Biomedical subjects

S O Franklin

Publications and source records attributed to S O Franklin.

16 recordsLinked to original sources

PubMed1999

Preproenkephalin mRNA and enkephalin levels in the adult Syrian hamster: the influence from glucocorticoids.

Proenkephalin (Penk) gene structure in hamsters and humans are similar but they differ from rats. In this study hamster Penk gene expression was examined after hypophysectomy+/-glucocorticoid receptor blockade with RU 486 (mifepristone). In contrast to rats, basal Penk gene expression in hamster adrenals did not change after treatments that reduced both the influence from glucocorticoids and phenylethanolamine-N-methyltransferase mRNA levels. Meanwhile, striatal preproenkephalin mRNA levels increased under these conditions.

Adrenal Glands↗

PubMed1998

Differences in delta opioid receptor antinociception, binding, and mRNA levels between BALB/c and CXBK mice.

Mu and delta opioid receptors have been demonstrated to mediate supraspinal opioid antinociception. Whereas the recombinant inbred CXBK mouse is notably deficient in mu opioid receptor antinociception, binding density, and mRNA (MOR-1) levels, little is known about delta opioid receptor processes in this strain. The present study thus compared CXBK mice and their BALB/c strain progenitors with respect to delta opioid antinociception, whole-brain receptor binding levels, and mRNA (DOR-1) levels. Following intracerebroventricular injections of the selective delta1 and delta2 opioids DPDPE and [d-Ala2]deltorphin II, respectively, CXBK mice displayed relatively lower antinociception on the tail-flick test, resulting in significantly increased ED50 values for both agonists in this strain. Decreased whole-brain specific binding of [3H][d-Ala2]deltorphin II, but not [3H]DPDPE, was also observed in CXBK mice. Solution hybridization with a probe for the DOR-1 revealed increased transcript levels in the caudate-putamen, frontal cortex, and spinal cord of this strain. The present data demonstrate a deficiency in delta1 and delta2 opioid antinociception in CXBK mice concomitant with reductions in whole-brain delta2 receptor binding and regional increases in DOR-1. Whether these observations are causally related remains to be clarified.

Analgesics↗

PubMed1997

Changes in proenkephalin gene expression in the developing hamster.

Proenkephalin (Penk) gene expression is high in the adult hamster adrenal medulla and it is comparable to that found in both the hamster and rat striatum. In addition, Penk gene expression in the hamster adrenal medulla is more typical of adult mammalian adrenals than the rat. Since the nature of Penk gene expression in the developing hamster adrenal is not known, it was examined and compared to that found in the striatum were adult levels in the adrenal and striatum are similar. The results show that Penk gene expression progressively increases in the developing hamster adrenal to peak on postnatal day 4. There is then a small decline to adult levels by postnatal day 12 when the morphology of the developing adrenal resembles the adult. Functional splanchnic nerve activity, as assessed by the ability of reserpine to induce increases in adrenal tyrosine hydroxylase mRNA, is not present until after postnatal day 4. Therefore, early increases in Penk gene expression are independent of splanchnic nerve activity. Adrenal EC peptides resulting from the developmental increases in Penk gene expression appear to be unprocessed and proenkephalin-like. This is based on the very low levels of free enkephalin (met-enkephalin) detected in the adrenals from both newborn and adult hamsters (1-5% of total EC peptide levels). In the developing hamster striatum, Penk gene expression remains low and unchanged until postnatal day 4 and increases six-fold by adulthood. Free enkephalin (met-enkephalin) levels remain high (between 36 and 88% of total EC peptide levels) in the developing and adult hamster striatum. Therefore the results show early increases in adrenal Penk gene expression in the developing hamster that are independent of splanchnic nerve activity and adult Penk gene expression which is high and dependent on splanchnic nerve activity. This differs from what is observed in the frequently studied rat. However, developmental changes in the hamster striatum are similar to those in the rat.

Adrenal Glands↗

PubMed1995

Differential regulation of c-fos, proenkephalin and tyrosine hydroxylase gene expression by metrazole in the hamster adrenal and hippocampus.

