Dynamic and static calcium gradients inside large snail (Helix aspersa) neurones detected with calcium-sensitive microelectrodes

We have used quartz Ca2+-sensitive microelectrodes (CASMs) in large voltage-clamped snail neurones to investigate the inward spread of Ca2+ after a brief depolarisation. Both steady state and [Ca2+]i transients changed with depth of penetration. When the CASM tip was within 20 μm of the far side of the cell the [Ca2+]i transient time to peak was 4.4 ± 0.5 s, rising to 14.7 ± 0.7 s at a distance of 80 μm. We estimate that the Ca2+ transients travelled centripetally at an average speed of 6 μm2 s−1 and decreased in size by half over a distance of about 45 μm. Cyclopiazonic acid had little effect on the size and time to peak of Ca2+ transients but slowed their recovery significantly. This suggests that the endoplasmic reticulum curtails rather than reinforces the transients. Injecting the calcium buffer BAPTA made the Ca2+ transients more uniform in size and increased their times to peak and rates of recovery near the membrane. We have developed a computational model for the transients, which includes diffusion, uptake and Ca2+ extrusion. Good fits were obtained with a rather large apparent diffusion coefficient of about 90 ± 20 μm2 s−1.This may assist fast recovery by extrusion.
1
Introduction
Changes in free intracellular calcium ([Ca2+]i) play a vital role in intracellular signalling, notably in coupling electrical excitation to secretion, contraction and other activation processes [1,2]. Many of the processes activated by a rise in [Ca2+]i are believed to occur in the nucleus [3,4], which in large neurones may be far from the cell membrane. It is well-established that a brief depolarization of most nerve cells causes an influx of Ca2+ ions across the plasma membrane and thus a local rise in [Ca2+]i. The spatiotemporal characteristics of this rise in calcium are defined by the interactions between Ca2+ ions and the cytoplasmic milieu. Calcium-induced calcium release (CICR) processes via either IP3 or ryanodine receptors can facilitate the regenerative spread of the calcium signal throughout the cell, whilst immobile calcium buffers will slow and attenuate the signal. If the increase in sub-membrane [Ca2+]i is to act as a signal at a distance, the speed at which the Ca2+ ions travel from the cell membrane to their target depends critically on the rate of diffusion of Ca2+ ions in the cytoplasm. Most studies of calcium homeostasis and signalling in nerve and muscle cells have been made using optical methods to measure [Ca2+]i via the binding of Ca2+ ions by fluorescent or other indicators [5]. Such indicators tend to increase intracellular Ca2+ buffering and mobility [6]. Many authors (for example [7–11]) have modelled the effect of mobile indicators on Ca2+ transients, but very few have produced data on intracellular Ca2+ movement from the periphery towards the centre inside real neurones in the absence of such alien additives. We now present such data, obtained with Ca2+-sensitive microelectrodes (CASMs), for the first time.
CASMs offer an alternative but rarely-used method for measuring [Ca2+]i. They have the advantage of measuring [Ca2+]i in a small volume without adding any extraneous compounds which might interact with Ca2+ throughout the cell. But they are rather slow to respond, and only a realistic choice for large cells since they tend to create a leak at the site of penetration [12–15].
In previous work, with CASMs made from aluminosilicate glass, the CASMs were rarely moved once a stable potential had been obtained from deep inside the neurone [e.g. 13,14,16]. Recently a modified electrode construction technique has allowed us to use CASMs with very slender tips and longer lives made from thin-walled quartz glass. We have also found it possible, with a motorized micromanipulator, to move the CASMs in repeatable steps up and down, usually without apparently causing damage to the cell.
We have sought to record Ca2+ transients at different distances from the inside of the membrane at the far side of the penetrated cell. Our recordings at different depths were quite repeatable, and enabled us to reconstruct the radial inward movement of the entering Ca2+ towards the centre of the cell. We have done this in large neurones with initially no added Ca2+ indicator or buffer. Although our method does tend to induce a leak of Ca2+ ions at the penetration site, we believe that the leak simply increases the steady state level without apparently influencing the kinetics of the Ca2+ transients. We investigated the role of the endoplasmic reticulum by blocking its Ca2+ uptake pump with cyclopiazonic acid. We also injected the mobile Ca2+ buffer BAPTA, which has very similar Ca-buffering properties to many fluorescent Ca2+ indicators, to measure its effects on Ca2+ diffusion. We have modelled the Ca2+ increases mathematically and have been able to model the transients closely by assuming that calcium signalling in these cells can be described by Ca2+ influx, diffusion, extrusion, sequestration and buffering. Good fits were obtained with a diffusion coefficient of about 90 ± 20 μm2 s−1, which is higher than reported in other cell types.
2
Materials and methods
2.1
General
All experiments were done on large (150–250 μm diameter) snail neurones in isolated sub-oesophageal ganglia [14,16]. Cells were voltage-clamped using two microelectrodes to −50 mV, and depolarized at intervals by 40 or 50 mV to generate an influx of Ca2+ ions. The resulting changes in [Ca2+]i were measured using CASMs. The main differences from previous methods were that we made the CASMs with quartz rather than aluminosilicate glass and used a motorized micromanipulator (Luigs & Neumann, Ratingen, Germany) to move the CASM up and down in reproducible steps. Rotating the Z-control wheel by 360° moved the CASM up or down by 40 μm, thus for a 20 μm step we rotated it manually by 180°.
2.2
Preparation
An aestivating snail, Helix aspersa, was killed humanely by rapid removal of the circumoesophageal ring of ganglia. These ganglia were mounted dorsal side uppermost on a plastic bath insert. The thick connective tissue covering the back of the visceral and right and left pallial ganglia was then removed and the bath insert with ganglia was mounted in the experimental chamber at an angle of 50° to the vertical. Once covered with snail Ringer (flow rate 1.0–1.2 ml min−1, bath volume 0.1 ml) the inner connective tissue was torn with a fine tungsten hook to expose some of the neurones. All experiments were carried out at room temperature, 18–22 °C, starting at least an hour after the dissection.
2.3
Solutions
The normal snail Ringer contained (mM): 80 NaCl, 4 KCl, 7 CaCl2, 5 MgCl2, 20 HEPES, titrated with NaOH to pH 7.5. Nominally Ca-free Ringers had the same composition but with no CaCl2 and 12 mM MgCl2. The EGTA Ca-free solution had an additional 1 mM EGTA. The CO2 Ringer was the same as normal Ringer except that it had 20 mM NaHCO3 instead of HEPES, was bubbled with 2.5% CO2 in air and contained 0.1 mM NaH2PO4. The high K low Ca solution for BAPTA iontophoresis calibration contained 100 KCl, 5 NaCl, 0.6 MgCl2, and 10 mM HEPES. The pH was adjusted to 7.2 with KOH. Cyclopiazonic acid (CPA) was kept frozen as a 50 mM stock solution in dimethyl sulphoxide and diluted in snail Ringer on the day of use. All these chemicals were from Sigma.
2.4
Microelectrodes
Conventional micropipettes were pulled from 1.2 mm filamented borosilicate glass tubing and backfilled with either 2 M CsCl (for passing current), or 2 M KCl (for recording membrane potential). Tips were broken if necessary by touching a pin in the bath to give resistances between 5 and 15 MΩ. BAPTA was iontophoresed from similar micropipettes filled with 100 mM BAPTA (K salt). CASMs were made by a method refined from those described previously [16,17]. Every few days we pulled 10–12 long, sharp micropipettes from unfilamented quartz tubing (1.0 mm o.d., 0.7 mm i.d.) using a Sutter P-2000 puller (Sutter Instrument Co, Novato, Cal.). We then broke the tips on a glass rod to o.d. 1.0–1.5 μm. We then left the micropipettes in air for 2–4 h before silanizing them with bis(dimethylamino)dimethylsilane (Fluka 14755) as described before [16].
Individual CASMs were prepared as follows on the day of the experiment. First we backfilled 2 or 3 silanized micropipettes with 1 mM CaCl2 (instead of the 100 mM KCl, 0.1 mM CaCl2 used before, which we realised shortened the CASM's useful life) using a 10 ml syringe connected to the back of each micropipette via a silicone rubber tube to force air out of the tips. We then sucked a column (150–250 μm) of sensor cocktail into the tip of each micropipette, taking care to avoid any air gap between cocktail and backfill. The cocktail composition was (mg/g): 12 Ca ionophore ETH 129 (Fluka 21193), 6 Na tetrakis[3,5-bis(trifluoromethyl)phenyl]borate, 200 2-nitrophenyloctylether, 17 high molecular weight PVC, and 765 tetrahydrofuran (all from Fluka). Filled microelectrodes were left in air to allow the tetrahydrofuran to evaporate for at least 2 h before use, and had useful lives of 2–3 days. During the experiment we applied 80–120 kPa of air pressure to the back of the CASM, since this maximizes its response [15]. Before use each microelectrode was tested in the experimental bath, initially perfused with normal snail Ringer. Once the potential had stabilised the perfusate was switched to Ca-free (EGTA) Ringer's solution. Any electrode showing a potential change of less than 150 mV was discarded. The potentials recorded from the CASMs during the experiments were displayed as VCa (Vm being automatically subtracted when both electrodes were in the same cell) and we have not converted them either to pCa or nM free [Ca2+]i except for modelling purposes. Calibration of CASMs is difficult because, in our hands, their responses tend to change when they are withdrawn from a cell at the end of an experiment. Previous work [14] suggests that their responses are essentially linear with pCa between 2 and 7(10 mM–100 nM), with a potential change of 28 mV per decade. Half-response times ranged from 0.2 to 0.5 s, but were not routinely measured. Results were discarded if on withdrawal the electrode potentials had changed by more than 7 mV.
