Biomedical subjects

J F Whitfield

Publications and source records attributed to J F Whitfield.

At least 55 records · Page 3Linked to original sources

PubMed1996

Ras is involved in gap junction closure in proliferating fibroblasts or preadipocytes but not in differentiated adipocytes.

A decrease in gap junctional, intercellular communication (GJIC) has been associated with cells neoplastically transformed by a variety of factors. To investigate the role of the Ras oncogene product in gap junction function, a panel of murine C3H10T1/2 (10T1/2) fibroblasts was constructed in which the levels of ras gene expression could be effectively up- or down-regulated. Intercellular communication was measured using a novel technique of in situ electroporation of adherent cells on a partly conductive slide. The introduction of increasing amounts of activated Ras(leu61) in mouse 10T1/2 fibroblasts proportionally reduced GJIC, while the downregulation of endogenous c-ras gene expression increased junctional permeability. These results indicate that Ras plays an important role in the junction closure pathway leading to the proliferation of normal cells. However, differentiation of c-Ras-deficient preadipocytes entirely abolished their initially extensive GJIC, indicating that junction closure in response to adipocytic differentiation is independent of Ras.

Adipocytes↗

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

Solution structure and adenylyl cyclase stimulating activities of C-terminal truncated human parathyroid hormone analogues.

Analogues of human parathyroid hormone (hPTH) truncated at the C-terminal end have been studied for adenylyl cyclase (AC) activity and for solution conformation by circular dichroism (CD) spectroscopy. Analogues of hPTH-(1-34)-NH2, containing the first 28-31 residues, had only a slightly diminished ability to stimulate AC in rat osteosarcoma (ROS) cells as compared to that of the parent analogue. CD data on hPTH-(16-34)-NH2 and C-terminal deletion mutants of hPTH-(1-34)-NH2 supported the presence of a partially stable alpha-helix over residues 17-28. A carboxyl-terminal mutant, hPTH-(1-30)-OH, showed both reduced helix and greatly reduced AC-stimulating activity as compared to the corresponding amide analogue. In contrast, both of these analogues, in the presence of palmitoyloleoylphosphatidylserine (POPS) vesicles, showed an equal stabilization of alpha-helix. All other analogues showed at least some enhancement of alpha-helix in the presence of POPS. However, both in neutral, aqueous buffer and in POPS, the relative amount of alpha-helix decreased greatly as the peptide was shortened below the 1-28 sequence. These data provide additional support for an amphiphilic alpha-helix over residues 21-28 being the conformation for receptor binding of hPTH for stimulation of AC activity. Modeling human parathyroid hormone-related peptide as an alpha-helix over this same region, and comparison to hPTH, suggests that both may bind via the hydrophobic face to the receptor.

Adenylyl Cyclases↗

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

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

Structure-function relationship of human parathyroid hormone in the regulation of vitamin D receptor expression in osteoblast-like cells (ROS 17/2.8).

Studies of the relationship between PTH structure and function in the activation of protein kinases have revealed that different regions within the biologically active PTH-(1-34) peptide are responsible for different functions. The first two N-terminal amino acids are required for plasma membrane adenylyl cyclase stimulation, and the C-terminal region 29-32 is necessary for the translocating activity of protein kinase C. In the present study, we explored the structure-function relationship of human (h) PTH in the regulation of the vitamin D receptor (VDR) in osteoblast-like cells (ROS 17/2.8). VDR-rich cytosol extract was prepared after the confluent cells were incubated with different hPTH fragments for 16 h. hPTH-(1-34) at concentrations of 10(-9)-10(-7) M caused a dose-dependent decrease in VDR content from a control level of 70.2 +/- 2.2 fmol/mg protein to 62.1 +/- 3.3 (-16%) at 10(-9) M, 52.3 +/- 5.3 (-25.5%; P < 0.02) at 10(-8) M, and 45.5 +/- 3.5 fmol/mg protein (-35.3%; P = 0.001) at 10(-7) M (n = 6). hPTH-(1-31) also decreased VDR content from 65.5 +/- 3.6 to 55.2 +/- 7.9 (-19.5%) at 10(-9) M, 44.3 +/- 5.8 (-32.4%; P < 0.05) at 10(-8) M, and 40.6 +/- 3.2 fmol/mg protein (-38.9%; P < 0.05) at 10(-7) M (n = 6). Incubation of ROS 17/2.8 cells with 0.5 nM 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] led to up-regulation of VDR content by 340-370% of the control value. hPTH-(1-34) decreased the VDR up-regulatory effect of 1,25-(OH)2D3 from 340% to 230% of the control value at 10(-8) M (P < 0.0001) and 170% of the control value (P < 0.0001) at 10(-7) M, respectively (n = 6). hPTH-(1-31) also decreased the receptor up-regulatory effect of 1,25-(OH)2D3 from 370% to 286% (P < 0.02) at 10(-8) M and 220% (P < 0.002) at 10(-7) M, respectively (n = 6). hPTH-(3-34) and -(13-34) at concentrations of 10(-9)-10(-7) M did not decrease VDR content in either the absence or presence of 1,25-(OH)2D3. Quantitation of VDR messenger RNA by reverse transcription-polymerase chain reaction showed that PTH-(1-34) and -(1-31) at 10(-7) M, but not PTH-(3-34) and -(13-34), inhibited ROS 17/2.8 cell VDR gene expression in both the absence and presence of 1,25-(OH)2D3.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenylyl Cyclases↗

