Biomedical subjects

P Morley

Publications and source records attributed to P Morley.

At least 91 records · Page 5Linked to original sources

PubMed1996

Glucose, potassium, and CCK-8 induce increases in membrane-associated PKC activity that correspond to increases in [Ca2+]i in islet cells from neonatal rats.

The effects of glucose, K+, and cholecystokinin octapeptide (CCK-8) on intracellular free Ca2+ concentration ([Ca2+]i) and membrane-associated protein kinase C (PKC) activity were examined in cultured islet cells from neonatal rats. Raising the glucose concentration from 2.8 to 22.2 mM or external K+ (from 5 to 45 mM), or adding CCK-8 (200 nM) all triggered a [Ca2+]i surge that peaked between 3 and 10 min afterward, depending on the stimulus, and then declined, either to a suprabasal plateau (glucose and K+) or to basal levels (CCK-8). These same manipulations triggered a burst of membrane-associated PKC activity that peaked between 5 and 10 min and then variously declined. Incubation in Ca(2+)-free medium abolished both the effects of glucose and K+ on [Ca2+]i and the stimulation of membrane-associated PKC activity. The K(+)-triggered stimulation of PKC activity was also inhibited by pretreating the cells with the general Ca2+ entry blocker lanthanum (1 mM). However, incubation in Ca(2+)-free medium did not affect the CCK-8-induced release Ca2+ from internal stores, although it abolished the burst of membrane-associated PKC activity, which showed the importance of Ca2+ influx as opposed to internal release for PKC activation. Thus, glucose, the principal stimulator of insulin secretion, rapidly stimulates Ca2+ influx into islet cells from neonatal rats, and it is probably this influx that stimulates membrane-associated PKC activity.

Animals↗

PubMed1996

Mechanisms of 1S,3R-ACPD-induced neuroprotection in rat hippocampal slices subjected to oxygen and glucose deprivation.

The efficacy and mechanisms of 1-amino-cyclopentyl-1S,3R-dicarboxylate (1S,3R-ACPD)-induced neuroprotection were investigated in rat hippocampal slices subjected to 10 min of oxygen and glucose deprivation. Neuronal viability was assessed by measuring both the amplitude of evoked population spike in the CA1 pyramidale and by imaging CA1 neurons using a live/dead fluorescence assay with confocal microscopy. CA1 pyramidal neurons in oxygen-glucose deprived slices remained viable for up to 120 min following the insult but were dead by 240 min. Pretreatment with 1S,3R-ACPD significantly protected the oxygen-glucose deprived slices in a concentration-dependent fashion. Oxygen-glucose deprived slices pretreated for the same period with the protein kinase C (PKC) activation phorbol 12-myristate 13-acetate (PMA; 1 microM) were significantly protected whereas oxygen-glucose deprived slices treated with the adenylyl cyclase activator, forskolin (30 microM) were not. Oxygen-glucose deprivation induced a rapid and persistent decrease (approximately 50%) in PKC activity and a > 6 fold increase in cyclic adenosine monophosphate (cAMP) levels in whole hippocampal slices. While 1S,3R-ACPD did not stimulate PKC activity and had no effect on basal cAMP in whole slices, it significantly enhanced the rate of return of cAMP to basal levels following reperfusion. Consistent with this observation, the 1S,3R-ACPD-induced neuroprotection was inhibited by forskolin (30 microM). These results suggest that in vitro neuroprotection of CA1 neurons by 1S,3R-ACPD involves metabotropic glutamate receptors negatively linked to cAMP and possibly those which increase PKC activity.

Animals↗

PubMed1996

An early loss in membrane protein kinase C activity precedes the excitatory amino acid-induced death of primary cortical neurons.

