The role of skeletal‐muscle‐based thermogenic mechanisms in vertebrate endothermy

Thermogenesis is one of the most important homeostatic mechanisms that evolved during vertebrate evolution. Despite its importance for the survival of the organism, the mechanistic details behind various thermogenic processes remain incompletely understood. Although heat production from muscle has long been recognized as a thermogenic mechanism, whether muscle can produce heat independently of contraction remains controversial. Studies in birds and mammals suggest that skeletal muscle can be an important site of non‐shivering thermogenesis (NST) and can be recruited during cold adaptation, although unequivocal evidence is lacking. Much research on thermogenesis during the last two decades has been focused on brown adipose tissue (BAT). These studies clearly implicate BAT as an important site of NST in mammals, in particular in newborns and rodents. However, BAT is either absent, as in birds and pigs, or is only a minor component, as in adult large mammals including humans, bringing into question the BAT‐centric view of thermogenesis. This review focuses on the evolution and emergence of various thermogenic mechanisms in vertebrates from fish to man. A careful analysis of the existing data reveals that muscle was the earliest facultative thermogenic organ to emerge in vertebrates, long before the appearance of BAT in eutherian mammals. Additionally, these studies suggest that muscle‐based thermogenesis is the dominant mechanism of heat production in many species including birds, marsupials, and certain mammals where BAT‐mediated thermogenesis is absent or limited. We discuss the relevance of our recent findings showing that uncoupling of sarco(endo)plasmic reticulum Ca²⁺‐ATPase (SERCA) by sarcolipin (SLN), resulting in futile cycling and increased heat production, could be the basis for NST in skeletal muscle. The overall goal of this review is to highlight the role of skeletal muscle as a thermogenic organ and provide a balanced view of thermogenesis in vertebrates.     

Chronic mitochondrial uncoupling treatment prevents acute cold-induced oxidative stress in birds

Endotherms have evolved two major types of thermogenesis that allow them to actively produce heat in response to cold exposure, either through muscular activity (i.e. shivering thermogenesis) or through futile electro-chemical cycles (i.e. non-shivering thermogenesis). Amongst the latter, mitochondrial uncoupling is of key importance because it is suggested to drive heat production at a low cost in terms of oxidative stress. While this has been experimentally shown in mammals, the oxidative stress consequences of cold exposure and mitochondrial uncoupling are clearly less understood in the other class of endotherms, the birds. We compared metabolic and oxidative stress responses of zebra finches chronically treated with or without a chemical mitochondrial uncoupler (2,4-dinitrophenol: DNP), undergoing an acute (24 h) and a chronic (4 weeks) cold exposure (12 °C). We predicted that control birds should present at least a transient elevation of oxidative stress levels in response to cold exposure. This oxidative stress cost should be more pronounced in control birds than in DNP-treated birds, due to their lower basal uncoupling state. Despite similar increase in metabolism, control birds presented elevated levels of DNA oxidative damage in response to acute (but not chronic) cold exposure, while DNP-treated birds did not. Plasma antioxidant capacity decreased overall in response to chronic cold exposure. These results show that acute cold exposure increases oxidative stress in birds. However, uncoupling mitochondrial functioning appears as a putative compensatory mechanism preventing cold-induced oxidative stress. This result confirms previous observations in mice and underlines non-shivering thermogenesis as a putative key mechanism for endotherms in mounting a response to cold at a low oxidative cost.     

Thermogenesis in Birds

The article discusses the importance of avian skeletal muscle as a source for heat generation by means of both shivering and non-shivering. Non-shivering thermogenesis in birds is still a polemic issue. Recent evidence at the molecular/cellular level indicates, however, that this type of heat generation may also exist among birds. The involvement of the sarcoplasmic reticulum calcium ATPase in non-shivering thermogenesis is discussed in-depth.     

