Prostaglandin F2 alpha stimulates inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate formation in bovine luteal cells.

The present studies were conducted to further evaluate inositol phosphate formation and metabolism in prostaglandin F2 alpha (PGF2 alpha)-stimulated bovine luteal cells. Corpora lutea were dispersed with collagenase, and luteal cells were prelabeled for 3 h with [3H]inositol. Inositol phosphates produced in response to PGF2 alpha were analyzed by ion exchange column chromatography and HPLC. Time-course experiments revealed that significant increases in inositol trisphosphate (InsP3) were apparent within 5 sec of incubation with PGF2 alpha. Increases in inositol bisphosphate (InsP2) were also apparent within 5 sec. InsP1 and InsP4 were observed after a short (5-sec) lag period. HPLC revealed that PGF2 alpha provoked rapid (5 sec) increases in inositol 1,4,5-trisphosphate (Ins 1,4,5-P3), which was rapidly converted to inositol 1,3,4,5-tetrakisphosphate (Ins 1,3,4,5-P4) and inositol 1,3,4-trisphosphate (Ins 1,3,4-P3). The primary inositol bisphosphate isomer present in PGF2 alpha-stimulated bovine luteal cells was inositol 1,4-bisphosphate (Ins 1,4-P2), with lesser amounts of Ins 1,3-P2. Inositol monophosphates were also increased. These findings were confirmed in studies in which the metabolism of purified [3H]Ins 1,4,5-P3 was followed temporally in saponin-permeabilized bovine luteal cells. Additional studies demonstrated the presence of an enzyme, InsP3-3-kinase, in the cytosolic fraction of bovine corpora lutea. InsP3-3-kinase phosphorylated Ins 1,4,5-P3 to form Ins 1,3,4,5-P4. The activity of InsP3-3-kinase was calcium dependent and was enhanced by calmodulin at low calcium concentrations. Calmidazolium, a calmodulin inhibitor, reduced InsP3-3-kinase activity in a concentration-dependent manner. These results demonstrate the presence of multiple polyphosphorylated inositol phosphates in PGF2 alpha-stimulated bovine luteal cells. The isomers were formed via the action of a specific calcium/calmodulin-regulated kinase (InsP3-3-kinase), which phosphorylated Ins 1,4,5-P3 during agonist-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate. These data suggest that the inositol tris/tetrakisphosphate pathway is an important sequelae to PGF2 alpha-stimulated inositol phospholipid hydrolysis, and that the pathway may be activated during agonist-mediated calcium mobilization.     

Prostaglandin F2 alpha-induced calcium transient in ovine large luteal cells: II. Modulation of the transient and resting cytosolic free calcium alters progesterone secretion

A previous study demonstrated that prostaglandin F2 alpha (PGF2 alpha) stimulates a transient increase in cytosolic free Ca2+ levels [( Ca2+]i) in ovine large luteal cells. In the present study, the magnitude of the PGF2 alpha (0.5 microM)-induced calcium transient in Hanks' medium (87 +/- 2 nM increase above resting levels) was reduced (P less than 0.05) but not completely eliminated in fura-2 loaded large luteal cells incubated in Ca2(+)-free or phosphate- and carbonate-free medium (10 +/- 1 nM, 32 +/- 6 nM, above resting levels; respectively). Preincubation for 2 min with 1 mM LaCl3 (calcium antagonist) eliminated the PGF2 alpha-induced calcium transient. The inhibitory effect of PGF2 alpha on secretion of progesterone was reduced in Ca2(+)-free medium or medium plus LaCl3. Resting [Ca2+]i levels and basal secretion of progesterone were both reduced (P less than 0.05) in large cells incubated in Ca2(+)-free medium (27 +/- 4 nM; 70 +/- 6% control, respectively) or with 5 microM 5,5'-dimethyl bis-(O-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid (40 +/- 2 nM; 49 +/- 1% control; respectively). In addition, secretion of progesterone was inhibited (P less than 0.05) by conditions that increased (P less than 0.05) [Ca2+]i; that is LaCl3 ([Ca2+]i, 120 +/- 17 nM; progesterone, 82 +/- 8% control) and PGF2 alpha ([Ca2+]i, 102 +/- 10 nM; progesterone, 82 +/- 3% control). In small luteal cells, resting [Ca2+]i levels and secretion of progesterone were reduced by incubation in Ca2(+)-free Hanks ([Ca2+]i, 28 +/- 2 nM; progesterone, 71 +/- 6% control), however, neither LaCl3 nor PGF2 alpha increased [Ca2+]i levels or inhibited secretion of progesterone. The findings presented here provide evidence that extracellular as well as intracellular calcium contribute to the PGF2 alpha-induced [Ca2+]i transient in large cells. Furthermore, whereas an adequate level of [Ca2+]i is required to support progesterone production in both small and large cells, optimal progesterone production in large cells depends upon an appropriate window of [Ca2+]i.     

