Dynamics of Ca2+ i and pHi in Ehrlich ascites tumor cells after Ca2+-mobilizing agonists or exposure to hypertonic solution

 Intracellular free calcium concentration ([Ca²⁺]_i) and intracellular pH (pH_i) were monitored in Ehrlich ascites tumor cells using Fura-2 or 2′,7′,-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF), or both probes in combination. An increase in [Ca²⁺]_i induced by thrombin or bradykinin, agonists known to elicit transient cell shrinkage in these cells, evoked a transient intracellular acidification, followed by an alkalinization. The latter was due to activation of a Na⁺/H⁺ exchanger and was inhibited under conditions preventing agonist-induced cell shrinkage without preventing the increase in [Ca²⁺]_i. In contrast, a smaller, slower increase in [Ca²⁺]_i elicited by thapsigargin did not cause cell shrinkage, and did not activate the Na⁺/H⁺ exchanger. Exposure to hypertonic solution was not associated with an increase in [Ca²⁺]_i, but elicited an intracellular alkalinization similar to that induced by thrombin or bradykinin, via activation of the Na⁺/H⁺ exchanger. Thus, activation of the exchanger by the Ca²⁺-mobilizing agonists is suggested to be secondary to the cell shrinkage induced by these compounds. NH₄Cl-induced intracellular alkalinization resulted in an increase in [Ca²⁺]_i, apparently via stimulation of Ca²⁺ influx, whereas shrinkage-induced intracellular alkalinization did not stimulate Ca²⁺ influx. Thus, cell shrinkage appears to inhibit the Ca²⁺ influx otherwise resulting from alkalosis. In agreement with that notion, thapsigargin-induced Ca²⁺ influx was inhibited by cell shrinkage. Received: 6 January 1998 / Received after revision: 10 March 1998 / Accepted: 11 March 1998     

Mechanism of intracellular calcium oscillations in fibroblasts expressing the ras oncogene

In NIH fibroblasts expressing the ras oncogene bradykinin leads to sustained, calcium-dependent oscillations of cell membrane potential by oscillating activity of calcium sensitive potassium channels. The present study has been performed to further analyse the underlying mechanisms. In cells expressing the oncogene, but not in NIH fibroblasts not expressing the oncogene, bradykinin elicits calcium oscillations, which are detected by fura-2 fluorescence and amplified by a decrease of extracellular sodium activity. These calcium oscillations are dependent on the presence of extracellular calcium and are inhibited by lanthanum ions. It is concluded that in cells expressing the ras oncogene, bradykinin activates lanthanum sensitive calcium entry from the extracellular space. Ras oncogene expression leads to enhanced bradykinin-induced formation of both, 1, 4, 5 inositoltrisphosphate and 1, 3, 4, 5 inositoltetrakisphosphate, an effect probably accounting for the oscillations of intracellular calcium activity.     

Effects of inhibitors and ion substitutions on oscillations of cell membrane potential in cells expressing the RAS oncogene

Previous studies revealed that in NIH fibroblasts expressing the ras oncogene but not in other NIH fibroblasts, bradykinin leads to sustained, calcium dependent oscillations of cell membrane potential by repetitive activation of calcium-sensitive K⁺ channels. The present study has been performed to test for ion and inhibitor sensitivity of these oscillations. Both, Lys-bradykinin (kallidin) and bradykinin, but not any shorter peptide tested, maintained the oscillations. The oscillations are abolished in the presence of the K⁺ channel blocker barium (10 nmol/l). The amplitude but not the frequency of the oscillations is dependent on the extracellular potassium concentration. The oscillations are not dependent on the presence of extracellular sodium, bicarbonate or chloride. The oscillations are abolished in the absence of extracellular calcium and their frequency is significantly decreased at reduced extracellular calcium (to 0.2 mmol/l). The oscillations are not inhibited by acute administration of ouabain (0.1 mmol/l), by dimethylamiloride (100 mol/l), furosemide (1 mmol/l) and hydrochlorothiazide (100 mol/l), by cobalt (100 mol/l), zinc (100 mol/l), gadolinium (100 mol/l), verapamil (10 mol/l) and diltiazem (10 mol/l), but are abolished in the presence of 100 mol/l lanthanum, 1 mmol/l cadmium, 10 mol/l nifedipine, 25 mol/l SK & F 96365 and 200 mol/l TMB-8. Stimulation of calcium entry by 10 mol/l ionomycin is frequently followed by oscillations of cell membrane potential even in the absence of bradykinin. In conclusion, in cells expressing the ras oncogene bradykinin leads to sustained activation of calcium channels at the cell membrane, which cause oscillations of the cell membrane potential by triggering intracellular calcium release.     

