SOME ASPECTS OF SECRETION : I. SECRETION OF WATER

If we increase the osmotic pressure at one end of a Nitella cell by applying a solution of sucrose and if we subsequently submerge the entire cell in water we find that water enters at the end where the osmotic pressure is higher and comes out of the cell at the other end. If similar inequalities of osmotic pressure should arise as the result of metabolism we can understand how a secreting cell might take up water at one spot on its surface and expel it in another spot and thus bring about the secretion of water. The Nitella cell can expel water from a region of the cell which is in contact with water, air, or mineral oil.     

Differences in the way potassium chloride and sucrose solutions effect osmotic potential of significance to stomata aperture modulation

Guard cell solution osmotic potential changes resulting in the opening and closing of stomata apertures follow an initial influx of potassium ions, their substitution with sucrose molecules and the subsequent reduction of the latter. To provide an insight into the osmotic mechanism of the changes, the new equation for calculating osmotic pressure, which equates the difference between the energy of pure water across a semi-permeable membrane interface with that of solution water, was used to compare the osmotic properties of KCl and sucrose. For sucrose solutions, the effect of the sucrose molecules in increasing the spacing of the solution water was mainly responsible for osmotic potential; this contrasted with K⁺ + Cl^? ions where their spacing effect was only a little higher to that of water held to those ions. At solute concentrations giving an osmotic potential level of ?3.0 MPa near that of turgid guard cells, the spacing effect on the potential of the unattached solution water molecules caused by sucrose, but in its theoretical absence, was estimated as ?2.203 MPa compared with ?1.431 MPa for KCl. In contrast, the potential attributed to water molecules firmly held to the K⁺ + Cl^? ions was ?1.212 MPa versus zero for sucrose. The potential to keep the sucrose molecules in solution was ?0.797 MPa compared with ?0.357 MPa for KCl. The findings illustrate that the way KCl effects osmotic pressure is very different to that of sucrose. It is concluded that stomata aperture modulation is closely linked to the osmotic properties of its guard cell solution solutes.     

Evaluation of water stress control with polyethylene glycols by analysis of guttation

The water relations of pepper plants (Capsicum frutescens L.) under conditions conducive to guttation were studied to evaluate the control of plant water stress with polyethylene glycols. The addition of polyethylene glycol 6000 to the nutrient solution resulted in water relations similar to those expected in soil at the same water potentials. Specifically, xylem pressure potential in the root and leaf became more negative during a 24-hour treatment period, while osmotic potential of the root xylem sap remained constant. The decrease in pressure potential was closely correlated with the decrease in osmotic potential of the nutrient solution. In contrast, the addition of polyethylene glycol 400 to the nutrient medium resulted in a reduction of osmotic potential in the root xylem sap; this osmotic adjustment in the xylem was large enough to establish an osmotic gradient for entry of water and cause guttation at a nutrient solution osmotic potential of −4.8 bars. Pressure potential in the root and leaf xylem became negative only at nutrient solution osmotic potentials lower than −4.8 bars. About half of the xylem osmotic adjustment in the presence of polyethylene glycol 400 was caused by increased accumulation of K⁺, Na⁺, Ca²⁺, and Mg²⁺ in the root xylem. These studies indicate that larger polyethylene glycol molecules such as polyethylene glycol 6000 are more useful for simulating soil water stress than smaller molecules such as polyethylene glycol 400.     

THE RECIPROCAL RELATION BETWEEN THE OSMOTIC PRESSURE AND THE VISCOSITY OF GELATIN SOLUTIONS

1. These experiments confirm the conclusion that protein solutions are true solutions consisting of isolated ions and molecules, and that these solutions may or may not contain in addition solid submicroscopic particles capable of occluding water. 2. The typical influence of electrolytes on the osmotic pressure of protein solutions is due to the isolated protein ions since these alone are capable of causing a Donnan equilibrium across a membrane impermeable to the protein ions but permeable to most crystalloidal ions. 3. The similar influence of electrolytes on the viscosity of protein solutions is due to the submicroscopic solid protein particles capable of occluding water since the amount of water occluded by (or the amount of swelling of) these particles is regulated by the Donnan equilibrium. 4. These ideas are supported by the fact that the more the submicroscopic solid particles contained in a protein solution or suspension are transformed into isolated ions (e.g., by keeping gelatin solution for 1 hour or more at 45°C.) the more the viscosity of the solution is diminished while the osmotic pressure is increased, and vice versa.     

