Evaluating the influence of marine protected areas on surf zone fish.

Marine protected areas (MPAs) globally serve conservation and fisheries management goals, generating positive effects in some marine ecosystems. Surf zones and sandy beaches, critical ecotones bridging land and sea, play a pivotal role in the life cycles of numerous fish species and serve as prime areas for subsistence and recreational fishing. Despite their significance, these areas remain understudied when evaluating the effects of MPAs. We compared surf zone fish assemblages inside and outside MPAs across 3 bioregions in California (USA). Using seines and baited remote underwater videos (BRUVs), we found differences in surf zone fish inside and outside MPAs in one region. Inside south region MPAs, we observed higher abundance (Tukey's honest significant difference [HSD] = 0.83, p = 0.0001) and richness (HSD = 0.22, p = 0.0001) in BRUVs and greater biomass (HSD = 0.32, p = 0.0002) in seine surveys compared with reference sites. Selected live-bearing, fished taxa were positively affected by MPAs. Elasmobranchs displayed greater abundance in BRUV surveys and higher biomass in seine surveys inside south region MPAs (HSD = 0.35, p = 0.0003 and HSD = 0.23, p = 0.008, respectively). Although we observed no overall MPA signal for Embiotocidae, abundances of juvenile and large adult barred surfperch (Amphistichus argenteus), the most abundant fished species, were higher inside MPAs (K-S test D = 0.19, p < 0.0001). Influence of habitat characteristics on MPA performance indicated surf zone width was positively associated with fish abundance and biomass but negatively associated with richness. The south region had the largest positive effect size on all MPA performance metrics. Our findings underscored the variability in species richness and composition across regions and survey methods that significantly affected differences observed inside and outside MPAs. A comprehensive assessment of MPA performance should consider specific taxa, their distribution, and the effects of habitat factors and geography.

Evaluación de la influencia de las áreas marinas protegidas sobre los peces de la zona de rompientes Resumen Las áreas marinas protegidas (AMP) cumplen los objetivos de conservación y manejo de pesquerías a nivel mundial, lo que genera efectos positivos en algunos ecosistemas marinos. Las zonas de rompientes y las playas arenosas, ecotonos importantes que conectan la tierra con el mar, tienen un papel esencial en el ciclo de vida de varios peces y fungen como áreas óptimas para la pesca recreativa y de sustento. A pesar de su importancia, estas áreas están poco estudiadas con respecto a la evaluación del efecto de las AMP. Comparamos la composición de peces del área de rompientes dentro y fuera de las AMP de tres bioregiones de California, EUA. Usamos chinchorros y videos submarinos con carnada (BRUVs) y descubrimos diferencias en los peces de la zona de rompientes dentro y fuera de las AMP en una región. Dentro de las AMP de la región sur observamos una mayor abundancia (diferencia significativa honesta de Tukey [DSH]  =  0.83, p = 0.0001) y riqueza (DSH  =  0.22, p = 0.0001) en los BRUV y una mayor biomasa (DSH  =  0.32, p = 0.0002) en los censos con chinchorro en comparación con los sitios de referencia. Los taxones seleccionados de peces de sustento fueron afectados de manera positiva por las AMP. Los elasmobranquios mostraron una mayor abundancia en los BRUV y una mayor biomasa en los censos con chinchorro dentro de las AMP de la región sur (DSH  =  0.35, p = 0.0003 y DSH  =  0.23, p = 0.008, respectivamente). Aunque no observamos una señal generalizada de las AMP para la familia Embiotocidae, la abundancia de Amphistichus argenteus juveniles y adultos, la especie pescada más abundante, fue mayor dentro de las AMP (prueba K­S D  =  0.19, p < 0.0001). La influencia de las características del hábitat sobre el desempeño de las AMP indicó que el ancho de la zona de rompientes está asociado de forma positiva con la abundancia y biomasa de los peces, pero de forma negativa con la riqueza. La región sur tuvo el mayor tamaño de efecto positivo sobre todas las medidas de desempeño de las AMP. Nuestros hallazgos destacan la variabilidad en la riqueza y composición de especies en todas las regiones y los censos que afectan significativamente las diferencias observadas dentro y fuera de las AMP. Una evaluación completa del desempeño de las AMP debe considerar taxones específicos, su distribución y el efecto de los factores de hábitat y la geografía.

