Biomedical subjects

B T Stokes

Publications and source records attributed to B T Stokes.

At least 37 records · Page 2Linked to original sources

PubMed1998

Assessment of the mechanism by which prolactin stimulates progesterone production by early corpora lutea of pigs.

Previously, we reported that administration of prolactin (PRL) during the early luteal phase in sows increases plasma progesterone concentrations. In the current study, we searched for the mechanisms by which PRL exerts this luteotrophic effect. The objectives of the study were (1) to examine the effect of PRL and/or low-density lipoproteins (LDL) on progesterone production by porcine luteal cells derived from early corpora lutea, and (2) to assess the ability of PRL to activate phosphoinositide-specific phospholipase C (PI-PLC) and protein kinase C (PKC) in these luteal cells. Ovaries with early corpora lutea (day 1-2 of the oestrous cycle) were obtained from the slaughterhouse. Progesterone production by dispersed luteal cells was measured after treatment with PRL, phorbol 12-myristate 13-acetate or inhibitors of PKC in the presence or absence of LDL. LDL increased progesterone concentration in the incubation medium (304.5 vs 178.6 ng/ml in control, P<0.05). PRL augmented LDL-stimulated progesterone secretion by luteal cells (to 416 ng/ml, P<0.05), but PRL alone did not affect progesterone production (209.6 ng/ml, P>0.05). Staurosporine, a PKC inhibitor, inhibited progesterone secretion stimulated by the combined action of LDL and PRL; however, such inhibition was not demonstrated when cells were treated with the PKC inhibitor, H-7. PKC activation was assessed by measuring the specific association of [H]phorbol dibutyrate (H-PDBu) with luteal cells after treatment with PRL or ionomycin (a positive control). PRL and ionomycin increased H-PDBu-specific binding in early luteal cells by 28+/-5.5% (within 5 min) and 70.2+/-19.3% (within 2 min) over control binding respectively (P<0.05). In addition, PRL did not augment the LDL-stimulated progesterone production in PKC-deficient cells. In contrast with PKC, total inositol phosphate accumulation, as well as intracellular free calcium concentrations, were not affected by PRL in the current study. We conclude that PRL, in the presence of LDL, stimulates progesterone production by early corpora lutea in vitro. Moreover, PRL appears to activate PKC, but not PI-PLC, in these cells. Thus intracellular transduction of the PRL signal may involve activation of PKC that is not dependent on PI-PLC.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1997

Cellular inflammatory response after spinal cord injury in Sprague-Dawley and Lewis rats.

The distribution of microglia, macrophages, T-lymphocytes, and astrocytes was characterized throughout a spinal contusion lesion in Sprague-Dawley and Lewis rats by using immunohistochemistry. The morphology, spatial localization, and activation state of these inflammatory cells were described both qualitatively and quantitatively at 12 hours, 3, 7, 14, and 28 days after injury. By use of OX42 and ED1 antibodies, peak microglial activation was observed within the lesion epicenter of both rat strains between three and seven days post-injury preceding the bulk of monocyte influx and macrophage activation (seven days). Rostral and caudal to the injury site, microglial activation plateaued between two and four weeks post-injury in the dorsal and lateral funiculi as indicated by morphological transformation and the de-novo expression of major histocompatibility class II (MHC II) molecules. Similar to the timing of microglial reactions, T-lymphocytes maximally infiltrated the lesion epicenter between three and seven days post-injury. Reactive astrocytes, while present in the acute lesion, were more prominent at later survival times (7-28 days). These cells were interspersed with activated microglia but appeared to surround and enclose tissue sites occupied by reactive microglia and phagocytic macrophages. Thus, trauma-induced central nervous system (CNS) inflammation, regardless of strain, occurs rapidly at the site of injury and involves the activation of resident and recruited immune cells. In regions rostral or caudal to the epicenter, prolonged activation of inflammatory cells occurs preferentially in white matter and primarily consists of activated microglia and astrocytes. Differences were observed in the magnitude and duration of macrophage activation between Sprague-Dawley (SD) and Lewis (LEW) rats throughout the lesion. Increased expression of complement type 3 receptors (OX42) and macrophage-activation antigens (ED1) persisted for longer times in LEW rats while expression of MHC class II molecules was attenuated in LEW compared to SD rats at all times examined. Variations in the onset and duration of T-lymphocyte infiltration also were observed between strains with twice as many T-cells present in the lesion epicenter of Lewis rats by 3 days post-injury. These strain-specific findings potentially represent differences in corticosteroid regulation of immunity and may help predict a range of functional neurologic consequences affected by neuroimmune interactions.

