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Biomedical subjects

A Sahai

Publications and source records attributed to A Sahai.

At least 73 records · Page 4Linked to original sources
PubMed1990

Prostaglandin F2 alpha inhibits the ammoniagenic response to acute acidosis in LLC-PK1 cells.

A kidney epithelial cell line, LLC-PK1, which does not synthesize prostaglandins, provides an ideal in vitro model system to investigate the effect of prostaglandins in the regulation of renal ammoniagenesis. Previous studies from our laboratory have demonstrated significant increases in glutamine-dependent ammonia and alanine production by rocked cultures of LLC-PK1 cells subjected to either acute metabolic or respiratory acidosis. In the study presented here, experiments were conducted to investigate the role of prostaglandin F2 alpha (PGF2 alpha) and prostaglandin E2 (PGE2) in the response of ammonia metabolism to acute metabolic acidosis by LLC-PK1 cells. A low dose of PGF2 alpha (0.1 ng/mL) dramatically inhibited the stimulatory effect of a low pH (pH 6.8) on ammonia production. In contrast, the inhibition of cytosolically generated alanine was less dramatic and averaged only 20% of the effect on ammonia production. Furthermore, PGF2 alpha increased cellular alpha-ketoglutarate concentration, suggesting an increase in intramitochondrial pH. Thus, the cellular mechanism of PGF2 alpha action appears to involve either interference with the cytosolic pH signal or its translation to the intramitochondrial compartment. The inhibitory response of PGF2 alpha on pH-stimulated ammoniagenesis was progressively lost at higher concentrations. Both low-dose (0.1 ng/mL) and high-dose (10 ng/mL) PGF2 alpha had no significant effect on the basal rates of ammonia and alanine production at pH 7.4. PGE2, on the other hand, did not exhibit any significant response on ammonia or alanine production at either pH 6.8 or 7.4 when given in a wide range of doses.(ABSTRACT TRUNCATED AT 250 WORDS)

Acidosis↗
PubMed1990

Biochemical pathways and modulators of renal ammoniagenesis.

The renal proximal tubule contains a variety of biochemical pathways, which can metabolize glutamine, the major substrate for renal ammoniagenesis. The intramitochondrially located phosphate-dependent glutaminase (PDG) pathway, rather than the various cytosolic pathways, appears to play the predominant role in regulating the rate of renal NH3 production. Acute acidosis stimulates NH3 production by activating alpha-ketoglutarate dehydrogenase and secondarily glutamate dehydrogenase; whereas the adaptation to chronic metabolic acidosis results primarily from enhanced glutamine transport into the mitochondria and possibly increased activity of PDG. There is no adaptation of ammoniagenesis to chronic respiratory acidosis, because the proximal tubular intracellular pH is not decreased. Alkalosis suppresses NH3 formation but the precise mechanism is not clarified. Ammoniagenesis can be modulated independent of acid-base status by a variety of factors including potassium homeostasis, TCA cycle intermediates, hormones which increase cAMP, prostaglandin F2 alpha, insulin, growth hormone, angiotensin II, corticosteroids, aldosterone, and tubular flow rate.

Acidosis, Renal Tubular↗
PubMed1989

Response of ammonia metabolism to acute acidosis: insights from cultured renal epithelium.

LLC-PK1 kidney epithelial cell lines cultured under the condition of continuous rocking exhibit both acute and adaptive changes in glutamine-dependent ammonia metabolism in response to acid-base manipulations in the media pH. Pulse-chase studies with 14C-glutamine as well as studies with various metabolic inhibitors of the ammoniagenic pathways suggest that glutamine in LLC-PK1 cells is metabolized via mitochondrial pathway and that intramitochondrial phosphate-dependent glutaminase pathway plays a predominant role in the regulation of ammoniagenesis to acute acidosis. Furthermore, the measurements of intracellular pH under both basal and acute low pH conditions resulted in the estimation of intracellular pH, which paralleled the alterations in media pH. Therefore, a change in intracellular pH appears to act as a direct signal for alterations in ammonia metabolism in LLC-PK1 cells. Thus, LLC-PK1 cultures provide an excellent model system to investigate renal ammoniagenesis and the intracellular signals that modulate this process.

Acidosis↗
PubMed1989

Rocking promotes differentiated properties in LLC-PK cells by improved oxygenation.

