The aging leydig cell: VII. Cytoplasmic estradiol receptors

Plasma estradiol and cytosolic estradiol receptor levels of testes were determined in a group of young (2–3 months) and old (24 months) Sprague-Dawley rats. Estradiol binding sites for the young rats averaged 5.6 ± 0.3 fmol/mg protein ($\text{x}\text{±}\text{SE, n}\text{=12}$), which was comparable to that of the old rats, 5.7 ± 0.3 fmol/mg protein (n=12). Using Scatchard analyses, the association constants at equilibrium of estradiol receptor binding of the old and young rats were the same, 6.1 × 10¹⁰M^?1. Plasma estradiol levels were also similar in both groups-19.6 ± 2.8 pg/ ml (n=14) for the young and 19.2 ± 2.6 pg/ml (n=10) for the old rats. Our results suggest that impaired testosterone biosynthesis in old rats was not due to elevated plasma estradiol levels or to differences in testicular estradiol receptor content.     

Enteric release of vasoactive intestinal peptide after a peptone meal in the dog

Vasoactive intestinal peptide (VIP) concentrations were measured by radioimmunoassay in plasma from portal and peripheral venous blood obtained from six alert, non-anesthetized dogs before and after gastric infusion of a 10% peptone meal. Mean basal portal and cephalic vein plasma VIP concentrations were $\text{42 ± 11.7}$ and $\text{42 ± 8.0}\text{(S.E.M.) pg/ml}$, respectively. No significant changes in peripheral venous plasma VIP concentrations were noted after the peptone meal throughout the duration of the collection period. In contrast, however, the mean VIP concentration in portal plasma increased promptly after the peptone meal with a peak of $\text{79 ± 8.2}\text{pg/ml}$ ($\text{P < 0.02}$) occurring 8 min after infusion of the meal. This was followed by a gradual decline in portal plasma VIP levels, with a return to prefeeding concentrations at 60 min ($\text{44 ± 6.3}\text{pg/ml}$). Results of these studies demonstrate that following gastric infusion of a peptone meal in the dog, portal, but not peripheral, plasma VIP concentrations increase significantly. Failure to detect augmentation of peripheral vein VIP levels after the meal is probably due to hepatic clearance of VIP.     

Effect of metabolic acidosis on phosphate transport by the renal brush-border membrane

Metabolic acidosis produces a phosphaturia which is independent of parathyroid hormone or dietary phosphorus intake. To study the underlying mechanism, inorganic phosphate (P_i) and glucose transport were studied in brush-border membrane vesicles prepared from the renal cortex of parathyroidectomized rats gavaged for three days with either 7.5 ml of 1.6% NaCl (control) or 1.5% NH₄Cl (acidosis). At killing, blood pH and plasma bicarbonate were $\text{7.36 ± 0.01}$ and $\text{21.8 ± 0.8}\text{mequiv./l}$, respectively, in control and $\text{7.12 ± 0.03}$ ($\text{P < 0.01}$) and $\text{11.1 ± 1.2}$ ($\text{P < 0.01}$) in acidotic rats. Serum P_i was similar in both groups, while 24 h urine P_i excretion was higher in the acidotic group ($\text{P < 0.01}$). Peak sodium-dependent uptake of P_i, measured after 1.5 min of incubation, was higher in controls than acidotic rats ($\text{4442 ± 464}$ vs. $\text{2412 ± 259}\text{pmol/mg}$ protein, $\text{P < 0.01}$), whereas peak glucose uptake at 1.5 min was not significantly different between the groups. Equilibrium values for P_i and glucose uptake were similar in the two groups. $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for P_i uptake in the control and acidotic animals were not different, 0.036 and 0.040 mM, respectively. By contrast, $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ was higher in controls than in the acidotic group, 3.13 vs. 1.15 nmol/mg protein per 15 s. These results suggest that metabolic acidosis directly inhibits P_i uptake by the brush border of the proximal tubule by decreasing the availability of P_i carriers of the renal brush-border membrane.     

