iCGRP Release Research Articles

Dexamethasone and activators of the protein kinase A and C signal transduction pathways regulate neuronal calcitonin gene-related peptide expression and release.

Primary cultures of adult rat dorsal root ganglia (DRG) neurons were used to determine if activation of either the protein kinase A or C signal transduction pathways or treatment with the synthetic glucocorticoid dexamethasone modulate neuronal calcitonin gene-related peptide (CGRP) synthesis and release. DRG are the sites of neuronal cell bodies known to produce abundant CGRP levels, and to send axons peripherally to blood vessels and centrally to the spinal cord. Using immunocytochemical techniques, we confirmed that synthesis of immunoreactive CGRP (iCGRP) is restricted to a subpopulation of DRG neurons. Subsequently, we determined that treatment (24 h) of the neurons with either dibutyryl cAMP (1 mM) or phorbol 12-myristate 13-acetate (2 microM) increased CGRP mRNA content 2.2 +/- 0.4 (n = 6, p < 0.03) and 3.0 +/- 0.6-fold (n = 6, P < 0.02) respectively, while secreted iCGRP levels were increased 1.8 +/- 0.2 (n = 14, P < 0.005) and 4.5 +/- 1.0 (n = 14, P < 0.001)-fold over control levels. Treatment of the neurons with dexamethasone alone had no effect on CGRP expression; however, this agent was able to significantly attenuate the stimulatory effects of NGF on both CGRP mRNA accumulation and release of iCGRP. Time course studies demonstrated that in the phorbol ester treated neurons CGRP mRNA levels continued to increase at 48 h, while maximal induction with dibutyryl cAMP occurred at approximately 12 h. These results indicate that local and/or circulating factors which act through the protein kinase A and C signal transduction pathways upregulate both CGRP expression and release, while glucocorticoids attenuate the stimulatory effects of NGF.

Prostaglandin E2 enhances bradykinin-stimulated release of neuropeptides from rat sensory neurons in culture

Prostaglandins are known to enhance the inflammatory and nociceptive actions of other chemical mediators of inflammation such as bradykinin. One possible mechanism for this sensitizing action is that prostanoids augment the release of neuroactive substances from sensory neurons. To initially test this hypothesis, we examined whether selected prostaglandins could enhance the resting or bradykinin-evoked release of immunoreactive substance P (iSP) and/or immunoreactive calcitonin gene-related peptide (iCGRP) from sensory neurons in culture. Bradykinin alone causes a concentration-dependent increase in the release of iSP and iCGRP from isolated sensory neurons, and this action is abolished in the absence of extracellular calcium. Pretreating the neurons with PGE2 (10 nM to 1 microM) potentiates the bradykinin-evoked release of both iSP and iCGRP by approximately two-to fourfold. At these concentrations, PGE2 alone did not significantly alter peptide release. Exposing the cultures to 1 microM PGF2 alpha is ineffective in altering either resting or bradykinin-evoked peptide release. Sensory neurons in culture contain cyclooxygenase-like immunoreactivity suggesting that the enzyme that converts arachidonic acid to prostaglandins is present. In addition, pretreating cultures with 14C-arachidonic acid yields radiolabeled eicosanoids that cochromatograph with known prostaglandin standards. Preexposing cultures to indomethacin abolishes the production of prostaglandins and attenuates the bradykinin-stimulated release of iSP and iCGRP. This implies that the synthesis of prostaglandins contributes to the bradykinin-evoked release of peptides. The augmentation of bradykinin-induced release of iSP and iCGRP by PGE2 may be one mechanism to account for the inflammatory and hyperalgesic actions of this eicosanoid.

Sodium nitroprusside evokes the release of immunoreactive calcitonin gene-related peptide and substance P from dorsal horn slices via nitric oxide-dependent and nitric oxide-independent mechanisms