Previous studies from our laboratory indicated that in contrast to cells cultured under still conditions, LLC-PK cells cultured under conditions of continuous rocking exhibit pH-modulated ammonia production and also manifest lower levels of lactate dehydrogenase activity. In the present studies, we assessed whether other metabolic features and parameters of differentiation are modified by rocking. Rocked, as contrasted with still, cultures exhibited decreased medium lactate production and increased cellular ATP content consistent with heightened oxidative metabolism. Still cultures did not exhibit either dome formation or sodium-dependent alpha-methylglucoside uptake until the cultures reached full confluency. By contrast, rocked cultures underwent dome formation as well as enhanced alpha-methylglucoside uptake during the growth phase, and these parameters were quantitatively greater than in still culture even after full confluency was achieved. Thus rocking promotes several functions, which can reflect differentiation in LLC-PK cells. To determine whether provision of adequate oxygen accounts for the enhancement of cellular metabolism and differentiated functions by rocking, still cultures grown in a high oxygen concentration of 36% were compared with standard cultures grown in 18% oxygen. The high oxygen environment resulted in the development of pH-modulated ammonia production, a decrease in lactate dehydrogenase activity, lower medium lactate formation, increased ATP levels, more copious dome formation, and increased alpha-methylglucoside uptake. Thus rocking promotes cellular metabolism and differentiated functions by the provision of adequate oxygenation.

Adenosine Triphosphate↗
PubMed1989

Pathways and regulation of ammoniagenesis by the LLC-PK1 cells in culture.

LLC-PK1 kidney epithelial cells grown under the condition of continuous rocking exhibit a variety of differentiated functions of proximal tubular epithelium, including pH-modulated ammoniagenesis. To further determine their value as a model system, we investigated the pathways of ammoniagenesis under both normal conditions and acid-base manipulations. Pulse-chase studies with carbon 14-labeled glutamine demonstrated a marked delay in glutamine conversion to glutamate, indicating that glutamine deamidation is a critical rate-limiting step, and also provided evidence for metabolism of the glutamine carbon skeleton by the tricarboxylic acid cycle. Ammonia and alanine were the predominant nitrogen metabolites of glutamine at all pH conditions, and the stoichiometry suggested that glutamate is metabolized through both glutamate dehydrogenase and glutamate transaminase at pH 7.4. Increased ammonia production in response to a low pH was associated with increased flux through phosphate-dependent glutaminase and the glutamate transamination pathway and was accompanied by a fall in intracellular glutamate and alpha-ketoglutarate concentrations, which was similar to events in the intact kidney. Studies with the inhibitors acivicin and amino oxyacetate suggested that the gamma-glutamyltranspeptidase and glutamine transamination pathways are inconsequential in LLC-PK1 cells. The phosphate-dependent glutaminase pathway appears to play a predominant role in the regulation of ammoniagenesis. The similarity in ammonia metabolism with other in vitro and in vivo models suggests that LLC-PK1 cells will be a useful system for investigating renal ammoniagenesis and the intracellular signals that modulate this process.

Alanine↗
PubMed1986

Effect of epidermal growth factor and 12-O-tetradecanoylphorbol-13-acetate on the phosphorylation of soluble acidic proteins in A431 epidermoid carcinoma cells.

Treatment of [32P]phosphate prelabeled intact human A431 epidermoid carcinoma cells with epidermal growth factor (EGF, 100 ng/ml) or 12-O-tetradecanoylphorbol-13-acetate (TPA, 10(-7)M) resulted in a selective enhancement in the phosphorylation of the following soluble acidic proteins: a phosphoprotein with a molecular weight of 17,000 (pp17; similar notation used throughout) pI approximately 5.5); pp27 (pI approximately 5.5); pp34 (pI approximately 6.2); and pp80 (pI approximately 4.5) as detected by two-dimensional gel electrophoresis. EGF or TPA induced a 4- to 6-fold increase in the phosphorylation of pp17 and a 2- to 4-fold increase in the phosphorylation of pp27, pp34, and pp80 within 15 min after treatment of subconfluent A431 cells. Alkali treatment of the gels removed most of the incorporated [32P] phosphate from the phosphoproteins, including pp27, pp34, and pp80; however, the phosphoester bond in pp17 was stable to alkaline hydrolysis since there was no removal of [32P]phosphate from this protein. Treatment of A431 cells with dibutyryl cyclic adenosine 3':5'-monophosphate (1 mM) also increased the phosphorylation of pp17, pp27, and pp34 but not of pp80. Activation of endogenous calcium- and phospholipid-dependent protein kinase C in the cytosol of A431 cells in a cell-free system resulted in the enhanced phosphorylation of pp27, pp34, and pp80 but not of pp17 while exogenous addition of the catalytic subunit of cyclic adenosine 3':5'-monophosphate-dependent protein kinase to cytosol preparations resulted in the phosphorylation of pp17, pp27, and pp34, but not of pp80. These results demonstrate that at least four soluble acidic proteins are phosphorylated in A431 cells in response to either EGF or TPA in vivo suggesting that these two agents may exert part of their biological effects on A431 cells through a biochemical pathway involving the phosphorylation of several common proteins; moreover, the studies suggest that these four acidic proteins may be substrates in vitro for protein kinase C and/or a cyclic adenosine 3':5'-monophosphate-dependent protein kinase.

