Pharmacological characterization of dopamine receptors in the corpus allatum of Manduca sexta larvae

Dopamine receptors previously identified in corpora allata (CA) of Manduca sexta last instars on the basis of dopamine effects on JH (juvenile hormone)/JH acid biosynthesis and cyclic AMP (cAMP) accumulation, were characterized pharmacologically. For this study, a broad spectrum of agonists or antagonists of D1, D2, D3 or D4 dopamine receptors, together with the dopamine metabolite N-acetyl-dopamine, other neurotransmitters and their agonists/antagonists, were tested for their effects on gland activity and cAMP production. The lack of effect of other neurotransmitters supports the specificity of the effect of dopamine and the dopamine specificity of the receptors. Only the D2 receptor antagonist spiperone had a potent effect on JH biosynthesis and cAMP formation by CA taken on day 0 of the last stadium, when dopamine stimulates both activities and thus appears to be acting via a D1-like receptor. Several other D2 receptor antagonists, and D1, D2/D1 and D4,3/D2 receptor antagonists were less effective. Thus, the D1-like receptor of the Manduca CA appears to be distinct pharmacologically from vertebrate D1 receptors. By contrast, a number of D2 agonists/antagonists had a significant effect on JH acid biosynthesis and cAMP production by the CA from day 6 of the last stadium, when dopamine inhibits both activities and thus appears to be acting via a D2-like receptor. Certain D1-specific agonists/antagonists were equally effective. The Manduca D2-like receptor therefore bears some pharmacological resemblance to vertebrate D2 receptors. N-acetyl dopamine acted as a dopamine agonist with day 6 CA, the first identified function for an N-acetylated biogenic amine in insects. Dopamine was found to have the same differential affect on the formation of cAMP in homogenates of day 0 and day 6 brains as it did with CA, and in the same concentration range. Dopamine receptor agonists/antagonists affecting cAMP formation by day 0 and day 6 CA homogenates had similar effects with brain homogenates. By contrast, dopamine only stimulated cAMP formation by homogenates of day 0 and day 6 abdominal or ventral nerve cord. These results suggest that D1- and D2-like dopamine receptors of Manduca are regionally as well as temporally localized.     

Cellular metamorphosis. II. The larval labial gland duct and its prospective adult fates in the tobacco hornworm.

The larval labial gland of the sphingid moth, Manduca sexta, produces a viscous secretion, presumably a lubricant, facilitating the burrowing which precedes pupation. During metamorphosis, the gland transforms into a salivary organ, producing an invertase-rich digestive secretion. The single-cell type found in the duct of the larval gland transforms into the four structurally and functionally distinct cell types found in the four sequentially arranged secretory and conductive regions of the adult salivary gland. Surgical experiments were performed to study the prospective fates of different parts of the larval gland. The glands were bisected and one or both fragments were left in situ to undergo metamorphosis. In addition, fragments of the larval gland were implanted in pupal hosts and went through metamorphosis free of their prior attachments. The four linearly arrayed adult regions originate from correspondingly positioned areas in the larval duct.     

The effects of dopamine agonists and antagonists on the secretory responses in the salivary glands of the locust (Locusta migratoria)

A study has been made on the effect of dopamine on salivary gland secretion rates from isolated locust salivary glands. Application of dopamine induced a concentration-dependent secretion with an IC(50) of approximately 0.3 microM. We investigated the pharmacological profile of this receptor using dopaminergic agonists and antagonists. The effects of dopamine could be mimicked by the selective D1 agonist SKF82958, but not by the D2 agonist TNPA-HCl. The receptor also showed selectively towards certain D1 agonists. SKF82958 was more potent at inducing secretion than SKF81297. We found that dopamine-induced salivary secretions were blocked by the selective D1 antagonist SCH23390, whereas the D2 antagonist sulpiride was relatively ineffective. The cAMP analogue 8-Bromo cAMP also increased secretion rates from isolated salivary glands. These data and the rank order of potency of the agonists and antagonists in this screen suggest that this receptor is a D1-type receptor.     

