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W H Barry

Publications and source records attributed to W H Barry.

At least 37 records · Page 2Linked to original sources
PubMed1998

Effects of overexpression of the Na+-Ca2+ exchanger on [Ca2+]i transients in murine ventricular myocytes.

We measured [Ca2+]i and [Na+]i in isolated transgenic (TG) mouse myocytes overexpressing the Na+-Ca2+ exchanger and in wild-type (WT) myocytes. In TG myocytes, the peak systolic level and amplitude of electrically stimulated (ES) [Ca2+]i transients (0.25 Hz) were not significantly different from those in WT myocytes, but the time to peak [Ca2+]i was significantly prolonged. The decline of ES [Ca2+]i transients was significantly accelerated in TG myocytes. The decline of a long-duration (4-s) caffeine-induced [Ca2+]i transient was markedly faster in TG myocytes, and [Na+]i was identical in TG and WT myocytes, indicating that the overexpressed Na+-Ca2+ exchanger is functionally active. The decline of a short-duration (100-ms) caffeine-induced [Ca2+]i transient in 0 Na+/0 Ca2+ solution did not differ between the two groups, suggesting that the sarcoplasmic reticulum (SR) Ca2+-ATPase function is not altered by overexpression of the Na+-Ca2+ exchanger. There was no difference in L-type Ca2+ current density in WT and TG myocytes. However, the sensitivity of ES [Ca2+]i transients to nifedipine was reduced in TG myocytes. This maintenance of [Ca2+]i transients in nifedipine was inhibited by Ni2+ and required SR Ca2+ content, consistent with enhanced Ca2+ influx by reverse Na+-Ca2+ exchange, and the resulting Ca2+-induced Ca2+ release from SR. The rate of rise of [Ca2+]i transients in nifedipine in TG myocytes was much slower than when both the L-type Ca2+ current and the Na+-Ca2+ exchange current function together. In TG myocytes, action potential amplitude and action potential duration at 50% repolarization were reduced, and action potential duration at 90% repolarization was increased, relative to WT myocytes. These data suggest that under these conditions, overexpression of the Na+-Ca2+ exchanger in TG myocytes accelerates the decline of [Ca2+]i during relaxation, indicating enhanced forward Na+-Ca2+ exchanger function. Increased Ca2+ influx also appears to occur, consistent with enhanced reverse function. These findings provide support for the physiological importance of both these modes of Na+-Ca2+ exchange.

Action Potentials↗
PubMed1998

Complications associated with rapid caffeine application to cardiac myocytes that are not voltage clamped.

The rapid application of caffeine to cardiac myocytes is commonly used to assess changes in the Ca2+ content of the sarcoplasmic reticulum (SR) and to study other parameters of intracellular Ca2+ regulation. Here we examined the effects of rapid caffeine application on membrane potential, intracellular Ca2+, and cell shortening in ventricular myocytes (rat, rabbit, guinea pig, dog) and atrial myocytes (rabbit) that were not voltage clamped. Conditioning pacing was used to achieve a steady-state level of SR Ca2+ loading prior to caffeine (10 mM) application. Caffeine transiently depolarized myocytes as expected from activation of forward Na+-Ca2+ exchange. However, we also found in each species (50% rat, 36% rabbit ventricular, 53% rabbit atrial, 56% guinea pig, 31% dog) that the caffeine-induced depolarization could also trigger an action potential. Caffeine-triggered potentials were completely blocked by thapsigargin (1 microM). The Ca2+ transient and contraction that accompanied caffeine-triggered action potentials had a larger magnitude and slower rate of decline (or relaxation) than occurred during caffeine-induced subthreshold depolarizations. Thus, the use of rapid caffeine application to study SR function and [Ca2+]i regulation in myocytes that are not voltage clamped can yield erroneous results.

Animals↗
PubMed1998

Sarcoplasmic reticulum function in murine ventricular myocytes overexpressing SR CaATPase.

