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J M Burt

Publications and source records attributed to J M Burt.

36 records · Page 2Linked to original sources
PubMed1991

Characterization of gap junction channels in A7r5 vascular smooth muscle cells.

Recent evidence suggest that coordination of blood flow in the microcirculation involves cell-to-cell coupling via gap junctions. In this study, using A7r5 cells as a model of vascular smooth muscle, we have characterized the gap junctions in terms of the unitary conductances of the observed channels, the responses to second messengers, and subunit protein composition. The cells were typically well coupled several hours after plating, with junctional conductances on the order 20-40 nS. Channels with mean conductances of 36 and 89 pS were observed in low-conductance cell pairs and in cell pairs whose macroscopic conductance was reduced by exposure to halothane. Connexin43 was the only known gap junction sequence detected by Northern blots (low and high stringency), immunoblots, or immunohistochemical studies. Junctional conductance was reduced 15% by 8-bromoadenosine 3',5'-cyclic monophosphate; 8-bromoguanosine 3',5'-cyclic monophosphate had no effect. The results suggest that connexin43 can form stable channels of at least two distinct conductances and gap junctions with differing responses to second messengers.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1990

Structure-activity relations of the cardiac gap junction channel.

Cardiac gap junction channels play the important roles of synchronizing pacemaker cells and allowing impulse propagation along the conduction system and throughout the ventricular myocardium. These channels, which support current flow in both longitudinal and tranverse directions, are permeable to anions and cations with radii less than approximately 0.5 nm and in rat heart have unitary conductances on the order of 50 pS. This unitary conductance is consistent with channel geometry described by a right cylindrical pore with diameter large enough for the brilliantly fluorescent dye molecule lucifer yellow to pass between cells. These channels, like others in biological systems, are opened and closed by various treatments, a process termed gating. Cytoplasmic acidification reduces junctional conductance (gj), an effect that is apparently potentiated by elevated myoplasmic Ca ions. Reduced gj also occurs in response to a variety of lipophilic molecules, including halothane, heptanol, and unsaturated fatty acids; the mechanism of action may involve disruption of the protein-lipid microenvironment of the gap junction channel. Arachidonic acid uncouples, and this effect is partially, but incompletely, blocked by an inhibitor of the lipoxygenase metabolic pathways. Cyclooxygenase inhibitors have no protective effects. Certain cyclic nucleotides can rapidly increase gj [adenosine 3',5'-cyclic monophosphate (cAMP)] or slightly decrease it [guanosine 3',5'-cyclic monophosphate (cGMP)], and agents that use these cyclic nucleotides as second messengers (isoproterenol and perhaps carbachol, respectively) produce consistent effects. Agents expected to cause protein kinase C activation (tumor-promoting phorbol esters and diacylglycerol) increase gj rapidly. The gap junction protein from rat heart has been cloned and sequenced. From the primary sequence for the protein, plausible sites of action within the putative cytoplasmic domains are proposed for each of these treatments. In response to gating stimuli that close the channel (halothane, CO2, heptanol), unitary channel conductance is unchanged, suggesting that these agents act by reducing open time probability. Together, these properties constitute the beginnings of our endeavor to define pharmacological agents that are potentially useful in therapeutic manipulation of synchronous discharge, conduction velocity, and isochronous wavefront propagation in cardiac tissue.

Animals↗
PubMed1989

Uncoupling of cardiac cells by doxyl stearic acids specificity and mechanism of action.

The influence of doxyl stearic acids (DSAs) on gap junctional conductance (gj) between pairs of neonatal rat heart cells was studied. DSAs are spin probes that perturb the membrane at different depths depending on position of the doxyl group on the fatty acyl chain. 16-DSA and 12-DSA rapidly and reversibly reduced gj to unmeasureable levels in a dose- and time-dependent manner. Single channel events observed when gj was low were of the same unitary size as those observed under control conditions. The methyl esters of 16- and 12-DSA, stearic acid itself, and TEMPO, an analogue of the doxyl group that has no fatty acyl chain, had no effect on gj. Protonation of the carboxyl head group (by acidifying the solution) reduced the potency of 16- or 12-DSA. Spontaneous beating activity and action potentials were observed at concentrations of the DSAs 15-20 times that necessary for uncoupling. These results indicate that uncoupling by the DSAs requires the presence of the charged carboxyl group and localized perturbation of the channel at the lipid-channel interface by the doxyl group. Furthermore, they predict that unsaturated free fatty acids, which accumulate during ischemia, may exert their arrhythmogenic effect by reducing gj, and thereby slowing conduction.

