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Kallikrein and salivary secretion in rats during heat exposure
Immunopharmacology ELSEVIER
Immunopharmacology32 (1996) 125-127
Kallikrein and salivary secretion in rats during heat exposure Jacques Damas Department of Human Physiology, UniversiO,of Liege, Facult3'of Medicine, Liege, B-4020, Belgium
Abstract RP 67580, a NKI receptor antagonist, inhibited the sialogogic effect of Substance P (SP). Heat-induced salivation was indirectly measured through the changes in body weight. Thermolytic salivation was reduced by atropine and RP 67580 and thus would be mainly controlled by acetylcholine and tachykinins. This salivation was associated with a large depletion of kallikrein in submaxillary glands. This depletion was not inhibited by atropine and RP 67580. Feeding has been reported to reduce amylase activity in parotid glands but did not modify the kallikrein content in submaxillary glands. Heat exposure did not modify amylase activity. There is a dissociation between the releases of amylase and kallikrein from salivary glands. Keywords: Heat; Salivation: Tachykinin;RP 67580; Kallikrein
1. Introduction During heat exposure, rats do not sweat nor pant but increase evaporative water losses by spreading saliva on their fur and skin. This thermolytic salivation mainly depends on a stimulation of the submaxillary glands (Hainsworth and Stricker, 1972) and was associated with a depletion of tissue kallikrein from these latter glands (Damas and Bourdon, 1994). In this work, a comparison between the release of kallikrein and amylase from salivary glands was performed. The involvement of substance P (SP) in the thermolytic salivation was also evaluated by the use of RP 67580, a NK1 receptor antagonist (Garret et al., 1991).
2. Materials and methods We used normal male Wistar rats and kininogendeficient male Brown Norway ( B N / M A Y Pfd Lg) rats from our breeding colony. Wistar rats were anesthetized with pentobarbital
(45 m g / k g i.p.) and salivary secretion was elicited by the intravenous injection of SP (1 /~g/kg) 5 min after the intravenous administration of saline or RP 67580. Saliva was collected for 5 min after SP injection. It was obtained from the mouth with preweighed strips of filter-paper. Salivary secretion was determined by weight. Heat exposure was performed in a large incubator for bird eggs with an adjustable temperature control and a large window through which the animals could be watched. Before heat exposure, the animals received an intraperitoneal injection of saline, atropine or of RP 67580. Their weight were determined also. Immediately after heat exposure, the animals were anesthetized with sodium pentobarbital. Their colonic temperature was measured and their new weight was determined. Twenty-four other male Wistar rats were divided in two groups. All the animals were fasted: food but not water was withheld for 32 h. Then, the diet (standard pelleted diet) was reintroduced to a group of 12 rats for 90 min. Afterwards, all the animals
0162-3109/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0162-3 109(95)00072-0
126
3".Damas / lmmunopharmacology 32 (1996) 125-127
were killed with pentobarbital. Their submaxillary glands were removed, weighed and homogenized in a solution containing NaC1 (0.15 M), NaH2PO 4 (0.015 M, pH 7.4), EDTA (0.01 M) and serum albumin (2 g/l), 1 / 5 ( w / v ) . After centrifugation, the supernatant fluid was collected and a part was frozen ( - 2 0 ° C ) for the measurement of tissue kallikrein by a specific assay already described (Damas and Bourdon, 1994). The glandular amylase activity was assayed by an enzymatic colorimetric test (Boebringer-Mannheim, Mannheim, Germany) using o~-4-nitrophenylmaltoheptanoside as substrate. Values are means + SEM. Statistical significances of difference were calculated either by Student's t-test for unpaired values or by one-way analysis of variance followed by Fisher's protected least significant difference. Probabilities of less than 5% were considered significant.
