Insulin increases near-membrane but not global Ca2+ in isolated skeletal muscle

It has long been debated whether changes in Ca2+ are involved in insulin-stimulated glucose uptake in skeletal muscle. We have now investigated the effect of insulin on the global free myoplasmic Ca2+ concentration and the near-membrane free Ca2+ concentration ([Ca2+]mem) in intact, single skeletal muscle fibers from mice by using fluorescent Ca2+ indicators. Insulin has no effect on the global free myoplasmic Ca2+ concentration. However, insulin increases [Ca2+]mem by approximately 70% and the half-maximal increase in [Ca2+]mem occurs at an insulin concentration of 110 microunits per ml. The increase in [Ca2+]mem by insulin persists when sarcoplasmic reticulum Ca2+ release is inhibited but is lost by perfusing the fiber with a low Ca2+ medium or by addition of L-type Ca2+ channel inhibitors. Thus, insulin appears to stimulate Ca2+ entry into muscle cells via L-type Ca2+ channels. Wortmannin, which inhibits insulin-mediated activation of glucose transport in isolated skeletal muscle, also inhibits the insulin-mediated increase in [Ca2+]mem. These data demonstrate a new facet of insulin signaling and indicate that insulin-mediated increases in [Ca2+]mem in skeletal muscle may underlie important actions of the hormone.     

Attenuation of Ca(2+)-activated ATPase and shortening velocity in hypertrophied fast twitch skeletal muscle from aged Japanese quail

The effect of aging on the in vitro contractile properties of the patagialis (PAT) muscle of 35 young adult (YA; 8 weeks of age) and 35 aged adult (AA, 110 weeks of age) Coturnix quails was examined after 0-30 days of stretch-overload. Overload was achieved by placing a weight equivalent to 12% of the birds' body weight on one wing. The contralateral wing served as the intra-animal control. Overload increased the weight of the PAT by 45.1+/-2.1% in YA, and 24.1+/-2.6% in AA. Twitch contraction time increased with loading from 43.2+/-1.2 to 67.3+/-2.2 ms in YA birds and 57.2+/-1.7 to 77.4+/-1.9 ms in AA birds. Unloaded shortening velocity (Vo) decreased by 40.1+/-2.2 and 38.8+/-3.2% in YA and aged birds, respectively. The decrease in fast myosin expression was greater in overloaded muscles of YA (20%) as compared to AA birds (12%). However, this was accompanied by a greater decrease in total muscle ATPase activity in aged birds (61%) compared to YA birds (40%). Myosin isozyme Ca(2+)-ATPase activity was 26% lower in FM1 but not other fast myosins in YA birds, but it was approximately 30% lower in all fast myosins in PAT muscles of aged birds. These data show that the reduction of Vo and the increase in twitch duration with aging may be due in part to reductions in ATPase activity in all myosin isoforms, as compared to myosin isoforms isolated from YA birds.     

Privileged coupling between Ca(2+) entry through plasma membrane store-operated Ca(2+) channels and the endoplasmic reticulum Ca(2+) pump

The sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) is the third element of capacitative calcium entry. It colocalizes with STIM1 and Orai1 at puncta, where couples plasma membrane store-operated Ca(2+) channels (SOC) to Ca(2+) pumping into the ER. The efficiency of this calcium entry-calcium refilling (CECR) coupling is comparable to the classic excitation-response transduction mechanisms. This allows efficient filling of the endoplasmic reticulum (ER) with the Ca(2+) entering through SOC channels with little progression of the Ca(2+) wave towards the cell core. CECR coupling is very sensitive to changes in stoichiometry among STIM, Orai and SERCA, with excess Orai antagonizing ER refilling. ER takes up most of the calcium load that enters through SOC, whereas mitochondria take up a very small fraction. This difference is due to the spatial positioning with regard to SOC, the amplitude of the high Ca(2+) microdomains, and the differences in the Ca(2+) affinity of the uptake mechanisms.     

Enhanced Ca(2+) mobilization in airway smooth muscle contributes to airway hyperresponsiveness in an inbred strain of rat.

