Single Sucrose-gap Technique Research Articles

A comparative electrophysiological study of enzymatically isolated single cells and strips of frog ventricle

Single heart cells were obtained from frog ventricle using an enzymatic dispersion technique. The whole cell variation of the patch clamp technique was used to monitor action potential and cell membrane currents. The clamp circuit could be switched electronically between voltage and current clamp modes. The effects of seal leakage currents were to depolarize the cell, reduce the amplitude of the plateau, and lengthen the action potential duration. A scheme to compensate for these currents is presented. The membrane currents obtained from the single cell under voltage clamp conditions were compared to those obtained from multicellular preparations using the single sucrose gap technique. Hyperpolarizing clamps showed time-dependent, depletion-related K+ currents for the multicellular preparation, whereas for the single cell no such currents were observed. The absence of extracellular accumulation or depletion of K+ in the single cell was confirmed by the lack of post-clamp afterpotentials or changes in resting potential following a train of frequently elicited action potentials. The TTX-insensitive inward current was relatively faster in the single cell, compared to that measured in the multicellular preparation. A delayed time-dependent outward current was observed in the positive potential range for both single and multicellular preparations. The isochronal current-voltage (I-V) relations obtained at 400 ms were N-shaped for both preparations, but was more negative for the single cell at potentials positive to -20 mV. The results indicate a strong similarity between membrane currents obtained in single and multicellular preparations. The differences in the currents in the two preparations are due in large part to accumulation or depletion of K+ in the extracellular space.

Evidence for a specific receptor site for lidocaine, quinidine, and bupivacaine associated with cardiac sodium channels in guinea pig ventricular myocardium.

According to the modulated receptor hypothesis, sodium channels have a specific receptor site for local anesthetic and antiarrhythmic drugs. Thus, in the presence of a high concentration of two drugs, competitive displacement of one drug by another may occur. Furthermore, if a drug that has relatively rapid post-stimulation recovery kinetics (e.g., lidocaine) displaces another drug with relatively slow recovery kinetics (i.e., quinidine or bupivacaine), then a net reduction in sodium channel blockade is expected at certain stimulation rates. We tested this prediction, using the maximum upstroke velocity of the ventricular action potential as an indicator of drug-free sodium channels. A single sucrose gap technique was used to stimulate guinea pig papillary muscles, and to control membrane voltage at all times except during the action potential upstroke. Drug-induced inhibition of maximum upstroke velocity increased as the stimulation rate was increased, and was significant (P less than 0.05) at stimulation rates between 2.5 and 4 Hz in the presence of 43 microM lidocaine (n = 5), and between 0.15 and 4 Hz in the presence of 3.5 microM bupivacaine (n = 4). The addition of 43 microM lidocaine to a perfusate containing 3.5 microM bupivacaine resulted in a net increase in maximum upstroke velocity that was significant at rates between 1 and 3.3 Hz, with a maximum increase of 25 +/- 6% at 1.6 Hz. In contrast, addition of 43 microM lidocaine to a perfusate containing 15 microM quinidine did not result in a significant change in maximum upstroke velocity at driving rates between 0.05 and 3.3 Hz (P greater than 0.2; n = 4). However, evidence for displacement of quinidine by lidocaine could be demonstrated by measuring post-stimulation recovery after a conditioning train of 19 10-msec pulses applied at 28 Hz. With this stimulation protocol, 41 +/- 4% of maximum upstroke velocity recovered slowly from block with a time constant of 3.7 +/- 1.2 seconds at - 100 mV in the presence of 15 microM quinidine (n = 5). In the presence of a mixture of 43 microM lidocaine and 15 microM quinidine, this slow component was significantly reduced to 16 +/- 7% (n = 5; P less than 0.01), while 71 +/- 13% of maximum upstroke velocity recovered with a time constant of 115 +/- 21 msec, typical of lidocaine-blocked channels. A two-drug version of the modulated receptor theory was formulated. The effects of drug mixtures could be accounted for by this model.(ABSTRACT TRUNCATED AT 400 WORDS)

DIFFERENTIATION OF THE ACTION POTENTIAL IN THE SMOOTH MUSCLE OF CANINE GASTRIC ANTRUM USING CALCIUM‐INHIBITORY DRUGS

Electrical and mechanical activity were recorded simultaneously in smooth muscle preparations from the antrum region of canine stomach by means of a single sucrose gap technique (SGT). The SGT was optimized to permit stable recording from multicellular smooth muscle preparations over several hours of electrical and mechanical activity with little disturbance of their normal properties. Acetylcholine (ACh, 10(-8) to 10(-6) M) induced or augmented dose-dependently the electrical and mechanical activity. The plateau of the action potential complex was elevated by ACh, while the contraction was increased in linear correlation to the magnitude of the plateau component. In spontaneously active (or in ACh-stimulated) preparations TEA (5 to 20 mM) magnified the plateau component, induced or strengthened spikes on the plateau ('secondary spikes'), and induced or strengthened phasic contractions. Nifedipine (10(-6) M) abolished secondary spikes, part of the plateau component of the action potential, and suppressed mechanical activity. The complex action potential of canine gastric antrum can be differentiated into (a) a basic action potential, consisting of an initial, primary spike and a plateau depolarization; this basic action potential is resistant to nifedipine and does not trigger any mechanical activity; and (b) a nifedipine-sensitive component (calcium component), which consists of an augmentation of the plateau depolarization and of secondary spikes, and which is responsible for the initiation of mechanical activity.

