PubMed Health⌕ Search

Biomedical subjects

Mark B Cannell

Publications and source records attributed to Mark B Cannell.

15 recordsLinked to original sources
PubMed2006

TRPC-like conductance mediates restoration of intracellular Ca2+ in cochlear outer hair cells in the guinea pig and rat.

Ca2+ signalling is central to cochlear sensory hair cell physiology through its influence on sound transduction, membrane filter properties and neurotransmission. However, the mechanism for establishing Ca2+ homeostasis in these cells remains unresolved. Canonical transient receptor potential (TRPC) Ca2+ entry channels provide an important pathway for maintaining intracellular Ca2+ levels. TRPC3 subunit expression was detected in guinea pig and rat organ of Corti by RT-PCR, and localized to the sensory and neural poles of the inner and outer hair cells (OHCs) by confocal immunofluorescence imaging. A cation entry current with a TRPC-like phenotype was identified in guinea pig and rat OHCs by whole-cell voltage clamp. This slowly activating current was induced by the lowering of cytosolic Ca2+ levels ([Ca2+]i) following a period in nominally Ca2+-free solution. Activation was dependent upon the [Ca2+]o and was sustained until [Ca(2+)]i was restored. Ca2+ entry was confirmed by confocal fluorescence imaging, and rapidly recruited secondary charybdotoxin- and apamin-sensitive K(Ca) currents. Dual activation by the G protein-coupled receptor (GPCR)-phospholipase C-diacylglycerol (DAG) second messenger pathway was confirmed using the analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG). Ion substitution experiments showed that the putative TRPC Ca2+ entry current was selective for Na+ > K+ with a ratio of 1: 0.6. The Ca2+ entry current was inhibited by the TRPC channel blocker 2-aminoethyl diphenylborate (2APB) and the tyrosine kinase inhibitor, erbstatin analogue. We conclude that TRPC Ca2+ entry channels, most likely incorporating TRPC3 subunits, support cochlear hair cell Ca2+ homeostasis and GPCR signalling.

Animals↗
PubMed2006

A buffering SERCA pump in models of calcium dynamics.

Many cells use oscillations in calcium concentration to transmit messages. The oscillations largely result from an influx of calcium into the cytosol from the endoplasmic reticulum (ER), followed by an efflux of calcium from the cytosol back into the ER. The sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) pump pumps calcium into the ER. It binds calcium on the cytosolic side and releases it on the ER side and in the delay between binding and release, calcium is buffered by the pump. We developed a model of a buffering SERCA pump and investigated whether including this in a model of calcium oscillations has any significant effects. We found that the oscillations produced when using the SERCA pump, which does not buffer calcium, have a larger amplitude and a slightly smaller period than when using the buffering SERCA pump. We show that the buffering SERCA pump shows adaptation to a stimulus, and we demonstrate that, by using a bidirectional SERCA pump, we are able to eliminate futile cycling of calcium between the cytosol and ER when the cell is at rest.

Biological Clocks↗
PubMed2006

Three-dimensional distribution of ryanodine receptor clusters in cardiac myocytes.

The clustering of ryanodine receptors (RyR2) into functional Ca2+ release units is central to current models for cardiac excitation-contraction (E-C) coupling. Using immunolabeling and confocal microscopy, we have analyzed the distribution of RyR2 clusters in rat and ventricular atrial myocytes. The resolution of the three-dimensional structure was improved by a novel transverse sectioning method as well as digital deconvolution. In contrast to earlier reports, the mean RyR2 cluster transverse spacing was measured 1.05 microm in ventricular myocytes and estimated 0.97 microm in atrial myocytes. Intercalated RyR2 clusters were found interspersed between the Z-disks on the cell periphery but absent in the interior, forming double rows flanking the local Z-disks on the surface. The longitudinal spacing between the adjacent rows of RyR2 clusters on the Z-disks was measured to have a mean value of 1.87 microm in ventricular and 1.69 microm in atrial myocytes. The measured RyR2 cluster distribution is compatible with models of Ca2+ wave generation. The size of the typical RyR2 cluster was close to 250 nm, and this suggests that approximately 100 RyR2s might be present in a cluster. The importance of cluster size and three-dimensional spacing for current E-C coupling models is discussed.

