Lindane, a gamma-isomer of hexachlorocyclohexane has largely been used as an insecticide and is widely spread in the environment due to the long life time of the molecule 1. Absorbed by the respiratory, digestive or cutaneous pathways, it accumulates in tissues in the following order: fat > brain > kidney > muscle > lung > heart > spleen > liver > blood 2. Lindane stimulates the synaptic transmission of a large number of muscular and nerve preparations, and suppresses the GABA-activated chloride current 3 by interacting with the receptor GABA-chloride channel complex 4. Due to the similarity between lindane and inositol 1, 4, 5 triphosphate (IP₃) 5, it has been suggested that lindane releases Ca²⁺ from IP₃-sensitive intracellular stores in macrophages 6 and smooth myometrial muscle cells 7. Lindane transiently depolarizes the membrane, opens Ca²⁺ channels thus increasing the intracellular Ca²⁺ concentration, and subsequently triggers Ca²⁺-activated K⁺ current (I_K-Ca) in human sperm 8. Lindane (1 microM – 100 microM) does not depress the peak of intracellular Ca²⁺ transient in guinea pig myocytes, and does not interact directly with the ryanodine receptor Ca²⁺ release channels from cardiac sarcoplasmic reticulum vesicles 9. A Ca²⁺ release from the endoplasmic reticulum, mitochondria and other Ca²⁺ stores has been reported in the presence of lindane (0.15 mM) in cat kidney cells 10. Lindane (30 microM) has no effect on the L-type Ca²⁺ current, but suppresses the activity of large conductance Ca²⁺-activated K⁺ channels and increases the firing rate of spontaneous action potentials in rat pituitary GH(3) cells 11.

Little is known about the effect of the pesticide on cardiac tissues. The aim of the present work was to study the effect of lindane on the action potential and transmembrane currents of frog auricular heart muscle.

Intracellular recordings of transmembrane potentials show that the addition of lindane (3.4 microM) to the Ringer solution did not alter the RP, decreased the amplitude of the OS and shortened the plateau duration (Fig. 1). The effects of lindane on the AP were dose-dependent. Table 1 shows that lindane (0.34 microM to 6.8 microM) did not significantly modify RP; lindane (0.34 microM) slightly but significantly (P < 0.05) shortened APD₄₀ and APD₁₀ by 6% and 3%, respectively. APD₄₀ and APD₁₀ shortening was not significantly increased by increasing the lindane concentration to 6.8 microM. APD₀ was only significantly shortened (P < 0.05) in the presence of lindane 3.4 microM in the Ringer solution (Table 1). Under voltage-clamp conditions, the remaining currents recorded in the Ringer solution containing TTX (0.6 microM), Cd²⁺ (1 mM) and TEA (10 mM) (control solution) mainly corresponded to the leak current and to the background inward rectifier K⁺ current (I_K1) (Fig. 2A). Current-voltage relationships plotted for the current measured at the end of the clamp step potential (V) show that the current was inward (I_in) at V more negative than HP and outward (I_out) at V more positive than HP (Fig. 2B). The addition of lindane (1.7 microM) to the control solution increased I_out but did not alter the tail current (Fig. 2A). Current-voltage relationships of Fig. 2B show that lindane (1.7 microM) increased the amplitude of I_out which developed at membrane potentials more positive than -70 mV. Subsequent addition of Sr²⁺ (5 mM) to the control solution containing lindane (1.7 μM) decreased the amplitude of I_out in the membrane potential range of -120 mV to +30 mV (Fig. 2B), whereas further addition of quinidine (0.5 mM) to the solution containing both, lindane and Sr²⁺, suppressed the remaining I_out whatever the membrane potential tested (Fig. 2B). Lindane (1.7 microM) increased the magnitude of I_out which developed when I_K1 was blocked by the addition of Ba²⁺ (2 mM) to the control solution (Fig. 3A). Current-voltage relationships show that the lindane-increased I_out developed at membrane potentials more positive than -20 mV (Fig. 3B). Subsequent addition of E-4031 (1 microM) to the control solution containing lindane blocked the lindane-increased I_out (Fig. 2A) whatever the membrane potential studied (Fig. 3B). The addition of E-4031 (2 microM) to the Ringer solution did not modify RP but prolonged APD (Fig. 4Aa) and further addition of lindane (3.4 microM) to the solution containing E-4031 (2 microM) did not modify the APD (Fig. 4Ab). Conversely, the addition of E-4031 (2 microM) to the Ringer solution containing lindane (3.4 microM) lengthened APD₀, APD₄₀ and APD₁₀ (Fig. 4B).

The present study shows that micromolar concentrations of lindane shortened the action potential duration APD and increases a quinidine and E-4031-sensitive outward current in frog auricle.

