Genistein inhibits contractile force, intracellular Ca2+ increase and Ca2+ oscillations induced by serotonin in rat aortic smooth muscle

The soy-derived isoflavones genistein and daidzein affect the contractile state of different kinds of smooth muscle. We describe acute effects of genistein and daidzein on contractile force and intracellular Ca2+ concentration ([Ca2+]i) in in situ smooth muscle of rat aorta. Serotonin (5-HT) (2 microM) or a depolarizing high K+ solution produced the contraction of aortic rings, which were immediately relaxed by 20 microM genistein and by 20 microM daidzein. Accordingly, both 5-HT and a high K+ solution increased the [Ca2+]i in in situ smooth muscle cells. Genistein strongly inhibited the [Ca2+]i increase evoked by 5-HT (74.0 +/- 7.3%, n = 11, p < 0.05), and had a smaller effect on high K+ induced [Ca2+]i increase (19.9 +/- 4.0%, n = 7, p < 0.05). The K+ channels blocker tetraethylammonium (TEA) (0.5 mM) diminished genistein effects on 5-HT-induced [Ca2+]i increase. Interestingly, during prolonged application of 5-HT, the [Ca2+]i oscillated and a short (90 s) preincubation with genistein (20 microM) significantly diminished the frequency of the oscillations. This effect was totally abolished by TEA. In conclusion, in rat aortic smooth muscle, genistein is capable of diminishing the increase in [Ca2+]i and in force evoked by 5-HT and high K+ solution, and of decreasing the frequency of [Ca2+]i oscillations induced by 5-HT. The short time required by genistein, and the relaxing effect of daidzein suggest that tyrosine kinases inhibition is not involved. The small inhibiting effect of genistein on the [Ca2+]i increase evoked by high K+ and the effect of TEA point to the activation by genistein of calcium-activated K+ channels.     

Facilitatory and inhibitory transmitters modulate calcium influx during action potentials in aplysia sensory neurons

Serotonin (5-HT) produces presynaptic facilitation and FMRFamide produces presynaptic inhibition in Aplysia sensory neurons. These effects may involve the modulation of Ca2+ influx into sensory neuron terminals during action potentials. Here, we have used the Ca2+ indicator dye fura-2 to monitor directly the effects of 5-HT and FMRFamide on internal Ca2+ concentration ([Ca2+]i). 5-HT caused a 50% increase in the transient rise in [Ca2+]i in response to action potentials, whereas FMRFamide decreased the [Ca2+]i transient by 40%. Neither transmitter altered the resting [Ca2+]i, the time course of recovery of the [Ca2+]i transient, or the [Ca2+]i transients produced by intracellular injection of CaCl2 or inositol 1,4,5-trisphosphate. We conclude that the effects of the transmitters on the action potential-induced [Ca2+]i transient are due to changes in Ca2+ influx and not in intracellular Ca2+ homeostasis.     

Attenuation of the serotonin-induced increase in intracellular calcium in rat aortic smooth muscle cells by sarpogrelate

Although serotonin (5-HT) induced proliferation of vascular smooth muscle cells is considered to involve changes in intracellular Ca2+ ([Ca2+]i), the mechanism of Ca2+ mobilization by 5-HT is not well defined. In this study, we examined the effect of 5-HT on rat aortic smooth muscle cells (RASMCs) by Fura-2 microfluorometry for [Ca2+]i measurements. 5-HT was observed to increase the [Ca2+]i in a concentration- and time-dependent manner. This action of 5-HT was dependent upon the extracellular concentration of Ca2+ ([Ca2+]e) and was inhibited by both Ca2+ channel antagonists (verapamil and diltiazem) and inhibitors of sarcoplasmic reticular Ca2+ pumps (thapsigargin and cyclopia zonic acid). The 5-HT-induced increase in [Ca2+]i was blocked by sarpogrelate, a 5-HT2A-receptor antagonist, but not by different agents known to block other receptor sites. 5-HT-receptor antagonists such as ketanserin, cinanserin, and mianserin, unlike methysergide, were also found to inhibit the 5-HT-induced Ca2+ mobilization, but these agents were less effective in comparison to sarpogrelate. On the other hand, the increase in [Ca2+]i in RASMCs by ATP, angiotensin II, endothelin-1, or phorbol ester was not affected by sarpogrelate. These results indicate that Ca2+ mobilization in RASMCs by 5-HT is mediated through the activation of 5-HT2A receptors and support the view that the 5-HT-induced increase in [Ca2+]i involves both the extracellular and intracellular sources of Ca2+.     

