Activation of protein kinase C does not cause desensitization in rat and rabbit mandibular acinar cells

We have examined whether activation of protein kinase C by phorbol esters decreases the responsiveness of rat and rabbit mandibular, and rat lacrimal, acinar cells to muscarinic stimulation. Intracellular free calcium concentration ([Ca²⁺]_i) was measured in isolated single acini and cell clusters by fura-2 microspectrofluorimetry. Accumulation of inositol phosphates was measured in acinar cell suspensions. All three cell types showed very similar changes in [Ca²⁺]_i in response to acetylcholine (ACh), although mobilization of Ca²⁺ required somewhat higher ACh concentrations in rat lacrimal acinar cells than in mandibular acinar cells. There was no evidence for different dose dependencies of the peak and plateau phases of the [Ca²⁺]_i response. The ACh-evoked [Ca²⁺]_i increase in rabbit mandibular acinar cells exhibited desensitization, since it declined in magnitude when cells were stimulated repeatedly with a maximal dose of agonist. The phorbol ester 12-O-tetradecanoylphorbo-l13-acetate (TPA) rapidly and irreversibly decreased the ACh-evoked [Ca²⁺]_i signals in rat lacrimal acinar cells and reduced ACh-stimulated inositol phosphate accumulation. This inhibitory effect of TPA was most marked in cells stimulated with low doses of ACh, implying that TPA treatment shifted the ACh dose response curve to higher ACh concentrations. In contrast to the results obtained with lacrimal acinar cells, TPA had no effect on the [Ca²⁺]i and inositol phosphate responses to ACh in either rat or rabbit mandibular acinar cells. These results suggest that, although ACh-evoked [Ca²⁺]_i signals, and hence presumably the stimulus-response coupling machinery, are very similar between different acinar cell types, acinar cells show marked differences in their sensitivity to phorbol esters. The insensitivity of mandibular acinar cell [Ca²⁺]_i signals to TPA also suggests that the secretory tachyphylaxis observed in perfused rat and rabbit mandibular salivary glands is unlikely to be a consequence of negative feedback mediated by protein kinase C.     

Involvement of intracellular calcium ions in the release of the fluorescent dye calcein by cholinergic and α-adrenergic agonists from rat parotid acinar cells

Effects of cholinergic and adrenergic agonists on the secretion of the fluorescent dye calcein were examined to clarify the involvement of calcium ions in the secretion of calcein from acinar cells dispersed from the rat parotid gland. Addition of carbachol (CCh) and noradrenalin (NA), but not isoproterenol (IPR), enhanced the net release of calcein from acinar cells during the subsequent 10 min in a dose range from 10^–8 M to 10^–6 M. The net release of calcein reached a maximum 7 min after the addition of CCh. The release of calcein was suppressed by the simultaneous additions of atropine with CCh, or phenoxybenzamine with NA. Addition of CCh induced a sustained dosedependent increase in the intracellular levels of calcium ions, ([Ca²⁺]_i). Addition of NA at 10^–6 M increased [Ca²⁺]_i. Phenoxybenzamine completely inhibited the NA-induced increase, but propranolol did not. The removal of extracellular calcium ions did not influence the release of calcein induced by 10^–6 M CCh, but it abolished the sustained increase in [Ca²⁺]_i. The transient increase in [Ca²⁺]_i induced by CCh was observed in the absence of extracellular calcium ions. A calcium ion chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA) inhibited the CCh-induced release of calcein. The calcium ionophore, A23187 (2.5×10^–6 M), but not 10^–3 M dibutyryl cAMP, evoked the release of calcein. It also increased [Ca²⁺]_i. Removal of extracellular calcium ions suppressed the A23187-induced release of calcein. These results suggest that the release of calcein from parotid acinar cells is transiently induced through an increase in [Ca²⁺]_i by muscarinic and -adrenergic agonists and may represent the initial process of salivary secretion.     

