Biomedical subjects

K W Spitzer

Publications and source records attributed to K W Spitzer.

At least 37 records · Page 2Linked to original sources

PubMed1997

Effects of the nitric oxide donor sodium nitroprusside on intracellular pH and contraction in hypertrophied myocytes.

BACKGROUND: We compared the effects of the nitric oxide donor sodium nitroprusside (SNP) on intracellular pH (pHi), intracellular calcium concentration ([Ca2+]i) transients, and cell contraction in hypertrophied adult ventricular myocytes from aortic-banded rats and age-matched controls. METHODS AND RESULTS: pHi was measured in individual myocytes with SNARF-1, and [Ca2+]i transients were measured with indo 1 simultaneously with cell motion. Experiments were performed at 37 degrees C in myocytes paced at 0.5 Hz in HEPES-buffered solution (extracellular pH = 7.40). At baseline, calibrated pHi, diastolic and systolic [Ca2+]i values, and the amplitude of cell contraction were similar in hypertrophied and control myocytes. Exposure of the control myocytes to 10(-6) mol/L SNP caused a decrease in the amplitude of cell contraction (72 +/- 7% of baseline, P < .05) that was associated with a decrease in pHi (-0.10 +/- 0.03 U, P < .05) with no change in peak systolic [Ca2+]i. In contrast, in the hypertrophied myocytes exposure to SNP did not decrease the amplitude of cell contraction or cause intracellular acidification (-0.01 +/- 0.01 U, NS). The cGMP analogue 8-bromo-cGMP depressed cell shortening and pHi in the control myocytes but failed to modify cell contraction or pHi in the hypertrophied cells. To examine the effects of SNP on Na(+)-H+ exchange during recovery from intracellular acidosis, cells were exposed to a pulse and washout of NH4Cl. SNP significantly depressed the rate of recovery from intracellular acidosis in the control cells compared with the rate in hypertrophied cells. CONCLUSIONS: SNP and 8-bromo-cGMP cause a negative inotropic effect and depress the rate of recovery from intracellular acidification that is mediated by Na(+)-H+ exchange in normal adult rat myocytes. In contrast, SNP and 8-bromo-cGMP do not modify cell contraction or pHi in hypertrophied myocytes.

Animals↗

PubMed1997

The restriction of diffusion of cations at the external surface of cardiac myocytes varies between species.

In cardiac muscle sarcolemmal structures such as T-tubules, caveolae and negatively charged protein-polysaccharides may affect the rate of cation exchange on the external surface of the cells. To test this hypothesis, we examined the rate of external cation exchange in adult rabbit and rat ventricular myocytes using a rapid solution switcher to change the bulk external solution within 4 ms. To assess the rate of diffusion of monovalent cations, we increased [K+]o from 4.4 to 6.6 or 8.8 mM and measured the time required to achieve a stable membrane depolarization. In rat myocytes, the mean time to 90% depolarization (t90) was significantly longer than that in rabbit myocytes (137 and 64 ms, respectively) and the difference in t90 was not associated with the cell size. To assess the time course of exchange of external Ca2+, we rapidly exposed the myocytes to 0 Ca2+-2 mM EGTA solution at specific time points before action potentials or voltage clamp steps, and measured the rate of alteration of the normalized peak [Ca2+]i transient (Fluo-3) or Ca2+ current. Exposure to 0 Ca2+-2 mM EGTA solution caused a decline in the intracellular calcium transient. In rat myocytes, the rate of decline in the [Ca2+]i transient was much slower (t90 > 1500 ms, the time required for 90% decline) than for the rabbit (t90 = 295 ms). Also, the rate of decline in the Ca2+ current was prolonged in rat myocytes (t90 = 910 ms) compared with rabbit myocytes (t90 = 241 ms). These data indicate that there is a restricted space on the external surface of sarcolemma which limits diffusion of divalent cations more markedly than monovalent cations. The extent of this limitation of cation diffusion varies between species, and may have functional significance.

