G. Tobin

Depletion of neuropeptides in rat parotid glands and declining atropine-resistant salivary secretion upon continuous parasympathetic nerve stimulation

In rats the parasympathetic auriculo-temporal nerve on one side was continuously stimulated at 40 Hz for 20-80 min in the presence of adrenergic blockers (dihydroergotamine and propranolol) +/- atropine. During the first 10 min this gave rise to a flow of saliva from the parotid gland that in the atropinized rats amounted to 35% of that found in rats not treated with atropine, while the protein and amylase outputs were 75% of those in non-atropinized rats. The atropine-resistant secretion of fluid and proteins declined to 5-10% of the initial value within 40 min but did not cease completely even after 80 min. The marked reduction in secretory responses was not due to desensitization or exhaustion of the gland cells. The nerve stimulation reduced the parotid gland content of vasoactive intestinal peptide (VIP) and substance P (SP) to approximately 60 and 25% of that of contralateral glands after 20 and 60 min, respectively. The probable explanation for the decline in secretory response seems to be depletion of non-adrenergic, non-cholinergic transmitter(s). The present results suggest that neuropeptides are involved in the regulation of salivary secretion but provide no direct evidence that either VIP or SP is responsible for the atropine-resistant salivary secretion.

Atropine-resistant submandibular responses to stimulation of the parasympathetic innervation in the anaesthetized ferret.

1. Submandibular salivary and vascular responses to stimulation of the peripheral end of the chorda‐lingual nerve at 20 Hz continuously for 60 min were investigated in anaesthetized ferrets, in which the sympathetic innervation to the gland was cut, in the presence and absence of atropine (2.0 mg kg‐1). 2. Both the increase in submandibular salivary flow and protein output, which occurred in response to nerve stimulation, were substantially reduced following the administration of atropine, the latency was greatly increased thereby, and both responses were more transient but neither was abolished by atropine. The fall in submandibular vascular resistance was not significantly affected by atropine, either in respect of extent or duration. 3. Chorda‐lingual stimulation produced an increase in the output of vasoactive intestinal peptide (VIP), substance P (SP) and calcitonin gene‐related peptide (CGRP) in the submandibular venous effluent blood. Each of these responses was maximal within the first 10 min after the onset of stimulation and declined thereafter. The time‐scales of both the CGRP and SP responses were similar to those of the atropine‐resistant secretory responses, both being quite short‐lived, whereas the output of VIP (like the atropine‐resistant vascular response) was significantly greater than the basal value throughout the whole of the 60 min period of stimulation. 4. The CGRP response was completely abolished by pre‐treatment with atropine, whereas the outputs of both VIP and SP were significantly enhanced thereby. Both the submandibular vascular and secretory responses to chorda‐lingual stimulation were almost completely suppressed following the administration of hexamethonium, and there was then no detectable release of peptidergic agonists from the gland. 5. The atropine‐resistant submandibular salivary secretory responses were completely abolished by pre‐treatment with a tachykinin inhibitor [( D‐Arg1, D‐Cl2 Phe5, Asn6, D‐Trp7,9, Nle11]‐SP; 0.75 mg kg‐1) without affecting the fall in submandibular vascular resistance. 6. Following pre‐treatment with hexamethonium, I.V. bolus injections of methacholine, SP and CGRP elicited increases in submandibular blood flow and secretion of saliva. VIP caused an increase in blood flow without overt secretion, although it is known to increase secretion of protein and to potentiate the secretory response to SP. Taken together, all these results are consistent with the contention that VIP contributes to the vasodilator response to stimulation of the para‐sympathetic innervation in this gland and that both SP and CGRP are likely to contribute to the secretory response.

Nitric oxide and release of the peptide VIP from parasympathetic terminals in the submandibular gland of the anaesthetized cat

The role of nitric oxide (NO) in mediating various submandibular responses to stimulation of the parasympathetic innervation has been investigated in anaesthetized cats, in which N omega‐nitro‐L‐arginine methyl ester (L‐NAME; 30 mg kg‐1 I.A.) was used to block the synthesis of NO. L‐NAME significantly reduced the vasodilator response and the flow of saliva, together with the output of salivary protein that occurred during stimulation of the chorda lingual nerve (20 Hz for 1 s at 10 s intervals), without significantly reducing the output of vasoactive intestinal peptide (VIP) from the gland. The results show that NO is implicated not only in the release of VIP, as established previously, but also in mediating its actions following release in the submandibular gland of the cat.

