Experimental Biology and Medicine

The dynamics of insulin secretion from pancreatic islets of the Zucker-obese rat were studied by in vitro perfusion of individual islets. Glucose and l-leucine were used as insulinogenic stimuli. Control pancreatic islets were obtained from both normal weight Zucker-thin littermates and equivalent weight Sprague-Dawley rats.

Our results demonstrate that pancreatic islets from 13-wk-old Zucker-obese rats hypersecrete insulin in the basal state and in response to both glucose and amino acid (l-leucine) stimuli. Neither pancreatic islets from control Zucker-thin littermate animals (matched with the Zucker-obese animals for age or for total body weight), nor islets from Sprague-Dawley rats of comparable age and weight demonstrate comparable hypersecretion of insulin.

These findings; i.e., hypersecretion of insulin from obese pancreatic islets, suggest that the plasma hyperinsulinism characteristic of the obese state is maintained, at least in part, by an inherent abnormality of β cell secretion. Whether this abnormality in β cell secretion results from a genetic trait in the Zucker-obese strain or is induced by the insulin resistance of the obese animal is not resolved by this study. In any case, the observed in vitro hyper-insulin secretion from these islets supports the postulation that in vivo peripheral insulin resistance characteristic of obesity may be a physiological response that protects the animal from insulin-induced hypoglycemia.

This investigation was supported by U. S. Public Health Service Grant 5 ROI HE12085, by Career Development Award 1 K04 35843, and by Grants from the KROC Foundation and from Ayerst Laboratories.

This work was done during tenure of D. S. Schade by a Public Health Service Special Post-Doctoral Fellowship HEW AM 55979-01.

Male Japanese quail castrated and treated with β-estradiol-3-benzoate for 3 wk were more resistant than sham-operated males to the necrogenic effects of massive doses of isoproterenol.

The estrogen-treated castrate also weighed significantly more and had significantly lower hematocrit ratios than either the sham-operated male or the intact male. These results indicate that there is a definite sex-related protective effect against this type of cardiac necrosis which cannot be explained on the basis of differences in body weight.

After an overnight fast and oral hydration with water, hypertensive subjects developed a significant natriuresis (mean urine sodium excretions increased from 130 to 291 μeq/min). The incidence of a natriuresis (>200 μeq sodium excreted per minute) was 75% in the hypertensive group (16 subjects) compared to 27% in a previously studied normotensive group (22 subjects). The incidence of a carbohydrate-induced antinatriuresis (>30% decrease in urinary sodium excretion) was 62% in the hypertensive group compared to 41% in the normotensive group. No decrease in plasma volume (¹³¹I-labeled albumin concentration) due to a shift of solute and water intracellularly could be documented to explain the antinatriuretic effect of glucose. An incidental observation was a significant decrease in plasma zinc concentrations after glucose ingestion.

The authors acknowledge the technical assistance of Mr. Carl Haygood, Mr. Howard Guiles, Mrs. Dorothy Shirey, Mrs. Norma Luster, and Mrs. Dee Hammarsten and the nursing assistance of Mrs. Fern Brandt, R.N.

Radiophosphate incorporation into phosphatidic acid is decreased in the red cells of lead-poisoned humans and rabbits. The decrease in activity is not related to the length of red cell survival nor to the differences in osmotic fragility observed in the lead-poisoned cells. Incorporation of radiophosphate into phosphatidic acid of normal red cells is sulfhydryl dependent and occurs at the cell surface. Lipid composition of the red cell membrane of the lead-poisoned humans and rabbits is normal.

The ability of 2 Br-α-ergocryptine (CB-154) to suppress serum prolactin levels was examined in animals under the influence of continuous estrogen stimulation. A single injection of polyestradiol phosphate (Estradurin) once every 21 days produced a constant elevation of serum prolactin. The simultaneous administration of 1 mg/day of CB-154 to estrogenized animals suppressed serum prolactin levels below that of Estradurin alone but the levels were significantly greater than those of animals receiving CB-154 alone. It was suggested that, while CB-154 prevents the release of prolactin, estrogen stimulates prolactin synthesis despite the block of its release. Ether anesthesia may be capable of partially overriding the block of CB-154 and released the stored hormone from the gland.

The authors thank Mrs. Cynthia Van De Walle for her skilled and dedicated technical assistance in the performance of the prolactin radioimmunoassay. We also acknowledge the Rat Pituitary Hormone Distribution Program of the NIAMDD for the rat prolactin used for iodination (RP-1-1) and standards (RP-1). The authors thank Dr. J. H. Trapold of Sandoz Pharmaceuticals, East Hanover, NJ, for the gift of 2 Br-α-ergocryptine (CB-154) used in this study.

Lamellar bodies in individual pulmonary granular pneumocytes in the species examined (mouse, rat, rabbit, and dog) have virtually the same volume density with respect to the cytoplasmic volume as estimated by stereological techniques. The surface-to-volume ratio for these structures also fail to show any interspecies variation in these species.

This investigation was supported in part by U.S. Public Health Service Grant HL-16031 and a grant from the American Lung Association. We thank Dr. D. B. Gail for reviewing the manuscript.

The metabolic inhibitors, actinomycin D, cycloheximide, puromycin dihydrochloride, puromycin aminonucleoside, and p-fluorophenylalanine did not inhibit the release of leukocytic pyrogen whether endotoxin was preincubated with cells for 20 min at 37°C before addition of inhibitor or inhibitor was preincubated with cells for 1 hr before addition of endotoxin. On the other hand, cortisol inhibited release of pyrogen under both experimental conditions. Poly (I): poly(C) was not effective in inducing rabbit leukocytes to produce an endogenous pyrogen.

Proliferation rates and cellular protein content have been measured in cultured fibroblasts derived from the skin of normal volunteers and cystic fibrosis patients. Three methods of measuring growth indicated that under our conditions, CF fibroblasts divide normally with a mean doubling time of 29 hr. During the logarithmic growth phase, however, lower cell protein/DNA ratios were observed consistently in CF cultures. This difference was not present in contact-inhibited, confluent fibroblasts. The finding of an apparent reduction in protein synthesis during rapid division, coupled with an observation by others that CF fibroblasts fail to normally induce collagen formation, suggests the possibility of a disturbance in the biochemical regulation of protein synthesis.

Special thanks are extended to Dr. Paul A. di Sant’Agnese, Chief, Pediatric Metabolism Branch, NIAMDD for his continued support, encouragement, and guidance in this study. In addition, we gratefully acknowledge the skillful assistance of Miss Judith M. Podskalny and Miss Jane M. Willison.

