Effects of acute and long-term atropine treatment on levels,release and response to VIP and PHI in the submandibular gland of cat and rat

We have studied the effects of acute and long-term treatment of cats and rats with atropine on the levels, release and effects of two peptides, vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI), that probably co-exist with acetylcholine in the parasympathetic nerves supplying the submandibular gland. Atropine treatment (progressively increasing doses from 2 to 15 mg kg^-1 injected s.c.) for 14 days did not alter the contents of VIP- or PHI-like immunoreactivity (-IR) in the cat submandibular gland or in three other tissues (nasal mucosa, trachea and tongue). Acute as well as long-term atropine treatment decreased the vasodilatation following low-, but not high-, frequency parasympathetic nerve stimulation. During prolonged stimulation (60 min) there was a decreased vasodilatation response following both acute and long-term atropine treatment. The overflow of VIP-IR and PHI-IR following parasympathetic nerve stimulation was markedly increased by acute, but not by long-term atropine treatment. The VIP- or PHI-induced stimulation of cyclic AMP (cAMP) accumulation in the cat submandibular gland was not altered after long-term atropine treatment. Similarly, treatment of male Sprague-Dawley rats with atropine (20 mg kg^-1) or imipramine (20 mg kg^-1) for 14 days did not alter the sensitivity to VIP or to PHI of cAMP accumulation in the submandibular gland, nor was there any change in VIP-IR or PHI-IR content. In conclusion, although atropine treatment causes an acute increase in the overflow of VIP and PHI evoked by parasympathetic nerve stimulation, there is no depletion of peptide stores upon long-term treatment, nor is there any change in the effect of exogenous VIP and PHI on cAMP-accumulation.     

Effects of acute and long-term atropine treatment on levels, release and response to VIP and PHI in the submandibular gland of cat and rat.

We have studied the effects of acute and long-term treatment of cats and rats with atropine on the levels, release and effects of two peptides, vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI), that probably co-exist with acetylcholine in the parasympathetic nerves supplying the submandibular gland. Atropine treatment (progressively increasing doses from 2 to 15 mg kg-1 injected s.c.) for 14 days did not alter the contents of VIP- or PHI-like immunoreactivity (-IR) in the cat submandibular gland or in three other tissues (nasal mucosa, trachea and tongue). Acute as well as long-term atropine treatment decreased the vasodilation following low-, but not high-, frequency parasympathetic nerve stimulation. During prolonged stimulation (60 min) there was a decreased vasodilatation response following both acute and long-term atropine treatment. The overflow of VIP-IR and PHI-IR following parasympathetic nerve stimulation was markedly increased by acute, but not by long-term atropine treatment. The VIP- or PHI-induced stimulation of cyclic AMP (cAMP) accumulation in the cat submandibular gland was not altered after long-term atropine treatment. Similarly, treatment of male Sprague-Dawley rats with atropine (20 mg kg-1) or imipramine (20 mg kg-1) for 14 days did not alter the sensitivity to VIP or to PHI of cAMP accumulation in the submandibular gland, nor was there any change in VIP-IR or PHI-IR content. In conclusion, although atropine treatment causes an acute increase in the overflow of VIP and PHI evoked by parasympathetic nerve stimulation, there is no depletion of peptide stores upon long-term treatment, nor is there any change in the effect of exogenous VIP and PHI on cAMP-accumulation.     

Complementary role of vasoactive intestinal polypeptide (VIP) and acetylcholine for cat submandibular gland blood flow and secretion I. VIP release

The effects of parasympathetic and sympathetic nerve stimulation on VIP release in relation to blood flow and secretion were studied in the cat submandibular salivary gland. Parasympathetic nerve stimulation caused a marked VIP overflow (over thousand fold increase in VIP output) into the venous effluent from the gland which was simultaneous with profuse salivation and an about 10–15 fold increase in blood flow. The VIP output was dependent on the stimulation frequency, the duration of the stimulation period as well as the glandular blood flow. At 15 Hz maximal VIP output (about 4 fmol per impulse) was obtained after about 5 min of stimulation simultaneously with the maximum of the maintained phase of vasodilation. About 50 pmol VIP was recovered in the venous effluent from the gland during 1 h of maximal nerve stimulation. The VIP output after 1 h, was only about 20% of maximal, however, suggesting that the storage reserves and/or resupply of VIP might be running out. Under physiological conditions (frequencies ≤ 6 Hz) it was estimated that the axonal transport mechanism should be sufficient for replacement of VIP. At lower frequencies (2 and 6 Hz) the VIP output was parallel to vasodilation and secretion, while at a high frequency (15 Hz) a much more pronounced VIP output was seen. The increased overflow at 15 Hz may either be due to an actual increase in release or to a saturation of local VIP inactivating mechanisms. When stimulated simultaneously, the parasympathetic vasodilator mechanism seemed much more potent than sympathetic vasoconstriction. Since VIP may be present in cholinergic neurons, data from the literature concerning acetylcholine release are discussed in relation to the observed VIP output.     

