Metabolism In Primary Cultures Research Articles

Effect of protein kinase C modulators on 14,15-epoxyeicosatrienoic acid incorporation into astroglial phospholipids.

Our previous studies have shown that 14,15-epoxyeicosatrienoic acid (14,15-EET) is a major product of arachidonic acid metabolism in astrocytes. The purpose of this study was to investigate cellular regulation of 14,15-EET incorporation, distribution, and metabolism in primary cultures of rat brain cortical astrocytes. Incorporation of 14,15-EET into astrocytes was lower (93,390 +/- 11,121 dpm/5 x 10(6) cells) than incorporation of 8,9-EET (226,500 +/- 5,567 dpm/5 x 10(6) cells) and arachidonic acid (321,600 +/- 1,200 dpm/5 x 10(6) cells). 14,15-EET was distributed in the order neutral lipids and free fatty acids (solvent front) >> phosphatidylcholine (PC) > phosphatidylinositol (PI) > phosphatidylethanolamine. In contrast, 8,9-EET and arachidonic acid were exclusively incorporated into PC. During incubation, astroglial epoxide hydrolase selectively metabolized 14,15-EET, but not 8,9-EET, to its vic-diol. Although 4-phenylchalcone oxide, a potent inhibitor of epoxide hydrolase, completely inhibited 14,15-EET metabolism, a large amount of cell-incorporated radioactivity remained as free 14,15-EET. Long-term exposure of astrocytes to 4 beta-phorbol 12-myristate 13-acetate (4 beta-PMA) resulted in a time-dependent incorporation of 14,15-EET into PI but not in control cells exposed to 4 alpha-phorbol 12,13-didecanoate. PKC down-regulation completely inhibited epoxide hydrolase metabolism of 14,15-EET. Following recovery of down-regulated PKC, 1 week after treatment with 4 beta-PMA, astrocytes regained their normal pattern of low incorporation of 14,15-EET. Protein kinase C (PKC) inhibition by staurosporine enhanced 14,15-EET incorporation without affecting its metabolism to 14,15-dihydroxyeicosatrienoic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin and Thyroid Hormones Stimulate Matrix Metabolism in Primary Cultures of Articular Chondrocytes from Young Rabbits Independently and in Combination

These studies examined the effects of heat-inactivated horse serum, insulin, triiodothyronine (T3), and thyroxine (T4), individually and in combination, on collagen and proteoglycan synthesis by primary cell cultures of articular chondrocytes from immature male rabbits. Insulin concentrations of 25 to 100 ng/ml (4.4 to 17.4 x 10(-9) M) increasingly stimulated collagen and proteoglycan synthesis in the absence of serum. The effects of 25 ng/ml (4.4 x 10(-9) M) insulin or 15% heat-inactivated horse serum on collagen synthesis were similar. Triiodothyronine (10(-10) to 10(-6) M) and T4 (10(-8) to 10(-4) M) also stimulated collagen synthesis in the absence of serum, with peak effects at 10(-8) and 10(-6) M, respectively. Biphasic stimulation of proteoglycan synthesis was obtained with 10(-11) to 10(-7) MT3 (maximum at 10(-8) M) and 10(-8) to 10(-5) M T4 (maximum at 10(-7) M). In these experiments, triiodothyronine was 10 to 100 times more potent than T4 in stimulating cartilage matrix production. The cells retained their chondrocytic phenotype under hormonal stimulation, secreting almost exclusively Type II collagen and large, chondroitin sulfate-rich proteoglycans. The addition of insulin to maximally-stimulating concentrations of either T3 or T4 in serum-free medium further stimulated matrix synthesis, suggesting that these hormones modulate chondrocyte metabolism via multiple biosynthetic/receptor pathways.

Synthesis and vectorial export of cGMP in airway epithelium: expression of soluble and CNP-specific guanylate cyclases.

