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Biomedical subjects

J F Whitfield

Publications and source records attributed to J F Whitfield.

At least 109 records · Page 6Linked to original sources
PubMed1990

Long-term culture of neonatal rat pancreatic endocrine cells as model for insulin-secretion and ion-channel studies.

So far, only freshly isolated cells or short-term cultures have been used to study ion-channel activity in pancreatic nontumor beta-cells. We report a procedure for the long-term cultivation of pancreatic endocrine cells to study the relationship between ion channels and insulin secretion. Using thimerosal to suppress fibroblastoid cell proliferation and a preliminary 2-day cell exposure to alternating normal (5.6 mM) and high (16.7 mM) glucose levels, we observed a significant secretory responsiveness of the cells to a glucose challenge for at least 4 wk in culture. Cells also responded to glucose or other secretagogues, such as quinine and the sulfonylurea glyburide, with membrane voltage oscillations. In the cell-attached configuration of the patch-clamp technique, a 65-pS-conductance K+ channel was observed, which was inhibited by glucose, quinine, and glyburide. In the inside-out configuration, the activity of this channel was suppressed by ATP applied to the cytoplasmic side of the membrane. A K+ channel with a conductance of 200 pS was also observed, which was activated by intracellular Ca2+. A 13-pS-conductance glucose-insensitive K+ channel was present in both cell-attached and inside-out patch recordings. Even after 3 wk, the characteristics of these currents and channels were comparable to those reported by other investigators with freshly dissociated or short-term-cultured beta-cells from neonatal and adult rats and adult mice. Therefore, the neonatal rat endocrine cell culture characterized herein provides an improved model for long-term investigations combining secretion and electrophysiological studies.

Animals↗
PubMed1989

A novel method for measuring protein kinase C activity in a native membrane-associated state.

Physiological activation of protein kinase C (PKC) is believed to occur by redistributing soluble enzyme to the phospholipid environment of membranes. Currently available in vitro methods of measuring PKC activation all involve prior extraction of membrane-associated enzyme and its reconstitution in an artificial phospholipid environment or modification (such as partial trypsinization) of the enzyme itself. Here we report a novel method which, for the first time, allows measurement of active PKC still in its native, membrane-associated state using a specific, physiological substrate. Thus, with this new method PKC activity can be measured while still in an environment that approximates the in vivo situation.

Animals↗
PubMed1989

Control of DNA polymerase-alpha activity in regenerating rat liver by calcium and 1 alpha,25(OH)2D3.

The late G1 surge of DNA polymerase-alpha activity and the initiation of DNA replication in the hepatocytes of partial hepatectomy-induced regenerating liver were severely reduced when the mitogenic partial hepatectomy was carried out in the hypocalcemic and 1,25(OH)2D3 (1 alpha,25-dihydroxycholecalciferol)-deficient environment of parathyroidectomized (PTX) or thyroparathyroidectomized (TPTX) rats. These inhibitions were prevented in TPTX rats by a postpartial hepatectomy injection of 1,25(OH)2D3, which also restored blood calcium to normocalcemic levels. Inhibition of active DNA polymerase-alpha accumulation and initiation of DNA synthesis in TPTX rats were also completely prevented by prefeeding the rats a low phosphorus diet, which stopped the lowering of the blood levels of calcium and 1,25(OH)2D3 following parathyroid removal. These studies indicate that the rise of DNA polymerase-alpha activity and the initiation of DNA replication in regenerating liver are controlled by cellular processes that rely on normal blood levels of calcium and 1,25(OH)2D3. Because DNA polymerase-alpha is the third DNA replication enzyme (the others are ribonucleotide reductase and thymidylate synthase) that has been shown to depend on parathyroid hormone and/or the circulating levels of calcium and 1,25(OH)2D3 that it controls, the authors concluded that the processes dependent on calcium and 1,25(OH)2D3 are parts of a mechanism that coordinately activates the DNA-replicating enzymes. The possibility that cyclic adenosine monophosphate (cAMP)-dependent protein kinases are involved in this replication mechanism is considered.

Animals↗
PubMed1989

Protein kinase C and a viral K-RAS protein cooperatively enhance the response of adenylate cyclase to stimulators.