Effects of antidromic trigeminal nerve stimulation in relation to parasympathetic vasodilation in cat nasal mucosa

High threshold stimulation of the trigeminal nerve in sympathectomized cats caused an atropine and hexamethonium resistant vasodilation in the nasal mucosa. Stimulation of efferent parasympathetic neurons to the nose caused a partially atropine sensitive vasodilation which was abolished by hexamethonium. Combined trigeminal and parasympathetic nerve stimulation did not reveal any obvious functional interactions between these two systems. Local intra-arterial infusions of substance P, VIP and acetylcholine caused a dose dependent vasodilation in the nasal mucosa. The relative vasodilatory potencies were substance P greater than to VIP greater than acetylcholine. Local infusions of capsaicin, known to release substance P from nerve endings, caused a marked longlasting biphasic vasodilation which was atropine and hexamethonium resistant. In conclusion, the present findings illustrate the presence of two vasodilator mechanisms of different nervous origin in the nasal mucosa. The trigeminal response is probably mediated via release of substance P, while the parasympathetic effect seems to be caused by acetylcholine and VIP.     

Complementary role of vasoactive intestinal polypeptide (VIP) and acetylcholine for cat submandibular gland blood flow and secretion

The effects of local intraarterial infusions of VIP, acetylcholine (ACh), substance P. iso-prenaline and bradykinin on submandibular gland blood flow and salivary secretion were studied in cats. It was found that VIP (lo^-14 to 10^--¹⁰ mol/min) caused an atropine resistant vasodilation but no salivary secretion. Several hundred fold higher doses of exogenous VIP had to be infused than the amounts of VIP seen in the venous outflow during maximal nerve stimulation at a similar vasodilatory response. ACh infusions (5×10–¹² to 5×10^--⁸ mol/min) caused both a muscarinic vasodilation and salivary secretion. ACh was about 100 times less potent than VIP as a vasodilating agent. Both ACh and VIP induced in high doses a vasodilatory response similar to that seen during parasympathetic nerve stimulation at 15 Hz. ACh by itself did in the present doses, however, only induce about 50–60% of the maximal secretory response. Combined infusions of ACh and VIP, had mostly an additive effect on vasodilation. The salivatory volume response to ACh was potentiated by VIP and to a smaller extent also by isoprenaline. This potentiating effect may be due to a direct effect on secretory elements as well as partly to the additional increase in blood flow. Bradykinin was about 1000 times less potent than VIP as a vasodilating agent. Substance P (10^--⁹mol/min) only caused a weak vasodilation. Since there is evidence that ACh and VIP coexist within the same neurons and are both released upon parasympathetic nervous activation, the present findings suggest that the secretory and vasodilatory responses may be caused by an interaction between these two agents.     

Carbachol stimulation of inositol phosphate accumulation in rat submandibular gland fragments is not modified by VIP

Formation of inositol phosphates in response to carbachol, phenylephrine and vasoactive intestinal polypeptide (VIP) was studied after labelling with [3H]myo-inositol in rat submandibular gland fragments. Carbachol enhanced the accumulation of inositol phosphates in a concentration-dependent manner. This effect was independent of calcium in the incubation medium and totally antagonized by atropine (IC50 = approx. I nM). Phenylephrine also induced an increase in inositol phosphate accumulation, which was totally antagonized by prazosin but not by atropine. Vasoactive intestinal polypeptide, isoproterenol and forskolin, compounds known to enhance the levels of cAMP in rat salivary gland, or addition of dibutyryl-cAMP (DB-cAMP) failed to alter basal or carbachol-evoked accumulation of inositol phosphates. It is concluded that the formation of inositol phosphates during muscarinic receptor stimulation with carbachol in rat submandibular gland fragments is not affected by adrenoceptor occupation or by cAMP. In particular, addition of VIP, which coexists with acetylcholine, did not alter the muscarinic inositol phosphate response.     

