Skip Navigation

Cardiovascular Research 1998 40(3):538-545; doi:10.1016/S0008-6363(98)00195-3
© 1998 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Fong Yew, S.
Right arrow Articles by Woodward, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Fong Yew, S.
Right arrow Articles by Woodward, B.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 1998, European Society of Cardiology

Effects of prostaglandin F2{alpha} on intracellular pH, intracellular calcium, cell shortening and L-type calcium currents in rat myocytes

Su Fong Yew, Katherine A. Reeves and Brian Woodward*

Department of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY, UK

* Corresponding author. Tel.: +44-1225-323-206; fax: +44-1225-826-114; e-mail: b.woodward@bath.ac.uk

Received 11 March 1998; accepted 1 May 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: We have studied the mechanisms underlying the positive inotropic action of prostaglandin F2{alpha} (PGF2{alpha}) by monitoring intracellular calcium transients, intracellular pH, L-type calcium currents and cell shortening in isolated ventricular myocytes. Methods: Rat myocytes were loaded with fura-2AM for intracellular calcium measurements, or BCECF-AM for pH measurements. Cell shortening was recorded using an edge detection system, and L-type calcium currents measured using whole cell patch clamping. Results: PGF2{alpha}(3 nmol l–1–3 µmol l–1) increased single myocyte shortening and reduced resting cell length in a concentration-dependent manner. While myocyte shortening was increased by PGF2{alpha}, this was not associated with any change in the amplitude of intracellular calcium transients, diastolic calcium, or L-type calcium currents. However, the same myocytes were capable of responding to catecholamines with increases in calcium transient amplitude and L-type calcium currents. PGF2{alpha} (3 µmol l–1) caused a reversible rise in intracellular pH of 0.08±0.01 pH units (n=5, p<0.05). The Na+–H+ exchanger inhibitor, HOE 694 (10 µmol l–1), abolished the PGF2{alpha}-induced rise in pH and the increase in cell shortening. PGF2{alpha}-induced increases in cell shortening and intracellular pH were also attenuated by the protein kinase C (PKC) inhibitor, chelerythrine (2 µmol l–1). Conclusion: The positive inotropic action of PGF2{alpha} appears to be mediated via activation of the Na+–H+ exchanger with the possible involvement of PKC. This suggests that PGF2{alpha}_produces intracellular alkalosis, which then sensitizes cardiac myofilaments to calcium.

KEYWORDS Calcium; Contractile function; Na/H–exchanger; Prostaglandins; Rat


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Prostaglandin F2{alpha} (PGF2{alpha}) has complex effects on the heart. It increases cardiac contractility [1–6], being the most potent of the prostaglandins studied [1–3]. Additionally, it depresses ventricular recovery of reperfused hearts [7], constricts coronary vessels [8], modulates cardiac rhythm [9, 10], releases atrial natriuretic factor [11, 12]and causes ventricular hypertrophy [13]. These effects may be important during myocardial ischaemia, myocardial infarction or acute pressure overload, where the cardiac release of PGF2{alpha} is substantially increased [13–15].

The mechanisms underlying the positive inotropic action of PGF2{alpha} are poorly understood but it is known to be an agonist at FP prostaglandin receptors that are linked to phosphoinositide metabolism [16]. PGF2{alpha} has been reported to increase inositol 1,4,5-trisphosphate levels in rat papillary muscle [5]. This action, together with its reported inhibition of Na+/K+ ATPase [4], and stimulation of calcium uptake by the sarcoplasmic reticulum [17], would directly or indirectly be expected to increase the amount of calcium available for contraction. PGF2{alpha} has been reported to prolong the duration of cardiac action potentials, one possible explanation being an increased inward calcium current [18]but this has not been studied experimentally. Otani et al. [5]showed that PGF2{alpha} was capable of initiating contractions in paced, potassium depolarised guinea-pig papillary muscles, suggesting that it increases calcium entry. In contrast, a brief report by Sperelakis et al. indicated that PGF2{alpha} inhibits slow action potentials in cultured chick heart cells [19]. Consequently it is not clear if PGF2{alpha} does affect free intracellular calcium levels on a beat to beat basis in cardiac myocytes or whether it affects L-type calcium currents.

An alternative mechanism for the inotropic action of PGF2{alpha} is that it may sensitize contractile filaments to calcium by causing an intracellular alkalosis [20]. In vascular smooth muscle, PGF2{alpha}-mediated contraction occurs partly by sensitization of the contractile filaments to calcium [21, 22]; it has also been shown to increase intracellular pH (pHi) of osteoblasts and kidney cells [23, 24]. In cardiac myocytes, intracellular alkalosis is known to contribute to the positive inotropic actions of angiotensin II [25]and phenylephrine [26].

