Biomedical subjects

P Morley

Publications and source records attributed to P Morley.

At least 55 records · Page 3Linked to original sources

PubMed1999

Stimulation of membrane-associated protein kinase-C activity in spleen lymphocytes by hPTH-(1-31)NH2, its lactam derivative, [Leu27]-cyclo(Glu22-Lys26)-hPTH-(1-31)NH2, and hPTH-(1-30)NH2.

Human parathyroid hormone, hPTH-(1-34), stimulates adenylyl cyclase and phosphatidylinositol-bisphosphate-specific phospholipase-C (PIP2-PLC), as indicated by increased membrane-associated protein kinase C (PKC) activity in ROS 17/2 rat osteosarcoma cells. The C-terminally truncated hPTH-(1-31)NH2 stimulates adenylyl cyclase as strongly as hPTH-(1-34) in these cells, but it does not stimulate PKC activity. Even [Leu27]-cyclo(Glu22-Lys26)-hPTH-(1-31)NH2, a 6-fold stronger adenylyl cyclase stimulator than hPTH-(1-34), cannot stimulate PKC activity in ROS cells. Therefore PTH required its 32-34 region to stimulate PIP2-PLC/PKCs in this osteosarcoma line. In contrast, hPTH-(1-31)NH2 [Leu27]-cyclo(Glu22-Lys26)-hPTH-(1-31)NH2 and even hPTH-(1-30)NH2 can stimulate PKC activity in freshly isolated rat spleen lymphocytes as strongly as hPTH-(1-34)NH2. The difference in the ability of membrane-associated PKC activity in spleen lymphocytes, but not in ROS cells, to be stimulated by C-terminally truncated PTH fragments might be due to different receptor densities or to the lymphocyte's atypical PTH/PTHrP receptor.

Animals↗

PubMed1999

Signalling events mediating the activation of protein kinase C by interleukin-2 in cytotoxic T cells.

Interleukin-2 (IL-2) plays a vital role in the generation and regulation of the immune response, including important aspects of T cell survival. IL-2-mediated survival of T cells appears to be dependent on the activation of a pool of membrane-associated protein kinase C (PKC) that occurs in the absence of detectable translocation of the enzyme from the cytosol to membranes. In this report we investigate the mechanism(s) responsible for this PKC activation after IL-2 stimulation in the cytotoxic T cell line, CTLL-2. Tyrosine kinase activity, activated after IL-2 stimulation, was found not to be linked to the activation of PKC by the cytokine. On the other hand, a pertussis toxin (PTX)-sensitive G protein did appear coupled to PKC activation since PTX effectively blocked IL-2 stimulated PKC activity. Diacylglycerols (DAG), but not inositol 1,3,5-triphosphate (IP3) and intracellular Ca2+, increased after IL-2 stimulation suggesting that DAGs were generated via the phosphatidylcholine-phospholipase C (PC-PLC) or phosphatidylcholine-phospholipase D (PC-PLD) pathways. The increase in DAG by IL-2 was probably necessary for activation of membrane-resident PKC since exogenously applied DAG stimulated this PKC pool in both intact cells and in isolated membranes. IL-2 also increased arachidonic acid (AA) production in CTLL-2 cells, probably via phospholipase A2 (PLA2) since the PLA2 inhibitors oleoyloxyethyl phosphocholine and AACOCF3 (AACF) effectively blocked IL-2 stimulated PKC activation. Exogenous AA also increased PKC activity in intact cells and isolated membranes, suggesting that AA produced by IL-2 receptor stimulation was probably linked to PKC activation. These results suggest that the activation of membrane-resident PKC by IL-2 involves multiple second messengers, including G proteins, DAG and AA.

Arachidonic Acid↗

PubMed1999

Evidence that brain-derived neurotrophic factor neuroprotection is linked to its ability to reverse the NMDA-induced inactivation of protein kinase C in cortical neurons.

