Identification of SNARE and cell trafficking regulatory proteins in the salivary glands of the lone star tick,Amblyomma americanum (L.)

Prostaglandin E(2) (PGE(2)) stimulates secretion of tick salivary gland proteins via a phosphoinositide signaling pathway and mobilization of intracellular Ca(2+) (). Highly conserved intracellular SNARE (soluble NSF attachment protein receptors) complex proteins are associated with the mechanism of protein secretion in vertebrate and invertebrate neuronal and non-neuronal cells. Proteins in the salivary glands of partially fed female lone star ticks cross-react individually with antibodies to synaptobrevin-2 (vesicle (v)-SNARE), syntaxin-1A, syntaxin-2 and SNAP-25 (target (t)-SNAREs), cytosolic alpha/beta SNAP and NSF (N-ethylmaleimide-sensitive fusion protein), Ca(2+) sensitive synaptotagmin, vesicle associated synaptophysin, and regulatory cell trafficking GTPases Rab3A and nSec1. V-SNARE and t-SNARE proteins form an SDS-resistant, boiling sensitive core complex in the salivary glands. Antibodies to SNARE complex proteins inhibit PGE(2)-stimulated secretion of anticoagulant protein in permeabilized tick salivary glands. We conclude that SNARE and cell trafficking regulatory proteins are present and functioning in the process of PGE(2)-stimulated Ca(2+) regulated protein secretion in tick salivary glands.     

Immunocytochemistry of myoepithelial cells in the salivary glands

MECs are distributed on the basal aspect of the intercalated duct and acinus of human and rat salivary glands. However, they do not occur in the acinus of rat parotid glands, and sometimes occur in the striated duct of human salivary glands. MECs, as the name implies, have structural features of both epithelial and smooth muscle cells. They contract by autonomic nervous stimulation, and are thought to assist the secretion by compressing and/or reinforcing the underlying parenchyma. MECs can be best observed by immunocytochemistry. There are three types of immunocytochemical markers of MECs in salivary glands. The first type includes smooth muscle protein markers such as -SMA, SMMHC, h-caldesmon and basic calponin, and these are expressed by MECs and the mesenchymal vasculature. The second type is expressed by MECs and the duct cells and includes keratins 14, 5 and 17, 1β1 integrin, and metallothionein. Vimentin is the third type and, in addition to MECs, is expressed by the mesenchymal cells and some duct cells. The same three types of markers are used for studying the developing gland.

Development of MECs starts after the establishment of an extensively branched system of cellular cords each of which terminates as a spherical cell mass, a terminal bud. The pluripotent stem cell generates the acinar progenitor in the terminal bud and the ductal progenitor in the cellular cord. The acinar progenitor differentiates into MECs, acinar cells and intercalated duct cells, whereas the ductal progenitor differentiates into the striated and excretory duct cells. Both in the terminal bud and in the cellular cord, the immediate precursors of all types of the epithelial cells appear to express vimentin. The first identifiable MECs are seen at the periphery of the terminal bud or the immature acinus (the direct progeny of the terminal bud) as somewhat flattened cells with a single cilium projecting toward them. They express vimentin and later -SMA and basic calponin. At the next developmental stage, MECs acquire cytoplasmic microfilaments and plasmalemmal caveolae but not as much as in the mature cell. They express SMMHC and, inconsistently, K14. This protein is consistently expressed in the mature cell. K14 is expressed by duct cells, and vimentin is expressed by both mesenchymal and epithelial cells.

After development, the acinar progenitor and the ductal progenitor appear to reside in the acinus/intercalated duct and the larger ducts, respectively, and to contribute to the tissue homeostasis. Under unusual conditions such as massive parenchymal destruction, the acinar progenitor contributes to the maintenance of the larger ducts that result in the occurrence of striated ducts with MECs. The acinar progenitor is the origin of salivary gland tumors containing MECs. MECs in salivary gland tumors are best identified by immunocytochemistry for -SMA. There are significant numbers of cells related to luminal tumor cells in the non-luminal tumor cells that have been believed to be neoplastic MECs.     

Control of salt gland activity in the hatchling green sea turtle, Chelonia mydas

 We studied the control of salt gland secretion in hatchling Chelonia mydas. The threshold salt load to activate salt secretion was between 400 μmol NaCl 100 g bodymass (BM)⁻¹ and 600 μmol NaCl 100 g BM⁻¹, which caused an increase in plasma sodium concentration of 13% to 19%. Following a salt load of 2700 μmol NaCl 100 g BM⁻¹, salt gland secretion commenced in 12 ± 1.3 min and reached maximal secretory concentration within 2–7 min. Maximal secretory rate of a single gland averaged 415 μmol Na 100 g BM⁻¹ h⁻¹. Plasma sodium concentration and total osmotic concentration after salt loading were significantly higher than pretreatment values within 2 min. Adrenalin (25 μg kg BM⁻¹) and the cholinergic agonist methacholine (1 mg kg BM⁻¹) inhibited salt gland activity. Atropine (10 mg kg BM⁻¹) reversed methacholine inhibition and stimulated salt gland secretion when administered with a subthreshold salt load. Arginine vasotocin produced a transient reduction in sodium secretion by the active gland, while atrial natriuretic factor, vasoactive intestinal peptide and neuropeptide Y had no measurable effect on any aspect of salt gland secretion. Our results demonstrated that secretion of the salt gland in C. mydas can be modified by neural and hormonal chemicals in vivo and that the cholinergic and adrenergic stimulation of an exocrine gland do not appear to have the typical, antagonist actions on the chelonian salt gland. Accepted: 28 September 1999     

