Thrombin-induced calcium movements in platelet activation

The thrombin-induced Ca2+ fluxes and their coupling to platelet aggregation of the human platelet were studied using quin2 as a measure of the cytoplasmic Ca2+ concentration [( Ca2+]cyt) and chlorotetracycline (CTC) as a measure of internally sequestered Ca2+. Evidence is given that the CTC fluorescence change is proportional to the free internal Ca2+ concentration in the dense tubular lumen. The intracellular quin2 concentration was 1 mM and analysis showed that it did not perturb the processes reported herein. The value of [Ca2+]cyt at rest and during thrombin activation was analyzed in terms of Ca2+ influx, Ca2+ release, Ca2+ sequestration, and Ca2+ extrusion. Influx was distinguished from internal release by removing extracellular Ca2+ 1 min before thrombin activation. In the presence of 2 mM external Ca2+, the thrombin-induced Ca2+ influx accounts for most of the increase in [Ca2+]cyt (over 80%). Thrombin-induced Ca2+ influx and release have somewhat different EC50 values (0.17 U/ml vs. 0.35 U/ml). The contribution of influx can be inhibited by verapamil, bepridil and Cd2+ (IC50 values of 19 microM, 2 microM and 50 microM). The influx results were analyzed in terms of a thrombin-activated channel. Indomethacin pretreatment experiments suggest that activation of the arachidonic pathway accounts for approx. 50% of the influx-related [Ca2+]cyt elevation. Elevation of [Ca2+]cyt by intracellular release is not inhibited by verapamil or Cd2+ but is inhibited by bepridil with a high IC50 (25 microM). It is only 15-20% inhibited by indomethacin and is thus not dependent on thromboxane A2 formation. The release reaction does not require Ca2+ influx. The rate of thrombin-activated platelet aggregation is shown to have an approximately fourth-power dependence on [Ca2+]cyt with an apparent Km of 0.4 microM. Comparisons of aggregation rates of the partially thrombin-activated vs. fully thrombin-activated, partially verapamil-inhibited conditions suggest that this dependence on [Ca2+]cyt is the major determinant of the aggregation behavior. Analysis shows that calcium influx is the major pathway for elevating [Ca2+]cyt by thrombin when physiological concentrations of external Ca2+ are present.     

Gonadotropin-releasing hormone (GnRH) receptor expression and membrane signaling in early embryonic GnRH neurons: role in pulsatile neurosecretion

The characteristic pulsatile secretion of GnRH from hypothalamic neurons is dependent on an autocrine interaction between GnRH and its receptors expressed in GnRH-producing neurons. The ontogeny and function of this autoregulatory process were investigated in studies on the properties of GnRH neurons derived from the olfactory placode of the fetal rat. An analysis of immunocytochemically identified, laser-captured fetal rat hypothalamic GnRH neurons, and olfactory placode-derived GnRH neurons identified by differential interference contrast microscopy, demonstrated coexpression of mRNAs encoding GnRH and its type I receptor. Both placode-derived and immortalized GnRH neurons (GT1-7 cells) exhibited spontaneous electrical activity that was stimulated by GnRH agonist treatment. This evoked response, as well as basal neuronal firing, was abolished by treatment with a GnRH antagonist. GnRH stimulation elicited biphasic intracellular calcium ([Ca2+]i) responses, and both basal and GnRH-stimulated [Ca2+]i levels were reduced by antagonist treatment. Perifused cultures released GnRH in a pulsatile manner that was highly dependent on extracellular Ca2+. The amplitude of GnRH pulses was increased by GnRH agonist stimulation and was diminished during GnRH antagonist treatment. These findings demonstrate that expression of GnRH receptor, GnRH-dependent activation of Ca2+ signaling, and autocrine regulation of GnRH release are characteristics of early fetal GnRH neurons and could provide a mechanism for gene expression and regulated GnRH secretion during embryonic migration.     

Autocrine regulation of calcium influx and gonadotropin-releasing hormone secretion in hypothalamic neurons.

