Spontaneous Ca2+ transients in rat pulmonary vein cardiomyocytes are increased in frequency and become more synchronous following electrical stimulation.

The pulmonary veins have an external sleeve of cardiomyocytes that are a widely recognised source of ectopic electrical activity that can lead to atrial fibrillation. Although the mechanisms behind this activity are currently unknown, changes in intracellular calcium (Ca2+) signalling are purported to play a role. Therefore, the intracellular Ca2+ concentration was monitored in the pulmonary vein using fluo-4 and epifluorescence microscopy. Electrical field stimulation evoked a synchronous rise in Ca2+ in neighbouring cardiomyocytes; asynchronous spontaneous Ca2+ transients between electrical stimuli were also present. Immediately following termination of electrical field stimulation at 3 Hz or greater, the frequency of the spontaneous Ca2+ transients was increased from 0.45 ± 0.06 Hz under basal conditions to between 0.59 ± 0.05 and 0.65 ± 0.06 Hz (P < 0.001). Increasing the extracellular Ca2+ concentration enhanced this effect, with the frequency of spontaneous Ca2+ transients increasing from 0.45 ± 0.05 Hz to between 0.75 ± 0.06 and 0.94 ± 0.09 Hz after electrical stimulation at 3 to 9 Hz (P < 0.001), and this was accompanied by a significant increase in the velocity of Ca2+ transients that manifested as waves. Moreover, in the presence of high extracellular Ca2+, the spontaneous Ca2+ transients occurred more synchronously in the initial few seconds following electrical stimulation. The ryanodine receptors, which are the source of spontaneous Ca2+ transients in pulmonary vein cardiomyocytes, were found to be arranged in a striated pattern in the cell interior, as well as along the periphery of cell. Furthermore, labelling the sarcolemma with di-4-ANEPPS showed that over 90% of pulmonary vein cardiomyocytes possessed T-tubules. These findings demonstrate that the frequency of spontaneous Ca2+ transients in the rat pulmonary vein are increased following higher rates of electrical stimulation and increasing the extracellular Ca2+ concentration.

Calcium signalling in sarcoplasmic reticulum, cytoplasm and mitochondria during activation of rabbit aorta myocytes.

This study investigated the relationship between cytoplasmic, mitochondrial, and sarcoplasmic reticulum (SR) [Ca(2+)] in rabbit aorta smooth muscle cells, following cell activation. Smooth muscle cells were loaded with the Ca(2+)-sensitive fluorescent indicator Mag-Fura-2-AM, and then either permeabilized by exposure to saponin, or dialyzed with a patch pipette in the whole-cell configuration to remove cytoplasmic indicator. When the intracellular solution contained millimolar EGTA or BAPTA, activation of SR Ca(2+)release through IP(3)or ryanodine receptors induced a decrease in the [Ca(2+)] reported by Mag-Fura-2. However, when EGTA was present at < or =100 microM, the same stimuli caused an increase in the [Ca(2+)] reported by Mag-Fura-2. The increase in [Ca(2+)] caused by phenylephrine or caffeine was delayed, and prolonged, with respect to the cytoplasmic Ca(2+)transient. Evidence is presented that this Mag-Fura-2 signal reflected a rise in mitochondrial [Ca(2+)]. Agents that inhibit mitochondrial function, such as FCCP or cyanide in combination with oligomycin B, converted the increase in organelle Mag-Fura-2 fluorescence to a decrease, while also prolonging the cytoplasmic Ca(2+)transient. There was considerable similarity between the localization of Mag-Fura-2 fluorescence and the mitochondria-selective indicator tetramethylrhodamine ethyl ester. Thus, we propose that there is close functional integration between the SR and mitochondria in aorta smooth muscle cells, with mitochondria taking up Ca(2+)from the cytoplasm following cell activation.

P2X7 purinoceptor expression in Xenopus oocytes is not sufficient to produce a pore-forming P2Z-like phenotype.

