Dissociation of Ca2+ entry and Ca2+ mobilization responses to angiotensin II in bovine adrenal chromaffin cells

In fura-2-loaded bovine adrenal chromaffin cells, 0.5 microM angiotensin II (AII) stimulated a 185 +/- 19 nM increase of intracellular-free calcium [( Ca2+]i) approximately 3 s after addition. The time from the onset of the response until achieving 50% recovery (t 1/2) was 67 +/- 10 s. Concomitantly, AII stimulated both the release of 45Ca2+ from prelabeled cells, and a 4-5-fold increase of [3H]inositol 1,4,5-trisphosphate [( 3H]Ins(1,4,5)P3) levels. In the presence of 50 microM LaCl3, or when extracellular-free Ca2+ [( Ca2+]o) was less than 100 nM, AII still rapidly increased [Ca2+]i by 95-135 nM, but the t 1/2 for recovery was then only 23-27 s. In medium with 1 mM MnCl2 present, AII also stimulated a small amount of Mn2+ influx, as judged by quenching of the fura-2 signal. When [Ca2+]o was normal (1.1 mM) or low (less than 60 nM), 1-2 microM ionomycin caused [Ca2+]i to increase 204 +/- 26 nM, while also releasing 45-55% of bound 45Ca2+. With low [Ca2+]o, ionomycin pretreatment abolished both the [Ca2+]i increase and 45Ca2+ release stimulated by AII. However, after ionomycin pretreatment in normal medium, AII produced a La3+-inhibitable increase of [Ca2+]i (103 +/- 13 nM) with a t 1/2 of 89 +/- 8 s, but no 45Ca2+ release. No pretreatment condition altered AII-induced formation of [3H]Ins(1,4,5)P3. We conclude that AII increased [Ca2+]i via rapid and transient Ca2+ mobilization from Ins(1,4,5)P3- and ionomycin-sensitive stores, accompanied (and/or followed) by Ca2+ entry through a La3+-inhibitable divalent cation pathway. Furthermore, the ability of AII to activate Ca2+ entry in the absence of Ca2+ mobilization (i.e. after ionomycin pretreatment) suggests a receptor-linked stimulus other than Ca2+ mobilization initiates Ca2+ entry.     

Intracellular alkalinization leads to Ca2+ mobilization from agonist-sensitive pools in bovine aortic endothelial cells

Receptor-stimulated phosphoinositide turnover leads to activation of Na+/H+ exchange and subsequent intracellular alkalinization. To probe the effect of increased intracellular pH (pHi) on Ca2+ homeostasis in cultured bovine aortic endothelial cells (BAEC), we studied the effect of weak bases, ammonium chloride (NH4Cl) and methylamine (agents which increase pHi by direct passive diffusion), on resting and ATP (purinergic receptor agonist)-induced Ca2+ fluxes. Changes in cytosolic free Ca2+ ([Ca2+]i) or pHi were monitored in BAEC monolayers using the fluorescent dyes, fura-2 or 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, respectively. NH4Cl-induced, dose-dependent (5-20 mM) increases in [Ca2+]i (maximum change = 195 +/- 26 nM) which were temporally similar to the NH4Cl-induced pHi increases. Methylamine (20 mM) induced a more sustained pHi increase and also stimulated a prolonged [Ca2+]i increase. When BAEC were bathed in HCO3- buffer, removal of extracellular CO2/bicarbonate caused pHi to increase and also induced [Ca2+]i to increase transiently. Extracellular Ca2+ removal did not abolish the rapid NH4Cl-induced rise in [Ca2+]i, although the response was blunted and more transient. NH4Cl addition to BAEC cultures resulted in an increase in 45Ca efflux and decrease in total cell 45Ca content. BAEC treatment with ATP (100 microM) to deplete inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pools completely blocked the NH4Cl (20 mM)-induced rise in [Ca2+]i. Likewise, prior NH4Cl addition partially inhibited ATP-induced increases in [Ca2+]i, as well as slowed the frequency of repetitive [Ca2+]i spikes in single endothelial cells due to agonist. NH4Cl augmented the rate of [Ca2+]i increase that occurs in response to the depletion of agonist-sensitive intracellular Ca2+ pools. However, the internal Ca2+ store remained depleted during the continued presence of NH4Cl, as indicated by a decreased [Ca2+]i response to ATP in Ca2(+)-free medium. Finally, NH4Cl exerted these actions without affecting basal or ATP-stimulated IP3 formation. These observations provide direct evidence that increased pHi leads to Ca2+ mobilization from an agonist-sensitive pool and impairs Ca2+ pool(s) refilling mechanisms without altering cellular IP3 levels.     