Gonadotropin-releasing hormone (GnRH) receptors are expressed in hypothalamic tissues from adult rats, cultured fetal hypothalamic cells, and immortalized GnRH-secreting neurons (GT1 cells). Their activation by GnRH agonists leads to an overall increase in the extracellular Ca2+-dependent pulsatile release of GnRH. Electrophysiological studies showed that GT1 cells exhibit spontaneous, extracellular Ca2+-dependent action potentials, and that their inward currents include Na+, T-type and L-type Ca2+ components. Several types of potassium channels, including apamin-sensitive Ca2+-controlled potassium (SK) channels, are also expressed in GT1 cells. Activation of GnRH receptors leads to biphasic changes in intracellular Ca2+ concentration ([Ca2+]i), with an early and extracellular Ca2+-independent peak and a sustained and extracellular Ca2+-dependent plateau phase. During the peak [Ca2+]i response, electrical activity is abolished due to transient hyperpolarization that is mediated by SK channels. This is followed by sustained depolarization and resumption of firing with increased spike frequency and duration. The agonist-induced depolarization and increased firing are independent of [Ca2+]i and are not mediated by inhibition of K+ currents, but by facilitation of a voltage-insensitive and store depletion-activated Ca2+-conducting inward current. The dual control of pacemaker activity by SK and store depletion-activated Ca2+ channels facilitates voltage-gated Ca2+ influx at elevated [Ca2+]i levels, but also protects cells from Ca2+ overload. This process accounts for the autoregulatory action of GnRH on its release from hypothalamic neurons.     

Naloxone evokes large-amplitude GnRH pulses in luteal-phase ewes

Ewes were sampled during the mid-late luteal phase of the oestrous cycle. Hypophysial portal and jugular venous blood samples were collected at 5-10 min intervals for a minimum of 3 h, before i.v. infusions of saline (12 ml/h; N = 6) or naloxone (40 mg/h; N = 6) for 2 h. During the 2-h saline infusion 2/6 sheep exhibited a GnRH/LH pulse; 3/6 saline infused ewes did not show a pulse during the 6-8-h portal blood sampling period. In contrast, large amplitude GnRH/LH pulses were observed during naloxone treatment in 5/6 ewes. The mean (+/- s.e.m.) amplitude of the LH secretory episodes during the naloxone infusion (1.07 +/- 0.11 ng/ml) was significantly (P less than 0.05) greater than that before the infusion in the same sheep (0.54 +/- 0.15 ng/ml). Naloxone significantly (P less than 0.005) increased the mean GnRH pulse amplitude in the 5/6 responding ewes from a pre-infusion value of 0.99 +/- 0.22 pg/min to 4.39 +/- 1.10 pg/min during infusion. This episodic GnRH secretory rate during naloxone treatment was also significantly (P less than 0.05) greater than in the saline-infused sheep (1.53 +/- 0.28 pg/min). Plasma FSH and prolactin concentrations did not change in response to the opiate antagonist. Perturbation of the endogenous opioid peptide system in the ewe by naloxone therefore increases the secretion of hypothalamic GnRH into the hypophysial portal vasculature. The response is characterized by a large-amplitude GnRH pulse which, in turn, causes a large-amplitude pulse of LH to be released by the pituitary gland.     

Actions of thapsigargin on the Ca(2+)-handling systems of the human platelet. Incomplete inhibition of the dense tubular Ca2+ uptake, partial inhibition of the Ca2+ extrusion pump, increase in plasma membrane Ca2+ permeability, and consequent elevation of resting cytoplasmic Ca2+.

Thapsigargin (Tg) effects on Ca2+ handling in the intact human platelet were studied using Quin2 and chlorotetracycline to measure free cytoplasmic and dense tubular (DT) Ca2+ concentrations ([Ca2+]cyt and [Ca2+]dt, respectively). Tg inhibits Ca2+ uptake by the DT Ca(2+)-ATPase pumps, but incompletely, lowering the Vm to 32% of control (IC50,Tg = 0.18 +/- 0.10 microM). The kinetics of loss of DT Ca2+, transient increases in [Ca2+]cyt, and lowered steady-state [Ca2+]dt after Tg addition are all explained by pump inhibition, with no effect on the rate constant of Ca2+ leakage across the DT membrane (kleak,DT = 1.14 min-1). Tg lowers by 30% the Vm of the Ca2+ extrusion pump located in the plasma membrane (PM), as shown by a Quin2-based method measuring active Ca2+ extrusion (Johansson, J. S., and Haynes, D. H. (1988) J. Membr. Biol. 104, 147-163). This effect (IC50,Tg = 0.45 +/- 0.06 microM), together with a 24 +/- 16% increase in kleak,PM,Ca (to 3 x 10(-4) min-1), accounts for a Tg-dependent sustained elevation [Ca2+]cyt (to 708 +/- 78 nM) which is independent of DT Ca2+ status or history. Thrombin and Tg release 30 and 70% (respectively) of the DT Ca2+ available at any instant, independent of order of challenge, consistent with a single class of DT with respect to these agents.     

