Simultaneous Measurement of Intracellular pH, Calcium, and Tension inRat Mesenteric Vessels: Effects of Extracellular pH

Clare Austin,1 Keith Dilly,* David Eisner,* and Susan Wray

Departments of Physiology and *Veterinary Preclinical Sciences, The University of Liverpool,Liverpool, L69 3BX, United Kingdom

Received April 9, 1996

In rat mesenteric vessels changes in external pH (pHo) alter tension. Changes in intracellular pH (pHi) andcalcium ([Ca2+]i) have both been suggested to underlie the tension changes. As no simultaneous measurementsof pHi and [Ca2+]i had been made, however, the relative importance and temporal relationship between pHi

[Ca2+]i and tension were unknown. In order therefore to gain a clearer understanding of the mechanismsinvolved, we have made simultaneous measurements of these parameters using carboxy-SNARF and INDO-1.Raising pHo caused rises in pHi, Ca

2+ and tension. The increases in pHi preceded increases in [Ca2+]i, whichpreceded the increases in tension. Similar but opposite effects were observed when pHo was decreased. Weconclude that the change in [Ca2+]i upon alteration of pHo is subsequent to that of pHi. © 1996 Academic Press, Inc.

Although it has been known for many years that changes in external pH (pHo) alter vascular tone,the mechanisms involved are still not fully understood. We have previously shown in rat mesentericvessels that changes in pHo cause large (approximately 70% change in pHo) and rapid (<2 mins.)changes in intracellular pH (pHi). By simultaneously measuring pHi and tone when pHo waschanged we have shown that the induced changes in pHi precede, and are essential for, the changesin tone (1,2). For pHo to alter tension it must also be altering either the intracellular calciumconcentration ([Ca2+]i), or the sensitivity of the contractile proteins to [Ca2+]. By simultaneouslymeasuring [Ca2+]i and tension we have also shown that changes in pHo cause changes in [Ca

2+]i,which preceded the changes in tension (3). Furthermore, if the changes in [Ca2+]i were prevented,the effects of pHo on tension were also prevented. This therefore suggests that it is a change in[Ca2+]i, and not calcium sensitivity, which is responsible for the effect on tone (3).In rat mesenteric vessels therefore changes in both pHi and [Ca

2+]i are involved in the changesin tone observed when pHo is altered and changes in both intracellular ions precede changes intension. The temporal relationship between the changes in the two ions, however, is unknown. Twohypotheses suggest themselves: (i) pHo induces a change in pHi which causes a subsequent changein [Ca2+]i, which then alters tone; (ii) pHo has a direct effect on [Ca2+]i i.e changes in [Ca2+]iprecede those of pHi. By obtaining simultaneous measurements of pHi, [Ca

2+]i and tension, therelationship between these parameters can be investigated. Although a few recent studies havesimultaneously measured pHi and [Ca

2+]i in isolated cells (5,9) no previous studies have measuredthe parameters in intact tissues from which tension measurements could also be obtained. This wastherefore the aim of the present study.

METHODS

Male Wistar rats (200–250g) were killed by chloroform and exsanguination. The mesentery was removed and mesentericbranch vessels (radius 200–400mm) were cleared of fat and dissected. Strips of vessel were mounted in a small bath on thestage of an epifluorescent microscope. One end of the strip was attached to the bath, the other to a SWEMA transducer fortension measurement. The endothelium was removed by gentle rubbing. Tissues were loaded initially with the acetoxy-methylester for of the pH-sensitive dye carboxy-SNARF (5mM) for 30–60 mins at room temperature. They were thenloaded with the calcium sensitive dye INDO-1 (20mM) (again the ester form) for a similar period of time.