Shifts in thermoregulatory strategy during ontogeny in harp seals (Pagophilus groenlandicus)

Heat balance can be difficult for young and/or small animals in polar regions because environmental conditions in combination with small body size or physiological immaturity can increase heat loss. We investigated how thermoregulatory patterns change with ontogeny in 5 age classes of harp seal (Pagophilus groenlandicus) from birth to post-molt to further understand the timing of thermoregulatory development in relation to their potential vulnerability to ongoing fluctuations in the extent and stability of Arctic pack ice. We measured changes in the amount, conductivity, and resistance of the seal pups׳ insulative layers (blubber and fur), the potential for endogenous heat-generation by shivering (muscle enzyme activity), and nonshivering thermogenesis (NST; brown adipose tissue (BAT) uncoupling protein 1 (UCP1) expression and mitochondrial density). There was no significant difference in blubber conductivity among age classes, though the amount of blubber insulation significantly increased from birth to weaning. Pelage conductivity was low (0.12±0.01 W m⁻¹ °C⁻¹) except in 9-day old pups (0.40±0.08 W m⁻¹ °C⁻¹); the significantly higher conductivity may signal the beginning of the molt, and this age group may be the most vulnerable to early water entry. Citrate synthase activity significantly increased (49.68±3.26 to 75.08±3.52 μmol min⁻¹ g wet weight⁻¹) in the muscle; however it is unlikely that increasing a single enzyme greatly impacts heat generation. BAT of younger pups contained UCP1, though expression and mitochondrial density quickly declined, and the ability of pups to produce heat via NST was lost by weaning. While total thermal resistance did not differ, neonatal and early nursing animals gained the majority of their thermal resistance from lanugo (82.5±0.03%); however, lanugo is not insulative when wet, and NST may be important to maintain euthermia and dry the coat if early immersion in water occurs. By late nursing, blubber seems sufficient as insulation (75.87±0.01% of resistance after 4 weeks), but high conductivity of fur may be responsible for retention of UCP1 expression. Weaned animals rely on blubber insulation, and no longer need NST, as wetted fur is no longer a threat to euthermia.     

Study of age-dependent structural and functional changes of mitochondria in skeletal muscles and heart of naked mole rats (<Emphasis Type="Italic">Heterocephalus glaber</Emphasis>)

Morphometric analysis of mitochondria in skeletal muscles and heart of 6- and 60-month-old naked mole rats (Heterocephalus glaber) revealed a significant age-dependent increase in the total area of mitochondrial cross-sections in studied muscle fibers. For 6- and 60-month-old animals, these values were 4.8 ± 0.4 and 12.7 ± 1.8%, respectively. This effect is mainly based on an increase in the number of mitochondria. In 6-month-old naked mole rats, there were 0.23 ± 0.02 mitochondrial cross-sections per μm² of muscle fiber, while in 60-month-old animals this value was 0.47 ± 0.03. The average area of a single mitochondrial cross-section also increased with age in skeletal muscles–from 0.21 ± 0.01 to 0.29 ± 0.03 μm². Thus, naked mole rats show a drastic enlargement of the mitochondrial apparatus in skeletal muscles with age due to an increase in the number of mitochondria and their size. They possess a neotenic type of chondriome accompanied by specific features of mitochondrial functioning in the state of oxidative phosphorylation and a significant decrease in the level of matrix adenine nucleotides.     

Participation of breast and leg muscles in shivering thermogenesis in young turkeys and guinea fowl

Turkey (Meleagris gallopavo) and guinea fowl (Numida meleagris) chicks (0–27 days posthatch) were exposed to decreasing or increasing ambient temperatures. Root mean square electromyographic activity of musculus pectoralis (m. pect.) and musculus iliotibialis (m. iliot.) was recorded simultaneously with O₂ consumption and CO₂ production. From both muscles, relative mass, water fraction and fibre type were determined. M. iliot. participated in shivering from hatching onwards. The relationship between its root mean square electromyographic activity and ambient temperature resembled that of metabolic rate and ambient temperature, and the shivering threshold temperature was indistinguishable from the lower critical temperature. This suggests that the leg muscles are major contributors to shivering thermogenesis. M. pect. participated in shivering only at days 6–20 in turkeys and at days 6–10 in guinea fowl. Both water fraction and histological analysis indicated that m. pect. was less developed than m. iliot. at hatching. We hypothesize that a minimal level of maturity is required before a muscle can participate in shivering, which is probably represented by a water fraction of about 0.85. Both species recruited the aerobic leg muscles first; the anaerobic breast muscle was recruited only when the rate of mass-specific heat loss was high. Accepted: 20 March 1997     