Bradykinin causes contraction in rat uterus through the same signal pathway as oxytocin

The signal pathway for bradykinin-induced contraction of the uterine smooth muscle was investigated by comparing the effect of blocking agents on bradykinin and oxytocin induced contractions of the isolated rat uterus in organ bath. The phospholipase C inhibitor U-73122 abolished the effect of both bradykinin and oxytocin. Inhibition of non-voltage-dependent Ca²⁺ influx by SK & F 96365 reduced the contraction induced by both agonists to about 20% of control. The tissues failed to contract when they were exposed to bradykinin or oxytocin in Ca²⁺-free Krebs–Henseleit buffer with 2 mM EDTA. Both bradykinin and oxytocin induced further contraction when the tissues were partially depolarized and partially contracted by 30 mM KCl. These observations suggest that bradykinin, like oxytocin, activates phospholipase C which generates IP₃ with a subsequent release of Ca²⁺ from intracellular stores followed by store-operated Ca²⁺ influx. Thus, membrane potential independent steps appear to be important in bradykinin-induced contraction in the rat uterus.     

Cell transformation induces a cytoplasmic Ca2+ oscillator in Madin-Darby canine kidney cells

Alkaline stress transforms Madin-Darby canine kidney (MDCK) cells as indicated by loss of epithelial structure, multilayer cell growth and formation of foci. In the present study we report that transformed MDCK cells (MDCK-F cells) exhibit spontaneous and lasting oscillations of intracellular Ca²⁺ concentration ([Ca²⁺]_i), which are absent in non-transformed cells. Oscillations, as revealed by Fura-2 video imaging, were due to the activity of an inositol 1,4,5-trisphosphate-(InsP ₃)-sensitive Ca²⁺ store since their frequency was dependent on bradykinin concentration and they were abolished by the phosphoinositidase C inhibitor U73122. Moreover, blockers of the cytoplasmic Ca²⁺-ATPase, thapsigargin and 2,5-di-(tetr-butyl)-1,4-benzohydroquinone inhibited oscillatory activity. In contrast, neither injection of ruthenium red, ryanodine nor caffeine had any effect on oscillations. Analysis of the spatial distribution of [Ca²⁺]_i showed that Ca²⁺ transients originated from an initiation site constant for a given cell and spread through the cell as an advancing Ca²⁺ wave. Oscillations started in a random manner from single cells and spread over neighbouring cells, suggesting a kind of intercellular communication. We conclude that MDCK-F cells have acquired the ability for endogenous Ca²⁺ release through transformation. Oscillations are primarily due to the activity of an InsP ₃-sensitive cytosolic Ca²⁺ oscillator.     

Cytoplasmic Ca2+ determines the rate of Ca2+ entry into Mardin-Darby canine kidney-focus (MDCK-F) cells

Transformed Mardin-Darby canine kidney-focus (MDCK-F) cells exhibit spontaneous Ca²⁺ oscillations from an inositol 1,4,5-trisphosphate-sensitive cytoplasmic Ca²⁺ store. In this study, Ca²⁺ entry from the extracellular space and its role in generation of oscillations were investigated by means of Ca²⁺ video imaging and the Fura-2/Mn²⁺ quenching technique. Oscillations were dependent on extracellular Ca²⁺ concentration and were inhibited by extracellularly applied La³⁺, Co²⁺ and Ni²⁺. Depolarization of the cell membrane with high K⁺ concentrations and the L-type Ca²⁺ channel blocker nifedipine had no effect on oscillations, indicating the lack of involvement of voltage-gated Ca²⁺ channels. Mn²⁺ quenching experiments disclosed significant Ca²⁺ influx into MDCK-F cells. The rate of this influx was constant between Ca²⁺ spikes, but markedly increased during the spontaneous Ca²⁺ spikes. Similar transient increases in Ca²⁺ entry could be mimicked by agents triggering intracellular Ca²⁺ release such as bradykinin and thapsigargin. We conclude that the plasma membrane of MDCK-F cells exhibits a marked voltage-independent Ca²⁺ permeability permitting Ca²⁺ entry into the cytoplasm. The rate of Ca²⁺ entry which determines the frequency of oscillations is most likely to be regulated by the cytoplasmic Ca²⁺ concentration.     

