Experimental Biology and Medicine

The diurnal fattening response to prolactin in the Golden Topminnow, Fundulus chrysotus, involves a circadian periodicity of fattening sensitivity to the hormone. Phasing of the rhythm in fish kept in continuous light can be obtained by injections of thyroxin. A phase angle of 18 hr between the time of thyroxin injection and the time of prolactin injection results in conditions favoring the accumulation of fat stores. Phase angles of 6 hr do not promote fattening.

Effective molecular titrations of C1, C4, and C2 revealed values ranging from 75,000 to 123,000 for C1, from 70,000 to 228,000 for C4, and from 160 to 250 for C2 in the serum of five mongrel dogs. A striking feature of these experiments has been the efficiency of the homologous system as compared to that which employs guinea pig components. Complete or partial functional incompatibilities were demonstrated when: (i) C1^gp was employed to make EAC1 intermediate used for the titration of C4^dog; (ii) when EAC1^gp was used in the formation of the EAC4^dog cells which were converted to EAC14^dog cells for the titration of C2^dog; (iii) when C2^gp was introduced to prepare EAC14^dog cells for lysis by C-EDTA, and (iv) when lysis of EAC142^dog cells was attempted with C^gp-EDTA.

Addition of lethally irradiated cells or “nuclei” prepared from them to tumor inocula increased the frequency of tumor takes and decreased latency and host survival. In previous studies, sonication of either cells or “nuclei” abolished this effect. It was postulated that the stimulatory effect is due to the transfer of nutrients from the membrane-bound structures in the lethally irradiated preparations to living tumor cells prior to the establishment of a vascular supply. In the current studies it was found that sonication of ⁷⁵Se-methionine-labeled lethally irradiated cells or “nuclei” resulted in a more rapid disappearance of radioactivity from the site of injection and reduced the uptake of the label into simultaneously injected viable tumor cells. Similarly, tumors developing from inocula containing sonicated ¹²⁵IUdR-labeled irradiated cells retained less radioactivity than tumors which developed from inocula containing unsonicated lethally irradiated cells.

Rats were exposed to 45 psig of oxygen until central nervous system toxicity was evident (approximately 3 hr). Oxygen consumption, lactic acid production, and ATP concentration were studied in the heart tissue of these rats after their hyperbaric oxygen experience. Of these 3 parameters only the lactic acid production proved to be significantly different in the experimental animal. Heart homogenates from the exposed rats produced a greater amount of lactic acid than control rats.

Surgery induced a significant and transient depression of the reticuloendothelial system (RES) as characterized by the impaired phagocytic clearance of colloidal carbon and the gelatinized “RP test lipid emulsion.” RE recovery was observed at approximately 2.5-hr postsurgery, and a phase of RE stimulation existed by 3- and 4-hr postsurgery. Circulating opsonic activity was decreased during RE depression after surgery, and recovered toward control levels during the period of RE recovery. Comparative phagocytic and opsonic determinations suggested that RE depression after surgery was due to the decrease in opsonin activity. These findings support the concept that the increased host susceptibility to disease after surgery may be mediated in part by an alteration of the opsonic system and reticuloendothelial function.

Pituitary tissue and rat pituitary extracts were incubated with dopamine in vitro. The results indicate that dopamine chemically inactivates ACTH and FSH when incubated in high concentration with these hormones for prolonged periods. The data fail to support a direct effect of dopamine on the pituitary release of ACTH and FSH, and dopamine does not alter the pituitary response to hypothalamic extract.

Rats were treated with norethynodrel with mestranol given orally mixed with their diet in amounts calculated to give 1 and 10 × the normal human amount on a weight basis. The animals which received 10 × normal norethynodrel-mestranol had a significant decrease in their blood pressure response to the largest amount of angiotensin and a significant increase in their blood pressure response to renin. These data indicate that renin substrate is rate-limiting in vivo.

Previous work by Dörr et al. (1) in this laboratory has shown that a hypertonic NaHCO₃ infusion increased extracellular water (ECW), measured as the rapidly equilibrating sucrose space, by 25% in hypothyroid dogs and only by 17% in euthyroid dogs. He suggested that this difference could be caused by an accumulation of mucopolysaccharides in the extracellular space of hypothyroids.

Substantially increased amounts of the mucopolysaccharide, hyaluronic acid, have been found (2) in the extracellular space of humans and animals with myxedema (the chronic form of hypothyroidism). This material has been localized in the ground substance of connective tissues. It has been demonstrated (3) that its polyelectrolyte characteristics make hyaluronic acid a very influential substance in determining water and electrolyte distribution in the body. Engel (4), Engel et al. (5) and Joseph et al. (6) studied the ion and water binding characteristics of the ground substance of connective tissues, and showed that under hormonal influences the macromolecules comprising the tissue would depolymerize. Their work demonstrated that ion and water shifts between the various body fluids occurred as a result of this change in the physicochemical state of the ground substance.

It is the hypothesis of this paper that the physicochemical state of the connective tissue ground substance is altered by changes in the hydrogen ion concentration of its environment (the interstitial fluid), and that these changes of state are particularly significant in the hypothyroid. Since a base infusion increased the extracellular and interstitial space in the hypothyroid as compared to the euthyroid, an acid infusion should yield comparable changes in the opposite direction. In the present study, a series of experiments was performed to measure the changes in water and electrolyte distribution in euthyroid and hypothyroid dogs as a result of an infusion of HCl.

Six compounds in a series of 3-substituted triazinoindoles were tested for inhibitory activity against a large number of rhinovirus strains. All of the compounds produced some inhibition of rhinovirus growth as measured by the suppression of viral CPE in WI-38 cell culture tubes. Two of the compounds, 2,2-dimethyl-3-[(5-methyl-5H-as-triazino [5,6-b | indole-3-yl)amino]-1-propanol (SK&F 28938) and 2-methyl-4-| (5-methyl-5 H-as-triazino [5,6-b] indole-3-yl)amino|-2-butanol (SK&F 30097), with paired methyl groups in the side chains were the most active. In gradient plate plaque reduction experiments using HeLa cells, SK&F 30097 had a minimum inhibitory capacity for rhino-viruses ranging from 21 to 32 μM (6–9 μg/ml). HeLa cell morphology was not altered by SK&F 30097 at a concentration of 350 μM (100 μg/ml). The mechanism of the antiviral action of the 3-substituted triazinoindoles is currently unknown.