The purinergic rP2X7 receptor expressed in a number of heterologous systems not only functions as a cation channel but also gives rise to a P2Z-like response, i.e. a reversible membrane permeabilization that allows the passage of molecules with molecular masses of > or = 300 Da. We investigated the properties of rP2X7 receptors expressed in Xenopus oocytes. In two-electrode voltage-clamp experiments, ATP or BzATP caused inward currents that were abolished or greatly diminished when NMDG+ or choline replaced Na+ as the principal external cation. In fluorescent dye experiments, BzATP application did not result in entry of the fluorophore YO-PRO-1(2+). Thus, rP2X7 expression in Xenopus oocytes does not by itself give rise to the pore-forming P2Z phenotype, suggesting that ancillary factors are involved.

The effect of danazol on tumour control and weight loss in patients on tamoxifen therapy for advanced breast cancer: a randomised double-blind placebo controlled trial.

To assess the effect of danazol in advanced breast cancer 183 patients were randomised to receive either tamoxifen plus danazol or tamoxifen plus placebo. Patients underwent systemic work-up pretreatment then every 12 weeks or sooner if they clinically progressed. There were no differences in objective response rates with tamoxifen plus danazol vs. tamoxifen plus placebo (27% vs. 24%), time to progression (median 6.4 vs. 6.2 months) or survival (median 22.6 vs. 23.5 months) when the two arms were compared (all P > 0.5). The addition of danazol to tamoxifen had no effect on time to progression when adjusted for significant prognostic factors in a multivariate analysis. However, it was found incidentally that weight was stable on tamoxifen plus danazol (average gain 0.6 kg, S.E. 0.6 kg) compared with a significant loss on tamoxifen plus placebo (average loss 2.0 kg, S.E. 0.6 kg, P = 0.003). The average weight was maintained on tamoxifen plus danazol even in patients who did not respond to treatment.

Investigation of the selectivity of alpha, beta-methylene ATP in inhibiting vascular responses of the rat in vivo and in vitro.

1. The proposal that alpha,beta-methylene adenosine 5'-triphosphate (mATP) inhibits pressor responses in the pithed rat by selective desensitization of P2x-purinoceptors was examined by comparing the selectivity of its inhibitory effect on vascular responses in vitro and in vivo. 2. In isolated ring preparations of rat femoral and tail artery, which had been denuded of endothelium, mATP markedly reduced the contractile response to exogenous ATP but had no effect on the response of the arteries to exogenous noradrenaline (NA). 3. In the pithed rat a substantial proportion of the pressor response to sympathetic nerve stimulation was resistant to alpha-adrenoceptor blockade, suggesting a non-adrenergic component to the sympathetic vasoconstriction. 4. In the pithed rat, repeated administration of desensitizing doses of mATP attenuated the pressor response to sympathetic nerve stimulation by approximately 80%, suggesting that a component of the sympathetic vasoconstriction is mediated by ATP acting on vascular P2x-purinoceptors. However, the same mATP treatment also attenuated, to a similar degree, the pressor responses to intravenous NA, angiotensin II and vasopressin, indicating that the desensitization procedure was non-selective. 5. These results demonstrate that while mATP can be used to desensitize selectively P2x-purinoceptors in vitro, its attenuation of the sympathetic nerve-mediated pressor response in vivo is non-selective.

In vitro effect of nifedipine on KCl and 5-hydroxytryptamine-induced contractions of the sheep coronary, cerebral and pulmonary arteries.

The aim of the present study was to compare the sensitivity to nifedipine of contraction, obtained with 5-hydroxytryptamine (5-HT) and high K+, of arteries from three vascular territories (coronary, cerebral and pulmonary), selecting arteries of equivalent diameter (0.4-0.8 mm). In the coronary and middle cerebral arteries, contraction produced by KCl was abolished by nifedipine 10(-7) M. However, in the pulmonary artery 39 +/- 2% (n = 5) of the KCl contraction remained in the presence of nifedipine 10(-6) M and a similar sized contraction remained in the presence of Ni2+ 10 mM, La3+ 1 mM or Ca(2+)-free conditions. Nifedipine caused less inhibition of 5-HT than of KCl in all three arteries. In the coronary and middle cerebral arteries 5-HT was inhibited to 55 +/- 9% and to 55 +/- 4% and in the pulmonary artery to 78 +/- 3% (n = 6) by nifedipine 10(-6) M. In conclusion, a comparison of coronary, cerebral and pulmonary arteries, of comparable size and from the same species, has shown that there is diversity in the sensitivity of 5-HT-induced contraction to inhibition by nifedipine. Moreover, the KCl-induced contraction in the sheep pulmonary artery is mediated, in part, by a mechanism independent of Ca2+ influx.

