Interaction of calcitonin gene-related peptide (CGRP), substance P (SP) and conventional autonomic agonists in rat submandibular salivary peroxidase release in vitro.

Our previous immunohistochemical studies reveal that several neuropeptides, such as substance P and calcitonin gene-related peptide, innervate the major salivary glands of the mouse, rat and human. The aim of the study was to clarify their interactions by measuring their effects alone or with conventional autonomic agonists (carbachol, phenylephrine and isoproterenol) on peroxidase secretion of incubated submandibular gland slices. Calcitonin gene-related peptide evoked significant increase in peroxidase activity of the secretion only when used at 10(-5) M concentration, while substance P evoked significant, dose-dependent increase at much lower concentrations (10(-10) M). Adrenergic and cholinergic agonists enhanced peroxidase activity. Interestingly, substance P inhibited both phenylephrine and isoproterenol induced increase in peroxidase activity. Calcitonin gene-related peptide did not affect the inhibition caused by substance P. Our results demonstrate that in the salivary gland tissue substance P alone or in conjunction with adrenergic agonists result in opposing secretory responses with the doses used in vitro. Conversely, the response mediated by adrenergic receptors may be critically affected by simultaneous occupation of substance P receptors.

Nitric oxide synthase in the autonomic and sensory ganglia innervating the submandibular salivary gland.

This article reviews the neuroanatomical studies on the distribution of nitric oxide synthase (NOS) in neurons and nerve fibers innervating the submandibular gland. Specificity of NADPH-diaphorase activity as a histochemical marker of neuronal NOS is discussed in light of corresponding NOS immunoreactivity. Anatomical data suggest that nitric oxide may affect neural regulation of the submandibular gland through both sympathetic, parasympathetic and sensory divisions of the autonomic nervous system. NOS-containing nerve terminals in the gland parenchyme are mainly vascular and either parasympathetic and/or sensory in nature, while sympathetic terminals lack NOS. Most postganglionic parasympathetic neurons are intensely NOS-immunoreactive. Some of the preganglionic parasympathetic neurons show vague reactivity, while their terminals in the submandibular ganglia stain heavily. The postganglionic sympathetic neurons normally show only barely visible reactivity, while manipulations interrupting axonal continuity increase neuronal NOS content. A subpopulation of the preganglionic sympathetic neurons and their terminals are intensely reactive. The observations summarized here suggest that nitric oxide participates in the control of blood flow through the gland, while direct effect on secretion is unlikely.

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

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.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistochemistry of enkephalins: model studies on hapten-carrier conjugates and fixation methods.

For immunohistochemical demonstration of the enkephalin octapeptide Met5-enkephalin-Arg6-Gly7-Leu8, the peptide was conjugated with a carrier protein using either glutaraldehyde or 1-ethyl-3 (3-dimethylaminopropyl)-carbodiimide as coupling agent. Antisera were raised in rabbits and their specificity was studied using the immunoblotting technique. The results suggest that glutaraldehyde selectively couples the amino terminus of the peptide to the carrier protein, while carbodiimide coupling produces a mixture of specificities. Accordingly, antiserum raised against the glutaraldehyde-induced conjugate specifically recognized the peptide carboxyl terminus and allowed immunohistochemical distinction of the octapeptide from other closely related opioid peptides, such as Leu5- and Met5-enkephalin, Met5-enkephalin-Arg6-Phe7, and Phe1-Met2-Arg3-Phe4-NH2. In contrast, antiserum raised against the carbodiimide-induced octapeptide conjugate showed a mixture of specificities. Addition of glutaraldehyde to the fixative enhanced octapeptide immunoreactivity in several tissues and revealed a previously unknown nerve system in the pituitary gland. These results support the idea that optimal immunohistochemical demonstration of small molecules, which requires conjugation to a carrier protein, is obtained when the coupling agent is included in the fixative so as to induce the actual coupling reaction during fixation.

Met5-enkephalin-Arg6-Gly7-Leu8-immunoreactive nerve fibers in the major salivary glands of the rat: evidence for both sympathetic and parasympathetic origin.

The localization of the proenkephalin A-derived octapeptide, Met5-enkephalin-Arg6-Gly7-Leu8 (MEAGL), was studied in the major salivary glands of Sprague-Dawley and Wistar rats with the indirect immunofluorescence method. MEAGL-immunoreactive nerve fibers were found around the acini, along intra- and interlobular salivary ducts and in close contact with blood vessels. In the parotid and submandibular glands tyrosine hydroxylase (TH) immunoreactivity was observed in nerve fibers around the acini, in association with intra- and interlobular salivary ducts and around blood vessels, while in the sublingual gland TH-immunoreactive nerve fibers were only seen around blood vessels. Parasympathetic neurons in submandibular ganglia contained MEAGL immunoreactivity. Moderate TH immunoreactivity was seen in some neurons of the submandibular ganglia. A subpopulation of sympathetic principal neurons in the superior cervical ganglion were immunoreactive for both MEAGL and TH. In the trigeminal ganglion, no MEAGL-immunoreactive sensory neurons or nerve fibers were observed. Superior cervical ganglionectomies resulted in a complete disappearance of TH-immunoreactive nerve fibers, while MEAGL-immunoreactive nerve fibers were still present in the glands. The presence of MEAGL immunoreactivity in neurons of both sympathetic superior cervical ganglia and parasympathetic submandibular ganglia and the results of superior cervical ganglionectomies suggest, that MEAGL-immunoreactive nerve fibers in the major salivary glands of the rat have both sympathetic and parasympathetic origin.

