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T Powell

Publications and source records attributed to T Powell.

At least 73 records · Page 4Linked to original sources
PubMed1991

The slowing of Ca2+ signals by Ca2+ indicators in cardiac muscle.

The use of high-affinity fluorescent probes for monitoring intracellular free Ca2+ in cardiac muscle is now widespread. We have investigated the consequences of introducing intracellular buffers with the properties of Fura-2 or Indo-1 on the action potential, Ca2+ transient and contractile activity of the myocardium. Our theoretical results suggest that, at the high intracellular concentrations of these fluorescent probes used on occasion to improve the signal-to-noise ratio of the emitted fluorescence, modulation of action potential profile and attenuation of the amplitudes of the Ca2+ transient and contraction can occur, together with subtle changes in the kinetics of these events.

Action Potentials↗
PubMed1991

Cytosolic free Ca2+ during operation of sodium-calcium exchange in guinea-pig heart cells.

1. Membrane current generated by the Na(+)-Ca2+ exchange mechanism was recorded in single guinea-pig ventricular myocytes using the whole-cell voltage-clamp technique and the intracellular free calcium concentration ([Ca2+]i) was monitored using the fluorescent probe Indo-1, applied intracellularly through a perfused patch pipette. The reversal potential of the exchanger (ENa, Ca) was measured from records of the 2 mM-Ni(2+)-sensitive current and used in an attempt to clamp [Ca2+]i at a level determined by the ionic compositions of the external and pipette solutions. 2. Measurements of ENa, Ca indicated that [Ca2+]i was close to that in the pipette solution when the holding potential was set at the ENa, Ca expected for a 3Na+:1Ca2+ exchanger. The measured value of ENa, Ca was more positive than the theoretical value when the membrane potential was held positive to ENa, Ca and the opposite was true when the holding potential was more negative than the expected ENa, Ca. 3. As Indo-1 diffused into the cell from the whole-cell clamp electrode, the intensities of the fluorescent signals measured at 405 and 480 nm increased with time, with no obvious saturation over a 10-45 min recording period. However, the ratio of these two signals reached a steady level within 5 min after rupture of the patch membrane, when the holding potential was set at the expected ENa, Ca of the exchanger. The intensity ratios measured using pipette solutions containing 600 and 803 nM [Ca2+] were almost equal to the ratios obtained extracellularly from internal solutions of identical compositions, but in experiments using pipette solutions having lower [Ca2+] the intensity ratios measured in myocytes were higher than those obtained extracellularly. 4. If the membrane was depolarized or hyperpolarized, the fluorescence ratio either increased or decreased, respectively. These changes in the fluorescence ratio were virtually blocked by the extracellular application of 2 mM-Ni2+. 5. When the concentration of bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) in the recording pipette was reduced from 30 to 1 mM, an increase in [Ca2+]i was observed during a depolarizing ramp pulse. The Ca2+ influx estimated by integrating the 2 mM-Ni(2+)-sensitive current during the pulse correlated with the increase in [Ca2+]i estimated from Indo-1 using the extracellular calibration curve, but the values of the influx determined directly from Indo-1 fluorescence were always larger than those calculated from the exchanger current.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Flash photolysis of intracellular caged GTP gamma S increases L-type Ca2+ currents in cardiac myocytes.

L-type calcium currents were recorded from isolated ventricular myocytes using standard patch-clamp methods. Rapid release of photolabile guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), tetralithium salt, by flash photolysis of intracellular caged-GTP gamma S increased the magnitude of the L-type calcium current: an effect independent of the ultraviolet flash per se or the production of photolytic by-products. This increase in calcium current was markedly reduced by intracellularly applied guanosine 5-O-(thiodiphosphate), trilithium salt or by an excess of GTP gamma S. It is therefore likely that rapid release of GTP gamma S intracellularly in the absence of an agonist can, via a G protein-mediated mechanism, cause L-type calcium current activation. In the presence of Rp-adenosine 3,5-mono-thionophosphate (Rp-cAMP), photoreleased GTP gamma S results in a transient and much reduced increase in the amplitude of the L-type calcium current. We conclude that activation of Gs coupled to adenylate cyclase and ultimately cAMP-dependent phosphorylation may be primarily responsible for L-type channel activation, although a fast membrane-delimited (direct) pathway, not involving cytoplasmic second messengers, may also contribute to this effect.