Animals↗

PubMed1997

Contributions of the specialized conduction system to the activation sequence in the canine pulmonary conus.

This study was designed to characterize the relative contributions of the specialized conduction system and the myocardial architecture to the ventricular activation sequence. In animal experiments, the activation sequence within a 14 x 14-mm region on each surface of the pulmonary conus from isolated canine hearts was determined from electrograms recorded during ventricular drives applied at the periphery of the measurement region. Recordings were obtained simultaneously from electrode arrays mounted on the endocardium and epicardium. Activation sequences were determined before and after the right ventricular cavity was bathed with a dilute Lugol-normal Tyrode (LNT) solution that selectively inhibited excitation of Purkinje cells. Simulations of action potential propagation in three-dimensional models (14.4 mm long x 7.2 mm wide x 3.6 mm thick) that included the major features of the midwall architecture were performed to aid in the interpretation of the experimental findings. During endocardial pacing (7 animals, 43 total drives), LNT application markedly prolonged the endocardial (13.7 +/- 1.3 ms) and epicardial (5.7 +/- 1.0 ms) activation sequences. However, epicardial isochrone maps constructed with electrograms recorded before LNT application showed no signs of multiple breakthrough sites and, with the exception of overall timing, closely resembled isochrone maps constructed with electrograms recorded after LNT application. During epicardial pacing (9 animals, 55 total drives), LNT application prolonged the endocardial (3.7 +/- 0.6 ms) and epicardial (1.9 +/- 0.6 ms) activation sequences much less dramatically than during endocardial pacing, suggesting a primary contribution of myocardial architecture. However, in those instances where nonuniform anisotropy slowed epicardial expansion of the depolarization wavefront, the specialized conduction system contributed to the activation sequence to a greater extent.

Action Potentials↗

PubMed1997

Electrotonic modulation of electrical activity in rabbit atrioventricular node myocytes.

Electrotonic effects of electrically coupling atrioventricular (AV) nodal cells to each other and to real and passive models of atrial and ventricular cells were studied using a technique that does not require functional gap junctions. Membrane potential was measured in each cell using suction pipettes. Mutual entrainment of two spontaneously firing AV nodal cells was achieved with a junctional resistance (Rj) of 500 M omega, which corresponds to only 39 junctional channels, assuming a single-channel conductance of 50 pS. Coupling of AV nodal and atrial cells at Rj of 50 M omega caused hyperpolarization of the nodal cell, decreasing its action potential duration and either slowing or blocking diastolic depolarization in the AV node myocyte. Opposite changes occurred in the atrial action potential. When AV nodal and ventricular cells were coupled at Rj of 50 M omega, nodal diastolic potential was markedly hyperpolarized and diastolic depolarization was completely blocked with little change in ventricular diastolic potential. However, coupling did elicit marked changes in the action potential duration of both cells, with prolongation in the nodal cell and shortening in the ventricular cell. Nodal maximum upstroke velocity was increased by both atrial and ventricular coupling, as expected from the hyperpolarization that occurred. With an Rj of 50 M omega, spontaneous firing was blocked in all single AV nodal pacemaker cells during coupling to a real or passive model of an atrial or ventricular cell. These results demonstrate that action potential formation and waveform in a single AV nodal cell is significantly affected by electrical coupling to other myocytes.

Animals↗

PubMed1997

Sarcoplasmic reticulum and Na+/Ca2+ exchanger function during early and late relaxation in ventricular myocytes.

