UC San Francisco researchers have identified a new molecular pathway through which chemical signals alert the body to pain, and inhibiting the key protein in this pathway could bring relief in a broad spectrum of pain syndromes, they say.
The finding, drawn from a study in mice and rats, applies to inflammatory pain associated with such conditions as arthritis and colitis, torn ligaments and sprained ankles, and post-operative pain. However, the researchers expect the finding will apply even more broadly.
"This discovery is extremely important," said the director of the National Institutes of Health Pain Center at UCSF, Jon Levine, PhD, a professor of oral and maxillofacial surgery and medicine and a senior author of the paper. "I think this signaling pathway will be shown to play a role in many kinds of pain."
The study, published in the Sept. 24 issue of Neuron, was funded by the Ernest Gallo Clinical and Research Center at UCSF and the National Institutes of Health.
The body's immune system responds to many forms of tissue injury by producing an inflammatory response, which includes the release of chemical signals into injured tissue, where they sensitize pain-sensing neurons. As a result, stimuli that normally would not cause pain, such as the brush of a shirt being drawn onto the body, become painful when the skin is sunburned; likewise, the movement of a joint, normally unnoticed, would cause pain in the presence of arthritis.
Chemical signals act on pain-sensing neurons by latching on to specific cell-surface receptors that convey the signals into the cell. Once inside, the chemical signal initiates a cascade of molecular events that culminates with the neurons transmitting pain signals out of the cell body and into the central nervous system, where pain is felt.
Current inflammatory-pain drugs -- the nonsteroidal anti-inflammatory drugs, or NSAIDS, including the new COX-2 inhibitors -- act by blocking the production of some of these chemical signals, or inflammatory "mediators." However, because these drugs block only a small percentage of these messages, their effectiveness is limited.
The significance of the UCSF finding is that the researchers have identified a protein enzyme inside pain-sensing neurons through which they believe many of these inflammatory mediators - including those targeted by NSAIDS - act, suggesting a possible target for broad-based pain therapy.
"Identifying the common signaling pathways inside these pain-sensing cells would prevent us from having to identify blocks for every inflammatory mediator," said Levine. "I think this enzyme will prove to be the central signaling pathway by which most chemical mediators act on pain-sensing neurons."
For more than a decade, researchers have thought that the protein kinase C (PKC) enzyme played a role in the pain-sensing neurons' activity, but they have not known which of the ten known forms of the enzyme might be involved. In the current study, the researchers discovered the role played by protein kinase C epsilon (PKC).
The researchers discovered the PKC signaling pathway by conducting studies in mice that lacked the enzyme and in rats in which the enzyme was inhibited by a drug.
In one study, they compared the responses of normal mice, and mice lacking the PKC enzyme, to painful stimuli, and determined that the mice responded equally to stimulation. However, when they added epinephrine, an inflammatory mediator that heightens the sensitivity of pain sensing neurons, those without the enzyme exhibited a "significantly reduced" reaction to stimulation.
In a second study, the researchers applied the chemical irritant acetic acid. The response to the painful stimulus, which causes inflammation, was "almost completely blocked" in the mice lacking PKC, they said. In a third study, the researchers examined rats in which PKC was inhibited. Predictably, both these animals and control animals responded to stimulation. However, when epinephrine was added to increase pain sensitivity, the animals with the inhibited enzyme became markedly less sensitive to the pain.
Epinephrine acts on pain-sensing neurons, or nociceptors, by enhancing an ion channel known as TTX- RINA, which sensitizes the pain-sensing neurons to previously innocuous stimuli. As a check on the animal study results, the researchers examined whether inhibiting PKC would blunt epinephrine's action in pain-sensory neurons in laboratory cultures. It did. In cultured cells in which the enzyme was inhibited, epinephrine's effect was decreased by half, demonstrating that epinephrine depends on PKC to prompt a full effect on the TTX-RI NA channel in a critical group of pain-sensing neurons, the researchers said.
The researchers further demonstrated PKC's role by examining the response of rats to a potent irritant known as carrageenan. When they applied the seaweed compound in rats exposed to stimulation, the animals exhibited pain. But when the animals were pretreated with the chemical that inhibits the PKC enzyme, the painful response was "almost completely reversed," the researchers report. Carrageenan is commonly used by the pharmaceutical industry as a model to screen for pain-reducing drugs.
Finally, the researchers showed that PKC modulates the pain response induced by the chemical known as nerve growth factor. When the factor was injected into normal rats exposed to stimulation, the animals experienced heightened pain. But when the factor was injected in animals in which PKC was inhibited, their pain threshold was higher.
"These results suggest that PKC plays a key role in regulating pain sensitivity," said a senior author of the UCSF paper, Robert Messing, MD, an associate professor of neurology. "The fact that inhibiting PKC reduced pain in response to several different sensitizing agents is significant."
Since absence or inhibition of PKC does not disturb basic pain-sensory thresholds, needed to help alert the body to possible danger, and the mice in which the enzyme was missing appeared normal, it may be possible, the researcher said, to develop PKC inhibitors that reduce pathologic pain without producing serious systemic side effects or interfering with normal pain responses.
Co-authors of the UCSF study were Sachia G. Khasar, PhD, an assistant research pharmacologist, K.O. Aley, PhD, an assistant research pharmacologist, William Isenberg, MD, PhD, an assistant reseach endocrinologist, Gordon McCarter, PhD, a post-doctoral fellow, Paul G. Green, PhD, an assistant professor, all in the Department of Internal Medicine and Oral Surgery and NIH/UCSF Pain Center; and Yu-Huei Lin, PhD, at the time a postdoctoral fellow, Annick Martin, PhD, a post-graduate research fellow, Jahan Dadgar, BS, a staff research associate, Thomas McMahon, BS, a staff research associate, Dan Wang, MS, BS, a a staff research associate, Bhupinder Hundle, PhD, at the time a postdoctoral fellow, and Clyde Hodge, PhD, an assistant adjunct professor in the Department of Neurology, Ernest Gallo Clinical and Research Center at UCSF.
Journal
Neuron