Biomedical subjects

R A Bouchard

Publications and source records attributed to R A Bouchard.

At least 19 recordsLinked to original sources

PubMed1999

Modulation of iron uptake in heart by L-type Ca2+ channel modifiers: possible implications in iron overload.

Heart failure is the leading cause of mortality in patients with transfusional iron (Fe) overload in which myocardial iron uptake ensues via a transferrin-independent process. We examined the ability of L-type Ca2+ channel modifiers to alter Fe2+ uptake by isolated rat hearts and ventricular myocytes. Perfusion of rat hearts with 100 nmol/L 59Fe2+ and 5 mmol/L ascorbate resulted in specific 59Fe2+ uptake of 20.4+/-1.9 ng of Fe per gram dry wt. Abolishing myocardial electrical excitability with 20 mmol/L KCl reduced specific 59Fe2+ uptake by 60+/-7% (P<0.01), which suggested that a component of myocardial Fe2+ uptake depends on membrane voltage. Accordingly, 59Fe2+ uptake was inhibited by 10 micromol/L nifedipine (45+/-12%, P<0.02) and 100 micromol/L Cd2+ (86+/-3%; P<0. 001) while being augmented by 100 nmol/L Bay K 8644 (61+/-18%, P<0. 01) or 100 nmol/L isoproterenol (40+/-12%, P<0.05). By contrast, uptake of 100 nmol/L ferric iron (59Fe3+) was significantly lower (1. 4+/-0.3 ng Fe per gram dry wt; P<0.001) compared with divalent iron. These data suggest that a component of Fe2+ uptake into heart occurs via the L-type Ca2+ channel in myocytes. To investigate this further, the effects of Fe2+ on cardiac myocyte L-type Ca2+ currents were measured. In the absence of Ca2+, noninactivating nitrendipine-sensitive Fe2+ currents were recorded with 15 mmol/L [Fe2+]o. Low concentrations of Fe2+ enhanced Ca2+ current amplitude and slowed inactivation rates, which was consistent with Fe2+ entry into the cell, whereas higher Fe2+ levels caused dose-dependent decreases in peak current. Fe3+ had no effect on current amplitude or decay. Combined, our data suggest that myocardial Fe2+ uptake occurs via L-type Ca2+ channels and that blockade of these channels might be useful in the treatment of patients with excessive serum iron levels.

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

PubMed1996

Action potential duration modulates calcium influx, Na(+)-Ca2+ exchange, and intracellular calcium release in rat ventricular myocytes.

The experimental work summarized in this paper and described in more detail in our previous publication demonstrates a very important functional role for Na(+)-Ca2+ exchange in intracellular Ca2+ homeostasis in ventricular myocytes from rat hearts. Ca2+ homeostasis in mammalian cardiac myocytes can be considered to be the result of four interactive processes: (i) Ca2+ influx through L-type Ca2+ channels, (ii) Ca2+ release from the SR and its subsequent re-uptake, (iii) intracellular Ca/+ buffering, and (iv) Ca2+ extrusion across the sarcolemma. Our results demonstrate a number of interesting features of these processes. (1) When the action potential voltage-clamp technique is used to identify the size and time-course of Ca2+ fluxes during the action potential, both the peak current and the associated influx of Ca2+ are relatively large as was previously demonstrated by Isenberg and his colleagues. (2) Nevertheless, this source of Ca2+ is unable, by itself, to produce a significant twitch, which is consistent with previous data from rat ventricle. (3) This Ca2+ influx, however, does represent the trigger for SR Ca2+ release. (4) The Na(+)-Ca2+ exchanger on the SR is able, on average, to extrude all the Ca2+ which enters through L-type Ca2+ channels, although it provides relatively little Ca2+, i.e., during the course of the normal action potential there is no significant reverse Na(+)-Ca2+ exchange activity, at least under our experimental conditions. Our results also suggest that although the L-type Ca2+ current cannot by itself trigger and control contraction its amplitude, frequency, and time-course can alter the rate and the extent of Ca2+ release from the SR. Recently, detailed mathematical formulations and a direct demonstration of some of these phenomena have been published. Stern and Stern and Lakatta predicted more than three years ago that the concentration and the time-course of change in concentration of Ca2+ very near the release sites of the SR may be critical determinants of the overall release process. Within the past year Wier and his colleagues and also Lederer et al. have combined electrophysiological measurements with recordings of localized intracellular Ca2+ (made using a confocal microscope) and have shown that rapid, and relatively large, but very localized changes in intracellular Ca2+ due to Ca2+ influx through L-type Ca2+ channels are responsible for triggering, and to some extent, controlling the release of Ca2+ from the SR. However, it has also been shown that this release depends importantly on the loading or priming state of the SR. Perhaps not surprisingly, the massive release of Ca2+ from the SR can, itself, alter the pattern of subsequent SR release events (cf. Ref. 46) and the time-course of Ca2+ influx through the L-type Ca2+ channels. Thus, although our relatively crude measurements have clearly demonstrated the relationship between L-type Ca2+ channel activity and Na+-Ca2+ exchanger function during a normal cardiac action potential in rat ventricle, they fall far short of any delineation of the functional roles of either of these processes in overall Ca2+ homeostasis. This additional information can, in principle, be obtained from studies in which cellular microanatomy can be visualized dynamically in conjunction with localized changes in intracellular Ca2+ as well as Ca2+ of L-type Ca2+ channels, SR release, and cell shortening.