The inositol 1,4,5-trisphosphate-forming agonist histamine activates a ryanodine-sensitive Ca2+ release mechanism in bovine adrenal chromaffin cells

The role of a Ca(2+)-induced Ca2+ release (CICR) mechanism in the generation of agonist-induced increases of intracellular free Ca2+ concentration ([Ca2+]i) was studied in bovine adrenal chromaffin cells. In single cells, repetitive stimulations with caffeine at 200-s intervals evoked reproducible spikes of [Ca2+]i. Ryanodine, an agent that interacts with the CICR channel of muscle, inhibited the caffeine-induced spikes of [Ca2+]i in a "use-dependent" way. High affinity binding sites for [3H]ryanodine (Kd 3.3 nM, Bmax 26 fmol/mg protein) were also detected in membranes from chromaffin cells, supporting the presence of a caffeine- and ryanodine-sensitive CICR channel. Pretreatment of single cells with caffeine + ryanodine to reduce the size of the caffeine-sensitive Ca2+ compartment inhibited a subsequent spike of [Ca2+]i evoked by histamine, a D-myo-inositol 1,4,5-trisphosphate-forming agonist. This demonstrates that a significant portion of the Ca2+ released by histamine comes from a caffeine- and ryanodine-sensitive pool. Ryanodine inhibited by 50% the size of [Ca2+]i spikes evoked by repetitive stimulation with histamine and did so in a use-dependent manner. These data suggest that, in addition to D-myoinositol 1,4,5-trisphosphate, activation of a caffeine- and ryanodine-sensitive CICR channel participates in the generation of histamine-induced release of intracellular Ca2+.     

Receptor density determines secretory response patterns mediate by inositol lipid-derived second messengers. Comparison of thyrotropin-releasing hormone and carbamylcholine actions in thyroid-stimulating hormone-secreting mouse pituitary tumor cells

Signal transduction by thyrotropin-releasing hormone (TRH) and carbamylcholine (CCH) in some cells is mediated by inositol lipid hydrolysis forming the second messengers, inositol 1,4,5-trisphosphate (I-1,4,5-P3) and 1,2-diacylglycerol, and causing elevation of cytoplasmic free Ca2+ concentration [( Ca2+]i). In mouse thyrotropic tumor (TtT) cells, maximally effective doses of TRH caused biphasic stimulation of thyroid-stimulating hormone (TSH) secretion, whereas CCH stimulated monophasic sustained TSH secretion without a burst phase. TRH, at maximally effective doses, stimulated a rapid marked increase in I-1,4,5-P3 which was associated with a rapid elevation of [Ca2+]i to approximately 1000 nM, whereas maximally effective doses of CCH caused little increase in I-1,4,5-P3 and no burst elevation of [Ca2+]i. Both TRH and CCH caused sustained modest (to 210-280 nM) elevations of [Ca2+]i which were inhibited by voltage-sensitive channel-blocking agents and stimulated sustained hydrolysis of inositol lipids. CCH-like responses were observed when TtT cells were stimulated by low doses of TRH. In TtT cells prepared from five tumors, the ratio of the number of TRH receptors to muscarinic receptors ranged from 10 to 40:1. Lastly, CCH-like responses were observed with maximally effective doses of TRH when the TRH receptor number was down-regulated to a level similar to that of muscarinic receptors. These data suggest that the kinetic pattern of stimulated TSH secretion caused by secretagogues that use the inositol lipid signal transduction pathway is determined by the density of receptors. In particular, there appears to be a minimal number of receptor-ligand complexes which is required to generate rapidly sufficient I-1,4,5-P3 to release intracellular Ca2+ and cause a secretory burst.     

PDGF-AA effectively stimulates early events but has no mitogenic activity in AKR-2B mouse fibroblasts.

