EGTA Solution Research Articles

Calcium in phase control of the Bulla circadian pacemaker

The circadian pacemaker in the retina of the eye of the marine snail Bulla gouldiana was examined using the whole eye in vitro preparation. Phase-response curves were generated to 6-h pulses of a low calcium EGTA solution and to a hyperpolarizing low potassium-low sodium solution. Both treatments yielded similar phase response curves with phase delays in the late subjective night/early subjective day and phase advances in the late subjective day. The similarity of the phase response curves to hyperpolarizing and low calcium solutions and the absence of additivity when both treatments are combined raises the possibility that both treatments affect the underlying pacemaker through a common mechanism. The persistence of phase shifts to low calcium pulses delivered in the presence of depolarizing light suggests that hyperpolarization is not required for low calcium phase shifting. However, it is possible that both treatments act by reducing a transmembrane calcium flux which is postulated to result from the periodic depolarization of the pacemaker cell membrane during the subjective day. Since a transmembrane calcium flux is known to be essential for both light and depolarization-induced phase shifting, we discuss the hypothesis that calcium fluxes play a pivotal role in the entrainment pathway of the circadian pacemaker.

Decay of the slow calcium current in twitch muscle fibers of the frog is influenced by intracellular EGTA.

The mechanism(s) of the decay of slow calcium current (ICa) in cut twitch skeletal muscle fibers of the frog were studied in voltage-clamp experiments using the double vaseline-gap technique. ICa decay followed a single exponential in 10 mM external Ca2+ and 20 mM internal EGTA solutions in all pulse protocols tested: single depolarizing pulses (activation protocol), two pulses (inactivation protocol), and during a long pulse preceded by a short prepulse (400 ms) to 80 mV (tail protocol). In single pulses the rate constant of ICa decay was approximately 0.75 s-1 at 0 mV and became faster with larger depolarizations. ICa had different amplitudes during the second pulses of the inactivation protocol (0 mV) and of the tail protocol (-20 to 40 mV) and had similar time constants of decay. The time constant of decay did not change significantly at each potential after replacing 10 mM Ca2+ with a Ca2+-buffered solution with malate. With 70 mM intracellular EGTA and 10 mM external Ca2+ solutions, ICa also decayed with a single-exponential curve, but it was about four times faster (approximately 3.5 s-1 at 0 mV pulse). In these solutions the rate constant showed a direct relationship with ICa amplitude at different potentials. With 70 mM EGTA, replacing the external 10 mM Ca2+ solution with the Ca2+-buffered solution caused the decay of ICa to become slower and to have the same relationship with membrane potential and ICa amplitude as in fibers with 20 mM EGTA internal solution. The mechanism of ICa decay depends on the intracellular EGTA concentration: (a) internal EGTA (both 20 and 70 mM) significantly reduces the voltage dependence of the inactivation process and (b) 70 mM EGTA dramatically increases the rate of tubular calcium depletion during the flow of ICa.

Influence of Magnesium and Extracellular Calcium Reduction on Ouabain‐treated Sinoatrial Node Cells in Rabbit Heart

The influence of the extracellular Mg on the slow regenerative potential induced by Ca removal was investigated by using rabbit SA node tissue treated with ouabain. When the SA node tissue was superfused with ouabain (10(-5) M), the preparation became quiescent. Further superfusion with Mg-free and Ca-free solution produced a small regenerative potential reaching 21 +/- 3 mV (n = 5). Superfusion with high Mg, Ca-free solution containing 5.19 mM Mg inhibited the regenerative potential. Superfusion of the ouabain-treated SA node tissue with Ca-free (2 mM EGTA) solution containing normal Mg or Mg-free, Ca-free (2 mM EGTA) solution produced a slow regenerative potential reaching 45 +/- 2 mV (n = 5) or 44 +/- 3 mV (n = 5). Increasing the external Mg to 3.6 mM or 6.18 mM in the Ca-free, EGTA solution caused a significant inhibition of slow regenerative potential. The addition of diltiazem (0.08 mM) blocked both normal action potential and slow regenerative potential induced by superfusion with Mg-free, Ca-free (2 mM EGTA) solution. These results suggest that Mg inhibits the development of the slow regenerative potential resulting from inward currents through the Ca channel.

