Biomedical subjects

R J Miller

Publications and source records attributed to R J Miller.

At least 271 records · Page 15Linked to original sources

PubMed1988

Regulation of calcium homeostasis in sensory neurons by bradykinin.

The nonapeptide bradykinin (BK) activates sensory neurons and stimulates the transmission of nociceptive information into the CNS. We investigated the effect of this peptide on rat dorsal root ganglion neurons (DRG) grown in vitro. BK stimulated the synthesis of inositol trisphosphate (IP3) and the breakdown of phosphatidylinositol bisphosphate, the synthesis of diacylglycerol, and the release of arachidonic acid from DRG cells. The release of IP3 and arachidonic acid was not inhibited by pretreatment of the cells with pertussis toxin. BK also mobilized intracellular Ca2+ stores in DRG cells as assessed by fura-2-based microfluorimetry. Two types of Ca2+ stores appeared to exist in DRG neurons. One type could be mobilized by caffeine (10(-2) M), and this effect could be blocked by ryanodine in a use-dependent manner. These stores occurred primarily in the cell soma and were virtually absent from cell processes. A second type of store could be mobilized by BK, presumably through the mediation of IP3. These latter stores were distributed equally between the cell soma and processes. Experiments with combinations of caffeine and BK suggested that the stores mobilized by these 2 agents may be separate entities. Both the caffeine and BK sensitive Ca2+ storage sites appeared to participate in buffering a Ca2+ load induced in DRG neurons by cell depolarization. The relevance of these observations to the mechanism of action of BK on sensory neurons is discussed.

Animals↗

PubMed1988

The role of caffeine-sensitive calcium stores in the regulation of the intracellular free calcium concentration in rat sympathetic neurons in vitro.

Intracellular Ca2+ stores were studied in sympathetic neurons grown in primary culture from the superior cervical ganglion of the rat. The [Ca2+]i was measured in single cells using the fluorescent Ca2+ indicator fura-2 and a sensitive microfluorimeter. Superfusion of the cells with 10 mM caffeine elicited a rapid and transient increase in [Ca2+]i in the absence of extracellular Ca2+, indicating the presence of a caffeine-sensitive intracellular Ca2+ storage site. After depletion of the store by mobilization of Ca2+ with caffeine, it could be refilled by elevating [Ca2+]i, allowing multiple caffeine-induced [Ca2+]i transients to be elicited from a single neuron. Ryanodine (1 microM), an alkaloid that promotes Ca2+ release from the sarcoplasmic reticulum, was an effective inhibitor of the caffeine-induced [Ca2+]i transients in sympathetic neurons. Exposure to ryanodine in the presence of caffeine was required to produce a subsequent inhibition of the caffeine-induced response, suggesting a "use-dependent" inhibition that may result from depletion of the Ca2+ stores. In contrast, dantrolene Na (10 microM), an agent known to interfere with Ca2+ release from the sarcoplasmic reticulum, also blocked the caffeine-induced [Ca2+]i transients, but in a time-dependent rather than a use-dependent manner. Electrophysiological measurements using the whole cell version of the patch-clamp technique were made simultaneously with [Ca2+]i microfluorimetric recordings. The magnitude of the [Ca2+]i transients elicited by step depolarizations closely paralleled the magnitude of Ca2+ influx via voltage-sensitive Ca2+ channels, regardless of whether the magnitude of the Ca2+ current was modified by varying the test pulse duration or potential. The relationship between the magnitude of Ca2+ influx and the resulting increase in [Ca2+]i saturated at large Ca2+ influxes resulting from long depolarizations, consistent with the activation of a large capacity, low affinity [Ca2+]i buffering mechanism. Caffeine (10 mM) and ryanodine (10 microM), applied singly or together, produced a small and variable decrease in the [Ca2+]i transient resulting from cell depolarization using the whole-cell patch-clamp technique. We conclude that mammalian sympathetic neurons possess intracellular Ca2+ stores with pharmacological characteristics that closely resemble those found in muscle but that these are relatively small and produce little amplification of [Ca2+]i transients resulting from Ca2+ influx through voltage-sensitive Ca2+ channels.

Animals↗

PubMed1988

The effect of down regulation of protein kinase C on the inhibitory modulation of dorsal root ganglion neuron Ca2+ currents by neuropeptide Y.

