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R J Miller

Publications and source records attributed to R J Miller.

At least 217 records · Page 12Linked to original sources
PubMed1990

Nitrendipine and omega-conotoxin modulate gonadotropin release and gonadotrope [Ca2+]i.

We have examined the pharmacology of the voltage-sensitive Ca2+ channels (VSCCs) that mediate gonadotropin secretion from primary cultures of rat pituitary cells, stimulated by either cell depolarization or by binding of gonadotropin-releasing hormone (GnRH). We also measured single-cell [Ca2+]i transients using fura-2 in gonadotropes identified by a reverse hemolytic plaque assay employing an antiserum to luteinizing hormone (LH). Cell depolarization evoked by either 50 mM K+ or 30 microM veratridine induced 2- to 6-fold increases in gonadotropin secretion over basal levels. GnRH caused 6- to 20-fold increases in follicle-stimulating hormone (FSH) and LH secretion, respectively, with maximal stimulation at 100 nM GnRH. K(+)- or GnRH-induced FSH release was largely prevented by co-incubation with 1 mM CdCl. Tetrodotoxin (TTX, 5 microM) prevented the veratridine-, but not the K(+)- or GnRH-induced, stimulation of FSH secretion. Nitrendipine (Ntd, 1 microM) produced 35-50% inhibition (NS) of both FSH and LH release stimulated by either 50 mM K+ or 100 nM GnRH. Ntd also inhibited the K(+)-induced [Ca2+]i rise (greater than 90%), as well as the secondary, plateau phase of the GnRH-induced elevation of [Ca2+]i (100% inhibition). Omega-conotoxin (omega-CgTx, 100 nM) partially suppressed FSH and LH release (NS) due to both K+ (33% each) and GnRH (44% and 18%, respectively). omega-CgTx showed variable effects on [Ca2+]i transients evoked by K+ or GnRH ranging from clear inhibition to no effect. We conclude that influx of extracellular Ca2+ is one of several fundamental events underlying the depolarization- or receptor-activated release of LH and FSH, and that this influx can be inhibited by dihydropyridine-sensitive ('L') Ca2+ channels. Two classes of L-channels may exist in gonadotropes, that differ in their sensitivity to omega-CgTx.

Animals↗
PubMed1990

Purification and characterization of Go alpha and three types of Gi alpha after expression in Escherichia coli.

Complementary DNAs for the G protein alpha subunits Gi alpha 1, Gi alpha 2, Gi alpha 3, and Go alpha were expressed in Escherichia coli, and the four proteins were purified to homogeneity. The recombinant proteins exchange and hydrolyze guanine nucleotide, are ADP-ribosylated by pertussis toxin, and interact with beta gamma subunits. The rates of dissociation of GDP from Gi alpha 1 and Gi alpha 3 (0.03 min-1) are an order of magnitude slower than that from rGo alpha; release of GDP from Gi alpha 2 is also relatively slow (0.07 min-1). However, the values of kcat for the hydrolysis of GTP by rGo alpha and the three rGi alpha proteins are approximately the same, about 2 min-1 at 20 degrees C. The recombinant proteins restore inhibition of Ca2+ currents in pertussis toxin-treated dorsal root ganglion neurons in response to neuropeptide Y and bradykinin, indicating that the proteins can interact functionally with all necessary components of at least one signal transduction system. The two different receptors function with different arrays of G proteins to mediate their responses, since all four G proteins restored responses to bradykinin, while Gi alpha 2 was inactive with neuropeptide Y. Despite these results, high concentrations of activated Gi alpha proteins are without effect on adenylyl cyclase activity, either in the presence or absence of forskolin or Gs alpha, the G protein that activates adenylyl cyclase. These results are consistent with the hypothesis that G protein beta gamma subunits are primarily responsible for inhibition of adenylyl cyclase activity.

