Net Water Flow Research Articles

Ion transport by primary cultures of Mongolian gerbil middle ear epithelium.

The transport properties of Mongolian gerbil middle ear epithelial cells grown in primary culture were studied. These cells formed polarized monolayers that exhibited domes on nonporous supports. On porous supports, monolayers developed an apical-negative transepithelial electric potential difference (VT = -37.2 +/- 2.7 mV) and a transepithelial resistance (RT = 519 +/- 56 omega.cm2). The short-circuit current equivalent (Ieq) was 62.4 +/- 6.2 microA/cm2 (mean +/- SE, n = 15). Na+ and Cl- accumulated in the basal bath and generated a basolateral hyperosmolarity that drove a net water flow. Amiloride (10 microM), when added to the apical but not to the basal bath, induced a 23.4 +/- 1.5 mV and 44.1 +/- 1.3 microA/cm2 decrease of VT and Ieq, respectively, while RT increased by 403 +/- 69 omega.cm2 (P less than 0.001, n = 15). Exposure of the monolayers to a low-Cl- solution (30 mM) enhanced the transepithelial potential, possibly by means of a Cl- secretion through apical Cl- channels. Isoproterenol (10(-4) M basolateral) increased intracellular adenosine 3',5'-cyclic monophosphate (cAMP) content (concentration of half-maximal response = 2.5 x 10(-7) M) and decreased VT, RT, and Ieq. The isoproterenol-induced fall of VT occurred even in the presence of low-Cl-solutions. This suggested an increase of the paracellular pathway conductance, although there was no significant modification of the mannitol permeability.(ABSTRACT TRUNCATED AT 250 WORDS)

GEWEX: The Global Energy and Water Cycle Experiment

The need for better quantitative understanding of energy fluxes and water exchanges is basic to all climate studies, yet our quantitative knowledge of the global hydrological cycle remains surprisingly poor. Evaporation minus precipitation or, similarly, the net flow of water from land to oceans and the net advection of moisture from the marine atmosphere to the terrestrial atmosphere is known at best to a factor of two to three (J. Schaake, personal communication, 1991). The regional and temporal distribution of the components of the hydrological budget are even more uncertain.Improved understanding of the global hydrological cycle has been limited in part by the absence of reliable global data on such essential quantities as precipitation, evaporation, and atmospheric transport; acquisition of these data is a prerequisite for the study of global energetics and hydrology. Space agencies in the United States, Europe, and Japan have announced plans to launch a series of global Earth observing satellites in the late 1990s to monitor these essential atmospheric and surface parameters and to complement existing operational meteorological and hydrological networks.

Cell Volume Regulation by Erythrocytes of the Euryhaline Fish, <i>Platichthys flesu</i><i>s</i>, after Hyposmotic Stress in Bicarbonate/Carbon-Dioxide-Buffered Medium

Subsequent to osmotic swelling, the nucleated erythrocytes of the flounder (Platichthys flesus) displayed a volume-regulatory decrease (RVD) which was similar in HEPES and HCO–3 media. This RVD was characterized by net K+, Cl– and water loss at constant Na+. During RVD, 46% of the net water flow was secondary to net inorganic ion flux in HEPES compared to about 77% in HCO-3 media, the resting might be attributed to organic osmolytes. In the presence of added bicarbonate ions at physiological concentrations and oxygen in the experimental medium, not only a K+/C1- cotransport seems to be involved during RVD, but also K+ and Cl– conductances. Moreover, transport pathways sensitive to N-phenylanthronilic acid or 4-acetamido-4’-isothyocyanate-2, 2-stilbene di-sulfonic acid play an important role in the volume-dependent ionic movements and in the volume-activated release of taurine. These results strongly suggest that the anion transporter (band 3) or another neighboring protein plays an important part in RVD in volume-activated osmolyte release after hyposmotic stress in flounder red blood cells.

