Caffeine-sensitive Calcium Stores Research Articles

Actions of thalidomide in producing vascular relaxations

We have investigated the cardiovascular actions of thalidomide in vivo and in vitro. Blood pressure was recorded in pentobarbitone anaesthetized rats. Isometric contractions were examined in rings of rat tail artery and aorta. Radioligand binding studies of α1A- and α1B-adrenoceptor sites were carried out in membranes of rat submandibular gland and spleen, respectively. In pentobarbitone anaesthetized rats, thalidomide and the T-type calcium channel blocker NNC55-0396 (both 1mg/kg, i.v.) significantly reduced blood pressure. In rat tail artery, thalidomide (10–100μM) produced relaxations of phenylephrine (1μM) induced contractions. Also in tail artery, thalidomide (100μM) significantly reduced the contraction to phenylephrine (1μM), but not KCl (40mM), produced by calcium restoration, and NNC55-0396 (100μM) had similar actions to thalidomide. Glibenclamide (10μM), calphostin C (1μM) or SB203580 (1μM) failed to affect the inhibitory actions of thalidomide, and thalidomide did not affect contractions to caffeine (10mM). Ligand binding studies found no evidence for α1A- or α1B-adrenoceptor affinity of thalidomide, and functional studies in rat aorta found no evidence for α1D-adrenoceptor affinity. It is concluded that thalidomide has previously unreported vascular relaxant actions. Relaxant actions in vitro do not seem to involve α1-adrenoceptors, caffeine sensitive calcium stores, glibenclamide sensitive potassium channels, protein kinase C (PKC) or P38 mitogen activated protein kinase (P38 MAP kinase). However, actions of thalidomide resembled those of the T-type calcium channel blocker NNC 55-0396. Further study is necessary to establish the mode of action of thalidomide in causing relaxations.

Calcium Handling in Human Embryonic Stem Cell-Derived Cardiomyocytes

The objective of the current study was to characterize calcium handling in developing human embryonic stem cell-derived cardiomyocytes (hESC-CMs). To this end, real-time polymerase chain reaction (PCR), immunocytochemistry, whole-cell voltage-clamp, and simultaneous patch-clamp/laser scanning confocal calcium imaging and surface membrane labeling with di-8-aminonaphthylethenylpridinium were used. Immunostaining studies in the hESC-CMs demonstrated the presence of the sarcoplasmic reticulum (SR) calcium release channels, ryanodine receptor-2, and inositol-1,4,5-trisphosphate (IP3) receptors. Store calcium function was manifested as action-potential-induced calcium transients. Time-to-target plots showed that these action-potential-initiated calcium transients traverse the width of the cell via a propagated wave of intracellular store calcium release. The hESC-CMs also exhibited local calcium events ("sparks") that were localized to the surface membrane. The presence of caffeine-sensitive intracellular calcium stores was manifested following application of focal, temporally limited puffs of caffeine in three different age groups: early-stage (with the initiation of beating), intermediate-stage (10 days post-beating [dpb]), and late-stage (30-40 dpb) hESC-CMs. Calcium store load gradually increased during in vitro maturation. Similarly, ryanodine application decreased the amplitude of the spontaneous calcium transients. Interestingly, the expression and function of an IP3-releasable calcium pool was also demonstrated in the hESC-CMs in experiments using caged-IP3 photolysis and antagonist application (2 microM 2-Aminoethoxydiphenyl borate). In summary, our study establishes the presence of a functional SR calcium store in early-stage hESC-CMs and shows a unique pattern of calcium handling in these cells. This study also stresses the importance of the functional characterization of hESC-CMs both for developmental studies and for the development of future myocardial cell replacement strategies. Disclosure of potential conflicts of interest is found at the end of this article.