Comparison of the role of protein kinase C in platelet functional responses induced by three different mechanisms, PAF, ionomycin and arachidonic acid.

The role of protein kinase C (PKC) in modulating platelet activation has been examined in platelets pre-incubated with either the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA) or the non-specific protein kinase inhibitor, staurosporine. In order to determine where in the signal transduction pathway PKC is exerting its effect platelets were activated either with a receptor-operated stimulus platelet activating factor (PAF) or by direct elevation of [Ca2+]i (ionomycin) or with arachidonic acid which is converted into thromboxane B2 (TxB2). In PAF-stimulated platelets activation of PKC inhibited both [Ca2+]i elevation and TxB2 generation but had no effect on 5-hydroxytryptamine (5-HT) release whilst staurosporine increased the duration of [Ca2+]i elevation and potentiated TxB2 generation but inhibited 5-HT release. In ionomycin-stimulated platelets modulation of PKC had no effect on [Ca2+]i elevation but in contrast to PAF-stimulated platelets PKC activation caused potentiation of TxB2 generation and 5-HT release whilst inhibition of PKC caused inhibition of TxB2 generation and 5-HT release. Modulation of PKC did not affect arachidonic acid-induced TxB2 generation. These findings suggest that in receptor activated platelets endogenously activated PKC is exerting a negative feedback role, however, when [Ca2+]i elevation is not modified by PKC activation or inhibition (such as in ionomycin stimulated platelets) the relationship between the state of PKC activation and subsequent platelet functional responses corresponds more closely. The findings from this study suggest a different relationship between PKC and TxB2 generation than between PKC and dense granule release in PAF-stimulated platelets.     

5-Hydroxytryptamine-induced phosphoinositide hydrolysis and Ca2+ mobilisation in canine cultured aorta smooth muscle cells.

The effect of 5-hydroxytryptamine (5-HT) on phospholipase C (PLC)-mediated phosphoinositide (PI) hydrolysis and intracellular Ca2+ ([Ca2+]i) changes was investigated in canine cultured aorta smooth muscle cells (ASMCs). 5-HT-stimulated inositol phosphate (IP) accumulation was time and concentration dependent with a half-maximal response (pEC50) and a maximal response at 6.4 and 10 microM, n = 6, respectively. Stimulation of ASMCs by 5-HT produced an initial transient peak followed by a sustained, concentration-dependent elevation in [Ca+]i. The half-maximal response (pEC50) values of 5-HT for the peak and sustained plateau were 7.1 and 6.9, respectively. Ketanserin and mianserin (1 and 3 nM), 5-HT2A antagonists, were equipotent and had high affinity in antagonising the 5-HT-induced IP accumulation and [Ca2+]i change with pK(B) values of 8.6-9.1 and 8.6-9.4, respectively. In contrast, the concentration-effect curves of 5-HT-induced IP and [Ca2+]i responses were not shifted until the concentrations of NAN-190 and metoctopramide (5-HT1A and 5-HT3 receptor antagonists, respectively) were increased to as high as 1 microM with pK(B) values of 5.7-6.3 and 6.1-6.6, respectively, indicating that the 5-HT receptor-mediated responses had low affinity for these antagonists. Pre-treatment of ASMCs with pertussis toxin (100 ng/mL, 24 h) caused a significant inhibition of 5-HT-induced IP accumulation and [Ca2+]i change in ASMCs. Depletion of external Ca2+ or removal of Ca2+ by addition of EGTA led to a significant attenuation of IP accumulation and [Ca2+]i change induced by 5-HT. Influx of external Ca2+ was required for the 5-HT-induced responses, because Ca2+-channel blockers--verapamil, nifedipine and Ni2+--partly inhibited the 5-HT-induced IP accumulation and Ca2+ mobilisation. The sustained elevation of [Ca2+]i response to 5-HT was dependent on the presence of external Ca2+. Removal of external Ca2+ by addition of 5 mM EGTA during the sustained phase caused a rapid decline in [Ca2+]i to lower than the resting level. The sustained elevation of [Ca2+]i could then be evoked by addition of 1.8 mM Ca2+ in the continued presence of 5-HT. These results demonstrate that 5-HT directly stimulates PLC-mediated PI hydrolysis and Ca2+ mobilisation, at least in part, through a pertussis toxin-sensitive G protein in canine ASMCs. 5-HT2A receptors may be predominantly mediating IP accumulation, and subsequently IP-induced Ca2+ mobilisation may function as the transducing mechanism for 5-HT-stimulated contraction of aorta smooth muscle.     