Animals↗
PubMed2005

Electrochemical detection of DNA hybridization amplified by nanoparticles.

Detection of specific oligonucleotide (ODN) fragments has become an important field in many areas of biomedicine. We describe a novel ODN sensor based on electropolymerization of a conducting polymer (polypyrrole) in the presence of a sample containing ODN(s). The resulting trapped ODN(s) are then probed by addition of complimentary sequence ODN. By incorporating CdS nanoparticles with the probe, a significant improvement in sensor sensitivity was observed. Impedance spectroscopy suggested that optimal detection of hybridization occurred at frequencies>or=3000 Hz (for a 0.07 cm2 85 nm thick film). At these frequencies, the impedance signal was almost linear with the logarithm of ODN concentration in the range 3.7-370 nM with a detection limit of approximately 1 nM ODN (for the sensor fabricated). Importantly, the sensor could be regenerated by removing hybridized ODN with NaOH suggesting possibility of the sensor re-use.

Biosensing Techniques↗
PubMed2005

Label-free electrochemical DNA sensor based on functionalised conducting copolymer.

A simple and label-free electrochemical sensor for recognition of the DNA hybridization event was prepared based on a new functionalised conducting copolymer, poly[pyrrole-co-4-(3-pyrrolyl) butanoic acid]. This precursor copolymer can be easily electrodeposited on the electrode surface and shows high electroactivity in an aqueous medium. An amino-substituted oligonucleotide (ODN) probe was covalently grafted onto the surface of the copolymer in a one step procedure and tested on hybridization with complementary ODN segments. The cyclic voltammogram of ODN probe-modified copolymer showed very little change when incubated in presence of non-complementary ODN, while a significant, and reproducible, modification of the voltammogram was observed after addition of complementary ODN. The AC impedance spectrum showed an increased charge transfer resistance (Rct) and double layer capacitance of the sensor film after hybridisation. Sensors with thinner films showed higher sensitivity than thicker films, suggesting that hybridisation at or near the surface of the film produces a larger change in electrical properties than that within the body of the film.

Biosensing Techniques↗
PubMed2004

Functional imaging: new views on lens structure and function.

1. We have developed an experimental imaging approach that allows the distribution of lens membrane proteins to be mapped with subcellular resolution over large distances as a function of fibre cell differentiation. 2. Using this approach in the rat lens, we have localized precisely histological sites of connexin 46 cleavage, quantitatively mapped changes in gap junction distribution and fibre cell morphology and correlated these changes to differences in intercellular dye transfer. 3. Profiling of glucose transporter isoform expression showed that lens epithelial cells express GLUT1, whereas deeper cortical fibre cells express the higher-affinity GLUT3 isoform. Near the lens periphery, GLUT3 was located in the cytoplasm of fibre cells, but it underwent a differentiation-dependent membrane insertion. 4. Similarly, the putative adhesion protein membrane protein 20 is inserted into fibre cell membranes at the stage when the cells lose their nuclei. This redistribution is strikingly rapid in terms of fibre cell differentiation and correlates with a barrier to extracellular diffusion. 5. Our imaging-orientated approach has facilitated new insights into the relationships between fibre cell differentiation and lens function. Taken together, our results indicate that a number of strategies are used by the lens during the course of normal differentiation to change the subcellular distribution, gross spatial location and functional properties of key membrane transport proteins.

Animals↗
PubMed2004

Gap junction processing and redistribution revealed by quantitative optical measurements of connexin46 epitopes in the lens.

