Ca2+-dependent capacitance increases in rat basophilic leukemia cells following activation of store-operated Ca2+ entry and dialysis with high-Ca2+-containing intracellular solution

 Ca²⁺-dependent vesicular fusion was studied in single whole-cell patch-clamped rat basophilic leukemia (RBL) cells using the capacitance technique. Dialysis of the cells with 10 μM free Ca²⁺ and 300 μM guanosine 5′-O-(3-thiotriphosphate) (GTP[γ-S]) resulted in prominent capacitance increases. However, dialysis with either Ca²⁺ (225 nM to 10 μM) or GTP[γ-S] alone failed to induce a capacitance change. Under conditions of weak Ca²⁺ buffering (0.1 mM EGTA), activation of Ca²⁺-release-activated Ca²⁺ (CRAC) channels by dialysis with inositol 1,4,5-trisphosphate (InsP ₃) failed to induce a capacitance increase even in the presence of GTP[γ-S]. However, when Ca²⁺ATPases were inhibited by thapsigargin, InsP ₃ and GTP[γ-S] led to a pronounced elevation in membrane capacitance. This increase was dependent on a rise in intracellular Ca²⁺ because it was abolished when cells were dialysed with a high level of EGTA (10 mM) in the recording pipette. The increase was also dependent on Ca²⁺ influx because it was effectively suppressed when external Ca²⁺ was removed. Our results demonstrate that I _CRAC represents an important source of Ca²⁺ for triggering a secretory response. Received: 1 May 1998 / Received after revision: 15 June 1998 / Accepted: 2 July 1998     

An examination of the role of intracellular ATP in the activation of store-operated Ca2+ influx and Ca2+-dependent capacitance increases in rat basophilic leukaemia cells

 The role of ATP in both the activation of store-operated Ca²⁺ current I _CRAC and in Ca²⁺-dependent vesicular fusion was examined in a study of rat basophilic leukaemia (RBL) cells using the whole-cell patch-clamp technique. Fusion was monitored via changes in plasma membrane capacitance. Following a decrease in the levels of intracellular ATP, achieved using the mitochondrial poison antimycin and the ATP synthase inhibitor oligomycin, as well as a reduction of glycolysis by removal of external glucose, I _CRAC activated in a manner similar to control cells when stores are depleted by dialysis with a pipette solution containing either inositol 1,4,5-trisphosphate (InsP ₃) or ionomycin together with a high concentration of EGTA. Dialysis of cells for 150 s with the non-hydrolysable ATP analogue 5′-adenylylimidodiphosphate (AMP-PNP) (2 mM) in addition to the mitochondrial inhibitors also failed to prevent activation of I _CRAC following external application of ionomycin and thapsigargin, when compared with control recordings obtained with 2 mM ATP instead. Ca²⁺-dependent vesicular fusion was triggered by dialysing cells with 10 μM Ca²⁺ and guanosine-5′-O-(3-thiotriphosphate (GTP[γ-S]). The capacitance increase was unaffected by inhibition of glycolysis, mitochondrial inhibitors or dialysis with either AMP-PNP or adenosine 5′-O-(3-thiotriphosphate) (ATP[γ-S]) instead of ATP. We conclude that ATP hydrolysis does not seem to be necessary for the activation of I _CRAC or for the capacitance increases elicited by high concentrations of intracellular Ca²⁺. Received: 1 May 1998 / Received after revision: 16 July 1998 / Accepted: 16 July 1998     

Na+/Ca2+ exchange during Ca2+ repletion is not a prerequisite for the Ca2+ paradox in isolated rat hearts