Action Potentials↗

PubMed1996

Maize seedlings show cell-specific responses to heat shock as revealed by expression of RNA and protein.

The cellular localization of heat-shock proteins has been described in a number of experimental animal systems but is not well defined in plant systems. Sense and antisense RNA transcripts from the open reading frame (ORF) of 18-kDa maize heat-shock protein genes were employed in in situ hybridizations of inbred Oh43 radicles and plumules to reveal the locations of their mRNAs. Localization of the specific mRNAs to the younger meristematic cells of the root-tips and shoot-tips and also to the densely cytoplasmic cells of the vasculature was observed routinely. The ORF of one of our 18-kDa genes was cloned into an expression vector, and the 161-amino acid polypeptide was used to raise antibodies. Using a Fast Red procedure, the cellular positions of the heat-shock protein-antibody conjugates were observed in sections similar to those employed in the antisense RNA in situ hybridizations. The localization of the antibody appears to parallel closely the patterns of distribution of the mRNAs.

Amino Acid Sequence↗

PubMed1995

Effects of action potential duration on excitation-contraction coupling in rat ventricular myocytes. Action potential voltage-clamp measurements.

Although each of the fundamental processes involved in excitation-contraction coupling in mammalian heart has been identified, many quantitative details remain unclear. The initial goal of our experiments was to measure both the transmembrane Ca2+ current, which triggers contraction, and the Ca2+ extrusion due to Na(+)-Ca2+ exchange in a single ventricular myocyte. An action potential waveform was used as the command for the voltage-clamp circuit, and the membrane potential, membrane current, [Ca2+]i, and contraction (unloaded cell shortening) were monitored simultaneously. Ca(2+)-dependent membrane current during an action potential consists of two components: (1) Ca2+ influx through L-type Ca2+ channels (ICa-L) during the plateau of the action potential and (2) a slow inward tail current that develops during repolarization negative to approximately -25 mV and continues during diastole. This slow inward tail current can be abolished completely by replacement of extracellular Na+ with Li+, suggesting that it is due to electrogenic Na(+)-Ca2+ exchange. In agreement with this, the net charge movement corresponding to the inward component of the Ca(2+)-dependent current (ICa-L) was approximately twice that during the slow inward tail current, a finding that is predicted by a scheme in which the Ca2+ that enters during ICa is extruded during diastole by a 3 Na(+)-1 Ca2+ electrogenic exchanger. Action potential duration is known to be a significant inotropic variable, but the quantitative relation between changes in Ca2+ current, action potential duration, and developed tension has not been described in a single myocyte. We used the action potential voltage-clamp technique on ventricular myocytes loaded with indo 1 or rhod 2, both Ca2+ indicators, to study the relation between action potential duration, ICa-L, and cell shortening (inotropic effect). A rapid change from a "short" to a "long" action potential command waveform resulted in an immediate decrease in peak ICa-L and a marked slowing of its decline (inactivation). Prolongation of the action potential also resulted in slowly developing increases in the magnitude of Ca2+ transients (145 +/- 2%) and unloaded cell shortening (4.0 +/- 0.4 to 7.6 +/- 0.4 microns). The time-dependent nature of these effects suggests that a change in Ca2+ content (loading) of the sarcoplasmic reticulum is responsible. Measurement of [Ca2+]i by use of rhod 2 showed that changes in the rate of rise of the [Ca2+]i transient (which in rat ventricle is due to the rate of Ca2+ release from the sarcoplasmic reticulum) were closely correlated with changes in the magnitude and the time course of ICa-L.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗

PubMed1993

The independent stage-specific expression of the 18-kDa heat shock protein genes during microsporogenesis in Zea mays L.

