Secondary traumatic stress increases expression of proteins implicated in peripheral and central sensitization of trigeminal neurons.

The pathology of migraine, a common neurological disease, involves sensitization and activation of trigeminal nociceptive neurons to promote hyperalgesia and allodynia during an attack. Migraineurs often exhibit characteristics of a hyperexcitable or hypervigilant nervous system. One of the primary reported risk factors for development of a hyperexcitable trigeminal system is chronic, unmanaged stress and anxiety. While primary traumatic stress is a commonly cited risk factor for many pain conditions, exposure to secondary traumatic stress early in life is also thought to be a contributing risk factor. The goal of this study was to investigate cellular changes within the spinal trigeminal nucleus and trigeminal ganglion mediated by secondary traumatic stress. Male Sprague Dawley rats (sender) were subjected to forced swim testing (primary traumatic stress) and were then housed in close visual, olfactory, and auditory proximity to the breeding male and female rats, pregnant female rats, or female rats and their nursing offspring (all receivers). In response to secondary stress, levels of calcitonin gene-related peptide, active forms of the mitogen activated protein kinases ERK, JNK, and p38, and astrocyte expression of glial fibrillary acidic protein were significantly elevated in the spinal trigeminal nucleus in day 45 offspring when compared to naïve offspring. In addition, increased nuclear expression of ERK and p38 was observed in trigeminal ganglion neurons. Our results demonstrate that secondary traumatic stress promotes cellular events associated with prolonged trigeminal sensitization in the offspring, and provides a mechanism of how early life stress may function as a risk factor for migraine.

Effects of cocoa-enriched diet on orofacial pain in a murine model.

OBJECTIVES: To investigate and discuss the effects of cocoa on orofacial pain. SETTING AND SAMPLE POPULATION: The Department of Orthodontics at the University of Florida (UF). Male and female hairless rats (N=20/group) were tested. MATERIALS AND METHODS: Rats were tested using the Orofacial Pain Assessment Device (OPAD) before and after changing their food from the standard chow to a cocoa-enriched or control-equivalent diet. RESULTS: Male rats fed the cocoa diet had a significantly higher operant pain index when tested at 37°C as compared to control diet-fed animals. Female rats on the cocoa diet had a significantly higher pain index when tested at 18°C and 44°C, as compared to animals fed the control diet. Capsaicin-induced pain was inhibited, with cocoa-diet male rats having a significantly higher pain index than control-diet male rats and cocoa-diet female rats at both 37°C and 44°C. Cocoa-diet female rats had a significantly higher pain index at 44°C than control-diet females. Mechanical sensitivity was affected following capsaicin cream, with a significantly decreased tolerated bottle distance in both cocoa- and control-diet animals, but there was no difference between cocoa- and control-diet groups. CONCLUSION: Using the OPAD operant system, we demonstrated that a diet rich in cocoa was effective in inhibiting neurogenic inflammatory pain in rats. This has implications for the use of novel alternative therapies such as diet modification for pain control.

Nicotine stimulates expression of proteins implicated in peripheral and central sensitization.

Pain patients who are nicotine dependent report a significantly increased incidence and severity of pain intensity. The goal of this study was to determine the effects of prolonged nicotine administration on inflammatory proteins implicated in the development of peripheral and central sensitization of the trigeminal system. Behavioral, immunohistochemical, and microarray studies were utilized to investigate the effects of nicotine administered daily for 14 days via an Alzet® osmotic pump in Sprague Dawley rats. Systemic nicotine administration caused a significant increase in nocifensive withdrawals to mechanical stimulation of trigeminal neurons. Nicotine stimulated expression of the pro-inflammatory signal transduction proteins phosphorylated-extracellular signal-regulated kinase (p-ERK), phosphorylated-c-Jun N-terminal kinase (p-JNK), and protein kinase A (PKA) in the spinal trigeminal nucleus. Nicotine also promoted elevations in the expression of glial fibrillary acidic protein (GFAP), a biomarker of activated astrocytes, and the microglia biomarker ionized calcium-binding adapter molecule 1 (Iba1). Similarly, levels of eleven cytokines were significantly elevated with the largest increase in expression of TNF-α. Levels of PKA, p-ERK, and p-JNK in trigeminal ganglion neurons were increased by nicotine. Our findings demonstrate that prolonged systemic administration of nicotine promotes sustained behavioral and cellular changes in the expression of key proteins in the spinal trigeminal nucleus and trigeminal ganglion implicated in the development and maintenance of peripheral and central sensitization.