Desensitization of swine arterial smooth muscle to transplasmalemmal Ca2+ influx.

1. The effect of transplasmalemmal Ca2+ influx on the [Ca2+]i dependence of smooth muscle contraction was evaluated by measuring intracellular [Ca2+] (as estimated by aequorin), myosin phosphorylation, and isometric stress in swine carotid media. 2. Extracellular Ca2+ was removed by incubation in physiological saline with 1 mM-EGTA and no added CaCl2 for 20 min (termed EGTA treatment). In some preparations, intracellular Ca2+ was released by a brief (5 min) histamine stimulation while in this Ca2(+)-free EGTA solution (termed histamine treatment). 3. Restoration of extracellular CaCl2 to EGTA and histamine-treated preparations in the continued presence of histamine was associated with an initial large aequorin light transient. However, this light transient was not initially associated with an increase in myosin phosphorylation or rapid stress development, suggesting that the contractile apparatus was desensitized to aequorin-estimated myoplasmic [Ca2+]. The desensitization was temporary, and resolved by 10 min after restoration of extracellular CaCl2. 4. The light transient observed upon restoration of extracellular CaCl2 was smaller in preparations only EGTA treated when compared to preparations treated with both EGTA and histamine, suggesting that histamine treatment further desensitized the contractile apparatus. 5. The stress development rate was not slowed when histamine and extracellular CaCl2 were simultaneously added to EGTA-treated preparations, suggesting that the desensitization was only to transplasmalemmal Ca2+ influx (from extracellular CaCl2 readdition), and not intracellular Ca2+ release (from the histamine stimulation). 6. In EGTA and histamine-treated preparations, restoration of extracellular CaCl2 in the presence of 109 mM-KCl was associated with a larger aequorin light signal than was observed upon readdition of CaCl2 in the presence of histamine, suggesting that depolarization also further desensitized the contractile apparatus. 7. Depolarization of EGTA-treated preparations did not increase [Ca2+] or stress, suggesting that depolarization did not release intracellular Ca2+ stores. 8. No significant light transient was observed upon addition of extracellular LaCl3, suggesting that tissue damage or leakage of aequorin into the extracellular space was not the cause of the Ca2(+)-reintroduction light signal. 9. These data suggest that removal of extracellular CaCl2 desensitizes the contractile apparatus of smooth muscle to transplasmalemmal Ca2+ influx. This desensitization is only to readdition of extracellular Ca2+; the contractile apparatus still responds to intracellular Ca2+ release. The desensitization is increased by prior depolarization or brief histamine treatment (potentially by depleting intracellular Ca2+). The source of activator Ca2+ appears to affect the relationship between aequorin light and phosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)

Ca 2+-activated K + channel in rat pancreatic islet B cells: permeation, gating and blockade by cations

Activation of Ca 2+-dependent K + conductance has long been postulated to contribute to the cyclical pauses in glucose-induced electrical activity of pancreatic islet B cells. Here we have examined the gating, permeation and blockade by cations of a large-conductance, Ca 2+-activated K + channel in these cells. This channel shares many features with BK (or maxi-K +) Ca 2+-activated K + channel in other cells. (1) Its ‘permeability’ selectivity sequence is P Tl + : P K + : P Rb + : P NH 4 + : P Na +, Li +, Cs + =1.3:1.0:0.5:0.17:<0.05. Permeant, as well as impermeant, cations reduce channel conductance. (2) Its conductance saturates at 325–350 pS with bath KCl > 400 mM (144 mM KCl pipette). (3) It shows asymmetric blockade by tetraethylammonium ion (TEA) and Na +. (4) It is sensitive to Ca 1 2+ over the range 5 nM–100 μM; over the range 50–200 nM, channel activity varies as [Ca 2+ free] 1–2. (5) It is sensitive to internal pH over the range 6.85–7.35, but the decrease in channel activity seen with reduced pH i may be partially compensated by the increase in free Ca 2+ concentration which occurs on acidification of buffered Ca 2+/EGTA solutions.

