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R L DeHaan

Publications and source records attributed to R L DeHaan.

At least 37 records · Page 2Linked to original sources
PubMed1984

Insulin-specific receptor-mediated slowing of beat rate in embryonic heart cells.

Spheroidal aggregates of embryonic heart cells showed their spontaneous beat rate when exposed to insulin. The concentration that produced a half-maximal response (1.7 nM) corresponded to the dissociation constant of binding to a specific high-affinity insulin receptor. The pace-maker phase of action potentials recorded during insulin perfusion was preceded by a prolonged or flattened after hyperpolarization, and its slope was less steep than controls. The action potential duration was also prolonged. These results indicate that physiological concentrations of insulin can regulate the embryonic heart rate.

Action Potentials↗
PubMed1983

Concanavalin A increases spontaneous beat rate of embryonic chick heart cell aggregates.

The plant lectin concanavalin A (Con A), at concentrations of 5-200 micrograms/ml, induced a twofold to fivefold increase in spontaneous beat rate of cultured aggregates of ventricular cells from seven-day chick embryos. This response was time, dose, and temperature dependent and was accompanied by a decrease in transmembrane potential. It could be blocked or reversed by alpha-methyl-D-mannoside but was not reversed by dilution alone. Binding of the lectin occurred in the cold, but a temperature-dependent process was also necessary to produce the response. Divalent (succinyl) Con A did not cause a beat rate increase. Whole heart aggregates responded similarly but less intensely than ventricular aggregates. Atrial aggregates, and whole heart aggregates treated with 5 microgram/ml of Con A, produced a biphasic chronotropic response, first decreasing then increasing their beat rates. These results suggest that saccharide-bearing macromolecules on the heart cell surface play a role in regulating spontaneous beat rate.

Action Potentials↗
PubMed1983

Caffeine-induced current in embryonic heart cells: time course and voltage dependence.

Abrupt exposure of 90- to 130-micron diameter chick embryonic myocardial cell aggregates to 10 mM caffeine has been shown to induce a transient inward current. In the present study, we recorded a similar current in small cell clusters (less than 10 cells) in which access of caffeine to each of the cells was rapid. The resulting inward current consisted of a single peak, which decayed exponentially (predominant time constant 335 +/- 130 ms at -40 mV) and had a peak amplitude of up to 15.5 microA/cm2. The caffeine-induced current persisted when the slow inward current was abolished by a 30-s pretreatment with 2 microM D 600 and could be observed at potentials where the fast sodium channels were fully inactivated. The current-voltage relation of the caffeine response was linear between -110 and -40 mV, giving an extrapolated voltage intercept of +12 mV. However, the inward current did not diminish or reverse with further depolarization. A substantial inward current occurred at potentials up to +60 mV, which is more positive than the reversal potential of the tetrodotoxin-sensitive inward current. We conclude that the caffeine-induced current is mediated in part by electrogenic Na+-Ca2+ exchange.

Animals↗
PubMed1981

Electrical coupling among heart cells in the absence of ultrastructurally defined gap junctions.

Cells from the ventricles of 7-day chick embryos were aggregated into spheroidal clusters by 48 hr of culture on a gyratory platform. All aggregates beat spontaneously and rhythmically. Microelectrode impalement of widely separated cells within aggregates indicated that they were coupled, as evidenced by a mean coupling ratio (delta V2/ delta V1) of 0.81 +/- 0.09, and by simultaneity of intrinsic electrical activity (action potentials and subthreshold voltage fluctuation). In freeze-fracture preparations, the cell surfaces contained numerous small groups of intramembrane protein (IMP) particles, arranged in macular clusters, and linear and circular arrays. Using the criterion of 4 clustered IMP particles to defined a minimal gap junction, 0.27% of the total P-face examined was devoted to gap junctional area. Within such clusters particles were packed at about 8200/micrometer2; in nonjunctional regions, particles were scattered at a density of about 2000/micrometer2. When exposed to cycloheximide (CHX: 50 micrograms/ml) for 24--48 hr, coupling ratio declined to 0.44. This decrease could be attributed largely to leakiness of the nonjunctional membrane. Aggregates continued to beat rhythmically and in a coordinated fashion even after 72 hr in inhibitor. However, between 3--21 hr in CHX gap junctional area declined to 0.10%, and all particle clusters disappeared from the P-faces of aggregates in CHX for 24 or 48 hr. Neither macular nor linear particle arrays were seen. We conclude that organized gap junctions are unnecessary for electrotonic coupling between embryonic heart cells. These findings support the idea that low-resistance cell-to-cell pathways may exist as isolated channels scattered throughout the area of closely apposed plasma membranes.