The mechanisms underlying airway hyperresponsiveness are still unknown but increased contractility of airway smooth muscle may play a role. This study sought to demonstrate a relationship between in vivo airway responsiveness and a number of measures of airway smooth muscle responsiveness ex vivo, including intracellular Ca(2+) signaling, by comparing three inbred strains of rat with different degrees of airways responsiveness to methacholine. Lewis, ACI, and Fisher strains of rat were characterized for their pulmonary responses to 5-hydroxytryptamine (5HT) in vivo and Fisher rats were found to be hyperresponsive to 5HT compared with ACI and Lewis rats. The responsiveness of the airways from these strains of rat ex vivo revealed that intraparenchymal airways from Fisher rats significantly narrowed to a greater degree and at a faster rate to 5HT than Lewis rat airways, consistent with their differences in vivo. Intraparenchymal ACI airways, however, narrowed to the same degree as Fisher airways but took longer to do so at a high concentration of 5HT. 5HT caused concentration-dependent increases in intracellular Ca(2+) in airway smooth muscle cells from all three strains of rat, but Fisher and ACI displayed higher responses than Lewis airway smooth muscle. Our results demonstrate that the degree of intracellular Ca(2+) mobilization by 5HT in airway smooth muscle parallels the rate and degree of intraparenchymal airway narrowing and suggest that the degree of intracellular Ca(2+) mobilization plays a role in determining airway smooth muscle contractility.     

Cellular adaptation contributes to calorie restriction-induced preservation of skeletal muscle in aged rhesus monkeys

We have previously shown that a 30% reduced calorie intake diet delayed the onset of muscle mass loss in adult monkeys between ~16 and ~22 years of age and prevented multiple cellular phenotypes of aging. In the present study we show the impact of long term (~17 years) calorie restriction (CR) on muscle aging in very old monkeys (27-33 yrs) compared to age-matched Control monkeys fed ad libitum, and describe these data in the context of the whole longitudinal study. Muscle mass was preserved in very old calorie restricted (CR) monkeys compared to age-matched Controls. Immunohistochemical analysis revealed an age-associated increase in the proportion of Type I fibers in the VL from Control animals that was prevented with CR. The cross sectional area (CSA) of Type II fibers was reduced in old CR animals compared to earlier time points (16-22 years of age); however, the total loss in CSA was only 15% in CR animals compared to 36% in old Controls at ~27 years of age. Atrophy was not detected in Type I fibers from either group. Notably, Type I fiber CSA was ~1.6 fold greater in VL from CR animals compared to Control animals at ~27 years of age. The frequency of VL muscle fibers with defects in mitochondrial electron transport system enzymes (ETS(ab)), the absence of cytochrome c oxidase and hyper-reactive succinate dehydrogenase, were identical between Control and CR. We describe changes in ETS(ab) fiber CSA and determined that CR fibers respond differently to the challenge of mitochondrial deficiency. Fiber counts of intact rectus femoris muscles revealed that muscle fiber density was preserved in old CR animals. We suggest that muscle fibers from CR animals are better poised to endure and adapt to changes in muscle mass than those of Control animals.     

Crucial role of type 1, but not type 3, inositol 1,4,5-trisphosphate (IP(3)) receptors in IP(3)-induced Ca(2+) release, capacitative Ca(2+) entry, and proliferation of A7r5 vascular smooth muscle cells

