Desensitization, as represented by the progressive decline in the electromotive effects of depolarizing agents at the neuromuscular junction, was studied by observing the time course of changes in effective transmembrane resistance during the prolonged application of 0.27 mM carbamylcholine to the postjunctional region of frog skeletal muscle fibers. The effective transmembrane resistance was measured by means of two intracellular microelectrodes implanted in the junctional region of single muscle fibers. When carbamylcholine was applied to the muscle there was an immediate decrease in the effective membrane resistance followed by a slower return toward control values which was identified as the phase of desensitization. When the calcium concentration was increased from 0 to 10 mM there was an approximately sevenfold increase in the rate of desensitization. On the other hand, an increase in the concentration of sodium from 28 to 120 mM caused a slowing of the rate of desensitization. Even in muscles depolarized by potassium sulfate, calcium increased the rate of desensitization while high concentrations of potassium tended to prolong the process. Some mechanisms by which calcium might exert these effects are discussed.

Insulin-like growth factor-I (IGF-I) stimulates the production of extracellular matrix by cartilage cells and this action is mediated through the Type 1 IGF receptor. Expression of the genes for the IGF receptor and for IGF-I was examined in normal and osteoarthritic (OA) human articular cartilage by in situ hybridization. RNA transcripts for Type 1 receptor were detected in all 73 tissue samples and in 80-100% of chondrocytes per section. Signal for the receptor was present in normal and OA cells, and the highest message levels were in the tissues exhibiting advanced pathology. Strong message signals in the ceUs of the more advanced lesionS were also noted for IGF-I, whereas little or no IGF-I mRNA was IntroductionArticular cartilage consists of chondrocytes embedded in an extensive extracellular matrix. These cells synthesize, organize, and regulate the deposition of their surrounding matrix, and in normal mature tissue they actively maintain a stable equilibrium between synthesis and degradation of matrix molecules. In disease states such as osteoarthritis (OA), the stable equilibrium is disrupted, leading eventually to complete loss of cartilage from the joint surface. Reports of altered phenotypic expression in human OA chondrocytes include production of Type 111 and Type X collagen (I), the detection of novel chondroitin sulfate epitopes attached to aggrecan (2), progressive loss of extracellular matrix, and the formation of clonal cell clusters in depleted regions (3).Insulin-like growth factors (IGFs) or somatomedins regulate the growth and differentiation of a variety of tissues and are implicated in their hypertrophy and repair (4). They are bound in vivo to specific binding proteins (IGF BP 1-6) which extend the half-life of the growth factor and modulate its activity (5). Traditionally, IGF-I Supported by the Nuffield Foundation (Oliyr Bird Fund) and by the Arthritis and Rheumatism Council.Correspondence tu James Middleton, PhD, Dept. of General Dermatology, Sandot Forschungsinstitut, Brunnerstr. 59, A-1235 Vienna, Austria. was considered to be mainly produced in the liver and to mediate the actions of growth hormone on target tissues (endocrine function). However, it is now clear that IGFI is synthesized by many cells of mesenchymal origin in different tissues, indicating a local function involving autocrinelparacrine mechanisms. IGFI is a major anabolic growth factor in the regulation of articular cartilage metabolism in vitro (6,7). It also stimulates the growth of epiphyseal cartilage in vivo (8). Rat epiphysial chondrocytes express the IGF-I gene, and locally produced IGFI is implicated in stimulating clonal expansion of these cells (9). In a previous study we showed that chondrocytes in human articular cartilage also express IGFI mRNA. Very low amounts were detected in normal samples, whereas enhanced levels, four-to fivefold higher, were evident in cells from OA cartilage that had formed clusters at fibrillated sites (10). The metabolic and mitogenic action of IGFs are believed to be mediated...