Action Potentials↗
PubMed1980

Ouabain-resistant hyperpolarization induced by insulin in aggregates of embryonic heart cells.

Spheroidal aggregates formed from trypsin-dissociated 14-day embryonic chicken hearts after 48 hr of rotation on a gyratory shaker. Intracellularly recorded resting membrane potentials of aggregates bathed in 1.3 mM K+ balanced salt solution had a mean (+/- SD) of 64 +/- 4 mV. After a stable potential was achieved, addition of 1-100 nM sodium bovine insulin caused a slow hyperpolarization of up to 19 mV after 4-5 min, followed, in some cases, by a further, more rapid, shift to a potential near EK. Equivalent hyperpolarizations were observed when insulin was added in the presence of 10 mM ouabain, indicating that enhanced Na+,K+ pump activity was not responsible for the change in membrane potential. The concentration of insulin that produced half-maximal hyperpolarization (2 nM) corresponded to the association constant of a high-affinity insulin receptor, suggesting that binding to this class of receptors led to the change in membrane potential. Steady-state current-voltage curves from current clamp experiments suggested that insulin produced an increase in slope conductance at potentials near rest by inducing an outward current with an apparent potential negative to -90 mV.

Animals↗
PubMed1980

Junctional resistance and action potential delay between embryonic heart cell aggregates.

Spheroidal aggregates of embryonic chick ventricle cells were brought into contact and allowed to synchronize their spontaneous beats. Action potentials were recorded with both intracellular and extracellular electrodes. The degree of electrical interaction between the newly apposed aggregates was assessed by measuring the delay or latency (L) between the entrained action potentials, and by determining directly interaggregate coupling resistance (Rc) with injected current pulses. Aggregate size, contact area between the aggregates, and extracellular potassium concentration (Ko+) were important variables regulating the time-course of coupling. When these variables were controlled, L and Rc were found to be linearly related after beat synchrony was achieved. In 4.8 mM Ko+ L/Rc = 3.7 ms/M omega; in 1.3 mM Ko+ L/Rc = 10.1 ms/M omega. We conclude that action potential delay between heart cell aggregates can be related quantitatively to Rc.

Action Potentials↗
PubMed1979

Development of electrical coupling and action potential synchrony between paired aggregates of embryonic heart cells.

Pairs of spheroidal aggregates of embryonic chick heart cells, held in suction pipettes were brought into contact and allowed to synchronize their spontaneous action potentials. Contractions were suppressed with cytochalasin B. Both intracellular and extracellular electrodes were used to analyze the development of synchrony. Electric coupling occurred in three phases. During phase I electrical interactions were absent despite close physical contact. Phase II was characterized by partial synchrony. Action potentials in the faster aggregate (F) induced small depolarizations in the other member of the pair (S). These depolarizations sometimes triggered action potentials in S depending on when during the diastolic depolarization in S they occurred. In these cases both the latency between the action potentials (L) and the fluctuations in latency (VL) were large. At the end of phase II the aggregates often passed through a brief period when functuation in interbeat interval in both increased noticeably. In phrase III, beginning about 8 min after initial contact, action potentials were completely entrained at a certain L. During the subsequent 20--40 min L fell along an approximately exponential time course from about 130 to less than 1 msec, while VL declined in parallel. When well-coupled aggregates were pulled apart and immediately pressed back together, they re-established synchronization according to the usual three-phase time course. Synchronized aggregates could be partially decoupled by separating them just far enough to reduce the area of mutual contact. Pairs joined only by cellular strands maintained entrained action potentials with long latencies for many minutes. These results indicate that electronic junctions form between the paired heart cell aggregates causing the gradual development of action potential synchrony.

Action Potentials↗
PubMed1979

Current noise parameters derived from voltage noise and impedance in embryonic heart cell aggregates.