2.5
Estimating the BAPTA intophoresis transport number
We first used CASMs to measure how much BAPTA was required to change the VCa of 1 ml of the high K, low Ca solution from −50 to −110 mV (CASM zeroed in normal snail Ringer). The BAPTA was injected into the 1 ml aliquots using an oocyte microinjector. The average quantity of BAPTA needed was 75 nmol. We then made droplets (volumes 15–25 nl) of the same solution under oil, measured VCa as with a snail neurone, and injected BAPTA iontophoretically. We found that to achieve the equivalent VCa change as in the 1 ml samples we had to inject an average 0.6 μC nl−1, giving a transport number of 0.01.
2.6
Data collection and analysis
Potentials from the microelectrodes were led via Ag/AgCl wires to preamplifiers in the cage. Outputs from these and the clamp amplifier were filtered (8-pole Bessel) and recorded at 20 Hz on a PC via a CED micro 1401 interface and Spike 2 data collection program (Cambridge Electronic Design, UK). The CASM potential was differentially recorded with the Vm microelectrode potential as reference. Figures were prepared from the CED data after loading into Microsoft EXCEL. Large spikes in the VCa records generated by electronic pickup were erased, and the clamp current records were restricted in range. For more details of most of the apparatus see [14,16]. Data are presented as means ± S.E.M. of n observations. The statistical significance of observed shifts in means was determined by a one-tailed Wilcoxon rank sum test. The difference between means was considered significant when the p-value was higher than 0.95. For baseline adjustment, selecting transients, time to peak and exponential fitting, a home made Matlab script with a graphical user interface was used.
2.7
Computational modelling
There is an extensive body of literature on the modelling of Ca2+-signalling in a wide variety of cell types, for example [18–21]. In this study we set out to develop a minimal computational model of Ca2+-signalling in snail neurones. We used the model to validate and explain the results obtained from the experiments. Furthermore, we used the model to estimate the mobility of Ca2+ in these large neuronal cell bodies, which have not been exposed to mobile Ca2+-indicators.
The model includes the following dynamic processes: Ca2+ diffusion, influx, extrusion, sequestration and buffering. We assume that influx and extrusion mechanisms at the plasma membrane are uniformly distributed over the cell's surface. The CASM and the axon attached to the soma only represent a small fraction of the cell's volume (<1%) and surface area (<5%), furthermore transients were measured near the far side of the cell, opposite to the site of penetration. Since we could not detect any major deformation of the cell looking through the microscope we assume that the Ca2+ transients measured close to the membrane can be modelled with spherical symmetry. Using these assumptions we can reduce the three-dimensional diffusion problem to a one-dimensional radial diffusion equation in a sphere. Similar computational models for Ca2+ diffusion in a spherical cell can be found elsewhere [9,22].
The removal of Ca2+ after a brief influx is relatively slow, with time constants ranging from 5 to 15 s, while Ca2+ buffering occurs on the micro- and millisecond time scale. We therefore can separate these different processes kinetically and remove the explicit buffering terms from the transport equation [20]. In so doing we have to replace the Ca2+ diffusion coefficient DCa (μm2 s−1) with an apparent diffusion coefficient Dapp:
Here we used the notation from [7]; κe andκb denote the endogenous and exogenous Ca2+ binding ratio, respectively; the associated diffusion coefficients are De and Db. The diffusion coefficient of free cytoplasmic Ca2+ is denoted by DCa and was assumed to be 220 μm2 s−1
[23]. Furthermore, all other transport processes have to be scaled down by the total Ca2+ binding ratio κtot = 1 + κe + κb.
The total Ca2+ binding ratio κtot depends on the Ca2+ concentration; however, when the concentration is much lower than the dissociation constant it may be approximated to be constant. Moreover, although some data are available on buffering in snail neurones [24], precise measurements that separate the buffering and sequestration processes are not. We have therefore worked with apparent fluxes and apparent parameters in most cases. Multiplication of these apparent parameters with the total buffering power should yield the real parameters.
The general transport equation that describes the calcium dynamics in the cell, taking into account the different aforementioned considerations then becomes:
The first term on the right hand side represents the radial diffusional process that is driven by the concentration gradient in the cell. The transmembrane fluxes jin,app and jout,app describe the apparent influx of calcium through channels and apparent efflux of calcium by the pump, respectively, Jseq,app denotes the apparent calcium flux associated with sequestration to stores and mitochondria in the cell's cytoplasm and finally jbasal,app represents the basal influx of Ca2+ at rest. The surface-to-volume ratio rsv (m−1) is necessary to convert the flux density j (mol m−2 s−1) into a concentration flux J (mM s−1). The concentration [Ca2+]i and all fluxes are functions of position r (m) and time t (s); however, for readability we do not write this explicitly.
There are few precise quantitative data available about the different transport processes involved. To prevent the introduction of many unknown parameters we therefore kept the mechanistic complexity to a bare minimum. Consequently we modelled the influx as a square pulse with the same duration as the depolarisation:where the apparent Ca2+-current denoted by ICa,app (A) relates to the real current as follows: ICa,app = ICa/κtot. S denotes the surface area (m2) of the cell and F is Faraday's constant. The extrusion of Ca2+ by the pump is modelled by the following linear equation (for example see [25]):where Pout,app denotes the apparent extrusion permeability (m s−1) and relates to the real permeability as follows: Pout,app = Pout/κtot. The flux associated with sequestration was also assumed to be linear:where kseq,app denotes the apparent sequestration rate constant (s−1) and relates to the real rate constant as follows: kseq,out = kseq/κtot·[Ca2+]s is the steady state Ca2+ concentration. This approximate model is also known as a pool model [26]. Finally the basal influx jbasal,app was directly calculated from the steady state Ca2+ concentration and the extrusion parameter: jbasal,app = Pout,app [Ca2+]s.
2.8
Microelectrode model
In this study we used CASMs to measure the Ca2+ concentration after short depolarisations. If we assume equal activity coefficients in the Ringer and cytoplasm, the steady state cytoplasmic Ca2+ concentration can be directly estimated from the Nernst equation applied to the CASM potential:where the steady state Nernst-potential is denoted by ECa (V), R is the gas constant and the temperature T was 293 K. The backfill Ca2+ concentration [Ca2+]CASM in the microelectrode was 1.0 mM. The reference potential E0 was manually adjusted prior to the experiment such that the measured potential was 0 mV in Ringer solution; the Ringer solution contained 7 mM Ca2+.
Were the resistance and capacitance of the CASMs to be infinitely small and VCa to follow the Nernst equation over the whole range it could be used directly to estimate the cytoplasmic Ca2+ concentration. VCa is indeed close to the Nernstian value at [Ca2+]i over 100 nM [14]. Unfortunately the resistance of these microelectrodes is very high, which will introduce a delayed microelectrode response. To allow for this delay we used a RC filter to calculate the model microelectrode potential VCa:The response time of the microelectrode is denoted by τCASM (s). We assume that it is constant and depends only weakly on the microelectrode potential and [Ca2+]i.
2.9
Integration and parameter fitting
Assuming a linear transport equation a rather complex analytical solution consisting of an infinite series of exponentials with different time constants can be obtained using standard methods [27]:
The amplitudes An and the time constants τn are:
The constants αn are the positive roots of the following equation:where the diffusion time constant τD = d2/(4D) and the extrusion time constant τp = d/(2P).
The slowest time constant determines the overall recovery time. When there is no sequestration this time constant depends on the cell diameter d, the extrusion rate constant and the diffusion coefficient in a complex way. However, when diffusion is much faster than extrusion (extrusion limited) the time constant is simply τ0 = d/(6Pout) and vica versa when diffusion is much slower than extrusion (diffusion limited) the time constant is simply τ0 = d2/(4πDapp).
To compare the model with the results obtained from the experiments, we solved the transport equation simultaneously with the differential equation for the microelectrode potential. Although the analytical solution is very useful for theoretical analysis and testing, it requires many terms to obtain an accurate solution and does not allow for non-linearity. Therefore, we used a numerical approach for solving both equations, allowing more flexibility. Time integration was done using the forward Euler scheme and diffusion was calculated using a finite volume scheme (see for example [22]). The value of surface-to-volume ratio rsv in Eq. (2) using this finite volume scheme is simply the ratio of the cell surface S and the volume of the first compartment at the membrane. Both solutions were calculated using a Matlab 6.5 mex-routine written in C++.
In a typical experiment several Ca2+ transients were measured in succession at different depths. The relative distance was reconstructed from the micromanipulator movements that were all either 10 or 20 μm. The diameter of the cell was estimated from all of these successive steps. Unfortunately the absolute position (offset) of the transient measured closest to the membrane was not known exactly. We therefore allowed the depth as an adjustable parameter within the limits of ±10 μm. The transients measured closer to the middle were therefore more reliable. Furthermore, the response time of the electrode was estimated to be about 1.5–2.5 s and was allowed to vary within these limits. The remaining four unknown parameters in the transport equation, together with the response time, were obtained from an optimal fit. A simplex search algorithm was used to find this least square fit. To increase reliability of the parameter estimates, several transients measured at different depths were fitted simultaneously. Also, any slow baseline drift was removed and the first two seconds of the transients, that show an electrical artefact, were not used during the fitting process.
3
Results
3.1
Changes in measured calcium with depth of penetration
At the start of all experiments the tip of a CASM was placed centrally on the top of the chosen neurone and moved down in 20 μm steps until there was a sudden fall in the potential, as shown in Fig. 1A. Inside the cell the CASM registers both the membrane potential (Vm) and the change in VCa (the voltage, set to zero outside the cell, proportional to pCa). In this representative example the CASM was moved down in four steps before its potential changed from zero to about −50 mV. During a further six steps downward the CASM potential gradually became more negative, eventually more negative than −150 mV. By this time the electrode tip was 200 μm from the top of the cell, which had a diameter of about 240 μm. We then inserted the membrane potential microelectrode (the output of which was electrically subtracted from the CASM potential to give VCa) and clamp current microelectrode, using manually-operated micromanipulators. To test that the CASM and Vm electrodes were recording the same change in Vm, we then hyperpolarised the cell by 30 mV for 20 s. There were only small transient changes in VCa, generated by electrical pickup and imperfect subtraction of rapid changes. We then (not shown in Fig. 1A) started to depolarize the cell by 50 mV for 1 s, repeated every 2 min, to allow an influx of Ca2+ ions. The recorded increase in [Ca2+]i was very small; VCa increased from −107.5 to −106.4 mV, and then recovered. After the first depolarisation we raised the CASM by 60 μm and left it unmoved for 15 min to allow recovery from the initial penetrations.
