The development of guidelines for drug and alcohol dependence treatment: affecting policy and practice

The rationale and methodology behind the Australian Quality Assurance Project is described. The Project aimed to develop guidelines for treatment content based on three sources of information: research findings, current practice and expert opinion. The issue of the gap between research and practice is discussed, as well as the role of dissemination in altering clinician behaviour.     

Modulation of action potential duration by inhibition of the transient outward current in sheep cardiac Purkinje fibers

In sheep cardiac Purkinje fibers concentration-dependent inhibition of transient outward current (i_to) by 4-aminopyridine (4-AP, 3-1000 mol/l) was recorded with the two-microelectrode voltage-clamp technique, and correlated effects on action potential duration measured at — 70 mV (APD-70) were investigatigated.Half-maximal inhibition of i_to-amplitude occurred at 15 mol/l 4-AP. The drug exhibited no major effect on voltage-dependent control of inactivation but reduced the maximally available i_to-current. At different activation frequencies (0.05 Hz, 0.25 Hz, 1 Hz) an equal amount of i_to-current, measured as percentage of the respective control, was inhibited by 4-AP. The APD-70 was on the average increased by 4-AP (3–500 mol/l) in a concentration-dependont manner up to 151 % of control. The drug-induced prolongation, measured as percentage of the respective control, was independent of stimulation frequency (0.05 Hz, 0.25 Hz, 1 Hz). Prolongation of APD-70 was on the average more pronounced for short action potentials (APD-70<150 ms: 169 % of reference) than for longer ones (APD-70 150–300 ms: prolongation to 117 % of reference; 500 mol/l 4-AP; 0.25 Hz stimulation rate). Few long control signals (APD-70 >300 ms) were shortened by 4-AP. These results indicate that inhibition of i_to-current by appropriate drugs will result in a reduction of inhomogeneity of action potential duration.     

Extracellular site of action of phenylalkylamines on L-type calcium current in rat ventricular myocytes

The effects of the phenylalkylamines verapamil, gallopamil, and devapamil on L-type calcium currents (I_Ca) were studied in ventricular myocytes from rat hearts using the whole-cell patch-clamp technique. In particular, the question was addressed, whether the pharmacological binding sites for these drugs were located at the inner and/or at the outer surface of the cell membrane. Therefore, tertiary verapamil, gallopamil, and devapamil and their corresponding quaternary derivatives were applied either from the outside or the inside of the cell membrane. Extracellular application of verapamil, gallopamil and devapamil (each at 3 M) reduced Ica to 16.1 ±8.6%, 11 ± 8.9 %, and 9.3 ± 6 % of control, respectively. Intracellular application of the same substances, via the patch pipette filled with 30 M of either verapamil, gallopamil, or devapamil, failed to depress I_Ca. The quaternary derivatives of the phenylalkylamines (30 M) were ineffective both when applied extracellularly or intracellularly. It is suggested that phenylalkylamines block I_Ca in ventricular myocytes by acting on a binding site of the calcium channel molecule located at the outer surface of the cell membrane.     

胺碘酮抑制缺血浦肯野纤维起搏离子流

目的观察正常及模拟“缺血”时胺碘酮对绵羊心室浦肯野纤维起搏离子流（Ｉｆ）的影响。方法双微电极电压钳制术,使膜电位过度极化以激活Ｉｆ。结果胺碘酮１×１０－５ｍｏｌ／Ｌ在正常台氏液中显著降低浦肯野纤维Ｉｆ幅值;模拟“缺血”也使Ｉｆ幅值降低,在此基础上,胺碘酮使Ｉｆ幅值进一步减小,加剧了“缺血”对Ｉｆ离子流的抑制作用。结论提示胺碘酮能抑制心肌缺血时心室正常自律性活动。     

Manipulation of synaptic sign and strength with divalent cations in the vertebrate retina: pushing the limits of tonic, chemical neurotransmission

