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Biomedical subjects

D O Carpenter

Publications and source records attributed to D O Carpenter.

At least 55 records · Page 3Linked to original sources
PubMed1997

Metabotropic GABA receptors regulate acetylcholine responses on snail neurons.

1. The A type of acetylcholine response of Helix neurons is downmodulated by low concentrations of GABA that do not elicit any measurable change in membrane potential or conductance. 2. We find that these physiological actions are associated with an increase in both intracellular cyclic AMP levels and 45Ca2+ influx. 3. The modulation of the acetylcholine response by GABA is blocked when the neurons are injected with EGTA to prevent a rise in intracellular Ca2+ concentration or when tolbutamide, an inhibitor of protein kinase A, is applied. 4. These results are consistent with the effects of GABA being mediated by a metabotropic GABA receptor that is activated at very low GABA concentrations and mediates modulation of the acetylcholine response via regulation of intracellular Ca2+ and cyclic AMP levels.

Acetylcholine↗
PubMed1997

Effects of inorganic mercury and methylmercury on the ionic currents of cultured rat hippocampal neurons.

1. The effects of inorganic Hg2+ and methylmercuric chloride in the ionic currents of cultured hippocampal neurons were studied and compared. We examined the effects of acute exposure to the two forms of mercury on the properties of voltage-activated Ca2+ and Na+ currents and N-methyl-D-aspartate (NMDA)-induced currents. 2. High-voltage activated Ca2+ currents (L type) were inhibited by both compounds at low micromolar concentrations in an irreversible manner. Mercuric chloride was five times as potent as methylmercury in blocking L-channels. 3. Both compounds caused a transient increase in the low-voltage activated (T-type) currents at low concentrations (1 microM) but blocked at higher concentrations and with longer periods of time. 4. Inorganic mercury blockade was partially use dependent, but that by methylmercury was not. There was no effect of exposure of either form of mercury on the I-V characteristics of Ca2+ currents. 5. Na(+)- and NMDA-induced currents were essentially unaffected by either mercury compound, showing only a delayed nonspecific effect at a time of overall damage of the membrane. 6. We conclude that both mercury compounds show a relatively selective blockade of Ca2+ currents, but inorganic mercury is more potent than methylmercury.

Animals↗
PubMed1997

Time course and involvement of protein kinase C-mediated phosphorylation of F1/GAP-43 in area CA3 after mossy fiber stimulation.

1. Protein kinase C (PKC) activity and phosphorylation of F1/growth associated protein (GAP)-43, a PKC substrate, have been proposed to play key roles in the maintenance of long-term potentiation (LTP) at the synapses of Schaffer collateral/commissural on pyramidal neurons in CA1 (Akers et al., 1986). We have studied in the involvement of PKC and PKC-dependent protein phosphorylation of F1/GAP-3 in in vitro LTP observed at the synapses of mossy fiber (MF) on CA3 pyramidal neurons of rat hippocampus by post hoc in vitro phosphorylation. 2. After LTP was induced in CA3 in either the presence or absence of D-2-amino-5-phosphonovaleric acid (AP5), an NMDA receptor antagonist, the CA3 region was dissected for in vitro phosphorylation assay. In vivo phosphorylation of F1/GAP-43 was increased in membranes at 1 and 5 min after tetanic stimulation (TS) but not at 60 min after TS. 3. The degree of phosphorylation of F1/GAP-43 in the cytosol was inversely related to that in membranes at each time point after LTP. 4. The similar biochemical changes obtained from either control slices or AP5-treated slices indicate that LTP and the underlying biochemical changes are independent of the NMDA receptor. Immunoreactivity of the phosphorylated F1/GAP-43 in LTP slices was not significantly different from control, indicating that results from western blotting and post hoc in vitro phosphorylation are consistent. 5. Post hoc in vitro phosphorylation of F1/GAP-43 was PKC-mediated since phosphorylation of F1/GAP-43 was altered by the PKC activation cofactors, Ca2+, phosphatidylserine and phorbol ester. 6. Calmodulin (CaM) at > 5 microM inhibited phosphorylation, consistent with the presence of CaM-binding activity at the site on F1/GAP-43 acted upon by PKC. 7. We conclude that phosphorylation of F1/GAP-43 is associated with the induction but not the maintenance phase of MF-CA3 LTP.