As shown in detail in Fig. 1B, we then moved the CASM down every 2 min, between the 9th and 16th depolarisations, in six steps of 20 μm and two steps of 10 μm. (The third of these steps made the clamp current increase from −3 to −7 nA, but this was not correlated with any change in [Ca2+]i.) As the CASM tip was moved down the transient increases in VCa after each depolarisation became larger, and the steady-state VCa after each recovery became more negative. After the sixth step VCa had fallen to −127 mV, although after the next two it had risen slightly to −126 mV. After the last step down VCa rose to −118 mV before any depolarisation, suggesting the CASM had gone too far; although there was no change in the clamp current. We therefore then withdrew the CASM in several steps—two each of 10 and 20 μm. After the first 20 μm step up, the VCa paradoxically decreased, as if the upward movement somehow allowed a better seal. Such paradoxical improvements on withdrawal were often seen in the early part of an experiment.
Fig. 2 shows the VCa responses to the first nine depolarisations of Fig. 1B in more detail. As the CASM tip was moved closer to the far side of the cell the responses to the depolarisations became larger and faster. Presumably this reflects the fact that during the depolarisations Ca2+ entered the cytoplasm only at the cell membrane. Once Vm is returned to −50 mV, entry ceased and the Ca2+ both diffused into the cell interior and was pumped out by the plasma membrane Ca ATPase, or PMCA. In some experiments we were able to make several different sequences of downward and upward movements in the same cell to a depth at which, as shown in Fig. 3A, the apparent [Ca2+]i rose rapidly. This experiment was done on a cell of diameter about 140 μm, and the depths at which the apparent [Ca2+]i began to increase were, respectively, 140, 150 and 140 μm. In other words, it appears that when the CASM tip reached the far side of the cell it suffered a loss of sensitivity or caused a local leak. We could see no change in the clamp current associated with the sudden increases in VCa. In many other cells, once the CASM created a sudden rise in [Ca2+]i it took many minutes to recover, if at all.
The simplest explanation of the decreases in apparent [Ca2+]i as the CASM was advanced deeper and deeper into a cell is that the electrode insertion induced a leak at the site of penetration. As the CASM was pushed in, its tip moved further away from the leak, which itself may be reduced by better sealing. During long experiments, as shown below, CASM movement had less dramatic effects on steady-state levels, suggesting that the leak declined with time.
In Fig. 3B we show mean steady-state VCa values against the estimated distance of the CASM tip from the bottom of the cell, from the early part of a total of six experiments in which the CASM was moved down in steps, like those of Figs. 1 and 3A. The bottom of the cell was taken as the point at which VCa rose rapidly without a change in membrane potential. Transients recorded within 20 μm of this were plotted as “close” to the membrane. The mean VCa close to the cell membrane was −126 mV, which corresponds to a pCa of 6.7; similar to the 6.76 (equivalent to a [Ca2+]i of 170 nM) measured in snail neurones which had not been injected with buffers [13].
The VCa transients became faster as the CASM tips were moved closer to the far side of the cell. Fig. 3C shows the mean times to peak of the VCa increases from the same six experiments. From a best-fit line through the points, we can calculate that the mean rate at which the VCa peak moved towards the centre of the cell was 6 μm s−1. We also show in Fig. 3C the average size of the same transients, in mV from base to peak. The distance over which the transients decreased to half their (extrapolated) initial size of about 16 mV was about 45 μm, assuming that close to the membrane is equivalent to about 10 μm.
3.2
The effect of removing external calcium
If calcium ions, driven by the large electrochemical gradient, normally leak into the top of the cell around the tips of the microelectrodes, reducing external calcium should reduce the apparent [Ca2+]i. We have tested the effect of a possible leak by exposing cells to either nominally Ca-free solutions or the same with added EGTA. Measured with a CASM, the [Ca2+] of the nominal solution was about 20 μM, that of the EGTA Ca-free Ringer less than 10 nM. Fig. 4 shows a representative experiment in which the cell was superfused with the Ca-free solutions with the CASM at three different depths.
For the first two exposures to Ca-free solution the CASM was 80 μm deep in a cell of diameter about 170 mm. The Ca-free solutions caused VCa to decrease by 11 mV from, respectively, −115 and −116 mV. There was little difference between the effect of the two Ca-free solutions except that the EGTA solution almost abolished the second depolarisation-induced rise in Ca. As the CASM was moved deeper into the cell the steady-state VCa fell to −129 mV, and the reduction seen with Ca-free Ringer decreased to 4 and 3 mV, respectively. While some of the decreases seen with the removal of external Ca may reflect increased pump activity, it seems most likely that the elevated VCa levels seen near the middle of the cell are due to leakage round the microelectrodes where they cross the cell membrane.
3.3
The effect of CO2-bicarbonate buffering
We have done most of our experiments in HEPES-buffered Ringer, although it is known that CO2-bicarbonate-buffered solutions greatly increase intracellular pH buffering while reducing pH buffering power extracellularly [28,29]. It is possible that such a change in pH buffering might influence the diffusion of Ca2+ ions. We have therefore recorded the effects of changing the extracellular buffer from HEPES to CO2-bicarbonate on Ca2+ transients recorded at different depths. While it is well established [16,30] that the intracellular pH (pHi) decrease caused by CO2 entry leads to a decrease in steady-state [Ca2+]i, the Ca2+ transients were little changed (not shown). By 20 min after changing the superfusate to CO2 the Na-dependent chloride/bicarbonate exchanger [28] should have returned pHi to normal, so we then again moved the CASM down in steps. This time the VCa baseline did not change, and the transient times to peak were very similar to those recorded in HEPES. In a total of four similar experiments, the average time to peak at distances of 0–20 μm was 4.20 ± 0.45 s in HEPES, and 4.35 ± 0.68 s in CO2-bicarbonate.
3.4
The role of calcium stores.
It seems possible that the VCa gradients described above, and the higher steady-state apparent [Ca2+]i than previously reported in cells which had added buffer, were partly due to mechanically-induced leakage from the endoplasmic reticulum calcium stores. Having penetrated the cell membrane the CASM must have been moved down through the endoplasmic reticulum and might have caused significant injury. We have therefore tested the effects of the SERCA pump inhibitor cyclopiazonic acid (CPA). It is well-established that CPA empties the Ca2+stores over a period of several minutes by blocking their uptake mechanism [31], and emptying the stores will presumably stop them leaking. Fig. 5 shows one of four similar experiments with CPA, in all of which (once the stores were empty) it had little effect on either the baseline VCa or the near-membrane VCa changes with CASM movement. The times-to-peak are trending to be later in CPA treated cells, although this was not very pronounced close to the membrane and statistically not significant (p-value assuming time-to-peak is later after CPA: 0.635 (n = 4)). For distances of 20, 40 and 60 μm from the membrane the times to peak were significantly longer (p-values assuming times-to-peak are later after CPA 0.952(5), 0.952(5) and 0.952(5)). The recovery time after the Ca2+ transients was clearly much longer in CPA treated cells (p-values assuming recovery times are longer after CPA: 0.984(4), 0.984(5), 0.972(5) and 0.996(5)), as shown in Fig. 5C. Interestingly the effect of CPA on recovery times was more pronounced for transients measured close to the membrane. Furthermore, the transient size showed a downward trend and decreased on average about 15% (nominal [Ca2+]i) in CPA treated cells. Although this percentage was statistically not significant (p-values assuming transients are smaller after CPA: 0.794(4), 0.845(5), 0.79(5) and 0.726(5)), it may suggest that a small fraction of the increase in [Ca2+]i is caused by release of Ca2+.
3.5
The effect of BAPTA injection
Exogenous calcium buffers have been widely shown to influence Ca2+ signalling in a variety of cells; for example in nerve terminals [32] and Xenopus oocytes [33]. The fast buffer BAPTA is particularly interesting since it has similar properties to many of the indicators used to study Ca2+ signalling. We have therefore examined the effects of BAPTA, injected iontophoretically, on both the steady-state VCa and the Ca2+ transients induced by depolarisations. Fig. 6A shows a representative part of one of four similar experiments. Before the BAPTA injection we recorded VCa transients at several different CASM depths. At the closest approach to the membrane the time to peak was 2.9 s and VCa reached a peak of −107 mV. The first two BAPTA injections loaded the cell to give an estimated BAPTA concentration of 9 μM. At the closest approach to the membrane the VCa transient then had a time to peak of 6.6 s. After two more BAPTA injections giving an estimated final concentration of 135 μm, the transient time to peak at the closest point was again slower, at 9.1 s. In three of the experiments we were able to measure times to peak and the size of the VCa transients at three different depths before and after injecting BAPTA to a final concentration of 100–150 μm. The collected data are plotted in Fig. 6B and C. This shows that BAPTA tended to make the Ca2+ transients more uniform throughout the cell, both in times to peak and overall sizes.
Although the first two BAPTA injections had little effect on the steady-state VCa, the second two lowered VCa from −128 to −131 mV. In a total of four similar experiments large BAPTA injections (raising the BAPTA concentration by over 100 μM) also lowered the steady state VCa by an average 4 ± 1.1 mV. This effect of BAPTA is similar to that reported before in snail neurones [14] in which intracellular CASM responses were calibrated by pressure-injecting BAPTA calibration solutions.
