Gavril W. Pasternak, MD, PhD

Although this laboratory has a wide range of interests, its primary focus is the molecular mechanisms of opioid action. Opioids have a rich pharmacology, which is easily studied in a wide variety of behavioral paradigms. The recent cloning of 4 genes encoding opioid receptors has led us to focus our attention upon correlating these various cloned receptors with pharmacological actions.

Mu Opioid Receptors

Using a series of opioid antagonists synthesized in our laboratory and other pharmacological models, we proposed 3 subtypes of mu receptors. However, only a single mu opioid receptor gene has been identified. MOR-1, the first cloned mu opioid receptor, contains 4 exons encoding a receptor within the G-protein-coupled receptor family. Soon after its identification, an alternatively spliced exon was identified.

Enlarge Image Figure 1 The MOR-1 gene and its splice variants

Pharmacological studies in our laboratory predicted additional mu receptor subtypes, leading us to embark upon an intensive cloning effort. We have now identified an additional 9 exons encoding more than a dozen different splice variants of the mu opioid receptor (Figure 1). Although alternative splicing of a G-protein-coupled receptor has been noted, the extensive splicing observed with MOR-1 is relatively unusual. The presence of 2 distinct promoters, one with exon 1 and another with exon 11, makes this gene even more unique.

Correlating these cloned variants with specific pharmacological actions has employed both antisense mapping studies as well as knockout mice. Antisense methodologies can downregulate protein levels, providing insights into their functions. Prior work from our laboratory has shown that antisense probes can be equally effective regardless of where along the mRNA they are targeted. Antisense mapping involves targeting different exons within the gene to define the functional signficance of the protein and possible splice variants. We have used this approach to explore the functional role of splice variants of all the opioid receptors and nitric oxide synthase and have shown that different splice variants of a gene may have distinct, even opposing, pharmacological actions.

Enlarge Image Figure 2 Confocal microscopy of MOR-1 and MOR-1C in the dorsal horn of the spinal cord

The variants encoded by the MOR-1 gene have unique regional distributions at both the mRNA and protein levels. Even when expressed within the same region, individual cells rarely express more than one variant, as revealed by confocal microscopy (Figure 2).

Thus, this gene demonstrates both region and cell-specific processing. A major goal of the laboratory is to understand the signals responsible for the processing of the various variants.

Other Opioid Receptors

We are also investigating several other cloned opioid receptors. Using antisense mapping and knockout strategies, we have been defining their pharmacology. We have also been studying the molecular biology of these receptors and have identified alternative splicing for these genes as well. As with the mu opioid receptor gene, a primary goal is the correlation of these variants with functions.

Opioid Pharmacology

Many aspects of opioid action are well described but poorly understood. These include the mechanisms of tolerance and the variability of subjects' sensitivity toward opioid analgesics.

Tolerance is mediated through a variety of mechanisms, starting at the level of the cell with receptor desensitization and internalization. However, the importance of the NMDA receptor/nitric oxide cascade in the production of opioid tolerance has opened many new avenues of investigation. We are very interested in these systems and their circuitry. Recent evidence has also implicated P-glycoprotein (Pgp) in morphine tolerance. Pgp is located within the choroid plexus and transports drugs, such as morphine, out of the brain, playing a significant role in the blood-brain barrier. Downregulation of Pgp using antisense strategies in both mice and rats has profound effects upon morphine tolerance and activity.

Opioid systems also are modulated by a number of other transmitter systems. Of these, the sigma receptors have proven important in helping to explain the wide range in sensitivity among many strains of mice and rats to various opioid analgesics. We have now cloned the sigma receptor from both mice and rats and are looking at its actions on opioid function.

Overview

The cloning of the opioid receptors has greatly facilitated studies on the molecular mechanisms of action of these clinically important drugs. Understanding how they work will greatly impact upon their clinical use. The major focus of these drugs has been in the management of pain, but as the field has matured it is becoming clear that opioid systems are important in a wide range of other physiological actions as well.