Action Potentials↗
PubMed1989

Volatile anesthetics block intercellular communication between neonatal rat myocardial cells.

The effects of halothane and ethrane on gap junction-mediated intercellular communication and on membrane excitability were examined in cultured neonatal rat cardiac myocytes using whole-cell voltage-clamp and current-clamp techniques. Excitability was maintained at doses of both anesthetics that reversibly abolished current flow through junctional membranes. The degree of reduction of junctional conductance was a steep function of the dose of anesthetic; complete block occurred at lower aqueous concentrations of halothane than ethrane. The time course for loss of communication was rapid; 90% reduction of initial junctional conductance occurred in less than 15 seconds after exposure to 2 mM halothane or 4 mM ethrane. Recovery of junctional conductance and junctional permeability to intracellularly injected Lucifer yellow was rapid and complete on washout of the anesthetics. As junctional conductance was reduced by halothane or ethrane exposure, unitary conductance of the gap junctional channels remained constant at about 50 pS. Uncoupling by these anesthetics is thus attributable to a decrease in the number of conducting channels rather than to reduction of the channel's unitary conductance. The data are discussed with regard to the possible role of this intercellular communication pathway in the arrhythmias and alterations of conduction velocity and contractility produced by volatile anesthetics.

Animals↗
PubMed1988

Single-channel events and gating behavior of the cardiac gap junction channel.

The activity of gap junction channels between pairs of neonatal rat heart cells in culture was studied under control conditions and during uncoupling procedures by using dual whole-cell voltage clamp techniques. Under control conditions gap junctional conductance ranged from 0.05 to 35 nS. In cell pairs exhibiting low gap junctional conductance (less than 500 pS), single-channel events with a unitary conductance of 53 +/- 2 pS (5 experiments; 186 events) were apparent. Event duration and open-time probability were estimated to be 0.95 sec and 0.17, respectively. When the junctional conductance in well-coupled cell pairs (with initial junctional conductance, greater than 5 nS) was reduced by cytoplasmic acidification or application of heptanol, single-channel events could be visualized. Compared to low-conductance controls, unitary channel conductance was unaltered (for acidification the conductance was 58 +/- 3 pS in 11 experiments with 253 events; for heptanol the conductance was 61 +/- 1 pS in 2 experiments with 171 events), while the probability of channels being open was decreased. The constancy of unitary channel conductance under control conditions and during uncoupling procedures suggests that opening and closing of the gap junction channel are all-or-none processes during which no stable subconductance states are formed.

Alcohols↗
PubMed1988

Inotropic agents modulate gap junctional conductance between cardiac myocytes.

Cardiac gap junction channels mediate the intercellular exchange of second messenger and small molecules. Through effects on conduction velocity, enhanced junctional conductance (gj) facilitates rapid and synchronous activation of the contractile myocardium, whereas reduced gj slows activation and could contribute to arrhythmogenesis. We report here that gj is enhanced by agents that elevate intracellular adenosine 3',5'-cyclic monophosphate (cAMP; 8-bromo-cAMP or isoproterenol) and is depressed by agents that elevate intracellular guanosine 3',5'-cyclic monophosphate (cGMP; 8-bromo-cGMP or carbachol). Both effects occur with a time course comparable to the inotropic events mediated by these agents. The effect of cAMP on gj is not dependent on simultaneous changes in intracellular calcium; however, during calcium-overload conditions cAMP can precipitate calcium-dependent uncoupling. These results indicate that cyclic nucleotide-dependent changes in gj may contribute to the inotropic effects of these agents. Furthermore, the results suggest that the inotropic effect of cAMP includes a calcium-independent component.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1987

Block of intercellular communication: interaction of intracellular H+ and Ca2+.

