- Email: [email protected]
Effect of parasympathetic denervation on K+ release by rat parotid slices
Gen. Pharmac. Vol. 18, No. 5, pp. 517-523, 1987
0306-3623/87 $3.00+0.00 Copyright © 1987 Pergamon Journals Ltd
Printed in Great Britain. All rights reserved
E F F E C T OF P A R A S Y M P A T H E T I C D E N E R V A T I O N O N K ÷ R E L E A S E BY R A T P A R O T I D SLICES N. ADHAM and D. TEMPLETON* Department of Physiology and Pharmacology, University of Southampton, Southampton SO9 3TU, England (Received 28 October 1986)
Almract--l. By measuring the release of 86Rb, potassium movement was compared in parasympathectomized (Px) and contralateral control parotid gland slices three weeks following surgical denervation. 2. Carbachol and phenylephrine elicited a biphasic increase in ~Rb release that was dose related and could be blocked by atropine and phentolamine respectively. The transient phase was of 2-4 min duration and Ca independent whereas the sustained phase of ~Rb release was greatly reduced by the omission of the external Ca. 3. Denervation caused a shift to the left of the S6Rb effiux dose-response curve to carbachol and phenylephrine (3.75- and 3.37-fold respectively). Pilocarpine had similar action to earbachol but it behaved as a partial agonist. 4. Parasympathectomy (Px) increased the intrinsic activity of the partial agonist pilocarpine and converted it to a full agonist. Results of the present study indicate the possibility of an altered receptor-signal transduction mechanism between the receptor and phospholipid turnover/Ca mobilization in the denervated rat parotid gland.
INTRODUCTION A survey of the relevant literature indicates that extensive studies on the mechanism of postsynaptic supersensitivity have been carried out in the skeletal-neuromuscular junction, the central nervous system and the peripheral sympathetic nervous system. At all these sites, denervation can be readily carried out by surgical, pharmacological or chemical means. To date, such intensive studies have not been performed in the parasynpathetic division of the autonomic nervous system because the ganglia of the parasympathetic system lie within the peripheral tissue, making denervation difficult and often impossible. Furthermore, chemical denervation is not possible due to the lack of an appropriate chemical. The rat parotid gland is one exception to the general description of the anatomy of the parasympathetic nervous system. Postganglionic parasympathectomy can be achieved successfully in this gland because the ganglion (otic) lies outside the effector tissue and the postganglionic fibre (the auriculo-temporal nerve) can be sectioned. In the rat parotid gland, at least 4 receptor systems have been identified, namely a-adrenergic,/~-adrenergic, muscarinic cholinergic and tachykinin receptors. In the gland the flow of watery saliva, rich in electrolytes is elicited by the stimulation of either muscarinic cholinergic, ~-adrenergic (Emmelin et al. 1965) or tachykinin receptors (Bertaccini and De Caro, 1965), whereas the secretion elicited by /~-adrenoceptor stimulation is scanty, viscous and rich in protein. Two to three weeks following denervation of either branch of the autonomic nerve
supply to the rat parotid gland postsynaptic supersensitivity develops to sialogogue agents in vivo, as judged by a decrease in the threshold dose of drug required to evoke secretion and increase in the amount of saliva secreted in response to supraliminal doses of drugs. Parasympathectomy (Px) of the rat parotid gland by means of avulsion of the auriculotemporal nerve causes marked sensitization mainly towards muscarinic eholinergic, ~t-adrenergic (Aim and Ekstrom, 1976; Ekstrom 1980) and tachykinin receptor agonists (Ekstrom and Wahlestedt, 1982). Most previous work on the supersensitivity resuiting from (Px) has been limited to those changes observed in vivo, in the anaesthetised rat by cannulating the parotid duct and measuring the volume of saliva produced in response to agonist administration. In the present study a method was developed to investigate supersensitivity of rat parotid gland in vitro as an increase in secretagogue-stimulated K ÷ efflux (using 86Rb as a tracer) from parotid gland slices, thus offering a more convenient system with which to analyze the cellular mechanisms of denervation supersensitivity in vitro.
*Present address: D. Templeton, Duphar, C. J. van Houten Laan 36, 1381 CP Weasp, Holland. 517
METHODS AND MATERIALS
Animals
Animals used in the experiments were male Wistar rats, (180-250 g) bred in the animal house of the department and were maintained in a control environment (12 hr day/night cycle, 21°C with free access to food and water). Denervation
The procedure for the avulsion of the postganglionic parasympathetic nerve to the rat parotid gland has been reported elsewhere (Adham and Templeton, 1986). Animals were used 3 weeks following denervation.
518
N. ADHAMand D. TEMPLETON
Release of potassium (S6Rb) from superfused parotid gland slices Rats were killed by cervical dislocation and the parotid glands removed, trimmed of fat and connective tissue and sliced with a razor blade. Slices were incubated in 4 ml of oxygenated Kreb's bicarbonate buffer (composition, mM: NaC1 118, KC1 5, Mg SO4 1, CaCI2 2.5, KH 2 PO 41, NaHCO 3 25, glucose 10) containing 3-5 gCi/ml of 86Rb for 30 rain at 37°C under an atmosphere of 95% 02: 5% CO 2. This procedure allowed the uptake of S6Rb into tissue slices. After this incubation period, the tissue slices were removed and approximately 25 mg portions were loaded onto rubber "O" tings fixed with fine mesh base and were then fitted into each of the eight channels (volume approximately 0.3 ml) of a superfusion apparatus. A second ring was placed above the first and a tight-fitting nylon piston was fitted into each channel. The slices were immediately perfused with Kreb's bicarbonate medium delivered at a rate of 6 ml/min by a Watson-Marlow peristaltic pump. The temperature of perfusate and chamber was maintained at between 36-38°C. The perfusate was collected at I rain intervals (6 ml) and the ~Rb counted by Cerenkov counting. Tissues were exposed to agonists for 5 min. In experiments designed to investigate the effect of antagonists, slices were exposed to the antagonist during both the equilibration period and the stimulation period. In experiments in which the effect of Ca removal was studied, CaC1 was omitted from the Kreb's solution and 20 mM EGTA [ethyleneglycol-bis-(ct-aminoethylether)N,N'-tetra acetic acid] was included throughout the experiment. The following protocol was used for the stimulation of the efflux of 86Rb by the Ca ionophore A-23187: slices were loaded with 86Rb, as mentioned previously. After loading, slices were transferred to the perfusion chamber as before and perfused for 10 min with Kreb's solution containing no added Ca but containing 20 mM EGTA, and 5 #M concentration of A-23187 dissolved in ethanol as solvent (to give a final concentration of ethanol of 0.5%); the control slices were exposed to the same solvent concentration but without the ionophore. After this equilibration period, all slices were exposed to normal Kreb's medium containing CaC1 (2.5 mM) for a further period of 20 min. The rate coefficient of 86Rb efflux was calculated for each collection period thus:
RESULTS
Effect of denervation on functional responsiveness of rat parotid gland basal efflux of S6Rb 86Rb effiux in the absence of secretagogues was not affected by denervation, as shown in Fig. 1. The average rate coefficients o f S6Rb efflux determined for the period immediately prior to drug administration (7, 8, 9 and 10 min) was found to lie between 3 - 5 % per min in both control and denervated parotid gland slices.