Several lines of evidence indicate that a rapid loss of neuronal protein kinase C (PKC) activity is a characteristic feature of cerebral ischemia and is a necessary step in the NMDA-induced death of cultured neurons. Exposing embryonic day 18 primary rat cortical neurons to 50 microM NMDA or 50 microM glutamate for 10 min caused approximately 80% cell death over the next 24 h, but excitotoxic death was largely averted, i.e., by 70-80%, in cells pretreated with brain-derived neurotrophic factor (BDNF). An 8-h preexposure to BDNF (50-100 ng/ml) maximally protected cortical cells from the effects of NMDA and glutamate, although the transient application of BDNF between 8 and 4 h before NMDA was equally protective. These effects of BDNF were abolished at supralethal, i.e., >100 microM, NMDA concentrations. It is significant that BDNF pretreatment prevented the inactivation of PKC in cortical cells normally seen 30 min to 2 h following lethal NMDA or glutamate exposure. This BDNF effect did not arise from changes in NMDA channel activity because neither whole-cell NMDA current amplitudes nor increases in intracellular free Ca2+ concentration were altered by the 8-h BDNF pretreatment. Furthermore, BDNF offered no neuroprotection to cells treated with the PKC inhibitors staurosporine (10-20 nM), calphostin C (1-2.5 microM), or GF-109203X (100 nM) at the time of NMDA addition. These results underscore the importance of PKC inactivation in glutamate-induced neuronal death. They also suggest that BDNF neuroprotection arises, at least in part, via its ability to block the mechanism by which pathophysiological Ca2+ influx through the NMDA receptor causes membrane PKC inactivation.

Animals↗

PubMed1999

Pain relief using smart technology: an overview of a new patient-controlled analgesia device.

A new adaptive patient-controlled analgesia (PCA) system designed to improve PCA through the use of a variable bolus dose and a variable background infusion is outlined here. The handset allows patients to rate their pain on a scale of 1-10. Data derived from the handset signals are used by an expert algorithm to repeatedly adapt the drug dosage of the bolus and the background infusion according to both pain intensity and patient response to previous dosages. A feasibility study of the system consisted of a small number of randomized, double-blind, crossover clinical trials. The new system was alternated with a conventional system every 12 h. When the new system was active, 12-h questionnaire pain scores were significantly lower and a trend toward fewer bolus requests was found in alternating intervals. In addition, when the new system was used to commence trials, the number of bolus requests were significantly lower and there was a trend toward lower handset scores, lower questionnaire pain scores, and lower verbal pain scores over the entire trial period. The new adaptive PCA system was well accepted by both patients and clinical staff. The trials successfully established the feasibility of the new system. Further development is being carried out as a result.

Analgesia, Patient-Controlled↗

PubMed1999

Cell-specific signaling and structure-activity relations of parathyroid hormone analogs in mouse kidney cells.

PTH is an 84-amino acid protein. Occupancy of its cognate receptor generally results in activation of adenylyl cyclase and/or phosphoinositide-specific phospholipase Cbeta (PLCbeta). In the kidney, PTH receptors are present on proximal and distal tubule cells. In proximal tubules, PTH induces calcium signaling, typified by a transient rise in intracellular calcium ([Ca2+]i) and inositol trisphosphate formation, but does not affect calcium absorption. By contrast, in distal tubules, PTH increases calcium absorption that is associated with a slow and sustained rise in [Ca2+]i, but does not stimulate phospholipase C (PLC) or cause inositol trisphosphate accumulation. Nonetheless, stimulation of distal calcium transport requires activation of protein kinase C (PKC) and protein kinase A. We now characterize the origin of the differential effects of ligand occupancy by using synthetic human PTH analogs that preferentially activate adenylyl cyclase and/or PLCbeta. We further tested the hypothesis that phospholipase D is responsible for PKC activation in distal tubule cells. PTH-(1-31) increased [Ca2+]i in distal tubule but not in proximal tubule cells, whereas PTH-(3-34) caused a partial increase in [Ca2+]i in proximal cells, but had no effect in distal cells. PTH-(7-34) blocked increases in [Ca2+]i in distal tubule cells stimulated by PTH-(1-34) and PTH-(1-31). The PLC inhibitor U73122 abolished the PTH-induced rise in [Ca2+]i and inositol trisphosphate formation by proximal tubule cells, but had no effect on PTH-stimulated Ca2+ uptake by distal tubule cells. These results support the view that activation of PKC by PTH in distal tubule cells does not involve PLCbeta. PTH did, however, activate phospholipase D with attendant formation of diacylglycerol in distal cells. As activation of PKC is required for induction of calcium transport by PTH, we conclude that PTH receptors are capable of activating multiple phospholipases and that the structural requirements for such activation differ in proximal and distal tubule cells.