 Ca²⁺ paradox damage has been suggested to be determined by Na⁺ entry during Ca²⁺ depletion and exchange of Na⁺ for Ca²⁺ during Ca²⁺ repletion. Since previously a Ca²⁺ paradox without prior increase of total intracellular [Na⁺] ([Na⁺]_i) has been observed, we investigated whether local accumulation of Na⁺ close to the inner side of the sarcolemma during Ca²⁺ depletion plays a role in the Ca²⁺ paradox by replacing all extracellular Na⁺ by Li⁺ 5 min before and during 10 min Ca²⁺-free perfusion (37°C) in isolated rat hearts (group I). Subsequently, hearts were perfused with a standard, Na⁺- and Ca²⁺-containing solution. Verapamil was used to prevent contracture due to the absence of Na⁺/Ca²⁺ exchange during Na⁺-free perfusion in the presence of Ca²⁺. In group II, the Ca²⁺-free period was omitted, and in group III normal extracellular [Na⁺] was used throughout. ²³Na-NMR was used to monitor intra- and extracellular Na⁺ signals. Total creatine kinase release was 2,977±413, 36±24 and 3170±297 IU/g dry weight in groups I, II and III respectively, indicating a full Ca²⁺ paradox in groups I and III. [Na⁺]_i decreased from 11.3±0.6 mM during control perfusion to 1.2±0.4 mM after 10 min Ca²⁺ depletion in group I, whereas in group III [Na⁺]_i was 10.9±2.2 mM during control perfusion and did not change significantly after 10 min Ca²⁺-free perfusion. It is concluded that accumulation of Na⁺ close to the inner side of the sarcolemma during Ca²⁺ depletion is not a prerequisite for the Ca²⁺ paradox. Received: 2 February 1998 / Received after revision: 31 March 1998 / Accepted: 9 April 1998     

Ca2+-channel-dependent and -independent inhibition of exocytosis by extracellular ATP in voltage-clamped rat adrenal chromaffin cells

 Membrane currents and capacitance were measured to examine the effects of extracellular ATP on exocytosis in voltage-clamped rat adrenal chromaffin cells. ATP reversibly inhibited Ca²⁺ current (I _Ca) and exocytosis. The dependency of exocytosis on I _Ca evoked by 1-s depolarizations was determined. However, inhibition of exocytosis was 2.6 times larger than that estimated from the reduction of I _Ca, implying the existence of a Ca²⁺-channel-independent pathway. This inhibition did not rely on a further reduction of the intracellular Ca²⁺ concentration spike. ATP reduced the rate of exocytosis induced by clamping the intracellular Ca²⁺ concentration. Pertussis toxin blocked the inhibitory effects of ATP on I _Ca and exocytosis. Although RB-2, a P_2Y antagonist, blocked the inhibitory effect of ATP on I _Ca, RB-2 itself produced large increase or decrease in membrane capacitance. Adenosine inhibited I _Ca via a pertussis-toxin-sensitive pathway but did not significantly inhibit exocytosis. Our data show that extracellular ATP inhibits exocytosis via inhibition of I _Ca by activation of a pertussis-toxin-sensitive G-protein linked to P_2Y receptors. Furthermore, our data strongly suggest that ATP activates another pathway, which is also G-protein dependent and accounts for the majority of the inhibitory effect of ATP on exocytosis. Received: 20 February 1997 / Received after revision: 10 July 1997 / Accepted: 23 July 1997     

A modified confocal laser scanning microscope allows fast ultraviolet ratio imaging of intracellular Ca2+ activity using Fura-2

