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.

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+.

Measurements of intracellular Ca2+ in cultured human myometrial smooth muscle cells bathed in low Na+ solutions.

In cultured human myometrial smooth muscle cells, removal of external Na+ activates repetitive increases in intracellular Ca2+ ([Ca2+]i). The Ca2+ transients persist in isotonic K+ solutions which suggests that the activity does not arise from regenerative changes in membrane potential. In nominally Ca(2+)-free solution, the activity disappears after a few cycles suggesting the involvement of internal stores. On Na+ removal, a background influx of Ca2+ may be responsible for activating the cyclical release of Ca2+ from internal stores. The absence of any effect of caffeine on resting [Ca2+]i suggests that the classical cardiac type of Ca(2+)-induced-Ca(2+)-release mechanism is not operating in these cells.

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.

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+.

The effect of the oxytocin antagonists, CAP 476 and F327, on calcium mobilisation in single cultured human myometrial cells.

OBJECTIVE: To investigate the mechanism of action of the oxytocin (OT) antagonists, CAP 476 and F327. DESIGN: A prospective descriptional study. SUBJECTS: Women undergoing caesarean section at term or hysterectomy. INTERVENTIONS: Myometrial cells were cultured from uterine biopsies. MAIN OUTCOME MEASURES: Intracellular calcium ([Ca2+]i), determined in single cells. RESULTS: Application of OT caused a transient increase in [Ca2+]i. CAP 476 abolished and F327 reduced the response to OT but neither reduced the [Ca2+]i transient induced by cell depolarisation with 120 mmol K+. CAP 476 did not reduce transients caused by prostaglandin E2. F327 reduced the frequency of repetitive [Ca2+]i transients occurring during continuous application of OT. CONCLUSIONS: The results demonstrate that the antagonists reduce the effect of OT and that their action is relatively specific. Their mechanism of action as clinical tocolytic agents is discussed.

The actions of caffeine and 2,3-butanedione monoxime on calcium transients in human vascular smooth muscle.

Ca2+ mobilization by membrane depolarization or histamine application, was measured in isolated human uterine artery smooth muscle cells. Nifedipine, a Ca2+ channel blocker, was found to inhibit the depolarization-induced increase in [Ca2+]i but not the histamine-induced increase. This suggests the presence of functional voltage-dependent Ca2+ channels and agonist-induced mobilization of Ca2+ via a different mechanism. Caffeine inhibited both the depolarization- and histamine-induced increases in intracellular calcium. The mechanisms of inhibition do not involve cAMP and point to a complex action of caffeine at multiple sites. The dephosphorylating agent 2,3-butanedione monoxime (BDM) was found to block voltage-dependent Ca2+ changes but not agonist-induced changes suggesting a role for phosphorylation in the regulation of the Ca2+ channels in these 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.

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.

Transients in intracellular free calcium in subconfluent and confluent cultures of a rat smooth muscle cell line.

The Ca2+ mobilizing mechanisms in the smooth muscle cell line A7r5 were found to undergo changes related to the degree of confluence of the cultures. In sparse cultures resting calcium was stable and exposure to arginine vasopressin (AVP) resulted in a single transient increase in intracellular free calcium (Ca2+i). In confluent cultures the cells could be divided into two general groups, those with a stable resting Ca2+i and those which demonstrated spontaneous brief elevations in Ca2+i of variable frequency. Application of AVP elevated Ca2+i, induced oscillations in quiescent confluent cells, increased the frequency of oscillatory activity in cells which were already active and, in cells which exhibited high frequency spontaneous fluctuations, inhibited this activity. Isotonic K+ depolarizing solution and normal solutions containing Co2+ inhibited Ca2+ spikes. These data suggest that the mechanism underlying the transients involves cyclical electrical phenomena at the cell membrane possibly utilizing calcium channels. There is no indication that the mechanism involves cytoplasmic oscillators.

Characteristics of patients aged 75 years and over who are discharged from hospital without district nursing support.

To investigate the state of health and needs of over-75-year-olds discharged from hospital and not referred to the District Nurse Service, patients in this age group who were discharged from hospital, who returned to their own or a relative's home and were not referred to the District Nurse Service were interviewed by Health Visitors during their normal post-discharge visit. Assessment was by a questionnaire on details of discharge, statutory, voluntary and informal support, physical status, disability level, ability to self-care and mental status. It was found that there was wide variation in timing of Health Visitors' post-discharge visits, and most patients had had some contact with their GP before this visit. Almost half the patients were discharged from General Surgery or surgical specialties. Most patients were fairly healthy but a minority had severe disabilities. Physical status, disability level and ability to self-care were related to age and sex of the patient, with older patients and women having poorer health status. Women had poorer mental status than men. It is concluded that the wide variety in health status and needs of this group of patients suggest that they should be taken into account in the planning of new discharge procedures. Closer links between the hospital, community teams and general practice are suggested.

