salivary secretion to stimulus frequency was ml min-' g-1 Hz-l. Hong Kong

Transcription

1 Journal of Physiology (199), 431, pp With 7 figures Printed in Great Britain VARIATIONS IN BLOOD FLOW ON MANDIBULAR GLANDULAR SECRETION TO AUTONOMIC NERVOUS STIMULATIONS IN ANAESTHETIZED DOGS BY MARY A. LUNG From the Department of Physiology, Faculty of Medicine, University of Hong Kong, Hong Kong (Received 28 March 199) SUMMARY 1. Continuous stimulation of the preganglionic parasympathetic nerve (the ramus communicans of the mandibular ganglion) for 1-2 min at supramaximal voltage (5 V) and pulse duration (1 ms) increased salivary gland arterial inflow and this was accompanied by copious salivary secretion. The responses were recorded continuously during the period of stimulation. The frequency for initiating the responses was 5 Hz. Maximal responses occurred at 16 Hz. The response coefficient of arterial inflow to stimulus frequency was 417 ml min-1 g-' Hz-1 and that of secretion to stimulus frequency was -16 ml min-' g-1 Hz-'. 2. The secretory response to low and moderate levels of parasympathetic nerve stimulation (below 8 Hz) was not affected by a reduction or cessation in arterial inflow whereas the response to high level parasympathetic nerve stimulation (above 8 Hz) was significantly alleviated if blood flow to the gland was maintained (via controlled vascular perfusion) at a level less than that of the resting arterial inflow. However, when the gland was already secreting near-maximally (stimulated at 8 Hz), sudden cessation of blood flow for a short period of time (-5-2 min) had no effect on the salivary flow. 3. Continuous stimulation of the cervical sympathetic nerve for 1-2 min at supramaximal voltage (2 V) and pulse duration (1 ms) decreased arterial inflow and this was accompanied by scanty salivary secretion. The vascular response persisted during the period of stimulation. The secretory response was 15 s late in onset and might continue for 1 min after stimulation. The frequency for initiating the responses was 1-4 Hz. Maximal responses occurred at Hz. The response coefficient of arterial inflow to stimulus frequency was - -4 ml min-' g-1 Hz-' and that of salivary secretion to stimulus frequency was 1 ml min-' g-1 Hz-l. 4. The secretory response to sympathetic nerve stimulation at different frequencies in glands with blood flow maintained at resting rate (via controlled vascular perfusion) resembled that in glands with spontaneous blood flow. 5. Sympathetic nerve stimulation was found to retard salivary secretion caused by parasympathetic stimulation, irrespective of whether the gland received spontaneous arterial inflow or controlled vascular perfusion at a resting flow rate. 6. The results suggest that the salivary secretion to stimulation of para- MS 834

2 48 M. A. LUNG sympathetic nerve is independent of blood flow over a wide range of stimulus frequencies; however, the response to high frequency stimulation of the parasympathetic nerve may be affected by fluctuations in blood flow. Retardation of parasympathetic-induced salivary flow by superimposed sympathetic nerve stimulation may not be related to blood flow changes. INTRODUCTION In experimental animals, mandibular salivary secretion evoked by supramaximal parasympathetic nerve stimulation is usually diminished by concurrent sympathetic nerve stimulation. This effect has been thought to be the result of a reduced blood flow brought about by sympathetic vasoconstriction (Langley, 1878; Emmelin, 1955a). Salivary secretion induced by pilocarpine is also retarded, as in the case of parasympathetic secretion, by procedures believed to reduce blood flow, such as carotid arterial occlusion, vagal stimulation, sympathetic stimulation or injection of adrenaline (Emmelin, 1955b). In the dog, the salivary response to sympathetic nerve stimulation is usually scanty. However, in the presence of an alpha-adrenoreceptor blockade the salivary response is enhanced. The adrenoreceptor blockade agent is believed to prevent sympathetic vasoconstriction and hence leading to the loss of the indirect inhibitory action on secretion (Emmelin & Gjorstrup, 1976). These studies suggest that salivary secretion is influenced by the volume of blood supply to the gland. Nevertheless, the procedures taken to reduce blood flow to the gland may modify salivation via direct or indirect actions on the secretory apparatus. Sympathetic stimulation or application of adrenoreceptor agonists and antagonists may act directly on the secretory cells, whereas carotid arterial occlusion or vagal stimulation often causes systemic changes which may result in a secondary reflex action on the gland (Hockman, Hagstrom & Hoff, 1965). No systematic study has yet been carried out to show the relationship between fluctuations in blood supply and salivary gland secretion. We have established a method of direct measurement of spontaneous arterial inflow and saliva secretion in the mandibular gland of the dog (Lung & Wang, 199). Recently, we also described a technique for vascular perfusion of the gland (Wang & Lung, 1989). These methods have been used in the present study which examines the relationship between acute changes in arterial inflow and salivary secretion during stimulation of the autonomic nerves. Some preliminary results have already been communicated in a scientific meeting (Wang & Lung, 1989). METHODS Mongrel dogs (body weight kg; n = 24) of either sex were anaesthetized by intravenously administered sodium pentobarbitone (25 mg kg-'); supplementary doses were given when necessary. Body temperature (rectal) was maintained at 37 C by means of an electric heating pad placed beneath the animal. A femoral artery was cannulated for the measurement of systemic arterial pressure. Heparin (1 units h-') was given via a cannulated femoral vein. With the animal lying in the supine position, a longitudinal incision was made along the lingual border of the body of the mandible, extending posteriorly to the angular process. The digastricus and mylohyoideus muscles were separated to expose the facial artery (or external maxillary artery) and its glandular branch which is the major arterial supply to the mandibular gland of the dog

