Flecainide exerts paradoxical effects on sodium currents and atrial arrhythmia in murine RyR2-P2328S hearts.

AIMS: Cardiac ryanodine receptor mutations are associated with catecholaminergic polymorphic ventricular tachycardia (CPVT), and some, including RyR2-P2328S, also predispose to atrial fibrillation. Recent work associates reduced atrial Nav 1.5 currents in homozygous RyR2-P2328S (RyR2(S/S) ) mice with slowed conduction and increased arrhythmogenicity. Yet clinically, and in murine models, the Nav 1.5 blocker flecainide reduces ventricular arrhythmogenicity in CPVT. We aimed to determine whether, and how, flecainide influences atrial arrhythmogenicity in RyR2(S/S) mice and their wild-type (WT) littermates. METHODS: We explored effects of 1 µm flecainide on WT and RyR2(S/S) atria. Arrhythmic incidence, action potential (AP) conduction velocity (CV), atrial effective refractory period (AERP) and AP wavelength (λ = CV × AERP) were measured using multi-electrode array recordings in Langendorff-perfused hearts; Na(+) currents (INa ) were recorded using loose patch clamping of superfused atria. RESULTS: RyR2(S/S) showed more frequent atrial arrhythmias, slower CV, reduced INa and unchanged AERP compared to WT. Flecainide was anti-arrhythmic in RyR2(S/S) but pro-arrhythmic in WT. It increased INa in RyR2(S/S) atria, whereas it reduced INa as expected in WT. It increased AERP while sparing CV in RyR2(S/S) , but reduced CV while sparing AERP in WT. Thus, RyR2(S/S) hearts have low λ relative to WT; flecainide then increases λ in RyR2(S/S) but decreases λ in WT. CONCLUSIONS: Flecainide (1 µm) rescues the RyR2-P2328S atrial arrhythmogenic phenotype by restoring compromised INa and λ, changes recently attributed to increased sarcoplasmic reticular Ca(2+) release. This contrasts with the increased arrhythmic incidence and reduced INa and λ with flecainide in WT.

Use of OsiriX in developing a digital radiology teaching library.

Widespread adoption of digital imaging in clinical practice and for the image-based examinations of the Royal College of Radiologists has created a desire to provide a digital radiology teaching library in many hospital departments around the UK. This article describes our experience of using OsiriX software in developing digital radiology teaching libraries.

Radiology of acute thoracic and lumbar spine injuries.

Thoracolumbar vertebral fractures typically involve high velocity trauma such as road traffic collisions, falls from height or sporting accidents, and are a major cause of spinal injury. Plain radiographs remain the first-line investigation, but interpretation can be difficult because of the variety of injury patterns and sometimes subtle radiographic signs. This article reviews the normal anatomy, systematic interpretation of thoracic and lumbar radiographs and typical injury patterns.

Prolongation of actions of Ca2+ early in phototransduction by 9-demethylretinal.

During adaptation Ca2+ acts on a step early in phototransduction, which is normally available for only a brief period after excitation. To investigate the identity of this step, we studied the effect of the light-induced decline in intracellular Ca2+ concentration on the response to a bright flash in normal rods, and in rods bleached and regenerated with 11-cis 9-demethylretinal, which forms a photopigment with a prolonged photoactivated lifetime. Changes in cytoplasmic Ca2+ were opposed by rapid superfusion of the outer segment with a 0Na+/0Ca2+ solution designed to minimize Ca2+ fluxes across the surface membrane. After regeneration of a bleached rod with 9-demethlyretinal, the response in Ringer's to a 440-nm bright flash was prolonged in comparison with the unbleached control, and the response remained in saturation for 10-15s. If the dynamic fall in Ca2+i induced by the flash was delayed by stepping the outer segment to 0Na+/0Ca2+ solution just before the flash and returning it to Ringer's shortly before recovery, then the response saturation was prolonged further, increasing linearly by 0.41 +/- 0.01 of the time spent in this solution. In contrast, even long exposures to 0Na+/0Ca2+ solution of rods containing native photopigment evoked only a modest response prolongation on the return to Ringer's. Furthermore, if the rod was preexposed to steady subsaturating light, thereby reducing the cytoplasmic calcium concentration, then the prolongation of the bright flash response evoked by 0Na+/0Ca2+ solution was reduced in a graded manner with increasing background intensity. These results indicate that altering the chromophore of rhodopsin prolongs the time course of the Ca2+-dependent step early in the transduction cascade so that it dominates response recovery, and suggest that it is associated with photopigment quenching by phosphorylation.

