Effects of calcium and salinity on the growth rate of Ephydatia fluviatilis (Porifera: Spongillidae)

The freshwater sponge, Ephydatia fluviatilis (Porifera: Spongillidae), was maintained in a continuous-flow laboratory culture system under several conditions of calcium ion (Ca⁺⁺) concentration and salinity. Experimental results suggest that sponge growth rate increases with increasing Ca⁺⁺ concentration, that sponge growth rate decreases with increasing salinity, and that the negative effect of higher salinity can be overcome by increasing Ca⁺⁺ concentration. The experimental results correlate well with field observations on the effects of salinity and Ca⁺⁺ on the distribution of E. fluviatilis.     

Active Calcium and Strontium Transport in Human Erythrocyte Ghosts

Both calcium and strontium could be transported actively from erythrocytes if adenosine triphosphate, guanosine triphosphate, or inosine triphosphate were included in the hypotonic medium used to infuse calcium or strontium into the cells. Acetyl phosphate and pyrophosphate were not energy sources for the transport of either ion. Neither calcium nor strontium transport was accompanied by magnesium exchange, and the addition of Mg⁺⁺ to the reaction medium in a final concentration of 3.0 mmoles/liter did not promote the transport of either ion. In the absence of nucleotide triphosphates, the addition of 1.5 mmoles/liter of Sr⁺⁺ to the reaction solution did not bring about active calcium transport and similarly 1.5 mmoles/liter of Ca⁺⁺ did not bring about active strontium transport. The inclusion of 1.5 mmoles/liter of Ca⁺⁺ or Sr⁺⁺ in the reaction medium did not interfere with the transport of the other ion when the erythrocytes were infused with adenosine triphosphate.     

Role of external calcium in sodium and chloride transport in the gills of seawater-adaptedMugil capito

Summary When the mulletMugil capito is transferred to medium lacking Ca⁺⁺ (either Ca⁺⁺-free seawater or distilled water) the passive permeability of the gill to Na⁺ and Cl^– is increased and the activating effect of external K⁺ on the Na⁺ and Cl^– effluxes in hyposaline media is inhibited. The permeability of the gill increases progressively in proportion to the time of Ca⁺⁺ deprivation; it declines when Ca⁺⁺ is added again to the external medium. The active mechanisms for ion excretion are not reversible. At external Ca⁺⁺ concentrations from 0.1 to 10 mM the Na⁺ permeability is constant but the activation of Na⁺ efflux by K⁺ shows a maximum at a Ca⁺⁺ concentration of about 1 mM. For activation of Cl^– efflux external bicarbonate must be present, in addition to Ca⁺⁺, suggesting the existence of a Cl^–/HCO ₃ ^– exchange. The mechanism by which Ca⁺⁺ controls the passive branchial permeability is thus probably different from that involved in K⁺ activation of ion excretion. The Ca⁺⁺ effect on the K⁺ sensitive ionic excretory mechanisms seems to be related to intracellular Ca⁺⁺ movements. Thus, on the one hand, substances such as Ruthenium Red and La⁺⁺⁺ which both inhibit Ca⁺⁺ exchange, in media containing Ca⁺⁺ and HCO ₃ ^– also inhibit K⁺ activation of Na⁺ and Cl^– effluxes; on the other hand, the ionophore A 23187, a stimulator of Ca⁺⁺ exchange, when added to these media, activates the Na⁺ and Cl^– effluxes; its maximal effect on the Na⁺ flux occurs at 2 mM Ca⁺⁺.Abbreviations ASW-Ca artificial seawater minus calcium - DW deionised water - DWCa deionised water with 1 mM Ca⁺⁺ added - DWCaHCO ₃ DW with calcium plus bicarbonate - DWHCO ₃ DW with 1 mM sodium bicarbonate added - FW freshwater (tap water) - FWK freshwater with K⁺ added - P. D. potential difference - SW seawater The experiments reported in this paper were done with Jean Maetz who tragically died in August 1977. It is the last report about several years of friendly collaboration     