Immunohistochemical localization of [Met5]enkephalin and [Met5]enkephalin-Arg6-Gly7-Leu8 in sympathetic and parasympathetic neurons and nerve fibers projecting to the rat submandibular gland.

The localization of [Met5]enkephalin, [Met5]enkephalin-Arg6-Gly7-Leu8, vasoactive intestinal polypeptide and tyrosine hydroxylase immunoreactivities was studied in the submandibular gland of adult Sprague-Dawley and Wistar rats using the indirect immunofluorescence technique. Immunoreactivities for [Met5]enkephalin and [Met5]enkephalin-Arg6-Gly7-Leu8, a proenkephalin A-derived octapeptide, showed identical distributions. A large number of enkephalin-immunoreactive nerve fibers were detected around secretory acini, along intercalated ducts, convoluted granular tubules, intra- and interlobular ducts, as well as in close contact with blood vessels. The submandibular ganglia contained several enkephalin-immunoreactive neurons and nerve fibers. In the superior cervical ganglion numerous enkephalin-immunoreactive neurons and nerve fibers were also detected. Immunohistochemical co-localization studies indicated that [Met5]enkephalin and [Met5]enkephalin-Arg6-Gly7-Leu8 immunoreactivities co-exist with vasoactive intestinal polypeptide in a subpopulation of neurons of the rat submandibular ganglia, in nerve trunks along the salivary ducts of the gland, and in nerve fibers around the acini. Uni- or bilateral superior cervical ganglionectomies for 1-4 weeks resulted in a complete disappearance of tyrosine hydroxylase immunoreactivity in the glandular parenchyma, while moderate tyrosine hydroxylase immunoreactivity was seen in some neurons of the submandibular ganglia. Abundant [Met5]enkephalin-Arg6-Gly7-Leu8-immunoreactive nerve fibers were still seen around the acini and blood vessels, as well as close to salivary ducts. These operations did not affect the [Met5]enkephalin-Arg6-Gly7-Leu8-immunoreactive neurons in the submandibular ganglia. Many principal neurons in the superior cervical ganglion contained both [Met5]enkephalin-Arg6-Gly7-Leu8 and tyrosine hydroxylase immunoreactivity. Nerve ligation experiments indicated that [Met5]enkephalin-Arg6-Gly7-Leu8-immunoreactive sympathetic fibers project along the external carotid nerve. Accordingly, nerve fibers were found around the acini and blood vessels as well as in nerve trunks along the salivary ducts of the submandibular gland, showing co-localization of [Met5]enkephalin-Arg6-Gly7-Leu8 and tyrosine hydroxylase. Taken together, these observations suggest that the nerve fibers of the rat submandibular gland containing proenkephalin A-derived peptides are of both sympathetic and parasympathetic origin.

CGRP-immunoreactive sensory nerve fibers in the submandibular gland of the rat.

Indirect immunofluorescence technique was used to study the occurrence and distribution of CGRP immunoreactivity in the submandibular gland of normal rats and after unilateral sensory and sympathetic denervations. In normal rats, CGRP-immunoreactive nerve fibers and nerve trunks were seen around or in close contact with interlobular salivary ducts as well as around small blood vessels of the gland. Occasionally, CGRP-immunoreactive nerve fibers were also detected between or around the acini of the gland. The submandibular ganglia contained CGRP-immunoreactive nerve fibers, but the ganglion cells were not immunoreactive for CGRP. The trigeminal ganglion contained a population of CGRP-immunoreactive, mainly small sized ganglion cells and nerve fibers distributed throughout the ganglion. Unilateral electrocoagulation of the trigeminal nerve caused a significant reduction in the number of immunoreactive nerve fibers in the gland, although some fibers still were present in the ipsilateral glandular tissue. Unilateral superior cervical ganglionectomy caused no detectable effect on the number of CGRP-immunoreactive nerve fibers in the gland. The present results suggest that the rat submandibular gland contains CGRP-immunoreactive nerve fibers both around blood vessels and in glandular secretory elements. Denervation experiments support the view that the majority, but perhaps not all of them originate from the trigeminal ganglion.