In skeletal muscle, it is now widely accepted that neuromuscular transmission is mediated by acetylcholine. On arrival of a nerve impulse at the motor neuron terminals, acetylcholine is released and it diffuses across the junctional cleft to reach and react with the postjunctional membrane (PJM). As a result of this interaction, the permeability of the PJM to Na and K ions is increased.'At the molecular level, it is commonly visualized that there are receptors sites on the PJM and that the permeability increase is brought about when these sites combine with acetylcholine (ACh). In our laboratory we have been interested in learning more about the nature of the receptive sites and the molecular mechanisms which control the ionic permeability of the PJM. In this paper, we wish to summarize some of our own work and that of other investigators whose publications bear on this subject. Location of Receptor SitesAn important point concerns the location of the receptor sites in skeletal muscle. The work of del Castillo an$. KatzZ has shown that these sites are found on the outer but not on the inner surface of the PJM. In addition, they can be found, more sparsely distributed, on the outer surface of the extrajunctional membrane of the muscle fiber. Extrajunctional receptors are easy to detect in chronically denervated muscle where the entire muscle fiber membrane becomes sensitive to acetylcholine. If we extend this thinking further, it is reasonable to speculate that chemosensitive receptor sites may also be located in the walls of the transverse tubules of each sarcomere because these tubules have been shown to be an inward extension of the plasma membrane. However, at the moment there is no direct evidence which indicates what kind of chemosensitivity exists in the transverse tubules. Activation of Receptor SitesSince the nature of membrane receptor sites is unknown we cannot directly measure their rate of reaction with an activating ion such as ACh. However, we can estimate the speed of onset of this interaction by measuring the changes in the PJM potential produced by the close range application of ACh. Several years ago there was originated in our laboratory an electrophoretic technique for applying ACh and similar ions to discrete sites at high speed.3 A diagrammatic representation of this technique is shown in FIGURE 1. With this arrangement, ACh can be rapidly applied to the PJM thereby causing it to undergo a short enduring depolarization. Increasing the amount of ACh applied increases the amplitude of the PJM depolarization and eventually an action potential can be initiated (FIGURE 2). If a still greater amount of ACh is ejected a train of action potentials is produced.

The rate at which the postjunctional membrane of muscle fibers becomes desensitized to the action of carbamylcholine is increased after the muscle has been soaked in solutions containing increased concentrations of calcium. Some further aspects of this effect of calcium were investigated by measuring changes in the input resistance of single fibers of the frog sartorius during local perfusion of the neuromuscular junction with 2.73 X 10 -3 M carbamylcholine in isolated muscles immersed in 165 mM potassium acetate. It was found that (a) sudden changes in the local concentration of calcium brought about by perfusing fibers with carbamylcholine solutions containing 20 mM calcium, 40 mM oxalate, or 40 mM EDTA were followed within 20 sec by marked changes in the rate of desensitization; (b) prior to 13 sec after the introduction of carbamylcholine, however, no effect on the input resistance could be detected even though the muscle had been presoaked in 10 mM calcium; (c) the ability of high concentrations of calcium to bring about rapid desensitization disappears when a lower concentration of carbamylcholine (0.137 X 10-3 M) is applied to the muscle fiber. These findings suggest that calcium present in the extracellular fluid can act directly on the postjunctional membrane to promote the desensitization process and that an increased permeability of the membrane to calcium brought about by the presence of carbamylcholine is a factor which contributes to this action.

Measurements of agonist-induced single-channel currents of nicotinic acetylcholine receptor channels (nAChR) have shown that addition of divalent metal cations (Ca2+, Mg2+, Ba2+) in millimolar amounts causes a decrease of 40% or more in currents carried by monovalent cations (Na+, Cs+). Conventional voltage-clamp measurements were made at frog (Rana pipiens) sartorius endplates to determine if this occlusive interaction between mono- and divalent cations is preserved in macroscopic currents produced by higher concentrations of nAChR agonists, as would be expected on the basis of simple summation of unitary currents. Single ion nAChR currents of Na+ (INa+ ) and Mg2+(IMg2+ ) and the composite current when both cations were present (INa+, Mg2+ ) were measured in high-K+, low ionic strength bathing media during local superfusion with various concentrations of carbamylcholine. It was found that, at lower test carbamylcholine levels of 27 and 54 microM, peak values of INa+, Mg2+ were 40% and 53% of the respective INa+ , in agreement with the effects observed from single-channel measurements. At all higher carbamylcholine levels, however, peak INa+, Mg2+ exceeded INa+ and increased instead with the sum of INa+ and IMg2+ as if there was an independent movement of these ions through the channels rather than the occlusive interaction found at lower carbamylcholine levels. This suggests that with exposure to increasing agonist concentrations above those commonly used in single-channel measurements there are changes in the open state of nAChR affecting cation selectivity possibly in the narrow pore region of the channels.