Ultrastructural localization of VIP-like immunoreactivity in large dense-core vesicles of ‘cholinergic-type’ nerve terminals in cat exocrine glands

Exocrine glands of the cat were analysed with the peroxidase-antiperoxidase method and routine electron microscopy. Vasoactive intestinal polypeptide (VIP)-like immunoreactivity was observed in certain nerve endings in the submandibular salivary gland, lacrimal gland and Harderian gland. The distribution of the VIP immunoreactive nerve fibres agreed well with earlier light microscopic findings. At the electron microscopic level electron-dense precipitates representing VIP-like immunoreactivity were seen in so-called large dense-core vesicles (median diameter about 990A?) in nerve fibres and varicosities also containing many small (‘immunonegative’) agranular vesicles. In conventional electron microscopy, the small agranular vesicles outnumbered the large dense-core vesicles by about 9 to 1. Immunoreactive fibres and varicosities could be seen close to the secretory acini (distance less than 400A?) and more distant (1500A?or more) to e.g. demilunes, ducts and blood vessels of the glands. The number and distribution of immunoreactive nerve fibres were not affected by sympathectomy. Furthermore, no typical ‘p-type’ bouton profiles, which are dominated by large opaque vesicles (dia. 800–2000A?), could be seen in the ultrastructural analysis of conventional preparations of the glands.The morphological features of the VIP immunoreactive nerve endings could not be distinguished from those often described as representing cholinergic fibres. These findings are in agreement with earlier suggestions of a possible coexistence of acetylcholine and VIP in neurons innervating exocrine glands and indicate possible functions for VIP in the roles of these nerves in evoking vasodilation and exocrine secretion.     

Membrane potential and input resistance in acinar cells from cat and rabbit submaxillary glands in vivo: effects of autonomic nerve stimulation

1. Membrane potential and input resistance measurements were made from acinar cells of cat and rabbit submaxillary glands in vivo, using intracellular glass micro-electrodes.2. The mean resting cell membrane potential was higher than previously reported, but ranged widely from -15 to -80 mV.3. Single shock electrical stimulation of the parasympathetic nerve fibres evoked characteristic potential changes. In some cases monophasic hyperpolarizations, in others biphasic responses (depolarization - hyperpolarization) were observed.4. The latency of the hyperpolarizing response was considerably longer (300-550 msec) than the latency of the biphasic response (about 150 msec).5. Hyperpolarizing and biphasic responses could be observed in the same cell at different levels of membrane potential. The initial depolarization of the biphasic response was dependent on the magnitude of the resting potential in such a manner that it was very small or absent at the lowest potentials and increased gradually with increasing level of the resting potential.6. Single-shock stimulation of the sympathetic nerve fibres to the gland did not evoke any response. In the cat, repetitive stimulation evoked hyperpolarizing or biphasic responses similar to those seen after repetitive stimulation of the parasympathetic nerve fibres. In the rabbit small hyperpolarizations were seen in a few cells only; mostly there was no response. Repetitive stimulation of the parasympathetic nerve fibres to the rabbit submaxillary gland evoked complex potential changes mostly of the depolarization-hyperpolarization type.7. Both single shock and repetitive stimulation of the parasympathetic nerve fibres evoked marked reductions in cell input resistance. In the hyperpolarizing cell type the conductance change sometimes preceded the potential change whereas they always occurred simultaneously in the biphasic cell type.8. It is concluded that both the hyperpolarizing and the biphasic secretory potentials are derived from the same type of acinar cells. The neurotransmitter released by the parasympathetic nerve endings (ACh) acts on the acinar cell membrane by increasing the ion permeability.     

