Hypoxic vasoconstriction of cyclostome systemic vessels: the antecedent of hypoxic pulmonary vasoconstriction?

Hypoxic vasoconstriction (HV) is an intrinsic response of mammalian pulmonary vascular smooth muscle (VSM). In the present study, HV was examined by myography of vessel rings from three primitive vertebrates: New Zealand hagfish (NZH), Pacific hagfish (PH), and sea lamprey (SL). Hypoxia dilated pre-gill arteries (ventral aorta, afferent branchial) from all species, whereas it contracted systemic arteries [dorsal aorta (DA), efferent branchial, celiacomesenteric]. DA HV was reproducible over several days, and it could be sustained in NZH for 8 h without adverse effects. Tension was proportional to PO(2), and half-maximal HV was obtained at PO(2) (mmHg) of 4.7 +/- 0. 2 (NZH), 0.8 +/- 0.1 (PH), and 10.7 +/- 1.9 (SL). HV did not require preconditioning (preexisting contractile stimulus) and was unaffected by elevated extracellular potassium (200 mM NZH; 80 mM SL); removal of the endothelium (NZH); or inhibitors of cyclooxygenase, lipoxygenase, cytochrome P-450 or antagonists of alpha-adrenergic, muscarinic, nicotinic, purinergic, or serotoninergic receptors. These results show that HV is an intrinsic feature of systemic VSM in cyclostomes and suggest that HV has been in the repertoire of VSM responses, since the origin of vertebrates. The exceptionally hardy HV in cyclostome DA may provide a useful model with which to examine both the phylogeny and mechanisms of this response.     

Slow sodium-free contractures in the frog heart mediated by the sodium-calcium exchange

1. Sodium-free contractures were studied in myocardial strips from R. pipiens when extracellular sodium (Na+o) was replaced by choline chloride and extracellular free calcium (Ca2+o) was defined with EGTA-buffer. 2. Resting membrane potentials (RMP) were normal in sodium-free solutions with Ca2+o calculated below 1.0 x 10(-9) mol/l. 3. When Ca2+o was subsequently increased from zero to 1.0 x 10(-3) mol/l Na+-free contractures developed slowly with unchanged RMP even at maximum contracture, at which the intracellular ultrastructure is grossly altered. 4. The contractures developed significantly faster in the presence of 3 x 10(-6) mol/l ouabain. 5. In sodium-free solutions La3+ did not influence Ca2+-dependent contractures, apart from causing an increase in time to maximum contracture. 6. It is concluded that sarcolemmal integrity is maintained in frog myocardium treated initially with Na+/Ca2+-free solutions and then with Na+-free medium containing 1 mmol/l Ca2+. 7. Our experiments indicate that sodium-free, Ca2+o-dependent contractures are mediated by the Na+/Ca2+-exchange, operation at higher rates when Na+i is increased. La3+ (1 mmol/l) probably does not compete with Ca2+ at extracellular binding sites of the exchanger. 8. The Na+/Ca2+-exchange may under certain experimental conditions be able to increase Ca2+i to cytotoxic concentrations.     

Characteristics of functioning of electromechanical coupling in striated muscles of higher and lower vertebrates

