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J H Bridge

Publications and source records attributed to J H Bridge.

At least 37 records · Page 2Linked to original sources
PubMed1993

The effect of exchanger inhibitory peptide (XIP) on sodium-calcium exchange current in guinea pig ventricular cells.

We investigated the effect of exchanger inhibitory peptide (XIP) on Na-Ca exchange current (INa-Ca) in guinea pig ventricular cells. Cells were voltage-clamped with microelectrodes containing 20 mM Na+ and 14.0 mM EGTA ([Ca]i = 100 nM). An outward putative exchange current was stimulated when extracellular Na+ was reduced from 144 mM to zero (Li+ replaced Na+). This outward current showed a significant dependence on extracellular Ca2+. When Na+ removal was delayed for up to 40 minutes (in the absence of extracellular K+ or the presence of 3.0 mM ouabain to block the Na+ pump), outward INa-Ca increased presumably because [Na]i increased. Time-dependent increases of outward current in the absence of K+ could be abolished by reapplication of K+, which presumably reactivates the Na+ pump and reduces intracellular Na+. This effect is blocked in the presence of 3.0 mM ouabain. The dependence of this current on extracellular Ca2+, its dependence on intracellular Na+, and activation by extracellular Na+ reduction, together with its resistance to ouabain all suggest that it is a Na-Ca exchange current. After dialyzing the cell with 10 microM XIP, outward INa-Ca was largely abolished. This indicates that XIP, which is a rather large molecule, can enter the heart cell via the microelectrode in sufficient quantities to inhibit exchange. Inward INa-Ca was blocked secondary to the blockade of outward INa-Ca. L-type Ca2+ current (ICa) was not measurably affected by XIP.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗
PubMed1992

Expression of the cardiac Na(+)-Ca2+ exchanger in insect cells using a baculovirus vector.

We constructed a recombinant baculovirus containing cardiac Na(+)-Ca2+ exchanger cDNA under control of the polyhedrin promoter. When either Sf9 or Sf21 insect cells are infected with the recombinant baculovirus, both Na(+)-Ca2+ exchanger protein and Na(+)-Ca2+ exchange activity are expressed at high level. The exchanger protein can be detected either by immunoblot or by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole cell lysate. At maximal expression, the exchanger protein comprises about 3-5% of total cell protein. The Na(+)-Ca2+ exchanger can be purified by alkaline extraction of infected cells followed by elution from a Bio-Rad Prep Cell. The expressed exchanger, in contrast to the native sarcolemmal exchanger, is not glycosylated. Sf9 cells expressing the exchanger are intensely stained by anti-exchanger antibodies as observed by immunofluorescence. The expressed exchanger is predominantly in the cell plasma membrane since it is susceptible to extracellular trypsin. In 45Ca2+ flux experiments, the expressed Na(+)-Ca2+ exchange activity is about 4-fold higher than that in cultured neonatal rat heart cells. The expressed exchanger was also analyzed electrophysiologically using whole cell patch clamp techniques. The characteristics of inward exchange currents in infected Sf21 cells are very similar to those of ventricular myocytes, although of a larger magnitude.

Animals↗
PubMed1992

Relationship between intracellular pH and tension development in resting ventricular muscle and myocytes.

Simultaneous measurements of intracellular pH (pHi) and tension development were made in resting cat papillary muscles and resting ventricular myocytes (cat, guinea pig). pH microelectrodes and the fluorescent indicator carboxy-seminaphthorhodafluor-1 (SNARF-1) were used to measure pHi in muscles and myocytes, respectively. pHi-induced changes in isometric muscle tension and myocyte length were elicited by variations in PCO2, HCO3-, and [NH4Cl]. Increased pHi elevated resting tension and decreased resting cell length, whereas decreased pHi decreased tension and increased cell length. The tension-pHi and cell length-pHi relationships were nonlinear and displayed hysteresis. A reduction in extracellular [Ca2+] from 2.7 to 0.5 mM caused little or no change in the tension and cell length responses to elevated pHi. Ca2+ uptake and/or release by the sarcoplasmic reticulum (SR) does not appear to be involved in the tension response to intracellular alkalosis because the response was unaffected by a postpacing rest period and was not inhibited by ryanodine (5 microM), which depletes SR Ca2+ stores. The cross-bridge inhibitor 2,3-butanedione monoxime (15 mM), but not internal dialysis with 14 mM ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid, inhibited myocyte contractures elicited by elevated pHi. The latter finding suggests that factors other than pHi-induced increase in myofilament Ca2+ sensitivity may contribute to the resting contractile response to elevated pHi.