Inhibitory effects of procaine on the contractile responses of the guinea-pig taenia caecum to acetylcholine, substance P and potassium chloride.

The effect of procaine on the contractile responses to acetylcholine, substance P and KCl was investigated using the isolated guinea-pig taenia caecum. In normal Tyrode solution (37 degrees C), procaine (10-100 mumol/l) caused a parallel shift to the right of only the dose-response curve of acetylcholine (pA2 value, 5.11). The pA2 value of procaine against acetylcholine was not significantly affected by increasing the Ca concentration in the bathing solution from 0.9 to 7.2 mmol/l. On the other hand, a high concentration of procaine (10 mmol/l) caused a transient contraction of the taenia caecum, but completely suppressed contractions to all concentrations of the agonists. In K-depolarized preparations, procaine (1-10 mmol/l) shifted the dose-response curve for the CaCl2-induced contraction to the right. Substance P (3 mumol/l)-induced contraction of the taenia caecum incubated with Ca-free EGTA (0.1 mmol/l) solution (20 degrees C) was markedly reduced by procaine (10 mmol/l). Using the single sucrose-gap technique, it was found that procaine (10 mmol/l) produced a membrane depolarization and increases in both amplitude and frequency of spontaneous spike discharge. These potential changes were still observed even after the procaine-induced contraction had disappeared. The spike discharges and contraction caused by procaine were abolished in the presence of a Ca-entry blocker, verapamil (10 mumol/l).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of the venom of the Brazilian scorpion (Tityus serrulatus) on the compound action potential of the rabbit vagus nerve fibres.

1 The effects of the venom of the scorpion (Tityus serrulatus) on nerve fibres of the rabbit cervical vagus were studied by the single sucrose-gap technique. Scorpion venom (1 microgram/ml) increased irreversibly the duration of the B component of the compound action potential of the vagus nerves, leaving the C component with its normal configuration. Tetrodotoxin (200 nM) suppressed the prolongation of the action potential duration in venom-treated B fibres. 2 At the same concentration (1 microgram/ml), scorpion venom reduced the amplitude and the rate constant of decay of the hyperpolarization produced by tetanic stimulation of non-myelinated nerve fibres. 3 A lower concentration (0.2 micrograms/ml) blocked completely the hyperpolarization of the potassium-activated response. After washing, the potassium-activated response partially recovered its amplitude but there was a significant increase in the time constant of the decay of hyperpolarization. 4 It is suggested that scorpion venom may modify the sodium pumping mechanism within fibres as well as affecting the passive and active sodium permeability systems.

The complex structure of mammalian heart contraction.

Guinea pig papillary muscle contraction at 25 degrees C and 2 mg/liter norepinephrine (NE) consists of two components (C1, C2). Their parameters were determined in an analysis of contraction after abrupt load shifts. The C1 amplitude increased by 81 +/- 22% (N = 9) when stimulation frequency was raised from 0.1 to 1.0 Hz; its time to peak was constant at 169 +/- 6 msec (N = 12). C1 was abolished by 2 mM caffeine. The amplitude of C2 was constant between 0.1 and 0.5 Hz but decreased to 19 +/- 10% (N = 4) at 1.0 Hz; its time to peak correlated with the duration of action potential (DAP) (r = 0.98) and decreased from 518 +/- 12 msec at 0.1 Hz to 314 +/- 20 msec at 1.0 Hz (N = 12). C2 was eliminated by 5 mg/liter verapamil. Increase of DAP by a current sent through the muscle in the single sucrose gap technique gave rise to a C3 with time to peak equal to the new DAP. Mammalian heart muscle contraction in the presence of NE is supposed to be the sum of components elicited by Ca release (and after-release) from sarcoplasmic reticulum and a tonic component evoked by Ca release from sarcolemmal binding sites.