Protein secretion in salivary glands of cats in vivo and in vitro in response to vasoactive intestinal peptide

In anaesthetized cats exogenous vasoactive intestinal peptide failed to elicit any secretion of saliva from the submandibular and parotid glands. However, protein release from both glands occurred in response to VIP in the presence of alpha- and beta-adrenoceptor blocking agents and was dose-dependent. This response was revealed by means of a subsequent washout flow of saliva evoked by intravenous injections of methacholine or stimulations of the parasympathetic innervation. The submandibular glands responded to vasoactive intestinal peptide at a lower dose than the parotid glands. In the presence of atropine (but in the absence of adrenoceptor blockers), stimulation of the parasympathetic chorda-lingual nerve, which of itself elicited no secretion of saliva, contributed to the release of protein within the submandibular gland, since the output of protein in response to a subsequent stimulation of the sympathetic innervation was increased. Vasoactive intestinal peptide administered in combination with methacholine or during ongoing parasympathetic nerve-induced salivary secretion revealed positive interactions, particularly with respect to protein release. In-vitro protein release in response to vasoactive intestinal peptide was also demonstrated by perfusing small pieces of the two glands in the presence of muscarinic and adrenoceptor blockers. As in vivo, submandibular tissue responded at a lower concentration of vasoactive intestinal peptide than the parotid tissue. One to two weeks after combined parasympathetic and sympathetic denervation of the parotid glands, the glands were sensitized to vasoactive intestinal peptide when tested in vitro. It is concluded that vasoactive intestinal peptide or a structurally related peptide is a potential transmitter in the parasympathetic control of protein secretion in salivary glands of cats.     

Carbachol-induced phosphatidylcholine hydrolysis and choline efflux in rat submandibular gland involves phospholipase D activation and is modulated by protein kinase C and calcium

The role of calcium and protein kinase C activation in carbachol-induced choline efflux from submandibular glands was investigated. The participation of phospholipase D in this signal transduction pathway was demonstrated by the formation of [¹⁴C]phospha-tidylethanol in [¹⁴C]lysophosphatidylcholine-labelled submandibular gland cells treated with carbachol or noradrenaline in the presence of ethanol. Chelation of the intracellular calcium with BAPTA/AM reduced the carbachol stimulated outflow of [³H]choline.* The calcium ionophore A23187 in a high concentration (10 μm) increased the basal [³H]choline outflow, but decreased the carbachol-induced outflow. Removal of the extracellular calcium enhanced the carbachol-stimulated outflow, which returned to control when calcium was re-added to the medium. Activation of protein kinase C by phorbol-12,13-dibutyrate (100 nM) or l-oleyI-2-acetyl-sn-glycerol (20 μM) was without effect per se, but enhanced the carbachol-mediated outflow of [³H]choline. Phorbol-12,13-dibutyrate in combination with 1//M A23187 induced a small efflux of [³H]choline. A 2h treatment with phorbol-12,13-dibutyrate (1 μM), causing down-regulation of protein kinase C, significantly decreased the carbachol-stimulated [³H]choline outflow. In conclusion, elevation of intracellular calcium levels and protein kinase C activation are of importance for the carbachol-stimulated outflow of [³H]choline. Inflow of calcium, if anything, reduces the carbachol-stimulated outflow of [³H]choline. Since phosphatidylcholine breakdown generates diacylglycerol and this could lead to activation of protein kinase C, activation of this signal transduction pathway may be important for the protein content of the saliva and for the known trophic effects of parasympathetic innervation.     

大鼠、小鼠颌下腺副交感神经节神经肽的免疫组织化学观察

本实验采用免疫组织化学 ABC法对大鼠、小鼠颌下腺副交感神经节细胞的神经肽进行了定性观察。结果证明 :在颌下腺小叶间结缔组织中存在着副交感神经节 ,神经节内神经元胞体呈圆形、椭圆形或不规则形 ;还可看到有发自神经元胞体的细长或短的突起 ,与邻近神经元似有联系。神经元胞体呈 SP、VIP、NPY、SOM和 CGRP免疫反应阳性 ,细胞核为阴性反应。本实验结果提示 :大鼠、小鼠颌下腺副交感神经节细胞内的神经肽可能以神经递质或神经调质的方式 ,调节其复杂的分泌活动     

Calcium channels controlling acetylcholine release from preganglionic nerve terminals in rat autonomic ganglia

