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PubMed1997

Effects of protein tyrosine kinase inhibitors on contractility of isolated bronchioles of the rat.

The role of protein tyrosine kinases (PTK) in modulating contractility has not been investigated in airway smooth muscle (ASM). We have examined the effects of the PTK inhibitors ST638, genistein, and tyrphostin A47 on contractions induced by carbachol, serotonin, ionomycin, and 75 mM KCl in isolated bronchioles of the rat with internal diameters of 614 +/- 16 microm (small, n = 143), and 1,433 +/- 39 microm (large, n = 57). ST638 caused a dose-dependent decrease in the maximum response to carbachol, and shifted the carbachol concentration-response curve to the right. This effect was greater in small bronchioles. Tyrphostin A47 (100 microM) and genistein (74 microM) had a similar effect to ST638. ST638 caused a concentration-dependent relaxation (EC50 approximately 7.2 microM) in bronchioles precontracted with 0.5 microM carbachol, and was maximally effective at 50 microM when tone was reduced by 82.5 +/- 3.8% in small bronchioles, and 57.2 +/- 2.8% in large bronchioles. ST638 also reduced the maximal response to serotonin, and caused a large shift to the right of the serotonin concentration-response curve. Pretreatment with ST638 (50 microM) reduced the response to 75 mM KCl in both small and large bronchioles in the presence of atropine (small: by 88.9 +/- 5.6%, n = 11; large: by 90.1 +/- 4.4%, n = 11). Tyrphostin A47 (100 microM) had a similar effect (91%). ST638 (50 microM) and tyrphostin A47 (100 microM) substantially relaxed small bronchioles contracted with 1.5 microM ionomycin (ST638: by 86.7 +/- 1.8%, n = 6; tyrphostin: by 89.3 +/- 1.7%, n = 5). We have therefore demonstrated that PTK inhibitors can suppress contraction induced by a number of different mechanisms in ASM. These results suggest that PTK signaling pathways are not only important for proliferation of ASM, but also fon contractile function.

Acetylcholine↗
PubMed1993

Explant culture of rabbit tracheobronchial epithelium: structure and prostaglandin metabolism.

This study examines the potential usefulness of explant culture of rabbit tracheal epithelium as a model for the study of epithelial function under normal and potentially pathologic conditions. Accordingly, we assessed the structure and prostaglandin E2 (PGE2) release of tracheal epithelial explants obtained from adult pathogen-free rabbits. Epithelial cells attached to their native connective tissue substratum were maintained in culture for 5 days in serum-free medium, under bipolar conditions (air-liquid interface) on a permeable membrane (pore size, 0.2 mm), and nourished from the basolateral surface. At 5 days in culture, scanning and transmission electron microscopy and light microscopy demonstrated a pseudostratified, ciliated columnar epithelium with prominent folds and mucus secretion identical in appearance to the mucosa before culture. On the day of dissection (day 0) and after 4 days in culture (day 4), explants released PGE2 into the medium spontaneously. However, day 4 explants produced 3- to 4-fold greater amounts of PGE2 than day 0 explants. Moreover, day 4 explants demonstrated increased PGE2 release in response to bradykinin, a receptor-dependent agonist, and ionomycin, a calcium ionophore, while day 0 explants did not. Primary tracheal epithelial cell cultures grown to confluence (day 9) on a collagen substrate demonstrated PGE2 responses to bradykinin and ionomycin that qualitatively resembled those of day 4 explants. We conclude that rabbit tracheal explants cultured in vitro under the above conditions maintain cellular differentiation, in situ three-dimensional organization, and PGE2 synthetic pathways over several days in culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2006

Roles of protein kinase C, Ca2+, Pyk2, and c-Src in agonist activation of rat lacrimal gland p42/p44 MAPK.