Afterhyperpolarization induced by the activation of nicotinic acetylcholine receptors in pelvic ganglion neurons of male rats

The electrophysiological mechanism underlying afterhyperpolarization induced by the activation of the nicotinic acetylcholine receptor (nAChR) in male rat major pelvic ganglion neurons (MPG) was investigated using a gramicidin-perforated patch clamp and microscopic fluorescence measurement system. Acetylcholine (ACh) induced fast depolarization through the activation of nAChR, followed by a sustained hyperpolarization after the removal of ACh in a dose-dependent manner (10 μM to 1 mM). ACh increased both intracellular Ca²⁺ ([Ca²⁺]_i) and Na⁺ concentrations ([Na⁺]_i) in MPG neurons. The recovery of [Na⁺]_i after the removal of ACh was markedly delayed by ouabain (100 μM), an inhibitor of Na⁺/K⁺ ATPase. Pretreatment with ouabain blocked ACh-induced hyperpolarization by 67.2 ± 5.4% (n = 7). ACh-induced hyperpolarization was partially attenuated by either the chelation of [Ca²⁺]_i with BAPTA/AM (20 μM) or the blockade of small-conductance Ca²⁺-activated K⁺ channels by apamin (500 nM). Taken together, the activation of nAChR increases [Na⁺]_i and [Ca²⁺]_i, which activates Na⁺/K⁺ ATPase and Ca²⁺-activated K⁺ channels, respectively. Consequently, hyperpolarization occurs after the activation of nAChR in the autonomic pelvic ganglia.     

Effects of external calcium on acetylcholine-evoked increases in membrane capacitance in rat pancreatic acinar cells

Effects of external calcium on acetylcholine-induced increases in membrane capacitance and conductance were investigated with the patch-clamp technique in combination with the phase-sensitive detection method, in single dialysed pancreatic acinar cells of rats. Both increases depended on an increase in [Ca²⁺]_i, and a high concentration of EGTA in the cell-dialysing solution made ACh ineffective. In acinar cells exposed to a bathing solution containing the normal concentration of Ca²⁺ (2.5 mM CaCl₂), the increase in membrane capacitance was transient and synchronous with that in membrane conductance (current) in response to 0.2 M acetylcholine. However, in a bathing solution without CaCl₂ and with EGTA (0.2 mM), the increase in membrane capacitance was sustained after the membrane conductance recovered to the original level during the ACh-stimulation. The evidence suggests that external calcium facilitates either the resealing of the fusion- or fission-pores formed at the contact between the secretory granule and the luminal cell membrane, or the membrane retrieval (endocytosis) in Ca²⁺-dependent exocytosis.     

Actions of growth-hormone-releasing hormone on rat pituitary cells: intracellular calcium and ionic currents

Actions of growth-hormone-releasing hormone (GHRH) on single rat anterior pituitary cells were studied using indo-1 fluorescence to monitor changes in intracellular calcium, [Ca²⁺]_i, and perforated-patch recording to measure changes in membrane potential and ionic currents. GHRH elevated [Ca²⁺]_i in non-voltage-clamped cells by a mechanism that was dependent upon extracellular Na⁺ and Ca²⁺ and was blocked by the dihydropyridine Ca²⁺-channel blocker, nitrendipine. Resting cells had a fluctuating membrane potential whose a mean value depolarized by 9 mV in response to GHRH. The membrane-permeant cAMP analogue, 8-(4-chlorophenylthio)cAMP, mimicked the action of GHRH on membrane potential. Under voltage clamping, GHRH activated a small inward current (1–5 pA). Two types of response could be distinguished. The type I response had an inward current that was largest at more negative potentials (–90 mV), and the type II response had inward current that was larger at more positive potentials (–40 to –70 mV). Both types of response were reversible and blocked by removal of extracellular Na⁺. These results suggest that the rise in [Ca²⁺]_i produced by GHRH in non-voltage-clamped cells results from the activation via cAMP of a Na⁺-dependent conductance, which depolarizes the cell and increases the Ca²⁺ influx through voltage-gated Ca²⁺ channels.Dedicated in memory of the late Alexander P. Naumov.     