Activation of different pathways for calcium elevation by bradykinin and ATP in rat pheochromocytoma (PC 12) cells

We have studied the pathways by which extra-cellular bradykinin and adenosine 5-triphosphate (ATP) elicit changes in intracellular free calcium ([Ca²⁺]_i) in nerve-growth-factor(NGF)- treated rat pheochromocytoma (PC 12) cells. Both substances caused a significant rise in [Ca²⁺]_i as assessed by fura-2 based micro-fluorimetry. The bradykinin-induced response consisted of an initial Ca²⁺ mobilization from an internal pool followed by a sustained increase in [Ca²⁺]_i, which was due to activation of a small inward current. The initial response always started at a localized site opposite to the cell nucleus. The inward current was partially carried by Ca²⁺ and began with a time lag of about 4 s after the start of the initial transient signal. Stepwise hyperpolarization of the plasma membrane, after activation of the inward current by bradykinin, caused a simultaneous increase in current amplitude and in [Ca²⁺]_i, due to an increase in the driving force for Ca²⁺ influx. With ATP as an agonist the onset of inward current coincided with an increase in [Ca²⁺]_i. Inward current and [Ca²⁺]_i were enhanced during hyperpolarizing steps indicating a substantial Ca²⁺ influx through ATP-activated channels. No release of Ca²⁺ from internal stores, but a large Na⁺ inward current, was observed in Ca²⁺-free external solution after addition of ATP. While the bradykinin-induced responses were much more pronounced in cell bodies than in growth cones, the ATP effects were somewhat variable in cell bodies and more homogeneous in growth cones.     

Endothelin-induced electrical activity and calcium dynamics in vascular smooth muscle cells: A model study

A model is proposed to describe the electrical activitity and intracellular calcium dynamics of vascular smooth muscle cells (SMC) induced by endothelin (ET1). The conductance of the nonselective channels (NSCs), proportional to the ET1-receptor complex (ET·R), is intracellular calcium dependent. Inositol (1,4,5)-trisphosphate (IP₃) produced by ET1 releases Ca²⁺ from the IP₃-sensitive Ca²⁺ store. The transient increase of intracellular Ca²⁺ triggers the release of Ca²⁺ from the Ca²⁺-sensitive store by a Ca²⁺-induced Ca²⁺ (CICR) mechanism and activates the Ca²⁺-activated K⁺ current (I _K,Ca) The inward current (I _in) via the NSC can depolarize the cell to a level at which the L-type Ca²⁺ current becomes activated (I _Ca). The level of depolarization is determined by the relative amplitude of (I _in+I _Ca+I _K.Ca) and the voltage- and time-dependent K⁺ current. The model simulations show that (a) in cells without a CICR mechanism, short-lasting stimulation by ET1 elicits higher membrane potential and Ca²⁺ than long-lasting stimulation; (b) in cells with or without a CICR mechanism, a reduction of normal membrane capacitance (1 μf/cm²) results in either significant and sustaining or oscillatory membrane potential and intracellular calcium concentration. The applicability of the model to the study of electrical activity and calcium dynamics associated with hypercholesterolemia is discussed.     

[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.     

Global,synchronous oscillations in cytosolic calcium and adherence in bradykinin‐stimulated Madin‐Darby canine kidney cells

Aims and Methods: Intercellular Ca²⁺ oscillations are a universal mode of signalling in both excitable and non‐excitable cells. Here, we study the relationship between Ca²⁺ signalling and coherent changes in adhesion properties by measuring the transepithelial impedance across bradykinin‐stimulated Madin‐Darby canine kidney (MDCK) cell layers grown on a microelectrode. During hormone stimulation, the impedance is found to oscillate, reflecting that the cells undergo morphological/adhesive alterations with high spatio‐temporal organization. The experiments are supplemented with parallel, digital imaging fluorescence microscopy of bradykinin‐induced single‐cell Ca²⁺ oscillations. Results: In agreement with previous experiments, MDCK cells are found to elicit synchronous, multicellular Ca²⁺ oscillations in response to hormone stimulus. The periods of the Ca²⁺ oscillations and the electrical fluctuations are found to coincide. Further, blocking of gap junctions by 18α‐glycyrrhetinic acid causes a loss of synchrony in Ca²⁺ signals and inhibition of impedance oscillations, emphasizing the importance of gap junctions in the signal transduction process. Conclusion: Based on these observations it is concluded that the co‐ordinated adhesive changes in MDCK cells are a direct consequence of synchronized Ca²⁺ oscillations. Calcium signalling represents an efficient way of organizing physiological responses in a tissue. A possible functional implication of the structural changes might be to modulate transportation of various substances across the cell sheet.     