3. Results The intravenous injection of SP (1 /zg/kg) in rats induced salivation. This sialogogic effect of SP was inhibited by RP 67580 in a dose-dependent manner (ID50:141 /xg/kg). Normal male Wistar rats were exposed at ambient temperature of 26 or 36°C for 1 h. After this time period, the weight of rats exposed to 26°C was decreased by 1.01 __+0.15% (n = 9). This reduction depends mostly from urination and defecation induced by the handling of the animals. The loss of body weight was increased in animals kept at 36°C and reached: 3.1 + 0.12% (n = 16). Thus, heat exposure induced a loss of water corresponding to about 2% of body weight. A great part of this water loss was due to salivation (Damas, 1993). This weight loss was reduced by atropine (3 m g / k g ) and RP 67580 (2.5 m g / k g ) to 1.42 _+ 0.12% ( P < 0.01; n = 14) and 2.30 _+ 0.21% ( P < 0.01; n = 16), respectively. Body temperature of Wistar rats exposed to 36°C reached a mean of 40.5°C after 1 h. This change was not modified by atropine and RP 66580. A loss of body weight was also recorded in kininogen-deficient Brown Norway rats exposed to 36°C. The body weight loss reached 2.56 _+ 0.33% (n = 6) and was reduced by atropine to 1.26 + 0.22% ( P < 0.01; n = 6). RP 67580 injected alone had no
significant effect on this change. However, the treatment of the animals with atropine and RP 67580 suppressed the body weight loss induced by heat exposure. Indeed, in these treated animals, the reduction of weight only reached: 0.63 +0.05% ( P < 0.005; n = 6). Mean body temperature of kininogen-deficient rats exposed to 36°C reached 40.5 + 0.1°C after 1 h. After atropine, mean body temperature was significantly increased up to 41.3 + 0.2°C ( P < 0.01; n = 6). Though RP 67580 alone had no influence on body temperature in the heat, it enhanced the hyperthermic effect of atropine: mean body temperature: 42.5 + 0.4°C ( P < 0.01, versus saline and atropine treated rats; n = 6). Heat exposure reduced the content in kallikrein of submaxillary glands from 4.02 ± 0.53 m g / g of tissue to 1.75+0.44 m g / g ( P < 0 . 0 1 ; n = 6 ) . This reduction was not inhibited by RP 67580 and atropine: kallikrein level = 1.68 + 0.37 and 1.10_+ 0.09 m g / g , respectively. Heat exposure did not modify the level of amylase activity in submaxillary glands of Wistar rats. In rats exposed to 26°C for 1 h, this activity was 102.5 + 33.1 U / g l a n d (n = 12) while after 1 h at 36°C, this activity was 108.9 + 26.4 U/gland (n = 8). According to Asztely et al. (1994), in fasted rats, feeding for 60-90 min reduced density of acinar secretory granules and total amylase activity in parotid glands. However, this procedure did not modify the kallikrein content of submaxillary glands (kallikrein level in fasted rats: 3.23 _+ 0.85 m g / g , n = 12; in fasted rats after refeeding: 3.56 + 0.73 m g / g , n = 12).
4. Discussion Depletion of kallikrein in submaxillary glands was observed after heat exposure but not after feeding. A reduction of amylase activity has been reported in parotid glands after feeding (Asztely et al., 1994) but was not recorded after heat exposure. Thus, there is a dissociation between the secretions of kallikrein and of amylase by salivary glands. Kallikrein depletion induced by heat exposure was not inhibited by atropine and RP 67580. It results from the stimulation of the glands by other mediators than acetylcholine and SP. Indeed, it was suppressed
J. Damas / lmmunopharmacology 32 (1996) 125-127
by c~- and /3-adrenergic inhibitors (Damas and Bourdon, 1994). During heat exposure, a large oedema in and around submaxillary glands develops (Damas, 1994). According to Garrett et al. (1995), this oedema might be the cause of the release of kallikrein in blood during heat exposure. The thermolytic salivation of rats exposed to heat was indirectly measured by the determination of the changes in body weight (Damas, 1993). This salivation is reduced by atropine and RP 67580. In fact, in kininogen-deficient rats, the thermolytic salivation was suppressed by the association of RP 67580 and atropine. These results suggest that beside acetylcholine, a tachykinin or tachykinins, like SP or neurokinin A, which induce salivation through the activation of N K l receptor, participate in the stimulation of salivary glands in the heat. Heat-induced salivation would result from a large stimulation of submaxillary glands by parasympathetic and sympathetic nerves. The former would be responsible of the salivary secretion. The latter would modulate this salivary response and would be responsible of the kallikrein release.