Stimulation of G protein- or tyrosine kinase-coupled receptors regulates cell proliferation through intracellular Ca(2+) ([Ca(2+)](i)) signaling. In A7r5 cells, we confirmed that inositol 1,4,5-trisphosphate (IP(3)) mediates vasopressin (VP)-evoked Ca(2+) release from intracellular stores and showed that types 1 (IP(3)R(1)) and 3 (IP(3)R(3)) IP(3) receptors were expressed. Using antisera selective for IP(3)R(1) or IP(3)R(3) and another that interacted equally well with both subtypes, together with membranes from SF:9 cells expressing only single IP(3)R subtypes to calibrate immunoblotting, we established that A7r5 cells express 81% IP(3)R(1) and 19% IP(3)R(3). To elucidate the contributions of IP(3)R(1) and IP(3)R(3) to Ca(2+) signaling and proliferation, stable clones expressing promoter-inducible antisense cDNA fragments (-90 to +9) corresponding to the two IP(3)R subtypes were selected. Mild inhibition of IP(3)R(1) (71+/-8% of control level) slightly attenuated the IP(3)-evoked Ca(2+) release (IICR) induced by VP but significantly decreased the subsequent capacitative Ca(2+) entry (CCE) and proliferation. Moderate inhibition (34+/-6%) strongly decreased both IICR and CCE and further blocked proliferation. Complete inhibition almost abolished IICR and CCE and arrested proliferation entirely. Complete inhibition of IP(3)R(3) expression slightly attenuated IICR without affecting CCE or proliferation. In cells microinjected with a low dose of heparin, VP-induced CCE was more susceptible than IICR to mild inhibition of both IP(3)R(1) and IP(3)R(3). A high dose of heparin had a similar effect to complete inhibition of IP(3)R(1) expression: it blocked VP-evoked IICR entirely and CCE by 90%. We conclude that IP(3)R(1), but not IP(3)R(3), is crucial for IICR, CCE, and proliferation of vascular smooth muscle cells.     

Store-operated Ca2+ entry and TRPC expression; possible roles in cardiac pacemaker tissue

Store-operated Ca(2+) channels (SOCCs) were first identified in non-excitable cells by the observation that depletion of Ca(2+) stores caused increased influx of extracellular Ca(2+). Recent studies have suggested that SOCCs might be related to the transient receptor potential (TRPC) gene family. The mechanism of cardiac pacemaking involves voltage-dependent pacemaker current; in addition there is growing evidence that intracellular sarcoplasmic reticulum (SR) Ca(2+) release plays an important role. In the present short review we assess preliminary evidence for Ca(2+) entry related to SR store depletion and expression of TRPCs in pacemaker tissue. These newer findings suggest that Ca(2+) entry and inward current triggered by store depletion might also contribute to the pacemaker current. Many hormones, drugs and interventions such as ischaemia and stretch, which alter Ca(2+) handling, will also modulate pacemaker firing thought their effect on SOCCs.     

Ca(2+) sparks operated by membrane depolarization require isoform 3 ryanodine receptor channels in skeletal muscle

Stimuli are translated to intracellular calcium signals via opening of inositol trisphosphate receptor and ryanodine receptor (RyR) channels of the sarcoplasmic reticulum or endoplasmic reticulum. In cardiac and skeletal muscle of amphibians the stimulus is depolarization of the transverse tubular membrane, transduced by voltage sensors at tubular-sarcoplasmic reticulum junctions, and the unit signal is the Ca(2+) spark, caused by concerted opening of multiple RyR channels. Mammalian muscles instead lose postnatally the ability to produce sparks, and they also lose RyR3, an isoform abundant in spark-producing skeletal muscles. What does it take for cells to respond to membrane depolarization with Ca(2+) sparks? To answer this question we made skeletal muscles of adult mice expressing exogenous RyR3, demonstrated as immunoreactivity at triad junctions. These muscles showed abundant sparks upon depolarization. Sparks produced thusly were found to amplify the response to depolarization in a manner characteristic of Ca(2+)-induced Ca(2+) release processes. The amplification was particularly effective in responses to brief depolarizations, as in action potentials. We also induced expression of exogenous RyR1 or yellow fluorescent protein-tagged RyR1 in muscles of adult mice. In these, tag fluorescence was present at triad junctions. RyR1-transfected muscle lacked voltage-operated sparks. Therefore, the voltage-operated sparks phenotype is specific to the RyR3 isoform. Because RyR3 does not contact voltage sensors, their opening was probably activated by Ca(2+), secondarily to Ca(2+) release through junctional RyR1. Physiologically voltage-controlled Ca(2+) sparks thus require a voltage sensor, a master junctional RyR1 channel that provides trigger Ca(2+), and a slave parajunctional RyR3 cohort.     