Dorsal root ganglion (DRG) neurons cultured from neonatal rats contained high concentrations of protein kinase C (PKC). Normally, the majority of the enzyme activity was found in the cytosol and considerably less was associated with the membrane fraction. Upon incubation with the phorbol ester phorbol dibutyrate (PDBu, 10(-6) M) for 20 min, PKC activity increased in the membrane-associated fraction and decreased in the cytoplasmic fraction. Longer incubations with phorbol ester also induced a decline in membrane-associated PKC activity. If incubations were continued for periods of over 10 hr, both membrane and cytosolic PKC activity declined essentially to zero. Down-regulation of PKC had no effect on the number or affinity of 125I-neuropeptide Y (NPY) binding sites on DRG cells or on the absolute magnitude of the DRG Ca2+ current. However, the ability of NPY to inhibit the DRG Ca2+ current was greatly reduced. When sustained Ca2+ currents were evoked from depolarized holding potentials (-40 mV), all concentrations of NPY (10(-10)-10(-7) M) were less effective. In contrast, higher concentrations of NPY still blocked the transient portion of the DRG Ca2+ current evoked from hyperpolarized holding potentials. These results support the suggestion that PKC is involved in the inhibitory modulation of DRG Ca2+ currents by neurotransmitters. The precise role of PKC may vary depending on the type of Ca2+ channel involved.

Animals↗

PubMed1988

Dislocations and fracture dislocations of the metacarpophalangeal joint of the thumb.

Due to its exposed position, the MCP joint of the thumb is particularly vulnerable to dislocations and fracture dislocations. Depending on the direction of the injuring force, injuries to the ulnar, radial, and volar aspect of the joint can occur. If high-grade lesions are not identified and treated appropriately during their acute phase, marked instability with associated long-term disability due to weakness and pain in pinch and grip can result. In the absence of a reliable method for diagnosing the Stener lesion, surgical repair of acute, third-degree lesions on the ulnar side of the joint remains the treatment of choice. In the presence of chronic instability, a variety of effective soft tissue reconstructive measures are available. The fact that both acute and chronic injuries enjoy a favorable prognosis with operative repair is due to the fact that operative intervention reliably restores stability to the joint. Mild to moderate loss of motion at the joint is well tolerated functionally. For this reason, arthrodesis remains an exceptionally satisfactory salvage for failed soft tissue reconstructions.

Acute Disease↗

PubMed1988

Arousal-related changes in dark focus accommodation and dark vergence.

Dual-innervation theory suggests that dark focus (DF; the accommodation in effect in total darkness) reflects a balance between sympathetic and parasympathetic inputs. Since it is assumed that relaxation of accommodation is sympathetically innervated, and that accommodative increases are parasympathetically innervated, stimulation that leads to sympathetic activation should produce lower DF values than is the case when there is greater parasympathetic influence. In addition, the synkinesis between accommodation and vergence, and the known effects of fatigue, injury and drugs on fusion, promote an interest in the effects of arousal on dark vergence (DV; the vergence in effect in total darkness). Twenty-three young adult males experienced a variety of stimulus conditions. The conditions, selected because of their likely autonomic effects, included deep muscle relaxation, cold pressor and mental activity, among others. During each condition, measurements were made of DF (with a laser optometer) and DV (with a Nonius alignment system). Results for DF were consistent with dual innervation theory; manipulations expected to induce sympathetic activation (eg, cold pressor) produced lower DF values than did conditions expected to induce more parasympathetic (or less sympathetic) activation (eg, relaxation). DV tended to be influenced in the opposite direction, in that relaxation led to smaller DV angles than did the other conditions. Several analyses confirmed a lack of synkinesis between accommodation and vergence during darkness.

Accommodation, Ocular↗

PubMed1987

Multiple calcium channels and neuronal function.

Recent investigations have demonstrated that neurons have a number of different types of calcium channels, each with their own unique properties and pharmacology. These calcium channels may be important in the control of different aspects of nerve activity. Some of the possibilities can now be discussed.

Animals↗

PubMed1987

3H-labeled MK-801 binding to the excitatory amino acid receptor complex from rat brain is enhanced by glycine.

