The function and roles of P450c17 in androgen excess states.

Cytochrome P450c17 is a multifunctional enzyme that converts C21 steroids to the C19 sex steroid precursor DHEA. Intuitively, increased expression of P450c17 in the adrenals and ovaries might be expected to accompany androgen excess states, but the function of P450c17 in androgen biosynthesis is not so simple. The abundance of cofactor proteins and the coexistence of other enzymes contribute to the flux of precursor along pathways to different classes of active steroids. This paper explores the function of P450c17 in its proper biological context, using biochemical studies of patients with isolated 17,20-lyase activity to demonstrate key concepts in P450c17 enzymology.

Angiotensin II infused intrarenally causes preglomerular vascular changes and hypertension.

The effects on the renal vasculature and on arterial blood pressure of chronic infusion of low doses of angiotensin II (Ang II) into the renal artery were studied. Sprague Dawley rats were infused continuously with Ang II (0.5, 1.5, or 4.5 ng. kg(-1). min(-1)) or vehicle into the right renal artery (contralateral nephrectomy). Intrarenal Ang II infusion for 25 days produced dose-dependent rises (P:<0.001) in awake mean arterial pressure (111+/-1, 119+/-5, and 130+/-3 mm Hg in rats receiving 0.5, 1.5, and 4.5 ng. kg(-1). min(-1) Ang II, respectively) compared with 105+/-1 mm Hg (vehicle). Renal vessel lumen characteristics were assessed with an established, maximally dilated, isosmotic perfused kidney preparation. This revealed a small dose-dependent right shift in the pressure-flow relation (P=0.05), as well as a dose-dependent right shift and a dose-dependent reduction in the slope of the pressure-glomerular filtration rate relation (P=0.04 and 0.03, respectively). The effects of Ang II infusion on arterial pressure were not affected by the timing of the contralateral nephrectomy but were reduced when the contralateral kidney remained in situ. Acute losartan administration (10 mg/kg IV bolus) produced similar effects on arterial pressure in rats infused with vehicle or Ang II (4.5 ng. kg(-1). min(-1)) for 14 days, P=0.89), indicating the lack of systemic spillover of Ang II. Intraperitoneal Ang II (0.5, 1.5, or 4.5 ng. kg(-1). min(-1) for 25 days) had no effect on arterial pressure. Thus, chronic intrarenal infusion of low doses of Ang II resulted in changes in the renal vasculature compatible with dose-related structural reductions in the lumen diameter of preglomerular vessels and produced dose-related increases in arterial pressure.

Probing structural and functional domains of human P450c17.

Human P450c17 performs at least six chemical transformations, but this spectrum of activity is differentially regulated by structural changes and by redox partner proteins. Furthermore, P450c17 isoforms from different species with approximately 90% amino acid identity exhibit markedly different relative rates for these transformations. Although this phenomenology has been recognized for nearly 20 years, the underlying chemistry and structural basis for these effects are poorly understood. We have constructed a structural model of human P450c17 using computational chemistry to understand informative, naturally occurring human mutations and to provide a rational basis for designing alterations in P450c17 that probe functional domains of the protein. We have mapped with considerable confidence key residues involved in the interaction with redox partner proteins, including K89, R347, and R358, which form positive charges on the "proximal" surface of P450c17. Neutralization of these charges selectively impairs 17, 20-lyase activity without large reductions in 17alpha-hydroxylase activity or 17alpha-hydroxypregnenolone binding. We are now directing our efforts to the identification of key residues in the active site that mediate the substrate specificity and catalytic selectivity of human P450c17.

Inhibitor regulation of tissue kallikrein activity in the synovial fluid of patients with rheumatoid arthritis.