Ultrastructural changes in rabbit heart mitochondria during the perinatal period: Neonatal transition to aerobic metabolism

Ultrastructural changes in mitochondria of rabbit left ventricular myocardial cells were measured during the interval from 3 days before to 4 days after birth. This interval is characterized by a transition from partially anaerobic to aerobic metabolism and by a concomitant increase in the work of contraction. Measurements were made by recently developed morphometric techniques [Smith, H. E., and Page, E. (1976). J. Ultrastruct. Res.55, 31–41]. It was shown that the perinatal transition was associated with a rapid and large accumulation of mitochondria and myofibrils, reflected by increases of 29–35% and 37–41% in the mitochondrial and myofibrillar fractions of cell volume. The mitochondria thus accumulated were packed more densely with cristae. The membrane area of cristae + inner membrane per unit of mitochondrial volume rose from an antenatal value of 47 μm²/μm³ to a peak of 64 μm²/μm³ 2 days after birth, then declined significantly. The relative and absolute increases in the membrane area and volume of respiratory membrane were associated with a relative decrease in matrix volume. These measurements also permitted the calculation of the area of mitochondrial respiratory membrane per unit myofibrillar volume. This physiologically important index, which relates the amount of ATP-producing membrane to the volume of the principal ATP-consuming organelle, increased progressively throughout the perinatal period.     

The effect of short-term fasting on shivering thermogenesis in Japanese quail chicks (Coturnix coturnix japonica): indications for a significant role of diet-induced/growth related thermogenesis

(1) The effect of short-term fasting on metabolism and shivering thermogenesis was studied in 9-day-old Japanese quail. (2) After 31 h of fasting, heat production decreased 39% and body temperature over 2°C in the thermoneutral zone. The difference in heat production between control and fasting groups decreased with decreasing ambient temperature. (3) Despite the lower metabolic rate, the amplitudes of shivering EMGs were higher in fasted chicks, especially in pectoralis. This indicates that fasted quails used shivering to compensate the decrease in diet-induced/growth related thermogenesis. (4) In cold, conductance of control birds decreased simultaneously with increasing heat production while in fasted chicks, conductance decreased to its minimum before heat production was activated. (5) Japanese quail chicks adapt quickly to short-term fasting by decreasing metabolism but they maintain their ability to thermoregulate in cold. Diet-induced/growth related thermogenesis has a significant role in thermoregulation since it reduces the need of shivering thermogenesis.     

The effects of light intensity and algae-induced turbidity on feeding behaviour of larval striped trumpeter

Striped trumpeter larvae reared in algal cell‐induced turbid water (greenwater) fed equally well in clearwater in a light intensity range of 1–10 μmol s^‐1 m^‐2, when evaluated in terms of both the proportion of larvae feeding and larval feeding intensity. An ontogenetic improvement in photopic visual sensitivity of larvae was indicated by improved feeding at 0·1 μmol s^‐1 m^‐2, from 26±5% of larvae feeding and 0·027±0·005 rotifers consumed per feeding larva min^‐1 on day 8, to 96±2% and 0·221±0·007 rotifers consumed larva^‐1 min^‐1 on day 23 post‐hatching. Algal cell‐induced turbidity was shown to reduce incident irradiance with depth, indicated by increasing coefficients of attenuation (1·4–33·1) with increasing cell densities (0–2×10⁶ cells ml^‐1), though light intensities in the feeding experiment test chambers, at the algal cell densities tested, were within the optimal range for feeding (1–10 μmol s^‐1 m^‐2). Algae‐induced turbidity had different effects on larval feeding response dependent upon the previous visual environment of the larvae. Young larvae (day 9 post‐hatching) reared in clearwater showed decreased feeding capabilities with increasing turbidity, from 98±1% feeding and 0·153±0·022 rotifers consumed larva^‐1 min^‐1 in clearwater to 61±10% feeding and 0·042±0·004 rotifers consumed larva^‐1 min^‐1 at 56 NTU, while older clearwater reared larvae fed well at all turbidities tested. Likewise, greenwater reared larvae had increased feeding capabilities in the highest algal cell densities tested (32 and 66 NTU) compared with those in low algal cell density (6 NTU), and clearwater (0·7 NTU) to which they were naïve.     

