Arch oralBid.Vol. 35.No. 6,pp.435441. 1990 Printed in Great Britain. A.8rights reserved
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THE EFFECT OF CHRONIC PROPRANOLOL TREATMENT ON SALIVARY COMPOSITION AND CARIES IN THE RAT G. E. WATSON, S. K. PEARSON,J. L. FALANY, L. A. TABAK and W. H. BOWEN* Department
of Dental Research, University of Rochester, 601 Elmwood Ave, Rochester, NY 14642, U.S.A. (Accepted I8 December 1989)
Summary-Many drugs are known to affect salivary secretion. The purpose of this study was to explore the chronic effects of a commonly used fl-adrenergic blocker, propranolol. Adult rats were desalivated or treated for 28days with propranolol HCI (IO or 20mg/kg, daily) or sterile buffer (sham-operated control) using osmotic pumps for delivery. The parotid and submandibular glands of each rat were cannulated and secretion elicited by pilocarpine (lOmg/kg, intravenous). There were no statistical differences in zsalivary protein content (Lowry) or output among the groups (ANOVA, p > 0.05). Analysis of salivary proteins by SDS-PAGE revealed a constant profile for submandibular secretions, but peak A and SP-3 proline-rich proteins were not detectable in parotid saliva of animals treated with propranolol for the entire experiment. Significantly increased smooth-surface (p = 0.0003) and sulcal (p = 0.001 I) caries scores were found within these propranolol groups (ANOVA). The findings provide further evidence that chronic administration of propranolol alters salivary composition by decreasing proline-rich proteins and concurrently enhances susceptibility to caries. Key words: hyposalivation, saliva, caries, propranolol.
IVTRODUCTION
environment of the body is maintained by autonomic nervous control of visceral muscles and glands of veg,etative organs (Schottelius and Schottehus, 1973). Disease can require treatment with medications that act on target tissues to correct imbalances, but this can also produce side-effects of varying consequences in other non-target or ‘innocent’ tissues. One of the more common side-effects of prescription and non-prescription drugs is xerostomia (Navazesh and Ship, 1983; Glass et al., 1984), a symptom especially prevalent in the elderly because of their increased use of medications (Handelman et nl., 1986). There are many drugs with hyposalivary effects (Sreebny and Schwartz, 1986), most of which interfere with the autonomic control of secretion. For example, the commonly prescribed anti-arrhythmic, propranolol, blocks p-adrenergic receptors in cardiac tissue and also in all areas of the body where the drug can gain access, including the salivary glands (Winer, 1985). Stimulation of the sympathetic nervous system in humans results in a proteinaceous secretion from salivary glands, while increased volume of secretion results from parasympathetic stimulation (Baum, 1987). A similar innervation is presented in the rat parotid (Spiers and Hodgson, 1976) and subThe internal
*Address correspond’ence to: Dr W. H. Bowen, Department of Dental Research. Universitv of Rochester. 601 Elmwood Ave, Rochester, NY 14642, U.S.A. Abbreuiarions: ANOVA, analysis of variance; SDSPAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis.
mandibular glands (Abe, 1987). Chronic administration of propranolol via an osmotic pump is known to alter rat parotid saliva by decreasing the expression of proline-rich proteins (Johnson and Cortez, 1988). However, when propranolol was administered chronically by intraperitoneal injection, neither the incidence of dental caries nor the composition of whole saliva was affected (Johansson and Ericson, 1987). Our purpose now was to examine the effect of propranolol on caries development, when administered to rats via osmotic pumps for 4 weeks. The effect of the drug on salivary output and protein composition was also determined, using pilocarpine as a secretagogue. MATERIALS
AND METHODS
Forty-one female Sprague-Dawley rats and their dams were acquired from the Charles River Kingston facility. They were screened for the presence of mutans streptococci using methods described by Bowen, Pearson and Young (1988). At the age of 20 days the animals were weaned from their dams and maintained on unmodified Diet 2000 (56% sucrose; Keyes, 1959) (Ziegler Brothers, Gardners, PA, U.S.A.) ad I&turn and water sweetened with sucrose (10% w/v). Streptococcus sobrinus 6715, grown in broth culture to exponential phase, was swabbed into the rats’ mouths with a cotton-tipped applicator (Bowen, Madison and Pearson, 1988). Infection with Strep. sobrinus was confirmed 3 days later by streaking swabs from rats’ mouths onto mitis salivarius agar + streptomycin. None of the animals required reinoculation. 435
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At age 37 days the animals were divided into 5 groups as follows: LOM. propranolol group-6 animals had osmotic pumps (Alza Corp., Palo Alto, CA, U.S.A.) delivering 10 mg/kg propranolol HCI daily implanted into their backs. High propranolol group-12 animals had osmotic pumps delivering 20 mg/kg propranolol HCI daily implanted into their backs. Sham-operated group-l 1 animals had osmotic pumps delivering sterile 0.1 M potassium phosphate solution, pH 6.0, implanted into their backs. Desalirated grozcp-6 animals had both parotid ducts ligated and both submandibular and sublingual glands removed (Bowen et al., 1985). These animals served as positive controls. Unoperated group-6 animals were not subjected to surgery. Rats undergoing surgical procedures were first anaesthetized with chloral hydrate (400 mg/kg intraperitoneally). The surgical site was shaved and then scrubbed with hydrogen