The action of dopamine receptor antagonists on the secretory response of the cockroach salivary gland in vitro

1. A study has been made of the potency of a number of dopamine antagonists to inhibit dopamine-induced secretion from the cockroach salivary gland in vitro. 2. Chlorpromazine (0.5-5 microM), SCH23390 (10-100 microM), haloperidol (10-100 microM) and metoclopramide (2 mM) competitively inhibited the secretory response to dopamine. In contrast (+/-)sulpiride (1-100 microM) and domperidone (1-100 microM) had no effect on either basal or dopamine-induced secretion. 3. Apparent dissociation constants (KDapp) were obtained using a 'three point assay'. The rank order of potency (KDapp in parentheses) was as follows: chlorpromazine (0.2 microM) greater than SCH23390 (2.2 microM) greater than haloperidol (17.5 microM) much greater than metoclopramide (1.2 mM). 4. It is concluded that the receptor mediating dopamine-induced secretion in the cockroach salivary gland is similar to the D1/DA1 dopamine receptor and distinct from the D2/DA2 receptor found in mammalian systems.     

Morphological changes in gland cells and axons resulting from stimulation of the salivary nerves of the Cockroach, Nauphoeta cinerea

The salivary glands of the cockroach, Nauphoeta cinerea (Olivier, 1789), are innervated and there is considerable evidence to suggest that dopamine is the neurotransmitter at the neuroglandular junction. As the gland is a bilaterally symmetrical structure it was possible to electrically stimulate the salivary nerve supplying the ipsilateral side of the gland whilst the contralateral side of the gland served as a convenient control. Saliva elicited from the glands by electrical stimulation of these nerves was collected and used to monitor the physiological state of the tissue. Glands were fixed for light and electron microscopy during secretion and it was observed that the ductules in peripheral acinar cells were distended in stimulated sides of the glands but not in contralateral unstimulated sides. This evidence implies that peripheral cells are responsible for the initiation of salivary fluid secretion. Changes were also observed in the catecholamine containing axons that innervate the glands. In stimulated axons a statistically significant reduction in numbers of small agranular vesicles was observed when compared with contralateral unstimulated controls and freshly fixed tissue. This was not the case with the larger granular vesicles of the same axons which showed no reduction in number as a result of stimulation. In addition it was also noted that the small agranular vesicles tended to aggregate and change their shapes in response to nerve stimulation. These results imply that the small agranular vesicles play a role in transmitter release.     

Comparative histochemical study of glycoconjugates of the major salivary glands and pancreas in humans

The human parotid, submandibular, sublingual salivary glands and pancreas have been studied with lectin--horseradish peroxidase conjugates (con A, PNA, SBA, WGA, LAL), aldehyde fuchsin and Bismark brown. Intercalated duct cells produce a specific aldehyde-fuchsin-reactive substance. These cells are found only in the submandibular and parotid, but not in the sublingual glands. Similar reactivity is found in B-insulocytes of the pancreas. Aldehyde-fuchsin marks cytoplasmic granularity of the striated duct cells of all large salivary glands. This specific granularity is also selectively stained with Bismark brown and con A. Using fucose-specific lectin from Laburum anagyroides bark (LAL), granularity in serocytes of the submandibular gland is demonstrated. Some individual variations are observed in PNA binding to serocytes of the submandibular gland. It reveals that thyroglobulin-peroxidase conjugate (previously reported as an available second-step reagent for indirect lectin histochemical methods) non-specifically binds to the striated duct cells of the submandibular gland. During control staining it is also found, that DAB-reaction for endogenous peroxidase can be used as a test-system for a selective histochemical exposure of nuclear regions of endotheliocytes, pericytes and striated duct epitheliocytes of the human salivary glands. Possible significance of the phenomena observed is discussed.     

Histochemical properties of axons associated with the salivary apparatus of the cockroach, Nauphoeta cinerea

A variety of techniques have indicated that dopamine is probably the neurotransmitter at the salivary gland of the cockroach, Nauphoeta cinerea (Olivier). It is known from a previous ultrastructural study that two types of axon are associated with the gland but it is not known which of these axons contain catecholamines. The present study, using permanganate fixation or incubation in 5-hydroxydopamine or 6-hydroxydopamine, shows that only one category of axon contains catecholamines.     

