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Effects of endothelin on submandibular salivary responses to parasympathetic stimulation in anaesthetized sheep
Autonomic Neuroscience: Basic and Clinical 99 (2002) 47 – 53 www.elsevier.com/locate/autneu
Effects of endothelin on submandibular salivary responses to parasympathetic stimulation in anaesthetized sheep A.P. Harrison, Morven E. Cunningham, A.V. Edwards* Physiological Laboratory, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK Received 23 August 2001; accepted 12 April 2002
Abstract Submandibular responses to stimulation of the parasympathetic chorda tympani nerve have been investigated in anaesthetized sheep before, during and after an intracarotid infusion of endothelin, which reduced the blood flow through the gland by 56F5%. Stimulation of the peripheral end of the chorda tympani nerve produced a frequency-dependent increase in the flow of submandibular saliva, and in sodium, potassium and protein output. The reduction in submandibular blood flow, which occurred in response to endothelin, was associated with a decrease in the flow of saliva at all frequencies tested amounting on average to 44F6% ( P < 0.01). The flow of saliva was linearly related to the blood flow before and after endothelin. Both parameters were also linearly related during the infusion of endothelin and the regression lines were parallel. Salivary sodium concentration was significantly increased at the lower frequencies (1 and 2 Hz). Protein output was generally reduced but the difference only achieved statistical significance during stimulation at 1 Hz ( P < 0.01). Thus, submandibular secretory responses to parasympathetic stimulation are significantly attenuated by reducing the blood flow through the gland in this way. D 2002 Published by Elsevier Science B.V. Keywords: Saliva; Submandibular; Blood flow; Endothelin; Protein secretion
1. Introduction Whereas the mechanism of salivary vasodilatation has been the subject of intense interest for over a century (see, for instance, Edwards, 1998), its importance has generally been assumed rather than investigated. It has been widely supposed that any reduction in salivary blood flow would lead to a fall in the rate of salivary secretion. This belief derives from a study in which stimulation of the sympathetic innervation to the submandibular gland of the cat was superimposed on a background of parasympathetic stimulation (Emmelin, 1955). This generally reduced the existing parasympathetic dilatation and, whenever it did, there was a reduction in the rate of flow of saliva; further, the greater the reduction in the blood flow, the greater the reduction in the flow of saliva. However, more recently, it has been shown that, following a small dose of atropine (just sufficient to block secretion of parasympathetic saliva), the flow of saliva in response to sympathetic stimulation is potentiated
*
Corresponding author. Tel.: +44-1223-333-834; fax: +44-1223-333840. E-mail address: [email protected] (A.V. Edwards). 1566-0702/02/$ - see front matter D 2002 Published by Elsevier Science B.V. PII: S 1 5 6 6 - 0 7 0 2 ( 0 2 ) 0 0 0 6 2 - 0
when superimposed on a background of parasympathetic stimulation, despite a reduction in the blood flow (Edwards et al., 1997). Emmelin proceeded to test the effects of reducing submandibular blood flow in other ways. These included carotid and venous occlusion, lowering the arterial blood (perfusion) pressure by vagal stimulation or haemorrhage and the injection of vasoconstrictor agents into the gland. In each case, decreasing submandibular blood flow produced a decrease in the flow of saliva during stimulation of the parasympathetic innervation. Lung (1990) has reported that, at frequencies of parasympathetic stimulation up to and including 8 Hz, the flow of submandibular saliva over 2-min periods is completely independent of blood flow in the anaesthetized dog. However, reducing the submandibular blood flow in anaesthetized cats, by withdrawing blood and so lowering the blood pressure (by about 50%) for 5 min, produced a substantial reduction in the flow of saliva (Hanna et al., 1999). Similar results were obtained when the blood flow was reduced by an intra-arterial infusion of endothelin (Rourke and Edwards, 2000). Either protocol alone is susceptible to criticism. The former on the grounds that severe, generalised hypotension might conceivably have resulted in some secondary effect on salivation and the fact that the animals
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A.P. Harrison et al. / Autonomic Neuroscience: Basic and Clinical 99 (2002) 47–53
The experiments were carried out on seven Welsh ewes (35 –44 kg body weight). Food but not water was withheld for 48 h prior to each experiment. Anaesthesia was induced and maintained with sodium pentobarbitone [Sagatal, Rhoˆne Me´rieux, Harlow, U.K.; 15 –20 mg kg 1 i.v. and then 0.1 – 0.3 mg min 1 kg 1 i.v. (adjusted to maintain a stable blood pressure)]. At the end of each experiment, the animal was given a lethal dose of barbiturate (Pentoject, Animalcare, York, U.K.; ca. 15 ml 20% w/v).
minimise spread of stimulus. Each of the tributaries of the ipsilateral linguofacial vein, except that draining the submandibular gland, was ligated. The animals were heparinized (Mutiparin, CP Pharmaceuticals, Wrexham, U.K.; 1000 i.u. kg 1 i.v.) and the linguofacial vein cannulated with a short length of polythene tubing. The submandibular venous effluent was thereby diverted through a second photoelectric drop-counter and returned to the animal by an electronically controlled pump, via the ipsilateral jugular vein, in such a way as to match input to output. Finally, a bipolar platinum stimulating electrode was placed under the duct and chorda tympani close to the hilum of the gland. The protocol involved comparing submandibular vascular and secretory responses to parasympathetic stimulation at 1, 2, 4 and 8 Hz continuously for 5 min (20 V squarewave; 10 ms pulse-width) before, during and after an ipsilateral intracarotid infusion of endothelin (Endothelin-1 (ET-1), Peninsula Laboratories Europe; 10– 20 pmol min 1 kg 1). The order in which the different stimulus frequencies were tested was varied from animal to animal. The rates of flow of blood and saliva were recorded photometrically drop by drop and also estimated gravimetrically. During stimulation events during the first min were disregarded to allow time for the response to stabilise and to ensure complete evacuation of the submandibular dead space. Samples of blood and saliva were then collected from 1 to 3 and 3 to 5 min. The samples of blood were weighed for gravimetric estimation of blood flow and then returned to the animal. The samples of saliva were weighed for gravimetric estimation of salivary flow and then sequestered at + 4 jC for cation and protein measurements. At postmortem examination, both submandibular glands were removed and weighed. Small representative pieces were fixed in formol sucrose. Later, they were processed to paraffin wax, sectioned (7-Am sections), stained with celestine blue, eosin and alcian blue, and examined microscopically.
