Ca2+ uptake by cardiac sarcoplasmic reticulum ATPase in situ strongly depends on bound creatine kinase

The role of creatine kinase (CK) bound to sarcoplasmic reticulum (SR), in the energy supply of SR ATPase in situ, was studied in saponin-permeabilised rat ventricular fibres by loading SR at pCa 6.5 for different times and under different energy supply conditions. Release of Ca²⁺ was induced by 5 mM caffeine and the peak of relative tension (T/T _max) and the area under isometric tension curves, S _T, were measured. Taking advantage of close localisation of myofibrils and SR, free [Ca²⁺] in the fibres during the release was estimated using steady state [Ca²⁺]/tension relationship. Peak [Ca²⁺] and integral of free Ca²⁺ transients (S[Ca²⁺]_f) were then calculated. At all times, loading with 0.25 mM adenosine diphosphate, Mg²⁺ salt (MgADP) and 12 mM phosphocreatine (PCr) [when adenosine triphosphate (ATP) was generated via bound CK] was as efficient as loading with both 3.16 mM MgATP and 12 mM PCr (control conditions). However, when loading was supported by MgATP alone (3.16 mM), T/T _max was only 40% and S[Ca²⁺]_f 31% of control (P < 0.001). Under these conditions, addition of a soluble ATP-regenerating system (pyruvate kinase and phosphoenolpyruvate), did not increase loading substantially. Both S _T and S[Ca²⁺]_f were more sensitive to the loading conditions than T/T _max and peak [Ca²⁺]. The data suggest that Ca²⁺ uptake by the SR in situ depends on local ATP/ADP ratio which is effectively controlled by bound CK. Received: 23 January 1996/Received after revision: 19 April 1996/Accepted: 3 May 1996     

Spontaneous tension oscillation in skinned bovine cardiac muscle

 Skinned fibres from bovine ventricles exhibited spontaneous tension oscillations when MgADP and inorganic phosphate (Pi) were added to the solution bathing fibres in the relaxed state (ADP-SPOC). A similar type of oscillation was observed at intermediate concentrations of free Ca²⁺ in the absence of MgADP and Pi (Ca-SPOC). To investigate the correlation between ADP-SPOC and Ca-SPOC, we constructed two-dimensional state diagrams of cardiac muscle using different concentrations of Pi (0–20 mM) and free Ca²⁺ [pCa=around 5 (+Ca²⁺), pCa=5.15–6.9 and +EGTA (–Ca²⁺)], with varying concentrations of MgADP (0–10 mM), with 2 mM MgATP and 2 mM free Mg²⁺ maintaining ionic strength at 0.15±0.01 M, pH 7.0, 25 °C. The three-dimensional (pCa-Pi-MgADP) state diagram thus obtained was divided into three regions, i.e. the contraction region in which tension oscillation was undetectable, the spontaneous tension oscillation (SPOC) region and the relaxation region. We found that the regions of ADP-SPOC and Ca-SPOC were continuously connected by a single oscillation region sandwiched between the contraction and relaxation regions. The state diagram, which encompasses physiological conditions, shows that the probability of SPOC is higher in cardiac muscle than in skeletal muscle. From these results, we suggest that, despite distinct ionic conditions, the molecular state of cross-bridges during SPOC is common to both ADP-SPOC and Ca-SPOC. Received 19 February 1996 / Received after revision: 16 July 1996 / Accepted: 14 August 1996     

Effect of alkalinization of cytosolic pH by amines on intracellular Ca2+ activity in HT29 cells

