Using atmospheric plasma to increase wettability, imbibition and germination of physically dormant seeds of Mimosa Caesalpiniafolia.

In this study, we analyzed seed wettability as well as imbibition and germination after treatment with atmospheric pressure cold plasma (APCP) using dielectric barrier discharge (DBD) in seeds that have very low germination rates. To aid industrial applications, several seeds were simultaneously treated with plasma within a space between two coaxial glass tubes sandwiched by two metal mesh screens that produced high-voltage pulses at 17.5kV with a frequency of 990Hz. Three treatment times (3min, 9min and 15min) as well as untreated seeds were used to conduct the wettability, imbibition and germination tests. The wettability and imbibition were found to be directly related to the treatment duration, but saturation of the imbibition was found for treatment durations greater than 9min. Plasma treatment was also effective in improving germination, but shorter treatment duration presented greater germination. This apparent contradiction is explained by the cell damage caused by the increased exposure to plasma, as observed in other studies. The results suggest that there must be an optimal wettability and imbibition condition that ensures that excessive moisture does not harm the germination process.

Morphometric changes associated with sex and development in the Malpighian tubules of Aedes aegypti.

The Malpighian tubules of Aedes aegypti showed significant differences in their diameters between male and female larvae, male and female pupae, male larvae and male adults and male pupae and male adults. In every case, female values were greater than in males. Measurements of mean nuclear areas of the principal and stellate cells from Malpighian tubules, taken in males and females during development, showed that this parameter in both types of cell was significantly greater in females than in male larvae, pupae and adult stages. In males, significant differences between developmental stages were observed only in comparison with the nuclear area of larvae and adults in the principal cells, but in females, every comparison between stages showed significant differences except between pupae and adults in stellate cells. The frequency distribution of nuclear area values, in development, for male stellate and principal cells, were mostly concentrated in the first seven classes among the 30 classes considered in every stage, while for females, the frequency dropped drastically in the same classes from larvae to pupae and adults, moving to classes of higher values. Considering the importance of Malpighian tubules in insect physiology, the meaning of the differences detected are discussed on the basis of different metabolic levels, between sexes and developmental stages.

Anion-induced dynamic behavior of apical water channels in vasopressin-sensitive epithelia exposed to mercury.

We showed recently that, in toad skins preexposed to Hg, water permeability is high in SO4-Ringer and low in Cl-Ringer. This anion effect was further investigated in Hg-treated skins and bladders of toads (Bufo marinus) in a variety of experimental conditions, including glutaraldehyde fixation and stimulation by vasopressin (VP) or isoproterenol (IP). In fixed bladders either unstimulated or stimulated with VP, net water flow (Jw) in SO4-Ringer [Jw (SO4)] was always significantly higher than Jw in Cl [Jw (Cl)]; the same applies to fixed toad skins, either unstimulated or stimulated with IP. In unfixed isolated toad epidermis challenged with IP before Hg exposure, Jw(SO4)/Jw(Cl) >> 1 approaching the ratio Jw (maximally stimulated)/Jw (basal). Therefore, anion-induced Jw changes were present whether Hg acted on epithelial water channels exocytosed by Hg itself or by hydrosomotic agents and suggest a switching between open and closed configurations of the channel protein. This anion effect was not abolished by glutaraldehyde and might be correlated with changes in intracellular chloride.

Mercury blockage of apical water channels in toad skin (Bufo marinus).

1. Net water flow (Jw) was continuously monitored across the abdominal skin of the toad Bufo marinus by means of a volumetric, automatic technique. Jw was either averaged over periods of 2 min or taken cumulatively (10 or 30 min periods). 2. The state of high water permeability induced by vasopressin or isoprenaline was reversed (88-89% inhibition of delta Jw after 1 h) by the addition of 10(-3) M HgCl2 (or CH3ClHg) to the external bathing medium. Similarly, pre-exposure of the skins to Hg2+, totally blocked the induction of the hydrosmotic response to the same agents. By itself, Hg2+ exerted only a minor (26%) stimulation of basal Jw. 3. There was a sigmoidal dose-response relationship between the reduction of the hydrosmotic effect of vasopressin (VP) and the concentration of Hg2+ in the external medium, with a half-maximal effect at 1.2 x 10(-4) M HgCl2. 4. Total replacement of Na+ by K+, Rb+ or Cs+ in the Ringer solution, caused a VP-like, hydrosmotic effect that was reversed, or prevented, by exposure to Hg2+ in a manner indistinguishable from that previously seen with vasopressin or isoprenaline. 5. The data point to the presence of a Hg(2+)-sensitive apical water pathway in stimulated epithelia, very probably constituted by water channels similar to those reported in red blood cells, amphibian bladder and mammalian kidney tubules.

