Circulating immunoreactivities of gastrin,glucagon and VIP after jejuno-ileal bypass

Seven Sprague-Dawley rats (404–440 g) underwent a 90% jejuno-ileal bypass (JIB); the functional loop consisted of $\text{1}\text{3}$ ileum and $\text{2}\text{3}$ jejunum with the bypassed loop being anastamosed to the ascending colon. Seven control rats were sham-operated. After 35 days, the rats were fasted 18 hours and venous blood was collected. Immunoreactivity of gastrin, measured with an antibody binding equally to G17 and G34, was higher in the plasma of the JIB (256±55 SEM pg/ml) than control (85±9 pg/ml) rats. This agrees with recent human studies but is in conflict with results in less mature rats. VIP levels were not significantly different. Glucagon-like immunoreactivity measured with antibodies specific for the C- and N-terminal regions of the hormone, respectively, were also higher in the JIB (510±40 and 129±15 pg/ml) rats.     

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.     

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.     

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.     

Evidence that 19-hydroxyandrostenedione is secreted by the adrenal cortex and is under the control of ACTH and the renin-angiotensin system in man

Plasma 19-hydroxyandrostenedione (19-OH-A-dione) concentrations in man were evaluated using a specific and sensitive radioimmunoassay. Plasma 19-OH-A-dione concentrations (mean ± SE) in normal subjects are 151 ± 14 pg/ml (n=13) in males and 141 ± 9 pg/ml (n=14) in females. Plasma 19-OH-A-dione (mean ± SE) rises significantly during ACTH stimulation (116 ± 25 pg/ml vs 288 ± 38 pg/ml; P<0.01; n=5), declines significantly during dexamethasone suppression (180 ± 30 pg/ml vs 36 ± 14 pg/ml; P<0.01; n=4) and rises significantly during angiotensin II infusion (89 ± 10 pg/ml vs 159 ± 27 pg/ml; P<0.05; n=5). Plasma 19-OH-A-dione in the adrenal vein is much higher than that in the inferior vena cava (2076–3076 pg/ml vs 115–184 pg/ml; n=2). These results demonstrate that 19-OH-A-dione is directly secreted by the adrenal cortex and is under the control of ACTH and the renin-angiotensin system.     

Reduction of LH-RH pituitary and estradiol uterine binding sites by a superactive analog of luteinizing hormone-releasing hormone

The ability of the Luteinizing Hormone-Releasing Hormone (LH-RH) analogs to displace LH-RH from its pituitary receptors was evaluated $\text{in}\text{vitro}$. The two superactive analogs tested showed higher potency than the antagonists and LH-RH itself, D-Trp⁶-LH-RH being the most potent. The LH-RH specific binding activity in the pituitary fluctuated throughout the age of the rats. The highest number of LH-RH binding sites were seen on day 35 of age (276 fmol × 10^?2/pit) and an increment was induced by 0.05 μg D-Trp⁶-LH-RH (400 fmol × 10^?2/pit). However, 1 μg D-Trp⁶-LH-RH reduced the binding of LH-RH at all the times studied. In the control animals the number of estradiol binding sites increased on day 42 of age, and 0.05 μg D-Trp⁶-LH-RH augmented them on day 35 of age. On the contrary, 1 μg D-Trp⁶-LH-RH diminished the estradiol uterine receptors at all the times studied. Similar results were obtained in the ovariectomized-hypophysectomized rats on day 35 of age. Our studies demonstrated a biphasic action of D-Trp⁶-LH-RH on LH-RH pituitary receptors and a direct effect on uterus which could be mediated through the uterine estradiol receptors.     

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.     

Response of two cows with mummified fetus to treatment with cloprostenol (ICI 80, 996)

Two cases of bovine fetal mummification were treated with a single intramuscular injection of 500 μg cloprostenol. Plasma progesterone levels fell rapidly within 24 hours following injection from a mean level of 10.45 ng/ml to 1.05 ng/ml. Extradiol-17β levels were variable and low ($\text{x}\text{= 3.04 pg/ml}$) during the 120-hour postinjection sampling period. The mummies were delivered at 104 and 120 hours postinjection. Both were expelled into the vagina and traction was required to complete removal from the genital tract. No adverse reactions which could be attributed to $\text{cloprostenol}$ treatment were observed.     