Contractile effects of substance P and other tachykinins on the mouse isolated distal colon

1. Substance P (SP), physalaemin, eledoisin and kassinin induced concentration-related contractions of the longitudinal muscle of the mouse distal colon. The responses were not antagonized by atropine (1.5 x 10(-7) M), mepyramine (2.5 x 10(-7) M), methysergide (5 x 10(-7) M), timolol (10(-6) M), phentolamine (10(-6) M) or naloxone (4 x 10(-7) M). They were enhanced by tetrodotoxin (TTX, 1.5 x 10(-7) M). These observations indicate that the contractile responses to the tachykinins result from a direct activation of smooth muscle cells. 2. The contractile activity provoked by SP and physalaemin was inhibited by nifedipine (a Ca2+-entry blocker) and was abolished in Ca2+-free EGTA solution. Such data suggest that the myogenic effects of SP and physalaemin are mainly dependent on their ability to promote Ca2+ influx. 3. Eledoisin and kassinin evoked a contractile response in the absence of external Ca2+ and their myogenic activity was, to some extent, resistant to the inhibitory effect of nifedipine. This may indicate that an additional process, probably the release of an intracellularly bound Ca2+ store, participates in the mechanism by which eledoisin and kassinin contract the mouse distal colon. 4. After desensitization of the mouse distal colon to SP, the contractile activity provoked by SP or physalaemin was totally abolished whilst the responses evoked by eledoisin and kassinin were barely affected. These observations and other experimental findings indirectly support the assumption that the mouse distal colon could possess different tachykinin-binding sites.

MRNA-induced expression of the cardiac Na+-Ca2+ exchanger in Xenopus oocytes.

Xenopus oocytes were injected with total mRNA isolated from hearts of 1-day-old chicks. After 5 days of incubation the follicular cell layers were removed and the oocytes were loaded with Na+ by incubation in hypertonic EGTA solution at 37 degrees C. The Na+-loaded oocytes accumulated 45Ca2+ from a Na+-free medium at a 3-18-fold higher rate than noninjected oocytes or oocytes injected with control solution containing no mRNA. Oocytes not subjected to the Na+-loading procedure showed no mRNA-dependent 45Ca2+ uptake. Size fractionation of the mRNA using sucrose density gradient centrifugation under denaturing conditions led to the identification of a 25 S fraction competent for induction of the Na+-Ca2+ exchange system.

Pharmacological analysis of the effects of Bay K 8644 and organic calcium antagonists on the mouse isolated distal colon

1. Bay K 8644 (10(-8) to 10(-6) M) induced concentration-related contractions of the longitudinal muscle of the mouse distal colon. The maximal responses were enhanced and the EC50 was lowered in the presence of tetrodotoxin (TTX; 1.5 x 10(-7) M). The responses were not affected by atropine (10(-7) M), mepyramine (2.5 x 10(-7) M), methysergide (5 x 10(-7) M), propranolol (10(-6) M), phentolamine (10(-6) M) or naloxone (4 x 10(-7) M). By contrast, the contractile responses were inhibited by Ca2+ entry blockers (verapamil, nifedipine) and abolished in Ca2+-free EGTA solution. These observations indicate that the contractile effects of Bay K 8644 are dependent on its ability to promote Ca2+ influx. 2. At 10(-4) M, Bay K 8644 provoked a slow relaxation of the preparation. Moreover, from 10(-5) M, Bay K 8644 markedly reduced the contractile responses to ACh and K+ depolarization. These inhibitory effects were comparable with those produced by nifedipine. Such data suggest that, at high concentrations, Bay K 8644 could act in part as a dihydropyridine Ca2+ channel antagonist. 3. Bay K 8644 (10(-9) M) preferentially enhanced, while nifedipine (10(-10) to 10(-8) M) as well as verapamil (3 x 10(-9) to 10(-6) M) preferentially inhibited, the tonic component of the contractile response evoked by K+ depolarizing solution. This may indicate that different populations of voltage-sensitive Ca2+ channels are involved in the biphasic response to K+ depolarization. 4. The biphasic contractile activity induced by ACh was barely enhanced by Bay K 8644 (10-9M) and was less sensitive to Ca2+ entry blockers than the responses to KCl. These findings are discussed in terms of receptor-operated channels and mobilization of bound calcium.

