Evidence for negative feedback control of cholesterogenesis from mevalonate in liver: Absence in the intestine of guinea pigs fed A 0,5 % cholesterol diet

The in vitro rate of incorporation of [2-¹⁴C]-acetate and [2-¹⁴C]-mevalonate into cholesterol of liver, ileum and caecum was determined in guinea pigs. In control animals, contrary to the situation observed when acetate was used as precursor, the rate of conversion of mevalonate to cholesterol was higher in liver than in intestine. In this latter tissue, the cholesterogenesis varied depending on the portion tested. The distribution of radiolabel derived from mevalonate between esterified and unesterified cholesterol differed among the various tissues. In cholesterol-fed guinea pigs, the plasma, liver, intestine and aorta cholesterol contents increased significantly. In addition, a negative feedback control existed for hepatic cholesterol synthesis for mevalonate and acetate. This control was absent in intestinal tissues.     

Respiration of 14CO2 by intact animals of various species given l-[1-14C]fucose or d-[1-14C]arabinose

The production of ¹⁴CO₂ from l-[1-¹⁴C]fucose and d-[1-¹⁴C]arabinose has been studied in five mammalian species.Cats, guinea pigs, mice, and rabbits respired about 22% of the label of l[1-¹⁴C]fucose or of d-[1-¹⁴C]arabinose within 6 h after intraperitoneal injection of the sugar. Rats respired only 1.5% of the l-fucose label and 5% of the d-arabinose label in the same time period.Liver homogenates from cat, guinea pig, and rabbit produced significantly more ¹⁴CO₂ from l-[1-¹⁴C]fucose or d-[1-¹⁴C]arabinose than mouse or rat liver homogenates. Unlike those of the other species, guinea pig liver homogenates had very low l-fucose dehydrogenase activity.The results suggest that substantial catabolism of l-fucose and d-arabinose occurs in the tissues of some animal species. Investigators wishing to employ l-fucose as a tracer of glycoprotein metabolism must, therefore, ensure that the species that they employ does not metabolize l-fucose to products interfering with their studies.     

Determination of tissue UDP-glucuronic acid levels by high-pressure liquid chromatography

A new and sensitive method to measure UDP-glucuronic acid extracted from as little as 25 mg wet weight tissue has been developed. This procedure employs high-pressure liquid chromatography and liquid scintillation spectrophotometry to measure p-[¹⁴C]nitrophenylglucuronide generated enzymatically from p-[¹⁴C]nitrophenol and UDP-glucuronic acid. The reaction was catalyzed by UDP-glucuronyltransferase obtained from rat liver microsomes. The tissue levels of UDP-glucuronic acid assayed were 2 to 20 μmol/100 g wet wt, which are well below the levels detectable by the widely used spectrophotometric method.     

Estimation of acyldihydroxyacetone phosphate and lysophosphatidate in animal tissues

Chemical and enzymatic methods have been developed to measure small quantities (10(-8) - 10(-10) mol) of acyldihydroxyacetone phosphate in animal tissues. Lipids extracted from tissue samples with acidic CHCl3/methanol were subjected to solvent partitioning at two different pH values for partial purification of this keto-lipid from other lipids. This lipid was then estimated radiometrically either by chemical reduction with NaB3H4 or by enzymatic reduction with [4B-3H]NADPH using a partially purified acyldihydroxyacetone-phosphate reductase (EC 1.1.1.101). Thin-layer chromatography revealed the presence of a number of 3H-labeled lipids in the NaB3H4-reduced product and further purification of the product was necessary to estimate the amount of acyl[2-3H]glycerol 3-phosphate formed. The enzymatic reduction was very specific for acyl/alkyldihydroxyacetone phosphate. The amounts (nmol/g) of these keto-lipids estimated in different tissues by the enzymatic method were 10.06 +/- 0.64 (guinea pig liver), 4.3 +/- 0.15 (rat liver), 2.1 (rat testis), 1.5 (rad kidney) and 1.2 (rat brain). Monoacylglycerol 3-phosphate, i.e., lysophosphatidic acid, which was co-purified with acyldihydroxyacetone phosphate, was found to be present in relatively larger amounts in tissues. The amounts (nmol/g) of this lipid, estimated by enzymatically measuring the amounts of sn-glycerol 3-phosphate released after alkaline methanolysis of the partially purified lipid extracts, were 143 (guinea pig liver), 58 (rat liver), 53 (rat kidney) and 92 (rat brain). Stearic acid (18:0) was found to be the major (65%) fatty acid present in the lysophosphatidate purified from guinea pig liver.     