Animals↗
PubMed1990

The relationship between charge movements associated with ICa and INa-Ca in cardiac myocytes.

Ventricular myocytes exhibit a nifedipine-sensitive inward calcium current (ICa) and contracture when they are voltage clamped from -40 to 0 millivolt in the presence of caffeine and in the absence of extracellular sodium. However, upon repolarization they fail to relax because neither the sarcoplasmic reticulum nor the sodium-calcium exchange can reduce intracellular calcium. Sudden application of extracellular sodium during the contracture (but after repolarization) causes immediate relaxation and activates a transient inward sodium-calcium exchange current (INa-Ca), whose peak slightly precedes mechanical relaxation. The total charge carried by the nifedipine-sensitive ICa is twice the total charge carried by the transient inward INa-Ca. Assuming an exchange stoichiometry of three sodium to one calcium, these results indicate that all the calcium entering the cell during the initial depolarization is extruded by the sodium-calcium exchange.

Animals↗
PubMed1989

Intracellular Ca2+ transients during rapid cooling contractures in guinea-pig ventricular myocytes.

1. We measured intracellular Ca2+ transients during rapid cooling contractures (RCCs) in guinea-pig ventricular myocytes using the fluorescent Ca2+ indicator, Indo-1. 2. Rapid cooling of myocytes from 22 to 0-1 degrees C induced a rapid increase in [Ca2+]i which preceded the peak of the contraction and was sometimes large enough to saturate Indo-1. This indicates that [Ca2+]i may reach greater than 10 microM during an RCC. 3. The [Ca2+]i during the RCC slowly declined from its peak value and most of this decline in [Ca2+]i can be attributed to slow reaccumulation of Ca2+ by the sarcoplasmic reticulum (SR) in the cold. RCCs induced in the absence of Cao2+, were not different from control, supporting previous conclusions that RCCs depend exclusively on intracellular Ca2+ stores. 4. RCCs are depressed by long rest periods (rest decay) or by exposure to ryanodine or caffeine, which supports conclusions that RCCs are due to Ca2+ release from the SR. The rest decay of RCCs can be almost completely prevented by applying Nao(+)-free solution during the rest period. This implies that the loss of SR Ca2+ during rest depends on the sarcolemmal Na(+)-Ca2+ exchange (and not the sarcolemmal Ca2(+)-ATPase pump). 5. Rapid rewarming during an RCC normally leads to an additional transient contraction (or rewarming spike), without any increase in [Ca2+]i. Thus, the rewarming spike might be attributable to an increase in myofilament Ca2+ sensitivity induced by rewarming. 6. A second RCC is used to assess the fraction of Ca2+ which is re-sequestered by the SR during relaxation from the first RCC. In control solution progressive RCCs decline in amplitude, but in Na(+)-free, Ca2(+)-free solution they are of constant amplitude. We conclude that the SR Ca2+ pump and Na(+)-Ca2+ exchange are responsible for relaxation and that the latter may account for 20-50% of relaxation. 7. These results support the use of RCCs as a useful means of assessing SR Ca2+ content in intact cardiac muscle cells.

Animals↗
PubMed1989

A simple device for rapidly exchanging solution surrounding a single cardiac cell.

This study describes the design and various physiological applications of a simple device to rapidly change the solution surrounding a single intact cardiac cell. It consists of a short length of double-barreled glass tubing (theta-tubing) attached to a miniature solenoid. A cell is positioned in one of two parallel streams of solution that simultaneously flow from each barrel. Rapid solution switching is achieved by activation of the solenoid that directs the adjacent stream over the cell, changing the bulk solution within 7 ms. Approximately 150 ms were required to change the solution at the membrane surface of guinea pig ventricular cells, judging from potassium-induced changes in resting membrane potential. This delayed response was probably due to, in part, restricted diffusion in the transverse tubular system. The switching speed of this device makes it possible to change extracellular solutions during action potentials and voltage-clamp pulses.

Action Potentials↗
PubMed1989

Relaxation of rabbit ventricular muscle by Na-Ca exchange and sarcoplasmic reticulum calcium pump. Ryanodine and voltage sensitivity.