3.6
Computational modelling of Ca2+ transients in large snail neurones
In this section we present a minimal computational model that agrees fairly well with our experimental results. In the model we assume that during a 1 s current pulse (ICa,app) Ca2+ enters the cell at the plasma membrane whence it diffuses into the interior with an apparent diffusion coefficient Dapp. Furthermore, Ca2+ is removed at the plasma membrane by a Ca2+ pump with extrusion permeability Pout,app; we also allow for sequestration everywhere in the cell with rate kseq,app. Further mathematical details can be found in Section 2.
In modelling Ca2+ transients at different depths we overcame our imprecise knowledge of the different parameters by estimating them using a least square fit. Fig. 7A shows eight simultaneously fitted transients that were measured in the middle of a long experiment at various depths ranging form close to the membrane at about 20 μm to about 70 μm away from the membrane. The depths estimated from micrometer movements are the lower values shown above the peaks. To fit the data these positions were allowed to vary within the limits of half a CASM step. The depths obtained from the fit (upper values) are reasonably close to the micrometer positions. Interestingly, modelled positions near the membrane are slightly shifted towards the centre of the cell, which might indicate lower mobility close to the membrane.
The optimal fit yielded an apparent diffusion coefficient of Dapp = 80 μm2 s−1. Furthermore, the fit required an apparent sequestration rate constant of kseq,app = 0.047 s−1, which corresponds to a time constant of ∼20 s. Attempts to fit the transients without sequestration failed.
The extrusion permeability was Pout,app = 11 μm s−1, which corresponds to a time constant of about 3.6 s, much shorter than the diffusional time constant of 18 s (see Section 2). This suggests that clearance is limited by diffusion rather than by the pump rate. In most cells we observed that after influx a rather steep gradient develops in the direction of the membrane, which fits the diffusion-limited case.
The response time was estimated to be τCASM = 2.1 s, which was slower than the 1 s that was measured in 10 μM Ca2+ Ringer. In most experiments the times-to-peak very close to the membrane ranged from 2.5 to 3.5 s. Because the time-to-peak near the membrane should equal the duration of the influx, the response time of the electrode in cytoplasm is about 1.5–2.5 s.
The estimated apparent diffusion coefficient for the recordings shown in Fig. 7 of Dapp = 80 μm2 s−1 is higher than reported in other studies: ∼14 μm2 s−1 in frog muscle [34], ∼13 μm2 s−1 in cytoplasmic extracts of Xenopus oocytes [24], ∼20 μm2 s−1 in the axoplasm of metacerebral cells of Aplysia californica
[23] and ∼10 μm2 s−1 in rod photoreceptor outer segments [35]. A diffusion coefficient of ∼10 μm2 s−1 has been reported in intact Myxicola axoplasm [36], but much higher values were seen in ATP-depleted axoplasm, and with high [Ca2+]i.
In most of these studies the diffusion coefficient was estimated using a simple model with only a few parameters. Our model contained more parameters that we varied to obtain a good fit. The parameters for influx, extrusion and sequestration and to some extent the positions and the response time may compromise the estimate of Dapp. We therefore calculated the mean error variance as a function of the apparent diffusion coefficient of four fits obtained from four different cells. While fixing the apparent diffusion coefficient, all other parameters were allowed to vary in order to obtain an optimal fit. In each cell we fitted at least three transients measured at distances ∼40, ∼60 and ∼80 μm and calculated the mean error variance as a function of Dapp. All cells gave a reasonable fit with a minimum error at 90 ± 20 μm2 s−1 (Fig. 7B). Since we allow all other parameters to compensate for an incorrect diffusion coefficient this value should be a conservative estimate. Attempts to fit the transients with Dapp values smaller than 50 μm2 s−1 failed. Some cells fitted better than others, since the expected transient size did not always match perfectly when multiple transients were used. However, perfect fits could be obtained with any individual transient. Close inspection of the traces revealed that baseline variations could affect the transient size. Possibly the Ca2+ influx is reduced or removal mechanisms are enhanced at higher basal [Ca2+]i levels. We still conclude that the diffusion coefficient is considerably higher (five to seven times) than reported for other cell types.
A large fraction of the cytoplasmic Ca2+ is bound to buffers. The properties and distribution of these Ca2+ buffers are therefore critical for Ca2+ mobility in the cytoplasm. Analysis of Ca2+ transients under differing free Ca2+ concentrations and exogenous Ca2+ buffering in snail neurones indeed showed that Ca2+ is quite well buffered in snail neurones [24]. To explain the high diffusion coefficient a large fraction of the Ca2+ buffers must be mobile. When we injected the cells with BAPTA, it clearly changed the VCa transients considerably, promoting spatially uniform global signals as described in Xenopus oocytes [33]. These were loaded with 48 μm Oregon Green to record [Ca2+]i, which may itself have influenced Ca2+ movement. Our Fig. 7C shows three transients measured at 50–60 μm from the membrane with different BAPTA concentrations; the nominal concentrations were 0, 50 and 150 μM. BAPTA slightly increased the times to peak (10.2, 10.6 and 12.6 s), reduced the size of the transients and slowed the recovery time significantly (13.6, 18.1 and 30.0 s). These values are consistent with a reduced clearance rate. Typically, if the Ca2+ mobility is not changed, reduction of the clearance rate slows the recovery time but also increases the time-to-peak. After normalisation of the times to peak we could only detect a slight reduction in the latency (<0.5 s) between 0 and 150 μM BAPTA, suggesting that the mobility of BAPTA is comparable to that of the endogenous Ca2+ buffers.
We fitted the three transients simultaneously to the model allowing a different exogenous Ca2+ binding ratio for each transient. The model gives a reasonable fit with an endogenous buffer diffusion coefficient of De = 100 μm2 s−1 and Ca2+ binding ratio of κe = 65. This suggests that the endogenous Ca2+-buffers have a comparable Ca2+ binding ratio to that in Aplysia axons [25] but appear to be much more mobile. The apparent diffusion coefficients for Ca2+ at the different BAPTA concentrations are: Dapp = 102 μm2 s−1 (0 μM BAPTA), Dapp = 12 μm2 s−1 (50 μM BAPTA) and Dapp = 151 μm2 s−1 (150 μM BAPTA). The estimated diffusion coefficient of BAPTA was Db = 183 μm2 s−1, which slightly increases the mobility of Ca2+ at higher BAPTA concentrations. Similar values for the BAPTA diffusion coefficient were reported by others [37–39].
4
Discussion
We have shown that with CASMs it is possible to record steady-state calcium levels and depolarisation-induced Ca2+ transients at different depths in large snail neurones. The decrease in calcium levels as the CASM was pushed deeper was probably due to increasing the distance from a site of leakage around the CASM and other microelectrode insertions at the top of the cell. We found that the sizes and kinetics of the Ca2+transients measured after brief (1 s) depolarisations to zero or −10 mV varied systematically and repeatably with distance from the plasma membrane at the bottom of the cell. Blocking the endoplasmic reticulum SERCA pump with CPA had little effect on the transient sizes and times to peak, but significantly slowed subsequent recovery times. Injecting BAPTA both reduced and slowed the transients close to the membrane while magnifying them deep inside the cell. By fitting our results to a mathematical model we calculate that the apparent Ca2+ diffusion coefficient is about 90 μm2 s−1.
4.1
The problem of leakage
Ion-sensitive and other non-patch microelectrodes inevitably make a hole in the cell membrane, which allows leakage in or out of the cell. Given the gradients, the ions whose leakage is most likely to distort the measured intracellular level are Na+ and Ca2+. Thus, Vaughan-Jones [40] recorded [Na+]i in crab muscle with a Na+-sensitive microelectrode (NASM) inserted radially (perpendicular to the long axis) and found that low external Na caused a surprisingly rapid fall in [Na+]i. He considered that this might have been caused by the low external Na reducing a leak. This was later confirmed by experiments in which it was shown that NASMs inserted along the long axis into a cannulated fibre recorded only small changes in [Na+]i when external Na was reduced, but when inserted radially the recorded [Na+]i was at first high but fell as the NASM was pushed deeper [12].
Perhaps because the gradient is small and pHi very well buffered no one has considered that pH-sensitive microelectrode measurements have been contaminated by leakage. On the other hand, [Ca2+]i is very low and only moderately buffered. In our hands CASMs unfortunately do not perform well unless their tips are at least 1 μm in diameter [15,17]. Furthermore, only after much manipulation did CASMs record a low [Ca2+]i which increased on depolarization. The initially high levels were recorded even in cells loaded with Fura-2 whose fluorescence indicated a normal low [Ca2+]i. Experiments in which CASMs were calibrated while inside neurones by injecting buffers led to the conclusion that the true [Ca2+]i in quiescent voltage clamped snail neurones with injected buffer was 40 nM (equivalent to a CASM VCa of about −140 mV) and that CASMs usually cause a leak at the point of insertion [14].
The early experiments with CASMs were all done with a manually-operated micromanipulator, so that systematic movements were not attempted once a stable VCa was obtained. The current results were obtained with a motor-driven and remote-controlled micromanipulator. Typically, the CASM was moved down in several 20 μm steps before its potential changed at all. This shows that the cell membrane is very extensible, since the cell as a whole did not visibly move as the CASM was inserted. For example in Fig. 1A, the CASM was moved down by 80 μm before the potential first fell. As the CASM tip was then moved deeper the potential became more and more negative. This was probably due to a combination of movement away from the site of leakage, and a reduction in the size of the leak. It is hard to distinguish between these two possibilities, but the reproducible effects of movement as shown for example in Fig. 3A suggest that the leak was relatively constant, although tending to reduce with time. In some experiments the steady-state VCa appeared to stabilize some distance from the apparent far side of the cell, as shown in the control part of Fig 6A, and was not reduced further as the tip approached the far side of the cell. Presumably the leakage was by then too small to have a significant effect.