We have studied the binding of the excitatory amino acid antagonist 3H-labeled MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohept-5,10-imine maleate] to extensively washed rat brain membranes. Binding of 3H-labeled MK-801 was inhibited by phencyclidine, Mg2+, and 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid in a manner dependent on the presence of L-glutamate or N-methyl-D-aspartate, suggesting that it labeled a site linked to the N-methyl-D-aspartate subtype of the glutamate receptor. Glycine also regulated 3H-labeled MK-801 binding, enhancing it in the concentration range of 0.01-10 microM. The actions of glutamate and glycine involved increases in binding affinity, without altering the number of 3H-labeled MK-801 binding sites. The effects of glycine in this system were mimicked by L- and D-alanine and L- and D-serine. However, beta-alanine and taurine were much less effective, and strychnine did not block the actions of glycine indicating that they were not mediated by the classical glycine receptors. Glycine also enhanced the ability of N-methyl-D-aspartate to increase Ca2+ influx into primary cultures of mouse striatal neurons measured using the Ca2+-sensitive fluorescent dye fura-2. These results support the suggestion that glycine may be an important regulator of the physiological actions of glutamate in vivo.

Amino Acids↗

PubMed1987

Nonius alignment apparatus for measuring vergence.

The present paper describes an apparatus designed for the assessment of vergence. Based on the Nonius alignment system of viewing dichoptic stimuli, this apparatus provides a compact, portable, easily constructed, and easily maintained piece of equipment. It can be used for the assessment of vergence under a wide variety of viewing conditions and laboratory demands. Also included are an example of one way in which the apparatus may be set up, the equations for finding vergence angle using that set-up, and data concerning the validity and reliability of measurements made with the apparatus.

Convergence, Ocular↗

PubMed1987

Neurochemical characteristics of a novel dorsal root ganglion X neuroblastoma hybrid cell line, F-11.

We investigated the properties of the novel dorsal root ganglion (DRG) hybrid cell line F-11 to see how closely these cells resembled normal DRG cells. Under normal growth conditions, F-11 cells appeared to contain several short neurite-like processes. However, these cells could also be grown under conditions in which they showed a much more extensive neuronal morphology, exhibiting many long neurites. Several differentiated features of DRG cells were present on F-11 cells. These included the presence of delta-opioid receptors, receptors for prostaglandins and bradykinin, and dihydropyridine-sensitive calcium channels. F-11 cells also synthesized and released a substance P-like compound, as determined by immunoreactivity. Both the number of bradykinin receptors and the voltage-sensitive calcium influx increased on cell differentiation. Opioid agonists (delta-specificity) were found to decrease cyclic AMP levels in F-11 cells in a naloxone- and pertussis toxin-reversible fashion. Bradykinin stimulated the synthesis of inositol-1,4-bisphosphate and inositol-1,4,5-trisphosphate. Ca2+ channel agonists stimulated voltage-sensitive Ca2+ influx in a dose-dependent, stereospecific manner, whereas Ca2+ channel antagonists inhibited Ca2+ influx. F-11 cells should, therefore, prove useful as models for authentic DRG neurons.

Animals↗

PubMed1987

Modulation of bradykinin-induced inositol trisphosphate release in a novel neuroblastoma x dorsal root ganglion sensory neuron cell line (F-11).

In the mouse neuroblastoma x dorsal root ganglion hybrid cell line F-11, bradykinin receptor stimulation induced the release of inositol-1,4,5-trisphosphate (IP3) and inositol-1,4-bisphosphate (IP2). Maximal stimulation of [2-3H]IP3 and [2-3H]IP2 release by bradykinin in the absence of LiCl occurred at 7 (or less) and 15 s, respectively, with average levels of 5.7-(IP3) and 3.4-(IP2) fold of control values. The EC50 for bradykinin was 33 +/- 5 nM. IP3 and IP2 concentrations returned to basal levels approximately 1 min after bradykinin addition. Bradykinin-induced IP3 release was blocked by several novel bradykinin analogues. In particular, [D-Arg0]-Hyp3-Thi5,8-[D-Phe7]-bradykinin [Hyp, hydroxyproline; Thi, beta-(2-thienyl)-L-alanine] blocked IP3 production in a dose-dependent fashion. Several of these analogues alone showed little or no agonist activity. The bradykinin receptor may be coupled to phospholipase C via a GTP-sensitive protein (Gi or Go), as preincubation for 18-20 h with pertussis toxin decreased IP3 concentrations by 45%. Bradykinin is also known to modulate the concentrations of other second messengers in neurons, increasing the concentrations of Ca2+, diacylglycerol (DG), and cyclic GMP and decreasing the concentration of cyclic AMP. These second messengers modulated bradykinin-dependent IP3 release to varying degrees. A23187, a Ca2+ ionophore, produced a 37% decrease in IP3 concentration. 12-O-Tetradecanoylphorbol-13-acetate, which mimics the effects of DG and activates protein kinase C, inhibited IP3 release by 80%. Dibutyryl cyclic GMP produced little or no inhibition of IP3. [D-Ala2,D-Leu5]Enkephalin (DADLE), an opioid peptide that decreases cyclic AMP concentrations, likewise had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