PURPOSE: Although p42/p44 mitogen-activated protein kinase (MAPK) negatively modulates protein secretion stimulated by cholinergic and alpha(1D)-adrenergic agonists, it does not play a role in epidermal growth factor (EGF)-stimulated protein secretion. Therefore, this study was conducted to determine the roles that protein kinase C (PKC), intracellular Ca(2+) ([Ca(2+)](i)), and nonreceptor tyrosine kinases Pyk2 and Src play in the activation of agonist- and EGF-stimulated MAPK activation. METHODS: Lacrimal gland acini were isolated by collagenase digestion and incubated with phorbol 12-myristate 13-acetate (PMA) to activate PKC or ionomycin, a Ca(2+) ionophore. Acini were preincubated with the PKC inhibitors calphostin C or Ro-31-8220, EGTA to chelate Ca(2+), or the c-Src inhibitor PP1 before stimulation with the cholinergic agonist carbachol, the alpha(1D)-adrenergic agonist phenylephrine, or EGF. Activated MAPK, Pyk2, and c-Src amounts were measured by Western blot analysis. RESULTS: PMA and ionomycin significantly increased the activation of MAPK in a time- and concentration-dependent manner. Inhibition of PKC partially inhibited carbachol-stimulated MAPK activation while completely inhibiting phenylephrine- and EGF-stimulated MAPK activation. Chelation of Ca(2+) also partially inhibited carbachol-stimulated MAPK with no effect on phenylephrine- and EGF-stimulated MAPK activation. Carbachol increased the phosphorylation of Pyk2 on tyrosine 402 and c-src on tyrosine 416 in a time-dependent manner. The c-src inhibitor PP1 inhibited carbachol-stimulated phosphorylation of Pyk2. CONCLUSIONS: It was concluded that cholinergic agonists use Ca(2+) and PKC to phosphorylate Pyk2 and c-Src, which subsequently stimulate MAPK activity. In contrast, alpha(1D)-adrenergic agonists and EGF do not use Pyk2 and Src but do use PKC to activate MAPK.

Adrenergic alpha-Agonists↗
PubMed1990

Endothelin-1 stimulates contraction of rat glomerular mesangial cells and potentiates beta-adrenergic-mediated cyclic adenosine monophosphate accumulation.

The newly isolated peptide, endothelin-1 (ET-1), is a potent pressor agent that reduces GFR and the glomerular ultrafiltration coefficient. Recent evidence demonstrates that ET-1 mobilizes intracellular Ca2+ [( Ca2+]i) in glomerular mesangial cells by activating the phosphoinositide cascade. The present experiments were designed to examine whether ET-1 stimulates mesangial cell contraction and regulates the synthesis of PGE2 and cAMP, which dampen vasoconstrictor-induced mesangial contraction. ET-1 (greater than or equal to 1 nM) reduced the cross-sectional area of rat mesangial cells cultured on three-dimensional gels of collagen type I. ET-1 also caused complex rearrangements of F-actin microfilaments consistent with a motile response. Contraction in response to ET-1 occurred only at concentrations that activate phospholipase C, and contraction was unaffected by blockade of dihydropyridine-sensitive Ca2+ channels. Elevation of [Ca2+]i with ionomycin, to equivalent concentrations of [Ca2+]i achieved with ET-1, also reduced mesangial cell cross-sectional area. ET-1 (0.1 microM) also evoked [3H]arachidonate release and a fivefold increase in PGE2 synthesis as well as increased synthesis of PGF2 alpha and small changes of TXB2. ET-1 caused a minor increase in intracellular cAMP accumulation only in the presence of 3-isobutyl-1-methylxanthine. ET-1 also amplified cAMP production in response to isoproterenol. TPA and ionomycin, alone and in combination, failed to mimic the potentiating effect of ET-1; however, indomethacin blocked ET-1-induced potentiation of isoproterenol-stimulated cAMP, which was restored by addition of exogenous 10 nM PGE2. Thus the present data demonstrate that ET-1 stimulates mesangial cell contraction via pharmacomechanical coupling and activates phospholipase A2 to produce PGE2, PGF2 alpha, and TXB2. ET-1 also amplified beta adrenergic-stimulated cAMP accumulation by a PGE2-dependent mechanism.

Animals↗
PubMed1991

Role of increased cytosolic free calcium in the pathogenesis of rabbit proximal tubule cell injury and protection by glycine or acidosis.

