[Effect of wrist arthroscopy with intraarticular hyaluronan substitution therapy: a randomised, controlled, prospective, non-blinded, single-centre, comparative trial]. / Auswirkungen einer intraartikulären Substitutionstherapie mit Hyaluronsäure im Rahmen handgelenksarthroskopischer Operationen. Eine randomisierte, kontrollierte, prospektive, unverblindete, monozentrische Vergleichsstudie.

PURPOSE: The present study investigates the effect of an intra- and postoperative intraarticular hyaluronan injection (HS) in patients undergoing wrist arthroscopy. PATIENTS AND METHODS: A total of 140 adults were included and prospectively randomised to one of 2 treatment groups. All patients presented wrist pain resistant to non-operative therapy. 69 patients were assigned to therapeutic wrist arthroscopy without additional treatment (A-group), another 70 patients were assigned to wrist arthroscopy and additional intraarticular instillation of a 1% HS solution (HS-group). The HS administration (2 mL of 1% HS solution each) was performed directly at the end of arthroscopic procedure and a second time 3 weeks after surgery. For outcome assessment, Mayo wrist score (modified according to Krimmer, MMWS), DASH questionnaire, absolute grip strength, VAS pain (visual analogue scale) and clinical global impression (CGI) of patients and investigators were used. The follow-up was 6 months. Furthermore, the correlation between severity of pathological findings and level of postoperative benefit was investigated. RESULTS: In both groups, pain decreased and the function of the wrist joint improved. While pa-tients with additional HS injection had significantly better values in MMWS than patients without additional HS injection, no significant differences could be observed for DASH score, absolute grip strength and pain intensity. 12 and 24 weeks after surgery, therapeutic success was rated better in HS-group than in A-group. The highest clinical benefit was obtained for patients in the HS-group with marginal to moderate pathological findings. CONCLUSION: The benefit of therapeutic wrist arthroscopy can be significantly improved by a 2-time intraarticular substitution of hyaluronan.

Multitasking with neurotensin in the central nervous system.

The 13-amino acid peptide neurotensin (NT) was discovered over 30 years ago and has been implicated in a wide variety of neurotransmitter and endocrine functions. This review focuses on four areas where there has been substantial recent progress in understanding NT signaling and several functions of the endogenous peptide. The first area concerns the functional activation of the high-affinity NT receptor, NTR-1, including the delineation of the NT binding pocket and receptor domains involved in functional coupling to intracellular signaling pathways. The development of NT receptor antagonists and the application of genetic and molecular genetic approaches have accelerated progress in understanding NT function in several areas, including the involvement of NT in antipsychotic drug actions, psychostimulant sensitization and the modulation of pain, and these are reviewed in that order. There is now substantial evidence indicating that NT is required for certain antipsychotic drug actions and that the peptide plays a key role in stress-induced analgesia.

Endogenous neurotensin facilitates visceral nociception and is required for stress-induced antinociception in mice and rats.

Central neurotensin (NT) administration can both facilitate and inhibit somatic and visceral nociception, depending on the dose and administration site. NT microinjection in the rostroventral medulla facilitates nociception at low doses, while NT antagonist microinjection can markedly attenuate nociception, supporting the hypothesis that endogenous NT facilitates nociception. However, higher doses of NT produce a mu-opioid receptor-independent analgesia, similar to that resulting from various intense stressors. Furthermore, intense stress results in increased NT expression in several hypothalamic nuclei that have been implicated in stress-induced antinociception (SIAN); however, there is little direct evidence that endogenous NT is required for SIAN. We have investigated the role of endogenous NT in both basal visceral nociception and SIAN using both NT knockout mice and pharmacological approaches in rats. Visceral nociception was monitored by measuring visceromotor responses during colorectal distension both prior to and following water avoidance stress. Visceral nociception was significantly attenuated in both NT knockout mice and rats pre-treated with the NT antagonist SR 48692. Disruption of NT signaling also blocked SIAN, revealing a novel stress-induced hyperalgesic response that was significantly greater in female than in male rats. NT was also required for acetic acid-induced hyperalgesia. These results indicate that endogenous NT normally facilitates visceral pain responses, is required for irritant-induced hyperalgesia, and plays a critical role in SIAN.