peroxide and betadine. A small incision was made in the animal’s back, the fascia beneath the skin was separated, the pump inserted and the wound closed with surgical clips (Clay Adams, Parsippany, NJ, U.S.A.). The osmotic pumps were designated to deliver 2.69 + 0.17 ~1 of solution/h over the 28 day experimental period. Propranolol solution was made by dissolving propranolol HCI (Sigma Corp., St Louis, MO, U.S.A.), 40 mg/ml, in 0.1 M citric acid buffer, pH 3.0. On average, low propranolol rats received 0.041 mg propranolol/h and high propranolol rats received 0.077 mg/h. The unused propranolol stock solution was stored at 37-C for the length of the experiment to expose it to thermal conditions similar to those experienced by solutions in implanted pumps. The stability of the stock solution was examined by removing a sample weekly and storing it frozen at -20-C for later ana!ysis. After recovery from surgery, animals were placed singly in suspended cages and fed Diet 2000 ad libitum and drinking water sweetened with sucrose (10% w/v). They were observed daily for untoward effects, and weighed weekly. Surgical clips were removed after 7 days. Blood was collected from the tail veins (tail bleeds) of 2 rats in each group weekly; the samples were taken from different rats within the group each week. The blood was centrifuged and the plasma stored at -20 C. The experiment was ended 24 days after surgery. Saliva was successfully collected from 19 animals by ductal cannulation as described by Robinovitch and Sreebny (1969). An average of 7 rats per day, picked at random, were cannulated until all had been sam-
Plate
pled. They were first anaesthetized as described above and 1 ml of blood was obtained from the tail vein (final tail bleed). The parotid, submandibular and sublingual ducts were cannulated extraorally and salivary flow stimulated by 10 mg/kg pilocarpine (Sigma Corp.) delivered into a femoral vein. Secretion was maintained by administering another single dose 15 min after initial secretion had been elicited. Saliva was collected for a total of 30 min. Samples were collected over ice into tared Eppendorf tubes containing 10 ~1 of protease inhibitor cocktail [O.l M tris-HCl, pH 7.4, 10 mM EDTA. 5 mM benzamidine HCI, 1 mM soybean trypsin inhibitor, 5 mM pepstatin with 1 mM phenylmethylsulphonyl fluoride (PMSF)]. After collection the tubes were capped, weighed and frozen at -2O’C. The volume of saliva was determined (1 mg equalled I ~1; Suddick and Shannon. 1970) and divided by 30 min to estimate salivary output. While still anaesthetized, the animals were killed by cardiac puncture: this blood was then centrifuged and the resultant plasma stored at -20°C. The rats were then decapitated and the heads defleshed. To measure the amount of propranolol in the plasma samples and in our stock solution we performed competition assays, as described by Bilezikian et al. (1979), using the /I-adrenergic antagonist [‘2’1]iodohydroxybenzylpindolol, and /I-adrenergic receptors from a membrane suspension of turkey erythrocytes. Levels of radioactivity in each sample were measured in a Gamma 4000 gamma counter (Beckman Instruments, Inc., Fullerton, CA, U.S.A.). Specific binding was determined by subtracting nonspecific binding from total binding. Protein determination was performed on saliva samples by the method of Lowry ef al. (1951) with bovine serum albumin as standard. Salivary proteins were resolved by SDS-PAGE [parotid (12% polyacrylamide); submandibular (10%); and sublingual (SO/,)] (Laemmli, 1970) and visualized with Coomassie brilliant blue R (Weber and Osborn, 1975) and periodic acid-Schiff (Fairbanks, Steck and Wallach, 1971) stains. The major components of the parotid proteins were identified by their electrophoretic mobility on SDS-PAGE gels as determined from previous studies (Keller et al., 1975; Johnson, 1984). A colour positive transparency of the destained gel of parotid saliva samples was scanned at 550 nm in a DU-8B Spectrophotometer equipped with a scanning system (Beckman Instruments, Irvine. CA, U.S.A.). Each lane was examined individually and the percentage of the total area attributable to each protein component was calculated. Coronal caries was scored with the method described by Keyes (1958), as modified by Larson (1981).
I
Fig. 1. SDS-PAGE of rat parotid saliva from sham-operated, low propranolol (10 mg/kg, daily), and high propranolol (20 mg/kg, daily) groups. Fifty microgrammes of salivay protein (Lowry) were loaded per lane, electrophoresed, stained with Coomassie blue R, and destained. Proteins were identified from previous work by Keller er al. (1975) and Johnson (1984): (1) peak A, a basic proline-rich protein; (2) amylase; (3) SP-I, a basic proline-rich protein; (4) fraction IV, an acidic proline-rich protein; (5) deoxyribonuclease: (6) fraction V; (7) SP-3, a basic proline-rich protein; (8) fraction I; (9) minor band A; and (IO) minor band B.
Propranolol influences saliva and caries
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ZOOK92.5K66_2K-
2
4,5K\ /
7
31K-
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9
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LOW 1 HIGH PROPRANOLOL Plate 1
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WAT~N
Statistical evaluations used the ANOVA and linear regression programs of the STATVIEW II statistical software package (Abacus Concepts, Berkeley, CA, U.S.A.). A p-value of less than 0.05 was regarded as statistically significant. All mean values in text are +SEM. When samples were obtained from both glands of the same type in one animal, the average value was used and counted as one observation.