Enteric bacteria and osmotic stress: Intracellular potassium glutamate as a secondary signal of osmotic stress?

Abstract Enteric bacteria have evolved an impressive array of mechanisms that allow the cell to grow at widely different external osmotic pressures. These serve two linked functions; firstly, they allow the cell to maintain in relatively constant turgor pressure which is essential for cell growth; and secondly they permit changes in cytoplasmic composition such that the accumulation of intracellular osmolytes required to restore turgor pressure does not impair enzyme function. The primary event in turgor regulation is the controlled accumulation of potassium and its counterion glutamate. At high external osmolarities the cytoplasmic levels of potassium glutamate can impair enzyme function. Rapid growth is therefore dependent upon secondary responses, principally the accumulation of compatible solutes, betaine ( N -trimethylglycine), proline and trehalose. The accumulation of these solutes is achieved by the controlled activity of transport systems and enzymes in response to changes in external osmotic pressure. It has been proposed that the accumulation of potassium glutamate during turgor regulation acts as a signal for the activation of these systems [1,2]. This brief review will examine the evidence that control over the balance of cytoplasmic osmolytes is achieved by sensing of the intracellular potassium (and glutamate) concentration.     

Enteric bacteria and osmotic stress: intracellular potassium glutamate as a secondary signal of osmotic stress?

Enteric bacteria have evolved an impressive array of mechanisms that allow the cell to grow at widely different external osmotic pressures. These serve two linked functions; firstly, they allow the cell to maintain a relatively constant turgor pressure which is essential for cell growth; and secondly they permit changes in cytoplasmic composition such that the accumulation of intracellular osmolytes required to restore turgor pressure does not impair enzyme function. The primary event in turgor regulation is the controlled accumulation of potassium and its counterion glutamate. At high external osmolarities the cytoplasmic levels of potassium glutamate can impair enzyme function. Rapid growth is therefore dependent upon secondary responses, principally the accumulation of compatible solutes, betaine (N-trimethylglycine), proline and trehalose. The accumulation of these solutes is achieved by the controlled activity of transport systems and enzymes in response to changes in external osmotic pressure. It has been proposed that the accumulation of potassium glutamate during turgor regulation acts as a signal for the activation of these systems [1,2]. This brief review will examine the evidence that control over the balance of cytoplasmic osmolytes is achieved by sensing of the intracellular potassium (and glutamate) concentration.     

Changes in ionic selectivity with changes in density of water in gels and cells

Gels equilibrated with aqueous solutions of impermeant solutes reached a steady state in which, in the absence of a pressure difference, the activity of water in the pores of the gel was higher than that of water in the external solution. The chemical potential of water in the gel/polymer solution slurry was higher than that in the supernatant polymer solution removed from the gel. Water in the pores of the gel decreased in density to 0.96 as increasing osmotic stress was applied. It is argued that at constant temperature and pressure water can equilibrate between two compartments of unequal osmolality only by adjusting its molar volume. Experiments showed that when gel water had a higher activity than external water it was K⁺ selective; when it had a lower activity it was Na⁺ selective. It is proposed that a continuous spectrum of water structures can exist in these two compartment systems from dense, reactive, weakly-bonded water which selects highly hydrated ions, to expanded, stretched, unreactive, viscous water which is strongly hydrogen bonded and selects K⁺ and univalent anions. These findings are related to the state and properties of cytoplasmic water which is probably held under osmotic stress by the activity of the sodium pump.     

