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Previous phase I DNA-vaccine based clinical trials using Mammaglobin-A (Mam-A), a human breast tumor associated antigen (TAA), demonstrated that this agent was safe and efficient at treating patients with stage IV breast cancer. The long-term success of cancer vaccines is limited by the diminished expression of human leukocyte antigen (HLA) class I molecules in the tumor microenvironment. The current study assessed the impact of various selenocompounds on the expression of HLA class I molecules in THP-1 cells, an apparent proficient antigen that presents a human monocyte-like cell line, and their eventual activation of MamA2.1 (HLA-A2 immunodominant epitope of Mam-A) specific cytotoxic CD8(+) T lymphocytes (CTLs). The results revealed that, following treatment with methylselenol producing compounds [methylselenic acid (MSA) and dimethylselenide (DMDSe)], the expression of HLA class-I was increased and components involved with the antigen presentation machinery of THP-1 cells were upregulated. Furthermore, CTLs activated by MamA2.1 peptide presenting THP-1 cells, pre-treated with MSA and DMDSe, demonstrated an enhanced cytotoxicity in HLA-A2(+)/Mam-A(+) AU565 and UACC-812 breast cancer cell lines when compared with CTLs activated by THP-1 cells without drug treatment. However, no significant cytotoxicity was observed under similar conditions in HLA-A2(+)/Mam-A(−) MCF-7 and MDA-MB-231 breast cancer cell lines. The results indicated that treatment with methylselenol producing compounds retained antigen-dependent activation of CD8(+) T cells. The data of the current study demonstrated that MSA and DMDSe potentiated effector cytotoxic responses following TAA specific activation of CTLs, indicating their future role as vaccine adjuvants in cancer immunotherapy.

Simple Summary Cell based immunotherapy is rapidly emerging as a promising cancer treatment. Salt (sodium chloride) treatment to immune cell cultures is known to induce inflammatory activation. In our current study, we analyzed the anti-cancer ability of salt treatment on immune cells outside the host followed by reinfusion into the host. Using a pre-clinical breast cancer model, we demonstrated that external salt treatment on T-cell subset of immune cells produced a viable anti-cancer response, which may have future human clinical application. Abstract Cell based immunotherapy is rapidly emerging as a promising cancer treatment. A modest increase in salt (sodium chloride) concentration in immune cell cultures is known to induce inflammatory phenotypic differentiation. In our current study, we analyzed the ability of salt treatment to induce ex vivo expansion of tumor-primed CD4 (cluster of differentiation 4)+T cells to an effector phenotype. CD4+T cells were isolated using immunomagnetic beads from draining lymph nodes and spleens from tumor bearing C57Bl/6 mice, 28 days post-injection of Py230 syngeneic breast cancer cells. CD4+T cells from non-tumor bearing mice were isolated from splenocytes of 12-week-old C57Bl/6 mice. These CD4+T cells were expanded ex vivo with five stimulation cycles, and each cycle comprised of treatment with high salt (Δ0.035 M NaCl) or equimolar mannitol controls along with anti-CD3/CD28 monoclonal antibodies for the first 3 days, followed by the addition of interleukin (IL)-2/IL-7 cytokines and heat killed Py230 for 4 days. Ex vivo high salt treatment induced a two-fold higher Th1 (T helper type 1) expansion and four-fold higher Th17 expansion compared to equimolar mannitol treatment. Importantly, the high salt expanded CD4+T cells retained tumor-specificity, as demonstrated by higher in vitro cytotoxicity against Py230 breast cancer cells and reduced in vivo syngeneic tumor growth. Metabolic studies revealed that high salt treatment enhanced the glycolytic reserve and basal mitochondrial oxidation of CD4+T cells, suggesting a role of high salt in enhanced pro-growth anabolic metabolism needed for inflammatory differentiation. Mechanistic studies demonstrated that the high salt induced switch to the effector phenotype was mediated by tonicity-dependent transcription factor, TonEBP/NFAT5. Using a transgenic murine model, we demonstrated that CD4 specific TonEBP/NFAT5 knock out (CD4cre/creNFAT5flox/flox) abrogated the induction of the effector phenotype and anti-tumor efficiency of CD4+T cells following high salt treatment. Taken together, our data suggest that high salt-mediated ex vivo expansion of tumor-primed CD4+T cells could induce effective tumor specific anti-cancer responses, which may have a novel cell-based cancer immunotherapeutic application.