Receptors for the neuropeptides,myoinhibitory peptide and SIFamide,in control of the salivary glands of the blacklegged tick Ixodes scapularis

Tick salivary glands are important organs that enable the hematophagous feeding of the tick. We previously described the innervation of the salivary gland acini types II and III by a pair of protocerebral salivary gland neurons that produce both myoinhibitory peptide (MIP) and SIFamide (?imo et al., 2009b). In this study we identified authentic receptors expressed in the salivary glands for these neuropeptides. Homology-based searches for these receptors in the Ixodes scapularis genome sequence were followed by gene cloning and functional expression of the receptors. Both receptors were activated by low nanomolar concentrations of their respective ligands. The temporal expression patterns of the two ligands and their respective receptors suggest that the SIFamide signaling system pre-exists in unfed salivary glands, while the MIP system is activated upon initiation of feeding. Immunoreactivity for the SIFamide receptor in the salivary gland was detected in acini types II and III, surrounding the acinar valve and extending to the basal region of the acinar lumen. The location of the SIFamide receptor in the salivary glands suggests three potential target cell types and their probable functions: myoepithelial cell that may function in the contraction of the acini and/or the control of the valve; large, basally located dopaminergic granular cells for regulation of paracrine dopamine; and neck cells that may be involved in the control of the acinar duct and its valve.     

Cell Junctions in Arthropod Ion-transport Systems

The epithelial cells involved in the movement of ions and waterform a major subset of all epithelial cell types. Both the formand the functions of cell junctions present in these cells areessentially the same as those found elsewhere. Gap junctionsare believed to regulate intercellular communication; desmosomesand hemidesmosomes provide mechanical anchorage to other cellsand the extracellular matrix; septate junctions play roles inproviding cell to cell anchorage, and perhaps in sealing thelateral surfaces of adjacent cells together to prevent paracellularfluid and solute movement; tight junctions (of limited distributionin insects) are seals between adjacent cells. They form a barrierto the paracellular movement of solutes and water. Examination of the junctions in salivary glands and midgut provideinsight into the roles of these junctions in the developmentand function of ion transport systems. In Manduca sexta (Johannsen)the cells of the salivary gland are joined by pleated septateand gap junctions. Individual salivary cells have numerous foldsand canaliculi. The walls of the canaliculi consist of extensivelyfolded plasma membrane in intimate association with mitochondria.Gap junctions connect adjacent parts of the same cell acrossmembrane folds, effectively shortening diffusion distances inthe cells. Hemidesmosomes are present in the walls of developingcanaliculi. They are attached to pore filaments that occupythe lumen of the developing canaliculi. The hemidesmosomes andpore filaments may have a morphogenetic role as they disappearafter the canaliculi are formed. In Manduca sexta the midgut cells are joined by gap and septatejunctions. These junctions differ in morphology from their counterpartsin the salivary gland; physiological studies show the gobletcells are not coupled to neighboring tall columnar cells. Wehave shown the gap junctions joining them are typical of non-couplingjunctions. Preliminary studies suggest that the gap junctionschange form when the cells are coupled.     

Characteristics of sialidase in the rat salivary glands

Using 4-methylumbelliferyl-N-acetylneuraminic acid (4MU-NeuAc) as substrate, we measured sialidase activity in the salivary glands and other organs of the rat. The pH optima of salivary gland sialidase were between 4.0 and 4.5, which were similar to those of the enzyme in the brain, liver and kidney. Among the salivary glands, the submandibular one showed the highest sialidase activity followed by the parotid and the sublingual glands. However, sialidase activity in these glands was lower when compared with the activity in the brain, liver and kidney. From the subcellular distribution study, salivary gland sialidase was found to be mainly localized in the lysosomes. The pH optima of the lysosomal sialidase of the salivary glands were between 4.0 and 4.5; and Km values for 4MU-NeuAc approximately 0.09 mmol/l. In the submandibular and parotid glands, a soluble sialidase with a different pH optimum (5.5) and Km value (0.25 mmol/l) was also detected.     

