Ca-EGTA affects the relationship between [Ca2+] and tension in α-toxin permeabilized rat anococcygeus smooth muscle

Summary The relationship between calcium concentration ([Ca²⁺]) and force in smooth muscle can be studied by permeabilizing the sarcolemma and bathing the preparation in a mock intracellular solution. Normally [Ca²⁺] is set in these solutions using the Ca²⁺ chelator EGTA in the concentration range of 4–10 mm. This study shows that lowering total EGTA concentration ([EGTA]_t) below 10 mm depresses Ca²⁺-activated force generated in 0.1 m Ca²⁺. The observed threshold for the effect of EGTA_t is 0.2 mm, and the effect is maximal at approximately 10 mm. BAPTA, another Ca²⁺ chelator, also produces this effect. Tension production in smooth muscle is controlled by acto-myosin interaction. This in turn is mediated by the relative activities of myosin light chain kinase (MLCK) and phosphatase (MLCP). Inhibiting MLCP with Microcystin LR (10 m), an increase [EGTA_t] from 0.2 mm to 10 mm still enhanced force. This suggests that EGTA promotes phosphorylation of myosin by the activation of MLCK and not by inhibition of MLCP.     

Cross-bridge movement and stiffness during the rise of tension in skeletal muscle – a theoretical analysis

Predictions for the time courses of cross-bridge attachment, N(t), stiffness, S(t), and force, T(t), during the tetanus rise were analysed for a special class of cross-bridge models where cross-bridges initially attach in a non-stereospecific weak-binding state, A _w. This state is in rapid equilibrium (equilibrium constant K) with detached states and the force generating transition (rate constant F ₊) is delayed. One model (model IA) which assumed step-function rise of activation at onset of tetanus, gave a poor fit to the experimental data (judged by root mean square error, RMSe 0.038) but the experimentally observed lead of N(t) over T(t) was reproduced qualitatively. An activation mechanism where K increased towards its maximum value according to an exponential function (Model IB) improved the fit considerably (RMSe 0.013). However, the activation time constant ( = 30 ms) derived in the fit was too high to reflect Ca²⁺ binding to troponin. In a further developed model (model II) both Ca²⁺-binding to troponin and cross-bridge attachment were assumed to be required for full activation. This more complex model gave a good fit to the experimental data (RMSe 0.013) with a realistic time constant for Ca²⁺ binding to troponin (9 ms). In both model IB and model II the best fit was obtained with F ₊ 40 s^–1 . An extended version of model IB, with distributed cross-bridge attachment and a series elastic element, gave a fit of similar quality (RMSe 0.009) as obtained with model IB and model II and with a similar value of F₊. The results support the view that weakly bound cross-bridges (state A _w) may account for the lead of cross-bridge movement over force during tension rise. It is also shown that, if the stiffness of the myofilaments is non-linear (stiffness increasing with tension) the experimentally observed lead of S(t) over T(t) may, to a significant degree, be attributed to cross-bridges in the state A _w.     

Effects of the PKA inhibitor H-89 on excitation–contraction coupling in skinned and intact skeletal muscle fibres

This study investigated the effects of the protein kinase A (PKA) inhibitor, H-89, in mechanically-skinned muscle fibres and intact muscle fibres, in order to determine whether PKA phosphorylation is essential for normal excitation–contraction (E–C) coupling. In skinned EDL fibres of the rat, force responses to depolarization (by ion substitution) were inhibited only slightly by 10M H-89, a concentration more than sufficient to fully inhibit PKA. Staurosporine (1 M), a potent non-specific kinase inhibitor, also had little if any effect on depolarization-induced responses. At 1–2 M, H-89 significantly slowed the repriming rate in rat skinned fibres, most likely due to it deleteriously affecting the T-system potential. With 100 M H-89, the force response to depolarization by ion substitution was completely abolished. This inhibitory effect was reversed by washout of H-89 and was not due to block of the Ca²⁺ release channel in the sarcoplasmic reticulum (SR). In intact single fibres of the flexor digitorum longus (FDB) muscle of the mouse, 1–3 M H-89 had no noticeable effect on action-potential-mediated Ca²⁺ transients. Higher concentrations (4–10 M) caused Ca²⁺ transient failure in fibres stimulated at 20 Hz in a manner indicative of action-potential failure. At 10–100 M, H-89 also inhibited net Ca²⁺ uptake by the SR and affected the Ca²⁺-sensitivity of the contractile apparatus in rat skinned fibres. All such effects were proportionately greater in toad muscle fibres. These results do not support the hypothesis that phosphorylation is essential for the Ca²⁺ release channel to open in response to voltage-sensor activation in skeletal muscle fibres.     