To examine the effects of the overexpression of sarcoplasmic reticulum (SR) CaATPase on function of the SR and Ca2+ homeostasis, we measured [Ca2+]i transients (fluo-3), and L-type Ca2+ currents (ICa.L), Na/Ca exchanger currents (INa/Ca), and SR Ca2+ content with voltage clamp in ventricular myocytes isolated from wild type (WT) mice and transgenic (SRTG) mice. The amplitude of [Ca2+]i transients was insignificantly increased in SRTG myocytes, while the diastolic [Ca2+]i tended to be lower. The initial and terminal declines of [Ca2+]i transients were significantly accelerated in SRTG myocytes, implying a functional upregulation of the SR CaATPase. We examined the functional contribution of only the SR CaATPase to the initial and the terminal phase of the decline of [Ca2+]i, by abruptly inhibiting Na/Ca exchange with a rapid switcher device. The rate of [Ca2+] decline mediated by the SR CaATPase was increased by 40% in SRTG compared with WT myocytes. The function of the L-type Ca2+ channel was unchanged in SRTG myocytes, while INa/Ca density was slightly (10%) decreased. Measured SR Ca2+ content was significantly increased by 29% in SRTG myocytes. Thus, overexpression of SR CaATPase markedly accelerates the decline of [Ca2+]i transients, and induces in increase in SR Ca2+ content, with some downregulation of the Na/Ca exchanger.

Animals↗
PubMed1998

Abnormal myocyte Ca2+ homeostasis in rabbits with pacing-induced heart failure.

To determine whether there are abnormalities in myocyte excitation-contraction coupling and intracellular Ca2+ concentration ([Ca2+]i) homeostasis in pacing-induced heart failure (PF), we measured L-type Ca2+ current (ICa,L) and Na+/Ca2+ exchanger current (INa/Ca) with voltage clamp and measured intracellular Na+ concentration ([Na+]i) and [Ca2+]i with the use of sodium-binding benzofuran isophthalate (SBFI) and fluo 3 in ventricular myocytes isolated from control and paced rabbits. The peak systolic and diastolic levels and the amplitude of electrically stimulated [Ca2+]i transients (0.25 Hz, extracellular Ca2+ concentration = 1.08 mM) were significantly less in PF myocytes. Also, there was prolongation of the times to peak and decline of [Ca2+]i transients. ICa,L density was markedly decreased in PF myocytes. INa/Ca at -40 mV elicited by rapid exposure to 0 Na+ solution with a rapid solution switcher was significantly reduced in PF myocytes, suggesting that the function of the Na+/Ca2+ exchanger is impaired in these myocytes. In PF myocytes the decline of the [Ca2+]i transient when the Na+/Ca2+ exchanger was abruptly disabled was markedly prolonged compared with the decline in control myocytes, consistent with depressed sarcoplasmic reticulum (SR) Ca2+-ATPase function. RNase protection assay showed decreased levels of Na+/Ca2+ exchanger and SR Ca2+-ATPase mRNA in PF hearts, consistent with the function studies. We conclude that the functions of L-type Ca2+ channels, Na+/Ca2+ exchanger, and SR Ca2+-ATPase are impaired in myocytes from rabbit hearts with failure induced by rapid pacing. These abnormalities result in reduced [Ca2+]i transients and systolic and diastolic dysfunction and appear to account for the abnormal ventricular function observed.

Animals↗
PubMed1998

Effect of ANG II on pHi, [Ca2+]i, and contraction in rabbit ventricular myocytes from infarcted hearts.

In this study we examined Na+/H+ exchange activity, Ca2+ transients, and contractility in rabbit ventricular myocytes isolated from normal and chronically (8-12 wk) infarcted left ventricles. Myocytes from infarcted hearts (post-MI myocytes) were isolated from the peri-infarcted region of the left ventricle. Intracellular pH (pHi) and Ca2+ concentration ([Ca2+]i) were measured with the fluorescent pH indicators seminaphthorhodafluor 1 and fluo 3, respectively, and contractility was assessed from changes in cell shortening during field stimulation. Experiments were performed at extracellular pH 7. 4 in the presence and absence (HEPES buffer) of CO2 and HCO-3. Our findings demonstrate that 1) myocytes after myocardial infarction (post-MI) were significantly larger than normal, 2) post-MI hypertrophy was not accompanied by changes in non-CO2 intracellular buffering power, 3) post-MI hypertrophy did not significantly affect the ability of Na+/H+ exchange to mediate pHi recovery from intracellular acidosis, 4) the stimulatory effect of ANG II (100 nM) on Na+/H+ exchange was significantly reduced in post-MI myocytes, 5) in HCO-3-buffered solutions, ANG II did not significantly stimulate pHi recovery from acidosis in post-MI myocytes, 6) the angiotensin AT1 receptor mediates the stimulatory action of ANG II on Na+/H+ exchange in normal and post-MI myocytes, and 7) the stimulatory effect of ANG II on the Ca2+ transient and contraction was blunted in post-MI myocytes bathed in HEPES-buffered solution. A suppressed ventricular responsiveness to ANG II may be beneficial in the intact myocardium by attenuating ATP consumption and by reducing intracellular Na+ accumulation during ischemia-reperfusion.

