The induction of intracellular calcium activity in cultured human myometrial smooth muscle cells.

Cells maintained in serum free medium for 24 hours were found to have a higher incidence of both spontaneous increases in intracellular calcium ([Ca2+]i) (Ca2+ spikes) and small random changes in [Ca2+]i (Ca2+ 'noise'). The spontaneous transient increases in [Ca2+]i and the Ca2+ 'noise' disappear in solutions containing nominally zero Ca2+.

Intracellular calcium 'signatures' evoked by different agonists in isolated bovine aortic endothelial cells.

Agonist induced increases in intracellular free calcium, [Ca2+]i, were measured in single Fura-2 loaded bovine aortic endothelial (BAE) cells by dual wavelength microspectrofluorimetry. Low doses of ATP (less than 10 microM) induced complex changes in [Ca2+]i. These changes usually consisted of a large initial transient peak with subsequent fluctuations superimposed upon a maintained rise in [Ca2+]i. Higher doses of ATP (greater than 50 microM) produced much simpler biphasic increases in [Ca2+]i in individual cells. Acetylcholine and bradykinin also elicited increases in [Ca2+]i in single cells in confluent monolayers of endothelial cells. However, only acetylcholine produced complex fluctuations. High doses of acetylcholine evoked simple rises in [Ca2+]i similar to those seen with high doses of ATP. In contrast, bradykinin evoked relatively simple rises in [Ca2+]i at all doses used. These results indicate that the mechanisms responsible for generating agonist induced increases in [Ca2+]i in BAE cells are not homogeneous. ATP and acetylcholine produced more complex Ca2+ changes or 'signatures' in single confluent bovine aortic endothelial cells than bradykinin. All three agonists appeared to release Ca2+ from intracellular stores as well as stimulating Ca2+ influx. The possible mechanisms underlying these phenomena are discussed.

Growth stimulation by bombesin does not involve activation of Na+/H+ exchange in chick embryo otic vesicles in vitro.

The effect of the mitogen bombesin on pHi in cells of the chick otic vesicle was studied using dual wavelength micro-spectrofluorimetric techniques. The results show that the bombesin did not produce an immediate alkalinization in quiescent otic vesicle cells nor a long-term change in pHi in vesicles reactivated to grow in culture. The recovery of pHi from an acid load, caused by an NH4 pulse, involved both Na+/H+ exchange and Na+:HCO3 influx mechanisms, neither of which was influenced by bombesin. These observations do not fit with a general model whereby pHi is a universal signal for DNA replication and cell proliferation.

An increase in intracellular free calcium is an early event during differentiation of cultured human keratinocytes.

The effect of [Ca2+]o on [Ca2+]i in cultured human keratinocytes was studied using dual-wavelength microspectrofluorometric techniques. The results show that increasing [Ca2+]o from 70 microM to 1 mM causes an early rise in [Ca2+]i complete by 2 h. Heterogeneity within cultures was demonstrated. The [Ca2+]i in spontaneously differentiated cells of low Ca2+ cultures was similar to that of Ca2+ induced differentiated cells. The increase in [Ca2+]i preceded the morphological changes and growth inhibition induced by increasing [Ca2+]o. These observations are consistent with an increase in [Ca2+]i mediating differentiation of human keratinocytes.

A novel ryanodine sensitive calcium release mechanism in cultured human myometrial smooth-muscle cells.

In cultured human myometrial cells application of caffeine (1-30 mM) did not result in an elevation of intracellular Ca2+ ([Ca2+]i). Caffeine was found to reversibly inhibit both spontaneous and agonist-induced repetitive rises in [Ca2+]i possibly as a consequence of its ability to interfere with the binding of inositol trisphosphate (IP3) to the receptor on the sarcoplasmic reticulum. Brief applications of ryanodine (1-10 microM) were observed to elevate [Ca2+]i and repeated exposures to ryanodine could elicit Ca2+ transients of similar magnitude. Ryanodine was also observed to mobilise Ca2+ in cells bathed in nominally Ca(2+)-free solution. These observations suggest the presence of a novel type of ryanodine-sensitive Ca(2+)-induced Ca2+ release (R-CICR) system in human myometrial cells.

Growth and differentiation stimuli induce different and distinct increases in intracellular free calcium in human keratinocytes.

