help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2006-0907
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wada, T.
Right arrow Articles by Sasaoka, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wada, T.
Right arrow Articles by Sasaoka, T.
Endocrinology Vol. 148, No. 2 790-799
Copyright © 2007 by The Endocrine Society

Chronic Nicotine Exposure Enhances Insulin-Induced Mitogenic Signaling via Up-Regulation of {alpha}7 Nicotinic Receptors in Isolated Rat Aortic Smooth Muscle Cells

Tsutomu Wada, Maiko Naito, Hiroki Kenmochi, Hiroshi Tsuneki and Toshiyasu Sasaoka

Department of Clinical Pharmacology, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan

Address all correspondence and requests for reprints to: Toshiyasu Sasaoka, M.D., Ph.D., Department of Clinical Pharmacology, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan. E-mail: tsasaoka{at}pha.u-toyama.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Insulin resistance and smoking are significant risk factors for cardiac and cerebral vascular diseases. Because vascular smooth muscle cells play a key role in the development and progression of atherosclerosis, we investigated the effect of nicotine on insulin-induced mitogenic signaling in aortic vascular smooth muscle cells isolated from Sprague Dawley rats. RT-PCR revealed the expression of {alpha}2–7, {alpha}10, ß1–3, {delta}, and {epsilon} subunits of the nicotinic acetylcholine receptor (nAChR) in the cells. Short-term nicotine treatment stimulated phosphorylation of p44/42-MAPK, p38-MAPK, and signal transducer and activator of transcription 3. However, an additive effect of nicotine pretreatment on insulin stimulation was only observed on p44/42-MAPK. The nicotine-induced phosphorylation of p44/42-MAPK and [methyl-3H]thymidine incorporation were effectively suppressed by a {alpha}7-nAChR-selective antagonist, methyllycaconitine, and the phosphorylation of p44/42-MAPK was stimulated by a {alpha}7-nAChR-specific agonist, GTS21. Furthermore, the phosphorylation was mediated via calmodulin kinase II, Src, and Shc. Interestingly, long-term (48-h) pretreatment with nicotine increased the amount of {alpha}7-AChR in the plasma membrane and insulin-induced phosphorylation of p44/42-MAPK. These results provide the first evidence that acute exposure to nicotine enhances insulin-induced mitogenesis predominantly by affecting the phosphorylation of p44/42-MAPK and that chronic exposure further augments the insulin signal via up-regulation of {alpha}7-nAChR, which may be crucial for the development and progression of atherosclerosis in large vessels.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DIABETES MELLITUS IS a significant risk factor for atherosclerotic diseases, including cardiovascular ischemia and cerebral infarction (1). A major pathological feature of type 2 diabetes is insulin resistance, one of the hallmarks of which is hyperinsulinemia. Insulin is known to facilitate the progression of atherosclerosis, because it promotes the proliferation of vascular smooth muscle cells (VSMCs) (1). Concerning the mitogenic properties of insulin signaling, the activated insulin receptor tyrosine phosphorylates receptor substrates, including the insulin receptor substrate family of proteins, and src homology and collagen (Shc) to signal downstream (2). There are four major pathways involved in insulin-induced mitogenesis in VSMCs. First, p44/42-MAPK has been identified as a key molecule of the mitogenic signaling activated by various growth factors, including insulin (1, 2, 3). Insulin-induced phosphorylation of Shc promotes the formation of a Shc/growth factor receptor-bound protein (Grb2)/Sos complex, leading to the activation of p44/42-MAPK through the activation of Ras, Raf, and MAPK kinase (MEK) (3, 4). Second, p38-MAPK is known to be phosphorylated and activated by insulin (5). Although the precise upstream signals for the insulin-induced activation of p38-MAPK are still unclear, MAPK kinase 3 and MAPK kinase 6 have been identified as upstream kinases of p38-MAPK in VSMCs (5). Third, Jak-signal transducer and activator of transcription (STAT) pathways are primarily activated by cytokines such as interleukins, interferon, and erythropoietin. Among them, insulin is known to activate STAT3 via Jak2 (6, 7). Fourth, insulin activates Akt via the insulin receptor substrate-phosphatidylinositol 3-kinase pathway (2). Among Akt isoforms, Akt1 is known to be involved in cell proliferation by protecting against apoptosis (8).

Recent epidemiological studies showed a clear association between smoking cigarettes and atherosclerosis. The United Kingdom Prospective Diabetes Study has shown that smoking is a significant and independent risk factor for cardiovascular disease (9), stroke (10), and peripheral vascular disease (11) in type 2 diabetes. Nicotine is a major constituent of tobacco (12) and may promote the progression of atherosclerosis by stimulating the secretion of catecholamine, activating sympathetic nerves, and inducing vasoconstriction (12, 13, 14). In addition, nicotine is known to be directly involved in cell proliferation by possibly stimulating the production of platelet-derived growth factor (PDGF)-BB in human VSMCs (15). Furthermore, it has been reported that nicotine mediates cell proliferation and enhances angiotensin II-induced mitogenesis by activating p44/42-MAPK, p38-MAPK, and STAT3 (16). Thus, insulin and nicotine appear to use, at least in part, common cellular signaling pathways leading to cell proliferation.

Because hyperinsulinemia and smoking are crucial risk factors for the development and progression of atherosclerosis (1, 9, 10, 11, 12), it is important to clarify the effect of nicotine on insulin-induced mitogenic action. In the present study, we investigated the molecular mechanisms of the cross talk of mitogenic signaling elicited by insulin and nicotine in isolated rat VSMCs and determined which signaling molecules are significantly implicated in nicotine’s enhancement of insulin signaling. In addition, we examined the nAChR subtype responsible for the nicotine-induced mitogenic signaling in VSMCs. Habitual smoking has been reported to cause a chronic elevation in the concentration of nicotine in serum, whereas acute smoking transiently elevates the serum nicotine concentration (17). Therefore, we further investigated the impact of long-term treatment with nicotine on insulin-induced mitogenic signaling in VSMCs by comparing it with that of short-term nicotine treatment.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Human recombinant insulin was provided by Novo Nordisk Pharmaceutical Co. (Copenhagen, Denmark). A polyclonal anti-Thr202/Tyr204 phospho-specific p44/p42-MAPK antibody, a polyclonal anti-p44/p42-MAPK antibody, a polyclonal anti-Tyr705 phospho-specific STAT3 antibody, a polyclonal anti-Ser727 phospho-specific STAT3 antibody, a polyclonal anti-STAT3 antibody, a polyclonal anti-Thr180Tyr182 phospho-specific p38-MAPK antibody, a polyclonal anti-p38-MAPK antibody, and a polyclonal anti-Ser473phospho-specific Akt antibody were purchased from Cell Signaling Technology Inc. (Beverly, MA). A monoclonal anti-Akt antibody, a monoclonal anti-phospho-tyrosine (PY99) antibody, and a polyclonal anti-{alpha}7-nAChR were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). A polyclonal anti-epidermal growth factor (EGF) receptor (EGFR) antibody and a polyclonal anti-PDGF receptor (PDGFR) antibody were purchased from Upstate Biotechnology (Lake Placid, NY). A polyclonal anti-Shc antibody, a monoclonal anti-Grb2 antibody, and a polyclonal anti-Flotillin 1 antibody were obtained from BD Bioscience (San Jose, CA). Nicotine and methyllycaconitine were purchased from Sigma (St. Louis, MO). Hexamethonium was from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). KN-93 and PP-2 were from Calbiochem (La Jolla, CA). GTS-21 and 4-OH-GTS-21 were provided by TAIHO Pharmaceutical Co. Ltd. (Tokyo, Japan). [metyl-3H]thymidine and enhanced chemiluminescence reagents were from GE Healthcare Bio-Science Corp. (Tokyo, Japan). DMEM, MEM vitamin mixtures, and MEM amino acid solutions were from Life Technologies, Inc. Japan (Tokyo, Japan). All other reagents were of analytical grade and purchased from Sigma or Wako Pure Chemical Industries, Ltd.