Characteristics responses
of
cholinergic
receptor-mediated
Effect of carbachol on StRb e~ux
where Ax is the amount of radioactivity collected in each collection interval, At is the length of the interval, and X~ is the total amount of 86Rb released up until the start of the interval A t . For each experiment results were expressed as follows: the four resting values, taken at times, 7, 8, 9 and 10 min of the experiment, were averaged and assumed as basal effiux. Values obtained at times, 12, 13, 14 and 15 min of the experiment were then expressed as a percentage increase above basal, averaged and subsequently used in log dose-response curves.
The effect of varying concentrations (0.1 # M 0.1 mM) o f carbachol on the fractional effiux of S6Rb from contralateral control and denervated rat parotid gland slices is illustrated in Fig. 1. Addition o f carbachol to the control and denervated parotid gland slice system resulted in a biphasic 86Rb effiux response which had the following pattern: phase 1 was rapid, concentration dependent and transient. M a x i m u m release was achieved within 2 m i n of stimulation with secretagogue. Phase 2 was sustained and fell gradually. Average net release in response to carbachol is depicted in Fig. 2. It must be emphasized that the dose-response curve shown in Fig. 2 is not a cumulative one, but a mean curve obtained from several slice systems exposed to a single given dose of carbachol. U n d e r these conditions, repeated exposure of the same slice system to different doses of secretagogue was therefore not possible. The lowest dose concentration of carbachol which gave a measurable net 86Rb release from both the contralateral control and denervated parotid gland slices was 0.1 # M . M a x i m u m response was achieved at 30 # M. In the denervated parotid gland slices, the dose-response curve of net StRb release to carbachol was shifted to the left. Statistically significant differences between control and denervated values were observed at carbachol concentrations of 0.1, 0.3, 1, 3 and 1 0 g M . There was no increase in the m a x i m u m response after denervation. The half maximal response (ECs0) in control slices occurred at a carbachol concentration o f 3 # M , whereas that in slices o f denervated animals was observed at a dose o f 0 . 8 # M . This implies that denervation caused a 3.75-fold shift to the left in the dose-response curve to carbacbol.
Statistical methods
Effect of pilocarpine on S6Rb eJ~ux
Experimental values are presented as the mean+ standard error of the mean statistical comparison was by Student's t-test for unpaired means. Probability values, P < 0.05 were considered to be significant.
The effect of varying concentrations (1 # M - 1 raM) of pilocarpine on the fractional elflux o f 86Rb from contralateral control and denervated rat parotid gland slices is illustrated in Fig. 3. Similar to the response obtained with carbachol above, addition o f pilocarpine to contralateral control and denervated parotid gland slices, elicited a biphasic response. In the contralateral control parotid gland, pilocarpine stimulation produced a much smaller response compared to stimulation with carbachol and appeared to be acting as a partial agonist in this particular cell system. In view of the shape o f the control d o s e response curve, it was not possible to obtain an
rate coefficient (/min) = Ax/A d X~
Commercial origin of drugs The following compounds were used and the sources are shown in brackets: A-23187 (Calbiochem), atropine sulphate, carbamylcholine chloride, L-phenylephrine hydrochloride (Sigma), phentolamine mesylate (Ciba), pilocarpine hydrochloride (BDH). Solutions of these drugs were normally made up fresh daily, in the appropriate buffer. ~Rb, specific radioactivity, (400-800mCi/mmol) was obtained from Amersham International, England.
Parasympathetic denervation
519
18
Cch OA mM
I
9
o
c~.~/~
o
Kfllb'$
c~_.., ~,,,
000 0
0
I
%
o
o o
o0%0 I 10 18
I 20
I 30
greb's
I0
I
,110
0
°%°o°°°°o Ooo~
~o
I 20
I 20
I 30
I 0
I 10
I 30
I 0
I Cc h'lO M
g: o
q) %0
o
o
~,~o~o0~o ° I
20
3/
Cch 0.1 mM
¢b
o
I
~10
o
o°0o~oo I
I
o%oo,;o° L:~
II 30 0
Time
oo
°o oo %
Ooo
%
%%%°
%oO~0oo0O %O0eo
I
I
10
20
I|
30 0
I
I
I
10
20
30
(min)
Fig. 1. S~Rb induced by carbachol (Cch) from innervated and denervated rat parotid gland slices. Traces show fractional efflux of S6Rb from superfused slices of denervated ( 0 ) and contralateral control (O) rat parotid gland slices under basal (unstimulated) and following stimulation with varying concentrations, (0.1 #M-0.1 m M ) o f carbachol. Each point is the fractional efflux for a one rain collection period plotted as a function of time. Horizontal bars indicate the duration of secretagogue stimulation. Each trace depicts one separate experiment that is representative of at least 3 others. For each experiment, slices from one rat were pre-equilibrated for 30 rain in the presence of ~Rb. Time zero refers to the beginning of washout in non-radioactive Kreb's solution.
accurate estimate of the ECs0. Nevertheless denervation did not appear to alter the ECs0 in the dencrvated parotid gland but to change the interaction of pilocarpine with the receptor so that pilocarpine stimulation exhibited characteristics similar to that of a full agonist, reaching a maximum net n=19
140 -o Control. • Denervated
n = q7 n=13
n =4 T
/ ~ , " ~
StRb release, comparable to that of carbachoi (see Fig. 2).
Characteristics of the g-adrenergic receptor-mediated responses effect of phenylephrine on StRb effiux The effect o f increasing c o n c e n t r a t i o n s (0.1 # M n=5 n=9 nx--x8 xexx
140-oControt
l
• Denervated
[
~
n~9 . / n=4 n~19
D/ n=7 I -7'
n=9
o I -6
I -5
I -4
log [Car tx]chol. ] Dose-response curves of carbachol-mediated StRb efflux from rat parotid gland slices. Graphs show doseresponse curves for net ~Rb cfflux (represented as a percentage increase above basal; protocol for determination of this value is described under "Methods") due to carbachol in dcnervated (O) and contralaterai control (O) parotid gland slices. Each point is the mean of at least 4 separate experiments (one of which is illustrated in Fig. I). Vertical bars indicate one SEM. Statistical significance *P < 0.05, **P < 0.01, between the response to carbachol in control and denervated glandswas calculated by unpaired student t-test, n Indicates number of experiments performed. Fig.
n=4 I
-6
I
I
-5 -4 Log [ Pitocarpine]
[
-3
2.
Fig. 3. Dose-response curves of pilocarpine-mediated 86Rb efflux from rat parotid gland slices. Graphs show dose-response curves from net 86Rb etttux (represented as a percentage increase above basal), induced by pilocarpine in denervated ( 0 ) and contralateral control (O) rat parotid gland slices. Each point is the mean of at least 4 separate experiments. Vertical bars represent one SEM. Statistical significance *P < 0.05, **P < 0.01, ***P < 0.001, between the response to pilocarpine in control and denervated glands was calculated by unpaired student t-test, n Indicates number of experiments performed.
N. ADHAM and D. TEMPLETON
520 n:4 140
o Controt • Oenervoted
n:4
n:5
Maximum response was achieved at 0.3 mM. In the denervated parotid gland slices, the dose-response curve of net S6Rb release to phenylephrine was shifted to the left. Statistically significant differences between control and denervated values were observed at phenylephrine concentrations of 0.1, 1, 3, 10 and 30 #M. The shape of the dose-response curves suggested the possibility of an increase in the maximum response in the denervated state. The half maximal response (ECs0) in control slices occurred at a phenylephrine concentration of 10 #M, whereas, that from slices of denervated gland was observed at a dose of 3 #M. This implies that denervation caused a 33-fold shift to the left in the dose-response curve to phenylephrine.