Asynchronous Ca(2+) waves in intact venous smooth muscle

The rabbit inferior vena cava (IVC) is a large-capacitance vessel that displays typical contractile dose-response curves for caffeine and phenylephrine (PE). Using confocal microscopy on the endothelium-denuded IVC, we undertook experiments to correlate these whole-tissue contractile dose-response curves with changes in subcellular [Ca(2+)](i) signals in the in situ vascular smooth muscle cells (VSMCs). We observed that both caffeine and PE initially elicited Ca(2+) waves in individual VSMCs. The [Ca(2+)](i) in cells challenged with caffeine subsequently returned to baseline whereas the [Ca(2+)](i) in cells challenged with PE exhibited repetitive asynchronous Ca(2+) waves. These [Ca(2+)](i) oscillations were related to Ca(2+) release from the sarcoplasmic reticulum as they were inhibited by ryanodine and caffeine. The lack of synchronicity of the [Ca(2+)](i) oscillations between VSMCs can explain the observed tonic contraction at the whole-tissue level. The nature of these Ca(2+) waves was further characterized. For caffeine, the amplitude was all-or-none in nature, with individual cells differing in sensitivity, leading to their recruitment at different concentrations of the agonist. This concentration dependency of recruitment appears to form the basis for the concentration dependency of caffeine-induced contraction. Furthermore, the speed of the Ca(2+) waves correlated positively with the concentration of caffeine. In the case of PE, we observed the same characteristics with respect to wave speed, amplitude, and recruitment. Increasing concentrations of PE also enhance the frequency of the [Ca(2+)](i) oscillations. We therefore conclude that PE stimulates whole-tissue contractility through differential recruitment of VSMCs and enhancement of the frequency of asynchronous [Ca(2+)](i) oscillations once the cells are recruited.     

Angiotensin II-induced Ca(2+) influx in renal afferent and efferent arterioles: differing roles of voltage-gated and store-operated Ca(2+) entry

Angiotensin II (Ang II)-induced Ca(2+) signaling was studied in isolated rat renal arterioles using fura-2. Ang II (10 nmol/L) caused a sustained elevation in [Ca(2+)](i), which was dependent on [Ca(2+)](o) in both vessel types. This response was blocked by nifedipine in only the afferent arteriole. Using the Mn(2+) quench technique, we found that Ang II stimulates Ca(2+) influx in both vessels. Nifedipine blocked the Ang II-induced Ca(2+) influx in afferent arterioles but not in efferent arterioles. In contrast to Ang II, KCl-induced depolarization stimulated Ca(2+) influx in only the afferent arteriole. Cyclopiazonic acid (CPA, 30 micromol/L) was used to examine the presence of store-operated Ca(2+) entry in myocytes isolated from each arteriole. In efferent myocytes, CPA induced a sustained Ca(2+) increase that was dependent on [Ca(2+)](o) and insensitive to nifedipine. This mechanism was absent in afferent myocytes. SKF 96365 inhibited Ang II-induced Ca(2+) entry in efferent arterioles and CPA-induced Ca(2+) entry in efferent myocytes over identical concentrations. Our findings thus indicate that Ang II activates differing Ca(2+) influx mechanisms in pre- and postglomerular arterioles. In the afferent arteriole, Ang II activates dihydropyridine-sensitive L-type Ca(2+) channels, presumably by membrane depolarization. In the efferent arteriole, Ang II appears to stimulate Ca(2+) entry via store-operated Ca(2+) influx.     

Scorpion toxins targeted against the sarcoplasmic reticulum Ca(2+)-release channel of skeletal and cardiac muscle.

We report the purification of two peptides, called "imperatoxin inhibitor" and "imperatoxin activator," from the venom of the scorpion Pandinus imperator targeted against ryanodine receptor Ca(2+)-release channels. Imperatoxin inhibitor has a M(r) of approximately 10,500, inhibits [3H]ryanodine binding to skeletal and cardiac sarcoplasmic reticulum with an ED50 of approximately 10 nM, and blocks openings of skeletal and cardiac Ca(2+)-release channels incorporated into planar bilayers. In whole-cell recordings of cardiac myocytes, imperatoxin inhibitor decreased twitch amplitude and intracellular Ca2+ transients, suggesting a selective blockade of Ca2+ release from the sarcoplasmic reticulum. Imperatoxin activator has a M(r) of approximately 8700, stimulates [3H]ryanodine binding in skeletal but not cardiac sarcoplasmic reticulum with an ED50 of approximately 6 nM, and activates skeletal but not cardiac Ca(2+)-release channels. These ligands may serve to selectively "turn on" or "turn off" ryanodine receptors in fragmented systems and whole cells.     