Endogenous neurotensin facilitates enterohepatic bile acid circulation by enhancing intestinal uptake in rats.

Initial studies on the digestive hormone neurotensin (NT) showing that intestinal NT mRNA expression and blood levels were altered in rats fed chow containing bile acid (BA) and the BA chelator cholestyramine led us to investigate the role of NT in the enterohepatic circulation of BA. In fasted, anesthetized rats with common bile ducts cannulated for bile collection, intravenous NT infusion (10 pmol. kg(-1). min(-1)) enhanced BA output relative to control over 3 h in animals administered donor bile into the duodenum (30 microl/min). This suggested that the effect of NT was on the return of BA from the intestine to the liver, which is rate determining in the normal process. In rats prepared as described above and administered [(3)H]taurocholate ([(3)H]TC; 5 mM, 1 ml) duodenally, NT infusion (3-10 pmol x kg(-1) x min(-1)) increased the [(3)H]TC recovery rate in bile approximately twofold, whereas sulfated CCK-8 (12-50 pmol x kg(-1) x min(-1)) had no effect. To investigate the roles of endogenous NT and CCK, we administered [(3)H]TC into the rat duodenum or lower jejunum and tested the effect of the NT antagonist SR-48692 (2 nmol x kg(-1) x min(-1)) or CCK-A antagonist lorglumide (100 nmol x kg(-1) x min(-1)). SR-48692 reduced the [(3)H]TC recovery rate by congruent with 50% and congruent with 24% in the duodenum and jejunum, respectively, whereas lorglumide had no effect. These results suggest that NT or a similar peptide is an endogenous regulator of enterohepatic BA cycling, which acts by enhancing BA uptake in the intestine.

Neurotensin-deficient mice show altered responses to antipsychotic drugs.

The peptide transmitter neurotensin (NT) exerts diverse neurochemical effects that resemble those seen after acute administration of antipsychotic drugs (APDs). These drugs also induce NT expression in the striatum; this and other convergent findings have led to the suggestion that NT may mediate some APD effects. Here, we demonstrate that the ability of the typical APD haloperidol to induce Fos expression in the dorsolateral striatum is markedly attenuated in NT-null mutant mice. The induction of Fos and NT in the dorsolateral striatum in response to typical, but not atypical, APDs has led to the hypothesis that the increased expression of these proteins is mechanistically related to the production of extrapyramidal side effects (EPS). However, we found that catalepsy, which is thought to reflect the EPS of typical APDs, is unaffected in NT-null mutant mice, suggesting that NT does not contribute to the generation of EPS. We conclude that NT is required for haloperidol-elicited activation of a specific population of striatal neurons but not haloperidol-induced catalepsy. These results are consistent with the hypothesis that endogenous NT mediates a specific subset of APD actions.

The neurotensin antagonist SR 48692 attenuates haloperidol-induced striatal Fos expression in the rat.

Neurotensin interacts with central dopamine systems and has been suggested to exert antipsychotic drug-like actions. Antipsychotic drugs such as haloperidol induce striatal immediate-early gene expression. In order to study neurotensin's role in antipsychotic drug actions, rats were pretreated with the neurotensin antagonist SR 48692 and then injected with haloperidol. SR 48692 dose-dependently decreased haloperidol-elicited immediate-early gene expression in the dorsolateral and central striatum but not other striatal areas. SR 48692 reduced Fos expression in the striatal patch (striosome) and matrix compartments, with a significantly greater effect in the patch. These data suggest that neurotensin may play a role in the actions of haloperidol. In view of proposed functional roles of the striatal patch and matrix, we suggest that neurotensin may be important in the therapeutic rather than side effects of antipsychotic drugs.