2-Amino-5-phosphonovalerate↗
PubMed1997

Biochemical and physiological evidence that carnosine is an endogenous neuroprotector against free radicals.

1. Carnosine, anserine, and homocarnosine are endogenous dipeptides concentrated in brain and muscle whose biological functions remain in doubt. 2. We have tested the hypothesis that these compounds function as endogenous protective substances against molecular and cellular damage from free radicals, using two isolated enzyme systems and two models of ischemic brain injury. Carnosine and homocarnosine are both effective in activating brain Na, K-ATPase measured under optimal conditions and in reducing the loss of its activity caused by incubation with hydrogen peroxide. 3. In contrast, all three endogenous dipeptides cause a reduction in the activity of brain tyrosine hydroxylase, an enzyme activated by free radicals. In hippocampal brain slices subjected to ischemia, carnosine increased the time to loss of excitability. 4. In in vivo experiments on rats under experimental hypobaric hypoxia, carnosine increased the time to loss of ability to stand and breath and decreased the time to recovery. 5. These actions are explicable by effects of carnosine and related compounds which neutralize free radicals, particularly hydroxyl radicals. In all experiments the effective concentration of carnosine was comparable to or lower than those found in brain. These observations provide further support for the conclusion that protection against free radical damage is a major role of carnosine, anserine, and homocarnosine.

Animals↗
PubMed1997

Flow cytometric measurements of neuronal death triggered by PCBs.

We have examined the effects of several individual polychlorinated biphenyl (PCB) congeners and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on viability and intracellular calcium concentration in acutely dissociated cerebellar granule cell neurons obtained from 7-14 day old rat pups, using a flow cytometer. Cell death was determined using the fluorescent dye, 7-aminoactinomycin-D (7-AAD), while intracellular calcium concentration was determined using Fluo-3-AM. When acutely isolated neurons were exposed to 2,4,4' trichlorobiphenyl (2,4,4', there was a dose- and time-dependent loss of viability beginning within 5 min. At a concentration of 100 microM, 80% of neurons were dead within 45 min. Loss of viability was preceded by an apparent increase in cell granularity. No significant cell death was observed upon exposure to the highly dioxin-like coplanar PCB, 3,4,5,3',4', at a concentration of 100 microM or by TCDD at a concentration of 0.1 microM. A slight loss of viability was seen with the ortho 2,6,2',6' and the coplanar 3,4,3',4', but for both it was small. All of the PCB congeners caused an early, transient increase in intracellular free calcium concentration, although TCDD did not. A small, late increase in intracellular free calcium was seen with 2,4,4', probably reflecting events leading to loss of viability. We conclude that at least one PCB congener, 2,4,4', is very neurotoxic and can cause rapid death of cerebellar granule cells.

Animals↗
PubMed1996

The translocation and involvement of protein kinase C in mossy fiber-CA3 long-term potentiation in hippocampus of the rat brain.

