Nerve Growth Factor Potentiates the Hormone-Stimulated Intracellular Accumulation of Inositol Phosphates and Ca2+ in Rat PC12 Pheochromocytoma Cells: Comparison with the Effect of Epidermal Growth Factor

The effects of nerve growth factor (NGF) and epidermal growth factor (EGF) on the intracellular accumulation of inositol phosphates and on cytosolic free Ca2+ concentrations were studied in rat PC12 pheochromocytoma cells. Both NGF and EGF potentiate in these cells the increase in the accumulation of inositol phosphates that is elicited by bradykinin and carbachol. A corresponding potentiation was also found for the agonist-induced increase of cytosolic Ca2+ concentrations. The effect of NGF, but not that of EGF, is abolished when the cells are preincubated with 5'-deoxy-5'-methylthioadenosine, an inhibitor of S-adenosylhomocysteine hydrolase. These results suggest that an increased response to hormones, which act via phosphoinositide-derived second messengers, may be important in the mechanism of action of NGF and EGF.     

Polyphosphoinositide hydrolysis in endothelial cells and carotid artery segments. Bradykinin-2 receptor stimulation is calcium-independent

Bovine aortic and cerebral microvascular endothelial cells and cultured segments of canine common carotid artery possess functional receptors for the nonapeptide bradykinin which mediate a rapid increase in the formation of [3H]inositol 1-phosphate, [3H]inositol 1,4-bisphosphate, and [3H]inositol 1,4,5-trisphosphate from cell membranes containing isotopically labeled myo-inositol. Bradykinin stimulated the formation of [3H]inositol phosphates from cells in culture or tissues at threshold concentrations of 0.1 nM and 1 nM, and with a half-maximal effective concentration of 0.6-1.0 nM and 30 nM, respectively. In cultured cells, the formation of [3H]inositol trisphosphate and [3H]inositol bisphosphate preceded the formation of [3H]inositol monophosphate. Similarly, [3H]inositol phosphate formation was not inhibited by addition of calcium channel blockers, a calcium chelator, or an intracellular calcium antagonist. Calcium ionophore A23187 did not promote [3H]inositol phosphate accumulation. The receptor selectivity of the bradykinin response in cultured cells was most compatible with a type-2 mediated response. Kallidin stimulated with the same potency as bradykinin but was more potent than methionyl-lysyl-bradykinin or des-Arg9-bradykinin. The B1 receptor antagonists des-Arg9-[Leu8]-bradykinin and des-Arg10-[Leu9]-kallidin were without effect. The rapidity of the inositol phosphate response as well as the close correspondence between the bradykinin type-2 receptor mediated hydrolysis of polyphosphoinositides and changes in prostacyclin synthesis, vessel dilation, and permeability suggests that breakdown products of inositol lipids serve as second messengers mediating the effects of bradykinin on the vascular endothelium.     

Cellular mechanisms of bradykinin-induced hyperpolarization in renal epitheloid MDCK-cells.

Previous studies have demonstrated that bradykinin hyperpolarizes the cell membrane of subconfluent MDCK cells by increase of the potassium conductance. The present study has been performed to elucidate the intracellular mechanisms involved. To this end, the effects of bradykinin on the potential difference across the cell membrane (PD), on formation of inositol phosphates, and on intracellular calcium concentration (Cai) have been analyzed in cells without or with pretreatment with pertussis toxin or 12-O-tetradecanoylphorbol 13-acetate diester (TPA). In untreated cells, bradykinin leads to a transient increase of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate, increase of Cai, activation of potassium channels and hyperpolarization of the cell membrane. The effects of bradykinin on PD and Cai are still present in the absence of extracellular calcium. In cells pretreated with pertussis toxin the effect of bradykinin on inositol trisphosphate formation is almost abolished but bradykinin still leads to a transient increase of Cai and PD in the presence and absence of extracellular calcium. In cells pretreated with TPA the bradykinin-induced increase of inositol trisphosphate formation is blunted, the bradykinin-induced increase of Cai abolished, but the bradykinin-induced hyperpolarization still present. The observations indicate that bradykinin increases Cai in part by phorbol ester and pertussis toxin sensitive activation of phospholipase C. In addition, bradykinin is capable of enhancing Cai by utilizing pertussis toxin insensitive mechanisms. Furthermore, bradykinin is able to transiently enhance the potassium conductance without a general increase of intracellular calcium.     