Disease-driven mass mortality event leads to widespread extirpation and variable recovery potential of a marine predator across the eastern Pacific.

The prevalence of disease-driven mass mortality events is increasing, but our understanding of spatial variation in their magnitude, timing and triggers are often poorly resolved. Here, we use a novel range-wide dataset comprised 48 810 surveys to quantify how sea star wasting disease affected Pycnopodia helianthoides, the sunflower sea star, across its range from Baja California, Mexico to the Aleutian Islands, USA. We found that the outbreak occurred more rapidly, killed a greater percentage of the population and left fewer survivors in the southern half of the species's range. Pycnopodia now appears to be functionally extinct (greater than 99.2% declines) from Baja California, Mexico to Cape Flattery, Washington, USA and exhibited severe declines (greater than 87.8%) from the Salish Sea to the Gulf of Alaska. The importance of temperature in predicting Pycnopodia distribution rose more than fourfold after the outbreak, suggesting latitudinal variation in outbreak severity may stem from an interaction between disease severity and warmer waters. We found no evidence of population recovery in the years since the outbreak. Natural recovery in the southern half of the range is unlikely over the short term. Thus, assisted recovery will probably be required to restore the functional role of this predator on ecologically relevant time scales.

Influence of protogynous sex change on recovery of fish populations within marine protected areas.

Marine protected areas (MPAs) are increasingly implemented as a conservation tool worldwide. In many cases, they are managed adaptively: the abundance of target species is monitored, and observations are compared to some model-based expectation for the trajectory of population recovery to ensure that the MPA is achieving its goals. Most previous analyses of the transient (short-term) response of populations to the cessation of fishing inside MPAs have dealt only with gonochore (fixed-sex) species. However, many important fishery species are protogynous hermaphrodites (female-to-male sex-changing). Because size-selective harvest will predominantly target males in these species, harvesting not only reduces abundance but also skews the sex ratio toward females. Thus the response to MPA implementation will involve changes in both survival and sex ratio, and ultimately reproductive output. We used an age-structured model of a generic sex-changing fish population to compare transient population dynamics after MPA implementation to those of an otherwise similar gonochore population and examine how different features of sex-changing life history affect those dynamics. We examined both demographically open (most larval recruitment comes from outside the MPA) and demographically closed (most larval recruitment is locally produced) dynamics. Under both scenarios, population recovery of protogynous species takes longer when fishing was more intense pre-MPA (as in gonochores), but also depends heavily on the mating function, the degree to which the sex ratio affects reproduction. If few males are needed and reproduction is not affected by a highly female-biased sex ratio, then population recovery is much faster; if males are a limiting resource, then increases in abundance after MPA implementation are much slower than for gonochores. Unfortunately, the mating function is largely unknown for fishes. In general, we expect that most protogynous species with haremic mating systems will be in the first category (few males needed), though there is at least one example of a fish species (though not a sex-changing species) for which males are limiting. Thus a better understanding of the importance of male fish to population dynamics is needed for the adaptive management of MPAs.

Fishing top predators indirectly affects condition and reproduction in a reef-fish community.

To examine the indirect effects of fishing on energy allocation in non-target prey species, condition and reproductive potential were measured for five representative species (two-spot red snapper Lutjanus bohar, arc-eye hawkfish Paracirrhites arcatus, blackbar devil Plectroglyphidodon dickii, bicolour chromis Chromis margaritifer and whitecheek surgeonfish Acanthurus nigricans) from three reef-fish communities with different levels of fishing and predator abundance in the northern Line Islands, central Pacific Ocean. Predator abundance differed by five to seven-fold among islands, and despite no clear differences in prey abundance, differences in prey condition and reproductive potential among islands were found. Body condition (mean body mass adjusted for length) was consistently lower at sites with higher predator abundance for three of the four prey species. Mean liver mass (adjusted for total body mass), an indicator of energy reserves, was also lower at sites with higher predator abundance for three of the prey species and the predator. Trends in reproductive potential were less clear. Mean gonad mass (adjusted for total body mass) was high where predator abundance was high for only one of the three species in which it was measured. Evidence of consistently low prey body condition and energy reserves in a diverse suite of species at reefs with high predator abundance suggests that fishing may indirectly affect non-target prey-fish populations through changes in predation and predation risk.