Metrazole (MTZ) induces sequential activation of c-fos, proenkephalin (Penk) and tyrosine hydroxylase (TH) gene expression in the rat adrenal and c-fos and Penk gene expression in the rat hippocampus. As in the rat, MTZ produced a dose-dependent induction of c-fos mRNA in the hamster adrenal and hippocampus together with an increase in adrenal TH mRNA. Although MTZ-induction of preproenkephalin (PPenk) mRNA was observed in the hippocampus of the hamster, the same treatment failed to induce PPenk mRNA in the hamster adrenal. These results indicate that Penk gene expression in the hamster is differentially regulated in the adrenal and hippocampus. Furthermore, the regulation of adrenal Penk gene expression differs significantly when rat and hamster are compared.

Adrenal Glands↗

PubMed1995

Quantitative distribution of the delta opioid receptor mRNA in the mouse and rat CNS.

We have used a sensitive solution hybridization assay that employs a riboprobe obtained from the mouse delta opioid receptor (DOR) coding sequence to quantitate the relative abundance of DOR mRNA in the central nervous system (CNS) of the adult mouse and rat. In brain Poly A+ RNA extracts this riboprobe hybridized to a single 10 kb transcript from mouse and two transcripts, one of 12 and the other of 4.5 kb in size from rat. In mouse CNS the highest levels of DOR mRNA were found in the caudate-putamen at 3.3 +/- 0.1 (SEM) pg/micrograms RNA. DOR mRNA levels in the range from 2.6 to 2.1 pg/micrograms RNA were measured in frontal cortex, nucleus accumbens, whole brain and olfactory tubercle. Spinal cord, periaqueductal gray and hippocampus had DOR mRNA levels in the range from 1.8 to 1.5 pg/micrograms RNA, while medial thalamus and cerebellum had the lowest levels (0.5 pg/micrograms RNA). These results correlate with the reported relative distribution of DOR mRNA in mouse using an in situ hybridization technique. In rat CNS, the highest levels of DOR mRNA were measured in caudate-putamen at 2.3 +/- 0.1 pg equivalents/micrograms RNA. Whole brain, cerebral cortex, olfactory bulb and brain stem had levels in the range from 1.5 to 0.9 pg equivalents/micrograms RNA while the lowest DOR mRNA levels were measured at 0.5 pg equivalents/micrograms RNA or less in thalamus, hippocampus, substantia nigra and cerebellum. This study demonstrates the ability of solution hybridization assays to quantitate homologous (mouse) as well as similar but heterologous (rat) DOR mRNA levels.

Animals↗

PubMed1994

Acute and persistent suppression of preproenkephalin mRNA expression in the striatum following developmental hypoxic-ischemic injury.

The striatum is vulnerable to hypoxic-ischemic injury during development. In a rodent model of perinatal hypoxia-ischemia, it has been shown that striatal neurons are not uniformly vulnerable. Cholinergic neurons and NADPH-diaphorase-positive neurons are relatively spared. However, it is unknown what classes of striatal neurons are relatively sensitive. One of the major classes of striatal neurons uses enkephalin as a neurotransmitter. We have studied the effect of early hypoxic-ischemic injury on this class of neurons using a quantitative solution hybridization assay for preproenkephalin mRNA in conjunction with in situ hybridization. Hypoxia-ischemia results in an early (up to 24 h) decrease in striatal preproenkephalin mRNA, which is shown by in situ hybridization to occur mainly in the dorsal portion of the striatum. By 14 days, whole striatal preproenkephalin mRNA and total enkephalin-containing peptide levels are normal. However, at 14 days, in situ hybridization reveals that regions of complete preproenkephalin mRNA-positive neuron loss remain in the dorsal region. Normal whole striatal levels are due to an up-regulation of preproenkephalin mRNA expression in the ventrolateral region of the injured striatum. Given the important role that the enkephalin-containing striatal efferent projection plays in regulating motor function, its relative loss may be important in the chronic disturbances of motor control observed in brain injury due to developmental hypoxic-ischemic injury.

Aging↗

PubMed1993

Quantitation of the levels of tyrosine hydroxylase and preproenkephalin mRNAs in nigrostriatal sites after 6-hydroxydopamine lesions.