The influence of elevated intracellular levels of H+ and Ca2+ on intercellular communication between cultured neonatal rat myocardial cells was examined by quantifying the percent of primary neighboring cells to which intracellularly injected Lucifer yellow had spread within 10 s of injection. Partial acidosis was induced by incubation in and then removal of NH4Cl. Intracellular Ca2+ was raised through the use of treatments that are standard in studies of heart muscle: reduction of the Na+ gradient, addition of caffeine, and combinations of these interventions. Under control conditions and during application of NH4Cl, cells exhibited spontaneous electrical and contractile activity and were well coupled (dye detectable in 100% of primary neighbors). Sustained intracellular acidosis without simultaneous elevation of intracellular Ca2+ (NH4Cl exposure followed by zero Na+, zero Ca2+) reduced the incidence of dye transfer to 90%. Elevation of intracellular Ca2+ (exposure to zero Na+, Ca2+-containing solution, with or without 10 mM caffeine) had no effect on coupling. These same interventions, when employed together, reduced the incidence of dye coupling to 18%. The results are consistent with a synergism of action of Ca2+ and H+ in the regulation of junctional permeability.

Ammonium Chloride↗
PubMed1986

Myocardial function after preservation for 24 hours.

The orthotopically transplanted heart undergoes several steps between harvest from the donor and reperfusion in the recipient: cardioplegic arrest and cooling, ischemia during the operation, in most cases a preservation or storage period of varying duration, and reperfusion. Each of these steps represents a period during which damage to the heart can occur. In this study we have quantified the degree of damage sustained by a donor heart during each of these steps. This objective was achieved by evaluating the function of rabbit hearts via Langendorff procedures following (Group 1) cooling and reperfusion; (Group 2) cardioplegic arrest, cooling, and reperfusion; (Group 3) arrest, cooling, 1 hour of ischemia (5 degrees or 25 degrees C), and reperfusion; (Group 4) arrest, cooling, 24 hours of preservation, and reperfusion; and (Group 5) arrest, cooling, 24 hours of preservation, 1 hour of ischemia (25 degrees C), and reperfusion. Comparisons were made between groups and to control hearts. Cooling and reperfusing the heart (Group 1) led to no loss of function, although recovery to precooling function levels required approximately 25 minutes. Hearts that were arrested before cooling (Group 2) regained full function without the slow recovery time. Hearts that were arrested, cooled, and made ischemic at 5 degrees C (Group 3) recovered 95% of preischemic contractile function: maximum systolic pressure and the maximum positive derivative of the systolic pressure curve. No change in diastolic compliance was detected. Hearts that were arrested, cooled, and made ischemic at 25 degrees C (Group 3) recovered 89% of preischemic contractile function (maximum systolic pressure and the maximum positive derivative of the systolic pressure curve). Again, no change in diastolic compliance was detected. Hearts that were arrested, cooled, preserved for 24 hours, and reperfused (Group 4) recovered 84% of control contractile function (maximum systolic pressure and the maximum positive derivative of the systolic pressure curve), whereas hearts that had the additional hour of ischemia at 25 degrees C (Group 5) recovered only 75% of control contractile function. In the latter two groups diastolic compliance was also compromised. Group 4 had a 20% decrease in the volume required to reach 10 mm Hg, and Group 5 had a 26% decrease. Pressure-volume curves suggest a loss of contractility and a loss of compliance in these hearts. These data indicate that while significant damage occurred as a result of ischemia and reperfusion, this damage was masked by the larger decrease in function occurring as a result of the preservation period.

Animals↗
PubMed1984

Phospholipase D increases cell surface Ca2+ binding and positive inotropy in rat heart.