The relative importance of the Na+/Ca2+ exchanger in the initial and terminal phases of relaxation and the decline in the [Ca2+]i transient was investigated in adult rabbit ventricular myocytes loaded with the Ca2+ indicator fluo 3. For electrically stimulated contractions, the peak intracellular Ca2+ concentration ([Ca2+]i) was 700 +/- 87 nM and end-diastolic [Ca2+]i was 239 +/- 30 nM (0.25 Hz, 37 degrees C, 1.08 mM extracellular Ca2+ concentration; n = 14). Abrupt inhibition of Na+/Ca2+ exchange was produced by removal of extracellular Na+ (KCl substitution) and Ca2+ [2 mM Ca(2+)-free ethylene glycol-bis(beta-aminoethyl either)-N,N,N',N'-tetraacetic acid] by means of a rapid switcher device (SW). Abrupt exposure to high K+ induced an action potential, although sufficient Ca2+ remained adjacent to the sarcolemma to induce a contraction (SW beat) and [Ca2+]i transient that were identical in amplitude to those induced by electrical stimulation (ES beat). The initial relaxation and decline in the [Ca2+]i transient was not significantly prolonged by abrupt elimination of the Na+/Ca2+ exchanger, but the rate and extent of the terminal phase of the decline in the [Ca2+]i transient were significantly reduced. The first derivative of [Ca2+]i with respect to time versus [Ca2+]i during the decline of the [Ca2+]i transient attributable to sarcoplasmic reticulum (SR) function was estimated from the average SW transients, and that attributable to Na+/Ca2+ exchange was estimated from the difference between SW and ES transients. By this analysis, the Na+/Ca2+ exchanger produces 13% of the first half of the decline in [Ca2+]i and 45% of the second half of the decline. We conclude that abrupt inhibition of forward Na+/Ca2+ exchange does not significantly affect the amplitude or the initial rate of decline of the [Ca2+]i transient and relaxation. However, its contribution to the reduction of [Ca2+]i becomes apparent late during the [Ca2+]i transient, when cytosolic [Ca2+]i has been reduced.

Aniline Compounds↗

PubMed1996

Effect on the indo-1 transient of applying Ca2+ channel blocker for a single beat in voltage-clamped guinea-pig cardiac myocytes.

1. We used rapid solution changes to investigate the mechanisms which trigger Ca2+ release from the sarcoplasmic reticulum (SR) in guinea-pig ventricular myocytes. We patch-clamped myocytes at 36 degrees C and used indo-1 to monitor intracellular Ca2+. Before each test pulse, we established a standard level of SR Ca2+ load by applying a train of conditioning pulses. 2. We switched rapidly to 32 microM nifedipine (an L-type Ca2+ current (ICa,L) blocker) 8 s before a test pulse, and just after applying nifedipine we applied a ramp depolarization to pre-block Ca2+ channels. We found that ICa,L elicited by the following test pulse was inhibited almost completely (98-99% inhibition). 3. The indo-1 transient elicited by an 800 ms depolarizing pulse showed a rapid initial rise which was inhibited by ryanodine-thapsigargin. This indicated that the rapid rise was due to Ca2+ release from the SR, and therefore provides an index of SR Ca2+ release. 4. In cells dialysed internally with 10 mM Na(+)-containing solution, nifedipine application before a +10 mV test pulse blocked 62% of the rapid initial phase of the indo-1 transient. Calibration curves of indo-1 for intracellular Ca2+ (using a KD of indo-1 for Ca2+ of either 250 or 850 nM, the reported range) indicated that between 67 and 76% of the Ca2+i transient was inhibited by nifedipine. Thus, in cells dialysed with 10 mM Na+ and depolarized to +10 mV, and in the absence of ICa,L, this suggests that another trigger mechanism for SR release is able to trigger between 33 and 24% of the Ca2+i transient. 5. For a given dialysing Na+ concentration, the fraction of indo-1 transient which was inhibited by nifedipine decreased as test potential became more positive. In cells dialysed with 10 mM Na+ and pulsed to +110 mV, 24% of the rapid phase of the indo-1 transient was inhibited by nifedipine (equivalent to between 27 and 37% of the Ca2+i transient). 6. For a given test potential, the fraction of the indo-1 transient which was inhibited by nifedipine decreased as dialysing Na+ concentration increased. In cells dialysed with Na(+)-free solution and pulsed to +10 mV, 84% of the indo-1 transient was inhibited by nifedipine (equivalent to between 88 and 91% of the Ca2+i transient). In contrast, in cells dialysed with 20 mM Na+ and pulsed to +10 mV, 41% of the indo-1 transient was inhibited by nifedipine (equivalent to between 47 and 57% of the Ca2+i transient). 7. Dialysing cells with different Na+ concentrations could lead to a different SR Ca2+ content. We therefore manipulated the conditioning train before each test pulse to change the extent of SR loading. For each dialysing Na+ concentration, we found no change in the degree to which nifedipine blocked the indo-1 transient when SR content was either increased or decreased. 8. The results support the idea that both ICa, L and a second mechanism are able to trigger SR release and the resulting Ca2+i transient. When ICa, L was blocked with nifedipine, the fraction of Ca2+i transient which remained increased with more positive test potential and higher internal Na+. This is consistent with the hypothesis that the second SR trigger mechanism is Ca2+ entry via reverse Na(+)-Ca2+ exchange, elicited by a step change in membrane potential.

