Increased steady-state VO2 and larger O2 deficit with CO2 inhalation during exercise

Aim: To examine whether inhalation of CO₂‐enriched gas would increase steady‐state during exercise and enlarge O₂ deficit. Methods: Ten physically active men ( 53.7 ± 3.6 mL min^?1 kg^?1; ± SD) performed transitions from low‐load cycling (baseline; 40 W) to work rates representing light (≈ 45%; 122 ± 15 W) and heavy (≈ 80%; 253 ± 29 W) exercise while inhaling normal air (air) or a CO₂ mixture (4.2% CO₂, 21% O₂, balance N₂). Gas exchange was measured with Douglas bag technique at baseline and at min 0–2, 2–3 and 5–6. Results: Inhalation of CO₂‐enriched air consistently induced respiratory acidosis with increases in PCO₂ and decreases in capillary blood pH (P < 0.01). Hypercapnic steady‐state was on average about 6% greater (P < 0.01) than with air in both light and heavy exercise, presumably because of increased cost of breathing (ΔVE 40–50 L min^?1; P < 0.01), and a substrate shift towards increased lipid oxidation (decline in R 0.12; P < 0.01). during the first 2 min of exercise were not significantly different whereas the increase in from min 2–3 to min 5–6 in heavy exercise was larger with CO₂ than with air suggesting a greater slow component. As a result, O₂ deficit was greater with hypercapnia in heavy exercise (2.24 ± 0.51 L vs. 1.91 ± 0.45 L; P < 0.05) but not in light (0.64 ± 0.21 L vs. 0.54 ± 0.20 L; ns). Conclusion: Inhalation of CO₂‐enriched air and the ensuing respiratory acidosis increase steady‐state in both light and heavy exercise and enlarges O₂ deficit in heavy exercise.     

Vacuolar H+-ATPase expression is increased in acid-secreting intercalated cells in kidneys of rats with hypercalcaemia-induced alkalosis

Aims: Hypercalcaemia is known to be associated with systemic metabolic alkalosis, although the underlying mechanism is uncertain. Therefore, we aimed to examine whether hypercalcaemia was associated with changes in the expression of acid–base transporters in the kidney. Methods: Rats were infused with human parathyroid hormone (PTH, 15 μg kg^?1 day^?1), or vehicle for 48 h using osmotic minipumps. Results: The rats treated with PTH developed hypercalcaemia and exhibited metabolic alkalosis (arterial HCO: 31.1 ± 0.8 vs. 28.1 ± 0.8 mmol L^?1 in controls, P < 0.05, n = 6), whereas the urine pH of 6.85 ± 0.1 was significantly decreased compared with the pH of 7.38 ± 0.1 in controls (P < 0.05, n = 12). The observed alkalosis was associated with a significantly increased expression of the B1‐subunit of the H⁺‐ATPase in kidney inner medulla (IM, 233 ± 45% of the control level). In contrast, electroneutral Na⁺‐HCO cotransporter NBCn1 and Cl^?/HCO anion exchanger AE2 expression was markedly reduced in the inner stripe of the outer medulla (to 26 ± 9% and 65 ± 6%, respectively). These findings were verified by immunohistochemistry. Conclusions: (1) hypercalcaemia‐induced metabolic alkalosis was associated with increased urinary excretion of H⁺; (2) the increased H⁺‐ATPase expression in IM may partly explain the enhanced urinary acidification, which is speculated to prevent stone formation because of hypercalciuria and (3) the decreased expression of outer medullary AE2 suggests a compensatory reduction of the transepithelial bicarbonate transport.     

Non‐linear dependence of interstitial fluid pressure on joint cavity pressure and implications for interstitial resistance in rabbit knee