Dual orexin receptor antagonist 12 inhibits expression of proteins in neurons and glia implicated in peripheral and central sensitization.

Sensitization and activation of trigeminal nociceptors is implicated in prevalent and debilitating orofacial pain conditions including temporomandibular joint (TMJ) disorders. Orexins are excitatory neuropeptides that function to regulate many physiological processes and are reported to modulate nociception. To determine the role of orexins in an inflammatory model of trigeminal activation, the effects of a dual orexin receptor antagonist (DORA-12) on levels of proteins that promote peripheral and central sensitization and changes in nocifensive responses were investigated. In adult male Sprague-Dawley rats, mRNA for orexin receptor 1 (OX1R) and receptor 2 (OX2R) were detected in trigeminal ganglia and spinal trigeminal nucleus (STN). OX1R immunoreactivity was localized primarily in neuronal cell bodies in the V3 region of the ganglion and in laminas I-II of the STN. Animals injected bilaterally with complete Freund's adjuvant (CFA) in the TMJ capsule exhibited increased expression of P-p38, P-ERK, and lba1 in trigeminal ganglia and P-ERK and lba1 in the STN at 2 days post injection. However, levels of each of these proteins in rats receiving daily oral DORA-12 were inhibited to near basal levels. Similarly, administration of DORA-12 on days 3 and 4 post CFA injection in the TMJ effectively inhibited the prolonged stimulated expression of protein kinase A, NFkB, and Iba1 in the STN on day 5 post injection. While injection of CFA mediated a nocifensive response to mechanical stimulation of the orofacial region at 2h and 3 and 5 days post injection, treatment with DORA-12 suppressed the nocifensive response on day 5. Somewhat surprisingly, nocifensive responses were again observed on day 10 post CFA stimulation in the absence of daily DORA-12 administration. Our results provide evidence that DORA-12 can inhibit CFA-induced stimulation of trigeminal sensory neurons by inhibiting expression of proteins associated with sensitization of peripheral and central neurons and nociception.

Treatment of mast cells with carbon dioxide suppresses degranulation via a novel mechanism involving repression of increased intracellular calcium levels.

BACKGROUND: Intranasal noninhaled delivery of carbon dioxide (CO2) is efficacious in the symptomatic treatment of seasonal allergic rhinitis. The goal of this study was to determine whether and how 100% CO2 inhibits mast cell degranulation, thereby possibly contributing to the reduction of symptoms in seasonal allergic rhinitis. METHODS: Peritoneal mast cells isolated from rats and labelled with sulforhodamine-B (SFRM-B) were used to determine whether CO2 treatment could block mast cell degranulation and histamine release in response to 48/80. In addition, the effect of CO2 on intracellular calcium levels in unstimulated and stimulated mast cells was determined by fluorescent microscopy. RESULTS: Treatment with 48/80 caused >90% of mast cells containing SFRM-B to degranulate, resulting in a marked decrease in the fluorescent intensity within the mast cells, and simultaneously causing a significant increase in histamine release. Significantly, the stimulatory effect of 48/80 on fluorescent intensity and histamine levels was greatly inhibited (>95%) to near control levels by pretreatment with 100% CO2. Treatment with 48/80 also caused a robust transient increase in intracellular calcium, whereas pretreatment with CO2 repressed the increase in calcium (>70%) in response to 48/80. CONCLUSIONS: Results from this study provide the first evidence of a unique regulatory mechanism by which CO2 inhibits mast cell degranulation and histamine release by repressing stimulated increases in intracellular calcium. Thus, our data provide a plausible explanation for the reported therapeutic benefit of noninhaled intranasal delivery of 100% CO2 to treat allergic rhinitis.