Intracellular β2-adrenergic receptor signaling specificity in mouse skeletal muscle in response to single-dose β2-agonist clenbuterol treatment and acute exercise

The aim of this study was to clarify the intracellular β₂-adrenergic receptor signaling specificity in mouse slow-twitch soleus and fast-twitch tibialis anterior (TA) muscles, resulting from single-dose β₂-agonist clenbuterol treatment and acute exercise. At 1, 4, and 24 h after single-dose treatment with clenbuterol or after acute running exercise, the soleus and TA muscles were isolated and subjected to analysis. The phosphorylation of p38 mitogen-activated protein kinase (MAPK) increased after single-dose clenbuterol treatment and acute exercise in the soleus muscle but not in the TA muscle. Although there was no change in the phosphorylation of Akt after acute exercise in either muscle, phosphorylation of Akt in the soleus muscle increased after single-dose clenbuterol treatment, whereas that in the TA muscle remained unchanged. These results suggest that p38 MAPK and Akt pathways play a functional role in the adaptation to clenbuterol treatment and exercise, particularly in slow-twitch muscles.     

Effects of clenbuterol and ICI118551, a selective β2-antagonist,on the growth of skeletal muscle of suckling rats

The ₂-adrenergic agonist, clenbuterol, was administered to lactating rats (4 mg/kg diet) from post-partum day 1 to day 19, or directly injected into neonate rats (0.1 and 1.0 mg/kg body weight) from post-partum day 3 until day 15. Changes in body weight and the skeletal muscles soleus (SOL) and extensor digitorum longus (EDL) were studied in both dams and suckling offspring. Drug treatment consistently increased body weight in dams whilst significantly reducing the growth of their suckling pups. In dams treated with clenbuterol (4 mg/kg of diet) muscle weights and protein contents were significantly increased. Total protein content increased by 16% in SOL and 47% in EDL after 19 days of treatment. In contrast, in their suckling pups, there was a 22% and 26% reduction in protein content of SOL and EDL respectively. Administration of the ₂-antagonist ICI1 18551 to these pups failed to prevent these reductions in body and muscle weights. Hence, if clenbuterol did reach the pups via the milk from treated mothers it did not act via conventional ₂-receptors. Injection of pups with clenbuterol (1.0 mg/kg every 12 h) from litters suckling from untreated dams also resulted in significant reductions in muscle weights and protein contents. Protein content was reduced by 10% in SOL and 13% in EDL after 12 days of treatment. No alteration in fibre type proportions in SOL or EDL resulted from this treatment. Further work is required to determine whether the growth suppression in the two situations occurs via the same mechanism.     

The rise of [Na+]i during ischemia and reperfusion in the rat heart—underlying mechanisms

Intracellular Na⁺ concentration ([Na⁺]_i) rises in the heart during ischemia, and on reperfusion, there is a transient rise followed by a return toward control. These changes in [Na⁺]_i contribute to ischemic and reperfusion damage through their effects on Ca²⁺ overload. Part of the rise of [Na⁺]_i during ischemia may be caused by increased activity of the cardiac Na⁺/H⁺ exchanger (NHE1), activated by the ischemic rise in [H⁺]_i. In support of this view, NHE1 inhibitors reduce the [Na⁺]_i rise during ischemia. Another possibility is that the rise of [Na⁺]_i during ischemia is caused by Na⁺ influx through channels. We have reexamined these issues by use of two different NHE1 inhibitors, amiloride, and zoniporide, in addition to tetrodotoxin (TTX), which blocks voltage-sensitive Na⁺ channels. All three drugs produced cardioprotection after ischemia, but amiloride (100 μM) and TTX (300 nM) prevented the rise in [Na⁺]_i during ischemia, whereas zoniporide (100 nM) did not. Both amiloride and zoniporide prevented the rise of [Na⁺]_i on reperfusion, whereas TTX was without effect. In an attempt to explain these differences, we measured the ability of the three drugs to block Na⁺ currents. At the concentrations used, TTX reduced the transient Na⁺ current (I _Na) by 11 ± 2% while amiloride and zoniporide were without effect. In contrast, TTX largely eliminated the persistent Na⁺ current (I _Na,P) and amiloride was equally effective, whereas zoniporide had a substantially smaller effect reducing I _Na,P to 41 ± 8%. These results suggest that part of the effect of NHE1 inhibitors on the [Na⁺]_i during ischemia is by blockade of I _Na,P. The fact that a low concentration of TTX eliminated the rise of [Na⁺]_i during ischemia suggests that I _Na,P is a major source of Na⁺ influx in this model of ischemia.     