Reduced expression of sarcalumenin and related Ca2+ -regulatory proteins in aged rat skeletal muscle

In skeletal muscle, Ca(2+)-cycling through the sarcoplasm regulates the excitation-contraction-relaxation cycle. Since uncoupling between sarcolemmal excitation and fibre contraction may play a key role in the functional decline of aged muscle, this study has evaluated the expression levels of key Ca(2+)-handling proteins in senescent preparations using immunoblotting and confocal microscopy. Sarcalumenin, a major luminal Ca(2+)-binding protein that mediates ion shuttling in the longitudinal sarcoplasmic reticulum, was found to be greatly reduced in aged rat tibialis anterior, gastrocnemius and soleus muscle as compared to adult specimens. Minor sarcolemmal components of Ca(2+)-extrusion, such as the surface Ca(2+)-ATPase and the Na(+)-Ca(2+)-exchanger, were also diminished in senescent fibres. No major changes were observed for calsequestrin, sarcoplasmic reticulum Ca(2+)-ATPase and the ryanodine receptor Ca(2+)-release channel. In contrast, the age-dependent reduction in the alpha(1S)-subunit of the dihydropryridine receptor was confirmed. Hence, this report has shown that downstream from the well-established defect in coupling between the t-tubular voltage sensor and the junctional Ca(2+)-release channel complex, additional age-related alterations exist in the expression of essential Ca(2+)-handling proteins. This may trigger abnormal luminal Ca(2+)-buffering and/or decreased plasmalemmal Ca(2+)-removal, which could exacerbate impaired signaling or disturbed intracellular ion balance in aged fibres, thereby causing contractile weakness.     

Ca(2+)-induced Ca(2+) release in the pancreatic beta-cell: direct evidence of endoplasmic reticulum Ca(2+) release

The role of the Ca(2+)-induced Ca(2+) release channel (ryanodine receptor) in MIN6 pancreatic beta-cells was investigated. An endoplasmic reticulum (ER)-targeted "cameleon" was used to report lumenal free Ca(2+). Depolarization of MIN6 cells with KCl led to release of Ca(2+) from the ER. This ER Ca(2+) release was mimicked by treatment with the ryanodine receptor agonists caffeine and 4-chloro-m-cresol, reversed by voltage-gated Ca(2+) channel antagonists and blocked by treatment with antagonistic concentrations of ryanodine. The depolarization-induced rise in cytoplasmic Ca(2+) was also inhibited by ryanodine, which did not alter voltage-gated Ca(2+) channel activation. Both ER and cytoplasmic Ca(2+) changes induced by depolarization occurred in a dose-dependent manner. Glucose caused a delayed rise in cytoplasmic Ca(2+) but no detectable change in ER Ca(2+). Carbamyl choline caused ER Ca(2+) release, a response that was not altered by ryanodine. Taken together, these results provide strong evidence that Ca(2+)-induced Ca(2+) release augments cytoplasmic Ca(2+) signals in pancreatic beta-cells.     

Calpain (Ca(2+)-dependent thiol protease) in erythrocytes of young and old individuals.