Involvement of calcium mobilization from caffeine-sensitive stores in mechanically induced cell cycle arrest in the dinoflagellate Crypthecodinium cohnii

Mechanical loads can profoundly alter cell growth and cell proliferation. The dinoflagellates are especially sensitive to mechanical stimulation. Many species will be arrested in cell cycle in response to turbulence or shear stress. We demonstrate here that mechanical shaking and caffeine, the ryanodine-receptor agonist, induced an elevation of cytosolic calcium in the dinoflagellate Crypthecodinium cohnii. Dantrolene, a ryanodine-receptor antagonist, dose-dependently inhibited both shaking-induced and caffeine-induced calcium release. Similar to the effect of mechanical shaking, caffeine alone dose-dependently and reversibly induced cell cycle arrest in dinoflagellates. Prolonged shaking substantially abolished the magnitude of caffeine-induced calcium release and vice-versa, suggesting that both agents released calcium from similar stores through ryanodine receptors. Fluorescence-conjugated ryanodine gave positive labeling, which could be blocked by ryanodine, in the cortice of C. cohnii cells. In addition, caffeine or shaking mobilized intracellular chlortetracycline (CTC)-positive membrane-bound calcium, which could be similarly depleted by t-BuBHQ, a SERCA pump inhibitor. Prior treatment with shaking or caffeine also inhibited the ability of the other agent in mobilizing CTC-positive calcium. CTC-positive microsomal fractions could also be induced to release calcium by caffeine and cADPR, the ryanodinee receptor modulator. t-BuBHQ, but not calcium ionophores, induced cell cycle arrest, and the calcium chelator BAPTA-AM was unable to rescue caffeine-induced cell cycle arrest. These data culminate to suggest that mobilization or depletion of caffeine-sensitive calcium stores, but not calcium elevation per se, is involved in the induction of cell cycle arrest by mechanical stimulation. The present study establishes the role of caffeine-sensitive calcium stores in the regulation of cell cycle progression.

Advancing age alters rapid and spontaneous refilling of caffeine-sensitive calcium stores in sympathetic superior cervical ganglion cells.

Intracellular calcium concentration ([Ca2+]i) release from smooth endoplasmic reticulum (SER) stores plays an important role in cell signaling. These stores are rapidly refilled via influx through voltage-gated calcium channels or spontaneously via store-operated calcium channels and subsequent pumping by SER Ca2+-ATPases. We measured [Ca2+]i transients in isolated fura 2-loaded superior cervical ganglion cells from 6-, 12-, 20-, and 24-mo-old Fischer 344 rats. For rapid refilling, [Ca2+]i transients were elicited by a 1) 5-s exposure to K+, 2) caffeine to release Ca2+ from SER stores, 3) K+ to refill SER Ca2+ stores, and 4) caffeine. The percent difference between the peak and rate of rise of the first and second caffeine-evoked [Ca2+]i transient significantly declined over the age range of 12-24 mo. To estimate spontaneous refilling, cells were depolarized for 5 s with 68 mM K+ (control), followed by a 10-s exposure to 10 mM caffeine "conditioning stimulus" to deplete [Ca2+]i stores. Caffeine was then rapidly applied for 5 s at defined intervals from 60 to 300 s. Integrated caffeine-evoked [Ca2+]i transients were measured and plotted as a percentage of the K+ response vs. time. The derivative of the refilling time curves significantly declined over the age range from 12-24 mo. Overall, these data suggest that the ability of superior cervical ganglion cells to sustain release of [Ca2+]i following rapid or spontaneous refilling declines with advancing age. Compromised ability to sustain calcium signaling may possibly alter the overall function of adrenergic neurons innervating the cerebrovasculature.

Pregnant rat myometrial cells show heterogeneous ryanodine- and caffeine-sensitive calcium stores.

Intracellular Ca(2+) release channels such as ryanodine receptors play crucial roles in the Ca(2+)-mediated signaling that triggers excitation-contraction coupling in muscles. Although the existence and the role of these channels are well characterized in skeletal and cardiac muscles, their existence in smooth muscles, and more particularly in the myometrium, is very controversial. We have now clearly demonstrated the expression of ryanodine receptor Ca(2+) release channels in rat myometrial smooth muscle, and for the first time, intracellular Ca(2+) concentration experiments with indo 1 on single myometrial cells have revealed the existence of a functional ryanodine- and caffeine-sensitive Ca(2+) release mechanism in 30% of rat myometrial cells. RT-PCR and RNase protection assay on whole myometrial smooth muscle demonstrate the existence of all three ryr mRNAs in the myometrium: ryr3 mRNA is the predominant subtype, with much lower levels of expression for ryr1 and ryr2 mRNAs, suggesting that the ryanodine Ca(2+) release mechanism in rat myometrium is largely encoded by ryr3. Moreover, using intracellular Ca(2+) concentration measurements and RNase protection assays, we have demonstrated that the expression, the percentage of cells responding to ryanodine, and the function of these channels are not modified during pregnancy.