Several lines of evidence indicate that a rapid loss of protein kinase C (PKC) activity may be important in the delayed death of neurons following cerebral ischemia. However, in primary neuronal cultures, cytotoxic levels of glutamate have been reported not to cause a loss in PKC as measured by immunoblot and conventional activity methods. This apparent contradiction has not been adequately addressed. In this study, the effects of cytotoxic levels of glutamate, NMDA, and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) on membrane PKC activity was determined in cortical neurons using an assay that measures only PKC that is active in isolated membranes, which can be used to differentiate active enzyme from that associated with membranes in an inactive state. A 15-min exposure of day 14-18 cortical neurons to 100 microM glutamate, AMPA, or NMDA caused a rapid and persistent loss in membrane PKC activity, which by 4 h fell to 30-50% of that in control cultures. However, the amount of enzyme present in these membranes remained unchanged during this period despite the loss in enzyme activity. The inactivation of PKC activity was confirmed by the fact that phosphorylation of the MARCKS protein, a PKC-selective substrate, was reduced in intact neurons following transient glutamate treatment. By contrast, activation of metabotropic glutamate receptors by trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid was not neurotoxic and induced a robust and prolonged activation of PKC activity in neurons. PKC inactivation by NMDA and AMPA was dependent on extracellular Ca2+, but less so on Na+, although cell death induced by these agents was dependent on both ions. The loss of PKC activity was likely effected by Ca2+ entry through specific routes because the bulk increase in intracellular free [Ca2+] effected by the Ca2+ ionophore ionomycin did not cause the inactivation of PKC. The results indicate that the pattern of PKC activity in neurons killed by glutamate, NMDA, and AMPA in vitro is consistent with that observed in neurons injured by cerebral Ischemia in vivo.

Animals↗

PubMed1996

The desglycinyl metabolite of remacemide hydrochloride is neuroprotective in cultured rat cortical neurons.

The neuroprotective actions of remacemide and its anticonvulsant metabolite 1,2-diphenyl-2-propylamine monohydrochloride (desglycinylremacemide; DGR) a low-affinity NMDA receptor antagonist, were investigated using primary rat cortical neuronal cultures. Exposure of cortical cultures to NMDA (100 microM) for 15 min killed 85% of the neurons during the next 24 h. This neurotoxicity was blocked in a concentration-dependent manner by adding DGR (5-20 microM), but not its remacemide precursor (10-100 microM), to the cultures during the time of NMDA exposure. This suggests that the neuroprotective, as well as the anticonvulsant, activity of remacemide is mediated by DGR. Neuroprotective concentrations of DGR also inhibited two of the principal acute effects of NMDA. DGR (5-20 microM) prevented the loss of membrane-associated protein kinase C (PKC) activity that developed by 4 h after transient exposure to 100 microM NMDA and reduced the NMDA-triggered increases in intracellular free Ca2+ concentration ([Ca2+]i) by up to 70%. By contrast, remacemide (50 and 100 microM) did not prevent the NMDA-induced loss of PKC activity or reduce the [Ca2+]i responses. These data suggest that DGR protection against NMDA-mediated toxicity in cultured cortical neurons is associated with a reduction of NMDA-triggered [Ca2+]i surges and a prevention of the loss of membrane-associated PKC activity. In addition, the inhibition of NMDA-triggered [Ca2+]i responses by DGR was qualitatively different from the inhibition of these responses by the high-affinity NMDA-receptor antagonists MK-801 and phencyclidine. This may be a consequence of DGR's lower affinity for the NMDA receptor.

Acetamides↗

PubMed1996

Role of protein kinase C in the regulation of ATP-triggered intracellular Ca2+ oscillations in chicken granulosa cells.

