The expression of calcitonin gene-related peptide in dorsal horn neurons of the mouse lumbar spinal cord.

Using immunohistochemistry and in situ hybridization the expression of calcitonin gene-related peptide (CGRP) was studied in the mouse spinal cord under normal conditions and after unilateral rhizotomy and after local colchicine treatment. Under normal conditions a dense plexus of CGRP-immunoreactive (IR) fibres was observed in the superfical layers of dorsal horns with lower numbers of fibers in deeper laminae. Seven days after unilateral rhizotomy, there was a marked reduction of CGRP-IR fibres in the ipsilateral superfical layers and distinctly CGRP-IR neurons could be detected in the ipsilateral lamina III. CGRP mRNA-positive neurons were observed in lamina III in both the ipsilateral and contralateral dorsal horn. Colchicine treatment did not markedly increase the number of CGRP-IR neurons. The results suggest that CGRP is synthesized in local dorsal horn neurons of the mouse, and these neurons presumably participate in sensory processing.

Glucocorticoids downregulate cyclooxygenase-1 gene expression and prostacyclin synthesis in fetal pulmonary artery endothelium.

Prostacyclin (prostaglandin I2 [PGI2]) is a key mediator of pulmonary vascular function during early postnatal life, and its production in the pulmonary vasculature rises markedly during that period because of increasing expression of cyclooxygenase type 1 (COX-1). The postnatal rise in COX-1 may be due to the release of inhibition by glucocorticoids, since plasma glucocorticoid levels fall after birth and glucocorticoids decrease PGI2 synthesis in certain nonpulmonary cell types. We therefore studied the direct effects of dexamethasone (DEX) on COX-1 expression in early-passage ovine fetal pulmonary-artery endothelial cells (PAECs). DEX (10(-10) to 10(-6) mol/L) caused a dose-related decrease in COX-1 mRNA expression that was evident by 24 hours, was maximal at 10(-6) mol/L (50% inhibition), and was not due to changes in mRNA stability. There was a parallel decline in COX-1 protein expression. COX-1 protein rose following DEX withdrawal, and DEX blunted the stimulatory effect of 17beta-estradiol on COX-1 expression. DEX alone (10(-8) mol/L for 48 hours) caused a 93% fall in basal PGI2 production, and bradykinin- and A23187-stimulated PGI2 were diminished 96% and 94%, respectively. Similarly, PGI2 synthesis from arachidonic acid fell 86% with DEX; all of the above effects are consistent with COX-1 downregulation. The glucocorticoid receptor (GR) antagonist mifepristone (RU-486; 10(-6) mol/L) blocked the inhibitory effect of DEX, and GR expression was evident by immunoblot analysis. These findings indicate that glucocorticoids downregulate COX-1 expression and PGI2 synthesis in fetal PAECs through the activation of PAEC GR and effects on COX-1 gene transcription. This mechanism may modulate pulmonary PGI2 production in the perinatal period, and it may also play a role in the effects of glucocorticoids on the systemic circulation at a variety of ages.

Effects of systemic hypothermia and selective brain cooling on ischemic brain damage and swelling.

The present study investigates the neuroprotective effects of temporary mild systemic hypothermia and selective brain cooling against focal cerebral infarction in the rat and the changes of cortical blood flow, and compares these two treatment modalities. In permanent middle cerebral artery (MCA) model, the treatments were induced 15 min following the artery occlusion. The animals were kept at the desired rectal or brain temperature (about 32 degrees C) for 30 min; (each, n = 6) and for 1 hr (each, n = 6), and then allowed to rewarm spontaneously, whereas control animals were kept at normothermia throughout the experiment. The volumes of brain infarction and edema were assessed 24 hr post-occlusion. The blood flow of the dorsolateral cortex was monitored by Laser-Doppler flowmetry (LDF) in the other experiments. Hemispheric infarct volume was attenuated only in the animals treated for 1 hr with systemic hypothermia (49.2%, P < 0.001) and selective brain cooling (26.7%, P < 0.01). The volume of brain swelling was diminished only in the animals treated with systemic hypothermia for 1 hr (23.6%, P < 0.05). LDF examination revealed a sharp drop in blood flow upon MCA occlusion and maintaining in low blood flow throughout the experiment in the control and systemic hypothermia. However, in the selective brain cooling, the reduced blood flow increased from 40% to 70% of baseline value while the brain was rewarmed. The present study indicates that mild systemic hypothermia has much stronger protective effects against focal cerebral infarction and edema than selective brain cooling. The lack of protective effects of selective brain cooling may be caused by post-cooling cerebral hyperemia in the ischemia area.

Estrogen upregulates cyclooxygenase-1 gene expression in ovine fetal pulmonary artery endothelium.

