Biomedical subjects

E C Johnson

Publications and source records attributed to E C Johnson.

At least 37 records · Page 2Linked to original sources

PubMed1999

Relationship between moderate intensity endurance training volume and natural killer cell cytolytic activity.

BACKGROUND: This study was designed to examine the relationship between endurance training volume and natural killer cell (NK) cytolytic activity. We hypothesized that a dose dependent relationship exists between forced treadmill training volume and training induced increases in NK cell cytolytic activity. METHODS EXPERIMENTAL DESIGN: female, Swiss Webster mice were assigned to treadmill control (TC) or treadmill trained groups (n = 10 per group). Trained mice ran at 12 m per min. (8 degrees grade) for: 15 (EX15), 30 (EX30), or 60 minutes (EX60) per day, five days per week for 11 weeks. Splenic NK cell activity was expressed as median lytic unit (LU), median LU per asialo GM1 (AsGM1+) cell, and median LU per spleen. RESULTS: Median NK activity was not significantly increased by training volume. A trend toward greater median LU per AsGM1+ cell was observed in EX30 group versus TC (p = 0.1). Training volumes less than or greater than this level produced smaller increases in NK cytolytic activity. CONCLUSIONS: These data provide preliminary evidence indicating that training induced increases in splenic NK cell cytolytic activity do not exhibit a dose dependent relationship with treadmill training volume.

Animals↗

PubMed1998

Artifact category membership and the intentional-historical theory.

Bloom has illustrated a number of complex and interesting phenomena that must be accounted for by any theory of categorization. We agree with Bloom about several important points: that the intended function of an object alone does not account for its categorization; that historical relations among objects can be important in assigning category membership; and that a name bestowed upon an object by a prior source can be an important contributor to categorization of the object. We also agree that intention may enter into category judgments in some cases. Still, we believe that the intentional-historical theory falls short of providing a compelling general account of artifact category membership.

Classification↗

PubMed1998

Functional characterization of human N-methyl-D-aspartate subtype 1A/2D receptors.

The human NMDAR2D subunit was cloned, and the pharmacological properties of receptors resulting from injection of transcripts encoding human NMDAR1A and NMDAR2D subunits in Xenopus oocytes were characterized by profiling NMDA receptor agonists and antagonists. We found that glutamate, NMDA, glycine, and D-serine were significantly more potent on hNMDAR1A/2D than on hNMDAR1A/2A or hNMDAR1A/2B. Also, the potencies of NMDA and glycine were higher for hNMDAR1A/2D than for hNMDAR1A/2C. Ifenprodil was more potent at hNMDAR1A/2B than at hNMDAR1A/2D, whereas 5,7-dichlorokynurenate was more potent at hNMDAR1A/2A than at hNMDAR1A/2D. As measured in transiently transfected human embryonic kidney 293 cells, the maximal inward current in the presence of external Mg2 occurred at -40 mV, and full block was not observed at negative potentials. Kinetic measurements revealed that the higher affinity of hNMDAR1A/2D for both glutamate and glycine relative to hNMDAR1A/2A and hNMDA1A/2B can be explained by slower dissociation of each agonist from hNMDAR1A/2D. The hNMDAR1A/2D combination represents a pharmacologically and functionally distinct receptor subtype and may constitute a potentially important target for therapeutic agents active in the human CNS.

Animals↗

PubMed1998

The human N-methyl-D-aspartate receptor 2C subunit: genomic analysis, distribution in human brain, and functional expression.

cDNAs encoding four isoforms of the human NMDA receptor (NMDAR) NMDAR2C (hNR2C-1, -2, -3, and -4) have been isolated and characterized. The overall identity of the deduced amino acid sequences of human and rat NR2C-1 is 89.0%. The sequences of the rat and human carboxyl termini (Gly925-Val1,236) are encoded by different exons and are only 71.5% homologous. In situ hybridization in human brain revealed the expression of the NR2C mRNA in the pontine reticular formation and lack of expression in substantia nigra pars compacta in contrast to the distribution pattern observed previously in rodent brain. The pharmacological properties of hNR1A/2C were determined by measuring agonist-induced inward currents in Xenopus oocytes and compared with those of other human NMDAR subtypes. Glycine, glutamate, and NMDA each discriminated between hNR1A/2C-1 and at least one of hNR1A/2A, hNR1A/2B, or hNR1A/2D subtypes. Among the antagonists tested, CGS 19755 did not significantly discriminate between any of the four subtypes, whereas 5,7-dichlorokynurenic acid distinguished between hNR1A/2C and hNR1A/2D. Immunoblot analysis of membranes isolated from HEK293 cells transiently transfected with cDNAs encoding hNR1A and each of the four NR2C isoforms indicated the formation of heteromeric complexes between hNR1A and all four hNR2C isoforms. HEK293 cells expressing hNR1A/ 2C-3 or hNR1A/2C-4 did not display agonist responses. In contrast, we observed an agonist-induced elevation of intracellular free calcium and whole-cell currents in cells expressing hNR1A/2C-1 or hNR1A/2C-2. There were no detectable differences in the macroscopic biophysical properties of hNR1A/2C-1 or hNR1A/2C-2.

