter in spermatogenic cells of transgenic mice. These studies define a totally new function for an SREBP as a transactivator of male germ cell-specific gene expression. We propose that SREBP2gc is part of a cadre of spermatogenic cell-enriched isoforms of ubiquitously expressed transcriptional coregulators that were specifically adapted in concert to direct differentiation of the male germ cell lineage.

Recently, we described the identification of a novel protein, nuclear receptor-associated protein 80 (RAP80), which is highly expressed in spermatocytes and appears to have a role in regulating gene expression. To identify proteins interacting with this protein, we performed yeast two-hybrid screening using full-length RAP80 as bait. This screen identified one in-frame clone encoding a novel testis-specific protein (Tsp), referred to as Tsp57. Tsp57 encodes a basic protein with a mass of 56.8 kDa. The amino acid sequence of Tsp57 is highly conserved (87%) between mouse and human. The mouse and human Tsp57 genes map to chromosomes 9A1 and 11q21, respectively. Northern blot analysis showed that the expression of Tsp57 mRNA was highly restricted to the testis and temporally regulated during testicular development. Tsp57 mRNA was greatly induced between Day 21 and Day 25 of postnatal testicular development. In situ hybridization analysis demonstrated that the hybridization signal for Tsp57 mRNA was strongest in sections of seminiferous tubules at stages VI-VIII of spermatogenesis, consistent with the conclusion that Tsp57 is most highly expressed in round spermatids. Study of Tsp57 expression in several purified subpopulations of spermatogenic cells confirmed maximum levels of expression in round spermatids. Consistently, Tsp57 expression was absent in testes from vitamin A-deficient mice, which do not have any round spermatids, and was reduced in RARalpha null mice, which have lowered numbers of round spermatids in their testes. These results indicate the possibility that Tsp57 protein plays a role in the postmeiotic phase of germ cell differentiation. Tsp57 contains two putative nuclear localization signals: NLS1 and NLS2. Examination of the cellular localization showed that the green fluorescent protein-Tsp57 fusion protein localized to both cytoplasm and nucleus. After deletion of NLS1 but not NLS2, Tsp57 localized solely to the cytoplasm, indicating a role for NLS1 in the nuclear localization of Tsp57. The localization suggests a nuclear function for Tsp57. Pull-down analysis demonstrated that Tsp57 and RAP80 form a complex in intact cells.

Most arterial mechanics studies have focused on excised non-coronary vessels, with few studies validating the application of ex-vivo results to in-vivo conditions. A method was developed for testing the mechanical properties of intact left anterior descending coronary arteries under a variety of conditions. Vascular deformation and pressure were simultaneously measured with intravascular ultrasound and a pressure transducer guidewire, respectively. Results suggest the importance of understanding in-vivo factors such as myocardial support, vascular tone and local pressure fluctuations when applying ex-vivo coronary characterization data. With further development, this method can more accurately characterize the true in-vivo constitutive behavior in normal and atherosclerotic coronaries.

Cholesterol biosynthesis in somatic cells is controlled at the transcriptional level by a homeostatic feedback pathway involving sterol regulatory element binding proteins (SREBPs). These basic helix-loop-helix (bHLH)-Zip proteins are synthesized as membrane-bound precursors, which are cleaved to form a soluble, transcriptionally active mature SREBP that regulates the promoters for genes involved in lipid synthesis. Homeostasis is conferred by sterol feedback inhibition of this maturation process. Previous work has demonstrated the expression of SREBP target genes in the male germ line, several of which are highly up-regulated during specific developmental stages. However, the role of SREBPs in the control of sterol regulatory element-containing promoters during spermatogenesis has been unclear. In particular, expression of several of these genes in male germ cells appears to be insensitive to sterols, contrary to SREBP-dependent gene regulation in somatic cells. Here, we have characterized a novel isoform of the transcription factor SREBP2, which is highly enriched in rat and mouse spermatogenic cells. This protein, SREBP2gc, is expressed in a stage-dependent fashion as a soluble, constitutively active transcription factor that is not subject to feedback control by sterols. These findings likely explain the apparent sterol-insensitive expression of lipid synthesis genes during spermatogenesis. Expression of a sterol-independent, constitutively active SREBP2gc in the male germ line may have arisen as a means to regulate SREBP target genes in specific developmental stages. This may reflect unique roles for cholesterol synthesis and other functional targets of SREBPs during spermatogenesis.

