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Salivation in the red kangaroo (Macropus rufus) during sympathetic nerve stimulation
Camp. Biochem.
Physiol. Vol. 92A, No. 4, pp. 495-491,
1989 0
Printed in Great Britain
0300.9629/89 $3.00 + 0.00 1989 Pergamon Press plc
SALIVATION IN THE RED KANGAROO (~~CR~~U~ RU‘US) DURING SYMPATHETIC NERVE STIMULATION A. M.
&Al_
School of Biological Science, University of New South Wales, PO Box 1, Kensington, NSW 2033, Australia. Telephone: 697-2222 (Received 12 August 1988) Abstract-l. Continuous electrical stimulation at low frequency (5 Hz) and short pulse duration (500 psec) of the cervical sympathetic trunk for periods up to 15 min caused no obvious flow from the parotid or mandibular glands of the red kangaroo. 2. Higher frequencies combined with longer pulse durations caused both glands to secrete. Flow reached maximum in less than 3 min and then declined but, on cessation of stimulation, flow increased again for a short period. This flow response may be caused by the interaction of the secretory response with myoepithelial contraption. 3. The parotid saliva had substantially higher protein, phosphate and hydrogen ion concentrations, and lower sodium concentrations than cholinergic parotid saliva. The low pH indicates bicarbonate concentrations far lower than in other sympathetic salivas. 4. The mandibular saliva had higher protein, urea and potassium, and lower chloride and hydrogen concentrations than cholinergic mandibular saliva
INTRODUCTION
Electrical stimulation of the sympathetic innervation of eutherian salivary glands results in responses varying from no secretion to flow rates equivalent to that of parasympathetic or chohnergic stimulation (see review, Young and Schneyer, 1981). This variability between glands of different species can be explained by the extent and distribution of the sympathetic innervation of the glands and by the classes of adrenergic receptor therein. Among the marsupials, sympathetic stimulation has been reported for the mandibular gland of the brush-tailed possum (Trichosurus vulpeculu) only. The gland did not secrete (Creed and McDonald, 1970; Compton, 1976) and it was subsequently shown that sympathetic fibres did not reach the secretory elements of the gland (Blood et al., 1977). Porter (1981) found that both the parotid and mandibular glands of the red kangaroo possessed sympathetic innervation of the secretory end pieces with the striated ducts of the mandibular also being innervated. This paper reports on the response to electrical stimulation of the parotid and mandibular glands of the red kangaroo. MATERIALS
AND METHODS
The experiments were done as an adjunct to the surgical preparation of carotid artery loops. Five adult red kangaroos were used, three males weighing 3141 kg and two females weighing 22.5 and 25.5 kg. The animals were maintained on a diet of hrcerne chaff, supplement pellets and drinking water containing 25 mmol/l NaCl + 25 mmol/l NaHCO,. Anaesthesia was induced by intravenous injection of sodium pentobarbitone (25-36 mg/kg) and maintained, using the cornea1 reflex as a guide, with additional injec-
tions. The animals were placed on an electrically-heated pad and had an air cushion under the hip to prevent pressure damage. The trachea was intubated with a cuffed endotracheai tube. Under sterile conditions, the carotid sheath was exposed and the sympathetic trunk was dissected clear for about 6cm. Both parotid and mandibular ducts were catheterized through their orifices in the mouth using vinyl (I.57 mmi.d.; 2.08mm o.d.)and polythene(O.5~.8mmi.d.; 0.8-1.2 mm o.d.; Dural Plastics, NSW) tubes, respectively. The trunk was stimulated with paired electrodes using square-wave pulses of 510 V, pulse durations of 0.5, 2 or 5msec and frequencies of 5, 10 or 20Hz. Continuous stimulation for periods up to 15 min was tried on each animal but gave a poor secretory response. Intermittent stimulation at the higher pulse durations and frequencies using a regime of 2-3 min stimulation followed by rest periods of equal length proved more effective at eliciting a salivary sample. No attempt to obtain saliva was longer than 65 min whereupon the experiment was terminated and the preparation of the loop completed. During saliva