Adenylyl Cyclases↗

PubMed1999

Comparison of the biochemical responses to human parathyroid hormone-(1-31)NH2 and hPTH-(1-34) in healthy humans.

The 1-31 fragment of human PTH [hPTH-(1-31)NH2] has been shown, like hPTH-(1-34), to have anabolic effects on the skeletons of ovariectomized rats when given intermittently, but, unlike hPTH-(1-34), it does so without affecting serum calcium concentrations and does not activate the protein kinase C second messenger pathway in some target cells. To investigate the biochemical responses to hPTH-(1-31) in humans, we have directly compared it to hPTH-(1-34) during the course of slow infusions of each. Ten healthy adults, five men and five women, aged 26+/-5 yr (range, 22-37), each received 8-h continuous infusions of 8 pmol/kg.h hPTH-(1-34) and hPTH-(1-31) given in random order at least 2 weeks apart. During the infusions there were significant increases in both plasma and urinary cAMP (P < 0.05), but there were no differences in the responses between the two peptides (P = 0.362 for plasma; P = 0.987 for urine). There were also significant phosphaturic and natriuretic responses to the two peptides, which again were not different between peptides. During the infusion of hPTH-(1-34) serum ionized calcium (Ca2+) increased from 1.21+/-0.033 to 1.29+/-0.046 mmol/L (P < 0.01), and endogenous hPTH-(1-84) decreased from 29.6+/-9 to 15.0+/-5.7 pg/mL (P < 0.01), such that there was a negative correlation between them (r2 = 0.45). However, when hPTH-(1-31) was infused, neither serum Ca2+ (1.24+/-0.03 vs. 1.25+/-0.03) nor hPTH-(1-84) (26.8+/-5 vs. 30.7+/-12 pg/mL) was affected. Circulating concentrations of 1,25-dihydroxyvitamin D3 increased from 92+/-42 to 131+/-63 pmol/L (P < 0.05) during infusion of hPTH-(1-34) and from 92+/-27 to 110+/-42 pmol/L (P = NS) during hPTH-(1-31) infusion. There was also a significant increase in the urinary measure of type I collagen degradation of aminoterminal telopeptides from 78+/-45 to 101+/-51 nmol/mmol creatinine (P < 0.05) when hPTH-(1-34) was infused, but it was not affected (68+/-30 vs. 66+/-24 nmol/mmol creatinine) by hPTH-(1-31). Therefore, hPTH-(1-31) appears to be equivalent and equipotent to hPTH-(1-34) in the release of cAMP from target tissues and the renal handling of phosphate and sodium. However, at the doses employed, it does not increase serum calcium, is a weaker stimulator of the 25-hydroxyvitamin D-1alpha-hydroxylase, and does not induce rapid bone resorption.

Adult↗

PubMed1999

Prolonged low-dose infusion of human parathyroid hormone does not increase femoral cancellous bone volume in ovariectomized rats.