Capillary Distribution in the Lateral Muscle of Axolotl (Ambystoma mexicanum Shaw)

The vascular supply of red, intermediate and white fibres in the axial muscle of axolotl (Ambystoma mexicanum Shaw) was visualized with Indian ink-injections and quantified with morphometrical methods on semithin sections. Red fibres were surrounded by 1.4 ± 0.6 capillaries (mean + SD), the intermediate fibres by 1.2 ± 0.9 capillaries and white fibres by 0.3 ± 0.6 capillaries. The mean vascularized surface area per unit volume of fibre was 0.0159, and 0.0068 and 0.0007 (μm²/μm³) for red, intermediate and white fibres, respectively. A unit volume of mitochondria within each type of fibre was supplied by 0.15, 0.17 and 0.08 μm² vascularized surface for red, intermediate and white fibres, respectively. This indicates that there exist a good balance between oxygen demand represented by mitochondrial content and oxygen supply represented by the vascularized surface.     

Exercise suppression of thermoregulatory thermogenesis in warm- and cold-acclimated rats

An evaluation was made of the effects of an acute exercise bout on nonshivering thermogenesis (NST) in cold-acclimated rats (4 degrees C for 6 weeks) and shivering thermogenesis in 24 degrees C-acclimated rats (24 degrees C for 6 weeks). Assessment techniques included indirect calorimetry during treadmill running and brown adipose tissue (BAT) mitochondrial guanosine diphosphate (GDP) binding immediately following a treadmill run. Calorimetric results for 24 degrees C-acclimated rats running at 4 degrees C indicated total substitution of shivering thermogenesis by exercise-derived heat. No difference in GDP-binding, an index of BAT nonshivering thermogenic activity, was observed between exercised and nonexercised 24 degrees C-acclimated rats. Calorimetric results for cold-acclimated rats running at 4 degrees C indicated a total suppression in the energy cost associated with NST, exercise-derived heat replacing or substituting for NST. Examining BAT properties in the exercised cold-acclimated rats revealed a significant 40% decrease in BAT mitochondrial GDP-binding. These results suggest that during running, metabolic heat due to the exercise totally replaces shivering in 24 degrees C-acclimated rats and totally replaces BAT nonshivering thermogenesis in cold-acclimated rats.     

Thermoregulation: What Role for UCPs in Mammals and Birds?

Mammals and birds are endotherms and respond to cold exposure by the means of regulatory thermogenesis, either shivering or non-shivering. In this latter case, waste of cell energy as heat can be achieved by uncoupling of mitochondrial respiration. Uncoupling proteins, which belong to the mitochondrial carrier family, are able to transport protons and thus may assume a thermogenic function. The mammalian UCP1 physiological function is now well understood and gives to the brown adipose tissue the capacity for heat generation. But is it really the case for its more recently discovered isoforms UCP2 and UCP3? Additionally, whereas more and more evidence suggests that non-shivering also exists in birds, is the avian UCP also involved in response to cold exposure? In this review, we consider the latest advances in the field of UCP biology and present putative functions for UCP1 homologues.     

