The fusion of synaptic vesicle membranes studied by lipid mixing: the R18 fluorescence assay validity

The various experimental approaches and octadecyl rhodamine B chloride (R18) assay's capability to meet the criteria for examining the Ca²⁺dependent synaptic vesicles (SVs) fusion with target membranes have been investigated. The existence of at least two simultaneous processes one of which attributed to real Ca²⁺-dependent membrane fusion, while another is considered to be non-specific probe transfer has been shown. The differences in response to temperature changes were found for R18 fluorescence dequenching upon stimulation of membrane fusion or nonspecific probe transfer. The temperature dependences of the probe dequenching rate were the same for heterotypic and homotypic membrane systems and increased with the temperature growth. The combination of R18 fluorescence studies with the data obtained by dynamic light scattering (DLS) offers a unique opportunity for the determination of SVs aggregation and the membrane fusion. The cholesterol content of the synaptosomal plasma membrane was modulated by methyl-β-cyclodextrin (MCD). The MCD molecule has proven to bind directly the membrane cholesterol and interact with lipophilic probe R18 that affects its fluorescence. The obvious distinctions in probe dequenching due to the membrane mixing or the MCD effect were observed. The cholesterol depletion from the synaptosomal plasma membranes was found to inhibit the process of Ca²⁺-induced membrane fusion with SVs. Thus, the manipulations with conditions of R18 probe dequenching at the model conditions, specific for the Ca²⁺-triggered fusion steps of regulated exocytosis, allowed us to determine the relative contribution of probe transfer and genuine membrane fusion to the overall fluorescence signal.     

Comparison of Ca uptake characteristics of microsomal fractions isolated from unfertilized and fertilized sea urchin eggs

The microsomal fraction isolated from sea urchin H. pulcherrimus eggs has the ability to actively accumulate Ca²⁺ in the presence of ATP. The Ca²⁺ uptake was sustained by addition of oxalate and was apparently insensitive to sodium azide. The sequestered microsomal Ca was readily released by the divalent cation ionophore A23187. The microsomal fraction obtained from fertilized eggs accumulated Ca²⁺ about five times more quickly than did that from unfertilized eggs. The increased Ca²⁺ uptake by microsomal fraction obtained from fertilized eggs was due to an increase in the maximum velocity of Ca²⁺ uptake and there was no difference in K_m for calcium between the two fractions.     

Calcium homeostasis and early embryotoxicity in marine invertebrates

Chemicals of various origins: chlorambucil, maitotoxin, sigmoidines, caulerpenyne, tributyltin, thapsigargin, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) and retinoid CD 367 were assayed on the cleavage of sea urchin eggs, their embryonic development and mechanisms regulating Ca²⁺ homeostasis. Compounds were used at therapeutic doses or at concentrations which were previously shown to be cytotoxic. These molecules did not affect the fertilization of Paracentrorus lividus eggs but all of them delayed the first cleavage. Only chlorambucil and CD 367 retarded hatching. All compounds provoked embryonic abnormalities if development was followed up to the pluteus stage, 72 hr after fertilization. Chemicals inhibited the ability of ATP-driven Ca²⁺ accumulation by the eggs in non-mitochondrial intracellular stores. Chlorambucil, maitotoxin and sigmoidines provoked a release of the Ca²⁺ sequestered with kinetics comparable to those provoked by the Ca²⁺ ionophore A23187. Ca²⁺ permeability of the plasma membrane was greatly increased by maitotoxin and 2,4,5-T whereas the other compounds were without effect. A drug-induced change in the Ca²⁺ storage capacity of sea urchin eggs resulting in retardation of cleaving stages and in further developmental defects is discussed in view to the possibility of relating changes in Ca²⁺-homeostasis with teratogenicity.     