Aims: Synovium retains lubricating fluid in the joint cavity. Synovial outflow resistance estimated as dP_j/d (P_j, joint fluid pressure and trans‐synovial flow) is greater, however, than expected from interstitial glycosaminoglycan concentration. This study investigates whether subsynovial fluid pressure increases with intra‐articular pressure, as this would reduce the estimated resistance estimate. Methods: Interstitial fluid pressure (P_if) was measured as a function of distance from the joint cavity in knees of anaesthetized rabbits, using servo‐null pressure‐measuring micropipettes and using an external ‘window’. Joint fluid pressure P_j was either endogenous (?2.4 ± 0.4 cmH₂O, mean ± SEM) or held at ～4, 8 or 15.0 cmH₂O by a continuous intra‐articular saline infusion that matched the trans‐synovial interstitial drainage rate. Results: At endogenous P_j the peri‐articular P_if was subatmospheric (?1.9 ± 0.3 cmH₂O, n = 19). At raised P_j the P_if values became positive. Gradient dP_if /dx was ～20 times steeper across synovium than subsynovium. P_if close to the synovium–subsynovium border () increased as a non‐linear function of P_j to 1.4 ± 0.2 cmH₂O (n = 23) at P_j = 4.3 ± 0.1 cmH₂O : 2.3 ± 0.2 cmH₂O (n = 17) at P_j = 7.6 ± 0.2 cmH₂O: and 3.0 ± 0.4 cmH₂O (n = 26) at P_j = 15 ± 0.2 cmH₂O (P = 0.03, anova ). Conclusions: Synovial resistivity is ～20× subsynovial resistivity. The increase in with P_j means that true synovial resistance d()/d is overestimated 1.5× by dP_j/d. This narrows but does not eliminate the gap between analysed glycosaminoglycan concentration, 4 mg ml^?1, and the net interstitial biopolymer concentration of 11.5 mg ml^?1 needed to generate the resistance.     

Effects of aerobic fitness on hypohydration-induced physiological strain and exercise impairment

Aim: Hypohydration exacerbates cardiovascular and thermal strain and can impair exercise capacity in temperate and warm conditions. Yet, athletes often dehydrate in exercise, are hypervolaemic and have less cardiovascular sensitivity to acute hypervolaemia. We tested the hypothesis that trained individuals have less cardiovascular, thermoregulatory and performance affect of hypohydration during exercise. Methods: After familiarization, six trained [O_{2 peak} = 64 (SD 8) mL kg⁻¹ min⁻¹] and six untrained [O_{2 peak} = 45 (4) mL kg⁻¹ min⁻¹] males cycled 40 min at 70%O_{2 peak} while euhydrated or hypohydrated by 1.5–2.0% body mass (crossover design), before a 40-min work trial with euhydration or ad libitum drinking (in Hypohydration trial), in temperate conditions (24.3 °C, RH 50%, v_a = 4.5 m s⁻¹). Baseline hydration was by complete or partial rehydration from exercise+heat stress the previous evening. Results: During constant workload, heart rate and its drift were increased in Hypohydration compared with Euhydration for Untrained [drift: 33 (11) vs. 24 beats min⁻¹ h⁻¹ (10), 95% CI 5–11] but not Trained [14 (3) vs. 13 beats min⁻¹ h⁻¹ (3), CI −2 to 3; P = 0.01 vs. Untrained]. Similarly, rectal temperature drift was faster in Hypohydration for Untrained only [by 0.57 °C h⁻¹ (0.25); P = 0.03 vs. Trained], concomitant with their reduced sweat rate (P = 0.05) and its relation to plasma osmolality (P = 0.03). Performance power tended to be reduced for Untrained (−13%, CI −35 to 2) and Trained (−7%, CI: −16 to 1), without an effect of fitness (P = 0.38). Conclusion: Mild hypohydration exacerbated cardiovascular and thermoregulatory strain and tended to impair endurance performance, but aerobic fitness attenuated the physiological effects.     

Identification of three isoforms of the InsP 3 receptor in human myometrial smooth muscle

The mechanisms responsible for the mobilisation of Ca²⁺ from intracellular stores sensitive to inositol trisphosphate (InsP ₃) were studied in saponin-permeabilised human myometrial cells in which the sarcoplasmic reticulum was pre-loaded with⁴⁵Ca²⁺. InsP ₃-induced⁴⁵Ca²⁺ release was measured over the InsP ₃ concentration range of 100 nM to 100 M and showed a graded response. InsP ₃-induced⁴⁵Ca²⁺ release was inhibited by heparin (20–40 g/ml) but not significantly affected by caffeine. The Ca²⁺ sensitivity of InsP ₃-induced Ca²⁺ release was measured under conditions which were designed to exclude interference with Ca²⁺ released by the ryanodine receptor/channel complex. The data showed a bell-shaped relationship with the InsP ₃ receptor (InsP ₃R) functional at 10 nM, becoming maximally activated at 300 nM but inhibited at 10 M Ca²⁺. Messenger RNA encoding for three isoforms of InsP ₃R, type I, II and type III, was shown to be present. The relative expression levels of these messengers were obtained by ratio-PCR analysis and the levels of expression of the different isoforms were found to differ between individual patients.     