Simultaneous recording of receptor current and intraciliary Ca2+ concentration in salamander olfactory receptor cells.

1. The suction pipette technique was combined with laser spot fluorescence microscopy to record simultaneously odour-induced current responses and intraciliary Ca2+ concentration from isolated salamander olfactory receptor cells loaded with the fluorescent Ca2+ indicator fluo-3. 2. When exposed for 1 s to increasing odour concentrations both the suction pipette current and fluo-3 fluorescence increased dynamically, rising with a similar time course. Thereafter, the fluorescence signal decayed more slowly, outlasting the current response by 0.56 +/- 0.12 s. 3. The fluo-3 fluorescence evoked by progressively increasing odour concentrations varied in an approximately linear manner with the magnitude of the suction pipette current. 4. Prolonged odour stimulation evoked synchronous oscillations in both suction pipette current and intraciliary calcium concentration with a mean period of 4.5 +/- 0.3 s. 5. When external Na+ was omitted from the stimulating solution the oscillation period for both the current and fluorescence signals was lengthened by a factor of 1.9 +/- 0.2 in comparison with the oscillation period when stimulated in Ringer solution. 6. These results support the currently accepted mechanism for Ca2+ homeostasis within the olfactory cilia, and are consistent with the notion that the oscillations induced by prolonged odour exposure represent the coupled oscillation of Ca2+ and cyclic nucleotide concentrations.

Responses to prolonged odour stimulation in frog olfactory receptor cells.

1. The suction pipette technique was used to record receptor current and spiking responses from isolated frog olfactory receptor cells during prolonged odour stimuli. 2. The majority (70 %) of cells displayed 'oscillatory' responses, consisting of repeated bursts of spikes accompanied by regular increases in receptor current. The period of this oscillation varied from 3.5 to 12 s in different cells. The remaining cells responded either with a 'transient' burst of spikes at the onset of stimulation (10 %), or by 'sustained' firing throughout the odour stimulus (20 %). 3. In cells with oscillatory responses, the Ca(2+)-activated Cl(-) channel blocker niflumic acid prolonged the period of oscillation only slightly, despite a 3.8-fold decrease in the receptor current. A 3-fold reduction in the external Cl(-) concentration nearly doubled the receptor current, but had little effect on the oscillation period. These results imply that the majority of the receptor current underlying these oscillatory responses is carried by the Ca(2+)-activated Cl(-) conductance, suggesting that the intracellular Ca(2+) concentration oscillates also. 4. In cells with oscillatory responses, the period of oscillation was prolonged 1.5-fold when stimulated in a low-Na(+) solution designed to incapacitate Na(+)-Ca(2+) exchange, irrespective of whether Na(+) was replaced by permeant Li(+) or impermeant choline. The dependence of the oscillation period upon external Na(+) suggests that it may be governed by the dynamics of Ca(2+) extrusion via Na(+)-Ca(2+) exchange. 5. Exposure to the membrane-permeable cyclic nucleotide analogue CPT-cAMP evoked a sustained rather than an oscillatory response even in cells with oscillatory responses to odour. The inability of CPT-cAMP to evoke an oscillatory response suggests that the cAMP concentration is likely to oscillate also. 6. Perforated-patch recordings revealed that oscillatory responses could only be evoked when the membrane potential was free to change, but not when it was clamped near the resting potential. Since substantial changes in Ca(2+)-activated Cl(-) current, and hence odour-induced depolarisation, had little effect upon the period of oscillation, changes in membrane potential are suggested to play only a permissive role in these oscillatory responses. 7. These results are interpreted in terms of the coupled oscillation of Ca(2+) and cyclic nucleotide concentrations within the olfactory cilia during prolonged odour stimulation.

