Jordan E. Warnick

Batrachotoxinin-A 20-alpha-benzoate: a new radioactive ligand for voltage sensitive sodium channels.

Batrachotoxinin-A 20-α-benzoate (BTX-B), an analog of the potent depolarizing agent batrachotoxin (BTX), was prepared by selective esterification of naturally occurring batrachotoxinin-A with benzoic acid. BTX-B depolarizes rat phrenic nerve-diaphragm preparations with a time course and concentration dependence virtually indistinguishable from that of BTX. A specific, saturable component of equilibrium binding of [3H]BTX-B to mouse cerebral cortex homogenates was measured, described by an equilibrium dissociation constant of 0.7 µM and a maximum number of binding sites of 90 pmol per gram of tissue (wet weight). Specific binding is inhibited by BTX and other BTX analogs, veratridine, and grayanotoxin but is unaffected by tetrodotoxin and cevine. Under conditions of this assay, neither crude Leiurus quinquestriatus scorpion venom nor purified sea anemone toxin have any effect on specific binding. The data support the conclusion that BTX-B interacts with a recognition site associated with voltage sensitive sodium channels which is identical to the recognition site for BTX.

Gender-specific action of thyrotropin-releasing hormone in the mammalian spinal cord.

Thyrotropin‐releasing hormone (TRH) potentiated the monosynaptic reflex in isolated spinal cords obtained from 7‐ to 9‐day‐old rats. A concentration‐dependent increase in the monosynaptic reflex was observed in spinal cords obtained from male but not from female or castrated male rats. In contrast, the magnitude of potentiation in cords from ovariectomized control female rats and in ovariectomized female rats treated with testosterone approached that seen in intact males. The results provide evidence that gender plays a prominent role in the variability of response both of humans with amyotrophic lateral sclerosis and of animal tissues to TRH. Furthermore, exposure to androgen during the neonatal period may determine the responsiveness of motoneurons to TRH. Thus the use of TRH in the treatment of amyotrophic lateral sclerosis may be more effective in males than in females.— Deshpande, S. B.; Pilotte, N. S.; Warnick, J. E. Gender‐specific action of thyrotropin‐releasing hormone in the mammalian spinal cord. FASEB J. 1: 478‐482; 1987.

The roles of disuse and loss of neurotrophic function in denervation atrophy of skeletal muscle

Abstract Denervated muscle fibers are characterized by a lowered resting membrane potential (RMP), increased extrajunctional acetylcholine (ACh) sensitivity, and decreased junctional acetylcholinesterase (AChE) activity. Whether these changes in denervated muscle result from cessation of contractile activity, from interruption of axonal transport, or from both is not known. Experiments were therefore designed to analyze whether or not the denervation changes could be ascribed solely to the loss of contractile activity. In one experiment, the hemidiaphragm of the rat was rendered quiescent for 1 to 3 weeks either by spinal hemisection at C2 (disuse) or by unilateral phrenicotomy (denervation). After denervation there was a spread of ACh sensitivity to extrajunctional regions, a decline in RMP, and a reduction in 16 S AChE (a measure of junctional AChE activity). Comparable changes did not occur after spinal hemisection, and we therefore conclude that inactivity alone does not induce these changes in denervated muscle. In another experiment, rats were chronically paralyzed by repeated administration of d-tubocurarine. During this time the extensor digitorum longus muscle of one hind limb was denervated. After 6 h of immobilization by d-tubocurarine, the RMP of denervated muscle fibers was significantly reduced whereas that of the contralateral innervated muscle fibers was unchanged. This result supports the previous interpretation, viz., that the change in RMP of denervated muscle fibers cannot be attributed solely to muscle inactivity. Experiments by others have shown that chronic disuse causes changes that are qualitatively but not quantitatively equivalent to those of denervation. Those observations, together with the present results, enable us to conclude that inactivity does not initiate the changes in extrajunctional ACh sensitivity, RMP, and junctional AChE activity seen in denervated muscle and that these properties of muscle are normally regulated by axonally transported neurotrophic influences.

The demonstration of neurotrophic function by application of colchicine or vinblastine to the peripheral nerve.

