Identification of mRNA for endocannabinoid biosynthetic enzymes within hippocampal pyramidal cells and CA1 stratum radiatum interneuron subtypes using quantitative real-time polymerase chain reaction.

The hippocampus is required for short-term memory and contains both excitatory pyramidal cells and inhibitory interneurons. These cells exhibit various forms of synaptic plasticity, the mechanism underlying learning and memory. More recently, endocannabinoids were identified to be involved in synaptic plasticity. Our goal was to describe the distribution of endocannabinoid biosynthetic enzymes within CA1 stratum radiatum interneurons and CA3/CA1 pyramidal cells. We extracted mRNA from single interneurons and pyramidal cells and used real-time quantitative polymerase chain reaction (RT-PCR) to detect the presence of 12-lipoxygenase, N-acyl-phosphatidylethanolamine-specific phospholipase D, diacylglycerol lipase α, and type I metabotropic glutamate receptors, all known to be involved in endocannabinoid production and plasticity. We observed that the expression of endocannabinoid biosynthetic enzyme mRNA does occur within interneurons and that it is coexpressed with type I metabotropic glutamate receptors, suggesting interneurons have the potential to produce endocannabinoids. We also identified that CA3 and CA1 pyramidal cells express endocannabinoid biosynthetic enzyme mRNA. Our data provide the first molecular biological evidence for putative endocannabinoid production in interneurons, suggesting their potential ability to regulate endocannabinoid-mediated processes, such as synaptic plasticity.

Thyroid hormone effects on LKB1, MO25, phospho-AMPK, phospho-CREB, and PGC-1alpha in rat muscle.

Expression of all of the isoforms of the subunits of AMP-activated protein kinase (AMPK) and AMPK activity is increased in skeletal muscle of hyperthyroid rats. Activity of AMPK in skeletal muscle is regulated principally by the upstream kinase, LKB1. This experiment was designed to determine whether the increase in AMPK activity is accompanied by increased expression of the LKB1, along with binding partner proteins. LKB1, MO25, and downstream targets were determined in muscle extracts in control rats, in rats given 3 mg of thyroxine and 1 mg of triiodothyronine per kilogram chow for 4 wk, and in rats given 0.01% propylthiouracil (PTU; an inhibitor of thyroid hormone synthesis) in drinking water for 4 wk (hypothyroid group). LKB1 and MO25 increased in the soleus of thyroid hormone-treated rats vs. the controls. In other muscle types, LKB1 responses were variable, but MO25 increased in all. In soleus, MO25 mRNA increased with thyroid hormone treatment, and STRAD mRNA increased with PTU treatment. Phospho-AMPK and phospho-ACC were elevated in soleus and gastrocnemius of hyperthyroid rats. Thyroid hormone treatment also increased the amount of phospho-cAMP response element binding protein (CREB) in the soleus, heart, and red quadriceps. Four proteins having CREB response elements (CRE) in promoter regions of their genes (peroxisome proliferator-activated receptor-gamma coactivator-1alpha, uncoupling protein 3, cytochrome c, and hexokinase II) were all increased in soleus in response to thyroid hormones. These data provide evidence that thyroid hormones increase soleus muscle LKB1 and MO25 content with subsequent activation of AMPK, phosphorylation of CREB, and expression of mitochondrial protein genes having CRE in their promoters.

Formation of transient non-protein calcium pores by lysophospholipids in S49 Lymphoma cells.

Palmitoyl-lysophosphatidylcholine promotes a transient calcium influx in lymphoma cells. Previously, it was observed that this influx was accompanied by a temporary increase in propidium iodide permeability that appeared linked to calcium entry. Those studies demonstrated that cobalt or nickel could block the response to lysophosphatidylcholine and raised the question of whether the calcium conductance involved specific channels. This communication describes a series of experiments to address that issue. The time dependence and structural specificity of the responses to lysophosphatidylcholine reinforced the hypothesis of a specific channel or transporter. Nevertheless, observations using patch clamp or calcium channel blockers suggested that this "channel" does not involve proteins. Alternative protein-mediated mechanisms such as indirect involvement of the sodium-calcium exchanger and the sodium-potassium ATPase were also excluded. Experiments with extracellular and intracellular calcium chelators suggested a common route of entry for calcium and propidium iodide. More directly, the ability of lysophosphatidylcholine to produce cobalt-sensitive permeability to propidium iodide was reproduced in protein-free artificial membranes. Finally, the transient nature of the calcium time course was rationalized quantitatively by the kinetics of lysophosphatidylcholine metabolism. These results suggest that physiological concentrations of lysophosphatidylcholine can directly produce membrane pores that mimic some of the properties of specific protein channels.

Endurance training increases LKB1 and MO25 protein but not AMP-activated protein kinase kinase activity in skeletal muscle.

LKB1 complexed with MO25 and STRAD has been identified as an AMP-activated protein kinase kinase (AMPKK). We measured relative LKB1 protein abundance and AMPKK activity in liver (LV), heart (HT), soleus (SO), red quadriceps (RQ), and white quadriceps (WQ) from sedentary and endurance-trained rats. We examined trained RQ for altered levels of MO25 protein and LKB1, STRAD, and MO25 mRNA. LKB1 protein levels normalized to HT (1 +/- 0.03) were LV (0.50 +/- 0.03), SO (0.28 +/- 0.02), RQ (0.32 +/- 0.01), and WQ (0.12 +/- 0.03). AMPKK activities in nanomoles per gram per minute were HT (79 +/- 6), LV (220 +/- 9), SO (22 +/- 2), RQ (29 +/- 2), and WQ (42 +/- 4). Training increased LKB1 protein in SO, RQ, and WQ (P < 0.05). LKB1 protein levels after training (%controls) were SO (158 +/- 17), RQ (316 +/- 17), WQ (191 +/- 27), HT (106 +/- 2), and LV (104 +/- 7). MO25 protein after training (%controls) was 595 +/- 71. Training did not affect AMPKK activity. MO25 but not LKB1 or STRAD mRNA increased with training (P < 0.05). Trained values (%controls) were MO25 (164 +/- 22), LKB1 (120 +/- 16), and STRAD (112 +/- 17). LKB1 protein content strongly correlated (r = 0.93) with citrate synthase activity in skeletal muscle (P < 0.05). In conclusion, endurance training markedly increased skeletal muscle LKB1 and MO25 protein without increasing AMPKK activity. LKB1 may be playing multiple roles in skeletal muscle adaptation to endurance training.