The effect of growth and differentiation stimuli on intracellular free calcium ([Ca2+]i) in cultured human keratinocytes was investigated using micro-spectrofluorimetric techniques and the calcium-sensitive dye FURA-2. The mean [Ca2+]i of keratinocytes in 70 microM calcium medium was 104 +/- 3 nM (mean +/- SEM), significantly lower than the transformed keratinocyte line SVK14 (128 +/- 2 nM). When cultured in 2.0 mM calcium medium the [Ca2+]i increased in both normal and transformed keratinocytes to 135 +/- 4 nM and 180 +/- 4 nM, respectively. Keratinocytes grew more slowly in the absence of EGF, but [Ca2+]i was unaltered. Stimulation with EGF (10 ng/ml) induced, over 4 min, a large transient rise in [Ca2+]i up to 230 nm, due to an influx of extracellular calcium. Heterogeneity of keratinocytes was observed with 46% (n = 13) responding, but confluent or differentiated keratinocytes did not respond. TGF--beta (1 ng/ml) reduced cell growth without inducing differentiation and was not associated with any change in [Ca2+]i. The phorbol ester TPA (50 nM) induced irreversible growth arrest and terminal differentiation and increased the [Ca2+]i from 102 +/- 2 nM to 126 +/- 3 nM at 2 h, an effect similar to that of 2 mM extracellular calcium. Addition of 500 nM TPA was associated with a rise in [Ca2+]i, over several minutes to a plateau of 200-300 nM, due to release from internal stores and an influx of extracellular calcium. In normal human keratinocytes an increase in [Ca2+]i appears to be an early event in differentiation, whether induced by calcium or TPA, but not during growth inhibition without differentiation.

The selective permeability of the pancreatic duct of the cat to monovalent ions.

In the anaethetised cat the electrical potential difference between the lumen of the main collecting duct of the pancreas and blood in the jugular vein was measured. The duct was perfused with isotonic solutions of monovalent ions and the recorded potential corrected for the liquid junction potentials. The data were fitted to the Shinagawa extension of the Goldman constant field equation and the relative permeabilities of the duct epithelium to the ions were determined. The duct showed negligible selectivity between the monovalent cations Li:Na:K:Rb:Cs = 1.08:1.10:1.09:1.12 in contrast to the definite selectivity sequence for the anions F:Br:Cl:I:HCO3 = 0.44:1.38:1.08:2.05:0.60. This halide selectivity sequence is the Eisenman sequence I and is indicative of the selectivity being due to weak positive charges on membrane bound sites surrounding a highly hydrated channel. It is argued that these highly hydrated channels may be identified with the "tight junctions" between cells and the selectivity properties of the pancreatic duct are determined by flow of ions through these areas rather than flow through the epithelial cells.

The effects of cholecystokinin-pancreozymin, acetylcholine and secretin on the membrane potentials of mouse pancreatic cells in vitro.

Cell membrane potentials in an in vitro preparation of mouse pancreas have been measured and the effects of cholecystokinin-pancreozymin, acetylcholine and secretin studied. The membrane potential-frequency histogram has four distinct peaks and was split into the sum of four Gaussian distributions with means of minus 14, minus 23, minus 32 and minus 41mV. The minus 32 mV peak is attributed to acinar cells which respond to cholecystokinin-pancreozymin or acetylcholine by depolarising to minus 15 to minus 20 mV, the response lasting up to 10 min. The minus 23 and minus 41 mV peaks are due to duct cells. A duct cell has a resting membrane potential of minus 23 mV and hyperpolarises to minus 41 mV in response to secretin stimulation, the response lasting for longer than 15 min. Duct cells regardless of their position in the duct system function electrophysiologically in identical fashion. The secretory sites for enzyme and electrolyte are distinct, the duct cells secrete only electrolyte while the acinar cells secrete only enzymes.

Action of caerulein, gastrin 17, pentagastrin, and secretin on the active transport of sodium by the frog skin.

Frog skin was mounted in an Ussing chamber and the actions of caerulein, gastrin, pentagastrin, and secretin on the active transport of sodium were studied using the short-circuit current method. All polypeptides exerted their effect when placed in the solution bathing the outside surface of the skin. The response was a transient dose-related increase in the transepithelial electrical potential difference and in the short-circuit current. Analysis of the response indicated that at submaximal doses the effect was due to an increase in the rate of entry of sodium through the outer barrier to active sodium transport. At supramaximal doses the passive permeability of the skin was also increased. Th ED50 concentrations of the hormones were: caerulein, 50 pM; gastrin, 53 pM; pentagastrin, 440 pM; and secretin, 30 pM. It is argued that the large quantity of caerulein or caerulein-like peptides stored in the skin may be required either to control the entry of sodium when the amphibian is undergoing maximum stress in a freshwater environment, or that it may have a protective function for the amphibian as it could elicit a noxious hypersecretion in the gastrointestinal tract of the predator together with a marked hypotension.