These studies were designed to investigate the role of protein kinase C (PKC) in the regulation of ATP-triggered intracellular Ca2+ ([Ca2+]i) oscillations in chicken granulosa cells. Granulosa cells were obtained from the two largest preovulatory follicles (F1 and F2) of hens and [Ca2+]i was measured in cells loaded with the Ca(2+)-responsive fluorescent dye fura-2. Adenosine triphosphate (100 mumol/l) triggered an immediate, large [Ca2+]i spike that was followed by oscillations that returned to the resting level between spikes. The ATP (100 mumols/l) also stimulated a 1.70 +/- 0.1-fold increase in membrane-associated PKC activity over control levels. The frequency of the ATP-triggered [Ca2+]i oscillations was reduced in a concentration-dependent (1-10 nmol/l) manner by treating the cells for 2 min with a PKC activator, 12-O-tetradecanoyl phorbol-13-acetate (TPA). A higher TPA concentration (100 nmol/l) completely prevented ATP from triggering the initial [Ca2+]i spike and oscillations. Adding TPA during the ATP-triggered [Ca2+]i oscillations immediately stopped the oscillatory activity. Interestingly, PKC inhibitors failed to amplify the ATP-triggered [Ca2+]i oscillations. Instead, adding the PKC inhibitors staurosporine (20 nmol/l), calphostin C (200 nmol/l) or 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7; 100 mumols/l), either before or during the ATP (100 mumols/l)-triggered [Ca2+]i response, also completely blocked the [Ca2+]i oscillations. Therefore, ATP-triggered [Ca2+]i oscillations in chicken granulosa cells appear to be regulated by a negative feedback loop requiring PKC, because the [Ca2+]i oscillations were prevented by either full activation or inhibition of PKC activity.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1995

Ca2+ x calmodulin prevents myristoylated alanine-rich kinase C substrate protein phosphorylation by protein kinase Cs in C6 rat glioma cells.

Ionomycin stimulated membrane-associated protein kinase Cs (PKCs) activity in C6 rat glioma cells as much as the potent PKCs stimulator 12-O-tetradecanoyl phorbol 13-acetate (TPA). However, while TPA, as expected, powerfully stimulated the phosphorylation of the PKCs' 85-kDa myristoylated alanine-rich protein kinase C substrate (MARCKS) protein, ionomycin unexpectedly did not. Instead, ionomycin reduced the basal MARCKS phosphorylation. Pretreating the glioma cells with ionomycin prevented TPA-stimulated PKCs from phosphorylating the MARCKS protein. The stimulation of membrane PKCs activity and the prevention of MARCKS phosphorylation by ionomycin required external Ca2+ because they were both abolished by adding 5 mM EGTA to the culture medium. Recently (Chakravarthy, B. R., Isaacs, R. J., Morley, P., Durkin, J. P., and Whitfield, J. F. (1995) J. Biol. Chem. 270, 1362-1368), we proposed that Ca2+ x calmodulin complexes block MARCKS phosphorylation by the activated PKCs in keratinocytes stimulated by raising the external Ca2+ concentration. In the present experiments calmodulin prevented MARCKS phosphorylation by TPA-stimulated PKCs in glioma cell lysates, and this blockade was lifted by a calmodulin antagonist, the calmodulin-binding domain peptide. But, physiologically more significant, pretreating intact glioma cells with a cell-permeable calmodulin antagonist, calmidazolium, prevented ionomycin from blocking MARCKS phosphorylation by PKCs in unstimulated and TPA-stimulated cells. The effect of ionomycin on MARCKS phosphorylation was not due to the stimulation of Ca2+ x calmodulin-dependent phosphoprotein phosphatase, calcineurin, because cyclosporin A, a potent inhibitor of this phosphatase, did not stop ionomycin from preventing MARCKS phosphorylation. The ability of ionomycin to prevent TPA-stimulated PKCs from phosphorylating MARCKS depended on whether ionomycin was added before, with, or after TPA. Maximum blockade occurred when ionomycin was added before TPA but was less effective when added with or after TPA. These results indicate that Ca2+ x calmodulin can profoundly affect PKCs' signaling at the substrate level.

Amino Acid Sequence↗

PubMed1995

Neuroprotective effects of omega-Aga-IVA against in vitro ischaemia in the rat hippocampal slice.

