Parasympathetic denervation increases responses to VIP in isolated rat parotid acini

Vasoactive intestinal peptide (VIP) is a putative neurotransmitter found in the salivary glands of many species, including the rat parotid gland. Parasympathetic denervation has been reported to deplete VIP in the rat parotid gland and to lead to supersensitivity to this peptide in vivo. We have compared the effects of VIP on acini isolated from parasympathetically denervated and unoperated parotid glands to examine possible supersensitivity to the peptide in vitro. VIP normally produced responses similar to those obtained with a low concentration of the beta adrenergic agonist isoproterenol (ISO), but strikingly different from the effects obtained with the muscarinic agonist carbachol (CARB). In parotid membrane preparations, VIP stimulated adenylate cyclase activity. Dissociated acini treated with VIP showed increases in cAMP accumulation and amylase release which were potentiated by forskolin and also by inhibition of phosphodiesterase. After parasympathetic denervation, maximal effects of VIP on adenylate cyclase, cAMP accumulation and amylase release in intact cells were increased two- to five-fold over contralateral control (or unoperated) parotid responses. The increase in adenylate cyclase-mediated responses after denervation was specific to VIP; there was no increased response nor increased sensitivity of any of these responses to ISO. Specific [125I]VIP binding to parotid acini increased two-fold per gland and three-fold per mg of protein after denervation; this probably explains the observed increases in the response to VIP.     

Beta-adrenergic and muscarinic receptors of parotid gland following maintenance of rats on liquid diet

Parotid gland of adult rats maintained exclusively on liquid (milk) diet for 7 or 13 days showed a 25% reduction in number of beta-adrenoceptors, and after 21 days, the reduction was 33%; with maintenance of rats on Metrecal for 7 days, the decrease was 24% for female rats and 22% for male rats. The decrease in number of muscarinic receptors after 7 or 13 days on milk was 32%, and 38% after 21 days; the decreases for rats on Metrecal for 7 days were 32% for females and 35% for males. In rats maintained on liquid (milk) diet for 7 days, and then denervated by unilateral removal of the parasympathetic and sympathetic innervations to parotid there were decreases of 39-42% in number of beta-receptors and 50-52% in muscarinic receptors at 6 or 14 days after denervation (maintenance on liquid diet for 13 and 21 days, respectively) from those of innervated glands of chow-fed rats; denervated glands of rats on chow diet showed the same reduction. Thus, it was concluded that absence of neurally mediated glandular activity, imposed by either diet or surgical removal of the nerves, caused marked decreases in number of both beta-adrenergic and muscarinic receptors, but that the presence of the nerves, even though inactive (liquid diet), provided a trophic influence that prevented the more marked decreases seen in the absence (surgical removal) of the nerves.     

Comparison of effects of surgical and chemical sympathectomy on beta adrenergic and muscarinic receptors of parotid gland of young and adult rats

Surgical (removal of a superior cervical ganglion) or chemical [(administration of a single dose of 6 hydroxydopamine (6 OHDA) (50 mg/kg dose body wt)] sympathectomy of rats at 2 or 8 days of age resulted in an increase in [3H]DHA binding of membranes of parotid gland of young rats (age range 21 days to 48 days). The increase progressed with postnatal age; at 21 days of age (surgical sympathectomy), it was 13%; at 32 days of age with 6 OHDA, it was as much as 34%, but only 26% at 42 days of age with surgical sympathectomy. No change in [3H]QNB binding was observed at any postnatal ages following neonatal sympathectomy. Conversely, surgical sympathectomy of the parotid of adult rat resulted in little or no change in [3H]DHA binding at 1, 2, 3, or 4 weeks postdenervation, but [3H]QNB binding was reduced at all periods, with the reduction from control values at 2 weeks being 34%, and at the subsequent intervals, 24-26%. The increase in number of beta adrenoceptors of the parotid gland was not related to the kind of sympathectomy (chemical or surgical) or neonatal age at which it was done; however, duration of the denervation for 2-3 weeks was necessary for the receptor increase to occur. In the adult, however, the duration of the denervation was of no importance since change in number of beta adrenoceptors did not occur at 1, 2, 3, or 4 weeks after surgical denervation but did occur after only 1 week after of reserpine-induced denervation. QNB binding was decreased with surgical sympathectomy as well as reserpine-induced sympathectomy of adult parotid gland; norepinephrine concentration was decreased to levels of a few percent of innervated glands. The relation between development of glandular supersensitivity and increase in beta adrenoceptors is discussed.     