ELECTRIFICATION OF WATER AND OSMOTIC PRESSURE

1. Amphoteric electrolytes form salts with both acids and alkalies. It is shown for two amphoteric electrolytes, Al(OH)₃ and gelatin, that in the presence of an acid salt water diffuses through a collodion membrane into a solution of these substances as if its particles were negatively charged, while water diffuses into solutions of these electrolytes, when they exist as monovalent or bivalent metal salts, as if the particles of water were positively charged. The turning point for the sign of the electrification of water seems to be near or to coincide with the isoelectric point of these two ampholytes which is a hydrogen ion concentration of about 2 x 10^–5 N for gelatin and about 10^–7 for Al(OH)₃. 2. In conformity with the rules given in a preceding paper the apparently positively charged water diffuses with less rapidity through a collodion membrane into a solution of Ca and Ba gelatinate than into a solution of Li, Na, K, or NH₄ gelatinate of the same concentration of gelatin and of hydrogen ions. Apparently negatively charged water diffuses also with less rapidity through a collodion membrane into a solution of gelatin sulfate than into a solution of gelatin chloride or nitrate of the same concentration of gelatin and of hydrogen ions. 3. If we define osmotic pressure as that additional pressure upon the solution required to cause as many molecules of water to diffuse from solution to the pure water as diffuse simultaneously in the opposite direction through the membrane, it follows that the osmotic pressure cannot depend only on the concentration of the solute but must depend also on the electrostatic effects of the ions present and that the influence of ions on the osmotic pressure must be the same as that on the initial velocity of diffusion. This assumption was put to a test in experiments with gelatin salts for which a collodion membrane is strictly semipermeable and the tests confirmed the expectation.     

Effect of the prolactin-inhibiting substances bromocripton and lergotrile on hydromineral regulation in rainbow troutSalmo gairdneri

Summary Plasma Na⁺, K⁺ and osmotic pressure were measured in rainbow trout (Salmo gairdneri) following the administration of the prolactin-inhibiting substances Lergotrile and Bromocripton. Both drugs elicited a significant fall in plasma Na⁺ concentrations although a significant response to Bromocripton was apparent only in trout acclimated to distilled water.The changes in plasma Na⁺ levels and in some cases of plasma osmotic pressure following administration of the prolactin-inhibiting substances were consistent with the hypothesis that prolactin acts to maintain plasma Na⁺ levels in this salmonid species in the same manner as in other teleosts. However, since the changes in plasma ions were small (albeit significant) is is proposed that prolactin may play a less important role in osmotic and/or ionic regulation in this species than it does in other teleostean species. Conversely, it may be that the drugs effects a less complete blockage of prolactin secretion than they appear to do in mammals.Ovine prolactin, administered with the drugs, effected a partial retention of plasma Na⁺ in Lergotrile-injected fish but did not significantly modify the effect of Bromocripton. These findings are discussed in light of the proposed action of the drugs, namely that of inhibiting the release of endogenous prolactin.Both Bromocripton and Lergotrile caused a significant fall in hematocrit values. Since plasma osmotic pressure values and plasma K⁺ concentrations were not markedly affected by the drugs (except for a significant (P<0.01) reduction in plasma osmotic pressure in the Bromocriptons-injected groups maintained in distilled water) it was thought that these changes were due to a reduction in the number of blood cells in the peripheral circulation rather than to an influx of water in response to the inhibition of prolactin.     

The Influence of the Cell Membrane on some Toxicity Effects of Growth Substances in Nitella

When cells of Nitella are placed in a solution of some plantgrowth substances there is a profound increase in the membraneresistance as measured by means of an internal silver/silverchloride micro-electrode. This impedance effect is accompaniedby some marked visible changes within the cell cytoplasm, thesechanges being dependent upon the concentration of undissociatedgrowth substance in the vicinity of the membrane, and upon thepH of the external environment. A detailed study is made of these visible changes and, takinginto account the negatively charged membrane, one possible interpretationof the results is that only undissociated molecules of the growthsubstance may enter the cytoplasm of Nitella from the environment,the diffusion obeying a simple Fick Law relationship. The substances are used in sufficient quantity to kill the Nitellacell, death following the visible effects, and they appear tobe unique in that their toxic effect produces a system of highmembrane impedance.     