OBJECTIVE: Daily injections of human parathyroid hormone (hPTH) increase bone volume in various animal species and in osteoporotic women. For hPTH to be widely accepted as an anabolic therapy for treating postmenopausal osteoporosis alternative delivery options need to be explored to replace the need for daily patient subcutaneous self-injection. Among these are inhalation, oral delivery and the use of programmable implanted minipumps to deliver the peptide. While infusion of high doses of PTH causes bone loss and hypercalcemia, no studies have assessed the effects of prolonged infusion of low doses of PTH on bone growth. DESIGN AND METHODS: [Leu(27)]-cyclo(Glu(22)-Lys(26))-hPTH-(1--31)NH(2) was delivered by Alzet minipumps to ovariectomized rats for 6 weeks after which histomorphometric indices (cancellous bone volume, trabecular thickness, mean trabecular number) of bone formation were measured in distal femurs. RESULTS: Infusing low doses (0.05 and 0.1 nmole/100g body weight/day) of the hPTH analog, [Leu(27)]-cyclo(Glu(22)-Lys(26))-hPTH-(1--31)NH(2), for 6 weeks does not prevent the ovariectomy-induced loss of rat femoral cancellous bone volume, trabecular thickness or trabecular number. CONCLUSION: These results support the absolute requirement of daily injections for the osteogenic action of hPTH on bone.

Animals↗

PubMed1999

The bone-building action of the parathyroid hormone: implications for the treatment of osteoporosis.

As populations age in the world's 7 major pharmaceutical markets, an increasing number of men and women, but especially women during their first postmenopausal decade, are falling victim to a bone disease that can result in hospitalisation, disability and death. This disease is osteoporosis. Indeed, by the year 2007, as many as 153,000,000 people will have experienced at least some significant osteopenia, if not osteoporosis. Although there are many different drugs, such as calcitonin, the bisphosphonates, estrogen and selective estrogen receptor modulators, that can slow or even stop further bone loss, there are currently few that can replace already lost bone by directly stimulating bone growth. However, a family of potent bone-building (or bone-anabolic) peptides is currently undergoing clinical development. These are the first-generation 84-amino acid native parathyroid hormone (PTH) and its 34- to 38-amino acid N terminal fragments, and the potent second-generation mini-PTHs. In this article, we briefly summarise what has so far been learned about how these molecules stimulate the production of new biomechanically strong bone in animals and humans.

Animals↗

PubMed1999

AR-R15896AR blocks the early NMDA-induced loss of MAP2 in primary cortical cultures.

The low-affinity use-dependent N-methyl-D-aspartate (NMDA) receptor antagonist AR-R15896AR is neuroprotective in primary rat cortical cultures exposed to toxic concentrations of NMDA and reduces the magnitude of NMDA-triggered increases in [Ca2+]i. Here we show using fluorescence staining and measurements of microtubule-associated protein-2 (MAP2) levels, that AR-R15896AR inhibits the NMDA-induced loss of MAP2 that occurs within 2 min following NMDA exposure. Understanding the multiple, Ca(2+)-triggered intracellular events that occur following NMDA receptor stimulation is important to the development of safe and effective neuroprotective agents.

Animals↗

PubMed1999

Design and applications of parathyroid hormone analogues.

The endocrine parathyroid hormone (PTH) is the major regulator of serum calcium levels. In contrast, the autocrine/paracrine parathyroid hormone-related peptide (PTHrP) has been associated with organism development. Both are secreted as much larger molecules but have their major functions associated with their N-terminal 34 residues. They share a common receptor expressed in organs critical to PTH function - bone, kidney, and intestine. PTH and PTHrP receptor activation stimulates adenylyl cyclase (AC), phospholipase C (PLC), and phospholipase D (PLD) in target cells. It has been possible to separate the AC-stimulation from that of PLC. AC-stimulation requires at least the N-terminal 28 residues of PTH and PLC-stimulation requires a minimum of residues 29-32-NH2. Intermittent administration of PTH stimulates bone growth and requires AC-stimulation. The shortest linear sequence of hPTH with essentially full anabolic activity for bone growth-stimulation is hPTH(1-31)NH2. Two applications are postulated for PTH and PTHrP-based pharmaceuticals - treatment of bone loss due to osteoporosis and reversal of the hypercalcemic effect of malignancy. PTHrP analogues which strongly inhibit PTHrP AC-stimulation showed promise for the treatment of malignancy-associated hypercalcemia in animal trials but failed in human ones. However, both animal and human trials of hPTH have shown significant bone growth-stimulating effects. New deletion, substitution and cyclized analogues of PTH show great promise both for greater in vitro activity and possibly for improved delivery and greater specificity as agents for restoration of bone loss in osteoporosis.

