Time-dependent changes in Bolton-Hunter-labeled 125I-substance P binding in rat spinal cord following unilateral adjuvant-induced peripheral inflammation.

Time-dependent changes in Bolton-Hunter-labeled 125I-substance P binding occurred in the dorsal horn of the spinal cord following unilateral adjuvant-induced inflammation in the hindpaw of the rat. Inflammation was characterized by measures of edema and hyperalgesia. Edema and hyperalgesia were both present 6 h after induction of inflammation. However, by eight days, hyperalgesia had dissipated while edema persisted. Six hours after the induction of inflammation, widespread decreases in Bolton-Hunter-labeled 125I-substance P binding occurred on both sides of the dorsal horn of spinal level L4 in comparison to the control group. However, by two days, widespread increases in Bolton-Hunter-labeled 125I-substance P binding occurred on both sides of the spinal cord at level L4 compared to the control group. The increase in radioligand binding was primarily due to a 10-fold increase in affinity of neurokinin-1 receptors for substance P. At later time-points of four and eight days, Bolton-Hunter-labeled 125I-substance P binding remained increased only in laminae I/II on the side of the spinal cord ipsilateral to inflammation. The changes in Bolton-Hunter-labeled 125I-substance P binding suggest that alterations in substance P synaptic transmission in the spinal cord may contribute to the increased excitability of spinal neurons that accompanies adjuvant-induced peripheral inflammation.

Plasticity in the synthesis and storage of substance P and calcitonin gene-related peptide in primary afferent neurons during peripheral inflammation.

Several indices of peptidergic, primary afferent neural transmission in rat at the level of the lumbar spinal cord exhibited differential changes over time in response to adjuvant-induced inflammation of the hindpaw. The indices were measurements of the production of messenger RNA encoding the precursors for substance P and calcitonin gene-related peptide in dorsal root ganglia, the storage of substance P and calcitonin gene-related peptide in the dorsal spinal cord and the release of the peptides evoked by application of capsaicin to the dorsal spinal cord. A 47% decrease in the content of immunoreactive substance P in the dorsal half of the lumbar spinal cord, as determined by radioimmunoassay, was measured at 6 h following the injection of complete Freund's adjuvant into the hindpaw. Decreased content of immunoreactive SP persisted for four days, but was no longer present at eight days after the adjuvant injection. The content of immunoreactive calcitonin gene-related peptide in the dorsal spinal cord was decreased by 29% at one day following the injection of adjuvant into the rat hindpaw and 43% at two days; the content then increased to a level greater than that of control animals at eight days. The amount of messenger RNA encoding preprotachykinin and prepro-calcitonin gene-related peptide in L4-L6 dorsal root ganglia was determined from northern blot analysis of the total messenger RNA extracted from the dorsal root ganglia. Each species of messenger RNA had increased compared to the control animals at two days following the injection of adjuvant into the rat hindpaws and remained elevated after eight days. Thus, an increase in the messenger RNAs encoding substance P and calcitonin gene-related peptide in the dorsal root ganglia preceeded the recovery of the content of the peptides in the spinal cord. Morphometric studies of calcitonin gene-related peptide-immunoreactive perikarya in the L4 dorsal root ganglia indicated that the increase in messenger RNA occurred in neurons of the size that normally express calcitonin gene-related protein. Radioimmunoassay of the superfusate of the dorsal half of the lumbar spinal cord was used to measure the release of immunoreactive substance P and immunoreactive calcitonin gene-related protein in vitro. Although the basal release of immunoreactive substance P and immunoreactive calcitonin-gene related protein from the dorsal spinal cord was constant throughout the time points examined, changes occurred in the release of peptide evoked by 10 microM capsaicin. The capsaicin-evoked release of immunoreactive substance P was decreased at 6 h and eight days post-injection of adjuvant.(ABSTRACT TRUNCATED AT 400 WORDS)

Islet amyloid polypeptide (IAPP) competes for two binding sites of CGRP.