Adenosine Diphosphate Ribose↗
PubMed1990

Neoadjuvant methotrexate, vinblastine, doxorubicin, and cisplatin for locally advanced transitional cell carcinoma of the bladder.

Nine patients with locally advanced transitional cell carcinoma (TCC) of the bladder were treated with neoadjuvant methotrexate, vinblastine, Adriamycin (doxorubicin; Adria Laboratories, Columbus, OH), and cisplatin (M-VAC) followed by radical cystoprostatectomy and modified pelvic lymphadenectomy. Five patients, including three with pelvic sidewall fixation, had clinical stage T4N0M0 tumors whereas the remaining patients had T3N0M0 tumors. All patients were pathologically restaged by a referee pathologist after surgery. The complete response rate was 22% and an additional 44% experienced a partial response. Neutropenia preventing a second cycle of M-VAC occurred in one patient. Downstaging of locally advanced TCC of the bladder was achieved in the majority of patients treated with neoadjuvant M-VAC.

Aged↗
PubMed1990

Toxicity comparison of neoadjuvant versus adjuvant methotrexate, vinblastine, doxorubicin, and cisplatin (M-VAC) in radical cystectomy patients.

A reduction in the toxicity of M-VAC chemotherapy has been postulated for patients who receive these drugs prior to radical cystectomy for muscle invasive bladder carcinoma. A review of the toxicity and patient dropout rates from a group of 9 patients who received neoadjuvant M-VAC was little different from 9 patients who were treated with M-VAC after radical cystectomy. This similarity suggests that preoperative M-VAC should not be favored on the basis of reduced patient morbidity.

Aged↗
PubMed1990

Effects of epidermal growth factor and androgen influences on accessory sex glands in prepubertal Swiss-Webster mice.

The effects of epidermal growth factor (EGF) on androgen stimulation of accessory sex gland growth and biochemistry were determined for prepubertal male Swiss-Webster mice. For the seminal vesicle and anterior prostate, 5 alpha dihydrotestosterone (5 alpha DHT) treatment significantly increased the quantitative levels of organ wet weight, DNA content, polyamine content, and stereologically determined epithelium and lumen volumes above the control group. EGF treatment alone slightly enhanced the levels of most measured parameters from control values. However, when a combined EGF&5 alpha DHT treatment was compared with 5 alpha DHT treatment alone, the 5 alpha DHT treatment effects on epithelial and lumen volumes and polyamine content were antagonized by the action of EGF.

Animals↗
PubMed1990

Excitatory amino acid receptors, second messengers and regulation of intracellular Ca2+ in mammalian neurons.

As we have learnt from earlier articles in our series on the pharmacology of excitatory amino acids, neurons and glia express several subtypes of excitatory amino acid receptor, the activation of which increases intracellular free calcium ion concentration. In this sixth article, Mark Mayer and Richard Miller detail the mechanisms and consequences of this activity. Ionotropic excitatory amino acid receptors, most notably those activated by NMDA, allow Ca2+ influx as a result of passive diffusion down its concentration gradient. Metabotropic receptors trigger an increase in polyphosphoinositide metabolism, and release of Ca2+ from intracellular stores. The increase in [Ca2+]i mediated by excitatory amino acids underlies a complex cellular physiology, including the activation of other second messenger systems, and contributes to the initiation of long-term potentiation.

Amino Acids↗
PubMed1990

Excitotoxicity induced by enhanced excitatory neurotransmission in cultured hippocampal pyramidal neurons.