The protein kinase C stimulator TPA (12-O-tetradecanoyl phorbol-13-acetate) enhanced the responsiveness of adenylate cyclase to IPR (isoproterenol) and PGE1 (prostaglandin E1) in quiescent tsKSV-NRK cells at the nonpermissive 41 degrees C. Reactivating the thermolabile mitogenic/oncogenic K-ras protein in tsKSV-NRK cells by dropping the temperature to 36 degrees C also enhanced the responsiveness of adenylate cyclase to IPR and PGE1. The enhancement was transient and peaked at 6 hours after the temperature shift. This enhanced responsiveness was specifically due to the reactivated viral K-ras protein rather than the temperature shift because the same temperature shift did not affect adenylate cyclase responsiveness in uninfected NRK cells, nor was it a result of the mitogenic stimulus since reacting the mitogenic pp60v-src protein in tsASV-NRK cells did not affect adenylate cyclase responsiveness. The increased responsiveness of adenylate cyclase at 6 hours after the temperature shift was not a result of elevated membrane-associated PKC activity. However, the reactivated viral K-ras protein strongly increased the stimulability of membrane-associated PKC by TPA and it further increased TPA's ability to enhance the responsiveness of adenylate cyclase to IPR and PGE1. Thus, a viral K-ras protein and membrane-associated protein kinase C can cooperate to increase the responsiveness of adenylate cyclase to agonists.

Adenylyl Cyclases↗
PubMed1989

Ribonucleotide reductase--new twists in an old tale.

Although they are proliferatively quiescent, the cells in the intact adult rat liver express the gene coding for the M1 subunit of ribonucleotide reductase. But since they do not need deoxyribonucleotides, they promptly inactivate the 88 to 90 kDa M1 products and degrade them into 40 kDa fragments. Partial hepatectomy signals the remaining cells to start proliferating. Two hours before the onset of DNA replication, around 16 to 18 hr after partial hepatectomy, the cells start accumulating a large pool of functional ribonucleotide reductase M2 subunits. Near the end of the G1 build-up the cells step up M1 gene expression, stop inactivating, and reduce the degradation of the M1 products. The accumulating functional 88 to 90 kDa M1 subunits, each with more than one catalytic site, couple with functional M2 subunits to produce active ribonucleotide reductase holoenzyme which accumulates in the outer nuclear membrane from which they supply deoxyribonucleotide precursors to intranuclear replication enzymes. At the end of the S phase, the cell reduces M1 gene expression and resumes degrading 88 to 90 kDa M1 subunits. At least some of the 40 kDa M1 fragments are still active and can form partially active "holoenzymes" when mixed with a standard preparation of functional M2 subunits. The M1 control mechanism appears not to operate in hepatoma cells and Ehrlich ascites tumor cells, both of which maintain a pool of undegraded 88 to 90 kDa M1 components.

Animals↗
PubMed1989

Regulation of the synthesis of DNA polymerase-alpha in regenerating liver by calcium and 1,25-dihydroxyvitamin D3.

Digestion of nuclei from normal or partially hepatectomised rat livers with endogenous nucleases liberated a pool of cryptic DNA polymerase-alpha activity which had previously gone unrecognised. Most of this activity is released into the supernatant as free enzyme molecules (11S), but a small fraction of it is released as a complex of 16S (probably with DNA primase). About 40% of the enzyme remains in the pellet, which contains undigested chromatin and components of the residual nuclear matrix and nucleoskeletal structures. Virtually all of this remaining activity is extracted by 2.0 M salt. The activity of DNA polymerase-alpha increases equally in all nuclear fractions during the period of DNA replication in regenerating rat liver. Lowering of the serum calcium level by thyroparathyroidectomy does not affect basal DNA polymerase-alpha activity, but prevents induction of the enzyme during the later stages of prereplicative development. However, an injection of 1 alpha,25-dihydroxy vitamin D3 into the rat during the first 6 h after parital hepatectomy restores the ability of the hepatocytes to induce DNA polymerase-alpha activity and initiate DNA synthesis. These results are discussed in terms of the role of calcium ions in the regulation of the critical stage of prereplicative development which commits the cells to DNA replication.

Animals↗
PubMed1989

T- and L-calcium channels in steroid-producing chicken granulosa cells in primary culture.

Steroidogenesis and other functions in granulosa cells are calcium dependent. Using the patch-clamp technique to record single ion channel activity, we have identified for the first time two kinds of calcium channels through which the divalent ion may enter chicken granulosa cells. The cells were maintained in primary culture whose basal and hormone-stimulated progesterone production was evaluated at different times in culture and at different temperatures. A small channel, with a conductance of 4-5 picosiemens (pS), had short openings and inactivated rapidly. A large channel had a conductance of 20-30 pS, a high activation threshold, and slow inactivation kinetics. The dihydropyridine compound Bay K-8644, a L-calcium channel agonist, significantly increased the activity of the large channel, and nifedipine, a dihydropyridine calcium channel blocker, inhibited it completely. Both types of channels were seen in functionally competent signal-responsive granulosa cells cultured for up to 48 h. Whether these channels are involved in steroidogenesis, protein production, and/or secretion remains to be established.

Animals↗
PubMed1988

Ionic currents in avian granulosa cells.

