Single Sucrose-gap Technique Research Articles

Characterization of the first two toxins isolated from the venom of the ancient scorpion Tityus (Archaeotityus) mattogrossensis (Borelli, 1901).

Background: Almost all Tityus characterized toxins are from subgenera Atreus and Tityus, there are only a few data about toxins produced by Archaeotityus, an ancient group in Tityus genus.Methods: Tityus (Archaeotityus) mattogrossensis crude venom was fractionated by high performance liquid chromatography, the major fractions were tested in a frog sciatic nerve single sucrose-gap technique. Two fractions (Tm1 and Tm2) were isolated, partially sequenced by MALDI-TOF/MS and electrophysiological assayed on HEK293 Nav 1.3, HEK293 Nav 1.6, DUM and DRG cells.Results: The sucrose-gap technique showed neurotoxicity in four fractions. One fraction caused a delay of action potential repolarization and other three caused a reduction in amplitude. An electrophysiological assay showed that Tm1 is active on HEK293 Nav 1.3, HEK293 Nav 1.6, DUM and DRG cells, and Tm2 on HEK293 Nav 1.3 and DRG cells, but not in HEK293 Nav 1.6. In addition, Tm1 and Tm2 did promote a shift to more negative potentials strongly suggesting that both are α-NaScTx. Conclusion: Although Tityus (Archaeotityus) mattogrossensis is considered an ancient group in Tityus genus, the primary structure of Tm1 and Tm2 is more related to Tityus subgenus. The patch clamp electrophysiological tests suggest that Tm1 and Tm2 are NaScTx, and also promoted no shift to more negative potentials, strongly suggesting that both are α-NaScTx. This paper aimed to explore and characterize for the first time toxins from the ancient scorpion Tityus (Archaeotityus) mattogrossensis.

Bioassay-Guided Evaluation of Antinociceptive Effect ofN-Salicyloyltryptamine: A Behavioral and Electrophysiological Approach

We investigated the antinociceptive and nerve excitability effects of the N-salicyloyltryptamine (NST) NST-treated mice exhibited a significant decrease in the number of writhes when 100 and 200 mg/kg (i.p.) were administered (i.p.). This effect was not antagonized by naloxone (1.5 mg/kg, i.p.). NST inhibited the licking response of the injected paw when 100 and 200 mg/kg were administered (i.p.) to mice in the first and second phases of the formalin test. Because the antinociceptive effects could be associated with neuronal excitability inhibition, we performed the single sucrose gap technique and showed that NST (3.57 mM) significantly reduced (29.2%) amplitude of the compound action potential (CAP) suggesting a sodium channel effect induced by NST. Our results demonstrated an antinociceptive activity of the NST that could be, at least in part, associated to the reduction of the action potential amplitude. NST might represent an important tool for pain management.

Rosewood oil induces sedation and inhibits compound action potential in rodents

Aim of the study Aniba rosaeodora is an aromatic plant which has been used in Brazil folk medicine due to its sedative effect. Therefore, the purpose of the present study was to evaluate the sedative effect of linalool-rich rosewood oil in mice. In addition we sought to investigate the linalool-rich oil effects on the isolated nerve using the single sucrose-gap technique. Materials and methods Sedative effect was determined by measuring the potentiation of the pentobarbital-induced sleeping time. The compound action potential amplitude was evaluated as a way to detect changes in excitability of the isolated nerve. Results The results showed that administration of rosewood oil at the doses of 200 and 300 mg/kg significantly decreased latency and increased the duration of sleeping time. On the other hand, the dose of 100 mg/kg potentiated significantly the pentobarbital action decreasing pentobarbital latency time and increasing pentobarbital sleeping time. In addition, the effect of linalool-rich rosewood oil on the isolated nerve of the rat was also investigated through the single sucrose-gap technique. The amplitude of the action potential decreased almost 100% when it was incubated for 30 min at 100 μg/ml. Conclusions From this study, it is suggested a sedative effect of linalool-rich rosewood oil that could, at least in part, be explained by the reduction in action potential amplitude that provokes a decrease in neuronal excitability.