At the first synaptic level of the vertebrate retina, photoreceptor light responses are transmitted to second order neurones through a chemical synapse based on a tonic release of neurotransmitter modulated by graded changes of presynaptic potential. The possibility that such synapses could work through a Ca2+-independent process had been proposed by previous authors, based on the persistence of transmission process in low Ca2+ media containing Co2+ or Ni2+ ions. Recently, we were able to explain these results within the framework of the classical calcium-hypothesis of synaptic transmission by taking into account the modifications of presynaptic surface potential brought about by changes of divalent cation concentrations. Here we report data showing how a surface-charge hypothesis could account for several apparently paradoxical effects of divalent cation manipulations such as: the enhancement of neurotransmitter release induced by low Ca2+ media; the transmission "unblocking" effect of Zn2+, Co2+ and Ni2+; and the reversal of transmission polarity induced by application of low Ca2+ media containing Cd2+ or Mg2+ ions.     

The effect of arachidonic acid on the M current of NG108-15 neuroblastoma × glioma hybrid cells

The M current, I _M, a voltage-dependent non-inactivating K⁺ current, was recorded in NG108-15 neuroblastoma × glioma hybrid cells, using the whole-cell mode of the patch-clamp technique. We studied the effect of arachidonic acid, other fatty acids and inhibitors of the arachidonic acid metabolism. In relatively high concentrations (25–50 M) arachidonic acid first increased and later decreased the current, I _h, which holds the membrane potential at –30 mV and mainly flows through open M channels. It shifted the midpoint potential, V _o, of the relation between M conductance, g _M, and membrane potential, V, to more negative values and decreased the maximum conductance ¯g _M and the time constant _M. In smaller concentrations (5–10 M) arachidonic acid merely decreased I _h and ¯g _M with little effect on V _o and _M. Eicosatetraynoic acid and docosa-hexaenoic acid acted similarly to arachidonic acid whereas stearic acid had no effect. Of the three enzyme inhibitors studied, nordihydroguaiaretic acid acted similarly to arachidonic acid. i. e. caused a biphasic change in I _h. Indomethacin and quinacrine caused, respectively, a pure increase and a pure decrease of I _h and ¯g _M. Possible explanations are build-up of internally produced arachidonic acid, depletion of eicosanoid products or an inhibitory effect unrelated to arachidonic acid metabolism.     

A loudspeaker-driven system for rapid and multiple solution exchanges in patch-clamp experiments

A new and inexpensive system allowing rapid and synchronized changes of solutions around a membrane patch or a cell under voltage-clamp conditions is described. Four plastic capillary tubings (OD 640 m; ID 430 m) were glued together horizontally and attached to a coil of a commercially available loudspeaker. Servo-control of the position of the coil allowed the mouth of any of the capillaries to be positioned near the pipette tip within 6 ms. A high flow speed of the test solution was crucial to achieve rapid solution exchange. At a flow speed of 5 cm/s, complete exchange of the external environment of a frog ventricular cell was achieved within 20–30 ms. The time course of solution change was found to be 3–5 times faster at the tip of an open patch pipette. To preserve the physical integrity of the cell, the cell was usually perfused by a control capillary at a slow velocity (0.2 –0.4 cm/s) and test solutions flowing out of adjacent capillaries at high velocity (4–5 cm/s) were applied to the cell only for short periods. Determination of the three-dimensional contamination profile around the mouth of the control capillary allowed the optimal conditions for the use of the system to be established and possible sources of contamination to be avoided between adjacent capillaries with unmatched flow speeds. Successive and multiple changes in external solutions could be easily synchronized with voltage-clamp depolarizations to examine the time course of the effect of drugs on voltage-operated ion channels. An example of this application is given with rapid applications of the dihydropyridine agonist (-)BayK 8644 to the L-type Ca²⁺ channel current in frog ventricular myocytes.     