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 phorbol ester on mitogen and orthovanadate stimulated responses of cultured human fibroblasts

Mitogenic stimulation of quiescent human fibroblasts (HSWP) with serum or a mixture of growth factors (consisting of vasopressin, bradykinin, EGF, and insulin) stimulates the release of inositol phosphates, mobilization of intracellular Ca, activation of Na/H exchange and subsequent incorporation of [3H]-thymidine. We have determined previously that pretreatment with the tumor-promoting phorbol ester 12-0-tetradecanoyl-phorbol-13-acetate (TPA) inhibits mitogen-stimulated Na influx in HSWP cells. We report herein that TPA pretreatment also substantially inhibits the mitogen-stimulated release of inositol phosphates in HSWP cells. Half maximal inhibition of mitogen-stimulated inositol phosphate release occurs at 1-2 nM TPA. Treatment of cells with TPA alone has no effect on inositol phosphate release. The effect of TPA pretreatment on inositol phosphate release induced by individual growth factors has also been determined. Orthovanadate, reported by Cassel et al. (1984) to increase Na/H exchange in A431 cells, has been demonstrated to stimulate both Na influx and inositol phosphate release in HSWP cells. TPA pretreatment also inhibits both orthovanadate-stimulated inositol phosphate release and Na influx. In addition, orthovanadate was determined to increase intracellular Ca activity by mobilizing intracellular calcium stores, as determined with the fluorescent intracellular calcium probe fura-2. TPA pretreatment blocks orthovanadate stimulated mobilization of intracellular Ca stores. It appears clear that in HSWP cells pretreatment of cells with phorbol ester is capable of artificially desensitizing the early cellular responses to mitogenic stimuli (growth factors, orthovanadate) by blocking the signal transduction mechanism involved at a point prior to the release of inositol phosphates. We hypothesize that in HSWP cells the normal desensitization of both inositol phosphate release and Na/H exchange is mediated via activation of protein kinase C subsequent to the stimulus-mediated activation of phospholipase C and release of protein kinase C activator diacylglycerol. However it is interesting to note that TPA-mediated inhibition of these early responses in HSWP cells does not inhibit their ability to be stimulated to incorporate [3H]-thymidine.(ABSTRACT TRUNCATED AT 400 WORDS)     

Calcium-Dependent Nerve Growth Factor-Stimulated Hydrolysis of Phosphoinositides in PC12 Cells

The effect of nerve growth factor (NGF) on the hydrolysis of phosphoinositides in PC12 cells was examined. Addition of NGF to PC12 cells prelabeled with [3H]-inositol resulted in an increase in the formation of labeled inositol trisphosphate ([3H]IP3), inositol bisphosphate ([3H]IP2), and inositol monophosphate ([3H]IP), an observation indicating that NGF stimulated hydrolysis of the polyphosphoinositides. The increase in these inositol phosphates was detected as early as 15 s after addition of NGF. In the presence of LiCl, the accumulation of [3H]IP was linear for at least 20 min. The NGF-stimulated accumulation of [3H]IP was dose-dependent with a Kact of 0.17 nM and was dependent on the presence of extracellular calcium. In a calcium-free buffer containing EGTA, the NGF-dependent increase in accumulation of [3H]IP was not seen, and the basal level of [3H]IP accumulation was lower than that observed in the presence of extracellular calcium. Lanthanum inhibited both the basal and NGF-stimulated accumulation of [3H]IP, whereas the calcium ionophore A23187, in the absence of NGF, stimulated an accumulation of [3H]IP. The maximal accumulation of [3H]IP in the presence of A23187 was the same as that observed in the presence of NGF. Incubation of the cells with both A23187 and NGF resulted in an accumulation of [3H]IP that was not significantly different from the effect of either agent alone. These results suggest that NGF rapidly stimulates the hydrolysis of phosphoinositides in PC12 cells and that this NGF-stimulated hydrolysis of phosphoinositides occurs by a calcium-dependent mechanism.     