Vasoactive intestinal peptide and substance P in salivary glands of the rat following denervation or duct ligation

Immunoreactive vasoactive intestinal peptide (VIP) and substance P (SP) were studied in parotid, submaxillary and sublingual glands of the rat. The concentration of VIP was highest in the submaxillary gland and lowest in the parotid gland. The concentration of SP was highest in the parotid gland; it was at, or below the limit of detection in the sublingual gland. In the parotid gland the total amounts of VIP and SP were reduced by 95% after parasympathetic denervation (section of the auriculo-temporal nerve). In the submaxillary gland the total amounts of the peptides were unchanged after parasympathetic decentralization (section of the chorda-lingual nerve). In this gland the total amount of SP was reduced by 92% and that of VIP by 50%, when the chorda tympani nerve fibres were cut deep into the hilum. Cutting the nerve fibres at the hilum left the total amounts of the peptides unchanged in the submaxillary gland, whereas in the sublingual gland the total amount of VIP was reduced by 70%. Sympathetic denervation did not reduce the total amounts of the peptides. Duct ligation caused gland atrophy. In the parotid gland the total amounts of VIP and SP were reduced by 40%. In the submaxillary gland the same percentage reduction occurred with regard to SP; however, the total amount of VIP was reduced by 99%. The VIP- and SP-containing nerve fibres reach the salivary glands by the parasympathetic nerves. In both submaxillary and sublingual glands a certain fraction of VIP originates within the glands.     

Secretory and structural changes in the parotid salivary gland of sheep and lambs after parasympathetic denervation.

The effect of the parasympathetic nerve supply on the development of the parotid gland in the immature lamb and its maintenance in the adult sheep has been investigated by unilateral postganglionic denervation. Seventy-seven to ninety-three days after denervation secretory activity of the gland was examined and material taken for histological examination. The adult denervated glands secreted at lower rates than the innervated and their atropine-resistant secretory flow was reduced to as low as one fifth of that of the innervated glands. In two lambs an atropine-resistant flow did not develop in the denervated glands: in another two, flows of saliva from the denervated glands were present but were much less than in the contralateral innervated glands. After denervation glands were, with one exception, smaller than the contralateral innervated glands. The acinar cells of the denervated adult and lamb glands were smaller than the cells of the innervated glands but similar in size to those of 7-14 day old unoperated control lambs. Acinar cells in denervated glands had periodic acid Schiff staining material but the staining reaction to pyronin-methyl green was similar in the innervated and denervated. The results indicate that the integrity of the parasympathetic innervation is essential for the development of the parotid gland of the sheep and for its maintenance in the adult animal.     

Development of muscarinic receptors and regulation of secretory responsiveness in rodent sweat glands

Sweat glands are innervated by sympathetic neurons which undergo a change in transmitter phenotype from noradrenergic to cholinergic during development. As soon as the glands begin to differentiate, M3 muscarinic receptor mRNA and binding sites are detectable. Receptor expression appears in the absence of innervation and is maintained after denervation. While receptor expression is not regulated by innervation, secretory responsiveness is. Muscarinic blockade during development or in adult animals results in the loss of responsiveness and its reappearance requires several days. Cholinergic muscarinic activation is most likely to regulate one or more steps in the signalling cascade that are downstream of calcium mobilization. The anterograde regulation of sweat gland responsiveness is one facet of the reciprocal interactions are required to establish a functional synapse in this system.     

Effects of chronic clonidine administration on parasympathetic-evoked rat saliva.