Excessive accumulation of Ca2+ in neurones and glutamate release are involved in neuropathological processes, including ischaemia. We investigated the neuroprotective effects of the Ca2+ channel antagonist, omega-Aga-IVA, in CA1 pyramidal neurones in rat hippocampal slices following an in vitro hypoxic-hypoglycaemic insult. Following this insult, evoked post-synaptic response amplitudes decreased from 3.7 +/- 0.5 mV to 0.6 +/- 0.2 mV and the CA1 neurones appeared dead using a live/dead fluorescence assay with confocal microscopy. Slices treated with 200 nM omega-Aga-IVA had evoked response amplitudes not significantly different from control (3.3 +/- 0.5 mV) and the CA1 neurones appeared viable using the live/dead fluorescence assay. The neuroprotective efficacy of omega-Aga-IVA suggests that omega-Aga-IVA-sensitive Ca2+ channels participate in ischaemic neuronal death and constitute a potential target of therapeutic intervention.

Animals↗

PubMed1995

Stimulation of protein kinase C during Ca(2+)-induced keratinocyte differentiation. Selective blockade of MARCKS phosphorylation by calmodulin.

Raising the external Ca2+ concentration from 0.05 to 1.8 mM stimulated membrane-associated protein kinase Cs (PKCs) activity as strongly as the specific PKCs activator, 12-O-tetradecanoyl phorbol-13-acetate (TPA) in BALB/MK mouse keratinocytes. This was indicated by the increased phosphorylation of a PKC-selective peptide substrate, Ac-FKKSFKL-NH2, by membranes isolated from the Ca(2+)- or TPA-stimulated keratinocytes. Raising the external Ca2+ concentration to 1.8 mM also triggered a 4-fold rise in the intracellular free Ca2+ concentration. As reported elsewhere (Moscat, J. Fleming, T. P., Molloy, C. J. Lopez-Barahona, M., and Aaronson, S. A. (1989) J. Biol. Chem. 264, 11228-11235), TPA stimulated the phosphorylation of the PKCs substrate, the 85-kDa myristoylated alanine-rich kinase C substrate (MARCKS) protein, in intact keratinocytes, but Ca2+ did not. Furthermore, Ca(2+)-pretreatment reduced the TPA-induced phosphorylation of the 85-kDa protein in intact cells. There was no significant increase in MARCKS phosphorylation when keratinocytes were treated with a Ca2+.CaM-dependent phosphatase inhibitor, cyclosporin A, before stimulation with 1.8 mM Ca2+.Ca2+.calmodulin suppressed the ability of isolated membranes to phosphorylate the 85-kDa MARCKS holoprotein in vitro in the presence of phosphatase inhibitors such as fluoride, pyrophosphate, and vanadate, and this inhibition was overcome by a calmodulin antagonist, the calmodulin-binding domain peptide. Thus, the ability of 1.8 mM Ca2+ to strongly stimulate the membrane PKCs activity without stimulating the phosphorylation of the MARCKS protein in keratinocytes is consistent with the possibility of Ca2+.calmodulin complexes, formed by the internal Ca2+ surge, binding to, and blocking the phosphorylation of, this PKC protein substrate.

Amino Acid Sequence↗

PubMed1995

The role of intracellular calcium and protein kinase C in endothelin-stimulated proliferation of rat type I astrocytes.