To assess the role of increased cytosolic free calcium (Caf) in the pathogenesis of acute proximal tubule cell injury and the protection afforded by exposure to reduced medium pH or treatment with glycine, fura-2-loaded tubules were studied in suspension and singly in a superfusion system. The Ca2+ ionophore, ionomycin, increased Caf to micromolar levels and rapidly produced lethal cell injury as indicated by loss of lactate dehydrogenase to the medium by suspended tubules and accelerated leak of fura and failure to exclude Trypan blue by superfused tubules. Decreasing medium Ca2+ to 100 nM prevented the ionomycin-induced increases of Caf and the injury. Reducing medium pH from 7.4 to 6.9 or adding 2 mM glycine to the medium also prevented the cell death, but did not prevent the increase of Caf to micromolar levels. Cells treated with 1799, an uncoupler of oxidative phosphorylation which produced severe adenosine triphosphate (ATP) depletion, did not develop increases of Caf until just before loss of viability. Preventing these increases of Caf with 100 nM Ca2+ medium did not protect 1799-treated cells. Reduced pH and glycine protected 1799-treated cells without ameliorating the increases of Caf. These data demonstrate the toxic potential of increased Caf in the proximal tubule and show that Caf does sharply increase prior to loss of viability in an ATP depletion model of injury, but this increase does not necessarily contribute to the outcome. The potent protective actions of decreased pH and glycine allow the cells to sustain increases of Caf to micromolar levels in spite of severe, accompanying cellular ATP depletion without developing lethal cell injury.

Acidosis↗
PubMed1993

Regulation of membrane chloride currents in rat bile duct epithelial cells.

This study examines the conductive properties of the plasma membrane of cells isolated from the intrahepatic portion of bile ducts. Membrane Cl- conductance was measured in single cells using whole-cell patch clamp recording techniques and in cells in short-term culture using 36Cl and 125I efflux. Separate Ca(2+)- and cAMP-dependent Cl- currents were identified. Ca(2+)-dependent Cl- currents showed outward rectification of the current-voltage relation, time-dependent activation at depolarizing potentials, and reversal near the equilibrium potential for Cl-. Ionomycin (2 microM) increased this current from 357 +/- 72 pA to 1,192 +/- 414 pA (at +80 mV) in 5:7 cells, and stimulated efflux of 125I > 36Cl in 15:15 studies. Ionomycin-stimulated efflux was inhibited by the Cl- channel blocker 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS) (150 microM). A separate cAMP-activated Cl- current showed linear current-voltage relations and no time dependence. Forskolin (10 microM) or cpt-cAMP (500 microM) increased this current from 189 +/- 50 pA to 784 +/- 196 pA (at +80 mV) in 11:16 cells, and stimulated efflux of 36Cl > 125I in 16:16 studies. cAMP-stimulated efflux was unaffected by DIDS. Because the cAMP-stimulated Cl- conductance resembles that associated with cystic fibrosis transmembrane conductance regulator (CFTR), a putative Cl- channel protein, the presence of CFTR in rat liver was examined by immunoblot analyses. CFTR was detected as a 150-165-kD protein in specimens with increased numbers of duct cells. Immunoperoxidase staining confirmed localization of CFTR to bile duct cells but not hepatocytes. These findings suggest that Ca(2+)- and cAMP-regulated Cl- channels may participate in control of fluid and electrolyte secretion by intrahepatic bile duct epithelial cells, and that the cAMP-regulated conductance is associated with endogenous expression of CFTR. Abnormal ductular secretion may contribute to the pathogenesis of cholestatic liver disease in cystic fibrosis.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2003

Sphingosine 1-phosphate is a novel inhibitor of T-cell proliferation.

Sphingosine 1-phosphate (S1P) is a pleiotropic lysosphingophospholipid stored and secreted by platelets. Using reverse transcription-polymerase chain reaction and flow cytometric analyses, we determined the expression of S1P receptors (S1P1, S1P3, S1P4, and S1P5) in peripheral blood T cells. T cells were induced to proliferate in the presence of phorbol 12-myristate 13-acetate (PMA) plus ionomycin, anti-CD3 plus anti-CD28, and allogeneic immature or mature dendritic cells. This activity was inhibited by the addition of S1P. Enhanced T-cell proliferation was observed when these cells were stimulated with the same stimuli, but were incubated in serum-free media (SFM). Addition of S1P to SFM inhibited the stimulation of T cells induced by T-cell stimuli, suggesting that S1P is an important inhibitory molecule present in the serum. T-cell proliferation was also inhibited by the addition of dihydrosphingosine 1-phosphate (DHS1P), sphingosine, and ceramide; however, the latter 2 sphingolipids required higher concentrations than S1P. Pretreatment of T cells with pertussis toxin (PTX) blocked the inhibitory effect of S1P on activation with PMA plus ionomycin, but not on activation with anti-CD3 plus anti-CD28. This is corroborated with the down-regulation of S1P1 in T cells stimulated with anti-CD3 plus anti-CD28. Similarly, PTX did not affect the inhibitory effect of S1P on T-cell proliferation when dendritic cells were used as stimuli. Further, S1P or DHS1P but not ceramide or sphingosine enhanced rather than decreased secretion of interleukin 2 and interferon gamma by T cells stimulated with anti-CD3 plus anti-CD28. These results show differential effects of S1P on polyclonal T-cell proliferation and cytokine secretion.

