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1.
Each of the four convulsants used significantly influenced the release of [3H]-GABA from brain slices, without affecting [3H]GABA uptake. Bicuculline (10?5M, but not 10-fold higher or lower concentrations) potentiated the electrically evoked release of [3H]GABA but not the resting release, whereas metrazol (10?4 to 10?6 M) was found to inhibit resting but not electrically evoked release. Strychnine (10?4 and 10?5 M) and picro-toxin (10?4 M) inhibited electrically evoked release.  相似文献   

2.
The saturable and specific high-affinity uptake of [3H]serotonin ([3H]5HT) (5 × 10?8 M) was studied in slices from the hippocampus, parietal cortex, septum-preoptic area, and hypothalamus of male 2, 6, 12 and 24–32 month old C57BL/6N mice. Hippocampal [3H]5-HT uptake showed a significant biphasic relationship to age, with lower values in the 2 and 24–32 month old mice compared to 6 month old mice. No significant age effects were seen in the other regions, or in [3H]norepinephrine high-affinity uptake in the hippocampus.Studies of the high-affinity uptake mechanism in synaptosomal preparations were made in a subgroup of 12 and 24 month old mice. A micro-assay using a tissue-harvester measured high-affinity uptake on 8–30 μl of the P2 suspension (crude-synaptosomal preparation). The high-affinity uptake was linear for 4 min at 37°C and inhibited in both the adult and aged tissue by 10?5 M cold 5-HT (83 and 78% respectively), 10?5 M fluoxetine (85 and 82% respectively) and 10?3 M NaCN (57 and 57% respectively). Kinetic analysis of the [3H]5HT high-affinity uptake in the hippocampus (3 min, 37°C) revealed the same apparent Km for serotonin at both ages (6.7 x 10?8 M), but a 44% decrease in Vmax in the aged hippocampal synaptosomal high-affinity uptake compared to adults (120 vs 215 pmol of 5-HT/g-tissue/3 min).These results are discussed in relationship to the reported age effects on the intrinsic neurons of the hippocampus.  相似文献   

3.
A superfusion system was used to study the effects of neuroexcitatory amino acids upon spontaneous and depolarization-evoked release of exogenously taken up and newly synthesized [3H]dopamine by rat striatal slices. Neither l-glutamate nor other aminoacids such as l-aspartate and d-glutamate (5 × 10?5 M) modified the spontaneous release of exogenous [3H]dopamine from rat striatal slices. In contrast, these neuroexcitatory aminoacids did potentiate spontaneous release of striatal [3H]dopamine newly synthesized from [3H]tyrosine. A different pattern of effects emerged when depolarization-evoked release of dopamine was studied. Only l-glutamate (5 × 10?6-1 × 10?4 M) potentiated dopamine release under these experimental conditions in a rather specific and stereoselective manner. In addition, similar results were obtained regardless of whether depolarization-induced release of exogenous or newly synthesized [3H]dopamine was studied. The effect of l-glutamate on depolarization-induced release depended both upon the degree of neuronal depolarization and upon the presence of external Ca2+ in the superfusion medium and it was blocked by l-glutamate diethylester. Furthermore, this effect of l-glutamate seemed quite specific with regard to regional localization within the brain as it was only demonstrated in slices from striatum and not in slices from olfactory tubercle or hippocampus. It is suggested that during depolarization a Ca2+-dependent event occurs at the striatal membrane level which changes the sensitivity of the dopamine release process to neuroexcitatory aminoacids in such a way as to render it relatively more specific and stereoselective towards l-glutamate stimulation. The findings reported have led us to propose that l-glutamic acid could play a role as a neuromodulator of dopaminergic transmission in the rat corpus striatum.  相似文献   