Fluorescence digital image analysis of thrombin and ADP induced rise in intracellular Ca2+ concentration of single blood platelets.

Cytoplasmic free Ca2+ concentration, [Ca2+]i, was estimated in single rabbit blood platelets by digital imaging microscopy with the use of the specific Ca(2+)-indicator dye Fura-2. Uneven distribution and low level of [Ca2+]i was found in the resting platelet even in the presence of extracellular 1 mM Ca2+. Thrombin at 1 unit/ml immediately caused a transient increase in [Ca2+]i, which was followed by a secondary and sustained increase in [Ca2+]i. The distribution of increased levels of [Ca2+]i was also shown to be uneven within the cell. The presence of 1 mM EGTA in the medium only slightly decreased the initial rise in [Ca2+]i, but completely inhibited the latter phase, a sustained rise in [Ca2+]i. This result shows that the initial rise of [Ca2+]i might not be caused by Ca2+ influx, but might be induced by mobilization of Ca2+ from intracellular Ca2+ storage sites. This speculation is further supported by the fact that the elevated [Ca2+]i induced by thrombin immediately decreased to the base line value when 3 mM EGTA was applied. Thus, thrombin induced elevation of [Ca2+]i is suggested to consist of two different processes, namely the mobilization of Ca2+ from the intracellular storage sites and the successive Ca2+ influx through the receptor activated Ca2+ channels. Stimulation with ADP also caused a rapid elevation of platelet [Ca2+]i, but this effect of ADP was different form that of thrombin. Thus, the ADP induced rise in [Ca2+]i was accompanied by oscillation and was inhibited by extracellular EGTA. Our present experiment is the first report that clearly and directly reveals the differences between the effects of thrombin and ADP on [Ca2+]i of platelets.     

Characterisation of the serotonin efflux induced by cytosolic Ca2+ and Na+ concentration increase in human platelets.