L • nx8 . . ~
~
!/-
o
.a "o 7 0
//!~5
n!4
n= 5
o
~x~.,~in=Lc~r7 n=5
n-4
0
n:6
;~-L4 n:4 I 7
I 6
I 5
I 4
I 3
Effect of antagonists
Log [Phenytephrine] Fig. 4. Dose-response curve of phenylephrine-mediated 86Rb effiux from rat parotid gland slices. Graphs show dos~response for net S6Rb efflux (represented as a percentage increase above basal), induced by phenylephrine, from denervated (0) and contralateral control (O) rat parotid gland slices. Each point is the mean of at least 4 separate experiments whereas each vertical line represents one SEM. Statistical significance *P <0.05, **P <0.01, between the response to phenylephrine in control and denervated glands was calculated by unpaired student t-test. n Indicates number of experiments performed.
0.3 mM) of phenylephrine (an ~-adrenergic agonist) on the fractional efflux of S6Rb from contralateral control and denervated rat parotid gland slices is illustrated in Fig. 4. Comparable to the cholinergic receptor-mediated responses, addition of phenylephrine to the control and denervated parotid gland slices initiated a biphasic response. The lowest concentration of phenylephrine which gave a measurable net "6Rb release from both contralateral control and denervated parotid gland slices was 0.1 #M. I
120
Figure 5 details the effects of atropine on the carbachol, and phentolamine on the phenylephrineinduced 86Rb etflux respectively, from slices of control and denervated rat parotid glands. In these experiments, the antagonists were added to the incubation medium at time 0-15 min of the experiment. The presence of either antagonists in the incubation medium had little effect on the basal (unstimulated) fractional efflux of 86Rb in both control and denervated parotid gland slices. The fractional efflux seen after the addition of either 10 # M carbachol in the presence of 0.1 mM atropine or 10#M phenylephrine in the presence of 0.1 mM phentolamine was reduced when compared to those obtained in the absence of the antagonist. Figure 5 depicts that in both denervated and contralateral control glands, atropine and phentolamine significantly blocked the net g~Rb release in response to carbachol and phenylephrine respectively. These results indicate that carbachol-evoked g6Rb release occurred specifically via stimulation of muscarinic receptors, whereas the release induced by phenylephrine was mediated by -adrenoceptors.
[ Controt Denervated
--
n=8
n=17
L /A
o
n=17 o o 60
T
I
z
o
~n=6 AtroO.lmM L _ _ J Cch IO/~M
/A /A /A
1.1=8 n=6 ooe Atro O,lmM t _ _ l Cch lOh~ M
1 I
n=7
Phent00.IrnM I I
Phr IO#M
//~
n=9
Phento 0.1raM I
Phr IO#M
Fig. 5. Receptor specificity of ~Rb efllux. Histograms show the effect of antagonism by atropine of carbachol (Cch) and by phentolamine of phenylephrine-induced (Phr) net 8~Rbrelease (represented as a percentage increase above basal) in denervated (hatched bars) and contralateral control (open bars) parotid gland slices. Each histogram represents the mean of at least 4 separate experiments. Vertical lines refer to SEM. Statistical significance,***P < 0.001, between the response to secretagogue in the absence and presence of antagonist was calculated by unpaired student t-test, n Indicates number of experiments performed.
Parasympathetic denervation I
i Controt
140 -17-7"'~ Denervatect n=8
n=17
°
-7-1
o 70 o
,
n=8 / ~ ~ T / //`
/1
•
/1
•
/ /
i
/ I
I
Cch IO#M
n=6 n =6 -r
Phr lOpM
I I A-23187 5pM
Fig. 6. Comparison of Rb efflux from rat parotid gland slices induced by ionophore A-23187 with cholinergic and ~-adrenergic stimulation. Histograms summarize the effect of carbachol (Cch), phenylphrine (Phr) and ionophore A-23187, on net 86Rb efflux, (represented as a percentage increase above basal) in denervated (hatched bars) and contralateral control (open bars) rat parotid gland slices. Vertical lines refer to one SEM. n Indicates number of experiments performed. Statistical significance, *P < 0.05, between the response to secretagogne in denervated and contralateral control parotid gland slices was calculated by unpaired student t-test. Effect o f the divalent cations ionophore, A-23187 The efflux of 86Rb from the denervated control rat parotid gland was assessed in the presence of the divalent cation ionophore, A-23187. S6Rb efflux is best elicited by the addition of Ca to parotid gland slices which have been pre-exposed to ionophore A-23187. The addition of CaC12 to the superfusion medium, to a final concentration of 2.5 mM, stimulated 86Rb effiux transiently with a maximum effect achieved within 2 rain, a time course similar to that of carbachol or phenylephrine. Control and denervated rat parotid gland slices were equally able to release 86Rb in the presence of the ionophore, and there was no significant difference between their net a6Rb effiux response (Fig. 6). The net 86Rb effiux elicited by carbachol and phenylephrine was compared to the response produced by 5 # M A-23187 (Fig. 6). Although there was a significant difference in 86Rb release between control and denervated glands to carbachol and phenylephrine, there was no similar difference in response to the ionophore (Fig. 6).
DISCUSSION
Several mechanisms have been proposed to explain the molecular mechanisms that underlie postjunctional supersensitivity. In skeletal muscle it has been demonstrated that an increase in the number of nicotinic cholinergic receptors following denervation, is the most important mechanism for causing supersensitivity (Fambrough, 1979). Whereas in other tissues such as cardiac and smooth muscle, alterations
521
in the properties of receptors are more involved in supersensitivity. We (Adham and Templeton, 1986) and others (Talamo et al., 1979) have shown that in the rat parotid gland supersensitive secretory responses to cholinergic muscarinic stimulation that follow (Px) are not due to an increase in the number of these receptors, measured by radioligand binding methods. However we have suggested that an increase in the affinity of the low affinity type of muscarinic receptor may be responsible. Although the delineation of the regulation of receptor binding sites using radioligand binding techniques is an important area of study, the significance of this regulation is apparent only by the concurrent study of the function of these receptors. The objective of the present study was to investigate whether (Px) alters the coupling of the receptor to the physiological response, in this case measured as K ÷ effiux from the gland. The results of this report demonstrate that an in vitro system of parotid gland slices can release K (monitored by 86Rb) in response to stimulation with muscarinic cholinergic and ~-adrenergic drugs and furthermore that these responses are dependent on the dose of the secretagogue. In the present study (Px) was found to cause a shift to the left of the 86Rb efflux dose-response curve to carbachol and phenylephrine (3.75- and 3.3-fold respectively). These results agree quite well with those reported by Martinez and Quissel (1977). They found that sympathetic denervation (using reserpine) of rat submaxillary gland (which normally causes a limited non-specific supersensitivity to ~-adrenergic and cholinergic muscarinic receptor stimulation in vivo) led to a shift to the left of K ÷ release dose-response curve to carbachol and norepinephrine (3.47- and 2.57-fold respectively), without a concomitant increase in the maximum response. Similarly, Martinez et al. (1979) demonstrated that sympathetic denervation (using reserpine) of the rat parotid gland caused approximately 3-fold increase in the net K ÷ release in response to a single dose of epinephrine, although it is more accurate to study supersensitivity changes using full dose-response curves. The above examples appear to be the only two that have been reported on the effect of sympathectomy on ion movements in salivary glands and they are used here for the sake of comparison as there is no literature available on the effect of (Px) on in vitro responses of the rat parotid gland. Martinez et al. (1979) and Martinez and Quissel (1977) suggested that in the rat submaxillary gland the supersensitivity of K ÷ release response to ~-adrenergic and cholinergic agonists following sympathectomy is due to an impairment in the handling of Ca by the salivary acinar cells and an alteration in the Na+/K + pump activity. These two possibilities were studied in the present study by investigating the effects of (1) removal of external Ca; (2) addition of ouabain on the secretagogue-stimulated S6Rb efflux in the control and denervated rat parotid gland slices (results now shown). To further investigate the hypothesis whether the mechanism coupling receptor occupation to Ca mobilization (and thus K release) is altered in (Px) parotid glands, the ability of the Ca ionophore
