585
Journal of Physiology (1990), 422, pp. 585-602 With 7 figures Printed in Great Britain
KINETICS OF THE CONDUCTANCE EVOKED BY NORADRENALINE, INOSITOL TRISPHOSPHATE OR Ca2l IN GUINEA-PIG ISOLATED HEPATOCYTES
BY D. C. OGDEN*t, T. CAPIOD, J. W. WALKER: AND D. R. TRENTHAM From the Department of Pharmacology, King's College London, Strand, London WC2R 2LS, and the Division of Physical Biochemistry, National Institute for Medical Research, London NW7 1AA
(Received 8 August 1989) SUMMARY
1. Guinea-pig hepatocytes respond to noradrenaline (NA, 5-10 /tM) with a large membrane conductance increase to K+ and Cl-. The response has a long initial delay (range 2-30 s). Following the delay, the K+ conductance (studied in Cl--free solutions) rises quickly to a peak in 1-2 s and is maintained in the continued presence of NA, though often with superimposed oscillations of conductance. The roles of intracellular Ca2+ and D-myo-inositol 1,4,5-trisphosphate (InsP3) in this complex response have been investigated by rapid photolytic release of intracellular Ca2+ (from Nitr5-Ca2+ buffers) or InsP3 from 'caged' InsP3. 2. A rapid increase of intracellular [Ca2+] produced an immediate membrane conductance increase which rose approximately exponentially to a new steady level, consistent with a direct activation of Ca2+-dependent ion channels. 3. Following a pulse of InsP3, conductance rose after a brief delay (range 70-1500 ms) which was shortest at high [InsP3] or if the initial cytosolic [Ca2+] had been raised above normal levels. The maximum conductance produced by InsP3 was similar in each cell to the peak recorded with NA and could be evoked by InsP3 concentrations of 0 5-1 atm. 4. The rates of rise of conductance increased with InsP3 concentration in the range 0-25-12-5 ,lM (range 10-90 %, rise times 90-1000 ms), indicating that InsP3-evoked Ca2+-efflux from stores increases with InsP3 concentration in this range. 5. Photochemically released InsP3 and Ca2+ activate at physiological concentrations the same membrane conductances as NA. The results indicate that the long initial delay in NA action occurs prior to or during generation of InsP3. The mechanism of the delay and the subsequent apparently all-or-none conductance increase during NA action are discussed in terms of the high co-operativity in InsP3 and Ca2+ actions and an additional positive feedback step. * Present address and address for correspondence: Division of Neurophysiology and Neuropharmacology, National Institute for Medical Research, The Ridgeway, London NW7 1AA. t Permanent address: INSERM U274, Bat. 443, Universite Paris Sud, 91405, Orsay Cedex, France. I Present address: Department of Physiology, University of Wisconsin, 1300 University Avenue, Madison, WI 53706, USA. MS 7867
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6. Evidence was found of a negative interaction between [Ca2+] and InsP3-evoked Ca2' release. The time course of the recovery of InsP3-evoked Ca2' release following a rise of cytosolic [Ca2+] suggests that this interaction may be important in regulating oscillatory responses of [Ca2+] during hormonal stimulation of guinea-pig hepatocytes. INTRODUCTION
Stimulation of liver cells from guinea-pig and many other mammalian species with noradrenaline (NA) causes elevation of the cytosolic free Ca2+ concentration, leading to activation of glycogen breakdown and efflux of glucose, Ca2+, K+ and Cl- from the cell (Haylett & Jenkinson, 1972; Haylett, 1976; Burgess, Claret & Jenkinson, 1981; Egashira, 1980; Field & Jenkinson, 1987). It has been shown that NA acts through ac-adrenoceptors to activate phospholipase C. This generates D-myo-inositol 1,4,5trisphosphate (InsP3) within the cell which in turn mediates the rise of Ca2+ concentration by release from intracellular stores (Burgess, Godfrey, McKinney, Berridge, Irvine & Putney, 1984a; Joseph, Thomas, Williams, Irvine & Williamson, 1984). It is thought that this source of Ca2+ can raise free cytosolic [Ca2+] to a level sufficient to activate phosphorylase b kinase, the Ca2+-ATPase pump and Ca2+activated ion channels in the plasma membrane (for a review see Exton, 1988). Recent electrophysiological studies have characterized both K+ and Cl- conductances in the surface membrane of guinea-pig and rabbit hepatocytes (Field & Jenkinson, 1987; Capiod & Ogden, 1989a) that are activated by internal application of either buffered Ca2+ of InsP3 (Capiod, Field, Ogden & Sandford, 1987). The Ca2+activated K+ conductance, when studied in isolation in solutions containing impermeant anions, is blocked by the bee venom toxin apamin (Banks, Brown, Burgess, Burnstock, Claret, Cocks & Jenkinson, 1979) and by (+)-tubocurarine (Cook & Haylett, 1985). The properties of single ion channels underlying this conductance have been examined in isolated membrane patches: the unitary conductance is 6 pS and channel activation depends steeply on [Ca2+] at the inside surface in the range 0-3-10/JM, reaching a maximum open probability of 85 % without indication of densensitization to Ca2+ (Capiod & Ogden, 1989b). The basic scheme that emerges from studies of the activation of the K+ conductance is that NA acts primarily to stimulate intracellular InsP3 production and that InsP3 influences the membrane conductance by mobilizing internal Ca2+. Several features of the membrane conductance increase evoked by NA indicate that it is regulated by complex intracellular mechanisms. After NA binding, there is a delay of several seconds before a change in membrane conductance occurs. The conductance then increases quickly to a maximum level in a manner suggestive of underlying processes involving a high degree of co-operativity or positive feedback. During prolonged application of NA the conductance often shows marked oscillations with a period of 10-20 s (Field & Jenkinson, 1987), presumably reflecting oscillations of intracellular [Ca2+] similar to those observed directly in rat hepatocytes (Woods, Cuthbertson & Cobbold, 1987). In order to investigate the intracellular mechanisms responsible for these complex phenomena, whole-cell patch clamp recording (Hamill, Marty, Neher, Sakmann & Sigworth, 1981) has been used to monitor membrane K+ and Cl- conductances
PHOTOLYTIC RELEASE OF InsP3 AND Ca2+ IN LIVER
587
(Capiod & Ogden, 1989a). Using this method, agents can be applied intracellularly by perfusion from the patch pipette. However, measurements of the time course of events are hampered by the slow rate of access of active agents by diffusion from the patch pipette (see Pusch & Neher, 1988). Moreover, it is possible that agents introduced into the cell by this means may be rapidly metabolized, so raising questions about the concentration at the site of action and also the identity of the active agent. To circumvent these limitations we have used inactive 'caged' precursors of InsP3 (caged InsP3; Walker, Somlyo, Goldman, Somlyo & Trentham, 1987; Walker, Feeney & Trentham, 1989) and Ca21 (Nitr5: Gurney, Tsien & Lester, 1987; Adams, Kao, Grynkiewicz, Minta & Tsien, 1988) that can be converted to active InsP3 or liberate free Ca2' respectively by pulse illumination with near UV light. The principal aim of these experiments was to probe the mechanism of hormone action by establishing the time course and concentration dependence of both InsP3 and Ca2+ actions in activating the K+ conductance of guinea-pig hepatocytes. A preliminary account of this work has been given (Capiod, Ogden, Trentham & Walker, 1988). METHODS