Antinociceptive Activity of (-)-Carvone: Evidence of Association with Decreased Peripheral Nerve Excitability

(-)-Carvone is a monoterpene ketone that is the main active component of Mentha plant species like Mentha spicata. This study aimed to investigate the antinociceptive activity of (-)-carvone using different experimental models of pain and to investigate whether such effects might be involved in the nervous excitability elicited by others monoterpenes. In the acetic acid-induced writhing test, we observed that (-)-carvone-treated mice exhibited a significant decrease in the number of writhes when 100 and 200 mg/kg was administered. It was also demonstrated that (-)-carvone inhibited the licking response of the injected paw when 100 and 200 mg/kg was administered (i.p.) to mice in the first and second phases of the formalin test. Since naloxone (5 mg/kg, s.c.), an opioid antagonist, showed no influence on the antinociceptive action of (-)-carvone (100 mg/kg), this suggested nonparticipation of the opioid system in the modulation of pain induced by (-)-carvone. Such results were unlikely to be provoked by motor abnormality, since (-)-carvone-treated mice did not exhibit any performance alteration on the Rota-rod apparatus. Because the antinociceptive effects could be associated with neuronal excitability inhibition, we performed the single sucrose gap technique and observed that (-)-carvone (10 mM) was able to reduce the excitability of the isolated sciatic nerve through a diminution of the compound action potential amplitude by about 50% from control recordings. We conclude that (-)-carvone has antinociceptive activity associated with decreased peripheral nerve excitability.

Study of anticonvulsant effect of citronellol, a monoterpene alcohol, in rodents

Citronellol is one monoterpene alcohol, which is present in the essential oils of various aromatic plant species. This study evaluated the neuroprotective activity of citronellol on pentylenetetrazol- and picrotoxin-induced convulsions and maximal electroshock-induced seizures in mice. Administration of citronellol significantly reduced the number of animals of convulsion induced by pentylenetetrazol and eliminated the extensor reflex of maximal electroshock-induced seizures test in about 80% of the experimental animals. In addition, administration of citronellol showed protection in the pentylenetetrazol and picrotoxin tests by increasing the latency of clonic seizures. We also investigated the effect of citronellol in the rat isolated nerve using the single sucrose-gap technique. We showed that the amplitude of the compound action potential decreased more than 90% when the monoterpene was incubated for 30 min at 6.4 mM and we did not verify any effect on the repolarization of the compound action potential. Taken together, our results demonstrated an anticonvulsant activity of the citronellol that could be, at least in part, explained by the diminution of the action potential amplitude.

The possible role of ATP and PACAP as mediators of apaminsensitive NANC inhibitory junction potentials in circular muscle of guinea-pig colon.