A light-dependent increase in free Ca2+ concentration in the salamander rod outer segment.

1. The Ca(2+) indicator dye fluo-5F was excited by an argon ion laser to measure changes in free Ca(2+) concentration ([Ca2+]i) in the outer segments of isolated salamander rods rapidly exposed to a 0 Ca(2+), 0 Na(+) solution designed to minimise surface membrane Ca(2+) fluxes. Over 30-60 s of laser illumination, the fluorescence first increased rapidly and then declined at a rate that was much slower than in Ringer solution and consistent with previous physiological evidence that 0 Ca(2+), 0 Na(+) solution greatly retards light-induced changes in [Ca(2+)]i. 2. The initial increase in fluorescence was investigated with a sequence of 100 ms laser flashes presented at 5 s intervals. The fluorescence evoked by the second laser flash was on average 30 % larger than the first, and subsequent responses exhibited a slow decline like that measured with continuous laser exposures. The initial increase in fluorescence did not depend upon the timing of exposure to 0 Ca(2+), 0 Na(+) solution but appeared to be evoked by exposure to the laser light. 3. Both the increase and subsequent decline in fluorescence measured with brief laser flashes could be reduced by incorporation of the Ca(2+) chelator BAPTA. This and other results indicate that the fluorescence increase was unlikely to have been caused by a change in the affinity of fluo-5F for Ca(2+) or an increase in the quantity of incorporated dye available to bind Ca(2+) but reflects an actual release of intracellular Ca(2+) within the outer segment. 4. The pool of Ca(2+) available to be released could be decreased if, before the first laser flash, the rod was exposed to light bright enough to bleach a substantial fraction of the photopigment. The releasable pool could also be depleted by exposure to saturating light of much lower intensity if delivered in Ringer solution but not if delivered in 0 Ca(2+), 0 Na(+) solution. We conclude that Ca(2+) can be released within the outer segment both by the bleaching of rhodopsin and by the reduction in [Ca(2+)]i which normally accompanies illumination in Ringer solution. 5. The activation of rhodopsin appears somehow to induce the release of Ca(2+) from a binding site or store within the outer segment. Substantial release, however, required stimulating light of an intensity sufficient to bleach a considerable fraction of the visual pigment. It therefore seems unlikely that such release contributes to the normal Ca(2+)-mediated modulation of transduction during light adaptation. The mechanism and physiological function of light-induced Ca(2+) release are unknown.

Regulation of Staphylococcus aureus pathogenesis via target of RNAIII-activating Protein (TRAP).

Staphylococcus aureus can cause disease through the production of toxins. Toxin production is autoinduced by the protein RNAIII-activating protein (RAP) and by the autoinducing peptide (AIP), and is inhibited by RNAIII-inhibiting peptide (RIP) and by inhibitory AIPs. RAP has been shown to be a useful vaccine target site, and RIP and inhibitory AIPs as therapeutic molecules to prevent and suppress S. aureus infections. Development of therapeutic strategies based on these molecules has been hindered by a lack of knowledge of the molecular mechanisms by which they activate or inhibit virulence. Here, we show that RAP specifically induces the phosphorylation of a novel 21-kDa protein, whereas RIP inhibits its phosphorylation. This protein was termed target of RAP (TRAP). The synthesis of the virulence regulatory molecule, RNAIII, is not activated by RAP in the trap mutant strain, suggesting that RAP activates RNAIII synthesis via TRAP. Phosphoamino acid analysis shows that TRAP is histidine-phosphorylated, suggesting that TRAP may be a sensor of RAP. AIPs up-regulate the synthesis of RNAIII also in trap mutant strains, suggesting that TRAP and AIPs activate RNAIII synthesis via distinct signal transduction pathways. Furthermore, TRAP phosphorylation is down-regulated in the presence of AIP, suggesting that a network of signal transduction pathways regulate S. aureus pathogenesis.

Response properties of isolated mouse olfactory receptor cells.

