Effects of midazolam, pentobarbital and ketamine on the mRNA expression of ion channels in a model organism Daphnia pulex

BACKGROUND: Over the last few decades intensive studies have been carried out on the molecular targets mediating general anesthesia as well as the effects of general anesthetics. The γ-aminobutyric acid type A receptor (GABA(A)R) has been indicated as the primary target of general anaesthetics such as propofol, etomidate and isoflurane, and sedating drugs including benzodiazepines and barbiturates. The GABA(A)R is also involved in drug tolerance and dependence. However, the involvement of other ion channels is possible. METHODS: Using reverse transcription and quantitative PCR techniques, we systematically investigated changes in the mRNA levels of ion channel genes in response to exposure to midazolam, pentobarbital and ketamine in a freshwater model animal, Daphnia pulex. To retrieve the sequences of Daphnia ion channel genes, Blast searches were performed based on known human or Drosophila ion channel genes. Retrieved sequences were clustered with the maximum-likelihood method. To quantify changes in gene expression after the drug treatments for 4 hours, total RNA was extracted and reverse transcribed into cDNA and then amplified using quantitative PCR. RESULTS: A total of 108 ion channel transcripts were examined, and 19, 11 and 11 of them are affected by midazolam (100 μM), pentobarbital (200 μM) and ketamine (100 μM), respectively, covering a wide variety of ion channel types. There is some degree of overlap with midazolam- and pentobarbital-induced changes in the mRNA expression profiles, but ketamine causes distinct changes in gene expression pattern. In addition, flumazenil (10 μM) eliminates the effect of midazolam on the mRNA expression of the GABA(A) receptor subunit Rdl, suggesting a direct interaction between midazolam and GABA(A) receptors. CONCLUSIONS: Recent research using high throughput technology suggests that changes in mRNA expression correlate with delayed protein expression. Therefore, the mRNA profile changes in our study may reflect the molecular targets not only in drug actions, but also in chronic drug addiction. Our data also suggest the possibility that hypnotic/anesthetic drugs are capable of altering the functions of the nervous system, as well as those non-nerve tissues with abundant ion channel expressions.