RESULTS
All animals in the control and low propranolol groups survived; one animal in the high propranolol group died before the end of the experiment. Shamoperated rats had mean weight gains of 89.2 f 3.0 g during the experiment; low propranolol and’ high propranolol rats gained 96.2 + 7.2 and 87.3 f 4.2 g, respectively-these weight gains were not significantly different (ANOVA, p = 0.42): The desalivated group gained the least weight over the course of the experiment (60.8 f 6.2 g), significantly less than all three other groups (ANOVA, p = 0.004; significantly different at the 95% level by Fisher PSLD and Sheffe F-tests). Mean sucrose-water consumption of shamoperated, low propranolol, high propranolol, and desalivated animals for the 4 days preceding death was 327.9 k28.2, 347.6 k42.7, 341.3 + 36.3 and 38 1.1 f 26.5 ml, respectively, and did not differ significantly (p > 0.05, ANOVA). Propranolol stock solution (40 mg/ml) was stable in buffer for at least 28 days at 37°C (data not shown). Plasma levels of propranolol during the first week averaged 111.3 and 233.5 ng/ml for low and high groups, respectively; these levels had decreased to 30.9 + 14.0 and 77.9 + 13.4 ng/ml by the end of the experiment. A slowly decreasing plasma propranolol concentration with time had been expected as pump output was constant to animals that were continually gaining weight. In total, 56 salivary ducts from 19 animals were successfully cannulated and produced measurable saliva. There were too few sublingual samples, however, to allow accurate comparisons to be made, and these samples were not included in the results. The effect of chronic administration of propranolol on output from pilocarpine-stimulated parotid and submandibular salivary glands is shown in Table 1; there were no statistical differences among the groups (ANOVA; parotid, p = 0.53; submandibular, p = 0.85). Protein determinations were performed on all salivary samples (Table 1). The results suggested a trend of decreasing protein concentration as the level of propranolol increased, but there were no statistical differences among the groups (ANOVA; parotid, p = 0.13; submandibular, p = 0.24). Qualitative differences in the pattern of parotid proteins were observed in propranolol-treated animals, when compared to controls (Plate Fig. 1). The protein bands equivalent to those missing in the parotid saliva from propranolol-treated animals displayed metachromasia in the control lanes suggestive of proline-rich proteins (Humphreys-Beher and Wells, 1984) and corresponded to the peak A and SP-3 bands de-
et al.
Table 1. Mean output and protein concentration of rat saliva Gland experimental group Parotid Sham (n = 5) Low propranolol (n = 4) High propranolol (n = 7) Submandibular Sham (n = 5) Low propranolol (n = 5) High propranolol (n = 4)
Output (P Umin)
Lowry protein (mgiml)
2.085 & 0.289 2.373 k 0.380
13.911 & 1.534 1I.308 + 1.703
I.757 + 0.402
9.059 ) 1.705
2.200 k 0.625 2.565 + 0.434
2.108 + 0.51 I I.883 + 0.543
2.183 k 0.662
0.833 + 0.254
There were no statistical differences among the groups (ANOVA). Numbers of animals contributing to the mean value indicated by n. Values are *SEM.
by Keller et al. (1975) and Johnson (1984). Protein bands SP-1, acidic proline-rich proteins, deoxyribonuclease, and fraction V were grouped together as a single area because resolution of each individual band was not precise in all samples. A significant, dose-related decrease in the percentage area attributable to these grouped proteins was seen (Table 2); there was a concomitant increase in the total area attributable to fraction I and minor B bands. The gels of submandibular salivas were remarkably constant in that there were no perceived differences among the different groups (data not shown). The influence of chronic administration of propranolo1 on caries is shown in Text Fig. 2. Regression tests indicated a significant positive trend in smoothsurface caries scores from sham to low to high dose groups (p = O.OOOl),and the differences among the groups were also significant (ANOVA, p = 0.0003). Similarly, regression tests indicated a positive trend in sulcal caries scores from sham to low to high dose (p = 0.001) and differences among the groups (ANOVA, p = 0.0011). Interestingly, the mean sulcal caries scores for the high propranolol group were higher than those for desalivated animals, but not significantly so. scribed
DISCUSSION
Clearly, our findings are dissimilar from those of Johansson and Ericson (1987), but perhaps the differences can be explained by differing routes of drug administration in the two studies. A drug injected intraperitoneally is absorbed into the gastrointestinal tract and transported through the liver via the hepatic portal system before being returned to the general circulation. When a large part of a drug is removed by the liver and metabolized before gaining access to the systemic vessels, it is said to exhibit a significant first-pass effect (Clark and Smith, 1986). Propranolol, when given by intraperitoneal injection in doses below 0.8 mg/kg, shows near complete first-pass metabolism and thus this is not an efficacious route for administering the drug to rats (Shand, Rangno
Propranolol
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Table 2. Protein composition of rat parotid saliva determined by gel scanning* Protein component
Control
Peak 4
1.70* 0.20”,b
Amylase SP- 1 acidic proline-rich proteins, deoxyribonuclease and fraction V SP-3 Fraction I Mine* A Minor B
Low propranolol 0.47 * 0.02”
High propranolol 0.37 + 0.12h
33.08 & 0.87
33.84 + 0.24
34.73 + 0.41
39.55 0.87 22.98 0.75 0.32
36.04 0.0” 27.16 0.90 0.73
3 I .02 + I .55”,b O.Ob 30.83 & 1.25” 1.26+0.13 0.98 + 0. I 1b
& 0.68” f 0. 16”,b k 0.38” + 0.03 + 0.06”,b _
f 0.71b k 0.09” + 0.17 f 0.02”
*Percl:ntage of total area attributable to each component. Means sharing the same superscript differ significantly (p < 0.05. ANOVA). In each group n = 2
and Evans, 1972). Shand ef al. (1972) also found that doses as high as 5 mg/kg (intraperitoneally) were 50% metabolized before reaching the general circulation. The once-daily dose administered to rats by Johansson and Ericson (1987) was 0.5 mg/kg (intraperitoneally). Thl.ts, our use of osmotic pumps provided two advantages for drug delivery. First, because the pumps were inserted subcutaneously with the delivery end posterior to the nape of the neck, most of the absorption of propranolol was through cutaneous vessels, so avoiding the portal system. Second, as delivery of the drug was by continuous infusion, steady-state blood levels could be reached and maintained, thus avoiding the fluctuations associated with single daily injections. In humans, propranolo1 is normally given by the less efficacious but convenient oral route to give a therapeutic plasma concentration ranging from 20 to 1000 ng/ml, varying with the type of heart arrhythmia being treated (Bigger and Hoffman, 1985). We found that administration of 10 mg propranolol/kg body weight and 20 mg/kg daily via osmotic pumps maintained levels within this range. The finding that salivary output was not statistically different amo’ng sham-operated, low propranolo1 and high propranolol rats (Table 1) was
W 50
q
SulcalCaries Smooth Caries
Sham
l
High Low Animal Group
Desallvated
Fig. 2. The influence of chronic propranolol administration on smooth and sulcul caries scores (mean _+ SEM). There were significant differences (*j in smooth-surface (p = 0.0003, ANOVA) and sulcal (p = 0.0011, ANOVA) scores among the groups. when compared with sham-operated controls.