Regulation of intracellular free calcium in human myometrial cells by prostaglandin F2 alpha: comparison with oxytocin

The effects of prostaglandin F2 alpha (PGF2 alpha) on intracellular Ca2+ concentration ([Ca2+]i) and inositol phosphate (IP) generation in human myometrial cells were evaluated and compared to the effects of oxytocin. Basal [Ca2+]i levels were 146 and 153 nM in the absence and presence of 1 mM extracellular Ca, respectively. In Ca-containing medium, both PGF2 alpha and oxytocin significantly (P less than 0.01) increased [Ca2+]i over control values, eliciting half-maximal stimulation (ED50) at 4 and 1 nM, respectively. In Ca-free medium the potency of PGF2 alpha to raise [Ca2+]i was drastically reduced (ED50, 2 microM), whereas that of oxytocin remained the same, although maximal responses were markedly decreased. PGF2 alpha had no effect on total IP production in the concentration range that significantly raised [Ca2+]i. However, at a 100 times higher concentration (10 microM), PGF2 alpha produced a maximum 48% increase in total IP, with a rapid (15-30 s) rise in IP3 and IP2, followed by IP1. A similar increase in IP production was obtained when [Ca2+]i levels were raised by A23187 to the same level as that obtained with 10-50 microM PGF2 alpha. The effect of PGF2 alpha was dependent on extracellular Ca and could be suppressed by verapamil, but not by pertussis toxin, or phorbol ester. In contrast, the potencies of oxytocin to raise IP and [Ca2+]i were similar and independent of extracellular Ca2+, and could be suppressed by pertussis toxin and phorbol ester, but not by verapamil. These data provide evidence that in isolated human myometrial cells, PGF2 alpha and oxytocin trigger an increase in [Ca2+]i by different mechanisms. The action of PGF2 alpha depends on extracellular Ca2+, whereas oxytocin activates the G-protein-dependent phospholipase-C-IP3-Ca2+ signal-transducing pathway, complemented by the influx of extracellular Ca2+.     

Effect of prostaglandin F2 alpha on cytosolic free calcium ion concentrations in rat luteal cells.

Changes in cytosolic free calcium concentrations [( Ca2+]i) in response to prostaglandin F2 alpha (PGF2 alpha) were measured in single rat luteal cells, using the calcium-sensitive fluorescent dye fura-2. A total of 112 cells were studied in 20 experiments. The average resting [Ca2+]i was 113 +/- 6.4 nM. In 59 cells (53%), there was a 4.6 +/- 0.2-fold increase in [Ca2+]i within 29.3 +/- 1.0 sec of PGF2 alpha administration, and the cells recovered by 78.0 +/- 4.5 sec. The magnitude of the increase in [Ca2+]i in response to PGF2 alpha was not changed by an increase in the concentration of PGF2 alpha. Perifusion with low calcium buffer reduced, then eliminated, the [Ca2+]i response to PGF2 alpha. Perifusion of cells with PGF2 alpha resulted in a single transient [Ca2+]i response, similar to that after short term exposure to PGF2 alpha. Many (67%) of the cells that responded to PGF2 alpha also responded to GnRH. No additive increase in [Ca2+]i was seen when PGF2 alpha and GnRH were administered together. The source of the calcium appears to be intracellular stores that are shared by GnRH and PGF2 alpha.     

High calcium and other divalent cations increase inositol trisphosphate in bovine parathyroid cells

Calcium and other divalent cations rapidly increase intracellular free Ca2+ ([Ca2+]i) in bovine parathyroid cells and inhibit PTH release. In other secretory cells, agonist-dependent generation of inositol trisphosphate (InsP3) through polyphosphoinositide turnover initiates the rise in [Ca2+]i by mobilizing Ca2+ from intracellular stores. To determine whether polyphosphoinositide breakdown is involved in mediating the response to Ca2+ and the divalent cations Ba2+, Mn2+, and Sr2+, we measured the production of inositol polyphosphates in parathyroid cells. Within 120 sec of increasing extracellular Ca2+ to 2.0 mM, InsP3, inositol bisphosphate (InsP2), and inositol monophosphate (InsP1) rose 95 +/- 37%, 87 +/- 17%, and 96 +/- 29%, respectively, vs. values in cells at 0.5 mM Ca2+ (n = 5). Raising extracellular Ca2+ from 0.5-3.0 mM produced even greater peak increments of 134 +/- 13%, 179 +/- 35%, and 313 +/- 65% in InsP3, InsP2, and InsP1, respectively, by 120 sec (n = 4). Similarly, within 10 sec of their addition, BaCl2 (2 mM), MnCl2 (2 mM), and SrCl2 (4 mM) stimulated the production of InsP3 56 +/- 2%, 152 +/- 31%, and 160 +/- 25%, respectively, vs. that in untreated cells at 0.5 mM Ca2+. At later time points, InsP2 and InsP1 were increased. The Ca2+ ionophore ionomycin at concentrations up to 500 nM had no effect on inositol phosphates, although it inhibited PTH release in a dose-dependent manner. Since high Ca2+ and other divalent cations depolarize parathyroid cells, we assessed the effect of high extracellular K+ on inositol polyphosphates. The addition of depolarizing concentrations of K+ (40 mM) did not change inositol phosphates. Thus, Ca2+ and other divalent cations increase the production of InsP3, InsP2, and InsP1 in parathyroid cells by a mechanism independent of increases in [Ca2+]i and of membrane depolarization. We conclude that parathyroid cells express membrane receptors or sensors for Ca2+ and other divalent cations linked to polyphosphoinositide turnover.     

