Polymorphisms in and around the Cholesteryl Ester Transfer Protein (CETP) gene have been associated with HDL levels, risk for coronary artery disease (CAD), and response to therapy. The mechanism of action of these polymorphisms has yet to be defined. We used mRNA allelic expression and splice isoform measurements in human liver tissues to identify the genetic variants affecting CETP levels. Allelic CETP mRNA expression ratios in 56 human livers were strongly associated with several variants 2.5–7 kb upstream of the transcription start site (e.g., rs247616 p = 6.4×10−5, allele frequency 33%). In addition, a common alternatively spliced CETP isoform lacking exon 9 (Δ9), has been shown to prevent CETP secretion in a dominant-negative manner. The Δ 9 expression ranged from 10 to 48% of total CETP mRNA in 94 livers. Increased formation of this isoform was exclusively associated with an exon 9 polymorphism rs5883-C>T (p = 6.8×10−10) and intron 8 polymorphism rs9930761-T>C (5.6×10−8) (in high linkage disequilibrium with allele frequencies 6–7%). rs9930761 changes a key splicing branch point nucleotide in intron 8, while rs5883 alters an exonic splicing enhancer sequence in exon 9.

The effect of these polymorphisms was evaluated in two clinical studies. In the Whitehall II study of 4745 subjects, both rs247616 and rs5883T/rs9930761C were independently associated with increased HDL-C levels in males with similar effect size (rs247616 p = 9.6×10−28 and rs5883 p = 8.6×10−10, adjusted for rs247616). In an independent multiethnic US cohort of hypertensive subjects with CAD (INVEST-GENE), rs5883T/rs9930761C alone were significantly associated with increased incidence of MI, stroke, and all-cause mortality in males (rs5883: OR 2.36 (CI 1.29–4.30), p = 0.005, n = 866). These variants did not reach significance in females in either study. Similar to earlier results linking low CETP activity with poor outcomes in males, our results suggest genetic, sex-dependent CETP splicing effects on cardiovascular risk by a mechanism independent of circulating HDL-C levels.

Because of the lack of information about effective analgesics in non-mammalian vertebrates, the potency of various non-opioid agents were tested in a model of analgesia by using Northern grass frogs (Rana pipiens). This alternative model has been used widely for investigating opioid analgesic action. Potential non-opioid analgesics tested included antipsychotic, benzodiazepine, barbiturate, antihistamine, non-steroidal anti-inflammatory (NSAID), and partial opioid agents. Northern grass frogs were acclimated to lab conditions in individual cages. Drugs were administered systemically through the dorsal lymph sac, and analgesic effects were estimated by using the acetic acid test (AAT). The AAT is done by placing logarithmic dilutions of acid dropwise on the dorsum of the animal’s thigh until a wiping response is obtained. At various doses, chlorpromazine and haloperidol (antipsychotics), chlordiazepoxide (a benzodiazepine), buprenorphine (a partial opioid agonist), and diphenhydramine (a histamine antagonist) produced moderate to strong analgesic effects. Indomethacin and ketorolac (NSAIDs), butorphanol (a partial opioid agonist), and pentobarbital (a barbiturate) produced weaker but noticeable analgesic effects. Our results are the first to document the effectiveness of a wide array of pharmacologically active agents in a novel amphibian model for analgesia. These findings provide needed data regarding the use of alternative, non-opioid agents for the treatment of pain in amphibians and other poikilothermic species.

Opioid agonists produce analgesia in mammals through the activation of µ, κ, or δ opioid receptors. Previous behavioral and binding studies from our laboratory using an amphibian model suggested that, µ, κ, or δ opioid agonists may activate a single type of opioid receptor in the grass frog, Rana pipiens. In the present study, kinetic, saturation, and competitive binding profiles for three opioid radioligands, [3H]DAMGO ([d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin) (µ-selective), [3H]U65953 [(5α,7α,8β)-(+)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]-benzeneacetamide] (κ-selective), and [3H]DPDPE ([d-Pen2,d-Pen5]-enkephalin) (δ-selective) were determined using frog whole brain homogenates. Kinetic analyses and experimentally derived values from saturation experiments gave affinity constants (KD) in the low nanomolar range. The density of opioid binding sites (Bmax) was 224.4, 118.6, and 268.9 fmol/mg for µ, κ, and δ opioid radioligands, respectively. The affinity values did not significantly differ among the three opioid radioligands, but the κ radioligand bound to significantly fewer sites than did the µ or δ radioligands. Ki values for unlabeled µ, κ, and δ competitors, including highly selective opioid antagonists, were consistent with each radioligand selectivity profile. The present data suggest that µ, κ, and δ opioid radioligands bind to distinct opioid receptors in amphibians that are surprisingly similar to those found in mammalian brain.

Opioids elicit antinociception in mammals through three distinct types of receptors designated as μ, κ and δ. However, it is not clear what type of opioid receptor mediates antinociception in non-mammalian vertebrates. Radioligand binding techniques were employed to characterize the site(s) of opioid action in the amphibian, Rana pipiens. Naloxone is a general opioid antagonist that has not been characterized in Rana pipiens. Using the non-selective opioid antagonist, [3H]-naloxone, opioid binding sites were characterized in amphibian spinal cord. Competitive binding assays were done using selective opioid agonists and highly-selective opioid antagonists. Naloxone bound to a single-site with an affinity of 11.3 nM and 18.7 nM for kinetic and saturation studies, respectively. A Bmax value of 2725 fmol/mg protein in spinal cord was observed. The competition constants (Ki) of unlabeled μ, κ and δ ranged from 2.58 nM to 84 μM. The highly-selective opioid antagonists yielded similar Ki values ranging from 5.37 to 31.1 nM. These studies are the first to examine opioid binding in amphibian spinal cord. In conjunction with previous behavioral data, these results suggest that non-mammalian vertebrates express a unique opioid receptor which mediates the action of selective μ, κ and δ opioid agonists.