Therefore, the purpose of our investigation was to examine the effects of PGF2{alpha} on basal intracellular calcium, and calcium transients of stimulated rat ventricular myocytes using the calcium probe fura-2. We have also studied the effects of PGF2{alpha} on whole cell L-type calcium currents with the whole cell patch clamp technique. In addition to the calcium studies, we have examined the effect of PGF2{alpha} on pHi in rat cardiac myocytes using the pH sensitive probe, BCECF. By using the Na+–H+ exchange inhibitor, HOE 694 [27, 28], and the protein kinase C inhibitor, chelerythrine [29], we have investigated the possibility that changes in the activity of the Na+– H+ exchanger and PKC are involved in the positive inotropic effect of PGF2{alpha}. In all cases parallel studies have been carried out to examine the effects of these drugs on PGF2{alpha}-induced myocyte shortening.

Our data show that PGF2{alpha} does not affect calcium transients or L-type calcium currents, however, it does produce an intracellular alkalosis mediated by the Na+–H+ exchanger, possibly with the involvement of PKC. This alkalosis sensitizes the cardiac myofilaments to calcium, and this is the likely cause of the positive inotropic effect of PGF2{alpha} in rat cardiac myocytes.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Myocyte isolation and cell shortening measurements
The investigation conforms with the Home Office Guidance on the Operation of Animals (Scientific Procedures) Act 1986 published by HMSO, reference HC182. Hearts were removed from terminally anaesthetised (pentobarbitone sodium 100 mg kg–1, i.p.) male Wistar rats weighing 250–300 g, and perfused retrogradely at a flow rate of 10 ml min–1 with a physiological buffer (composition in mmol l–1: NaCl 118.5, NaHCO3 14.5, KCl 2.6, KH2PO4 1.2, MgSO4 1.2, D-Glucose 11.1, HEPES 10.0, made up to pH 7.4 with NaOH) gassed with 95% O2/5% CO2, for 4 min. Perfusion was then switched to a 50 ml recirculating system, composition as above, containing collagenase (50 units), protease (25 units) and 25 µmol l–1 CaCl2. This was continued for 15 min. The ventricles were then sliced into sections, teased apart, and triturated in 20 ml of enzyme solution at 37°C. The myocyte suspension was filtered through a 100 µm nylon mesh gauze and the filtrate centrifuged at 300 rpm for 5 min followed by re-suspension in calcium- and enzyme-free buffer. This centrifugation and re-suspension procedure was carried out twice. Calcium was then gradually added to 0.1, 0.6 and 1 mmol l–1 to produce calcium tolerant myocytes.

Myocytes were allowed to attach to a 0.2 ml volume laminin (15 µg ml–1) pre-coated perfusion chamber and observed with an inverted microscope. Only rod-shaped quiescent myocytes which responded to stimulation with a rapid twitch were selected for single cell recordings. Cells were paced at 1 Hz (1 ms pulse width, supramaximal voltage) using silver chloride electrodes placed 1–2 mm apart, and superfused at 2 ml min–1 at room temperature (24–26°C) with the physiological buffer described above containing 1 mmol l–1 calcium. Myocyte shortening was measured with a video camera and edge detection system as used by Harding et al. [30]. Cell shortening changes are expressed as percentages of control shortening value. As we were unable to measure cell shortening and fura-2 fluorescence at the same time, some of the cell shortening studies were carried out in cells that had been loaded with fura-2 AM. This was done to check that fura-2 loading did not affect cell shortening. Time to peak tension development, and to 50% and 90% relaxation were measured using a software supplied by Dr. S.E. Harding (National Heart and Lung Institute, London).

2.2 Intracellular calcium and pH measurements
For intracellular calcium measurements, myocytes were loaded in nominally calcium free solution for 30 min with 5 µmol l–1 fura-2 AM and 0.5% bovine serum albumin at 37°C. For pHi measurements, myocytes were loaded with 2 µmol l–1 BCECF-AM at room temperature for 20 min. Removal of the extracellular fluorescent dyes and gradual exposure of the myocytes to extracellular calcium was then performed with three centrifugation steps and re-suspension with 0.1, 0.6 and 1 mmol l–1 calcium buffer. A Photon Technologies International Deltascan spectrofluorimeter was used to detect dye fluorescence. Single, electrically paced, fura-2 loaded myocytes were alternately excited with UV light of 340 and 380 nm wavelengths and the emission wavelength at 510 nm detected. For measurements of pHi, single, electrically paced, BCECF-loaded myocytes were alternately excited with UV light of 440 nm and 490 nm wavelengths with the emission wavelength at 535 nm detected. To limit photobleaching, myocytes loaded with BCECF were exposed with excitation wavelengths of 10 s duration at 1 min intervals. While calcium transients were not calibrated, pHi was calibrated for each myocyte. The calibration procedure involved perfusing myocytes with high K+-nigericin calibration solutions of different pH (composition in mmol l–1: KCl 140, MgCl 1, HEPES 20, nigericin 0.01, made up to pH 6.8, 7.2 and 7.6 with NaOH). Linear regression of the fluorescence ratio versus the pH value of the calibration buffer determined the pHi of each cell. Nigericin was washed out with 100 ml 90% ethanol and 0.1 mol l–1 HCl followed by 100 ml hot water before the next myocyte was studied.