E2Fs play a central role in cell proliferation and growth arrest through their ability to regulate genes involved in cell cycle progression, arrest and apoptosis. Recent studies further indicate that this family of transcriptional regulators participate in cell fate/differentiation events. They are thus likely to have a prominent role in controlling the terminal differentiation process and its irreversibility. Here we have specifically examined the role of E2F2 in neuronal differentiation using a gain-of-function approach. Endogenous E2F2 increased in PC12 cells in response to nerve growth factor (NGF) and was also expressed in cerebellar granule neurons undergoing terminal differentiation. While PC12 cells normally undergo reversible dedifferentiation and cell cycle re-entry upon NGF removal, forced expression of E2F2 inhibited these events and induced apoptosis. Thus, E2F2 converted PC12-derived neurons from a reversible to a 'terminally' differentiated, NGF-dependent state, analogous to postmitotic sympathetic neurons. This contrasts with the effects of E2F4, which enhances the differentiation state of PC12 cells without affecting cell cycle parameters or survival. These results indicate that E2F2 may have a unique role in maintaining the postmitotic state of terminally differentiated neurons, and may participate in apoptosis in neurons attempting to re-enter the cell cycle. It may also be potentially useful in promoting the terminally arrested/differentiated state of tumor cells.

Spermatogenesis occurs in a series of well-defined stages and serves as an excellent model for lineage-specific cell development. Yet, little is known regarding the transcriptional mechanisms responsible for cell- and stage-dependent gene regulation in the male germ line. The rat and mouse proenkephalin genes are expressed from an alternative, spermatogenic cell-specific promoter specifically in meiotically-active pachytene spermatocytes and early post-meiotic spermatids. This promoter thus serves as an excellent model for defining transcriptional regulators involved in germ line-specific gene expression in meiotic cells. Previous transgenic studies identified a proximal, 51 bp 5'-flanking sequence containing two direct repeat elements that are absolutely required for in vivo proenkephalin promoter activity in spermatocytes and spermatids. Here, footprinting analyses were used to further delineate the specific interactions of a spermatogenic cell nuclear factor with the repeat elements within the proximal promoter region. This repeat-binding factor was also shown to be developmentally upregulated specifically in pachytene spermatocytes. Using Southwestern analysis, we have identified a unique nuclear protein enriched in pachytene spermatocytes that specifically recognizes the repeat elements within the proximal 5'-flanking sequence. We propose that this DNA binding factor, termed PACH1, is a key transcriptional regulator of the proenkephalin and potentially other gene promoters, uniquely expressed during meiosis in the male germ line.

E2F transcription factors play a critical role in cell cycle progression through the regulation of genes required for G(1)/S transition. They are also thought to be important for growth arrest; however, their potential role in the cell differentiation process has not been previously examined. Here, we demonstrate that E2F4 is highly upregulated following the neuronal differentiation of PC12 cells with nerve growth factor (NGF), while E2F1, E2F3, and E2F5 are downregulated. Immunoprecipitation and subcellular fractionation studies demonstrated that both the nuclear localization of E2F4 and its association with the Rb family member p130 increased following neuronal differentiation. The forced expression of E2F4 markedly enhanced the rate of PC12 cell differentiation induced by NGF and also greatly lowered the rate at which cells lost their neuronal phenotype following NGF removal. Importantly, this effect occurred in the absence of any significant change in the growth regulation of PC12 cells by NGF. Further, the downregulation of E2F4 expression with antisense oligodeoxynucleotides inhibited NGF-induced neurite outgrowth, indicating an important role for this factor during PC12 cell differentiation. Finally, E2F4 expression was found to increase dramatically in the developing rat cerebral cortex and cerebellum, as neuroblasts became postmitotic and initiated terminal differentiation. These findings demonstrate that, in addition to its effects on cell proliferation, E2F4 actively promotes the neuronal differentiation of PC12 cells as well as the retention of this state. Further, this effect is independent of alterations in cell growth and may involve interactions between E2F4 and the neuronal differentiation program itself. E2F4 may be an important participant in the terminal differentiation of neuroblasts.