The measurement of 18-hydroxy-11-deoxycorticosterone in human plasma by radioimmunoassay

A radioimmunoassay method for the measurement of plasma levels of 18-hydroxy-11-deoxycorticosterone (18-OH-DOC) has been developed. The antiserum against 18-OH-DOC was produced in rabbits immunized against 18-OH-DOC-3-oxime-bovine serum albumin. Plasma (1–2 ml) was extracted with dichloromethane and chromatographed on paper. The purified extracts were incubated with antiserum at a $\text{1}\text{22,000}$ dilution for $\text{1}\text{2}$ hour at 37°C and for 2 hours at 4°C. Saturated ammonium sulfate was used to separate free from bound 18-OH-DOC. 1, 2-³H-18-OH-DOC was added to all samples to correct for losses and to determine the percent free. Pyridine (0.1%) was added to solvents to maintain the stability of 18-OH-DOC. Recovery after extraction was 58 ± 8 (S.D.)%. The accuracy and precision of the method were acceptable, and a sensitivity of 2 pg per sample enabled the measurement of very low levels of 18-OH-DOC. High specificity was demonstrated by a low blank value (0 ± 0.2 pg) and by demonstrating that alternative paper chromatography separation systems gave results not differing significantly from those obtained by the present method. The mean 8AM plasma 18-OH-DOC level was 8.5 ± 1.2 ng per 100 ml in 18 normotensive control subjects. There was a marked response of plasma 18-OH-DOC to ACTH stimulation and dexamethasone suppression and a significant increase after 3 hours upright posture.     

Influence of caloric intake on gastric inhibitory polypeptide,VIP and gastrin release in man

Blood glucose, gastric inhibitory polypeptide (GIP), vasoactive intestinal polypeptide (VIP) and gastrin secretions were measured over a three-hour period following the ingestion by normal subjects of a mixed meal with two different caloric levels (1055 Kcal and 1192 Kcal). No VIP secretion was observed after either meal. Gastrin release was not modified by the increase of caloric intake (mainly carbohydrates and lipids), whereas GIP secretion was significantly more important after the meal with the highest caloric value (peak at 30 mn: $\text{499.5±250.4}$ vs. $\text{273.4±128.7}\text{pg/ml}$ and integrated response $\text{53.3±20.5}$ vs. $\text{28.2±9.9}\text{ng}\text{×}\text{ml}{}^{\mathrm{?1}}\text{×180}\text{min}{}^{\mathrm{?1}}\text{?p<0.05}$). This difference could not be attributed to glucose since the blood glucose levels were not significantly different. It is more probably related to the total amount of ingested food. This suggests the existence of rapid mechanisms of adaptation to the incoming load of the GIP-producing cells.     

Renal metabolism of substance P in the dog.

Substance P is a potent vasodilatory, diuretic, and natriuretic agent. Since subcellular fractions of the kidney rapidly inactivate substance P in vitro, the present study was designed to examine this observation $\text{in}$$\text{vivo}$ in anesthetized dogs. Arterial, renal venous, and urinary levels of immunoreactive substance P were determined by radioimmunoassay and were found to be 117±11, 128±12 and 659±104 pg/ml, respectively. The urinary and fractional excretion of immunoreactive substance P were 122±22 pg/min and 6.6±2.0%, respectively. When substance P was infused intravenously, the arterial and renal venous plasma levels of immunoreactive material increased whereas the urinary levels did not change. Infusions of 50 ng/kg/min of substance P significantly decreased mean arterial pressure, urinary volume, creatinine clearance as well as the urinary excretion, clearance, and fractional excretion of immunoreactive substance P. During intrarenal infusion of ¹²⁵I-(8-Tyr) substance P, high levels of radioactive material were found in the urine and renal venous plasma which failed to migrate on thin layer chromatography with intact ¹²⁵I-(8-Tyr) substance P. Thus under these conditions, intact substance P was not released from the kidney into the urine or renal venous blood, but instead circulating substance P was rapidly and completely metabolized, probably by both vascular and tubular elements of the kidney.     

Effect of active immunisation against testosterone-3-BSA on circulating levels of testosterone, LH, prolactin and testosterone antibody titre in the male rat

Male Sprague-Dawley rats were actively immunised against testosterone-3-bovine serum albumin (T-3-BSA) and on appearance of detectable anti-testosterone antibodies, elevated serum testosterone and LH concentrations were observed. These concentrations reached values of >28 μg/100ml testosterone and 16 μg/100ml LH in some animals after 5 months of immunisation. The corresponding prolactin values did not appear to differ significantly from controls. The circulating bound testosterone fraction as determined by equilibrium dialysis, rose from 65.0 ± 2.75% before immunisation to 98.7 ± 0.75% in those animals possessing high titre antisera. This entailed a nett $\text{decrease}$ in the concentration of unbound steroid from 144 ± 49 ng/100 ml to 78 ± 25 ng/100ml.     

