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R J Miller

Publications and source records attributed to R J Miller.

At least 109 records · Page 6Linked to original sources
PubMed1996

Leptin, the obese gene product, rapidly modulates synaptic transmission in the hypothalamus.

We examined the effects of leptin, the product of the obese gene, on synaptic transmission in the arcuate nucleus in rat hypothalamic slices. Both leptin and neuropeptide Y (NPY) reduced the evoked glutamatergic excitatory postsynaptic current in the arcuate nucleus. NPY also depressed the GABAergic inhibitory postsynaptic current, although leptin had no effect. Leptin also decreased the input resistance of arcuate neurons, and this was accompanied by the activation of an outward current at depolarized potentials. Leptin modulated Ca2+ signals in acutely isolated arcuate neurons. In some cells, the intracellular calcium concentration rise produced by 50 mM K+ was decreased, whereas in others it was increased. However, leptin produced no effects on synaptic transmission and little or no effect on Ca2+ signaling in the hypothalamus of Zucker fatty rats that contain mutated leptin receptors. On the other hand, NPY exhibited synaptic modulatory effects in Zucker lean and fatty rats. These data suggest that leptin can produce rapid synaptic modulatory effects in the arcuate nucleus, which may contribute to its effects on food intake.

Animals↗
PubMed1996

Isoform expression of the voltage-dependent calcium channel alpha 1E.

The alpha 1E voltage-dependent calcium channel has not been clearly identified with a specific neuronal calcium current. To help identify the role of alpha 1E, we examined differential expression of alpha 1E splice variants in mouse brain and cultured cell lines and examined the gene structure of the region encoding the amino terminal. Three splice variants were analyzed by a ribonuclease protection assay, and a fourth variant reported previously in a fetal human alpha 1E sequence was also detected in mouse brain and a pituitary cell line. Whole brain, telencephalon, and olfactory bulb contained predominantly the splice variant corresponding to alpha 1E-1 although other known variants could be detected. Neuroendocrine cells in vitro (beta TC3 insulinoma cells and AtT-20 pituitary cell lines) expressed predominantly one alpha 1E isoform. The existence of a 5' exon accounting for the origin of variant 5' ends reported in different species was suggested by the sequence of the mouse alpha 1E gene in the region encoding the amino terminal.

Alternative Splicing↗
PubMed1995

Functional expression of an epitope-tagged G protein-coupled K+ channel (GIRK1).

An epitope-tagged form of an inwardly rectifying and G protein-coupled K+ channel (GIRK1-cp) was expressed at high levels in transfected mammalian cells. Immunoblot analysis of transfected human embryonic kidney cells (HEK293) and mouse insulinoma cells (beta TC3) revealed several GIRK1-cp polypeptides, including the major 59-kDa band, corresponding to the predicted mass of the GIRK1 polypeptide plus the epitope tag. Immunohistochemical staining using two anti-tag antibodies showed abundant immunoreactive material, which was predominantly concentrated in the perinuclear area in both transfected cell types. While functional GIRK1-cp message was present in poly(A)+ RNA prepared from HEK293 cells expressing GIRK1-cp protein, appropriate K+ currents could not be detected. In contrast, whole cell recordings made directly from transfected beta TC3 cells expressing GIRK1-cp revealed inwardly rectifying, pertussis toxin-sensitive currents activated by norepinephrine and galanin. Single channel recordings in excised patches of beta TC3 cells expressing GIRK1-cp showed rectifying K+ currents when activated by 50 microM guanosine 5'-O-(thiotriphosphate), with a slope conductance of 39.1 +/- 1.0 picosiemens. This is the first report of stable heterologous expression of a functional G protein-coupled K+ channel in mammalian cells. The activity of an epitope-tagged channel in insulinoma cells demonstrates the utility of this system for further biochemical and biophysical analyses of G protein-K+ channel interactions.

Animals↗
PubMed1995

Regulation of excitatory transmission at hippocampal synapses by calbindin D28k.

Distinct subpopulations of neurons in the brain contain one or more of the Ca(2+)-binding proteins calbindin D28k, calretinin, and parvalbumin. Although it has been shown that these high-affinity Ca(2+)-binding proteins can increase neuronal Ca2+ buffering capacity, it is not clear which aspects of neuronal physiology they normally regulate. To investigate this problem, we used a recently developed method for expressing calbindin D28k in the somatic and synaptic regions of cultured hippocampal pyramidal neurons. Ninety-six hours after infection with a replication-defective adenovirus containing the calbindin D28k gene, essentially all cultured hippocampal pyramidal neurons robustly expressed calbindin D28k. Our results demonstrate that while calbindin D28k does not alter evoked neurotransmitter release at excitatory pyramidal cell synapses, this protein has a profound effect on synaptic plasticity. In particular, we show that calbindin D28k expression suppresses posttetanic potentiation.