Neurological mechanisms of migraine: potential of the gap-junction modulator tonabersat in prevention of migraine.

Migraine is a neurovascular disorder characterized by recurrent episodic headaches, and is caused by abnormal processing of sensory information due to peripheral and/or central sensitization. The exact pathophysiological mechanism underlying migraine is not fully understood; however, cortical spreading depression (CSD) is thought to provide the basis for migraine aura and may serve as a trigger of migraine pain. CSD depends on neuronal-glial cell communication, which is mediated by intercellular transfer of messengers through connexin-containing gap junctions, as well as messengers released into the extracellular space by non-junctional connexin-containing hemichannels. These processes are believed to be important in peripheral sensitization within the trigeminal ganglion and to lead to central sensitization. The novel benzopyran compound tonabersat binds selectively to a unique site in the brain. In preclinical studies, tonabersat markedly reduced CSD and CSD-associated events and inhibited gap-junction communication between neurons and satellite glial cells in the trigeminal ganglion. Together, these findings suggest that tonabersat should have clinical application in preventing migraine attacks.

CGRP stimulation of iNOS and NO release from trigeminal ganglion glial cells involves mitogen-activated protein kinase pathways.

Clinical and basic science data support an integral role of calcitonin gene-related peptide (CGRP) in the pathophysiology of temporomandibular joint disorders. Recently, we have shown that CGRP can stimulate the synthesis and release of nitric oxide (NO) from trigeminal ganglion glial cells. The goal of this study was to determine the role of mitogen-activated protein kinase (MAPK) signaling pathways in CGRP regulation of iNOS expression and NO release from cultured trigeminal ganglion glial cells from Sprague-Dawley rats. CGRP treatment for 2 h significantly increased activity of the MAPK reporter genes, Elk, ATF-2, and CHOP. In addition, CGRP increased nuclear staining for the active forms of the MAPKs: extracellular signal-regulated kinase, c-Jun amino-terminal kinase, and p38. This stimulatory event was not observed in cultures pre-treated with the CGRP receptor antagonist peptide CGRP(8-37). Similarly, pre-treatment with selective MAPK inhibitors repressed increases in reporter gene activity as well as CGRP-induced increases in iNOS expression and NO release mediated by MAPKs. In addition, over-expression of MAPK kinase 1 (MEK1), MEK3, MEK6, and MEK kinase significantly increased iNOS expression and NO production in glial cells. Results from our study provide evidence that CGRP binding to its receptor can stimulate iNOS gene expression via activation of MAPK pathways in trigeminal ganglion glial cells.

Nitric oxide-proton stimulation of trigeminal ganglion neurons increases mitogen-activated protein kinase and phosphatase expression in neurons and satellite glial cells.

Elevated nitric oxide (NO) and proton levels in synovial fluid are implicated in joint pathology. However, signaling pathways stimulated by these molecules that mediate inflammation and pain in the temporomandibular joint (TMJ) have not been investigated. The goal of this study was to determine the effect of NO-proton stimulation of rat trigeminal neurons on the in vivo expression of mitogen-activated protein kinases (MAPKs) and phosphatases (MKPs) in trigeminal ganglion neurons and satellite glial cells. Low levels of the active MAPKs extracellular signal-regulated kinase (ERK), Jun amino-terminal kinase (JNK), and p38 were localized in the cytosol of neurons and satellite glial cells in unstimulated animals. However, increased levels of active ERK and p38, but not JNK, were detected in the cytosol and nucleus of V3 neurons and satellite glial cells 15 min and 2 h following bilateral TMJ injections of an NO donor diluted in pH 5.5 medium. While ERK levels returned to near basal levels 24 h after stimulation, p38 levels remained significantly elevated. In contrast to MKP-2 and MKP-3 levels that were barely detectable in neurons or satellite glial cells, MKP-1 staining was readily observed in satellite glial cells in ganglia from unstimulated animals. However, neuronal and satellite glial cell staining for MKP-1, MKP-2, and MKP-3 was significantly increased in response to NO-protons. Increased active ERK and p38 levels as well as elevated MKP levels were also detected in neurons and satellite glial cells located in V2 and V1 regions of the ganglion. Our data provide evidence that NO-proton stimulation of V3 neurons results in temporal and spatial changes in expression of active ERK and p38 and MKPs in all regions of the ganglion. We propose that in trigeminal ganglia these cellular events, which are involved in peripheral sensitization as well as control of inflammatory and nociceptive responses, may play a role in TMJ pathology.

