Neurotensin in diabetes and obesity

Basal and postprandial concentrations of immunoreactive neurotensin were measured in insulin dependent diabetic patients and lean and obese noninsulin dependent diabetic patients when partially withdrawn from subcutaneous (s.c.) insulin treatment and again when near normoglycemia had been achieved from insulin infusion by an artificial endocrine pancreas (AEP). Neither basal nor postprandial neurotensin differed among the 3 groups of diabetic patients during s.c. insulin treatment nor from weight matched nondiabetic subjects. In addition, AEP resulted in no significant change in postprandial neurotensin responses. No differences in neurotensin levels were observed between lean and obese nondiabetic subjects. In contrast to observations in experimental diabetes, these observations do not support the presence of an abnormality of neurotensin in human diabetes.     

Plasma somatostatin in normal subjects and in various diseases: increased levels in somatostatin-producing tumors

The plasma levels of somatostatin (SRIF) were studied in normal subjects and patients with various disorders by a sensitive and specific radioimmunoassay. In 45 normal subjects, the fasting plasma SRIF concentrations were 13.3 +/- 5.3 pg/ml (mean +/- SD). Very high concentrations of plasma SRIF, ranging from 125.0 pg/ml to 400.0 pg/ml, were found in all four patients with medullary carcinoma of the thyroid examined and the SRIF levels were changed in parallel with their clinical course after resection of the tumor. A case of pheochromocytoma also showed a relatively high SRIF concentration in plasma (47.0 pg/ml), but the plasma SRIF level decreased to 8.7 pg/ml after removal of the tumor. In normal subjects, plasma SRIF levels did not fluctuate during 2 hr-observation period in basal state. Glucagon (1 mg, iv) and secretin (3 CHRU/kg B.W., iv infusion over 30 min) had no effect on the SRIF levels in the peripheral blood plasma of normal subjects. On intravenous infusion of arginine (0.5 g/kg B.W.) over 30 min, all 6 normal subjects showed a significant increase in plasma SRIF 30-45 min after the start of the infusion (basal value, 11.6 +/- 1.5 pg/ml; peak value, 27.2 +/- 3.0 pg/ml; p less than 0.005). Two cases of medullary thyroid carcinoma showed exaggerated responses after the arginine administration (increases of 103 pg/ml and 157 pg/ml, respectively), suggesting that SRIF was released from the tumor. The findings indicate that plasma SRIF determination in the basal state and after arginine administration is useful for detecting and following up SRIF-producing tumors.     

The influence of plasma triglycerides on human growth hormone response to arginine and insulin: a study in hyperlipemics and normal subjects.

Human growth hormone (HGH) response to arginine (25 gm IV in 30 min) and to insulin (0.1 U/kg B.W.) was studied in 12 male patients (mean age 36 +/- 2 years), with normal glucose tolerance and normal body weight, affected with Fredrickson's Type IV primary hyperlipemia. The patients were examined both when plasma triglycerides (TG) were elevated and following clofibrate (2 gm/die for 30-60 days) induced TG reduction. No variations in glucose or FFA behaviour or in body weight were observed after clofibrate. HGH response to arginine was absent, while that to insulin was only inhibited, when plasma TG were elevated. A significant increase in HGH peaks after arginine (from 1.99 +/- 0.59 to 9.34 +/- 1.58 ng/ml) and a slight increment in HGH peaks after insulin (from 23.09 +/- 7.19 to 31.46 +/- 7.95 ng/ml) were observed following reduction in plasma TG. Arginine test was carried out in 7 normal subjects during saline infusion and at the 3rd hour of lipid infusion (Intralipid 20%). HGH response to arginine was absent in all of the subjects during lipid infusion. The HGH response to insulin test, carried out in 9 other normal subjects during saline infusion and at the 3rd hour of lipid infusion (Lipiphysan 15%) was significantly inhibited during lipid infusion. Since lipid infusion provoked an increment, not only in plasma TG but also in FFA, the inhibition of HGH release could be correlated with the elevated plasma levels of both TG and FFA. The results obtained in both spontaneous and experimental hyperlipemia not only confirm the role played by FFA in the regulation of HGH secretion, but also support the hypothesis that elevated TG levels could inhibit HGH response to some stimuli.     