A method for quantitative assay of oxytocin using strips from the mammary gland of lactating mice has been studied. The method is suitable for determinations of small amounts of oxytocin. Doses as small as 10 μU/ml can be estimated. The precision of the method is high and it is highly specific.

The level of cytochrome P-450 and the activity of taurochenodeoxycholate 6β-hydroxylase of rat liver microsomes were determined after the administration of phenobarbital, cholestyramine, thyroxine and chenodeoxycholate. Treatment with phenobarbital caused increases in both the 6β-hydroxylase activity and P-450 level, while the other compounds caused decreases in both. Quantitatively there was a lack of parallelism between the hydroxylase activity and the level of P-450, indicating that P-450 is not a rate-limiting factor in the 6β-hydroxylase system. An extract of the microsomes was prepared in 1.0 M phosphate (pH 7.6) as previously described. The extract was active in 6β-hydroxylation, with an apparent Michaelis-Menten constant towards taurochenodeoxycholate of 1.2 × 10^-4M, and a maximum rate of hydroxylation of approximately 1 nmole/min/mg protein. The ratio of P-450 to protein of the extract was 2 to 2.5 times greater than that in the microsomes.

Young Fischer rats allowed to exercise grow at a faster rate than litter mates confined to small quarters. The bodies of both groups of rats contain the same amount of water. The growth of the parts studied is proportional to body size except for the adrenal glands and the quadriceps-femoris group of muscles. These grow at a greater rate than the body as a whole. The basal metabolism in the rats allowed to exercise is higher than that of the controls.

Japanese quail fed diets containing 0.5% nitrite developed a methemoglobinemia together with reduced food consumption and depressed body weights. Nitrite also was deposited in eggs, and continued to be present for about 140 hr after nitrite feeding stopped. Female Japanese quail, either controls or nitrite treated, were observed to have less blood hemoglobin, with approximately a twofold increase in blood methemoglobin than males from the equivalent treatments. Ascorbic acid added at 0.5% to the diet had no effect on methemoglobinemia or egg nitrite deposition.

Local anesthetics inhibited the clearance of 4-iodoantipyrine when injected into rat subcutaneous tissue. The order of potency for the inhibition was cocaine > prilocaine > lidocaine > procaine > tetracaine. This order is not the same as their local anesthetic potencies. The inhibition of clearance suggests that the local anesthetics have an effect on capillary uptake in the rat which is not related to their well-known vasodilator effect.

Epididymal sperm have high trypsin-like enzyme (TLE) activity, CPE activity and neuraminidase activity whereas these enzyme activities are much lower in ejaculated sperm. Incubation of epididymal sperm with seminal plasma caused a decrease in each of these enzyme activities, indicating that seminal plasma inhibitors may transfer to the acrosomal enzymes during ejaculation. Boiled and centrifuged seminal plasma did not inhibit the TLE activity of epididymal sperm but did decrease the CPE and neuraminidase activity.

Thoracic duct lymphocytes from male rats with delayed sensitivity to PPD were cultured together with lymphocytes from syngeneic unsensitized female rats. The addition of PPD to the cultures caused a significant increase in mitotic activity of roughly equal magnitude in both sensitized and unsensitized cells. Assuming a clonal organization of the immunological memory, these observations make possible that most cells dividing in response to antigen stimulation are not committed cells [i.e., members of the stimulated clone] but ldquo;recruited” uncommitted cells, thus lowering the concentration of the clone in the population of lymphoid cells.

These studies revealed that cell strains derived from the least mature human embryos were the least sensitive to interferon and those derived from neonatal tissues were the most sensitive to IF. The significance of these observations was not demonstrated; the possible implications of the findings are obvious and were briefly discussed.

This report describes a relatively simple apparatus for the investigation of infectious agents propagated in intact, perfused, mammalian organs. Results of pilot experiments with parainfluenza virus in perfused isolated human embryonic lung preparations are summarized.

Direct passive Arthus reactions were performed in guinea pigs with homologous γ₁ and γ₂ antibody preparations fully reactive in precipitation, passive hemagglutination, and less than 0.1% reciprocally contaminated (as verified with passive cutaneous anaphylaxis and passive hemolysis). Under these conditions, a definite role for the elicitation of the reaction could be ascribed to each of these two immunoglobulins: γ₁ antibodies alone build up a strongly edematous reaction with vascular permeability increase but no hemorrhage; the hemorrhage is conferred to the preceding reaction by γ₂ antibodies; γ₂ antibodies alone are practically incapable of producing a visible reaction. The lack of reaction with γ₂ antibodies alone was not subsequent to a drastic elimination of this type of immunoglobulin since a similar combination of results was obtained when large quantities of γ₂ antibodies were given 20 min after, 20 min or 48 hr before antigen. In the latter case, their persistence in blood with full biological capacities was verified.

Specific myelin binding antibodies have been demonstrated in the serum and globulin fractions of guinea pigs with EAE. This myelin binding antibody is present only in the 7S gamma₂ fraction and appears to be specific for the encephalitogenic fractions of myelin since the immunofluorescence can be inhibited by absorption of the 7S gamma₂ fraction with purified EF.

To determine if the fall in serum alkaline phosphatase (SAP) in rats after whole-body or abdominal X-irradiation was due largely to decreased ability to produce intestinal alkaline phosphatase (IAP), young adult male Sprague-Dawley rats were exposed to 800 R whole-body or regional irradiation. Determinations were made of SAP with or without the addition of L-phenylalanine. The reduction in value of SAP by this inhibitor was used to indicate the relative amount of IAP contributed to the serum. Administration of olive oil by stomach tube, which stimulates production of IAP, caused a sharp rise in the L-phenylalanine-inhibited fraction of SAP in normal starved rats, but virtually no rise 4 days after whole-body or abdominal irradiation. Shielding the abdomen or its upper or middle portions during irradiation gave fairly complete protection and permitted a normal response to olive oil, while shielding the lower abdomen or duodenum gave only slight to moderate protection. Irradiating the entire abdomen or the midportion gave the same effect as whole-body irradiation, while irradiating the upper or the lower abdomen or duodenum caused a moderate suppression of the normal response to olive oil. These findings indicate that injury to the duodenum and journal mucosa is mainly responsible for the fall in IAP in the serum following irradiation and that the injury is sufficient to diminish or prevent synthesis of intestinal aklaline phosphatase even after stimulation by olive oil. This appears to be due primarily to a reduced population of differentiated cells lining the villi.