Effects of anions and/or cell volume on the permeance of an apical water pathway induced by Hg in toad skin epithelium.

Hg compounds block membrane transport units behaving as water channels. Here we show that Hg induces an apical water pathway in toad skins pretreated with 10(-3) M CH3ClHg or HgCl2, added to the outer bathing medium. Washing with SO4-Ringer caused a several-fold increase in net water flow (Jw) and osmotic permeability coefficient (Pf) that was reversed by re-exposure to Cl- or NO3-Ringer and mimicked by gluconate-Ringer. These Pf changes could be elicited repeatedly and were present if, and only if, anion replacements took place in the inner bathing solution. Such inner polarity was related to the anion permeability of the epidermal basolateral membrane: impermeant anions (SO4, gluconate) increased Pf; permeant anions (Cl, NO3) did not change basal Pf but reversed the high Pf induced by impermeant anions. Hg induced the appearance of aggregates that persisted despite repeated washings of the skins during 4-5 h, and whether Pf was high (SO4-Ringer) or low (Cl-Ringer) before skin fixation. The Hg-induced apical water pathway in toad skin appears to be a unique model for studying the interplay between cell volume, cell ionic composition and water permeability.

Effects of vasopressin on epithelial transport.

Vasopressin increases the permeability of responsive epithelia for a variety of chemical species, such as sodium, urea, water, and lipophilic molecules. The analysis of the mechanisms leading to the stimulation of water transport (hydrosmotic action) is the main topic of this review. A model implying the quantal insertion of water channels into the apical epithelial membrane by an exocytosislike process is discussed with reference to biophysical, biochemical, and ultrastructural results obtained in the past few years. It is also shown that, in nonexcitable cells, insertion of transport units packed in intracytoplasmic structures may be a rather general mechanism for controlling membrane transport rates.

Forskolin mimics the hydrosmotic action of vasopressin in the urinary bladder of toads Bufo marinus.

Net water flow JW was measured across the urinary bladder of toads Bufo marinus and averaged over periods of 1 min by means of a volumetric, automatic technique. The diterpene forskolin, an activator of adenylate cyclase bypassing the hormonal receptor subunit, induced a rapid, reversible, dose-dependent increase in osmotic water permeability, Pf, very similar to that induced by vasopressin. At 1.1 microM, forskolin induced a half-maximal response. At 5 microM forskolin caused a near maximal response and Pf increased from 1.66 +/- 0.15 to 66.6 +/- 2.99 microns s-1. In bladders pre-exposed to 5 microM-forskolin, further significant increases in Pf were obtained by their subsequent exposure to vasopressin, cyclic AMP, theophylline or serosal hypertonicity. The similarity of the forskolin and vasopressin actions was further demonstrated by the finding that substances causing enhancement (quercetin) or inhibition (trifluoperazine, vanadate, silver, cobalt, manganese and Ca2+-free Ringer solution) of the vasopressin response, induced parallel changes in the forskolin response. Three agents, however, induced dissimilar effects on vasopressin and forskolin: high K+ potentiated vasopressin but inhibited forskolin; methohexital and diamide inhibited vasopressin but had no effect on forskolin. The forskolin-induced hydrosmotic response can be viewed as a new criterion for ascertaining the messenger role of cycle AMP in the the hydrosmotic effect of vasopressin.

High [K+] alters the stimulus-hydrosmotic response coupling in toad bladder.

Substitution of K+ for Na+ in the Ringer solution bathing the inner surface of toad urinary bladders (Bufo marinus) had no effect on basal water permeability but significantly altered the stimulus-hydrosmotic response of this epithelium. In chloride-Ringer, high [K+] increased the hydrosmotic responses to submaximal stimulations induced by vasopressin or exogenous cAMP, while the responses to theophylline or serosal hypertonicity were decreased. In sulfate-Ringer, all these responses were enhanced but for that induced by serosal hypertonicity which was actually diminished. As a step towards determining if Ca2+ might mediate the K+-induced effects on water flow, experiments were conducted either in the presence of a Ca2+ "antagonist" (cobalt) or in nominally Ca2+-free Ringer. In both conditions the hydrosmotic effects of vasopressin and cAMP were markedly reduced. The results raise the possibility that a transient Ca2+ influx through voltage-sensitive, Co2+-blockade Ca2+ channels may play a role in the stimulus-hydrosmotic response of toad urinary bladder.

Cellular and membrane events involved in the K-induced increase in water permeability of toad skin.