Regulation of Ca2+-sensitive adenylyl cyclase in gonadotropin-releasing hormone neurons

In immortalized GnRH neurons, cAMP production is elevated by increased extracellular Ca2+ and the Ca2+ channel agonist, BK-8644, and is diminished by low extracellular Ca2+ and treatment with nifedipine, consistent with the expression of adenylyl cyclase type I (AC I). Potassium-induced depolarization of GT1-7 neurons causes a dose-dependent monotonic increase in [Ca2+]i and elicits a bell-shaped cAMP response. The inhibitory phase of the cAMP response is prevented by pertussis toxin (PTX), consistent with the activation of G(i)-related proteins during depolarization. Agonist activation of the endogenous GnRH receptor in GT1-7 neurons also elicits a bell-shaped change in cAMP production. The inhibitory action of high GnRH concentrations is prevented by PTX, indicating coupling of the GnRH receptors to G(i)-related proteins. The stimulation of cAMP production by activation of endogenous LH receptors is enhanced by low (nanomolar) concentrations of GnRH but is abolished by micromolar concentrations of GnRH, again in a PTX-sensitive manner. These findings indicate that GnRH neuronal cAMP production is maintained by Ca2+ entry through voltage-sensitive calcium channels, leading to activation of Ca2+-stimulated AC I. Furthermore, the Ca2+ influx-dependent activation of AC I acts in conjunction with AC-regulatory G proteins to determine basal and agonist-stimulated levels of cAMP production.     

Platelet activating factor raises intracellular calcium ion concentration in macrophages

Peritoneal cells from thioglycollate-stimulated mice were allowed to adhere to coverglasses for 2 h to give a dense monolayer of adherent cells greater than 95% of which were macrophages. After incubation with the tetra-acetoxymethyl ester of quin2, coverglasses were rinsed with Ca2+-free saline, oriented at a 45 degree angle in square cuvettes containing a magnetically driven stir bar, and analyzed for changes in quin2 fluorescence in a spectrofluorimeter. Such fluorescence, taken as an indication of intracellular calcium ion concentration ([Ca2+]i), increased as exogenous calcium ion concentration ([Ca2+]o) was raised to 1 mM. At [Ca2+]o approximately equal to 10 microM, [Ca2+]i = 72 +/- 14 nM (n = 26); at [Ca2+]o = 1 mM, [Ca2+]i = 140-220 nM, levels not increased by N, N, N', N'-tetrakis (2-pyridylmethyl) ethylenediamine, a membrane-permeant chelator of heavy metals than can quench quin2. Addition of mouse alpha + beta fibroblast interferon, lipopolysaccharide, thrombin, collagen, vasopressin, ADP, compound 48/80, or U46619 did not change [Ca2+]i. However, addition of platelet activating factor (PAF) (2-20 ng/ml) raised [Ca2+]i by 480 nM within 1 min if [Ca2+]o = 1 mM. In the presence of 5 mM EGTA, PAF raised [Ca2+]i by 25 nM. This suggests that PAF causes influx of exogenous Ca2+, as well as releasing some Ca2+ from intracellular stores. Consistent with these results, when PAF was added to 1 mM Ca2+ in the presence of 100 microM Cd2+ or Mn2+ to block Ca2+ influx, [Ca2+]i increased by only intermediate amounts; at the times of such dampened peak response, [Ca2+]i could be raised within 1 min to normal PAF-stimulated levels by chelation of the exogenous heavy metals with diethylenetriaminepentaacetic acid. Normal PAF responses were observed in the presence of indomethacin. The lowest dose of PAF observed to raise [Ca2+]i was 0.1 ng/ml. Response of [Ca2+]i to 2-20 ng/ml PAF was transient, and second applications had no effect. The PAF response also was seen in cell suspensions. These results suggest that an increase in [Ca2+]i may be an early event in PAF activation of macrophages.     

Mitochondria exert a negative feedback on the propagation of intracellular Ca2+ waves in rat cortical astrocytes.

We have used digital fluorescence imaging techniques to explore the interplay between mitochondrial Ca2+ uptake and physiological Ca2+ signaling in rat cortical astrocytes. A rise in cytosolic Ca2+ ([Ca2+]cyt), resulting from mobilization of ER Ca2+ stores was followed by a rise in mitochondrial Ca2+ ([Ca2+]m, monitored using rhod-2). Whereas [Ca2+]cyt recovered within approximately 1 min, the time to recovery for [Ca2+]m was approximately 30 min. Dissipating the mitochondrial membrane potential (Deltapsim, using the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxy-phenyl-hydrazone [FCCP] with oligomycin) prevented mitochondrial Ca2+ uptake and slowed the rate of decay of [Ca2+]cyt transients, suggesting that mitochondrial Ca2+ uptake plays a significant role in the clearance of physiological [Ca2+]cyt loads in astrocytes. Ca2+ signals in these cells initiated either by receptor-mediated ER Ca2+ release or mechanical stimulation often consisted of propagating waves (measured using fluo-3). In response to either stimulus, the wave traveled at a mean speed of 22.9 +/- 11.2 micrometer/s (n = 262). This was followed by a wave of mitochondrial depolarization (measured using tetramethylrhodamine ethyl ester [TMRE]), consistent with Ca2+ uptake into mitochondria as the Ca2+ wave traveled across the cell. Collapse of Deltapsim to prevent mitochondrial Ca2+ uptake significantly increased the rate of propagation of the Ca2+ waves by 50%. Taken together, these data suggest that cytosolic Ca2+ buffering by mitochondria provides a potent mechanism to regulate the localized spread of astrocytic Ca2+ signals.     

