Experimental Biology and Medicine

Bicarbonate titration curves were determined in 15 neonatal dogs, 5 to 30 days of age, under conditions of isovolemia and with the administration of various doses of acetazolamide to inhibit renal carbonic anhydrase. Under each experimental condition used, no T_M for bicarbonate reabsorption could be found. With no acetazolamide, essentially all of the bicarbonate filtered was reabsorbed even at filtered loads of 50 μeq/ml g.f.r. Acetazolamide at a dose of 5 mg/kg inhibited less renal bicarbonate reabsorption than at doses of 50, 100, and 500 mg/kg but there was no difference among the latter three doses. At the higher acetazolamide dose levels, 5.3 μeq of bicarbonate was inhibited per ml g.f.r., amounting to only 12–24% of the total bicarbonate reabsorption (depending upon plasma bicarbonate levels) indicating that the major portion of the renal bicarbonate reabsorption in the neonatal dog is independent of the effects of carbonic anhydrase.

Platelet production rates were measured in thrombopoietin (TSF) assay mice by use of both [³⁵S]sodium sulfate and [⁷⁵Se]selenomethionine (⁷⁵SeM) as platelet labels. Although significantly higher values were found using Na₂ ³⁵SO₄ than ⁷⁵SeM, the incorporation patterns for both isotopes showed similarities in dose-response experiments.

The authors thank Marilyn Cottrell for expert technical assistance and Pat Taylor for stenographic aid.

A major calcium-binding protein of laying hen plasma was fractionated and partially characterized. After lipid flotation, approximately 80% of the total binding activity was localized in a high-density plasma fraction. Chromatography on Sepha-dex G-200 or Sepharose 6B gave a bimodal elution pattern. Sucrose gradient centrifugation yielded a similar bimodal pattern; the fast- and slow-sedimenting ultraviolet (UV) absorption peaks appeared identical to the first and second chromatographic elution peaks, respectively. Equilibrium dialysis localized the calcium-binding activity to the faster-sedimenting material. An orcinol-sulfuric acid test indicated a total carbohydrate content of 5% for the fast peak.

Analytical velocity ultracentrifugation measurements of this calcium-binding component yielded varying amounts of two entities with sedimentation coefficients (S_20,w) of approximately 16 and 8 S. The 16 S material was shown by equilbrium centrifugation (meniscus depletion) to have a 5.4 × 10⁵ M _r. Dialysis of the 16 S moiety against citrate or ethylene diamine tetraacetic acid (EDTA) buffer produced 8S material having a 2.7 × 10⁵ M _r and this monomer-dimer association was partially reversible by subsequent dialysis against calcium ions. The calcium-binding molecule appears to be vitellogenin. Calcium-binding protein was found at elevated levels in plasma from long-term estrogenized birds but not from nonlaying birds.

This research was supported in part by U.S. Public Health Service Grants R01-AM ES 05970 and R01 AM ES 17217.

Although heparin has been known to release lipoprotein lipase (diacylglycerol acylhydrolase, EC 3.1.1.34) since 1943 (1), the quantitative aspects related to dosage have never been fully determined. Furthermore, only the data of Robinson et al. (2) are available concerning the half-life of enzyme action, once released into the blood. Olsson et al. (3) and Estes et al. (4) published data on the volume of distribution of heparin and kinetic data on heparin's anticoagulant action in man. More recently, McAvoy (5) determined the biologic half-life of heparin. In this paper, we present quantitative dose-response data on the release of lipoprotein lipase by heparin in mice, as well as information on time-duration action of released lipoprotein lipase in serum.

Materials and Methods. Seventy male Swiss-Webster mice (weighing 20–30 g) were used in this investigation. Heparin sodium was injected intravenously in the tail vein in a volume of 0.1 ml. Axillary blood was obtained from etherized mice, allowed to clot, and then spun down in a refrigerated centrifuge at 2500 rpm for 30 min at 5°. (In previous experiments we found that etherization did not affect lipoprotein lipase release.) The clear serum was either tested immediately or frozen at −10° and used on subsequent days. The enzyme was found to be quite stable and showed no discernible diminution in activity on storage at −10° for 20 days. It was always tested either immediately or within a few days after collection.