522
N. ADHAMand D. TEMPLETON
A-23187 to induce 86Rb efflux from gland slices was tested. In the present study control and denervated parotid gland slices were equally responsive to release 86Rb in the presence of the ionophore A-23187 at the external Ca concentration of 2.5 mM. Simultaneous comparison of phenylephrine- and carbacholmediated S6Rb efflux by denervated gland slices demonstrated a significant enhancement of 86Rb release compared to the control gland. On the contrary Martinez et al. (1979) found that K ÷ release in response to the ionophore A-23187 was significantly greater in the sympathectomized parotid glands. An interesting finding is that of Ito et al. (1982), who demonstrated that parotid gland acinar cells, prepared from aged rats (12 and 24 months old) showed decreased responsiveness to ~-adrenergic stimulation of K ÷ release. However, when the ~t-adrenoceptors were by-passed by using the Ca ionophore A-23187 to elicit K ÷ release, young and old parotid cells were equally responsive. This is in agreement with the present study since it suggests that alterations in the physiological responsiveness of the parotid gland are not due to changes in the handling of Ca by the acinar cells. The K released through the Ca activated channels into the extracellular space is then taken back up into the cell via a K - N a - C i cotransport system, the activity of which is dependent on the Na gradient and eventually on the N a / K pump activity. Thus in the presence of ouabain, K release is expected to be enhanced (Exeley and Gallacher, 1984). In the present study, when the N a / K pump activity was inhibited in the presence of ouabain (1 mM final concentration) 86Rb efflux elicited by either carbachol or phenylephrine was still greater in the denervated gland. This finding indicates that the supersensitivity was not due to a decrease in the rate of K ÷ uptake. These results disagree with those of Martinez et al. (1979), who have demonstrated that ouabain enhanced K ÷ release in response to epinephrine by 66% in the control, but by only 36% in the sympathectomized parotid glands. Some of the disagreements between the results of the present study and those obtained by Martinez et al. (1979) could be due to the difference in the denervation procedure (these authors have used pharmacological methods, namely, reserpine treatment, whereas surgical procedures were employed in the present report). They have sympathectomized the parotid glands, whereas parasympathectomized parotid glands were used here. Reserpine treatment in addition to causing supersensitivity, may have other toxic effects, therefore it is not clear whether the supersensitivity that is observed following reserpine is due to a non-specific toxic effect of the drug. Surgical methods are by far the best procedures for the study of supersensitivity phenomenon. The findings of the present study of the ability of the ionophore A-23187 to promote K release in denervated parotid gland slices and our previous results (Adham and Templeton, 1986) showing the lack of an increase in the number of muscarinic receptors but an increase in the affinity of agonist carbachol following denervation, strongly suggest that supersensitivity is due to some alteration between receptor occupation and Ca entry. This alter-
ation could reside at the Ca entry mechanism or at a transduction step linking receptor occupation to Ca entry. Several authors have suggested that alterations in stimulated inositol phospholipid metabolism, induced by denervation may be important in supersensitivity. It has been demonstrated that agonist stimulated inositol phosphate metabolism is enhanced following surgical denervation of the rat iris smooth muscle (Abdel-Latif et al., 1979), the rat vas deferens (Takenawa et al., 1983) .and the rat pineal gland (Zatz, 1985). An interesting finding of the present study was that following (Px), pilocarpine which was shown above to be a partial agonist in the control glands, stimulated 86Rb efflux with a similar maximum as that obtained with carbachol or phenylephrine, indicating that Px results in the conversion of pilocarpine form a partial agonist to a full agonist. Similarly Kendall et al. (1985) demonstrated that sympathetic denervation of the rat cerebral cortex by 6-OH dopamine administration caused an increase in the intrinsic activity of phenylephrine. In conclusion the possibility of an altered receptor-signal transduction mechanism between the receptor and phospholipid turnover/Ca mobilization following Px appears to offer the most probable explanation and requires further investigation. REFERENCES
Abdel-Latif A. A., Green K. and Smith J. P. (1979) Sympathetic denervation and the triphosphoinositide effect in the iris smooth muscle: a biochemical method for the determination of ct-adrenergic receptor denervation supersensitivity. J. Neurochem. 32, 225-228. Adham N. and Templeton D. (1986) Parasympathetic denervation of the rat parotid gland. Br. J. Pharmac. Proceedings Supplement, Amsterdam. Alm P. and Ekstrom J. (1976) Cholinergic nerves of unknown origin in the parotid glands of rats. Archs oral Biol. 21, 417~;21. Bertaccini G. and De Caro G. (1965) The effect of physalaemin and related peptides on salivary secretion. J. Physiol. 181, 68-81. Ekstrom J. (1980) Sensitization of the rat parotid gland to secretagogues following either parasympathetic denervation or sympathetic denervation or decentralization. Acta physiol, scand. 108, 223-261. Ekstrom J. and Wahlestedt C. (1982) Supersensitivity to substance P and physalaemin in rat salivary glands after denervation or decentralization. Acta physioL scand. 115, 437-446. Emmelin N., Holmberg J. and Ohlin P. (1965) Receptors for catecholamines in the submaxillary glands of rats. Br. J. Pharmac. 25, 134--138. Exley P. M. and Gallacher D. V. (1984) Chloridedependent, diuretic-sensitive potassium re-uptake in mouse submandibular gland. Evidence for a potassiumsodium-chloride co-transmitter. J. PhysioL 354, 94P. Fambrough D. M. (1979) Control of acetylcholine receptors in skeletal muscle. Physiol. Rev. 59, 165-227. Ito H., Hoopes T., Baum B. J. and Roth G. S. (1982) K release from rat parotid cells: an ~t-adrenergic mediated event. Biochem. Pharmac. 31, 567-573. Kendall D. A., Brown E. and Nahorski S. (1985) ~-Adrenoceptor-mediated inositol phospholipid hydrolysis in rat cerebral cortex: relationship between receptor occupancy and response and effects of denervation. Eur. J. Pharmac. 114, 41-52. Martinez J. R. and Quissel D. O. (1977) Potassium release
Parasympathetic denervation from the rat submaxillary gland /n vitro III. Effect of pretreatment with reserpine. J. Pharmac. exp. Ther. 200, 206-217. Martinez J. R., Braddock M. E., Martinez A. M. and Cooper C. (1979) Effect of chronic reserpine administration on K and amylase release from the rat parotid gland. Pediat. Res. 13, 1024-1029. Takenawa T., Masaki T. and Goto K. (1983) Increase in norepinephrine-induced formation of phosphatidic acid
G.P. 18/~-E
523
in rat vas deferens after denervation. J. Biochem. 83, 303-306. Talamo B. R., Chona Adler S. and Burr D. R. (1979) Parasympathetic denervation decreases musearinic recep' tor binding in rat parotid. Life $ci. 24, 1573-1580. Zatz M. (1985) Denervation supersensitivity of the rat pineal to norepinephrine-stimulated (3H)inositol turnover revealed by lithium and a convenient procedure. J. Neurochem. 45, 95-100.
