Jörgen Ekström

Calcitonin gene-related peptide in rat salivary glands: neuronal localization, depletion upon nerve stimulation, and effects on salivation in relation to substance P.

Calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibres occurred predominantly around blood vessels and large ducts and, to a minor extent, around acini and small ducts in the parotid, sublingual and submaxillary glands of the rat. Double immunostaining showed most of the CGRP-containing nerve fibres to contain substance P. However, the vast majority of substance P-immunoreactive periacinar nerve fibres in the parotid and submandibular glands lacked CGRP. After parasympathetic denervation of the parotid gland by section of the auriculotemporal nerve these periacinar substance P-immunoreactive nerve fibres disappeared almost completely, whereas the number of substance P/CGRP-immunoreactive nerve fibres seemed unchanged. After this operation the total amount of substance P in the parotid gland was reduced by about 90% as judged by radioimmunoassay; in denervation experiments the facial nerve was found to contribute to the residual substance P content. In contrast, the contribution of the auriculotemporal nerve to the CGRP content of the gland was small; the reduction in CGRP after section of the nerve was 20%. The facial nerve and the dorsal root nerves (C3 and C4) contributed to the CGRP content with about 50%. The source of the remaining 30% of the parotid gland CGRP is unknown. It is not the sympathetic nerve: sympathetic denervation resulted in a marked increase in CGRP, regardless of whether the auriculotemporal nerve was intact or not. Upon long-lasting electrical stimulation of the auriculotemporal nerve at a high frequency the parotid gland content of CGRP was gradually reduced, indicating depletion of this peptide in response to nerve stimulation. Intravenous injections of CGRP evoked no salivary flow; however, a release of amylase was revealed. Also, when CGRP was tested on isolated parotid gland lobules amylase was released into the medium. When, in vivo, CGRP was injected in combination with substance P, the substance P-evoked flow of parotid and submaxillary saliva was markedly enhanced. In addition, CGRP enhanced the in vivo secretory response to parasympathomimetics and to vasoactive intestinal peptide. The localization of CGRP-containing nerve fibres suggests that CGRP is involved in the regulation of secretion and blood flow of salivary glands. CGRP may interact positively with acetylcholine and certain nonclassical transmitters, and it may be involved (together with other neuropeptides) in the atropine-resistant parasympathetic secretion occurring in the glands under study.

NITRIC OXIDE SYNTHASE-CONTAINING NEURONS IN RAT PARASYMPATHETIC, SYMPATHETIC AND SENSORY GANGLIA : A COMPARATIVE STUDY

SummaryIn rats, the distribution of nerve structures staining for NADPH-diaphorase, and showing immunoreactivities for nitric oxide synthase (NOS), tyrosine hydroxylase and various neuropeptides was studied in sensory ganglia (dorsal root, nodose and trigeminal ganglia), in sympathetic ganglia (superior cervical, stellate, coeliac-superior and inferior mesenteric ganglia), parasympathetic ganglia (sphenopalatine, submandibular, sublingual and otic ganglia), and in the mixed parasympathetic/ sympathetic ganglia (major pelvic ganglia). The coincidence of neuronal cell bodies with strong NOS-immunoreactivity and strong NADPH diaphorase reactivity was almost total. The relative proportions of NOS-immunoreactive nerve cell bodies were largest in parasympathetic ganglia and major pelvic ganglia followed by sensory ganglia. In sympathetic ganglia no NOS-immunoreactive neuronal cell bodies could be detected. In parasympathetic and major pelvic ganglia, there was a very significant neuronal co-localization of immunoreactivities for NOS and vasoactive intestinal polypeptide (VIP). This was almost total in major pelvic ganglia, in which NOS-/VIP-immunoreactive nerve cell bodies were separate from sympathetic (tyrosine hydroxylase-/neuropeptide Y-immunoreactive), suggesting that NOS-/VIP-immuno-reactive neurons might also be parasympathetic.

Neuropeptides and Secretion

In the rat parotid gland, an atropine-resistant parasympathetic-nerve-evoked secretion was demonstrated in vivo. In the absence of atropine, the non-adrenergic, non-cholinergic transmitter release seemed to contribute to the fluid secretion and to be largely responsible for the secretion of amylase and acinar secretory granules. The gland was reached by nerve fibers containing substance P (SP), vasoactive intestinal peptide (VIP), and, to some extent, calcitonin-gene-related peptide (CGRP) via the parasympathetic auriculo-temporal nerve. Upon electrical stimulation of the nerve, these peptides were released. SP and substance K (SK), a novel tachykinin, induced a profuse watery secretion when injected i.v., while VIP caused a sparse but amylase-rich secretion. CGRP caused no secretion on its own. The tachykinin-evoked secretory response was enhanced by VIP and CGRP. A SPanalogue almost abolished the SP-evoked response, while the atropine-resistant parasympathetic response was only halved. None of the peptides under study can on its own account for the atropine-resistant parasympathetic secretion. The neuropeptides may play complementary roles in the regulation of the exocrine functions of the gland.

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.

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.