Lipoprotein lipase assay. To determine the level of lipoprotein lipase, 0.6 ml of serum was added to a flask in which 5.4 ml of [¹⁴C]triolein substrate (see below) was placed; it was incubated in a shaking incubator for 20 min at 27° (according to a modification of the method of Schotz (6, 7)). At intervals of 1.5, 10, and 20 min, 0.5-ml samples were removed from the flask and added to isopropanol-sulfuric acid (40:1) solution to stop the reaction. Then 5 ml of hexane and 1 ml of water were added to each aliquot and the tubes were shaken for 1 min in a mechanical shaker. The upper layer containing the hexane phase was separated and shaken with 2 ml of 0.1 N KOH for 10 min. The lower phase contains the potassium oleate generated by enzymic action on the triolein substrate. The 1-ml aliquots of the lower phase were added to 10 ml Aquasol and counted in an Intertechnique liquid scintillation spectrometer, Model SL-30.

Cold-induced pain threshold and pain tolerance were measured in drug-free ex-addicts (DFE), in methadone-maintained ex-addicts (MME), and in age-matched nonaddict siblings (NAS). Multiple responses to an ice-water stimulus were obtained for each hand. Resting systolic and diastolic blood pressures were determined across a 15-min period both before and immediately after the pain-testing session. Pain threshold of both ex-addict groups (DFE and MME) was lower than that of NAS. Pain tolerance was slightly but not significantly lower. Ex-addicts further differed from non-addicts in an autonomic response to the experimental situation: The diastolic blood pressure of nonaddicts rose following cold pressor stimulation but there was no rise in the ex-addict groups. Methadone-maintained ex-addicts do not differ from drug-free ex-addicts in pain threshold, pain tolerance, or blood pressure response.

We thank the directors, particularly Dr. Mitchell S. Rosenthal of Phoenix House, the staff, and patients of various drug-free programs and methadone clinics for their cooperation in contacting or serving as subjects. The study was supported by NIDA Grant DA-00390-01 and by New York State Office of Drug Abuse Services Grant C-107653.

Human sera or plasma containing acid phosphatases were mixed with rabbit antiserum specific for prostatic acid phosphatase (PAP) and the resulting primary immune complexes were precipitated by ammonium sulfate. The amount of specifically precipitated PAP was estimated directly by its enzyme activity. Using this assay, as little as 10 ng/ml of the PAP could be determined. Excellent correlation of this assay method with other immunoassays of PAP was demonstrated.

This work was supported in part by PHS Research Grant CA 18748 and CA 16426 from the National Cancer Institute.

Cycloleucine, chemically known as 1-aminocyclopentane-1-carboxylic acid (ACPC) is not a naturally occurring amino acid (see Figure 1). Metabolic studies of ACPC in mice and rats have shown that it is not metabolized nor excreted rapidly; therefore, blood and peripheral tissue levels are maintained for several days (1, 2). Ross et al (3) reported a decreased growth rate in animals fed a diet containing ACPC and suggested it may function as an amino acid antagonist. Clark et al (4) proposed that its effect on growth rate and amino acid pools were secondary to a depressed food intake.

The results obtained in the present study suggest yet another mechanism of action of ACPC on reducing food efficiency in rats fed a purified high starch diet.

Materials and methods. Male Wistar rats (160–170 g) were given a standard laboratory Purina Chow or high carbohydrate diet 2 in powdered form ad libitum to facilitate estimation of daily food consumption. They were housed individually in open wire bottom cages. Water was available at all times. In experiment I, 5 days after commencement of the high carbohydrate diet, groups of animals received either the basal ration or ACPC incorporated into the diet at the following levels: 0.0125, 0.025, 0.05, or 0.1%. In experiment II, ACPC and its chemically related amino acids (l-(–)-proline, l-4-hydroxyproline, glycine, l-leucine, or l-isoleucine) were admixed separately to the Purina Chow diet at 0.5 mg/g diet (0.05%) or 1 mg/g diet (0.1%), respectively. Animals were given ad libitum for a period of 3 days.

