Biomedical subjects

K Emanuel

Publications and source records attributed to K Emanuel.

12 recordsLinked to original sources

PubMed2003

Voltage dependent activation of tonic contraction in cardiac myocytes.

UNLABELLED: Contractions of isolated single myocytes of guinea pig heart stimulated by rectangular depolarizing pulses consist of a phasic component and a voltage dependent tonic component. In this study we analyzed the mechanism of activation of the graded, sustained contractions elicited by slow ramp depolarization and their relation to the components of contractions elicited by rectangular depolarizing pulses. Experiments were performed at 37 degrees C in ventricular myocytes of guinea pig heart. Voltage-clamped myocytes were stimulated by the pulses from the holding potential of -40 to +5 mV or by ramp depolarization shifting voltage within this range within 6 s. [Ca2+]i was monitored as fluorescence of Indo 1-AM and contractions were recorded with the TV edge-tracking system. Myocytes responded to the ramp depolarization between -25 and -6 mV by the slow, sustained increase in [Ca2+]i and shortening, the maximal amplitude of which was in each cell similar to that of the tonic component of Ca2+ transient and contraction. The contractile responses to ramp depolarization were blocked by 200 microM ryanodine and Ca2+-free solution, but were not blocked by 20 microM nifedipine or 100-200 microM Cd2+ and potentiated by 5 mM Ni2+. The responses to ramp depolarization were with this respect similar to the tonic but not to the phasic component of contraction: both components were blocked by 200 microM ryanodine, and were not blocked by Cd2+ or Ni2+ despite complete inhibition of the phasic Ca2+ current. However, the phasic component but not the tonic component of contraction in cells superfused with Ni2+ was inhibited by nifedipine. Both components of contraction were inhibited by Ca2+-free solution superfused 15 s prior to stimulation. CONCLUSIONS: In myocytes of guinea pig heart the contractile response to ramp depolarization is equivalent to the tonic component of contraction. It is activated by Ca2+ released from the sarcoplasmic reticulum by the ryanodine receptors. Their activation and inactivation is voltage dependent and it does not depend on the Ca2+ influx by the Ca2+ channels or reverse mode Na+/Ca2+ exchange, however, it may depend on Ca2+ influx by some other, not yet defined route.

Action Potentials↗

PubMed2001

On the source of Ca(2+) activating the tonic component of contraction of myocytes of guinea pig heart.

OBJECTIVE: Contractions of isolated, single myocytes of guinea pig heart stimulated at 37 degrees C consist of a phasic component and a voltage dependent tonic component. In this study we investigated the source of Ca(2+) activating the tonic component. METHODS: Experiments were performed at 37 degrees C in ventricular myocytes of guinea pig heart. Voltage-clamped cells were stimulated by the pulses from the holding potential of -40 to +5 mV. [Ca(2+)](i) was monitored as fluorescence of Indo 1-AM and contractions were recorded with the TV edge-tracking system. RESULTS: Superfusion of 5 mmol/l Ni(2+) during 30 s pause did not inhibit subsequent biphasic Ca(2+) transients and contractions despite inhibition of Ca(2+) current and Na(+)/Ca(2+) exchange. KB-R7943 (5 micromol/l) or intracellular dialysis with 0 Na(+) solution, both of which inhibit reversed Na(+)/Ca(2+) exchange, decreased amplitude of Ca(2+) transients and contractions by approximately 40%. The ratio of amplitudes of tonic to phasic component was increased by Ni(2+) and was not changed by KB-R7943 or 0 Na(+)(i). Ryanodine (200 micromol/l) inhibited both components of contractions in cells superfused with Ni(2+). The phasic component but not the tonic component was inhibited by 20 micromol/l nifedipine in cells superfused with Ni(2+). CONCLUSIONS: Tonic component of contraction of single myocytes of guinea pig heart is not activated by Ca(2+) current or by the reverse mode Na(+)/Ca(2+) exchange as currently proposed in literature. Rather, it is activated by Ca(2+) released from the sarcoplasmic reticulum. However, kinetics and mechanism of release seem to be quite different from those of Ca(2+) fraction activating the phasic component of contraction.