Arteriolar smooth muscle Ca2+ dynamics during blood flow control in hamster cheek pouch.

Intracellular calcium concentration ([Ca2+]i) governs the contractile status of arteriolar smooth muscle cells (SMC). Although studied in vitro, little is known of SMC [Ca2+]i dynamics during the local control of blood flow. We tested the hypothesis that the rise and fall of SMC [Ca2+]i underlies arteriolar constriction and dilation in vivo. Aparenchymal segments of second-order arterioles (diameter 35 +/- 2 microm) were prepared in the superfused cheek pouch of anesthetized hamsters (n = 18) and perifused with the ratiometric dye fura PE-3 (AM) to load SMC (1 microM, 20 min). Resting SMC [Ca2+]i was 406 +/- 37 nM. Elevating superfusate O2 from 0 to 21% produced constriction (11 +/- 2 microm) that was unaffected by dye loading; [Ca2+]i increased by 108 +/- 53 nM (n = 6, P < 0.05). Cycling of [Ca2+]i during vasomotion (amplitude, 150 +/- 53 nM; n = 4) preceded corresponding diameter changes (7 +/- 1 microm) by approximately 2 s. Microiontophoresis (1 microm pipette tip; 1 microA, 1 s) of phenylephrine (PE) transiently increased [Ca2+]i by 479 +/- 64 nM (n = 8, P < 0.05) with constriction (26 +/- 3 microm). Flushing blood from the lumen with saline increased fluorescence at 510 nm by approximately 45% during excitation at both 340 and 380 nm with no difference in resting [Ca2+]i, diameter or respective responses to PE (n = 7). Acetylcholine microiontophoresis (1 microA, 1 s) transiently reduced resting SMC [Ca2+]i by 131 +/- 21 nM (n = 6, P < 0.05) with vasodilation (17 +/- 1 microm). Superfusion of sodium nitroprusside (10 microM) transiently reduced SMC [Ca2+]i by 124 +/- 18 nM (n = 6, P < 0.05), whereas dilation (23 +/- 5 microm) was sustained. Resolution of arteriolar SMC [Ca2+]i in vivo discriminates key signaling events that govern the local control of tissue blood flow.     

Egg activation events are regulated by the duration of a sustained [Ca2+]cyt signal in the mouse

Although the dynamics of oscillations of cytosolic Ca2+ concentration ([Ca2+]cyt) play important roles in early mammalian development, the impact of the duration when [Ca2+]cyt is elevated is not known. To determine the sensitivity of fertilization-associated responses [i.e., cortical granule exocytosis, resumption of the cell cycle, Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity, recruitment of maternal mRNAs] and developmental competence of the parthenotes to the duration of a [Ca2+]cyt transient, unfertilized mouse eggs were subjected to a prolonged [Ca2+]cyt change for 15, 25, or 50 min by means of repetitive Ca2+ electropermeabilization at 2-min intervals. The initiation and completion of fertilization-associated responses are correlated with the duration of time in which the [Ca2+]cyt is elevated, with the exception that autonomous CaMKII activity is down-regulated with prolonged elevated [Ca2+]cyt. Activated eggs from 25- or 50-min treatments readily develop to the blastocyst stage with no sign of apoptosis or necrosis and some implant. Ca2+ influx into unfertilized eggs causes neither Ca2+ release from intracellular stores nor rapid removal of cytosolic Ca2+. Thus, the total Ca2+ signal input appears to be an important regulatory parameter that ensures completion of fertilization-associated events and oocytes have a surprising degree of tolerance for a prolonged change in [Ca2+]cyt.     

Stimulation of dense tubular Ca2+ uptake in human platelets by cAMP.