Effect of diesel fuel hydrocarbons on embryogenesis and 45Ca2+ uptake by unfertilized eggs of sea urchin,Strongylocentrotus intermedius

1. The quality of unfertilized eggs of the sea urchin Strongylocentrotus intermedius, kept for a long time (50 days) in a sea water containing water soluble hydrocarbons of diesel fuel in sublethal concentrations (0.3–0.04 mg/l), was assessed through observation of embryogenesis and the intensity of ⁴⁵Ca²⁺ uptake.2. It has been shown that such treatment led to delay and asynchronism of embryonal and larval development and to appearance of a greater number of abnormalities compared to the control.3. Unfertilized eggs of sea urchins exposed to the hydrocarbons in sublethal concentrations accumu- lated 30–60% more ⁴⁵Ca²⁺ than those of control animals. Short-term incubation (2 hr) of eggs at the same hydrocarbon concentrations did not change ⁴⁵Ca²⁺ uptake by unfertilized eggs of control animals.4. The increase of hydrocarbon concentration up to 1 mg/l (i.e. to a concentration causing disturbance of embryogenesis in acute experiments) in short-term experiments caused a small elevation in the ⁴⁵Ca²⁺ uptake by unfertilized eggs of control animals (30% more than in untreated eggs).5. Ionomycin-induced (concentration 10⁻⁸−10⁻⁹) increase of ⁴⁵Ca²⁺ uptake by unfertilized eggs (50–100% more than the untreated eggs) caused the same disturbance of embryogenesis as under hydrocarbon exposure.6. It is suggested that one of the mechanisms inducing the deleterious effect of hydrocarbons in sea urchin gametes is related to the increase of membrane permeability to calcium ions.     

Free intracellular cations in echinoderm oocytes and eggs

The concentrations of Ca²⁺, Na⁺ and H⁺ in echinoderm oocytes and eggs were measured during maturation and activation using ion-selective microelectrodes. In both oocytes and eggs, from three species of starfish and two species of sea urchin, the resting level of cytosolic Ca²⁺ was about 10^-7 M. We did not detect any change in Ca²⁺ concentration either during hormone-induced oocyte maturation (starfish) or during egg activation (starfish and sea urchin) induced by spermatozoa or chemical agents. During 1-methyl-adenine induced maturation of starfish oocytes the intracellular level of Na⁺ increased from 12–35 mM to 40–90 mM, while the pH changed from 6.6–6.8 to 7.0–7.2 Aged oocytes, with intact germinal vesicles, also had elevated levels of Na⁺ and pH.     

Nitric oxide (NO) increase at fertilization in sea urchin eggs upregulates fertilization envelope hardening

Previous studies indicate that the nitric oxide (NO) increase at fertilization in sea urchin eggs is Ca²⁺-dependent and attributed to the late Ca²⁺ rise. However, its role in fertilization still remains unclear. Simultaneous measurements of the activation current, by a single electrode voltage clamp, and NO, using the NO indicator DAF-FM, showed that the NO increase occurred at the time of peak current (t_p) which corresponds to peak [Ca²⁺]_i, suggesting that NO is not related to any other ionic changes besides [Ca²⁺]_i. We measured O₂ consumption by a polarographic method to examine whether NO regulated a respiratory burst for protection as reported in other biological systems. Our results suggested NO increased O₂ consumption. The fluorescence of reduced pyridine nucleotides, NAD(P)H was measured in controls and when the NO increase was eliminated by PTIO, a NO scavenger. Surprisingly, PTIO decreased the rate of the fluorescence change and the late phase of increase in NAD(P)H was eliminated. PTIO also suppressed the production of H₂O₂ and caused weak and high fertilization envelope (FE). Our results suggest that NO increase upregulates NAD(P)H and H₂O₂ production and consolidates FE hardening by H₂O₂.     

