The increase in the intracellular Ca2+ concentration induced by mechanical stimulation is propagated via release of pyrophosphorylated nucleotides in mammary epithelial cells

Mechanical stimulation of one mammary tumor cell in culture induced an increase in its intracellular calcium concentration which spread to surrounding cells. The increase in calcium can also be induced by addition of a solution in which cultured mammary tumor cells were stimulated by repeated pipetting (solution after pipetting cells, SAPC). The activity of the SAPC was completely abolished by treatment with snake venom phosphodiesterase or pyrophosphatase. Uridine triphosphate (UTP), uridine diphosphate (UDP) and ATP (1 M each) were detected in the SAPC, whereas 5-UMP and 5-AMP were produced by phosphodiesterase digestion. A mixture of UTP, UDP and ATP (1 M each) elicited a calcium response which was comparable to that induced by SAPC, while UTP, UDP or ATP alone at 1 M elicited a small increase in calcium concentration in mammary tumor cells. Suramin, a competitive antagonist of P₂ purinoceptors, diminished the spreading of the calcium wave induced by mechanical stimulation. It also blocked the responses to SAPC, UTP, UDP and ATP. These findings suggest that the mechanical stimulation results in the release of UTP, UDP and ATP into the extracellular space which mediates induction of the spreading calcium response via P_2U-type purinoceptors.     

Involvement of P2-purinergic receptors in intracellular Ca2+ responses and the contraction of mammary myoepithelial cells

 Mammary myoepithelial cells were isolated and cultured to characterize their properties. The intracellular calcium concentration (Ca_i ²⁺) was measured using the ratio of fura-2 fluorescence at 340 nm to that at 360 nm (F ₃₄₀/F ₃₆₀), and the contraction was simultaneously monitored by the change of fluorescence intensity at 360 nm (F ₃₆₀). Cultured myoepithelial cells retained their contractile machinery as in the intact tissue. NBD-phallacidin fluorescence, which marks F-actin, and electron microscopy showed abundant bundles of microfilaments in the cytoplasm. Oxytocin (> 0.1 nM) induced an increase in Ca_i ²⁺ and contraction. The amplitude and time course of the Ca_i ²⁺ increase were not markedly affected in the Ca²⁺-free solution. Nifedipine (10 μM) did not affect the response to oxytocin. ATP (>1 μM) induced an increase in Ca_i ²⁺ and contraction. The response to ATP was not affected by Ca²⁺ removal, but was suppressed by suramin (100 μM), an antagonist of P₂-purinergic receptors. The order of potency of nucleotides to increase Ca_i ²⁺ was ATP = ADP > UTP > UDP. These findings indicate the presence of P₂-purinergic receptors in mammary myoepithelial cells. The results suggest that stimulant-induced Ca_i ²⁺ increases and contractions are due to release of Ca²⁺ from intracellular stores in these cells. In addition to the hormonal action of oxytocin, extracellular nucleotides may function as paracrine agents to contract myoepithelial cells in the mammary gland. Received: 17 March 1997 / Received after revision: 10 July 1997 / Accepted: 21 July 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     

Chemically, mechanically, and hyperosmolarity-induced calcium responses of rat cortical capillary endothelial cells in culture

 The purpose of the present work was to characterize calcium responses of brain-capillary endothelial cells (BCEC), the cells forming the blood-brain barrier, to chemical, hyperosmolar and mechanical stimulation. Confluent BCEC cultures were grown from capillary fragments isolated from rat cerebral cortex. Intracellular free calcium ([Ca²⁺]_i) was measured using fura-2 and digital imaging. Our experiments show large endothelial calcium responses to substance P and ATP, up to a peak value of approximately 1000 and 600 nM, respectively, and these responses were observed in 2/3 of the cells. Calcium responses to bradykinin, histamine, and hyperosmolar sucrose or mannitol were smaller, attaining a peak in the range 180–340 nM, and were observed in a smaller fraction of the cells. No calcium responses were observed to high-potassium, l-glutamate, serotonin, carbachol, noradrenalin, and nitric-oxide donors. Consecutive superfusion of the cultures with ATP, bradykinin, and histamin showed that cells with a certain response pattern were spatially grouped; the response pattern itself varied widely between experiments. Mechanical stimulation of a single cell caused a calcium response in the stimulated cell in primary cultures and triggered an intercellularly propagating calcium wave in passaged cultures. Given the important effect of endothelial [Ca²⁺]_i on blood-brain barrier permeability and transport, we conclude that substance P and ATP are potential modulators of blood-brain barrier function. Hyperosmolarity-induced blood-brain barrier opening is probably not mediated through endothelial [Ca²⁺]_i. Received: 21 September 1998 / Accepted: 8 February 1999     