An adverse effect of hypothermia for 18 hr to the immune response was observed in hamster sera up to 10 days after primary immunization with Influenza A virus vaccine. Hypothermia caused less inhibition to the secondary response than did radiation, while 2000 R completely destroyed the primary response of both active and hypothermic hamsters. Hypothermia under the conditions described did not afford any protection to the radiosensitivity of either the primary or the secondary immune response. The combination of hypothermia and radiation seemed to act synergistically in lowering the immune response. The presence of both IgG and IgM was shown in the sera of all the groups used in the primary response studies. IgM seemed to be the predominant immunoglobulin in the HI test.

Observations were made on the hydrolytic effect of human serum and saliva, as well as hog pancreatic α-amylase, on a new chromogenic substrate, Cibachron blue F3GA-amylose (CBA). Maltose markedly inhibited hydrolysis of CBA while sucrose did not. This obtained whether the enzyme source was human serum, saliva, or crystalline α-amylase. Rabbit liver glycogen and shellfish glycogen also inhibited hydrolysis by either serum or crystalline amylase. The various findings were interpreted to indicate that hydrolysis of CBA reflects amylase activity.

The present experiments demonstrate that the isotonic saline absorption of cecal sacs in vivo is significantly larger in germfree than in conventionally-raised rats living on the same diet. The osmotic permeability of the cecal wall, on the other hand, is not statistically different in the two groups under the experimental in vivo conditions employed. While other findings are compatible with the assumption that the retention of water in the cecal lumen of the intact germfree animal is due to the presence of osmotically active substances to which the cecal mucosa is impermeable, the sodium-coupled water transport seems to be increased as a compensatory mechanism.

Six days after unilateral ureteral ligation, serum colony-stimulating factor (CSF) is four times as high as in nonoperative germfree mice. The elevation in serum CSF is associated with a significant increase in peripheral blood granulocytes, but not lymphocytes, monocytes, or eosinophils. This finding supports the view that CSF not only is capable of stimulating proliferation of granulocytes and mononuclear cells in vitro, but also promotes granulopoiesis in vivo, and can be regarded as a leukopoietin.

The effects of nicotine on H⁺ secretion, transmucosal PD, and resistance were studied in isolated bullfrog gastric mucosa. It was found that nicotine produced a reversible dose-related inhibition of H⁺ secretion, with an increase in transmucosal PD, resistance, and calculated short-circuit current. The results indicate that nicotine may selectively inhibit H⁺ secretory mechanism.

Investigations were carried out to determine the influence of concurrent treatment with uridine on the antileukemic, toxic, and immunosuppressive effects of 5-AzaCyd, a synthetic analog of cytidine. A single treatment on day 1 after tumor implantation with 5-AzaCyd alone (20–90 mg/kg) increased the MST of leukemic mice by 57–79% over the untreated controls. Combination treatment with uridine reduced the antileukemic activity of 5-AzaCyd. Treatment with 150 or 250 mg/kg of 5-AzaCyd was lethal to leukemic as well as nonleukemic mice. Concurrent treatment with uridine reduced the toxic effect of 5-AzaCyd in these mice.

A single treatment with 5-AzaCyd alone (20–250 mg/kg) diminished the surviving bone marrow cells 30 to 6% and CFU to 2% of the untreated control values. Treatment with uridine reduced the toxic effect of 5-AzaCyd on the bone marrow cells and CFU. Similarly, a single treatment with 5-AzaCyd (33–150 mg/kg) inhibited the hemagglutinin synthesis and hemolytic plaque-forming ability of the spleen cells of nonleukemic mice. In hemagglutination tests simultaneous administration of uridine reduced the immunosuppressive effect of 5-AzaCyd to a greater extent when the drugs were administered after, rather than before, SRBC administration, Furthermore, treatment with uridine partially reversed the effect of 5-AzaCyd on hemolytic plaque-forming cells.

DL-Tryptophan was chromatographically resolved into the D- and L-isomers on powdered paper columns. Both isomers of tryptophan were metabolized to ¹⁴CO₂ equally well by the rat. A load of either isomer increased the ¹⁴CO₂ production from the opposite labeled isomer almost twofold, but did not affect the urinary ¹⁴C levels. The kynurenic acid levels were slightly elevated when the D-isomer was given, but nothing like the 30% concentrations seen when labeled D-kynurenine was given to rats in previous studies. The loading doses of either isomer slightly increased the urinary kynurenic acid, quinolinic acid, and picolinic acid levels and the similarities in urinary levels of these components showed that comparable quantities of either isomer were metabolized via similar pathways. The concentration of activity from either isomer in the tissues decreased in the order of liver, plasma, kidney, testes, heart, spleen, and brain. The level of ¹⁴C in the tissues was depressed when a load of either unlabeled isomer was given in the presence of the opposite labeled isomer. No detectable activity appeared in urinary 3-hydroxykynurenine when L-tryptophan was given. Urinary levels of 5-hydroxyindoleacetic acid indicated that this pathway was of minor importance quantitatively.

Bacterial arylamidase has an absolute requirement for an unsubstituted α-amino group of the L-connguration on the N-terminal residue of substrates hydrolyzed by this enzyme. The penultimate residue must also be of the L-connguration. This enzyme acts as an exopeptidase beginning at the N-terminal residue and cleaving one residue at a time.

The surface or interface of bone mineral is an important parameter in bone metabolism. In particular, this surface must play a role in the interchange of ions between the body fluids and bone mineral. This is a report on a study of the specific surfaces obtained on a series of synthetic hydroxyapatites. synthetic amorphous calcium phosphates, and bone samples, treated in different ways. The surface measurements have been made with gas adsorption and small angle X-ray scattering techniques. A comparison of the results obtained by these contrasting methods adds to our knowledge of the interface between bone mineral and the remainder of bone which is essentially water and collagen. In addition, this study gives a comparison of surface measurements made by these fundamentally different methods.

Materials and Methods. 1. Synthetics. Large batches of a series of samples of poorly crystallized hydroxyapatite and amorphous calcium phosphate were prepared by the method described by Eanes et al. (1). Surface area measurments were obtained using low temperature adsorption of N₂ and the application of the BET method (2). Concomitant surface measurements by small angle X-ray scattering were performed.

It was ascertained by wide angle X-ray diffraction (3), that the hydroxyapatites were 100% crystalline (i.e., contained no amorphous fraction) and the amorphous calcium phosphates were free of any crystalline apatite. This assay was necessary because the amorphous phase is a precursor in the chemical precipitation of hydroxyapatite (1) and one may contaminate the other.

