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[The morphological structure of salt gland and salt secretion in Aeluropus littoralis var. sinensis Debeaux].

The leaves of Aeluropus littoralis var. sinensis Debeaux were scanned with a scanning electron microscope, it showed that the upper-epidermis had almost the same number of salt glands as the lower-epidermis (Plate I-1, 2), and the salt gland is the typical bicelluar gland, which consists of a large basal cell inlaid into the epidermis and a small cap cell (Plate I-6). These salt glands were distributed mainly on the leaf veins, which favors the rapid collection of salts from the roots. Ion X-ray microanalysis indicated that the salt glands could effectively absorb Na(+) from the epidermal cells and mesophyllous cells (Table 1), then secreted Na(+) from the cap cells (Plate I-4), which would decrease the salinity of plant. After the plants were treated with various salts for 17 d, the ion contents of the leaves and the secretion were measured, and the results implied that salt glands had different selection in absorbing and secreting Na(+) , K(+) and Ca(2+), that is, the plants first selected K(+) when absorbing ions, while first selected Na(+) when sending ions out, but Ca(2+) was fewer in both absorbed and secreted, and the order of secretion of the three ions was found to be Na(+)>K(+)>Ca(2+) (Figs. 1-6). The secretion of Na(+) or three ions were respectively higher than that of leaves within 24 h (Figs. 1, 2, 7, 8), while K(+) situation was completely opposite with them (Figs. 3, 4). At the same time, the total ion content and composition inside the leaves remained more or less constant (Fig. 8).

Biological Transport↗

Identification and analysis of HD-ZIP transcription factors that regulate salt gland development and salt tolerance in Limonium bicolor.

Soil salinity severely constrains agricultural production. Elucidating the salt-tolerance mechanisms of halophytes can provide innovative approaches for improving the salt tolerance of crop plants. In this study, we performed genome-wide identification and analysis of 36 LbHDZ genes encoding homeodomain-leucine zipper (HD-ZIP) transcription factors in Limonium bicolor, a typical recretohalophyte that excretes excess salt ions through specialized salt glands. Expression profiling across different stages of salt gland development, as well as in various tissues under salt stress, indicated that multiple LbHDZ genes are involved in regulating salt gland development and salt tolerance. Among these genes, LbHDZ14 (a member of the HD-ZIP II subfamily) exhibited sustained high expression during the critical period of salt gland formation, while its transcript levels were significantly downregulated in leaves and roots under salt stress. Subsequent experiments demonstrated that LbHDZ14 is localized in the nucleus and negatively regulates salt gland density and salt tolerance by directly binding to the promoter of LbGDSL, a positive regulator of salt gland development. In conclusion, this study reveals the expression patterns of LbHDZ genes in L. bicolor, characterizes the functional mechanism of LbHDZ14, further elucidates the regulatory network underlying salt gland development, and provides candidate genes for enhancing crop salt tolerance.

Plumbaginaceae↗

Transcription factor LbUBC positively regulates salt gland development and salt tolerance by directly binding to the LbTTG1 promoter and repressing its transcription.

KEY MESSAGE:: LbUBC enhances salt tolerance by promoting salt gland development via repressing LbTTG1, revealing a synergisticregulatory mechanism in Limonium bicolor. In the context of increasingly severe soil salinization, salt-tolerant genetic resources from halophytes show great application potential. In particular, the recretohalophyte Limonium bicolor, which possesses specialized salt gland structures, has become a key model for deciphering the molecular mechanisms underlying salt tolerance and salt gland development. In this study, using LbTTG1-overexpressing and -silenced lines, we demonstrate that LbTTG1 negatively regulates salt-gland development and salt tolerance. Through yeast one-hybrid, EMSA, and dual-luciferase assays, Lb7G33228 (LbUBC) was screened and verified as an upstream transcriptional regulator of LbTTG1. LbUBC enhances salt tolerance in L. bicolor by positively regulating salt-gland development, verified using LbUBC silence and overexpression strains. Interestingly, LbUBC represses the expression of its downstream target LbTTG1, thereby releasing the inhibitory effect of LbTTG1 on salt-gland development. In this manner, LbUBC positively regulates salt-gland development, achieving a dynamic balance in the regulation of salt-gland development and salt tolerance in L. bicolor. This study reveals a synergistic regulatory mechanism involving multiple genes, offering new insights for comprehensively dissecting the molecular regulatory network of salt-gland development.

