Biomedical subjects

J F Whitfield

Publications and source records attributed to J F Whitfield.

At least 19 recordsLinked to original sources

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

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↗

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

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↗

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

Extracellular calcium modulates prereplicative cyclic AMP surges in EGF-stimulated primary neonatal rat hepatocytes.

The cells in nearly pure (96-98%) primary cultures of hepatocytes from neonatal rat liver in high (1.0 mM)-Ca2+, serum-free, synthetic HiWo5Ba2000 medium initiated DNA synthesis and entered mitosis between 11 and 30 h after the addition of 10 ng/ml EGF. During the 10-h prereplicative period, the cultured hepatocytes, like regenerating rat liver cells, generated two large cyclic AMP transients, one peaking between 30 min and 2 h and the other around 6 h. Hepatocytes stimulated by the same concentration of EGF in low (0.02 mM)-Ca2+ medium increased cyclic AMP synthesis as much as the EGF-treated hepatocytes in high-Ca2+ medium, but they released the additional cyclic AMP into the medium and could not generate prereplicative internal cyclic AMP surges, initiate DNA replication, or enter mitosis. These results suggest that one of the ways external Ca2+ controls prereplicative development of hepatocytes is to restrain the release of cyclic AMP and thus enable the cell to accumulate enough internal cyclic AMP to stimulate events required to initiate DNA replication.

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↗

PubMed1994

Insulin secretion and intracellular Ca2+ rises in monolayer cultures of neonatal rat beta-cells.

Glucose-induced insulin release, glucose-induced rises in intracellular free Ca2+ concentration ([Ca2+]i), and voltage-dependent Ca2+ channel activity were assessed in monolayer cultures of beta-cells from 3-5-day-old rats. The glucose-stimulated insulin secretory responses and [Ca2+]i rises were like those in adult rat beta-cells rather than fetal rat beta-cells. Voltage-dependent Ca2+ channel antagonists decreased glucose-induced insulin secretion, aborted the [Ca2+]i rise and, like deprivation of extracellular Ca2+, prevented the glucose-induced rise in [Ca2+]i when added before the glucose challenge. The presence of nifedipine-sensitive, voltage-dependent Ca2+ channels was demonstrated directly by measuring Ca2+ currents using the whole-cell configuration of the patch-clamp technique and indirectly by measuring [Ca2+]i after membrane depolarization by 45 mM K+ or 200 microM tolbutamide. Thus, in cultured beta-cells of 3-5-day-old rats the coupling of glucose stimulation to Ca2+ influx is essentially mature, in contrast to what has been reported for fetal or very early neonatal cells.

Age Factors↗

PubMed1994

Purinergic receptor-mediated intracellular Ca2+ oscillations in chicken granulosa cells.

These studies were designed to investigate the effects of extracellular ATP on intracellular calcium ion concentration ([Ca2+]i) and progesterone secretion in granulosa cells obtained from the two largest preovulatory follicles (F1 and F2) of hens. [Ca2+]i was measured in cells loaded with the Ca(2+)-responsive fluorescent dye fura-2. The resting [Ca2+]i in these cells was 99 +/- 7 nM (n = 22). There was a 5.7 +/- 0.7-fold increase in [Ca2+]i in all (n = 140) of the cells within 5 sec of adding a maximally stimulatory concentration (100 microM) of extracellular ATP. The initial spike was followed by [Ca2+]i oscillations that returned to the resting level between spikes. The frequency and amplitude of the [Ca2+]i oscillations were varied and persisted for 1-40 min. [Ca2+]i oscillations were also triggered by 100 microM UTP, UDP, GTP, GDP, ADP, and the nonhydrolyzable analog ATP gamma S. Adenosine, AMP, GMP, and UMP (all at 100 microM) were ineffective. The lowest ATP concentration to trigger a [Ca2+]i response was 1 microM. The sustained oscillatory phase of the response, but not the initial spike, was inhibited by incubating the cells in Ca(2+)-free medium containing 2 mM EGTA. The nucleotide-triggered [Ca2+]i oscillations were not affected by adding the dihydropyridine Ca2+ channel blockers verapamil (100 microM), methoxy-verapamil (D600; 100 microM), or nifedipine (10 microM), before or during the response. However, the oscillations, but not the initial spike, were prevented by pretreating the cells with a general Ca2+ channel blocker, lanthanum (1 mM) or cobalt (5 mM). Lanthanum and cobalt also promptly stopped the [Ca2+]i oscillations when added during the oscillatory phase. The nucleotide-triggered [Ca2+]i response was also abolished by pretreating the cells with an inhibitor of inositol phospholipid hydrolysis, neomycin (1.5 mM). In 3-h incubations, adenosine (100 microM) or ATP (100 microM) did not affect basal or LH (20 or 100 ng/ml)-stimulated progesterone production. These studies demonstrate that chicken granulosa cells display P2 purinergic receptors on their surfaces. Activation of these receptors triggers [Ca2+]i oscillations that follow the release of Ca2+ from internal stores and depend on Ca2+ influx through dihydropyridine-insensitive Ca2+ channels. The physiological function(s) of P2 purinergic receptors on granulosa cells is not known.

Adenosine↗

PubMed1993

Evidence that an additional conserved element with the consensus C/GAGA/C is essential for maximal responsiveness of the cyclic AMP enhancer.

We analyzed the ability of cyclic AMP-response element binding proteins (CREBs) to interact with the CRE sequences derived from different genes and examined the role of sequences flanking the core CRE element in rendering cAMP-responsiveness to the enhancer. We were able to detect reproducibly, sing the Southwestern blotting technique, five major CREB factors of molecular weights 56, 47, 40, and 36-34 kDa which were present in various rat tissues and cultured cells. The 34-40 kDa proteins (CREB-327/341) were able to bind to the CRE of cAMP-inducible genes (somatostatin, c-fos, E2A), but not to genes whose expression is not controlled by cAMP (glucagon, parathyroid hormone). The novel 47 kDa CREB had a high specificity for the core octameric CRE sequence and it bound equally well to the consensus CRE of cAMP-inducible and noninducible genes. On the other hand, the 47 kDa CREB did not bind at all to the phorbol ester response element (TRE), whereas the 56 kDa protein, reminiscent of the CRE-BP1 protein, could bind to both elements. A computer aided sequence analysis of cAMP-inducible gene promoters revealed the presence of an additional conserved element starting 4-6 nucleotides 3' to the octomer with the consensus C/GAGA/C. We have shown this element to be essential for maximal cAMP-responsiveness of the enhancer in transient expression assays of CRE-CAT plasmid constructs indicating that the functional interaction of CREB proteins with the cAMP-inducible enhancer involves an additional 8-10 base pairs immediately downstream from the CRE core element.

Animals↗