Dopamine induces hyperpolarization of locust salivary gland acinar cells via D(1)-like receptors

The effect of dopamine on the salivary gland acinar cells of the locust was examined using conventional intracellular recording techniques. Application of dopamine induced a reversible, dose-dependent hyperpolarization of the acinar cells, with an EC(50) of 0.1 &mgr;M dopamine. We investigated the pharmacology of the dopamine receptor mediating hyperpolarization of the acinar cells using a range of dopaminergic agonists and antagonists. The effect of dopamine could be mimicked by the selective D(1) receptor agonist SKF82958, whilst the D(2) receptor agonists PPHT-HCl and TNPA-HBr were far less potent at inducing hyperpolarization. The receptor also showed selectivity to certain synthetic D(1)-like agonists. SKF82958 was much more effective at inducing a hyperpolarization than SKF81297. The dopamine-induced hyperpolarization of locust acinar cells could be blocked using the selective D(1) receptor antagonist SCH23390 whilst the D(2) receptor antagonists sulpiride and spiperone were inactive. The rank order of potency of several dopaminergic agonists and antagonists was obtained and suggests that the dopamine receptor mediating the hyperpolarization in locust salivary gland acinar cells is similar to a mammalian D(1) receptor. Stimulation of the salivary nerve mimicked the effect of dopamine on the acinar cells, inducing a rapid reversible hyperpolarization. This neurally-evoked hyperpolarization of the locust acinar cells was suppressed using 1.0 &mgr;M SCH23390, whilst 10 &mgr;M sulpiride was inactive. This demonstrated that both exogenously applied dopamine and endogenously released dopamine are probably acting on the same receptor.     

Plant growth-promoting factor in the salivary gland of the bug, Lygus disponsi

In the salivary gland of Lygus disponsi, the factor promoting the activity of 3-indoleacetic acid (IAA), or the factor inhibiting the activity of IAA-oxidase, was present. It showed a maximum activity in fraction III of the salivary gland solution, 1·6 times as high as that of the IAA control. The significance of the factor in the salivary gland is discussed with respect to injury (malformation). Auxin was not found in the salivary gland.     

Phospholipase A2 in salivary glands isolated from tobacco hornworms, Manduca sexta

We describe a phospholipase A2 (PLA2) associated with the salivary glands of tobacco hornworms, Manduca sexta. This enzyme is able to hydrolyze arachidonic acid from the sn-2 position of PLs. Addition of the calcium chelator, EGTA, or calcium, to the Tris reaction buffer impaired the PLA2 activity, from which we infer the enzyme requires very low concentrations of calcium or perhaps other ions for optimal activity. PLA2 activity was sensitive to protein concentration (highest activity at 25 microg protein per microl), reaction time (optimal at 30 min), buffer pH (optimal at pH 8-10), and reaction temperature (optimal range 18-38 degrees C). The salivary gland PLA2 was sensitive to the site-specific inhibitor, oleyloxyethylphosphorylcholine and stable to freezing at -80 degrees C, but not -20 degrees C. The biological significance of this enzyme may relate to hydrolysis of fatty acid moieties from dietary PLs for absorption by midgut epithelia. This salivary gland enzyme may also be responsible for killing food-borne bacteria.     

Aberrant Activation of the RANK Signaling Receptor Induces Murine Salivary Gland Tumors

Unlike cancers of related exocrine tissues such as the mammary and prostate gland, diagnosis and treatment of aggressive salivary gland malignancies have not markedly advanced in decades. Effective clinical management of malignant salivary gland cancers is undercut by our limited knowledge concerning the key molecular signals that underpin the etiopathogenesis of this rare and heterogeneous head and neck cancer. Without knowledge of the critical signals that drive salivary gland tumorigenesis, tumor vulnerabilities cannot be exploited that allow for targeted molecular therapies. This knowledge insufficiency is further exacerbated by a paucity of preclinical mouse models (as compared to other cancer fields) with which to both study salivary gland pathobiology and test novel intervention strategies. Using a mouse transgenic approach, we demonstrate that deregulation of the Receptor Activator of NFkB Ligand (RANKL)/RANK signaling axis results in rapid tumor development in all three major salivary glands. In line with its established role in other exocrine gland cancers (i.e., breast cancer), the RANKL/RANK signaling axis elicits an aggressive salivary gland tumor phenotype both at the histologic and molecular level. Despite the ability of this cytokine signaling axis to drive advanced stage disease within a short latency period, early blockade of RANKL/RANK signaling markedly attenuates the development of malignant salivary gland neoplasms. Together, our findings have uncovered a tumorigenic role for RANKL/RANK in the salivary gland and suggest that targeting this pathway may represent a novel therapeutic intervention approach in the prevention and/or treatment of this understudied head and neck cancer.     