Antibodies↗
PubMed2002

Inhibition of the MEK1/ERK pathway reduces arachidonic acid release independently of cPLA2 phosphorylation and translocation.

BACKGROUND: The 85-kDa cytosolic phospholipase A2 (cPLA2) mediates arachidonic acid (AA) release in MDCK cells. Although calcium and mitogen-activated protein kinases regulate cPLA2, the correlation of cPLA2 translocation and phosphorylation with MAPK activation and AA release is unclear. RESULTS: MEK1 inhibition by U0126 inhibited AA release in response to ATP and ionomycin. This directly correlated with inhibition of ERK activation but not with phosphorylation of cPLA2 on Ser505, which was only partially inhibited by ERK inhibition. Inhibition of AA release by U0126 was still observed when stoichiometric phosphorylation of cPLA2 on Ser505 was maintained by activating p38 with anisomycin. Translocation kinetics of wild-type cPLA2 and cPLA2 containing S505A or S727A mutations to Golgi were similar in response to ATP and ionomycin and were not affected by U0126. CONCLUSIONS: These results suggest that the ability of cPLA2 to hydrolyze membrane phospholipid is reduced by inhibition of the MEK1/ERK pathway and that the reduction in activity is independent of cPLA2 phosphorylation and translocation to membrane. The results also demonstrate that cPLA2 mutated at the phosphorylation sites Ser505 and Ser727 translocated with similar kinetic as wild-type cPLA2.

Adenosine Triphosphate↗
PubMed1986

Measurement of cytosolic free Ca2+ concentrations in human and rat osteosarcoma cells: actions of bone resorption-stimulating hormones.

Influx of extracellular Ca++ into bone cells has been postulated as an early action of PTH and other bone resorption-stimulating factors. To test this hypothesis directly, we measured the cytosolic free Ca2+ concentration ([Ca2+]i) in two hormone-responsive human (SaOS-2 and G-292) and two rat osteosarcoma cell lines (Ros 25/1 and Ros 17/2.8) and in primary cultures of bone cells from neonatal mouse calvaria using the fluorescent Ca2+ indicator Quin 2. Actions of bovine PTH-(1-34), vasoactive intestinal peptide, epidermal growth factor, prostaglandin E2, and ionomycin were studied. Medium cAMP (20 min; 37 C; 25 microM 3-isobutyl-1-methylxanthine) was quantitated by RIA. Basal [Ca2+]i was: SaOS-2, 126 +/- 8 nM; G-292, 61 +/- 6 nM; Ros 25/1, 109 +/- 15 nM; Ros 17/2.8, 363 +/- 42 nM; and primary cultures, 266 +/- 39 nM (mean +/- SE; n = 3-14). In each cell type, no acute (1 sec to 20 min) spike in [Ca2+]i was observed in response to PTH (24-120 nM), vasoactive intestinal peptide (100 nM), epidermal growth factor (17 nM), or prostaglandin E2 (2.8 microM). However, in SaOS-2 cells only, PTH reproducibly increased [Ca2+]i 10-15% above basal values beginning about 3 min after hormone addition, and this small increase returned to baseline at 15-20 min. Ionomycin (100 nM) elicited an immediate spike in [Ca2+]i to levels 2- to 4-fold above basal in all cells; the peak [Ca2+]i decayed rapidly (within 4-5 min) to baseline in G-292, Ros 25/1, and Ros 17/2.8 cells. The decay of peak [Ca2+]i in SaOS-2 was prolonged. To test for intact hormone responses in Quin 2-loaded cells, cAMP accumulation was measured. In SaOS-2 and Ros 17/2.8, both control and Quin 2-loaded cells showed similar increases in cAMP in response to PTH. Considering the limitations of the Quin 2 technique, we conclude that in the four hormone-responsive bone cell lines and primary cultures of bone cells tested, acute elevation of [Ca2+]i is not an inevitable consequence of receptor occupancy and/or adenylate cyclase activation by bone resorption-stimulating hormones.

Aminoquinolines↗
PubMed1989

Regulation of cytosolic pH in bovine parathyroid cells: effect of fluoride.

