Involvement of calcium and cyclic AMP in controlling ixodid tick salivary fluid secretion.

Catecholamine-stimulated salivary fluid secretion (in vitro) by ixodid ticks is reduced by deletion or lowering the concentration of exogenous bathing medium Ca++. The Ca++ antagonist, verapamil, reversibly inhibits dopamine-stimulated secretion. Ionophore A-23187 is unable to induce glands to secrete. Studies in which labeled and unlabeled Ca++ flux were measured indicate that catecholamines induce release of calcium from intracellular stores during secretion. Cyclic AMP/theophylline-stimulated secretion is inhibited by verapamil, and the exclusion of calcium from the support medium. It is concluded that the primary catecholamine stimulus induces cyclic AMP formation and mobilization of Ca++ (intra- and extracellular). Cyclic AMP and calcium are thought to interact to control secretion within the fluid transporting cells of types II and III alveoli.     

Brain factor induced formation of inositol phosphates in tick salivary glands

A factor from tick brain increases inositol phosphates in isolated, whole tick salivary glands. The factor is sensitive to trypsin and heat (5 min, 100°C) suggesting that it may be a neuropeptide or protein. The salivary glands undergo growth and differentiation accompanied by considerable proliferation of plasma membranes during tick feeding. Salivary glands from ticks in later stages of feeding produce higher levels of inositol phosphates than glands from ticks in early stages of feeding. Cyclic AMP modulates the formation of inositol phosphates suggesting interaction of salivary gland function by the transducing system that employs cyclic AMP as a “second messenger” and that which employs inositol phosphates.     

Protein phosphorylation and control of tick salivary gland function

Tick salivary glands are controlled by nerves, dopamine being a neurotransmitter at the neuroeffector junction. Dopamine and cyclic AMP (cAMP) stimulate fluid secretion by isolated salivary glands. Dopamine activates an adenylate cyclase to increase intracellular cAMP within the female salivary glands. Phosphoproteins whose levels of phosphate are affected by cAMP-dependent protein kinase have been identified in subcellular fractions. Protein(s) phosphorylated by cAMP appears to activate protein phosphatase in the salivary glands.Another phosphorylation pathway appears to act through protein kinase C because of an ability of phorbol esters (known activators of protein kinase C) to stimulate the phosphorylation of proteins, and an ability of a peptide factor in tick brain to metabolize salivary-gland phosphoinositides, an event that often precedes activation of protein kinase C. Because cAMP modulates brain-factor-stimulated formation of inositol phosphates (products of phosphoinositide breakdown) an interrelationship between the two pathways seems likely.Evidence of regulatory processes, including protein phosphorylation'dephosphorylation reactions, will provide a basis for helping asses the physiological significance of secretory products and the role of the salivary glands in disease transmission.     

Salivary fluid secretion in the ixodid tick Rhipicephalus appendiculatus is inhibited by Thogoto virus infection

Adult Rhipicephalus appendiculatus ticks, infectedwith Thogoto (THO) virus or control, were fed on guinea pigs and removed atintervals throughout the feeding cycle. Salivary fluid secretion was measuredbyan in vitro technique. The salivary glandsof infected, partially-fed ticks secreted fluid in vitro at about 75% the rateof controls, but the difference between infected and controls among engorgedticks was not statistically significant. Basal and DA-stimulated levels ofcyclic AMP (cAMP) were determined in isolated glands and were significantlyaffected by THO virus infection. The differences in secretory rate amongcontroland infected ticks could not be explained in terms of altered cAMP levels.Haemolymph volume was measured by a tracer-dilution technique using³H-inulin. The mean haemolymph volume for both THO-infected andcontrol groups was between 23–24% body weight throughout the feedingcycle, indicating that infection by this arbovirus did not influence salivaryfluid secretion via altered haemolymph volume. The mechanism by which THO virusaffects secretory activity of its tick vector remains unknown.     

Changes in cyclic AMP and cyclic GMP concentrations during the action of 5-hydroxytryptamine on an insect salivary gland.