Action Potentials↗

PubMed2001

Agonist of dihydropyridine receptors, BayK8644 depresses excitation-contraction coupling in myocytes of guinea pig heart.

BayK8644(-)(BayK), an agonist of L-type Ca2+ channels has been recently shown to impair excitation-contraction coupling in cardiac myocytes by increasing Ca2+ leak from the sarcoplasmic reticulum (SR) and by decreasing the gain factor of calcium induced release of calcium. It has been proposed that BayK affects the properties of ryanodine receptors (RyRs) of SR by binding to the sarcolemmal dihydropyridine receptors (DHPRs). This would suggest that the linkage between these receptors is more direct than currently sought. However, it has been recently found that BayK may also directly affect the RyRs increasing their open probability. In this paper we tested the effect of BayK on excitation-contraction coupling in single ventricular myocytes of guinea-pig heart superfused with 5 mM Ni2+ which blocks the L-type Ca2+ current and Na+/Ca2+ exchange. We have previously shown that it is possible to activate in these cells nearly normal Ca2+ transients and contractions despite total inhibition of ICa. This eliminated the effect of ICa increased by BayK on excitation contraction coupling thus simplifying the studied system. 0.5 microM BayK increased the diastolic [Ca2+]i and decreased the diastolic length in stimulated or rested cells superfused with Ni2+, decreased by approximately 50% amplitude of Ca2+ transients and contractions and decreased by approximately 70% the responses of cells to rapid superfusion of 15mM caffeine used as an indirect index of the SR Ca2+ content. The effects on diastolic length and [Ca2+]i in rested cells were not affected by 20 microM nifedipine. We conclude that under our experimental conditions the dominating mechanism of suppression of excitation-contraction coupling by BayK was depletion of the SR Ca2+ by the direct effect on the RyRs.

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

PubMed2000

Dihydropyridine receptors functioning as voltage sensors in cardiac myocytes.

Isolated, superfused with [Ca2+]0 = 2.5-3 mM (T = 37 degrees C) and voltage clamped ventricular myocytes of guinea-pig hearts were stimulated by pulses from a holding potential -40 mV to +5 mV (duration 300 ms). They activated L-type Ca2+ current and a biphasic contractile response: a phasic component of amplitude approximately 7% of resting cell length (duration approximately 150 ms) and a tonic component of amplitude approximately 3% of resting cell length. The phasic component was inhibited by 10(-6) M thapsigargin (Tg). Pulses from -40 mV to +5 mV stimulated a similar bi-phasic contractile response in 74% of cells (n = 126) superfused from the beginning of a 30 s period of rest with 5-10 mM Ni2+ which blocked the Ca2+ current and Na+ -Ca2+ exchanger (Ni2+-contractions). Thus, the Ni2+-contractions could be activated only by intracellular Ca2+ release. The phasic component of control contractions showed the bell-shaped voltage relation at [Ca]0 = 2 mM and sigmoid relation at [Ca]0 = 3 mM. The phasic component o Ni2+-contractions showed a sigmoid relation at voltages from -40mV to +100 mV and could not be activated at [Ca]0 = 2 mM. It was inhibited by 20 microM nifedipine, a blocker of dihydropyridine receptors, even when activated by the pulses to +70 mV, during which the Ca2+ current does not flow. We proved that nifedipine does not affect Na+-Ca2+ exchange. The phasic component of Ni2+-contractions was also inhibited by 2 nM indolizinesulphone SR33557, another dihydropyridine receptor blocker, which halved the phasic component of contractions in sontrol cells without any significant effect on the Ca2+ current. Stimulation did not activate contraction in any of 19 cells in which 20 microM nifedipine was superfused from the beginning of 30 s rest instead of 5 mM Ni2+. These cells were depolarized to +5 mV over the rest period in order to prevent intracellular Ca2+ loss by Na+ -Ca2+ exchange. Residual Ca2+ currents were much stronger in cells superfused with nifedipine than residual currents in cells superfused with Ni2+ (hardly visible in the records). Our results suggest that a vestigial remnant of a voltage-sensing mechanism similar to that in the skeletal muscle may trigger the Ca2+ release from the SR of cardiac myocytes under specific experimental conditions. In normal cells it may be complementary to calcium induced calcium release (CICR).

