^

(?) 1981 Emily A. Barron I

THE EFFECT OF NEURAMINIDASE ON CELL SURFACE AND BEAT RATE

OF AGGREGATED MYOCARDIAL CELLS

by

Emily Ann Barron, B.A.

A THESIS

IN

ANATOMY

Submitted to the Graduate Faculty of Texas Tech University Health Sciences Centers in Partial Fulfillment of the Requirements for the Degree of

MASTER OF SCIENCE I -—S}> r-3 , ACKNO\^^LEDGEMENTS hi

:•> ^*- !

I would like to thank Dr. Roger Markwald and Dr. Rick Nathan for

their direction on this thesis and to others. Dr. Jim Hutson, Dr.

Penelope Coates, Dr. W. G. Seliger, and Dr. Jack Yee, for their help­

ful criticism. I would also like to thank Mr. and Mrs. George F.

Barron for their encouragement during my graduate studies.

11 \

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ii

ABSTRACT iv

LIST OF FIGURES vi

I. INTRODUCTION 1

II. MATERIAL AND METHODS ^

Tissue Culture Techniques ^

Enzyme Preparations ^

Transmission Electron Microscopy 6

Scanning Electron Microscopy and

Energy Dispersive Analysis ^

Beat Rate Measurements °

III. RESULTS 10

Purity of Enzyme Preparations 10

Physiological Responses to Neuraminidase 10

Morphological Responses to Neuraminidase H

IV. DISCUSSION 30

V. CONCLUSIONS 35

LIST OF REFERENCES 36

111 ABSTRACT

Morphological as well as physiological changes occur in aggre­ gated embryonic chick myocardial cells after treatment with the sialic acid removing enzyme, neuraminidase CNMDase). Aggregates of seven day embryonic chick ventricular cells were prepared according to a proce­ dure developed by DeHaan, including a plating sequence to remove most of the contaminating cell types. While muscle cells made up the ma­ jority (90%) of the core of the aggregate, there was an incomplete layer of fibroblasts ClQ%) covering the periphery. These beating spherical climips of cells were then treated with two highly purified preparations of Clostridium perfringens neuraminidase. Roth prepara­ tions of enzyme were contaminated with variable amounts of a protein which electrophoretically comigrated with a known standard of

C^. perfringens phospholipase C. Therefore, phospholipase C was used as a control in both the beat rate and cell surface studies. The morphological effect of NMDase on the available myocardial cells pre­ sent at the aggregate's periphery was visualized using polycationic ferritin controlled by pH and acid methylation procedures. NMDase treatment (1.2 Units/ml for 2 hrs. at 37° C) on both prefixed and postfixed aggregates showed a reduction in stain at the cell surface.

Although lower concentrations or shorter treatment times with NMDase showed little morphological change from controls, physiological dif­ ferences in aggregate beat rate could be detected with only 0.03 U/ml after ten minutes. NMDase concentrations, 0.03, 0.5, and 1.2 U/ml, showed net decreases in beat rates over a sixty minute incubation. iv Within this period, the first thirty minutes displayed definitive de­ creases in aggregate beat rate while the rest of the hour was typified by arrhythmic contractions. This study demonstrates that NMDase re­

duces the number of anionic sites at the cell surface of embryonic myocardial cells and also reduces the aggregate beat rate.

KEY WORDS: Neuraminidase, Sialic Acid, Beat Rate, Cell Surface,

Myocardium LIST OF FIGURES

Figure 1. Seven day embryonic chick myocardial aggregate 5

Figure 2. Morphological and analytical experiments using Worthington neuraminidase 14

Figure 3. Control experiments using a contaminating enzyme, a sialic acid rich tissue, and an acid methylation treatment 17

Figure 4. Effects of low concentrations of Sigma neuraminidase on myocardial cell surfaces 20

Figure 5. Effects of a high concentration of Sigma neuraminidase on myocardial cell surfaces 22