2.2. Surgical and experimental procedures
2.3. Estimations
The trachea was intubated and then exposed via a midline incision low in the neck. The right ascending cervical sympathetic nerve was exposed and cut, and a narrow bore needle, suaged to a length of fine polythene tubing, was inserted into the lumen of the ipsilateral carotid artery for the subsequent infusion of endothelin. An arterial catheter was introduced into the abdominal aorta via a femoral artery and later employed to monitor arterial blood pressure and heart rate; samples of arterial blood were also collected periodically for measurements of the packed cell volume. A femoral vein was cannulated to provide a conduit for the continuous infusion of sodium pentobarbitone. The right chorda tympani nerve was exposed and the ipsilateral submandibular duct was cannulated with the widest bore nylon tubing practicable. The free end was then positioned above a photoelectric drop-counter. A neighbouring length of the right hypoglossal nerve was excised in order to
The concentrations of sodium and potassium in saliva were measured using a Corning 435 Flame Photometer. Salivary protein was measured using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Munchen, Germany). Submandibular vascular resistance was estimated by dividing the perfusion (arterial blood) pressure by the submandibular blood flow. All the results presented are those of the 3– 5min samples because salivary flow at the lower frequencies was sometimes too low to clear the dead space completely. They are expressed as mean valuesFS.E.M. and were assessed statistically by means of paired or unpaired Student’s t-test with n = number of animals. Statistical significance between gradients and analysis of covariance were calculated as described by Armitage and Berry (1994). All flows and outputs are expressed per unit weight of the contralateral gland to eliminate any error arising from oedema in the experimental gland.
were pretreated with large doses of adrenergic blocking agents. The latter on the grounds that endothelin might conceivably have some effect on the submandibular gland, other than vasoconstriction, although none such has been reported. Taken together, they provide compelling evidence that reducing submandibular blood flow in this way compromises the secretion of saliva from the gland in the cat; they also validate the use of endothelin as a tool with which to manipulate submandibular blood flow. In the present study we have capitalised on this to investigate the effects of reducing the blood flow through the gland in sheep, which are noncarnivorous and could not survive a 50% reduction in blood pressure induced by haemorrhage with total adrenergic blockade. This is the first study of the effect of blood flow on submandibular function in any ruminant species, and was undertaken in order to establish the effects of endothelin-induced vasoconstriction on the volume and composition of saliva secreted in response to parasympathetic stimulation. The results have been reported previously in a preliminary form (Cunningham et al., 2001).
2. Materials and methods 2.1. Animals
A.P. Harrison et al. / Autonomic Neuroscience: Basic and Clinical 99 (2002) 47–53
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3. Results 3.1. Cardiovascular effects of ET-1 Mean arterial blood pressure during chorda tympani stimulation was closely similar at all frequencies tested before, during and after the infusion of ET-1 (10 – 20 pmol min 1 kg 1 i.c., Fig. 1.). It seems that any rise in total peripheral resistance was compensated for by a reduction in cardiac output as there was a significant fall in mean heart rate during the infusion, from 100F6 to 92F4 beats min 1 ( P < 0.05, Fig. 1). There was a frequency-dependent fall in submandibular vascular resistance over the range 1– 8 Hz and the relation was strictly linear over the range 1 – 4 Hz (Fig. 2). During the infusion of ET-1, there was a substantial and significant rise in these values ( P < 0.05) but the linear relation over the range 1– 4 was preserved (r2=0.99). This resulted in a significant fall in the flow of blood through the gland at each frequency tested as had been intended ( P < 0.01, Fig. 3) and amounted, on average, to 56F5%. Periodic measurements of arterial packed cell volume showed that this remained constant throughout the experiments. 3.2. Secretory effects of ET-1 The reduction in submandibular blood flow, during the infusion of ET-1, was associated with a significant decrease in the flow of saliva, in response to chorda tympani stimulation at all frequencies tested (Fig. 3). Thus, the rate of flow of saliva in response to stimulation at 1 Hz was 14F2 Al min 1 (g gland) 1 before ET-1 compared with a
Fig. 1. Mean aortic blood pressure and heart rate in seven anaesthetized sheep during parasympathetic stimulation before, during and after ET-1 (10 – 20 pmol min 1 kg 1 i.c.). Vertical bars: S.E. of each mean value.
Fig. 2. The relation between submandibular vascular resistance and the frequency of parasympathetic chorda tympani stimulation in seven anaesthetized sheep before and after (o) and during (.) ET-1 (10 – 20 pmol min 1 kg 1 i.c.). Regression lines estimated by method of least squares. The (o) data points at 1 Hz overlap precisely.
value of 4 F 1 Al min 1 (g gland) 1 in the presence of the peptide ( P < 0.01). At the higher frequencies of stimulation that were employed, the corresponding values before and during the infusion of ET-1 were 33F5 and 16 F 5 Al min 1 (g gland) 1 at 2 Hz ( P < 0.05), 74F7 and 39 F 8 Al min 1 (g gland) 1 at 4 Hz ( P < 0.01) and 97F10 and 62F 11 Al min 1 (g gland) 1 at 8 Hz ( P < 0.05). On average, the reduction in salivary flow during the infusion of ET-1 amounted to 44 F 6%. There was also a closely linear relation between the flow of blood through and the secretion of saliva from the gland during parasympathetic stimulation before and after ET-1 (r2= 0.98; Fig. 4). In
Fig. 3. Submandibular blood and salivary flow in response to parasympathetic stimulation in seven anaesthetized sheep before, during and after ET-1 (10 – 20 pmol min 1 kg 1 i.c.). Vertical bars: S.E. of each mean value. * P < 0.05; *** P < 0.01.