Actin,more than just a housekeeping protein at the scene of fertilization

Since the first demonstration of sperm entry into the fertilized eggs of Mediterranean sea urchin Paracentrotus lividus by Hertwig (1876), enormous progress and insights have been made on this topic. However, the precise molecular mechanisms underlying fertilization are largely unknown. The two most dramatic changes taking place in the zygote immediately after fertilization are: (i) a sharp increase of intracellular Ca²⁺ that initiates at the sperm interaction site and traverses the egg cytoplasm as a wave, and (ii) the concomitant dynamic rearrangement of the actin cytoskeleton. Traditionally, this has been studied most extensively in the sea urchin eggs, but another echinoderm, starfish, whose eggs are much bigger and transparent, has facilitated experimental approaches using microinjection and fluorescent imaging methodologies. Thus in starfish, it has been shown that the sperm-induced Ca²⁺ increase in the fertilized egg can be recapitulated by several Ca²⁺-evoking second messengers, namely inositol 1,4,5-trisphosphate (InsP3), cyclic ADP-ribose (cADPr) and nicotinic acid adenine dinucleotide phosphate (NAADP), which may play distinct roles in the generation and propagation of the Ca²⁺ waves. Interestingly, it has also been found that the dynamic rearrangement of the actin cytoskeleton in the fertilized eggs plays pivotal roles in guiding monospermic sperm entry and in the fine modulation of the intracellular Ca²⁺ signaling. As it is well known that Ca²⁺ regulates the structure of the actin cytoskeleton, our finding that Ca²⁺ signaling can be reciprocally affected by the state of the actin cytoskeleton raises an intriguing possibility that actin and Ca²⁺ signaling may form a ‘positive feedback loop’ that accelerates the downstream events of fertilization. Perturbation of the cortical actin networks also inhibits cortical granules exocytosis. Polymerizing actin bundles also compose the ‘acrosome process,’ a tubular structure protruding from the head of fertilizing sperm. Hence, actin, which is one of the most strictly conserved proteins in eukaryotes, modulates almost all major aspects of fertilization.     

NH3-treatment of unfertilized sea urchin eggs turns on the Ca2+-ATPase cycle.

Cyclic fluctuations of a Ca²⁺-ATPase activity are turned on in NH₃-activated unfertilized sea urchin eggs, although no mitotic apparatus is formed. Subsequent fertilization of the activated eggs and the concomitant formation of spindle-like structures does not change drastically the course of the enzymatic fluctuations.     

Ca-transport in sea urchin unfertilized eggs: Regulation by endogenous sulfated polysaccharides and K

Previous data from our laboratory showed that the reticulum of the sea cucumber smooth muscle body wall retains both a sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) and a sulfated polysaccharide. In this invertebrate, the transport of Ca²⁺ by the SERCA is naturally inhibited by these endogenous sulfated polysaccharides. The inhibition is reverted by K⁺ leading to an enhancement of the Ca²⁺ transport rate. We now show that vesicles derived from the endoplasmic reticulum of unfertilized eggs from the sea urchin Arbacia lixula retain a SERCA that is able to transport Ca²⁺ at the expense of ATP hydrolysis. As described for the sea cucumber SERCA isoform, the enzyme from the sea urchin is activated by K⁺ but not by Li⁺ and is inhibited by thapsigargin, a specific inhibitor of SERCA. A new sulfated polysaccharide was identified in the sea urchin eggs reticulum composed mainly by galactose, glucose, hexosamine and manose. After extraction and purification, this sulfated polysaccharide was able to inhibit the mammal SERCA isoform found in rabbit skeletal muscle and the inhibition is reversed by K⁺. These data suggest that the regulation of the SERCA pump by K⁺ and sulfated polysaccharides is not restricted to few marine invertebrates but is widespread.     