Age-related alterations in caffeine-sensitive calcium stores and mitochondrial buffering in rat basal forebrain

The properties of caffeine- and thapsigargin-sensitive endoplasmic reticulum calcium stores were compared in acutely dissociated basal forebrain neurons from young and aged F344 rats by ratiometric microfluorimetry. The ability of these stores to sequester and release calcium resembles that observed in other central neurons, with an important role of mitochondrial calcium buffering in regulating the response to caffeine. An age-related reduction in the filling state of the stores in resting cells appears to be mediated by increased rapid calcium buffering, which reduces the availability of calcium for uptake into the stores. An age-related decrease in the amplitude of maximal caffeine-induced calcium release was attributed to increased mitochondrial buffering. There were no age-related differences in the sensitivity to caffeine or in the calcium sequestration/release process at the level of the endoplasmic reticulum per se. These findings demonstrate the importance of interactions between cellular calcium buffering mechanisms and provide details regarding age-related changes in calcium homeostasis which have been thought to occur in these and other neurons associated with age-related neuronal dysfunctions.

Refilling of caffeine-sensitive intracellular calcium stores in bovine airway smooth muscle cells.

The goal of this study was to assess the mechanisms by which the caffeine-sensitive calcium stores of airway smooth muscle cells are refilled. Bovine trachealis cells were loaded with fura 2-AM (0.5 microM) for imaging of cytosolic calcium concentrations ([Ca2+]i) in the inner cytosol. After a first stimulation (S1) with caffeine, the response to a second stimulation (S2) depended on the presence of extracellular calcium during an intervening 80-s-long refilling phase. The S2-to-S1 ratio (S2/S1) was 0.11 +/- 0.05 (n = 13 cells) during calcium-free refilling but 0.72 +/- 0.04 (n = 36 cells) within 80 s of exposure to extracellular calcium. Maximum mean [Ca2+]i during the 80 s of refilling was not different for calcium-free (116 +/- 19 nM; n = 13 cells) versus extracellular calcium plus nickel (2 mM) (121 +/- 12 nM; n = 21 cells); despite this, significantly greater refilling (S2/S1 0.58 +/- 0.06; n = 24 cells) occurred in the presence of extracellular calcium plus nickel. The protein tyrosine kinase inhibitors genistein (100 microM) and ST-638 (50 microM) significantly decreased refilling over 80 s (S2/S1 0.35 +/- 0.06, n = 14 cells and 0.51 +/- 0.07, n = 14 cells, respectively). Daidzein (100 microM) had no effect on S2/S1. We concluded that [Ca2+]i of the inner cytosol during refilling correlated poorly with S2/S1 values and that, therefore, additional compartments not well detected by fura 2 contribute to refilling. The findings suggest that calcium influx for refilling is segregated from the inner cytosol of the cell, relatively insensitive to nickel, and regulated or modulated by protein tyrosine kinase activity.

Calcium in sympathetic boutons of rat superior cervical ganglion during facilitation, augmentation and potentiation