Abstract Application of Silastic cuffs containing either colchicine or vinblastine to the sciatic nerves of rats produced electrophysiological signs of denervation (i.e., membrane depolarization, extrajunctional acetylcholine sensitivity, and tetrodotoxin-resistant action potentials) without altering electrical activity along the nerve, spontaneous or evoked transmitter release, or the contractile activity of the muscles examined. Fibrillation potentials were conspicuously absent in all muscles. Eighty percent of all colchicine-treated rats did not show paresis of the hind limb, and only these animals were used in this study. In these rats after 5 to 7 days, 80% of the surface fibers in extensor muscles which were depolarized by 15 mV and which had extrajunctional acetylcholine sensitivity (mean of about 145 mV/nC) contracted in response to indirect stimulation. Prior hyperpolarization of the remaining muscle cells allowed action potential generation to occur. Similar results were obtained in the soleus muscle. The acetylcholine sensitivity observed in colchicine-treated and vinblastine-treated preparations exceeded that seen in tetrodotoxin-immobilized extensor and soleus muscles by about 10-fold and in pin-immobilized soleus muscles by 17-fold, but was about 20% of that in chronically denervated extensor and soleus muscles. The appearance of denervation-like responses in muscles whose nerves are exposed to colchicine or vinblastine is therefore not due to disuse or denervation but to an interruption of axonal flow and trophic influence.

Macromolecular Sites for Specific Neurotoxins and Drugs on Chemosensitive Synapses and Electrical Excitation in Biological Membranes

The present review deals with the molecular mechanisms and elementary phenomena underlying the activation of the voltage- and chemo-sensitive membrane macromolecules: sodium- and potassium-ion channels and nicotinic ACh receptors and their associated ion channel. To achieve an understanding of their various kinetics and conformational states, a number of novel alkaloids, BTX, HTXs, gephyrotoxins, and certain psychotomimetic drugs such as phencyclidine, and many other pharmacologically active agents have been used. Biochemical assays and various electrophysiological techniques have been used in a number of biological preparations--e.g., Torpedo membranes, brain synaptosomes, amphibian and mammalian neuromuscular preparations--to describe the action of such agents. The availability of BTX and scorpion toxins together with aconitine and veratridine as activators and TTX and STX as antagonists of the voltage-sensitive sodium channels, made possible the identification and the physiological and pharmacological characterization of these channels. These studies provided the basis for understanding the mechanisms underlying electrical excitability and culminated, more recently, in the purification and reconstitution of sodium channels from rat brain and in the successful cloning of these channels with the elucidation of their primary structure. We now know that the sodium channel has a molecular mass of 316,000 daltons, consists of five subunits, and has multiple sites for various ligands. In contrast to sodium channels, various classes of potassium channels (inward and outward rectifier potassium channels and Ca(2+)-activated potassium channels) have been described. Unlike the sodium channels, there are no known specific activators for potassium channels. However, a number of potassium channel blockers such as 4-aminopyridine, HTX, histamine, and norepinephrine have been identified which complement the varying types of potassium channels in different neurons. One class of potassium channel blockers with profound medical and social implications comprises PCP and its analogues. The blockade of the potassium-induced 86Rb+ efflux from brain cells, the resulting prolongation of muscle and nerve action potentials, and the increase in transmitter release observed with PCP and some analogues are all highly suggestive of a role for the potassium channel in the behavioral effects of these drugs and its potential involvement in schizophrenia. A number of toxic principles of both plant and animal origin played a significant role in the development of our knowledge about the nAChR.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunosorbent chromatography of sea nettle (Chrysaora quinquecirrha) venom and characterization of toxins

A lethal toxic fraction from nematocysts of the sea nettle (Chrysaora quinquecirrha) fishing tentacle was partially purified by immunochromatography using an immobilized monoclonal antibody column. Elution from the immunosorbent was accomplished under mild conditions which conserved the biological activity of the toxin. The isolated fraction, which contained two purified protein bands with molecular weights of 100,000 and 190,000 daltons on SDS polyacrylamide gels, was both cardiotoxic and neurotoxic and exhibited an intravenous lethal activity (LD50) of 0.37 microgram/g in mice.

Effect of vinblastine on neural regulation of metabolism in rat skeletal muscle

Abstract Chronic application of vinblastine, a substance known to disrupt axoplasmic flow, to nerves innervating the fast extensor digitorum longus and slow soleus muscles of the rat, produces electrophysiological signs of denervation (depolarization and extrajunctional acetylcholine sensitivity), but does not alter motor activity. We therefore examined the effects of vinblastine treatment on those metabolites and enzymes that are known to change after denervation of fast and slow skeletal muscle. A silastic cuff containing 0.1% vinblastine was placed around sciatic nerves of adult rats for 5 days. Glucose-6-P decreased 68% in the extensor, but did not change in soleus muscles. Phosphocreatine also decreased slightly, but significantly, in the extensor. Thus, intracellular levels of glucose-6-P and phosphocreatine in the extensor may be controlled, in part, by a factor (s) transported to the muscle by axoplasmic flow. Other metabolites known to change 5 days after denervation, namely glucose, glycogen, lactate, and α-ketoglutarate, were not altered in extensor and soleus muscles innervated by vinblastine-treated nerves. The activities of glucose-6-phosphate dehydrogenase and hexokinase increased in denervated extensor muscles, but not in denervated soleus muscles. Thus, metabolism in extensor muscles may be more readily altered after disruption of neural influences than is metabolism in soleus muscles. In contrast to denervation, exposure of sciatic nerves to vinblastine did not alter enzyme activities. These results provide evidence that certain metabolic processes, as well as membrane properties in skeletal muscle, are influenced by separate and distinct neural factors.