The effect of secondary, tertiary and quaternary methyl- and ethylamines on intracellular pH (pH_i) and intracellular Ca²⁺ activity ([Ca²⁺]_i) of HT₂₉ cells was investigated microspectrofluorimetrically using pH- and Ca²⁺- sensitive fluorescent indicators, [i.e. 2′,7′-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) and fura-2 respectively]. Membrane voltage (V _m) was studied by the patch-clamp technique. Secondary and tertiary amines led to a rapid and stable concentration-dependent alkalinization which was independent of their pK _a value. Trimethylamine (20 mmol/l) increased pH_i by 0.78 ± 0.03 pH units (n = 9) and pH remained stable for the application time. Removal led to an undershoot of pH_i and a slow and incomplete recovery: pH_i stayed 0.26 ± 0.06 pH units more acid than the resting value. The quaternary amines, tetramethyl- and tetraethylamine were without influence on pH_i. All tested secondary and tertiary amines (dimethyl-, diethyl-, trimethyl-, and triethyl-amine) induced a [Ca²⁺]_i transient which reached a peak value within 10–25 s and then slowly declined to a [Ca²⁺]_i plateau. The initial Δ[Ca²⁺]_i induced by trimethylamine (20 mmol/l) was 160 ± 15 nmol/l (n = 17). The [Ca²⁺]_i peak was independent of the Ca²⁺ activity in the bath solution, but the [Ca²⁺]_i plateau was significantly lower under Ca²⁺-free conditions and could be immediately interrupted by application of CO₂ (10%; n = 6), a manoeuvre to acidify pH_i in HT₂₉ cells. Emptying of the carbachol- or neurotensin-sensitive intracellular Ca²⁺ stores completely abolished this [Ca²⁺]_i transient. Tetramethylamine led to higher [Ca²⁺]_i changes than the other amines tested and only this transient could be completely blocked by atropine (10⁻⁶ mol/l). Trimethylamine (20 mmol/l) hyperpolarized V _m by 22.5 ± 3.7 mV (n = 16) and increased the whole-cell conductance by 2.3 ± 0.5 nS (n = 16). We conclude that secondary and tertiary amines induce stable alkaline pH_i changes, release Ca²⁺ from intracellular, inositol-1,4,5-trisphosphate-sensitive Ca²⁺ stores and increase Ca²⁺ influx into HT₂₉ cells. The latter may be related to both the store depletion and the hyperpolarization. Received: 11 September 1995/Received after revision and accepted: 18 December 1995     

Modulation of Ca2+ channels by intracellular Mg2+ ions and GTP in frog ventricular myocytes

Under conditions of low intracellular [Mg²⁺] ([Mg²⁺]_i), achieved by dialysis with pipette solutions containing ethylenediamine tetraacetic acid (EDTA), 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) and adenosine triphosphate (ATP) as chelator, calcium currents through the L-type calcium channels (I _Ca) were increased in frog ventricular myocytes. Total suppression of phosphorylation by depleting the cell of ATP with a cocktail of β,γ-methyleneadenosine 5′-triphosphate (AMP-PCP) 2-deoxyglucose and carboxylcyanide-M-chlorophenylhydrazone (CCCP) did not inhibit the increase in I _Ca in the Mg²⁺-deficient medium. Thus, the involvement of phosphorylation process in the increase in I _Ca was not likely. Effective suppression of this enhancement of I _Ca was achieved by the application of guanosine triphosphate (GTP). From the dose-response curve for GTP, the GTP concentration required for half-maximal inhibition (IC₅₀) was estimated to be 4.0 μM at pMg 6. This GTP-induced suppression of I _Ca is not due to the guanine nucleotide binding protein (G-protein) cascade, because both activators and inhibitors of G-protein, which are structural analogues of GTP, suppressed I _Ca similarly. Treatment with pertussis toxin (PTX) did not affect the inhibitory action of Mg²⁺ and GTP on I _Ca. GTP is therefore assumed to bind directly to the Ca²⁺ channel. Interaction of Mg²⁺ and GTP with the Ca²⁺ channel activated in the Mg²⁺-deficient medium was examined by comparing the dose/response curves for GTP at two different [Mg²⁺]. The IC₅₀ for GTP suppression was estimated to be 5.7 μM at pMg 6 and 6.9 μM at pMg 5. The results suggest strongly that Mg²⁺ and GTP independently bind and control Ca²⁺ channels. Received: 22 December 1995/Received after revision and accepted: 11 March 1996     

The contribution of the sarcoplasmic reticulum Ca2+-transport ATPase to caffeine-induced Ca2+ transients of murine skinned skeletal muscle fibres

The present study was carried out to investigate the contribution of the Ca²⁺-transport ATPase of the sarcoplasmic reticulum (SR) to caffeine-induced Ca²⁺ release in skinned skeletal muscle fibres. Chemically skinned fibres of balb-C-mouse EDL (extensor digitorum longus) were exposed for 1 min to a free Ca²⁺ concentration of 0.36 μM to load the SR with Ca²⁺. Release of Ca²⁺ from the SR was induced by 30 mM caffeine and recorded as an isometric force transient. For every preparation a pCa/force relationship was constructed, where pCa = −log₁₀ [Ca²⁺]. In a new experimental approach, we used the pCa/force relationship to transform each force transient directly into a Ca²⁺ transient. The calculated Ca²⁺ transients were fitted by a double exponential function: Y ₀ + A ₁⋅exp (−t/t ₁) + A ₂⋅exp(t/t ₂), with A ₁ < 0 < A ₂, t ₁ < t ₂ and Y ₀, A ₁, A ₂ in micromolar. Ca²⁺ transients in the presence of the SR Ca²⁺-ATPase inhibitor cyclopiazonic acid (CPA) were compared to those obtained in the absence of the drug. We found that inhibition of the SR Ca²⁺-ATPase during caffeine-induced Ca²⁺ release causes an increase in the peak Ca²⁺ concentration in comparison to the control transients. Increasing CPA concentrations prolonged the time-to-peak in a dose-dependent manner, following a Hill curve with a half-maximal value of 6.5 ± 3 μM CPA and a Hill slope of 1.1 ± 0.2, saturating at 100 μM. The effects of CPA could be simulated by an extended three-compartment model representing the SR, the myofilament space and the external bathing solution. In terms of this model, the SR Ca²⁺-ATPase influences the Ca²⁺ gradient across the SR membrane in particular during the early stages of the Ca²⁺ transient, whereas the subsequent relaxation is governed by diffusional loss of Ca²⁺ into the bathing solution. Received: 2 February 1996/Accepted: 1 April 1996     