The precipitated samples were separated from the solution by freeze-drying and then stored in stoppered vessels at room temperature. Prior to the adsorption measurements the samples were evacuated to 10^-6 Torr at 300° for periods of 18–36 hr. Weight losses on these samples due to loss of water varied from 10–20% as determined by weighing the samples prior to and after evacuation.

A cyclic nonapeptide has been isolated in homogeneous form from an extract of the bovine pineal gland. Structural elucidation, involving amino acid analysis and mass spectrometry, has shown the compound to be 8-arginine vasotocin. Comparing synthetic 8-arginine vasotocin to the pineal hormone by mouse uterine and ventral prostate assays, shows their activities to be identical. The possibility that the pineal peptide is exhibiting an oxytocic effect is discussed.

In earlier papers (1, 2) we reported chemically defined medium CMRL-1415 1 and described the preparation and use of certain macromolecular supplements, namely, α₁-acid glycoprotein (orosomucoid) from the supernatant solution of Cohn's fraction V (method 6), sometimes referred to as fraction VI, of human plasma together with α₂-macroglobulin from horse serum or with commercial dextran (mol wt 100,000–200,000). The latter combination (CMRL-1415-DSCV 1 ) yielded a population doubling time of 2.5 days for newly-isolated mouse embryo cells, whereas in replicate cultures prepared with unsupplemented CMRL-1415 it was 4.33 days.

Although many established cell lines have been adapted to serum-free, chemically defined media, newly-isolated mammalian cells in continuous culture require certain proteins that are usually supplied in the form of a native or dialyzed serum supplement in a suitable defined medium. The nature of these proteins and their nutritional role have not been fully elucidated. It is generally agreed that normal sera contain several proteins that greatly influence cell growth; and these proteins include both activators (3, 4, 6–10) and inhibitors (3–6) of growth.

In view of the multiplicity of these factors in serum, no single technique such as column chromatography or gel filtration will resolve them without certain preliminary separations. It is the purpose of this report to present a method for the separation of growth-active globulins from fetal calf serum. All operations were performed at room temperature unless otherwise specified. A few crystals of chlorobutanol were added as a preservative to all solutions during fractionation (except during the Rivanol 1 treatment). The method of separation is outlined in Table I.

Bentonite adsorption removes certain lipoproteins and clotting components (11, 12). The potassium phosphate precipitates the globulins, leaving most of the albumin in solution (13).

At levels of 1, 5, and 10%, sodium alginate, guar gum, carboxymethyl guar gum, and Questran were tested in diets fed to rats to determine their effects on radiostrontium skeletal retention. Alginate was the only effective agent against radiostrontium retention, approaching, at the 10% level, 70% less ⁸⁹Sr skeletally retained than radio-control animals and a 3-fold improvement over natural biological strontium-calcium discrimination. The strontium-binding ability shown by alginate was probably clue to its functional carboxyl groups.

Numbers of unimmunized CBA spleen cells, too small to give a PFC response in culture by themselves, can produce PFC if they are cultured in the presence of a large number of heavily irradiated cells. The survival after irradiation of the PFC response by normal spleen cells has a D ₀ of 95–105 rads. In contrast, inhibition of the capacity of heavily irradiated spleen cells to facilitate PFC production by normal cells requires doses greater than 2.5 krads. Attempts to demonstrate the provision of a conditioning factor by the heavily irradiated cells were not successful. However, mixtures of normal and heavily irradiated cells in limiting dilution did result in PFC production. These results are in keeping with the hypothesis that cell—-cell interaction between the two cell populations is required for generation of PFC responses in culture. Since it is known that in thymus-marrow synergism in intact animals proliferation of both the thymus- and marrow-derived cell populations is required for PFC production, it is suggested that in culture a third, nonproliferating class of cells is also required. This class of cells can be detected in the spleen and lymph nodes but not in the thymus or bone marrow.

Bradykinin increases the progression of rat thymocytes (suspended in vitro) into mitosis by a cyclic AMP-mediated reaction which stimulates the initiation of deoxyribonucleic acid synthesis.

The effect of partial pancreatectomy, hypophysectomy, and hypophysectomy plus pancreatectomy on the incorporation of ³⁵S sulfate into the aortic mucopolysaccharides of rats was studied. There was a marked peak in sulfate³⁵ uptake 4 hr after the injection of the label in pancreatectomized and in hypophysectomized animals, and the appearance of this peak preceded the development of hyperglycemia in the pancreatectomized animals. In hypophysectomized-pancreatectomized animals, this peak was abolished. The findings suggest that insulin has an important role in vivo in the regulation of sulfate metabolism in vascular connective tissue. The results are discussed in relation to a possible interaction between growth hormone and insulin in the regulation of sulfate uptake and incorporation.

The influence of various chemicals on the proliferation of pigeon crop-sac in response to prolactin was studied. A 1% concentration of ATP or GTP significantly enhanced the crop-sac response of low levels of prolactin. ADP, 3′,5′-cyclic AMP, dibutyryl cyclic AMP, bovine serum albumin and gamma globulin had no beneficial effect. ATP added in increasing concentrations to a constant dose of prolactin (1.0 μg) caused further increases in the crop-sac response.

It would seem that both phentolamine blockade and denervation affected the series-coupled circuits by lowering precapillary resistance and opening precapillary sphincters, thus making a larger capillary surface area available. Capillary hydrostatic pressure probably was also increased. The parallel-coupled circuits were affected by making A-V shunts available to elevated flow. These shunts are apparently in the skin and one could, therefore, expect blood flow to pass through the skin at an increasingly greater percentage of the total limb flow as the flow rate is increased.

The RLP (radiation-leukemia protection) activity of sheep spleen extracts is sharply eluted from the first Sephadex G-200 peak with the proteins of higher molecular weight (“19S peak”), whereas in serum the activity is more diffuse and considerable amounts appear in the lighter fractions (1). Berenblum and his co-workers have suggested that RLP is an α2-macroglobulin (α₂M) (2). As α₂M is readily detectable by its ability to bind trypsin causing protection of the esterase activity of the enzyme from inactivation by trypsin inhibitor (3), we have determined the distribution of the trypsin-esterase binding activity (TEBA) of serum and spleen extracts. The activity follows the RLP elution pattern for spleen but not for serum. Furthermore, the TEBA of serum and spleen exhibit differences in stability to heat and pH changes. In addition to the TEBA of serum and spleen, another characteristic property of α₂-macroglobulin, the trypsin inhibiting capacity (TIC) has also been investigated.