Free calcium transients and oscillations in nerve cells

Summary Changes in the intracellular level of free calcium induced by different influences in neurones of the snail Helix pomatia have been measured by changes in Fura-2 fluorescence. Thymol in submillimolar concentrations induced the release of stored intracellular calcium. This effect was similar to xantine-induced release. IP₃ and Gpp[NH]p injections also released intracellular calcium. The response to cAMP injections was more complicated and included, probably, both the release of stored calcium and its influx through membrane channels. Oscillations of intracellular free calcium are described. It has been suggested that oscillations can occur only in cases where the mechanism of Ca-dependent calcium release is activated.     

Single calcium-activated potassium channel in cultured mammary epithelial cells

The properties of Ca²⁺-activated K⁺ channels in mouse mammary epithelial cells in primary culture were studied by the patch-clamp technique. In cell-attached patches, spontaneous channel openings were sometimes observed; the slope conductance of the currents was about served; the slope conductance of the currents was about 12 pS at negative membrane potentials with a physiological solution (152 mM Na⁺, 5.4 mM K⁺) in the pipette. External application of A23187, a calcium ionophore, activated this channel. In excised inside-out patches, the channel was activated by increasing the internal Ca²⁺ concentration (10^–7 to 10^–6 M). No voltage dependence of the channel activity was observed. Internal Na⁺ blocked the outward K⁺ current in a voltage dependent manner and this block led to the non-linear I–V relationship at positive membrane potentials. The channel was blocked by internal Ba²⁺ (0.1 mM) and tetracthylammonium (TEA⁺, 20–50 mM). Ba²⁺ reduced the open probability but not the single channel conductance, whereas TEA⁺ reduced the single channel conductance. The single channel conductance of this channel, measured from the inward current with a high-K⁺ solution (150 mM K⁺) in the pipette, was large (about 40 pS), and showed inward rectification. These results suggest that this channel is different from the usual small conductance Ca²⁺-activated K⁺ channels observed in many other cells.     

Intracellular calcium responses and shape conversions induced by endothelin in cultured subepithelial fibroblasts of rat duodenal villi

Subepithelial fibroblasts of rat duodenal villi were cultured and the physiological characteristics were studied using fura-2 fluorescence. The intracellular calcium concentration (Ca _i ²⁺ ) responded to various substances, i.e., endothelins (ET1 and ET3), substance P, serotonin, angiotensin II, ATP, and bradykinin. The Ca _i ²⁺ responses to ET1 (>0.1 nM) and ET3 (>1 nM) were transient and sometimes followed oscillations that consisted of an initial Ca²⁺ release from the intracellular store and a sustained Ca²⁺ influx. Simultaneously with Ca _i ²⁺ measurement, changes in the cell shape were monitored using fluorescence intensity upon 360-nm excitation. Stellate cells (with thick cell body and slender processes), formed as a result of 1 mM dibutyryl(Bt₂)-cAMP treatment, began to change immediately after the short-term application of the endothelin and became flat about 20 min later. This process was not affected by the depletion of extracellular Ca²⁺ or by the treatment with BAPTA acetoxymethyl ester that completely suppressed the Ca _i ²⁺ response. Substance P (>100 nM) increased Ca _i ²⁺ , but did not induce any morphological changes. The conversion of the shape from flat to stellate, induced by Bt₂cAMP treatment, was not accompanied by any Ca _i ²⁺ change. BQ-123, a specific blocker of the ET_A-type receptor, did not block either Ca _i ²⁺ change or shape conversion at low (100 nM) concentration. The results indicated that shape conversion in subepithelial fibroblasts did not require any Ca _i ²⁺ response. Our findings regarding the characteristics of subepithelial fibroblasts in intestinal villi imply a functional similarity to astrocytes in the brain.     