Animals↗

PubMed1997

Phosphorus-31 magnetic resonance spectroscopy studies of pig spinal cord injury. Myelin changes, intracellular pH, and bioenergetics.

RATIONALE AND OBJECTIVES: Phosphorus-31 (31P) nuclear magnetic resonance (NMR) spectroscopy was used to monitor changes in phosphocreatine (PCr), adenosine triphosphate (ATP), inorganic phosphate (Pi), intracellular pH (pHi), and free magnesium in the in vivo pig spinal cord after injury. METHODS: Phosphorus-31 NMR spectra were acquired from healthy (n = 4) and injured pig spinal cords (n = 8) under in vivo conditions using a 4.7-tesla spectrometer. Spinal cords were injured by dropping a 20-g weight from 20 cm onto the surgically exposed cord surface. RESULTS: In vivo spectra of injured cords revealed a reduction in ATP, PCr, pHi, and an increase in Pi. In addition, a broad resonance that is likely to arise from myelin phospholipids was reduced significantly after injury. CONCLUSIONS: Phosphorus-31 NMR can be used to follow in vivo changes in high energy phosphates after injury and may have the potential to follow changes in myelin structure. This technique may prove important in the study of myelin breakdown after secondary, nonreversible spinal cord injury. Changes in high energy phosphates and pHi did not seem to parallel these putative changes in myelin structure.

Adenosine Triphosphate↗

PubMed1997

Purinergic Ca2+ signaling in myenteric neurons via P2 purinoceptors.

Fura 2 microfluorimetry was used to test the hypothesis that ATP acts at P1 and P2 purinoceptors to elevate cytosolic free Ca2+ concentrations [Ca2+]i) in calbindin-immunoreactive cultured myenteric neurons from adult guinea pig small intestine. Local "micro-puff" application of ATP or ATP(gamma)S caused an increase in [Ca2+]i in 99% of 200 multipolar neurons. The potency profile of agonists for the rise in [Ca2+]i was ATP(gamma)S = ATP >> ADP >> AMP, adenosine, 5'-(N-ethylcarboxamido)adenosine, and 2-chloro-N(6)-cyclopentyladenosine. Tetrodotoxin-sensitive synaptic transmission could contribute as much as 25% to the ATP response. The P1 antagonist 8-cyclopentyl-1,3-dipropylxanthine blocked 50% of the peakATP Ca2+ response. P2 antagonists blocked the ATP response: pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid > reactive blue 2 > suramin. Suramin enhanced the ATP response in 27.5% of neurons. Some neurons (<15%) displayed distinct multiphasic Ca2+ signatures. About 54% of ATP-responsive neurons expressed calbindin. The data support the following hypotheses: 1) two distinct P2 purinoceptors are linked to the rise in [Ca2+]i in myenteric neurons; 2) purinergic Ca2+ signaling is not restricted to one neuronal phenotype; and 3) intraneuronal Ca2+ is not involved in adenosinergic hyperpolarization in AH/type 2 neurons.

Adenosine↗

PubMed1996

Concept of autoimmunity following spinal cord injury: possible roles for T lymphocytes in the traumatized central nervous system.