A comparative pharmacological analysis of relative contributions of different signal transduction pathways in the activation of contraction (excitation-contraction coupling, ECC) in intact fast striated muscles of frog and lamprey was performed. It was found that the major mechanism responsible for the ECC in muscles of both animals is Ca2+ release from the sarcoplasmic reticulum through the ryanodine-sensitive channels. However, the ECC in lamprey muscle displays some important differences in the units of electromechanical coupling, which precede the calcium release from sarcoplasmic reticulum. The maximum contraction force in frog muscle develops during caffeine-induced contracture, which indicates that all Ca2+ stored in sarcoplasmic reticulum is released through ryanodine-sensitive channels. In contrast, in lamprey muscle, the maximum force develops not in response to high caffeine concentration, but in response to repetitive electrical stimulation. Hence, in addition to stores liberated by ryanodine-sensitive channels, some other sources of calcium ions should exist, which contribute to the contraction activation. A source of this additional Ca2+ ions can be external medium, because acetylcholine contracture is abolished in a calcium-free medium. In frog muscle, the acetylcholine contracture was abolished in a Na(+)-free solution. It was concluded that in frog muscle ECC can be triggered by changes in the transmembrane potential (depolarization-induced calcium release), while in lamprey muscle the entry of calcium ions into myoplasm as the trigger in ECC (calcium-induced calcium release). The lamprey muscle was found to be more resistant to tetrodotoxin and tetracaine, which is indicative of a role in the activation of contraction of tetrodotoxin-resistant Na+ and/or Ca2+ channels. It was concluded, that ECC mechanism in striated muscles of low vertebrates is not limited by the generally accepted scheme of depolarization-induced calcium release but can include some other schemes, which require the Ca2+ influx into the cell.     

Na<Superscript>+</Superscript>/Ca<Superscript>2+</Superscript> exchange and regulation of cytoplasmic concentration of calcium in rat cerebellar neurons treated with glutamate

In the present work, the forward and/or reversed Na+/Ca2+ exchange in cerebellar granular cells was suppressed by substitution of Na+o by Li+ before, during, and after exposure to glutamate for varied time and also using the inhibitor KB-R7943 of the reversed exchange. After glutamate challenge for 1 min, Na+o/Li+ substitution did not influence the recovery of low [Ca2+]i in a calcium-free medium. A 1-h incubation with 100 microM glutamate induced in the neurons a biphasic and irreversible [Ca2+]i rise (delayed calcium deregulation (DCD)), enhancement of [Na+]i, and decrease in the mitochondrial potential. If Na+o had been substituted by Li+ before the application of glutamate, i.e. the exchange reversal was suppressed during the exposure to glutamate, the number of cells with DCD was nearly fourfold lowered. However, addition of the Na+/K+-ATPase inhibitor ouabain (0.5 mM) not preventing the exchange reversal also decreased DCD in the presence of glutamate. Both exposures decreased the glutamate-caused loss of intracellular ATP. Glucose deprivation partially abolished protective effects of the Na+o/Li+ substitution and ouabain. KB-R7943 (10 microM) increased 7.4-fold the number of cells with the [Ca2+]i decreased to the basal level after the exposure to glutamate. Thus, reversal of the Na+/Ca2+ exchange reinforced the glutamate-caused perturbations of calcium homeostasis in the neurons and slowed the recovery of the decreased [Ca2+]i in the post-glutamate period. However, for development of DCD, in addition to the exchange reversal, other factors are required, in particular a decrease in the intracellular concentration of ATP.     

Distribution of Mammalian-Like Melanopsin in Cyclostome Retinas Exhibiting a Different Extent of Visual Functions

Mammals contain 1 melanopsin (Opn4) gene that is expressed in a subset of retinal ganglion cells to serve as a photopigment involved in non-image-forming vision such as photoentrainment of circadian rhythms. In contrast, most nonmammalian vertebrates possess multiple melanopsins that are distributed in various types of retinal cells; however, their functions remain unclear. We previously found that the lamprey has only 1 type of mammalian-like melanopsin gene, which is similar to that observed in mammals. Here we investigated the molecular properties and localization of melanopsin in the lamprey and other cyclostome hagfish retinas, which contribute to visual functions including image-forming vision and mainly to non-image-forming vision, respectively. We isolated 1 type of mammalian-like melanopsin cDNA from the eyes of each species. We showed that the recombinant lamprey melanopsin was a blue light-sensitive pigment and that both the lamprey and hagfish melanopsins caused light-dependent increases in calcium ion concentration in cultured cells in a manner that was similar to that observed for mammalian melanopsins. We observed that melanopsin was distributed in several types of retinal cells, including horizontal cells and ganglion cells, in the lamprey retina, despite the existence of only 1 melanopsin gene in the lamprey. In contrast, melanopsin was almost specifically distributed to retinal ganglion cells in the hagfish retina. Furthermore, we found that the melanopsin-expressing horizontal cells connected to the rhodopsin-containing short photoreceptor cells in the lamprey. Taken together, our findings suggest that in cyclostomes, the global distribution of melanopsin in retinal cells might not be related to the melanopsin gene number but to the extent of retinal contribution to visual function.     