expected and consistent with the accepted view that the volume of secretion is mainly under parasympathetic (cholinergic) control. In a similar experiment, using osmotic pumps to deliver propranolol (2.6 mg/day), Johnson and Cortez (1988) found no significant differences in parotid salivary output or in protein concentration of stimulated secretions between control and experimental rats. We also found no statistical differences in protein (Lowry) concentrations among our groups (Table 1): however, the electrophoretic pattern of equal amounts (50 pg, Lowry) of parotid saliva from the animals that received propranolol (Plate Fig. 1) showed a loss of basic proline-rich proteins, peak A and SP-3. The grouped area corresponding to SP-1 (a basic prolinerich protein) and fraction IV (an acidic proline-rich protein) (and including deoxyribonuclease and fraction V) was also decreased when compared with sham-operated controls. The Lowry protein assay does not accurately measure proline-rich proteins (Oppenheim, Hay and Franzblau, 1971) and thus using this assay may have underestimated their normal levels in control animals. Johnson and Cortez (1988) also discussed this possibility in their study, which suggested that the expression of proline-rich proteins (and other salivary proteins) was under fi,-adrenergic control. If the loss of the proline-rich proteins from propranolol-treated animals were to be specifically assayed, significant differences in levels of protein concentration might be found. Another interesting finding was the apparent, doserelated increase in fraction I and minor b protein bands in parotid saliva from propranolol-treated rats (Table 2). However, when a protein (or proteins) is missing from a scan, one expects the total area attributable to each unaffected protein to increase by equal proportions so that the total area will equal 100%. The percentage area attributable to amylase remained constant in all scanned samples, but fraction I and minor B increased. These findings would be consistent with an actual decrease in amylase expression with increased propranolol dosage, rather than increased expression of fraction V and minor B proteins. However, because of the inaccuracies of protein staining by Coomassie blue, quantitation by a more direct method (e.g. immunochemical) would be necessary to confirm this observation. Even so, the other changes in the proportions of parotid proteins in propranolol-treated rats suggest that non-parallel
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G. E. WATSONet al.
secretion of these was taking place. Parotid proteins have been considered to be secreted in parallel or in equal proportions irrespective of the stimulus (Abe and Dawes, 1978; Kanagasuntheram and Lim, 1981), except in certain circumstances such as with chronic isoproterenol treatment (Johnson, 1984) or 1 week after the removal of the superior cervical ganglion (Asking et al., f987). There is other evidence, however, to suggest that non-parallel secretion is possible, even with acute stimulation alone (Cooper, Watson and Tabak, 1989; Procter, Asking and Garrett, 1989). We are aware of only one other animal study that examined caries developed concurrent with propranolo1 administration (Johansson and Ericson, 1987). They found no differences in caries scores among their various groups after 9 weeks, but different routes of administration and dosage may explain the conflict between these and our results, as previously discussed. We found statistically significant, doserelated increases in sulcal and smooth-surface caries scores in rats that received low and high doses of propranolol, as compared with controls (Text Fig. 2), suggesting that chronic treatment with propranolol, such as for arrhythmias and hypertension, could
predispose a patient to increased caries development. Because the stimulated salivary output of experimental rats was not significantiy different from that of controls, it may be that the increased caries scores were related to the quality of saliva rather than volume of secretion. The most evident compositional change was the decreased or missing proline-rich proteins in saliva from animals receiving propranolol. An altered expression of acidic proline-rich protein after minipump delivery for one week of isoproterenol, a j?-adrenergic agonist, has been reported by Kousvelari, Ciardi and Bowers (1988). Potential alterations in expression of other proteins cannot be ruled out. However, the levels of total salivary proteins appear not to differ significantly in our study, with the notable exception of the various proline-rich moieties, leading us to suggest that the development of caries is either directly or indirectly influenced by the levels of proline-rich proteins. This speculation assumes that the resting secretion, which we did not measure, was affected similarly to the stimulated secretion. We conclude that chronic administration of propranolol to rats via osmotic pumps is an effective way of maintaining steady-state blood levels of the drug. In comparison with controls, rats receiving propranolo1 had normal levels of salivary output and salivary protein concentration in response to a measured dose of pilocarpine, but an increased development of caries and a decreased expression of parotid prolinerich proteins. This suggests that further investigations are needed to determine the relationship between various salivary proteins and development of dental caries during chronic administration of hyposalivary drugs. Acknowledgements-This investigation was supported in Dart bv USPHS P50-DE 07703 and TE 32 DE07165. LAT is supported by research career development award K04 DE 00217.We thank Dr Herbert Bandemer for his expert assistance in obtaining fresh turkey blood.