Guinea-pig hepatocytes were isolated by perfusion with collagenase followed by mechanical dispersal. Cells in suspension were plated onto 35 mm Falcon dishes and whole-cell voltage clamp recordings made after 2-8 h (Capiod & Ogden, 1989a). For experiments in the presence of Cl- the external solution contained (mM): NaCl, 145; KCl, 5-6; CaCl2, 1-8; MgSO4, 0-8; HEPES, 8; pH 7-3; the internal solution contained (mM): KCl, 153; Na-ATP, 1; MgSO4, 3; Na-GTP, 0 05; HEPES, 8; pH 7-3. External Cl--free solutions contained (mM): sodium gluconate, 145; potassium gluconate, 5-6; CaSO4, 5; MgSO4, 0-8; HEPES, 8; pH 7-3; internal Cl--free solutions contained (mM): potassium gluconate, 153; Na-ATP 1: MgSO4, 3; Na-GTP, 0 05; HEPES, 8; pH 7-3. Propranolol (2 /SM) was present throughout to block fl-adrenoceptors. Experiments were made at room temperature (ca 25 °C). Noradrenaline and other agents were applied externally by pressure ejection from a pipette near the cell. Data were recorded on FM tape and replayed on a fibre-optic recorder. Delays were measured as the time following the flash or NA application before the trace deviated noticeably (about 5-10 pA) from the baseline. Current records were differentiated electronically to display rates of l7ise and facilitate measurement of delays if the baseline was unsteady. Caged InsP3 (the P-4 1-(2-nitrophenyl)ethyl ester of InsP3), caged InsP2 (the P-1 ester of D-myoinositol 1,4-bisphosphate) (Walker et al. 1989) or Nitr5 (complexed with Ca2+) were introduced into the cell via the patch pipette, allowing 2-3 min for equilibration. Photolysis was produced by a 1 ms pulse from a xenon arc flashlamp (Rapp & Guth, 1988) focused to a spot of about 2 x 3 mm around the cell from an incident angle of 38 deg to the horizontal; this arrangement resulted in minimal (about 8 %) energy loss due to reflection from the fluid surface. Light was bandpassfiltered with a UG 11 glass between 300-350 nm. Set to maximum power, the energy output was 100-120 mJ in this band. This produced 58% (± 6% S.D.) conversion of caged ATP (the P3-1(2-nitrophenyl) ethyl ester of ATP; Kaplan, Forbush & Hoffman, 1978) to ATP at full power, or less at reduced power. The efficiencies of caged ATP and caged InsP3 photolysis have been shown to be similar under the experimental conditions used (Walker et al. 1989) and photolysis of a droplet of 10 1cl 0 4 mM-caged ATP after each experiment followed by HPLC analysis was used to assess to proportion of caged InsP3 photolysed. Making allowance for reflection, photolysis of intracellular caged InsP3 was 50 % (S.D. + 6 %) at full power. The rate of photolysis of caged InsP3 is estimated as 200 s-5 in these conditions, corresponding to a half-time for InsP3 formation of about 3 ms (Walker et al, 1989). Photolysis of Nitr5 releases Ca2+ ions within the 1 ms duration of the light pulse (Adams et al. 1988). The experiments described here indicate that the biological properties of caged InsP3 are as previously found in vascular smooth muscles (Walker et al. 1987, 1989). Caged InsP3 esterified on
588
D. C. OGDEN AND OTHERS
the 4 or 5 phosphates shows no InsP3-like Ca2+-mobilizing activity at concentrations up to 50 /M. In addition Walker et al. (1989) found that neither isomer is metabolized by or interferes with the hydrolysis of InsP3 by InsP3 5-phosphatase; the 5-isomer has some inhibitory activity towards InsP3 3-kinase. The experiments reported here were made mainly with the 4-isomer. Control photolysis experiments were made with caged InsP2 at a concentration of 37 /LM to test the toxicity of the by-product 2-nitrosoacetophenone. These showed that photolysis to liberate 18 ,#M-InsP2 and nitrosoacetophenone produced no response and did not prevent subsequent activation by NA. Noradrenaline and propranolol were obtained from Sigma (St Louis, MO, USA) and Nitr5 from Calbiochem (La Jolla, CA, USA). Other reagents were from Sigma or BDH. RESULTS
The increase in membrane conductance evoked by NA in voltage clamped guineapig hepatocytes is illustrated in Fig. 1. In Fig. 1A conditions were chosen to show both K+ and Cl- conductances. The responses to 5 /tM-NA showed a delay of 8 s before the conductance increased, first to K+ seen as a brief outward K+ current (upward deflection) followed by a large conductance increase to Cl- with net inward current (downwards deflection) and finally a net outward current as NA was removed. The properties of these conductances have been described in detail elsewhere (Field & Jenkinson, 1987; Capiod & Ogden, 1989a). Figure lB shows the response under similar conditions to a pulse of InsP3 photolytically released inside the cell at the time indicated by the arrow. After a short delay (100 ms), an inward current due to the increase in Cl- conductance was seen. In contrast to the activation by NA, temporal separation of K+ and Cl- conductances was rarely seen following a pulse ofInsP3 (cf. responses to NA; Capiod & Ogden 1989a). In the record shown in Fig. 1 C the K+ conductance alone was activated by NA, generating an outward current. The peak current amplitudes and rise times under these conditions varied little between responses, whereas the delay between NA application and the conductance increase ranges from 2-5 s to more than 30 s, with a mean value of 10 s in the present experiments (see Table 1, row 1). Delays were similar in Cl--containing or Cl--free solution. A comparison between the K+ conductance evoked by NA and InsP3 may be made in Fig. 1D, which shows on the same scale as Fig. 1 C current evoked in the same cell by photolysis of 2 saM-caged InsP3, releasing a pulse of1 /IM-InsP3 in the cytosol. The amplitudes of the responses are similar, close to the maximum seen in these cells, and most likely reflect maximal activation of the K+ conductance by Ca2+ in each case. The responses differ first in the rise times, that for NA being longer than that for photolytically released InsP3. Secondly, the delay before the current rises are strikingly different, 5 s for NA compared to 300 ms for InsP3. The responses of the Cl- and K+ conductances to rapid changes of free[Ca2+] were examined with release from photolysed Nitr5 and are illustrated in Fig. 2. For cells inCl--containing solutions (Fig. 2A) the calculated free [Ca2+] before photolysis was 015 /tM, close to the resting [Ca2+] in hepatocytes (Burgess, McKinney, Fabiato, Leslie & Putney, 1983; Berthon, Binet, Mauger & Claret, 1984). Three consecutive pulses of UV light produced inward Cl- currents which began immediately after the flash, approaching steady levels with half-times of 10-100ms. The responses were faster with each successive flash as the freeLCa2+] increased. Responses to the activation of the K+ conductance alone by Ca2+ are shown in Fig.
PHOTOLYTIC RELEASE OF InsP3 AND Ca2+ IN LIVER
589
5 gM-NA
A
m
500 pA
L10 s
12*5 uM-insP3
B
500 pA 1 s
10 gM-NA
I
200 pAL 2s
C
200 pA L 2s
D
Fig. 1. Membrane current evoked by noradrenaline (NA) or by photolysis of caged InsP3 in isolated guinea-pig hepatocytes. A, 5 1uM-NA was applied during the bar in Cl--containing solution. Cell voltage clamped at a membrane potential of -20 mV. Pulses of 10 mV were applied to monitor the membrane conductance. After a delay of 8 s an initial brief outward K+ current (upward deflection, EK = -85 mV) was followed by a large inward Cl- current (Ec1 = 0 mV) which declined to leave a K+ current and finally decayed to the baseline. B, conductance increase at -20 mV in Cl--containing solutions evoked by pulse photolysis of caged InsP3 at the point indicated by the arrow in B and D. 12-5 ,#M-InsP3 was released and produced a net inward current after a delay of 100 ms. C, conductance increase to K+ alone evoked by 10 ,uM-NA (applied during the bar), recorded with Cl--free solutions at 0 mV. Delay was 5 s, peak current 330 pA. D, conductance increase to K+ following photolytic release of 1 #uM-InsP3, recorded at 0 mV in the same cell as C. Delay was 300 ms and peak current 350 pA. Time of flash is indicated by the arrow.
2B. These were obtained by photolysis of 5 mM-Nitr5 equilibrated with 2-5, 3-25 and 3.75 mM-Ca2+. In each case outward K+-current rose approximately exponentially to approach a new level, and this was frequently followed by a further much slower rise of conductance. Half-times for the rapid phase of the relaxations were 250 ms at
D. C. OGDEN AND OTHERS
590
TABLE 1. Amplitude, delay and rise time (10-90 %) for K+ current increase evoked at 0 mV by; row 1: external noradrenaline (NA, 5 or 10, M) and rows 2-7: photolytic release of InsP3 from internal caged InsP3. Data are means+s.E.M. Delay Amplitude Rise time (pA) (s) (s) 10-0 NA 1P0+0-2 460+75 (range 2 5-30) [InsP3]
(/#M) 05 1.0 2-5 5 12-5 25
233+ 129 280+99 445+68 570+100 483+92 621+73
A -20 mV Cl- solutions Initial [Ca2+ = 0-15 ,UM
1
0-64+0-12 037+0*10 0-38+0-10 0-37+0-13 0-18+0-03 0-21+0'05
0419+0-06
B 0 mV Cl1-free solutions Initial [Ca2+l = 0.15 PM Final [Ca2+1 = 0_5 pM
t
J 200 pA is Initial [Ca2+] = 0.3 pM Final [Ca2 1 = 1.4 uM
J 200 pA
I
1P14+0-35 0-37+0-10 0-41+0412 037+009 0-16+0-02
1 s
I
-J 200 pA 0.5 s
Initial [Ca2+1 = 0.6 pM Final [Ca2J1 = 1.9 MM
J 200 pA 0.5 s
Fig. 2. Conductance increase following photolysis of Ca2+-loaded Nitr5. A, Cl--containing solutions at a membrane potential of -20 mV. 5 mM-Nitr5 with 2-5 mM-Ca2 , giving an initial [Ca2+] of 0 15 ,UM and a final [Ca2+] of 0 54 fIM after 1 pulse. Subsequent flashes to same cell, shown in the lower traces, produced further increases of [Ca2+] and conductance. Time of flashes indicated by arrows. B, K+ currents recorded in Cl--free solutions at 0 mV. 5 mM-Nitr5 loaded with 2-5 mM-Ca2+ (upper trace), 3-25 mM-Ca2+ (middle trace) and 3.75 mM-Ca2+ (lower trace). Initial and final [Ca2+] were calculated from the equation given by Gurney et al. (1987) and are shown above each trace.