BACKGROUND/AIM: The present study aimed at elucidating the mechanism(s) of serotonin (5-HT) efflux induced by thapsigargin from human platelets in the absence of extra-cellular Ca2+. METHODS: Efflux of pre-loaded radiolabeled serotonin was generally determined by filtration techniques. Cytosolic concentrations of Ca2+, Na+ and H+ were measured with appropriate fluorescent probes. RESULTS: 5-HT efflux from control or reserpine-treated platelets--where reserpine prevents 5-HT transport into the dense granules--was proportional to thapsigargin evoked cytosolic [Ca2+]c increase. Accordingly factors as prostacyclin, aspirin and calyculin which reduced [Ca2+]c-increase also inhibited the 5-HT efflux. Thapsigargin, which also caused a remarkable increase in cytosolic [Na+]c, promoted less 5-HT release, in parallel to lower [Na+]c and [Ca2+]c increase, when added to platelet suspensions containing low [Na+]. The Na+/H+ exchanger monensin increased the [Na+]c and induced 5-HT efflux without affecting the Ca2+ level. The 5-HT efflux induced by both [Ca2+] or [Na+]c increase did not depend on pH or membrane potential changes, whereas it decreased in the absence of extra-cellular K+, and increased in the absence of Cl- or Na+. CONCLUSION: Increases in [Ca2+]c and [Na+]c independently induce serotonin efflux through the outward directed plasma membrane serotonin transporter SERT. This event might be physiologically important at the level of capillaries or narrowed arteries where platelets are subjected to high shear stress which causes [Ca2+]c increase followed by 5-HT release which might exert vasodilatation.     

Involvement of inositol 1,4,5-trisphosphate in Ca2+ influx in thrombin stimulated human platelets

By incubating platelets at low temperature (10 degrees C), the relationship between Ca2+ mobilization and formation of inositol 1,4,5-trisphosphate (IP3) in thrombin stimulated platelets could be precisely investigated. In the presence of 1 mM EGTA, time dependent changes in the intracellular free calcium concentration [( Ca2+]i) were closely related to those in IP3 formation. Time course of the influx of external Ca2+, estimated by delta [Ca2+]i obtained by subtracting [Ca2+]i in the presence of 1 mM EGTA from that in the presence of 1 mM CaCl2 was also very similar to that of IP3 formed. Furthermore, the increase in delta [Ca2+]i was extremely well correlated with the amount of IP3 formed (Y = 49X - 34, r = 0.99). Thus, these data indicate that IP3 might be involved not only in intracellular Ca2+ mobilization but in Ca2+ influx of human platelets stimulated by thrombin.     

Feedback inhibition of Ca2+ release by Ca2+ is the underlying mechanism of agonist-evoked intracellular Ca2+ oscillations in pancreatic acinar cells.

Oscillations of free intracellular Ca2+ concentration ([Ca2+]i) are known to occur in many cell types during physiological cell signaling. To identify the basis for the oscillations, we measured both [Ca2+]i and extracellular Ca2+ concentration ([Ca2+]o) to follow the fate of Ca2+ during stimulation of [Ca2+]i oscillations in pancreatic acinar cells. [Ca2+]i oscillations were initiated by either t-butyloxycarbonyl-Tyr(SO3)-Nle-Gly-Tyr-Nle-Asp-2-phenylethyl ester (CCK-J), which mobilized Ca2+ from the inositol 1,4,5-trisphosphate (IP3)-insensitive pool, or low concentration of cholecystokinin octapeptide (CCK-OP), which mobilized Ca2+ from the IP3-sensitive internal pool. Little Ca2+ efflux occurred during the oscillations triggered by CCK-J or CCK-OP in spite of a large average increase in [Ca2+]i. When internal store Ca2+ pumps were inhibited with thapsigargin (Tg) during [Ca2+]i oscillations, a rapid Ca2+ efflux occurred similar to that measured in intensely stimulated, nonoscillatory cells. Tg also stimulated 45Ca efflux from internal pools of cells stimulated with CCK-J or a low concentration of CCK-OP. Hence, a large fraction of the Ca2+ released during each spike is reincorporated by the internal store Ca2+ pumps. Surprisingly, when the increase in [Ca2+]i during stimulation of oscillations was prevented by loading the cells with 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid, a persistent activation of Ca2+ release and Ca2+ efflux occurred. This was reflected as a persistent increase in [Ca2+]o in cells suspended at low [Ca2+]o or persistent efflux of 45Ca from internal stores of cells maintained at high [Ca2+]o. Since agonist-stimulated Ca2+ release evidently remains activated when [Ca2+]i is highly buffered, the primary mechanism determining Ca2+ oscillations must include an inhibition of Ca2+ release by [Ca2+]i. Loading the cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid had no apparent effect on the levels or kinetics of IP3 formation in agonist-stimulated cells. This suggests that [Ca2+]i regulated the oscillation by inhibition of Ca2+ release independent of its possible effects on cellular levels of IP3.     