Transmembrane ionic currents have been recorded in single granulosa cells from the laying hen using the whole-cell patch-clamp technique. Under voltage-clamp conditions, depolarizing voltage steps evoked currents composed of a fast inactivating inward component and a delayed outward component. The former was activated at voltages more positive than -50 mV and was fully inactivated within 500 ms. It was blocked by D600 (methoxyverapamil) and by cobalt, suggesting that it is a calcium current. The latter displayed inward rectification and did not inactivate during long duration pulses. It was blocked by tetraethylammonium indicating that it is a potassium current. This is the first evidence of the existence of potassium and calcium transmembrane currents in granulosa cells.

Animals↗
PubMed1988

Evidence for two distinct potassium channels in avian granulosa cells.

Single-channel potassium currents were recorded in avian granulosa cells using the patch-clamp technique. Two types of channel were observed. The smaller of the two channels, gK1, had a conductance of 15 to 30 picosiemens (pS) and was voltage- and calcium-independent. Its null-current potential was -50 mV in the cell-attached recording mode. The other channel, gK2, was infrequently observed in the cell-attached configuration. Its conductance was between 160 and 195 pS. It could be activated by calcium on the cytoplasmic side of the membrane patch in the inside-out configuration. It was also voltage-dependent. These results suggest that fast transmembrane potassium movements may be involved in the membrane voltage regulation of granulosa cells, which in turn may play an important role in the modulation of steroidogenesis and other metabolic activities.

Animals↗
PubMed1988

Protein kinase C increases the activity of the polyoma virus middle T antigen-associated phosphatidylinositol kinase.

Exposure of polyoma virus-transformed fibroblasts to the protein kinase C-stimulating phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) is known to increase the transforming potential of the virus's middle T antigen. Here it is shown that this TPA treatment also stimulates an 85 kDa phosphatidylinositol kinase associated with the middle T antigen. Since activation of this kinase is known to be necessary, although not by itself sufficient for the transformation of cells by polyoma virus, bursts of protein kinase C activity, triggered by TPA or various cellular receptors, might enhance the oncogenicity of polyoma virus by stimulating this middle T antigen-associated phosphatidylinositol kinase.

Animals↗
PubMed1988

The regulatory and catalytic subunits of cAMP-dependent protein kinases are associated with transcriptionally active chromatin during changes in gene expression.

Changes in the association of the catalytic subunit and the regulatory subunits of isozymes I and II of cAMP-dependent protein kinases (RI and RII, respectively) with the transcriptionally active chromatin fraction from rat liver were examined after a glucagon/theophylline injection and also after partial hepatectomy. Chromatin was partitioned into transcriptionally active and bulk, transcriptionally inactive fractions by digestion with micrococcal nuclease under appropriate conditions. In both experimental models, an increased content of catalytic and both RI and RII subunits was observed in chromatin fractions that were enriched in transcriptionally active DNA, particularly in the fraction associated with the residual nuclear matrix-lamina. The changes in the association of the subunits with these fractions paralleled the increases in intracellular cAMP levels and occurred in a time frame compatible with the changes in gene expression. The catalytic subunits could be removed from the nuclear matrix-lamina fraction by salt, whereas the two regulatory subunits remained tightly bound. The data support the concept of a direct role of the regulatory subunits of cAMP-dependent protein kinases in the induction of gene expression. However, we were unable to confirm that RII possessed an intrinsic topoisomerase activity.

Animals↗
PubMed1987

The effects of corticotrophin (ACTH1-24), cyclic AMP and TPA (12-O-tetradecanoyl phorbol-13-acetate) on DNA replication and proliferation of primary rabbit adrenocortical cells in a synthetic medium.

ACTH1-24 stimulated the parenchymal cells in cultures of rat adrenal cortex in serum-free synthetic HiWoBa 2000 medium to replicate DNA, enter mitosis and divide. But ACTH's principal mediator, cyclic AMP, was not a complete mitogen: the adenylate cyclase-stimulating cholera toxin and dibutyryl cyclic AMP stimulated parenchymal cells to replicate DNA but not to enter mitosis. Thus, there must have been an additional mediator of the response to ACTH1-24 that enabled the parenchymal cells to enter mitosis. This additional mediator might have been protein kinase C because a protein kinase C activator and cyclic AMP elevator, TPA, stimulated the adrenocortical parenchymal cells to replicate DNA, enter mitosis and divide.

Adrenal Cortex↗
PubMed1987

Evidence that the viral Ki-ras protein, but not the pp60v-src protein of ASV, stimulates proliferation through the PDGF receptor.

Protamine sulfate (PS), a specific blocker of PDGF action, inhibited the proliferative response of tsKSV-NRK cells to a reactivated, temperature-sensitive viral Ki-RAS protein, but it did not affect the proliferative response of tsASV-NRK cells to a reactivated pp60v-src protein kinase. The inhibition by PS of the proliferation response of tsKSV-NRK cells to reactivated Ki-RAS protein was overcome by serum growth factors, notably EGF, and concentrated serum-free conditioned medium from cultured NRK cells infected with wild-type KSV, but not by a combination of PDGF and insulin. These observations suggest that the viral Ki-RAS protein, but not pp60v-src, stimulates proliferation exclusively by inducing the host cells to produce PDGF or PDGF-like mitogenic factors.