Elimination kinetics and protein binding of theophylline in the rabbit

The detailed elimination kinetics of theophylline were studied in 27 rabbits. Each received a 10 mg/kg intravenous bolus of aminophylline. The theophylline half-life ($\text{T}\text{1}\text{2}$) was 3.8 ± 0.63 hr. The apparent volume of distribution (V_D) and total body clearance (TBC) for theophylline were 439 ± 60 ml/kg and 81.0 ± 17.3 ml/kg·hr respectively. Theophylline protein binding was determined in 10 animals. The mean bound fraction was 74.3 ± 3.9% (range, 68.3–78.0%); the fraction bound was concentration indifferent over a serum concentration range of 5–20 μgm/ml.     

In. vitro evaluation of corpus luteum function of cycling and pregnant rhesus monkeys: Progesterone production by dispersed luteal cells

Corpus luteum function in the cycling and the pregnant rhesus monkey (Macaca mulatta) was evaluated through short term $\text{in vitro}$ studies of progesterone production by suspensions of collagenase-dispersed luteal cells in the presence and absence of exogenous gonadotropin (human chortonic gonadotropin, HCG). Cells from mid-luteal phase of the menstrual cycle secreted progesterone, as measured by accumulation of this hormone in the incubation medium, and responded to the addition of 100 ng HCG/ml with a marked increase in progesterone secretion above basal level ($\text{63.7 ± 13.1}\text{versus}\text{24.7 ± 5.5}\text{ng progesterone}\text{/}\text{ml}\text{/5 × 10}{}^4\text{cells}\text{/ 3}\text{hr}\text{,}\text{X}\text{± S.E.,}\text{n}\text{= 6;}\text{p}\text{< 0.05}$). However, luteal cells from early pregnancy (23–26 days after fertilization) secreted significantly less progesterone than cells of the non-fertile menstrual cycle (3.6 ± 2.4 versus 24.7 ± 5.5 ng/ml/5 × 10⁴ cells/3 hr, n = 3; p < 0.05) and did not respond to HCG with enhanced secretion. By mid-pregnancy (108–118 days gestation) luteal cells exhibited partially renewed function, and near the time of parturition (163–166 days gestation) basal and HCG-stimulated progesterone secretion (30.2 ± 5.6 and 63.0 ± 13.0 ng/ml/5 × 10⁴ cells/3 hr, respectively; n = 3) was equivalent to that of cells from the luteal phase of the non-fertile menstrual cycle. The data suggest that following a period around the fourth week of gestation, when steroidogenic activity is markedly diminished, the corpus luteum of pregnancy progressively reacquires its functional capacity and at term exhibits gonadotropin-sensitive steroidogenesis similar to that of the corpus luteum of the menstrual cycle.     

Heterogeneity in the performance of outbred sprague-dawley rats in an elevated-plus maze test: A possible animal model for anxiety disorder

A wide variation in the performance of inbred rats measured in the elevated plus maze test suggests a possible genetic basis for anxiety response (AR). To gain further insight into the role of genetics in AR, we have characterized AR in male outbred S-D rats. Rats were placed in the black compartment (BC) facing the wall opposite the aperture and time needed for the animal to exit BC was noted. All rats underwent 3 successive trials 1–1.5 hrs apart. Naive rats showed a wide variation in their AR in trial 1(mean = 89 ± 19 sec, range = 5–360 sec). Sixty-eight % of the rats exhibiting low AR exited BC in <30sec, whereas 16% stayed in for the entire 360 sec (high AR). On successive testing, there was a progressive increase in AR which reached to max on second trial (Trial 1: 89±19, Trial 2: 171± 23, Trial 3: 210± 22 sec, p<0.0001). The time spent in BC on successive trials increased for most rats ($\text{33}\text{44}$), decreased for some ($\text{2}\text{44}$), showed min to no change ($\text{5}\text{44}$) or erratic response ($\text{4}\text{44}$) for others. In conclusion wide variation in the AR in outbred rats could be exploited to study genetic and neurochemical mechanisms of anxiety.     