2.3 Whole cell L-type calcium current measurements
Myocytes were placed in a superfusion chamber pre-treated with laminin and superfused at 2 ml min–1, 32°C, using a physiological solution of the following composition (mmol l–1): NaCl 121.1, NaHCO3 14.5, NaH2PO4 1.2, MgSO4 1.2, D-glucose 11.1, HEPES 10, CaCl2 1.8. Myocytes were then patch clamped in the whole cell configuration, using a patch pipette (resistance 2–2.5 M{Omega}) filled with the following solution (mmol l–1): CsCl 110, EGTA 11, MgCl2 2, HEPES 10, K2ATP 5, CaCl2 1. Calcium current recordings were elicited by depolarising the cell every 20 s from a holding potential of –80 mV to 0 mV for 200 ms, and measured using an AXOPATCH 200A amplifier/pClamp6 software. Fast sodium currents were eliminated using a pre-pulse to –40 mV for 40 ms prior to the test pulse.

2.4 Protocols
In the cell shortening studies PGF2{alpha} was added cumulatively to the perfusate. The concentration was increased once cell shortening had reached a stable value following the previous drug addition. This normally took about 10 min. Therefore, when single concentrations were used in later studies to measure intracellular calcium, L-type calcium currents and pHi, a contact time of at least 10 min was usually used. In some experiments where PGF2{alpha} did not induce any change in intracellular free calcium, L-type calcium currents or cell shortening, either alone or in the presence of an inhibitor, noradrenaline or isoprenaline was added at the end of the experiment. This was done to check that the cells were capable of responding in the expected way to β-adrenergic stimulation, i.e. increases in cell shortening, calcium current and calcium transients. When the protein kinase C inhibitor, chelerythrine, was used it was added 10 min prior to the addition of PGF2{alpha}. The Na+–H+ exchange inhibitor, HOE 694, was added at the same time as PGF2{alpha}. Solvent controls were carried out as appropriate.

2.5 Drugs and enzymes
Collagenase Type II was supplied by Worthington, while protease Type XIV and laminin were obtained from Sigma. Stock solutions of the following chemicals were prepared and stored at –20°C. PGF2{alpha} tris salt (ICN Biomedicals, Thame) was dissolved in 3:1 ethanol:water. HOE 694 (gift from Dr. H.-J. Lang, Hoechst A.G., Germany) was prepared in saline. Chelerythrine chloride (Calbiochem, Nottingham) was dissolved in 1:4 dimethylsulphoxide:water. Nigericin (Sigma, Poole) was dissolved in 95% ethanol. Fura-2 AM and BCECF-AM (Calbiochem, Nottingham) were dissolved in anhydrous dimethylsulphoxide. These solutions were diluted to the appropriate concentrations in buffers when required.

2.6 Statistics
Results are expressed as mean±standard error of means for n number of experiments. All data were first analysed for homogeneity of variance with Bartlett's test for normal distribution. For analysis of parametric paired data, the paired Student's t-test was used, and Wilcoxon signed rank test was used for non-parametric data. Data between groups were analysed with either one way ANOVA (parametric data) or Mann Whitney U test (non-parametric data). A value of p<0.05 was considered significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Effect of PGF2{alpha} on cell shortening of isolated myocytes
PGF2{alpha}, over the range 3 nmol l–1 to 3 µmol l–1, increased cell shortening of myocytes in a concentration dependent manner (Fig. 1A). Basal cell length was reduced in a concentration dependent fashion (Fig. 1B). At these concentrations PGF2{alpha} did not affect the time to peak contraction, nor the time to 50% and 90% relaxation (data not shown). In fura-2 loaded myocytes, PGF2{alpha} (3 µmol l–1) increased the contractile amplitude by 40.4±10.7% (n=7). This value was not significantly different from that obtained with 3 µmol l–1 PGF2{alpha} in myocytes which were not loaded with fura-2 (39.6±3.4%, n=4, Fig. 1A).


Figure 1
View larger version (15K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Cumulative data showing (A), effects of PGF2{alpha} on cell shortening in rat cardiac myocytes expressed as a percentage increase in basal cell shortening, and (B), effects of PGF2{alpha} on diastolic cell length, n=4. Basal cell shortening was 12.2±1.1% of cell length.

 
3.2 Effects of PGF2{alpha} on L-type calcium currents
In ten cells, perfusion with 100 nmol l–1 PGF2{alpha} for 10 min had no significant effect on whole cell L-type calcium currents (Fig. 2). In three of these cells, isoprenaline (100 nmol l–1) was added at the end of the experiment in the continued presence of PGF2{alpha}. In all three cells, isoprenaline produced the expected increase in calcium current amplitude as shown in Fig. 2B. When a higher concentration of PGF2{alpha} (3 µmol l–1) was used, it was difficult to maintain a good seal with the patch pipette for the 10-min duration of drug application. However, prior to losing the seal PGF2{alpha} had no effect on the calcium current.


