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J K Randolph

Publications and source records attributed to J K Randolph.

26 records · Page 2Linked to original sources
PubMed1985

Induction of methotrexate release from rat hepatocytes in suspension by alpha-adrenergic agents: involvement of calcium and metabolic energy.

Hepatocytes in suspension which have accumulated [3H]methotrexate release the antifolate compound into the medium upon exposure to alpha-adrenergic agents. In the presence of metabolic poisons, such as sodium azide, dinitrophenol, or dicumarol, the release of methotrexate is attenuated, indicating that integrity of the cellular metabolic apparatus is required for response to the hormonal stimulus. In the presence of millimolar concentrations of the organic acid, probenecid, release of cellular methotrexate may be reduced (1 mM probenecid) or eliminated (2 mM probenecid), suggesting the involvement of a "membrane carrier." Microtubule poisons such as vincristine, vinblastine, and griseofulvin do not modify epinephrine + isobutyl methyl xanthine (IBMX)-induced release of methotrexate. The involvement of calcium in release of methotrexate from the hepatocyte is substantiated by a dose-dependent response to the calcium ionophore, A23187, in the presence of calcium, with a lack of response in the absence of calcium. These effects of A23187 are not related to inhibition of methotrexate influx. Other putative "calcium antagonists" such as tetracaine, neomycin sulfate, and 3,4,5-trimethoxybenzoic acid [8-(diethylamino)octyl ester], do not interfere with epinephrine + IBMX-induced release of [3H]methotrexate, suggesting that these agents may not be effective probes of calcium flux in the liver cell.

1-Methyl-3-isobutylxanthine↗
PubMed1984

Interaction of probenecid with methotrexate transport and release in the isolated rat hepatocyte in suspension.

Probenecid has been shown to delay the plasma clearance of methotrexate in the rat and to reduce both hepatic and renal excretion of methotrexate in this animal model. In order to probe the mechanism by which probenecid alters hepatic excretion of the antifolate, studies assessed the effects of probenecid on transport, accumulation, distribution, and release of methotrexate in the rat hepatocyte in suspension. Probenecid was found to effectively inhibit methotrexate influx with a Ki of approximately 100 microM. Inhibition of methotrexate influx was accompanied by a reduction in methotrexate accumulation; with 200 microM probenecid, the levels of exchangeable and nonexchangeable intracellular methotrexate were reduced by 43.4 +/- 2.4 (S.E.) and 41.8 +/- 7.7%, respectively. As a consequence of reduced accumulation of the methotrexate substrate, the formation of cellular polyglutamate derivatives of methotrexate was likewise reduced. Concentrations of probenecid which inhibited methotrexate influx and accumulation by 70 to 80% did not markedly alter methotrexate efflux under conditions where efflux was effected by a washout procedure or by the presence of inducing agents, such as N6,O2'-dibutyryl cyclic adenosine 3':5'-monophosphate or alpha-agonists. These studies suggest that the inhibition of hepatic methotrexate secretion by probenecid in vivo is likely to be a consequence of interference with hepatic uptake of the antifolate rather than an interaction of probenecid and methotrexate at a hepatic "secretory" site.

Animals↗
PubMed1980

Transport, binding, and polyglutamation of methotrexate in freshly isolated rat hepatocytes.

