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Abstract
It is known that marijuana use decreases saliva secretion. Therefore, we hypothesized that cannabinoid receptors (CBs) are located in salivary glands to mediate that effect. In these experiments, we used the submandibular gland (SMG) of male rats, which is one of the major salivary glands. Mammalian tissues contain at least two types of CBs, CB1 and CB2, mainly located in the nervous system and peripheral tissues, respectively. Both receptors are coupled to Gi protein and respond by inhibiting the activity of adenylyl cyclase. We demonstrated that both CB1 and CB2 are present in the SMG, each showing specific localizations. The best-known endocannabinoid is anandamide (AEA), which binds with high affinity to CB1 and CB2. We showed that AEA markedly reduced forskolin-induced increase of cAMP content in vitro. This effect was blocked by AM251 and AM630 (CB1 and CB2 antagonists, respectively), indicating that both receptors are implicated in SMG physiology. In addition, we showed that AEA injected intraglandularly to anesthetized rats inhibited norepinephrine (NE)— and methacholine (MC)—stimulated saliva secretion in vivo and that both AM251 or AM630 prevented the inhibitory action of AEA. Also, the intraglandular injection of AM251 increased saliva secretion induced by lower doses of NE or MC. This increase was synergized after coinjection with AM630. Therefore, we concluded that AEA decreases saliva secretion in the SMG acting through CB1 and CB2 receptors.
Introduction
It is well known that Δ9-tetrahydrocannabinol (Δ9-THC), the psychoactive principle isolated from Cannabis sativa, alters different physiologic parameters in biologic systems. Mammalian tissues contain at least two types of cannabinoid receptors (CBs), CB1 and CB2, mainly located in the nervous system (1—3) and in peripheral tissues or immune cells (4), respectively. Both receptors are coupled to Gi protein and respond by inhibiting the activity of adenylyl cyclase (AC) (5). The best-known endocannabinoids are arachidonoyl ethanolamide (anandamide [AEA]) and arachidonoyl glycerol, both derivatives of arachidonic acid that bind with high affinity to CBs (6). Specific and selective antagonists for CBs have been developed, such as AM251 (7), AM281 (8), SR141716A (9, 10), and LY320135 (11) for CB1 and AM630 (12) and SR144528 (13) for CB2. For our experiments, we used AM251 because this is a potent selective CB1 antagonist that has a Ki of 7.49 nM for CB1 and is 306-fold selective over CB2. We also used AM630, a CB2 antagonist/inverse agonist (Ki, 31.2 nM), which is 165-fold selective over CB1 and behaves as a weak partial/inverse agonist at CB1 (14).
The submandibular gland (SMG) is one of the major salivary glands, together with the sublingual and parotid glands. End secretory units, called acini, are continuous with a duct system that in rodents has four sequential segments: intercalated ducts (IDs), granular convoluted tubules (GCTs), striated ducts (SDs), and excretory ducts that release the saliva to the oral cavity (15).
Saliva formation is thought to involve a two-stage process (16). First, acinar cells secrete an isotonic "primary fluid" similar in ionic composition to plasma, and second, the ducts then modify this primary fluid by removing sodium and chloride and adding potassium and bicarbonate to produce a final hypotonic solution that enters the mouth (17), without great changes in the volume of secretion.
The secretion of saliva is controlled by the autonomic nervous system. The parasympathetic nervous system exerts its function through the activation of muscarinic receptors via impulses in the chorda tympani nerve that releases acetylcholine. Salivation depends on the contraction of myoepithelial cells embracing the acini and IDs (18). The sympathetic nervous system controls salivary secretion by releasing norepinephrine (NE) that acts on α- and β-adrenergic receptors (15). Agonist-stimulated acinar secretion is initiated by the opening of K+ and Cl− channels located in the basolateral and apical membranes, respectively. Luminal accumulation of ions generates a trans-epithelial osmotic gradient that drives the movement of water into the lumen (19). An increase in the intracellular Ca2+ is the primary fluid secretion signal in salivary acinar cells.
It has been demonstrated that cannabinoids modulate autonomic neurotransmission. Activation of CB1 by Δ9-THC and AEA causes hypotension via sympathoinhibitory action in rats (20). These effects are related to presynaptic inhibition of NE exocytosis, as AEA and Δ9-THC abolished NE release from isolated rat atria and vasa deferentia, whereas SR141716A, a selective CB1 antagonist, competitively reversed that effect. Moreover, WIN55212—2 and CP55940, synthetic CB agonists, presynaptically inhibited the sympathetic cardioaccelerator response in the rabbit heart by activating CB1 (21). The same agonist also inhibited the bradycardia evoked by stimulation of the vagus nerve.
In vitro studies on rat SMG slices with 3H-Δ9-THC showed that this drug was transported into and accumulated by salivary tissue (22). Also, it has been demonstrated that Δ9-THC decreased electrically stimulated salivary flow in dogs by a mechanism involving a decrease in acetylcholine release, which results in reduction of blood flow to the SMG (23).
Because cannabinoid effects were shown in salivary glands and it is known that marijuana use decreases saliva secretion, we had 3 study objectives. We aimed (i) to determine the localization of CBs in the SMG, (ii) to investigate whether both CB1 and CB2 are activated by AEA in this gland, and (iii) to determine the effect of AEA on saliva secretion using a novel model of intraglandular injection.
Materials and Methods
Chemicals.