Store depletion triggers the calcium release-activated calcium current (I CRAC) in macrovascular endothelial cells: a comparison with Jurkat and embryonic kidney cell lines

 In endothelial cells, different types of Ca²⁺ conductances have been described, but none of them has been clearly identified as I _CRAC, the Ca²⁺ release-activated Ca²⁺ current originally described in mast and lymphoma cells. Here we show that in bovine pulmonary artery endothelial cells (CPAE) depletion of intracellular Ca²⁺ stores by inositol 1,4,5-trisphosphate (InsP ₃), Ca²⁺ ionophores and Ca²⁺ pump inhibitors activates a Ca²⁺-selective conductance in the presence of the Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). The current shows inward rectification, a highly positive reversal potential and is blocked by micromolar concentrations of La³⁺. The conditions used in studies of endothelial cells were also employed in those of HEK-293, an embryonic kidney cell line commonly used to express putative store-operated channels, and Jurkat cells, the reference cell model. Similar to CPAE, HEK cells also have an I _CRAC-like current. At 0 mV holding potential the estimated current density is –0.1 and –0.2 pA/pF in CPAE and HEK cells respectively, i.e. 15 and 30% of that measured in Jurkat cells. As shown in studies of Jurkat cells, larger Na⁺ currents are detectable in CPAE and HEK cells following store depletion in Ca²⁺- and Mg²⁺-free medium. The current carried by Na⁺ ions is similarly blocked by micromolar La³⁺, is inwardly rectifying and has a positive reversal potential. Received: 12 November 1997 / Received after revision: 11 February 1998 / Accepted: 12 February 1998     

Contraluminal para-aminohippurate (PAH) transport in the proximal tubule of the rat kidney

In order to study the characteristics of contraluminal para-aminohippurate transport into proximal tubular cells the stopped flow capillary perfusion method was applied. The disappearance of³H-paraaminohippurate from the capillary perfusate at different concentrations and contact times was measured and saturation type behaviour was found with aK _m of 0.08±0.01 (SE) mmol/l,J _max of 1.1±0.1 pmol·s^–1·cm^–1 andr, the final extracellular/intracellular distribution ratio of 0.93±0.03. Omission of Na⁺ from the capillary test perfusate caused a small reduction of contraluminal PAH uptake at small transport rates (0.1 mmol/l PAH in the test perfusate) but not at high transport rates (1.0 mmol/l PAH in the test perfusate). Change of K⁺ between 0 and 40 mmol/l and pH between 6.0 and 8.0 did not influence contraluminal PAH uptake. Isotonic replacement of chloride by gluconate, nitrate, sulfate, phosphate, methanesulfonate or increase in bicarbonate to 50 mmol/l did not influence PAH uptake at small transport rates. But isotonic sulfate and phosphate, as well as 50 mmol/l HCO ₃ ^– and 25 mmol/l Hepes in isotonic solutions reduced PAH uptake at high transport rates. Addition of 5 mmol/l Ca²⁺, Mg²⁺, Mn²⁺, Ba²⁺, Cd²⁺ to isotonic Na⁺-gluconate solution did not influence PAH uptake except for Mg²⁺ and Mn²⁺ which inhibited uptake at small transport rates only. Preperfusion of the peritubular capillaries with rat serum, Na⁺ gluconate (Ca²⁺-+Mg²⁺-free), Na⁺ gluconate (Ca²⁺-+Mg²⁺-free) plus 10 mmol/l lactate or pyruvate or 0.1 mmol/l 2-oxoglutarate did not influence PAH uptake at small PAH transport rates, but inhibited at high transport rates. Preperfusion of the capillaries for 10 s with Na⁺-, Ca²⁺- and Mg²⁺-free solutions reduced PAH uptake in the presence of Na⁺ at both transport rates. A second 10 s preperfusion — after the first 10 s Na⁺-, Ca²⁺-, Mg²⁺-free preperfusion — with serum or solutions which contained Na⁺ and Ca²⁺ or Mg²⁺ restored the PAH fluxes to control values. The data are compatible with the hypothesis that contraluminal PAH uptake occurs by a saturable transport mechanism in exchange for other intracellular anions rather than in cotransport with Na⁺ ions. It was, however, not possible to identify the type of counteranions involved. The large effect of cation replacement on para-aminohippurate transport, which was reported in many previous studies with kidney slices, is not a direct effect on the para-aminohippurate transporter, but is rather caused indirectly via cell metabolism and/or changed ion gradients.     