Materials and Methods. The preparation of sheep spleen extracts (40% homogenates in phosphate-buffered saline, pH 7.4, centrifuged 1 hr at 25,000 rpm in a Spinco rotor No. 30 in the cold) and fractionation of extracts and serum have been described (1). Fractionations were performed on a column of Sephadex G-200, equilibrated with phosphate-buffered saline. Both TEBA and TIC were determined by a modification of the methods of Ganrot (3, 4). The determinations were performed on 5-μl samples and the change in absorbancy at 410 mμ was recorded for at least 10 min in a Gilford spectrophotometer at 25°. Immunoelectrophoresis was performed in a Gelman apparatus in 1% agar at pH 8.6 in 0.1 M buffer.

Results. The fractionation on Sephadex of the substances precipitable upon 40% saturation of spleen extract with ammonium sulfate differs from that of the comparable serum preparation.

The data indicate that in the sera from the white Pekin duck with spontaneously occurring amyloidosis there is an elevation in the β-globulins. Electrophoretic data using two buffers are reported. Variation exists in the electrophoretic patterns of plasmas from ducks with and without amyloidosis. Components faster than albumin occur in the sera and plasmas from some ducks—not all, and seem to be in larger concentrations in ducks without amyloidosis.

Benzylpenicilloyl (BPO) haptens of widely different molecular size were compared for their efficiency in eliciting PCA reactions in guinea pigs sensitized with rabbit anti-BPO antibodies. Equimolar doses of different sized haptens were found to be equipotent in eliciting the PCA reactions. Therefore, in this system the elicitation of PCA reactions by multivalent haptens could also be compatible with a simple bridging hypothesis.

Hepatic and splenic uptakes of ⁵¹Cr-labeled sheep red cells (SRC) were significantly depressed in adult rats which had previously been immunized with heterophilic antigen (HA, boiled homogenate of guinea pig kidney). Uptake of ⁵¹Cr-labeled HA was not affected by preimmunization. Administration of HA to newborn rats significantly depressed uptake of ⁵¹Cr-SRC, but not of ⁵¹Cr-HA, given 10–13 weeks later. Administration of SRC to newborn rats failed to interfere with uptake of ⁵¹Cr-SRC or ⁵¹Cr-HA later in life. Since these observations parallel the reduced formation of antibodies for SRC in rats preimmunized with HA, the changes in phagocytosis and immune responses may be attributable to imprinting of numerous macrophages with receptors specific for HA. Subsequently introduced SRC may thus be diverted to HA-specific macrophages and be prevented from contact with macrophages better equipped to phagocytose and process this substrate containing both heterophilic and isophilic antigens.

Administration of morphine sulfate in water-loaded rats exerts an antidiuretic effect that is dose dependent. In animals not waterloaded, the narcotic exerts a diuretic effect at a dose of 3 mg/kg sc but not at 6 and 12 mg/kg. The diuresis is characterized by an increase in urea and calcium excretion. Since, in the past, experiments dealing with the antidiuretic effect of morphine were usually carried out under hydrated conditions, the clinical significance of the effect of the narcotic on urine must be reevaluated, especially under nonwater-loading conditions.

The effects of the prostaglandins E1 and F_1α on the in vitro biosynthesis of cholesteryl palmitate, oleate, and linoleate by rat liver microsomes from cholesterol and free fatty acids was studied. Both prostaglandins exerted an inhibitory action on the enzymatic esterification of cholesterol. The degree of inhibition increased with increasing concentrations of PGE₁ and PGF_1α in the incubation medium. At the lowest concentration used (4.6 × 10^-8 M) cholesteryl linoleate synthesis showed the least inhibition (about 15%) while cholesteryl palmitate formation was inhibited by about 25%. At the higher concentrations of PGE₁ and PGF_1α used, no difference in the degree of inhibition of the individual esters was observed.

The effects of inhibition of monoamine oxidase (MAO) by pargyline on the concentrations of homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) in the cerebrospinal fluid (CSF) of dogs are reported. Administration of pargyline reduced control levels of HVA and 5-HIAA and inhibited the probenecid-induced rise of these acid metabolites. The magnitude of this inhibition was related to the dose of pargyline. Pargyline inhibited the probenecid-induced rise in HVA more effectively than the rise in 5-HIAA, suggesting the existence of different monamine oxidases in dog brain. After completion of the pargyline treatment, the probenecid-induced rise of 5-HIAA and HVA returned to normal in about 1 month. Measurement of the acid metabolites in the CSF is a direct index not only of the MAO activity in brain but of the degree to which the metabolism of a specific monoamine is inhibited. Probenecid treatment makes this method sensitive enough that it might be applied for measurement of brain MAO activity in clinical practice.

The effects of 8-iso-prostaglandin E₁ on the systemic and pulmonary circulations were studied and compared with those of PGE₁ and PGF_2α in anesthetized dogs. It was found that the iv administration of 8-iso-PGE₁ decreased systemic arterial pressure slightly and increased heart rate and myocardial contractile force slightly. The magnitude of the systemic hypotensive effect of 8-iso-PGE₁ was equivalent to approximately 1/125 to 1/250 of that of PGE₁ in dogs. On the other hand, the pulmonary hypertensive action of 8-iso-PGE₁ was 5 times greater than that of PGE₁. The present study indicates that 8-iso-PGE₁ increases pulmonary arterial pressure through its vasoconstrictor action on the pulmonary vascular bed.

Seventy-two female rats were randomly allotted to three groups and mated at average ages of 44, 63, and 82 days. One half of each age group was killed at parturition and the other one half at day 16 of lactation. Although rats in the youngest age group weighed less than those in the other two groups at each weighing, these age and weight differences did not significantly affect litter weight or number of pups per litter at birth. The oldest rats had more total mammary DNA at parturition, but no significant difference existed by the 16th day of lactation. Total RNA doubled during lactation for all three age groups, but there was no significant difference among age groups at parturition or at day 16. Average litter weight gains were not significantly affected by age at mating, indicating that rats bred as early as 40 days of age lactated and raised litters as well as rats bred after 60 days of age.

Addition of microgram amounts of highly purified porcine LH-RH twice daily for 3 days to female rat anterior pituitaries in vitro significantly increased the quantities of LH and FSH released. Total LH and FSH contents in stimulated tissue and medium exceeded those of controls as measured by radioimmunoassays for LH and FSH and by bioassay for FSH.