Stimulation of cellular sphingomyelin import by the chemokine connective tissue-activating peptide III.

The selective import of phospholipids into cells could be mediated by proteins secreted from the cells into the extracellular compartment. We observed that the supernatants obtained from suspensions of thrombin-activated platelets stimulated the exchange of pyrene (py)-labeled sphingomyelin between lipid vesicles in vitro. The proteins with sphingomyelin transfer activity were purified and identified as the chemokine connective tissue-activating peptide III (CTAP-III) and platelet basic protein. Isolated CTAP-III stimulated the exchange of py-sphingomyelin between lipid vesicles but did not affect the translocations of py-labeled phosphatidylcholine and phosphatidylethanolamine. CTAP-III rapidly increased the transfer of py-sphingomyelin from low density lipoproteins into peripheral blood lymphocytes, other immune cells, and fibroblasts. In the presence of heparin, CTAP-III was unable to insert sphingomyelin into the peripheral blood lymphocytes. The activation energy of the py-sphingomyelin transfer suggested that the translocation proceeded entirely in a hydrophobic environment. [(3)H]Sphingomyelin transferred to the cells by CTAP-III was hydrolyzed to [(3)H]ceramide and [(3)H]sphingosine after activation with tumor necrosis factor alpha. The generation of the [(3)H]sphingolipid messengers was catalyzed by acid sphingomyelinase. Our results identify CTAP-III as the first mediator of the selective (endocytosis-independent) cellular import of sphingomyelin allowing the paracrine modulation of the sphingolipid signaling.

Sequences required for induction of neurotensin receptor gene expression during neuronal differentiation of N1E-115 neuroblastoma cells.

The promoter region of the mouse high affinity neurotensin receptor (Ntr-1) gene was characterized, and sequences required for expression in neuroblastoma cell lines that express high affinity NT-binding sites were characterized. Me(2)SO-induced neuronal differentiation of N1E-115 neuroblastoma cells increased both the expression of the endogenous Ntr-1 gene and reporter genes driven by NTR-1 promoter sequences by 3-4-fold. Deletion analysis revealed that an 83-base pair promoter region containing the transcriptional start site is required for Me(2)SO activation. Detailed mutational analysis of this region revealed that a CACCC box and the central region of a large GC-rich palindrome are the crucial cis-regulatory elements required for Me(2)SO induction. The CACCC box is bound by at least one factor that is induced upon Me(2)SO treatment of N1E-115 cells. The Me(2)SO effect was found to be both selective and cell type-restricted. Basal expression in the neuroblastoma cell lines required a distinct set of sequences, including an Sp1-like sequence, and a sequence resembling an NGFI-A-binding site; however, a more distal 5' sequence was found to repress basal activity in N1E-115 cells. These results provide evidence that Ntr-1 gene regulation involves both positive and negative regulatory elements located in the 5'-flanking region and that Ntr-1 gene activation involves the coordinate activation or induction of several factors, including a CACCC box binding complex.

Platelet high affinity low density lipoprotein binding and import of lipoprotein derived phospholipids.

The binding of low density lipoprotein (LDL) to the platelet cell membrane could facilitate the transfer of phospholipids from LDL to the platelets. A polyclonal antibody against the platelet glycoproteins IIb/IIIa inhibited the high affinity binding of 125I-LDL by up to 80%. The transfer of pyrene (py)-labeled sphingomyelin (SM), phosphatidylcholine and phosphatidylethanolamine from LDL to the platelets was unaffected by the antibody. The lectin wheat germ agglutinin (WGA) reduced the binding of 125I-LDL to the platelets by approximately 80%. In contrast, the lectin stimulated the transfer of SM from LDL into the platelets by about three-fold. WGA also specifically augmented the transfer of py-SM between lipid vesicles and the platelets, the stimulation being abolished in the presence of N-acetylglucosamine. Dextran sulfate (DS) increased the specific binding of 125I-LDL to the platelets by up to 2.8-fold. On the other hand, the import of LDL-derived py-phospholipids was unaffected by DS. Together, the results indicate that the phospholipid transfer from LDL to the platelets is independent of the high affinity LDL binding to the platelets and is specifically stimulated by WGA. Thus, the interactions of platelets with LDL phospholipids differ markedly from those with the apoprotein components of the lipoproteins.