In the present investigation, sodium fluoride (NaF) was employed to explore the role of guanine nucleotide-binding proteins (G-proteins), protein kinase-C, or cytosolic calcium [( Ca]i) in the regulation of cytosolic pH [( pH]i) in dispersed bovine parathyroid cells, using the pH-sensitive fluorescent dye BCECF. When cells acidified by nigericin in Na-free medium were resuspended in Na-containing buffer, [pH]i returned to basal levels. This recovery was blocked by continued removal of Na+ or the addition of amiloride. NaF (10 mM) increased [32P]phosphate incorporation into phosphatidylinositol bisphosphate, suggesting an increase in phosphatidylinositol bisphosphate turnover. NaF caused an initial acidification, followed by an alkaline recovery in a dose-dependent manner (1-10 mM). Amiloride blocked the NaF-induced alkaline recovery. The protein kinase-C activator phorbol 12-myristate 13-acetate (10(-7) M) caused cytosolic alkalinization, while the protein kinase-C inhibitor H7 (6 x 10(-5) M) significantly inhibited the NaF-induced alkaline recovery. Pertussis toxin (1 microgram/ml) did not affect the NaF-induced changes in [pH]i. Removal of extracellular Ca2+ with EGTA blocked the NaF-induced increase in [Ca]i and alkaline recovery. Ionomycin (5 x 10(-7) M) caused cytosolic alkalinization, but pretreatment with EGTA inhibited the ionomycin-induced cytosolic alkalinization. The present studies clearly demonstrated the presence of an amiloride-sensitive Na+/H+ exchanger in parathyroid cells. Our findings suggest that the NaF-induced cytosolic alkaline recovery was via two complementing pathways: 1) activation of protein kinase-C, followed by stimulation of a Na+/H+ exchanger, and 2) existence of extracellular calcium and/or an increase in [Ca]i.

Amiloride↗
PubMed1989

The antigonadotropic action of prostaglandin F2 alpha is not mediated by elevated cytosolic calcium levels in rat luteal cells.

We have investigated the role of intracellular calcium in the mechanism of action of prostaglandin F2 alpha (PGF2 alpha) in cultured rat luteal cells. PGF2 alpha (1 microM) maximally inhibited LH-stimulated cAMP accumulation and also initiated a transient release of intracellular calcium. Low doses of the calcium ionophore ionomycin also increased intracellular calcium to a similar extent as PGF2 alpha (1 microM), but did not inhibit LH-stimulated cAMP accumulation. Chelation of intracellular calcium with dimethyl bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) (10 microM) attenuated the transient calcium rise stimulated by PGF2 alpha, but did not affect the inhibitory characteristics of PGF2 alpha on LH-stimulated cAMP accumulation. Treatment of luteal cells with EGTA (1 mM) and ionomycin (500 nM) resulted in depletion of intracellular calcium to such an extent that a subsequent exposure of the luteal cells to PGF2 alpha (1 microM) did not elicit any change in intracellular calcium. Depletion of intracellular calcium and ablation of the calcium response to PGF2 alpha, however, did not affect either the dose response or the time course of inhibition of LH-stimulated cAMP accumulation. We conclude that although intracellular calcium is mobilized by PGF2 alpha in cultured rat luteal cells, the antigonadotropic action of PGF2 alpha on LH-stimulated cAMP accumulation is not mediated by this mechanism.

Animals↗
PubMed1989

The role of Ca2+ and calmodulin in the regulation of atrial natriuretic peptide-stimulated guanosine 3',5'-cyclic monophosphate accumulation by isolated mouse Leydig cells.

We have investigated the role of Ca2+ and calmodulin in the stimulation of cGMP formation by mouse Leydig cells in response to rat atriopeptin-II (rAP-II). Removal of extracellular Ca2+ had no influence on the levels of cGMP accumulated by the cells stimulated with rAP-II. The amounts of testosterone produced by unstimulated and rAP-II-stimulated cells were, however, reduced by 50% in the absence of Ca2+ from the incubation medium. Addition of ionomycin to the Leydig cells led to a dose-related inhibition of rAP-II-stimulated cGMP formation, but the basal cGMP level was not affected. These experiments were carried out in the presence of a phosphodiesterase inhibitor. The inhibitory effect of ionomycin was absolutely dependent upon the presence of Ca2+ in the medium. The guanylate cyclase activity required the presence of a cation, and Mn2+, Mg2+, or Ca2+ could function as the required cation. There was no direct inhibition of the cyclase activity by Ca2+ up to as high a concentration as 8 mM. Furthermore, three structurally unrelated calmodulin antagonists, W7, trifluoperazine, and calmidazolium, but not W5, caused a dose-related inhibition of rAP-II-stimulated cGMP accumulation by the cells. The inhibitory effect of calmodulin antagonists was not exerted directly at the level of guanylate cyclase activity, since the particulate enzyme was not inhibited by any of these drugs. We conclude, therefore, that extracellular Ca2+ is not essential for rAP-II-mediated stimulation of cGMP formation by mouse Leydig cells, at least under the short term incubation conditions used. An excessive ionophoretic influx of Ca2+ into the cells impairs the ability of rAP-II to stimulate cGMP formation. Therefore, it appears that a finely regulated level of intracellular Ca2+ is required for optimal activation of atrial natriuretic peptide-responsive guanylate cyclase in mouse Leydig cells, and calmodulin plays an important role in this process.