The small (18-kDa) heat shock proteins (hsps) of maize are encoded by a complex multigene family. In a previous report, we described the genetic information from cDNAs encoding two different members of the family. In this communication, we report the isolation and characterization of cDNA and genomic clones encoding information for a third member of this hsp family (c/gMHSP18-1). DNA fragments containing nucleotide sequences common to, or specific for, each of these characterized 18-kDa genes were prepared and used as probes to assess the expression of these genes during microsporogenesis and development of the gametophyte in an inbred line of maize (Oh43). Our results demonstrate (1) that mRNA transcripts encoding the 18-kDa hsps are expressed and/or accumulate during microsporogenesis, and (2) that genes encoding two of the characterized 18-kDa hsps are expressed and/or accumulate independently, in a stage-specific manner during microsporogenesis. These observations imply that the stage-specific expression of particular 18-kDa hsp genes results from gene-specific regulation during microsporogenesis and gametophyte development rather than from an overall activation of the heat shock or stress response.

DNA↗

PubMed1993

Characterization of two maize HSP90 heat shock protein genes: expression during heat shock, embryogenesis, and pollen development.

We have isolated two genes from Zea mays encoding proteins of 82 and 81 kD that are highly homologous to the Drosophila 83-kD heat shock protein gene and have analyzed the structure and pattern of expression of these two genes during heat shock and development. Southern blot analysis and hybrid select translations indicate that the highly homologous hsp82 and hsp81 genes are members of a small multigene family composed of at least two and perhaps three or more gene family members. The deduced amino acid sequence of these proteins based on the nucleotide sequence of the coding regions shows 64-88% amino acid homology to other hsp90 family genes from human, yeast, Drosophila, and Arabidopsis. The promoter regions of both the hsp82 and hsp81 genes contain several heat shock elements (HSEs), which are putative binding sites for heat shock transcription factor (HSF) commonly found in the promoters of other heat shock genes. Gene-specific oligonucleotide probes were synthesized and used to examine the mRNA expression patterns of the hsp81 and hsp82 genes during heat shock, embryogenesis, and pollen development. The hsp81 gene is only mildly heat inducible in leaf tissue, but is strongly expressed in the absence of heat shock during the pre-meiotic and meiotic prophase stages of pollen development and in embryos, as well as in heat-shocked embryos and tassels. The hsp82 gene shows strong heat inducibility at heat-shock temperatures (37-42 degrees C) and in heat shocked embryos and tassels but is only weakly expressed in the absence of heat shock. Promoter-GUS reporter gene fusions made and analyzed by transient expression assays in Black Mexican Sweet (BMS) Maize protoplasts also indicate that the hsp82 and hsp81 are regulated differentially. The hsp82 promoter confers strong heat-inducible expression of the GUS reporter gene in heat-treated cells (60- to 80-fold over control levels), whereas the hsp81 promoter is only weakly heat inducible (5- to 10-fold over control levels).

Amino Acid Sequence↗

PubMed1993

Heterogeneity of action potential waveforms and potassium currents in rat ventricle.

OBJECTIVE: The ionic mechanisms for differences in action potential waveforms in rat left ventricle were studied by recording L-type Ca2+ current, transient outward K+ current, and inwardly rectifying background K+ current in single myocytes. METHODS: Single cells were obtained from adult rat hearts by enzymatic dispersion of tissue segments from the epicardium at the apex and the endocardium at the base of the left ventricle. Whole cell voltage clamp methods together with cell shortening measurements were used to identify the K+ currents involved in early and late repolarisation and to correlate changes in action potential shape with inotropic responses. 4-Aminopyridine was used to block the transient outward K+ current, I(t), to evaluate the contribution of this current to repolarisation. RESULTS: Action potential recordings demonstrated that cells from endocardial tissue at the base of the left ventricle have a considerably longer action potential than those from epicardial tissue at the apex. 4-Aminopyridine had a much more pronounced action potential lengthening and inotropic effects on cells from epicardium than on myocytes from endocardium suggesting that I(t) is larger in the epicardium. Voltage clamp measurements confirmed this. In contrast, the L-type Ca2+ current, the resting membrane potential, and the inwardly rectifying background K+ current were very similar in these two regions of left ventricle. CONCLUSIONS: One significant factor contributing to the heterogeneity of action potential waveforms in rat left ventricle is a differential distribution of a Ca+ independent transient outward K+ current, I(t). Regional differences in action potential duration have important implications for the gradient of repolarisation in rat left ventricle, for the genesis of the T wave of the electrocardiogram, and for both electrical and mechanical restitution (refractoriness).