Effect of Quin-2 on 45Ca2+ uptake mediated by Na+i/Ca2+o exchange and 45Ca2+ efflux in rat brain synaptosomes: a requirement for [Ca2+]i

The Na+/Ca2+ exchanger of squid axons, barnacle muscle and sarcolemma requires micromolar intracellular calcium for activation in the Na+i/Ca2+o exchange mode ('reverse' Na+/Ca2+ exchange). The requirement for [Ca2+]i has been demonstrated with the use of intracellular calcium buffers, such as Quin-2, to inhibit Na+i/Ca2+o exchange. However, the inhibition of Na+i/Ca2+o exchange in mammalian nerve terminals loaded with Quin-2 has not been observed [7], suggesting a lower sensitivity to low [Ca2+]i for this system. In contrast, the results reported herein indicate that 45Ca2+ uptake in synaptosomes through Na+i/Ca2+o exchange is inhibited by Quin-2 much in the same way as it is in the squid, provided that synaptosomes are preincubated in low Ca2+ medium to avoid saturation of Quin-2. Under these conditions, 45Ca2+ efflux via Ca2+i/Ca2+o exchange is also inhibited. Our results indicate that the Na+i/Ca2+o and Ca2+i/Ca2+o modes of the Na+/Ca2+ exchanger from rat brain synaptosomes require intracellular calcium for activation. However, because no clear relationship between the observed [Ca2+]i values and the inhibition of Na+i/Ca2+o exchange has been found, it is suggested that localised submembrane calcium concentrations not detected by the [Ca2+]i probe might regulate the exchanger.     

In squid nerve fibers monovalent activating cations are not cotransported during Na+/Ca2+ exchange

Squid axons display a high activity of Na+/Ca2+ exchange which is largely increased by the presence of external K+, Li+, Rb+ and NH+4. In this work we have investigated whether this effect is associated with the cotransport of the monovalent cation along with Ca2+ ions. 86Rb+ influx and efflux have been measured in dialyzed squid axons during the activation (presence of Ca2+i) of Ca2+o/Na+i and Ca2+i/Ca2+o exchanges, while 86Rb+ uptake was determined in squid optic nerve membrane vesicles under equilibrium Ca2+/Ca2+ exchange conditions. Our results show that although K+o significantly increases Na+i-dependent Ca2+ influx (reverse Na+/Ca2+ exchange) and Rb+i stimulates Ca2+o-dependent Ca2+ efflux (Ca2+/Ca2+ exchange), no sizable transport of rubidium ions is coupled to calcium movement through the exchanger. Moreover, in the isolated membrane preparation no 86Rb+ uptake was associated with Ca2+/Ca2+ exchange. We conclude that in squid axons although monovalent cations activate the Na+/Ca2+ exchange they are not cotransported.     

Sodium-free contractures in frog myocardium damaged by catecholamines

1. Sodium-free contractures were studied in myocardial strips from R. pipiens with extracellular sodium (Na+o) replaced by choline chloride and extracellular calcium (Ca2+o) varied with EGTA-buffer. Normal myocardium was compared with that damaged by adrenaline (ADR) or isoproterenol (ISO). 2. Frog myocardium, damaged by in vivo injections of catecholamines, remained relaxed when exposed to Na+/Ca2+-free solutions. Only in 2 out of 18 experiments were small contractures observed after several hours. 3. Addition of KCN to the Na+/Ca2+-free solution caused small contractures after several hours in 7 out of 10 experiments. 4. The time to maximum Na+-free contractures was correlated to Ca2+o in a dose-dependent manner, but not influenced by catecholamine-induced myocardial damage. 5. Cell injury in the frog heart after in vivo injections of catecholamines does not affect the sarcolemmal Na+/Ca2+-exchange and is not associated with passive leakage of Ca2+ from the extracellular to the intracellular space.     

