The interaction between the activator agents batrachotoxin and veratridine and the gating processes of neuronal sodium channels

The depolarization of frog sciatic nerves by the Na channel-activating toxins, batrachotoxin and veratridine, was studied using the sucrose-gap technique. To study the interaction between the activators and the gating processes of Na channels, we measured the depolarizations of unstimulated nerves, of nerves during repetitive stimulation, and of nerves whose Na channel inactivation process had been pharmacologically modified. Stimulation enhanced the rates of depolarization by the activators but did not effect the steady state depolarization values. Of the three inhibitors of Na channel inactivation that were tested (Leiurus alpha-scorpion toxin, chloramine T, and Ni2+), only Leiurus toxin enhanced the potencies of the activators. Neither chloramine T nor Ni2+ had any effect on the steady state level of depolarization produced by either activator. Both chloramine T and Ni2+, however, enhanced the rate of batrachotoxin action, although neither affected the rate of veratridine action. Leiurus toxin also potentiated the effects of the activators in chloramine T-treated nerves. We tested the interaction between the Na channel activators and a class of agents, local anesthetics, that stabilize a non-conducting state of the Na channel. The presence of lidocaine inhibited the depolarization produced by addition of either activator, although the addition of lidocaine subsequent to the development of batrachotoxin-induced depolarization produced repolarization very weakly and slowly. We also found that the lidocaine homologue, RAC 109I, was about 3 times as potent as its stereoisomer, RAC 109II, in its ability both to reduce the compound action potential amplitude and to inhibit the veratridine-induced depolarization.     

Perfluorochemicals as US contrast agents for tumor imaging and hepatosplenography: preliminary clinical results

In animals, perfluorochemicals (PFCs) are effective ultrasound (US) contrast agents that produce hepatic, splenic, and tumor enhancement. The use of Fluosol-DA 20%, an emulsion of perfluorodecalin and perfluorotripropylamine, was studied in nine non-critically ill patients with cancer who had liver lesions. US studies without Fluosol were compared with studies obtained 24, 48, and 72 hours after Fluosol infusion. Vital signs and extensive laboratory analyses are performed before and after Fluosol infusion. Liver metastases from colonic, pancreatic, and gastric carcinoma exhibited rim or diffuse enhancement after a Fluosol dose of 1.6 g/kg or greater. Fluosol produced echogenic enhancement of the liver and spleen relative to kidney at a dose of 2.4 g/kg, allowing the detection of nonenhancing lesions. In addition, Fluosol at a dose of 1.6 g/kg or greater allowed detection of lesions not seen before contrast medium was administered in three of the seven patients studied. There was a mild increase in the level of serum glutamic oxaloacetic transaminase in two patients, one given 2.4 and the other 3.2 g/kg of Fluosol. Mild and transient allergic reactions without change in vital signs were experienced by two patients.     

The nitric oxide pathway in pre-eclampsia: pathophysiological implications

Pre-eclampsia, one of the most significant health problems inhuman pregnancy, complicates 6-7% of all gestations and is theleading cause of fetal growth retardation, infant morbidityand mortality, premature birth and maternal death. Recent researchimplicates free radicals in the pathophysiology of pre-eclampsia.This review covers the biochemistry of nitric oxide (NO) andpossible interactions with other free radicals. Studies in therat show that pregnancy is associated with enhanced productionand responsiveness to NO in both reproductive tissues and bloodvessels. Rats infused with N^G-nitro-L-arginine methyl ester(L-NAME, a NO synthase inhibitor) have been used as an animalmodel of pre-eclampsia, and the effects of steroid hormoneson blood pressure in this model have been tested. Results suggestthat pre-eclampsia may be a state of NO deficiency. However,in humans there seem to be contradictions regarding the involvementof NO in maternal adaptation to pregnancy. It is suggested thatNO may be one of several systems that act in concert to maintaina symbiotic relationship between mother and fetus. However,the input of each system may be genetically determined.     

Surgical sperm retrieval and intracytoplasmic sperm injection as treatment of obstructive azoospermia

Male genital tract obstructions may result from infections, previous inguinal and scrotal surgery (vasectomy) and congenital bilateral absence of the vas deferens (CBAVD). Microsurgery can sometimes be successful in treating the obstruction. In other cases and in cases of failed surgical intervention, the patient can be treated by microsurgical or percutaneous epididymal sperm aspiration (MESA, PESA) or testicular sperm extraction (TESE) and intracytoplasmic sperm injection (ICSI). We present the results of 39 ICSI procedures for obstructive azoospermia in 24 couples. The aetiology of the obstruction was failed microsurgery in 11 patients, CBAVD in nine and genital infections in four. Sperm retrieval was accomplished via MESA in four cases, PESA in 18 cases and via TESE in 11 cases. TESE was only applied when PESA failed to produce enough spermatozoa for simultaneous ICSI. In six patients, the ICSI procedure was performed with cryopreserved spermatozoa after an initial PESA procedure. Fertilization occurred in 47% of the metaphase II oocytes; embryo transfer was performed in 92% of procedures and resulted in a clinical pregnancy in 13/39 procedures. Ongoing pregnancy was achieved in 10/39 procedures. One pregnancy was terminated early after prenatal investigation showed a cytogenetic abnormality (47,XX+18, Edwards syndrome). The other nine pregnancies resulted in the live birth of 10 children, without any congenital abnormalities. Epididymal and testicular retrieved spermatozoa were successfully used for ICSI to treat obstructive azoospermia, and resulted in an ongoing pregnancy in 10 of 24 couples (41.6%) after 39 ICSI procedures, a success rate of 25.6% per treatment cycle and of 27.7% per embryo transfer.      