Amino Acid Sequence↗

PubMed1998

Characterization of human recombinant neuronal nicotinic acetylcholine receptor subunit combinations alpha2beta4, alpha3beta4 and alpha4beta4 stably expressed in HEK293 cells.

Human embryonic kidney (HEK293) cells were transfected with cDNA encoding the human beta4 neuronal nicotinic acetylcholine (ACh) receptor subunit in pairwise combination with human alpha2, alpha3 or alpha4 subunits. Cell lines A2B4, A3B4.2 and A4B4 were identified that stably express mRNA and protein corresponding to alpha2 and beta4, to alpha3 and beta4 and to alpha4 and beta4 subunits, respectively. Specific binding of [3H]epibatidine was detected in A2B4, A3B4.2 and A4B4 cells with Kd (mean +/- S.D. in pM) values of 42 +/- 10, 230 +/- 12 and 187 +/- 29 and with Bmax (fmol/mg protein) values of 1104 +/- 338, 2010 +/- 184 and 3683 +/- 1450, respectively. Whole-cell patch-clamp recordings in each cell line demonstrated that (-)nicotine (Nic), ACh, cytisine (Cyt) and 1, 1-dimethyl-4-phenylpiperazinium iodide (DMPP) elicit transient inward currents. The current-voltage (I-V) relation of these currents showed strong inward rectification. Pharmacological characterization of agonist-induced elevations of intracellular free Ca++ concentration revealed a distinct rank order of agonist potency for each subunit combination as follows: alpha2beta4, (+)epibatidine (Epi) > Cyt > suberyldicholine (Sub) = Nic = DMPP; alpha3beta4, Epi > DMPP = Cyt = Nic = Sub; alpha4beta4, Epi > Cyt = Sub > Nic > DMPP. The noncompetitive antagonists mecamylamine and d-tubocurarine did not display subtype selectivity. In contrast, the Kb value for the competitive antagonist dihydro-beta-erythroidine (DHbetaE) was highest at alpha3beta4 compared with alpha2beta4 or alpha4beta4 receptors. These data illustrate that the A2B4, A3B4.2 and A4B4 stable cell lines are powerful tools for examining the functional and pharmacological properties of human alpha2beta4, alpha3beta4 and alpha4beta4 neuronal nicotinic receptors.

Bridged Bicyclo Compounds, Heterocyclic↗

PubMed1998

Pharmacological characterization of the human ionotropic glutamate receptor subtype GluR3 stably expressed in mammalian cells.

We have cloned the human ionotropic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluR3 flip splice variant (hGluR3i) and developed a stable cell line expressing this receptor in HEK293 cells. Electrophysiological recordings demonstrated that glutamate-evoked currents desensitize rapidly, with a mean desensitization time constant of 5.4 ms. Robust glutamate-evoked increases in intracellular Ca++ ([Ca++]i) were observed in the presence of cyclothiazide, which attenuated receptor desensitization. [Ca++]i measurements were used to perform a detailed pharmacological characterization of hGluR3i with reference agonists and antagonists. The results of these studies showed that kainate and domoate were not fully efficacious agonists relative to glutamate. The binding affinities of agonists and competitive antagonists were determined in a [3H]AMPA competition binding assay. There was a good correlation between the functional data and the binding affinities obtained for competitive antagonists. However, the binding affinities of the agonists did not correlate with their functional EC50 values from [Ca++]i data, possibly because the binding assay predominantly measures the desensitized high-affinity state of the receptor. [3H]AMPA binding also was performed on membranes prepared from rat forebrain, and comparison of the data from HEK293 cells expressing hGluR3i and rat forebrain suggest that nearly all of the reference compounds show similar binding activities between the two membrane preparations, with the exception of fluoro-willardiine, kainate and 6-nitro-7-sulfamoylbenzo(f)quinoxaline-2-3-dione (NBQX). These data suggest that cells stably expressing recombinant hGluR3i represent pharmacologically valid experimental systems to study human AMPA receptors.