Transient elevation of spontaneous release at the frog neuromuscular junction following acetylcholine iontophoresis

Miniature end-plate currents (m.e.p.c.s) were recorded from frog neuromuscular junctions using a two-electrode voltage clamp. The m.e.p.c. frequency was temporarily elevated following 10s iontophoretic applications of acetylcholine (ACh) when the junctions were clamped at 100 mV. This post-ACh "burst" of quanta was also observed at unclamped junctions. At-100 mV, the intensity of the burst was proportional to the amount of current flowing across the end-plate during ACh iontophoresis, but usually did not reach its peak until the end-plate receptor channels were almost completely closed. Addition of 0.5 microM TTX to the Ringer's solution, or total replacement of sodium with guanidine, lithium, or methylamine did not inhibit the burst. No burst was observed in Ca2+-free, EGTA solutions, or in Ca2+-free solutions containing 2 mM Mn2+. Sr2+ effectively substituted for Ca2+. Addition of 2 mM Co2+ or Mn2+ to normal Ringer's did not inhibit the burst. Presynaptic muscarinic receptors did not obviously contribute to the burst, since it was not blocked by atropine, nor produced by oxotremorine or pilocarpine. The ACh analogs carbachol and acetyl-beta-methylcholine also produced the burst. The burst was highly dependent on the muscle membrane potential during the period of ACh iontophoresis, becoming more intense at potentials negative to -100 mV and disappearing at -60 mV. The critical importance of the post-synaptic membrane potential suggests that the burst may be due to an action of the muscle end-plate on the motor nerve terminal, possibly by the movement of an anionic substance through open end-plate receptor channels, but this hypothesis does not account for the delay of the burst until near the end of the ACh-induced end-plate current.

Calcium dependence of voltage sensitivity in adenosine 3',5'‐cyclic phosphate‐stimulated sodium current in Pleurobranchaea.

1. Ionophoretic injection of cyclic AMP into a voltage-clamped molluscan neurone caused a transient slow inward current (Isi) whose amplitude was enhanced by depolarization. Na+-replaced salines abolished the current, placing it with cyclic AMP-stimulated Na+ currents of other gastropod species. 2. Isi amplitude was suppressed by extracellular Ca2+. The amplitude increased up to 4-fold at holding potentials of -50 mV in nominally Ca2+-free saline. Ion substitutions showed that Ca2+ suppressed Isi more effectively than Mg2+, Co2+, Cd2+, Mn2+, Ba2+ or Sr2+. 3. Voltage sensitivity of Isi was abolished by low-Ca2+ salines, by the Ca2+ current blocker Co2+ and by substitution of Ba2+ or Sr2+ as Ca2+ channel current carriers. In such salines Isi showed no appreciable change in amplitude at holding potentials between -70 and -25 mV. 4. Intracellular injection of the Ca2+ chelator EGTA both augmented the amplitude of the current and its duration. EGTA injection failed to suppress the Ca2+-dependent voltage sensitivity of Isi. Intracellular injection of concentrated 3-N-(morpholino) propanesulphonic acid (MOPS) pH buffer to inhibit secondary, Ca2+-dependent intracellular acidification also failed to suppress the voltage sensitivity, as did injections of a mixed EGTA and MOPS solution. 5. While the data indicate a requirement for extracellular Ca2+ in conferring voltage sensitivity, they do not support a role for an intracellular action. An extracellular binding site for Ca2+ could mediate the voltage sensitivity, either by local depolarization-dependent changes in extracellular Ca2+ concentration or through direct voltage-sensitive block of the Isi channel.