Body weight and food consumption were monitored throughout experimental period. Blood was obtained by cardiac puncture and portions of the pancreas, small intestine, and liver were collected and chilled rapidly from anesthetized rats at the end of the studies.

Calcium concentration of saliva, when electrical stimulation of the parasympathetic postganglionic fibers to rat parotid is used to evoke secretion, increases with increases in flow rate, but both calcium concentration and flow rate reach maximal levels at frequencies of 16 Hz. The concentration of calcium at highest flow rate is three times that of serum. Amylase activity of the saliva remains consistently low at all flow rates. Therefore, these data show that while calcium and amylase are secreted together, some calcium may be secreted without amylase. By inference it is also possible to suggest that the concentrating mechanism for calcium is at the acinar level, and that as the precursor fluid, rich in calcium, flows through the duct system, very little absorption of calcium by ductal cells occurs at high flow rates, but as contact time is increased with decreasing flow rate, more calcium is reabsorbed, resulting in a final saliva low in calcium. It is not likely that the increased calcium concentration is the result of reabsorption of water by ductal cells since in perfused main submaxillary duct at least, the duct cells are relatively impermeable to water. The present data suggest this interpretation, but micropuncture analysis of calcium concentration of the precursor fluid is required to establish this as a fact. Flow rate differences found with different kinds of autonomic stimulation cannot be used to assess relationships between calcium concentration and flow rate of saliva, and only when the flow rate and calcium concentration are compared with a particular kind of autonomic stimulation are such comparisons meaningful.

Sprague-Dawley female rats were placed on a low-fat diet, a high saturated fat diet, or a high polyunsaturated fat diet at time of weaning (21 days of age). Half of the rats in each dietary group were given an intraperitoneal injection of MER (0.5 mg) on Day 28 and again on Day 35. On Day 50, all rats were given 10 mg of DMBA in 1 ml of stripped corn oil via stomach tube. Tumor incidence was determined weekly by palpation and confirmed by histological analysis upon completion of the experiment. In each dietary group, rats immunized with MER had a lower tumor incidence, a longer latent period, and smaller tumors compared to non-immunized rats. In other experiments, rats were placed on the high polyunsaturated fat diet at time of weaning, exposed to DMBA on Day 50, and maintained on this diet for either 3 or 4 weeks after DMBA. At those times, some rats were placed on the low-fat diet while other rats were maintained on the high polyunsaturated fat diet. Half of the rats from each group were treated with MER. Results indicate that the low-fat diet provided significant therapeutic benefit. Further benefit was observed if these rats had been treated with MER. However, MER was not beneficial if given to rats maintained on the high polyunsaturated fat diet.

Phosphate depletion in the rat is associated with the rapid development of marked magnesuria (1, 2). This phenomenon has been attributed to a decrease in the renal tubular transport of magnesium since the magnesuria occurred in the face of falling filtered load of this ion.

However, it is also possible that magnesium secretion by the renal tubule occurs during phosphate depletion and contributes to the magnesuria. The present study was undertaken to examine this question.

Methods. Male Sprague-Dawley rats weighing 220–280 g and 7 to 10 weeks old were housed individually in metabolic cages. During an adaptation period of 7 days, all rats were fed control diet containing 0.44% phosphate, 0.4% sodium, 0.41% calcium, and 0.03% magnesium (I.C.N. Pharmaceutical Co., Cleveland, Ohio). The rats were then randomly allocated to the phosphate-depleted group or the control group. The phosphate-depleted group (PD) received a diet identical to the control diet except that its phosphate content was reduced to 0.03%. Control animals continued to receive the control diet ad libitum. The rats had free access to deionized water.