Angiotensin II↗
PubMed1998

Influence of prior Na+ pump activity on pump and Na+/Ca2+ exchange currents in mouse ventricular myocytes.

We examined the dependence of peak Na+ pump and Na+/Ca2+ exchanger currents on prior Na+ pump inhibition induced by exposure to zero extracellular K+ in voltage-clamped adult murine ventricular myocytes. Abrupt activation of the Na+ pump by reexposure of myocytes to extracellular K+ with a rapid solution switcher resulted in the development of a transient peak current at approximately 500 ms, followed by a decline over 1-2 min to a steady-state level. The magnitudes of both the peak Na+ pump current (Ip) and the peak outward Na+/Ca2+ exchange current, activated by rapidly reducing extracellular Na+ to zero with the solution switcher, were dependent on previous Na+ pump activity. [Na+] gradients (Na+-binding benzofuran isophthalate fluorescence) between the patch pipette and the bulk cytosol were relatively small and could not account for the large differences between peak and steady-state Ip and reverse Na+/Ca2+ exchanger currents. Our results are consistent with the presence of a subsarcolemmal Na+ concentration gradient, which is similar for the Na+ pump and the Na+/Ca2+ exchanger. These findings also support the hypothesis that the Na+ pump and the Na+/Ca2+ exchanger are colocalized in the sarcolemma.

Animals↗
PubMed1998

Effects of angiotensin II on intracellular calcium and contracture in metabolically inhibited cardiomyocytes.

Angiotensin II (A-II) is known to potentiate ischemic dysfunction during ischemia, but the mechanisms involved are not completely established. We examined the effects of A-II on intracellular calcium concentration ([Ca++]i) and cell contracture caused by metabolic inhibition in isolated adult rabbit ventricular myocytes. [Ca++]i was assessed by flow cytometry, using the Ca(++)-sensitive fluorescent probe, fluo-3. After 90 min of exposure to 2 mM cyanide (CN) and 0 glucose, there was a significant increase in myocyte [Ca++]i. This increase was slightly augmented in the presence of 100 nM A-II. In the presence of partial Na+/K+ ATP pump inhibition ([K+]o = 0.8 mM), there was a more significant increase in [Ca++]i associated with exposure to CN + A-II vs. CN alone. Similar results were obtained with CN plus 2-deoxyglucose, and the effect of A-II was inhibited by 10 microM 5-(N-ethyl-N-isopropyl)amiloride. Myocytes exposed to 2 mM CN and 0 glucose gradually developed contracture over a 3-hr period. Addition of 100 nM A-II significantly (P < .01) enhanced loss of rod shape morphology during 3 hr of CN exposure. Partial inhibition of the Na+ pump by exposure to 0.8 mM K+ had no effect on myocyte survival in the absence of CN, but augmented the harmful effect of A-II on cell contracture caused by CN exposure. This effect of A-II was completely reversed by the addition of 1 mM amiloride, a Na+/H+ exchange inhibitor. We conclude that A-II directly enhances cell injury during CN exposure in isolated rabbit ventricular myocytes. We postulate that this effect of A-II is mediated by stimulation of Na+/H+ exchange with resultant increased [Na+]i and subsequent [Ca++]i loading, possibly via reverse Na+/Ca++ exchange.

Angiotensin II↗
PubMed1997

Endothelin and angiotensin II stimulation of Na+-H+ exchange is impaired in cardiac hypertrophy.