3 BLOOD FLOW AND SALIVARY SECRETION (Miller, Christensen & Evans, 1964). From previous dissection studies on the gland, we found that a minor arterial supply may arise from some small branches of the lingual artery. To ensure that the gland received its blood supply solely from the glandular branch, all small branches supplying the gland were ligated. Care was also taken to exclude the presence of any collateral arterial supply; this included the monitoring of the occluded glandular arterial pressure and haematocrit measurements at rest and during secreting activity (see Discussion). To monitor the occluded arterial pressure, a catheter was inserted retrogradely into the facial artery and its tip advanced to the origin of the glandular artery. When arterial inflow was stopped by a snare placed proximal to the origin of the glandular artery, the pressure monitored by the catheter reflected the occluded glandular arterial pressure. Samples of glandular arterial and venous blood were taken for haematocrit measurements. When determining the arterial inflow, an electromagnetic flow sensor (SL-7515, Statham) of appropriate size was placed around the facial artery just proximal to the origin of the glandular branch. For vascular perfusion of the mandibular gland, the facial artery proximal to the origin of the glandular branch was closed with a snare. The glandular artery was then perfused, via the facial arterial catheter, with blood from a reservoir which was continuously replenished from a cannulated femoral artery. Perfusion was carried out by means of a peristaltic pump (MHRE, MK4, Watson-Marlow). The perfusion pressure was measured from a point between the facial arterial catheter and the pump. Perfusion rate was normally adjusted to give a perfusion pressure approximating the systemic arterial pressure, although its rate could be varied according to the experimental protocol. To expose the mandibular duct, the posterior part of the mylohyoideus muscle was severed latero-medially. The duct was retrogradely cannulated and the catheter connected to a bottle in which the secreted saliva displaced a saline solution. A cannulating-type electromagnetic flow sensor (U-21, Medicon) was inserted along the outlet tube of the bottle to measure the salivary flow. Drops of saline, of -25 ml in volume, displaced from this bottle were measured by means of a drop-counter (92-1-7, E & M Instr.); salivary flow was also calculated from the time interval between falling drops. The two methods were used simultaneously to measure salivary secretion in order to evaluate if there is any discrepancy in the results obtained by electromagnetic flowmetry and drop-recording (see Discussion). All pressure and flow variables were recorded on magnetic tape (Store 14, Racal) and an oscillographic chart-recorder (28S, Gould), and mean values were calculated with the use of Universal amplifiers ( , Gould). Gould P231D transducers were used for arterial pressure measurement. All pressure transducers were zeroed to atmospheric pressure and set at the level of the mid-chest. The U-21 electromagnetic flow sensor was connected to a Statham M41 flowmeter whereas the SL-7515 Statham sensor was connected to a Statham SP-222 flowmeter. Determination of zero baseline and calibration of the sensors were carried out as previously described (Lung & Wang, 1987). In the dog, the preganglionic parasympathetic fibres to the mandibular gland follow the chorda tympani nerve and then the ramus communicans to synapse in the mandibular ganglion located in the hilus of this gland. The preganglionic sympathetic fibres pass to the cervical vagosympathetic trunk to synapse in the superior cervical ganglion (Miller et al. 1964). The ramus communicans to the mandibular ganglion, which courses alongside the mandibular duct, was exposed. The cervical sympathetic trunk just cranial to the caudal cervical ganglion was also exposed as previously described (Lung & Wang, 1986). The tied peripheral ends of both nerves were stimulated separately by bipolar platinum electrodes. Square-wave pulses were provided by a Grass S4 stimulator through an isolation unit (SI U4). Stimulation voltage ranging from 1 to 3 V, frequency from 5 to 64 Hz and pulse duration from 1 to 1-5 ms were tested as described in a previous study (Lung & Wang, 1989). Steady-state vascular and secretory responses were attained within 3 s after stimulation. Maximal vascular and salivary responses to parasympathetic nerve stimulation were obtained with an average voltage of 5 V, frequencies of 8-16 Hz and pulse durations of 8-1O ms; maximal responses to sympathetic nerve stimulation were obtained with an average voltage of 2 V, frequencies of Hz and pulse durations of 8-1 ms. Since the parasympathetic and sympathetic fibres are carried in separate nerves, the strength of stimulation could be altered by varying the frequency of stimulation. Hence, in all subsequent experiments, electrical stimulation was performed with varying frequency at fixed supramaximal voltage (5 V for parasympathetic nerve and 2 V for sympathetic nerve) and pulse duration (1 ms) for a period of 1-2 min PHY 431