Guanosine 5'-cyclic monophosphate (cGMP) is an important modulator of fluid balance in many epithelia. We examined its metabolism in primary cultures of human airway epithelia. Sodium nitroprusside increased cGMP levels 30-fold, suggesting that the respiratory epithelium expresses a soluble guanylate cyclase; however, endogenous nitric oxide production was not detected. cGMP levels could also be increased by C-type natriuretic peptide (CNP), but not by atrial natriuretic peptide, brain natriuretic peptide, or Escherichia coli heat-stable enterotoxin, indicating expression of a CNP-specific membrane-bound guanylate cyclase. The one-half effective concentration for CNP was 40 nM and the maximal velocity was 56.7 pmol cGMP.mg protein-1.h-1. After CNP stimulation, approximately 60% of the total synthesized cGMP was preferentially exported from the polarized epithelial cells across the basolateral membrane by a probenecid-sensitive process. Isoproterenol-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) export revealed a similar export pattern and probenecid sensitivity, although a lower efficiency of export (27% of total cAMP was exported). Consistent with previous reports, export of neither cyclic nucleotide was saturable at the concentrations tested. We conclude that the respiratory epithelium expresses a soluble guanylate cyclase, a CNP-specific receptor, and a novel vectorial cyclic nucleotide export mechanism.

Excitatory amino acid-induced phosphoinositide hydrolysis in Müller glia.

The presence of excitatory amino acid (EAA) receptors coupled to phosphoinositide metabolism in primary cultures of Müller (glial) cells from the chick retina was established. The order of potency of analogues for stimulating [3H]inositol phosphate (IP) accumulation was quisqualate (QA) > L-glutamate (L-Glu) = kainate (KA) > N-methyl-D-aspartate (NMDA) > L-aspartate (L-Asp) with EC50 in the range of 1-100 microM. 1-Aminocyclopentane-1,3-dicarboxylate (trans-ACPD), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), 2-amino-3-phosphonopropionate (AP3), and 2-amino-4-phosphonobutyrate (AP4) showed no effect either on basal concentration or on stimulated accumulation of [3H]IPs. The effect of EAA was potently inhibited by the ionotropic NMDA receptor antagonists 2-amino-5-phosphonopentanoate (AP5), 3-[(RS)-2-carboxy-piperazin-4-yl)]-propyl-1-phosphonate (CPP), and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5-10- imine (MK-801); L-Glu antagonists at non-NMDA receptors, the quinoxalines NBQX and DNQX, inhibited weakly the response to L-Glu, KA, and NMDA, and more potently that to QA. The translocation of protein kinase C was also stimulated by EAA with the same pharmacological profile, and was partially inhibited by kynurenate (KYN). L-Glu and KA induced 45Ca2+ influx, which was decreased by KYN and CNQX. EAA-induced [3H]IPs accumulation was decreased by verapamil but not by nifedipine, and slightly diminished by dantrolene. Results demonstrate that EAA-induced phosphoinositide hydrolysis in Müller cells shows pharmacological differences with that in astrocytes and neuronal cells and could be triggered by a different mechanism.

The role of short chain fatty acid substrates in aerobic and glycolytic metabolism in primary cultures of renal proximal tubule cells

This study examined the role of odd and even short-chain fatty acid substrates on aerobic and glycolytic metabolism in well-aerated primary cultures of rabbit renal proximal tubule cells (RPTC). Increasing oxygen delivery to primary cultures of RPTC by shaking the dishes (SHAKE) reduced total lactate levels and lactate dehydrogenase (LDH) activity and reduced net glucose consumption compared to RPTC cultured under standard conditions (STILL). The addition of butyrate, valerate, heptanoate, or octanoate to SHAKE RPTC produced variable effects on glycolytic metabolism. Although butyrate and heptanoate further reduced total lactate levels and net glucose consumption during short-term culture (< 24 h), no fatty acid tested further reduced total lactate levels, net glucose consumption, or LDH activity during long-term culture (7 days). During the first 12 h of culture, maintenance of aerobic metabolism in SHAKE RPTC was dependent on medium supplementation with fatty acid substrates (2 mM). However, by 24 h, SHAKE RPTC did not require fatty acid substrates to maintain levels of aerobic metabolism equivalent to freshly isolated proximal tubules and greater than STILL RPTC. This suggests that SHAKE RPTC undergo adaptive changes between 12 and 24 h of culture, which give RPTC the ability to utilize other substrates for mitochondrial oxidation, therefore allowing greater expression of mitochondrial oxidative potential in SHAKE RPTC than in STILL RPTC.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on protein kinase C and inositol phosphate metabolism in primary cultures of rat hepatocytes.