Salivary glands from adult blowflies (Calliphora erythrocephala Meigen) were studied in vitro. The time course of changes in cyclic AMP content of the glands was followed at different concentration of 5-hydroxytryptamine. There was an immediate biphasic rise and fall in cyclic AMP content, following by a slower rise and subsequent gradual decline. The initial rise preceded the onset of fluid secretion by the glands. Rises in cyclic AMP content were inhibited by compound RMI 12330 A (an adenylate cyclase inhibitor) and were halted after about 15-20s if the glands were deprived of Ca2+. Theophylline (a phosphodiesterase inhibitor) abolished the decline phase of the fast response, Losses of cyclic AMP from the glands either to the bathing medium or to the saliva were small and could not account for the rapid fall found. Evidence is presented that cyclic GMP is not involved in the process of initiating secretion in the blowfly salivary gland.     

Rhipicephalus (Boophilus) microplus (Canestrini, 1887) (Acari: Ixodidae): acid phosphatase and ATPase activities localization in salivary glands of females during the feeding period

This study investigates the presence and the localization of acid phosphatase and ATPase in the salivary glands of Rhipicephalus (Boophilus) microplus female ticks during feeding. Semi-engorged females showed a larger amount of acid phosphatase compared to those at beginning of feeding, localized mainly in the apical portion of the secretory cells, and in the basal labyrinth of the interstitial cells. Ultrastructural observations also demonstrated its presence in secretion granules and inside some nuclei of secretory cells at beginning of feeding. Acid phosphatase in a free form probably has a hemolymph and/or ribosomal origin and participates in salivary gland secretion control. ATPase was detected in basal membrane of all types of acini and/or in the cytoplasm of the secretory cells at both feeding stages. The enzyme activities found strongly suggests that cell death by apoptosis occurs during the degenerative process.     

Effects of octopamine on fluid secretion by isolated salivary glands of a feeding ixodid tick

Octopamine elicited a dose-related secretory response by salivary glands isolated from the feeding female tick Amblyomma americanum. Half-maximal stimulation occurred at about 60 μM. Phentolamine (10 μM) failed to inhibit the octopamine-mediated response; however, thioridazine (50 μM) inhibited both octopamine (1,000 μM) and dopamine-stimulated (0.1 μM) secretion. Maximal stimulation by dopamine (1.0 μM) showed no further increase in the rate of secretion after adding octopamine (1,000 or 0.1 μM). Glands responded to octopamine (100 μM) with rates significantly lower than controls following exposure to amphetamine (1,000 μM). Octopamine receptors do not appear to mediate the secretory response, and octopamine may stimulate secretion by releasing catecholamines from presynaptic neurons. These results support the hypothesis that dopamine is the natural transmitter mediating fluid secretion in the feeding tick salivary gland.     

Cyclic AMP and calcium modulated ATPase activity in the salivary glands of the lone star tick Amblyomma americanum (L.)

Na⁺,K⁺-activated ATPase activity in tick salivary glands increases during the rapid stage of tick feeding paralleling similar increases in dopamine and cAMP-stimulated fluid secretion. High concentrations of cyclic AMP increase Na⁺,K⁺-ATPase activity in a plasma membrane-enriched fraction from the salivary glands of rapidly feeding ticks. Cyclic AMP-dependent protein kinase inhibitor protein blocks activation of Na⁺,K⁺-ATPase activity at low but not high concentrations of cAMP indicating that both activator and inhibitor modulator phosphoproteins of Na⁺,K⁺-ATPase activity exist in the plasma membrane-enriched fraction.ATPase activity in the plasma membrane-enriched fraction is not measurable in the absence of Mg²⁺, Ca²⁺ and Na⁺. Ca-stimulated nucleotidase activity is highest with ATP serving as the preferred substrate in a series including CTP, UTP, GTP and ADP. Calcium, Mg²⁺ stimulated ATPase activity is activated further by calmodulin and partially inhibited by low concentration of vanadate, trifluoperazine and oligomycin. Results suggest that the plasma membrane-enriched fraction of tick salivary glands contains both Ca²⁺-ATPase activity and oligomycin-sensitive Ca²⁺, Mg²⁺-ATPase activities, the latter likely from a small amount of mitochondria in the partially purified organelle fraction.     