Water and ion regulation in cicadas in relation to xylem feeding

Cicadas feed on xylem fluid. This is hypotonic to the haemolymph and contains high concentrations of potassium, sodium, calcium, magnesium, chloride, and phosphate ions. The urine contains the same ions in the same proportions but in slightly lower concentrations. Amino acids and sucrose are present in xylem fluid and traces of amino acids are also found in urine.Water is rapidly shunted from foregut to hindgut via the filter chamber. Injection of xylem fluid into the oesophagus results in an immediate tenfold increase in flow rate in the ileum. The osmotic pressure of xylem fluid in the filter chamber rapidly rises whilst the osmotic pressure in the anterior part of the ileum rapidly falls.Absorption of nutrients and ions into the haemolymph probably occurs in the conical segment and anterior tubular midgut. Storage excretion of divalent ions occurs in the mid-midgut and ions may be transported from the haemolymph into the posterior tubular midgut.The Malpighian tubules secrete a fluid slightly hypertonic to blood containing K⁺ (42 mM/l.] and Na⁺ (14 mM/l.).The osmotic pressures within the internal Malpighian tubules and internal midgut in the filter chamber are considerably higher than the osmotic pressure of the xylem fluid when it first enters the filter chamber proper. Passive osmosis will occur and water will be shunted into the ileum.Reabsorption of K⁺ and Na⁺ occurs in the ileum.     

Ionic and osmotic regulation of the haemolymph of the dragonfly, Libellula quadrimaculata (Odonata : Libellulidae)

Larvae of the widespread dragonfly, Libellula quadrimaculata, were adapted to a series of salt solutions, and the osmotic pressure, and sodium, potassium and chloride concentrations in the haemolymph measured. The regulation of potassium is extremely efficient over the range 0–50 m-mole/l. external concentration. Above this, larvae die. Sodium and chloride are regulated to a lesser extent, the larvae being able to withstand considerable changes in the concentration of these ions in the haemolymph. However, at higher external concentrations, the haemolymph concentration of these ions is maintained below that of the external medium. The osmotic pressure is regulated in parallel with sodium concentration over most of the range tested. However, in higher salinities, the osmotic pressure of the haemolymph does not fall below that of the external medium. This is seen as a strategy to limit the amount of drinking in saline media. Overall, the osmoregulatory system of L. quadrimaculata resembles that of brackish-water insects, rather than that of the more strictly freshwater dragonflies that have been studied.     

APPARENT VIOLATIONS OF THE ALL-OR-NONE LAW IN RELATION TO POTASSIUM IN THE PROTOPLASM

In Vol. 37, No. 6, July 20, 1954, page 814, in the third paragraph, delete the first sentence and insert the following: This equation gives excellent results with Nitella for example with 0.001 M KCl outside and 0.05 M KCl in the sap we may use concentrations in place of activities in the equation and put a_s = 0.05 and a_o = 0.001. No allowance is made for any change in the concentration of incoming ions due to their combination with carrier molecules since this change would be small and difficult to estimate. If we put U = 73 and V = 1 we obtain P = 97 mv. which is close to the usual observed value. The expression (73 –1) ÷ (73 + 1) = 0.973.     