Expression of the striatal proenkephalin gene is modulated by dopaminergic input from the substantia nigra (SN). We have used rapid, specific and sensitive solution hybridization assays for the quantitation of tyrosine hydroxylase (TH) mRNA, preproenkephalin (PPenk) mRNA and total cellular RNA to compare ipsilateral and contralateral levels of these RNAs in tissue dissected from the origin and termination of the nigrostriatal pathway of individual rats following sham (vehicle) or 6-hydroxydopamine (6-OHDA) induced lesions of the SN. Three weeks after treatment the rats that had received 6-OHDA, but not sham treated controls, demonstrated a characteristic contralateral rotation in response to apomorphine. Four weeks after 6-OHDA treatment, TH mRNA levels were reduced below the limits of sensitivity of the assay (1 pg/ug RNA) in ipsilateral SN while the levels of TH mRNA in contralateral SN (4.8 pg/ug RNA) did not differ from that in sham treated animals. PPenk mRNA levels in striatum were increased 3 fold to 64.9 pg/ug RNA on the side of the 6-OHDA lesions while the contralateral PPenk mRNA levels (21.6 pg/ug RNA) did not differ from sham treatment. The 6-OHDA treatment did not alter the levels of total cellular RNA in either SN or striatum. These results provide quantitative evidence for the tonic inhibition of striatal proenkephalin gene expression by the dopaminergic nigrostriatal pathway.

Animals↗

PubMed1993

Quantitation of NMDA receptor (NMDAR1) mRNA levels in the adult and developing rat CNS.

A rapid and sensitive solution hybridization assay was used to quantitate N-methyl-D-aspartate (NMDA) receptor mRNA levels in the central nervous system (CNS) of rat, mouse and human. A riboprobe labelled with 32P was prepared from a plasmid containing a 1413 base sequence from the cDNA for the functional rat NMDA receptor subunit, NMDAR1. Using a full length sense transcript as the calibration standard, the assay reliably measures 8 pg of NMDAR1 mRNA. When expressed as pg of NMDAR1 mRNA/micrograms total cellular RNA, the highest levels in the adult rat CNS are in the olfactory bulb (20.9 pg/micrograms RNA) and the lowest levels are in the spinal cord (5.2 pg/micrograms RNA). Intermediate levels were found in frontal cortex, hippocampus, cerebellum and whole brain. In the mouse CNS the highest levels of NMDAR1 mRNA were found in the olfactory bulb (12.9 pg equivalents/micrograms RNA), followed closely by hippocampus, frontal cortex and cerebellum. Mouse spinal cord (4.4 pg equivalents/micrograms RNA) had the lowest levels of NMDAR1 mRNA. The NMDAR1 riboprobe hybridizes with the same size transcripts in Poly(A)+ RNA from rat, mouse and human brain. In the developing rat, NMDAR1 mRNA levels in frontal cortex and hippocampus increased nearly 3 fold from postnatal day 3 to day 15 and approximately doubled from day 15 to day 67 (adult). Therefore, from postnatal day 3 to adult (day 67) frontal cortex and hippocampus levels of NMDAR1 mRNA increased nearly 6 fold.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

PubMed1993

Metrazole induces the sequential activation of c-fos, proenkephalin, and tyrosine hydroxylase gene expression in the rat adrenal gland: modulation by glucocorticoid and adrenocorticotropic hormone.

The immediate-early gene c-fos (a nuclear transcription factor) has been viewed as a nuclear "third messenger" or cellular "master switch." Both in vitro and in vivo studies have suggested that the proenkephalin (Penk) and tyrosine hydroxylase (TH) genes are potential targets of this immediate-early gene. We investigated the relationships between the activation of the c-fos gene and the activation of the Penk and TH genes in both rat hippocampus and adrenal using a commonly used model, metrazole (MTZ)-induced convulsions. The administration of MTZ produced a sequential elevation in c-fos, preproenkephalin (PPenk), and TH mRNAs. One hour after MTZ administration, c-fos mRNA was increased about 10-fold in rat hippocampus and about 5-fold in rat adrenal, without a significant change in spinal cord levels. Immunocytochemistry revealed that Fos-like immunoreactivity was greatly increased in both hippocampus and adrenal medulla at 3 hr after MTZ administration. The levels of PPenk and TH mRNAs were significantly increased (5-fold and 3-fold, respectively) in the adrenal 6 hr after MTZ treatment. The effects of MTZ on c-fos, PPenk, and TH mRNAs were dose dependent in both adrenal and hippocampus. In the adrenal, both the basal levels and the MTZ induction of PPenk mRNA were significantly attenuated by hypophysectomy (hypox) and were partially reinstated by adrenocorticotropic hormone (ACTH) replacement. In contrast, the basal levels of c-fos and TH mRNAs were not altered in hypox rat adrenal. ACTH treatment completely blocked the MTZ induction of adrenal c-fos mRNA and the subsequent induction of Fos-like immunoreactivity, whereas MTZ increased PPenk and TH mRNAs nearly 3-fold. Thus, in hypox rats MTZ can increase adrenal c-fos and TH mRNA levels without a corresponding increase in PPenk mRNA, whereas in ACTH-treated rats PPenk and TH mRNA levels in adrenal can be increased by MTZ without a preceding increase in c-fos mRNA. The MTZ induction of c-fos appears neither sufficient nor always necessary for the subsequent MTZ induction of Penk and TH gene expression. We conclude that c-fos, Penk, and TH genes can be differentially regulated in the adrenal of hypox rats or animals treated with ACTH, although they are co-localized in the same medullary cells.