The response of AKR-2B mouse fibroblasts, which express approximately equal numbers of platelet-derived growth factor (PDGF)-alpha and -beta receptors on their surface (V. Hoppe et al. Eur. J. Biochem. 187, 207-214, 1990) to all three isoforms of PDGF, was studied. All isoforms stimulated early events, i.e., receptor autophosphorylation on tyrosine, total cellular phosphorylation, increase in 32P-labeled phospholipid content, but there was no correlation between the extents measured for the different effects. Although rPDGF-AA effectively stimulated these early events, it was unable to induce [3H]thymidine incorporation and cell growth whereas rPDGF-BB and -AB stimulated the division of more than 90% of the cells. This activity was restored by addition of insulin-like growth factor I (IGF-I), which itself exhibited only a low mitogenic activity. rPDGF-AB or -BB did not require the presence of IGF-I to fully stimulate cells for [3H]thymidine incorporation and cell division. Apparently, rPDGF-AA induced only a "competence" state of the cells whereas rPDGF-AB or -BB was also able to initiate "progression". It is speculated that some early events occurring during the competence phase might be part of a "maintenance" program elicited by growth factors.     

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.     

Partial purification and reconstitution of inositol 1,4,5-trisphosphate receptor/Ca2+ channel of bovine liver microsomes.

The binding of inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] to bovine liver microsomes was characterized. The Ins(1,4,5)P3 receptor of the microsomes was solubilized by 1% Triton X-100 and purified by sucrose density gradient, Heparin-Sepharose, DEAE-Toyopearl, ATP-Agarose, and Ins(1,4,5)P3-Sepharose column chromatographies. More than 1,000-fold enrichment of the Ins(1,4,5)P3-binding activity was achieved. Kd values of the binding activity were 2.8 nM in microsomes and 3.0 nM in the partially purified receptor, respectively, and the binding activity was optimal in the medium containing 100 mM KCl and at pH between 7.5 and 8.5. The presence of Ca2+ failed to inhibit the binding. Phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PtdIns), and phosphatidylinositol-4-monophosphate [PtdIns(4)P] showed no effect on the Ins(1,4,5)P3 binding. However, soybean phospholipids asolectin and phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] strongly inhibited the binding activity. PtdIns(4,5)P2 inhibited the activity competitively with a half-maximal inhibitory concentration of 30 micrograms/ml. The partially purified Ins(1,4,5)P3 receptor was reconstituted into proteoliposomes. Fluorescence measurements using Quin 2 indicated that Ins(1,4,5)P3 stimulated Ca2+ influx into the proteoliposomes. The EC50 of Ins(1,4,5)P3 on Ca2+ influx was 50 nM. This result strongly suggest that Ins(1,4,5)P3 binding protein of liver microsomes acts as a physiological Ins(1,4,5)P3 receptor/Ca2+ channel.     

Muscarinic receptor-induced phosphoinositide hydrolysis at resting cytosolic Ca2+ concentration in PC12 cells

In PC12 cells, cultured in the presence of nerve growth factor to increase their complement of muscarinic receptors, treatment with carbachol induces muscarinic receptor-dependent rises in free cytosolic Ca2+ as well as hydrolysis of membrane phosphoinositides. Experiments were carried out to clarify the relationship between these two receptor-triggered events. In particular, since inositol-1,4,5-trisphosphate (the hydrophilic metabolite produced by the hydrolysis of phosphatidylinositol-4,5-bisphosphate) is believed to mediate intracellularly the release of Ca2+ from nonmitochondrial store(s), it was important to establish whether it can be generated at resting cytoplasmic concentration of Ca2+ (approximately 0.1 microM). Cells incubated in Ca2+-free medium were depleted of their cytoplasmic Ca2+ stores by pretreatment with ionomycin. When these cells were then treated with carbachol, their cytosolic concentration of Ca2+ remained at the resting level, whereas inositol-1,4,5-trisphosphate generation was still markedly stimulated. Our results demonstrate that an increase in the concentration of cytosolic Ca2+ is not a necessary intermediate between receptor activation and phosphoinositide hydrolysis, and therefore support the second-messenger role of inositol-1,4,5-trisphosphate.     