CHARACTERIZATION OF LYMPHOCYTE TRANSFORMATION INDUCED BY ZINC IONS

Lymphocyte cultures from all normal human adults are stimulated by zinc ions to increase DNA and RNA synthesis and undergo blast transformation. Optimal stimulation occurs at 0.1 mM Zn⁺⁺. Examination of the effects of other divalent cations reveals that 0.01 mM Hg⁺⁺ also stimulates lymphocyte DNA synthesis. Ca⁺⁺ and Mg⁺⁺ do not affect DNA synthesis in this culture system, while Mn⁺⁺, Co⁺⁺, Cd⁺⁺, Cu⁺⁺, and Ni⁺⁺ at concentrations of 10^-7–10^-3 M are inhibitory. DNA and RNA synthesis and blast transformation begin to increase after cultures are incubated for 2–3 days with Zn⁺⁺ and these processes reach a maximum rate after 6 days. The increase in Zn⁺⁺-stimulated lymphocyte DNA synthesis is prevented by rendering cells incapable of DNA-dependent RNA synthesis with actinomycin D or by blocking protein synthesis with cycloheximide or puromycin. Zn⁺⁺-stimulated DNA synthesis is also partially inhibited by 5'-AMP and chloramphenicol. Zn⁺⁺ must be present for the entire 6-day culture period to produce maximum stimulation of DNA synthesis. In contrast to its ability to independently stimulate DNA synthesis, 0.1 mM Zn⁺⁺ inhibits DNA synthesis in phytohemagglutinin-stimulated lymphocytes and L1210 lymphoblasts.     

Cytoplasmic calcium response to fluid shear stress in cultured vascular endothelial cells

Summary Vascular endothelial cells modulate their structure and functions in response to changes in hemodynamic forces such as fluid shear stress. We have studied how endothelial cells perceive the shearing force generated by blood flow and the substance(s) that may mediate such a response. We identify cytoplasmic-free calcium ion (Ca⁺⁺), a major component of an internal signaling system, as a mediator of the cellular response to fluid shear stress. Cultured monolayers of bovine aortic endothelial cells loaded with the highly fluorescent Ca⁺⁺-sensitive dye Fura 2 were exposed to different levels of fluid shear stress in a specially designed flow chamber, and simultaneous changes in fluorescence intensity, reflecting the intracellular-free calcium concentration ([Ca⁺⁺] _i ), were monitored by photometric fluorescence microscopy. Application of shear stress to cells by fluid perfusion led to an immediate severalfold increase in fluorescence within 1 min, followed by a rapid decline for about 5 min, and finally a plateau somewhat higher than control levels during the entire period of the stress application. Repeated application of the stress induced similar peak and plateau levels of [Ca⁺⁺] _i but at reduced magnitudes of response. These responses were observed even in Ca⁺⁺-free medium. Thus, a shear stress transducer might exist in endothelial cells, which perceives the shearing force on the membrane as a stimulus and mediates the signal to increase cytosolic free Ca⁺⁺. This work was partly supported by a grant-in-aid, for Special Project Research no. 61132008, from the Japanese Ministry of Education, Science and Culture and a research fund from the Atherosclerosis Study Association.     

Effects of Cd++ on short-circuit current across epithelial membranes

Summary Cadmium ion (Cd⁺⁺) significantly increased potential difference (PD) and short-circuit current (SCC) across isolated frog skin when added to the outside Ringer's solution at 10^–4, 10^–3 and 5×10^–3 m concentration. Resistance was reduced by 10^–4 m Cd⁺⁺ but not significantly changed by the higher concentrations. When SCC was first stimulated by vasopressin, 10^–4 and 10^–3 m Cd⁺⁺ produced additive stimulation which was reversible by washing with Cd⁺⁺-free Ringer's. If SCC was first stimulated by Cd⁺⁺, further stimulation by vasopressin was additive with 10^–4 m Cd⁺⁺ but completely inhibited by 10^–3 m Cd⁺⁺. Elevating the calcium ion (Ca⁺⁺) concentration of the outer Ringer's from 10^–3 m to 5×10^–3 m or 10^–2 m prior to Cd⁺⁺ treatment did not reduce the magnitude of SCC stimulation by Cd⁺⁺. Removal of Ca⁺⁺ from the outside Ringer's with 2×10^–3 m EDTA increased SCC as predicted. Subsequent addition of 5×10^–3 m Cd⁺⁺ drastically reduced SCC below control levels while equimolar concentrations of Cd⁺⁺ and EDTA reduced SCC only to control levels. These results suggest that Cd⁺⁺ interacts with the components of the apical plasma membranes of epithelial cells which are associated with the stimulation of SCC by vasopressin and Ca⁺⁺ removal and may be a useful probe for elucidating these components.     