0306-3623/87 $3.00+0.00 Copyright © 1987 Pergamon Journals Ltd
Printed in Great Britain. All rights reserved
E F F E C T OF P A R A S Y M P A T H E T I C D E N E R V A T I O N O N K ÷ R E L E A S E BY R A T P A R O T I D SLICES N. ADHAM and D. TEMPLETON* Department of Physiology and Pharmacology, University of Southampton, Southampton SO9 3TU, England (Received 28 October 1986)
Almract--l. By measuring the release of 86Rb, potassium movement was compared in parasympathectomized (Px) and contralateral control parotid gland slices three weeks following surgical denervation. 2. Carbachol and phenylephrine elicited a biphasic increase in ~Rb release that was dose related and could be blocked by atropine and phentolamine respectively. The transient phase was of 2-4 min duration and Ca independent whereas the sustained phase of ~Rb release was greatly reduced by the omission of the external Ca. 3. Denervation caused a shift to the left of the S6Rb effiux dose-response curve to carbachol and phenylephrine (3.75- and 3.37-fold respectively). Pilocarpine had similar action to earbachol but it behaved as a partial agonist. 4. Parasympathectomy (Px) increased the intrinsic activity of the partial agonist pilocarpine and converted it to a full agonist. Results of the present study indicate the possibility of an altered receptor-signal transduction mechanism between the receptor and phospholipid turnover/Ca mobilization in the denervated rat parotid gland.
INTRODUCTION A survey of the relevant literature indicates that extensive studies on the mechanism of postsynaptic supersensitivity have been carried out in the skeletal-neuromuscular junction, the central nervous system and the peripheral sympathetic nervous system. At all these sites, denervation can be readily carried out by surgical, pharmacological or chemical means. To date, such intensive studies have not been performed in the parasynpathetic division of the autonomic nervous system because the ganglia of the parasympathetic system lie within the peripheral tissue, making denervation difficult and often impossible. Furthermore, chemical denervation is not possible due to the lack of an appropriate chemical. The rat parotid gland is one exception to the general description of the anatomy of the parasympathetic nervous system. Postganglionic parasympathectomy can be achieved successfully in this gland because the ganglion (otic) lies outside the effector tissue and the postganglionic fibre (the auriculo-temporal nerve) can be sectioned. In the rat parotid gland, at least 4 receptor systems have been identified, namely a-adrenergic,/~-adrenergic, muscarinic cholinergic and tachykinin receptors. In the gland the flow of watery saliva, rich in electrolytes is elicited by the stimulation of either muscarinic cholinergic, ~-adrenergic (Emmelin et al. 1965) or tachykinin receptors (Bertaccini and De Caro, 1965), whereas the secretion elicited by /~-adrenoceptor stimulation is scanty, viscous and rich in protein. Two to three weeks following denervation of either branch of the autonomic nerve
supply to the rat parotid gland postsynaptic supersensitivity develops to sialogogue agents in vivo, as judged by a decrease in the threshold dose of drug required to evoke secretion and increase in the amount of saliva secreted in response to supraliminal doses of drugs. Parasympathectomy (Px) of the rat parotid gland by means of avulsion of the auriculotemporal nerve causes marked sensitization mainly towards muscarinic eholinergic, ~t-adrenergic (Aim and Ekstrom, 1976; Ekstrom 1980) and tachykinin receptor agonists (Ekstrom and Wahlestedt, 1982). Most previous work on the supersensitivity resuiting from (Px) has been limited to those changes observed in vivo, in the anaesthetised rat by cannulating the parotid duct and measuring the volume of saliva produced in response to agonist administration. In the present study a method was developed to investigate supersensitivity of rat parotid gland in vitro as an increase in secretagogue-stimulated K ÷ efflux (using 86Rb as a tracer) from parotid gland slices, thus offering a more convenient system with which to analyze the cellular mechanisms of denervation supersensitivity in vitro.
*Present address: D. Templeton, Duphar, C. J. van Houten Laan 36, 1381 CP Weasp, Holland. 517
METHODS AND MATERIALS
Animals
Animals used in the experiments were male Wistar rats, (180-250 g) bred in the animal house of the department and were maintained in a control environment (12 hr day/night cycle, 21°C with free access to food and water). Denervation
The procedure for the avulsion of the postganglionic parasympathetic nerve to the rat parotid gland has been reported elsewhere (Adham and Templeton, 1986). Animals were used 3 weeks following denervation.
518
N. ADHAMand D. TEMPLETON
Release of potassium (S6Rb) from superfused parotid gland slices Rats were killed by cervical dislocation and the parotid glands removed, trimmed of fat and connective tissue and sliced with a razor blade. Slices were incubated in 4 ml of oxygenated Kreb's bicarbonate buffer (composition, mM: NaC1 118, KC1 5, Mg SO4 1, CaCI2 2.5, KH 2 PO 41, NaHCO 3 25, glucose 10) containing 3-5 gCi/ml of 86Rb for 30 rain at 37°C under an atmosphere of 95% 02: 5% CO 2. This procedure allowed the uptake of S6Rb into tissue slices. After this incubation period, the tissue slices were removed and approximately 25 mg portions were loaded onto rubber "O" tings fixed with fine mesh base and were then fitted into each of the eight channels (volume approximately 0.3 ml) of a superfusion apparatus. A second ring was placed above the first and a tight-fitting nylon piston was fitted into each channel. The slices were immediately perfused with Kreb's bicarbonate medium delivered at a rate of 6 ml/min by a Watson-Marlow peristaltic pump. The temperature of perfusate and chamber was maintained at between 36-38°C. The perfusate was collected at I rain intervals (6 ml) and the ~Rb counted by Cerenkov counting. Tissues were exposed to agonists for 5 min. In experiments designed to investigate the effect of antagonists, slices were exposed to the antagonist during both the equilibration period and the stimulation period. In experiments in which the effect of Ca removal was studied, CaC1 was omitted from the Kreb's solution and 20 mM EGTA [ethyleneglycol-bis-(ct-aminoethylether)N,N'-tetra acetic acid] was included throughout the experiment. The following protocol was used for the stimulation of the efflux of 86Rb by the Ca ionophore A-23187: slices were loaded with 86Rb, as mentioned previously. After loading, slices were transferred to the perfusion chamber as before and perfused for 10 min with Kreb's solution containing no added Ca but containing 20 mM EGTA, and 5 #M concentration of A-23187 dissolved in ethanol as solvent (to give a final concentration of ethanol of 0.5%); the control slices were exposed to the same solvent concentration but without the ionophore. After this equilibration period, all slices were exposed to normal Kreb's medium containing CaC1 (2.5 mM) for a further period of 20 min. The rate coefficient of 86Rb efflux was calculated for each collection period thus:
RESULTS
Effect of denervation on functional responsiveness of rat parotid gland basal efflux of S6Rb 86Rb effiux in the absence of secretagogues was not affected by denervation, as shown in Fig. 1. The average rate coefficients o f S6Rb efflux determined for the period immediately prior to drug administration (7, 8, 9 and 10 min) was found to lie between 3 - 5 % per min in both control and denervated parotid gland slices.
Characteristics responses
of
cholinergic
receptor-mediated
Effect of carbachol on StRb e~ux
where Ax is the amount of radioactivity collected in each collection interval, At is the length of the interval, and X~ is the total amount of 86Rb released up until the start of the interval A t . For each experiment results were expressed as follows: the four resting values, taken at times, 7, 8, 9 and 10 min of the experiment, were averaged and assumed as basal effiux. Values obtained at times, 12, 13, 14 and 15 min of the experiment were then expressed as a percentage increase above basal, averaged and subsequently used in log dose-response curves.