Cultures of rat hippocampal pyramidal neurons were used to examine the roles of excitatory synaptic transmission, NMDA receptors, and elevated [Ca2+]i in the production of excitotoxicity. In integral of 70% of the cells observed, perfusion with Mg2(+)-free, glycine-supplemented medium induced large spontaneous fluctuations or maintained plateaus of [Ca2+]i. [Ca2+]i fluctuations could be blocked by tetrodotoxin, NMDA receptor antagonists, dihydropyridines, or compounds that inhibit synaptic transmission in the hippocampus, but not by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione. When cells were treated with Mg2(+)-free, glycine-supplemented medium and examined 24 hr later, integral of 30% of the neurons were found to have died. Cell death could be inhibited by the same agents that reduced [Ca2+]i fluctuations. These results support a role for direct excitatory synaptic transmission, as opposed to the general release of glutamate, in excitotoxicity. A major role for synaptically activated NMDA receptors, rather than kainate/quisqualate receptors, is also indicated. Neuronal death may be produced by abnormal changes in neuronal [Ca2+]i.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1990

Allosteric modulation of N-methyl-D-aspartate receptors.

In this review we have attempted to describe the basis for current models of the NMDA receptor, and justify the need for the various binding sites that have been proposed. The NMDA receptor is clearly a complex molecule with a number of modulatory sites, any of which may have great functional significance. From the data presented above it is apparent that the NMDA recognition site is closely coupled with the glycine site, and can also be regulated by Zn2+. The glycine site is reciprocally coupled to the NMDA site, and may also be coupled to a divalent-cation site outside the channel. However, the glycine site is insensitive to Zn2+. The Zn2+ site is probably not inside the channel to any degree, but can profoundly affect the ability of NMDA site ligands to operate the channel. However, the determination of reciprocal effects at the Zn2+ site await the development of a suitably potent and selective ligand for this site. Several lines of evidence suggest that the phencyclidine and channel-blocking Mg2+ site are located within the NMDA-operated ion channel. Glutamate, glycine, and Zn2+ alter the binding of ligands to these sites. However, this is most likely to be due to alteration of access of the ligands to their sites rather than a direct allosteric coupling. It does appear that phencyclidine site drugs and Mg2+ bind to separate sites within the channel, and that these separate sites are allosterically coupled. This complex series of interactions, many of which are mediated by endogenous agents, may allow very fine control over the expression of NMDA receptor-mediated synaptic transmission. In addition to these ligand-produced modulatory effects, there may also be covalent modification of the channel by receptor phosphorylation. Furthermore, the voltage sensitivity of some of the effects allows control of NMDA receptor-mediated signaling by alteration of the membrane potential in the postsynaptic cell, which can be achieved in a wide variety of ways. The level of sophistication possible in adjusting the responsiveness of this receptor seems entirely appropriate given its central involvement in a wide variety of fundamental neurobiological events, and underscores the deleterious pathological sequelae of the system tilting out of balance. At the same time, the wide array of possible therapeutic targets raises hopes that it may soon be possible to treat effectively some severely debilitating and currently untreatable diseases.

Allosteric Regulation↗
PubMed1990

Glutamate receptors activate Ca2+ mobilization and Ca2+ influx into astrocytes.

We measured changes in the molar concentration of cytosolic Ca2+ ([Ca2+]i) in individual astrocytes in culture produced by the glutamate analog quisqualate (QA) and related substances by using fura-2 digital fluorescence microscopy. In cells cultured from the cortex, hippocampus, and cerebellum, the QA analog alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA; 10 microM) produced a slow increase in [Ca2+]i that was modest in amplitude (approximately 200 nM). These effects were completely abolished by 10 microM 6-nitro-7-cyano-quinoxaline-2,3-dione (CNQX). In cerebellar astrocytes, similar effects were produced by QA. However, in cortical and hippocampal astrocytes, the response to QA was much more complex. In these cells, QA produced an initial [Ca2+]i spike that was followed by a sustained influx of Ca2+ ("plateau"). In the absence of extracellular Ca2+, this plateau was abolished but the spike remained. CNQX did not block the spike and only slightly reduced the size of the plateau in some cells. Ni2+ (10 microM) but not nimodipine (10 microM) reduced the amplitude of the plateau. Pretreatment with 100 nM phorbol 12-myristate 13-acetate for 15 min abolished the spike but not the plateau portion of the QA response. Treatment with pertussis toxin at 250 ng/ml for 12-16 hr failed to alter the response. In some instances, the latency of the QA response differed considerably for individual cells in a group. It appeared that the response began in one cell and then spread to neighboring cells. Thus, QA appears to trigger a complex response in some astrocytes consisting of Ca2+ mobilization from intracellular stores and also Ca2+ influx resulting from the activation of AMPA-sensitive and -insensitive pathways.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1990

The effect of capsaicin on voltage-gated calcium currents and calcium signals in cultured dorsal root ganglion cells.