Calcium entry in rabbit corneal epithelial cells: Evidence for a nonvoltage dependent pathway

We performed experiments to elucidate the calcium influx pathways in freshly dispersed rabbit corneal epithelial cells. Three possible pathways were considered: voltage-gated Ca⁺⁺ channels, Na⁺/Ca⁺⁺ exchange, and nonvoltage-dependent Ca⁺⁺-permeable channels. Whole cell inward currents carrying either Ca⁺⁺ or Ba⁺⁺ were not detected using voltage clamp techniques. We also used imaging technology and the Ca⁺⁺-sensitive ratiometric dye fura 2 to measure changes in intracellular Ca⁺⁺ concentration ([Ca]_i). Bath perfusion with NaCl Ringer's solution containing the calcium channel agonist Bay-K-8644 (1 m), or Ni⁺⁺ (40 m), a blocker of many voltage-dependent calcium channels, did not affect [Ca⁺⁺]_i. Membrane depolarization with a KCl Ringer's bath solution resulted in a decrease in [Ca⁺⁺]_i. These results are inconsistent with the presence of voltage gated Ca⁺⁺ channels. Nonvoltage gated Ca⁺⁺ entry, on the other hand, would be reduced by membrane depolarization and enhanced by membrane hyperpolarization. Agents which hyperpolarize via stimulation of K⁺ current, such as flufenamic acid, resulted in an increase in ratio intensity. The cells were found to be permeable to Mn⁺⁺ and bath perfusion with 5 mm Ni⁺⁺ decreased [Ca⁺⁺]_i suggesting that the Ca⁺⁺ conductance was blocked. These results are most consistent with a nonvoltage gated Ca⁺⁺ influx pathway. Finally, replacing extracellular Na⁺ with Li⁺ resulted in an increase in [Ca⁺⁺]_i if the cells were first Na⁺-loaded using the Na⁺ ionophore monensin and ouabain, a Na⁺-K⁺-ATPase inhibitor. These results suggest that Na⁺/Ca⁺⁺ exchange may also regulate [Ca⁺⁺] in this cell type.The authors are grateful to Chris Bartling for expert technical assistance with the imaging experiments, Helen Hendrickson for cell preparation, and Jonathon Monck for helpful discussions regarding imaging technology. This work was supported by National Institutes of Health grants EYO3282, EYO6005, DK08677, and an unrestricted award from Research to Prevent Blindness.     

Calcium-stimulated respiration and active calcium transport in the isolated chick chorioallantoic membrane

Summary Calcium markedly stimulates the respiration of the isolated chick chorioallantoic membrane. This stimulation of oxygen uptake appears to be closely associated with the membrane's active transcellular calcium transport mechanism. In the presence of 1mm Ca⁺⁺ the rate of uptake increases from 9.3±0.15 to 13.0±0.2 liters O₂/cm²/hr, an increase of about 40%. The calcium-stimulated respiration is specific for the ectodermal layer of cells, the known location of the calcium transport mechanism, and only occurs when the calcium transport mechanism is operative. Sr⁺⁺ and Mn⁺⁺ are transported by the tissue at a lower rate than Ca⁺⁺ and cause a smaller stimulation of oxygen consumption. Mg⁺⁺ and La³⁺ have no effect on tissue respiration. In the presence of Ca⁺⁺, the organic mercurialp-chloromercuribenzene sulfonate (PCMBS) inhibits calcium transport and specifically decreases the oxygen uptake of the ectoderm to a rate identical to that obtained in a calcium-free medium. Stripping the inner shell membrane away from the chorioallantoic membrane mimics these effects. The specificity and locus of action of these two inhibitors suggest that a vital component of the active transcellular calcium transport mechanism resides on or near the outer surface of the plasma membrane of the ectodermal cells and that sulfhydryl groups are important to the normal function of this component.     