Preparations of rat aortic smooth muscle cells and cell cultures
All procedures involving animals were conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Research Committee at the University of Toyama. Aortic VSMCs were prepared from thoracic aorta of male Sprague Dawley rats (8 wk old) as described previously (18). Rat aortic VSMCs were cultured in DMEM supplemented with 10% fetal calf serum, and the growth medium was changed every 2 d. Cultured cells were confirmed to be VSMCs by immunostaining with smooth muscle {alpha}-actin. Subcultures of VSMCs at two to five passages were used for experiments.

RT-PCR
Total RNA was isolated from rat aortic VSMCs with an ISOGEN RNA extraction kit (Nippon Gene Co. Ltd., Toyama, Japan). The identification of nicotinic receptor subtypes was carried out by using the SuperScript II One-Step RT-PCR system with a Platinum Taq DNA polymerase kit (Invitrogen, Carlsbad, CA) as described previously (19). In brief, 0.5 µg of template RNA was mixed with 25 µl of 2x reaction mixture buffer, 0.2 µM sense and antisense primer, and 2 µl SuperScript II RT/Platinum Taq Mix. Reverse transcription and denaturing were conducted for 30 min at 50 C and for 2 min at 94 C, respectively. Subsequently, 30 or 40 cycles of PCR amplification were performed (denaturing for 15 sec at 94 C, annealing for 45 sec at 46–60 C, and extension for 45 sec at 72 C). The final extension was conducted for 5 min at 72 C. RT-PCR products were subjected to electrophoresis on a 2% agarose gel to determine the expression of nAChR subtypes. Primer pairs for mammalian nAChR subunits and annealing temperatures are listed in Fig. 1BGo. PCR products were confirmed not to be amplified when the template was replaced with water. RT-PCR products of glyceraldehyde-3-phosphate dehydrogenase were obtained as a positive control for efficient RNA isolation and cDNA synthesis (data not shown).


Figure 1
View larger version (46K):
[in this window]
[in a new window]

  		FIG. 1. Expression of nicotinic receptor subunits in rat aortic VSMCs. VSMCs were isolated from 8-wk-old male Sprague Dawley rats and cultured in DMEM. A, Total mRNA was extracted from VSMCs with a ISOGEN mRNA purification kit and mixed with various specific primers of rat nicotinic subunits ({alpha}1–7, {alpha}9, {alpha}10, ß1–4, {gamma}, {delta}, and {epsilon}). All samples were subjected to RT-PCR to determine the nicotinic receptor expressed in VSMCs. MW, Molecular weight standards. B, Primer pairs for mammalian nAChR subunits and annealing temperatures are listed.

 
Immunoprecipitation and Western blotting
VSMCs grown in six-well multiplates were serum starved for 2 d in DMEM. The cells were pretreated with 10–9 M nicotine for specific periods of time and stimulated with or without 170 nM insulin for 10 min at 37 C. The cells were lysed in a buffer containing 20 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium deoxycholate, 1 mM ß-glycerophosphate, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, 50 mM sodium fluoride, 10 µg/ml of aprotinin, and 10 µM leupeptin (pH 7.4) for 30 min at 4 C. Lysates obtained from the same number of cells were centrifuged to remove insoluble materials. The supernatants (100 µg of protein) were immunoprecipitated with antibodies overnight at 4 C and protein G Sepharose (GE Healthcare Bio-Science Corp.) for 2 h at 4 C. The precipitates or whole-cell lysates were then separated by 7.5% SDS-PAGE and transferred onto polyvinylidene difluoride membranes using a Bio-Rad Transblot apparatus (Bio-Rad Laboratories Inc., Hercules, CA). The membranes were blocked in a buffer containing 50 mM Tris, 150 mM NaCl, 0.1% Tween 20, and 2.5% BSA or 5% nonfat milk (pH 7.5) for 2 h at 20 C. The membranes were then probed with antibodies for 2 h at 20 C or for 16 h at 4 C. After the membranes were washed in a buffer containing 50 mM Tris, 150 mM NaCl, and 0.1% Tween 20 (pH 7.5), blots were incubated with a horseradish peroxidase-linked secondary antibody and subjected to enhanced chemiluminescence detection using the ECL reagent according to the manufacturer’s instructions (GE Healthcare Bio-Science Corp.) (18).

Measurement of [methyl-3H]thymidine incorporation into VSMCs
The effects of {alpha}7-nAChR antagonist and MEK inhibitor on nicotine- and insulin-induced DNA synthesis were measured by counting of [3H]thymidine incorporation as previously described with minor modification (16, 20). VSMCs grown in six-well dishes were serum starved for 48 h, pretreated with or without 50 nM methyllycaconitine or 20 nM PD98059 for 30 min, followed by stimulation with 10–8 M nicotine and/or 170 nM insulin for 12 h. The cells were further incubated with 2 µCi of [methyl-3H]thymidine for an additional 24 h. The cells were washed twice with PBS and subsequently incubated with 5% trichloroacetic acid for 20 min at 4 C. The cells were washed twice with 5% trichloroacetic acid, then once with PBS, and lysed with 0.5 N NaOH. The radioactivity of the cell lysates was measured by liquid scintillation counter.

Subcellular fraction of VSMCs
VSMCs were incubated with 10–9 M nicotine in serum-free starvation medium for 1 or 2 d. Cells were rinsed twice with PBS, and the medium was replaced with DMEM. After 30 min, cells were restimulated with 10–9 M nicotine, GTS-21, or 4-OH-GTS-21 for 2 min followed by 170 nM insulin for 10 min. For subcellular fraction, cells were harvested as described for the Western blot analysis. The cell lysates (two 10-cm-diameter dishes per condition) were frozen in liquid nitrogen and thawed three times. Then, samples were centrifuged at 250 x g for 5 min to remove the nucleus. The supernatants were centrifuged at 100,000 x g for 30 min at 4 C yielding a high-speed pellet containing plasma membrane fraction. Remaining supernatants were used as the cytosol. All fractions were adjusted to a final protein concentration of 1–3 mg/ml, which was measured by the Bradford method, and separated by 7.5% SDS-PAGE and immunoblotted with anti-{alpha}7-nAChR antibody.

Statistical analysis
The data are presented as the means ± SE. P values were determined using the Student’s t test for two groups or one-way ANOVA (Scheffé’s multiple comparison test) for comparisons among three or more groups. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of nicotinic acetylcholine receptors (nAChRs) in rat aortic vascular smooth muscle cells
Nicotine exerts various pharmacological actions for synaptic transmission via nAChRs in neuronal cells (21, 22, 23). The nAChR possesses a ligand-gated channel composed of a homo- or hetero-pentamer of genetically distinct receptor subunits, and the characteristics of receptors differ in composition (21, 23). Although it has recently been reported that nicotine promotes mitogenesis and angiogenesis, the precise molecular mechanism by which nicotine exerts its effect in VSMCs is unclear. In particular, the nAChR subtypes involved in the mitogenesis remain unknown. First, we exclusively examined the possible expression of neuronal nAChR subunits ({alpha}2–7, {alpha}9, {alpha}10, and ß2–4) as well as muscle nAChR subunits ({alpha}1, ß1, {gamma}, {delta}, and {epsilon}) in VSMCs obtained from Sprague Dawley rats by using RT-PCR. Total RNA extracted from the VSMCs was mixed with various sense and antisense primers listed in Fig. 1BGo, and RT-PCR was conducted. As shown in Fig. 1AGo, we identified the RNA expression of {alpha}2–7, {alpha}10, ß2, and ß3 among neuronal nAChR subunits and ß1, {delta}, and {epsilon} among muscle nAChR subunits in rat aortic VSMCs. A lack of expression of {alpha}1 and ß4 in the VSMC sample was confirmed by the fact that the expression of these two subunits was seen in the total RNA extract obtained from skeletal muscle and hypothalamus in the same rat (data not shown).