Ethanol protects the heart against the calcium paradox injury

Rat hearts were depleted of Ca2+ (less than 10(-9) M) for 10 min, followed by 15 min of Ca2+-repletion. The calcium paradox injury occurs during Ca2+-repletion, after a period of calcium depletion. The calcium paradox injury was assessed by percent recovery (hemodynamics, [Ca2+]i, and energy levels) during Ca2+-repletion. A decrease in Na+ concentration during Ca2(+)-depletion did not allow for recovery during Ca2(+)-repletion, however 2.5% and 5% ethanol during Ca2(+)-depletion allowed for an approximate 50% recovery during Ca2(+)-repletion. A combination of ethanol (2.5% or 5%) with a low extracellular Na+ concentration (88 mM) allowed for complete recovery. Ethanol prevented a depletion of diastolic [Ca2+]i during Ca2(+)-depletion, and allowed for a return of normal diastolic [Ca2+]i during Ca2(+)-repletion. Ethanol modulates the activity of the Na+/Ca2+ exchanger and protects against the Ca2(+)-paradox injury.     

Effects of low extracellular sodium on cytosolic ionized calcium. Na+-Ca2+ exchange as a major calcium influx pathway in kidney cells

The effects of extracellular Na+ (Na+o) on cytosolic ionized calcium (Ca2+i) and on calcium and sodium fluxes were measured in monkey kidney cells (LLC-MK2). Ca2+i was measured with aequorin and the ion fluxes with 45Ca and 22Na. Na+-free media rapidly increased Ca2+i from 60 to a maximum of about 700 nM in 2-3 min. After the peak, Ca2+i declined and reached a plateau of about twice the resting Ca2+i. The peak Ca2+i was inversely proportional to Na+o and directly proportional to the extracellular calcium concentration (Ca2+o). On the other hand, a pH of 6.8 reduced and Ca2+o substitution with Sr2+ completely blocked the Ca2+i response to low Na+o. A Na+-free medium stimulated calcium efflux from the cells 4-5-fold, a response which was abolished in the absence of extracellular Ca2+. Na+-free media also stimulated calcium influx and sodium efflux. The cell calcium content, however, was not increased. These results indicate that removal of extracellular Na+ increases Ca2+i by stimulating calcium influx and not by inhibiting calcium efflux; the increased calcium influx takes place on the Na+-Ca2+ antiporter operating in the reverse mode in exchange for sodium efflux. The increased calcium efflux occurs as a consequence of the rise in Ca2+i and presumably takes place on the (Ca2+-Mg2+) ATPase-dependent calcium pump.     

Trypanosoma cruzi: mechanisms of intracellular calcium homeostasis.

