Ken H. Tachiki

Tacrine in the Treatment of Alzheimer’s Disease

The initial report of the effectiveness of tacrine or tetrahydroaminoacridine (THA) in the treatment of some patients with Alzheimers disease has been confirmed by further study of additional subjects and by preliminary reports from other investigators. The major side effect, elevation of liver enzymes, is shown to be reversible, dose-dependent and without significant hepatic pathology. Therapeutic serum concentrations of THA vary between 7 and 20 ng/ml. In addition to its presumed action as an acetylcholinesterase inhibitor, data are presented demonstrating marked effects on other brain neurotransmitters in animal models.

A Targeted Thyroid Hormone Receptor α Gene Dominant-Negative Mutation (P398H) Selectively Impairs Gene Expression in Differentiated Embryonic Stem Cells

Thyroid hormone and retinoic acid (RA) are essential for normal neural development in vivo, yet all in vitro differentiation strategies of embryonic stem (ES) cells use only RA. We developed a novel differentiation strategy of mouse ES cells using T(3). A dominant-negative knock-in point mutation (P398H) was introduced into the thyroid hormone receptor alpha gene to determine the influence of T(3) on ES cell differentiation. Differentiation promoted by T(3) (1 nM), RA (1 microM), or combined T(3)/RA was assessed in wild-type (wt) and mutant (m) ES cells on the basis of neuronal-specific gene expression and cell cycle. T(3) alone stimulated neural differentiation in a similar fashion as that seen with RA in both wtES and mES cells. Expression of neurogranin and Ca(2+)/calmodulin-dependent kinase IV mRNA (identified in vivo as T(3)-regulated genes), however, was markedly reduced in mES, compared with wtES cells. RA treatment enhanced apoptosis, significantly greater than that seen with T(3) stimulation. T(3) treatment given with RA significantly reduced the apoptotic effects of RA, an effect not seen in mES cells. T(3)-induced ES cell neural differentiation of thyroid hormone alpha mutant and wtES cells provides an in vitro model to study T(3)-dependent gene regulation in neural development. This system could also be used to identify novel T(3)-regulated genes. The modulation of the apoptotic effects of RA by T(3) may have implications for stem cell therapy.

PAC1 and PACAP expression, signaling, and effect on the growth of HCT8, human colonic tumor cells.

The pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor (PAC1) is a heptahelical, G protein-coupled receptor that has been shown to be expressed by non-squamous lung cancer and breast cancer cell lines, and to be coupled to the growth of these tumors. We have previously shown that PACAP and its receptor, PAC1, are expressed in rat colonic tissue. In this study, we used polyclonal antibodies directed against the COOH terminal of PAC1, as well as fluorescently labeled PACAP, Fluor-PACAP, to demonstrate the expression of PAC1 on HCT8 human colonic tumor cells, using FACS analysis and confocal laser scanning microscopy. Similarly, anti-PACAP polyclonal antibodies were used to confirm the expression of PACAP hormone by this cell line. We then investigated the signal transduction properties of PAC1 in these tumor cells. PACAP-38 elevated intracellular cAMP levels in a dose-dependent manner, with a half-maximal (EC(50)) stimulation of approximately 3 nM. In addition, PACAP-38 stimulation caused an increase in cytosolic Ca(2+) concentration [Ca(2+)](i), which was partially inhibited by the PACAP antagonist, PACAP-(6-38). Finally, we studied the potential role of PACAP upon the growth of these tumor cells. We found that PACAP-38, but not VIP, increased the number of viable HCT8 cells, as measured by MTT activity. We also demonstrated that HCT8 cells expressed the Fas receptor (Fas-R/CD95), which was subsequently down-regulated upon activation with PACAP-38, further suggesting a possible role for PACAP in the growth and survival of these tumor cells. These data indicate that HCT8 human colon tumor cells express PAC1 and produce PACAP hormone. Furthermore, PAC1 activation is coupled to adenylate cyclase, increase cytosolic [Ca(2+)](i), and cellular proliferation. Therefore, PACAP is capable of increasing the number of viable cells and regulating Fas-R expression in a human colonic cancer cell line, suggesting that PACAP might play a role in the regulation of colon cancer growth and modulation of T lymphocyte anti-tumoral response via the Fas-R/Fas-L apoptotic pathway.

Acyl-Coenzyme A Causes Ca2+ Release in Pancreatic Acinar Cells

The regulation of cytosolic Ca2+ is important for a variety of cell functions. One non-inositol 1,4,5-trisphosphate (IP3) compound that may regulate Ca2+ is palmitoyl-coenzyme A (CoA), a fatty acid-CoA that is reported to cause Ca2+ release from intracellular stores of oocytes, myocytes, and hepatocytes. To study the role of palmitoyl-CoA in the pancreatic acinar cell, rat pancreatic acini were isolated by collagenase digestion, permeablized with streptolysin O, and the release of Ca2+ from internal stores was measured with fura-2. Palmitoyl-CoA released Ca2+ from internal stores (EC50 = 14 μm). The palmitoyl-CoA-sensitive pool was distinct from, and overlapping with the IP3-sensitive Ca2+pool. The effects of submaximal doses of IP3 or cyclic ADP-ribose plus palmitoyl-CoA were additive. Fatty acid-CoA derivatives with carbon chain lengths of 16–18 were the most potent and efficacious. Ryanodine and caffeine or elevated resting [Ca2+] sensitized the Ca2+ pool to the actions of palmitoyl-CoA. Fatty acid-CoA levels in pancreatic acini were measured by extraction with 2-propanol/acetonitrile, followed by separation and quantification using reverse phase high performance liquid chromatography, and were found to be 10.17 ± 0.93 nmol/mg protein. These data suggest the presence of an IP3-insensitive palmitoyl-CoA-sensitive Ca2+store in pancreatic acinar cells and suggest that palmitoyl-CoA may be needed for Ca2+-induced Ca2+ release.