Differential regulation of mitogen-activated protein kinase-responsive genes by the duration of a calcium signal.

We have investigated the cellular mechanisms by which changes in intracellular calcium (Ca2+) can differentially regulate gene expression. Two Ca2+ paradigms, involving prolonged and transient Ca2+ increases, were used. As a starting point, we studied the slow, prolonged elevation of Ca2+ caused by activation of 5-HT1 receptors. We had previously shown that 5-HT1 agonists inhibit calcitonin gene-related peptide (CGRP) transcription and secretion. The Ca2+ ionophore, ionomycin, was used to produce a prolonged elevation of the Ca2+ signal similar to that generated by 5-HT1 receptor agonists. Ionomycin treatment of the neuronal-like CA77 cell line specifically inhibited mitogen-activated protein (MAP) kinase stimulation of the CGRP enhancer and two synthetic MAP kinase-responsive reporter genes (4- to 10-fold). We then showed that ionomycin repression of promoter activity involved selective induction of MAP kinase phosphatase-1 (MKP-1), but not MKP-2, and that overexpression of MKP-1 was sufficient to repress CGRP enhancer activity. These effects were then compared with a Ca2+ paradigm involving a transient elevation in Ca2+ as seen after depolarization. At 4 h after the transient increase in Ca2+, the CGRP enhancer and synthetic MAP kinase-responsive reporter genes were stimulated. In contrast, exposure to depolarizing stimuli overnight caused only a less than 2-fold inhibition of promoter activity. We propose that the duration of the Ca2+ signal can determine the magnitude of a negative feedback loop that leads to differential regulation of MAP kinase-responsive genes.

Regulation of calcitonin gene-related peptide secretion by a serotonergic antimigraine drug.

We have investigated the regulation of calcitonin gene-related peptide (CGRP) release from trigeminal neurons by the serotonergic antimigraine drug sumatriptan. Serum levels of the neuropeptide CGRP are elevated during migraine. Treatment with the drug sumatriptan returns CGRP levels to normal coincident with the alleviation of headache. However, despite this clinical efficacy, the cellular target and mechanism of sumatriptan action are not well understood beyond the pharmacology of its recognition of the 5-HT1 class of serotonin receptors. We have used cultured trigeminal neurons to demonstrate that sumatriptan can directly repress CGRP secretion from sensory neurons. The stimulated secretion in response to depolarization or inflammatory agents was inhibited, but not the basal secretion rate. Unexpectedly, sumatriptan did not lower cAMP levels, in contrast to the classical role ascribed to the 5-HT1 receptors. Instead, activation of 5-HT1 receptors caused a slow and remarkably prolonged increase in intracellular calcium. The inhibition of CGRP secretion is attenuated by the phosphatase inhibitor okadaic acid, suggesting that sumatriptan action is mediated by calcium-recruited phosphatases. These results suggest that 5-HT1 agonists may block a deleterious feedback loop in migraine at the trigeminal neurons and provide a general mechanism by which this class of drugs can attenuate stimulated neuropeptide release.

Serotonergic repression of mitogen-activated protein kinase control of the calcitonin gene-related peptide enhancer.