Bucladesine↗
PubMed1985

Modulation of type-IV procollagen and laminin production in A431 human squamous epidermoid carcinoma cells by 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor (EGF).

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor (EGF) on type-IV-procollagen (basement-membrane collagen) and laminin synthesis, turnover, and secretion was studied in human A431 squamous epidermoid carcinoma cells. Type-IV procollagen and laminin were biochemically and immunologically identified in the medium and cell extracts using immuno-precipitation followed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. EGF or TPA produced a sixfold increase in type-IV-procollagen and laminin secretion within 2 h; this was accompanied by a three- to fourfold increase in the levels of cell-associated type-IV procollagen and laminin, respectively. The level of type-IV-procollagen and laminin synthesis and secretion remained elevated for at least 16 h after the administration of either EGF or TPA. A combination of EGF and TPA was more effective than either agent alone in promoting the secretion of laminin but not of type-IV procollagen. EGF and/or TPA did not, however, produce a selective increase in the synthesis of collagen and laminin, since total protein synthesis was also increased to the same degree by these agents. As determined by labeling and chase studies, neither EGF nor TPA had any appreciable effect upon type-IV-procollagen or laminin degradation. These results indicate that the synthesis of components associated with the basement membrane in A431 cells (i.e., type-IV procollagen and laminin) can be rapidly modulated by EGF and a tumor promoter, i.e., TPA.(ABSTRACT TRUNCATED AT 250 WORDS)

Basement Membrane↗
PubMed1984

Peroneal tubercle and its variations in the Indian calcanei.

A total number of 1,410 calcanei of either sex belonging to people of Agra and Lucknow region were studied and classified into 4 types depending upon the pattern of peroneal tubercle. A significant difference in the incidence of various types in the two regions were observed.

Adult↗
PubMed1984

Differential phosphorylation events associated with phorbol ester effects on acceleration versus inhibition of cell growth.

Phorbol-12-myristate-13-acetate (PMA) in some systems causes inhibition of growth; in others, PMA exerts a mitogenic effect. We have previously reported that the PMA-induced growth arrest and differentiation in leukemic cells are associated with a rapid increase in phosphorylation-dephosphorylation of two cytosolic proteins, phosphoprotein with molecular weights of 17,000 to 20,000 (pp 17-20) and phosphoprotein with a molecular weight of 27,000 (pla approximately 5.5). The phosphorylation of these proteins was found to be catalyzed directly by cyclic adenosine 3':5'-monophosphate-dependent protein kinase. To elucidate whether these phosphorylation events are specific to certain cellular functions induced by PMA, we compared the effect of PMA on phosphorylation of proteins in several cell systems. We found that, in cells where PMA accelerates growth (NIH/3T3 mouse fibroblasts, JB-6 mouse epidermal cells, and human peripheral lymphocytes) as well as in human platelets, the phosphorylation of pp 17-20 was only slightly affected by PMA, while phosphorylation of other proteins at Mr approximately 80,000 (pl approximately 5) and Mr 20,000 (pl approximately 5.2) was markedly increased. We provide evidence to suggest that the Mr 20,000/pl 5.2 protein might be myosin light chain (Mr 20,000). In contrast to these cell systems, in the human malignant cell A431 epidermoid carcinoma, the growth of which is inhibited by PMA, the pattern of protein phosphorylation by PMA exhibited striking similarity to that of leukemic cells; phosphorylation of pp 17-20 was dramatically increased while the phosphorylation of the putative myosin light chain (Mr 20,000/pl 5.2) was not affected. The identity of pp 17-20 in A431 and HL-60 leukemic cells was demonstrated by the unique stability of its phosphoester bond to alkali treatment. These results suggest that phosphorylation of different proteins and possibly activation of different protein kinases mediate the effects of PMA on inhibition versus acceleration of growth. Specifically, phosphorylation-dephosphorylation of pp 17-20 emerges as a signal which might be preferentially involved in PMA effect in certain neoplastic cells.

Animals↗
PubMed1983

Effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the growth inhibitory and increased phosphatidylinositol (PI) responses induced by epidermal growth factor (EGF) in A431 cells.