The effect of immunological activation on the neuropathologic sequelae and neurologic outcome from spinal cord injury is unclear. Similar to models of neuroinflammatory disease (e.g., experimental autoimmune encephalomyelitis; EAE), injury to the spinal cord precipitates the activation of resident microglia and the recruitment of circulating inflammatory cells (e.g., macrophages and lymphocytes). In EAE, these cells are known to cause tissue damage and loss of neurological function via autoimmune reactions to myelin proteins. The role these cells play in the pathology of traumatic injury to the spinal cord has not been clarified. In this review, data are presented that indicate that T cells isolated from spinal-injured rats are capable of causing neurologic deficits and histopathologic changes similar to EAE when injected intravenously into naive animals. These data are consistent with the concept of trauma-induced autoimmune reactions. However, disease transfer was only possible when T cells were obtained from animals at 1 week post-injury. Thus, the encephalitogenic T-cell repertoire appears to be rapidly regulated. It is possible that trauma-induced autoimmunity evolves into a mechanism by which the autoreactive repertoire regulates ongoing central nervous system (CNS) immunologic responses. Similar immunoregulatory networks have been proposed in EAE and are discussed here in the context of CNS trauma and neurodegenerative disease.

Animals↗

PubMed1996

Responsiveness to ATP with an increase in intracellular free Ca2+ is not a distinctive feature of calbindin-D28 immunoreactive neurons in myenteric ganglia.

The aim of this study was to test the hypothesis that ATP elevates cytosolic free Ca2+ levels ([Ca2+]i) in myenteric neurons expressing the Ca2+ binding protein, calbindin-D28. A laser microbeam marked the location of cultured neurons on coverslips and provided unequivocal relocation of ATP-responsive neurons after immunocytochemistry. All myenteric multipolar neurons displayed ATP Ca2+ transients, and 42% also expressed calbindin-D28 reactivity. Statistical analysis of the kinetics and shape of ATP Ca2+ transients revealed no differences between calbindin and non-calbindin neurons. The identity of other responsive neurons is unknown. Less than 8% of ganglion cells with ATP Ca2+ transients were immunopositive for the glial protein S-100. We conclude that one of the actions of ATP in myenteric ganglia is to increase [Ca2+]i which may activate gKCa leading to membrane hyperpolarization in AH, Dogiel Type II neurons expressing calbindin-D28. An efficient buffering mechanism for handling large purinergic Ca2+ loads is a common feature of all types of myenteric ganglion cells.

Adenosine Triphosphate↗

PubMed1996

Quantitative analysis of vascularization and cytochrome oxidase following fetal transplantation in the contused rat spinal cord.

In the normal adult central nervous system, a coupling between energy consumption and vascular density is well established. Likewise, the survival of fetal neural tissue grafts is highly dependent on the establishment of functional vascular integration with the host. However, to what degree graft vascularization and tissue metabolism influence the normal host response to traumatic injury has not been extensively studied. In the present report, embryonic day 14 fetal spinal cord suspension grafts were made into the lesion epicenter of subchronic (10 days) contusion-injured rats. Three months later, intraspinal transplants were analyzed using correlative cytochrome oxidase histochemistry and vascular morphometric analysis. The same approaches were applied to the host spinal cord and injured, non-transplanted animals in order to determine the ability of a graft to alter the level of post-injury vascularization and/or metabolism. In general, graft vascular density was increased over that measured in normal or injured gray matter. Vascular density in gray matter near the host/graft interface was markedly increased when compared to either gray matter of the same spinal level in injured non-grafted animals or normal control spinal gray matter. Vascular changes were not noted in gray matter 3 mm distal to the lesion epicenter (rostral or caudal) in all groups analyzed. Cytochrome oxidase was up-regulated at this time in the graft and gray matter at the host/graft interfaces when compared to either gray matter of the same spinal level in injured, non-grafted animals or that of uninjured controls. These data indicate that an intraspinal transplant placed into the contused adult rat spinal cord reaches a metabolic capacity that is likely to be associated with high levels of oxidative metabolism in the well-vascularized graft neuropil. In addition, transplantation chronically alters vascularization and metabolic patterns of adjacent spinal gray matter following contusion injury.

Animals↗

PubMed1996

A quantitative spatial analysis of the blood-spinal cord barrier. II. Permeability after intraspinal fetal transplantation.