We have investigated the mechanisms underlying regulation of the calcitonin gene-related peptide (CGRP) cell-specific enhancer. Recently, we reported that this enhancer is inhibited by serotonin type-1 (5-HT1) agonists, similar to currently used antimigraine drugs. We have now tested whether this repression involves a mitogen-activated protein (MAP) kinase pathway. We first demonstrate that the CGRP enhancer is strongly (10-fold) activated by a constitutively active MAP kinase kinase (MEK1), yielding reporter activities 100-fold above the enhancerless control. The involvement of a MAP kinase pathway was confirmed by down-regulation of reporter activity upon cotransfection of a dominant negative Ras. Activation of the enhancer by MEK1 was blocked in a dose-dependent manner by the 5-HT1 receptor agonist CGS 12066A (CGS). Since it is not known whether the CGRP enhancer factors are immediate targets of MAP kinases, we then used EIk-1- and c-Jun-dependent reporter genes that are directly activated by the ERK (extracellular signal-regulated kinases) and JNK (c-Jun N-terminal kinase) MAP kinases. CGS treatment repressed the activation of both of these reporters, suggesting that at least two MAP kinases are the immediate targets of CGS-mediated repression. We further demonstrate that 5-HT1 agonists inactivate ERK by dephosphorylation, even in the presence of constitutively activated MEK1. This inactivation appears to be due to a marked increase in the level of MAP kinase phosphatase-1. These results have defined a novel and general mechanism by which 5-HT1 receptor agonists can repress MAP kinase activation of target genes, such as CGRP.

Repression of the calcitonin gene-related peptide promoter by 5-HT1 receptor activation.

We have investigated the control of calcitonin gene-related peptide (CGRP) expression by a serotonergic agonist that is related pharmacologically to currently used antimigraine drugs. During migraines, CGRP levels are elevated but then returned to normal by a 5-HT1 receptor agonist, sumatriptan. However, neither the molecular nor cellular targets of this drug are known. Trigeminal neurons are the major source of cerebrovascular CGRP, and thus we have used trigeminal primary cultures and the neuronal-like CA77 thyroid C-cell line as a model. We first demonstrate that sumatriptan and another 5-HT1 agonist, CGS 12066A (CGS), cause a robust and prolonged increase with oscillations in intracellular calcium in CA77 cells. CGS caused a similar increase in trigeminal cultures. We then show that CGS treatment leads to a decrease in CGRP mRNA levels in the CA77 cells. This decrease is attributable to the repression of promoter activity through two discrete elements: (1) the cAMP-responsive region, via a cAMP-independent mechanism; and (2) the cell-specific enhancer, which binds the upstream stimulatory factor helix-loop-helix protein and a cell-specific activator. These results demonstrate that activation of the endogenous 5-HT1 receptor is coupled to calcium signaling pathways and leads to inhibition of CGRP gene transcription.

Thyroid parafollicular cells. An accessible model for the study of serotonergic neurons.

Serotonergic neurons play key roles in modulating a wide variety of behavioral and homeostatic processes. However, there is a paucity of good model systems to study these neurons at a molecular level. In this review we will present evidence that cell lines derived from an unexpected source, thyroid parafollicular cells (PF) (also called C cells), fit the criteria for use as models for the study of serotonergic neurons. A strength of PF cell lines over other cell lines is that the parental PF cells have serotonergic properties and a neuronal potential that is consistent with their neural crest origin. Furthermore, PF cells and PF cell lines are capable of expressing the fundamental properties of serotonergic neurons, including: (1) serotonin (5-HT) biosynthesis by tryptophan hydroxylase (TPH), (2) vesicular 5-HT storage and regulated release, (3) expression of a 5-HT autoreceptor, and (4) expression of the 5-HT transporter. In this review, we will focus primarily on the serotonergic and neuronal properties of the rat CA77 PF cell line and the parental rat PF cells. The applicability of CA77 cells for molecular analyses will be described. First, their use for studies on the glucocorticoid regulation of the TPH gene will be discussed. Second, control of the calcitonin/calcitonin gene-related peptide (CT/CGRP) gene will be discussed, with particular emphasis on the application of serotonergic drugs in treating migraine headaches. These examples highlight the versatility of thyroid PF cell lines as a system for studying the control of both serotonin biosynthesis and physiological actions.

Regulation of the beta 2 subunit chain of laminin in developing rabbit fetal lung tissue.