Intracellular calcium in mammalian brain cells: fluorescence measurements with quin2

Summary Dispersed brain cells from 12–14 day old mouse embryos were loaded with the Ca²⁺-sensitive fluorescent probe, quin2 and shown to have a resting intracellular Ca²⁺ concentration ([Ca²⁺]_i) of 158 nM (SE ± 5) in the presence of 1 mM [Ca²⁺]_o. When external [Ca²⁺] was raised from 0 to 1 mM there was an increase of [Ca²⁺]_i of 70 nM; with further additions of Ca to >10 mM [Ca²⁺]_o the level of [Ca²⁺]_i increased by <25 nM. Releasable intracellular Ca²⁺ stores, estimated from the increase in [Ca²⁺] produced by 4Br A23187 in the absence of extracellular Ca²⁺, were 24 fmol/10⁶ cells. A small increase in [Ca²⁺]_i could be produced by the mitochondrial inhibitor, carbonyl cyanide m-chlorophenylhydrazone (CCCP). When extracellular K⁺ was raised by 10–20 mM, intracellular Ca²⁺ levels increased from 152 (SE ± 7) to 204 nM (SE ± 10). These K⁺-induced increases in [Ca²⁺]_i were blocked by verapamil, did not occur in the absence of extracellular Ca²⁺, and presumably reflect the activation of voltage-dependent Ca²⁺ channels. N-methyl-D-aspartic acid (NMDA) evoked an increase in [Ca²⁺]_i, while the kainate-like lathyrus sativus neurotoxin, L-3-oxalyl-amino-2aminopropionic acid (L-3,2-OAP) did not; this is consistent with previous observations of different and respectively Ca²⁺-dependent and -independent mechanisms of action of these excitatory amino acids.     

Effect of contraction on lymphatic,venous, and tissue electrolytes and metabolites in rabbit skeletal muscle

Summary The effect of muscle contraction on lymphatic and plasma [K⁺], [Na⁺], [Ca²⁺], [Mg²⁺], [Cl^–], [P_i], [lactate] ([Lac^–]); [creatine] ([Cr]), ideal osmolality (OSM), and [protein] was evaluated in femoral venous blood and lymph specimens sampled from the calf muscles of rabbits before, in the course of, and after contractions. In addition, total [K⁺], [Na⁺], [Ca²⁺], [Mg²⁺], [Cl^–], and [H₂O] were analyzed in the muscle tissue. To facilitate lymph sampling both hind limbs were passively flexed and extended, in imitation of natural running movements, by an electrically driven crank. The muscles of one side also performed superimposed rhythmic isotonic contractions. Before contractions, lymphatic [K⁺], [Na⁺], [Ca²⁺], [Mg²⁺], [Lac^–], [Cr], and OSM did not significantly differ from corresponding femoral venous concentrations, [Cl^–], and [P_i] were significantly higher, [protein] significantly lower in the lymph than in the plasma. During contractions lymphatic [K⁺], OSM, [Lac^–], and [P_i] were raised significantly more in the lymph compared with the plasma concentrations. [Na⁺], [Cl^–], [Ca²⁺], and [Mg²⁺] showed only small changes in the course of contractions and thereafter, and they were altered in a similar way in the lymph and plasma. It was suggested that lymphatic and interstitial concentrations were in equilibrium. Comparing inactive with active muscles, the latter lost K⁺ but gained Na⁺, Cl^–, and H₂O, whereas minimal changes occurred in the [Ca²⁺] and [Mg²⁺]. The changes were discussed in connection with the hypothesis that electrolyte shifts might be involved in the activation of the muscular non-proprioceptive interstitial nerve endings which appear to play a role in reflexogenic cardiovascular and respiratory control.A preliminary report of this work has been given elsewhere [33]Supported by Deutsche Forschungsgemeinschaft     

Na/Ca exchange in the basolateral membrane of the A6 cell monolayer: role in Cai homeostasis