In previous experiments we utilized quantitative autoradiography to temporally describe vascular permeability of a radiolabeled vascular tracer following spinal contusion injury in the rat. In the present report we compare these findings with permeability assessments following fetal grafting in the contused rat spinal cord. At 10 days postinjury, Embryonic Day 14 spinal tissue was grafted into the lesioned spinal cord of Sprague-Dawley rats. At 7, 14, and 28 days postgrafting the alpha-aminoisobutyric acid (AIB) technique was used to assess blood-to-tissue transfer rates in graft and host tissue over several segments of the injured spinal cord. Regional changes in permeability were assessed using four distinct image analysis techniques. Using these methods, we have previously shown that contusion injury alone results in a chronic relapse in vascular permeability. The present data indicate that fetal transplants at 7 days postgrafting have AIB transfer rates that are significantly above uninjured control levels and are similar in magnitude to neighboring host spinal tissue. In addition, permeability in 14- and 28-day intraspinal grafts decreased relative to that of the 7-day transplant group, but remained significantly elevated at and rostral to the injury epicenter. Alternately, graft and host tissue in regions caudal to the injury epicenter (e.g., T10--L2) acquired a functional barrier to AIB as early as 14 days posttransplantation. These experiments suggest that graft development occurs in a different manner or at a different rate in segments of the injured spinal cord rostral and caudal to the injury site. Additionally, it appears that vascular permeability of the injured spinal cord can be influenced by the process of intraspinal transplantation.

Aminoisobutyric Acids↗

PubMed1996

A quantitative spatial analysis of the blood-spinal cord barrier. I. Permeability changes after experimental spinal contusion injury.

Blood-spinal cord barrier (BSB) permeability was measured using quantitative autoradiography following contusion injury to the rat spinal cord. Permeability was assessed by calculating blood-to-tissue transfer constants (Ki values) for the vascular tracer [14C]-alpha-aminoisobutyric acid (AIB) in injured (3, 7, 14, and 28 days postinjury), laminectomy control, and uninjured control animals. Permeability was quantitated using four separate imaging techniques in gray and white matter throughout the rostro-caudal extents of the forming lesion. Away from the epicenter, gray matter permeability was further differentiated within discrete spinal lamina using computerized templates. Regardless of the type of analysis used, increased AIB permeability (Ki values) was noted at all survival times in all tissue regions with respect to both uninjured and laminectomy control groups. The data indicate a large increase in individual Ki values throughout the dorsoventral axis of the spinal cord at 3 days postinjury (approximately 6-9 ml/kg/min). By 7 days, Ki values were quantitatively smaller (approximately 4-5 ml/kg/min) in all regions compared with 3-day tissues. Despite further attenuation of AIB uptake in the gray matter at 14 and 28 days postinjury, circumferential white matter tracts showed a secondary increase in permeability compared to 7-day tissue. Permeability in the white matter at 14-28 days postinjury (approximately 5-6 ml/kg/min) was comparable to that at 3 days postinjury (6-7 ml/kg/min). Measurements of the axial distribution of AIB permeability indicate increased BSB permeability over several segments rostral and caudal to the lesion epicenter (approximately 3 cm in both directions). Secondary elevations of AIB transfer in the spinal white matter between 14 and 28 days were colocalized with zones of immunohistochemically defined microglial clusters. The known plasticity of this cell type in response to changes in the extracellular microenvironment suggests that the spinal white matter at later survival times (14-28 days postinjury) is an area of dynamic vascular and/or axonal reconstruction. The implications of increased permeability to both tissue injury and neural regeneration are discussed.

Aminoisobutyric Acids↗

PubMed1996

Operant conditioning of H-reflex in spinal cord-injured rats.

Operant conditioning of the spinal stretch reflex or its electrical analog, the H-reflex, is a new model for exploring the mechanisms of supraspinal control over spinal cord function. Both rats and primates can gradually increase (HRup conditioning mode) or decrease (HRdown conditioning mode) soleus H-reflex magnitude when exposed to an operant conditioning task. This study used H-reflex operant conditioning to assess and modify spinal cord function after injury. Soleus H-reflexes were elicited and recorded with chronically implanted electrodes from rats that had been subjected to calibrated contusion injuries to the spinal cord at T8. From 18 to 140 days after injury, background EMG, M response amplitude, and initial H-reflex amplitude were not significantly different from those of normal rats. HRdown conditioning was successful in some, but not all, spinal cord-injured rats. The H-reflex decrease achieved by conditioning was inversely correlated with the severity of the injury as assessed histologically or by time to return of bladder function. It was not correlated with the length of time between injury and the beginning of conditioning. The results confirm the importance of descending control from supraspinal structures in mediating operantly conditioned change in H-reflex amplitude. In conjunction with recent human studies, they suggest that H-reflex conditioning could provide a sensitive new means for assessing spinal cord function after injury, and might also provide a method for initiating and guiding functional rehabilitation.