 A confocal, ultraviolet laser scanning microscope (LSM) for reliable ratio measurements of localized intracellular Ca²⁺ gradients using the Ca²⁺-sensitive dye Fura-2 was developed. In a commercial LSM, the filter wheels for the excitation band-pass filters and the grey filters were replaced by acousto-optic tunable filters (AOTF) for rapid switching (≤1.5 μs) of the ultraviolet (351 and 364 nm) and the visible (457, 476, 488, 514 nm) excitation light. This enabled dual wavelength excitation of Fura-2, or 2’7’-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) for pH measurements. Changing to a transmitted-light detector of high sensitivity allowed for simultaneous recording of differential interference contrast images of the preparation with the excitation light. The AOTF fine control of the intensity of the excitation light and improvements in the emission detector sensitivity enabled the acquisition of up to 120 ratio pairs of high-quality images from a single cell. The optical capabilities and limitations of the instrument were evaluated with fluorescent beads and dye-loaded cultured cells. Agonist-induced intracellular Ca²⁺ transients in HT₂₉ cells were recorded to test for the instrument’s ability to measure changes in [Ca²⁺]_i. Ratio z-sections from Fura-2-loaded cells showed an inhomogeneity of the Fura-2 loading with an accumulation of the dye mostly in the mitochondria. We show, as an example of the microscope’s achievable resolution, the spatial and temporal heterogeneity of [Ca²⁺]_i signals in mitochondria and the cytosol in response to agonist-evoked stimulation of HT₂₉ cells. In addition, we show that the lipophilic, membrane-bound Fura-2 derivative Fura-C₁₈, for measurements of near-membrane Ca²⁺ changes, can be used with this confocal microscope. This new LSM is expected to deepen our understanding of localized [Ca²⁺]_i signals; for example, the nuclear Ca²⁺ signalling or the [Ca²⁺]_i changes that occur during stimulation of ion secretion in polarized epithelial cells. Received: 10 October 1996 / Received after revision and accepted: 4 November 1996     

Characterisation of a mutant of barnacle troponin C lacking Ca2+-binding sites at positions II and IV

 This study investigates a mutant barnacle troponin C (TnC) protein (BTnC2–4-) in which the Ca²⁺-binding sites (sites II and IV) have been rendered non-functional. Eliminating Ca²⁺ binding at both Ca²⁺-binding sites of barnacle TnC did not prevent the incorporation of BTnC2–4- into TnC-depleted myofibrillar bundles, although, as expected, the mutant was not able to effect muscle regulation. We conclude that the Mg²⁺ involved in stabilising the interaction between TnC and TnI in the barnacle must bind at a separate location to the Ca²⁺-binding sites. Competition experiments between BTnC2–4- and wild-type barnacle TnC (BTnCWT) or the native isoform BTnC₂ indicate that BTnC2–4- has an approximately fourfold higher affinity for barnacle TnI than BTnCWT but a lower affinity for TnI compared to BTnC₂. These results indicate that disabling sites II and IV changes the affinity of BTnC2–4- for TnC-denuded barnacle myofibrils, altering the stability of the bond formed between TnC and the thin filament. Received: 30 September 1998 / Received after revision: 12 February 1999 / Accepted: 15 February 1999     

Estimation of myofibrillar responsiveness to Ca2+ in isolated rat ventricular myocytes

 To estimate myofibrillar responsiveness to Ca²⁺, we used the relation between cell length and intracellular [Ca²⁺] ([Ca²⁺]_i) during tetanic contractions of isolated ventricular myocytes. Enzymatically isolated rat ventricular myocytes were loaded with fura-2 AM (4 μM for 10 min) and excited alternately at 340 nm and 380 nm. The ratio (R) of fura-2 fluorescence at these wavelengths [F(340)/F(380), an index of [Ca²⁺]_i] and cell length (L) were measured simultaneously. Following treatment with thapsigargin (0.2 μM), myocytes were stimulated at 10 Hz for 10 s to produce a tetanic contraction every min and an instantaneous plot of R vs L (R-L trajectory) was constructed. The R-L trajectory followed the same path during cell shortening and re-lengthening, suggesting that dynamic equilibrium between R and L was achieved during tetanus. Increasing the extracellular [Ca²⁺] from 1 to 8 mM extended the R-L trajectory without a substantial shift of the relation. The Ca²⁺-sensitizing thiadiazinone derivative, EMD57033 (1 μM), shifted the R-L trajectory to the left (sensitization of the myofibrils to Ca²⁺), whereas the non-selective phosphodiesterase inhibitor, 3-isobutyl-1-methylxantine (200 μM), shifted the R-L trajectory to the right (desensitization of the myofibrils to Ca²⁺), in agreement with previous results obtained using skinned preparations. We conclude that the R-L trajectory is useful for estimating the myofibrillar responsiveness to Ca²⁺ in isolated myocytes and may be beneficial for the evaluation of inotropic agents. Received: 13 March 1998 / Received after revision: 4 May 1998 / Accepted: 2 June 1998     