The nerve growth factor receptor, TrkA, has a critical role in the survival, differentiation, and function of neurons in the peripheral and central nervous systems. Recent studies have demonstrated a strong correlation between abundant expression of TrkA and a favorable prognosis of the pediatric tumor, neuroblastoma. This correlation suggests that TrkA may actively promote growth arrest and differentiation of neuroblastoma tumor cells and may be an important therapeutic target in the treatment of this disease. In the present study, we have examined the mechanistic basis for TrkA gene expression in human neuroblastoma cells. Northern blotting and nuclear run-on analyses demonstrated that transcription is a primary determinant of both cell-specific and variable expression of the TrkA gene in neuroblastoma cell lines that express it to different degrees. Cell-specific and variable transcription in neuroblastoma cells was recapitulated by transient transfection of TrkA promoter-luciferase reporter constructs, and regulatory sequences mediating these processes were localized to a 138-base pair region lying just upstream of the transcription initiation region. This neuroblastoma regulatory region formed multiple DNA-protein complexes in gel shift assays that were highly enriched in neuroblastoma cells exhibiting abundant TrkA expression. Thus, TrkA-positive neuroblastoma cells are distinguished by differential expression of putative transcription factors that ultimately may serve as targets for up-regulating TrkA expression in tumors with poor prognosis.

In mammals, cytosine methylation is important for the regulation of gene expression and chromatin structure. Recently, we have found evidence indicating that the maintained DNA methyltransferase activity is critical for neuronal cell differentiation. In the present study, we have investigated the effect of the DNA methyltransferase inhibitor 5-azacytidine on gene regulation during nerve growth factor (NGF)-induced neuronal differentiation of PC12 cells. Expression of the helix-loop-helix proteins Id1, Id2 and Id3 was specifically reduced by NGF and this effect was blocked in 5-azacytidine-treated cells, concomitant with the inhibition of NGF-induced neuronal differentiation. Nuclear run-on and Id2 promoter analyses further demonstrated that the decreased transcription of Id proteins is at least in part dependent on the DNA methyltransferase activity. These findings indicate that Id proteins are downstream targets of the NGF transduction pathway. Moreover, these results suggest that therapeutic strategies using 5-azacytidine against certain types of tumors should be reconsidered because of the possible deleterious effects on neuronal cell function.

The Gli family of DNA binding proteins has been implicated in multiple neoplasias and developmental abnormalities, suggesting a primary involvement in cell development and differentiation. However, to date their specific roles and mechanisms of action remain obscure, and a drawback has been the lack of a model system in which to study their normal function. Here we demonstrate that Gli family members are differentially expressed during spermatogenesis in mice. Specifically, Gli and Gli3 mRNAs were detected in mouse germ cells, while Gli2 was not. Further, both Gli and Gli3 exhibited stage-dependent patterns of expression selectively in type A and B spermatogonia. Gli expression was somewhat higher in type B spermatogonia while the abundance of Gli3 transcripts was similar in type A and B cells. Gel-shift analyses also demonstrated the enrichment of DNA binding activity specific for the Gli target sequence in spermatogonial cells. These results indicate a selective role for Gli and Gli3 during mitotic stages of male germ cell development. Spermatogenesis may thus provide a unique opportunity to identify downstream targets and explore the normal function of Gli family proteins.