Mitochondria contribute to Ca2+ removal in smooth muscle cells.

Recent evidence, from a variety of cell types, suggests that mitochondria play an important role in shaping the change in intracellular calcium concentration ([Ca2+]i) that occurs during physiological stimulation. In the present study, using a range of inhibitors of mitochondrial Ca2+ uptake, we have examined the contribution of mitochondria to Ca2+ removal from the cytosol of smooth muscle cells following stimulation. In voltage-clamped single smooth muscle cells, we found that following a 8-s train depolarizing pulses, the rate of Ca2+ extrusion from the cytosol was reduced by more than 50% by inhibitors of cytochrome oxidase or exposure of cells to the protonophore carbonyl cyanide P-trifluoromethoxy-phenylhydrazone. Using the potential-sensitive indicator-tetramethyl rhodamine ethyl ester, we confirmed that the effect of these agents was associated with depolarization of the mitochondrial membrane. Since, the primary function of the mitochondria is to provide the cell's ATP, it could be argued that it is the ATP supply to the ion pumps which is limiting the rate of Ca2+ removal. However, experiments carried out with the mitochondrial Ca2+ uniporter inhibitor ruthenium red produced similar results, while the ATP synthetase inhibitor oligomycin had no effect, suggesting that the effect was not due to ATP insufficiency. These results establish that mitochondria in smooth muscle cells play a significant role in removing Ca2+ from the cytosol following stimulation. The uptake of Ca2+ into mitochondria is proposed to stimulate mitochondrial ATP production, thereby providing a means for matching increased energy demand, following the cell's rise in [Ca2+]i, with increased cellular ATP production.

Contraction of the sheep middle cerebral, pulmonary and coronary arteries initiated by release of intracellular calcium.

The aim of the study was to compare contraction initiated by intracellular Ca2+ release in the middle cerebral, coronary and pulmonary arteries of the sheep. With all three arteries from the sheep, incubation in Ca(2+)-free physiological salt solution (PSS) reduced agonist-induced contraction much more than occurred with the rabbit aorta. The intracellular Ca2+ store appeared to be of limited capacity, since contraction was transient in Ca(2+)-free conditions with most agonists. In the middle cerebral artery, contraction in Ca(2+)-free conditions was much reduced if a previous contraction had been obtained (for 5-hydroxytryptamine, 5-HT, from 11 +/- 4 to 1 +/- 0.5% of control contraction in 2.5 mM Ca2+), suggesting that the previous contraction had partly discharged the intracellular Ca2+ store. Contraction was less affected in the pulmonary artery and almost unaffected in the coronary artery (for 5-HT, from 15 +/- 1 to 11 +/- 1%) by a previous contraction in Ca(2+)-free conditions. Rings prepared from small branches of the pulmonary and coronary arteries were affected by Ca2+ deprivation in a similar manner to large diameter pulmonary and coronary artery rings. In Ca(2+)-free PSS, contraction induced by prostaglandin E2 was almost eliminated (3 +/- 1% of control contraction in 2.5 mM Ca2+), contractions induced by 5-HT and noradrenaline were reduced, and contraction induced by the thromboxane mimetic U46619 was least affected (up to 73 +/- 8%). Increasing agonist concentration from EC50 to the maximally effective concentration raised the percentage contraction remaining in the middle cerebral artery (for noradrenaline from 7 +/- 2% to 12 +/- 3%) but not in the pulmonary artery (for noradrenaline from 22 +/- 2% to 24 +/- 6%). The present study has revealed notable differences, in coupling to intracellular Ca2+ release between the three vascular territories studied.

Release of Ca2+ from the sarcoplasmic reticulum increases mitochondrial [Ca2+] in rat pulmonary artery smooth muscle cells.