Evidence for the existence of inositol (1,4,5)-trisphosphate- and ryanodine-sensitive pools in bovine endothelial cells. Ca2+ releases in cells with different basal level of intracellular Ca2+

In single bovine aortic endothelial (BAE) cells pre-loaded with Fura-2, Ca²⁺ transients in a Ca²⁺-free medium have been revealed, which evidently reflects Ca²⁺ release from intracellular stores. In cells with different levels of resting basal cytoplasmic Ca²⁺ ([Ca²⁺]_i) from about 50 to 110 nM, a biphasic dependence of the Ca²⁺ transients on resting [Ca²⁺]_i was shown and spontaneous Ca²⁺ oscillations were observed. At a [Ca²⁺]_i level over 110 nM, a pronounced rise in Ca²⁺ transients occurred and only single transients were observed. Ryanodine (10 μM) produced a transient [Ca²⁺]_i elevation, suggesting the presence of ryanodine receptors in intracellular store membranes. The results imply that both inositol 1,4,5-trisphosphate-sensitive Ca²⁺ release (IICR) and Ca²⁺-sensitive Ca²⁺ release (CICR) take place in BAE cells. Only IICR seems to be sufficient for generating baseline Ca²⁺ oscillations in BAE cells, whereas the ATP-induced (5–100 μM) Ca²⁺ response involves the CICR set in motion by an oscillatory IICR of high frequency. The completion of both the spontaneous and ATP-induced Ca²⁺ transients was associated with a [Ca²⁺]_i decrease to a level below the initial resting [Ca²⁺]_i (undershoot). Its depth biphasically depended on the resting [Ca²⁺]_i from 50 to 110 nM, suggesting that the lack of a Ca²⁺ leak from inositol 1,4,5-trisphosphate-sensitive stores is responsible for the undershoot in this range. The Ca²⁺ leak is concluded to play a key role in the initiation and termination of regenerative IICR both in spontaneous oscillations and in ATP-induced transients. Received: 13 November 1995/Received after revision and accepted 27 March 1996     

Interactions between epithelial cells and dendritic cells in airway immune responses: lessons from allergic airway disease

Micro-organisms constantly invade the human body and may form a threat to our health. Traditionally, concepts of defence mechanisms have included a protective outer layer of epithelia and a vigilant immune system searching for areas where the integrity of the outer layer may be compromised. Instead of considering these elements as two independent mechanisms, we should be treating them as a single integrated system. This review will present and discuss the role of local immune-competent cells and local epithelia in the recognition of potential pathogens and how the interaction between the two components may affect the initiation of the airway immune response. A concept emerges where airway mucosal dendritic cells act as integrators of both immunostimulatory and immunosuppressive signals that act within actively-involved mucosal tissue.     

Changes of endoplasmic reticulum and mitochondria in mammary epithelial cells during mammogenesis in Chinese Holstein dairy cows

The objective of the research was to study the changes of the major organelles, endoplasmic reticulum (ER) and mitochondria, in mammary epithelial cells of the Chinese Holstein dairy cow during mammogenesis. For this purpose, a mammary epithelial cell bank was established from 9 selected Chinese Holstein dairy cows using collagenase I digestion and attachment culture biotechniques. This cell bank included 9 samples at stages of pregnancy, lactation and involution. The changes of ER and mitochondria in the mammary cells were observed at the subcellular level using living cell fluorescent labeling and laser confocal microscopy. Subsequently, the area of integrated optical density of each sample was calculated to determine changes of ER and mitochondria in the mammary epithelial cells. The results showed clear differences in the epithelial major organelles during the various mammary gland development stages. The ER and mitochondria, as an indicator of lactogenic activity of alveolar secretory cells, increased in number from pregnancy to lactation by an average 37.32% and 18.44%, respectively, which was followed by a reduction at involution by an average 38.04% and 22.91% compared to lactation. Our study shows that the stages of mammogenesis are accompanied by changes in activity of the major organelles of the mammary epithelial cells.     