The presence of a Na/Ca exchanger in A6 cells was investigated by measuring intracellular calcium (Ca_i) fluctuations and the ⁴⁵Ca fluxes through the basolateral membranes (blm) of the cell monolayer. Removal of Na⁺ from the medium produced a transient increase in Ca_i followed by a regulatory phase returning Ca_i to control levels in 3–4 min, this phase being greatly accelerated (< 60 s) by NaCl addition (apparent K _m of approximately 5 mM Na⁺). The Ca_i increase was only found with the Na⁺-free medium on the basolateral side of the cell monolayer. A twofold increase in the ⁴⁵Ca influx was observed under these conditions. In Ca²⁺- depleted cells, the initial Ca_i increase after Ca²⁺ addition to the medium was greater when the putative Na/Ca exchanger was not functioning (i.e. in a Na⁺-free medium). ⁴⁵Ca effluxes through the blm of the monolayer were greatly and transiently increased by a Na⁺-free medium on the serosal side and blocked by orthovanadate (1 mM). The Ca_i increase induced by a hypo-osmotic shock was greater in cells bathed in a Na⁺-medium, conditions expected to block the activity of the Na/Ca exchanger. These findings support the hypothesis that a Na/Ca exchanger is present on the blm of A6 cells and affirm its role in Ca_i homeostasis in steady-state conditions and following osmotic shock. In addition, a Ca²⁺ pump also located on the blm and Ca²⁺ stores sensitive to inositol 1,4,5-trisphosphate were found to be implicated in Ca_i homeostasis.     

Action of ANG II and ANP on colon epithelial cells

The interaction of angiotensin II (ANG II) and atrial natriuretic peptide (ANP) on intracellular pH (pH_i) and calcium ([Ca²⁺]_i) was investigated in T84 cells (a permanent cell line derived from human colon epithelium) using the fluorescent stains BCECF/AM and Fluo 4/AM, respectively. pH_i recovery rate mediated by the Na⁺/H⁺ exchanger (NHE) was examined following an NH₄Cl pulse. Under control conditions pH_i recovered at 0.114±0.005 pH units/min (n=35). ANG II (10^–12 or 10^–9 M) increased this value, whilst ANG II (10^–7 M) decreased it. These effects of ANG II were impaired by simultaneous addition of 1 M or 25 M HOE-694, indicating that the stimulatory and inhibitory effects of ANG II on pH_i recovery are mediated in part via the NHE1 and NHE2 isoforms. ANG II increased [Ca²⁺]_i concentration-dependently. ANP (10^–6 M) or dimethyl-BAPTA/AM (50 M) blocked the effects of ANG II on [Ca²⁺]_i and on the rate of pH_i recovery. Thapsigargin (10^–5 M) enhanced the effect of ANG II on [Ca²⁺]_i and reversed its stimulatory effect on the rate of pH_i recovery to an inhibitory one. External Ca²⁺-free solution did not affect the effects of ANG II on these parameters. These data suggest that the [Ca²⁺]_i increase induced by ANG II is dependent on intracellular calcium stores. They are compatible with the demonstration of two sites on the C-terminal of the Na⁺/H⁺ exchanger, one stimulating Na⁺/H⁺ activity by increases of [Ca²⁺]_i in the lower range (at 10^–12 or 10^–9 M ANG II) and the other inhibiting this activity at high [Ca²⁺]_i levels (at 10^–7 M ANG II). ANP or dimethyl-BAPTA/AM, by impairing the pathway mediating the increase in [Ca²⁺]_i, block both the stimulatory and inhibitory effects of ANG II.     

Cyanide inhibits the Na+/Ca2+ exchanger in isolated cardiac pacemaker cells of the cane toad

