Epidermal growth factor (EGF) produces inositol phosphates and increases cytoplasmic free calcium in cultured porcine thyroid cells

The initial signal for thyroid cell proliferation is unknown. This is the first report to show that epidermal growth factor (EGF) produces inositol phosphates and increases cytoplasmic free calcium ([Ca2+]i) in the thyroid gland. In cultured porcine thyroid cells, 10 nM EGF produces a breakdown of phosphatidylinositol and stimulates inositol phosphate production. Ten nM EGF increases [Ca2+]i, measured using fura-2, a fluorescent Ca2+ indicator; the EGF-induced [Ca2+]i response occurs immediately, reaches a maximum within several seconds, and then slowly declines. EGF stimulates production of inositol phosphates, which seem to increase [Ca2+]i. Inositol phosphate production and an increase in [Ca2+]i after EGF-stimulation may function as an initial signal for thyroid cell proliferation.     

Interrelationship between insulin-like growth factor I-induced activation of the Na+/H(+)-antiporter and intracellular Ca(2+)-mobilization in thyroid cells.

Insulin-like growth factor I (IGF-I) increased cytoplamic pH (pHi) and cytoplasmic Ca2+ [( Ca2+]i) in cultured porcine thyroid cells. Inhibition of the Na+/H(+)-antiporter by dimethylamiloride or a reduction of external Na(+)-concentrations attenuates the increases in pHi and [Ca2+]i. The [Ca2+]i response to IGF-I is a pHi-dependent process. IGF-I activates Na+/H(+)-antiporter and alkalinizes thyroid cells. The resulting increase in pHi facilitates the [Ca2+]i response by adjusting the pHi closer to the pHi-optimum of the intracellular Ca(2+)-mobilizing system. One of the biological functions of IGF-I-induced activation of the Na+/H(+)-antiporter is to shift the pHi to an optimal value for the [Ca2+]i response.     

Involvement of Ca2+ influx in F(-)-stimulated pepsinogen release from guinea pig gastric chief cells

In isolated guinea pig gastric chief cells, pepsinogen release was stimulated by NaF in a dose-dependent manner. Cholecystokinin (CCK) and Ca2+ ionophore A23187 had no additional effect on NaF-stimulated pepsinogen release. CCK caused a rapid increase in intracellular free Ca2+ concentration ([Ca2+]i) monitored by Quin-2 and markedly stimulated inositol phosphate accumulation in chief cells. By contrast, NaF did not cause any change in [Ca2+]i. NaF, even at a maximal concentration for pepsinogen release, appeared to be relatively ineffective on inositol phosphate accumulation. On the other hand, NaF markedly stimulated Ca2+ influx into chief cells. These results suggest that F- stimulates pepsinogen release probably by increasing Ca2+ influx into chief cells. Since F- is a well known activator of guanine nucleotide regulatory proteins (G proteins), it is proposed that there may exist a G protein regulating the opening of Ca2+ channel in gastric chief cells.     

Lack of correlation between the activation of the Ca(2+)-phosphatidylinositol cascade and the regulation of DNA synthesis in the dog thymocyte.