Further studies on the actions of salicylates on nerve membranes

The effects of sodium salicylate, sodium benzoate and 5-bromo salicylate on nerve fibres of the rabbit cervical vagus and of the frog sciatic nerve ‘in vitro’ were studied using the single sucrose gap technique. The application of 5-bromo salicylate in concentrations between 2 × 10 −3 to 5 × 10 −3 M induced a slow fall in spike height accompanied by a small hyperpolarization (0.5−2.0 mV). Higher concentrations (1 × 10 −2 − 2 × 10 −2 M) produced depolarization and a rapid block of nervous conduction. Concentrations of 1 × 10 −2−2 × 10 −2 M of sodium salicylate were needed to induce the same hyperpolarizing and slow blocking effects in non-myelinated nerve fibres. Myelinated fibres of the frog sciatic nerve were also blocked by concentrations greater than 1 × 10 −2 M of 5-bromo salicylate, but showed an increase in the amplitude of the compound action potential and a prolonged falling phase when superfused with concentrations around 5 × 10 −3 M. A frequency-dependent decrease in the action potential amplitude was observed with sodium salicylate and 5-bromo salicylate and was shown to be more easily induced in non-myelinated than in myelinated fibres. 5-Bromo salicylate (2 × 10 −2 M) and 2,4-dinitrophenol (1 × 10 −4 M) reduced the amplitude and the rate constant of decay of the hyperpolarization produced by tetanic stimulation of non-myelinated nerve fibres. Sodium salicylate (2 × 10 −2 M) increased the time constant of decay of the hyperpolarization of the potassium-activated response and slightly decreased its amplitude. Sodium salicylate was 3–5 times less potent than 5-bromo salicylate; while sodium benzoate was practically ineffective in producing the phenomena under study.

The local anesthetic effect of cyproheptadine on mammalian nerve fibres

The local anesthetic effect of cyproheptadine on nerve fibres in the rabbit's cervical vagus and sciatic nerve was studied by the single sucrose-gap technique. Local anesthetics such as procaine and tetracaine, and an antihistaminic with local anesthetic activity, diphenhydramine, were studied for comparison. Increasing concentrations of cypropheptadine, starting from 5 × 10 −5 M, produced a dose-related fall in the amplitude of the compound action potential of the vagus nerve without significant change in the resting membrane potential. A complete reversibility of the local anesthetic effect was difficult or impossible to obtain when doses greater than 1 × 10 −4 M were used. Cyproheptadine was more potent than procaine and diphenhydramine, and less potent than tetracaine in producing nerve conduction block. Frequency-dependent block was observed with cyproheptadine and the other agents at frequencies that can be considered low (1–5 Hz). Myelinated fibres of the sciatic nerves were also blocked by cyproheptadine within the same range of concentrations (1 × 10 −4 to 1 × 10 −3 M). Our results provide an additional explanation for the mechanisms underlying the actions of cyproheptadine as an antiarrhythmic and an antipruritic agent.

The depolarizing action of 5-HT on mammalian non-myelinated nerve fibres

The effects of 5-hydroxytryptamine (5-HT) on nerve fibres in the rabbit cervical vagus and on the sciatic nerve “in vitro” were studied by the single sucrose-gap technique. The addition of 5-HT to the Locke solution bathing the vagus nerve induced rapid depolarization and a fall in spike height at the threshold concentration of 1 × 10 −7 M. In most cases the depolarizing effect was completely reversed by washing for 5–10 min while in about 40% of the preparations the action potential amplitude remained 10–30% below the control level. These effects were dose-related up to a maximum concentration of 3 × 10 −5 M. Tachyphylaxis was not observed when the drug was added at 12–15 min intervals. Depolarization was abolished by perfusing the nerve with sodium-free medium or by previous exposure to lidocaine (10 −3). External hyperpolarizing currents (10 −7 to 10 −6A) were not able to restore the action potential amplitude. Cyproheptadine (50 μM), which was found to have a slight local anesthetic action, reduced the 5-HT-induced depolarization by 20–30%. Methysergide (50 μM), a more specific 5-HT antagonist, did not affect the action of 5-HT. 5-HT was inactive when applied to the myelinated fibres of the sciatic nerve. Our results indicate that 5-HT-induced depolarization appears to be related to an increase in the resting sodium permeability of nerve fibres.

Voltage Clamp of Cardiac Muscle: A Theoretical Analysis of Early Currents in the Single Sucrose Gap

A theoretical model is presented for the early currents in the voltage clamp of cardiac muscle using the single sucrose gap technique. The preparation is represented by a single one-dimensional active cable with modified Hodgkin-Huxley membrane and the interent imperfections in the technique are also included, e.g., leakage through the sucrose gap and resistance in series with the membrane in the test compartment. The stability of the control system was found to depend on the position of the control point with respect to the sucrose gap border. Computed currents for a stable system closely resembled those in the literature and those from a near-ideal system (e.g., squid axon.) The potential immediately across the membrane, however (not including potential drops across the series resistance external to the membrane), was found to be essentially uncontrolled and the "current-voltage" relationship was shown to be almost independent of membrane properties.