Platelet free calcium concentrations measured with fura-2 are influenced by the transmembrane sodium gradient

The influence of the transmembrane Na+ gradient on the intracellular free calcium concentration, [Ca2+]i, was studied in Sepharose gel-filtered platelets from healthy human subjects, using the Ca-sensitive fluorescent dye, fura-2. Raising the internal Na+ concentration, [Na+]i, by Na+ pump inhibition with 0.05 mM ouabain, without changing external Na+ did not cause a significant increase in [Ca2+]i. Substitution of extracellular Na+ by iso-osmolar sucrose induced a rapid (half-time about 2 min) and significant rise in [Ca2+]i; this effect was amplified in Na-loaded platelets. Partial restitution of external Na+ in these cells with increased [Ca2+]i promoted a significant and rapid Na+ concentration-dependent fall in [Ca2+]i; little decline in [Ca2+]i was observed if K+ was used instead of Na+. These observations represent in vitro evidence for the existence of a Na/Ca exchange mechanism in human platelets that may, in vivo, participate in the control of [Ca2+]i.     

Intracellular calcium oscillations regulate ciliary beat frequency of airway epithelial cells

The effect of ATP-induced Ca2+ oscillations on ciliary activity was examined in airway epithelial cells by simultaneously measuring the ciliary beat frequency (CBF) and the intracellular Ca2+ concentration ([Ca2+]i) near the base of the cilia. Exposure to extracellular ATP (ATPo) induces a rapid and large increase in both [Ca2+]i and CBF, followed by oscillations in [Ca2+]i and a sustained elevation in CBF. After each Ca2+ oscillation, the [Ca2+]i returned to near basal values. By contrast, the CBF remained elevated during these Ca2+ oscillations, although each Ca2+ oscillation induced small variations in CBF. During Ca2+ oscillations, increases in CBF closely followed the rising phase of increases in [Ca2+]i, but declines in CBF lagged behind declines in [Ca2+]i. Higher frequency Ca2+ oscillations reduced variations in CBF, producing a stable and sustained elevation in CBF. The maximal CBF was induced by Ca2+ oscillations and was 15% greater than the CBF induced by the substantially larger initial [Ca2+]i increase. These data demonstrate that the rate of CBF is not directly dependent on the absolute [Ca2+]i, but is dependent on the differential changes in [Ca2+]i and suggest that CBF in airway epithelial cells is regulated by frequency-modulated Ca2+ signaling.     

Effects of AlF4 – and ATP on intracellular calcium dynamics of crypt epithelial cells in mouse small intestine

Previous digital imaging analysis of intracellular calcium ion ([Ca2+]i) dynamics in the crypt of the small intestine showed little response by most columnar cells to cholinergic and adrenergic agonists. The objective of the present study was to demonstrate whether G-protein activators and other transmitters elicit [Ca2+]i changes in crypt cells. We used digital imaging to analyze spatiotemporal dynamics of [Ca2+]i in Fura-2/AM-loaded isolated crypts of mouse duodenum and ileum. A1F4- increased [Ca2+]i in crypt columnar cells. In many cases, we observed [Ca2+]i oscillations, which were synchronized throughout the entire crypt. The oscillations were blocked by octanol. ATP, but not adenosine, caused a [Ca2+]i increase in middle crypt-regions of the duodenum and upper regions of the ileum, and the [Ca2+]i wave propagated towards the crypt bottom. The ATP-induced [Ca2+]i increase was prevented by pretreatment with thapsigargin or suramin, but not by La3+ or an extracellular Ca(2+)-free environment. Neither dopamine, 5-hydroxytryptamine (5-HT), histamine, vasoactive intestinal peptide, substance P. cholera toxin, nor guanylin had significant effects. The [Ca2+]i dynamics of Paneth cells were independent of the AlF4(-)-induced synchronous oscillations of columnar cells and of the ATP-induced [Ca2+]i wave. In conclusion, crypt columnar cells have [Ca2+]i-dependent intracellular signaling mechanisms that are linked with G proteins, and by which the cells communicate with each other. ATP elicited [Ca2+]i mobilization from columnar cells via P2 receptors, although some regional differences were noted between the duodenum and ileum.     