Growth hormone responses to sleep, insulin hypoglycaemia and arginine infusion

This study compares the peak serum growth hormone (GH) concentration during slow wave sleep with the serum GH responses to insulin-induced hypoglycaemia and intravenous arginine infusion in 23 children referred because of short stature (20) or precocious puberty (3). Peak serum GH concentration during sleep correlated significantly with peak GH response to insulin hypoglycaemia (r = 0.64, p less than 0.01) and arginine infusion (r = 0.57, p less than 0.01). 3 children had subnormal (less than 15 mU/l) peak serum GH concentrations during sleep but normal responses to either insulin-induced hypoglycaemia or intravenous arginine infusion. 1 child had a normal peak serum GH response to sleep but subnormal responses to insulin and arginine. Sleep studies of GH secretion may be indicated when the GH responses to pharmacological stimuli are inconsistent with the observed growth pattern.     

Use of sulfate production as a measure of short-term sulfur amino acid catabolism in humans

There is no fully satisfactory method for measuring amino acid catabolism in the nonsteady state that follows normal protein consumption. Because sulfate is the major product of sulfur amino acid catabolism, we tested whether its production can be accurately depicted using simple tracer or nontracer approaches under basal conditions and after the intravenous administration of a known amount of sulfate. In the basal postabsorptive state, serum sulfate concentration and urinary sulfate excretion remained constant for many hours, but the apparent steady-state serum sulfate rate of appearance achieved with primed continuous oral administration of sodium [(34)S]sulfate was 20% higher than urinary sulfate excretion. By contrast, after magnesium sulfate infusion, the increase in sulfate production above basal accounted for 95% over 6 h and 98% over 9 h of the administered dose when measured simply as urinary inorganic sulfate excretion corrected for changes in its extracellular fluid content. Using the latter method, we measured sulfate production after oral methionine and intravenous infusion of methionine in a mixture of other essential amino acids. Sulfate production above basal accounted for 59% over 6 h and 75% over 9 h of the oral methionine dose. Similar results were obtained with the mixed amino acid infusion, but interpretation of the latter experiment was limited by the mild protein sparing (and, hence, reduced endogenous sulfate production) induced by the amino acid infusion. We conclude that a simple nontracer method can provide an accurate measure of sulfate production and, hence, sulfur amino acid catabolism over collection periods as short as 6 h after a test meal. A significant portion of the sulfur derived from methionine appears to be retained in nonprotein compounds immediately after its ingestion.     

Hyperfunction of the entero-PP axis in non-insulin dependent diabetes mellitus

In a previous study we have found that the plasma pancreatic polypeptide (PP) response to oral glucose loading is exaggerated in diabetic patients compared with normal subjects. We have investigated, therefore, the effects of a protein-rich meal or meat extract ingestion on plasma PP secretion and examined also the effects of intravenous arginine administration on PP levels in normal subjects and in patients with noninsulin-dependent diabetes mellitus (NIDDM). Following a 600 Kcal meal ingestion, plasma PP levels increased immediately and showed biphasic secretion in normal subjects and in NIDDM, but the response was exaggerated in NIDDM. A 50 g meat extract administration also produced an exaggerated PP response in NIDDM compared with normal subjects. In NIDDM and normal subjects, plasma PP levels did not change significantly during an arginine infusion (30 g for 45 min) but after the end of the infusion PP levels increased significantly compared with basal levels. In normal subjects, plasma PP rose abruptly after a bolus arginine injection (4 g for 2 min) and then remained at significantly high levels even 30 min after the injection. In NIDDM, however, plasma PP levels tended to increase, but not significantly, after the bolus arginine injection. Since in NIDDM the protein-rich meal and meat extract ingestion produced an exaggerated rise in plasma PP while the PP responses to the intravenous arginine administration were rather impaired compared with normal subjects, we suggest that the entero-PP axis is overactive in NIDDM.     