Prostaglandin F2 alpha and gonadotropin-releasing hormone increase intracellular free calcium in rat granulosa cells.

Changes in cytosolic free calcium concentration ([Ca2+]i) in response to prostaglandin F2 alpha (PGF2 alpha) and gonadotropin-releasing hormone (GnRH) were measured in single rat granulosa cells, using the calcium-sensitive fluorescent dye, fura-2AM. In 90 out of 135 granulosa cells (67%), there was a 3- to 4-fold increase in resting [Ca2+]i within 30 s of administration of PGF2 alpha (10(-6) M). The resting [Ca2+]i returned to pre-stimulation levels in approximately 80 s. Granulosa cells were responsive to PGF2 alpha at concentrations ranging from 10(-7) M to 10(-4) M (n = 7). Within this range of concentrations, the magnitude of the calcium response did not differ. In another series of experiments, the majority (93%, n = 57) of the granulosa cells which responded to PGF2 alpha also responded to GnRH. Neither the magnitude of the [Ca2+]i response nor the time to response differed between PGF2 alpha and GnRH. Furthermore, simultaneous treatment of granulosa cells with both hormones did not generate a larger response than with either hormone alone. During perifusion with low calcium media, the characteristic [Ca2+]i response to PGF2 alpha decreased, and was eliminated within 10 min (n = 9). Similar observations were made in response to GnRH under these conditions. These data confirm that PGF2 alpha and GnRH stimulate a transient increase in [Ca2+]i in rat granulosa cells, the source of which may be shared intracellular stores.     

Oxytocin activates the inositol-phospholipid-protein kinase-C system and stimulates prostaglandin production in human amnion cells

It has been postulated that fetal hormonal signals act upon amnion to trigger labor via prostaglandin (PG) production. Human amnion epithelial cell cultures were established to test the effects of potential activators of the inositol phospholipid-protein kinase-C effector system on intracellular inositol phosphate turnover, intracellular free calcium ([ Ca2+]i), and PGE2 production. Oxytocin provoked 3-, 2.5-, and 4-fold increases in inositol triphosphate, inositol bisphosphate, and inositol monophosphate, respectively. [Ca2+]i, measured with the fluorescent dye fura-2, was stimulated by oxytocin and vasopressin (oxytocin greater than vasopressin) in a dose-dependent manner. The [Ca2+]i transient produced by oxytocin reached a peak in 15 sec, followed by a slow return to baseline over 10 min. Preincubation with phorbol 12-myristate-13 acetate (PMA) markedly blunted the oxytocin-induced transient. No [Ca2+]i transient was seen with leukotrienes, PG, serotonin, angiotensin, or alpha- or beta-adrenergic agents. PGE2 production increased 30- to 50-fold with phospholipase-C and PMA, and 10-fold with the calcium ionophore A23187. Oxytocin and vasopressin produced 10- and 3-fold PGE2 increases, respectively. Increased PGE2 production induced by PMA, oxytocin, and A23187 was first seen after 8 hr of incubation and reached maximal levels at 24 h. Minimal PGE2 stimulation occurred with agents that produced no [Ca2+]i transient. Direct activators of the inositol phospholipid-protein kinase-C system in human amnion induce large increases in PGE2 in human amnion cells. Oxytocin and vasopressin are hormonal activators of this system in these cells, as demonstrated by their effects on inositol phosphate turnover and [Ca2+]i. These hormones also increase PGE2 production and may influence labor by stimulating PGE2 production in amnion through the inositol phospholipid-protein kinase-C system.     

Activation of the phosphatidylinositol pathway in the primate corpus luteum by prostaglandin F2 alpha.

The current study was designed to investigate the ability of prostaglandin F2 alpha (PGF2 alpha) to activate a second messenger system (phosphatidylinositol pathway) in corpora lutea (CL) of rhesus monkeys. Activation of this pathway was assessed by monitoring the hydrolysis of phosphatidylinositol to inositol phosphates. Since inositol triphosphate mobilizes intracellular Ca2+, intracellular free calcium concentrations ([Ca2+]i) were also assessed in individual cells by fura-2 fluorescence photometry. These responses to PGF2 alpha were measured in luteal cells collected from nonpregnant rhesus monkeys. CL were collected during the early (days 4-5 after estimated LH surge; n = 4), mid (days 8-9; n = 4), and late (days 13-14; n = 5) luteal phase and 1 day after in vivo hCG treatment (15 IU/dose, morning and evening), which began during the midluteal phase (n = 5). PGF2 alpha significantly increased the accumulation of inositol phosphates in all groups (P less than 0.05), except the midluteal phase (P = 0.07). The luteal sensitivity to PGF2 alpha, judged by phosphatidylinositol hydrolysis, was low in the early to midluteal phase compared to that in the late luteal phase and after in vivo hCG treatment. PGF2 alpha also caused a rapid, yet transient, increase in [Ca2+]i in a large proportion of primate luteal cells. The proportion of luteal cells that responded to PGF2 alpha with an increase in [Ca2+]i was smaller (P less than 0.05) in CL collected during the early luteal phase than in the other groups. Luteal progesterone production was inhibited by PGF2 alpha in CL collected after in vivo hCG. CL treated in vivo with hCG also displayed in vitro the largest increases in phosphatidylinositol hydrolysis and [Ca2+]i in response to PGF2 alpha. Therefore, this study demonstrates that PGF2 alpha is a potent activator of the phosphatidylinositol pathway in the primate CL. This activation is augmented as the luteal phase progresses and is influenced by in vivo hCG treatment. This study also provides evidence that the inhibitory effects of PGF2 alpha on progesterone production are associated with the activation of the phosphatidylinositol pathway.     