E–C coupling and contractile characteristics of mechanically skinned single fibres from young rats during rapid growth and maturation

The postnatal growth of rats involves a developmental phase (0 to approximately 3 weeks), a rapid growth phase ( approximately 3 to approximately 10 weeks), and a slower maturation phase ( approximately 10 weeks+). In this study, we investigated the age-related changes in excitation-contraction (E-C) coupling characteristics of mammalian skeletal muscle, during rapid growth (4-10 weeks) and maturation (10-21 weeks) phases, using single, mechanically skinned fibres from rat extensor digitorum longus (EDL) muscle. Fibres from rats aged 4 and 8 weeks produced lower maximum T-system depolarization-induced force responses and fewer T-system depolarization-induced force responses to 75% run-down than those produced by fibres from rats aged 10 weeks and older. The sensitivity of the contractile apparatus to Ca(2+) in fibres from 4-week rats was significantly higher than that in fibres from 10-week rats; however, the maximum Ca(2+)-activated force per skinned fibre cross-sectional area (specific force) developed by fibres from 4-week rats was on average approximately 44% lower than the values obtained for all the other age groups. In agreement with the age difference in specific force, the MHC content of EDL muscles from 4-week rats was approximately 29% lower than that of 10-week rats. Thus, mechanically skinned fibres from rats undergoing rapid growth are less responsive to T-system depolarization and maximal Ca(2+) activation than fibres from rats at the later stage of maturation or adult rats. These results suggest that during the rapid growth phase in rats, the structure and function of elements involved in E-C coupling in fast-twitch skeletal muscle continue to undergo significant changes.     

Long-term depolarization regulates the α1s subunit of skeletal muscle Ca2+ channels and the amplitude of L-type Ca2+ currents

The effects of long-term depolarization on the level of alpha1s and on L-type Ca2+ currents of skeletal muscle were investigated. Long-term depolarization (14 h) caused a 50% decrease of alpha1s, revealed with the Western blot technique. This decline was prevented by preincubation with the Ca2+ channel blocker nifedipine. Electrophysiological experiments using the voltage-clamp technique were performed to measure the actions of long-term depolarization on Ca2+ currents and charge movement. A progressive decline in the amplitude of the Ca2+ currents by depolarizations lasting 0.5-14 h was observed. Similar to Western blot results, the fall in current amplitude was prevented by nifedipine, and it depended on external Ca2+. The nonlinear charge mobilized by step pulses was also significantly reduced (50%) by long-term depolarization. It is suggested that alpha1s subunit is down-regulated by long-term depolarization by a very stringent mechanism and that, in this process, Ca2+ ions permeating through L-type channels play a key role. A new role for the L-type Ca2+ current in skeletal muscle fibers in which the channels are self-regulated is proposed.     

The effect of 4 weeks β-alanine supplementation and isokinetic training on carnosine concentrations in type I and II human skeletal muscle fibres

Seven male students were supplemented with β-alanine (β-ALG) for 4 weeks (6.4 g day⁻¹) and seven with a matching placebo (PLG). Subjects undertook 4 weeks of isokinetic training with the right leg (T) whilst the left leg was untrained (UT), serving as a control. Each training session consisted of 10 × 10 maximal 90° extension and flexion contractions at 180°/s using a Kin-Com isokinetic dynamometer, with 1 min rest between bouts. Muscle biopsies were taken from the vastus lateralis immediately before and at the end of the supplementation period. Following freeze drying muscle fibres were dissected and characterised by their MHC profile, as type I, IIa, IIx, or as hybrids of these. Carnosine was measured by HPLC. There was a significant increase in carnosine in both T and UT legs of the β-ALG (9.63 ± 3.92 mmol kg⁻¹ dry muscle and 6.55 ± 2.36 mmol kg⁻¹ dry muscle respectively). There was a significant increase in the carnosine content of all fibre phentotypes, with no significant difference between types. There were no significant differences in the changes in muscle or in fibres between the T and UT legs. In contrast there was no significant change in the carnosine content in either the T or UT legs with placebo. The results indicate that 4 weeks training has no effect on the muscle carnosine content. Whilst an increase was seen with β-alanine supplementation, this was not further influenced by training. These findings suggest that β-alanine availability is the main factor regulating muscle carnosine synthesis.     