The effects of the metabolic inhibition on the activity of the Na⁺/Ca²⁺ exchanger (NCX) were studied in single isolated pacemaker cells from the cane toad. Ca²⁺ influx on NCX (reverse mode) was estimated by measuring the increase in intracellular calcium concentration ([Ca²⁺]_i) in response to extracellular Na⁺-free solution. After application of 2 mM sodium cyanide for 3–5 min, the peak [Ca²⁺]_i in Na⁺-free solution was significantly decreased from 377±42 nM to 260±46 nM, suggesting inhibition of NCX. To study Ca²⁺ efflux on NCX (forward mode), we recorded the tail currents on repolarization which were abolished by Ni²⁺ and by Na⁺-free solution. Cyanide decreased the amplitude of tail currents by 36±3%. To investigate the intrinsic properties of NCX during the metabolic inhibition, we used rapid application of caffeine to trigger sarcoplasmic reticulum Ca²⁺ release, which then stimulates NCX current (I_NCX ). Both the caffeine-induced peak [Ca²⁺]_i and the peak I_NCX were reduced by cyanide exposure. When I_NCX was plotted against [Ca²⁺], the slope of the decay phase was decreased in the presence of CN^– to 44±8% of control, indicating that for a given [Ca²⁺]_i there was less I_NCX produced. These results show that cyanide (CN^–) inhibits NCX activity at least partly through changes in the intrinsic properties of NCX. The inhibition of NCX probably contributes to the slower firing rate of pacemaker cells in CN^–.     

Spatial and temporal control of intracellular free Ca2+ in chick sensory neurons

Digital imaging of fura-2 fluorescence and the voltage-clamp technique were combined to study cytoplasmic free Ca²⁺ concentration, [Ca]_i, in neurons cultured from chick dorsal root ganglia. Depolarizing pulses raised [Ca]_i to a new steady-state level which was achieved earlier in neurites than in the soma. The rise in [Ca]_i during stimulated bursting or rhythmic activity was also faster in neurites. After stimulation [Ca]_i recovered monoexponentially in the soma and biexponentially in neurites. Application of 50 mM KCl produced membrane depolarization and a concomitant increase of [Ca]_i. During wash-out [Ca]_i often declined to an intermediate steady-state level at which it stayed for several minutes. Thereafter the resting level of [Ca]_i was quickly restored. [Ca]_i recovery was delayed after treating the cell with 2 M thapsigargin, an inhibitor of the Ca²⁺ pump of internal Ca²⁺ stores. Caffeine (10 mM) transiently increased [Ca]_i. A second caffeine application produced smaller [Ca]_i changes due to the prior depletion of Ca²⁺ stores, which could be replenished by brief exposure to KCl. Thapsigargin (2 M) transiently increased [Ca]_i both in the standard and Ca²⁺-free solution. [Ca]_i transients due to caffeine and thapsigargin started in the cell interior, in contrast to [Ca]_i changes evoked by membrane depolarization, which were noticed first at the cell edge. Caffeine and thapsigargin induced a transient inward current which persisted in the presence of 1 mM La³⁺ and in Ca²⁺-free solutions, but which was greatly diminished in Na⁺-free solutions. The effects of caffeine and thapsigargin were mutually exclusive both in the generation of [Ca]_i transients and in the inward current induction.     

Intracellular Na+ measurements in smooth muscle using SBFI – changes in [Na+], Ca2+ and force in normal and Na+-loaded ureter

 Our understanding of the control and effects of intracellular [Na⁺] ([Na⁺]_i) in intact smooth muscle is limited by the lack of data concerning [Na⁺]_i. The initial aim of this work was therefore to investigate the suitability of using the Na⁺-sensitive fluorophore SBFI in intact smooth muscle. We find this to be a good method for measuring [Na⁺]_i in ureteric smooth muscle. Resting [Na⁺]_i was found to be around 10 mM and rose to 25 mM when the Na⁺-K⁺-ATPase was inhibited by ouabain. This relatively low [Na⁺]_i in the absence of Na⁺-K⁺-ATPase suggests that other cellular processes, such as Na⁺-Ca²⁺ exchange, play a role in maintaining [Na⁺]_i under these conditions. Simultaneous measurements of [Na⁺]_i or [Ca²⁺] _i and force showed that Na⁺-Ca²⁺ exchange can play a functional role in ureteric smooth muscle. We found that the greater the driving force for Na⁺ exit and hence Ca²⁺ entry, the larger the contraction. In addition the Na⁺-Ca²⁺ exchanger activity under these conditions was found to be pH sensitive: acidification reduced the contraction and concomitant changes in [Ca²⁺] and [Na⁺]_i. We conclude that SBFI is a useful method for monitoring [Na] in smooth muscle and that Na⁺-Ca²⁺ exchange may play a functional role in the ureter. Received: 26 August 1997 / Received after revision: 27 October 1997 / Accepted: 28 October 1997     