Animals↗

PubMed1997

Functional coupling of a human retinal metabotropic glutamate receptor (hmGluR6) to bovine rod transducin and rat Go in an in vitro reconstitution system.

The cDNA encoding hmGluR6, appended with a 15-amino acid antibody epitope (1D4), was transiently transfected in COS-7 cells. The receptor was purified from COS cell membranes using an antibody affinity column. The purified receptor was then reconstituted into lipid vesicles, and its ability to activate either transducin, the rod photoreceptor-specific GTP-binding protein, or the alpha subunit of Go was assayed in vitro using a guanosine 5'-3-O-(thio)triphosphate binding assay. Activation of both transducin and Go was observed. The rate of Go activation was 18-fold greater than the rate of transducin activation. This indicates that the coupling of mGluR6 to Go is more efficient and suggests that Go may be involved in coupling to mGluR6 in ON-bipolar cells.

Amino Acid Sequence↗

PubMed1997

A rat model of chronic pressure-induced optic nerve damage.

To develop unilateral, chronically elevated intraocular pressure in rats, episcleral veins were injected with hypertonic saline and the intraocular pressure was monitored with a Tono-Pen XL tonometer. Histologic analyses of eyes with differing degrees and durations of intraocular pressure elevation were performed to ascertain the effects of these pressures on the optic nerve. Out of 20 consecutive animals, nine had elevations of intraocular pressure following a single injection, while subsequent injections raised intraocular pressure in seven others. One eye became hypotonous. In the remaining animals, subsequent injections sufficient to raise intraocular pressure were deliberately withheld, to determine the possible direct effects of injections on the optic nerve. Mean sustained pressure elevations ranged from 7 to 28 mm Hg and the retinal vasculature remained perfused in all eyes. Optic nerve cross sections from eyes without intraocular pressure elevation appeared identical to those from uninjected eyes, while nerves from eyes with the greatest intraocular pressure rise demonstrated axonal damage that involved 100% of the neural area. Eyes with either less severe pressure elevations or shorter durations showed partial damage, ranging from 0.5% to 10.4% of the neurla area. In 70% of these nerves, damage was concentrated in the superior temporal region. Within the optic nerve head, often associated with astrocytes, axons contained abnormal accumulations of membrane-bound vesicles and mitochondria. The anterior chamber angles showed sclerosis of the trabecular meshwork with anterior synechiae, but Schlemm's canal, collector channels and aqueous veins appeared patent. Unilateral sclerosis of the trabecular meshwork produces sustained elevation of intraocular pressure in rats with optic nerve damage that in many ways resembles that seen in human glaucoma. Understanding the mechanism of nerve damage in this model may provide new insights into the pathogenesis of human glaucoma.

Animals↗

PubMed1997

Effects of inhaled nitric oxide on gas exchange in lungs with shunt or poorly ventilated areas.

Inhaled nitric oxide (NO) is a selective pulmonary vasodilator with beneficial effects on some lung diseases, yet conflicting results, particularly in chronic obstructive pulmonary disease, have been reported. We hypothesized that although inhaled NO would improve gas exchange in the presence of shunt (by increasing blood flow to normal areas), it could worsen gas exchange when areas of low ventilation-perfusion (VA/Q) ratio were present since these areas could be preferentially vasodilated by NO. We examined how approximately 80 ppm inhaled NO altered pulmonary gas exchange in anesthetized ventilated dogs with the following: (1) normal lungs (n = 8), (2) shunt (n = 9, 24.7% shunt) produced by complete obstruction of one lobar bronchus, and (3) VA/Q inequality (n = 8) created by partial obstruction of one lobar bronchus resulting in a bimodal VA/Q distribution with 13% perfusion of low VA/Q areas (0.005 < VA/Q < 0.1) without shunt. Inhaled No significantly reduced pulmonary arterial (p < 0.001) and wedge pressures (p < 0.01) and pulmonary vascular resistance (p < 0.01) without changing cardiac output in each group. In normal lungs, NO did not alter PaO2 or VA/Q inequality. However, with complete obstruction, shunt fell slightly (p < 0.001) with NO. In lungs with VA/Q inequality, NO variably affected VA/Q matching, which was improved in some dogs and worsened in others. In these lungs, changes in pulmonary vascular resistance of the abnormal area of the lung were negatively correlated with changes in VA/Q dispersion (logSDQ) (R = -0.85, p < 0.01) and positively correlated with PaO2 (R = 0.79, p < 0.05). We conclude that NO has net effects on pulmonary gas exchange, depending on the underlying lung pathology consistent with competing vasodilatory effects on the normal and abnormal areas that receive the gas.