Intracellular Ca2+ transients in HT29 cells induced by hypotonic cell swelling

Cell swelling induced by hypotonic solution led to an osmolality-dependent increase in intracellular Ca²⁺ activity ([Ca²⁺]_i) in HT₂₉ cells. At moderate reductions in osmolality from 290 to 240 or 225 mosmol/l in most cases only a small monophasic increase of [Ca²⁺]_i to a stable plateau of 10–20 nmol/l above resting [Ca²⁺]_i was observed. Lower osmolalities resulted in a triphasic increase of [Ca²⁺]_i to a peak value. In a first phase after the volume change, lasting 20–40 s, [Ca²⁺]_i increased slowly by about 30 nmol/l. Thereafter [Ca²⁺]_i increased more rapidly within 20–30 s to a peak value. This peak was 189±45 nmol/l (190 mosmol/l, n=9) and 243±41 nmol/l (160 mosmol/l, n=20) above resting [Ca²⁺]_i. The peak was then followed by a decline of [Ca²⁺]_i over the next 2–3 min to a stable plateau value of 28±6 (n=6) and 32±11 nmol/l (n=11) above resting [Ca²⁺]_i at 190 and 160 mosmol/l, respectively. The plateau lasted as long as the hypotonic solution was present. Under Ca²⁺-free bath conditions the peak value for the cell-swelling-induced [Ca²⁺]_i transient was reached significantly later (60–100 s, compared to 40–60 s under control conditions). The peak values under Ca²⁺-free conditions were not significantly lower. This indicates that the [Ca²⁺]_i peak was mostly of intracellular origin. No [Ca²⁺]_i plateau phase was observed under Ca²⁺-free bath conditions. With the use of the fura-2-Mn ²⁺ quenching technique an increased Ca²⁺ influx induced by hypotonic cell swelling was shown (160 mosmol/l; n=4). This influx started immediately after or simultaneously with the cell swelling and preceded the [Ca²⁺]_i peak for more than 50 s.This study was supported by DFG grant Gr 480/10.     

Contribution from P2X and P2Y purinoreceptors to ATP-evoked changes in intracellular calcium concentration on cultured myotubes

Although the alteration of purinoreceptor pattern on skeletal muscle is known to accompany physiological muscle differentiation and the pathogenesis of muscle dystrophy, the exact identity of and the relative contribution from the individual receptor subtypes to the purinergic signal have been controversial. To identify these subtypes in cultured myotubes of 5–10 nuclei, changes in intracellular calcium concentration and surface membrane ionic currents were detected and calcium fluxes calculated after the application of the subtype-specific agonists 2′3′-O-(benzoyl-4-benzoyl)-ATP (BzATP), 2-methyltio-ADP and UTP. The effectiveness of these agonists together with positive immunocytochemical staining revealed the presence of P2X₄, P2X₅, P2X₇, P2Y₁ and P2Y₄ receptors. siRNA-reduced protein expression of P2X₅, P2X₇ and P2Y₁ receptors was accompanied by reduction in the ATP-evoked calcium transients. Furthermore, anti-P2X₇ siRNA caused a significant drop in the early peak and delayed steady component of the calculated calcium flux. The use of its antagonist, oxidized ATP, similarly to transfection with anti-P2X₇ siRNA caused significant reduction in the agonist-elicited ionic currents I _ATP and I _BzATP, with a greater drop in the latter. Our results demonstrate that the activation of ionotropic P2X₄, P2X₅ and P2X₇ and metabotropic P2Y₁ and P2Y₄ purinoreceptors participates in forming the calcium transients of multinucleated myotubes.     