Response properties of isolated mouse olfactory receptor cells were investigated using the suction pipette technique. Cells were exposed to the odour cineole or to solutions of modified ionic content by rapidly changing the solution superfusing the cilia. All experiments were performed at 37 degrees C. Mouse olfactory receptor cells displayed a steep dependence of action potential frequency on stimulus concentration, a 3-fold increase in stimulus concentration often saturating the firing frequency at 200-300 Hz. The receptor current increased more gradually with increasing cineole concentration and did not saturate within the 100-fold range of cineole concentrations applied. When stimulated for 30 s with a low odour concentration, cells responded with sporadic spike firing. Higher concentrations led to the generation of a large receptor current at the onset of stimulation which returned to baseline levels within a few seconds, accompanied during its rising phase by a short burst of action potentials. Thereafter an oscillating response pattern was observed during the remainder of the stimulus, consisting of repetitive increases in receptor current of around 1 s duration accompanied by short bursts of action potentials. Olfactory adaptation was studied by comparing the responses to two closely spaced odour stimuli. The response to the second odour stimulus recovered to 80% of its original magnitude when the cell was superfused with Ringer solution during the 5 s interval between odour exposures. In contrast, exposure to a choline-substituted low Na+ solution between odour stimuli had two effects. First, the receptor current response to the first odour stimulus did not terminate as quickly as in the presence of Na+, suggesting the presence of a Na+ -Ca2+ exchanger. Second, the response to the second stimulus only recovered to 55% of its original magnitude, demonstrating the involvement of Na+-Ca2+ exchange in the recovery of sensitivity in mouse olfactory receptor cells following stimulation.

Adaptation in vertebrate photoreceptors.

When light is absorbed within the outer segment of a vertebrate photoreceptor, the conformation of the photopigment rhodopsin is altered to produce an activated photoproduct called metarhodopsin II or Rh(*). Rh(*) initiates a transduction cascade similar to that for metabotropic synaptic receptors and many hormones; the Rh(*) activates a heterotrimeric G protein, which in turn stimulates an effector enzyme, a cyclic nucleotide phosphodiesterase. The phosphodiesterase then hydrolyzes cGMP, and the decrease in the concentration of free cGMP reduces the probability of opening of channels in the outer segment plasma membrane, producing the electrical response of the cell. Photoreceptor transduction can be modulated by changes in the mean light level. This process, called light adaptation (or background adaptation), maintains the working range of the transduction cascade within a physiologically useful region of light intensities. There is increasing evidence that the second messenger responsible for the modulation of the transduction cascade during background adaptation is primarily, if not exclusively, Ca(2+), whose intracellular free concentration is decreased by illumination. The change in free Ca(2+) is believed to have a variety of effects on the transduction mechanism, including modulation of the rate of the guanylyl cyclase and rhodopsin kinase, alteration of the gain of the transduction cascade, and regulation of the affinity of the outer segment channels for cGMP. The sensitivity of the photoreceptor is also reduced by previous exposure to light bright enough to bleach a substantial fraction of the photopigment in the outer segment. This form of desensitization, called bleaching adaptation (the recovery from which is known as dark adaptation), seems largely to be due to an activation of the transduction cascade by some form of bleached pigment. The bleached pigment appears to activate the G protein transducin directly, although with a gain less than Rh(*). The resulting decrease in intracellular Ca(2+) then modulates the transduction cascade, by a mechanism very similar to the one responsible for altering sensitivity during background adaptation.

Adaptation-induced changes in sensitivity in frog olfactory receptor cells.

The suction pipette technique was used to study simultaneously the odour-induced action potential and receptor current responses in frog olfactory receptor cells, which were exposed to the odour cineole for 1 s by rapidly exchanging the solution bathing their cilia. The frequency of action potential firing increased as the odour concentration was raised and saturated within a 15-fold elevation above the odour threshold, while the number of spikes fired initially grew at low-to-intermediate concentrations but then declined at higher concentrations. The receptor current response rose steadily and showed no clear sign of saturation over the 300-fold range of cineole concentration employed. The effect of adaptation on the sensitivity of olfactory receptor cells was investigated by first exposing the cell for 4 s to an adapting pre-pulse and then stimulating with a 1 s test pulse. As the pre-pulse concentration was increased, adaptation led to a progressive shift of the dose-response relationships towards higher test pulse concentrations. This resulted in a steep decline in the sensitivity of the receptor current response, combined with an even more dramatic fall in the sensitivity of the spiking responses, since the higher pre-pulse concentrations prevented the generation of action potentials at test pulse concentrations which still evoked a receptor current response.