Effects of parathyroid hormone on plasma zinc concentration in rat with chronic renal failure

Although both secondary hyperparathyroidism (HPT) and hypozincemia are commonly observed in humans and animals with chronic renal failure (CRF), the relationship between secondary HPT and hypozincemia is little delineated. The present study was designed to examine whether the elevated plasma parathyroid hormones (PTH) levels do affect the disposition of extrarenal zinc and decrease plasma zinc level in CRF rats. The experiment was performed in normal and CRF rats with intact parathyroid gland and parathyroidectomized (PTX), using an acute zinc load alone or in combination with PTH infusion in five groups of rats: normal control, CRF control, CRF + PTH, CRF + PTX and CRF + PTX + PTH. Five sixths nephrectomy was used to produce CRF. All rats were infused with 0.05 mg/kg/min ZnSO4 alone or in combination with 10 microg/kg/min PTH through intravenous infusion for 90 min with serial monitoring of plasma zinc levels every 30 min. The alteration of plasma interleukin-6 (IL-6) levels and the effect of zinc levels in red blood cells (RBCs), as well as the output of bile juice zinc and urinary zinc excretion during the 90-min infusion were also examined. After 90-min infusion, liver tissue was harvested to determine its contents of zinc and metallothionein (MT). During zinc sulfate infusion, the responses of plasma zinc concentration in PTH-combined infusion groups markedly decreased as compared with those of the non-PTH-combined infusion groups, especially in the CRF rats with PTX. However, when zinc sulfate alone was infused, the response of plasma zinc concentration was found to increase in CRF rats with PTX as compared with that of the CRF control rats. PTH infusion groups significantly increased the levels of plasma IL-6 (P < 0.05), but it did not alter the levels of RBC zinc and the secretion of bile zinc during the 90-min infusion. After 90-min zinc sulfate infusion, higher liver zinc and MT contents were found in CRF control, CRF + PTH and CRF + PTX + PTH rats, but were [corrected] not found in the CRF + PTX rats. Zinc sulfate infused alone was found to increase the excretion of basal zinc in bile juice and urine, in both normal and CRF rats. The percentage of zinc load translocated out from the plasma during 90-min zinc sulfate infusion significantly rises in CRF rats and CRF rats with PTH-combined infusion as compared with normal control rats. However, in CRF rats with PTX, the percentage of zinc load translocated out from plasma during 90-min zinc sulfate infusion was similar to that in the normal control rats. Therefore, we suggested that in CRF rats, the excessive secretion of PTH may play a role in the pathogenesis of hypozincemia because PTH enhanced extrarenal zinc disposal.     

Effects in chicks of arsenic,arginine, and zinc and their interaction on body weight,plasma uric acid,plasma urea,and kidney arginase activity

The interaction among arsenic, zinc, and arginine was studied in chicks using two fully crossed, three-way, two-by-two-by-two experiments. Arsenic at levels of 0 and 2 μg/g zinc at levels of 2.5 (zinc-deficient) and 25 (zinc-adequate) μg/g, and arginine at levels of 0 and 16 mg/g were supplemented to the diet. After 28 d in both experiments, growth was depressed in chicks fed diets either supplemented with arginine or deficient in zinc. Arsenic deprivation depressed growth of chicks fed diets containing the basal level of arginine and 25 μg supplemental Zn/g. Arsenic deprivation had little or no effect on growth of zinc-deprived chicks fed diets containing the basal level of arginine, or in zinc-deprived or zinc-adequate chicks fed supplemental arginine. Zinc-deficiency elevated urea in plasma and arginase activity in kidney. Those elevations, however, were more marked in arsenic-supplemented than in arsenic-deprived chicks. Also, plasma urea and kidney arginase activity were markedly elevated in chicks fed supplemental arginine; the elevations were more marked in zinc-deficient chicks. These findings support the concept that arsenic has a physiological role, associated with zinc, that can influence arginine metabolism in the chick.     