Calcium and contractions of isolated smooth muscle cells from rat myometrium

Smooth muscle cells were isolated from estrogenized rat myometrium by collagenase digestion. Electron microscopic examination and measurement of cell lengths by image-splitting micrometry were carried out after fixation with acrolein. Mean lengths of cells before and after isolation were 81.7 and 66.9 micron, respectively. Responses of cells were compared with contractions of isolated strips recorded isometrically. Effects of carbachol and KCl were examined in 2 mM Ca, 2 mM Ca + 4 mM EGTA, and 2 mM Ca + 10(-8) M nitrendipine solution. Carbachol and KCl produced concentration-dependent shortening of isolated cells maximal at 30 s after addition. The concentrations of carbachol required to produce shortenings were about 100-fold less than those required to produce isometric contractions; but no major difference was observed in the concentration dependence of cell shortening and isometric contraction produced by potassium-induced depolarization. In 2 mM Ca solution, there was a phasic response, followed by a tonic response such that more than 50% of maximum cell shortening was maintained for 10 min. However, in 2 mM Ca + 4 mM EGTA or 10(-8) M nitrendipine, the tonic contraction was abolished and cells rapidly relaxed after 30 s. If carbachol was added to cells after varying times in the EGTA-containing solution, the ability to initiate a contraction declined exponentially with a half-time of 160 s. Effects of depolarization by KCl were examined in 2 mM Ca plus nitrendipine and 2 mM Ca + 4 mM EGTA solution. Shortening occurred in 2 mM Ca solution by depolarization but not if nitrendipine was added. Though shortening was not observed in 2 mM Ca + 4 mM EGTA solution by KCl, subsequent addition of carbachol induced shortening. These results suggested that there was an intracellular Ca store site from which Ca was released by carbachol and which was not affected by depolarization in the absence of external Ca. No evidence was obtained that the contraction persists in Ca2+-free medium in isolated cells, which is in agreement with previous findings in small muscle strips in which only a similar transient response was obtained.

Cytoplasmic Ca2+ during differentiation of 3T3-L1 adipocytes. Effect of insulin and relation to glucose transport.

The cytoplasmic concentration of ionized Ca2+ [( Ca2+]i) was determined in 3T3-L1 cells during their differentiation from fibroblasts to adipocytes, suspended and loaded with the fluorescent Ca2+ indicators quin2 or indo-1. In undifferentiated fibroblasts, as well as in differentiated adipocytes up to day 9, [Ca2+]i was steady around 170 nM, and it increased significantly only in old adipocytes (day 12). During differentiation, stimulation of glucose uptake by insulin increased from a few percent to severalfold. Stimulation of uptake was already apparent after 10 min of addition of the hormone, and 10 nM insulin produced maximal stimulation in 30 min. Insulin (10(-6) M) added to quin2- or indo-1-loaded, suspended adipocytes had no detectable effect on [Ca2+]i for at least 10 min. In contrast, addition of the general anesthetic halothane increased [Ca2+]i from 172 to 251 nM in 3 min. In EGTA solution, the Ca2+ ionophore ionomycin elicited release of Ca2+ from intracellular stores that resulted in a transient increase in [Ca2+]i. A smaller but measurable Ca2+ release from intracellular stores (increasing [Ca2+]i by 20 nM) resulted upon addition of 20 micrograms/ml phosphatidic acid. In contrast, insulin did not produce any detectable release of Ca2+ from intracellular stores. Incubation of 3T3-L1 adipocytes with insulin in the presence of EGTA (the latter in excess over the Ca2+ concentration of the medium) did not prevent the stimulation of hexose uptake by the hormone, indicating that extracellular Ca2+ does not play a role in the insulin response. Furthermore, incubation of cells with quin2/AM in EGTA medium during exposure to insulin did not prevent stimulation of hexose uptake. Under these conditions it is demonstrated that intracellular quin2 suffices to chelate cytoplasmic Ca2+ even if releasable Ca2+ from intracellular stores were to pour into the cytoplasm. Thus, quin2 effectively lowers [Ca2+]i without impairing insulin action. It is concluded that insulin does not produce changes in [Ca2+]i and that chelating intracellular Ca2+ does not prevent stimulation of hexose uptake by insulin. These results suggest that it is unlikely that changes in [Ca2+]i may play a role in the transduction of information in insulin stimulation of glucose uptake in 3T3-L1 adipocytes.