Exposure of the inner surface of toad skin (Bufo marinus) to high [K+] resulted in a marked (up to 7-fold) increase in water permeability (Pf) that was more marked in KC1-Ringer than in K2SO4-Ringer. Although high [K+] did not elicit a maximal increase in Pf, it blunted the hydrosmotic responses to vasopressin, isoproterenol and cAMP. Both "post-cAMP" inhibitors of stimulated water flow, such as diamide and vanadate, and "pre-cAMP" inhibitors, such as methohexital and propranolol, markedly reduced the K response, while exposure to Ca2+-free, KC1-Ringer did not inhibit water flow. Intramembrane particle aggregates, similar to those induced by cAMP-mediated hydrosmotic agents, were seen in the apical membrane of granular cells, just beneath the stratum corneum, in skins exposed to KC1. Available evidence indicates that cAMP might mediate, at least partially, the hydrosmotic effect of high [K+]. In contrast, a role of voltage-dependent Ca2+ channels, described in other cell systems depolarized with K, was not apparent in toad skin.

Osmotic water flow across the abdominal skin of the toad bufo marinus: effect of vasopressin and isoprenaline.

1. Net water flow J(w), was measured across the abdominal skin of the toad Bufo marinus with a volumetric, automatic technique that allows for averaging J(w) over time intervals as short as 1 sec.2. Basal J(w) was very stable and corresponded to a coefficient of osmotic flow, L(PD), of ca. 15 x 10(-7) cm sec(-1) atm(-1) (or to an osmotic water permeability coefficient, P(f), of 20 mum sec(-1)).3. Both vasopressin and the beta-adrenergic agonist, isoprenaline, triggered high hydrosmotic responses that could lead to P(f) values exceeding 250 mum sec(-1). The effect of isoprenaline was very reproducible while that of vasopressin varied considerably.4. Methohexital and propranolol selectively inhibited the hydrosmotic effects of vasopressin and isoprenaline, respectively, whereas amiloride and ouabain had no effect.5. Mutual inhibition was found between vasopressin and isoprenaline in skins very sensitive to vasopressin. In less sensitive skins isoprenaline further increased J(w) despite exposure of the epithelia to supramaximal concentrations of vasopressin.6. Differential reactivity to vasopressin was found between the skin and the bladder taken from the same toad. In some instances, the bladder responded normally to vasopressin while the skin was totally unresponsive, suggesting the presence of osmoregulatory mechanisms exerting a local modulation of the vasopressin action in different target epithelia of the same animal.

Evidence for a role of calmodulin in the hydrosmotic action of vasopressin in toad bladder.

1. The informational role of cytosolic Ca2+ appears to be mediated by a ubiquitous protein--calmodulin--in most cell systems. 2. Evidence has been accumulating that not only cAMP, but also Ca2+, behaves as an intracellular messenger in the stimulation of water transport by vasopressin (hydrosmotic effect). 3. To examine whether calmodulin plays a role in the hydrosmotic effect of vasopressin, we used a specific antagonist of calmodulin--trifluoperazine--and looked at its effects on water transport in the urinary bladder of toads Bufo marinus. 4. The results showed that trifluoperazine, at micromolar concentrations, blocked the hydrosmotic effects of vasopressin or cAMP, thus indicating a post-cAMP site of action. 5. Two other psychotropic drugs--amitriptyline and harmaline--had similar effects, but higher concentrations were required to induce the same degree of inhibition of water flow. 6. Calmodulin was detected in the membrane and in the cytosolic fractions of isolated epithelial cells of toad bladder by means of the phosphodiesterase test. The content of both fractions was similar to that found in bovine brain. 7. The results suggest that calmodulin plays a regulatory role in the hydrosmotic action of vasopressin by possibly interacting with proteins associated with microfilaments and/or microtubules.

The amphibian epidermis: distribution of mitochondria-rich cells and the effect of oxytocin.

It is known that the ion-transporting capacity and the permeability to water of amphibian skins vary greatly both between and within species. Furthermore, the extent to which different skins respond to hormonal stimulation of these parameters also shows considerable inter- and intra-specific variation. As a first step towards defining a possible morphological basis for this physiological heterogeneity, we examined different regions of skins from 3 anurans, Bufo bufo, Rana ridibunda and Xenopus laevis, that are species with widely differing habitats. The mitochondria-rich cell population of the epidermis was counted and the epidermal thickness was measured. There were large differences in the mitochondria-rich cell content and in the epidermal thickness of the skins from different species and from different regions of skin from the same animal. In a second set of studies, the same morphological features were examined and, in addition, routine functional parameters were measured to monitor some transport properties of the skins used. The skins also varied considerably with respect to short-circuit current, potential difference, water permeability and sensitivity to oxytocin. Although no apparent relationship was noted between either basal or hormone-stimulated physiological parameters and the morphological features of the individual skins, the striking variation in the density of mitochondria-rich cells in amphibian epidermis merits further studies, including the use of techniques or experimental designs that allow the movement of individual species of ion across the skin to be followed.