Elevation of intracellular cAMP is shown to increase the rate (V) and maximal extent of Ca2+ uptake by the dense tubules in intact human platelets. Elevation of [cAMP] was accomplished by preincubation with the adenylate cyclase activator forskolin or with dibutyryl-cAMP (Bt2-cAMP). The free concentration of Ca2+ in the dense tubular lumen ([Ca2+]dt) was monitored using the fluorescence of chlorotetracycline (CTC) according to protocols developed in this laboratory. The free cytoplasmic Ca2+ concentration ([Ca2+]cyt) was monitored in parallel experiments with quin2. Both [Ca2+]cyt and [Ca2+]dt were analyzed in terms of competition between pump and leak mechanisms in the plasma membrane (PM) and dense tubular membrane (DT). When platelets are incubated in media with approx. 1 microM external Ca2+, [Ca2+]cyt is approx. 50 nM and [Ca2+]dt is very low. When 2 mM external Ca2+ is added, [Ca2+]cyt rises to approx. 100 nM and the process of dense tubular Ca2+ uptake can be resolved. Forskolin (10 microM) and Bt2-cAMP increase the rate of dense tubular Ca2+ uptake (V) to 2.1 +/- 0.60 and 1.70 +/- 40 times control values (respectively). The agents also increase the final [Ca2+]dt to 1.70 +/- 0.21 and 1.72 +/- 0.60 times control values (respectively). Titrations with ionomycin (Iono) showed that the increase was due to an increase in the Vm of the dense tubular Ca2+ pump. With [Iono] = 500 nM, [Ca2+]cyt was raised to greater than or equal to 1.0 microM and Vm of the dense tubular pump was elicited. (At [Iono] = 1.0 microM, the final [Ca2+]dt values were degraded 15% due to shunting of Ca2+ uptake.) Analysis showed that forskolin (10 microM) and Bt2-cAMP (1 mM) increase the Vm by a factors of 1.56 +/- 40 and 1.56 +/- 40, respectively. Analysis showed that neither agent changed the Km of the pump significantly from its control value of 180 nM. Neither agent changed the rate constant for passive leakage of Ca2+ across the DT membrane (1.7 min-1).     

Relationship between serum luteinizing hormone and estradiol in prepubertal boars

Effects of estradiol on serum luteinizing hormone (LH) were studied in prepubertal boars. In Exp. 1, 15-wk-old boars were given (iv) 50 mug estradiol, 1 mg testosterone or 1.5 ml ethanol. Estradiol (P<0.05) decreased LH over a 2.5-hr period, but testosterone did not. In Exp. 2, an estradiol implant reduced LH sample variance (P<0.01) while LH (547 +/- 96 vs 655 +/- 43 pg/ml) and estradiol (14.2 +/- 3.3 vs 18.4 +/- 1.0 pg/ml; control vs implant) were unchanged in 12-wk-old boars. Pulsatile LH releases (4.3 +/- 1.1 vs 3.0 +/- 0.4 pulses/pig/8 hr; control vs treated) and pulse amplitude (272 +/- 34 vs 305 +/- 40 pg/ml) were not affected. The implant tended to decrease serum testosterone (4.86 +/- 0.75 vs 7.66 +/- 1.51 ng/ml; P<0.10). In Exp. 3, LH was higher after zero implants than after four implants (279 +/- 7 vs 227 +/- 9 pg/ml; P<0.01), and LH after two implants was also higher than after four implants (263 +/- 7 pg/ml; P<0.01) in 14-wk-old boars in a Latin square design. Peak LH after 40 mug gonadotropin releasing hormone (GnRH) was less after two and four implants (1,100 +/- 126 and 960 +/- 167 pg/ml, respectively; P<0.01) than after zero implants (1,742 +/- 126 pg/ml). Slope of the first 20 min of LH response to GnRH was greater after zero implants (45.3 pg/min; P<0.05) than after either two or four implants (20.6 and 16.9 pg/min, respectively). Implant treatment decreased serum testosterone (P<0.025) but increased estradiol (P<0.10). Small changes in serum estradiol resulted in changes in LH. These changes in sample variance and mean LH were recognized by boars as different from normal because serum testosterone decreased. Changes in LH may result from estradiol's negative effect on pituitary responsiveness to endogenous GnRH because response to exogenous GnRH was depressed by estradiol.     

Lack of inhibition of thrombin-induced rise in intracellular Ca2+ levels and 5-hydroxytryptamine secretion by 1-oleoyl-2-acetylglycerol in human platelets

The effect of 1-oleoyl-2-acetylglycerol (OAG) on the thrombin-induced rise in intracellular Ca2+ levels ([Ca2+]i) and 5-hydroxy[14C]tryptamine ([14C]5HT) secretion was studied. In washed human platelets prelabelled with [14C]5HT and quin 2, OAG (10-50 micrograms/ml) induced no significant aggregation, [14C]5HT secretion or rise in [Ca2+]i in the presence or absence of fibrinogen. However, addition of OAG (10-50 micrograms/ml) 10 s to 5 min before or 10-60 s after addition of threshold concentrations of thrombin (less than 0.03 U/ml) resulted in a significant potentiation of aggregation and [14C]5HT secretion without any effect on the thrombin-induced rise in [Ca2+]i. Both EGTA, which abolished the latter and creatine phosphate/creatine phosphokinase, the ADP scavenger, totally inhibited the aggregation but only partially reduced [14C]5HT secretion in response to thrombin plus OAG. At higher concentrations of thrombin, neither the rise in [Ca2+]i nor the extent of [14C]5HT secretion was significantly altered by OAG addition. The results demonstrate that, unlike phorbol esters, OAG has no inhibitory effect on thrombin-induced [Ca2+]i mobilisation but can synergize with low concentrations of thrombin in potentiating [14C]5HT secretion even at basal [Ca2+]i.     