Regulation of intracellular Ca2+ homeostasis was characterized in epimastigote forms of Trypanosoma cruzi using the fluorescence probe Fura-2. Despite an increase in extracellular Ca2+, [Ca2+]o, from 0 to 2 mM, cytosolic Ca2+, [Ca2+]i, increased only from 85 +/- 9 to 185 +/- 21 nM, indicating the presence of highly efficient mechanisms for maintaining [Ca2+]i. Exposure to monovalent Na+ (monensin)-, K+ (valinomycin, nigericin)-, and divalent Ca2+ (ionomycin)-specific ionophores, uncouplers of mitochondrial respiration (oligomycin), inhibitors of Na+/K(+)-ATPase (ouabain), and Ca(2+)-sensitive ATPase (orthovanadate) in 0 or 1 mM [Ca2+]o resulted in perturbations of [Ca2+]i, the patterns of which suggested both sequestration and extrusion mechanisms. Following equilibration in 1 mM [Ca2+]o, incubation with orthovanadate markedly increased [Ca2+]i, results which are compatible with an active uptake of [Ca2+]i by endoplasmic reticulum. In contrast, equilibration in 0 or 1 mM [Ca2+]o did not influence the relatively smaller increase in [Ca2+]i following incubation with oligomycin, suggesting a minor role for the mitochondrial compartment. In cells previously equilibrated in 1 mM [Ca2+]o, exposure to monensin or ouabain, conditions known to decrease the [Na+]o/[Na+]i gradient, upon which the Na+/Ca2+ exchange pathways are dependent, markedly increased [Ca2+]i. In a complementary manner, decreasing the extracellular Na+ gradient with Li+ increased [Ca2+]i in a dose-dependent manner. Finally, the calcium channel blockers verapamil and isradipine inhibited the uptake of Ca2+ by greater than 50%, whereas diltiazem, nifedipine, and nicardipine were ineffective. The results suggest that epimastigote forms of T. cruzi maintain [Ca2+]i by uptake, sequestration, and extrusion mechanisms, with properties common to eukaryotic organisms.     

Calcium export by sodium-calcium exchange in bovine chromaffin cells

Calcium efflux from bovine chromaffin cells in tissue culture has been examined after loading them with small amounts of Ca2+ by brief depolarization in media containing 20 mumol/l to 1 mmol/l Ca2+ and 45Ca2+ in trace amounts. In the presence of normal external Na+ and Ca2+ concentrations cells depolarized in media containing up to 200 mumol/l Ca2+ exported nearly 100% of their accumulated Ca2+ loads within 10 min and 20% within the first 5 s. In the absence of external Na+ and Ca2+ the proportion of a small (i.e., depolarization in 20 mumol/l calcium) Ca2+ load exported at any time point in the range to 10 min was approximately two thirds of the total efflux measured in their presence indicating that under these conditions the external Na+/Ca(2+)-dependent and Na+/Ca(2+)-independent mechanisms both contribute significantly to the export of calcium. At higher cellular loads of calcium (i.e., depolarization in 200 mumol/l to 1 mmol/l calcium) the Na+/Ca(2+)-dependent mechanism exported a progressively greater proportion of the accumulated Ca2+. Both sodium and calcium alone promoted a component of Ca2+ efflux; Ca2+ (i.e. calcium-calcium exchange) was as effective as Na+ (i.e. sodium-calcium exchange). The Km for Na+ stimulation of Ca(2+)-efflux (KNa) was approximately 65 mM. Increased external Mg2+ (from 1.2 to 10 mmol/l) increased the apparent KNa to 90 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+-activated Na+ fluxes in human red cells. Amiloride sensitivity

The effect of Ca2+ on the ouabain- and bumetanide-resistant Na+ fluxes in intact red cells was studied at relatively constant internal Ca2+, membrane potential, and cell volume. The red cell calcium concentration was modified using the ionophore A23187. In fresh red cells, the Na+ influx and efflux (1.2 +/- 0.13 and 0.26 +/- 0.07 mmol/liter cells x h, respectively) were not affected by amiloride (1 mM). When external Ca2+ was raised from 0 to 150 microM, in the presence of A23187, both the Na+ influx and efflux were stimulated (about 3.5-fold). The Ca2+-activated Na+ efflux and influx had an apparent Km for activation by Ca2+o of about 25 microM. The Ca2+-dependent Na+ transport was inhibited 30-60% by amiloride (ID50 = 17.3 +/- 8 microM). Amiloride, however, had no effect on the Ca2+-dependent K+ influx. The amiloride-sensitive (AS) transport pathway was a linear function of the Na+o concentration in the range from 0 to 75 mM. The Ca2+i activation seems to depend on the metabolic integrity of red cells. 1) It does not take place in ATP-depleted red cells; 2) ATP-repletion of ATP-depleted red cells fully restored AS Na influx; and 3) ATP-enrichment (ATP-red cells) enhanced the AS Na influx by about 100%. The Ca2+-activated AS Na+ influx was not affected by either DIDS or trifluoperazine. The present results indicate that in human erythrocytes an increase in internal Ca2+ activates on otherwise silent AS Na+-transport system, which is dependent on the metabolic integrity of the red cells.     

