PubMed Health⌕ Search

Biomedical subjects

R D Nathan

Publications and source records attributed to R D Nathan.

At least 19 recordsLinked to original sources
PubMed2002

Ischemia alters the electrical activity of pacemaker cells isolated from the rabbit sinoatrial node.

The purpose of this study was to investigate the mechanisms responsible for ischemia-induced changes in spontaneous electrical activity. An ischemic-like Tyrode solution (pH 6.6) reversibly depolarized the maximum diastolic potential (MDP) and reduced the action potential (AP) overshoot (OS). We used SNARF-1, which is an indicator of intracellular pH (pH(i)), and perforated-patch techniques to test the hypothesis that acidosis caused these effects. Acidic but otherwise normal Tyrode solution (pH 6.8) produced similar effects. Basic Tyrode solution (pH 8.5) hyperpolarized the MDP, shortened the AP, and slowed the firing rate. In the presence of "ischemic" Tyrode solution, hyperpolarizing current restored the MDP and OS to control values. HOE-642, an inhibitor of Na/H exchange, did not alter pH(i) or electrical activity and did not prevent the effects of ischemic Tyrode solution or recovery after washout. Time-independent net inward current but not hyperpolarization-activated inward current was enhanced by ischemic Tyrode solution or by 30 microM BaCl(2), a selective blocker of inward-rectifying K currents at this concentration. The results suggest that 1) acidosis was responsible for the ischemia-induced effects but Na/H exchange was not involved, 2) the OS was reduced because of depolarization-induced inactivation of inward currents that generate the AP upstroke, and 3) reduction of an inward-rectifying outward K current contributed to the depolarization.

Action Potentials↗
PubMed1997

Ionic basis of ryanodine's negative chronotropic effect on pacemaker cells isolated from the sinoatrial node.

Spontaneous electrical activity and indo 1 fluorescence ratios were recorded simultaneously in cultured pacemaker cells isolated from the rabbit sinoatrial node. Ryanodine (10 microM) reduced the amplitude of action potential-induced intracellular Ca2+ (Ca2+i) transients by 19 +/- 3%, increased the time constant for their decay by 51 +/- 5%, and slowed spontaneous firing by 32 +/- 3%. 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-acetoxymethyl ester (AM; 25 microM) inhibited the Ca2+i transients and slowed spontaneous firing by 28 +/- 4%. Ryanodine did not alter hyperpolarization-activated or time-independent inward current, but it reduced the sum of L- and T-type Ca2+ currents (ICa,L and ICa,T) in both the presence and absence of BAPTA-AM. In contrast, ICa,L was unchanged by ryanodine. Slow inward current tails, presumed to be Na/Ca exchange current (INa/Ca), were abolished by BAPTA or ryanodine. The results suggest that a decrement of ICa,T, due to reduction of the intracellular Ca2+ concentration or a direct effect of ryanodine on T-type Ca2+ channels, contributes to the negative chronotropic effect. Another possibility, based primarily on theory and results in other preparations, is that a reduction of INa/Ca, as a consequence of the smaller action potential-induced Ca2+i transients, contributes to the effect of ryanodine.

Action Potentials↗
PubMed1996

Characterization of a hyperpolarization-activated inward current in cultured pacemaker cells from the sinoatrial node.

The perforated-patch, whole-cell voltage-clamp technique was used to characterize a hyperpolarization-activated inward current (if) in cultured pacemaker cells isolated from the rabbit sinoatrial (S-A) node. A modified Tyrode solution was used to block interfering currents. After correcting for uncompensated series resistance, leakage current and liquid-junction potentials between the pipette and bath, we obtained the following values for the maximum conductance and reversal potential of (i(f)): 16.2 +/- 2.8 nS or 0.26 +/- 0.07 nS/pF (0.37 +/- 0.10 mS/cm2); and -27.7 +/- 1.2 mV (n = 10). In the modified Tyrode solution, the threshold for activation of i(f) and the half-activation potential and slope factor for Boltzmann fits were -66 +/- 1 mV (n = 53), -77.2 +/- 2.0 mV and 4.2 +/- 0.4 mV (n = 16), respectively. The time course of i(f) was best fit by a sum of two exponentials. Between -110 and -5 mV, time constants ranged from 0.06 to 0.42 s for the rapid component and from 0.65 to 4.39 s for the slow component. Using these characteristics of i(f) and a short segment of spontaneous firing as the membrane potential, we computed the expected time course of i(f) during normal pacemaker activity. It reached a maximum of -3 pA during the diastolic depolarization and +4 pA during the action potential. We conclude that most of the characteristics of i(f) in cultured S-A node cells are similar to those measured previously in freshly isolated pacemaker cells.