The detailed mechanisms underlying long-term potentiation (LTP) are not known. In hippocampal CA1, translocation of protein kinase C (PKC) activity from cytosol to membrane and subsequent phosphorylation of growth associated protein (GAP)-43 have been demonstrated to be critical events for the maintenance phase of LTP. LTP in mossy fiber (MF)-CA3 pathway and the Schaffer collateral/commissural (SC)-CA1 pathway differ in a number of ways: SC-CA1 LTP depends on NMDA receptors while MF-CA3 LTP does not, and SC-CA1 LTP is primarily postsynaptic while MF-CA3 LTP is primarily presynaptic. The role of PKC in MF-CA3 LTP has not been studied. We investigated the role of PKC in CA3 and show that PKC inhibitors prevent LTP, but that PKC activators produce a reversible synaptic potentiation, indicating that PKC activation is an essential but not sufficient component of LTP in CA3. Then using antibodies against specific PKC isozymes we have determined the membrane vs. cytosolic distribution of various PKC isozymes in slices subjected to low or tetanic stimulation, or perfused with phorbol esters (PDAc). Compared with control, LTP and PDAc slices show greater PKC-alpha and -epsilon immunoreactivity in the membrane fraction, indicating that both LTP and phorbol ester treatment induce translocation of PKC-alpha and -epsilon from cytosol to membrane. However, with PKC-beta and PKC-gamma the only detectable translocation from cytosol to membrane was in the phorbol ester-treated slices. Thus, while phorbol ester treatment causes translocation of PKC-alpha, -beta, -gamma and -epsilon, the only detectable translocation associated with CA3 LTP is that of PKC-alpha and -epsilon.

Analysis of Variance↗
PubMed1996

Flow-cytometric estimation on glutamate- and kainate-induced increases in intracellular Ca2+ of brain neurons: a technical aspect.

Effects of glutamate and kainate on the intracellular Ca2+ concentration ([Ca2+]i) in a large population (several thousand) of dissociated cerebellar granule cell neurons were evaluated using a flow-cytometer and a combination of two fluorescent dyes, fluo-3-AM for estimating [Ca2+]i and ethidium bromide for removing neurons that had compromised membranes from the cell population examined. The number of neurons responding to glutamate or kainate in augmenting the fluo-3 fluorescence increased in a dose-dependent manner. The number of neurons responding to kainate was much greater than that to glutamate. CNQX, a blocker of non-NMDA receptors, completely blocked the response elicited by kainate while the complete blockade of this glutamate-induced response was made by a combination of MK-801, a NMDA receptor blocker, and CNQX. Nicardipine, a calcium antagonist, decreased the number of neurons responding to glutamate and kainate, suggesting involvement of voltage-dependent calcium channels. These results indicate that the flow-cytometric measurement of glutamate and kainate responses has the potential to provide answers to such questions as what percentage of the population of neurons respond to these amino acids and what is the resulting distribution of [Ca2+]i.

Aniline Compounds↗
PubMed1996

Propylthiouracil treatment reduces long-term potentiation in area CA1 of neonatal rat hippocampus.

Rat pups were made hypothyroid by exposure to propylthiouracil in drinking water beginning at 1 week of age, and the degree of long-term potentiation (LTP) in hippocampal area CA1 determined from brain slices of animals ranging in age from 2 to 6 weeks. Serum T3 levels were less than 20% of that of age matched controls after 3 weeks of treatment, and remained at that level. Relative to the age-matched controls, LTP was reduced significantly after 2 weeks of treatment. These observations are consistent with the conclusion that LTP magnitude is a reflection of cognitive function, which is known to be depressed in hypothyroid conditions in both animals and man.

Administration, Oral↗
PubMed1996

Low power laser irradiation reduces ischemic damage in hippocampal slices in vitro.

BACKGROUND AND OBJECTIVE: Low power laser irradiation has been reported to reduce injury, promote regeneration, and produce analgesia. While the mechanism is unknown, one hypothesis is that light produces free radicals, which have a beneficial effect at low concentrations. STUDY DESIGN/MATERIALS AND METHODS: We have investigated the effects of low power laser irradiation on the loss of electrical excitability of hippocampal brain slices after a transient exposure to a perfusion medium lacking oxygen and containing reduced glucose concentrations. Injury in this system is known to result at least in part from free radical production. RESULTS: Low power laser irradiation increased the time required for loss of excitability and increased recovery from the ischemic injury. CONCLUSIONS: Low power laser irradiation has acute protective effects against ischemic damage in brain slices.