Activation of K+ and Cl− channels by Ca2+ and cyclic AMP in dissociated kidney epithelial (MDCK) cells

In dissociated MDCK cells, activators of the cyclic AMP system cause depolarization detectable by changes in fluorescence of the membrane potential sensitive dye bisoxonol. Addition of forskolin (60 M), vasopressin (2 M), 8-bromo-cyclic AMP (0.5 mM) or l-epinephrine (10 M) depolarized the cells substantially in low Cl^– (5 mM) but had little effect in high Cl^– (140 mM) solution. These results are consistent with cyclic AMP activation of Cl^– channels. The Ca²⁺-ionophore ionomycin (1 M) produced a rapid hyperpolarization in low and high Cl^– solutions, consistent with K⁺ channel opening. Using a clonal subline, MDCK-14, the magnitude of the ionomycin hyperpolarization was roughly proportional to the concomitant rise in [Ca²⁺]_i as measured with the intracellular Ca²⁺ probe indo-I. Both l-epinephrine and isoproterenol appeared to activate the Cl^– channels. However only l-epinephrine produced a [Ca²⁺]_i rise and a transient hyperpolarization (due to K⁺ channel opening), which preceeded the depolarization due to Cl^– channel opening. The l-epinephrine-induced [Ca²⁺]_i response of the heterogeneous MDCK cell population but not of the clonal subline MDCK-14 was inhibited by removal of extracellular Ca²⁺. In the latter only the slow secondary phase of the [Ca²⁺]_i rise was affected by Ca²⁺ removal. It is concluded that l-epinephrine activates K⁺ and Cl^– channels in a sequential manner in MDCK cells by Ca²⁺ and cAMP signals, presumably via - and -adrenergic receptors located on the same cell.Abbreviations MDCK cells Madin Darby Canine Kidney cells - [Ca²⁺]_i intracellular calcium concentration - [Cl^–]_i intracellular chloride concentration - [Cl^–]_o extracellular chloride concentration - [Na⁺+K⁺]_i intracellular concentration of Na⁺ and K⁺ - [Na⁺+K⁺]_o extracellular concentration of Na⁺ and K⁺ - E_M transmembrane potential - E_Cl chloride equilibrium potential - E_K potassium equilibrium potential - bis-oxonol [bis(1,3-diethylthio-barbiturate)] trimethine oxonol - DMSO dimethylsulfoxide - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol bis (-aminoethyl ether) N,N-tetraacetic acid - Hepes 4-(2-hydroxyethyl)-1 piperazineethanesulfonic acid - NMG⁺ N-methylglucamine - RPMI medium Rosewell Park Memorial Institute medium     

Intracellular Na+ modulates large conductance Ca2+-activated K+ currents in human umbilical vein endothelial cells

We studied the effects of Na⁺ influx on large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels in cultured human umbilical vein endothelial cells (HUVECs) by means of patch clamp and SBFI microfluorescence measurements. In current-clamped HUVECs, extracellular Na⁺ replacement by NMDG⁺ or mannitol hyperpolarized cells. In voltage-clamped HUVECs, changing membrane potential from 0 mV to negative potentials increased intracellular Na⁺ concentration ([Na⁺]_i) and vice versa. In addition, extracellular Na⁺ depletion decreased [Na⁺]_i. In voltage-clamped cells, BK_Ca currents were markedly increased by extracellular Na⁺ depletion. In inside-out patches, increasing [Na⁺]_i from 0 to 20 or 40 mM reduced single channel conductance but not open probability (NPo) of BK_Ca channels and decreasing intracellular K⁺ concentration ([K⁺]_i) gradually from 140 to 70 mM reduced both single channel conductance and NPo. Furthermore, increasing [Na⁺]_i gradually from 0 to 70 mM, by replacing K⁺, markedly reduced single channel conductance and NPo. The Na⁺–Ca²⁺ exchange blocker Ni²⁺ or KB-R7943 decreased [Na⁺]_i and increased BK_Ca currents simultaneously, and the Na⁺ ionophore monensin completely inhibited BK_Ca currents. BK_Ca currents were significantly augmented by increasing extracellular K⁺ concentration ([K⁺]_o) from 6 to 12 mM and significantly reduced by decreasing [K⁺]_o from 12 or 6 to 0 mM or applying the Na⁺–K⁺ pump inhibitor ouabain. These results suggest that intracellular Na⁺ inhibit single channel conductance of BK_Ca channels and that intracellular K⁺ increases single channel conductance and NPo. GH Liang and MY Kim contributed equally to this publication and therefore share the first authorship.     