Homologous desensitization to prostaglandins in rabbit ileum.

Prostaglandins (PG's) increase short-circuit current (Isc), inhibit NaCl absorption, and stimulate Cl secretion in rabbit ileum. These changes occur with the following PGs; E2, E1, nitrilo-I2 and, to a lesser extent, with A2, D2, and F2 alpha. Arachidonic acid (AA) also stimulates secretion. The PG- or AA-stimulated Isc does not persist, however, and on prolonged exposure tachyphylaxis develops. Resensitization of the Isc response to PGE2 is rapid, being essentially complete in 15 min after the PG is removed. Desensitization to AA is not reflected by diminished PG generation. PGE2 release from the mucosa after AA addition is constant, although the AA-stimulated Isc decreases. Isc measurements indicate that PGE2 at slightly below its EC50 partially desensitizes and a near-maximal concentration completely desensitizes to PGE2 but does not, however, inhibit the subsequent change in Isc caused by theophylline or vasoactive intestinal peptide (VIP). Adenosine 3',5'-cyclic monophosphate (cAMP) measurements suggest that desensitization applies to cAMP production. PGE2 (10(-5) M) increases mucosal cAMP three- to sevenfold, but this elevation is transient; a second challenge dose, which fails to elicit a Isc change, also fails to increase mucosal cAMP. Adenylate cyclase measurements from untreated and PGE2-treated enterocytes demonstrate a decrease in stimulation by PGE2 but not in stimulation by VIP, fluoride, or 5-guanylylimidodiphosphate.

Adenylyl Cyclases↗

PubMed1987

Distribution of multiple types of Ca2+ channels in rat sympathetic neurons in vitro.

Ca2+ influx through voltage sensitive Ca2+ channels produces a rise in intracellular-free Ca2+, [Ca2+]i, that serves as a trigger for the release of neurotransmitters. We measured [Ca2+]i in primary cultures of superior cervical ganglion (SCG) neurons of the rat using 2-(6-(bis(carboxymethyl)amino)-5-methylphenoxy)ethoxy-2-benzofuranyl)5- oxazole carboxylic acid-based microfluorimetry. Recordings were obtained from either single or small bundles of neuronal processes and compared with recordings from single neuronal cell bodies. Depolarization with 50 mM K+ produced a rapid increase in [Ca2+]i consisting of both transient and sustained components. This response pattern was seen in recordings from both the soma and processes of SCG neurons. The entire response could be reversibly blocked by 30 microM La3+. Nitrendipine, 1 microM, inhibited the response by 52 +/- 7% and 49 +/- 7% in the soma and processes, respectively. The dihydropyridine (DHP) agonist 1,4-dihydro-2,-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine- 5-carboxylic acid methyl ester enhanced depolarization-induced increases in [Ca2+]i in both regions of the neuron. The transient component of the response was greatly reduced by predepolarization, and the remaining sustained component was inhibited 77 +/- 7% by nitrendipine (1 microM). These data demonstrate that both DHP-sensitive and -insensitive Ca2+ channels are present in processes as well as cell bodies of SCG neurons. The importance of these findings is discussed in relation to the insensitivity of neurotransmitter release from sympathetic neurons to DHP antagonists.

Animals↗

PubMed1987

Voltage-dependent nitrendipine binding to cardiac sarcolemmal vesicles.