Our data show that the shortening of the duration of the repolarizing phase (APD₄₀ and APD₁₀) of the AP is the first significant event occurring in response to the application of a lindane concentration as low as 0.34 microM. This effect is then followed by a shortening of the plateau duration APD₀ which is clearly visible only at a ten times higher concentration.

Voltage-clamp experiments indicate that lindane increases an outward current (I_out). This current develops in the presence of TEA, known to block the delayed K⁺ current, in the control solution and under conditions where Ca²⁺ current has previously been blocked by Cd²⁺, suggesting that a lindane-increased Ca²⁺ influx may not be directly involved in the development of I_out. The lindane-increased I_out cannot be attributed to the opening of lindane-induced ionic channels since lindane has been shown to be devoid of ionophoretic properties in planar lipid bilayers 9. Our data show that the lindane-increased I_out still persists in the presence of Sr²⁺ which is known to block the background I_K112 and I_K-Ca13 currents in cardiac tissues. Our findings reveal that quinidine inhibits the effect of lindane on I_out. Quinidine is an open channel blocker of the cardiac rapid delayed rectifier K⁺ current (I_Kr) 14151617. In addition, they show that micromolar concentrations of E-4031, a specific blocker of I_Kr 1415, prolong the APD in frog auricle, are able to prevent or to reverse the APD shortening induced by lindane and in addition suppressed the lindane-increased I_out. These observations indicate that I_Kr participates to the AP repolarization in frog auricular cells, as in mammalian cardiac tissues 1819. This current is sensitive to quinidine and E-4031 but, as reported in rabbit ventricular cells 20, it is not sensitive to Sr²⁺ or Ba²⁺. The data also reveal that lindane increases I_Kr. The mechanism by which lindane increases I_Kr is probably not the result of a direct activation of the channel I_Kr, believed to be encoded by the human ether-a-go-go related gene (HERG) 2122232425, and which is involved in long QT syndrome, a cardiac disorder characterized by syncope, seizure and sudden death which can be congenital, idiopathic or iatrogenic 26. HERG K⁺ channel regulation depends on protein-kinase (PK)-dependent pathways. In guinea pig ventricular myocytes, the shift of the activation of HERG K⁺ channel induced by phorbol ester involves a PKA-dependent pathway 27. A PKC-dependent pathway links a G protein-coupled receptor that activates phospholipase C to modulate the Herg channel in Xenopus oocytes co-expressing the channel and tyrotropin releasing hormone receptor 28. According to Heath and Terrar 29, I_Kr is thought to be regulated by PKC which is activated by beta-adrenoceptors stimulation in guinea-pig ventricular myocytes. Lindane activates PKC activity in rat brain and liver tissues 30. In addition, it has been shown that dynamic regulation of the Herg K⁺ channels may be achieved via receptor-mediated changes in phosphatidyl inositol bisphosphate (PIP2) concentrations; elevated PIP2 accelerated activation and slowed inactivation kinetics 31. But single exposure of rats to lindane (100 mg / kg) did not cause any significant change in phosphoinositide levels in erythrocyte membrane and cerebrum 2 or 24 h after exposure 32.

In conclusion, the results presented show for the first time that the rapid delayed outward current I_Kr, involved in the repolarization of the cardiac AP, is increased by micromolar concentrations of lindane and may be responsible for the alterations of the AP duration induced by the pesticide. Although the mechanism by which lindane may increase I_Kr remains to be elucidated, the consequences of the effect of lindane on I_Kr are of toxicological interest since this current is involved in cardiac disorder.

Experiments were performed at 20–21°C on quiescent whole auricle isolated from frog heart and on myocytes isolated enzymatically from the auricle.

AP: action potential

APD: action potential duration

APD₀: duration of the plateau measured at 0 mV

APD₄₀: duration of the AP at the end of the plateau measured at a membrane potential + 40 mV higher than RP

APD₁₀: duration of the AP at the end of the repolarization phase measured at a membrane potential + 10 mV higher than RP

EGTA: ethylene glycol tetra acetic acid

HP: holding potential

IP₃: inositol 1, 4, 5 triphosphate

I_K-Ca: Ca²⁺-activated K⁺ current

I_in: inward current

I_K1: inward rectifying K⁺ current

I_out: outward current

I_Kr: rapid delayed outward current

microM: micromolar

mM: millimolar

mm: millimeter

ms: millisecond

mV: millivolt

mU / ml: milliunit per milliliter

OS: overshoot

pA: picoampere

PIP2: phosphatidyl inositol bisphosphate

PK: protein kinase

RP: resting membrane potential

TEA: tetraethylammonium

TTX: tetrodotoxin

V: clamp step potential