Inorganic phosphate transport in matrix vesicles from bovine articular cartilage

Aims: In mineralizing tissues such as growth plate cartilage extracellular organelles derived from the chondrocyte membrane are present. These matrix vesicles (MV), possess membrane transporters that accumulate Ca²⁺ and inorganic phosphate (P_i), and initiate the formation of hydroxyapatite crystals. MV are also present in articular cartilage, and hydroxyapatite crystals are believed to promote cartilage degradation in osteoarthritic joints. This study characterizes P_i transport in MV derived from articular cartilage. Methods: Matrix vesicles were harvested from collagenase digests of bovine articular cartilage by serial centrifugation. P_i uptake by MV was measured using radioactive phosphate (³³[P]HPO). The Na⁺ dependence, pH sensitivity and effects of P_i analogues that inhibit P_i transport were determined. Results: P_i uptake was temperature‐sensitive and comprised Na⁺‐dependent and Na⁺‐independent components. The Na⁺‐dependent component saturated at high extracellular P_i concentrations, with a K_m of 0.16 mm . In Na⁺‐free solutions, uptake did not fully saturate implying that carrier‐mediated uptake is supplemented by a diffusive pathway. Uptake was inhibited by phosphonoacetate and arsenate, although a fraction of Na⁺‐independent P_i uptake persisted. Total P_i uptake was maximal at pH 6.5, and reduced at more acidic or alkaline values, representing inhibition of both components. Conclusion: These properties are highly similar to those of P_i uptake by chondrocytes, suggesting that MV inherit P_i transporters of the chondrocyte membrane from which they are derived. Na⁺‐independent P_i uptake has not previously been described in MV from growth plate cartilage and is relatively uncharacterized, but warrants further attention in articular cartilage, given its likely role in initiating inappropriate mineral formation.     

Endothelin‐1‐induced proliferation of human endothelial cells depends on activation of K+ channels and Ca2+ influx

Aims: Endothelin‐1 (ET‐1) promotes endothelial cell growth. Endothelial cell proliferation involves the activation of Ca²⁺‐activated K⁺ channels. In this study, we investigated whether Ca²⁺‐activated K⁺ channels with big conductance (BK_Ca) contribute to endothelial cell proliferation induced by ET‐1. Methods: The patch‐clamp technique was used to analyse BK_Ca activity in endothelial cells derived from human umbilical cord veins (HUVEC). Endothelial proliferation was examined using cell counts and measuring [³H]‐thymidine incorporation. Changes of intracellular Ca²⁺ levels were examined using fura‐2 fluorescence imaging. Results: Characteristic BK_Ca were identified in cultured HUVEC. Continuous perfusion of HUVEC with 10 nmol L^?1 ET‐1 caused a significant increase of BK_Ca open‐state probability (n = 14; P < 0.05; cell‐attached patches). The ET_B‐receptor antagonist (BQ‐788, 1 μmol L^?1) blocked this effect. Stimulation with Et‐1 (10 nmol L^?1) significantly increased cell growth by 69% (n = 12; P < 0.05). In contrast, the combination of ET‐1 (10 nmol L^?1) and the highly specific BK_Ca blocker iberiotoxin (IBX; 100 nmol L^?1) did not cause a significant increase in endothelial cell growth. Ca²⁺ dependency of ET‐1‐induced proliferation was tested using the intracellular Ca²⁺‐chelator BAPTA (10 μmol L^?1). BAPTA abolished ET‐1 induced proliferation (n = 12; P < 0.01). In addition, ET‐1‐induced HUVEC growth was significantly reduced, if cells were kept in a Ca²⁺‐reduced solution (0.3 mmol L^?1), or by the application of 2 aminoethoxdiphenyl borate (100 μmol L^?1) which blocks hyperpolarization‐induced Ca²⁺ entry (n = 12; P < 0.05). Conclusion: Activation of BK_Ca by ET‐1 requires ET_B‐receptor activation and induces a capacitative Ca²⁺ influx which plays an important role in ET‐1‐mediated endothelial cell proliferation.     