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53.00+ 0.00 0003-9969190
Copyright Q 1990Pergamon
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THE EFFECT OF CHRONIC PROPRANOLOL TREATMENT ON SALIVARY COMPOSITION AND CARIES IN THE RAT G. E. WATSON, S. K. PEARSON,J. L. FALANY, L. A. TABAK and W. H. BOWEN* Department
of Dental Research, University of Rochester, 601 Elmwood Ave, Rochester, NY 14642, U.S.A. (Accepted I8 December 1989)
Summary-Many drugs are known to affect salivary secretion. The purpose of this study was to explore the chronic effects of a commonly used fl-adrenergic blocker, propranolol. Adult rats were desalivated or treated for 28days with propranolol HCI (IO or 20mg/kg, daily) or sterile buffer (sham-operated control) using osmotic pumps for delivery. The parotid and submandibular glands of each rat were cannulated and secretion elicited by pilocarpine (lOmg/kg, intravenous). There were no statistical differences in zsalivary protein content (Lowry) or output among the groups (ANOVA, p > 0.05). Analysis of salivary proteins by SDS-PAGE revealed a constant profile for submandibular secretions, but peak A and SP-3 proline-rich proteins were not detectable in parotid saliva of animals treated with propranolol for the entire experiment. Significantly increased smooth-surface (p = 0.0003) and sulcal (p = 0.001 I) caries scores were found within these propranolol groups (ANOVA). The findings provide further evidence that chronic administration of propranolol alters salivary composition by decreasing proline-rich proteins and concurrently enhances susceptibility to caries. Key words: hyposalivation, saliva, caries, propranolol.
IVTRODUCTION
environment of the body is maintained by autonomic nervous control of visceral muscles and glands of veg,etative organs (Schottelius and Schottehus, 1973). Disease can require treatment with medications that act on target tissues to correct imbalances, but this can also produce side-effects of varying consequences in other non-target or ‘innocent’ tissues. One of the more common side-effects of prescription and non-prescription drugs is xerostomia (Navazesh and Ship, 1983; Glass et al., 1984), a symptom especially prevalent in the elderly because of their increased use of medications (Handelman et nl., 1986). There are many drugs with hyposalivary effects (Sreebny and Schwartz, 1986), most of which interfere with the autonomic control of secretion. For example, the commonly prescribed anti-arrhythmic, propranolol, blocks p-adrenergic receptors in cardiac tissue and also in all areas of the body where the drug can gain access, including the salivary glands (Winer, 1985). Stimulation of the sympathetic nervous system in humans results in a proteinaceous secretion from salivary glands, while increased volume of secretion results from parasympathetic stimulation (Baum, 1987). A similar innervation is presented in the rat parotid (Spiers and Hodgson, 1976) and subThe internal
*Address correspond’ence to: Dr W. H. Bowen, Department of Dental Research. Universitv of Rochester. 601 Elmwood Ave, Rochester, NY 14642, U.S.A. Abbreuiarions: ANOVA, analysis of variance; SDSPAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis.
mandibular glands (Abe, 1987). Chronic administration of propranolol via an osmotic pump is known to alter rat parotid saliva by decreasing the expression of proline-rich proteins (Johnson and Cortez, 1988). However, when propranolol was administered chronically by intraperitoneal injection, neither the incidence of dental caries nor the composition of whole saliva was affected (Johansson and Ericson, 1987). Our purpose now was to examine the effect of propranolol on caries development, when administered to rats via osmotic pumps for 4 weeks. The effect of the drug on salivary output and protein composition was also determined, using pilocarpine as a secretagogue. MATERIALS
AND METHODS
Forty-one female Sprague-Dawley rats and their dams were acquired from the Charles River Kingston facility. They were screened for the presence of mutans streptococci using methods described by Bowen, Pearson and Young (1988). At the age of 20 days the animals were weaned from their dams and maintained on unmodified Diet 2000 (56% sucrose; Keyes, 1959) (Ziegler Brothers, Gardners, PA, U.S.A.) ad I&turn and water sweetened with sucrose (10% w/v). Streptococcus sobrinus 6715, grown in broth culture to exponential phase, was swabbed into the rats’ mouths with a cotton-tipped applicator (Bowen, Madison and Pearson, 1988). Infection with Strep. sobrinus was confirmed 3 days later by streaking swabs from rats’ mouths onto mitis salivarius agar + streptomycin. None of the animals required reinoculation. 435
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At age 37 days the animals were divided into 5 groups as follows: LOM. propranolol group-6 animals had osmotic pumps (Alza Corp., Palo Alto, CA, U.S.A.) delivering 10 mg/kg propranolol HCI daily implanted into their backs. High propranolol group-12 animals had osmotic pumps delivering 20 mg/kg propranolol HCI daily implanted into their backs. Sham-operated group-l 1 animals had osmotic pumps delivering sterile 0.1 M potassium phosphate solution, pH 6.0, implanted into their backs. Desalirated grozcp-6 animals had both parotid ducts ligated and both submandibular and sublingual glands removed (Bowen et al., 1985). These animals served as positive controls. Unoperated group-6 animals were not subjected to surgery. Rats undergoing surgical procedures were first anaesthetized with chloral hydrate (400 mg/kg intraperitoneally). The surgical