Calcium regulation of skeletal myogenesis. I. Cell content critical to myotube formation

Summary Primary cultures of embryonic chick pectoral skeletal muscle were used to study calcium regulation of myoblast fusion to form multinucleated myotubes. Using atomic absorption spectrometry to measure total cellular calcium and the⁴⁵Ca-exchange method to determine free cellular Ca⁺⁺, our data suggest that only the free cellular calcium changes significantly during development under conditions permissive for myotube formation (0.9 mM external Ca⁺⁺). Increases in calcium uptake occurred before and toward the end of the period of fusion with the amount approximating 2 to 4 pmol per cell in mass cultures. If the medium [Ca⁺⁺] is decreased to 0.04 mM, as determined with a calcium electrode, a fusion-block is produced and free cell Ca⁺⁺ decreased 5- to 10-fold. Removal of the fusion-block by increasing medium [Ca⁺⁺] results in a release of the fusion-block and an increase in cellular Ca⁺⁺ to approximately 1 pmol per cell during fusion, and higher thereafter. Cation ionophore A23187 produced transient increases in cellular calcium and stimulated myoblast fusion and the final extent of myotube formation only when added at the onset of culture. Results suggest that transient increased calcium uptake alone is insufficient for fusion because critical cellular content in conjunction with permissive amounts of medium [Ca⁺⁺] must exist. The latter suggests further that cell surface Ca⁺⁺ was also critical.     

Influx of Ca ion in cultured excitable cells studied by fluorescence microscope imaging technique

The spatial distribution and temporal variation of intracellular Ca ion in differentiated Neuroblastoma-Glia Hybridoma 108–15 cells (NG108–15) were investigated using a fluorescence microscope imaging technique. Fura-2 was used as a probe. Electrical current pulses of 10–20 µA were applied to axons connecting to NG cells in order to elicit the influx of Ca ion. The concentration of intracellular Ca is usually 50–80 nM in NG cells in the resting state. Upon stimulation, the Ca level increases by a factor of 2–5. The entry of Ca⁺⁺ across cell membranes is followed by intracellular diffusion and the propagation of a wave front is clearly seen in digital images. The diffusion constant was calculated to be approximately 1.66×10^–6 cm²/sec. This value is about one-fifth of the free diffusion coefficient of Ca ion in aqueous solution (7.82 × 10^–6 cm²/sec). Cd ion, at the concentration of 1–2 mM, blocks the influx of Ca as expected whereas the influx is unaffected by TTX at the concentration of 0.1 – 0.2µM.     

Extracellular calcium dependence of contracture and modulation by serotonin in buccal muscle E1 of Aplysia

Serotonin [5-hydroxytryptamine (5-HT)] enhances acetyl choline (ACh)-elicited contractures of Aplysia buccal muscles E1 and I5. The possible role of external calcium in regulating the magnitude of ACh contracture in the presence and absence of 5-HT was investigated. Superfusion of E1 with zero calcium medium caused ACh contractures to fail within one to two minutes. Recovery of ACh contracture upon restoring normal medium occurred within two to four minutes. In the absence of 5-HT, ACh contracture decreased proportionally to external [Ca⁺⁺] in the concentration range of 0–10 mM; however, the amount of enhancement of of ACh contracture following 5-HT treatment did not decrease with external [Ca⁺⁺] as long as [Ca⁺⁺] was above a threshold concentration that varied from preparation to preparation. For most preparations, the enhancement of ACh contracture by 5-HT was dependent on the presence of external calcium during 5-HT treatment. Calcium influx into muscles E1 and I5 increased approximately two and a half fold in the presence of 10^?6 M 5-HT. A model in which 5-HT brings about calcium “loading” of an ACh releasable intracellular storage site is discussed.     

A study of the intracellular transport of calcium in rat heart

The distribution of in vivo injected ⁴⁵Ca⁺⁺ in the subcellular fractions of rat heart has been studied. Most of the radioactivity of the cell was found to be associated with the subcellular organelles; only a small fraction was recovered in the soluble phase. Mitochondria contained the greatest part of the total radioactivity associated with the subcellular organelles. After injection of ⁴⁵Ca⁺⁺ the specific activity of the mitochondrial calcium pool was several times higher than that of the calcium of the sarcoplasmic reticulum. Pentachlorophenol has been administered to rats to uncouple oxidative phosphorylation in heart mitochondria in vivo and its effect on the distribution of ⁴⁵Ca⁺⁺ in the heart studied. Under these conditions, it has been found that mitochondria contained much less ⁴⁵Ca⁺⁺ than the controls; this decrease was paralleled by an increase of the radioactivity associated with the microsomes and with the final supernatant. Experiments in which ⁴⁵Ca⁺⁺ was added to heart homogenates at 0° indicated that ⁴⁵Ca⁺⁺ also became bound to mitochondria and the other subcellular structures at 0°. However, PCP had no effect on the distribution of radioactivity among the subcellular fractions under these conditions. The results suggest that (1) energy-linked movements of Ca⁺⁺ take place in mitochondria of the intact rat heart, (2) a part of the uptake of ⁴⁵Ca⁺⁺ by mitochondria does not depend on metabolism, and, (3) the movements of Ca⁺⁺ in heart mitochondria in vivo are probably more active than those in the sarcoplasmic reticulum.     