Animals↗
PubMed1995

Calcium and sodium currents evoked by action potential waveforms in rat sympathetic neurones.

1. Calcium channel currents evoked by action potential waveforms were recorded from rat sympathetic neurones using the whole-cell variant of the patch-clamp technique. A voltage template was created in which the unmodified action potential (AP) was played back followed by a rectangular pulse. 2. The inhibitory effects of noradrenaline (NA) and neuropeptide Y (NPY) on the AP and rectangular pulse-evoked currents, were compared. The percentage inhibition of the Ca2+ current produced by NA and NPY was significantly greater in the case of currents evoked using AP waveforms. 3. A train of APs was applied in the voltage command (40 or 75 Hz) and the inhibition produced by NPY on the AP-evoked Ca2+ currents was examined. The inhibitory effect of NPY on the Ca2+ currents evoked by the high frequency APs did not change significantly during the train. 4. The AP falling phase was artificially prolonged and the resulting Ca2+ currents were compared. AP prolongation increased the amount of Ca2+ entering into the cell. However, the peak value of the Ca2+ current was primarily determined by the AP height. The AP plateau phase was also prolonged in defined voltage ranges. Prolongation in the positive voltage region was most effective in increasing the Ca2+ current. 5. Ion substitution studies were used to isolate Na+ and Ca2+ currents evoked by AP waveforms. The inhibitory effects of NA and oxotremorine (OXO-M) on Ca2+ currents evoked by AP waveforms were examined in the presence and absence of the Na+ current.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗
PubMed1995

The Ca2+ influx induced by beta-amyloid peptide 25-35 in cultured hippocampal neurons results from network excitation.

Although a neurotoxic role has been postulated for the beta-amyloid protein (beta AP), which accumulates in brain tissues in Alzheimer's disease, a precise mechanism underlying this toxicity has not been identified. The peptide fragment consisting of amino acid residues 25 through 35 (beta AP25-35), in particular, has been reported to be toxic in cultured neurons. We report that beta AP25-35, applied to rat hippocampal neurons in culture, caused reversible and repeatable increases in the intracellular Ca2+ concentration ([Ca2+]i), as measured by fura 2 fluorimetry. Furthermore, beta AP25-35 induced bursts of excitatory potentials and action potential firing in individual neurons studied with whole cell current clamp recordings. The beta AP25-35-induced [Ca2+]i elevations and electrical activity were enhanced by removal of extracellular Mg2+, and they could be blocked by tetrodotoxin, by non-N-methyl-D-aspartate (NMDA) and NMDA glutamate receptor antagonists, and by the L-type Ca2+ channel antagonist nimodipine. Similar responses of bursts of action potentials and [Ca2+]i increases were evoked by beta AP1-40. Responses to beta AP25-35 were not prevented by pretreatment with pertussis toxin. Excitatory responses and [Ca2+]i elevations were not observed in cerebellar neuron cultures in which inhibitory synapses predominate. Although the effects of beta AP25-35 depended on the activation of glutamatergic synapses, there was no enhancement of kainate- or NMDA-induced currents by beta AP25-35 in voltage-clamp studies. We conclude that beta AP25-35 enhances excitatory activity in glutamatergic synaptic networks, causing excitatory potentials and Ca2+ influx. This property may explain the toxicity of beta AP25-35.

Amyloid beta-Peptides↗
PubMed1995

Presynaptic metabotropic glutamate receptors modulate omega-conotoxin-GVIA-insensitive calcium channels in the rat medulla.