Animals↗

PubMed1996

Activation of elements of the phosphatidylinositol pathway in the primate corpus luteum by prostaglandin E2.

The current study was designed to examine the effects of prostaglandin (PG) E2 on progesterone production by primate luteal cells collected during the late luteal phase. PGE2 inhibited basal and human chorionic gonadotrophin (HCG)-stimulated progesterone production (P < 0.01) in late luteal phase corpora lutea. The ability of PGE2 to activate a second messenger system (phosphatidylinositol pathway) in corpora lutea of rhesus monkeys was also assessed. PGE2 significantly increased the accumulation of inositol phosphates (P < 0.05). This stimulation was not apparent in the early luteal phase but was manifested in the mid-late luteal phase. PGE2 also caused a rapid, yet transient, increase (P < 0.01) in intracellular free calcium ion concentrations ([Ca2+]i) in a large proportion of primate luteal cells. The proportion of luteal cells that responded to PGE2 with an increase in [Ca2+]i was smaller (P < 0.05) in corpora lutea collected during the early luteal phase (12%) in comparison with those collected during the latter half of the luteal phase (63-66%). Changes in [Ca2+]i in response to PGE2 were similar in small and large luteal cells. This study demonstrates that PGE2 activates elements of the phosphatidylinositol pathway in primate corpora lutea. This activation is augmented as the luteal phase progresses. Thus, the inhibitory effects of PGE2 on luteal progesterone production observed in the late luteal phase are associated with activation of elements of the phosphatidylinositol pathway.

Animals↗

PubMed1996

Phosphorus-31 nuclear magnetic resonance spectroscopy of the spinal cord in the pig, rat, and rabbit.

RATIONALE AND OBJECTIVES: To ensure that contamination-free phosphorus-31 nuclear magnetic resonance (31P-NMR) spectra of the spinal cord could be obtained, a porcine model was adopted that provided a large cord sample and a greater area free from adjacent muscle tissue. METHODS: Phosphorus-31 NMR spectra were acquired from the porcine spinal cord under in vivo conditions using a 4.7-T spectrometer. Spectra also were collected from perchloric acid and lipid extracts, and excised freeze trapped samples of the rat, rabbit, and pig spinal cord. RESULTS: The in vivo spectrum showed resonances corresponding to adenosine triphosphate, phosphocreatine, inorganic phosphate, phosphomonoesterase, and phosphodiesterase as confirmed by extracts. In addition, a broad resonance was observed that was assigned to myelin phospholipids. CONCLUSION: Phosphorus-31 NMR spectra of the spinal cord revealed resonances common to brain tissue. Importantly, the existence of a previously undetected resonance, which is likely to correspond to myelin phospholipids, also is reported. This resonance may prove important in future studies monitoring changes in myelin in response to trauma and ischemia.

Animals↗

PubMed1995

Staurosporine affects calcium homeostasis in cultured bovine adrenal chromaffin cells.