AEA, forskolin (FRSK), and NE-HCl were purchased from Sigma Chemical Co. (St. Louis, MO). Chloralose and methacholine (MC) were obtained from FLUKA (Laborchemikalien, Berlin, Germany). AM251 [N-(piperidin-1-yl)-1-(2,4-dichlorophenyl)-5-(4-chlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] and AM630 6-Iodo-2-methyl-1-[2-(4-morpholinyl)ethyl-1H-indol-3-yl](4-methoxyphenyl)methanone were obtained from Tocris (Ellisville, MO).
Animals.
Adult male Wistar rats (250—300 g) from our own colony were kept in group cages in an animal room having a photoperiod of 12 hrs of light (0700—1900 hrs), room temperature at 22°C—25°C, and free access to rat chow and tap water. The animals were divided into several experimental groups with six to eight animals each and were kept in the laboratory area. The experimental procedures reported herein were approved by the Animal Care Committee of the Center of Experimental Pharmacology and Botanicals of the National Council for Research of Argentina and were carried out in accord with the Declaration of Helsinki.
Immunocytochemical Studies.
The rat SMGs were fixed by intracardial perfusion using 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The SMGs were post-fixed for 1 hr and were cryoprotected using graded sucrose solutions (24). Tissue blocks were frozen in N2-cooled acetone and were sectioned at 16 μm. Primary antibodies against CB1 (Cayman Chemical, Ann Arbor, MI) and CB2 (Affinity Bioreagents Antibodies, Golden, CO) were used. After overnight incubation in primary antibody, sections were developed using biotinylated secondary antibodies followed by avidin-biotin-peroxidase complex (Vectastain Elite ABC, Vector Laboratories, Burlingame, CA). A color reaction was obtained using nickel-enhanced diaminobenzidine staining (25). In other sections, bound primary antibodies were labeled with fluorescein-5-isothio-cyanate-conjugated goat anti-rabbit IgG and/or goat lissamine rhodamine-conjugated anti-mouse IgG (Jackson Immunoresearch Laboratories, West Grove, PA). Glycerol-mounted specimens were observed with a Laser Scanning System Radiance 2000 (BioRad, Hemel Hempstead, UK) using the 488 line of the argon laser and followed by the 543 line of a helium-neon laser, and the emission filters HQ515/30 and HQ590/70, respectively. Optical sections separated by 1 μm were performed in the z axis and were processed using Confocal Assistant Software (BioRad).
In Vitro Studies.
The animals were killed by cervical dislocation, and the SMGs were removed. The SMGs were cut in half to enhance the penetration of the different substances into the tissue and were preincubated in 500 μl of Krebs-Ringer bicarbonate buffer medium (pH 7.4) containing 0.1% glucose within a Dubnoff metabolic shaker (50 cycles/min, 95% O2/5% CO2) for 15 mins before replacement with fresh medium containing the compounds to be tested. The incubation was continued for 30 mins with FRSK (7.6 × 10−5 M), an AC activator, or FRSK plus different doses of AEA (1 × 10−10 M to 1 × 10−7 M). AEA was first diluted in ethyl alcohol (1 × 10−2 M), and further dilutions were made in Krebs-Ringer bicarbonate. FRSK was added to the buffer to increase cAMP content because AEA showed no effect on cAMP basal levels.
In other experiments, the SMGs were preincubated for 15 mins in 500 μl of Krebs-Ringer bicarbonate buffer medium alone, with AM630 (6 × 10−5 M), a specific antagonist of CB2, or with AM251 (6 × 10−5 M), a specific antagonist of CB1. AM251 and AM630 were dissolved in dimethyl sulfoxide (2 × 10−3 M), and further dilutions were made in saline to reach 6 × 10−5 M of AM251 and AM630 before sonication. The incubation was continued for 30 mins with Krebs-Ringer (control), FRSK, FRSK+AEA (1 × 10−9 M), FRSK+AEA+AM630 (6 × 10−5 M), AM630 alone, FRSK+AEA+AM251 (6 × 10−5 M), or AM251 alone.
After that, the SMGs were homogenized in 1 ml of H2O and centrifuged at 6000 g for 10 mins at 4°C. Supernatants were collected, and samples were stored at −20°C before assays. cAMP was measured by radioimmunoassay (RIA).
In Vivo Studies.
Salivary Secretion Studies.
Salivary responses were determined in anesthetized rats (100 mg/kg of chloralose, 0.5 ml of 0.9% NaCl, iv). The ducts of the SMGs were cannulated, and the different sialogogues (NE, NA, and MC) were sequentially injected via the right femoral vein, as previously described (26). No resting (unstimulated) flow of saliva was observed. The secretion induced by NE (1, 3, 10, and 30 mg/kg in saline) and MC (1, 3 and 10 mg/kg in saline) during 3 mins after the administration of each dose was colleted on aluminum foil and weighed. Results were expressed as milligrams of saliva per gland.
Effect of AEA and CB Antagonists on Salivary Secretion.
To evaluate the participation of the endocannabinoid system on the salivary secretion, intraglandular injections (50 μl) of agonists and/or antagonists were made 15 mins before the measurement of salivary secretion with increasing doses of NE or MC in different animals (4—5 per group). The experimental groups were: 1) saline; 2) AEA (6 × 10−5 M); 3) AEA+AM630 (6 × 10−4 M), a specific antagonist of CB2; 4) AM630 alone; 5) AEA+AM251 (6 × 10−4 M), a specific antagonist of CB1; 6) AM251 alone; and 7) AM630+AM251.