Osmotic water transport with glucose in GLUT2 and SGLT

Carrier-mediated water cotransport is currently a favored explanation for water movement against an osmotic gradient. The vestibule within the central pore of Na⁺-dependent cotransporters or GLUT2 provides the necessary precondition for an osmotic mechanism, explaining this phenomenon without carriers. Simulating equilibrative glucose inflow via the narrow external orifice of GLUT2 raises vestibular tonicity relative to the external solution. Vestibular hypertonicity causes osmotic water inflow, which raises vestibular hydrostatic pressure and forces water, salt, and glucose into the outer cytosolic layer via its wide endofacial exit. Glucose uptake via GLUT2 also raises oocyte tonicity. Glucose exit from preloaded cells depletes the vestibule of glucose, making it hypotonic and thereby inducing water efflux. Inhibiting glucose exit with phloretin reestablishes vestibular hypertonicity, as it reequilibrates with the cytosolic glucose and net water inflow recommences. Simulated Na⁺-glucose cotransport demonstrates that active glucose accumulation within the vestibule generates water flows simultaneously with the onset of glucose flow and before any flow external to the transporter caused by hypertonicity in the outer cytosolic layers. The molar ratio of water/glucose flow is seen now to relate to the ratio of hydraulic and glucose permeability rather than to water storage capacity of putative water carriers.     

Transport of water from concentrated to dilute solutions in cells of Nitella

The transport of water from concentrated to dilute solutions which occurs in the kidney and in a variety of living cells presents a problem of fundamental importance. If the cell acts as an osmometer we may expect to bring about such transport by creating an inwardly directed osmotic drive which is higher in one part of the cell than in other regions of the same cell. The osmotic drive is defined as the difference between internal and external osmotic pressure. Experiments with Nitella show that this expectation is justified. If water is placed at one end of the cell (A) and 0.4 M sucrose with an osmotic pressure of 11.2 atmospheres at the other end (B) water enters at A, passes along inside the cell, and escapes at B leaving behind at B the solutes which cannot pass out through the protoplasm. Hence the internal osmotic pressure becomes much higher at B than at A. When 0.4 M sucrose at B is replaced by 0.3 M sucrose with an osmotic pressure of 8.1 atmospheres we find that water enters at B, passes along inside the cell, and escapes at A so that water is transported from a concentrated to a dilute solution although the difference in osmotic pressure of the 2 solutions is more than 8 atmospheres. The solution at B thus becomes more concentrated. It is evident that if metabolism produces a higher osmotic pressure and consequently a higher inwardly directed osmotic drive in one region of the cell as compared with other parts of the same cell water may be transferred from a concentrated to a dilute solution so that the former solution becomes still more concentrated.     

The role of membrane in osmotic flow

Osmosis as a phenomenon caused by internal forces goes on without the necessity for the presence of any external forces. Therefore pressure gradient plays no special role in osmotic flow. Membrane as a component of solution with its molecules possessing some kind of mobility ceases to be a passive obstacle to the flow of other components, but becomes also a co-determining factor in osmotic flow. This has been shown by using the methods of irreversible thermodynamics. In the state of osmotic equilibrium osmosis does not occur. So also the mobility of water molecules which may then be found in tracer experiment does not determine the osmotic permeability coefficient. The coefficientsσ andω as defined by the parameters of the system under the condition of zero volume flow are not directly connected to L_p.     

Barrier components acting against osmotic water flow inNitella

In order to check whether or not the layer of chloroplasts densely arranged in the cortical gel of aNitella internode offers substantial resistance to osmotic water flow, a material was prepared which had the cortical gel layer freed from chloroplasts by centrifugation either longitudinal or lateral to the cell axis. The water permeability of the cell remained the same as normal even though the chloroplasts were exfoliated from the cell cortex to the extent of 50% of the total area, showing that the chloroplast layer plays hardly any significant part as a barrier to osmotic flow. Since it is known that the layer of the streaming endoplasm is also negligible as resistance against osmotic water flow (Tasawa and Kamiya, 1965), it is concluded that the major barrier components against osmotic flow in theNitella internode are the cell wall, plasmalemma and/or tonoplast.     

Osmotic regulation in the marine alga, Codium decorticatum. I. Regulation of turgor pressure by control of ionic composition.

