Relationship between water transfers and the salinity of the environment in the isopod crustacean Sphaeroma serratum (Fabricius)]

The isopod Crustacean Sphaeroma serratum is isotonic to the medium in sea water and hypertonic in diluted media. The drinking rate is 15.9 microliter in SW and 3.2 microliter 24 h-1 100 mg-1 wet weight in 50% SW. The extracellular space is 28.4% in SW and 27.0% of the wet weight in 50% SW. The rate of urine production, calculated from the half time for the loss of sodium diatriazoate is 11.8 mg in SW and 42.9 mg 24 h-1 100 mg-1 wet weight in 50% SW. 95% of the diffusion fluxes of water take place through the pleopods: their surface is about 62.6 mm2 in a 100 mg weighing animal. The activation energy of water molecules is 15.0 kcal/mol between +5 degrees C and +15 degrees C and 6.7 kcal/mol between +15 degrees C and +25 degrees C. The diffusion permeability coefficient Pd is 1.71 X 10(-4) cm/sec in SW and 0.70 X 10(-4) cm/sec in 50% SW. The osmotic permeability coefficient Pos has a mean value of 1.91 X 10(-4) cm/sec in SW and 1.24 X 10(-4) cm/sec in 50% SW. Our results are compared with those obtained in other Crustaceans and in Fishes. Their validity is discussed (influence of unstirred layers, solute-solvent interaction). They are explained according to the different theories dealing with the water transit through the membranes.     

Exchange diffusion effect and euryhalinity in teleosts

The sea water (SW)-adapted euryhaline Platichthys flesus, and the marine Serranus exchange about 50% of their internal sodium with the external sodium per hour. This rate of exchange decreases with decreasing salinity of the adaptation medium. When the flounder is transferred from SW to FW an instantaneous 90% reduction of the Na and Cl outflux is observed. About 30 min later a second, progressive, reduction occurs. The outflux reductions appear to result from two types of regulatory mechanisms reducing gill permeability and preventing excessive salt loss. The first reduction corresponds to independent "Na- and Cl-free effects" as shown by transfers to artificial media containing either Na or Cl with an impermeant co-ion. The pattern of simultaneous rapid variations of Na influx and outflux for a range of salinity changes in flounder adapted to SW, 1/2 SW, or 1/4 SW has been studied. The data are compatible with the hypothesis of an exchange diffusion mechanism characterized by a coupling of both unidirectional fluxes. The affinity of the exchange diffusion carrier for sodium has been measured (Km approximately equal to 400 mM). The delayed reduction would result from a progressive diminution of the quantity of carrier available but without modification of its affinity for sodium. When the stenohaline marine perch is transferred from SW to FW, a 40% reduction of the outflux is observed. But it is not the result of an exchange diffusion effect as it is related to the external osmolarity change and not to the NaCl concentration change. Furthermore no delayed reduction is observed after transfer into FW. This transfer is accompanied by a heavy loss of electrolytes resulting in a rapid decline of the plasma electrolyte level and death. A comparative survey of the relative importance of these regulatory mechanisms has been made.     

Branchial Na,K-ATPase and osmoregulation in the purple shore crab,Hemigrapsus nudus (Dana)

The purple shore crab, Hemigrapsus nudus, controls its hemolymph osmolality over a wide range of external salinities: it is a strong hyperosmoregulator in 25%, 50% and 75% sea water (SW) and is isosmotic in 100% SW. The role of branchial sodium + potassium-activated, magnesium-requiring adenosine triphosphatase (NA, K-ATPase) in osmoregulation was investigated by assaying enzyme-specific activity (SEA) in gills from crabs acclimated for 14 d in the four sea water media. Assay conditions were characterized for optimal ESA with crude homogenates of gills; ion and cofactor requirements were found to be similar to those of other crustacean Na, K-ATPases. Branchial ESA was highest in crabs acclimated for 2 weeks in 50% SW and was significantly correlated with the osmotic gradient across the body wall in 50%, 75% and 100% SW. Gills 6, 7 and 8 had the highest ESA in all media and possessed approximately 70% of the total branchial Na, K-ATPase activity, but all gills showed significant, approximately twofold increases of ESA in 50% SW compared with values in 100% SW. The time courses of increased branchial Na, K-ATPase ESA and decreased hemolymph osmotic pressure in crabs transferred from 100% SW to 50% SW are consistent with both increased in vivo activity of existing enzyme in the short term and a longer-term synthesis of new enzyme by the gills which is measured by our in vitro assay.     