Adult rat hepatocytes, after maintenance for 24 h in serum-free culture, were treated with the tumor promoters, 12-O-tetradecanoylphorbol-13-acetate (TPA) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Short-term treatment (15 min) with TPA, 1 microM, increased protein kinase C (PKC) activity in the particulate fraction of hepatocytes and, concomitantly, decreased the vasopressin (100 nM)-stimulated synthesis of inositol phosphates. The latter effect of TPA could be prevented by prior addition of the PKC inhibitor, H7 (100 microM). After short-term treatment (15 min) with TCDD, 1 pM, no effects on PKC or inositol phosphate metabolism were observed. However, after prolonged exposure to TCDD (3-48 h), the particulate PKC was significantly activated (1.5-fold). In contrast to the effect of TPA (24 h), no down-regulation was found. Moreover, long-term treatment with TCDD significantly enhanced vasopressin-stimulated inositol 1,3,4,5-tetrakisphosphate synthesis, while TPA treatment (24 h) stimulated the synthesis of inositol trisphosphates and inositol 1,3,4,5-tetrakisphosphate. The results suggest that the tumor promoters, TPA and TCDD, act differently on the signal transduction pathways in hepatocytes. Thus, the effects of TCDD on PKC and inositol phosphate metabolism might be mediated by a yet unknown mechanism rather than by direct activation of PKC as seen with TPA.

Isoenzymes of carbohydrate metabolism in primary cultures of hepatocytes from thioacetamide-induced rat liver necrosis: responses to growth factors.

Hepatocytes isolated from the liver of rats after a necrotizing dose of thioacetamide (6.6 mmol/kg) were used to study the postnecrotic process of liver regeneration. Flow cytometry analysis revealed populations of dedifferentiated hepatocytes exhibiting physical properties (size and fluorescence emission at 530 nm) similar to those found in fetal (22 days old) liver cells. The percentage of these cells increased progressively from 24 to 48 and 72 hr after thioacetamide administration. In primary cultures of hepatocytes the effects of phorbol 12-myristate 13-acetate, bombesin and insulin were investigated on the 6-phosphofructo 2-kinase/fructose 2,6 bisphosphate system. Bombesin and insulin stimulated 6-phosphofructo 2-kinase activity and fructose 2,6-bisphosphate content both in control and in thioacetamide-treated hepatocytes. However, phorbol 12-myristate 13-acetate stimulated 6-phosphofructo 2-kinase activity and increased fructose 2,6-bisphosphate concentration in thioacetamide-treated liver cells, whereas no similar response was found in hepatocytes from control rats. The response of postnecrotic thioacetamide-treated hepatocytes to phorbol 12-myristate 13-acetate was similar to that obtained from 22-day-old fetal liver cells, which reveals that different methods might control fructose 2,6-bisphosphate content and therefore the mechanisms of glycolysis and gluconeogenesis at this regulatory step. The lack of response to glucagon of glycogen phosphorylase a and 6-phosphofructo 2-kinase from thioacetamide-treated hepatocytes may indicate that the expression of specific enzymes of carbohydrate metabolism undergoes transitions to less-differentiated isoenzymatic forms. Moreover, the isoenzyme pattern of hexokinases elicits a complete disturbance in glucokinase and hexokinases activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of parathyroid hormone on arachidonic acid metabolism in mouse osteoblasts: permissive action of dexamethasone.