Figure 6. Effects of neuraminidase on fixed myocardial cell surfaces 24

Figure 7. Summary diagram of PCF staining on myo­ cardial cell surfaces treated with low doses of Sigma neuraminidase 27

Figure 8. Summary diagram of PCF staining on prefixed and postfixed myocardial cell surfaces treated with a high dose of Sigma neuraminidase 29

VI INTRODUCTION

Neuraminidase (NMDase) is a hydrolase which cleaves the strongly anionic monosaccharide, sialic acid, from the nonreducing end of oligo­ saccharides and of the carbohydrate portion of glycoconjugates such as glycoproteins and glycolipids (3,10,18). The release of NMDase susceptible sialic acid from their terminal positions in sialocompounds may alter the behavior of these macromolecules. One example is the enzyme, hyaluronidase, which when extracted from chick embryo fibro­ blast culturing medium and treated with NMDase shows a decrease in thermal stability and a shift in elution profile that is characteristic of the cellular (probably lysosomal) form of the enzyme (25).

Sialic acids include a current total of twenty variably substi­ tuted N-acylated neuraminic acids, each of which contains a low pk carboxyl group (30,32). This strongly anionic group is the prime contributor of the net negative charge characteristic of sialoglyco- conjugates. The most common sialic acid, N-acetylneuraminic acid

(NANA), has been shown to have a high affinity for calcium ions at physiological pH (16). Therefore, by deduction, loss of sialic acid

(mainly NANA) from sialocompounds should result in a reduction of negative charge and loss of calcium binding potential.

When sialoglycoconjugates are members of the cell membrane, re­ moval of sialic acid engenders polymorphic consequences to living cells. Visual changes after NMDase treatment can be shown by stain­ ing membranes with cationic dyes such as lanthanum (9,13), colloidal iron (9,35), and polycationic ferritin (1,6,24). Functional changes 1 have been correlated with sialic acid liberation, also. Neuramini­ dase, previously known as the receptor destroying enzyme, has been re­ ported to inhibit antigen induced histamine release in rat peritoneal cells (2), increase lymphoid cell susceptibility to cytolysis by com­ plement (28), and inhibit glucose induced insulin release in pancreatic

6 cells (11).

In cardiac muscle cells, increasing evidence shows that calcium bound to anions at the cell surface may be important for activating

contraction (19). Langer and coworkers have reported that removal of

sialic acid in cultured rat heart cells selectively increases calcium

exchange (21). Recently, Nathan ^ _al_. (24) have observed that neura­ minidase modifies the electrical properties of cultured heart cells

possibly as a result of an increase in calcium influx.

Experiments have been designed in this study to determine 1) whether removing sialic acid from reaggregated chick myocardial cells will produce an effect on the spontaneous beat rate and if, at similar

enzyme concentrations, the myocardial cells will morphologically show

a qualitative decrease in negatively charged membrane sites, 2) whether

NMDase affected only peripheral cells of the aggregate or internal myocardial cells as well, and 3) whether NMDase preparations contain

contaminants which also alter aggregate beat rate and myocardial cell

surfaces. MATERIAL AND METHODS

TISSUE CULTURE TECHNIQUES

White Leghorn chick embryos were collected after incubation at

37 C for seven days. Hearts were surgically removed and ventricles excised. These ventricular cells were dissociated using the multiple- cycle trypsinization method of DeHaan (7a). Cells were plated thirty minutes in 818A medium (7b) to allow fibroblast attachment. Non- attached cells, primarily myocytes, were transferred to Erlenmeyer flasks. Cultures were kept at physiological pH with a 5% CO_/95% air mixture and gyrated in a shaker water bath at 37° C for three days.

Aggregates 100-250 microns in diameter were formed consisting of a

core of myocardial cells and a surrounding discontinuous layer of

fibroblasts (one to three cells deep) (Figure 1).