Effect of nicotine pretreatment on insulin-induced mitogenic signaling
Insulin promotes atherosclerosis by stimulating the proliferation of VSMCs (1, 3). It is known that insulin’s stimulation of p44/42-MAPK, p38-MAPK, STAT3, and Akt is potentially important for VSMC proliferation (2, 3, 5, 6, 7, 8). Therefore, the effect of nicotine, a major constituent of tobacco, which is a significant risk factor for atherosclerosis, on insulin-induced mitogenic signaling was studied. We first confirmed the positive effect of nicotine on mitogenic signaling at various concentrations and selected 10–9 M as the stimulatory concentration (data not shown), which almost corresponds to the concentration of nicotine in serum after smoking (17). Insulin increased the phosphorylation of p44/42-MAPK 1.6-fold. Short-term pretreatment with 10–9 M nicotine also significantly increased the amount of phosphorylated p44/42-MAPK. The pretreatment for 2 min enhanced the insulin-induced phosphorylation of p44/42-MAPK 3.0-fold (Fig. 2AGo). We next examined the insulin-induced phosphorylation of p38-MAPK by using an anti-Thr180/Tyr182 phospho-specific p38-MAPK antibody. Insulin increased the phosphorylation of p38-MAPK 3-fold. Treatment with nicotine for short term also enhanced phosphorylation of p38-MAPK 3.0-fold. Although the short-term pretreatment appeared to enhance the insulin-induced phosphorylation of p38-MAPK, the effect was not significant (Fig. 2BGo). STAT3 is also known to be involved in insulin-induced mitogenesis (6, 7). STAT3 is activated via phosphorylation at Tyr705 by which nuclear translocation and DNA binding are promoted (7). The transcriptional activation appears to be regulated by phosphorylation at Ser727 (7). Insulin treatment for 10 min induced the phosphorylation of STAT3 at both Tyr705 and Ser727, albeit to a lesser extent. On the other hand, short-term nicotine treatment strongly induced the phosphorylation of STAT3 at Tyr705 and Ser727for up to 10 min with a peak at 2 min of 7.1- and 6-fold, respectively. The pretreatment with nicotine did not enhance the insulin-induced phosphorylation of STAT3 (Fig. 2CGo). Akt is known to be involved in proliferation by protecting cells from apoptosis (8). Because the phosphorylation of Akt at Thr307 and Ser473 is required for the activation, we further examined the effect of insulin and nicotine on the phosphorylation of Akt. In contrast to STAT3, insulin strongly increased the phosphorylation of Akt at Ser473 3.8-fold, whereas short-term nicotine treatment only slightly induced the phosphorylation. In addition, the pretreatment with nicotine did not affect the insulin-induced phosphorylation of Akt (Fig. 2DGo). Results for insulin- and nicotine-induced phosphorylation of Akt at Thr307 were similar to those for Akt’s phosphorylation at Ser473 (data not shown). These results indicate that short-term nicotine treatment stimulated the phosphorylation of p44/42-MAPK, p38-MAPK, and STAT3, whereas insulin preferentially induced the phosphorylation of p44/42-MAPK, p38-MAPK, and Akt in rat aortic VSMCs. Importantly, only the insulin-induced phosphorylation of p44/42-MAPK was significantly enhanced by the pretreatment with nicotine.


Figure 2
View larger version (69K):
[in this window]
[in a new window]

  		FIG. 2. Effect of nicotine on insulin-induced mitogenic signaling. VSMCs were isolated from 8-wk-old male Sprague Dawley rats and cultured in DMEM. Cells were pretreated with 10–9 M nicotine for the periods indicated and stimulated with 170 nM insulin for 10 min. The cell lysates were subjected to SDS-PAGE, and immunoblotted with (A) anti-phospho-specific p44/42-MAPK antibody or anti-p44/42-MAPK antibody, (B) anti-phospho-p38-MAPK antibody or anti-p38-MAPK antibody, (C) anti-phospho-Tyr180/Tyr182 STAT3 antibody or anti-phospho-Ser727 STAT3 antibody or anti-STAT3 antibody, and (D) anti-phospho-Ser 473 Akt antibody or anti-Akt antibody. The phosphorylation of these molecules was quantified by densitometry. Results are means ± SE of five separate experiments. {dagger}{dagger}, P < 0.01 vs. control, determined with Student’s t test. *, P < 0.05; **, P < 0.01 determined by one-way ANOVA with Scheffé’s test. ns, Not significant.

 
Identification of nAChR subtypes involved in nicotine-induced mitogenic signaling
We next studied which nicotinic receptor subtypes are responsible for the nicotine-induced mitogenesis. Because methyllycaconitine (MLY) is a well-characterized {alpha}7-nAChR-selective antagonist at a concentration of 10–50 nM (24, 25), we examined its effect on the nicotine-induced phosphorylation of p44/42-MAPK, p38-MAPK, and STAT3. As shown in Fig. 3AGo, the nicotine-induced phosphorylation of p44/42-MAPK was effectively inhibited by pretreatment with MLY in a concentration-dependent manner. Treatment with 50 nM MLY almost completely inhibited the nicotine-induced phosphorylation of p44/42-MAPK. In contrast, pretreatment with MLY at 10–50 nM only partly inhibited the nicotine-induced phosphorylation of STAT3 and p38-MAPK. To further examine the involvement of {alpha}7-nAChR in nicotine-induced mitogenic signaling, we used two types of {alpha}7-nAChR-specific agonists, GTS-21 and 4-OH-GTS-21 (25, 26). Cells were stimulated with the {alpha}7-nAChR-specific agonist instead of nicotine, and the phosphorylation of p44/42-MAPK, p38-MAPK, and STAT3 was examined (Fig. 3BGo). Consistent with the result obtained with the {alpha}7-nAChR antagonist, GTS-21 significantly stimulated the phosphorylation of p44/42-MAPK and only minimally induced phosphorylation of STAT3 and p38-MAPK. Similar effects were observed when cells were stimulated with 4-OH-GTS-21 instead of GTS-21 (data not shown). We next examined the effect of another antagonist, hexamethonium, which antagonizes most of the major types of neuronal nAChRs except for {alpha}7-nAChR at a concentration of 10–50 µM (27). Importantly, nicotine-induced phosphorylation of p44/42-MAPK was not inhibited by treatment with hexamethonium (Fig. 3CGo). These results indicate that the nicotine-induced phosphorylation of p44/42-MAPK is induced mainly through {alpha}7-nAChR. Although {alpha}7-nAChR may also mediate the nicotine-induced phosphorylation of p38-MAPK and STAT3, at least in part, it is possible that other nAChR subtypes contribute to the phosphorylation.


Figure 3
View larger version (56K):
[in this window]
[in a new window]

  		FIG. 3. Effects of an {alpha}7-nAChR antagonist, {alpha}7-nAChR agonist, and non{alpha}7-nAChR antagonist on the phosphorylation of p44/42-MAPK, p38-MAPK, and STAT3. VSMCs isolated from 8-wk-old male Sprague Dawley rats were pretreated with (A) methyllycaconitine or (C) hexamethonium for 30 min. Cells were treated with 10–9 M nicotine for 12 min. The cell lysates were subjected to SDS-PAGE and immunoblotted with anti-phospho-p44/42-MAPK antibody, anti-p44/42-MAPK antibody, anti-phospho-p38-MAPK antibody, anti-phospho-Tyr180/Tyr182 STAT3 antibody, or anti-phospho-Ser727 STAT3 antibody. A and C, The amount of phosphorylated p44/42-MAPK was quantified by densitometry. Results are means ± SE of four separate experiments. *, P < 0.05; **, P < 0.01, determined by one-way ANOVA with Scheffé’s test. B, VSMCs isolated from 8-wk-old male Sprague Dawley rats were treated with 10–12 to 10–6 M GTS-21 ({alpha}7-nAChR-specific agonist) for 12 min. The cell lysates were subjected to SDS-PAGE and immunoblotted with anti-phospho-p44/42-MAPK antibody, anti-phospho-Tyr180/Tyr182 STAT3 antibody, anti-phospho-Ser727 STAT3 antibody, or anti-phospho-p38-MAPK antibody.