collection, blood samples from the leg vein were taken into heparinized syringes. Blood plasma and saliva were analysed for protein, urea, sodium, potassium, chloride, phosphate and hydrogen ions. Duplicate estimations of total protein in plasma and saliva were made by the FolinLowry method (Daughaday et al., 1952) using standards composed of a 50:50 mixture of bovine albumin and human globulin. Urea concentrations in the samples were estimated by enzymatic cleavage of urea and calorimetric determination of ammonia (Fawcett and Scott, 1960). Salivary pH was measured at 36°C under anaerobic conditions using thermostated Radiometer microelectrodes. Saliva and plasma were analysed in duplicate for sodium and potassium by atomic absorption spectroscopy using appropriate ionization suppressants. Duplicate estimations of the chloride concentration in plasma and saliva were made using a Radiometer chloride titrator (model CMT 10). Total inorganic phosphate concentrations in saliva and plasma were determined in duplicate using the method of Baginski et al. (1967). 495
496
A. M. BEAL
Table I. Summary of the mean flow rates and electrolyte concentration of saliva obtained from the parotid (n = 5) and mandibular elands of red kanearoos usine electrical stimulation of the cervical svmoathetic trunk (SEM eiven in oarentheses) Saliva flow (ml/min) Parotid
(s/l)
Urea (mmol/l)
Na (mmol/l)
K (mmol/l)
Cl (mmol/l)
(mmoljl)
PO4
H+ (nmol/l)
0.033 (0.0022)
3.42 (0.702) 74.90 (3.432)
14.61 (1.161) 11.38 (I ,258)
105.4 (7.19) 145.3 (1.30)
23.8 (4.70) 4.10 (0.164)
40.4 (5.24) 100.4 (0.99)
49.6 (4.02) 2.51 (0.222)
129.2 (25.80) 43.96 fl.556)
0.025 (0.0075)
14.62 (3.507) 78.57 (4.693)
23.38 (0.246 12.24 (2.050)
4.12 (1.593) 144.5 (2.1 I)
86.2 (11.04) 4.20 (0.266)
17.8 (I .79) 100.4 (1.37)
6.21 (2.497) 2.33 (0.352)
15.76 (1.063) 46.12 (2.309)
Plasma Mandibular
Protein
(n = 3)
Plasma
RESULTS
Salivary flow was obtained from the parotid glands of all five animals whereas mandibular saliva was obtained for three animals only. Continuous stimulation with a pulse width of 500 psec combined with a frequency of 5 Hz produced no obvious secretion of thud. Under continuous stimulation at higher frequencies and longer pulse durations, flow commenced with increasing rapidity, reached maximum within 3 min and then decreased to a very low secretion rate. After cessation of stimulation, flow increased again for a short period. Consequently to maximise the volume obtained without substantially lengthening the duration of the surgery, intermittent stimulation of the nerve trunk was used over most of the collection period. Flow rates given in Table 1 are averages for this period and take no account of the many short periods in which negligible flow occurred. Under these conditions, maximum ilow for the parotid was 0.037 ml/min and for the mandibular, 0.038 ml/min-’ (same animal). Mean electrolyte, protein and urea concentrations for the samples are given in Table 1.
DISCUSSION
Salivary flow was not sustained by continuous stimulation and, using intermittent stimulation, showed peaks of flow after onset and after cessation of stimulation. Since both glands possess myoepithelia (Porter, 1981), this pattern of flow may have resulted from contraction of these cells, as occurs in the sheep parotid gland (Coats et al., 1956). At onset of stimulation of the sympathetic trunk, adrenergic receptors (CIandjor 8) in the end organs were activated causing secretion and the myoepithelia caused to contract, resulting in squeezing of the secretory elements. If, at cessation of stimulation, the myoepithelia relaxed before the secretory effect had abated, then filling of the ducts and return to flow would occur for a short period. The essential difference between the kangaroo glands and the sheep parotid (Coats et al., 1956; Patterson and Titchen, 1979) in their response to sympathetic stimulation may be the absence of spontaneous secretion by the kangaroo glands. Alpha-adrenergic stimulation of the vasculature would be expected to cause constriction which could also influence salivary secretion. The absence of flow by the mandibular of two animals can be explained by blockage of a small calibre duct/catheter with mucous when the gland is generating little secretion pressure.