Aggregates were cultured in medium 818A which consisted of 72.5% modified Earle's balanced salt solution, 20% M199 (Grand Island Bio­

logical) , 4% heat inactivated fetal calf serum (Grand Island Biologi­

cal) , 2% heat inactivated horse serum (KG Biological), 1% glutamine

(Grand Island Biological) and 0.5% Gentamycin (Schering Corp.).

ENZYME PREPARATIONS

Two commercially available C^. perfringens neuraminidase prepara­

tions were used in this study. One chromatographically purified, dia- lyzed, and lyophilized powder preparation (NEUA) was obtained from

Worthington Biochemical Corp. The other enzyme preparation (type IX) purified by affinity chromatography came from Sigma Chemical Co. 3

Type IX was suspended in ammonium sulfate which proved to be detri­ mental to beating myocardial aggregates. This preparation was dia- lyzed against water and lyophilized in our lab. Enzyme activity was then determined by measuring the amount of sialic acid released per minute (37 C, pH 5.5) from bovine submaxillary mucin using the fluo- rometric thiobarbituric acid assay procedure of Hammond and Paper- master (12).

Degrees of protease, aldolase, glycosidases, and phospholipase C have been discovered in most, if not all, C^. perfringens neuraminidase preparations. Sigma Chemical Co. reported less than 0.009 U/mg pro­ tein of protease activity (using Casein as substrate) and less than

0.002 U/mg protein NANA-aldolase (using NANA as substrate). Frank found 0.0025 U/ml phospholipase C activity in 0.25 U/ml Worthington neuraminidase (9). In light of these reports, the enzymes were analyzed by SDS-polyacrylamide gel electrophoresis to determine im­ purity levels, particularly that of phospholipase C. Using a 3% stacking gel and 10% separation gel at room temperature for twelve hours, the proteins were separated.

Electrophoresis showed that a protein band comigrated with a known standard of phospholipase C (Figure 2). Therefore, phospholi­ pase C, chromatographically purified from £. perfringens (type X

Sigma Chemical Co.), was also used as a control in both physiological and morphological studies.

TRANSMISSION ELECTRON MICROSCOPY

Two procedures were used for examining the effect of NMDase on myocardial cell surfaces. One group of aggregates plus a control tissue (colon) was fixed with 3% glutaraldehyde containing 1% tannic acid buffered with 0.067 M sodium cacodylate pH 7.2-7.4 for two hours.

After fixation the aggregates and colon were treated with type IX

Sigma neuraminidase (1.2 U/ml) under optimal conditions, 37° C and at pH 5.5, for either 2 or 4 hours. The other group was treated with type IX or NEUA neuraminidase in 818A medium and subsequently fixed two hours with 3% glutaraldehyde-1% tannic acid or 6% glutaraldehyde containing hydrogen peroxide buffered with 0.067 M sodium cacodylate

(pH 7.2-7.4). This group included the aggregates used in the beat rate experiments.

After enzyme treatments (varying times and concentrations) and

fixation, both groups were stained en bloc for one hour with 115 yg/ml polycationic ferritin (Miles Laboratories) in 0.067 M cacodylate buffer at pH 3.4 and pH 7.2. The samples were then rinsed and postfixed in 1%

OsO. for one hour. Routine procedures were used to dehydrate and em­ bed (23). Sections were cut on a Reichert ultramicrotome, stained with uranyl acetate, and viewed on a Zeiss lOA electron microscope.

Possibly due to differences in culturing, prior trypsinization, or numbers and influences of fibroblasts, staining inconsistencies occurred between cultures. As a result, aggregates from one large

culture were used for as many experiments as possible and were all fixed and stained with the same fixative and PCF solutions.