PURPOSE: To map changes in the structure and function of fiber cell gap junctions that occur with lens differentiation. METHODS: Equatorial lens sections were fluorescently labeled with antibodies to the gap junction protein connexin (Cx)46, the membrane marker wheat germ agglutinin, and the nuclear stain propidium iodide. Two-photon microscopy and digital image analysis were used to quantify label and cell morphology as a function of radial distance (r/a) across the lens. Loop- and tail-specific Cx46 antibodies were used to identify regions of posttranslational modification. Local fiber cell coupling was imaged in situ using two-photon flash photolysis of caged fluorescein. RESULTS: Antibody labeling showed that the cytoplasmic tail of Cx46 was removed in two zones (r/a approximately 0.9 and r/a approximately 0.7). In addition, with increasing depth, the large radially aligned plaques of peripheral fiber cells became fragmented and dispersed around the cell membrane, and cells became more circular in cross section. Fluorescein transfer between peripheral fiber cells was highly anisotropic and occurred predominantly within a column of fiber cells, resulting in radially directed transport. In regions beyond the zone of nuclear loss, transport was more isotropic and occurred across columns of fiber cells. CONCLUSIONS: The cleavage of Cx46 is associated with a spatial redistribution of gap junction plaques. The distribution of gap junction plaques around the cell membrane can explain the observed directionality of intercellular solute transfer. The findings suggest that the processing and redistribution of gap junction proteins is central to controlling radial and circumferential solute gradients in different regions within the lens.

Animals↗
PubMed2003

Resolving morphology and antibody labeling over large distances in tissue sections.

Protein expression patterns are a primary determinant of tissue function and in this study we developed methods to study protein expression over macroscopic distances at subcellular levels of detail. Using the mammalian lens as a model tissue system, we show that by combining two-photon microscopy with novel image montage methods (fast beam blanking coupled with mathematical alignment tools) we have extended the limited field of view of laser scanning microscopes. To illustrate the utility of our approach, the distribution of connexin-46 was visualized across equatorial sections of the rat mammalian lens. By optimizing fixation protocols, good morphological preservation could be achieved over the thickness of the lens (approximately 4 mm) while preserving antigenicity of lens proteins. Using the same image data, changes in lens fiber cell morphology were mapped quantitatively by automatic image analysis routines. The methods presented should be generally applicable to any tissue system where changes in antibody labeling and tissue structure occur over large and small distances.

Animals↗
PubMed2003

Noise induces up-regulation of P2X2 receptor subunit of ATP-gated ion channels in the rat cochlea.

Regulation of P2X(2) receptor (P2X(2)R) expression in the rat cochlea in response to noise was analysed. Sustained loud sound (90-120 dB white noise, > 6 h), increased P2X(2)R mRNA and protein levels in rat organ of Corti and spiral ganglion (primary auditory neurones). P2X(2)R expression by the type I spiral ganglion neurones, which innervate the inner hair cells via the inner spiral plexus, was confirmed by confocal immunofluorescence. This also revealed increased P2X(2)R labelling of outer hair cell (OHC) stereocilia and cuticular plates, reflecting trafficking of greater numbers of ATP-gated ion channels assembled with P2X(2)R subunits to the transducer site. Whole-cell voltage clamp of OHC confirmed the noise-induced up-regulation of ATP-gated inward currents. These data indicate that regulation of P2X(2) receptor gene expression in the cochlea is adaptive, with sustained loud sound promoting increased transcription and translation specifically at sites regulating hearing sensitivity and auditory neurotrans-mission.

Adenosine Triphosphate↗
PubMed2003

Reduced contraction strength with increased intracellular [Ca2+] in left ventricular trabeculae from failing rat hearts.