Chronic inflammation is known to play a critical role in the development of cancer. Recent evidence suggests that high salt in the tissue microenvironment induces chronic inflammatory milieu. In this report, using three breast cancer-related cell lines, we determined the molecular basis of the potential synergistic inflammatory effect of sodium chloride (NaCl) with interleukin-17 (IL-17). Combined treatment of high NaCl (0.15 M) with sub-effective IL-17 (0.1 nM) induced enhanced growth in breast cancer cells along with activation of reactive nitrogen and oxygen (RNS/ROS) species known to promote cancer. Similar effect was not observed with equi-molar mannitol. This enhanced of ROS/RNS activity correlates with upregulation of γENaC an inflammatiory sodium channel. The similar culture conditions have also induced expression of pro-inflammatory cytokines such as IL-6, TNFα etc. Taken together, these data suggest that high NaCl in the cellular microenvironment induces a γENaC mediated chronic inflammatory response with a potential pro-carcinogenic effect.

Recent evidence from our laboratory has demonstrated that high salt (Δ0.05 M NaCl) induced inflammatory response and cancer cell proliferation through salt inducible kinase-3 (SIK3) upregulation. As calcium influx is known to effect inflammatory response and drug resistance, we examined the impact of high salt on calcium influx in breast cancer cells. Treatment of MCF-7 and MDA-MB-231 cells with high salt induced an enhanced intracellular calcium intensity, which was significantly decreased by store operated calcium entry (SOCE) inhibitor co-treatment. Further, high salt induced P-glycoprotein (P-gp) mediated paclitaxel drug resistance in breast cancer cells. Murine tumor studies demonstrated that injection of MCF-7 cells cultured in high salt, exerted higher tumorigenicity compared to the basal cultured counterpart. Knock down of SIK3 by specific shRNA inhibited tumorigenicty, expression of SOCE regulators and P-gp activity, suggesting SIK3 is an upstream mediator of SOCE induced calcium influx. Furthermore, small molecule inhibitor, prostratin, exerted anti-tumor effect in murine models through SIK3 inhibition. Taken together, we conclude that SIK3 is an upstream regulator of store operated calcium entry proteins, Orai1 and STIM1, and mediates high salt induced inflammatory cytokine responses and P-gp mediated drug resistance. Therefore, small molecule inhibitors, such as prostratin, could offer novel anti-cancer approaches.

The identities of genes that underlie population variation in adipose tissue development in farm animals are poorly understood. Previous studies in our laboratory have suggested that increased fat tissue involves the expression modulation of an array of genes in broiler chickens. Of special interest are eight genes,FGFR3, EPHB2, IGFBP2, GREM1, TNC, COL3A1, ACBD7, andSCD. To understand their expression regulation and response to dietary manipulation, we investigated their mRNA levels after dietary manipulation during early development. Chickens were fed either a recommended standard or a high caloric diet from hatch to eight weeks of age (WOA). The high caloric diet markedly affected bodyweight of the broiler birds. mRNA levels of the eight genes in the abdominal adipose tissue were assayed at 2, 4, 6, and 8 WOA using RT-qPCR. Results indicate that (1)FGFR3mRNA level was affected significantly by diet, age, and diet:age interaction; (2)COL3AmRNA level was repressed by high caloric diet; (3) mRNA levels ofEPHB2, ACBD7, andSCDwere affected by age; (4) mRNA level ofTNCwas modulated by age:diet interaction; (5) changes inGREM1andIGFBP2mRNA levels were not statistically different.