Limited proteolysis by calpain (Ca(2+)-activated protease; EC 3.4.22.17) is believed to regulate the function of membrane enzymes and modify the behavior of membrane structural proteins. Calpain is activated by autolysis. The degradation of band 3 protein by mu-calpain is known to be enhanced in erythrocyte membranes from human individuals > 70 years old (old) as compared with that from individuals 20-30 years old (young). In the present study, monoclonal antibody to mu-calpain was used to study the behavior of calpain in erythrocytes of young and old individuals. Less calpain was found in erythrocyte cytosol and membranes from old than in those from young. Increasing the erythrocyte Ca2+ induced translocation of calpain to the cell membrane and autolysis of the enzyme. Alkylation of erythrocyte thiols also promoted translocation of calpain to the membrane, especially in the presence of Ca2+. When calpain was added to erythrocyte membranes, initial binding was greater and subsequent autolysis faster in old than in young individuals, possibly arising from alterations in cell membranes of old individuals. The enhanced calpain autolysis was accompanied by enhanced degradation of band 3 protein in the old. The results suggest that calpain in old individuals is translocated to the cell membrane and is activated by autolysis, resulting in degradation of certain membrane proteins and loss of calpain. Enhanced calpain-induced membrane proteolysis may play a role in abnormal cell destruction (e.g., shortening the life span of erythrocytes in the aged, neuronal degeneration, etc). The erythrocyte membrane provides a convenient model for the study of age-associated alterations in cell membranes and in calpain behavior.     

Cooperation of two-domain Ca(2+) channel fragments in triad targeting and restoration of excitation- contraction coupling in skeletal muscle

The specific incorporation of the skeletal muscle voltage-dependent Ca(2+) channel in the triad is a prerequisite of normal excitation-contraction (EC) coupling. Sequences involved in membrane expression and in targeting of Ca(2+) channels into skeletal muscle triads have been described in different regions of the alpha(1S) subunit. Here we studied the targeting properties of two-domain alpha(1S) fragments, green fluorescent protein (GFP)-I x II (1-670) and III x IV (691-1873) expressed alone or in combination in dysgenic (alpha(1S)-null) myotubes. Immunofluorescence analysis showed that GFP-I x II or III x IV expressed separately were not targeted into triads. In contrast, on coexpression the two alpha(1S) fragments were colocalized with one another and with the ryanodine receptor in the triads. Coexpression of GFP-I x II and III x IV also fully restored Ca(2+) currents and depolarization-induced Ca(2+) transients, despite the severed connection between the two channel halves and the absence of amino acids 671-690 from either alpha(1S) fragment. Thus, triad targeting, like the rescue of function, requires the cooperation and coassembly of the two complementary channel fragments. Transferring the C terminus of alpha(1S) to the N-terminal two-domain fragment (GFP-I x II x tail), or transferring the I-II connecting loop containing the beta interaction domain to the C-terminal fragment (III x IV x beta in) did not improve the targeting properties of the individually expressed two-domain channel fragments. Thus, the cooperation of GFP-I.II and III.IV in targeting cannot be explained solely by a sequential action of the beta subunit by means of the I-II loop in releasing the channel from the sarcoplasmic reticulum and of the C terminus in triad targeting.     

Ca(2+) signaling in cardiac myocytes overexpressing the alpha(1) subunit of L-type Ca(2+) channel

Voltage-gated L-type Ca(2+) channels (LCCs) provide Ca(2+) ingress into cardiac myocytes and play a key role in intracellular Ca(2+) homeostasis and excitation-contraction coupling. We investigated the effects of a constitutive increase of LCC density on Ca(2+) signaling in ventricular myocytes from 4-month-old transgenic (Tg) mice overexpressing the alpha(1) subunit of LCC in the heart. At this age, cells were somewhat hypertrophic as reflected by a 20% increase in cell capacitance relative to those from nontransgenic (Ntg) littermates. Whole cell I(Ca) density in Tg myocytes was elevated by 48% at 0 mV compared with the Ntg group. Single-channel analysis detected an increase in LCC density with similar conductance and gating properties. Although the overexpressed LCCs triggered an augmented SR Ca(2+) release, the "gain" function of EC coupling was uncompromised, and SR Ca(2+) content, diastolic cytosolic Ca(2+), and unitary properties of Ca(2+) sparks were unchanged. Importantly, the enhanced I(Ca) entry and SR Ca(2+) release were associated with an upregulation of the Na(+)-Ca(2+) exchange activity (indexed by the half decay time of caffeine-elicited Ca(2+) transient) by 27% and SR Ca(2+) recycling by approximately 35%. Western analysis detected a 53% increase in the Na(+)-Ca(2+) exchanger expression but no change in the abundance of ryanodine receptor (RyR), SERCA2, and phospholamban. Analysis of I(Ca) kinetics suggested that SR Ca(2+) release-dependent inactivation of LCCs remains intact in Tg cells. Thus, in spite of the modest cardiac hypertrophy, the overexpressed LCCs form functional coupling with RyRs, preserving both orthograde and retrograde Ca(2+) signaling between LCCs and RyRs. These results also suggest that a modest but sustained increase in Ca(2+) influx triggers a coordinated remodeling of Ca(2+) handling to maintain Ca(2+) homeostasis.     