Administration, Inhalation↗

PubMed1997

Pharmacological characterization of recombinant human neuronal nicotinic acetylcholine receptors h alpha 2 beta 2, h alpha 2 beta 4, h alpha 3 beta 2, h alpha 3 beta 4, h alpha 4 beta 2, h alpha 4 beta 4 and h alpha 7 expressed in Xenopus oocytes.

Human neuronal nicotinic acetylcholine receptors (nAChRs) h alpha 2 beta 2, h alpha 2 beta 4, h alpha 3 beta 2, h alpha 3 beta 4, h alpha 4 beta 2, h alpha 4 beta 4 and h alpha 7 were expressed in Xenopus oocytes and tested for their sensitivities to the nicotinic agonists acetylcholine (ACh), nicotine, cytisine (CYT) and 1,1-dimethyl-4-phenylpiperazinium (DMPP) and the nAChR. antagonists mecamylamine (MEC), d-tubocurarine and dihydro-beta-erythroidine. CYT was the least efficacious agonist at hnAChRs containing beta 2 subunits, but it displayed significant activity at h alpha 2 beta 4, h alpha 3 beta 4, h alpha 4 beta 4 and h alpha 7 nAChRs. ACh was one of the most efficacious agonists at all hnAChRs, except at h alpha 3 beta 2, where DMPP was markedly more efficacious than ACh. ACh was among the least potent agonists at all hnAChRs. The rank order of potency displayed by h alpha 3 beta 2 and h alpha 3 beta 4 nAChRs (DMPP approximately CYT approximately nicotine > ACh and DMPP > CYT approximately nicotine > ACh, respectively), differs from that reported for their rat homologs (Luetje and Patrick, 1991; Covernton et al., 1994). The agonist profile observed in h alpha 7 also differs from that reported for its rat homolog (Seguela et al., 1993). Human alpha 4 beta 2 and h alpha 4 beta 4 nAChRs were more sensitive to dihydro-beta-erythroidine than d-tubocurarine, whereas h alpha 7 and h alpha 3 beta 4 were more sensitive to d-tubocurarine than dihydro-beta-erythroidine. These antagonists were equipotent at h alpha 2 beta 2, h alpha 3 beta 2 and h alpha 2 beta 4 nAChRs. MEC (3 microM) inhibited h alpha 2 beta 4 and h alpha 4 beta 4 nAChRs by > 80%, whereas h alpha 2 beta 2, h alpha 4 beta 2 and h alpha 7 nAChRs were inhibited by approximately 50%. Taken together, the differential sensitivities observed at various recombinant hnAChR subtypes indicate that both alpha and beta subunits contribute to the pharmacology of these ligand-gated channels. The unique selectivity profiles displayed by human nAChRs constitute a valuable tool for the development of selective nicotinic analogs as potential therapeutic drugs.

Animals↗

PubMed1996

The effect of chronically elevated intraocular pressure on the rat optic nerve head extracellular matrix.