Atrotoxin: a specific agonist for calcium currents in heart.

A specific label for voltage-dependent calcium channels is essential for the isolation and purification of the membrane protein that constitutes the calcium channel and for a better understanding of its function. A fraction of Crotalus atrox that increases voltage-dependent calcium currents in single, dispersed guinea pig ventricular cells was isolated. In the doses used, neither sodium nor potassium currents were changed. The fraction was active in the absence of detectable phospholipase or protease activity, and the active component, designated atrotoxin, produced its effect rapidly and reversibly. The effect was produced by extracellular but not intracellular application of the agent. The increase in Ca2+ current was blocked by the Ca2+ channel blockers cobalt and nitrendipine. The active fraction completely blocked specific [3H]nitrendipine binding to guinea pig ventricular membrane preparations. The inhibition of nitrendipine binding by atrotoxin was apparently via an allosteric mechanism. Thus atrotoxin was shown to bind to the Ca2+ channel and to act as a specific Ca2+ channel agonist.

Diseases associated with altered ryanodine receptor activity.

Mutations in two intracellular Ca2+ release channels or ryanodine receptors (RyR1 and RyR2) are associated with a number of human skeletal and cardiac diseases. This chapter discusses these diseases in terms of known mechanisms, controversies, and unanswered questions. We also compare the cardiac and skeletal muscle diseases to explore common mechanisms.

Functional interactions of cytoplasmic domains of the skeletal muscle Ca2+ release channel.

The skeletal muscle Ca(2+) release channel (RYR1) is a homotetramer with subunits of 565 kD. Although the channel part of this protein is probably formed by the C-terminal one fifth of the protein, most of the regulation of channel activity is likely to arise from intermolecular and intramolecular interactions of its large cytoplasmic domain. This cytoplasmic region of the protein is thought to contain binding sites for a variety of modulators, including the t-tubule voltage sensor, and mutations in some cases of malignant hyperthermia and central core disease. Regulation of channel activity must, therefore, involve long-distance allosteric modulation arising from changes in both intersubunit and intrasubunit interactions within the cytoplasmic domain of the RYR1 tetramer. Biochemical studies are beginning to elucidate some of the sites within the cytoplasmic domains important for modulting channel activity in the membrane-spanning domain. This review summarizes these findings and presents a working model for the regulation of the channel by the interactions of its cytoplasmic domains.

The survival motor neuron protein interacts with the transactivator FUSE binding protein from human fetal brain.

To identify interacting proteins of survival motor neuron (SMN) in neurons, a fetal human brain cDNA library was screened using the yeast two-hybrid system. One identified group of SMN interacting clones encoded the DNA transactivator FUSE binding protein (FBP). FBP overexpressed in HEK293 cells or endogenously expressed in fetal and adult mouse brain bound specifically in vitro to recombinant SMN protein. Furthermore, an anti-FBP antibody specifically co-immunoprecipitated SMN when both proteins were overexpressed in HEK293 cells. These results demonstrate that FBP is a novel interacting partner of SMN and suggests a possible role for SMN in neuronal gene expression.

RyR1 modulation by oxidation and calmodulin.

Alteration of skeletal muscle function by reactive oxygen species and nitric oxide (NO) may involve regulation of the activity of the skeletal muscle Ca2+ release channel (also known as RyR1). We have shown that oxidants can activate RyR1 and produce inter-subunit disulfide bonds. Both effects are prevented by pretreatment with either NO donors or N-ethylmaleimide under conditions that modify less than 5% of the total sulfhydryls on RyR1. Oxidation-induced intersubunit crosslinking can also be prevented by the binding of either Ca2+ calmodulin or apocalmodulin to RyR1. Also, both Ca2+ calmodulin and apocalmodulin binding are blocked by oxidation of RyR1. In contrast, alkylation with N-ethylmaleimide or reaction with NO donors preferentially blocks apocalmodulin binding to RyR1, suggesting the existence of a regulatory cysteine within the apocalmodulin binding site. We have demonstrated that Ca2+ calmodulin and apocalmodulin bind to overlapping, but nonidentical, sites on RyR1 and that cysteine 3635 is close to or within the apocalmodulin-binding site on RyR1. This cysteine is also one of the cysteines that form the intersubunit disulfide bonds, suggesting that calmodulin binds at an intersubunit contact site. Our findings are consistent with a model in which oxidants regulate the activity of RyR1 directly by altering subunit-subunit interactions and indirectly by preventing the binding of either Ca2+-bound calmodulin or apocalmodulin. NO also has both a direct and an indirect effect: it blocks the ability of oxidants to generate intersubunit disulfide bonds and prevents apocalmodulin binding.