We examined the regulation of arachidonic acid (AA) metabolism in primary cultures of mouse osteoblasts under steady state and after acute stimulation by PTH. Both dexamethasone-treated and untreated cells were evaluated, as glucocorticoids are known to modulate some actions of PTH in osteoblasts and to affect arachidonic acid metabolism in cells in general. Cells were labeled with [3H]AA for 3 h, followed by a 20-h equilibrium period, then exposed to 10(-8) M PTH for different time periods ranging from 2-60 min. The results showed that although osteoblasts maintained in vitro in the absence of dexamethasone were responsive to PTH, as measured by cAMP production (50-fold increase), PTH had no effect on the distribution of AA in phospholipids and did not induce the release of free AA from phospholipid pools. After 7-day treatment of the cells with 4 x 10(-7) M dexamethasone, 15-min PTH stimulation resulted in a significant increase in free AA within the cells (mean +/- SE, +142 +/- 11%; n = 9) and a short-lived change in the distribution of AA within cellular phospholipids (phosphatidylethanolamine, -63 +/- 3%; phosphatidylinositol, +168 +/- 7%; phosphatidylserine, +296 +/- 50%; sphingomyelin, +220 +/- 20%; lysophosphatidylcholine, +634 +/- 31%; mean +/- SE; n = 3), despite the fact that no changes in PTH-induced cAMP production were observed. Because the release of free AA is an essential step in the production of eicosanoid metabolites that could act as second messengers, these findings suggest that corticosteroid treatment may activate signal transduction pathways for PTH which are latent in untreated cells, and thereby explain at least in part the profound effects of corticosteroids on osteoblast function.

Regulation of glycolytic metabolism during long-term primary culture of renal proximal tubule cells

Adequate oxygenation was a major factor regulating the induction of glycolytic metabolism in primary cultures of rabbit renal proximal tubule cells during short-term (less than 1 day) and long-term (1-7 day) culture. As measured by cellular lactate content, glucose consumption, and lactate dehydrogenase activity, less glycolytic metabolism was induced in cultured cells that were constantly aerated than in cells that were held stationary. When oxidative metabolism is supported by providing 2-10 mM heptanoate (HEP) as a metabolic substrate glycolytic metabolism further decreased during short-term, but not long-term culture. Cellular proliferation did not play a major role in regulating the induction of glycolytic metabolism, since glycolytic metabolism increased before cell growth had occurred, did not decline once logarithmic cell growth had ceased, and was stimulated less by cell growth than by inadequate oxygenation. Fructose-1,6-bisphosphatase and alkaline phosphatase, representative markers of gluconeogenic and brush-border membrane enzyme activities, respectively, declined during culture regardless of culture conditions or the presence of HEP. Therefore, glycolytic metabolism can be effectively minimized by constantly aerating cultured proximal tubule cells and can be further reduced by the addition of HEP during short-term culture.

Protein kinase C activation modulates arachidonic acid metabolism in cultured alveolar epithelial cells.

Cultured alveolar type II cells can liberate esterified arachidonic acid (AA) and metabolize it predominantly via the cyclooxygenase pathway, and their capacity to do so increases as they alter their phenotype over time in culture. Little is known, however, about the regulation of AA metabolism in alveolar pneumocytes. We have examined the effects of protein kinase C (PKC) activation on arachidonate metabolism in primary cultures of rat alveolar epithelial cells studied at 2 and 7 days following isolation. The potent PKC activator phorbol myristate acetate (PMA) stimulated dose-dependent increases in free AA levels in both day 2 and day 7 cultures, with optimal stimulation at 50 nM. Greater stimulation was demonstrated for day 7 cells, and this was associated with greater prostanoid synthesis in response to PMA by day 7 than by day 2 cells. The capacity of PMA to "prime" epithelial cells for augmented AA liberation and metabolism in response to calcium ionophore A23187 (5 microM) was examined also. Significant priming by PMA was observed in both day 2 and day 7 cells; once again, augmentation of both free AA levels as well as prostaglandin E2 levels was greater for day 7 cells than for day 2 cells. That the capacity of PMA to modulate AA metabolism was mediated by activation of PKC was confirmed by demonstrating that (1) phorbol didecanoate, which lacks the ability to activate PKC, failed to activate AA metabolism; (2) pretreatment for 18 h with 1 microM PMA, which depletes cellular PKC, abolished subsequent AA metabolism activated by 50 nM PMA; and (3) the PKC inhibitor staurosporine abrogated increases in the quantities of both free AA and prostaglandin E2 in response to PMA. We conclude that activation of PKC increases the availability of AA for prostanoid synthesis in alveolar pneumocytes, and that this effect is more evident as type II cell differentiation is modeled during prolonged cultivation.