Adrenal Glands↗

PubMed1992

Time course of enkephalin mRNA and peptides in cultured rat adrenal medulla.

Explantation of rat adrenal medullae to organ culture results in dramatic changes in enkephalins and catecholamines that are similar to the changes seen in vivo in response to denervation, which eliminates transsynaptic impulse activity. We have used rapid and sensitive solution hybridization methods to measure preproenkephalin (PPenk) mRNA and total cellular RNA in samples from rat tissues and adrenal medullary explants. The profiles of adrenal medullary PPenk mRNA, enkephalin-containing (EC) peptides, total cellular RNA and catecholamines [epinephrine (epi) and norepinephrine (norepi)] were measured during 14 days of organ culture. After 8 h in culture, total RNA had declined by 60%, epi and norepi declined 80 to 85% and EC peptides by 50% while the amount of PPenk mRNA per gland increased by 400%. Between 8 h and 14 days total RNA and catecholamine levels remained constant while PPenk mRNA increased to a peak of 85 +/- 10 (S.E.M.) pg/gland at 2-4 days, a value that was 80 times greater than the zero time (preculture) values. EC peptide levels lagged behind the increase in PPenk mRNA and reached a peak of 25 +/- 4 (S.E.M.) pmol Met-enkephalin equivalents/gland at 4 days that was 80 times greater than zero time values. Both PPenk mRNA and EC peptides declined in parallel between 4 and 14 days. The ratio of the copies of proenkephalin (Penk) peptide to PPenk mRNA was estimated to be 25,000 at the time of explantation and after 4 days in culture. From steady-state kinetics half-life estimates of 9.6 h for PPenk mRNA and 14.7 h for Penk peptide were obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Medulla↗

PubMed1991

Preproenkephalin mRNA and enkephalin in normal and denervated adrenals in the Syrian hamster: comparison with central nervous system tissues.

The distribution and characteristics of preproenkephalin (PPenk) mRNA and enkephalin-containing (EC) peptides are compared in CNS and adrenal tissues from Syrian hamsters and Sprague-Dawley rats. Total cellular RNA extracts from both rat and hamster tissues produce a single hybridization band of PPenk mRNA of approximately 1500 bases when analyzed by Northern blot hybridization. Quantitation by solution hybridization reveals that in the hamster the highest levels of PPenk mRNA are found in adrenal (16.3 +/- 1.4 pg equivalents/micrograms RNA (mean +/- S.E.M.)) and striatum (13.3 +/- 0.7), followed by hypothalamus (0.8 +/- 0.2), and hippocampus (0.4 +/- 0.2). In the rat the highest levels of PPenk mRNA are in the striatum (35 +/- 2 pg/micrograms RNA) followed by the hypothalamus (3.0 +/- 0.5), hippocampus (0.3 +/- 0.1) and adrenal (0.18 +/- 0.04). Thus, the rank order of abundance of PPenk mRNA is similar in these CNS tissues for rat and hamster. The hamster adrenal levels are more than 90-fold greater than those of the rat. The abundance of EC peptides in both hamster and rat tissues mirror the rank order found with PPenk mRNA. Hamster adrenal contains the highest level of EC peptides (441 +/- 37 pmol/mg protein (mean +/- S.E.M.)) which is more than 400-fold greater than that of the rat adrenal and 8- to 12-fold greater than that found in rat and hamster striatum or hypothalamus. Both size exclusion chromatography and Western blot analysis indicate that EC peptides in hamster adrenal are predominantly large proenkephalin-like peptides with approximately 6 copies of Met- and 1 copy of Leu-enkephalin and that included in their number is a prominent EC peptide with a molecular weight of 34 kDa. Unilateral denervation of the hamster adrenal results in a time-dependent ipsilateral decrease in EC peptide and PPenk mRNA levels. Thus, by day 8 postsurgery, PPenk mRNA levels have declined by an average of 80% while EC peptides are reduced by 68% when compared to the innervated contralateral adrenal. These results demonstrate the great abundance of PPenk mRNA and EC peptides in the hamster adrenal. They also demonstrate the apparent need for transsynaptic impulse activity to maintain the high steady-state levels of PPenk and EC peptides. These characteristics of the hamster adrenal system provide opportunities for physiological and pharmacological investigations of the regulation of proenkephalin gene expression.

