Measurements and observations of five early events of fertilization, singly and in pairs, from single sea urchin eggs have revealed the precise temporal sequence and spatial distribution of these events. In the Arbacia punctulata egg, a wave of surface contraction occurs coincident with membrane depolarization (t = 0). These two earliest events are followed by the onset of a rapid, propagated increase in cytoplasmic-free calcium at ~23 s as measured by calcium-aequorin luminescence. The luminescence reaches its peak value by 40 s after the membrane depolarization. The luminescence remains uniformly elevated for some time before its decay over several minutes. The onset of an increase in the pyridine nucleotide (NAD(P)H) fluorescence follows the membrane depolarization at ~51 s. The fertilization membrane begins its elevation in a wave-like fashion coincidentally with the increase in NAD(P)H fluorescence. Similar results are observed in the Lytechinus variegatus egg.The results suggest that while the increase in cytoplasmic-free calcium may be important for many changes occurring in the egg, the elevated-free calcium is not directly responsible for the propagated wave of cortical granule exocytosis.Fertilization initiates various physiological, biochemical, and structural changes within the egg that occur in a well-orchestrated manner both temporally and spatially. These events are requisite for normal embryonic development (8, 9). Included among the early events of fertilization are sperm-egg fusion, membrane depolarization, a surface contraction, an increase in cytoplasmic free calcium, the exocytosis of cortical granules, and a change in the pyridine nucleotide redox state. To some extent the sequence of these early events is known (8,12,19,26).That we might better understand the significance and casual relationships among the various events, we have examined simultaneously several of the events that occur within single sea urchin eggs at fertilization. The use of single eggs allows for the precise determination of the sequence of events which is unobtainable from studies that employ a population of eggs. Specifically, we have examined the temporal and spatial relationships among the fertilization potential, surface contraction, increase in cytoplasmic free calcium, NAD(P)H fluorescence, and elevation of the fertilization membrane.The relationships among the various early events have been largely inferential although some work has been reported from single cells (12,25,26). We are principally concerned with when the increase in cytoplasmic-free calcium occurs relative to the other events. The increase in cytoplasmic-free calcium has been associated with sperm-egg fusion, initiation of the fertilization potential, the surface contraction, and, of course, the cortical reaction and fertilization membrane elevation (4, 8-11, 18, 20, 28, 29, 32). A precise determination of the temporal sequence of the early events in single eggs would help clarify the role, if any, for calcium in each of the other events....

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As in previous studies in cell culture, altered mRNA and/or the functional expression of a specific subunit of the sodium channel (SkM2) was found in biopsy sections of muscle from all 12 persons examined who were susceptible to malignant hyperthermia but in none of the 16 nonsusceptible participants. Human malignant hyperthermia is a heterogeneous disorder, and the down-regulation of SkM2 may be involved in the final common pathway through which mutations in any one of several proteins, including the ryanodine receptor, could render a person susceptible.

Voltage-activated sodium channels of cultured skeletal muscle show diametrically divergent responses to intracellular vs. extracellular exposure to free fatty acids. Intracellular exposure to 1-20 microM arachidonic acid increased the magnitude of voltage-activated sodium currents, but not potassium currents, in whole cell recordings of human primary muscle cells and in the C2C12 mouse cell line. Oleic and stearic acids also stimulated increased sodium currents. In contrast, extracellular exposure to 5-10 microM arachidonic acid reversibly inhibited inward currents. Externally applied oleic acid was a less effective inhibitor, and stearic acid (up to 20 microM) produced no inhibition. The difference in sodium current responses to intracellular vs. extracellular exposure indicates that fatty acids can modulate skeletal muscle sodium channel function by at least two different pathways.

Free fatty acids (FFAs), including arachidonic acid (AA), are implicated in the direct and indirect modulation of a spectrum of voltage-gated ion channels. Skeletal muscle sodium channels can be either activated or inhibited by FFA exposure; the response is dependent on both FFA structure and site of exposure. Recombinant human skeletal muscle sodium channels (hSkM1) were transfected into heterologous human renal epithelium HEK293t cells. Cytoplasmic delivery of 5 M AA augmented the voltage-activated sodium current of hSkM1 channels by 190% (؎54 S.E., n ‫؍‬ 7) over a 20-min period. Similar results were seen with 5 M oleic acid. Sodium currents in HEK293t cells transfected with human cardiac muscle sodium channels (hH1) were insensitive to AA treatment, and exposure to oleic acid inhibited the hH1 currents over a 20-min period by 29% (؎13 S.E., n ‫؍‬ 5). The increase in hSkM1 current was not accompanied by shifts in voltage dependence of activation, steady-state inactivation, or markedly altered kinetics of inactivation of the macroscopic current. The FFA-induced increase in sodium currents was not dependent on protein kinase C activity. In contrast, both isoforms were reversibly inhibited by external application of unsaturated FFA. Thus, the differential effects of FFA on skeletal muscle sodium channels first noted in cultured muscle cells can be reproduced by expressing recombinant sodium channels in epithelial cells. Although the responses to applied FFAs could be direct or indirect, we suggest that: 1) SkM1 has two classes of response to FFA, one which produces augmentation of macroscopic currents with intracellular FFA, and a second which produces inhibition with extracellular FFA; 2) H1 has only one class of response, which produces inhibition with extracellular FFA. A testable hypothesis is that the presence or absence of each response is due to a specific structure in SkM1 or H1. These specific structures may directly interact with FFA or may interact with intermediate components.