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Fig. 4. The relation between submandibular blood and salivary flow in response to parasympathetic stimulation in seven anaesthetized sheep before and after (o) and during (.) ET-1 (10 – 20 pmol min 1 kg 1 i.c.).
the presence of ET-1, the values obtained during stimulation at the higher frequencies (2– 8 Hz) were displaced to the left, showing that the gland is capable of secreting more saliva than corresponds to the normal blood flow under these experimental conditions. Over the range of frequencies that were employed in this study, this difference amounted to about 150 Al min 1 (g gland) 1. The raw data from a representative experiment in which the chorda tympani was stimulated at 4 Hz are illustrated in Fig. 5. As commonly occurred, both submandibular blood flow and salivary flow rose to initial peaks within the first min of the onset of chorda tympani stimulation. They then
Fig. 5. Submandibular saliva and blood flow, heart rate and aortic blood pressure in response to stimulation of the peripheral end of the chorda tympani (4 Hz for 5 min) before, during and after an infusion of ET-1 (10 – 20 pmol min 1 (g gland) 1 i.c.) in an anaesthetized sheep.
Fig. 6. Submandibular salivary sodium and potassium concentrations in response to parasympathetic stimulation in seven anaesthetized sheep before, during and after ET-1 (10 – 20 pmol min 1 kg 1 i.c.). Vertical bars: S.E. of each mean value. * P < 0.05; ** P < 0.02; *** P < 0.01.
declined to lower plateau values, which were usually well maintained until stimulation was discontinued. The pattern was preserved when the blood flow was reduced during the
Fig. 7. Submandibular salivary sodium and potassium outputs in response to parasympathetic stimulation in seven anaesthetized sheep before, during and after ET-1 (10 – 20 pmol min 1 kg 1 i.c.). Vertical bars: S.E. of each mean value. * P < 0.05; ** P < 0.02; *** P < 0.01; **** P < 0.001.
A.P. Harrison et al. / Autonomic Neuroscience: Basic and Clinical 99 (2002) 47–53
infusion of endothelin but the overall rate of secretion was diminished. The concentration of sodium ions in the submandibular saliva increased steadily with frequency of chorda tympani stimulation over the range 2 –8 Hz before, during and after the infusion of ET-1. The peptide had the effect of raising the salivary sodium concentration at each of the frequencies tested and the differences were statistically significant at both 1 and 2 Hz ( P < 0.05 and 0.02, respectively; Fig. 6). This effect was more persistent than the reduction in flow and the values for the tests carried out after the infusion of ET-1 had been discontinued were uniformly significantly higher than the initial values (Fig. 6). The elevated sodium concentrations tended to mitigate the effect of the reduction in flow on the output of sodium and in no case did this differ significantly during the infusion of ET-1 from the corresponding initial value (Fig. 7). ET-1 significantly reduced the concentration of potassium in saliva produced in response to chorda tympani stimulation at 1 Hz (from 24.6F4.6 to 14.6F2.5 mmol l 1, P < 0.05; Fig. 6) but not at the other frequencies tested. Nevertheless, the output of potassium in the submandibular saliva was generally significantly reduced in the presence of ET-1 (Fig. 7), reflecting the reduction in the flow of saliva. Like the change in sodium concentration, the effect on potassium output was persistent and significantly greater than it had been initially at all frequencies when tested after the infusion of ET-1 had been discontinued. The output of protein in the submandibular saliva increased steadily with the frequency of chorda tympani stimulation over the range 1– 8 Hz (Fig. 8). In the presence of ET-1, the output of protein in response to chorda tympani stimulation was significantly reduced at 1 Hz (from 33.7F4.5 to 12.6F4.6 Ag min 1 (g gland) 1, P < 0.01; Fig. 8) but not at the higher frequencies that were employed. Histological examination postmortem failed to reveal any gross differences between glands that had been tested and those on the contralateral side, both appearing quite normal.
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4. Discussion In embarking on this study, it has been assumed that the infusion of ET-1, at the low dose (10 –20 pmol min 1 kg 1 i.c.) that was employed, produced constriction of the submandibular vasculature, as it does throughout the body (Yanagisawa et al., 1988; Ais et al., 1989), without eliciting any other effects in the gland that might affect secretion in response to parasympathetic stimulation. This is justified by the fact that there have been no reports that the peptide exerts any effect in salivary glands other than vasoconstriction in any species either in vivo or in vitro. Furthermore, its effect on submandibular secretory responses to parasympathetic stimulation in the cat can be mimicked precisely, when the blood flow is reduced by simply by lowering the perfusion pressure (Hanna et al., 1999; Rourke and Edwards, 2000). The results show that decreasing the flow of blood through the gland produces a concomitant reduction in the secretion of saliva in response to parasympathetic stimulation and that the effect is reversible within a few minutes. In this respect, the submandibular gland of the sheep resembles that of the cat (Rourke and Edwards, 2000). Likewise, the flow of saliva was linearly related to blood flow both before and after the infusion of ET-1 (Fig. 4; r = 0.99), as it is in the cat (Rourke and Edwards, 2000). However, although all the values during the infusion of ET-1 fell to the left of the regression line for the values obtained before and after the infusion, the shift was less pronounced than in the cat. It would therefore appear that the ovine submandibular secretory mechanisms are less robust, in the face of a reduced blood flow, than those in the feline gland. In fact, over the whole range of stimulus frequencies that were employed, the flow of blood was about 150 Al min 1(g gland) 1 less for any given flow of saliva (Fig. 4). When the rate of flow of saliva is reduced, there is normally an associated reduction in salivary sodium concen-
Fig. 8. Submandibular salivary protein output in response to parasympathetic stimulation in seven anaesthetized sheep before, during and after ET-1 (10 – 20 pmol min 1 kg 1 i.c.). Vertical bars: S.E. of each mean value. *** P < 0.01.