Low-density lipoproteins supply phospholipid-bound arachidonic acid for platelet eicosanoid production.

After the rapid extracorporal reduction of plasma low-density lipoprotein (LDL) by LDL apheresis, the percentages of arachidonic acid (AA)-containing species of phosphatidylcholine (PC) were lowered in the plasma of patients with hypercholesterolemia. The same PC species with AA were also decreased in the patient's platelets. Thus the supply of phospholipid-bound AA from LDL to the platelets was probably diminished after the apheresis. We therefore analyzed the concentration dependence of the transfer of phospholipid-bound AA from LDL to the platelets under in vitro conditions. The amount of [14C]AA-PC transferred to platelets strongly increased upon elevation of LDL from 0.1 to 1 mg protein/ml, with a less marked elevation being noted at higher LDL concentrations. After stimulation with thrombin (0.5 U/ml), 7.1% ([14C]AA-PC) and 10.6% ([14C]AA-phosphatidylethanolamine) of the 14C transferred from LDL to the platelets were recovered in the eicosanoids [14C]thromboxane B2 (TxB2) plus 12-[14C]hydroxyeicosatetraenoic acid. Experimental increases and reductions of the [14C]AA-PC import were associated with comparable modifications in the [14C]TxB2 production of the platelets. Accordingly, the import of phospholipid-bound [14C]AA is a necessary prerequisite for the formation of 14C-labeled eicosanoids. In summary, the transfer of phospholipids from LDL to the platelets markedly varies within the physiological range of lipoprotein concentrations. LDL contributes to platelet eicosanoid formation by supplying platelets with phospholipid-bound AA.

Platelet agonists enhance the import of phosphatidylethanolamine into human platelets.

It is unknown whether the endocytosis-independent transfer of phospholipids from lipoproteins to platelets is regulated by platelet agonists such as thrombin. The movements of the choline phospholipids phosphatidylcholine and sphingomyelin (labeled with either 14C or the fluorescent pyrenedecanoic acid) between low density lipoproteins and platelets were unaffected by thrombin (0.5 unit/ml). In contrast, thrombin accelerated the import of diacyl phosphatidylethanolamine (PE) and alkenylacyl phosphatidylethanolamine into platelets by about 4-fold. Similarly, thrombin receptor-activating peptide (15 microM), collagen (10 microgram/ml), and ADP (10 microM) enhanced PE uptake. High density lipoprotein particles and egg phosphatidylcholine vesicles were also donors for stimulation of platelet PE import. Part of the [14C]arachidonic acid-labeled PE transferred from low density lipoprotein to platelets activated by thrombin and collagen was metabolized to 14C-eicosanoids. Inhibitors of protein kinase C partially prevented thrombin-induced [14C]PE uptake, while direct activators of protein kinase C increased incorporation of [14C]PE into platelets. Proteinaceous factor(s) recovered in the extracellular medium from ADP- and thrombin-activated platelet suspensions were found to accelerate the transfer of pyrenedecanoic acid-labeled PE between donor and acceptor lipid vesicles. The stimulation of import of ethanolamine phospholipids led to a 2-fold enhancement of the prothrombinase activity of thrombin-activated platelets. Our study demonstrates that physiological platelet stimuli increase specifically the transfer of ethanolamine phospholipids from lipoproteins to platelets through a secretion-dependent mechanism. This might contribute to the increase of procoagulant activity of stimulated platelets.

