Low Concentrations Of Caffeine Research Articles

Modulation of caffeine contractures in mammalian skeletal muscles by variation of extracellular potassium.

Caffeine contractures were induced after K(+)-conditioning of skeletal muscles from pigs and mice. K(+)-conditioning is defined as the partial depolarization caused by increasing external potassium (K+0) with [K+]x[Cl-] constant. Conditioning depolarizations that rendered muscles refractory to brief electrical stimulation still enhanced the contracture tension elicited by subsequent direct caffeine stimulation of sarcoplasmic reticulum (SR) calcium release. The effects of K(+)-conditioning on caffeine-induced contractures of intact cell bundles reached a maximum at 15-30 mM K+0 and then progressively declined at higher [K+]0. Conditioning with 30 mM K+ for 5 min, which inactivates excitation-contraction (EC) coupling in response to action potentials, both increased the magnitude of caffeine contractures 2-10-fold and shifted the contracture threshold toward lower caffeine concentrations. Enhanced sensitivity to caffeine was inhibited by dantrolene (20 microM) and its watersoluble analogue azumolene (150 microM). These drugs decreased caffeine-induced contractures following depolarization with 4-15 mM K+ to 25-50% of control tension. The inorganic anion perchlorate (CIO-4), which like caffeine potentiates twitches, increased caffeine-induced contractures approximately twofold after K(+)-conditioning (> 4 mM). The results suggest that CIO-4 and dantrolene, in addition to caffeine, also influence SR calcium release either directly or by mechanism(s) subsequent to depolarization of the sarcolemma. Moreover, since CIO-4 is known to shift the voltage-dependence of intramembrane charge movement, CIO-4 may exert effects on the transverse-tubule voltage sensors as well as the SR.

Electrophysiologic effects of cocaine on subendocardial Purkinje fibers surviving 1 day of myocardial infarction.

Cocaine has been shown to have broad cardiovascular effects that could be life threatening. Most of the reported electrophysiologic effects of cocaine have been studied in normal but not infarcted myocardium. Using microelectrode techniques, we investigated the electrophysiologic effects of cocaine on endocardial canine Purkinje fibers that survived 1 day of myocardial infarction. In quiescent infarcted preparations, stimulated trains were followed by subthreshold delayed afterdepolarizations (DADs), in the presence of propranolol (1 microM). Cocaine (10 microM) decreased the amplitude of DADs from 6.1 +/- 1.8 mV to 3.0 +/- 1.3 mV (P < 0.05, n = 6). When stimulated preparations (n = 23) showing no triggered activity during control (+propranolol) were superfused with a low concentration of caffeine (1 mM) or high extracellular Ca2+ (8.1 mM), triggered activity was induced. Subsequent cocaine (10 microM) superfusion prevented the induction of caffeine- and high Ca(2+)-induced triggered activity. Cocaine's effects were reversible upon washout. In preparations that showed triggered activity during control conditions (+propranolol), the mean cycle length of triggered activity was 755 +/- 45 msec. Cocaine (10 microM) superfusion lengthened the cycle length to 1030 +/- 141 msec and terminated triggered activity with a subthreshold DAD (n = 12). In addition, cocaine and ryanodine (10 microM) suppressed triggered activity in a similar manner when tested in the same preparations (n = 4). During control conditions, cocaine did not cause any significant change on the rate of rise of action potential upstroke (from 55.6 +/- 24.3 to 54.5 +/- 28.6 V/sec, n = 8) and maximum diastolic potential (from -58.4 +/- 4.3 to -56.6 +/- 6.5 mV, n = 8). In the absence of propranolol, 50 microM but not 10 microM cocaine induced early afterdepolarizations in 62% of the preparations exhibiting triggered activity during control conditions. The results suggest that cocaine modulates DADs and triggered activity in infarcted endocardial fibers via direct inhibition of cyclic release of Ca2+ from sarcoplasmic reticulum (SR) independently from a local anesthetic or sympathomimetic effect. This SR inhibition could account for the myocardial depressant effect of cocaine. However, while cocaine suppressed DADs, its induction of EADs can precipitate malignant ventricular arrhythmias in the setting of cocaine overdose and infarction.