4.
Concanavalin A-induced proliferation of rat T-lymphocytes is completely inhibited by 10?5 M pyrazofurin, a potent inhibitor of pyrimidine de novo synthesis, as judged by cell viability and [3H]thymidine incorporation. Proliferation is completely restored by 5 × 10?5 M uridine. Cytidine, deoxycytidine, deoxyuridine and thymidine 10 × 10?5 M each, fail to re-establish proliferation but produce an isotropic dilution of [3H]thymidine uptake in DNA. Bases (cytosine, uracil and thymine) neither restore proliferation nor induce isotopic dilution. The unexpected inability of cytidine to reverse de novo pyrimidine synthesis inhibition suggests a lack of cytidine deaminase activity in rat T-lymphocytes. This is confirmed by a direct sensitive radioisotopic assay (<0.001 nmol.min?1.10?6 cells).  相似文献   

5.
The elucidation of factors that support human mesenchymal stem cells (hMSCs) growth has remained unresolved partly because of the reliance of many researchers on ill‐defined, proprietary medium formulation. Thus, we investigated the effects of high glucose (D ‐glucose, 25 mM) on hMSCs proliferation. High glucose significantly increased [3H]‐thymidine incorporation and cell‐cycle regulatory protein expression levels compared with 5 mM D ‐glucose or 25 mM L ‐glucose. In addition, high glucose increased transforming growth factor‐β1 (TGF‐β1) mRNA and protein expression levels. High glucose‐induced cell‐cycle regulatory protein expression levels and [3H]‐thymidine incorporation, which were inhibited by TGF‐β1 siRNA transfection and TGF‐β1 neutralizing antibody treatment. High glucose‐induced phosphorylation of protein kinase C (PKC), p44/42 mitogen‐activated protein kinases (MAPKs), p38 MAPK, Akt, and mammalian target of rapamycin (mTOR) in a time‐dependent manner. Pretreatment of PKC inhibitors (staurosporine, 10?6 M; bisindolylmaleimide I, 10?6 M), LY 294002 (PI3 kinase inhibitor, 10?6 M), Akt inhibitor (10?5 M), PD 98059 (p44/42 MAPKs inhibitor, 10?5 M), SB 203580 (p38 MAPK inhibitor, 10?6 M), and rapamycin (mTOR inhibitor, 10?8 M) blocked the high glucose‐induced cellular proliferation and TGF‐β1 protein expression. In conclusion, high glucose stimulated hMSCs proliferation through TGF‐β1 expression via Ca2+/PKC/MAPKs as well as PI3K/Akt/mTOR signal pathways. J. Cell. Physiol. 224:59–70, 2010 © 2010 Wiley‐Liss, Inc.  相似文献   

6.
The release of [3H]GABA which is newly synthesized from [3H]l-glutamic acid (GLU) has been examined using striatal slices obtained from the rat brain. It was found that 8–10% of [3H]GLU transported was converted to [3H]GABA during the incubation of striatal slices in the presence of nipecotic acid (5 × 10?5 M). Nipecotic acid was added to the medium in order to prevent possible reuptake of [3H]GABA released during its synthesis, and it was found to have no significant effect on the formation of [3H]GABA from [3H]GLU as well as on the uptake of [3H]GLU. The application of high potassium (60 mM) stimulation exhibited a significant enhancement of the release of this newly synthesized [3H]GABA in a Ca2+ dependent manner. Kinetic analysis revealed that the evoked release of newly synthesized [3H]GABA was approximately two times greater than that of previously-loaded [3H]GABA, whereas no significant difference was observed in the spontaneous release. An immobilization stress in water failed to affect the release of newly synthesized [3H]GABA from striatal slices despite the occurrence of a significant enhancement of GABA formation in this structure.These results suggest that newly synthesized GABA may be preferentially released from its nerve terminals in response to the excitation of neurons at least in the striatum as compared with previously accumulated GABA.  相似文献   