Salt Tolerance↗

Morphology of isolated crustacean larval salt glands.

Larval salt glands isolated from the naupliar brine shrimp (Artemia salina) were examined using light microscopy and scanning and transmission electron microscopy. These methods demonstrated that most cellular and subcellular features of the in vitro organ compared favorably with those seen in vivo. This salt gland measures 130 micron in diameter and is comprised of 50-70 secretory cells, which are of a single epithelial cell type. Characteristic ultrastructural features that are well preserved include apical to basal cell polarity, apical plasma membrane projections, and the extent of the basolateral tubular labyrinth and its association with numerous mitochondria. Some features that have been altered are a decrease in cell-cell contact, separation of septate junctions, and expansion of tubular labyrinth lumens and mitochondrial cristae. Use of this preparation has allowed examination of the salt gland cell's hemocoelic surface for the first time and provided information about the ultrastructure of the tufts formed by the apical plasma membrane.

Animals↗

Changes in Na+/K+-ATPase expression during adaptive cell differentiation in avian nasal salt gland

Chronic salt stress in ducklings (Anas platyrhynchos) resulted in a sustained accumulation of cyclic AMP in the secretory cells of the nasal salt glands. Adaptive increases in the activity of the Na+/K+-ATPase, measured as ATP hydrolysis rates in freshly isolated tissue, were observed after 12 h of salt stress. This change in enzyme activity was associated with increases in protein abundance in the - as well as in the ss-subunit of Na+/K+-ATPase and an increase in ss-subunit glycosylation. We investigated whether the increase in the cytosolic cyclic AMP concentration and the adaptive changes in Na+/K+-ATPase activity were causally related. Using an organotypic tissue culture system for salt gland slices from unstressed (naive) ducklings, we produced similar changes in Na+/K+-ATPase activity and subunit abundance by treating cultured tissue with drugs that elevate cytosolic cyclic AMP levels (forskolin, 8-CPT-cAMP) during a 15 h culture period. Protein synthesis assays using cultured tissue revealed that elevations in cytosolic cyclic AMP level mediate increases in Na+/K+-ATPase subunit abundance by slowing down the degradation of ATPase subunits. This increase in the amount of enzyme protein was associated with a significant increase in Na+/K+-ATPase activity in tissue homogenates. The time course of these changes in cyclic-AMP-treated cultured tissue resembled that observed in salt-stressed intact animals, indicating that the elevation in cyclic AMP level in salt gland tissue may constitute a portion of the signalling events ultimately leading to the adaptive increase in Na+/K+-ATPase activity in vivo.

Journal Article↗

Vasoactive intestinal peptide stimulates ion transport in avian salt gland.

Avian salt glands are considered to be under the control of cholinergic nerve fibers. Here we report evidence that vasoactive intestinal peptide (VIP) also regulates ion transport. Nerve fibers stained immunocytochemically with anti-VIP were distributed throughout the tissue within the peritubular connective tissue and were in close proximity to the secretory tubules. VIP applied to primary cultures of the secretory cells elicited active ion transport as assayed by short-circuit current (Isc) analysis. The mucosal-to-serosal positive Isc was produced in a dose-dependent fashion [(EC50) = 3.1 X 10(-9) M], was potentiated by theophylline, and was inhibited by either ouabain or furosemide. This Isc was independent of activation by cholinergic agonists. VIP also increased ouabain-sensitive respiration 14-18% in acutely isolated cells from salt-stressed and unstressed animals. These data demonstrate for the first time that VIP is present in the avian salt gland and can act as a secretagogue by directly affecting the secretory cells. In addition, the results provide evidence for direct control of ion transport by an adenosine 3',5'-cyclic monophosphate-linked neurohormone in both adult unstressed and fully salt-stressed animals.