Pressure-induced changes in the isometric contractions of single intact frog muscle fibres at low temperatures

Effects of increased hydrostatic pressure (range 0.1-10 MPa) on isometric twitch and tetanic contractions of single intact muscle fibres, isolated from frog tibialis anterior muscle, were examined at 4-12 degrees C. The tension changes produced on exposure to steady high pressures are compared with those produced on exposure to low concentrations of caffeine (0.5 mM, subthreshold for contracture) and when pressure is rapidly released during a contraction. The peak twitch tension was potentiated by pressure accompanied by increased rate of tension rise and increased duration; the pressure sensitivity of twitch tension was approximately 8% MPa-1. The correlation between the rate of tension rise and peak tension in caffeine-induced twitch tension potentiation was quantitatively similar to that in pressure-induced twitch potentiation. Experiments involving the rapid release of pressure (approximately 2 ms) during twitch contractions demonstrate that high pressure need only be maintained for a brief period during the early part of tension development to elicit full twitch potentiation. The tetanic tension was depressed by pressure (approximately 1% MPa-1). Results demonstrate that the major effect of increased hydrostatic pressure on intact muscle fibres, which results in tension potentiation, is complete very early during contraction and is similar to that of caffeine.

Regenerative caffeine‐induced responses in native rabbit aortic endothelial cells

1. Single native aortic endothelial cells obtained by enzymatic dispersion of the rabbit aortic endothelium were held under voltage clamp using patch pipette and whole-cell membrane currents were measured. In parallel experiments performed on cells from the same batches, the free internal calcium concentration, [Ca2+]i, in the cell was estimated by use of the Ca(2+)-sensitive fluorescent dye, fura-2. 2. Caffeine (20 mM) applied to the cell evoked an outward current and an initial peak in [Ca2+]i followed by a lower sustained rise (plateau). Ca(2+)-free, EGTA-containing solution applied outside the cells did not reduce these responses. 3. Following caffeine stimulation there was a biphasic rising phase of outward current both in the presence and absence of extracellular Ca2+. 4. Application of graded doses of caffeine revealed all-or-none type responses of both the outward current and the rise in [Ca2+]i. 5. Preincubation with lower doses of caffeine reduced the magnitude of both the outward current and the [Ca2+]i transient evoked by 20 mM caffeine. 6. Tetraethylammonium (3 mM) applied to the bathing solution blocked unitary and spontaneous transient outward currents (STOCs) stimulated by Ca(2+)-free solution, but only reduced the outward current evoked by caffeine (20 mM). 7. In conclusion, our results reveal the all-or-none nature of Ca2+ release from the endoplasmic reticulum (ER) in native aortic endothelial cells. Lower concentrations of caffeine (0.4-0.5 mM) may deplete intracellular Ca2+ stores. Extracellular Ca2+ is not necessary for maintaining the activity of spontaneous and caffeine-induced outward currents in native aortic endothelial cells. Spontaneous outward currents are believed to represent the sporadic release of calcium from store sites independent of both extracellular Ca2+ and the caffeine-sensitive Ca2+ stores which stimulate the outward current.

Effects of caffeine on cytoplasmic free Ca2+ concentration in pancreatic beta-cells are mediated by interaction with ATP-sensitive K+ channels and L-type voltage-gated Ca2+ channels but not the ryanodine receptor.