Activation of the Na-K(NH4)-2Cl-cotransporter from rat submandibular glands in response to VIP

A cellular suspension from rat submandibular glands was prepared with collagenase. The intracellular pH (pH_i) was estimated with 2′,7′-bis-(2-carboxy-ethyl)-5(6)-carboxyfluorescein (BCECF). After exposure to NH₄Cl, the pH_i transiently increased (diffusion of NH₃) and then dropped (influx of NH₄⁺). Isoproterenol increased 2.5-fold the rate of NH₄⁺ influx; bumetanide, an inhibitor of the Na⁺-K⁺-2Cl⁻-cotransporter blocked the response to isoproterenol, confirming that the beta-adrenergic agonist stimulated the cotransporter. Forskolin (1 μmol/L) mimicked the response to isoproterenol. VIP (1 nmol/L-1 μmol/L) also increased the activity of the cotransporter. Cyclic AMP rather than calcium was the mediator of this activation since 1) carbachol which increased the [Ca²⁺]_i fivefold increased the uptake of NH₄⁺ by only 50%; 2) only high concentrations of VIP significantly increased the [Ca²⁺]_i; 3) incubation in the presence of EGTA had no effect on the response to VIP; 4) low concentrations (nmol/L) of the neuropeptide increased the intracellular level of cAMP; and 5) the stimulation of the cotransporter by VIP, forskolin, and isoproterenol was inhibited by H8, an inhibitor of cAMP-dependent protein kinase. It is concluded that the Na⁺-K⁺-2Cl⁻-cotransporter of rat submandibular glands is activated by isoproterenol, forskolin, and neuropeptides of the VIP family by a mechanism involving cAMP-dependent processes. The activation of the cotransporter by VIP could partly explain the potentiating effect of VIP on the response to sialagogues like substance P or muscarinic agonists.¹     

Antioxidant enzymatic defense in salivary glands of streptozotocin‐induced diabetic rats: a temporal study

Hyperglycemia induces overproduction of superoxide and it is related to diabetic complications. In this study, we analyzed the antioxidant enzymatic defense and the lipid peroxidation of rat salivary glands in six different periods of diabetic condition. Ninety‐six rats were divided into 12 groups: C7/14/21/28/45/60 (non‐diabetic animals) and D7/14/21/28/45/60 (diabetic animals). Diabetes was induced by streptozotocin and the rats were euthanized after 7, 14, 21, 28, 45, or 60 days. Their parotid (PA) and submandibular (SM) glands were removed soon after the sacrifice and the total protein and malondialdehyde (MDA) concentrations, as well as, the superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) activities were determined. Twenty‐one days after the diabetes induction, the SM glands showed an increase in SOD, CAT, and GPx activities, as well as, MDA concentration. Concerning the PA glands, an increase in the CAT activity and MDA content was observed throughout the observation period. The results suggest that diabetes can cause alterations on the salivary glands and that PA and SM glands react differently when exposed to diabetes condition. However, no impairment of antioxidant system was observed in the group whose diabetic condition had been induced 60 days earlier, herein named 60‐day group. Copyright © 2010 John Wiley & Sons, Ltd.     

Accessory sex glands as a tool to measure the efficacy of immunocastration in male pigs

The use of an anti-gonadotropin-releasing hormone vaccine for immunocastration of male pigs has been recently approved in the European Union. This technique is potentially useful for avoiding both castration-associated pain for the animal and boar taint in pork. However, some animals may escape immunocastration and be slaughtered as entire males, potentially exhibiting boar taint. Therefore, it is important to check the efficacy of immunocastration on the slaughter line. To achieve that, the currently proposed method, based on testis weight, is not fully reliable because there is some overlap in the distributions of testis weight between immunocastrates and entire males. On the basis of literature data on the effect of immunocastration on the development of accessory sex glands, this paper provides evidence that the weight of seminal vesicles might be a much better criterion for checking the efficacy of immunocastration, because their size decreases more rapidly, and to a greater extent, than that of the testis.     

Shoot anatomy and secretory structures in Hypericum species (Hypericaceae)

The anatomy and ultrastructure of internodes, leaves and petals were compared in Hypericum elegans, H. inodorum, H. olympicum, H. forrestii and two genotypes of H. perforatum. Internode anatomy was variable between species with respect to the structure of the cortical and pith parenchyma, including the presence of secretory reservoirs. Also, the secondary growth was more extensive in shrubs, i.e. H. inodorum and H. forrestii. In leaves, phloem secretory reservoirs were formed in all species, mesophyll secretory reservoirs were absent only in H. elegans and internal nodules were present only in H. elegans and H. perforatum. The petals differed between species in the mesophyll structure and the occurrence and location of secretory structures. The phloem secretory reservoirs lacked sheaths, whereas these were distinct in the mesophyll reservoirs. Other ultrastructural traits of the reservoirs were similar in all the species studied, with the exception of the leucoplast ultrastructure. In internal nodules, the inner cells vs. sheath cells differed in the number of vesicles and other membranous structures and plastid ultrastructure. © 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 163 , 70–86.