Animals↗

PubMed1996

Evidence that reverse Na-Ca exchange can trigger SR calcium release.

Several results suggest that the Na-Ca exchange can function as a trigger promoting SR Ca release and ensuing contractions. First, if the Ca current was the sole trigger for contraction we would expect the relationship between triggered contractions and voltage to be similar to the relationship between Ca current and contraction. When Na is present in the pipette this is not observed. Between -40 and +10 mV the relationships between contractions and voltage and current and voltage are similar. At potentials positive to 10 mV the Ca current declines as expected but contractions either decline much more slowly or continue to increase depending upon the concentration of intracellular Na. In addition, we have observed that contractions can be activated when Ca current is largely or completely blocked. Since these contractions are sensitive to the presence of ryanodine and thapsigargin they appear to be triggered by Na-Ca exchange. Also, contractions that are activated in the presence of nifedipine are sensitive to the Na-Ca exchange inhibitor XIP. Finally, rapid removal of extracellular Na apparently stimulates enough reverse exchange triggering of SR Ca release without affecting the SR content. It is clear that the shape of the shortening voltage relationship depends upon the concentration of dialyzing Na. This is likely to occur for two reasons. Either the shape of the shortening voltage relationship depends upon the extent to which Na-Ca exchange contributes a trigger for SR Ca release or alternatively the shape of the shortening voltage relationship depends upon SR Ca content. The latter is known to depend upon the Na concentration. In addition it is now established that the gain of SR Ca release is influenced by SR content. However, we studied triggered contractions in the absence of a Na gradient when the only available trigger is the Ca current. We measured triggered contractions over a range of voltages between -30 and +60 mV. Between each measurement we reestablished the Na gradient and activated a series of conditioning pulses to standardize the SR Ca content. Just before a test pulse we removed extracellular Na and activated either 3 or 6 pulses to produce two different SR Ca loads (in the absence of a Na gradient entering Ca cannot be extruded and therefore changes the SR Ca content). Regardless of the number of prepulses in the absence of a Na gradient the shortening voltage relationship was similar and bell shaped. From this we conclude that the shape of the relationship between shortening and voltage does not depend upon SR Ca content. Therefore, we conclude that the asymmetry in the shortening voltage relationship that depends upon intracellular Na is due to a contribution of reverse Na-Ca exchange. It is too early to say what the physiological significance (if any) of triggering by reverse exchange actually is. However, it does seem likely that it might provide a powerful inotropic mechanism. For example intracellular Na might be expected to change with heart rate and to be elevated at higher heart rates. Presumably this increased intracellular Na would tend to favor triggering by reverse exchange and would therefore enhance contractility at a time when it would be most required.

Animals↗

PubMed1996

Effect of internal sodium and cellular calcium load on voltage-dependence of the Indo-1 transient in guinea-pig ventricular myocytes.