Tissue kallikrein (TK) and alpha 1-antitrypsin (AT)/TK complexes can be detected in SF from patients with RA if components of the fluids which interfere with the detection of TK are removed. alpha 2-Macroglobulin (alpha 2-M) in SF was demonstrated to contain trapped proteases which were still active in amidase assays. Removal of alpha 2-M from RA SF reduced their amidase activity. However, at least some of the remaining activity was due to TK because it was soya bean trypsin inhibitor resistant and trasylol sensitive and was partly removed by affinity chromatography on anti-TK sepharose. Removal of RF from the fluids reduced the values obtained for TK levels by ELISA. Addition of SF to human urinary kallikrein (HUK) considerably reduced the levels of TK detected suggesting the presence of a TK ELISA inhibitor in the fluids. Removal of components of > 300 kDa from SF markedly reduced the TK ELISA inhibitory activity and increased the values for both the TK and alpha 1-AT/TK levels in fluids as measured by ELISA. It is considered this novel inhibitor does not bind to the active site of TK but rather binds to the site reactive with anti-TK antibodies.

Determination of tissue kallikrein and alpha 1-antitrypsin-tissue kallikrein complexes in synovial fluid of patients with rheumatoid, osteo and psoriatic arthritis.

An enzyme-linked immunosorbant assay was developed to determine tissue kallikrein and alpha 1-antitrypsin-tissue kallikrein complexes in pooled synovial fluid of patients with rheumatoid, osteo and psoriatic arthritis. Even though basal values could be determined, the addition of synovial fluid shifted the standard curves for both tissue kallikrein and alpha 1-antitrypsin-tissue kallikrein complex to the right, because of the presence of a novel inhibitor.

Comparison of plasma prekallikrein levels in human skin blister and chamber fluids from volunteer subjects and psoriatic patients.

Suction blisters were successfully raised on lesional, perilesional and non-lesional skin of psoriatic patients. Similar blisters were created on the skin of volunteer subjects. A specific method has been established for the measurement of plasma prekallikrein, and validated for blister fluid, skin chamber fluid, plasma and synovial fluid. Combined levels of plasma prekallikrein and its active form were compared in blister fluids obtained from psoriatic patients and the volunteers. Significantly higher values for plasma prekallikrein were found in the non-lesional blister fluids of psoriatic patients when compared to the volunteer subjects.

Metabolism and characterisation of kinins and Hoe 140 (kinin antagonist) in the synovial fluid of patients with inflammatory joint diseases.

Methods have been optimised for the collection of synovial fluid and the chromatographic separation of individual kinins (bradykinin and kallidin) in the fluid by HPLC. In addition, the stability of the kinin antagonist, Hoe 140, in synovial fluid was compared with that of synthetic bradykinin. Although bradykinin was completely degraded after incubation for only 6 h in pooled synovial fluid obtained from patients with rheumatoid arthritis, Hoe 140 was stable for as long as 2 weeks under the same conditions. These studies will provide quantitative information regarding levels of kinins in inflamed joints and an insight into the therapeutic potential of kinin antagonists.

Effects of bromocriptine and perphenazine on prolactin and progesterone concentrations in pregnant pony mares during late gestation.

Pregnant pony mares in Group A (n = 4) received i.m. injections at 07:00 and 17:00 h of 0.8 mg bromocriptine/kg body weight 0.75 per day beginning on Day 295 of gestation and continuing until parturition. Group B (n = 4) was treated similarly, but perphenazine was administered orally at 0.375 mg/kg body weight twice a day beginning on Day 305 of gestation and continuing until parturition. Mares in Group C (n = 3) received i.m. injections of saline. Mean plasma prolactin and progesterone concentrations were greater (P less than 0.05) for mares in Group C than in Groups A and B from 295 to 309 days of gestation. From 305 days of gestation, plasma prolactin and progesterone concentrations were greater (P less than 0.05) in Group B and C than in Group A mares. Progesterone and prolactin concentrations increased over this period for Group B and Group C mares, but remained constant in Group A mares. From 10 days pre partum through foaling, mares in Group A had lower progesterone (P less than 0.05) and prolactin (P less than 0.01) concentrations than Group B and C mares. All mares in Group A were agalactic at foaling, while all mares in Groups B and C had normal milk secretion. Gestation was longer (P less than 0.05) in Group A than in Group C mares. In Group A, 2 mares retained the placenta for greater than 3 h, 3 mares had dystocia and all 4 mares had thickened, haemorrhagic placentae.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of phenothiazine on plasma prolactin levels in non-pregnant mares.