Ca2+-transients induced by K+ channel openers in isolated coronary capillaries

 To study the role of endothelial ATP-sensitive K⁺ channels in the regulation of vascular tone we examined the intracellular calcium concentration ([Ca²⁺]_i) in coronary capillaries consisting only of endothelial cells. Coronary capillary fragments were isolated enzymatically from the guinea-pig heart and [Ca²⁺]_i was determined by microfluorometry of fura-2 loaded cells. Low concentrations of the K⁺ channel opener diazoxide, which caused pronounced glibenclamide-sensitive hyperpolarization in capillaries, induced a rapid, transient rise in [Ca²⁺]_i followed by a sustained elevation of [Ca²⁺]_i (19 of 40 experiments). [Ca²⁺]_i in the endothelial cells increased from 32 ± 7 nM at rest to 66 ± 11 nM at the peak (n = 19). One third of the [Ca²⁺]_i-transients showed irregular oscillations of [Ca²⁺]_i. No significant difference in the [Ca²⁺]_i-response induced by 100 nM or 1 μM diazoxide was found. Similar results were obtained with the K⁺ channel opener rilmakalim. Simultaneous measurements of the membrane potential and [Ca²⁺]_i with fluorometric methods indicated that the hyperpolarization but not the [Ca²⁺]_i-transient could be repeatedly induced in a single capillary by the K⁺ channel openers. Electrophysiological recordings of the membrane potential using the ”perforated patch” method (n = 4), showed that rilmakalim (1 μM) induced hyperpolarization of capillaries towards the K⁺ equilibrium potential, confirming our fluorometric measurements. In conclusion, for the first time, these data indicate that K⁺ channel openers induce [Ca²⁺]_i-transients in microvascular endothelial cells. This raises the possibility that these drugs not only act as synthetic vasoactive factors via hyperpolarizing smooth muscle cells but also via NO release of microvascular endothelial cells. Interestingly, only 100 nM diazoxide was sufficient for a maximal response, suggesting the expression of a new type of K_ATP-channel in coronary capillaries characterised by high sensitivity to diazoxide. Received: 22 August 1997 / Received after revision and accepted: 7 November 1997     

A hypo-osmotically induced increase in intracellular Ca2+ in lactating mouse mammary epithelial cells involving Ca2+ influx

 Using a fluorescent Ca²⁺-sensitive dye, we studied the effect of hypo-osmotic stress on the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in acini freshly isolated from lactating mouse mammary gland. The basal [Ca²⁺]_i of mammary acini was unaffected by a 50% (v/v) dilution of suspensions with isotonic or hypertonic buffer, or after ionic (iso-osmotic) dilution (external Ca²⁺ was 3 mM). Hypo-osmotic dilution (50%) elicited a rapid increase in [Ca²⁺]_i comprising a large, transient elevation, followed by a maintained plateau phase. No hypo-osmotically induced rise in [Ca²⁺]_i was observed in the absence of extracellular Ca²⁺. Neither microtubule disassembly using nocodazole nor actin disruption with cytochalasin D prevented hypo-osmotically evoked stimulation of [Ca²⁺]_i. Pre-incubation of acini with nifedipine did not prevent hypo-osmotically induced stimulation of [Ca²⁺]_i, whereas a non-specific cation channel blocker, gadolinium, partially inhibited the increases in [Ca²⁺]_i induced by hypo-osmotic stress. Furthermore, the transient component was still apparent, and not diminished in magnitude, after [Ca²⁺]_i had been elevated by mobilisation of Ca²⁺ from intracellular stores using thapsigargin. The results demonstrate that hypo-osmotic stress generates an increase in [Ca²⁺]_i in lactating mammary epithelial cells, the major, transient component of which appears to be due to influx of extracellular Ca²⁺. Received: 15 October 1996 / Received after revision and accepted: 1 November 1996     

Capillary force-driven cell perfusion microchamber: calcium transients in isolated smooth muscle cells