Salivary glands in ixodid ticks: control and mechanism of secretion

The salivary glands are vital to the biological success of ixodid ticks and the major route for pathogen transmission. Important functions include the absorption of water vapor from unsaturated air by free-living ticks, excretion of excess fluid for blood meal concentration, and the secretion of bioactive protein and lipid compounds during tick feeding. Fluid secretion is controlled by nerves. Dopamine is the neurotransmitter at the neuroeffector junction regulating secretion via adenylate cyclase and an increase in cellular cAMP. Dopamine also affects the release of arachidonic acid which is subsequently converted to prostaglandins. Prostaglandin E(2) (PGE(2)) is secreted at extremely high levels into tick saliva for export to the host where it impacts the host physiology. Additionally, PGE(2) has an autocrine or paracrine role within the salivary gland itself where it interacts with a PGE(2) receptor to induce secretion (exocytosis) of bioactive saliva proteins via a phosphoinositide signalling pathway and an increase in cellular Ca(2+). Regulation of fluid secretion has been extensively studied, but little is known about the mechanism of fluid secretion. Continuing advances in tick salivary gland physiology will be made as key regulatory and secretory gland proteins are purified and/or their genes cloned and sequenced.     

Structural and functional aspects of salivary fluid section in Calliphora

Calliphora salivary glands are described, emphasizing correlations between structure and physiology. In vitro studies show that the distal part of each gland produces a potassium-rich primary saliva when stimulated with 5-hydroxytryptamine or cyclic AMP. The secretory cells have elaborate canaliculi opening into the lumen. Stimulation of the secretory region causes a 60-fold increase in fluid secretion rate without affecting cell structure. The proximal part of the gland reabsorbs potassium when stimulated with cyclic AMP, but 5-HT has no effect. Potassium reabsorption from the primary saliva results in formation of a dilute saliva. The structure of the secretory and reabsorptive cells is discussed with regard to the functional role of long narrow channels in transport.     

Cell biology of human salivary secretion

We treated surgical specimens of human parotid and submandibular glands in vitro to manipulate the receptor-signaling cascade pharmacologically and analyzed cellular responses by light microscopy on epoxy embedded sections. Treatment of specimens with the b-agonist, isoproterenol, and with the second messenger analog, dibutyryl cyclic AMP, stimulated serous acinar cells to engage in exocytosis and degranulation. The muscarinic agonist, carbachol, and the calcium ionophore, A23187, on the other hand, elicited formation of "vacuoles" in the cytoplasm of serous acinar cells. Taking previous in vivo human and animal studies into account, these changes are suggested as the morphological expression of enzyme release and fluid secretion, respectively. Specimens obtained from patients over 70 years old exhibited poor response even though their morphological appearance remained intact. Aged salivary glands are thus suggested to experience a decline in their secretory activity at the cellular level, probably by impairment of the signaling processes downstream to the receptor activation and second messenger production.     

Molecular cloning of cAMP-dependent protein kinase catalytic subunit isoforms from the lone star tick, Amblyomma americanum (L.)

The salivary glands of ixodid ticks are central to tick feeding and to survival during off-host periods. They produce and secrete a number of molecules critical to maintaining the complex host-vector interface and to maintaining osmotic balance. We have previously shown that a cyclic AMP-dependent protein kinase (cAPK) is involved in the mechanism of salivary gland secretion. We have now cloned cDNAs encoding three isoforms of the catalytic subunit (cAPK-C) of the cAPK from Amblyomma americanum, which are probably produced from alternative RNA processing of a single cAPK-C gene. The cDNAs contain unique N-termini of variable lengths that are linked to a common region containing the alpha A helix, catalytic core, and a C-terminal tail. The common region is highly similar to both insect and vertebrate cAPK-Cs. We have examined mRNA profiles in whole ticks and in isolated salivary glands throughout feeding and find that a single cAPK-C isoform is expressed in the salivary glands of both unfed and feeding females.     