4 482 M. A. LUNG The results are displayed as means + S.E. of the means. Student's t test was used to determine the level of significance of differences between the means. RESULTS Under resting conditions, the arterial in-flow was found to be ml min-' g-' (n = 24) and saliva secretion was absent. Table 1 shows the TABLE 1. The occluded mandibular arterial pressure and haematocrit at rest and during parasympathetic nerve stimulations OPma (mmhg) Hctv (%) At Rest Parasympathetic nerve stimulation (1 min) Atl Hz At 2Hz At 4 Hz * At 8Hz * At 16Hz * Values are given as means+ S.E. of the means. Number of animals = 6. Value of arterial haematocrit = % (n = 6). pma, occluded mandibular arterial pressure. Hctv, venous haematocrit. *P < -5, when compared to corresponding value at rest. occluded arterial pressure and venous haematocrit when the gland was at rest or in an active state (due to stimulation of the parasympathetic nerve). The occluded arterial pressure at rest was similar to that seen when the gland was stimulated. There was no difference between the resting arterial and venous haematocrit values. The venous haematocrit reading rose progressively, in parallel with the level of secreting activity induced by increasing parasympathetic nerve stimulation. Effects of parasympathetic nerve stimulation The average frequency for initiating vascular and secretory responses was -5 Hz and that for maximal responses was 16 Hz (Fig. 1). Arterial inflow increased and saliva secretion started immediately, reaching a maximum within 1 s and with a slight decline to a steady level within 3 s (Fig. 2). The magnitude of the steady responses was in direct proportion to the frequency of stimulation; the response coefficient of spontaneous arterial inflow to stimulus frequency was 17 ml min-1 g-1 Hz-' (r = 99, P < 1) and that of salivary secretion to stimulus frequency was -16 ml min-1 Hz-1 (r = -96, P < -1). Figure 3 summarizes the effects of changes in blood flow (via variations in vascular perfusion rate) on the secretory response to different levels of parasympathetic nerve stimulation in a group of six dogs. Changes in perfusion rate were effected 1-2 min prior to nerve stimulation. The duration of nerve stimulation lasted for 1-2 min. For stimulations with frequencies less than 8 Hz, the secretory response obtained under different perfusion conditions, such as normal perfusion rate, double perfusion rate, half perfusion rate or even complete cessation of perfusion, was not significantly

