A new oil-gap method for internal perfusion and voltage clamp of single cardiac cells.

(1.) We designed a new technique to achieve fast voltage clamp, combined with internal perfusion. The single guinea-pig cardiac cell, dissociated by collagenase treatment, was stretched across an oil-gap (30-40 micron wide) from a pool of Tyrode solution to a pool of internal solution.

Voltage-dependent magnesium block of adenosine-triphosphate-sensitive potassium channel in guinea-pig ventricular cells.

1. The adenosine-5'-triphosphate (ATP)-sensitive K+ channel of guinea-pig ventricular cells was examined in the presence and absence of internal Mg2+ or Na+ using an open cell-attached configuration of the patch-clamp technique. 2.

Identification of sodium-calcium exchange current in single ventricular cells of guinea-pig.

1. The Na-Ca exchange current was investigated in single ventricular cells from guinea-pig hearts by combining the techniques of whole-cell voltage clamp and intracellular perfusion. 2.

Separation and quantitation of plasma free fatty acids as phenacyl esters by HPLC.

We have developed a rapid method for the separation of plasma free fatty acids as their phenacyl esters by high-performance liquid chromatography (HPLC) using a reversed-phase (C18) column. The derivatives of series of both saturated and unsaturated fatty acids (C12:0-C22:6) are simultaneously separated within 45 min and detected with ultraviolet at 241 nm. The limit of detection of fatty acids was approximately 0.

Dependence of junctional conductance on proton, calcium and magnesium ions in cardiac paired cells of guinea-pig.

1. The dependence of gap junctional conductance on the intracellular concentrations of H+, Ca2+ and Mg2+ was studied in paired myocytes dissociated enzymatically from guinea-pig ventricle. To apply an internal solution buffered to specific H+, Ca2+ or Mg2+ concentration directly to one aspect of the gap junction, the non-junctional membrane of one of the pair was mechanically ruptured.

Direct measurement of the gap junctional conductance under the influence of Ca2+ in dissociated paired myocytes of guinea-pig.

A new method to measure the gap junctional conductance under the influence of a given pCa solution was described. The paired ventricular cells were enzymatically dissociated from the guinea pig and were voltage clamped. One of the pair was crushed to expose one aspect of the gap junction to the bath solution, which contained different pCa.

Na-Ca exchange current in mammalian heart cells.

Electrogenic Na-Ca exchange has been known to act in the cardiac sarcolemma as a major mechanism for extruding Ca ions. Ionic flux measurements in cardiac vesicles have recently suggested that the exchange ratio is probably 3 Na:1 Ca, although a membrane current generated by such a process has not been isolated. Using the intracellular perfusion technique combined with the whole-cell voltage clamp, we were able to load Na+ inside and Ca2+ outside the single ventricular cells of the guinea pig and have succeeded in recording an outward Na-Ca exchange current while blocking most other membrane currents.

Effects of intracellular acidification on membrane currents in ventricular cells of the guinea pig.

The membrane currents of single ventricular cells were measured under whole cell voltage clamp using a giga-sealed patch electrode, and the effects of intracellular acidification were examined by perfusing the electrode pipette with different pH solutions. The plateau of the action potential was shortened when the pH of the pipette solution was lowered from the control of 7.2 to 6, and finally to 5.

Membrane current through adenosine-triphosphate-regulated potassium channels in guinea-pig ventricular cells.

The question whether activation of the ATP-regulated K channel is responsible for macroscopic anoxia-induced outward currents was examined in ventricular cells isolated enzymatically from guinea-pig heart. Gigaseal patch-clamp electrodes were used for a whole-cell voltage clamp. Membrane currents were compared in the same cell while the cell interior was dialysed by perfusing the electrode with different solutions.

Properties of adenosine-triphosphate-regulated potassium channels in guinea-pig ventricular cells.