Adrenal Glands↗

PubMed1991

Transsynaptic activity regulates proenkephalin and tyrosine hydroxylase gene expression and the response to reserpine in the hamster adrenal.

Transsynaptic neurogenic activity and reserpine are two signals that cause the proenkephalin (Penk) gene to alter the levels of preproenkephalin (PPenk) mRNA and enkephalin-containing (EC) peptides. In the Syrian hamster adrenal, but not in rat adrenal, both of these signals appear to be positive activators of Penk gene expression. The separate and combined effects of reserpine and denervation on EC peptides and catecholamine systems were investigated in the adrenal of the hamster, a species with relatively high medullary PPenk mRNA and EC peptide levels. Unilateral adrenal denervation resulted in a rapid decrease in PPenk mRNA levels of 54% after 2 days, and by 11 days 90% of Penk mRNA had disappeared. After 4 days both EC peptide and PPenk mRNA levels fell in parallel, whereas total RNA and soluble protein levels were unchanged. Denervation had no effect on TH mRNA levels until 8 days after surgery, and after 11 days both TH mRNA and catecholamine levels had decreased by 35-45%. Reserpine produced a dose- and time-dependent depletion of EC peptides and catecholamines. One day after 5 mg/kg reserpine (given subcutaneously on each of 2 consecutive days), EC peptides were reduced by 80%, norepinephrine by 79%, and epinphrine by greater than 95%. By 4 days after treatment, EC peptides and catecholamines slightly exceeded or had returned to control (concurrent vehicle treatment) values. PPenk mRNA levels, as measured by solution hybridization, were doubled (206 +/- 17%, mean +/- standard error) by day 4. Tyrosine hydroxylase (TH) mRNA levels were increased nearly 7-fold (686 +/- 71%) 24 hr after the first reserpine dose and declined thereafter. Northern blot analysis demonstrated that reserpine did not alter the size of either PPenk or TH mRNAs. Size exclusion chromatography showed a small (20%) reserpine-induced increase in processing of high molecular weight Penk-like peptides. The effects of reserpine, which increases PPenk mRNA, EC peptides, and TH mRNA, were completely blocked by unilateral denervation, whereas the contralateral innervated gland showed the expected responses. The co-localized EC peptide and catecholamine systems, as reflected in their mRNAs, respond differently in both time sequence and magnitude to reserpine and to denervation. Our results support a critical role, in vivo, for transsynaptic mechanisms in the maintenance of the high levels of Penk gene expression in this species and for the positive activation (mediated by reflex neurogenic stimulation) of reserpine on Penk and TH gene expression.

Adrenal Glands↗

PubMed1988

Characterization of enkephalins in rat adrenal medullary explants.

In the rat, removal of depolarizing stimuli to the adrenal medulla by surgical denervation in vivo or by explanting adrenal medullae has been shown to dramatically increase preproenkephalin mRNA, and enkephalin-containing (EC) peptides. To further elucidate the cellular basis of these effects and the role of transsynaptic influences on post-translational processing, we have defined the time course, and characterized EC peptides in rat adrenal medullary explants in control and depolarized states. The rise in EC peptides begins after 1 day in culture and reaches a peak at 4-7 days. Although the onset of the increase in EC peptides in culture is delayed by 12-24 h compared to the changes seen in vivo, following surgical denervation, the time course of peak and duration is remarkably similar. Size exclusion chromatography (SEC) revealed that the major species of newly appearing EC peptides in explanted glands is a high molecular weight peptide of approximately 18,000 with a Met-/Leu-enkephalin ratio of approximately 6. These results suggest that proenkephalin, the initial precursor of the EC peptide family, is the major EC peptide that accumulates in rat adrenal medullary explants. A low-molecular weight EC peptide, found by high-performance liquid chromatography to be free Met-enkephalin, is a minor component of the culture induced increase in EC peptides. Culturing of medullae in the presence of depolarizing concentrations of K+ prevents the accumulation of the proenkephalin-like EC peptides and free enkephalins.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Medulla↗

PubMed1988

Glucocorticoid regulation of enkephalins in cultured rat adrenal medulla.