Trinucleotide repeat sequences have become of great interest due to their association with specific genetic disorders. Here we report the identification of a cDNA containing opa trinucleotide repeats from mouse testis, termed t-OPA. The opa repeat is contained within the longest open reading frame within the cDNA. Northern analysis demonstrated that four distinct t-OPA transcripts (1.6, 2.5, 3.6, 4.0 kilobases) are preferentially expressed in mouse and rat testis, with low expression in the pituitary, brain, and adrenal gland. Further, t-OPA RNAs were highly abundant in both pachytene spermatocytes and round spermatids and decreased in cytoplasts. Polysome profile analysis indicated that t-OPA mRNAs are translated in mouse testis with efficiencies similar to other transcripts expressed in late meiotic/early post-meiotic spermatogenic cells. These findings thus suggest a role for cell-specific mRNAs containing opa repeats during mouse spermatogenesis.

The developmental program controlling sperm formation occurs in multiple stages that sequentially involve mitosis, meiosis, and spermiogenesis. The transcriptional mechanisms regulating these distinct phases are poorly understood. In particular, while a required role for the germ cell transcription factor cyclic AMP response element modulator-tau during spermiogenesis has recently been demonstrated, the transcriptional mechanisms leading to early haploid cell formation are unknown. The rat and mouse proenkephalin genes are selectively expressed from an alternate, germ cell-specific promoter in meiotic and early haploid cells. In this study, the minimal rat proenkephalin germ line promoter was localized to a 116-bp region encompassing the transcriptional start site region. Further, a proximal 51-bp sequence located in the 5'-flanking region is absolutely required for germ line promoter activity. This 51 bp sequence corresponds to a previously characterized binding element (GCP1) that forms cell-specific complexes with rat spermatogenic cell nuclear factors distinct from cyclic AMP response element binding proteins. Further, GCP1 contains novel direct repeat sequences required for factor binding and transgene expression in spermatogenic cells. These repeat elements are highly similar to sequences within the active regions of other male germ line promoters expressed during meiosis. GCP1 may therefore contain transcriptional elements that participate more generally during meiosis in the differentiation of spermatocytes and early haploid spermatids.

Cell differentiation in the nervous system is dictated by specific patterns of gene expression. We have investigated the role of gene methylation during differentiation of PC12 pheochromocytoma cells in response to nerve growth factor (NGF). Here we present evidence that NGF-induced neuronal differentiation is dependent on gene methylation and that this process is not associated with inhibition of cell cycle arrest. The DNA methylation inhibitor 5-azacytidine is able to block the neurite outgrowth of NGF-treated PC12 cells. Inhibition of neuronal differentiation is accompanied by significant changes in the protein and mRNA expression pattern of the high-affinity NGF receptor (trkA). These studies reveal a new growth factor receptor-mediated mechanism of cellular differentiation dependent on gene methylation. The results indicate that DNA methyltransferase is necessary for the initiation phase of NGF-induced neurite formation in PC12 cells and has a role in growth factor-dependent cellular responses distinct from cell proliferation.

TATA-binding protein (TBP) and its associated factors are required for transcriptional initiation by all three RNA polymerases, and evidence for regulation of their activities during early development has been recently reported. In the present study, we have investigated the regulation of TBP gene expression during male germ cell development. TBP mRNA was found to be increased more than 40-fold in rat and mouse testis and spermatogenic cells relative to somatic tissues. This up-regulation was stage-dependent, occurring specifically in meiotic and postmeiotic cells. Nuclear run-on analysis further demonstrated that transcription of the TBP gene was markedly elevated in the adult mouse testis relative to somatic tissue and prepuberal testis, indicating that transcriptional induction accounts, in large part, for the increased abundance of TBP mRNA in spermatogenic cells. In contrast to TBP mRNA, levels of TBP protein were elevated only 2.5-fold in mouse spermatogenic cells relative to somatic tissues. Polysome gradient analysis suggested that translational repression is an important determining factor in the unexpectedly low ratio of TBP protein-mRNA in male germ cells. These findings raise the possibility that transcriptional induction of TBP during spermatogenesis reflects a cell-specific homeostatic mechanism that maintains TBP protein concentrations at sufficiently high levels throughout male germ cell development.