Animals↗

PubMed1999

Properties of ventricular myocytes isolated from the hypertrophied and failing hearts of spontaneously hypertensive rats.

OBJECTIVE: To investigate how the morphological and physiological properties of single myocytes isolated from the hypertrophied, failing left ventricles (LV) differ from those of normal or hypertrophied not failing ventricles. METHOD: Single myocytes were isolated separately from right (RV) and left ventricles (LV) of male spontaneously hypertensive rats (SHR) or Wistar-Kyoto (WKY) rats at the age of 6 and 12 months and of SHRs which developed or not developed heart failure at the age of 20-24 months. We measured cells dimensions, range and kinetics of electrically stimulated or initiated by caffeine contractions and Ca2+ transients, and investigated the response of cells to thapsigargin. RESULTS: The transversal dimensions of the LV myocytes of 6 months old SHRs showed approximately 20% increase with respect to transversal dimensions of their RV myocytes and LV and RV myocytes of WKY rats. The difference did not change with progressing age and in the heart failure. The LV myocytes of 6 or 12 months old SHRs showed slowed kinetics of the Ca2+ transients and of contraction and relaxation and decreased contractile response to 2 s superfusion with 15 mM caffeine preceded by 5 mM Ni2+ used as an index of the sarcoplasmic reticulum (SR) Ca2+ content. Despite of this the range of shortening and relative contribution of the SR to contraction (assessed by measuring of the residual contractile response to electrical stimulation in cells poisoned with thapsigargin) or relaxation (assessed by calculation of the ratio of rate constants of the electrically stimulated and stimulated by 30 s superfusion with caffeine Ca2+ transients) was not altered in the hypertrophied myocytes. Properties of the LV myocytes of the 20-24 old SHRs with or without heart failure did not differ from those of LV myocytes of younger SHRs. The contractile response to caffeine of their RV myocytes dropped to the level of that in the LV myocytes. CONCLUSION: Our results suggest that transition from the compensated hypertrophy to the heart failure in 20-24 months old SHRs did not result from the further changes in properties of the surviving myocytes. Data from literature suggest that myocyte apoptosis and remodeling of the extramyocyte space is the more likely reason.

Animals↗

PubMed1998

Effects of mibefradil, a blocker of T-type Ca2+ channels, in single myocytes and intact muscle of guinea-pig heart.

We investigated the effects of a relatively selective blocker of the T-type Ca2+ channels, mibefradil (MBF), in the isovolumic left ventricles of the isolated, perfused hearts of guinea-pigs and single myocytes isolated from the ventricles of this species. In the myocytes superfused with 0 Na+ solution containing 200 microM lidocaine and pulsed from -90 mV to -40 mV to +5 mV, MBF proved to be about 3 times more potent inhibitor of the T-type than of the L-type Ca2+ current. The effect on the L-type current was strongly voltage and use dependent. In the ventricles and in the myocytes contraction was reduced by 50% by about 1 microM MBF, the concentration 12 times higher than this increasing the coronary flow by 50%. In myocytes the decrease in unloaded shortening paralleled inhibition of the T-type rather, than of the L-type Ca2+ current. Inhibition of electrically stimulated contraction of the myocytes was three times stronger than inhibition of the caffeine contractures regarded as an index of sarcoplasmic reticulum (SR) Ca2+ content. These findings are consistent with the hypothesis that the T-type Ca2+ channels may contribute to release of Ca2+ from the SR. It is concluded that MBF has a definite negative inotropic effect in the ventricular myocardium of guinea-pig heart at the concentrations found in the blood of the patients submitted to the clinical trials.

Animals↗

PubMed1998

Dibucaine displaceable sarcolemmal Ca2+ fraction in guinea-pig cardiac myocytes.