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tration (Gregersen and Ingalls, 1931; Langstroth et al., 1938), which is generally attributed to increased reabsorption of sodium in the ducts when the time taken to traverse them increases (Thaysen et al., 1954; Miyoshi, 1963). In the present study, the values during the infusion of ET-1 were consistently higher than they had been initially and the differences were statistically significant at both the lowest frequencies. It therefore appears that, as reported previously in the cat (Hanna et al., 1999; Rourke and Edwards, 2000), reducing the blood flow impairs tubular sodium reabsorption at least to the same extent as it inhibits the flow of saliva. The concentration of potassium in the saliva was generally unaffected by the rate of flow, in agreement with observations in other species (Gregersen and Ingalls, 1931; Langstroth et al., 1938), though not the cat, using a precisely similar protocol (Rourke and Edwards, 2000). During the plateau phase of salivary secretion, potassium is derived from the plasma through the intracellular pool (Burgen, 1956). During the ET-1 infusion in sheep, it may be that the decreased blood flow through the submandibular gland limits the availability of potassium and so prevents a rise in the concentration in the saliva. Unlike the cat, in which the output of protein was significantly reduced during the infusion of ET-1 at all frequencies of parasympathetic stimulation tested (Rourke and Edwards, 2000), this was only observed during stimulation at the lowest frequency (1 Hz) in sheep. Vasoactive intestinal peptide (VIP) has been found to enhance the output of protein in submandibular saliva in several species, including the cat and the sheep, with an action that depends upon the presence of nitric oxide (Reid and Heywood, 1988; Buckle et al., 1995; Hanna and Edwards, 1998). VIP is known to be released from the submandibular gland of the sheep during parasympathetic stimulation (Edwards and Edwards, 2001). However, release of effective amounts of this peptide is demanding of relatively high frequencies of stimulation ( > 2 Hz; Andersson et al., 1982; Lundberg and Ho¨kfelt, 1983; Edwards and Edwards, 2001). Accordingly it seems that, in this gland, secretion of protein in response to chorda tympani stimulation at a frequency below that which releases effective amounts of VIP is more susceptible to inhibition when the blood flow is reduced than the response to stimulation at a high frequency such as would elicit release of VIP. It is concluded that an adequate blood flow is required to maintain salivary secretion in the sheep, as established previously in the cat (Hanna et al., 1999; Rourke and Edwards, 2000). These complementary results in two very different species reinforce the conclusion that the gland is unable to sustain the normal rate of flow of saliva over more than a few minutes in the face of a substantial reduction in blood flow. However, the sheep differs from the cat with respect to the associated changes in the output of submandibular protein. In the cat, reducing the submandibular blood flow with i.c. infusions of ET-1 produced a highly significant fall in the output of salivary protein ( 67F7%; P < 0.01)
during parasympathetic stimulation at 2, 4, 8 and 16 Hz (Rourke and Edwards, 2000). The results of the present study show that precisely the same procedure has no significant effect on submandibular protein output in sheep during parasympathetic stimulation within this range of frequencies, although there was a significant fall during stimulation at a lower frequency (1 Hz; P < 0.01). Thus, whereas the flow of saliva is more sensitive to this constraint in the sheep than it is in the cat, the opposite obtains with regard to protein output. A rate of submandibular blood flow that is adequate for one secretory function may therefore be insufficient for another and the relations vary between species.
Acknowledgements It is a particular pleasure to acknowledge the skilled technical assistance provided by Mr. P.M.M. Bircham.
References Ais, G., Novo, C., Lo´pez-Farre´, A., Romeo, J.M., Lo´pez-Novoa, J.M., 1989. Effect of endothelin on systemic and regional hemodynamics in rats. Eur. J. Pharmacol. 170, 113 – 116. Andersson, P.-O., Bloom, S.R., Ja˚rhult, J., Edwards, A.V., 1982. Effects of stimulation of the chorda-tympani in bursts on submaxillary responses in the cat. J. Physiol. (London) 322, 469 – 483. Armitage, P., Berry, G., 1994. Statistical methods. In: Armitage, P., Berry, G. (Eds.), Medical Research, 3rd edn. Blackwell, Oxford, Ch. 9. Buckle, A.D., Parker, S.J., Bloom, S.R., Edwards, A.V., 1995. The role of nitric oxide in the control of protein secretion in the submandibular gland of the cat. Exp. Physiol. 80, 1019 – 1030. Burgen, A.S.V., 1956. The secretion of potassium in saliva. J. Physiol. 132, 20 – 39. Cunningham, M.E., Harrison, A.P., Edwards, A.V., 2001. Effects of endothelin-1 on submandibular vascular and secretory responses to parasympathetic stimulation in anaesthetized sheep. J. Physiol. (London) 531P, 100P – 101P. Edwards, A.V., 1998. Autonomic control of salivary blood flow. In: Garrett, J.R., Ekstro¨m, J., Anderson, L.C. (Eds.), Glandular Mechanisms of Salivary Secretion. Frontiers of Oral Biology. Karger, Basle, pp. 101 – 117, Ch. 10. Edwards, C.M.B., Edwards, A.V., 2001. Release of vasoactive intestinal peptide (VIP) from the submandibular gland in the anaesthetized cats. J. Physiol. (London) 531P, 100P. Edwards, A.V., Garrett, J.R., Proctor, G.B., 1997. Secretory interactions between the sympathetic and parasympathetic innervations of the submandibular gland in the anaesthetized cat. Exp. Physiol. 82, 697 – 708. Emmelin, N., 1955. Blood flow and secretion in the salivary gland. Acta Physiol. Scand. 34, 22 – 28. Gregersen, M.I., Ingalls, E.M., 1931. The influence of rate of secretion on the concentration of potassium and sodium in the dog submaxillary saliva. Am. J. Physiol. 98, 441 – 446. Hanna, S.J., Edwards, A.V., 1998. The role of nitric oxide in the control of protein secretion in the parotid gland of anaesthetized sheep. Exp. Physiol. 83, 533 – 544. Hanna, S.J., Brelen, M.A., Edwards, A.V., 1999. Effects of reducing submandibular blood flow on secretory responses to parasympathetic stimulation in anaesthetized cats. Exp. Physiol. 84, 677 – 687. Langstroth, G.O., McRae, D.R., Stavraky, G.W., 1938. The secretion of protein material in the parasympathetic submaxillary saliva. Proc. R. Soc., Ser. B 125, 335 – 347.