Animals↗
PubMed1996

Characterization of a TTX-sensitive Na+ current in pacemaker cells isolated from rabbit sinoatrial node.

A tetrodotoxin (TTX)-sensitive Na+ current (iNa) was investigated in single pacemaker cells after 1-4 days in culture. Ruptured-patch and perforated-patch whole cell recording techniques were used to record iNa and spontaneous electrical activity, respectively. For seven cells exposed to 20 mM Na+ (22-24 degrees C) and held at -98 mV (25% of the channels inactivated), the uncorrected maximum iNa was -39 +/- 10 pA/pF at -29.1 +/- 2.4 (SE) mV, maximum conductance was 0.9 +/- 0.2 nS/pF (1.6 +/- 0.2 mS/cm2). Half-activation and inactivation potentials were -41.4 +/- 2.0 and -90.6 +/- 2.5 mV, and the corresponding slope factors were 6.0 +/- 0.4 and 6.4 +/- 0.6 mV. Inactivation and recovery from inactivation were best fit by sums of two exponentials. During action potential clamp, a TTX-sensitive compensation current accounted for 55% of the upstroke velocity. The results suggest that iNa contributes significantly to the action potential in some nodal pacemaker cells, and the characteristics of iNa are similar to those of atrial and ventricular myocytes.

Action Potentials↗
PubMed1991

Removal of sialic acid alters both T- and L-type calcium currents in cardiac myocytes.

The whole cell configuration of the patch-clamp technique was used to test the hypothesis that the presence of sialic acid residues influences both T- and L-type Ca2+ currents (ICa,T and ICa,L) in cultured pacemaker cells isolated from the rabbit sinoatrial node. Removal of these anionic sugar moieties by neuraminidase (1.0 U/ml for 5-20 min) increased ICa,T in five of nine cells (by a factor of 2.2-5.1) and ICa,L in three of six cells (by a factor of 1.2-1.6). In cells that did not exhibit such an increase, the enzyme reduced ICa,T but had no significant effect on ICa,L. In cells that exhibited an increase in ICa,T, exposure to neuraminidase also shifted the activation curve to more negative potentials and increased the slope of the inactivation curve. The enzyme did not influence the gating of ICa,L or the rates of inactivation of either ICa,T or ICa,L. The enhancement of ICa,T and ICa,L could not be mimicked by including neuraminidase in the patch pipette or by adding a contaminant of the enzyme preparation, phospholipase C, to the bath. When external Ca2+ was replaced by Ba2+, neither ICa,T nor ICa,L was increased significantly by neuraminidase. It is proposed that by removing sialic acid residues neuraminidase might directly alter the gating of T-type Ca2+ channels. On the other hand, the increased amplitudes of ICa,T and ICa,L might be due to a rise in intracellular Ca2+.

Animals↗
PubMed1990

Sialic acid and the surface charge associated with hyperpolarization-activated, inward rectifying channels.

The whole-cell configuration of the patch-clamp technique was used with cultured pacemaker cells from the rabbit sinoatrial node to test the hypothesis that sialic acid residues (NANA) constitute much of the negative surface charge associated with hyperpolarization-activated, inward rectifying channels. Activation-voltage relationships (between -70 and -140 mV) were determined for hyperpolarization-activated (inward rectifying) current (i(f)). Addition of 10 mM Ca2+ shifted the half-activation potential (V 1/2) from -89.5 +/- 0.9 mV to -77.9 +/- 2.6 mV (P less than 0.01), confirming the presence of negative fixed charges on the myocytes after 3 to 5 days in culture. Addition of 20 mM dimethonium, an organic divalent cation that "screens" but does not bind to negative surface charge, shifted V 1/2 from -86.8 +/- 1.4 mV to -75.0 +/- 1.7 mV (P less than 0.001) without affecting the amplitude of the current. In contrast, 10 mM Ca2+ reduced the amplitude of i(f) significantly. Incubation of cells with a highly purified preparation of neuraminidase (0.1-2.0 U/ml, 1 hr, 37 degrees C), an enzyme that selectively removes NANA from glycoproteins and glycolipids, failed to alter V 1/2 or the amplitude of i(f) significantly. Pretreatment of cells with neuraminidase (1.0 U/ml, 1 hr, 37 degrees C) failed to alter the positive shift of V 1/2 produced by dimethonium. The results suggest that NANA does not constitute the negative surface charge associated with hyperpolarization-activated, inward rectifying channels.