Toxins that affect voltage-dependent calcium channels.

At this time, there are five potential candidates for calcium channel specific toxins. All five of these toxins appear to affect the function of voltage-dependent calcium channels. Atrotoxin, beta-leptinotarsin-h and maitotoxin activate channels, whereas both taicatoxin and omega-conotoxin are inhibitors. Neither maitotoxin nor omega-conotoxin alters the binding of dihydropyridines to membranes derived from the cells upon which the toxins exert their effects. In contrast, both atrotoxin and taicatoxin inhibit the binding of dihydropyridines to ventricular membranes. It is not currently known whether beta-leptinotarsin-h affects the binding. The effects of maitotoxin and atrotoxin are blocked by dihydropyridines and verapamil. Direct binding studies with radiolabeled toxins have been performed only with omega-conotoxin, and the binding site density for this toxin appears to be at least one order of magnitude greater than the density of dihydropyridine binding sites in synaptosomes. Studies to examine the third and fourth criteria which we have listed (i.e. that the effects are not via a second messenger, or an enzyme activity) have not been reported for either beta-leptinotarsin-h or omega-conotoxin. Atrotoxin and taicatoxin, added outside a patch pipette, have no effects on calcium channels within the patch and are, therefore, probably not affecting calcium channels via a second messenger. Maitotoxin, however, affects the formation of inositol phosphates and, hence, could be affecting the channel indirectly. The fractions containing the toxic components atrotoxin and taicatoxin have no phospholipase or protease activity, and this is presumably true also for omega-conotoxin since it has been purified to homogeneity. Although all of the toxins have the potential to be important tools with which to study calcium channel structure and function, a number of experiments remain to be done in order to establish conclusively that these five toxins bind specifically to the voltage-dependent calcium channel. In conclusion, we would like to briefly mention why so much effort is being devoted to the search for these calcium channel specific toxins. Such a toxin would provide a very valuable tool in the study of calcium channels for a number of reasons. First, the toxin would be another ligand for the channel and, as such, would provide an alternative to organic ligands such as the dihydropyridines which are lipophilic and, in many tissues, have more than one binding site.(ABSTRACT TRUNCATED AT 400 WORDS)

Low affinity binding sites for 1,4-dihydropyridines in mitochondria and in guinea pig ventricular membranes.

In this paper, we describe the occurrence of both high and low affinity sites for dihydropyridines in crude membrane preparations from guinea pig ventricular tissue. The physiological significance of the low affinity site (apparent dissociation constant = 76 +/- 9 nM) is not currently known; it has, however, a binding capacity which was 300-1000 times that of the high affinity site and was resistant to heat denaturation. The magnitude of the binding to the low affinity site was affected by both the ionic strength of the medium and by the presence of divalent ions. Both unlabeled nitrendipine and nimodipine inhibited [3H]nitrendipine binding at both sites, but verapamil and diltiazem only affected binding at the high affinity site. We also characterized, both kinetically and by equilibrium binding, a low affinity, heat-stable nitrendipine binding site in purified mitochondria. The Bmax for this site was also dependent on ionic strength. This suggests the possibility that the low affinity site in crude membranes is due to mitochondrial contaminants and hence not directly related to voltage-dependent calcium channels.

An analysis of UVA emissions from sunlamps and the potential importance for melanoma.

Exposure to solar UV radiation is a risk factor for cutaneous malignant melanoma (CMM). Epidemiologic studies have also considered the use of sunlamps as a possible contributor to CMM. We measured and analyzed the emission spectra of six different currently marketed sunlamps and a historical sunlamp, the UVB-emitting FS lamp, and compared the results to solar exposure. For a typical tanner (20 sessions @ 2 minimal erythema doses (MED)/session), the annual UVA doses from commonly used fluorescent sunlamps were 0.3-1.2 times that received from the sun. For a frequent tanner (100 sessions @ 4 MED/session), the annual UVA doses from fluorescent sunlamps were 1.2-4.7 times that received from the sun and 12 times for recently available, high-pressure sunlamps. To determine biologically effective doses, action spectra for squamous cell carcinoma (SCC) in humans and for melanoma in the Xiphophorus fish (XFM) were applied to the sunlamps' emission spectra. The results for the effective doses using the SCC action spectrum tracked the UVB doses, while the results using the XFM action spectrum tracked the UVA doses. When combined with UV exposure received from the sun, typical sunlamp use results in an approximate doubling of annual effective dose, if the XFM action spectrum is applied. Frequent use, however, can increase the annual effective XFM dose by as much as 6 times what would be received from the sun alone for fluorescent sunlamps and as much as 12 times for newer, high-pressure sunlamps.