Noncyclooxygenase metabolites of arachidonic acid amplify the vasopressin-induced Ca2+ signal in glomerular mesangial cells by releasing Ca2+ from intracellular stores

Noncyclooxygenase metabolites of arachidonic acid may be potent modulators of the mitogenic response of renal mesangial cells to the mitogenic vasoactive peptide arginine vasopressin (AVP). Since Ca2+ is a critical second messenger in the response of mesangial cells to AVP, and Ca2+ has been implicated in the regulation of growth, we determined whether noncyclooxygenase metabolites altered the phospholipase C-Ca2+ signalling cascade which is activated by AVP. Pretreatment of mesangial cells for 10 min with lipoxygenase and cytochrome P450 monooxygenase inhibitors, nordihydroguaiaretic acid (NDGA, 10(-5) M) or SKF-525A (2.5 x 10(-5) M), but not the cyclooxygenase inhibitor indomethacin (2 x 10(-5) M), reduced the magnitude of the AVP (10(-8) and 10(-7) M)-induced increase in cytosolic free Ca2+ concentration ([Ca2+]i) without affecting inositol trisphosphate production. With 10(-8) M AVP, [Ca2+]i increased to 250 +/- 47 nM in NDGA-treated cells versus 401 +/- 59 nM in control cells (p less than 0.01). [Ca2+]i, measured 2 min after exposure to AVP, was also lower with NDGA (152 +/- 21 nM) when compared with AVP alone (220 +/- 22 nM, p less than 0.01). 14,15-epoxyeicosatrienoic acid (EET) (10(-8) M), which had no effect on inositol trisphosphate production, completely reversed the NDGA-induced inhibition of the [Ca2+]i transient, whereas 5-hydroperoxyeicosatetraenoic acid (HPETE) (5 x 10(-7) M) did not. Pretreatment with higher concentrations of 14,15-EET (10(-7)-10(-6) M) markedly potentiated the AVP-induced increase in [Ca2+]i. NDGA-induced inhibition of the AVP-generated [Ca2+]i transient was also observed when cells were incubated in low Ca2+ media ([Ca2+] less than 5 x 10(-8) M), suggesting that NDGA pretreatment impaired intracellular release of Ca2+. Since NDGA had no direct effect on inositol 1,4,5-trisphosphate-induced Ca2+ release, we postulated that NDGA blocked production of a metabolite that releases Ca2+ from intracellular stores. 14,15-EET and 15-HPETE, but not 15-hydroxyeicosatetraenoic acid (each at 3 x 10(-7) M), raised [Ca2+]i when added directly to cells in low Ca2+ media. In permeabilized cells 14,15-EET and 15-HPETE (10(-7) M) potently released Ca2+ from intracellular stores. In summary, noncyclooxygenase metabolites of arachidonic acid, and in particular P450 metabolites, are potent endogenous amplifiers of the AVP-induced [Ca2+]i signal by mechanisms not directly involving phospholipase C activation. This effect is mediated, at least in part, by enhanced release of Ca2+ from intracellular storage sites by an inositol 1,4,5-trisphosphate-independent mechanism.     

Extracellular Ca2+ mobilization in potential-dependent contraction of trachealis of maturing swine.

We studied the effect of maturation on potassium-induced parasympathetic activation and Ca2+ entry in tracheal smooth muscle (TSM) from fifteen 2-wk-old (2ws) and sixteen 10-wk-old (10ws) male domestic farm swine. Atropine (10(-7) M) caused inhibition of the maximal contraction elicited by potassium to 50.3 +/- 2.6% maximum of control response (P less than 0.001) in TSM from 2ws but had no significant effect in TSM from 10ws (94.6 +/- 4.2% maximum; P = NS vs. control). Verapamil (10(-7) M) plus 10(-7) M atropine reduced contraction elicited by potassium in both 2ws (23.7 +/- 5.8% maximum; P less than 0.001 vs. control) and 10ws (50.6 +/- 6.3% maximum; P less than 0.001 vs. control, P less than 0.05 vs. 2ws); 10(-6)M verapamil caused greater than 95% blockade of contraction caused by potassium in both 2ws and 10ws. In separate studies, atropine-treated strips were equilibrated with extracellular Ca2+ concentrations ([Ca2+]o) ranging from normal (1X [Ca2+]o) to four times normal (4x [Ca2+]o). Increasing [Ca2+]o increased maximal contractile response in atropine-treated TSM strips from 68.7 +/- 3.8% maximum for 1x [Ca2+]o to 100.8 +/- 4.8% maximum for 4x [Ca2+]o (P less than 0.001) in 2ws. Neither atropine nor [Ca2+]o affected maximal responses of TSM in 10ws (103.5 +/- 3.0% maximum for 1x [Ca2+]o; P = NS vs. control). However, in the presence of atropine and verapamil, 4x [Ca2+]o augmented KCl-elicited contraction of TSM from both 2ws (46.9 +/- 6.3% maximum; P less than 0.01 vs. control) and 10ws (78.6 +/- 2.3% maximum; P less than 0.005 vs. control).(ABSTRACT TRUNCATED AT 250 WORDS)     