Increased Ca2+ sensitivity and protein expression of SERCA 2a in situations of chronic β3-adrenoceptor deficiency

This study investigated the influence of chronic β₃-adrenoceptor deficiency on myocardial function. Therefore, we investigated Ca²⁺-regulatory proteins, SERCA 2a activity, and myofibrillar and mitochondrial function in hearts of wild-type (WT, n=7) and β₃-adrenoceptor knockout mice (β₃-KNO, n=7). Morphometric heart analysis showed no difference between WT and β₃-KNO. No alterations were observed for the protein expression of the ryanodine receptor or phospholamban. However, in β₃-KNO mice, protein expression of SERCA 2a and phospholamban phosphorylation were significantly increased. These changes were accompanied by an increased SERCA 2a activity in β₃-KNO. Alterations in phospholamban phosphorylation were independent of alterations in β₁/β₂-adrenoceptor distribution and protein expression of G proteins in β₃-KNO. Measurement of myofibrillar Ca²⁺ sensitivity showed no difference in the Ca²⁺/force relation for WT and β₃-KNO. The same seems to hold true for mitochondrial function since the protein expressions of cytochrome c, uncoupling protein 3 and cytochrome c oxidase subunit IV were similar in WT and β₃-KNO. The conclusion is that depression of β₃-adrenergic stimulation may modulate the protein expression of SERCA 2a and phospholamban phosphorylation, thereby improving sarcoplasmic reticulum Ca²⁺ uptake. Thus, β₃-adrenergic depression may be a therapeutic aim in situations of impaired SERCA 2a activity, e.g. for the treatment of heart failure.     

Changes in muscle size, architecture, and neural activation after 20 days of bed rest with and without resistance exercise

Nine healthy men carried out head-down bed rest (BR) for 20 days. Five subjects (TR) performed isometric, bilateral leg extension exercise every day, while the other four (NT) did not. Before and after BR, maximal isometric knee extension force was measured. Neural activation was assessed using a supramaximal twitch interpolated over voluntary contraction. From a series cross-sectional magnetic resonance imaging scans of the thigh, physiological cross-sectional areas (PCSA) of the quadriceps muscles were estimated (uncorrected PCSA, volume/estimated fibre length). Decrease in mean muscle force after BR was greater in NT [−10.9 (SD 6.9)%, P < 0.05] than in TR [0.5 (SD 7.9)%, not significant]. Neural activation did not differ between the two groups before BR, but after BR NT showed smaller activation levels. Pennation angles of the vastus lateralis muscle, determined by ultrasonography, showed no significant changes in either group. The PCSA decreased in NT by −7.8 (SD 0.8)% (P < 0.05) while in TR PCSA showed only an insignificant tendency to decrease [−3.8 (SD 3.8)%]. Changes in force were related more to changes in neural activation levels than to those in PCSA. The results suggest that reduction of muscle strength by BR is affected by a decreased ability to activate motor units, and that the exercise used in the present experiment is effective as a countermeasure. Accepted: 18 September 2000     

Activation of an apical Cl– conductance by extracellular ATP in Necturus gallbladder is mediated by cAMP and not by [Ca2+]i

Necturus gallbladder epithelium (NGE) expresses a CFTR-like apical Cl- conductance that can be activated by cAMP. Here, we show that extracellular ATP (100 microM), which is known to elevate intracellular Ca2+ and to hyperpolarize cells by stimulating apical and basolateral K+ conductances, also stimulates an apical Cl- conductance (Ga,Cl), however with a much slower time course. The selectivity sequence of Ga,Cl was SCN- > I- > NO3- > Br- > Cl- > isethionate (ISE-), but SCN- and I- partially blocked it, which is analogous to observations of CFTR Cl- channels. To disclose a possible role for intracellular Ca2+, gallbladders were incubated with the Ca2+ chelator BAPTA/AM or bathed in solutions containing only submicromolar Ca2+ concentrations. BAPTA partially inhibited the Ca(2+)-mediated hyperpolarization, but did not reduce the ATP-dependent activation of Ga,Cl and the latter was also seen in low extracellular Ca2+. On the other hand, the cAMP-antagonist Rp-8-Br-cAMPS strongly inhibited the stimulation of Ga,Cl by ATP (as well as by forskolin), but left the ATP-induced hyperpolarization unchanged. Preincubation with a low concentration of forskolin markedly enhanced the stimulatory effect of ATP, and this effect was not modified by the selective inhibition of protein kinase C. These data suggest the involvement of different signal transduction pathways in the ATP-dependent activation of K+ and Cl- conductances in NGE. The stimulation of the Ga,Cl appears to be mediated by cAMP but not by elevation of intracellular Ca2+.     