Sixteen non-pregnant pony mares were divided into four groups of similar age and bodyweight (bwt). Groups were randomly assigned to one of four treatments consisting of oral administration of perphenazine (0.5 and 1.0 mg/kg bwt, phenothiazine (10 mg/kg bwt) and a control group. Blood samples were taken by jugular venepuncture and plasma prolactin concentrations measured using an homologous assay for equine prolactin. Analysis of variance was conducted on data designed as a split plot over time. Perphenazine given orally (0.5 and 1.0 mg/kg bwt) increased plasma prolactin concentrations when measured 3 and 6 h following feeding (P less than 0.05). Prolactin concentrations returned to normal by 11 h post drug administration. There was no response in plasma prolactin concentrations following oral phenothiazine treatment (10 mg/kg bwt). Perphenazine at the 1.0 mg/kg bwt level was discontinued after two days due to two mares exhibiting signs of hyperesthesia.

Plasma concentrations of progestagens, oestrone sulphate and prolactin in pregnant mares subjected to natural challenge with equid herpesvirus-1.

Multiparous pregnant mares, on two studfarms, were studied following natural challenge with equid herpesvirus-1 (EHV-1). They were divided into three groups according to serum complement fixation titres: Group A (N = 11) were not challenged and delivered normal foals; Group B (N = 13) were challenged but delivered normal foals; Group C (N = 23) were challenged and delivered infected foals which were stillborn or lived for less than 31 h. In Groups A and B mean (+/- s.d.) gestational age at delivery was 343 (+/- 8) and 339 (+/- 8) days respectively, whereas in Group C it was significantly (P less than 0.01) shorter (294 +/- 44 days). Group C mares had significantly lower pre-partum concentrations of progestagens except for 2 mares in which the values were comparable to those of mares in Groups A and B; one of these 2 mares delivered a live foal which survived for 31 h. Thin-layer chromatography showed that substances eluting in RF positions similar to 20 alpha-dihydroprogesterone and 5 alpha-pregnane-3,20-dione, were the main progestagen metabolites in plasma at 275-295 and 305-335 days gestation respectively. Incubation of liver, placenta, gonads, kidney and brain revealed no significant difference in the ability of virus-infected and non-infected fetal tissues to metabolize labelled progesterone in the presence of NADPH. Concentrations of plasma oestrone sulphate pre partum, from all groups of mares, were related to gestational age. Plasma prolactin concentrations were elevated at parturition in mares in Groups A and B but lower values were observed in Group C. We conclude that (1) endocrine patterns in maternal peripheral blood of mares with EHV-1 infected fetuses are similar to those of mares carrying normal fetuses and probably are related to gestational age at the time of delivery; and (2) substantial quantities of progestagen metabolites are found in the plasma of late-term pregnant mares.

Plasma prolactin concentrations in non-pregnant mares at different times of the year and in relation to events in the cycle.

Plasma prolactin concentrations were measured in mares using an homologous radioimmunoassay. An annual rhythm in plasma prolactin was found, with concentrations higher during the summer than during the winter. In addition to this seasonal pattern, occasional high concentrations of prolactin were seen when concentrations were otherwise basal. Blood samples taken from mares during an oestrous cycle in October-November showed that prolactin values were basal for most of the cycle, with a marked rise in prolactin shortly before the onset of oestrus. This prolactin peak was associated with an increase in the size of the largest follicle, and with a peak of PGFM in some mares, but did not appear to be related to the LH surge. In oestrous cycles in March and May-June, there was a wide variation in the baseline of prolactin secretion, in accordance with the seasonal pattern already mentioned. However, the peak of prolactin seen around oestrus in October-November was less obvious in March and May-June. Post-partum mares showed a high but irregular profile of prolactin concentrations with no clear-cut pattern in relation to the oestrous cycle.