We studied relaxation during rapid rewarming of rabbit ventricular muscles that had been activated by rapid cooling. Rewarming from 1 degree to 30 degrees C (in less than 0.5 second) activates mechanisms that contribute to the reduction of intracellular calcium concentration and thus relaxation (e.g., sarcoplasmic reticulum [SR] calcium pump and sarcolemmal Na-Ca exchange and calcium pump). Rapid rewarming in normal Tyrode's solution induces relaxation with a half-time (t1/2) of 217 +/- 14 msec (mean +/- SEM). During cold exposure, changing the superfusate to a sodium-free, calcium-free medium with 2 mM CoCl2 (to eliminate Na-Ca exchange) slightly slows relaxation upon rewarming in the same medium (t1/2 = 279 +/- 44 msec). Addition of 10 mM caffeine (which prevents SR calcium sequestration) to normal Tyrode's solution during cold superfusion slows relaxation somewhat more (t1/2 = 376 +/- 31 msec) than sodium-free, calcium-free solution. However, if both interventions are combined (sodium-free + caffeine) during the cold exposure and rewarming, the relaxation is greatly slowed (t1/2 = 2,580 +/- 810 msec). These results suggest that either the SR calcium pump or, to a lesser extent, sarcolemmal Na-Ca exchange can produce rapid relaxation, but if both systems are blocked, relaxation is very slow. If muscles are equilibrated with 500 nM ryanodine before cooling, relaxation upon rewarming is not greatly slowed (t1/2 = 266 +/- 37 msec) even if sodium-free, calcium-free solution is applied during the cold and rewarming phases (t1/2 = 305 +/- 66 msec). This result suggests that ryanodine does not prevent the SR from accumulating calcium to induce relaxation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Relaxation of isolated ventricular cardiomyocytes by a voltage-dependent process.

Cell contraction and relaxation were measured in single voltage-clamped guinea pig cardiomyocytes to investigate the contribution of sarcolemmal Na+-Ca2+ exchange to mechanical relaxation. Cells clamped from -80 to 0 millivolts displayed initial phasic and subsequent tonic contractions; caffeine reduced or abolished the phasic and enlarged the tonic contraction. The rate of relaxation from tonic contractions was steeply voltage-dependent and was significantly slowed in the absence of a sarcolemmal Na+ gradient. Tonic contractions elicited in the absence of a Na+ gradient promptly relaxed when external Na+ was applied, reflecting activation of Na+-Ca2+ exchange. It appears that a voltage-dependent Na+-Ca2+ exchange can rapidly mechanically relax mammalian heart muscle.

Animals↗
PubMed1988

Effect of acetylstrophanthidin on twitches, microscopic tension fluctuations and cooling contractures in rabbit ventricle.

1. We have measured the effect of the aglycone acetylstrophanthidin (ACS) on twitches, cooling contractures and microscopic tension fluctuations in rabbit ventricular muscle. 2. Both developed twitches and cooling contractures are strengthened by applications of ACS in the range 1-4 microM. This positive inotropy averages 150-160% of control (zero ACS) in both twitches and cooling contractures. Cooling contracture magnitude is assumed to reflect the availability of sarcoplasmic reticulum (SR) Ca2+ for contraction (Bridge, 1986). We infer that ACS increases the availability of SR Ca2+ by enlarging SR Ca2+ stores and this may contribute to the positive inotropy. 3. However, twitches appear to increase at lower concentrations of ACS than those required to increase cooling contractures. This observation suggests that the initial ACS inotropy may be achieved without an increase in SR Ca2+. Furthermore, low doses of ACS produce positive inotropy in the presence of 10.0 mM-caffeine where cooling contractures are abolished. This also suggests that positive inotropy occurs in the absence of SR Ca2+ accumulation. 4. Rest decay of both cooling contractures and twitches is significantly slowed in 4 and 8 microM-ACS. We infer that ACS slows the rate of decline of SR Ca2+ available for contraction by slowing the rate at which Ca2+ is lost from the cell during rest. This suggests that ACS produces a net slowing of Ca2+ efflux during activity which in the absence of altered Ca2+ influx will result in net Ca2+ gain and presumably enlarged SR Ca2+ stores. 5. Increasing the concentration of ACS (6-10 microM) results in a decline in developed twitch tension, total tension and an increase in rest tension. Measurement of microscopic tension fluctuations indicates that as developed twitches decline, the root mean square (r.m.s.) of the tension fluctuations increases in a reciprocal manner. This supports the suggestion of others that the decline in developed twitch tension and the appearance of tension fluctuations are causally related. 6. Although ACS (6-10 microM) causes a decline in twitch tension, rapid cooling contractures remain elevated. We suggest that in the presence of Ca2+ oscillations the magnitude of cooling contractures reflects the sum of cytosolic Ca2+ and Ca2+ that is available for release. If microscopic tension fluctuations do represent Ca2+ moving between the SR and cytosol the sum of SR and cytosolic Ca2+ and hence cooling contracture might not decline.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

Alterations in cation homeostasis in cultured chick ventricular cells during and after recovery from adenosine triphosphate depletion.