Animals↗

NASAL SALT GLAND: INDEPENDENCE OF SALT AND WATER TRANSPORT.

The nasal salt-secreting gland of the domestic duck can produce a fluid with a sodium chloride concentration about three times that in blood plasma. To study the cellular mechanism responsible for the formation of the highly concentrated fluid, the gland was poisoned by retrograde injection of mercuric chloride into the lumen, decreasing the salt concentration to that in plasma while the volume of secretion was unchanged. Carbonic anhydrase inhibitor (acetazoleamide) caused a moderate decrease in salt concentrations, again with maintenance of volume of secretion. The results suggest that concentration and volume of the secreted fluid depend on two different cellular mechanisms.

Acetazolamide↗

Salt gland and kidney responses to intracerebral osmotic stimulation in salt- and water-loaded ducks.

Saltwater-adapted ducks with functioning supraorbital salt glands were chronically implanted with a device for perfusion of the third cerebral ventricle (icv perfusion) with artificial cerebrospinal fluid (CSF) of different tonicities. The osmoregulatory responses to icv stimulation were studied at conditions of salt and water loading in which only the salt glands, both salt glands and urinary fluid excretion, or only urinary fluid excretion were stimulated; in the latter experiments plasma antidiuretic hormone (ADH) was measured with a radioimmunoassay. Hypertonic icv stimulation enhanced salt gland secretion and caused antidiuresis, due to the increase of plasma ADH. Hypotonic icv stimulation inhibited salt gland activity and caused diuresis, due to the decrease of plasma ADH. Salt gland activity, urine formation, and plasma ADH reacted more sensitively to changes of icv tonicity in the hypertonic than in the hypotonic range. The effect of icv hypotonic stimulation could be obtained also with icv perfusion of isosmotic artificial CSF deficient in NaCl content. Perfusion with artificial CSF exceeding plasma tonicity by 50 mosmol X kg-1 or more caused inhibition of salt gland secretion associated with enhanced urinary output in several experiments.

Afferent Pathways↗

The ultrastructure of Cynodon salt glands: the apoplast.

The salt glands of Bermuda grass were found to be similar to those of many dicotyledonous plants with respect to the relatively large quantities of chloride that accumulate in the subcuticular collecting chamber of secreting glands. The salt glands of grasses lack the suberized or cuticularized zone that is present in the walls of the glands of dicotyledonous plants that has been hypothesized to prevent the apoplastic backflow of accumulated salts from the collecting chamber to the leaf mesophyll. An ionic lanthanum solution was used to determine whether or not the apoplastic pathway is blocked between the collecting chamber and the leaf mesophyll. The results of the lanthanum tracer study revealed that this pathway is significantly restricted. It was determined that the restriction of the apoplastic pathway was due to the highly lignified condition of the lateral cell walls in the outer portion of the basal gland cell. The lanthanum tracer study also revealed that an apoplastic continuum exists between the leaf mesophyll and a system of membranous extracellular channels that occur in the basal cell. Based on this finding, it was suggested that the extracellular channels may function in the absorption of solutes from the apoplast.

Chlorides↗

Alpha-melanocyte-stimulating hormone stimulates sodium excretion in the salt gland of the duck.

Salt glands of ducks were induced to secrete sodium through the ingestion of salt water. In salt-adapted animals the administration of melanocyte-stimulating hormone (MSH) produced a rise in the sodium excreted by the salt gland, an effect which was not mimicked by adrenocorticotropin. Studies in vitro using incubations of gland slices and radioactive sodium ion showed that MSH increased sodium efflux, indicating that it acted directly upon the gland. We have previously observed that MSH has no effect on the pigmentary system of the duck. It is proposed that in the evolutionary process this hormone has acquired new target tissues in these birds.