Role of Ca(2+)-ATPase in spontaneous oscillations of cytosolic free Ca2+ in GH3 rat pituitary cells.

The relative contribution of voltage-sensitive Ca2+ channels, Ca(2+)-ATPases, and Ca2+ release from intracellular stores to spontaneous oscillations in cytosolic free Ca2+ concentration ([Ca2+]i) observed in secretory cells is not well characterized owing to a lack of specific inhibitors for a novel thapsigargin (Tg)-insensitive Ca(2+)-ATPase expressed in these cells. We show that spontaneous [Ca2+]i oscillations in GH3 cells were unaffected by Ca2+ depletion in inositol-1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores by the treatment of Tg, but could be initiated by application of caffeine. Moreover, we demonstrate for the first time that these spontaneous [Ca2+]i oscillations were highly temperature dependent. Decreasing the temperature from 22 to 17 degrees C resulted in an increase in the frequency, a reduction in the amplitude, and large inhibition of [Ca2+]i oscillations. Furthermore, the rate of ATP-dependent 45Ca2+ uptake into GH3-derived microsomes was greatly reduced at 17 degrees C. The effect of decreased temperatures on extracellular Ca2+ influx was minor because the frequency and amplitude of spontaneous action potentials, which activate L-type Ca2+ channels, was relatively unchanged at 17 degrees C. These results suggest that in GH3 secretory cells, Ca2+ influx via L-type Ca2+ channels initiates spontaneous [Ca2+]i oscillations, which are then maintained by the combined activity of Ca(2+)-ATPase and Ca(2+)-induced Ca2+ release from Tg/IP3-insensitive intracellular stores.     

Desynchronising effect of the endothelium on intracellular Ca2+ concentration dynamics in vascular smooth muscle cells of rat mesenteric arteries

The kinetics of the intracellular Ca2+ concentration ([Ca2+]i) of vascular smooth muscle cells (VSMCs) in rat small mesenteric arteries was investigated by confocal laser scanning microscopy using the fluorescent Ca2+ indicator fluo-3 AM. One micromole noradrenaline (NA) induced randomly distributed transient elevations of [Ca2+]i in several single VSMCs which were weakly temporally coupled. Higher NA concentrations of 3 or 10 microM, however, induced strongly synchronised [Ca2+]i oscillations in VSMCs. In preparations with intact endothelium, the synchronisation of [Ca2+]i signals was attenuated by acetylcholine (ACh) but augmented by the NO synthase antagonist L-NAME, pointing to a desynchronising effect of the endothelium even under basal conditions. In preparations with or without intact endothelium sodium nitroprusside (SNP) as well as the gap-junction uncoupler heptanol reversibly desynchronised the [Ca2+]i transients. The effect of ACh but not that of SNP was influenced by L-NAME. Propagated intracellular [Ca2+]i waves had a velocity of 25 microm/s. The phase shift of [Ca2+]i oscillations between single VSMCs were maximally 2s and independent of the distance of up to 90 microm between individual cells. Therefore, we consider intercellular [Ca2+]i waves to be too slow to account for the synchronisation of [Ca2+]i oscillations.We conclude that the coupling of [Ca2+]i signals in vascular smooth muscle cells is not constant but highly regulated by NA and by endothelium derived NO. Oscillations of vessel contraction at high sympathetic tone may be induced by synchronisation of [Ca2+]i transients of distinct VSMCs whereas endothelium derived NO inhibits vasomotion by desynchronising [Ca2+]i transients of single VSMCs.     

