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M M Salpeter

Publications and source records attributed to M M Salpeter.

At least 19 recordsLinked to original sources
PubMed2001

Two distinct effects on neurotransmission in a temperature-sensitive SNAP-25 mutant.

Vesicle fusion in eukaryotic cells is mediated by SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). In neurons, the t-SNARE SNAP-25 is essential for synaptic vesicle fusion but its exact role in this process is unknown. We have isolated a SNAP-25 temperature-sensitive paralytic mutant in Drosophila, SNAP-25(ts). The mutation causes a Gly50 to Glu change in SNAP-25's first amphipathic helix. A similar mutation in the yeast homologue SEC9 also results in temperature sensitivity, implying a conserved role for this domain in secretion. In vitro-generated 70 kDa SNARE complexes containing SNAP-25(ts) are thermally stable but the mutant SNARE multimers (of approximately 120 kDa) rapidly dissociate at 37 degrees C. The SNAP-25(ts) mutant has two effects on neurotransmitter release depending upon temperature. At 22 degrees C, evoked release of neurotransmitter in SNAP-25(ts) larvae is greatly increased, and at 37 degrees C, the release of neurotransmitter is reduced as compared with controls. Our data suggest that at 22 degrees C the mutation causes the SNARE complex to be more fusion competent but, at 37 degrees C the same mutation leads to SNARE multimer instability and fusion incompetence.

Amino Acid Sequence↗
PubMed1999

Rate constants of acetylcholine receptor internalization and degradation in mouse muscles.

The rate constants for internalization and subsequent extrusion of acetylcholine receptors (AChRs) during degradation in adult innervated and denervated mouse diaphragm muscles were determined using proteinase K (PK) digestion. This procedure separated (125)I-alpha-bungarotoxin (Bgt)-labeled AChRs into PK-sensitive and PK-resistant compartments. The time course of the residual radioactivity in these two compartments suggested that they represented surface membrane and internalized compartments, respectively. The data were compatible with a mathematical model based on the assumption that during degradation of AChRs a surface compartment, A, fed an internal compartment, B, with an internalization rate constant (k(i)), and that B is drained from the cell with an extrusion rate constant (k(o)). Using the mathematical model, we were able to determine that k(i) and k(o) were, respectively, 0.068 (t(1/2) approximately 10.2 days) and 0.69-0.55 (t(1/2) approximately 1.0- 1.25 days) for innervated muscle and were, respectively, 0.69 (t(1/2) approximately 1.0 day) and 6.93 (t(1/2) approximately 0.1 day) for denervated muscle. Thus, the rate for internalization was about 8-10 times slower than that for extrusion from the cell for both the slowly degrading innervated (Rs) AChRs and for the rapidly degrading denervated (Rr) AChRs. This inequality between k(i) and k(o) therefore allows the combined quantity of A(t) + B(t), usually measured in AChR degradation studies, to approximate a single exponential.

Animals↗
PubMed1999

The temperature sensitivity of miniature endplate currents is mostly governed by channel gating: evidence from optimized recordings and Monte Carlo simulations.

The temperature dependence of miniature endplate current (MEPC) amplitude (A(c)), 20-80% rise time (t(r)), and 90-33% fall-time (t(f)) was determined for lizard (Anolis carolinensis) intercostal muscle using broadband extracellular (EC) and voltage clamp (VC) recordings. Voltage clamp methods were optimized for the fast MEPC rising phase using custom electronics. From 0-43 degrees C, A(c) increased by approximately 4.2-fold, while t(r) and t(f) decreased by approximately 3.6- and approximately 9.5-fold, respectively. Arrhenius plots were smoothly curved, with small apparent Q(10) (A(c)) or (Q(10))(-1) (t(r) and t(f)) values mostly well below 2.0. Nearly identical extracellular and voltage clamp results ruled out measurement artifacts, even for the shortest t(r) values (<60 microseconds). Monte Carlo simulation of MEPCs showed that a single underlying rate cannot determine the observed temperature dependence. To quantitatively reproduce the experimental t(f) results, a minimal model required activation energies of 46.0 (Q(10) approximately 2.0) and 63.6 (Q(10) approximately 2.5) kJ mol(-1) for channel opening and closing, respectively, and accounted for most of the observed changes in A(c) and t(r) as well. Thus, relatively large but offsetting temperature sensitivities of channel gating mostly govern and minimize the temperature dependence of MEPCs, preserving the safety factor for neuromuscular transmission. Additional temperature-sensitive parameters that could fine-tune the minimal model are discussed.