Adenosine triphosphate activates the phospholipase-C cascade system in human amnion cells without increasing prostaglandin production

Human amnion is hypothesized to be a target tissue for hormone messages from the fetus regarding labor. We have previously demonstrated prostaglandin E2 (PGE2) release in amnion after treatment with phorbol and oxytocin, but other potential agonists of the inositol phospholipid/protein kinase-C system have not been investigated. The effects of extracellular ATP on cytosolic calcium concentration [( Ca2+])i) inositol phosphate (IP) accumulation, and PGE2 production were studied in cultured human amnion cells. Intracellular free calcium [Ca2+]i was measured using the fluorescent dye fura-2. Addition of 0.01-30 microM ATP resulted in a [Ca2+]i transient which peaked within 15 sec and returned to baseline over 10 min. UTP (1 microM) was more effective than ATP (1 microM); [Ca2+]i levels rose from 233 to 2880 nM (UTP) and 2320 nM (ATP). A reduced effect was observed with other nucleotides in a rank order of agonist potency of ITP greater than CTP greater than ADP greater than GTP greater than TTP. No effect was seen with AMP, cAMP, or adenosine. This is consistent with P2 purinoceptors, as described in other tissues. ATP (100 microM) also dramatically increased IP accumulation. Inositol triphosphate, inositol bisphosphate, and inositol monophosphate were increased 7-, 9-, and 16-fold respectively. The agonist potency order of other nucleotides for IP accumulation was the same as that of [Ca2+]i. Pharmacological concentrations of ATP (1 mM) were required to increase PGE2 production. Many other nucleotides were equally effective at this concentration. ATP activates the phospholipase-C system in human amnion, as demonstrated by the increase in [Ca2+]i and inositol phosphates. The physiological significance of purinergic stimulation of this tissue remains unclear.     

Effects of phosphatidic acid on parathyroid hormone release, intracellular free Ca2+, and inositol phosphates in dispersed bovine parathyroid cells

The observation that increases in extracellular Ca2+ or the addition of divalent cations, such as Ba2+, Mg2+, Mn2+, or Sr2+, stimulate the accumulation of inositol trisphosphate (InsP3) and its breakdown products in parathyroid cells strongly supports the idea that polyphosphoinositides are hydrolyzed under these conditions. Since phosphatidic acid is produced as a result of polyphosphoinositide hydrolysis, and it has been proposed that phosphatidic acid may be a second messenger for Ca2+ mobilization, we examined the effects of this compound on parathyroid cells. We assessed PTH release, intracellular free Ca2+ ([Ca2+]i), and inositol polyphosphate accumulation in response to phosphatidic acid. Natural phosphatidic acid reduced PTH release at 1.0 mM extracellular Ca2+ by 18 +/- 6%, 48 +/- 5%, 59 +/- 10%, and 79 +/- 6% at concentrations of 1, 10, 50, and 100 micrograms/ml, respectively (n = 5-11). The effect was not dependent on the presence of extracellular Ca2+, since phosphatidic acid (100 micrograms/ml) inhibited PTH secretion by 39 +/- 3% in medium with no added Ca2+ and 1.0 mM EGTA (n = 3). This agent rapidly and transiently increased [Ca2+]i in a dose-dependent manner, as determined by fura-2 fluorescence. At 1.0 mM extracellular Ca2+, [Ca2+]i rose from 309 +/- 8 to a peak of 356 +/- 26, 454 +/- 22, and 587 +/- 57 nM with the addition of 1, 10, and 100 micrograms/ml phosphatidic acid, respectively (n = 2-14). In the absence of extracellular Ca2+ (i.e. medium with 1 or 2 mM EGTA and no added Ca2+), phosphatidic acid produced a quantitatively smaller peak increment of 38 +/- 4% in [Ca2+]i, indicating that this compound could mobilize Ca2+ from intracellular stores (n = 3). At 1.0 mM extracellular Ca2+, phosphatidic acid (200 micrograms/ml) stimulated the accumulation of Inositol trisphosphate (InsP3), Inositol bisphosphate (InsP2), and Inositol monophosphate (InsP1) by 46 +/- 9%, 37 +/- 9%, and 59 +/- 11% after 60 sec, respectively (n = 5-7). Phosphatidic acid had no significant effect on forskolin-stimulated cAMP accumulation. We further determined whether the specific fatty acid composition of phosphatidic acid might influence its effects in parathyroid cells by testing several synthetic compounds. Dipalmitoyl phosphatidic acid (greater than or equal to 50 micrograms/ml) inhibited PTH release in a dose-dependent manner without significantly changing [Ca2+]i. Dioleoyl phosphatidic acid had modest biphasic effects on secretion, with 20 +/- 5% inhibition observed at lower doses (10 micrograms/ml) and a 27 +/- 8% stimulation of secretion at 100 micrograms/ml (n = 6).(ABSTRACT TRUNCATED AT 400 WORDS)     

Divergent differentiation of rat adrenocortical cells is associated with an interruption of angiotensin II-mediated signal transduction.