7.
Incorporation of [3H]leucine and [3H]valine into proteins of freshwater bacteria was studied in two eutrophic lakes. Incorporation of both amino acids had a saturation level of about 50 nM external concentration. Only a fraction of the two amino acids taken up was used in protein synthesis. At 100 nM, the bacteria respired 91 and 78% of leucine and valine taken up, respectively. Respiration of 3H and 14C isotopes of leucine gave similar results. Most of the nonrespired leucine was recovered in bacterial proteins, while only up to one-half of the nonrespired valine occurred in proteins. In intracellular pools of the bacteria, [3H]leucine reached an isotope saturation of 88 to 100% at concentrations of >40 nM. For [3H]valine, an isotope equilibrium of about 90% was obtained at concentrations of >80 nM. Within an incubation period of typically 1 h, tritiated leucine and valine incorporated into proteins of the bacteria reached an isotope saturation of 2 to 6%. In a 99-h batch experiment, bacterial protein synthesis calculated from incorporation of leucine and valine corresponded to 31 and 51% (10 nM) and 89 and 97% (100 nM), respectively, of the chemically determined protein production. Measured conversion factors of 100 nM leucine and valine were 6.4 × 1016 and 6.6 × 1016 cells per mol, respectively, and fell within the expected theoretical values. The present study demonstrates that incorporation of both valine and leucine produces realistic measurements of protein synthesis in freshwater bacteria and that the incorporation can be used as a measure of bacterial production.  相似文献   

8.
[3H]norepinephrine was shown to bind to specific sites on isolated fat cells. A Scatchard plot of norepinephrine binding showed two apparent Ka of 1.9 · 106 and 1.2 · 105 LM?. 1.4 · 10?4 M Norepinephrine covalently-linked to agarose beads reduced [3H]norepinephrine binding by over 50%. Several structurally related drugs were compared as inhibitors of [3H]norepinephrine binding and as stimulators of lipolysis in preparations of similarly prepared cells. Dose-response curves for norepinephrine, epinephrine and isoproterenol showed the affinities for binding inhibition and for stimulation of lipolysis to be in the same range of 6 · 10?7-2 · 10?6 M. Dopamine and dopa were potent inhibitors of [3H]norepinephrine binding at 8.5 · 10?7 M and 2.0 · 10?6 M respectively, but did not stimulate lipolysis even at 10?4 M. Propranolol, a β-adrenergic antagonist, had no effect on [3H]norepinephrine binding at 10?4 M but completely inhibited catecholamine-stimulated lipolysis at 10?5 M. Phentolamine, an α-adrenergic antagonist, did not inhibit binding or catecholamine-stimulated lipolysis at 10?4 M. Ephedrine, metaraminol, phenylephrine and normetanephrine were also ineffective both as [3H]norepinephrine binding inhibitors and as stimulators of lipolysis. The results suggested the catechol ring of catecholamines is more important than the ethanolamine side chain as a requirement for binding, while both an intact catechol moiety and ethanolamine function appear necessary for physiological effect.  相似文献   

9.
We have observed that preincubation of 48 hour-fasted or alloxan diabetic rat liver slices, with no exogenous energy supply, for 3 hours resulted in an increased rate of incorporation of [1-14C] acetate into fatty acids and cholesterol during the following 2 hours. This preincubation effect was enhanced by the presence of glucose (25mM) in or prevented by the addition of dibutyryl cyclic adenosine 3′,5′ monophosphate (10?4M) to the preincubation medium. Preincubation of normal rat liver slices did not change their rate of incorporation of [1-14C] acetate into fatty acids or cholesterol. The rate of 14CO2 synthesized by normal, fasted or diabetic liver slices was little affected by preincubation. The preincubation effect, i.e. enhanced fatty acid synthesis was also observed in suspensions of hepatocytes from fasted and diabetic rats, preincubated for 2 hours, followed by a 1 hour incubation with either [1-14C] acetate or [3H] H2O as precursor. We conclude from these data that there is concurrent and coordinated short- and long-term regulation of fatty acid biosynthesis in fasted and diabetic rat livers. Further, we suggest that the release of inhibition by preincubation of these tissues provides a useful tool for studying the coordinated control  相似文献   