Relationship of urinary kallikrein excretion to urinary potassium excretion during furosemide administration with and without amiloride

The relationship of urinary kallikrein excretion to urine volume, and to urinary sodium and potassium excretions was studied in normal rats during furosemide diuresis and superimposed injection of amiloride, a K+-sparing diuretic. Continuous infusion of furosemide increased urinary kallikrein, sodium and potassium excretions and the urine volume. Amiloride injection during furosemide diuresis caused further increase in diuresis and natriuresis, but a prompt decrease in urinary kallikrein excretion to basal level, and potassium excretion to below the basal level. The significant correlation of urinary kallikrein excretion to urinary potassium excretion, but not to urine volume and urinary sodium excretion after amiloride injection suggests that the major determinant of urinary kallikrein excretion is renal potassium secretion through a mechanism that is affected by amiloride.     

Arginine deprivation and tumour cell death: Arginase and its inhibition

Arginase treatment of cell cultures reduced arginine in the medium to ~ micromolar levels within 5–30 min, and proved as effective as arginine-free medium (AFM) prepared by formulation. The enzyme was heat stable and as active at pH 7.2 as at pH 9.9. It persisted in culture for at least 3 days with only a small diminution in its speed of action, and still actively destroyed arginine after 6 days, since arginine supplementation failed to rescue viable cells.Addition of L-norvaline, an inhibitor of arginase, rescued cells from arginase-induced deprivation. Its efficacy at low concentrations was short-lived (probably < 1 day), while at higher concentrations it did not appear to inhibit completely the enzyme. However, L-norvaline at these same levels also slowed the growth of positive non-enzyme treated controls receiving the normal arginine levels. Thus the difference in this growth indicated that arginase was more inhibitory than cursory examination of initial kinetic data suggested. It also agreed with the inhibition of arginase in the ornithine assay used to measure biochemically enzyme activity. We conclude that norvaline partially but not completely antagonises arginase activity, which allows cell rescue in a dose-dependent manner between 0.4 and 4 mM, but cannot be used above about 2 mM without exhibiting a general non-specific interference of cell growth of its own, although no evidence of cell toxicity was observed in either AFM or arginine-containing medium. L-ornithine, the product of arginase that inhibits the enzyme by a feedback mechanism, had no inhibitory effect on arginase over a similar concentration range.     

The short-term effect of cortisol, dexamethasone and ACTH administration on the serum levels of hexuronic acids and monosaccharides in man

The levels of serum monosaccharides (SMO) and hexuronic acids (SHA) were measured in subjects without any metabolic or endocrine disease after a short-time administration of cortisol, dexamethasone and ACTH. The effects of the three hormones were evaluated in regard to the urinary excretion of free cortisol and cortisone at basal conditions. In thirteen subjects a significant increase of SMO during cortisol treatment was registered after 24 hours. A distinct difference in the response of SMO to cortisol treatment was observed in patients with normal or increased cortisol excretion, respectively. In the subjects with high urinary free corticoids a peak of SMO occurred soon after 4 hours after cortisol administration, in the next 48 hours no tendency of return towards basal levels was observed. In the subjects with normal urinary free cortisol excretion only a slight increment was seen after 24 hours. Soon after 4 hours in eight subjects dexamethasone administration resulted in an increase of SMO without regard to the excretion of urinary free corticoids. The highest values were obtained after 28 hours of dexamethasone treatment. Ten hours after cessation of dexamethasone the levels of SMO reached the basal values. In the study in which ACTH was administered, an increment of SMO was registered only in the first four hours. In the group of subjects treated with ACTH a slight difference between subjects with normal and increased corticoid excretion was seen. The levels of SHA successively increased after the administration of all three hormones, without regard to the basal excretion of urinary free corticoids. This increase persisted also 10 hours after cessation of cortisol and dexamethasone, and 40 hours after the last dosis of ACTH, respectively. The possibility of an altered metabolism of glucose through the glucuronate pathway under conditions of glucocorticoid excess is discussed.     