Computed resolution and relative specific radioactivities of radiolabelled proteins synthesized by isolated gastric mucosal cells.

1. By radiolabelling, polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate and fluorography, more than 50 soluble proteins in the molecular-weight range 15000-100000 were shown to continue to be synthesized after cells had been isolated from rat gastric mucosa. 2. Densitometric measurements of stained gels and fluorographic films were processed by computer to resolve individual overlapping Gaussian peaks corresponding to the protein bands. 3. Comparison of resolved peak areas of radioactivity and staining showed certain bands to have characteristically high relative specific radioactivities. 4. The computer programs (in FORTRAN) permit the analysis of a single densitometric trace or the simultaneous comparison of a corresponding pair of densitometric records of stained gels, or of fluorographic films, or a combination. Central processing unit time is used economically. 5. The programs identify the Gaussian components that contribute to the records and estimate their means, standard deviations and enclosed areas. These estimates are improved by a piecewise iterative method that minimizes the errors between the calculated and the experimental data. 6. Relative specific radioactivities are calculated as the normalized ratio of the area of a fluorographic film peak and the area of the corresponding stained gel peak. The computer programs have been deposited as Supplementary Publication SUP 50094 (55 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms given in Biochem. J. (1978) 169, 5.

The kinetics of pancreatic amylase secretion and its relationship to volume flow and electrical conductance in the anaesthetized cat.

1. Enzyme secretion in response to short duration vagal stimulation or to rapid I.V. injections of cholecystokinin-pancreozymin (CCK-PZ) or gastrin or to intra-arterial injections of acetylcholine is a function of the volume of juice secreted and not primarily a function of time.2. The output of amylase in response to each stimulus occurred in a constant volume of approximately 15 drops (0.5 ml.) regardless of the rate of background secretin stimulated flow of water and electrolyte.3. It is argued that because amylase secretion occurs in this constant volume, it is due to the rapid secretion of enzyme into the duct system from the acini, and subsequently the secretin stimulated secretion of water and bicarbonate washes the enzyme from the duct system.4. When enzyme secretion is stimulated an increase in the electrical conductance (measured at 1.592 kHz) occurs across the tail of the pancreas. This increased conductance has two components. An early peak associated with the extrusion of enzyme from the acinar cell and a later peak which is probably due to vasodilatation.5. Atropine was without effect on both enzyme secretion and the conductance record when the stimulant was CCK-PZ. Atropine blocked enzyme secretion and both peaks of the conductance record in response to stimulation by acetylcholine. Atropine blocked enzyme secretion and abolished the early phase of the conductance record, on vagal stimulation. It was without effect on the later peak which probably indicates an atropine resistant vasodilation of the pancreatic vessels.6. When the vagus is stimulated on a background of submaximal electrolyte secretion caused by the intravenous infusion of secretin, the volume rate of secretion and the rate of amylase secretion follow a similar time course. The maximal volume response occurred between 7 Hz and 15 Hz and the maximal amylase output per impulse was at 5 Hz.

Changes in intracellular pH and pH regulating mechanisms in somitic cells of the early chick embryo: a study using fluorescent pH-sensitive dye.

1. Measurements of intracellular pH (pHi) have been made using the fluorescent dye dicyano-hydroquinone (DCH) in somites isolated from 2-day-old chick embryos. Measurements of pHi, from freshly dissected somites, gave values of 7.18 +/- 0.02 (mean +/- S.D., n = 12) while in somites kept in media containing 10% fetal calf serum for 2-5 h pHi was 7.36 +/- 0.02 (n = 11). 2. In freshly dissected control tissue recovery from an acid load (NH4Cl pre-pulse), in the nominal absence of HCO3-, was 0.007 +/- 0.003 pH units/min (n = 11). In the presence of 5 mM-HCO3- the rate of recovery was increased to 0.132 +/- 0.003 pH units/min (n = 11). This HCO3(-)-dependent recovery was inhibited by pre-treatment with DIDS (5 X 10(-4) M) and stopped when Na+ was replaced in the bathing medium with K+ or N-methylglucamine. Amiloride (10(-3) M) had no effect. 3. Replacement of Cl- with gluconate had little effect on pHi in control somites suggesting that absence of Cl(-)-HCO3- exchange. 4. These observations are consistent with the presence of a coupled Na+-HCO3- entry and the absence of Na+-H+ exchange. 5. In serum-treated cells recovery from an acid load was 0.101 +/- 0.01 pH units/min (n = 11) in the nominal absence of HCO3-. Increasing the HCO3- concentration (5-10 mM) or pre-treatment with DIDS had no effect on the rate of acid extrusion. Recovery is Na+ dependent and inhibitable with amiloride, indicating the presence of Na+-H+ exchange. 6. These results suggest that during somitic development the mechanisms regulating pHi and recovery from acid loading are modified from a Na+-HCO3- influx to a Na+-H+ exchanger. This transition allows the cells to increase pHi by up to 0.2 pH units and may have a role in somitic cell development.