Figure 2
View larger version (19K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 (A) Time course of peak calcium current density in the presence of 100 nmol l–1 PGF2{alpha} (O, added where indicated, n=10), compared with time matched controls ({blacksquare}, n=6) in rat ventricular myocytes. (B) A typical example of L-type calcium currents in a cell which did not respond to PGF2{alpha} (100 nmol l–1) but which did respond to a subsequent application of isoprenaline (100 nmol l–1). One of three similar recordings.

 
3.3 Effects of PGF2{alpha} on intracellular calcium transients in fura-2 loaded myocytes
At a concentration which increased cell shortening, PGF2{alpha} (3 µmol l–1) did not increase the amplitude of the calcium transients, (control fura-2 340/380 ratio 0.66±0.09, compared with PGF2{alpha} 0.71±0.1 units, n=9, p0.05), neither did it alter the basal fluorescence ratio of fura-2 loaded myocytes. Fig. 3 shows mean data for three cells which did not respond to PGF2{alpha} to which noradrenaline was subsequently added. It can be seen that these cells were capable of responding to noradrenaline (300 nmol l–1) with an increase in calcium transient amplitude. In these cells maximal amplitude of the fura-2 340/380 ratio, 100 seconds after noradrenaline addition, increased from 0.68±0.09 to 1.40±0.09 (n=3).


Figure 3
View larger version (22K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Effects of PGF2{alpha}, (3 µmol l–1) on calcium transient amplitude, i.e. systolic minus diastolic Ca2+ (upper panel), and diastolic Ca2+ level (lower panel), as measured by fura-2 340/380 ratios. Points averaged from ten Ca2+ transients from each time point in each of the myocytes. Noradrenaline (300 nmol l–1) was added at the end of each experiment, n=3.

 
3.4 Effect of PGF2{alpha} on myocyte pHi
PGF2{alpha} (3 µmol l–1) increased the pHi of BCECF loaded myocytes by 0.08±0.01 pH units (Fig. 4Fig. 5A, n=5); this effect was reversible upon washout (Fig. 5A). This intracellular alkalosis occurred over a similar time course to the increase in cell shortening seen in the experiments described above (Fig. 4). In contrast, time matched vehicle control cells showed a slow decline in myocyte pHi (Fig. 5A).


Figure 4
View larger version (23K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Mean data from separate experiments showing the parallel time course of the effects of PGF2{alpha} (3 µmol l–1) on cell shortening (bullet n=7) and intracellular pH ({circ} n=5).

 

Figure 5
View larger version (23K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 (A) Effects of PGF2{alpha}±HOE 694 on pHi changes. Time matched control ({circ}); PGF2{alpha} 3 µmol l–1 (bullet); HOE 694 10 µmol l–1 ({triangleup}); HOE 10 µmol l–1 plus PGF2{alpha} 3 µmol l–1 ({blacktriangleup}). Solid bar represents duration of drug administration. * p<0.05 significant differences comparing data with and without PGF2{alpha}, Mann Whitney U test. (B) Effects of PGF2{alpha}±HOE 694 on cell shortening changes. Open columns: time matched control (TMC), or HOE alone 0.1 µmol l–1 or 10 µmol l–1. Filled columns: PGF2{alpha} alone, or PGF2{alpha} plus HOE 694 0.1 µmol l–1 or 10 µmol l–1, n=5–6 per group. Significant differences compared with appropriate control * p<0.05, *** p<0.001 paired t-test; # p<0.05 one way ANOVA.

 
3.5 Effects of HOE 694 on pHi and cell shortening changes induced by PGF2{alpha}
The addition of HOE 694 did not significantly affect basal pHi or cell shortening. However, when added at the same time as PGF2{alpha}, it produced a concentration-dependent inhibition of the PGF2{alpha}-induced increase in cell shortening (Fig. 5B). The PGF2{alpha}-induced alkalosis was also significantly attenuated by 10 µmol l–1 HOE 694 (Fig. 5A). In all myocytes tested, washout of the highest concentration of HOE 694 (10 µmol l–1) caused a marked increase in cell shortening that reversed within 30 min. This was paralleled by an increase in pHi (Fig. 5A). In four cells which did not respond to PGF2{alpha} in the presence of HOE 694 (10 µmol l–1), noradrenaline (300 nmol l–1) was added at the end of the experiment. In these cells, noradrenaline increased contractile amplitude to 213±46% of the baseline shortening value.

3.6 Effects of chelerythrine on PGF2{alpha}-induced changes in cell shortening and pHi
The PKC inhibitor, chelerythrine (2 µmol l–1), had no effect on basal pH or cell shortening, however, when it was added to the perfusate 10 min before PGF2{alpha}, it produced a significant attenuation of both the PGF2{alpha}-induced increase in intracellular alkalosis (Fig. 6A) and cell shortening (Fig. 6B). In the presence of chelerythrine, cells were still capable of responding to noradrenaline (300 nmol l–1) with an increase in cell shortening to 254±33% of the basal value (n=6).