 
Identification of nicotinic signaling associated with insulin-induced activation of p44/42-MAPK
We showed that the reinforcement of insulin-induced mitogenic signaling by nicotine is mainly caused by enhancement of p44/42-MAPK activation, and this nicotinic effect appears to be mediated by {alpha}7-nAChR. We next investigated the key molecules downstream of {alpha}7-nAChR connected to the insulin-induced activation of p44/42-MAPK. The nAChR is a ligand-gated channel, which functions to increase the intracellular levels of Ca2+. The homo-pentamer of {alpha}7-nAChR is highly permeable to Ca2+ compared with other nAChR subtypes not composed of {alpha}7-nAChR subunits in neural cells (21, 22). Recently, it was reported that the elevation of intracellular Ca2+ levels by ionomycin, angiotensin II, and thrombin leads to the activation of nonreceptor proline-rich tyrosine kinase (PYK2) and Src via the activation of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) (28, 29). Activated PYK2 and/or Src phosphorylated the EGFR, which in turn stimulated p44/42-MAPK in VSMCs (30). Therefore, to address the possible involvement of a Ca2+-dependent signaling pathway mediated by {alpha}7-nAChR after nicotine treatment, we used a CaM kinase II inhibitor, KN93, and a nonspecific Src inhibitor, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2).

Pretreatment with KN93 had no apparent effect on the insulin-induced phosphorylation of p44/42-MAPK. In contrast, the phosphorylation of p44/42-MAPK seen after short-term treatment with nicotine alone was abolished after pretreatment with 10 µM and 30 µM KN93. In addition, pretreatment with 30 µM KN93 effectively inhibited the enhancement by nicotine of the insulin-induced phosphorylation of p44/42-MAPK (Fig. 4AGo). Pretreatment with KN92, an inactive analog of KN93, did not affect the nicotine-induced phosphorylation of p44/42-MAPK (data not shown). Consistent with this result, pretreatment with PP2 did not affect the insulin-induced phosphorylation of p44/42-MAPK. In contrast, the nicotine-induced phosphorylation of p44/42-MAPK and the enhancement by nicotine of the insulin-induced phosphorylation were effectively inhibited by pretreatment with 1 µM and 10 µM PP2, respectively (Fig. 4BGo). These results indicate that the effect of nicotine via {alpha}7-nAChR is mediated by activation of CaM kinase II and Src, resulting in stimulation of p44/42-MAPK.


Figure 4
View larger version (36K):
[in this window]
[in a new window]

  		FIG. 4. Effect of KN93 and PP2 on nicotine- and insulin-induced phosphorylation of p44/42-MAPK. VSMCs isolated from 8-wk-old male Sprague Dawley rats were pretreated with (A) KN93 (calmodulin kinase II inhibitor) or (B) PP2 (Src inhibitor) for 30 min. Subsequently, cells were treated with 10–9 M nicotine for 2 min and then stimulated with 170 nM insulin for 10 min. The cell lysates were subjected to SDS-PAGE and immunoblotted with anti-phospho-p44/42-MAPK antibody or anti-p44/42-MAPK antibody. Results are means ± SE of four separate experiments. *, P < 0.05; **, P < 0.01 determined by one-way ANOVA with Scheffé’s test.

 
Src has the ability to activate membrane tyrosine kinase receptor for cell proliferation (30). Because EGFR and PDGFR potentially play a key role in the proliferation of VSMCs, we next examined the effect of insulin and nicotine on the phosphorylation of EGFR and PDGFR. However, neither nicotine nor insulin induced the phosphorylation of EGFR and PDGFR (Fig. 5Go, A and B).


Figure 5
View larger version (41K):
[in this window]
[in a new window]

  		FIG. 5. Effect of nicotine treatment on tyrosine phosphorylation of EGF and PDGF receptors. VSMCs isolated from 8-wk-old male Sprague Dawley rats were pretreated with 10–9 M nicotine for the periods indicated and then stimulated with 170 nM insulin for 10 min. The cell lysates were immunoprecipitated with anti-phosphotyrosine (PY99) antibody and immunoblotted with (A) anti-EGFR (RGFR) antibody or (B) anti-PDGF receptor (PDGFR) antibody.

 
To clarify whether the nicotine-induced activation of Ca2+-dependent kinases, including Src, in turn lead to phosphorylation of MAPK (31), we examined the effect of PP2 on the phosphorylation of Shc and Shc/Grb2 association during short-term treatment with nicotine. As shown in Fig. 6Go, short-term nicotine treatment significantly increased tyrosine phosphorylation of Shc and subsequent formation of the Shc/Grb2 complex. Pretreatment with PP2 effectively inhibited the nicotine-induced phosphorylation of Shc and Shc/Grb2 association. These results indicate that short-term treatment with nicotine activates p44/42-MAPK via CaM kinase II, Src, and Shc/Grb2 in rat aortic VSMCs.


Figure 6
View larger version (27K):
[in this window]
[in a new window]

  		FIG. 6. Effect of PP2 on nicotine-induced phosphorylation of Shc and association of Grb2 with Shc. VSMCs isolated from 8-wk-old male Sprague Dawley rats were pretreated with or without PP2 (Src inhibitor) for 30 min. Cells were then treated with 10–9 M nicotine for 12 min. The cell lysates were immunoprecipitated with anti-Shc antibody. The precipitates were subjected to immunoblot analysis with (A) anti-phospho tyrosine (PY99) antibody or (B) anti-Grb2 antibody, respectively. The amount of phosphorylated Shc and Shc-associated Grb2 was quantified by densitometry. Results are means ± SE of three separate experiments. **, P < 0.01 determined by one-way ANOVA with Scheffé’s test.

 
Long-term pretreatment with nicotine further enhanced insulin-induced mitogenic signaling and DNA synthesis
Habitual smoking results in a chronic elevation of the serum nicotine concentration (17). In addition, chronic nicotine treatment elicits a dose-dependent increase in the number of nAChRs in neuronal cells (32, 33). We examined the effect of nicotine treatment on insulin-induced mitogenic signaling after long-term treatment with nicotine (24 or 48 h). VSMCs were treated with nicotine for 24 h or 48 h, washed with PBS, and incubated in starvation medium to reverse the desensitization of nicotinic receptors. After 30 min, the cells were retreated with nicotine, and then the insulin-induced phosphorylation of p44/42-MAPK, p38-MAPK, and STAT3 was examined. Under these conditions, nicotine induced a 1.6-fold increase in the phosphorylation of p44/42-MAPK (Fig. 7AGo, lane 3 vs. 11), and the insulin-induced the phosphorylation of p44/42-MAPK was increased 1.8-fold by nicotine (Fig. 7AGo, lane 4 vs. 12). In contrast, long-term treatment with nicotine did not influence the subsequent effect of nicotine on the insulin-induced phosphorylation of p38-MAPK and STAT3 (Fig. 7Go, B and C). The amounts of p44/42-MAPK, p38-MAPK, and STAT3 were not altered during 48-h treatment with nicotine.