Animals↗
PubMed1990

Sialic acid and the surface charge of delayed rectifier potassium channels.

We used the whole-cell configuration of the patch-clamp technique and cultured ventricular myocytes from 7-day embryonic chicks to test the hypothesis that sialic acid residues (NANA) constitute the negative surface charge associated with delayed rectifier potassium channels. Delayed rectifier current (iK) was elicited at potentials between -40 and +60 mV. The existence of negative fixed charges close to the "gating sensor" was confirmed by a 6.8-mV negative shift of the half-activation potential (V1/2) following a 10-fold reduction of divalent cations and a 22.6-mV position shift following the addition of 10 mM NiCl2. An 8.4-mV increase in the Boltzmann equation slope factor (k) in the former experiment and a 5.5-mV decline in the latter suggested that the surface charge is not uniformly distributed. We used a high performance liquid chromatography procedure to detect freed sarcolemmal NANA and found that 71-88% was released by neuraminidase (0.2-2.0 U/ml) during 1-h treatments. Such treatments had no significant effect upon the amplitudes of iK or V1/2. On the other hand, k was increased significantly by the enzyme (2.0 U/ml), but only when Ca2+ was present. Finally, 1-h pre-treatments with neuraminidase (2.0 U/ml) had no effect on the positive shift of V1/2 induced by Ni2+. We conclude that although sarcolemmal NANA may bind Ca2+, it does not constitute the surface charge of delayed rectifier potassium channels.

Animals↗
PubMed1988

Selective block of calcium current by lanthanum in single bullfrog atrial cells.

A single suction microelectrode voltage-clamp technique was used to study the actions of lanthanum ions (La3+) on ionic currents in single cells isolated from bullfrog right atrium. La3+, added as LaCl3, blocked the "slow" inward Ca2+ current (ICa) in a dose-dependent fashion; 10(-5) M produced complete inhibition. This effect was best fitted by a dose-response curve that was calculated assuming 1:1 binding of La3+ to a site having a dissociation constant of 7.5 x 10(-7) M. La3+ block was reversed (to 90% of control ICa) following washout and, in the presence of 10(-5) M La3+, was antagonized by raising the Ca2+ concentration from 2.5 to 7.5 mM (ICa recovered to 56% of the control). However, the latter effect took approximately 1 h to develop. Concentrations of La3+ that reduced ICa by 12-67%, 0.1-1.5 x 10(-6) M, had no measurable effect upon the voltage dependence of steady state ICa inactivation, which suggest that at these concentrations there are no significant surface-charge effects of La3+ on this gating mechanism. Three additional findings indicate that doses of La3+ that blocked ICa failed to produce nonspecific effects: (a) 10(-5) M La3+ had no measurable effect on the time-independent inwardly rectifying current, IK1; (b) the same concentration had no effect on the kinetics, amplitude, or voltage dependence of a time- and voltage-dependent K+ current, IK; and (c) 10(-4) M La3+ did not alter the size of the tetrodotoxin-sensitive inward Na+ current, INa, or the voltage dependence of its steady state inactivation. Higher concentrations (0.5-1.0 mM) reduced both IK1 and IK, and shifted the steady state activation curve for IK toward more positive potentials, presumably by reducing the external surface potential. Our results suggest that at a concentration of less than or equal to 10(-5) M, La3+ inhibits ICa selectively by direct blockade of Ca channels rather than by altering the external surface potential. At higher concentrations, La3+ exhibits nonspecific effects, including neutralization of negative external surface charge and inhibition of other time- and voltage-dependent ionic currents.

Action Potentials↗
PubMed1987

Feasibility of electrical recordings from unconnected vertebrate CNS neurons cultured in a three-dimensional extracellular matrix.