Animals↗
PubMed1991

Acute effects of adriamycin on the macromolecular organization of the cardiac muscle cell plasma membrane.

Adriamycin is a potent chemotherapeutic agent used in the treatment of human neoplastic disease. A major side effect limiting the use of this drug is its toxic effect on the heart, and congestive heart failure becomes an increasingly common complication as the cumulative dose of the drug rises. To learn more about the mechanism of adriamycin cardiotoxicity and, in particular, to investigate its initial effects on the cardiac muscle cell plasma membrane, isolated guinea pig myocytes were exposed to the drug in vitro. Plasma membrane macromolecular structure was examined by freeze-fracture electron microscopy of myocytes exposed to 0.1, 1 and 2 mM adriamycin for periods up to 105 minutes. The principal effect of adriamycin was rapid induction of smooth (protein-poor) domains in the membrane, with displacement and clustering of intramembrane particles (the structures representing integral membrane proteins). These effects, which are time-dependent and dose-dependent, culminate in the formation of saucer-shaped lesions in the membrane, which broadly resemble deformations of the membrane induced by polymyxin B. Both adriamycin and polymyxin are known to have the ability to interact selectively with anionic phospholipids. It is concluded that an important initial effect of adriamycin on cardiac muscle cells is alteration of the macromolecular architecture of the plasma membrane, probably through interaction with anionic phospholipids, and that this may represent the underlying cause of a range of sarcolemmal dysfunctions associated with exposure to the drug.

Animals↗
PubMed1990

Mitochondrial import of cytochrome c oxidase subunit VIIa in Saccharomyces cerevisiae. Identification of sequences required for mitochondrial localization in vivo.

Subunit VIIa of yeast cytochrome c oxidase is a small (59 amino acids) protein of the inner mitochondrial membrane that lacks a cleavable amino-terminal presequence. To identify regions within this polypeptide that are essential for its import, gene fusions were constructed using a leader peptide substitution vector (pLPS) developed in this laboratory (Glaser, S. M., Trueblood, C. E., Dircks, L. K., Poyton, R. O., and Cumsky, M. G. (1988) J. Cell. Biochem. 36, 275-287). In this vector, oligonucleotide sequences encoding all or part of subunit VIIa were fused in-frame with the coding region of mature cytochrome c oxidase subunit Va. The plasmid pLPS is ideal for assaying protein sequences for their ability to direct mitochondrial import in vivo since subunit Va's leader peptide is essential for import and because subunit V is required for cytochrome c oxidase activity and respiration. Strains containing these fusions but lacking both subunit V genes (COX5a and COX5b) were analyzed to determine whether the chimeric protein is directed to mitochondria. Our findings indicate that the amino-terminal 17 amino acids of subunit VIIa are sufficient to localize subunit Va to the mitochondrion and that a 6-amino acid-long region within the amino terminus (Gly8-Arg13) is essential. In addition, some import (approximately 10% of wild type) is observed with the highly charged carboxyl terminus of subunit VIIa, suggesting that the subunit may contain redundancy in its import information.

Amino Acid Sequence↗
PubMed1990

Integrity of the dissociated adult cardiac myocyte: gap junction tearing and the mechanism of plasma membrane resealing.

