Frances V. McCann

Ultrastructure of the myocardium of the moth, Hyalophora cecropia

Abstract The tubular heart of the moth, Hyalophora cecropia , is composed of a single layer of striated muscle cells linked together by intercalated disks. The intercalated disk can be resolved into two components: interfibrillar junction and septate desmosome. The width of the cells varies from 10 to 30 μ and the cell length is 60 to 100 μ. The A Bands of the heart muscle are 1·8 μ long. The diameter of the thick filaments is 200 A and that of the thin filaments 60 A. Each thick filament in the overlap region in the A Band is surrounded by an orbit of ten to twelve thin filaments. Transverse tubules and sarcoplasmic reticulum vesicles are irregularly spaced throughout the cells. Sections of the heart from different abdominal segments were studied, and no significant differences were observed in cell size, sarcomere structure, distribution of mitochondria, transverse tubules and sarcoplasmic reticulum, or in the components of the intercalated disk in any of the cells. The physiological significance of these observations is discussed.

UNIQUE PROPERTIES OF THE MOTH MYOCARDIUM

Because of certain peculiar features, viz., a simple structure, the periodic reversal of direction of heart beat, and an unusual ionic milieu, the myocardium of the adult moth is an intriguing subject for the study of fundamental problems of cardiac excitability. Research attention has thus far been focused in three general areas: the anatomical and histological features of the muscular organization of the tubular heart, the nature of excitation and conduction in the forward and reverse directions, and the roles of various cations and anions in maintaining and regulating the excitability of the myocardial cell. The adult moth used in these studies, Samia cecropia, is pictured in FIGL’RE 1A. The pupal forms are collected in the early autumn and then placed under refrigeration and high humidity during the period of arrested development or “diapause.” Two to three months later the diapause is broken by placing the pupae in a warm moist atmosphere. The progression of the life-span through complete metamorphosis reaches its terminal stages as the adult form emerges from the cocoon and attains a fully developed wing-spread of about six inches. The adult stage is ephemeral, lasting about five to seven days. During this time the adult moth ingests neither food nor water, and as the life-span runs its course the insect becomes remarkably dehydrated. The moth heart preparation is shown in FIGURE 1B. The insect has been anesthetized with carbon dioxide, decapitated, and the legs and wings removed. A midventral incision has exposed the heart which is shown with a cannula inserted into the caudal end and threaded forward in the cephalad direction. Great care is taken during dissection in order to disturb as little as possible the tracheolar system which serves the respiratory needs of the tissues. Thus, the moth heart can be studied in situ and the preparation remains viable for many hours. The tubular heart courses a distance of 3 to 4 cm. from the midthoracic region to the caudal end of the abdomen and is suspended by muscle fibers attached in a fan-like overlay at segmental intervals. These alary muscles run laterally to the body wall and aid in re-expanding the heart following systole. The heart is perforated by eight pairs of ostia through which the hemolymph circulates. The open circulatory system does not require very large pressures and, since no respiratory function is assigned to the hemolymph, maximum regular contractions of the heart do not seem essential.

Ultrastructure of moth alary muscles and their attachment to the heart wall

Abstract Evidence is presented to show that the alary muscles of the adult moth Hyalophora cecropia have ultrastructural characteristics of striated muscle cells. A-Band width averages 5·5 μ and ten to twelve thin filaments are localized around each thick filament. There is, however, a wide variation in the diameter of the thick filaments, i.e. 100 to 325 A. The alary muscles share certain structural features with ‘slow’ striated muscles in the diminutive number of transverse tubules and reduced sarcoplasmic reticulum. The muscle fibres send out branches which are joined to the heart wall by means of a myomuscular junction, the physiological significance of which is discussed.

Fine structure of the pericardial cells of the moth, Hyalophora cecropia, and their rôle in protein uptake

Abstract Structural characteristics of pericardial cells of the moth, Hyalophora cecropia , include multiple nuclei, an extensive network of extracellular channels, and a profusion of coated vesicles, vacuoles, and tubular elements. Pericardial cells exposed to ferritin take up the protein via coated vesicles present in the cell. Ferritin granules are also found in the tubular elements and vacuoles even though only the coated vesicles are observed attached to the plasmalemma and only tubular elements are found attached to vacuoles. It is suggested that coated vesicles can be converted to tubular elements which then transport the ingested protein to vacuoles.

Calcium action potentials in insect myocardial fibers

Abstract 1. 1. Electrical activity in single myocardial cells of an insect was measured in relation to the amount of calcium ([Ca2+]o) in physiological test salines. 2. 2. Action potentials are reduced to 0 mV in zero [Ca2+]o and increase 22 mV per tenfold increase in [Ca2+]o. 3. 3. Action potentials are inhibited by 2 mM Cl2 while the resting potential is not altered. 4. 4. It is concluded that action potentials in the myocardial cells of the moth Hyalophora cecropia are “calcium-spikes”.