Ionic mechanism of the outward current induced by intracellular injection of inositol trisphosphate into Aplysia neurons

Inositol 1,4,5-trisphosphate (InsP3) has been proposed to be the intracellular second messenger in the mobilization of Ca2+ from intracellular stores in a variety of cell types. The ionic mechanism of the effect of intracellularly injected InsP3 on the membrane of identified neurons (R9-R12) of Aplysia kurodai was investigated with conventional voltage-clamp, pressure-injection, and ion-substitution techniques. Brief pressure injection of InsP3 into a neuron voltage-clamped at -40 mV reproducibly induced an outward current (10-60 sec in duration, 20-60 nA in amplitude) associated with a conductance increase. The current was increased by depolarization and decreased by hyperpolarization up to -80 mV, where it disappeared. Extracellular application of tetraethylammonium (TEA; 5 mM) blocked the InsP3-induced outward current, and the current was not affected by the presence of bath-applied 4-aminopyridine (4-AP; 5 mM). The InsP3-induced outward current recorded at a holding potential of -40 mV increased in amplitude in low-K+ solutions and decreased in amplitude in high-K+ solutions. Alteration of [Cl-]0, as well as perfusion with Ca2+ free plus 2 mM EGTA solution, did not affect the outward current. The InsP3-induced outward current was found to disappear when the neuron was injected with the Ca2+ chelator EGTA. The outward current evoked by repeated InsP3 injection at low doses exhibited summation and facilitation and, at high doses, was shown to desensitize. The calmodulin inhibitor N-(6-amino-hexyl)-5-chloro-1-naphthalene sulfonamide (W-7; 20-50 microM), inhibited both the InsP3-induced and the Ca2+-activated outward currents. An intracellular pressure injection of Ca2+ ions into the same identified neuron was shown to produce an outward current associated with a K+ conductance increase similar to the InsP3-induced current, and the current was blocked by bath-applied TEA (5mM). These results suggest that brief pressure injection of InsP3 into certain identified neurons of Aplysia induces a 4-AP-resistant, TEA-sensitive K+ current activated by increased intracellular free Ca2+ concentration, and this increase might be the result of the mobilization of Ca2+ from intracellular stores by InsP3.

Preparation and some properties of giant liposomes and proteoliposomes.

Optimal conditions for formation of giant liposomes and proteoliposomes were investigated. A suspension of small unilamellar vesicles made of various phospholipids in a buffer of 0-3 M KCl, 0.1 mM EDTA, and 20 mM MOPS (pH 7.0) was subjected to a freeze-thaw treatment. Giant multilamellar liposomes of diameter ranging from 10 to 60 microns were found to form from phospholipid mixtures containing phosphatidylethanolamine as a major component and phosphatidylserine as a minor component. The concentration of KCl optimal for the giant vesicle formation was 30-500 mM. By applying a patch-pipette to a giant liposome, suitable conditions for obtaining a high-resistance (giga-ohm) seal were sought. It was found that use of a patch-pipette of relatively small tip diameter (less than 1 micron), the presence of divalent metal cations in the suspension medium and inflation of vesicles in a hypotonic solution facilitated giga-seal formation. In a suspension of asolectin (soybean phospholipid) vesicles which had been subjected to the freeze-thaw treatment, giant unilamellar vesicles were found. They could be held on the tip of a suction pipette and impaled with a microelectrode filled with an EGTA solution. Small unilamellar proteoliposomes were prepared by the cholate-dialysis method from asolectin and sarcoplasmic reticulum vesicles, and were subjected to a freeze-thaw cycle. When the ratio of exogenous phospholipid to protein was larger than 10, giant multilamellar vesicles were formed.(ABSTRACT TRUNCATED AT 250 WORDS)