The effect of varying concentrations (0.1 # M 0.1 mM) o f carbachol on the fractional effiux of S6Rb from contralateral control and denervated rat parotid gland slices is illustrated in Fig. 1. Addition o f carbachol to the control and denervated parotid gland slice system resulted in a biphasic 86Rb effiux response which had the following pattern: phase 1 was rapid, concentration dependent and transient. M a x i m u m release was achieved within 2 m i n of stimulation with secretagogue. Phase 2 was sustained and fell gradually. Average net release in response to carbachol is depicted in Fig. 2. It must be emphasized that the dose-response curve shown in Fig. 2 is not a cumulative one, but a mean curve obtained from several slice systems exposed to a single given dose of carbachol. U n d e r these conditions, repeated exposure of the same slice system to different doses of secretagogue was therefore not possible. The lowest dose concentration of carbachol which gave a measurable net 86Rb release from both the contralateral control and denervated parotid gland slices was 0.1 # M . M a x i m u m response was achieved at 30 # M. In the denervated parotid gland slices, the dose-response curve of net StRb release to carbachol was shifted to the left. Statistically significant differences between control and denervated values were observed at carbachol concentrations of 0.1, 0.3, 1, 3 and 1 0 g M . There was no increase in the m a x i m u m response after denervation. The half maximal response (ECs0) in control slices occurred at a carbachol concentration o f 3 # M , whereas that in slices o f denervated animals was observed at a dose o f 0 . 8 # M . This implies that denervation caused a 3.75-fold shift to the left in the dose-response curve to carbacbol.
Statistical methods
Effect of pilocarpine on S6Rb eJ~ux
Experimental values are presented as the mean+ standard error of the mean statistical comparison was by Student's t-test for unpaired means. Probability values, P < 0.05 were considered to be significant.
The effect of varying concentrations (1 # M - 1 raM) of pilocarpine on the fractional elflux o f 86Rb from contralateral control and denervated rat parotid gland slices is illustrated in Fig. 3. Similar to the response obtained with carbachol above, addition o f pilocarpine to contralateral control and denervated parotid gland slices, elicited a biphasic response. In the contralateral control parotid gland, pilocarpine stimulation produced a much smaller response compared to stimulation with carbachol and appeared to be acting as a partial agonist in this particular cell system. In view of the shape o f the control d o s e response curve, it was not possible to obtain an
rate coefficient (/min) = Ax/A d X~
Commercial origin of drugs The following compounds were used and the sources are shown in brackets: A-23187 (Calbiochem), atropine sulphate, carbamylcholine chloride, L-phenylephrine hydrochloride (Sigma), phentolamine mesylate (Ciba), pilocarpine hydrochloride (BDH). Solutions of these drugs were normally made up fresh daily, in the appropriate buffer. ~Rb, specific radioactivity, (400-800mCi/mmol) was obtained from Amersham International, England.
Parasympathetic denervation
519
18
Cch OA mM
I
9
o
c~.~/~
o
Kfllb'$
c~_.., ~,,,
000 0
0
I
%
o
o o
o0%0 I 10 18
I 20
I 30
greb's
I0
I
,110
0
°%°o°°°°o Ooo~
~o
I 20
I 20
I 30
I 0
I 10
I 30
I 0
I Cc h'lO M
g: o
q) %0
o
o
~,~o~o0~o ° I
20
3/
Cch 0.1 mM
¢b
o
I
~10
o
o°0o~oo I
I
o%oo,;o° L:~
II 30 0
Time
oo
°o oo %
Ooo
%
%%%°
%oO~0oo0O %O0eo
I
I
10
20
I|
30 0
I
I
I
10
20
30
(min)
Fig. 1. S~Rb induced by carbachol (Cch) from innervated and denervated rat parotid gland slices. Traces show fractional efflux of S6Rb from superfused slices of denervated ( 0 ) and contralateral control (O) rat parotid gland slices under basal (unstimulated) and following stimulation with varying concentrations, (0.1 #M-0.1 m M ) o f carbachol. Each point is the fractional efflux for a one rain collection period plotted as a function of time. Horizontal bars indicate the duration of secretagogue stimulation. Each trace depicts one separate experiment that is representative of at least 3 others. For each experiment, slices from one rat were pre-equilibrated for 30 rain in the presence of ~Rb. Time zero refers to the beginning of washout in non-radioactive Kreb's solution.
accurate estimate of the ECs0. Nevertheless denervation did not appear to alter the ECs0 in the dencrvated parotid gland but to change the interaction of pilocarpine with the receptor so that pilocarpine stimulation exhibited characteristics similar to that of a full agonist, reaching a maximum net n=19
140 -o Control. • Denervated
n = q7 n=13
n =4 T
/ ~ , " ~
StRb release, comparable to that of carbachoi (see Fig. 2).
Characteristics of the g-adrenergic receptor-mediated responses effect of phenylephrine on StRb effiux The effect o f increasing c o n c e n t r a t i o n s (0.1 # M n=5 n=9 nx--x8 xexx
140-oControt
l
• Denervated
[
~
n~9 . / n=4 n~19
D/ n=7 I -7'
n=9
o I -6
I -5
I -4
log [Car tx]chol. ] Dose-response curves of carbachol-mediated StRb efflux from rat parotid gland slices. Graphs show doseresponse curves for net ~Rb cfflux (represented as a percentage increase above basal; protocol for determination of this value is described under "Methods") due to carbachol in dcnervated (O) and contralaterai control (O) parotid gland slices. Each point is the mean of at least 4 separate experiments (one of which is illustrated in Fig. I). Vertical bars indicate one SEM. Statistical significance *P < 0.05, **P < 0.01, between the response to carbachol in control and denervated glandswas calculated by unpaired student t-test, n Indicates number of experiments performed. Fig.
n=4 I
-6
I
I
-5 -4 Log [ Pitocarpine]
[
-3
2.
Fig. 3. Dose-response curves of pilocarpine-mediated 86Rb efflux from rat parotid gland slices. Graphs show dose-response curves from net 86Rb etttux (represented as a percentage increase above basal), induced by pilocarpine in denervated ( 0 ) and contralateral control (O) rat parotid gland slices. Each point is the mean of at least 4 separate experiments. Vertical bars represent one SEM. Statistical significance *P < 0.05, **P < 0.01, ***P < 0.001, between the response to pilocarpine in control and denervated glands was calculated by unpaired student t-test, n Indicates number of experiments performed.
N. ADHAM and D. TEMPLETON
520 n:4 140
o Controt • Oenervoted
n:4
n:5
Maximum response was achieved at 0.3 mM. In the denervated parotid gland slices, the dose-response curve of net S6Rb release to phenylephrine was shifted to the left. Statistically significant differences between control and denervated values were observed at phenylephrine concentrations of 0.1, 1, 3, 10 and 30 #M. The shape of the dose-response curves suggested the possibility of an increase in the maximum response in the denervated state. The half maximal response (ECs0) in control slices occurred at a phenylephrine concentration of 10 #M, whereas, that from slices of denervated gland was observed at a dose of 3 #M. This implies that denervation caused a 33-fold shift to the left in the dose-response curve to phenylephrine.