Epithelial- Stromal Interactions in the Regulation of Rat Ventral Prostate Function: Identification and Characterization of Pathways for Androgen Metabolism in Isolated Cell Types*

Androgen metabolism plays a significant role in the androgen regulation of prostate cell function. In this report the various pathways for androgen metabolism in primary cultures of rat ventral prostate epithelial and stromal cells were identified and characterized by in vitro whole cell assays, using HPLC. Confluent cultures of both cell types were incubated with supraphysiological concentrations (50 nM) of tritiated androgens (testosterone, 5 alpha-dihydrotestosterone, 5 alpha-androstane-3 alpha(and 3 beta), 17 beta-diols, and delta 4-androstene-3,17-dione), and the metabolites were analyzed at several time points over a 24-h period. The metabolism studies indicated that 5 alpha-reductase activity, the oxidative reactions of 3 alpha-, 3 beta-, and 17 beta-hydroxysteroid oxidoreductases, and the reductive reaction of 3 beta-hydroxysteroid oxidoreductase were expressed at significantly higher levels in epithelial cells compared to stromal cells. The reductive reactions of 3 alpha- and 17 beta-hydroxysteroid oxidoreductases were similar in both cell types. In contrast, stromal cells exhibited substantially higher levels of 6 alpha/7 alpha-hydroxylase activity. In addition, stromal cells were capable of metabolizing 5 alpha-dihydrotestosterone directly to a new unidentified polar androgen metabolite (HO5 alpha-DHT). Overall, epithelial cells were approximately 29 times more capable than stromal cells of forming the biologically active androgen 5 alpha-dihydrotestosterone. Conversely, stromal cells were more capable of forming biologically inactive polar androgen metabolites.

Relationship between proliferation and glucose metabolism in primary cultures of rabbit proximal tubules

Primary cultures of rabbit proximal tubules, which revert to a glycolytic profile as reflected by increased activity of pyruvate kinase (PK) paralleled by increased glucose consumption and lactate formation, were utilized to explore the relationship between glycolytic metabolism and proliferation. Tubules placed in serum-free, hormonally defined Dulbecco's modified Eagle's medium with 5 mM glucose exhibited logarithmic growth beginning on day 3 in culture. The increase in PK activity lagged approximately 1 day behind, suggesting that the reversion to glycolysis is a consequence of rather than a prerequisite for cellular proliferation. Tubules cultured in 0.5 mM as contrasted with 25 mM glucose exhibited heightened proliferation reflected by an increase in protein content and cell number on day 5 in culture. The heightened proliferation was accompanied by increased PK activity. On day 9, after confluency had been achieved, no differences in protein content or PK activity were detected between tubules cultured in different glucose concentrations. These findings indicate that a low glucose concentration is mitogenic for renal proximal tubules and that the proliferative process in some fashion up-regulates the activity of the glycolytic enzyme PK. Furthermore, because accelerated growth proceeds in the presence of glucose restriction, the energy from glycolysis is not required for the proliferative process.

Arachidonic acid metabolism in cultured astrocytes from rat embryo and in C 6 glioma cells

Arachidonic acid metabolism in primary cultures of astroglial cells prepared from cerebra of rat embryos was examined. Arachidonic acid was mainly metabolized through the lipoxygenase pathway and the major metabolites formed were 12-hydroxyeicosatetraenoic acid (12-HETE), 11-HETE and 15-HETE. By contrast, in C 6 cells, which are considered to be of astroglial origin, arachidonic acid was mainly metabolized through the cyclooxygenase pathway and the major metabolites formed were prostaglandin (PG)E 2, PGF 2α and throm☐ane B 2.

Attenuation of anterior pituitary phosphoinositide phosphorylase activity by the D2 dopamine receptor.

