Autoantibodies to acetylcholine receptor in myasthenia gravis: light chains.

We studied the light chain type of autoantibodies to acetylcholine receptor (AChR) by affinity chromatography with monoclonal anti-kappa and anti-lambda antibodies. The autoantibodies in four of eight myasthenic patients were of a single light chain type; the others comprised both types. In Graves' disease and cold-reactive hemolytic anemia, the pathogenic autoantibodies are confined to a single light chain type in individual patients, and in other diseases, doubtfully pathogenic autoantibodies are invariably mixtures of both light chain types. AChR antibodies may comprise both pathogenic and nonpathogenic types of autoantibody.

Loss of antigenic properties of acetylcholine receptors in rat skeletal muscle after birth.

Antibodies in a myasthenic serum were used to follow the changes in antigenic properties of acetylcholine receptors in rat skeletal muscle during development. In binding assays at saturating concentrations of antigen or antibody, the antibodies reacted with extrajunctional receptors of fetal and denervated adult muscle but showed little binding to junctional receptors of adult rat muscle. They did, however, bind to junctional receptors of adult chicken muscle which, unlike rat receptors, do not appear to undergo a change in their channel properties during development. Binding studies with acetylcholine receptors of developing rat muscle carried out at saturating concentrations of antibody showed that the loss of antigenic determinant(s) begins at 1-2 days after birth.

Axonal transport and distribution of cyclophilin A in chicken neurones.

In the course of pulse-label studies on the axonal transport of the small, basic, actin-binding proteins--actin depolymerizing factor, cofilin and profilin--in chicken motor neurones, we observed a heavily labelled protein of M(r) 18 kDa and pI 8.2 on fluorographs of two-dimensional polyacrylamide gels. On the basis of its M(r), pI and amino acid composition, we tentatively identified it by database searching as cyclophilin A and subsequently confirmed its identity by immunostaining. Like actin and its associated proteins, cyclophilin A was transported in slow component b of axonal transport, but unlike these proteins, cyclophilin A did not copurify with actin on DNase I. It was not found amongst labelled proteins transported by fast axonal transported by fast axonal transport. Immunostaining of chicken dorsal root ganglion cells revealed that it accumulated in neurites at points of branching, varicosities and growth cones. Our results raise the possibility that cyclophilin A is important in maintaining the native folding of actin and associated proteins during transit in axons and assembly in growth cones.

Localization of immunoreactive angiotensin II in the hippocampus and striatum of rat brain.

Angiotensin II (ANG II) was estimated by radioimmunoassay in extracts of rat brain. In extracts of whole brain the mean content was 108 +/- 16 fmol/g and estimates of ANG II in kidney and plasma were similar to previous reports. A regional distribution of ANG II was determined. Hippocampus had the highest concentration and cortex the lowest. The concentrations relative to cortex were: hippocampus, 8; striatum, 5; cerebellum, 4; hypothalamus:thalamus:septum:midbrain (HTSM), 3; and medulla, 3.

Distribution of angiotensin II receptors in rat brain.

Angiotensin II binding activity of rat brain particles was examined using [125I]-angiotensin II (0.1-0.3 nM) in the presence and absence of excess unlabelled angiotensin II. Certain features of the binding suggested that physiological receptors were involved. The binding activity was temperature dependent and was increased 3-fold by the addition of 0.5 M EDTA. The binding appeared specific as judged by inhibition with angiotensin II agonists and antagonists. The "specific" binding was saturable, two-thirds reversible and occurred with high affinity. The equilibrium dissociation constant (Kd) of the "specific" binding was 0.9 nM. Subcellular fractionation studies indicated that over 90% of the binding was associated with particulate matter and was concentrated in the crude microsomal fraction. Binding was localized to the midbrain, thalamus, septum, hypothalamus and medulla; Very low levels of binding were found in the cortex, hippocampus and striatum; The lateral septum had the highest binding activity of all the tissues examined. Subdivision of the medulla showed that the highest binding activity was associated with the area postrema and medullary regions ventral to this organ.

Effects of electrical stimulation and tetrodotoxin paralysis on antigenic properties of acetylcholine receptors in rat skeletal muscle.

To examine the role of muscle activity in the expression of fetal- and adult-type acetylcholine receptors (AChRs), we studied the effects of muscle stimulation in cell culture and of tetrodotoxin (TTX)-induced paralysis and denervation in adult rat muscles. The AChR content of these muscles was determined using [125I]alpha-bungarotoxin and the proportion of fetal-type receptors was estimated using a radioimmunoprecipitation assay with a myasthenic serum that was highly specific for fetal-type receptors. We found that both stimulated, aneural muscle cells in vitro and inactive muscles in vivo produced predominantly fetal-type AChRs. However the TTX-paralysed muscles had a lower proportion of fetal-type receptors than the denervated muscles. We conclude that neither muscle activity nor innervation alone, but a combination of both, is required for full regulation of AChR antigenicity.