site was shaved and then scrubbed with hydrogen peroxide and betadine. A small incision was made in the animal’s back, the fascia beneath the skin was separated, the pump inserted and the wound closed with surgical clips (Clay Adams, Parsippany, NJ, U.S.A.). The osmotic pumps were designated to deliver 2.69 + 0.17 ~1 of solution/h over the 28 day experimental period. Propranolol solution was made by dissolving propranolol HCI (Sigma Corp., St Louis, MO, U.S.A.), 40 mg/ml, in 0.1 M citric acid buffer, pH 3.0. On average, low propranolol rats received 0.041 mg propranolol/h and high propranolol rats received 0.077 mg/h. The unused propranolol stock solution was stored at 37-C for the length of the experiment to expose it to thermal conditions similar to those experienced by solutions in implanted pumps. The stability of the stock solution was examined by removing a sample weekly and storing it frozen at -20-C for later ana!ysis. After recovery from surgery, animals were placed singly in suspended cages and fed Diet 2000 ad libitum and drinking water sweetened with sucrose (10% w/v). They were observed daily for untoward effects, and weighed weekly. Surgical clips were removed after 7 days. Blood was collected from the tail veins (tail bleeds) of 2 rats in each group weekly; the samples were taken from different rats within the group each week. The blood was centrifuged and the plasma stored at -20 C. The experiment was ended 24 days after surgery. Saliva was successfully collected from 19 animals by ductal cannulation as described by Robinovitch and Sreebny (1969). An average of 7 rats per day, picked at random, were cannulated until all had been sam-
Plate
pled. They were first anaesthetized as described above and 1 ml of blood was obtained from the tail vein (final tail bleed). The parotid, submandibular and sublingual ducts were cannulated extraorally and salivary flow stimulated by 10 mg/kg pilocarpine (Sigma Corp.) delivered into a femoral vein. Secretion was maintained by administering another single dose 15 min after initial secretion had been elicited. Saliva was collected for a total of 30 min. Samples were collected over ice into tared Eppendorf tubes containing 10 ~1 of protease inhibitor cocktail [O.l M tris-HCl, pH 7.4, 10 mM EDTA. 5 mM benzamidine HCI, 1 mM soybean trypsin inhibitor, 5 mM pepstatin with 1 mM phenylmethylsulphonyl fluoride (PMSF)]. After collection the tubes were capped, weighed and frozen at -2O’C. The volume of saliva was determined (1 mg equalled I ~1; Suddick and Shannon. 1970) and divided by 30 min to estimate salivary output. While still anaesthetized, the animals were killed by cardiac puncture: this blood was then centrifuged and the resultant plasma stored at -20°C. The rats were then decapitated and the heads defleshed. To measure the amount of propranolol in the plasma samples and in our stock solution we performed competition assays, as described by Bilezikian et al. (1979), using the /I-adrenergic antagonist [‘2’1]iodohydroxybenzylpindolol, and /I-adrenergic receptors from a membrane suspension of turkey erythrocytes. Levels of radioactivity in each sample were measured in a Gamma 4000 gamma counter (Beckman Instruments, Inc., Fullerton, CA, U.S.A.). Specific binding was determined by subtracting nonspecific binding from total binding. Protein determination was performed on saliva samples by the method of Lowry ef al. (1951) with bovine serum albumin as standard. Salivary proteins were resolved by SDS-PAGE [parotid (12% polyacrylamide); submandibular (10%); and sublingual (SO/,)] (Laemmli, 1970) and visualized with Coomassie brilliant blue R (Weber and Osborn, 1975) and periodic acid-Schiff (Fairbanks, Steck and Wallach, 1971) stains. The major components of the parotid proteins were identified by their electrophoretic mobility on SDS-PAGE gels as determined from previous studies (Keller et al., 1975; Johnson, 1984). A colour positive transparency of the destained gel of parotid saliva samples was scanned at 550 nm in a DU-8B Spectrophotometer equipped with a scanning system (Beckman Instruments, Irvine. CA, U.S.A.). Each lane was examined individually and the percentage of the total area attributable to each protein component was calculated. Coronal caries was scored with the method described by Keyes (1958), as modified by Larson (1981).
I
Fig. 1. SDS-PAGE of rat parotid saliva from sham-operated, low propranolol (10 mg/kg, daily), and high propranolol (20 mg/kg, daily) groups. Fifty microgrammes of salivay protein (Lowry) were loaded per lane, electrophoresed, stained with Coomassie blue R, and destained. Proteins were identified from previous work by Keller er al. (1975) and Johnson (1984): (1) peak A, a basic proline-rich protein; (2) amylase; (3) SP-I, a basic proline-rich protein; (4) fraction IV, an acidic proline-rich protein; (5) deoxyribonuclease: (6) fraction V; (7) SP-3, a basic proline-rich protein; (8) fraction I; (9) minor band A; and (IO) minor band B.
Propranolol influences saliva and caries
437
1
ZOOK92.5K66_2K-
2
4,5K\ /
7
31K-
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9
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LOW 1 HIGH PROPRANOLOL Plate 1
438
G. E.
WAT~N
Statistical evaluations used the ANOVA and linear regression programs of the STATVIEW II statistical software package (Abacus Concepts, Berkeley, CA, U.S.A.). A p-value of less than 0.05 was regarded as statistically significant. All mean values in text are +SEM. When samples were obtained from both glands of the same type in one animal, the average value was used and counted as one observation.