Dissociation of adult cardiac myocytes by collagenase perfusion techniques requires separation of the junctional contacts that link the cells physically, electrically and metabolically in the intact heart. Gap junctions, one of three types of intercellular junction present at the cardiac intercalated disc, are not split into their component membranes when myocytes are dissociated; they are ripped from the plasma membrane of one cell, to be retained by its neighbour. Partitioning of junctions in this way might be expected to constitute a serious threat to the ionic integrity of dissociated myocytes, but in practice, high yields of functionally intact cells, suitable for experimental studies, are routinely obtained. To explain this apparent paradox, repair mechanisms, operating to seal the membrane lesions caused by gap junction tearing, have been hypothesized, but evidence for their existence has previously been lacking. Using freeze-fracture electron microscopy, the present study identifies repair sites as smooth membrane domains that are continuous with the neighbouring plasma membrane, thus forming intact seals. That these structures are not chemically-induced artefacts is demonstrated by their presence in myocytes that were frozen directly from the living state. Subsarcolemmal vesicle clusters, detected in thin sections and freeze-fracture replicas, are associated with the smooth sealing domains. These structures may represent either rounded-up fragments of mechanically disrupted membrane or structures concerned with the synthesis of new lipid. From their freeze-fracture morphology, the sealing domains appear to be lipid-rich and protein-poor. Cytochemical studies using Ruthenium Red, cationized ferritin and lectins show in addition that they have a lower content of negatively-charged membrane components than the neighbouring plasma membrane, and that the carbohydrate residues normally associated with plasma membrane glycolipids and glycoproteins are absent.

Animals↗
PubMed1990

Chronic carbamazepine down-regulates adenosine A2 receptors: studies with the putative selective adenosine antagonists PD115,199 and PD116,948.

Carbamazepine (CBZ), an anticonvulsant with psychotropic and anti-pain properties, has been reported to displace ligands at adenosine binding sites. This paper describes biochemical and behavioural studies in rodents comparing CBZ to the adenosine agonists L-phenylisopropyl-adenosine (L-PIA) and N-ethylcarboxamido-adenosine (NECA), the new antagonists PD116,948 and PD115,199 which are also relatively A1 and A2 specific respectively, and the mixed antagonists theophylline and caffeine, attempting to determine functional correlates of the binding studies. Changes in cAMP synthesis and behavioural syndromes produced by the drugs, alone and in combination, were monitored. Classification of the observed effects in terms of A1 and A2 activity was complex, probably due to functional interactions between A1 and A2 subtypes. Nevertheless, it was found that chronic CBZ administration (0.25% in food for 3 days, followed by 0.5% for 11 days) produced a pattern of interaction identical to that of PD115,199 (10-100 mg/kg IP). Thus, both treatments attenuated the behavioural syndrome produced by L-PIA (0.1 or 0.5 mg/kg SC), but did not affect that produced by NECA (0.03 mg/kg SC). CBZ mildly increased hypoactivity after clonidine (0.2 mg/kg IP) which was used as a control. By contrast, the A1 antagonist PD116,948 (0.1-10 mg/kg IP) antagonised both behavioural syndromes. Similarly in the biochemical experiments both chronic CBZ and PD115,199 (10-100 microM) reduced stimulation of cAMP synthesis by L-PIA (confirming that this is mediated by A2 receptors), while only basal cAMP synthesis was affected by PD116,948 (10 microM) and theophylline (60 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗
PubMed1990

Comparison of intracellular pH transients in single ventricular myocytes and isolated ventricular muscle of guinea-pig.

1. Intracellular pH was recorded (double-barrelled pH-selective microelectrodes) in single ventricular myocytes and whole papillary muscles isolated from guinea-pig heart. Both preparations were acid-loaded by various manoeuvres (addition and removal of external NH4Cl or CO2) in order that a comparison could be made of the size and speed of intracellular pH changes and hence of the apparent intracellular buffering power (beta). 2. For the same acid-loading procedure, the size of intracellular pH (pHi) changes was about threefold larger in the isolated myocyte than in whole papillary muscle. The rate of initial acid loading as well as the subsequent rate of pHi recovery (caused by acid extrusion from the cell) were also threefold faster in the myocyte. Estimates of apparent intrinsic (non-CO2) buffering power, based upon the size of pHi changes during acid loading, were 15-20 mmol l-1 for the myocyte and about 70 mmol l-1 for whole muscle. This latter value is similar to previous estimates of beta in heart. 3. When acid extrusion was reduced by applying a high dose of amiloride (1 mmol l-1), then the size of the pHi change during acid loading increased greatly in papillary muscle but changed much less in the myocyte; beta now appeared to be about 30 mmol l-1 in whole muscle but remained essentially unchanged in the myocyte. 4. We conclude that previous values for beta in cardiac muscle have been greatly overestimated because of the presence of sarcolemmal acid extrusion. Paradoxically, this error in estimating beta is far less evident in the isolated myocyte. We suggest that this is because a much more rapid acid loading is achievable in the myocyte so that acid loading will be blunted less by acid extrusion than in whole muscle. We present a simple mathematical model that demonstrates this phenomenon. We conclude that beta in ventricular muscle is likely to resemble that measured in the isolated myocyte, i.e. 15-20 mmol l-1. 5. Slow acid loading in whole ventricular muscle will also affect the kinetics of pHi changes. The model indicates that the rate of pHi recovery from an acid load in papillary muscle does not reflect the pHi dependence of acid extrusion. Instead, it is heavily influenced by the slow rate of acid loading. This emphasises that great care should be taken when interpreting the kinetics of pHi changes in multicellular ventricular preparations.