ADH-induced water permeability and particle aggregates: alteration by a synthetic estrogen

In the amphibian urinary bladder, the increase in water permeability induced by antidiuretic hormone (ADH) is accompanied by the appearance of apical intramembrane particle (IMP) aggregates that are believed to contain specific channels for water. In a previous work, we have shown that 3,3'-diallyldiethylstilbestrol (DADES), a synthetic estrogen which is a blocker of the glucose transporter, also inhibits the hydrosmotic response to ADH in the bladder. Our aim in the present study was to analyze the alterations of the membrane fine structure further and to correlate them with the water permeability changes. The results point to a selective inhibition of the ADH-induced net water flow, probably due to an interference with one of the last steps of the response to the hormone. This inhibition is associated with an increase in the density of the apical IMP aggregates, which are thus probably not operational. The resting net water flow is not inhibited and, surprisingly, typical IMP aggregates are frequently observed in the apical membrane after DADES treatment. The compound also induces the appearance of unusual loose IMP clusters that can only be seen on the apical membrane of the granular cells and that share several ultrastructural similarities with the ADH-induced aggregates. These results suggest that 1) apical DADES treatment stimulates the insertion of IMP aggregates in the apical membrane of the urinary bladder and 2) DADES inhibits the ADH-induced water flow by interfering with the aggregates and thus probably by blocking the specific water channels.

Flotation of coarse particles in a counter-current column cell

The effect of particle size on the flotation of coarse particles is analyzed. It is concluded that turbulence and the presence of the froth layer are detrimental to the recovery of coarse particles during conventional flotation. A counter-current column cell is proposed to avoid these two factors. In this column, a net upwards flow of water is prevalent, such that no froth is allowed to form on the top. Also, the levitation of heavy bubble-particle aggregates is helped by the ascending water current. Preliminary work at laboratory level with kilogram-size samples indicated that under similar conditions recoveries in the column were much higher than in conventional cells. Flotation results obtained with coarsely sized phosphate and quartz samples are presented. One interesting feature of this type of column is very fast flotation kinetics. Thus, a short column (only 55-cm collection zone) yielded full recovery of floatable values.

Feeding and orientation of mottled sculpin, Cottus bairdi, to water jets

Mottled sculpin, Cottus bairdi, from Lake Michigan are primarily nocturnal feeders that use the mechanoreceptive lateral line system to locate prey. Signals from swimming and suspension feeding prey are due to rhythmic movements of the body and/or appendages which produce a current with a net water flow (DC) and a vibratory water flow component (AC) superimposed on it. To determine whether the feeding response of the mottled sculpin is elicited by AC and/or DC components, we tested the response of mottled sculpin to water jets which were either an unmodulated flow (DC) or had a superimposed modulated flow (AC: 10 and 20 Hz). Both free-roaming fish and fish that had been pre-positioned relative to the stimulus were tested. Results indicate that sculpins are more likely to bite at an AC jet nozzle and will bite at the jet nozzle at a greater distance for AC jets than for DC. However, fish can be conditioned to turn toward the DC flow. A fish trained to respond by turning toward a DC jet will nearly cease to respond to the DC jet after a few trials with an AC jet (which mainly elicit bites). The results suggest that there is an unconditioned bite response to AC signals and that training can produce a conditioned turn response to DC signals.

Mechanism of calcium ionophore stimulated Cl secretion from frog skin glands

The aim of the present study was to investigate the mechanism by which the calcium ionophore A23187 stimulates Cl and water secretion from exocrine glands in the frog skin. The Cl secretion was visualized as changes in short-circuit current (SCC) in skins where the Na absorption was blocked by amiloride applied to the apical membrane. Measurements of A23187 stimulated ion fluxes showed that the ionophore induced a net secretion of Cl, Na and K. The active Cl secretion was enhanced more than the Na and K secretion, resulting in a net secretion of negative ions which closely resembled the A23187-stimulated SCC. The effect of A23187 was abolished in skins pretreated with indomethacin, implying the involvement of prostaglandins in the response. Furthermore, the effect of A23187 was inhibited in the presence of quinacrine, indicating that the activation of the cyclooxygenase pathway is dependent on phospholipase A2 activity. In addition, the A23187, but not the arachidonic acid stimulated Cl secretion was abolished in the presence of trifluoperazine, suggesting that the effect of the ionophore may be mediated via a Ca2+ -calmodulin-dependent step located before the activation of the cyclooxygenase. The net water flow and the Cl secretion were measured simultaneously under the conditions outlined above. The stimulation, inhibition, and time-course of the water secretion were similar to the changes observed for the Cl secretion. The A23187 stimulated Cl secretion was enhanced by the phosphodiesterase inhibitor, theophyllin, indicating that the effect of A23187 was caused by an increase in the intracellular cAMP level in the gland cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydrophilic solute transport across rat alveolar epithelium.