Caffeine induces periodic oscillations of Ca2+-activated K+ current in pulmonary arterial smooth muscle cells

The periodic oscillations of outward currents were studied in smooth muscle cells of the rabbit pulmonary artery. The combined stimuli of superfusion with 1 mM caffeine and depolarization of the membrane potential to 0 mV evoked periodic oscillations of outward currents with fairly uniform amplitudes and intervals. The oscillating outward currents induced by caffeine were dependent on intracellular Ca²⁺ concentration ([Ca²⁺]_i) and had a reversal potential near to the equilibrium potential for K⁺. So the oscillating outward currents are carried by K⁺ through Ca²⁺-dependent K⁺ channels (I _K(Ca)), and may reflect the oscillations of [Ca²⁺]_i. The oscillating outward currents were abolished, or their frequency reduced, by lowering external [Ca²⁺], Ca²⁺ channel blockers, or by 1 M ryanodine, indicating that: (1) there is a continuous influx of Ca²⁺ through the plasma membrane at a holding potential of 0 mV; (2) the periodic transient increases of [Ca²⁺]_i are ascribed to the rhythmic release of Ca²⁺ from ryanodinesensitive intracellular store by the mechanism of Ca²⁺-induced Ca²⁺ release (CICR). On the basis of the above results, we simulated the oscillation of [Ca²⁺]_i induced by caffeine, which is known to lower the threshold of CICR. The patterns of peak amplitude histograms of spontaneous transient outward currents (STOC) in the oscillating cells were different from those in non-oscillating cells. The amplitudes of STOC in the latter were more variable than those in the former. The oscillating outward currents were modulated by 1 M forskolin and 1 M sodium nitroprusside, but STOC were little affected. The above differences between STOC and oscillating outward currents suggest that the two currents are activated by the Ca²⁺ originating from different intracellular Ca²⁺ stores which are functionally heterogeneous.     

Action of stimulatory and inhibitory α-MSH secretagogues on spontaneous calcium oscillations in melanotrope cells of Xenopus laevis

The secretion of α-melanophore-stimulating hormone (α-MSH) from melanotrope cells in the pituitary gland of Xenopus laevis is regulated by various neural factors, both classical neurotransmitters and neuropeptides. The majority of these cells (80%) display spontaneous Ca²⁺ oscillations. In order to gain a better understanding of the external regulation of intracellular Ca²⁺ ([Ca²⁺]_i) in the melanotrope cell, we have examined the action of well known α-MSH secretagogues on the Ca²⁺ oscillations. It is shown that all secretagogues tested also control the oscillatory state of Xenopus melanotropes, that is, the secreto-inhibitors dopamine, isoguvacine (γ-aminobutyric acid, GABA_A agonist), baclofen (GABA_B agonist) and neuropeptide Y evoked a rapid quenching of the spontaneous Ca²⁺ oscillations, whereas the secreto-stimulant sauvagine, an amphibian peptide related to corticotropin releasing hormone, induced oscillatory activity in non-oscillating cells. Supporting argument is given for the idea that the regulation of Ca²⁺ oscillations is a focal point in the regulation of secretory activity of melanotrope cells. There was considerable heterogeneity among melanotrope cells in the threshold of their Ca²⁺ response to secretagogue treatment. This heterogeneity may be the basis for melanotrope cell recruitment observed during physiological adaptations of the animal to the light intensity of its background.     

The role of intracellular Ca2+ pool in sodium nitroprusside-induced relaxation of rat aorta smooth muscle cells

A relaxation effect of sodium nitroprusside on smooth muscle cells of rat aorta due to intracellular Ca²⁺-store refilling is demonstrated using the double sucrose gap technique. It is suggested that sodium nitroprusside-induced repolarization of the smooth muscle cell membrane is associated with inhibition of Ca permeability and/or Ca-dependent K⁺ permeability of the plasma membrane. Translated fromByulleten' Eksperimental'noi Biologii I Meditsiny, Vol. 127, No. 2, pp. 177–179, February 1999     

Cell membrane stretch in osteoclasts triggers a self-reinforcing Ca2+ entry pathway