127
References Asztely A, Tobin G, Ekstr6m J. Masticatory-salivaryreflexes mobilize non-adrenergic, non-cholinergic secretory mechanisms in parotid glands of conscious rats. Acta Physiol Stand 1994: 151: 373-376. Damas J. Water losses induced by heat exposure and the kinin system in the rat. Arch. int. Physiol Biochim 1993: 101: 227-232. Damas J. Kallikrein, nitric oxide and the vascular responses of submaxillary glands in rats exposed to heat. Arch Int Physiol Biochim 1994: 102: 139-146. Damas J, Bourdon V. The release of glandular kallikrein from submaxillary glands of rats exposed to heat. Arch Int Physiol Biochim 1994; 102: 183-188. Garret C, Carruette A, Fardin V, Moussaoui S. Peyronel JF, Blanchard JC, Laduron P. Pharmacological properties of a potent and selective nonpeptide substance P antagonist. Proc Natl Acad Sci USA 1991; 88: 10208-10212. Garrett JR, Chao J, Proctor GB, Wang C, Zhang XS, Chan KM, Shori DK. Influencesof secretory activitiesin rat submandibular glands on tissue kallikrein circulating in the blood. Exp Biol 1995; 80: 429-440. Hainsworth FR, Stricker EM. Evaporative cooling in the rat: further consideration of functional differences between salivary glands. Can. J. Physiol.. 1972: 50:494 497.
Immunopharmacology32 (1996) 125-127
Kallikrein and salivary secretion in rats during heat exposure Jacques Damas Department of Human Physiology, UniversiO,of Liege, Facult3'of Medicine, Liege, B-4020, Belgium
Abstract RP 67580, a NKI receptor antagonist, inhibited the sialogogic effect of Substance P (SP). Heat-induced salivation was indirectly measured through the changes in body weight. Thermolytic salivation was reduced by atropine and RP 67580 and thus would be mainly controlled by acetylcholine and tachykinins. This salivation was associated with a large depletion of kallikrein in submaxillary glands. This depletion was not inhibited by atropine and RP 67580. Feeding has been reported to reduce amylase activity in parotid glands but did not modify the kallikrein content in submaxillary glands. Heat exposure did not modify amylase activity. There is a dissociation between the releases of amylase and kallikrein from salivary glands. Keywords: Heat; Salivation: Tachykinin;RP 67580; Kallikrein
1. Introduction During heat exposure, rats do not sweat nor pant but increase evaporative water losses by spreading saliva on their fur and skin. This thermolytic salivation mainly depends on a stimulation of the submaxillary glands (Hainsworth and Stricker, 1972) and was associated with a depletion of tissue kallikrein from these latter glands (Damas and Bourdon, 1994). In this work, a comparison between the release of kallikrein and amylase from salivary glands was performed. The involvement of substance P (SP) in the thermolytic salivation was also evaluated by the use of RP 67580, a NK1 receptor antagonist (Garret et al., 1991).