The extracellular matrix of the optic nerve head is altered in both human glaucoma and in experimental primate models of this disease. However, the relationship of this change to glaucomatous optic nerve degeneration is unknown. This report describes similar matrix alterations in rats with unilateral elevated intraocular pressure. Brown Norway rats received episcleral vein injections of hypertonic saline to produce prolonged elevations of intraocular pressure. After up to 6 months of pressure elevation, optic nerve head sections from the rats were evaluated by light microscopic immunohistochemistry using antibodies to collagens I, III, IV and VI, laminin, elastin and chondroitin and dermatan sulfate proteoglycans. In experimental eyes with 11 days or more of pressure elevation, depositions of collagen IV, collagen VI and laminin were found within regions of the optic nerve head that, in normal eyes, are occupied solely by nerve bundles. Collagen I and III deposition appeared to be more dependent on the level and duration of the pressure rise. Eyes with lower mean intraocular pressures showed deposits of interstitial collagens primarily at the level of the sclera, while eyes with higher mean pressure elevations had depositions in the neck regions as well. Chondroitin and dermatan sulfate proteoglycans were deposited in a pattern similar to that of collagen I. No extracellular matrix deposition was seen in the orbital optic nerve in any experimental eye. These extracellular matrix changes in rats replicate previous findings in human glaucomatous eyes and monkey eyes with experimentally elevated pressures. They also suggest a sequence of extracellular matrix protein deposition in response to pressure elevation. The optic nerve head deposition of matrix materials in response to elevated intraocular pressures may affect the susceptibility of remaining axons to pressure by changing the physical properties of their support tissues, by affecting the support functions of astrocytes and by changing the microenvironment of injured axons. This model may be useful for studying these and other aspects of the process of axonal injury resulting from elevated intraocular pressure.

Animals↗

PubMed1996

Comparative structure of human neuronal alpha 2-alpha 7 and beta 2-beta 4 nicotinic acetylcholine receptor subunits and functional expression of the alpha 2, alpha 3, alpha 4, alpha 7, beta 2, and beta 4 subunits.

cDNA clones encoding human neuronal nicotinic acetylcholine receptor alpha 2, alpha 3, alpha 4, alpha 5, alpha 6, alpha 7, beta 2, beta 3, and beta 4 subunits were isolated from brainstem, hippocampus, prefrontal cortex, substantia nigra, thalamus, and IMR32 libraries. Human alpha 2 and alpha 6 and full-length beta 3 and beta 4 clones have not been previously reported. Deduced amino acid sequences of the alpha 2, alpha 6, beta 3, and beta 4 predicted mature peptides are 503 residues (56.9 kDa), 464 residues (53.7 kDa), 440 residues (50.8 kDa), and 477 residues (54.1 kDa), respectively. These sequences show 84 (alpha 2), 87 (alpha 6), 89 (beta 3), and 84% (beta 4) identity to the corresponding rat sequences. The amino termini of the human alpha 2 and beta 3 mature peptides contain 23 and six additional residues, respectively, compared to those of rat alpha 2 and beta 3. Recombinant receptors were expressed in Xenopus laevis oocytes injected with in vitro transcripts encoding either alpha 7 alone or alpha 2, alpha 3, or alpha 4 in pairwise combination with beta 2 or beta 4. Inward currents were elicited by the application of acetylcholine (1-100 microM) and other agonists; these responses were blocked 65-97% by application of 10 microM d-tubocurare, confirming functional expression of human nicotinic receptors.

Acetylcholine↗

PubMed1996

Angiogenic growth factor mRNA responses in muscle to a single bout of exercise.

A major adaptation to exercise is new capillary formation in skeletal muscle. On the basis of angiogenesis in tumors and during development, several angiogenic growth factors may be involved, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and transforming growth factor-beta 1 (TGF-beta 1). In 9-wk-old female Wistar rats, mRNA expression for these three growth factors in gastrocnemius muscle was examined by quantitative Northern analysis after a single 1-h run at 15 or 20 m/min at 10 degrees incline in room air. A third group ran at 15 m/min in 12% O2, and resting control groups were included at inspired O2 fractions of 0.21 and 0.12. Exercise significantly increased mRNA levels two- to fourfold, which was evident over the first 4 h postexercise; by 8 and 24 h, mRNA levels returned to baseline. For all three factors, mRNA levels were significantly higher after exercise at 20 than at 15 m/min. Hypoxia at rest doubled VEGF and TGF-beta 1 message but had no effect on bFGF. Hypoxic exercise further raised VEGF mRNA levels but had no effect on the other factors. We suggest that VEGF, bFGF, and TGF-beta 1 may be involved in the angiogenic response to exercise and that reduced intracellular PO2 (as occurs during normoxic exercise) may be part of the stimulus to such growth factor production.

Animals↗

PubMed1996

Pulmonary hemodynamic response to exercise in subjects with prior high-altitude pulmonary edema.