The increased expression of immunoreactive endothelin-1 (ET-1) in reactive astrocytes and its mitogenic effects on astrocytes and glioma cell lines, have implicated endothelins in the development of reactive gliosis. In this study, an increase in DNA synthesis in rat type I astrocytes was observed after cultures were transiently exposed to ET-1 for 15 min, suggesting that early signal transduction events are essential and sufficient for the propagation of the ET-1-induced mitogenic signal. Prompt increases in inositol triphosphate (IP3) formation and [Ca2+]i were observed upon the addition of ET-1 to these cells. The ET-1-evoked increase in [Ca2+]i consisted of an initial peak which was preserved in Ca(2+)-free medium, and a sustained phase which was abolished in Ca(2+)-free medium and partly attenuated by nifedipine. ET-1 also increased the activity of membrane-associated protein kinase C (PKC) and induced the in vivo phosphorylation of the 85 kD MARCKS protein, an endogenous PKC-specific substrate. The ET-1-evoked increases in DNA synthesis, IP3, [Ca2+]i, membrane PKC, and 85 kD MARCKS protein phosphorylation in rat cortical astrocytes were prevented by either the selective endothelin ETA receptor antagonist, BQ-123, or the phospholipase C (PLC)-specific inhibitor, U-73122. However, the inhibition of PKC activity did not affect ET-1-induced DNA synthesis in rat cortical astrocytes. These results suggest that ET-1-induced IP3 and/or [CA2+]i responses, but not the activation of PKC, are essential for the growth-factor like actions of ET-1 in rat cortical astrocytes.

Animals↗

PubMed1995

Restoration of severely depleted femoral trabecular bone in ovariectomized rats by parathyroid hormone-(1-34).

It is commonly believed that the parathyroid hormone's (PTH's) main function in bone is to stimulate osteoclastic resorption. However, intermittent injections of small doses of PTH holoprotein, but more often its bioactive hPTH-(1-34) fragment, have been shown to stimulate bone growth in animals and humans through their ability to stimulate adenylyl cyclase and not their ability to independently activate a protein kinases-C stimulating mechanism. This anabolic action suggests that PTH might be an effective therapeutic for osteoporosis. If so, the hormone must be able to restore severely depleted trabecular bone, and the goal of this study was to find out if it can. To do this, we started a multiweek program of daily subcutaneous injections of 0.8 nmoles of hPTH-(1-34)/100 g body weight into rats at 4, 8, or 16 weeks after ovariectomy (OVX) and the increasingly severe selective loss of trabecular bone. These injections strongly stimulated femoral trabecular bone to grow and mineralize at the same rate regardless of how much of it had been lost before the injections were started. Thus, the progressively depleting trabecular bone in the femurs of OVX rats does not lose its anabolic responsiveness to PTH. This finding is another indication of the likelihood of small, adenylyl cyclase-stimulating PTH fragments being effective therapeutics for osteoporosis.

Analysis of Variance↗

PubMed1995

Do pancreatic islet cells from neonatal rats have surface receptors or sensors for divalent cations?

The effects of extracellular divalent cations on the intracellular Ca2+ concentration ([Ca2+]i) in neonatal rat islet cells were investigated to determine whether these cells, like several others, have signal-generating surface cation sensors. Raising the external Ca2+ concentration by 1 mM increments triggered either sustained increases in [Ca2+]i or large sharp [Ca2+]i spikes followed by return to a suprabasal level. The external Ca(2+)-triggered [Ca2+]i responses were abolished by treating the cells with the inhibitor of inositol phospholipid hydrolysis, neomycin (1.5 mM), but not by another phospholipase C inhibitor, U-73,122 (2.5 microM), or the voltage-sensitive Ca2+ channel blockers nifedipine (20 microM) and methoxyverapamil (D600; 50 microM). [Ca2+]i responses were also triggered by barium (Ba2+; 1 mM) and cobalt (Co2+; 1 mM). The Ba2+ responses were also inhibited by neomycin and unaffected by nifedipine or D600 and the Co2+ response required external Ca2+. Therefore, neonatal rat pancreatic islet cells may display divalent cation receptors/sensors on their surfaces. Activation of these putative receptors, which are coupled to neomycin-sensitive, voltage-independent, dihydropyridine-insensitive channels, by Ca2+, Ba2+ or Co2+ would trigger [Ca2+]i responses by opening these channels to admit external Ca2+ into the cell. The physiological function(s) of such cell-surface divalent cation receptors/sensors and the [Ca2+]i surges they generate in pancreatic islet cells is not known.