Changes in the [Ca2+]i and/or activation of phospholipase C are thought to participate in the control by several growth factors of the mammalian cell proliferation. It has even been claimed that activation of the Ca(2+)-phosphatidylinositol cascade is sufficient to elicit cell proliferation [Jackson et al. (1988) Nature 335, 437-440; Julius et al. (1989) Science 244, 1057-1062]. In this work, we have evaluated the control of DNA synthesis by this cascade in a differentiated epithelial cell model: the dog thyrocyte in primary culture. We first observed that potent activators of the dog thyrocyte (2+)-phosphatidylinositol cascade such as carbachol or bradykinin failed to promote the onset of DNA synthesis in these cells. Moreover, carbachol inhibited the mitogenic effect of thyroid stimulating hormone (TSH) and of epidermal growth factor (EGF). The mitogenic effect of EGF was also reduced by bradykinin. Nevertheless, carbachol enhanced the expression of the protooncogenes c-fos and c-myc mRNAs. The time course of this enhancement was identical to the time course for the induction of c-fos and c-myc mRNAs by phorbol esters or EGF. On the other hand, in most experiments, TSH and EGF were able to trigger the onset of dog thyrocyte DNA synthesis without affecting their intracellular free Ca2+ concentration [Ca2+]i, 45Ca2+ efflux, or inositol phosphate generation. In several experiments, TSH increased the dog thyrocyte 45Ca2+ release and promoted a rise in the [Ca2+]i or the inositol phosphate accumulation but these effects were weak. In contrast to the effect of carbachol, the TSH effects on the [Ca2+]i and the 45Ca2+ efflux appeared slowly, were sustained, and were extremely sensitive to extracellular Ca2+ depletion. They were observed at hormone concentrations higher than the concentration achieving maximal stimulation of DNA synthesis. Similarly, in a few experiments, a slight increase in the [Ca2+]i or in the inositol trisphosphate generation were provoked by EGF. However, these modifications were not associated with an increased mitogenic potency of EGF. Finally, in all experiments, fetal calf serum slightly accelerated the dog thyrocyte 45Ca2+ efflux and increased their inositol phosphate generation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Different patterns of inositol polyphosphate production are seen in B lymphocytes after cross-linking of sIg by anti-Ig antibody or by a multivalent anti-Ig antibody dextran conjugate

Anti-delta antibody conjugated to 2 x 10(6) m.w. dextran (dex) stimulates B lymphocyte proliferation at 10,000-fold lower concentrations than that required by the unconjugated antibody. Dex conjugated antibody also stimulates a greater and more sustained increase in intracellular ionized calcium [( Ca2+]i) than does the unconjugated anti-Ig antibody. Inasmuch as inositol phosphate metabolites have been linked to rises in [Ca2+]i, we analyzed by FPLC the relative amounts of the inositol polyphosphates (IP) in these cells. Anti-Ig-dextran induced a threefold greater increase in total IP than did the unconjugated anti-Ig. Furthermore, in cells stimulated by unconjugated anti-Ig there was a transient induction of I(1,4,5)P3 followed by a rapid accumulation of the I(1,3,4)P3 isomer with little accumulation of I(1,4)P2, whereas in anti-Ig-dex-stimulated cells there was prolonged elevation of I(1,4,5)P3 with more accumulation of I(1,4)P2. In addition, levels of I(1,3,4,5)P4 were maintained over a longer period of time in B cells stimulated by anti-Ig-dex than in those stimulated by unconjugated anti-Ig. The enhanced ratio of I(1,4,5)P3/I(1,3,4)P3 was also seen when suboptimal concentrations of anti-Ig-dex were used which stimulated a level of total inositol phosphate that was similar to that stimulated by the unconjugated anti-Ig. The possibility that the greater stimulation of increased [Ca2+] by anti-Ig-dex than by unconjugated anti-Ig was a predominant factor in influencing the metabolic pathway of I(1,4,5)P3 was excluded. These results show that 1) stimulation of increases in the various IP isomers occurs in anti-Ig stimulated normal B cells as has been shown in B cell lines and 2) that signal transduction and consequent PIP2 hydrolysis that is stimulated by Ag-mediated cross-linking of sIg is strongly influenced by the extent and type of cross-linking that is induced.     