Amiloride↗
PubMed1990

Growth failure secondary to moyamoya syndrome.

We describe a boy who presented at the age of 7 years with short stature due to hypopituitarism. Six months after starting appropriate hormone replacement treatment at the age of 8 he suffered his first generalised convulsion. Further neuroradiological investigation led to the diagnosis of moyamoya syndrome.

Arterial Occlusive Diseases↗
PubMed1990

Current trends in the radiology of the head and neck.

In the head and neck, the changes in radiological practice resulting from the introduction of high resolution computed tomography and magnetic resonance imaging have been far reaching. They relate principally to the greatly improved soft tissue information which is now obtainable, and to the additional possibilities for characterization of soft tissues by intravenous contrast enhancement.

Angiography↗
PubMed1989

Calcium movements during each heart beat.

Transsarcolemmal calcium movements are closely related to force generation in the heart. It is important to understand the transport pathways that control these movements of calcium across the sarcolemmal membrane. In the normal, beating heart, sodium-calcium exchange appears to be an important mechanism for the extrusion of calcium from the cell. The kinetics of this exchange are dependent upon the characteristics of the cell action potential. Calcium efflux via sodium-calcium exchange may be sufficient to balance calcium entry through calcium channels during the action potential.

Action Potentials↗
PubMed1989

Li+ inhibition of membrane current responses to epinephrine in guinea-pig ventricular cells.

Membrane currents of guinea-pig ventricular myocytes were recorded using the whole-cell voltage clamp method. The epinephrine-induced increase in Ca2+ current (2.9 +/- 0.5 times control) was reduced (1.8 +/- 0.3 times) by replacing Na+ with Li+ in the bathing solution. In addition, 0.5 microM epinephrine increased a time-independent membrane conductance in the Na+ external solution, having a reversal potential of -19 +/- 3 mV (epinephrine-induced current). In the Li+ external solution, however, 0.5 microM epinephrine failed to induce the epinephrine-induced current. The findings are consistent with the reported Li+ inhibition of GTP-binding protein and/or adenylate cyclase.

Animals↗
PubMed1989

Prolapsed thoracic intervertebral disc: the importance of CT assisted myelography.

We present six new cases of prolapsed thoracic intervertebral disc (PTIVD), and review the clinical and radiological features, particularly with regard to the role of computed tomography (CT) examination in the diagnosis and management. In patients with atypical lower limb pain, particularly when associated with sphincter disturbances, lumbar myelography is incomplete unless supplemented by an examination of the conus region and lower thoracic cord. The supplementary use of CT in suspected PTIVD is of considerable value to the surgeon in determining the size, position, and volume of the sequestration, and the extent of excavation of the spinal cord by prolapsed disc material.

Aged↗
PubMed1989

Evidence of reduced 'cognitive inhibition' in schizophrenia.

An experiment is described which investigated cognitive inhibition in schizophrenia. It is noted that both the abnormal and cognitive literatures use the concept of inhibition. Frith (1979) suggests that the more cognitive symptoms of schizophrenia may be due to the failure to limit the current contents of consciousness due to a failure adequately to inhibit the output of preconscious processes. Current thinking in cognitive psychology suggests that in the process of selective attention there is active inhibition of distractor information. A technique used to investigate this is termed negative priming (Tipper, 1985). The general nature of this paradigm is as follows: if a distractor, which has been previously ignored, is response, due to inhibition of the information when it was originally a distractor. It was found that inhibition of such distracting information was reduced in schizophrenics. This finding is seen as providing some support for Frith's (1979) theory that the cognitive symptoms of schizophrenia are due to awareness of processes that normally occur preconsciously.