The effects of chlorphentermine and phentermine on certain metabolic parameters associated with development of rat kidney and liver.

Daily oral administration of the anorexigenic agents chlorphentermine or phentermine (60 mg/kg) to rats for either 1, 3, 5 or 7 days resulted in a significant fall in the incorporation of [¹⁴C]thymidine into renal and hepatic DNA throughout the course of the experiment. Although 24 h after treatment with either drug there was no dramatic change in the incorporation of [¹⁴C]orotic acid into liver RNA, a statistically significant reduction was noted after 3, 5 and 7 days. In rat kidney, the incorporation of [¹⁴C]orotic acid into RNA was only significantly depressed by chlorphentermine at 5 days and by phentermine at 3 days. In general, treatment with either anorexigenic agent tended to significantly lower or not affect the endogenous concentrations of renal and hepatic putrescine, spermidine and spermine. The chlorphentermine-induced decrease in liver and kidney nucleic acid synthesis was accompanied by depression in the levels of cyclic AMP in both tissues as well as a reduction in the activity of adenylate cyclase in renal tissue. In contrast, chlorphentermine produced a rise in hepatic adenylate cyclase at 5 days followed by a return to control values after 7 days. The phentermine-induced alterations in nucleic acid metabolism appeared generally to occur independent of any changes in the adenylate cyclase-cyclic AMP system of renal and hepatic tissues. In view of the fact that nucleic acids, polyamines and cyclic AMP constitute integral components of the growth process, our data suggest that chlorphentermine and phentermine interfere with certain biochemical parameters associated with the development of kidney and liver.     

Inhibition of mouse brain synaptosomal gamma-aminobutyric acid transport by pyridoxal 5'-phosphate

Crude lysates from a strain of enterotoxigenic $\text{E. coli}$ have been shown to catalyse the incorporation of [³²P] from [adenylate-³²P] NAD⁺ into an 11,000 dalton protein in rat liver membranes. [³²P] incorporation paralleled adenylate cyclase activation and the results suggest that the mechanism of action of the heat-labile $\text{E. coli}$ enterotoxin may involve ADP-ribosylation of an intracellular acceptor protein.     

The metabolism of d-galactosamine and N-acetyl-d-galactosamine in rat liver

d-[1-¹⁴C]Galactosamine appears to be utilized mainly by the pathway of galactose metabolism in rat liver, as evidenced by the products isolated from the acid-soluble fraction of perfused rat liver. These products were eluted in the following order from a Dowex 1 (formate form) column and were characterized as galactosamine 1-phosphate, sialic acid, UDP-glucosamine, UDP-galactosamine, N-acetylgalactosamine 1-phosphate, N-acetylglucosamine 6-phosphate, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine and an unidentified galactosamine-containing compound. In addition, [1-¹⁴C]glucosamine was found in the glycogen, an incorporation previously shown to result from the substitution of UDP-glucosamine for UDP-glucose in the glycogen synthetase reaction. Analysis of the [1-¹⁴C]glucosamine-containing disaccharides released from glycogen by β-amylase provided additional evidence that they consist of a mixture of glucose and glucosamine in a 1:1 ratio, but with glucose predominating on the reducing end. UDP-N-acetylgalactosamine was shown to result from the reaction of UTP with N-acetylgalactosamine 1-phosphate in the presence of a rat liver extract.     

Long - lasting synchrony of the division of enteric bacteria

Recent finding of α-N-acetylglucosamine(1)phospho(6)mannose diesters in lysosomal enzymes suggested that formation of mannose 6-phosphate residues involves transfer of N-acetylglucosamine 1-phosphate to mannose. Using dephosphorylated β-hexosaminidase as acceptor and [β-³²P]UDP-N-acetylglucosamine as donor for the phosphate group, phosphorylation of β-hexosaminidase by microsomes from rat liver, human placenta and human skin fibroblasts was achieved. The reaction was not affected by tunicamycin. Acid hydrolysis released mannose 6-[³²P]phosphate from the phosphorylated β-hexosaminidase. Our results suggest that lysosomal enzymes are phosphorylated by transfer of N-acetylglucosamine 1-phosphate from UDP-N-acetylglucosamine. The transferase activity was deficient in fibroblasts from patients affected with l-cell disease. This deficiency is proposed to be the primary enzyme defect in l-cell disease.     