In the pancreatic beta-cell, an increase in the cytoplasmic free Ca2+ concentration ([Ca2+]i) by caffeine is believed to indicate mobilization of Ca2+ from intracellular stores, through activation of a ryanodine receptor-like channel. It is not known whether other mechanisms, as well, underlie caffeine-induced changes in [Ca2+]i. We studied the effects of caffeine on [Ca2+]i by using dual-wavelength excitation microfluorimetry in fura-2-loaded beta-cells. In the presence of a non-stimulatory concentration of glucose, caffeine (10-50 mM) consistently increased [Ca2+]i. The effect was completely blocked by omission of extracellular Ca2+ and by blockers of the L-type voltage-gated Ca2+ channel, such as D-600 or nifedipine. Depletion of agonist-sensitive intracellular Ca2+ pools by thapsigargin did not inhibit the stimulatory effect of caffeine on [Ca2+]i. Moreover, this effect of caffeine was not due to an increase in cyclic AMP, since forskolin and 3-isobutyl-1-methylxanthine (IBMX) failed to raise [Ca2+]i in unstimulated beta-cells. In beta-cells, glucose and sulphonylureas increase [Ca2+]i by causing closure of ATP-sensitive K+ channels (KATP channels). Caffeine also caused inhibition of KATP channel activity, as measured in excised inside-out patches. Accordingly, caffeine (> 10 mM) induced insulin release from beta-cells in the presence of a non-stimulatory concentration of glucose (3 mM). Hence, membrane depolarization and opening of voltage-gated L-type Ca2+ channels were the underlying mechanisms whereby the xanthine drug increased [Ca2+]i and induced insulin release. Paradoxically, in glucose-stimulated beta-cells, caffeine (> 10 mM) lowered [Ca2+]i. This effect was due to the fact that caffeine reduced depolarization-induced whole-cell Ca2+ current through the L-type voltage-gated Ca2+ channel in a dose-dependent manner. Lower concentrations of caffeine (2.5-5.0 mM), when added after glucose-stimulated increase in [Ca2+]i, induced fast oscillations in [Ca2+]i. The latter effect was likely to be attributable to the cyclic AMP-elevating action of caffeine, leading to phosphorylation of voltage-gated Ca2+ channels. Hence, in beta-cells, caffeine-induced changes in [Ca2+]i are not due to any interaction with intracellular Ca2+ pools. In these cells, a direct interference with KATP channel- and L-type voltage-gated Ca(2+)-channel activity is the underlying mechanism by which caffeine increases or decreases [Ca2+]i.

Properties of the caffeine-sensitive intracellular calcium stores in mammalian neurons

Using indo-1- and fura-2-based microfluorometry for measuring the cytoplasmic free calcium concentration ([Ca2+]in), the properties of caffeine-induced Ca2+ release from internal stores were studied in rat cultured central and peripheral neurons, including dorsal root ganglion (DRG) neurons, neurons from then. cuneatus, CA1 and CA3 hippocampal regions, and pyramidal neocortical neurons. Under resting conditions, the Ca2+ content of internal stores in DRG neurons was high enough to produce caffeine-triggered [Ca2+]in transients. Prolonged exposure of caffeine depleted the caffeine-sensitive stores of releasable Ca2+; the degree of this depletion depended on caffeine concentration. The depletion of the caffeine-sensitive internal stores to some extent was linked to calcium extrusion via La3+-sensitive plasmalemmal Ca2+-ATPases. Caffeine-induced Ca2+ release deprived internal stores in DRG neurons, but they refilled themselves spontaneously within 10 min. Pharmacological manipulation with caffeine-sensitive stores interferred with the depolarization-induced [Ca2+]in transients. In the presence of low caffeine concentration (0.5–1.0 mM) in the extracellular solution, the rate of rise of the depolarization-triggered [Ca2+]in transients significantly increased (by a factor of 2.15 ± 0.29) suggesting the occurrence of Ca2+-induced Ca2+ release. When the caffeine-sensitive stores were emptied by prolonged application of caffeine, the amplitude and rate of rise of the depolarization-induced [Ca2+]in transients decreased. These findings suggest the involvement of internal caffeine-sensitive calcium stores in generation of calcium signal in sensory neurons. In contrast, in all types of central neurons tested the resting Ca2+ content of internal stores was low, but the stores could be charged by transmembrane Ca2+ entry through voltage-operated calcium channels. After charging, the stores in central neurons spontaneously lost releasable calcium content and within 10 min they became completely empty again. We suggest that internal Ca2+ stores in peripheral and central neurons, although having similar pharmacological characteristics, handle Ca2+ ions in a different manner. Calcium stores in sensory neurons are continuously filled by releasable calcium and after discharging they can be spontaneously refilled, whereas in central neurons internal calcium stores can be charged by releasable calcium only transiently. Caffeine-evoked [Ca2+]in transients in all types of neurons were effectively blocked by 10 mM ryanodine, 5 mM procaine, 10 mM dantrolene, or 0.5 mM Ba2+, thus sharing the basic properties of the Ca2+-induced Ca2+ release from endoplasmic reticulum.

Quantal Ca2+ mobilization by ryanodine receptors is due to all-or-none release from functionally discrete intracellular stores.

