Theodore A. Hall

X-ray microanalysis of elements in frozen-hydrated sections of an electrogenic K+ transport system: the posterior midgut of tobacco hornworm (Manduca sexta) in vivo and in vitro

SummaryThe lepidopteran midgut is a model for the oxygendependent, electrogenic K+ transport found in both alimentary and sensory tissues of many economically important insects. Structural and biochemical evidence places the K+ pump on the portasome-studded apical plasma membrane which borders the extracellular goblet cavity. However, electrochemical evidence implies that the goblet cell K+ concentration is less than 50mm. We used electron probe X-ray microanalysis of frozenhydrated cryosections to measure the concentration of Na, Mg, P, S, Cl, K, Ca and H2O in several subcellular sites in the larval midgut ofManduca sexta under several experimental regimes. Na is undetectable at any site. K is at least 100mm in the cytoplasm of all cells. Typicalin vivo values (mm) for K were: blood, 25; goblet and columnar cytoplasm, 120; goblet cavity, 190; and gut lumen, 180. The high K concentration in the apically located goblet cavity declined by 100mm under anoxia. Both cavity and gut fluid are Cl deficient, but fixed negative charges may be present in the cavity. We conclude that the K+ pump is sited on the goblet cell apical membrane and that K+ follows a nonmixing pathway via only part of the goblet cell cytoplasm. The cavity appears to be electrically isolated in alimentary tissues, as it is in sensory sensilla, thereby allowing a PD exceeding 180 mV (lumen positive) to develop across the apical plasma membrane. This PD appears to couple K+ pump energy to nutrient absorption and pH regulation.

The localization and assay of chemical elements by microprobe methods

The principle of the microprobe analysis of chemical elements is illustrated in Fig. i. Some kind of radiation is directed on to the specimen, generating signals characteristic of the elements present. Local analysis in situ is achieved in one of two ways. Most often the impinging beam is finely focused so that the signal at any moment comes only from a selected microregion. Alternatively, in some instruments, the impinging beam floods a larger region but the emergent signals characteristic of a particular element may be selected and focused to give an elemental ‘map’ or ‘image’.

Fine structure and X-ray microanalysis of mineralized concretions in the Malpighian tubules of the housefly, Musca domestica

The epithelium and the lumen of the Malpighian tubules of the housefly contains mineralized dense bodies called concretions. The morphological characteristics, mode of origin, nature of the sequestered elements and the age-associated changes in the distribution of concretions are reported. There are three types of concretions in the cytoplasm, which have been designated as type A, type B, and type C. Type A concretions are membrane-bound spherical structures which may arise by the gradual intravacuolar accumulation of dense material. Type B concretions appear to be related to multivesicular bodies. Type C concretions are heteromorphic and morphologically resemble the residual bodies. They show a positive localization of acid phosphatase reaction product. X-ray microanalysis of intracytoplasmic and intraluminal concretions revealed the presence of phosphorus, sulphur, chlorine, calcium, iron, zinc and copper. There was no evidence suggesting the extrusion of the intracytoplasmic concretions into the lumen of the Malpighian tubules. There is an age-associated increase in the distribution of type C concretions. It is hypothesized that the sequestration of metal ions within the concretions may provide a means for the effective excretion of these elements.

Origin, structure, composition and age-dependence of mineralized dense bodies (concretions) in the midgut epithelium of the adult housefly, Musca domestica

The epithelial cells of the midgut in 1-40 day old adult female houseflies were examined by electron microscopic X-ray microalnalytic and histochemical techniques in order to study the mode of genesis, chemical nature and age-associated distribution of dense bodies. Dense bodies contain high concentration of phosphorus sulphur, chlorine, calcium, iron and copper; they are therfore termed concretions. Concretionary material is initially deposited within Golgi vesicles, lamellar bodies and residual bodies. The average size of the concretion granules and the concentration of the sequestered material increases with age, while new concretions are continually formed throughout life. With advancing age, concretions accumulate in the epithelial cells and occupy a considerable proportion of the cytoplasm in old flies. It is postulated that the concretions sequester superfluous minerals and may play an important role in the excretory system of the adult housefly.

ELECTRON MICROPROBE X-RAY ANALYSIS OF CALCIUM*

For a proper understanding of the mechanisms of Ca2+ transport and the regulatory role of Ca2+ in cell function one needs to know (a) the Ca2+ activity in the external media and in the cytosol. and (b) the total concentration and distribution of calcium in various microcompartments in the cell as well as around the cell in complex tissues. Reliable measurements of the total concentration of calcium are difficult to obtain by the conventional methods of chemical microanalysis and impossible for intracellular organelles and narrow extracellular spaces without disrupting the cell organization and hence destroying the distribution of diffusible elements in vivo. To obtain such information one needs a cytochemical method which can provide a “snap shot” view with a quantitative “contour map” of elemental distribution of cells and tissues in vivo. Any cytochemical technique suitable to provide such information for a diffusible element like calcium must meet the following minimum requirements.’,* ( 1 ) The procedure to prepare the tissues should quantitatively preserve and should not allow redistribution of the element over distances more than the desired resolution of analysis. (2) It should adequately preserve the cellular fine structure and allow subsequent imaging at a suitable resolution. ( 3 ) It should be specific for the analyzed element (Ca) and permit reliable quantitative measurements. Electron microprobe x-ray analysis is a method that allows such measurements as the mass of total calcium (free as well as bound) per unit mass of the microvolume within the specimen in a nondestructive fashion. potentially at the morphological resolution of an electron microscope (EM) and with an x-ray analytical resolution down to 50 nm. The general principles of x-ray microanalysis as well as its application in biology have been extensively reviewed in several recent publications The quality of the analytical information depends to some extent on the instrumental arrangements used? but more critically on the procedures employed to prepare the biological samples. A recent survey of literature by Chandler12 reveals that there are well over 300 published studies on microprobe analysis of biological tissues in which Ca is one of the elements analyzed. At least 50% of these studies deal mainly with Ca. However, in most cases the cells and tissues have been prepared by some method of chemical fixation with or without the inclusion of a calcium-precipitating agent such as pyroantimonate, oxalate, and lead followed by