Similar mechanisms regulate protein exocytosis from the salivary glands of ixodid and argasid ticks

Numerous bioactive compounds are secreted from large dense core granules in tick salivary glands during feeding in response to an external stimulus. Investigations into the signalling pathways regulating secretion indicated that they are similar for Argasidae (fast-feeding ticks) and Ixodidae (slow-feeding ticks), but differ in their sensitivity to prostaglandin E(2). In both cases, dopamine is the external signal for inducing exocytosis. Dopamine-induced exocytosis was shown to be strongly calcium dependant. Firstly, it requires extracellular calcium via a L-type voltage-gated calcium channel located on the plasma membrane and, secondly, intracellular calcium which is released presumably in response to inositol 1,4,5-triphosphate (IP(3)). Pathways such as the activation of phospholipase C, inositol-phosphate kinases, G-proteins, GTPases and Na(+)-K(+)-ATPases have been shown to be essential.     

Amblyomma americanum (L.): protein kinase C-independent fluid secretion by isolated salivary glands.

Protein kinase C activity was partially purified from tick salivary glands by fast protein liquid chromatography anion-exchange chromatography. Enzyme activity was stimulated by Ca2+, phosphatidylserine, and diacylglycerol with the highest activity observed in the presence of all three modulators. Enzyme activity was inhibited by a synthetic pseudosubstrate peptide with an amino acid sequence resembling the protein kinase C substrate phosphorylation site. The protein kinase C activator, 1-oleoyl-2-acetyl-sn-glycerol (OAG), when added to whole in vitro salivary glands previously prelabeled with 32P, stimulated the phosphorylation of salivary gland proteins. Activators of protein kinase C (phorbol ester or OAG) did not stimulate fluid secretion by isolated tick salivary glands. OAG and phorbol ester had only minimal affects on the ability of dopamine to stimulate secretion by isolated salivary glands and dopamine's ability to increase salivary gland cyclic AMP.     

Molecular crowding as a mechanism for tick secretory granule biogenesis

During feeding ticks secrete bioactive components into the host to counter-act its immune and hemostatic defense systems. These bioactive components are stored in secretory granules that are secreted during feeding in an exocrine stimulus-response type of mechanism. All proteins destined for secretion are packaged into these granules during granule biogenesis. Up to date no mechanism for granule biogenesis has been proposed, except to note that biogenesis occurs under conditions of high protein and calcium concentrations in an acidic environment. Previously, the most abundant proteins (TSGPs) found in the salivary glands of the soft tick, Ornithodoros savignyi, were suggested to play a part in granule biogenesis, based on their high abundance. The TSGPs are part of the lipocalin family, of which numerous members have been identified in ticks. We consider here the high concentrations of the TSGPs in salivary glands and what effect this will have on the crowded environment inside the secretory granules. It is shown that the TSGPs occur at concentrations that will lead to molecular crowding of which one result is the non-specific aggregation of components to reduce crowding effects. Aggregation of proteins as a mechanism of granule biogenesis has been proposed before, but not in terms of molecular crowding. We thus propose molecular crowding as the general mechanism of granule biogenesis, in tick secretory granules, but can also be extended to other forms of secretory granules in general.     

Effects of dopamine on prolactin secretion and cyclic AMP accumulation in the rat anterior pituitary gland