Laminins are a family of basement membrane-associated heterotrimeric proteins that are important in mediating the growth, migration, and differentiation of a variety of cell types. The beta 2 subunit chain is a component of several laminin isoforms, e.g., laminin-3, laminin-4, laminin-7, and possibly other, as yet uncharacterized laminin isoforms. Utilizing monoclonal antibodies directed against the beta 2 subunit chain of laminin, we detected this protein in fetal, neonatal, and adult lung tissues. The relative amount of laminin beta 2 subunit chain in fetal lung tissue increased as gestation proceeded, reaching its peak around the time of alveolar type II cell differentiation in the rabbit. The laminin beta 2 subunit chain was localized in early gestational age rabbit fetal lung tissue primarily in basement membranes of prealveolar ducts, airways, and smooth muscle cells of airways and arterial blood vessels. A rabbit laminin beta 2 cDNA was generated using RT-PCR and utilized as a probe in northern blot analysis to characterize the levels of laminin beta 2 mRNA in developing rabbit lung tissue. Similar to the pattern of laminin beta 2 protein induction observed in fetal lung tissue, laminin beta 2 mRNA levels were maximal late in gestation. Utilizing a laminin beta 2 chain cRNA probe and in situ hybridization, we detected laminin beta 2 mRNA in the epithelial cells of prealveolar ducts, the alveolar wall, and airways, as well as in connective tissue cells, and the smooth muscle cells of airways and blood vessels in fetal and adult lung tissues. In addition, using an in vitro explant model, we determined that alveolar type II cells are capable of synthesizing laminin beta 2 subunit mRNA and depositing this laminin subunit chain in the basement membrane beneath type II cells. The results of this study are suggestive that the laminin beta 2 chain may be involved in alveolar epithelial cell differentiation.

Characterization of alpha 1, beta 1, and gamma 1 laminin subunits during rabbit fetal lung development.

Laminin-1 is an extracellular matrix protein composed of three polypeptide chains that are designated alpha 1, beta 1, and gamma 1. We investigated the expression of laminin alpha 1, beta 1, and gamma 1 subunit chains during several stages of rabbit fetal lung development. Utilizing polyclonal antibodies directed against human placental laminin and immunoblot analysis, we found that the highest levels of laminin alpha 1, beta 1, and gamma 1 subunit chains in the fetal lung were present on day 26 of gestation (term = 31 days), coincident with the initiation of alveolar epithelial cell differentiation. Levels of the laminin chains were approximately five times higher in fetal lung at day 26 of gestation than in adult lung tissue. Different temporal patterns of laminin alpha 1, beta 1, and gamma 1 subunit chain expression were observed, data suggestive that the chains are independently regulated during lung development. Laminin was localized to the basement membranes of bronchi, bronchioles, prealveolar ducts, and blood vessels in fetal lung tissue, as shown by immunostaining with polyclonal laminin antibodies. A similar staining pattern was observed in adult lung tissue, but the alveolar wall was also stained. Laminin was also observed surrounding a few mesenchymal cells in fetal lung on day 19 of gestation; the number of positive mesenchymal cells increased with lung development. Laminin alpha 1 subunit chains, detected using a monoclonal antibody, were present in the basement membranes of bronchi, bronchioles, prealveolar ducts, and blood vessels in fetal lung tissue. No laminin alpha 1 chain staining was observed in the mesenchyme of early fetal lung tissue. Using a monoclonal antibody, laminin beta 1 subunit chains were immunolocalized in the basement membranes of bronchi, bronchioles, in prealveolar ducts, and surrounding some mesenchymal cells in fetal lung tissue. Laminin alpha 1 and beta 1 subunit chains in adult lung tissue were present in basement membranes of airways, blood vessels, and alveoli. Thus, changes in the localization and accumulation of laminin near the time of alveolar type I and type II epithelial cell differentiation suggest that laminin may play a role in mediating the differentiation of these cell types during rabbit fetal lung development.

Glucocorticoid regulation of surfactant-associated proteins in rabbit fetal lung in vivo.