Individuals with a prior history of (susceptible to high altitude pulmonary edema (HAPE-S) have high resting pulmonary arterial pressures, but little data are available on their vascular response to exercise. We studied the pulmonary vascular response to exercise in seven HAPE-S and nine control subjects at sea level and at 3,810 m altitude. At each location, both normoxic (inspired PO2 = 148 Torr) and hypoxic (inspired PO2 = 91 Torr) studies were conducted. Pulmonary hemodynamic measurements included pulmonary arterial and pulmonary arterial occlusion pressures. A multiple regression analysis demonstrated that the pulmonary arterial pressure reactivity to exercise was significantly greater in the HAPE-S group. This reactivity was not influenced by altitude or oxygenation, implying that the response was intrinsic to the pulmonary circulation. Pulmonary arterial occlusion pressure reactivity to exercise was also greater in the HAPE-S group, increasing with altitude but independent of oxygenation. These findings suggest an augmented flow-dependent pulmonary vasoconstriction and/or a reduced vascular cross-sectional area in HAPE-S subjects.

Adult↗

PubMed1996

Exercise-induced VA/Q inequality in subjects with prior high-altitude pulmonary edema.

Ventilation-perfusion (VA/Q) mismatch has been shown to increase during exercise, especially in hypoxia. A possible explanation is subclinical interstitial edema due to high pulmonary capillary pressures. We hypothesized that this may be pathogenetically similar to high-altitude pulmonary edema (HAPE) so that HAPE-susceptible people with higher vascular pressures would develop more exercise-induced VA/Q mismatch. To examine this, seven healthy people with a history of HAPE and nine with similar altitude exposure but no HAPE history (control) were studied at rest and during exercise at 35, 65, and 85% of maximum 1) at sea level and then 2) after 2 days at altitude (3,810 m) breathing both normoxic (inspired Po2 = 148 Torr) and hypoxic (inspired Po2 = 91 Torr) gas at both locations. We measured cardiac output and respiratory and inert gas exchange. In both groups, VA/Q mismatch (assessed by log standard deviation of the perfusion distribution) increased with exercise. At sea level, log standard deviation of the perfusion distribution was slightly higher in the HAPE-susceptible group than in the control group during heavy exercise. At altitude, these differences disappeared. Because a history of HAPE was associated with greater exercise-induced VA/Q mismatch and higher pulmonary capillary pressures, our findings are consistent with the hypothesis that exercise-induced mismatch is due to a temporary extravascular fluid accumulation.

Adult↗

PubMed1996

Circadian rhythm of intraocular pressure in the rat.

To define the characteristics of the diurnal variation of intraocular pressure (IOP) in eyes of awake rats, ten male brown Norway rats were entrained to a 12-hour light:12-hour dark (12L:12D) lighting schedule and were conditioned to IOP measurement with the TonoPen XL tonometer while awake, using only 0.5% proparacaine HCl anesthesia. The IOP measurements were performed in 4 experiments: Preliminary-IOP was measured at 6-hour intervals in both eyes of each animal, to determine correlation between right and left eyes; Light:Dark-lighting remained the same as in the preliminary experiment, but the measurement schedule was altered so that measurements were obtained at 4-hour intervals in alternating eyes, over two 24-hour light cycles; Dark:Dark-animals were placed in constant dark (0L:24D) and, after 72 h, measurements were obtained at 4-hour intervals in alternating eyes. Animals were then re-entrained to the previous 12L:12D schedule for 7 days, after which they were returned to constant dark and the experiment was repeated; and Dark:Light-animals were entrained to a reversed light:dark cycle (12D:12L) for 28 days, after which measurements were obtained in the same fashion as in the Light:Dark experiment. Close agreement was found between right- and left-eye IOPs. Animals on a 12L:12D schedule exhibited lowest IOP while the lights were on (19.3 +/- 1.9 mm Hg), and highest (31.3 +/- 1.3 mm Hg) while the lights were off. Pressure changes anticipated the change from light to dark and dark to light. This pattern persisted in constant dark, and was reversed when the cycle was changed to 12D:12L. Brown Norway rats possess a regular rhythm of IOP that is entrained by the cycle of light and dark, and persistence of this rhythm in constant dark establishes it as a circadian rhythm. Furthermore, our results indicate that reliable and physiologically meaningful IOP measurements can be obtained in awake rats using the TonoPen XL tonometer.

Animals↗

PubMed1996

Cloning and functional characterization of human heteromeric N-methyl-D-aspartate receptors.