1 Current address: Department of Medicine, Manchester Royal Infirmary, Oxford Road, Manchester, M13 9WL.

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Following incubation and washing the tissues were alternatively excited at 530 nm and 340 nm, the excitation wave-lengths for the two dyes, by means of a spinning wheel system (50 Hz, Cairn). A dual-band reflection (350 and 540 nm)multi-band transmission multi-wavelength dichroic mirror (Chroma Technology) was placed in the usual place for theepifluorescence dichroic mirror (Wiegmannet al.,1993). Sequentially light was reflected via a 410 nm dichroic mirror toallow measurement of INDO-1 emission at 400 nm and via a 530 nm dichroic for measurement at 500 nm. Light >530 nmwas transmitted for measurement of SNARF emissions at 580 and 640 nm which were separated by a 610 nm dichroic. Theratio of SNARF emissions was calibrated by use of either the K+-H+ ionophore nigericin, in situ or with free acid SNARF,in vitro. We have previously shown a good agreement between these methods (2). Due to problems associated withcalibrating INDO-1 the ratio of the 400 and 500 nm emissions was used as an indication of [Ca2+]i. Previous work hasshown that there is a good agreement between changes in this ratio and changes in [Ca2+]i in vascular smooth muscle (3).We have also previously calculated that the fluorescence of INDO-1 is not greatly affected by pHi changes over the rangesencountered in this study (see 3). Tissues were constantly perfused with a Krebs solution of the following composition(mM): NaCl, 154; KCl, 5.4; MgSO4, 1.2; glucose, 12; CaCl2, 3 at 37°C, gassed with 100% O2 and buffered to pH 7.4 with11 mM HEPES. pHo was altered by addition of NaOH or HCl.

StatisticsAll results are expressed as mean ± standard errors of means. Then values indicate the number of animals. Differences

were taken to be significant ifP < 0.05 in paired or unpaired students t-tests, as appropriate.

RESULTS

Simultaneous measurement of pHi, [Ca2+]i and vascular tension were obtained by this method.

Tissues were found to be well loaded with both carboxy SNARF and INDO-1 after incubating.Resting pHi in tissues was found to be 7.28 ± 0.06 (n4 14). Addition of a high K+ solution todepolarize tissues produced a rapid contraction which was accompanied by an increase in [Ca2+]ias previously shown. The mean steady state pHi of depolarized tissues was significantly lower thancontrols (7.21 ± 0.06, n4 14).Increasing pHo from 7.4 to 7.9 produced an increase in pHi (0.36 ± 0.05 pH units), and a steady

increase in [Ca2+]i and tension. When normalized to the changes in response to KCl (100%), [Ca2+]irose by 91 ± 12% and tension by 102 ± 24% (n4 10). Figure 1 show a typical example. Whenthe temporal relationship between the parameters was investigated it was found in every case that,following a change in pHo, the change in pHi preceded that of [Ca

2+]i (n4 10, see fig. 1). A change

FIG. 1. Simultaneous traces of tension (top trace), [Ca2+]i (400:500 nm ratio, middle) and pHi (bottom trace) in responseto elevation of pHo from 7.4 to 7.9. The traces were obtained from strips of rat mesenteric resistance vessels loaded withboth the pH-sensitive dye carboxy SNARF and the Ca2+-sensitive dye INDO-1, and pre-contracted with 40 mM KCl. Inset:expanded part of the record shown in the dotted box to highlight the faster pHi response compared to [Ca

2+]i, a and c indicatethe points at which Ca2+ and pHi responses began and b and d indicate the times to half-maximal responses.

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in pHi was first observed 17.4 ± 2.3 secs after the solution was changed. This was significantlyfaster than that of [Ca2+]i which was 28.1 ± 3.7 s. The times taken for half maximal response werealso different; 48.5 ± 1.5 and 54.6 ± 5.3 s. for pHi and [Ca2+]i respectively following the changein pHo. These responses were both faster than that of tension (58.2 ± 3.7 s). The speed of theindividual responses, measured from when a response was first observed, were also examined. Thefollowing values were obtained; 27.4 ± 2.4 s for pHi, 30.3 ± 4.8 s for [Ca

2+]i and 32.4 ± 3.5 s fortension. Thus the consistent temporal sequence of events was; pHi then [Ca

2+]i, then tension. Thetemporal dissociation between pH, Ca and contraction is emphasised in the hysteresis plots offigure 2. Figure 2A shows that, during the application of alkaline solution the relationship betweenpHi and [Ca

2+]i differs from that on return to control. There is a similar hysteresis between calciumand tension.When pHo was decreased from 7.4 to 6.9, decreases in pHi (0.26 ± 0.04 pH units), [Ca2+]i and

tension were observed. When the decreases in [Ca2+]i and tension were again normalized to theincreases observed in response to KCl, the following changes were observed; 20 ± 20% for [Ca2+]iand 56 ± 12% for tension (n4 7). A similar temporal sequence of changes to that described abovewas again seen, pHi changing before [Ca

2+]i (28.3 ± 9.0 and 32.2 ± 8.3 s). Times to half maximalresponse after the initial solution change and after the first detectable change were; 61.5 ± 10.1 and

FIG. 2. Hysteresis plots of A; pH against Ca2+ and B; Ca2+ against tension, for the mesenteric vessel. Changes producedby elevating pHo from 7.4 to 7.9. The SNARF (pH) ratio is plotted as the reciprocal so that the direction of the changes arethe same as those for the Indo (Ca2+) ratio. The filled circles show the data points when tension was increasing and the opencircles were obtained as tension was relaxing.