4.2
Ca2+ influx rather than Ca2+ release determines the increase in [Ca2+]i
Our finding that the peak of the VCa transient diffuses away from the membrane at a speed of about 6 μm s−1 confirms the long-held view that Ca2+ signalling which relies purely on diffusion will inevitably have a slow response time. The lack of effect of CPA, and the decline of the signal size with distance, rather unexpectedly both rule out significant regeneration of the signal as it spreads inwards. This is surprising, since snail neurones respond well to caffeine and certainly have the capacity for Ca-induced Ca2+ release (CICR) [41,16]. For example, the addition of a low concentration of caffeine slightly enhanced Ca2+ transients [41], while the removal of caffeine temporarily reduced the peak amplitudes suggesting that the stores were at least partly emptied [16].
Either the signal is large enough without any store contribution, or something about our experimental conditions has inhibited CICR. We depolarised the cell for 1 s to ensure a large Ca2+ load every 2 min, but this may have in some way have inhibited store release. On the other hand in other experiments we have avoided depolarising the cell for long periods, and not found that the first subsequent response was different. The distance over which the VCa transients fell to half their extrapolated initial size was about 45 μm, similar to the distance between the cell membrane and the nucleus. In normal-sized neurones of course the nucleus would be much closer.
We conclude that the main mechanism in our experiments that leads to an increase in [Ca2+]i is transmembrane Ca2+ influx through voltage-gated Ca2+ channels opened by depolarisation and not massive release triggered by a small Ca2+ influx as in vertebrate cardiac muscle. The only previous direct measurements of Ca2+ movement in nerve cells without added buffers were described by Gorman et al. [42]. They were able to detect a rise in [Ca2+]i only within about 40 μm of the membrane in Aplysia neurones after a 5 s depolarisation. Their CASMs were made with a different sensor (ETH 1001) to that which we used, and appear to have been relatively insensitive at low values of [Ca2+]i.
4.3
The transmembrane pump and sequestration by cytoplasmic organelles determine Ca2+ clearance
The depolarisation-induced Ca2+ influx is presumably cleared by extrusion across the cell membrane and sequestration to cytoplasmic organelles. The PMCA or Ca2+:H+ ATPase in snail neurones appears to exchange two extracellular protons for one intracellular Ca2+ at the expense of ATP [43]. When it is blocked by ortho-vanadate, the [Ca2+]i does not fully recover from an influx but ends in a plateau. This suggests that the PMCA is the essential removal mechanism, since the endoplasmic-reticulum and mitochondrial uptake mechanisms are not inhibited by cytoplasmic o-vanadate [16].
Nevertheless, for many different cell types it has been shown that mitochondria participate in Ca2+ clearance when Ca2+ concentrations are high [44,45]. It is very difficult to measure the action of mitochondria in snail neurones directly. However, an increased muffling power at high Ca2+ concentrations [24] suggests that mitochondria may play a role close to the membrane where high transients occur.
Instead of reinforcing the Ca2+ signals, and thereby generating a fast travelling Ca2+ wave into the cell, the stores clearly play a role in the recovery of the Ca2+ transients. CPA significantly increased the Ca2+ clearance time from ∼13 to ∼18 s; these were measured in the centre of the cell and were corrected for the CASM response time. From this we estimate that the stores contribute about 25% to the clearance. This is lower but comparable to the result found in Purkinje cell somata from rat cerebellar slices, where blocking of the endoplasmic Ca2+ pump also significantly slowed the recovery time after influx [46].
4.4
Calcium transients change systematically with depth
Although the steady-state VCa levels were probably elevated by continuous Ca2+ entry, the recorded transients and the way they changed with depth were, we believe, physiologically significant. It seems unlikely that the kinetics of the diffusion of Ca2+ from the far side of the cell towards the centre would be changed by a constant or slowly-declining leak. Our average value for the speed at which a depolarisation-induced Ca2+ transient flows from the periphery to the centre of a nerve cell with no added mobile buffer was ∼6 μm s−1. We can find no previous measurements of this parameter in excitable cells, although several groups have modelled the process; (for example [8,12]). The only real measurement we have found was done with 100 μm Fluo-3 in an isolated atrial myocyte with inhibited sarcoplasmic reticulum Ca2+ pumps [10]. Our estimate from their Fig. 1 is that the calcium peak moved at least 10 times faster than in our observations, but the authors’ model showed that this was probably due to the Fluo-3. Their modelled data as shown in their Fig. 4 suggest that with no added mobile buffer their Ca2+ transients might travel to the centre of their model myocyte at a similar speed to that which we measure, although their time axis does not extend far enough. Furthermore, when the speed at which Ca2+ transients travel depends on diffusion, the velocity is not constant but varies with distance; typically velocity first increases and then decreases when the centre of the cell is approached. In contrast, true waves are constantly regenerated and tend to move with a constant speed [47].
4.5
Calcium diffusion in large snail neurones without added buffer
If recovery from a brief Ca2+ influx relies on extrusion at the plasma membrane, diffusion will become important for large cells because Ca2+ has to diffuse to the plasma membrane to be extruded. Moreover, the fastest possible clearance without sequestration is the diffusion limit. When the extrusion rate is much faster than diffusion, the recovery time constant is proportional to the square of the cell's diameter and inversely proportional to the diffusion coefficient (see Section 2). Because large snail neurones mainly rely on transmembrane extrusion mechanisms and do not seem to reinforce Ca2+-signals, the diffusion coefficient becomes a critical parameter for Ca2+ clearance and spatial signalling. Fig. 8 shows the recovery times, assuming that diffusion is the rate-limiting process, for various different diffusion coefficients as function of the cell's diameter. The recovery time of large snail neurones is about 10–20 s (dashed line), which requires a diffusion coefficient between 60 and 120 μm2 s−1. However, most snail and other animal nerve cell bodies are much smaller, with diameters between 5 and 50 μm. The recovery times of these cells is typically in the order of seconds (dotted line). Hence, with diffusion coefficients <20 μm2 s−1 the cell can still be cleared readily by the pump.
4.6
Why is Ca2+ more mobile in large snail neurones?
It is possible that large cells, that do not use Ca2+release mechanisms for signalling and rely on extrusion at the membrane for reasonably fast recovery, require a high Ca2+ mobility to function. A high mobility will also ensure fast signal transmission to the nucleus. This is quite different from large cells that use release mechanisms to trigger rapid on/off responses such as muscle, oocytes and chromaffin cells. These cells typically have low Ca2+ mobilities and do not rely as much on extrusion, but recycle internally released Ca2+. Small neurones and axonal regions with high surface to volume ratios represent another class of geometries, which do not require high Ca2+ mobility for fast recovery.
Because Ca2+ is well buffered with an estimated binding ratio of 65, the mobility of the endogenous mobile buffers must be high to explain the high apparent mobility of Ca2+. Possibly the ratio of mobile buffers to immobile buffers is higher in large snail neurones, or the diffusion coefficient of the mobile buffer fraction is much higher than in other cells. This may be related to less cytoplasmic crowding, for example by reduced cytoskeleton, causing the movement of proteins to be less constricted [48]. Axonal regions and muscle cells contain high concentrations of cytoskeletal components that may restrict or slow considerably the movement of larger calcium binding proteins compared to large snail neurones.
The great majority of studies on intracellular Ca2+ signalling have been done with indicators such as Fura 2, which can be used on all sizes and types of cell. But the interaction of the Ca2+ signals with the indicator may have subtle effects, which are hard to predict. It is to be regretted that CASMs so far have such large tips and slow responses that they can be used only on exceptionally large cells.


Microtitre plate-based antibacterial assay incorporating resazurin as an indicator of cell growth, and its application in the in vitro antibacterial screening of phytochemicals

The resazurin assay utilising microtitre-plate, described by Drummond and Waigh in 2000, has been modified to achieve more accuracy in the determination of the minimum inhibitory concentration (MIC) values of natural products, including crude extracts, chromatographic fractions or purified compounds against various bacterial strains. This modified resazurin method is simple, sensitive, rapid, robust and reliable, and could be used successfully to assess antibacterial properties of natural products.
1
Introduction
Antibiotics have revolutionised mankind’s health status, allowing treatment of life threatening infections. However with the increasing occurrence of bacterial resistance against available antibiotics, it has now become essential to look for newer antibiotics. Most of the antibiotics available today come from natural origin, especially from various microbial or marine sources. Plants also produce compounds to protect themselves from microbial attacks. For screening natural products, e.g. crude extracts, chromatographic fractions or purified compounds for antibacterial activities, it is essential to employ an in vitro antibacterial assay that is simple, rapid, efficient, reliable, sensitive, safe and cost-effective. Moreover, most often the small quantities of natural products, especially purified compounds, that are available for antibacterial screening, can be a limiting factor in any viable screening programme. The conventional methods, e.g. disc diffusion method, may be time consuming and require significant quantities of the test materials, and there are also a few other problems associated with this method as discussed by Drummond and Waigh [3].
At the beginning of our research project, aimed at screening Scottish plants for antibacterial activities [1,2], the resazurin assay utilising a microtitre-plate as described by Drummond and Waigh in 2000 [3] seemed useful. This was because this method uses an indicator, resazurin, which allows the detection of microbial growth in extremely small volumes of solution in microtitre plates without the use of a spectrophotometer. However, it was soon realised that this published method incorporated changes in the concentration of both the test material as well as the bacterial suspension. Thus, although there is a serial dilution of the test materials, this method results in a decrease in the bacterial concentration serially, and as a consequence cannot be a ‘true’ indicator of the minimum inhibitory concentration (MIC) which is supposed to be assessed in this assay. Moreover, in a number of other antibacterial assays, the bacterial concentration is only approximate as they are compared to the Macfarland standard. This approach is subjective and results are often not reproducible in two different laboratories. To overcome these drawbacks, we have modified the resazurin assay, especially the dilution protocols, and utilised a standard concentration of bacterial suspension so that a ‘true’ MIC value can be obtained. By challenging the test materials with a standardised inoculum, reproducible and meaningful results can be obtained. We now present a detailed description of the modified resazurin method, which is simple, sensitive, rapid, robust and reliable, and could be used successfully to assess antibacterial properties of natural products.