5 BLOOD FLOW AND SALIVARY SECRETION n= 12.3 c E E i- 2 m ?----s~~~~~~~~1. 1 -E A? 3 "I ~~~~~~~~~~~~~~~~~~.1 /~~~~~~~~~~~~~~~~~~~~~~~~~~~~~/ 4 m m a a m ma. I n VIA Stimulus frequency (Hz) Fig. 1. Actions of parasympathetic (ramus communicans to the mandibular ganglion) stimulations (5 V and 1 ms) on the mandibular arterial blood flow (@) and salivary secretion (). Values are means+s.e. of the means. n indicates number of animals. BP 15 (mmhg) 5 Qma 3 (ml min ) ol _o QSS 2[ (ml min1) Dss (4 m r') ll11! 1'---- I_i1 1 Wll1111HII _iii 1 min Stimulations Hz Fig. 2. An experimental record illustrating the effects of varying the frequency (1-8 Hz) of parasympathetic stimulation on blood flow and salivary secretion. Traces from above downwards: BP, systemic arterial pressure. Qm8, mandibular arterial inflow. QS, salivary secretion monitored by the electromagnetic flow sensor. D.., salivary drops monitored by the 'drop-recorder'. Bar, the period of stimulation. 16-2

6 484 M. A. LUNG different from that obtained with spontaneous blood flow. For stimulations with frequencies higher than 8 Hz, the secretory response under the conditions of normal perfusion rate and double perfusion rate was also not significantly different from that obtained with spontaneous blood flow; however, halving the perfusion rate or complete cessation of perfusion significantly decreased the response (P < 5). A.3 n=6 -~ I o.1 1 I I I CB...O2[ l Il l C CE.2. D.1 2IIII n=e ji i Stimulus frequency (Hz) Fig. 3. Response of salivary secretion to varying frequency (O5-32 Hz) of parasympathetic stimulation in glands with spontaneous blood flow (A ), vascular perfusion at normal resting flow rate (B), double resting flow rate (C) and half resting flow rate (D), and cessation of vascular perfusion (E). Values are given as means+ 5.E. of the means. n indicates number of animals. *P < O5, when compared to glands with spontaneous blood flow (A). Figure 4 illustrates the immediate effect of a short period of cessation of arterial inflow (O 5 min) on the secretory response to near-maximal parasympathetic nerve stimulation at 8 Hz. The glandular secretory volume was similar, either in the presence of spontaneous blood flow (A) or controlled vascular perfusion at a resting flow rate (B). Effects of sympathetic nerve stimulation The average frequency for initiating a vascular response to sympathetic nerve stimulation was 1 Hz whereas the frequency for starting the secretory response was 4 Hz. Maximal vascular and secretory responses occurred at about Hz (Fig. 5).

7 BLOOD FLOW AND SALIVARY SECRETION Arterial inflow decreased immediately and reached a steady state within 1 s, but the onset of the secretory response was usually delayed by 15-3 s with the steady state being reached within 3-6 s (Fig. 6A and B). The magnitude of the steady responses was in direct proportion to the frequency of stimulation; the response coefficient of 485 A B BP 15 M W (mmhg) 5 Qma 2 15 (mlmini) (mmhg) I t Oss 2[ 5i (ml min1).5 min - (41) mlm I Fig. 4. Experimental records illustrating the vascular and secretory responses to a short period of sudden cessation of blood flow during parasympathetic stimulation at 8 Hz in glands with spontaneous blood flow (A) and under vascular perfusion at a normal flow rate (B). Plm., mandibular vascular perfusion pressure. Other abbreviations for traces as in Fig. 2. Bar, period of stimulation. spontaneous arterial inflow to stimulus frequency was - 4 ml min-1 g-' Hz-1 (r = --91, P < -5) and that of salivary secretion to stimulus frequency was 1 ml min-' g-1 Hz-' (r = 95, P < 5). If sympathetic stimulation was performed shortly after parasympathetic stimulation, the secretory response was immediate, with the duration and volume of the initial secretion being in direct proportion to the strength of the previous parasympathetic stimulation; however, the secretory response fell gradually and reached a steady state within 3-6 s. The steady-state secretory response to sympathetic nerve stimulation was not significantly different when blood flow to the gland was maintained at resting level via controlled vascular perfusion (Figs 5B and 6B). Interaction between parasympathetic and sympathetic nerve stimulation Figure 7 illustrates the effects of interaction between parasympathetic and sympathetic nerve stimulation on arterial inflow (or perfusion pressure) and salivary secretion. In glands with spontaneous blood flow or controlled vascular perfusion at resting flow rate, steady state vascular and secretory responses were induced with a near maximal level of parasympathetic nerve stimulation (8 Hz). Concurrent maximal sympathetic nerve stimulation (16 Hz) immediately reduced the vascular response; however, the secretory response was initially enhanced before falling to a steady low level within 3-6 s. When sympathetic nerve stimulation was stopped,