We are employing recent advances in the understanding of the cell cycle to study the inverse relationship between proliferation and neuronal differentiation. Nerve growth factor and aphidicolin, an inhibitor of DNA polymerases, synergistically induce neuronal differentiation of SH-SY5Y neuroblastoma cells and the expression of p21WAF1, an inhibitor of cyclin-dependent kinases. The differentiated cells continue to express p21WAF1, even after removal of aphidicolin from the culture medium. The p21WAF1 protein coimmunoprecipitates with cyclin E and inhibits cyclin E-associated protein kinase activity. Each of three antisense oligonucleotides complementary to p21WAF1 mRNA partially blocks expression of p21WAF1 and promotes programmed cell death. These data indicate that p21WAF1 expression is required for survival of these differentiating neuroblastoma cells. Thus, the problem of neuronal differentiation can now be understood in the context of negative regulators of the cell cycle.

Gene expression during spermatogenesis is highly cell- and stage-specific and involves the complex interplay of multiple developmentally regulated transcription factors. Recent evidence suggests that the DNA-binding protein Sp1 functions as an important trans-activator during cell development and differentiation. In the present study, the developmental expression of Sp1 was characterized during mouse spermatogenesis. Three distinct Sp1 transcripts were detected in mouse spermatogenic cells, each with a distinct developmental pattern; an 8.2-kilobase (kb) messenger RNA (mRNA) identical in size to the somatic mRNA expressed in spermatogonial cells, a larger mRNA approximately 8.8 kb in size present in meiotic cells, and a 2.4 kb mRNA in meiotic and postmeiotic germ cells. The 8.8- and 2.4-kb Sp1 transcripts were not observed in somatic cells and, thus, are male germ cell specific. Northern, ribonuclease protection, and RT-PCR assays revealed that the 2.4-kb Sp1 transcript is truncated in both the 5'- and 3'-untranslated regions relative to the somatic mRNA and lacks a short segment of the N-terminal coding region. Polysome analysis further indicated that these germ cell-specific Sp1 mRNAs are translated, albeit with a lower efficiency than the somatic transcript. Consistent with these results, spermatogenic cells were shown to contain approximately 9-fold lower concentrations of Sp1 proteins that are approximately the same size as the somatic form. Of particular interest, the apparent affinity of Sp1 DNA-binding activity in nuclear extracts from mouse germ cells was 5-fold greater than that in extracts from mouse somatic tissues. This may reflect the existence of mechanisms within mouse spermatogenic cells that compensate for the lower nuclear concentrations of Sp1 protein. These results suggest that cell- and stage-specific regulation of Sp1 gene expression and activity may be an important component of the mouse spermatogenic cell developmental program.

Protein-protein interactions involving specific transactivation domains play a central role in gene transcription and its regulation. The promoter-specific transcription factor Sp1 contains two glutamine-rich transcriptional activation domains (A and B) that mediate direct interactions with the transcription factor TFIID complex associated with RNA polymerase II and synergistic effects involving multiple Sp1 molecules. In the present study, we report the complementary DNA sequence for an alternatively spliced form of mouse Sp1 (mSp1-S) that lacks one of the two glutamine-rich activation regions present in the full-length protein. Corresponding transcripts were identified in mouse tissues and cell lines, and an Sp1-related protein identical in size to that predicted for mSp1-S was detected in mouse nuclear extracts. Cotransfection analysis revealed that mSp1-S lacks appreciable activity at promoters containing a single Sp1 response element but is active when multiple Sp1 sites are present, suggesting synergistic interactions between multiple mSp1-S molecules. The absence of a single glutamine-rich domain does not fully explain the properties of the smaller protein and indicates that additional structural features account for its unique transcriptional activity. The functional implications of this alternatively spliced form of Sp1 are discussed.