OBJECTIVE: To investigate the effect of altering internal Na and cell Ca load on the voltage-dependence of the intracellular Ca transient. METHODS: Ventricular myocytes were isolated enzymatically from the guinea-pig heart. They were patch clamped and dialysed internally with pipette solutions which contained either 0 or 10 or 20 mM Na. Intracellular Ca was monitored with Indo-1 and experiments were carried out at 36 degrees C. A standard level of Ca loading was established before each test pulse by applying a train of conditioning pulses. The voltage-dependence of the Cai (Indo-1) transient provided information about the mechanisms which trigger Ca release from the sarcoplasmic reticulum (SR). RESULTS: The voltage-dependence of L-type Ca current (ICa.L) was assessed in separate experiments by dialysing myocytes with a Cs-based solution. ICa.L had a maximum amplitude at 0 mV, declined at more positive potentials and there was little net inward ICa.L at +100 mV. The rapid initial phasic component of the Indo-1 transient was abolished by ryanodine/thapsigargin; therefore, this component reflected the magnitude of SR Ca release. In cells dialysed with 10 mM Na, the voltage-dependence of the Indo-1 transient was different from ICa.L. The Indo-1 transient became maximal at +20 mV, and the decline of the Indo-1 transient at positive potentials was less steep than the decline of ICa.L. A large proportion of the phasic Indo-1 transient could remain at positive potentials where there was no detectable ICa.L. Increasing dialysing Na from 10 to 20 mM led to a marked change in voltage-dependence of the Indo-1 transient. With 20 mM Na, the amplitude of the phasic Indo-1 transient remained large between +20 and +100 mV. Removing Na from the pipette dialysis solution had the opposite effect on voltage-dependence of the transient. For each dialysing [Na], the level of cellular and SR Ca content was altered by varying the potential of conditioning pulses applied before each test pulse. There was no significant effect on voltage-dependence of the Indo-1 transient of either increasing or reducing the cellular Ca content. CONCLUSION: These data are consistent with the hypothesis that the voltage-dependence of the Cai transient results from the sum of the voltage-dependencies of the two main trigger mechanisms--Ica.L and reverse Na/Ca exchange. When a myocyte was dialysed with Na-free solution, the voltage-dependence of the Cai transient became more similar (but not identical) to that for ICa.L. With 20 mM Na dialysis, the altered voltage-dependence of the Cai transient may reflect an increased trigger influence of reverse Na/Ca exchange.

Animals↗

PubMed1995

Effects of premature beats on repolarization of postextrasystolic beats.

BACKGROUND: A short-long-short sequence of cycle lengths predisposes to reentrant tachyarrhythmias. There is limited information about the effects of premature ventricular contractions (PVCs) on repolarization of postextrasystolic depolarizations (PEDs). Such information would contribute to understanding the mechanism for facilitating reentry with short-long-short cycle lengths. METHODS AND RESULTS: We introduced PVCs, over a range of coupling intervals and during a range of basic drive cycle lengths (BCLs), and determined PED repolarization. Our results from whole-animal experiments, isolated cell studies, and computer simulations are reported. In the whole-animal experiments, PED refractory periods (RPs) were longer than RPBCL. The greatest difference between RPPED and RPBCL (delta RPmax) occurred after short coupling interval PVCs and was 4.3 +/- 0.8, 4.2 +/- 0.8, and 2.1 +/- 0.5 ms (mean +/- SEM) during drives with short, intermediate, and long BCLs, respectively. The diastolic interval preceding the PED (DIPED) was inversely related to the coupling interval between the basic drive beat and the PVC and directly related to RPPED. PED action potential durations (APDs) of isolated canine myocytes were 9.8 +/- 4.9 ms (mean +/- SEM) longer than APD BCL (n = 19). The DiFrancesco-Noble membrane equations were used in simulations of action potential propagation in a one-dimensional cable, with stimulation protocols duplicating those in the animal experiments. PVCs prolonged APDPED, and APDPED was prolonged more during short than during long BCL drives. There was a direct relation between DIPED and APDPED. Analysis of the membrane currents over the time course of the PVCs and PEDs suggested that the ionic basis for PED repolarization prolongation was the interaction of Ito and Ik. Hyperpolarizing constant-current injections introduced immediately after the spike of isolated myocyte action potentials caused APD prolongation. This observation is consistent with the Ito and Ik interaction causing PED repolarization prolongation. CONCLUSIONS: PED repolarization prolongation could provide sites for unidirectional block to propagation of PVCs after PEDs and could facilitate initiation of reentrant tachyarrhythmias after short-long-short sequences of cycle lengths.