1. In the presence of atropine (1 microM), guanethidine (3 microM), indomethacin (3 microM), nifedipine (1 microM), L-nitroarginine (L-NOARG, 100 microM), and the selective tachykinin NK1 and NK2 receptor antagonists, SR 140,333 and GR 94,800, respectively (0.1 microM each), a single pulse of electrical field stimulation (EFS) produced a monophasic non-adrenergic non-cholinergic (NANC) inhibitory junction potential (i.j.p., about 10 mV in amplitude) in the circular muscle of guinea-pig proximal colon, recorded by the modified single sucrose gap technique. 2. The P2 purinoceptor agonist, alpha, beta methylene ATP (alpha, beta mATP, 100 microM) and the pituitary adenylyl cyclase activating peptide (PACAP, 1 microM) both produced hyperpolarization (11 +/- 0.8 mV, n = 14 and 10.2 +/- 0.8 mV, n = 19, respectively) and relaxation (1.1 +/- 0.2 mV, n = 14 and 1.5 +/- 0.2 mN, n = 19, respectively) of the circular muscle. 3. Apamin (0.1 microM) nearly abolished (about 90% inhibition) the NANC i.j.p. and the alpha, beta mATP-induced hyperpolarization, markedly reduced the alpha, beta mATP-induced relaxation (73% inhibition) and the PACAP-induced hyperpolarization (65% inhibition), while the PACAP-induced relaxation was unaffected. 4. Tetraethylammonium (TEA, 10 mM) increased the EFS-evoked i.j.p. and revealed an excitatory junction potential (e.j.p.). In the presence of TEA, alpha, beta mATP induced a biphasic response: transient depolarization and contraction followed by hyperpolarization and relaxation. The hyperpolarization to PACAP was reduced by TEA (45% inhibition) but the relaxation was unaffected. 5. The combined application of apamin (0.1 microM) and TEA (10 mM) abolished the i.j.p. and single pulse EFS evoked a pure e.j.p. with latency three times longer than that of the i.j.p. In the majority of strips tested, alpha, beta mATP and PACAP elicited a biphasic response : depolarization and small contraction followed by hyperpolarization and relaxation. 6. The P2 purinoceptor antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) inhibited the NANC i.j.p. in concentration-dependent manner and inhibited the alpha, beta mATP-induced hyperpolarization and relaxation, without affecting the hyperpolarization and relaxation induced by PACAP. On the other hand, the P2 purinoceptor antagonist, suramin (100 microM) inhibited to a similar extent (60-80%) the NANC i.j.p. and the hyperpolarization and relaxation induced by alpha, beta mATP or PACAP. 7. PPADS and suramin reduced the NANC e.j.p. evoked by a single pulse EFS in the presence of apamin and TEA (100 microM of PPADS and 300 microM of suramin inhibited the e.j.p. by about 40%). 8. We conclude that ATP, but not PACAP, mediates the apamin-sensitive NANC i.j.p. in the circular muscle of the guinea-pig colon. After blockade of the NANC i.j.p., ATP may act as an excitatory transmitter by activating excitatory P2 purinoceptors. The subtypes of P2 purinoceptor involved in the inhibitory and excitatory responses remain to be established. The data suggest that excitatory P2 purinoceptors may be located extrajunctionally.

Apamin‐sensitive and ‐insensitive components of inhibitory junction potentials in rat caecum: role of nitric oxide

1. The non-adrenergic non-cholinergic (NANC) inhibitory response to electrical field stimulation (EFS) in circular muscle from rat caecum was investigated using the single sucrose-gap technique. EFS with single pulses evoked hyperpolarization oral inhibitory function potential (IJP) of the membrane associated with muscular relaxation or with transient inhibition of spontaneous contractile activity. 2. The amplitude and the duration of the IJPs were enhanced by using train stimulation at increasing frequency. 3. Apamin (10(-7) M) reduced the amplitude of IJPs at all frequencies tested. 4. N omega-nitro-L-arginine methyl ester (L-NAME) (10(-4) M, 5 x 10(-4) M), but not D-NAME, caused a concentration dependent decrease in the amplitude of IJPs at all frequencies tested. L-Arginine (10(-3) M) prevented these effects. 5. L-NAME (5 x 10(-4) M) caused the disappearance of the apamin-resistant IJP-component, evoked by single pulse or by low frequency trains. 6. Sodium nitroprusside (SNP) (10(-4) M), a nitric oxide (NO) donor, induced hyperpolarization of membrane potential and muscular relaxation. SNP-induced effects were not affected by pretreatment of the muscle strips with effective concentrations of tetrodotoxin, apamin, and L-NAME. 7. P2-purinergic antagonists, reactive blue 2 (up to 5 x 10(-4) M) and suramin (up to 3 x 10(-4) M), failed to affect the evoked IJPs. 8. These results show that, in rat caecum, the NANC response to electrical stimulation is composed of two distinguishable components: an apamin-resistant and an apamin-sensitive component. NO or a related compound is mainly involved in the mediation of the apamin-resistant component, while ATP is not the mediator responsible for the apamin-sensitive component.