1. The effects of capsaicin on voltage-gated Ca2+ currents (ICa), and intracellular Ca2+ concentrations [( Ca2+]i) in cultured dorsal root ganglion (DRG) neurones of the rat were examined in vitro by use of combined patch clamp-microfluorometric recordings. 2. Under voltage-clamp conditions, capsaicin (0.1-10 microM) caused a concentration-dependent decrease in the magnitude of the ICa, an elevation in the holding current (Ih) and a concomitant rise in the [Ca2+]i in most cells examined. Repeated application of capsaicin produced marked desensitization. 3. Some decrease in the ICa produced by capsaicin was also observed when the rise in [Ca2+]i was buffered with EGTA or BAPTA and when Ba2+ was used as the charge carrier; under these conditions the desensitization previously observed was smaller. 4. The decrement in voltage-gated current was smaller in Ba2+ containing solutions than in Ca2+ containing solutions suggesting that the capsaicin-induced influx of Ca2+ partially mediated the observed decrease in the voltage-gated current. In cells which showed a marked response to capsaicin an outward (positive) current was sometimes observed upon depolarization from -80 to 0 mV. This effect was consistent with an outward movement of cations through the capsaicin conductance pathway which may also account, in part, for the apparent reduction in ICa by capsaicin. 5. The effects of capsaicin under voltage-clamp conditions were prevented by ruthenium red (1 microM). 6. Under current clamp conditions, capsaicin depolarized and caused a rise in [Ca2+]i in the majority of DRG cells examined. Both of these effects could be prevented by ruthenium red (500 nM). 7. It is concluded that capsaicin reduces the Ic. of rat DRG neurones primarily by indirect mechanisms.

Animals↗
PubMed1990

Regulation of the intracellular free calcium concentration in single rat dorsal root ganglion neurones in vitro.

1. Simultaneous whole-cell patch-clamp and Fura-2 microfluorimetric recordings of calcium currents (ICa) and the intracellular free Ca2+ concentration ([Ca2+]i) were made from neurones grown in primary culture from the dorsal root ganglion of the rat. 2. Cells held at -80 mV and depolarized to 0 mV elicited a ICa that resulted in an [Ca2+]i transient which was not significantly buffered during the voltage step and lasted long after the cell had repolarized and the current ceased. The process by which the cell buffered [Ca2+]i back to basal levels could best be described with a single-exponential equation. 3. The membrane potential versus ICa and [Ca2+]i relationship revealed that the peak of the [Ca2+]i transient evoked at a given test potential closely paralleled the magnitude of the ICa suggesting that neither voltage-dependent nor Ca2(+)-induced Ca2+ release from intracellular stores made a significant contribution to the [Ca2+]i transient. 4. When the cell was challenged with Ca2+ loads of different magnitude by varying the duration or potential of the test pulse, [Ca2+]i buffering was more effective for larger Ca2+ loads. The relationship between the integrated ICa and the peak of the [Ca2+]i transient reached an asymptote at large Ca2+ loads indicating that Ca2(+)-dependent processes became more efficient or that low-affinity processes had been recruited. 5. Inhibition of Ca2+ influx with neuropeptide Y demonstrated that inhibition of a large ICa produced minor alterations in the peak of the [Ca2+]i transient, while inhibition of smaller currents produced corresponding decreases in the [Ca2+]i transient. Thus, inhibition of the ICa was reflected by a change in the peak [Ca2+]i only when submaximal Ca2+ loads were applied to the cell, implying that modulation of [Ca2+]i is dependent on the activation state of the cells. 6. Intracellular dialysis with the mitochondrial Ca2+ uptake blocker Ruthenium Red in whole-cell patch-clamp experiments removed the buffering component which was responsible for the more efficient removal of [Ca2+]i observed when large Ca2+ loads were applied to the cell. 7. When cells were superfused with 50 mM-K+, [Ca2+]i transients recorded from the cell soma returned to control levels very slowly. Pharmacological studies indicated that mitochondria were cycling Ca2+ during this sustained elevation in [Ca2+]i. In contrast, [Ca2+]i transients recorded from cell processes returned to basal levels relatively rapidly. 8. Extracellular Na(+)-dependent Ca2+ efflux did not significantly contribute to buffering [Ca2+]i transients in dorsal root ganglion neurone cell bodies.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1990