Current therapies to enhance CNS cholinergic function rely primarily on extracellular acetylcholinesterase (AChE) inhibition, a pharmacotherapeutic strategy that produces dose-limiting side effects. The Na(+)-dependent, high-affinity choline transporter (CHT) is an unexplored target for cholinergic medication development. Although functional at the plasma membrane, CHT at steady-state is localized to synaptic vesicles such that vesicular fusion can support a biosynthetic response to neuronal excitation. To identify allosteric potentiators of CHT activity, we mapped endocytic sequences in the C-terminus of human CHT, identifying transporter mutants that exhibit significantly increased transport function. A stable HEK-293 cell line was generated from one of these mutants (CHT LV-AA) and used to establish a high-throughput screen (HTS) compatible assay based on the electrogenic nature of the transporter. We established that the addition of choline to these cells, at concentrations appropriate for high-affinity choline transport at presynaptic terminals, generates a hemicholinium-3 (HC-3)-sensitive, membrane depolarization that can be used for the screening of CHT inhibitors and activators. Using this assay, we discovered that staurosporine increased CHT LV-AA choline uptake activity, an effect mediated by a decrease in choline K(M) with no change in V(max). As staurosporine did not change surface levels of CHT, nor inhibit HC-3 binding, we propose that its action is directly or indirectly allosteric in nature. Surprisingly, staurosporine reduced choline-induced membrane depolarization, suggesting that increased substrate coupling to ion gradients, arising at the expense of nonstoichiometric ion flow, accompanies a shift of CHT to a higher-affinity state. Our findings provide a new approach for the identification of CHT modulators that is compatible with high-throughput screening approaches and presents a novel model by which small molecules can enhance substrate flux through enhanced gradient coupling.

The pro-inflammatory cytokines, tumor necrosis factor (TNF)-α, and interleukin (IL)-17, have been implicated in the pathogenesis of liver fibrosis. In this study, we investigated the role of TNFα and IL-17 toward induction of profibrotic factor, periostin.HepG2 cells were cultured and treated with inflammatory cytokines, TNFα and IL-17. Computational promoter sequence analysis of the periostin promoter was performed to define the putative binding sites for transcription factors. Transcription factors were analyzed by Western blot and Chromatin Immunoprecipitation. Periostin and transcription factor expression analysis was performed by RT-PCR, Western blot, and fluorescence microscopy. Type I collagen expression from fibroblast cultures was analyzed by Western blot and Sircol soluble collagen assay.Activation of HepG2 Cells with TNFα and IL-17 enhanced the expression of periostin (3.5 and 4.4 fold, respectively p0.05) compared to untreated cells. However, combined treatment with both TNFα and IL-17 at similar concentration demonstrated a 13.3 fold increase in periostin (p0.01), thus suggesting a synergistic role of these cytokines. Periostin promoter analysis and specific siRNA knock-down revealed that TNFα induces periostin through cJun, while IL-17 induced periostin via STAT-3 signaling mechanisms. Treatment of the supernatant from the cytokine activated HepG2 cells on fibroblast cultures induced enhanced expression of type I collagen (9.1 fold, p0.01), indicative of a direct fibrogenic effect of TNFα and IL-17.TNFα and IL-17 induced fibrogenesis through cJun and STAT-3 mediated expression of profibrotic biomarker, periostin. Therefore, periostin might serve as a novel biomarker in early diagnosis of liver fibrosis.

Basement membrane, a specialized ECM that underlies polarized epithelium of eumetazoans, provides signaling cues that regulate cell behavior and function in tissue genesis and homeostasis. A collagen IV scaffold, a major component, is essential for tissues and dysfunctional in several diseases. Studies of bovine and Drosophila tissues reveal that the scaffold is stabilized by sulfilimine chemical bonds (S = N) that covalently cross-link methionine and hydroxylysine residues at the interface of adjoining triple helical protomers. Peroxidasin, a heme peroxidase embedded in the basement membrane, produces hypohalous acid intermediates that oxidize methionine, forming the sulfilimine cross-link. We explored whether the sulfilimine cross-link is a fundamental requirement in the genesis and evolution of epithelial tissues by determining its occurrence and evolutionary origin in Eumetazoa and its essentiality in zebrafish development; 31 species, spanning 11 major phyla, were investigated for the occurrence of the sulfilimine cross-link by electrophoresis, MS, and multiple sequence alignment of de novo transcriptome and available genomic data for collagen IV and peroxidasin. The results show that the cross-link is conserved throughout Eumetazoa and arose at the divergence of Porifera and Cnidaria over 500 Mya. Also, peroxidasin, the enzyme that forms the bond, is evolutionarily conserved throughout Metazoa. Morpholino knockdown of peroxidasin in zebrafish revealed that the cross-link is essential for organogenesis. Collectively, our findings establish that the triad - a collagen IV scaffold with sulfilimine cross-links, peroxidasin, and hypohalous acids - is a primordial innovation of the ECM essential for organogenesis and tissue evolution.