Diffusional fluxes of a series of hydrophilic nonelectrolytes (molecular radii ranging from 0.15 to 0.57 nm) were measured across the alveolocapillary barrier in the isolated perfused fluid-filled rat lung. Radiolabeled solutes were lavaged into the distal air spaces of isolated Ringer-perfused lungs, and apparent permeability-surface area products were calculated from the rates of isotope appearance in the recirculating perfusate. These data were used to estimate theoretical equivalent pore radii in the alveolar epithelium, with the assumption of diffusive flow through water-filled cylindrical pores. The alveolar epithelium is best characterized by two pore populations, with small pores (radius 0.5 nm) occupying 98.7% of total pore area and larger pores (radius 3.4 nm) occupying 1.3% of total pore area. Net water flow out of the alveolar space was measured by including an impermeant solute (dextran) in the lavage fluid and measuring its concentration in the alveolar space as a function of time. Under control conditions, net water flow averaged 167 nl/s. When 24 microM terbutaline was added to the perfusate, net water flow increased significantly to 350 nl/s (P less than 0.001). Terbutaline had no effect on the fluxes of either glycerol (which traverses the small pore pathway) or sucrose (which traverses the large pore pathway). These findings indicate that the intact mammalian alveolar epithelium is complex and highly resistant to the flow of solutes and water.

Dynamic circulation of interstitial seawater in a Jamaican fringing reef

Twelve 7.62 cm diameter cores were taken along three and a half kilometers of coastline in the vicinity of Discovery Bay, Jamaica, to document the distribution of dolomite previously described (Mitchellet al., 1987). Water was observed to flow actively in and out of the open holes, which range in water depths from 6 to 13 meters. Most of the shallowest holes, and the single deepest one, “exhale” slowly and continuously, little affected by the wind-driven surge. Holes at intermediate depths exhibit a net flow of water into the reef, “breathing” asymmetrically with the wind-driven surge. In one case an integrated current velocity of 86 cm sec−1 was measured over a five minute interval into a three meter deep open hole. Astronomical tides, local wind-driven surge, and dispersion due to meteoric water discharge are apparently not direct causes of the phenomenon.

Responses of a CAM Plant to Drought and Rainfall: Capacitance and Osmotic Pressure Influences on Water Movement

Daily and seasonal patterns in water flow and water potential were investigated for the Crassulacean acid metabolism succulent Agave deserti during an extended summer drought and for a period following rainfall. Field measurements of transpiration and of osmotic pressure changes over selected 24 h periods were used as input variables for a computer model of water flow that was based on an electrical circuit analog of the whole plant. Parameters such as root resistance and tissue capacitance were also varied to reflect the effects of changing plant or soil water status. The model predicted internal water flow and water potential during the drought cycle and was used to assess the role of tissue osmotic properties in water uptake from the soil and in internal water redistribution. For plants under wet soil conditions, 55% of the night-time transpiration was derived from water storage, this storage being recharged during the day. As drought progressed, transpiration and the nocturnal increase in osmotic pressure declined, although the osmotic pressure itself increased. The difference in osmotic pressure between the water storage tissue and the chlorenchyma caused a net flow of water into the chlorenchyma after 3 weeks of drought, thereby increasing chlorenchyma turgor pressure. Simulations also indicated that a large increase in root resistance must occur to prevent substantial water loss from the plant to the dry soil. After rainfall, recharge of plant water storage was complete within one week, although full recovery in the amplitude of daily osmotic pressure variations took longer.