Protein concentrations in both salivas were elevated, being some 10 and 30 times the concentration in cholinergic saliva (Beal, 1987) from the parotid and mandibular glands, respectively. The high protein concentrations may indicate the presence of padrenoreceptors in the kangaroo glands since it is stimulation of /I rather than 01receptors which usually evokes protein rich saliva from eutherian glands. Parotid sodium concentrations were lower and mandibular potassium concentrations were higher than those reported for cholinergic saliva (Beal, 1984, 1986a). Increased secretion of adrenal steroids might be caused by the experimental manipulations but would be unlikely to lower parotid sodium since the parotid of the sodium-replete red kangaroo does not respond to aldosterone or DOCA for at least 4 hr after administration (Beal, 1986b). The elevated potassium of mandibular saliva could be caused by endogenous steroid levels (Beal, 1986a) but could equally well be an effect of /I-adrenergic stimulation as seen in rat and rabbit mandibular salivas (Young and Martin, 1971; Case et al., 1980). Since the pH of the parotid saliva averaged about 6.9, salivary bicarbonate must have been very low (about IOmmol/l, assuming a Pco, of 60mmHg), much lower than that of cholinergic saliva (Beal, 1984), and consequently phosphate was the major anion in the sympathetic parotid saliva. In contrast, mandibular salivary pH was much higher and its chloride concentration much lower than in cholinergically-evoked saliva (Beal, l986a) and thus with phosphate levels being low, the major anion in sympathetically-evoked mandibular saliva was probably bicarbonate. Rat and rabbit mandibular glands produce high bicarbonate salivas during sympathetic and sympathomimetic stimulation (Yoshida et al., 1967; Young and Martin, 1971; Case et al., 1980). At the onset and termination of stimulation, saliva produced during the transition between flow states frequently differs in composition from that produced during steady-state flow (Burgen, 1956; Coats and Wright, 1957; Petersen and Poulsen, 1968; Schneyer, 1975; Beal, 1986a). The protocol necessary to obtain the samples from the kangaroos obviously has a number of transition periods and these transients may have influenced the composition of the samples obtained. Consequently, sympathomim~tic stimulation using specific a and fi adrenergic agonists may be a better approach to investigating the influence of the sympathetic nervous system on the composition of kangaroo saliva.
Sympathetic Acknowledgemenrs-I am skilful technical assistance diligent husbandry of the Abbott Australia Pty Ltd barbitone.
salivation
indebted to Mr David Hair for and to Mrs Glenys Forsyth for kangaroos. I would like to thank for the gift of the sodium pento-
REFERENCES
Baginski E. S., Foa P. P. and Zak B. (1967) Microdetermination of inorganic phosphate, phospholipids and total phosphate in biological materials. Clin. Chem. 13, 326332. Beal A. M. (1984) Electrolyte composition of parotid saliva from sodium-replete red kangaroos (Macropus rufus). J. exp. Biol. 111, 225-237. Beal A. M. (1986a) Effects of flow rate, duration of stimulation, and mineralocorticoids on the electrolyte concentrations of mandibular saliva from the red kangaroos (Macropus rufus). J. exp. Biol. 126, 315-339. Beal A. M. (1986b) Responsiveness of the parotid salivary gland of red kangaroos (Macropus rufus) to mineralocorticoids. J. exp. Biol. 120, 309-324. Beal A. M. (1987) Amylase activity, protein and urea in saliva of red kangaroos (Macropus rufus). Arch. oral Biol. 32, 825-832. Blood L. A., Kennerson A. R. and van Lennep E. W. (1977) Morphology and histochemistry of the mandibular gland of the Australian brush-tail possum Trichosurus vulpecula (Marsupialia). Anal. Rec. 188, 4899508. Burgen A. S. V. (1956) The secretion of potassium in saliva. J. Physiol., Lond. 132, 2&39. Case R. M., Conigrave A. D., Novak I. and Young J. A. (1980) Electrolyte and protein secretion by the perfused
in kangaroos
497