Characterization of a large conductance non-selective anion channel in B lymphocytes

Studies on ion channel currents in freshly isolated murine B lymphocytes with the patch clamp technique revealed the presence of a non-selective anion channel of large conductance in inside-out (i/o) patches. This channel is characterized here according to its unitary conductance, ion selectivity, regulatory factors, distribution and kinetic behaviour. With a unitary conductance of 348 +/- 4.4 pS in a normal physiological ion gradient, it exhibited an indiscriminate selectivity to cations (Na+ and K+). Selectivity to chloride over sodium was established by substitution of high concentrations of NaCl (450 mM) in the bath (i/o patches), resulting in a selectivity ratio (PCl/PNa) of 33. Selectivity to chloride over potassium was confirmed in a similar manner by substitution of TEA-Cl for KCl, yielding a selectivity ratio (PCl/Pk) greater than 80. Conductance of aspartate through the channel demonstrated the non-selective nature of this anion channel. Voltage proved to be a regulatory factor but other influences on channel activity were also present, including the configuration of the patch (channel is inactive in cell attached patches), and the enhancement of activity at negative membrane voltages by previous pulsing. Intracellular levels of calcium (i/o patches) did not appear to control channel conductances or regulate kinetic activity. Kinetic behaviour of this channel was complex, with periods of bursting and flickering activity interspersed with prolonged closed/open intervals. Multiple subconductance states were also present. The complex properties and behaviour of this channel suggest a possible role in signal transduction in B cell activation.

MERCURY SUPPRESSION OF A POTASSIUM CURRENT IN HUMAN B LYMPHOCYTES

Mercury is a recognized environmental toxin. Several organ systems are targeted by this substance and impairment of immune function is known to result from exposure to mercury. Using the patch clamp technique in the whole cell configuration on resting human B lymphocytes we have identified an outward potassium current and studied the effects of mercury on this current. We present data that demonstrate: (i) the absence of inward currents; (ii) a time and voltage dependent outward current with a threshold of -40 mV and reversal potential near EK+; (iii) blocking of this current by TEA (tetraethylammonium chloride) in a dose dependent manner; (iv) a slow time course for recovery from inactivation of this outwardly rectifying K+ current and, (v) the diminution and final block of this potassium current by mercury. These data supplement the findings from our laboratories that demonstrate inhibitory effects on B cell activation by mercury. We propose that the movement of potassium ions across the B cell membrane, an event presumed to be one of the first signals in the mitogenic process, is a target of mercury toxicity.

Ion channels in human macrophages compared with the U-937 cell line

SummaryThe human cell line U-937 has been used extensively to model many macrophage functions. We have examined the cell membranes of human monocyte-derived macrophages (HMDM) and U-937 cells to compare membrane properties as expressed by single ion channel currents. The patch-clamp technique was applied to isolated, nonactivated, inside-out patches of cell membranes obtained from HMDM and from the U-937 cell line. Voltage-gated potassium channels of similar conductance but different kinetics are present in both types of cells, and a calcium-activated potassium channel is present only in the HMDM. These differences in ion channel properties suggest fundamentally different behavior between these two cell types at the level of the cell membrane.

Neuromuscular transmission in insects: effect of injected chemical agents.

Abstract Electrical activity was measured in single muscle fibers of the fly Sarcophaga bullata during treatmnet with several pharmacological agents known to modify vertebrate neuromuscular transmission. Drugs were administered by two routes, viz. topical application and intra-abdominal injection. 1. 1. Acetylcholine, eserine and GABA had no effect when topically applied. 2. 2. Acetylcholine and glutamic acid caused depolarization when injected, while eserine provoked repetitive spontaneous firing. 3. 3. In view of the differential effects observed with respect to the route of drug administration, the role of acetylcholine in neuromuscular transmission in insects may require reconsideration.

PRESENCE OF FUNCTIONAL NMDA RECEPTORS IN A HUMAN NEUROBLASTOMA CELL LINE

Data are presented that provide convincing evidence for the expression of structurally normal and functional NMDA receptors by acetylcholine-producing human LA-N-2 neuroblastoma cells in culture. Reverse transcription and polymerase chain reaction (RT-PCR), followed by cloning and DNA sequencing, revealed the presence in these cells of mRNA representing the key subunit, NMDAR1, of the receptor. This mRNA was further demonstrated by Northern analysis to be the same size as that described for human neurons. The neutral red cytotoxicity assay was utilized to examine the influence on these neuroblastoma cells of a 48-h incubation with either L-glutamic acid or the specific NMDA agonist N-phthalamoyl-L-glutamic acid (NPG). Cell cytotoxicity was shown by this assay to be increased through incubation with glutamate at 1 and 10 mM by 27 and 37%, and through incubation with NPG at 0.1 and 1 mM by 28 and 46%. A possible mechanism of these toxic effects was further evaluated using the whole-cell configuration of the patch-clamp technique and the specific NMDA agonists (+/-)1-aminocyclobutane-cis-1,3-dicarboxylic acid (ACDA) and NPG. Using this procedure, a voltage-dependent tetrodotoxin-sensitive inward sodium current was found to be increased (x 1.5) by L-glutamic acid and by both NMDA agonists in the presence of glycine. Another voltage-gated inward current, probably carried by calcium ions, was increased three- to fourfold. Hence, these glutamate activities observed in human LA-N-2 neuroblastoma cells appear to occur through the activation of functional NMDA receptors in much the same way as reported for neurons, and both glutamate and NMDA agonists can be toxic to these neuroblastoma cells. Our findings, therefore, suggest this cell line will provide a model suitable for investigating the mechanisms of NMDA-related long-term potentiation (LTP) in neurons and of the NMDA-related neurotoxic effects of glutamate in disease states that involve a reduction in cholinergic function.