The effect of naproxen-sodium on the prostaglandin concentrations of the menstrual blood and uterine “jet-washings” in dysmenorrehic women

A group of dysmenorrheic women was treated during two consecutive menstrual bleedings, once with placebo and once with naproxen-sodium (naproxen-Na), a potent inhibitor of prostaglandin synthesis. Concentrations of prostaglandins F and E (PGF, PGE) were assayed in the menstrual blood collected into cervical cups, and in uterine “jet-wash” specimens.In the $\text{menstrual blood}$ the high PGF concentrations of patients receiving placebo were significantly reduced following treatment with naproxen-Na (from ± S.E. 227±78.9 ng/ml to 42±19.5 ng/ml; p=0.03). A significant decrease of PGE concentrations was also observed during naproxen-Na treatment (from 10.8±2.1 ng/ml to 3.4±1.7 ng/ml; p=0.03).In the $\text{uterine “jet washings”}$ naproxen-Na significantly reduced PGE concentrations (p=0.03) while the decrease of PGF concentrations was close to statistical signficance (p=0.06). These results strenthened the premise of causal relationships between naproxen-Na treatment, decreased uterine prostaglandins, reduction of intrauterine pressure, and relief from dysmenorrehic pain.     

An ultrastructural examination of everted rat jejunum

Male, albino, Sprague-Dawley rats were sacrificed by cervical separation. Segments of jejunum were excised, everted and examined with the electron microscope. Examination of tissue fixed immediately after eversion revealed the following changes as compared to non-everted segments fixed $\text{in}$$\text{situ}$ and $\text{in}$$\text{vitro}$: 1) an increase in the length of microvilli from (mean ± S. E.) 0.991 ± 0.011μ for normal tissue to 1.389 ± 0.023μ for everted tissue, 2) an increase in width of microvilli from (mean ± S. E.) 0.089 ± 0.001μ for normal tissue to 0.097 ± 0.001μ for everted tissue, 3) an increase in length and number of lateral membrane interdigitations, and 4) the appearance of intercellular “lakes” in the lateral spaces. The above changes are in those structures hypothesized to be involved with salt and water transport across epithelia and may reflect altered transport rates $\text{in}$$\text{vitro}$ as compared to $\text{in}$$\text{vivo}$.     

Elevation of plasma tyrosine after a single oral dose of L-tyrosine.

Plasma tyrosine concentrations in twelve normal, fasting human subjects were significantly elevated 2–8 hours after they ingested 100 mg/kg or 150 mg/kg tyrosine. Mean plasma tyrosine levels were maximal after 2 hours, rising from $\text{69 ± 3.9}\text{to}\text{154 ± 9.5}\text{nmols/ml}\text{(}\text{X}\text{±}\text{SEM}\text{)}$ after the 100 mg/kg dose and to 203 ± 31.5 nmols/ml after the 150 mg/kg dose (p ≤ 0.001 for both doses). The mean tyrosine ratio (defined as the ratio of plasma tyrosine concentration to the sum of the concentrations of six other neutral amino acids that compete for the same blood-brain barrier uptake system) increased from $\text{0.10 ± 0.02}\text{to}\text{0.28 ± 0.04 (}\text{X}\text{±}\text{SEM}\text{)}$ 2 hours after the 100 mg/kg dose (p ≤ 0.001) and to 0.35 ± 0.05 2 hours after the 150 mg/kg dose (p ≤ 0.005). No side effects of orally-administered L-tyrosine were noted.     

Pregnancy diagnosis in the domestic horse through direct urinary estrone conjugate analysis

Urinary estrone conjugates were measured directly by radioimmunoassay (RIA) in 20 pregnancies from preconception diestrus to day 78 of pregnancy. High performance liquid chromatography separation defined estrone sulfate (ES) as the predominant immunoreactive peak which accounted for 94% to 97% of the total immunoreactivity after chromatography. Diestrous values indexed by creatinine were 0.15 ± 0.07 micrograms/mg Cr, $\text{x}\text{± SEM}$ as compared to estrous values which rose to 0.47 ± 0.14 micrograms/mg Cr, $\text{x}\text{± SEM}$. Urinary ES concentrations significantly increased (P = 0.0001 in pregnant mares from day 35 to day 47 (1.21 ± 0.12 micrograms/mg Cr) as compared to day 25 to day 34 (0.27 ± 0.01 micrograms/mg Cr). Measurement of urinary ES may provide an alternate or augmentive method of pregnancy diagnosis in the domestic mare.     