Studies on the biosynthesis of protein and lipid components of rat liver mitochondria

1. Male rats were injected intraperitoneally with l-[³⁵S]methionine, [³²P]-phosphate and [2-¹⁴C]acetate. The animals were killed at various times up to 72hr. after injection, and liver mitochondria were prepared and fractionated into soluble protein, insoluble protein and lipid for assay of the radioactivity of each fraction. 2. The maximal specific radioactivity of total mitochondrial phospholipid with respect to both ³²P and ¹⁴C was attained after approx. 6hr. 3. ³²P was incorporated most rapidly into phosphatidylethanolamine, maximal incorporation being attained after approx. 6hr.; maximal incorporation into lecithin occurred after 6–12hr. The specific radioactivity of cardiolipin was still slowly increasing at the end of the experiment (72hr.). 4. There were no major differences between the rates of incorporation of ¹⁴C into the lecithin, phosphatidylethanolamine and cardiolipin fractions of mitochondrial phospholipid, maximal incorporation in each case occurring after approx. 6hr. 5. Maximal incorporation of ³⁵S into both soluble and insoluble protein fractions was attained less than 12hr. after injection, the maximal specific radioactivity of soluble protein being higher than that of insoluble protein.     

Action of Miracil D and related compounds on histone synthesis and phosphorylation in regenerating liver

The effect of Miracil D and hycanthone on ³H-amino acid incorporation into histones was studied under conditions known to cause a greater than 90% inhibition of thymidine incorporation into DNA of regenerating rat liver. A dose level of 50 mg of either drug per kg body weight administered 8 h after partial hepatectomy caused an approximate 50% inhibition of amino acid incorporation into fl, f2b and combined f2a plus f3 histone in 24-h regenerating liver. There was little or no effect on amino acid nitrogen concentration or incorporation of ³H-amino acid into the acid-soluble fraction, cytoplasmic proteins or acid-insoluble nuclear proteins. Under the same conditions, Miracil D caused a 65% inhibition of ³²P incorporation into lysirierich f1 histone whereas a structurally related compound, GE-99, did not have a significant inhibitory effect on this parameter nor on [³H]thymidine incorporation into DNA. Temporal studies with hycanthone revealed a suppression of the increased phosphorylation of fl histone in regenerating rat liver without influencing the phosphorylation of other histones. The data support the concept of coordinated control of DNA synthesis and phosphorylation of fl histone.     

Studies on the specific activity of [γ-32P]ATP in adipose and other tissue preparations incubated with medium containing [32P]phosphate

1. The specific activity of the γ-³²P position of ATP was measured in various tissue preparations by two methods. One employed HPLC and the enzymatic conversion of ATP to glucose 6-phosphate and ADP. The other was based on the phosphorylation of histone by catalytic subunit of cAMP-dependent protein kinase (Hawkins, P.T., Michell, R.H. and Kirk, C.J. (1983) Biochem. J. 210, 717–720). The HPLC method also allowed the incorporation of ³²P into the (α + β)-positions of ATP to be determined. 2. In rat epididymal fat-pad pieces and fat-cell preparations the specific activity of [γ-³²P]ATP attained a steady-state value after 1–2 h incubation in medium containing 0.2 mM [³²P]phosphate. Addition of insulin or the β-agonist isoprenaline increased this value by 5–10% within 15 min. 3. Under these conditions the steady-state specific activity of [γ-³²P]ATP was 30–40% of the initial specific activity of the medium [³²P]phosphate. However, if allowance was made for the change in medium phosphate specific activity during incubations the equilibration of the γ-phosphate position of ATP with medium phosphate was greater than 80% in both preparations. The change in medium phosphate specific activity was a combination of the expected equilibration of [³²P]phosphate with exchangeable intracellular phosphate pools plus the net release of substantial amounts of tissue phosphate. At external phosphate concentrations of less than 0.6 mM the loss of tissue phosphate to the medium was the major factor in the change in medium phosphate specific activity. 4. It is concluded that little advantage is gained in employing external phosphate concentrations of less than 0.6 mM in experiments concerned with the incorporation of phosphate into proteins and other intracellular constituents. Indeed, a low external phosphate concentration may cause depletion of important intracellular phosphorus-containing components.     