Coupling of a voltage‐gated Ca2+ channel homologue with a plasma membrane H+‐ATPase in yeast

Yeast has a homologue of mammalian voltage‐gated Ca²⁺ channels (VGCCs), enabling the efficient uptake of Ca²⁺. It comprises two indispensable subunits, Cch1 and Mid1, equivalent to the mammalian pore‐forming α₁ and auxiliary α₂/δ subunits, respectively. Unlike the physiological roles of Cch1/Mid1 channels, the regulatory mechanisms of the yeast VGCC homologue remain unclear. Therefore, we screened candidate proteins that interact with Mid1 by an unbiased proteomic approach and identified a plasma membrane H⁺‐ATPase, Pma1, as a candidate. Mid1 coimmunoprecipitated with Pma1, and Mid1‐EGFP colocalized with Pma1‐mCherry at the plasma membrane. The physiological relevance of their interaction was determined using the temperature‐sensitive mutant, pma1‐10. At the nonpermissive temperature, the membrane potential was less negative and Ca²⁺ uptake was lower in pma1‐10 than in wild‐type cells. Increased extracellular H⁺ increased the rate of Ca²⁺ uptake. Therefore, H⁺ extrusion by Pma1 may be important for Ca²⁺ influx through Cch1/Mid1. These results suggest that Pma1 interacts physically with Cch1/Mid1 Ca²⁺ channels to enhance their activity via its H⁺‐pumping activity.     

Prolonged exercise reduces Ca2+ release in rat skeletal muscle sarcoplasmic reticulum

Prolonged exercise decreased the rate of Ca⁺ release in sarcoplasmic reticulum (SR) vesicles isolated from rat muscle by 20–30% when release was initiated by 5, 10, and 20 M AgNO₃. [³H]Ryanodine binding was also depressed by 20% in SR vesicles isolated from the exercised animals. In contrast, the maximum amount of Ca²⁺ released by Ag⁺ remained unaffected by exercise. The passive permeability of SR vesicles and the rate of Ca²⁺ release in the presence of ruthenium red, a known inhibitor of the Ca²⁺ release mechanism, was not affected by prolonged exercise. These results suggest that exercise depressed Ca²⁺ release from SR by directly modifying the Ca²⁺ release channel. Current address: Department of Physics, Portland State University, Portland, OR 97207, USA     

Contraction-induced changes in skeletal muscle Na+,K+ pump mRNA expression – importance of exercise intensity and Ca2+-mediated signalling

Aim: To investigate if exercise intensity and Ca²⁺ signalling regulate Na⁺,K⁺ pump mRNA expression in skeletal muscle. Methods: The importance of exercise intensity was evaluated by having trained and untrained humans perform intense intermittent and prolonged exercise. The importance of Ca²⁺ signalling was investigated by electrical stimulation of rat soleus and extensor digitorum longus (EDL) muscles in combination with studies of cell cultures. Results: Intermittent cycling exercise at ∼85% of VO_2peak increased (P < 0.05) α1 and β1 mRNA expression ∼2-fold in untrained and trained subjects. In trained subjects, intermittent exercise at ∼70% of VO_2peak resulted in a less (P < 0.05) pronounced increase (∼1.4-fold; P < 0.05) for α1 and no change in β1 mRNA. Prolonged low intensity exercise increased (P < 0.05) mRNA expression of α1 ∼3.0-fold and α2 ∼1.8-fold in untrained but not in trained subjects. Electrical stimulation of rat soleus, but not EDL, muscle increased (P < 0.05) α1 mRNA expression, but not when combined with KN62 and cyclosporin A incubation. Ionomycin incubation of cultured primary rat skeletal muscle cells increased (P < 0.05) α1 and reduced (P < 0.001) α2 mRNA expression and these responses were abolished (P < 0.05) by co-incubation with cyclosporin A or KN62. Conclusion: (1) Exercise-induced increases in Na⁺,K⁺ pump α1 and β1 mRNA expression in trained subjects are more pronounced after high- than after moderate- and low-intensity exercise. (2) Both prolonged low and short-duration high-intensity exercise increase α1 mRNA expression in untrained subjects. (3) Ca²⁺_i regulates α1 mRNA expression in oxidative muscles via Ca²⁺/calmodulin-dependent protein kinase (CaMK) and calcineurin signalling pathways.     