An increase in the content of anionic phospholipid (AP) in the sarcolemma may lead to increased Ca2+ binding and a concomitant inotropic response. To test this hypothesis we treated cultured neonatal rat myocardial cells with phospholipase D (PLD), an enzyme that converts membrane phospholipids to phosphatidic acid. PLD treatment resulted in an increase in total exchangeable Ca2+ of 36%, or 1.56 +/- 0.27 mmol Ca2+/kg dry wt, 79 +/- 3% of which remained displaceable by La3+. The quantity of Ca2+ displaced by polymyxin B, a drug that displaces Ca2+ preferentially from anionic phospholipid sites, increased by 85% from a predicted 0.74 +/- 0.07 to 1.37 +/- 0.19 mmol Ca2+/kg dry wt. Simultaneously the contractility of neonatal rat ventricular tissue increased by 1.7- to 2.5-fold. Spontaneous electrical activity of treated cells was not significantly altered. Thus PLD treatment, which increases the quantity of AP in the membrane, resulted in an 85% increase in Ca2+ bound to AP and concomitantly resulted in a significant increase in contractility. Present results, in association with published results on the effect of PLD on Na+-Ca2+ exchange (J. Biol. Chem. 259: 16-19, 1984), indicate that Ca2+ bound to anionic sarcolemmal phospholipids plays a major role in the control of transsarcolemmal Ca2+ flux and force development.

Animals↗
PubMed1983

Ca2+ displacement by Polymyxin B from sarcolemma isolated by 'gas dissection' from cultured neonatal rat myocardial cells.

Amphiphilic, cationic Polymyxin B is shown to displace Ca2+ from 'gas dissected' cardiac sarcolemma in a dose-dependent, saturable fashion. The Ca2+ displacement is only partially reversible, 57% and 63%, in the presence of 1 mM or 10 mM Ca2+, respectively. Total Ca2+ displaced by a non-specific cationic probe, lanthanum (La3+), at maximal displacing concentration (1 mM) was 0.172 +/- 0.02 nmol/microgram membrane protein. At 0.1 mM, Polymyxin B displaced 42% of the total La3+-displaceable Ca2+ or 0.072 +/- 0.01 nmol/microgram protein. 5 mM Polymyxin displaced Ca2+ in amounts equal to those displaced by 1 mM La3+. Pretreatment of the membranes with neuraminidase (removal of sialic acid) and protease leads to a decrease in La3+-displaceable Ca2+ but to an increase in the fraction displaced by 0.1 mM Polymyxin from 42% to 54%. Phospholipase D (cabbage) treatment significantly increased the La3+-displaceable Ca2+ to 0.227 +/- 0.02 nmol/microgram protein (P less than 0.05), a gain of 0.055 nmol. All of this phospholipid specific increment in bound Ca2+ was displaced by 0.1 mM Polymyxin B. The results suggest that Polymyxin B will be useful as a probe for phospholipid Ca2+-binding sites in natural membranes.

Animals↗
PubMed1983

Influence of polymyxin B, a probe for anionic phospholipids, on calcium binding and calcium and potassium fluxes of cultured cardiac cells.

Polymyxin B, an amphiphilic, cationic peptidolipid, which is thought to bind to anionic phospholipids in cell membranes, is shown to interact with the cellular calcium of cultured neonatal rat myocardial cells in a dose-dependent, partially reversible manner. At concentrations of less than or equal to 0.1 mM, it has two distinct effects. First, it results in displacement of 1.4 +/- 0.3 mmol Ca/kg dry weight, which is equivalent to 18.1 +/- 3.4% of the total exchangeable cellular calcium. Total calcium displaced by polymyxin B and a nonspecific cationic probe, lanthanum, at its maximal displacing concentration (1 mM), was 5.9 +/- 1.3 mmol/kg dry weight. Thus, the total displaceable calcium represented 76.3 +/- 2.5% of the total exchangeable calcium. Second, polymyxin B (less than or equal to 0.1 mM) causes a reduction in net uptake of calcium, and slows the efflux of both calcium and potassium. Concentrations of polymyxin B higher than 0.1 mM result in an initial displacement of calcium, followed by an irreversible and sustained period of enhanced net calcium uptake. Efflux of calcium is slowed at the higher polymyxin B concentrations, whereas efflux of potassium is enhanced. Cellular contractile activity and electrical activity are irreversibly altered only by the higher concentrations. The results suggest that polymyxin B is a useful probe for the role of membrane phospholipids in control of ion binding and fluxes.