The electrogenic action of carbamylcholine on the postjunctional membrane of muscle fibers disappears gradually over a period of several seconds even when this agent is applied by constant perfusion. This waning of drug action, termed "desensitization" by some investigators, occurs also at other cholinergic synapses. It has been found in earlier studies that increased amounts of calcium in the extracellular medium can cause the desensitization process to occur more rapidly and that potassium tends to oppose this action of calcium. In the present investigation, it is found that the relative effects of these ions can be expressed in terms of the quantity (Ca)/(K)(1.7). A simple ion-exchange mechanism is proposed in which one calcium ion or two potassium ions are able to combine with an anionic site near the cell surface. A general relation between the time course of conductance changes and the extracellular concentration of the ions is derived which appears to be consistent with the conductance measurements over a period of 60 seconds and in the range of calcium concentrations from 0.05 to 20 millimoles/liter and of potassium from 33 to 165 millimoles/liter. Further experiments are presented which suggest that the time course of conductance changes under these conditions is limited primarily by the rate of increase of the calcium concentration at the exchange site.

The effect of changes in extracellular and intracellular magnesium content on desensitization rate in frog (Rana pipiens) sartorius muscle was studied using measurements of input conductance of single fibers during local superperfusion of the postjunctional region with carbamylcholine chloride (0.27-2.7 mM). Two intracellular KCl-filled glass capillary microelectrodes were used for current injection and recording in fibers equilibrated in high-K+ media (33-165 mM). In low-ionic strength solutions (33 mM K), time to half-decline of conductance during desensitization to carbamylcholine (T1/2) was decreased by increasing extracellular Mg2+ concentration throughout range 0-100 mM. Equivalent effects on T1/2 were produced by increased extracellular Ca2+ in lower concentrations (0-10 mM). Increase in K+ concentration or decrease of carbamylcholine concentration resulted in increased T1/2 in both Mg2+ and Ca2+ media. Increase of intracellular Mg2+ by soaking in high-Mg2+ solutions (100 mM) or by intracellular iontophoresis caused decrease in T1/2. It is concluded that Mg2+, when introduced either in the extracellular or intracellular phase, can promote increased desensitization rate and that this action is similar to but weaker than that of calcium.

The possibility that increases in agonist concentration beyond threshold levels may force changes in the character of high-conductance open states of skeletal muscle nicotinic acetylcholine receptor channels (nAChR) was examined by seeing whether differences in several critical ionic properties of nAChR currents could be detected with changes in agonist level. Single- and bi-ionic whole-cell currents of Na+ and Li+ in voltage-clamped frog (Rana pipiens) muscle fibers were measured during local superfusion of endplates with carbamylcholine (carb) at concentrations of 54 microm (low-carb) and 270 microM (high-carb). Three ionic properties that would be affected by changes in the open-state configuration of channel subunits were tested. First, ion-saturation characteristics. Peak Na+ and Li+ currents in low-carb trials showed sublinear dependence on ion concentrations from 0 to 60 mM with Km values of 78 (Na+) and 49 (Li+) mM and a power function slope of 0. 75 on double-log plot. In contrast, the concentration dependence of Na+ and Li+ currents in high-carb tests was linear through the origin with a power function slope of 1.02. Second, Na+/Li+ selectivity. The ratio of peak Na+ and Li+ currents in low-carb tests varied from 1.86 to 2.28 for ion concentrations of from 20 to 60 mM [mean = 2.02 +/- 0.06 (SEM)] whereas the ratio for high-carb trials ranged from only 1.29 to 1.52 [mean = 1.42 +/- 0.40 (SEM)]. Third, competitive interactions of Na+ and Li+ currents. Equimolar mixtures of Na+ and Li+ in low-carb tests produced bi-ionic inward currents which were never larger than the single-ion Na+ current alone, but bi-ionic currents at the high-carb level were always greater than the single-ion Na+ current, approximating the sum of the single-ion Na+ and Li+ currents in most cases. The results are consistent with a decrease in ion-channel binding at the high-carb level and support the possibility of agonist-induced changes in the high-conductance open-state configuration of nAChR subunits which result in a weakening of constraints on cation movements through the channel.