Incorporation of intravenously injected (2-(14)C) acetate into tissue lipids of hamsters during the last hour of 3-, 6-, and 24-hour periods of hypothermia.

The in vivo incorporation of total lipid ¹⁴C from [2-¹⁴C]acetate is decreased in kidney, liver, and small intestine tissue from 3-, 6-, and 24-hr hypothermic hamsters compared to tissues from normothermic animals. The length of time in hypothermia affects hamster tissues differently; thus, ¹⁴C activity: decreases with time in kidney; increases with time in liver; and increases at 3 and 6 hr but decreases from 6 to 24 hr of hypothermia in small intestine.Tissues from hypothermic hamsters incorporated a greater percentage of [2-¹⁴C] acetate into free sterols and diglycerides and a smaller percentage into phospholipid than did corresponding tissues from normothermic hamsters.The percentage of total fatty acid ¹⁴C activity found as polyunsaturated fatty acid ¹⁴C activity increases in hypothermic kidney, liver, and small intestine with a decrease in the percentage of ¹⁴C activity measured in the saturated fatty acids. Esterification of fatty acid was inhibited in all tissues taken from hypothermic hamsters.     

An easy and rapid method for the measurement of [γ-32P]ATP specific radioactivity in tissue extracts obtained from in vitro rat heart preparations labeled with 32Pi

A rapid method for the measurement of [γ-³²P]ATP specific radioactivity in tissue extracts containing other ³²P-labeled compounds is described. The neutralized acid extract is incubated with cyclic AMP-dependent protein kinase, cyclic AMP and casein. The incorporation of ³²P into casein from [γ-³²P]ATP is measured by perchloric acid precipitation of the protein on filter paper. ³²P-Casein formation is linearly related to the specific radioactivity of the [γ-³²P]ATP. Separation of ATP from other ³²P-labeled compounds is not required for the assay. Application of this method in the evaluation of [γ-³²P]ATP specific radioactivity in two rat cardiac muscle preparations exposed to ³²P_i is demonstrated.     

Metabolism of the plant sulfolipid—Sulfoquinovosyldiacylglycerol: Degradation in animal tissues

Metabolism of the plant sulfolipid—sulfoquinovosyldiacylglycerol (SQDG)—was studied in animal tissues. In vivo experiments with [³⁵S]SQDG in guinea pigs showed that this lipid is not absorbed intact in the gastrointestinal tract. In these experiments, 3 h after administration of [³⁵S]SQDG, the intestinal mucosa contained 1 to 5% of the radioactivity as SQDG, while the remainder was in a water-soluble form. Analysis of the water-soluble components showed that about 60% of the radioactivity was present as sulfoquinovosylglycerol (SQG) and the remainder was present as free SO²⁻₄. In the blood, 99% of the radioactivity was present as SO²⁻₄, SQG was not observed. In liver, only very little radioactivity was observed and appeared to be mainly in the form of SO²⁻₄. Experiments with everted intestinal sacs of guinea pigs confirmed the formation of SQG, SO²⁻₄, and, in addition, sulfoquinovosylmonoacylglycerol (SQMG) in this tissue. In vitro experiments with saline extracts of acetone powders of pancreas and intestinal mucosa of guinea pig, sheep, and rat showed that [³⁵S]SQDG was deacylated to SQMG (sulfolipase A activity) and SQG (sulfolipase B activity). It is concluded that animal tissues deacylate SQDG in a stepwise manner to SQG. It is further metabolized to yield free SO²⁻₄ by cleavage of the C-S bond which appears to be brought about by the intestinal microflora. Sheep pancreatic sulfolipases were characterized. Bile salts, sodium dodecyl sulfate, and Triton X-100 inhibited the pancreatic sulfolipases, while CaCl₂ activated them. Substrate competition experiments and investigations on substrate specificity with a partially purified preparation indicated that relatively specific sulfolipase(s) may exist in pancreas. Among the species tested, guinea pig tissues showed the highest sulfolipase A and B activities followed sheep and rat tissues. Pancreatic enzymes were 18 to 60 times more active than intestinal enzymes.     