2
Method
2.1
General
Incubator at 35 and 37 °C; pipettes of various sizes (Gilson); sterile tips, 100, 200, 500, and 1000 μL; 5 mL multichannel pipette (Fischer Supplies); centrifuge tubes (Fischer Supplies); vortex mixer (Fischer Supplies); centrifuge (Fisons); petridishes (Fischer Supplies); sterile universal bottles (Fischer Supplies); UV spectrophotometer (Shimadzu); sterile resazurin tablets (BDH Laboratory Supplies); sterile normal saline; sterile isosensitest broth (Oxoid); sterile isosensitest agar (Southern Group Laboratory, SGL); antibiotic solutions (Sigma–Aldrich); sterile solution of 10% (v/v) DMSO in water (Sigma–Aldrich).
2.2
Medium
Isosensitest medium was used throughout this assay, as it is pH buffered. Although NCCLS recommends the use of Mueller Hinton medium for susceptibility testing [4], the isosensitest medium had comparable results for most of the tested bacterial strains [5].
2.3
Use of standardised bacterial colony numbers
The method wherein turbidity is compared to Macfarland standards usually 0.5 is not able to give a standardised number of CFU for all strains, because this is operator driven and is thus subjective. It also makes it difficult to compare different bacterial species as they have differing optical densities. Thus to ensure that a uniform number of bacteria were always used, a set of graphs of killing/viability curves for each strain of bacterial species was prepared. A final concentration of 5 × 105 cfu/mL was adopted for this assay. Thus different strains and different bacterial species could be compared.
2.4
Preparation of bacterial culture
Using aseptic techniques a single colony was transferred into a 100 mL bottle of isosensitest broth, capped and placed in incubator overnight at 35 °C. After 12–18 h of incubation, using aseptic preparation and the aid of a centrifuge, a clean sample of bacteria was prepared. The broth was spun down using a centrifuge set at 4000 rpm for 5 min with appropriate aseptic precautions. The supernatant was discarded into an appropriately labelled contaminated waste beaker. The pellet was resuspended using 20 mL of sterile normal saline and centrifuged again at 4000 rpm for 5 min. This step was repeated until the supernatant was clear. The pellet was then suspended in 20 mL of sterile normal saline, and was labelled as Bs. The optical density of the Bs was recorded at 500 nm, and serial dilutions were carried out with appropriate aseptic techniques until the optical density was in the range of 0.5–1.0. The actual number of colony-forming units was calculated from the viability graph. The dilution factor needed was calculated and the dilution was carried out to obtain a concentration of 5 × 106 cfu/mL.
2.5
Preparation of resazurin solution
The resazurin solution was prepared by dissolving a 270 mg tablet in 40 mL of sterile distilled water. A vortex mixer was used to ensure that it was a well-dissolved and homogenous solution.
2.6
Preparation of the plates
Plates were prepared under aseptic conditions. A sterile 96 well plate was labelled (Fig. 1). A volume of 100 μL of test material in 10% (v/v) DMSO or sterile water (usually a stock concentration of 1 mg/mL for purified compounds, and 10 mg/mL for crude extracts) was pipetted into the first row of the plate. To all other wells 50 μL of nutrient broth or normal saline was added. Serial dilutions were performed using a multichannel pipette. Tips were discarded after use such that each well had 50 μL of the test material in serially descending concentrations. To each well 10 μL of resazurin indicator solution was added. Using a pipette 30 μL of 3.3× strength isosensitised broth was added to each well to ensure that the final volume was single strength of the nutrient broth. Finally, 10 μL of bacterial suspension (5 × 106 cfu/mL) was added to each well to achieve a concentration of 5 × 105 cfu/mL. Each plate was wrapped loosely with cling film to ensure that bacteria did not become dehydrated. Each plate had a set of controls: a column with a broad-spectrum antibiotic as positive control (usually ciprofloxacin in serial dilution), a column with all solutions with the exception of the test compound, and a column with all solutions with the exception of the bacterial solution adding 10 μL of nutrient broth instead (see Fig. 2).
The plates were prepared in triplicate, and placed in an incubator set at 37 °C for 18–24 h. The colour change was then assessed visually. Any colour changes from purple to pink or colourless were recorded as positive (Fig. 1). The lowest concentration at which colour change occurred was taken as the MIC value. The average of three values was calculated and that was the MIC for the test material and bacterial strain.
3
Results
Resazurin is an oxidation–reduction indicator used for the evaluation of cell growth, particularly in various cytotoxicity assays [6]. It is a blue non-fluorescent and non-toxic dye that becomes pink and fluorescent when reduced to resorufin by oxidoreductases within viable cells. Resorufin is further reduced to hydroresorufin (uncoloured and nonfluorescent). A resazurin reduction test has also been used for decades to demonstrate bacterial and yeast contamination of milk [6,7].
The effectiveness of this modified resazurin assay has been demonstrated with methanol extracts of selected Scottish plants, purified compounds (Fig. 1), and the positive control ciprofloxacin (Table 1), and a direct comparison of the MIC determination of the antibiotics using the old and modified resazurin methods (Table 2) using the antibiotics norfloxacin, cefotaxime, and amoxicillin.
4
Concluding remarks
Choosing the correct assay to assess the antimicrobial potential of extracts and compounds is important for generating high-quality data with the greatest accuracy, speed and efficiency, enabling the addition of potential new antimicrobial compounds and extracts to our armamentarium. This modified resazurin assay corrected the dilution inaccuracies, especially in relation to the MIC determinations, as described by Drummond and Waigh [3], and enabled results to be comparable for the test material for different bacterial strains. The method described here is easy to follow and accurate. The generation of an accurate MIC value, which can be compared to existing antibiotics, empowers the scientist with the knowledge to decide whether the extracts and compounds are worth pursuing further in terms of their antimicrobial potential.

antibacterial assay
resazurin
serial dilution
plant extract


Experimental assessment of bioenergetic differences caused by the common European mitochondrial DNA haplogroups H and T

Studies of both survival after sepsis and sperm motility in human populations have shown significant associations with common European mitochondrial DNA haplogroups, and have led to proposals that mitochondria bearing haplogroup H have different bioenergetic capacities than those bearing haplogroup T. However, the validity of such associations assumes that there are no non-random influences of nuclear genes or other factors. Here, we removed the effect of any differences in nuclear genes by constructing transmitochondrial cybrids harbouring mitochondria with either haplogroup H or haplogroup T in cultured A549 human lung carcinoma cells with identical nuclear backgrounds. We compared the bioenergetic capacities and coupling efficiencies of mitochondria isolated from these cells, and of mitochondria retained within the cells, as a critical experimental test of the hypothesis that these haplogroups affect mitochondrial bioenergetics. We found that there were no functionally-important bioenergetic differences between mitochondria bearing these haplogroups, using either isolated mitochondria or mitochondria within cells.
1
Introduction
Analysis of the variation in normal human mitochondrial DNA (mtDNA) has identified many haplogroups–specific patterns of polymorphisms that have arisen over the last 150,000–200,000 years–that have been used to study the origin, radiations and evolution of human populations (Cann et al., 1987; Wallace et al., 1999; Wallace, 2005). These mtDNA haplogroups have been less studied bioenergetically than mtDNA mutations that lead to disease (Dimauro and Davidzon, 2005; Taylor and Turnbull, 2005), but normal variation in mtDNA may affect disease susceptibility (Herrnstadt and Howell, 2004) and longevity (Santoro et al., 2006). These effects could be explained by differences in mitochondrial coupling efficiency (the percentage of oxygen consumption used for ATP synthesis rather than heat generation) or mitochondrial production of ROS (reactive oxygen species). It has been proposed that particular haplogroups cause inefficient oxidative phosphorylation and greater heat production and were therefore selected during the radiations of humans into Arctic environments (Mishmar et al., 2003; Ruiz-Pesini et al., 2004; Wallace, 2005; Montiel-Sosa et al., 2006), but this proposal is controversial (Elson et al., 2004; Kivisild et al., 2006; Ruiz-Pesini and Wallace, 2006) and is not supported by direct measurement of mitochondrial coupling efficiencies in mitochondria carrying representative “arctic” and “tropical” mtDNAs (Amo and Brand, 2007; Elson et al., 2007).
Two lines of evidence suggest that common European haplogroups may affect bioenergetic function: there are associations of both survival after sepsis and sperm motility with haplogroup H. In a prospective study of intensive care patients in Newcastle-upon-Tyne, U.K., individuals with haplogroup H (about 40% of the population studied) were more than twice as likely to survive 180 days after sepsis than those with non-H haplogroups (primarily the closely-related haplogroups J and T; about 30% of the population) (Baudouin et al., 2005). Mitochondrial dysfunction may be linked to sepsis-induced multiple organ failure (Protti and Singer, 2007), so altered mitochondrial bioenergetics might be the causal link between haplogroup and survival. Two possibilities have been proposed: haplogroup H might protect through greater heat generation (because of higher electron transport rates or looser coupling) (Baudouin et al., 2005), or through greater ROS production (because of tighter coupling and raised protonmotive force), which could reduce bacterial infection (Wallace, 2005). There have been no direct experimental tests of these possibilities.
