Robert Grundin

Induction of microsomal aryl hydrocarbon (3,4-benzo(α)pyrene) hydroxylase and cytochrome P-450k in rat kidney cortex: I. Characteristics of the hydroxylase system☆

Abstract Both the rat kidney cortex aryl hydrocarbon hydroxylase activity and cytochrome P-450 K are induced by benzo(α)pyrene treatment. Following a single injection of benzo(α)pyrene, maximal hydroxylase activity and cytochrome P-450 K content occur at 24 hr, returning to control levels within 72–96 hr. Induction of both the enzyme activity and hemoprotein is inhibited by cycloheximide. The enzyme system is localized in the microsomal fraction, has an absolute requirement for NADPH and molecular oxygen, and a pH optimum at 7.4; the induced activity is linear with microsomal protein concentration up to 0.8 mg and with time up to 20 min. Both the hydroxylase activity and cytochrome P-450 K follow the same pattern of inactivation with increasing temperature. The apparent K m for the induced hydroxylase was 7.7 μ m and V was increased fourfold above control value. In the presence of laurate, a substrate for the kidney microsomal cytochrome P-450 K -dependent monooxygenase system, the amount of inhibition of hydroxylase activity corresponded to the level of activity present in untreated kidney cortex microsomes. α-Naphthoflavone (10 −5 m ), a type I inducer (36) produced a greater inhibitory effect on the induced hydroxylase activity than on the control (55% vs 20%). The presence of cytochrome c or carbon monoxide markedly decreased hydroxylase activity. This evidence in addition to aforementioned characteristics of the enzyme suggests a cytochrome P-450 K -dependent aryl hydroxylase activity which differs from that present in the control rat.

Spectral studies on drug-cytochrome P-450 interaction in isolated rat liver cells

Abstract Various drugs including hexobarbital, lidocaine and nortriptyline were added to suspensions of liver cells isolated from untreated and phenobarbital-treated male rats. Upon drug addition, there was a fast binding to cytochrome P-450, as revealed by the appearance of a rapidly growing type I spectral change in the difference spectrum. When this had reached optimal magnitude, an absorption peak at 437 nm could often be seen to appear and quickly disappear, followed by yet another increase in absorption at about 446 nm; the latter and the type I spectral change then rapidly disappeared. These spectral changes were most pronounced with liver cells from phenobarbital-treated rats which contained markedly increased levels of cytochrome P-450. Also the rate of hexobarbital binding to cytochrome P-450 seemed to be increased after phenobarbital pretreatment. Finally, evidence was obtained that the major part of cytochrome P-450 in the isolated liver cells is present in an oxidized, non-substrate-bound form.

Spectral studies on the rapid uptake and subsequent binding of drugs to cytochrome P-450 in isolated rat liver cells

Summary The rate of formation of the type I spectral change upon drug addition to a suspension of isolated rat liver cells was used to study factors that influence drug uptake by the hepatocytes. Although considerably slower than in liver homogenates and microsomes, drug combination with cellular cytochrome P-450 was still rapid and occurred within a few seconds. The effects of varying temperature and concentration and lipid solubility of the drugs studied as well as the lack of effect of preincubation of the cells with rotenone on the rate of formation of the type I spectral change, lead us to suggest that drug uptake into the hepatocytes occurs by a non-energy requiring diffusion process.

Drug metabolism in isolated rat liver cells.

Although considerable knowledge has been gathered on the functional aspects of microsomal monooxygenation, comparatively little has so far been known about the intracellular regulation of this process. For such studies, we have found the isolated rat liver cell system to be a very useful model, combining the convenience of an in vitro system with the access to the complex mechanisms of the intact in vivo system. This model has the advantage over the perfused liver that it readily lends itself to the study of rapid reaction sequences and makes quantitation of short-term drug metabolic reactions easier. It is also superior to liver slices which often show considerable leakage of adenine and pyridine nucleotides and where substrate penetration and oxygen diffusion may present problems depending on the relative thickness of the slice.

Induction of microsomal aryl hydrocarbon (3,4-benzo(α)pyrene) hydroxylase and cytochrome P-450k in rat kidney cortex: II. Interactions with the induced hemoprotein☆

Abstract Benzo(α)pyrene treatment resulted in stimulation of only cytochrome P-450 K and benzo(α)pyrene hydroxylase activity in rat kidney cortex microsomes. Spectral properties of cytochrome P-450 K showed that the 452 nm peak of the reduced hemoprotein CO-complex was not shifted in benzo(α)pyrene-treated rats. The off-balance absolute spectrum of oxidized cytochrome P-450 K displayed an absorption maximum at 414 nm, another band at 385 nm, and a distinct shoulder at 398 nm. Addition of benzo(α)pyrene to kidney microsomes resulted in a type I spectral change seen only in benzo(α)pyrene-treated rats. The addition of ethyl isocyanide to dithionitetreated microsomes from control rats gave rise to two Soret peaks, 432 nm and 458 nm. These peaks were proportionately increased in benzo(α)pyrene-treated rats; furthermore, the 458 nm peak was not shifted. The relative heights of the two peaks were in a pH-dependent equilibrium similar to that observed in liver; however, in contrast to liver, the pH, at which the ratio of the peak heights equals one, was the same for both benzo(α)pyrene-treated and control microsomes. These data indicate that the newly induced hemoprotein has spectral properties markedly different from those of the benzo(α)pyrene-induced liver hemoprotein, yet similar to those of the “noninduced” kidney hemoprotein. α-Naphthoflavone, an inhibitor of the aryl hydroxylase system, induced a type I spectral change, suggesting the mode of action of α-naphthoflavone to be its interaction with cytochrome P-450 K probably at or near the active site. Finally, the rate of reduction of cytochrome P-450 K was not affected by the presence of benzo(α)pyrene.

Anticholinergic potency of psychoactive drugs in human and rat cerebral cortex and striatum

The affinity of selected antipsychotic and antidepressant drugs for the muscarinic receptor was studied in membranes from both human and rat striatum and cerebral cortex. While there are regional differences in the anticholinergic potency of the drugs, there is good agreement between the obtained inhibition constants from the corresponding human and rat striatum (r: 0.98) and from human and rat cerebral cortex (r: 0.96). There is also good agreement between the obtained Ki values within one species: human cerebral cortex versus human striatum (r: 0.99) and for rat cerebral cortex and rat striatum (r: 0.87). Thus, the previously published quantitative estimates of the antimuscarinic activity of psychoactive drugs which were derived from studies on membranes from rat brain give an accurate estimate of the antimuscarinic activity in human brain. The drugs tested in this study include chlorpromazine acetophenazine, haloperidol, sulpiride, remoxipride (FLA-731 (-), a substituted benzamide), amitriptyline and two serotonin uptake blockers: norzimelidine and alaproclate.