Four-week pedometer-determined activity patterns in normal weight and overweight UK adults.

OBJECTIVE: To assess pedometer-determined physical activity levels and activity patterns in a sample of free-living normal weight and overweight UK adults. DESIGN: Pedometer-based 4-week observational study. PARTICIPANTS: One hundred and twenty-two healthy participants, recruited from two regions in the UK, classified as normal weight (33 females and 26 males) or overweight (31 females and 32 males), in the age range of 18 to 65 years, completed the study. MEASUREMENTS: Daily step counts were measured using a Yamax SW-200 pedometer, and were then recorded in an activity log. Comparisons were made between activity patterns occurring over different days of the week for the normal weight and overweight groups. Measurements of height, weight and percentage body fat, by bioelectrical impedance, were taken pre- and post-study. RESULTS: A consistent reduction in activity was observed on a Sunday in the overweight group, and mean daily step counts accumulated on Sundays were significantly lower, by an average of 2221 steps/day, when compared with all other days of the week (all P<0.001). In comparison, no day-of-the-week effect was observed in the normal weight group. Mean step counts reported on each day of the week did not differ significantly between the two groups, with the exception of Sunday when the overweight group reported significantly fewer steps than the normal weight participants (8093 versus 10 538, P<0.001). CONCLUSIONS: Activity levels dropped dramatically in the sample of overweight adults on a Sunday. Simple instructions to at-risk individuals, to increase their general activity levels on a Sunday, via general practitioners and public health messages could prove to be a subtle, but effective, strategy to tackle obesity.

The structure of the nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor of the electric ray is currently one of the most extensively studied integral membrane proteins. A very speculative model for the structure of acetylcholine receptor in the membrane is shown in Fig. 1. This model does not take into account the extremely high density of receptor in the subsynaptic region of the membrane. The receptor is an oligomeric glycoprotein composed of five subunits, all of which are exposed externally, span the lipid bilayer, and are exposed cytoplasmically. Although the subunits have considerable sequence homology, they arise from the translation of different messenger RNAs. The naturally occurring form of the Torpedo receptor is a dimer, but monomer is also functionally active. Each receptor monomer has two binding sites for agonists and competitive antagonists but probably only one site for specific binding of local anesthetics. This latter site appears to be in the open ion channel near the center of the membrane.

Functional anatomy of the second visual area (V2) in the macaque.

To study the functional organization of secondary visual cortex (V2) in the primate, 14C-2-deoxy-d-glucose (DG) was injected while macaque monkeys were shown specific visual stimuli. Wherever possible, patterns of DG uptake were compared with the position of dark and light cytochrome oxidase (cytox) stripes (Tootell et al., 1983). Often, the DG effects of 2 different stimuli were compared in the same hemisphere to eliminate ambiguities inherent in between-animal comparisons. Data were obtained from a large number of animals in conjunction with related DG studies in area V1 (primary visual or striate cortex). The following conclusions were reached: (1) in some macaque monkeys, dark cytox stripes were faint or absent. Although this could conceivably be due to poor staining technique, some evidence suggests that the lack of enzyme stripe pattern is real. In all animals, including those that showed poor or no cytox staining evidence for stripes, the functional architecture revealed by the DG was consistently present and robust. (2) Uniform gray stimuli produce a relatively uniform pattern and minimal stimulus-related DG uptake. (3) Eye movements per se produce some uptake in the V2 stripes. (4) Very generalized visual stimulation conditions (e.g., binocular stimulation with a grating of varied orientation and varied spatial frequency) produced a pattern of uptake that is greatest in both sets of dark cytox stripes and lighter in the light cytochrome stripes. (5) In both the DG and cytox results, the V2 "stripes" are more accurately described as stripe-shaped collections of patches. (6) In almost all cases, DG patterns were columnar in shape, extending from white matter to cortical surface. The boundaries of the columns were most sharply defined, and the contrast was highest, in layers 3B/4, becoming slightly more blurry and lower in contrast in other layers. Laminar differences between DG patterns in V2 were almost negligible, compared with the profound laminar differences in macaque V1. (7) There is no DG evidence for, and much against, the possibility of an ocular dominance architecture in V2. (8) There are orientation columns in macaque V2. DG-labeled orientation columns are spaced further apart than those in V1, by a factor of about 1.6, but the columns are not correspondingly wider. (9) Spatially diffuse variations in color produce high uptake confined, at least largely, to the thin cytox stripes. (10) There is evidence for spatially antagonistic color surrounds in color cells in the thin stripes.(ABSTRACT TRUNCATED AT 400 WORDS)

