Release in Single Smooth Muscle Cells of Guinea-Pig Small Intestine A

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MS 2932, pp. 97-109 Journal of Physiology (1994), 481.1 97 Ca2" inhibition of inositol trisphosphate-induced Ca2" release in single smooth muscle cells of guinea-pig small intestine A. V. Zholos*, S. Komorit, H. Ohashit and T. B. Bolton* *Department ofPharmacology and Clinical Pharmacology, St George 's Hospital Medical School, London SW17 ORE, UK and t University Laboratory of Pharmacology, Department of Veterinary Medicine, Gifu University, Gifu 501-11, Japan

1. Single smooth muscle cells from the longitudinal muscle layer of guinea-pig small intestine were voltage clamped using patch pipettes in the whole-cell mode. 2. When D- myo-inositol 1,4,5-trisphosphate (InsP3) was released at intervals, by photolysis of 'caged' InsP3 within the cell, increases in [Ca2+]i in many cells, as judged from Ca2+_ activated K+-current, were all-or-none; release of InsP3 before a critical interval had elapsed, which was quite stable for an individual cell, resulted in no response. After Ca2+-induced Ca2+ release had been evoked by depolarization, the InsP3 response was inhibited. Oscillations in [Ca2+]i evoked by muscarinic receptor activation were unaffected by Ruthenium Red; during these oscillations exogenous InsP3 was not effective close to, or shortly after, peak [Ca2+]i but was effective at other times. 3. Reproducible release of Ca2+ and elevation of [Ca2+]i could be produced by brief (up to 0 5 s) pressure applications of 10 mM caffeine at intervals of 10 s or greater but caffeine itself rarely evoked oscillations in [Ca2+]i. Responses to flash release of InsP3 were reduced after caffeine-induced responses and recovery of caffeine-induced Ca2+ release was faster than recovery ofInsP3-induced Ca2+ release. 4. The results support the idea that InsP3-induced Ca2+-store release can be inhibited by a certain level of [Ca2+]i at a time when Ca2+ stores have refilled and can be released by caffeine; they also support the suggestion that during oscillations of [Ca2+] evoked by muscarinic receptor activation, Ca2+ inhibition of InsP3-induced Ca2+ release at some critical level of [Ca2+]i allows Ca2+ stores to refill and leads to a fall in [Ca2+]i so contributing to the oscillations which are observed.