Receptor coupled events in bradykinin action: rapid production of inositol phosphates and regulation of cytosolic free Ca2+ in a neural cell line.

The addition of bradykinin to NG115-401L cells grown on coverslips results in the generation of rapid transient increases in intracellular [Ca2+] and inositol phosphates. Changes in intracellular Ca2+, measured using the fluorescent indicator dye Fura-2, show two components; an initial rapid peak in [Ca2+]i which is essentially independent of extracellular Ca2+, and a sustained plateau dependent on the presence of extracellular Ca2+. Analysis of bradykinin stimulated production of [3H]inositol phosphates, by h.p.l.c., shows a rapid biphasic production of inositol 1,4,5-trisphosphate, inositol tetrakisphosphate and inositol bisphosphates, followed by a sustained rise in inositol 1,3,4-trisphosphate production. Quantitative measurements have indicated the presence of other, more polar, [3H]inositol-labelled metabolites which do not show major changes on bradykinin stimulation. The initial phase of inositol phosphate production parallels the rapid transient increase in intracellular [Ca2+], however, the second phase of inositol phosphate production occurs when intracellular [Ca2+] is declining and implies a complex series of regulatory events following receptor stimulation. Similar time courses of inositol 1,4,5-trisphosphate and Ca2+ signals provides supporting evidence that inositol 1,4,5-trisphosphate is the second messenger coupling bradykinin receptor stimulation to release of Ca2+ from intracellular stores.     

Parathyroid hormone inhibition of Na+/phosphate cotransport in OK cells: intracellular [Ca2+] as a second messenger

Parathyroid hormone increases cellular cAMP, 1,2-diacylglycerol, inositol 1,4,5-trisphosphate and cytosolic Ca2+ concentration ([Ca2+]i) in OK cells. In the present study, we determined the importance of the PTH-dependent increase in [Ca2+]i in the control of sodium-dependent phosphate (Na+/Pi) cotransport. PTH (10(-7) M) results in a transient increase in [Ca2+]i from basal levels of 67 +/- 4 nM to maximal concentrations of 190 +/- 9 nM. The increase in [Ca2+]i was dose-dependent with half-maximal increases at about 5.10(-8) M PTH. These hormone levels were 10(3)-fold higher than that required for half-maximal inhibition of Na+/Pi cotransport. Clamping [Ca2+]i with either intracellular Ca2+ chelators or by ionomycin in the presence of high concentrations of extracellular Ca2+ did not alter PTH-dependent inhibition of Na/Pi cotransport. Nor did indomethacin, an inhibitor of the cyclooxygenase pathway, influence the hormonal inhibition of cotransport. Accordingly, these data suggest that changes in [Ca2+]i and/or activation of the phospholipase A2 and the cyclooxygenase pathways are not involved in signal induction of the PTH-mediated control of Na+/Pi cotransport.     

Ca2+ Mobilized by Caffeine from the Inositol 1,4,5-Trisphosphate-Insensitive Pool of Ca2+ in Somatic Regions of Sympathetic Neurons Does Not Evoke [3H]Norepinephrine Release

The effects of electrical stimulation, muscarinic and serotonergic agonists, and caffeine on [3H]inositol 1,4,5-trisphosphate ([3H]Ins(1,4,5)P3) content, intracellular free Ca2+ concentration ([Ca2+]i), and release of [3H]norepinephrine ([3H]NE) were studied in cultured sympathetic neurons. Neuronal cell body [Ca2+]i was unaffected by muscarinic or serotonergic receptor stimulation, which significantly increased [3H]Ins(1,4,5)P3 content. Stimulation at 2 Hz and caffeine had no effect on [3H]Ins(1,4,5)P3, but caused greater than two-fold increase in [Ca2+]i. Only 2-Hz stimulation released [3H]NE. Caffeine had no effect on the release. When [Ca2+]i was measured in growth cones, only electrical stimulation produced an increase in [Ca2+]i. The other agents had no effect on Ca2+ at the terminal regions of the neurons. We conclude that Ins(1,4,5)P3-insensitive, but caffeine-sensitive Ca2+ stores in sympathetic neurons are located only in the cell body and are not coupled to [3H]NE release.     