The time course of intracellular calcium movements in single human umbilical vein smooth muscle cells

Intracellular Ca²⁺ ([Ca²⁺]_i) was measured in single isolated human umbilical vein smooth muscle cells. Stimulation with histamine, in the absence of external Ca²⁺, mobilised Ca²⁺ from intracellular stores. When repeated brief applications of agonist were used, the time to onset, amplitude and rate of rise of the Ca²⁺ transients were found to change. Two components could often be discerned in the rising phase of the transients, an initial slow pacemaker and a second faster and larger component. Following the first histamine-activated transient the basal level of [Ca²⁺]_i was invariably lower than that prior to stimulation. This lower value was maintained whilst the cell remained in Ca²⁺-free solution, but could be returned to a higher level if the cell was exposed to external Ca²⁺. When the mobilisation of the intracellular store was reduced to undetectable levels, re-exposure to Ca²⁺-containing medium reactivated responses. In the absence of external Ca²⁺, continuous application of histamine activated a series of transient increases in intracellular Ca²⁺, which decreased progressively in amplitude and rate of rise. The interval between transients also increased. These findings are discussed in terms of the activation of inositol trisphosphate-sensitive intracellular Ca²⁺ stores and their sensitivity to cytoplasmic Ca²⁺ and intrasarcoplasmic reticulum Ca²⁺.     

Thapsigargin discharges intracellular calcium stores and induces transmembrane currents in human endothelial cells

We have measured the effects of thapsigargin, a specific inhibitor of endoplasmic Ca²⁺-adenosine 5-triphosphatase (Ca²⁺-ATPase), on membrane currents and on the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in single endothelial cells from the human umbilical cord vein. Currents were recorded by means of the patchclamp technique in the whole-cell mode and [Ca²⁺]_i was measured using Fura II. Application of thapsigargin at concentrations between 0.2 and 2 mol/l induced a slow increase in [Ca²⁺]_i to a peak value of 400±110 nmol/l above a resting level of 120±35 nmol/l, and then slowly declined to a new steady-state level of 315±90 nmol/l (n=33). The thapsigargin-induced increase in [Ca²⁺]_i depended on the extracellular Ca²⁺ concentration ([Ca²⁺]_o: it declined after removal of extracellular Ca²⁺, but increased again when [Ca²⁺]_o was augmented, indicating that the response depends on a transmembrane influx of Ca²⁺ ions. The peak amplitude of the histamine-induced Ca²⁺ transient was reduced in the presence of thapsigargin. This reduction was more pronounced when histamine was applied at the peak of the increase in [Ca²⁺]_i induced by thapsigargin than during the rising phase of the changes in [Ca²⁺]_i. The decline of the Ca²⁺ transient induced by histamine after washing out the agonist was also affected by thapsigargin. Before application of thapsigargin, this decline could be described by a single exponential with a time constant equal to 24.5±5 s (n=7). In the presence of thapsigargin, the decline was much slower (n =8 cells), although in four cells a fraction of about 23% still exchanged with a similar fast value of 29.4±4 s. Thapsigargin also induced a slowly developing inward current in endothelial cells at a holding potential of –40 mV. Voltage ramps applied before and during the development of this current indicated that a non-selective cation channel with a reversal potential near 0 mV was activated. In contrast with the Ca²⁺ transients, these currents did not show a declining phase. These results indicate that inhibition of the endoplasmic Ca²⁺ pump in endothelial cells increases [Ca²⁺]_i. The tonic component of this increase might be partly due to opening of non-selective Ca²⁺-permeable cation channels activated by depletion of intracellular stores.     

Effect of ageing on CA3 interneuron sAHP and gamma oscillations is activity-dependent

Normal ageing-associated spatial memory impairment has been linked to subtle changes in the hippocampal network. Here we test whether the age-dependent reduction in gamma oscillations can be explained by the changes in intrinsic properties of hippocampal interneurons.Kainate-induced gamma oscillations, but not spontaneous gamma oscillations, were reduced in slices from aged mice. CA3 interneurons were recorded in slices from young and aged mice using Fura-2-filled pipettes. Passive membrane properties, firing properties, medium- and slow-afterhyperpolarisation amplitudes, basal [Ca²⁺]_i and firing-induced [Ca²⁺]_i transients were not different with ageing. Kainate caused a larger depolarisation and increase in [Ca²⁺]_i signal in aged interneurons than in young ones. In contrast to young interneurons, kainate increased the medium- and slow-afterhyperpolarisation and underlying [Ca²⁺]_i transient in aged interneurons.Modulating the slow-afterhyperpolarisation by modulating L-type calcium channels with BAY K 8644 and nimodipine suppressed and potentiated, respectively, kainate-induced gamma oscillations in young slices.The age-dependent and stimulation-dependent increase in basal [Ca²⁺]_i, firing-induced [Ca²⁺]_i transient and associated afterhyperpolarisation may reduce interneuron excitability and contribute to an age-dependent impairment of hippocampal gamma oscillations.     