Action Potentials↗

PubMed1995

Excitation-contraction coupling in ventricular myocytes: effects of angiotensin II.

The effects of the vasoactive peptide angiotensin II (AII) on contractility and excitation-contraction coupling in isolated adult rabbit ventricular myocytes were investigated. In most ventricular myocytes, AII (10(-8) M) induced a significant increase in fractional shortening which was not associated with an increase in the calcium transient measured with indo-1. AII did increase the intracellular pH by approximately 0.2 5 pH units coincident with the positive inotropic effect. Effects of AII on pH and contractility were blocked by inhibitors of Na+/H+ exchange. AII also increased the rate of pHi recovery from intracellular acidosis at pHi values above 6.9. AII was shown not to affect the L-type inward calcium current. However, in an occasional cell, AII was observed to cause a slight increase in the calcium transient. We hypothesize that this response may reflect an increase of calcium influx on the sodium calcium exchanger, as a consequence of an increase in subsarcolemmal sodium concentration resulting from enhanced Na(+)-H+ exchange.

Angiotensin II↗

PubMed1995

Angiotensin II stimulates sodium-hydrogen exchange in adult rabbit ventricular myocytes.

OBJECTIVE: The aim was to characterise the effects of angiotensin II on Na+/H+ exchange in adult ventricular myocytes. METHODS: Intracellular pH (pHi) was continuously measured with the fluorescent pH indicator, SNARF-1, in single resting myocytes obtained from adult rabbits by enzymatic dissociation. In some experiments cells were electrically paced to elicit contractions. All experiments were performed at 36 degrees C in HEPES buffered solution containing no added CO2 or HCO3- (pHo 7.4). RESULTS: Rapid application of angiotensin II caused pHi to rise. The initial rate of rise and initial net H+ efflux responded to angiotensin II in a concentration dependent manner, EC50 = 7.8. Buffering of cytosolic calcium with the calcium chelator BAPTA did not affect the initial net H+ efflux elicited by 1 microM angiotensin II. The increase in steady state pHi was blocked by inhibitors of Na+/H+ exchange, amiloride (1 mM) and EIPA (10 microM). Angiotensin II also increased the rate of pHi recovery from intracellular acidosis at pHi values above approximately 6.9. During inhibition of Na+/H+ exchange the application of angiotensin II decreased steady state pHi. This acidosis was blocked by preincubation in dextrose-free solution containing 20.0 mM 2-deoxy-D-glucose and 10 microM EIPA. The positive inotropic effect of angiotensin II was markedly suppressed by amiloride. CONCLUSIONS: Angiotensin II exerts a concentration dependent stimulatory effect on Na+/H+ exchange in adult rabbit ventricular myocytes. This effect does not appear to involve changes in cytosolic calcium. During inhibition of Na+/H+ exchange, angiotensin II causes pHi to fall, perhaps by stimulating metabolic acid production. The positive inotropic action of angiotensin II depends, in part, on stimulation of Na+/H+ exchange.

Amiloride↗

PubMed1994

Effects of activation sequence on the spatial distribution of repolarization properties.