Many different cells often exhibit more or less regular 1990). On the other hand, Ca2+-dependent inhibition of the spike-like increases in cytosolic Ca2+ ([Ca2+]i) which occur InsP3-gated Ca2+-release channel (InsP3 receptor) (Worley, as a result of Ca2+ release from the intracellular stores both Baraban, Supattapone, Wilson & Snyder, 1987; Joseph, spontaneously and particularly during receptor activation Rice & Williamson, 1989; Berridge & Potter, 1990; Jino, and increased production of the Ca2+-releasing messenger 1990; Parker & Ivorra, 1990; Zhao & Muallem, 1990; D- myo-inositol 1,4,5-trisphosphate (InsP3). Several Bezprozvanny, Watras & Ehrlich, 1991; Finch, Turner & mechanisms have been proposed so far to explain this Goldin, 1991; Fohr, Ahnert-Hilger, Stecher, Scott & phenomenon; one of the suggested models is based on the Gratzl, 1991; Keizer & De Young, 1992; Zhang & Muallem, idea that InsP3 formation is itself oscillatory. Some other 1992; Levy & Payne, 1993) can also play an important role models assume a more steady level of InsP3 during as a negative feedback control in giving rise to the Ca2+ phospholipase C activation and different types of positive oscillations. and/or negative feedback control of some part of the Ca2+- Recently, oscillations of receptor-operated cationic release process to explain the [Ca2+]i oscillations. Positive current were observed in single smooth muscle cells feedback mechanisms include potentiation by Ca2+ of its (Desilets, Driska & Baumgarten, 1989; Komori, Kawai, own release from the stores, so-called Ca2+-induced Ca2+ Takewaki & Ohashi, 1992; Komori, Kawai, Pacaud, release (CICR) which occurs via a Ca2+-gated channel Ohashi & Bolton, 1993). They are associated with [Ca2+]i (ryanodine receptor). In fact caffeine, a potentiator of oscillations (Pacaud & Bolton, 1991). There is evidence that CICR, or Ca2+ infusion has been demonstrated to set up activation of a G-protein results in opening of cationic [Ca2+]i 'spikes' in some cells (Marty & Tan, 1989; Malgaroli, channels and that this is further potentiated by increases Fesce & Meldolesi, 1990; Wakui, Osipchuk & Petersen, in [Ca2+]i. However, the mechanism of [Ca2+]i oscillations 98 A. V Zholos, S. Komori, H. Ohashi and T. B. Bolton J. Physiol. 481.1 in smooth muscles in response to receptor activation is not Data were stored on a disk for later analysis and illustration understood. using the pCLAMP program. Leakage compensation was not In the present study on single isolated intestinal smooth used. Values are given as means + S.E.M. muscle cells, we used the patch-clamp technique to record Solutions and drugs ionic currents through membrane channels dependent on PSS contained (mM): NaCl, 120; KCl, 6; CaCl2, 25; MgC12, 1M2; intracellular Ca2+ (Ca2+-dependent K+ channels and glucose, 12; Hepes, 10; pH adjusted to 7-35 with NaOH. Ca2+- muscarinic receptor-activated cationic channels) to free PSS was prepared by replacing CaCl2 with MgCl2. The measure [Ca2+]i oscillations indirectly and the flash pipette solution had the following composition (mm): KCl, photolysis technique to liberate InsP3 from its inactive 130; MgCl2, 1; Na2ATP, 1; creatine, 5; glucose, 20; EGTA, precursor at desired moments. We aimed to see (i) whether 0 05; caged InsP3 (when added), 0 03; Hepes, 10; pH adjusted to 7-35 with NaOH. In some experiments CsCl-based solution CICR plays a role as a positive feedback mechanism and was used (KCl replaced by CsCl) to block K+ currents. In this (ii) whether there are indications of a negative feedback case KCl in the external solution was replaced with NaCl. control during [Ca2+]i oscillations in these cells. The chemicals and drugs used were: collagenase (Type IA), Our results suggest that CICR via ryanodine (caffeine) soybean trypsin inhibitor (Type II-S), bovine serum albumin, receptors is not involved, at least as a primary adenosine-5'-triphosphate (ATP, disodium salt), creatine, N-2- mechanism, in [Ca2+]i oscillations during receptor hydroxyethylpiperazine-N'-2-ethanesulphonic acid (Hepes), stimulation in intestinal smooth muscle cells. However, ethyleneglycol-bis-(f-aminoethylether)-N,N,N,N'-tetraacetic acid (EGTA), carbamylcholine chloride (carbachol), caffeine we have found that when Ca2+ was released from the store, and Ruthenium Red (all from Sigma Chemical Co., Poole, regardless of what mechanism has been involved (Ca2+_ or Dorset, UK). D-myo-inositol-1,4,5-triphosphate,P4(5)-1-(2-nitro- caffeine-induced Ca2+ release or InsP3-induced Ca2+ release phenyl)ethyl ester, sodium salt (caged InsP3) was from as a result of either carbachol (CCh) or flash application in Calbiochem (Beeston, Nottingham, UK). For fast exchange of caged InsP3-loaded cells), this resulted in significant the external solution we used a system similar to that desensitization or inactivation of cell response to described previously (Zholos, Baidan & Shuba, 1991) or, subsequent InsP3. This inactivation lasted longer than was alternatively, a blunt micropipette filled with test solution was positioned 50-100 ,sm away from the cell and this necessary for Ca2+-store refilling so it did not arise due to solution was superfused over the cell using pressure pulses depletion of the Ca2+ stores. This mechanism may, at least (200 mmHg) of the required duration applied to the pipette in part, account for [Ca2+]i oscillations in stimulated interior. smooth muscle cells. The flash photolysis technique The flash photolysis technique was similar to that used METHODS previously (Komori & Bolton, 1991). Light emitted from a Cell preparation xenon arc lamp as a flash of approximately 1 ms duration was filtered (300-380 nm) and focused for photolysis of caged Male adult guinea-pigs were killed by decapitation after compounds. The light intensity could be regulated by dislocation of the neck. The longitudinal layer of the ileum adjusting the capacitor charge voltage (CV). In control was dissected and placed in physiological salt solution (PSS). experiments, the direct inhibitory effect of light intensity on It was cut into small pieces which were transferred to Ca2+_ calcium current (ICa) was studied. In these experiments Cs+- and Mg2+-free PSS for several minutes. To bring about cell based solution was used for ICa separation (no caged dispersal, the pieces were incubated in this solution substances added). A sigmoidal relationship between ICa containing collagenase, soybean trypsin inhibitor and bovine amplitude and the CV was observed with a CV of 152 V serum albumin (all at 1 mg mnl-) at 36 °C for 25-35 min. Single producing 50% inhibition of Ica. At higher voltages some cells were obtained through agitation of pieces in enzyme-free increase in leakage current was also observed immediately solution until it became cloudy. The cell suspension was after flash application. However, at a CV of 50 V the effect on diluted using normal PSS and small aliquots were placed on Ica was barely detectable with only 13-5 + 2-7% (n= 7) coverslips and kept at 4 °C until use. inhibition at 100 V. Thus, we used CVs not exceeding 75 V. These also provided a lower coefficient of caged compound Membrane current recordings conversion which was beneficial in producing several Whole-cell membrane current was recorded at room reproducible increases in InsP3 concentration. The efficiency temperature (20-25 °C) using the standard patch-clamp of the light source used to produce photolysis of the caged technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). compound when repeated flashes were applied at intervals Borosilicate patch pipettes had resistances of 1-3 MQ. A List down to approximately 5 s was tested on microdrops EPC-7 voltage clamp amplifier was used (List Electronic, containing 'caged' ATP (adenosine-5'-triphosphate,P3-1-(2- Darmstadt, Germany). Voltage-clamp pulses were generated nitrophenyl)ethyl ester). No reduction in efficiency of and data were captured using a Labmaster DMA TL-1-125 photolysis was observed. We are grateful to Dr S. A. Prestwich (Axon Instruments, Foster City, CA, USA) interfaced to a (St George's Hospital Medical School, London) for measurement computer running the pCLAMP program (Axon Instruments). of ATP by HPLC. J. Physiol. 481.1 Ca2+ inhibits action of inositol trisphosphate 99

RESULTS generated outward currents evoked by the agonist usually Carbachol-induced oscillations of Ca2+_ decreased in amplitude with time even in the presence of activated K+ and cationic currents CCh (Fig. IA and B, see also Fig. 2A). In some cells, particularly in those challenged with a lower In smooth muscle cells of guinea-pig small intestine the concentration ofCCh (1 or 2 /IM), IkCh,O gradually increased muscarinic agonist carbachol (CCh) induced oscillations in in amplitude to reach a maximum and then either [Ca2+]i due to periodic Ca2+ release from the SR decreased or varied in amplitude in an irregular manner (sarcoplasmic reticulum; Komori et al. 1993). As Ca2+- (Fig. IC). The frequency of ICcCho varied from 0-02 to dependent K+ channels are numerous in the membrane of 0 3 Hz (n = 8) and, at a higher CCh concentration (100 ,UM), these cells, under appropriate conditions CCh was also it ranged between 0 3 and 0 6 Hz in another four cells expected to produce oscillations of outward Ca2+_activated studied. Independently of CCh concentration, the interval K+ current (IK(ca)). Single cells dialysed with K+-based between two successive peaks in 'cch,Owas prolonged with pipette solution were voltage clamped at a holding time. At the same time STOCs, if present, had low potential (Vh) of 0 mV, close to the reversal potential for amplitude and frequency (Fig. 1, see also Fig. 2A). In a the CCh-activated cationic current, Ihat (Benham, Bolton fraction of cells which exhibited ICch,o with intervals & Lang, 1985; Inoue, Kitamura & Kuriyama, 1987). CCh longer than 30s, STOC discharge was inhibited briefly (1-100 j/M) evoked a brief outward current only once, as after each peak and then gradually recovered until the described previously (Komori et al. 1992), or repeatedly, as next peak of ICch,Ooccurred (Fig. 1D). Fig. 1 shows. The duration of the brief outward currents It is well known that Ca2+-dependent K+ channels are (Icch,o) ranged from 0 5 to 5 s, which was longer than that also voltage dependent. They are deactivated at more of spontaneous transient outward currents (STOCs, negative membrane potentials at the same [Ca2+]i whereas 50-100 ms; Benham & Bolton, 1986). The repeatedly inward Ihat is expected to increase due to the increase in