PHOTOLYTIC RELEASE OF InsP3 AND Ca2+ IN LIVER
591
0-5 /M final free [Ca2"], 1 10 ms at 1-4/tM and 20 ms at 1P9 /gm. These time courses most probably reflect the rate of channel activation by free Ca2 . Several control experiments were made. The K+ conductance increase evoked by NA, internally perfused Ca21 or InsP3, is blocked by apamin or (+ )-tubocurarine (Cook & Haylett, 1985; Capiod et al. 1987). The K+ conductance produced by photolytic release of Ca2+ or by InsP3 (12 /tM) was rapidly blocked by 10 /M (+ )tubocurarine applied during the response (data not shown). The response to photoreleased InsP3 (1-2 aM) could be completely blocked by including bis(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid (BAPTA) in the internal solution in the patch pipette; responses were blocked by 0 75 or 1P0 ,M-BAPTA but not by 05 ,aM (four trials at each concentration). Removing extracellular Ca2+ had little effect on the InsP3 responses after 1-2 min exposure, consistent with an internal origin of the [Ca2"] increase. These results show that InsP3 acts on the surface membrane conductance mainly by intracellular Ca2+ release and not by a direct action on the membrane K+ conductance channels. Figure 3 shows K+ currents following release of InsP3 by pulse photolysis of 5 or 10/M caged InSp3in three cells, represented by traces in A, B and C. The upper trace in each panel represents the K+ current and the lower trace the current differentiated with respect to time to emphasize rates of change. In order to make comparisons in the same cell, the lamp output powder was reduced to produce photolysis which could be set in the range 5-14 %, leaving sufficient caged InsP3 unphotolysed for consecutive responses to different concentrations of InsP3 in the same cell. The responses were separated by 1-2 min. In a number of experiments twin closely spaced pulses of InsP3 were used to test recovery of InsP3 sensitivity, for reasons described below. Considering first the onset of the responses shown in Fig. 3, both the peak amplitude and the kinetics of the K+ current were found to depend on the InsP3 concentration released. In Fig. 3A 0 25 /tM-InsP3 produced a small outward current of 30 pA after a delay of 1 4 s following photolysis. Increasing the concentration released to 0 7 aM gave a current of maximal amplitude, 400 pA in this cell, with a delay of 300 ms and peak rate of rise of 1000 pA s-'. In Fig. 3B the currents evoked in another cell by 0 35 and 05 /tm-InsP3 were both of maximal amplitude (720 pA) but the delay was longer (700 ms) and the rise slower (800 pA s-') at 0-35 /IM than at 0 5 /M (delay 350 ms and rise 1340 pA s-', respectively). Similar effects of increasing the concentration of InsP3 from 0 5 to 1 UM, decreasing the delay and rise time at the same current amplitude, are seen with the cell of Fig. 3 C. The rise of the K+ current following InsP3 release was sigmoidal after the initial delay, and the duration of responses was from about 3 to 20 s. In prolonged recordings the delay, the time-topeak and the duration of the responses usually increased with four or five consecutive similar pulses by a factor of 2-5 over 10 min. When comparing the InsP3 responses of different cells a degree of variability from cell to cell was encountered in the parameters described in connection with Fig. 3. The K+ conductances, represented by the current at 0 mV membrane potential, are summarized for the first responses of a number of cells in Table 1, rows 2-7, for different concentrations of photolytically released InsP3. The maximum current amplitude of these cells, averaging about 530 pA, was obtained consistently with InsP3 concentrations of 2 /tM or more, indicating that this concentration released
_~ ~ ~ ~I
592
D. C. OGDEN AND OTHERS
Ca2+ from the stores at a rate sufficient to achieve a free [Ca2+] adjacent to the membrane which fully activated the K+ conductance. Data with buffered Ca2+ has shown that maximal steady-state activation of the K+ conductance occurs with concentrations of 1-2 /M-free Ca21 (Capiod & Ogden, 1989b). The variability of maximum amplitude from cell to cell is largely due to differences in surface area A 0.25 pM-IfnSP3
0.7 pM-InsP3
J 200 pA
is B
0.35 M-IlnSP3
C 0.5 M-IlnSP3
0-5 M-IlnSP3
1 uM-InsP3
200 pA
2s
Fig. 3. K+ currents evoked by photolysis of caged InsP3 in Cl--free solutions at a membrane potential of 0 mV. Data are from three cells shown in panels A, B and C, loaded with 5 /M (A and B) or 10/tM (C) caged InsP3. Each section shows two consecutive responses separated by 1-2 min evoked by the InsP3 concentrations given above each trace, released at the arrows. In A (second response) and C twin pulses separated by 4-5 s were applied. The upper trace in each record is K+ current and the lower trace the differentiated current to show the rate of rise. Vertical calibration bar corresponds to 200 pA for the upper trace and 3200 pA s-' for the differentiated trace. Time calibration 1 sforA andB, 2s for C.
(reflected by the range of cell capacitances recorded of 26-70 pF). At lower InsP3 concentrations, 0-25-10 tM, a number of responses were of submaximal amplitude and in this concentration range current amplitudes varied widely from cell to cell, from zero to maximal activation of the K+ conductance. The cell-to-cell variability observed may arise in several processes, including the photolysis reaction (S.D. for photolysis of caged ATP 6%, see Methods), the rates of ihnsP3 metabolism, Ca2+
593 PHOTOLYTIC RELEASE OF InsP3 AND Ca2+ IN LIVER release and reuptake by stores, Ca2+ buffering and the activation of K+ conductance by Ca2+. The delay between the light flash and initiation of the conductance change decreased as the InsP3 concentration was increased. The longest delay observed was about 1-5 s at 0-25 ,uM-InsP3 and the shortest 70 ms for a response to 25 ftM-InsP3. A 2 piM-InsP3
t|
1 ,UM) to activate the K+ conductance in a nearly all-or-none manner. However, the InsP3-evoked Ca2+ efflux from the stores, as monitored by the peak rate of rise of conductance, showed a more graded dependence on the InsP3 concentration. These results are consistent with a report by Meyer, Holowka & rate of Ca2+ flux from permeabilized rat basophilic leukaemia cells showed a graded yet steep dependence on [InsP3]. Taken together with the narrow Ca2+ concentration range required for activation of the K+ conductance (Capiod & Ogden, 1989b), co-operativity in the InsP3-evoked Ca2+ release may be sufficient to account for the sigmoidal rise of the conductance and the almost all-or-none conductance increase seen with InsP3 and NA. However, the occurrence of an additional autocatalytic step within the reaction sequence leading to Ca2+ release cannot be excluded on the basis of the experiments reported here.