Alterations in cation homeostasis during and after recovery from myocardial ischemia may account for some of the reversible and irreversible components of myocardial cell injury. To investigate possible mechanisms involved, we exposed cultured layers of spontaneously contracting chick embryo ventricular cells to media containing 1 mM cyanide (CN) and 20 mM 2-deoxyglucose (2-DG), and zero glucose for up to 6 h, and then allowed cultured cells to recover in serum-free culture medium for 24 h. Changes in Na, K, and Ca contents, 42K uptake and efflux, ATP content, cell water content, and lactate dehydrogenase (LDH) release were measured, and compared with changes produced by exposure to 10(-3) M ouabain and severe hypoxia. Exposure to CN and 2-DG caused marked increase in cell Na (sevenfold) and Ca (fivefold) contents, and a decrease in K content (one-fifth normal), coincident with ATP depletion to one-tenth normal levels. This produced only slight cell injury, evidenced by increased LDH release. Recovery for 24 h resulted in return to near normal values (expressed in nanomoles per milligram of protein) of Na, Ca, and ATP contents. However, there was failure of cell K content to return to normal, associated with a persistent reduced net uptake of 42K, and an increase in the rate of 42K efflux. These abnormalities in K homeostasis were associated with a decrease in cell volume and water content per milligram of protein. More marked ATP depletion (to 1/100 normal values) was produced by hypoxia plus 2-DG and zero glucose, and was associated with much more severe cell injury manifested by LDH loss. Ouabain exposure resulted in a much greater Ca gain (20-30-fold), relative to increase in Na content, than did either CN and 2-DG or hypoxia; and ouabain effects were not reversible (after a 15-fold or greater increase in Ca content was produced) and were associated with significant LDH release. We conclude that these cells are resistant to cell injury caused by moderately severe Ca overload and ATP depletion produced by exposure to CN and 2-DG. However, metabolic inhibition of ATP production produces persistent abnormalities in K homeostasis, associated with functional abnormalities.

Adenosine Triphosphate↗
PubMed1987

The mechanism of ryanodine action in rabbit ventricular muscle evaluated with Ca-selective microelectrodes and rapid cooling contractures.

Cellular Ca uptake and efflux in rabbit ventricular muscle was measured using double-barreled Ca microelectrodes in the extracellular space. When repetitive stimulation was stopped there was a slow loss of cellular Ca. Upon resumption of stimulation Ca was taken up by the cells. These Ca movements are thought to represent the loss of Ca from the sarcoplasmic reticulum and the cell during rest and the refilling of the sarcoplasmic reticulum during stimulation. Ryanodine (100 nM) greatly enhanced both the efflux of Ca during rest and the uptake of Ca induced by stimulation. These results are consistent with the conclusions drawn below, but they are dependent upon the interpretation that these extracellular Ca depletions are indicative of sarcoplasmic reticulum Ca movements. To examine further this process, contractures induced by rapid cooling to 0 degrees C were used as an independent assay of sarcoplasmic reticulum Ca content. These rapid cooling contractures were smaller after longer rest intervals (declining with a half time of 1.5 min). In the presence of ryanodine, the rapid cooling contracture immediately after a contraction was greater than that seen under control conditions. However, in the presence of ryanodine these rapid cooling contractures decline as a function of rest duration with a half time of about 1 s. These results suggest that in the presence of ryanodine the sarcoplasmic reticulum can still take up Ca, but that it also loses this Ca very rapidly at the onset of rest.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaloids↗
PubMed1987

Simultaneous measurement of calcium transients and motion in cultured heart cells.

The fluorescent Ca2+ probe indo-1 is a new intracellular Ca2+ concentration [( Ca2+]i) indicator that may be suitable for measurement of [Ca2+]i transients in intact heart cells. We exposed spontaneously contracting cultured chick embryo ventricular cells (37 degrees C) to the membrane-permeable indo-1-acetoxymethyl ester (indo-1 AM). Indo-1 loading was associated with a decrease in the amplitude of contraction measured with a video motion detector, but contractility returned to control levels during a subsequent 30-min wash. Analysis of emission spectra of dye obtained by digitonin permeabilization of cells loaded in indo-1 AM showed that the active intracellular dye was not pure indo-1 but probably includes partially deesterified molecules. With the use of an inverted X40 objective epifluorescence system, washed cells containing indo-1 were excited at 360 nm, and fluorescence intensity was measured at 410 nm (increases with increasing [Ca2+]) and 480 nm (decreases with increasing [Ca2+]). Calibration of the [Ca2+]i signals, reflected by the ratio of 410 to 480 nm fluorescence, was achieved by use of ethylen-glycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA)-Ca2+ buffered solutions containing the nonfluorescent Ca2+ ionophore Bromo-A23187. Average end-diastolic and peak-systolic [Ca2+]i were 328 +/- 32 and 813 +/- 72 nM (means +/- SE, n = 8). The onset of the [Ca2+]i transient preceded motion by 27 +/- 5 ms (means +/- SE, n = 4), but generally resembled the motion signals in contour. These findings indicate that indo-1 may be used to detect [Ca2+]i transients in isolated ventricular cells without causing significant alterations in mechanical performance.