Effects of capsaicin pretreatment on neuropeptides and salivary secretion of rat parotid glands

1 ‘Atropine‐resistant’ secretion of saliva in response to parasympathetic stimulation may reflect antidromic activation of sensory nerve fibres. In this investigation, the effect of pretreatment in the rat with capsaicin (total dose of 125 mg kg−1, s.c.), was determined. 2 In the parotid glands substance P (SP)/calcitonin gene‐related peptide (CGRP)‐containing nerve fibres around ducts and blood vessels disappeared after capsaicin, while periacinar SP‐containing fibres (devoid of CGRP) and CGRP‐containing fibres (devoid of SP) remained. Vasoactive intestinal peptide (VIP)‐containing nerve fibres seemed to be unaffected. The parotid content of SP and CGRP was reduced by 11 and 36% respectively, while that of VIP remained unchanged. 3 The weights of the parotid glands and their sensitivity to the secretagogues methacholine and SP, injected intravenously, were unchanged as was the response to stimulation of the auriculo‐temporal nerve in the presence and absence of atropine. 4 In contrast to capsaicin pretreatment, parasympathetic denervation of the parotid gland reduced the weight of the gland and produced an increase in the response to methacholine and SP. 5 For comparison, the effectiveness of the capsaicin treatment on neuropeptide content was determined in the urinary bladder. The bladder of capsaicin‐pretreated rats increased in weight (21%) and in VIP content (31%), while the content of SP and CGRP was reduced by 86 and 94%, respectively. SP‐ and CGRP‐containing nerve fibres were virtually eliminated, while VIP‐containing nerve fibres seemed unaffected. 6 In conclusion, antidromic activation of primary afferent (capsaicin‐sensitive) C‐fibres does not contribute significantly to the ‘atropine‐resistant’ secretory response of the parotid gland to stimulation of the parasympathetic nerve.

Secretion of protein from salivary glands in the ferret in response to vasoactive intestinal peptide

1. Secretory responses of ferret parotid and submandibular glands were investigated in the presence of muscarinic, alpha‐ and beta‐adrenoceptor blocking agents. 2. In pentobarbitone‐anaesthetized animals I.V. doses of vasoactive intestinal peptide (VIP) failed to elicit overt secretion of saliva from either gland. 3. However, an occult secretion of protein occurred in response to VIP from both types of gland. This was revealed by means of a subsequent I.V. wash‐out injection of substance P, which is a potent secretagogue in ferrets. This effect of VIP was much more marked in the submandibular than in the parotid gland. 4. Electrical stimulation of the chorda‐lingual nerve gave rise to protein secretion in the submandibular gland at subthreshold frequencies for overt non‐cholinergic, non‐adrenergic secretion of saliva, as revealed by subsequent wash‐out I.V. injection of substance P. 5. Protein secretion in response to VIP was also demonstrated in vitro by perifusing small pieces of the two glands. The sensitivity of submandibular tissue to VIP greatly exceeded that of the parotid tissue. 6. It is concluded that VIP, or a structurally related peptide, may be involved in the non‐cholinergic, non‐adrenergic secretory response of ferret salivary glands evoked by parasympathetic nerve stimulation.

Relative secretory contributions of the three major salivary glands of the rat in response to substance P and super-sensitivity

In vivo salivation in the rat in response to a range of intravenous doses of substance P was studied. The ducts of the parotid, submandibular and sublingual glands were cannulated. The secretory threshold dose of substance P, in microgram/kg, was 0.05-0.1 in the submandibular glands, 0.2 in the parotid glands and 0.2-0.5 in the sublingual glands. The maximal secretory response in all three types of glands was obtained at a dose level of 5-10 micrograms/kg. The total amount of saliva secreted at this dose level from the three pairs of glands was calculated to about 300 mg; the submandibular glands were responsible for 65 per cent, the parotid glands for 32 per cent and the sublingual glands for 3 per cent. Parasympathetic decentralization but not sympathetic denervation caused the sublingual glands to develop a super-sensitivity to substance P. The secretory effect of substance P was not exerted via cholinergic, alpha-adrenergic or beta-adrenergic receptors.

Nitric oxide in the control of submandibular gland function in the anaesthetized ferret

Stimulation of parasympathetic innervation of the submandibular gland (2 or 20 Hz continuously or 20 Hz for 1 s at 10 s intervals), in the ferret, produced secretion of fluid and protein and a fall in vascular resistance. The responses following the administration of N omega‐nitro‐L‐arginine methyl ester (L‐NAME; 2 mmol kg‐1 i.a.) to block the synthesis of nitric oxide (NO) were reduced, and the persisting responses were abolished (at 2 Hz continuously and 20 Hz intermittently) or further reduced (at 20 Hz continuously) by the additional administration of atropine. The output of vasoactive intestinal peptide (VIP) from the gland was not affected. Neither the secretory nor the vascular response to intra‐arterial infusions of acetylcholine (1.25 nmol kg‐1) was affected by L‐NAME, whereas the vascular responses to both VIP (10 pmol kg‐1) and pituitary adenylate cyclase‐activating peptide (1‐38) (PACAP) (0.5 pmol kg‐1) were reduced thereby. Neither peptide evoked a fluid secretion per se. However, when infused during parasympathetic stimulation of saliva secretion, VIP increased both flow rate and the output of protein. These effects of VIP were abolished by L‐NAME. The experiments were performed in the presence of sodium nitroprusside at doses (4‐8 nmol min‐1 kg‐1 i.v.) aimed to counterbalance the systemic effects of L‐NAME. The results show that, in the ferret, parasympathetic nerve activity increases submandibular blood flow, and elicits the flow of saliva and output of protein by mechanisms that involve in situ generation of NO, upon which the effects of VIP and PACAP but not acetylcholine are largely dependent.