The effects of dopamine on pituitary prolactin secretion and pituitary cyclic AMP accumulation were studied by using anterior pituitary glands from adult female rats, incubated in vitro. During 2h incubations, significant inhibition of prolactin secretion was achieved at concentrations between 1 and 10nm-dopamine. However, 0.1–1μm-dopamine was required before a significant decrease in pituitary cyclic AMP content was observed. In the presence of 1μm-dopamine, pituitary cyclic AMP content decreased rapidly to reach about 75% of the control value within 20min and there was no further decrease for at least 2h. Incubation with the phosphodiesterase inhibitors theophylline (8mm) or isobutylmethylxanthine (2mm) increased pituitary cyclic AMP concentrations 3- and 6-fold respectively. Dopamine (1μm) had no effect on the cyclic AMP accumulation measured in the presence of theophylline, but inhibited the isobutylmethylxanthine-induced increase by 50%. The dopamine inhibition of prolactin secretion was not affected by either inhibitor. Two derivatives of cyclic AMP (dibutyryl cyclic AMP and 8-bromo cyclic AMP) were unable to block the dopamine (1μm) inhibition of prolactin secretion, although 8-bromo cyclic AMP (2mm) significantly stimulated prolactin secretion and both compounds increased somatotropin (growth hormone) release. Cholera toxin (3μg/ml for 4h) increased pituitary cyclic AMP concentrations 4–5-fold, but had no effect on prolactin secretion. The inhibition of prolactin secretion by dopamine was unaffected by cholera toxin, despite the fact that dopamine had no effect on the raised pituitary cyclic AMP concentration caused by this factor. Dopamine had no significant effect on either basal or stimulated somatotropin secretion under any of the conditions tested. We conclude that the inhibitory effects of dopamine on prolactin secretion are probably not mediated by lowering of cyclic AMP concentration, although modulation of the concentration of this nucleotide in some other circumstances may alter the secretion of the hormone.     

Effect of 20-hydroxyecdysone on the salivary glands of the male tick,Amblyomma hebraeum

Female ticks (Acari: Ixodidae) feed only once in the adult stage, dying after laying a large batch of eggs. During the early post-engorgement stage, haemolymph ecdysteroid titre rises, which is probably responsible for autolysis of the salivary glands that takes place at this time. Males, on the other hand, can re-attach and feed numerous times during the adult stage. Males were fed on rabbits for either 7 or 14 days. Haemolymph was collected either the day of removal from the host or 4 days later, and ecdysteroid titre was measured by radioimmunoassay. The approximate titre in all 4 groups was 20 ng of 20-hydroxyecdysone (20-OHE) equivalents/ml haemolymph. Fluid secretory competence in vitro can be used as an index of salivary-gland degeneration. The glands dissected from fed males which had been left off the host for 4 days lost 62% of their fluid secretory competence compared to glands dissected shortly after the males were removed. This loss in fluid secretory competence was reversed by allowing ticks left off the host of 4 days to resume feeding. Male salivary glands lost fluid secretory competence when exposed for 4 days in organ culture to 20-OHE; the effect was maximal at the lowest concentration tested (20 ng/ml). Thus, although male salivary glands were highly sensitive to 20-OHE, it is still not clear whether this hormone causes the tissue to degenerate.     

The salivary glands of Hyalomma anatolicum anatolicum: structural changes during attachment and feeding

The histology and ultrastructure of the salivary glands of male and female H.a. anatolicum ticks have been examined m unfed and feeding ticks with special emphasis on aspects related to the feeding process. The salivary glands of H.a. anatolicum consisted of three types of acinus (acinus I, II and III) in females and an additional type IV acinus in males. The type I acinus was agranular and showed slight morphologic changes during feeding. The presence of cells with ultrastructural features characteristic of epithelia involved in the secretion of hyperosmotic fluids supports the hypothesis that these acini secrete hygroscopic saliva during questing stages to absorb water from an unsaturated atmosphere. There were five granular cell types (a, b, c₁–c₃) in type II acinus, three granular cell types (d, e, and f) in type III acinus, and one granular cell type (g) in type IV acinus. The cells a, d and e secreted most of their granules early in feeding and are considered to be cement precursors. The b and c cells appeared to synthesise and secrete their products throughout feeding and so are likely to secrete anticoagulants, enzymes and other pharmacologically active agents required during feeding. The interstitial cells, which were insignificant in acinar types II, III and IV of unfed ticks, became more distinct during feeding. The type III acinus in females showed remarkable cell transformations, during the course of feeding. The ablumenal interstitial cells of type III acinus, in females formed a basal labyrinth of extraordinary complexity by interdigitating with the basolateral membranes of transformed f cells to form a network of extracellular channels to excrete fluid during feeding. There was an enormous increase in the secretory granules of g cells as the feeding advanced. The secretory granules were released by a process of exocytosis, by direct fusion with the apical membranes and through channels connecting several granules.