PubMed1995

Alterations in cell-cell communication in human papillomavirus type 16 (HPV16) transformed rat myoblasts.

A reduction of gap-junctional intercellular communication (GJIC) often accompanies neoplastic transformation. The present work demonstrates that transformation by the oncogenic human DNA virus, human papilloma virus 16(HPV16), also reduces GJIC between L6 rat myoblasts. HPVs are associated with anogenital cancers, the incidence of which is increasing in HIV positive patients of both sexes. Using videofluorescence imaging of Fura-2 loaded cells a lack of GJIC between transformed HPV16-L6 cells was first indicated by uncoordinated brief [Ca2+]i spikes in clusters of DMSO-treated HPV16-L6 cells instead of the synchronous, sustained [Ca2+]i surges in clusters of DMSO-treated L6 cells. Reduced GJIC between HPV16-L6 cells was demonstrated directly by a much reduced transfer of lucifer yellow dye from HPV16-L6 cells, which had been loaded with the dye through electroporation with an EPIZAP II in situ electroporator, to neighbouring nonelectroporated HPV16-L6 cells. One reason for this reduced GJIC between HPV16-L6 cells could have been their dramatically enhanced activity of membrane-associated PKC which is known to phosphorylate connexins and down-regulate gap junctions. However, the main reason was the viral-induced inhibition of the expression of a major gap junction component, Cx43 (Connexin 43), in the transformed myoblasts.

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

Inactive membrane protein kinase Cs: a possible target for receptor signalling.

The activation of the multifunctional cell signalling enzymes, the protein kinase Cs (PKCs), is generally thought to result from the translocation of inactive cytosolic enzymes to activation sites in cell membranes. However, recent studies suggest that PKCs may also be stimulated in cells by processes independent of translocation. One possible mechanism is the modulation of the activity of PKCs already resident in membranes. A PKC assay that measures enzyme activity directly in isolated native membranes has revealed the presence of an activatable pool of PKCs resident in native membranes of various cells and tissues. In 3T3-L1 cells, some or all of this pool of membrane PKCs was stimulated within 10 min of exposing the cells to 10 ng/ml epidermal growth factor or 100 ng/ml fibroblast growth factor. Similar increases in PKC activity were observed in native membranes isolated from CTLL-2, WEHI-231 and S49 lymphoma cells that had been exposed to interleukin-2. These growth factors all stimulated membrane PKC activity without detectably translocating cytosolic enzymes to the membranes. In intact WEHI cells, low concentrations (5-10 microM) of a diacylglycerol, 1-oleoyl-2-acetyl-sn-glycerol (OAG), or low concentrations (2-10 nM) of phorbol 12-myristate 13-acetate sufficed to activate PKCs already resident in membranes, but much higher concentrations (50-100 microM and 50-100 nM respectively) were needed to detectably stimulate the translocation of cytosolic PKCs. A phosphatidylcholine-specific phospholipase C also selectively stimulated membrane PKCs in WEHI cells at concentrations that were much less than those needed to induce the translocation of cytosolic enzymes. Furthermore, interleukin-2 and low concentrations of OAG both stimulated the phosphorylation of the 85 kDa PKC-selective substrate protein in intact WEHI cells in which translocation of PKCs was not evident. These results suggest that the membranes of some cells maintain a pool of activatable PKCs that respond to lower levels of extracellular stimuli than cytosolic PKCs, and that can be stimulated by signals which produce diacylglycerols through the hydrolysis of phospholipids other than polyphosphoinositides.

