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The involvement of cAMP- and Ca2+-mediated pathways in the activation of tyrosine hydroxylase (TH) gene expression by nicotine was examined in PC-12 cells. Extracellular Ca2+ and elevations in intracellular Ca2+ concentration ([Ca2+]i) were required for nicotine to increase TH mRNA. The nicotine-elicited rapid rise in [Ca2+]iwas inhibited by blockers of either L-type or N-type, and to a lesser extent P/Q-, but not T-type, voltage-gated Ca2+ channels. With continual nicotine treatment, [Ca2+]ireturned to basal levels within 3–4 min. After a lag of ∼5–10 min, there was a smaller elevation in [Ca2+]ithat persisted for 6 h and displayed different responsiveness to Ca2+ channel blockers. This second phase of elevated [Ca2+]iwas blocked by an inhibitor of store-operated Ca2+ channels, consistent with the observed generation of inositol trisphosphate. 1,2-Bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid-AM (BAPTA-AM), when added before or 2 h after nicotine, prevented elevation of TH mRNA. Nicotine treatment significantly raised cAMP levels. Addition of the adenylyl cyclase inhibitor 2′,5′-dideoxyadenosine (DDA) prevented the nicotine-elicited phosphorylation of cAMP response element binding protein. DDA also blocked the elevation of TH mRNA only when added after the initial transient rise in [Ca2+]iand not after 1 h. This study reveals that several temporal phases are involved in the induction of TH gene expression by nicotine, each of them with differing requirements for Ca2+ and cAMP.

Nicotine treatment increases intracellular free Ca 2ϩ concentration [Ca 2ϩ ] i , stimulates catecholamine release, and elevates gene expression for the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH) and dopamine ␤-hydroxylase (DBH). However, the type of nicotinic acetylcholine receptors (nAChRs) mediating these events is unclear. The nAChR receptor antagonists ␣-bungarotoxin (␣BTX) and methyllycaconitine greatly reduced the nicotine-triggered initial transient rise in [Ca 2ϩ ] i and prevented the second prolonged elevation of [Ca 2ϩ ] i , suggesting the involvement of ␣7 nAChRs. Two specific ␣7 nicotinic agonists, 3-(2,4-dimethoxybenzilidene)anabaseine (DMXB) and E,E-3-(cinnamylidene)anabaseine (3-CA), were found to elicit a small, delayed increase in [Ca 2ϩ ] i with kinetics and magnitude similar to the second elevation observed with nicotine. This increase was inhibited by the inositol trisphosphate receptor antagonist xestospongin C. Exposure to 3-CA or DMXB for 6 or 24 h elevated TH and DBH mRNA levels two-to fourfold over control levels. These agonists were more effective than nicotine alone in increasing TH and DBH gene expression and significantly elevated [Ca 2ϩ ] i for up to 6 h. The increase in [Ca 2ϩ ] i or the elevation in TH mRNA by 3-CA was completely inhibited by ␣BTX. This study, for the first time, implicates stimulation of ␣7 nAChRs in the activation of TH and DBH gene expression. Key Words: Nicotinic acetylcholine receptors-Tyrosine hydroxylase -Dopamine ␤-hydroxylase -Nicotine -Catecholamine biosynthesis.

Previous studies have indicated that the receptor for urokinase-type plasminogen activator, uPAR, can form functional complexes with integrin receptors thereby modulating integrin activity. In the present study, the role of uPAR in the regulation of ␣ 5 ␤ 1 -dependent polymerization of the fibronectin matrix was investigated. Incubation of fibroblast monolayers with the P-25 peptide, a uPAR ligand, resulted in a 12-15-fold increase in the accumulation of exogenous fibronectin in the cell layer. The exogenous fibronectin co-localized in the extracellular matrix with endogenous cell-derived fibronectin, and its deposition into the matrix was inhibited by blocking antibodies against the ␤ 1 integrin receptor. The P-25-dependent increase in fibronectin assembly was associated with a 7-8-fold increase in the expression of matrix assembly sites as well as a 37-fold increase in the rate of transfer of cell surface-bound fibronectin into a detergent-insoluble matrix. The effects of P-25 on the matrix assembly were attenuated by incubating cells with either phospholipase C or with antibodies against uPAR, confirming a role for uPAR in the P-25-dependent increase in matrix assembly. P-25-treated cells exhibited a 10-fold increase in the binding of the 120-kDa cell-binding fragment of fibronectin suggesting an increase in ␣ 5 ␤ 1 affinity for fibronectin. Consistent with this, treatment of cells with P-25 also resulted in a 6 -10-fold increase in the binding of two different monoclonal antibodies that recognize the active conformation of the ␤ 1 integrin. These results indicate that P-25 increases matrix assembly by altering the activation state of the ␣ 5 ␤ 1 integrin receptor and suggest that changes in integrin activation affect both the number of matrix assembly sites as well as the rate of transfer of cell-bound fibronectin into a detergent-insoluble matrix. These data provide direct evidence that uPAR and integrin receptors synergistically regulate the levels of fibronectin in the extracellular matrix.