Ca Mobilization from the Intracellular Ca Store in Spontaneously Hypertensive Rats

To evaluate the size of the intracellular Ca store in spontaneously hypertensive rats (SHR), caffeine-induced contractions were investigated in mesenteric artery preparation. Twelve-week-old SHR and age-matched Wistar-Kyoto (WKY) rats were used. The isolated mesenteric artery was prepared by the method of McGregor. Caffeine (2 mg)- and norepinephrine (2 micrograms)-induced pressor responses were measured in both normal (Ca, 2 mM) and Ca-free solution (1 mM EGTA solution). In the presence of Ca, pressor responses to exogenous norepinephrine in SHR were higher than those in WKY rats. In Ca-free solution, pressor responses to norepinephrine were markedly reduced in SHR compared to WKY rats and caffeine-induced contractions in SHR were lower than those in WKY rats. These results suggest that pressor responses to norepinephrine in SHR may be more dependent on extracellular Ca than in WKY rats and the caffeine-sensitive intracellular Ca store might be less in SHR.

Full and partial relaxing CCK-C-terminal fragments in hog duodenal circular muscle

The essential active moieties in the cholecystokinin (CCK) ligand for relaxing duodenal circular muscles of pigs were investigated. The decrease in isotonic tension from the normal tone was expressed as a percentage of the relaxation obtained with Ca 2+-free EGTA (2 mM) solution. Inhibition was expressed as a percentage of the relaxation induced by CCK-C-terminal tetrapeptide (CCK-4, 4.23 μM) without antagonists. Amino acid sequences from CCK-(27–28) to CCK-(27–31) increased the potency. CCK-(27–30) or CCK-(27–31) attained the same extent of relative intrinsic activity as CCK-8. The amino acid sequence from Tyr 27, CCK-(27), to Trp 30, CCK-(30), is another prerequisite for relaxation, in addition to the sequence of CCK-4. Newly synthesized CCK-(27–30)-R derivatives with increasingly bulky substituents (R = H, ethyl, phenyl, n-butyl and allyl) were changed from full agonists to partial agonists. Phenyl, n-butyl and allyl derivatives of CCK-(27–30)-R, in particular, had a mixed agonist and antagonist action. CCK-(27–30)-allyl antagonized competitively the CCK-4-induced relaxation (IC 50 = 123 (75.9–200) μM).

The role of membrane depolarization in norepinephrine-induced contractions of the rabbit mesenteric resistance artery.

Changes in membrane potential during norepinephrine-induced contractions in the rabbit mesenteric resistance artery (3rd or 4th branch) were investigated using microelectrodes. Norepinephrine at concentrations greater than 10(-6) M depolarized the membrane and induced contractions dose-dependently. Maximum effects were produced by 10(-4) M norepinephrine. Depolarization was maintained at almost steady level during 15 min application of norepinephrine. During the same period, contractions continued with slight decay. Oscillatory contractions were observed at more than 3 X 10(-6) M norepinephrine, and occasionally persisted throughout the application of norepinephrine. Treatments with Ca2+-rich 1 mM EGTA solution, 10(-5) M diltiazem, 3 X 10(-6) M D600 and 1 mM La3+ did not significantly affect the amount of depolarization induced by 10(-4) M norepinephrine; however, contractions were greatly inhibited by these treatments. Replacement of Na+ by choline markedly reduced depolarization while contractions were not affected. In Ca2+ -free Na+-free solution, no depolarization was induced, while contractions were still produced by norepinephrine, indicating that Cl- was not essential for membrane depolarization. These results suggest that contractions of the rabbit mesenteric resistance artery to norepinephrine are mainly due to the enhanced influx of extracellular Ca2+ which is not dependent on potential-sensitive mechanisms. Depolarization is thought to be due to the increase in the membrane permeability to Na+ and Cl- which is coincidentally produced by norepinephrine. The membrane potential oscillations were dependent on Ca entry but could not be shown to be the result of fluctuations in Ca current.