Little is known about the nature of the calcium channels controlling neurotransmitter release from preganglionic parasympathetic nerve fibres. In the present study, the effects of selective calcium channel antagonists and amiloride were investigated on ganglionic neurotransmission. Conventional intracellular recording and focal extracellular recording techniques were used in rat submandibular and pelvic ganglia, respectively. Excitatory postsynaptic potentials and excitatory postsynaptic currents preceded by nerve terminal impulses were recorded as a measure of acetylcholine release from parasympathetic and sympathetic preganglionic fibres following nerve stimulation. The calcium channel antagonists ω-conotoxin GVIA (N type), nifedipine and nimodipine (L type), ω-conotoxin MVIIC and ω-agatoxin IVA (P/Q type), and Ni²⁺ (R type) had no functional inhibitory effects on synaptic transmission in both submandibular and pelvic ganglia. The potassium-sparing diuretic, amiloride, and its analogue, dimethyl amiloride, produced a reversible and concentration-dependent inhibition of excitatory postsynaptic potential amplitude in the rat submandibular ganglion. The amplitude and frequency of spontaneous excitatory postsynaptic potentials and the sensitivity of the postsynaptic membrane to acetylcholine were unaffected by amiloride. In the rat pelvic ganglion, amiloride produced a concentration-dependent inhibition of excitatory postsynaptic currents without causing any detectable effects on the amplitude or configuration of the nerve terminal impulse.These results indicate that neurotransmitter release from preganglionic parasympathetic and sympathetic nerve terminals is resistant to inhibition by specific calcium channel antagonists of N-, L-, P/Q- and R-type calcium channels. Amiloride acts presynaptically to inhibit evoked transmitter release, but does not prevent action potential propagation in the nerve terminals, suggesting that amiloride may block the pharmacologically distinct calcium channel type(s) on rat preganglionic nerve terminals.     

Nerve-evoked secretion of immunoglobulin A in relation to other proteins by parotid glands in anaesthetized rat

Secretion of fluid and proteins by salivary cells is under the control of parasympathetic and sympathetic autonomic nerves. In a recent study we have shown that, in the rat submandibular gland, autonomic nerves can also increase the secretion of IgA, a product of plasma cells secreted into saliva as SIgA (IgA bound to Secretory Component, the cleaved poly-immunoglobulin receptor). The present study aimed to determine if parotid secretion of SIgA is increased by autonomic nerves and to compare SIgA secretion with other parotid proteins stored and secreted by acinar and ductal cells. Assay of IgA in saliva evoked by parasympathetic nerve stimulation immediately following an extended rest period under anaesthesia indicated that it had been secreted into intraductal saliva in the absence of stimulation during the rest period. The mean rate of unstimulated IgA secretion (2.77+/-0.28 microg min(-1) g(-1)) and the 2.5-fold increase in IgA secretion evoked by parasympathetic stimulation were similar to results found previously in the rat submandibular gland. Sympathetic nerve stimulation increased SIgA secretion 2.7-fold, much less than in the submandibular gland. SDS-PAGE and Western blot analysis with anti-IgA and anti-Secretory Component antibodies confirmed that SIgA was the predominant form of IgA in saliva. Acinar-derived amylase and ductal-derived tissue kallikrein were more profoundly increased by parasympathetic and particularly sympathetic stimulation than SIgA. Overall, the results of the present study indicate that SIgA forms a prominent component of unstimulated parotid salivary protein secretion and that its secretion is similarly increased by stimulation of either autonomic nerve supply. The secretion of other parotid salivary proteins that are synthesized and stored by acinar or ductal cells is upregulated to a much greater extent by parasympathetic and particularly sympathetic stimulation.     

Autonomic nerve stimulation, kallikrein content anc acinar cell granules of the cat''s submandibular gland.

The parasympathetic and sympathetic nerves to the submandibular gland of the anaesthetized cat were stimulated under specific conditions. 1. It was possible to decrease the kallikrein (kininogenase) content of the gland by as much as 90-95% by sympathetic nerve stimulation. In such a gland the appearance and concentration of secretory granules in the acinar cells were indistinguishable from an unstimulated gland. 2. Parasympathetic nerve stimulation, in contrast to sympathetic nerve stimulation, whilst having no significant effect on the kininogenase content of the gland, resulted in the disappearance of a great majority of the acinar granules. 3. These results demonstrate that the acinar granules in the submandibular gland of the cat are not a significant source of kallikrein. 4. Our experiments also failed to indicate any obvious correlation between the granules of the demilune cells and the kallikrein content of the gland. 5. The possibility is raised that kallikrein is located in the cells of the striated ducts.     