Stimulation of Inositol Incorporation into Lipids of PC 12 Cells by Nerve Growth Factor and Bradykinin

The effects of bradykinin (BK) and lithium on the phosphatidylinositol cycle were examined in PC12 cells cultured for 20 h in the presence [PC12(+)] or in the absence [PC12(-)] of nerve growth factor (NGF). BK (1 microM) induced a small stimulation of the incorporation of myo-[2-3H]inositol into the lipids of PC12(-) cells and a three- to fourfold stimulation of such incorporation into the lipids of PC12 (+) cells. About 15 h of incubation with NGF and greater than 10 min of incubation with BK were needed for maximal stimulation of inositol incorporation by BK. In the presence of 25 mM LiCl, BK stimulated the inositol monophosphate levels nine-fold in PC12 (-) and 30-fold in PC12 (+) cells. After incubation for 20 h with NGF, an increased binding of [3H]BK to the PC12 (+) cells was observed at 4 degrees C. Exposure of the cells for 30 min to 25 mM LiCl enhanced the effect of BK on the inositol incorporation into total inositol lipids, especially in PC12(+) cells. In these cells, LiCl in the presence of BK also increased several-fold the intracellular levels of inositol bisphosphate and inositol trisphosphate.     

Mitogen-stimulated release of inositol phosphates in human fibroblasts

Mitogenic stimulation of quiescent human fibroblasts (HSWP) with a growth factor mixture (consisting of epidermal growth factor (EGF), insulin, bradykinin, and vasopressin) rapidly induces an increase in Na influx via a Ca-mediated activation of an amiloride-sensitive Na/H exchanger. Inositol phosphates (specifically inositol-1',4',5'-phosphate) have been implicated in mediating the mobilization of intracellular Ca stores in other cell types and we have now completed a detailed analysis of the mitogen-induced release of inositol phosphates in HSWP cells. Stimulation of inositol trisphosphate release is rapid (within 5 s) and reaches a maximum level (416-485% basal) within 10-15 s after the addition of growth factor mixture. Inositol bisphosphate and inositol monophosphate reach maximum levels by 30 s (1257% basal) and 60 s (291% basal), respectively. Levels of all three compounds then decay toward basal levels but remain elevated (150-350% of basal levels) after 10 min of incubation with mitogens. The effects of different combinations of these growth factors and of the bee venom peptide, melittin, have also been determined. We have also found that 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate, which prevents the mitogen-induced rise in intracellular calcium activity and activation of Na influx, does not alter the mitogen-stimulated accumulation of inositol trisphosphate. In addition, the calcium ionophore A23187, which increases cytosolic Ca activity and induces a Na influx, does not stimulate the release of inositol trisphosphate. Assays performed in the presence of lithium, which inhibits inositol phosphate monophosphatase, promotes the prolonged and enhanced accumulation of inositol monophosphate. Treatment with the phospholipase inhibitor mepacrine or pretreatment with dexamethasone reduces the amount of inositol phosphates released upon mitogenic stimulation. Hence mitogenic stimulation of HSWP cells leads to the rapid stimulation of inositol phosphate release via a calcium-independent mechanism and suggests inositol trisphosphate as a candidate to mediate the release of intracellular calcium stores which is involved in the processes responsible for the activation of the Na/H exchanger.     