Amino Acid Sequence↗

PubMed1999

Differences in degree of trapping of low-affinity uncompetitive N-methyl-D-aspartic acid receptor antagonists with similar kinetics of block.

This study characterizes the trapping of block of N-methyl-D-aspartic acid (NMDA)-induced currents by three structurally distinct, use-dependent NMDA receptor antagonists with similar rapid on-off rates. The antagonism of whole-cell currents in cultured rat cortical neurons by AR-R15896AR, ketamine, and memantine was examined. All three compounds produced a steady-state block after a 30-s coapplication, which was fully relieved after 50 s of NMDA exposure. The amplitudes of block caused by 50 microM AR-R15896AR, 10 microM ketamine, or 10 microM memantine were not significantly different, being 82 +/- 1%, 80 +/- 2%, and 81 +/- 2%, respectively. All three NMDA receptor antagonists exhibited trapping of block that was not significantly increased by extending the agonist/antagonist coapplication beyond 30 s. Although the initial blocks were similar, after 120 s of washout without agonist present, there were significant differences in trapping of block between antagonists, as only 54 +/- 3% of the AR-R15896AR block, 86 +/- 1% of the ketamine block, and 71 +/- 4% of the memantine block remained trapped. The lack of complete trapping is consistent with closed-channel egress by these compounds. Higher antagonist concentrations produced larger initial blocks, but the degree of trapping block was not significantly different from that at lower antagonist concentrations. The results demonstrate that differences in the degree of trapping exist among use-dependent NMDA receptor antagonists even when on and off rates are similar. These differences are correlated with measures of therapeutic index.

Animals↗

PubMed1998

Selective coupling of mu-calpain activation with the NMDA receptor is independent of translocation and autolysis in primary cortical neurons.

Excessive mu-calpain activation has been linked to several cellular pathologies including excitotoxicity and ischemia. In erythrocytes and other non-central nervous system (CNS) cells, calpain activation is thought to occur following a Ca2+-induced translocation of inactive cytosolic enzyme to membranes and subsequent autolysis. In the present report, we show that transiently exposing primary rat cortical neurons to lethal (50 microM) N-methyl-D-aspartic acid (NMDA) caused protracted calpain activation, measured as increased spectrin hydrolysis, but this was independent of translocation or autolysis of the protease. An anti-mu-calpain antibody showed that calpain was largely membrane associated in cortical neurons, and, consequently, neither translocation nor autolysis of the protease was observed following ionomycin or lethal NMDA treatment. By contrast, in rat erythrocytes, calpain was largely cytosolic and underwent rapid translocation and autolysis in response to ionomycin. Calpain-mediated spectrin hydrolysis was specifically coupled to Ca2+ entry through the NMDA receptor because nonspecific Ca2+ influx via ionomycin or KCl-mediated depolarization failed to activate the enzyme. Thus, calpain appears selectively linked to glutamate receptors in cortical neurons and regulated by mechanisms distinct from that occurring in many non-CNS cells. The data suggest that intracellular signals coupled to the NMDA receptor are responsible for activating calpain already associated with cellular membranes in cortical cells.

Animals↗

PubMed1998

Brain derived neurotrophic factor induction of N-methyl-D-aspartate receptor subunit NR2A expression in cultured rat cortical neurons.

N-methyl-D-aspartate (NMDA) receptor subunit expression changes during development and following injury in several brain regions. These changes may be mediated by neurotrophic factors, such as brain derived neurotrophic factor (BDNF). Exposure of cultured cortical neurons to BDNF (100 ng/ml) for 24 h produced a significant decrease in the NMDA-induced whole-cell currents sensitive to the NR2B subunit selective NMDA receptor antagonist, CP-101,606, suggesting a relative decrease in NR2B subunit expression. There was a significant increase in NR2A by Western blot analysis. Consistent with the electrophysiology and Western blot analysis, reverse transcriptase-polymerase chain reaction (RT-PCR) amplification revealed that BDNF caused a significant increase in relative NR2A subunit expression, a significant decrease in relative NR2B subunit expression and no change in relative NR2C subunit expression. These results suggest that BDNF enhances NMDA receptor maturation, warranting further study of the mechanism of BDNF effects on NMDA receptor subunit expression and the role these effects play in development and neuronal injury.