Chemical and endocrine parameters were investigated in commercially available fetal bovine sera intended for use as culture media supplements. A high degree of serum variability was present both within and between suppliers in all major categories investigated. It is suggested that caution be employed in the interpretation of results from experiments utilizing serum supplements without specific quantitation of possible interfering or modulating factors.

The authors thank Dr. H. A. Tucker for the bovine growth hormone and bovine prolactin RIA and Dr. P. Foa for the total and pancreatic glucagon RIA.

The effect of high-molecular-weight levan on skin graft rejection was studied. Daily ip administration of 15-30 mg levan was shown to delay rejection in Balb/c and C57BL recipient mice. An increase in MST value by 3.6 days was obtained in Balb/c mice and of 5.6 days for C57BL.

Since the availability of the synthetic decapeptide, LH-RH, several groups of investigators have produced iodine-labeled hormone for immunological studies. Both the chloramin-T (1-3) and the lactoperoxidase iodination methods have been used (3). However, few data are available on the biological activity of the iodinated product, or on the contamination of ¹²⁵I LH-RH with unlabeled hormone. In 1973, a group (3) reported that the ¹²⁵I LH-RH possessed 13% of the bioactivity of the native hormone, but did not comment on possible contamination with unlabeled hormone. Again in 1973, another group (4) produced bioactive ¹²⁵I LH-RH using a lactoperoxidase technique, and were able to separate labeled from unlabeled hormone by means of polyacrylamide gel electrophoresis.

The aim of the present study was to prepare biologically active ¹²⁵I LH-RH free of carrier hormone which would be suitable for both immunological and pituitary cell membrane-binding studies.

Materials and Methods. Preparation of ¹²⁵I LH-RH. The iodination system was based on the lactoperoxidase method using a hydrogen peroxide generating system described by Miyachi et al. (4). Reagents were added to the iodination vial in the following order: 15 μl 0.01 M PBS (0.01 M phosphate buffer, 0.15 M saline, pH 7.5); 5 μg synthetic LH-RH (Abbott) in 5 μl of 0.01 M PBS; 1.5 mCi of ¹²⁵I (Industrial Nuclear); Lactoperoxidase (Sigma) 50 ng in 10 μl of 0.1 M sodium acetate (pH 5.6); 5 μl glucose oxidase (Miles); 25 μl of 0.1% glucose. All reagents were at room temperature (21°C) when added except for the glucose oxidase (4°C). The reaction was allowed to continue for 1 1/2 min at room temperature, mixing being achieved by finger-flicking. Then, 0.5 ml of 0.01 M phosphate (pH 7.5) was added, and the iodination mixture transferred to 200 mg of Dowex I × 10 (200-400 mesh) in 0.5 ml of 0.01 M phosphate to remove excess unreacted iodine. This was then mixed for 10 sec and centrifuged.

The prostaglandins are a group of closely related naturally occurring acidic lipids which possess diverse biological activity (1-3). The effects of the prostaglandins on the peripheral circulation have been studied extensively and in most peripheral vascular beds E series prostaglandins are potent vasodilators whereas F type prostaglandins are weak vasoconstrictor agents (3-8). In the canine pulmonary vascular bed PGE₁ is a moderately active vasodilator whereas PGE₂ is a weak vasoconstrictor agent (9-11). In contrast PGF_2α is one of the most potent pressor substances known in the canine pulmonary vascular bed, and infusion rates which establish concentrations of less than 9 ng/ml in lobar arterial blood increase pulmonary vascular resistance by more than 100% (9, 10). Although the effects of PGF_2α on the pulmonary vascular bed have been documented, nothing is known about the direct effects of PGF_1α, PGF_1β, and PGF_2β on the pulmonary circulation. The purpose of the present investigation was to compare the effects of PGF_1α, PGF_1β, PGF_2α, and PGF_2β on the canine pulmonary vascular bed under conditions of controlled blood flow in the intact spontaneously breathing dog.

Methods. Thirty-five mongrel dogs (16-25 kg) were anesthetized with pentobarbital sodium (30 mg/kg iv) and were strapped in the supine position to a Philips fluoroscopic table. The trachea was intubated with a cuffed endotracheal tube and the animals spontaneously breathed room air. A specially designed 20F double-lumen balloon catheter was positioned in the artery of the left lower lung lobe from the external jugular vein under fluoroscopic guidance (Philips 6-in. intensifier). A Teflon catheter with its tip positioned 2 cm distal to the balloon catheter was used to measure perfusion pressure in the lobar artery. Catheters with side holes near the tip were passed into the main pulmonary artery and the aorta and into a small pulmonary lobar vein and the left atrium transseptally.

Sreter et al. (1) observed that the Ca²⁺ transport function of the FSR 2 was impaired in the muscles of dystrophic animals. Previous work in this laboratory (2, 3) confirmed the discovery of Sreter and further showed that the lipid composition of the FSR obtained from the dystrophic chickens was different from FSR of normal controls. In this study experimental evidence has been obtained that the Ca²⁺-activated ATPase enzyme in the FSR of the dystrophic chickens is also different from that of the normal control. On the other hand, no alterations were found in the activity of the natural tropomyosin (tropomyosin plus troponin) in the muscles of the dystrophic chickens.

Materials and Methods. All reagents used were of analytical grade. Chickens inflicted with genetically controlled muscular dystrophy were obtained from the Department of Avian Sciences of the University of California, Davis, CA. The FSR was prepared using the method of Martonosi and Feretos (4), and the natural tropomyosin was isolated by the method of Ebashi and Ebashi (5). The yield of natural tropomyosin from the breast muscles of both the normal and the dystrophic chicken (2 mg natural tropomyosin per g of muscle protein) was approximately the same. Troponin-free actin was prepared according to the method of Bailin and Barany (6), and myosin A and myosin B were prepared as described by Mommaerts (7). The ATPase enzyme of the FSR was solubilized by the method of Ikemoto et al. (8) except that we omitted glycerol from the mixtures. The ATPase activity was measured as described previously (3) except that the incubation time was 1 min. The composition of each assay mixture will be specified in the text.

Results. The Ca²⁺-activated “extra” ATPase activity was studied first using the FSR of the normal and the dystrophic chickens as the source of the enzyme.