We compared the effects of endothelin-1 (ET-1) on intracellular pH, intracellular [Ca2+]i, and cell contraction in hypertrophied adult ventricular myocytes from ascending aortic banded rats and age-matched controls. Intracellular pH (pH(i)) was measured in individual myocytes with SNARF-1, and [Ca2+]i was measured with indo-1, simultaneous with cell motion. Experiments were performed at 36 degrees C in myocytes paced at 0.5 Hz in Hepes-buffered solution (pH(o) 7.40) containing 1.2 mM CaCl2. At baseline, calibrated pH(i), 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 nM ET-1 caused an increase in the amplitude of cell contraction to 163+/-22% of baseline (P < 0.05), associated with intracellular alkalinization (pH(i) + 0.08+/-0.02 U, P < 0.05) and a slight increase in peak systolic [Ca2+]i (104+/-11% of baseline, P < 0.05). In contrast, in the hypertrophied myocytes, exposure to ET-1 did not increase the amplitude of cell contraction or cause intracellular alkalinization (-0.01+/-0.02 U, NS). Similar effects were observed in the hypertrophied and control myocytes in response to exposure to 10 nM angiotensin II. ET-1 also increased the rate of recovery from intracellular acidosis induced by the washout of NH4Cl in the control cells, but did not do so in the hypertrophied cells. In the presence of 10 microM 5-(N-ethyl-N-isopropyl)-amiloride, which inhibits Na+-H+ exchange, ET-1 did not cause a positive inotropic effect or intracellular alkalinization in control cells. The activation of protein kinase C by exposure to phorbol ester caused intracellular alkalinization and it increased the rate of recovery from intracellular acidification induced by an NH4Cl pulse in control cells but not in hypertrophied cells. ET-1, as well as angiotensin II, and phorbol ester, fail to stimulate forward Na+-H+ exchange in adult hypertrophied myocytes. These data suggest a defect in the coupling of protein kinase C signaling with Na+-H+ exchange in adult hypertrophied myocytes.

Amiloride↗
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

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

Long-term angiotensin-converting enzyme inhibition with fosinopril improves depressed responsiveness to Ca2+ in myocytes from aortic-banded rats.

BACKGROUND: We have previously shown that long-term ACE inhibition with fosinopril prolongs survival and improves ventricular function despite persistent severe left ventricular pressure overload in ascending aortic-banded rats with left ventricular hypertrophy during the transition from compensation to failure. METHODS AND RESULTS: To study the cellular mechanism of the effects of long-term ACE inhibition on the modification of the transition to failure in pressure-overload hypertrophy, we measured simultaneous intracellular Ca2+ transients and myocyte shortening in isolated left ventricular myocytes from fosinopril-treated aortic-banded rats (n = 9), untreated aortic-banded rats (n = 9), and normal age-matched control rats (n = 10). Fosinopril therapy was begun 6 weeks after banding and was continued until week 21 after banding, when the animals were killed. Collagenase-dissociated myocytes loaded with indo 1-AM were paced at 3 Hz at 36 degrees C and superfused at [Ca2+]o of 0.6, 1.2, and 3.0 mmol/L. In myocytes from untreated aortic-banded rats, peak systolic [Ca2+]i was higher than in control myocytes, and the relationship between myocyte shortening and [Ca2+]i was depressed relative to control myocytes, implicating impaired responsiveness to Ca2+. Long-term fosinopril treatment improved both myocyte shortening and the relationship of shortening to [Ca2+]i (P < .05 versus myocytes from untreated aortic-banded rats). Maximal Ca(2+)-activated force was depressed in chemically skinned left ventricular fibers from untreated aortic-banded hypertrophied rats relative to age-matched controls but not in the fosinopril-treated aortic-banded rats. CONCLUSIONS: Long-term ACE inhibition improves responsiveness to Ca2+ in the presence of normalization of maximal Ca(2+)-activated force in aortic-banded rats subjected to persistent pressure overload. This may contribute to the favorable effects whereby ACE inhibition modifies the transition from compensated hypertrophy to failure.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1996

Lysis of adult ventricular myocytes by cells infiltrating rejecting murine cardiac allografts.