Effect of intracellular delivery of energy metabolites on intracellular Ca2+ in mouse islets of Langerhans

Regulation of glucose-induced oscillations in intracellular Ca2+ concentration ([Ca2+]i) was investigated by using a novel technique, electroporation from an electrolyte-filled capillary, to deliver energy metabolites to the intracellular compartment of mouse islets. Intracellular application of ATP resulted in a nifedipine-sensitive increase in [Ca2+]i, consistent with a KATP-channel dependent mechanism of Ca2+ influx. [Ca2+]i in islets exposed to 10 mM glucose oscillated with a period of approximately 3 min, often superimposed with faster oscillations. Electroporation of ATP blocked all types of oscillations and elevated [Ca2+]i while delivery of ADP had no effect on oscillations. Intracellular delivery of glucose-6-phosphate or fructose-1,6-bisphosphate tended to transform slow oscillations to fast oscillations. These results demonstrate that modulation of ATP concentrations and glycolytic flux are important in development of slow oscillations.     

Oscillations of intracellular calcium induced by vasopressin in individual fura-2-loaded mesangial cells. Frequency dependence on basal calcium concentration, agonist concentration, and temperature

Intracellular free calcium concentration ([Ca2+]i) was measured in fura-2-loaded single rat mesangial cells by dual wavelength spectrofluorometry. Stimulation with arginine vasopressin (AVP) caused an initial sharp rise of [Ca2+]i followed by repetitive spikes. The frequency of the oscillations was dependent on the concentration of AVP. At 0.1, 1.0, 10.0, and 100.0 nM AVP, the frequencies of oscillations were 0.17 +/- 0.05 (n = 6), 0.32 +/- 0.05 (n = 6), 0.49 +/- 0.05 (n = 6), and 0.48 +/- 0.05 min-1 (n = 5), respectively. Reduction in extracellular [Ca2+] reduced the frequency of AVP-induced oscillations but did not abolish the oscillations. The frequency of calcium oscillations, upon stimulation with 1.0 nM AVP, was directly correlated with the basal [Ca2+]i prior to stimulation. Oscillation frequency increased with increasing temperature. An Arrhenius plot between 24 and 37 degrees C indicated a strong temperature dependency of the oscillations with a Q10 of 3.0. Protein kinase C stimulation by active phorbol esters inhibited AVP-induced calcium oscillations but not the initial [Ca2+] response to AVP. These observations are consistent with a model incorporating a feedback loop linking [Ca2+]i to the mechanism of [Ca2+]i increase. Ca(2+)-induced Ca2+ release may be involved, whereby inositol 1,4,5-trisphosphate (inositol 1,4,5-P3) formation releases Ca2+ from an inositol 1,4,5-P3-sensitive pool, with subsequent Ca2+ uptake and release from an inositol 1,4,5-P3-insensitive pool.     

Two thrombin-activated Ca2+ channels in human platelets.

The regulation of extracellular Ca2+ entry into fura-2-loaded human platelets was examined following stimulation with thrombin. In the presence of external Ca2+, stimulation of platelets with thrombin resulted in a rapid increase, followed by a plateau, in intracellular Ca2+ concentration ([Ca2+]i). Pretreatment with wortmannin, a specific inhibitor of myosin light chain kinase, suppressed only the plateau phase and had no effect on the initial rapid increase in [Ca2+]i. In Ca(2+)-free EGTA buffer, thrombin induced a transient and relatively small increase in [Ca2+]i caused by Ca2+ release from internal stores. When Ca2+ was added subsequently to the Ca(2+)-free medium within 10 min after thrombin activation, a marked increase in [Ca2+]i was seen, reflecting thrombin-stimulated external Ca2+ entry. With the Ca(2+)-free medium, wortmannin did not affect either the Ca2+ mobilization from the internal stores or the rapid external Ca2+ entry at early time points (within 5 s) after thrombin stimulation, whereas it significantly inhibited Ca2+ entry when Ca2+ was added later (at 3 min). Wortmannin inhibition of this late Ca2+ entry and that of 20-kDa myosin light chain phosphorylation after thrombin stimulation were dose- and preincubation time-dependent and correlated well with each other. These results suggest that two different channels are responsible for Ca2+ entry in human platelets at the early and late phases of thrombin stimulation and that the channel responsible for the late phase of Ca2+ entry may be activated by a mechanism involving myosin light chain kinase.     