Tachykinin involvement in parasympathetic nerve-evoked salivation of the ferret

1 The tachykinin antagonist (D‐Arg1, D‐Cl2Phe5, Asn6, D‐Trp7.9, Nle11)‐substance P, injected intravenously, blocked salivary secretion from the ferret parotid and submandibular glands in response to subsequent i.v. injections of the tachykinins, substance P and neurokinin A. 2 The tachykinin antagonist reduced the parasympathetic nerve‐evoked secretion of parotid and submandibular saliva by 15–20% and 35–40%, respectively. Atropine abolished the remaining secretory response. 3 The ‘atropine‐resistant’ parasympathetic nerve‐evoked secretion of saliva from the parotid and submandibular glands (about 5 and 30%, respectively, of that before administration of atropine) was abolished by the tachykinin antagonist. 4 The tachykinin antagonist was without effect on the protein concentration of parotid and submandibular saliva secreted in response to parasympathetic nerve stimulation. Parotid and submandibular saliva lacked amylase. 5 Atropine reduced the protein concentration of the submandibular saliva secreted in response to parasympathetic nerve stimulation by 50%; this was the protein concentration of substance P‐evoked saliva. 6 The secretory response to methacholine and to stimulation of preganglionic sympathetic nerve fibres, tested in rats, was unaffected by the tachykinin antagonist, contra‐indicating an unspecific action of the antagonist. 7 The results suggest that the neuronal release of tachykinins is probably important in the nerve‐evoked secretory response of the parotid and submandibular glands.

The effects of atropine and chronic sympathectomy on maximal parasympathetic stimulation of parotid saliva in rats.

1. The effects of stimulating the postganglionic parasympathetic nerve to the parotid gland, the auriculo‐temporal nerve, continuously at 40 Hz for 80 min have been assessed on the flow of saliva and its amylase content during each 10 min period and on the glandular morphology at the end of the stimulation, in female Sprague‐Dawley rats under chloralose anaesthesia. Adrenergic responses were blocked by prior administration of dihydroergotamine (1 mg/kg I.P.) and propranolol (1 mg/kg I.P.). The contralateral unstimulated parotid gland from each animal was used for morphological control purposes. 2. In normal animals there was a copious flow of saliva reaching 3 ml or more in 80 min. It was well maintained after a gradual small decline and the flow in the last 10 min period was still 60% of that in the initial period. Amylase output was relatively high in the initial period but then declined more rapidly than the flow. The acini were densely packed with granules on the control side and showed a moderate amount of degranulation on the parasympathetically stimulated side. 3. After atropine (2 mg/kg I.V.), normal animals initially showed a moderate flow of saliva, being 41% of the non‐atropinized animals, but there was a rapid decline and the total flow over 80 min was only 15% of that in the non‐atropinized animals. The amylase secreted was also high initially and showed a rapid decline; the total amount secreted was similar to that in non‐atropinized animals. The morphology was similar to that in non‐atropinized animals and showed a similar loss of acinar granules on the stimulated side. 4. Chronic bilateral postganglionic sympathectomy (4‐6 weeks duration) caused a drastic reduction in the capacity of the gland to secrete saliva in response to parasympathetic stimulation, reaching only one‐third of that from normal animals. The initial output of amylase was greater than in normal animals but the total output was similar. The control unstimulated sympathectomized glands appeared similar morphologically to normal resting glands. However, on the parasympathetically stimulated side, besides the usual amount of acinar degranulation, there was also a conspicuous development of acinar vacuolation, not seen in the other groups of animals.(ABSTRACT TRUNCATED AT 400 WORDS)

Submandibular responses to stimulation of the parasympathetic innervation in bursts in the anaesthetized ferret.

1. Submandibular salivary and vascular responses to different patterns of stimulation of the parasympathetic innervation have been investigated in anaesthetized ferrets in the presence and absence of atropine. 2. At low stimulus frequencies likely to fall within the physiological range (0.5‐2.0 Hz continuously; 5.0‐20.0 Hz in 1 s bursts at 10 s intervals) secretion of fluid and protein were both potentiated by the bursting pattern of stimulation and the latency of the secretory response was reduced. Over a somewhat lower range (0.5‐1.0 Hz continuously; 5.0‐10.0 Hz in bursts) the submandibular vascular response was also significantly potentiated by employing this intermittent pattern of stimulation. Above these frequency ranges no such potentiation occurred. 3. Pre‐treatment with atropine (2.0 mg kg‐1) blocked the submandibular secretory responses to stimulation of the chorda‐lingual nerve at these low frequencies and the residual responses at higher frequencies were not significantly affected by changing the pattern of stimulation. The vascular response was somewhat reduced after atropine but that which persisted was enhanced by stimulating in bursts. 4. It is concluded that the release of some transmitter from postganglionic terminals in the submandibular gland of the ferret must be potentiated by the arrival of action potentials at short intervals and possible mechanisms are considered.