RESULTS
All animals in the control and low propranolol groups survived; one animal in the high propranolol group died before the end of the experiment. Shamoperated rats had mean weight gains of 89.2 f 3.0 g during the experiment; low propranolol and’ high propranolol rats gained 96.2 + 7.2 and 87.3 f 4.2 g, respectively-these weight gains were not significantly different (ANOVA, p = 0.42): The desalivated group gained the least weight over the course of the experiment (60.8 f 6.2 g), significantly less than all three other groups (ANOVA, p = 0.004; significantly different at the 95% level by Fisher PSLD and Sheffe F-tests). Mean sucrose-water consumption of shamoperated, low propranolol, high propranolol, and desalivated animals for the 4 days preceding death was 327.9 k28.2, 347.6 k42.7, 341.3 + 36.3 and 38 1.1 f 26.5 ml, respectively, and did not differ significantly (p > 0.05, ANOVA). Propranolol stock solution (40 mg/ml) was stable in buffer for at least 28 days at 37°C (data not shown). Plasma levels of propranolol during the first week averaged 111.3 and 233.5 ng/ml for low and high groups, respectively; these levels had decreased to 30.9 + 14.0 and 77.9 + 13.4 ng/ml by the end of the experiment. A slowly decreasing plasma propranolol concentration with time had been expected as pump output was constant to animals that were continually gaining weight. In total, 56 salivary ducts from 19 animals were successfully cannulated and produced measurable saliva. There were too few sublingual samples, however, to allow accurate comparisons to be made, and these samples were not included in the results. The effect of chronic administration of propranolol on output from pilocarpine-stimulated parotid and submandibular salivary glands is shown in Table 1; there were no statistical differences among the groups (ANOVA; parotid, p = 0.53; submandibular, p = 0.85). Protein determinations were performed on all salivary samples (Table 1). The results suggested a trend of decreasing protein concentration as the level of propranolol increased, but there were no statistical differences among the groups (ANOVA; parotid, p = 0.13; submandibular, p = 0.24). Qualitative differences in the pattern of parotid proteins were observed in propranolol-treated animals, when compared to controls (Plate Fig. 1). The protein bands equivalent to those missing in the parotid saliva from propranolol-treated animals displayed metachromasia in the control lanes suggestive of proline-rich proteins (Humphreys-Beher and Wells, 1984) and corresponded to the peak A and SP-3 bands de-
et al.
Table 1. Mean output and protein concentration of rat saliva Gland experimental group Parotid Sham (n = 5) Low propranolol (n = 4) High propranolol (n = 7) Submandibular Sham (n = 5) Low propranolol (n = 5) High propranolol (n = 4)
Output (P Umin)
Lowry protein (mgiml)
2.085 & 0.289 2.373 k 0.380
13.911 & 1.534 1I.308 + 1.703
I.757 + 0.402
9.059 ) 1.705
2.200 k 0.625 2.565 + 0.434
2.108 + 0.51 I I.883 + 0.543
2.183 k 0.662
0.833 + 0.254
There were no statistical differences among the groups (ANOVA). Numbers of animals contributing to the mean value indicated by n. Values are *SEM.
by Keller et al. (1975) and Johnson (1984). Protein bands SP-1, acidic proline-rich proteins, deoxyribonuclease, and fraction V were grouped together as a single area because resolution of each individual band was not precise in all samples. A significant, dose-related decrease in the percentage area attributable to these grouped proteins was seen (Table 2); there was a concomitant increase in the total area attributable to fraction I and minor B bands. The gels of submandibular salivas were remarkably constant in that there were no perceived differences among the different groups (data not shown). The influence of chronic administration of propranolo1 on caries is shown in Text Fig. 2. Regression tests indicated a significant positive trend in smoothsurface caries scores from sham to low to high dose groups (p = O.OOOl),and the differences among the groups were also significant (ANOVA, p = 0.0003). Similarly, regression tests indicated a positive trend in sulcal caries scores from sham to low to high dose (p = 0.001) and differences among the groups (ANOVA, p = 0.0011). Interestingly, the mean sulcal caries scores for the high propranolol group were higher than those for desalivated animals, but not significantly so. scribed
DISCUSSION
Clearly, our findings are dissimilar from those of Johansson and Ericson (1987), but perhaps the differences can be explained by differing routes of drug administration in the two studies. A drug injected intraperitoneally is absorbed into the gastrointestinal tract and transported through the liver via the hepatic portal system before being returned to the general circulation. When a large part of a drug is removed by the liver and metabolized before gaining access to the systemic vessels, it is said to exhibit a significant first-pass effect (Clark and Smith, 1986). Propranolol, when given by intraperitoneal injection in doses below 0.8 mg/kg, shows near complete first-pass metabolism and thus this is not an efficacious route for administering the drug to rats (Shand, Rangno
Propranolol
influences
saliva and caries
439
Table 2. Protein composition of rat parotid saliva determined by gel scanning* Protein component
Control
Peak 4
1.70* 0.20”,b
Amylase SP- 1 acidic proline-rich proteins, deoxyribonuclease and fraction V SP-3 Fraction I Mine* A Minor B
Low propranolol 0.47 * 0.02”
High propranolol 0.37 + 0.12h
33.08 & 0.87
33.84 + 0.24
34.73 + 0.41
39.55 0.87 22.98 0.75 0.32
36.04 0.0” 27.16 0.90 0.73
3 I .02 + I .55”,b O.Ob 30.83 & 1.25” 1.26+0.13 0.98 + 0. I 1b
& 0.68” f 0. 16”,b k 0.38” + 0.03 + 0.06”,b _
f 0.71b k 0.09” + 0.17 f 0.02”
*Percl:ntage of total area attributable to each component. Means sharing the same superscript differ significantly (p < 0.05. ANOVA). In each group n = 2
and Evans, 1972). Shand ef al. (1972) also found that doses as high as 5 mg/kg (intraperitoneally) were 50% metabolized before reaching the general circulation. The once-daily dose administered to rats by Johansson and Ericson (1987) was 0.5 mg/kg (intraperitoneally). Thl.ts, our use of osmotic pumps provided two advantages for drug delivery. First, because the pumps were inserted subcutaneously with the delivery end posterior to the nape of the neck, most of the absorption of propranolol was through cutaneous vessels, so avoiding the portal system. Second, as delivery of the drug was by continuous infusion, steady-state blood levels could be reached and maintained, thus avoiding the fluctuations associated with single daily injections. In humans, propranolo1 is normally given by the less efficacious but convenient oral route to give a therapeutic plasma concentration ranging from 20 to 1000 ng/ml, varying with the type of heart arrhythmia being treated (Bigger and Hoffman, 1985). We found that administration of 10 mg propranolol/kg body weight and 20 mg/kg daily via osmotic pumps maintained levels within this range. The finding that salivary output was not statistically different amo’ng sham-operated, low propranolo1 and high propranolol rats (Table 1) was
W 50
q
SulcalCaries Smooth Caries
Sham
l
High Low Animal Group
Desallvated
Fig. 2. The influence of chronic propranolol administration on smooth and sulcul caries scores (mean _+ SEM). There were significant differences (*j in smooth-surface (p = 0.0003, ANOVA) and sulcal (p = 0.0011, ANOVA) scores among the groups. when compared with sham-operated controls.