Plasma prolactin concentrations and cyclic activity in pony mares during parturition and early lactation.

Five pony mares were blood sampled during late pregnancy, foaling and early lactation. An homologous assay for horse prolactin was used to measure plasma prolactin concentrations in these samples. Regular estimates of cyclic activity were also made. Plasma prolactin concentrations rose markedly in the last week of pregnancy and remained high although variable in early lactation, before declining to basal levels by 1-2 months post partum. All mares showed a post-partum oestrus 7.0 +/- 0.9 days after parturition. One mare whose foal died shortly after birth showed a rapid decline in plasma prolactin values after death of the foal and an early oestrous period (4 days after parturition). The pattern of prolactin changes reported for the mare are in agreement with those reported for other mammalian species.

Evidence that the onset of the breeding season in the ewe may be independent of decreasing plasma prolactin concentrations.

Ten ewes of each of two breeds, Dorset Horn (long breeding season) and Welsh Mountain (short breeding season), were given subcutaneous oestradiol-17 beta implants and then ovariectomized. Another 10 ewes of each breed were left intact. On 3 May 1982, all the ewes were housed in an artificial photoperiod of 16L:8D. After 4 weeks, half of the ewes of each breed and physiological state were abruptly exposed to a short-day (8L:16D) photoperiod while the others remained in long days (16L:8D). The time of onset of the breeding season was significantly (P less than 0.05) advanced in ewes switched to short days (12 August +/- 10 days) compared to those maintained in long days (4 September +/- 14 days). Dorset Horn ewes began to cycle (20 July +/- 7 days) significantly (P less than 0.001) earlier than Welsh Mountain ewes (19 September +/- 6 days). Disparities in the time of onset of cyclic activity in ewes of different breeds and daylength groups were echoed in disparities in the time at which plasma LH and FSH concentrations rose in oestrogen-implanted, ovariectomized ewes of the same light treatment group. Prolactin concentrations showed an immediate decrease in ewes switched to short days, but remained elevated in long-day ewes. Since the breeding season started in the presence of high prolactin concentrations in long-day ewes, it seems unlikely that prolactin is an important factor determining the timing of the onset of cyclic activity.

Evidence that the onset of seasonal anoestrus in the ewe may be independent of increasing prolactin concentrations and daylength.

Nine ewes of each of two breeds, Dorset Horn (long breeding season) and Welsh Mountain (short breeding season), were ovariectomized after insertion of subcutaneous implants containing oestradiol-17 beta. A further 9 ewes of each breed were left entire. All of the ewes were placed in an artificial photoperiod of 8L : 16D on 12 December 1980. After 5 weeks half of the ewes of each breed and physiological state were abruptly changed into a long-day photoperiod (16L :8D) while the rest remained in short days. The time of onset of seasonal anoestrus in entire animals was significantly advanced in ewes changed to long days, with the end of the breeding season coming at the normal time of year in ewes maintained in short days. These differences in oestrous cycle activity were reflected by differences in the time at which LH concentrations in ovariectomized, oestrogen-treated ewes on the two light treatments fell to basal values. Prolactin concentrations showed an immediate and sustained rise in ewes changed to long days, but remained low in ewes maintained in short days. Since the onset of seasonal anoestrus occurred in the absence of high levels of prolactin (in short-day ewes), it is concluded that prolactin is not the major vehicle by which seasonal changes in hypothalamic responsiveness to the negative feedback effects of oestradiol are produced. The results suggest that anoestrus may be due to photorefractoriness.