Radioimmunoassays.
cAMP was measured by RIA by using the highly specific cAMP antibody kindly provided by Dr. A. F. Parlow (National Hormone & Peptide Program, Harbor-UCLA Medical Center, Torrance, CA). The sensitivity of the assay was 0.061 pmol/ml. Intraassay and interassay coefficients of variation were 8.1% and 10.5%.
Statistical Analysis.
Data are presented as the mean ± SEM. For in vitro studies, comparisons between groups were performed using a one-way analysis of variance, followed by the Student-Newman-Keuls multiple comparisons test for unequal replicates. For in vivo studies, when two groups were compared, Student's t test was used. All analyses were conducted using the GraphPad Instat software (GraphPad Software, Inc., San Diego, CA). Differences with P < 0.05 were considered statistically significant.
Results
Immunohistochemical Localization of CBs.
In SMG cryosections labeled with the immunoenzymatic procedure, acini and different kinds of ducts were identified by the characteristic structure in cross-sectional and longitudinal sections. As shown in Figure 1⇓, CB1 immunoreactivity could not be detected in acini, but it was strongly present in the ductal system. CB1 antiserum homogeneously labeled ID cells, but it showed a distinct basal distribution in GCTs. By contrast, SD cells exhibited moderate immuno-labeling of their cytoplasm and a very strong staining of their apical border. A different distribution was found for CB2 immunoreactivity (Fig. 2⇓). It was present in the periphery of acinar cells but could not be detected in IDs. GCTs exhibited basal CB2 immunoreactivity in most cells but contained a distinct subpopulation with staining of the whole cytoplasm. Distribution of CB2 immunoreactivity in SDs was almost identical to that of CB1 immunoreactivity, with very strong labeling of the apical border and less intense labeling of the cytoplasm. Double immunofluorescent studies combining CB2 and smooth muscle actin (SMA) labeling demonstrated that peripheral immunostaining of acinar cells reflected the expression of CB2 in the basal cytoplasm of acinar cells and in surrounding myoepithelial cells.
Inhibitory Effects of AEA in Vitro on cAMP Content.
Effect of Different Concentrations of AEA on FRSK-Stimulated cAMP Content.
The cAMP content was increased (P < 0.01) by FRSK (7.6 × 10−5 M), an activator of AC. This stimulatory effect was significantly blocked (P < 0.01, P < 0.01, and P < .001) by AEA (10−7 M, 10−8 M, and 10−9 M, respectively), with the highest inhibitory effect occurring at 10−9 M (Fig. 3⇓). However, 10−10 M AEA had no effect on FRSK-stimulated cAMP content.
Effect of AEA Plus AM630 or AM251 on FRSK-Stimulated cAMP Content.
To investigate whether the inhibition of AEA on cAMP content was mediated by CB1 or CB2, the specific antagonists of CB1 and CB2, AM251 (10−5 M) and AM630 (10−5 M), respectively, were added to the media concomitantly with AEA (10−9 M)+FRSK (7.6 × 10−5 M) or alone. The concentration of the antagonists used was 10−5 M because lower concentrations were not capable of blocking the inhibitory effect of AEA. AM251 partially blocked (P < 0.01) the inhibition of AEA on FRSK-stimulated cAMP content (Fig. 4⇓). Similarly, AM630 blocked (P < 0.05), but not totally, the inhibition of AEA on FRSK-stimulated cAMP content. Neither AM251 nor AM630 alone had any effect on cAMP content.
Inhibitory Effects of AEA in Vivo on Salivary Secretion.
Effect of AEA on MC-Stimulated Salivary Secretion.
The intra-SMG injection of AEA (6 × 10−5 M) inhibited the saliva secretion stimulated by three different doses of MC (Fig. 5⇓). At a dose of 3 μg/kg of MC, AEA reduced salivation 2.5-fold (P < 0.01). At a dose of 10 μg/kg of MC, AEA reduced salivation 2-fold (P < 0.01). The inhibitory effect of AEA on MC-induced saliva secretion was prevented by injecting AM251 (6 × 10−4 M) or AM630 (6 × 10−4 M) at three MC doses. The injection of AM251 alone had a stimulatory effect (P < 0.05) on the saliva secretion induced by the lowest MC dose (1 μg/kg), whereas AM630 alone did not modify MC-induced salivation. However, simultaneous injection of both antagonists increased the salivation (P < 0.01) induced by the lowest MC dose over the volume obtained with AM251 injected alone. The injection of saline had no significant effect on any dose (data not shown). These results are also given in more detail in Table 1⇓.
Effect of AEA on NE-Stimulated Salivary Secretion.
At the dose of 1 μg/kg of NE, AEA reduced salivation 9-fold (P < 0.01) in comparison to the control curve (Fig. 6⇓). At a dose of 3 μg/kg of NE, AEA reduced salivation 4-fold (P < 0.001). At a dose of 10 μg/kg of NE, AEA reduced salivation 2.5-fold (P < 0.01). At a dose of 30 μg/kg of NE, AEA reduced salivation 2-fold (P < 0.001). The inhibitory effect of AEA on saliva secretion was prevented by injecting AM251 (6 × 10−4 M) or AM630 (6 × 10−4 M) at all the doses observed. The injection of AM251 alone had a stimulatory effect (P < 0.05) on the saliva secretion induced by the lowest dose of NE utilized (1 μg/kg), which was synergized (P < 0.01) after the coinjection with AM630. The injection of AM630 alone as well as saline (data not shown) had no significant effect at any dose. These results are also given in more detail in Table 2⇓.