The zones of the adrenal cortex contain distinct populations of cells which share a common developmental origin and steroidogenic template. In the rat, zona glomerulosa cells respond to angiotensin II (Ang II) with increased steroidogenesis while zona fasciculata/reticularis cells do not. We have examined Ang II-mediated signal transduction in homogeneous cellular sub-populations derived from either the zona glomerulosa (GLOM) or the zona fasciculata (FASC). In both of these sub-populations Ang II treatment significantly increased the levels of 3H-labelled inositol phosphates as well as the total mass of inositol 1,4,5-triphosphate. In contrast, the two cell types exhibited very different Ang II-mediated changes in free intracellular calcium ([Ca2+]i). Ang II (10 nM), induced [Ca2+]i increases of > 50 nM in 90% of individual GLOM cells (53/58), but in only 28% of FASC cells (11/39). These [Ca2+]i responses occurred after a transient Ang II stimulation ( < 1 min), in the presence of verapamil and in the absence of extracellular calcium, indicating an intracellular release. In small groups of 10-30 cells, stimulation with 1, 10 and 100 nM Ang II induced [Ca2+]i increases of 78, 178 and 215 nM respectively in GLOM cultures compared to only 35, 64, and 65 nM in FASC cultures. Thapsigargin treatment, which releases intracellular calcium in an inositol phosphate independent manner, elicited [Ca2+]i increases in both populations. Importantly, a calcium ionophore induced elevation of [Ca2+]i increased steroidogenesis in both cell types. These results suggest that an interruption of the signaling cascade at the level of intracellular calcium release contributes to the lack of a steroidogenic response to Ang II by the FASC cells. Therefore, in the rat adrenal cortex, divergent differentiation of related cell types may involve alterations within signal transduction pathways distal to initial receptor-mediated events (i.e. inositol phosphate production) and proximal to downstream effector events (i.e. steroidogenesis).     

Cytosolic free calcium increased by prostaglandin F2 alpha (PGF2 alpha), gonadotropin-releasing hormone, and angiotensin II in rat granulosa cells and PGF2 alpha in human granulosa cells.

Cytosolic [Ca2+]i was measured using a microspectrofluorimetric technique. Prostaglandin F2 alpha (PGF2 alpha, 10(-6) M) transiently increased the concentration of free cytosolic Ca2+ ([Ca2+]i) in individual rat and human granulosa cells. In a study examining a total of 170 individual rat and human granulosa cells, approximately 100% of rat granulosa cells and 80% of human granulosa cells tested responded to PGF2 alpha (10(-6) M). In a dose-response trial, the magnitude of the [Ca2+]i response did not vary, although a decreasing number of cells responded to decreasing PGF2 alpha concentrations (10(-5) to 10(-9) M). PGE2 (10(-4) to 10(-6) M) did not affect [Ca2+]i in rat or human granulosa cells. GnRH (10(-6) M) increased [Ca2+]i in rat but not human granulosa cells. Over 90% of rat granulosa cells tested responded. Angiotensin II (ANG II, 10(-5) M) increased [Ca2+]i in approximately 25% of rat, but not human granulosa cells. Individual rat granulosa cells which responded to GnRH responded to PGF2 alpha and vice versa. Individual rat granulosa cells which responded to ANG II responded to PGF2 alpha and GnRH. Conversely, less than 30% of individual rat granulosa cells which responded to PGF2 alpha and GnRH responded to ANG II. Desensitization (pretreatment) of rat granulosa cells by continuous hormone perifusion suggested that effects of PGF2 alpha, GnRH, and ANG II on [Ca2+]i were receptor specific. However, the effects of combined hormone treatments on [Ca2+]i were not additive. The transient increase in [Ca2+]i in response to PGF2 alpha or GnRH, alone, may be maximal. Results of this study suggested that effects of PGF2 alpha, GnRH, and ANG II receptor-ligand interactions may be at least partially mediated by transient increases in [Ca2+]i in rat granulosa cells. Similarly, effects of PGF2 alpha, but not GnRH or ANG II, receptor-ligand interactions may be mediated by transient increases in [Ca2+]i in human granulosa cells.     

Differential effects of luteinizing hormone on intracellular free Ca2+ in small and large bovine luteal cells