Low caffeine concentrations were unable to completely release the caffeine- and ryanodine-sensitive intracellular Ca2+ pool in intact adrenal chromaffin cells. This 'quantal' Ca2+ release is the same as that previously observed with inositol Ins(1,4,5)P3-induced Ca2+ release. The molecular mechanism underlying quantal Ca2+ release from the ryanodine receptor was investigated using fura-2 imaging of single chromaffin cells. Our data indicate that the intracellular caffeine-sensitive Ca2+ pool is composed of functionally discrete stores, that possess heterogeneous sensitivities to caffeine. These stores are mobilized by caffeine in a concentration-dependent fashion, and, when stimulated, individual stores release their Ca2+ in an 'all-or-none' manner. Such quantal Ca2+ release may be responsible for graded Ca2+ responses in single cells.

Ca2+ and Sr2+ entry induced Ca2+ release from the intracellular Ca2+ store in smooth muscle cells of rat portal vein.

1. Changes in cytosolic free Ca2+ concentration ([Ca2+]i) induced by membrane depolarizations were investigated using indo-1 microspectrofluorimetry in single patch-clamped smooth muscle cells of rat portal vein at room temperature (20-21 degrees C) and in the presence of 2 mM Ca2+. 2. During a 1 s depolarization from -50 to -30 mV [Ca2+]i rose, but, although the Ca2+ current was terminated by repolarization to -50 mV, [Ca2+]i continued to increase in a regenerative manner. The delay between the end of the voltage step and the peak of the [Ca2+]i rise was reduced by increasing the depolarization. 3. When a second identical depolarization was rapidly applied (8-13s) after the first one, it induced an identical Ca2+ current but a smaller increase in [Ca2+]i which started to decay upon repolarization. 4. A low concentration of caffeine (0.05 mM), applied to cells showing a small depolarization-induced [Ca2+]i transient which reached a peak at the end of the voltage step, produced an increase in amplitude and in duration of the [Ca2+]i rise without changing the amplitude of the depolarization-induced Ca2+ current. 5. The depolarization-induced [Ca2+]i rise was shortened and reduced in amplitude after noradrenaline- (NA 10 microM) or caffeine- (5 mM) induced release of Ca2+ store and when the patch pipette solution contained ryanodine (100 microM). Under these conditions, the depolarization-induced [Ca2+]i transient was maximal at the end of the voltage step and declined immediately when the membrane was repolarized at -50 mV. 6. Experiments were done by replacing extracellular Ca2+ by Sr2+. Depolarization-induced Sr2+ entry through voltage-dependent Ca2+ channels could evoke an increase in indo-1 fluorescence which occurred after the termination of the voltage step. This delayed component of fluorescence increase displayed properties similar to those of the regenerative [Ca2+]i rise recorded in the Ca(2+)-containing solution. 7. The inefficiency of the second of two successive depolarizations to produce the delayed component of [Ca2+]i rise was not due to the emptiness of the intracellular Ca2+ store since, under these conditions, caffeine was still able to induce a Ca2+ release. 8. It is concluded that depolarization-evoked Ca2+ or Sr2+ entry through voltage-dependent Ca2+ channels induced the release of Ca2+ from an intracellular store, which could occur in a regenerative manner, independent of the termination of the triggering current.

Caffeine-induced calcium release from internal stores in cultured rat sensory neurons

Free intracellular calcium concentration [Ca 2+] in was recorded at 22°C by means of Indo-1 or Fura-2 single-cell microfluorometry in cultured dorsal root ganglion neurons obtained from neonatal rats. The resting [Ca 2+] in in dorsal root ganglion neurons was 73 ± 21nM (mean ± S.D., n = 94). Fast application of 20 mM caffeine evoked [Ca 2+] in transient which reached a peak of 269 ± 64nM within 5.9 ± 1.1 s. After reaching the peak the [Ca 2+] in level started to decline in the presence of caffeine and for 87.2 ± 10.6s cytoplasmic calcium returned to an initial resting value. In 40% of neurons tested [Ca 2+] in decreased to subresting levels following the washout of caffeine (the so-called post-caffeine undershoot). On average, the undershoot level was 19 ± 2.5nM below the resting [Ca 2+] in value. Prolonged exposure of caffeine depleted the caffeine-sensitive stores of releasable Ca 2+; the degree of this depletion depended on caffeine concentration. The depletion of the caffeine-sensitive internal stores to some extent was linked to calcium extrusion via La 3+-sensitive plasmalemmal Ca 2+-ATPases. The stores could be partially refilled by the uptake of cytoplasmic Ca 2+, but the complete recovery of releasable Ca 2+ content of the caffeine-sensitive pools required the additional calcium entry via voltage-operated calcium channels. Caffeine-evoked [Ca 2+] in transients were effectively blocked by 10 μM ryanodine, 5mM procaine, 10 μM dantrolene or 0.5 mM Ba 2+, thus sharing the basic properties of the Ca 2+-induced-Ca 2+ release from endoplasmic reticulum. Pharmacological manipulation with caffeine-sensitive stores interfered with the depolarization-induced [Ca 2+] in transients. In the presence of low caffeine concentration (0.5–1 mM) in the extracellular solution the rate of rise of the depolarization-triggered [Ca 2+] in transients significantly increased (by a factor 2.15 ± 0.29) suggesting the occurrence of Ca 2+-induced Ca 2+ release. When the caffeine-sensitive stores were emptied by prolonged application of caffeine, the amplitude and the rate of rise of the depolarization-induced [Ca 2+] in transients were decreased. These facts suggest the involvement of internal caffeine-sensitive calcium stores in the generation of calcium signal in sensory neurons.