Six different Balb/c IgA myeloma proteins have been found which bind multiple β-D (16)-linked β-galactopyranose residues. They are J1, S10, X24, X44, J539, and T191B (1). The independent production of different myeloma proteins with the same activity suggests that common natural antigens are present in the environment of the Balb/c mouse and that plasmacytomas may have their origin from precursor cells responding to these natural antigens (2). In fact, it has shown been that S10 and T191B myeloma proteins precipitate with antigenic materials present in food and bedding of mice (3). We now report additional galactans present in the environment of the mouse which precipitate with T191B. One of them, a phosphogalactan from Sporobolomyces sp. consists of approximately equal amounts of α1, 6- and α1, 3-galactosyl linkages (4).

Materials and Methods. Myeloma tumor T191B was obtained from Dr. Michael Potter (National Cancer Institute, Bethesda, MD) and was maintained in Balb/c female mice by subcutaneous passage of tumor fragments. Animals were bled from the ophthalmic plexus after the appearance of a palpable mass and the sera from these animals were pooled.

Quantitative microprecipitin reactions were carried out by adding various concentrations of polysaccharide antigens to 70-μl aliquots of T191B serum and incubating the reactants for 24 hr at 4°C. The N content of the precipitates which formed was determined by Nesslerization (5). Inhibition assays were performed by mixing the inhibiting hapten with T191B serum before the addition of an amount of polysaccharide antigen which gave maximal precipitation.

Screening tests for pollen extracts which would precipitate with T191B serum were carried out by adding 0.025 ml of extract to 0.025 ml of serum, adjusting the volume to 2 ml with isotonic saline, and incubating the mixture at 37°C for 15 min and then overnight at 4°C. In most cases precipitation occurred within minutes.

DPH has a colchicine-like action on metaphase arrest of cultured human lymphocytes. The first step in detoxification of DPH increased its power to accumulate metaphases 3-fold. This hydroxy derivative [5-[4-hydroxyphenyl]-5-phenylhydantoin, HPPH] 3.6 × 10^-4 M was equivalent to colchicine 1 × 10^-5 M in its power to inhibit metaphase completion. The effect of HPPH on mitosis was reversible; colchicine effect was not reversed and vincristine effect was partially reversed by washing drug from the medium. Hydroxylation of DPH did not change its inhibition of DNA synthesis and enhanced inhibition of protein synthesis to a minor degree. Detoxification increases the colchicine-like action of DPH.

Virgin mice were treated with gonadotropic hormones in order to induce superovulation; at term the embryos were removed by cesarean section.

This treatment induced malformations (mainly forelimb defects and to a smaller extent central nervous system anomalies), as well as an altered sex ratio in the offspring. Both phenomena were statistically significant. These hormone-induced effects on the progeny were significantly dependent on both the dosage of hormones and the time (season) of administration.

Because the time of administration (before mating) of gonadotropic hormones does not coincide with the critical period during embryogenic development for the teratogenic induction of malformations in the limbs and in the central nervous system (ca. 8th-12th day of gestation), the investigated defects are interpreted as of mutagenic origin.

Microtubules were successfully illustrated in thyrotrophs and thyroidectomy cells of rat pituitary glands. In contrast, microfilaments were mostly seen in the nonglandular follicular cells. Numerous microtubules were observed in the early stages of development of the thyroidectomy cell. In thyroidectomy cells microtubules were located in close proximity to mitochondria, endoplasmic reticula, secretory granules, and membranes of Golgi complexes. Consequently, it is suggested that microtubules may play a role in degranulation or other processes associated with the hypersecretory state.

Fig. 1. An example of a thyroidectomy cell thought to represent an early stage in the cellular modification which follows thyroidectomy. Arrows indicate microtubules (X37,000).

Fig. 2. Microtubules (arrows) located among swollen endoplasmic reticula and secretory granules of a thyroidectomy cell. In the lower right portion of the figure is a cytoplasmic process of a follicular cell. This cell process contains numerous microfilaments as well as microtubules (X 45,000).

Fig. 3. Microtubules (arrows) in close proximity to two mitochondria of a thyroidectomy cell (X 55,000).

Fig. 4. Microtubules (arrows) in the vicinity of the Golgi complex of a thyroidectomy cell. G: Golgi zone M: Mitochondria. (X 44,000).

In our previous experiments (4) we demonstrated that L1210 leukemic lymphoblasts were capable of taking up vitamin B-12 when bound to transcobalamin II. Intracellular B-12 was also found to be bound to a protein with the characteristics of TCII. In the present study we have demon-strated that L-1210 cells do not themselves contain a TCII-like protein in the cytoplasmic soluble phase. We have further demonstrated that a portion of this protein-bound B-12 has been converted to the adenosyl and methyl forms and that such coenzyme forms may be countertransported to the exterior of the cell. These observations are consistent with our original suggestion that the entire B-12 complex crosses the plasma membrane. TCII may deliver B-12 to mitochondria where conversion to the coenzyme forms occurs. A portion of the converted vitamin appears to be transported out of the cell where it is free to recirculate.

The Re chemotype mutant of Salmonella minnesota R595 has a cell-wall glycolipid composed principally of 2-keto, 3 deoxyoctonate and Lipid A, which is an antigen widely shared by Enterobacteriaceae. High-titered antiserum against this antigen can be conjugated with fluorescein isothiocyanate for direct detection of this antigen in heterologous bacteria and staining of bacteria in tissue. Alternatively, the indirect immunofluorescence technique can be used for antigen detection on bacterial surfaces and in tissues, and this method can quantitate glycolipid antibody in mammalian sera. The latter may be particularly useful in serologic studies because, although the glycolipid antigen is a surface antigen and purified extracts can be used to coat latex particles, high-titered antisera will not agglutinate bacteria or coated latex particles.

The expert technical assistance of Shahnaz K. Raza is gratefully acknowledged.

In order to investigate the role of C3 in host defense in vivo, normal AKR/J mice, genetically deficient in C5, were depleted of serum C3 by the injection of purified cobra venom factor (CoVF). Concurrent with their C3 depletion, their serum opsonizing activity decreased to a level less than 20% of normal. When these mice were challenged with an intraperitoneal injection of pneumococci 2 hr after the CoVF treatment, the LD₅₀ was from 30 to 80 times lower than the LD₅₀ in saline-treated control animals. When the CoVF was given only 6 hr after the pneumococcal challenge, the LD₅₀ was the same as in the control mice. If the pneumococci were first preopsonized in vitro and then injected into CoVF-treated animals, the LD₅₀ was the same as that in control animals.

These experiments demonstrate that C3 plays a significant role in vivo in the host's defense against infection and that a major part of that role is through its action as an opsonin. Furthermore, these experiments demonstrate that the role of C3 is most significant during the early stages of bacterial invasion.