Effects of phyto-estrogenic alfalfa consumption on plasma LH levels in cycling ewes

Plasma luteinizing hormone (LH) concentrations were determined in five Dorset ewes fed orchard grass hay (Dactylus glomerata) and five ewes fed alfalfa (Medicago sativa). Total phyto-estrogen content ($\text{X}\text{±}\text{SEM}$ genistein equivalents) of the orchard grass hay and alfalfa was 16.9 ± 2.9 and 118 ± 12.3 ppm respectively. LH was determined at regular intervals during the estrous cycles synchronized with progesterone impregnated pessaries and characterized by marker ram and vaginal cytology.Peak LH levels in control ewes (40.1 ± 5.5 ng/ml) were lower (P<0.05) than in ewes fed phyto-estrogenic alfalfa (66.0 ± 16.8 ng/ml). Results also indicate that the LH peak may occur later (P<0.05) in the estrus period of ewes fed phyto-estrogenic alfalfa (15.4 ±4.5 h). These experiments may suggest that peak LH concentrations are elevated and delayed further into the estrus period in ewes fed phyto-estrogenic alfalfa.     

The relationship between 13, 14-dihydro-15-keto PGF2α and LH secretion in bulls

Half-life ($\text{t}\text{1}\text{2}$), volume of distribution (Vd)_and total body clearance (TBC) of 13, 14-dihydro-15-keto PGF_2α (PGFM) were measured in order to determine optimal sampling frequency for accurate measurement of PGFM. Three yearling Holstein bulls (349.2 ± 6.7 kg) and 3 yearling Holstein steers (346.7 ± 7.0 kg) were utilized in a 3 × 3 Latin square design. Animals were given 0, 25 or 50 μg PGF_2α I.V.; blood samples collected every 2 min and plasma PGFM determined. The $\text{t}\text{1}\text{2}$, Vd and TBC of PGFM were 2.3 ± .2 min, 43.3 ± 3.3 liters and 13.7 ± 1.9 liters/min, respectively and were similar for 25 and 50 μg doses. To determine the relationship between endogenous PGFM and LH secretion in bulls, blood samples were collected every 2 min for 12 h in 4 yearling Angus bulls (489.1 ± 11.6 kg). All animals elicited at least one LH surge and PGFM concentrations were measured in samples coincident with the LH surge. Mean plasma PGFM concentrations were greater prior to the LH surge than during the LH surge. In addition, mean plasma PGFM concentration and frequency of PGFM peaks appeared to increase prior to the LH surge suggesting an association between PGFM and pulsatile LH secretion in the bull.     

Variations in the number of pituitary LHRH receptors correlated with altered responsiveness to LHRH

Isolated pituitary cells from metestrous, ovariectomized (OVX), and ovariectomized-estradiol treated (OVX-EB) rats were employed to study the gonadotropin response to luteinizing hormone-releasing hormone (LHRH) challenge and to quantitate LHRH receptors, using a labeled LHRH analog. Ovariectomy (3–4 weeks post castration) resulted in a reduction of LHRH receptor concentration from 34.4 ± 2.1 in metestrous females to 14.3 ± 0.9 fmoles/10⁶ cells. Concomitantly, the luteinizing hormone (LH) response to a near-maximal dose of LHRH (5 ng/ml) decreased from a 3-fold stimulation in intact females to 1.13-fold stimulation in cells from OVX rats. Replacement therapy with EB (50 ug/rat for 2 days) to OVX rats restored LH response and LHRH binding sites (a 2.5-fold stimulation in LH secretion and 32.0 ± 2.1 fmoles/10⁶ cells, respectively). The LH response to LHRH stimulation was not altered after one day of EB treatment although the number of LHRH binding sites was increased. The changes in the number of LHRH binding sites were not accompanied by any alterations in the affinity of the LHRH analog ($\text{K}{}_{\mathrm{d}}\text{? 0.5 × 10}{}^{\mathrm{?9}}\text{M}$). It is concluded that variations in LHRH receptor number reflect the degree of pituitary sensitivity to LHRH and it may suggest that LHRH and estradiol modulation of gonadotropin release is mediated by these receptors.     