Figure 6
View larger version (19K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Effects of PGF2{alpha}±chelerythrine on pHi changes. Time matched DMSO/chelerythrine control ({triangleup}); PGF2{alpha} 3 µmol l–1(bullet); chelerythrine 2 µmol l–1 plus PGF2{alpha} 3 µmol l–1 ({blacktriangleup}); n=6 per group. * p<0.05 compared with PGF2{alpha}, # p<0.05 compared with chelerythrine control, Mann Whitney U test. (B) Effects of PGF2{alpha}±chelerythrine on cell shortening. Open columns, DMSO solvent or chelerythrine (2 µmol l–1) 10-min controls. Filled columns, subsequent 15 min with PGF2{alpha} (3 µmol l–1) alone, or PGF2{alpha} (3 µmol l–1) plus chelerythrine (2 µmol l–1), n=6/group, * p<0.05 Wilcoxon signed rank test, # p<0.05 Mann Whitney U test.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
We have confirmed earlier studies that PGF2{alpha} has a positive inotropic action in rat ventricular tissue. In addition we have presented data showing that PGF2{alpha} does not activate L-type calcium channels or increase intracellular levels of free calcium. However, the inotropic action of PGF2{alpha} is associated with an intracellular alkalosis and both effects are attenuated by chelerythrine, a PKC inhibitor, and by HOE 694, an inhibitor of the Na+–H+ exchanger.

Previous studies have presented conflicting data on the possible role of L-type calcium channel activation as a mediator of the positive inotropic action of PGF2{alpha}. Some of these differences may relate to species variation but, in these earlier studies, no direct measurement of L-type calcium currents was made. In our study, measurement of L-type calcium currents in rat ventricular myocytes showed that PGF2{alpha} (100 nmol l–1), which produced a marked positive inotropic action as measured by an increase in cell shortening, had no significant effect on calcium entry via L-type calcium channels. This concentration of PGF2{alpha} has also been shown to be at the higher end of the dose response range that increases the force of contraction of rat papillary muscles [5]and Langendorff perfused rat hearts [3]. The fact that a subsequent addition of isoprenaline to cells that had not responded to PGF2{alpha} was capable of increasing calcium current amplitude shows that calcium currents could be increased in a predictable manner in these cells. Therefore, the lack of a response to PGF2{alpha} is not due to the viability of the myocytes. The patch clamp studies were carried out using a submaximal inotropic concentration of PGF2{alpha} (100 nmol l–1), and it could be argued that higher concentrations of PGF2{alpha} might have increased calcium currents. However, we were unable to carry out experiments using higher concentrations of PGF2{alpha} because a good seal could not be maintained for the 10-min drug application in these experiments.

In contrast to our observations, Otani et al. [5]showed that PGF2{alpha} restored the contraction–relaxation cycle in rat papillary muscle that had been depolarised with 25 mmol l–1 potassium, and this was blocked by nifedipine, suggesting a role for L-type calcium channels in this response. It is difficult to reconcile these two observations as the concentration of nifedipine used by Otani et al., 100 nmol l–1, was not excessive and would be expected to be reasonably selective for calcium channels. However, in that paper there was a clear dissociation in the time to maximal inotropic action in control preparations and the time to restoration of contraction in depolarised preparations, which suggests that the mechanisms of these two effects may differ.

In addition to showing no effect of PGF2{alpha} on calcium currents, our fura-2 studies were also unable to detect any effect of PGF2{alpha} on basal levels of intracellular free calcium, or pacing- induced calcium transients. As the intracellular calcium transients are due to calcium-induced calcium release, this provides further evidence that PGF2{alpha} is not affecting calcium entry via the L-type calcium channels. If PGF2{alpha} did increase calcium influx via the L-type calcium channels, this would be expected to increase calcium release from the sarcoplasmic reticulum, and the calcium transients [31]. This did not occur. Despite this, these cells were capable of responding in a predictable way to the addition of noradrenaline with an increase in calcium transient amplitude, demonstrating their viability. In the fura-2 studies, it is possible that fura-2 could have been buffering any PGF2{alpha}-induced change in intracellular calcium. We feel this is unlikely because when cell shortening measurements were made using fura-2 loaded cells, the response of these cells to PGF2{alpha} was the same as in the absence of fura-2. Additionally, this lack of effect on the calcium transient amplitude is probably not due to the PGF2{alpha}-induced increase in intracellular pH of 0.08 units. Grynkiewicz et al. showed that changes in pH from 6.75 to 7.05 at a physiological calcium concentration had little effect on fura-2 signals [32], while Martinez-Zaguilan et al. reported that the effect of pH on fura-2 signals is minor when pH is more than 7.0 [33]. Therefore, in rat ventricular myocytes, the inotropic action of PGF2{alpha} does not appear to be mediated by an increase in intracellular free calcium concentration.