Pitfalls in the conception, manipulation, and measurement of visual accommodation.

Reports of applied research in visual accommodation frequently contain points of confusion, suggesting that there may be some widespread misunderstanding regarding the nature of accommodation, the complexities involved in its measurement, and the multiple meanings inherent in its terminology. The present paper reviews several of these problem areas. Included are discussions of the effect of refractive error on accommodation assessment; the dangers involved in assuming equivalence among physiological, refractive power, and conjugate distance changes; and multiple meanings in the conception of resting accommodation. A variety of manipulation and measurement problems also are reviewed, including the specification of accommodative target distance, the use of lenses, and the correction of refractive error. The review concludes with recommendations regarding conceptions and procedures useful in avoiding the difficulties described.

Accommodation, Ocular↗
PubMed1990

Direct neuropeptide Y-induced modulation of gonadotrope intracellular calcium transients and gonadotropin secretion.

Neuropeptide Y (NPY) has been shown to be capable of both the enhancement and suppression of gonadotropin secretion from pituitary cells. In order to elucidate the underlying cellular mechanisms which might account for these actions, we have examined the effects of NPY on gonadotropin secretion stimulated by either cell depolarization or by GnRH from primary cultures of rat pituitary cells. In one set of experiments, we measured single-cell [Ca2+]i using the Ca2(+)-sensitive intracellular fluorescent indicator Fura-2, in gonadotropes which had been identified using a reverse hemolytic plaque assay employing an antiserum to LH. In another group of investigations, we measured FSH and LH secretion in response to depolarization or stimulation with GnRH, and examined the influence of NPY on these patterns of secretion. NPY was active in inhibiting the amplitude of the [Ca2+]i signal induced by depolarization with 20 mM K+, as well as in substantially blocking the secondary plateau phase of the GnRH-induced elevation of [Ca2+]i. However, the peak [Ca2+]i transients occurring in response to either depolarization with 50 mM K+ or the initial phase of the GnRH-induced response, were not sensitive to blockade by NPY. Moreover, treatment of the cells for 24 h with pertussis toxin prevented the NPY-mediated inhibition of the GnRH-stimulated [Ca2+]i plateau. Cell depolarization by 50 mM K+ induced 3-fold increases in FSH and LH release over 2-h incubations. GnRH (100 nM) elicited a 9-fold increase in FSH and a 14-fold stimulation of LH over the same time period. NPY had insignificant effects upon depolarization-induced hormone release, but at 1 microM partially suppressed LH release elicited by 100 nM GnRH over 2 h. We conclude that NPY is capable of inhibiting [Ca2+]i signals in gonadotropes that are stimulated by GnRH, and that these effects are mediated through activation of a pertussis toxin-sensitive G-protein. These effects on [Ca2+]i may underly the inhibitory effects of NPY on gonadotropin secretion.

Animals↗
PubMed1990

Obsessive-compulsive disorder: an exploration of some unresolved clinical issues.