Agonist concentration influences the pattern and time course of intracellular Ca2+ oscillations in human arterial smooth muscle cells.

Oscillations in intracellular Ca2+ were recorded in cultured human uterine artery vascular smooth muscle cells. In the absence of external Ca2+, prolonged application of 3 microM histamine activated a large transient increase in Ca2+ followed by a burst of Ca2+ spikes. The time course and frequency of the spikes were approximately constant until the last two to three spikes, when the inter-spike interval progressively increased. At 30 microM histamine the response was different; the amplitude of the spikes decreased rapidly to zero, the rate of rise of successive transients fell and the time between spikes increased. The cessation of oscillatory activity was not associated with the depletion of intracellular Ca2+ stores, since increased doses of agonist or the sulphydryl reagent thimerosal could reactivate Ca2+ release. The changes in the pattern of intracellular Ca2+ spikes seen with increasing agonist concentration may reflect the involvement of different inactivation mechanisms in the termination of Ca2+ transients. In the presence of external Ca2+, histamine (3-30 microM) activated regular Ca2+ oscillations. The frequency, but not the amplitude, of the oscillations was dependent on agonist concentration, the highest frequency of spiking was observed at 30 microM histamine. In cells depolarised with 30 mM K+, histamine was still able to activate Ca2+ oscillations, but the dependence of spike frequency upon agonist concentration was abolished. Ca2+ oscillations could be activated in the presence of verapamil and nifedipine (10 microM). These data suggest that in human uterine artery vascular smooth muscle cells histamine-induced Ca2+ oscillations are generated largely by a "cytosolic oscillator" and are modified by the influx of Ca2+ across the surface membrane.

Secretin-regulated chloride channel on the apical plasma membrane of pancreatic duct cells.

Using the patch clamp technique we have identified a small conductance ion channel that typically occurs in clusters on the apical plasma membrane of pancreatic duct cells. The cell-attached current/voltage (I/V) relationship was linear and gave a single channel conductance of about 4 pS. Since the reversal potential was close to the resting membrane potential of the cell, and unaffected by changing from Na+-rich to K+-rich pipette solutions, the channel selects for anions over cations in cell-attached patches. The open state probability was not voltage-dependent. Adding 25 mM-bicarbonate to the bath solution caused a slight outward rectification of the I/V relationship, but otherwise, the characteristics of the channel were unaffected. In excised, inside-out, patches the I/V relationship was linear and gave a single channel conductance of about 4 pS. A threefold chloride concentration gradient across the patch (sulphate replacement) shifted the single channel current reversal potential by -26 mV, indicating that the channel is chloride selective. Stimulation of duct cells with secretin (10 nM), dibutyryl cyclic AMP (1 mM) and forskolin (1 microM) increased channel open state probability and also increased the number of channels, and/or caused disaggregation of channel clusters, in the apical plasma membrane. Coupling of this channel to a chloride/bicarbonate exchanger would provide a mechanism for electrogenic bicarbonate secretion by pancreatic duct cells.

Measurement of intracellular calcium and pH in avian neural crest cells.

1. Intracellular pH (pHi) and calcium (Cai2+) were studied in freely migrating neural crest cells and in closely packed non-migrating cells derived from avian neural tubes in vitro, using the fluorescent dyes 2,3-dicyanohydroquinone (DCH) and Indo-1 to measure pHi and Cai2+ respectively. 2. In freely migrating crest cells the pHi was approximately 0.2 pH units more alkaline and Cai2+ 90 nM lower than in closely packed cells. 3. Experiments to establish the cellular mechanisms regulating pHi in isolated neural crest cells demonstrate the presence of Na(+)-H+ exchange in 66% of the cells and Na(+)-HCO3(-)-dependent pHi-regulating mechanisms in all cells examined. 4. Interactions between pHi and Cai2+ were examined. pHi was altered using either NH4Cl pulses resulting in small changes in Cai2+ or using a weak acid and base (propionate and trimethylamine), which produced a fall and a rise in Cai2+ respectively. 5. Exposure to Ca2(+)-free media caused a lowering of Cai2+ and induced a transient acidification. 6. Application of BAPTA-AM (50 microM), a cell-permeant analogue of EGTA, resulted in a fall in Cai2+ and an intracellular acidification. 7. Co2+ and La3+ (2 mM) each induced a reversible fall in Cai2+ that was accompanied by intracellular acidification. These data suggest the presence of a transmembrane flux of Ca2+ in the resting cells. 8. It would appear that the mechanisms influencing Cai2+ and pHi are linked. This idea is discussed in terms of possible mechanisms and roles for Ca2+ and pH as modulators of neural crest cell behaviour.