Evidence that tachykinin NK1 and NK2 receptors mediate non-adrenergic non-cholinergic excitation and contraction in the circular muscle of guinea-pig duodenum.

1. In the presence of atropine (1 microM), guanethidine (3 microM), indomethacin (3 microM), apamin (0.1 microM) and L-nitroarginine (L-NOARG, 30 microM), electrical field simulation (EFS) produced a nonadrenergic, noncholinergic (NANC) excitatory junctional potential (e.j.p.), action potentials and contraction of the circular muscle of the guinea-pig proximal duodenum, recorded by the single sucrose gap technique. 2. The selective tachykinin (TK) NK1 receptor antagonist, GR 82,334 (30 nM-3 microM) produced a concentration-dependent inhibition of the EFS-evoked NANC e.j.p. and contraction. Similarly, the selective NK2 receptor antagonists, MEN 10,627 (30 nM-3 microM) and GR 94,800 (100 nM-10 microM), both produced a concentration-dependent inhibition of the EFS-evoked NANC e.j.p. and contraction. GR 82,334 inhibited the electrical and mechanical NANC responses to EFS in an almost parallel manner, while MEN 10,627 and GR 94,800 were more effective in inhibiting the mechanical than the electrical response to EFS. 3. Activation of the NK1 or NK2 receptor by the selective agonists, [Sar9]substance P (SP) sulphone and [beta Ala8]neurokinin A (NKA) (4-10), respectively (0.3 microM each), produced depolarization, action potentials and contractions. GR 82,334 selectively inhibited the responses to [Sar9]SP sulphone, without affecting the responses to [beta Ala8]NKA (4-10). MEN 10,627 and GR 94,800 inhibited or abolished the responses to [beta Ala8]NKA (4-10), without affecting the responses to [Sar9]SP sulphone. 4. Nifedipine (1 microM) abolished the action potentials and contraction produced either by EFS or by the TK receptor agonists [Sar9]SP sulphone or [beta Ala8]NKA (4-10). 5. In the presence of nifedipine, the NANC e.j.p. produced by EFS was biphasic: in the majority of strips tested (21 out of 29) an early fast phase of depolarization was followed by a second slow component. The combined administration of GR 82,334 and GR 94,800 (3 microM each) reduced both components, the slow phase being inhibited to a greater extent than the fast phase. 6. The P2 purinoreceptor antagonist, suramin (100 microM) reduced the fast phase of the e.j.p. produced by EFS in the presence of nifedipine, without affecting the slow phase. The combined administration of suramin, GR 82,334 and GR 94,800 produced a nearly complete blockade of the e.j.p. produced by EFS in the presence of nifedipine. 7. When tested in the absence of apamin and L-NOARG, EFS induced a NANC inhibitory junction potential (i.j.p.) followed by an e.j.p., and the selective P2Y receptor agonist, adenosine-5'-O-(2-thiodiphosphate) (ADP beta S, 10 microM), produced membrane hyperpolarization. After addition of apamin and L-NOARG, the ij.p. was blocked, and EFS produced a pure NANC e.j.p.; ADPPS produced depolarization, action potentials and contraction.8. Suramin (100 microM) blocked the depolarization, action potentials and contractions produced by ADP beta S in the presence of apamin and L-NOARG, without affecting the responses produced by the NK1receptor agonist, [Sar9}SP sulphone.9. We conclude that NK1 and NK2 receptors cooperate in producing NANC excitation and contraction of the circular muscle in the guinea-pig proximal duodenum. Activation of either TK receptor produces membrane depolarization and both receptors contribute to generate action potentials which are essential for producing muscle contraction, via nifedipine-sensitive calcium channels. It appears that endogenous ATP chiefly acts as an inhibitory transmitter but, after blockade of NANC inhibitory mechanism(s),ATP may act as a fast signalling excitatory transmitter.