8 486 M. A. LUNG both the vascular and secretory values gradually returned to the parasympatheticinduced steady-state levels. DISCUSSION For more than a century, the physiology of the salivary gland has been investigated by monitoring the venous outflow and salivary secretion with drop- ) A I E E 4-3. B CD I EE.5r.41'.31.2 j.1 j 4 r n = 6 n= O.2 E.2 X en co I... C It ) L Stimulus frequency (Hz) Fig. 5. Vascular (@) and secretory () responses to cervical sympathetic stimulations (2 V and 1 ms) in glands with spontaneous blood flow (A) and vascular perfusion at normal resting flow rate (B). Values are given as means + S.E. of the means. n indicates the number of animals. O (U U) X._ itn recorders; the venous blood or salivary secretion had to be connected to an extracorporal circuit and the rate of flow was estimated either from the time interval between falling drops or gravimetrically over a certain period of time (Langley, 1878; Emmelin, 1955a; Bloom & Edwards, 198). The present study describes the first preparation which demonstrates simultaneous measurement of arterial inflow and

9 BLOOD FLOW AND SALIVARY SECRETION secretion of the salivary gland with the use of electromagnetic flow sensors. Droprecording was also used for monitoring the salivary flow in order to evaluate the results obtained by these two methods. We found that the salivary flow rate measured by calculating the time interval between the falling drops was not significantly different from that measured by electromagnetic flowmetry (see Figs 2, 487 BP 15 (mmhg) 5 A Qma (ml min 1) 1 Dssl l l I (4 ml-') BP 15 (mmhg) 51 Ppma 25 B (mmhg) 5 Dss l_l_ l l_l (4 ml-') -5 min Fig. 6. Experimental records illustrating the vascular and secretory responses to cervical sympathetic stimulations in glands with spontaneous blood flow (A) and vascular perfusion at normal resting flow rate (B). Abbreviations for traces as in Figs 2 and 4. Bar, period of stimulation. 4 and 7). However, electromagnetic flow sensor is superior to a drop-recorder in the measurement of fluid flow because it permits instantaneous and direct recording of events. Thus, more accurate analysis of the time-related changes of the variable as well as its relation to other simultaneously measured parameters is possible. Furthermore, there is no need to divert the blood or saliva extracorporally if cufftype electromagnetic flow sensor is used; the flow rate is, therefore, measured in situ. In all previous studies on the vascular control of the salivary secretion, the measurement of vascular and secretory responses to an experimental stimulus has been performed under conditions of spontaneous arterial inflow (Emmelin 1955a,

10 488 M. A. LUNG Emmelin & Gjorstrup, 1976; Ferreira & Smaje, 1976; Andersson, Bloom, Edwards & Jarhult, 1982 a). Hence, the changes in the measured variables reflect not only the primary responses to the stimulus but also the secondary effects caused by systemic changes. This is also the first study to demonstrate vascular perfusion of the salivary BP 15[ 5 (mmnhg) A (m min ).. oss 3[ (ml min~1 l Dss (4 ml') BP 15 (mmhg) 5 Ppma 25 1 (mmhg) 5 Qss 3r (ml min-') Dss (4 ml1) B 1 min Fig. 7. Experimental records illustrating the effects of superimposed maximal cervical sympathetic stimulation (16 Hz, 2 V and 1 ms) on the vascular and secretory responses to near-maximal parasympathetic stimulation (8 Hz, 5 V, 1 ms) in glands with spontaneous blood flow (A) and vascular perfusion at normal flow rate (B). Abbreviations for traces as in Figs 2 and 4. Continuous bar, period of parasympathetic stimulation. Broken bar, period of sympathetic stimulation. gland under a controlled flow rate, enabling only the primary changes to a stimulus to be measured. Also, this vascular perfusion preparation permits actual variations in the arterial supply of the salivary gland and, thus, makes possible an accurate evaluation of the relationship between blood flow and salivary secretion. Since the aim of the study is to evaluate the effects of changes in blood flow on salivary secretion, it is essential that the mandibular gland should receive its blood supply only from the glandular artery. Hence, all small arteries to the gland were ligated. Precautions were also taken to check for the presence of any collateral arterial supply to the gland; firstly, by monitoring the occluded arterial pressure and, secondly, the haematocrit reading. When the arterial supply to a vascular bed is suddenly occluded, the arterial pressure normally falls to the level of the capillary pressure. If collateral arterial channels are present, the occluded arterial pressure will rise gradually; the magnitude of the pressure rise depends on the size of the collateral