The sympathetic preganglionic nerve terminals of the rat superior cervical ganglion were loaded with the calcium indicator oregon green 488 BAPTA-1 to measure the change in calcium concentration in the terminal boutons, (Δ[Ca 2+] b) following short (1 or 5 impulses) and long (200 impulses) trains at 30 Hz. The Δ[Ca 2+] b after a single action potential or a short train declined in two phases: a fast phase with a time constant of 530±30 ms and a moderate phase with a time constant of 4.0±0.2 s. The Δ[Ca 2+] b following a long train eventually declined with a time constant of 127±34 s (slow phase). The addition of either ω-agatoxin TK (100 nM), ω-conotoxin GVIA (100 nM) or nifedipine (20 μM) to block P-type, N-type or L-type calcium channels respectively showed that the rise in Δ[Ca 2+] b in boutons was predominantly mediated by an influx of calcium through P-type (53±7%) and N-type (46±4%) calcium channels. Experiments with caffeine, ryanodine and thapsigargin indicate that intracellular caffeine-sensitive calcium stores have a small but statistically significant effect on the fast and moderate phases. The mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP; 2 μM) significantly decreased the amplitude of the slow phase of Δ[Ca 2+] b relaxation, and sped its time course, suggesting that mitochondria normally dump calcium during this phase. Adenosine reduced the amplitude of Δ[Ca 2+] b in response to single action potentials by 30±6%, suggesting that adenosine-mediated autoinhibition in these boutons reduces Ca 2+ influx. Spontaneous increases in Δ[Ca 2+] b demonstrated Ca 2+ coupling between adjacent boutons. The Δ[Ca 2+] b kinetics are compared with F2 facilitation, augmentation and post-tetanicpotentiation.

Depletion of caffeine-sensitive calcium store results in diminution of ATP-induced metabotropic calcium responses in rat neocortical neurons

ATP receptor-mediated changes in the Ca2+ concentration were recorded from neurons of the sensorimotor cortex in brain slices from 3-week-old rats. To measure the cytoplasmic concentration of Ca2+, slices were incubated with Fura-2/AM, and a microfluorimetry system was focused on an individual cell. Possible glutamatergic signals resulting from ATP-evoked glutamate release were excluded. After elimination of calcium from the extracellular solution, the first ATP-induced [Ca2+] i transient decreased to 62±9% of a similar response in the normal solution, suggesting the participation of metabotropic purinoreceptor-triggered Ca release in transient generation. Depletion of the caffeine-sensitive calcium store results in diminution of ATP-induced [Ca2+] i transient in the Ca2+-free solution by 31.4±7.0% (P<0.01). This may indicate that in pyramidal neurons of the sensorimotor cortex InsP3- and Ca-induced Ca-releases demonstrate noticeable functional interaction. Nevertheless, there is no single compartment in the endoplasmic reticulum bearing both IICR and CICR channels.

Dopamine (D2) receptor regulation of intracellular calcium and membrane capacitance changes in rat melanotrophs.

1. Indo-1 microfluorimetry and patch clamp techniques were used to study the decrease in cytosolic [Ca2+] ([Ca2+]i) caused by dopamine (D2) receptor activation and the calcium dependence of membrane capacitance changes in single rat melanotrophs. 2. [Ca2+]i decreased when extracellular calcium was removed or when the calcium channel blockers nickel (2 mM) or cadmium (100 microM) were applied by bath perfusion. 3. Quinpirole, a dopamine (D2) receptor agonist, reduced [Ca2+]i by 55 +/- 9 nM and hyperpolarized membrane potential by 29 +/- 9 mV simultaneously. 4. Quinpirole-induced [Ca2+]i decrease required deactivation of voltage-dependent calcium channels. Voltage clamping the membrane potential at -25 mV prevented the quinpirole-induced [Ca2+]i decrease. Nickel (2 mM) reduced [Ca2+]i without hyperpolarization and precluded additional [Ca2+]i decrease by quinpirole. 5. Membrane capacitance measurement of secretion rates in cells dialysed with buffered calcium solutions showed that secretion began at approximately 400 nM Cai2+. 6. Melanotrophs have IP3-sensitive calcium stores, but no caffeine-sensitive calcium stores. Calcium released from IP3-sensitive calcium stores also stimulated secretion. 7. Secretion in melanotrophs is modulated by protein kinase activators. cAMP (200 microM) enhanced secretion at [Ca2+]i > 1000 nM. Phorbol myristate acetate (PMA; 200 nM) enhanced secretion at [Ca2+]i < 400 nM, but not in the absence of calcium. 8. Dopamine receptor activation can reduce secretion by reducing the calcium influx through calcium channels with hyperpolarization of the membrane potential. However downregulation of either cAMP or protein kinase C activity may also contribute to the decrease in secretion.

