Water and solute regulation in Procephalothrix spiralis Coe and Clitellio arenarius (Müller) III. Long-term acclimation to diluted seawaters and effect of putative neuroendocrine structures

When acutely transferred to diluted seawater (SW), Procephalothrix spiralis and Clitellio arenarius regulate water content (g H₂O/g solute free dry wt = s.f.d.w.) via loss of Na and Cl (µmoles/g.s.f.d.w.). The present study extends these observations to a greater range of salinities and determines the effects of long-term, stepwise acclimation to diluted seawaters. Final exposure to a given experimental seawater (70, 50, 30, 15%) was 48 hours. Osmolality (mOsm/kg H₂O) and Na, K, and Cl ion concentrations (mEq/l) were determined in total tissue water and in the extracellular fluid of C. arenarius. Extracellular volume was determined as the ¹⁴C-polyethylene glycol space. Both species behaved as hyperosmotic conformers in diluted seawaters. However, reduction of the osmotic gradient between worm and medium occurred in P. spiralis, but not C. arenarius, in 30 and 15% SW. In both species, osmolality and Na, Cl, and K concentrations in total tissue water decreased with increased dilution of the SW. Water content increased with dilution of the medium but was lower than that which would be predicted based on approximation of the van't Hoff relation. This indicated the occurrence of regulatory volume decrease (RVD). In P. spiralis, in 70 or 50% SW, RVD was accompanied by loss of Na and Cl contents. However, in 30 or 15% SW, Na and Cl contents increased and in worms in 15% SW K content decreased. The latter movements of Na, Cl and K are indicative of cellular hysteresis and were associated with decreased viability, indicating the lower limits of regulatory ability in this species. In comparison, RVD in C. arenarius occurred in all diluted seawaters and was accompanied by loss of Na and Cl contents. In C. arenarius, evidence for reduced viability was absent. Removal of the supra- and subesophageal ganglia of C. arenarius resulted in retention of water, Na and Cl (g H₂O or µmoles/g s.f.d.w.) in worms acclimated to 70% SW. Removal of the cerebral ganglia and cephalic glands of P. spiralis did not significantly influence regulation of water content.     

Separate,Ca2+-activated K+ and Cl− transport pathways in Ehrlich ascites tumor cells

Summary The net loss of KCl observed in Ehrlich ascites cells during regulatory volume decrease (RVD) following hypotonic exposure involves activation of separate conductive K⁺ and Cl^– transport pathways. RVD is accelerated when a parallel K⁺ transport pathway is provided by addition of gramicidin, indicating that the K⁺ conductance is rate limiting. Addition of ionophore A23187 plus Ca²⁺ also activates separate K⁺ and Cl^– transport pathways, resulting in a hyperpolarization of the cell membrane. A calculation shows that the K⁺ and Cl^– conductance is increased 14-and 10-fold, respectively. Gramicidin fails to accelerate the A23187-induced cell shrinkage, indicating that the Cl^– conductance is rate limiting. An A23187-induced activation of⁴²K and³⁶Cl tracer fluxes is directly demonstrated. RVD and the A23187-induced cell shrinkage both are: (i) inhibited by quinine which blocks the Ca²⁺-activated K⁺ channel. (ii) unaffected by substitution of NO ₃ ^– or SCN^– for Cl^–, and (iii) inhibited by the anti-calmodulin drug pimozide. When the K⁺ channel is blocked by quinine but bypassed by addition of gramicidin, the rate of cell shrinkage can be used to monitor the Cl^– conductance. The Cl^– conductance is increased about 60-fold during RVD. The volume-induced activation of the Cl^– transport pathway is transient, with inactivation within about 10 min. The activation induced by ionophore A23187 in Ca²⁺-free media (probably by release of Ca²⁺ from internal stores) is also transient, whereas the activation is persistent in Ca²⁺-containing media. In the latter case, addition of excess EGTA is followed by inactivation of the Cl^– transport pathway. These findings suggest that a transient increase in free cytosolic Ca²⁺ may account for the transient activation of the Cl^– transport pathway. The activated anion transport pathway is unselective, carrying both Cl^–, Br^–, NO ₃ ^– , and SCN^–. The anti-calmodulin drug pimozide blocks the volume- or A23187-induced Cl^– transport pathway and also blocks the activation of the K⁺ transport pathway. This is demonstrated directly by⁴²K flux experiments and indirectly in media where the dominating anion (SCN^–) has a high ground permeability. A comparison of the A23187-induced K⁺ conductance estimated from⁴²K flux measurements at high external K⁺, and from net K^– flux measurements suggests single-file behavior of the Ca²⁺-activated K⁺ channel. The number of Ca²⁺-activated K⁺ channels is estimated at about 100 per cell.     