2. Materials and methods We used normal male Wistar rats and kininogendeficient male Brown Norway ( B N / M A Y Pfd Lg) rats from our breeding colony. Wistar rats were anesthetized with pentobarbital
(45 m g / k g i.p.) and salivary secretion was elicited by the intravenous injection of SP (1 /~g/kg) 5 min after the intravenous administration of saline or RP 67580. Saliva was collected for 5 min after SP injection. It was obtained from the mouth with preweighed strips of filter-paper. Salivary secretion was determined by weight. Heat exposure was performed in a large incubator for bird eggs with an adjustable temperature control and a large window through which the animals could be watched. Before heat exposure, the animals received an intraperitoneal injection of saline, atropine or of RP 67580. Their weight were determined also. Immediately after heat exposure, the animals were anesthetized with sodium pentobarbital. Their colonic temperature was measured and their new weight was determined. Twenty-four other male Wistar rats were divided in two groups. All the animals were fasted: food but not water was withheld for 32 h. Then, the diet (standard pelleted diet) was reintroduced to a group of 12 rats for 90 min. Afterwards, all the animals
0162-3109/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0162-3 109(95)00072-0
126
3".Damas / lmmunopharmacology 32 (1996) 125-127
were killed with pentobarbital. Their submaxillary glands were removed, weighed and homogenized in a solution containing NaC1 (0.15 M), NaH2PO 4 (0.015 M, pH 7.4), EDTA (0.01 M) and serum albumin (2 g/l), 1 / 5 ( w / v ) . After centrifugation, the supernatant fluid was collected and a part was frozen ( - 2 0 ° C ) for the measurement of tissue kallikrein by a specific assay already described (Damas and Bourdon, 1994). The glandular amylase activity was assayed by an enzymatic colorimetric test (Boebringer-Mannheim, Mannheim, Germany) using o~-4-nitrophenylmaltoheptanoside as substrate. Values are means + SEM. Statistical significances of difference were calculated either by Student's t-test for unpaired values or by one-way analysis of variance followed by Fisher's protected least significant difference. Probabilities of less than 5% were considered significant.
3. Results The intravenous injection of SP (1 /zg/kg) in rats induced salivation. This sialogogic effect of SP was inhibited by RP 67580 in a dose-dependent manner (ID50:141 /xg/kg). Normal male Wistar rats were exposed at ambient temperature of 26 or 36°C for 1 h. After this time period, the weight of rats exposed to 26°C was decreased by 1.01 __+0.15% (n = 9). This reduction depends mostly from urination and defecation induced by the handling of the animals. The loss of body weight was increased in animals kept at 36°C and reached: 3.1 + 0.12% (n = 16). Thus, heat exposure induced a loss of water corresponding to about 2% of body weight. A great part of this water loss was due to salivation (Damas, 1993). This weight loss was reduced by atropine (3 m g / k g ) and RP 67580 (2.5 m g / k g ) to 1.42 _+ 0.12% ( P < 0.01; n = 14) and 2.30 _+ 0.21% ( P < 0.01; n = 16), respectively. Body temperature of Wistar rats exposed to 36°C reached a mean of 40.5°C after 1 h. This change was not modified by atropine and RP 66580. A loss of body weight was also recorded in kininogen-deficient Brown Norway rats exposed to 36°C. The body weight loss reached 2.56 _+ 0.33% (n = 6) and was reduced by atropine to 1.26 + 0.22% ( P < 0.01; n = 6). RP 67580 injected alone had no
significant effect on this change. However, the treatment of the animals with atropine and RP 67580 suppressed the body weight loss induced by heat exposure. Indeed, in these treated animals, the reduction of weight only reached: 0.63 +0.05% ( P < 0.005; n = 6). Mean body temperature of kininogen-deficient rats exposed to 36°C reached 40.5 + 0.1°C after 1 h. After atropine, mean body temperature was significantly increased up to 41.3 + 0.2°C ( P < 0.01; n = 6). Though RP 67580 alone had no influence on body temperature in the heat, it enhanced the hyperthermic effect of atropine: mean body temperature: 42.5 + 0.4°C ( P < 0.01, versus saline and atropine treated rats; n = 6). Heat exposure reduced the content in kallikrein of submaxillary glands from 4.02 ± 0.53 m g / g of tissue to 1.75+0.44 m g / g ( P < 0 . 0 1 ; n = 6 ) . This reduction was not inhibited by RP 67580 and atropine: kallikrein level = 1.68 + 0.37 and 1.10_+ 0.09 m g / g , respectively. Heat exposure did not modify the level of amylase activity in submaxillary glands of Wistar rats. In rats exposed to 26°C for 1 h, this activity was 102.5 + 33.1 U / g l a n d (n = 12) while after 1 h at 36°C, this activity was 108.9 + 26.4 U/gland (n = 8). According to Asztely et al. (1994), in fasted rats, feeding for 60-90 min reduced density of acinar secretory granules and total amylase activity in parotid glands. However, this procedure did not modify the kallikrein content of submaxillary glands (kallikrein level in fasted rats: 3.23 _+ 0.85 m g / g , n = 12; in fasted rats after refeeding: 3.56 + 0.73 m g / g , n = 12).