10.
In the presence of tracer concentrations of extracellular leucine (5 μM), treatment of rat splenic lymphocyte suspensions in vitro with 1 μM dexamethasone for 2.5–4 h caused a 30–35% inhibition of [3H]leucine incorporation into protein. As the extracellular leucine concentration was raised to 5 mM, this inhibition was progressively reduced to 0–12%. This phenomenon correlated with a marked dependence on extracellular leucine concentration of the dexamethasone-dependent enlargement of free intracellular leucine pools in splenic lymphocytes: a 123% increase in pool size with tracer extracellular leucine; a 10% increase with 5 mM leucine. Varying extracellular leucine had no effect on: (1) nuclear [3H]dexamethasone binding by the cells; (2) the concentration of dexamethasone needed for half-maximal inhibition of [3H]leucine incorporation; (3) the time course of onset and maximal expression of the hormonal inhibition of [3H]leucine incorporation; or (4) the magnitude of dexamethasone-dependent inhibition of [3H]uridine incorporation into RNA by these cells. There was no detectable effect of dexamethasone on uptake and retention of [3H]leucine by the cells, regardless of the extracellular leucine concentration. Treatment of splenic lymphocytes for 4 h in vitro with 1 μM dexamethasone caused a small shift of ribosomes from larger aggregate polysomes to smaller forms. Thus, glucocorticoid-induced inhibition of amino acid incorporation in splenic lymphocytes is a multicomponent response, of which an actual decrease in protein synthesis is only a small part. Enlargement of free intracellular amino acid pools, probably resulting from increased protein degradation, is the major contributing factor to the hormonal inhibition of amino acid incorporation.  相似文献   

11.
1. The incorporation of [14C]valine and [3H]threonine has been studied in mucosa of goldfish acclimatized to different temperatures. 2. [14C]Valine was incorporated more rapidly than [3H]threonine at acclimatization temperatures below 15°; this distinction disappeared at higher temperatures. 3. The rate of amino acid incorporation rose as the environmental temperature was increased. 4. The number of valine residues in mucosal protein fell and the number of methionine residues rose as the acclimatization temperature was raised. 5. The concentration of free methionine was closely related to the amount in protein in goldfish mucosa; both increased at higher acclimatization temperatures. 6. An attempt is made to show that these changes are integrated with possible homoeostatic mechanisms in the goldfish intestine.  相似文献   

12.
The effects of some local anesthetics on plasma protein secretion by rat liver slices have been studied and have been compared with those of colchicine. Rat liver slices were pulse-labelled with l-[14C]leucine for 9 min at 37°C, collected on filter paper, washed with non-radioactive leucine and reincubated in the presence or absence of the drug to be tested. The radioactive plasma proteins produced were obtained by immunoprecipitation from either the chase medium or from the washed slices. Chlorpomazine, (3 · 10?5 M), dibucaine (10?5 M), lidocaine (10?3 M) and procaine (5 · 10?5 M) inhibited both the synthesis and secretion of plasma protein but did not affect the uptake of l-leucine into the slices nor the incorporation of phosphate into intracellular nucleotide phosphates or into phopholipids. The inhibition of secretion elicited by these drugs is probably not due to the inhibition of protein synthesis since cycloheximide, when added to the chase medium at a concentration which completely inhibits protein synthesis, did not inhibit plasma protein secretion, while cycloheximide plus procaine did inhibit secretion and also caused a retention of non-secreted plasma proteins within the slices. Unlike colchicine, howover, procaine did not cause the retained plasma proteins to accumulate in Goli-derived secretory vesicles, but showed a more general effect causing a distribution among several cell fractions.  相似文献   