Simultaneous recording of ATP-sensitive K+ current and intracellular Ca2+ in anoxic rat ventricular myocytes. Effects of glibenclamide

We investigated the temporal relationship between the adenosine triphosphate-sensitive K current (K _ATP current), hypoxic shortening and Ca accumulation in cardiomyocytes exposed to anoxia or metabolic inhibition. Whole-cell, patch-clamp experiments were performed with nonstimulated isolated rat heart ventricular muscle cells loaded with the Ca-sensitive fluorescent dye 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2′-amino-5′-methylphenoxy) ethane-N,N,N′,N′-tetraacetic acid (fura-2) via the patch pipette. After approximately 8 min anoxia, the K _ATP current started to rise and reached a maximum of 21.3 ± 3.7 nA (n = 5, recorded at 0 mV clamp potential) within 1–3 min. At that time hypoxic contracture also occurred. Resting cytoplasmic free calcium (Ca_i) did not change significantly before hypoxic shortening. After hypoxic contracture, the K _ATP current decreased and Ca_i started to rise, reaching about 1 μmol/l. The presence of glibenclamide (10 μmol/l) in the bath reduced the anoxia-induced K _ATP current by more than 50%, but did not significantly influence the time dependence of current, hypoxic shortening and Ca_i, or the magnitude of Ca_i. Metabolic inhibition with 1.5 mmol/l CN resulted in K _ATP current increase and hypoxic shortening, occurring somewhat earlier than under anoxia, but all other parameters were comparable. In non-patch-clamped cells loaded with fura-2 AM ester and field-stimulated with 1 Hz, 1 μmol/l glibenclamide had no significant effect on the magnitude of the Ca_i increase caused by exposure of the cells to 1.5 mmol/l CN⁻. After CN⁻ wash-out in non-patch-clamped cells, Ca_i declined, oscillated and finally returned to control values. It can be concluded that glibenclamide inhibits anoxia-induced K _ATP currents only partially and has no significant effect on anoxia-induced rise in resting Ca_i. Received: 3 November 1995/Received after revision: 9 January 1996/Accepted: 16 January 1996     

Attenuation of stimulated Ca2+ influx in colonie epithelial (HT29) cells by cAMP

In HT₂₉ colonic epithelial cells agonists such as carbachol (CCH) or ATP increase cytosolic Ca²⁺ activity ([Ca²⁺]_i) in a biphasic manner. The first phase is caused by inositol 1,4,5-trisphophate-(Ins P ₃-) mediated Ca²⁺ release from their respective stores and the second plateau phase is mainly due to stimulated transmembraneous Ca²⁺ influx. The present study was undertaken to examine the effect of increased adenosine 3′,5′-cyclic monophasphate (cAMP) (forskolin 10 μmol/l = FOR) on the Ca²⁺ transient in the presence of CCH (100 μmol/l). In unpaired experiments it was found that FOR induced a depolarization and reduced cytosolic Ca²⁺ ([Ca²⁺]_i, measured as the fura-2 fluorescence ratio 340/380 nm) significantly. Dideoxyforskolin had no such effect. The effect of FOR was abolished when the cells were depolarized by a high-K⁺ solution. In further paired experiments utilizing video imaging in conjunction with whole-cell patch-clamp, [Ca²⁺]_i was monitored separately for the patch-clamped cell and three to seven neighbouring cells. In the presence of CCH, FOR reduced [Ca²⁺]_i uniformly from a fluorescence ratio (345/380) of 2.9 ± 0.12 to 1.8 ± 0.07 in the patch-clamped cell and its neighbours (n = 48) and depolarized the membrane voltage (V _m) of the patch-clamped cells significantly and reversibly from −54 ± 7.4 to −27 ± 5.9 mV (n = 6). In additional experiments V _m was depolarized by 15–54 mV by various increments in the bath K⁺ concentration. This led to corresponding reductions in [Ca²⁺]_i. Irrespective of the cause of depolarization (high K⁺ or FOR) there was a significant correlation between the change in V _m and change in [Ca²⁺]_i. These data indicate that the cAMP-mediated attenuation of Ca²⁺ influx is caused by the depolarization produced by this second messenger. Received: 12 March 1996/Accepted: 2 April 1996     

Effect of chloride on Ca2+ release from the sarcoplasmic reticulum of mechanically skinned skeletal muscle fibres