Quantal Ca2+ release from caffeine-sensitive stores in adrenal chromaffin cells.

In populations of fura-2-loaded chromaffin cells, caffeine caused a concentration-dependent increase in the intracellular Ca2+ concentration ([Ca2+]i), in the presence or absence of external Ca2+ ([Ca2+]o), that was saturable, reversible, and inhibited in a use-dependent fashion by ryanodine. These data confirm that caffeine mobilizes Ca2+ from the ryanodine-sensitive intracellular stores in chromaffin cells. In nominally Ca(2+)-free medium, sustained stimulation of cell populations or single cells with low caffeine concentrations failed to completely empty the caffeine-sensitive stores. In each case, there was a transient [Ca2+]i elevation, but a subsequent challenge with a higher caffeine concentration evoked a further [Ca2+]i rise, indicating that Ca2+ stores within individual cells were heterogeneous in their sensitivities to caffeine and that caffeine-induced Ca2+ release was quantal. The heterogeneous sensitivity was also demonstrated using ryanodine; pretreatment of cell populations with increasing caffeine concentrations with a constant ryanodine concentration, caused a dose-dependent irreversible inhibition of the response to the subsequent addition of a maximal caffeine concentration. We conclude that, within single chromaffin cells, intracellular Ca2+ stores are heterogeneous in their sensitivity to caffeine and the fraction of Ca2+ stores mobilized by caffeine increases in direct proportion to the caffeine concentration.

The effect of caffeine on the contractile response of the rabbit urinary bladder to field stimulation

1. 1. Smooth muscle contraction is mediated by an increase in the concentration of cytoplasmic free calcium. 2. 2. Low concentrations of caffeine can induce transient contraction of smooth muscle through the release of intracellular calcium from intracellular storage sites. 3. 3. The current study determined the effect of caffeine (0.4–100 μM) on the response of the in vitro whole bladder preparation to field stimulation (FS) at 0.6, 1.8, and 5.4 mM extracellular calcium. 4. 4. The following parameters were determined: basal pressure (BP), peak pressure response (PP), maximal rate of pressure generation (PR), maximal rate of bladder emptying (RE), and percent of volume emptied (VE). 5. 5. The results demonstrated that in the absence of caffeine, BP, PP, and PR were increased progressively as a function of both frequency of stimulation and the concentration of calcium. 6. 6. In addition, caffeine significantly increased the rate of pressure generation at 0.6 and 1.8 mm calcium.

Potentiation of calcium- and caffeine-induced calcium release by cyclic ADP-ribose.