Acetylcholinesterase↗
PubMed1998

125I-labeled fasciculin 2: a new tool for quantitation of acetylcholinesterase densities at synaptic sites by EM-autoradiography.

Radio-iodinated fasciculin 2 (Fas2), a polypeptide anticholinesterase toxin from Mamba venom, was used as a new probe for localizing and quantifying acetylcholinesterase (AChE) at mouse neuromuscular junctions (NMJs) by quantitative electron microscope autoradiography. We demonstrate that 125I-Fas2 binds very specifically to the NMJs of mouse sternomastoid muscles, with very little binding to other regions in the muscles. Junctional AChE-site densities obtained from the autoradiograms were similar to those previously obtained for the same muscles using 3H-DFP. The use of 125I-Fas2 with EM-autoradiography is simpler and provides higher resolution and sensitivity, as well as considerably lower non-specific binding than previously attainable with 3H-DFP. The advantages and limitations of this procedure are discussed.

Acetylcholinesterase↗
PubMed1997

Epsilon subunit-containing acetylcholine receptors in myotubes belong to the slowly degrading population.

Two types of muscle acetylcholine receptors (AChRs) can be distinguished on the basis of their degradation rates and sensitivities to innervation, muscle activity, and agents elevating intracellular cAMP. The first type (Rs), is present in a stable form (degradation t1/2 = approximately 10 d) at the adult innervated neuromuscular junctions (NMJs). Rs can also exist in a less stable form (called accelerated Rs; t1/2 = approximately 3-5 d) at denervated NMJs and in aneurally cultured myotubes; agents that increase intracellular cAMP reversibly modulate Rs stability. The second type of AChR is a rapidly degrading receptor (Rr) expressed only in embryonic and noninnervated muscles. Rr can be stabilized by ATP and not by cAMP. This study tested the hypothesis that the degradation properties unique to the Rs are attributable to the presence of the epsilon subunit. Immunoprecipitation and Western blot analysis of AChRs extracted from rat muscle cells in tissue culture showed that AChRs recognized by antibodies against the epsilon subunit degraded as a single population with a half-life similar to that of the slow component, Rs, in these cells. In addition, as for Rs receptors in denervated NMJs and cultured muscle cell, the degradation rate of these epsilon-containing AChRs was stabilized by dibutyryl-cAMP. The data indicate that the epsilon-containing AChRs behave like Rs. Thus, the presence of the epsilon subunit is sufficient for selecting an AChR molecule to the Rs pool.

Animals↗
PubMed1997

Acetylcholine receptors in innervated muscles of dystrophic mdx mice degrade as after denervation.

Acetylcholine receptors (AChRs) are present at the top of the postsynaptic membrane of the neuromuscular junction (NMJ) at very high density, possibly anchored to cytoskeletal elements. The present study investigated whether AChR degradation is affected in animals lacking dystrophin, a protein that is an integral part of the cytoskeletal complex and is missing in Duchenne muscular dystrophy. The animal model for Duchenne muscular dystrophy, the mutant mdx mouse, was used to determine whether disruption of the cytoskeleton, caused by the absence of dystrophin, affects AChR degradation. Of the two populations of junctional AChRs, Rs (expressed in innervated adult muscles) and Rr (expressed in embryonic or denervated muscles), only Rs are affected in mdx animals. In innervated mdx soleus, diaphragm, and sternomastoid muscles, the AChRs have an accelerated degradation rate (t1/2 of approximately 3-5 d), similar to that acquired by Rs in control muscles after denervation. The Rs in mdx NMJs do not accelerate further when the muscles are denervated. The absence of dystrophin does not affect the degradation rate of the Rr AChRs (t1/2 of 1 d), which are expressed after denervation in mdx as in control muscles. These results suggest that dystrophin or an intact cytoskeletal complex may be required for neuronal stabilization of Rs receptors at the adult neuromuscular junctions.