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33.4 ± 4.2 s. for pHi, 79.9 ± 17.4 and 47.6 ± 11.8 s for [Ca2+]i and 81.8 ± 14.6 and 67.2 ± 24.3s for tension. Thus again pHi changed before [Ca2+]i.

DISCUSSIONWe have demonstrated that pHi, [Ca

2+]i and tension can be simultaneously measured by loadingisolated strips of rat mesenteric resistance vessels with both carboxy-SNARF and INDO-1. Wehave used this methodology to show that when pHo is altered in these vessels, pHi changes precedethose of [Ca2+]i, which precede changes in tension. This therefore supports the suggestion that itis pHi that alters [Ca

2+]i and hence tension (see below).Although other workers have previously measured pHi and [Ca

2+]i simultaneously this is the firststudy where tension has also been measured and the temporal relationships examined. The fluo-rescence spectra of the two indicators used are sufficiently separate to allow simultaneous mea-surement of pHi and [Ca2+]i. It was also noted that the behaviour of pHi and [Ca2+]i e.g. restingvalues and magnitude of changes, in the present study was in agreement with that anticipated fromour previous work (1), where they were measured independently - indicating the lack of interfer-ence between the dyes with dual loading (see also 9). Compared to previous measurements of thetwo ions separately, there was a reduction in the signal to noise ratio when simultaneous mea-surements were obtained, presumably due to the lack of continuous excitation. As shown in figure1 this did not, however, prevent useful data from being obtained.By simultaneously measuring the pHi, [Ca

2+]i and tension, we have shown that all the parameterschange over a similar time course in response to alteration of pHo. Irrespective of the time-pointchosen or the direction of the pHo change, we found that, changes in pHi preceded changes in[Ca2+]i which in turn, preceded changes in tension. It has previously been shown, in isolatedvascular smooth muscle cells, that changes in pHi, induced by addition of weak acids or bases, canalter [Ca2+]i; increases in pHi increasing [Ca2+]i (6). In view of this, and the fact that changes inpHi precede changes in [Ca2+]i, it seems likely that it is the induced changes in pHi which areresponsible for the changes in [Ca2+]i observed when pHo is altered. These changes in [Ca

2+]i maybe due to effects of pHi on Ca2+ movement across either the surface or sarcoplasmic reticulummembranes. It could be that the slower calcium response was coincidental rather than consequentialand other studies have shown that pHo can have a direct effect on L-type Ca

2+ currents (8). As wehave already shown, however, in experiments where [Ca2+]i and pHi were measured separately, ifthe effects of pHo on either parameter were prevented, the effect of pHo on tension was alsoprevented. Thus it seems that it is the changes in the intracellular ions, and their relationshipbetween each other, which is important in the determination of vascular tone in mesenteric vessels.Whether the relationship is similar in vessels where pHo causes smaller changes in pHi e.g. portalvein (7) is yet to be determined but clearly such simultaneous measurements will allow furtherstudy of the relationships between pHo, pHi, [Ca

2+]i and vascular tone, or indeed contraction inother muscles.

ACKNOWLEDGMENTSWe are grateful to the BHF and Wellcome Trust for support.

REFERENCES

1. Austin, C., and Wray, S. (1993)J. Physiol.466,1–8.2. Austin, C., and Wray, S. (1994)Pflugers Arch.427,270–276.3. Austin, C., and Wray, S. (1995)J. Physiol.488,281–291.4. Crichton, C. A., Templeton, A. G. B., and Smith, G. L. (1994)Cardiovasc. Res.28, 1378–1384.5. Martinez-Zaguilan, R., Martinez, G. M., Lattanzio, F., and Gillies, R. J. (1991)Am. J. Physiol.260,C297–C307.6. Siskind, M. S., McCoy, C. E., Chobanian, A., and Schwartz, J. H. (1989)Am. J. Physiol.393,57–71.7. Taggart, M., Austin, C., and Wray, S. (1994)J. Physiol.475,285–292.8. West, G. A., Leppla, D. C., and Simard, J. M. (1992)Circ. Res.71, 201–209.9. Wiegmann, T. B., Welling, L. W., Beatty, D. M., Howard, D. E., Vamos, S., and Morris, S. J. (1993)Am. J. Physiol.

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