We have previously demonstrated that the metabotropic glutamate receptor (mGluR) agonist (1S,3R)-1 aminocyclopentane-1,3-dicarboxylate (ACPD) presynaptically inhibits evoked glutamatergic EPSCs and GABAergic IPSCs in patch clamped rat nucleus tractus solitarius (NTS) neurons recorded in this slices. The present study investigated the pharmacology of the presynaptic mGluRs, the the voltage dependent Ca2+ channel (VDCC) subtypes supporting neurotransmitter release, and possible interactions between the two. Monosynaptic EPSCs or IPSCs were evoked by electrical stimulation in the region of the tractus solitarius (TS). The effects of the mGluR agonists ACPD, (2S,3S,4S)-alpha-(carboxycyclopropyl)glycine (L-CCG-I) and L-2-amino-4-phosphonobutyrate (AP4) were examined upon EPSCs. The effects of the above compounds and quisqualate (QUIS) were examined upon IPSCs. L-CCG-I proved the most potent inhibitor of EPSCs and IPSCs. The VDCC blockers omega-AGA-IVA (AGA), omega-conotoxin GVIA (GVIA), omega-conotoxin MVIIC (MVIIC) and nimodipine (NIM) were assessed for their ability to inhibit monosynaptic EPSCs and IPSCs. EPSCs were inhibited by GVIA >> AGA > or = MVIIC. IPSCs were inhibited by AGA > or = MVIIC >> GVIA. NIM was without effect on the EPSC or IPSC. The potency of mGluR inhibition of evoked synaptic transmission was assessed in the absence and following treatment with VDCC blockers. mGluR agonists blocked a greater percentage of the EPSC or IPSC following treatment with GVIA, but not the other VDCC antagonists, than under control conditions. We have previously demonstrated that the postsynaptic inhibitory effects of mGluR activation upon GABAA mediated currents can be mimicked by cyclic guanosine monophosphate (cGMP) analogs. The cGMP-dependent protein kinase (PKG) inhibitors H8 and Rp-8-4-chlorophenylthio-guanosine-3',5'-cyclic monophosphorothioate (Rp-cG) blocked mGluR inhibition of GABAA mediated currents without blocking the ability of mGluR agonists to inhibit the IPSC. The effect of L-CCGI was enhanced following treatment with GVIA in the presence of Rp-cG, confirming a presynaptic locus of mGluR mediated inhibition of the IPSC. In contrast, cGMP analogues potentiate postsynaptic responses to glutamate agonists but depress the EPSC. As with the mGluR agonists, the inhibition of the EPSC by cGMP was potentiated following treatment with GVIA. These results suggest that presynaptic mGluR reduce both glutamate release from afferent fibers and GABA release from inhibitory interneurons following electrical stimulation in the region of the TS. Although different VDCCs support the majority of glutamate and GABA release and mGluR effects on release appear to utilize differing intracellular pathways, presynaptic GVIA-insensitive VDCCs are favorably targeted for inhibition by mGluR agonists.

Animals↗
PubMed1995

Characteristics of a human N-type calcium channel expressed in HEK293 cells.

The human alpha 1B-1 alpha 2b beta 1-2 Ca2+ channel was stably expressed in HEK293 cells producing a human brain N-type voltage-dependent calcium channel (VDCC). Whole cell voltage-clamp electrophysiology and fura-2 based microfluorimetry have been used to study its characteristics. Calcium currents (ICa) recorded in transfected HEK293 cells were activated at potentials more depolarized than -20 mV with peak currents occurring at approx + 10 mV in 5 mM extracellular CaCl2. ICa and associated rises in intracellular free calcium concentrations ([Ca2+]i) were sensitive to changes in both the [Ca2+]o and holding potential. Steady-state inactivation was half maximal at a holding potential of -60 mV. Ba2+ was a more effective charge carrier than Ca2+ through the alpha 1B-1 alpha 2b beta 1-2 Ca2+ channel and combinations of both Ba2+ and Ca2+ as charge carriers resulted in the anomalous mole fraction effect. Ca2+ influx into transfected HEK293 cells was irreversibly inhibited by omega-conotoxin-GVIA (omega-CgTx-GVIA; 10 nM-1 microM) and omega-conotoxin-MVIIA; 100 nM-1 microM) whereas 1 microM) whereas no reductions were seen with agents which block P or L-type Ca2+ channels. The inorganic ions, gadolinium (Gd3+), cadmium (Cd2+) and nickel (Ni2+) reduced the ICa under voltage-clamp conditions in a concentration-dependent manner. The order of potency of the three ions was Gd3+ > Cd2+ > Ni2+. These experiments suggest that the cloned and expressed alpha 1B-1 alpha 2b beta 1-2 Ca2+ channel subunits form channels in HEK293 cells that exhibit properties consistent with the activity of the native-N-type VDCC previously described in neurons.

Cadmium↗
PubMed1995

Capsaicin-induced neurotoxicity in cultured dorsal root ganglion neurons: involvement of calcium-activated proteases.