Animals↗
PubMed1996

Interactions among paired-pulse facilitation and post-tetanic and long-term potentiation in the mossy fiber-CA3 pathway in rat hippocampus.

Long-term potentiation (LTP) observed at the synapses of mossy fiber-CA3 (MF-CA3) pathway differs from that observed at the Schaffer collateral-CA1 pathway (SC-CA1), in being independent of N-methyl-D-aspartate (NMDA) receptors. The induction and expression mechanisms of MF-CA3 LTP remain to be determined. We have compared the occurrence and magnitude of LTP with that of two other indicators of presynaptic plasticity, post-tetanic potentiation (PTP) and paired-pulse facilitation in control brain slices from young rats and in slices treated with phorbol-12, 13-diacetate (PDAc), a protein kinase C activator. Paired-pulse facilitation is a potentiation of the second of two responses at intervals of tens of milliseconds and is due to a presynaptic calcium increase. Tetanic stimulation of mossy fibers induced LTP is area CA3 in only 64% of slices. In those slices that showed LTP, the size of the PTP was significantly greater than in those slices that did not, and the degree of correlation between LTP and PTP amplitude overall was r = 0.7. The degree of paired-pulse facilitation before tetanic stimulation was also positively correlated to the occurrence and magnitude of LTP and PTP after tetanic stimulation. The correlation coefficient between PTP and PPF was 0.749 for all slices studied, while that between LTP and PPF was 0.835 overall. Application of PDAc potentiated synaptic transmission and abolished paired-pulse facilitation (control ratio of second to first response, 2.1; after PDAc ratio 0.8) and LTP. PTP was absent at the control stimulus intensity in PDAc, but was apparent if the stimulus intensity was reduced to give a response of the same amplitude as before administration of PDAc. Stable LTP was also accompanied by a marked decrease in paired-pulse facilitation. These data suggest that MF-CA3 LTP, PTP and paired-pulse facilitation share common mechanisms and are all at least primarily of presynaptic origin. The occurrence of large paired-pulse facilitation or PTP is a predictor of a preparation which will show LTP. It is likely that presynaptic [Ca2+]i is an essential factor in LTP, PTP and paired-pulse facilitation, as well as the potentiation induced by application of PDAc, but the factors which determine whether or not [Ca2+]i rises following these various stimuli are not clear from the techniques used in these investigations.

Animals↗
PubMed1996

Mitochondrial oxidation in rat hippocampus can be preconditioned by selective chemical inhibition of succinic dehydrogenase.

Repeatedly it was reported that a short ischemic episode may ameliorate biochemical and morphological impairment upon succeeding severe ischemia. We investigated whether the pattern of respiratory enzyme activity (RA), adenine nucleotides, and membrane potential in hippocampal slices following low-dose in vivo (20 mg/kg) and high-dose in vitro (1 mM) application of 3-nitropropionic acid (3-np), a specific inhibitor of succinic dehydrogenase (SDH), indicates a similar tolerance phenomenon. One hour in vivo treatment decreased RA, spectrophotometrically quantitated by intensity of staining with 2,3,5-triphenyltetrazolium chloride (TTC), to 48 +/- 5% (mean +/- SE; P<0.01). Intermittent increase after 2 h (79 +/- 5%; P<0.05) was followed by gradual decline to 48 +/- 16% (P<0.01) after 8 h. The intermittent increase predominated in stratum pyramidale of hippocampal region CA1 (CA1sp) vs CA3 (CA3sp) (89 +/- 6% vs 57 +/- 6% of control; P <0.01). ATP levels paralleled the intensity of average (CA1sp, CA3sp, plus CA1 stratum radiatum) TTC staining (r=0.93). After pretreatment of 3-np in vivo for 1 h, no further decrease of RA upon 30-min in vitro treatment was seen in any region. At all other times, RA declined further upon in vitro treatment (P<0.01). Compared to 1-h in vivo treatment, hyperpolarization of CA1sp pyramidal cells upon in vitro application of 1 mM 3-np was reduced after 8-h pretreatment in vivo (P<0.04). At this time, depolarization upon glibenclamide (10 muM), an antagonist at KATP-channels, was reduced. We conclude that the severity of impairment of oxidative phosphorylation upon repeated inhibition of SDH in vivo and in vitro is not increased in an additive manner. At appropriate times, relative protection against further decrease of energy metabolism is observed-chemical preconditioning. Activation of KATP-channels is associated with chemical preconditioning.