The effect of LH on the intracellular free Ca2+ concentration ([Ca2+]i) was investigated in highly purified small and large bovine luteal cell populations. Luteal cells were obtained from midcycle corpora lutea dispersed with collagenase and separated by flow cytometry into large and small cells. Resting levels of Ca2+ were higher (P less than 0.05) in the large than small cells [314 +/- 25 nM (n = 5) vs. 186 +/- 13 nM (mean +/- SE; n = 13) for large and small cells, respectively]. LH rapidly increased [Ca2+]i in both small and large cells loaded with the fluorescent Ca2+ probe fura-2. In the small cells, [Ca2+]i was immediately increased 2- to 6-fold (from 176 +/- 8 to 468 +/- 8 nM; n = 5) after adding LH. The LH induced [Ca2+]i rise occurred in two phases: an initial peak due to intracellular Ca2+ mobilization and a secondary rise due to Ca2+ influx from extracellular sources. Preincubation of the small cells with EGTA reduced the initial phase and abolished the secondary rise in [Ca2+]. Both forskolin and 8-bromo-cAMP increased [Ca2+]i in the small cells. In contrast, only a single phase of [Ca2+]i rise was observed in LH-treated large cells, and the response was 1.5- to 2-fold greater than the resting Ca2+ levels [314 +/- 25 vs. 435 +/- 60 nM (n = 4), for resting vs. LH-treated values, respectively]. The addition of both LH and prostaglandin F2 alpha (PGF2 alpha) to the large cells resulted in increases in [Ca2+]i that were greater than those induced by each hormone separately (2.0-fold for LH and 2.7-fold for PGF2 alpha vs. 7- to 9-fold in the presence of both hormones). These findings demonstrate that LH induces rapid increases in intracellular [Ca2+]i that differ in magnitude and profile between the small and large bovine luteal cells. Furthermore, LH and PGF2 alpha interacted to promote increases in [Ca2+]i in the large cells, that were higher than the sum of [Ca2+]i induced by each hormone separately.     

Endothelin ETA receptors mediate the signaling and secretory actions of endothelins in pituitary gonadotrophs.

Specific receptors for endothelin (ET), localized by autoradiographic studies with [125I]ET in frozen sections of the rat pituitary gland, were abundant in the adenohypophysis, but not in the neurohypophysis. Specific binding of [125I]ET-1 and [125I]ET-3 was also demonstrable in 3-day-old primary cultures of anterior pituitary cells. The binding of [125I]ET-1 to its receptors was time and temperature dependent and was followed by rapid internalization of the receptor-ligand complex. Binding of [125I]ET-1 and [125I]ET-3 to pituitary tissues and cells was more effectively displaced by ET-1 and ET-2 than by ET-3. In cultured pituitary cells, ET-1 caused a rapid increase in polyphosphoinositide hydrolysis, and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] production, with a prompt rise in the cytoplasmic calcium concentration ([Ca2+]i) and LH secretion. The Ins(1,4,5)P3 response to 100 nM ET-1 was transient, with a spike at 10 sec followed by an exponential decrease toward the low steady state level. Ins(1,3,4)P3 and inositol bisphosphate (InsP2) increased more slowly, reaching peak values 30-40 sec after stimulation. The kinetics of the [Ca2+]i response to ET-1 were similar to those of the Ins(1,4,5)P3 response and more rapid than those of the Ins(1,3,4)P3 and InsP2 responses. In perifused cells, ET-stimulated increases in LH release showed the same biphasic patterns as the Ins(1,4,5)P3 and [Ca2+]i responses. ET-1 was more potent than ET-3 in stimulating [Ca2+]i and LH responses, consistent with its higher affinity for the pituitary ET receptors. The initial activation of Ca2+ signaling and LH exocytosis by ETs was followed by prolonged refractoriness to both ET-1 and ET-3. The development of desensitization occurred more rapidly in ET-1- than ET-3-stimulated cells and correlated temporally with endocytosis of the receptor-ligand complex. These findings indicate that stimulation of gonadotropin release by ETs occurs via activation of ETA-type receptors, which are coupled to polyphosphoinositide hydrolysis and [Ca2+]i mobilization, and undergo rapid internalization and profound desensitization.     

Effects of calcitonin on 3',5'-cyclic adenosine monophosphate and calcium second messenger generation and osteoblast function in UMR 106-06 osteoblast-like cells.

The UMR 106-06 rat osteosarcoma osteoblast-like cell line possesses calcitonin (CT) receptors in addition to expressing PTH receptors and a highly osteoblast-like phenotype, and may represent an intermediate developmental stage between early osteoblast precursors and mature osteoblasts. Therefore, we examined the effects of CT and PTH on second messenger generation and osteoblastic function in these cells. In UMR-106-06 cells, 10-1000 nM CT produced a dose-dependent stimulation of intracellular free calcium concentration ([Ca2+]i), which reached a plateau between 2-3 min. This stimulatory effect was abolished in the absence of extracellular Ca2+ ([Ca2+]o) and was mimicked by forskolin and (Bu)2cAMP. One hundred nanomolar CT also produced a slight but significant increase in inositol triphosphate production (13%, P less than 0.05) but did not produce a rapid, transient increase in [Ca2+]i. In contrast, PTH produced a rapid, transient increase in [Ca2+]i, which reached a maximum within 30 sec. This stimulatory effect of PTH on [Ca2+]i signal was dose-dependent and accompanied by a parallel stimulation of inositol triphosphate production. PTH, forskolin, and (Bu)2cAMP all produced a marked dose-related suppression of both DNA and collagen synthesis, which paralleled their stimulatory effects on intracellular cAMP levels. In marked contrast, CT only minimally reduced DNA and collagen synthesis despite producing comparable increases in intracellular cAMP. One hundred nanomolar CT also stimulated alkaline phosphatase specific activity by 33% (P less than 0.05). Thus, CT stimulates cAMP, [Ca2+]i, and inositol phosphate second messengers in UMR 106-06 cells. However, in contrast to other agents which elevate intracellular cAMP levels, CT does not suppress DNA synthesis. These results suggest that the linkage of CT receptor second messengers to effects on cell function differ from those of PTH and/or that CT may produce additional second messenger(s) which antagonize the antiproliferative effect of increased cAMP levels in UMR-106-06 cells.     