rabbit mandibular gland stimulated with acetylcholine or catecholamines. J. Physiol., Land. 300, 467487. Coats D. A. and Wright R. D. (1957) Secretion by the parotid gland of the sheep: the relationship between salivary flow and composition. J. Physiol., Lond. 135, 61 l-622. Coats D. A., Denton D. A.. Godinn J. R. and Wright R. D. (1956) Secretion by the parotih gland of the sheep. J. Phvsiol.. Land. 131. 13-31. Compton J. S. (1976) A’micropuncture study of the effect of mineral active steroids on normal salivary gland function. Honours Thesis, University of Sydney, Sydney. Creed K. E. and McDonald I. R. (1970) Salivary secretory potentials in the marsupial Trichosurus vulpecuia. Camp. gen. Pharmacol. 1, 285-292. Daughaday W. H., Lowry 0. H., Rosebrough N. J. and Fields W. S. (1952) Determination of cerebrospinal fluid protein with the Folin phenol reagent. J. lab. clin. Med. 39, 663465. Fawcett J. K. and Scott J. E. (1960) A rapid and precise method for the determination of urea. J. clin. Pathol. 13, 15&159. Patterson J. and Titchen D. A. (1979) Sympathetic and fi-adrenergic stimulation of parotid salivary secretion in sheep. Q.-J1 exp. Physiol. 64, 175-184. Petersen 0. H. and Poulsen J. H. (1968) The secretion of sodium and potassium in cat submandibular saliva during the first period after start of stimulation. Acfa Physiol. Stand. 73, 93-100. Porter B. (198 1) The structure and function of macropodid salivary glands. PhD thesis, University of New South Wales, Australia, pp. 5455. Young J. A. and Schneyer C. A. (1981) Composition of saliva in mammalia. Aust. J. exp. Biol. med. Sci. 59, l-53.
Physiol. Vol. 92A, No. 4, pp. 495-491,
1989 0
Printed in Great Britain
0300.9629/89 $3.00 + 0.00 1989 Pergamon Press plc
SALIVATION IN THE RED KANGAROO (~~CR~~U~ RU‘US) DURING SYMPATHETIC NERVE STIMULATION A. M.
&Al_
School of Biological Science, University of New South Wales, PO Box 1, Kensington, NSW 2033, Australia. Telephone: 697-2222 (Received 12 August 1988) Abstract-l. Continuous electrical stimulation at low frequency (5 Hz) and short pulse duration (500 psec) of the cervical sympathetic trunk for periods up to 15 min caused no obvious flow from the parotid or mandibular glands of the red kangaroo. 2. Higher frequencies combined with longer pulse durations caused both glands to secrete. Flow reached maximum in less than 3 min and then declined but, on cessation of stimulation, flow increased again for a short period. This flow response may be caused by the interaction of the secretory response with myoepithelial contraption. 3. The parotid saliva had substantially higher protein, phosphate and hydrogen ion concentrations, and lower sodium concentrations than cholinergic parotid saliva. The low pH indicates bicarbonate concentrations far lower than in other sympathetic salivas. 4. The mandibular saliva had higher protein, urea and potassium, and lower chloride and hydrogen concentrations than cholinergic mandibular saliva
INTRODUCTION
Electrical stimulation of the sympathetic innervation of eutherian salivary glands results in responses varying from no secretion to flow rates equivalent to that of parasympathetic or chohnergic stimulation (see review, Young and Schneyer, 1981). This variability between glands of different species can be explained by the extent and distribution of the sympathetic innervation of the glands and by the classes of adrenergic receptor therein. Among the marsupials, sympathetic stimulation has been reported for the mandibular gland of the brush-tailed possum (Trichosurus vulpeculu) only. The gland did not secrete (Creed and McDonald, 1970; Compton, 1976) and it was subsequently shown that sympathetic fibres did not reach the secretory elements of the gland (Blood et al., 1977). Porter (1981) found that both the parotid and mandibular glands of the red kangaroo possessed sympathetic innervation of the secretory end pieces with the striated ducts of the mandibular also being innervated. This paper reports on the response to electrical stimulation of the parotid and mandibular glands of the red kangaroo. MATERIALS
AND METHODS
The experiments were done as an adjunct to the surgical preparation of carotid artery loops. Five adult red kangaroos were used, three males weighing 3141 kg and two females weighing 22.5 and 25.5 kg. The animals were maintained on a diet of hrcerne chaff, supplement pellets and drinking water containing 25 mmol/l NaCl + 25 mmol/l NaHCO,. Anaesthesia was induced by intravenous injection of sodium pentobarbitone (25-36 mg/kg) and maintained, using the cornea1 reflex as a guide, with additional injec-