Discussion
The present work shows for the first time, to our knowledge, the presence of CBs in the SMG. Both CB1 and CB2 were immunohistochemically detected in acinar and ductal components of the gland, and evidence of their role in saliva secretion was obtained.
CB1 was mainly localized in the ductal system, from IDs to SDs. CB2 immunoreactivity was also detected in the acini, cellular structures that are responsible for the production and release of the primary saliva, and in myoepithelial cells, cells that are located close to the acini and whose contraction facilitates the release of the acinar secretion. The acini have rounded nuclei, typical of serous acini and basal cytoplasmic basophilia. Two different morphologic classes of acini as described in humans are not observed in the rat. CB2 immunoreactivity was not detected in IDs, but it was present in more distal ducts in the same localizations as CB1. Therefore, CBs could be associated with the two fundamental mechanisms of salivary secretion: acinar release of protein and fluid and ductal modification of primary secretion.
Immunohistochemical studies have shown the presence of CB1 immunoreactivity in the epithelia of the prostate, where it also negatively regulates AC activity (27). CB1 immunoreactivity has also been demonstrated in follicular and parafollicular cells of the thyroid, where they can decrease the release of triiodothyronine and thyroxine (28). Although in the different organs along the gastrointestinal tract, CB1 has been mainly found on neurons and nerve fibers (29, 30), no evidence of the colocalization of CBs with neuronal markers was found in the SMG.
There are two major signal transduction pathways implicated in salivary gland cells. One involves the generation of cAMP, and the other involves the breakdown of plasma membrane polyphosphoinositides (31). It is known that cAMP mediates the mobilization of intracellular Ca+2 by β-adrenergic stimulation in acinar cells as well as GCTs (32). Moreover, an increase in the intracellular level of cAMP stimulates some salivary functions, such as salivary flow rate and secretion of proteins (33).
Taking into account the importance of AC activity for salivary function, and because the mechanism of action of cannabinoids acting on its receptors is by inhibition of AC with consequent decrease in cAMP (5), we measured the effect of AEA on FRSK-induced cAMP levels. This endocannabinoid markedly reduced FRSK-induced increase of cAMP, and this effect was blocked by the CB1 and CB2 antagonists, indicating that both receptors are implicated in SMG physiology. The inhibitory effect of AEA on cAMP accumulation was not blocked completely by incubating the selective antagonists separately, constituting additional evidence for the presence and function of both CB1 and CB2 in the SMG.
The cAMP response element (CREB) located at the 5′-promoter region of cAMP-regulated genes is phosphorylated by cAMP protein kinase inducing their transcription (34). In the SMG, CREB elements are concentrated in GCTs (35), where we detected the highest immunoreactivity for CB1 and CB2. This localization suggests that CBs could modulate hormonal effects on these specialized ducts.
Salivation is controlled by the autonomic nervous system not only by direct activation of salivary gland receptors but also by activation of brain mechanisms (36—39). Moreover, it has been reported that atropine, a muscarinic antagonist, injected intracerebroventricularly reduced the salivation induced by pilocarpine, a muscarinic agonist, injected ip, suggesting that pilocarpine enters the brain and acts on central muscarinic receptors, activating autonomic efferents to induce salivation (37). In addition, it was shown that the injection intracerebroventricularly of isoproterenol, a β-adrenoreceptor agonist, produced an increased salivary flow, demonstrating the participation of a central adrenergic system in the regulation of the salivary flow. Therefore, it is possible that the sialogogues injected through the femoral vein acted not only on their receptors in the SMG but also on their receptors at the brain level, stimulating efferent responses in the periphery that could be decreased by endocannabinoids. Therefore, the AEA injected intraglandularly, acting mainly through CB1, could inhibit the release of neurotransmitters from presynaptic terminals on the SMG, as it has been demonstrated that the endocannabinoid system decreases autonomic neurotransmission. Moreover, it was shown that THC decreased salivary flow from the SMG during electrical stimulation by a mechanism involving a decrease in the release of acetylcholine (23). Also, evidence exists that AEA and THC abolished NE release in other systems by presynaptic inhibition (20). Although CB immunoreactivities have been detected in the enteric innervation (40), we were not able to detect them in SMG nerves. Our results showed that AEA injected intraglandularly inhibited NE- and MC-stimulated saliva secretion in vivo and that both AM251 and AM630 prevented that inhibition. These results provided evidence that both CB1 and CB2 are implicated in the modulation of saliva secretion. The CB2 mainly located peripherally to the acini could regulate the release of saliva from these cells to the salivary ducts. The function of CB1 in the SMG is more difficult to explain, as these receptors were not immunodetected in the vicinity of the acini. However, the immunodetection of CB1 at the ductal system suggests a paracrine effect from the duct cells to the acinar cells to reduce the volume of saliva.
In addition, CB1 and CB2 antagonists increased the induced salivation at lower doses of sialogogues. These findings indicate that there is an endogenous endocannabinoid tone that could regulate salivary secretion.
In summary, the inhibitory effects of AEA on saliva secretion could be exerted at different levels 1) as a relaxant of myoepithelial cells or 2) within the cytoplasm of the secretory cells, blocking the intracellular signaling pathways activated by NE or MC. 3) Finally, because intravenous sialogogues could also induce some central activation of salivation, AEA may also control the release of cholinergic, adrenergic, or peptidergic neurotransmitters from the presynaptic terminals to the salivary glands.