Stryer (1988) in which the
PHOTOL YTIC RELEASE OF InsP3 A ND Ca2+ IN LIVER
599
Evidence of a feedback mechanism has been described in the form of conductance oscillations in response to prolonged stimulation by NA (Field & Jenkinson, 1987), high intracellular concentrations of InsP3 (Capiod et al. 1987) or bile acids applied externally (Capiod et al. 1989). Oscillatory conductance changes were seen in some responses initiated by photolysis of caged InsP3 (e.g. in the declining phases of Figs 1D and 6). These occurred particularly with high concentrations of InsP3, had a similar time course to those seen with NA but were often of low amplitude. Large amplitude oscillations with a period of 10-20 s were seen in the present experiments with D-myo-inositol 2,4,5-trisphosphate applied directly from the patch pipette (see Fig. 7). As suggested previously (Capiod et al. 1987) the presence of oscillations with high (and presumably maximal) concentrations of InsP3, and with the bile acids (Capiod et al. 1989) which elevate [Ca21] without InsP3 production (Combettes et al. 1988), suggests that in the guinea-pig liver oscillations of cytosolic [Ca2+] in response to hormones can be generated by mechanisms which do not depend on fluctuations of [lInsP3]. Similar conclusions have been reached for parotid and pancreatic acinar cells (Gray, 1988; Wakui, Potter & Petersen, 1989) and Xenopus oocytes (Berridge, 1988). A possible clue to the mechanism is in the observations that InsP3-evoked Ca21 release is considerably reduced shortly after a conditioning response and recovers with a time course of half-time 10 s, similar to the period of oscillations observed with NA or intracellular perfusion with InsP3. Depression of the InsP3 response following Ca2+ release by TLCS indicates that the effect is not InsP3-specific and therefore not analogous to, for example, desensitization of nicotinic receptors by acetylcholine. It is unlikely that the reduced Ca2' release is due simply to Ca2+ depletion of the stores, because it is seen following small responses and when the [Ca2+] presumably has been restored back to the resting level (or near it) by reuptake into stores (e.g. Fig. 3C, first trace). The most likely explanation is a reduced sensitivity of the Ca2+ release mechanism to InsP3, and possibly other agents, induced by the raised [Ca2+], and which recovers with a slow time course even when the [Ca2+] has been restored to resting levels (see Fig. 5A). This explanation is supported by the reduced InsP3 binding observed in brain membranes with [Ca2+] in the range 01-1 JIM (Worley, Baraban, Supattapone, Wilson & Snyder, 1987) and the inhibition by injected Ca2+ of the InsP3-mediated photoresponse in Limulus photoreceptors (Payne, Walz, Levey & Fein, 1988). Studies of Ca2+ oscillations in fibroblasts by Harootunian, Kao & Tsien (1988) show that a pulse of InsP3 during the period between Ca2+ spikes produces a phase shift of the subsequent oscillations as if the spikes were controlled by InsP3 fluctuations. The stimulation by Ca2+ of InsP3 production by phospholipase C has been proposed by Meyer & Stryer (1988) as a mechanism of positive feedback which could produce this effect. A difference in the periodicity of oscillations in different cells and conditions may be noted; in the exocrine cells and in the present experiments the period was 10-20 s, whereas the fibroblasts, endothelial cells and rat hepatocytes stimulated with low concentrations of hormones have much longer periods of 60-90 s between Ca2+ spikes (for reviews see Berridge & Gallione, 1988; Rink & Jacob, 1989). The reduced sensitivity of the release mechanism to InsP3 following a Ca2+ pulse, observed in the experiments reported here, may also be involved in the termination of Ca2+ spikes in slowly oscillating systems.
600
6D. C. OGDENV AiND OTHERS
In summary, the experimental approach used here has provided time-resolved measurements of the conductance increase, normally elicited by extracellular NA, in response to known intracellular concentrations of free Ca2+ and InsP3. The data are consistent with a model in which InsP3 activates K+ and Cl- conductances in the hepatocyte plasma membrane by releasing Ca21 from intracellular stores rapidly and with a high degree of co-operativity. Calcium ions released from the stores activate the membrane conductance channels directly. The delay in the response to NA and other hormones is associated with a step prior to the production of InsP3. Photolysis of caged InsP3 does not produce a sustained conductance increase, probably because InsP3 produced is readily metabolized, free Ca2' released is rapidly removed from the cytosol and the Ca2+ release mechanism quickly becomes refractory after stimulation by InsP3. The latter mechanism and the role of external Ca21 in the response to NA are currently being investigated. We thank Dr R. F. Irvine for kindly providing inositol phosphates, Professor D. H. Jenkinson for discussion and allowing use of facilities for cell production and Dr M. Claret for discussion. Meera Prasad and R. J. Congram gave excellent technical help. The work was supported by the MRC, an MRC-INSERM exchange Fellowship and by NIH Grant 15835 to the Pennsylvania Muscle Institute. REFERENCES
ADAMS, S. R., KAO, J. P., GRYNKIEWICZ, G., MINTA, A. & TSIEN, R. Y. (1988). Biologically useful chelators that release Ca upon illumination. Journal of the American Chemical Society 110, 3212-3220. BANKS, B. E. C., BROWN, C., BURGESS, G. M., BURNSTOCK, G., CLARET, M., COCKS, T. M. & JENKINSON, D. H. (1979). Apamin blocks certain neurotransmitter induced increases in potassium permeability. Nature 282, 415-417. BERRIDGE, M. J. (1988). Inositol trisphosphate induced membrane potential oscillations in Xenopus oocytes. Journal of Physiology 403, 589-599. BERRIDGE, M. J. & GALLIONE, A. (1988). Cytosolic Ca oscillators. FASEB Journal 2, 3074-3082. BERTHON, B., BINET, A., MAUGER, J. P. & CLARET, M. 1984). Cytosolic free Ca in isolated rat hepatocytes as measured by quin2. Effects of noradrenaline and vasopressin. FEBS Letters 167, 19-24. BURGESS, G. MI., CLARET, M. & JENKINSON, D. H. (1981). Effects of quinine and apamin on the calcium-dependent potassium permeability of mammalian hepatocytes and red cells. Journal of Physiology 317, 67-90. BURGESS, G. M., GODFREY, P. P., MCKINNEY, J. S., BERRIDGE, M. J., IRVINE, R. F. & PUTNEY, J. W. (1984a). The second messenger linking receptor activation to internal Ca release in liver. Nature 309, 63-66. BURGESS, G. AI., IRVINE, R. F., BERRIDGE, M. J., MCKINNEY, J. S. & PUTNEY, J. W. (1984 b). Actions of inositol phosphates on Ca2" pools in guinea-pig hepatocytes. Biochemical Journal 224, 741-746. BURGESS, G. M., MCKINNEY, J. S., FABIATO, A., LESLIE, B. A. & PUTNEY, J. W. (1983). Calcium pools in saponin permeabilised guinea-pig hepatocytes. Journal of Biological Chemistry 258, 15336-15345. CAPIOD, T., COMBETTES, L. & CLARET, M. (1989). Natural bile acids mimic hormonal action on Kconductance in guinea-pig liver cells. Pfiuigers Archiv 414, S162-163. CAPIOD, T., FIELD, A. C., OGDEN, D. C. & SANDFORD, C. A. (1987). Internal perfusion of guinea-pig hepatocytes with buffered Ca2+ or inositol 1,4,5-trisphosphate mimics noradrenaline activation of K+ and Cl- conductances. FEBS Letters 217, 247-252. CAPIOD, T. & OGDEN, D. C. (1989a). Properties of membrane ion conductances evoked by hormonal
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stimulation of guinea-pig and rabbit isolated hepatocytes. Proceedings of the Royal Society B 236, 187-201. CAPIOD, T. & OGDEN, D. C. (1989b). The properties of calcium-activated potassium ion channels in guinea-pig isolated hepatocytes. Journal of Physiology 409, 285-295. CAPIOD, T., OGDEN, D. C., TRENTHAM, D. R. & WALKER, J. W. (1988). Activation of membrane conductance in isolated guinea-pig hepatocytes by photochemically generated intracellular inositol 1,4,5-trisphosphate or free calcium ions. Journal of Physiology 406, 124P. CHAMPEIL, P., COMBETTES, L., BERTHON, B., DOUCET, E., ORLOWSKI, S. & CLARET, M. (1989). Fast kinetics of calcium release induced by myo-inositol trisphosphate in permeabilized rat hepatocytes. Journal of Biological Chemistry 264, 17665-17673. COMBETTES, L., BERTHON, B., DOUCET, E., ERLINGER, S. & CLARET, M. (1989). Characteristics of bile acid mediated calcium release from permeabilized liver cells and liver microsomes. Journal of Biological Chemistry 264, 157-167. COMBETTES, L., DUMOND, M., BERTHON, B., ERLINGER, S. & CLARET, M. (1988). Release of Ca from endoplasmic reticulum by bile acids in rat liver cells. Journal of Biological Chemistry 263, 2299-2306. COOK, N. S. & HAYLETT, D. G. (1985). Effects of apamin, quinine and neuromuscular blockers on calcium-activated potassium channels in guinea-pig hepatocytes. Journal of Physiology 358, 373-394. DOLPHIN, A. C., WOOTTON, J. F., SCOTT, R. H. & TRENTHAM, D. R. (1988). Photoactivation of intracellular guanosine triphosphate analogues reduces the amplitude and slows the kinetics of voltage-activated calcium channel current in sensory neurones. PflJugers Archiv 411, 628-636. EGASHIRA, K. (1980). Biphasic response to noradrenaline in guinea-pig liver cells. Japanese Journal of Physiology 30, 81-91. EXTON, J. H. (1988). Role of phosphoinositides in the regulation of liver function. Hepatology 