Intestinal calcium transport in spontaneously hypertensive rats (SHR) and their genetically matched WKY rats

Calcium transport across the basolateral membranes of the enterocyte represents the active step in calcium translocation. This step occurs by two mechanisms, an ATP-dependent pump and a Ca2+/Na+ exchange process. These studies were designed to investigate these two processes in jejunal basolateral membrane vesicles (BLMV) of the spontaneously hypertensive rats (SHR) and their genetically matched controls, Wistar-Kyoto (WKY) rats. The ATP-dependent calcium uptake was stimulated several-fold compared with no ATP condition in both SHR and WKY, but no differences were noted between rate of calcium uptake in SHR and WKY. Kinetics of ATP-dependent calcium uptake at concentrations between 0.01 and 1.0 microM revealed a Vmax of 0.67 +/- 0.03 nmol/mg protein/20 sec and a Km of 0.2 +/- 0.03 microM in SHR and Vmax of 0.69 +/- 0.12 and a Km of 0.32 +/- 0.14 microM in WKY rats. Ca2+/Na+ exchange in jejunal BLMV of SHR and WKY was investigated in two ways. First, sodium was added to the incubation medium (cis-Na+). Second, Ca2+ efflux from BLMV was studied in the presence of extravesicular Na+ (trans-Na+). Both studies suggest a decreased exchange of calcium and Na+. Kinetic parameters of Na(+)-dependent Ca2+ uptake at concentrations between 0.01 and 1.0 microM exhibited Vmax of 0.05 +/- 0.01 nanmol/mg protein/5 sec and a Km of 0.21 +/- 0.13 microM in SHR and Vmax of 0.11 +/- 0.02 nanmol/mg protein/5 sec and a Km of 0.09 +/- 0.05 in WKY, respectively. These results confirm that the intestinal BLMV of SHR and WKY rats have two mechanisms for calcium extrusion, an ATP-dependent Ca2+ transport process and a Na+/Ca2+ exchange process. The ATP-dependent process appears to be functional in SHR; however, the Ca2+/Na+ exchange mechanism appears to have a marked decrease in its maximal capacity. These findings suggest that calcium extrusion via Ca2+/Na+ is impaired in the SHR, which may lead to an increase in intracellular calcium concentration. These findings may have relevance to the development of hypertension.     

Recovery from blood alkalosis in the Pacific hagfish (Eptatretus stoutii): involvement of gill V-H+-ATPase and Na+/K+-ATPase

To investigate the base secretory mechanisms in the Pacific hagfish (Eptatretus stoutii), we injected animals with NaHCO3 into the subcutaneous sinus. In the first series of experiments, hagfish were injected with 6000 micromol kg(-1) NaHCO3 (base-infused hagfish, BIH) or NaCl (controls). Blood pH increased significantly 1 h after injection in BIH (8.05+/-0.05 vs. 7.82+/-0.03 pH units), but returned to control values by t=6 h. Plasma total CO2 (TCO2) followed the same pattern. Immunolabeled sections revealed that Na+/K+-ATPase and V-H+-ATPase were usually located in the same cells. Western blotting revealed that the abundance of both proteins remained unchanged in whole gill homogenates and in a fraction enriched in cell membranes 6 h after the injections. The second experimental series was to induce long-term alkalosis by serially injecting 6000 micromol kg(-1) NaHCO3 every 6 h for 24 h. Blood pH completely recovered from the base loads within 6 h after each injection. Moreover, plasma TCO2 was not elevated 3 h after the second infusion, suggesting that HCO3(-) secreting mechanisms had been upregulated by that time. Na+/K+-ATPase and V-H+-ATPase cellular localizations did not change in the 24 h base infusion protocol. Na+/K+-ATPase abundance was similar in gill homogenates from fish from both treatments. However, Na+/K+-ATPase abundance in the membrane fraction was significantly lower in BIH, while V-H+-ATPase was greater both in whole gill and membrane fractions. Our results suggest that differential insertion of V-H+-ATPase and Na+/K+-ATPase into the basolateral membrane is involved in recovering from alkalotic stress in hagfish.     