Intracellular calcium ([Ca2+](i)) and isometric force were measured in left ventricular (LV) trabeculae from spontaneously hypertensive rats (SHR) with failing hearts and normotensive Wistar-Kyoto (WKY) controls. At a physiological stimulation frequency (5 Hz), and at 37 degrees C, the peak stress of SHR trabeculae was significantly (P < or = 0.05) reduced compared to WKY (8 +/- 1 mN mm(-2) (n = 8) vs. 21 +/- 5 mN mm(-2) (n = 8), respectively). No differences between strains in either the time-to-peak stress, or the time from peak to 50 % relaxation were detected. Measurements using fura-2 showed that in the SHR both the peak of the Ca2+ transient and the resting [Ca2+](i) were increased compared to WKY (peak: 0.69 +/- 0.08 vs. 0.51 +/- 0.08 microM(P < or = 0.1) and resting: 0.19 +/- 0.02 vs. 0.09 +/- 0.02 microM(P < or = 0.05), SHR vs. WKY, respectively). The decay of the Ca2+ transient was prolonged in SHR, with time constants of: 0.063 +/- 0.002 vs. 0.052 +/- 0.003 s (SHR vs. WKY, respectively). Similar results were obtained at 1 Hz stimulation, and for [Ca2+ ](o) between 0.5 and 5 mM. The decay of the caffeine-evoked Ca2+ transient was slower in SHR (9.8 +/- 0.7 s (n = 8) vs. 7.7 +/- 0.2 s (n = 8) in WKY), but this difference was removed by use of the SL Ca2+ -ATPase inhibitor carboxyeosin. Histological examination of transverse sections showed that the fractional content of perimysial collagen was increased in SHR compared to WKY (18.0 +/- 4.6 % (n = 10) vs. 2.9 +/- 0.9 % (n = 11) SHR vs. WKY, respectively). Our results show that differences in the amplitude and the time course of the Ca2+ transient between SHR and WKY do not explain the reduced contractile performance of SHR myocardium per se. Rather, we suggest that, in this animal model of heart failure, contractile function is compromised by increased collagen, and its three-dimensional organisation, and not by reduced availability of intracellular Ca2+.

Animals↗
PubMed2002

Estimation of the sarcoplasmic reticulum Ca2+ release flux underlying Ca2+ sparks.

Using a combination of experimental and numerical approaches, we have tested two different approaches to calculating the sarcoplasmic reticulum (SR) Ca2+ release flux, which gives rise to cardiac muscle Ca2+ sparks. By using two-photon excited spot photolysis of DM-Nitrophen, known Ca2+ release flux time courses were generated to provide the first experimental validation of spark flux reconstruction algorithms. These artificial Ca2+ sparks show that it is possible to calculate the SR Ca2+ release waveform with reasonable accuracy, provided the flux equations reasonably reflect the properties of the experimental system. Within cardiac muscle cells, we show that Ca2+ flux reconstruction is complicated by the substantial dye binding to proteins, a factor that has not been adequately addressed in previous flux reconstruction algorithms. Furthermore, our numerical experiments suggest that the calculated time course of release flux inactivation based on conventional flux reconstruction algorithms is likely to be in error. We therefore developed novel algorithms based on an explicit dye binding scheme. When these algorithm were applied to evoked Ca2+ sparks in rat cardiac ventricular myocytes, the reconstructed Ca2+ release waveform peaked in ~5 ms and decayed with a halftime of approximately 5 ms. The peak flux magnitude was 7-12 pA, suggesting that sparks must arise from clusters of >15 ryanodine receptors.

Algorithms↗
PubMedDate not supplied

Analysing cardiac excitation-contraction coupling with mathematical models of local control.

Cardiac excitation-contraction (E-C) coupling describes the process that links sarcolemmal Ca2+ influx via L-type Ca2+ channels to Ca2+ release from the sarcoplasmic reticulum via ryanodine receptors (RyRs). This process has proven difficult to study experimentally, and complete descriptions of how the cell couples surface membrane and intracellular signal transduction proteins to achieve both stable and sensitive intracellular calcium release are still lacking. Mathematical models provide a framework to test our understanding of how this is achieved. While no single model is yet capable of describing all features of cardiac E-C coupling, models of increasing complexity are revealing unexpected subtlety in the process. In particular, modelling has established a general failure of 'common-pool' models and has emphasized the requirement for 'local control' so that microscopic sub-cellular domains can separate local behaviour from the whole-cell average (common-pool) behaviour. The micro-architecture of the narrow diadic cleft in which the local control takes place is a key factor in determining local Ca2+ dynamics. There is still considerable uncertainty about the number of Ca2+ ions required to open RyRs within the cleft and various gating models have been proposed, many of which are in reasonable agreement with available experimental data. However, not all models exhibit a realistic voltage dependence of E-C coupling gain. Furthermore, it is unclear which model features are essential to producing reasonable gain properties. Thus, despite the success of local-control models in explaining many features of cardiac E-C coupling, more work will be needed to provide a sound theoretical basis of cardiac E-C coupling.

Adaptation, Physiological↗