Turnover of internal water during the intermoult cycle of Sphaeroma serratum (Crustacea, Isopoda)]

1. The relation between the wet weight and the tritiated water outflux could be expressed by the following equation: M = 5.67 W0.75 2. The diffusion water outflux undergoes variations along the molting cycle : stage C :320 microliter H2O/h/100 mg; D1: 459; D2 :477, E: 1900; A :1 551; B :544 microliter H2O/h/100 mg wet weight. 3. The internal water turnover rises sharply and heavily (lambda stage D2: 805%; lambda stage E :2621%) during the ecdysis of the back of the body. After ecdysis of the fore-part of the body it returns approximately toward the intermoult level within 5 or 6 days.     

Adaptation of trout to seawater. Effects on sodium plasma concentration, gill exchange and intestinal transport]

10. Trout Salmo irideus are adapted to sea water (S W) within four weeks by submitting them to a stepwise increase in external salinity (successively: 1/3 SW, 1/2 SW, 3/4 SW, full SW). 20. In completely adapted individuals mean plasma electrolyte concentrations vary only slightly (by a few %) from one medium to another. Thus, the trout may be regarded as a euryhaline, eventually homeosmotic species. 30. With increasing outside salinity there is a progressive diminution in the overall gill permeability to ions which is suggested by saturation curves obtained for sodium fluxes (maximum at about 500 muEq/h. 100 g at 15 degrees C, for unshocked fish). 40. Disturbance of the animals provokes a striking elevation and desequilibrium in these exchanges and this in turn induces an abnormal rise in plasma concentrations and a subsequent failure to adapt to hypertonic media. 50. In vitro absorption of water and sodium by intestinal everted-sacs increases only after transfer to full sea water. Mucosal entry of ions into intestinal epithelial cells measured by the technique of Schultz et al. (1967), is diminished in sea water-adapted animal (by 42% in the case of sodium). 60. These results demonstrate that Salmo irideus possesses efficient osmoregulatory mechanisms which operate with minimal energy expenditure in hypertonic media.     

Measurement of blood volume in the elasmobranch fish Scyliorhinus canicula following acute and long‐term salinity transfers

A technique using ⁵¹chromium‐labelled erythrocytes was used to measure blood volume in Scyliorhinus canicula following long‐term and acute salinity transfers. Basal whole‐blood volume was 5·6 ± 0·2 ml 100 g^?1 (mean ±s .e .), this increased (6·3 ± 0·2 ml 100 g^?1) following +14 day acclimation to 80% sea water (SW) and decreased (4·6 ± 0·2 ml 100 g^?1) following acclimation to 120% SW. These changes were shown to be primarily due to changes in plasma volume, with no significant changes in extrapolated red‐cell volume being demonstrated. Blood volume was also measured in the same animals during 10 h acute transfer to 100% SW. Plasma volume in S. canicula during acclimation from 80% SW was significantly reduced (4·5 ± 0·3 ml 100 g^?1) after 6 h of transfer to 100% SW. Blood volume in animals during acclimation from 120% SW was significantly increased (4·8 ± 0·2 ml 100 g^?1) after 4 h of acute transfer. The osmoregulatory implications of these different timeframes during hyposaline and hypersaline transfer are discussed, along with the importance of this in vivo technique as context for in vitro studies with haemo‐dynamic stimuli.     

Dexamethasone modulates the activity of the eel branchial Na+/K+ATPase in both chloride and pavement cells

In fish, gills actively accumulate ions in freshwater (FW) with Na+ absorption taking place at the level of pavement cells, and excrete monovalent ions, mainly Na+ and Cl-, through the chloride cells in sea water (SW). The Na+/K+ATPase plays a crucial role in the functionality of osmoregulatory cells and we showed previously that angiotensin II modulates its activity in the eel gill (1). We here show the effects of synthetic steroid dexamethasone (DEX) on the activity of Na+/K+ATPase in both gill pavement and chloride cells from FW- and SW-adapted animals. Results showed that in the chloride cells 100 nM DEX provoked a significant increment in the activity of Na+/K+ATPase in both SW- and FW-adapted animals. This effect was greatest at 2 hours in SW, and at 6 hours in FW. The increment in the activity of the Na+/K+ATPase was dose-dependent in both environmental adaptations. Conversely, in pavement cells from FW-adapted eels 100 nM DEX decremented the activity of Na+/K+ATPase (4-fold reduction after 6 hour incubations), while in SW, DEX increased the enzyme activity of about 25% at 2 hours, and of about 55% at 6 hours. These results are consistent with the different physiological roles that pavement and chloride cells have in the two different adaptive conditions.     