Animals↗
PubMed1997

Stabilization of acetylcholine receptors by exogenous ATP and its reversal by cAMP and calcium.

Innervation of the neuromuscular junction (nmj) affects the stability of acetylcholine receptors (AChRs). A neural factor that could affect AChR stabilization was studied using cultured muscle cells since they express two distinct populations of AChRs similar to those seen at the nmjs of denervated muscle. These two AChR populations are (in a ratio of 9 to 1) a rapidly degrading population (Rr) with a degradation half-life of approximately 1 d and a slowly degrading population (Rs) that can alternate between an accelerated form (half-life approximately 3-5 d) and a stabilized form (half-life approximately 10 d), depending upon the state of innervation of the muscle. Previous studies have shown that elevation of intracellular cAMP can stabilize the Rs, but not the Rr. We report here that in cultured rat muscle cells, exogenous ATP stabilized the degradation half-life of Rr and possibly also the Rs. Furthermore, pretreatment with ATP caused more stable AChRs to be inserted into the muscle membrane. Thus, in the presence of ATP, the degradation rates of the Rr and Rs overlap. This suggests that ATP released from the nerve may play an important role in the regulation of AChR degradation. Treatment with either the cAMP analogue dibutyryl-cAMP (dB-cAMP) or the calcium mobilizer ryanodine caused the ATP-stabilized Rr to accelerate back to a half-life of 1 d. Thus, at least three signaling systems (intracellular cAMP, Ca2+, and extracellular ATP) have the potential to interact with each other in the building of an adult neuromuscular junction.

Adenosine Triphosphate↗
PubMed1997

Absence of nerve-dependent conversion of rapidly degrading to stable acetylcholine receptors at adult innervated endplates.

It has been suggested that acetylcholine receptors newly inserted into adult innervated endplates have a rapid degradation rate, but are normally converted to a stable, slowly degrading form in a nerve-dependent fashion. Denervation therefore should eliminate conversion and cause pre-existing unconverted receptors to continue degrading rapidly. We tested this model of nerve-dependent conversion in mouse sternomastoid muscle, using quantitative electron microscopic autoradiography in order to specifically examine degradation of receptors at identified endplate membrane. Prior to denervation, we labelled the receptors with sequential alpha-bungarotoxin exposures, using conditions designed to maximize the predicted effect of denervation. However, we observed no difference in the rate of receptor degradation at innervated and denervated endplates up to seven days after denervation (at which time accelerated degradation of pre-existing stabilized receptors is known to begin in this muscle). The regulation of endplate acetylcholine receptor metabolic turnover is a complex and still largely undefined issue, related to many factors such as subunit composition, cytoskeleton and basement membrane composition, muscle activity, and neural influences. In particular, the nerve's influence on the normal stabilization of receptors at innervated adult endplates has been controversial. Our data indicate that slow degradation is probably an inherent property of newly inserted junctional receptors, and argue against nerve-dependent conversion and stabilization. Based on the present data, however, we cannot rule out the presence of a small nerve-independent subpopulation that degrades rapidly. The molecular mechanisms involved in establishing and maintaining a stable population of adult endplate acetylcholine receptors remain to be established.

Animals↗
PubMed1996

Miniature endplate current rise times less than 100 microseconds from improved dual recordings can be modeled with passive acetylcholine diffusion from a synaptic vesicle.