The effects of a maternally administered synthetic glucocorticoid, betamethasone, on the levels of mRNA for the surfactant proteins SP-A, SP-B, and SP-C and on the levels of SP-A protein were investigated in day 27 gestational age rabbit fetal lung tissue. Betamethasone administration to the pregnant rabbit caused approximately a twofold increase in the fetal lung level of SP-A protein and a threefold increase in fetal lung SP-A mRNA levels when compared to levels in fetuses obtained from saline-treated or uninjected animals. SP-B mRNA was increased fourfold in fetal lung tissue obtained from glucocorticoid-treated pregnant does when compared to levels in fetuses of uninjected pregnant does. However, SP-B mRNA levels in fetal lung tissue from saline-injected controls were also significantly elevated, approximately twofold, when compared to fetal lung SP-B mRNA levels in the uninjected control condition. SP-C mRNA levels in lung tissue of fetuses from both saline-injected and betamethasone-injected pregnant does were increased similarly, approximately twofold, over SP-C mRNA levels in fetal lung tissue obtained from uninjected control does. These data are suggestive that betamethasone treatment increases fetal lung SP-A and SP-B mRNA levels and that maternal stress alone can increase the expression of SP-B and SP-C mRNA in rabbit fetal lung tissue. Using in situ hybridization, SP-A mRNA was shown to be present primarily in alveolar type II cells in fetuses of control and saline-injected does. However, SP-A mRNA was easily detected in both alveolar type II cells and bronchiolar epithelial cells of rabbit fetal lung tissue following maternal betamethasone treatment. In contrast, SP-B and SP-C mRNA were present only in alveolar type II cells of lung tissue obtained from fetuses of control, saline, or betamethasone-treated does. Thus maternal administration of glucocorticoids increased SP-A protein as well as SP-A and SP-B mRNA levels in rabbit fetal lung tissue. SP-A mRNA was localized to both alveolar type II cells and in smaller amounts in bronchiolar epithelial cells of rabbit fetal lung tissue. However, SP-B and SP-C mRNA were detected only in alveolar type II cells.

Developmental regulation of surfactant-associated proteins in rabbit fetal lung in vivo.

The developmental regulation of the rabbit surfactant-associated proteins, SP-A, SP-B, and SP-C, was investigated using Northern blot analysis. These proteins comprise approximately 10% by weight of pulmonary surfactant, a lipoprotein secreted by type II cells that reduces surface tension at the air-alveolar interface. SP-A mRNA and SP-B mRNA were first detected in rabbit fetal lung at day 24 of gestation (term = 31 days), i.e., approximately two days prior to the appearance of lamellar bodies within differentiated alveolar type II cells. The relative abundance of SP-B mRNA detected on day 24 of gestation was greater than that of SP-A mRNA. Fetal lung SP-A mRNA and SP-B mRNA levels increased rapidly during the remainder of gestation, reaching a maximum at day 31 of gestation. The relative concentrations of SP-A mRNA and SP-B mRNA were decreased in day 2 neonatal and adult lung tissues when compared to the levels present in fetal lung tissue late in gestation. A 0.5-kb rabbit SP-C cDNA was generated using the reverse transcriptase-polymerase chain reaction and was found to have high sequence homology to the human and rat SP-C cDNA nucleotide sequences. The predicted amino acid sequence for the rabbit SP-C cDNA revealed strong conservation of a hydrophobic region close to the amino terminus of the SP-C protein. Fetal lung SP-C mRNA was detected at day 19 of gestation, the earliest time point examined in this study. SP-C mRNA levels gradually increased in fetal lung tissue until day 28 of gestation and then remained level throughout the remainder of gestation and in the day 2 neonatal and adult rabbit lung tissue. These results suggest that the developmental pattern of induction of mRNA for the surfactant-associated proteins, SP-A, SP-B, and SP-C, differ from each other and are different in several respects from the developmental patterns observed in fetal lung tissue of the rat and human species.

Localization of surfactant-associated protein C (SP-C) mRNA in fetal rabbit lung tissue by in situ hybridization.