In vivo distribution of cryogenically preserved guinea pig granulocytes

Granulocytes were isolated from whole blood of guinea pigs by counterflow centrifugation and labeled with [¹⁴C]diisopropylfluorophosphate ([¹⁴C]DFP). One-half of the labeled cells was injected intravenously via the femoral vein into a guinea pig, while the other half was cryogenically preserved with 5% dimethyl sulfoxide (DMSO), 6% hydroxyethyl starch (HES), and 4% human albumin, at a rate of 4 °C per minute by storage at ?80 °C and then stored for 3 days at ?80 °C. Ninety percent of the isolated granulocytes were recovered after cryogenic preservation, thawing, and washing. Aliquots before injection all produced fluorescein from fluorescein diacetate and excluded ethidium bromide. Latex ingestion was 78% and yeast ingestion was 75%. The frozen-thawed-washed-resuspended labeled granulocytes were injected into a second guinea pig. Paired animals sacrificed 35 min after injection were examined in whole-body sections for distribution of radiolabeled granulocytes to the tissues. In two pairs of animals, activity was found in the lung, liver, spleen, and kidney. The technique does not permit a distinction between fresh and cryopreserved granulocytes although there was a greater deposition of fresh cells in the liver and spleen. No activity was found in the blood of the vena cava in animals with either fresh or frozen cells. An animal injected with free [¹⁴C]DFP revealed a vascular distribution with high activity in blood, lung, and kidney, and less activity in the liver and spleen. The data indicate that radiolabeled, cryogenically preserved guinea pig granulocytes exhibited a tissue distribution qualitatively similar to fresh granulocytes, and free [¹⁴C]DFP infused without granulocytes differed qualitatively and quantitatively from fresh and cryopreserved granulocytes.     

An easy and rapid method for the measurement of [γ-P]ATP specific radioactivity in tissue extracts obtained from in vitro rat heart preparations labeled with Pi

A rapid method for the measurement of [γ-³²P]ATP specific radioactivity in tissue extracts containing other ³²P-labeled compounds is described. The neutralized acid extract is incubated with cyclic AMP-dependent protein kinase, cyclic AMP and casein. The incorporation of ³²P into casein from [γ-³²P]ATP is measured by perchloric acid precipitation of the protein on filter paper. ³²P-Casein formation is linearly related to the specific radioactivity of the [γ-³²P]ATP. Separation of ATP from other ³²P-labeled compounds is not required for the assay. Application of this method in the evaluation of [γ-³²P]ATP specific radioactivity in two rat cardiac muscle preparations exposed to ³²P_i is demonstrated.     

Effects of formamidoxime on macromolecular synthesis in regenerating liver and hepatomas

Formamidoxime caused an inhibition of [³H]thymidine incorporation into DNA in regenerating liver and transplanted hepatomas of different growth rates when administered by i.p. injection to rats. A dose level of formamidoxime (500 mg/kg body weight) which caused at least a 75% inhibition of DNA synthesis in these tissues had little or no effect on the incorporation of [³H]orotate into total RNA. After administration of formamidoxime there was no significant effect on amino acid nitrogen concentration in the tissues. The incorporation of ³H-labeled amino acids into acid-soluble material, cytoplasmic proteins and acid-insoluble nuclear proteins were either unaffected or showed only small changes after treatment of rats with the drug. In regenerating rat liver and Morris hepatomas 7787 and 7777, formamidoxime caused an inhibition of incorporation of ³H-labeled amino acids into both lysine-rich and arginine-rich histones. In the host livers of rats bearing the transplanted hepatomas, histone synthesis was less affected. The data indicated that formamidoxime causes inhibitory effects which are similar in nature and extent to those previously shown for the structurally related compound, hydroxyurea, in the regenerating rat liver and demonstrated that these effects can also be observed in liver tumors.     