Prostaglandin F2α can modulate the growth and the differentiation of bovine corneal epithelial cells cultured in vitro

The effects of PGF_2α on the growth, morphology, morphometry and keratinization pattern of bovine corneal epithelial cells cultured in vitro were studied. The cells were grown with a basal medium or, in the presence of keratocytes and/or their products, using a keratocyte-conditioning medium. Cell growth was evaluated by MTT assay. Daily treatments with exogenous PGF_2α at concentrations equal to or lower than 10⁻⁶ M induced significant increases in cell proliferation when the epithelial cells were cultured on a keratocyte feeder-layer or with the conditioning medium. No variations were observed in cultures grown with the basal medium. 10⁻⁵ M PGF_2α induced a decrease in cell growth under all culturing conditions. PGF_2α did not affect cell morphology and modified only nuclear dimensions among the cells grown under different culturing conditions. No variations of any parameters were observed between cells cultured on feeder-layer, with conditioning or basal medium and the corresponding cultures supplemented with the autacoid. Moreover, PGF_2α induced only the disappearance of 43 kDa keratin in cells grown on basal medium, while the keratin pattern of epithelial cells cultured on feeder-layer or with the conditioning medium was not modified by the autacoid. From these data we can suppose that a cooperation could exist between PGF_2α and fibroblasts and their products for the modulation of cell growth. Finally, it was observed that the autacoid had no effect on cell morphology and morphometry, except for nuclear dimensions, despite the presence of other prostaglandins, such as PGE₂.     

ATP-induced Ca2+ signals in bronchial epithelial cells

 Ca²⁺-dependent Cl^– secretion in the respiratory tract occurs physiologically or under pathophysiological conditions when inflammatory mediators are released. The mechanism of intracellular Ca²⁺ release was investigated in the immortalized bronchial epithelial cell line 16HBE14o-. Experiments on both intact and permeabilized cells revealed that only inositol 1,4,5-trisphosphate (InsP ₃) receptors and not ryanodine receptors are involved in intracellular Ca²⁺ release. The expression pattern of the three InsP ₃ receptor isoforms was assessed both at the mRNA and at the protein level. The level of expression at the mRNA level was type 3 (92.5%) >> type 2 (5.4%) > type 1 (2.1%) and this rank order was also observed at the protein level. The ATP-induced Ca²⁺ signals in the intact cell, consisting of abortive Ca²⁺ spikes or fully developed [Ca²⁺] rises and intracellular Ca²⁺ waves, were indicative of positive feedback of Ca²⁺ on the InsP ₃ receptors. Low Ca²⁺ concentrations stimulated and high Ca²⁺ concentrations inhibited InsP ₃-induced Ca²⁺ release in permeabilized 16HBE14o- cells. We localized a cytosolic Ca²⁺-binding site between amino acid residues 2077 and 2101 in the type-2 InsP ₃ receptor and between amino acids 2030 and 2050 in the type-3 InsP ₃ receptor by expressing the respective parts of these receptors as glutathione S-transferase fusion proteins in bacteria. We conclude that the InsP ₃ receptor isoforms expressed in 16HBE14o- cells (mainly type-3 and type-2) are stimulated by Ca²⁺ and that this phenomenon contributes to the ATP-induced Ca²⁺ signals in intact 16HBE14o- cells. Recieved: 11 September 1997 / Received after revision: 2 January 1998 / Accepted: 21 January 1998     

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

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

Activation of calcium influx by ATP and store depletion in primary cultures of renal proximal cells