The electrotonic effects of activation spread on the spatial distribution of repolarization properties were studied in animal experiments and with computer simulations. Refractory periods (RPs) were measured at 36 sites within a 1.0 cm2 region of the epicardial surface of the canine pulmonary conus during 37 drives in 11 experiments. In each experiment three or four sites along the perimeter of the region bounding the RP test sites were driven. Activation propagated uniformly during some and nonuniformly during other drives in the same animals. In general, RPs were distributed uniformly when activation spread uniformly and nonuniformly when activation spread nonuniformly. The authors observed RP differences as large as 16 ms between sites with 2 mm separation during drive from some epicardial sites in these normal canine hearts. Indices of nonuniformity of activation and of relative RP values were used to quantify the relation between nonuniformity of activation spread and the spatial distribution of the RP. There was a significant negative correlation between nonuniformity of activation and RP indices during the 19 drives in which activation spread nonuniformly. This indicated that RPs were relatively long at sites where activation spread decelerated and relatively short at sites where activation spread accelerated. When nonuniform activation spread was simulated by introducing high-resistance barriers in a model with fixed anisotropic conductivities, there were marked spatial variations in action potential duration. The spatial variations in action potential duration were negatively correlated to acceleration and deceleration of activation spread. The major new finding of this study is that the spatial distributions of RPs are markedly affected by activation spread. Since both characteristics of activation sequence and nonuniformity of RP distributions have roles in reentrant arrhythmias, the findings suggest that some sites of origin of premature activity may be more arrhythmogenic than others. The findings may also explain why ventricular tachycardia can sometimes be initiated from one but not from other sites in patients undergoing electrophysiologic testing.

Action Potentials↗

PubMed1994

Depolarization-induced Ca entry via Na-Ca exchange triggers SR release in guinea pig cardiac myocytes.

In mammalian heart muscle, Ca entry through L-type Ca channels is thought to be the primary trigger for the sarcoplasmic reticulum (SR) Ca release, which initiates contraction. The results of this study show that, in guinea pig myocytes with a normal internal Na (10 mM Na in pipette), another trigger mechanisms for SR release and contraction exists. A crucial feature of these experiments was the ability to change rapidly the extracellular environment of a single myocyte so that alterations of intracellular Ca and SR Ca load were minimized for each solution change. We found the following results. 1) A switch to Na-free solution 50 ms before depolarization led to an increase of phasic contraction without increasing L-type Ca current (Ica) or Ca loading of the SR. 2) Although rapid application of 20 microM nifedipine 3 s before a + 10-mV pulse blocked ICa completely, 43 +/- 11 (SE) % of the phasic contraction remained. Similar results were obtained by rapid switching to 150 microM Cd to block ICa. 3) Phasic contraction and ICa had different voltage dependence. With steps to positive potentials there was little ICa but still a substantial phasic contraction. 4) Under action potential conditions, 64.6 +/- 7.9% of the control phasic contraction remained after switching to 20 microM nifedipine to block ICa. 5) The contraction remaining with nifedipine was unaffected by adding 100 microM Ni. Because 100 microM Ni blocks T-type Ca channels, this shows that Ca entry via T-type Ca channels is not involved in triggering SR release. 6) The phasic contraction remaining after a rapid switch to nifedipine was blocked completely by adding 5 mM Ni. Because this concentration of Ni is known to block the Na-Ca exchange, this result suggests that the exchange plays a role in triggering SR release. Taken together, the present results indicate that depolarization-induced Ca entry on the Na-Ca exchange is able to trigger SR release and phasic contraction. This explanation can account for increased phasic contraction after a rapid switch to Na-free solution, persistence of a phasic contraction in the complete absence of ICa, substantial phasic contraction at positive test potentials where there is no ICa, and abolition of nifedipine-resistant contraction by 5 mM Ni.

Action Potentials↗

PubMed1994

Na-independent Cl(-)-HCO3- exchange mediates recovery of pHi from alkalosis in guinea pig ventricular myocytes.