Prostacyclin (PGI2) is a key mediator of pulmonary vasodilation in the perinatal period and its synthesis in the pulmonary vasculature increases markedly during late gestation due to enhanced expression of the rate-limiting enzyme cyclooxygenase-1 (COX-1). The hormone estrogen may play a role in COX-1 upregulation since fetal estrogen levels rise dramatically during late gestation and estrogen enhances PGI2 synthesis in nonpulmonary vascular cells. We therefore studied the direct effects of estrogen on COX-1 expression in ovine fetal pulmonary artery endothelial cells (PAEC). Exposure to estradiol-17beta (E2beta, 10(-)10 to 10(-)6 M) caused a dose-related increase in COX-1 mRNA expression that was evident after 48 h and maximal at 10(-)8 M (fourfold increase). COX-1 mRNA stability was unchanged, suggesting that the upregulation is mediated at the level of transcription. E2beta treatment (10(-)8 M for 48 h) also caused a threefold increase in COX-1 protein expression and a threefold increase in PGI2 synthesis stimulated by bradykinin, the calcium ionophore A23187, or arachidonic acid. The estrogen receptor (ER) antagonist ICI 182,780 fully reversed the effects of the hormone on COX-1 protein expression and on arachidonic acid-stimulated PGI2 synthesis, and ER expression was evident in the PAEC by immunoblot analysis. These findings indicate that physiologic levels of estrogen cause upregulation of COX-1 expression and PGI2 synthesis in fetal PAEC via activation of PAEC ER. This process may play a critical role in optimizing the capacity for PGI2-mediated pulmonary vasodilation at birth, and it may also be involved in estrogen responsiveness in other vascular beds.

Estrogen upregulates endothelial nitric oxide synthase gene expression in fetal pulmonary artery endothelium.

NO, produced by endothelial NO synthase (eNOS), is a key mediator of pulmonary vasodilation during cardiopulmonary transition at birth. The capacity for NO production is maximal at term because pulmonary eNOS expression increases during late gestation. Since fetal estrogen levels rise markedly during late gestation and there is indirect evidence that the hormone enhances nonpulmonary NO production in adults, estrogen may upregulate eNOS in fetal pulmonary artery endothelium. Therefore, we studied the direct effects of estrogen on eNOS expression in ovine fetal pulmonary artery endothelial cells (PAECs). Estradiol-17beta caused a 2.5-fold increase in NOS enzymatic activity in PAEC lysates. This effect was evident after 48 hours, and it occurred in response to physiological concentrations of the hormone (10(-10) to 10(-6) mol/L). The increase in NOS activity was related to an upregulation in eNOS protein expression, and eNOS mRNA abundance was also enhanced. Estrogen receptor antagonism with ICI 182,780 completely inhibited estrogen-mediated eNOS upregulation, indicating that estrogen receptor activation is necessary for this response. In addition, immunocytochemistry revealed that fetal PAECs express estrogen receptor protein. Furthermore, transient transfection assays with a specific estrogen-responsive reporter system have demonstrated that the endothelial estrogen receptor is capable of estrogen-induced transcriptional transactivation. Thus, estrogen upregulates eNOS gene expression in fetal PAECs through the activation of PAEC estrogen receptors. This mechanism may be responsible for pulmonary eNOS upregulation during late gestation, thereby optimizing the capacity for NO-mediated pulmonary vasodilation at birth.

Assessment of the relationship between ischemic damage and brain swelling in frozen brain slices.

The purpose of the study was to verify a method for the measurement of both cerebral infarction and brain swelling in frozen brain slices for histology. The animals were divided into two groups sham-operated control (n = 10) and focal cerebral ischemia group (n = 10). Focal cerebral ischemia was produced by permanent occlusion of the left middle cerebral artery. The rats were sacrificed 24 hours postocclusion. The brain was divided into two through the corpus callosum. Each hemisphere was weighted and frozen. Cerebral infarction and brain swelling were each assessed at 8 predetermined coronal planes. The volume of brain swelling was obtained by subtracting the total volume of nonischemic hemisphere from the total volume of the ischemic one. In the MCA occlusion group, brain infarction and the differences of hemispheric volume and weight between the right and left hemispheres were consistently observed, whereas sham-operated rats demonstrated no brain infarction or significant differences between two hemispheres. There were good corelationships not only between the volumes of brain edema and infarction (p < 0.05) but between the volume of brain edema and the difference in weight (p < 0.01) also. The results indicate that the measurement of the volume of ischemic brain edema in frozen brain slices may be useful in elusidating relationship with ischemic brain damage.

Interaction of the FAF B-containing subunit with the Photosystem 1 core heterodimer.

The structure of the predicted amino acid sequence in the FX domain of Photosystem 1 was studied by molecular modeling and a working hypothesis was developed for the functional interaction of PsaC with the core heterodimer. We propose that the intervening sequences between homologous cysteines in the FX cluster form two flexible loops and participate in the binding of PsaC, and that the arginine residues in the two surface-exposed loops may promote the interaction between the P700-FX core and the subunit. The model was tested experimentally; chemical modification of arginine residues in the P700-FX core using phenylglyoxal prevented reconstitution of the core with PsaC and PsaD after insertion of FeS clusters in vitro. Treatment of the P700-FX core with trypsin also prevented reconstitution of terminal electron transfer to FAFB, although neither treatments affected the electron transfer to FX as judged by flash kinetic spectrophotometry. Electron transfer in the P700-FAFB complex was not impaired by either phenylglyoxal or trypsin treatment indicating that the small subunit(s) protect the arginine residues that become chemically modified or cleaved. The data are consistent with the working model and point to additional experiments designed to identify the specific residues involved in the interaction between the P700-FX core and PsaC.