 The effect of intracellular Cl^– on Ca²⁺ release in mechanically skinned fibres of rat extensor digitorum longus (EDL) and toad iliofibularis muscles was examined under physiological conditions of myoplasmic [Mg²⁺] and [ATP] and sarcoplasmic reticulum (SR) Ca²⁺ loading. Both in rat and toad fibres, the presence of 20 mM Cl^–in the myoplasm increased Ca²⁺ leakage from the SR at pCa (i.e. –log₁₀ [Ca²⁺]) 6.7, but not at pCa 8. Ca²⁺ uptake was not significantly affected by the presence of Cl^–. This Ca²⁺-dependent effect of Cl^– on Ca²⁺ leakage was most likely due to a direct action on the ryanodine receptor/Ca²⁺ release channel, and could influence channel sensitivity and the resting [Ca²⁺] in muscle fibres in vivo. In contrast to this effect, acute addition of 20 mM Cl^– to the myoplasm caused a 40–50% reduction in Ca²⁺ release in response to a low caffeine concentration both in toad and rat fibres. One possible explanation for this latter effect is that the addition of Cl^– induces a potential across the SR (lumen negative) which might reduce Ca²⁺ release via several different mechanisms. Received: 20 October 1997 / Received after revision: 1 December 1997 / Accepted: 2 December 1997     

Measurement of SR Ca2+ content in the presence of caffeine in permeabilised rat cardiac trabeculae

 This study was designed to measure the Ca²⁺ content of rat cardiac sarcoplasmic reticulum (SR) after equilibration with normal diastolic levels of Ca²⁺ (100 nM), in the absence and presence of caffeine. Measurements of [Ca²⁺] based on Fura-2 fluorescence were made from a limited bath volume (230 nl) containing individual saponin-permeabilised rat cardiac trabeculae. Injection of caffeine (5–40 mM) into this volume caused an initial release of Ca²⁺ from the SR, but within 30 s the SR was able to re-accumulate a significant proportion of the Ca²⁺. Ca²⁺ re-accumulation into the SR could be prevented by removal of ATP to inhibit the SR Ca²⁺ pump. Incubation of the preparation in an ATP-containing solution containing caffeine (5–40 mM) and 100 nM Ca²⁺ indicated that the SR’s ability to retain Ca²⁺ depends inversely on the dose of caffeine. The relative Ca²⁺ content of the SR after preincubation with caffeine was 86.7±3.5% at a caffeine concentration of 5 mM, 62.5±5.1% at 10 mM caffeine, 37.8±8.1% at 20 mM caffeine and 7.1±1.9% at 40 mM caffeine. Measurement of the SR Ca²⁺ release in the presence of different BAPTA concentrations was used to calculate (1) the Ca²⁺-binding capacity of the preparation (equivalent to 245±10 μM BAPTA) and (2) the Ca²⁺ content of the SR accessed by caffeine after equilibration with 100 nM Ca²⁺ (186±11 μmol/l cell volume or 5.6 mmol/l SR volume). Received: 9 June 1998 / Received after revision: 29 July 1998 / Accepted: 31 July 1998     

Potassium channels of adult locust (Schistocerca gregaria) muscle

Two types of K⁺ channels have been identified in patches of plasma membrane of metathoracic extensor tibiae muscle fibres of adult locust, Schistocerca gregaria. One channel had a maximum conductance of 170 pS, fast open-closed kinetics, and a linear current/ voltage relationship. In inside-out patches it was activated by ‘‘internally applied’’ Ca²⁺, but at unexpectedly low levels (between 10⁻¹⁰ and 10⁻⁹M). The other channel had a maximum conductance of 35 pS, slower open-closed kinetics, and was not activated by Ca²⁺. In cell-attached patches, its channel conductance measured in symmetrical salines was about three times greater for hyperpolarisations than for depolarisations. This inward rectification was proved to be due to block by intracellular Mg²⁺. For both channels, open probability (P _o) and mean open time increased during depolarisations and decreased during hyperpolarisations, resulting in outward rectifications in terms of net current (I _n , product of the single-channel current and P _o). For both channels, the K⁺ conductance was 10 times greater than that for Na⁺. Internally applied tetraethylammonium or tetramethylammonium ions blocked both channels. Received: 12 June 1995/Received after revision and accepted: 30 January 1996     

Regulation of L- and N-types of Ca2+ channels by intracellular ATP4– in frog dorsal root ganglion neurons

