Anesthesiology, Perioperative and Pain Medicine

ß2 Adrenoreceptor: The Focus of Multi-Investigator, Multidisciplinary Research



By Frances Davies, PhD

Name this receptor: it is currently being investigated by several pain researchers in our Department, it is involved in pain systems and ischemic neural injury, and it may play a significant role in opioid-induced hyperalgesia (OIH). If you guessed the opiate receptor, you would be wrong. The beta adrenergic receptor (ßAR) is of course best known for receiving messages from the sympathetic nervous system, driving heart rate, and being a major force behind cellular cyclic AMP production, thereby mediating the “fight or flight” response. However, several new functions for the ßAR are under investigation in our Department, both here at Stanford and the Palo Alto VA, by multiple investigators, supported by NIH funding, and cutting across different disciplines. The work of these researchers is leading to unexpected results both about ßAR function in the heart and elsewhere in the body.

THE PATTERSON LAB

Dr. Drew Patterson has devoted his research career to studying intracellular signaling in different ßAR subtypes. When he began his investigations in the mid-1990s, ß1ARs were known to be primarily responsible for regulating heart rate and contractility. The role of ß2ARs was less clear, but it was thought to duplicate that of ß1ARs. Since that time, in vitro and in vivo studies have demonstrated that activating ß1AR can produce both helpful and harmful effects. For example, acutely, ß1AR stimulation enhances cardiac performance. However, continuous ß1AR activation leads to myocyte toxicity. Hence, clinicians now administer ß1AR blockers to patients with ischemic heart disease and congestive heart failure, in part for myocardial protection.
During the past decade, Patterson and his collaborators have zerßoed in on the role of ß2AR. They have learned it plays a protective role in the heart during periods of continuously elevated catecholamine levels (as occur in congestive heart failure and after stressful events like surgical procedures). However, the mechanism of this protection is not entirely clear.

When Patterson first began his research with Dr. Brian Kobilka, it was thought that the ßAR signal-transduction pathway had a traditional linear design—ßARs associated with stimulatory G proteins (Gs), which activated adenylate cyclase to produce cAMP, which thereby activated protein kinase A (PKA). However, a decade of research has shown a more complex situation. Now we think that the signaling schema for ß1ARs and ß2ARs are multidimensional, involving promiscuous G protein signaling (stimulatory and inhibitory G proteins), time-dependent changes, and even G protein-independent pathways. We also know that scaffolding proteins play an important role in ßAR signaling. Specific motifs on ßAR subtypes influence which scaffolding proteins they associate with. ß2AR’s ability to switch its intracellular coupling from stimulatory to inhibitory G protein during continuous activation is thought to play a particularly important role in ß2AR-mediated cardiac protection, though other mechanisms also appear to be involved.

You can see that the roles of ß1AR and ß2AR do in fact differ. In fact, some envision ß2AR as a molecular pop-off valve when ß1AR activation continues for too long. Indeed, ß1ARs and ß2ARs are activated by the same endogenous catecholamine neurotransmitters. However, when the signal continues to the point of causing ß1AR-mediated toxicity in myocytes, ß2AR goes into protection mode. Unfortunately, developing drugs that selectively activate ß2ARs has proved difficult, primarily because the agonist-binding sites on ß1ARs and ß2ARs are very similar. Consequently, at concentrations used clinically, ß2AR agonists become non-selective and begin activating ß1ARs. However, Patterson and others are investigating the details of the ß2AR-mediated protective signaling pathways, hoping they can eventually develop pharmaceutical agents that selectively activate these pathways and bypass the receptors altogether. Their strategy could lead to better therapies for disease states like congestive heart failure.

One technique Patterson uses is analyzing complex gene- and protein-expression in genetically engineered “knockout” mice. Some of the mice lack ß1AR, others lack the ß2AR, and still others lack only segments of the ß2AR (such as the PDZ binding motif). Many of the mice are conventional knockout animals who develop without the receptors or receptor segments. Others are conditional knockout animals who develop with the receptors until they are adults, whereupon they receive inducer agents that cause their heart cells to stop generating the receptors of interest.