Usefulness of Mycobacterium tuberculosis genomic mutations in the genes katG and inhA for the prediction of isoniazid resistance.

SETTING: Mutations in two genes of Mycobacterium tuberculosis, inhA and katG, are known to correlate with resistance to isoniazid (INH). OBJECTIVE: To determine which mutation or mutations are the most predictive for INH resistance and the most frequent ones in such isolates. Further, to propose a simple and generally applicable method for their detection. DESIGN: Codons 94 and 95 in the inhA gene and codons 315 and 463 in the katG gene were characterized in 50 INH-resistant and 12 INH-sensitive isolates from Germany and Sierra Leone. RESULTS: Mutations in codon 315 of the katG gene were detected in 27 of the INH-resistant and none of the INH-sensitive isolates. All mutations in this codon altered an AciI restriction enzyme site. No mutations were found in the investigated codons of the inhA gene. CONCLUSION: We propose that most INH resistances can be rapidly predicted by a simple AciI restriction enzyme digest of a polymerase chain reaction (PCR)-amplified katG fragment.

Disequilibria in the distribution of rpoB alleles in rifampicin-resistant M. tuberculosis isolates from Germany and Sierra Leone.

The effectiveness of culture-independent resistance predictions by molecular techniques is dependent on the number and the frequency of accessible resistance-associated genomic mutations. We have characterized an rpoB gene region involved in rifampicin resistance in 49 Mycobacterium tuberculosis isolates resistant to rifampicin from Germany and Sierra Leone. The determined frequencies of mutations differed between both countries of origins as well as with respect to previously reported distributions of resistance mutations. It is concluded that at least for some isolates the acquisition of mutations leading to rifampicin resistance in clinical samples of M. tuberculosis is a non-random process which may lead to a geographical and temporal dependence of the sensitivities of molecular typing techniques for rifampicin resistance predictions.

Neurotensin receptor expression in prostate cancer cell line and growth effect of NT at physiological concentrations.

BACKGROUND: Neurotensin (NT), a neuroendocrine peptide, exerts trophic effects in vivo and stimulates growth of some tumor cells in vitro. Androgen-sensitive prostate cells derived from lymph node carcinoma of the prostate (LNCaP) secrete NT and exhibit growth responses to NT. This study examines NT secretion, NT receptor and NT-growth responses in androgen-independent prostatic carcinoma (PC3) cells derived from prostate adenocarcinoma metastatic to bone. METHODS: Binding of 125I-NT to PC3 membranes was studied by filtration. NT was measured by RIA. Reverse transcriptase polymerase chain reaction (RT-PCR) was used for NT and NT receptor mRNA. Growth was measured as 3H-thymidine incorporation into DNA. RESULTS: Scatchard analyses gave two binding components (Kd1 = 40 pM and Kd2 = 300 pM) in equal amounts (15-30 x 10(3) sites/cell). The bioactive region of NT was essential and the specific, non-peptide NT antagonist, SR48692, inhibited (IC50 = 3 nM). GTP analogs, sodium ion and SH-directed alkylating agents also inhibited. Glutaraldehyde crosslinking labeled two substances (M(r) of 23 and 46 kDa). RT-PCR indicated robust expression of authentic NT receptor but little for NT precursor. NT was stable in PC3 cultures but it was not found in cells or conditioned media. Incubated with PC3 cells, NT exhibited a mitogenic effect with bell-shaped dose-response and maximum at 100 pM NT. CONCLUSIONS: PC3 cells expressed genuine NT receptors and generated growth responses to physiologic levels of NT which were blocked by SR48692. If NT contributes to the survival of prostate tumor cells upon androgen deprivation therapy, NT antagonists might be useful agents in further treatment.

Geographic variation of the predictive values of genomic mutations associated with streptomycin resistance in Mycobacterium tuberculosis.