11 BLOOD FLOW AND SALIVARY SECRETION blood supply. Collateral arterial flow, if present, becomes very prominent during parasympathetic nerve stimulation when vasodilatation occurs, and this results in an abrupt secondary rise in the occluded arterial pressure. We found, in our preparation, that the occluded glandular arterial pressure dropped to the level of the capillary pressure without any secondary rise in pressure, irrespective of whether the gland was at rest or under the influence of parasympathetic stimulation (Table 1 and Fig. 5). This indicates that collateral blood supply, even if present, has a negligible influence. In some experiments, haematocrit readings of the glandular arterial and venous blood were taken before and immediately after parasympathetic nerve stimulation. Should the collateral arterial supply be substantial, the venous haematocrit value would remain constant despite increasing secretory activity induced by increasing levels of parasympathetic stimulation. This effect is due to dilution by blood flow via the dilated collateral channels. We found that the venous haematocrit value increased gradually with increasing level of parasympathetic stimulation, thus, confirming that the collateral blood supply to the gland was absent or insignificant. We have already shown that the total venous outflow, when monitored from the main glandular vein when all the other minor venous channels are ligated, is always lower than the arterial inflow via the glandular artery (Lung & Wang, 199). Thus, there is reasonable evidence to indicate that, in our preparations, the major or sole blood supply to the mandibular gland is from the glandular artery. Autonomic nerve fibres normally discharge their activity in bursts. Stimulation of autonomic nerves in the form of bursts has been reported to be more effective than a continuous mode of stimulation in eliciting vascular and secretory responses in the salivary gland of animals (Andersson, Bloom & Edwards, 1982b; Andersson et al. 1982a; Bloom, Edwards & Garrett, 1987). In a series of preliminary studies, we studied the effects of continuous and intermittent electrical stimulation of the autonomic nerves on the canine mandibular gland. The pattern of stimulation used by Edwards and his co-workers in cats and lambs (1 s bursts at 1 s intervals) was tested. We found that the mean vascular and secretory responses to intermittent stimulation at 2-16 Hz were even weaker than when the same total number of impulses were delivered in continuous mode (2-16 Hz). Moreover, the measured variables fluctuated greatly during the period of stimulation (Lung, unpublished observations). Hence it is questionable whether or not the tested intermittent pattern of stimulation is physiological. A species difference in the bursts pattern of autonomic nerve stimulation of a particular organ obviously cannot be excluded. Because of the uncertainty regarding the natural or physiological bursts pattern of the autonomic nerves to the canine mandibular gland, we have, therefore, used the conventional continuous mode of stimulation in this study. Effects of parasympathetic nerve stimulation We found that both the vascular and secretory responses to parasympathetic nerve stimulation were frequency dependent with maximal responses occurring at 16 Hz. The maximal secretory response was about -24 ml min-' g-1, and is within the range found by other workers (Terroux, Sekelj & Burgen, 1959; Emmelin & Holmberg, 1967). The maximal vascular response was ten-times the resting blood 489