Influence of sodium intake on circulating levels of neuropeptide Y

Neuropeptide Y (NPY) is present in the adrenal medulla, in sympathetic neurons as well as in the circulation. This peptide not only exerts a direct vasoconstrictor effect, but also potentiates the vasoconstriction evoked by norepinephrine and sympathetic nerve stimulation. The vasconstrictor effect of norepinephrine is also enhanced by salt loading and reduced by salt depletion. The purpose of this study was therefore to assess whether there exists a relationship between dietary sodium intake and the levels of circulating NPY. Uninephrectomized normotensive rats were maintained for 3 weeks either on a low, a regular or a high sodium intake. On the day of the experiment, plasma levels of NPY and catecholamines were measured in the unanesthetized animals. There was no significant difference in plasma norepinephrine and epinephrine levels between the 3 groups of rats. Plasma NPY levels were the lowest (65.4 ± 8.8 fmol/ml, n=10, Mean ± SEM) in salt-restricted and the highest (151.2 ± 25 fmol/ml, n=14, p < 0.02) in salt-loaded animals. Intermediate values were obtained in rats kept on a regular sodium intake (117.6 ± 20.1 fmol/ml). These findings are therefore compatible with the hypothesis that sodium balance might to some extent influence blood pressure regulation via changes in circulating NPY levels which in turn modify blood pressure responsiveness.     

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.     

Pharmacokinetic study of exogenously administered ß-endorphin using a rapid radioreceptor assay in rats

A rapid radioreceptor assay for measuring ß-endorphin (ß-EP) in unextracted serum has been developed. The method is based upon the inhibition by ß-EP of ³H-naloxone binding to the specific receptors on rat brain membranes, prepared in a stable form of pellets. Effect of serum on the assay was minimized by adding pooled serum to the equal dilution of total serum in the assay mixture. Pharmacokinetic analysis of pharmacologically active ß-EP equivalents (ß-EP eq.) in rats was performed using this method. The serum disappearance of ß-EP eq. after iv administration followed a biexponential decline and pharmacokinetic parameters were calculated by a two-compartment open model. The half-lives of α-phase and ß-phase were 2.6 ± 0.5 min and 6.2 ± 1.6 hr (mean ± SE; n=6), respectively. The volume of the central compartment (V₁) and that of steady-state (Vd_ss) were 67 ± 16 and 480 ± 75 ml/kg (mean ± SE; n=6), respectively. The total body serum clearance (CL_tot) was 2.1 ± 0.9 ml/min/kg (mean ± SE; n=6). The serum disappearance curve of ß-EP eq. obtained in the present study was similar to that previously reported by Houghten et al. (Proc. Natl. Acad. Sci. U.S.A. $\text{77}$, 4588–4591 (1980)), in which the disapperance of total radioactivity of tritiated ß-EP in rats was examined.     

Age-related increase of β1-adrenergic receptor gene expression in rat liver: a potential mechanism contributing to increased β-adrenergic receptor density and responsiveness during aging

In this study we examined whether the levels of gene expressions of the three β- adrenergic receptor (βAR) subtypes, β₁, β₂, and β₃, contribute to age-related increase in βAR density. Liver membranes and total RNA were prepared from young (4- to 6-month-old) and old (24-month-old) male Fischer 344 rats. βAR density (B_max) in liver membranes was measured by a radioligand receptor binding assay using the receptor subtype nonselective βAR antagonist ¹²⁵I-pindolol as the radioligand. Steady-state levels of β₂AR mRNA in rat liver were measured by Northern blot analysis; because of the low abundance of β₁AR and β₃AR mRNA in rat liver, the expressions of these genes were measured by a semiquantitative RT-PCR or an RT-PCR. Scatchard analysis of saturation binding curves of the binding assay confirmed an age-related increase in B_max (young: 7.1?±?0.8?fmol/mg protein vs. old: 18.1?±?4.3?fmol/mg protein). No age-related differences were found in the levels of β₂AR mRNA. However, semiquantitative RT-PCR revealed an approximately twofold increase in β₁AR mRNA level between young and old rats (P?<?0.05). β₁AR mRNA levels were also correlated with B_max values for ¹²⁵I-pindolol binding sites in individual rats (r = 0.67; P?=?0.012). β₃AR mRNA, which was demonstrable in rat white adipose tissue by RT-PCR, was generally not detected in livers from young or old rats, with the exception of two old rats with the highest B_max. These results suggest that an age-related increase of β₁AR gene expression contributes to increased βAR density and β adrenergic responsiveness in rat liver during aging.     