A class of K channels in cardiac muscle is reversibly blocked by intracellular adenosine 5'-triphosphate (ATP). The characteristics of this K channel were studied by recording single-channel currents in ventricular cells isolated enzymatically from guinea-pig heart. The reversal potential of single-channel currents agreed well with the K equilibrium potential.

Voltage dependence of Na/K pump current in isolated heart cells.

The Na/K pump usually pumps more Na+ out of the cell than K+ in, and so generates an outward component of membrane current which, in the heart, can be an important modulator of the frequency and shape of the cardiac impulse. Because it is electrogenic, Na/K pump activity ought to be sensitive to membrane potential, and it should decline with hyperpolarization. However, such voltage dependence of outward pump current has yet to be demonstrated, one reason being the technical difficulty of accurately measuring pump current over a sufficiently wide voltage range.

Isolation of calcium current and its sensitivity to monovalent cations in dialysed ventricular cells of guinea-pig.

The ion selectivity of the Ca2+ channels in single ventricular cells of guinea-pig was studied using a 'giga-ohm seal' patch electrode for voltage clamp and internal dialysis. To isolate the Ca2+ channel current, currents through the Na+ channel and K+ channels were minimized by replacing external Na+ with Tris+ and removing K+ from both sides of the membrane. With 5 mM-ATP and 5 mM-EGTA in the pipette solution, the Ca2+ current was well maintained for more than 30 min in K+- and/or Na+-free external solution.

Action potential and membrane currents of single pacemaker cells of the rabbit heart.

Single, viable pacemaker cells were isolated from sinoatrial (S-A) and atrioventricular (A-V) nodes by treating with collagenase. In normal Tyrode solution containing 1.8 mM Ca2+, these pacemaker cells had a round configuration and contracted rhythmically at a frequency of about 150-260/min.

Automated measurement of amylase isoenzymes with 4-nitrophenyl-maltoheptaoside as substrate and use of a selective amylase inhibitor.

We automated a kinetic procedure for determining amylase isoenzymes in serum and urine samples. We used 4-nitro-phenylmaltoheptaoside as substrate and a selective amylase inhibitor with the Abbott-VP bichromatic system. By use of the maximum differences between pancreatic (P) and salivary (S) amylase activities remaining after inhibition by the selective inhibitor and by use of the linear range, a one-point standard method for calibration is proposed for determining amylase activities between about 50 and 1500 U/L when the P/S ratio exceeds 0.

Adenosine-5'-triphosphate-sensitive single potassium channel in the atrioventricular node cell of the rabbit heart.

The patch-clamp method was applied to single atrioventricular (a.v.) node cells of the rabbit heart to study the characteristics of the K+ channel.

Effect of pantethine on post-heparin plasma lipolytic activities and adipose tissue lipoprotein lipase in rats.

The lipid-lowering effect of pantethine, a new drug affecting lipid metabolism, had been evaluated in carbohydrate-induced hyperlipidemic rats. Administration of the drug raised post-heparin lipolytic activities, the change being due to an increase in lipoprotein lipase activity, whereas hepatic lipase activity remained virtually unchanged. Total lipoprotein lipase activity per g of adipose tissue increased in pantethine-treated rats compared with controls.

Mode of regulation of the ACh-sensitive K-channel by the muscarinic receptor in rabbit atrial cells.

The mechanism underlying the regulation of the K-channel by the muscarinic receptor was examined with patch-clamp experiments in atrial cells isolated enzymatically from the rabbit heart. The patch-electrode and the recording chamber were perfused with various solutions while the activity of the K-channels in the membrane-patch was recorded continuously. In the absence of muscarinic agonists, opening of K-channels occurred at a low frequency (basal activity).

Resting K conductances in pacemaker and non-pacemaker heart cells of the rabbit.

Currents through the inward rectifier K channel (iK X rec) and the ACh-operated K channel (iK X ACh) were recorded in isolated heart cells of rabbit using the patch clamp technique with electrodes having 0.5-1 micron tip inner diameter. The maximum number of overlaps of open iK X rec channels per patch was measured over 347 experiments.