4-Aminopyridine↗

PubMed1993

Regulation of unloaded cell shortening by sarcolemmal sodium-calcium exchange in isolated rat ventricular myocytes.

1. Regulation of unloaded cell shortening and relaxation by sarcolemmal Na(+)-Ca2+ exchange was investigated in rat ventricular myocytes. Contraction of single cells at 22 +/- 1 degrees C was measured simultaneously with membrane current and voltage using the whole-cell voltage clamp technique in combination with a video edge-detection device. 2. The extent of mechanical activation (cell shortening amplitude) was strongly dependent on diastolic membrane potential over the voltage range -140 to -50 mV. This voltage sensitivity of contraction was abolished completely when a recently described inhibitory peptide of the cardiac Na(+)-Ca2+ exchanger (XIP, 2 x 10(-5) M) was present in the recording pipette, demonstrating that in rat ventricular cells Na(+)-Ca2+ exchange is modulated by diastolic membrane potential. 3. Possible influences of Na(+)-Ca2+ exchange on contraction were studied from a holding potential of -80 mV. Depolarizations (-50 to +60 mV) resulted in a bell-shaped shortening-voltage (S-V) relationship. These contractions were suppressed completely by either Cd2+ (10(-4) M) or verapamil (10(-5) M), but remained unchanged during superfusion with tetrodotoxin (TTX, 1.5 x 10(-5) M), when [NA+]o was reduced from 140 to 10 mM by substitution with either Li+ or Cs+ ions or when pipette Na+ was varied between 8 and 13 mM. XIP (2 x 10(-5) M) increased the magnitude and duration of twitch contractions, but had no effect on the shape of the S-V relationship. Thus, the Ca2+ current but not the Na+ current or Ca2+ influx due to reversed Na(+)-Ca2+ exchange can release Ca2+ from the sarcoplasmic reticulum (SR) under these experimental conditions. 4. The effect of the rate of repolarization on cell shortening was studied under voltage clamp by applying ramp waveforms immediately following the depolarizations which activated contraction. Although slowing of the rate of repolarization had no effect on the first contraction following a train of conditioning depolarizations, a positive inotropic effect developed thereafter. 5. Caffeine (10 mM) was applied to determine whether Na(+)-Ca2+ exchange and/or Ca2+ sequestration/buffering by the sarcoplasmic reticulum were primarily responsible for these inotropic effects. In the presence of caffeine the positive inotropic effect developed fully during the first test depolarization. Changes in the rate of repolarization had much less effect on shortening in cells dialysed intracellularly with XIP (2 x 10(-5) M). In combination, these results suggest that the changes in the inotropic effects resulting from changes in rate of repolarization may be due to altered loading and release of Ca2+ from the SR.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1993

Role of sodium-calcium exchange in activation of contraction in rat ventricle.