B-ETA-Endorphin immunoreactivity in rat and human blood: radioimmunoassay, comparative levels and physiological alterations.

Antiserum against human β-Endorphin (β_hEP) has been obtained from rabbit. The antiserum, diluted $\text{1}\text{1500}$ bound I¹²⁵ β_h-EP, demonstrating an effective range from 10pM to 10nM. The sensitivity of the assay is 2–3 fmoles. This antibody exhibits 10–15% cross-reactivity with human β-Lipotropin (β_h-LPH). β-EP-like immunoreactivity in rat blood has been detected in unextracted samples when compared to blood from hypophysectomized rats. The whole assay and calibration curves are carried out in plasma from hypophysectomized animals. β-EP-like immunoreactivity can be detected in normal rat plasma (75 ± 15 fmole/ml), and exhibits substantial increases with adrenalectomy (287 ± 32 fmoles/ml). In contrast, samples from five healthy normal human males gave values near the limits of detection of the assay (12 fmoles ± 3.9 per ml of plasma). Such values may be due to cross-reactivity of the antiserum with β_h-LPH or other circulating hormones. In contrast, patients with elevated ACTH production and normal pregnant humans exhibit significantly elevated levels of β-EP immunoreactivity in plasma.     

N-acetylation of dopamine and tyramine by mosquito pupae (Aedes togoi)

The $\text{N-}\text{acetyltransferase}$ (NAT) activity of mosquito pupae was measured by a radioenzymatic assay, using [¹⁴C]-, [³H]dopamine, [¹⁴C]tyramine or [¹⁴C]acetyl-CoA. The pupal extract could also generate acetyl-CoA from ATP, acetate and CoA for this acetylation reaction. Both the dopamine- and tyramine-NAT reactions proceeded linearly up to 20 min at an optimum pH of 8.4. It is possible that the same enzyme is involved in the acetylation of both biogenic amines as shown by the competitive inhibition kinetics obtained, and the similarities of the NAT reaction with both amines, in the presence of metal chelators, metal ions, SH reagents and MAO inhibitors. Mn²⁺ stimulated and Zn²⁺ inhibited the reaction. The specific activity of NAT in individual pupae measured soon after pupation showed no significant difference between the male and female pupae: the values obtained were, respectively, $\text{893 ± 57}$ and $\text{861 ± 30}$ pmol [¹⁴C]NAcT formed/min per mg protein and $\text{21.9 ± 1.2}$ and $\text{22.0 ± 1.4}$ pmol [³H]NADA formed/min per mg protein.     

Studies in antifertility agents — Part XLI: Secosteroids — X: Syntheses of various stereoisomers of (±) 2,6β-diethyl-7α-ethynyl-3-(p-hydroxyphenyl)-trans-bicyclo[4.3.0]nonan-7β-ol

The syntheses of (±) 2α,6β-diethyl-7α-ethynyl-3α-($\text{p}$-hydroxyphenyl)-$\text{trans}$-bicyclo[4.3.0]nonan-7β-ol ($\text{8}$), (±)2β,6β-diethyl-7α-ethynyl-3β-($\text{p}$-methoxyphenyl)-$\text{trans}$-bicyclo[4.3.0]nonan-7β-ol ($\text{12}$) and (±) 2α,6β-diethyl-7α-ethynyl-3β-($\text{p}$-hydroxyphenyl)-$\text{trans}$-bicyclo[4.3.0]nonan-7β-ol ($\text{18}$) and their derivatives, which are essentially B-seco-steroids having $\text{cis-anti-trans}\text{,}\text{cis-syn-trans}$ and $\text{trans-anti-trans}$ geometries have been carried out. A study of their antiimplantation activities (AI) and receptor binding affinities (RBA) show that $\text{trans-anti-trans}$ compounds are biologically most potent, followed by the corresponding $\text{cis-anti-trans}$ and $\text{cis-syn-trans}$ compounds. The most potent compound $\text{18}$ is active at 1 mg/kg in rats. Introduction of 7α-ethynyl group increases their AI activity; however, no significant effect on their RBA is observed.