Cyclic ADP-ribose (cADPR) is a naturally occurring metabolite of NAD+ that is as potent as inositol 1,4,5-trisphosphate (IP3) in mobilizing Ca2+ in sea urchin eggs. Previous pharmacological evidence suggests that cADPR acts through a system similar to the Ca(2+)-induced Ca2+ release (CICR). Here I showed that in the presence of low concentrations of cADPR addition of Ca2+ to egg homogenates stimulated further release of Ca2+ in a concentration-dependent manner. In the absence of cADPR, no induced release was seen, and the added Ca2+ was, instead, sequestered by a thapsigargin-sensitive transport system. High concentrations of strontium (> 50 microM) could also induce Ca2+ release. The effective concentrations of Sr2+, however, were reduced 10-20-fold in the presence of low concentrations of cADPR. Barium, at up to 0.4 mM, did not stimulate Ca2+ release with or without cADPR. The potentiation between divalent cations and cADPR was mutual since the Ca2+ releasing activity of cADPR was also increased in the presence of strontium. Ionomycin and thapsigargin both released Ca2+ but neither potentiated Ca2+ release induced by divalent cations. Caffeine also released Ca2+ in a concentration-dependent manner, and its potency was greatly increased by low concentrations of cADPR, while no such simulation was seen with IP3. Conversely, low concentrations of caffeine that were not sufficient to release Ca2+ increased the effectiveness of cADPR 10-fold. Isocaffeine, an isomer of caffeine, was four to five times less effective, demonstrating the specificity of the caffeine effect. These results suggest that cADPR can function as an endogenous regulator of CICR in eggs.

Effect of caffeine and zinc on DNA and protein synthesis of neonatal rat cardiac muscle cell in culture.

The effect of caffeine and/or zinc on DNA and protein synthesis of purified neonatal-rat ventricular cardiac myocytes was studied. Caffeine (0.2-2 mM) inhibited both DNA and protein synthesis of the cells. Addition of EDTA in the growth medium inhibited both DNA and protein synthesis. Without caffeine and in the presence of lower concentrations of caffeine (0.2 mM) in the growth medium, 10 microM of zinc concentration reversed DNA synthesis, which was inhibited by the chelating agent (EDTA). Higher concentrations of caffeine (2 mM) in the growth medium completely abolished sensitivity of cardiac myocytes to zinc. Additional zinc supplementation to the growth medium of cardiac myocytes did not alter the rate of protein synthesis. The present results suggest that the effect of caffeine on cardiac myocytes may be associated with the zinc-dependent enzymes involved in DNA synthesis.

BAY K 8644 and nifedipine alter halothane but not caffeine contractures of malignant hyperthermic muscle fibers

The purpose of these experiments was to determine if the Ca2+ agonist BAY K 8644 and the Ca2+ antagonist nifedipine alter the mechanical responses of malignant hyperthermia-susceptible (MHS) skeletal muscle to halothane and caffeine. Muscle fiber bundles were dissected from MHS porcine skeletal muscle and exposed to BAY K 8644 (10 microM), nifedipine (1 microM), low-Ca2+ media [Ca2+ replaced by 1 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid], or diltiazem (30 microM) administered alone and with halothane (3%) or caffeine (0.5-0.8 mM). When administered alone, both halothane and BAY K 8644 evoked a significant change in resting tension (i.e., contracture) of 193.7 +/- 61.0 and 51.9 +/- 21.5 mN/cm2, respectively. When administered in combination, BAY K 8644 had no effect on the magnitude of the halothane contracture (195.2 +/- 58.6 mN/cm2) but reduced its onset time from 306.7 +/- 36.3 to 105.9 +/- 8.9 s. Nifedipine, low Ca2+, and diltiazem significantly reduced the halothane contracture (103.1 +/- 30.3, 123.1 +/- 20.6, and 112.6 +/- 16.2 mN/cm2, respectively) but had no effect on its onset time. In addition, low Ca2+ reduced the magnitude of the BAY K 8644 contracture (8.2 +/- 2.1 mN/cm2). BAY K 8644 also increased contractures induced by low caffeine concentrations (0.5-2.0 mM) but did not alter contractures induced by 4.0 and 8.0 mM caffeine, whereas nifedipine, low Ca2+, and diltiazem had no effect on these contractures. These results suggest that extracellular Ca2+ influx may have some influence on halothane but not on caffeine contractures of MHS skeletal muscle.

Effects of BAY K 8644, nifedipine, and low Ca2+ on halothane and caffeine potentiation

The purpose of this investigation was to examine the effects of the Ca2+ agonist BAY K 8644 and the Ca2+ antagonist nifedipine on halothane- and caffeine-induced twitch potentiation of mammalian skeletal muscle. Muscle fiber bundles were taken from normal Landrace pigs and exposed to BAY K 8644 (10 microM), nifedipine (1 microM), and low Ca2+ media administered alone and in combination with halothane (3%) or with increasing concentrations of caffeine (0.5-8.0 mM). Both BAY K 8644 and halothane potentiated twitches by approximately 80%; when they were administered in combination, twitch potentiation was nearly double that caused by either drug alone. In the presence of nifedipine, halothane increased twitches by less than 30%. Low Ca2+ significantly depressed twitches by approximately 25% but also inhibited halothane's inotropic effect. BAY K 8644 augmented caffeine potentiation but only at low caffeine concentrations (0.5-2.0 mM). Nifedipine and low Ca2+ failed to inhibit caffeine's inotropic effects. These results suggest that halothane potentiates twitches via a mechanism that involves or is influenced by extracellular Ca2+.