The effects of extracellular calcium removal on sino-atrial node cells treated with potassium-depleted solutions.

The effect of extreme calcium reduction was studied in rabbit sino-atrial node cells treated with K-free media. Ca removal (Ca = 0, EGTA = 6 mM) suppressed spontaneous miniature fluctuations elicited by exposure to the K-free media. Further, successive exposure to the Ca-free, EGTA solutions produced a propagated action potential and suppressed a contracture. The action potential was not noticeably altered by tetrodotoxin (TTX). In the presence of normal Ko, reducing the bathing calcium concentration with EGTA prolonged the duration of the action potential. The potential induced by Ca removal was similar to that observed in the K-free media. From these results, we have concluded that a drastic reduction in the extracellular Ca concentration produces a decrease in the overloaded Ca in the cell exposed to the K-free media, resulting in an increase in the amplitude of action potentials.

Potassium channels in cultured bovine adrenal chromaffin cells.

K channels of bovine adrenal chromaffin cells were studied using patch-clamp techniques. Whole-cell K currents measured near +10 mV were much larger in 1 mM-external Ca than in Ca-free saline. Noise analysis suggested that this Ca-dependent current was carried by a large unitary conductance channel, called BK channel, which was previously described in inside-out patches (Marty, 1981). The Ca-dependent K current near +10 mV declined with time due to 'run-down' of Ca channels. At the same time, a fraction of the outward current observed above +50 mV was also eliminated. This outward current component probably represents K efflux through Ca channels. Whole-cell Ca-dependent K currents were studied using various Ca buffers. EGTA buffers were surprisingly inefficient: in order to block the current entirely, it was necessary to use an isotonic EGTA solution and to increase internal pH. 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) was at least five times more efficient than EGTA. In isolated patches three types of single-channel K currents were observed. Under normal ionic conditions (140 mM-K inside, 140 mM-Na outside), the unitary conductances measured between -20 and +40 mV were 96 pS, 18 pS and 8 pS. The 96 pS channels are the Ca-dependent BK channels. 18 pS and 8 pS channels were both activated and then inactivated by membrane depolarization. Both displayed complex kinetics; single-channel currents were grouped in bursts. Activation and inactivation kinetics were faster for the 18 pS channel (therefore termed FK channel, for fast K channel) than for the 8 pS channel (SK channel, for slow or small amplitude channel). The voltage dependence of opening probability was steeper for the FK channel as compared to the SK channel.

Calcium channel currents in isolated guinea-pig ventricular cells superfused with Ca-free EGTA solution.

Changes in membrane currents seen in Ca-free, EGTA (1 mM)-containing Tyrode solution (EGTA Tyrode), were studied in isolated guinea-pig ventricular cells, under the voltage clamp performed with a "G omega seal" patch electrode. Application of the EGTA Tyrode (calculated [Ca]0 = 1.3 X 10(-9) M) first eliminated the usual calcium current, but induced an extra inward current within 2 min. The reversal potential of this current, as judged by the direction of the current change, was about +25 mV (without correction of a liquid junction potential of -12 mV), but above this voltage a decaying outward current was observed. The decay of these inward and outward currents during depolarization was slow, but a large, nearly time-independent component was evident. These currents, regardless of their polarity and time course, were reduced by application of verapamil (10(-5) M) and Mg (5 mM), and were inactivated by pre-depolarizations. In Na-free EGTA Tyrode, the inward current disappeared but the outward current persisted at high voltages. These results suggest that in ventricular cells, reduction of external Ca concentrations to a nanomolar range induces a Ca channel current composed of an inward current carried by Na, and an outward current, presumably carried by K ions. Because of the persistence of the apparently non-inactivating Ca channel current, the net membrane current evoked at voltages around 0 mV remained close to zero, or even inward, after the decay of the time-dependent component, which was completed within a few hundreds ms. This characteristic I-V relation was considered to be linked to the development of the long-lasting action potentials, with a plateau maintained at around 0 mV, in EGTA Tyrode.