In previous work we found that Ca2+ bound to the internal leaflet of sarcolemma of the neonatal cultured rat cardiomyocytes may be displaced by a local anesthetic, dibucaine (DBC). This resulted in inhibition of Na/Ca exchange. Now we found that the DBC displaceable Ca2+ fraction may be demonstrated also in single, enzymatically isolated cardiomyocytes of adult guinea pigs. Only cell length was used as an index of the free sarcoplasmic Ca2+ concentration since we found that DBC is fluorescent when irradiated with UV light, the wave length overlapping that of Indo 1 fluorescence. DBC (0.5 mM) induced a contracture whose amplitude ranged from 30%-100% of that of the electrically stimulated twitch. Blocking of Na/Ca exchange with 0.5 mM Ni2+, or 0Na+, 0Ca2+ solution did not affect the amplitude nor the time course of the contracture. Caffeine (15 mM) superfused during exposure of the cell to DBC initiated contraction which relaxed despite continued caffeine superfusion. DBC also initiated contracture in cells pretreated with thapsigargin, a blocker of sarcoplasmic reticulum Ca2+ uptake. DBC did not initiate contracture in skinned myocytes and decreased their sensitivity to Ca2+. These results suggest that DBC displaces Ca2+ from sarcolemma and that the displacement results in inhibition of Na/Ca exchange. Inhibition may however, be overcome by high subsarcolemmal Ca2+ concentration induced by its SR release with caffeine. DBC rapidly blocked sodium and calcium currents in voltage clamped cells. Calcium current recovered within about a minute upon washout of DBC, whereas recovery of sodium current started after about 8 min and was completed within about 12 min. Rapid depolarisation from a holding potential of -80 mV to 0 or +20 mV potentiated the response of the cell to DBC. This suggests that the Ca2+ fraction which is displaced by DBC is distinct from that released by depolarisation and therefore does not contribute to activation of a normal, electrically stimulated contraction.

Anesthetics, Local↗

PubMed1992

The mammalian iris-ciliary complex affects organization and synthesis of cytoskeletal proteins of organ and tissue cultured lens epithelial cells.

A water soluble growth inhibitor was isolated from the mammalian ocular iris-ciliary complex. The molecular weight of this protein is 10 kD or lower as determined by ultrafiltration fractionation. The iris-ciliary (IC) complex water soluble protein(s) significantly inhibits synthesis of lower molecular weight proteins of the epithelial cells of the organ cultured mammalian ocular lens. It was also found that this inhibitory effect of IC is mediated via the structural organization of the lens. Monolayer cultures of the lens epithelial cells exposed to IC did not manifest any inhibition of their protein synthesis. Moreover, these tissue cultured lens epithelial (TCLE) cells showed a significant increase in their protein synthetic activities in response to the presence of IC factors in the culture medium. It is postulated that the IC activity is modulated via either the lens capsule, an extracellular matrix, or due to the specific organization of the intact lens. The specific effects of IC on the cytoskeletal organization and synthesis in the organ cultured lens epithelial (OCLE) and TCLE cells were also examined. Both groups, treated with IC factors, manifested significant alterations in their protein synthetic activities and cytoskeletal architecture. The 3H-leucine incorporation experiments showed that alpha-actin and alpha-tubulin synthesis is partially inhibited by IC factors in OCLE cells but vimentin synthesis is not, whereas in TCLE cells all of them showed increased synthesis in response to IC factors. Turnover rates of these proteins in both OCLE and TCLE cells were also computed. The immunofluorescence and microscopic evaluation of OCLE and TCLE cells exposed to IC factors illustrated significant alteration in the cytoarchitecture of the filaments. We demonstrate that an inhibitor(s) molecule of 10 kD or lower size isolated from IC inhibited protein synthesis of OCLE cells and stimulated protein synthesis in TCLE cells. The IC factor also affects the synthesis and organization of cytoskeletal filaments of both the OCLE and TCLE cells.

Actins↗

PubMed1991

Studies on the possible role of vitreous humor on protein synthesis and morphology of organ cultured adult rabbit lens. II. Epithelial cells.

The protein synthetic activities of epithelial cells of lenses organ-cultured without adhered vitreous humor manifest significant increase compared to the epithelial cells of lenses incubated with attached vitreous humor. This effect is not due to trauma of vitreous removal, as the addition of freeze-dried vitreous humor to the culture medium of lenses without attached vitreous humor could inhibit protein synthesis. However, the protein synthesis inhibitor in the vitreous humor has no visible effect on the lens morphology. It was also found that the factor from vitreous humor has no effect on mRNA production or cell-free protein synthesis. Thus, it seems that the effect on protein synthesis must be mediated via some other pathway.