Effects of repeated infusions of substance P and vasoactive intestinal peptide on the weights of salivary glands subjected to atrophying influences in rats

1 The long‐term influence of substance P (SP) and vasoactive intestinal peptide (VIP) on rat salivary gland weight was investigated after parasympathetic denervation or on feeding soft food. 2 The parotid gland lost about one‐third of its weight within 4–5 days following parasympathetic postganglionic denervation or change in dietary regimen, from pellets to liquid diet, thought to reduce nerve reflex activity. 3 Daily i.v. infusions with SP or VIP diminished or largely prevented the fall in parotid gland weight, whereas infusions with pentagastrin, bethanechol and saline had no effect. The infusions were preceded by administration of α‐ and β‐adrenoceptor antagonists; these antagonists were also given to the control animals. 4 The effect of SP and VIP on the parotid gland weight appeared to be related to cell size rather than to cell number, as judged by measurements of RNA and DNA. 5 Observations on the two other major salivary glands underlined the fact that different gland types in the same animal behave differently. Parasympathetic preganglionic denervation (decentralization) lowered the weights of the sublingual and submandibular glands, whereas liquid diet only reduced the weight of the sublingual gland. SP and VIP did not affect the weights of the submandibular glands, but VIP prevented the slight fall in sublingual gland weight induced by liquid diet. 6 The present results suggest a trophic role in rats for SP and VIP on parotid glands and for VIP on sublingual glands. Such an influence may be exerted naturally as a result of their release from nerves containing these peptides around acini.

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.

Presence and effects of pituitary adenylate cyclase activating peptide in the submandibular gland of the ferret

Pituitary adenylate cyclase activating peptide (PACAP), a recently described vasoactive intestinal peptide-like neuropeptide, was found to be present in neurons in the submandibular gland of the ferret, where PACAP-immunoreactive nerve fibers were distributed around blood vessels, acini and ducts. Most of the PACAP-immunoreactive fibres were distinct from those storing vasoactive intestinal peptide. PACAP occurs in tissues as PACAP1-38 and PACAP1-27. PACAP1-38 and PACAP1-27 but not PACAP16-38 displayed biological activity with about the same potency. They exerted vasodilator effects on the submandibular vasculature, which resulted in a greater fall in vascular resistance than an equimolar dose of vasoactive intestinal peptide. The vasodilator response was independent of muscarinic receptor activation. Neither vasoactive intestinal peptide nor PACAP alone evoked any flow of saliva. However, both vasoactive intestinal peptide and PACAP enhanced the fluid response to acetylcholine, and the flow of saliva as well as the output of protein in response to parasympathetic nerve stimulation, vasoactive intestinal peptide being more potent than PACAP. In vitro, protein was released from submandibular gland tissue in response to both vasoactive intestinal peptide and PACAP, vasoactive intestinal peptide being more potent than PACAP. PACAP (and vasoactive intestinal peptide) exerted its in vitro effect following adrenoceptor and muscarinic blockade and following degeneration of sympathetic nerves. Sympathetic denervation combined with parasympathetic preganglionic denervation resulted in supersensitivity to both vasoactive intestinal peptide and PACAP. The fact that PACAP and vasoactive intestinal peptide occur in different nerve fibre populations suggests different roles for the two peptides in the submandibular gland.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of long-term treatment with estrogen and progesterone on in vitro muscle responses of the female rabbit urinary bladder and urethra to autonomic drugs and nerve stimulation

Ovariectomized virginal rabbits were treated with either estrogen or progesterone for 4 to 6 months. In vitro responses of muscle strips of the bladder and circular urethra were examined. Estrogen resulted in bladder contractions in response to noradrenaline and phenylephrine, whereas these agonists were without effect or evoked weak relaxations in castrated animals (and normals). Estrogen also caused a rightward shift of the frequency-contraction curve to nerve stimulation. Progesterone increased bladder sensitivity to contraction-evoking bethanechol. Contractile urethral responsiveness to bethanechol increased after both steroids. Urethral sensitivity to noradrenaline, evoking contraction, increased following estrogen. Further, estrogen abolished the marked relaxatory urethral response to nerve stimulation of castrated (and normal) rabbits and caused contraction only, which was abolished by a combination of alpha-adrenoceptor antagonists and scopolamine. When comparing the present results with those of other studies, it is evident that hormone-induced changes become manifest at an early stage. The present animal study gives support for the use of estrogen in the therapy of stress incontinence and, further, it provides no objections to the use of progesterone in combination with estrogen in this condition.

Cholinergic nerves of unknown origin in the parotid glands of rats

Abstract The effectiveness of the parasympathetic denervation of the gland caused by avulsion of the auriculo-temporal nerve was investigated. In the first type of experiment, the cholinesterase inhibitor, eserine, was injected through the salivary duct. The substance is thought to preserve acetylcholine leaking from nerve endings, thereby causing secretion of saliva; a secretory response was thus taken as evidence for the presence of cholinergic nerves. In the second type of experiment, the acetylcholinesterase activity of the gland was examined histochemically. From the denervated glands, eserine caused secretion and acetylcholinesterase positive nerves were found in the glands. Both the amount of secretion and the number of nerves were greatly diminished when compared with the intact glands. The results indicate that the denervation procedure was incomplete, leaving some cholinergic nerves unsevered. That cholinergic nerves remain offers an explanation to the residual choline acetyltransferase activity, which has earlier been shown in the gland after avulsion of the auriculo-temporal nerve. To extend the denervation procedure, either avulsion of the facial nerve or avulsion of the superior cervical ganglion was included. Neither procedure abolished the response to eserine or the acetyl-cholinesterase activity, indicating that the routes for the remaining nerves are to be found elsewhere.