Dose-response data are reported for three serum antigens evaluated in a rapid screening test suitable for evaluation of anti-inflammatory compounds. Canine serum, when administered subcutaneously, was found to be a more useful antigen than bovine or equine sera for producing an immune response with rapid onset and relatively short duration. The test model described may be completed in 12 days. Dexamethasone, dexamethasone 21-phosphate, prednisolone, and indomethacin were successful in reducing the canine serum uveitis.

The inhibitory effects of aspirin on platelet function persist during long-term administration. This drug would therefore appear to hold promise as an antithrombotic agent.

In an attempt to clarify the mechanism by which biological effects of the polynucleotide complex, I:C, are enhanced by the polycation, DEAE-D, the dose response of the polycation on cell protection and on the induction of interferon was determined for various concentrations of I:C Utilizing kinetic studies at 37 and 4° it was possible to show that attachment of I:C to cells is depressed by DEAE-D. In addition, as much as 100 μg/ml of DEAE-D did not inhibit the function of pancreatic RNase in digesting attached I:C. Pretreatment of cells alone before I:C attachment, gave definite, although reduced enhancement of I:C function, and this enhancement could be negated with the polyanion heparin.

ALS administration failed to influence the clinical, biochemical, morphological including ultrastructural and immuno-histochemical features of the so-called homolous, and autologous phases of nephrotoxic serum nephritis in rats. This indicates that progression of this form of experimental renal disease is not necessarily dependent upon immunologic functions abrogated by ALS.

Changes which occur in hepatic lipid metabolism of hypophysectomized and bovine growth hormone-treated hypophysectomized rats were studied. Synthesis of lipids, as measured by the ability of tissue slices to incorporate ³H-glycerol into lipids, decreased rapidly following hypophysectomy. Three weeks after hypophysectomy, glycerol incorporation was approximately 40% of that in normal rats and remained depressed over the experimental period. Synthesis of acidic lipids was not appreciably affected by hypophysectomy but labeling of all other classes of lipids, particularly triglycerides, was diminished. Daily administration of bovine growth hormone over a 10-day period to animals 4-weeks posthypophysectomy resulted in a 75% increase in total lipid synthesis. Incorporation of label into the lecithin and cephalin fractions exhibited the greatest increase of those lipid fractions studied during the first 5 days of bovine growth hormone treatment.

⁷⁵Se-selenomethionine turnover rate was used as an index for protein metabolism in rats ranging in age from 30 to 600 days of age. The data indicated that protein turnover rate was more rapid in young (150 days or less) than in old rats (600 days of age) and this rate is stable beyond 240 and 360 days of age.

Weanling female Sprague-Dawley strain rats were given sc injections of 50 μg of mestranol, 1250 μg of norethynodrel or their combination three times weekly for 10 weeks, controls received vehicle. Anterior pituitary glands (AP) of each of the steroid-treated groups of donors were pooled for assay of GH content by measuring tibial epiphyseal cartilage width of hypophysectomized recipients. Recipients received four ip injections of AP homogenates containing a total of 1/4 AP, 1/2 AP, or 1 AP; control recipients received saline solution alone. As judged by tibial cartilage width of the recipients, the GH content of the AP of any of the three steroid-treated groups of donors decreased significantly as compared to that of the AP of the control donors.

Chronic treatment with methyldopa, for 4 to 7 days, produced augmentation of pressor responses to angiotensin in the denervated hindleg of dogs, whereas vasoconstrictor responses to norepinephrine were unaffected. The administration of a single iv injection of methyldopa did not alter the hindleg pressor responses to either angiotensin or norepinephrine. Since chronic treatment with methyldopa has been shown to decrease plasma renin activity in man, it is possible that the potentiation of the vasoconstrictor effect of angiotensin seen in dogs after chronic treatment with methyldopa may be related to an effect of this drug on the renin-angiotensin system.

Shortly after polypeptide hormones act on target tissues, the tissues undergo a series of morphologic changes that can be related to the rearrangement of vacuolar systems. Thus thyroid-stimulating hormone induces endocytosis of thyroglobulin, parathyroid hormone induces the bulk exocytosis of lysosomal hydrolases and hydrogen ions from osteoclasts, glucagon stimulates the formation of autophagic vacuoles in liver, and melanophore-stimulating hormone induces the rearrangement of melanosomes in skin (1, 2). Since cyclic 3′, 5′-adenosine monophosphate (cA MP) has been implicated as the “second messenger” for these effects of hormones upon target tissues, it appeared possible that adenine nucleotides might regulate other functions of the vacuolar system in various cell types. Evidence suggesting that increments in the level of intracellular cAMP may inhibit granule flow and merger has been obtained by Lichtenstein and Margolis (3), who found that both theophylline and dibutyryl cyclic AMP (dcAMP) inhibited the release of histamine from leukocytes. Indeed, further experiments with peripheral blood leukocytes showed that these compounds inhibited both the antigenic release of histamine and the phagocytic release of β-glucuronidase (4).

Since redistribution of acid hydrolases follows enhanced endocytosis in cultures of human lymphocytes exposed to phytohemagglutinin (PHA) (5, 6), these findings suggested that the effects of PHA on lymphocytes might also be mediated by cyclic AMP. We have therefore studied the effect of cyclic AMP, its dibutyryl derivative and theophylline (an inhibitor of cyclic AMP degradation) (1, 2) upon the stimulation of lymphocytes.

Materials and Methods. Separation and culturing of cells. Human peripheral blood lymphocytes were obtained by methods previously described. Briefly, heparinized blood was allowed to sediment spontaneously at 37°, the supernatant plasma was removed, treated with adenosine diphosphate to aggregate platelets and the filtrate was passed through a prewarmed nylon fiber column at 37°.

An immunoassay procedure based on the use of specific antibody coupled to polyaminostyrene is described. Quantitation of human IgG in the range of 0.1 to 1.5 μg was possible and by minor adjustments the sensitivity could be increased severalfold.

Stimulation of single rat anterior pituitary glands has been obtained in a continuously superfused system as demonstrated by radioimmunoassay of LH in the superfusate. The results show the possibility of repeatedly stimulating a single pituitary gland, with approximately graded responses to hypothalamic extracts, in presence of a constant basal release rate. The data indicate that a relatively constant basal release rate is obtained under these conditions and that intermittent administration of hypothalamic extracts results in repeated acute release of LH. The period of release was largely limited to the time during which hypothalamic extract was administered. Equivalent amounts of cerebral cortex extract only produced slight increase of LH concentration in the superfusate.