 We present a new design for a sub-microlitre chamber which enables perfusion of individual cells. No equipment is required for it to be operational, as the exchange of solutions in the chamber is driven solely by capillary forces. Its active volume consists of a 30-μm-thin layer between two coverslips (separated by a couple of spacers) and is adjustable down to 0.1 μl. This slimline design (1) guarantees that all cells are kept in one (focal) plane during recording/perfusion (thus minimizing movement artefacts when intracellular fluorescence is monitored); (2) facilitates fixing of cells to a coverslip (often even without the use of poly-L-lysine, especially when small cell clusters are used); and (3) makes it possible to perfuse individual cells on a specimen stage of an upright microscope even under high-power objectives with a short working distance, which is of potential use in field studies where the smaller size of an upright microscope (compared to the inverted one) can be advantageous. As an example, we present a chamber with an active volume of approx. 0.5 μl and perfusion rate high enough to enable complete exchange of solution within 250 ms in an area of 500 μm × 500 μm. Only 1 μl of perfusing solution is required for exchanging the entire volume of the chamber. We present an example of intracellular free calcium transients in isolated smooth muscle cells upon release of intracellular sequestered calcium. Received: 13 May 1997 / Received after revision: 18 July 1997 / Accepted: 21 July 1997     

Group III metabotropic glutamate receptor agonists modulate high voltage-activated Ca2+ currents in pyramidal neurons of the adult rat

In pyramidal neurons of the rat sensorimotor cortex, we have investigated the modulation of high voltage-activated calcium currents by agonists at group III metabotropic glutamate receptors (mGluRs). l-2-Amino-4-phosphonobutyrate (l-AP4) and l-serine-O-phosphate (l-SOP) reduced calcium currents in the vast majority of cells isolated from the adult animal. Interestingly, this modulation was negligible in the young animals (2–14 postnatal days), becoming prominent only after full development (more than 21 days). The efficacy of l-SOP mimicked l-AP4 in reducing calcium currents. Yet, l-SOP produced saturating responses at about 3 μM and significant modulation at nanomolar concentrations (EC₅₀=923 nM). The voltage-dependence of the group III mGluR-mediated responses was evaluated by comparing the inhibition of “standard” and “facilitated” conductances. On the calcium currents facilitated by depolarizing prepulse, 3 μM l-SOP produced a mean 13.4% inhibition compared with 19.6% in control condition, supporting the proposition that part of the modulation was voltage-dependent. The calcium current inhibition caused by the activation of group III metabotropic glutamate receptors was only partially sensitive to ω-conotoxin GVIA, but largely inhibited by ω-agatoxin IVA, at concentrations (100 nM) known to block P- and Q-type channels. Conversely, the dihydropyridine antagonists nifedipine and nimodipine (50–500 nM) failed to prevent the group III mGluR-mediated response in the majority of tested cells (more than 65%). Furthermore, the long-lasting tail promoted by the inclusion of the dihydropyridine agonist Bay K 8644 was not consistently affected by l-SOP and l-AP4. These findings imply that the observed modulation involves different channel subtypes, namely N- and P- or Q-type channels, and suggests that group III mGluRs play an important role in the intrinsic and synaptic functions of adult cortical pyramidal neurons. Received: 10 April 1997 / Accepted: 10 September 1997     

The basolateral Ca2+-dependent K+ channel in rat colonic crypt cells

 Previous studies have indicated that a 16-pS K⁺ channel (KC_ca) in the basolateral membrane is responsible for the acetylcholine-induced whole-cell K⁺ conductance in these cells. In the present study we have examined this channel in excised inside-out patches of the basolateral membrane. Over a wide voltage range this channel showed inward rectification. The Ca²⁺ sensitivity was very marked, with a Hill coefficient of three and with half-maximal activation at 330 nmol/l. After several minutes most channels showed a slow run-down. Channel activity could be refreshed by addition of ATP (1 mmol/l) to the bath solution. The non-metabolizable derivative 5’-adenylylimidodiphosphate (AMP-PNP) had no such effect. In contrast, it inhibited channel activity by some 50%. ATP and its derivatives had no effect on the Ca²⁺ sensitivity. Channels activated by ATP were subsequently studied in the presence of alkaline (10 kU/l) or acidic (1 kU/l) phosphatase. Both phosphatases reduced channel activity significantly. These data suggest that the 16-pS K⁺ channel is directly controlled by cytosolic Ca²⁺. This regulatory step is probably distal to an activation produced by protein-kinase-C-dependent phosphorylation. As is the case for several other K⁺ channels, high concentrations of non-metabolizable ATP analogues inhibit this channel. Received: 23 July 1997 / Accepted: 17 September 1997     