Adrenocorticotropic Hormone↗

Ultrastructure of the purified and reconstituted Na/K-ATPase of the avian salt gland.

Na/K-ATPase of salt-stressed salt glands of the domestic duck (Anas platyrhynchos) was purified in membrane-bound form by incubation of the microsomal fraction with sodium dodecylsulphate and ATP followed by discontinuous sucrose gradient centrifugation. Gel electrophoresis of the purified plasma membrane preparation substantially showed the two polypeptide subunits of the Na/K-ATPase both of which stained with the periodic acid-Schiff reagent. About 99% of the total ATPase activity was ouabain-inhibitable amounting to 1300 mumol Pi/(mg protein X h) of specific activity. The anion-stimulated, ouabain-insensitive ATPase increased parallel to the Na/K-ATPase up to the microsomal fraction until it totally vanished during SDS incubation. Electron microscopy of thin sections revealed that the purified fraction consisted of flat and cup-shaped triple-layered membrane fragments. Particles arranged into clusters and strands were visible as 3 to 5 nm surface particles in negatively stained suspensions and as 8 to 10 nm intramembraneous particles in freeze fracture replicas. The differential distribution of the intramembraneous particles on the fracture faces reflected the structural membrane asymmetry. Solubilization of Na/K-ATPase led to the disappearance of intramembraneous particles. Incorporation of the solubilized enzyme into phosphatidylcholine vesicles again showed 8 to 10 nm particles apparently orientated at random in the artificial membrane. Control liposomes prepared in the absence of solubilized enzyme were devoid of intramembraneous particles. These results clearly demonstrate that the avian salt gland Na/K-ATPase exists as 8 to 10 nm particles in both the purified plasma membrane and the artificial phospholipid membrane.

Animals↗

Regulation of salt gland, gut and kidney interactions.

Marine birds can drink seawater because their cephalic 'salt' glands secrete a sodium chloride (NaCl) solution more concentrated than seawater. Salt gland secretion generates osmotically free water that sustains their other physiological processes. Acclimation to saline induces interstitial water and Na move into cells. When the bird drinks seawater, Na enters the plasma from the gut and plasma osmolality (Osm(pl)) increases. This induces water to move out cells expanding the extracellular fluid volume (ECFV). Both increases in Osm(pl) and ECFV stimulate salt gland secretion. The augmented intracellular fluid content should allow more rapid expansion of ECFV in response to elevated Osm(pl) and facilitate activation of salt gland secretion. To fully utilize the potential of the salt glands, intestinally absorbed NaCl must be reabsorbed by the kidneys. Thus, Na uptake at gut and renal levels may constrain extrarenal NaCl secretion. High NaCl intake elevates plasma aldosterone concentration of Pekin ducks and aldosterone stimulates intestinal and renal water and sodium uptake. High NaCl intake induces lengthening of the small intestine of adult Mallards, especially males. High NaCl intake has little effect on glomerular filtration rate or tubular sodium Na uptake of birds with competent salt glands. Relative to body mass, kidney mass and glomerular filtration rate (GFR) are greater in birds with salt glands than in birds that do not have them. Birds with salt glands do not change GFR, when they drink saline. Thus, their renal filtrate contains excess Na that is, in some species, almost completely renally reabsorbed and excreted in a more concentrated salt gland secretion. Na reabsorption by kidneys of other species, like mallards is less complete and their salt glands make less concentrated secretion. Such species may reflux urine into the hindgut, where additional Na may also be reabsorbed for extrarenal secretion. During exposure to saline, marine birds maintain elevated aldosterone levels despite high Na intake. Marine birds are excellent examples of physiological plasticity.