Reversion by polyclonal antibodies of α effects of Tityus serrulatus venom on frog sciatic nerve

Fraction T 2 from Tityus serrulatus venom produced a marked lengthening of action potentials recorded with the single sucrose-gap technique, a characteristic effect of α scorpion toxins. This effect was not reversed by thorough washing of the nerve. On the other hand, T 2 fraction deactivated by complete iodination did not cause any alteration of the compound action potential, even if applied in concentration as high as 4000 times the half saturation dose of the unmodified T 2 fraction. This high dose of deactivated T 2 did not hinder the onset of the full effect of a single dose of T 2 fraction applied subsequently. Polyclonal antibodies against native or against iodinated venom reverted the action of T 2 fraction, restoring normal electrical response. We conclude that both types of antibodies may remove the effect of α toxins on sodium channel.

Purification of protein toxins from Leiurus Quinquestriatus Hebraeus that modify Na channels

J. Borneman and R. Hahin. Purification of protein toxins from Leiurus quinquestriatus hebraeus that modify Na channels. Toxicon31, 1019–1038, 1993.—Two protein toxins (Lqh1 and Lqh2) were purified from crude venom obtained from Middle Eastern scorpions, Leiurus quinquestriatus hebraeus, by using cationic exchange chromatography. Lqh1 and Lqh2 were compared to toxin V (Lqq5) obtained from the venom of the North African scorpion Leiurus quinquestriatus quinquestriatus. Lqh1 and Lqh2 were purified to homogeneity; they had mol. wts of 6390 and 5870, respectively; thus both toxins differ in size from Lqq5 (7462). Electrophysiological experiments also suggested that all three toxins are different. In a dose-dependent manner, Lqh1, Lqh2 and Lqq5 lengthened and attenuated propagated compound action potentials (AP) recorded from frog sciatic nerves using the single sucrose-gap technique. Toxins Lqh1 and Lqh2 were found to be more effective than Lqq5 in both lengthening and blocking APs. Voltage-clamp experiments using the vaseline-gap technique on frog skeletal muscle fibres showed that Lqh1 and Lqh2 attenuated the Na current amplitude and slowed inactivation, while Lqq5 primarily lengthened the Na current duration. Increases in the holding potential increase the current attenuation caused by all three toxins. Evidence from sucrose-gap and voltage-clamp experiments suggests that all three toxins bind to Na channels and block them, besides their well-known ability to slow inactivation kinetics. The increased effectiveness of Lqh1 appears to be produced by a slowed rate of exit of the toxin from its binding site.

Non-adrenergic, non-cholinergic inhibitory responses to nerve stimulation in rat colonic circular muscle.

The nerve-mediated response to electrical field stimulation (EFS) in rat colonic circular muscle was investigated using the single sucrose-gap technique. EFS with a single pulse (0.4 ms, supramaximal voltage) elicited transient TTX-sensitive hyperpolarization (IJP) often followed by an 'off' depolarization associated with muscular contraction. No relaxation associated with the IJP could be seen unless tone was pharmacologically induced by carbachol (10(-6) M). IJPs were due to non-adrenergic, non-cholinergic (NANC) nerve activation since they were not affected by atropine (10(-7) M) or guanethidine (10(-6) M) superfusion. The mechanism underlying the IJP was presumably an increase in K+ conductance, and the NANC neurotransmitter might open largely apamin-sensitive, Ca(2+)-dependent K+ channels. Purines or vasoactive intestinal polypeptide (VIP) did not mimic the effects of NANC nerve stimulation. Therefore, the NANC inhibitory system, producing IJPs, in rat colonic circular muscle is not purinergic or VIPergic in nature.