Functional and molecular characterization of neuronal nicotinic ACh receptors in rat CA1 hippocampal neurons.

The molecular and functional properties of neuronal nicotinic acetylcholine receptors (nAChRs) were characterized from CA1 neurons in rat hippocampal slices using single-cell reverse-transcription polymerase chain reaction (RT-PCR) in conjunction with whole-cell patch-clamp recordings. We analysed the presence of the neuronal nAChR subunit mRNAs alpha2-7 and beta2-4, along with the mRNA for the GABAergic markers GAD (glutamic acid decarboxylase) 65 and 67 isoforms, and VGAT (vesicular GABA transporter) in interneurons from the stratum radiatum and stratum oriens, and in CA1 pyramidal neurons. Functional nAChR-mediated currents were detected in both interneuron populations, but not in pyramidal neurons. The neuronal nAChR subunit mRNAs detected in > 20 % of the populations examined were alpha4, alpha5, alpha7 and beta2-4 in stratum radiatum interneurons; alpha2, alpha3, alpha4, alpha7, beta2 and beta3 subunits in stratum oriens interneurons; and beta2-4 in pyramidal neurons. High levels of GABAergic marker mRNAs were detected in both interneuron populations, but not in pyramidal neurons. Significant co-expression of nAChR subunits within individual neurons was detected for alpha3 + alpha5, alpha4 + beta2, alpha4 + beta3, alpha7 + beta2, beta2 + beta3 and beta3 + beta4. The kinetics of the nAChR-mediated currents in response to the application of 100 microM ACh were significantly correlated with the expression of particular nAChR subunits. The alpha3, alpha7 and beta2 nAChR subunits were individually correlated with a fast rise time, the alpha2 nAChR subunit was correlated with a medium rise time, and the alpha4 nAChR subunit was correlated with a slow rise time. The alpha2 and alpha4 nAChR subunits were also significantly correlated with slow desensitization kinetics.

MTSEA potentiates 5-HT3 receptors containing the nicotinic alpha4 subunit.

In order to study the subunit composition of 5-HT3 receptors (5-HT3R), we report that (2-aminoethyl)methanethiosulfonate (MTSEA) can enhance the function of both nicotinic ACh receptors (nAChRs) comprised of alpha4/beta2 subunits, and heteromeric channels assembled from serotonin 5-HT3R and alpha4 nAChR subunits. MTSEA has no effect on homomeric 5-HT3 receptor channels.

Nicotinic receptors in the brain: correlating physiology with function.

Nicotinic ACh receptors (nAChRs) have been implicated in a variety of brain functions, including neuronal development, learning and memory formation, and reward. Although there are substantial data indicating that nAChR subunits are found in many brain regions, the precise cellular roles of these subunits in neuronal functions have remained elusive. Until recently, nAChRs were thought primarily to serve a modulatory role in the brain by regulating neurotransmitter release from nerve terminals. However, new evidence has revealed that nAChRs also function in a postsynaptic role by mediating fast ACh-mediated synaptic transmission in the hippocampus and in the sensory cortex, and are found at somatodendritic as well as nerve terminal sites in the reward system. It is possible that presynaptic and postsynaptic nAChRs mediate changes in the efficacy of synaptic transmission in these brain regions. These changes could underlie the proposed functions of nAChRs in cognitive functions of the hippocampus and cerebral cortex, in neuronal development in the sensory cortex, and in reward.

The nicotinic alpha4 receptor subunit contributes to the lining of the ion channel pore when expressed with the 5-HT3 receptor subunit.

To understand the wide variation of calcium permeability seen in native and recombinant 5-HT3 receptor (5-HT3R) channels, we reported previously the novel hypothesis that the serotonin 5-HT3R subunit can co-assemble with the alpha4 subunit of the nicotinic acetylcholine receptor (van Hooft, J. A., Spier, A. D., Yakel, J. L., Lummis, S. C. R. & Vijverberg, H. P. M. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 11456-11461). To test the hypothesis that the alpha4 subunit contributes to the lining of the pore of the resulting 5-HT3R channel, a mutant nicotinic alpha4 subunit with a reactive cysteine residue engineered into the putative pore region was constructed by substituting the leucine at position 285 (alpha4-L285C). The sulfhydryl-modifying reagent [2-(trimethylammonium) ethyl]methanethiosulfonate (MTSET) reduced the acetylcholine-induced current in oocytes expressing this mutant nicotinic alpha4-L285C subunit along with the nicotinic beta2 subunit by approximately 60%. When the alpha4-L285C subunit was co-expressed with the 5-HT3R subunit, both MTSET and silver nitrate (AgNO3), another cysteine-modifying reagent, significantly reduced the serotonin-induced current. No reduction was seen when the 5-HT3R was expressed alone or with the wild-type alpha4 subunit. These data provide direct molecular evidence that the nicotinic alpha4 subunit co-assembles with the 5-HT3R subunit and forms an integral part of the ion channel pore.