Exocrine and endocrine release of kallikrein after reflex-induced salivary secretion

Exocrine and endocrine release of rat submandibular gland kallikrein has been shown to be low after parasympathetic and beta-adrenergic stimulation but greatly increased after alpha-adrenergic stimulation. In the present study, release of glandular kallikrein was investigated under conditions known to give a reflex-induced salivary gland response. Heat stress induced a rich flow of saliva originating in the submandibular glands. Salivary kallikrein secretory rate was higher than after parasympathetic stimulation but lower than after sympathetic stimulation (P less than 0.005). Only heat stress increased circulating glandular kallikrein (12.7 +/- 0.8 ng ml-1 before heat exposure and 53.3 +/- 14.1 ng ml-1 40 min afterwards, P less than 0.005). There were no indications that the endocrine release of kallikrein was due to non-specific leakage. Atropine abolished heat-induced salivation and endocrine kallikrein secretion, possibly through interference with central pathways (P less than 0.05). However, phentolamine did not, which may indicate as an yet unidentified mediator of endogenous kallikrein release. The salivary gland response to acid and ether was comparable to that observed after parasympathetic nerve stimulation and was abolished by atropine (P less than 0.005). Stimuli known to influence other salivary gland ductal cells, such as aggression and starvation followed by drinking, also did not increase the plasma concentration of glandular kallikrein. The fact that various conditions which induce salivation did not increase circulating glandular kallikrein, coupled with the fact that kallikrein concentration was the highest in animals that died from heat stress, may suggest that the increase in circulating glandular kallikrein seen after heat stress may be pathological and could contribute to the development of heat shock.     

Release and metabolism of [3H]dopamine in the neurointermediate lobe of the rat pituitary gland

Neurointermediate lobes of the rat pituitary gland were incubated with [3H]dopamine in the presence of desipramine and then superfused with radioactivity-free medium. The outflow of tritium was studied and in most experiments [3H]dopamine and its metabolites were separated by column chromatography. After 60-70 min of superfusion, the spontaneous rate of tritium outflow was 1.2%/min. The spontaneously released radioactivity consisted of 52% O-methylated and deaminated metabolites, 28% 3,4-dihydroxyphenylacetic acid, 18% dopamine and 2% 3-methoxytyramine. In the presence of pargyline (10 microM) the spontaneous rate of total tritium outflow decreased by 46%, that of the O-methylated and deaminated metabolites by 72% and that of 3,4-dihydroxyphenylacetic acid by 79%. The spontaneous rate of outflow of dopamine was unchanged and that of 3-methoxytyramine increased 3-fold. Further addition of nomifensine (10 microM) doubled the rate of outflow of dopamine and 3-methoxytyramine, but had no effect on the other metabolites. Electrical stimulation of the pituitary stalk (0.2 ms, 80 V, 3 Hz, 2 min) caused a tritium release of 8.5% of the tissue tritium. The evoked tritium release was only partially dependent on the extracellular calcium and not affected by tetrodotoxin. In contrast, vasopressin release evoked by stimuli of the same strength was completely calcium-dependent and blocked by tetrodotoxin. After modification of the stimulation conditions (1 ms, 10 V, 10 Hz, 2 min) the evoked tritium release was 4.1% of the tissue tritium. This tritium release was reduced by 73% in the presence of tetrodotoxin. The total evoked tritium release was decreased by 30% in the presence of pargyline and increased by 150% after further addition of nomifensine. Under the latter conditions, tetrodotoxin reduced the evoked tritium release by 67%, but nearly all of the tetrodotoxin-resistant tritium release could be identified as dopamine metabolites. Thus, the electrical stimulation appears to liberate some [3H]dopamine metabolites from an extraneuronal compartment. In conclusion, oxidative deamination and O-methylation are important pathways of the catabolism of dopamine in the neurointermediate lobe of the pituitary gland. After labelling of the transmitter stores with [3H]dopamine, the total tritium release is a poor indicator of [3H]dopamine release from the nerve terminals. Only the isolated [3H]dopamine fraction appears to reflect the release of neuronal [3H]dopamine.     