Amino Acid Sequence↗

PubMed1994

Cyclic AMP triggers large [Ca2+]i oscillations in glucose-stimulated beta-cells from ob/ob mice.

The modulation of intracellular free calcium concentration ([Ca2+]i) by cAMP was compared in pancreatic beta-cells of lean (+/+) and obese (ob/ob) mice. Neither forskolin nor 8-bromo-cAMP significantly affected basal [Ca2+]i in unstimulated lean and obese mouse beta-cells. In obese, but not in lean mouse beta-cells, adding forskolin or 8-bromo-cAMP during the glucose-induced [Ca2+]i response triggered external Ca(2+)-dependent [Ca2+]i oscillations with a duration of 5-11 s and a frequency of 2.3-4.8 min-1. The induction of oscillations by cAMP required both a stimulatory glucose concentration and membrane depolarization. (Sp)-cAMPS, did induce oscillations in lean mouse beta-cells. However, these oscillations were different from those seen in obese mouse beta-cells and required higher concentrations of (Sp)-cAMPS. The inducibility of fast oscillations in obese mouse beta-cells indicates hypersensitivity of these cells to cAMP, and suggests an abnormal behavior of K+ and/or Ca2+ channels.

8-Bromo Cyclic Adenosine Monophosphate↗

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

Further definition of the protein kinase C activation domain of the parathyroid hormone.

The protein kinase C (PKC) activation domain of the parathyroid hormone (PTH) was believed to be the 28-34 region of the molecule. We have now shown that PTH-(29-32) is the smallest PTH fragment that can stimulate significantly membrane-associated PKC activity in ROS 17/2 rat osteosarcoma cells. As was previously shown for full-length PTH-(1-84) and the fully bioactive PTH-(1-34) fragment, there were two peaks in the PKC response to PTH-(29-32): one peak was obtained with low picomolar concentrations and the other with much higher nanomolar concentrations of the fragment. The PKC-activating ability was unaffected by the loss of Asn33 and Phe34, but it was abolished by removing His32. Thus, the PTH-(28-31) and PTH-(29-31) fragments did not stimulate membrane-associated PKC activity. The much larger PTH-(1-31) fragment also did not stimulate membrane-associated PKC activity, although it stimulated adenylyl cyclase as strongly as PTH-(1-34). This functional sensitivity to the loss of the polar His32 was not caused by a specific need for His or another polar amino acid in this position because replacing it with the apolar Leu did not abolish adenylyl cyclase or PKC activation. It is concluded that the minimum, fully functional PKC activation domain of the PTH molecule is Gln29-Asp30-Val31-His32.

Adenylyl Cyclases↗

PubMed1994

Parathyroid hormone fragments may stimulate bone growth in ovariectomized rats by activating adenylyl cyclase.

PTH is regarded conventionally as a catabolic hormone that stimulates osteoclastic resorption of bone. However, it has been known since 1932 that intermittent pulses of PTH stimulate bone formation in animals and humans. PTH independently activates two signal mechanisms: one that stimulates adenylyl cyclase and one that stimulates protein kinase C (PKC). The goal of this study was to use the 3- to 5-month-old ovariectomized (OVX) rat model to determine which of the two signal mechanisms is responsible for the anabolic action of PTH on bone. OVX triggered a large loss of trabecular bone without significantly affecting the normal slow growth of cortical bone in the distal halves of the femora. Daily injections of human hPTH(1-34) fragment (1 nmol/100 g body weight), which stimulated both adenylyl cyclase and membrane-associated PKC activity in osteoblast-like ROS 17/2 rat osteosarcoma cells, stimulated the growth of both cortical and trabecular bone in the OVX rats. Daily injections of the same dose of hPTH(1-31), which stimulated adenylyl cyclase but not PKC in ROS 17/2 cells, stimulated trabecular bone growth in the OVX rats less effectively than hPTH(1-34), but it stimulated cortical bone growth as rapidly and as dramatically as hPTH(1-34). Injections of equimolar amounts of desamino-hPTH(1-34) [N-propionyl(2-3)hPTH-amide], which stimulated PKC as strongly as hPTH(1-34) in ROS 17/2 cells but had a drastically reduced ability to stimulate adenylyl cyclase, or injections of recombinant hPTH(8-84) which stimulated PKC only in the ROS 17/2 cells, did not stimulate cortical or trabecular bone growth in the OVX animals. Thus, cyclic AMP and cyclic AMP-dependent protein kinases may be the primary mediators of the anabolic action of intermittent pulses of PTH on bone in OVX rats.

Adenylyl Cyclases↗