Codium decorticatum regulates its internal ionic composition and osmotic pressure in response to changes in external salinity. Over a salinity range of 23 to 37% (675 to 1120 mosmol/kg) Codium maintains a constant turgor pressure of 95 mosmol/kg (2.3 atm), observed as a constant difference between internal and external osmotic pressures. The changes in internal osmotic pressure are due to changes in intracellular inorganic ions. At 30 0/00 salinity the major intracellular ions are present in the following concentrations (mmol/kg cell H20): K+, 295; Na+, 255; Cl-, 450. At different salinities intracellular ion concentrations remain in constant proportion to the external ion concentrations, and thus the equilibrium potentials are approximately constant. The potential difference between the vacuole and seawater (-76 mV), whici is predominantly a K+ diffusion potential, is also constant with changing salinity. Comparison of the equilibrium potentials with the vacuole potential suggests that Cl- is actively absorbed and Na+ actively extruded, whereas K+ may be passively distributed between the vacuole and seawater. Turgor pressure does not change with environmental hydrostatic pressure, and increasing the external osmotic pressure with raffinose elicits a response similar to that obtained by increasing the salinity. These two results suggest that the stimulus for turgor regulation is a change in turgor pressure rather than a change in internal hydrostatic pressure or ion concentrations.     

Responses of Atriplex spongiosa and Suaeda monoica to Salinity

The growth and tissue water, K⁺, Na⁺, Cl⁻, proline and glycinebetaine contents of the shoots and roots of two Chenopodiaceae, Atriplex spongiosa and Suaeda monoica have been measured over a range of external NaCl salinities. Both species showed some fresh weight response to low salinity mainly due to increased succulence. S. monoica showed both a greater increase in succulence (at low salinities) and tolerance of high salinities than A. spongiosa. Both species had high affinities for Na⁺ and maintained constant but low shoot K⁺ contents with increasing salinity. These trends were more marked with S. monoica in which Na⁺ stimulated the accumulation of K⁺ in roots. An association between high leaf Na⁺ accumulation, high osmotic pressure, succulence, and a positive growth response at low salinities was noted. Proline accumulation was observed in shoot tissues with suboptimal water contents. High glycinebetaine contents were found in the shoots of both species. These correlated closely with the sap osmotic pressure and it is suggested that glycinebetaine is the major cytoplasmic osmoticum (with K⁺ salts) in these species at high salinities. Na⁺ salts may be preferentially utilized as vacuolar osmotica.     

Osmoadaptation in Representatives of Haloalkaliphilic Bacteria from Soda Lakes

The adaptation of microorganisms to life in brines allows two strategies: the accumulation of organic osmoregulators in the cell (as in many moderate halophiles, halomonads in particular) or the accumulation of inorganic ions at extremely high intracellular concentrations (as, for example, in haloanaerobes). To reveal the regularities of osmoregulation in haloalkaliphiles developing in soda lakes, Halomonas campisalis Z-7398-2 and Halomonas sp. AIR-2 were chosen as representatives of halomonads, and Natroniella acetigena, as a representative of haloanaerobes. It was established that, in alkaliphilic halomonads, the intracellular concentrations of inorganic ions are insufficient for counterbalancing the environmental osmotic pressure and balance is attained due to the accumulation of organic osmoregulators, such as ectoine and betaine. On the contrary, the alkaliphilic haloanaerobe N. acetigena employs K⁺, Na⁺, and Cl^? ions for osmoregulation. High intracellular salt concentrations increasing with the content of Na⁺ in the medium were revealed in this organism. At a concentration of 1.91 M Na⁺ in the medium, N. acetigena accumulated 0.83 M K⁺, 0.91 M Na⁺, and 0.29 M Cl^? in cells, and, with an increase in the Na⁺ content in the medium to 2.59 M, it accumulated 0.94 M K⁺, 1.98 M Na⁺, and 0.89 M Cl^?, which counterbalanced the external osmotic pressure and provided for cell turgor. Thus, it was shown that alkaliphilic microorganisms use osmoregulation strategies similar to those of halophiles and these mechanisms are independent of the mechanism of pH homeostasis.