Cholinergic neurons have both a low-affinity and a high-affinity choline transport process. The high-affinity choline transport is sodium dependent and thus it can be referred to as choline cotransport. Choline cotransport has been shown to be up-regulated by neuronal activity. Protein kinase C has also been shown to regulate choline cotransport. Both forms of regulation appear to modulate transport by altering the numbers of choline cotransporters in the nerve terminal membrane. The present study centers on choline cotransporter trafficking in Limulus brain hemi-slice preparations. The competitive, reversible, non-permeant ligand, [3H]hemicholinium-3, was used in binding studies to estimate the relative number of choline cotransporters in plasma membranes. The hemicholinium-3 mustard derivative has been shown to be an irreversible, highly selective, non-permeant ligand for the choline cotransporter, and was also used. Hemicholinium-3 mustard binding to the choline cotransporter blocked [3H]choline transport and [3H]hemicholinium-3 binding. Antecedent elevated potassium exposure of cholinergic tissues has been shown to up-regulate choline transport by the recruitment of additional choline cotransporters to surface membranes. This treatment was also effective in the recruitment of cotransporters following maximal inhibition by hemicholinium-3 mustard of brain hemi-slices. Long-term washout of hemicholinium-3 mustard in hemi-slices resulted in a time-dependent restoration of choline cotransport. Full recovery occurred within 2 h. In uninhibited slice preparations, both staurosporine and chelerythrine, protein kinase C inhibitors, stimulated choline uptake. However, within a 1-h washout recovery of uptake following hemicholinium-3 mustard inhibition, the staurosporine responsive but not chelerythrine responsive transport had returned. On the basis of these findings, we hypothesize the existence of two distinct populations of cycling choline cotransporters, which includes inactive or “silent” transporters.

The involvement of protein kinase C (PKC) in the regulation of [3H]choline cotransport was studied in Limulus brain hemi-slice preparations. The PKC activators, phorbol 12-myristate 13-acetate (PMA) or phorbol 12,13-dibutyrate (PDBu), significantly decreased [3H]choline cotransport. Conversely, the PKC inhibitors, staurosporine (STAURO) and polymyxin B (PMB), each increased [3H]choline cotransport. These PKC inhibitors prevented the phorbol ester-induced reduction of transport. Both the PMA induced decrease and the STAURO induced increase in [3H]choline cotransport were paralleled by respective and comparable changes in [3H]hemicholinium-3 (HC-3) specific binding. Pre-exposure of brain hemi-slices to elevated potassium chloride (120 mM KCl) resulted in a doubling of [3H]choline cotransport and [3H]HC-3 binding. The enhancement of [3H]choline cotransport by STAURO and antecedent 120 mM KCl treatment were additive. PMA did not significantly alter elevated potassium stimulated transport. Moreover, arachidonyltrifluoromethyl ketone (AACOCF3) and quinacrine (QUIN), both phospholipase A2 (PLA2) inhibitors, markedly decreased enhanced [3H]choline transport and [3H]HC-3 binding induced by antecedent exposure to depolarizing concentrations of potassium. These results suggest that PKC and PLA2 are involved in the regulation of [3H]choline cotransport but at different regulatory sites.

The cholinergic neurotoxin AF64A (ethylcholine aziridinium) has been used to selectively destroy the cholinergic system. Due to its structural similarity to choline, this compound may be selectively taken up by the cholinergic terminal via the high-affinity choline transport (HAChT) system to produce persistent and selective cholinergic deficits. The mechanism by which it exerts its cholinotoxicity remains to be elucidated. We have examined the effects of AF64A in the human neuroblastoma cell line, LA-N-2, which has an intact sodium-coupled choline uptake system, and is capable of synthesizing acetylcholine (ACh). AF64A (25, 50 and 100 μM) produced dose-dependent increases in cell kill as measured by colony formation assay. The addition of increasing concentrations (10 −5 , 10 −4 and 10 −3 M) of choline and hemicholinium-3 (HC-3) protected the cells from the cytotoxic effects of AF64A. At the same doses, AF64A also decreased choline acetyltransferase (ChAT) activity. In the presence of the highest concentration of choline or HC-3 (10 −3 M) which produced complete protection against AF64A's cytotoxicity in the colony formation assay, ChAT activity was restored to control values. These results demonstrate that agents that utilize (i.e., choline) or inhibit (i.e., HC-3) the choline uptake system prevented AF64A-induced cytotoxicity and decreases in ChAT activity, in a manner similar to that which has been observed in chick and rat primary cholinergic cultures in vitro. The LA-N-2 neuroblastoma cell line thus serves well as an in vitro model of the cholinergic neuron and provides a useful system to study the mode of cholinotoxicity induced by AF64A.