Adult↗
PubMed1989

Sodium-calcium exchange during the action potential in guinea-pig ventricular cells.

1. Slow inward tail currents attributable to electrogenic sodium-calcium exchange can be recorded by imposing hyperpolarizing voltage clamp pulses during the normal action potential of isolated guinea-pig ventricular cells. The hyperpolarizations return the membrane to the resting potential (between -65 and -88 m V) allowing an inward current to be recorded. This current usually has peak amplitude when repolarization is imposed during the first 50 ms after the action potential upstroke, but becomes negligible once the final phase of repolarization is reached. The envelope of peak current tail amplitudes strongly resembles that of the intracellular calcium transient recorded in other studies. 2. Repetitive stimulation producing normal action potentials at a frequency of 2 Hz progressively augments the tail current recorded immediately after the stimulus train. Conversely, if each action potential is prematurely terminated at 0.1 Hz, repetitive stimulation produces a tail current much smaller than the control value. The control amplitude of inward current is only maintained if interrupted action potentials are separated by at least one full 'repriming' action potential. These effects mimic those on cell contraction (Arlock & Wohlfart, 1986) and suggest that progressive changes in tail current are controlled by variations in the amplitude and time course of the intracellular calcium transient. 3. When intracellular calcium is buffered sufficiently to abolish contraction, the tail current is abolished. Substitution of calcium with strontium greatly reduces the tail current. 4. The inward tail current can also be recorded at more positive membrane potentials using standard voltage clamp pulse protocols. In this way it was found that temperature has a large effect on the tail current, which can change from net inward at 22 degrees C to net outward at 37 degrees C. The largest inward currents are usually recorded at about 30 degrees C. It is shown that this effect is attributable predominantly to the temperature sensitivity of activation of the delayed potassium current, iK, whose decay can then mask the slow tail current at high temperatures. 5. Studies of the relationship between the tail current and the membrane calcium current, iCa, have been performed using a method of drug application which is capable of perturbing iCa in a very rapid and highly reversible manner. Partial block of iCa with cadmium does not initially alter the size of the associated inward current tail. When iCa is increased by applying isoprenaline, the percentage augmentation of the associated tail current is much greater but occurs more slowly.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1989

Fate of gap junctions in isolated adult mammalian cardiomyocytes.

The fate of gap junctions in dissociated adult myocytes, maintained for up to 22 hours in culture medium, was investigated by semiquantitative analysis of thin sections and by freeze-fracture electron microscopy. Gap junctions in the dissociated myocyte are intact bimembranous structures seen either as invaginated surface-located structures or as annular profiles in the cytoplasm. Surface-located junctions are sealed from the exterior by a sheet of nonjunctional membrane originating (together with the "outer" junctional membrane) from the formerly neighboring cell. Serial sectioning was used to establish that at least part of the annular gap junction population in the freshly isolated myocyte represents truly discrete cytoplasmic vesicles; thus, some gap junctions are rapidly endocytosed after myocyte separation. Analysis of the surface-located-to-annular gap junction ratio suggested that no further endocytosis occurred in rabbit and cat myocytes maintained for 22 and 15 hours, respectively. Guinea pig myocytes, by contrast, did appear to continue endocytosis in culture. Analysis of the distance of gap junctional structures from the cell surface suggested that little if any inward migration of gap junction vesicles occurred. Hypoxia had no detectable effect on the internalization or inward movement of gap junctions. The quantity of ultrastructurally detectable gap junction membrane appeared to remain constant over time, as did the incidence of "complex structures" (i.e., annular gap junction profiles with features previously suggested to represent degradation). New gap junction formation was negligible, and a reappraisal of the nature of "complex structures" led to the conclusion that the origin of these structures need not be related to degradation. Taken together, the findings suggest that degradation and disappearance of gap junctional membrane after isolation of the mature myocyte constitute a much slower process than previously believed, and the possibility that the cardiac gap junction protein has a longer half-life than its counterpart in liver remains open.

Animals↗