We recorded miniature endplate currents (mEPCs) using simultaneous voltage clamp and extracellular methods, allowing correction for time course measurement errors. We obtained a 20-80% rise time (tr) of approximately 80 micros at 22 degrees C, shorter than any previously reported values, and tr variability (SD) with an upper limit of 25-30 micros. Extracellular electrode pressure can increase tr and its variability by 2- to 3-fold. Using Monte Carlo simulations, we modeled passive acetylcholine diffusion through a vesicle fusion pore expanding radially at 25 nm x ms(-1) (rapid, from endplate omega figure appearance) or 0.275 nm x ms(-1) (slow, from mast cell exocytosis). Simulated mEPCs obtained with rapid expansion reproduced tr and the overall shape of our experimental mEPCs, and were similar to simulated mEPCs obtained with instant acetylcholine release. We conclude that passive transmitter diffusion, coupled with rapid expansion of the fusion pore, is sufficient to explain the time course of experimentally measured synaptic currents with trs of less than 100 micros.

Acetylcholine↗
PubMed1995

Protein kinase A regulates the degradation rate of Rs acetylcholine receptors.

Acetylcholine receptors at the neuromuscular junction of innervated vertebrate muscle (called Rs AChRs) have a stable degradation rate (t1/2 approximately 8-12 days) which accelerates after denervation to a half-life of approximately 3 days, but can be restabilized by reinnervation or by cAMP. We examined the mechanism by which cAMP regulates the Rs degradation rate. When dibutyryl cAMP (DB-cAMP) was applied to denervated mouse diaphragms in organ culture, it stabilized the accelerated degradation rate of the Rs. We found that this stabilization is reversible upon removal of the DB-cAMP, is cAMP specific and is mediated by intracellular cAMP. A major observation of this study is that the cAMP-induced stabilization of Rs AChRs is via protein kinase A (PKA), since H89, a PKA inhibitor, blocked the DB-cAMP induced stabilization of Rs, and H85, an analog of H89, which does not inhibit PKA but does inhibit other kinases as efficiently as H89, did not prevent the DB-cAMP-induced stabilization of Rs degradation. These results suggest that the cAMP messenger system via a PKA-dependent pathway could be among the mechanisms whereby the nerve regulates AChR degradation.

Animals↗
PubMed1994

Mouse muscle epsilon- and gamma-containing acetylcholine receptors expressed in Xenopus laevis oocytes do not differ in their degradation half-lives.

Mouse acetylcholine receptors (AChRs) consisting of either the embryonic form (2 alpha, 1 beta, 1 delta and 1 gamma) or the adult form (2 alpha, 1 beta, 1 delta and 1 epsilon) were expressed in Xenopus laevis oocytes. As expected, the single channel conductance was approximately 52 pS and the exponential decay time constants were 2.2 and 8.2 ms for the gamma-AChR and respectively 65 pS and 0.6 and 2.8 ms for the epsilon-AChR. No difference was seen in the degradation rate between the gamma- and epsilon-containing AChRs, both having a half-life of about 5 days.

Animals↗
PubMed1994

Acetylcholinesterase density and turnover number at frog neuromuscular junctions, with modeling of their role in synaptic function.

Acetylcholinesterase (AChE) density at the neuromuscular junction of frog cutaneous pectoris muscle was determined by electron microscope autoradiography and biochemistry to be approximately 600 sites micron-2 of postsynaptic area, approximately 4-fold lower than all previous reports (mouse), whereas the hydrolytic turnover number was 9,500 s-1, well within the range (2,000-16,000 s-1) for AChE from other species. Monte Carlo computer simulations of miniature endplate currents showed that for vertebrate neuromuscular junctions with different morphologies, an AChE density of only approximately 400 sites microns-2 and a turnover number of only approximately 1,000 s-1 are sufficient for normal quantal currents. Above these critical lower limits, miniature endplate currents were essentially insensitive to AChE density and turnover number values up to 5,000 sites microns-2 and 16,000 s-1, respectively.

Acetylcholine↗
PubMed1994

In situ hybridization and cytochemistry: localization of mRNA at stained neuromuscular junctions with 33P-labeled probes.