Figure 7
View larger version (54K):
[in this window]
[in a new window]

  		FIG. 7. Effect of long-term pretreatment with nicotine on insulin-induced phosphorylation of p44/42-MAPK. VSMCs isolated from 8-wk-old male Sprague Dawley rats were pretreated with 10–9 M nicotine for 2 min, 24 h, and 48 h. Thereafter, the cells were washed with PBS and the medium was replaced with DMEM for 30 min (washout of nicotine). The cells were again treated with 10–9 M nicotine for 2 min and then stimulated with 170 nM insulin for 10 min. The cell lysates were subjected to SDS-PAGE and immunoblotted with (A) anti-phospho-p44/42-MAPK antibody or anti-p44/42-MAPK antibody, (B) anti-phospho-p38-MAPK antibody or anti-p38-MAPK antibody, or (C) anti-phospho-Tyr180/Tyr 182 STAT3 antibody, anti-phospho-Ser727STAT3 antibody, or anti-STAT3 antibody. The amount of phosphorylated protein was quantified by densitometry. Results are means ± SE of six separate experiments. **, P < 0.01 determined by one-way ANOVA.

 
To further clarify whether the elevation of p44/42-MAPK phosphorylation correlates with cell proliferation, we examined the effect of nicotine on insulin-induced DNA synthesis (Fig. 8Go). The VSMCs treated with nicotine enhanced thymidine incorporation 1.4-fold, whereas pretreatment of MLY almost completely abolished this nicotine’s effect. Insulin also stimulated thymidine incorporation 1.8-fold. Although MLY did not affected insulin-induced DNA synthesis, pretreatment with PD98059, MEK inhibitor, completely abolished nicotine- and insulin plus nicotine-induced phosphorylation of p44/42-MAPK (data not shown) and DNA synthesis (Fig. 8Go). Consistent with the results of MLY on nicotine-induced DNA synthesis, long-term nicotine treatment also enhanced the phosphorylation of p44/42-MAPK induced by either GTS-21 (Fig. 9AGo) or 4-OH-GTS-21 (data not shown), two types of {alpha}7-nAChR-specific agonists, to 2-fold and 1.4-fold (with insulin), respectively (Fig. 9AGo, lane 3 vs. 9, lane 4 vs. 10).


Figure 8
View larger version (22K):
[in this window]
[in a new window]

  		FIG. 8. Effect of methyllycaconitine and MEK inhibitor on nicotine and insulin-induced DNA synthesis in VSMC. VSMCs isolated from 8-wk-old male Sprague Dawley rats were pretreated without or with methyllycaconitine or PD98059 for 30 min. Then, cells were stimulated with 10–8 M nicotine and/or 170 nM insulin for 12 h. The cells were further incubated with 2 µCi of [methyl-3H]thymidine for 24 h. The [methyl-3H]thymidine incorporation into VSMCs was examined by liquid scintillating counting as described in Materials and Methods. Results are means ± SE of five separate experiments. *, P < 0.05 determined by one-way ANOVA with Scheffé’s test.

 

Figure 9
View larger version (30K):
[in this window]
[in a new window]

  		FIG. 9. Effect of long-term pretreatment with nicotine on up-regulation of {alpha}7-nAChR at the membrane fraction. VSMCs isolated from 8-wk-old male Sprague Dawley rats were pretreated with 10–9 M nicotine for 2 min, 24 h, and 48 h. Thereafter, the cells were washed with PBS and the medium changed to DMEM for 30 min (washout of nicotine). A, Cells were treated with 10–9 M GTS-21 for 2 min and stimulated with 170 nM insulin for 10 min. The cell lysates were subjected to SDS-PAGE and immunoblotted with anti-phospho-p44/42-MAPK antibody or anti-p44/42-MAPK antibody. B, Cells were fractionated into the nucleus, high-speed pellet or cytosol fraction by differential centrifugation as described in Materials and Methods. The membrane and cytosol fractions were separated by 7.5% SDS-PAGE and immunoblotted with anti-{alpha}7-nAChR antibody. Existence of plasma membrane fraction in the high-speed pellet was determined by immunoblotting with an antibody against Flotillin-1, a plasma membrane marker protein. Results are means ± SE of six separate experiments. *, P < 0.01 determined by one-way ANOVA with Scheffé’s test.

 
Finally, we directly examined the cellular distribution of {alpha}7-nAChR after long-term treatment with nicotine, because it is known that chronic nicotine treatment increases the number of nAChRs at the cell surface in neural cells (32, 33). The cell lysates were differentially centrifuged and divided into cytosol and the high-speed pellet, including plasma membrane fraction, and the amount of {alpha}7-nAChR was analyzed. As shown in Fig. 9BGo, the amount of {alpha}7-nAChR at the high-speed pellet was markedly increased 2.4-fold by 48 h of treatment with nicotine.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Insulin resistance with hyperinsulinemia and smoking are risk factors for atherosclerotic diseases, including cerebral infarction and cardiac ischemia (1, 4, 9, 10, 11, 12). VSMCs play an important role in the development and progression of atherosclerosis (1, 4, 5, 6, 7). Insulin and nicotine as a major component of tobacco possess growth-promoting effects in VSMCs (1, 2, 3, 4, 5, 6, 7, 12). Therefore, we precisely examined the effect and the molecular mechanism behind the effect of nicotine-treatment on insulin-induced mitogenic signaling. The concentration of nicotine used for the pretreatment in this study was 10–9 M similar to the concentration in serum seen in smokers (17). The present results showed that insulin stimulated the phosphorylation of p44/42-MAPK, p38-MAPK, STAT3, and Akt, and that nicotine-induced transient phosphorylation of p44/42-MAPK, STAT3, and p38-MAPK but not Akt in rat VSMCs. Thus, insulin and nicotine use a relatively similar signaling machinery in mitogenesis, although the degree of their relative importance appears to differ. Importantly, the short-term treatment with nicotine enhanced insulin-induced phosphorylation of p44/42-MAPK, but not STAT3 or p38-MAPK. Based on the results, p44/42-MAPK appears to be a key mediator among these signaling molecules of the coordinated effect of insulin and nicotine. Thus, p44/42-MAPK may play a crucial role in the proliferation of VSMCs leading to the progression of atherosclerosis in subjects with insulin resistance and a smoking habit.

nAChRs are pentameric ligand-gated ion channels made up of various subunits (21, 22, 23). nAChRs can be classified into two groups: muscle nAChRs that mediate neuromuscular transmission and neuronal receptors that mediate or modulate synaptic transmission in the central and peripheral nervous systems. The muscle nAChRs are composed of ({alpha}1)2ß1{gamma}{delta} in fetal muscle, and the {gamma} subunit is replaced by the {epsilon} subunit to give an ({alpha}1)2ß1{epsilon}{delta} composition in adult muscle (34). The neuronal receptors are composed of 11 types of subunits, {alpha}2–10 [{alpha}8 has only been detected in chick (35)], and ß2–4 (21, 22, 23). The subunit composition of nAChR determines ligand specificity, ligand affinity, cation permeability, and channel kinetics. Recently, the expression of neuronal nAChRs in many kinds of nonneural cells, including vascular endothelial cells and aortic smooth muscle cells, was reported (36, 37). These nAChRs might be involved in cellular growth, migration, and differentiation, although their specific function is not clear. Therefore, we comprehensively examined the expression of all nAChR subunits by RT-PCR and demonstrated the expression of nine neuronal nAChR subunits ({alpha}2–7, {alpha}10, ß2, and ß3) and three muscle nAChR subunits (ß1, {delta}, and {epsilon}) in aortic VSMCs obtained from Sprague Dawley rats. Our results are consistent with a recent report showing the partial clarification of the expression of {alpha} subunits in aortic VSMCs derived from Wister rats in which the expression of ß subunits and muscle receptor subunits has not been studied (37).