Two distinct forms of proenkephalin messenger RNA (mRNA) are present in the murine testis, a family of 1.7 kilobases (kb), germ cell-specific transcripts and a 1.45-kb form that is also found in somatic tissues. In situ hybridization and molecular analysis of purified spermatogenic cell types were used to characterize the cellular localization of these different transcripts during development of the mouse testis. Both forms of proenkephalin mRNA were observed in isolated germ cells by RNA gel-blot analysis, but in distinct developmental patterns; the 1.7-kb transcripts were present in cells undergoing meiosis and spermiogenesis, whereas the 1.45-kb mRNA was detected primarily in type B spermatogonia. In contrast, in situ hybridization analysis did not detect significant amounts of the 1.45-kb transcript in any spermatogenic cell type. Using transcript-specific probes, distinct patterns of developmental expression were evident for the two mRNAs. The 1.45-kb transcript was the only form detected in the prepubertal testis, where it was localized mainly in interstitial cells. In contrast, the 1.7-kb transcripts were the major mRNAs observed in the adult testis and were localized to spermatogenic cells. A transition from the prepubertal to the adult pattern occurred on or about postnatal day 21, when proenkephalin-expressing pachytene spermatocytes begin to populate the seminiferous tubules. In situ hybridization analysis further demonstrated that proenkephalin gene expression in mutant (at/at) mice, which lack germ cells, was identical to that observed in the early prepubertal testis. These results suggest that the 1.45-kb proenkephalin mRNA is developmentally down-regulated in mouse interstitial cells and that this process requires ongoing spermatogenesis.

Rat and mouse spermatogenic cells contain a family of 1700-nucleotide (nt) proenkephalin mRNAs that are generated from an alternate, germ cell-specific promoter. This promoter is located approximately 350 base pairs (bp) downstream of the promoter used in somatic cells, within the first intron for the somatic transcript. In a previous study, rat proenkephalin-chloramphenicol acetyltransferase fusion genes containing both promoters were shown to be transcribed selectively from the germ cell promoter and in the correct developmental pattern in spermatogenic cells of transgenic mice. In the present study it was found that spermatogenic cell-specific transgene expression was maintained after deletion of the upstream somatic promoter. This result establishes that the rat proenkephalin germ-line promoter is capable of functioning independently of transcriptional elements associated with the somatic promoter and localizes the requisite spermatogenic cell cis-elements to a 500-bp region encompassing the germ cell initiation sequences. A comprehensive analysis of binding sites for rat spermatogenic cell nuclear factors within this 500-bp region was performed using gel-shift and DNAse I footprinting techniques. Eight distinct binding regions were identified, each of which formed one or more cell-specific complexes with nuclear proteins from rat spermatogenic cells. These results suggest that multiple cis-acting elements may cooperate in the cell-specific and developmental regulation of rat proenkephalin gene transcription during spermatogenesis.

The rat and mouse proenkephalin genes each contains two distinct promoters, one of which is utilized exclusively by spermatogenic cells. The germ cell-specific promoter lacks TATA sequences, is G+C rich, and contains multiple initiation sites. To investigate the nature of the cis-acting elements that determine selective transcription of the proenkephalin gene in male germ cells, two rat proenkephalin-chloramphenicol acetyltransferase fusion genes containing the two different promoter regions as well as 1.6 or 0.3 kilobases, respectively, of 5'-flanking sequence were expressed in transgenic mice. Multiple transgenic lines were developed which expressed the fusion genes in testis, brain, and heart but not in tissues that do not normally express the proenkephalin gene. Fusion gene transcripts in transgenic mouse testes were localized to those spermatogenic cell types that utilize the spermatogenic cell promoter and were selectively and accurately initiated from the multiple rat germ cell start sites. Transgenic mice thus provide a useful model for the localization and characterization of cis-acting elements mediating transcription of the proenkephalin gene from its germ cell-specific promoter.