1. The functional role of reverse Na(+)-Ca2+ exchange in the activation of contraction of rat ventricular myocytes has been studied. Mechanical activity of single cells, measured as unloaded cell shortening, was recorded simultaneously with membrane current and voltage using a single microelectrode voltage clamp and a video edge detection device. 2. The voltage dependence of contraction was studied by applying trains of depolarizations. At test potentials between +20 and +80 mV (under conditions where large outward currents were activated) a plateau on the shortening vs. voltage (S-V) relationship was observed. Significant cell shortening also occurred at test potentials between -70 and -40 mV; and these contractions were accompanied by large inward Na+ currents. We have investigated the ionic mechanisms for three components of the S-V relation in rat ventricle: (i) shortening which occurs between -70 and -40 mV and is thought to be dependent on the sodium current; (ii) phasic contractions in the voltage range -40 to +40 mV where the L-type Ca2+ current is present; (iii) the plateau of the S-V relation at strongly depolarized voltages where reverse Na(+)-Ca2+ exchange may occur. 3. Experiments in which two independent microelectrode impalements were made in a single myocyte showed that during activation of contraction at test potentials between -70 and -40 mV, and during very large depolarizations (+20 to +80 mV), there were significant deviations of the measured membrane potential from the applied voltages. Activation of cell shortening in these voltage ranges could be eliminated by electronic series resistance compensation, which significantly reduced these voltage errors. Consistent with these findings, when tetrodotoxin (TTX) and 4-aminopyridine (4-AP) were used to block inward Na+ and transient outward K+ currents, respectively, no significant voltage errors were present and a bell-shaped shortening-voltage (S-V) relationship was obtained. 4. When Na+ and K+ currents were blocked, depolarizations from holding potentials of either -80 or -50 mV demonstrated that the threshold for activation of contraction was about -30 mV, and that the voltage dependence of peak shortening was very similar to that of the L-type Ca2+ current (ICa,L). These contractions were suppressed completely by either Cd2+ or ryanodine, showing that activation of cell shortening was due to Ca2+ influx through L-type channels which induced release of Ca2+ from the sarcoplasmic reticulum (SR). No T-type calcium currents were observed.(ABSTRACT TRUNCATED AT 400 WORDS)

4-Aminopyridine↗

PubMed1992

Contribution of sarcolemmal sodium-calcium exchange and intracellular calcium release to force development in isolated canine ventricular muscle.

The aim of this work was to determine the relationship between peak twitch amplitude and sarcoplasmic reticulum (SR) Ca2+ content during changes of stimulation frequency in isolated canine ventricle, and to estimate the extent to which these changes were dependent upon sarcolemmal Na(+)-Ca2+ exchange. In physiological [Na+]o, increased stimulation frequency in the 0.2-2-Hz range resulted in a positive inotropic effect characterized by an increase in peak twitch amplitude and a decrease in the duration of contraction, measured as changes in isometric force development or unloaded cell shortening in intact muscle and isolated single cells, respectively. Action potentials recorded from single cells indicated that the inotropic effect was associated with a progressive decrease of action potential duration and a marked reduction in average time spent by the cell near the resting potential during the stimulus train. The frequency-dependent increase of peak twitch force was correlated with an increase of Ca2+ uptake into and release from the SR. This was estimated indirectly using the phasic contractile response to rapid (less than 1 s) lowering of perfusate temperature from 37 degrees C to 0-2 degrees C and changes of twitch amplitude resulting from perturbations in the pattern of electrical stimulation. Lowering [Na+]o from 140 to 70 mM resulted in an increase of contractile strength, which was accompanied by a similar increase of apparent SR Ca2+ content, both of which could be abolished by exposure to ryanodine (1 x 10(-8) M), caffeine (3 x 10(-3) M), or nifedipine (2 x 10(-6) M). Increased stimulation frequency in 70 mM [Na+]o resulted in a negative contractile staircase, characterized by a graded decrease of peak isometric force development or unloaded cell shortening. SR Ca2+ content estimated under identical conditions remained unaltered. Rate constants derived from mechanical restitution studies implied that the depressant effect of increased stimulation frequency in 70 mM [Na+]o was not a consequence of a decreased rate of refilling of a releasable pool of Ca2+ within the cell. These results demonstrate that frequency-dependent changes of contractile strength and intracellular Ca2+ loading in 140 mM [Na+]o require the presence of a functional sarcolemmal Na(+)-Ca2+ exchange process. The possibility that the negative staircase in 70 mM [Na+]o is related to inhibition of Ca(2+)-induced release of Ca2+ from the SR by various cellular mechanisms is discussed.