Force production following transient potential changes in voltage‐clamped myocardium

Inter-relationships between force, membrane voltage and currents were studied in ferret and guinea-pig papillary muscles using the single sucrose gap technique (37 degrees C). The preparations were held at -90 or -40 mV and depolarized (excited) to 0 mV for 180 ms at 1.0 Hz. At regular intervals the shape of a single clamp pulse (called '1') was varied and its effects were investigated during the same test cycle and in two subsequent test cycles ('2' and '3'). Peak force of contraction 1 (F1) increased with the duration of the test clamp up to 90 ms and was constant thereafter. F1 increased with clamp amplitude (V1) between -30 and 10 mV and decreased at greater amplitudes. This relation was similar to the relation between peak second inward current (I1) and V1. The peak force of contractions 2 and 3 rose with the clamp duration and clamp amplitudes of cycle 1. The relation between F3 and F2 was linear (slope 0.40), except at the lowest and highest F2 values where there was a small deviation. There was an inverse relation between I2 and F2. The results support the idea that increased duration or amplitude of the voltage clamp pulse leads to a greater calcium entry which is manifested in the following potentiated contraction. The relation between F3 and F2 implies that about 40% of calcium recirculates between the contractions. The inverse relationship between F2 and I2 indicates that the second inward current is regulated by release from the sarcoplasmic reticulum via negative feedback.

Depressant effect of magnesium on early afterdepolarizations and triggered activity induced by cesium, quinidine, and 4-aminopyridine in canine cardiac Purkinje fibers

Magnesium chloride has been shown to terminate torsades de pointes in some patients with the acquired long QT syndrome. The mechanism for this effect is unknown. Recently early afterdepolarizations (EADs) and triggered activity (TA) have been proposed as causes of torsades de pointes. The purpose of the present study was to examine whether magnesium suppressed EADs that were initiated in vitro by different agents and if so its mechanism of action. TA arising from EADs was induced by quinidine (1 to 4 μmol/L, n = 5) at high temperature (38.5 to 40° C), cesium chloride (5 to 12 mmol/L, n = 6), and 4-aminopyridine (1.5 to 5 mmol/L, n = 7) in canine cardiac Purkinje fibers superfused with modified Tyrode's solution (KCl = 2.7 mmol/L). MgCl 2 (2 to 7 mmol/L) reversibly ablished TA and suppressed EADs. Tetrodotoxin (TTX; 1 to 5 μmol/L) also abolished TA elicited by 4-aminopyridine (n = 6). We then examined the effects of MgCl 2, TTX, and verapamil on depolarization-induced automaticity by means of a single sucrose gap technique to gain insight into the mechanism of action of magnesium. MgCl 2 (5 mmol/L) abolished automaticity arising from membrane potentials more negative than −70 mV and prolonged the spontaneous cycle length at less negative membrane potentials. The effects of TTX (1 to 5 μmol/L) resembled those of MgCl 2. Verapamil (1 μmol/L) prolonged the cycle length of the initial automatic response at high levels of membrane potential and progressively reduced the amplitude of the subsequent automatic potentials. It abolished automaticity arising from less negative membrane potentials. Thus MgCl 2 suppressed EADs induced by three diverse agents that reduce potassium conductance. Similarities between the effects of MgCl 2 and TTX suggest that magnesium may suppress EADs and TA by reducing the TTX-sensitive sodium “window” current.

Pacemaker activity in the proximal lower oesophageal sphincter of the dog.

1. The electrical and mechanical activities of different regions of the canine lower oesophageal sphincter were measured using the single sucrose gap technique. 2. Spontaneous electrical activity was found in the region 0-6 mm oral to the squamocolumnar border. 3. The electrical activity consisted of bursts of spikes superimposed on slow waves. The slow-wave frequency ranged from 0.6 to 5 min-1 in different muscle strips. 4. The slow wave-spike complex and associated contraction were insensitive to tetrodotoxin and atropine. 5. In the pacemaker region, electrical stimulation of intrinsic nerves evoked excitatory junction potentials (atropine sensitive), inhibitory junction potentials (non-adrenergic) and post-stimulus excitation. 6. Increase in the frequency of the slow waves was obtained by muscarinic receptor stimulation (carbachol 10(-7) M) and 10 mM-KCl. 7. The distal lower oesophageal sphincter exhibited a high basal tension but did not show spontaneous electrical activity and stimulation of intrinsic nerves revealed only non-cholinergic, non-adrenergic inhibition. 8. The electrical slow-wave activity observed in the proximal sphincter may constitute the control mechanism for the phasic nature of the contractile activity seen during both the postprandial period and phase III of the interdigestive migrating myoelectric complex. 9. The neural cholinergic activity present in the proximal lower oesophageal sphincter suggests the possibility of neural modulation of the myogenic control activity.