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.

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.

Properties of spontaneous inward currents in rabbit pulmonary artery smooth muscle cells

Spontaneous inward and outward currents were studied with perforated patch recording in freshly dispersed rabbit pulmonary artery smooth muscle cells. With physiological potassium concentrations, spontaneous outward and inward currents were recorded at negative membrane potentials. Ion substitution experiments revealed that the outward and inward currents were respectively potassium and chloride conductance increases. Both conductances were abolished by bath application of caffeine (2-10 mM), which releases calcium from internal stores. The rise time and half-decay time of spontaneous potassium currents were both about 25 ms. The spontaneous chloride current has a rise time of 30 ms and decayed exponentially with a time constant (tau) of 70 ms at -50 mV. The tau value was increased by depolarization and increased e-fold for a change of 99 mV in membrane potential. In every cell examined when the spontaneous currents occurred as biphasic events, typically between -20 mV and -40 mV, outward currents preceded inward currents in over 90% of these events whereas the inward current always preceded the outward current in caffeine- and noradrenaline-evoked responses. An explanation for these data is that there may be localization of some chloride channels with respect to the caffeine-sensitive calcium store.

Calcium entry through nicotinic receptor channels and calcium channels in cultured rat superior cervical ganglion cells.

1. Patch-clamp techniques in conjunction with indo-1 fluorescent measurements were used to measure increases in intracellular free calcium concentration and membrane conductance induced by the activation of nicotinic and calcium channels in cultured rat sympathetic neurons. 2. Under voltage-clamp conditions, pressure application of the nicotinic agonist DMPP (1,1-dimethyl-4-phenylpiperazinium iodide, 100 microM, 100 ms) increased [Ca2+]i by 193 +/- 26 nM at a clamp potential of -60 mV. This was accompanied by an inward current of -4.53 +/- 0.89 nA, giving a mean ratio of the delta (Ca2+]i to the total inward charge transfer of 42.7 nmoles per litre of free calcium per nanocoulomb of charge (M/q ratio). 3. The DMPP-induced current and associated delta [Ca2+]i were reduced by mecamylamine (100 nM-10 microM) but were unaffected by alpha-bungarotoxin (100 nM) or cadmium (100 microM). 4. The M/q ratio was not affected by the holding potential (from -80 to -40 mV) but was a function of the external calcium concentration. 5. The M/q ratio was reduced by increasing the intracellular calcium buffering capacity and increased by heparin but not affected by ryanodine or by depletion of the caffeine-sensitive calcium store. 6. Under the same recording conditions, we quantified the increase in [Ca2+]i associated with activation of the voltage-dependent calcium current. On average at -60 mV, the M/q ratio of this highly calcium-selective permeability was 1961 mM nC-1, which is 46 times that obtained for the nicotinic channel. 7. Assuming constant-field theory, ion-substitution experiments suggest that in 2.5 mM external calcium, the permeability sequence for the nicotinic conductance was Cs+ < Li+ < Na+ < K+ < Ca2+. 8. We conclude that the nicotinic channels in rat sympathetic neurones are significantly permeant to Ca2+ and that the influx of Ca2+ through these channels is the principal cause of the rise in [Ca2+]i seen under voltage clamp.

Calcium binding to the cytosol and calcium extrusion mechanisms in intact synaptosomes and their alterations with aging.