Animals↗
PubMed1990

Evidence for distinct glucocorticoid and guanine 3',5'-monophosphate-effected inhibition of stimulated adrenocorticotropin release in vitro.

Previous work has shown that corticosterone, cell-membrane permeant analogs of cGMP, as well as activators of guanylyl cyclase inhibit secretagogue-stimulated ACTH release. In the present study we have examined whether cGMP mediates the inhibitory effect of corticosterone in perifused isolated rat anterior pituitary cells. A brief 22.5-min exposure to corticosterone strongly inhibited ACTH secretion evoked by arginine vasopressin (AVP), 48 mM KC1, and two types of combined stimuli, i.e. 41-residue CRF and AVP (0.05 and 0.5 nM, respectively; CRF/AVP), or ionomycin and phorbol-dibutyrate (200 and 10 nM, respectively; PdBu/IM). The time course of inhibition by corticosterone was similar in all cases; a rapid approximately 30% reduction in ACTH was evident within 25 min, which increased to 60% by 50-70 min and will be referred to as the delayed effect. The corticosteroid inhibition of PdBu/IM-induced ACTH release was fully antagonized by the glucocorticoid/progestin antagonist RU 38486, indicating that it is exerted through type II glucocorticoid receptors. In contrast to corticosterone, the cGMP derivative 8-bromo-cGMP failed to suppress ACTH release evoked by PdBu/IM, whereas it effectively inhibited the action of CRF/AVP. Furthermore, ionomycin reversed the reduction of CRF/AVP-stimulated ACTH release by 8-bromo-cGMP, but had no effect on the delayed inhibition caused by corticosterone. These data indicate that there are two distinct cellular pathways of inhibiting stimulus-evoked ACTH secretion in vitro. One of these is activated by corticosterone, whereas the other involves cGMP as a cellular messenger.

Adrenocorticotropic Hormone↗
PubMed1990

Further studies on the involvement of protein kinase C in human sperm flagellar motility.

Addition of the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) or the membrane-permeable diacylglycerol analog, 1-oleoyl-2-acetylglycerol to human sperm resulted in increased motility. The biologically inactive 4 alpha-phorbol 12,13 didecanoate had no effect on flagellar motility. Basal motility was markedly reduced in the absence of Ca2+ in the incubation medium, but TPA-induced sperm motility persisted even in the absence of Ca2+. Sperm motility was also enhanced by the Ca2+ ionophore ionomycin in a Ca2(+)-dependent, protein kinase c (PKC)-independent fashion. Although all stimulants examined here reached maximal response at about 15 min of incubation, nevertheless whereas the effect of TPA and 1-oleoyl-2-acetylglycerol declined at 60 min of incubation, that of ionomycin still persisted. Human sperm PKC activity is extremely low and represents only about 20% and 25% of the specific activity recovered from PC-12 and rat pituitary cells, respectively. Immunohistochemical analysis using various type-specific PKC antibodies revealed staining only in the equatorial segment and the principal piece of the tail. Thus, PKC is present in human ejaculated sperm and is involved in flagellar motility.

Adult↗
PubMed1992

pH homeostasis in pituitary GH4C1 cells: basal intracellular pH is regulated by cytosolic free Ca2+ concentration.