Previous studies have shown that spermatogenic cells are a major source of testicular RNA encoding the opioid peptide precursor proenkephalin, suggesting that proenkephalin-derived peptides may function as intratesticular paracrine factors produced by male germ cells. However, direct evidence for the production of proenkephalin by spermatogenic cells has been lacking. In this report, we have used polysome profile analysis, peptide quantitation, and immunocytochemistry to show that proenkephalin products are synthesized during spermatogenesis and are retained within spermatozoa of humans, hamsters, rats, and sheep. We further show that these peptides are stored in the sperm acrosome and are depleted from sperm following the acrosome reaction, an exocytotic event required for fertilization. Proenkephalin products thus may serve a dual function as sperm acrosomal factors released during the fertilization process as well as intratesticular regulators secreted by spermatogenic cells.

During spermatogenesis, several genes are expressed in a germ cell-specific manner. Previous studies have demonstrated that rat and mouse spermatogenic cells produce a 1,700-nucleotide proenkephalin RNA, while somatic cells that express the proenkephalin gene contain a 1,450-nucleotide transcript. Using cDNA cloning, RNA protection, and primer extension analyses, we showed that transcription of the rat and mouse spermatogenic-cell RNAs is initiated downstream from the proenkephalin somatic promoter in the first somatic intron (intron As). In both species, the germ cell cap site region consists of multiple start sites distributed over a length of approximately 30 base pairs. Within rat and mouse intron As, the region upstream of the germ cell cap sites is GC rich and lacks TATA sequences. A consensus binding site for the transcription factor SP1 was identified in intron As downstream of the proenkephalin germ cell cap site region. These features are characteristic of several previously described promoters that lack TATA sequences. Homologies were also identified between the proenkephalin and rat cytochrome c spermatogenic-cell promoters, including the absence of a TATA box, a multiple start site region, and several common sequences. This promoter motif thus may be shared with other genes expressed in male germ cells.

The opioid peptides have been implicated as potential regulators of cell development in nervous and reproductive tissues. A survey of proenkephalin gene expression during rat development showed that the mRNA for this opioid precursor is present at substantial concentrations in several developing tissues (kidney, liver, skin, skeletal muscle, and lung) that have essentially undetectable levels in adults. In neonatal rats, skeletal muscle has greater concentrations of this transcript than brain. Polysomal analysis further demonstrated that proenkephalin mRNA is actively translated in skeletal muscle from newborn rats. These results raise the possibility that proenkephalin and its products perform a general regulatory role in cell proliferation or differentiation.

The mammalian proenkephalin-derived peptides [Leu]enkephalin, [Met]enkephalin, and [Met]enkephalin-Arg6-Phe7 were identified in acid extracts of the brain of Bufo marinus by using reversed-phase HPLC and specific radioimmunoassays. [Met]Enkephalin was the predominant opioid peptide present (270 pmol/g). In contrast, the octapeptide [Met]enkephalin-Arg6-Gly7-Leu8, which is also derived from mammalian proenkephalin, was not detected. The ratio of free [Met]enkephalin to [Met]enkephalin-Arg6-Phe7 was found to be 3.5 to 1, which is similar to that observed in mammalian proenkephalin-containing tissues. Together these data (i) indicate that amphibian brain contains a proenkephalin related to the mammalian precursor and (ii) establish the existence of enkephalins and proenkephalin-derived enkephalin-containing peptides in a submammalian species.

The functional stability of primary cultures of adrenal medulla cells was investigated. Isolated cells were prepared by treatment of bovine adrenal glands with collagenase followed by purification on Percoll density gradients and were maintained in Dulbecco's medium containing 10% fetal calf serum. Within 12 h after plating on plastic culture dishes, the cells became firmly attached and exhibited good survival for periods of time up to 3 weeks, as indicated by their morphology using light and electron microscopy, by maintenance of their content of catecholamines, tyrosine hydroxylase, dopamine-beta-hydroxylase, and phenylethanolamine N-methyltransferase, and their ability to respond to secretagogues. During the first 10 days to 2 weeks in culture there was little or no change in any of these parameters. During the 3rd week there were progressive losses of catecholamine and enzyme activities and increased vacuolization of medullary cells. The cells synthesized protein and RNA with no apparent loss in activities over the period studied, but did not incorporate [3H]thymidine into PCA-precipitable material. The cells responded to secretagogues and secretory antagonists similarly to isolated perfused adrenal glands. The studies described here demonstrate that primary cultures of adrenal medulla cells provide an excellent experimental system for obtaining more detailed information on stimulus-secretion coupling and other functional aspects of the adrenal medulla.