Trypsin affects basal and stimulated osmotic water permeability in isolated toad skin

1. 1. We investigated the effect of trypsin (Tryp) on basal, stimulated and fluphenazine (FPZ)-inhibited net water flow ( J w) through isolated toad skin ( Bufo arenarum). 2. 2. Epidermal Tryp (20 min) promoted an increase in basal J w which was dose-dependent (maximal with 0.5 mg/ml) and was prevented by a Tryp inhibitor (SBTI). 3. 3. Tryp treatment inhibited the subsequent response to substances known to act before (oxytocin, Oxy) or after cyclic AMP (cAMP) generation (theophylline). 4. 4. Tryp-induced J w was not additive with the maximal response to Oxy or theophylline and did not modify FPZ's inhibitory effect on stimulated J w. 5. 5. Dermal Tryp (0.5 mg/ml, 20 min) did not modify basal, but inhibited Oxy and isoproterenol-stimulated J w, without altering the response to theophylline or db-cAMP. 6. 6. Collectively, our results show a differential action for epidermal and dermal Tryp. Tryp's side-selective action enables its use as a pharmacological tool in the functional dissection of J w across toad skin.

Inhibition of the hydrosmotic response to antidiuretic hormone by 3,3′‐diallyldiethylstilbestrol (DADES)

3,3'-diallyldiethylstilbestrol (DADES), a blocker of the facilitated diffusion of glucose, was found to interfere markedly with the hydrosmotic response to antidiuretic hormone and its related agonists. Frog urinary bladders were isolated and monitored for transmural net water flow. DADES was added either to the serosal or to the apical medium at concentrations ranging from 10(-4) M to 10(-6) M. Pretreatment for 30 min with apical 10(-4) M DADES drastically reduced the subsequent hydrosmotic response: (a) to oxytocin (4.4 x 10(-8) M) by 91.7 +/- 17.6% versus 6.2 +/- 7.8 in control; (b) to 8-bromo 3',5'-cyclic AMP by 93.5 +/- 19.4% versus 19.4 +/- 11.4%; (c) to serosal hyperosmolarity (mannitol 220 mOsm) by 99.3 +/- 0.5% versus 12.3 +/- 18.2%. This effect was dose-dependent. Inhibitory action of DADES was more effective on the apical side than on the serosal side (97.0 +/- 1.5 versus 45.8 +/- 10.8). Freeze-fracture studies revealed a modified distribution of the particles and unusual endocytotic pits and vesicles in the apical membrane of both granular and mitochondria-rich epithelial cells. These observations point to multiple and complex effects of the drug. Thus, it seems that DADES has numerous effects on urinary epithelium, which makes it a nonspecific inhibitor of water permeation. Conclusions on its use should therefore be drawn with suitable caution.

To what extent is microtubular network involved in antidiuretic response?

Antimitotic drugs markedly interfere with antidiuretic response, strongly implying that cytoskeleton integrity is essential to this function. This role of the cytoskeleton in controlling the epithelial transport has been seen as a necessary step in the translocation of the water channel containing particle aggregates and in their delivery to the apical membrane. We have now reexamined the exact role of the microtubular network by appropriate time course determinations, by the use of microtubule disruptive agents that lack of the side effects of colchicine, and by trying to visualize the apparent modifications of the microtubular network that accompany water permeability alterations using immunocytochemical techniques. Our results fully confirm that after microtubular network disruption, antidiuretic hormone-induced water permeability variations undergo typical alterations consisting in both a reduction in peak net water flow and a slowing down of its onset. At the same time, the microtubular network disappears in all the epithelial cells. We also show that colchicine-induced inhibition can still be observed in the presence of a prostaglandin synthetase inhibitor and that this inhibition is most likely to occur at a post-adenosine 3',5'-cyclic monophosphate level. These data, as well as results from other series with nocodazole, indicate that the reduction of the net water flow directly results from microtubular network disruption and not from side effects of the disrupting drugs. They also show that the hydrosmotic response is only partially dependent on the microtubular network, which probably has only a guidance role in the translocation of particle aggregates and their exocytotic fusion to the apical membrane.