The effect of [Na+]i and [Na+]o on Ca2+ mobilization in the rat aortic smooth muscle cell line A7r5.

Measurements of intracellular calcium (Ca2+i) and sodium (Na+i) have been made in single smooth muscle cells in confluent cultures of the A7r5 cell line using the Ca(2+)- and Na(+)-sensitive dyes Fura-2 and sodium-binding benzofuran isophthalate (SBFI). Reversal of the Na+ gradient in control cells results in a small increase in Ca2+i and slows the rate of recovery in Ca2+i following agonist stimulation. This suggests that a Na(+)-Ca2+ exchange mechanism may be functioning in these cells. In ouabain-pretreated cells, Na+i is elevated and the recovery from agonist stimulation is significantly slowed. This suggests that the elevation of Na+i alters Ca2+ homeostasis. Reversal of the Na+ gradient in ouabain-pretreated cells results in a transient increase in Ca2+i which was larger than in control cells. This response is reduced during a second or third exposure to zero Na+o.NA+i, in Na(+)-loaded cells, falls in the absence of external Na+. This fall is slowed in the absence of external Ca2+ supporting the idea that the Na+ loss is via Na(+)-Ca2+ exchange. The possible modulation of the Na(+)-Ca2+ exchanger and Ca2+ mobilization by internal Na+ is discussed.

Actions of the phorbol ester, PMA, on ATP-induced fluctuations in cytoplasmic calcium in single isolated bovine aortic endothelial cells.

Agonist-evoked rises in [Ca2+]i were recorded from single bovine aortic endothelial cells in the middle of confluent monolayers by dual-wavelength microspectrofluorimetry. Low doses of ATP (1-5 microM) induced a transient rise in [Ca2+]i followed by a maintained plateau phase upon which were superimposed irregular fluctuations in [Ca2+]i. The mechanism underlying these fluctuations is not known. Addition of the phorbol ester, phorbol 12-myristate 13-acetate (PMA), to single cells displaying ATP-induced fluctuations reduced the amplitude and frequency of these fluctuations but the maintained plateau phase was unaffected. Elevation of intracellular cAMP by activation of adenylate cyclase with forskolin, or application of dibutyryl cAMP did not affect the ATP-induced fluctuations. These results suggest a possible role for the diacylglycerol limb of the phosphoinositide hydrolysis pathway, via activation of protein kinase C, but not cAMP, in the mechanism responsible for generating ATP-induced fluctuations in [Ca2+]i.

The care of people over 75 years old after discharge from hospital: an evaluation of timetabled visiting by Health Visitor Assistants.

The objective of this study was to evaluate a programme of timetabled visiting by Health Visitor Assistants (HVAs) to patients over 75 years old who were recently discharged from hospital. An outcome evaluation examined whether patients benefited in measured health status and use of services over the year of the programme. A randomly selected group of patients (the intervention group) who were allocated to the programme of visiting were compared with an equal-sized randomly selected group of patients (the control group) who had no timetabled visiting. A process evaluation examined the actions taken by HVAs during their visits and related the actions taken to patients' measured health status and other characteristics. No overall benefit from the programme of visiting was found in the outcome evaluation. There was wide variation in the numbers of actions recorded for different patients. Numbers of HVAs' actions were related to patient's health status and sex, with more actions being initiated for those in poorer health and women. Neither age nor whether the patient lived alone were found to be related to numbers of HVAs' actions. It was concluded that the lack of demonstrated overall benefit and the wide variation in actions taken on patients' behalf suggest that this type of service cannot be recommended for all discharged patients over 75 years. Poor health at the time of discharge is recommended as a first choice of measure for need of support. Exchange of information between general practice and hospital about previous dependency may aid the identification of patients who need post-discharge support.

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.

Mobilization of calcium by the brief application of oxytocin and prostaglandin E2 in single cultured human myometrial cells.