Characterization of the Electrogenic Sodium Channel from Rat Brain Membranes Using Neurotoxin-Dependent 22Na Uptake

The sodium channel was studied in osmotically-sensitive membrane preparations from rat brain and in innervated and chronically denervated rat soleus and extensor digitorum longus muscles. These experiments were undertaken in order to define a set of parameters for sodium channel function at the subcellular level to be used as a measure of retention of channel integrity upon subsequent isolation of the channel. Various neurotoxins and drugs were employed to control the permeability of the brain membranes to 22Na and the sodium-conductance properties of the muscles. Batrachotoxin (ED50 = 0.2 micrometer), veratridine (ED50 = 1 micrometer), or grayanotoxin I (ED50 = 30 micrometers) stimulated 22Na uptake in brain membranes is inhibited in an apparently uncompetitive manner by the sodium channel blocking agents tetrodotoxin and saxitoxin in a simple competitive manner by Ca2+ and in a partial or allosteric competitive manner by lidocaine and procaine. This 22Na uptake assay, which can be equated to a measure of equilibrium toxin binding, shows dependence on the concentration of the membranes and is sensitive to pH, temperature, ionic strength, and the ionic composition of the media. Parallel biophysical studies on sodium channels in rat muscle show that the properties of the sodium channel are similarly affected by these agents.

Sea nettle (Chrysaora quinquecirrha) toxin on electrogenic and chemosensitive properties of nerve and muscle

Abstract Rat and frog nerves and skeletal muscles and identified neurons of Aplysia californica were exposed in vitro to the toxin of Chrysaora quinquecirrha (sea nettle). All tissues examined were depolarized by the toxin. In skeletal muscle the postsynaptic depolarization had a delayed onset. The effect of the toxin was not accompanied by any alteration of the action potential generating mechanism, delayed rectification, or quantal release of transmitter. The postsynaptic depolarization was followed by a transient increase in miniature endplate potential frequency. Action potential amplitude, overshoot and rate of rise in both rat skeletal muscle and the amplitude and conduction velocity of frog sciatic nerve were reduced by the toxin-induced depolarization but spike generation in rat skeletal muscle was restored by hyperpolarizing the fibers. The depolarizing effects of the toxin were not altered by tetrodotoxin, d-tubocurarine or by changing the external Ca2+ level, although lowering the Ca2+ level delayed the toxin effect on transmitter release. Heating the toxin to 100°C destroyed its action. In Aplysia central neurons, the depolarization was immediate, accompanied by an increase in spontaneous synaptic activity, and an alteration of resting membrane conductance; the toxin effects were reversible. Neither hyperpolarizations induced by histamine or by acetylcholine nor synaptic cholinergic inhibitory potentials were affected by the toxin. The toxin appears to induce a non-specific membrane depolarization although an increase in sodium conductance resistant to tetrodotoxin cannot be ruled out.

Biphasic action of sarin on monosynaptic reflex in the neonatal rat spinal cord in vitro

The action of sarin, an organophosphorus (OP) compound, was examined in vitro for its effects on the spinal monosynaptic reflex (MSR) in neonatal rats. The effects of sarin were biphasic, i.e. facilitation at lower concentrations (2–20 nM) followed by depression of the MSR at concentrations above 30 nM. Facilitation of MSR was maximal (150% of control) at 20 nM sarin. The depression of MSR was maximal (70% of control) at 200 nM sarin, with half maximal inhibition occurring at 90 nM sarin. Atropine (200–500 nM) effectively reversed the depression caused by sarin, while pretreatment with low concentrations of atropine (10 nM) completely blocked the depression otherwise observed with sarin. Benactyzine was also effective in preventing sarin-induced depression, while pirenzepine was less effective. The nicotinic blocking agents tubocurarine and mecamylamine were, however, ineffective in preventing or reversing sarin-induced depression. The facilitation of MSR seen with lower concentrations (2–20 nM) correlated well with the blockade of late phase inhibition (between 30 and 50 ms conditioning-test interval) elicited in spinal cord by stimulating the adjacent dorsal root at various condition-test intervals, which has been shown elsewhere to be sensitive to bicuculline (Deshpande and Warnick 1988). Thus it is speculated that sarin at lower concentrations blocks GABA transmission, producing facilitation, and at higher concentrations activates the muscarinic receptors producing depression of MSR. The beneficial action of pretreatment with antimuscarinic agents may be attributed to the protection of the muscarinic receptors.