SCANNING ELECTRON MICROSCOPY and ENERGY DISPERSIVE ANALYSIS

Electron microscopic analyses were limited to the aggregate 8 surface because polycationic ferritin (PCF) could not penetrate past the outer cell layer. To determine whether the enzyme, NMDase, had access into the interior muscle cells, aggregates were enzyme digested

(0.5 u/ml, Worthington NEUA), fixed, microdissected into halves and stained with PCF. The sliced spheres were dehydrated in a graded series of acetone and finally rinsed thoroughly with two washes of

100% acetone. The acetone was then directly substituted with liquid

CO^ during a critical point drying procedure. The aggregate slices were carefully placed on carbon studs with their cut edges exposed and were carbon coated using a Denton vacuum evaporator. A Princeton

Gamma Tech energy dispersive detector joined to a Hitachi S500 scanning electron microscope was used to detect emissions from elements above atomic number 11 and was standardized to count a constant one thousand phosphorus X-ray emissions. All samples were counted at 20 kV excita­ tion current and observed at a constant magnification (20,000X), tilt angle, and detector take off angle. The iron present in polycationic ferritin readily emitted X-rays to be counted. Comparisons of iron counts were made between cut edges of NMDase treated aggregates and nontreated controls.

BEAT RATE MEASUREMENTS

One hundred to two hundred aggregates were plated in 2 mis medi um

818A and incubated at 37° C and pH 7.3 for thirty minutes allowing attachment to the culture dish. The dish was transferred to an in­ verted microscope (Leitz Diavert) equipped with a camera and televi sion monitor. Temperature was maintained at 35-37° C by a heated coil, and pH was kept within a 1.1-1.6 range by the flow of 100% CO2 through a toroidal gassing ring surrounding the petri dish. Three to six aggregates, with no interconnections, were viewed simultaneously on the television screen. Using a stopwatch, the time necessary for the aggregates to contract twenty times was recorded. Due to the large intrinsic variation in spontaneous beat rates among aggregates identically prepared, the baseline beat rate of each individual aggre­ gate was established before any enzyme was added. Counts were contin­ uously registered after addition of enzyme to the culture dish as were the temperature and pH. The percent change in beat rates was calcu­ lated using the baseline rate as the control. RESULTS

PURITY OF ENZYME PREPARATIONS

Electrophoresis of two commercially available C^. perfringens neuraminidase preparations revealed the presence of various proteins.

Of the two preparations employed, the affinity chromatographically purified Sigma type IX proved to contain fewer impurities. Bands co- migrating with standards of phospholipase C were present in both neu­ raminidase preparations. The Sigma type IX preparation, having the least contamination, was primarily used with results routinely com­ pared with those obtained with phospholipase C and the Worthington preparation of NMDase (Figure 2).

PHYSIOLOGICAL RESPONSES TO NEURAMINIDASE

Before neuraminidase was tested, the effect of time on aggregate spontaneous contraction was examined. Beat rates were recorded over an hour period from aggregates not treated with enzyme. These time controls exhibited a constant or net increase in beat rate. Of those controls, six aggregates were closely observed and none showed any arrhythmias within the total sixty minute experiment (Table 1).

A net decrease in the aggregate beat rate over a sixty minute incubation was observed when Sigma neuraminidase at 0.03 U/ml was added to the culture medium of seven day chick myocardial aggregates.

Increasing the dose to 0.5 U/ml or 1.2 U/ml did not potentiate this effect. The maximum response to neuraminidase usually occurred after ten to twenty minutes in enzyme. Thereafter, further inhibition 10 11 of beat rate did not occur but rather, after thirty minutes to one hour incubation, the contraction rates began to variably fluctuate

(i.e. became arrhythmic). Periods of stopping, starting, racing, and slowing made interpretation of the rates difficult. Mean percent changes over a sixty minute incubation (which includes the arrhythmic periods) were as follows: 0.03 U/ml Sigma neuraminidase produced a

9.84% decrease, 0.5 U/ml a 6.68% decrease, and 1.2 U/ml a 9.86% de­ crease. The non-arrhythmic time period (10-30 minutes) alone showed a 7.88% decrease for 0.03 U/ml, 10.42% for 0.5 U/ml, and 7.32% for

1.2 U/ml. In contrast, the less pure Worthington enzyme at a low concentration of 0.03 U/ml showed a continual decrease in beat fre­ quency over a forty minute incubation with a total average decrease of 18.38% (Table 1).