12 49 M. A. L UNG flow rate, which is also in agreement with the findings of others (Terroux et al. 1959), although a smaller increase in blood flow has been reported (about 4-5-times the resting flow; Ferreira & Smaje, 1976). For decades, it has been believed that the rate of salivary secretion is affected greatly by blood flow (Langley, 1878; Emmelin 1955b; Emmelin & Gjorstrup, 1976). Surprisingly, we found that the secretory response to low and moderate levels of parasympathetic nerve stimulation (less than 8 Hz) was completely independent of blood flow. As long as the blood flow was maintained (via controlled vascular perfusion) at the resting level, secretory responses to maximal parasympathetic nerve stimulation (above 8 Hz) were not significantly affected. These findings suggest that, although parallel changes in blood flow and secretion occur during parasympathetic stimulation, the blood flow response is not essential for the secretory response; this could imply that the vascular and secretory changes may be separate or coincidental phenomena. Studies by Barcroft and his collaborators (Barcroft & Muller, 1912) in cats and by Terroux et al. (1959) in dogs have shown that although the rate of salivary secretion and oxygen consumption are linearly related during activity of the salivary gland, oxygen consumption and blood flow are poorly correlated. Furthermore, there is also a poor correlation between oxygen consumption and blood flow in resting conditions. Hence, it seems that metabolism is not the major controller of blood flow to the salivary gland; any increased metabolic demand caused by increased activity could be satisfied by more oxygen extraction from the blood perfusing the gland. Besides, evidence is available which suggests that the mandibular gland is able to contract an oxygen debt. The period of increased oxygen consumption accompanying secretory activity has been found to outlast the secretory response; the duration of oxygen deficit appears to be in proportion to the period of activity (Barcroft & Piper, 1912; Stromblad, 1959). Hence, when the salivary gland is active, although the blood supply is inadequate, e.g. under conditions of controlled vascular perfusion, normal secretory activity can still be maintained over a short period of time of 1-2 min through increased oxygen extraction from the blood, or by developing an oxygen debt. However, if the increased metabolic demand is in excess of the compensatory mechanisms, the secretory ability will then be impaired. This event may have caused the significant decrease in secretory response to maximal stimulation when blood flow was halved or completely stopped in our experiments. There is no doubt that salivary secretion involves an active electrolyte and water transport mechanism. However, we cannot exclude the fact that the intravascular (or capillary) hydrostatic pressure is the ultimate source of fluid supply to the secreting cells. When arterial supply is occluded (e.g. cessation of vascular perfusion in our experiments), arterial pressure drops to the level of capillary pressure (Table 1), which is sometimes even lower than the osmotic pressure of the plasma proteins. According to the Starling-Landis' concept of capillary fluid dynamics, for a normal capillary under such conditions, fluid absorption instead of filtration occurs (Landis & Pappenheimer, 1963). In this study, we found that the cessation of vascular perfusion did not affect salivary response to low and moderate stimulations. This is only possible if the secreting cells are able to incur a fluid debt, and that fluid replenishment can be made later. The ability of the secreting cells to withstand a

13 BLOOD FLOW AND SALIVARY SECRETION fluid debt of course depends on the size of the debt, which is related to the volume and duration of secretion. This could explain why there is a significant reduction of saliva secretion to maximal stimulation when the vascular perfusion is halved or stopped. Whether or not the secreting cells are indeed capable of building a fluid debt requires further investigation. Effects of sympathetic nerve stimulation Previous studies have shown that the vascular response to sympathetic stimulation is variable, being dependent on the strength and duration of stimulation. Some workers have observed an increased blood flow with brief stimulation of low intensity, and an opposite response to stimulation of high intensity; these findings suggest the presence of both vasodilating and vasoconstricting fibres (Carlson, 197). Others later found a decrease in blood flow after prolonged stimulation with low frequency and an elevated blood flow following maximal stimulation; the inhibitory response was attributed to vasoconstriction whereas the stimulatory response was thought to be due to the presence of vasodilating metabolites (Hilton & Lewis, 1956; Bhoola, Morley, Schachter & Smaje, 1965). In the present study, we found that sympathetic stimulation lowered arterial inflow and increased vascular perfusion pressure; the magnitude of the responses was greater with increasing strength of stimulation, suggesting that the primary vascular response was vasoconstriction. In glands with spontaneous blood flow, arterial inflow immediately after stimulation very often was greater than the pre-stimulation resting level (Fig. 6A). However, in glands with controlled vascular perfusion, perfusion pressure right after stimulation normally returned to pre-stimulation level (Fig. 6B). These results confirm the view that, at least in the dog, sympathetic-induced vasodilatation is probably due to the action of metabolites which accumulate because of low blood flow during stimulation. The secretory response to sympathetic stimulation is species dependent (Langley, 1878). In the dog, secretion is scanty and late in onset, and this response is confirmed by the results of the present study. Some workers believe that such a weak and delayed secretory response is due to dominant vasoconstriction since removal of vasoconstriction by adrenergic blockade results in immediate copious secretion (Emmelin & Gjorstrup, 1976). However, we found that the magnitude of the secretory response to sympathetic stimulation was the same, whether the mandibular gland was receiving spontaneous blood flow or was under controlled vascular perfusion, implying that the scanty secretion is not due to a reduced blood flow following vasoconstriction. A pharmacological antagonist, apart from its action on the blood vessels, may affect directly the secreting cells and, hence, it is difficult to predict whether the change in the secretory response after its administration is due to its effect on the secreting cells or on the blood vessels or both. If the secreting cells are capable of incurring oxygen and fluid debts (as discussed above), a short period of inadequate blood supply caused through sympathetic vasoconstriction is unlikely to have a significant effect on secretion which is so small in volume. We have confirmed the reports of others who have shown that the secretory response to sympathetic stimulation can be increased by a previous parasympathetic stimulation (Bradford, 1888; Langley, 1889). We have also found that the onset of the response is quicker and that the initial response is augmented, but the late steady 491