The apparent rate constant for the dissociation of force generating myosin crossbridges from actin decreases during Ca2+ activation of skinned muscle fibres

Summary The effect of Ca²⁺ activation on the apparent rate constant governing the dissociation of force generating myosin cross-bridges was studied in skinned rabbit adductor magnus fibres (fast-twitch) at 21±1 °C. Simultaneous measurements of Ca²⁺-activated isometric force and ATPase activity were conducted in parallel with simultaneous measurements of DANZ-labelled troponin C (TnC_DANZ) fluorescence and isometric force in fibres whose endogenous troponin C had been partially replaced with TnC_DANZ. The Ca²⁺ activation of isometric force occurred at approximately two times higher Ca²⁺ concentration than did actomyosin ATPase activity at 2.0 mM MgATP. Since increases in both TnC_DANZ fluorescence and ATPase activity occurred over approximately the same Ca²⁺ concentration range at substantially lower concentrations of Ca²⁺ than did force, this data suggests that the TnC_DANZ fluorescence is associated with the Ca²⁺ activation of myosin crossbridge turnover (ATPase) rather than force. According to the model of Huxley (1957) and assuming the hydrolysis of one molecule of ATP per cycle of the crossbridge, the apparent rate constantg _app for the dissociation of force generating myosin crossbridges is proportional to the actomyosin ATPase/isometric force ratio. This measure ofg _app shows approximately a fivefold decrease during Ca²⁺ activation of isometric force. This change ing _app is responsible for separation of the Ca²⁺ sensitivity of the normalized ATPase activity and isometric force curves. If the MgATP concentration is reduced to 0.5 mM, the change ing _app is reduced and consequently the difference in Ca²⁺ sensitivity between normalized steady state ATPase and force is also reduced.The abbreviations used are TnC troponin C - DANZ 5-dimethylaminonapthalene-2-sulphonyl aziridine - TnC_DANZ DANZ-labelled TnC - a the number of half sarcomeres - A the cross-sectional area of the fibre - AP₅A P¹,P⁵-di(adenosine-5)pentaphosphate - EGTA ethyleneglycolbis-(betaaminoethyl ether)-N,N,N,N-tetraacetic acid - F force a muscle fibre develops - f_app apparent rate of formation of force generating myosin crossbridges - F_av the average force per myosin head - Fs steady-state fraction of cycling myosin crossbridges in the force generating state - g _app apparent rate of dissociation of force generating myosin crossbridges - L_1/2s the length of a half sarcomere - LDH lactate dehydrogenase - [M] is the concentration of myosin per fibre volume - NAD nicotinamide adenine dinucleotide - NADH reduced form of NAD - pCa -log₁₀ of the free Ca²⁺ concentration - PEP phosphenol pyruvate - PK pyruvate kinase     

Regulation and possible physiological role of the Ca2-dependent K+ channel of cortical collecting ducts of the rat

In the luminal membrane of rat cortical collecting duct (CCD) a big Ca²⁺-dependent and a small Ca²⁺-independent K⁺ channel have been described. Whereas the latter most likely is responsible for the K⁺ secretion in this nephron segment, the function of the large-conductance K⁺ channel is unknown. The regulation of this channel and its possible physiological role were examined with the conventional cell-free and the cell-attached nystatin patch-clamp techniques. Patch-clamp recordings were obtained from the luminal membrane of isolated perfused CCD segments and from freshly isolated CCD cells. Intracellular calcium was measured using the calcium-sensitive dye fura-2. The large-conductance K⁺ channel was strongly voltage- and calcium-dependent. At 3 mol/l cytosolic Ca²⁺ activity it was half-maximally activated. At 1 mmol/l it was neither regulated by cytosolic pH nor by ATP. At 1 mol/l Ca²⁺ activity the open probability (P _o) of this channel was pH-dependent. At pH 7.0 P _o was decreased to 4±2% (n=9) and at pH 8.5 it was increased to 425±52% (n=9) of the control. At this low Ca²⁺ activity the P _o of the channel was reduced by 1 mmol/l ATP to 8±4% (n=6). Cell swelling activated the large-conductance K⁺ channel (n=14) and hyperpolarized the membrane potential of the cells by 9±1 mV (n=23). Intracellular Ca²⁺ activity increased after hypotonic stress. This increase depended on the extracellular Ca²⁺ activity. A possible physiological function of the large-conductance K⁺ channel in rat CCD cells may be the reduction of the intracellular K⁺ concentration after cell swelling. Once this channel is activated by increases in the cytosolic Ca²⁺ activity it can be regulated by changes in cellular pH and ATP.Supported by DFG Schl 277/2-3     

Expression and function of Na+/Ca2+ exchangers 1 and 3 in human macrophages and monocytes