Degeneration secretion from parotid glands after section of the auriculotemporal nerves at different levels

1. In cats under ether or hexobarbitone anaesthesia the auriculotemporal nerve was cut near the parotid gland on one side and 12-20 mm more proximally on the other. After 22-64(1/2) hr the cats were anaesthetized with chloralose and the parotid ducts cannulated. Degeneration secretion of saliva which appears after post-ganglionic parasympathetic denervation was found to start 2-5(1/2) hr later in the gland denervated proximally than in that denervated distally. It ceased, on the other hand, later in the former than in the latter gland.2. Before degeneration secretion had started spontaneously it could be provoked by intravenous injection of acetylcholine, methacholine, carbachol or eserine and the effect was more pronounced on the gland denervated distally. When it had ceased spontaneously it could also be provoked, and the effect on the other gland was now more marked.3. Earlier it has been assumed that while a nerve is degenerating there is a period when the nerve endings are unable to retain in a normal way acetylcholine still being synthesized. It is now suggested that this period starts later after proximal than after distal denervation because more of the material required for the normal function of the endings is available in a long piece than in a short piece of nerve.     

Substance P: Indirect and direct effects on parotid acinar cell membrane potential

Direct effects of exogenously applied substance P on salivary acinar cells have been previously reported. This electro-physiological study confirms these direct effects in rat but not mouse parotid gland. We demonstrate that in the absence of autonomic blockade the peptide evokes marked responses which are blocked by atropine (10^–6 M). These effects cannot be attributed to direct activation of acinar cells and are presumably due to release of acetylcholine from parasympathetic nerve endings. We consider that substance P, in addition to direct effects, could act to modulate neuronal activity in salivary glands; a role already assigned to the peptide in the central nervous system.     

Glucocorticoid treatment increases inhibitory m(2) muscarinic receptor expression and function in the airways

M(2) muscarinic receptors on parasympathetic nerve endings inhibit acetylcholine release in the airways. In this study, the effects of dexamethasone on M(2) receptors in vivo and in primary cultures of airway parasympathetic neurons were tested. Treating guinea pigs with dexamethasone (0.1 mg/kg, daily for 2 d) substantially increased inhibitory M(2) muscarinic receptor function, decreasing airway responsiveness to electrical stimulation of the vagi. At the same time, dexamethasone decreased the response to acetylcholine but not to methacholine, suggesting that cholinesterase activity was increased. When both cholinesterase and M(2) receptors were blocked (using physostigmine and gallamine, respectively) vagally induced bronchoconstriction was increased to control values. In primary cultures of airway parasympathetic neurons, dexamethasone significantly decreased the release of acetylcholine in response to electrical stimulation. Blocking inhibitory M(2) receptors using atropine (10(-5) M) increased acetylcholine release. After the M(2) receptors were blocked there was no difference in acetylcholine release between control and dexamethasone-treated cultures. M(2) receptor gene expression was increased by more than fivefold in dexamethasone-treated cultures. Immunostaining of dexamethasone-treated neurons demonstrated more intense staining. Thus, decreased vagally mediated reflex bronchoconstriction after glucocorticoid treatment may be the result on increased M(2) receptor expression and function as well as increased degradation of acetylcholine by cholinesterase.     

In Vitro Studies of Frog Mucous Glands

The ionic outflow, mainly consisting of Na⁺ and Cl^-, from the mucous glands in an excised nerve-skin preparation of frog has been determined by recording the conductance changes occurring in a fluid layer covering a small area of the skin surface. In the main series of experiments the glands were activated by stimulation of sympathetic nerve fibers in the skin nerve. The relationship between the ionic outflow and the number of nerve volleys was studied over a wide range. The outflow per impulse was found to be fairly constant during the first tens of impulses but diminished gradually with increasing number of stimuli up to a certain maximum value—varying in different preparations—after which the outflow ceased completely. During the initial phase of stimulation the outflow is most likely caused by an ejection of preformed secretion due to the contractions of the glandular myoepithelium. The continued outflow in the later stages of the stimulation periods must be due to production of new secretion. Since the glandular epithelium is devoid of nerve terminals a nervous control of the ionic secretion can only be explained by an indirect influence mediated either by transmitter diffusion from the myoepithelial nerve endings or by a close electric coupling between the contractile and the secretory gland cells. Adrenaline and noradrenaline induce ionic outflows which like those evoked by nerve stimulation are inhibited by the β-adrenoreceptor blocker propranolol, α-adrenoreceptor blockers being without effect. A serendipitous finding of tonus changes in the frog skin during nerve stimulation is also described.