Cultured bovine adrenal medulla cells have been shown to contain several different ion channels (Na+, Ca2+, acetylcholine receptor regulated) whose activation leads to the secretion of catecholamines. The pharmacology of these ion channels and their interactions during secretion have been examined. The mechanisms of agonist-induced calcium influx are of particular interest since this is an early obligatory event during secretion from the adrenal medulla. Data obtained on catecholamine release and 45Ca2+ uptake indicate that both voltage-dependent and voltage-independent calcium influx mechanisms operate in cultured bovine adrenal medulla cells. The significance of these results in understanding the mechanism of action of the physiological stimulus acetylcholine (Ach) will be discussed. The alkaloid channel neurotoxins D-600, batrachotoxin, veratridine, and aconitine were shown to exert a noncompetitive inhibitory effect on Ach-induced ion flux in adrenal medulla cells, presumably through an interaction with the nicotinic receptor regulated channel. Lipid-soluble neurotoxins may interact with multiple ion channels in nerve and muscle membrane.

The tridecapeptide NH2-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Gln-Phe-Lys-Val-Val-Thr-COOH has been purified from extracts of bovine posterior pituitary glands. This unique peptide, which has been given the name "rimorphin," is a major [Leu]enkephalin-containing peptide in all tissues examined that contain dynorphin and alpha-neo-endorphin. However, except for the initial hexapeptide sequence, it is structurally unrelated to the other two peptides.

The effect of denervation on the expression of rat adrenal proenkephalin has been examined. Following splanchnicectomy there was a several-fold increase in the steady-state levels of preproenkephalin mRNA, which became maximal after 24-48 hr (greater than 10-fold). These results indicate that the previously observed increase in rat adrenal enkephalin-containing peptides following denervation occurs entirely by a pretranslational mechanism. The increase in preproenkephalin mRNA was accompanied by a 50-75% decrease in rat adrenal poly(A)+ RNA. Neural input thus exerts a profound trophic influence on proenkephalin gene expression and RNA metabolism in rat adrenals.

The role of Na+ channels and membrane potential in stimulus secretion coupling in adrenal medulla cell cultures was investigated. Veratridine, aconitine, batrachotoxin (BTX), and scorpion venom, which increase the flux of ions through tetrodotoxin(TTX)-sensitive Na+ channels, all evoke secretion of catecholamines that is blocked by TTX. TTX partially inhibits secretion induced by low concentrations of nicotine in Locke's solution but has no effect on high concentrations of nicotine (20 microM). In Ca2+-sucrose media TTX has no effect on secretion at either high or low concentrations of nicotine. Replacement of Na+ with Li+ in Locke's solution reduces the response to nicotine and to veratridine. Complete replacement of Na+ with hydrazine, diethanolamine, TRIS, and choline completely inhibits the response to nicotine and almost completely inhibits the response to veratridine. Following exposure of cells to 50 mM-100 mM-K+, nicotine does not stimulate catecholamine secretion unless the cells are resuspended in media containing less than 50 mM-K+. Neither dibutyryl-cyclic AMP nor dibutyryl-cyclic GMP evokes secretion. alpha-Bungarotoxin (1 microM) did not inhibit nicotine-induced secretion. These studies indicate that Na+ channels and acetylcholine (ACh) receptor ion channels are independently coupled to the influx of Ca2+. The membrane potential appears to affect nicotine- and veratridine-evoked secretion.