Phosphorylation (P-) of cAMP-response element-binding protein (CREB) by protein kinase A or mitogen-activated protein kinases was implicated in mediating the increased tyrosine hydroxylase (TH) gene expression after prolonged exposure to nicotine in vivo and in cell culture. We examined the time course and signaling pathways for phosphorylation of CREB and possible involvement of ATF-2. Treatment of PC12 cells with 200 M nicotine triggered rapid but transient elevation of P-CREB followed by a second sustained rise after 2-5 h of continuous nicotine. In contrast, ERK1/2 was only phosphorylated with short term nicotine exposure. MEK inhibitor U0126 abolished nicotine-induced rise in P-ERK1/2, but not P-CREB, nor did it inhibit nicotine-evoked elevation in TH promoter activity, indicating that ERK1/2 was not needed for induction of TH gene expression by nicotine. In contrast, protein kinase A inhibitor H-89 or Ca 2؉ / calmodulin-activated protein kinase inhibitor KN-93 reduced the nicotine-triggered rise in P-CREB and TH promoter activity. There was a delayed elevation of P-ATF-2 after 1 h of nicotine treatment, accompanied by increased ATF-2 protein. Upstream kinase JNK, but not p38, was phosphorylated especially after 5 min to 2 h of nicotine exposure. To examine the requirement for CREB and ATF-2, cells were transfected with dominant negative forms of ATF-2 or CREB. Both reduced the basal TH promoter activity and the response to nicotine. Knockdown of ATF-2 or CREB with siRNA did not alter basal TH promoter activity or mRNA but greatly attenuated the response to nicotine. The results suggest that both ATF-2 and CREB mediate activation of TH gene transcription by nicotine.Administration of nicotine in vivo and in cell culture triggers increased expression of a number of genes related to neurosecretion including the catecholamine biosynthetic enzymes. These changes in gene expression may be involved in neurochemical and cardiovascular effects of smoking. Nicotine is a potent sympathomimetic agent, and its administration, in doses similar to those obtained in smoking, increases heart rate and systolic and diastolic blood pressure in humans and many animal species (for review, see Ref. 1). These cardiovascular effects are largely attributed to the direct stimulation of release of catecholamines, epinephrine and norepinephrine, from the adrenal medulla and peripheral sympathetic nerve endings (2-4). In this regard a polymorphism in the gene for tyrosine hydroxylase (TH), 2 the rate-limiting enzyme in catecholamine biosynthesis, has been associated with tobacco use (5).Transcriptional mechanisms are at least partially responsible for the nicotine triggered changes in TH gene expression. The response of TH to nicotine has been proposed to be mediated by CREB. Nicotine treatment both in vivo and in vitro induces phosphorylation of CREB (6 -8).The cAMP/calcium response element (CRE/CaRE) in the TH promoter is required for its transcriptional activation by nicotine in PC12 cells (6). CREB is activated through the phosphor...

1. Stress elicits activation of several transcription factors involved in the regulation of catecholamine biosynthetic enzyme gene expression depending on its duration or repetition. However, the dynamic of the conversion of transient transcriptional activation with acute stress to sustained changes in transcription in response to repeated exposure to stress in adrenomedullary catecholaminergic systems is not clear. 2. Here, we analyzed changes in levels of phospho-CREB (P-CREB), phospho-ERK1/2 (P-ERK1/2) and Fra-2 by Western Blot analysis in adrenal medulla of Sprague Dawley male rats exposed to single or repeated immobilization stress (IMO). For single stress, rats were immobilized for 5 min, 30 min, or 2 h and sacrificed immediately afterwards. In the repeated stress conditions, animals were immobilized for 2 h daily on each consecutive day prior to the final day (day 2 for 2x IMO, day 6 for 6x IMO) in which the rats were immobilized for a session lasting 5 min, 30 min or 2 h. There were two control groups, an absolute control (AC) not exposed to stress, and a handled control (HC) gently handled daily for 6 days. 3. Phosphorylation of CREB was rapid and elevated at the earliest time examined, even with single stress. However, with a second daily episode of stress the increase in P-CREB was observed for at least the entire duration of the stress. In contrast, phosphorylation of ERK1/2 was only significant after brief exposure to a single IMO. The elevation of Fra-2 protein level was slower, but was significant after 2 h of a single IMO. With repeated IMO, there were marked elevations of Fra-2 throughout the 2 h IMO, which were especially pronounced at the end of the immobilization. 4. The transient nature of the phosphorylation of CREB may be responsible for the short-lived induction of transcription of catecholamine biosynthetic enzymes after brief exposure to a single immobilization stress. The sustained phosphorylation of CREB throughout the repeated stress coupled with induction of Fra-2 may mediate the longer lasting responses to repeated stress.

Classically, upon hypothalamic stimulation, adrenocorticotropic hormone (ACTH) is released from the pituitary and acts on melanocortin 2 receptors (MC2R) in the adrenal cortex, stimulating glucocorticoid synthesis and release. Our earlier studies suggested that ACTH might have a direct effect on sympathetic ganglia. To analyze further the involvement of ACTH in regulation of gene expression of norepinephrine (NE) biosynthetic enzymes, we examined the effect of bilateral adrenalectomy (ADX) of Sprague-Dawley male rats. Fourteen days post-ADX, as expected, plasma ACTH was elevated, and levels of tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH) and MC2R mRNAs in superior cervical ganglia (SCG), and TH mRNA in locus coeruleus (LC) were increased compared with sham-operated animals. To determine effect of pulsatile elevation of ACTH, corticosterone pellets were implanted to ADX rats. Similar to immobilization (IMO) stress ACTH injections to these animals caused a rise in ACTH in plasma and triggered elevation of TH and DBH mRNAs in SCG and in LC with single and repeated daily injections, and MC2R mRNA in SCG with single injections. To study the effect of ACTH in isolated cells, primary cultures of rat SCG were transfected with TH and DBH promoter constructs and treated with ACTH. In agreement with the in vivo data, ACTH elevated their promoter activities similar to levels triggered by cyclic AMP analog. ACTH in the human SK-N-SH neuroblastoma cells increased TH and DBH promoter activity and endogenous DBH mRNA levels. The results show that ACTH can have a direct effect on transcription and gene expression of NE biosynthetic enzymes even without contribution of adrenal hormones.

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