Phosphate depletion lasted for 6 to 8 days. During this period both phosphate-depleted rats and the control animals were placed in plastic restraining cages several hours each day for adaptation. The day prior to the experiment, silastic catheters were placed in the left carotid artery and the right jugular vein and a blood sample was taken from the vein for the analysis of phosphate. Only PD animals with plasma phosphates less than 6.5 mg% were used in the study. On the morning of the experiment, the rats were anesthetized with halothane (Surital, Ayerst Laboratories, New York), a bladder catheter was placed and was connected to an extension which led out through the abdominal wall.

We have recently reported the isolation and identification of leucine from the plasma of shocked animals which exerted significant cardiodepressant activity in vitro. This study tested the hypothesis that if leucine is a circulating-factor in vivo and if this has any significant deleterious effects in the shocked animal, then the exogenous administration of leucine should accelerate the shock syndrome. We infused leucine, isoleucine (7.6 mmole/kg), or KH into endotoxin-shocked rats and measured survival times and blood concentrations of leucine and isoleucine. Following endotoxin administration, the leucine-administered rats died significantly sooner (P < 0.01) than the KH-infused animals; the isoleucine animals also died sooner (P = 0.06). Terminal leucine concentrations were elevated in all animals following endotoxin administration compared to preshock concentrations. The terminal serum leucine concentration in the leucine- and isoleucine-infused groups was significantly higher (P < 0.06) than the KH-infused group. In contrast, terminal serum isoleucine concentrations were significantly elevated only in the isoleucine-infused group. These data suggest that the elevated serum leucine concentration may contribute to the development of irreversible shock.

This research was supported by Grant HL 19977 from the National Institutes of Health. Dr. Goldfarb is the recipient of Career Research Development Award HL-00947. We thank Mrs. Joanne Bayreuther for her assistance in the preparation of this manuscript.

Peripheral, medullary, and splenic erythroid parameters were quantitatively assessed in mice during the course of virulent malarial (P. berghei) infection. Mice became progressively anemic, with circulating erythrocytes, hematocrit, and hemoglobin levels declining to approximately 20% of control by Day 21. The anemia was further characterized by a 34% increase in mean corpuscular volume, a 16% elevation in mean corpuscular hemoglobin, and a 14% decrease in mean corpuscular hemoglobin concentration by Day 21. A meager 1.5-fold reticulocytosis was noted on Day 7, which declined thereafter. Tibial nucleated cellularity was depressed to 85 and 72% of control values on Days 7 and 21, respectively, while erythroid precursor numbers remained at 74% of control levels throughout the course of infection. In contrast, splenic nucleated cells were elevated 2-fold on Day 7 and increased approximately 4-fold on Days 14 and 21. Although nearly 20-fold elevations in erythroid precursor levels were apparent, these increases were below what one might expect in noninfected mice with a similar degree of anemia.

These data support the contention that the erythropoietic capability of mice infected with malaria is impaired. Accordingly, it is suggested that this situation contributes to the severe anemic status of malarial mice, in addition to the well-documented mechanisms leading to the destruction of parasitized and nonparasitized erythrocytes.

The research participation of P. Desrochers, R. Gundersen, M. Hardy, M. Haviland, D. Lynch, S. McGinnis, and M. Parlee is greatly appreciated.

Captopril reduced blood pressure and increased PRA in both SHR and NTR. Both propranolol and indomethacin caused significant reductions in resting PRA of both SHR and NTR but only propranolol caused an inhibition of the PRA changes caused by captopril. Furthermore, since propranolol was highly effective only in SHR and captopril decreased blood pressure much more in SHR than NTR, the increase in PRA caused by captopril in SHR was probably reflexly mediated by compensatory sympathetic activation, all of which actions were a consequence of reducing angiotensin II formation.