Calcium overload in endotoxemia

E.coli endotoxin stimulates endogenous lipolysis in the $\text{in vitro}$ perfused rat heart. Verapamil^® inhibits endotoxin- (as well as glucagon-) stimulated lipolysis. This suggests that the endotoxin used increases the availability of Ca⁺⁺ to the lipolytic system in the cardiocytes. This conclusion is supported by the observed stimulation of contractility of the heart, especially during perfusion at a low Ca⁺⁺ concentration.The endotoxin was found to inhibit ATP-dependent Ca⁺⁺ accumulation in sarcolemma vesicles prepared from rat heart. A direct Ca⁺⁺ ionophoric action of the endotoxin on these vesicles could be excluded.It is discussed that Ca⁺⁺ overload may not be confined to the cardiovascular system during endotoxemia.     

Comparison of the Ca ++ requirement for the steroidogenic effect of ACTH acid dibutyryl cyclic AMP in rat adrenal cell suspension

Calcium requirement for ACTH and Dibutyryl cyclic AMP (DBCAMP) stimulation of steroidogenesis was compared in rat adrenal cell suspensions. In the absence of added calcium ACTH at low concentrations (< 1 mU/ml) was ineffective; however, the calcium requirement decreased when higher concentrations of ACTH were used. This was not the case with DBCAMP. At all levels of the nucleotide tested, the Ca⁺⁺ requirement was about the same. When the cells were preincubated with EGTA, the Ca⁺⁺ requirement became more pronounced for ACTH than for DBCAMP. The results indicate that the events before the formation of cyclic AMP show a greater dependence on Ca⁺⁺ than the events following its formation.     

The binding of thyrotropin to liposomes containing gangliosides.

The relationship between uptake of Ca⁺⁺ and incorporation of sn-[¹⁴C]-glycerol-3-phosphate into phosphatidate, diglyceride, and triglyceride was evaluated in microsomes isolated from livers of normal fed male rats. Uptake of Ca⁺⁺ was dependent on concentration of Ca⁺⁺ (0.1 – 2.5 mM), and accompanied by a decrease in the rate of glycerolipid synthesis. The quantity of Ca⁺⁺ ion taken up at 20 μM CaCl₂ in the presence of ATP was equivalent to that observed with 2.5 mM CaCl₂ in the absence of ATP. The ATP dependent uptake of Ca⁺⁺, like the passive uptake at higher concentrations of Ca⁺⁺, was correlated with inhibition of incorporation of sn-glycerol-3-phosphate into phosphatidate. Accumulation of Ca⁺⁺ in hepatic microsomes, therefore, appears to result in a calcium-dependent decrease in biosynthesis of phosphatidate and other glycerolipids.     

Calcium ion,a putative intracellular messenger for light-adaptation in Limulus ventral photoreceptors

Calcium ion fulfills several criteria for identifying an intracellular messenger for light-adaptation in Limulus photoreceptors. Direct injection of Ca⁺⁺ mimicks two aspects of light-adaptation; sequestration of intracellular calcium tends to prevent light-adaptation; and light induces an increase in intracellular Ca⁺⁺ as demonstrated by two independent techniques.Presented at the EMBO-Workshop on Transduction Mechanism of Photoreceptors, Jülich, Germany, October 4–8, 1976     

Rapid alteration in Ca++ content and fluxes in phorbol 12-myristate 13-acetate treated myoblasts

The tumor promoter phorbol 12-myristate 13-acetate rapidly induces alterations in both Ca⁺⁺ content and transport in cultured differentiated chick myoblasts. At 4 ng/ml (6nM), the promoter caused a 25 ± 12% decrease in total intracellular Ca⁺⁺ within 5 h after its addition. Measurement of ⁴⁵Ca⁺⁺ transport at this time revealed a 15 ± 6% decrease in the rate constants for both efflux and influx. Values of $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for the cytosolic Ca⁺⁺ pool in control and treated cells were 9.1 and 10.7 min, respectively, for efflux and 8.6 and 10.4 min, respectively, for influx. Ca⁺⁺ influx was decreased maximally within 90 sec after promoter addition. No effect was observed on ⁸⁶Rb⁺ uptake or intracellular concentration at equilibrium. The Ca⁺⁺ response is among the most rapid yet reported and may play a primary role in altering cellular metabolism.     