13.
Rat brain synaptosomes preincubated with [3H]5-HT. were further incubated and the release of [3H]5-HT from the preparation was studied. The spontaneous release consisted of an initial rapid phase followed by slower release. Incubation with 60 mM-KCl increased the release while 60 mw-NaCl did not affect it. The effect of KG was abolished when NaCl was omitted from the medium. The potassium-induced release was Ca2+ -dependent while that induced by tyramine (10?5-10?4M) and the spontaneous release did not depend on Ca2+. Vinblastine (10?5–2.5 X 10?4 M) caused an increase in the spontaneous release and an decrease in the potassium-induced release, while it completely inhibited the release by tyramine at 2.5 X 10?4 M. Colchicine (5 X 10?5–10?3M) and cytochalasin D (10?5, 10?4 M) failed to produce any change in the release. Cytochalasin B (10?5, 10?4M) increased the spontaneous release and decreased the potassium-induced release but it did not affect the release by tyramine. Colchicine (10?3 M). vinblastine (10?4 M) and cytochalasin B (10?4 M) did not affect significantly Na+.K+-. Mg2- and Ca2+ -ATPase activities. These results suggest that none of microtubules. microfilaments and contractile protein participates in the mechanism of [3H]5-HT release from synaptosomes, in vitro.  相似文献   

14.
Rat cerebral cortex slices were incubated in vitro with [3H]dopamine (DA) or [3H]noradrenaline (NA) (10?7M), superfused by fresh buffer and stimulated by an electric field. The stimulation-induced overflow of [3H]DA and [3H]NA was determined. In slices from untreated rats about 16 ng [3H]NA/g tissue was formed from [3H]DA, corresponding to about 5 per cent of the endogenous NA concentration. Stimulation markedly enhanced the overflow of [3H]NA. The [3H]NA newly formed from [3H]DA was overflowing to a greater extent than [3H]NA previously taken up from the incubation medium, indicating a preferential release of newly synthesized transmitter. The stimulation-induced overflow of [3H]DA and [3H]NA was increased in slices of rats pretreated with a tyrosine hydroxylase inhibitor (H44/68). It seems that depletion of the endogenous NA stores of central NA neurons by tyrosine hydroxylase inhibition makes the [3H]cate-cholamines more available for release. Pretreatment of the rats with the DA-β-hydroxylase inhibitors FLA63 or FLA69 considerably diminished the formation of [3H]NA from [3H]DA. Stimulation markedly enhanced the overflow of [3H]DA indicating that DA can act as a ‘false transmitter’ in central NA neurons after DA-β-hydroxylase inhibition.  相似文献   

15.
The rate of [3H]dopamine binding to crude synaptic membranes from canine caudate nucleus was considerably increased by 2 mM ATP, 5′-adenylylimidodiphosphate and GTP or by 1 mM 5′-guanylyl-imidodiphosphate, while strongly inhibited by 2 mM ADP and GDP. Half maximal concentrations of [3H]dopamine to bind to the membranes were 1.11 × 10?7M and 8.75 × 10?6M in the absence of 4 mM ATP, indicating a negative cooperativity of the dopamine receptor, and 9.25 × 10?7 M in its presence. Hill coefficient was increased from 0.70 to 1.04 by addition of 4 mM ATP. The optimal concentration of ATP for [3H]dopamine binding was in the range of 0.5 to 5 mM.  相似文献   

16.
The effects of different concentrations of exogenously supplied dThd on DNA replication were investigated in seedlings of Pisum sativum. Nascent DNA was labeled with either [3H]dThd or [3H]dAdo in the presence of 1·10?6, 1·10?5 or 1·10?4 M unlabeled dThd. The rate of DNA synthesis was determined by measuring the kinetics of radioactivity incorporation into trichloroacetic acid-precipitable material and the size of the nascent molecules was investigated using alkaline sucrose gradients. The results obtained showed that high concentrations of exogenously supplied dThd accelerated the joining of completed nascent replicons without affecting the rate of DNA synthesis. This observation strengthens the hypothesis that the dTTP pool size is one of the factors controlling the timing of nascent replicon maturation.  相似文献   

