The messenger RNA (mRNA) that encodes alpha subunit of the guanosine triphosphate-binding protein transducin (T alpha) and T alpha immunoreactivity were localized and measured in the rat retina during the light-dark cycle with in situ hybridization and immunohistochemistry. Both T alpha mRNA and T alpha immunoreactivity were observed only in photoreceptors. Within the photoreceptor T alpha mRNA was present primarily in the inner segments and to a lesser extent in the outer nuclear layer at all times during the day and night. However, the distribution of T alpha immunoreactivity varied profoundly with the light-dark cycle; during the day, T alpha immunoreactivity was highest in the inner segments, and at night the outer segments were more immunoreactive. The amounts of T alpha mRNA and T alpha immunoreactivity also depended on the light-dark cycle. Levels of T alpha mRNA were high immediately before and after lights on; levels were low for the rest of the light-dark cycle. During the day, T alpha immunoreactivity increased in the inner segments following the increase in T alpha mRNA. After the lights were turned off, T alpha immunoreactivity decreased in the inner segments and increased in the outer segments. Thus, it appears that T alpha is synthesized in the inner segments after a morning increase in T alpha mRNA. Newly synthesized T alpha remains in the inner segments until it is transported to the outer segments at night, where it may be involved in the increase in the sensitivity of photoreceptor rods at night.
Dynorphin, an opioid peptide, is thought to play an important role in the modulation of nociceptive neural circuits at the level of the spinal cord. In a model of peripheral inflammation and hyperalgesia, an oligodeoxyribonucleotide probe complementary to a portion of preprodynorphin mRNA and antisera to dynorphin A-(1-8) were used to localize changes in dynorphin mRNA and peptide to individual spinal cord neurons. Intraplantar injection in rats of complete Freund's adjuvant resulted in edema and hyperalgesia to radiant heat stimulation of the in ected hind paw that reached a peak at 4 days. At the same time, in situ hybridization histochemistry and immunocytochemistry identified an increase in transcription of preprodynorphin mRNA that was paralleled by an increase in dynorphin peptide. These changes were seen in spinal neurons in the medial two-thirds of laminae I and II and in laminae V and VI of lumbar segments receiving innervation from the inflamed paw. Since neurons demonstrating the increase in dynorphin biosynthesis are located in both the superficial and deep dorsal horn laminae, our data provide evidence for opioid modulation of nociceptive neural circuits in these two distinct spinal locations.Dynorphin, an opioid peptide (1), is encoded by the preprodynorphin gene (2). It has been localized to neurons in laminae I-VII of the spinal cord (3-5) and has been implicated in the response to peripheral nociceptive stimuli at the level of the spinal cord following inflammation (6, 7) and in chronic arthritic rats (8-10). In a model of Freund's adjuvant-induced inflammation and hyperalgesia of the rat hind paw, RIA identified a rapid increase in dynorphin A-(1-8) peptide that was unilateral and confined to the spinal segments that received innervation from the affected limb (6). An increase in preprodynorphin mRNA has been shown to parallel that of the peptide in terms of onset and location (7). Those (6,11). This model differs from chronic inflammatory arthritic models in that it is rapid in onset, and only the injected limb demonstrates the inflammation and hyperalgesia. Although the animals guarded the affected limb from additional stimulation, their general behavior indicated that they were exposed to minimal discomfort. They groomed properly, showed less than 6% weight loss, and socially interacted with littermates. The condition of the animals was observed by the National Institute of Dental Research Animal Care and Use Committee, which approved these experiments. Some animals (n = 11) received an intrathecal injection (12) of colchicine (100 ,ug in 100 1.d of sterile saline) 2 days before sacrifice. This colchicine treatment was near threshold to visualize, by immunocytochemistry, cell bodies in untreated rats. Animals were killed 4 days after hind paw injection either by decapitation, in which case the lumbar spinal cords were quickly removed and frozen, or by cardiac perfusion with 4% paraformaldehyde in 0.1 M P04 buffer (2.65 g of NaH2PO4H20/14.0 g of K2HPO4 per liter). Spinal cords from...
Hypertrophic scar is the result of abnormal healing that often follows thermal injury. Hypertrophic scar is characterized by excessive dermal fibrosis and scarring. Five cases of human hypertrophic scar were compared with normal skin using in situ hybridization to localize mRNAs for procollagen types I and III and transforming growth factor-beta 1. Expression of type I procollagen and TGF-beta 1 were also examined with immunohistochemistry. The results demonstrated a significant increase in the expression of mRNA for types I and III procollagen and type I procollagen protein by fibroblasts in hypertrophic scar compared with normal skin. In all cases of hypertrophic scar, significant numbers of cells expressed TGF-beta 1 mRNA or peptide. Neither TGF-beta 1 mRNA nor protein was detected in control tissues. These results suggest a profound increase in production and expression of types I and III collagen mRNA by the fibroblasts in hypertrophic scar. This may result from increased TGF-beta 1 production, through paracrine and autocrine pathways, as have been described for this fibrogenic cytokine.
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