A.P. Harrison et al. / Autonomic Neuroscience: Basic and Clinical 99 (2002) 47–53 Lundberg, J.M., Ho¨kfelt, T., 1983. Coexistence of peptides and classical neurotransmitters. Trends Neurosci. 6, 325 – 333. Lung, M.A., 1990. Variations in blood flow on mandibular glandular secretion to autonomic nervous stimulations in anaesthetized dogs. J. Physiol. (London) 431, 479 – 493. Miyoshi, M., 1963. Studies on nervous control of the salt composition in saliva. Jpn. J. Physiol. 13, 541 – 563. Reid, A.M., Heywood, L.H., 1988. A comparison of the effects of vasoactive intestinal polypeptide on secretion from the submaxillary gland of sheep and goats. Regul. Pept. 20, 211 – 221.
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Rourke, K., Edwards, A.V., 2000. Submandibular secretory and vascular responses to stimulation of the parasympathetic innervation in anaesthetized cats. J. Appl. Physiol. 89, 964 – 1970. Thaysen, J.H., Thorn, N.A., Schwartz, I.L., 1954. Excretion of sodium, potassium, chloride and carbon dioxide in human parotid saliva. Am. J. Physiol. 178, 155 – 159. Yanagisawa, A.M., Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, M., Mitsui, Y., Yazaki, Y., Goto, K., Masaki, T., 1988. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332, 411 – 415.
Effects of endothelin on submandibular salivary responses to parasympathetic stimulation in anaesthetized sheep A.P. Harrison, Morven E. Cunningham, A.V. Edwards* Physiological Laboratory, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK Received 23 August 2001; accepted 12 April 2002
Abstract Submandibular responses to stimulation of the parasympathetic chorda tympani nerve have been investigated in anaesthetized sheep before, during and after an intracarotid infusion of endothelin, which reduced the blood flow through the gland by 56F5%. Stimulation of the peripheral end of the chorda tympani nerve produced a frequency-dependent increase in the flow of submandibular saliva, and in sodium, potassium and protein output. The reduction in submandibular blood flow, which occurred in response to endothelin, was associated with a decrease in the flow of saliva at all frequencies tested amounting on average to 44F6% ( P < 0.01). The flow of saliva was linearly related to the blood flow before and after endothelin. Both parameters were also linearly related during the infusion of endothelin and the regression lines were parallel. Salivary sodium concentration was significantly increased at the lower frequencies (1 and 2 Hz). Protein output was generally reduced but the difference only achieved statistical significance during stimulation at 1 Hz ( P < 0.01). Thus, submandibular secretory responses to parasympathetic stimulation are significantly attenuated by reducing the blood flow through the gland in this way. D 2002 Published by Elsevier Science B.V. Keywords: Saliva; Submandibular; Blood flow; Endothelin; Protein secretion
1. Introduction Whereas the mechanism of salivary vasodilatation has been the subject of intense interest for over a century (see, for instance, Edwards, 1998), its importance has generally been assumed rather than investigated. It has been widely supposed that any reduction in salivary blood flow would lead to a fall in the rate of salivary secretion. This belief derives from a study in which stimulation of the sympathetic innervation to the submandibular gland of the cat was superimposed on a background of parasympathetic stimulation (Emmelin, 1955). This generally reduced the existing parasympathetic dilatation and, whenever it did, there was a reduction in the rate of flow of saliva; further, the greater the reduction in the blood flow, the greater the reduction in the flow of saliva. However, more recently, it has been shown that, following a small dose of atropine (just sufficient to block secretion of parasympathetic saliva), the flow of saliva in response to sympathetic stimulation is potentiated
*
Corresponding author. Tel.: +44-1223-333-834; fax: +44-1223-333840. E-mail address: [email protected] (A.V. Edwards). 1566-0702/02/$ - see front matter D 2002 Published by Elsevier Science B.V. PII: S 1 5 6 6 - 0 7 0 2 ( 0 2 ) 0 0 0 6 2 - 0
when superimposed on a background of parasympathetic stimulation, despite a reduction in the blood flow (Edwards et al., 1997). Emmelin proceeded to test the effects of reducing submandibular blood flow in other ways. These included carotid and venous occlusion, lowering the arterial blood (perfusion) pressure by vagal stimulation or haemorrhage and the injection of vasoconstrictor agents into the gland. In each case, decreasing submandibular blood flow produced a decrease in the flow of saliva during stimulation of the parasympathetic innervation. Lung (1990) has reported that, at frequencies of parasympathetic stimulation up to and including 8 Hz, the flow of submandibular saliva over 2-min periods is completely independent of blood flow in the anaesthetized dog. However, reducing the submandibular blood flow in anaesthetized cats, by withdrawing blood and so lowering the blood pressure (by about 50%) for 5 min, produced a substantial reduction in the flow of saliva (Hanna et al., 1999). Similar results were obtained when the blood flow was reduced by an intra-arterial infusion of endothelin (Rourke and Edwards, 2000). Either protocol alone is susceptible to criticism. The former on the grounds that severe, generalised hypotension might conceivably have resulted in some secondary effect on salivation and the fact that the animals
48
A.P. Harrison et al. / Autonomic Neuroscience: Basic and Clinical 99 (2002) 47–53
The experiments were carried out on seven Welsh ewes (35 –44 kg body weight). Food but not water was withheld for 48 h prior to each experiment. Anaesthesia was induced and maintained with sodium pentobarbitone [Sagatal, Rhoˆne Me´rieux, Harlow, U.K.; 15 –20 mg kg 1 i.v. and then 0.1 – 0.3 mg min 1 kg 1 i.v. (adjusted to maintain a stable blood pressure)]. At the end of each experiment, the animal was given a lethal dose of barbiturate (Pentoject, Animalcare, York, U.K.; ca. 15 ml 20% w/v).