Animals↗

PubMed1992

Mechanisms for the positive inotropic effect of alpha 1-adrenoceptor stimulation in rat cardiac myocytes.

alpha 1-Adrenoceptor activation can enhance myocardial contractility, and two possible inotropic mechanisms are an increase in myofilament Ca2+ sensitivity and action potential prolongation, which can increase net Ca2+ entry into cells. In adult rat ventricular myocytes (bath Ca2+, 1 mM; stimulated at 0.2-0.5 Hz), the drug 4-aminopyridine and the whole-cell voltage clamp have been used to control Ca2+ entry and differentiate between the two mechanisms. At 22-23 degrees C the specific alpha 1-adrenoceptor agonist methoxamine (100 microM) prolonged action potential duration at 50% repolarization from 55 +/- 2 to 81 +/- 5 msec, delayed time to peak contraction, and increased shortening amplitude from 5.3 +/- 0.6 to 7.8 +/- 1 microns (n = 18). Reduction of the transient outward current and other K+ currents by methoxamine was the major cause of action potential prolongation in rat myocytes with little change in the L-type calcium current. Block of the transient outward current with 2 mM 4-aminopyridine prolonged action potential duration from 52 +/- 6 to 98 +/- 12 msec and increased unloaded cell shortening from 2.9 +/- 0.4 to 6.6 +/- 0.6 microns (n = 4). Subsequently, methoxamine no longer increased cell shortening, although significant potentiation of twitch amplitude was still seen after a brief rest interval. In voltage-clamp experiments, with 70-500-msec pulses, although membrane currents were reduced, methoxamine had no positive inotropic effect and reduced cell shortening from 5.3 +/- 0.7 to 4.97 +/- 0.8 microns at pulse potentials positive to -40 mV. Similar alpha 1-adrenoceptor responses were observed at 35 degrees C during action potential and voltage-clamp experiments, which could be blocked by 10 microM prazosin. In myocytes loaded with the Ca2+ indicator indo-1, alpha 1-adrenoceptor stimulation or 4-aminopyridine both increased cell contraction and intracellular Ca2+ transients by similar amounts. As in unloaded cells, prior exposure to 4-aminopyridine prevented any inotropic effect of methoxamine without changing the systolic intracellular Ca2+ transient. The results indicated that under our experimental conditions positive inotropy in rat cardiomyocytes on exposure to alpha 1-adrenoceptor agonists was strongly correlated with the action potential prolongation that accompanied K+ current reduction. In addition, modulation of K+ channels could occur independent of changes in contractility and/or [Ca2+]i.

4-Aminopyridine↗

PubMed1991

Isolation and characterization of a small heat shock protein gene from maize.

A maize (Zea mays L.) genomic clone (Zmempr 9') was isolated on the basis of its homology to a meiotically expressed Lilium sequence. Radiolabeled probe made from the maize genomic clone detected complementary RNA at high fidelity. Furthermore, it hybridized to RNA isolated from staged (an interval that is coincident with meiotic prophase) maize tassel spikelets. Complimentary RNA was strongly (at least 50-fold) induced during heat shock of maize somatic tissue and appeared as a single size class in Northern blot hybridizations. Sequencing of the complete coding region of Zmempr 9' confirmed the homology of the inferred amino acid sequence to other small heat shock proteins. Consensus sequences found in the flanking regions corresponded to the usual signals for initiation of RNA transcription, polyadenylate addition, and the induction of heat shock genes. The latter sequences conferred heat shock-specific transient expression in electroporated protoplasts when cloned into promoterless reporter gene plasmid constructs. Hybrid-selected translations revealed specific translation products ranging from 15 to 18 kilodaltons, providing evidence that this gene is a member of a related multigene family. We therefore conclude that this maize genomic DNA clone, recovered through its homology to clones for meiotic transcripts in lily, represents a genuine maize small heat shock protein gene.

Journal Article↗

PubMed1991

Influence of experimental diabetes on sarcoplasmic reticulum function in rat ventricular muscle.

We examined whether the decrease in cardiac contractility in streptozotocin-induced diabetes in the rat is accompanied by reduced or excessive loading of the sarcoplasmic reticulum (SR) with Ca2+. Pooled SR Ca2+ content and fractional release on stimulation were estimated with rapid cooling contracture (RCC) and twitch height measurements, respectively. Interval-force relation was studied to assess the ability of diabetic tissue to alter the relative contribution of SR Ca2+ for contraction. Two months after injection with streptozotocin, peak isometric contraction and steady-state RCC decreased in parallel to approximately 50% of control values. The time to peak force development and complete relaxation was prolonged to 156 and 161% in diabetes in the presence of 1.25 and 2.5 mM extracellular Ca2+ concentration [Ca2+]o, respectively. A stepwise increase in the rate of stimulation from 0.2 to 0.5 and 1.0 Hz resulted in a negative force staircase, the slope of which was identical in control and diabetic animals in each [Ca2+]o tested. Postrest contractions and RCC, after variable test intervals, were significantly depressed after 0.2 and 0.5 Hz stimulation in diabetic muscles at 1.25 mM [Ca2+]o. This defect of SR Ca2+ availability was reversed by increasing the stimulation frequency to 1.0 Hz or by elevating [Ca2+]o to 2.5 mM. The results suggest that the marked reduction of developed tension in diabetic tissues was a consequence of depleted SR Ca2+ stores, rather than a result of chronic SR Ca2+ overloading. The maintained integrity of the interval-force relation in the presence of diabetes implies that the cellular mechanisms responsible for frequency- and time-dependent alterations in SR Ca2+ availability are not disturbed at this stage of disease.