Urinary N tau-methylimidazole acetic acid excretion in respiratory disease

N tau-methylimidazole acetic acid (N tau-MIAA) is the principal urinary metabolite of histamine. The basal urinary excretion rate of N tau-MIAA was determined as 0.117 +/- 0.008 (SE) mg/h, with a renal clearance for N tau-MIAA of 273 +/- 27 ml/min implying active secretion. After subpharmacological infusion of histamine (50 ng.kg-1.min-1 over 2 h) in five volunteers that increased plasma histamine from 0.28 +/- 0.04 to 0.71 +/- 0.15 ng/ml, urinary excretion of N tau-MIAA over 8 h was increased by less than 17% compared with a control saline infusion. Urinary N tau-MIAA excretion in normal controls (273 +/- 14 micrograms/mmol creatinine) was similar to that observed in patients with severe acute asthma (253 +/- 22 micrograms/mmol), antigen-induced bronchoconstriction (269 +/- 21 micrograms/mmol), seasonal allergic rhinitis (304 +/- 31 micrograms/mmol), and clinically stable bronchiectasis (270 +/- 22 micrograms/mmol). In contrast, large increases in metabolite excretion (greater than 7,000 micrograms/mmol creatinine) were observed in a patient with systemic mastocytosis where very high plasma histamine levels were recorded (greater than 500 ng/ml) and marked systemic hemodynamic effects occurred. We conclude that urinary N tau-MIAA will only be increased in pathologies where sustained hyperhistaminemia occurs and that increased local histamine production in the lung or the upper airway does not cause a measurable change in the basal urinary excretion of this metabolite.     

Morphological changes in the skin and wool fibres of Merino sheep infused with mouse epidermal growth factor

Intravenous infusion of 4.5-4.7 mg of mouse epidermal growth factor (mEGF) into nine castrated male Merino sheep for 26 h resulted in complete casting of the fleeces 6-8 days later. The morphological changes which occurred in the skin were studied in skin samples taken before infusion and at intervals between 1 h and 42 days after the infusion had begun. Wool fibres from the shed fleeces were examined with the scanning electron microscope. Increased cell proliferation occurred in the epidermis and sebaceous glands, whereas the wool follicles regressed. Transient dermal haemorrhages occurred during the first 3 h of infusion. The fibre and inner root sheath in the keratogenous zone of 30-40% of the follicles were partially disrupted within the first 6 h of mEGF infusion; catagen began in all follicle bulbs within 24 h. Fibre and inner root sheath production, although markedly reduced, continued in about 60% of follicles which had partially regressed, but production ceased in the remainder in which tapered ends formed on the fibres prior to shedding. Follicles began to regenerate asynchronously 4-8 days after the beginning of infusion and completed their development during the next 3 weeks. The follicle regression and fleece casting induced by mEGF infusion, and subsequent follicle regeneration were completed more rapidly than observed previously with other depilatory agents, and, except for prolonged epidermal thickening, there was no lasting cutaneous abnormality.     

Rat endometrial stromal-epithelial response to estrogen infusion

Morphologic changes at the interface of rat endometrial luminal epithelial cells and the stromal cells immediately adjacent were examined and correlated with hypertrophy of the epithelial cells during estradiol (E2) infusion (1 microgram E2/24 h). While the lamina densa in castrate endometrium was thread-like, it became thicker and apparently more granular in some areas below the luminal epithelium during E2 infusion. However, no changes were seen in the intensity of laminin-like immunoreactivity at various time points up to 96 hours after beginning infusion, suggesting that these alterations were due to changes in nonlaminin components. The stromal cells adjacent to the basal lamina in the castrate state had cell processes extending toward the epithelium that terminated on the basal lamina. Under estrogen infusion, stromal cell bodies migrated close to and became oriented along the basal lamina. No interruptions were seen in the lamina densa or in the laminin-like immunoreactivity in the basal lamina. Thus, there were no direct morphologic interactions between epithelial and stromal cells induced by estrogen. Some of the stromal cells developed a dilated rough endoplasmic reticulum and some developed multiple elaborate processes within 41 hours after minipump implantation. Within 28 hours, nuclear hypertrophy had occurred in 15% of the epithelial cell layer. If interactions occur between stromal and epithelial cells, and morphologic evidence presented here suggests they do, then all such interactions are through an intact lamina densa-laminin layer, and any chemical mediators affecting cells on opposite sides of the lamina densa must migrate through it.     

Effects of acute administration of taurocholic and taurochenodeoxycholic acid on biliary lipid excretion in the rat.