Among these nAChR subtypes, nicotine-induced phosphorylation of p44/42-MAPK was effectively inhibited by pretreatment with an {alpha}7-nAChR-selective antagonist, MLY. In addition, the {alpha}7-nAChR-specific agonists GTS-21 and 4-OH-GTS-21 effectively induced phosphorylation of p44/42-MAPK. In contrast, pretreatment with hexamethonium, a broad-spectrum neuronal nAChR antagonist not acting on {alpha}7-nAChR, did not affect the nicotine-induced phosphorylation of p44/42-MAPK. These results indicate that the effects of nicotine and its enhancement of insulin action on the phosphorylation of p44/42-MAPK are mainly mediated via {alpha}7-nAChR in rat VSMCs. In fact, recent studies showed the crucial involvement of {alpha}7-nAChR in angiogenesis in endothelial cells (36, 38).

In contrast, pretreatment with MLY only partly inhibited the nicotine-induced phosphorylation of STAT3 and p38-MAPK. Neither GTS-21 nor 4-OH-GTS-21 stimulated the phosphorylation of STAT3 and p38-MAPK. These results indicate that the nicotine-induced phosphorylation of STAT3 and p38-MAPK is mediated largely by receptors other than {alpha}7-nAChR. Our results are consistent with a recent report that GTS-21 only phosphorylates p44/42-MAPK, not p38-MAPK, in PC12 neuronal model cells (39). Further study will be needed to clarify which nAChR subtypes are responsible for the nicotine-induced phosphorylation of STAT3 and p38-MAPK.

Among nAChR subtypes, {alpha}7-nAChR possesses unique functional characteristics with high Ca2+ permeability and a rapid onset of desensitization (21, 22, 23). Therefore, one can speculate that nicotine stimulates the influx of Ca2+ into cells via {alpha}7-nAChR and activates Ca2+-dependent pathways including CaM kinase II (28). In fact, Ginnan and Singer (30) showed that an ionomycin-induced elevation of intracellular Ca2+ levels resulted in the activation of p44/42-MAPK via Ca2+/CaM, CaM kinase II, PYK2/Src, and EGFR in rat VSMCs. The present results demonstrated that pretreatment with pharmacological inhibitors of CaM kinase II abolished nicotine-induced phosphorylation of p44/42-MAPK, and Src inhibitor also abolished the nicotine-induced phosphorylation of Shc, Shc/Grb2 association, leading to the p44/42-MAPK phosphorylation. However, the phosphorylation of EGFR was not affected by nicotine. Although it is difficult to explain the discrepancy between these studies, it is possible that the degree of the increase in the intracellular Ca2+ concentration induced by ionomycin or nicotine differs. Further studies appear to be required to clarify this issue.

Another possible mechanism of nicotine-induced phosphorylation of p44/42-MAPK might involve the PDGF receptor. PDGF is one of the keys to cell proliferation, because the PDGF receptor is highly expressed in VSMCs, and cell growth is markedly promoted by PDGF secreted from the endothelium neighboring atherosclerotic lesions (1, 40). In addition, a recent report showed that long-term treatment with nicotine increased the cellular level of the PDGF-BB transcript and the amount of PDGFß-receptor protein in human aortic VSMCs (15). However, this was not the case in our studies. Pretreatment with nicotine for a short period, up to 60 min, did not change the amount of PDGF receptor or the degree of phosphorylation.

We further examined DNA synthesis to clarify the impact of nicotine on insulin-induced p44/42-MAPK phosphorylation. Long-term nicotine treatment stimulated thymidine incorporation in VSMCs, and the effect was markedly suppressed by pretreatment with PD98059 and MLY. Nicotine treatment also had a tendency to enhance insulin-induced DNA synthesis, and the effect was again diminished by treatment with these inhibitors. These results indicate that nicotine promotes cell proliferation via {alpha}7nAChR-mediated p44/42-MAPK activation in VSMCs.

Chronic treatment with agonists results in a decrease in number of cognate neurotransmitter receptors or growth factor receptors through down-regulation. In contrast, chronic nicotine administration in animal models is known to evoke a dose-dependent increase in the number of nicotinic receptors, especially in the brains of rats (32) and mice (33) as well as humans who have a lifelong history of smoking (41). Although this unique characteristic is considered to affect sensitivity to nicotine and the development of nicotine addiction, it has so far been reported only in neuronal cells. Based on these findings, we hypothesized that habitual smoking may also affect the function and/or number of vascular nAChRs in VSMCs during the progression of atherosclerosis. In the present study, we examined the effect of nicotine treatment for long periods (24 and 48 h) on insulin-induced mitogenic signals to determine whether habitual chronic exposure to nicotine has an impact greater than a short-term exposure on the cell proliferation regulated by insulin signaling in VSMCs. We showed that pretreatment with nicotine for 24 and 48 h followed by a brief washout further augmented the subsequent nicotine-induced phosphorylation of p44/42-MAPK, but not the phosphorylation of p38-MAPK and STAT3. Thus, the important role p44/42-MAPK plays in nicotine- and insulin-induced mitogenic signaling is more evident in the effect of chronic exposure to nicotine. Furthermore, we showed that chronic treatment with nicotine induced the redistribution of {alpha}7-nAChR from the cytosol to the high-speed pellet containing plasma membrane fraction. Apparent existence of plasma membrane in the high-speed pellet preparation was confirmed by two plasma membrane markers, flotillin-1 and caveolin (data not shown) in this fraction. We cannot rule out the possibility that the high-speed pellet obtained from our cell fractionation method also contains endoplasmic reticulum and Golgi apparatus. However, studies in neuronal cells have shown that the up-regulation of nAChR is a posttranscriptional event, and the amount of cell surface nAChRs is increased on long-term nicotine treatment (42, 43, 44, 45, 46). These results further support the involvement of {alpha}7-nAChR in the enhancement of p44/42-MAPK phosphorylation after chronic nicotine treatment.

We cannot completely rule out the possible up-regulation of other nAChR subtypes. It is known that {alpha}7-nAChR needs less time than other nAChRs to recover from desensitization after chronic nicotine treatment (32, 33, 44). According to reports, we adopted a 30-min washout for the recovery from desensitization of {alpha}7-nAChR in the present study. It remains therefore possible that the washout period is not long enough for the recovery from desensitization of certain nAChR subtypes that mediate the nicotine-induced phosphorylation of p38-MAPK and STAT3. Although the precise mechanism of nAChR’s up-regulation is still unclear, it is possible that long-term nicotine treatment could induce a structural modification and desensitization of nAChRs (45) resulting in a reduction in the turnover of nAChRs recruited from the cell surface to cytosol followed by an increase in cell surface nAChRs (46). Alternatively, maturation of nAChRs that promote subunit assembly (42, 47) or the isomerization of low-affinity surface receptors into high-affinity receptors may also be involved in the increase at the cell surface (48).

In summary, we demonstrated the effect of nicotine on insulin-induced mitogenic signaling in rat aortic VSMCs. Although nicotine and insulin both activated p44/42-MAPK, p38-MAPK, and STAT3, nicotine only the enhanced insulin-induced phosphorylation of p44/42-MAPK through {alpha}7-nAChR, which stimulates CaM kinase II, Src, and Shc-dependent pathways. In addition, chronic nicotine treatment further enhanced the insulin-induced phosphorylation of p44/42-MAPK via the up-regulation of {alpha}7-nAChR at the cell surface. This is a novel finding that may explain the molecular mechanism by which habitual smoking promotes mitogenesis in concert with hyperinsulinemia leading to the development and progression of atherosclerosis. Thus, the present findings that insulin and nicotine (chronic nicotine, in particular) together promote cell proliferation would be valuable for further understanding the prevention of macrovascular diseases in patient with type 2 diabetes and smoking habit.


   Acknowledgments
 
We thank Drs. Takashi Suzuki, Mariko Ikubo, Makoto Kadowaki, and Masashi Kobayashi (University of Toyama, Toyama, Japan) for assistance in the study.


   Footnotes
 
This work was supported in part by a grant from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (T.W., H.T., and T.S.).

Disclosure Statement: The authors have nothing to declare.