USE OF FLUORESCENT DYES FOR MEASUREMENT AND LOCALIZATION OF ORGANELLES ASSOCIATED WITH CA2+ STORE RELEASE IN HUMAN NEUTROPHILS

Fura-2 and its lipid analogue, FFP-18, were used to measure changes in cytosolic free Ca²⁺concentration within human neutrophils. Whereas fura-2 was employed to monitor cytosolic Ca²⁺increases throughout the cytosol, FFP-18 was used to monitor Ca²⁺changes only near the membrane. This latter probe was incorporated into the plasma membrane as its acetoxymethyl ester (FFP-18-AM) but as de-esterification was catalysed by cytosolic esterases, the Ca²⁺-sensing probe (FFP-18 acid) accumulated on the inner face of membrane. The fluorescence of esterified probe on the extracellularly facing membrane leaflet was quenched by the membrane-impermeant ion Ni²⁺. Under these conditions, near membrane Ca²⁺changes which resulted from the release of Ca²⁺from intracellular stores was possible by conventional ratio fluorescence measurement of FFP-18. From the timing of arrival of Ca²⁺at the plasma membrane, it was proposed that there were two Ca²⁺storage sites, liberated by different stimuli, one close to the plasma membrane and the other more distant. In order to discover whether organelles within the neutrophil had distributions which correlate with the Ca²⁺release sites, fluorescent dyes for structures within the cytosol were employed. We have previously shown that the location of the intracellular membrane stain, DiOC₆(3) corresponds to the distant Ca²⁺release site. Here a second stain, BODIPY-C₅ceramide, has also been used and is shown to stain a peripheral region of the neutrophil, in a similar pattern to the near membrane Ca²⁺storage site. These data therefore raise the question of whether these stains mark the organelles in neutrophils which are the two Ca²⁺storage and release sites.     

Sea urchin eggs in the acid reign

Sea urchin eggs have been an indispensable model system for studying egg activation and ionic signalling for at least a century. Instrumental in the discovery of two Ca²⁺-mobilizing second messengers, cyclic ADP-ribose and NAADP, the sea urchin has revolutionized cell biology for all phyla. This review attempts to summarize what we currently know about egg acidic vesicles in the context of Ca²⁺ signalling. The dynamics of Ca²⁺ storage, Ca²⁺ mobilization, proton fluxes and two-pore channels will be discussed.     

CHANGE IN THE ACTIVITY OF LIPASE IN SEA URCHIN EGGS AND EMBRYOS DURING EARLY DEVELOPMENT*

During the early development of the sea urchin, Anthocidaris crassispina, the activity of lipase was maintained at the same level as in unfertilized eggs until the mesenchymal blastula stage (20 hr culture at 20°C) and then increased gradually after gastrulation. The activity in the embryos kept in SO^2?₄-free artificial sea water changed in a similar manner to that in those kept in normal sea water, during the development until 36 hr of fertilization. At 48 hr, the activity in the embryos, which had developed to the permanent blastulae in SO^2?₄-free sea water, was markedly lower than in normal plutei and was similar to that in unfertilized eggs. The lipase activity in fertilized eggs 30 min after fertilization, which was almost the same as that in unfertilized eggs was found mainly to be localized in the precipitate fraction obtained by the centrifugation at 12,000 x g for 20 min, whereas the activity in unfertilized eggs was found in the precipitate by the centrifugation at 105,000 x g for 60 min. Ca²⁺, adenosine 3′, 5′-cyclic monophosphate (cAMP) and guanosine 3′, 5′-cyclic monophosphate (cGMP) had no effect on the lipase activity.     

Polar lobe formation and cytokinesis in fertilized eggs of Ilyanassa obsoleta. II. Large bleb formation caused by high concentrations of exogenous calcium ions