The second line of evidence comes from studies of sperm motility, which is correlated with mitochondrial enzymatic activities and depends on the activity of the mitochondrial electron transport chain (Ruiz-Pesini et al., 1998; Ruiz-Pesini et al., 2000). Haplogroup T was over-represented in men with asthenozoospermia (reduced sperm motility), whereas haplogroup H was over-represented in men with other fertility problems. Sperm with haplogroup H performed better in a test of motility and had higher cytochrome oxidase activity than those with haplogroup T (Ruiz-Pesini et al., 2000), suggesting that mitochondria carrying haplogroup H make more ATP than those with haplogroup T. This conclusion has been extended to sublineages of haplogroup U (Montiel-Sosa et al., 2006), but associations between haplogroups and reduced male fertility and sperm motility have been disputed by others (Pereira et al., 2005; Pereira et al., 2007). Less well-coupled mitochondria make less ROS (Korshunov et al., 1997; Liu, 1997; Lambert and Brand, 2004), so people having such mitochondria might suffer less from neurodegenerative diseases caused by ROS (Wallace, 2005). Some epidemiological studies support this prediction; e.g. haplogroup T is under-represented in Alzheimer's disease patients (Chagnon et al., 1999). However, other studies have not replicated this finding (Elson et al., 2006), and correlations between mitochondrial haplogroups and neurodegenerative diseases are controversial (Raule et al., 2007). Again, there have been no direct experimental tests of possible differences in the bioenergetic properties of mitochondria that might underlie the reported associations of haplogroup and phenotype.
In the present study, we analyse bioenergetic capacity and coupling efficiency in mitochondria isolated from cytoplasmic hybrids (cybrids) carrying haplogroups H and T with identical nuclear DNA. Furthermore, to investigate the effects of haplogroups H and T on the bioenergetic status of mitochondria at the cellular level, we analyse the respiratory capacities and mitochondrial coupling efficiencies of intact cybrids.
2
Materials and methods
2.1
Subjects
Healthy volunteers were recruited by advertisement and written informed consent was obtained. Ethical approval was obtained from the Cambridge Research Ethics Committee. DNA was extracted from buccal swab samples using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). Mitochondrial haplogroups were determined by PCR-RFLP analysis according to published criteria (Wallace et al., 1999). Entire mtDNAs were amplified in several overlapping fragments by PCR (Torroni et al., 1997). Each fragment was digested by restriction enzymes and resolved on agarose gels. 15 ml of blood were taken from each of three volunteers who had haplogroup H, and three who had haplogroup T, for platelet preparation and cybrid construction. Haplogroup H (Achilli et al., 2004) is subdivided into at least 15 sub-haplogroups (H1-H15). In the Newcastle sepsis study, there were no significant differences in survival of sub-haplogroups H1 (− 3008 TaqI), H2 (− 4769 AluI), H3 (+ 6773 NlaIII) and the remaining H sub-haplogroups combined (Baudouin et al., 2005). Our haplogroup H volunteers were H1, H3 and (H, not H1, H2 or H3). Haplogroup T (Macaulay et al., 1999) is subdivided into at least 5 sub-haplogroups (T1–T5) (Pike 2006). Our haplogroup T volunteers were one T (sub-haplogroup not checked), one T1 (− 12629 AvaII) and one (T, not T1). No major differences in cybrid phenotypes between sub-haplogroups were observed.
2.2
Generation of cybrid cell lines
Cybrid cell lines are constructed by repopulation of mtDNA-less (ρ0) cells with exogenous mitochondria (King and Attardi, 1989). A549.B2 ρ0 (mtDNA-less) cells derived from human lung carcinoma A549 (originally carrying mitochondrial DNA of haplogroup H) were cultivated in Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g/l glucose, 110 µg/ml sodium pyruvate, 10% (v/v) fetal bovine serum (FBS) and 50 µg/ml uridine. Platelets (which have no nuclei) were isolated from the volunteers' blood samples and fused with A549.B2 ρ0 cells as described elsewhere (Chomyn, 1996). The resultant cybrids had mtDNA from the different donors, but their nuclear DNA was identical. After many generations of cybrid growth (diluting platelet-derived nuclear-encoded subunits), all nuclear-encoded mitochondrial protein subunits will be specified by the host cell, but all mitochondrial-encoded subunits will be specified by the donor mtDNA. The cybrid cell lines were constructed and routinely maintained in DMEM with 10% dialysed FBS. Stocks of cell lines were frozen and kept at − 80 °C until required. DNA was extracted from cultured cybrid cells as described previously (Laird et al., 1991) to confirm mitochondrial haplogroups by PCR-RFLP analysis.
2.3
Mitochondrial respiration and membrane potential
Human A549 cell mitochondria were prepared from cultured cybrids as previously described (Amo and Brand, 2007). Mitochondrial oxygen consumption was measured at 37 °C using a Clark electrode (Rank Brothers, Cambridge, UK) calibrated with air-saturated medium comprising 0.115 M KCl, 10 mM KH2PO4, 3 mM Hepes (pH 7.2), 2 mM MgCl2, 1 mM EGTA and 0.3% (w/v) defatted BSA, assumed to contain 406 nmol atomic oxygen·ml− 1 (Reynafarje et al., 1985). No correction was made for consumption of oxygen by the electrode, so oxygen consumption at low rates will be slightly overestimated. Mitochondrial membrane potential (Δψ) was measured simultaneously with respiratory activity using an electrode sensitive to the lipophilic cation TPMP+ (triphenylmethylphosphonium) (Brand, 1995). Mitochondria were incubated at 1.0 mg·ml− 1 (for succinate respiration) or 1.5 mg·ml− 1 (for 2-oxoglutarate + malate respiration) in the presence of 80 ng·ml− 1 nigericin (to collapse the pH gradient so that the protonmotive force was expressed exclusively as Δψ) and 4 µM rotenone (to inhibit complex I). The TPMP+-sensitive electrode was calibrated with sequential additions of TPMP+ up to 2 µM, then 4 mM succinate or 3.2 mM 2-oxoglutarate + 0.8 mM malate (with rotenone omitted) was added to initiate respiration. Experiments were terminated with 1.6 µM FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone), allowing correction for any small baseline drift. Δψ was calculated from the distribution of TPMP+ across the mitochondrial inner membrane using a binding correction factor of 0.35 mg protein·µl− 1. Respiratory rates and respiratory control ratios (the state 3 respiration rate with 0.8 mM ADP divided by the state 4 rate with oligomycin) with 2-oxoglutarate + malate as substrate were determined in the absence of nigericin.
2.4
Modular kinetics
To investigate differences in oxidative phosphorylation caused by mitochondrial DNA variants, we applied a systems approach: modular kinetic analysis (Amo and Brand, 2007). This analyses the kinetics of the whole of oxidative phosphorylation divided into three modules connected by their common substrate or product, Δψ. The modules are (i) the reactions that produce Δψ, consisting of the substrate translocases, dehydrogenases and other enzymes and the components of the respiratory chain, called ‘substrate oxidation’, (ii) the reactions that consume Δψ and synthesize, export and dephosphorylate ATP, consisting of the ATP synthase, the phosphate and adenine nucleotide translocases and any ATPases that may be present, called the ‘phosphorylating system’, and (iii) the reactions that consume Δψ without ATP synthesis, called the ‘proton leak’ (Brand, 1990). The analysis reports changes anywhere within oxidative phosphorylation that are functionally-important, but is unresponsive to changes that have no functional consequences. Comparison of the kinetic responses of each of the three modules to Δψ obtained using mitochondria isolated from different cybrids reveals any effects of mitochondrial haplogroup on the kinetics of oxidative phosphorylation. Oxygen consumption and Δψ were measured simultaneously using mitochondria incubated with 80 ng·ml− 1 nigericin and 4 µM rotenone. Respiration was initiated by 4 mM succinate or 3.2 mM 2-oxoglutarate + 0.8 mM malate (with rotenone omitted). The kinetic behaviour of a ‘Δψ-producer’ can be established by specific modulation of a Δψ-consumer and the kinetics of a consumer can be established by specific modulation of a Δψ-producer (Brand, 1998). To measure the kinetic response of proton leak to Δψ, the state 4 (non-phosphorylating) respiration of mitochondria in the presence of oligomycin (0.8 µg·ml− 1; to prevent any residual ATP synthesis), which was used solely to drive the proton leak, was titrated with malonate (up to 0.5 mM). In a similar way, state 4 respiration was titrated by FCCP (up to 0.8 µM) for measurement of the kinetic response of substrate oxidation to Δψ. State 3 (maximal rate of ATP synthesis) was obtained by addition of excess ADP (0.8 mM). Titration of state 3 respiration with malonate (up to 1.15 mM) allowed measurement of the kinetics of the Δψ-consumers (the sum of the phosphorylating system and proton leak). The coupling efficiencies of oxidative phosphorylation were calculated from the kinetic curves as the percentage of mitochondrial respiration rate at a given Δψ that was used for ATP synthesis and was therefore inhibited by oligomycin. Note that any slip reactions will appear as proton leak in this analysis (Brand et al., 1994).
2.5
Cell respiration
Cell respiration was measured at 37 °C using a Seahorse XF24 Extracellular Flux Analyzer (Seahorse Bioscience, Billerica, MA, USA), which is fully described elsewhere (Wu et al., 2007). Cybrids were thawed and subcultured in Petri dishes in DMEM to which had been added 4.5 g/l glucose, 110 µg/ml sodium pyruvate, 0.02 vol of penicillin/streptomycin (5000 units/5000 µg/ml), 0.02 vol of 200 mM l-glutamine and 0.1 vol Fetal Bovine Serum (heat inactivated). Cybrids were seeded at 20,000–50,000 cells per well in Seahorse XF24 24-well plates and grown for 3–5 d in a 37 °C incubator under 95% air/5% CO2, then washed and incubated for about 1 h in a 37 °C incubator under air in 700 µl of assay medium (comprising 120 mM NaCl, 3.5 mM KCl, 2 mM MgCl2, 1.3 mM CaCl2, 1.2 mM Na2SO4, 0.4 mM KH2PO4, 15 mM d-glucose and 0.4% (w/v) BSA, pH adjusted to 7.4 with NaOH). Experiments were carried out to a paired design: each plate was seeded half with one H-haplotype cybrid line and half with one T-haplotype cybrid line. Appropriate amounts of concentrated stocks of oligomycin, FCCP, rotenone and myxothiazol dissolved in 70% ethanol were diluted into 75 µl of assay medium and loaded into the appropriate addition ports in a measuring cartridge. The plate and measuring cartridge (containing waveguides and fluorescent oxygen sensors together with reagents for automated pneumatic additions of inhibitors and uncouplers) was loaded into the machine and after the calibration procedure (30 min calibration, 10 min rest), measurement cycles of 1 min sample mixing, 2 min waiting and 3 min measurement of oxygen consumption rate were initiated. All respiration rates were calculated as a percentage of the rate in the same well for the fourth measurement point — the basal rate just preceding addition of oligomycin (see Fig. 4). This corrected for differences in cell density between wells. In the range measured, absolute rates of oxygen consumption were linearly related to cell numbers seeded. Absolute basal oxygen consumption rates for the parental A549 cell line are reported to be about 1.7 nmol/min/106 cells (Wu et al., 2007). Respiration rates at each time-point from three or four replicate wells were averaged.