Animals↗

PubMed1998

Neuroprotective effects of a novel AMPA receptor antagonist, YM872.

Quinoxalinediones such as NBQX are neuroprotective in most models of cerebral ischemia but their poor solubility results in nephrotoxicity limiting their clinical utility. We have investigated the neuroprotective effects of a water soluble AMPA receptor antagonist, YM872, using two in vitro models. The viability of cortical cultures exposed to 400 microM AMPA for 15 min (16.4 +/- 2.6%; n = 10) was significantly (p < 0.05) increased (84.7 +/- 4.6%; n = 6) with YM872 (10 microM) in a concentration-dependent manner. Evoked post-synaptic response amplitudes in oxygen-glucose deprived hippocampal slices treated with 10 microM YM872 (3.5 +/- 0.3 mV; n = 27) were significantly different from untreated deprived slices (0.3 +/- 0.1 mV; n = 31, p < 0.05) and the CA1 neurons appeared viable using a confocal live/dead fluorescence assay with confocal microscopy. The neuroprotection seen with YM872 in vitro warrants further investigation in vivo.

Animals↗

PubMed1998

Comparison of the abilities of human parathyroid hormone (hPTH)-(1-34) and [Leu27]-cyclo(Glu22-Lys26)-hPTH-(1-31)NH2 to stimulate femoral trabecular bone growth in ovariectomized rats.

hPTH-(1-31)NH2, so far the smallest of the potently anabolic N-terminal fragments of the human parathyroid hormone, stimulates trabecular growth in the distal femurs of ovariectomized (OVX) rats as strongly as hPTH-(1-34) when injected at a high daily dose such as 1 nmol/100 g of body weight, but it is only about 70% as effective as hPTH-(1-34) when injected at the suboptimal 0.6 nmol/100 g of body weight. A lactam derivative of hPTH-(1-31)-NH2, [Leu27]-cyclo(Glu22-Lys26)-hPTH-(1-31)NH2, is a much more effective stimulator of adenylyl cyclase in ROS 17/2 rat osteoblast-like cells and a significantly more effective stimulator of femoral trabecular growth in OVX rats than hPTH-(1-31)NH2. We have now shown that [Leu27]-cyclo(Glu22-Lys26)-hPTH-(1-31)NH2 prevents the OVX-induced loss of femoral trabeculae significantly more effectively than hPTH-(1-34) and stimulates the thickening of the trabeculae remaining in severely depleted femoral trabecular bone of OVX rats as effectively as hPTH-(1-34) when injected at 0.6 nmol/100 g of body weight.

Adenylyl Cyclases↗

PubMed1998

A regulated environmental perfusion system for the study of anoxic or hypoxic cultured neurons using microfluorescence imaging and electrophysiology.

We describe the design and performance of a newly developed regulated environmental perfusion system (REPS). This system allows study of the effects of anoxia or hypoxia in cultured cells at physiological temperature, without the use of oxygen-scavenging compounds or metabolic inhibitors. The REPS incorporates a "canoe-shaped" flow-through chamber with access from above to allow positioning of pipettes for patch-clamp, microinjection, rapid-application perfusion, or microprobes for monitoring physical parameters. The combination of laminar flow and complete washout of perfusate within the chamber, and the use of a gas-tight perfusate delivery system and pressurized reservoirs containing media with pre-stabilized oxygen tensions (pO2 values) allow rapid production of accurate perfusate pO2 within the chamber. Perfusate pO2 in the chamber declined monoexponentially with time constants of </= 20 s to stable, pre-determined levels of 0 or 2 kPa (15 Torr). Shielding the gas/liquid interface of the chamber with an argon curtain only minimally decreased time constants at flow rates >/= 2 ml/min. The perfusion chamber of the REPS is easily mounted on the stage of an inverted microscope, for use with fluorescence imaging or electrophysiological studies of cultured cells. In tests with cultured rat cortical neurons, intracellular calcium concentration increased exponentially to values exceeding 1 microM during 10 min of anoxic insult, and returned to baseline values within 1 min after restoring normoxia.