L • nx8 . . ~
~
!/-
o
.a "o 7 0
//!~5
n!4
n= 5
o
~x~.,~in=Lc~r7 n=5
n-4
0
n:6
;~-L4 n:4 I 7
I 6
I 5
I 4
I 3
Effect of antagonists
Log [Phenytephrine] Fig. 4. Dose-response curve of phenylephrine-mediated 86Rb effiux from rat parotid gland slices. Graphs show dos~response for net S6Rb efflux (represented as a percentage increase above basal), induced by phenylephrine, from denervated (0) and contralateral control (O) rat parotid gland slices. Each point is the mean of at least 4 separate experiments whereas each vertical line represents one SEM. Statistical significance *P <0.05, **P <0.01, between the response to phenylephrine in control and denervated glands was calculated by unpaired student t-test. n Indicates number of experiments performed.
0.3 mM) of phenylephrine (an ~-adrenergic agonist) on the fractional efflux of S6Rb from contralateral control and denervated rat parotid gland slices is illustrated in Fig. 4. Comparable to the cholinergic receptor-mediated responses, addition of phenylephrine to the control and denervated parotid gland slices initiated a biphasic response. The lowest concentration of phenylephrine which gave a measurable net "6Rb release from both contralateral control and denervated parotid gland slices was 0.1 #M. I
120
Figure 5 details the effects of atropine on the carbachol, and phentolamine on the phenylephrineinduced 86Rb etflux respectively, from slices of control and denervated rat parotid glands. In these experiments, the antagonists were added to the incubation medium at time 0-15 min of the experiment. The presence of either antagonists in the incubation medium had little effect on the basal (unstimulated) fractional efflux of 86Rb in both control and denervated parotid gland slices. The fractional efflux seen after the addition of either 10 # M carbachol in the presence of 0.1 mM atropine or 10#M phenylephrine in the presence of 0.1 mM phentolamine was reduced when compared to those obtained in the absence of the antagonist. Figure 5 depicts that in both denervated and contralateral control glands, atropine and phentolamine significantly blocked the net g~Rb release in response to carbachol and phenylephrine respectively. These results indicate that carbachol-evoked g6Rb release occurred specifically via stimulation of muscarinic receptors, whereas the release induced by phenylephrine was mediated by -adrenoceptors.
[ Controt Denervated
--
n=8
n=17
L /A
o
n=17 o o 60
T
I
z
o
~n=6 AtroO.lmM L _ _ J Cch IO/~M
/A /A /A
1.1=8 n=6 ooe Atro O,lmM t _ _ l Cch lOh~ M
1 I
n=7
Phent00.IrnM I I
Phr IO#M
//~
n=9
Phento 0.1raM I
Phr IO#M
Fig. 5. Receptor specificity of ~Rb efllux. Histograms show the effect of antagonism by atropine of carbachol (Cch) and by phentolamine of phenylephrine-induced (Phr) net 8~Rbrelease (represented as a percentage increase above basal) in denervated (hatched bars) and contralateral control (open bars) parotid gland slices. Each histogram represents the mean of at least 4 separate experiments. Vertical lines refer to SEM. Statistical significance,***P < 0.001, between the response to secretagogue in the absence and presence of antagonist was calculated by unpaired student t-test, n Indicates number of experiments performed.
Parasympathetic denervation I
i Controt
140 -17-7"'~ Denervatect n=8
n=17
°
-7-1
o 70 o
,
n=8 / ~ ~ T / //`
/1
•
/1
•
/ /
i
/ I
I
Cch IO#M
n=6 n =6 -r
Phr lOpM
I I A-23187 5pM
Fig. 6. Comparison of Rb efflux from rat parotid gland slices induced by ionophore A-23187 with cholinergic and ~-adrenergic stimulation. Histograms summarize the effect of carbachol (Cch), phenylphrine (Phr) and ionophore A-23187, on net 86Rb efflux, (represented as a percentage increase above basal) in denervated (hatched bars) and contralateral control (open bars) rat parotid gland slices. Vertical lines refer to one SEM. n Indicates number of experiments performed. Statistical significance, *P < 0.05, between the response to secretagogne in denervated and contralateral control parotid gland slices was calculated by unpaired student t-test. Effect o f the divalent cations ionophore, A-23187 The efflux of 86Rb from the denervated control rat parotid gland was assessed in the presence of the divalent cation ionophore, A-23187. S6Rb efflux is best elicited by the addition of Ca to parotid gland slices which have been pre-exposed to ionophore A-23187. The addition of CaC12 to the superfusion medium, to a final concentration of 2.5 mM, stimulated 86Rb effiux transiently with a maximum effect achieved within 2 rain, a time course similar to that of carbachol or phenylephrine. Control and denervated rat parotid gland slices were equally able to release 86Rb in the presence of the ionophore, and there was no significant difference between their net a6Rb effiux response (Fig. 6). The net 86Rb effiux elicited by carbachol and phenylephrine was compared to the response produced by 5 # M A-23187 (Fig. 6). Although there was a significant difference in 86Rb release between control and denervated glands to carbachol and phenylephrine, there was no similar difference in response to the ionophore (Fig. 6).
DISCUSSION
Several mechanisms have been proposed to explain the molecular mechanisms that underlie postjunctional supersensitivity. In skeletal muscle it has been demonstrated that an increase in the number of nicotinic cholinergic receptors following denervation, is the most important mechanism for causing supersensitivity (Fambrough, 1979). Whereas in other tissues such as cardiac and smooth muscle, alterations
521
in the properties of receptors are more involved in supersensitivity. We (Adham and Templeton, 1986) and others (Talamo et al., 1979) have shown that in the rat parotid gland supersensitive secretory responses to cholinergic muscarinic stimulation that follow (Px) are not due to an increase in the number of these receptors, measured by radioligand binding methods. However we have suggested that an increase in the affinity of the low affinity type of muscarinic receptor may be responsible. Although the delineation of the regulation of receptor binding sites using radioligand binding techniques is an important area of study, the significance of this regulation is apparent only by the concurrent study of the function of these receptors. The objective of the present study was to investigate whether (Px) alters the coupling of the receptor to the physiological response, in this case measured as K ÷ effiux from the gland. The results of this report demonstrate that an in vitro system of parotid gland slices can release K (monitored by 86Rb) in response to stimulation with muscarinic cholinergic and ~-adrenergic drugs and furthermore that these responses are dependent on the dose of the secretagogue. In the present study (Px) was found to cause a shift to the left of the 86Rb efflux dose-response curve to carbachol and phenylephrine (3.75- and 3.3-fold respectively). These results agree quite well with those reported by Martinez and Quissel (1977). They found that sympathetic denervation (using reserpine) of rat submaxillary gland (which normally causes a limited non-specific supersensitivity to ~-adrenergic and cholinergic muscarinic receptor stimulation in vivo) led to a shift to the left of K ÷ release dose-response curve to carbachol and norepinephrine (3.47- and 2.57-fold respectively), without a concomitant increase in the maximum response. Similarly, Martinez et al. (1979) demonstrated that sympathetic denervation (using reserpine) of the rat parotid gland caused approximately 3-fold increase in the net K ÷ release in response to a single dose of epinephrine, although it is more accurate to study supersensitivity changes using full dose-response curves. The above examples appear to be the only two that have been reported on the effect of sympathectomy on ion movements in salivary glands and they are used here for the sake of comparison as there is no literature available on the effect of (Px) on in vitro responses of the rat parotid gland. Martinez et al. (1979) and Martinez and Quissel (1977) suggested that in the rat submaxillary gland the supersensitivity of K ÷ release response to ~-adrenergic and cholinergic agonists following sympathectomy is due to an impairment in the handling of Ca by the salivary acinar cells and an alteration in the Na+/K + pump activity. These two possibilities were studied in the present study by investigating the effects of (1) removal of external Ca; (2) addition of ouabain on the secretagogue-stimulated S6Rb efflux in the control and denervated rat parotid gland slices (results now shown). To further investigate the hypothesis whether the mechanism coupling receptor occupation to Ca mobilization (and thus K release) is altered in (Px) parotid glands, the ability of the Ca ionophore