We have recorded membrane impedance and voltage noise in the pacemaker range of potentials (-70 to -59 mV) from spheroidal aggregates of 7-d embryonic chick ventricle cells made quiescent by exposure to tetrodotoxin in medium containing 4.5 mM K+. The input capacitance is proportional to aggregate volume and therefore to total membrane area. The specific membrane capacitance is 1.24 microF/cm2. The input resistance at constant potential is inversely proportional to aggregate volume and therefore to total membrane area. The specific membrane resistance in 18 k omega . cm2 at -70 mV and increases to 81 k omega . cm2 at -59 mV. The RC time constant is 22 ms at -70 mV and increases to 146 ms at -59 mV. The aggregate transmembrane small-signal impedance can be represented by a parallel RC circuit itself in parallel with an inductive branch consisting of a resistor (rL) and an inductor (L) in series. The time constant of the inductive branch (L/rL) is 340 ms, and is only weakly dependent on potential. Correlation functions of aggregate voltage noise and the impedance data were modeled by a population of channels with simple open-close kinetics. The time constant of a channel (tau s) derived from the noise analysis is 300 ms. The low frequency limit of the pacemaker current noise (SI[0]), derived from the voltage noise and impedance, increases from 10(-20) A2/Hz . cm2 at -67 mV to 10(-19) A2/Hz . cm2 at -61 mV.

Animals↗
PubMed1979

Fluctuations in interbeat interval in rhythmic heart-cell clusters. Role of membrane voltage noise.

Small clusters of ventricular cells prepared from 7-d chick heart maintain spontaneous, stationary, rhythmic beating in culture for many hours. For clusters containing I-125 cells, mean interbeat interval (IBI) is 0.45 +/- 0.08 s and is independent of cell number (N), whereas, the coefficient of variation of IBI (C) is proportional to N-1/2. Because membrane voltage noise in such clusters would also be expected to vary as N-1/2, we propose a model relating fluctuation in IBI (sigma IBI) to voltage noise (sigma v). A simplified model consisting of random voltage fluctuations superimposed upon a linear pacemaker depolarization of slope a is used to analyze the N-dependent shape of the IBI histogram. Values of sigma v derived from the relation sigma IBI = sigma v/a, or calculated from the skewness of the measured IBI histograms, both agree well with those extrapolated from steady-state noise recorded from resting heart-cell aggregates.

Animals↗
PubMed1979

Voltage clamp analysis of embryonic heart cell aggregates.

The double-microelectrode voltage clamp technique was applied to small spheroidal aggregates of heart cells from 7-d chick embryos. A third intracellular electrode was sometimes used to monitor spatial homogeneity. On average, aggregates were found to deviate from isopotentiality by 12% during the first 3--5 ms of large depolarizing voltage steps, when inward current was maximal, and by less than 3% thereafter. Two components of inward current were recorded: (a) a fast, transient current associated with the rapid upstroke of the action potential, which was abolished by tetrodotoxin (TTX); and (b) a slower inward current related to the plateau, which was not affected by TTX but was blocked by D600. The magnitudes, kinetics, and voltage dependence of these two inward currents and a delayed outward current were similar to those reported for adult cardiac preparations. From a holding potential of -60 mV, the peak fast component at the point of maximal activation (-20 mV) was -185 microA/cm2. This value was about seven times greater than the maximal slow component which peaked at 0 mV. The ratio of rate constants for the decay of the two currents was between 10:1 and 30:1.

Action Potentials↗
PubMed1979

Relationship of insulin binding to amino acid transport by cultured 14-day embryonic chick heart cells.

