Estrogen alters spinal NMDA receptor activity via a PKA signaling pathway in a visceral pain model in the rat

Abstract

Pain symptoms in several chronic pain disorders in women, including irritable bowel syndrome, fluctuate with the menstrual cycle suggesting a gonadal hormone component. In female rats, estrogens modulate visceral sensitivity although the underlying mechanism(s) are unknown. In the present study the effects of 17-beta estradiol on N-methyl-D-aspartate (NMDA) receptor signaling of colorectal nociceptive processing in the spinal cord were examined. Estrogen receptor alpha and the NR1 subunit of the NMDA receptor are co-expressed in dorsal horn neurons, supporting a direct action of estradiol on NMDA receptors. Intrathecal administration of the NMDA receptor antagonist D(-)-2-amino-5-phosphonopentanoic acid (APV) dose-dependently attenuated the visceromotor response with greater potency in ovariectomized (OVx) rats compared to OVx with estradiol replacement (E2) rats. Estradiol significantly increased protein expression of NR1 in the lumbosacral spinal cord compared to OVx rats. Colorectal distention significantly increased phosphorylation of NR1ser-897, a PKA phosphorylation site on the NR1 subunit in E2, but not OVx rats. Intrathecal administration of a PKA inhibitor significantly attenuated the visceromotor response, decreased NR1 phosphorylation and increased the potency of APV to attenuate the visceromotor response compared to vehicle-treated E2 rats. These data suggest that estradiol increases spinal processing of visceral nociception by increasing NMDA receptor NR1 subunit expression and increasing site-specific receptor phosphorylation on the NR1 subunit contributing to an increase in NMDA receptor activity.

Figure 1. Colocalization of ERα and NR1 in the dorsal spinal cord

A: Low power photomontage (4 images using a 20× objective) showing double labeled cells (arrows) in the dorsal horn. NR1 is labeled red, ERα is labeled green. B,C,D: High power (60× objective) images of the cells in the box in A. B: NR1; C: ERα; D: merged image.

Figure 2. The effect of intrathecal APV on the visceromotor response (VMR) to CRD in OVx and E2 rats

A: Time course of attenuation of the visceromotor response following 100 nmol APV. The peak effect occurred starting 15 min following APV administration. # p<0.01 vs. OVx (baseline). * p<0.001 vs. 15 min post APV for each treatment group. B: % inhibition of the visceromotor response as a function of dose of APV. * p<0.001 vs. E2. § p<0.01 vs. same dose in E2 rats.

Figure 3. Total NR1 protein expression in estradiol treated rats is significantly greater than OVx rats

A: representative Western blot showing the NR1 band from two OVx and two E2 rats. β-actin was used as the loading control. B: quantified Western blot data. Values represent the NR1/actin ratio normalized to OVx rats. * p<0.001.

Figure 4

Phosphorylation of NR1 ser-897 (pNR1) is increased in the LS spinal dorsal horn in E2, but not OVx rats, following CRD (A,B). Representative Western blots are shown in the upper panels and quantified results in the lower panels. Values represent the pNR1/NR1 ratio normalized to nondistended rats. * p<0.01. C: NR1 ser-897 phosphorylation was significantly greater in E2 rats than OVx rats following CRD. Values represent the pNR1/NR1 ratio normalized to OVx + CRD rats. NR1 was used as the loading control in all groups. * p<0.001.

Figure 5. Intrathecally administered PKA inhibitors attenuated the effect of estradiol

A: the time course of the effect of intrathecally injected Rp-8-Br-cAMP (200 nmol) on the visceromotor response (VMR) to CRD in OVx and E2 rats. b: baseline. * p<0.05, **p<0.01 vs. baseline response in E2 rats. B: the visceromotor response to CRD was atttenuated by Rp-8-Br-cAMP (Rp), but not vehicle in E2 rats. # p<0.05 vs. saline treated group. * p<0.05, ** p<0.01 vs. same time point in saline group. C: representative Western blots (left) and quantification of Western blot data (right) showing Rp-8-Br-cAMP attenuated the phosphorylation of NR1 ser-897 in the rats shown in B. Values represent the pNR1/NR1 ratio normalized to saline treated rats. * p<0.01. D, E: the peak attenuation of the visceromotor response by intrathecal H-89 (D) or chelerythrine chloride (E) compared to the appropriate vehicle (V). * p<0.05 vs. vehicle.

Figure 6. Intrathecally injected Rp-8-Br-cAMP (Rp) potentiates the effect of APV in E2 rats

A: the time course of the effect of intrathecally injected Rp-8-Br-cAMP (200 nmol) on the visceromotor response to CRD in E2 rats. * p<0.05 vs. 10 minutes. B: the magnitude of the visceromotor response before any intrathecal drug injection (b), following pretreatment with saline or Rp-8-Br-cAMP (Sal/Rp) and post APV. C: The percent inhibition of the visceromotor response by intrathecally injected APV in saline or Rp-8-Br-cAMP pretreated rats. * p<0.01.