Limited nerve impulse blockade by "leashed" local anesthetics

To measure the depth of the local anesthetic binding site within the neuronal membrane, biotin-containing polyethylene glycols having zero, three, and six ethylene glycol subunits were added to the p-amino termini of tetracaine and procaine, thereby interposing a pharmacologically inert "spacer" molecule between the local anesthetic and the biotin moiety. These biotinyl-local anesthetic derivatives produced "tonic" inhibition of the compound action potential of split, desheathed frog sciatic nerves in a concentration-dependent, reversible manner. However, no inhibition of the action potential occurred when sufficient avidin, a 66,000-MW protein that binds four biotins, was present to bind and anchor the biotin-containing end of each derivative outside the plasma membrane. Increasing the "leashed" anesthetic derivative's concentration to 4 times that which reduced impulse height by 50% in the absence of avidin still produced no detectable block when equimolar avidin was present. Apparently, the "spacer" in the derivative compound was too short to permit the avidin-complexed anesthetic to reach its site of action on the sodium channel. In a similar fashion, the local anesthetic derivatives produced "use-dependent" block when drug-treated nerves were stimulated at 40 Hz in the absence of equimolar avidin, but failed to produce "use-dependent" block when equimolar avidin was present. In common with others, we assume that tertiary amine local anesthetics may reach their binding site via hydrophobic (transmembrane) pathways without necessarily entering the cytoplasm. Thus, since our longest local anesthetic derivative, that containing six ethylene glycol subunits, placed the local anesthetic group a maximum of 15-18 A from the surface of the avidin moiety, we conclude that the local anesthetic binding site for block of sodium channels of amphibian nerve must be greater than or equal to 15 A from the outer surface of the plasma membrane.     

Use-dependent block of atrial sodium current by ethylisopropylamiloride.

Ethylisopropylamiloride (EIPA) is a potent inhibitor of Na(+)-H+ exchange in many tissues and is frequently used to study cellular regulation of pH, but the electrophysiologic effects of EIPA on cardiac cells have not been studied previously. The use-dependent effects of EIPA on the sodium current (INa) of cultured embryonic chick atrial myocytes were investigated using standard whole-cell patch-clamp techniques. With 150-ms depolarizations from -140 to 0 mV, applied at 1-3 Hz in the presence of 10 microM EIPA, a decrement in INa was observed. This use-dependent reduction equaled 31 +/- 6% of control INa at steady state during 1-Hz stimulation. Inhibition increased with stimulation rate and with depolarization of the holding potential to -100 mV, but there was no effect of pulse duration on the EIPA-induced inhibition over the range of 20-500 ms. Moreover, repetitive depolarizations to potentials that did not activate macroscopic current but that did yield pronounced channel inactivation did not result in a decrement in INa. The effect of EIPA increased over the concentration range of 1-30 microM so that with 3-Hz stimuli steady-state inhibition increased from 3 +/- 1 to 85 +/- 5%. Amiloride, which slows repolarization of the cardiac action potential, was at least 100-fold less potent than EIPA in reducing INa. We conclude that EIPA is an "open-channel" blocker of the cardiac sodium current at concentrations comparable to those of many type I antiarrhythmic agents.     

Stereoselective inhibition of neuronal sodium channels by local anesthetics. Evidence for two sites of action?

The objective of this study was to determine if the "tonic," resting inhibition of Na+ channels by local anesthetics results from binding at a site different from that for "phasic," use-dependent inhibition. Stereoselective actions of four local anesthetics were examined in isolated frog peripheral nerve and single Na+ channels. Using the sucrose-gap method on desheathed nerves, four actions of local anesthetics were assayed: 1) tonic depression of compound action potentials at low stimulation frequency (one per minute); 2) phasic depression of the compound action potential during trains of stimulation at 5, 10, and 20 Hz; 3) competitive antagonism of the reversible Na+ channel activator veratridine assayed through the depolarization of the compound resting membrane potential; and 4) depression of the depolarization of the compound resting membrane potential initially induced by the irreversible channel activator batrachotoxin. For assays 1, 2, and 3, all local anesthetics showed a stereoselectivity, where rectus, or (+), enantiomers were more potent than sinister, or (-), enantiomers. In contrast, for the noncompetitive antagonism of veratridine's action and the depression of batrachotoxin-induced depolarization, also a noncompetitive interaction between anesthetic and activator, the (-) enantiomer was more potent than the corresponding (+) enantiomer. Blockade of single Na+ channels activated by batrachotoxin in planar lipid bilayers was also stereoselective for the (-) enantiomer. These findings, along with previously reported voltage-clamp results, can be applied to infer properties of a local anesthetic binding site in activator-free channels. Local anesthetic molecules with more sharply angled shapes have stronger stereoselectivities than less angled, more planar drugs. The inversion of the stereopotency induced by the activators can be explained by either of two mechanisms. There may be two binding sites for local anesthetics, one of high and one of low affinity and of opposite stereoselectivity; activators may change the conformation at the high affinity site, reducing its local anesthetic affinity below that of the usual low affinity site and thereby revealing the pharmacology of the weaker site. Alternatively, only a single binding site may exist and be conformationally altered by activators such that both anesthetic affinity and stereopotency are modified. In activator-free channels, however, a single, high-affinity binding site with a constant stereoselectivity can account for both tonic and phasic inhibition by local anesthetics.