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.     

Absence of regulation of the T-type calcium current by Cav1.1, β1a and γ1 dihydropyridine receptor subunits in skeletal muscle cells

The subunit structure of low voltage activated T-type Ca²⁺ channels is still unknown. Co-expression of dihydropyridine receptor (DHPR) auxiliary subunits with T-type α₁ subunits in heterologous systems has produced conflicting results. In developing foetal skeletal muscle fibres which abundantly express DHPR subunits, Ca_v3.2 (α_1H) subunits are believed to underlie T-type calcium currents which disappear 2 to 3 weeks after birth. Therefore, a possible regulation of foetal skeletal muscle T-type Ca²⁺ channels by DHPR subunits was investigated in freshly isolated foetal skeletal muscle using knockout mice, which provide a powerful tool to address this question. The possible involvement of α_1S (Ca_v1.1), β₁ and γ₁ DHPR subunits was tested using dysgenic (α_1S-null), β_1a and γ₁ knockout mice. The results show that the absence of α_1S, β₁ or γ₁ DHPR subunits does not significantly affect the electrophysiological properties of T-type Ca²⁺ currents in skeletal muscle, suggesting that (1) native Ca_v3.2 is not regulated by β₁ or γ₁ DHPR subunits; (2) T-type and L-type currents have distinct and not interchangeable roles.     

Distribution of cGMP-dependent and cGMP-independent Ca2+-activated Cl− conductances in smooth muscle cells from different vascular beds and colon

In the present patch-clamp study we have, for the first time, shown the tissue distribution of a recently characterized cGMP-dependent Ca²⁺-activated Cl^– conductance [18] in smooth muscle cells freshly isolated from different regions: aorta, pulmonary artery, tail artery, femoral artery, femoral vein, middle cerebral artery, renal artery, portal vein, superior mesenteric artery, mesenteric small artery and colon. The cGMP-dependent Cl^– conductance has properties distinct from those of the classical Ca²⁺-activated Cl^– conductances; their different sensitivities to niflumic acid and zinc were here utilized to distinguish them. They were found to be co-expressed in different patterns in smooth muscle cells of different origins. The cGMP-dependent conductance was greater in myocytes from cerebral artery and femoral vein and was greater in the renal artery, aorta, mesenteric small artery, femoral artery and the superior mesenteric artery. The presence of the cGMP-dependent Ca²⁺-activated Cl^– current in smooth muscle cells isolated from the colon demonstrates that this conductance is not limited to the vasculature. The classical Ca²⁺-activated Cl^– conductance was strongly expressed in smooth muscle cells from the portal vein and the tail artery, and noticeably higher in the pulmonary artery.     

Differential effects of a green tea-derived polyphenol (−)-epigallocatechin-3-gallate on the acidosis-induced decrease in the Ca2+ sensitivity of cardiac and skeletal muscle

(-)-Epigallocatechin-3-gallate (EGCg), a green tea-derived polyphenol, has received much attention as a protective agent against cardiovascular diseases. In this study, we determined its effects on the acidosis-induced change in the Ca(2+) sensitivity of myofilaments in myofibrils prepared from porcine ventricular myocardium and chicken pectoral muscle. EGCg (0.1 mM) significantly inhibited the decrease caused by lowering the pH from 7.0 to 6.0 in the Ca(2+) sensitivity of myofibrillar ATPase activity in cardiac muscle, but not in skeletal muscle. Studies on recombinant mouse cardiac troponin C (cTnC) and chicken fast skeletal troponin C (sTnC) using circular dichroism and intrinsic and extrinsic fluorescence spectroscopy showed that EGCg bound to cTnC with a dissociation constant of approximately 3-4 muM, but did not bind to sTnC. By presumably binding to the cTnC C-lobe, EGCg decreased Ca(2+) binding to cTnC and overcame the depressant effect of protons on the Ca(2+) sensitivity of the cardiac contractile response. To demonstrate isoform-specific effects of the action of EGCg, the pH sensitivity of the Ca(2+) response was examined in cardiac myofibrils in which endogenous cTnC was replaced with exogenous sTnC or cTnC and in skeletal myofibrils in which the endogenous sTn complex was replaced with whole cardiac Tn complex (cTn). The results suggest that the binding of EGCg to the cardiac isoform-specific TnC or Tn complex alters the effect of pH on myofilament Ca(2+) sensitivity in striated muscle.     