Receptor-stimulated phospholipase A2 activation is coupled to influx of external calcium and not to mobilization of intracellular calcium in C62B glioma cells

C62B rat glioma cells respond to muscarinic cholinergic stimulation with transient inositol phosphate formation and phospholipase A2-dependent arachidonic acid liberation. Since phospholipase A2 is a Ca2+-sensitive enzyme, we have examined the role of the agonist-stimulated Ca2+ response in production of the arachidonate signal. The fluorescent indicator fura-2 was used to monitor changes in cytoplasmic Ca2+ levels ([Ca2+]i) of C62B cells following acetylcholine treatment. In the presence of extracellular Ca2+, acetylcholine induces a biphasic [Ca2+]i response consisting of an initial transient peak that precedes arachidonate liberation and a sustained elevation that outlasts the phospholipase A2 response. The initial [Ca2+]i peak is not altered by the absence of external Ca2+ and therefore reflects intracellular Ca2+ mobilization. The sustained elevation phase is dependent on the influx of external Ca2+; it is lost in Ca2+-free medium and restored on the addition of Ca2+. Pretreating cells with phorbol dibutyrate substantially inhibits acetylcholine-stimulated inositol phosphate formation and the peak [Ca2+]i response without affecting the sustained elevation in [Ca2+]i. This suggests that the release of internal Ca2+ stores by inositol 1,4,5-trisphosphate can be blocked without interfering with Ca2+ influx. Pretreatment with phorbol also fails to affect acetylcholine-stimulated arachidonate liberation, demonstrating that phospholipase A2 activation does not require normal intracellular Ca2+ release. Stimulated arachidonate accumulation is totally inhibited in Ca2+-free medium and restored by the subsequent addition of Ca2+. Pretreatment with verapamil, a voltage-dependent Ca2+ channel inhibitor, also blocks both the sustained [Ca2+]i elevation and arachidonate liberation without altering peak intracellular Ca2+ release. We conclude that the influx of extracellular Ca2+ is tightly coupled to phospholipase A2 activation, whereas large changes in [Ca2+]i due to mobilization of internal Ca2+ stores are neither sufficient nor necessary for acetylcholine-stimulated phospholipase A2 activation.     

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.     

Effects of insulin-like growth factor-II on proliferation and differentiation of ovarian granulosa cells.

The effects of insulin-like growth factor-II (IGF-II) on the proliferation and differentiation of ovarian granulosa cells were studied in cultured human and porcine granulosa cells. IGF-II significantly increased basal progesterone secretion in granulosa cells at concentrations of 1-100 ng/ml. A stimulatory effect was also observed in gonadotropin-stimulated porcine granulosa cells treated with IGF-II. The secretion of estradiol by basal and gonadotropin-stimulated porcine granulosa cells was also significantly increased by adding IGF-II. IGF-II led to dose-dependent increases in [3H]thymidine incorporation into DNA and in the number of granulosa cells. To further characterize the cellular mechanisms underlying the stimulatory effects of IGF-II on the proliferation and differentiation of granulosa cells, we investigated the intermediary roles of cyclic AMP and intracellular Ca2+ concentration ([Ca2+]i). Treatment with 100 ng/ml IGF-II produced a significant increase in the basal accumulation of cyclic AMP in porcine granulosa cells. However, no change of [Ca2+]i by IGF-II was noted. IGF-II produced effects in accumulation that were similar to those of IGF-I. Our findings suggest that IGF-II may be a general stimulator in the proliferation and differentiation of granulosa cells, and that cyclic AMP may be a second messenger for the effects of IGF-II in ovarian granulosa cells.     

Interactions between inositol phosphates and cytosolic free calcium following bradykinin stimulation in cultured human skin fibroblasts