Effects of chronic administration of clonidine on parasympathetic-evoked saliva from both parotid and submandibular glands were investigated. Clonidine at 1 mg/kg/day for 5 or 7 days caused a significant reduction in the salivary secretion (flow rate and total volume) evoked by parasympathetic nerve stimulation of parotid but not submandibular glands. Ion concentrations (Na, K and Ca) of parasympathetically nerve-evoked parotid saliva were not altered. However, the total protein concentration as well as output, amylase activity, and output of such saliva were markedly increased. Possible mechanisms for clonidine-induced increase in nerve-elicited salivary protein concentration include release of neuropeptides, and changes in adrenergic receptor binding which need further study.     

Changes in striatal specific 3-H-atropine binding after unilateral 6-hydroxydopamine lesions of nigrostriatal dopaminergic neurones.

Specific binding of ³H-atropine to crude synaptosomal membrane fractions of the rat striatum was measured at different times after unilateral 6-hydroxydopamine lesions of the nigrostriatal dopaminergic neurones. In a group of rats killed between 4 to 15 days after lesioning the right side, specific ³H-atropine binding was reduced by 20 percent compared to the right side of unlesioned rats. There was a concomitant increase (20 percent) of specific ³H-atropine binding in the contralateral side compared to control animals. These changes in muscarinic receptor binding depended on the time after which the lesions were made : maximum effects occured about 8 days after lesioning but almost completely disappeared 13 days later. Dissociation constants for ³H-atropine in the right and left striata of control and lesioned rats were not significantly different. The decrease in muscarinic receptor binding in the ipsilateral striatum of lesioned animals may result from an activation of cholinergic neurones produced by removal of the inhibitory dopaminergic terminals.     

Cl(-)/HCO(3)(-) exchange is acetazolamide sensitive and activated by a muscarinic receptor-induced [Ca(2+)](i) increase in salivary acinar cells

Large volumes of saliva are generated by transepithelial Cl(-) movement during parasympathetic muscarinic receptor stimulation. To gain further insight into a major Cl(-) uptake mechanism involved in this process, we have characterized the anion exchanger (AE) activity in mouse serous parotid and mucous sublingual salivary gland acinar cells. The AE activity in acinar cells was Na(+) independent, electroneutral, and sensitive to the anion exchange inhibitor DIDS, properties consistent with the AE members of the SLC4A gene family. Localization studies using a specific antibody to the ubiquitously expressed AE2 isoform labeled acini in both parotid and sublingual glands. Western blot analysis detected an approximately 170-kDa protein that was more highly expressed in the plasma membranes of sublingual than in parotid glands. Correspondingly, the DIDS-sensitive Cl(-)/HCO(3)(-) exchanger activity was significantly greater in sublingual acinar cells. The carbonic anhydrase antagonist acetazolamide markedly inhibited, whereas muscarinic receptor stimulation enhanced, the Cl(-)/HCO(3)(-) exchanger activity in acinar cells from both glands. Intracellular Ca(2+) chelation prevented muscarinic receptor-induced upregulation of the AE, whereas raising the intracellular Ca(2+) concentration with the Ca(2+)-ATPase inhibitor thapsigargin mimicked the effects of muscarinic receptor stimulation. In summary, carbonic anhydrase activity was essential for regulating Cl(-)/HCO(3)(-) exchange in salivary gland acinar cells. Moreover, muscarinic receptor stimulation enhanced AE activity through a Ca(2+)-dependent mechanism. Such forms of regulation may play important roles in modulating fluid and electrolyte secretion by salivary gland acinar cells.     