8, 152-166. FIELD, A. C. & JENKINSON, D. H. (1987). The effect of noradrenaline on the ion permeability of isolated mammalian hepatocytes, studied by intracellular recording. Journal of Physiology 392, 493-512. GRAY, P. T. A. (1988). Oscillations of free cytosolic calcium evoked by cholinergic and catecholaminergic agonists in rat parotid acinar cells. Journal of Physiology 406, 35-53. GURNEY, A. M., TSIEN, R. Y. & LESTER, H. A. (1987). Activation of a potassium current by rapid photochemically generated step decreases of intracellular calcium in rat sympathetic neurons. Proceedings of the National Academy of Sciences of the USA 84, 3496-3500. HAMILL, 0. P., MARTY, A., NEHER, E., SAKMANN, B. & SIGWORTH, F. J. (1981). Improved patchclamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Archiv 391, 85-100. HAROOTUNIAN, A. T., KAO, J. P. Y. & TSIEN, R. Y. (1988). Agonist induced oscillations in depolarized fibroblasts and their manipulation by photoreleased Ins(1,4,5)P3 Ca and Ca buffer. Cold Spring Harbour Symposium 53, 935-943. HAYLETT, D. G. (1976). The effects of sympathomimetic amines on 45Ca efflux from liver slices. British Journal of Pharmacology 57, 158-160. HAYLETT, D. G. & JENKINSON, D. H. (1972). Effects of noradrenaline on potassium efflux, membrane potential and electrolyte levels in tissue slices prepared from guinea-pig liver. Journal of Physiology 225, 721-750. IRVINE, R. F. & MOORE, R. M. (1986). Microinjection of inositol 1,3,4,5-tetrakis phosphate activates sea urchin eggs by a mechanism dependent on external calcium. Biochemical Journal 240, 917-920. JOSEPH, S. K., THOMAS, A. P., WILLIAMS, R. J., IRVINE, R. F. & WILLIAMSON, J. R. (1984). Myo inositol 1,4,5-trisphosphate. A second messenger for the hormonal mobilization of intracellular calcium in liver. Journal of Biological Chemistry 259, 3077-3081. KAPLAN, J. H., FORBUSH, B. III, & HOFFMAN, J. F. (1978). Rapid photolytic of adenosine-5triphosphate from a protected analogue: utilization by the Na-K exchange pump of human red cell ghosts. Biochemistry 17, 1929-1935. MEYER, T., HOLOWKA, D. & STRYER, L. (1988). Highly cooperative opening of calcium channels by inositol 1,4,5-trisphosphate. Science 240, 653-656.
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MEYER, T. & STRYER, L. (1988). Molecular model for receptor-stimulated calcium spiking Proceedings of the NVational Academy of Sciences of the 7USA 85, 5051-5055. PAYNE, R., WALZ, B., LEVY, S. & FEIN, A. (1988). The localization of Ca release in Limulu photoreceptors and its control by negative feedback. Philosophical Transactions of the Royo Society B 320, 359-370. PUSCH, M. & NEHER, E. (1988). Rates of diffusional exchange between small cells and a measurinl patch pipette. Pflugers Archiv 411, 204-21 1. RAPP, G. & GUTH, K. (1988). A low cost intensity flash device for photolysis experiments. Pfiiger Archiv 411, 200-203. RINK, T. J. & JACOB, R. (1989). Calcium oscillations in non-excitable cells. Trends in Neuroscienc 12, 43-46. RODBELL, M., LIM, M. C. & SALOMON, Y. (1974). Evidence for interdependent action of glucagot and nucleotides on the hepatic adenylate cyclase system. Journal of Biological Chemistry 249 59-65. STOREY, D. G., SHEARS, S. B., KIRK, C. J. & MICHEL, R. M. (1984). Stepwise enzymatic degradatior of inositol 1,4,5-trisphosphate to inositol in liver. Nature 312, 374-376. WAKUI, M., POTTER, B. V. P. & PETERSEN, 0. H. (1989). Pulsatile intracellular calcium release does not depend on fluctuations in inositol trisphosphate concentration. NVature 339, 317-320. WALKER, J. W., FEENEY, J. & TRENTHAM, D. R. (1989). Photolabile precursors of inositol phosphates. Preparation and properties of 1(2-nitrophenyl)ethyl esters of myo-inositol 1,4,5-
trisphosphate. Biochemistry 28, 3272-3280. WALKER, J. W., SOMLYO, A. V., GOLDMAN, Y. E., SOMLYO, A. P. & TRENTHAM, D. R. (1987). Kinetics of smooth and skeletal muscle activation by laser pulse photolysis of caged inositol 1,4,5-trisphosphate. Nature 327, 249-252. WOODS, N. M., CUTHBERTSON, K. S. R. & COBBOLD, P. H. (1987). Agonist induced oscillations in cytoplasmic free calcium ion concentration. Cell Calcium 8, 79-100. WORLEY, P. F., BARABAN, J. M., SUPATTAPONE, S., WILSON, V. S. & SNYDER, S. H. (1987). Characterisation of inositol trisphosphate receptor binding in brain; regulation by pH and calcium. Journal of Biological Chemistry 262, 12132-12136.
Journal of Physiology (1990), 422, pp. 585-602 With 7 figures Printed in Great Britain
KINETICS OF THE CONDUCTANCE EVOKED BY NORADRENALINE, INOSITOL TRISPHOSPHATE OR Ca2l IN GUINEA-PIG ISOLATED HEPATOCYTES
BY D. C. OGDEN*t, T. CAPIOD, J. W. WALKER: AND D. R. TRENTHAM From the Department of Pharmacology, King's College London, Strand, London WC2R 2LS, and the Division of Physical Biochemistry, National Institute for Medical Research, London NW7 1AA
(Received 8 August 1989) SUMMARY
1. Guinea-pig hepatocytes respond to noradrenaline (NA, 5-10 /tM) with a large membrane conductance increase to K+ and Cl-. The response has a long initial delay (range 2-30 s). Following the delay, the K+ conductance (studied in Cl--free solutions) rises quickly to a peak in 1-2 s and is maintained in the continued presence of NA, though often with superimposed oscillations of conductance. The roles of intracellular Ca2+ and D-myo-inositol 1,4,5-trisphosphate (InsP3) in this complex response have been investigated by rapid photolytic release of intracellular Ca2+ (from Nitr5-Ca2+ buffers) or InsP3 from 'caged' InsP3. 2. A rapid increase of intracellular [Ca2+] produced an immediate membrane conductance increase which rose approximately exponentially to a new steady level, consistent with a direct activation of Ca2+-dependent ion channels. 3. Following a pulse of InsP3, conductance rose after a brief delay (range 70-1500 ms) which was shortest at high [InsP3] or if the initial cytosolic [Ca2+] had been raised above normal levels. The maximum conductance produced by InsP3 was similar in each cell to the peak recorded with NA and could be evoked by InsP3 concentrations of 0 5-1 atm. 4. The rates of rise of conductance increased with InsP3 concentration in the range 0-25-12-5 ,lM (range 10-90 %, rise times 90-1000 ms), indicating that InsP3-evoked Ca2+-efflux from stores increases with InsP3 concentration in this range. 5. Photochemically released InsP3 and Ca2+ activate at physiological concentrations the same membrane conductances as NA. The results indicate that the long initial delay in NA action occurs prior to or during generation of InsP3. The mechanism of the delay and the subsequent apparently all-or-none conductance increase during NA action are discussed in terms of the high co-operativity in InsP3 and Ca2+ actions and an additional positive feedback step. * Present address and address for correspondence: Division of Neurophysiology and Neuropharmacology, National Institute for Medical Research, The Ridgeway, London NW7 1AA. t Permanent address: INSERM U274, Bat. 443, Universite Paris Sud, 91405, Orsay Cedex, France. I Present address: Department of Physiology, University of Wisconsin, 1300 University Avenue, Madison, WI 53706, USA. MS 7867
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6. Evidence was found of a negative interaction between [Ca2+] and InsP3-evoked Ca2' release. The time course of the recovery of InsP3-evoked Ca2' release following a rise of cytosolic [Ca2+] suggests that this interaction may be important in regulating oscillatory responses of [Ca2+] during hormonal stimulation of guinea-pig hepatocytes. INTRODUCTION
Stimulation of liver cells from guinea-pig and many other mammalian species with noradrenaline (NA) causes elevation of the cytosolic free Ca2+ concentration, leading to activation of glycogen breakdown and efflux of glucose, Ca2+, K+ and Cl- from the cell (Haylett & Jenkinson, 1972; Haylett, 1976; Burgess, Claret & Jenkinson, 1981; Egashira, 1980; Field & Jenkinson, 1987). It has been shown that NA acts through ac-adrenoceptors to activate phospholipase C. This generates D-myo-inositol 1,4,5trisphosphate (InsP3) within the cell which in turn mediates the rise of Ca2+ concentration by release from intracellular stores (Burgess, Godfrey, McKinney, Berridge, Irvine & Putney, 1984a; Joseph, Thomas, Williams, Irvine & Williamson, 1984). It is thought that this source of Ca2+ can raise free cytosolic [Ca2+] to a level sufficient to activate phosphorylase b kinase, the Ca2+-ATPase pump and Ca2+activated ion channels in the plasma membrane (for a review see Exton, 1988). Recent electrophysiological studies have characterized both K+ and Cl- conductances in the surface membrane of guinea-pig and rabbit hepatocytes (Field & Jenkinson, 1987; Capiod & Ogden, 1989a) that are activated by internal application of either buffered Ca2+ of InsP3 (Capiod, Field, Ogden & Sandford, 1987). The Ca2+activated K+ conductance, when studied in isolation in solutions containing impermeant anions, is blocked by the bee venom toxin apamin (Banks, Brown, Burgess, Burnstock, Claret, Cocks & Jenkinson, 1979) and by (+)-tubocurarine (Cook & Haylett, 1985). The properties of single ion channels underlying this conductance have been examined in isolated membrane patches: the unitary conductance is 6 pS and channel activation depends steeply on [Ca2+] at the inside surface in the range 0-3-10/JM, reaching a maximum open probability of 85 % without indication of densensitization to Ca2+ (Capiod & Ogden, 1989b). The basic scheme that emerges from studies of the activation of the K+ conductance is that NA acts primarily to stimulate intracellular InsP3 production and that InsP3 influences the membrane conductance by mobilizing internal Ca2+. Several features of the membrane conductance increase evoked by NA indicate that it is regulated by complex intracellular mechanisms. After NA binding, there is a delay of several seconds before a change in membrane conductance occurs. The conductance then increases quickly to a maximum level in a manner suggestive of underlying processes involving a high degree of co-operativity or positive feedback. During prolonged application of NA the conductance often shows marked oscillations with a period of 10-20 s (Field & Jenkinson, 1987), presumably reflecting oscillations of intracellular [Ca2+] similar to those observed directly in rat hepatocytes (Woods, Cuthbertson & Cobbold, 1987). In order to investigate the intracellular mechanisms responsible for these complex phenomena, whole-cell patch clamp recording (Hamill, Marty, Neher, Sakmann & Sigworth, 1981) has been used to monitor membrane K+ and Cl- conductances