An early study by Metsa-Ketela showed that PGF2{alpha} increased 45Ca levels in rat atria [2]. However, in that study which compared the positive inotropic action of a number of prostaglandins, all of the prostaglandins produced similar increases in 45Ca despite having differing inotropic effects. In each case the prostaglandin concentration used was high (50 µmol l–1), and from that study, it was not possible to distinguish between effects on calcium influx and efflux from the tissue. The lipid nature of the prostaglandins, the high concentration used in these experiments, together with the dissociation between calcium accumulation and the inotropic response, suggest that these effects on calcium levels may be non-specific in nature. In another study using rat papillary muscle [5], PGF2{alpha} was shown to increase tissue levels of inositol 1,4,5-trisphosphate (IP3) and this might be expected to increase calcium release from the sarcoplasmic reticulum via stimulation of IP3 receptors. However, in this study the increased levels of IP3 did not correlate with the inotropic action of PGF2{alpha}, suggesting that the change in contractility was not directly related to IP3-induced calcium release. Also, although there is good evidence that IP3 receptor stimulation increases calcium release in smooth muscle [34], it does not appear to be an important mediator of calcium release in cardiac tissue [35–37]. Therefore our studies on L-type calcium channels and calcium transients show that PGF2{alpha} is unlikely to be affecting calcium entry and mobilisation in rat ventricular myocytes. The lack of effect on intracellular calcium indicates that the positive inotropic action of PGF2{alpha} could be due to sensitization of the contractile elements to calcium.

We have shown for the first time that PGF2{alpha} increases intracellular pH in cardiac myocytes. While PGF2{alpha} had no significant effect on calcium mobilisation, it did increase intracellular pH, and the change in pH paralleled its positive inotropic action. This increase in pHi of 0.08 units and change in myocyte shortening of 40% is similar to that induced by endothelin-1, which increases pHi by 0.08 units and cell shortening by 59% [38]. The inotropic action of angiotensin II is also associated with an intracellular alkalosis [25]. Fabiato and Fabiato have shown that an increase in intracellular pH is capable of increasing the sensitivity of contractile proteins to calcium [20].

The fact that a selective Na+–H+ exchange inhibitor, HOE 694 [27, 28], prevented the PGF2{alpha}-induced alkalosis and its inotropic action provides good evidence that the PGF2{alpha}-induced inotropic response is mediated via activation of the Na+–H+ exchanger and the intracellular alkalosis. This action of HOE 694 was not due to a non-specific inhibitory effect as cells were still capable of responding to β-adrenoceptor stimulation in a positive manner. The highest concentration of HOE 694 that we used was 10 µmol l–1, which is close to the concentration of 30 µmol l–1 reported to inhibit the Na+–H+ exchanger completely in cardiac myocytes [28]. Interestingly, although HOE 694 did not significantly affect basal pHi and cell shortening, there was a large increase in cell shortening when the drug was washed out. This also correlated with an increase in pHi. Under basal conditions in the absence of drugs, a gradual drift in pHi occurred. The reason for this is not clear, but this effect has also been reported by Sun et al. [39].

In addition to being inhibited by HOE 694, the effects of PGF2{alpha} on cell shortening and intracellular pH were attenuated by the PKC inhibitor, chelerythrine [29]. The concentration of chelerythrine used was 2 µmol l–1, about three times above the IC50 for PKC inhibition of 0.7 µmol l–1 [29]. However, at the concentration used, chelerythrine did not completely inhibit the effects of PGF2{alpha} and its effect was not as great as that of HOE 694. We did not wish to use a higher concentration of chelerythrine as we have shown that it can increase cell shortening on its own, and this would complicate interpretation of the data [40]. Although PKC has been shown to phosphorylate the Na+–H+ exchanger in other cell types [41], its ability to activate the cardiac Na+–H+ exchanger is controversial, and is likely to be indirect [26, 42, 43]. PGF2{alpha} may be using a similar signalling pathway to mediate its positive inotropic action.

Therefore, our data provide evidence that the positive inotropic action of PGF2{alpha} in rat cardiac myocytes is not due to calcium mobilisation, but it is due to activation of the Na+–H+ exchanger and an intracellular alkalosis, and that this alters the sensitivity of the contractile proteins to calcium. The partial inhibition of the inotropic response by a concentration of chelerythrine which would be expected to produce a good inhibition of PKC makes it difficult to draw firm conclusions on the precise role of PKC in this response, also, our studies do not indicate which isoform(s) of PKC may be involved and they do not rule out a role for other intracellular signalling mechanisms. However, the fact that HOE 694 completely inhibited the inotropic action of PGF2{alpha} indicates that activation of the Na+–H+ exchanger is a major factor in this response.

Time for primary review 16 days.