Metabolic changes associated with sustained 48-Hr shivering thermogenesis in the newborn pig

Metabolic changes associated with sustained 48-hr shivering thermogenesis were studied in piglets maintained at 34 (thermoneutrality) or 25°C (cold) between 6 and 54 hr of life. Despite their high shivering activity and elevated heat production, cold-exposed piglets exhibited a slightly lower rectal temperature than thermoneutral animals (-1.1°C; P < 0.01) at the end of the treatment. The enhancement of heat production and shivering activity were associated with a decrease in muscle glycogen (− 47%; P < 0.05) and total lipid content (− 23%; P < 0.05), a reduction of blood lactate levels (P < 0.05) and an enhancement of muscle cytochrome oxidase activity (+20%;P < 0.05), which suggests that muscle oxidative potential was increased by cold exposure. Potential for capturing lipids (lipoprotein lipase activity) was also higher in the redrhomboideus muscle (+ 71%; P < 0.01) and lower in adipose tissue (−58%; P < 0.01) of the cold-exposed piglets. Measurements performed at the mitochondrial level show no changes inrhomboideus muscle, but respiratory capacities (state IV and FCCP-stimulated respiration) and intermyofibrillar mitochondria oxidative and phosphorylative (creatine kinase activity) capacities were enhanced inlongissimus dorsi muscle (P < 0.05). These changes may contribute to provide muscles with nonlimiting amount of readily oxidable substrates and ATP necessary for shivering thermogenesis. A rise in plasma norepinephrine levels was also observed during the second day of cold exposure (P < 0.05).     

Effect of photoperiod and melatonin on cold resistance,thermoregulation and shivering/nonshivering thermogenesis in Japanese quail

Summary The effect of photoperiod and melatonin treatment on cold resistance and thermogenesis of quails was studied. The birds were acclimated for 8 weeks to short day (8L:16D) or long day (16L:8D) conditions, and 8 of 16 quails in each group were implanted with melatonin capsules. One group of quails was maintained outside in an aviary during winter. Oxygen consumption ( ) body temperature (T _b, recorded with temperature transmitters) and shivering (integrated pectoral EMG) were recorded continuously, and samples of heart rate and breathing rate were picked up when ambient temperature was decreased stepwise from 27 down to –75 °C. Heat production maximum (HP_max), cold limit, lower critical temperature, basal metabolic rate (BMR) and thermal conductance were determined.The results show that short day, cold and melatonin treatment improved cold resistance and thermal insulation of quils when compared with quails acclimated to long day conditions. An increase in HP_max was induced only by melatonin treatment. The results suggest that the acclimatization of quails is under control of the pineal gland.The linear increase of shivering intensity with at moderate cold load shows that shivering is the primary source for thermoregulatory heat production in the quail. AtT _a's below –40 °C shivering remained constant although , heart rate and breathing rate continued to increase with increasing cold load. This could indicate the existence of a nonshivering thermogenesis in birds. Unlike to mammals, this non-shivering thermogenesis in birds would serve as secondary source of heat supporting shivering thermogenesis in severe coldAbbreviations BMR basal metabolic rate - ECG electrocardiogram - EMG electromyogram - NST nonshivering thermogenesis - SMR standard metabolic rate     

Facultative and obligatory thermogenesis in young birds: a cautionary note

A brief overview on thermogenic mechanisms in young precocial birds is given. While shivering thermogenesis is well documented in these birds, evidence for a facultative non-shivering component of heat production, comparable to that found in the brown adipose tissue of mammals, is ambiguous. One reason for this is the confusion between thermoregulatory and obligatory thermogenesis. In particular, the existence of a thermogenic reaction, even a futile one, does not by itself constitute proof of true thermoregulatory non-shivering thermogenesis. More probably, such a reaction is another obligatory component of heat production. Heat increment of feeding and motor activity are classical examples of such mechanisms. Thermogenesis arising from such mechanisms can often be adaptively used by the thermoregulatory systems in young birds, as well as in adults.     

