Jennifer K. Stewart

Catecholamines in a macrophage cell line

This study provides the first evidence for catecholamine synthesis and release in the RAW264.7 cell line, an important macrophage model. Although catecholamines were low in unstimulated cells, activation with lipopolysaccharide (LPS) induced tyrosine hydroxylase (TH) mRNA and increased extracellular norepinephrine and intracellular dopamine within 48 h. The catecholamine synthesis inhibitor alpha-methyl-para-tyrosine (alpha-mpt) decreased extracellular norepinephrine levels, suggesting release and rapid turnover of newly synthesized norepinephrine. High concentrations of dopamine or norepinephrine (>/=100 microM) decreased proliferation and increased apoptosis of macrophages. These anti-proliferative effects were prevented by simultaneous treatment with the anti-oxidant ascorbic acid. Pre-incubation with a glutathione synthesis inhibitor (L-buthionine-[S,R]-sulfoximine [L-BSO]) increased sensitivity to catecholamine-stimulated apoptosis, suggesting that glutathione protects macrophages from both endogenous and exogenous catecholamines.

Catecholamines in murine bone marrow derived mast cells

Cultured murine bone marrow derived mast cells (BMMC) were found to store high levels of dopamine (3753+/-844 pg/10(7) cells) and occasionally produce norepinephrine and epinephrine. The catecholamine synthesis inhibitor, alpha-methyl-para-tyrosine, decreased intracellular catecholamine concentrations, and activation with ionomycin stimulated dopamine release. Neither dopaminergic receptor antagonists nor exogenous dopamine < or =10 microM affected IL-3-induced cell proliferation. High exogenous dopamine (20-100 microM) decreased proliferation and increased apoptosis, and the anti-oxidant ascorbic acid prevented these effects. Increased expression of the anti-apoptotic factor Bcl-2 or loss of pro-apoptotic Bax expression attenuated dopamine-induced apoptosis, suggesting the apoptosis proceeds through a mitochondrial pathway.

Autocrine actions of macrophage-derived catecholamines on interleukin-1β

Previous studies indicate that norepinephrine and epinephrine modulate production of interleukin-1(beta) (IL-1(beta)) by activated macrophages, but it is not known if macrophage-derived catecholamines affect IL-1(beta). In this study, recruited peritoneal macrophages from CBA/J female mice were activated with lipopolysaccharide (LPS) and treated with vehicle or adrenergic receptor antagonists for 24 h. Extracellular and intracellular levels of IL-1(beta) were measured with ELISA. Treatment with the beta-adrenergic receptor antagonists propranolol or ICI 118,551 increased LPS-induced production of IL-1(beta), whereas treatment with the alpha-adrenergic antagonists phentolamine or yohimbine decreased IL-1(beta). These findings demonstrate that adrenergic receptor antagonists unmask autocrine actions of macrophage-derived catecholamines on IL-1(beta) that may influence the inflammatory response.

Peritoneal macrophages express the serotonin transporter.

Although it is known that macrophages take up serotonin, a specific monoamine transporter has not been identified in macrophages. In this study, mRNA coding for the serotonin transporter (SERT) was detected with the reverse transcription-polymerase chain reaction (RT-PCR) in recruited mouse peritoneal macrophages. Sequencing confirmed the identity of the RT-PCR product to mouse SERT mRNA. SERT protein was detected by Western blotting. Macrophage activation with lipopolysaccharide had no effect on expression of SERT mRNA or protein. Consistent with expression of a functional SERT, specific uptake of (3)H-serotonin in macrophages was sodium dependent and inhibited by fluoxetine (IC(50) 6.9 nM) and desipramine (IC(50) 32 nM) but not by nisoxetine or reserpine.

Phenylethanolamine N-methyltransferase mRNA in rat spleen and thymus.

Phenylethanolamine N-methyltransferase (PNMT), the final enzyme in the biosynthesis of epinephrine, has been detected in rat and human spleen with radioenzymatic assays, but the presence of PNMT has not been examined in other lymphoid tissues. Using the reverse transcription polymerase chain reaction (RT-PCR) and Southern blot analysis, we tested for PNMT mRNA in rat spleen and thymus. A single PCR fragment from spleen, thymus, adrenal and brainstem gave a strong hybridization signal with a PNMT cDNA probe, whereas a PCR fragment from liver was only faintly visible on Southern blots. These findings indicate that the PNMT gene is expressed in spleen and thymus and raise the possibility that lymphoid organs synthesize epinephrine as an intrinsic regulator.