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Figure 1. Oscillatory outward current (Icch O) responses to carbachol (CCh) Cells were dialysed with a KCl-based solution and voltage clamped at 0 mV. A, current oscillations evoked by application of 100,lM CCh and the effect of adding 10 mm caffeine. B-D, current oscillations evoked by 2 /SM CCh in three different cells. The oscillations waned with time (A and B), varied in amplitude (C) or continued with a relatively low discharge rate (D). The calibration bars in B and D apply to A and C, respectively. too A. V. Zholos, S. Komori, H. Ohashi and T. B. Bolton J. Phy8ioL. 481.1 the driving force. Figure 2A shows that when the Vh was InsP3 with those evoked by exogenous InsP3 (flash displaced from 0 to -40 mV, ICchO was replaced by ICat released). We also examined the effects of CICR activators which was repeatedly generated at about twice the and blockers. frequency at 0 mV due to the change in potential (Komori et al. 1993). Sometimes the inward current trace was InsP3-induced Ca2" release is all-or-none complex in shape or blunt at the top. An unusual type of Figure 3A shows that in K+-filled cells held at -20 mV activity was observed when CCh was applied at an where caged InsP3 was present at a concentration of 30 ,UM intermediate Vh of -20 mV: an outward component was in the pipette solution, STOCs were generated before superimposed on the inward component producing a W- flashes (triangles). A flash resulted in the generation of a shaped current response (Fig. 2B). With 10 or 20 nm large outward current suggesting substantial InsP3- EGTA and no added Ca2+ in the pipette, CCh at induced Ca2+ release from the stores. Flash-induced concentrations up to 100 uM evoked almost no current current was measured to be 91 + 14% of that evoked by (data not illustrated). ICch,o arose from a flat baseline caffeine in the same cells (n = 8) at +10 mV. Flash-induced during CCh application presumably because some critical outward current was followed by a short period during level of [Ca2+]i must be exceeded before channel opening which STOCs were inhibited (compare with Fig. IC and D). occurs; however, Ihat continuously changed (oscillated) It was notable that during this period two subsequent because cationic channel opening is sensitive to [Ca2+]i over flashes were ineffective except that they probably slightly a wide range and has a threshold below the resting level accelerated discharge of the STOCs. Flash number 4, (hence the W-shape at -20 mV). Thus Icat, unlike ICChO, however, again elicited a full response. This pattern was shows no threshold behaviour but seems to increase reproducible (Fig. 3A, upper trace). Later, flashes of the monotonically with [Ca2+]1. same intensity were applied in the same cell at a higher To test possible mechanisms involved in the oscillatory frequency as the lower trace of Fig. 3A shows. This activity of both ICCho and Icat, we first attempted to experimental protocol would be expected to produce more compare these currents during endogenous production of or less sustained elevation of [InsP3]i with some A Vh 0 mV

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Figure 2. Oscillatory outward (lCCh o) and inward current (Ihat) responses to CCh A, outward current oscillations induced by 1 ,UM CCh at a Vh of 0 mV and inward current oscillations at a Vh of -40 mV (indicated by horizontal bar). B, repetitive discharge of a W-shaped current induced by 100 /M CCh in a cell held at -20 mV consisting of an inward component with an outward component superimposed. The inset (b) shows a time- and voltage-expanded trace of section a as indicated by the dashed box. High-K+ pipette solution was used. J. Physiol. 481.1 Ca2" inhibits action of inositol trisphosphate 101

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Figure 3. Membrane current responses to flash-evoked A intracellular application of InsP3 in a cell loaded with * * * 30 EM caged InsP3 (high-K+ pipette solution) A, flashes (A) were applied with long (upper trace) or short (lower trace) intervals between them in the same cell. Upper and lower traces are continuous. Note that a transient period of STOC inhibition followed each flash 300 pA and preceded the moment when the cell could again respond to a flash. B, in another cell studied with a similar protocol, reduced responses to InsP3 could be 50 s evoked before full recovery of the response occurred. In AAA AA AAAAAA A AA AA AA*AA both experiments flashes at a CV of40 V were applied at a Vh of -20 mV. Several responses in A and B were truncated by the recorder (asterisks).

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A 130mM K+

0-2 nA Figure 4. Membrane current responses to flash- released intracellular InsP3 at negative membrane 5 s potentials A, effect of high-K+ (130 mM) external solution B application, as shown by the bar, to a cell held at -50 mV. STOCs reversed in accordance with the shift of , . . . . . the K+ equilibrium potential to 0 mV. B, when flashes w.l-1ifrr were applied (0 05 Hz; V) at a Vhof-50 mV in the high- l l K+ external solution to evoke inward K+ current the pattern of responses was similar to that shown in Fig. 3. C, in the same cell 7 min later the inward Ca2+-activated l K+ current increased, suggesting an increase in [Ca2+]I, 2 nA r and in parallel the responses to flashes (02 Hz; CV, 40 V; 50 V) were significantly reduced and became graded in size. Vertical calibration in B applies also to C.