A simple method to measure cytosolic calcium binding in intact presynaptic nerve terminals (synaptosomes) from rat brain, which is based on the simultaneous determination of [Ca2+]i and total [45Ca2+] in quin2-loaded synaptosomes undergoing a switch from high- to low-calcium containing medium, is presented. Binding to the cytosolic compartment alone was obtained following depletion of calcium storing organelles in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone/oligomycin plus caffeine. Synaptosomes, as compared to various cells types, have a high calcium binding capacity to the cytosolic compartment; maximum binding, Ca.Bmax, was 4.76 mM and calculated s0.5 was 218 nM. Calcium binding to the cytosolic compartment as a function of aging was also determined; Ca.Bmax was reduced to 1.84 mM and s0.5 increased to 492 nM in 30-month-old rats, indicating that the buffering of high calcium loads is impaired in old animals. The results obtained for binding of calcium to mitochondria and caffeine-sensitive calcium stores are consistent with an age-dependent reduction in calcium bound to mitochondria, whereas caffeine-sensitive calcium stores were unaffected. Finally, we have estimated the net rates of calcium extrusion in intact synaptosomes, and found that calcium efflux through the Na/Ca exchanger and Ca(2+)-ATPase was markedly reduced in old rats.

Endothelin in perinatal pharmacology.

E ndothelin (ET) is a potent vasoconstrictor peptide that was initially isolated from the conditioned medium of the cultured porcine aortic endothelial cells.’ It contains 21 amino acidresidues including two intramolecular disulfide linkages. Subsequently, analysis of the human endothelin gene showed existence of three human endothelin genes, predicting the three distinct isotypes of ET designated ET-l, ET-2 and ET-3 (Fig l).’ Interestingly, an amino acid sequence very similar to ET was found in the sequence of a snake venom sarafotoxin S6b.3 Human, porcine, dog, and rat ET-1s have the identical amino acid sequences. It is now well-known that the cluster of the hydrophobic amino acid residues at the carboxy1 terminus of the peptide, particularly the terminal Trp21, aminoterminus carboxyl groups of Asp8 and GlulO and the aromatic group of Phel4 are very important in exerting the potent vasoconstriction.4,5 Further investigations have demonstrated widespread binding sites for ET-1 not only in vascular bed but also in lung, adrenal gland, nervous systems, and other organs.6 In addition, widespread transcription of ET-l mRNA was found in various tissues including neurons,7,8 suggesting a variety of physiological roles of ET. ET causes a slow-developing and long-lasting vasoconstriction in almost all arteries and veins.’ The concentration to induce vasoconstriction is very low, although ET exhibits different potencies depending on the vessel and species employed.1,g ET binds to a specific receptor on smooth muscle membrane and activates phospholipase C, producing phosphoinositol 1, 4, 5,-trisphosphate. The latter elicits the release of calcium ion from the caffeine-sensitive calcium store, inducing constriction. On the other hand, ET stimulates calcium channels of the smooth muscle membrane, inducing influx of exterior calcium ion in porcine coronary artery.“’ The activated influx of calcium ion prolongs the duration of intracellular calcium signal. However, several reports suggested no responsibility of extracellular calcium influx for endothelininduced vasoconstriction. There is still a controversy on this problem. ET elicits a constriction not only in vascular smooth muscle but also in nonvascular smooth muscle.g ET causes rhythmic contractions in nonpregnant human and rat uterine muscles. The contractile frequency increases. Concomitantly sustained tonic contraction appears. In the presence of dihydropyridine calcium antagonist, the increase in frequency of rhythmic contraction induced by ET is completely abolished whereas the sustained increase in tonic contraction is insensitive to external calcium ion.i1,12 This phenomenon is very similar to the oxytotin-induced contraction. Oxytocin stimulates both frequency and force of contractile activity in uterine smooth muscle. Oxytocin initiates spike discharges and increases the frequency. ET also elicits an increase in the number of spikes in a similar manner in rat portal vein.i3 However, WC have no information on uterine muscle. In case of oxytocin, these effects are highly dependent on the stage of gestation. We have no further information on the hormonal effect on the ET-induced contraction of uterine smooth muscle. In this connection, it is interesting that ET was detected in supra-optic and paraventricular nuclei and posterior lobe of pituitary.‘* Distribution of ET in this region is very similar to vasopressin. In addition, water deprivation depleted completely ET from the posterior lobe of pituitary, suggesting the role of ET in water balance.14 Oxytocin is also synthesized in the same region. However, neurons containing oxytocin are distinct from those of vasopressin. Recently Stojilbovic et ali5 demonstrated that ET releases luteinizing hormone (LH) and follicle-