Factors influencing [3H]ryanodine binding to the skeletal muscle Ca2+ release channel.

Optimal [3H]ryanodine binding to skeletal muscle sarcoplasmic reticulum membranes is dependent on a number of factors such as Ca2+ concentration, ionic strength, and the presence of modulators of the Ca2+ release channel. The rate of association of [3H]-ryanodine with its binding site is slower than a diffusion limited process, and often the binding reaches a peak value which is followed by a slow decline. This phenomenon makes it extremely difficult to determine kinetic constants for [3H]ryanodine binding. The inclusion of bovine serum albumin (BSA) or the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps) in the incubation buffer prevents the decrease in [3H]ryanodine binding observed in association studies. BSA or Chaps slows this decline in binding partially by preventing a conversion to a more rapidly dissociating component. Pretreatment of the membranes with Chaps does not prevent the decrease in [3H]ryanodine binding, suggesting that Chaps is not exerting its effect by extracting a lipid or peripheral membrane protein. The decrease in affinity observed in the absence of BSA and Chaps appears to require the occupation of the high-affinity ryanodine binding site. Incubation for extended times in the absence of [3H]ryanodine prior to the initiation of the association produced similar curves to those obtained without preincubation. These combined results suggest that Chaps and BSA stabilize the ryanodine-modified Ca2+ release channel by preventing an alteration in the ryanodine binding site which leads to decreased affinity, thus allowing for a more quantitative interpretation of binding data.

Functional anatomy of macaque striate cortex. IV. Contrast and magno-parvo streams.

Macaque monkeys were shown achromatic gratings of various contrasts during 14C-2-deoxy-d-glucose (DG) infusion in order to measure the contrast sensitivity of different subdivisions of primary visual cortex. DG uptake is essentially saturated at stimulus contrasts of 50% and above, although the saturation contrast varies with layer and with different criteria. Following visual stimulation with gratings of 8% contrast, stimulus-driven uptake was relatively high in striate layer 4Ca (which receives primary input from the magnocellular LGN layers), but was absent in layer 4Cb (which receives primary input from the parvocellular layers). In this same (magnocellular-specific) stimulation condition, striate layers 4B, 4Ca, and 6 showed strong stimulus-induced DG uptake, and layers 2, 3, 4A, and 5 showed only light or negligible uptake. By comparison to other cases that were shown stimuli of systematically higher contrast, and to a wide variety of DG cases shown very different stimuli, it is evident that information derived from the magnocellular and parvocellular layers in the LGN remains partially, or largely, segregated in its passage through striate cortex, and projects in a still somewhat segregated fashion to different extrastriate areas. The sum of all available evidence suggests that the magnocellular information projects strongly through striate layers 4Ca, 4B, and 6, with moderate input into the blobs in layers 2 + 3, and to blob-aligned portions of layer 4A. Parvocellular-dominated regions of striate cortex include both the blob and interblob portions of layers 2 + 3, 4A, 4Cb, and 5. Because the major striate input to V2 arrives from striate layers 2 + 3, and because the major striate input to MT originates in layer 4B and 6, it appears that area V2 receives information derived largely from the parvocellular LGN layers, and that area MT receives information derived mainly from the magnocellular layers.

Diphenylhydantoin blocks cardiac calcium channels and binds to the dihydropyridine receptor.