Animals↗
PubMed1986

External Na-independent Ca extrusion in cultured ventricular cells. Magnitude and functional significance.

The relative magnitudes and functional significance of Ca extrusion by Na-Ca exchange and by an Nao-independent mechanism were investigated in monolayer cultures of chick embryo ventricular cells. Abrupt exposure of cells in 0-Nao, nominally 0-Cao solution to 20 mM caffeine produced a large contracture (3.94 +/- 0.90 micron of cell shortening) that relaxed with a t1/2 of 8.60 +/- 1.22 s. An abrupt exposure to caffeine plus 140 mM Na resulted in a contracture that was smaller in amplitude (1.53 +/- 0.50 micron) and relaxed much more rapidly (t1/2 = 0.77 +/- 0.09 s). An abrupt exposure to caffeine in 0-Nao solutions produced an increase in 45Ca efflux that persisted for 20 s, and a net loss of Ca content, determined by atomic absorption spectroscopy (AAS), of approximately 4 nmol/mg protein, within 35 s. A comparable net loss of Ca was demonstrated in the presence of 100 microM [Ca]o. The abrupt exposure of cultured cells to 0 Nao in 1.8 mM Ca produced a Ca uptake, estimated with 45Ca, of 3.2 nmol/mg protein X 15 s, but produced no increase in cell Ca content (AAS). In cells in which a 30% increase in Nai was produced by 5 min exposure to 10(-6) M ouabain, the abrupt exposure to 0 Nao produced a Ca uptake of 6 nmol/mg protein X 15 s and an increase in Ca content (AAS) of 4 nmol/mg protein. We conclude that there is an Nao-independent mechanism for Ca extrusion in these cells, presumably a Ca-ATPase Ca pump, with a limited Ca transport capacity of no more than 2 nmol/mg protein X 15 s. This is five times smaller than the demonstrated maximum capacity of the Na-Ca exchanger in these cells. The relaxation of twitch tension in these cells seems to be dependent primarily on sarcoplasmic reticulum uptake of Ca, with a secondary role provided by the Na-Ca exchanger. The Ca pump appears to contribute little to beat-to-beat relaxation.

Animals↗
PubMed1986

Relationships between the sarcoplasmic reticulum and sarcolemmal calcium transport revealed by rapidly cooling rabbit ventricular muscle.

Rabbit right ventricular papillary muscles were cooled from 30 to approximately 1 degree C immediately after discontinuing electrical stimulation (0.5 Hz). This produced a contracture that was 30-50% of the preceding twitch magnitude and required 20-30 s to develop. The contractures were identical in cooling solutions with normal (144 mM) or low (2.0 mM) Na. They were therefore not Na-withdrawal contractures. Contracture activation was considerably slower than muscle cooling (approximately 2.5 s to cool below 2 degrees C). Cooling contractures were suppressed by caffeine treatment (10.0 mM). Rapid cooling did not cause sufficient membrane depolarization (16.5 +/- 1.2 mV after 30 s of cooling) to produce either a voltage-dependent activation of contracture or a gated entry of Ca from the extracellular space. Contractures induced by treating resting muscles with 5 X 10(-5) M strophanthidin at 30 degrees C exhibited pronounced tension noise. The Fourier spectrum of this noise revealed a periodic component (2-3 Hz) that disappeared when the muscle was cooled. Cooling contractures decayed with rest (t1/2 = 71.0 +/- 9.3 s). This decay accelerated in the presence of 10.0 mM caffeine and was prevented and to some extent reversed when extracellular Na was reduced to 2.0 mM. 20 min of rest resulted in a net decline in intracellular Ca content of 1.29 +/- 0.38 mmol/kg dry wt. I infer that cooling contractures are principally activated by Ca from the sarcoplasmic reticulum (SR). The properties of these contractures suggest that they may provide a convenient relative index of the availability of SR Ca for contraction. The rest decay of cooling contractures (and hence the decay in the availability of activating Ca) is consistent with the measured loss in analytic Ca during rest. The results suggest that contraction in heart muscle can be regulated by an interaction between sarcolemmal and SR Ca transport.

Animals↗