The inositol triphosphate (IP3) that results from hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) is generally accepted to be responsible for the mobilization of intracellular calcium. However, some studies suggest that low concentrations of agonists elevate cytosolic free calcium concentration ([Ca2+]i) without IP3 formation. Thus, in the present studies, a comparison of the temporal response of inositol phosphates (IP3, IP2 and IP) and [Ca2+]i to a wide range of bradykinin concentrations was used to examine the relation of these two signal transduction events in cultured human skin fibroblasts (GM3652). In addition, the effects of alterations in internal or external calcium on the response of these second messengers to bradykinin were determined. Bradykinin stimulated accumulation of inositol phosphates and a rise of [Ca2+]i in a time- and dose-dependent manner. Decreasing the bradykinin concentration from 1 microM to 0.1 microM increased the time until the IP3 peak, and when the bradykinin concentration was reduced to 0.01 microM IP3 was not detected. [Ca2+]i was examined under parallel conditions. As the bradykinin concentration was reduced from 1 microM to 0.01 microM, the time to reach the peak of [Ca2+]i increased progressively, but the magnitude of the peak was unaltered. These two second messengers were variably dependent on external calcium. Although the bradykinin-stimulated initial spike of [Ca2+]i did not depend on extracellular calcium, the subsequent sustained levels of [Ca2+]i were abolished in calcium free medium. The bradykinin-stimulated inositol phosphate formation was not dependent on the extracellular calcium nor on the elevation of [Ca2+]i that was produced with Br-A23187. These results demonstrate that bradykinin-induced IP3 formation can be independent of [Ca2+]i and of external calcium, whereas changes in [Ca2+]i are partially dependent on external calcium.     

Changes in free cytosolic calcium and accumulation of inositol phosphates in isolated hepatocytes by [Leu]enkephalin.

Isolated hepatocytes from fed rats were used to study the effects of the opioid peptide [Leu]enkephalin on intracellular free cytosolic Ca2+ ([Ca2+]i) and inositol phosphate production. By measuring the fluorescence of the intracellular Ca2+-selective indicator quin-2, [Leu]enkephalin was found to increase [Ca2+]i rapidly from a resting value of 0.219 microM to 0.55 microM. The magnitude of this response was comparable with that produced by maximally stimulating concentrations of either vasopressin (100 nM) or phenylephrine (10 microM). The opioid-peptide-mediated increase in [Ca2+]i showed a dose-dependency comparable with the activation of phosphorylase, but it preceded the increase in phosphorylase alpha activity. Addition of [Leu]enkephalin to hepatocytes prelabelled with myo-[2-3H(n)]inositol resulted in a significant stimulation of inositol phosphate production. At 10 min after hormone addition, there were increases in the concentrations of inositol mono-, bis- and tris-phosphate fractions of 12-, 9- and 14-fold respectively. No effect was apparent on the glycerophosphoinositol fraction. The effect of 10 microM-[Leu]enkephalin on inositol phosphate production was significantly greater than that obtained with 10 microM-phenylephrine, but marginally smaller than that induced by 100 nM-vasopressin. However, at these concentrations all three agonists gave a comparable increase in [Ca2+]i and activation of phosphorylase a. These data provide evidence for [Leu]enkephalin acting via a mechanism involving a mobilization of Ca2+ as a result of increased phosphatidylinositol turnover.     

Calcium influx mediated by nicotinic receptors and voltage sensitive calcium channels in SK-N-SH human neuroblastoma cells

When SK-N-SH human neuroblastoma cells were exposed to nicotine (NIC) or KCl they showed a dose-dependent transient increase (2- to 4-fold) in intracellular Ca2+ concentration ([Ca2+])i as detected by quin-2 fluorescence, with half maximal effects (EC50) observed at 13 microM and 26 mM, respectively. Tubocurarine and 1-isodihydrohistrionicotoxin potently blocked the NIC-evoked (IC50 congruent to 1 microM and 0.3 microM, respectively), but not the high [K+]o-evoked [Ca2+]i accumulation. The KCl-induced response was inhibited by verapamil and diltiazem (IC50 = 1.4 and 10.9 microM, respectively). Tetrodotoxin (3 microM) and tetraethylammonium (10 microM) had no effect on [Ca2+]i accumulation induced by either agent. Increases in [Ca2+]i could be evoked sequentially by NIC and KCl in the same cells suggesting independent mechanisms of Ca2+ entry. In a Ca2+-free medium, no response to either KCl or NIC was observed. However, when Ca2+ ions were restored, [Ca2+]i accumulation was enhanced to the same extent as cells suspended in a Ca2+-containing buffer. Long-term (18 hr) pretreatment of SK-N-SH cells with pertussis (100 ng/ml) or cholera toxins (10 nM) had no effect on NIC or KCl-induced [Ca2+]i accumulation. Together, these data demonstrate the presence of NIC receptors and voltage-sensitive Ca2+ channels on SK-N-SH neuroblastoma cells, through which [Ca2+]i may be modulated.     