   Acknowledgements
 
S.F. Yew is funded by Universiti Kebangsaan Malaysia. K.A. Reeves is funded by the British Heart Foundation.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

  1. Vergroesen A.J., de Boer J., Gottenbos J.J. Effects of prostaglandins on perfused isolated rat hearts. Acta Physiol Pharmacol Neer (1967) 15:72.
  2. Metsa-Ketela T. Cyclic AMP-dependent and -independent effects of prostaglandins on the contraction-relaxation cycle of spontaneously beating isolated rat atria. Acta Physiol Scand (1981) 112:481–485.[Web of Science][Medline]
  3. Karmazyn M., Dhalla N.S. Effect of adenosine on the cardiac actions of prostaglandins E2, I2 and F2{alpha}. Can J Physiol Pharmacol (1981) 60:819–824.[Web of Science]
  4. Karmazyn M., Tuana B.S., Dhalla N.S. Effect of prostaglandins on rat heart sarcolemmal ATPases. Can J Physiol Pharmacol (1981) 59:1122–1127.[Web of Science][Medline]
  5. Otani H., Otani H., Das D.K. Positive inotropic effect and phosphoinositide breakdown mediated by arachidonic acid and prostaglandin F2{alpha}. J Pharmacol Exp Ther (1988) 244:844–851.[Abstract/Free Full Text]
  6. Nutter D.O., Ratts T. Direct actions of prostaglandins E1, A1, and F2{alpha} on myocardial contraction. Prostaglandins (1973) 3:323–336.[CrossRef][Web of Science][Medline]
  7. Karmazyn M. Contribution of prostaglandins to reperfusion-induced ventricular failure in isolated rat hearts. Am J Physiol (1986) 251:H133–H140.[Web of Science][Medline]
  8. Balwierczak J.L. The relationship of KCL- and prostaglandin F2{alpha}-mediated increases in tension of the porcine coronary artery with changes in intracellular calcium measured with fura-2. Br J Pharmacol (1991) 104:373–378.[Web of Science][Medline]
  9. Forster W., Mest H.-J., Mentz P. The influence of PGF2{alpha} on experimental arrhythmias. Prostaglandins (1973) 3:895–903.[CrossRef][Web of Science][Medline]
  10. Moffat M.P., Karmazyn M., Ferrier G.R. Role of prostaglandins in the arrhythmogenic effects of ouabain on isolated guinea pig hearts. Eur J Pharmacol (1987) 141:383–393.[CrossRef][Web of Science][Medline]
  11. Rayner T.E., Chen B.N., McLoughlin J.W., et al. Prostaglandin F2{alpha} mediates platelet-activating factor-stimulated atrial natriuretic factor release from the isolated rat heart. Endocrinology (1993) 133:1108–1115.[Abstract/Free Full Text]
  12. Gardner D.G., Schultz H.D. Prostaglandins regulate the synthesis and secretion of the atrial natriuretic peptide. J Clin Invest (1990) 86:52–59.[Web of Science][Medline]
  13. Lai J., Jin H., Yang R., et al. Prostaglandin F2{alpha} induces cardiac myocyte hypertrophy in vitro and cardiac growth in vivo. Am J Physiol (1996) 271:H2197–H2208.[Web of Science][Medline]
  14. Berger H.J., Zarat B.L., Speroff L., Cohen L.S., Wolfson S. Cardiac prostaglandin release during myocardial ischaemia induced by atrial pacing in patients with coronary artery disease. Am J Cardiol (1977) 39:481–486.[CrossRef][Web of Science][Medline]
  15. Chazov E.I., Pomoinetsky V.D., Geling N.G., et al. Heart adaptation to acute pressure overload. An involvement of endogenous prostaglandins. Circ Res (1979) 45:205–211.[Abstract/Free Full Text]
  16. Coleman R.A., Smith W.L., Narumiya, VII S. International Union of Pharmacology classification of prostanoid receptors: properties, distribution, and structure of the receptors and their subtypes. Pharmacol Rev (1994) 46:205–229.[Web of Science][Medline]
  17. Sabatini-Smith S. Action of prostaglandins E1 and F2{alpha} on calcium (Ca) flux in the isolated guinea pig atria and fragmented cardiac sarcoplasmic reticulum (Abstract). Pharmacologist (1970) 12:239.
  18. January C.T., Schottelius B.A. Electrophysiologic and inotropic actions of prostaglandin F2{alpha} in rat myocardium. Proc Soc Exp Biol Med (1974) 147:403–406.[CrossRef][Medline]
  19. Sperelakis N. Regulation of calcium slow channels. Curr Top Memb Transport (1985) 1 (25):43–76.
  20. Fabiato A., Fabiato F. Effects of pH on the myofilaments and the sarcoplasmic reticulum of skinned cells from cardiac and skeletal muscles. J Physiol (1978) 276:233–255.[Abstract/Free Full Text]
  21. Hori M., Sato K., Miyamoto S., Ozaki H., Karaki H. Different pathways of calcium sensitization activated by receptor agonists and phorbol esters in vascular smooth muscle. Br J Pharmacol (1993) 110:1527–1531.[Web of Science][Medline]
  22. Suematsu E., Resnick M., Morgan K.G. Change of Ca2+ requirement for myosin phosphorylation by prostaglandin F2{alpha}. Am J Physiol (1991) 261:C253–C258.[Web of Science][Medline]