Source, Graphs and Figures: Inhibition of Salivary Secretion by Activation of Cannabinoid Receptors
It is known that marijuana use decreases saliva secretion. Therefore, we hypothesized that cannabinoid receptors (CBs) are located in salivary glands to mediate that effect. In these experiments, we used the submandibular gland (SMG) of male rats, which is one of the major salivary glands. Mammalian tissues contain at least two types of CBs, CB1 and CB2, mainly located in the nervous system and peripheral tissues, respectively. Both receptors are coupled to Gi protein and respond by inhibiting the activity of adenylyl cyclase. We demonstrated that both CB1 and CB2 are present in the SMG, each showing specific localizations. The best-known endocannabinoid is anandamide (AEA), which binds with high affinity to CB1 and CB2. We showed that AEA markedly reduced forskolin-induced increase of cAMP content in vitro. This effect was blocked by AM251 and AM630 (CB1 and CB2 antagonists, respectively), indicating that both receptors are implicated in SMG physiology. In addition, we showed that AEA injected intraglandularly to anesthetized rats inhibited norepinephrine (NE)— and methacholine (MC)—stimulated saliva secretion in vivo and that both AM251 or AM630 prevented the inhibitory action of AEA. Also, the intraglandular injection of AM251 increased saliva secretion induced by lower doses of NE or MC. This increase was synergized after coinjection with AM630. Therefore, we concluded that AEA decreases saliva secretion in the SMG acting through CB1 and CB2 receptors.
Introduction
It is well known that Δ9-tetrahydrocannabinol (Δ9-THC), the psychoactive principle isolated from Cannabis sativa, alters different physiologic parameters in biologic systems. Mammalian tissues contain at least two types of cannabinoid receptors (CBs), CB1 and CB2, mainly located in the nervous system (1—3) and in peripheral tissues or immune cells (4), respectively. Both receptors are coupled to Gi protein and respond by inhibiting the activity of adenylyl cyclase (AC) (5). The best-known endocannabinoids are arachidonoyl ethanolamide (anandamide [AEA]) and arachidonoyl glycerol, both derivatives of arachidonic acid that bind with high affinity to CBs (6). Specific and selective antagonists for CBs have been developed, such as AM251 (7), AM281 (8), SR141716A (9, 10), and LY320135 (11) for CB1 and AM630 (12) and SR144528 (13) for CB2. For our experiments, we used AM251 because this is a potent selective CB1 antagonist that has a Ki of 7.49 nM for CB1 and is 306-fold selective over CB2. We also used AM630, a CB2 antagonist/inverse agonist (Ki, 31.2 nM), which is 165-fold selective over CB1 and behaves as a weak partial/inverse agonist at CB1 (14).
The submandibular gland (SMG) is one of the major salivary glands, together with the sublingual and parotid glands. End secretory units, called acini, are continuous with a duct system that in rodents has four sequential segments: intercalated ducts (IDs), granular convoluted tubules (GCTs), striated ducts (SDs), and excretory ducts that release the saliva to the oral cavity (15).
Saliva formation is thought to involve a two-stage process (16). First, acinar cells secrete an isotonic "primary fluid" similar in ionic composition to plasma, and second, the ducts then modify this primary fluid by removing sodium and chloride and adding potassium and bicarbonate to produce a final hypotonic solution that enters the mouth (17), without great changes in the volume of secretion.
The secretion of saliva is controlled by the autonomic nervous system. The parasympathetic nervous system exerts its function through the activation of muscarinic receptors via impulses in the chorda tympani nerve that releases acetylcholine. Salivation depends on the contraction of myoepithelial cells embracing the acini and IDs (18). The sympathetic nervous system controls salivary secretion by releasing norepinephrine (NE) that acts on α- and β-adrenergic receptors (15). Agonist-stimulated acinar secretion is initiated by the opening of K+ and Cl− channels located in the basolateral and apical membranes, respectively. Luminal accumulation of ions generates a trans-epithelial osmotic gradient that drives the movement of water into the lumen (19). An increase in the intracellular Ca2+ is the primary fluid secretion signal in salivary acinar cells.
It has been demonstrated that cannabinoids modulate autonomic neurotransmission. Activation of CB1 by Δ9-THC and AEA causes hypotension via sympathoinhibitory action in rats (20). These effects are related to presynaptic inhibition of NE exocytosis, as AEA and Δ9-THC abolished NE release from isolated rat atria and vasa deferentia, whereas SR141716A, a selective CB1 antagonist, competitively reversed that effect. Moreover, WIN55212—2 and CP55940, synthetic CB agonists, presynaptically inhibited the sympathetic cardioaccelerator response in the rabbit heart by activating CB1 (21). The same agonist also inhibited the bradycardia evoked by stimulation of the vagus nerve.
In vitro studies on rat SMG slices with 3H-Δ9-THC showed that this drug was transported into and accumulated by salivary tissue (22). Also, it has been demonstrated that Δ9-THC decreased electrically stimulated salivary flow in dogs by a mechanism involving a decrease in acetylcholine release, which results in reduction of blood flow to the SMG (23).
Because cannabinoid effects were shown in salivary glands and it is known that marijuana use decreases saliva secretion, we had 3 study objectives. We aimed (i) to determine the localization of CBs in the SMG, (ii) to investigate whether both CB1 and CB2 are activated by AEA in this gland, and (iii) to determine the effect of AEA on saliva secretion using a novel model of intraglandular injection.
Materials and Methods
Chemicals.