Fertilized eggs of the mollusk Ilyanassa obsoleta (Nassarius obsoletus) form large blebs resembling polar lobes within 12 min of exposure to solutions of isotonic CaCl₂, whereas control eggs in sea water remain spherical. Under identical conditions, fertilized eggs of the sea urchin, Strongylocentrotus purpuratus, an organism which normally does not form polar lobes, do not form blebs upon exposure to solutions of isotonic CaCl₂. The calcium-induced blebbing in Ilyanassa still occurs if other cations such as Na⁺, Mg²⁺, or Mn²⁺ are present in addition to Ca²⁺, but not if comparable concentrations of K⁺ are present. Cytochalasin B prevents the calcium-induced blebbing, whereas colchicine does not. Cytokinesis in both Ilyanassa and Strongylocentrotus and normal polar lobe formation in Ilyanassa appear to require exogenous K⁺ but not exogenous Ca²⁺. Preliminary electron microscopy of Ilyanassa eggs exposed to isotonic solutions of CaCl₂ has shown microfilaments in the cortical cytoplasm in the region of the bleb constriction but no microfilaments in spherical control eggs in sea water. These data suggest that high concentrations of exogenous Ca²⁺ trigger the polymerization and contraction of a ring of microfilaments in the cortical cytoplasm of the Ilyanassa egg which results in the formation of a lobelike bleb of cytoplasm. The observation that K⁺ antagonizes this Ca²⁺-induced blebbing has led to the formulation of a theory which postulates that the ratio of intracellular Ca²⁺ to intracellular K⁺ is critical in the control of polar lobe formation and cytokinesis.     

The isolation of intact cortical granules from sea urchin eggs: calcium lons trigger granule discharge.

A simple procedure is described for isolating preparations of cortical granule layers from sea urchin eggs. The isolated granules are structurally intact as revealed by scanning and transmission electron microscopy. When Ca²⁺ is present, the isolated granules instantaneously discharge their contents. The site of action of Ca²⁺ may reside in the membrane of the granule. Procaine, a competitive inhibitor of Ca²⁺ binding to membranes, completely blocks the discharge of cortical granules that normally occurs at fertilization. The results support the hypothesis that once initiated, the propagation of cortical granule discharge spreads as an autocatalytic wave in which discharging granules release Ca²⁺ through their membranes which in turn triggers the discharge of adjacent granules.     

Selective Cytochrome c Displacement by Phosphate and Ca2+ in Brain Mitochondria

In brain mitochondria, phosphate- and Ca²⁺-dependent cytocrome c (cyt c) release reveals pools that interact differently with the inner membrane. Detachment of the phosphate-dependent pool did not influence the pool released by Ca²⁺. Cyt c pools were also detected in a system of cyt c reconstituted in cardiolipin (CL) liposomes. Gradual binding of cyt c (1 nmol) to CL/2–[12-(7-nitrobenz- 2-oxa-1,3-diazol-4-yl)amino]dodecanoyl-1-hexadecan oyl-sn-glycero-3-phosphocholine (NBDC₁₂-HPC) liposomes (10 nmol) produced NBD fluorescence quenching up to 0.4 nmol of added protein. Additional bound cyt c did not produce quenching, suggesting that cyt c-CL interactions originate distinct cyt c pools. Cyt c was removed from CL/NBDC₁₂-HPC liposomes by either phosphate or Ca²⁺, but only Ca²⁺ produced fluorescence dequenching and leakage of encapsulated 8-aminonaphthalene-1,3,6-trisulfonic acid/p-xylene-bis-pyridinium bromide. In mitochondria, complex IV activity and mitochondrial membrane potential (Δψ_m) were not affected by the release of the phosphate-dependent cyt c pool. Conversely, removal of cyt c by Ca²⁺ caused inhibition of complex IV activity and impairment of Δψ_m. In a reconstituted system of mitochondria, nuclei and supernatant, cyt c detached from the inner membrane was released outside mitochondria and triggered events leading to DNA fragmentation. These events were prevented by enriching mitochondria with exogenous CL or by sequestering released cyt c with anti-cyt c antibody.     