1. The distribution of several cholinergic components generally accepted as being associated with cholinergic transmission was studied in Limulus nerve and muscle tissue. 2. Acetylcholine (ACh) and choline were distributed among all the tissues assayed, with abdominal ganglia showing the highest concentration of both. 3. Corpora pedunculata showed the greatest enrichment of choline acetyltransferase (ChAT) and the brain nerve roots had the highest levels of acetylcholinesterase (AChE) activity. No direct correlation was found between levels of ChAT and the amount of measurable ACh in a tissue. 4. Tissue/medium (T/M) ratios indicative of high affinity [ 3 H]choline uptake were selectively distributed, ranging from 13.22 to 4.62 in corpora pedunculata, circumoesophageal ring, abdominal ganglia and cardiac ganglion. All these tissues, with the exception of cardiac ganglion, released [ 3 H]ACh in a Ca 2+ dependent fashion in response to treatment with elevated potassium (90 mM). 5. Cardiac ganglion was found not to synthesize [ 3 H]ACh and had no detectable levels of ChAT activity. 6. These results suggest that high affinity [ 3 H]choline uptake is not exclusively associated with the synthesis of [ 3 H]ACh and hence is not a unique property of cholinergic neurons and/or terminals.

The intent of this study was to determine whether the drug 2-(4-phenylpiperidino)cyclohexanol (AH 5183 or vesamicol) might inhibit the veratridine-induced increase in acetylcholine (ACh) synthesis by reducing the veratridine-induced activation of a detergent-soluble choline-O-acetyltransferase (EC 2.3.1.6; ChAT) fraction associated with a vesicle-bound store of ACh. When minces of rat hippocampal tissue were loaded with [14C]choline and subsequently depolarized with veratridine, an increase in the synthesis of [14C]ACh occurred that could be abolished by l-AH 5183 (75 nM). When minces were depolarized with veratridine in the presence of l-AH 5183 (75 nM), the depolarization-induced activation of a detergent-soluble ChAT fraction associated with a vesicle-bound store of ACh was blocked. Conversely, the veratridine-induced activation of a water-soluble ChAT fraction believed to be cytosolic was not. AH 5183 also blocked the repletion of the vesicle-bound store with newly synthesized ACh following veratridine-induced depletion of ACh, a result that appeared to be mediated by an effect on the synthesis of ACh at the vesicular surface. These results suggest that veratridine depolarization of rat hippocampal nerve terminals stimulates the synthesis of ACh by activating a detergentsoluble fraction of ChAT closely associated with synaptic vesicle release sites. ACh synthesis and transport at the vesicular surface may be influenced by a common AH 5183-sensitive regulatory protein.

The effect of L- and D-stereoisomers of 2-(4-phenylpiperidino) cyclohexanol (AH 5183) on the spontaneous release of acetylcholine (ACh) from rat hippocampal tissue was studied. L-AH 5183 was approximately 100 times more potent than was D-AH 5183 in reducing spontaneous ACh release. Spontaneous ACh release was also temperature dependent. These results may suggest that the spontaneous release of ACh from brain tissue is carrier-mediated.

1. The transport of [3H]choline by the corpora pedunculata of the circumoesophageal ring gland (brain) of Limulus polyphemus was studied. Corpora pedunculata slices were incubated individually in Chao's solution containing 0.01 μM [3H]choline at room temperature (25 ± 2°C) and readily accumulated the radiolabel from the extracellular environment. 2. The corpora pedunculata uptake of [3H]choline was linear over 60 min. 3. The kinetic analysis indicated the existence of dual uptake systems for choline within the corpora pedunculata, a high affinity choline uptake process (Km = 0.54 μM and Vmax = 0.037 pmoles/mg/min) and a low affinity process (Km = 137 μM and Vmax = 6.3 pmoles/mg/min). 4. The high affinity choline transport system was dependent on sodium ions and was inhibited by micromolar concentrations of hemicholinium-3. 5. The pre-exposure of the corpora pedunculata to Chao's solution containing 90 mM potassium for 15 min resulted in a 24% increase in the velocity of the high affinity choline uptake process (Vmax = 0.046 pmoles/mg/min). 6. The 90 mM potassium Chao's pretreatment stimulated a substantial increase in the synthesis of [3H]acetyleholine by the corpora pedunculata. 7. The results suggest that the high affinity choline uptake process within the Limulus corpora pedunculata is associated with the synthesis of the transmitter acetylcholine, presumably within cholinergic terminals in this tissue.