AEA, forskolin (FRSK), and NE-HCl were purchased from Sigma Chemical Co. (St. Louis, MO). Chloralose and methacholine (MC) were obtained from FLUKA (Laborchemikalien, Berlin, Germany). AM251 [N-(piperidin-1-yl)-1-(2,4-dichlorophenyl)-5-(4-chlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] and AM630 6-Iodo-2-methyl-1-[2-(4-morpholinyl)ethyl-1H-indol-3-yl](4-methoxyphenyl)methanone were obtained from Tocris (Ellisville, MO).
Animals.
Adult male Wistar rats (250—300 g) from our own colony were kept in group cages in an animal room having a photoperiod of 12 hrs of light (0700—1900 hrs), room temperature at 22°C—25°C, and free access to rat chow and tap water. The animals were divided into several experimental groups with six to eight animals each and were kept in the laboratory area. The experimental procedures reported herein were approved by the Animal Care Committee of the Center of Experimental Pharmacology and Botanicals of the National Council for Research of Argentina and were carried out in accord with the Declaration of Helsinki.
Immunocytochemical Studies.
The rat SMGs were fixed by intracardial perfusion using 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The SMGs were post-fixed for 1 hr and were cryoprotected using graded sucrose solutions (24). Tissue blocks were frozen in N2-cooled acetone and were sectioned at 16 μm. Primary antibodies against CB1 (Cayman Chemical, Ann Arbor, MI) and CB2 (Affinity Bioreagents Antibodies, Golden, CO) were used. After overnight incubation in primary antibody, sections were developed using biotinylated secondary antibodies followed by avidin-biotin-peroxidase complex (Vectastain Elite ABC, Vector Laboratories, Burlingame, CA). A color reaction was obtained using nickel-enhanced diaminobenzidine staining (25). In other sections, bound primary antibodies were labeled with fluorescein-5-isothio-cyanate-conjugated goat anti-rabbit IgG and/or goat lissamine rhodamine-conjugated anti-mouse IgG (Jackson Immunoresearch Laboratories, West Grove, PA). Glycerol-mounted specimens were observed with a Laser Scanning System Radiance 2000 (BioRad, Hemel Hempstead, UK) using the 488 line of the argon laser and followed by the 543 line of a helium-neon laser, and the emission filters HQ515/30 and HQ590/70, respectively. Optical sections separated by 1 μm were performed in the z axis and were processed using Confocal Assistant Software (BioRad).
In Vitro Studies.
The animals were killed by cervical dislocation, and the SMGs were removed. The SMGs were cut in half to enhance the penetration of the different substances into the tissue and were preincubated in 500 μl of Krebs-Ringer bicarbonate buffer medium (pH 7.4) containing 0.1% glucose within a Dubnoff metabolic shaker (50 cycles/min, 95% O2/5% CO2) for 15 mins before replacement with fresh medium containing the compounds to be tested. The incubation was continued for 30 mins with FRSK (7.6 × 10−5 M), an AC activator, or FRSK plus different doses of AEA (1 × 10−10 M to 1 × 10−7 M). AEA was first diluted in ethyl alcohol (1 × 10−2 M), and further dilutions were made in Krebs-Ringer bicarbonate. FRSK was added to the buffer to increase cAMP content because AEA showed no effect on cAMP basal levels.
In other experiments, the SMGs were preincubated for 15 mins in 500 μl of Krebs-Ringer bicarbonate buffer medium alone, with AM630 (6 × 10−5 M), a specific antagonist of CB2, or with AM251 (6 × 10−5 M), a specific antagonist of CB1. AM251 and AM630 were dissolved in dimethyl sulfoxide (2 × 10−3 M), and further dilutions were made in saline to reach 6 × 10−5 M of AM251 and AM630 before sonication. The incubation was continued for 30 mins with Krebs-Ringer (control), FRSK, FRSK+AEA (1 × 10−9 M), FRSK+AEA+AM630 (6 × 10−5 M), AM630 alone, FRSK+AEA+AM251 (6 × 10−5 M), or AM251 alone.
After that, the SMGs were homogenized in 1 ml of H2O and centrifuged at 6000 g for 10 mins at 4°C. Supernatants were collected, and samples were stored at −20°C before assays. cAMP was measured by radioimmunoassay (RIA).
In Vivo Studies.
Salivary Secretion Studies.
Salivary responses were determined in anesthetized rats (100 mg/kg of chloralose, 0.5 ml of 0.9% NaCl, iv). The ducts of the SMGs were cannulated, and the different sialogogues (NE, NA, and MC) were sequentially injected via the right femoral vein, as previously described (26). No resting (unstimulated) flow of saliva was observed. The secretion induced by NE (1, 3, 10, and 30 mg/kg in saline) and MC (1, 3 and 10 mg/kg in saline) during 3 mins after the administration of each dose was colleted on aluminum foil and weighed. Results were expressed as milligrams of saliva per gland.
Effect of AEA and CB Antagonists on Salivary Secretion.
To evaluate the participation of the endocannabinoid system on the salivary secretion, intraglandular injections (50 μl) of agonists and/or antagonists were made 15 mins before the measurement of salivary secretion with increasing doses of NE or MC in different animals (4—5 per group). The experimental groups were: 1) saline; 2) AEA (6 × 10−5 M); 3) AEA+AM630 (6 × 10−4 M), a specific antagonist of CB2; 4) AM630 alone; 5) AEA+AM251 (6 × 10−4 M), a specific antagonist of CB1; 6) AM251 alone; and 7) AM630+AM251.