The effect of dexamethasone on enkephalin-containing (EC) peptide levels and preproenkephalin mRNA levels was determined in adrenal medullary explants (glands) from sham and hypophysectomized (hypox) rats. Culture for 4 days in serum-free medium without dexamethasone resulted in a 13- and 4-fold increase in EC peptide levels in sham and hypox glands, respectively. The addition of dexamethasone (10(-5) M) produced a 20- to 26-fold increase in EC peptides in sham and hypox glands. In serum free medium, hypox glands showed a concentration dependent increase in EC peptides with the ED50 for dexamethasone equal to 5.7 x 10(-7) M. Since the glucocorticoid antagonist RU486 partially blocked the rise in EC peptides in sham glands, it appears that the increase in EC peptides in sham glands in the absence of dexamethasone is a result of a higher concentration of endogenous corticosterone in sham compared to hypox glands. Dexamethasone resulted in a 6-fold increase in preproenkephalin mRNA in hypox glands cultured for 2 days. This increase was approximately proportional to the increase in EC peptides seen at 4 days. In serum free medium progesterone, testosterone, and deoxycorticosterone failed to increase EC peptides in hypox glands. These results indicate that glucocorticoid treatment is required for maximal proenkephalin gene expression and EC peptide biosynthesis in cultured glands.

Adrenal Medulla↗

PubMed1987

Regulation of rat adrenal medullary enkephalins by glucocorticoids.

Opioid peptides and their precursors of the proenkephalin family are found in the chromaffin cells of the rat adrenal medulla in low quantities. However, if the gland is denervated, there is a 10 to 20-fold increase in enkephalin-containing (EC) peptides consisting mostly of the precursor proenkephalin. The denervation-induced rise in medullary EC peptides is blocked by hypophysectomy, and partially reinstated by corticosterone, dexamethasone or ACTH treatment. In the intact rat, intermediate doses of corticosterone or dexamethasone reduce the denervation-induced increase in EC peptides, while a high dose of dexamethasone restores this response. These results indicate that glucocorticoids exert a permissive effect in vivo on the denervation-induced stimulation of EC peptide biosynthesis.

Adrenal Medulla↗

PubMed1987

Further evidence that there is more than one adrenal 21-hydroxylase system.

The 21-hydroxylase activity of microsomes isolated from bovine adrenal cortex have been assayed using [21-3H]17-hydroxypregnenolone and [1,2-3H]17-hydroxyprogesterone as substrates. When the assays are performed in the presence of an NADH regenerating system, to inhibit steroid 3 beta-hydroxy isomerase-dehydrogenase activity, the microsomes oxidize the 3 beta-hydroxy-5-ene steroid at a rate of 0.37 nmol/min.nmol cytochrome P-450 and the 3-keto-4-ene steroid at a rate of 6.4 nmol/min.nmol. When the microsomes are solubilized with Triton CF-54 they lose the ability to oxidize the 3-hydroxy-5-ene steroid, while the specific activity of the microsomes for the 3-keto-4-ene steroid is enhanced 3-fold. In contrast, when the microsomes are solubilized with sodium cholate, their specific activity towards the 4-ene steroid is decreased by 50% while the specific activity for a low concentration of the 5-ene steroid, 1 microM, is unchanged. In addition, when the oxidations of the labeled steroids (at 1 microM) by the microsomes, are examined in the presence of unlabeled 17-hydroxyprogesterone (at 20 microM) the oxidation of the 3-keto-4-ene steroid is inhibited by 92% while the oxidation of the 3 beta-hydroxy-5-ene steroid is only inhibited by 20%. These results all suggest that there are at least two 21-hydroxylases in bovine adrenal tissue, one of which can utilize the 3-keto-4-ene steroids only, the other of which, in addition, can utilize the 3 beta-hydroxy-5-ene steroids as substrates.

17-alpha-Hydroxypregnenolone↗