Pharmacological control of muscular activity in the sea urchin larva. II. Role of calcium in nicotinic stimulation and paralysis,and the modulatory role of muscarinic agents

1. Ca²⁺-antagonists counteract the muscular activity of the sea urchin pluteus. Agents that block rapid Na⁺-channels have no effect.2. High muscular activity is induced by increasing the sea water concentration of Ca²⁺ or K⁺ and by a Ca²⁺-ionophore. The stimulatory effects tend to decline.3. Muscarinic agents counteract the effects of Ca²⁺ and K⁺.4. Variation in the concentration of Ca²⁺ or K⁺ has profound effects on the response to nicotinic agents.5. It is suggested that Ca²⁺ plays the role as a charge-carrier and in the release of monoamines from an inner source, and that an excessive Ca²⁺-influx induces an outflux of K⁺ leading to hyperpolarization and abolition of the impulse activity.     

Salinity tolerance of the guppy, Poecilia reticulata Peters

Experiments were conducted to determine the adaptability of the guppy to various salt concentrations. The guppy, Poecilia reticulata , (total length 11–40 mm) were subjected to abrupt and gradual changes from fresh water (salinity=0.1%) to various salinities (%). No mortality occurred when the fish were transferred from fresh water to 50% sea water (19.5%). Through gradual adaptation from 50% sea water to 80% of the fish were able to tolerate 100% (39%) sea water for 7 days. After a 7 day stay in sea water, fish were readapted to fresh water during a 3 h period. Through gradual adaptation fish were also able to tolerate salinities ranging from 39.0% (100% sea water) to 58.5%. After a 30 day stay in 150% sea water (58.5%), fish were readapted to fresh water over a 5 h period. The results indicate that they were well able to tolerate the abrupt change from 100 or 150% sea water to fresh water. Females that stayed in 150% sea water (58.5%) for 30 days had embryos in their gonads.     

Osmoregulatory action of 17beta-estradiol in the gilthead sea bream Sparus auratus

The osmoregulatory action of 17beta-estradiol (E2) was examined in the euryhaline teleost Sparus auratas. In a first set of experiments, fish were injected once with vegetable oil containing E2 (1, 2 and 5 microg/g body weight), transferred 12h after injection from sea water (SW, 38 ppt salinity) to hypersaline water (HSW, 55 ppt) or to brackish water (BW, 5 ppt salinity) and sampled 12h later (i.e. 24 h post-injection). In a second experiment, fish were injected intraperitoneally with coconut oil alone or containing E2 (10 microg/g body weight) and sampled after 5 days. In the same experiment, after 5 days of treatment, fish of each group were transferred to HSW, BW and SW and sampled 4 days later (9 days post-implant). Gill Na+,K+ -ATPase activity, plasma E2 levels, plasma osmolality, and plasma levels of ions (sodium and calcium), glucose, lactate, protein, triglyceride, and hepatosomatic index were examined. Transfer from SW to HSW produced no significant effects on any parameters assessed. E2 treatment did not affect any parameter. Transfer from SW to BW resulted in a significant decrease in plasma osmolality and plasma sodium but did not affect gill Na+,K+ -ATPase activity. A single dose of E2 attenuated the decrease in these parameters after transfer from SW to BW, but was without effect on gill Na+,K+ -ATPase activity. An implant of E2 (10 microg/g body weight) for 5 days significantly increased plasma calcium, hepatosomatic index, plasma metabolic parameters, and gill Na+,K+ -ATPase activity. In coconut oil-implanted (sham) fish, transfer from SW to HSW or BW during 4 days significantly elevated gill Na+,K+ -ATPase. Gill Na+,K+ -ATPase activity remained unaltered after transfer of E2-treated fish to HSW or BW. However, in E2-treated fish transferred from SW to SW (9 days in SW after E2-implant), gill Na+,K+ -ATPase activity decreased with respect to HSW- or BW-transferred fish. Shams transferred to HSW showed increased levels of lactate, protein, and trygliceride in plasma, while those transferred to BW only displayed increased trygliceride levels. E2-treated fish transferred to HSW showed higher protein levels without any change in other plasmatic parameters, while those transferred to BW displayed elevated plasma glucose levels but decreased osmolality and protein levels. These results substantiate a chronic stimulatory action of E2 on gill Na+,K+ -ATPase activity in the euryhaline teleost Sparus auratas.     