Quercetin enhances water transport in toad bladder.

A highly significant enhancement of the hydrosmotic actions both of vasopressin and of exogenous cAMP was seen in the presence of quercetin. The hypothesis is advanced that quercetin affects the intracellular coupling between Ca++ in cAMP.

The mode of action of vasopressin: membrane microstructure and biological transport.

Vasopressin affects a variety of cell systems. This review is focused on permeability changes induced by vasopressin in tight epithelia such as the collecting duct of the mammalian kidney and the skin and the bladder of anurans. These vasopressin effects are discussed with reference to current concepts and models of the microstructure of the plasma membrane. The transport of three major chemical species--Na, urea and water--is analyzed. In each instance, the hormone appears to activate selective membrane pathways situated at the rat-limiting barrier of the epithelium, i.e., the apical membrane. Available data suggest that two intra-cellular messengers -- cAMP and calcium -- plan a key role in the coupling between stimulus (receptor occupancy) and biological effect (permeability change). The enhancement of Na transport (natriferic effect) depends on the opening and/or the insertion of Na channels, the biophysical and biochemical characteristics of which have been investigated by fluctuation analysis and by means of several chemical blockers of Na transport, particularly the amiloride molecule and its congeners. Likewise, the finding of inhibitors and activators of urea transport, which do not cause any appreciable change in Na or water permeability, led to the notion of selective urea channels or pores. Finally, the enhancement of water transport (hydrosmotic effect) possibly results from the insertion in the apical membrane of water channels already present in vesicular cytoplasmic structures. The restructuring of the apical membrane underlying the transition from a low to a higher state of water permeability is very likely related to the appearance of intramembrane particle aggregates detectable with the freeze-fracture technique in epithelia exposed to vasopressin. The putative water channels (or pores) appear to be so narrow that trans-apical water movement is constrained to single-file diffusion. Recent data also suggest that, in addition to cAMP, microtubules and microfilaments, the calmodulin-Ca complex is a major element in the hydrosmotic effect of vasopressin.

Isoproterenol-induced intramembrane particle aggregation and water flux in toad epidermis.

Stimulation of toad skin with isproterenol resulted in a dramatic increase in water flow, and in the appearance of aggregates of intramembrane particles in the apical membrane of granular cells of the replacement layer, just beneath the stratum corneum. This membrane structural modification appears to be a general prerequisite for the change in water permeability of vasopressin-sensitive epithelia.

Inhibitory and stimulatory effects of amiloride analogues on sodium transport in frog skin.

Effects of amiloride analogues on Na transport were studied in isolated skins of the frog Rana ridibunda. The pattern of structure-activity relationship of these compounds showed that both the -NH2 group at position 5 and Cl at position 6 of the pyrazine ring of the amiloride molecule were important for their biological activity. The paramount role of the groups at position 5 was further demonstrated by the striking properties of an analogue resulting from dimethylation of that -NH2 group. A stimulation of Na transport, opposite to the effect of amiloride itself, was observed in this instance. The increase in Na transport could already be seen at 10(-6) M and was equivalent to the measured increase in Na influx, reversible, dose-dependent, and additive to the natriferic action of oxytocin. Such characteristics resemble those reported with "external" agents like propranolol and La3+. Furthermore, mutual inhibition was observed between the stimulatory effects of this analogue and those of propranolol or La3+. These results suggest that the analogue may be considered as another "external" agent acting at sites of the external membrane distinct from those activated by cAMP but similar to the Ca sites described by Herrera and Curran (Herrera, F.C., Curran, P.F. 1963. J. Gen. Physiol. 46:999).

Cyclic AMP levels in isolated frog skin epithelium: effects of phosphodiesterase inhibitors, oxytocin and catecholamines.

Direct measurements of cyclic AMP were performed in the isolated epithelium of frog skin. Phosphodiesterase inhibitors (methylxanthines, papaverine) and activators of adenylyl cyclase (oxytocin, catecholamines) significantly increased the cyclic AMP content. Propranolol completely blocked the generation of cAMP induced by beta-adrenergic agonists but had little or no effect on that induced by oxytocin. Phentolamine enhanced the cAMP production by adrenalin and noradrenalin. At supramaximal concentrations, oxytocin and isoproterenol produced similar increments in cAMP, while exposure to both agents roughly doubled the increase in cAMP. The results suggest the presence of independent receptors for oxytocin and catecholamines in frog skin, with additive effects on cAMP generation.