1. The Ca2+-sensitive fluorescent indicator rhod-2 was used to measure mitochondrial [Ca2+] ([Ca2+]m) in single smooth muscle cells from the rat pulmonary artery, while simultaneously monitoring cytosolic [Ca2+] ([Ca2+]i) with fura-2. 2. Application of caffeine produced an increase in [Ca2+]i and also increased [Ca2+]m. The increase in [Ca2+]m occurred after the increase in [Ca2+]i, and remained elevated for a considerable time after [Ca2+]i had returned to resting values. 3. The protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), which causes the mitochondrial membrane potential to collapse, markedly attenuated the increase in [Ca2+]m following caffeine application and also increased the half-time for recovery of [Ca2+]i to resting values. 4. Activation of purinoceptors with ATP also produced increases in both [Ca2+]i and [Ca2+]m in these smooth muscle cells. In some cells, oscillations in [Ca2+]i were observed during ATP application, which produced corresponding oscillations in [Ca2+]m and membrane currents. 5. This study provides direct evidence that Ca2+ release from the sarcoplasmic reticulum, either through ryanodine or inositol 1,4, 5-trisphosphate (InsP3) receptors, increases both cytosolic and mitochondrial [Ca2+] in smooth muscle cells. These results have potential implications both for the role of mitochondria in Ca2+ regulation in smooth muscle, and for understanding how cellular metabolism is regulated.

The temporal profile of calcium transients in voltage clamped gastric myocytes from Bufo marinus.

1. Decay in intracellular calcium concentration ([Ca2+]i) was recorded following step depolarizations in voltage clamped gastric myocytes from Bufo marinus. 2. Depolarizations (300 ms) to +10 mV were followed by three phases of [Ca2+]i decay with repolarization to both -110 and -50 mV. The decline was initially rapid (mean fractional decay rate = 81 +/- 11%s-1 at -110 mV), then slowed (decay rate = 14 +/- 2%s-1) and finally accelerated again (decay rate = 24 +/- 3%s-1; n = 19). 3. The initial phase of rapid decay became shorter as the length of the depolarizing pulse increased but was unaffected by changes in pulse voltage. 4. The delayed acceleration in [Ca2+]i decay was no longer seen when the duration of the depolarizing pulses was reduced to 100 ms, but was clearly evident following 500 ms pulses. This phase was abolished when the depolarizing voltage was altered to minimize the rise in [Ca2+]i. 5. Ryanodine and caffeine had no effect on the temporal profile of [Ca2+]i decay. 6. Removal of extracellular Na+ decreased the decay rate during all three phases at -110 mV, but this effect was particularly marked for the initial rapid phase of decay, the rate of which was reduced by 75%. A delayed increase in decay rate was still seen. 7. Inhibition of mitochondrial Ca2+ uptake with cyanide, carbonyl cyanide p-trifluoromethoxy-phenylhydrazone or Ruthenium Red had no effect on the initial rate of [Ca2+]i decay but blocked the delayed acceleration. 8. These results are discussed in terms of a model in which rapid influx of Ca2+ produces a high subsarcolemmal [Ca2+], favouring rapid Ca2+ removal by near-membrane mechanisms, particularly Na(+)-Ca2+ exchange. Mitochondrial Ca2+ removal produces a delayed increase in [Ca2+]i decay if the global [Ca2+]i is raised high enough for long enough.

Calcium-calmodulin-dependent mechanisms accelerate calcium decay in gastric myocytes from Bufo marinus.

1. [Ca2+] was recorded in voltage-clamped gastric myocytes from Bufo marinus. Repolarization to -110 mV following a 300 ms depolarization to +10 mV led to triphasic [Ca2+]i decay, with a fast-slow-fast pattern. After a conditioning train of repetitive depolarizations the duration of the second, slow phase of decay was shortened, while the rate of decay during the third, faster phase was increased by 34 +/- 6% (mean +/- S.E.M., n = 21) when compared with unconditioned transients. 2. [Ca2+]i decay was biphasic in cells injected with the calmodulin-binding peptide RS20, with a prolonged period of fast decay followed by a slow phase. There was no subsequent increase in decay rate during individual transients and no acceleration of decay following the conditioning train (n = 8). Decline of [Ca2+]i in cells injected with the control peptide NRS20 was triphasic and the decay rate during the third phase was increased by 50 +/- 19% in conditioned transients (n = 6). 3. Cell injection with CK3AA, a pseudo-substrate inhibitor of calmodulin-dependent protein kinase II, prevented the increase in the final rate of decay following the conditioning train (n = 6). In cells injected with an inactive peptide similar to CK3AA, however, there was a 45 +/- 17% increase after the train (n = 5). 4. Inhibition of Ca2+ uptake by the sarcoplasmic reticulum with cyclopiazonic acid or thapsigargin did not prevent acceleration of decay. 5. These results demonstrate that [Ca2+]i decay is accelerated by Ca(2+)-calmodulin and calmodulin-dependent protein kinase II. This does not depend on Ca2+ uptake by the sarcoplasmic reticulum but may reflect upregulation of mitochondrial Ca2+ removal.