First Published Online October 26, 2006

Abbreviations: CaM, Calmodulin-dependent protein; EGF, epidermal growth factor; EGFR, EGF receptor; Grb2, growth factor receptor-bound protein; MEK, MAPK kinase; MLY, methyllycaconitine; nAChR, nicotinic acetylcholine receptor; PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; PP2, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; PYK2, proline-rich tyrosine kinase; Shc, src homology and collagen; STAT, signal transducer and activator of transcription; VSMC, vascular smooth muscle cells.

Received July 7, 2006.

Accepted for publication October 16, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Ross R 1993 The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 362:801–809[CrossRef][Medline]
  2. Virkamäki A, Ueki K, Kahn CR 1999 Protein-protein interaction in insulin signaling and the molecular mechanisms of insulin resistance. J Clin Invest 103:931–943[Medline]
  3. Sasaoka T, Rose DW, Jhun BH, Saltiel AR, Draznin B, Olefsky JM 1994 Evidence for a functional role of Shc proteins in mitogenic signaling induced by insulin, insulin-like growth factor-1, and epidermal growth factor. J Biol Chem 269:13689–13694[Abstract/Free Full Text]
  4. Johnson GL, Vaillancourt RR 1994 Sequential protein kinase reactions controlling cell growth and differentiation. Curr Opin Cell Biol 6:230–238[CrossRef][Medline]
  5. Igarashi M, Yamaguchi H, Hirata A, Daimon M, Tominaga M, Kato T 2000 Insulin activates p38 mitogen-activated protein (MAP) kinase via a MAP kinase kinase (MKK) 3/MKK 6 pathway in vascular smooth muscle cells. Eur J Clin Invest 30:668–677[CrossRef][Medline]
  6. Gottardello ZH, De Souza CT, Oliveira PP, Campello CJB, Augusto VL, Abdalla Saad MJ 2005 Effect of obesity on insulin signaling through JAK2 in rat aorta. Vasc Pharmacol 43:346–352[CrossRef]
  7. Ceresa BP, Horvath CM, Pessin JE 1997 Signal transducer and activator of transcription-3 serine phosphorylation by insulin is mediated by a Ras/Raf/MEK-dependent pathway. Endocrinology 138:4131–4137[Abstract/Free Full Text]
  8. Cho H, Horvaldsen JL, Hu Q, Feng F, Birnbaum MJ 2001 Akt1/PKB{alpha} is required for normal growth but dispensable for maintenance of glucose homeostasis in mice. J Biol Chem 276:38349–38352[Abstract/Free Full Text]
  9. Turner RC, Millns H, Neil HA, Stratton IM, Manley SE, Matthews DR, Holman RR 1998 Risk factors for coronary artery disease in non-insulin dependent diabetes mellitus: United Kingdom Prospective Diabetes Study (UKPDS:23). BMJ 316:823–828[Abstract/Free Full Text]
  10. Kothari V, Stevens RJ, Adler AI, Stratton IM, Manley SE, Neil HA, Holman RR 2002 UKPDS 60: risk of stroke in type 2 diabetes estimated by the UK Prospective Diabetes Study risk engine. Stroke 33:1776–1781[Abstract/Free Full Text]
  11. Adler AI, Stevens RJ, Neil A, Stratton IM, Boulton AJ, Holman RR 2002 UKPDS 59: Hyperglycemia and other potentially modifiable risk factors for peripheral vascular disease in type 2 diabetes. Diabetes Care 25:894–899[Abstract/Free Full Text]
  12. Kapoor D, Jones TH 2005 Smoking and hormones in health and endocrine disorders. Eur J Endocrinol 152:491–499[Abstract/Free Full Text]
  13. Cryer PE, Haymond MW, Santiago JV, Shah SD 1976 Norepinephrine and epinephrine release and adrenergic mediation of smoking-associated hemodynamic and metabolic events. N Engl J Med 295:573–577[Abstract]
  14. Niedermaier ON, Smith ML, Beightol LA, Zukowska-Grojec Z, Goldstein DS, Eckberg DL 1993 Influence of cigarette smoking on human autonomic function. Circulation 88:562–571
  15. Pestana IA, Vazquez-Padron RI, Aitouche A, Pham SM 2005 Nicotinic and PDGF-receptor function are essential for nicotine-stimulated mitogenesis in human vascular smooth muscle cells. J Cell Biochem 96:986–995[CrossRef][Medline]
  16. Li JM, Cui TX, Shiuchi T, Liu HW, Min LJ, Okumura M, Jinno T, Wu L, Iwai M, Horiuchi M 2004 Nicotine enhances angiotensin II-induced mitogenic response in vascular smooth muscle cells and fibroblasts. Arterioscler Thromb Vasc Biol 24:80–84[Abstract/Free Full Text]
  17. Benowitz NL, Porchet H, Sheiner L, Jacob 3rd P 1988 Nicotine absorption and cardiovascular effects with smokeless tobacco use; comparison with cigarettes and nicotine gum. Clin Pharmacol Ther 44:23–28[Medline]
  18. Sasaoka T, Kikuchi K, Wada T, Sato A, Hori H, Murakami S, Fukui K, Ishihara H, Aota R, Kimura I, Kobayashi M 2003 Dual role of SRC homology domain 2-containing inositol phosphatase 2 in the regulation of platelet-derived growth factor and insulin-like growth factor I signaling in rat vascular smooth muscle cells. Endocrinology 144:4204–4214[Abstract/Free Full Text]
  19. Tsuneki H, Ito K, Sekizaki N, Ma EL, You Y, Kawakami J, Adachi I, Sasaoka T, Kimura I 2004 Nicotinic enhancement of proliferation in bovine and porcine cerebral microvascular endothelial cells. Biol Pharm Bull 27:1951–1956[CrossRef][Medline]
  20. Wada T, Sasaoka T, Ishiki M, Hori H, Haruta H, Ishihara H, Kobayashi M 1999 Role of the Src homology 2 (SH2) domain and C-terminus tyrosine phosphorylation sites of SH2-containing inositol phosphatase (SHIP) in the regulation of insulin-induced mitogenesis. Endocrinology 140:4585–4594[Abstract/Free Full Text]
  21. Hogg RC, Raggenbass M, Bertrand D 2003 Nicotinic acetylcholine receptors: from structure to brain function. Rev Physiol Biochem Pharmacol 147:1–46[Medline]
  22. Dajas-Bailador F, Wonnacott S 2004 Nicotinic acetylcholine receptors and the regulation of neuronal signalling. Trends Pharmacol Sci 25:317–324[CrossRef][Medline]
  23. Alkondon M, Albuquerque EX 2004 The nicotinic acetylcholine receptor subtypes and their function in the hippocampus and cerebral cortex. Prog Brain Res 145:109–120[Medline]
  24. Ward JM, Cockcroft VB, Lunt GG, Smillie FS, Wonnacott S 1990 Methyllycaconitine: a selective probe for neuronal {alpha}-bungarotoxin binding sites. FEBS Lett 270:45–48[CrossRef][Medline]
  25. Meyer EM, Tay ET, Papke RL, Meyers C, Huang GL, de Fiebre CM 1997 3-[2,4-Dimethoxybenzylidene] anabaseine (DMXB) selectively activates rat {alpha}7 receptors and improves memory-related behaviors in a mecamylamine-sensitive manner. Brain Res 768:49–56[CrossRef][Medline]
  26. Meyer EM, Kuryatov A, Gerzanich V, Lindstrom J, Papke RL 1998 Analysis of 3-(4-hydroxy, 2-methoxybenzylidene) anabaseine selectivity and activity at human and rat {alpha}7 nicotinic receptors. J Pharmacol Exp Ther 287:918–925[Abstract/Free Full Text]
  27. Bertrand D, Bertrand S, Ballivet M 1992 Pharmacological properties of the homomeric {alpha}7 receptor. Neurosci Lett 146:87–90[CrossRef][Medline]
  28. Abraham ST, Benscoter HA, Schworer CM, Singer HA 1997 A role for Ca2+/calmodulin-dependent protein kinase II in the mitogen-activated protein kinase signaling cascade of cultured rat aortic vascular smooth muscle cells. Circ Res 81:575–584[Abstract/Free Full Text]
  29. Brinson AE, Harding T, Diliberto PA, He Y, Li X, Hunter D, Herman B, Earp HS, Graves LM 1998 Regulation of a calcium dependent tyrosine kinase in vascular smooth muscle cells by angiotensin II and platelet-derived growth factor. Dependence on calcium and the actin cytoskeleton. J Biol Chem 273:1711–1718[Abstract/Free Full Text]
  30. Ginnan R, Singer HA 2002 CaM kinase II-dependent activation of tyrosine kinases and ERK1/2 in vascular smooth muscle. Am J Physiol Cell Physiol 282:C754–C761
  31. McGlade J, Cheng A, Pelicci G, Pelicci PG, Pawson T 1992 Shc proteins are phosphorylated and regulated by the v-Src and v-Fps protein-tyrosine kinases. Proc Natl Acad Sci U S A 89:8869–8873[Abstract/Free Full Text]
  32. Schwartz RD, Kellar KJ 1983 Nicotinic cholinergic receptor binding sites in the brain: regulation in vivo. Science 220:214–216[Abstract/Free Full Text]
  33. Marks MJ, Pauly JR, Gross SD, Deneris ES, Hermans-Borgmeyer I, Heinemann SF, Collins AC 1992 Nicotine binding and nicotinic receptor subunit RNA after chronic nicotine treatment. J Neurosci 12:2765–2784[Abstract]
  34. Mishina M, Takai T, Imoto K, Noda M, Takahashi T, Numa S, Methfessel C, Sakmann B 1986 Molecular distinction between fetal and adult forms of muscle acetylcholine receptor. Nature 321:406–411[CrossRef][Medline]
  35. Keyser K, Britto L, Schoepfer R, Whiting P, Cooper J, Conroy W, Brozozowska-Prechtl A, Karten H, Lindstrom J 1993 Three subtypes of {alpha}-bungarotoxin-sensitive nicotinic acetylcholine receptors expressed in chick retina. J Neurosci 13:442–454[Abstract]
  36. Heeschen C, Jang JJ, Weis M, Pathak A, Kaji S, Hu RS, Tsao PS, Johnson FL, Cooke JP 2001 Nicotine stimulates angiogenesis and promotes tumor growth and atherosclerosis. Nat Med 7:833–839[CrossRef][Medline]
  37. Bruggmann D, Lips KS, Pfeil U, Haberberger RV, Kummer W 2002 Multiple nicotinic acetylcholine receptor {alpha}-subunits are expressed in the arterial system of the rat. Histochem Cell Biol 118:441–447[CrossRef][Medline]
  38. Heeschen C, Weis M, Aicher A, Dimmeler S, Cooke JP 2002 A novel angiogenic pathway mediated by non-neuronal nicotinic acetylcholine receptors. J Clin Invest 110:527–536[CrossRef][Medline]
  39. Ren K, Puig V, Papke RL, Itoh Y, Hughes JA, Meyer EM 2005 Multiple calcium channels and kinases mediate {alpha}7 nicotinic receptor neuroprotection in PC12 cells. J Neurochem 94:926–933[CrossRef][Medline]
  40. Zhan Y, Kim S, Izumi Y, Izumiya Y, Nakao T, Miyazaki H, Iwao H 2003 Role of JNK, p38, and ERK in platelet-derived growth factor-induced vascular proliferation, migration, and gene expression. Arterioscler Thromb Vasc Biol 23:795–801[Abstract/Free Full Text]
  41. Breese CR, Marks MJ, Logel J, Adams CE, Sullivan B, Collins AC, Leonard S 1997 Effect of smoking history on [3H]nicotine binding in human postmortem brain. J Pharmacol Exp Ther 282:7–13[Abstract/Free Full Text]
  42. Wang F, Nelson ME, Kuryatov A, Olale F, Cooper J, Keyser K, Lindstrom J 1998 Chronic nicotine treatment up-regulates human {alpha}3ß2 but not {alpha}3ß4 acetylcholine receptors stably transfected in human embryonic kidney cells. J Biol Chem 273:28721–28732[Abstract/Free Full Text]
  43. Buisson B, Bertrand D 2001 Chronic exposure to nicotine upregulates the human {alpha}4ß2 nicotinic acetylcholine receptor function. J Neurosci 21:1819–1829[Abstract/Free Full Text]
  44. Kawai H, Berg DK 2001 Nicotinic acetylcholine receptors containing {alpha}7 subunits on rat cortical neurons do not undergo long-lasting inactivation even when up-regulated by chronic nicotine exposure. J Neurochem 78:1367–1378[CrossRef][Medline]
  45. Fenster CP, Whitworth TL, Sheffield EB, Quick MW, Lester RA 1999 Upregulation of surface {alpha}4 ß2 nicotinic receptors is initiated by receptor desensitization after chronic exposure to nicotine. J Neurosci 19:4804–4814[Abstract/Free Full Text]
  46. Peng X, Gerzanich V, Anand R, Whiting PJ, Lindstrom J 1994 Nicotine-induced increase in neuronal nicotinic receptors results from a decrease in the rate of receptor turnover. Mol Pharmacol 46:523–530[Abstract]
  47. Sallette J, Pons S, Devillers-Thiery A, Soudant M, Prado de Carvalho L, Changeux JP, Corringer PJ 2005 Nicotine upregulates its own receptors through enhanced intracellular maturation. Neuron 46:595–607[CrossRef][Medline]
  48. Buisson B, Bertrand D 2002 Nicotine addiction: the possible role of functional upregulation. Trends Pharmacol Sci 23:130–136[CrossRef][Medline]