The authors are grateful to Andrea Swift for her expert technical assistance.

Successful long-term culture of isolated pancreatic islet as well as dissociated islet cells was accomplished with the use of Microtest II trays which provides the very small amount of culture medium that is easily conditioned. In the culture of isolated islet, the ultrastructure of the islet cells was completely preserved up to the 30th day and vigorous secretion of insulin was maintained even on the 75th day. While in the islet cell culture, insulin secretion gradually decreased from the 12th day. The latter procedure makes inoculation of a definite number of dissociated pure islet cells possible, thus opening the way to the quantitative evaluation of B cell function in vitro.

The effect of various concentrations of concanavalin A (Con A) on the in vitro secondary antibody response of rabbit lymph node and spleen cells to sheep red blood cells (SRBC) was studied. Complete suppression of the IgM plaque-forming cell (PFC) response of both lymph node and spleen cultures was observed when 10 μg/ml of Con A was added at the time of initiation of the cultures whereas only partial suppression was observed when 1 μg/ml of Con A was added. Moreover, marked suppression of the immune responses of both spleen and lymph node cultures was observed when 10 μg/ml of Con A was added at 24 hr after antigenic challenge and to a lesser extent when added at 48 hr. Suppression of the IgM PFC response was also detected when spleen cultures were exposed to 10 μg/ml of Con A for as little as 2 hr after antigenic challenge. However, substantial increases in DNA synthesis were observed only in those cultures which were in contact with Con A for at least 24 hr. Finally evidence is presented that the Con A-induced suppression is mediated by a soluble substance (s).

The androgen content of amniotic fluid, plasma, and gonads from 107 fetal lambs was determined by radioimmunoassay in an attempt to understand the ontogeny of gonadal function. Testosterone (T) was too low to be reliably measured in the amniotic fluid from fetuses of either sex. Ovaries were without T activity at an ofy the stages of gestation studied. Testicular T-5α-dihydrotestosterone (T-DHT) concentration steadily decreased from 1.4 ± 0.2 to 0.08 ± 0.01 μg T-DHT/g testis during the last 80 days of gestation. This was due to both increasing gonadal size as well as a decline in the absolute amount of T-DHT/testis. T was detected in the plasma of fetuses of both sexes. In females, levels rose steadily from 112 ± 20 to 459 ± 223 pg/ml during the last 80 days of fetal development. The pattern in the male was different in that plasma T at 70 days of gestation was 658 ± 145 pg/ml; then fell to 279 ± 51 pg/ml at about 100 days of gestation. Plasma T then rose to 866 ± 141 pg/ml near term. It is suggested that nongonadal testosterone production increases during fetal life and that T secretion by the fetal testis may contribute steadily less to the plasma pool of T as gestation proceeds.

The John R. Morrell Packing Company, Sioux Falls, South Dakota, was most cooperative in allowing us to obtain tissue and blood samples. H. Eiler and R. Curnock assisted in the collection of the samples. Mrs. Lei-Hwa Yen performed much of the technical work reported here.

Oral contraceptive steroids, norethynodrel and mestranol, were fed to 11-wk-old female Sprague-Dawley rats, in combination and in quantities proportional to those used by women for contraceptive purposes. Three experiments were performed. The first experiment, demonstrated that 10 wk of treatment, impaired the animal's glucose tolerance, but not its insulin response to glucose. The second experiment demonstrated that 6 wk of steroid feeding, decreased the in vivo conversion of blood U-¹⁴C-labeled glucose into adipose tissue fatty acids and into diaphragm glycogen, although the effect on the diaphragm was not statistically significant. In the third experiment, it was found that the uptake of 2-deoxyglucose-l-¹⁴C by the adipose tissue removed from rats after 6 wk of treatment, was not different from that of control tissue, but the uptake by the hemidiaphragms was slightly lower.

Assessment of certain parameters of renal function were carried out before and 1 wk after total denervation of the heart by a method which leaves nerves to other organs intact. No changes in mean blood pressure, central venous pressure, cardiac output, GFR, or RPF were noted after cardiac denervation. U_NaV after a low sodium diet was similar during a control period before and after denervation, but in response to expansion of the plasma volume a 3-fold greater natriuresis was seen in the denervated group. Alterations in the filtered load of sodium, the secretion of aldosterone, or most of the recently described physical and compositional factors known to influence sodium excretion cannot adequately explain this natriuresis. Expansion of an already augmented plasma volume after denervation or the possibility of a natriuretic or antinatriuretic factor with afferents interrupted in the process of cardiac denervation must be considered as etiologic factors.

During attempts to eliminate Mycoplasma hominis from a monkey kidney BSC-1 cell line with antibiotics, the mycoplasmas were isolated repeatedly. However, the organisms ultimately failed to grow on medium although electron microscopy confirmed that the cell culture still contained mycoplasmas. Thus, the mycoplasma had adapted to an environment in which viable cells were required for growth. Budding mycoplasmas which are indicative of replication were seen associated with viable cells extracellularly. Moreover, structures resembling mycoplasmas were observed budding from the cells which suggests that the mycoplasmas replicate within the cells and are similar to many viruses in their manner of release from the cells.

The authors are pleased to acknowledge the very capable technical assistance of Mrs. M. DeMaggio-Cioffi and Mr. G. Gomayko. This research was supported by NIH Grant RO1 A108282 from the National Institute of Allergy and Infectious Diseases.

The production of neuraminidase by the recombinant influenza virus X7(F₁) in human, monkey, rabbit, hamster, mouse, and chicken cell cultures is inhibited by interferon. Described is a new enzyme assay for neuraminidase that can be applied to the bioassay of interferons. The advantages of this interferon bioassay are its sensitivity, reproducibility, rapidity, and convenience.

We thank Mary Dixon, Joyce F. Otto, and Christine Schoenherr for their excellent technical assistance.

Note added in proof: Experiments subsequent to this manuscript have revealed that the production of neuraminidase in the RK-13 cell line is variable and at times too low for use in an interferon assay.

Note added in proof: Low yields of neuraminidase have recently been obtained in the RK-13 rabbit cell line, an observation which makes this line unsuitable for the neuraminidase bioassay. However, high neuraminidase yields are obtained in another interferon-sensitive line of rabbit skin (RS) cells.