We have examined the influences of dopamine and the D2 receptor agonist bromocriptine on phosphoinositide metabolism in primary cultures of rat anterior pituitary cells, monitoring changes in the levels of phosphatidylinositol (PtdIns), phosphatidylinositol-4-phosphate [PtdIns(4)P], and phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2]. Basal incorporation of [3H]inositol ([3H]Ins) into phosphoinositides was progressive, and radioisotopic equilibrium was attained in all three species within 48 h. The inclusion of dopamine or bromocriptine in the incubation medium promoted concentration-dependent reductions in the rate, but not the magnitude, of phosphoinositide radiolabeling. The onset of this effect was rapid; inhibition of [3H]Ins incorporation by dopamine (500 nM) and bromocriptine (100 nM) could be detected within 2 h. This treatment also produced a comparable reduction in the incorporation of [32P]orthophosphate into PtdIns(4,5)P2. In extended time-course studies, bromocriptine dramatically retarded the radiolabeling of PtdIns(4)P and PtdIns(4,5)P2, and apparent equilibria in these species were attained only after 96 h. We also assessed the ability of dopamine to modify the concentration-response characteristics of [3H]Ins-labeled inositol phosphate ([3H]InsPx) production by TRH, angiotensin II (AII), neurotensin (NTS), bombesin (BBS), and vasoactive intestinal polypeptide (VIP). Neither dopamine nor bromocriptine altered the rate or magnitude of TRH-, AII-, NTS-, or BBS-related InsPx generation. VIP was completely ineffective in stimulating InsPx generation. PRL release was significantly reduced in all dopamine-treated groups. That the InsPx concentration-response relationships for each of these peptides remained unimpaired by exposure to dopamine or bromocriptine extends our previous observation that the phosphoinositide-specific phospholipase-C is insensitive to dopaminergic tone. Consistent with our earlier findings, these data indicate that activation of the D2 dopamine receptor attenuates the activity of mechanisms associated with the serial phosphorylations of PtdIns and PtdIns(4)P, reactions that give rise to PtdIns(4)P and PtdIns(4,5)P2, respectively. It is our conclusion that dopamine, in addition to its other actions, attenuates the phosphorylation, rather than the hydrolysis, of anterior pituitary phosphoinositide. This attenuation appears to be mediated by an inhibitory coupling of the D2 receptor with the phosphoryltransferase activities that catalyze PtdIns(4)P and PtdIns(4,5)P2 formation.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of serum proteins on haem uptake and metabolism in primary cultures of liver cells

A role of haemopexin in transporting haem to hepatocytes for degradation has been inferred from the high affinity of haemopexin for haem. We have examined this question in primary cultures of chick-embryo and adult rat liver cells. We present here the results of four sets of experiments which indicate that haemopexin retarded haem uptake by hepatocytes in culture. (1) Haem bound to bovine serum albumin is known to repress the activity of delta-aminolaevulinate synthase in chick cultures as indicated by decreased porphyrin accumulation. When haem-albumin was added in the presence of excess purified or freshly secreted chicken haemopexin, no haem-mediated repression of porphyrin production was observed. The haem-mediated repression of porphyrin accumulation was partially prevented when human, but not chicken, albumin was added to cultures. This finding reflected the higher affinity of human albumin for haem compared with that of chicken albumin. (2) Haemopexin inhibited the ability of haem to be incorporated into cytochrome P-450 induced in the chick cultures in the presence of the iron chelator desferrioxamine. (3) The rate of association of [55Fe]haem with cultured rat hepatocytes when [55Fe]haem-haemopexin was added was one-eighth of the rate observed when [55Fe]haem-bovine serum albumin was used as the haem donor. (4) The presence of haemopexin also diminished the catabolism of haem by both rat and chick-embryo liver cell cultures. It is concluded that the uptake and subsequent metabolic effects of haem are inhibited in cultured hepatocytes by proteins such as haemopexin which have a high affinity for haem.

Free and protein-conjugated polyamines in mouse epidermal cells. Effect of high calcium and retinoic acid.

We have investigated polyamine metabolism in primary cultures of mouse epidermal cells. These cells, which grow at low Ca2+ levels as a monolayer with characteristics of basal cells, terminally differentiate when the extracellular Ca2+ level is raised above 1 mM. The cellular levels of free polyamines were measured, and, after incubation of cell cultures with [3H]putrescine, the distribution of label in both acid-soluble and acid-insoluble cellular components was examined. Free polyamine levels were reduced in cells induced to differentiate. Treatment with retinoic acid, which prevents differentiation and causes increased proliferation, resulted in an increase in free putrescine. Upon adjustment of the calcium concentration to a level that induces differentiation, the enzyme transglutaminase was activated, and a concomitant increase in the level of both protein-bound mono- and bis-gamma-glutamyl derivatives of putrescine and spermidine was observed. Isolation of a material of apparent molecular weight about 6000 which contains only mono-gamma-glutamylpolyamines and the finding of both mono- and bis-gamma-glutamylpolyamines in the protein fraction containing cornified cell envelopes provided the basis for speculation on polyamines in envelope formation. Our data suggest that polyamines play a role during epidermal cell differentiation through transglutaminase-mediated post-translational modification.