An examination of the method of sizing the spermatozoa of the domestic fowl and duck with an electronic counter

Although many estimations have been made electronically of mammalian sperm volume, detailed investigations have not been reported for avian spermatozoa with an electronic counter. In the present study, sizing of spermatozoa of fowls and Muscovy and Pekin drakes was examined using a Coulter counter (model ZB). In our preliminary work on fowl sperm volumes, we found mono- or di-morphic distribution displays that were modified depending on the combination of amplification (AMP) and aperture current (APC). Therefore, methodology to estimate the fowl and drake sperm volume was examined. Dilution of semen had no effect on the dimorphic distribution pattern of the sperm volume. Density-gradient centrifugation did not separate two kind of particles in the semen in either continuous or discontinuous Percoll gradients; therefore, we varied settings of AMP and APC, and found that the most suitable settings for measuring sperm volumes of these birds are 1 for AMP and 8 for APC. With these settings, mean volumes of spermatozoa were 5.1 μm³ for fowls, 5.7 μm³ for Muscovy drakes, and 5.6 μm³ for Pekin drakes.     

Effects of irradiation level on leaf growth of sunflower

Sunflower, Helianthus annuus L. cv. INRA 6501, plants were grown in a gravel culture subirrigated with Hoagland nutrient solution, at photosynthetically active radiation levels of 15, 30 and 60 W m^-2 at a daylength of 16 h, a temperature of 20°C and a relative humidity of 60% throughout. Development of the plant and growth of the leaves were measured. High irradiance accelerated development proportionally in all phases from germination, through leaf initiation, primordial flower formation and the maturation of all plant organs until anthesis. High irradiance levels stimulated the expansion of the growing shoot, which produced more and larger primordia. Under constant conditions the ratio between leaf initiation rate and mature length of a leaf remained constant, although the growth patterns [relationship between relative growth rate (RGR) and organ age] of successive leaves were not similar. Consequently, it may be assumed that, as in poplar, the increasing size of the growing shoot reflects the increase of the vascular system of sunflower. The growth patterns of the leaves depend on the developmental stage of the plant and, in the young primordial stage, also on irradiance level. In the linear phase of growth the growth pattern is independent of irradiance level.     

Aedes aegypti: model for blood finding strategy and prediction of parasite manipulation

Aedes aegypti mosquitoes salivate during intradermal probing of vertebrate prey before ingesting blood (Griffiths and Gordon 1952). Nonsalivating mosquitoes locate blood more slowly; this difference was ascribed to an anti-platelet activity found in the mosquito's saliva (Ribeiro et al. 1984). Mosquitoes infected with Plasmodium gallinaceum suffer pathology that specifically impairs saliva anti-hemostatic activity but without reducing volume of output (Rossignol et al. 1984). The complexity of the feeding apparatus of mosquitoes provides opportunity for a variety of strategies in which pathogens may produce specific lesions that enhance their transmission, but the variables that affect the duration of probing by mosquitoes have not been defined. We sought to resolve this complexity by identifying and quantifying relevant parameters of probing behavior. Mosquitoes thrust their mouthparts repeatedly through their host's skin while searching for blood. Female A. aegypti thrust at 7-sec intervals. If this search results in success, feeding ensues. Alternatively, the mosquito "desists," the mouthparts stylets are withdrawn, and the mosquito attempts to feed at another site. Even after previous desistance, the probability of finding blood remains undiminished. Functions for the probability of feeding success and desistance over time were derived using data from observations on 300 mosquitoes. The probability of feeding success was interpreted as being a function of the density of vessels in the skin, their geometric distribution, and the conditions locally affecting hemostasis. During each probe, the probability of desisting increased linearly with time, and after desisting once, mosquitoes tended to desist more rapidly. A model was developed incorporating Monte Carlo simulation which closely fit observed data. By changing values for the several parameters of the probability functions, we predicted modes in which parasites may manipulate their hosts to enhance transmission, both to and from the vector. In particular, parasite strategies in the vector would include induced salivary pathology; increased duration of probing thrusts; decreased desistance time; and inhibited phagoreception. Predicted parasite strategies in the reservoir host would include increased skin vascular volume and impaired host hemostasis. Our model supports the hypothesis of a mutualistic interaction of malaria and mosquitoes.     