Single, i.e. unconnected spinal or cerebral neurons from chick embryos rapidly regenerate morphologically identifiable axons and dendrites when cultured in a three-dimensional (3-D) extracellular matrix (ECM) consisting of a hydrated native collagen lattice. We now show that it is possible to study the intrinsic electrophysiological properties of such neurons as early as 2 days in culture. Cells were plated at 10(5) cells/dish using Medium 199 containing 10% fetal calf serum but no other supplements or antibiotics-antimycotics. A patch-clamp/whole-cell voltage clamp system was used to record single-channel currents from cell-attached patches, transmembrane potentials during the injection of rectangular currents, and whole-cell currents during voltage clamp. After 2-4 days in culture, isolated cerebral and spinal neurons exhibited single-channel currents. Within 7 days, rectangular currents injected through the recording electrode evoked action potentials. These results demonstrate that unconnected CNS neurons quickly display at least some properties of excitability when cultured in a 3-D ECM. This culture system should facilitate investigation of intrinsic electrical properties of single CNS neurons, and how extrinsic factors including neurotransmitters, hormones, pharmacological agents and contacts with other cells influence electrical activity.

Animals↗
PubMed1987

Highly enriched preparations of cultured myocardial cells for biochemical and physiological analyses.

Cultures of enzyme-dissociated myocardial cells contain myocytes as well as other cell types (e.g. fibroblasts); therefore, cell separation is necessary to interpret accurately biochemical measurements of substances or physiological measurements of processes common to more than one type of cell. Previous methods of cell separation have resulted in cultures enriched with, at most, about 80% myocytes. In the present study, a monoclonal antibody to cell-surface adhesion factors, CSAT, was used to obtain four highly enriched preparations: cultures of 7-day embryonic chick ventricular cells that contained either 99.1 +/- 0.2% (n = 10) myocytes or 96.5 +/- 0.8% (n = 6) fibroblasts; and cultures of 17-day chick cells that contained 99.1 +/- 0.2% (n = 11) myocytes or 92.5 +/- 0.8% (n = 8) fibroblasts. Following the removal of CSAT from myocyte-enriched cultures, 96.9 +/- 0.4% (n = 7) of the myocytes obtained from 7-day embryos and 93.8 +/- 1.4% (n = 6) of the myocytes obtained from 17-day embryos attached to culture dishes. Using these four preparations, we found the sialic acid content of myocytes to be significantly less than that of fibroblasts at both 7 and 17 days of development. In fact, the sialic acid content of myocytes did not change during this period, whereas that of fibroblasts increased significantly. These differences underscore the necessity of using highly enriched cell preparations. The antibody/differential adhesion method described provides an opportunity to evaluate and correlate the biochemical, physiological and pharmacological properties of myocytes without interference from other cell types.

Animals↗
PubMed1986

A time- and voltage-dependent K+ current in single cardiac cells from bullfrog atrium.

Individual myocytes were isolated from bullfrog atrium by enzymatic and mechanical dispersion, and a one-microelectrode voltage clamp was used to record the slow outward K+ currents. In normal [K+]o (2.5 mM), the slow outward current tails reverse between -95 and -100 mV. This finding, and the observed 51-mV shift of Erev/10-fold change in [K+]o, strongly suggest that the "delayed rectifier" in bullfrog atrial cells is a K+ current. This current, IK, plays an important role in initiating repolarization, and it is distinct from the quasi-instantaneous, inwardly rectifying background current, IK. In atrial cells, IK does not exhibit inactivation, and very long depolarizing clamp steps (20 s) can be applied without producing extracellular K+ accumulation. The possibility of [K+]o accumulation contributing to these slow outward current changes was assessed by (a) comparing reversal potentials measured after short (2 s) and very long (15 s) activating prepulses, and (b) studying the kinetics of IK at various holding potentials and after systematically altering [K+]o. In the absence of [K+]o accumulation, the steady state activation curve (n infinity) and fully activated current-voltage (I-V) relation can be obtained directly. The threshold of the n infinity curve is near -50 mV, and it approaches a maximum at +20 mV; the half-activation point is approximately -16 mV. The fully activated I-V curve of IK is approximately linear in the range -40 to +30 mV. Semilog plots of the current tails show that each tail is a single-exponential function, which suggests that only one Hodgkin-Huxley conductance underlies this slow outward current. Quantitative analysis of the time course of onset of IK and of the corresponding envelope of tails demonstrate that the activation variable, n, must be raised to the second power to fit the sigmoid onset accurately. The voltage dependence of the kinetics of IK was studied by recording and curve-fitting activating and deactivating (tail) currents. The resulting 1/tau n curve is U-shaped and somewhat asymmetric; IK exhibits strong voltage dependence in the diastolic range of potentials. Changes in the [Ca2+]o in the superfusing Ringer's, and/or addition of La3+ to block the transmembrane Ca2+ current, show that the time course and magnitude of IK are not significantly modulated by transmembrane Ca2+ movements, i.e., by ICa. These experimentally measured voltage- and time-dependent descriptors of IK strongly suggest an important functional role for IK in atrial tissue: it initiates repolarization and can be an important determinant of rate-induced changes in action potential duration.