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.     

Ca2+-dependent unidirectional vesicular release detected with a carbon-fibre electrode in rat pancreatic acinar cell triplets

An amperometric constant-voltage method for detection of serotonin oxidation currents was applied to pancreatic acinar cell triplets to determine the site of release of granular content following an increase in [Ca²⁺]_i. The carbon fibre electrode, fabricated to be compatible with a conventional patch-clamp amplifier, was voltage-clamped at 600 mV exceeding the serotonin oxidation voltage, 300 mV. The electrode was placed on the different regions of cell surface of acinar cell triplets loaded with exogenous serotonin. Transient oxidation currents were detected only when the electrode was placed on the acinar lumen after stimulation with a Ca²⁺ ionophore, A23187, but never observed on the basal or lateral cell surface, or paracellular clefts. No such current responses were observed in the acinar cells without serotonin loading. The results indicate that the A23187-induced sustained increase in [Ca²⁺]_i discharges serotonin specifically into the lumen, and provides direct evidence for the presence of Ca²⁺-dependent unidirectional release of granular contents in pancreatic acinar cells.     

Sodium-sensitive, calcium-independent potassium conductance in the leech giant glial cell

 We have measured membrane current, membrane potential and intracellular Na⁺ and Ca²⁺ concentrations, [Na⁺]_i and [Ca²⁺]_i, of the giant glial cell in the nervous system of the leech Hirudo medicinalis using conventional microelectrodes and the fluorescent dyes sodium-binding benzofuran isophthalate (SBFI) and fura-2. When the Na⁺ was removed from the saline, the membrane conductance increased twofold from 1.29±0.1 μS to 2.57±0.18 μS (mean ± SEM; n=27). The rise in membrane conductance was accompanied by a current, which reversed around –74 mV, and the amplitude of K⁺-induced depolarizations or currents increased during Na⁺ removal, suggesting an increase in the K⁺ conductance of the glial membrane. We also monitored [Ca²⁺]_i when removing external Na⁺ in the presence and absence of external Ca²⁺, and during injection of the Ca²⁺-chelator BAPTA into the cells. Our results indicate that Na⁺ modulates a K⁺ conductance of these glial cells, independent of intra- and extracellular Ca²⁺. Received: 1 April 1998 / Received after revision and accepted: 22 May 1998     

[Ca2+]i-dependent membrane currents in guinea-pig ventricular cells in the absence of Na/Ca exchange