Purified phospholipase C also showed an inhibitory effect upon aggregate beat rate. Using an amount of phospholipase C estimated

to be present in 0.25 U/ml NEUA neuraminidase (Worthington), a 10.96%

change was observed over a forty minute incubation period. After

that time, beating became irregular and eventually stopped CTable 1).

MORPHOLOGICAL RESPONSES TO NEURAMINIDASE

The non-penetrating, positively charged ferritin stain was visu­ alized by transmission electron microscopy as a uniform 11.3 nm dot (6).

Variations in PCF staining patterns could be accounted for by addi­

tives and percentages of glutaraldehyde in each of the fixatives.

The heavy clumping of polycationic ferritin dots, which was seen 12

TABLE 1

Mean Percent Change From Baseline Beat Rate

of Myocardial Aggregates Treated with

Preparations of Neuraminidase and Phospholipase C.

ENZYME PREARRHYTHMIC ARRHYTHMIC TOTAL PREPARATION PERIOD PERIOD TIME U/ml (10-30 min.)^ (40-60 min.)^ (10-60 min.)' mean A% mean A% mean A% ±std. error istd. error ±std. error

CONTROL • (N=6)'' 0.96 ±0.98 0.31 ±1.45 0.64 ±0.85

NEURAMINIDASE Sigma 0.03 U/ml (N=6) 7.88 ±1.36 11.69 ±2.07 9.84 ±1.27 0.5 U/ml (N=5) 10.42 ±2.62 2.94 ±0.93 6.68 ±1.55 1.2 U/ml (N=6) 7.32 ±4.03 12.74 ±3.58 9.86 ±2.76 Worthington 0.03 U/ml (N=6) 16.30 ±2.34 30.90 ±0.69^ 18.38 ±2.29^

PHOSPHOLIPASE C 0.002 U/ml (N=6) 7.09 ±1.11 26.46 ±1.60 10.96 ±2.21

^Each aggregate's beat rate was measured before addition of enzyme. After addition, rates were continuously measured at 10 min. inter­ vals. Percent changes were made using the baseline rate as the con­ trol. The means A% represented values for the entire group.

Non-enzyme treated aggregates

*^N = number of aggregates observed

Values at 40 minutes

^Total time = 10-40 minutes

15 when tissue was fixed in 6% glutaraldehyde plus HO CFigure 2), was not present when 3% glutaraldehyde together with 1% tannic acid was used. Instead a uniform halo of PCF was seen (Figure 4) when the latter fixative was employed.

The presence of sialic acid on myocardial cells was supported by the results of low pH polycationic ferritin staining and by acid methylation of aggregates. Polycationic ferritin at pH 7.2 stains all available anionic sites (sulfate, carboxyl, and phosphate) while

PCF at pH 3.4 labels only those negatively charged groups whose pk is 3.4 or less (primarily sulfates and carboxyls). An earlier pilot experiment (not shown) using high iron diamine (specific for sulfate esters) determined that few sulfate groups are present at the myocar­ dial cell surface. Therefore, polycationic ferritin at pH 3.4 is decidedly labelling rayocaridal surface carboxyl groups, as shown below, contributed primarily by sialic acid. Myocardial cells at the aggre­ gate's periphery bound PCF at the low pH and showed a distinct decrease in stain after enzyme treatment (Figures 5&6). Conversely, acid meth­ ylation at 37° C esterifies carboxyl groups rendering them incapable of binding polycationic ferritin CPCF) (33). Acid methylated aggre­ gates showed complete absence of PCF binding (Figure 3) indicating that a) there are few anionic groups except carboxyls present on myocardial cell surfaces, b) if there are other groups present, they are not accessible to PCF, or c) the acid methylation, contrary to expected results, affected all anionic sites. The most probable conclusions to be drawn are that there are very few sulfate groups, that the phosphate groups which are components of the membrane are difficult to reach.. 16

18 and that the majority of the PCF binding is to carboxyl groups. One further experiment was run using colon, a periodic acid-Schiff (PAS) staining tissue rich in sialomucins (4), as a positive tissue control.