Animals↗

Circulatory and osmoregulatory effects of angiotensin II perfusion of the third ventricle in a bird with salt glands.

In Pekin ducks adapted to salt water, 1Asp - 5Val -angiotensin II, 1Asp - 5Ile -angiotensin II and 1Asp - 5Ile -tetradecapeptide were applied intracerebroventricularly (I.C.V.) during steady-state conditions evoked by continuous intravenous loading with 200 mosmol kg-1 saline. Each of the angiotensin II (AII) analogues caused a dose-dependent antidiuresis with a concomitant rise in urine osmolality and electrolyte concentration. Antidiuresis was linearly correlated with plasma arginine vasotocin (AVT). The elevation of plasma AVT occurred rapidly during I.C.V. stimulation with AII and declined exponentially to the pre-stimulation level. Under conditions of salt loading with 1000 mosmol kg-1 saline in which the ducks excreted the salt and water by their supraorbital salt glands, AII applied I.C.V. in a concentration of 1 nmol ml-1, inhibited the NaCl excretion via the salt glands. Arterial blood pressure and heart rate increased after I.C.V. microperfusion with 1 nmol ml-1 AII. This was not due to leakage of I.C.V. AII into the circulation because systemic application of AII required a 100-fold higher dose to elicit similar effects. Respiration rate remained constant. Systemically applied AVT which produced plasma levels similar to, or greater than, those caused by centrally acting AII resulted in the same antidiuretic responses but did not mimic the circulatory effects of I.C.V. AII. Specific AVT antiserum, injected intravenously, totally suppressed the renal response to I.C.V. AII and reduced the rise in blood pressure and heart rate by more than 50%. The anterior part of the third ventricle was more sensitive than the posterior part in eliciting the antidiuretic responses to I.C.V. applied AII. The particular combination of effects on renal excretion, salt gland secretion and cardiovascular function of centrally applied AII in the duck supports the idea that AII plays a major role as a central modulator of volume homeostasis.

Angiotensin II↗

The effects of saltwater acclimation on neurotransmitters in the lingual salt glands of the estuarine crocodile, Crocodylus porosus.

INTRODUCTION: Most avian and reptilian salt glands display marked phenotypic plasticity when animals are exposed to hyperosmotic conditions. In addition, the activity of most salt glands is under considerable control by the nervous system and nerves containing cholinergic, adrenergic and peptidergic neurotransmitters have been identified in avian and reptilian salt gland tissues. The present study sought to determine whether the salt glands of the estuarine crocodile, Crocodylus porosus contain the peptidergic neurotransmitters SP, CGRP, VIP, and PACAP and the gaseous neurotransmitter, NO. In addition, we sought to determine whether there was any evidence for the adaptation of the C. porosus salt gland nervous system to hyperosmotic conditions. METHODS: Salt glands from freshwater- and saltwater-acclimated C. porosus hatchlings were sectioned and examined immunohistochemically for neurotransmitters within the tissue. RESULTS: Neurons containing SP, CGRP, VIP, PACAP and NO synthase were identified within C. porosus salt glands. There was no difference in the overall number (density) of neurons within SW-acclimated tissues when compared with FW-acclimated animals. However, there was a significant reduction in density of neurons containing SP and PACAP in SW-acclimated animals. CONCLUSION: C. porosus salt glands display phenotypic plasticity following exposure to hyperosmotic conditions. In addition to cholinergic and adrenergic neurons, they contain a variety of peptidergic neurotransmitters and the gaseous neurotransmitter NO. Additionally, there appears to be some evidence of acclimation of the nervous system of C. porosus to hypersaline conditions, although the functional significance of these changes remains to be determined.

Acclimatization↗

Correlation of Na+,K+-ATPase content and plasma membrane surface area in adapted and de-adapted salt glands of ducklings.