Microprobe study of toad urinary bladder in absence of serosal K

SummaryThe bulk of the intracellular potassium in mucosal epithelial cells from toad urinary bladder has been previously reported to exchange very slowly with the serosal medium, with a half-time of some 9 hr. This observation, based on chemical analyses of mucosal cell scrapings, has been reexamined with simultaneous diffractive and energy dispersive electron probe X-ray microanalysis. Fifty-three intracellular sites in hydrated sections and 286 sites in dehydrated sections were studied in bladders from eight toads under baseline conditions and after removal of serosal K+ for 83–133 min, with or without 10−2m ouabain. The baseline data confirm and extend previous examinations of the intracellular ionic composition, and provide the most direct measure of intracellular water thus far available for this tissue. Removal of serosal K+ reduced the intracellular K+ content by 20%, increased intracellular Na+ content threefold, and slightly reduced the intracellular Cl− and water contents, qualitatively consistent with published chemical analyses. The intracellular Na+ content of mucosal origin, measured by radioactive tracers and chemical analyses of cell scrapings, has been reported to be unchanged under these conditions Simultaneous addition of ouabain and removal of external K+ produced a dramatic fall in intracellular K+ of more than 80% in a third of the cells and reduced the mean intracellular K+ content by 60%; 20% of the cells appeared to retain K+ more effectively than the bulk of the epithelial cell population. We conclude that: (i) the low rate of net exchange of intracellular K+ with the serosal bulk solution primarily reflects recycling of K+ across the basolateral membranes, (ii) radioactive tracer and chemical measurements of the intracellular Na+ pool of mucosal origin substantially underestimate the total intracellular Na+ content under certain experimental conditions, and (iii) the epithelial cells display a functional heterogeneity of response to the effects of adding ouabain and withdrawing external K+.

Electron probe x-ray microanalysis of calcium and other elements in meiotic spindles, in frozen sections of spermatocytes from crane fly testes.

Abstract Crane fly spermatocytes were quench-frozen in Freon and 1 μm sections cut at −80 °C were studied using electron probe X-ray microanalysis. Light microscopic controls showed that the cells were normal at the instant they were quench-frozen. Two sections were analysed in detail: one was of a cell in prometaphase, the other was of a cell in late prophase, containing an aster. Ca, Na, K, Cl and P concentrations were determined for chromosomes, spindle, aster, cytoplasm, and nucleoplasm. In the spindle, Ca was about 6 mM/kg wet weight, which seems incompatible with the presence of microtubules: hence it was concluded that the Ca was not “free” but rather was sequestered. The data illustrate the potential of electron probe X-ray microanalysis for analysing Ca concentrations in meiotic and mitotic spindles to determine if Ca 2+ might be involved in the control of spindle activity.

Calcium and sulphur in neurosecretory granules and calcium in mitochondria as determined by electron microscope X-ray microanalysis

SummarySections of neurosecretory cells fixed in glutaraldehyde and osmium tetroxide were studied by means of an EMMA-4 analytical microscope. Secretory granules in neurosecretory cells of the corpus cardiacum and of the brain, both in the desert locust Schistocerca and in the blowfly Calliphora, as well as neurosecretory granules in posterior pituitaries of the frog Rana and of the albino rat all contain a high concentration of calcium. A distinct sulphur peak was also a constant feature.In neurosecretory cells of the corpus cardiacum of Schistocerca the chromatin contained a high concentration of calcium. The mitochondria also contained much calcium, but part of this disappeared during preparation except when fixative and wash contained calcium chloride. By block staining with uranyl acetate most calcium is displaced from the mitochondria, whereas most of the calcium remains in the neurosecretory granules. Since the calcium peaks in spectra from neurosecretory granules appear of similar size, regardless of variations in the preparative procedure, this calcium must be firmly bound. The possible role of the calcium bound to the neurosecretory substance is discussed.The presence of sulphur in insect neurosecretory granules indicates the presence of a protein besides the hormone, i.e., an insect neurophysin.

Effects of the high-molecular-weight cryoprotectant dextran on fluid secretion by Calliphora salivary glands

Abstract The rate of fluid secretion by isolated salivary glands of Calliphora was inhibited as a linear function of dextran and poly vinyl pyrrolidone concentrations in the range 15–35% w/w. This inhibition was not overcome by supramaximal concentrations of 5-hydroxytryptamine, nor was it caused by a decreased availability of K + from the medium. Although the polymers caused large decreases of freezing point (and vapor pressure) of the incubation medium, the glands did not respond to this by secreting a more K + -rich saliva. When dextran and polyvinyl pyrrolidone were added as powders to salt solutions, the total freezing-point depression of the mixture was equal to the sum of that exerted by the pure salt solution and that expected for the polymer concentration. The activities of K + and Cl − , as measured by ion-selective electrodes, were not increased in solutions by the addition of dextran. Dextran was demonstrated by electron microscopy to penetrate into the basal clefts and intercellular spaces of the isolated glands. These results demonstrate that addition of dextran (and probably of polyvinyl pyrrolidone) does not decrease the solvent activity of water in physiological salt solutions. The inhibition of fluid secretion by isolated salivary glands of Calliphora seems therefore due only to the altered physical characteristics of the medium.