Adaptation of the odour-induced response in frog olfactory receptor cells.

1. Receptor current and spiking responses were recorded simultaneously from isolated frog olfactory receptor cells using the suction pipette technique. Cells were stimulated with the odour cineole by rapid exchange of the solution bathing the olfactory cilia. 2. The receptor current response to a 1 s odour stimulus increased in a graded manner over a 300-fold range of odour concentration without clear saturation, and was accompanied by a train of action potentials. As the concentration of the odour stimulus increased, the frequency of firing increased also, until it saturated at the highest concentrations. The number of spikes evoked by the stimulus first increased and then decreased with increasing concentration, reaching a maximum at intermediate odour concentrations. The dose-response relation for spike firing rose at lower odour concentrations than the dose-response relation for the receptor current response. 3. Adaptation to steady odour stimuli was investigated by exposing the cilia to a 4 s odour pre-pulse and then to a 1 s odour test pulse. As the pre-pulse concentration was increased the dose-response relations derived from the receptor current and spiking responses shifted to higher absolute test pulse concentrations. However the number of spikes fired in response to a given test pulse was little affected by the pre-pulse until, at the highest pre-pulse concentrations spike firing was abolished despite the continued presence of a receptor current response. 4. The sensitivity of the receptor-current response to incremental stimuli fell with increasing pre-pulse concentration, declining with a limiting slope of 2.4 in double logarithmic co-ordinates. The sensitivity determined from the spiking responses declined to zero at a lower pre-pulse concentration, reflecting the abolition of spike firing at pre-pulse concentrations which still evoked a graded receptor-current response.

Expression, characterization, and mutagenesis of the Yersinia pestis murine toxin, a phospholipase D superfamily member.

A phospholipase D (PLD) superfamily was recently identified that contains proteins of highly diverse functions with the conserved motif HXKX4DX6G(G/S). The superfamily includes a bacterial nuclease, human and plant PLD enzymes, cardiolipin synthases, phosphatidylserine synthases, and the murine toxin from Yersinia pestis (Ymt). Ymt is particularly effective as a prototype for family members containing two conserved motifs, because it is smaller than many other two-domain superfamily enzymes, and it can be overexpressed. Large quantities of pure recombinant Ymt allowed the formation of diffraction-quality crystals for x-ray structure determination. Dimeric Ymt was shown to have PLD-like activity as demonstrated by the hydrolysis of phosphatidylcholine. Ymt also used bis(para-nitrophenol) phosphate as a substrate. Using these substrates, the amino acids essential for Ymt function were determined. Specifically, substitution of histidine or lysine in the conserved motifs reduced the turnover rate of bis(para-nitrophenol) phosphate by a factor of 10(4) and phospholipid turnover to an undetectable level. The role of the conserved residues in catalysis was further defined by the isolation of a radiolabeled phosphoenzyme intermediate, which identified a conserved histidine residue as the nucleophile in the catalytic reaction. Based on these data, a unifying two-step catalytic mechanism is proposed for this diverse family of enzymes.

Light-dependent changes in outer segment free-Ca2+ concentration in salamander cone photoreceptors.