Replacement of Cl^- with SO₄ ^2- gives a Cl^- diffusion potential of about 19 mV across the nutrient membrane per 10-fold change in Cl^- concentration, suggesting, on the basis of the Nernst equation, that the Cl^- conductance pathway contributes about one-third to the total conductance of this membrane in regular Cl^- solutions bathing the frog gastric mucosa. Isethionate ion gives a change in PD of about 17 mV per 10-fold change which is quite close to the value obtained with SO₄ ^2-.

Bombesin was administered to young fasted rats, and blood levels of immunoreactive gastrin and calcitonin (CT) were measured. Doses of bombesin up to 100 μg/rat i.v. did not affect serum calcium or phosphorus. Five minutes after bombesin, 10 or 50 μg/rat, serum gastrin increased 1- to 2-fold and, by 30 min, it was elevated 5- to 10-fold. Time-course and dose-response studies showed that doses of 10–100 μg/rat increased gastrin and that gastrin rose within 5 min after 50 μg/rat and remained elevated for at least 2 hr. Despite bombesin-induced hyper-gastrinemia, CT remained low or undetectable (<50–100 pg/ml) at all times. In one study, somatostatin (50 μg/rat i.v.), given together with bombesin, inhibited the rise in serum gastrin by 50%, but serum CT, which was detectable, was unaffected by either bombesin or somatostatin. We conclude that bombesin stimulates gastrin release in the rat, but the elevated gastrin does not promote CT secretion. The results support our earlier findings which indicate that gastrin is weak or ineffective in the rat as a CT secretagogue.

The technical assistance of Ms. Deloris B. Alston and Ms. Lillie T. Williams is gratefully acknowledged.

The effects of three highly bound acids, viz salicylic acid, indomethacin, and dicloxacillin, on the binding of phenylbutazone to human plasma proteins were studied by equilibrium dialysis. Salicylic acid was found to displace phenylbutazone from its binding sites to a degree which might be clinically significant. Indomethacin caused a slight decrease in the percentage binding of phenylbutazone which is not likely to be of clinical importance. Dicloxacillin, however, did not appear to affect the plasma protein binding of phenylbutazone suggesting that the two drugs could be bound at mutually noninteracting sites in the protein molecule.

The influence of experimental lathyrism generated by β-aminopropionitrile (BAPN) treatment on matrix-induced endochondral ossification was investigated. Although the mesenchymal cell proliferation was not affected, BAPN was inhibitory to chondrogenesis as quantitated by ³⁵SO₄ incorporation. Both cartilage and bone mineralization, as indicated by ⁴⁵Ca incorporation, were profoundly inhibited by BAPN treatment. The initial rate and the final extent of mineralization was impaired by the lathyrism. Matrices prepared from lathyritic bone and tooth were not as active as controls in induction of new bone as quantitated by alkaline phosphatase activity and ⁴⁵Ca incorporation.

Bilaterally adrenodemedullated rats were successfully cold acclimated at 5°C for 5 weeks. This was evidenced by (i) successful survival, (ii) elevated oxygen uptake measured at 28°C, (iii) enhanced calorigenic response to tyramine although demedullated rats had significantly lower oxygen uptakes than the intact rats, and (iv) an increased tolerance to cold (−10°C) similar to that observed in intact rats after 5 weeks of acclimation. Non-cold-acclimated demedullated rats were unable to maintain their colonic temperature to the same extent as the intact non-cold-acclimated rats during exposure to −10°C. All cold-acclimated rats had greater response to tyramine and better tolerance to −10°C than non-cold-acclimated rats.

This work was supported in part by the National Institutes of Health under Grant NIH AG00021.

Serum levels of LH and FSH were measured in the pregnant rabbit utilizing homologous assays for rabbit gonadotropins. Results show that following mating, serum LH rose to peak levels by 2 hr postcoitum, returned to estrous levels by 12 hr, and remained at low levels through the duration of pregnancy. Serum FSH also exhibited a preovulatory surge at 2–3 hr after mating, returning to precoital levels by 12 hr. In contrast to serum LH, however, a second surge of serum FSH occurred at 1–2 days of pregnancy at a time when serum LH was not elevated. Following the second peak, serum FSH also remained low through the remainder of pregnancy.