Specific mutations associated with resistance to streptomycin (SM) in Mycobacterium tuberculosis suggest themselves for its rapid prediction. However, as with any diagnostic test, their predictive values are dependent on their prevalences. In this report, SM resistance associated mutations in the rrs and rpsL genes of 25 SM resistant isolates from Germany and 25 SM resistant isolates from Sierra Leone were characterized and compared. Mutations in the rrs gene were infrequent in isolates from both localities (20% and 12%, respectively) and thus of limited predictive values. In contrast, rpsL mutations were found in 48% of the German isolates but only in 24% of the isolates from Sierra Leone. It is concluded that the predictive values of mutations in this gene may vary significantly with the origin of the samples under investigation.

Developmental expression and activities of specific fos and jun proteins are functionally related to osteoblast maturation: role of Fra-2 and Jun D during differentiation.

Developmental studies of oncogene expression implicate the Fos and Jun family of transcription factors in the regulation of bone growth and differentiation. Promoters of many developmentally regulated genes, including osteocalcin, a marker of osteoblast differentiation, contain AP-1 sites that bind Fos/Jun dimers. Here, we demonstrate that the selective expression of fos- and jun-related genes is functionally related to the stage of osteoblast growth and differentiation in vitro. During osteoblast proliferation, nuclear protein levels of all seven activating protein-1 (AP-1) members are maximal. Subsequently, during the period of extracellular matrix maturation, levels decline. In fully differentiated osteoblasts, Fra-2 and (to a lesser extent) Jun D are the principal AP-1 members detectable by Western blot analysis. AP-1 complex composition and binding activity also exhibit developmental changes. All Fos and Jun family members are involved in AP-1 complex formation in proliferating cells, whereas Fra-2 and Jun D predominate in AP-1 complexes in differentiated osteoblasts. Overexpression of Fos and Jun family members in ROS 17/2.8 cells markedly affects the expression of an osteocalcin promoter-chloramphenicol acetyltransferase construct. Coexpression of only one AP-1 pair, Fra-2 and Jun D, stimulated reporter expression, whereas coexpression of other AP-1 pairs down-regulated expression (i.e. c-jun and any Fos family member) or had no effect (i.e. Fra-1 and Jun B). Promoter deletion analyses indicate that these effects are site specific. Consequential effects of Fra-2 on osteoblast differentiation are further demonstrated by antisense studies in which osteoblast differentiation and the development of a bone tissue-like organization were suppressed. Consistent with recent findings suggesting that AP-1 complex composition can selectively regulate gene transcription, our findings demonstrate that differential expression of Fos and Jun family members could play a role in the developmental regulation of bone-specific gene expression and, as a result, may be functionally significant for osteoblast differentiation.

Rapid identification of mycobacterial species by PCR amplification of hypervariable 16S rRNA gene promoter region.

A total of 0.3 to 0.4 kb of the promoter region of the 16S rRNA genes from Mycobacterium tuberculosis, M. gordonae, M. xenopi, and M. leprae was PCR amplified, cloned, and sequenced. The observed number of substitutions, insertions, and deletions exceeded those found in previously used target sequences, including the entire 16S coding region. A simple and generally applicable restriction fragment length polymorphism method that can be used to distinguish between mycobacterial species is described.

Synergistic activation of neurotensin/neuromedin N gene expression by c-Jun and glucocorticoids: novel effects of Fos family proteins.