The roles of free Mg²⁺ ions, ATP^4– ions and Mg-ATP complexes in the regulation of N- and L-types of Ca²⁺ channels were studied in frog dorsal root ganglion (DRG) neurons using the whole-cell patch-clamp technique. Because Mg²⁺ ions interact with ATP^4– ions to form Mg-ATP complexes, addition of one species can influence the concentrations of the other two. In this study their concentrations were carefully controlled by varying the concentrations of two constituents at a time while keeping the third constant. The effects of each of the three species on barium currents through L-type (I _BaL) and N-type (I _BaN) Ca²⁺ channels were plotted against its concentrations. The dose-response curves for ATP^4– show that I _BaL and I _BaN proportionally increased with ATP^4– concentrations up to 1 mM at three different Mg²⁺ concentrations. At a fixed concentration of ATP^4–, I _BaL and I _BaN remained unchanged even when pMg changed from 3 to 5. Dose-response curves for I _BaL and I _BaN plotted against Mg-ATP concentration did not show a consistent pattern. H-7 and Mg²⁺ ions did not exert any blocking effect on the activity of either Ca²⁺ channel type, and neither dibutyryl-cAMP nor NKH-477 had any stimulating effect, suggesting that phosphorylation is not likely to be involved in ATP-induced potentiation. From these observations, it is concluded that L-type and N-type Ca²⁺ channels in frog DRG neurons are regulated by ATP^4– ions alone, and that the neuronal Ca²⁺ channels are regulated by mechanisms that are different from those regulating the cardiac Ca²⁺ channels. Received: 30 October 1998 / Received after revision: 19 February 1999 / Accepted: 8 March 1999     

Effects of glucose,forskolin and tolbutamide on membrane potential and insulin secretion in the insulin-secreting cell line INS-1

Membrane voltages (V _m) of INS-1 cells, an insulin-secreting cell line, were measured mostly using the cell-attached mode of the patch-clamp method. The cell-attached configuration allowed the cell to be kept intact. Measurement of V _m was possible because seal resistances were very high and because the membrane obviously had a sufficiently high conductance (probably via K⁺ channels). Resting V _m was −80 ± 1mV (n = 42) and was mainly determined by sulphonylurea-sensitive K⁺ _ATP channels since tolbutamide depolarized the plasma membrane in a concentration-dependent manner and generated action potentials at 50 and 100 μmol/l. D-Glucose, tested between 0.5 and 16.7 mmol/l, also depolarized the plasma membrane in a concentration-dependent manner and induced action potentials at concentrations higher than 5.6 mmol/l. Similarly, forskolin (5 μmol/l) depolarized the cells and increased the frequency of Ca²⁺-mediated action potentials. Insulin secretion was measured from cells growing in culture dishes, by radioimmunoassay. Glucose doubled secretion in INS-1 cells, whereas tolbutamide had no significant effect on secretion in the presence of 0.5 mmol/l and 16.7 mmol/l glucose. At 3 mmol/l glucose, tolbutamide increased insulin release slightly. Forskolin elevated secretion twofold at a low glucose concentration. In contrast, when glucose or tolbutamide were added together with forskolin secretion was potentiated five- to tenfold. These results show that glucose induces membrane activation in INS-1 cells. Furthermore, the potent effect of tolbutamide, i.e. to depolarize the plasma membrane without inducing insulin release, leads to the conclusion that effects distal to depolarization are pivotal for secretion in INS-1 cells. Received: 21 November 1995/Accepted: 18 April 1996     

Presence of two Ca2+ influx components in internal Ca2+-pool-depleted rat parotid acinar cells

The molecular mechanism(s) involved in mediating Ca²⁺ entry into rat parotid acinar and other non-excitable cells is not known. In this study we have examined the kinetics of Ca²⁺ entry in fura-2-loaded parotid acinar cells, which were treated with thapsigargin to deplete internal Ca²⁺ pools (Ca²⁺-pool-depleted cells). The rate of Ca²⁺ entry was determined by measuring the initial increase in free cytosolic [Ca²⁺] ([Ca²⁺]_i) in Ca²⁺-pool-depleted, and control (untreated), cells upon addition of various [Ca²⁺] to the medium. In untreated cells, a low-affinity component was detected with K _Ca = 3.4 ± 0.7 mM (where K _Ca denotes affinity for Ca²⁺) and V _max = 9.8 ± 0.4 nM [Ca²⁺]_i/s. In thapsigargin-treated cells, two Ca²⁺ influx components were detected with K _Ca values of 152 ±  79 μM (V _max = 5.1 ± 1.9 nM [Ca²⁺]_i/s) and 2.4 ±  0.9 mM (V _max = 37.6 ± 13.6 nM [Ca²⁺]_i/s), respectively. We have also examined the effect of Ca²⁺ and depolarization on these two putative Ca²⁺ influx components. When cells were treated with thapsigargin in a Ca²⁺-free medium, Ca²⁺ influx was higher than into cells treated in a Ca²⁺-containing medium and, while there was a 46% increase in the V _max of the low-affinity component (no change in K _Ca), the high-affinity component was not clearly detected. In depolarized Ca²⁺-pool-depleted cells (with 50 mM KCl in the medium) the high-affinity component was considerably decreased while there was an apparent increase in the K _Ca of the low-affinity component, without any change in the V _max. These results demonstrate that Ca²⁺ influx into parotid acinar cells (1) is increased (four- to five-fold) upon internal Ca²⁺ pool depletion, and (2) is mediated via at least two components, with low and high affinities for Ca²⁺. Received: 30 October 1995/Received after revisionand accepted: 13 December 1995     