The binding of the calcium channel antagonist [3H]nitrendipine (3[H]Ntd) to purified bovine cardiac sarcolemmal vesicles was examined at different membrane potentials. The vesicles were loaded with 150 mM K and exposed to a range of external K concentrations to induce negative internal membrane potentials using N-methyl-D-glucamine or choline to substitute for K. The lipophilic cation [3H]tetraphenylphosphonium was used to quantitate the membrane potential, and the vesicles were capable of maintaining a relatively stable negative inside membrane potential for at least 15 min. Equilibrium binding studies of [3H]Ntd performed on vesicles maintained at a depolarized potential (0 mV) revealed 24.7 +/- 5.6% more high affinity [3H]Ntd-binding sites than were present on vesicles maintained at a hyperpolarized potential (approximately -40 to -50 mV) with N-methyl-D-glucamine substituting for K. There was no significant change in Kd values which were 0.398 +/- 0.069 and 0.388 +/- 0.032 nM, respectively. When choline was used to substitute for K, comparable results were obtained with 19.01 +/- 3.4% more high affinity [3H]Ntd-binding sites under depolarized conditions and no significant change in Kd (0.356 +/- 0.025 and 0.379 +/- 0.030 nM, respectively). Varying the external K concentration resulted in a graded change in [3H]Ntd binding over the same range of external K concentrations which resulted in the greatest change in membrane potential. These findings suggest that the observed effect was due to changes in membrane potential rather than changes in the composition of the incubation medium. Additional control experiments employing choline-loaded vesicles also support the hypothesis that the observed effect was primarily due to changes in membrane potential. The present results are compared with those from electrophysiological studies.

Animals↗

PubMed1987

The effects of excitatory amino acids on intracellular calcium in single mouse striatal neurons in vitro.

Using microspectrofluorimetry and the calcium-sensitive dye fura-2, we examined the effect of excitatory amino acids on [Ca2+]i in single striatal neurons in vitro. N-methyl-D-aspartic acid (NMDA) produced rapid increases in [Ca2+]i. These were blocked by DL-2-amino-5-phosphonovaleric acid (AP5), by Mg2+, by phencyclidine, and by MK801. The block produced by Mg2+ and MK801 could be relieved by depolarizing cells with veratridine. When external Ca2+ was removed, NMDA no longer increased [Ca2+]i. Furthermore, the effects of NMDA were not blocked by concentrations of La3+ that blocked depolarization induced rises in [Ca2+]i. Substitution of Na+o by Li+ did not block the effects of NMDA. Concentrations of L-glutamate greater than or equal to 10(-6) M also increased [Ca2+]i. The effects of moderate concentrations of glutamate were blocked by AP5 but not by La3+ or by substitution of Na+ by Li+. The effects of glutamate were blocked by removal of external Ca2+ but were not blocked by concentrations of Mg2+ or MK801 that completely blocked the effects of NMDA. The glutamate analogs kainic acid (KA) and quisqualic acid also increased [Ca2+]i. The effects of KA were blocked by removal of external Ca2+ but not by La3+, Mg2+, MK801, or replacement of Na+ by Li+. Although AP5 was able to block the effects of KA partially, very high concentrations were required. These results may be explained by considering the properties of glutamate-receptor-linked ionophores. Excitatory amino acid induced increases in [Ca2+]i are consistent with the possibility that Ca2+ mediates excitatory amino acid induced neuronal degeneration.

Amino Acids↗

PubMed1987

The chromatic aberration adjustment in laser optometry.

The issues involved in designing chromatic aberration corrections in laser optometry are reviewed. There is considerable disagreement regarding both the magnitude of human eye chromatic aberration and the value that should be assumed for the reference wavelength. Also important are the observer's accommodative state, target luminance wavelength, laser wavelength, intersubject dioptric variations, and several other factors. The appropriate solution for most experiments appears to be to report no chromatic aberration adjustment at all; rather, the investigator should provide basic technical information from which interested readers can make their own corrections, depending on the assumptions of importance to them.

Accommodation, Ocular↗

PubMed1987

Down regulation of protein kinase C in neuronal cells: effects on neurotransmitter release.