Effects of fasting and aminophylline on norepinephrine-stimulated non-shivering thermogenesis

In an attempt to further elucidate the mechanisms of fasting-depressed maximum thermogenesis and cold tolerance, norepinephrine (NE)-stimulated non-shivering thermogenesis (NST) in cold-acclimated rats was used as a functional index of possible alterations in adrenergic efficacy after fasting. Fasting decreased the magnitude of maximum NE-Stimulated NST by 18.2% [6.87±0.47 Kcal (Kg^.75.min)^?1 well-fed vs. 5.81±0.39 Kcal (Kg^.75.min)^?1 fasted], but the apparent adrenergic binding affinity was not affected [Ke=0.43 μg NE min^?1 well-fed vs 0.55 μg NE min^?1 fasted]. Pretreatment with aminophylline [15 mg Kg^?1, i.p.], a phosphodiesterase inhibitor, restored the fasting-depressed NE-stimulated NST to the fed level. The results suggest that the depression of maximum thermogenesis after fasting is not due to changes in adrenergic binding characteristics but to alteration in cAMP production/degradation, resulting in decreased substrate mobilization for thermogenesis.     

Radioimmunoassay of vasoactive intestinal polypeptide

A sensitive radioimmunoassay for plasma vasoactive intestinal polypeptide (VIP) has been developed based on preparations of fully immunoreactive ¹²⁵I-labeled VIP and hightiter specific antiserum as well as elimination of plasma interference substance(s). Fully immunoreactive ¹²⁵I-labeled VIP (specific activity = 520 μCi/nmol) was prepared by lactoperoxidase iodination and purified by gel filtration followed by chromatography on an O-(carboxymethyl) (CM)-Sephadex C-25 column. Specific anti-VIP serum produced from New Zealand white rabbits had a titer of 1:500,000 and the following binding parameters: effective affinity constant (K_eff), 2.9 × 10¹¹m^?1; heterogeneity index (α), 0.57; average affinity constant (K₀), 2.4 × 10¹⁰m^?1. Interfering substance(s) in plasma samples was proved to be present by direct radioimmunoassay and eliminated by an XAD-2 resin adsorption technique, leading to a minimal overall sensitivity of 0.48 pm for plasma samples. The average plasma VIP concentration of 78 normal fasting human subjects was 5.7 ± 3.4 (SD) pm, and that of 5 patients with watery diarrhea syndrome was 359 ± 93 pm, which reduced gradually to the normal basal value after clinical treatment.     

Coefficients for Active Transport and Thermogenesis of Ca-ATPase Isoforms

Coefficients for active transport of ions and heat in vesicles with Ca²⁺-ATPase from sarcoplasmic reticulum are defined in terms of a newly proposed thermodynamic theory and calculated using experiments reported in the literature. The coefficients characterize in a quantitative manner different performances of the enzyme isoforms. Four enzyme isoforms are examined, namely from white and red muscle tissue, from blood platelets, and from brown adipose mitochondria. The results indicate that the isoforms have a somewhat specialized function. White muscle tissue and brown adipose tissue have the same active transport coefficient ratio, but the activity level of the enzyme in white muscle is higher than in brown adipose tissue. The thermogenesis ratio is high in both white muscle and brown adipose tissue, in agreement with a specific role in nonshivering thermogenesis. Other isoforms do not have this ability to generate heat. A calcium-dependence of the coefficients is found, which can be understood as being in accordance with the role of this ion as a messenger in muscle contraction as well as in thermogenesis. The investigation points to new experiments related to structure as well as to function of the isoforms.     

Inhibitory control of shivering and nonshivering thermogenesis by preoptic warm-sensitive neurons

To investigate the characteristics of efferent projections from the preoptic area for the control of shivering and nonshivering thermogenesis, we tested the effects of thermal stimulation and injecting excitatory substances into the preoptic area on shivering and nonshivering thermogenesis in anesthetized rats. Preoptic warming and injection of glutamate suppressed shivering at ambient temperatures of 15–21°C. Likewise, preoptic warming and d,l-homocysteic acid injection suppressed nonshivering thermogenesis elicited by electrical stimulation of the ventromedial hypothalamic nucleus. Inhibitory signals from warm-sensitive neurons, thus, contribute a larger efferent signal for heat production than do signals from cold-sensitive neurons.     