The relation between the current underlying pacemaker activity and beta-adrenoceptors in cardiac Purkinje fibres: A study using adrenaline,procaine, atenolol and penbutolol

Summary The pacemaker current — i _K2 — in cardiac Purkinje fibres was analysed using the voltage clamp technique described by Deck et al. (1964). (–)-Adrenaline (5.5 · 10^–6 M) causes the wellknown shift of the Hodkin-Huxley kinetics in the depolarizing direction. Procaine (7.3·10^–4 M) does not cause any further shift of s in the presence of adrenaline. Atenolol (3.8·10^–5 M) causes a backshift of the kinetics in the negative direction in the presence of adrenaline and procaine. The instantaneous current-voltage relationship ( ) is altered neither with adrenaline, nor with procaine or atenolol. The results exclude the possibility that the local anaesthetic side effect of many beta-adrenoceptor blocking agents may be involved in the backshift of the s-kinetics. The voltage dependence of the reciprocals of the time constants is shifted in a similar way as s by the sympathomimetic or blocking drugs. Following the application of (–)-adrenaline (5.5·10^–6 M) the (–)-isomere of penbutolol (1.7 and 3.5·10^–6 M) is about equally effective in shifting the kinetics back as the (+)-isomere (3.5·10^–5 M). In the presence of (–)-adrenaline, the (+)- and (–)-forms of penubutolol cause virtually no change of the instantaneous current-voltage relationship, . Thus, (–)-adrenaline and (+)- and (–)-penbutolol are aiming for the s-kinetics whose voltage dependence is controlled by the electric field near the i _K2-channel of the membrane and do not influence the number of the i _K2-channels. These findings suggest that the sympathomimetic or blocking agents influence the s-kinetics of the pacemaker current i _K2 by altering the electric field; the fully activated current-voltage relationship which is proportional to the number of the open i _K2-channels is not subject to any appreciable modification. The results conclusively show that the kinetics of the pacemaker current can be controlled by beta-adrenoceptors.     

Enhancement of persistent Na+ current by sea anemone toxin (ATX II) exerts dual action on hippocampal excitability

We used anemone toxin II (ATX II) to study how a selective enhancement of persistent Na+ current (INaP) would affect the excitability of CA1 pyramidal neurons in the hippocampal slice. In whole-cell recordings from CA1 cell somata, local application of ATX II (10 microM) into the stratum pyramidale invariably depolarized the neurons and produced sustained burst discharges with depolarizing plateau potentials of variable amplitude and length. However, the strong excitatory action of ATX II, observed on the single cell level, was not mirrored in field potential recordings from the same hippocampal subfield. The amplitude of the electrically evoked population spike declined, reflecting the decreased availability of fast Na+ channels, and the intracellulary recorded burst discharges were not detected by the field electrode. The lacking synchronization of cellular bursting activity was seen during both local and bath application of ATX II, suggesting that the toxin, in addition to promoting burst discharges of individual neurons, simultaneously dampens network excitability. In fact, ATX II reduced afferent fibre volleys (reflecting axonal excitability) and field excitatory postsynaptic potentials (EPSPs) in a similar fashion. As the expression of different Na+ channel subtypes appears to be compartmentalized within hippocampal neurons, we propose that point mutations leading to pathologically enhanced INaP might exert quite opposite effects, depending on the type and location of the Na+ channel affected. Whereas alterations of somatodendritic Na+ channels would give rise to bursting activity, alterations of axonal Na+ channels would primarily decrease network excitability.     

Cloning, distribution and functional analysis of the type III sodium channel from human brain

The type III voltage-gated sodium channel was cloned from human brain. The full-length cDNA has 89% identity with rat type III, and the predicted protein (1951 amino acids) has 55 differences. The expression pattern of human type III mRNA was determined in adult brain tissue and, in contrast to rat, was detected in many regions, including caudate nucleus, cerebellum, hippocampus and frontal lobe. The human type III channel was stably expressed in Chinese hamster ovary (CHO) cells and its biophysical properties compared to the human type II channel using identical conditions. The voltage dependence and kinetics of activation were found to be similar to that of type II. The kinetics of inactivation of the two human subtypes were also similar. However, type III channels inactivated at more hyperpolarized potentials and were slower to recover from inactivation than type II. When expressed in human embryonic kidney (HEK293T) cells, type III channels produced currents with a prominent persistent component, which were similar to those reported for rat type II [Ma et al. (1997) Neuron, 19, 443-452]. However, unlike type II, this was prominent even in the absence of coexpressed G-proteins, suggesting type III may adopt this gating mode more readily. The distinct properties of the channel, together with its wide distribution in adult brain, suggest that in humans, type III may have important physiological roles under normal, and perhaps also pathological conditions.