tions. The animals were placed on an electrically-heated pad and had an air cushion under the hip to prevent pressure damage. The trachea was intubated with a cuffed endotracheai tube. Under sterile conditions, the carotid sheath was exposed and the sympathetic trunk was dissected clear for about 6cm. Both parotid and mandibular ducts were catheterized through their orifices in the mouth using vinyl (I.57 mmi.d.; 2.08mm o.d.)and polythene(O.5~.8mmi.d.; 0.8-1.2 mm o.d.; Dural Plastics, NSW) tubes, respectively. The trunk was stimulated with paired electrodes using square-wave pulses of 510 V, pulse durations of 0.5, 2 or 5msec and frequencies of 5, 10 or 20Hz. Continuous stimulation for periods up to 15 min was tried on each animal but gave a poor secretory response. Intermittent stimulation at the higher pulse durations and frequencies using a regime of 2-3 min stimulation followed by rest periods of equal length proved more effective at eliciting a salivary sample. No attempt to obtain saliva was longer than 65 min whereupon the experiment was terminated and the preparation of the loop completed. During saliva collection, blood samples from the leg vein were taken into heparinized syringes. Blood plasma and saliva were analysed for protein, urea, sodium, potassium, chloride, phosphate and hydrogen ions. Duplicate estimations of total protein in plasma and saliva were made by the FolinLowry method (Daughaday et al., 1952) using standards composed of a 50:50 mixture of bovine albumin and human globulin. Urea concentrations in the samples were estimated by enzymatic cleavage of urea and calorimetric determination of ammonia (Fawcett and Scott, 1960). Salivary pH was measured at 36°C under anaerobic conditions using thermostated Radiometer microelectrodes. Saliva and plasma were analysed in duplicate for sodium and potassium by atomic absorption spectroscopy using appropriate ionization suppressants. Duplicate estimations of the chloride concentration in plasma and saliva were made using a Radiometer chloride titrator (model CMT 10). Total inorganic phosphate concentrations in saliva and plasma were determined in duplicate using the method of Baginski et al. (1967). 495
496
A. M. BEAL
Table I. Summary of the mean flow rates and electrolyte concentration of saliva obtained from the parotid (n = 5) and mandibular elands of red kanearoos usine electrical stimulation of the cervical svmoathetic trunk (SEM eiven in oarentheses) Saliva flow (ml/min) Parotid
(s/l)
Urea (mmol/l)
Na (mmol/l)
K (mmol/l)
Cl (mmol/l)
(mmoljl)
PO4
H+ (nmol/l)
0.033 (0.0022)
3.42 (0.702) 74.90 (3.432)
14.61 (1.161) 11.38 (I ,258)
105.4 (7.19) 145.3 (1.30)
23.8 (4.70) 4.10 (0.164)
40.4 (5.24) 100.4 (0.99)
49.6 (4.02) 2.51 (0.222)
129.2 (25.80) 43.96 fl.556)
0.025 (0.0075)
14.62 (3.507) 78.57 (4.693)
23.38 (0.246 12.24 (2.050)
4.12 (1.593) 144.5 (2.1 I)
86.2 (11.04) 4.20 (0.266)
17.8 (I .79) 100.4 (1.37)
6.21 (2.497) 2.33 (0.352)
15.76 (1.063) 46.12 (2.309)
Plasma Mandibular
Protein
(n = 3)
Plasma
RESULTS
Salivary flow was obtained from the parotid glands of all five animals whereas mandibular saliva was obtained for three animals only. Continuous stimulation with a pulse width of 500 psec combined with a frequency of 5 Hz produced no obvious secretion of thud. Under continuous stimulation at higher frequencies and longer pulse durations, flow commenced with increasing rapidity, reached maximum within 3 min and then decreased to a very low secretion rate. After cessation of stimulation, flow increased again for a short period. Consequently to maximise the volume obtained without substantially lengthening the duration of the surgery, intermittent stimulation of the nerve trunk was used over most of the collection period. Flow rates given in Table 1 are averages for this period and take no account of the many short periods in which negligible flow occurred. Under these conditions, maximum ilow for the parotid was 0.037 ml/min and for the mandibular, 0.038 ml/min-’ (same animal). Mean electrolyte, protein and urea concentrations for the samples are given in Table 1.