Platinum anticancer agents and antidepressants: desipramine enhances platinum-based cytotoxicity in human colon cancer cells

A unique synergistic effect on platinum drug cytotoxicity is noted in the presence of the tricyclic antidepressant desipramine. Desipramine is used for treating neuropathic pain, particularly in prostate cancer patients. The clinically used drugs cisplatin (cis-[PtCl2(NH3)2]), oxaliplatin [1,2-diaminocyclohexaneoxalatoplatinum(II)], and the cationic trinuclear agent BBR3464 [{trans-PtCl(NH3)2}2-μ-(trans-Pt(NH3)2(H2N(CH2)6NH2)2)]4+, which has undergone evaluation in phase II clinical trials for activity in lung and ovarian cancers, were evaluated. Surprisingly, desipramine greatly augments the cytotoxicity of all the platinum-based chemotherapeutics in HCT116 colorectal carcinoma cell lines. Desipramine enhanced cellular accumulation of cisplatin, but had no effect on the accumulation of oxaliplatin or BBR3464, suggesting that enhanced accumulation could not be a consistent means by which desipramine altered the platinum-drug-mediated cytotoxicity. The desipramine/cisplatin combination resulted in increased levels of p53 as well as mitochondrial damage, caspase activation, and poly(ADP ribose) polymerase cleavage, suggesting that desipramine may synergize with cisplatin more than with other platinum chemotherapeutics partly by activating distinct apoptotic pathways. The study argues that desipramine may be a means of enhancing chemoresponsiveness of platinum drugs and the results warrant further investigation. The results emphasize the importance of understanding the differential pharmacological action of adjuvants employed in combinations with cancer chemotherapeutics.

Axonal localization of delta Ca2+/calmodulin-dependent protein kinase II in developing P19 neurons

Ca2+/calmodulin‐dependent protein kinase, type II (CaMK‐II) is an enzyme encoded by four genes (α, β, γ and δ) and traditionally associated with synaptic function in the adult central nervous system, but also believed to play a role during neuronal development. P19 mouse embryonic cells are a model system for neurogenesis and primarily express isozymes of δ CaMK‐II. It is not yet known whether or where δ CaMK‐II is expressed in P19 neurons. Using an antibody specific for the δ CaMK‐II C‐terminal tail, we detected a 20‐fold increase in levels of δ CaMK‐II along axons after 8 days of development. This coincides with increased mRNA and protein levels of δC CaMK‐II, which contains the alternative tail. This follows the initial stages of neurite outgrowth and β3 tubulin expression, which occur after 4 days. δ CaMK‐II co‐localizes with the axonal protein GAP‐43, but not the dendritic microtubule‐associated protein MAP‐2, a known substrate of α CaMK‐II. Like δ CaMK‐II, GAP‐43 shows increased expression after 8 days. These findings demonstrate developmental regulation of the alternative C‐terminal δ CaMK‐II exon and implicate endogenous δ CaMK‐II in axonal development in embryonic cells.

LPS-stimulated tumor necrosis factor-alpha and interleukin-6 mRNA and cytokine responses following acute psychological stress

The purpose of this study was to examine the effect of acute psychological stress on LPS-stimulated TNF-α and IL-6 mRNA expression. Twenty-one healthy male subjects participated in 20 min of acute stress. Blood samples for norepinephrine and LPS-stimulated TNF-α and IL-6 cytokines and mRNA were drawn prior to, immediately after and 1-h after stress. Stress-induced increases in anxiety scores, cortisol, plasma norepinephrine, and heart rate demonstrated that the experimental protocol elicited an acute stress response. LPS-stimulated TNF-α mRNA decreased significantly immediately post-stress and partially recovered at 1h post-stress, whereas LPS-stimulated IL-6 mRNA exhibited a significant change across time, with an increase immediately after stress and a decrease 1h after stress. Trends in LPS-stimulated TNF-α and IL-6 cytokine concentrations followed the patterns of mRNA expression. A negative correlation of body mass index (BMI) and percent change of LPS-stimulated TNF-α mRNA was observed immediately post-stress, and BMI positively correlated with percent change of LPS-stimulated IL-6 cytokine levels immediately following stress. These findings demonstrated that acute psychological stress affects LPS-stimulated IL-6 and TNF-α gene expression. These results also indicate that BMI may impact the effects of psychological stress on cytokine responses to immune challenge. Further examination of the effects of stress on synthesis of other cellular cytokines and investigation of the association of BMI and stress responses will provide a more clear representation of the cytokine responses to acute psychological stress. In addition, studies examining the influence of gender on the response of immune cell subsets to acute stress and the possible mediating effect of BMI are warranted.

Phenylethanolamine N-methyltransferase mRNA in rat hypothalamus and cerebellum

Using the reverse transcription polymerase chain reaction, we detected a single form of phenylethanolamine N-methyltransferase (PNMT) mRNA in hypothalamus and medulla/pons and two forms in cerebellum. These findings indicate that the PNMT gene is expressed in these brain areas and suggest that tissue specific splicing of PNMT mRNA may occur.

Effects of gonadal steroids on catecholamine levels in the brain of the oyster toadfish

We studied the effects of gonadol steroid implants on catecholamine levels in the brain of ovariectomized female toadfish (Opsanus tau L.). Control values for dopamine (DA), norepinephrine (NE) and epinephrine (EPI) in the brain were 2181 +/- 226, 1112 +/- 80 and 6.5 +/- 2.02 ng/g, respectively. Compared to controls, 17 beta-estradiol decreased brain levels of DA to 547 +/- 261, and dihydrotestosterone decreased DA to 444 +/- 290 ng/g (P less than 0.001). Furthermore, dihydrotestosterone increased brain levels of EPI to 15.0 +/- 4.7 ng/g. The steroids had no effect on brain levels of NE. These results suggest a role for both androgens and estrogens in regulating brain levels of DA and EPI in toadfish.