Coupling of a P2Z-like purinoceptor to a fatty acid-activated K(+) channel in toad gastric smooth muscle cells.

1. Extracellular application of ATP generates two whole-cell currents in toad gastric smooth muscle cells: an immediate inward non-selective cation current (due to the activation of a P2X or P2Z-like receptor) and a slowly developing outward K(+) current. The inward non-selective cation current depends on the continuous presence of ATP while the outward K(+) current can last for minutes after ATP application ceases. 2. In cell-attached patches, application of ATP to the extra-patch membrane can activate K(+) channels in the patch indicating that a diffusible cellular messenger may be involved. The characteristics of these K(+) channels are similar to those of a previously described fatty acid-activated K(+) channel that is also a stretch-activated channel. 3. This whole-cell K(+) current can be induced by ATP in the absence of extracellular Ca(2+) (with EGTA present to chelate trace amounts). However, the current generated in the presence of extracellular Ca(2+) is considerably larger. 4. The pharmacological profiles for the activation of the non-selective cation current and the K(+) current are similar, suggesting that the same P2Z-like receptor could be mediating both responses. This type of plasma membrane receptor/channel-channel coupling by a process that does not appear to involve Ca(2+) flow through the receptor/channel or a subsequent membrane potential change may be representative of a new class of signalling mechanisms.

An ATP-gated cation channel with some P2Z-like characteristics in gastric smooth muscle cells of toad.

1. Whole-cell and single-channel currents elicited by extracellular ATP were studied in freshly dissociated smooth muscle cells from the stomach of the toad Bufo marinus using standard patch clamp and microfluorimetric techniques. 2. This ATP-gated cation channel shares a number of pharmacological and functional properties with native rat myometrium receptors, certain native P2Z purinoceptors and the recently cloned P2X7 purinoceptor. But, unlike the last two, the ATP-gated channel does not mediate the formation of large non-specific pores. Thus, it may represent a novel member of the P2X or P2Z class. 3. Extracellular application of ATP (> or = 150 microM) elicited an inward whole-cell current at negative holding potentials that was inwardly rectifying and showed no sign of desensitization. Na+, Cs+ and, to a lesser degree, the organic cation choline served as charge carriers, but Cl- did not. Ratiometric fura-2 measurements indicated that the current is carried in part by Ca2+. The EC50 for ATP was 700 microM in solutions with a low divalent cation concentration. 4. ATP (> or = 100 microM) at the extracellular surface of cell-attached or excised patches elicited inwardly rectifying single-channel currents with a 22 pS conductance. Cl- did not serve as a charge carrier but both Na+ and Cs+ did, as did choline to a lesser extent. The mean open time of the channel was quite long, with a range in hundreds of milliseconds at a holding potential of -70 mV. 5. Mg2+ and Ca2+ decreased the magnitude of the ATP-induced whole-cell currents. Mg2+ decreased both the amplitude and the activity of ATP-activated single-channel currents. 6. ADP, UTP, P1, P5-di-adenosine pentaphosphate (AP5A), adenosine and alpha, beta-methylene ATP (alpha, beta-Me-ATP) did not induce significant whole-cell current. ATP-gamma-S and 2-methylthio ATP (2-Me-S-ATP) were significantly less effective than ATP in inducing whole-cell currents, whereas benzoylbenzoyl ATP (BzATP) was more effective. BzATP, alpha, beta-Me-ATP, ATP-gamma-S and 2-Me-S-ATP induced single-channel currents, but a higher concentration of alpha, beta-Me-ATP was required. 7. BzATP did not induce the formation of large non-specific pores, as assayed using mag-fura-2 as a high molecular mass probe.

Mitochondrial Ca2+ homeostasis during Ca2+ influx and Ca2+ release in gastric myocytes from Bufo marinus.