Effects of environmental water salinity on blood acid-base status in juvenile turbot (Scophthalmus maximus L.)

The time courses of extracellular ionic and acid-base adjustments were studied in juvenile turbot (Scophthalmus maximus) following a decrease of water salinity, either abruptly from 32 to 10%. or after a first step (4 weeks) in 19%. salinity followed by a direct transfer to 10%. brackish water (BW). Net exchanges of acid-base equivalents with the external water were also determined after transfer from 32%. SW to 10%. BW. Direct transfer from seawater (SW) to 10%. BW induced a transient decrease in plasma osmolarity, plasma sodium and chloride concentrations, associated with a marked and transient metabolic alkalosis in the blood. A significant net outflux of acidic equivalents was also measured only during the first day in BW. Four weeks preadaptation in 19%. BW reduced the intensity of the osmotic disturbances elicited by a subsequent abrupt transfer to 10%. BW. These ionic readjustments were also coupled with minimal acid-base changes, of lesser magnitude than those described after directly from SW to 10%. BW.     

Behaviour of Scrobicularia plana (Da Costa) in water of various salinities

Valve movements, heart and pumping rates have been recorded from Scrobicularia plana (Da Costa) when transferred stepwise from natural sea water to sea water of S = 30% (termed 100%) and then to 80, 60, 40, and 20% sea water, and after direct transfer from 100 to 20% Scrobicularia exhibits short periods of pumping alternating with short ventilatory pauses down to 40% sea water. These are termed activity cycles and contrast with periods of complete quiescence each ≈ 4–12 h long. The heart rate of animals in these salinities is fairly constant during activity cycles. When transferred to 20% sea water, either directly or gradually, short pumping phases alternate with long ventilatory pauses during the initial 72 h. At the same time the heart rate shows wide variation during activity cycles.On transference from 100 to 60% sea water, the valves opened and valve activity pattern returned to normal within 30 min and within 6 h of transfer from 60 into 40% SW. Transfer to 20%, whether stepwise or directly, resulted in valves remaining closed initially for ≈ 10 or 34 h, respectively before opening gradually to expose the mantle margin. The valve adductions were then of reduced frequency and extent.     

Adenylate cyclase activity in fish gills in relation to salt adaptation

The influence of salt adaptation on specific adenylate cyclase activity (measured by conversion of [alpha-32p]-ATP into [alpha-32p]-cAMP) was investigated in gill plasma membranes of rainbow trout (Salmo gairdneri) adapted to various salinities (deionized water, DW; fresh water, FW; 3/4 sea water, 3/4 SW; sea water, SW) and in sea water adapted-mullet (Mugil sp.). Basal activity declined by a factor of 2 in trout with increasing external salinity (pmoles cAMP/mg protein/10 min: 530 in DW, 440 in FW, 340 in 3/4 SW; 250 in SW) and was very low in SW adapted-mullet: 35. The Km for ATP was similar (0.5 mM) in both FW adapted- and SW adapted- trout in either the absence (basal activity) or in the presence of stimulating agents (isoproterenol; NaF) while the Vm varied. Analysis of stimulation ratios with respect to basal levels of the enzyme showed that hormones (glucagon, VIP) and pharmacological substances (isoproterenol, NaF) display a greater potency in high salt than in low salt adapted- fish gills. In contrast, salt adaptation did not have any effect on the regulation of adenylate cyclase by PGE1. These results are interpreted in relation to the general process of osmoregulation.     

Response of calcium-sensitive cells in the pars intermedia of the goldfish adapted to diluted sea water with different calcium and magnesium contents

Summary Goldfish kept in diluted Ca-free sea water (SW) (23 or 30 %) or in Ca-Mg-deficient SW (23 %) have a limited survival, release large amounts of mucus, and show spasmodic seizures. Plasma calcium decreases. The PAS-positive calcium-sensitive (Ca-s) cells of the pars intermedia show a low activity in diluted SW and in 23 % Mg-free SW. In diluted Ca-free SW, Ca-s cells are stimulated, but cell hypertrophy is not uniform and often restricted to an area adjacent to the proximal pars distalis. Nuclear hypertrophy is significant in the reactive area, although less pronounced or even absent at the periphery of the lobe. Mitotic activity occurs in the Ca-s cells of goldfish gradually adapted to diluted Ca-free SW and Ca-Mg-deficient SW, and sacrificed after 19 and 28 days, respectively. A general stimulation of the Ca-s cells, which remains less intense than that in goldfish kept in deionized water (DW), appears unable to ensure the survival of the goldfish in an isosmotic Ca-free environment containing Mg²⁺ (0.1, 12 or 16.8 mM). These data are compared with those obtained in the eel kept in Ca-free SW.     