Results showed that colon stains with polycationic ferritin; however, after neuraminidase treatment, this tissue like the aggregates was un­ able to bind the stain (Figure 3).

Neuraminidase was tested comparing viable and glutaraldehyde fixed aggregates. Representative samples of each group were stained with polycationic ferritin at pH 3.4 or at physiological pH (all anionic groups ionized). Of those treated with PCF at pH 7.2, only after a NMDase (type IX, Sigma) concentration of 1.2 U/ml was there any marked decrease in stain on either prefixed and postfixed myocardial cell surfaces. Diminution of PCF stain at pH 7.2 occurred after one to two hours incubation in enzyme and complete removal took place after four hours (Figures 5&6). The same pattern was established using poly­ cationic ferritin at the low pH, except that the plasmalemma was never completely clean of PCF stain. Figures 7 and 8 diagramatically show the results of PCF staining on both fixed and unfixed after treatment with Sigma neuraminidase. Myocardial cell surfaces treated with Worth­ ington NEUA neuraminidase, on the other hand, showed considerable re­ duction in polycationic ferritin staining using less than half the effective concentration of the Sigma enzyme (Figure 2).

Neuraminidase was shown to affect not only the peripheral myo­ cardial cells, but also the interior cells. X-ray microanalysis on aggregates treated with enzyme, fixed, dissected, PCF stained, and compared with nontreated aggregates verified that neuraminidase does 19 pH3.4 pH7.2

.^ •V* Vi, 1 • -2;^' / •4

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23

25 remove sialic acid from the membrane associated carbohydrates belonging to core cells of the aggregate (Figure 2).

Phospholipase C, found at varying levels in both neuraminidase preparations was tested to determine whether it had an effect on anionic sites of the myocardial cell surface. At a 0.002 U/ml concentration

(a dose found in a low concentration of Worthington NMDase), decreased staining occurred when compared to the controls. However, a dose

(0.2 u/ml phospholipase C), much greater than found in either neura­ minidase preparation, cleaved all sites which bind PCF at pH 7.2

(Figure 3). 26 PREFIXATION TREATED MYOCARDIAL CELL SURFACES TREATED WITH DIFFERENT CONCENTRATIONS OF NEURAMINIDASE (pH 7.4,37<>C1 BEFORE FIXATION POLYCATIONIC FERRITIN POLYCATIONIC FERRITIN pH 3.4 pH 7.2

mmi^t;<^mM CONTROL

..'.•..V-i..«; ^jg;[^^i^a^^ 0.03 U/ml 28 PREFIXATION TREATED MYOCARDIAL CELL SURFACES TREATED WITH NEURAMINIDASE IpH 7.4, 37"* CI BEFORE FIXATION

POLYCATIONIC FERRITIN POLYCATIONIC FERRITIN pH 3.4 pH 7.2

/'u.- .. •'.•:• i-i,/.-.-i-i''t'i:'.i/{.'iji •iJi/'e^.i./i^^J'/tU. CONTROL ^^£&;^^#^te^^v^v^^:^::?•^:#^^^^^ ^*S»^«^wi«iiiJ

1.2 U/ml Ihr.

1.2 U/ml

POSTFIXATION TREATED MYOCARDIAL CELL SURFACES TREATED WITH NEURAMINIDASE (pH 5.5, 37*^0 AFTER FIXATION

POLYCATIONIC FERRITIN POLYCATIONIC FERRITIN pH3.4 pH 7.2

^ijb^i^^^^s^ili^^^^ii^ CONTROL

--C.'t-iJ<. 1.2 U/ml 'i^"\vV;irJf^--f 2 hrs.