BACKGROUND: Immunologic mechanisms that mediate myocardial cell injury during rejection are not fully understood. We therefore investigated whether cells that infiltrate rejecting cardiac allografts are capable of directly injuring myocytes and whether this injury resembles that produced by cytotoxic T lymphocytes (CTLs) that are generated in a mixed lymphocyte reaction (MLR). METHODS AND RESULTS: Heart-infiltrating cells (HICs) were isolated from murine heterotopic BALB/c cardiac allografts undergoing rejection 6 to 8 days after transplantation into C57BL/6 mice. An in vitro model system of cultured adult murine ventricular myocytes was developed to facilitate investigation of cell-mediated myocyte injury. Isolated adult myocytes were incubated with either HICs or MLR effector cells, and myocyte death was quantified by counting the number of rod-shaped myocytes excluding trypan blue. The frequency of donor-reactive CTLs was similar in the HIC and MLR populations, as assessed by limiting dilution analysis. However, HICs were less efficient at killing donor-strain myocytes than were MLR cells. CTL-mediated cell lysis occurred by 6 hours, whereas myocyte injury produced by HICs was more gradual, with considerable cytotoxicity occurring between 12 and 24 hours. Furthermore, whereas MLR cells lysed only donor-strain myocytes, HIC lysed donor, third-party, and syngeneic myocytes. Treatment of MLR cells and HICs with anti-CD8 antibody plus complement produced a much greater inhibition of MLR cytotoxicity than of HIC cytotoxicity. CONCLUSIONS: These data demonstrate that only a small component of myocyte injury mediated by allograft-infiltrating cells can be ascribed to CTLs within the infiltrating cell population. These findings suggest that cell types associated with a delayed-type hypersensitivity response, as well as CTLs, cause myocyte injury during cardiac rejection.

Animals↗
PubMed1996

Management of intracoronary thrombosis complicating percutaneous transluminal coronary angioplasty.

With technological advances in equipment and increased experience of operators, the success rates of percutaneous transluminal coronary angioplasty (PTCA) now exceed 90%. However, acute periprocural occlusion continues to complicate approximately 6% of all procedures, and many of these occlusions are due to intracoronary (IC) thrombus. Patients at highest risk for this complication include those with acute ischemic syndromes or with angiographically apparent thrombus. These individuals may be candidates for the use of prolonged heparin infusions prior to dilatation, intracoronary thrombolytic therapy, or monoclonal antibody directed against the platelet glycoprotein IIb/IIIa receptor. All patients undergoing PTCA should receive adequate antiplatelet therapy, including aspirin, and heparin with dosing monitored by activated clotting times (ACT). In addition, some recommend the use of ionic contrast material. When IC thrombus accumulates following intervention, initial therapy should include IC nitroglycerin followed by a combination of redilatation and IC urokinase infusion. Prolonged balloon inflations may be useful, particularly with the use of autoperfusion catheters. Platelet glycoprotein IIb/IIIa receptor antagonists may prove to be beneficial in this situation as well. If the patient's clinical status deteriorates in spite of these measures, emergency coronary artery bypass graft surgery may be required.

Angioplasty, Balloon, Coronary↗
PubMed1996

Survival of metabolically inhibited ventricular myocytes is enhanced by inhibition of rigor and SR Ca2+ cycling.

During severe ATP depletion, sarcolemmal rupture resulting from rigor- and/or Ca(2+)-induced myofilament force development is considered to be an important cause of irreversible cell injury. Recent experiments in our laboratory demonstrated that during prolonged metabolic inhibition (MI) in adult rabbit ventricular myocytes, in which rigor was prevented by exposure to 30 mM 2,3-butanedione monoxime (BDM), cyclic uptake and release of cystolic Ca2+ occurred and was associated with strong phasic contractions. To investigate the relative contribution of this sarcoplasmic reticulum Ca2+ cycling and associated force development to energy depletion injury, the effects of BDM together with 7 mM caffeine were examined in isolated rabbit ventricular myocytes subjected to MI with 2 mM NaCN and 20 mM 2-deoxyglucose (2-DG). During 90 min of MI with CN and 2-DG, no cells retained a rod shape in the absence of BDM or caffeine. In the presence of both 30 mM BDM and 7 mM caffeine during MI, preservation of rod morphology was enhanced, and 52 +/- 6.2% of cells retained a rod shape 48 h after metabolic inhibition and had normal ATP content and resting membrane potential. Both systolic and diastolic functions of cells that survived MI, however, were impaired. We conclude that exposure to caffeine together with BDM markedly enhances survival of myocytes during severe prolonged ATP depletion. After recovery, these isolated myocytes show some characteristics of stunning.