The Na⁺/Ca²⁺ exchanger (NCX) is a membrane transporter that can switch Na⁺ and Ca²⁺ in either direction to maintain the homeostasis of intracellular Ca²⁺. Three isoforms (NCX1, NCX2, and NCX3) have been characterized in excitable cells, e.g. neurons and muscle cells. We examined the expression of these NCX isoforms in primary human lung macrophages (HLM) and blood monocytes. NCX1 and NCX3, but not NCX2, are expressed in HLM and monocytes at both mRNA and protein levels. Na⁺‐free medium induced a significant increase in intracellular calcium concentration ([Ca²⁺]_i) in both cell types. This response was completely abolished by the NCX inhibitor 5‐(N‐4‐chlorobenzyl)‐20,40‐dimethylbenzamil (CB‐DMB). Moreover, inhibition of NCX activity during Ca²⁺‐signaling induced by histamine caused a delay in restoring baseline [Ca²⁺]_i. Na⁺‐free medium induced TNF‐α expression and release in HLM comparable to that caused by LPS. TNF‐α release induced by Na⁺‐free medium was blocked by CB‐DMB and greatly reduced by RNAi‐mediated knockdown of NCX1. These results indicate that human macrophages and monocytes express NCX1 and NCX3 that operate in a bidirectional manner to restore [Ca²⁺]_i, to generate Ca²⁺‐signals, and to induce TNF‐α production. Therefore, NCX may contribute to regulate Ca²⁺ homeostasis and proinflammatory functions in human macrophages and monocytes.     

Histamine-evoked Ca2+ oscillations in HeLa cells are sensitive to methylxanthines but insensitive to ryanodine

The relative contribution of inositol-trisphosphate(InsP ₃)-sensitive and InsP ₃-insensitive Ca²⁺ stores to the agonist-evoked oscillatory release of Ca²⁺ in HeLa cells was investigated using fura-2 cytosolic Ca²⁺ measurements and whole-cell recordings of Ca²⁺-activated K⁺ currents [K(Ca²⁺)]. The experimental approach chosen consisted in studying the effects on Ca²⁺ oscillations of a variety of pharmacological agents such as ryanodine, ruthenium red, caffeine and theophylline, which are known to affect the Ca²⁺ channels responsible for Ca²⁺-induced Ca²⁺ release (CICR) in excitable cells. The results obtained essentially indicate (a) that neither ryanodine nor ruthenium red affects the generation of periodic K(Ca²⁺) current pulses in whole-cell experiments, and (b) that histamine-induced Ca²⁺ oscillations are inhibited by caffeine and theophylline in a dose-dependent manner. However, these methylxanthines were unable, at concentrations ranging from 0.1 mM to 10 mM, either to mobilize Ca²⁺ from internal stores or to block the initial Ca²⁺ rise evoked by histamine. In addition, both methylxanthines showed at high concentrations (10–20 mM) a moderate inhibitory action on the production of InsP ₃ induced by histamine. This effect was not essential to the action of caffeine on the oscillatory release of Ca²⁺, since an inhibition by caffeine of InsP ₃-induced Ca²⁺ oscillations was still observed in whole-cell experiments where the InsP ₃ concentration was kept constant. The results also show (c) that the application of either caffeine or theophylline during histamine stimulation leads systematically to an increased Ca²⁺ sequestration in InsP ₃-sensitive Ca²⁺ pools, the effect observed with theophylline being stronger than that resulting from the application of caffeine, and finally (d) that the action of caffeine and theophylline is not related to an increase in cAMP concentration since neither forskolin (10–50 M) nor 8-Br-cAMP (1 mM) caused an inhibition of the InsP ₃-induced Ca²⁺ oscillations. It is concluded on the basis of these results that the agonist-evoked Ca²⁺ oscillations in HeLa cells do not involve directly or indirectly a ryanodine-sensitive Ca²⁺-release channel with CICR properties, but rather arise from a control by Ca²⁺ of the InsP ₃ Ca²⁺-release process.     

Acetylcholine induces Ca2+ oscillations via m3/m4 muscarinic receptors in the mouse oocyte