Simultaneous measurements of photocurrent and outer segment Ca2+ were made from isolated salamander cone photoreceptors. While recording the photocurrent from the inner segment, which was drawn into a suction pipette, a laser spot confocal technique was employed to evoke fluorescence from the outer segment of a cone loaded with the Ca2+ indicator fluo-3. When a dark-adapted cone was exposed to the intense illumination of the laser, the circulating current was completely suppressed and fluo-3 fluorescence rapidly declined. In the more numerous red-sensitive cones this light-induced decay in fluo-3 fluorescence was best fitted as the sum of two decaying exponentials with time constants of 43 +/- 2.4 and 640 +/- 55 ms (mean +/- SEM, n = 25) and unequal amplitudes: the faster component was 1.7-fold larger than the slower. In blue-sensitive cones, the decay in fluorescence was slower, with time constants of 140 +/- 30 and 1,400 +/- 300 ms, and nearly equal amplitudes. Calibration of fluo-3 fluorescence in situ from red-sensitive cones allowed the calculation of the free-Ca2+ concentration, yielding values of 410 +/- 37 nM in the dark-adapted outer segment and 5.5 +/- 2.4 nM after saturating illumination (mean +/- SEM, n = 8). Photopigment bleaching by the laser resulted in a considerable reduction in light sensitivity and a maintained decrease in outer segment Ca2+ concentration. When the photopigment was regenerated by applying exogenous 11-cis-retinal, both the light sensitivity and fluo-3 fluorescence recovered rapidly to near dark-adapted levels. Regeneration of the photopigment allowed repeated measurements of fluo-3 fluorescence to be made from a single red-sensitive cone during adaptation to steady light over a range of intensities. These measurements demonstrated that the outer segment Ca2+ concentration declines in a graded manner during adaptation to background light, varying linearly with the magnitude of the circulating current.

Na+-dependent Ca2+ extrusion governs response recovery in frog olfactory receptor cells.

To study the mechanism by which Ca2+, which enters during the odor response, is extruded during response recovery, recordings were made from isolated frog olfactory receptor cells using the suction pipette technique, while superfusing the olfactory cilia with solutions of modified ionic composition. When external Na+ was substituted with another cation, the response to odor was greatly prolonged. This prolongation of the response was similar irrespective of whether Na+ was replaced with Li+, which permeates the cyclic nucleotide-gated conductance, or choline, which does not. The prolonged current was greatly reduced by exposure to 300 microM niflumic acid, a blocker of the calcium-activated chloride channel, indicating that it is carried by this conductance, and abolished if Ca2+ was omitted from the external solution, demonstrating that Ca2+ influx is required for its generation. When the cilia were exposed to Na+-free solution after odor stimulation, the recovery of the response to a second stimulus from the adaptation induced by the first was greatly reduced. We conclude that a Na+-dependent Ca2+ extrusion mechanism is present in frog olfactory cilia and that it serves as the main mechanism that returns cytoplasmic Ca2+ concentration to basal levels after stimulation and mediates the normally rapid recovery of the odor response and the restoration of sensitivity after adaptation.

Catalytic mechanism of the phospholipase D superfamily proceeds via a covalent phosphohistidine intermediate.

The phospholipase D (PLD) superfamily includes enzymes of phospholipid metabolism, nucleases, as well as ORFs of unknown function in viruses and pathogenic bacteria. These enzymes are characterized by the invariant sequence motif, H(X)K(X)4D. The endonuclease member Nuc of the PLD family was over-expressed in bacteria and purified to homogeneity. Mutation of the conserved histidine to an asparagine in the endonuclease reduced the kcat for hydrolysis by a factor of 10(5), suggesting that the histidine residue plays a key role in catalysis. In addition to catalyzing hydrolysis, a number of phosphohydrolases will catalyze a phosphate (oxygen)-water exchange reaction. We have taken advantage of this observation and demonstrate that a 32P-labeled protein could be trapped when the enzyme was incubated with 32P-labeled inorganic phosphate. The phosphoenzyme intermediate was stable in 1 M NaOH and labile in 1 M HCl and 1 M hydroxylamine, suggesting that the enzyme forms a phosphohistidine intermediate. The pH-stability profile of the phosphoenzyme intermediate was consistent with phosphohistidine and the only radioactive amino acid found after alkaline hydrolysis was phosphohistidine. These results suggest that the enzymes in the PLD superfamily use the conserved histidine for nucleophilic attack on the substrate phosphorus atom and most likely proceed via a common two-step catalytic mechanism.