Animals↗

PubMed1998

A fluorescence confocal assay to assess neuronal viability in brain slices.

Hippocampal slice models are used to study the mechanisms of ischemia-induced neurotoxicity and to assess the neuroprotective potential of novel therapeutic agents. A number of morphological and functional endpoints are available to assess neuronal viability. The slice model also allows the study of selectively vulnerable neuronal populations within the same preparation. The fluorescence procedure described here provides a method of assessing the viability of neurons in rat hippocampal slices exposed to hypoxic-hypoglycemic conditions. Control and/or treated slices that had been subjected to a 10 min oxygen-glucose deprivation insult are double stained with calcein-AM (4 microM), which stains live cells green, and ethidium homodimer (6 microM), which stains the nucleus of dead cells red. The stained slices are then imaged using confocal microscopy. Vulnerable neurons in the CA1 region of slices deprived of oxygen and glucose became increasingly permeant to ethidium homodimer over the 4 h reperfusion period. Exposure to low Ca2+ concentration (0.3 mM) or the N-, P- and Q-type Ca2+ channel antagonist MVIIC (100 nM), which have been shown to be neuroprotective in this model of ischemia using field evoked post-synaptic potential (EPSP) measures as an endpoint, were also shown to be protective using the fluorescence assay.

Animals↗

PubMed1998

Evidence that functional glutamate receptors are not expressed on rat or human cerebromicrovascular endothelial cells.

Excitatory amino acids can modify the tone of cerebral vessels and permeability of the blood-brain barrier (BBB) by acting directly on endothelial cells of cerebral vessels or indirectly by activating receptors expressed on other brain cells. In this study we examined whether rat or human cerebromicrovascular endothelial cells (CEC) express ionotropic and metabotropic glutamate receptors. Glutamate and the glutamate receptor agonists N-methyl-d-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA), and kainate failed to increase [Ca2+]i in either rat or human microvascular and capillary CEC but elicited robust responses in primary rat cortical neurons, as measured by fura-2 fluorescence. The absence of NMDA and AMPA receptors in rat and human CEC was further confirmed by the lack of immunocytochemical staining of cells by antibodies specific for the AMPA receptor subunits GluR1, GluR2/3, and GluR4 and the NMDA receptor subunits NR1, NR2A, and NR2B. We failed to detect mRNA expression of the AMPA receptor subunits GluR1 to GluR4 or the NMDA receptor subunits NR1(1XX); NR1(0XX), and NR2A to NR2C in both freshly isolated rat and human microvessels and cultured CEC using reverse transcriptase polymerase chain reaction (RT-PCR). Cultured rat CEC expressed mRNA for KA1 or KA2 and GluR5 subunits. Primary rat cortical neurons were found to express GluR1 to GluR3 and NR1, NR2A, and NR2B by both immunocytochemistry and RT-PCR and KA1, KA2, GluR5, GluR6, and GluR7 by RT-PCR. Moreover, the metabotropic glutamate receptor agonist 1-amino-cyclopentyl-1S, 3R-dicorboxylate (1S,3R-trans-ACPD), while eliciting both inositol trisphosphate and [Ca2+]i increases and inhibiting forskolin-stimulated cyclic AMP in cortical neurons, was unable to induce either of these responses in rat or human CEC. These results strongly suggest that both rat and human CEC do not express functional glutamate receptors. Therefore, excitatory amino acid-induced changes in the cerebral microvascular tone and BBB permeability must be affected indirectly, most likely by mediators released from the adjacent glutamate-responsive cells.

Animals↗