Animals↗

PubMed1995

Small bone-building fragments of parathyroid hormone: new therapeutic agents for osteoporosis.

The brittle, fracture-prone bones of an osteoporotic postmenopausal woman are the products of an excessive uncompensated resorption of trabecular bone by osteoclasts. Osteoporosis is currently treated with the osteoclast suppressors calcitonin, bisphosphonates, or oestrogen, which stop further bone resorption without stimulating new bone growth. Here, James Whitfield and Paul Morley review the growing evidence that small adenylate cyclase-stimulating fragments of the parathyroid hormone are promising therapeutic agents for osteoporosis that potently stimulate osteoblasts to make mechanically strong or supranormally strong bone.

Adenylyl Cyclases↗

PubMed1995

Glutamate receptor-mediated calcium surges in neurons derived from P19 cells.

Retinoic acid-treated murine P19 embryonal carcinoma cells differentiate into cells with neuronal morphology that display typical neuronal markers. In this study, the presence of glutamate receptors linked to Ca(2+)-signaling mechanisms on these neurons was demonstrated by testing the effects of glutamate agonists and antagonists on the intracellular calcium ion concentration ([Ca2+]i). Glutamate (1 mM) induced either sustained or transient increases in [Ca2+]i. The sustained glutamate-induced increase in [Ca2+]i was mimicked by NMDA (40 microM). The NMDA-triggered [Ca2+]i response was abolished by incubating the cells in Ca(2+)-free medium or by pretreating them with Mg2+ (2 mM) or MK-801 (0.1 microM). These responses were unaffected by the non-NMDA antagonist CNQX (10 microM), but they required glycine (3-30 microM). Kainate (40 microM) and AMPA (40 microM) did not affect [Ca2+]i. Without external Ca2+, glutamate triggered transient, sometimes oscillating, increases in [Ca2+]i. These responses were mimicked by the metabotropic agonist trans-(1S, 3R)-1-amino-1,3-cyclopentanedicarboxylic acid (300 microM). These results suggest that neurons derived from P19 embryonal carcinoma cells have NMDA and metabotropic, but not AMPA/kainate receptors, which are linked to Ca(2+)-signaling mechanisms. These cells could provide a consistent and reproducible model with which to study neuronal differentiation, neurotoxicity, and glutamate receptor-signaling mechanisms.

Animals↗

PubMed1995

N-methyl-D-aspartate- or glutamate-mediated toxicity in cultured rat cortical neurons is antagonized by FPL 15896AR.

The neuroprotective action of (S)-alpha-phenyl-2-pyridineethanamine dihydrochloride (FPL 15896AR), a novel noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, was examined in primary rat cortical neuronal cultures. Exposure of cortical cultures to NMDA (50 microM) or glutamate (50 microM) for 15 min resulted in the death of 85-95% of the neurons during the next 24 h. This neurotoxicity was completely eliminated by adding FPL 15896AR (50 microM) to the cultures during the time of NMDA or glutamate exposure. Neuroprotective concentrations of FPL 15896AR also inhibited other acute effects of NMDA. FPL 15896AR (50 microM) prevented the loss of membrane-associated protein kinase C activity that developed by 4 h after transient exposure to 50 microM NMDA or 50 microM glutamate. FPL 15896AR also reduced by approximately 35% the magnitude of NMDA-triggered increases in intracellular free Ca2+ concentration in the cortical cultures. These data indicate that NMDA-mediated toxicity in cultured cortical neurons can be blocked by the NMDA antagonist FPL 15896AR.

Animals↗

PubMed1995

Calcium-cell cycle regulator, differentiator, killer, chemopreventor, and maybe, tumor promoter.