Animals↗

PubMed1991

Effect of vitreous humor on the organ cultured rabbit ocular lens. III. Morphology and elemental analysis.

Distribution of ions and volume regulation of epithelial cells of lenses cultured in the presence and absence of vitreous humor were examined. Ion levels of the epithelial cells were measured using energy dispersive x-ray analysis(EDX) and computer-assisted morphometry was used for volume measurements. Data from these experiments revealed that the epithelial cells in the pre-equatorial region of lenses cultured without attached vitreous humor are larger and have significantly altered ionic balance. The cell volumes and ionic balances of central epithelial cells of the lenses cultured without attached vitreous humor also displayed discernible changes. However, overall data indicated that the epithelial cells in the pre-equatorial regions of the lenses are most susceptible to the effects of vitreous humor. Experiments performed in our laboratory have demonstrated that vitreous humor contains some factor(s) which can effectively inhibit protein synthesis. It was also reported that this factor(s) is not a direct inhibitor of protein synthesis. Therefore it was assumed that the inhibition of protein synthetic activity was mediated via some other pathway. One of these possible routes could be altered cytoplasmic ion fluxes. Earlier we reported that the ion levels of the lenses cultured with or without adhered vitreous humor were similar. Ion levels were measured from intact lenses. It is possible that there were regional differences in ion levels, which could be masked in whole lenses. It is also known that the epithelial cells are major participant in ion-pump activity, whereas cortical and nuclear areas of the lens may not contribute significantly to the ion transport. It has also been proposed that the epithelial cells of the pre-equatorial region are the main site of the Na+, K+ pump. Variation in ion levels of the epithelial cells could also affect protein synthesis activities of the lens. To test these possibilities, experiments were performed to measure ion levels in various areas of the lenses by energy dispersive x-ray analysis techniques. Altered cytoplasmic ion level can also change the volumes of the epithelial cells. This possibility was investigated by employing computer-assisted morphometry in measurements of two- and three-dimensional parameters of the lens epithelial cells and capsule.

Animals↗

PubMed1991

The effects of iris-ciliary complex on the organ cultured rabbit ocular lens.

Freshly isolated rabbit lenses were cultured with and without attached iris-ciliary (IC)-complex for 24 hours in TC-199 medium. Subsequent morphological analysis revealed that the IC-complex cannot be maintained in serum-free medium. In addition an observed effect of the IC-complex on the co-cultured lenses could not be due only because of the cellular degeneration of the IC-complex. To test this possibility lenses with attached IC-complexes were incubated in 20% serum-containing TC-199 medium. The IC-complex cultured in 20% serum containing medium retained its normal morphology. However co-cultured lens cells displayed vacuoles and other signs of degeneration. The protein synthetic and Na+/K+ pump activities of these lenses were also significantly depressed. These data indicated that the observed effects of IC-complex on the lens were not due to its cellular degradation. Preliminary experiments showed that the IC-complex contains water soluble factor(s) which could effectively inhibit lens protein synthesis and Na+/K+ pump.

Animals↗

PubMed1991

Elemental profiles in Emory mouse lens.

Energy dispersive x-ray microprobe analysis was used to determine the distribution of chloride, potassium, phosphorus and sulfur in the epithelial cells of the lenses obtained from 3 to 7 month old Emory mice and 7 month old cataract resistant strain of Emory mice. Rapidly frozen lenses were fractured in the frozen state and lyophilized. The anterior epithelial cells were analyzed from equator to equator. The results show that the epithelial cells of the 7 month old Emory mouse lens have considerably higher amounts of chloride, sulfur, potassium and phosphorus. Presence of increased amount of potassium in the epithelial cells is intriguing. The data obtained from these experiments show that the changes in the elemental levels of epithelial cells are similar to observed alteration found in the lens fiber mass of 7 month old Emory mouse.

Animals↗