Simultaneous presence of cAMP and cGMP exert a co-ordinated inhibitory effect on the agonist-evoked Ca2+ signal in pancreatic acinar cells

The stimulation of the pancreatic acinar cells by physiological secretagogues, such as acetycholine (ACh), activates a well-established intracellular signalling pathway, which involves the generation of Inositol 1,4,5-trisphosphate (InsP₃) and the release of Ca²⁺ from intracellular stores. Caffeine, which inhibits this agonist-evoked Ca²⁺ response reversibly and competitively also blocks the Ca²⁺ signal generated by the non-specific activation of the membrane guanine nucleotide-binding proteins (G-proteins). Removal of caffeine is associated with an increase of intracellular [Ca²⁺] ([Ca²⁺]_i) and the spatial and temporal characteristics of this Ca²⁺ signal are identical to those of the signal generated by the initial agonist stimulation. Caffeine is also a potent non-specific inhibitor of various cellular phosphodiesterases (PDE) and its inhibitory effect can be reproduced by other PDE inhibitors, chemically related (theophylline) or not (papaverine). Various protocols designed to increase the concentration of either of the major intracellular cyclic nucleotides [adenosine 3′,5′-cyclic monophosphate (cAMP) and guanosine 3′,5′-cyclic monophosphate (cGMP)] failed to reproduce the full extent of the caffeine inhibition: at maximal agonist concentration (1 μM ACh) increases of either cAMP or cGMP did not affect the Ca²⁺ signal, whereas at submaximal doses of agonist (0.1–0.3 μM ACh) they induced partial inhibition. Here we show that only the simultaneous increase of the cellular concentrations of both cyclic nucleotides (either simultaneous or sequential) are effective in mimicking the blocking effect of caffeine and other non-specific PDE inhibitors. These data indicate, thus, that, in addition to other independent intracellular effects, cAMP and cGMP can exert a co-ordinated inhibitory effect of the agonist-evoked Ca²⁺ signal in pancreatic acinar cells. Received: 25 March 1996/Accepted: 23 May 1996     

Intracellular free Mg2+ concentration in skeletal muscle fibres of frog and crayfish

The free intracellular Mg²⁺ concentration ([Mg²⁺]_i) was investigated in frog sartorius and crayfish phasic and tonic skeletal muscle fibres, using a new Mg²⁺-sensitive microelectrode based on the ionophore ETH 5214 [Hu et al. (1989) Anal Chem 61:574–576]. In Ringer solution containing 0.5 mmol/l MgCl₂, the mean [Mg²⁺]_i of the frog muscle fibres was 1.3 mmol/l. In phasic crayfish muscle fibres, [Mg²⁺]_i was about twice as high (mean 3.5 mmol/l) as in tonic fibres (mean 1.5 mmol/l), measured in van Harreveld solution containing 1.2 mmol/l MgCl₂. Long-lasting (3–12 h) incubation of frog skeletal muscle fibres in Na⁺-free solution produced a reversible increase of [Mg²⁺]_i by a factor of about 1.7. A tenfold rise of extracellular Mg²⁺ led to an increase in [Mg²⁺]_i in the presence as well as in the absence of Na⁺. In these experiments, mean [Mg²⁺]_i values of 3.2 mmol/l were never exceeded. Thus, [Mg²⁺]_i remained at least 60 times lower than predicted from a passive distribution across the cell membrane. The results suggest the existence of a Na⁺-dependent and a Na⁺-independent Mg²⁺ extrusion mechanism, which is regulated by actual Mg²⁺ concentrations.     