Effect of pimozide on the cytology of the eel pituitary

Pimozide, a specific blocker of dopaminergic receptors, was injected for 4 to 9 days in freshwater (FW) eels or eels acclimated to sea water (SW), for 10 to 30 days. The daily dose was 100 or 200 microgram/100 g. In FW, pimozide induces a nuclear hypertrophy in the prolactin (PRL) cells of eels; these elongated cells increase in height. The amount of erythrosinophilic granules in the cytoplasm, initially reduced, increases. Plasma electrolyte values are not modified: only the plasma sodium level slightly rises with the higher dose. In SW, PRL cells appear less active. After 10 days, this hypoactivity is not yet fully evident; pimozide stimulates PRL cells without affecting electrolyte values. After 1 month in SW, PRL cells are stimulated with pimozide and a slight regranulation may occasionally occur. The response in SW is never as marked as it is in FW; a high dose is not more effective than a low one. The higher dose significantly raises Na+, Ca2+ and Cl- plasma levels. These data suggest that prolactin synthesis and release increase with pimozide. They corroborate the hypothesis of a hypothalamic inhibitory control on PRL secretion mediated through dopaminergic fibers in the eel, but other factors may also be involved in this regulation in addition to the effect of salinity.     

Changes in chloride secretion rate and vascular perfusion in the rectal gland of the European lesser-spotted dogfish in response to environmental and hormonal stimuli

Chloride secretion rates of rectal glands taken from the European lesser-spotted dogfish Scyliorhinus canicula adapting to 70% and 120% sea water (SW) were significantly greater and less than, respectively, those in the control 100% SW group. C-type natriuretic peptide (CNP) significantly increased chloride secretion rates above basal values in 100% SW although angiotenisn II (ANG II) had no effect. Perfusion of the secretory epithelia in rectal glands from 70% SW lesser-spotted dogfish was significantly higher than in rectal glands from 100% and 120% SW lesser-spotted dogfish. Perfusion of rectal glands with ANG II had no effect on perfusion of the secretory epithelia, although CNP perfusion induced significantly greater perfusion of the secretory epithelia than all other treatments. It remains to be determined if a reduction in environmental salinity induces an increase in plasma concentration of CNP and hence an increase in rectal gland activity.     

Beta-adrenergic control of the water permeability of the skin during rehydration in the toad Bufo arenarum.

1. Toads dehydrated to 80% of their standard weight (% SW) were rehydrated during 3 hr in distilled water. 2. Water permeability of the skin was positively correlated with the degree of dehydration in the range 80-100% SW. 3. Systemic administration of the beta-adrenergic agonist isoproterenol (5 mg/kg) 90 min after rehydration started (animals fully hydrated) increased skin permeability to the values observed in 80% SW dehydrated animals. 4. The administration of the beta-adrenergic blocker propranolol (5 mg/kg) 15 min before rehydration started produced a long-lasting decrease in water permeability during the 3 hr of rehydration. 5. The results are consistent with the hypothesis of a beta-adrenergic control of the water permeability of the skin during rehydration.     

Effects of Sperm-Activating Peptide I on Hemicentrotus pulcherrimus Spermatozoa in High Potassium Sea Water

The effects of sperm-activating peptide I (SAP-I: Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) on Hemicentrotus pulcherrimus spermatozoa in high [K⁺] sea water were examined. In high [K⁺] sea water, the respiration rates and motility of H. pulcherrimus spermatozoa were lower than those in normal sea water. SAP-I did not stimulate the lowered respiration rate or motility, although the peptide bound to the spermatozoa as it does in normal sea water. SAP-I elevated the sperm cGMP level in 100 mM K⁺ sea water (from 0.37 to 4.81 pmol/mg wet weight spermatozoa) more than those in normal sea water (from 0.21 to 0.93 pmol/mg wet weight). A phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX) and SAP-I synergistically elevated the cGMP level from 0.35 to 33.08 pmol/mg wet weight in 100 mM K⁺ sea water. However, in high [K⁺] sea water, SAP-I did not increase the cAMP level even in the presence of IBMX. SAP-I caused rapid, transient elevation of the intracellular pH and Ca²⁺ concentration of spermatozoa in normal sea water but not in 100mM K⁺ sea water. SAP-I did not decrease the apparent molecular weight of sperm guanylate cyclase from 131,000 to 128,000 in high [K⁺] sea water. These results suggest that the SAP-I-induced elevation of the cGMP level in sea urchin spermatozoa occurs before or independently of membrane hyperpolarization induced by the opening of K⁺ channels.     