Detection of leukotriene C4-like immunoreactivity in tear fluid from subjects challenged with specific allergen

Tear fluid was obtained from allergic subjects from control eyes and eyes challenged with specific allergen and levels of leukotriene C₄ (LTC₄)-immunoreactivity determined by radioimmunoassay. Formal identification of the leukotrienes released was not possible but the levels of LTC₄-immunoreactive material in allergen-challenged tear fluid (4.9 ± 2.3 ng/ml, n = 9) were significantly higher (p < 0.01) than those in control tear fluid (0.07 ± 0.06 ng/ml, n = 9). These results provide evidence that leukotrienes, which account for the biological activity of slow reacting substance of anaphylaxis, may be released in allergic reactions $\text{in vivo}$ in man.     

[DES-ASP1] angiotensin II in the sheep: Blood levels and its effect on plasma renin concentration

The present study describes an improved method for measuring angiotensin III in arterial blood. This was accomplished by SE-sephadex column to separate angiotensin II from angiotensin III prior to radioimmunoassay. The arterial concentration of angiotensin III measured before and after 24 to 48 hours sodium depletion by acute cannulation of parotid gland was 12.4 ± 1.7 fmol/ml (SEM, n=7) and 49.8 ± 10.3 fmol/ml (SEM, n=7) respectively. The arterial concentration of Val⁴-angiotensin III obtained from continuous infusion of Val⁴-angiotensin III at rates of 24 and 48 nmol/h in sodium deficient sheep were 245 ± 32.5 fmol/ml (n=6) and 330 ± 11.4 fmol/ ml (n=7) respectively. The clearance rate of exogenous Val⁴-angiotensin III in sodium deficient sheep after correction for endogenous level was calculated to be 140 ± 13.6 L/h (SEM, n=13). This was in the same order as Ile⁵-angiotensin II and Ile⁴-angiotensin III reported earlier in sodium replete sheep. Prolonged intravenous infusion of Val⁴-angiotensin III at a rate of 48 nmol/h in sodium- deficient sheep suppressed plasma renin concentration to the same extent as equimolar infusions of angiotensin II. This suggests that angiotensin III may inhibit renin secretion by a similar mechanism to angiotensin II.     

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.     

Melatonin Binding Sites in Senegal Sole: Day/Night Changes in Density and Location in Different Regions of the Brain

We localized melatonin binding sites in different brain regions (optic tectum, telencephalon, cerebellum, hypothalamus, olfactory bulbs, and medulla oblongata) of Senegal sole, a species of aquaculture interest, and checked day/night changes in density (B_max) at mid‐light (ZT06) and mid‐dark (ZT18). Plasma melatonin was measured using a radioimmunoassay, while binding assays were performed using 2‐[¹²⁵I]iodomelatonin as a radioligand. Plasma melatonin concentrations were significantly lower at mid‐light (189.5±46 pg/ml) than mid‐dark (455.5±163 pg/ml). Values of B_max were statistically significantly higher in the optic tectum (5.6±0.6 and 12.3±1 fmol/mg prot, at mid‐light and mid‐dark, respectively) and in the cerebellum (7.7±1.1 and 10.6±1.3 fmol/mg prot, at mid‐light and mid‐dark, respectively). Significant day/night differences were only observed in these two tissues. These results show for the first time the distribution of melatonin binding sites within the brain of a flatfish species and their lack of down‐regulation.     

Binding of gonadotropin releasing hormone agonist to rat ovarian granulosa cells

We have previously shown that gonadotropin releasing hormone (GnRH) and its agonists inhibit ovarian functions by a direct action on ovarian granulosa cells $\text{in vitro}$. A labeled GnRH agonist, [des-Gly¹⁰, D-Ser (TBu)⁶, Pro⁹-NHEt]GnRH, was used here to examine the possibility that these inhibitory actions of GnRH were mediated through specific receptors which recognize GnRH. Ovarian membrane fractions obtained from immature, hypophysectomized diethylstilbesterol-treated rats were incubated with the ¹²⁵I-GnRH agonist and specific binding was determined by a filtration assay. Stereospecific, high affinity binding was detected in the ovarian membranes; the dissociation constant for the labeled GnRH agonist was determined to be 0.84 ± 0.33 × 10^?10 M and the binding capacity was calculated to be 12.9 fmol/mg protein, or 0.142 fmol/μg DNA. The binding affinity for the GnRH decapeptide was 3.3 times lower than that of the GnRH agonist whereas two GnRH partial peptides did not compete for the ¹²⁵I-agonist binding. After sequential treatment with FSH, LH and prolactin to the hypophysectomized female rats, the ovarian GnRH binding capacity increased per ovary, but decreased per mg ovarian protein.Furthermore, ovarian granulosa cells were isolated and their binding capacity was determined to be 25.2 fmol/mg protein, or 0.133 fmol/μg DNA, suggesting that the granulosa cells contain GnRH binding sites. Thus, this report demonstrates the presence of stereospecific, high affinity GnRH binding sites in the rat ovarian granulosa cells.     