DISCUSSION
Salivary flow was not sustained by continuous stimulation and, using intermittent stimulation, showed peaks of flow after onset and after cessation of stimulation. Since both glands possess myoepithelia (Porter, 1981), this pattern of flow may have resulted from contraction of these cells, as occurs in the sheep parotid gland (Coats et al., 1956). At onset of stimulation of the sympathetic trunk, adrenergic receptors (CIandjor 8) in the end organs were activated causing secretion and the myoepithelia caused to contract, resulting in squeezing of the secretory elements. If, at cessation of stimulation, the myoepithelia relaxed before the secretory effect had abated, then filling of the ducts and return to flow would occur for a short period. The essential difference between the kangaroo glands and the sheep parotid (Coats et al., 1956; Patterson and Titchen, 1979) in their response to sympathetic stimulation may be the absence of spontaneous secretion by the kangaroo glands. Alpha-adrenergic stimulation of the vasculature would be expected to cause constriction which could also influence salivary secretion. The absence of flow by the mandibular of two animals can be explained by blockage of a small calibre duct/catheter with mucous when the gland is generating little secretion pressure.
Protein concentrations in both salivas were elevated, being some 10 and 30 times the concentration in cholinergic saliva (Beal, 1987) from the parotid and mandibular glands, respectively. The high protein concentrations may indicate the presence of padrenoreceptors in the kangaroo glands since it is stimulation of /I rather than 01receptors which usually evokes protein rich saliva from eutherian glands. Parotid sodium concentrations were lower and mandibular potassium concentrations were higher than those reported for cholinergic saliva (Beal, 1984, 1986a). Increased secretion of adrenal steroids might be caused by the experimental manipulations but would be unlikely to lower parotid sodium since the parotid of the sodium-replete red kangaroo does not respond to aldosterone or DOCA for at least 4 hr after administration (Beal, 1986b). The elevated potassium of mandibular saliva could be caused by endogenous steroid levels (Beal, 1986a) but could equally well be an effect of /I-adrenergic stimulation as seen in rat and rabbit mandibular salivas (Young and Martin, 1971; Case et al., 1980). Since the pH of the parotid saliva averaged about 6.9, salivary bicarbonate must have been very low (about IOmmol/l, assuming a Pco, of 60mmHg), much lower than that of cholinergic saliva (Beal, 1984), and consequently phosphate was the major anion in the sympathetic parotid saliva. In contrast, mandibular salivary pH was much higher and its chloride concentration much lower than in cholinergically-evoked saliva (Beal, l986a) and thus with phosphate levels being low, the major anion in sympathetically-evoked mandibular saliva was probably bicarbonate. Rat and rabbit mandibular glands produce high bicarbonate salivas during sympathetic and sympathomimetic stimulation (Yoshida et al., 1967; Young and Martin, 1971; Case et al., 1980). At the onset and termination of stimulation, saliva produced during the transition between flow states frequently differs in composition from that produced during steady-state flow (Burgen, 1956; Coats and Wright, 1957; Petersen and Poulsen, 1968; Schneyer, 1975; Beal, 1986a). The protocol necessary to obtain the samples from the kangaroos obviously has a number of transition periods and these transients may have influenced the composition of the samples obtained. Consequently, sympathomim~tic stimulation using specific a and fi adrenergic agonists may be a better approach to investigating the influence of the sympathetic nervous system on the composition of kangaroo saliva.