Changes in Inositol 1,4,5-Trisphosphate and Inositol 1,3,4,5-Tetrakisphosphate Mass Accumulations in Cultured Adrenal Chromaffin Cells in Response to Bradykinin and Histamine

In previous studies it has been shown that both bradykinin and histamine increase the formation of 3H-labeled inositol phosphates in adrenal chromaffin cells prelabelled with [3H]inositol and that both these agonists stimulate release of catecholamines by a mechanism dependent on extracellular calcium. Here, we have used mass assays of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] to investigate changes in levels of these two candidates as second messengers in response to stimulation with bradykinin and histamine. Bradykinin increased the mass of Ins(1,3,4,5)P4 despite the failure in earlier studies with [3H]inositol-labelled cells to observe a bradykinin-mediated increase in content of [3H]InsP4. Bradykinin elicited a very rapid increase in level of Ins(1,4,5)P3, which was maximal at 5-10 s and then rapidly decreased to a small but sustained elevation at 2 min. The bradykinin-elicited Ins(1,3,4,5)P4 response increased to a maximum at 30-60 s and at 2 min was still elevated severalfold above basal levels. Histamine, which produced a larger overall total inositol phosphate response in [3H]inositol-loaded cells, produced significantly smaller Ins(1,4,5)P3 and Ins(1,3,4,5)P4 responses compared with bradykinin. The bradykinin stimulation of Ins(1,4,5)P3 accumulation was partially dependent on a high (1.8 mM) extracellular Ca2+ concentration, whereas the Ins(1,3,4,5)P4 response was almost completely lost when the extracellular Ca2+ concentration was reduced to 100 nM. Changes in the inositol polyphosphate second messengers are compared with the time course of bradykinin-stimulated increases in free intracellular Ca2+ concentrations and noradrenaline release.     

Trypanosoma cruzi: infection of cultured human endothelial cells alters inositol phosphate synthesis

Infection of cultured endothelial cells with Trypanosoma cruzi alters intracellular Ca2+ homeostasis. To help understand the biochemical basis for this phenomenon, we determined the influence of infection on inositol phosphate formation in a broken cell preparation. Inositol phosphates participate in the regulation of cytosolic Ca2+. In uninfected endothelial cells, bradykinin guanosine 5'-O-thiophosphate (GTP tau S), and calcium all stimulated inositol phosphate (IP1), inositol bisphosphate (IP2), and inositol trisphosphate (IP3) formation within 5 sec of incubation. At longer periods of incubation with GTP tau S and bradykinin, formation of IP1 was linear for 30 sec, whereas the rate of IP2 and IP3 generation was maximal at 20 and 5 sec, respectively. Second, infection markedly changed these aspects of inositol phosphate generation. First, unstimulated (basal) levels of IP1 and IP3 were markedly increased over those levels in membranes of uninfected cells. Infection decreased the rate of formation for the three inositol phosphates in response to GTP tau S and bradykinin. Finally, infection diminished the magnitude of inositol phosphate synthesis in response to Ca2+ for IP1, IP2, and IP3, respectively. Studies on G proteins using cholera and pertussis toxin were carried out to determine if the infection-associated changes in inositol phosphate generation could be attributed to functional changes in these regulatory proteins known to participate in the activation of phospholipase C. Infection markedly decreased the magnitude of cholera and pertussis toxin-dependent ADP ribosylation, as compared to control uninfected cells. Incubation of uninfected endothelial cells with cholera and pertussis toxin also decreased the magnitude of cholera and pertussis toxin ADP ribosylation. Despite the similar effects of infection and toxin treatment on subsequent toxin-catalyzed ADP ribosylation, toxin treatment did not influence inositol phosphate generation. Collectively, these results demonstrate an influence of infection on receptor-dependent and -independent synthesis of inositol phosphates, possibly by an action on phospholipase C. The results help to explain the apparent infection-associated increase in basal Ca2+ previously observed and suggest that interference with signal transduction may be a consequence of the presence of the parasite.     