Cytoplasmic calcium changes and calcium influx evoked by adenosine triphosphate (ATP) were investigated in primary cultures of rabbit proximal convoluted tubule cells. Extracellular ATP (50 M) induced a biphasic increase of [Ca²⁺]_i measured with the calcium probe fura-2. In the early phase, the mobilization of intracellular pools resulted in a transient increase of [Ca²⁺]_i from 106±11 nM (n=36) to 1059±115% (n=29) of the resting level within 10 s. In the presence of external calcium, [Ca²⁺]_i then decreased within 3 min to a sustained level (398±38%,n=8). Measurements of fura-2 quenching by external manganese revealed that this phase was the result of an increased Ca²⁺ uptake, blocked by lanthanum (10 M) and verapamil (100 M) but not by the nifedipin (25 M). Internal calcium store depletion by ATP induced an increased calcium influx through lanthanum- and verapamil-sensitive, nifedipininsensitive calcium channels, located on the apical membrane of the cells. As indicated by⁸⁶Rb⁺ efflux measurements, ATP activated a potassium efflux that was blocked by barium andLeiurus quinquestriatus hebraeus (LQH) venom (containing charybdotoxin) indicating the involvement of Ca²⁺-sensitive K⁺ channels. Moreover, in the presence of the LQH venom, the internal calcium stores were not replenished after being depleted by ATP. Our results indicate that an ATPevoked hyperpolarization of the plasma membrane leads to increased Ca²⁺ influx, which facilitates the replenishment of the internal stores.     

Antidiuretic hormone acts via V1 receptors on intracellular calcium in the isolated perfused rabbit cortical thick ascending limb

The effect of antidiuretic hormone ([Arg]vasopressin, ADH) on intracellular calcium activity [Ca²⁺]_i of isolated perfused rabbit cortical thick ascending limb (cTAL) segments was investigated with the calcium fluorescent dye fura-2. The fluorescence emission ratio at 500–530 nm (R) was monitored as a measure of [Ca²⁺]_i after excitation at 335 nm and 380 nm. In addition the transepithelial potential difference (PD _te) and transepithelial resistance (R _te) of the tubule were measured simultaneously. After addition of ADH (1–4 nmol/l) to the basolateral side of the cTAL R increased rapidly, but transiently, from 0.84±0.05 to 1.36±0.08 (n = 46). Subsequently, within 7–12 min R fell to control values even in the continued presence of ADH. The increase in R evoked by the ADH application corresponded to a rise of [Ca²⁺]_i from a basal level of 155±23 nmol/l [Ca²⁺]_i up to 429±53 nmol/l [Ca²⁺]_i at the peak of the transient, as estimated by intra- or extracellular calibration procedures. The electrical parameters (PD _te and R _te) of the tubules were not changed by ADH. The ADH-induced Ca²⁺ transient was dependent on the presence of Ca²⁺ on the basolateral side, whereas luminal Ca²⁺ had no effect. d(CH₂)₅[Tyr(Me)²]²,Arg⁸vasopressin, a V₁ antagonist (Manning compound, 10 nmol/l), blocked the ADH effect on [Ca²⁺]_i completely (n = 5). The V₂ agonist 1-desamino-[d-Arg⁸]vasopressin (10 nmol/l, n=4), and the cAMP analogues, dibutyryl-cAMP (400 mol/l, n = 4), 8-(4-chlorophenylthio)-cAMP (100 mol/l, n = 1) or 8-bromo-cAMP (200 mol/1, n = 4) had no influence on [Ca²⁺]_i. The ADH-induced [Ca²⁺]_i increase was not sensitive to the calcium-channel blockers nifedipine and verapamil (100 mol/l, n = 4). We conclude that ADH acts via V₁ receptors to increase cytosolic calcium activity transiently in rabbit cortical thick ascending limb segments, possibly by an initial Ca²⁺ release from intracellular stores and by further Ca²⁺ influx through Ca²⁺ channels in the basolateral membrane. These channels are insensitive to L-type Ca²⁺ channel blockers, e.g. nifedipine and verapamil.Supported by DFG GR 480/10