The acute antihyperalgesic action of nonsteroidal, anti-inflammatory drugs and release of spinal prostaglandin E2 is mediated by the inhibition of constitutive spinal cyclooxygenase-2 (COX-2) but not COX-1.

Western blots show the constitutive expression of COX-1 and COX-2 in the rat spinal dorsal and ventral horns and in the dorsal root ganglia. Using selective inhibitors of cyclooxygenase (COX) isozymes, we show that in rats with chronic indwelling intrathecal catheters the acute thermal hyperalgesia evoked by the spinal delivery of substance P (SP; 20 nmol) or NMDA (2 nmol) and the thermal hyperalgesia induced by the injection of carrageenan into the paw are suppressed by intrathecal and systemic COX-2 inhibitors. The intrathecal effects are dose-dependent and stereospecific. In contrast, a COX-1 inhibitor given systemically, but not spinally, reduced carrageenan-evoked thermal hyperalgesia but had no effect by any route with spinal SP hyperalgesia. Using intrathecal loop dialysis catheters, we showed that intrathecal SP would enhance the release of prostaglandin E(2) (PGE(2)). This intrathecally evoked release of spinal PGE(2) was diminished by systemic delivery of nonspecific COX and COX-2-selective inhibitors, but not a COX-1-selective inhibitor. Given at systemic doses that block SP- and carrageenan-evoked hyperalgesia, COX-2, but not COX-1, inhibitors reduced spinal SP-evoked PGE(2) release. Thus, constitutive spinal COX-2, but not COX-1, is an important contributor to the acute antihyperalgesic effects of spinal as well as systemic COX-2 inhibitors.

Evidence for differences in cultured left ventricular fibroblast populations isolated from spontaneously hypertensive and Wistar-Kyoto rats.

OBJECTIVE: To compare fibroblast populations derived from spontaneously hypertensive rats (SHRLJ) and normotensive Wistar-Kyoto rats (WKYLJ) for angiotensin II receptor binding, gene expression of the AT1 receptor and angiotensinogen, hormone responsiveness and phenotypic changes. METHODS: Fibroblasts were isolated by either collagenase B or collagenase P and grown to confluency in the presence of 10% fetal bovine serum. Angiotensin II receptor binding was assessed under both serum and serum-free conditions. Hormonal treatment of cells was conducted in a serum-free background. The concentrations of AT1 receptor and angiotensinogen messenger RNA (mRNA) were determined by liquid hybridization. Phenotypic changes in fibroblast populations were analysed by visualization of lipid-containing vacuoles (oil red O stain) or of alpha-smooth muscle actin-containing fibres (immunostain). RESULTS: SHRLJ collagenase-B cells grew more slowly and had nearly twofold fewer angiotensin II receptors than WKYLJ cells as measured by both radioligand binding and AT1 mRNA content (SHRLJ 1.34 +/- 0.05 versus WKYLJ 5.94 +/- 0.41 pg mRNA per microgram total RNA) but contained significantly more angiotensinogen mRNA (SHRLJ 147 +/- 12 versus WKYLJ 98 +/- 8 fg mRNA per microgram total RNA). Collagenase-P cells from the two strains exhibited similar binding and growth properties. Collagenase-B fibroblasts also exhibited greater responses to exogenous steroids, including a greater shift towards an adipocyte phenotype, than collagenase-P cells. Exogenous angiotensin II promoted transformation towards a myofibroblast cell type, especially in collagenase-P SHRLJ cells. CONCLUSION: Our results indicate that subsets of fibroblasts that differ in growth rate, angiotensin II receptor binding, AT1 and angiotensinogen mRNA levels, structure and steroid responsiveness may be isolated from the left ventricle. The potential importance of these altered phenotypes to cardiac remodelling and hypertrophy warrants further examination.

In vitro prostanoid release from spinal cord following peripheral inflammation: effects of substance P, NMDA and capsaicin.