Many cell types respond to mechanical membrane perturbation with intracellular Ca²⁺ responses. Stretch-activated (SA) ion channels may be involved in such responses. We studied the occurrence as well as the underlying mechanisms of cell membrane stretche-voked responses in fetal chicken osteoclasts using separate and simultaneous patch-clamp and Ca²⁺ imaging measurements. In the present paper, evidence is presented showing that such responses involve a self-reinforcing mechanism including SA channel activity, Ca²⁺-activated K⁺ (K_Ca) channel activity, membrane potential changes and local and general intracellular Ca²⁺ ([Ca²⁺]_i) increases. The model we propose is that during membrane stretch, both SA channels and K_Ca channels open at membrane potential values near the resting membrane potential. SA channel characterization showed that these SA channels are permeable to Ca²⁺. During membrane stretch, Ca²⁺ influx through SA channels and hyperpolarization due to K_Ca channel activity serve as positive feedback, leading ultimately to a Ca²⁺ wave and cell membrane hyperpolarization. This self-reinforcing mechanism is turned off upon SA channel closure after cessation of membrane stretch. We suggest that this Ca²⁺entry mechanism plays a role in regulation of osteoclast activity.     

Transient membrane hyperpolarizations due to spontaneous fluctuations of the cytosolic Ca2+ in osteoblast-like cells

Summary The recently developed nystatin modification of the patch clamp technique allows stable whole-cell recordings without affecting the intracellular Ca²⁺ buffering capacity and thereby may provide a means to indirectly monitor spontaneous changes in the intracellular Ca²⁺ concentrations. To test this hypothesis, we applied the nystatin method to the well-characterized ROS 17/2.8 osteoblast-like cell system, where rises of the intracellular Ca²⁺ are known to cause transient hyperpolarizations via activation of Ca²⁺ -dependent K⁺ channels. Additionally to minor fluctuations (10–20 mV) around a mean potential of –42.1±4.2 mV, we observed spontaneously occurring, transient hyperpolarizations to membrane potentials as negative as –80 mV. These transient hyperpolarizations were not eliminated by Ca²⁺ entry blockers but abolished by intracellular infusion of 10 mM EGTA. Thapsigargin, a specific inhibitor of the endoplasmic reticulum Ca²⁺ -ATPase, hyperpolarized the cells close to the K⁺ reversal potential. Moreover, voltage-clamp studies revealed an intermittendly activating Ca²⁺ -dependent K⁺ conductance. These results strongly suggest that the nystatin method is particularly suitable to study Ca²⁺ -dependent channels and thereby spontaneous changes in the intracellular Ca²⁺.     

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     

Bradykinin and inositol 1,4,5-trisphosphate-stimulated calcium release from intracellular stores in cultured bovine endothelial cells

The relative importance of intracellular and extracellular Ca²⁺ in the release of endothelium-derived relaxing factor (EDRF) and the mechanisms involved in the release of intracellular Ca²⁺ were investigated in cultured bovine endothelial cells. The release of EDRF by bradykinin, determined by bioassay, was dose-dependent showing an EC₅₀ of 4×10^–10 M. The bradykinin-induced EDRF release from endothelial cells was maintained in the presence of extracellular Ca²⁺. However, in the absence of external Ca²⁺, bradykinin-induced EDRF release was both attenuated and transient. In cells loaded to isotopic equilibrium with⁴⁵Ca, bradykinin increased the⁴⁵Ca efflux into both calcium-containing and calcium-free solutions, with an EC₅₀ for the increase in⁴⁵Ca efflux induced by bradykinin of 1.3×10^–9 M. The involvement of an intracellular Ca²⁺ store and the participation of a second messenger in its release were investigated in saponin-permeabilized endothelial cells. In saponin-permeabilized cells, ATP-sensitive calcium uptake was Ca²⁺,Mg²⁺-ATPase-dependent. The ATP-sensitive uptake of calcium at different free Ca²⁺ concentrations showed at least two compartments involved in the uptake of Ca²⁺. The⁴⁵Ca uptake into the compartment with the lowest affinity and highest capacity could be inhibited by sodium azide, suggesting that this uptake was into mitochondria. The majority of the⁴⁵Ca uptake into the azide-insensitive store could be released by inositol-1,4,5-trisphosphate (IP₃). The IP₃-induced release was not affected by apyrase or exogenous GTP. The EC₅₀ for the release of Ca²⁺ by IP₃ was 1.0 M and was unaffected by an inhibitor of IP₃ breakdown (2,3-diphosphoglyceric acid). The results suggest that the release of EDRF is dependent on extracellular Ca²⁺ influx and the release of intracellular Ca²⁺. The release of calcium from one of the high affinity intracellular Ca²⁺ stores is mediated by the intracellular second messenger, IP₃.     