PACAP regulation of secretion and proliferation of pure populations of gastric ECL cells.

The gastric enterochromaffin-like (ECL) cell plays a major role in the regulation of gastric acid secretion. We have previously described that Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) is present on myenteric neurons in the rat and colocalizes with its high-affinity receptor, PAC1, expressed on the surface of gastric ECL cells. The study of ECL cell physiology has been hampered by the inability to isolate and purify ECL cells to homogeneity. Density gradient elutriation alone yields only 65–70% purity of ECL cells. In the present study, we used fluorescence-activated cell sorting (FACS) with a novel fluorescent ligand, Fluor-PACAP-38, for isolating pure ECL cells. FACS was used to isolate ECL cells based on their relatively small size, low density, and ability to bind the fluorescent ligand Fluor-PACAP-38. The sorted cells were unambiguously identified as ECL cells by immunohistochemical analysis using anti-PACAP type-I (PAC1), anti-histidine decarboxylase (HDC), and anti-somatostatin antibodies. Further confocal microscopy demonstrated that Fluor-PACAP-38, a ligand with a higher affinity for PAC1, bound to extracellular receptors of these FACS-purified cells. FACS yielded an average of 2 million ECL cells/4 rat stomachs, and >99% of the sorted cells were positive for PAC1 receptor and HDC expression. The absence of immunohistochemical staining for somatostatin indicated lack of contamination by gastric D cells, which are similar in size and shape to the ECL cells. Internalization of PACAP receptors and a rapid Ca2+ response in purified ECL cells were observed upon PACAP activation, suggesting that these cells are viable and biologically active. These ECL cells demonstrated a dose-dependent stimulation of proliferation in response to PACAP, with a maximum of 30% proliferation at a concentration of 10−7M. Microarray studies were perfor med to confirm the expression of genes specific for ECL cells. These results demonstrate that rat gastric ECL cells can be isolated to homogeneity by using a combination of density gradient centrifugation, followed by cell sorting using Fluor-PACAP. These techniques now allow microarray studies to be performed in ECL cells to characterize their functional gene expression and will facilitate pharmacological, biochemical, and molecular studies on ECL cell function.

Autonomic and Skeletal Muscle Responses to Nonelectrical Cutaneous Stimulation

Cutaneous stimulation has had a long history as a method of pain control. While there is general agreement that modern techniques such as electrical stimulation and massage often provide relief from acute pain and may in some cases significantly affect chronic pain, the mechanism by which these techniques affect pain remains unclear. Significant attention has been focused on the effects of stimulation on the autonomic nervous system (ANS) along with the increasing evidence of important ANS modulation of nociceptive activity throughout the pain pathway. However, inconsistent results on the presence and direction of ANS changes from cutaneous stimulation characterize the recent literature. The present study investigated a nonelectrical cutaneous stimulation device, the Dermapoints Massageroller, as well as an active placebo massage. The results indicate that the Dermapoints Massageroller has both general effects associated with simple skin stimulation (such as increased skin temperature), as well as specific effects from increased stimulation by the toothed design of the roller. These specific effects include decreased muscle tension (at least for some muscle sites) and increased sympathetic activation. The results are consistent with a model of activation of Pacinian receptors as a possible mechanism for the antinociceptive properties of cutaneous stimulation.

Phosphatidylserine inhibition of monoamine oxidase in platelets of schizophrenics

Phosphatidylserine (PS) has recently been reported to be a specific inhibitor of B-type monoamine oxidase (MAO-B). The effect of added PS liposomes on platelet MAO-B activity was examined in two schizophrenic groups (paranoid and a mixture of residual/undifferentiated) and in normal controls. PS was a potent partial-mixed-uncompetitive inhibitor of the platelet enzyme, whereas other phospholipids tested were without effect. The PS concentration required for 50% inhibition was significantly higher for the paranoid relative to the residual/undifferentiated group and controls. This correlated with a lower mean basal MAO-B activity in this group. Hill plots, as a measure of the concentration dependence of PS sensitivity, however, revealed a similarity between the two schizophrenic groups in regard to inhibitor binding properties. Mean Hill coefficients for both groups were significantly different from the controls. The results were consistent with an in vivo role for PS as an allosteric regulator of platelet MAO-B.

Electroencephalographic Cartography of Conscious States

We analyzed monopolar recordings obtained from a brain area implicated in the implementation of conscious intention--the Supplementary Motor Area. Other monopolar recordings were taken at F3, F4, C3, C4, P3, P4, O1, O2, F7, F8, T3, T4, T5, T6, and Cz using the standard international (10/20) pattern with linked ears as reference. Using on line fast-fourier transforms of brainwave signals and computer displays with one-second updates of 1Hz wide brainwave bands of 5, 7, 10, 12, 14, 16, 20 and 28 Hz, specific brainwave signatures were identified for attentional, cognitive, imaginal, and somatosensory states. This first, and thus limited, cartography of consciousness is discussed in light of the cognitive descriptions clinically ascribed to the commonly used brainwave frequency bands designated as Delta, Theta, Alpha, and Beta.