Adrenoceptor-mediated regulation of myofibrillar Ca2+ sensitivity through the GTP-binding protein-related mechanisms: tension recording in β-escin-skinned single rat cardiac cells with preserved receptor functions

 To investigate the mechanisms of receptor-mediated regulation of heart muscle contraction, we developed a tension-recording system using β-escin-skinned single cardiac cells of rats and studied the effects of agonists on myofibrillar Ca²⁺ sensitivity and Ca²⁺ release from the sarcoplasmic reticulum (SR). In pCa/tension relations, 1 μM isoproterenol plus 100 μM guanosine 5′-triphosphate (GTP) decreased the myofibrillar Ca²⁺ sensitivity (pCa₅₀, the [Ca²⁺] required for half-maximal tension, as an indicator of the sensitivity; from 6.07 to 5.92); this effect was blocked by 1 μM metoprolol or 1 mM guanosine 5′-O-(2-thiodiphosphate) (GDPβS). Phenylephrine (10 μM) plus 100 μM GTP increased the Ca²⁺ sensitivity (pCa₅₀; from 6.12 to 6.28), and this effect was blocked by 1 μM phentolamine or 1 mM GDPβS. After Ca²⁺ loading into the SR, 10 μM phenylephrine plus 100 μM GTP in a low-ethylene- glycol-bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA, 0.1 mM) relaxing solution induced oscillatory contractions that were attenuated by either 1 μM phentolamine or pre-treatment with 10 μM inositol 1,4,5-trisphosphate. Our results demonstrate that β₁-adrenergic stimulation decreases myofibrillar Ca²⁺ sensitivity and that α₁-adrenergic stimulation both increases the Ca²⁺ sensitivity and activates Ca²⁺ release from the agonist-sensitive SR through GTP-binding protein-related mechanisms. Received: 17 August 1998 / Received after revision: 23 October 1998 / Accepted: 15 December 1998     

Anti-inflammatory activity of the extracts from Rodgersia podophylla leaves through activation of Nrf2/HO-1 pathway,and inhibition of NF-κB and MAPKs pathway in mouse macrophage cells

Inflammation Research - Recently, Rodgersia podophylla has been reported to exhibit anti-inflammatory activity. However, little is known about the potential mechanisms about its anti-inflammatory...     

Direct effects of tolbutamide on mitochondrial function, intracellular Ca2+ and exocytosis in pancreatic β-cells

 Using the whole-cell voltage-clamp method to measure ATP-sensitive K⁺(K_ATP) currents, changes in cell capacitance to measure secretion and microfluorimetry to monitor intracellular Ca²⁺ and mitochondrial function, we have investigated the direct effect of sulphonylureas on exocytosis in pancreatic β-cells. Tolbutamide (100 μM) and 100 nM 4-β-12-phorbolmyristate-13-acetate (PMA), which activates the protein kinase C (PKC) isoforms found in β-cells, potentiated exocytosis in a non-additive manner. These effects were blocked by down-regulation of PKC. Our data support the idea that tolbutamide can potentiate secretion from β-cells via a PKC-dependent pathway. Because PKC and sulphonylureas can modulate the activity of K_ATP channels, we explored whether the above effects are caused by inhibition of this channel. PMA increased whole-cell K_ATP currents but did not affect their sensitivity to tolbutamide. Down-regulation of PKC affected neither the magnitude nor the tolbutamide sensitivity of the K_ATP current. Both tolbutamide and the mitochondrial uncoupler FCCP (1 μM) mobilized intracellular Ca²⁺ and prolonged Ca²⁺ transients elicited by cholinergic mobilization of intracellular Ca²⁺ stores. Tolbutamide (0.1–0.5 mM), like FCCP, depolarized the mitochondrial membrane potential and activated K_ATP currents. We suggest that sulphonylureas can directly potentiate exocytosis by impairing mitochondrial function and Ca²⁺ handling, which ultimately leads to activation of Ca²⁺-dependent enzymes such as PKC. Received: 1 September 1998 / Received after revision: 9 November 1998 / Accepted: 10 November 1998