1. Spinal prostanoids are implicated in the development of thermal hyperalgesia after peripheral injury, but the specific prostanoid species that are involved are presently unknown. The current study used an in vitro spinal superfusion model to investigate the effect of substance P (SP), N-methyl-d-aspartate (NMDA), and capsaicin on multiple prostanoid release from dorsal spinal cord of naive rats as well as rats that underwent peripheral injury and inflammation (knee joint kaolin/carrageenan). 2. In naive rat spinal cords, PGE2 and 6-keto-PGF1alpha, but not TxB2, levels were increased after inclusion of SP, NMDA, or capsaicin in the perfusion medium. 3. Basal PGE2 levels from spinal cords of animals that underwent 5-72 h of peripheral inflammation were elevated relative to age-matched naive cohorts. The time course of this increase in basal PGE2 levels coincided with peripheral inflammation, as assessed by knee joint circumference. Basal 6-keto-PGF1alpha levels were not elevated after injury. 4. From this inflammation-evoked increase in basal PGE2 levels, SP and capsaicin significantly increased spinal PGE2 release in a dose-dependent fashion. Capsaicin-evoked increases were blocked dose-dependently by inclusion of S(+) ibuprofen in the capsaicin-containing perfusate. 5. These data suggest a role for spinal PGE2 and NK-1 receptor activation in the development of hyperalgesia after injury and demonstrate that this relationship is upregulated in response to peripheral tissue injury and inflammation.

Characterization of variables defining hindpaw withdrawal latency evoked by radiant thermal stimuli.

We have examined the stability and sources of variation within the nociceptive model of rat hind paw withdrawal from an under-glass radiant stimulus (Hargreaves et al., 1988) using a system where stimulus intensity and floor temperature can be controlled and reproducibly changed. The current study demonstrates that: (i) increased stimulus intensity with a fixed surface temperature is associated with a monotonic decrease in mean response latency and its variance; (ii) for a fixed stimulus intensity, the mean paw withdrawal latency and variance increased as the glass floor temperature is lowered from 30 degrees C to room temperature (25 degrees C). Using subcutaneously-implanted thermocouples and a 30 degrees C glass surface, the subcutaneous paw temperature observed at an interval corresponding to the time at which the animal displayed a paw withdrawal did not differ across multiple heating rates (41-42.5 degrees C). This finding is in agreement with human studies of pain thresholds and C-fiber activity. These studies emphasize the importance of maintaining a fixed surface temperature to reduce experimental variability and the utility of this apparatus across multiple stimulus intensities to define agonist efficacy.

Effect of spinal cyclooxygenase inhibitors in rat using the formalin test and in vitro prostaglandin E2 release.

Spinally delivery of the non-specific cyclooxygenase inhibitor, S(+)-ibuprofen, reduces the second phase of the formalin test and the evoked release of prostaglandin E2 (prostaglandin E2) from rat spinal cord in vitro. Using two selective cyclooxygenase-2 inhibitors, SC58125 (1-[(4-methysufonyl)phenyl]-3-tri-fluoromethyl-5-(4-fluorophenyl)p yrazole) and SC-236 (4-[5-(4-chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfon amide), we observed that neither agent at the highest dose/concentration employed altered the second phase of the formalin test after intrathecal delivery or K+-evoked prostaglandin E2 release from spinal cord in vitro, although ibuprofen was effective in both models. These observations suggest that cyclooxygenase-2 may not be associated with spinal prostanoid synthesis acutely or with facilitated nociception which occurs within the limited time frame of the formalin test.

Thermal hyperalgesia in rat evoked by intrathecal substance P at multiple stimulus intensities reflects an increase in the gain of nociceptive processing.

Substance P (SP) is associated with primary afferent depolarization after a noxious or tissue-damaging peripheral stimulus and is proposed to be a "pain transmitter'. However, current work has shown that activation of spinal neurokinin receptors evokes few signs of "pain behavior', but rather decreases nociceptive thresholds, suggesting a modulatory role through additive or multiplicative mechanisms. The current study addressed this question by examining the change in nociceptive threshold under three different radiant thermal stimuli after intrathecal SP administration in unanesthetized rats. With increasing stimulus intensity, the numerical change in withdrawal latency after intrathecal SP decreased, but the fractional change from baseline latency did not change, suggesting that SP increases the gain of spinal nociceptive processing.

Intrathecal baclofen and muscimol, but not midazolam, are antinociceptive using the rat-formalin model.