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(Capiod & Ogden, 1989a). Using this method, agents can be applied intracellularly by perfusion from the patch pipette. However, measurements of the time course of events are hampered by the slow rate of access of active agents by diffusion from the patch pipette (see Pusch & Neher, 1988). Moreover, it is possible that agents introduced into the cell by this means may be rapidly metabolized, so raising questions about the concentration at the site of action and also the identity of the active agent. To circumvent these limitations we have used inactive 'caged' precursors of InsP3 (caged InsP3; Walker, Somlyo, Goldman, Somlyo & Trentham, 1987; Walker, Feeney & Trentham, 1989) and Ca21 (Nitr5: Gurney, Tsien & Lester, 1987; Adams, Kao, Grynkiewicz, Minta & Tsien, 1988) that can be converted to active InsP3 or liberate free Ca2' respectively by pulse illumination with near UV light. The principal aim of these experiments was to probe the mechanism of hormone action by establishing the time course and concentration dependence of both InsP3 and Ca2+ actions in activating the K+ conductance of guinea-pig hepatocytes. A preliminary account of this work has been given (Capiod, Ogden, Trentham & Walker, 1988). METHODS
Guinea-pig hepatocytes were isolated by perfusion with collagenase followed by mechanical dispersal. Cells in suspension were plated onto 35 mm Falcon dishes and whole-cell voltage clamp recordings made after 2-8 h (Capiod & Ogden, 1989a). For experiments in the presence of Cl- the external solution contained (mM): NaCl, 145; KCl, 5-6; CaCl2, 1-8; MgSO4, 0-8; HEPES, 8; pH 7-3; the internal solution contained (mM): KCl, 153; Na-ATP, 1; MgSO4, 3; Na-GTP, 0 05; HEPES, 8; pH 7-3. External Cl--free solutions contained (mM): sodium gluconate, 145; potassium gluconate, 5-6; CaSO4, 5; MgSO4, 0-8; HEPES, 8; pH 7-3; internal Cl--free solutions contained (mM): potassium gluconate, 153; Na-ATP 1: MgSO4, 3; Na-GTP, 0 05; HEPES, 8; pH 7-3. Propranolol (2 /SM) was present throughout to block fl-adrenoceptors. Experiments were made at room temperature (ca 25 °C). Noradrenaline and other agents were applied externally by pressure ejection from a pipette near the cell. Data were recorded on FM tape and replayed on a fibre-optic recorder. Delays were measured as the time following the flash or NA application before the trace deviated noticeably (about 5-10 pA) from the baseline. Current records were differentiated electronically to display rates of l7ise and facilitate measurement of delays if the baseline was unsteady. Caged InsP3 (the P-4 1-(2-nitrophenyl)ethyl ester of InsP3), caged InsP2 (the P-1 ester of D-myoinositol 1,4-bisphosphate) (Walker et al. 1989) or Nitr5 (complexed with Ca2+) were introduced into the cell via the patch pipette, allowing 2-3 min for equilibration. Photolysis was produced by a 1 ms pulse from a xenon arc flashlamp (Rapp & Guth, 1988) focused to a spot of about 2 x 3 mm around the cell from an incident angle of 38 deg to the horizontal; this arrangement resulted in minimal (about 8 %) energy loss due to reflection from the fluid surface. Light was bandpassfiltered with a UG 11 glass between 300-350 nm. Set to maximum power, the energy output was 100-120 mJ in this band. This produced 58% (± 6% S.D.) conversion of caged ATP (the P3-1(2-nitrophenyl) ethyl ester of ATP; Kaplan, Forbush & Hoffman, 1978) to ATP at full power, or less at reduced power. The efficiencies of caged ATP and caged InsP3 photolysis have been shown to be similar under the experimental conditions used (Walker et al. 1989) and photolysis of a droplet of 10 1cl 0 4 mM-caged ATP after each experiment followed by HPLC analysis was used to assess to proportion of caged InsP3 photolysed. Making allowance for reflection, photolysis of intracellular caged InsP3 was 50 % (S.D. + 6 %) at full power. The rate of photolysis of caged InsP3 is estimated as 200 s-5 in these conditions, corresponding to a half-time for InsP3 formation of about 3 ms (Walker et al, 1989). Photolysis of Nitr5 releases Ca2+ ions within the 1 ms duration of the light pulse (Adams et al. 1988). The experiments described here indicate that the biological properties of caged InsP3 are as previously found in vascular smooth muscles (Walker et al. 1987, 1989). Caged InsP3 esterified on
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the 4 or 5 phosphates shows no InsP3-like Ca2+-mobilizing activity at concentrations up to 50 /M. In addition Walker et al. (1989) found that neither isomer is metabolized by or interferes with the hydrolysis of InsP3 by InsP3 5-phosphatase; the 5-isomer has some inhibitory activity towards InsP3 3-kinase. The experiments reported here were made mainly with the 4-isomer. Control photolysis experiments were made with caged InsP2 at a concentration of 37 /LM to test the toxicity of the by-product 2-nitrosoacetophenone. These showed that photolysis to liberate 18 ,#M-InsP2 and nitrosoacetophenone produced no response and did not prevent subsequent activation by NA. Noradrenaline and propranolol were obtained from Sigma (St Louis, MO, USA) and Nitr5 from Calbiochem (La Jolla, CA, USA). Other reagents were from Sigma or BDH. RESULTS
The increase in membrane conductance evoked by NA in voltage clamped guineapig hepatocytes is illustrated in Fig. 1. In Fig. 1A conditions were chosen to show both K+ and Cl- conductances. The responses to 5 /tM-NA showed a delay of 8 s before the conductance increased, first to K+ seen as a brief outward K+ current (upward deflection) followed by a large conductance increase to Cl- with net inward current (downwards deflection) and finally a net outward current as NA was removed. The properties of these conductances have been described in detail elsewhere (Field & Jenkinson, 1987; Capiod & Ogden, 1989a). Figure lB shows the response under similar conditions to a pulse of InsP3 photolytically released inside the cell at the time indicated by the arrow. After a short delay (100 ms), an inward current due to the increase in Cl- conductance was seen. In contrast to the activation by NA, temporal separation of K+ and Cl- conductances was rarely seen following a pulse ofInsP3 (cf. responses to NA; Capiod & Ogden 1989a). In the record shown in Fig. 1 C the K+ conductance alone was activated by NA, generating an outward current. The peak current amplitudes and rise times under these conditions varied little between responses, whereas the delay between NA application and the conductance increase ranges from 2-5 s to more than 30 s, with a mean value of 10 s in the present experiments (see Table 1, row 1). Delays were similar in Cl--containing or Cl--free solution. A comparison between the K+ conductance evoked by NA and InsP3 may be made in Fig. 1D, which shows on the same scale as Fig. 1 C current evoked in the same cell by photolysis of 2 saM-caged InsP3, releasing a pulse of1 /IM-InsP3 in the cytosol. The amplitudes of the responses are similar, close to the maximum seen in these cells, and most likely reflect maximal activation of the K+ conductance by Ca2+ in each case. The responses differ first in the rise times, that for NA being longer than that for photolytically released InsP3. Secondly, the delay before the current rises are strikingly different, 5 s for NA compared to 300 ms for InsP3. The responses of the Cl- and K+ conductances to rapid changes of free[Ca2+] were examined with release from photolysed Nitr5 and are illustrated in Fig. 2. For cells inCl--containing solutions (Fig. 2A) the calculated free [Ca2+] before photolysis was 015 /tM, close to the resting [Ca2+] in hepatocytes (Burgess, McKinney, Fabiato, Leslie & Putney, 1983; Berthon, Binet, Mauger & Claret, 1984). Three consecutive pulses of UV light produced inward Cl- currents which began immediately after the flash, approaching steady levels with half-times of 10-100ms. The responses were faster with each successive flash as the freeLCa2+] increased. Responses to the activation of the K+ conductance alone by Ca2+ are shown in Fig.