1.2 U/ml • • •" -Viir^-^^- 4 hrs. DISCUSSION

Although aggregates intrinsically exhibit variable natures, the effect of neuraminidase is evident. Removal of sialic acid by NMDase concomitantly reduces the frequency of contraction. It is the strong anionic charge of the monosaccharide, sialic acid, that is implicated in calcium storage and perhaps regulation of contraction in cultured heart cells (19). Moreover, Langer et_ ail. found that sialic acid liberation increases permeability to Ca"*"^ (.21). Consequently, a suf­ ficient concentration of neuraminidase could result in an overload of calcium within a muscle cell. Elevated levels of intracellular cal­ cium produced by the ionophore A23187 or by 5-10 mM extracellular calcium have been shown to decrease beat rates in myocardial aggre­ gates (.24) . Such slowing of myocardial contractions together with other alterations in electrophysiological function have been attri­ buted to one or more of four mechanisms: 1) a hyperpolarizing shift of voltage-dependent conductance and kinetic parameters, 2) an in­ crease in potassium ion permeability, 3) a larger transmembrane po­ tassium gradient, and 4) stimulation of an electrogenic pump C24).

Another explanation is that abnormally high intracellular calcium causes myofilament degradation or possibly affects depolymerization- polymerization equilibrium resulting in an initial slow down then cessation of muscle contraction (8). It has been reported that mam­ malian muscle treated with ionophore A23187 undergoes myofilament degeneration (26). It is also interesting that elevated calcium levels have been found to be raised in degenerative mammalian 30 31 myopathies (37) some of which are characterized by loss or disorien­ tation of myofilaments in myofibrils (15).

Removal of sialic acid by neuraminidase is visualized with the use of polycationic ferritin. This cationized derivative labels nega­ tive charges on cell surfaces (5). Neuraminidase treatment at the highest concentration used in the beat rate studies reduces surface

PCF staining of myocytes in both fixed and unfixed aggregates. Not only is this reduction witnessed on the cells at the aggregate surface but also on those in the interior. Other cell types have shown dif­ ferences in PCF staining after neuraminidase treatment (1,5); however, some investigators have seen no differences (17,36). These contra­ dictions may be due to the particular cell surface, the activity and purity of the neuraminidase, the masking of small differences by other staining groups, or masking due to the clumping of PCF induced by glutaraldehyde fixation.

One puzzle in this work with polycationic ferritin is that neura­ minidase removes all negative sites on fixed aggregates when the stain­ ing solution is at physiological pH; yet, when the staining solution is pH 3.4, the enzyme treated cell surfaces show residual stain.

Ideally, neuraminidase should not affect carboxyl groups of any mole­ cule other than sialic acid. Those non-sialic acid carboxyl groups along with a few sulfate groups and, at pH 7, the phosphate groups should bind PCF. It is also possible that at low pH the hydroxyl groups in polysaccharide chains are experiencing some protonation and are binding the cationic stain (27). Considering these other possible anions which might be present, the results of the low pH 32 polycationic ferritin seem more reliable . However, acid methylation

° aggregates which render carboxyl groups incapable of binding cat­

ionic dyes results in complete removal of all PCF binding sites. The

acid methylation and the physiological pH polycationic ferritin ex­ periments indicate that PCF binding is primarily due to the carboxyl groups of sialic acid.

Results from this study show that neuraminidase concentrations necessary for a visual difference in polycationic ferritin labeling do not correspond with those required for a physiological response.

The intent of the PCF studies is to qualitatively show that indeed neuraminidase would remove anions from myocardial cell surfaces. The fact that the dosages needed for morphological results do not exactly correlate with the physiological doses possibly demonstrates that the sensitivities of the two probes are not equivalent. Compared to doses used in other strictly morphological works, the highest dose of enz5ane applied in this study was minimal (5,6). Another interpreta­ tion of the data could be that there is no relation between beat rate and removal of sialic acid residues.

In addition, increasing amounts of neuraminidase do not propor­ tionally change the beat rates in myocardial aggregates. One inter­ pretation of a lack of a dose response is that perhaps only a small percentage of the sialic acid pool is responsible for calcium ex­ change. Possibly many sialic residues are tied up with other macro- molecular components, such as proteins, functioning as part of a re­ ceptor or with other cations. Removal of these sialic acid residues would not necessarily have any effect on contraction. On the contrary. 33

0.03 U/ml NMDase maybe a saturating dose and thus to detect a dose response on beat rates, smaller amounts of highly purified neuramini­ dase and more sensitive recording techniques would be necessary.

Increasing the incubation times in neuraminidase up to one hour, which would proportionally remove more sialic acid (as seen in the PCF studies), results in a different physiological response. Intermittent stopping and starting and eventual complete cessation of aggregate contraction occurs. Without knowing the corresponding action poten­ tials of this arrhythmic period, suggestions explaining these results cannot be made. Further electrophysiological studies are necessary to determine the basis for these myocardial aggregate arrhythmias.

One important observation is that Worthington NEUA neuraminidase, at a low concentration, displays a much more dynamic effect physio­ logically and morphologically than the Sigma type IX neuraminidase.

Comparing the compositions of the two neuraminidase preparations based on electrophoretic data, Worthington type NEUA contains more contaminating proteins than does the affinity chromatographically purified Sigma neuraminidase. Evidence points to the presence of contaminants, possibly phospholipase C, to explain the added effects on beat rate and reduction of PCF binding sites of the NEUA pre­ paration.

. Accordingly, phospholipase C run through the same morphological and physiological tests exhibits results similar to those of the enzyme, neuraminidase. A reduction in beat rate very close to that seen using Sigma neuraminidase is observed. Furthermore, a decrease in PCF staining is seen at a dose equal to the activity suggested in 34

•25 U/ml Worthington neuraminidase while an extreme dose removes all

binding sites. A more complete cell surface study was performed by Lesseps who reported that phospholipase C (Clostridium perfringens) stripped the lanthanum staining matrix of embryonic chick heart cells at concentrations from 0.001 mg/ml to 5.0 mg/ml after twenty minute incubations (22).

It is known that the mode of action of phospholipase C is the hydrolysis of the phospholipid, phosphatidylcholine, into a diglyceride and choline phosphate. Moreover, various investigators have shown that acidic phospholipids including cardiolipin, phosphatidic acid, phospha- tidylserine, phosphatidylethylamine, and phosphatidylcholine (substrate of phospholipase C) bind calcium (14,31,34). As stated, calcium, bound to sites outside or associated with the sarcolemma, may be important in activating contraction. Therefore, since both preparations of NMDase contain varying amounts of phospholipase C, phospholipids cannot be excluded from the role of calcium regulation across myocardial mem- branes. Another possibility is that glycosylated phospholipids are present in membranes of myocardial cells and contain sialic acid on the terminal position of their carbohydrate side chains. Hydrolysis of the macromolecules by phospholipase C could result in the loss of a phosphate group and also a carboxyl group. Furthermore, the combi­ nation of both enzymes, phospholipase C and neuraminidase, makes it increasingly difficult to discern whether carboxyl groups of sialic acid or the phosphate groups of phospholipids or both control calcium exchange. CONCLUSIONS

The effects of neuraminidase on aggregates of embryonic chick

eart cells were 1) a decrease in the spontaneous beat rate, 2) time related arrhythmias, and 3) a decrease in anionic sites on the myo­ cardial cell surfaces both at the periphery and the interior of the aggregate. These effects cannot be definitively proven to be solely the consequences of neuraminidase since degrees of phospholipase C and other contaminants are present in both neuraminidase preparations,

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