We examined the mechanism by which capsaicin produces its toxic effects on cultures of rat sensory neurons. Capsaicin caused a robust increase in [Ca2+]i in a subpopulation of cultured rat dorsal root ganglion neurons. Similarly, a brief exposure to capsaicin resulted in delayed degeneration of a subpopulation of the cells. This subpopulation (about 35% of the cells present) was characterized by a capsaicin-induced uptake of Co2+, which could be detected cytochemically. Both capsaicin-induced Co2+ uptake and capsaicin-induced cell death were blocked by the capsaicin antagonist Ruthenium Red. Cell death was also prevented by removal of external calcium or by inhibiting calcium-activated proteases such as calpain. Evidence that calpain activity was increased was provided by examining the amount of degradation of the preferred calpain substrate alpha-spectrin. Capsaicin treatment produced a significant increase in the levels of the 150,000 molecular weight spectrin breakdown product. Furthermore, applying the protease inhibitors E64 or MDL 28,170 reduced capsaicin-mediated cell death. It is concluded that capsaicin kills a subpopulation of sensory neurons by activating a receptor-operated channel. The consequent Ca2+ ion influx causes large increases in [Ca2+]i and subsequent activation of Ca(2+)-sensitive proteases. This model provides support for the role of [Ca2+]i as the orchestrator of delayed neuronal degeneration.

Animals↗
PubMed1995

Urethral warming catheter for use during cryoablation of the prostate.

Cryoablation of the prostate has been shown to be a useful tool in the eradication of prostate cancer. Sloughing of necrotic tissue per urethra was one of the major complications of prostate cryosurgery during its initial application and led to its disuse by urologists. A catheter was then needed until sloughing was complete and the patient again able to void. In a previous report, we have demonstrated that thermal protection of the urethra could be obtained by flowing warm irrigant through a thin-walled urethral conduit. To simplify the application of this heat, a dual lumen urethral catheter has been designed that can be passed transurethrally to circulate warm saline and prevent the destruction of the urethral epithelium. This prevents transmural necrosis, maintaining the epithelial barrier for containment of necrotic prostate tissue after treatment. This urethral warming device is made of materials commonly found in operative stockrooms. To date, the device has functioned well with a urethral sloughing rate of 12% during its initial use.

Cryosurgery↗
PubMed1995

An initial experience with a posterior intrahepatic approach for liver resections.

This is a report of an initial experience using a recently devised posterior approach to the intrahepatic Glissonian sheaths of the liver, for the purpose of hepatic resections. Between February 1991 and October 1993, 22 patients, median age 58 years (range 36-77) underwent either a right or a left hepatectomy or a segmentectomy procedure of the liver using this technique. Seventeen of these patients underwent this operation for malignant disease of the liver. Median operating time was 230 min (range 100-285) with nine of the 22 patients requiring an intra-operative blood transfusion. There was one postoperative death, from liver failure, and 17 of the patients were alive at the early time of reporting. It is concluded that this technique is a useful addition to the surgeon's armamentarium for operating on the liver.

Adult↗
PubMed1995

Triangular fibrocartilage complex: normal appearance on coronal three-dimensional gradient-recalled-echo MR images.

PURPOSE: To evaluate the usefulness of three-dimensional gradient-recalled-echo (GRE) sequences in demonstration of the triangular fibrocartilage complex (TFCC). MATERIALS AND METHODS: Appearance of the TFCC in wrists of 11 cadavers, 17 patients, and six volunteers at three-dimensional GRE magnetic resonance (MR) imaging was evaluated and compared with that at dissection. RESULTS: MR appearance of the TFCC was similar for all wrists. Volar and dorsal radioulnar ligaments were identified. The TFCC had two types of attachments to the ulna: The more common consisted of two striated attachments, one to the tip and the other to the base of the styloid; the less common consisted of a broad-based striated attachment along the styloid. MR appearance of the ulnotriquetral and ulnolunate ligaments and meniscus homologue varied but correlated with observations at dissection. CONCLUSION: The three-dimensional GRE sequence consistently demonstrated most TFCC components. Dissection helped confirm MR-demonstrable variability in the ulnar aspect of the TFCC.

Adult↗
PubMed1995

Increase in [Ca2+]i by CCh in adult rat sympathetic neurons are not dependent on intracellular Ca2+ pools.