Two products, thought to be metabolites of dieldrin, have been detected in extracts of the liver and of various organs of the sailfin mollie P. latipinna, after the fish had been exposed to dilute solutions of dieldrin in sea water for 1 hr. Some properties of these substances are different from those described for dieldrin metabolites in other animals. We suggest that they may be partially dechlorinated cyclo-diene fragments of the dieldrin originally administered.

Increased enzyme activity in serum is found in several nondisease states such as exercise (7) as well as under pathological conditions (5) including delirium tremens (19). The cellular mechanism of this elevation is obscure but may be related to the control, by divalent cations, of the passive permeability properties of several membrane types. Examples of this mode of regulation have been observed as an increased passage of macromolecules across mammalian intestine (17), the leakage of protein from rat liver slices (10), and the uptake of actinomycin by bacteria (12). Accordingly this study was designed to elucidate cellular enzyme loss from muscle with the intent of demonstrating the role of calcium and magnesium in the maintenance of membrane permeability and the functional integrity of skeletal muscle. The ionic permeability status of the preparation was monitored by assessing the transmembrane ionic gradient for sodium and potassium under the experimental conditions. Disodium dihydrogen ethylenediamine-tetraacetate (Na₂H₂EDTA) was the chelating agent used to promote enzyme loss from rat diaphragm muscle in vitro. Aldolase was selected for study because it is present in high concentration in muscle; it is believed to be a soluble intracellular enzyme; its leakage from rat diaphragm has been studied under other circumstances (18); and, unlike the metalloenzymes, the assay procedure for aldolase may be undertaken successfully in the presence of chelating agents (9).

Methods. Entire diaphragms of adult, 300–400 g, Sprague-Dawley rats maintained on commercial chow were removed under ether anesthesia and washed in ice-cold, physiologic saline solution. Each diaphragm was cut into segments of approximately 50 mg and placed in fresh, ice-cold saline to remove blood clots from the surface. The segments were blotted, weighed, and placed immediately into the appropriate incubation medium.

A simple physicochemical assay for the quantitation of enhanced vascular permeability in inflammation was described. It was shown that the assay is applicable to the study of inflammatory lesions induced with known chemical mediators, to the study of enhanced vessel permeability associated with the Arthus reaction, and that associated with thermal injury.

There is abundant evidence that selenomethionine is an analogue of methionine. We have utilized whole-body counting of ⁷⁵Se after the administration of ⁷⁵Se-selenomethionine to determine whether this technique might be used to evaluate overall changes in protein metabolism in animals fed different amounts of protein or methionine. Changes in overall body counts were not sensitive to changes in these dietary variables, but the excretion of ⁷⁵Se was greatly affected by the selenium content of the diet. Thus, whole-body counting is apparently not a useful technique for following the metabolism of tissue proteins.

Antibody production in response to skin allografts in mice was studied with an isotopic antiglobulin technique. This technique has been shown to be sensitive and capable of detecting antibodies not demonstrable by the cytotoxicity technique. Antibodies were first found in most animals at 9–10 days after grafting, at the time of onset of graft rejection. Experiments were performed to determine whether antibodies were being formed earlier, but were absorbed in vivo by the grafts. When primary skin grafts were removed before rejection, antibodies were not detected earlier. Lower levels of antibodies occurred, indicating that continued presence of the alloantigens were important for maximal antibody production.

Endotoxic lipopolysaccharides (LPS) have potent ability to consume complement (C) components and generate biologically active peptides from the C system in normal mammalian serum. The present investigation shows that LPS derived from Escherichia coli has potent ability to consume hemolytic C in all vertebrate species in which C activity is detectable, including species as low phylogenetically as representative Chon-drichthyes (horned shark, nurse shark), Os-teichthyes (paddlensh, carp), Amphibia (bull frog, marine frog), and Reptilia (cobra, turtle). Hence, the consumption of C components in response to this ubiquitous bacterial product is a characteristic of vertebrate serum virtually throughout vertebrate evolution.

Earlier studies indicated that the carcinogen, benzo[rst]pentaphene (more commonly called 3, 4, 9, 10-dibenzpyrene), remained at the site of subcutaneous injection. However, experiments with ¹⁴C-labeled compound demonstrated that 85% of the carcinogen is removed from the injection site. This removal is accomplished in 2 stages: first, removal of significant quantities which can be detected at other body sites and which is associated with the trauma of the injection, and secondly, a chronic removal, nearly complete in 10 weeks. No relationship was found between the rate of tumor formation and the amount of carcinogen remaining at the injection site.

Some processed baby foods were lethal to hypertension-prone rats. Among 25 rats from a genetically hypertension-prone strain fed solely on such baby foods, all developed significant hypertension (averaging 180–190 mm Hg in the last 3 months of observation), 12 died, and 2 others became seriously ill during the 8 months of study. In contrast, the IS control rats maintained on a low sodium chow were all alive and their average pressure at 8 months was 141.4 mm Hg. Considerable evidence suggests that the difference in response of test and control groups was due to the high NaCl content added to the processed baby foods. This added NaCl is unnecessary for the health of infants. It may contribute to the later development of hypertension in genetically predisposed individuals.

Preparations from human breast tumors which are inactive in the formation of steroid sulfates are shown to be lacking the two sulfate-activating enzymes, sulfate adenylyltransferase, and adenylylsulfate kinase, and to have measurable amounts of 3-β-hydroxysteroid sulfotransferase and estrone sulfotransferase. The study also suggests that the sulfate-activating enzymes are the limiting factor in determining the level of steroid conjugating activity of the tumor preparations that have the ability to form steroid sulfates.

Cardiac norepinephrine content was determined in rapidly and slowly growing preweaning rats. Between 10 and 20 days of age, when the heart rate normally increases from approximately 400 to 500 beats/min, norepinephrine concentration and heart rate increased simultaneously and the rises of both were greater in rapidly than in slowly growing animals. Rate of growth was directly related to heart rate and norepinephrine content at 15 days of age but not at other times during ontogeny.