Na+/H+ exchange modulates Ca2+ mobilization in human platelets stimulated by ADP and the thromboxane mimetic U 46619

According to recent observations ADP stimulates platelets via activation of Na+/H+ exchange which increases cytosolic pH (pHi). This event initiates formation of thromboxane A2 (via phospholipase A2) and, thereafter, inositol 1,4,5-trisphosphate (via phospholipase C) which is known to mobilize Ca2+ from intracellular storage sites. We investigated changes in pHi and cytosolic free Ca2+, [Ca2+]i, activating platelets with ADP and the thromboxane mimetic U 46619. We found that ADP (5 microM) increased pHi from 7.15 +/- 0.08 to 7.35 +/- 0.04 (n = 8) in 2'-7'-bis-(carboxyethyl)-5,6-carboxyfluorescein-loaded platelets, whereas thromboxane A2 formation was inhibited by indomethacin. ADP also induced a dose-dependent Ca2+ mobilization in fura2-loaded platelets which again was not affected by indomethacin. [Ca2+]i increased by 54 +/- 10 nM (n = 8) at 1 microM and by 170 +/- 40 nM (n = 7) at 10 microM ADP above the resting value of 76 +/- 12 nM (n = 47). Inhibition of Na+/H+ exchange by ethylisopropylamiloride (EIPA) reduced ADP-induced Ca2+ mobilization by more than 65% in indomethacin-treated platelets. This inhibition could be completely overcome by artificially raising pHi using either NH4Cl or the Na+/H+ ionophore monensin. We found that U 46619 increased pHi by 0.18 +/- 0.05 at 0.1 microM and by 0.29 +/- 0.07 (n = 7) at 1.0 microM above the resting value via an EIPA-sensitive mechanism. In conflict with the proposed role of the Na+/H+ exchange we found that U 46619 raised [Ca2+]i via a mechanism that for more than 50% depended on intact Na+/H+ exchange. Again, artificially elevating pHi restored U 46619-induced Ca2+ mobilization despite the presence of EIPA. Thus, our data show that Na+/H+ exchange is a common step in platelet activation by prostaglandin endoperoxides/thromboxane A2 and ADP and enhances Ca2+ mobilization independently of phospholipase A2 activity.     

Inositol lipid hydrolysis contributes to the Ca2+ wave in the activating egg of Xenopus laevis.

We have used fluorescence ratio-imaging of fura-2 in the activating egg of Xenopus laevis to study the wave of increased intracellular free Ca2+ concentration ([Ca2+]i) while monitoring that of cortical granule exocytosis. Naturally matured eggs were dejellied, injected with fura-2, and activated by the iontophoresis of 1-30 nCoul of inositol-1,4,5-trisphosphate which triggers an immediate increase in free [Ca2+]i at the injection site. The Ca2+ rise spreads throughout the egg, reaching the opposite side in 5-8 min, and is followed by elevation of the fertilization envelope about 20-30 sec behind the [Ca2+]i wave. [Ca2+]i returns to preactivation levels within about 20 min after activation. We further studied the role of phosphatidylinositol-4,5-bisphosphate (PIP2) hydrolysis by microinjecting antibodies to PIP2 into the egg. PIP2 antibodies did not alter the propagation velocity of the wave but greatly reduced the amount of Ca2+ released in the egg cortex. These data suggest that PIP2 hydrolysis plays a role in the release of [Ca2+]i in the outer regions of the egg following activation.     

The T-cell antigen receptor regulates sustained increases in cytoplasmic free Ca2+ through extracellular Ca2+ influx and ongoing intracellular Ca2+ mobilization.