4. Discussion Depletion of kallikrein in submaxillary glands was observed after heat exposure but not after feeding. A reduction of amylase activity has been reported in parotid glands after feeding (Asztely et al., 1994) but was not recorded after heat exposure. Thus, there is a dissociation between the secretions of kallikrein and of amylase by salivary glands. Kallikrein depletion induced by heat exposure was not inhibited by atropine and RP 67580. It results from the stimulation of the glands by other mediators than acetylcholine and SP. Indeed, it was suppressed
J. Damas / lmmunopharmacology 32 (1996) 125-127
by c~- and /3-adrenergic inhibitors (Damas and Bourdon, 1994). During heat exposure, a large oedema in and around submaxillary glands develops (Damas, 1994). According to Garrett et al. (1995), this oedema might be the cause of the release of kallikrein in blood during heat exposure. The thermolytic salivation of rats exposed to heat was indirectly measured by the determination of the changes in body weight (Damas, 1993). This salivation is reduced by atropine and RP 67580. In fact, in kininogen-deficient rats, the thermolytic salivation was suppressed by the association of RP 67580 and atropine. These results suggest that beside acetylcholine, a tachykinin or tachykinins, like SP or neurokinin A, which induce salivation through the activation of N K l receptor, participate in the stimulation of salivary glands in the heat. Heat-induced salivation would result from a large stimulation of submaxillary glands by parasympathetic and sympathetic nerves. The former would be responsible of the salivary secretion. The latter would modulate this salivary response and would be responsible of the kallikrein release.
127
References Asztely A, Tobin G, Ekstr6m J. Masticatory-salivaryreflexes mobilize non-adrenergic, non-cholinergic secretory mechanisms in parotid glands of conscious rats. Acta Physiol Stand 1994: 151: 373-376. Damas J. Water losses induced by heat exposure and the kinin system in the rat. Arch. int. Physiol Biochim 1993: 101: 227-232. Damas J. Kallikrein, nitric oxide and the vascular responses of submaxillary glands in rats exposed to heat. Arch Int Physiol Biochim 1994: 102: 139-146. Damas J, Bourdon V. The release of glandular kallikrein from submaxillary glands of rats exposed to heat. Arch Int Physiol Biochim 1994; 102: 183-188. Garret C, Carruette A, Fardin V, Moussaoui S. Peyronel JF, Blanchard JC, Laduron P. Pharmacological properties of a potent and selective nonpeptide substance P antagonist. Proc Natl Acad Sci USA 1991; 88: 10208-10212. Garrett JR, Chao J, Proctor GB, Wang C, Zhang XS, Chan KM, Shori DK. Influencesof secretory activitiesin rat submandibular glands on tissue kallikrein circulating in the blood. Exp Biol 1995; 80: 429-440. Hainsworth FR, Stricker EM. Evaporative cooling in the rat: further consideration of functional differences between salivary glands. Can. J. Physiol.. 1972: 50:494 497.