Bradykinin transiently activates protein kinase C in Swiss 3T3 cells. Distinction from activation by bombesin and vasopressin

The results presented here demonstrate that bradykinin, acting through a B2 subtype receptor, induces a unique pattern of early signals in quiescent Swiss 3T3 cells. Bradykinin caused a rapid mobilization of calcium from internal stores, as judged by measurements of intracellular Ca2+ concentration in fura-2-loaded cells and by 45Ca2+ efflux from radiolabeled cells. Analysis of phosphoproteins from 32P-labeled Swiss 3T3 cells by one- and two-dimensional gel electrophoresis revealed that bradykinin stimulated transient phosphorylation of an acidic cellular protein migrating with an apparent Mr = 80,000 (termed 80K), identified as a major and specific substrate of protein kinase C. Down-regulation of protein kinase C by pretreatment with phorbol 12,13-dibutyrate (PDBu) completely abolished the increase in 80K phosphorylation. In contrast to the sustained effect induced by bombesin, vasopressin, or PDBu, the stimulation of 80K phosphorylation by bradykinin reached a maximum after 1 min of incubation, and then it rapidly decreased to almost basal levels. Furthermore, bradykinin did not induce protein kinase C-mediated events such as inhibition of 125I-epidermal growth factor binding or enhancement of cAMP accumulation. Bombesin and vasopressin elicited both responses in parallel cultures. Bradykinin induced rapid accumulation of total inositol phosphates in cells labeled with myo-[3H]inositol. In contrast to bombesin and vasopressin which stimulated a linear increase in inositol phosphate accumulation over a 10-min period, the effect of bradykinin reached a plateau after 2.5 min of incubation with no further increase up to 10 min. The results demonstrate that the early signaling events triggered by bradykinin can be distinguished from those elicited by bombesin and vasopressin in Swiss 3T3 cells.     

Epidermal growth factor stimulates the rapid accumulation of inositol (1,4,5)-trisphosphate and a rise in cytosolic calcium mobilized from intracellular stores in A431 cells

Exposure of A431 human epidermoid carcinoma cells to epidermal growth factor (EGF), bradykinin, and histamine resulted in a time- and concentration-dependent accumulation of the inositol phosphates (InsP) inositol monophosphate, inositol bisphosphate, and inositol trisphosphate (InsP3). Maximal concentrations of EGF (316 ng/ml; approximately 50 nM), bradykinin (1 microM), and histamine (1 mM) resulted in 3-, 6-, and 3-fold increases, respectively, in the amounts of inositol phosphates formed over a 10-min period. The K0.5 values for stimulation were approximately 10 nM, 3 nM, and 10 microM for EGF, bradykinin, and histamine, respectively. EGF and bradykinin stimulated the rapid accumulation of the two isomers of InsP3, Ins(1,3,4)P3, and Ins(1,4,5)P3 as determined by high performance liquid chromatography analysis; maximal accumulation of Ins(1,4,5)P3 occurred within 15 s. EGF and bradykinin also stimulated a rapid (maximal levels attained within 30 s after addition of hormone) and a sustained 4- and 6-fold rise, respectively, in cytosolic free Ca2+ levels as measured by Fura-2 fluorescence. EGF and bradykinin also produced a rapid, although transient, 3- and 5-fold increase, respectively, in cytosolic free Ca2+ after chelation of extracellular Ca2+ with 3 mM EGTA. These data are consistent with the idea that EGF elevates intracellular Ca2+ levels in A431 cells, at least in part, as a result of the rapid formation of Ins(1,4,5)P3 and the consequential release of Ca2+ from intracellular stores.     

The thapsigargin-sensitive intracellular Ca2+ pool is more important in plasma membrane Ca2+ entry than the IP3-sensitive intracellular Ca2+ pool in neuronal cell lines

In NG108-15 cells, bradykinin (BK) and thapsigargin (TG) caused transient increases in a cytosolic free Ca2+ concentration ([Ca2+]i), after which [Ca2+]i elevated by TG only declined to a higher, sustained level than an unstimulated level. In PC12 cells, carbachol (CCh) evoked a transient increase in [Ca2+]i followed by a sustained rise of [Ca2+]i, whereas [Ca2+]i elevated by TG almost maintained its higher level. In the absence of extracellular Ca2+, the sustained elevation of [Ca2+]i induced by each drug we used was abolished. In addition, the rise in [Ca2+]i stimulated by TG was less affected after CCh or BK, whereas CCh or BK caused no increase in [Ca2+]i after TG. TG neither increased cellular inositol phosphates nor modified the inositol phosphates format on stimulated by CCh or BK. We conclude that TG may release Ca2+ from both IP3-sensitive and -insensitive intracellular pools and that some kinds of signalling to link the intracellular Ca2+ pools and Ca2+ entry seem to exist in neuronal cells.     