Both gamma-aminobutyric acid (GABA)A and GABAB receptor subtypes have been implicated in spinally mediated antinociception in acute pain models. In the current study, the formalin test was used as a model of protracted nociception to examine the effect of intrathecally (i.t.) administered baclofen (GABAB agonist), muscimol (GABAA agonist) or midazolam (a benzodiazepine) on antinociception. At doses that did not affect motor function, baclofen (0.3 and 1.0 micrograms, i.t.) decreased the flinch response in a dose-dependent manner during Phase 1 and Phase 2. This effect was reversible by the GABAB-specific antagonist, CGP35348 ([P-(3-aminopropyl)-P-diethoxymethyl-phosphinic acid]). Muscimol (0.3 and 1.0 microgram i.t.) evoked a dose-dependent, bicuculline-reversible decrease in flinching during Phase 1 and Phase 2, but midazolam had no effect on either phase. No attenuation of the quiescent period between Phase 1 and Phase 2 was seen upon administration of baclofen, muscimol or midazolam. Additionally, no increase in nocifensive behavior was observed upon administration of either GABAA or GABAB antagonists alone. Therefore, our conclusions are that both GABAA and GABAB agonists are antinociceptive at the spinal cord level and that endogenous spinal GABA levels are insufficient for a GABA potentiator to act alone in an antinociceptive manner.

Temperature dependency of basal and evoked release of amino acids and calcitonin gene-related peptide from rat dorsal spinal cord.

Moderate hypothermia significantly diminishes consequences of spinal and cerebral anoxia. One component of this neuroprotection has been hypothesized to be suppression of excitotoxic transmitter release. Whether this suppression is attributable to reduced hypoxic injury that induces release or an alteration of the release process itself is unclear. We sought to characterize the temperature sensitivity (Q10) of basal and evoked calcitonin gene-related peptide (CGRP) and amino acid release from dorsal horn slices of rat spinal cord over a range of temperatures from 40 to 8 degrees C. At 40 degrees C, potassium (60 mM) and capsaicin (10 microM) evoked a 21- and 32-fold increase in basal CGRP concentrations, respectively. Capsaicin had no effect on glutamate release, but potassium evoked a 2.7-fold increase. Release evoked by either potassium or capsaicin was reduced in a biphasic fashion with declining temperature. Over the range of 40 to 34 degrees C, the Q10 values for evoked release for CGRP were 11.3 (potassium) and 39.7 (capsaicin) and for glutamate, 5. 5 (potassium). Over the range of 34 to 8 degrees C, Q10 values were near unity for all evoked release (0.8 and 1.3 for CGRP and 1.2 for glutamate). Although serine, glycine, glutamine, taurine, and citrulline showed no evoked release, basal levels were reduced at temperatures below 34 degrees C. The pronounced temperature dependency of evoked transmitter release between 40 and 34 degrees C is consistent with the profound cerebral protection observed with mild hypothermia in which metabolic activity is only slightly depressed.

Effect of COX-1 and COX-2 inhibition on induction and maintenance of carrageenan-evoked thermal hyperalgesia in rats.

Intrathecal administration of nonsteroidal anti-inflammatory drugs in the rat blocks the thermal hyperalgesia induced by tissue injury, which suggests a role for spinal cyclooxygenase (COX) products in this facilitated state. Two isozymes of the COX enzyme have been reported, COX-1 and COX-2, but the agents thus far examined are not isozyme selective. We examined the effects of intrathecally (i.t.) or systemically (i.p.) administered S(+)-ibuprofen (a nonselective COX inhibitor) or 1-[(4-methysulfonyl)phenyl]-3-tri-fluoromethyl-5-(4-fluorophenyl) pyrazole (SC58125; a COX-2 selective inhibitor) on carrageenan-induced thermal hyperalgesia (reduced hindpaw-withdrawal latency). The following observations were made: 1) Thermal hyperalgesia otherwise observed during the first 170 min was blocked in a dose-dependent manner by S(+)-ibuprofen or SC58125 administered i.t. or i.p. before carrageenan treatment. 2) Intraperitoneal, but not i.t., administration of either inhibitor after the establishment of hyperalgesia (170 min after carrageenan injection) reversed thermal hyperalgesia in a dose-dependent manner. Thus, the initial component of thermal hyperalgesia after tissue injury was blocked by systemic or spinal administration of both COX inhibitors, whereas established hyperalgesia was reversed only by systemic inhibitors. This study demonstrates that at least spinal COX-2, if not both COX-1 and COX-2, are necessary for the initiation of thermal hyperalgesia, whereas nonspinal sources of prostanoids (synthesized by COX-2 and perhaps also COX-1) are important for the maintenance of thermal hyperalgesia associated with tissue injury and inflammation.