minimise spread of stimulus. Each of the tributaries of the ipsilateral linguofacial vein, except that draining the submandibular gland, was ligated. The animals were heparinized (Mutiparin, CP Pharmaceuticals, Wrexham, U.K.; 1000 i.u. kg 1 i.v.) and the linguofacial vein cannulated with a short length of polythene tubing. The submandibular venous effluent was thereby diverted through a second photoelectric drop-counter and returned to the animal by an electronically controlled pump, via the ipsilateral jugular vein, in such a way as to match input to output. Finally, a bipolar platinum stimulating electrode was placed under the duct and chorda tympani close to the hilum of the gland. The protocol involved comparing submandibular vascular and secretory responses to parasympathetic stimulation at 1, 2, 4 and 8 Hz continuously for 5 min (20 V squarewave; 10 ms pulse-width) before, during and after an ipsilateral intracarotid infusion of endothelin (Endothelin-1 (ET-1), Peninsula Laboratories Europe; 10– 20 pmol min 1 kg 1). The order in which the different stimulus frequencies were tested was varied from animal to animal. The rates of flow of blood and saliva were recorded photometrically drop by drop and also estimated gravimetrically. During stimulation events during the first min were disregarded to allow time for the response to stabilise and to ensure complete evacuation of the submandibular dead space. Samples of blood and saliva were then collected from 1 to 3 and 3 to 5 min. The samples of blood were weighed for gravimetric estimation of blood flow and then returned to the animal. The samples of saliva were weighed for gravimetric estimation of salivary flow and then sequestered at + 4 jC for cation and protein measurements. At postmortem examination, both submandibular glands were removed and weighed. Small representative pieces were fixed in formol sucrose. Later, they were processed to paraffin wax, sectioned (7-Am sections), stained with celestine blue, eosin and alcian blue, and examined microscopically.
2.2. Surgical and experimental procedures
2.3. Estimations
The trachea was intubated and then exposed via a midline incision low in the neck. The right ascending cervical sympathetic nerve was exposed and cut, and a narrow bore needle, suaged to a length of fine polythene tubing, was inserted into the lumen of the ipsilateral carotid artery for the subsequent infusion of endothelin. An arterial catheter was introduced into the abdominal aorta via a femoral artery and later employed to monitor arterial blood pressure and heart rate; samples of arterial blood were also collected periodically for measurements of the packed cell volume. A femoral vein was cannulated to provide a conduit for the continuous infusion of sodium pentobarbitone. The right chorda tympani nerve was exposed and the ipsilateral submandibular duct was cannulated with the widest bore nylon tubing practicable. The free end was then positioned above a photoelectric drop-counter. A neighbouring length of the right hypoglossal nerve was excised in order to
The concentrations of sodium and potassium in saliva were measured using a Corning 435 Flame Photometer. Salivary protein was measured using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Munchen, Germany). Submandibular vascular resistance was estimated by dividing the perfusion (arterial blood) pressure by the submandibular blood flow. All the results presented are those of the 3– 5min samples because salivary flow at the lower frequencies was sometimes too low to clear the dead space completely. They are expressed as mean valuesFS.E.M. and were assessed statistically by means of paired or unpaired Student’s t-test with n = number of animals. Statistical significance between gradients and analysis of covariance were calculated as described by Armitage and Berry (1994). All flows and outputs are expressed per unit weight of the contralateral gland to eliminate any error arising from oedema in the experimental gland.
were pretreated with large doses of adrenergic blocking agents. The latter on the grounds that endothelin might conceivably have some effect on the submandibular gland, other than vasoconstriction, although none such has been reported. Taken together, they provide compelling evidence that reducing submandibular blood flow in this way compromises the secretion of saliva from the gland in the cat; they also validate the use of endothelin as a tool with which to manipulate submandibular blood flow. In the present study we have capitalised on this to investigate the effects of reducing the blood flow through the gland in sheep, which are noncarnivorous and could not survive a 50% reduction in blood pressure induced by haemorrhage with total adrenergic blockade. This is the first study of the effect of blood flow on submandibular function in any ruminant species, and was undertaken in order to establish the effects of endothelin-induced vasoconstriction on the volume and composition of saliva secreted in response to parasympathetic stimulation. The results have been reported previously in a preliminary form (Cunningham et al., 2001).
