Molecular Neuropharmacology

K.M. Standifer, C.C. Chien, C. Wahlestedt, G.P. Brown, G.W. Pasternak, "Selective loss of delta-opioid analgesia and binding by antisense oligodeoxynucleotide to a delta-opioid receptor," Neuron, 12:805-10, 1994. (Cited in more than 30 publications as of February 1996) Comments by Gavril W. Pasternak, Memorial Sloan-Kettering Cancer Center Unzip a chunk of DNA. Two strands result. One, dubbed the "sense" strand, carries genetic information. The other, the antisense strand, says nothing. In theory, scientists can use antisense molecules to block the production of a particular protein. Scientists construct antisense strands by stringing together DNA bases that are complementary to the messenger RNA (mRNA) encoding the protein. This mirror-image piece of genetic material, in binding to the mRNA, halts it from hooking up with a cell's protein-manufacturing machinery. SENSIBLE: Gavril W. Pasternak uses antisense to study opioid receptors In this paper, Gavril W. Pasternak, head of the laboratory of molecular neuropharmacology at Memorial Sloan-Kettering Cancer Center in New York, and his coworkers use antisense techniques to demonstrate that the recently cloned opioid receptors are responsible for the analgesia of pain-killing opiates such as morphine and codeine and that each one works by distinct molecular mechanisms. Several different classes of opiate receptors exist, each with several distinct subclasses, he notes. Mu, for example, works with morphine; delta, the enkephalins. "An analgesic like morphine works through mu receptors," he explains. "You can produce analgesia by activating any of these types of opioid receptors-they produce the same response, but through different receptors." Pasternak and his colleagues used tissue- culture methods to examine a type of cell called NG108, which expresses the delta opiate receptor. This receptor is one of four known cloned opiate receptors that sit on the cell surface of neurons. The researchers showed that treating the cells with antisense resulted in a "down-regulation"-fewer receptors were available. "This meant that the antisense was interfering with synthesis of the receptors," Pasternak says. "We subsequently showed there was a corresponding loss of mRNA and protein." He explains that many scientists had assumed antisense works by blocking the translation of the mRNA into a protein by the cell's ribosomes. That meant targeting the antisense to the specific part of the mRNA from which the message was starting to be translated by the ribosome. Instead, his group showed that targeting any one of three coding regions, or exons, for the delta opiate receptor would "down-regulate receptor binding to the same degree." The scientists moved on to study this phenomenon in a living model, the mouse. They discovered that antisense also blocked the analgesic action of several different delta opiates in the mouse spinal cord. "That proved that the receptor mediating delta analgesia at the spinal cord level was the same receptor that had been cloned from the NG108 cells," Pasternak states. "We also showed that the analgesic actions of morphine through mu receptors and analgesic effects through kappa-1 receptors were similarly affected by their antisense probes. By blocking the delta analgesia without blocking the mu analgesia, we also were able to show this was a separate system." The next step is to try to understand the receptor-analgesia mechanism in humans, he points out. "It's amazing how animal studies have been very predictive of how these drugs may work in people. "This work is giving us some potential insights into the analgesic mechanisms through which opiates can act, and it may be giving us new targets for analgesic drugs," Pasternak comments. "Many of the side effects of morphine, such as respiratory depression, are mediated by specific receptors," he says. "If you could induce analgesia through alternative receptors, you might avoid some of those side effects." Pasternak thinks the paper "might interest those working in the neurotransmitter field. It demonstrates that you can selectively knock down a receptor using the antisense approach. This can be extrapolated to a variety of receptors." He explains that all classes of opioids relieve pain, acting on their own selective receptor at the cell surface. "Opioids are localized to specific regions of the brain, but probably only a small fraction of receptors are involved in pain perception. They probably have more generalized functions. We haven't figured them out yet." He points to several subsequent papers continuing the antisense work. In G.C. Rossi et al., FEBS Letters, 369:192-6, 1995, Pasternak and his colleagues describe antisense mapping for the mu receptor. In a review article (G.W. Pasternak et al., Trends in Pharmacological Sciences, 16:344-50, 1995), the authors demonstrate how strategies using antisense molecules can provide a selective approach to correlating the properties of the cloned receptors with their pharmacological effects.

Molecular Neuropharmacology

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