Changes in intracellular Ca²⁺ concentration are required for the activation of mammalian oocytes. They are caused mainly by Ca²⁺ release from the endoplasmic reticulum (ER) via InsP ₃ receptors (InsP ₃R). Several studies have reported that acetylcholine (ACh) is capable of triggering early activation events in mouse oocytes over-expressed with the m1 muscarinic ACh receptor (m1AChR). Here we examined which subtypes of the mAChR (m1 to m4) are involved in the generation of Ca²⁺ oscillations in native mouse oocytes. ACh (10 M) elicited regular Ca²⁺ oscillations similar to those induced by sperm in their temporal characteristics. The Ca²⁺ oscillations were abolished by application with atropine, the mAChR inhibitor. Within 1 min after treatment of ACh, intracellular Fluo-3 fluorescence intensity increased from 794±119 to 2023±755 (increase to 250% of original value), indicating a strong rise of cytosolic Ca²⁺ concentration. 4-DAMP mustard and Tropicamide, specific antagonists of m3AChR and m4AChR, completely abolished ACh-induced Ca²⁺ oscillations. In the ovulated oocytes, the expression of m3/m4 AChR was clearly detected by RT-PCR analysis. Furthermore, ACh-induced Ca²⁺ oscillations were also abolished or decreased by PLC inhibitors (U73122 or D609) and an InsP ₃-receptor antagonist (xestospongin C), confirming that ACh generates Ca²⁺ oscillations via the PLC-InsP ₃ (PI) pathway. These results strongly suggest that m3/m4AChR is coupled to the generation of Ca²⁺ oscillations mainly via the PI pathway in mouse oocytes.     

Contribution of systemic arterial compliance and systemic vascular resistance to effective arterial elastance changes during exercise in humans

Background: Effective arterial elastance (Ea), an index of arterial load, increases with elevations in left ventricular elastance to maximize the efficiency of left ventricular stroke work during exercise. Systemic arterial compliance (C) and vascular resistance (R) are the primary components contributing to Ea, and R plays a greater role in determining Ea at rest. We hypothesized that the contribution of C to Ea increases during exercise to maintain an optimal balance between arterial load and ventricular elastance, and that the increase in Ea is due primarily to a reduction in C. Aim: The aim of this study was to investigate the contributions of C and R to Ea during exercise. Methods: Ea (0.9 × systolic blood pressure/stroke volume), C (stroke volume/pulse pressure), R (mean blood pressure/cardiac output), and cardiac cycle length (T) were measured at rest and during exercise of 40%, 60% and 80% maximal oxygen uptake (O_2max) using Doppler echocardiography in 45 healthy men. Results: Ea did not differ between rest and 40%O_2max, but it was greater at 60% and 80%O_2max. C markedly decreased during exercise in an exercise intensity‐dependent manner. The changes in R/T during exercise were small, whereas it decreased at 40%O_2max and gradually increased at 60% and 80%O_2max. Conclusions: The present results suggest that the contribution of systemic arterial compliance to effective arterial elastance increases during exercise. Therefore, we propose that the increase in arterial load during exercise is mainly driven by a reduction in systemic arterial compliance.     

Ion transport in human skeletal muscle cells: disturbances in myotonic dystrophy and Brody's disease

After excitation of skeletal muscle, the disturbed ion homeostasis is restored by Na⁺, K⁺ ATPase of the sarcolemma and Ca²⁺ ATPase of the sarcoplasmic reticulum (SR). Contrary to Na⁺, K⁺ ATPase, the concentration and isoenzyme distribution of SR Ca²⁺ ATPase in human skeletal muscle depend on fibre type and age. In cultured human muscle cells the concentration and activity of Na⁺, K⁺ ATPase and SR Ca²⁺ ATPase increase with maturation. In skeletal muscle and cultured muscle cells of patients suffering from myotonic dystrophy (MyD), the activity and the concentration of both Na⁺, K⁺ ATPase and SR Ca²⁺ ATPase are decreased by about 40%. In addition, we measured in cultured MyD muscle cells at rest an increased cytosolic Ca²⁺ concentration ([Ca²⁺]_i) caused by active voltage-operated Ca²⁺ channels, which are inactive in resting control cells. However, the restoration of a stimulus-induced Ca²⁺ transient is unaffected. A differentiation-related disturbance of membranes or a modulation defect of membrane proteins may play a role in MyD. In skeletal muscle and cultured muscle cells of patients suffering from Brody's disease, which is characterized by impaired muscle relaxation, the SR Ca²⁺ ATPase activity is reduced by about 50%, but the concentrations of total SR Ca²⁺ ATPase and the predominant SERCA1 isoform are normal. Diseased muscle cells show a delayed restoration of [Ca²⁺]_i after stimulation, which might be explained by structural modifications of SERCA1. Reduction of the Ca²⁺ release by drugs balances the excitation–relaxation cycle of the pathological cells.     