Agonist-induced fluctuations in cytoplasmic calcium in primary cultures of bovine endothelial cells.

Endothelial cells play an important role in the vascular responsiveness to many stimuli by releasing locally active agents. The intracellular signal which links the external stimulus to the release of the active compounds is almost certainly an elevation in cytoplasmic calcium (Cai2+). Thus a detailed knowledge of Cai2+ regulation is central to an understanding of the physiology and pharmacology of endothelial cells. The present experiments, on single bovine aortic endothelial cells, demonstrate that agonists stimulate complex changes in Cai2+. These include rapid and regular fluctuations in Cai2+ which are different from the oscillations reported in other endothelial cells and non-excitable cells. The fluctuations are completely abolished in media containing low calcium, 2 mM-cobalt or caffeine but are not affected if the cells are bathed in isotonic potassium solutions. The hypothesis is put forward that the fluctuations in Cai2+ are associated with localized influxes of calcium and are possibly involved with the recycling of calcium between the internal stores, the cytoplasm and the external medium.

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 Ca(2+)-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 Ca(2+)-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+.

Interactions between intracellular pH and calcium in single mouse neuroblastoma (N2A) and rat pheochromocytoma cells (PC12).

Intracellular pH (pHi) was measured at the tips of extending neurites and in the corresponding cell bodies of single cultured mouse neuroblastoma (N2A) and rat pheochromocytoma cells (PC12) using the fluorescent dye 2,3-di-cyanohydroquinone (DCH). It was observed that pHi at the tip of an extending neurite was consistently 0.2-0.3 pH units higher than pHi in the cell body. Experiments performed on whole cells to establish the types of cellular mechanism which could be responsible for such regional differences demonstrate the presence of Na+-H+ exchange and Cl- HCO3- exchange in these cells. Since regional variations in Ca2i+ have been reported between neurites and the cell body, experiments were performed to examine the possible interactions between pHi and Ca2i+. Intracellular calcium was measured using the fluorescent Ca2+-sensitive dye Indo-1. An increase in pHi, on application of NH4Cl, resulted in a transient elevation of Ca12i+. On subsequent acidification, on removal of NH4Cl, there was a further transient increase in Ca2i+. These changes in Ca2i+ were also present in solutions with low calcium suggesting that Ca2i+ is mobilized from within the cell. The results are discussed in terms of possible mechanisms whereby the extension and retraction of cell processes could be influenced by Ca2i+ and modulated by pHi.

Repetitive transients in intracellular Ca2+ in cultured human vascular smooth muscle cells.

Human uterine vascular smooth muscle cells have been isolated and maintained in culture. When these cells are exposed to bathing solutions with nominally zero sodium, using potassium, N-methyl-D-glucamine or Tris as substitutes, repetitive transient increases in intracellular calcium are observed. These transients are abolished when the calcium concentration of the bathing solution is reduced to nominally zero suggesting a role for extracellular calcium in the activation or maintenance of the transients. The hypothesis is proposed that the underlying mechanism involves a calcium influx through the reversed operation of a sodium-calcium exchange mechanism and the cyclical activation of calcium-induced calcium release from the sarcoplasmic reticulum. Noradrenaline (10(-6) M) and caffeine (20-30 mM) reversibly inhibited the transients. The inhibitory action of these agents could not be mimicked by dibutyryl cAMP suggesting that cAMP does not mediate the inhibition. Caffeine alone had no effect on resting calcium. Thimerosal (1-100 microM), an agent thought to activate a second type of calcium-induced calcium release mechanism activated repetitive transient increases in intracellular calcium which behave in a similar manner to those activated by sodium removal. These data are consistent with the presence of a thimerosal-activated calcium-induced calcium release mechanism in these cultured human cells. It is proposed that this mechanism is different from the calcium-induced calcium release mechanism, described in other cell types, which is activated by caffeine.