Diphenylhydantoin was studied for its effects on Ca currents in single isolated guinea pig ventricular cells. The whole-cell patch-clamp technique was used, and Ca currents were studied after suppressing Na and K currents. At low frequencies (0.1 Hz) and negative holding potentials (-50 mV), diphenylhydantoin produced a concentration-dependent decrease in Ca currents without any significant change in the current-voltage relations. Half blocking effect occurred at 2 X 10(-4) M. The effects of diphenylhydantoin on Ca currents were dependent upon the holding potential. Inactivation curves for Ca currents were shifted to more negative potentials by the drug. The recovery of Ca currents from inactivation was prolonged by diphenylhydantoin, and the repriming of the current displayed an additional component, attributed to slow release of the drug from the channels. The voltage-dependent block was attributed to preferred binding by the inactivated channel state. Diphenylhydantoin also blocked specific [3H]-nitrendipine binding to guinea pig ventricular membrane preparations. The inhibition of [3H]-nitrendipine binding by diphenylhydantoin was competitive. Diphenylhydantoin also blocks cardiac Na channels in a voltage-dependent manner. We suggest that diphenylhydantoin binding sites exist on both Ca and Na channels.

Arrangement of the subunits of the nicotinic acetylcholine receptor of Torpedo californica as determined by alpha-neurotoxin cross-linking.

[3H]Methyl-alpha-neurotoxin prereacted with dithiobis(succinimidyl propionate) (DTSP) can be covalently linked to each of the subunits of the nicotinic acetylcholine receptor in membranes from the electric tissue of Torpedo californica. Pronounced changes in the cross-linking pattern are observed upon prior incubation with receptor specific ligands and upon reduction and/or alkylation of the receptor. d-Tubocurarine has been shown to bind to two different sites in receptor-rich membranes. These sites are present in equal numbers but have different affinities [Neubig, R. R., & Cohen, J. B. (1979) Biochemistry 18, 5464-5475; Sine, S., & Taylor, P. (1981) J. Biol. Chem. 256, 6692-6699]. Using d-tubocurarine inhibition of [3H]-methyl-alpha-neurotoxin binding, we demonstrate two inhibitory constants for d-tubocurarine of 67 +/- 21 nM and 4.9 +/- 1.7 microM in unreduced membranes. We utilize the large difference in Ki's to preferentially block toxin cross-linking at the high affinity site for d-tubocurarine. Low concentrations of this competitive antagonist selectively block the cross-linking of toxin to the beta and gamma subunits of the receptor, suggesting that these subunits are located close to the toxin binding site which is also the high-affinity binding site for d-tubocurarine. Reduction of disulfide bonds alters the affinity of the receptor for alpha-neurotoxin. Alterations are also seen in the cross-linking pattern of DTSP-activated [3H]methyl-alpha-neurotoxin to reduced and alkylated membranes in the presence of tubocurarine. The constants for d-tubocurarine inhibition of [3H]methyl-alpha-neurotoxin binding to reduced and alkylated membranes are 172 +/- 52 nM and 2.4 +/- 0.4 microM. The effects of bromoacetylcholine, carbamoylcholine, gallamine, and procaine on the cross-linking pattern are also examined. Our observations are consistent with an arrangement of the subunits in the membrane of alpha beta alpha gamma delta.

A plasma membrane-enriched preparation from giant barnacle muscle fibers.

We describe a procedure for obtaining a highly enriched plasma membrane (sarcolemmal) preparation from muscle fibers of the giant barnacle (Balanus nubilus). The sarcolemmal-enriched portion migrated as a light fraction (F1) at the 10-24% sucrose interface. This fraction displayed saturable ouabain binding (Kd = 0.119 microM) that was enriched 10 times compared to that in the original homogenate. F1 was also prepared using muscle fibers previously labeled with 1,2-ditritio-1,2(2,2'-disulfo-4,4'-diisothiocyano)diphenylet hane, disodium salt [( 3H]-H2DIDS). F1 was enriched 25-fold in [3H]H2DIDS binding sites with respect to the homogenate. Ca2+-ATPase and succinic dehydrogenase-activities were low in F1, as was oxalate-supported Ca2+ uptake. Compared to membranes of sarcoplasmic reticulum origin, F1 was enriched in sarcolemma membranes by about 45-fold while it was enriched approximately 30-fold over mitochondrial membranes. Thus, F1 provides an extremely pure source of external muscle membranes.