We are indebted to the National Pituitary Agency for the rabbit LH and FSH radioimmunoassay kits. We also wish to thank Mrs. Kathy Maxwell and Miss Karyle Di Crispino for preparation of the manuscript. This work was supported in part by NIH grant 5 SO1 RR05365-13.

A 10% soybean oil emulsion (Intralipid) when given intravenously was found to prolong pentobarbital-induced sleep time in rats. The prolongation of sleep time was dose and time dependent. Heparin increased the clearance of Intralipid emulsion from the plasma and decreased the amount of time by which sleep was prolonged. It was further demonstrated that pentobarbital anesthesia decreased the clearance of intravenously administered Intralipid emulsion from the plasma. Thus, a reciprocal interaction was observed whereby pentobarbital prolonged the half-life of Intralipid emulsion and Intralipid emulsion prolonged the narcosis induced by pentobarbital.

Zinc ions, at concentrations which were shown to inhibit guanylate cyclase activity, abolished the actions of prolactin on RNA and casein synthesis in cultured mouse mammary gland explants. These observations are compatible with the hypothesis that certain of the actions of prolactin in the mammary gland may be mediated by cyclic GMP.

In the microcytotoxicity assay, a stimulatory effect exerted by nonsensitized spleen cells derived from nude mice was observed on the growth of tumorigeneic human tumor cells. In contrast, the growth of non-tumor-producing cell lines (malignant and nonmalignant) was inhibited by identical effector cells. Two out of four non-tumor-producing cell lines were of malignant origin. This lack of tumorigenicity, despite a derivation from tumor tissue, might be due to a selection of stromal cells during cultivation or might reflect a variation in the sensitivity to natural cytotoxicity independent of malignancy vs nonmalignancy. Consequently, lack of tumorigenicity does not exclude the possibility that the cells are malignant.

The technical assistance of Vibeke Ahrenkiel is gratefully acknowledged. I thank Lone Atke for careful secretarial assistance. This work was supported by Danish Medical Research Council Grants 512–5274 and 512-3547.

The effect of dopamine (DOP), dopamine-receptor antagonist, and β-adrenergic receptor antagonist agents on simultaneously measured renin release (RR) and cyclic adenosine 3':5'-monophosphate (cAMP) content of renal cortical slices from sodium-deficient rats was studied in vitro. A DOP dose of 10^-5 M significantly increased RR while a higher (10^-3 M) dose significantly stimulated both RR and cAMP content in the slice preparation. Addition of the DOP-receptor blocker pimozide (PIMO) at 10^-6 M effectively prevented stimulation of RR and tissue cAMP content by DOP. However, PIMO added alone also significantly decreased resting RR and tissue cAMP content levels. In subsequent experiments, the DOP-β-hydroxylase (DBH) enzyme inhibitor FLA-63 (10^-4 M) was used in conjunction with the other agents to prevent conversion of DOP to norepinephrine (NE) in the tissue preparation. In the presence of FLA-63, 10^-3 M DOP again significantly stimulated both RR and cAMP content as previously seen without FLA-63. Conversely, a previously inhibitory effect on RR was converted to significant stimulation by several PIMO doses in the presence of the DBH enzyme inhibitor. Tissue cAMP content appeared significantly inhibited by all PIMO doses added alone or together with 10^-3 M DOP whether FLA-63 was present or not. FLA-63 was ineffective by itself. A 10^-4 M dose of the β-adrenergic receptor blocker dl-propranolol (PROP), added together with 10^-3 M DOP and FLA-63, significantly prevented the stimulatory effect of DOP on RR and tissue cAMP content. These data indicate that (i) DOP itself can directly stimulate RR from the renal juxtaglomerular cells, (ii) a direct stimulatory effect of DOP on RR is at least partially mediated by a β-adrenergic receptor mechanism involving intracellular cAMP changes, and (iii) further studies are needed before the possibility of the existence of dopamine-specific renal receptors mediating the action of DOP on RR can be properly evaluated.