Influx of [3H]methotrexate into freshly isolated hepatocytes in suspension is mediated by two routes, one with a high affinity (Km = 5.9 microM) and another with a low affinity for methotrexate. Both transport routes are equally sensitive to the sulfhydryl group inhibitor, p-chloromercuriphenylsulfonic acid, alterations in temperature, substitution of extracellular Na+ with choline, and inhibition by ouabain or azide. The high-affinity pathway for methotrexate shows specificity for the 4-amino group of the pteridine moiety as methotrexate and aminopterin similarly inhibit influx of the labeled drug. On the other hand, 100 microM concentrations of the naturally occurring folates, folic acid, 5-methyltetrahydrofolate, and 5-formyltetrahydrofolate, are not inhibitory to influx of 1 microM methotrexate. Once in the cell, methotrexate rapidly reaches molar equivalence with dihydrofolate reductase following which both exchangeable and nonexchangeable intracellular methotrexate accumulates. The exchangeable component reaches steady state within 0.5 hr while the nonexchangeable component increases for at least 1 hr. The nonexchangeable component represents both bound methotrexate and methotrexate polyglutamates. Polyglutamates of methotrexate are a trivial component of total 3H within the cell until about 15 min, but thereafter, their rate of accumulation is constant so that by 1 hr they represent approximately 30% of total intracellular 3H. At steady state, there is a transmembrane chemical gradient for exchangeable methotrexate of 2.4:1; this is 24 times greater than the chemical gradient predicted for equilibrium when the transcellular membrane potential is considered. These results indicate that there are multiple routes for methotrexate transport in the rat hepatocyte that appear to be, at least in part, distinct from the routes for folic acid and the tetrahydrofolate cofactors. The data suggest that transport is energy and Na+ dependent and that the transport carrier requires intact sulfhydryl groups. Net association of methotrexate with the cells is a complex process determined by transport and binding to multiple sites within the cell and metabolism to polyglutamate derivatives that are retained within the cell.

4-Chloromercuribenzenesulfonate↗
PubMed1979

Formation of methotrexate polyglutamates in rat hepatocytes.

Polyglutamate derivatives of [3H]methotrexate (MTX) were detected in freshly isolated rat hepatocytes in suspension within 15 min after exposure to the folate analog. The rate of polyglutamate synthesis remained constant for at least one hr, and the polyglutamate derivatives accounted for an increasing proportion of the intracellular radiolabel with time. After initial exposure to 1 micron [3H]MTX, polyglutamate derivatives of Mtx continued to be synthesized even after the extracellular [3H]-MTX concentration had been reduced 20-fold. Prolonged exposure of hepatocytes in primary culture to 1 micron [3H]MTX resulted in the formation of longer-chain polyglutamate derivatives of MTX. The present studies demonstrate another important biosynthetic capacity of the freshly isolated hepatocyte and suggest the usefulness of this system for studying the mechanism of, and controlling factors in, the synthesis of polyglutamate derivatives of MTX. The ramifications of the formation of MTX polyglutamates on drug cytotoxicity in general and hepatotoxicity in particular are considered.

Animals↗
PubMedDate not supplied

Induction of taurocholate release from isolated rat hepatocytes in suspension by alpha-adrenergic agents and vasopressin: implications for control of bile salt secretion.

Hepatocytes incubated with 25 muM [3H] taurocholate rapidly deplete the extracellular medium of [3H] taurocholate and achieve a steady-state level of intracellular bile salt within 15 min. Exposure of cells at steady state with extracellular taurocholate to the catecholamines norepinephrine or epinephrine results in release of 3H from the cells into the incubation medium; the 3H released represents almost exclusively unmetabolized [3H] taurocholate. The hierarchy of effectiveness of the catecholamines, norepinephrine congruent to epinephrine greater than phenylephrine much greater than isoproterenol, is indicative of an alpha-adrenergic mechanism. Induction of [3H] taurocholate release by norepinephrine is inhibited by the alpha-antagonists phenoxybenzamine and phentolamine and by chlorpromazine, but is not affected by the beta-antagonist propranolol, further supporting an alpha-adrenergic basis for this phenomenon. Arginine vasopressin, at concentrations of 1 X 10(-9) M and greater, also induces bile salt release. Classical alpha- and beta-antagonists have minimal effects on vasopressin induced bile salt release. While the peptide hormones angiotensin and oxytocin are, alone, relatively ineffective inducers of bile salt release, oxytocin potentiates the induction of bile salt release by vasopressin, suggesting complex interactions with membrane receptor function. Further studies assessing the interaction of sympathetic neurotransmitters and peptide hormones with bile salt transport and release in the hepatocyte may provide insight into the regulation of hepatic secretory function in the intact animal.

Adrenergic alpha-Agonists↗