Adenine Nucleotides↗
PubMed1996

Mercury (Hg 2+) enhances the depressant effect of kainate on Ca-inactivated potassium current in telencephalic cells derived from chick embryos.

The effect of HgCl2 on kainate (KA)-induced depression of voltage-gated potassium (K+) current in chick embryo telencephalic cells was studied using conventional and nystatin-perforated whole-cell patch-clamp recordings, fluorescence imaging, and flow cytometry techniques. Hg2+ (1 microM) alone did not effect the 4-aminopyridine-(4-AP)-sensitive transient K+ current in immature cells (Embryonic Day 5), but irreversibly potentiated the depressant effect of KA on this K+ current. A 50% potentiation of KA-induced depression of the K+ current was produced by an application of 0.19 microM Hg2+. Application of ionomycin (5 microM) or calcium ionophore A23187 (2 microM) suppressed the K+ current. To test the possibility that the 4-AP-sensitive transient K+ current is a Ca-inactivated current, the effect of intracellular Ca2+ concentration ([Ca2+]i) in the range of 30 nM to 2 microM was determined. The amplitude of the K+ current was sensitive to [Ca2+]i with half-maximal inactivation at 370 nm at +60 mV. The concentration-response curve of the K+ current inhibition by [Ca2+]i was shifted to lower [Ca2+]i and the slope of the curve was reduced in the presence of KA. Hg2+ potentiated these effects of KA. The Ca-dependence of the K+ current was maximal at the 5th embryonic day, declined to the 9th embryonic day, and was absent at the 11th embryonic day. Application of Hg2+ (0.1-1 microM) had no effect on the basal [Ca2+]i of freshly dissociated cells (10th day in ovo) and cells in culture (the 4-day cultures from the telencephalon of 5-day-old embryos), but potentiated KA-induced increase of [Ca2+]i in a Ca-free-EGTA solution in a concentration-dependent manner. Moreover 1 microM Hg2+ delayed and reduced the recovery to basal [Ca2+]i after washout of KA. Exposure to 5-30 microM H2+ caused an irreversible decline of membrane resistance, an increased cell size, and reduced cell granularity and complexity. Intracellular recording of spontaneous neuronal activity and immunocytochemical identification showed that the KA/Hg2+-sensitive Ca-inactivated K+ current exists in early differentiating telencephalic neurons. Because depression of the K+ current by KA and Hg2+ decreases the interspike interval and irreversibly perturbs the frequency code of information in the nervous system, the expression of this current during early neuroembryogenesis may be one of the reasons for the developmental toxicity of inorganic mercury.

Animals↗
PubMed1996

Low concentrations of ouabain stimulate Na/Ca exchange in neurons.

1. The effects of low concentrations of ouabain on 22Na efflux, 86Rb influx, 45Ca uptake and cyclic AMP levels were studied in snail ganglia. Ouabain, at concentrations below that which inhibits the Na-K pump as monitored by 86Rb influx, activated "reverse mode" Na/Ca exchange, as indicated by an increased 22Na efflux and 45Ca influx. 2. With electrophysiologic recordings ouabain, in the presence of K(+)-free saline to block Na/K transport, caused a membrane hyperpolarization. These concentrations of ouabain also caused elevation of intracellular cyclic AMP levels. 3. We suggest that the ouabain-induced stimulation of Na efflux is due to a stimulation of reverse Na/Ca exchange. since Na/Ca exchange is electrogenic, these observations are most consistent with ouabain stimulation of Na/Ca exchange in a reversed direction (intracellular Na for extracellular Ca). 4. The effect on Na/Ca exchange may be secondary to a rise in intracellular cyclic AMP.