Epidermal growth factor (EGF) inhibited the growth of A431 human epidermoid carcinoma cells. The tumor promoting, phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) also retarded A431 cell growth. Addition of both TPA and EGF inhibited cell growth in an additive or synergistic manner depending upon the initial plating density of the cultures. EGF increased the production of diacylglycerol (60-70%) and stimulated the synthesis of phosphatidylinositol (PI) from 3H-inositol (three- to fourfold increase). Both of these responses were attenuated in the presence of TPA. TPA alone stimulated the production of diacylglycerol (DG) but had little effect on PI synthesis. The biological effect of TPA appeared to be mediated by the presence of a high-affinity receptor for phorbol esters on A431 cells. Moreover, the binding of 125I-EGF to A431 cells was unaffected by TPA, suggesting that the antagonistic effects of TPA were occurring distal to the EGF receptor. These findings also indicated that although TPA and EGF both inhibited A431 cell growth, this effect could be dissociated from changes in PI synthesis but may be dependent upon transient changes in DG production.

Carcinoma, Squamous Cell↗
PubMed1982

Ultracytochemical localization of estrogen-stimulated guanylate cyclase in rat uterus.

Estrogens are known to increase cyclic guanosine monophosphate (cGMP) levels in the uterus of rats by enhancing guanylate cyclase (GC) activity. In the present study, the cytochemical localization of GC activity was studied in the uteri of immature and ovariectomized rats after treatment with diethylstilbestrol (DES), progesterone, estrogen antagonist (CI628), and a combination of DES and CI628. Twenty-four hours after the first dose of DES, moderate to strong guanylate cyclase activity was indicated by lead phosphate precipitate on the luminal microvillar and basolateral surfaces of epithelial cells, whereas strong activity was found on the plasma membranes of fibroblasts, endothelial cells, and myometrial cells. The enzyme activity in the epithelial cells declined slightly 24 hr after the second daily dose of DES. Uterine tissues from DES-treated rats that were preheated at 60 degrees C for 30 min or preincubated with a GC inhibitor showed no reaction product. Guanylate cyclase activity was not observed cytochemically in the uterine tissues of the vehicle control (immature or ovariectomized) or progesterone-and CI628-treated animals. Weak guanylate cyclase activity was observed on the plasma membranes of epithelial cells and endothelial cells after doses of DES and CI628 were given simultaneously. The biochemical assays of the total homogenate in vitro indicated that uterine GC showed about a twofold increase after one dose of DES and a 1.3-fold increase following two doses (one dose per day) of DES when compared with their respective nontreated controls, or with progesterone-treated uteri. GC was found in particulate (09%) and cytosol (10%) fractions. These data demonstrated that DES stimulated uterine guanylate cyclase activity, while progesterone and CI628 were ineffective at the doses used. Estrogen antagonist CI628 doses not completely suppress the effect of DES.

Animals↗
PubMed1982

Identification and characterization of endogenous inhibitors of polyamine-responsive protein kinase activity.

Polyamine-responsive protein kinase activity was inhibited by two heat stable inhibitors which were purified from Morris hepatoma 3924A. They were of low molecular weight (inhibitor I-1,600 to 2,000, inhibitor II-600 to 800 daltons). The inhibitors, when purified, passed through a DEAE-cellulose column; however, the crude complex of inhibitors and protein kinase activity bound to the DEAE-cellulose column. This suggests that the inhibitors are bound to the polyamine-responsive protein kinase in vivo. Both inhibitors completely inhibited the polyamine stimulated activity, but did not affect the basal enzymatic activity. They were not affected by treatment at 90 degrees for 2 min. These results demonstrate the presence of two new protein kinase inhibitors in mammalian cells.

Animals↗
PubMedDate not supplied

The bipolar spectrum: a review of current concepts and implications for the management of depression in primary care.

Family physicians inevitably encounter patients with bipolar disorders, often when the patient is depressed. For most of these patients, the attendant elevations in mood fall short of mania. Such milder periods of expansive mood, hypomanias, may go unrecognized unless the physician specifically queries the patient to uncover them. In addition, patients with bipolar disorders often manifest other distinctive characteristics. An understanding of these hints of bipolarity is helpful to clinicians treating depressive illness. Patients with bipolar disorders are at risk for treatment complications caused by the administration of antidepressants without the concurrent use of mood stabilizers, such as lithium carbonate, valproate sodium, and carbamazepine. Such complications include exacerbation of hypomania or mania, induction of refractory states, and, perhaps, rapid cycling or mixed states. We review current issues in classification of bipolar disorders and emphasize the importance of identifying hypomania. An introduction to the concept of affective temperaments and a brief review of treatment strategies and treatment complications are included.

Bipolar Disorder↗