Comparison of the effects of biliary lipid excretion produced by infusion of taurochenodeoxycholate and taurocholate showed no significant difference when the bile acids were infused for a relatively short period of time. Cholesterol excretion rates measured during depletion of the bile acid pool were significantly higher than cholesterol excretion rates measured during infusion of bile acids at various rates. These data indicate that there is some mechanism in addition to bile acid excretion that is responsible for biliary excretion of cholesterol when the enterohepatic circulation is intact.     

Changes in urinary water and electrolyte excretion in sodium-loaded sheep in response to intravenous infusion of arginine vasopressin.

Mature sheep receiving supplements of sodium chloride into the rumen were given intravenous infusions of arginine vasopressin at rates varying from 4-6-23 pmol/min (2-10 mU/min). Infusion of the hormone led to an increase in urine flow and to increases in the amounts of sodium and chloride excreted, the effect on flow was, however, the greater so that the osmolality of the urine fell during the infusions. In sheep given intravenous infusions of a hypertonic sodium chloride solution addition of vasopressin to the infusate led to the formation of a larger volume of urine containing a higher proportion of the infused salt load compared to when the salt solution alone was given. As before the effect on flow was the greater and hence the osmolality of the urine was lower when the hormone was given. In other experiments intravenous infusion of a hypertonic sodium chloride solution at rates providing 2-8 mmol NaCl/min led to increases in urine flow and increases in sodium and chloride excretion, the size of these increases being proportional to infusion rate. Plasma vasopressin levels markedly increased during these infusions, the levels seen being similar to those seen in sheep given vasopressin in amounts which increased both urine flow and electrolyte excretion. This suggests that during hypertonic salt loading vasopressin probably contributes directly to the increases in urine flow and the increases in electrolyte excretion which are seen. Further evidence in support of this was obtained in experiments in which a greater natriuretic response was seen in sheep given a hypertonic sodium chloride solution into the carotid artery as opposed to the given a hypertonic sodium chloride solution into the carotid artery as opposed to the jugular vein and where it was shown that plasma vasopressin levels were indeed higher when the solution was given into the artery.     

Effect of glucagon antiserum on somatostatin,glucagon and insulin release from the isolated perfused rat pancreas

In order to elucidate the effect of glucagon antiserum on the endocrine pancreas, the release of somatostatin, glucagon, and insulin from the isolated perfused rat pancreas was studied following the infusion of arginine both with and without pretreatment by glucagon antiserum. Various concentrations of arginine in the presence of 5.5 mM glucose stimulated both somatostatin and glucagon secretion. However, the responses of somatostatin and glucagon were different at different doses of arginine. The infusion of glucagon antiserum strongly stimulated basal secretion in the perfusate total glucagon (free + antibody bound glucagon) and also enhanced its response to arginine, but free glucagon was undetectable in the perfusate during the infusion. On the other hand, the glucagon antiserum had no significant effect on either insulin or somatostatin secretion. Moreover, electron microscopic study revealed degrannulation and vacuolization in the cytoplasm of the A cells after exposure to glucagon antiserum, suggesting a hypersecretion of glucagon, but no significant change was found in the B cells or the D cells. We conclude that in a single pass perfusion system glucagon antiserum does not affect somatostatin or insulin secretion, although it enhances glucagon secretion.     

Immunohistochemical comparative analysis of transforming growth factor alpha, epidermal growth factor, and epidermal growth factor receptor in normal, hyperplastic and neoplastic human prostates.

Immunoreaction to TGF-alpha was limited to the basal epithelial cells of focal areas in the normal prostates. In benign prostatic hyperplasia (BPH) the immunostained areas were more widespread and immunolabelling was observed in both basal and columnar (secretory) cells of the epithelium. Some cells in the connective tissue stroma were also stained. In prostatic adenocarcinoma, epithelial immunostaining was even more extensive and intense than in BPH, and some stromal cells were also stained. Epidermal growth factor (EGF) immunostaining was only present in some basal cells in normal prostates. In BPH, this immunoreaction was strong in the basal cells and even stronger in the secretory cells. In prostatic cancer, the intensity of epithelial cell immunoreactivity was intermediate between that of normal prostates and that of BPH specimens. EGF-receptor immunostaining was focal and located in the basal cells in normal prostates. In BPH, labelling was also localized in basal cells but extended to wider areas. Some stromal cells appeared weakly labelled. In the prostatic carcinoma, both basal and columnar cells appeared stained and the number of immunolabelled stromal cells was higher than in BPH. The results presented suggest that, in normal conditions, EGF and TGF-alpha act as autocrine growth factors for the basal cells of the prostatic epithelium. In BPH this action is maintained and, in addition, the columnar cells start to secrete both factors which are bound by the basal cell receptors, giving rise to a paracrine regulation which probably overstimulates basal cell proliferation. In prostatic carcinoma, besides these regulatory mechanisms, the acquisition of EGF-receptors by the secretory cells develops an autocrine regulation which might induce their proliferation.     