Each ßAR subtype is composed of motifs—amino-acid sequences that bind to other proteins or molecules. The PDZ motif determines which other proteins the ßAR will bind to and how it will move to other areas of the cell following cell-membrane activation. In ß1ARs this motif keeps the receptor close to the cell surface after agonist activation, whereas in ß2ARs the motif helps move (“traffic”) the activated receptor to internal cellular sites that may facilitate the association of ß2ARs with other proteins that modify their function. Dr. Patterson’s in vivo work and the in vitro studies of other investigators suggest that this ß2AR PDZ trafficking may play an important role in ß2AR-mediated cardiac protection. Dr. Patterson’s collaborators at Stanford, include Drs. Brian Kobilka, Marco Conti, and Dan Bernstein. He also collaborates with Dr. Robert Lefkowitz at Duke University and Dr. Rui-Ping Xiao at the National Institutes of Health.

THE GIFFARD LAB

Dr. Rona Giffard, in collaboration with Dr. Patterson, researches the role of ß2ARs in cerebral ischemia. Dr. Ruquan Han in Dr. Giffard’s lab has compared the effects of ß2AR loss in both knockout and wild mice subjected to stroke, finding that although ß2ARs activation protects the heart, it appears to have the opposite effect in the brain. Others showed that blocking ß2ARs with antagonists had neuroprotective effects in experimental models of cerebral ischemia during cardiac surgery. The ß2ARs agonist clenbuterol, on the other hand, had a neuroprotective activity after transient forebrain ischemia, suggested to be mediated by increasing nerve-growth factor (NGF) expression.
However, because these studies did not fully distinguish receptor subtypes, Giffard and Patterson decided to study which receptor subtype might be responsible, so that they could tailor treatment. Researchers produced strokes by transiently occluding the middle cerebral artery in two types of mice—ß2AR knockout mice and wild type mice. The extent of brain injury was smaller in ß2AR knockout mice than in normal mice. The total infarct volume was decreased by 25% in ß2AR knockout mice when assessed 23 hours after a 1-hour period of ischemia. Neurological scores were better in ß2AR knockout mice than in control mice. The mechanism of this neuroprotective effect is currently unknown, but Giffard and Patterson are currently looking at changes in gene expression to determine possible candidate pathways.

THE CLARK LAB

Dr. David Clark and his colleagues pursue a different tack—studying what appears to be a strong link between ß2AR, opioid-induced hyperalgesia (OIH), and tolerance to opioids’ analgesic effects. OIH is a condition in which previously non-painful stimuli become painful. Although opioids remain the mainstay for treating moderate to severe pain for acute and chronic conditions, their effectiveness can be limited by dependence, sedation, tolerance, respiratory depression, and OIH (usually observed with sustained use of narcotics but also occurring even with short-term administration). So far, no specific therapies or interventions are available to limit or prevent OIH in humans. By understanding OIH’s underlying mechanisms, Clark hopes to design and test strategies to reduce its impact on OIH.

Funded by NIH’s Cutting-Edge Basic Research Award (CEBRA), Clark’s team placed 15 strains of inbred mice under a morphine-administration protocol and measured each strain’s propensity for developing OIH’s characteristic thermal and mechanical sensitization. In subsequent genetic mapping, they identified several, highly associated blocks that corresponded to known genes, learning that ß2AR gene coding was the most strongly genetically linked to OIH, thus pointing to ß2AR’s functional role in mechanically induced OIH.

Like the researchers in Patterson’s and Giffard’s labs, Clark and his colleagues have collaborators within the Department of Anesthesia, Dr. Martin Angst and Dr. Brooks Rohlen. Together they aim to learn if blocking ß2AR alleviates mechanical- and heat-induced pain in humans. Clark hopes all of these studies will lead to human translational studies using currently available ß2AR drugs.

IN SUM

We are very excited about the promise in our multi-disciplinary, multi-institutional research into ß2AR’s complex functions. Our hope is to learn not only how ß2AR controls heart rate, cardiomyopathy, and related “fight-or-flight” responses, but also how it affects postoperative pain and even ischemic brain injury. The future looks bright!

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