Joseph F. Gennaro

Reversible depletion of synaptic vesicles induced by application of high external potassium to the frog neuromuscular junction.

1. Reversible depletion of synaptic vesicles from frog cutaneous pectoris neuromuscular junctions was studied by application of a Ringer solution containing 115 m M‐K propionate.

Noncholinergic, Saturable Binding of (±)-[3H]Nicotine to Mouse Brain

(+/-)-[3H]Nicotine was bound saturably to crude particulate, and synaptosomal-mitochondrial fraction from mouse brain. Scatchard and Hill plots of the binding data are in agreement with the existence of two independent classes of binding sites with high (Kd of 0.1-0.4 microM) and low(Kd is 20 microM) affinities, although negative cooperativity or a two-step model of ligand-receptor interaction cannot be ruled out. Nicotinic or muscarinic agonists and antagonists had little or no affinity for the nicotine binding sites, suggesting that nicotine binds in brain to noncholinergic sites. The binding did not display stereospecificity; this is consistent with the similarity in the pharmacological effects of (-)- and (+)-nicotine. Our results indicate that binding studies with [3H]nicotine should be interpreted with extreme caution.

Superoxide Radical-Mediated Alteration of Synaptosome Membrane Structure and High-Affinity γ-[14C]Aminobutyric Acid Uptake

Abstract: Mouse cortical synaptosomal structure and function are altered when exposed to hypoxanthine/xanthine oxidase (HPX/XOD)‐generated active oxygen/free radical species. The structure of both the synaptic vesicle and plasma membrane systems are altered by HPX/XOD treatment. The alteration of synaptic vesicle structure is exhibited by a significant increase in the cumulative length of nonsynaptic vesicle membrane per nerve terminal. With respect to the nerve terminal plasma membrane, the length of the perimeter of the synaptosome is increased as the membrane pulls away from portions of the terminal in blebs. The functional lesion generated by HPX/XOD treatment results in a reduction in selective high‐affinity γ‐[14C]aminobutyric acid (GABA) uptake. Kinetic analysis of the reduction in high‐affinity uptake reveals that the V.max is significantly altered whereas the Km is not. Preincubation with specific active oxygen/free radical scavengers indicates that the superoxide radical is directly involved. This radical, most probably in the protonated perhydroxyl form, initiates lipid peroxidative damage of the synaptosomal membrane systems. Low‐affinity [14C]GABA transport is unaltered by the HPX/XOD treatment. The apparent ineffectiveness of free radical exposure on low‐affinity [14C]GABA transport coupled with its effectiveness in reducing high‐affinity transport supports the idea that two separate and different amino acid uptake systems exist in CNS tissue, with the high‐affinity being more sensitive (lipid‐dependent) and/or more energy‐dependent (Na+, K+‐ATPase) than the low‐affinity system.

Effect of cigarette smoke on protein synthesis in brain and liver.

Abstract The rates of protein synthesis in brain and liver were determined in 2 inbred mouse strains (BALB/c and CXBH) during 1 hr of smoke exposure, and after 3 and 6 days of cigarette smoke treatment. Exposure to cigarette smoke reduced valine incorporation into brain and liver protein by 12 and 30% respectively. The greatest part of this reduction in synthesis was due to the hypothermie effect of smoke exposure, which was an approx. 8% change in the synthesis rate for each degree of reduction in body temperature, though a significant smoke effect was still evident. Two major components of cigarette smoke, nicotine and carbon monoxide, were individually tested. Injections of nicotine produced a similar inhibition of brain protein synthesis, with no effect on liver protein synthesis. After extraction of nicotine from the smoke by use of a Cambridge filter, only slight inhibition of brain protein synthesis was observed, which was due to the decrease in body temperature; there was still a significant inhibition in the liver. Incorporation measured, not during but after smoke exposure, was still significantly reduced, although reduction in the liver was smaller than that measured during smoke exposure. Carboxyhemoglobin at levels 50% higher than that achieved by smoke exposure had no effect on brain or liver protein synthesis; higher carboxyhemoglobin levels (300–400% higher than levels during smoke exposure) produced inhibition of liver protein synthesis. The results suggest that the slight and significant inhibition of brain protein synthesis is due to nicotine, whereas the effect on the liver is probably due to anoxia. Smoke treatment for 3–6 days suggests that there is no adaptation to these effects. Strain differences in smoke sensitivity are not related to the effect of nicotine on protein synthesis, suggesting that other mechanisms are involved in smoke sensitivity.