Radioimmunoassays.
cAMP was measured by RIA by using the highly specific cAMP antibody kindly provided by Dr. A. F. Parlow (National Hormone & Peptide Program, Harbor-UCLA Medical Center, Torrance, CA). The sensitivity of the assay was 0.061 pmol/ml. Intraassay and interassay coefficients of variation were 8.1% and 10.5%.
Statistical Analysis.
Data are presented as the mean ± SEM. For in vitro studies, comparisons between groups were performed using a one-way analysis of variance, followed by the Student-Newman-Keuls multiple comparisons test for unequal replicates. For in vivo studies, when two groups were compared, Student's t test was used. All analyses were conducted using the GraphPad Instat software (GraphPad Software, Inc., San Diego, CA). Differences with P < 0.05 were considered statistically significant.
Results
Immunohistochemical Localization of CBs.
In SMG cryosections labeled with the immunoenzymatic procedure, acini and different kinds of ducts were identified by the characteristic structure in cross-sectional and longitudinal sections. As shown in Figure 1⇓, CB1 immunoreactivity could not be detected in acini, but it was strongly present in the ductal system. CB1 antiserum homogeneously labeled ID cells, but it showed a distinct basal distribution in GCTs. By contrast, SD cells exhibited moderate immuno-labeling of their cytoplasm and a very strong staining of their apical border. A different distribution was found for CB2 immunoreactivity (Fig. 2⇓). It was present in the periphery of acinar cells but could not be detected in IDs. GCTs exhibited basal CB2 immunoreactivity in most cells but contained a distinct subpopulation with staining of the whole cytoplasm. Distribution of CB2 immunoreactivity in SDs was almost identical to that of CB1 immunoreactivity, with very strong labeling of the apical border and less intense labeling of the cytoplasm. Double immunofluorescent studies combining CB2 and smooth muscle actin (SMA) labeling demonstrated that peripheral immunostaining of acinar cells reflected the expression of CB2 in the basal cytoplasm of acinar cells and in surrounding myoepithelial cells.
Inhibitory Effects of AEA in Vitro on cAMP Content.
Effect of Different Concentrations of AEA on FRSK-Stimulated cAMP Content.
The cAMP content was increased (P < 0.01) by FRSK (7.6 × 10−5 M), an activator of AC. This stimulatory effect was significantly blocked (P < 0.01, P < 0.01, and P < .001) by AEA (10−7 M, 10−8 M, and 10−9 M, respectively), with the highest inhibitory effect occurring at 10−9 M (Fig. 3⇓). However, 10−10 M AEA had no effect on FRSK-stimulated cAMP content.
Effect of AEA Plus AM630 or AM251 on FRSK-Stimulated cAMP Content.
To investigate whether the inhibition of AEA on cAMP content was mediated by CB1 or CB2, the specific antagonists of CB1 and CB2, AM251 (10−5 M) and AM630 (10−5 M), respectively, were added to the media concomitantly with AEA (10−9 M)+FRSK (7.6 × 10−5 M) or alone. The concentration of the antagonists used was 10−5 M because lower concentrations were not capable of blocking the inhibitory effect of AEA. AM251 partially blocked (P < 0.01) the inhibition of AEA on FRSK-stimulated cAMP content (Fig. 4⇓). Similarly, AM630 blocked (P < 0.05), but not totally, the inhibition of AEA on FRSK-stimulated cAMP content. Neither AM251 nor AM630 alone had any effect on cAMP content.
Inhibitory Effects of AEA in Vivo on Salivary Secretion.
Effect of AEA on MC-Stimulated Salivary Secretion.
The intra-SMG injection of AEA (6 × 10−5 M) inhibited the saliva secretion stimulated by three different doses of MC (Fig. 5⇓). At a dose of 3 μg/kg of MC, AEA reduced salivation 2.5-fold (P < 0.01). At a dose of 10 μg/kg of MC, AEA reduced salivation 2-fold (P < 0.01). The inhibitory effect of AEA on MC-induced saliva secretion was prevented by injecting AM251 (6 × 10−4 M) or AM630 (6 × 10−4 M) at three MC doses. The injection of AM251 alone had a stimulatory effect (P < 0.05) on the saliva secretion induced by the lowest MC dose (1 μg/kg), whereas AM630 alone did not modify MC-induced salivation. However, simultaneous injection of both antagonists increased the salivation (P < 0.01) induced by the lowest MC dose over the volume obtained with AM251 injected alone. The injection of saline had no significant effect on any dose (data not shown). These results are also given in more detail in Table 1⇓.
Effect of AEA on NE-Stimulated Salivary Secretion.
At the dose of 1 μg/kg of NE, AEA reduced salivation 9-fold (P < 0.01) in comparison to the control curve (Fig. 6⇓). At a dose of 3 μg/kg of NE, AEA reduced salivation 4-fold (P < 0.001). At a dose of 10 μg/kg of NE, AEA reduced salivation 2.5-fold (P < 0.01). At a dose of 30 μg/kg of NE, AEA reduced salivation 2-fold (P < 0.001). The inhibitory effect of AEA on saliva secretion was prevented by injecting AM251 (6 × 10−4 M) or AM630 (6 × 10−4 M) at all the doses observed. The injection of AM251 alone had a stimulatory effect (P < 0.05) on the saliva secretion induced by the lowest dose of NE utilized (1 μg/kg), which was synergized (P < 0.01) after the coinjection with AM630. The injection of AM630 alone as well as saline (data not shown) had no significant effect at any dose. These results are also given in more detail in Table 2⇓.