Protease-activated receptor-1-induced calcium signaling in gingival fibroblasts is mediated by sarcoplasmic reticulum calcium release and extracellular calcium influx

Thrombin is a serine protease activated during injury and inflammation. Thrombin and other proteases generated by periodontal pathogens affect the behavior of periodontal cells via activation of protease-activated receptors (PARs). We noted that thrombin and PAR-1 agonist peptide stimulated intracellular calcium levels ([Ca2+]i) of gingival fibroblasts (GF). This increase of [Ca2+]i was inhibited by EGTA and verapamil. U73122 and neomycin inhibited thrombin- and PAR-1-induced [Ca2+]i. Furthermore, 2-APB (75-100 microM, inositol triphosphate [IP3] receptor antagonist), thapsigargin (1 microM), SKF-96365 (200 microM) and W7 (50 and 100 microM) also suppressed the PAR-1- and thrombin-induced [Ca2+]i. However, H7 (100, 200 microM) and ryanodine showed little effects. Blocking Ca2+ efflux from mitochondria by CGP37157 (50, 100 microM) inhibited both thrombin- and PAR-1-induced [Ca2+]i. Thrombin induced the IP3 production of GF within 30-seconds of exposure, which was inhibited by U73122. These results indicate that mitochondrial calcium efflux and calcium-calmodulin pathways are related to thrombin and PAR-1 induced [Ca2+]i in GF. Thrombin-induced [Ca2+]i of GF is mainly due to PAR-1 activation, extracellular calcium influx via L-type calcium channel, PLC activation, then IP3 binding to IP3 receptor in sarcoplasmic reticulum, which leads to intracellular calcium release and subsequently alters cell membrane capacitative calcium entry.     

The role of Ca2+ in steroidogenesis in Leydig cells. Stimulation of intracellular free Ca2+ by lutropin (LH), luliberin (LHRH) agonist and cyclic AMP.

The requirements of purified rat Leydig cells for intra- and extra-cellular Ca2+ during steroidogenesis stimulated by LH (lutropin), cyclic AMP analogues and LHRH (luliberin) agonist were investigated. The intracellular Ca2+ concentrations ([Ca2+]i) were measured by using the fluorescent Ca2+ chelator quin-2. The basal [Ca2+]i was found to be 89.4 +/- 16.6 nM (mean +/- S.D., n = 25). LH, 8-bromo cyclic AMP and dibutyryl cyclic AMP increased [Ca2+]i, by 300-500 nM at the highest concentrations of each stimulator, whereas LHRH agonist only increased [Ca2+]i by a maximum of approx. 60 nM. Low concentrations of LH (less than 1 pg/ml) and all concentrations of LHRH agonist increased testosterone without detectable changes in cyclic AMP. With amounts of LH greater than 1 pg/ml, parallel increases in cyclic AMP and [Ca2+]i occurred. The steroidogenic effect of the LHRH agonist was highly dependent on extracellular Ca2+ concentration ([Ca2+]e), whereas LH effects were only decreased by 35% when [Ca2+]e was lowered from 2.5 nM to 1.1 microM. No increase in [Ca2+]i occurred with the LHRH agonist in the low-[Ca2+]e medium, whereas LH (100 ng/ml) gave an increase of 52 nM. It is concluded that [Ca2+]i can be modulated in rat Leydig cells by LH via mechanisms that are both independent of and dependent on cyclic AMP, whereas LHRH-agonist action on [Ca2+]i is independent of cyclic AMP. The evidence obtained suggests that, at sub-maximal rates of testosterone production, Ca2+, rather than cyclic AMP, is the second messenger, whereas for maximum steroidogenesis both Ca2+- and cyclic-AMP-dependent pathways may be involved.     