Ca2+ and Ca(2+)-binding proteins are involved in running the cell cycle. Ca2+ spikes and signals from integrin-activated focal adhesion complexes and Ca2+ receptors on the cell surface along with cyclic AMP begin the cycle of cyclin-dependent protein kinases (PKs). These transiently expressed PKs stimulate the coordinate expression of DNA-replicating enzymes, activate replication enzymes, inactivate replication suppressors (e.g., retinoblastoma susceptibility protein), activate the replicator complexes at the end of the G1 build-up, and when replication is complete they and a Ca2+ spike trigger mitotic prophase. Another Ca2+ surge at the end of metaphase triggers the destruction of the prophase-stimulating PKs and starts anaphase. Ca2+ finally stimulates cytoplasmic division (cytokinesis). However, Ca2+ does more than this in epithelial cells, such as those lining the colon, and skin keratinocytes. These cells also need Ca2+, integrin signals, and only a small amount (e.g., 0.05-0.1 mM) of external Ca2+ to start DNA replication. Signals from their surface Ca2+ receptors trigger a combination of differentiation and apoptosis ("diffpoptosis") when external Ca2+ concentration reaches their setpoints. The skin's steep, upwardly directed, Ca2+ gradient has a low concentration in the basal layer to allow stem and precursor keratinocytes to proliferate, and higher concentrations in the suprabasal layers to trigger the differentiation-apoptosis ("diffpoptosis") mechanism that converts granular cells into protective, hard-shelled, dead corneocytes. A similar Ca2+ gradient may exist in the colon crypt allowing the stem cell and its amplifying transit or precursor offspring to cycle in the lower parts of the crypt, while stopping proliferation and stimulating terminal differentiation in the upper crypt and flat mucosa. Raising the amount of Ca2+ in fecal water above a critical level reduces proliferation and thus colorectal carcinogenesis in normal rats and some high-risk humans. But during carcinogenesis the Ca2+ sensors malfunction or their signals become ineffective: high Ca2+ does not stop, and may even stimulate, the proliferation of initiated mutants. Therefore, Ca2+ may either not affect, or even promote, the growth of epithelial cells in carcinogen-initiated rat colon and human adenoma patients. Clearly, a much greater understanding of how Ca2+ controls the proliferation and differentiation of epithelial cells and why initiated cells lose their responsiveness to Ca2+ are needed to assess the drawbacks and advantages of using Ca2+ as a chemopreventor.

Animals↗

PubMed1994

Effect of tamoxifen on carbachol-triggered intracellular calcium responses in chicken granulosa cells.

The effect of the nonsteroidal antiestrogen tamoxifen on carbachol (CCh)-triggered intracellular Ca2+ surges was determined in granulosa cells from the two largest preovulatory follicles of laying hens. The intracellular calcium ion concentration ([Ca2+]i) was measured in cells loaded with the Ca(2+)-responsive fluorescent dye fura-2. Resting [Ca2+]i was 96 +/- 5 nM (n = 20), and CCh (1 mM) triggered a large initial [Ca2+]i spike to 600-800 nM, due to the mobilization of Ca2+ from internal stores. Following the spike, the [Ca2+]i dropped to a lower, suprabasal level with super-imposed oscillations, which depended on Ca2+ influx, and returned to the resting level by 2 to 4 min. Tamoxifen (10 microM) did not by itself affect [Ca2+]i but pretreating granulosa cells with tamoxifen (10 microM) prolonged the CCh-triggered [Ca2+]i surge and oscillations by as much as 10 to 30 min. Pretreatment with much higher concentrations of tamoxifen (e.g., 0.5 mM) also had no effect by themselves, but caused a prolonged rise in [Ca2+]i following CCh (1 mM) stimulation. The effect of tamoxifen on CCh-triggered [Ca2+]i responses was mimicked by the tamoxifen metabolite 4-hydroxytamoxifen (10 microM), but not by the structurally related antiestrogens nafoxidine (10 microM) or clomiphene citrate (10 microM). The tamoxifen effect on the CCh-triggered [Ca2+]i response was not mediated through estrogen receptors since pretreating granulosa cells with 17 beta-estradiol (10(-6) M) did not mimic the tamoxifen response. The effect of tamoxifen was inhibited by pretreating granulosa cells with the Ca2+ channel blocker, lanthanum (1 mM), or by incubating the cells in Ca(2+)-free medium. Tamoxifen did not affect [Ca2+]i surges triggered by 17 beta-estradiol (10(-6) M) or dimethyl sulfoxide (1%) which mobilize Ca2+ from internal stores. Pretreating granulosa cells with tamoxifen (10 microM) or 4-hydroxytamoxifen (10 microM) before inducing Ca2+ influx through voltage-dependent Ca2+ channels by depolarizing the cells with 45 mM external K+, caused a prolonged rise of [Ca2+]i, with oscillations, similar to the CCh response. These studies demonstrate that tamoxifen affects the activation of chicken granulosa cell Ca2+ channels by CCh or by raising the external K+ concentration, resulting in a prolongation of the sustained [Ca2+]i elevation and oscillations, which result from the influx of extracellular Ca2+. These observations suggest that tamoxifen interacts with open Ca2+ channels in chicken granulosa cells and keeps them open for prolonged periods of time.