Type IV collagen stimulates an increase in intracellular calcium. Potential role in tumor cell motility.

Type IV collagen (Coll IV), a component of the extracellular matrix, stimulates motility in the A2058 human melanoma cell line, a response that is inhibited by pertussis toxin (PT). Fibronectin (FN)-induced chemotaxis in this cell line is not affected by PT. To understand the mechanism of cellular signaling, single cell intracellular Ca2+ responses to Coll IV and FN were studied using Fura-2 and digital imaging fluorescence microscopy. Coll IV, at a dose that stimulates motility (100 micrograms/ml, 185 nM), induces a significant rise in cytosolic free Ca2+ concentration ([Ca2+]i) within 100 s. This response is not inhibited by PT. Treatment of the cells with FN 30 micrograms/ml (70 nM), a dose that stimulates near-maximal chemotaxis, does not increase [Ca2+]i appreciably. Removal of extracellular Ca2+ fails to inhibit the Coll IV-stimulated rise in Ca2+ in all cells. Depletion of extracellular Ca2+ and pretreatment of cells with Ca2+ channel blockers only partially inhibits Coll IV-induced motility. Depletion of intracellular Ca2+ inhibits both chemotaxis and the Coll IV-induced increase in intracellular Ca2+. Coll IV does not stimulate membrane phosphoinositide hydrolysis. We conclude that Coll IV treatment induces an inositol 1,4,5-trisphosphate-independent release of intracellular Ca2+ stores which appears to play a necessary role in the chemotactic response of A2058 cells but is not mediated by a PT-sensitive G-protein. This response is not seen in cells exposed to FN, suggesting different intracellular signaling mechanisms for stimulated motility between these two extracellular matrix molecules.     

Mobilization of extracellular Ca2+ by prostaglandin F2 alpha can be modulated by fluoride in 3T3-L1 fibroblasts.

Changes in the intracellular concentration of calcium [( Ca2+]i) have been shown to mediate the physiological effects of certain agonists. Ca2+ mobilization occurs through multiple mechanisms which involve both influx and internal release of Ca2+. Prostaglandin F2 alpha (PGF2 alpha) caused a transient mobilization of intracellular Ca2+ in 3T3-L1 fibroblasts. This effect was characterized by fluorescence measurements of trypsin-treated cells loaded with fura-2/AM. In the absence of extracellular Ca2+, the peak amount of Ca2+ mobilized by PGF2 alpha was decreased by 70%, a lag time before the onset of [Ca2+]i increase was observed, and the rate of rise of [Ca2+]i was slowed. Addition of NaF (10 mM) to fura-2-loaded 3T3-L1 cells caused a dose-dependent increase in [Ca2+]i after a brief (approximately 10 s) lag. Maximal effects (approximately 300 nM) were observed at 5-10 mM-NaF. This effect was dependent on the presence of extracellular Ca2+ and appeared to be independent of inositol phosphate production. After reaching a peak at around 40 s after fluoride addition, [Ca2+]i returned to near-baseline within 120 s. This return of [Ca2+]i to near-baseline after fluoride stimulation and the inability of the cells to respond to a subsequent addition of fluoride indicated that the response to fluoride underwent desensitization. Similarly, the pathway used by PGF2 alpha to mobilize Ca2+ underwent desensitization. Exposure of the cells to a maximally effective concentration of fluoride and subsequent addition of PGF2 alpha produced a [Ca2+]i response to PGF2 alpha which was similar in magnitude and kinetics to that seen for PGF2 alpha in the absence of extracellular Ca2+. Conversely, prior exposure of cells to PGF2 alpha diminished the ability of fluoride to mobilize Ca2+. PGF2 alpha also increased inositol phosphate formation, with a time course and dose-response consistent with its ability to increase [Ca2+]i. Prior exposure of cells to fluoride did not change the time course or dose-response characteristics of PGF2 alpha-induced generation of inositol phosphates. These data suggest that PGF2 alpha and fluoride share a common mechanism of activating Ca2+ influx in 3T3-L1 cells.     