Two distinct binding sites for [125I]human calcitonin gene-related peptide (hCGRP) were found in rat brain, skeletal muscle, and liver. Each tissue had a high affinity site with an average Kd of 46 pM and a low affinity site with an average Kd of 22 nM. Islet amyloid polypeptide (IAPP), which has N- and C-terminal sequence homology to CGRP and is produced by islet beta-cells, bound to both sites but had a potency closer to that of CGRP at the low affinity binding site. A C-terminal fragment of IAPP competed for [125I]hCGRP binding at the low affinity site with potency comparable to that of hIAPP. No specific binding to membrane preparations was found in experiments using [125I]rIAPP, which was iodinated at the C-terminal tyrosyl residue. These results suggest that some of the previously reported biological effects occurring at nM or microM concentrations of IAPP may be mediated by IAPP binding to low affinity CGRP receptors. This study further indicates that the C-terminal region of IAPP is important for binding to low affinity CGRP receptors, and suggests that C-terminal fragments of IAPP may be of biological importance.

Changes in [125I]hCGRP binding in rat spinal cord in an experimental model of acute, peripheral inflammation.

[125I]Human calcitonin gene-related peptide ([125I]hCGRP) binding in the dorsal horn of the spinal cord exhibited differential changes among laminae over time in response to unilateral adjuvant-induced inflammation. In laminae I/II, 4 days after induction of inflammation, the binding decreased 36% on the side of the spinal cord ipsilateral to the inflammation, while there was no change on the contralateral side. The decrease ipsilateral to inflammation was due primarily to a decrease in the Bmax of the high affinity binding site for CGRP. In lamina V, the binding increased 18% on both sides of the spinal cord at the same time point. In lamina X, the binding increased 16% on both sides of the spinal cord at 2 days after induction of inflammation and remained increased at 8 days. The increases in [125]hCGRP binding in laminae V and X were primarily due to a decrease in the Kd of the low affinity binding site for CGRP. the accompanying hyperalgesia was first measured at 2 days after induction of inflammation and persisted at 8 days. Because the changes in [125I]hCGRP binding did not parallel the hyperalgesia accompanying the unilateral adjuvant-induced inflammation, we believe that CGRP receptors are not directly involved with the hyperalgesia but may be involved with other plastic changes observed in the spinal cord during unilateral adjuvant-induced inflammation.

Plasticity of calcitonin gene related peptide neurotransmission in the spinal cord during peripheral inflammation.

Injection of complete Freund's adjuvant (CFA; 75 microL) into the plantar surface of the hind paw of the rat results in a mild inflammation that lasts for several days and is accompanied by hyperalgesia. Multiple components of calcitonin gene related peptide (CGRP) neurotransmission in the spinal cord are altered during the course of this peripheral inflammation. The content of immunoreactive (i) CGRP in the dorsal horn of the spinal cord, where primary afferent neurons terminate, is significantly decreased within 2 days after injection of CFA but increases to a level greater than that of the control at 8 days. The early decrease in iCGRP in the spinal cord suggests that the release of CGRP from primary afferent neurons is increased during the period of maximal hyperalgesia that accompanies peripheral inflammation. Changes in the mRNA for CGRP suggest that the increase in spinal content of iCGRP is due to an increase in synthesis of the peptide as the level of mRNA for CGRP is increased from 2 to 8 days after injection of CFA. Despite the decrease in the content of iCGRP in the spinal cord, there is no apparent decrease in the amount of iCGRP that can be released from the dorsal spinal cord by capsaicin; in fact, capsaicin-evoked release is increased at 4 days. Measurements of the binding of 125I-labelled CGRP in the dorsal spinal cord indicate that high affinity binding sites for CGRP are downregulated at 4 days after injection of CFA. In total, these data support the hypothesis that the activity of CGRP-containing primary afferent neurons is increased during peripheral inflammation. CGRP released from primary afferent neurons in the spinal cord may contribute to cellular changes that accompany peripheral inflammation.