14 492 M. A. LUNG response is not much different from the one without previous parasympathetic stimulation. The initial augmentation in secretion may be due to contraction of the myoepithelial cells (Emmelin, Garrett & Ohlin, 1969) or heightened excitability of the secretory cells caused by a previous parasympathetic stimulation (Langley, 1889; Babkin, 195). Interaction between sympathetic and parasympathetic nerve stimulation We found that superimposition of strong electrical stimulation of the sympathetic nerve (at 16 Hz) during the course of near-maximal stimulation by the parasympathetic nerve caused the previously good flow of secretion to increase further initially but to decrease significantly later (Fig. 7A). The initial short augmentation in secretion may be related to stimulation of myoepithelial cells or heightened excitability of the secreting cells caused by parasympathetic stimulation (as discussed above). The late decrease in secretion is believed to be due to vasoconstriction which leads to reduced blood flow and diminished secretion (Emmelin, 1955a, b). However, the late retardation in secretion also occurs in the gland with controlled vascular perfusion, suggesting that the cause is not related to a change in blood flow (Fig. 7B). We have already demonstrated that vascular perfusion at normal resting rate is adequate for supporting maximal secretory activity (Fig. 3). Besides, we have shown in Fig. 4 that when a gland is secreting at near-maximal level (with parasympathetic nerve stimulation at 8 Hz), sudden complete cessation of blood flow for a very short period of time does not lead to a significant impairment in secretion; this impairment in secretion is very much smaller than that which is evoked by superimposed sympathetic stimulation of similar duration. Hence, our findings strongly suggest that inhibition of parasympathetic-induced salivary secretion by sympathetic nerve activation is not due to vasoconstriction but to some presently unknown mechanism(s). Further studies are needed. This work has been supported by the University of Hong Kong Research Fund (337/34/11) and Lee Wing Tat Medical Research Fund (377/3/784). The author is grateful to Professor C. W. Ogle and Dr J. C. C. Wang for helpful advice, and to Mr K. K. Tsang and Mr Y. M. Lo for technical assistance. REFERENCES ANDERSSON, P. O., BLOOM, S. R. & EDWARDS, A. V. (1982b). Parotid responses to stimulation of the parasympathetic innervation in weaned lambs. Journal of Physiology 33, ANDERSSON, P. O., BLOOM, S. R., EDWARDS, A. V. & JARHULT, J. (1982a). Effects of stimulation of the chorda tympani in bursts on submaxillary responses in the cat. Journal of Physiology 322, BABKIN, B. P. (195). Secretory Mechanism of the Digestive Glands, 2nd edn. Hoeber, New York. BARCROFT, J. & MULLER, F. (1912). The relation of blood flow to metabolism of salivary gland. Journal of Physiology 44, BARCROFT, J. & PIPER, H. (1912). The gaseous metabolism of the submaxillary gland with reference especially to the effect of adrenaline and the time relation of the stimulus to the oxidation. Journal of Physiology 44, BHOOLA, K. D., MORLEY, J., SCHACHTER, M. & SMAJE, L. H. (1965). Vasodilatation in the submaxillary gland of the cat. Journal of Physiology 179,

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