The cyanobacterium Synechococcus R-2 (Anacystis nidulans, S. leopoliensis) PCC 7942 has a sodium-dependent chloride transporter

Synechococcus R-2 (PCC 7942) actively accumulated Cl^? in the light and dark, under control conditions (BG-11 media: pH_o, 7·5; [Na⁺]_o, 18 mol m^?3; [Cl^?]_o, 0·508 molm^?3). In BG-11 medium [Cl^?], was 17·2±0·848 mol m^?3 (light), electrochemical potential of Cl^? (ΔμCl^?_i,o) =+211±2mV; [Cl^?]_i= 1·24±0·11 mol m^?3(dark), ΔμCl^?_i,o=+133±4mV. Cl^? fluxes, but not permeabilities, were much higher in the light: ?Cl^?_i,o= 4·01±5·4 nmol m^?2 s^?1, ^PCl^?_i,o= 47±5pm s^?1 (light); ?Cl^?_i,o= 0·395±0·071 nmol m^?2 s^?1, _PCl^?_i,o= 69±14 pm s^?1 (dark). Chloride fluxes are inhibited by acid pH_o (pH_o 5; ?Cl^?_i,o= 0·14±0·04 nmol m^?2 s^?1); optimal at pH_o 7·5 and not strongly inhibited by alkaline pH_o (pH_o 10; ?Cl^?1_i,o= 1·7±0·14 nmol m^?2 s^?1). A Cl^?_in/2H⁺_in coporter could not account for the accumulation of Cl^? alkaline pH_o. Permeability of Cl^? is very low, below 100pm s^?1 under all conditions used, and appears to be maximal at pH_o 7·5 (50–70 pm s^?1) and minimal in acid pH_o (20pm s^?1). DCCD (dicyclohexyl-carbodiimide) inhibited ?Cl^?_i,o in the light about 75% and [Cl^?]_i fell to 2·2±0·26 (4) mol m^?3. Valinomycin had no effect but monensin severely inhibited Cl^? uptake ([Cl^?]_i= 1·02±0·32 mol m^?3; ?Cl^?_i,o= 0·20±0·1 nmol m^?2 s^?1). Vanadate (200 mmol m^?3) accelerated the Cl^? flux (?Cl^?_i,o= 5·28±0·64 nmol m^?2 s^?1) but slightly decreased accumulation of Cl^? ([Cl^?], = 13·9±1·3 mol m^?3) in BG-11 medium but had no significant effect in Na⁺-free media. DCMU (dichlorophenyldimethylurea) did not reduce [Cl^?], or ?Cl^?_i,o to that found in the dark ([Cl^?]_i= 8·41±0·76 mol m^?3; ?Cl^?_i,o= 2·06±0·36 nmol m^?2 s^?1). Synechococcus also actively accumulated Cl^? in Na⁺-free media, [Cl^?]_i was lower but ΔΨ_i,o hyperpolarized in Na⁺-free media and so the ΔμCl^?_i,o was little changed ([Cl^?]_i= 7·98±0·698 mol m^?3; ΔμCl^?_i,o=+203±3 mV). Net Cl^? uptake was stimulated by Na⁺; Li⁺ acted as a partial analogue for Na⁺. Synechococcus has a Na⁺ activated Cl^? transporter which is probably a primary 2Cl^?/ATP pump. The Cl^? pump is voltage sensitive. ΔμCl^?_i,o is directly proportional to ΔΨ_i,o(P»0·01%): ΔμCl^?_i,o= -1·487 (±0·102) ×ΔΨ_i,o, r= -0·983, n= 31. The ΔμCl^?_i,o increased (more positive) as the Δμ_i,o became more negative. The ΔμCl^?_i,o has no known function, but might provide a driving force for the uptake of micronutrients.