Effects of environmental dilution on body fluid regulation in the yellow stingray, Urolophus jamaicensis

Adult yellow stingrays (Urolophus jamaicensis), collected off the southeast Florida coast, were maintained in filtered and re-circulated synthetic sea water (33 per thousand) for 5-13 days at 30 degrees C. Animals exposed to 82%, 74% and 66% SW in gradual steps exhibited rapid and significant weight gains followed by recovery to pre-dilution levels in 2-6 days. Acclimated animals at each salinity [100% (N=12), 82% (7), 74% (4) and 66% SW (3)] were anesthetized (MS222) and bled from the caudal vein. In 100% SW, stingray plasma was slightly hypo-osmotic to the external medium. Plasma osmolality decreased with stepwise dilutions, but became increasingly hyperosmotic to the bathing media. Plasma [Na] and [Cl] each decreased by approximately 13%, 23% and 16%, respectively, in 82%, 74% and 66% SW. Plasma [urea] decreased by 21%, 25% and 59%, respectively. Changes in plasma [K] and [Ca] were minor. Mean corpuscular [Hb] measurements suggest that stingray red cells swelled less at each dilution than predicted for a passive erythrocyte osmometer. RBC [K] decreased by 12%, 36% and 29%, respectively, in 82%, 74% and 66% SW. Quantitatively, the other measured electrolytes (Cl, Na and Ca) changed by lesser amounts. Results suggest that for mild and moderate dilutions (82% and 74% SW), yellow stingrays release both ions and urea from intracellular and extracellular compartments. With further dilution (66% SW), the elasmobranchs retain electrolytes at the expense of urea.     

Effects of calcium antagonists, calmodulin antagonists, and methylated xanthines on polar lobe formation and cytokinesis in fertilized eggs of Ilyanassa obsoleta

We have studied the ability of fertilized eggs of Ilyanassa obsoleta to undergo polar lobe formation and cytokinesis in the presence of Ca2+ antagonists (Ca2+ channel blockers, Ca2+ uptake inhibitors). Earlier work had suggested little need for exogenous Ca2+ during these cellular shape changes. Again it appears that exogenous Ca2+ probably is not required, based on cell ability to undergo the shape changes with no, or only minor, delay in the presence of 50 mM La3+ at pH 6.5, 10 mM concentrations of Ni2+ or Co2+, 1 mM Cd2+, and 100 microM concentrations of Mn2+, papaverine, verapamil, D600, or diltiazem. In nominally Ca2+-free seawater (containing approximately 10 microM Ca2+) (CFSW), there still is no effect of Cd2+ (up to 100 microM), Ni2+, Co2+, Mn2+, or diltiazem; however, papaverine, verapamil, and D600 in CFSW cause longer delays in the shape changes than they do in the presence of normal levels of Ca2+ (SW). In 10-50 microM nifedipine, shape changes are progressively delayed to the same extent in both SW and CFSW, but more so in CFSW at concentrations above 50 microM nifedipine. Among calmodulin antagonists, trifluoperazine up to 100 microM was without effect, but chlorpromazine at 25-100 microM and calmidazolium at 50-100 microM caused substantial, concentration-dependent delays in the starting times for the shape changes. Methylxanthines caused a substantial speed-up in the starting times for both polar lobe formation and cytokinesis. The most effective of these, caffeine, at optimal concentrations of 0.7-10 mM in SW or CFSW caused shape changes to occur 12-15 min earlier than in controls undergoing a normal 50-min cycle. Caffeine is known to cause release of Ca2+ from muscle sarcoplasmic reticulum. A putative antagonist of intracellular Ca2+ mobilization, TMB-8, significantly inhibited the shape changes of the Ilyanassa cells, whereas a variety of inhibitors of exogenous Ca2+ uptake noted above did not inhibit. We conclude that Ca2+ may be necessary for polar lobe formation and cytokinesis in Ilyanassa cells, but that it may be released from intracellular, sequestered stores rather than derived from exogenous sources.