v V V Tv

1 0 s v 102 A. V. Zholos, S. Komori, H. Ohashi and T B. Bolton J. Physiol. 481.1 increments superimposed depending on how rapidly InsP3 elapsing after the previous response. These small outward was metabolized. Despite this, all of the responses current responses often had a considerable and very observed seemed to be of the all-or-none type, recovery variable latency following the flash (Fig. 3B), which may after a response taking up to 1 min or more. Thus, an indicate that they arise as a result of some spontaneous elevated level of InsP3 inside the cell obviously did not release event triggered by raised InsP3 levels. prevent the Ca2+ stores from refilling, so that it was To exclude the possibility that a less negative Vh of possible for a [Ca2+]i 'spike' to occur. In fact, the period -20 mV could affect, for example, the refilling of the Ca2+ between releases became even shorter when multiple stores, similar experiments were also performed at a Vh of flashes were applied (compare upper and lower traces in -50 mV, the same as was used to observe CCh-induced I,.at Fig. 3A). What seemed to be an all-or-none phenomenon oscillations. However, in this case external K+ was not necessarily so in all cases (compare with results concentration was increased in order to increase the size of from Missiaen, Taylor & Berridge, 1992). Figure 3B shows the Ca2+-activated K+ current, thus making it a more that in another cell greatly reduced responses to flashes sensitive indicator of changes in [Ca2+]i at this negative were evoked after a large response with a shorter time potential. Figure 4A demonstrates that at -50 mV STOCs

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Figure 5. Membrane current responses to caffeine Cells were dialysed with KCl-based solution and voltage clamped at the Vh indicated. Caffeine was applied by replacing the bathing medium with PSS containing caffeine at the concentrations shown (A, B and C) or by pressure ejection from a micropipette filled with 5 mm caffeine (D). A-C, current records from three different cells in response to application of caffeine at increasing concentrations. D, current record obtained during repetitive 50 ms applications of caffeine (A) at intervals of between 5 and 10 s duration. J. Physiol. 481.1 Ca"2 inhibits action of inositol trisphosphate 103 were reversed and greatly increased in amplitude in between 0 5 and 10 mm. When applied at concentrations of accordance with shifting the equilibrium potential for K+ 3-10 mm it evoked a brief outward current with a peak towards 0 mV. When flashes were applied, the pattern of amplitude of 1-5 nA followed usually by STOC abolition as responses observed was similar to that shown in Fig. 3. described previously (Bolton & Lim, 1989; Komori & Again, though flashes between full amplitude responses Bolton, 1990; Komori et al. 1992). However, at lower mostly failed to produce any response, occasionally small concentrations (0-5-2 mM) caffeine increased the discharge graded responses could occur (flash 5 in Fig. 4B). In the rate of STOCs with a reduction in their size. The effects same cell, about 7 min later, at the same intensity and were often preceded by a brief outward current with even higher frequency of flashes, each started to produce STOCs superimposed (Fig. 5A and B). In only two of small responses (Fig. 4C). This change in behaviour was thirteen cells tested was there any indication of associated with an increase in holding current suggesting oscillations similar to those evoked by muscarinic receptor some sustained increase in [Ca2+]1. It is possible that this activation. An example is shown in Fig. 5C. It should also increase in basal [Ca2+]i, probably due to deterioration in be noted that 2-10 mm caffeine was significantly less the ability of the Ca2+ stores to accumulate Ca2+ rapidly, effective in evoking the outward current when 0 5 or 1 mm was responsible for a partial (not complete) steady-state caffeine was already present (Fig. 5A-C). inhibition ofInsP3 action. If caffeine was applied briefly and repeatedly from a micropipette (5 mm caffeine, 50 ms pressure pulses), as Channels opened by ryanodine or caffeine are shown by triangles in Fig. 5D, reproducible current not involved in [Ca2+]i oscillations responses could be obtained at intervals of 10 s or longer. The effects of caffeine were tested to see whether it could Applied at shorter intervals, caffeine produced reduced produce oscillations in [Ca2+]i through sensitization of the responses. CICR mechanism to Ca2+ and, in turn, modify CCh- To test the effects of caffeine on oscillatory h,at evoked induced [Ca2+]i oscillations. by CCh, Cs+-loaded cells were held at -50 mV while K+-filled cells were held at -20 or 0 mV and caffeine caffeine (0f5-2 mM) was applied in the presence of CCh. was applied in the bathing solution at concentrations This resulted in a decrease in both frequency and A Vh -50 mV 7YffrvYrr(Tvrrr(yYrYyYYwvYn I 0-2 nA 1 uM CCh lT 0-5 mM caffeine 1-5 mM caffeine - 110 s B

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Figure 6. Effects of caffeine on oscillations of Icat Cells were dialysed with CsCl-based solution and held at -50 mV. Caffeine was applied in the bathing solution in the presence of CCh. The period of application of both drugs and the concentrations used are indicated by the horizontal bars. A-C, current records obtained from three different cells. Caffeine reduced the frequency of oscillations without (A) or with (B) an initial brief inward current, or abolished oscillations (C). 104 A. V Zholos, S. Komori, H. Ohashi and T. B. Bolton J. Phy-siol. 481.1

A Vh -50 mV 30 uM Ruthenium Red

Figure 7. CCh-induced It oscillations in cells 2,uM CCh pretreated with Ruthenium Red B 30 sM Ruthenium Red The cells were dialysed with CsCl-based pipette solution containing the drug at the concentrations indicated and held at -50 mV. A and B, an oscillatory Ihat in the same cell following 6 and 13 min pretreatment with 0-4 nA 5 /M CCh Ruthenium Red, respectively. Note the difference in CCh concentration. C, in another cell 2 uM CCh was

In -q applied after 5 min pretreatment. No substantial C I b 50 uM Ruthenium Red difference was observed compared with normal cells (see Fig. 6 of Komori et al. 1993, for comparison).