Animals↗
PubMed1986

Two electrophysiologically distinct types of cultured pacemaker cells from rabbit sinoatrial node.

Previous investigations employing multicellular nodal preparations (i.e., mixtures of dominant and subsidiary pacemaker cells) have suggested that the fast transient inward sodium current (iNa) either is not present in dominant pacemaker cells or is present but inactivated at the depolarized take-off potentials that these cells exhibit. In the present study, this question was resolved by voltage clamp analysis of single pacemaker cells isolated from the sinoatrial node and maintained in vitro for 1-3 days. Two types of cells, each with a different morphology, exhibited two modes of electrophysiological behavior. Type I cells (presumably dominant pacemakers) displayed only a tetrodotoxin (TTX)-resistant (but cadmium-sensitive) slow inward current, whereas type II cells (presumably subsidiary pacemakers) exhibited two components of inward current, a TTX-sensitive, fast transient inward current and a TTX-resistant (but cadmium-sensitive) slow inward current. Three other voltage-gated currents, 1) a slowly developing inward current activated by hyperpolarization (if, ih, delta ip), 2) a transient outward current activated by strong depolarization (ito, iA), and 3) a delayed outward current, were recorded in both types of pacemaker cells.

Action Potentials↗
PubMed1984

Perturbations in the membrane potential of cultured heart cells: role of calcium.

Intracellular recordings were obtained from spheroidal aggregates of 7-day embryonic chick heart cells after 3 days in gyratory culture. Three types of perturbations in the membrane potential were observed under experimental conditions expected to increase intracellular calcium: 1) multiple oscillations (of 5-20 mV peak-to-peak amplitude) during diastole in aggregates exposed to 10-15 mM Ca, 5 microM strophanthidin, or K-free solutions; 2) less periodic spontaneous voltage fluctuations (of less than 1.5 mV peak-to-peak amplitude) in aggregates exposed to solutions containing 22% of the normal [Na], and 3) depolarizing afterpotentials (DAPs), following repolarization of the action potential, in aggregates treated with 20-50 microM A23187, a Ca ionophore, or 5-10 mM caffeine. The oscillations were reduced markedly by 0.03-3.0 microM tetrodotoxin (TTX) and were blocked by 5-10 mM caffeine. Spontaneous voltage fluctuations were increased by raising external Ca, were unaffected by 30 microM TTX, and were blocked by 5 mM caffeine. DAPs were not blocked by 5 mM caffeine or by 0.1 microM TTX and 1 microgram/ml D 600, concentrations that greatly reduced the action potential upstroke velocity and plateau, respectively. Two intracellular electrodes were employed to test for electrotonic coupling between cells within an aggregate. An electrotonic response in one cell could be recorded when current was injected into another cell during recordings of each of the perturbations but was somewhat less during spontaneous voltage fluctuations. Possible ionic mechanisms for the perturbations and for concomitant changes in the configuration of the action potential are discussed.

Animals↗
PubMed1983

Calmodulin, Ca2+-antagonists and Ca2+-transporters in nerve and muscle.

Calcium is of fundamental importance in the regulation of both muscle contraction and neurosecretion. Its control of these processes is achieved by its binding and activation of various Ca2+-binding proteins (CBP), including those in the Ca2+ channel, the Na+-Ca2+ antiporter, and intracellular calmodulin (CDR). Generally, Ca2+-binding to regulatory CBP exposes hydrophobic sites on their surface at which the CBP interfaces with its receptor or binds inhibitory hydrophobic ligands. We find that some Ca2+-antagonist drugs (Ca-ANT) bind to and inhibit calmodulin and that some calmodulin antagonists (CDR-ANT) block Ca2+ channels. This suggests that CDR and the CBP that regulate the Ca2+ channel may be quite homologous proteins, Ca-ANT and CDR-ANT are not effective inhibitors of the Na+-Ca2+ antiporters of heart sarcolemma and brain synaptosomes, suggesting that these antiporters are fundamentally different from the antiporter of heart mitochondria. These results are discussed in terms of Ca2+-binding proteins being potential targets for pharmacological interventions designed to block specific aspects of the action of calcium.

Action Potentials↗