Cytosolic alkalinization alone is not sufficient for Ca2+ mobilization, phosphatidic acid formation, and protein phosphorylation in human platelets

One of the earliest events following stimulation of human platelets with thrombin is a rise in the cytosolic pH, pHi, mediated by Na+/H+ exchange, and an increase in the cytosolic free Ca2+ concentration, [Ca2+]i. In the present study we investigated whether an increase in pHi alone, induced by the Na+/H+ ionophore monensin, is sufficient for platelet activation. Although monensin (20 microM) raised pHi from 7.10 +/- 0.05 (n = 21) to 7.72 +/- 0.17 (n = 13), neither Ca2+ influx nor mobilization were detectable upon this treatment in fura2-loaded platelets. In contrast, thrombin (0.05 U/ml) raised pHi to 7.31 +/- 0.10 (n = 10) and increased [Ca2+]i by more than 250 nM both in the presence and absence of extracellular Ca2+. Thrombin also caused the formation of phosphatidic acid and phosphorylation of the 20 kDa and 47 kDa proteins in platelets labeled with 32P. Monensin, however, induced none of these responses. It is concluded that an increase in pHi alone is not a sufficient trigger for platelet activation but enhances intracellular signal transduction in platelets stimulated by natural agonists.     

Pattern of gonadotropin-releasing hormone (GnRH)-like stimuli sufficient to induce follicular growth and ovulation in ewes passively immunized against GnRH.

The pattern of GnRH-like stimuli capable of inducing follicular growth, ovulation, and luteal function was evaluated in ewes passively immunized against GnRH. The estrous cycles of 30 regularly cyclic sheep were synchronized using vaginal pessaries impregnated with a synthetic progestogen. Animals were passively immunized against GnRH (groups 2-5, n = 6) or the carrier protein, keyhole limpet hemocyanin (KLH; group 1, n = 6), at the time of pessary removal (PR). Circhoral delivery of saline (groups 1, 2, and 5) or low amplitude GnRH agonist (des-Gly10 GnRH ethylamide [100 ng/hourly pulse]; groups 3 and 4) was initiated at PR and continued for 3 (groups 4 and 5) or 12 days (groups 1-3). In groups 4 and 5, the amplitude of the GnRH-like stimulus was increased to 800 ng/hourly pulse (stimulus-shift) during the 24-h period beginning 72 h after PR. The amplitude of the hourly stimulus was adjusted to 100 ng/pulse 96 h after PR and continued at that level to Day 12. The endocrine changes associated with follicle growth and maturation (serum concentrations of estradiol [E2] above 10 pg/ml), ovulation (surge-like secretion of LH and FSH), and normal luteal function (serum concentrations of progesterone [P] above 2 ng/ml) were evident in ewes passively immunized against KLH (group 1). In this group, the preovulatory surge of gonadotropins was noted 48.7 +/- 1.2 h after PR. These endocrine events were blocked by passive immunization against GnRH (group 2).(ABSTRACT TRUNCATED AT 250 WORDS)     

Synergistic potentiation of 5-hydroxytryptamine secretion by platelet agonists and phorbol myristate acetate despite inhibition of agonist-induced arachidonate/thromboxane and beta-thromboglobulin release and Ca2+ mobilization by phorbol myristate acetate.

Previous studies have demonstrated an inhibition of agonist-induced inositol phospholipid breakdown and intracellular Ca2+ ([Ca2+]i) mobilization by phorbol esters in platelets. In this study, we have examined the effect of phorbol 12-myristate 13-acetate (PMA) on agonist-induced granule secretion and correlated it with agonist-induced [Ca2+]i mobilization, arachidonate and thromboxane (Tx) release in human platelets. With increasing times of incubation with PMA (10 s-5 min), the rise in [Ca2+]i induced by thrombin and the TxA2 mimetic, U46619, was increasingly inhibited (90-100% with 5 min incubation) and, correlating with this, thrombin-induced [3H]arachidonate, TxB2 and beta-thromboglobulin (beta TG) release were also inhibited. In addition, the conversion of exogenously added arachidonate to TxB2 was inhibited (50-80%) by a 10 s-5 min pretreatment with PMA. However, secretion of 5-hydroxy[14C]tryptamine (5HT) induced by thrombin or U46619 was not inhibited by 10 s-2 min incubations with PMA and, on the contrary, with low agonist concentrations, was potentiated by PMA in the absence of a significant rise in [Ca2+]i or endogenous Tx formation, to levels significantly greater than or equal to the sum of that obtained when agonist and PMA were added separately. With longer times of incubation with PMA (5 min), these synergistic effects became less pronounced as inhibitory effects of PMA on agonist-induced [14C]5HT secretion became apparent. The results indicate that, while PMA may cause an inhibition of agonist-induced [Ca2+]i mobilization resulting in an inhibition of agonist-induced arachidonate, TxB2 and beta TG release, its effects on agonist-induced 5HT secretion may be complicated by [Ca2+]i-independent synergistic effects of agonist and PMA.