2. Materials and methods 2.1. Animals
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3. Results 3.1. Cardiovascular effects of ET-1 Mean arterial blood pressure during chorda tympani stimulation was closely similar at all frequencies tested before, during and after the infusion of ET-1 (10 – 20 pmol min 1 kg 1 i.c., Fig. 1.). It seems that any rise in total peripheral resistance was compensated for by a reduction in cardiac output as there was a significant fall in mean heart rate during the infusion, from 100F6 to 92F4 beats min 1 ( P < 0.05, Fig. 1). There was a frequency-dependent fall in submandibular vascular resistance over the range 1– 8 Hz and the relation was strictly linear over the range 1 – 4 Hz (Fig. 2). During the infusion of ET-1, there was a substantial and significant rise in these values ( P < 0.05) but the linear relation over the range 1– 4 was preserved (r2=0.99). This resulted in a significant fall in the flow of blood through the gland at each frequency tested as had been intended ( P < 0.01, Fig. 3) and amounted, on average, to 56F5%. Periodic measurements of arterial packed cell volume showed that this remained constant throughout the experiments. 3.2. Secretory effects of ET-1 The reduction in submandibular blood flow, during the infusion of ET-1, was associated with a significant decrease in the flow of saliva, in response to chorda tympani stimulation at all frequencies tested (Fig. 3). Thus, the rate of flow of saliva in response to stimulation at 1 Hz was 14F2 Al min 1 (g gland) 1 before ET-1 compared with a
Fig. 1. Mean aortic blood pressure and heart rate in seven anaesthetized sheep during parasympathetic stimulation before, during and after ET-1 (10 – 20 pmol min 1 kg 1 i.c.). Vertical bars: S.E. of each mean value.
Fig. 2. The relation between submandibular vascular resistance and the frequency of parasympathetic chorda tympani stimulation in seven anaesthetized sheep before and after (o) and during (.) ET-1 (10 – 20 pmol min 1 kg 1 i.c.). Regression lines estimated by method of least squares. The (o) data points at 1 Hz overlap precisely.
value of 4 F 1 Al min 1 (g gland) 1 in the presence of the peptide ( P < 0.01). At the higher frequencies of stimulation that were employed, the corresponding values before and during the infusion of ET-1 were 33F5 and 16 F 5 Al min 1 (g gland) 1 at 2 Hz ( P < 0.05), 74F7 and 39 F 8 Al min 1 (g gland) 1 at 4 Hz ( P < 0.01) and 97F10 and 62F 11 Al min 1 (g gland) 1 at 8 Hz ( P < 0.05). On average, the reduction in salivary flow during the infusion of ET-1 amounted to 44 F 6%. There was also a closely linear relation between the flow of blood through and the secretion of saliva from the gland during parasympathetic stimulation before and after ET-1 (r2= 0.98; Fig. 4). In
Fig. 3. Submandibular blood and salivary flow in response to parasympathetic stimulation in seven anaesthetized sheep before, during and after ET-1 (10 – 20 pmol min 1 kg 1 i.c.). Vertical bars: S.E. of each mean value. * P < 0.05; *** P < 0.01.
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A.P. Harrison et al. / Autonomic Neuroscience: Basic and Clinical 99 (2002) 47–53
Fig. 4. The relation between submandibular blood and salivary flow in response to parasympathetic stimulation in seven anaesthetized sheep before and after (o) and during (.) ET-1 (10 – 20 pmol min 1 kg 1 i.c.).
the presence of ET-1, the values obtained during stimulation at the higher frequencies (2– 8 Hz) were displaced to the left, showing that the gland is capable of secreting more saliva than corresponds to the normal blood flow under these experimental conditions. Over the range of frequencies that were employed in this study, this difference amounted to about 150 Al min 1 (g gland) 1. The raw data from a representative experiment in which the chorda tympani was stimulated at 4 Hz are illustrated in Fig. 5. As commonly occurred, both submandibular blood flow and salivary flow rose to initial peaks within the first min of the onset of chorda tympani stimulation. They then
Fig. 5. Submandibular saliva and blood flow, heart rate and aortic blood pressure in response to stimulation of the peripheral end of the chorda tympani (4 Hz for 5 min) before, during and after an infusion of ET-1 (10 – 20 pmol min 1 (g gland) 1 i.c.) in an anaesthetized sheep.
Fig. 6. Submandibular salivary sodium and potassium concentrations in response to parasympathetic stimulation in seven anaesthetized sheep before, during and after ET-1 (10 – 20 pmol min 1 kg 1 i.c.). Vertical bars: S.E. of each mean value. * P < 0.05; ** P < 0.02; *** P < 0.01.
declined to lower plateau values, which were usually well maintained until stimulation was discontinued. The pattern was preserved when the blood flow was reduced during the
Fig. 7. Submandibular salivary sodium and potassium outputs in response to parasympathetic stimulation in seven anaesthetized sheep before, during and after ET-1 (10 – 20 pmol min 1 kg 1 i.c.). Vertical bars: S.E. of each mean value. * P < 0.05; ** P < 0.02; *** P < 0.01; **** P < 0.001.
A.P. Harrison et al. / Autonomic Neuroscience: Basic and Clinical 99 (2002) 47–53
infusion of endothelin but the overall rate of secretion was diminished. The concentration of sodium ions in the submandibular saliva increased steadily with frequency of chorda tympani stimulation over the range 2 –8 Hz before, during and after the infusion of ET-1. The peptide had the effect of raising the salivary sodium concentration at each of the frequencies tested and the differences were statistically significant at both 1 and 2 Hz ( P < 0.05 and 0.02, respectively; Fig. 6). This effect was more persistent than the reduction in flow and the values for the tests carried out after the infusion of ET-1 had been discontinued were uniformly significantly higher than the initial values (Fig. 6). The elevated sodium concentrations tended to mitigate the effect of the reduction in flow on the output of sodium and in no case did this differ significantly during the infusion of ET-1 from the corresponding initial value (Fig. 7). ET-1 significantly reduced the concentration of potassium in saliva produced in response to chorda tympani stimulation at 1 Hz (from 24.6F4.6 to 14.6F2.5 mmol l 1, P < 0.05; Fig. 6) but not at the other frequencies tested. Nevertheless, the output of potassium in the submandibular saliva was generally significantly reduced in the presence of ET-1 (Fig. 7), reflecting the reduction in the flow of saliva. Like the change in sodium concentration, the effect on potassium output was persistent and significantly greater than it had been initially at all frequencies when tested after the infusion of ET-1 had been discontinued. The output of protein in the submandibular saliva increased steadily with the frequency of chorda tympani stimulation over the range 1– 8 Hz (Fig. 8). In the presence of ET-1, the output of protein in response to chorda tympani stimulation was significantly reduced at 1 Hz (from 33.7F4.5 to 12.6F4.6 Ag min 1 (g gland) 1, P < 0.01; Fig. 8) but not at the higher frequencies that were employed. Histological examination postmortem failed to reveal any gross differences between glands that had been tested and those on the contralateral side, both appearing quite normal.