Transient inward currents (I _ti) during oscillations of intracellular [Ca²⁺] ([Ca²⁺]_i) in ventricular myocytes have been ascribed to Na/Ca exchange. We have investigated whether other Ca²⁺-dependent membrane currents contribute to I _ti in single guinea-pig ventricular myocytes, by examining membrane currents during [Ca²⁺]_i oscillations and during caffeine-induced Ca²⁺ release from the sarcoplasmic reticulum in the absence of Na⁺. Membrane currents were recorded during whole-cell voltage clamp and [Ca²⁺]_i measured simultaneously with fura-2. In the absence of Na/Ca exchange, i.e., with Li⁺, Cs⁺ or N-methyl-D-glucamine (NMDG⁺) substituted for Na⁺, the cell could be loaded with Ca²⁺ by repetitive depolarizations to +10 mV, resulting in spontaneous [Ca²⁺]_i oscillations. During these oscillations, no inward currents were seen, but instead spontaneous Ca²⁺ release was accompanied by a shift of the membrane current in the outward direction at potentials between –40 mV and +60 mV. This [Ca²⁺]_i-dependent outward current shift was not abolished when NMDG⁺ was substituted for internal monovalent cations, nor was it sensitive to substitution of external Cl^–. It was however, sensitive to the blockade of I_Ca by verapamil. These results suggest that the transient outward current shift observed during spontaneous Ca²⁺ release represents [Ca²⁺]_idependent transient inhibition of I _Ca. Similarly, during the [Ca²⁺]_i transients induced by brief caffeine (10 mM) applications, we could not detect membrane currents attributable to a Ca²⁺-activated nonselective cation channel, or to a Ca²⁺-activated Cl^– channel; however, transient Ca²⁺-dependent inhibition of I _Ca was again observed. We conclude that neither the Ca²⁺-activated nonselective cation channel nor the Ca²⁺-activated Cl^– channel contribute significantly to the membrane currents during spontaneous [Ca²⁺]_i oscillations in guineapig ventricular myocytes. However, in the voltage range between –40 mV and +60 mV Ca²⁺-dependent transient inhibition of I _Ca will contribute to the oscillations of the membrane current.     

Subcellular gradients of intracellular free calcium concentration in isolated lacrimal acinar cells

The spatial distribution of intracellular free calcium concentration ([Ca²⁺]_i) was measured in small clusters of isolated rat lacrimal acinar cells by imaging the fluorescence of the Ca²⁺-sensitive dye fura-2. In the absence of extracellular Ca²⁺, stimulation with acetylcholine (ACh) caused an increase in [Ca²⁺]_i, due to release of intracellular Ca²⁺ stores, which was maximal at the luminal pole of the cell. In contrast, the organellar Ca²⁺-ATPase inhibitor 2,5-di(tert-butyl)-hydroquinone caused an increase in [Ca²⁺]_i, which was most marked in the basolateral region of the cell. When the cells were stimulated with ACh in a medium containing Ca²⁺, the gradients of [Ca²⁺]_i (with [Ca²⁺]_i most elevated at the luminal pole) were maintained for the duration of agonist stimulation. The possible implications of these results concerning the location and identity of intracellular Ca²⁺ stores, and the location of the sites that underlie agonist-stimulated Ca²⁺ influx, are considered. In particular, it seems likely that intracellular inositol-1,4,5-trisphosphate (InsP₃) binding sites may be concentrated in the luminal region of the cell. It is not clear, however, whether this implies that there is a distinct luminally located InsP ₃-sensitive organelle.     

Does adrenaline modulate the Na+-Ca2+ exchanger in isolated toad pacemaker cells?

β-Adrenergic stimulation of pacemaker cells from the sinus venosus of the cane toad (Bufo marinus) increases intracellular calcium ([Ca²⁺]_i) and firing rate. The increase in [Ca²⁺]_i could contribute to the increased firing rate by increasing the inward Na⁺-Ca²⁺ exchange current (I _Na-Ca) during diastole. In this study we measured [Ca²⁺]_i and membrane currents in single, isolated, voltage-clamped pacemaker cells. We show that I _Na-Ca increases during β-adrenergic stimulation. To test whether this increase in I _Na-Ca is caused by elevated [Ca²⁺]_i or by changes in the properties of the Na⁺-Ca²⁺ exchanger, we made rapid applications of caffeine and plotted the I _Na-Ca against [Ca²⁺]_i. This relationship was linear during the declining phase of the [Ca²⁺]_i signal caused by caffeine and was not significantly different in the presence or absence of β stimulation. These results show that I _Na-Ca is increased during β-adrenergic stimulation and will contribute to the increased firing rate. However the increase in I _Na-Ca appears to be a consequence of the increase in [Ca²⁺]_i and is not caused by changes in the intrinsic properties of the Na⁺-Ca²⁺ exchanger. Received: 18 January 1999 / Received after revision: 9 April 1999 / Accepted: 22 April 1999