Isolation of Highly Enriched Preparations of Two Types of Mucosal Cells of the Turtles Urinary Bladder

Summary Enriched preparations of the two major cell types of the mucosal epithelium of the turtles urinary bladder can be obtained by density gradient centrifugation. Carbonic anhydrase activity is much greater in the mitochondria-rich than in the granular cell, suggesting the former cell type is the site of acetazolamide-sensitive transport. This technique should be useful in further defining the biochemical and transport properties of epithelial tissues composed of heterogeneous mucosal cells.

Quantitative Structural Aspects of the Renal Glomeruli of Hypertensive Mice

Kidneys of genetically hypertensive and normotensive mice were studied with respect to kidney weight, number and volume of glomeruli, and filtration surface area. The hypertensive animals have a larger kidney weight to body weight ratio and possess fewer glomeruli per kidney. The superficial cortical glomeruli of the hypertensives are slightly smaller in volume than those of the normotensives although the filtration surface area is similar. The juxtamedullary glomeruli of the hypertensives are both smaller in volume and have less filtration surface area. The possible implications of these findings in the development and/or maintenance of hypertension are discussed.

ELECTROKINETIC PROPERTIES AND MORPHOLOGIC CHARACTERISTICS OF AMPHIBIAN GASTRULA CELLS

Morphogenetic movements, the patterned translocation of cells, cell masses, or epithelia in the form-building processes, constitute a major feature of animal development. In the study of morphogenetic movements of amphibian gastrulation, the contributions of H~l t f re te r l -~ established the basis for much of contemporary experimental approaches to the analysis of these movements. Holtfreter proposed that gastrula and neurula morphogenesis is related to differential and selective affinities among the presumptive germ layer cell populations. Steinberg’s “Differential Adhesion Hypothesis”‘ suggests that quantitative differences in intercellular adhesiveness of each of the presumptive germ layers is the basis for Holtfreter’s differential and selective affinities and that such differences could account for gastrula and neurula morphogenetic movements. Both Holtfreter’s and Steinberg’s conceptions focused attention on the cell periphery as the site of important morphogenetic properties. I t was in this context that we initiated some investigations of the cell periphery of amphibian embryo cells prior to, and during, the onset of gastrula morphogenetic movements. Among the characteristics of the cell periphery that can be investigated, we have examined two: electrical potential and morphologic features of the surfaces of cells involved in gastrula morphogenetic movements. These two aspects of the cell periphery possess some relationship to intercellular contact and adhesion. Among the many factors involved in cell migration, two that are indispensable are adhesion to a cellular or noncellular substratum5 and intrinsic locomotor capability. Although there may be several modes of cell locomotion,e it would be necessary, in any case, for part of the cell periphery to be stabilized, via adhesion, so that more mobile regions could extend in one direction or another and establish adhesive contact at some point distal to the cell! The initially stabilized region could then be released from adherent restraint, and the cell could translocate toward the new point of attachment. Continued movement would involve repetition of this sequence. Failure of a cell to migrate could be due, therefore, to an inability to make, break, or alter adhesive contact or to an inactive intrinsic locomotor system. Conversely, the onset of movement could involve acquiring the ability to make and break adhesive contacts, alter the strengths of existing contacts, and activate the cell’s locomotor apparatus. The amphibian blastula is a sphere that consists of an epithelium of uneven thickness, which surrounds an eccentric cavity, the blastocoel (see FIGURE 1). In terms of migration, the blastula is quiescent. With the onset of gastrulation, exten-

Ultrastructural localization in the mouse lung of venom from the western diamondback (Crotalus atrox) rattlesnake

Procedures which make immune complexes between venom antigens and their complementary antibodies visible have been applied to detect the site of deposition of rattlesnake venom in the lung tissue of mice after in vivo envenomation. Lung tissue, from mice envenomated with reconstituted but otherwise unmodified Crotalus atrox venom, was incubated in commercially available polyvalent antiserum (against North American crotalid snakes) which had been conjugated to the enzyme horseradish peroxidase. The enzyme reaction was developed for visualization by transmission electron microscopy. The enzyme reaction products were located along alveolar surfaces and were associated with multilamellar bodies in cytosomes of type II pulmonary epithelial cells. It was concluded that the venom has a specific affinity towards the extracellular surfactant in the lung and towards intracellular sites of surfactant synthesis.