Animals↗
PubMed1987

A viral K-RAS protein increases the stimulability of adenylate cyclase by cholera toxin in NRK cells.

Incubation at 41 degrees C stops the proliferation of tsK-NRK rat kidney cells in serum-deficient medium by inactivating the mitogenic/oncogenic thermolabile viral K-RAS protein that is produced in these cells. Dropping the temperature to 36 degrees C reactivates the viral K-RAS protein which stimulates the serum-starved quiescent cells to resume proliferating without added serum factors. Here it is shown that while the reactivated viral protein does not by itself significantly stimulate adenylate cyclase, it greatly increases the stimulability of adenylate cyclase by cholera toxin. The data suggest that the viral K-RAS protein directly or indirectly affects adenylate cyclase by inactivating the Gi inhibitory component of the membrane associated enzyme.

1-Methyl-3-isobutylxanthine↗
PubMed1987

A complete complementary DNA for the oncodevelopmental calcium-binding protein, oncomodulin.

RNA from a rat liver tumor (Morris hepatoma 5123tc) was used to construct cDNAs together comprising the complete coding sequence of rat oncomodulin mRNA. Information obtained from these cDNAs as well as from primer extension analysis gave a deduced length for the complete oncomodulin mRNA of approximately 680 nucleotides (excluding the poly(A) tail) including a 5'-untranslated region of 97 +/- 2 nucleotides, a 324-nucleotide-coding sequence and a 259-nucleotide 3'-noncoding region. Comparison of the oncomodulin cDNA sequence with those coding for other members of the calcium-binding protein family shows little homology with the exception of a recently reported parvalbumin cDNA where the oncomodulin and parvalbumin nucleotide sequences are 59% identical in the protein-coding region. RNA blot analysis of poly(A+) RNA from normal adult rat liver gave no evidence of oncomodulin expression in this tissue. A single RNA species was detected, however, in RNA extracts from the hepatoma and from rat and human placentas. A probe prepared from one of the rat oncomodulin cDNAs hybridized with a single DNA species in restriction digests of hepatoma and normal DNA from rat and sequences in DNA of humans and other mammals. A 38-nucleotide sequence spanning the 5'-untranslated region and the first seven codons of the oncomodulin cDNA, was far less homologous than was the same region of a parvalbumin cDNA, to a chicken calmodulin cDNA sequence coding for the first calcium-binding domain. The oncomodulin gene appears to have diverged more from that of calmodulin than has the parvalbumin gene.

Amino Acid Sequence↗
PubMed1987

AEV-transformed chicken erythroid cells secrete autocrine factors which promote soft agar growth and block erythroleukemia cell differentiation.

LSCC HD3 chicken erythroleukemia cells, transformed by a temperature-sensitive avian erythroblastosis virus (tsAEV), secreted into the medium several transforming factors which after separation by Bio-Cel P-60 chromatography, stimulated quiescent (G0) chicken embryo fibroblasts and NIH 3T3 mouse cells to replicate DNA in serum-free medium and to form colonies in soft agar. Most of these factors were also mitogenic for the LSCC HD3 cells themselves when they were rendered phenotypically untransformed by incubation at 42 degrees C to inactivate the ts AEV. The transformed LSCC HD3 cells also secreted a non-mitogenic 40 kDa factor which blocked the erythropoietin-induced differentiation of untransformed LSCC HD3 (at 42 degrees C) and the DMSO-induced differentiation of Friend murine erythroleukemia cells into hemoglobin-synthetizing erythroid cells.

Alpharetrovirus↗
PubMed1987

Viral p21 Ki-RAS protein: a potent intracellular mitogen that stimulates adenylate cyclase activity in early G1 phase of cultured rat cells.

Rat kidney (NRK) cells infected with a temperature-sensitive mutant of the Kirsten sarcoma virus were arrested in the G0/G1 phase of their cell cycle by incubation in serum-deficient medium at a p21-inactivating temperature of 41 degrees C. These quiescent ts K-NRK cells were then stimulated to transit G1 and initiate DNA replication by lowering the temperature to 36 degrees C, which rapidly reactivated p21. Reactivating the viral Ki-RAS protein by temperature shift led to an increase in adenylate cyclase activity in early G1 phase. The Ki-RAS protein increased the sensitivity of adenylate cyclase to guanyl nucleotides by a mechanism that seemed to involve inactivation of the enzyme's inhibitory G1 regulatory protein.

Adenylyl Cyclases↗