  23. Kawase T., Orikasa M., Suzuki A. Effects of prostaglandin E2 and prostaglandin F2{alpha} on cytoplasmic pH in a clonal osteoblast-like cell-line, MOB 3-4. J Cell Physiol (1991) 146:141–147.[CrossRef][Web of Science][Medline]
  24. Mene P., Dubyak G.R., Scarpa A., Dunn M.J. Regulation of cytosolic pH of cultured mesangial cells by prostaglandin F2{alpha} and thromboxane-A2. Am J Physiol (1991) 260:C159–C166.[Medline]
  25. Ikenouchi H., Barry W.H., Bridge J.H.B., et al. Effects of angiotensin II on intracellular calcium and pH in isolated beating rabbit hearts and myocytes loaded with the indicator indo-1. J Physiol (1994) 480:203–215.[Abstract/Free Full Text]
  26. Puceat M., Clement-Chomienne O., Terzic A., Vassort G. {alpha}1-Adrenoceptor and purinoceptor agonists modulate Na-H antiport in single cardiac cells. Am J Physiol (1993) 264:H310–H319.[Web of Science][Medline]
  27. Sholz W., Albus U., Lang H.J., et al. Hoe 694, a new Na+/H+ exchange inhibitor and its effects in cardiac ischaemia. Br J Pharmacol (1993) 109:562–568.[Web of Science][Medline]
  28. Loh S.-H., Sun B., Vaughan-Jones R.D. Effect of Hoe 694, a novel Na+-H+ exchange inhibitor, on intracellular pH regulation in guinea pig ventricular myocyte. Br J Pharmacol (1996) 118:1905–1912.[Web of Science][Medline]
  29. Herbert J.M., Augereau J.M., Gleye J., et al. Chelerythrine is a potent and specific inhibitor of protein kinase C. Biochem Biophys Res Commun (1990) 172:993–999.[CrossRef][Web of Science][Medline]
  30. Harding S.E., Vescovo G., Kirby M., et al. Contractile responses of isolated adult rat heart and rabbit cardiac myocytes to isoproterenol and calcium. J Mol Cell Cardiol (1988) 20:635–647.[CrossRef][Web of Science][Medline]
  31. Fabiato A. Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum. Am J Physiol (1983) 245:C1–C14.[Web of Science][Medline]
  32. Grynkiewicz G., Poenie M., Tsien R.Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem (1985) 260:3440–3450.[Abstract/Free Full Text]
  33. Martinez-Zaguilan R., Martinez G.M., Lattanzio F., Gillies R.J. Simultaneous measurement of intracellular pH and Ca2+ using the fluorescence of SNARF-1 and fura-2. Am J Physiol (1991) 260:C297–C307.[Web of Science][Medline]
  34. Orallo F. Regulation of cytosolic calcium levels in vascular smooth muscle. Pharmacol Ther (1996) 69:153–171.[CrossRef][Web of Science][Medline]
  35. Berridge J. Elementary and global aspects of calcium signalling. J Physiol (1997) 499:291–306.[Free Full Text]
  36. Movsesian M.A., Thomas A.P., Selak M., Williamson J.R. Inositol trisphosphate does not release calcium from permeabilised cardiac myocytes and sarcoplasmic reticulum. FEBS Lett (1985) 185:328–334.[CrossRef][Web of Science][Medline]
  37. Kentish J.C., Barsotti R.J., Lea T.J., et al. Calcium release from cardiac sarcoplasmic reticulum induced by photorelease of calcium or Ins(1,4,5)P3. Am J Physiol (1990) 258:H610–H615.[Web of Science][Medline]
  38. Kohmoto O., Ikenouchi H., Hirata Y., et al. Variable effects of endothelin-1 on [Ca2+]i transients, pHi and contraction in ventricular myocytes. Am J Physiol (1993) 265:H793–H800.[Web of Science][Medline]
  39. Sun B., Leem C.H., Vaughan-Jones R.D. Novel chloride-dependent acid loader in the guinea-pig ventricular myocyte: part of a dual acid-loading mechanism. J Physiol (1996) 495:65–82.[Abstract/Free Full Text]
  40. Yew S.F., Woodward B. Chelerythrine influences basal and prostaglandin F2{alpha}-induced increases in rat cardiomyocyte contractility (Abstract). Br J Pharmacol (1997) 122:129.
  41. Kandasamy R.A., Yu F.H., Harris R., et al. Plasma membrane Na+–H+ exchanger isoforms (NHE-1, -2, and -3) are differentially responsive to second messenger agonists of the protein kinase A and C pathways. J Biol Chem (1995) 270:29209–29216.[Abstract/Free Full Text]
  42. Fliegel L., Walsh M.P., Singh D., Wong C., Barr A. Phosphorylation of the C-terminal domain of the Na+–H+ exchanger by Ca2+/calmodulin-dependent protein kinase II. Biochem J (1992) 282:139–145.[Web of Science][Medline]
  43. MacLeod K.T., Harding S.E. Effects of phorbol ester on contraction, intracellular pH and intracellular calcium in isolated mammalian ventricular myocytes. J Physiol (1991) 444:481–498.[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Fong Yew, S.
Right arrow Articles by Woodward, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Fong Yew, S.
Right arrow Articles by Woodward, B.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?