Effects of prostaglandin F2 alpha and a gonadotropin-releasing hormone agonist on inositol phospholipid metabolism in isolated rat corpora lutea of various ages

The sensitivity of rat corpora lutea to luteolytic agents increases with luteal age. We examined the effect of prostaglandin F2 alpha (PGF2 alpha) and [D-Ala6,Des-Gly10]GnRH ethylamide (GnRHa) on inositol phospholipid metabolism in day 2 and day 7 corpora lutea from PMSG-treated rats. Isolated corpora lutea were incubated with 32PO4 or [3H]inositol and were treated with LH, PGF2 alpha, or GnRHa. Phospholipids were purified by TLC, and the water-soluble products of phospholipase-C activity (inositol phosphates) were isolated by ion exchange chromatography. In day 2 corpora lutea, PGF2 alpha, (10 microM) and GnRHa (100 ng/ml) significantly increased 32PO4 incorporation into phosphatidic acid (PA) and phosphatidylinositol (PI), but not into other fractions. LH provoked slight increases in PA. Results were similar with 30 min of prelabeling or simultaneous addition of 32PO4 and stimulants. In other experiments, PGF2 alpha and GnRHa provoked rapid increases (1-5 min) in the accumulation of inositol mono-, bis-, and trisphosphates. LH did not significantly increase inositol phosphate accumulation, but stimulated cAMP accumulation in 2-day-old corpora lutea. Inositol phospholipid metabolism was increased in day 7 corpora lutea compared to that in day 2 corpora lutea. This increase was associated with increased incorporation of 32PO4 into PA and PI and increased accumulation of [3H]inositol phosphates. In day 7 corpora lutea, which are very sensitive to the luteolytic effect of PGF2 alpha, the PG-induced increase in PA labeling was small and inconsistent, whereas PI labeling was unaffected in 30-min incubations. GnRHa was without effect in such corpora lutea. LH, PGF2 alpha, or GnRHa did not increase inositol phosphate accumulation in 7-day-old corpora lutea. These studies demonstrate that the transformation of young (day 2) to mature (day 7) corpora lutea is associated with an increase in luteal inositol phospholipid metabolism. The results also show that PGF2 alpha and GnRHa stimulate phospholipase-C activity in young corpora lutea, but are ineffective in mature corpora lutea, and suggest that an increase in inositol phospholipid metabolism by itself is not sufficient to explain the acute luteolytic action of PGF2 alpha and GnRH in vitro. However, phospholipase-C-derived second messengers may be involved in the action of hormones that control luteal function.     

Effects of protein kinase C activation on inositol phosphate generation and intracellular Ca2+ mobilization in bovine parathyroid cells

Activators of protein kinase C, such as phorbol myristate acetate (PMA) and the synthetic diacylglycerol dioctanoylglycerol (diC8), either stimulate or inhibit PTH release depending on the extracellular Ca2+ concentration. By increasing PTH release at high extracellular Ca2+, these agents, in effect, block high Ca2(+)-induced inhibition of secretion. Since raising extracellular Ca2+ increases intracellular free Ca2+ ([Ca2+]i) and inositol trisphosphate (InsP3) formation in parathyroid cells, we assessed the effects of PMA pretreatment on [Ca2+]i and InsP3 to ascertain whether these second messengers might be altered by protein kinase C activation. Preincubation of parathyroid cells with PMA (10(-6) M) significantly lowered the intracellular Ca2+ response to raising extracellular Ca2+ from 0.5-2.0 mM. The peak increase in [Ca2+]i averaged 475 +/- 11 nM in PMA-treated cells compared to 703 +/- 44 nM in control cells. High extracellular Ca2(+)-induced InsP3 accumulation was also reduced after incubating the cells with PMA. To determine whether intracellular Ca2+ stores and/or transmembrane Ca2+ uptake were affected by activating protein kinase C, we examined intracellular Ca2+ responses to the Ca2+ ionophore ionomycin after PMA pretreatment. At 0.5 mM Ca2+, ionomycin (10(-6) M) increased [Ca2+]i to an initial peak of 738 +/- 49 nM followed by a sustained increase to 501 +/- 30 nM in control cells (n = 15). After exposure to PMA (greater than or equal to 20 min), however, peak and sustained increments in [Ca2+]i were significantly lower at 550 +/- 32 and 394 +/- 16 nM, respectively (P less than 0.02, n = 8). In the absence of extracellular Ca2+, basal [Ca2+]i was 197 +/- 5 and peaked at 323 +/- 15 nM with ionomycin (10(-6) M) in PMA-treated cells (n = 16). The latter value was significantly less than the peak increase in [Ca2+]i to 461 +/- 19 nM observed with ionomycin (10(-6) M) in control cells (P less than 0.001, n = 15). With respect to secretion, either of the protein kinase C agonists (i.e. PMA or diC8) or the Ca2+ ionophore ionomycin inhibited PTH release at 0.5 mM Ca2+. To determine whether the concomitant activation of protein kinase C- and Ca2(+)-dependent pathways could additively suppress PTH release, we assessed the effects of ionomycin and either PMA or diC8 on secretion. PTH release at 0.5 mM Ca2+ was reduced in an additive manner by either of these protein kinase C agonists plus ionomycin. At 2 mM Ca2+, protein kinase C agonists stimulated PTH release.(ABSTRACT TRUNCATED AT 400 WORDS)     

Gonadotropin-releasing hormone-induced calcium signaling in clonal pituitary gonadotrophs.