In GH4C1 cells, membrane depolarization induces a rapid and sustained increase in the cytosolic free calcium concentration ([Ca2+]i). In the present study we have investigated the role of [Ca2+]i in the regulation of basal intracellular pH (pHi). Depolarizing GH4C1 cells in buffer containing 0.4 mM extracellular Ca2+ decreased basal pHi from 7.02 +/- 0.04 to 6.85 +/- 0.03 (P less than 0.05). If the depolarization-induced influx of Ca2+ was inhibited by chelating extracellular Ca2+ or blocking influx through voltage-operated Ca2+ channels with nimodipine, no acidification was observed. Addition of TRH induced a rapid activation of Na+/H+ exchange in acidified cells, increasing pHi by 0.14 +/- 0.03 U. The action of TRH was blunted if extracellular Ca2+ was chelated; however, if influx of Ca2+ via voltage-operated channels was blocked by nimodipine, TRH still increased pHi. To deplete ATP, we incubated cells with 2-deoxy-D-glucose for 15-20 min and observed a decrease in basal pHi to 6.75 +/- 0.03 (P less than 0.05). No additional acidification was obtained when 2-deoxy-D-glucose-treated cells were depolarized, and no TRH-induced activation of Na+/H+ exchange was observed. Addition of ionomycin or 12-O-tetradecanoyl-phorbol-13-acetate separately to acidified cells had only modest effects on pHi; however, addition of 12-O-tetradecanoyl-phorbol-13-acetate and ionomycin together increased pHi markedly. We conclude that in GH4C1 cells, increasing [Ca2+]i reduces basal pHi through a mechanism dependent on influx of extracellular Ca2+ and independent of Na+/H+ exchange. In addition, elevation of [Ca2+]i and activation of protein kinase C act synergistically to enhance Na+/H+ exchange and increase pHi in acidified cells. Finally, normal cellular ATP is necessary for the activation of Na+/H+ exchange.

Adenosine Triphosphate↗
PubMed1995

Role of inositol trisphosphate-sensitive calcium stores in the regulation of adrenocorticotropin secretion by perifused rat anterior pituitary cells.

Intracellular Ca2+ (Cai2+) stores contribute significantly to Ca2+ signaling in many types of cells. We studied the role of inositol trisphosphate (InsP3)-sensitive Ca2+ stores, a principal Cai2+ store that presumably is within the endoplasmic reticulum (ER), in cell signaling by examining the effect of thapsigargin (Tg), an ER Ca2+ pump inhibitor that depletes the ER Ca2+ pool, on ACTH secretion. Preincubation for 6-24 h with 2-20 nM Tg had no effect on the resting cytosolic free Ca2+ concentrations ([Cai2+]) but inhibited the ionomycin-stimulated spike-type increase in [Cai2+], which is mediated by InsP3-independent Cai2+ release from the ER, in a dose-dependent (IC50, 4 nM) and time-dependent manner. In ER Cai(2+)-depleted cells, the spike phase (initial 5 min) of the ACTH secretory response to arginine vasopressin (AVP), which is mediated by InsP3-induced Cai2+ release, was also attenuated (IC50, 7.3 nM). However, the spike phase of the ACTH secretory response to AVP was inhibited to a much greater degree than the spike-type response to ionomycin, suggesting that ER Cai2+ stores might have functions other than simply providing Ca2+ for InsP3-stimulated Cai2+ release. Tg pretreatment (IC50, 12 nM) also markedly inhibited the sustained plateau (final 15-min) phase of the ACTH secretory response to AVP, which is mediated by diacylglycerol-induced activation of protein kinase C and subsequent influx of extracellular Ca2+ via L-type voltage-sensitive Ca2+ channels (VSCC), but had no effect on the sustained (full 20 min) response to dioctanoylglycerol that directly activates protein kinase C. Tg had no effect on specific cell binding of [125I]AVP or on specific cell binding of [3H]phorbol 12,13-dibutyrate (except at 20 nM Tg), an index of protein kinase C concentration, or on protein kinase C activity. AVP significantly stimulated inositol trisphosphate accumulation, but pretreatment with Tg completely abolished this effect of AVP, whereas [3H]myoinositol incorporation into membrane-associated inositol lipids and inositol phosphates was unaffected. Thus, Tg-induced depletion of ER Cai2+ stores inhibited both the spike and plateau phases of the ACTH secretory response to AVP, presumably by inhibiting phospholipase C activity and the resulting generation of InsP3 and diacylglycerol. Preincubation with Tg inhibited, in a dose-dependent (IC50, 13 nM) and time-dependent manner, the sustained ACTH secretory response to corticotropin-releasing hormone (CRH) that is mediated by cAMP-induced activation of protein kinase A and Cae2+ influx via L-type VSCC, and the sustained response to forskolin, which directly activates adenylate cyclase.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenocorticotropic Hormone↗
PubMed1992

The regulation of endothelin production in human umbilical vein endothelial cells: unique inhibitory action of calcium ionophores.