Effects of thapsigargin and cyclopiazonic acid on sarcoplasmic reticulum Ca2+ uptake,spontaneous force oscillations and myofilament Ca2+ sensitivity in skinned rat ventricular trabeculae

Thapsigargin (TG) and cyclopiazonic acid (CPA) have been reported to be potent inhibitors of the sarcoplasmic reticulum (SR) Ca²⁺ uptake in isolated SR vesicles and cells. We have examined the effect of TG and CPA on (1) the Ca²⁺ uptake by the SR in saponin-skinned rat ventricular trabeculae, using the amplitude of the caffeine-induced contraction to estimate the Ca²⁺ content loaded into the SR, (2) the spontaneous Ca²⁺ oscillations at pCa 6.6 using force oscillation as the indicator, and (3) the myofilament Ca²⁺ sensitivity in Triton X-100-treated preparations. Inhibition of Ca²⁺ loading by TG and CPA increased with time of exposure to the inhibitor over 18–24 min. TG and CPA produced half inhibition of Ca²⁺ loading at 34.9 and 35.7 μM respectively, when 18–24 min were allowed for diffusion. The spontaneous force oscillations were more sensitive to the inhibitors: 10 μM TG and 30 μM CPA both abolished the oscillations in this time. The myofilament Ca²⁺ sensitivity was not affected by 10 and 300 μM TG or CPA. The results show that the concentrations of TG and CPA necessary to inhibit the SR Ca²⁺ uptake of skinned ventricular trabeculae are much higher than the reported values for single intact myocytes. One reason for this may be slow diffusion of the inhibitors into the multicellular trabecula preparation. Received: 28 July 1995/Received after revision: 11 December 1995/Accepted: 18 December 1995     

Early effects of denervation on sarcoplasmic reticulum properties of slow-twitch rat muscle fibres

 The Ca²⁺ release activity of the sarcoplasmic reticulum (SR) in chemically skinned single slow-twitch fibres from control, 2-day and 7-day denervated rat soleus muscle was studied. Histochemical fibre type composition of the whole muscle, electrophysiological properties and the Ca²⁺ sensitivity of tension development by single muscle fibres were also studied. All the data were correlated with contractile properties of the in vitro muscle. In the 2-day denervated muscle the SR Ca²⁺ capacity and the rate of Ca²⁺ uptake decreased from the control values of 0.384 ± 0.030 μmol (mg fibre protein)^–1 and 19.8 ± 1.9 nmol min^–1 (mg fibre protein)^–1, respectively, to 0.210 ± 0.016 μmol (mg fibre protein)^–1 and 13.5 ± 0.9 nmol min^–1 (mg fibre protein)^–1; the calculated amount of Ca²⁺ released upon stimulation by caffeine decreased from the control value of 0.148 to 0.078 μmol (mg fibre protein)^–1. In the 7-day denervated muscle, the SR Ca²⁺ capacity and the rate of Ca²⁺ uptake increased to 0.517 ± 0.06 μmol (mg fibre protein)^–1 and 21.6 ± 2.3 nmol min^–1 (mg fibre protein)^–1, respectively; the calculated amount of Ca²⁺ released increased to 0.217 μmol (mg fibre protein)^–1. Both contraction time and tension of the isometric twitch decreased in 2-day denervated and increased in 7-day denervated muscles. Electrophysiological and histochemical changes, as well as changes in the Ca²⁺ sensitivity of the muscle fibres did not show any apparent correlation with mechanical changes. It is therefore concluded that the SR plays a prominent role in the early changes of contraction time and tension following denervation. Received: 15 October 1996 / Received after revision: 28 March 1997 / Accepted: 8 April 1997     

Replacement of troponin-I in slow-twitch skeletal muscle alters the effects of the calcium sensitizer EMD 53998

 We extracted troponin-I (TnI) from skinned rat and rabbit soleus muscle fibres using a modification of the method described by Strauss et al. (FEBS Lett 310:229–234, 1992) for replacement of TnI in cardiac preparations. Incubation of soleus muscle fibres with 10 mmol/l vanadate virtually completely abolished the Ca²⁺dependence of force. Immunoblot analysis revealed that more than 80% of TnI had been extracted from the preparations. The Ca²⁺dependence of force was restored by incubation with a complex of cardiac TnI (cTnI) and troponin-C (cTnC). We examined the effects of the Ca²⁺-sensitizing compound EMD 53998 on isometric tension in native porcine cardiac and rabbit soleus skinned fibres as well as soleus in which the endogenous slow skeletal TnI (ssTnI) had been replaced by cTnI (soleus–cTnI). It was found that 10 μmol/l EMD 53998 in native soleus increased maximum Ca²⁺-activated force to 120±1.4% of control. In soleus–cTnI fibres, maximum force was increased to only 105±0.9%, which was similar to the effect observed in cardiac muscle (108±0.6%). In cardiac muscle, 10 μmol/l EMD 53998 induced a leftward shift of the pCa-tension relation by 0.65 log units. In native soleus, ΔpCa was only 0.40. Again, the effect of EMD 53998 on soleus–cTnI (ΔpCa=0.56) more closely resembled the response found in cardiac muscle than that observed in native soleus muscle. The apparent TnI-isoform dependence of the effects elicited by EMD 53998 suggests that its actions are modulated by the regulatory proteins of the thin filament. Received: 15 December 1997 / Accepted: 16 March 1998