Damage to the high-affinity γ-aminobutyric acid (GABA) uptake system in mouse brain by horseradish peroxidase (HRP)

Presynaptic nerve terminals when depolarized are sensitive to morphological and functional alteration by horseradish peroxidase. Mouse brain slices, 0.1 mm, depolarized by a K(+)-HEPES buffer and exposed to horseradish peroxidase exhibited alterations in both synaptic vesicle membrane structure and in high-affinity [(14)C]?-aminobutyric acid uptake. The post stimulatory retrieval of synaptic vesicles from the nerve terminal plasma membrane in the presence of horseradish peroxidase resulted in a decrease in the synaptic vesicle population with a concurrent increase in non-synaptic vesicle membrane structures. High-affinity [(14)C]?-aminobutyric acid uptake into 0.1-mm slices of mouse cerebral cortex and ponsmedulla-spinal cord was inhibited by 31% and 24%, respectively, after incubation for 60 min in K(+)-HEPES buffer containing horseradish peroxidase. Superoxide dismutase protected both the synaptic vesicle membrane and the high-affinity uptake system from the deleterious effects of horseradish peroxidase, pointing to the possible involvement of superoxide anion radicals in the horseradish peroxidase-related effects. These horseradish peroxidase induced alterations appear to be directed towards the exposed synaptic vesicle membrane, since non-stimulated brain slices exposed to horseradish peroxidase do not exhibit a reduction in either high- or low-affinity [(14)C]?-aminobutyric acid uptake. Low-affinity uptake of [(14)C]?-aminobutyric acid and [(14)C]?-aminoisobutyric acid into cortical slices was not affected after incubation in K(+)-HEPES with horseradish peroxidase. Low-affinity uptake, however, is reduced by the high-K(+)/Na(+)-free stimulatory incubation prior to uptake. It appears, thus, that high- and low-affinity uptake are distinct and different systems, with the high-affinity transport system structurally associated with synaptic vesicle membrane.

PROPERTIES OF A TRANSPORTING EPITHELIUM ISOLATED FROM THE CANINE KIDNEY

The mammalian kidney is composed of a number of different types of cells which function as a unit to maintain the volume and composition of the body fluids within narrow limits. This morphologic complexity and the anatomical configuration of the nephron have compelled investigators to use a number of simpler model systems including: kidney slices,3 micropuncture and microperfusion of specific tubular segment~ ,~ , drugs with a high specificity for certain tubular segments and/ or physiological processes,6 urinary bladders of amphibia and reptilia,? and most recently, kidney epithelial cells in tissue culture.8, One of the attractions to the use of epithelial cells grown in culture is the greater control of the external milieu and the higher degree of cell homogeneity. We have used a hormonally defined serum-free medium to isolate and grow canine kidney tubular epithelioid cells in culture as reported previously.1° Briefly, small pieces of the kidney cortex of adult dogs were minced and the cells were dissociated using a collagenase mixture. Cell suspensions were plated either in hormonally defined serum-free medium ( 5 pg/ml insulin, 25 ng/ml prostaglandin E,, 5 X M hydrocortisone, 5 x 10-l2 M triiodothyronine, and 5 pg/ml transferrin) or medium containing 5 % fetal calf serum. In order to determine cell doubling times, 2 X 1oj dissociated cells per 25-cm2 flask were plated in either hormonally defined serum-free medium or in serum-containing medium. Cells were removed by trypsinization and counted using a hemocytometer. Each point was the average of three measurements. Scanning electron microscopy of the cultured cells was performed on cells which were fixed for 20 min in 3% glutaraldehyde, PIPES buffer (pH 7.40), and 0.05 M calcium chloride and then post-fixed with 1% osmium tetroxide for 20 min. Cells were dehydrated in a graded series of ethanols. critical-point dried, and coated with gold-palladium. Serum-containing cell cultures grew more rapidly than the hormonally defined serum-free medium grown cells (FIGURE 1 ) with cell doubling times of 23 h and 40.6 h, respectively. This difference in growth rates can be attributed to the isolation and growth of two different cell populations. Cells grown in hormonally defined serum-free medium form “tight” epithelioid colonies ( RGURE 2) while serum-grown cells rarely form a “tight” epithelium and are eventually overgrown by fibroblastic cells (FIGURE 3). The major epithelioid