Intracellular calcium ([Ca2+]i) mobilization was studied in single cultured human myometrial cells in response to the agonists oxytocin and prostaglandin E2 (PGE2) using the fluorescent dye Fura-2. Oxytocin and PGE2 applications were associated with an increase in [Ca2+]i, although there was a marked intercell variation in the amplitude of the agonist-induced response. Removal of extracellular calcium ([Ca2+]o) reduced the oxytocin-induced rise and abolished the PGE2-induced rise in [Ca2+]i, thereby demonstrating that oxytocin but not PGE2 can mobilize intracellular stores of calcium. In nominally calcium-free medium, [Ca2+]i was not increased by PGE2 but subsequent application of oxytocin increased [Ca2+]i, thereby demonstrating that, within a single cell, calcium stores were mobilized by oxytocin and not PGE2. The intracellular calcium stores were completely depleted by a single application of oxytocin and not replenished in the absence of [Ca2+]o. Perfusion with calcium-containing medium for 100 s enabled store refilling. Cell depolarization by 140 mM-K+ caused a transient increase followed by a sustained elevation of [Ca2+]i on which were superimposed small fluctuations. Oxytocin caused an influx of calcium in cells depolarized by K+. This was more marked than that obtained with PGE2.

Oxytocin-evoked repetitive rises of intracellular calcium in single cultured human myometrial cells.

Repetitive transient increases in intracellular calcium were recorded in single cultured human myometrial cells exposed continuously to oxytocin (1 pM-1 nM). Each transient was preceded by a pacemaker-like gradual increase in baseline [Ca2+]i. Removal of extracellular Ca2+ reversibly stopped the transients although small fluctuations in [Ca2+]i were observed in seven out of eleven cells studied. In a proportion of cells (1-2%) repetitive Ca2+ transients were observed in the absence of exogenous oxytocin. The pattern of activity was similar to that seen in cells exposed to oxytocin. These spontaneous elevations in [Ca2+]i were reversibly inhibited by removing extracellular calcium. These experiments demonstrate for the first time repetitive agonist-induced and spontaneous transient increases in [Ca2+]i in single cultured human myometrial cells.

Is there a calcium paradox in isolated bovine aortic endothelial cells?

When bovine aortic endothelial (BAE) cells are superfused with a solution containing low calcium (approximately 10 nM) and subsequently exposed to a solution containing normal Ca2+ (2 mM) a large transient increase in intracellular calcium is seen. This elevation in [Ca2+]i is similar to that described as the calcium paradox in cardiac cells. If the cells are exposed to the agonist, ATP, during the period in low-Ca2+ solution the paradoxical rise in [Ca2+]i is increased. Removal of external Na+ from the low-Ca2+ solution reduces the rise in [Ca2+]i on returning to 2mM-Ca2+ solution. These data are consistent with the presence of a calcium paradox in these cells and with the hypothesis that the underlying mechanism involves the loading of the cell with Na+ during the period in low Ca2+. This process may occur as a result of the altered selectivity of the ATP-activated Ca2+ influx mechanism.

Evidence for Na(+)-Ca2+ exchange and Ca(2+)-induced Ca2+ release in a cultured vascular smooth muscle cell line from the rat.

Measurements of intracellular calcium (Cai2+) and sodium (Nai+) have been made in single smooth muscle cells from the rat aortic cell line (A10) using the Ca(2+)- and Na(+)-sensitive dyes Fura-2 and SBFI (sodium-binding benzofuran isophthalate). The effects of manipulation of intracellular and extracellular Na+ on Cai2+ have been investigated. Reversal of the Na+ gradient in control cells does not result in any measurable increase in Cai2+ or change in the rate of recovery of the cells from agonist stimulation, suggesting that there is little functional Na(+)-Ca2+ exchange. In ouabain-pre-treated cells however, the recovery from agonist stimulation is significantly slowed, suggesting that in the presence of an elevated intracellular Na+ concentration there is an alteration in the Ca(2+)-handling mechanisms. Reversal of the Na+ gradient in ouabain-pre-treated cells results in a transient increase in Cai2+ followed by a slow secondary rise. The transient component of this rise is absent on a second activation of the cell or by prior mobilization of the intracellular stores of Ca2+ by agonist. Data presented in this paper suggest the possibility that the transient component is due to a Ca(2+)-induced Ca(2+)-release mechanism triggered by an initial influx of Ca2+. The mechanism underlying this influx is not known but may involve the Na(+)-Ca2+ exchanger operating in reverse. The possible modulation of the Na(+)-Ca2+ exchanger and Ca(2+)-induced Ca2+ release by internal Na+ is discussed.

Calcium oscillations in single isolated human vascular smooth muscle cells.

When cultured human vascular smooth muscle cells are exposed to sodium-free solutions oscillations in intracellular calcium concentration are observed. In some cells the oscillations are maintained at constant amplitude throughout the exposure to Na(+)-free solution, in others the amplitude of the oscillations falls to a lower, steady-state level. The oscillations are also activated and maintained in isotonic, Na(+)-free, K+ solutions suggesting that the underlying mechanism does not involve the opening and closing of voltage-dependent channels. The possible mechanisms responsible for this behaviour are discussed.