Studies on the mechanism of swelling-induced lysosomal alkalinization in vascular smooth muscle cells

Previous studies in renal cells and hepatocytes have shown that cell swelling leads to a rapid and reversible increase in pH in acidic cellular compartments, including lysosomes. Among the consequences are an inhibition of proteolysis. The present study shows that a similar lysosomal alkalinization occurs upon osmotic swelling of vascular smooth muscle cells, as evidenced by acridine orange and fluorescein isothiocyanate fluorescence. Furthermore, we have studied the mechanism underlying lysosomal alkalinization, which had remained unclear. The lysosomal alkalinization was not abolished by inhibition of vacuolar H⁺-ATPases (100 nM bafilomycin), Cl^– channels [100 M] 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), carbonic anhydrase (100 M acetazolamide) or Na⁺/H⁺ exchange (10 M HOE 694). The Ca²⁺ ionophore A23187 (10 M) led to a slight increase in lysosomal pH, but removal of extracellular Ca²⁺ and depletion of cellular Ca²⁺ stores (100 nM thapsigargin) did not appreciably blunt the swelling-induced lysosomal alkalinization. In the presence of bafilomycin the alkalinizing effect of osmotic cell swelling was not reversible, in contrast to that of NH₄Cl. In conclusion, osmotic swelling of vascular smooth muscle cells leads to lysosomal alkalinization, presumably in large part through activation of a hydrogen ion leak.     

Membrane rectification in single smooth muscle cells from the rat tail artery

Membrane rectification to depolarization was studied by voltage recording with patch electrodes in freshly isolated cells from the rat tail artery. Injection of depolarizing currents elicited electrotonic potentials that developed with a single-exponential time course (time constant of 94.8 ms). When the cell was depolarized beyond –30 mV, delayed rectification was observed. A second type of rectification, characterized by oscillations, was observed when the cell was depolarized positive to + 30 mV. The threshold of this rectification and the oscillations were sensitive to changes in intracellular Ca²⁺. Delayed rectification was more sensitive to 4-aminopyridine but more resistant to tetraethylammonium and charybdotoxin than the Ca²⁺-sensitive rectification. A 4-aminopyridine-sensitive outward current (I _K,dr) with a threshold of around –30 mV and a second Ca²⁺-sensitive outward current (I _K,Ca) activated at around + 30 mV were observed from whole-cell voltage clamp recordings. I _K,Ca was blocked by tetraethylammonium and charybdotoxin. An 11-pS and a 122-pS channel, having characteristics similar to I _K,dr and I _K,Ca respectively, were identified from single-channel recordings. These observations showed how membrane depolarization of vascular smooth-muscle cells was regulated by these two populations of K⁺ channels under various conditions.     

Alkaline stress transforms Madin-Darby canine kidney cells

Similar to growth factors aldosterone stimulates Na⁺/H⁺ exchange in renal target cells leading to cytoplasmic alkalinization. An alkaline intracellular pH reduces the H⁺ bonds between repressor proteins and DNA leading to the destabilization of the nuclear chromatin. We observed that sustained alkaline stress per se can lead to malignant transformation of Madin-Darby canine kidney (MDCK) cells. Cells grown for two weeks in alkaline culture medium (pH 7.8) developed multiple foci composed of spindle-shaped pleomorphic cells lacking contact inhibition and exhibiting poor adhesion to the culture support, typical characteristics of dedifferentiated tumor cells. Focus cells were cloned and grown in standard medium (pH 7.4). Cells maintained their abnormal growth pattern, indicating stable pH-induced genetic transformation. Cells were fused with polyethylene glycol to giant cells and impaled with microelectrodes. In contrast to non-transformed giant MDCK cells the plasma membrane potential showed spontaneous oscillations that could be virtually abolished by the omission of extracellular Ca²⁺ or by the addition of the K⁺ channel blocker Ba²⁺. We conclude that sustained alkaline stress can induce malignant transformation in MDCK cells indicated by an abnormal growth pattern and by membrane potential oscillations most likely due to Ca²⁺ activated K⁺ channels in the plasma membrane.