A mechanism for the effects of caffeine on Ca2+ release during diastole and systole in isolated rat ventricular myocytes.

1. The fluorescent indicator Indo-1 was used to measure both [Ca2+]i and [caffeine]i in single ventricular myocytes. 2. Caffeine (at concentrations of 1 mM or above) produced a transient increase of resting [Ca2+]i attributed to the release of Ca2+ ions from the sarcoplasmic reticulum (SR). Simultaneous measurement of [caffeine]i showed that the Ca2+ release only began when [caffeine]i had risen to about 1 mM. Subsequently the rate of release was a steep function of [caffeine]i. It is suggested that this results from a positive feedback as the Ca2+ released activates further release. 3. If external Ca2+ was removed the release of Ca2+ produced by caffeine was delayed such that [caffeine]i rose to a greater concentration before release was initiated. This suggests that an increase of [Ca2+]i increases the efficacy of caffeine to release Ca2+ ions from the SR. 4. Lower concentrations of caffeine (50-500 microM) had no effect on diastolic [Ca2+]i. In contrast they increased systolic [Ca2+]i and contraction. This increase was most obvious if the systolic contraction had previously been decreased either by reducing [Ca2+]o from 1 to 0.25 mM or (in voltage-clamped cells) by decreasing the magnitude of the depolarizing pulse. 5. If the exposure to caffeine was prolonged, this increase of systolic [Ca2+]i and contraction was completely transient. On removal of caffeine, systolic [Ca2+]i and contraction decreased to below control before recovering. 6. During these transient changes of systolic [Ca2+]i and contraction there was no change of the sarcolemmal Ca2+ current. 7. It is suggested that the increase of systolic [Ca2+]i is due to caffeine increasing the fraction of the SR Ca2+ content released during the twitch. 8. The above results concerning both diastolic and systolic [Ca2+]i can be explained by a model in which caffeine increases the affinity with which Ca2+ ions activate Ca2(+)-induced Ca2+ release. At high enough [caffeine], the threshold [Ca2+]i for regenerative Ca2(+)-induced Ca2+ release will be reduced to below the resting [Ca2+]i thus producing a diastolic increase of [Ca2+]i. At lower [caffeine] the threshold is higher than resting [Ca2+]i and caffeine only serves to enhance the release produced during systole.

Effects of caffeine and ryanodine on delayed afterdepolarizations and sustained rhythmic activity in 1-day-old myocardial infarction in the dog.

Caffeine and ryanodine are known to modulate oscillatory release of Ca2+ from the sarcoplasmic reticulum. The effects of caffeine and ryanodine on delayed afterdepolarizations (DADs) and sustained rhythmic activity in subendocardial Purkinje fibers surviving 1-day-old myocardial infarction in the dog were studied with standard microelectrode techniques. In preparations that showed sustained rhythmic activity, a high concentration of caffeine (10 mM) and ryanodine (10(-7) and 10(-6) M) slowed and terminated the sustained rhythmic activity and markedly suppressed DADs. An increase in the temperature of the tissue bath from 37 degrees to 39 degrees C did not change these results. In quiescent normal and infarcted preparations, a low concentration of caffeine (0.5 mM) differentially induced DADs in ischemic but not in normal Purkinje fibers, increased the amplitude of existing DADs, and brought subthreshold DADs to threshold potential that caused triggered activity. Our results are consistent with the hypothesis that triggered activity arising from DADs characterizes the sustained rhythmic activity in endocardial preparations 1 day after infarction and indicate an important role for the sarcoplasmic reticulum in the genesis of DADs and triggered activity in this model.

Mechanisms of caffeine activation of single calcium‐release channels of sheep cardiac sarcoplasmic reticulum.