2.6
Statistics
Values are given as mean ± SEM for n cell clones (n = 15 cell clones for Figs. 1–3, five different clones for each donor and three donors for each haplogroup, and n = 6 cell clones for Fig. 4, two different clones for each donor and three donors for each haplogroup). To test for significant differences in modular kinetics between haplogroups, we interpolated the mean respiration rates of mitochondria from the individual clones at a range of different potentials between the highest and lowest common membrane potential. The significance of differences between means was assessed by unpaired Student's t-test using Microsoft Excel X; P values < 0.05 were taken to be significant.
3
Results
3.1
Modular kinetic analysis of oxidative phosphorylation
Haplogroup H is a subgroup of haplogroup HV, and haplogroup T is a subgroup of haplogroup JT; HV and JT diverged about 40,000 years ago (Kivisild et al., 2006). Haplogroup H is found in about 41% of Europeans and haplogroup T is found in about 15% (Wallace et al., 1999). Haplogroup H differs from haplogroup T by four non-synonymous mutations in proteins (one each in the complex I subunits, ND1 and ND2, and two in the complex III subunit, cytochrome b), two mutations in tRNAs (tRNAarg and tRNAthr) and three mutations in rRNA (two in 16S rRNA and one in 12S rRNA) (Kivisild et al., 2006), so differences in oxidative phosphorylation between these haplogroups are plausible.
To compare mitochondrial functions between haplogroup H and T, we measured the kinetics and coupling efficiency of oxidative phosphorylation using mitochondria isolated from cybrid cells. Fig. 1A, B and C shows the kinetics of the three modules of oxidative phosphorylation using succinate as respiratory substrate. Fig. 1A shows the kinetic response of substrate oxidation to its product, Δψ; Fig. 1B shows the kinetic response of proton leak to its driving force, Δψ, and Fig. 1C shows the kinetic response of the ATP phosphorylating pathway to its driving force, Δψ. The three sets of kinetics curves using succinate were indistinguishable between mtDNA haplogroup H (closed symbols) and T (open symbols). We also measured the kinetics of substrate oxidation using 2-oxoglutarate + malate as substrate instead of succinate to check for any effects of differences in the mitochondrial complex I genes. Once again, the kinetic response of the substrate oxidation system to Δψ using 2-oxoglutarate + malate as substrate was not significantly different between haplogroups H and T (Fig. 1D).
From the kinetic curves shown in Fig. 1, we can analyse the coupling efficiency of oxidative phosphorylation. The classic measure is the respiratory control ratio: respiration at maximal rate of ATP synthesis (state 3) divided by respiration with no ATP synthesis (state 4) (Fig. 2A and B). A more precise measure of coupling efficiency is given by the percentage of respiration that is coupled to ATP synthesis. This is calculated by subtracting the rate of proton leak (Fig. 1B) from the total rate of respiration (Fig. 1A) at any chosen membrane potential, and expressing the difference as a percentage of the total rate. If this percentage is lower, then a greater proportion of the redox energy is diverted to heat production. Mitochondria in cells normally operate at less than maximal rates and values of Δψ. Fig. 2C reports the coupling efficiency across the whole range of rates of ATP synthesis and values of Δψ from state 3 (maximal rate of ATP synthesis) to close to state 4 (where ATP synthesis is minimal). As expected (Brand et al., 1993), coupling efficiency decreased as mitochondria moved from state 3 towards state 4. There were no significance differences in rates, respiratory control ratio or coupling efficiencies between haplogroups H and T.
We also independently measured the respiratory control ratio using 2-oxoglutarate + malate as substrate and calculated coupling efficiency from the data in Fig. 1 (Fig. 3). Once again, there was no clear difference between haplogroup H and T, either in respiratory rates (Fig. 3A), respiratory control ratio (Fig. 3B) or coupling efficiencies across a range of rates and potentials (Fig. 3C).
In summary, at the mitochondrial level we observed no significant functional differences between mitochondria isolated from cybrids of haplogroup H and haplogroup T.
3.2
Coupling efficiency of respiration in intact cybrid cells
To check whether differences in mitochondrial function between haplogroups were lost when mitochondria were isolated from the cells and measured at a standardised protein concentration in an artificial incubation medium, we measured the respiration rates and coupling efficiencies of mitochondria in intact cybrids. Cybrids were grown on 24-well plates and oxygen consumption was measured using a Seahorse XF24 Extracellular Flux Analyzer. Fig. 4A shows that oxygen consumption was inhibited when ATP synthesis was prevented by addition of oligomycin, stimulated when oxidation was uncoupled from phosphorylation by addition of the uncoupler FCCP, and inhibited again when mitochondrial electron transport was abolished by addition of rotenone (to inhibit complex I of the electron transport chain) and myxothiazol (to inhibit complex III).
The residual rate in the presence of rotenone and myxothiazol represents non-mitochondrial oxygen consumption. This comprised 16.7 + 2% of basal respiration rate in H-haplotype cybrids and 18.2 + 2.7% in T-haplotype cybrids. These values were not significantly different. Subtraction of this non-mitochondrial respiration allowed assessment of the rates and coupling efficiency of the remaining mitochondrial respiration.
Oligomycin inhibits mitochondrial ATP synthesis, so mitochondrial respiration that is insensitive to oligomycin is due to uncoupled respiration, i.e. to proton leak pathways through the mitochondrial inner membrane, and respiration that is sensitive to oligomycin is due to ATP synthesis occurring in the cells. Because oligomycin prevents the energy-utilizing pathway of ATP synthesis, the mitochondrial proton motive force will rise after oligomycin addition, and the proton leak rate will increase, leading to an overestimate of the proton leak rate and an underestimate of the ATP synthesis rate that occurred before oligomycin was added (Brand, 1990). With this proviso, Fig. 4B shows the estimates of the percentages of basal respiration used for ATP synthesis and proton leak within the cells. There was no difference between the haplogroups. The percentage of respiration used for ATP synthesis is the coupling efficiency, and, with a value of 85% of mitochondrial respiration, it was comparable to the coupling efficiency at intermediate respiration rates found in mitochondria isolated from the cells (Figs. 2 and 3), similar to the value of 80% measured previously using the parental cell line (Wu et al., 2007), and similar to values found in a range of other cell types (Brand, 2005).
Respiration rates in the presence of FCCP are not directly limited by ATP synthesis, and represent the maximal capacity of substrate oxidation by the cells under the conditions of measurement. In some cells this rate is equivalent to the maximum rate of the electron transport chain, but in others there is strong rate-limitation imposed by the supply of substrates to the mitochondria, particularly when ATP levels drop as a secondary result of uncoupling and substrates like glucose and fatty acids cannot be activated to glucose-6-phosphate and fatty acyl CoA. Fig. 4A shows that the FCCP-uncoupled respiration rates of A549 cybrids were initially high and rather variable, but then diminished with time, consistent with increasing limitation by substrate availability. There was no significant difference in uncoupled rates between haplogroups (Fig. 4B).
In summary, at the intact cell level we observed no significant functional differences between mitochondria within cybrids of haplogroup H and haplogroup T.
4
Discussion
There are published associations between mitochondrial haplogroups H and T and phenotypes that might be expected to have a strong bioenergetic component. However, our results show no significant bioenergetic differences in mitochondria with H or T-haplogroup mtDNAs in a constant nuclear background, at either the mitochondrial or the cellular level. We have previously shown that our methodology can pick up 10% differences in respiratory chain activity (Amo and Brand, 2007), so if haplogroup does affect mitochondrial bioenergetics, any effects must be very small. The lack of effect of haplogroup on mitochondrial coupling efficiency in intact cybrids shows that even if haplogroup affects mitochondrial proliferation or retrograde signalling, such changes do not affect coupling efficiency in the cells under basal conditions.
The strengths of our approach are (i) we used a direct empirical measurement, rather than inference from genetic and physiological traits, (ii) we separated the effects of mitochondrial haplogroup from any confounding effects of nuclear DNA background and (iii) we used a simple in vitro system with the powerful modular kinetic approach where the relevant variables could be tightly controlled and manipulated. However, our approach also has some weaknesses. (i) A small effect of mitochondrial haplogroup operating over hours or days might still be too small to be detected by biochemical experiments. (ii) We measured the bioenergetic capacity using isolated mitochondria and cybrid cells in simple defined media, but effects of mitochondrial haplogroups might only emerge under special conditions (for example, they might require triggers such as sepsis or energetic stress or might require particular nuclear genes to be expressed, and so be tissue-specific or suppressed in the lung carcinoma cell line we employed).
Our results fail to provide support for the hypothesis that the common European mitochondrial haplogroups H and T have significant effects on bioenergetics that cause the associations with survival after sepsis and sperm motility that have been reported. If such associations stand up to further scrutiny, they presumably operate through more subtle mechanisms than respiratory rates, mitochondrial coupling efficiencies or ATP supply.

fccp, carbonyl cyanide p-trifluoromethoxyphenylhydrazone