Calcium as a limiting factor in the biology of Biomphalaria pfeifferi (Krauss), (Gastropoda: Planorbidae)

The minimum calcium requirements, relative importance of buffering and optimum ratio of calcium to magnesium, calcium to sodium, and calcium to potassium ions were determined for laboratory populations ofBiomphalaria pfeifferi and related to suggested limiting factors for the natural distribution of this species. Snails were reared in a range of concentrations of both calcium bicarbonate and unbuffered calcium sulphate from 0.5 to 20 mg/l as Ca⁺⁺ and also in a series of media with a constant concentration of 2 mg/l as Ca⁺⁺ but with a range of Ca/Mg, Ca/Na and Ca/K ratios of 4.0 to 0.1. Shell growth, survivorship, fecundity, egg fertility, and the net reproductive rate were compared. In calcium bicarbonate cultures a concentration of 2mg/l Ca⁺⁺ appeared to be the lower limit for the survival of laboratory populations but a concentration of 4 mg/l Ca⁺⁺ was needed for a population to thrive. The calcium sulphate salt gave much poorer results, emphasizing the importance of the bicarbonate buffer. In the cationic ratio experiments the low Ca/Mg ratios proved to have the most damaging effects on snail populations but the effects of very low Ca/Na and Ca/K ratios could also be measured. A parallel experiment on the hatching rate of snail eggs, using similar experimental solutions, gave comparable results. The significance of these findings to snail ecology is discussed.     

Two calcium currents in the somatic membrane of mollusc neurons

Two new types of calcium channels were discovered during research in ionic currents in the somatic membrane ofHelix pomatia neurons, using an intracellular perfusion technique. Apart from the principal calcium current described in the literature with a holding potential of about –110 mV, an additional calcium current was observed activated at depolarizations of –40 to –80 mV and was not reduced when the cell was perfused with solutions containing fluoride anions. The kinetics of this current were well described in the context of the Hodgkin and Huxley model with a time constant of activation of 6–8 msec and of inactivation of 300–600 msec. It increased in amplitude as the Ca⁺⁺ rose in the cellular environment but was reduced by extracellular addition of the Ca⁺⁺ antagonists Co⁺⁺, Ni⁺⁺, and Cd⁺⁺, and the organic blockers nifedipine and verapamil. The association constants of these substances with corresponding channels determined from the maximum of the current-voltage relationship were 2 (Ca⁺⁺), 3 (Co⁺⁺), 0.06 (nifedipine), and 0.2 mM (verapamil). The properties detected in this component of calcium conductance are compared with those of calcium channels in other excitatory formations and its possible functional role is discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 17, No. 5, pp. 627–633, September–October, 1985.     

Influence of calcium on alginate production and composition in continuous cultures of Azotobacter vinelandii

Summary Azotobacter vinelandii strain E was cultivated in PO ₄ ^-- limited continuous cultures. The influence of growth medium Ca⁺⁺ levels on dry cell weight and alginate production and composition was examined. Low Ca⁺⁺ concentrations (<0.34 mM) were observed to inhibit growth, particularly in cultures maintained at a high dilution rate (D=0.32 hr^-1). In cultures with high levels of polysaccharide (>1.0 g l^-1), the production of alginate with a predominantly heteropolymeric structure was favoured by increasing Ca⁺⁺ levels. In cultures containing less polysaccharide (<1.0 g l^-1) increasing Ca⁺⁺ levels (0.068–0.34 mM Ca⁺⁺) resulted in the production of alginates high in polyguluronate. With further increases in Ca⁺⁺ levels (0.34–2.72 mM Ca⁺⁺) synthesis of alginates with a more heteropolymeric structure occurred. It is proposed that extracellular epimerisation of alginate is influenced by intermolecular associations, the formation of which is mediated by both Ca⁺⁺ concentration and the concentration of the polymer itself.