1. Calcium-release channels of sheep cardiac junctional sarcoplasmic reticulum were incorporated into planar phospholipid bilayers. Single-channel current fluctuations were recorded under voltage clamp conditions. 2. Channels incorporate into the bilayer with a fixed orientation and channel open probability is regulated by the calcium concentration at the cytosolic face of the membrane. 3. Addition of caffeine (0.5-2.0 mM) to the cytosolic side of the membrane increased the open probability of the calcium-activated calcium-release channel by increasing the frequency of opening without significant alteration to the durations of open events. This effect was observed at both 0.1 and 10 microM-activating cytosolic calcium. 4. Caffeine (0.5-2.0 mM) did not activate the channel at a subactivating cytosolic calcium concentration (80 pM). 5. At subactivating calcium concentrations, channels could be activated by higher concentrations of caffeine (greater than 5.0 mM) revealing a second, calcium-independent, mechanism for channel activation. Channel openings induced by these high concentrations of caffeine at subactivating calcium concentrations displayed different kinetics from those observed with calcium as the sole activating ligand or with combinations of calcium and low concentrations of caffeine. 6. Activation of channel opening by caffeine in the presence of calcium did not affect single-channel conductance. Channel openings produced by caffeine at subactivating cytosolic calcium concentrations had identical conductance and relative permeability to those seen on calcium activation. 7. Channels activated by caffeine at both activating and subactivating calcium concentrations were characteristically modified by ryanodine, Ruthenium Red, ATP and magnesium, implying that the same channel is involved under both conditions.

Direct inhibition of chicken gizzard smooth muscle contractile apparatus by caffeine

The mechanism of the inhibitory effect of caffeine on smooth muscle contraction was examined using chicken gizzard. Caffeine (0.1-5 mmol/l) inhibited the KCl-induced contraction of the muscle with an IC50 of 1.1 mmol/l. Forskolin (0.01-10 mumol/l) also inhibited KCl-induced contraction. The inhibitory effect of caffeine was potentiated by a low concentration of forskolin (0.3 mumol/l) and the inhibitory effect of forskolin was potentiated by a low concentration of caffeine (0.1 mmol/l). Although caffeine and forskolin increased tissue cyclic AMP levels, caffeine inhibited the KCl-induced contraction more strongly than forskolin at a given cyclic AMP level. Caffeine (1-40 mmol/l) inhibited the contractions induced by 3 mumol/l Ca2+ in Triton X-100-permeabilized muscle. Caffeine (1-40 mmol/l) inhibited the phosphorylation of 20 kDa myosin light chain (MLC) in native actomyosin preparation and the inhibition was enhanced by decreasing the ATP concentration in the reaction medium. Calmodulin (CaM) activity, as monitored by Ca2+/CaM-dependent erythrocyte membrane (Ca2+ + Mg2+)-ATPase, was not affected by 20 mmol/l caffeine. Time-dependent dephosphorylation of MLC upon removal of Ca2+, an indicator of phosphate activity, was not affected by caffeine. Caffeine also inhibited the Ca2(+)-independent contraction in thiophosphorylated permeabilized muscle. These results indicate that caffeine inhibits smooth muscle contraction by a direct inhibition of MLC kinase and actin-myosin interaction. A part of the inhibitory effect may be mediated by cyclic AMP-dependent mechanism(s).

Effects of doxorubicin and ruthenium red on intracellular Ca 2+ stores in skinned rabbit mesenteric smooth-muscle fibres

Several agents are known to influence the contraction of skeletal and cardiac muscle via a modification of the Ca 2+ release mechanism of the sarcoplasmic reticulum, e.g. caffeine, ryanodine, ruthenium red and doxorubicin. Of these substances, only the effects of caffeine and ryanodine have been described in smooth muscle. In this paper we describe the action of ruthenium red and doxorubicin on saponin-skinned mesenteric arteries of the rabbit. A high concentration (20 μM) of ruthenium red inhibited the Ca 2+ release induced by low concentrations of caffeine, but had little effect on Ca 2+ release induced by high concentrations (20 mM) of caffeine. This result indicates that the Ca 2+ release channel of the internal Ca 2+ store of smooth muscle cells is less sensitive to inhibition by ruthenium red than that of striated muscle. Doxorubicin in the micromolar range elicited a Ca 2+ release and a concomitant contraction, essentially similar to its effect on skinned skeletal muscle cells. This work reveals further similarities between the Ca 2+ release mechanisms of smooth and striated muscle, but the results also indicate that important differences between both systems may exist.