Physiological roles of Na+/Ca2+ exchange in Limulus ventral photoreceptors

In previous work we have presented evidence for electrogenic Na+/Ca2+ exchange in Limulus ventral photoreceptors (1989. J. Gen. Physiol. 93:473-492). This article assesses the contributions to photoreceptor physiology from Na+/Ca2+ exchange. Four separate physiological processes were considered: maintenance of resting sensitivity, light-induced excitation, light adaptation, and dark adaptation. (a) Resting sensitivity: reduction of [Na+]o caused a [Ca2+]o-dependent reduction in light sensitivity and a speeding of the time courses of the responses to individual test flashes; this effect was dependent on the final value to which [Na+]o was reduced. The desensitization caused by Na+ reduction was dependent on the initial sensitivity of the photoreceptor; in fully dark-adapted conditions no desensitization was observed; in light-adapted conditions, extensive desensitization was observed. (b) Excitation: Na+ reduction in fully dark-adapted conditions caused a Ca2+o-dependent depolarizing phase in the receptor potential that persisted beyond the stimulus duration and was evoked by a bright adapting flash. (c) Light adaptation: the degree of desensitization induced by a bright adapting flash was Na+o dependent, being larger with lower [Na+]o. Na+ reduction enhanced light adaptation only at intensities brighter than 4 x 10(-6) W/cm2. In addition to being Na+o dependent, light adaptation was Ca2+o dependent, being greater at higher [Ca2+]o. (d) Dark adaptation: the recovery of light sensitivity after adapting illumination was Na+o dependent. Dark adaptation after bright illumination in voltage-clamped and in unclamped conditions was faster in normal-Na+ saline than in reduced Na+ saline. The final sensitivity to which photoreceptors recovered was lower in reduced-Na+ saline when bright adapting illumination was used. The results suggest the involvement of Na+/Ca2+ exchange in each of these physiological processes. Na+/Ca2+ exchange may contribute to these processes by counteracting normal elevations in [Ca2+]i.     

The stoichiometry of the exchange catalysed by the mitochondrial calcium/sodium antiporter.

Rat heart mitochondria respiring on succinate in the presence of Ruthenium Red (to inhibit uptake on the Ca2+ uniporter) released Ca2+ on the calcium/sodium antiporter until a steady state was reached. Addition of the ionophore A23187 (which catalyses Ca2+/2H+ exchange) did not perturb this steady state. Thermodynamic analysis showed that if a Ca2+/nNa+ exchange with any value of n other than 2 was at equilibrium, addition of A23187 would cause an obvious change in extramitochondrial free [Ca2+]. Therefore the endogenous calcium/sodium antiporter of mitochondria catalyses electroneutral Ca2+/2Na+ exchange.     