Sympathetic Acknowledgemenrs-I am skilful technical assistance diligent husbandry of the Abbott Australia Pty Ltd barbitone.
salivation
indebted to Mr David Hair for and to Mrs Glenys Forsyth for kangaroos. I would like to thank for the gift of the sodium pento-
REFERENCES
Baginski E. S., Foa P. P. and Zak B. (1967) Microdetermination of inorganic phosphate, phospholipids and total phosphate in biological materials. Clin. Chem. 13, 326332. Beal A. M. (1984) Electrolyte composition of parotid saliva from sodium-replete red kangaroos (Macropus rufus). J. exp. Biol. 111, 225-237. Beal A. M. (1986a) Effects of flow rate, duration of stimulation, and mineralocorticoids on the electrolyte concentrations of mandibular saliva from the red kangaroos (Macropus rufus). J. exp. Biol. 126, 315-339. Beal A. M. (1986b) Responsiveness of the parotid salivary gland of red kangaroos (Macropus rufus) to mineralocorticoids. J. exp. Biol. 120, 309-324. Beal A. M. (1987) Amylase activity, protein and urea in saliva of red kangaroos (Macropus rufus). Arch. oral Biol. 32, 825-832. Blood L. A., Kennerson A. R. and van Lennep E. W. (1977) Morphology and histochemistry of the mandibular gland of the Australian brush-tail possum Trichosurus vulpecula (Marsupialia). Anal. Rec. 188, 4899508. Burgen A. S. V. (1956) The secretion of potassium in saliva. J. Physiol., Lond. 132, 2&39. Case R. M., Conigrave A. D., Novak I. and Young J. A. (1980) Electrolyte and protein secretion by the perfused
in kangaroos
497
rabbit mandibular gland stimulated with acetylcholine or catecholamines. J. Physiol., Land. 300, 467487. Coats D. A. and Wright R. D. (1957) Secretion by the parotid gland of the sheep: the relationship between salivary flow and composition. J. Physiol., Lond. 135, 61 l-622. Coats D. A., Denton D. A.. Godinn J. R. and Wright R. D. (1956) Secretion by the parotih gland of the sheep. J. Phvsiol.. Land. 131. 13-31. Compton J. S. (1976) A’micropuncture study of the effect of mineral active steroids on normal salivary gland function. Honours Thesis, University of Sydney, Sydney. Creed K. E. and McDonald I. R. (1970) Salivary secretory potentials in the marsupial Trichosurus vulpecuia. Camp. gen. Pharmacol. 1, 285-292. Daughaday W. H., Lowry 0. H., Rosebrough N. J. and Fields W. S. (1952) Determination of cerebrospinal fluid protein with the Folin phenol reagent. J. lab. clin. Med. 39, 663465. Fawcett J. K. and Scott J. E. (1960) A rapid and precise method for the determination of urea. J. clin. Pathol. 13, 15&159. Patterson J. and Titchen D. A. (1979) Sympathetic and fi-adrenergic stimulation of parotid salivary secretion in sheep. Q.-J1 exp. Physiol. 64, 175-184. Petersen 0. H. and Poulsen J. H. (1968) The secretion of sodium and potassium in cat submandibular saliva during the first period after start of stimulation. Acfa Physiol. Stand. 73, 93-100. Porter B. (198 1) The structure and function of macropodid salivary glands. PhD thesis, University of New South Wales, Australia, pp. 5455. Young J. A. and Schneyer C. A. (1981) Composition of saliva in mammalia. Aust. J. exp. Biol. med. Sci. 59, l-53.