Pyrimidine biosynthesis in normal and transformed cells

We have developed procedures for sensitive measurement of specific radioactivities of pyrimidine nucleosides excreted from cells in culture. The changes in the observed values reflect dilution of the added isotope through de novo biosynthesis of nonradioactive pyrimidine nucleosides or by shifting and equilibration of other nucleotide pools into the free uridine pool. It is thus possible to monitor uridine biosynthesis occurring in intact cells without destroying or disrupting the cell population. On comparing a series of normal and transformed lines, we have observed several growth-dependent patterns of change in specific activity and levels of uridine excretion and the temporal appearance of these changes. Hamster embyro fibroblasts slows pyrimidine biosynthesis at mid-growth while the hamster cell line V79 continues to dilute the pyrimidine pool at about 7% of the rate observed during exponential growth at confluence. Both cells exhibit Urd excretion beginning at one-half maximal growth. Passageable normal rat liver cells (IARC-20) also show a cessation of pyrimidine biosynthesis with a prior increase in uridine excretion. Two chemically transformed lines IARC-28 and IARC-19 derived from IARC-20 show different patterns. IARC-19 begins uridine excretion in early log growth and the specific activity continues to decrease at about 2% of the rate observed during exponential growth at confluence. The IARC-28 cells also begin excretion in early log growth but pyrimidine biosynthesis stops at about midlog. This method may prove to be an additional aid in recognizing and differentiating transformed cells in culture that do not exhibit the transformed phenotype.     

Role of the hepatocyte microtubular system in the excretion of bile salts and biliary lipid: implications for intracellular vesicular transport

The role of the hepatocyte microtubular system in the transport and excretion of bile salts and biliary lipid has not been defined. In this study the effects of microtubule inhibition on biliary excretion of micelle- and non-micelle-forming bile salts and associated lipid were examined in rats. Low-dose colchicine pretreatment had no effect on the baseline excretion of biliary bile salts and phospholipid in animals studied 1 hr after surgery (basal animals), but slightly retarded the excretion of tracer [14C]taurocholate relative to that of lumicolchicine-pretreated (control) rats. However, colchicine pretreatment resulted in a marked reduction in the excretion of 2 mumol/100 g doses of a series of four micelle-forming bile salts of differing hydrophilicity, but had no significant effect on the excretion of the non-micelle-forming bile salt, taurodehydrocholate. Continuous infusion of 0.2 mumol of taurocholate/(100 g.min) following 24 hr of biliary drainage (depleted/reinfused animals) resulted in physiologic bile flow with biliary excretion rates of bile salts, phospholipid, and cholesterol that were markedly inhibited (mean 33, 39, and 42%, respectively) by colchicine or vinblastine pretreatment. Excretion of tracer [14C]taurocholate also was markedly delayed by colchicine in these bile salt-depleted/reinfused animals. In contrast, colchicine did not inhibit bile salt excretion in response to reinfusion of taurodehydrocholate. Thus, under basal conditions, the microtubular system appears to play a minor role in hepatic transport and excretion of bile salts and biliary lipid. However, biliary excretion of micelle-forming bile salts and associated phospholipid and cholesterol becomes increasingly dependent on microtubular integrity as the transcellular flux and biliary excretion of bile salts increases, in both bile salt-depleted and basal animals. We postulate that cotransport of micelle-forming bile salts and lipids destined for biliary excretion, via an intracellular vesicular pathway, forms the basis for this microtubule dependence.