Lidocaine blocks open and inactivated cardiac sodium channels

Guinea-pig papillary muscles were voltage-clamped using the single sucrose gap technique. The maximum upstroke velocity of the action potential (Vmax) was used as an indicator of the sodium conductance. Lidocaine (5 mumol/l to 40 mumol/l) reduced Vmax in a use-dependent fashion. Block of sodium channels developed during channel opening and while the channels were inactivated. Block of inactivated channels was not voltage-dependent over the -40 mV to +40 mV range. Recovery from block occurs upon repolarization, and for a given diastolic interval the recovery is more complete as the membrane potential is hyperpolarized over the -80 mV to -150 mV range. These results can be accounted for in terms of the modulated receptor hypothesis, where lidocaine has a low affinity for rested sodium channels, but a high affinity for open and inactivated channels.

Changes in passive electrical properties of guinea-pig ventricular muscle exposed to low K + and high Ca 2+ conditions

Changes in passive electrical properties of guinea-pig papillary muscle exposed to low K+, high Ca2+ conditions were examined using a single sucrose gap technique. While quiescent preparations exposed for 5 mins did not develop delayed afterdepolarizations, those placed in the test solution for 30 mins with or without stimulation developed afterdepolarizations. Changes occurring during a short exposure to low K+, high Ca2+ solution were increases in membrane resistance, membrane time constant and space constant by 47%, 83% and 17% compared with the control, respectively. There were no significant changes in internal longitudinal resistance and membrane capacity. During long exposure to the test solution (30 mins), delayed afterdepolarizations developed. There were similar increases in membrane resistance and in time constant as found during the short exposures. Internal longitudinal resistance was calculated to have increased by 24% during the long exposure. A 19% increase in membrane capacity was also found during the test condition. High Ca2+ or low K+ alone did not cause a significant increase in internal longitudinal resistance. The conduction velocity in the longitudinal direction decreased from 107 +/- 23 cm/s during the control to 80 +/- 7 cm/s during the test period for 30 mins. These results suggest that, in addition to the abnormal impulse formation based on afterdepolarizations, low K+, high Ca2+ solution changes the passive electrical properties of the fibers, resulting in a lower rate of impulse conduction.

Electromechanical effects of okadaic acid isolated from black sponge in guinea-pig ventricular muscles.

Okadaic acid is a monocarboxylic acid with a unique molecular structure of C44H66O13. Okadaic acid, at concentrations above 10(-5) M, caused a dose-dependent increase of contractile force in guinea-pig isolated ventricular muscles. The increase of contractile force by okadaic acid (1-4 X 10(-5) M) was accompanied by a prolongation of action potential duration (a.p.d.), while a.p.d. was shortened by okadaic acid at above 10(-4) M. The okadaic-acid-induced changes in contractile force and a.p.d. were abolished by pre-treatment with verapamil (2 X 10(-6 M) or nifedipine (10(-6) M). In a low-Ca2+ medium (0.12 mM), the contractile response to okadaic acid (4 X 10(-5) M) was inhibited, whereas the a.p.d. was more prolonged by the drug than in a control medium (containing 1.2 mM-Ca2+). In a low-Na+ (70 mM) medium or in a low-Na+ (70 mM) and low-Ca2+ (0.12 mM) medium both the contractile force and a.p.d. were unaffected by okadaic acid (4 X 10(-5) M). The positive inotropic effect of okadaic acid (4 X 10(-5) M) was not inhibited by treatment with tetrodotoxin (TTX, 2 X 10(-6) M) or with ryanodine (2 X 10(-6) M). In voltage-clamp experiments using a single sucrose-gap technique, okadaic acid (4 X 10(-5) M), caused a significant increase of slow inward current, while it did not affect the net outward current. These findings suggest that okadaic acid has a positive inotropic effect on the heart, probably due to the enhancement of slow Ca2+ and/or Na+ inward current through the cell membrane.