17.
Abstract: Primary cultures of chromaffin cells from bovine adrenal medulla were used to evaluate the ability of several opiates to reduce the release of catecholamines induced by stimulation of nicotinic receptors. Etorphine, β-endorphin, Met-enkephalin[Arg6,Phe7], and the synthetic peptide [d -Ala2,Me-Phe4,Met(O)s-ol]enkephalin inhibited the acetylcholine-induced release of catecholamines with an IC30 varying from 10?7 to 1 × 10?6M. The effect was stereospecific because levorphanol (IC30= 7.5 × 10?7M) was approximately two orders of magnitude more potent than dextrorphan. Morphine (μ-receptor agonist), [d -Ala2, d -Leu5]enkephalin (δ-receptor agonist), ethylketazocine (k -receptor agonist), and N-allylnormetazocine (σ-receptor agonist) were at least 100–1000 times less potent than etorphine. Diprenorphine (IC50= 5 × 10?7M) and naloxone (IC50= 10?6M) antagonized the effect of etorphine. High-affinity, saturable, and stereospecific binding sites for [3H]etorphine, [3H]dihydromorphine, [3H-d -Ala2,d -Leu5]enkephalin, [3H]ethylketazocine, and for [3H]N-allylnormetazocine, [3H]diprenorphine, and [3H]naloxone were detected in chromaffin cell membranes and in membranes obtained from adrenal medulla homogenates. However, the number of binding sites for [3H]etorphine and [3H]diprenorphine was 10–70 times higher than the number of sites measured with the other 3H ligands. The rank order of potency of these compounds for the displacement of [3H]etorphine binding correlates (r = 0.90) with the rank order of potency of the same compounds for the inhibition of acetylcholine-induced catecholamine release. These data suggest that a stereoselective opiate receptor (different from the classic μ-, δ-, k -, or σ-receptor) with high affinity for etorphine, diprenorphine, β-endorphin, and Met-enkephalin[Arg6,Phe7] modulates the function of the nicotinic receptor in adrenal chromaffin cells.  相似文献   

18.
The effects of phenylpyruvate and hyperphenylalaninemia on the incorporation of [6-3H]glucose into lipids, proteins and nucleic acids were examined in differentiating and adult rat brain. Foetal brain was most sensitive to inhibition by phenylpyruvate in vitro, with significant effects occurring at 2·5 mM for labelling of lipids and proteins and at 5 mM for labelling RNA and DNA. Older age groups were less affected, and cortical slices from adult brain were slightly or not at all affected by phenylpyruvate. The inhibition by phenylpyruvate of incorporation of [6-3H]glucose into nucleic acids, proteins, and lipids could be further distinguished by the reversibility of the effect on nucleic acid and protein synthesis at high levels of glucose and the irreversibility of the effect on lipid synthesis. Lipid synthesis was most sensitive to inhibition by phenylpyruvate at the stage of fatty acid synthesis, with lesser effect on the formation of glyceride glycerol. Exposure in utero of the foetal brain to maternal hyperphenylalaninemia resulted in reduction of 26–38 per cent in the subsequent incorporation in vitro of [6-3H]glucose into lipids, proteins, RNA and DNA of brain slices from foetal animals. Feeding hyperphenylalaninemic pregnant rats a high-glucose diet significantly protected the foetal brain from the neurotoxicity accompanying the hyperphenylalanemia.  相似文献   