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.     

Myometrial LH/hCG receptors during the estrous cycle and pregnancy in pigs

Receptors for luteinizing hormone/human chorionic gonadotropin (LH/hCG) have been identified in porcine, rabbit, rat, and human myometrium. To determine the estrous cycle and pregnancy related changes in the receptor capacity and affinity, radioreceptor assays were performed with membrane homogenates of porcine uterine tissues. Cycling gilts were divided into four experimental groups: I (n=6), day 1–2; II (n=5), day 6–7; III (n=5), day 11–12; and IV (n=6), day 18–20 of the estrous cycle. Pregnant pigs were divided into three experimental groups: I (n=5), day 35–40; II (n=5), day 65–70; and III (n=4), day 95–105 of pregnancy. The concentrations [femtomoles/mg protein (fmol/mg protein)] and affinities of unoccupied LH/hCG binding sites were characterized in all samples of myometrium. Receptor concentrations were highest (P<0.01) in groups II and III (19.3±2.5 and 35.8±2.1 fmol/mg protein, respectively), and was lowest in groups I and IV (5.3±1.4 and 7.5±0.7 fmol/mg protein, respectively). Receptor affinity constants (K_a) were consistent (P>0.05) throughout the estrous cycle [I, (5.1±1.5)×10⁹; II, (3.0±0.8)×10⁹; III, (3.2±0.9)×10⁹; IV, 5.5±0.7×10⁹ lm⁻¹]. Plasma hormone concentrations of progesterone, estrogen and LH were typical of values noted at these times. During pregnancy, receptor concentrations were greatest (P<0.05) in group II (85.4±18.5 fmol/mg protein). In groups I and III receptor numbers were 10.8±2.3 and 26.7±6.6 fmol/mg protein, respectively. The K_a in group I was 10 times greater (P<0.05) than K_a in groups II and III, (I, 3.1±0.9×10¹⁰ lm⁻¹; II, 3.4±0.3×10⁹ lm⁻¹; III, 3.3±1.1×10⁹ lm⁻¹). Plasma hormone concentrations typically found during pregnancy were noted. The function of these LH/hCG binding sites remains unknown; however, changes in receptor capacity during the estrous cycle and pregnancy support a role for modulation of the receptor by hormonal factors.     

Greatly extended viability of rat brain storage vesicles in an intracellular medium based upon a non-permeant polyanion

The MgATP-stimulated accumulation of (-)-³H-nor- epinephrine (NE) by rat brain neuronal storage vesicles has been characterized in a new medium based upon polyacrylic acid (avg. MW 5,000). The medium allows careful regulation of K⁺ concentration (140 mM), has a large buffer capacity, and is non-permeant to membranes. Light scattering measurements have confirmed the osmotic stability of vesicles suspended in this medium. Vesicular accumulation of (-)-³ H-NE (Km 1 × 10^?6 M) in this system (37°) was examined under saturating (10^?5 M) and non-saturating (2 × 10^?7 M) concentrations of NE. At 10^?5 M NE, uptake saturated at 5 min and remained stable for periods up to one hour, with maximal uptake levels (pmol/mg protein) of 15.7±0.30 (37°), 3.0±0.49 (0°), 4.4±0.22 (reserpine pretreated $\text{in}$$\text{vivo}$) and 6.0±0.79 (without MgATP). At 2×10^?7 M NE uptake was biphasic with maximal uptake levels (pmol/mg protein) of 4.04±0.14 (37°), 0.19±0.01 (0°), 0.95±0.01 (reserpine) and 0.83±0.08 (without MgATP). Vesicle preparations refrigerated in this medium for 24 hrs displayed properties quite similar to those measured acutely (NE = 2.2x10^?7 M).