These studies show that the potent, non-specific, protein kinase inhibitor, staurosporine, disrupts Ca2+ homeostasis in cultured bovine adrenal chromaffin cells. Staurosporine treatment reduces basal and A23187-stimulated catecholamine release from chromaffin cells, but does not inhibit activated Ca2+ influx. Furthermore, pretreatment with staurosporine also reduces Ca(2+)-stimulated catecholamine release from digitonin-permeabilized cells (t1/2, 40.6 min; IC50, 66.0 nm). However, staurosporine does not inhibit the rise in intracellular Ca2+ ([Ca2+]i) in response to nicotine stimulation as measured by fura-2 photometry. These studies demonstrate that staurosporine interferes with the secretory process at some step at or after the rise in [Ca2+]i in adrenal chromaffin cells. Examination of the effects of staurosporine on 45Ca2+ movement shows that staurosporine produces a slowly developing basal 45Ca2+ accumulation; after 30 min no significant change is observed, but by 120 min, 45Ca2+ accumulation is increased by 29.5%. Thapsigargin and 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBHQ), inhibitors of Ca(2+) ATPases, were used to determine whether staurosporine induced 45Ca2+ accumulation results from sequestration of 45Ca2+ within intracellular stores. While thapsigargin has no significant effect, concomitant treatment with tBHQ prevents the increase in 45Ca2+ uptake associated with staurosporine treatment. Therefore, the tBHQ-sensitive Ca2+ store, but not the thapsigargin/inositol 1,4,5-triphosphate-sensitive Ca2+ store, appears to be staurosporine-sensitive. Overall, these studies indicate that staurosporine reduces catecholamine release by interfering with Ca2+ homeostasis. Furthermore, this work suggests that a staurosporine-sensitive phosphoprotein(s) is involved with the regulation of Ca2+ homeostasis in bovine adrenal chromaffin cells.

Alkaloids↗

PubMed1995

Fetal transplantation following spinal contusion injury results in chronic alterations in CNS glucose metabolism.

Glucose utilization of the injured rat spinal cord was determined using the autoradiographic technique of Sokoloff et al. (33). Animals were analyzed chronically (2 and 3 months) after spinal contusion injury alone or when a spinal lesion was followed by subchronic (10-day) intraparenchymal fetal spinal transplantation. At 2 and 3 months postinjury, spinal glucose utilization was reduced in dorsal gray and white matter above and below the lesion site. In addition, sensory regions of the forebrain and brain stem (e.g., nucleus gracilis and ventral posterior medial nucleus of the thalamus) had a lower basal metabolic rate than control animals. Decreased metabolic rates in supraspinal regions were reversed by the presence of a spinal graft at 3 but not at 2 months postinjury. Furthermore, gray matter in animals receiving an intraspinal transplant had elevated glucose utilization rates for several spinal segments rostral and caudal to the lesion epicenter. Graft glucose utilization was higher at 2 months (80-90 mumol/100 g/min) than at 3 months (60-70 mumol/100 g/min) posttransplantation. These data are the first quantitative metabolic imaging of spinal and brain metabolism following spinal contusion injury and fetal transplantation. The study suggests that intraspinal transplants can become functionally integrated with adjacent host gray matter and can chronically alter specific postinjury metabolic patterns.

Animals↗

PubMed1995

Gangliosides inhibit PDGF-induced signal transduction events in U-1242 MG human glioma cells.

In this study we investigated the responses of intracellular calcium ([Ca2+]i) and protein kinase C (PKC) to PDGF in U-1242 MG cells. PDGF-BB stimulated [3H]PDBu binding approximately 2-3 fold. This response was inhibited by preincubating the cells with an inhibitor of phospholipase C (PLC), U73122, suggesting that PLC mediates the induction of PKC translocation by PDGF. PDGF also increased the concentration of [Ca2+]i that was attenuated in a calcium-free medium. This indicates that PDGF-induced elevation of [Ca2+]i is mainly due to influx of extracellular calcium. PDGF-stimulated translocation of PKC was inhibited by the intracellular calcium buffer BAPTA/AM. All gangliosides studied except GM3 inhibited these responses with similar efficacy. Collectively, these results indicate that the signal transduction pathway initiated by PDGF leading to PKC translocation in U-1242 MG cells is intact, and this pathway is inhibited by several gangliosides.

Calcium↗

PubMed1995

Arachidonic acid inhibits hCG-stimulated progesterone production by corpora lutea of primates: potential mechanism of action.