The pH-sensitive fluorescent indicator, carboxy-seminaphthorhodafluor 1 (SNARF 1) was used to assess the contribution of forward Na-independent Cl(-)-HCO3- exchange (1 external Cl- exchanged for 1 internal HCO3-) to intracellular pH (pHi) recovery from alkalosis in adult ventricular myocytes (guinea pig). Intracellular alkalosis was elicited by external application of the weak base, trimethylamine. In the absence of CO2-HCO3- (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered solution) the initial rate of pHi recovery from alkalosis (pHi = 7.25-7.75) was slow and independent of pHi, yielding an apparent net HCO3- efflux of 0.36 +/- 0.11 mM/min. In CO2-HCO3(-)-buffered solution, the initial rate of pHi recovery and net HCO3- efflux were much faster and markedly increased by raising pHi. At pHi approximately 7.25, net HCO3- efflux was approximately 2 mM/min and rose to 9 mM/min at pHi approximately 7.6. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (0.4 mM) decreased net HCO3- efflux by 78.1 +/- 8.9% in CO2-HCO3(-)-buffered solution. Reduction in extracellular Cl- concentration from 135 to 20 mM markedly slowed the rate of pHi recovery from alkalosis and reduced net HCO3- efflux. pHi recovery from alkalosis was unaffected by removal of external sodium or exposure to 1 mM amiloride. These results indicate that forward Na-independent Cl(-)-HCO3- exchange mediates pHi recovery from alkalosis in guinea pig ventricular myocytes.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

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↗

PubMed1993

Computer simulations of three-dimensional propagation in ventricular myocardium. Effects of intramural fiber rotation and inhomogeneous conductivity on epicardial activation.

Three-dimensional membrane-based simulations of action potential propagation in ventricular myocardium were performed. Specifically, the effects of the intramural rotation of the fiber axes and inhomogeneous conductivity on the timing and pattern of epicardial activation were examined. Models were built, with approximately 400,000 microscopic elements arranged in rectangular parallelepipeds in each model. Simulations used the nonlinear Ebihara and Johnson membrane equations for the fast sodium current. Constructed models had histological features of ventricular myocardium. All models were anisotropic. In a subset of the models, an abrupt intramural rotation of the fiber axes was included. This feature was also combined with randomly distributed inhomogeneous conductivity and regions of high transverse resistance to represent nonuniform anisotropy in a further subset of the models. Epicardial stimuli were applied for each simulation. Three-dimensional activation patterns and epicardial isochron maps were constructed from the simulations. We noted that the rotation of fiber axes accelerated epicardial activation distant from the stimulus site. The inhomogeneous conductivity caused regional acceleration and deceleration of activation spread. We also noted features of epicardial activation that resulted from the fiber rotation, and the inhomogeneous conductivity corresponded to that observed in maps from experimental animals.

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↗

PubMed1991

Modulation of collision-induced changes in canine heart repolarization by cycle length.

The possibility that cycle length modulates the electronic effect of activation sequence on repolarization was investigated in experiments using isolated canine cardiac Purkinje strands, in situ canine ventricular myocardium, and computer simulations. Action potential durations and refractory periods during one-way propagation were compared to those obtained during action potential collision. In both the computer simulations and the Purkinje strand experiments, collision decreased action potential duration more at long cycle lengths than at short cycle lengths. Comparably, collision of activation fronts in ventricular myocardium was associated with greater reductions in refractory period during pacing at long cycle lengths than at short cycle lengths. Theoretic considerations indicate that the magnitude of electrotonic effects of activation sequence on repolarization are directly related to action potential height and the square root of membrane resistance during repolarization and are inversely related to conduction velocity. In computer simulations and Purkinje strand experiments, changes in conduction velocity and action potential height elicited by decreasing cycle length could not fully account for the cycle length dependence of collision-induced changes in repolarization. Time-varying membrane resistance of a single cell was calculated in the simulations by briefly hyperpolarizing the membrane and determining the change in total ionic current. Membrane resistance during repolarization was less at short cycle lengths than at long cycle lengths. The results suggest the cycle length dependence of collision-induced changes in repolarization results largely from the effect of cycle length on membrane resistance during action potential repolarization, with changes in action potential height and conduction velocity playing a lesser role.

Action Potentials↗