2,uM CCh amplitude of Ihat oscillations or their abolition (Fig. 6) pipette solution, strongly inhibited caffeine-induced probably caused by reduction in store Ca2+ or inhibition of outward current (data not shown). However, in all of five InsP3 production. The inhibitory effect was sometimes cells pre-treated with Ruthenium Red (30 or 50 /bM) for at preceded by a noticeable but brief inward current (Fig. 6B least 5 min CCh could still evoke oscillatory Ihat (Fig. 7). and C). In cells which responded to CCh with a small I,,t frequency varied from 0 05 to 0-2 Hz being higher at sustained inward current, application of 1-2 mm caffeine larger concentrations of the agonist. These effects of CCh failed to trigger oscillations. Instead, in some of these cells were similar to those observed in cells not treated with only a single brief inward current was generated in Ruthenium Red. response to,caffeine. These data suggested that CICR via the ryanodine Ruthenium Red is a potent blocker of the ryanodine receptor had a minor, if any, role in CCh-induced receptors of the SR. In these cells it has been shown to oscillations of [Ca2+]i but InsP3 was probably always block CICR when 10 /M was added to the bathing solution necessary. If pulsatile production of InsP3 does not occur (Zholos et al. 1991). We also observed that 50 /lM then feedback loops, necessary to explain [Ca2+]i Ruthenium Red in the bathing solution, or added to the oscillations at constant [InsP3]i, could be attributed to

Figure 8. Effect of flash release of InsP]3 at different phases of CCh-evoked Ihat oscillations A, CCh (3/uM) was applied 3 min after cell dialysis with CsCl-based pipette solution containing 30/uM caged InsP3 of a cell held at -50 mV. Flashes (triangles) were applied at a CV of 75 V in this cell. Note that flashes marked Fl and F3 were without any effect whereas F2 and F4 produced premature B oscillations similar to those due to endogenous InsP3 F2 but inward current developed more rapidly. v B, parts of the record are shown on an expanded time base. Note that at the instant of flash application a short artifact appears on the current trace.

I F3

Fl 5 s J. Physiol. 481.1 Ca2+ inhibits action of inositol trisphosphate 105 fluctuations in InsP3-induced Ca2+ release (IICR). Thus, Figure 8 illustrates that when two flashes (F3 and Fl) of the sensitivity of the SR to InsP3 at different phases of the same intensity were applied, one close to the peak and [Ca2+]i oscillations was tested by flash release of InsP3, the other during the descending phase of an Ihat oscillation particularly following [Ca2+]i elevations. just after its peak, both were ineffective in releasing additional Ca2+. On the other hand, flashes F2 and F4 Effects of flash release of InsP3 during applied at later times between peaks produced large oscillatory I-at responses. CCh (3-5 #M) applied to Cs+-filled cells loaded with caged It was unlikely that at the peak of an oscillation (i.e. at InsP3 and held at -50 mV resulted in Ihat oscillations maximum inward current) all Ca2+ was released from the which were measured as a maximum deflection of the stores as caffeine could still produce some response at this current trace at the first, second, or third oscillation. Their point (data not shown). An alternative explanation of mean amplitude was -181 + 26 pA (range, -59 to these observations could be that InsP3-mediated Ca2+ -364 pA; n = 16) and oscillation frequency 0-18 + 0 05 Hz release from the stores became inactive, or desensitized, (n = 12). When a flash was applied to liberate an additional for a period, due either to increase in cytosolic [Ca2+]i or amount of InsP3, the response varied greatly depending decrease in SR luminal [Ca2+]i (compare Figs 3 and 4). We on the phase of the oscillation. If a flash was applied close further explored these possibilities. to the peak of hCat the response was generally small, or absent, but if a flash was applied during the trough Ca2`-induced Ca2+ release and sensitivity to between two peaks this resulted in a large Ca2+ release as flash-released InsP3 judged from the change in I1Ct (Fig. 8). In the latter case, It was of interest to see whether a rise in [Ca2+]i due to peak amplitude of the flash-induced current was Ca2+-induced Ca2+ release could inactivate InsP3-induced -222 + 41 pA (range, -100 to -386 pA in 7 cells). The half- Ca2+ release. Late transient outward current (ILTO) is decay time, 1076 + 163 ms (n = 5), was also similar to that generated in these cells upon a depolarizing step as a result for the preceding CCh-induced oscillation, 1856 + 336 ms of activation of Ca2+-dependent K+ channels during (n = 5). The only substantial difference observed was that regenerative Ca2+-induced Ca2+ release following voltage- the flash-induced current reached a maximum more activated Ca2+ entry (Zholos et al. 1991; Zholos, Baidan & quickly (460 + 111 ms, n = 7) when compared with the Shuba, 1992). In caged InsP3-loaded cells, if the potential preceding CCh-induced ICat oscillations (2152 + 406 ms, was stepped to +10 mV and then was held at this level for n = 5). several seconds, ILTO was first generated and then decayed

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2 nA

A A A A A 10 mV 15 s 15 s L -50 mV

Figure 9. An inhibitory effect of CICR on the subsequent IICR Al ILTOwas generated during membrane depolarization to +10 mV, as the voltage protocol at the bottom shows, indicating that CICR was triggered by voltage-activated Ca2+ entry. Immediately following the ILTO decay, flash (A) was ineffective in evoking Ca2+ release despite substantial Ca2+ store refilling as judged by the effect of caffeine (A). B, the control for this cell showed considerable IICR immediately following the depolarizing pulse but before CICR developed (outward current was initially less than with the pulse in A and developed more slowly). Note that a second flash was again ineffective, an effect which was not due to the SR depletion as tested by caffeine application shortly after the flash. Flashes were applied at a CV of 40 V. Caffeine was applied by pressure ejection for 500 ms at 10 mM. 106 A. V Zholos, S. Komori, H. Ohashi and T. B. Bolton J. Physiol. 481.1 to a small current. Outward current in response to a flash obtained in five other cells tested. When a flash was was 4f6 + 0 4 nA (n = 22) compared with 4f3 + 0 7 nA applied immediately before ILTO developed (Fig. 9B, for (n = 13) in response to pressure ejection of 10 mM caffeine example) the response developed much faster than the after a prolonged period at +10 mV. The size of the ILTO- For comparison, mean time to peak for flash-induced responses to flash release of InsP3 or to caffeine varied current was 521 + 101 ms (n = 21) and 1837 + 176 ms depending on the phase of ILTO when they were applied; (n = 18) for ILTO. Thus, it is likely that Ca2+-induced Ca2+ application of either agent before peak ILTO was achieved release being severalfold slower did not contribute resulted in large, full-sized responses to both agents. After significantly during the response to InsP3-induced Ca2+ the peak of ILTO had been reached, responses to a flash or release. to caffeine were severely depressed; full recovery of the Elevation of [Ca2+]i by caffeine application alone also responses to both occurred within 30 s at +10 mV but the seemed to depress responses to flash-released InsP3 after a caffeine response recovered considerably more quickly short delay. As already mentioned, short applications of than the flash response (Fig. 9A). The recovery of the 10 mm caffeine (100-500 ms pressure pulses) repeatedly caffeine response at a time when the flash response was applied with intervals of 20 s or longer produced outward small suggests that the SR stores are refractory to release currents of fairly uniform size suggesting complete by InsP3 at a time when they are refilled with Ca2+ and reuptake of released Ca2+ during the interval between able to be discharged by caffeine. If a flash was applied applications. The minimal interval varied somewhat in before the peak of ILTO0 a large outward current occurred different cells but was reasonably constant in a particular (Fig. 9B) but the response to a second flash a few seconds cell during an experiment; it was probably determined by later was inhibited despite the fact that substantial the intrinsic ability of the SR to accumulate Ca2+ and was uptake of Ca2+ in the SR had occurred by this time, as not, or was only slightly, affected by caffeine itself because judged from the effect of caffeine. Similar results were a much reduced response to a second application ofcaffeine