Human cDNAs encoding N-methyl-D-aspartate receptor type (NMDAR)1A, NMDAR2A and NMDAR2B subunits were cloned and receptors encoded by these cDNAs were functionally expressed by injection of the respective mRNAs in Xenopus oocytes. The pharmacological properties of recombinant human N-methyl-D-aspartate (NMDA) receptors were characterized by profiling two agonists and four antagonists at both the NMDA and glycine sites in voltage-clamped oocytes. NMDA, glycine and D-serine were significantly more potent at human NMDAR (hNMDAR)1A/2B receptors than at nNMDAR1A/2A, whereas there was no detectable subtype-dependent difference in the potency of glutamate. Of the NMDA-site antagonists tested, CGP 43487 and 3-(2-carboxypiperazin-4-yl) propyl-1-phosphonate exhibited 5.8- and 3.9-fold greater potency, respectively, at hNMDAR1A/2A receptors than at hNMDAR1A/2B. Of the four glycine-site competitive antagonists tested, L-689,560 displayed 5-fold greater potency at hNMDAR1A/2A, whereas 5,7-dichlorokynurenic acid, HA-966 and CGP 58411 did not discriminate between hNMDAR1A/2A and hNMDAR1A/2B. Receptors resulting from injection of hNMDAR1A, hNMDAR2A and hNMDAR2B transcripts in a 1:1:1 ratio were indistinguishable from hNMDAR1A/2B receptors in terms of their sensitivity to NMDA, glycine, D-serine, CGS 19755 and CGP 40116. Ifenprodil was approximately 350-fold more potent at hNMDAR1A/2B than at hNMDAR1A/2A receptors. Ifenprodil sensitivities of receptors formed in oocytes injected with a constant amount of hNMDAR1A mRNA but varying ratios of hNMDAR2A or hNMDAR2B mRNAs were compared. The receptors expressed at a 10:1 ratio of 2A:2B transcripts displayed an ifenprodil sensitivity that would be predicted for a population in which 51% was represented by hNMDAR(1A)2(2A)3 complexes. Our results underscore the need for subtype-selective compounds acting at novel sites to sufficiently probe the pharmacological differences between NMDA receptor subtypes formed by different subunit combinations.

Animals↗

PubMed1996

Stable expression and characterization of recombinant human heteromeric N-methyl-D-aspartate receptor subtypes NMDAR1A/2A and NMDAR1A/2B in mammalian cells.

The electrophysiological and pharmacological properties of two mammalian cell lines stably transfected with cDNAs encoding recombinant human N-methyl-D-aspartate (NMDA) receptor subtypes NMDAR1A/2A and NMDAR1A/2B are described. In whole-cell electrophysiological recordings, application of NMDA/glycine elicited inward currents at negative holding potentials in human NMDAR1A/2A (hNMDAR1A/2A)- and hNMDAR1A/2B-expressing cells. The current-voltage relationships determined in both cell lines in the presence and absence of external Mg++ were similar to those observed with recombinant rat NMDA receptors. Power spectra calculated from NMDA/glycine-induced currents for both NMDA receptor-expressing cell lines suggested a kinetically homogeneous population of channels. Immunoprecipitation with an anti-NMDAR1A antibody coprecipitated the corresponding NMDAR2 subunit with the NMDAR1A, suggesting that heteromeric complexes are formed in these stable cell lines. Stimulation of NMDA receptors evoked an increase in intracellular Ca++, which was used to characterize their pharmacological properties. NMDA displayed less intrinsic activity than did glutamate in both NMDA receptor-expressing cell lines and was a 4-fold more potent agonist at hNMDAR1A/2B than hNMDAR1A/2A. NMDA/glycine-evoked increases in Ca++ levels were inhibited by CGS 19755, (+/-)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonate, MK-801, ketamine and ifenprodil. (+/-)-3-(2-Carboxypiperazin-4-yl)propyl-1-phosphonate was a 3-fold more potent antagonist at hNMDAR1A/2A than hNMDAR1A/2B, whereas ifenprodil was markedly more selective toward hNMDAR1A/2B, being 250-fold more potent than against hNMDAR1A/2A. These data suggest that cells stably expressing recombinant heteromeric hNMDAR1A/2A and hNMDAR1A/2B represent pharmacologically valid experimental systems to study human NMDA receptors.

Animals↗