Stimulus-Induced Oscillations in Guard Cell Cytosolic Free Calcium

Ca2+ is implicated as a second messenger in the response of stomata to a range of stimuli. However, the mechanism by which stimulus-induced increases in guard cell cytosolic free Ca2+ ([Ca2+]i) are transduced into different physiological responses remains to be explained. Oscillations in [Ca2+]i may provide one way in which this can occur. We used photometric and imaging techniques to examine this hypothesis in guard cells of Commelina communis. External Ca2+ ([Ca2+]e), which causes an increase in [Ca2+]i, was used as a closing stimulus. The total increase in [Ca2+]i was directly related to the concentration of [Ca2+]e, both of which correlated closely with the degree of stomatal closure. Increases were oscillatory in nature, with the pattern of the oscillations dependent on the concentration of [Ca2+]e. At 0.1 mM, [Ca2+]e induced symmetrical oscillations. In contrast, 1.0 mM [Ca2+]e induced asymmetric oscillations. Oscillations were stimulus dependent and modulated by changing [Ca2+]e. Experiments using Ca2+ channel blockers and Mn2+-quenching studies suggested a role for Ca2+ influx during the oscillatory behavior without excluding the possible involvement of Ca2+ release from intracellular stores. These data suggest a mechanism for encoding the information required to distinguish between a number of different Ca2+-mobilizing stimuli in guard cells, using stimulus-specific patterns of oscillations in [Ca2+]i.     

Effect of calcium concentration and inhibitors on the responses of platelets stimulated with collagen: contrast between human and rabbit platelets.

1. Variations in the concentration of Ca2+ [Ca2+] in the suspending medium have different effects on the responses of human and rabbit platelets to collagen. 2. When rabbit platelets are stimulated with a low concentration of collagen (0.5 micrograms/ml), aggregation, release of granule contents, and formation of thromboxane are maximal when the suspending medium contains [Ca2+] in the physiological range (0.5-2.0 mM), and very slight in a medium with no added Ca2+. 3. In contrast, human platelets respond most strongly when the suspending medium contains no added Ca2+ [( Ca2+] approx. 20 microM); this is attributable to the enhanced formation of thromboxane A2 (TXA2) upon close platelet-to-platelet contact in this medium. 4. When TXA2 formation is blocked by inhibition of cyclo-oxygenase with aspirin or indomethacin, rabbit platelet aggregation and release in response to 1.25-10 micrograms/ml collagen is also maximal at [Ca2+] of 0.5-2.0 mM and least at 20 microM; human platelets do not aggregate and the extent of release is relatively independent of [Ca2+]. 5. In 1 mM [Ca2+], use of apyrase and/or ketanserin with rabbit platelets in which TXA2 formation is blocked shows that released ADP and serotonin make large contributions to aggregation and release in response to high concentrations of collagen; human platelet aggregation is largely dependent on TXA2. 6. Use of fura-2-loaded platelets shows that the collagen-induced rise in cytosolic [Ca2+] is only slightly inhibited by aspirin or indomethacin in rabbit platelets, but almost completely inhibited in human platelets. 7. Responses of rabbit platelets to collagen are less dependent on TXA2 than those of human platelets. Released ADP and serotonin make major contributions to the responses of rabbit platelets to collagen.