1. The Ca(2+)-sensitive fluorescent indicator rhod-2 was used to monitor mitochondrial Ca2+ concentration ([Ca2+]m) in gastric smooth muscle cells from Bufo marinus. In some studies, fura-2 was used in combination with rhod-2, allowing simultaneous measurement of cytoplasmic Ca2+ concentration ([Ca2+]i) and [Ca2+]m, respectively. 2. During a short train of depolarizations, which causes Ca2+ influx from the extracellular medium, there was an increase in both [Ca2+]i and [Ca2+]m. The half-time (t1/2) to peak for the increase in [Ca2+]m was considerably longer than the t1/2 to peak for the increase in [Ca2+]i. [Ca2+]m remained elevated for tens of seconds after [Ca2+]i had returned to its resting value. 3. Stimulation with caffeine, which causes release of Ca2+ from the sarcoplasmic reticulum (SR), also produced increases in both [Ca2+]i and [Ca2+]m. The values of t1/2 to peak for the increase in [Ca2+] in both cytoplasm and mitochondria were similar; however, [Ca2+]i returned to baseline values much faster than [Ca2+]m. 4. Using a wide-field digital imaging microscope, changes in [Ca2+]m were monitored within individual mitochondria in situ, during stimulation of Ca2+ influx or Ca2+ release from the SR. 5. Mitochondrial Ca2+ uptake during depolarizing stimulation caused depolarization of the mitochondrial membrane potential. The mitochondrial membrane potential recovered considerably faster than the recovery of [Ca2+]m. 6. This study shows that Ca2+ influx from the extracellular medium and Ca2+ release from the SR are capable of increasing [Ca2+]m in smooth muscle cells. The efflux of Ca2+ from the mitochondria is a slow process and appears to be dependent upon the amount of Ca2+ in the SR.

Symptom duration and delay in referral for palliative radiotherapy in cancer patients: a pilot study.

OBJECTIVE: To estimate the frequency of delay in referral for palliative radiotherapy (PRT), and to identify factors associated with delay. DESIGN: Prospective survey over three months in 1997. SETTING: Radiotherapy department of a cancer centre in Melbourne, Victoria. PARTICIPANTS: 158 consecutive patients prescribed PRT in the lung, breast, urology and haematology units. MAIN OUTCOME MEASURES: Duration of symptoms; incidence of "unreasonable" delay in referral; and incidence of negative clinical outcome associated with referral delay. RESULTS: The median duration of symptoms before prescription of radiotherapy was four weeks. Thirty-eight patients (24%) were considered to have had an unreasonable delay in referral, with median symptom duration of 15 weeks, and median delay in referral of 12 weeks. Causes of delay were classified as "diagnostic uncertainty" (29%), "other treatment given" (18%), "patient related" (18%), "language difficulty" (3%), and "unexplained" (32%). Twenty-seven of these 38 patients (71%) had negative outcomes, including persistent pain, neurological deterioration and persistent respiratory symptoms. CONCLUSIONS: These data suggest that delay in referral for PRT is not uncommon, has a variety of causes and can result in negative clinical outcomes. There appears to be a need for greater awareness of patients' symptoms and of the role of PRT among clinicians caring for patients with cancer.

Signaling pathways underlying eosinophil cell motility revealed by using caged peptides.

Insights into structure-function relations of many proteins opens the possibility of engineering peptides to selectively interfere with a protein's activity. To facilitate the use of peptides as probes of cellular processes, we have developed caged peptides whose influence on specific proteins can be suddenly and uniformly changed by near-UV light. Two peptides are described which, on photolysis of a caging moiety, block the action of calcium-calmodulin or myosin light chain kinase (MLCK). The efficacy of theses peptides is demonstrated in vitro and in vivo by determining their effect before and after photolysis on activities of isolated enzymes and cellular functions known to depend on calcium-calmodulin and MLCK. These caged peptides each were injected into motile, polarized eosinophils, and when exposed to light promptly blocked cell locomotion in a similar manner. The results indicate that the action of calcium-calmodulin and MLCK, and by inference myosin II, are required for the ameboid locomotion of these cells. This methodology provides a powerful means for assessing the role of these and other proteins in a wide range of spatio-temporally complex functions in intact living cells.