A serum-free culture medium, supplemented with 1% bovine serum albumin, supported the growth of both primary and continuous suspension-type cultures of various mammalian tumor cells. The role of albumin added to the medium was also studied. Defatted albumin failed to support cell growth, unless reconstituted with its lipid extract. Similarly, defatted albumin, when combined with oleic and linoleic acids, also supported cell growth. Therefore, albumin-bound fatty acids play an important growth-promoting role in serum-free medium.

We thank the Nissui Saiyaku Company for supplying the fatty acids. This work was partly supported by a Grant-in-Aid from the Educational Ministry of Japan.

Spleen cells from nonlethally MCMV-infected weanling and adult DBA/2 mice had diminished responses to Con A stimulation. In contrast, only lethal MCMV infections were associated with a complete suppression of the Con A response. The immune response to SRBC was depressed even in asymptomatic infections of weanling and adult mice. A marked maturation of resistance to the lethal effects of MCMV infection was found to occur during the fourth week of life.

We thank Wilhelmina Dost, Julia Wirth, Ken Rosenberg, Joan Bellinger, and Rita Bell for their excellent assistance in this work.

Fusion rate of Cl-1D cells mediated by Sendai virus was decreased by preincubating the cells or the virus in the presence of lysolecithin. However, a significant increase occurred when the cells were preincubated in the presence of amphotericin B. This increase was suppressed by exogenous cholesterol.

Fluoxymesterone, a C-17 methylated derivative of testosterone employed in the treatment of renal-failure patients with refractory anemia, was administered to Sprague-Dawley rats and the effects on plasma triglyceride (TG) and immunoreactive insulin (IRI) levels and the rate of hepatic triglyceride secretion (TGSR) into plasma determined (Triton). Animals treated with fluoxymesterone demonstrated significantly lower TG (<0.05) and no alteration in TGSR. These findings are consistent with other observations which suggest that the triglyceride-lowering effect of androgens results from an accelerated rate of removal of triglyceride-rich lipoproteins from plasma.

This study was supported by Contract 1-AM-3-2212 from the Kidney Disease Control Branch of the National Institute of Arthritis and Metabolic Diseases, and Grant HL 16219-01-A1 from the National Institutes of Health.

The relative role of specific liver cells in the uptake of sulfobromophthalein (BSP) was ascertained by utilizing enzymatically isolated rat hepatic Kupffer and parenchymal cells. Kupffer cells demonstrated the ability neither to remove BSP from the incubation medium nor to form a BSP-glutathione conjugate. In contrast, parenchymal cells removed BSP from the medium and formed a BSP-glutathione conjugate. The rate and maximum uptake of BSP by the parenchymal cells were inversely related to the concentration of serum or albumin in the incubation medium. In an effort to evaluate the influence of ethanol on BSP uptake, parenchymal cells were incubated in the presence of varying concentrations of ethanol. No alteration in BSP uptake was induced by the prior addition of ethanol to the incubation medium. The uptake and conjugation of BSP are exclusive functional expressions of the hepatic parenchymal cell population.

Within 30 min of starting continuous iv infusion of 333 μg thyrotropin-releasing hormone (TRH)/hr into 12 cows, serum prolactin (PRL) increased more than 10-fold and growth hormone (GH) increased 2.6- to 4-fold above basal concentrations. Constant infusions of 30 mg/hr of prostaglandin F_2α (PGF_2α) increased serum PRL and GH to maxima within 30-40 min which were 64- and 5-fold greater than basal concentrations. Despite continuous infusion of TRH or PGF_2α, PRL declined throughout the 6- to 13-hr infusion interval although it remained well above preinfusion or saline-infusion control values. Serum GH declined more rapidly than PRL in the face of TRH or PGF_2α infusions, reaching basal concentrations in one experiment within 1-3 hr. Application of milking stimuli during the 5th hr of TRH infusion caused an additional increase of 23 ng/ml of PRL above the TRH-stimulated concentrations. Similarly, intravenous injection of 5 mg PGF_2α during the 5th or 12th hr of TRH infusion increased serum PRL an additional 582-682 ng/ml and further increased serum GH 9-91 ng/ml. When 200 μg TRH was injected during the 5th hr of a PGF_2α infusion, serum PRL increased another 267 ng/ml and GH increased an additional 61 ng/ml. Administration of 10 doses of TRH of 200 μg each in 2 hr did not increase PRL or GH in the serum above that observed when 2.2 mg TRH was infused over a 6-hr period. Collectively, the data suggest that a ceiling exists in cows for secretion of PRL and GH, but this ceiling may be overcome with application of a second heterologous stimulus.

A radioactive microsphere technique has been developed for the measurement of renal blood flow and intrarenal blood flow distribution in the dog (1, 2). Various techniques have been proposed (3-5) for the measurement of renal blood flow (RBF) in the rat in addition to the conventional PAH clearance method. This study presents a simple method for the assessment of RBF in rats using radioactive microspheres.

Method. Eight male Sprague-Dawley rats weighing 200-240 g were fed with Purina Lab Chow and given tap water ad lib. All animals were lightly anesthetized with ether and cannulated with polyethylene tube No. 10 (i.d. 0.011 in., o.d. 0.024 in.) through the femoral artery for blood collection and through the carotid artery into the left ventricle for injection of microspheres. After awakening from anesthesia, animals were placed in restraining cages and allowed to recover for 45-60 min prior to injection. Radioactive microspheres, 15 μm ± 5 μm diameter, (3m Co., St. Paul, MN) were utilized to measure renal blood flow (RBF) and its intracortical distribution. Two different nuclides were used, ⁸⁵Sr and ¹⁴¹Ce, for two separate measurements taken 1 hr apart. For each measurement, approximately 0.1 ml of 2 mg/ml concentration of microspheres in 10% dextran was injected through the carotid artery catheter within 5-7 sec. Just prior to injection, the spheres were sonicated for 5-10 min and then vigorously agitated with a vortex mixer for at least 3 min. Immediately upon injection of the spheres into the left ventricle, the femoral catheter was opened and blood was allowed to flow freely into a preweighed tube for exactly 1 min. Approximately 0.2 ml of blood was collected from the femoral artery in each minute. The adequacy of 1-min collection periods, vis à vis complete removal of microspheres from the circulation, was tested in a separate group of five animals.

This study was undertaken because of uncertainties regarding the influence of hormones on myocardial glycogen metabolism of fed and fasted rats. The results indicate that adrenal hormones exert a stabilizing effect on myocardial glycogen levels in fed animals but are not necessary for synthesis to occur. Hypophysectomy eliminates the glycogen increase that occurs from fasting in normal animals while insulin deficiency leads to elevated glycogen stores in both fed and fasted conditions. These findings suggest that changes in myocardial glycogen metabolism are the result of a synergetic relationship between a variety of hormonal and nutritional factors.