Neuromuscular physiology of Grillotia erinaceus metacestodes (Cestoda: Trypanorhyncha) in vitro

SUMMARYIsometric tension recordings and the single sucrose-gap technique were used to record mechanical and electrical activity from Grillotia erinaceus metacestodes in vitro. Both complete preparations and those cut longitudinally (to allow access of the bathing medium to the region of the somatic muscle) showed spontaneous contractions of frequency ≃ 1/min in Hanks’ Balanced Salt Solution (HBSS) at 13 °C. Changing the pH, temperature or osmolarity of the HBSS did not improve the activity but rather abolished it at extremes of these variables, so confirming the adequacy of the experimental environment for further work. The neurotoxin tetrodotoxin (TTX; 3 × 10−6 M) failed to abolish or modify these spontaneous contractions although excitatory and inhibitory responses to 5-hydroxytryptamine (5-HT) and acetylcholine (ACh) respectively were demonstrated. Electrical recordings showed that spontaneous contractions were mediated by slow membrane depolarizations upon which spike-like potentials were often superimposed. 5-HT was shown to mediate its excitatory effect via membrane depolarization and an increase in the frequency of both slow depolarization and spikes. In contrast, inhibition by ACh was accompanied by membrane hyperpolarization and loss of all regenerative electrical activity. Electric field stimulation of the preparation using a range of stimulus parameters failed to demonstrate a functional motor innervation to the somatic muscle.

Action of praziquantel on Grillotia erinaceus metacestodes (Cestoda: Trypanorhyncha) in vitro

SUMMARYIsometric tension recordings and the single sucrose-gap technique were used to record mechanical and electrical activity of somatic muscle from G. erinaceus metacestodes in vitro. Praziquantel (≥ 10−6 M) raised baseline tension and largely abolished spontaneous fluctuations in tension. The effect of the drug was critically dependent on the presence and availability of calcium in the bathing medium. In calcium-free solution or in the presence of manganese or lanthanum (10 mm) praziquantel failed to induce a rise in baseline tension. Electrical recordings of somatic muscle activity showed the action of praziquantel to be accompanied by membrane depolarization and loss of slow depolarizations and spikes. Tegumental membrane potential, as measured by microelectrodes, was not affected by the drug. These results suggest that in vitro praziquantel causes contraction of the somatic muscle of G. erinaceus by membrane depolarization and influx of extracellular calcium.

A new single sucrose-gap method allowing current to be injected and the resulting potential change to be recorded.

A novel single sucrose-gap technique which allows the injection of current and the recording of the resulting change in the gap potential has been developed. Current injection is achieved discontinuously at a chopping rate of between 500 Hz and 5 kHz and the gap potential is recorded during the off-period of current injection and held until the next sampling period. This approach eliminates the potential drop in the sucrose solution and makes the measurement of electrical properties possible with a single sucrose-gap technique. Membrane properties of the dorsal roots of the bullfrog were examined using this technique. An outward-going rectification was evident around the resting potential and an inward-going rectification appeared on hyperpolarization below -70 mV. This rectification gave rise to a sigmoidal relationship between voltage and current. Hyperpolarization in response to current injection revealed a very long charging time constant. These observed membrane characteristics were in general agreement with those intracellulary recorded from the thick myelinated fiber in the frog dorsal roots.