Stimulation of Formation of Inositol Phosphates in Primary Cultures of Bovine Adrenal Chromaffin Cells by Angiotensin II, Histamine, Bradykinin, and Carbachol

Histamine, bradykinin, and angiotensin II stimulate release of catecholamines from adrenal medulla. Here we show, using bovine adrenal chromaffin cells in culture, that these agonists as well as carbachol (with hexamethonium) stimulate production of inositol phosphates. The histamine response was mepyramine sensitive, implicating an H1 receptor, whereas bradykinin had a lower EC50 than Met-Lys-bradykinin, and [Des-Arg9]-bradykinin was relatively inactive, implicating a BK-2 receptor. Total inositol phosphates formed in the presence of lithium were measured, with histamine giving the largest response. The relative contribution of chromaffin cells and nonchromaffin cells in the responses was assessed. In each case chromaffin cells were found to be responding to the agonists; in the case of histamine the response was solely on chromaffin cells. When the inositol phosphates accumulating over 2 or 5 min, with no lithium present, were separated on Dowex anion-exchange columns, bradykinin gave the greatest stimulation in the inositol trisphosphate fraction, whereas histamine gave a larger inositol monophosphate accumulation. On resolution of the isomers of stimulated inositol trisphosphate after 2 min of stimulation, the principal isomer present was inositol 1,3,4-trisphosphate in each case. Two hypotheses for the differential responses to histamine and bradykinin are discussed.     

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.     

5-hydroxytryptamine-evoked [Ca]i oscillations in rat C6 glioma cells

We have investigated the modulation of the intracellular calcium concentration ([Ca²⁺]_i) in rat C6 glioma cells following their activation by the agonists 5-hydroxytryptamine·HCl (5-HT) and bradykinin, using single cell imaging of [Ca²⁺]_i with the calcium-sensitive dye Fura-2. The majority of the signals observed involved release of calcium from intracellular stores, and after prolonged application of 5-HT, but not bradykinin, the cells exhibited oscillations in [Ca²⁺]_i levels. These calcium oscillations were dependent on the presence of extracellular calcium, and were unaffected by the calcium channel antagonists nifedipine and verapamil. Caffeine, which in other cell types is able to release calcium from inositol trisphosphate-insentive stores, had very little effect on [Ca²⁺]_i levels in C6 cells. On the other hand, bradykinin, although able to elevate [Ca²⁺]_i probably by acting via the B₂-receptor subtype, was unable to induce any calcium oscillations in these cells.     

Reassessment of the Ca2+ sensing property of a type I metabotropic glutamate receptor by simultaneous measurement of inositol 1,4,5-trisphosphate and Ca2+ in single cells

Transient transfection of Chinese hamster ovary or baby hamster kidney cells expressing the Group I metabotropic glutamate receptor mGlu1alpha with green fluorescent protein-tagged pleckstrin homology domain of phospholipase Cdelta1 allows real-time detection of inositol 1,4,5-trisphosphate. Loading with Fura-2 enables simultaneous measurement of intracellular Ca(2+) within the same cell. Using this technique we have studied the extracellular calcium sensing property of the mGlu1alpha receptor. Quisqualate, in extracellular medium containing 1.3 mm Ca(2+), increased inositol 1,4,5-trisphosphate in all cells. This followed a typical peak and plateau pattern and was paralleled by concurrent increases in intracellular Ca(2+) concentration. Under nominally Ca(2+)-free conditions similar initial peaks in inositol 1,4,5-trisphosphate and Ca(2+) concentration occurred with little change in either agonist potency or efficacy. However, sustained inositol 1,4,5-trisphosphate production was substantially reduced and the plateau in Ca(2+) concentration absent. Depletion of intracellular Ca(2+) stores using thapsigargin abolished quisqualate-induced increases in intracellular Ca(2+) and markedly reduced inositol 1,4,5-trisphosphate production. These data suggest that the mGlu1alpha receptor is not a calcium-sensing receptor because the initial response to agonist is not sensitive to extracellular Ca(2+) concentration. However, prolonged activation of phospholipase C requires extracellular Ca(2+), while the initial burst of activity is highly dependent on Ca(2+) mobilization from intracellular stores.     