The secretion of lysosomal enzymes from rabbit peritoneal polymorphonuclear leukocytes caused by either cytochalasin B, or cytochalasin B plus chemotactic factors, or by the divalent ionophore, A23187, is associated with a decrease in the volume of these same cells. No quantitative correlation could be found between the extent of lysosomal enzyme secretion and volume decrease for any of the three stimuli. This suggests that the volume decrease is neither the direct cause nor the result of the secretion.

Glucose production was studied in isolated hepatocytes using various substrates and with increasing substrate concentrations (0-10 mM). Fructose was the best gluconeogenic substrate while other substrates studied stimulated net glucose production in the following decreasing order: lactate, pyruvate, glycerol, galactose, alanine, and succinate. Studies on oxygen consumption showed that endogenous respiration was linear for 60 min and was not altered by extracellular calcium. Studies on the incorporation of ¹⁴C-leucine into protein was linear for only 3-4 hr in cells containing low glycogen. However, cells containing high glycogen incorporated ¹⁴C-leucine into protein linearly for 8-10 hr. About 3 mg of protein per g per hr was synthesized by isolated cells when incubated for 4 hr with amino acids mixture, glucose, lactate, and insulin.

The activity of the sialyl ecto-transferase system of normal chick embryo fibroblasts (CEF) and chick embryo fibroblasts transformed with the Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV) have been compared. Neuraminidase treatment of the intact cells increased the sialyl ecto-transferase system activity of control and transformed cells two to three times. The ectotransferase system activity increased as the pH was decreased from 7.8 to 6.0. The temperature optimum for both systems was 40°C. Approximately 60% of the ¹⁴C-sialic acid incorporated at pH 6.5 or above could be removed with neuraminidase. The activity of the transformed cell system with or without neuraminidase treatment was more stimulated by addition of Mn²⁺ ions, particularly above pH 7.0. This difference in ion sensitivity indicates that a different cell surface phenomenon is being studied after transformation.

We thank Lorraine Lorenz and Nancy Fraser for technical assistance.

The hereditary polymyopathy and cardiomyopathy of inbred Syrian hamsters (UM X 7.1) are associated with a marked elevation of serum creatine phosphokinase (CPK) and aldolase levels. Fasting (overnight) further increases (+100-500%) the serum concentration of these enzymes in myopathic hamsters; however, no such effect is demonstrable in normal hamsters, or in first generation offspring produced by cross-breeding the two strains (myopathic and normal). The changes are reversible, and the enzyme values return to previous levels within 72 hr. Neither phenobarbital nor hydrocortisone prevents the rises, as shown by CPK determinations; on the contrary, hydrocortisone elicits even greater serum enzyme increases.

This work was supported in part by the Medical Research Council of Canada (MA-4669).

The authors thank the companies listed in the Materials and Methods section for the compounds used in this study.

The competent technical assistance of Mrs. C. Jubert and Miss L. Robitaille, the secretarial help of Miss L. Faulkner, and the editing by Mr. O. M. Da Silva are also gratefully acknowledged.

The effects of a 500-μg injection of T₃ on the renal handling of citrate by the albino rat was studied by measuring citrate synthase activity, NADP-isocitrate dehydrogenase activity, and plasma, kidney, and urine citrate concentrations 12, 18, 24, 36, and 48 hr after injection. Kidney citrate synthase activity of the T₃-injected rats was significantly lower than the controls in the 24- and 36-hr treatment groups, while NADP-IDH activity was significantly lowered only in the 36-hr treatment group. The injection of T₃ resulted in hypercitricemia in the 12-, 18-, and 48-hr experimental animals while there was no significant change in citrate between the control values and treated values in the 24- and 36-hr experiments. There was no significant change in renal citrate levels in any of the treatment groups, and hypercitrauria was not observed.

The results of the present study suggest that T₃ can control citrate utilization by increasing the levels of circulating citrate and then increasing the utilization of citrate by the kidney. This is facilitated by a decrease in NADP-IDH activity resulting in a decrease in biosynthesis and a decrease in citrate synthase activity resulting in a decrease in FFA metabolism. It is proposed that this system functions in providing fuel (citrate) for the increased Krebs cycle flux occurring in hyperthyroidism.

Experiments were designed to determine how varying levels of plasma lipids affect tissue deposition of α-tocopherol (vitamin E). Hypolipemia was induced by feeding orotic acid, and hyperlipemia was obtained using genetically obese rats. With equal dietary intakes of α-tocopherol, hypolipemic rats had lower plasma and tissue concentrations than rats with normal plasma lipids. An exception was liver, which due to fatty enlargement from orotic acid had more α-tocopherol. Hyperlipemic obese rats had plasma total lipids and α-tocopherol three times those of normal rats with the same intake of α-tocopherol. Tissue concentrations of the vitamin, however, were considerably lower in obese rats. Due to their large adipose mass, obese rats had considerably more total body α-tocopherol than normal rats. It was concluded that both plasma lipid levels and degree of adiposity are important factors in determining tissue deposition of α-tocopherol.

Nascent (newly synthesized) DNA obtained from the regenerating len-system of Triturus is first isolated as single-stranded molecules. These early intermediates are later converted into double-stranded molecules.

We have studied several parameters of prostate function in two inbred lines (2.4 and SsLak) of young and old LSH hamsters. These included weight, acid phosphatase, and [³H]testosterone uptake as influenced by age, castration, and androgen treatment. In the hamster, prostatic acid phosphatase concentration was found to vary inversely with androgen levels, contrary to the usual assumption that this enzyme is androgen dependent. Prostatic uptake of tritiated testosterone was enhanced by castration and by treatment of castrates with doses of androgen which induced a moderate increase in gland size. With advancing age, the prostates of LSH hamsters (both strains) became atrophic rather than hyperplastic, in contrast with a previous report (1). This atrophy appeared to be a consequence of decreased testicular function. The LSH hamsters appear to be a suitable model for the study of senescent changes in the male reproductive system.