Signal transduction by the T-cell antigen receptor involves the turnover of polyphosphoinositides and an increase in the concentration of cytoplasmic free Ca2+ ([Ca2+]i). This increase in [Ca2+]i is due initially to the release of Ca2+ from intracellular stores, but is sustained by the influx of extracellular Ca2+. To examine the regulation of sustained antigen-receptor-mediated increases in [Ca2+]i, we studied the relationships between extracellular Ca2+ influx, the mobilization of Ca2+ from intracellular stores, and the contents of inositol polyphosphates after stimulation of the antigen receptor on a human T-cell line, Jurkat. We demonstrate that sustained antigen-receptor-mediated increases in [Ca2+]i are associated with ongoing depletion of intracellular Ca2+ stores. When antigen-receptor-ligand interactions are disrupted, [Ca2+]i and inositol 1,4,5-trisphosphate return to basal values over 3 min. Under these conditions, intracellular Ca2+ stores are repleted if extracellular Ca2+ is present. There is a tight temporal relationship between the fall in [Ca2+]i, the return of inositol 1,4,5-trisphosphate to basal values, and the repletion of intracellular Ca2+ stores. Reversal of the increase in [Ca2+]i preceeds any fall in inositol tetrakisphosphate by 2 min. These studies suggest that sustained antigen-receptor-induced increases in [Ca2+]i, although dependent on extracellular Ca2+ influx, are also regulated by ongoing inositol 1,4,5-trisphosphate-mediated intracellular Ca2+ mobilization. In addition, an elevated concentration of inositol tetrakisphosphate in itself is insufficient to sustain an increase in [Ca2+]i within Jurkat cells.     

Contribution of external and internal Ca2+ to changes in intracellular free Ca2+ produced by mitogens in Swiss 3T3 fibroblasts: the role of dihydropyridine sensitive Ca2+ channels

Changes in intracellular free Ca2+ concentration [( Ca2+]i) produced by growth factors and mitogens have been studied using aequorin-loaded Swiss 3T3 cells. Decreasing free Ca2+ in the external medium by using EGTA had no significant effect on the increase in [Ca2+]i produced by vasopressin, bradykinin, bombesin or prostaglandin E2, but reduced the increase in [Ca2+]i produced by platelet derived growth factor (PDGF) by 58%, by prostaglandin E1 44% and by prostaglandin F2 alpha 47%. The dihydropyridine Ca2+-channel antagonist nifedipine at 10 microM inhibited the [Ca2+]i response to PDGF by 41% in both the presence of and in the absence of external Ca2+. Methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl) pyridine-5-carboxylate (BAY K8644), a Ca2+-channel agonist, at 10 microM produced an increase in [Ca2+]i and decreased the [Ca2+]i response to PDGF by 39%. Nifedipine did not block 45Ca2+ uptake or release by inositol 1,4,5-trisphosphate in saponin-permeabilized Swiss 3T3 fibroblasts but BAY K8644 inhibited 45Ca2+ release by inositol 1,4,5-trisphosphate. The results suggest that the increase in [Ca2+]i caused by PDGF in Swiss 3T3 fibroblasts is due to the influx of external Ca2+ through dihydropyridine sensitive Ca2+ channels, as well as release of internal Ca2+.     

Epidermal growth factor and angiotensin II stimulate formation of inositol 1,4,5- and inositol 1,3,4-trisphosphate in hepatocytes. Differential inhibition by pertussis toxin and phorbol 12-myristate 13-acetate

The ability of epidermal growth factor (EGF) and angiotensin II to stimulate production of inositol trisphosphate and mobilize intracellular Ca2+ in hepatocytes was compared using quin2 fluorescence to monitor changes in Ca2+ levels and high performance liquid chromatography to resolve the inositol trisphosphate (InsP3) isomers. Both EGF and angiotensin II stimulated an increase in free intracellular Ca2+ concentration ([Ca2+]i) as well as a rapid increase in the production of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). Concentrations of angiotensin II which gave a rise in [Ca2+]i equivalent to that seen with maximal doses of EGF produced an equivalent increase in Ins(1,4,5)P3 formation. Both EGF and angiotensin II stimulated the formation of the Ins(1,3,4)P3 and inositol 1,3,4,5-tetrakisphosphate isomers. The formation of the Ins(1,3,4)P3 isomer lagged behind production of Ins(1,4,5)P3 but eventually reached higher levels in the cell. The initial rise in [Ca2+]i and InsP3 levels stimulated by EGF and angiotensin II was not affected by reducing the external Ca2+ concentration below 30 nM with an excess of [ethylenebis(oxyethylenenitrilo)] tetraacetic acid. Treatment of hepatocytes for 30-180 s with 1 micrograms/ml phorbol 12-myristate 13-acetate prior to the addition of EGF blocked the EGF-stimulated production of Ins(1,4,5)P3 and the increase in [Ca2+]i. Phorbol 12-myristate 13-acetate attenuated the production of Ins(1,4,5)P3 generated by angiotensin II over the concentration range of 10(-10) to 10(-8) M; however, the Ca2+ signal was only inhibited at the 10(-10) M dose of angiotensin II. Treatment of rats with pertussis toxin for 72 h prior to isolating hepatocytes blocked the ability of EGF to increase Ins(1,4,5)P3 and Ins(1,3,4)P3 but did not inhibit the ability of any concentration of angiotensin II to stimulate formation of InsP3 or inositol tetrakisphosphate. The observation that pertussis toxin selectively abolishes EGF-stimulated inositol lipid breakdown suggests that EGF and angiotensin II use different mechanisms to activate phospholipase C in hepatocytes.     