We modified the Karnovsky and Roots method of staining sites of acetylcholinesterase (AChE) activity at neuromuscular junctions (NMJs) to survive the lengthy, multiple steps of in situ hybridization and autoradiography. When the original method of Karnovsky and Roots is used to identify the muscle endplates, the stain does not survive the in situ hybridization procedures and association of mRNA to specific endplates can be inferred only indirectly. The successful modification involves secondary staining with diaminobenzidine (DAB) and H2O2 using the Karnovsky-Roots staining reaction product as a catalyst. Mounted longitudinal cryosections of mouse sternocleidomastoid muscle were fixed and stained in one step on the slide with paraformaldehyde plus the Karnovsky-Roots stain, followed by DAB-H2O2 secondary staining. The tissues were then processed for in situ hybridization and probed for the acetylcholine receptor (AChR) epsilon-subunit mRNA, known to be localized at the NMJ. The probe was labeled with 33P, which is ideal for in situ hybridization. By this procedure, the endplate stain was retained even after the hybridization and autoradiographic procedures, and the developed grains due to radiolabeling of the AChR epsilon-subunit mRNA were localized at readily identified endplates.

Acetylcholinesterase↗
PubMed1993

Two populations of AChR in rat myotubes have different degradation rates and responses to cAMP.

Acetylcholine receptors (AChRs) of rat muscle cells grown in culture for 4 days were labeled with 125I-alpha-bungarotoxin and their degradation rate measured. Two AChR populations, a rapidly degrading one (Rr,t1/2 approximately 1 day) and a slowly degrading one (Rs, t1/2 approximately 4 days) were identified at a ratio of approximately 9 to 1. The degradation rate of the Rs was slowed to a t1/2 of approximately 10 days by cAMP while that of the Rr remained unchanged. These data provide further evidence that two AChR populations with different degradation rates can exist in noninnervated muscle and that they can be further distinguished by their differing response to cAMP. We suggest that the two AChR populations seen in myotubes may be physiologically equivalent to the Rs and Rr components seen at the neuromuscular junction of denervated adult muscle and could thus provide a good model for characterizing the Rr and Rs AChRs.

Animals↗
PubMed1993

Secreted collagen induced by ascorbic acid in L5 cloned muscle cultures does not affect acetylcholine receptor expression.

Previous studies have shown that ascorbic acid increases both the total surface acetylcholine receptor (AChR) expression and the mRNA for the alpha-subunit of this receptor in myotubes of cloned L5 muscle cultures. Since ascorbic acid increases collagen synthesis in fibroblasts, we studied the effect of ascorbic acid on collagen secretion in L5 muscle cells and investigated the possibility that the effects of ascorbic acid on collagen and AChR are related. We report that L5 muscle cells secrete collagen types I, III, and V, with collagen type I being the most abundant species, and that accumulation of secreted collagens increased in the medium approximately two- to ninefold within 3 h of ascorbic acid treatment. The increase in surface AChRs, on the other hand, developed more slowly, and was detected only about 20-24 h after ascorbic acid treatment. A short (5 h) treatment with ascorbic acid is, however, sufficient to trigger an increase in AChRs 24 h later. Since ascorbic acid caused a rapid increase in collagen secretion, whereas the effect on total surface AChRs occurs more slowly, we tested the possibility that an increase in secreted collagen might be necessary for the increase in AChRs. However, when the L5 cultures were treated with bacterial collagenase, the ascorbic acid-induced increase in secreted collagen was abolished but its inductive effect on AChRs was unchanged. The increase in secreted collagen is therefore not necessary for the increase in AChRs to occur.

Animals↗
PubMed1993

Degradation of two AChR populations at rat neuromuscular junctions: regulation in vivo by electrical stimulation.

The effect of electrical stimulation on the stability of junctional ACh receptors (AChR) on soleus muscles of Wistar rats was compared to that of denervation and reinnervation. Denervation causes the degradation rate of the slowly degrading AChRs (Rs) at the neuromuscular junction to accelerate and be replaced by rapidly degrading AChRs (Rr), while reinnervation restabilizes the accelerated Rs. Electrical stimulation initiated at the time of denervation prevented the acceleration of the Rs. It could not, however, reverse the effect of denervation if initiated after the AChRs became destabilized, nor could it slow the degradation rate of the Rr. We conclude that electrical stimulation of denervated muscle downregulates the expression of the Rr and prevents the destabilization of Rs.

Animals↗