Nitric oxide synthase immunoreactive nerves in rat and ferret salivary glands, and effects of denervation

Nitric oxide has been implicated in mechanisms mediating nerve-evoked vasodilatory and secretory responses in salivary glands. In the present study, the occurrence and distribution of nitric oxide synthase (NOS)-immunoreactive nerves in ferret and rat salivary glands were investigated using immunocytochemistry with rabbit and sheep NOS antisera, and using NADPH-diaphorase enzyme histochemistry. In the parotid, submandibular and sublingual glands of the rat and the ferret, NOS-immunoreactive varicose terminals encircled acini and arteries of various sizes. In the ferret, collecting ducts were also supplied with NOS-immunoreactive fibres. In the rat, only the granular ducts of the submandibular gland were supplied with such fibres. The NOS-immunoreactive innervation of acinar cells was more abundant in the rat than in the ferret, whereas the opposite was true for the innervation of blood vessels. No NOS immunoreactivity was observed in the vascular endothelium. In both species, NOS-positive ganglionic cell bodies were found in the hilar regions of the submandibular and sublingual glands, whereas none could be detected in the parotid glands. NADPH-diaphorase reactivity had the same neuronal distribution as NOS immunoreactivity and, in addition, NADPH-diaphorase reactivity was expressed in ductal epithelium. Neither sympathetic denervation (by removal of the superior cervical ganglion) nor treatment with the sensory neurotoxin capsaicin reduced the NOS-immunoreactive innervation of the parotid gland. However, parasympathetic denervation (by cutting the auriculo-temporal nerve) caused an almost total disappearance of the NOS-immunoreactive innervation. The present findings provide a morphological background to the suggested role of nitric oxide in parasympathetic secretory and vascular responses of salivary glands. This revised version was published online in November 2006 with corrections to the Cover Date.     

Parasympathetic degeneration secretion of saliva in rats.

In rats under chloralose anaethesia saliva was found to flow from the submandibular and parotid glands previously subjected to (partial) postganglionic parasympathetic denervation. Secretion started in the submandibular glands 8.8-11.8 hours, and in the parotid glands 14.0-12.6 hours after the denervation and lasted about 7 hours in both glands. It was not abolished by sympatholytic drugs but by atropine. It is regarded as an example of the "degeneration activity" described in many organs and species and provides a method for prolonged stimulation of salivary glands in rats.     

Sympathetic and parasympathetic regulation of cystatin S gene expression

The autonomic nervous system plays a regulatory role in the differentiation and growth of salivary glands, and in the expression of salivary specific genes. Cystatin S, a member of the evolutionarily conserved family 2 of the cysteine proteinase inhibitor superfamily, expressed in submandibular and parotid glands of rats during development, can be induced in adults by the beta-adrenergic agonist isopreterenol (IPR). It was shown previously that unilateral sympathectomy or bilateral parasympathectomy reduces IPR-induced cystatin S expression. The present experiments demonstrate that IPR-induced cystatin S gene expression in submandibular glands is reduced as early as 3 days post bilateral denervation of both branches of the autonomic nervous system. The reduction is nearly equal to that of either sympathectomy or parasympathectomy alone, suggesting that factor(s) in both sympathetic and parasympathetic fibers are simultaneously required for IPR-induced cystatin S gene expression.     

Sphingosine inhibits muscarinic cholinergic receptor binding in rat parotid acinar cells

1. Sphingosine inhibited the binding of [³H]quinuclidinyl benzilate (QNB), a potent and specific muscarinic antagonist, in dispersed rat parotid acinar cells.2. The inhibition of [³H]QNB binding was expressed as decrease in affinity without significant change of a number of membrane sites.3. The effect of Sphingosine on the binding was not affected by the chelation of extracellular Ca²⁺.4. H-7, an inhibitor of protein kinase C, failed to decrease [³H]QNB binding.5. Stearylamine, an analogue of Sphingosine, was as effective as Sphingosine in inhibiting [³H]QNB binding.6. These results suggest that Sphingosine inhibits muscarinic cholinergic receptor binding by a mechanism that is independent on extracellular Ca²⁺ and protein kinase C.     