The cis-regulatory region of the neurotensin/neuromedin N (NT/N) gene integrates diverse environmental signals in the neuroendocrine PC12 cell line, resulting in remarkable synergistic regulation. An AP-1 site appears to play a pivotal role in cooperative NT/N gene activation, as mutations in this site decrease responses to all inducer combinations by at least an order of magnitude. Here we report that c-Jun acts synergistically with glucocorticoids to activate the NT/N promoter, and that Fos family proteins have novel regulatory effects on this interaction. Cotransfection of individual pCMV-AP-1 expression plasmids revealed that c-Jun most potently activates the NT/N promoter and that costimulation with dexamethasone results in a further 6- to 12-fold increase in expression. Unlike its general inhibitory effects on glucocorticoid regulation in other systems, c-Fos potentiated activation by glucocorticoids when coexpressed with c-Jun, and Fos B had a similar, but more limited, positive effect. In contrast, Fra-1 reversed the direction of glucocorticoid regulation, and Fra-2 abolished synergism. AP-1, cAMP response element, and glucocorticoid response element motifs are required for full cooperative activation by either c-Jun or c-Jun/c-Fos and glucocorticoids. These results indicate that NT/N promoter activation involves synergistic interactions between specific AP-1 complexes and ligand-activated glucocorticoid receptor, and similar mechanisms may regulate NT/N gene expression in central neurons.

Characterization of promoter elements required for cell-specific expression of the neurotensin/neuromedin N gene in a human endocrine cell line.

Expression of the gene encoding neurotensin/neuromedin N (NT/N) is mostly limited to the brain and specialized enteroendocrine cells (N cells) of the distal small intestine. We have analyzed the NT/N DNA sequences upstream of the RNA start site that direct cell-specific expression using a novel human endocrine cell line, BON, that resembles intestinal N cells in several important aspects, including NT/N precursor protein processing, ratios of different NT/N mRNA forms, and high levels of constitutive expression of the NT/N gene. Transient transfection assays with plasmids with progressive 5' deletions of the rat NT/N promoter identified the proximal 216 bp of 5' flanking sequences as essential for high-level constitutive NT/N expression in BON cells. In addition, a detailed mutational analysis defined multiple regions within the proximal 216 bp that contribute to cell-specific NT/N expression. These elements include a proximal cyclic AMP response element (CRE)/AP-1-like motif (TGACATCA) that binds c-Jun, JunD, CRE-binding (CREB), and ATF proteins, a near-consensus glucocorticoid response element, and a distal consensus AP-1 site that binds c-Fos, Fra-1, and JunD. In addition, elements contained within two 21-bp imperfect direct repeats play an important role in NT/N expression in BON cells and may bind novel factors that act as positive regulators of NT/N expression. DNase I footprinting and gel shift analyses demonstrate that the sites identified by mutational analysis, and at least one additional site, specifically bind BON cell nuclear proteins in vitro. We speculate that a complex pattern of regulation requiring interaction between a proximal CRE/AP-1-like motif and other upstream control elements play an important role in the high-level constitutive expression of NT/N in the human endocrine cell line BON. In addition, the BON cell line provides a unique model to further characterize the factors regulating cell-specific NT/N expression and to better understand the mechanisms responsible for the terminal differentiation of the N-cell lineage in the gut.

Synergistic induction of neurotensin gene transcription in PC12 cells parallels changes in AP-1 activity.

A consensus AP-1 site in the promoter of the rat neurotensin/neuromedin N (NT/N) gene is a critical regulatory element required for synergistic regulation by combinations of nerve growth factor (NGF), lithium, glucocorticoids, and adenylate cyclase activators. A rapid RNase protection assay was developed to examine the kinetics of NT/N gene activation and to determine whether activation requires newly synthesized proteins. Either NGF or lithium in combination with dexamethasone and forskolin transiently activated NT/N gene expression, but with distinct kinetics. Protein synthesis was not required for activation when NGF was used as the permissive inducer, but was required for the rapid down-regulation of the response. In contrast, lithium responses were attenuated in the absence of protein synthesis, consistent with a requirement for newly synthesized AP-1 complexes in activation. In all cases, increases in NT/N gene expression closely paralleled increases in AP-1 binding activity. Lithium in combination with other inducers caused delayed increases in both AP-1 binding activity and c-jun, c-fos and fra-1 gene expression. These results indicate that NGF and lithium exert their effects on NT/N gene expression through distinct pathways. The lithium pathway is active in neuronally-differentiated PC12 cells and could potentially be involved in the regulation of NT/N gene expression in the nervous system.