Animals↗

PubMed1990

Characterization of expressed meiotic prophase repeat transcript clones of Lilium: meiosis-specific expression, relatedness, and affinities to small heat shock protein genes.

The inserts of plasmid cDNA clones for transcripts showing meiotic prophase specific expression show cross reassociation to varying degrees of intensity with one another. These clones were recovered from a cDNA library made from Lilium microsporocyte poly(A)+ RNA. RNA-dot and Northern-blot analyses indicate that these clones represent transcripts specific to the meiotic prophase interval in microsporocytes. The transcripts appear to constitute the most abundant class of meiosis-specific poly(A)+ RNAs. At least two subgroups can be distinguished by examining cloned transcripts from genes of this expressed meiotic prophase repeat (EMPR) sequence family. Members of each subgroup have similar although not identical restriction maps and show relatively high but varying fidelities of DNA cross reassociation between members. However, consensus restriction maps of the two subgroups are largely dissimilar and, except at low stringencies, cross reassociation is readily detected only at restriction fragments from a particular conserved internal segment. The DNA sequence of a representative EMPR clone has been determined, and the inferred peptide product has been found to show extensive sequence homology to that of a small heat-shock gene of Glycine max, particularly in the conserved region. Alignment of the sequences for the conserved regions of two EMPR subgroup representatives with the soybean sequence suggests that selection has acted to conserve similar blocks of amino acids in this area. These observations suggest that a major portion of the transcripts produced during the apparently unrelated processes of meiosis and heat shock in higher plants are derived from related gene sequences encoding similar products.

Amino Acid Sequence↗

PubMed1989

Cloning and characterization of NSP1, a locus encoding a component of a CDC25-dependent, nutrient-responsive pathway in Saccharomyces cerevisiae.

The NSP1 gene in Saccharomyces cerevisiae has been identified by its ability, when expressed at high levels, to bypass the CDC25 requirement for growth. Sequence analysis of the cloned NSP1 locus suggests that the NSP1 product contains 269 amino acids and has a membrane-spanning domain at its carboxyl terminus. The NSP1 protein does not have sequence similarity to other known proteins, and is not related to the CDC25 protein, or to any of the previously described suppressors of CDC25 mutants. Phosphoprotein analysis of NSP1-suppressed cells indicates that the NSP1 product controls the phosphorylation of two 31 kD proteins whose phosphorylation and dephosphorylation are strongly correlated with cell-cycle arrest and proliferation, respectively, and suggests that the NSP1 product is an important downstream element of a CDC25-dependent, nutrient-responsive, phosphorylation pathway.

Amino Acid Sequence↗

PubMed1989

Effects of caffeine and ryanodine on depression of post-rest tension development produced by Bay K 8644 in canine ventricular muscle.