During salt-water adaptation, an increase occurs in Na+,K+-ATPase content and surface area of the basolateral plasma membrane of the principal cell of the duck salt gland. To determine the degree to which these changes are correlated, accepted morphometric methods were used to determine numerical cell densities and plasma membrane surface densities of peripheral and principal cells. After adaptation, the plasma membrane surface area per principal cell was five times greater than in controls. Following de-adaptation, the plasma membrane content in principal cells returned to 1.9 times control levels. Two other cell constituents, mitochondria and lipid droplets, displayed similar quantitative changes. Na+,K+-ATPase content increased about fourfold with adaptation and decreased to near control levels with de-adaptation. Thus, changes in Na+,K+-ATPase content and basolateral plasma membrane surface area in adapting and de-adapting secretory epithelia of the salt gland occur nearly in parallel. These quantitative data enable Na+,K+-ATPase synthesis and degradation to be investigated in relation to membrane biogenesis.

4-Nitrophenylphosphatase↗

Partial uncoupling of salt gland blood flow and secretion in the Pekin duck (Anas platyrhynchos).

1. The aim of this study was to investigate the relationship between the blood flow through and the secretion by the salt glands of conscious, salt-water-adapted Pekin ducks. 2. Intravenous loading with hypertonic saline induced a steady-state secretion from the salt glands with a concomitant increase in whole-organ blood flow. The distribution of elevated local glandular blood flow was, however, uneven and in addition demonstrated vasomotor patterns that ranged from constant to rhythmic. 3. During on-going salt gland secretion, the infusion of three vasoactive agents, 5Val-angiotensin II (ANG II), 8Arg-vasotocin (AVT) and noradrenaline, via the carotid artery had differential effects on salt gland blood flow and secretion. 4. ANG II (80 pmol min-1 (kg body wt)-1) had no effect on mean arterial blood pressure (MABP), produced a transient 30% decrease in glandular blood flow and strongly diminished salt gland secretion (retention of 6.4 mosmol NaCl). 5. AVT (20 pmol min-1 (kg body wt)-1) had no effect on MABP and did not alter salt gland secretion despite a 35% reduction in blood flow. 6. Noradrenaline (20 nmol min-1 (kg body wt)-1) elevated MABP by 15 mmHg, reduced salt gland blood flow by more than 50%, but diminished salt gland secretion only slightly (retention of 2.7 mosmol NaCl). 7. Using ANG II, AVT and noradrenaline as hormonal tools, integrated changes in blood flow rate did not correspond with integrated changes in salt gland excretion. The partial dissociation between both parameters shows that control of secretion by the salt gland is more complex than simply being linearly dependent upon blood flow through it.

Angiotensin II↗

Vasoactive intestinal peptide stimulates blood flow and secretion of avian salt glands.

The neuromodulatory role of vasoactive intestinal peptide (VIP) in avian salt gland secretion and blood flow was investigated in conscious saltwater-acclimated Pekin ducks. Glandular blood flow was measured by laser-Doppler flowmetry or the radioactive microspheres technique. Osmolal excretion was closely related to salt gland blood flow during salt loading. At threshold conditions of salt gland secretion, VIP infused intracarotidally induced both osmolal excretion and arteriolar vasodilation dose dependently (30-240 pmol.min-1.kg body wt-1). The VIPergic effect on the secretory process for NaCl was enhanced by simultaneous intracarotid application of acetylcholine (5 nmol.min-1.kg body wt-1), whereas the intrinsic vasodilatory potency of acetylcholine appeared to be nonadditive in coinfusion experiments. Ongoing secretion induced by systemic infusion of hypertonic saline could be suppressed by muscarinic antagonists, with salt gland blood flow being sustained at the reduced level of atropine-resistant vasodilation. Subsequent intracarotid infusion of VIP stimulated glandular blood perfusion and also, to a minor extent, osmolal excretion, suggesting an independent, functional VIP system in efferent salt gland control.

Acclimatization↗