初代培養肝細胞における化学物質代謝

初代培養肝細胞における化学物質代謝

Regulation of glycogen metabolism in primary cultures of rat hepatocytes. Restoration of acute effects of glucose in cells from diabetic rats involves protein synthesis.

Defective acute regulation of hepatic glycogen synthase by glucose and insulin, caused by severe insulin deficiency, can be corrected in adult rat hepatocytes in primary culture by inclusion of insulin, triiodothyronine, and cortisol in a chemically defined serum-free culture medium over a 3-day period (Miller, T. B., Jr., Garnache, A. K., Cruz, J., McPherson, R. K., and Wolleben, C. (1986) J. Biol. Chem. 261, 785-790). Using primary cultures of hepatocytes isolated from normal and diabetic rats in the same serum-free chemically defined medium, the present study addresses the effects of cycloheximide and actinomycin D on the chronic actions of insulin, triiodothyronine, and cortisol to facilitate the direct effects of glucose on the short-term activation of glycogen synthase. The short-term presence (1 h) of the protein synthesis blockers had no effect on acute activation of glycogen synthase by glucose in primary hepatocyte cultures from normal rats. Normal cells maintained in the presence of cycloheximide or actinomycin D for 2 and 3 days exhibited unimpaired responsiveness to glucose activation of synthase. The protein synthesis inhibitors were effective at blocking the restoration of glucose activation of synthase in diabetic cells in media which restored the activation in their absence. Restoration of glycogen synthase phosphatase activity by insulin, triiodothyronine, and cortisol in primary cultures of diabetic hepatocytes was also blocked by cycloheximide or actinomycin D. These data clearly demonstrate that restoration of acute glycogen synthase activation by glucose and restoration of glycogen synthase phosphatase activity in primary cultures of hepatocytes from adult diabetic rats are dependent upon the synthesis of new protein.

Maintenance of steroid metabolism in primary cultures of adult rat hepatocytes in serum-free medium

Maintenance of steroid metabolism in primary cultures of adult rat hepatocytes in serum-free medium

The activation of inositol phospholipid metabolism as a signal- transducing system for excitatory amino acids in primary cultures of cerebellar granule cells

L-Glutamic, L-aspartic acids and a number of their structural analogs, including quisqualic, kainic, ibotenic, quinolinic, and N-methyl-D-aspartic (NMDA) acids, increase inositol phospholipid hydrolysis when added to primary cultures of cerebellar granule cells, as is reflected by an enhanced formation of 3H-inositolmonophosphate (3H-IP1) in the presence of Li+. L-Glutamic acid also enhances the formation of the initial products of inositol phospholipid hydrolysis, 3H-inositol di-(3H-IP2) and triphosphate (3H-IP3). In the absence of extracellular Ca2+, L-glutamic acid fails to enhance 3H-IP1 formation, but still increases 3H-IP2 and 3H-IP3 formation. The stimulation of 3H-IP1 formation elicited by L-glutamic acid is reduced by DL-2-amino-5-phosphonovaleric acid (APV) and gamma-glutamylglycine and, to a lesser extent, by 2,3-cis-piperidindicarboxylic acid (PDA). The stimulation of 3H-IP1 formation by kainic acid is antagonized by PDA and gamma-glutamylglycine, but it is almost unaffected by APV. The increase in 3H-IP1 formation elicited by quisqualic acid is not reduced by any of the dicarboxylic amino acid receptor antagonists that we have tested. We conclude that different subtypes of excitatory amino acid recognition sites are associated with inositol phospholipid metabolism in primary cultures of cerebellar granule cells.