Animals↗
PubMed1996

Protein kinase C activation is necessary but not sufficient for induction of long-term potentiation at the synapse of mossy fiber-CA3 in the rat hippocampus.

The involvement of protein kinase C in long-term potentiation was investigated in the mossy fiber-CA3 pathway in an in vitro slice preparation of rat hippocampus. Tetanic stimulation induced stable long-term potentiation in the mossy fiber-CA3 pathway which was not affected by N-methyl-D-aspartate receptor antagonists. Long-term potentiation was not induced in the presence of a protein kinase C inhibitor, sphingosine. Application of 1 microM phorbol-12, 13-diacetate, an activator of protein kinase C, potentiated the synaptic response by about 400% and this potentiation was completely reversible upon washing. Sphingosine blocked the potentiation when it was applied before protein kinase C activation by phorbol-12, 13-diacetate. However, sphingosine had no effect on the potentiation when it was applied after the synaptic response was potentiated to a plateau following phorbol-12,13-diacetate perfusion. Long-term potentiation and phorbol ester-induced potentiation were not additive when phorbol-12,13-diacetate was applied after induction of long-term potentiation, suggesting that long-term potentiation and phorbol-12, 13-diacetate activate the same protein kinase C pool. The enhanced response caused by phorbol-12,13-diacetate returned to the long-term potentiation level after wash-out of phorbol-12,13-diacetate. Thus the cellular changes underlying long-term potentiation are long-lasting or permanent, while those caused by phorbol-12,13-diacetate are not. However, if tetanic stimulation was induced during prolonged phorbol-12,13-diacetate application (1 h), a potentiation similar in amplitude to long-term potentiation was induced but the population response returned to the control pre-long-term potentiation level after 2 h of washing. The potentiation following tetanic stimulation during prolonged application of phorbol-12,13-diacetate was blocked in the presence of D-2-amino-5-phosphonovaleric acid, a N-methyl-D-aspartate receptor antagonist. Thus, in the presence of phorbol esters the N-methyl-D-aspartate-independent long-term potentiation is occluded but a transient potentiation appears, presumably due to hyperexcitability and activation of N-methyl-D-aspartate receptors in recurrent pathways of area CA3. Normal N-methyl-D-aspartate-independent long-term potentiation could be induced after the 2 h washout period and now was maintained. In conclusion, protein kinase C activation is essential but not sufficient for long-term potentiation in the mossy fiber-CA3 pathway and when stimulated by application of phorbol esters produces a large and reversible synaptic potentiation. These investigations show that long-term potentiation in CA3 is a complex event involving several steps, and that activation of protein kinase C is only one of them.

2-Amino-5-phosphonovalerate↗
PubMed1996

Optimizing professional education in public health.

Traditional public health education has lacked required practicums such as are the rule in other health professions. Described is a model professional education program that is built around required course work in all areas of public health and a series of mandatory internships at sites of public health practice, utilizing state and local health departments, other federal and state agencies, and public and private sites where students are supervised in public health rotations similar to those a medical, dental, nursing, or veterinary student would experience in a clinic or ward.

Curriculum↗
PubMed1996

Hazardous wastes in Eastern and Central Europe [meeting report].

The countries of Eastern and Central Europe have emerged from a political system which for decades has ignored protection of human health from hazardous wastes. While the economies of the countries in this region are stretched, awareness and concern about hazardous waste issues are a part of the new realities. At a recent conference sponsored in part by the National Institute of Environmental Health Sciences, representatives of seven countries in the region described the status of hazardous waste programs, issues of major concern, and steps being taken to protect human health. This report summarizes the deliberations, outlines some of the problems remaining in dealing with the legacy of the past, addressing the problems of the present, and providing a framework for future research and collaborative efforts.

Environmental Pollutants↗