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4. Discussion In embarking on this study, it has been assumed that the infusion of ET-1, at the low dose (10 –20 pmol min 1 kg 1 i.c.) that was employed, produced constriction of the submandibular vasculature, as it does throughout the body (Yanagisawa et al., 1988; Ais et al., 1989), without eliciting any other effects in the gland that might affect secretion in response to parasympathetic stimulation. This is justified by the fact that there have been no reports that the peptide exerts any effect in salivary glands other than vasoconstriction in any species either in vivo or in vitro. Furthermore, its effect on submandibular secretory responses to parasympathetic stimulation in the cat can be mimicked precisely, when the blood flow is reduced by simply by lowering the perfusion pressure (Hanna et al., 1999; Rourke and Edwards, 2000). The results show that decreasing the flow of blood through the gland produces a concomitant reduction in the secretion of saliva in response to parasympathetic stimulation and that the effect is reversible within a few minutes. In this respect, the submandibular gland of the sheep resembles that of the cat (Rourke and Edwards, 2000). Likewise, the flow of saliva was linearly related to blood flow both before and after the infusion of ET-1 (Fig. 4; r = 0.99), as it is in the cat (Rourke and Edwards, 2000). However, although all the values during the infusion of ET-1 fell to the left of the regression line for the values obtained before and after the infusion, the shift was less pronounced than in the cat. It would therefore appear that the ovine submandibular secretory mechanisms are less robust, in the face of a reduced blood flow, than those in the feline gland. In fact, over the whole range of stimulus frequencies that were employed, the flow of blood was about 150 Al min 1(g gland) 1 less for any given flow of saliva (Fig. 4). When the rate of flow of saliva is reduced, there is normally an associated reduction in salivary sodium concen-
Fig. 8. Submandibular salivary protein output in response to parasympathetic stimulation in seven anaesthetized sheep before, during and after ET-1 (10 – 20 pmol min 1 kg 1 i.c.). Vertical bars: S.E. of each mean value. *** P < 0.01.
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tration (Gregersen and Ingalls, 1931; Langstroth et al., 1938), which is generally attributed to increased reabsorption of sodium in the ducts when the time taken to traverse them increases (Thaysen et al., 1954; Miyoshi, 1963). In the present study, the values during the infusion of ET-1 were consistently higher than they had been initially and the differences were statistically significant at both the lowest frequencies. It therefore appears that, as reported previously in the cat (Hanna et al., 1999; Rourke and Edwards, 2000), reducing the blood flow impairs tubular sodium reabsorption at least to the same extent as it inhibits the flow of saliva. The concentration of potassium in the saliva was generally unaffected by the rate of flow, in agreement with observations in other species (Gregersen and Ingalls, 1931; Langstroth et al., 1938), though not the cat, using a precisely similar protocol (Rourke and Edwards, 2000). During the plateau phase of salivary secretion, potassium is derived from the plasma through the intracellular pool (Burgen, 1956). During the ET-1 infusion in sheep, it may be that the decreased blood flow through the submandibular gland limits the availability of potassium and so prevents a rise in the concentration in the saliva. Unlike the cat, in which the output of protein was significantly reduced during the infusion of ET-1 at all frequencies of parasympathetic stimulation tested (Rourke and Edwards, 2000), this was only observed during stimulation at the lowest frequency (1 Hz) in sheep. Vasoactive intestinal peptide (VIP) has been found to enhance the output of protein in submandibular saliva in several species, including the cat and the sheep, with an action that depends upon the presence of nitric oxide (Reid and Heywood, 1988; Buckle et al., 1995; Hanna and Edwards, 1998). VIP is known to be released from the submandibular gland of the sheep during parasympathetic stimulation (Edwards and Edwards, 2001). However, release of effective amounts of this peptide is demanding of relatively high frequencies of stimulation ( > 2 Hz; Andersson et al., 1982; Lundberg and Ho¨kfelt, 1983; Edwards and Edwards, 2001). Accordingly it seems that, in this gland, secretion of protein in response to chorda tympani stimulation at a frequency below that which releases effective amounts of VIP is more susceptible to inhibition when the blood flow is reduced than the response to stimulation at a high frequency such as would elicit release of VIP. It is concluded that an adequate blood flow is required to maintain salivary secretion in the sheep, as established previously in the cat (Hanna et al., 1999; Rourke and Edwards, 2000). These complementary results in two very different species reinforce the conclusion that the gland is unable to sustain the normal rate of flow of saliva over more than a few minutes in the face of a substantial reduction in blood flow. However, the sheep differs from the cat with respect to the associated changes in the output of submandibular protein. In the cat, reducing the submandibular blood flow with i.c. infusions of ET-1 produced a highly significant fall in the output of salivary protein ( 67F7%; P < 0.01)
during parasympathetic stimulation at 2, 4, 8 and 16 Hz (Rourke and Edwards, 2000). The results of the present study show that precisely the same procedure has no significant effect on submandibular protein output in sheep during parasympathetic stimulation within this range of frequencies, although there was a significant fall during stimulation at a lower frequency (1 Hz; P < 0.01). Thus, whereas the flow of saliva is more sensitive to this constraint in the sheep than it is in the cat, the opposite obtains with regard to protein output. A rate of submandibular blood flow that is adequate for one secretory function may therefore be insufficient for another and the relations vary between species.
Acknowledgements It is a particular pleasure to acknowledge the skilled technical assistance provided by Mr. P.M.M. Bircham.
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