Inositol trisphosphate enhances Ca2+ oscillations but not Ca2+-induced Ca2+ release from cardiac sarcoplasmic reticulum

The role of inositol 1,4,5-trisphosphate [Ins(1,4,5)P ₃] in excitation-contraction coupling in cardiac muscle is still unclear, although many laboratories are beginning to assume a critical role for this putative second messenger. Earlier studies from this laboratory [Nosek et al. (1986) Am J Physiol 250:C807] found that Ins(1,4,5)P ₃ enhanced spontaneous Ca²⁺ release and the caffeine sensitivity of Ca²⁺ release from myocardial sarcoplasmic reticulum (SR) and proposed an increase in the Ca²⁺ sensitivity of the release as a possible mechanism. In order to clarify the phyisological relevance of these actions of Ins(1,4,5)P ₃ and specifically to test the effect of Ins(1,4,5)P ₃ on the Ca²⁺ sensitivity of Ca²⁺ release, we compared the effects of Ins(1,4,5)P ₃ on Ca²⁺ oscillations and on Ca²⁺-induced Ca²⁺ release (CICR) from the SR in saponin-skinned rat papillary muscle. We found that: (a) 30 M Ins(1,4,5)P ₃ enhanced the Ca²⁺ oscillations (measured by tension oscillations) from the rat cardiac SR, consistent with the previous report on guinea pig tissue; (b) both GTP and GTP[S] enhanced Ca²⁺ oscillations. The effect was not additive to that of Ins(1,4,5)P ₃ indicating that two different Ca²⁺-release pools do not exist in cardiac SR; (c) 30 M Ins(1,4,5)P ₃ had no effect on the Ca²⁺ sensitivity of CICR; (d) Ins(1,4,5)P ₃ (up to 30 M) had no effect on SR Ca²⁺ loading. The studies were performed in the presence of Cd²⁺ or 2,3-bisphosphoglycerate, agents that inhibit Ins(1,4,5)P ₃ hydrolysis. These results suggest that: (a) two different mechanisms underlie Ca²⁺ oscillations and CICR, Ins(1,4,5)P ₃ influencing Ca²⁺ oscillations but not CICR; (b) Ins(1,4,5)P ₃ does not increase the Ca²⁺ sensitivity of Ca²⁺ release from the SR; (c) cardiac muscle is different from smooth muscle where Ca²⁺ release from the SR is dependent upon GTP; (d) the physiological role of Ins(1,4,5)P ₃ in excitation-contraction coupling in cardiac muscle is minimal. In contrast, Ins(1,4,5)P ₃ may play a pathological role in cardiac arrhythmogenesis by enhancing spontaneous Ca²⁺ ocsillations.     

Effects of different types of K+ channel modulators on the spontaneous myogenic contraction of guinea‐pig urinary bladder smooth muscle

In the present study, effects of different types of K⁺ channel modulators on the spontaneous rhythmic contractile activity were examined in guinea‐pig urinary bladder smooth muscle (UBSM). Guinea‐pig UBSM exhibited myogenic rhythmic contraction in the presence of atropine (1 μM ), phentolamine (1 μM ), propranolol (1 μM ), suramin (10 μM ) and tetrodotoxin (1 μM ). Nisoldipine (100 nM ) or diltiazem (10 μM ) substantially diminished UBSM contractile activity. Nisoldipine‐resistant component of UBSM rhythmic contraction was further inhibited by gadolinium (200 μM ). Iberiotoxin (50 nM ), a selective blocker of large‐conductance, voltage‐gated Ca²⁺‐activated K⁺ (K_Ca) (BK) channel, dramatically increased both contraction amplitude and frequency whereas NS‐1619 (30 μM ), which increases BK channel activity, decreased them. Apamin (100 nM ), a selective blocker of small‐conductance, K_Ca (SK) channel, increased contraction amplitude but decreased frequency. A blocker of voltage‐gated K⁺ (K_v) channel, 4‐aminopyridine (100 μM ), significantly increased contraction frequency. E‐4031, a blocker of a novel inwardly rectifying K⁺ channel, i.e. the human ether‐a‐go‐go‐related gene (HERG) K⁺ channel, significantly increased contraction amplitude. Glibenclamide (1–10 μM ) (K_ATP channel blocker) and Ba²⁺ (10 μM ) (conventional K_ir channel blocker) did not exhibit conspicuous effects on spontaneous contractile activity of UBSM. These findings imply that two types of K_Ca (BK and SK) channels have prominent roles as negative feedback elements to limit extracellular Ca²⁺ influx‐mediated guinea‐pig UBSM contraction by regulating both amplitude and frequency. It was also suggested that both non‐K_Ca type of K⁺ (K_v and HERG‐like K⁺) channels may contribute to the regulation of UBSM myogenic rhythmic contraction.