Behavioral measures were obtained for rats administered a zinc deficient diet, (8.0 mg/kg) and adequate in all other constituents, for 48 days beginning at 30 days of age. Pair-fed controls received a zinc-supplemented diet (70 mg/kg of total zinc). Lethargy and reduced weight gain were characteristic of all zinc-deficient subjects. Performance on two measures of learning ability (viz., a one-way conditioned avoidance test and an eight-blind water maze) and an test of activity-emotionality (viz., the open-field test) revealed zinc-deficient animals to be inferior when compared to zinc-supplemented subjects. The relationship between zinc and protein utilization was posited as a possible mechanism explaining the effects of zinc insufficiency.

Epinephrine (E), norepinephrine (NE), and isoproterenol (ISO), contract isolated strips of rabbit aorta and vena cava. ISO is less potent than either E or NE. In all cases, the contractions are considerably reduced by phenoxybenzamine and not significantly altered by pronethalol. It is concluded that the contractile response to the three catecholamines is initiated by alpha-adrenergic receptors in venous as well as arterial strips.

Antibodies to the Australia antigen were shown to be present in the ascitic fluid of mice hyperimmunized with Australia antigen-Freund's adjuvant mixtures. As much as 100 ml can be removed from individual mice if they are restimulated by intraperitoneal injections of adjuvant-saline mixtures. The antibodies contained in these ascitic fluids were shown to be both of the gamma G and gamma M types. The specificities of the antibodies contained in the mouse ascitic fluid were shown to be similar but not identical to human antibody to Australia antigen.

Changes in canine renal blood flow in response to intra-arterial injections of angiotensin were studied in innervated and chronically denervated kidneys under a variety of conditions. In all cases, angiotensin decreased renal blood flow. Angiotensin was equally effective in reducing renal blood flow in innervated kidneys, reserpinized kidneys, kidneys treated with lidocaine, and in chronically denervated kidneys. These results suggest that the renal vasoconstrictor action of angiotensin is largely independent of an intact renal vasomotor innervation.

Changes in canine renal blood flow in response to intra-arterial bolus injections of isoproterenol were studied in the presence and absence of adrenergic blocking agents. In the absence of blocking drugs, isoproterenol produced small increases in renal blood flow. Alpha-adrenergic blockade with phenoxybenzamine potentiated the vasodilator action of isoproterenol. In contrast, beta-adrenergic blockade with propranolol reversed the dilator action of isoproterenol to a marked constrictor action, which in turn was abolished by alpha-blockade. These results support the view that few responsive beta-receptors are present in renal vascular smooth muscle, and that isoproterenol can stimulate alpha-receptors, resulting in vasoconstriction.

Vaccinia virus yields were decreased from cells previously chronically infected with M. arginini but not M. hyorhinis. This decrease was found to be a result of utilization of arginine from the medium by this mycoplasma. When added arginine was incorporated, vaccinia yields were the same as in cells not infected by mycoplasma. Thus, vaccinia virus was found to be arginine dependent.

Autologous plasma was demonstrated both to sustain and inhibit human lymphocyte transformation in vitro. A plasma concentration of less than 1% was unable to adequately sustain PHA-induced transformation. However, by careful adjustments of PHA concentration, a plasma concentration of from 1 to 10% yielded optimum PHA-induced stimulation in the subjects tested. Optimum plasma levels were higher (10–25%) for candida-stimulated and for unstimulated lymphocyte transformation. Sub-optimum stimulation resulted when greater concentrations of plasma than the optimum were employed regardless of the stimulant. Considerable variation occurs between normal individuals in autologous plasma suppression of in vitro lymphocyte transformation.

In studies made of its general behavior in laboratory animals MVM was found to induce lethal infections in suckling rats, and runting accompanied by proliferations of virus to high titers in many organs in mice. MVM attacked the cerebellum of infant mice, but without sufficient damage to induce clinical ataxia. When H-l virus was studied in a comparable manner, however, it was found to induce marked ataxia accompanied by an extensive cerebellar hypoplasia. A survey of wild rats revealed that Hi antibodies to MVM (69.5%) were more prevalent than those to either H-l (61%) or to rat virus (34%).

Walker 256 tumor cells were injected subcutaneously in an X-irradiated area of the right hip of Sprague-Dawley rats and in a corresponding nonirradiated area of controls. The tumors which developed in the injected areas were much larger in the controls than in the irradiated animals. The tumors were heavily encapsulated by fibrous tissue in the controls, but with little or no encapsulation in the irradiated animals. More viable cells and necrosis were present in the tumors of the controls. Special characteristics of the irradiated animals and not of the controls were invasiveness of the tumor and the presence of metastases.

The effect on passive anaphylaxis and delayed hypersensitivity in guinea pigs and immune tolerance in rabbits of injections of heat-denatured bovine serum albumin (HDBSA) and its dialyzable and non-dialyzable peptides, formed by the action of crystalline trypsin, chymotrypsin, and pepsin has been presented. In some experiments, a comparison has been presented with the effect of native bovine serum albumin (BSA). A direct relationship was observed between the molecular size of the peptides and their in vivo immunological activities. There were also some differences in the immunological activity of peptides formed by the different enzymes. Chymotryptic peptides were most effective in eliciting passive anaphylaxis; whereas, there were no significant differences in the ability of nondialyzable products of various enzymes in causing Arthus and delayed hypersensitivity reactions in guinea pigs. Heat denaturation did not affect the ability of BSA to cause Arthus and delayed reactions. In fact, HDBSA and BSA behaved very similarly and also cross reacted equally well in the above reactions. Dialyzable peptides produced some Arthus reactions but no delayed reactions, whereas nondialyzable peptides elicited good Arthus reaction and some delayed hypersensitivity. Under the conditions in which tolerance to BSA was induced in rabbits, neither HDBSA nor its tryptic digests were able to establish complete tolerance in rabbits.

The effects of microinjection of adrenergic and cholinergic drugs and hypertonic saline into the third ventricle on the intake of water, hypertonic salt (NaCl) solution, and food were studied. Carbachol induced a dramatic, rapid, 15-fold increase in water intake, whereas none of the other drugs were active. Both carbachol and isoproterenol evoked large increases in salt intake. Again, all other drugs failed to produce significant effects. Food intake was increased by the following adrenergic compounds: epinephrine, norepinephrine, metaraminol, isoproterenol, and dopamine. Carbachol was also effective in augmenting food intake but the effect was delayed. Hypertonic saline produced a delayed increase in both water and food intake but did not alter salt intake. The results are interpreted to mean that a cholinergic synapse lies in the pathways which mediate water intake, whereas both cholinergic and adrenergic synapses may be involved in the mediation of salt and food intake.