1. Post-rest inotropy in canine ventricular myocardium has proved a useful indicator of sarcoplasmic reticular calcium release. This phenomenon is converted to rest depression by the calcium channel activator (agonist), Bay K 8644 as well as other chemically diverse agents such as caffeine and ryanodine. 2. Rapid cooling contractures and post-rest contraction amplitude were used as independent measures of sarcoplasmic reticular calcium content and release. Simultaneous recordings of transmembrane action potentials and their accompanying contractions were obtained to determine the association between electrophysiological and mechanical events. The present study was designed to elucidate the mechanism by which Bay K 8644, caffeine and ryanodine alter force production after variable periods of rest. 3. Bay K 8644 (1 microM) increased steady state contraction in response to a constant train of stimulation, caused rest-depression after 2 and 8 min rest, prolonged action potential duration and increased action potential plateau amplitude. Augmented steady state tension was not accompanied by any change in time to peak tension or rapid cooling contracture amplitude. However, the post-rest rapid cooling contracture was greatly diminished compared to that observed prior to Bay K 8644 treatment. 4. Caffeine (3 and 5 mM) caused rest-depression with an increase in steady state contraction amplitude. Along with this there was a slight decrease in action potential duration and plateau amplitude and an increase in time to peak tension. The rapid cooling contractures were virtually abolished at all conditioning intervals. The effect of caffeine on twitch tension and cooling contracture is consistent with the ability of this compound to inhibit calcium sequestration by the sarcoplasmic reticulum. 5. A combination of Bay K 8644 and caffeine caused significantly less rest-depression than that seen with Bay K 8644 alone. The augmented twitch tension was accompanied by a long time to peak tension and action potential duration. However, there was no increase in the amplitude of the rapid cooling contracture, either after a regular train of stimulation or after rest, compared to that seen after Bay K 8644. 6. Ryanodine (10 nM), produced rest-depression, reduced steady state twitch tension and augmented the rest-depression produced by Bay K 8644. The steady state rapid cooling contracture remained unchanged when both agents were present simultaneously, while the post-rest rapid cooling contracture was significantly depressed compared to that observed with Bay K 8644 alone. 7. Bay K 8644 and ryanodine appear to have similar actions with respect to promoting diastolic loss of calcium from the sarcoplasmic reticulum. Although caffeine also decreases post-rest potentiation, it antagonizes rest-depression caused by Bay K 8644. The data from these experiments suggest that this reversal is a result of depressed intracellular calcium buffering and enhanced myofilament sensitivity produced by caffeine in the presence of increased transmembrane calcium influx promoted by Bay K 8644.

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

PubMed1989

Analysis of the effects of (-) and (+) isomers of the 1,4-dihydropyridine calcium channel agonist BAY k 8644 on postrest potentiation in the canine ventricular muscle.

Contraction of canine ventricular trabeculae were recorded stimulation at a frequency of 0.5 Hz and after rest periods of 2 and 8 min to analyze the effect of the Ca channel agonist BAY k 8644, on sarcoplasmic reticular function. Short periods of rest interposed between steady trains of stimuli caused a potentiation of the postrest beat. This is believed to be due to the mobilization of activator Ca from the sarcoplasmic reticulum (SR). Racemic BAY k 8644 and its Ca channel agonist enantiomer, (-) BAY k 8644, both produced an increase in contraction in response to a steady train of stimuli but converted rest potentiation into rest depression. This has been interpreted as increased loss of Ca from the SR during diastole. Addition of Ca channel antagonists, (+) BAY k 8644, nitrendipine, or nifedipine, to reverse the agonistic effect of (-) and racemic BAY k 8644 on the Ca channel did not convert the rest depression into rest potentiation. In the presence of stimuli but converted rest potentiation into rest depression. This has been interpreted as increased loss of Ca from the SR during diastole.(ABSTRACT TRUNCATED AT 250 WORDS)

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

PubMed1989

Analysis of the interval-force relationship in rat and canine ventricular myocardium.

The mechanism of the negative force staircase in thin rat ventricular trabeculae was investigated and compared with the positive force staircase in dog ventricular muscles of comparable diameter. Increasing stimulus frequency from 0.2 to 0.5 and 1 Hz resulted in a stepwise reduction of twitch amplitude that was demonstrated in both 1.25 and 2.5 mM external calcium concentration ([Ca]o). The negative staircase was associated with no change in the amplitude of postrest contraction or rapid-cooling contracture at either [Ca]o investigated. Hence, neither decreased loading nor overloading of the rat sarcoplasmic reticulum with calcium during the steady state can be invoked as a likely explanation for the negative staircase. The results are consistent with a frequency-dependent increase in the refractoriness of the sarcoplasmic reticular calcium release process or a decrease in the amount of trigger for calcium release, assuming that the amount of calcium present in the release pool is constant from one frequency to the next. In contrast to the rat, canine ventricular muscle exhibited a positive force staircase, the slope of which depended on [Ca]o, as well as a frequency-dependent increase in postrest contraction and rapid-cooling contracture. Data obtained from this series of experiments suggests that increased filling of the release pool within the sarcoplasmic reticulum with calcium underlies the inotropic effect of high-frequency stimulation in canine ventricular muscle.

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