In agonist-stimulated clonal pituitary gonadotrophs (alpha T3-1 cells), cytoplasmic calcium ([Ca2+]i) exhibited rapid and prominent peak increases, followed by lower, but sustained, elevations for up to 15 min. The [Ca2+]i response to GnRH was rapidly inhibited by prior addition of a potent GnRH antagonist. In the absence of extracellular Ca2+ the initial peak [Ca2+]i response was only slightly decreased, but the prolonged increase in [Ca2+]i was abolished, indicating that the peak is derived largely from intracellular calcium mobilization and the sustained phase from Ca2+ influx. Application of the endoplasmic reticulum Ca(2+)-ATPase blocker thapsigargin caused progressive and dose-dependent elevation of [Ca2+]i and decreased the peak amplitude of the GnRH-induced Ca2+ response. On the other hand, addition of dihydropyridine calcium channel antagonists before or after GnRH treatment prevented or terminated the plateau phase, respectively, consistent with entry of Ca2+ through L-type voltage-sensitive Ca2+ channels (VSCC) as the major Ca2+ influx pathway during GnRH action. The presence of L-type VSCC in alpha T3-1 cells was further indicated by the ability of elevated extracellular K+ levels and the dihydropyridine calcium channel agonist Bay K 8644 to elevate [Ca2+]i in an extracellular calcium-dependent manner. These actions of depolarization and Bay K 8644 were inhibited by nifedipine, with an IC50 of 10 nM. High extracellular K(+)- and GnRH-induced Ca2+ entry was also attenuated by phorbol esters and permeant diacylglycerols, indicating that protein kinase-C exerts inhibitory modulation of VSCC activity. In contrast to normal pituitary gonadotrophs, in which GnRH induces a frequency-modulated oscillatory [Ca2+]i response, single alpha T3-1 cells exhibited a nonoscillatory amplitude-modulated signal during agonist stimulation. The [Ca2+]i responses observed in alpha T3-1 gonadotrophs indicate that the immortalized cells retain functional GnRH receptors and their coupling to the Ca2+ signaling pathway. Ca2+ influx through L-type channels maintains the plateau phase of the [Ca2+]i response during agonist stimulation and is inhibited by activation of protein kinase-C.     

1,25-dihydroxyvitamin D3 inhibits Na(+)-H+ exchange by stimulating membrane phosphoinositide turnover and increasing cytosolic calcium in CaCo-2 cells.

We have examined the effects of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] on the phosphoinositol signal transduction pathway in the human colon cancer-derived cell line CaCo-2 and have studied the regulation of intracellular calcium ([Ca2+]i) and pH (pHi) by this secosteroid. CaCo-2 cells were prelabeled with [3H]myoinositol and treated with 10(-8) M 1,25-(OH)2D3 or vehicle for 90 sec. 1,25-(OH)2D3 caused a decrease in labeled phosphatidylinositol-4-5-bis-phosphate and an increase in labeled inositol 1,4,5-trisphosphate. Treatment with 10(-8) M 1,25-(OH)2D3 for 90 sec also raised the cellular content of diacylglycerol. In a dose-dependent manner, 1,25-(OH)2D3 caused the translocation of protein kinase-C activity from the cytosolic to the membrane fraction, which occurred after as little as 15 sec of exposure to the secosteroid, peaked at about 1-5 min, and then returned toward baseline values. In these CaCo-2 cells, baseline [Ca2+]i was 258 +/- 2 nM (mean +/- SE), as assessed using the fluorescent dye fura-2. After exposure to 10(-8) M 1,25-(OH)2D3, [Ca2+]i rapidly increased to 392 +/- 14 nM after 100 sec, fell, and then subsequently rose to a plateau of 350 +/- 3 nM after 400 sec. In Ca(2+)-free buffer, 1,25-(OH)2D3 caused only a transient rise in [Ca2+]i, indicating that 1,25-(OH)2D3 stimulated both the release of intracellular calcium stores and calcium influx. 1,25-(OH)2D3 caused a dose-dependent decrease in pHi in CaCo-2 cells, as assessed by the fluorescent dye BCECF, which was not observed in cells suspended in Na(+)-free buffer or pretreated with amiloride, indicating that the secosteroid inhibited Na(+)-H+ exchange. No effect of 1,25-(OH)2D3 on pHi was observed in cells in a Ca(2+)-free buffer or pretreated with the phospholipase-C inhibitor U-73,122, which also blocked the rise in [Ca2+]i, or in cells pretreated with the Ca2+/calmodulin inhibitor calmidazolium. Taken together, these studies indicate that 1,25-(OH)2D3 rapidly stimulates membrane phosphoinositide breakdown in CaCo-2 cells, generating the second messengers inositol 1,4,5-trisphosphate and diacylglycerol, causing translocation of protein kinase-C to the membrane, and increasing [Ca2+]i by both releasing calcium stores and promoting calcium influx. Secondary to the rise in [Ca2+]i, Na(+)-H+ exchange is inhibited by a calcium/calmodulin-dependent pathway.