ATP regulates cardiac Ca2+ channel activity via a mechanism independent of protein phosphorylation

 The role of adenosine triphosphate (ATP) in the regulation of L-type Ca²⁺ channel activity was investigated in inside-out patches from guinea-pig ventricular cells, in which the Ca²⁺ channel activity had been reprimed by application of cytoplasm from bovine heart. Passing the cytoplasm through a diethylaminoethyl (DEAE)-sepharose column or heating at 60°C for 20 min attenuated the induction Ca²⁺ channel activity to 6–13% of that in the preceding cell-attached patch. Addition of 10 mM MgATP to the cytoplasm greatly improved the potency of cytoplasm in restoring Ca²⁺ channel activity (to 83 ± 22%, mean ± SE). This effect of MgATP was also produced, although with lower potency, by K₂ATP (61 ± 20%) or 5′-adenylylimidodiphosphate (AMP-PNP, 39 ± 7%), a non-hydrolyzable ATP analogue, suggesting that hydrolysis of ATP is not required for the stimulatory effect on channel activity. A non-specific protein kinase inhibitor H8 (50–100 μM) did not inhibit the effect of cytoplasm + MgATP on channel activity, suggesting the involvement of a pathway independent of phosphorylation. We conclude that ATP regulates Ca²⁺ channel activity in dual pathways: one with, and the other without, protein phosphorylation. Received: 7 June 1996 / Accepted: 15 November 1996     

Evidence for a Na+/Ca2+ exchange mechanism in rat peritoneal mast cells

 Mast cells lose their ability to secrete when incubated in nominally Ca²⁺-free medium, but the Na⁺/K⁺ pump inhibitor ouabain prevents this loss, suggesting a Na⁺ dependence of the Ca²⁺ gradient in rat mast cells. The present study includes measurements of histamine release from cell suspensions, and fura-2/AM and current-clamp experiments on single cells. KB-R7943, an inhibitor of the reverse mode of the Na⁺/Ca²⁺ exchanger, 2,4-dichlorobenzamil and La³⁺ counteracted the increase in histamine release induced by ouabain in a dose-dependent manner. The Ca²⁺ response to compound 48/80 was reduced by preincubation of the mast cells for 30 min in nominally Ca²⁺-free medium. This reduction was partly prevented by ouabain or by a low extracellular Na⁺ concentration. Superfusion of cells with a medium containing a low Na⁺concentration resulted in a hyperpolarization of the cells of 38.6±8.6 mV, n=8, followed by a repolarization after the superfusion had ceased (45.7±5.9 mV, n=4). KB-R7943 reduced the hyperpolarization and repolarization induced by a low extracellular Na⁺ concentration to 15.5±2.9 mV (n=7) and 0.2±3.4 mV (n=3), respectively. These results are consistent with the presence of a Na⁺/Ca²⁺ exchanger in rat peritoneal mast cells. Received: 3 July 1998 / Received after revision and accepted: 11 August 1998     

GABAB-mediated upregulation of the high-voltage-activated Ca2+ channels in rat dorsal root ganglia