Identification of uterine ion transporters for mineralisation precursors of the avian eggshell

ABSTRACT: BACKGROUND: In Gallus gallus, eggshell formation takes place daily in the hen uterus and requires large amounts of the ionic precursors for calcium carbonate (CaCO3). Both elements (Ca2+, HCO3-) are supplied by the blood via trans-epithelial transport. Our aims were to identify genes coding for ion transporters that are upregulated in the uterine portion of the oviduct during eggshell calcification, compared to other tissues and other physiological states, and incorporate these proteins into a general model for mineral transfer across the tubular gland cells during eggshell formation. RESULTS: A total of 37 candidate ion transport genes were selected from our database of overexpressed uterine genes associated with eggshell calcification, and by analogy with mammalian transporters. Their uterine expression was compared by qRTPCR in the presence and absence of eggshell formation, and with relative expression levels in magnum (low Ca2+/HCO3- movement) and duodenum (high rates of Ca2+/HCO3- trans-epithelial transfer). We identified overexpression of eleven genes related to calcium movement: the TRPV6 Ca2+ channel (basolateral uptake of Ca2+), 28 kDa calbindin (intracellular Ca2+ buffering), the endoplasmic reticulum type 2 and 3 Ca2+ pumps (ER uptake), and the inositol trisphosphate receptors type 1, 2 and 3 (ER release). Ca2+ movement across the apical membrane likely involves membrane Ca2+ pumps and Ca2+/Na+ exchangers. Our data suggests that Na+ transport involved the SCNN1 channel and the Na+/Ca2+ exchangers SLC8A1, 3 for cell uptake, the Na+/K+ ATPase for cell output. K+ uptake resulted from the Na+/K+ ATPase, and its output from the K+ channels (KCNJ2, 15, 16 and KCNMA1).We propose that the HCO3- is mainly produced from CO2 by the carbonic anhydrase 2 (CA2) and that HCO3- is secreted through the HCO3-/Cl- exchanger SLC26A9. HCO3- synthesis and precipitation with Ca2+ produce two H+. Protons are absorbed via the membrane's Ca2+ pumps ATP2B1, 2 in the apical membrane and the vacuolar (H+)-atpases at the basolateral level. Our model incorporate Cl- ions which are absorbed by the HCO3-/Cl- exchanger SLC26A9 and by Cl- channels (CLCN2, CFTR) and might be extruded by Cl-/H+ exchanger (CLCN5), but also by Na+ K+ 2 Cl- and K+ Cl- cotransporters. CONCLUSIONS: Our Gallus gallus uterine model proposes a large list of ion transfer proteins supplying Ca2+ and HCO3- and maintaining cellular ionic homeostasis. This avian model should contribute towards understanding the mechanisms and regulation for ionic precursors of CaCO3, and provide insight in other species where epithelia transport large amount of calcium or bicarbonate.     

Calcium entry in squid axons during voltage clamp pulses

Squid giant axons were injected with aequorin and tetraethylammonium and were impaled with sodium ion sensitive, current and voltage electrodes. The axons were usually bathed in a solution of varying Ca2+ concentration ([Ca2+]o) containing 150mM each of Na+, K+ and an inert cation such as Li+, Tris or N-methylglucamine and had ionic currents pharmacologically blocked. Voltage clamp pulses were repeatedly delivered to the extent necessary to induce a change in the aequorin light emission, a measure of axoplasmic Ca2+ level, [Ca2+]i. The effect of membrane voltage on [Ca2+]i was found to depend on the concentration of internal Na+ ([Na+]i). Voltage clamp hyperpolarizing pulses were found to cause a reduction of [Ca2+]i. For depolarizing pulses a relationship between [Ca2+]i gain and [Na+]i indicates that Ca2+ entry is sigmoid with a half maximal response at 22 mM Na+. This Ca2+ entry is a steep function of [Na+]i suggesting that 4 Na+ ions are required to promote the influx of 1 Ca2+. There was little change in Ca2+ entry with depolarizing pulses when [Ca2+]o is varied from 1 to 10mM, while at 50mM [Ca2+]o calcium entry clearly increases suggesting an alternate pathway from that of Na+/Ca2+ exchange. This entry of Ca2+ at high [Ca2+]o, however, was not blocked by Cs+o. The results obtained lend further support to the notion that Na+/Ca2+ exchange in squid giant axon is sensitive to membrane voltage no matter whether this is applied as a constant change in membrane potential or as an intermittent one.