Depolarization promotes caffeine induced [ 3H]-noradrenaline release in calcium-free solution from peripheral sympathetic nerves

The transmitter releasing action of caffeine was studied in the absence of extracellular Ca 2+ from the peripheral sympathetic nerves of the rabbit main pulmonary artery. Caffeine (10 mM) increased the release of [ 3H]-noradrenaline moderately, but not significantly in Ca 2+-free (+1 mM EGTA) Krebs solution. When peripheral nerve endings/varicoslties were depolarized by elevating extracellular K + to 47.2 mM and 70.8 mM in Ca 2+-free solution, the transmitter releasing effect of 10 mM caffeine became significant. Ca 2+ removal itself transiently increased the [ 3H]-noradrenaline outflow. In the individual experiments the amount of the caffeine evoked transmitter release at 47.2 mM and 70.8 mM K +-depolarization was inversely correlated to the release evoked by Ca 2+-removal. Our results suggest that caffeine-sensitive calcium stores are present in peripheral nerve terminals of rabbit pulmonary artery, and part of the caffeine sensitive calcium stores may discharge during Ca 2+-removal from the extracellular solution.

Mechanism of potentiation of contraction by depolarization during action potentials in guinea-pig ventricular muscle.

Action potentials were recorded from guinea-pig ventricular cells and contraction recorded by an optical technique. When the plateau of a single action potential was depolarized (by 70-120 pA applied 100 ms after the upstroke for 100 ms), contraction associated with the following normal action potential was potentiated. This potentiation was not seen in cells exposed to 10 mM-caffeine. The observations are consistent with potentiation of subsequent contraction by increased loading of caffeine-sensitive calcium stores, as a consequence of reduced Ca2+ extrusion or possibly Ca2+ entry via Na+-Ca2+ exchange during a depolarized plateau.

Effects of caffeine on the vascular smooth muscles isolated from two-kidney, one-clip renovascular hypertension in rabbits.

To examine the intracellular calcium content of the vascular smooth muscle in the chronic phase of renovascular hypertension, the effects of caffeine on isolated aortic, renal and iliac arterial strips were evaluated in two-kidney, one-clip renovascular hypertension in rabbits. 10 weeks after left renal artery constriction in the constricted group, systemic blood pressure (BP) was significantly higher than in the control group, but plasma renin activity was similar to the control value. The amplitude of caffeine-induced contraction of various vascular strips in the constricted group gradually increased and, at 10 weeks, this was higher in the constricted group than in the control group. In the constricted group, sodium loading produced BP elevation and increased the amplitude of caffeine-induced contraction, whereas sodium constriction decreased BP and the amplitude. In the control group, altered sodium intake did not affect BP and the amplitude of caffeine-induced contraction. These results suggest that in the chronic phase of renovascular hypertension, the increased caffeine-sensitive intracellular calcium store on vascular smooth muscle, which is increased by sodium loading and is decreased by sodium restriction, may contribute to the maintenance of hypertension.

Ryanodine: its possible mechanism of action in the caffeine-sensitive calcium store of smooth muscle.

The caffeine-sensitive intracellular Ca store was characterized and the mechanism of action of ryanodine in the store was studied using K-depolarized guinea-pig taenia caecum. (1) After incubation of the preparation with CaCl2 (Ca loading), caffeine was applied in Ca-deprived medium, to produce a transient contraction and to monitor the amount of the stored Ca. As duration of Ca deprivation was prolonged, the amplitude of the caffeine-induced contraction was decreased. When ryanodine was applied during Ca deprivation, the rate of the decrease was remarkably accelerated. (2) The rate of rise of the contraction induced by external Ca [Ca)o) was slowed by preceding depletion of the stored Ca by caffeine, compared with that observed in the Ca loaded preparation. However, in the presence of ryanodine, even if stored Ca was depleted by caffeine, the rate of rise of the (Ca)o-induced contraction remained at a higher level. (3) These results suggest that ryanodine stimulates a leak of the stored Ca, and that the contraction induced by the transmembrane influxed Ca could be modulated by the amount of Ca in, or leakiness of, the caffeine-sensitive Ca store.