The influence of a narrow transverse canyon on initially geostrophic flow

The interaction of a geostrophic current with a transverse submarine canyon is considered with an analytical, two‐level model of a homogeneous fluid on an f plane. The dynamical hypothesis is that the flow in the canyon is forced by the pressure gradient due to a sloping free surface. The interaction of flow in the canyon with the overlying fluid is through the vertical velocity forced by divergence in the canyon. The mathematical problem considers a flat‐bottom ocean with an infinitely long canyon having a rectangular cross section and uniform depth. The dynamics are rotationally modified, shallow‐water equations for each level. The initial time behavior of the model is considered with a power series solution which shows that flow in the canyon responds quickly, in about 0.1 of the inertia period, to an imposed surface pressure gradient. The time‐dependent model is solved by means of a Laplace transform in time in three spatial domains: over the canyon and on either side. For a canyon of arbitrary width, the flow over the canyon is composed of standing waves, while radiating waves exist on either side of the canyon. In the absence of friction, the oscillations continue indefinitely, since there are standing gravity modes in the canyon. In steady state, there is no net flow of water across the canyon, so such submarine features should act as barriers to barotropic geostrophic flow. The limit of a narrow canyon is considered. Arguments are presented to show that a canyon is narrow if its width is less than half of the smaller of the radius of deformation or the width scale of the overlying current. For a forcing current with a width about equal to the radius of deformation, the oscillations in the canyon have periods of about 0.1 up to 1. times the inertia period. Such oscillations have been observed in most canyons for which current meter observations exist.

Na-K-ATPase in the enamel organ: localization and possible roles in enamel formation.

Ouabain-sensitive, K-dependent p-nitrophenyl phosphatase (p-NPPase) activity was localized ultra-Ocytochemically in the lateral plasma membranes of secretory ameloblasts and the stratum intermedium and principally in the papillary layer cells of aldehyde-fixed rat incisor enamel organs by the one-step lead method. Daily intraperitoneal injection of ouabain (250 μg, 500 μg, and 1 mg/100 g body weight) for two weeks reduced p-NPPase activity in the enamel organ cells. However, the degree to which this activity was reduced appeared to vary among the experimental animals. Addition of ouabain to the cytochemical incubation medium completely inhibited p-NPPase activity in the tissues. Although long-term ouabain injection did not result in any morphological alterations of the enamel organ cells, it caused, in part, an appearance of electron-dense, homogeneous matrix-like substances (MS) in the extracellular spaces of the ameloblast layers at both the secretion and maturation stages. In addition, long-term ouabain injection appeared to have resulted in delayed maturation of enamel as measured by energy-dispersive x-ray analysis of Ca and P in surface enamel. These results suggest that Na-K-ATPase of enamel organ cells may participate in the net flow (removal) of organic matrix components and water from the enamel during the maturation stage of enamel formation. It is suggested that this flow is maintained by local osmotic gradients generated by Na-K-ATPase within the papillary layer.

Synthesis and apical and basolateral secretion of thyroglobulin by thyroid cell monolayers on permeable substrate: modulation by thyrotropin.

In the thyroid glands, thyroglobulin (Tg) is specifically synthesized by follicular cells and then secreted into the apical lumen where it is concentrated and used as a substrate for thyroid hormone synthesis. The presence of Tg in the circulation has been reported in normal and pathological situations. To determine the domains of the plasma membrane, apical and/or basolateral, involved in Tg secretion, porcine thyroid epithelial cells were cultured as a monolayer on the porous bottom of a culture chamber in which both apical and basal media are independently accessible. Control experiments using labeled Tg ascertained the tightness of the monolayer and showed that within 48 h only 0.2-0.5% of the Tg introduced in the apical medium was transferred through the cell layer into the basal compartment. For kinetic studies of Tg synthesis and secretion, monolayers were cultured for up to 72 h in the presence of 35S-methionine and with or without 100 microU/ml thyrotropin (TSH) in the basal medium. Labeled Tg was measured by double immunoprecipitation and by fluorography of polyacrylamide gel electrophoresis. We showed that 80-95% of total secreted Tg was recovered in the apical medium. The remainder was secreted through the basolateral membranes in the basal medium. The amount of tg secreted into the apical compartment was stimulated two- to threefold by TSH whereas no TSH effect was observed on secretion in the basal compartment. Moreover, measuring apical and basal volumes, we observed a net water flow from the apical to the basal side. It was stimulated threefold by TSH, increasing the Tg concentration in the apical compartment of the stimulated cell layer. During the culture time, the amount of Tg synthesized and secreted was increased by TSH, as was the Tg mRNA content, as determined by the dot-blot hybridization method.