This article has been cited by other articles:


Home page
 DiabetesHome page
B. C. Bergman, L. Perreault, D. M. Hunerdosse, M. C. Koehler, A. M. Samek, and R. H. Eckel
Intramuscular Lipid Metabolism in the Insulin Resistance of Smoking
Diabetes, October 1, 2009; 58(10): 2220 - 2227.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
T. Wada, S. Ohshima, E. Fujisawa, D. Koya, H. Tsuneki, and T. Sasaoka
Aldosterone Inhibits Insulin-Induced Glucose Uptake by Degradation of Insulin Receptor Substrate (IRS) 1 and IRS2 via a Reactive Oxygen Species-Mediated Pathway in 3T3-L1 Adipocytes
Endocrinology, April 1, 2009; 150(4): 1662 - 1669.
[Abstract] [Full Text] [PDF]

Home page
 Pharmacol. Rev.Home page
S. D. Buckingham, A. K. Jones, L. A. Brown, and D. B. Sattelle
Nicotinic Acetylcholine Receptor Signalling: Roles in Alzheimer's Disease and Amyloid Neuroprotection
Pharmacol. Rev., March 1, 2009; 61(1): 39 - 61.
[Abstract] [Full Text] [PDF]

Home page
 Toxicol SciHome page
R.-J. Chen, Y.-S. Ho, H.-R. Guo, and Y.-J. Wang
Rapid Activation of Stat3 and ERK1/2 by Nicotine Modulates Cell Proliferation in Human Bladder Cancer Cells
Toxicol. Sci., August 1, 2008; 104(2): 283 - 293.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
J. Arredondo, A. I. Chernyavsky, D. L. Jolkovsky, K. E. Pinkerton, and S. A. Grando
Receptor-mediated tobacco toxicity: acceleration of sequential expression of {alpha}5 and {alpha}7 nicotinic receptor subunits in oral keratinocytes exposed to cigarette smoke
FASEB J, May 1, 2008; 22(5): 1356 - 1368.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wada, T.
Right arrow Articles by Sasaoka, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wada, T.
Right arrow Articles by Sasaoka, T.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals