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Neuraminidase activity in the salivary glands of rats and human saliva
Archs oralBiol.Vol.15,pp.89-92,1970.PergamonPress. Printed inGreat Britain.
NEURAMINIDASE ACTIVITY IN THE SALIVARY GLANDS OF RATS AND HUMAN SALIVA M. S. NIJJAR,E. T. PRITCHARD,C. DAWES and S. R. PHILIPS Faculty of Dentistry, University of Manitoba, Winnipeg, Canada NEURAMINIDASEactivity (N-acetylneuraminate glycohydrolase, E.C. 3.2.1.18) is present in human saliva (ROLLA, 1966; PERLITSHand GLICKMAN, 1966, 1966a, 1967; LEACH and HAYES, 1967). It has been suggested that it could have a role in plaqueformation and, consequently, in periodontal disease (LURA, 1961; LEACH, 1963). PERLITSHand GLICKMAN (1967) noted that both caries-active and periodontallydiseased persons had significantly higher free N-acetylneuraminic acid (NANA) levels in their salivas than did healthy subjects, but these authors could not demonstrate any significant differences in salivary neuraminidase among the groups. Although oral bacteria would seem to be the prime source of salivary neuraminidase, there is a possibility that salivary gland secretions contribute some activity, perhaps of a different type or form (PERLITSH and GLICKMAN,1966, 1967). The present study was undertaken to examine human saliva for neuraminidase activity. In conjunction with these studies, rodent saliva and rodent submandibular and sublingual glands were investigated for their enzymic activity. Rodent salivary glands were chosen because it is obviously difficult to obtain human salivary glands. Rodent and human salivary glands, while having some dissimilar features, both morphologically and biochemically, do have many similarities (SHACKLEFORD and WILRORN, 1968). Lemon drop-stimulated parotid and submandibular salivas were collected by means of a modified Lashley cannula (DAWES and JENKINS,1964) into chilled vessels and kept at O”-3°C until used. These preparations were shown to be free from bacterial contamination by phase-contrast microscopy. Rodent whole saliva was aspirated from the sublingual region of the oral cavity under light ether anaesthesia after subcutaneous injection of pilocarpine (7 mg/kg) into the dorsal neck region. Human whole saliva was obtained by paraffin stimulation. For the preparation of tissue homogenates, Long-Evans rats, 6 to 8 weeks of age, of either sex, were carefully decapitated, the submandibular-sublingual salivary glands quickly removed, cleaned, separated, weighed and homogenized (5-10 per cent w/v) in cold 0.154 M-KC1 $0.05 M-acetate buffer, pH 5.8. Salivas were incubated in stoppered tubes at 37°C after 50/50 dilution with O-308 M-KC1 + 0.1 M-acetate buffer. Incubation volume was O-2 ml. Similar portions of gland homogenates were incubated without dilution. In most instances samples contained 1 mM-CaCl, as an enzyme activator (GOTTSCHALK, 1958 ; WARREN and SPEARING,1961). When required, 40 pmoles of N-acetylneuramin-lactose (NAN-LAC) were added as exogenous substrate. As a check on the 89
M. S. NIJJAR,E. T. PRITCHARD, C. DAWESANDS. R. PHILIPS
90
non-enzymic release of NANA, tubes containing equivalent amounts of heated tissue (1OO’C for 5 min) were incubated in an identical manner to those of the viable test tissues. The stability of NAN-LAC was also tested during each experiment. Reactions were terminated by the addition of 0.1 ml of sodium periodate for the estimation of free NANA (WARREN, 1959). Total NANA was estimated on sepalate samples,
before
and after incubation,
followed by hydrolysis to liberate tissue (LOWRY et al., 1951).
by the addition
of 0.2
ml of 0.2
I+H$O,,
NANA. Protein determinations were done on fresh
Percent increase in the release of free NANA in the presence of exogenous substrate compared to the release in its absence was greater with submandibular glands than with sublingual glands (Table 1). The latter result may have been due to the much higher content of sialoprotein in the sublingual glands (Table 2), which could provide sufficient substrate to saturate the enzyme. Sublingual salivary gland neuraminidase activity, however, was higher both in the presence and absence of substrate than that of the submandibular gland. Both preparations demonstrated a very low level of activity, the maximum rate of NANA release being less than 0.01 per cent of the total sialoprotein per hour in homogenates of either gland. Estimation of total sialoprotein in homogenates both before and after incubation did not show any significant differences. The hydrolytic rate, even if operative in z:ivo, would be very much less than the rate of sialoprotein formation (BYRT and GLAVILL, 1967), strongly supporting a predominately secretory fate for salivary gland sialoprotein. The level of innate gland neuraminidase activity relative to that of rodent whole saliva is high enough to allow some contribution to whole saliva but, as yet, there is no evidence that this enzymic activity is secreted from salivary glands of the rat. It does not appear to be secreted in man. Human parotid and submandibular salivas contain appreciable sialoprotein (Table 2). Because of the well-known fluctuation in protein content of saliva with TABLE1. RELEASE OFFREE NANA DURING INCUBATION in vitro*
Tissue preparation
Rodent submandibular gland homogenate Rodent sublingual gland homogenate Rodent whole saliva Human parotid saliva Human submandibular saliva Whole human saliva
NAN-LAC substrate added
~8 iI+
-t
Neuraminidase activity (rmoles NANA released/mg protein/hr) Average Range
0.362-0.456 0*067+094 0*45@0*540 0~180-0*220 0.0 -7.0 0.0 0.0 2.0 -20.0
0.430 0.081 0.524 0.209 1.93 0.0 0.0 7.0
*Incubation for 6 hr in O-2 ml of 0.05 M-acetate buffer containing 0.154 M-KC1 and 1 mM-CaCla. 40 pmoles of NAN-LAC added as shown.
91
SALIVARYNEURAMINIDASE TABLE2. ANALYIICALRESULTS
Tissue preparation
Rodent Rodent Rodent Human Human Whole
submandibular salivary gland sublingual salivary gland whole saliva parotid saliva submandibular saliva human saliva
pmoles NANA/ g fresh tissue or /ml saliva
mg protein/ g fresh tissue or /ml saliva
pmoles NANA/ mg protein
6945-7931 18200-23600 50-l 50 53-59 99-l 74 73-304
169-189 95-131 8-15 l-1.2 1.3-2.4 2.94.5
42 184 4-20 47-58 69-78 25-70
changing flow rate (DAWES, 1967, 1969) only range values have been given for saliva composition in Table 2, as flow rates were not studied. Neither parotid nor submandibular secretions obtained from human subjects showed any neuraminidase activity (Table l), whether incubated alone or in the presence of NAN-LAC. Activity was present in human whole saliva (bacteria present). Sunlingual gland saliva was not tested, so it is possible that this secretion contributes some activity to human whole saliva. A definitive answer must await the analysis of human sublingual salivary secretion.
Acknowledgement-This work was supported by the Associate Committee for Dental Research of the National Research Council of Canada (DT-109: ETP). REFERENCES BYRT, P. M. and GLANVILL,S. 1967. Effect of isoprenaline on the secretion of sialoproteins from rat salivary glands. Biochim. biophys. Acta 148, 215-221. DAWES, C. and JENKINS,G. N. 1964. The effects of different stimuli on the composition of saliva in man. J. Physiol., Lond. 170,86-100. DAWES, C. 1967. The effect of flow rate and length of stimulation on the protein concentration in human parotid saliva. Archs oral Biol. 12, 783-788. DAWES, C. 1969. The effect of flow rate, duration of stimulation, on the concentration of protein and the main electrolytes in human saliva. Archs oral Biol. 14,277-294. GOTTSCHALK,A. 1958. Neuraminidase, its substrate and mode of action. Adu. Enzymol. 20,135-146. LEACH, S. A. 1963. Release and breakdown of sialic acid from human salivary mucin and its role in the formation of dental plaque. Nature, Lond. 19!3,486-487. LEACH, S. A. and HAYES,M. L. 1967. Isolation in pure culture of human oral organisms capable of producing neuraminidase. Nature, Lond. 216, 599-600. LOWRY,0. H., ROSEBROUGH, N. J., FARR, A. L. and RANDALL,R. J. 1951. Protein measurement with Folin phenol reagent. J. biol. Chem. 193,265-275. LURA, H. E. 1961. An investigation into the relation between sialic acid of saliva and dental caries. Archs oral Biol. 4, 141-146. PERLITSH,M. J. and GLICKMAN,I. 1966. Salivary neuraminidase I. The presence of neuramidase in human saliva. J. Periodont. 37, 368-373. PERLIIXH,M. J. and GLICKMAN,I. 1966a. Salivary neuraminidase 2. Its source in whole human saliva. J. dent. Rex 45, 1239. PERLITSH,M. J. and GLICKMAN, 1. 1967. Salivary neuraminidase 3. Its relation to oral disease. J. Periodont. 38, 189-192.
92
M. S. NIJJAR,E. T. PRITCHARD, C. DAWESAND S. R. PHILIPS
RP)LLA,G. 1966. Neuraminidase in human sputum. ACIUodont. scund. 24,431-442. SHACKLEFORD, J. M. and WILLBORN,W. H. 1968. Structural and histochemical
diversity in mammalian salivary glands. AIa. J. Med. Sci. 5, 180-203. WARREN,L. 1959. The thiobarbituric acid assay of sialic acids. J. biol. Chem. 234, 1971-1975. WARREN,L. and SPEARING, C. W. 1961. Mammalian sialidase(neuraminidase).Biochem. biophyr. Res. Commun. 3,489492.
NEURAMINIDASE ACTIVITY IN THE SALIVARY GLANDS OF RATS AND HUMAN SALIVA M. S. NIJJAR,E. T. PRITCHARD,C. DAWES and S. R. PHILIPS Faculty of Dentistry, University of Manitoba, Winnipeg, Canada NEURAMINIDASEactivity (N-acetylneuraminate glycohydrolase, E.C. 3.2.1.18) is present in human saliva (ROLLA, 1966; PERLITSHand GLICKMAN, 1966, 1966a, 1967; LEACH and HAYES, 1967). It has been suggested that it could have a role in plaqueformation and, consequently, in periodontal disease (LURA, 1961; LEACH, 1963). PERLITSHand GLICKMAN (1967) noted that both caries-active and periodontallydiseased persons had significantly higher free N-acetylneuraminic acid (NANA) levels in their salivas than did healthy subjects, but these authors could not demonstrate any significant differences in salivary neuraminidase among the groups. Although oral bacteria would seem to be the prime source of salivary neuraminidase, there is a possibility that salivary gland secretions contribute some activity, perhaps of a different type or form (PERLITSH and GLICKMAN,1966, 1967). The present study was undertaken to examine human saliva for neuraminidase activity. In conjunction with these studies, rodent saliva and rodent submandibular and sublingual glands were investigated for their enzymic activity. Rodent salivary glands were chosen because it is obviously difficult to obtain human salivary glands. Rodent and human salivary glands, while having some dissimilar features, both morphologically and biochemically, do have many similarities (SHACKLEFORD and WILRORN, 1968). Lemon drop-stimulated parotid and submandibular salivas were collected by means of a modified Lashley cannula (DAWES and JENKINS,1964) into chilled vessels and kept at O”-3°C until used. These preparations were shown to be free from bacterial contamination by phase-contrast microscopy. Rodent whole saliva was aspirated from the sublingual region of the oral cavity under light ether anaesthesia after subcutaneous injection of pilocarpine (7 mg/kg) into the dorsal neck region. Human whole saliva was obtained by paraffin stimulation. For the preparation of tissue homogenates, Long-Evans rats, 6 to 8 weeks of age, of either sex, were carefully decapitated, the submandibular-sublingual salivary glands quickly removed, cleaned, separated, weighed and homogenized (5-10 per cent w/v) in cold 0.154 M-KC1 $0.05 M-acetate buffer, pH 5.8. Salivas were incubated in stoppered tubes at 37°C after 50/50 dilution with O-308 M-KC1 + 0.1 M-acetate buffer. Incubation volume was O-2 ml. Similar portions of gland homogenates were incubated without dilution. In most instances samples contained 1 mM-CaCl, as an enzyme activator (GOTTSCHALK, 1958 ; WARREN and SPEARING,1961). When required, 40 pmoles of N-acetylneuramin-lactose (NAN-LAC) were added as exogenous substrate. As a check on the 89
M. S. NIJJAR,E. T. PRITCHARD, C. DAWESANDS. R. PHILIPS
90
non-enzymic release of NANA, tubes containing equivalent amounts of heated tissue (1OO’C for 5 min) were incubated in an identical manner to those of the viable test tissues. The stability of NAN-LAC was also tested during each experiment. Reactions were terminated by the addition of 0.1 ml of sodium periodate for the estimation of free NANA (WARREN, 1959). Total NANA was estimated on sepalate samples,
before
and after incubation,
followed by hydrolysis to liberate tissue (LOWRY et al., 1951).
by the addition
of 0.2
ml of 0.2
I+H$O,,
NANA. Protein determinations were done on fresh
Percent increase in the release of free NANA in the presence of exogenous substrate compared to the release in its absence was greater with submandibular glands than with sublingual glands (Table 1). The latter result may have been due to the much higher content of sialoprotein in the sublingual glands (Table 2), which could provide sufficient substrate to saturate the enzyme. Sublingual salivary gland neuraminidase activity, however, was higher both in the presence and absence of substrate than that of the submandibular gland. Both preparations demonstrated a very low level of activity, the maximum rate of NANA release being less than 0.01 per cent of the total sialoprotein per hour in homogenates of either gland. Estimation of total sialoprotein in homogenates both before and after incubation did not show any significant differences. The hydrolytic rate, even if operative in z:ivo, would be very much less than the rate of sialoprotein formation (BYRT and GLAVILL, 1967), strongly supporting a predominately secretory fate for salivary gland sialoprotein. The level of innate gland neuraminidase activity relative to that of rodent whole saliva is high enough to allow some contribution to whole saliva but, as yet, there is no evidence that this enzymic activity is secreted from salivary glands of the rat. It does not appear to be secreted in man. Human parotid and submandibular salivas contain appreciable sialoprotein (Table 2). Because of the well-known fluctuation in protein content of saliva with TABLE1. RELEASE OFFREE NANA DURING INCUBATION in vitro*
Tissue preparation
Rodent submandibular gland homogenate Rodent sublingual gland homogenate Rodent whole saliva Human parotid saliva Human submandibular saliva Whole human saliva
NAN-LAC substrate added
~8 iI+
-t
Neuraminidase activity (rmoles NANA released/mg protein/hr) Average Range
0.362-0.456 0*067+094 0*45@0*540 0~180-0*220 0.0 -7.0 0.0 0.0 2.0 -20.0
0.430 0.081 0.524 0.209 1.93 0.0 0.0 7.0
*Incubation for 6 hr in O-2 ml of 0.05 M-acetate buffer containing 0.154 M-KC1 and 1 mM-CaCla. 40 pmoles of NAN-LAC added as shown.
91
SALIVARYNEURAMINIDASE TABLE2. ANALYIICALRESULTS
Tissue preparation
Rodent Rodent Rodent Human Human Whole
submandibular salivary gland sublingual salivary gland whole saliva parotid saliva submandibular saliva human saliva
pmoles NANA/ g fresh tissue or /ml saliva
mg protein/ g fresh tissue or /ml saliva
pmoles NANA/ mg protein
6945-7931 18200-23600 50-l 50 53-59 99-l 74 73-304
169-189 95-131 8-15 l-1.2 1.3-2.4 2.94.5
42 184 4-20 47-58 69-78 25-70
changing flow rate (DAWES, 1967, 1969) only range values have been given for saliva composition in Table 2, as flow rates were not studied. Neither parotid nor submandibular secretions obtained from human subjects showed any neuraminidase activity (Table l), whether incubated alone or in the presence of NAN-LAC. Activity was present in human whole saliva (bacteria present). Sunlingual gland saliva was not tested, so it is possible that this secretion contributes some activity to human whole saliva. A definitive answer must await the analysis of human sublingual salivary secretion.
Acknowledgement-This work was supported by the Associate Committee for Dental Research of the National Research Council of Canada (DT-109: ETP). REFERENCES BYRT, P. M. and GLANVILL,S. 1967. Effect of isoprenaline on the secretion of sialoproteins from rat salivary glands. Biochim. biophys. Acta 148, 215-221. DAWES, C. and JENKINS,G. N. 1964. The effects of different stimuli on the composition of saliva in man. J. Physiol., Lond. 170,86-100. DAWES, C. 1967. The effect of flow rate and length of stimulation on the protein concentration in human parotid saliva. Archs oral Biol. 12, 783-788. DAWES, C. 1969. The effect of flow rate, duration of stimulation, on the concentration of protein and the main electrolytes in human saliva. Archs oral Biol. 14,277-294. GOTTSCHALK,A. 1958. Neuraminidase, its substrate and mode of action. Adu. Enzymol. 20,135-146. LEACH, S. A. 1963. Release and breakdown of sialic acid from human salivary mucin and its role in the formation of dental plaque. Nature, Lond. 19!3,486-487. LEACH, S. A. and HAYES,M. L. 1967. Isolation in pure culture of human oral organisms capable of producing neuraminidase. Nature, Lond. 216, 599-600. LOWRY,0. H., ROSEBROUGH, N. J., FARR, A. L. and RANDALL,R. J. 1951. Protein measurement with Folin phenol reagent. J. biol. Chem. 193,265-275. LURA, H. E. 1961. An investigation into the relation between sialic acid of saliva and dental caries. Archs oral Biol. 4, 141-146. PERLITSH,M. J. and GLICKMAN,I. 1966. Salivary neuraminidase I. The presence of neuramidase in human saliva. J. Periodont. 37, 368-373. PERLIIXH,M. J. and GLICKMAN,I. 1966a. Salivary neuraminidase 2. Its source in whole human saliva. J. dent. Rex 45, 1239. PERLITSH,M. J. and GLICKMAN, 1. 1967. Salivary neuraminidase 3. Its relation to oral disease. J. Periodont. 38, 189-192.
92
M. S. NIJJAR,E. T. PRITCHARD, C. DAWESAND S. R. PHILIPS
RP)LLA,G. 1966. Neuraminidase in human sputum. ACIUodont. scund. 24,431-442. SHACKLEFORD, J. M. and WILLBORN,W. H. 1968. Structural and histochemical
diversity in mammalian salivary glands. AIa. J. Med. Sci. 5, 180-203. WARREN,L. 1959. The thiobarbituric acid assay of sialic acids. J. biol. Chem. 234, 1971-1975. WARREN,L. and SPEARING, C. W. 1961. Mammalian sialidase(neuraminidase).Biochem. biophyr. Res. Commun. 3,489492.