 The mechanism underlying the enhancement of the high-voltage-activated (HVA) Ca²⁺ current (I _Ca) after application of baclofen, a GABA_B agonist, in neurones of the rat dorsal root ganglia was studied by a combined use of the nystatin perforated patch clamp recording and our rapid superfusion system. Baclofen (50 μM) decreased the peak amplitude of HVA I _Ca and slowed the onset of the current, i.e. produced a typical G-protein-mediated inhibition of I _Ca. However, when baclofen was rapidly removed from the medium, the amplitude of the current was rather augmented, exceeding the control value obtained before application of the drug. This enhancement was not due to a shift of the voltage dependence of Ca²⁺ channel activation or a change in ionic permeability to other ions. The enhancement of HVA I _Ca by baclofen was sensitive to pertussis toxin treatment. The enhancement was evident during superfusion of baclofen. Since the inhibitory effect of baclofen on HVA I _Ca was not attenuated, even after a continuous application of baclofen for 10 min, the enhancement was not due to relief from tonic G-protein-mediated inhibition of the current or a desensitization of the GABA_B receptor–effector system. An extremely prolonged time course of the enhancement of HVA I _Ca by baclofen strongly suggests an involvement of some intracellular signal transduction system. Received: 22 May 1996 / Received after revision: 17 October 1996 / Accepted: 7 January 1997     

Effects of the protein kinase A inhibitor H-89 on Ca2+ regulation in isolated ferret ventricular myocytes

 We investigated the effects of a protein kinase A (PKA) inhibitor, H-89 {N-[2-(p-bromocinnamylamino)ethyl]-5-iso-quinolinesulphonamide}, on Ca²⁺ regulation in Fura-2-loaded ferret myocytes. H-89 (10 μmol/l) decreased the amplitude of the Fura-2 transient to 28.2±4.3% (P<0.001) of control and prolonged its duration, characterized by a decrease in the rate of decline of Ca²⁺ to diastolic levels: t _1/2 increased from 311±35 ms to 547±43 ms (P<0.001, n=7). Reduced Ca²⁺ uptake by the sarcoplasmic reticulum (SR) in the presence of H-89 was also indicated by a decrease in the SR Ca²⁺ content, as assessed with caffeine. The apparent slowing of the SR Ca²⁺-ATPase was not caused by changes in phosphorylation of phospholamban (PLB). However, Ca²⁺ uptake in microsomal vesicles prepared from canine hearts and fast-twitch rat skeletal muscle (which lacks PLB) was decreased by 34.1 and 46.8% (n=3), respectively, suggesting that H-89 has a direct inhibitory effect on the SR Ca²⁺-ATPase. In electrophysiological experiments, 5.0 μmol/l H-89 decreased the L-type Ca²⁺ current (I _Ca) by 39.5% (n=6) and slowed the upstroke of the action potential and, in some cases, caused loss of excitability without changes in the resting membrane potential. In summary, data show that [Ca²⁺ ]_i regulation, and hence contraction, is sustained by PKA-mediated phosphorylation, even in the absence of β-agonists. However, the use of H-89 as a tool to study the role of this signalling pathway is limited by the non-specific effects of H-89 on the SR Ca²⁺-ATPase. Received: 4 September 1998 / Received after revision: 19 October 1998 / Accepted: 20 October 1998     

Ca2+ and Ba2+ effects on basolateral tetraethylammonium transport in isolated snake renal proximal tubules

 Previous work on snake renal tubules suggested that basolateral transport of tetraethylammonium (TEA) is symmetrical. To examine regulation of this transport step more closely, we determined the effects of (1) reductions in the extracellular Ca²⁺ concentration and basolateral Ca²⁺ entry, and (2) the presence of extracellular Ba²⁺ on TEA uptake and efflux across the basolateral membrane of isolated snake renal proximal tubules. Removal of extracellular Ca²⁺ reduced initial TEA uptake and enhanced TEA efflux. Blocking Ca²⁺ entry also reduced initial TEA uptake. Extracellular Ba²⁺ depolarized the basolateral membrane and reduced both TEA uptake and efflux. Inhibition of basolateral TEA uptake with a reduced membrane potential supports previous data indicating that uptake involves potential-driven facilitated diffusion. Inhibition of basolateral TEA efflux by Ba²⁺ even with a reduced membrane potential not only supports previously obtained data indicating that efflux is not influenced by the potential difference and that basolateral TEA transport is asymmetrical, but also suggests that TEA uptake and efflux may occur by separate pathways. Received: 22 May 1997 / Received after revision: 24 July 1997 / Accepted: 28 July 1997     