Surfactant is a lipoprotein substance that is synthesized and secreted by alveolar type II epithelial cells and acts to reduce surface tension at the air-alveolar interface. SP-C is a 5,000-D molecular weight, hydrophobic, surfactant-associated protein. In the present study, we used a ribonuclease protection assay to show that SP-C mRNA is induced in rabbit fetal lung tissue early in development, increases in relative concentration as development proceeds, and is present in maximal concentration at term (31 days of gestation). We also used the technique of in situ hybridization to localize SP-C mRNA in fetal, neonatal, and adult rabbit lung tissue. SP-C mRNA was present in all of the epithelial cells of the prealveolar region of day 19 gestational age rabbit fetal lung tissue, i.e., about 7 days before the appearance of differentiated alveolar type II cells in the fetal lung tissue. By day 27 of gestation, SP-C mRNA was restricted to epithelial cells with the morphologic characteristics of alveolar type II cells. SP-C mRNA was not detected in bronchiolar epithelium at any stage of lung development. The intensity of SP-C mRNA hybridization in the prealveolar and alveolar type II epithelial cells increased as a function of gestational age and was maximal at term. The pattern of SP-C mRNA localization in neonatal and adult rabbit lung tissue was consistent with the restriction of SP-C gene expression to differentiated alveolar type II cells. Our data are suggestive that SP-C may serve some as yet unknown function early in lung development because it is present in fetal lung prealveolar epithelial cells much earlier in gestation than are differentiated, surfactant-producing alveolar type II cells.

Glucocorticoid effects on rabbit fetal lung maturation in vivo: an ultrastructural morphometric study.

Maternal administration of glucocorticoids is known to stimulate fetal lung maturation. In the present study, we used microscopy and stereology to evaluate the morphological effects of maternal glucocorticoid treatment on rabbit fetal lung tissue. Betamethasone was administered to pregnant rabbits on days 25 and 26 of gestation at a dose of 0.2 mg/kg body weight. The animals were sacrificed on day 27 of gestation. Glucocorticoid treatment significantly increased the presumptive airspace in the fetal lung tissue but did not alter the relative proportion of epithelium, connective tissue, or vasculature in the tissue. In addition, glucocorticoid treatment significantly increased the proportion of type II cells in the prealveolar epithelium, increased the rate of phosphatidylcholine synthesis, and increased the content of the major surfactant-associated protein, SP-A, in the fetal lung tissue. We could detect no effect of betamethasone on lamellar body cross-sectional area, numerical density, or volume density within fetal lung type II cells. Glucocorticoid treatment of the pregnant doe caused a decrease in the volume density of intracellular glycogen and an increase in the volume density of mitochondria in fetal lung type II cells. Betamethasone treatment did not alter the distance between fetal lung epithelial cells and subadjacent connective tissue cells. However, glucocorticoid treatment increased the number of connective tissue foot processes that pierced the epithelial basal lamina. Thus, glucocorticoid treatment of the pregnant doe results in structural changes in the fetal lung tissue, an acceleration of some aspects of type II cell differentiation, and a concomitant increase in epithelial-mesenchymal interactions.

Effect of Castanospermine and Related Polyhydroxyalkaloids on Purified Myrosinase from Lepidium sativum Seedlings.

Myrosinase (beta-thioglucoside glucohydrolase, EC 3.2.3.1) was purified to apparent homogeneity from light-grown cress (Lepidium sativum L.) seedlings. This enzyme, which catalyzes hydrolysis of the glucosinolate sinigrin (K(m), 115 micromolar) at an optimum pH of 5.5 in sodium citrate buffer, had a native molecular weight of 130 +/- 5 kilodaltons and an isoelectric point of 4.7 to 4.9. SDS-PAGE revealed two polypeptides with molecular weights of 62 and 65 kilodaltons. Both subunits contained carbohydrate as shown by periodic acid-Schiff staining. The purified enzyme hydrolyzed p-nitrophenyl-beta-d-glucoside (K(m), 2.0 millimolar) at an optimum pH of 6.5 in phosphate buffer. The indolizidine alkaloid castanospermine, a known inhibitor of O-glycosidases, competitively inhibited the hydrolyses of sinigrin (thioglucosidase activity) and p-nitrophenyl-beta-d-glucoside (O-glucosidase activity) with K(i) values of 5 and 6 micromolar, respectively. In contrast, the related polyhydroxyalkaloids swainsonine and deoxynojirimycin were without effect upon these hydrolyses.