L5 Li A A A A A A A

1 nA

A A A A A A A A 10 mV -50 mV

Figure 10. Inhibitory effects of caffeine- or InsP.-induced Ca2+ release (I1CR) on subsequent IICR A, caffeine (A) and flashes (A) were applied as indicated at 25 s intervals and in different combinations at a Vh of -20 mV. B, in another cell flashes and caffeine were applied at 18 s intervals during 27 s depolarizing pulses from -50 to +10 mV (current trace of 3 s duration at the Vh is also shown before pulse). The interval after onset of depolarization and before first application of either caffeine or flash was 3 s. In A and B high-K+ pipette solution was used. Flashes were applied at a CV of 40 V. Caffeine was applied by pressure ejection at 10 mm for 100 ms in A and 200 ms in B. The gaps between records represent 45-80 s in A and 70-90 s in B. J. Physiol 481.1 Ca2+ inhibits action of inositol trisphosphate 107 could be elicited even during the descending phase of Ca2+ store (Missiaen et al. 1992), a result which makes it outward current evoked by a previous caffeine less likely that Ca2+ in the present experiments inhibits application. This interval was assumed to represent the the InsP3-Ca2+ channel from within the store after uptake time when SR stores had fully refilled with Ca2+ and it was into it. Even when the Ca2+ store was filled and able to be used to avoid uncertainty about SR refilling in the released by caffeine, InsP3 was unable to release Ca2+ if experiment illustrated in Fig. 10A in which caffeine and cytoplasmic (or, strictly, subplasmalemmal) [Ca2+]i was, or flashes were applied in all possible combinations. Whereas had recently been, at a high level. InsP3- or caffeine-induced Ca2+ release did not prevent The Ca2+ store in these smooth muscle cells exhibits at subsequent caffeine-induced Ca2+ release (first and second least three types of Ca2+-release phenomenon, two of traces, Fig. 10A) InsP3-induced Ca2+ release was inhibited which are cyclical and lead to cyclical membrane currents by previous caffeine-induced release (third trace, Fig. 10A) viz. STOCs and receptor-evoked I'at oscillations. In or by previous InsP3 application (fourth trace, Fig. 10A). addition caffeine, or more slowly ryanodine, can release This was not because release of Ca2+ in the third trace was stored Ca2+ via a channel receptor which may also be greater than in the first trace, as the reverse situation involved in CICR brought about by Ca2+ entry through occurred in Fig. lOB, but InsP3-evoked release was still voltage-dependent Ca2+ channel opening. It is possible inhibited; here flashes and caffeine were alternated several that a STOC arises due to spontaneous Ca2+ release times with a delay of 18 s during pulses to +10 mV (Fig. through ryanodine receptor-channels brought about by a lOB), producing similar results. These results suggest that facilitatory action of Ca2+ on these from within the Ca2+ InsP3 receptors are inactivated by elevated [Ca2+]i store when Ca2+ stores become overloaded (Benham & probably in a concentration-dependent manner and stay Bolton 1986; Missiaen et al. 1992). Thus, caffeine, which in an inactivated state longer than the period necessary opens these channels, not only releases Ca2+ through them for Ca2+-store refilling. but also reduces STOC size in lower concentrations, perhaps because it sensitizes the channel to Ca2+ in the store so that channel opening occurs at a lower DISCUSSION concentration of store Ca2+. However, in contrast, CICR The above results support the view that InsP3-induced seems to involve Ca2+ acting from the cytoplasmic side and Ca2+ release can be inhibited by a rise in [Ca2+]i above a so the mechanism must be envisaged to be essentially certain level and that following the release of stored Ca2+, different. inhibition of the response to flash-released InsP3 persists Oscillations in [Ca2+]i brought about by muscarinic after repletion of the Ca2+ store since caffeine responses are receptor activation or by GTPyS introduced into the cell of normal size. It is notable in these single smooth muscle (Komori et al. 1992, 1993) require InsP3 and presumably cells that InsP3- and caffeine-releasable stores are the same involve InsP3-Ca2+ channels. The opening of these (Komori & Bolton, 1990, 1991). It would seem that the channels will cause [Ca2+]i to rise to a point at which relationship between inhibition of InsP3-induced Ca2+- inhibition of opening will occur. This inhibitory effect of store release and [Ca2+]i is rather steep such that only [Ca2+]i, once it occurs, persists for a short period, the rarely (approximately in 10-20) was partial, rather than duration of which may depend somewhat on [Ca2+]i. While almost complete, inhibition of InsP3-induced Ca2+-store the opening of InsP3-Ca2+ channels is inhibited, Ca2+ responses observed. Inhibition of InsP3-induced Ca2+-store stores refill to a greater or lesser extent: loss of the release by [Ca2+]i has been observed in oocytes (Parker & inhibition with time and with the fall in [Ca2+]i which Ivorra, 1990) synaptosomal vesicles (Finch et al. 1991) and results from the closing of the InsP3-Ca2+ channels, allows planar bilayers (Bezprozvanny et al. 1991) but evidence has InsP3 to act and the cycle begins again. In this way Ca2+ not previously been found for its existence in intact inhibition of InsP3-Ca2+ channels contributes to the smooth muscle cells. repetitive oscillations of [Ca2+]i, and of Ihat, during Inhibition of InsP3-induced Ca2+ release was observed muscarinic receptor activation. following elevation of [Ca2+]i by previous InsP3-induced The ryanodine (caffeine) SR Ca2+ channel does not seem release, caffeine-induced Ca2+ release, or Ca2+_induced to be directly involved in the oscillations of [Ca2+]i Ca2+ release brought about by Ca2+ entry through opening produced by muscarinic receptor activation. On the few of voltage-dependent Ca2+ channels. The site of inhibition occasions when periodic Ca2+-activated K+ currents seems likely to be on the cytoplasmic face of the appeared which were reminiscent of those evoked by sarcoplasmic reticulum (or Ca2+ store if this is elsewhere or muscarinic receptor activation (Fig. 5C) it may be that for a specialized part of the SR) and related to the InsP3 some reason resting InsP3 levels (Prestwich & Bolton, 1991) receptor-channel. A protein, calmedin, has been were higher than usual. Caffeine was observed to raise implicated in transduction of the inhibitory action of Ca2+ [Ca2+]i temporarily (Icat increased) and oscillations of on InsP3 binding (Danoff, Supattapone & Synder, 1988). It [Ca2+]i were abolished, presumably due to Ca2+ store has recently been described that InsP3-induced Ca2+ depletion; ryanodine, although it acted more slowly, release is promoted by an action of Ca2+ from within the produced a similar effect (Komori et al. 1993). In the cases 108 A. V. Zholos, S. Komori, H. Ohashi and T B. Bolton J. Physiol. 