Endothelins (ETs) are a recently discovered family of small proteins that have potent long-lasting vasoconstrictive activities. Increased circulating concentrations of ETs have been found in hypertensive and renal disorders, including pregnancy-induced hypertension (PIH). PIH has been postulated to be the end result of endothelial cell damage and aberrant calcium metabolism. We evaluated the effects of calcium ionophores, calcium channel blockers, and two forms of cellular damage on ET production by human umbilical vein endothelial cells (HUVEC). Cells were grown to confluence and then incubated for 16 h with these treatments: physical trauma ("scratching"), oxidant damage (hydrogen peroxide, 1-20 mM), ionomycin (0.25-2.0 microM), A-23187 (10(-9)-10(-5) M), verapamil (0.22-22.0 microM), and nifedipine (2-200 micrograms/mL). ET production was determined using a commercial RIA that detects ET-1 and ET-2. Physical trauma enhanced ET production, whereas oxidant damage had the opposite effect. Both ionomycin and A-23187 caused concentration-dependent inhibition of ET production. Neither verapamil nor nifedipine consistently altered ET production. We conclude that specific forms of cellular damage can stimulate HUVEC ET production, although oxidant damage may be slightly inhibitory. Thus, enhanced ET levels in PIH may represent endothelial cell activation, rather than damage. HUVEC ET production is regulated in an inverse manner by intracellular calcium concentrations, suggesting a negative feedback from mediators of ET action on cells.

6-Ketoprostaglandin F1 alpha↗
PubMed1991

Similar actions of glucocorticoids and calcium on the regulation of apoptosis in S49 cells.

Glucocorticoid-induced lymphocyte cell death is a programmed process which is thought to involve the calcium-dependent degradation of DNA into multiples of 180 basepairs, characteristic of internucleosomal degradation. We have used the glucocorticoid-sensitive mouse lymphoma cell line S49.1 [wild-type (wt)] and the glucocorticoid-resistant cell line S49.22r (nt-) to evaluate the role of both glucocorticoid receptors and calcium in the regulation of internucleosomal DNA degradation and expression of calcium-dependent deoxyribonuclease activity. DNA was isolated from untreated (control) and dexamethasone (dex)-treated viable cells and analyzed for internucleosomal DNA degradation by agarose gel electrophoresis, followed by ethidium bromide staining. Glucocorticoid treatment resulted in substantial internucleosomal DNA degradation in wt cells, but not in nt- cells. This effect was inhibited by coincubation of cells with dex and the glucocorticoid receptor antagonist RU486. In contrast to the glucocorticoid response, administration of either of two calcium ionophores, ionomycin or A23187, produced internucleosomal degradation of DNA in both wt and nt- cells, although the latter were less sensitive to ionophore treatment. Interestingly, A23187 treatment also resulted in a loss of cell viability in HeLa S3 cells, a cell line that does not exhibit glucocorticoid-induced apoptosis. No internucleosomal DNA degradation was detected in HeLa S3 cells killed by A23187. To determine whether similar nucleases are associated with this internucleosomal DNA degradation resulting from both glucocorticoid and calcium ionophore treatment, 0.3 M NaCl nuclear protein extracts were prepared from control and treated cells and analyzed for protein composition or nuclease activity. To assay for nuclease activity, nuclear extracts were electrophoresed in sodium dodecyl sulfate-polyacrylamide gels impregnated with [32P]DNA. Nuclease activity was detected by removal of sodium dodecyl sulfate from the gel, activation with calcium, and subsequent visualization of the loss of [32P]DNA by autoradiography. Dex treatment of wt cells resulted in the appearance of several proteins within the mol wt range of 12-18 kDa, only one of which (16-18 kDa) exhibited calcium-dependent nuclease activity. The appearance of these proteins in nuclear extracts was inhibited by coincubation of glucocorticoid-treated cells with RU 486. Glucocorticoid treatment did not result in the appearance of nuclease activity in nuclear extracts from nt- cells. Interestingly, A23187 or ionomycin treatment resulted in an increase in activity of the 16- to 18-kDa nuclease in both wt and nt- cells. These findings indicate that both glucocorticoid receptors and calcium may share common features in the regulation of apoptosis in lymphoid cells.

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