The characteristics of insulin receptors were studied in cultured embryonic chick heart cells which demonstrated insulin-responsive amino acid transport. Binding of [125I]iodoinsulin was time dependent, reversible, saturable, species specific, and proportional to cell number. Optimum binding occurred at pH 7.8 in the presence of 0.1% bovine serum albumin. Curvilinear Scatchard plots were found for chicken and bovine insulin binding at 15 and 30 C. Equilibrium association constants (Kas) and maximum capacities were calculated based on a two-receptor model. When studied at 15 C, chicken insulin was bound with Kas of 5.0 and 0.026 nM-1 for the high and low affinity receptors, respectively. Bovine insulin bound with Kas of 2.1 and 0.03 nM-1. The binding capacities of 600 and 9000 molecules/cell for the high and low affinity receptors, respectively, were the same for both species of ligand. At 30 C, the Ka of the high affinity chicken insulin receptor interaction decreased to 1.6 nM-1, whereas the low affinity Ka was not changed. In competitive binding assays, chicken insulin was 4 and 250 times more potent than bovine and guinea pig insulin, respectively. Human GH and mouse epidermal growth factor did not compete with chicken insulin. Although mouse epidermal growth factor did not compete with insulin, it did stimulate 2-aminoisobutyric acid accumulation. The maximal stimulation by this hormone was less than and additive to that produced by the maximal stimulation of insulin. These data indicate that insulin and epidermal growth factor acted through different receptors to stimulate amino acid transport. When insulin-stimulated functional response was compared to calculated receptor occupancy in this two-receptor model, stimulation of amino acid transport paralleled occupancy of the low affinity receptor such that at half-maximal transport stimulation, approximately 50% of these receptors were occupied. Half-maximal stimulation of 2-aminoisobutyric acid transport occurred at 18- and 20-nM concentrations of bovine and chicken insulins, respectively. Guinea pig insulin failed to stimulate 2-aminoisobutyric acid uptake even at 350 nM. Both the affinity of binding in the low affinity class and the insulin concentration for half-maximal stimulation of 2-aminoisobutyric acid transport were independent of temperature in the temperature ranges studied. We conclude that 14-day embryonic chick heart cells possess at least two classes of receptors which bind insulin. Occupancy of the lower affinity class of insulin receptors correlates quantitatively with insulin stimulation of 2-aminoisobutyric acid transport.

Aminoisobutyric Acids↗
PubMed1978

In vitro differentiation of a fast Na+ conductance in embryonic heart cell aggregates.

Aggregates of 3-day chick cardiac myocytes, after 2 days of gyration culture, lack a tetrodotoxin-sensitive, fast Na+ conductance. After 2--3 additional days in vitro these aggregates were shown by three different criteria to have developed such a conductance mechanism: (a) action potential upstroke velocities greater than 90 V/sec; (b) suppression of spontaneous activity by tetrodotoxin; and (c) presence of a fast component of inward current recorded during voltage clamp. These membrane properties were similar to those of aggregates derived from 7-day hearts that had developed in ovo. The differentiation of fast Na+ conductance channels was a dependent upon active protein synthesis but not upon the presence of exogenous macromolecules.

Action Potentials↗
PubMed1978

Reduction of intercellular adhesiveness of chick heart cells by herpes simplex viruses 1 and 2.

Infection with herpes simplex viruses type 1 or 2 prevented the aggregation of 7-day-old chick heart cells into smooth, spheroidal, spontaneously beating aggregates. Virus infection also caused a loosening of peripheral cells in aggregates formed from initially uninfected cells. Measurements of rate of attachment of labelled single heart cells to a monolayer of like cells (homotypic), to HEp-2 cells (heterotypic), or to plastic substrata (nonspecific adhesion) indicated that virus infection caused a significant but differential loss of homotypic and nonspecific adhesiveness, but no alteration in heterotypic attachment rates. These observations indicate that those cells surface changes induced by viruses which are related to cell adhesion can be quantified by techniques measuring attachment rates.

Animals↗
PubMed1978

Herpes virus infection alters electrical parameters of heart cell aggregates.

The spontaneous beat of embryonic heart cell aggregates in culture grew gradually weaker and stopped about 18 h after inoculation with herpes simplex virus 1 (HSV-1). Before electrical activity ceased, maximal action potential upstroke velocity fell to about 20% of control values, whereas overshoot and plateau duration declined about 50%. Maximal diastolic potential was reduced by only about 10%. Eighteen hours after viral inoculation, specific membrane resistance and intercellular coupling were measured in quiescent aggregates with injected current pulses passed between two widely spaced intracellular electrodes. These parameters were unaltered as compared with control aggregates. However, pairs of infected aggregates brought into contact required 8 h to synchronize their beats; mock-inoculated aggregates coupled in less than 1 h. It is concluded that the cell surface alterations caused by HSV-1 infection specifically reduce both fast and slow inward currents and interfere with the formation of new nexal junctions.

Action Potentials↗