Effects of surgical denervations on the autonomic nerves in parotid glands of dogs

Summary The innervation of the dog's parotid has been studied by cholinesterase staining and catecholamine fluorescence. In normal glands cholinergic and adrenergic nerves are plentiful around acini, muscular blood vessels, and to a lesser extent striated ducts. The main ducts, although surrounded by many cholinesterase-positive nerves, are associated with few adrenergic nerves. Severance of the classical parasympathetic post-ganglionic nerve to the gland, the auriculo-temporal, caused a moderate loss of cholinesterase-positive nerves. When this procedure was combined with section of the nerves on the internal maxillary artery there was a greater loss. Fewest cholinesterase-positive nerves remained when, in addition to these two procedures, the facial nerve was cut. These findings support the concept that all three sets of nerves contain some post-ganglionic parasympathetic fibres for the dog's parotid. The source of the remaining nerves is unknown. Preganglionic parasympathetic denervation by section of the tympanic branch of the glossopharyngeal nerve did not reduce the number of cholinesterase-positive nerves. None of these parasympathetic denervations caused reduction of adrenergic nerves, indicating that they do not travel to the gland with the parasympathetic nerves. After superior cervical ganglionectomy a few scattered fluorescent nerves remained in the gland; their origin is unknown.     

Distinct alpha-noradrenergic receptors differentiated by binding and physiological relationships.

Adult mice received two 70 μg doses of 6-hydroxydopamine intracisternally 72 hours apart, and the muscarinic binding properties of discrete brain regions were then investigated at various time intervals. Three days after the second injection, ³H-norepinephrine uptake was drastically reduced in all brain regions studied, and a distinct decrease in muscarinic receptor density was observed in the striatum (?18%), medulla-pons (?17%) and cerebellum (?15%) of lesioned animals as compared with controls. No changes were detected in muscarinic receptor density in the cortex or the hippocampus of treated animals, nor were any changes seen in the affinity of the labelled ligand for its receptor or in the displacement properties of the muscarinic binding by agonists in any of the regions studied. These effects still persisted after 60 days, with a further reduction in striatal muscarinic density to 74% of control values. Data are interpreted with respect to the proposed model for cholinergic modulation of central catecholamine release and cholinergic-catecholaminergic interactions in the striatum.     

Amitriptyline and imipramine inhibit the release of acetylcholine from parasympathetic nerve terminals in the rat iris

The electrically stimulated release of [3H]acetylcholine from the parasympathetic nerve terminals of the rat iris in vitro is increased in a dose-dependent manner by scopolamine but is decreased by the tricyclic antidepressants amitriptyline and imipramine. The increased release in the presence of scopolamine seems to be due to the blockade of a presynaptic muscarinic autoreceptor that, in the drug-free state, inhibits the release of acetylcholine. However, at drug concentrations that should have comparable antimuscarinic potency, the antidepressants inhibit the release of acetylcholine. This suggests that the anticholinergic side effects of the antidepressants may be due to the reduced release of acetylcholine from parasympathetic nerve terminals as well as a possible direct postsynaptic muscarinic receptor blocking action. Whatever the mechanism of this action, the antidepressants do not have the same effect as scopolamine at the presynaptic muscarinic autoreceptor in the rat iris.     

The sympathetic and parasympathetic nature of neuropeptide Y-immunoreactive nerve fibres in the major salivary glands of the rat

Summary The distribution and origin of neuropeptide Y in the major salivary glands of the rat was studied by indirect immunofluorescence technique. Numerous nerve fibres immunoreactive for the peptide were seen in the parotid and sublingual glands. Most of the fibres were located around blood vessels and salivary acini. In the submandibular gland the number of immunoreactive nerve fibres around the acini was lower in comparison with that in the parotid and sublingual glands. Some immunoreactive nerve fibres were also found around or along intra- and interlobular ducts in all major salivary glands.A large number of the neuropeptide-containing neuronal cell bodies and nerve fibres were detected in the sympathetic superior cervical ganglion. Sympathetic postganglionic nerve trunks of this ganglion contained numerous immunoreactive nerve fibres as well. A subpopulation of the neuronal cell bodies in the submandibular ganglion were immunoreactive to neuropeptide Y.Both uni- and bilateral superior cervical ganglionectomies caused a significant decrease in the number of immunoreactive nerve fibres around the blood vessels in all the major salivary glands. However, these denervations did not affect the density of nerve fibres around the acini and ducts. On the contrary, unilateral parasympathetic denervation by sectioning the auriculotemporal nerve reduced the fibres around the secretory acini in the parotid gland remarkably, while only a minor reduction in the density of immunoreactive fibres associated with the blood vessels of the gland was detected. Unilateral electrocoagulation of the trigeminal nerve branches caused no detectable change in the density of immunoreactive nerve fibres in any of the major salivary glands.On the basis of the present findings it is concluded that neuropeptide Y-reactive nerve fibres present in all major salivary glands around the blood vessels seem to be mainly sympathetic, whereas those around the acini and ducts seems to be of parasympathetic origin.     