Adenosine Triphosphate↗
PubMed1996

Effect of intracellular pH on ferret pulmonary arterial smooth muscle cell calcium homeostasis and pressure.

In this study, we investigated the role of Na+/H+ antiport in regulating cytosolic (intracellular) pH (pHi) in isolated and cultured ferret pulmonary arterial smooth muscle cells (PSMC). We also studied the effects of modulating pHi on the cytosolic (intracellular) calcium concentration ([Ca2+]i) in the PSMC and on the pulmonary arterial pressure (Ppa) of isolated ferret lungs. pHi was modulated by the NH4Cl washout method. To eliminate the contribution of Cl-/HCO3- exchangers, the PSMC and isolated lungs were perfused in HCO3- free buffer. Blocking the Na+/H+ antiporter decreased baseline pHi and prevented the recovery from NH4Cl washout-induced intracellular acidosis. Intracellular alkalinization caused an initial transient increase in both [Ca2+]i and Ppa that were dependent on extracellular Ca2+ entry. Maintaining cytosolic alkalinization caused another increase in Ppa that was not associated with an increase in [Ca2+]i. Intracellular acidosis also caused an increase in [Ca2+]i and Ppa. The cytosolic acidosis-induced increase in [Ca2+]i and Ppa were mediated by both extracellular Ca2+ influx and release of stored intracellular Ca2+. Cytosolic acidosis also appears to have a direct effect on the smooth muscle contractile elements. Both cytosolic alkalosis and acidosis increased vascular reactivity.

Acidosis↗
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

Regulated expression of a contractile protein gene correlates with recovery of contractile function after reversible metabolic inhibition in cultured myocytes.

Little is known of the relation between recovery of contraction and the regulation of contractile protein gene expression in ventricular myocytes after severe ATP depletion. We have examined alterations in activation of an MLC-2 luciferase fusion gene in cultured neonatal rat ventricular myocytes produced by exposure to 2 mM Na CN and 20 mM 2-deoxyglucose, and after recovery is serum or serum free medium. The effects of metabolic inhibition followed by recovery on expression on an RSV-luciferase activity were also investigated. Myocytes were co-transfected with a CMV beta-galactosidase fusion gene, and luciferase activities were normalized relative to beta-galactosidase activity to control for transfection efficiency. Two hours of metabolic inhibition produced significant cell injury, as documented by disorganization of myofilaments, and reduction in luciferase and beta-galactosidase activity within transfected cells. Cells allowed to recover for 48 h in serum free hormone supplemented medium showed a further decline in corrected luciferase activity, consistent with a marked reduction in MLC-2 gene transcription. Cells recovered from severe metabolic inhibition in serum free medium also showed failure to redevelop contractile activity, and failure of redevelopment of organized myofibrils. In contrast, myocytes exposed to serum during the 48 h recovery period had a marked increase in luciferase activity, resumed contractile activity and re-established organized myofilaments. There were no significant differences between RSV luciferase activities in cells recovered in serum versus serum free media. In ventricular myocytes in which contraction was inhibited by exposure to 10 microM verapamil, MLC-2 luciferase activity declined by 87%. However, even when contractile activity was inhibited by exposure to verapamil during recovery from metabolic inhibition, exposure to serum containing medium caused a significantly greater increase in MLC-2 luciferase activity than did serum free medium. Thus, the effects of serum on MLC-2 gene expression were not solely due to an effect of serum on recovery of contractile activity. Verapamil had no consistent effect on expression of RSV luciferase. These results suggest that expression of the MLC-2 gene is markedly reduced following recovery from severe metabolic inhibition, an effect largely due to cessation of myocyte contractile activity. Resupply of growth factors present in fetal calf serum reactivate expression of this gene, and this is associated with resumption of contractile activity and redevelopment of organized myofibrils. These results suggest that reactivation of contractile protein gene expression during recovery from metabolic inhibition may be beneficial in allowing cells to recover from this insult.

Analysis of Variance↗