We have examined the effects of the muscarinic agonists, carbachol (CCh) and oxotremorine (Oxo), on the intracellular free Ca2+ concentration ([Ca2+]i) in acutely dissociated sympathetic neurons from adult rats using fura 2-based microfluorometry. The drugs increased [Ca2+]i by 86 +/- 7 and 38 +/- 10 nM for CCh and Oxo, respectively (both 10 microM). Basal [Ca2+]i was 52 +/- 3 nM. Depletion of the caffeine-sensitive Ca2+ store or blockade of the Ca(2+)-adenosinetriphosphatase with thapsigargin did not alter the effect of either agonist on the rise in [Ca2+]i. On the other hand, the omission of Ca2+ from the perfusion solution or the use of TA-3090, a Ca2+ channel antagonist, blocked the effects of CCh and Oxo. In whole cell current-clamp recordings, the muscarinic agonists elicited a depolarization and action potential firing, which probably explained the rise in [Ca2+]i observed with microfluorimetric recording. In addition to their direct effects on the [Ca2+]i, muscarinic agonists also reduced the rise in [Ca2+]i induced by a nicotinic agonist. This inhibitory effect, observed in 68% of cells that responded to the nicotinic agonist, was blocked by atropine and pertussis toxin, whereas the muscarinic agonist-induced increase in [Ca2+]i was blocked by atropine but was pertussis toxin insensitive. These results suggest that at least two muscarinic receptors are present on sympathetic neurons and that they mediate opposite effect on the fluctuation of [Ca2+]i.

Animals↗
PubMed1995

Mechanisms of hemolysate-induced [Ca2+]i elevation in cerebral smooth muscle cells.

The effects of hemolysate on free cytosolic [Ca2+] ([Ca2+]i) homeostasis were studied in freshly isolated rat basilar artery smooth muscle cells using fura 2 and dual excitation wavelength microfluorimetry. Hemolysate reversibly produced a transient [Ca2+]i peak followed by a slowly decaying plateau which was absent in Ca(2+)-free solution. This effect of hemolysate was attenuated by 1) the sarcoplasmic reticulum Ca2+ pump inhibitors thapsigargin and cyclopiazonic acid, 2) the Ca2+ release-blocking agents ryanodine and dantrolene, 3) the cytochrome P-450 inhibitor econazole, and 4) the inorganic Ca2+ channel blocker lanthanum but was not significantly attenuated by 1) the receptor-regulated Ca2+ channel blocker SKF-96365 or 2) the voltage-dependent Ca2+ channel blocker nimodipine. Fractionation of hemolysate using membranes with specific pore sizes (0.5, 1, and 12-14 kDa) indicated that a component(s) > 0.5 but < 1 kDa could produce a similar [Ca2+]i peak and plateau while fractions > 1 and > 12-14 kDa produced a small and slow [Ca2+]i rise without a significant peak. ATP, which was found in hemolysate, produced a [Ca2+]i response similar to that of hemolysate. P2-purinoceptor antagonists significantly attenuated the effect of ATP, hemolysate, and the fractions < 1 and < 12-14 kDa. We conclude that hemolysate elevates [Ca2+]i by both releasing Ca2+ from internal stores and triggering Ca2+ entry, possibly from a voltage-independent Ca2+ influx pathway, an effect apparently identical to that of ATP.

2,3-Diphosphoglycerate↗
PubMed1995

Delayed antagonism of calpain reduces excitotoxicity in cultured neurons.

BACKGROUND AND PURPOSE: Glutamate receptor antagonists can produce protection against the neurotoxicity of excessive glutamate stimulation. However, antagonism of the postreceptor processes that produce cell damage may provide a longer window of opportunity for protecting neurons after the initiation of excitotoxic injury. Among various processes that have been thought to mediate the toxic effects of glutamate are activation of the Ca(2+)-dependent proteases calpain I and II and the activation of nitric oxide synthase. We tested the potential for neuroprotection by delayed application of calpain antagonists after excitotoxic treatment. METHODS: Primary cultures of cerebellar and hippocampal neurons were exposed to the glutamate receptor agonists kainate and N-methyl-D-aspartate (NMDA) for 20-minute periods, and survival was examined by fluorescent assay after 24 hours. Enzyme antagonists were applied at various time points during this interval. RESULTS: The neurotoxic effects of NMDA in cultured hippocampal neurons and of kainate in cultured cerebellar neurons have been previously shown to be Ca2+ dependent. Here we show that in both of these examples of glutamate receptor-mediated toxicity, activation of a calpainlike proteolytic activity occurred, which was blocked by the calpain inhibitor MDL-28170. This inhibitor also limited the toxicity, even when applied at times up to 1 hour after the onset of the toxic exposure. Another protease inhibitor, E-64, also blocked the proteolysis and toxicity produced by kainate in cerebellar neurons. Blocking nitric oxide synthase activity after 1 hour with the antagonist NG-nitro-L-arginine was also protective of cerebellar and hippocampal neurons, as was the combination of MDL-28170 and NG-nitro-L-arginine. CONCLUSIONS: The activation of calpain is among several enzymatic processes that contribute to the toxicity of glutamate receptor stimulation, and blocking these postreceptor mechanisms can be effective in protecting neurons from excitotoxicity at delayed time points.

Amino Acid Oxidoreductases↗