The expert technical assistance of J. Cottrell, M. Kennedy, K. Kellner, G. Kristy, and J. Quero is gratefully acknowledged. The authors also wish to thank Dr. I. A. Reid, Department of Physiology, University of California Medical Center in San Francisco, for kindly supplying us with 24-hr nephrectomized dog plasma and angiotensin I antibody. Special thanks are also expressed to Dr. R. V. Gallo, Department of Physiology, University of California Medical Center in San Francisco, for his generous donation of pimozide.

Proteins and phospholipids but not zinc were released into the medium containing hemoglobin-free ghosts from dog erythrocytes incubated in peroxidation-inducing agents (ascorbic acid, Fe²⁺, O₂). The content of zinc in the pure ghost depends on the purification procedure; using phosphate buffer it amounted to 62 ± 22 μg/g protein, and using barbital buffer-extracted ghost it was three times higher.

Although most of the zinc was linked to the lipid phase of the membrane, there is a certain protein moiety of membrane proteins binding more zinc than the other protein fractions. Among various fractions of lipids, zinc is linked mainly to phospholipids and this linkage is not broken by phosphate buffer.

It is concluded that zinc is part of the erythrocyte ghost, linked to protein as well as lipid phase.

This work is supported in part by NIH Research Grants AM 16489, ES 00790, and ES 01570. The professional assistance of Mrs. Waltraud W. Nichols is highly appreciated.

Heart rate (HR), mean arterial pressure (MAP), and mean right atrial pressure (MRAP) were measured in 15 pentobarbital-anesthetized rabbits, with vascularly isolated carotid sinuses, during increases in positive end expiratory pressures (PEEP) of 2.5, 5.0, and 7.5 cm H₂O. Also, peak reflex bradycardia (ΔHR) and depression of arterial blood pressure (ΔMAP) to left aortic nerve stimulation (LANS) were recorded during the PEEP changes in aortic and subsequently vagal denervated animals. Responses were measured with the carotid sinus receptors detecting systemic pressure (closed loop (CL) condition) and with sinus region pressures held constant (open loop (OL) condition). Results demonstrated that both HR and MAP were altered by an increase in PEEP. Analysis of HR, and ΔHR to LANS responses during OL conditions, suggested the reflex involvement of cardiopulmonary afferent to cardiac efferent vagal fibers during PEEP changes. Vascular responses to increased PEEP occurred in the presence or absence of arterial baroreceptors and, while reduced, were not abolished following vagotomy.

Our results demonstrate extensive metabolism of GDCA in the dog and rat by a pathway similar to that for DCA in man (7). Assignment of reduced metabolites and relative ratios was made by tlc comparison of tritiated metabolites with known compounds. Trace bolus doses of GDCA result in complete reductive metabolism, and increasing molar doses result in the excretion of only small amounts (<5%) of unchanged GDCA in bile. Thus our in vivo experiments are in contrast to the absence of GDCA metabolism reported in the isolated, perfused rat liver (12).

The authors are indebted to Ruthie Pettigrew and Becky Backof for invaluable technical assistance and to Jo Ellen Shockley for preparation of the manuscript.

Concordance of polymorphic enzyme phenotypes in various comparisons of human normal and malignant cells in vitro and in vivo establishes isozyme stability after change to the malignant state and after long-term tissue culture. Such enzymes, therefore, can be used with considerable confidence to individually distinguish human tumor cell lines. The stability validates the use of phenotype frequency data obtained from surveys of normal human populations in the analysis of data on human tumor cell lines.

We gratefully acknowledge the collaboration of Dr. Francis E. Sharkey and the Department of Pathology, Memorial Hospital, New York, for providing tissue specimens.