Animals↗

PubMed1994

C-terminal fragments of parathyroid hormone-related protein, PTHrP-(107-111) and (107-139), and the N-terminal PTHrP-(1-40) fragment stimulate membrane-associated protein kinase C activity in rat spleen lymphocytes.

Membrane-associated protein kinase C (PKC) activity in lymphocytes freshly isolated from rat spleen was stimulated by the C-terminal parathyroid hormone-related protein fragments, PTHrP-(107-111) and PTHrP-(107-139), at concentrations from 10(-3) to 10(4) pM. By contrast, the same concentrations of PTHrP-(120-139), without the 107-111 TRSAW (-Thr-Arg-Ser-Ala-Trp-) sequence of the other C terminal fragments, did not stimulate spleen lymphocyte PKC. Low concentrations of the N-terminal PTHrP-(1-40) fragment also stimulated membrane-associated PKC activity in the spleen lymphocytes. These results suggest that PTHrP might be an important physiological regulator of the immune response.

Animals↗

PubMed1994

Granulosa cells have calcium-dependent action potentials and a calcium-dependent chloride conductance.

We have found chicken granulosa cells to be excitable. Experiments using the whole-cell patch-clamp technique showed that they had membrane resting potentials of -62 +/- 3 mV (n = 8) and generated action potentials, either in response to 10-ms depolarizing current pulses or, on occasion, spontaneously. The action potentials persisted in a Na(+)-free bath and were reversibly blocked by 4 mM Co2+. They lasted 0.9-3.0s with 64 mM Cl- in the pipette, were shortened 67 +/- 8% by the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 20 microM), and lengthened to 8.7 +/- 2.2 when the Cl- equilibrium potential (Vcl) was changed from -20 mV to -2 mV by using 134 mM Cl- in the pipette. With conventional whole-cell voltage-clamp, slowly activating and inactivating currents, which reached maximum amplitude after 0.35-1.40 s, were evoked by depolarizing voltage steps. These slow currents activated between voltage steps of -60 mV and -50 mV and reached a maximum inward amplitude at about -40 mV. Changing the Cl- concentration in the pipette (VCl of -2MV or -20 mV) or bath (VCl of -2 mV or + 18 mV) shifted their reversal potential in a direction consistent with a Cl- electrode. They were inhibited by the Cl- channel antagonists 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS; 0.5 mM), NPPB (20 microM), and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS; 0.5 mM). The slow currents were blocked by Ca2+ deprivation, or by CO2+ (4 mM), or by replacing external Ca2+ with Ba2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