Transport complexes associated with slow axonal flow.

Cytoskeletal proteins--neurofilament polypeptides, tubulin and actin--are transported along axons by slow transport. How or in what form they are transported is not known. One hypothesis is that they are assembled into the cytoskeleton at the cell body and transported as intact polymers down the axon. However, recent radiolabeling and photobleaching studies have shown that tubulin and actin exist in both a mobile phase and a stationary phase in the axon. Consequently, it is more likely that cytoskeletal proteins move along the axon in some form of transport complex and are assembled into a cytoskeleton which is stationary. In this overview we discuss these topics and consider the evidence for the existence of transport complexes associated with slow axonal flow. Such evidence includes the slow transport of particulate complexes containing tubulin and neurofilament polypeptides along reconstituted microtubules in vitro, and the coordinate slow transport of actin with actin-binding proteins in vivo.

Flow of protein and ribonucleic acid in peripheral nerve.

Radioactively labelled leucine and orotic acid were injected into the ventral horn of the lumbar region of the spinal cord. The outflow of labelled products into the sciatic nerve was studied. Leucine is rapidly incorporated into protein and to a lesser extent into lipid. The labelled protein is transported down the nerve, apparently by axoplasmic flow. This labelled protein was present in all sub-cellular fractions of nerve although the soluble fraction had the highest specific activity. Orotic acid is converted to a number of nucleotide derivatives and RNA. Both the nucleotides and RNA move down the sciatic nerve, but the pattern of movement is diffuse whereas protein appears to move as a discrete band. The results suggest that two systems may be operating, one in which part of the RNA is transported by axoplasmic flow along the nerve and the other involving a synthesis of RNA in the nerve from precursors which flow down the nerve. This labelled RNA was membrane bound and on sedimentation analysis, proved to be predominantly of ribosomal type.

A slow-release technique for inducing prolonged paralysis by tetrodotoxin.

A technique is described for the slow-release of tetrodotoxin in peripheral nerves using a constriction capillary. The capillary, which was implanted under the epineurium of the sciatic nerve, released tetrodotoxin from a 25 micrometer pore. Nerve block was complete after approximately 20 min and lasted 6--9 days. Replacement of the capillary enabled paralysis of the rat hindlimb to be maintained for periods of 21 days and longer. Studies with radioactively labelled compounds demonstrated that the efflux rate from the capillary was dependent on the size of the pore and the relative molecular mass of the compound.

Dissociation between nerve-muscle transmission and nerve trophic effects on rat diaphragm using type D botulinum toxin.

Small doses of botulinum toxin can produce partial blockage of transmitter release at the nerve--muscle junction. 2. Subthreshold e.p.p.s, 3--10 days after poisoning, show a distribution of amplitudes that is fitted by Poisson statistics. Successive e.p.p.s. in a short train show a marked facilitation. 3. Two weeks or more after poisoning with a dose of toxin that paralyses the whole muscle, when nerve--muscle transmission is in course of recovery, subthreshold e.p.p.s have an amplitude distribution that is fitted by binomial statistics. This property of transmission is similar to those described in newly formed nerve--muscle junctions, during embryogenesis or regeneration. 4. Muscle fibres with subthreshold transmission in the 5--10 day group of muscles were all supersensitive to ACh, as were a number of fibres in which nerve stimulation still produced an action potential. 5. Two weeks or more after poisoning, muscle fibres with subthreshold transmission had lost their extrajunctional ACh-sensitivity, as had many fibres with m.e.p.p.s of roughly normal frequency but no response to nerve stimulation. 6. In diaphragm muscles poisoned with botulinum toxin between 1 and 4 days previously, the rate of fast axonal transport of radioactively labelled proteins down the phrenic nerve is not greatly affected, but the amount of materials carried is reduced to about one quarter of normal. These labelled proteins accumulate in the intramuscular portion of the phrenic nerve, in or near the nerve terminals, to a much greater extent than in controls, showing that the normal release of some of these materials has been prevented by the toxin. 7. It is concluded that the blockage of the trophic effects of nerves by botulinum toxin is due to a blockage of release of trophic factors other than ACh. 8. The muscle nerve cannot maintain a muscle in its normal state simply by activation of contraction, and a regenerating nerve terminal can restore a muscle towards its normal state before it can release enough ACh to produce muscle contraction.