PHOTOLYTIC RELEASE OF InsP3 AND Ca2+ IN LIVER
589
5 gM-NA
A
m
500 pA
L10 s
12*5 uM-insP3
B
500 pA 1 s
10 gM-NA
I
200 pAL 2s
C
200 pA L 2s
D
Fig. 1. Membrane current evoked by noradrenaline (NA) or by photolysis of caged InsP3 in isolated guinea-pig hepatocytes. A, 5 1uM-NA was applied during the bar in Cl--containing solution. Cell voltage clamped at a membrane potential of -20 mV. Pulses of 10 mV were applied to monitor the membrane conductance. After a delay of 8 s an initial brief outward K+ current (upward deflection, EK = -85 mV) was followed by a large inward Cl- current (Ec1 = 0 mV) which declined to leave a K+ current and finally decayed to the baseline. B, conductance increase at -20 mV in Cl--containing solutions evoked by pulse photolysis of caged InsP3 at the point indicated by the arrow in B and D. 12-5 ,#M-InsP3 was released and produced a net inward current after a delay of 100 ms. C, conductance increase to K+ alone evoked by 10 ,uM-NA (applied during the bar), recorded with Cl--free solutions at 0 mV. Delay was 5 s, peak current 330 pA. D, conductance increase to K+ following photolytic release of 1 #uM-InsP3, recorded at 0 mV in the same cell as C. Delay was 300 ms and peak current 350 pA. Time of flash is indicated by the arrow.
2B. These were obtained by photolysis of 5 mM-Nitr5 equilibrated with 2-5, 3-25 and 3.75 mM-Ca2+. In each case outward K+-current rose approximately exponentially to approach a new level, and this was frequently followed by a further much slower rise of conductance. Half-times for the rapid phase of the relaxations were 250 ms at
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TABLE 1. Amplitude, delay and rise time (10-90 %) for K+ current increase evoked at 0 mV by; row 1: external noradrenaline (NA, 5 or 10, M) and rows 2-7: photolytic release of InsP3 from internal caged InsP3. Data are means+s.E.M. Delay Amplitude Rise time (pA) (s) (s) 10-0 NA 1P0+0-2 460+75 (range 2 5-30) [InsP3]
(/#M) 05 1.0 2-5 5 12-5 25
233+ 129 280+99 445+68 570+100 483+92 621+73
A -20 mV Cl- solutions Initial [Ca2+ = 0-15 ,UM
1
0-64+0-12 037+0*10 0-38+0-10 0-37+0-13 0-18+0-03 0-21+0'05
0419+0-06
B 0 mV Cl1-free solutions Initial [Ca2+l = 0.15 PM Final [Ca2+1 = 0_5 pM
t
J 200 pA is Initial [Ca2+] = 0.3 pM Final [Ca2 1 = 1.4 uM
J 200 pA
I
1P14+0-35 0-37+0-10 0-41+0412 037+009 0-16+0-02
1 s
I
-J 200 pA 0.5 s
Initial [Ca2+1 = 0.6 pM Final [Ca2J1 = 1.9 MM
J 200 pA 0.5 s
Fig. 2. Conductance increase following photolysis of Ca2+-loaded Nitr5. A, Cl--containing solutions at a membrane potential of -20 mV. 5 mM-Nitr5 with 2-5 mM-Ca2 , giving an initial [Ca2+] of 0 15 ,UM and a final [Ca2+] of 0 54 fIM after 1 pulse. Subsequent flashes to same cell, shown in the lower traces, produced further increases of [Ca2+] and conductance. Time of flashes indicated by arrows. B, K+ currents recorded in Cl--free solutions at 0 mV. 5 mM-Nitr5 loaded with 2-5 mM-Ca2+ (upper trace), 3-25 mM-Ca2+ (middle trace) and 3.75 mM-Ca2+ (lower trace). Initial and final [Ca2+] were calculated from the equation given by Gurney et al. (1987) and are shown above each trace.
PHOTOLYTIC RELEASE OF InsP3 AND Ca2+ IN LIVER
591
0-5 /M final free [Ca2"], 1 10 ms at 1-4/tM and 20 ms at 1P9 /gm. These time courses most probably reflect the rate of channel activation by free Ca2 . Several control experiments were made. The K+ conductance increase evoked by NA, internally perfused Ca21 or InsP3, is blocked by apamin or (+ )-tubocurarine (Cook & Haylett, 1985; Capiod et al. 1987). The K+ conductance produced by photolytic release of Ca2+ or by InsP3 (12 /tM) was rapidly blocked by 10 /M (+ )tubocurarine applied during the response (data not shown). The response to photoreleased InsP3 (1-2 aM) could be completely blocked by including bis(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid (BAPTA) in the internal solution in the patch pipette; responses were blocked by 0 75 or 1P0 ,M-BAPTA but not by 05 ,aM (four trials at each concentration). Removing extracellular Ca2+ had little effect on the InsP3 responses after 1-2 min exposure, consistent with an internal origin of the [Ca2"] increase. These results show that InsP3 acts on the surface membrane conductance mainly by intracellular Ca2+ release and not by a direct action on the membrane K+ conductance channels. Figure 3 shows K+ currents following release of InsP3 by pulse photolysis of 5 or 10/M caged InSp3in three cells, represented by traces in A, B and C. The upper trace in each panel represents the K+ current and the lower trace the current differentiated with respect to time to emphasize rates of change. In order to make comparisons in the same cell, the lamp output powder was reduced to produce photolysis which could be set in the range 5-14 %, leaving sufficient caged InsP3 unphotolysed for consecutive responses to different concentrations of InsP3 in the same cell. The responses were separated by 1-2 min. In a number of experiments twin closely spaced pulses of InsP3 were used to test recovery of InsP3 sensitivity, for reasons described below. Considering first the onset of the responses shown in Fig. 3, both the peak amplitude and the kinetics of the K+ current were found to depend on the InsP3 concentration released. In Fig. 3A 0 25 /tM-InsP3 produced a small outward current of 30 pA after a delay of 1 4 s following photolysis. Increasing the concentration released to 0 7 aM gave a current of maximal amplitude, 400 pA in this cell, with a delay of 300 ms and peak rate of rise of 1000 pA s-'. In Fig. 3B the currents evoked in another cell by 0 35 and 05 /tm-InsP3 were both of maximal amplitude (720 pA) but the delay was longer (700 ms) and the rise slower (800 pA s-') at 0-35 /IM than at 0 5 /M (delay 350 ms and rise 1340 pA s-', respectively). Similar effects of increasing the concentration of InsP3 from 0 5 to 1 UM, decreasing the delay and rise time at the same current amplitude, are seen with the cell of Fig. 3 C. The rise of the K+ current following InsP3 release was sigmoidal after the initial delay, and the duration of responses was from about 3 to 20 s. In prolonged recordings the delay, the time-topeak and the duration of the responses usually increased with four or five consecutive similar pulses by a factor of 2-5 over 10 min. When comparing the InsP3 responses of different cells a degree of variability from cell to cell was encountered in the parameters described in connection with Fig. 3. The K+ conductances, represented by the current at 0 mV membrane potential, are summarized for the first responses of a number of cells in Table 1, rows 2-7, for different concentrations of photolytically released InsP3. The maximum current amplitude of these cells, averaging about 530 pA, was obtained consistently with InsP3 concentrations of 2 /tM or more, indicating that this concentration released
_~ ~ ~ ~I
592
D. C. OGDEN AND OTHERS
Ca2+ from the stores at a rate sufficient to achieve a free [Ca2+] adjacent to the membrane which fully activated the K+ conductance. Data with buffered Ca2+ has shown that maximal steady-state activation of the K+ conductance occurs with concentrations of 1-2 /M-free Ca21 (Capiod & Ogden, 1989b). The variability of maximum amplitude from cell to cell is largely due to differences in surface area A 0.25 pM-IfnSP3
0.7 pM-InsP3
J 200 pA
is B
0.35 M-IlnSP3
C 0.5 M-IlnSP3
0-5 M-IlnSP3
1 uM-InsP3
200 pA
2s
Fig. 3. K+ currents evoked by photolysis of caged InsP3 in Cl--free solutions at a membrane potential of 0 mV. Data are from three cells shown in panels A, B and C, loaded with 5 /M (A and B) or 10/tM (C) caged InsP3. Each section shows two consecutive responses separated by 1-2 min evoked by the InsP3 concentrations given above each trace, released at the arrows. In A (second response) and C twin pulses separated by 4-5 s were applied. The upper trace in each record is K+ current and the lower trace the differentiated current to show the rate of rise. Vertical calibration bar corresponds to 200 pA for the upper trace and 3200 pA s-' for the differentiated trace. Time calibration 1 sforA andB, 2s for C.