1. The Limulus cardiac ganglion high affinity choline uptake system (HAChUS) was inhibited 40, 51 and 64% following pre-exposure to 10, 100 and 500 μM vinblastine, respectively. 2. In contrast, high affinity uptake of choline in the Limulus corpora pedunculata and abdominal ganglia, tissues in which a cholinergic function has been described, were unaffected. 3. In pulse-chase experiments, the cardiac ganglion was incubated in 0.1 μM [ 3 H]choline for 60 min and then switched to an incubation medium containing 1 mM unlabelled choline for varying periods of time. 4. Under these conditions, a 3-fold increase of radiolabel above basal level was measured in the pellet fraction within 2 hr of post-labelling incubation. 5. Prior exposure of the ganglion to 500 μM vinblastine completely eliminated this increase of radioactivity in the pellet fraction. 6. Treatment of the radiolabelled pellet fraction with phospholipase C resulted in the solubilization of 72% of the radiolabel. 7. Ten (10) μM 5-hydroxytryptamine (5-HT), a concentration previously shown to inhibit spontaneous electrical activity within the cardiac ganglion, resulted in a 40% decrease in high affinity choline uptake in this tissue selectively. 8. These results are consistent with the view that a probable role of the Limulus cardiac ganglion HAChUS is the supply of choline subserving the synthesis of membrane phospholipid. 9. It is further speculated that this membrane phospholipid synthesis may be associated with synaptic vesicle turnover.

1. 1. The cardiac ganglion of the horseshoe crab, Limulus polyphemus , was incubated in Chao's solution containing 0.01 μM [ 3 H]choline at room temperature (25 ± 2°C) and the ganglion readily accumulated the radiolabel. 2. 2. The ganglion uptake of [ 3 H]choline was linear over 60 min. 3. 3. Kinetic analysis revealed dual choline uptake systems within the cardiac ganglion, a high affinity uptake system ( K m = 2.2 μ M, V max = 0.16pmoles/mg/min) and a low affinity system ( K m = 92.3 μ M, V max = 3.08 prnoles/mg/min). 4. 4. The high affinity uptake system was sodium-dependent and inhibited by micromolar concentrations of hemicholinium-3. 5. 5. A 15 min pre-exposure of the ganglion to Chao's solution containing 90 mM potassium stimulated a significant increase in choline uptake. 6. 6. There was no detectable synthesis of [ 3 H]acetylcholine from the [ 3 H]choline taken up by the cardiac ganglion. The major portion of the extraetable label appeared in a fraction which co-electrophoresed with phosphorylcholine. 7. 7. These results suggest that the sodium-dependent high affinity [ 3 H]choline uptake system of the cardiac ganglion subserves a specific requirement for choline which is unrelated to a cholinergic function.

1. Comparative studies of [ 3 H]choline accumulation were done in the Limulus corpora pedunculata, abdominal ganglia and cardiac ganglion. Dual uptake processes for choline were found in all three tissues. 2. In acute experiments, the corpora pedunculata high affinity choline uptake system showed exclusive sensitivity to ouabain. Prolonged exposure to ouabain revealed that the HAChUS of all three tissues were significantly inhibited. 3. The metabolism of [ 3 H]choline transported via the high affinity process in the three tissues was studied. [ 3 H]Acetylcholine was a major product of the [ 3 H]choline taken up by the corpora pedunculata and the abdominal ganglia. 4. Phosphorylcholine was the major product seen in cardiac ganglion extracts and occurred in significant proportions in abdominal ganglia extracts. 5. [ 3 H]Acetylcholine was not detected in cardiac ganglion extracts. 6. Treatment with either lithium chloride or hemicholinium-3 markedly inhibited high affinity uptake of [ 3 H]choline in all three tissues.