A novel tumour promoter, thapsigargin, transiently increases cytoplasmic free Ca2+ without generation of inositol phosphates in NG115-401L neuronal cells.

Thapsigargin, a sesquiterpene lactone with potent irritant and tumour-promoting activities, stimulates a rapid (within 15 s) transient increase in intracellular [Ca2+] in the NG115-401L neural cell line, as measured by the fluorescent indicator dye fura-2. This increase in cytoplasmic free [Ca2+] is concentration-dependent (ED50 around 20 nM) and occurs in the absence of extracellular Ca2+. Activation of NG115-401L cells by the inflammatory peptide bradykinin generates inositol phosphates, which parallel increases in intracellular [Ca2+]. However, the rise in cytoplasmic [Ca2+] stimulated by thapsigargin occurs in the absence of detectable production of inositol phosphates. Thapsigargin is unlike phorboid tumour promoters in that it has no action on two non-invasive indicators of phorbol stimulation of these cells, i.e. [3H]choline metabolite production and rise in intracellular pH. These data suggest that thapsigargin releases Ca2+ from an intracellular store by a novel mechanism, independent of the hydrolysis of phosphoinositides and concomitant activation of protein kinase C. Thus thapsigargin may provide a valuable tool for the analysis of intracellular signalling mechanisms.     

Increase in cytosolic calcium and phosphoinositide metabolism induced by angiotensin II and [Arg]vasopressin in vascular smooth muscle cells

Effects of angiotensin II and [Arg]vasopressin on cytosolic free Ca2+ concentration ([Ca2+]i) and phosphoinositide metabolism were studied in cultured aortic smooth muscle cells obtained from Wistar-Kyoto rats and their spontaneously hypertensive substrain. [Ca2+]i was measured using the fluorescent Ca2+ indicator quin2. No clear differences in basal [Ca2+]i were detected between cells derived from the two strains. High concentrations of angiotensin II (greater than or equal to 10 nM) and [Arg]vasopressin (greater than or equal to 100 nM) elicited large and rapid increases in [Ca2+]i, followed by a rapid return to control values. Low concentrations of these peptides (less than or equal to 1.0 nM) elicited small and slow increases in [Ca2+]i that persisted for minutes. These responses were blocked by specific antagonists for each of these peptides. Only high concentrations of angiotensin II caused [Ca2+]i increases in "Ca2+-free" medium, which suggested that high concentrations of angiotensin II could release Ca2+ from intracellular pools. A high concentration of angiotensin II and [Arg]vasopressin elicited progressive accumulations of inositol phosphates. Only high concentrations of angiotensin II caused inositol phosphate accumulation in Ca2+-free medium. Maximal accumulation of inositol phosphate elicited by angiotensin II and [Arg]vasopressin was found to be additive. A desensitization to the effects of both peptides on Ca2+ mobilization occurred despite the continued accumulation of inositol phosphates. These observations indicated that angiotensin II and [Arg]vasopressin interacted with independent receptors, both of which are linked to phosphoinositide breakdown and Ca2+ mobilization.     

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.     

Phorbol ester modulates serotonin receptor-mediated increases in inositol phosphate production and calcium mobilization in cultured rat vascular smooth muscle cells

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on serotonin-induced inositol phosphate (IP) accumulation and intracellular free Ca2+ concentrations [( Ca2+]i) was investigated in cultured rat vascular smooth muscle cells. Pretreatment with TPA had no effect on basal levels of both IP production and [Ca2+]i, whereas it significantly attenuated serotonin-induced increases in both IP production and [Ca2+]i. These data suggest that protein kinase C is involved in the negative feedback control of serotonin-induced rises in both IP production and [Ca2+]i.     