19.
Abstract: In this study, the interaction between 3′,5′-cyclic adenosine monophosphate (cAMP) and 3′,5′-cyclic guanosine monophosphate (cGMP) in [3H]adenine-or [3H]-guanine-prelabelled adult guinea-pig cerebellar slices was investigated. Basal levels of [3H]cGMP were enhanced by forskolin, although no plateau was reached over the concentration range tested (0.1-100 μM). However, forskolin elicited a concentration-dependent, saturable potentiation of sodium nitroprusside (SNP)-stimulated [3H]cGMP accumulation (forskolin EC50 value of 0.98 β 0.23 μM; 10 μM forskolin produced a 1.8 β 0.3-fold potentiation of the SNP response at 2.5 min). The forskolin potentiation was observed at all concentrations of SNP tested (0.001-10 mM). forskolin also elicited a large stimulation of [3H]-cAMP in [3H]adenine-prelabelled guinea-pig cerebellar slices; however, 1,9-dideoxyforskolin failed to elicit either a [3H]cAMP response or a potentiation of the SNP-induced [3H]cGMP response at concentrations up to 100 μM. Pretreatment with oxyhaemoglobin (50 μM) inhibited the response to SNP (1 mM) and forskolin (10 μM), as well as the response evoked by the combination of SNP and forskolih. AG-Nitro-l -arginine (100 μM) inhibited the response to forskolin alone, but did not change the response to SNP or the potentiation induced by forskolin on SNP-induced [3H]cGMP levels. The protein kinase inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7; 100 μM), staurosporine (10 μM), polymyxin B (100 μM), and Ro 31-8220 (10 μM) had no effect on the [3H]cGMP response to either SNP or the combination of SNP plus forskolin. N6,2′-Dibutyryl cAMP, at concentrations up to 10 mM, was also without effect on [3H]cGMP levels induced by SNP. 3-lso-butyl-1-methylxanthine reproduced the effect of forskolin on SNP-induced [3H]cGMP levels, but a less-than-additive effect was observed when the response to SNP was studied in the presence of forskolin and 3-isobutyl-1-methylxanthine. Taken together, these results infer that crosstalk between cyclic nucleotides takes place in guinea-pig cerebellar slices, and that cAMP may regulate cGMP-mediated responses in this tissue.  相似文献   

20.
Primary keratinocyte cultures free of dermal fibroblasts were used to investigate the effect of varying cyclic AMP (cAMP) concentrations on epidermal cell function. Addition of 10?3, 10?4 or 10?5 M dibutyryl cAMP to plated cells (day 1) results by day 5 in a dose dependent increase of [3H]TdR incorporation into DNA as determined by increases in both the labeling index and incorporation of 3H label into an isolated DNA fraction. 8-Bromo cAMP, another cAMP analogue, likewise induced keratinocyte proliferation. The proliferative response was dose and time dependent, and 5- to 6-fold increases in 3H label incorporated into DNA were seen at day 6, 8 and up until day 15 of culture. Moreover, elevation of cellular cAMP by addition of cholera toxin, an irreversible stimulator of adenylate cyclase, also demonstrated a time dependent stimulation of [3H]TdR uptake into DNA and increased the labeling index. Specific histochemical staining for keratinaceous protein (Kreyberg technique) demonstrated that elevated cAMP levels also enhance the production of specialized (differentiated) epidermal cells. Determination of the level of cAMP and cyclic GMP (cGMP) by RIA of partially purified fractions of the cultures revealed that addition of 8-bromo cAMP or cholera toxin to the cultures increased the levels of cAMP but not of cGMP. Addition of 8-bromo cGMP to the keratinocytes on day 1 at concentrations of 10?6, 10?7 or 10?8 M had no effect on culture proliferation on days 4, 6 and 8, although qualitative changes in the electron microscopic pattern of the culture stratification and specialization were observed. The results indicate (1) both large and moderate increases in cellular cAMP levels induce keratinocyte culture proliferation and specialization in the absence of fibroblasts or dermal influences, (2) the quantitative enhancement of keratinocyte growth and specialization occurs without apparent participation of cGMP, (3) cGMP may be a qualitative effector of epidermal cell differentiation.  相似文献   

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