(reflected by the range of cell capacitances recorded of 26-70 pF). At lower InsP3 concentrations, 0-25-10 tM, a number of responses were of submaximal amplitude and in this concentration range current amplitudes varied widely from cell to cell, from zero to maximal activation of the K+ conductance. The cell-to-cell variability observed may arise in several processes, including the photolysis reaction (S.D. for photolysis of caged ATP 6%, see Methods), the rates of ihnsP3 metabolism, Ca2+
593 PHOTOLYTIC RELEASE OF InsP3 AND Ca2+ IN LIVER release and reuptake by stores, Ca2+ buffering and the activation of K+ conductance by Ca2+. The delay between the light flash and initiation of the conductance change decreased as the InsP3 concentration was increased. The longest delay observed was about 1-5 s at 0-25 ,uM-InsP3 and the shortest 70 ms for a response to 25 ftM-InsP3. A 2 piM-InsP3
t|
1 ,UM) to activate the K+ conductance in a nearly all-or-none manner. However, the InsP3-evoked Ca2+ efflux from the stores, as monitored by the peak rate of rise of conductance, showed a more graded dependence on the InsP3 concentration. These results are consistent with a report by Meyer, Holowka & rate of Ca2+ flux from permeabilized rat basophilic leukaemia cells showed a graded yet steep dependence on [InsP3]. Taken together with the narrow Ca2+ concentration range required for activation of the K+ conductance (Capiod & Ogden, 1989b), co-operativity in the InsP3-evoked Ca2+ release may be sufficient to account for the sigmoidal rise of the conductance and the almost all-or-none conductance increase seen with InsP3 and NA. However, the occurrence of an additional autocatalytic step within the reaction sequence leading to Ca2+ release cannot be excluded on the basis of the experiments reported here.
Stryer (1988) in which the
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599
Evidence of a feedback mechanism has been described in the form of conductance oscillations in response to prolonged stimulation by NA (Field & Jenkinson, 1987), high intracellular concentrations of InsP3 (Capiod et al. 1987) or bile acids applied externally (Capiod et al. 1989). Oscillatory conductance changes were seen in some responses initiated by photolysis of caged InsP3 (e.g. in the declining phases of Figs 1D and 6). These occurred particularly with high concentrations of InsP3, had a similar time course to those seen with NA but were often of low amplitude. Large amplitude oscillations with a period of 10-20 s were seen in the present experiments with D-myo-inositol 2,4,5-trisphosphate applied directly from the patch pipette (see Fig. 7). As suggested previously (Capiod et al. 1987) the presence of oscillations with high (and presumably maximal) concentrations of InsP3, and with the bile acids (Capiod et al. 1989) which elevate [Ca21] without InsP3 production (Combettes et al. 1988), suggests that in the guinea-pig liver oscillations of cytosolic [Ca2+] in response to hormones can be generated by mechanisms which do not depend on fluctuations of [lInsP3]. Similar conclusions have been reached for parotid and pancreatic acinar cells (Gray, 1988; Wakui, Potter & Petersen, 1989) and Xenopus oocytes (Berridge, 1988). A possible clue to the mechanism is in the observations that InsP3-evoked Ca21 release is considerably reduced shortly after a conditioning response and recovers with a time course of half-time 10 s, similar to the period of oscillations observed with NA or intracellular perfusion with InsP3. Depression of the InsP3 response following Ca2+ release by TLCS indicates that the effect is not InsP3-specific and therefore not analogous to, for example, desensitization of nicotinic receptors by acetylcholine. It is unlikely that the reduced Ca2' release is due simply to Ca2+ depletion of the stores, because it is seen following small responses and when the [Ca2+] presumably has been restored back to the resting level (or near it) by reuptake into stores (e.g. Fig. 3C, first trace). The most likely explanation is a reduced sensitivity of the Ca2+ release mechanism to InsP3, and possibly other agents, induced by the raised [Ca2+], and which recovers with a slow time course even when the [Ca2+] has been restored to resting levels (see Fig. 5A). This explanation is supported by the reduced InsP3 binding observed in brain membranes with [Ca2+] in the range 01-1 JIM (Worley, Baraban, Supattapone, Wilson & Snyder, 1987) and the inhibition by injected Ca2+ of the InsP3-mediated photoresponse in Limulus photoreceptors (Payne, Walz, Levey & Fein, 1988). Studies of Ca2+ oscillations in fibroblasts by Harootunian, Kao & Tsien (1988) show that a pulse of InsP3 during the period between Ca2+ spikes produces a phase shift of the subsequent oscillations as if the spikes were controlled by InsP3 fluctuations. The stimulation by Ca2+ of InsP3 production by phospholipase C has been proposed by Meyer & Stryer (1988) as a mechanism of positive feedback which could produce this effect. A difference in the periodicity of oscillations in different cells and conditions may be noted; in the exocrine cells and in the present experiments the period was 10-20 s, whereas the fibroblasts, endothelial cells and rat hepatocytes stimulated with low concentrations of hormones have much longer periods of 60-90 s between Ca2+ spikes (for reviews see Berridge & Gallione, 1988; Rink & Jacob, 1989). The reduced sensitivity of the release mechanism to InsP3 following a Ca2+ pulse, observed in the experiments reported here, may also be involved in the termination of Ca2+ spikes in slowly oscillating systems.
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In summary, the experimental approach used here has provided time-resolved measurements of the conductance increase, normally elicited by extracellular NA, in response to known intracellular concentrations of free Ca2+ and InsP3. The data are consistent with a model in which InsP3 activates K+ and Cl- conductances in the hepatocyte plasma membrane by releasing Ca21 from intracellular stores rapidly and with a high degree of co-operativity. Calcium ions released from the stores activate the membrane conductance channels directly. The delay in the response to NA and other hormones is associated with a step prior to the production of InsP3. Photolysis of caged InsP3 does not produce a sustained conductance increase, probably because InsP3 produced is readily metabolized, free Ca2' released is rapidly removed from the cytosol and the Ca2+ release mechanism quickly becomes refractory after stimulation by InsP3. The latter mechanism and the role of external Ca21 in the response to NA are currently being investigated. We thank Dr R. F. Irvine for kindly providing inositol phosphates, Professor D. H. Jenkinson for discussion and allowing use of facilities for cell production and Dr M. Claret for discussion. Meera Prasad and R. J. Congram gave excellent technical help. The work was supported by the MRC, an MRC-INSERM exchange Fellowship and by NIH Grant 15835 to the Pennsylvania Muscle Institute. REFERENCES
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