Mitochondrial inhibitors activate influx of external Ca in sea urchin sperm

Sea urchin sperm have a single mitochondrion which, aside from its main ATP generating function, may regulate motility, intracellular Ca²⁺ concentration ([Ca²⁺]_i) and possibly the acrosome reaction (AR). We have found that acute application of agents that inhibit mitochondrial function via differing mechanisms (CCCP, a proton gradient uncoupler, antimycin, a respiratory chain inhibitor, oligomycin, a mitochondrial ATPase inhibitor and CGP37157, a Na⁺/Ca²⁺ exchange inhibitor) increases [Ca²⁺]_i with at least two differing profiles. These increases depend on the presence of extracellular Ca²⁺, which indicates they involve Ca²⁺ uptake and not only mitochondrial Ca²⁺ release. The plasma membrane permeation pathways activated by the mitochondrial inhibitors are permeable to Mn²⁺. Store-operated Ca²⁺ channel (SOC) blockers (Ni²⁺, SKF96365 and Gd²⁺) and internal-store ATPase inhibitors (thapsigargin and bisphenol) antagonize Ca²⁺ influx induced by the mitochondrial inhibitors. The results indicate that the functional status of the sea urchin sperm mitochondrion regulates Ca²⁺ entry through SOCs. As neither CCCP nor dicycloexyl carbodiimide (DCCD), another mitochondrial ATPase inhibitor, eliminate the oligomycin induced increase in [Ca²⁺]_i, apparently oligomycin also has an extra mitochondrial target.     

The Role of Phospholipase D in Regulated Exocytosis

There are a diversity of interpretations concerning the possible roles of phospholipase D and its biologically active product phosphatidic acid in the late, Ca²⁺-triggered steps of regulated exocytosis. To quantitatively address functional and molecular aspects of the involvement of phospholipase D-derived phosphatidic acid in regulated exocytosis, we used an array of phospholipase D inhibitors for ex vivo and in vitro treatments of sea urchin eggs and isolated cortices and cortical vesicles, respectively, to study late steps of exocytosis, including docking/priming and fusion. The experiments with fluorescent phosphatidylcholine reveal a low level of phospholipase D activity associated with cortical vesicles but a significantly higher activity on the plasma membrane. The effects of phospholipase D activity and its product phosphatidic acid on the Ca²⁺ sensitivity and rate of fusion correlate with modulatory upstream roles in docking and priming rather than to direct effects on fusion per se.     

Quenching of a proton gradient and concomitant increase of intragranular calcium in interstitial cells of Mytilus retractor muscle

Summary The content of specific glio-interstitial granules in situ was studied in Mytilus retractor muscle using fluorescent probes and X-ray microanalysis. The granules readily take up the fluorescent monoamine dye acridine orange added to sea water (2.7×10^-6 M) and appear red in fluorescence microscopy. The addition of ammonium chloride (10 mM) or various proton ionophores results in extinction of the granule fluorescence. In addition, a step-wise decrease in granule fluorescence is observed when the tissue is perfused with artificial sea water of decreasing pH. These granules thus appear to be acidic inside. The animals were maintained in artificial sea water containing 8.36 mM Ca²⁺ and 528.90 mM Na⁺, the ratio R=[Ca²⁺]₀/[Na⁺]² ₀ being thus equal to 3x10^-5. Perfusions of the tissue with artificial sea water containing a higher calcium concentration (12.2 mM) and/or a higher [Ca²⁺]₀/[Na⁺]² ₀ ratio (R=4.5×10^-5) result in a drastic reduction of the proton gradient, evidenced by a quenching of the acridine orange fluorescence. Under the same conditions, a significant increase of the total intragranular calcium concentration was demonstrated by quantitative X-ray micro-analysis of the tissue processed by quick freezing and freeze-substitution in the presence of oxalic acid. The fluorescence of the probe Fluo-3/AM, indicative of ionized calcium, is higher in the granules than in the surrounding cytoplasm; this suggests that calcium is accumulated in the granule against its concentration gradient. The acidic gradient of specific glio-interstitial cell granules could provide the energy needed for this calcium accumulation through a Ca²⁺/H⁺ exchange. These results are discussed with regard to the hypothesis that the glio-interstitial tissue can regulate pericellular calcium and/or hydrogen ion ioncentration in the vicinity of nerve and muscle cells.     