Prostaglandin E2-stimulated glandular ion and water secretion in isolated frog skin (Rana esculenta).

Prostaglandins are known to stimulate the active sodium absorption in frog skin. In this paper it is shown that prostaglandin E2 (PGE2) stimulates an active secretion of Cl-, Na+, and K+ from the skin glands in Rana esculenta. The active Cl secretion is enhanced more than the Na and K secretion. Therefore, in skins where the Na absorption is inhibited by amiloride, the addition of PGE2 results in an increase in the short-circuit current (SCC). The PGE2-stimulated Cl secretion could be inhibited by the presence of ouabain or furosemide in the basolateral solution or diphenylamine-2-carboxylate in the apical solution. The PGE2-stimulated Cl secretion was enhanced by the phosphodiesterase inhibitor, theophylline, indicating that the effect of PGE2 was caused by an increase in the intracellular cAMP level in the gland cells. The calcium ionophore A23187, which increases the PGE2 synthesis in frog skin, stimulated the glandular Cl secretion. This secretion could be blocked by the prostaglandin synthesis inhibitor indomethacin, indicating that A23187 acts by increasing the prostaglandin synthesis and not by a direct action of Ca2+ ions per se. The net water flow (Jw) and the Cl secretion were measured simultaneously under the conditions outlined above. The stimulation, inhibition, and the time-course of the outward-directed Jw were similar to the change observed for the Cl secretion. These results show that PGE2 stimulates a glandular secretion of Cl and water in frog skin, probably by increasing the cAMP level in the gland cells.

Performance study of a solar water heater incorporating a bed of solid particulates

An investigation of a solar water heater with built-in storage is presented. The experimental apparatus consists, essentially, of a rectangular insulated tank packed with solid particulates. The packed system is examined with no net water flow. An equation based on a heat balance is used to predict the theoretical temperature history of the water. Comparisons demonstrate that the model agrees well with experimental values. Predictions are also made regarding the effect of using various packing materials.

The Effects of Mechanical Work on Electrolyte and Water Distribution in Amphibian Skeletal Muscle

ABSTRACT The present experiments were undertaken to confirm whether the increase in haematocrit that consistently accompanies the build-up of lactate in amphibian muscle cells during exercise can be explained in terms of a movement of water from the blood into the active muscles. Electrically stimulated sartorius and gastrocnemius muscles isolated from Rana ridibunda and Xenopus laevis had consistently higher total water contents than their paired control muscles. In both instances, it was the intracellular water volume which gave rise to the increase in total muscle water. These results were corroborated in vivo by sampling gastrocnemius muscles from exercising and resting Xenopus laevis. Analyses of tissue electrolyte levels in the working muscles of each experimental series showed an increase in intracellular [lactate−] and [Na+]. A corresponding decline in cellular [K+] occurred in concert with increases in extracellular [K+], In saline-perfused gastrocnemii of Xenopus, the uptake of vascular water was proportional to the total mechanical work performed. Saline leaving the femoral vein of isotonically contracting gastrocnemius muscles had a greater osmotic pressure than that of the arterial perfusate, whereas arterio-venous osmolality differences of control muscles were negligible. Calculations show that the haemoconcentration during exercise in vivo can be attributed at least in part to a net flow of plasma water to osmotically enriched muscle cells.

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.