We investigated the effects of phorbol esters on protein kinase C (PKC) activity and on neurotransmitter release from cultured neuronal cells. Both differentiated and undifferentiated PC12 pheochromocytoma cells contained high levels of protein PKC. Under normal conditions all the enzyme activity was found in the cytoplasm. Addition of the phorbol esters phorbol 12-myristate-13-acetate (TPA) or phorbol 12,13-dibutyrate (PDBu) caused a rapid translocation of PKC from the cytoplasm to the particulate fraction. Continued culture of cells with these phorbol esters resulted in the decline of total PKC activity. After 10-20 hr of culture, both membrane and cytoplasmic PKC activity had declined to background levels. cAMP-dependent and Ca2+/calmodulin-dependent protein kinase activities were only slightly affected by chronic phorbol ester treatment. Addition of active phorbol esters to PC12 cells produced an enhancement of the depolarization-induced release of 3H-norepinephrine. Following chronic phorbol ester treatment, the ability of these substances to enhance evoked catecholamine release was lost. Furthermore, depolarizing stimuli released considerably less 3H-norepinephrine than in control untreated cells. Phorbol esters also enhanced depolarization-induced 3H-norepinephrine release from primary cultures of rat sympathetic neurons. Chronic treatment of these neurons with phorbol esters also resulted in the loss of their ability to enhance transmitter release and in a large reduction in the extent of depolarization-evoked transmitter release. Chronic phorbol ester treatment also resulted in the disappearance of PKC from sympathetic neurons, but had little effect on cAMP-dependent or Ca2+/calmodulin-dependent kinase activities. These results demonstrate that PKC-deficient neurons can be prepared. The data also demonstrate that depolarization-induced neurotransmitter release is mediated by both protein kinase C-dependent and independent pathways.

Animals↗

PubMed1987

Effects of relaxation and aversive visual stimulation on dark focus accommodation.

There is evidence that sympathetic innervation induces decreases in accommodation or accommodation amplitude. If so, emotional stress that produces sympathetic activation should induce similar changes in accommodation. In two experiments, dark focus accommodation was assessed following deep relaxation and after subjects looked at gruesome slides. Results showed that stress exposure led to lower dark focus values than did relaxation. Further, the degree to which dark focus was affected depended on subjects' levels of perceived arousal, and on the degree of stressfulness represented by the slides. It was concluded that psychological stress can produce predictable changes in accommodation, changes most parsimoniously understood in terms of autonomic innervation of the ciliary body.

Accommodation, Ocular↗

PubMed1986

Separation of cardiac plasmalemma into cell surface and T-tubular components. Distribution of saxitoxin- and nitrendipine-binding sites.

To compare surface sarcolemmal with T-tubular distributions of [3H]saxitoxin (STX)- and [3H]nitrendipine (NTD)-binding sites, we centrifuged membrane vesicles from sheep and bovine ventricles on a 10-40% linear sucrose gradient from which fractions were assayed for STX and NTD binding; for markers of surface sarcolemma (ouabain-sensitive Na,K-ATPase activity, [3H]quinuclidinyl benzilate binding); and for markers of junctional sarcoplasmic reticulum known to be preferentially associated with T-tubules (ryanodine-sensitive Ca2+ uptake, calsequestrin, an Mr 300,000 putative phosphorylatable "foot" protein, and electron microscopically visible junctional sarcoplasmic reticulum-plasmalemma complexes). We identified three distinct peaks in the sucrose gradient, each characterized by significant high and low affinity STX- and high affinity NTD-binding: Peak I (approximately 19% sucrose), highly enriched in surface sarcolemma; Peak III (approximately 36% sucrose), enriched in junctional sarcoplasmic reticulum markers and hence in junctional sarcoplasmic reticulum complexes with T-tubule; and Peak II (approximately 27% sucrose), showing greatest specific STX binding and only moderate NTD binding, enriched in T-tubular membrane, unassociated with junctional sarcoplasmic reticulum. For ventricular myocytes, the ratio NTD sites/STX sites was 2.5 for surface sarcolemma, but only approximately 1.0 for T-tubules. Unlike data published for mammalian skeletal muscle, sheep and beef cardiac NTD receptors were not significantly more concentrated in T-tubular than in surface plasmalemma.

Amphibian Proteins↗