Regulation of diacylglycerol production and protein kinase C stimulation during sperm- and PLCzeta-mediated mouse egg activation

Background information. At fertilization in mammalian eggs, the sperm induces a series of Ca²⁺ oscillations via the production of inositol 1,4,5‐trisphosphate. Increased inositol 1,4,5‐trisphosphate production appears to be triggered by a sperm‐derived PLCζ (phospholipase C‐ζ) that enters the egg after gamete fusion. The specific phosphatidylinositol 4,5‐bisphosphate hydrolytic activity of PLCζ implies that DAG (diacylglycerol) production, and hence PKC (protein kinase C) stimulation, also occurs during mammalian egg fertilization. Fertilization‐mediated increase in PKC activity has been demonstrated; however, its precise role is unclear. Results. We investigated PLCζ‐ and fertilization‐mediated generation of DAG in mouse eggs by monitoring plasma‐membrane translocation of a fluorescent DAG‐specific reporter. Consistent plasma‐membrane DAG formation at fertilization, or after injection of physiological concentrations of PLCζ, was barely detectable. However, when PLCζ is overexpressed in eggs, significant plasma‐membrane DAG production occurs in concert with a series of unexpected secondary high‐frequency Ca²⁺ oscillations. We show that these secondary Ca²⁺ oscillations can be mimicked in a variety of situations by the stimulation of PKC and that they can be prevented by PKC inhibition. The way PKC leads to secondary Ca²⁺ oscillations appears to involve Ca²⁺ influx and the loading of thapsigargin‐sensitive Ca²⁺ stores. Conclusions. Our results suggest that overproduction of DAG in PLCζ‐injected eggs can lead to PKC‐mediated Ca²⁺ influx and subsequent overloading of Ca²⁺ stores. These results suggest that DAG generation in the plasma membrane of fertilizing mouse eggs is minimized since it can perturb egg Ca²⁺ homoeostasis via excessive Ca²⁺ influx.     

Ca2+ Uptake Through Voltage-gated L-type Ca2+ Channels by Polarized Enterocytes from Atlantic Cod Gadus morhua

The presence and localization of voltage-gated Ca²⁺ channels of L-type were investigated in intestinal cells of the Atlantic cod. Enterocytes were loaded with the fluorescent Ca²⁺ probe, fure-2/AM and changes in intracellular Ca²⁺ concentrations ([Ca²⁺] _i ) were measured, in cell suspensions, in the presence of high potassium levels (100 mm), BAY K-8644 (5 μm), nifedipine (5 μm) or ω-conotoxin (1 μm). L-type Ca²⁺ channels were visualized on intestinal sections using the fluorescent dihydropyridine (-)-STBodipy. Depolarization of the plasma membrane produced a rapid (within 5 sec) and transient (at basal levels after 21 sec) increase in [Ca²⁺] _i . BAY K-8644 increased the [Ca²⁺] _i by 7.2%. Cells in a Ca²⁺-free buffer increased [Ca²⁺] _i after addition of 10 mm Ca²⁺, and this increase was abolished by nifedipine in both depolarizing and normal medium but not by ω-conotoxin. Single cell experiments using video microscopy revealed that enterocytes remained polarized several hours after preparation and that the Ca²⁺ entry and extrusion occurred at specific and different regions of the enterocyte outer membrane. Fluorescent staining of L-type Ca²⁺ channels in the intestinal mucosa showed the most intense staining at the brushborder membrane. These results demonstrate the presence of voltage gated L-type Ca²⁺ channels in enterocytes from the Atlantic cod. The channels are mainly located at the apical side of the cells, and there is a polarized uptake of Ca²⁺ into the enterocytes. This suggests that the L-type Ca²⁺ channels are involved in the transcellular Ca²⁺ entry into the enterocytes. Received: 21 August 1997/Revised: 15 April 1998