522
N. ADHAMand D. TEMPLETON
A-23187 to induce 86Rb efflux from gland slices was tested. In the present study control and denervated parotid gland slices were equally responsive to release 86Rb in the presence of the ionophore A-23187 at the external Ca concentration of 2.5 mM. Simultaneous comparison of phenylephrine- and carbacholmediated S6Rb efflux by denervated gland slices demonstrated a significant enhancement of 86Rb release compared to the control gland. On the contrary Martinez et al. (1979) found that K ÷ release in response to the ionophore A-23187 was significantly greater in the sympathectomized parotid glands. An interesting finding is that of Ito et al. (1982), who demonstrated that parotid gland acinar cells, prepared from aged rats (12 and 24 months old) showed decreased responsiveness to ~-adrenergic stimulation of K ÷ release. However, when the ~t-adrenoceptors were by-passed by using the Ca ionophore A-23187 to elicit K ÷ release, young and old parotid cells were equally responsive. This is in agreement with the present study since it suggests that alterations in the physiological responsiveness of the parotid gland are not due to changes in the handling of Ca by the acinar cells. The K released through the Ca activated channels into the extracellular space is then taken back up into the cell via a K - N a - C i cotransport system, the activity of which is dependent on the Na gradient and eventually on the N a / K pump activity. Thus in the presence of ouabain, K release is expected to be enhanced (Exeley and Gallacher, 1984). In the present study, when the N a / K pump activity was inhibited in the presence of ouabain (1 mM final concentration) 86Rb efflux elicited by either carbachol or phenylephrine was still greater in the denervated gland. This finding indicates that the supersensitivity was not due to a decrease in the rate of K ÷ uptake. These results disagree with those of Martinez et al. (1979), who have demonstrated that ouabain enhanced K ÷ release in response to epinephrine by 66% in the control, but by only 36% in the sympathectomized parotid glands. Some of the disagreements between the results of the present study and those obtained by Martinez et al. (1979) could be due to the difference in the denervation procedure (these authors have used pharmacological methods, namely, reserpine treatment, whereas surgical procedures were employed in the present report). They have sympathectomized the parotid glands, whereas parasympathectomized parotid glands were used here. Reserpine treatment in addition to causing supersensitivity, may have other toxic effects, therefore it is not clear whether the supersensitivity that is observed following reserpine is due to a non-specific toxic effect of the drug. Surgical methods are by far the best procedures for the study of supersensitivity phenomenon. The findings of the present study of the ability of the ionophore A-23187 to promote K release in denervated parotid gland slices and our previous results (Adham and Templeton, 1986) showing the lack of an increase in the number of muscarinic receptors but an increase in the affinity of agonist carbachol following denervation, strongly suggest that supersensitivity is due to some alteration between receptor occupation and Ca entry. This alter-
ation could reside at the Ca entry mechanism or at a transduction step linking receptor occupation to Ca entry. Several authors have suggested that alterations in stimulated inositol phospholipid metabolism, induced by denervation may be important in supersensitivity. It has been demonstrated that agonist stimulated inositol phosphate metabolism is enhanced following surgical denervation of the rat iris smooth muscle (Abdel-Latif et al., 1979), the rat vas deferens (Takenawa et al., 1983) .and the rat pineal gland (Zatz, 1985). An interesting finding of the present study was that following (Px), pilocarpine which was shown above to be a partial agonist in the control glands, stimulated 86Rb efflux with a similar maximum as that obtained with carbachol or phenylephrine, indicating that Px results in the conversion of pilocarpine form a partial agonist to a full agonist. Similarly Kendall et al. (1985) demonstrated that sympathetic denervation of the rat cerebral cortex by 6-OH dopamine administration caused an increase in the intrinsic activity of phenylephrine. In conclusion the possibility of an altered receptor-signal transduction mechanism between the receptor and phospholipid turnover/Ca mobilization following Px appears to offer the most probable explanation and requires further investigation. REFERENCES
Abdel-Latif A. A., Green K. and Smith J. P. (1979) Sympathetic denervation and the triphosphoinositide effect in the iris smooth muscle: a biochemical method for the determination of ct-adrenergic receptor denervation supersensitivity. J. Neurochem. 32, 225-228. Adham N. and Templeton D. (1986) Parasympathetic denervation of the rat parotid gland. Br. J. Pharmac. Proceedings Supplement, Amsterdam. Alm P. and Ekstrom J. (1976) Cholinergic nerves of unknown origin in the parotid glands of rats. Archs oral Biol. 21, 417~;21. Bertaccini G. and De Caro G. (1965) The effect of physalaemin and related peptides on salivary secretion. J. Physiol. 181, 68-81. Ekstrom J. (1980) Sensitization of the rat parotid gland to secretagogues following either parasympathetic denervation or sympathetic denervation or decentralization. Acta physiol, scand. 108, 223-261. Ekstrom J. and Wahlestedt C. (1982) Supersensitivity to substance P and physalaemin in rat salivary glands after denervation or decentralization. Acta physioL scand. 115, 437-446. Emmelin N., Holmberg J. and Ohlin P. (1965) Receptors for catecholamines in the submaxillary glands of rats. Br. J. Pharmac. 25, 134--138. Exley P. M. and Gallacher D. V. (1984) Chloridedependent, diuretic-sensitive potassium re-uptake in mouse submandibular gland. Evidence for a potassiumsodium-chloride co-transmitter. J. PhysioL 354, 94P. Fambrough D. M. (1979) Control of acetylcholine receptors in skeletal muscle. Physiol. Rev. 59, 165-227. Ito H., Hoopes T., Baum B. J. and Roth G. S. (1982) K release from rat parotid cells: an ~t-adrenergic mediated event. Biochem. Pharmac. 31, 567-573. Kendall D. A., Brown E. and Nahorski S. (1985) ~-Adrenoceptor-mediated inositol phospholipid hydrolysis in rat cerebral cortex: relationship between receptor occupancy and response and effects of denervation. Eur. J. Pharmac. 114, 41-52. Martinez J. R. and Quissel D. O. (1977) Potassium release
Parasympathetic denervation from the rat submaxillary gland /n vitro III. Effect of pretreatment with reserpine. J. Pharmac. exp. Ther. 200, 206-217. Martinez J. R., Braddock M. E., Martinez A. M. and Cooper C. (1979) Effect of chronic reserpine administration on K and amylase release from the rat parotid gland. Pediat. Res. 13, 1024-1029. Takenawa T., Masaki T. and Goto K. (1983) Increase in norepinephrine-induced formation of phosphatidic acid
G.P. 18/~-E
523
in rat vas deferens after denervation. J. Biochem. 83, 303-306. Talamo B. R., Chona Adler S. and Burr D. R. (1979) Parasympathetic denervation decreases musearinic recep' tor binding in rat parotid. Life $ci. 24, 1573-1580. Zatz M. (1985) Denervation supersensitivity of the rat pineal to norepinephrine-stimulated (3H)inositol turnover revealed by lithium and a convenient procedure. J. Neurochem. 45, 95-100.