Ethanol-induced increases in [Ca2+]i and inositol (1,4,5) triphosphate in rat hepatocytes

Rat hepatocytes were studied for [Ca2+]i with Fura-2 at the single cell level using a microfluorometer-imaging system which showed that both the number of cells elevating [Ca2+]i and the magnitude of [Ca2+]i increase were directly dependent upon ethanol concentration between 50 mM and 1 M. Peak [Ca2+]i increases ranged from 27 nM with 50 mM ethanol to 57 nM after 1 M ethanol. Ethanol appeared to initiate calcium release from intracellular stores and caused a dose dependent production of inositol(1,4,5) triphosphate (Ins(1,4,5)P3) in hepatocytes. Low concentrations of ethanol (50-100 mM) did not significantly raise Ins(1,4,5)P3 although 300 mM-1 M increased Ins(1,4,5)P3 comparable to that found with vasopressin (5 nM). In summary, physiologic amounts of ethanol raise [Ca2+]i in rat hepatocytes, although at lower levels (50-100 mM) the changes may or may not be related to an Ins(1,4,5)P3 pathway.     

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.     

Spatial and temporal organization of calcium signalling in hepatocytes.

Treatment of hepatocytes with agonists which act via the second messenger inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), results in increases of cytosolic free Ca2+ [( Ca2+]i) which are manifest as a series of discrete [Ca2+]i transients or oscillations. With increasing agonist dose [Ca2+]i oscillation frequency increases and the initial latent period decreases, but the amplitude of the [Ca2+]i oscillations remains constant. Studies of these [Ca2+]i oscillations at the subcellular level have indicated that the [Ca2+]i changes do not occur synchronously throughout the cell, but initiate at a specific subcellular domain, adjacent to a region of the plasma membrane, and then propagate through the cell as a [Ca2+]i wave. For a given ceil, the locus of [Ca2+]i wave initiation is constant for every oscillation in a series and is also identical when the cell is sequentially stimulated with different agonists or when the phospholipase C-linked G protein is activated directly using AIF4-. The kinetics of the [Ca2+]i waves indicate that a Ca(2+)-activated mechanism is involved in propagating the oscillatory [Ca2+]i increases throughout the cell, and the data appear to be most consistent with a process of Ca(2+)-induced Ca2+ release. It is proposed that the ability to propagate [Ca2+]i oscillations into regions of the cell distal to the region in which the signal transduction apparatus is localized could serve an important function in allowing all parts of the cell to respond to the stimulus.     

Inositol-1,4,5-trisphosphate releases calcium from skinned cultured smooth muscle cells

We examined the effects of inositol-1,4,5-trisphosphate on 45Ca uptake and 45Ca efflux in the saponin skinned primary cultured rat aortic smooth muscle cells. 10 microM inositol-1,4,5-trisphosphate induced a rapid (half time less than 10 sec) and large quantity of Ca release in both 45Ca uptake and 45Ca efflux in the skinned cells preloaded with 1 microM free Ca. Dose response curves showed that 100 microM inositol-1,4,5-trisphosphate produced a maximal Ca release of 97.3% of the MgATP dependent 45Ca uptake or 289 mumoles/liter cells, which was much greater than the maximal caffeine induced Ca release and would be sufficient to produce maximal tension.