expected and consistent with the accepted view that the volume of secretion is mainly under parasympathetic (cholinergic) control. In a similar experiment, using osmotic pumps to deliver propranolol (2.6 mg/day), Johnson and Cortez (1988) found no significant differences in parotid salivary output or in protein concentration of stimulated secretions between control and experimental rats. We also found no statistical differences in protein (Lowry) concentrations among our groups (Table 1): however, the electrophoretic pattern of equal amounts (50 pg, Lowry) of parotid saliva from the animals that received propranolol (Plate Fig. 1) showed a loss of basic proline-rich proteins, peak A and SP-3. The grouped area corresponding to SP-1 (a basic prolinerich protein) and fraction IV (an acidic proline-rich protein) (and including deoxyribonuclease and fraction V) was also decreased when compared with sham-operated controls. The Lowry protein assay does not accurately measure proline-rich proteins (Oppenheim, Hay and Franzblau, 1971) and thus using this assay may have underestimated their normal levels in control animals. Johnson and Cortez (1988) also discussed this possibility in their study, which suggested that the expression of proline-rich proteins (and other salivary proteins) was under fi,-adrenergic control. If the loss of the proline-rich proteins from propranolol-treated animals were to be specifically assayed, significant differences in levels of protein concentration might be found. Another interesting finding was the apparent, doserelated increase in fraction I and minor b protein bands in parotid saliva from propranolol-treated rats (Table 2). However, when a protein (or proteins) is missing from a scan, one expects the total area attributable to each unaffected protein to increase by equal proportions so that the total area will equal 100%. The percentage area attributable to amylase remained constant in all scanned samples, but fraction I and minor B increased. These findings would be consistent with an actual decrease in amylase expression with increased propranolol dosage, rather than increased expression of fraction V and minor B proteins. However, because of the inaccuracies of protein staining by Coomassie blue, quantitation by a more direct method (e.g. immunochemical) would be necessary to confirm this observation. Even so, the other changes in the proportions of parotid proteins in propranolol-treated rats suggest that non-parallel
440
G. E. WATSONet al.
secretion of these was taking place. Parotid proteins have been considered to be secreted in parallel or in equal proportions irrespective of the stimulus (Abe and Dawes, 1978; Kanagasuntheram and Lim, 1981), except in certain circumstances such as with chronic isoproterenol treatment (Johnson, 1984) or 1 week after the removal of the superior cervical ganglion (Asking et al., f987). There is other evidence, however, to suggest that non-parallel secretion is possible, even with acute stimulation alone (Cooper, Watson and Tabak, 1989; Procter, Asking and Garrett, 1989). We are aware of only one other animal study that examined caries developed concurrent with propranolo1 administration (Johansson and Ericson, 1987). They found no differences in caries scores among their various groups after 9 weeks, but different routes of administration and dosage may explain the conflict between these and our results, as previously discussed. We found statistically significant, doserelated increases in sulcal and smooth-surface caries scores in rats that received low and high doses of propranolol, as compared with controls (Text Fig. 2), suggesting that chronic treatment with propranolol, such as for arrhythmias and hypertension, could
predispose a patient to increased caries development. Because the stimulated salivary output of experimental rats was not significantiy different from that of controls, it may be that the increased caries scores were related to the quality of saliva rather than volume of secretion. The most evident compositional change was the decreased or missing proline-rich proteins in saliva from animals receiving propranolol. An altered expression of acidic proline-rich protein after minipump delivery for one week of isoproterenol, a j?-adrenergic agonist, has been reported by Kousvelari, Ciardi and Bowers (1988). Potential alterations in expression of other proteins cannot be ruled out. However, the levels of total salivary proteins appear not to differ significantly in our study, with the notable exception of the various proline-rich moieties, leading us to suggest that the development of caries is either directly or indirectly influenced by the levels of proline-rich proteins. This speculation assumes that the resting secretion, which we did not measure, was affected similarly to the stimulated secretion. We conclude that chronic administration of propranolol to rats via osmotic pumps is an effective way of maintaining steady-state blood levels of the drug. In comparison with controls, rats receiving propranolo1 had normal levels of salivary output and salivary protein concentration in response to a measured dose of pilocarpine, but an increased development of caries and a decreased expression of parotid prolinerich proteins. This suggests that further investigations are needed to determine the relationship between various salivary proteins and development of dental caries during chronic administration of hyposalivary drugs. Acknowledgements-This investigation was supported in Dart bv USPHS P50-DE 07703 and TE 32 DE07165. LAT is supported by research career development award K04 DE 00217.We thank Dr Herbert Bandemer for his expert assistance in obtaining fresh turkey blood.
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