The interaction of thyrotropinreleasing hormone (TRH) and ergocryptine or apomorphine in affecting prolactin secretion was examined. Ergocryptine and apomorphine inhibited the release of ³H-prolactin labeled in an in vitro incubation of the rat anterior pituitary and led to a retention within the gland of ³H-prolactin. TRH (1.4 × 10^-8 M) partially blocked the inhibitory effect of 4 × 10^-10 M ergocryptine on ³H-prolactin release and totally blocked the effect on radioimmunoassayable prolactin release. TRH also completely blocked the inhibitory effects of 5 × 10^-8 M apomorphine on both ³H-prolactin and radioimmunoassayable prolactin release. Since TRH can also completely block the inhibitory effects of dopamine upon prolactin secretion, these results are consistent with apomorphine and ergocryptine stimulating “dopaminergic” sites and also further support the role of TRH as a prolactin-releasing factor.

This investigation was supported by USPHS 1-R01-AM-13776 and NCI-CA-07535. The authors are grateful to Mrs. Pamela Orcutt and Mr. Ronald C. Pace for excellent technical assistance.

In dexamethasone-treated dogs, both antiogensin II and its heptapeptide fragment (des-asp¹-angiotensin II) stimulated the secretion of the adrenal steroids aldosterone, corticosterone, and cortisol. Further, there was no statistically significant difference in the steroidogenic potency of the two peptides. An interesting incidental finding was the decrease in PRA with both peptides, again with no demonstrable difference in the magnitude of the responses. The present data are consistent with the hypothesis that the heptapeptide mediates responses produced by the renin-angiotensin system in both the adrenals and the kidneys.

We thank Lois Hoover, Sandy Anderson, Dale Welch, and John Thomas for their technical assistance.

To study limb vascular responses in man to elevations in plasma calcium concentrations, we infused test isosmolar solutions of CaCl₂ (0.115, 0.230, and 0.460 meq calcium/min) and NaCl and control isosmolar solutions of NaCl into the brachial arteries of 10 normotensive men and eight men with essential hypertension of mild to moderate severity. Limb blood pressures were monitored, limb blood flow was measured by indicator-dilution, and limb vascular resistance was calculated as mm Hg/ml flow/min/100 cm³ limb volume. Measured concentration of calcium in limb venous plasma during infusion of 0.460 meq calcium/min was 11.5 ± 0.8 meq/liter (mean ± SEM) with individual values ranging up to 20 meq/liter. Changes in limb venous serum sodium, potassium, magnesium, and osmolality were similar during control and CaCl₂ infusions. Decreases in limb venous blood hematocrit during CaCl₂ infusions were the same or greater than those during control infusions. The infusions did not significantly change systemic blood calcium concentration or blood pressures. Limb blood flow decreased and resistance increased in response to CaCl₂. Increments averaging as little as 2.2 meq/liter elevated limb resistance by about 45%. Log dose-response curves were linear. Responses did not differ in normotensives and hypertensives (P > 0.8). We conclude that the vascular response to acute elevation of plasma calcium concentrations up to 20 meq/liter in the limb of man is an impressive vasoconstriction. We found no evidence for abnormal vascular responses to calcium in essential hypertensive men.

We appreciate the cooperation and assistance of the staff of the Veterans Administration Hospital, Saginaw, MI, especially Mrs. Mollie Wheatley, Dr. Kent Bradley, and the nursing staff of the medical wards. Dr. Francis J. Haddy aided us in preparation of the manuscript. Mr. Donald L. Anderson's technical assistance was invaluable.

We determined both basal and induced AHH activity in livers of six partially inbred strains of rabbits. Strain III rabbits had the highest enzyme activity upon induction by 3-MCA, i.e., four to five times that in strain WH (noninducible), which has the lowest enzyme activity. AHH induction was also “low” in strains X, OS, ACEP, and AC. F₁ hybrids between strains III and WH revealed a differential response to the induction of liver AHH activity by MCA: the levels of induced hydroxylase activity were consistently higher in (III X WH)F₁ rabbits than in the reciprocal (WH X III)F₁ hybrids. All possible crosses between these two “extreme” strains are now being analyzed to estimate the number of genes involved in their response difference to MCA.

This work was supported in part by a grant from the Council for Tobacco Research, USA, research contract N01 CP 33255 within the Special Virus Cancer Program of the National Cancer Institute, NIH Research Grant RR 00251 from the Division of Research Facilities and Resources, and by an allocation from NIH General Research Support Grant RR 05545 from the Division of Research Resources to the Jackson Laboratory. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.

Human diploid fibroblasts cultured in vitro were monitored for amino acid uptake in preconfluent and confluent cultures under conditions amenable to growth and in confluent cultures arrested in an essentially nonmitotic state. Preconfluent and confluent cultures in growth medium showed similar uptake patterns for leucine and α-aminoiso-butyrate; arrested cultures exhibited a reduced uptake of both amino acids. Kinetic measurements revealed a 4-fold reduction in apparent V _max for α-aminoisobutyrate influx in arrested cultures. These results suggest that the culture conditions used in this study to produce restrictions in mitotic activity likewise influence amino acid accumulation.

These experiments demonstrate the dependency of cuprozinc superoxide dismutase activity in red cells and liver on an adequate dietary intake of copper. The superoxide dismutase activity in red cells decreased to 15% of control values and, therefore, these cells may be used as a convenient model for studying the physiologic consequences of free radicals.

The authors are grateful for the technical assistance of Mr. George Trappett, Mr. Dale Chlarson, Ms. Jacqueline E. Thomas, and Mrs. Alice W. Tustison.

The uptake and retention of ³H-estradiol by the ovine corpus luteum (induced by superovulation) was maximal through days 8-12 of the cycle with little or no uptake and retention during luteinization or regression. LH, but not FSH or prolactin, inhibited the uptake and retention of estradiol via stimulation of progesterone synthesis.

Progesterone-containing implants were generously provided by Dr. R. E. Mauer, Abbott Laboratories. PMS was generously supplied by the Hormone Distribution Program of NIH and Ayerst Laboratories. HCG-Gonamone was generously supplied by the Fort Dodge Laboratories, Inc. LH, FSH and Prolactin were kindly supplied by the NIH Hormone Distribution Program. Aminoglutethimide was generously supplied by Dr. J. J. Chart, Ciba-Geigy.

The growth and metabolic activity of cultured cells derived from human adipose tissue (CAT cells) were studied and compared to cultured skin fibroblasts. The morphological appearance of the CAT cells was distinctly different from that of fibroblasts. The growth rate of CAT cells as measured by ³H-thymidine incorporation was much slower than the fibroblast growth rate. Cultured CAT cells synthesized significantly more ¹⁴C-lipid from ¹⁴C-glucose, while fibroblast cultures had a higher metabolic rate as measured by CO₂ production. Insulin stimulated ³H-thymidine incorporation in both CAT and fibroblast cultures. The CAT cells did not show a consistent insulin response of lipid or CO₂ production, but this may be a reflection of donor age or nutritional status. Even though the CAT cell may be a type of stromal cell peculiar to adipose tissue rather than a preadipocyte or adipocyte, it may prove useful in studies of human obesity.