The study data were obtained from a questionnaire survey of a South Australian community support group for obsessive-compulsive disorder (OCD). The response rate was 47%, yielding 81 questionnaire sets completed by members who, on the basis of their questionnaire responses, were judged to meet DSM-III-R criteria for OCD. The clinical and demographic characteristics of the respondents were very similar to those of previously reported clinical populations. Mean age at onset of OCD was 18 years and mean duration 17 years; 55% of respondents rated their current OCD as extremely or very severe. Seventy-seven percent had received psychiatric treatment (mean 55 occasions) and 47% had attended clinical psychologists or professional counsellors (mean 20 occasions). Those who reported prominent fears of losing control of motor behaviours had received a significant excess of outpatient and inpatient psychiatric treatment. Most respondents reported the presence of all 4 identified components of OCD, of which the obsession/rumination component was central. Levels of OCD correlated strongly with levels of overall psychopathology, and fertility rates were significantly reduced in those patients who reported the most symptoms.

Adult↗
PubMed1990

Acute- and long-term glutamate-mediated regulation of [Ca++]i in rat hippocampal pyramidal neurons in vitro.

We used a fura 2-based digital imaging technique to analyze the effects of glutamate (GLU) and GLU agonists on intracellular free calcium ([Ca++]i) in cultures of rat hippocampal pyramidal neurons. Depolarization of cells with 50 mM K+ raised [Ca++]i in all parts of the cell (e.g., soma and dendrites). [Ca++]i was also increased in these cells by GLU, kainate, quisqualate, N-methyl-D-aspartate (NMDA) and caffeine (CAF). Multiple challenges of a neuron with GLU gave rise to high "plateau" levels of [Ca++]i that were maintained over the entire length of an experiment (up to 1 hr). In the presence of the NMDA receptor antagonist 2-amino-5-phosphonovalerate multiple applications of GLU only produced multiple transient increases in [Ca++]i. Multiple challenges of a cell with NMDA (0 Mg++, 1 microM glycine) also produced maintained plateau responses in [Ca++]i. Multiple challenges with kainate or quisqualate only produced multiple transient responses in [Ca++]i. Plateau responses induced by GLU or NMDA could be reversibly reduced by removal of extracellular Ca++. Co++ and Ni++ (500 microM) also reduced the magnitude of the plateau, but nitrendipine and tetrodotoxin were generally ineffective. The kinase inhibitor staurosporine also reversibly reduced the magnitude of the plateau. The initiation of a [Ca++]i plateau could be blocked by 2-amino-5-phosphonovalerate although this compound was ineffective at reducing a plateau once it had formed. Thus, activation of NMDA receptors in these neurons leads to a maintained influx of Ca++ that could be responsible for certain long-term effects of GLU.

Animals↗
PubMed1990

Receptor-mediated regulation of calcium channels and neurotransmitter release.

Ca2+ influx into the nerve terminal is normally the trigger for the release of neurotransmitters. Many neurons possess presynaptic receptors whose activation results in changes in the quantity of neurotransmitter released by an action potential. This paper reviews studies that show that presynaptic receptors can regulate the activity of Ca2+ channels in the nerve terminal, resulting in changes in the influx of Ca2+ and in neurotransmitter release. Neurons possess several different types of voltage-sensitive Ca2+ channels. Ca2+ influx through N-type channels appears to trigger transmitter release in many instances. In other cases Ca2+ influx through L channels can influence transmitter release. Neurotransmitters can inhibit N channels through a G protein-mediated transduction mechanism. The G proteins are frequently pertussis toxin substrates. Inhibition of N channels appears to involve changes in their voltage dependence. Neurotransmitters can also regulate neuronal K+ channels. Activation of these K+ channels can lead to a reduction in Ca2+ influx and neurotransmitter release; these effects are also mediated by G proteins. Thus neurotransmitters may often regulate both presynaptic Ca2+ and K+ channels. These two effects may be synergistic mechanisms for the regulation of Ca2+ influx and neurotransmitter release.

Animals↗