Effect of intracellular pH on agonist-induced [Ca2+]i transients in HT29 cells

 In this study we examined the influence of intracellular pH (pH_i) on agonist-induced changes of intracellular Ca²⁺ activity ([Ca²⁺]_i) in HT₂₉ cells. pH_i and [Ca²⁺]_i were measured microspectrofluorimetrically using BCECF and fura-2, respectively. Buffers containing trimethylamine (TriMA), NH₃/NH₄ ⁺ and acetate were used to clamp pH_i to defined values. The magnitudes of the peak and plateau of [Ca²⁺]_i transients induced by carbachol (CCH, 10^–6 mol/l) were greatly enhanced by an acidic pH_i and nearly abolished by an alkaline pH_i. The relationship between pH_i and the [Ca²⁺]_i peak was nearly linear from pH_i 7.0 to 7.8. This effect of pH_i was also observed at higher CCH concentrations (10^–4 and 10^–5 mol/l), at which the inhibitory effect of an alkaline pH_i was more pronounced than the stimulatory effect of an acidic pH_i. An acidic pH_i shifted the CCH concentration/response curve to the left, whereas an alkaline pH_i led to a rightward shift. The influence of pH_i on [Ca²⁺]_i transients induced by neurotensin (10^–8 mol/l) or ATP (5 × 10^–7 mol/l) was similar to its influence on those induced by CCH, but generally not as pronounced. Measurements of cellular inositol 1,4,5-trisphosphate (InsP ₃) showed no changes in response to acidification with acetate (20 mmol/l) or alkalinization with TriMA (20 mmol/l). The InsP ₃ increase induced by CCH was unaltered at an acidic pH_i, but was augmented at an alkaline pH_i. Confocal measurements of cell volume showed no significant changes induced by TriMA or acetate. Slow-whole-cell patch-clamp experiments showed no additional effect of CCH on the membrane voltage (V _m) measured after TriMA or acetate application. We conclude that pH_i is a physiological modulator of hormonal effects in HT₂₉ cells, as the [Ca²⁺]_i responses to agonists were significantly changed at already slightly altered pH_i. The measurements of InsP ₃, cell volume and V _m show that pH_i must act distally to the InsP ₃ production, and not via changes of cell volume or V _m. Received: 21 March 1997 / Received after revision: 14 May 1997 / Accepted: 15 May 1997     

Modal gating transitions in cardiac ryanodine receptors during increases of Ca2+ concentration produced by photolysis of caged Ca2+

Channel adaptation is a basic property of the sarcoplasmic reticulum Ca²⁺-release channels/ryanodine receptors (RyRs). It allows channel activity to decay during sustained increases in the concentration of activating Ca²⁺. Despite the potential physiological importance of this self-confining process, its molecular mechanism is not well understood. To define the mechanism of adaptation we studied the dynamics of cardiac Ca²⁺-release channel (RyR) gating using the planar lipid bilayer technique in combination with photolysis of caged Ca²⁺ (DM-nitrophen). Channels activated by rapid and sustained increases in Ca²⁺ concentration (from 0.1 to 0.5 μmol/l) displayed three distinct gating modes, manifested as current records with frequent and long openings (H-mode), with rare and short openings (L-mode), and with no openings (I-mode). H-mode channel activity occurred primarily at early times while L- and I-modes predominated at late times after the rapid Ca²⁺ concentration increase. The decrease in probability of H-mode, mirrored by an increase in the probability of the I-mode, proceeded with a time constant similar to that observed for spontaneous decay in channel activity (i.e., adaptation) in ensemble average records. These results indicate that RyR adaptation transpires by a shift of channel gating from a high open probability mode to low open probability and inactivated modes of the channel. Received: 1 March 1999 / Received after revision: 16 April 1999 / Accepted: 19 April 1999