Discussion
The present work shows for the first time, to our knowledge, the presence of CBs in the SMG. Both CB1 and CB2 were immunohistochemically detected in acinar and ductal components of the gland, and evidence of their role in saliva secretion was obtained.
CB1 was mainly localized in the ductal system, from IDs to SDs. CB2 immunoreactivity was also detected in the acini, cellular structures that are responsible for the production and release of the primary saliva, and in myoepithelial cells, cells that are located close to the acini and whose contraction facilitates the release of the acinar secretion. The acini have rounded nuclei, typical of serous acini and basal cytoplasmic basophilia. Two different morphologic classes of acini as described in humans are not observed in the rat. CB2 immunoreactivity was not detected in IDs, but it was present in more distal ducts in the same localizations as CB1. Therefore, CBs could be associated with the two fundamental mechanisms of salivary secretion: acinar release of protein and fluid and ductal modification of primary secretion.
Immunohistochemical studies have shown the presence of CB1 immunoreactivity in the epithelia of the prostate, where it also negatively regulates AC activity (27). CB1 immunoreactivity has also been demonstrated in follicular and parafollicular cells of the thyroid, where they can decrease the release of triiodothyronine and thyroxine (28). Although in the different organs along the gastrointestinal tract, CB1 has been mainly found on neurons and nerve fibers (29, 30), no evidence of the colocalization of CBs with neuronal markers was found in the SMG.
There are two major signal transduction pathways implicated in salivary gland cells. One involves the generation of cAMP, and the other involves the breakdown of plasma membrane polyphosphoinositides (31). It is known that cAMP mediates the mobilization of intracellular Ca+2 by β-adrenergic stimulation in acinar cells as well as GCTs (32). Moreover, an increase in the intracellular level of cAMP stimulates some salivary functions, such as salivary flow rate and secretion of proteins (33).
Taking into account the importance of AC activity for salivary function, and because the mechanism of action of cannabinoids acting on its receptors is by inhibition of AC with consequent decrease in cAMP (5), we measured the effect of AEA on FRSK-induced cAMP levels. This endocannabinoid markedly reduced FRSK-induced increase of cAMP, and this effect was blocked by the CB1 and CB2 antagonists, indicating that both receptors are implicated in SMG physiology. The inhibitory effect of AEA on cAMP accumulation was not blocked completely by incubating the selective antagonists separately, constituting additional evidence for the presence and function of both CB1 and CB2 in the SMG.
The cAMP response element (CREB) located at the 5′-promoter region of cAMP-regulated genes is phosphorylated by cAMP protein kinase inducing their transcription (34). In the SMG, CREB elements are concentrated in GCTs (35), where we detected the highest immunoreactivity for CB1 and CB2. This localization suggests that CBs could modulate hormonal effects on these specialized ducts.
Salivation is controlled by the autonomic nervous system not only by direct activation of salivary gland receptors but also by activation of brain mechanisms (36—39). Moreover, it has been reported that atropine, a muscarinic antagonist, injected intracerebroventricularly reduced the salivation induced by pilocarpine, a muscarinic agonist, injected ip, suggesting that pilocarpine enters the brain and acts on central muscarinic receptors, activating autonomic efferents to induce salivation (37). In addition, it was shown that the injection intracerebroventricularly of isoproterenol, a β-adrenoreceptor agonist, produced an increased salivary flow, demonstrating the participation of a central adrenergic system in the regulation of the salivary flow. Therefore, it is possible that the sialogogues injected through the femoral vein acted not only on their receptors in the SMG but also on their receptors at the brain level, stimulating efferent responses in the periphery that could be decreased by endocannabinoids. Therefore, the AEA injected intraglandularly, acting mainly through CB1, could inhibit the release of neurotransmitters from presynaptic terminals on the SMG, as it has been demonstrated that the endocannabinoid system decreases autonomic neurotransmission. Moreover, it was shown that THC decreased salivary flow from the SMG during electrical stimulation by a mechanism involving a decrease in the release of acetylcholine (23). Also, evidence exists that AEA and THC abolished NE release in other systems by presynaptic inhibition (20). Although CB immunoreactivities have been detected in the enteric innervation (40), we were not able to detect them in SMG nerves. Our results showed that AEA injected intraglandularly inhibited NE- and MC-stimulated saliva secretion in vivo and that both AM251 and AM630 prevented that inhibition. These results provided evidence that both CB1 and CB2 are implicated in the modulation of saliva secretion. The CB2 mainly located peripherally to the acini could regulate the release of saliva from these cells to the salivary ducts. The function of CB1 in the SMG is more difficult to explain, as these receptors were not immunodetected in the vicinity of the acini. However, the immunodetection of CB1 at the ductal system suggests a paracrine effect from the duct cells to the acinar cells to reduce the volume of saliva.
In addition, CB1 and CB2 antagonists increased the induced salivation at lower doses of sialogogues. These findings indicate that there is an endogenous endocannabinoid tone that could regulate salivary secretion.
In summary, the inhibitory effects of AEA on saliva secretion could be exerted at different levels 1) as a relaxant of myoepithelial cells or 2) within the cytoplasm of the secretory cells, blocking the intracellular signaling pathways activated by NE or MC. 3) Finally, because intravenous sialogogues could also induce some central activation of salivation, AEA may also control the release of cholinergic, adrenergic, or peptidergic neurotransmitters from the presynaptic terminals to the salivary glands.
Source, Graphs and Figures: Inhibition of Salivary Secretion by Activation of Cannabinoid Receptors
