Surfactant protein D (SP-D) is a collagenous, surfactant-associated, carbohydrate-binding protein that is synthesized by pulmonary epithelial cells. In the present studies, we examined the expression of SP-D and SP-D mRNA during late fetal (day 17, 19, 21) and early postnatal (day 5) rat lung development using immunochemical, cell-free translation, and Northern hybridization assays. SP-D mRNA and immunoreactive SP-D protein were first detected in guanidine extracts of whole rat lung at 21 days of gestation and reached even higher concentrations during the postnatal period. Likewise, immunoperoxidase studies of rat lung using affinity-purified antibodies to SP-D showed no staining at day 17 or 19, but demonstrated strong cytoplasmic staining of cuboidal epithelial cells lining immature airspaces at day 21 and strong cytoplasmic staining of type II and nonciliated bronchiolar cells in adult lung. SP-D also appeared in amniotic fluid by day 21 and was partially purified by affinity chromatography on maltosyl-agarose under conditions used for the isolation of rat lung SP-D. These studies indicate that the production of SP-D is increased shortly prior to birth, and that the increases in total lung SP-D and SP-D mRNA are temporally correlated with SP-D secretion and the appearance of SP-D in amniotic fluid.

Cannabinoid compounds have been shown to produce antinociception and antihyperalgesia by acting upon cannabinoid receptors located in both the CNS and the periphery. A potential mechanism by which cannabinoids could inhibit nociception in the periphery is the activation of cannabinoid receptors located on one or more classes of primary nociceptive neurons. To address this hypothesis, we evaluated the neuronal distribution of cannabinoid receptor type 1 (CB1) in the trigeminal ganglion (TG) of the adult rat through combined in situ hybridization (ISH) and immunohistochemistry (IHC). CB1 receptor mRNA was localized mainly to medium and large diameter neurons of the maxillary and mandibular branches of the TG. Consistent with this distribution, in a de facto nociceptive sensory neuron population that exhibited vanilloid receptor type 1 immunoreactivity, colocalization with CB1 mRNA was also sparse (<5%). Furthermore, very few neurons (approximately 5%) in the peptidergic (defined as calcitonin gene-related peptide-or substance P-immunoreactive) or the isolectin B 4 -binding sensory neuron populations contained CB1 mRNA. In contrast, and consistent with the neuron-size distribution for CB1, nearly 75% of CB1-positive neurons exhibited N52-immunoreactivity, a marker of myelinated axons. These results indicate that in the rat TG, CB1 receptors are expressed predominantly in neurons that are not thought to subserve nociceptive neurotransmission in the noninjured animal. Taken together with the absence of an above background in situ signal for CB2 mRNA in TG neurons, these findings suggest that the peripherally mediated antinociceptive effects of cannabinoids may involve either as yet unidentified receptors or interaction with afferent neuron populations that normally subserve non-nociceptive functions. Keywordscalcitonin gene-related peptide; vanilloid receptor type 1; substance P; isolectin B 4 ; in situ hybridizationThe primary psychoactive component of Cannabis sativa, Δ 9 -tetrahydrocannabinol, has long been recognized for its medicinal properties. Over the last two decades, a large body of work has elucidated the receptor-mediated actions of natural and synthetic cannabinoid compounds (for review, see Khanolkar et al., 2000). Two G protein-coupled cannabinoid receptors have been cloned, the cannabinoid receptor type 1 (CB1; Matsuda et al., 1990), found primarily in neurons, and the cannabinoid receptor type 2 (CB2; Munro et al., 1993) (Felder et al., 1993), as well as the modulation of neurotransmitter release through the inhibition of calcium currents (Twitchell et al., 1997;Shen and Thayer, 1998) and the activation of G protein-coupled inwardly-rectifying potassium channels (Mackie et al., 1995). On a whole organism level, endogenous cannabinoids as well as exogenously administered cannabinoids have been shown to, among other things, alleviate pain and inflammation.While there is ample evidence for antinociceptive effects of cannabinoids in the CNS (Lichtman and Martin, 1991;Martin et al., 1993Martin et ...

SUMMARY Purpose Increased activity of mTOR Complex 1 (mTORC1) has been demonstrated in cortical dysplasia and tuberous sclerosis complex, as well as in animal models of epilepsy. Recent studies in such models revealed that inhibiting mTORC1 with rapamycin effectively suppressed seizure activity. However, seizures can recur after treatment cessation, and continuous rapamycin exposure can adversely affect animal growth and health. Here, we evaluated the efficacy of an intermittent rapamycin treatment protocol on epilepsy progression using neuron subset-specific-Pten (NS-Pten) conditional knockout mice. Methods NS-Pten knockouts were treated with a single course of rapamycin during postnatal weeks four and five, or intermittently over a period of five months. Epileptiform activity was monitored using video-EEG recordings, and mossy fiber sprouting was evaluated using Timm staining. Survival and body weight were assessed in parallel. Key Findings NS-Pten knockouts treated with a single course of rapamycin had recurrence of epilepsy four to seven weeks after treatment ended. In contrast, epileptiform activity remained suppressed, and survival increased if knockout mice received additional rapamycin during weeks 10–11 and 16–17. Aberrant mossy fiber sprouting, present by four weeks of age and progressing in parallel with epileptiform activity, was also blocked by rapamycin. Significance These findings demonstrate that a single course of rapamycin treatment suppresses epileptiform activity and mossy fiber sprouting for several weeks before epilepsy recurs. However, additional intermittent treatments with rapamycin prevented this recurrence and enhanced survival without compromising growth. Thus, these studies add to the growing body of evidence implicating an important role for mTORC1 signaling in epilepsy.

Surfactant protein D (SP-D, CP4) is a collagenous surfactant-associated carbohydrate binding protein that was initially characterized as a biosynthetic product of type II pneumocytes. Immunoperoxidase studies of formaldehyde solution-fixed and paraffin-embedded rat lung demonstrated staining for SP-D in the cytoplasm of a subpopulation of bronchiolar epithelial cells as well as type II cells. Accordingly, immunogold-labeling techniques were used to further examine the cellular distribution and subcellular localization of SP-D in the small airways. Lung tissues were fixed with 0.5% glutaraldehyde-3% paraformaldehyde and embedded in LR White resin. Sections were reacted with affinity purified polyclonal antibodies to SP-D, and sites of antibody binding were demonstrated using a biotinylated secondary antibody-streptavidin-gold detection system. Anti-SP-D selectively decorated secretory compartments of nonciliated bronchiolar cells (Clara cells) with strong and specific labeling of apical electron-dense secretory granules. Almost all of the granules in nonciliated columnar cells were labeled; however, labeling was typically nonuniform, with preferential decoration of the periphery of the granule. The largest numbers of immunoreactive epithelial cells were observed in the distal membranous bronchioles, with progressively smaller numbers of cells in more proximal bronchioles. There was no detectable labeling of cells lining the large cartilagenous airways or trachea. These studies provide evidence that SP-D is a secretory product of nonciliated bronchiolar cells. We suggest that Clara cell-derived SP-D is a component of bronchiolar lining material, consistent with our hypothesis that SP-D contributes to surfactant metabolism and/or host defense within small airways.