Agents that raise cAMP in human T lymphocytes release an intracellular pool of calcium in the absence of inositol phosphate production

Prostaglandins (PGs) of the E series are recognized by specific receptors on T lymphocytes which lead to an increase in cAMP. The role of cAMP in modulation of T lymphocyte function is unknown. Here, we demonstrate that agents which increase cAMP in human T cells raise the intracellular free calcium concentration ([Ca2+]i). This increase in [Ca2+]i occurred following receptor stimulation with PGEs or by bypassing the receptor with the cell-permeant analog 8-(4-chlorophenylthio)-cAMP or forskolin, a direct activator of adenylyl cyclase. The calcium response to a submaximally stimulatory concentration of PGE2 was potentiated by the cAMP phosphodiesterase inhibitor isobutylmethylxanthine. A time course of cAMP production in response to PGE2 stimulation closely resembled the calcium response and suggested that the two events were coincident. The PGE2 concentrations required to achieve 50% maximum effect of cAMP production and increases in [Ca2+]i were similar, 0.07 and 0.15 microM respectively. Chelation of extracellular Ca2+ did not abolish the PGE2-stimulated Ca2+ response, suggesting that an intracellular source of calcium was sensitive to cAMP. Significant inositol phosphate production was not detected in response to PGE2 over a wide concentration range. The PGE2-induced calcium response curves were of lesser magnitude with shorter times to peak than those of a known inositol 1,4,5 trisphosphate-producing agonist, anti-CD3, suggesting distinct Ca2+ release mechanisms. However, the cAMP-releasable store appeared to be contained within the inositol trisphosphate-releasable store since no response could be seen with cAMP-elevating agents following emptying of the inositol trisphosphate-sensitive pool of Ca2+.     

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.     

Modulation of bombesin-induced phosphatidylinositol hydrolysis in a small-cell lung-cancer cell line.

Bombesin is an amphibian tetradecapeptide whose mammalian homologue, gastrin-releasing peptide (GRP), is produced by many small-cell lung-cancer (SCLC) cells, and which can function in an autocrine growth-promoting manner in SCLC. Studies reported here show that [Tyr4]bombesin and its congeners increase inositol 1,4,5-trisphosphate within seconds in NCI-H345, a SCLC cell line that constitutively produces GRP. After 30 min in the presence of 0.01 M-Li+ and [Tyr4]bombesin, there is marked accumulation of inositol monophosphates and inositol tetrakisphosphate. Pretreatment with phorbol 12-myristate 13-acetate (PMA) for 20 min inhibited the ability of [Tyr4]bombesin to induce phosphatidylinositol (PtdIns) turnover and to increase intracellular free Ca2+ ([Ca2+]i). Pretreatment with PMA for 48 h attenuated the ability of subsequently added PMA to decrease the response to [Tyr4]bombesin. Pretreatment with pertussis toxin (PT; 1 microgram/ml for 18-24 h) decreased by less than 30% [Tyr4]bombesin-induced increases in [Ca2+]i and PtdIns metabolites. However, interpretation of this result is complicated by the inability of PT to ADP-ribosylate completely its substrates in intact NCI-H345 cells. In contrast, pretreatment with cholera toxin (1 microgram/ml for 18-24 h) lowered basal [Ca2+]i and basal inositol phosphate concentrations, attenuated the response of NCI-H345 to subsequently added [Tyr4]bombesin, and was not mimicked by treatments that increase cellular cyclic AMP. These data demonstrate the activation of phospholipase C in SCLC by bombesin congeners. In addition, the results suggest a regulatory role for protein kinase C, a cholera-toxin substrate, and perhaps a pertussis-toxin substrate in the response of SCLC to bombesin.