General method for the derivation and numerical solution of epithelial transport models

Summary A general method is presented for the formulation and numerical evaluation of mathematical models describing epithelial transport. The method is based on the principles of conservation of mass, and maintenance of electroneutrality within the cells and bathing solutions. It is therefore independent of the specific membrane transport mechanisms, and can be used to evaluate different models describing arbitrary transport processes (including passive, active and cotransport processes). Detailed numerical methods are presented that allow computation of steady-state and transient responses under open-circuit, current-clamp and voltage-clamp conditions, using a general-purpose laboratory minicomputer. To evaluate the utility of this approach, a specific model is presented that is consistent with the Koefoed-Johnson and Ussing hypothesis of sodium transport in tight epithelia (Acta Physiol. Scand. 42:298–308, 1958). This model considers passive transport of an arbitrary number of permeant solutes, active transport of sodium and potassium, and osmotically induced water transport across the apical and basolateral membranes. Results of the model are compared to published experimental measurements in rabbit urinary bladder epithelium.     

Transient breakdown in the selective permeability of the plasma membrane ofChlorella emersonii in response to hyperosmotic shock: Implications for cell water relations and osmotic adjustment

Summary In osmotic experiments involving cells of the euryhaline unicellular green algaChlorella emersonii exposed to hyperosmotic stress by immersion in a range of low molecular weight organic and inorganic solutes, a temporary breakdown in the selective permeability of the plasma membrane was observed during the initial phase of transfer to media of high osmotic strength (up to 2000 mosmol kg^–1). Thus, although the cells appeared to obey the Boyle-van't Hoff relationship in all cases, showing approximately linear changes in volume (at high salinity) as a function of the reciprocal of the external osmotic pressure, the extent of change was least for the triitols, propylene glycol and glycerol, intermediate for glucose, sorbitol, NaCl and KCl, with greatest changes in media containing the disaccharides sucrose and maltose. In NaCl-treated cells, uptake of external solute and loss of internal ions was observed in response to hyperosmotic treatment while sucrose-treated cells showed no significant uptake of external solute, although loss of intracellular K⁺ was observed. These observations suggest that the widely used technique of estimating cellular turgor, and osmotic/nonosmotic volume by means of the changes in volume that occur upon transfer to media containing increasing amounts of either a low molecular weight organic solute or an inorganic salt may be subject to error. The assumption that all algal cells behave as ideal osmometers, with outer membranes that are permeable to water but not to solutes, during the course of such experiments is therefore incorrect, and the data need to be adjusted to take account of hyperosmotically induced external solute penetration and/or loss of intracellular osmotica before meaningful estimates of cell turgor and osmotic volume can be obtained.     

Osmotic responses of unicellular blue-green algae (cyanobacteria): changes in cell volume and intracellular solute levels in response to hyperosmotic treatment

Abstract Changes in cell volume and solute content upon hyperosmotic shock have been studied for six unicellular blue-green algae (cyanobacteria): Synechococcus PCC 6301, PCC 6311; Synechocystis PCC 6702, PCC 6714, PCC 6803 and PCC 7008. The extent of change in volume was shown to be dependent upon the solute used to establish the osmotic gradient, with cells in NaCl showing a reduced shrinkage when compared to cells in media containing added sorbitol and sucrose. Uptake of extracellular solutes during hyperosmotic shock was observed in Synechocystis PCC 6714, with maximum accumulation of external solutes in NaCl and minimum solute uptake in sucrose solutions. Conversely, solute loss from the cells (K⁺ and amino acids) was greatest in sucrose-containing media and least in NaCl. The results show that these blue-green algae do not behave as ‘ideal osmometers’ in media of high osmotic strength. It is proposed that short-term changes in plasmalemma permeability in these organisms may be due to transient membrane instability resulting from osmotic imbalance between the cell and its surrounding fluid at the onset of hyperosmotic shock.