Arachidonic acid (AA) is a precursor of metabolites known to affect the corpus luteum (CL) in many species, including primates. We have shown that some of these products (prostaglandins F2 alpha and E2) inhibit pro-gesterone (P4) production and activate the phosphatidylinositol (PI) pathway in CL of rhesus monkeys. A direct role of AA in luteal function has also been suggested. The current experiments were designed to investigate the effect of AA on P4 synthesis and to examine the ability of AA to activate the PI pathway in CL of rhesus monkeys. Basal and hCG-stimulated P4 production by luteal cells collected during the midluteal phase was measured after treatment with AA (1, 5, and 10 microM) or linoleic acid (1, 5, and 10 microM). Dispersed cells (50,000/tube) were incubated at 37 degrees C for 2 h. AA elicited a dose-dependent decrease in hCG-stimulated, but not in basal, P4 production. hCG-stimulated P4 production was reduced (P < 0.01) at AA doses of 5 microM (12.1 +/- 1.5 ng/mL) and 10 microM (8.6 +/- 1.8 mg/mL) to hCG alone (18 +/- 1.6 ng/mL). There was no significant effect of 1 microM AA (15.2 +/- 1.6). Response to linoleic acid was dissimilar and was not dose-dependent. Viability of cells was not affected by any treatment. Indomethacin, a prostaglandin synthesis inhibitor, and nordihydroguaiaretic acid, an inhibitor of lipoxygenase, did not interfere with the inhibitory effect of AA. Activation of the PI pathway was assessed by monitoring the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2) to inositol phosphates and by monitoring increases in intracellular free calcium concentrations ([Ca2+]i) in individual cells. Moreover, the ability of AA to activate protein kinase C (PKC) in luteal cells was measured using a [3H]phorbol dibutyrate (PDBu) binding assay. AA did not alter PIP2 hydrolysis or [Ca2+]i, however, AA (10 microM) increased specific binding of [3H]PDBu to luteal cells (P < 0.05). We conclude that AA inhibits hCG-stimulated P4 production by primate luteal cells. AA exerts this action without being converted to prostaglandins or leukotrienes. This inhibition may be mediated through the activation of PKC. These results suggest a possible role for AA in the regulation of luteal function in primates, and that PKC-activation by AA may promote its effects.

Animals↗

PubMed1995

Analysis of TGF-beta 1 gene expression in contused rat spinal cord using quantitative RT-PCR.

We have used northern blot analysis and quantitative reverse transcription polymerase chain reaction (RT-PCR) to determine the postinjury expression profile of the transforming growth factor-beta 1 (TGF-beta 1) gene in the contused rat spinal cord. Spectrophotometric estimates of total sample RNA and quantitative analyses of cyclophilin mRNA using RT-PCR served as controls for comparisons between samples. No changes in cyclophilin gene expression were found at any postinjury survival times. The results of the TGF-beta 1 analyses, which were carried out on spinal cord samples taken at postinjury intervals ranging from 6 h to 10 days, show that the amount of TGF-beta 1 mRNA present in spinal cord increases rapidly following injury, reaching maximum levels 7 days postinjury. Unoperated control samples contained approximately 2 x 10(8) molecules of TGF-beta 1 mRNA/0.5 microgram total RNA. By 1 day postinjury, the amount of TGF-beta 1 mRNA in the cord had increased by a factor of 2.5 to 5 x 10(8) molecules/0.5 microgram total RNA. At 7 days postinjury, there were approximately 15 x 10(8) molecules of TGF-beta 1 mRNA/0.5 microgram total RNA. By 10 days postinjury the amount of TGF-beta 1 mRNA present in the spinal cord had declined to 8 x 10(8) molecules of TGF-beta 1 mRNA/0.5 microgram total RNA, a value similar to that observed at 3 days postinjury. The roles that TGF-beta 1 might play in modifying cellular responses in injured spinal cord are discussed.

Animals↗

PubMed1994

Elevation of the neurotoxin quinolinic acid occurs following spinal cord trauma.

Excitatory amino acid neurotoxicity and the inflammatory response are suspected as mediators of some of the pathological sequelae occurring as a result of spinal cord injury. Here we report temporal and regional increases of the NMDA receptor agonist, quinolinic acid (QUIN), in an experimental model of spinal contusion injury. These changes occurred at a time when the blood-brain barrier is known to be dysfunctional and the activation state and density of microglia and macrophages are increased. Thus, alterations in tissue QUIN levels may occur as a result of secondary activation of CNS inflammatory cells or from peripherally derived sources across a damaged blood-brain barrier.

Animals↗