481.1 of both caffeine and ryanodine their actions can be DANOFF, S. K., SUPATTAPONE, S. & SNYDER, S. H. (1988). explained simply by their ability to deplete the Ca2+ store Characterization of a membrane protein from brain mediating and no evidence for a direct involvement of the ryanodine the inhibition of inositol 1,4,5-trisphosphate receptor binding by calcium. Biochemical Journal 259, 701-705. SR Ca2+ channel in receptor-evoked oscillations was DESILETS, M., DRISKA, S. P. & BAUMGARTEN, C. M. (1989). obtained. STOCs were temporarily abolished following Current fluctuations and oscillations in smooth muscle cells caffeine, InsP3 or peaks in [Ca2+]i during receptor-evoked from hog carotid artery (role of the sarcoplasmic reticulum). oscillations (Fig. ID). This result provides support for the Circulation Research 65, 708-722. argument that FINCH, E. A., TURNER, T. J. & GOLDIN, S. M. (1991). Calcium is a all phenomena are properties of a single coagonist of inositol 1,4,5-trisphosphate-induced calcium Ca2+ store in these cells, in contradistinction to other release. Science 252, 443-446. smooth muscle (Jino, Kobayashi & Endo, 1988; Jino, 1990). FOHR, K. J., AHNERT-HILGER, G., STECHER, B., SCOTT, J. & Ca2+-activated K+ channels, and the cationic channels GRATZL, M. (1991). GTP and Ca2+ modulate the inositol 1,4,5- giving rise to are both sensitive to presumably trisphosphate-dependent Ca2+ release in streptolysin 'cat' [Ca2+]i 0-permeabilized bovine adrenal chromaffin cells. Journal of immediately in the vicinity of these channels. However, Neurochemistry 56, 665-670. the former open only when [Ca2+]i exceeds some threshold HAMILL, 0. P., MARTY, A., NEHER, E., SAKMANN, B. & which depends on potential (Benham, Bolton, Lang & SIGWORTH, F. J. (1981). Improved patch-clamp techniques for Takewaki, 1986). Oscillations of and [Ca2+]i, estimated high-resolution current recording from cells and cell-free h,at membrane patches. Pfliugers A rchiv 391, 85-100. by introducing indo-1 into the cell, occur at the same rate IINO, M. (1990). Biphasic Ca2+ dependence of inositol 1,4,5- but may differ very slightly in phase, possibly because the trisphosphate-induced Ca release in smooth muscle cells of the cationic channels and indo-1 respond to [Ca2+]i in different guinea pig taenia caeci. Journal of General Physiology 95, regions of the cytoplasm (Komori et al. 1993). 1103-1122. Unfortunately, although is a sensitive indicator of IINO, M. & ENDO, M. (1992). Calcium-dependent immediate h,at feedback control of inositol 1,4,5-trisphosphate-induced Ca2+ changes in [Ca2+]i (probably subplasmalemmal) both above release. Nature 360, 76-78. and below the resting level, it cannot indicate the absolute IINO, M., KOBAYASHI, T. & ENDO, M. (1988). Use of ryanodine for concentration of [Ca2+]i. The indo-1 light signal from a functional removal of calcium store in smooth muscle cells of volume of cytoplasm (Komori et al. 1993) has contributions the guinea-pig. Biochemical and Biophysical Research Communications 152, 417-422. from intracellular organelles as well as from superficial and INOUE, R., KITAMURA, K. & KURIYAMA, H. (1987). Acetylcholine deep cytoplasm. Thus, neither signal is suitable for activates single sodium channels in smooth muscle cells. studying the relationship between [Ca2+]i and InsP3 Pfliugers Archiv 410, 69-74. channel opening. In addition, it seems likely that, besides JOSEPH, S. K., RICE, H. L. & WILLIAMSON, J. R. (1989). The effect the inhibitory effect of on of external calcium and pH on inositol trisphosphate-mediated [Ca2+]i InsP3-Ca2+ channel calcium release from cerebellum microsomal fractions. opening, there is also a potentiating action early on which Biochemical Journal 258, 261-265. promotes their opening and accelerates the rise in [Ca2+]i KEIZER, J. & DE YOUNG, G. W. (1992). Two roles of Ca2+ in agonist (Bezprozvanny et al. 1991; Finch et al. 1991, Iino & Endo, stimulated Ca2+ oscillations. Biophysical Journal 61, 649-660. 1992). Experiments are in progress to investigate this KOMORI, S. & BOLTON, T. B. (1990). Role of G-proteins in muscarinic receptor inward and outward currents in rabbit possibility. jejunal smooth muscle. Journal of Physiology 427, 395-419. KOMORI, S. & BOLTON, T. B. (1991). Calcium release induced by inositol 1,4,5-trisphosphate in single rabbit intestinal smooth REFERENCES muscle cells. Journal of Physiology 433, 495-517. BENHAM, C. D. & BOLTON, T. B. (1986). Spontaneous transient KOMORI, S., KAWAI, M., PACAUD, P., OHASHI, H. & BOLTON, T. B. outward currents in single visceral and vascular smooth muscle (1993). Oscillations of receptor-operated cationic current and cells of rabbit. Journal of Physiology 381, 385-406. internal calcium in single guinea-pig ileal smooth muscle cells. BENHAM, C. D., BOLTON, T. B. & LANG, R. J. (1985). Acetylcholine Pflugers Archiv 424, 431-438. activates an inward current in single mammalian smooth KOMORI, S., KAWAI, M., TAKEWAKI, T. & OHASHI, H. (1992). muscle cells. Nature 316, 345-347. GTP-binding protein involvement in membrane currents BENHAM, C. D., BOLTON, T. B., LANG, R. J. & TAKEWAKI, T. evoked by carbachol and histamine in guinea-pig ileal muscle. (1986). Calcium-activated potassium channels in single smooth Journal of Physiology 450,105-126. muscle cells of rabbit jejunum and guinea-pig mesenteric LEVY, S. & PAYNE, R. (1993). A lingering elevation of Cai artery. Journal of Physiology 371, 45-67. accompanies inhibition of inositol 1,4,5-trisphosphate-induced BERRIDGE, M. J. & POTTER, B. V. (1990). Inositol trisphosphate Ca release in Limulus ventral photoreceptors. Journal of analogues induce different oscillatory patterns in Xenopus General Physiology 101, 67-84. oocytes. Cell Regulation 1, 675-681. MALGAROLI, A., FESCE, R. & MELDOLESI, J. (1990). Spontaneous BEZPROZVANNY, I., WATRAS, J. & EHRLICH, B. E. (1991). Bell- [Ca2+]i fluctuations in rat chromaffin cells do not require shaped calcium response curves of Ins(1,4,5)P3- and calcium- inositol 1,4,5-trisphosphate elevations but are generated by a gated channels from endoplasmic reticulum of cerebellum. caffeine- and ryanodine-sensitive intracellular Ca2+ store. Nature 351, 751-754. Journal of Biological Chemistry 265, 3005-3008. BOLTON, T. B. & LIM, S. P. (1989). Properties of calcium stores and MARTY, A. & TAN, Y. P. (1989). The initiation of calcium release transient outward currents in single smooth muscle cells of following stimulation in rat lacrimal glands. Journal of rabbit intestine. Journal of Physiology 409, 385-401. Physiology 419, 665-687. J. Physiol. 481.1 Ca2+ inhibits action of inositol trisphosphate 109