The results of behavioral studies suggest that nitric oxide (NO) participates in certain spinal mechanisms that contribute to hyperalgesia. Additionally, previous studies indicate that the release of immunoreactive calcitonin gene-related peptide (iCGRP) and substance P (iSP) is increased in the dorsal horn of the spinal cord during hyperalgesia. Therefore, the aim of this study was to determine whether NO acts to enhance peptide release in the dorsal horn of rats using an in vitro superfusion technique. Sodium nitroprusside (SNP) was used as an NO donor. The results of this study indicate that SNP caused a dose-related, calcium-dependent increase in the release of iCGRP and iSP from dorsal horn slices of the rat spinal cord. Furthermore, pretreatment with SNP reduced the ability of capsaicin to evoke the release of either peptide, suggesting that a target for SNP exists on certain capsaicin-sensitive primary afferent terminals. In addition to increasing peptide release, SNP also caused a significant five to sixfold increase in the levels of immunoreactive guanosine 3',5'-monophosphate (i-cGMP) in the dorsal horn. This SNP-evoked increase was significantly decreased by the guanylate cyclase inhibitor methylene blue in a dose-dependent manner. In addition, the release of iCGRP was also significantly reduced in the presence of methylene blue, although the relationship between peptide release and i-cGMP production remains unclear. Sodium nitroprusside-evoked peptide release was significantly reduced in the presence of hemoglobin (an oxide radical scavenger), suggesting that the drug effect was due to the generation of NO. However, the release of iCGRP and iSP was also evoked by sodium ferricyanide (the coproduct of SNP) and by 7-d-old, photoinactivated SNP.(ABSTRACT TRUNCATED AT 250 WORDS)

An in vitro method to evaluate regulation of neuropeptide release from dental pulp

Although pulpal neuropeptides such as calcitonin gene-related peptide and substance P may mediate neurogenic inflammation, little is known about the regulation of neuropeptide release from dental pulp. This article describes an in vitro method for superfusing dental pulp which permits the study of mechanisms regulating the release of immunoreactive CGRP (iCGRP). Tissue extracts from bovine dental pulp dilute in parallel to authentic calcitonin gene-related peptide and substance P peptide standards when assayed by radioimmunoassay. Pulpal levels of iCGRP were 17-fold greater than levels of immunoreactive substance P. Administration of a potassium pulse evoked a significant release of iCGRP from dental pulp (155 +/- 21 fmol/g/9 min) as compared with iCGRP spontaneously released from concurrent control chambers (18 +/- 11 fmol/g/9 min). The in vitro superfusion of pulp tissue may serve as a useful method for identifying peripherally acting drugs which modulate nociceptor secretory activity and for determining their mechanisms of action.

Enhanced release of immunoreactive CGRP and substance P from spinal dorsal horn slices occurs during carrageenan inflammation

In vitro superfusion was used to examine the effect of inflammation on the release of immunoreactive calcitonin gene-related peptide (iCGRP) and substance P(iSP) from the dorsal horn of rats. Three hours after carrageenan, hindpaws exhibited hyperalgesia, edema, and hyperthermia. Spontaneous and capsaicin-evoked release of iCGRP and iSP were significantly increased following inflammation. The enhanced release of iCGRP and iSP in the dorsal horn may serve as a biochemical marker for the development of hyperalgesia.

Influence of heat stimulation on the amount of calcitonin gene-related peptide and neurokinin A in the subcutaneous space of the rat hind instep.

The release of tachykinins and calcitonin gene-related peptide (CGRP) into subcutaneous perfusates was investigated. Immersion of the hind paw of rats in water at 47 degrees C for 30 min led to a marked increase of immunoreactive CGRP (iCGRP) release as well as immunoreactive substance P release, but no detectable increase of immunoreactive neurokinin A release. Neonatal pretreatment with capsaicin or section of the sciatic and the saphenous nerves significantly inhibited the heat stimulation-induced release of iCGRP.

Influence of Heat Stimulation on the Amount of Calcitonin Gene-Related Peptide and Neurokinin A in the Subcutaneous Space of the Rat Hind Instep

The release of tachykinins and calcitonin gene-related peptide (CGRP) into subcutaneous perfusates was investigated. Immersion of the hind paw of rats in water at 47°C for 30 min led to a marked increase of immunoreactive CGRP (iCGRP) release as well as immunoreactive substance P release, but no detectable increase of immunoreactive neurokinin A release. Neonatal pretreatment with capsaicin or section of the sciatic and the saphenous nerves significantly inhibited the heat stimulation-induced release of iCGRP.

Capsaicin-induced release of calcitonin gene-related peptide from dorsal horn slices is enhanced in adjuvant arthritic rats

The capsaicin (3 μM)-evoked release of the immunoreactive calcitonin gene-related peptide (iCGRP) from dorsal-half slices of the rat lumbar spinal cord was substantially reduced in slices from rats which had undergone dorsal rhizotomy. This suggests that the release is elicited mainly from the nerve terminals of primary afferents. The release was significantly increased in slices from adjuvant arthritic rats compared with those from control rats. This suggests that the amount of iCGRP in a capsaicin-sensitive pool of the nerve terminals of primary afferents is elevated by adjuvant arthritis. Since adjuvant arthritis is known to cause an enhancement of biosynthesis and axonal flow of iCGRP in primary sensory neurons 6, the present results imply an increased release of iCGRP from the central terminals of the primary afferents in the adjuvant arthritic rat.