Muscarinic receptor binding in the mouse heart: the selective modulatory effect of fluoride ion on agonist binding

The effect of fluoride ion on the binding of the specific muscarinic agonist ligand [³H]c is methyldioxolane ([³H]CD) to the mouse cardiac muscarinic receptor was investigated. Utilizing equilibrium ligand binding experiments, sodium fluoride (10mM) was shown to decrease [³H]CD binding, measured at a concentration of 2 nM, by 52%. Studies with several different ions demonstrated that the reduction in [³H]CD binding was a specific effect of fluoride. This fluoride modulation was selective for agonist binding, as no effect of fluoride on the binding of the muscarinic antagonist [³H](?) quinuclidinyl benzilate (QNB) was observed.     

麻醉剂氟烷对心脏毒蕈碱型钾通道的影响

神经递质乙酰胆碱（ＡＣｈ）调节心脏功能最重要的离子通道就暗毒蕈碱型钾通道（ｉＫ,ＡＣｈ）,该通道由ＡＣｈ经鸟苷酸调节蛋白（Ｇ蛋白）的βγ亚单位而激活。本实验彩全细胞膜片箝方法,观察了麻醉药氟烷对豚鼠心房肌细胞ｉＫ,ＡＣｈ的影响。氟烷对ｉＫ,ＡＣｈ电流具抑制效应,灌注之后可使ＡＣｈ激活的ｉＫ,ＡＣｈ速率减慢,峰植下降。但其抑制ｉＫ,ＡＣｈ的程度依激活方式而异：经正常激活途径,即由ＡＣｈ激活毒蕈碱Ｍ样     

Bilateral changes in glutamate uptake,muscarinic receptor binding and acetylcholinesterase level in the rat hippocampus after unilateral entorhinal cortex lesions

This report examines the effects of unilateral electrolytic and knife-cut lesions of entorhinal cortex on glutamate uptake, the muscarinic receptor [³H]QNB binding and acetylcholinesterase (AChE) activity in the dorsal and ventral parts of the ipsi- and contralateral hippocampus of the rat.We found that (1) in unoperated, control rats there are no pre-existing differences in the level of the investigated markers between the right and left hippocampus, (2) both electrolytic and knife-cut lesions of the entorhinal cortex evoke bilateral changes in the investigated markers and (3) the character of the response is dependent on the survival time and on the hippocampal part involved. Four days after operation a substantial reduction in glutamate uptake was found in both the dorsal and ventral parts of the ipsi- and contralateral hippocampus. At the same time there was a drop in muscarinic receptor binding, while AChE activity was not affected. The decrease in glutamate uptake persisted on the 21st postoperative day, whereas muscarinic receptor binding was enhanced, in comparison with the control level, in the ventral part of both the ipsi- and contralateral hippocampus. This overshoot was not so evident on the 30th postoperative day; glutamate uptake at that time reached or even surpassed the control level. Enhancement of AChE activity on the ipsi- and contralateral sides was noted on both the 21st and 30th day after operation.We suggest the following interpretation of these results: (1) glutamatergic projections from the entorhinal cortex to the hippocampus are bilateral, (2) some transneuronal changes probably contribute to the decline in glutamate uptake, particularly on the contralateral side, (3) neuronal depolarization does not seem to be the only mechanism responsible for the decrease in muscarinic receptor binding and (4) some compensatory mechanisms occur in the hippocampus at a later time after the lesion.Moreover, we believe that the use of the contralateral side as a control should be considered with caution in studies with unilaterally lesioned animals.