The ability of penicillin pre-treatment to increase PAH accumulation by slices of newborn rabbit renal cortex was dissected into two components, uptake and runout. The oxygen-requiring component of the uptake process was significantly enhanced by penicillin treatment, whereas runout was unaffected. Kinetically, the data suggest that penicillin alters the affinity of the transport system for PAH. Due to the limitations of such a kinetic analysis, no conclusions may be drawn from such a suggestion. However, it may be concluded that penicillin pretreatment increases renal accumulation of PAH solely by stimulating the uptake process. Elucidation of the molecular changes involved will require techniques more sophisticated than uptake into renal cortical slices.

Instillation of liver extract into antral pouches produced an increase in the serum concentrations of both little (G-17) and big (G-34) gastrins. The molar fraction of G-17 plus G-34 represented by G-17 was about 0.9 in antral mucosa and about 0.3 in serum 3 hr after initiating release with liver extract. The predominance of G-34 in serum can be accounted for only in part by its slower rate of removal from the blood so other factors probably also contribute. Although G-17 contributed only about 30% of the total molar concentration of gastrins in serum, it accounted for about 70% of the acid stimulatory activity because on a molar basis it is about five times more bioactive than G-34.

This work was supported by Grant AM 17328 of the NIAMDD for CURE (Center for Ulcer Research and Education), by USPHS Grant AM 17294, and by a Veterans Administration Senior Medical Investigatorship (Dr. M. I. Grossman). G. J. Dockray was in receipt of USPHS International Postdoctoral Fellowship.

Bod Weld gave technical assistance.

The hepatic K⁺-mobilizing effects of phenylephrine and isoproterenol were studied in dogs equipped with chronic indwelling portal vein catheters. Animals anesthetized with sodium pentobarbital, received intraportal injections of these sympathomimetic amines, alone or in combination, before and after α, or β, or combined adrenergic blockade. Hepatic K⁺ movements were assessed by measuring systemic arterial and hepatic venous K⁺ levels. It was concluded that adrenergic blockade exerted no significant influence on the ability of these agents to provoke the initial release and subsequent uptake of K⁺ by the liver.

Removal of the pericardium in combination with a mild exercise program of swimming resulted in a significant increase in heart weight and heart weight/body weight ratio of young rats. Either exercise or pericardiectomy alone did not significantly alter these parameters as compared to the control group of rats. This finding further substantiates the pericardium's physiologic effect on the heart. The effects of pericardiectomy should be considered in experimental studies of cardiac hypertrophy and in clinical studies involving cardiac surgery.

The mixed lymphocyte reactivity of thymus cells from normal and cortisone-treated, young and old C57BL/6J mice was evaluated. The results indicated that while in the young mice cortisone treatment caused a 12-fold increase in the reactivity of thymus cells, it had no effect on the response of thymus cells from old mice. The significance of the disappearance of cortisone-resistant mature thymus cells in old mice is discussed regarding the general decline in immunocompetence and the increase of autoimmune manifestations in old age.

The authors acknowledge with thanks the technical assistance of Rosalie Hooper.

When FSH is chromatographed on G-100 Sephadex in the absence of Blue Dextran, it elutes as a single peak in the region of monomeric FSH. The addition of Blue Dextran causes a significant portion of FSH, iodinated or uniodinated, to elute in the region of the void volume. When standard curves for FSH are performed under identical conditions, with tracer ¹²⁵I-FSH from the void volume, and from the FSH monomer region, maximal immunoreactivity was significantly greater for the Blue Dextran-associated tracer.

This work was supported by Chief Scientist, Israeli Ministry of Health, Mifal Hapayis, and Population Council.

Within 24 hr after intravenous administration of isotopic 25-hydroxyvitamin D₃ to three normal adults for kinetic studies, one-third of the radioactivity was secreted into the lumen of the duodenum, probably with the bile. The subsequent intestinal reabsorption of over 85% of secreted radioactivity suggests that this major metabolite of vitamin D has a hitherto unrecognized enterohepatic circulation.

Our observation of a dynamic hepatic secretion and intestinal reabsorption of radioactivity administered as ³H-labeled 25-hydroxyvitamin D₃ to vitamin D-replete man is indicative of an enterohepatic circulation that may be of physiologic importance. It is conceivable that interruption in the recycling of 25-OH-D₃ may be an important mechanism of acquired deficiency of vitamin D in gastrointestinal disease.

This investigation was supported in part by Research Grants AM-69-2168, RR 585, and AM-6908 from the National Institutes of Health, Public Health Service. We gratefully acknowledge the technical assistance of Jane Bailey, Pamela A. Bonnes, Diane J. Brinck, Mary C. Matthusen, Garry V. Mussmann, and the nursing staffs of the Russell M. Wilder Clinical Study Unit and the Gastroenterology Unit.

LiCl was found to stimulate RNA synthesis in the mammary gland in a manner similar to that of prolactin. Since LiCl is known to inhibit adenyl cyclase and thus to reduce levels of cyclic AMP, it is concluded that a reduced level of cyclic AMP may be one step in the mechanism whereby prolactin regulates the metabolism of the mammary gland.

Cycling hamsters were injected sc with 750 μg of d-Phe²-d-Leu⁶-LH-RH, an inhibitory analog of LH-RH at 3, 4, 5, and 6 pm on the day of proestrus. Serum LH levels were determined by radioimmunoassay in samples collected throughout the afternoon of proestrus. Treatment with d-Phe²-d-Leu⁶-LH-RH led to 82.8% suppression of the physiological surge of LH as compared with the integrated LH levels of diluent-treated animals. When ova were counted, treatment with d-Phe²-d-Leu⁶-LH-RH was shown to produce 29.6% (P < 0.05) inhibition of ovulation as compared with diluent-treated animals. No rebound phenomenon in serum LH was observed in the evening of proestrus in animals treated with the analog. Incomplete inhibition of ovulation may be due to the possibility that the small LH surge was well in excess of that needed to produce ovulation or could be synergized with an incomplete FSH surge which was not measured in this study. The small peaks of LH still present in the analog-treated animals may be explained by the intrinsic gonadotropin-releasing activity of the analog and/or the schedule of treatment used.