MISSIAEN, L., TAYLOR, C. W. & BERRIDGE, M. J. (1992). Luminal Ca"+ promoting spontaneous Ca + release from inositol trisphosphate-sensitive stores in rat hepatocytes. Journal of Physiology 455, 623-640. PACAUD, P. & BOLTON, T. B. (1991). Relation between muscarinic receptor cationic current and internal calcium in guinea-pig jejunal smooth muscle cells. Journal of Physiology 441, 477-499. PARKER, I. & IVORRA, I. (1990). Inhibition by Ca2+ of inositol trisphosphate-mediated Ca2+ liberation: A possible mechanism

for oscillating release of Ca2 . Proceedings of the National Academy of Sciences of the USA 87, 260-264. PRESTWICH, S. A. & BOLTON, T. B. (1991). Measurement of picomole amounts of any inositol phosphate isomer separable by h.p.l.c. by means of a bioluminescence assay. Biochemical Journal 274, 663-672. WAKUI, M., OSIPCHUK, Y. V. & PETERSEN, 0. H. (1990). Receptor- activated cytoplasmic Ca2+ spiking mediated by inositol trisphosphate is due to Ca2+-induced Ca2+ release. Cell 63, 1025-1032. WORLEY, P. F., BARABAN, J. M., SUPATTAPONE, S., WILSON, V. S. & SNYDER, S. H. (1987). Characterization of inositol trisphosphate receptor binding in brain (Regulation by pH and calcium). Journal of Biological Chemistry 262, 12132-12136. ZHANG, B. X. & MUALLEM, S. (1992). Feedback inhibition of Ca2+ release by Ca2+ is the underlying mechanism of agonist-evoked intracellular Ca2+ oscillations in pancreatic acinar cells. Journal of Biological Chemistry 267, 24387-24393. ZHAO, H. & MUALLEM, S. (1990). Inhibition of inositol 1,4,5- trisphosphate-mediated Ca2+ release by Ca2+ in cells from peripheral tissues. Journal of Biological Chemistry 265, 21419-21422. ZHOLOS, A. V., BAIDAN, L. V. & SHUBA, M. F. (1991). Properties of the late transient outward current in isolated intestinal smooth muscle cells of the guinea-pig. Journal of Physiology 443,555-574. ZHOLOS, A. V., BAIDAN, L. V. & SHUBA, M. F. (1992). Some properties of Ca2+-induced Ca2+ release mechanism in single visceral smooth muscle cell of the guinea-pig. Journal of Physiology 457, 1-25.

Acknowledgements This work was supported by the British Medical Research Council.

Received 6 December 1993; accepted 18 March 1994.