Distribution of lysozyme and protease, and amino acid concentration in the guts of a wood-feeding termite, Reticulitermes speratus (Kolbe): possible digestion of symbiont bacteria transferred by trophallaxis

Distribution of lysozyme and protease, and amino acid concentration in the guts of a wood‐feeding termite, Reticulitermes speratus (Kolbe) (Isoptera, Rhinotermitidae) were studied to examine the possibility that termites digest symbiont bacteria transferred by trophallaxis. Total lysozyme activity was found predominantly in the salivary gland and to a minor extent in the digestive tracts. However, specific lysozyme activity was high in the foregut as well as in the salivary gland. The similarity of the lysozyme pH profile of the salivary gland and of the foregut suggested that the foregut lysozyme came from the salivary gland. Major protease activity having the optimum pH of 7.5 was found in the midgut. Total free amino acid amount and concentration in the midgut was higher than elsewhere in the digestive tract. The possibility that lysozyme secreted from the salivary gland into the foregut digests hindgut bacteria transferred by trophallaxis was discussed.     

Fine structure of axons associated with the salivary apparatus of the cockroach, Nauphoeta cinerea

Previous studies have shown that the neurotransmitter at the salivary gland of the cockroach is probably dopamine. An ultrastructural study of the innervation of these glands was made. It was concluded that there are two types of axon present designated type A and type B. Type A axons possess small agranular elliptical vesicles and large granular vesicles, whereas type B possess only large granular vesicles of larger diameter than those found in type A. The relationship of these types of axon to the cells of the acini is discussed. It is concluded that type A axons are catecholaminergic.     

Human salivary gland branching morphogenesis: morphological localization of claudins and its parallel relation with developmental stages revealed by expression of cytoskeleton and secretion markers

Development of salivary glands is a highly complex and dynamic process termed branching morphogenesis, where branched structures differentiate into mature glands. Tight junctions (TJ) are thought to play critical roles in physiological functions of tubular organs, contributing to cell polarity and preventing lateral movement of membrane proteins. Evidence demonstrated that claudins are directly involved in TJ formation and function. Using immunohistochemistry and immunofluorescence we have mapped the distribution of claudins-1, 2, 3, 4, 5, 7 and 11 and compared it with the expression of differentiation markers in human salivary glands obtained from foetuses ranging from weeks 4 to 24 of gestation. Expression of all claudins, except claudin-2 was detected in the various phases of human salivary gland development, up to fully mature salivary gland. The expression of all claudins increased according to the progression of salivary gland maturation evidenced by the classical markers-cytokeratin 14, cytokeratin low molecular weight, smooth muscle actin and human secretory component. Tight junction proteins-claudins appear to be important in the final shape and physiological functions of human salivary glands and are parallel related with markers of salivary gland differentiation.     

The role of endogenous regulators in the compensatory process after salivary gland resection

The ways of penetration of submandibular salivary degeneration products into the blood after partial gland resection have been studied on 97 rats. Using isoenzyme LDH spectrum of the blood, as well as of gland and regional lymph node tissue, the acid-base balance values and morphological data, it has been shown that degeneration products of salivary glands penetrate into the general blood flow through regional lymph nodes, where they are partly modified. The true changes in LDH spectrum can be already registered half an hour after the injury of the gland. The influence of the degeneration products of gland tissue on the initiation of the compensation processes in cellular hyperplasia is discussed.