The trophic influence of tetrodotoxin-inactive nerves on normal and reinnervated rat skeletal muscles.

1. Nerve impulses in the rat sciatic nerve were blocked for long periods by tetrodotoxin (TTX) released from capillary implants. The TTX capillaries did not block axonal transport, nor did they cause any sign of nerve degeneration. 2. A comparison of the effects of TTX paralysis and denervation was made on both extensor digitorium longus (e.d.l.) and soleus muscles over 21 days, a time when the products of nerve degeneration were unlikely to contribute to the changes associated with denervation. The resting membrane potential of TTX-paralysed muscles was significantly different (P less than 0.005) from that of the denervated muscles at all periods and at 21 days the decrease that can be attributed to inactivity was 61% (e.d.l.) and 49% (soleus) of that which follows denervation. This disparity was even more pronounced for the ACh receptor density where the increase in receptors due to inactivity was only 34% (e.d.l.) and 21% (soleus) of that due to denervation. 3. A similar comparison was made on muscles which had been reinnervated by TTX-inactive nerves. These muscles were found to have a significantly higher resting membrane potential and lower ACh receptor density than the denervated muscles (P less than 0.05). 4. The experiments on reinnervated muscles preclude the possibility that nerve degeneration products are solely responsible for the difference between the TTX-paralysed and denervated muscles and suggest that the difference can be attributed to the trophic influence of the nerve. 5. An observed increase in the m.e.p.p. frequency of the TTX-paralysed muscles indicated that nerve action potentials play a role in regulating the spontaneous release from nerve terminals.

An autoradiographic study of the flow of protein and RNA along peripheral nerve.

Both chicken spinal cord and the posterior chamber of the rat eye have been injected with either [3H]leucine or [3H]orotic acid. The movement of newly synthesized protein and RNA down the sciatic and optic nerves was followed by means of autoradiography. Protein moved down both nerves by axoplasmic flow, the protein being confined to intra-axonal spaces. RNA appeared to move at about the same rate as protein, but much of the newly formed RNA appeared in structures outside the axons, particularly in Schwann and glial cells. There was, however a considerable proportion of the RNA inside the axons. This RNA may be involved in the direction of protein synthesis at sites along the axonal processes.

Evidence for the role of non-quantal acetylcholine in the maintenance of the membrane potential of rat skeletal muscle.

1. Resting membrane potentials of rat diaphragm muscles cultured in Trowell T8 medium were measured in vitro. After 3 hr in culture the resting membrane potential of muscle fibres within 2.5 mm of nerve section (;near') was -68.3 +/- 0.4 mV (nineteen preparations). This was significantly lower (P < 0.001) than the resting potential (-74.0 +/- 0.4 mV) measured in muscle fibres 8-10 mm from the site of nerve section (;far') in the same preparations. A difference between the ;near' and the ;far' fibres was maintained in muscles cultured for 6 and 12 hr. Miniature end-plate potentials were present in both ;near' and ;far' fibres cultured for 3 and 6 hr and ceased after 12-15 hr.2. The presence of carbamylcholine (10(-7) or 10(-8) M) maintained the resting membrane potential of ;near' fibres close to that of ;far' fibres at 3, 6 and 12 hr. For example, at 3 hr in the presence of 10(-8) M-carbamylcholine the mean resting potential was 75.6 +/- 0.5 mV in ;near' fibres and 76.1 +/- 0.4 mV in ;far' fibres (four preparations). A similar effect was produced in preparations exposed to anticholinesterases: diisopropylphosphorofluoridate (DFP) (10(-7) M), neostigmine (10(-7) M) or physostigmine (10(-5) M).3. Agents that blocked acetylcholine receptors had the reverse effect. In the presence of alpha-bungarotoxin (1 mug/ml.) or d-tubocurarine (10(-5) M) the resting membrane potential of ;far' fibres was reduced to the level of ;near' fibres over the 24 hr period of observation. For example, at 3 hr in the presence of alpha-bungarotoxin the mean resting potential was 67.2 +/- 0.5 mV in ;near' fibres and 68.5 +/- 0.6 mV in ;far' fibres (six preparations). The effect of d-tubocurarine was reversible.4. When muscles were cultured in Ca(2+)-free medium containing 1 mM-EGTA and 10 mM-Mg(2+), there was no difference in membrane potential between ;near' and ;far' fibres and physostigmine (10(-5) M) was ineffective in raising the membrane potential of ;near' fibres.5. It is suggested that non-quantal acetylcholine released from nerve terminals maintains the membrane potential of muscle fibres through a Ca(2+)-dependent mechanism.