Inhibition of early platelet-derived growth factor responses in BALB/c-3T3 cells by interferon

Stimulation of total inositol phosphate production, alteration of cytosolic free calcium [( Ca++]i), vinculin disruption from adhesion plaques, and DNA synthesis caused by PDGF were examined in normal and INF pretreated density arrested BALB/c-3T3 fibroblasts. In normal cells, PDGF caused an increase in total inositol phosphates, a rapid, transient increase in [Ca++]i, disappearance of vinculin from adhesion plaques, and stimulation of DNA synthesis. Pretreatment of cells with INF inhibited PDGF-stimulated increases in [Ca++]i, vinculin disruption from adhesion plaques, and DNA synthesis, but had no effect on PDGF-induced increase in total inositol phosphate levels. These findings suggest that INF prevents entry of quiescent BALB/c-3T3 cells into G1 by inhibiting PDGF-induced release of Ca++ from intracellular stores.     

Salmonella typhimurium induces an inositol phosphate flux in infected epithelial cells

Salmonella typhimurium, like many other intracellular pathogens, is capable of inducing its own uptake into non-phagocytic cells by a process termed invasion, and residing within a membrane-bound inclusion. During invasion it causes significant rearrangement of the host cytoskeleton, indicating that signals are transduced between the bacterium and the host cell cytoplasm, across the eukaryotic cell membrane. We found that intracellular inositol phosphate concentrations in HeLa cells increased during S. typhimurium entry and returned to normal levels after bacterial internalization. A chelator of intracellular calcium (BAPTA/AM) blocked S. typhimurium uptake into HeLa epithelial cells, but extracellular calcium chelators (BAPTA, EGTA, EDTA) had no effect on bacterial invasion. These results indicate that S. typhimurium may activate host cell phospholipase C activity to form inositol phosphates which in turn stimulate release of intracellular calcium stores to facilitate bacterial uptake.     

Cyclic AMP potentiates bFGF-induced neurite outgrowth in PC12 cells.

We report here that basic fibroblast growth factor (bFGF)-elicited neurite outgrowth in PC12 cells is potentiated by dibutyryl cyclic adenosine monophosphate (dbcAMP) or forskolin. This property was also described for nerve growth factor (NGF), suggesting that both NGF and bFGF may share common intracellular events leading to neurite outgrowth and synergism with dbcAMP and forskolin. The synergistic effect of dbcAMP and forskolin is specific, since treatment of PC12 cells with bFGF and dibutyryl cyclic guanosine monophosphate (dbcGMP) or phorbol ester did not change the neurite outgrowth response of cells treated with bFGF alone. Furthermore, neurite outgrowth depends on cellular adhesion. Increasing adhesion by plate treatment with poly-d-lysine increases the neurite outgrowth elicited by bFGF alone or bFGF plus dbcAMP. On the other hand, decreasing cellular adhesiveness by plating PC12 cells in semi-solid agarose renders the cells unable to develop neuritic processes. In addition, 3H-methylthymidine incorporation studies showed that bFGF-treated PC12 cells cease growth only when they become fully differentiated after 3-5 days of treatment. In contrast, dbcAMP, which is a poor differentiation factor, is able to block cellular growth after 24 hour treatment. These results suggest that when PC12 cells become differentiated, they stop growing. However, growth inhibition does not necessarily lead to differentiation.