We developed novel methods for analyzing the concentrationresponse curve of an agonist to estimate the product of observed affinity and intrinsic efficacy, expressed relative to that of a standard agonist. This parameter, termed intrinsic relative activity (RA i ), is most applicable for the analysis of responses at G protein-coupled receptors. RA i is equivalent to the potency ratios that agonists would exhibit in a hypothetical, highly sensitive assay in which all agonists behave as full agonists, even those with little intrinsic efficacy. We investigated muscarinic responses at the M 2 receptor, including stimulation of phosphoinositide hydrolysis through G Drug discovery often involves testing compounds in isolated tissues and high throughput assays to determine activity at target receptors. In the case of agonists, the measured parameters are usually EC 50 and E max , whereas the parameters of greater relevance to drug design are observed affinity and intrinsic efficacy.1 If the receptor is a ligand-gated ion channel, then receptor activation can be measured directly as whole cell current, and the EC 50 and E max are reasonable estimates of observed affinity and intrinsic efficacy provided that desensitization is not excessive. It would seem that, in most instances, the observed affinity and intrinsic efficacy of an agonist for a ligand-gated ion channel would be constant, regardless of the tissue or cell in which the receptor is expressed.The situation is more complex for a G protein-coupled receptor (GPCR). First, GPCRs are inactive in isolation and must interact with a G protein to elicit a response. It is possible that the G protein with which the receptor interacts selects for a receptor conformation having a unique agonist profile and that the observed affinity and intrinsic efficacy of the agonist-receptor complex may be G protein-specific or influenced by other proteins interacting with the receptor (Leff et al., 1997;Berg et al., 1998). Second, because it is difficult to measure receptor activation directly, most assays involve measuring a downstream response, and the corresponding EC 50 and E max values may vary, depending on the point in the signaling cascade at which the experimenter measures the response. Therefore, the magnitude of the response elicited by a GPCR is usually not proportional to receptor activation, and EC 50 and E max are not equivalent to observed affinity and intrinsic efficacy. Rather, EC 50 and 1 Here and throughout the article, we use the term "observed intrinsic efficacy" to refer to Furchgott's definition of "intrinsic efficacy" (Furchgott, 1966), which denotes the amount of activated receptors. As described previously (Ehlert, 2000), it is useful to discriminate between the latter definition of observed intrinsic efficacy and the ratio of agonist affinity constants for ground and active conformations of the receptor (intrinsic efficacy).

To explore cellular changes in sensory neurons after nerve injury and identify potential target genes contributing to different stages of neuropathic pain development, we used Affymetrix oligo arrays to profile gene expression patterns in L5/6 dorsal root ganglia (DRG) from the neuropathic pain model of left L5/6 spinal nerve ligation at different stages of neuropathic pain development. Our data indicated that nerve injury induced changes in expression of genes with similar biological functions in a temporal specific manner that correlates with particular stages of neuropathic pain development, indicating dynamic neuroplasticity in the DRG in response to peripheral nerve injury and during neuropathic pain development. Data from post-array validation indicated that there was a temporal correlation between injury-induced expression of the glial fibrillary acidic protein (GFAP), a marker for activated astrocytes, and neuropathic pain development. Spinal nerve ligation injury in GFAP knockout mice resulted in neuropathic pain states with similar onset, but a shortened duration compared with that in age, and gender-matched wild-type littermates. Intrathecal GFAP antisense oligonucleotide treatment in injured rats with neuropathic pain states reversed injury-induced behavioral hypersensitivity and GFAP upregulation in DRG and spinal cord. Together, these findings indicate that injury-induced GFAP upregulation not only serves as a marker for astrocyte activation, but it may play a critical, but yet identified, role in the maintenance of neuropathic pain states.

We measured the intrinsic relative activity (RA i ) of muscarinic agonists to detect possible selectivity for receptor subtypes and signaling pathways. RA i is a relative measure of the microscopic affinity constant of an agonist for the active state of a GPCR expressed relative to that of a standard agonist. Our results show that the RA i estimate is a useful receptordependent measure of agonist activity.

Previous studies have shown that peripheral nerve injury in rats induces increased expression of the voltage gated calcium channel (VGCC) alpha-2-delta-1 subunit (Ca v α 2 δ 1 ) in spinal dorsal horn and sensory neurons in dorsal root ganglia (DRG) that correlates to established neuropathic pain states. To determine if injury discharges trigger Ca v α 2 δ 1 induction that contributes to neuropathic pain initiation, we examined allodynia onset and Ca v α 2 δ 1 levels in DRG and spinal dorsal horn of spinal nerve ligated rats after blocking injury induced neural activity with a local brief application of lidocaine on spinal nerves before the ligation. The lidocaine pretreatment blocked ligation-induced discharges in a dose-dependent manner. Similar pretreatment with the effective concentration of lidocaine diminished injury-induced increases of the Ca v α 2 δ 1 in DRG and abolished that in spinal dorsal horn specifically, and resulted in a delayed onset of tactile allodynia post injury. Both dorsal horn Ca v α 2 δ 1 upregulation and tactile allodynia in the lidocaine pretreated rats returned to levels similar to that in saline pretreated controls two weeks post the ligation injury. In addition, preemptive intrathecal Ca v α 2 δ 1 antisense treatments blocked concurrently injury-induced allodynia onset and Ca v α 2 δ 1 upregulation in dorsal spinal cord. These findings indicate that injury induced discharges regulate Ca v α 2 δ 1 expression in the spinal dorsal horn that is critical for neuropathic allodynia initiation. Thus, preemptive blockade of injury-induced neural activity or Ca v α 2 δ 1 upregulation may be a beneficial option in neuropathic pain management.