Animals↗
PubMed1982

Permeability and structural studies of heart cell gap junctions under normal and altered ionic conditions.

The permeability and ultrastructure of communicating junctions of cultured neonatal rat ventricular cells are examined under control conditions and during treatments which raise intracellular Ca2+. Lucifer Yellow (487 mol wt) is used to examine junctional permeability. Under normal ionic conditions dye transfer from an injected muscle cell to neighboring muscle cells occurs rapidly (in less than 6 sec) while transfer to neighboring fibroblasts occurs more slowly. Application of monensin, which results in a partial contracture with superimposed asynchrony, or A23187, which results in a partial contracture, do not inhibit the transfer of dye between the muscle cells. A23187 did result in junctional blockade between muscle cells and fibroblasts. Freeze-fractured gap junctions from control and monensin-treated cells exhibit no distinguishable differences. Center-to-center spacing was not significantly different, 9.0 nm +/- 1.4 SD versus 9.2 nm +/- 1.3 SD, respectively; and particle diameters were virtually unchanged, 8.69 nm +/- 0.9 SD versus 8.61 nm +/- 1.07 SD, respectively. These results suggest that concentrations of intracellular Ca2+ sufficient to support a partial contracture and asynchronous contractile activity do not result in a block of intercellular junctions in cultured myocardial cells. These results are discussed in terms of intracellular Ca2+ -buffering and junctional sensitivity to Ca2+.

Animals↗
PubMed1982

Ca++ distribution after Na+ pump inhibition in cultured neonatal rat myocardial cells.

The influence of inhibition of the Na+ pump, with secondary stimulation of Na+-Ca++ exchange, on cellular Ca++ distribution is examined using the on-line scintillation disk technique and cultured neonatal rat myocardial cells. Under control conditions, La+++ displaced 78.1 +/- 1.13% (SEM) of the total cell associated 45Ca. Application of 1 mm ouabain or reduction of [K+]o to 0.5 mM resulted in a net increase of 10.6 +/- 1.3% and 13.8 +/- 2%, respectively, in total cell-associated Ca++. Of this added 45Ca, 75.9 +/- 2.7% and 78.4 +/- 2.1%, respectively remained La+++-displaceable. The 45Ca-binding characteristics of isolated sarcolemma, prepared from the cultured neonatal rat myocardial cells using the gas dissection technique, were examined. When treated with either ouabain or low [K+]o solutions, sarcolemmal 45Ca binding did not change. This result indicates that functional, intact tissue is necessary to observe the Ca++ increase. Treatment of the cells with verapamil before and during ouabain exposure failed to inhibit the ouabain-induced increase in cell-associated 45Ca. The evidence indicates that inhibition of the Na+-pump, and secondary stimulation of Na+-Ca++ exchange, result in a net increase of 11-15% in cell-associated Ca++, 78% of which remains La+++-displaceable and is, therefore, localized to the sarcolemma-glycocalyx complex.

Adenosine Triphosphatases↗
PubMedDate not supplied

Recovery of heart function following 24 hours preservation and ectopic transplantation.

We examined the capacity of hearts injured during 24 hours of preservation to recover function over 96 hours when placed in an ectopic transplant configuration. Standard Langendorff techniques employing an intraventricular balloon and fixed perfusion pressure of 80 mm Hg were used to measure parameters of myocardial function, including maximal systolic pressure, left ventricular end diastolic pressure, and left ventricular balloon volume. Hearts preserved for 24 hours and held ischemic for 1 hour at room temperature exhibited significantly depressed contractility (maximal systolic pressure decreased by 26%) and diastolic compliance (left ventricular balloon volume decreased by 61%). During 96 hours in the ectopic transplant configuration, 24-hour preserved hearts recovered 52% of the lost diastolic compliance. These data demonstrate that 24-hour preserved and transplanted hearts recovered function over 96 hours.

Animals↗