Passive transport pathways for Ca(2+) and Co(2+) in human red blood cells. (57)Co(2+) as a tracer for Ca(2+) influx

The passive transport of calcium and cobalt and their interference were studied in human red cells using (45)Ca and (57)Co as tracers. In ATP-depleted cells, with the ATP concentration reduced to about 1μM, the progress curve for (45)Ca uptake at 1mM rapidly levels off with time, consistent with a residual Ca-pump activity building up at increasing [Ca(T)](c) to reach at [Ca(T)](c) about 5μmol(lcells)(-1) a maximal pump rate that nearly countermands the passive Ca influx, resulting in a linear net uptake at a low level. In ATP-depleted cells treated with vanadate, supposed to cause Ca-pump arrest, a residual pump activity is still present at high [Ca(T)](c). Moreover, vanadate markedly increases the passive Ca(2+) influx. The residual Ca-pump activity in ATP-depleted cells is fuelled by breakdown of the large 2,3-DPG pool, rate-limited by the sustainable ATP-turnover at about 40-50μmol(lcells)(-1)h(-1). The apparent Ca(2+) affinity of the Ca-pump appears to be markedly reduced compared to fed cells. The 2,3-DPG breakdown can be prevented by inhibition of the 2,3-DPG phosphatase by tetrathionate, and under these conditions the (45)Ca uptake is markedly increased and linear with time, with the unidirectional Ca influx at 1mM Ca(2+) estimated at 50-60μmol(lcells)(-1)h(-1). The Ca influx increases with the extracellular Ca(2+) concentration with a saturating component, with K(?(Ca)) about 0.3mM, plus a non-saturating component. From (45)Ca-loaded, ATP-depleted cells the residual Ca-pump can also be detected as a vanadate- and tetrathionate-sensitive efflux. The (45)Ca efflux is markedly accelerated by external Ca(2+), both in control cells and in the presence of vanadate or tetrathionate, suggesting efflux by carrier-mediated Ca/Ca exchange. The (57)Co uptake is similar in fed cells and in ATP-depleted cells (exposed to iodoacetamide), consistent with the notion that Co(2+) is not transported by the Ca-pump. The transporter is thus neither SH-group nor ATP or phosphorylation dependent. The (57)Co uptake shows several similarities with the (45)Ca uptake in ATP-depleted cells supplemented with tetrathionate. The uptake is linear with time, and increases with the cobalt concentration with a saturating component, with J(max) about 16μmol(lcells)(-1)h(-1) and K(?(Co)) about 0.1mM, plus a non-saturating component. The (57)Co and (45)Ca uptake shows mutual inhibition, and at least the stochastic Ca(2+) influx is inhibited by Co(2+). The (57)Co and (45)Ca uptake are both insensitive to the 1,4-dihydropyridine Ca-channel blocker nifedipine, even at 100μM. The (57)Co uptake is increased at high negative membrane potentials, indicating that the uptake is at least partially electrogenic. The (57)Co influx amounts to about half the (45)Ca influx in ATP-depleted cells. It is speculated that the basal Ca(2+) and Co(2+) uptake could be mediated by a common transporter, probably with a channel-like and a carrier-mediated component, and that (57)Co could be useful as a tracer for at least the channel-like Ca(2+) entry pathway in red cells, since it is not itself transported by the Ca-pump and, moreover, is effectively buffered in the cytosol by binding to hemoglobin, without interfering with Ca(2+) buffering. The molecular identity of the putative common transporter(s) remains to be defined.