Effects of dopamine, gamma-aminobutyric acid and 5-hydroxytryptamine on incorporation of 32P into phosphatides in slices from the guinea pig brain

(1) Dopamine–In slices from guinea pig corpus striatum, dopamine significantly inhibited incorporation of ³²P into phosphatidylethanolamine-plus-phosphatidylserine at a concentration of 0001 mM, and into phosphatidylinositol and phosphatidylcholine at 001 mM. In eight areas of the guinea pig brain in which the effects of 01 mM-dopamine were studied, the only significant increase in incorporation of ³²P into phosphatides was into phosphatidic acid in the hypothalamus; there was significant inhibition of incorporation of ³²P into phosphatidylcholine in cerebellar cortex and thalamus, and into phosphatidylethanolamine-plus-phosphatidylserine in the olfactory bulbs. (2) Gamma-aminobutyric acid—In slices of guinea pig cerebral cortex, GABA (1 mM) significantly inhibited incorporation of ³²P into only phosphatidic acid, diphosphoinositide and phosphatidylinositol and did not significantly affect the level or the specific activity of the nucleotide ～P. GABA (10 mM), significantly inhibited incorporation of ³²P into diphosphoinositide, phosphatidylinositol and phosphatidylcholine, and significantly lowered the specific activity of the nucleotide ～P. (3) 5-Hydroxytryptamine—In slices of guinea pig cerebral cortex, 5HT, (1 mM) significantly increased incorporation of ³²P into phosphatidic acid; in a concentration of 10 mM, 5HT increased incorporation of ³²P into phosphatidic acid four-fold and into both diphosphoinositide and phosphatidylinositol two-fold; other phosphatides were not significantly affected and the specific activity of the nucleotide ～P was not significantly different. In eight brain areas studied, 5HT (10 mM) significantly increased incorporation of ³²P into phosphatidic acid in all areas; into phosphatidylinositol in six areas (excepting cerebellar cortex and hypothalamus); and into diphosphoinositide in the olfactory bulbs, cerebral cortex, hypothalamus and corpus striatum. Incorporation of ³²P into triphosphoinositide was not significantly affected in any area. Incorporation of ³²P into phospha-tidylethanolamine-plus-phosphatidylserine was significantly greater than the control in the olfactory bulbs and incorporation of ³²P into phosphatidylcholine was significantly less than the control in the cerebellar cortex, olfactory bulbs and hypothalamus. (4) The possibility is discussed that increased incorporation of ³²P into phosphatidic acid and/or phosphatidylinositol in response to neurotransmitters might be associated with excitatory, but not inhibitory, neurotransmission; and that inhibition of incorporation of ³²P into various phosphatides may be associated with inhibitory neurotransmission or neuromodulation.     

Preincubation stimulates fatty acid synthesis by liver slices and isolated hypatocytes from fasted and diabetic rats.

We have observed that preincubation of 48 hour-fasted or alloxan diabetic rat liver slices, with no exogenous energy supply, for 3 hours resulted in an increased rate of incorporation of [1-¹⁴C] acetate into fatty acids and cholesterol during the following 2 hours. This preincubation effect was enhanced by the presence of glucose (25mM) in or prevented by the addition of dibutyryl cyclic adenosine 3′,5′ monophosphate (10^?4M) to the preincubation medium. Preincubation of normal rat liver slices did not change their rate of incorporation of [1-¹⁴C] acetate into fatty acids or cholesterol. The rate of ¹⁴CO₂ synthesized by normal, fasted or diabetic liver slices was little affected by preincubation. The preincubation effect, i.e. enhanced fatty acid synthesis was also observed in suspensions of hepatocytes from fasted and diabetic rats, preincubated for 2 hours, followed by a 1 hour incubation with either [1-¹⁴C] acetate or [³H] H₂O as precursor. We conclude from these data that there is concurrent and coordinated short- and long-term regulation of fatty acid biosynthesis in fasted and diabetic rat livers. Further, we suggest that the release of inhibition by preincubation of these tissues provides a useful tool for studying the coordinated control     

An assay for δ-aminolevulinic acid synthetase based on a specific, semiautomatic determination of picomole quantitites of δ-[C]aminolevulinate

δ-Aminolevulinic acid (ALA) synthetase activities in the range of 0.1 to 100 U/ml of enzyme are routinely assayed using a modified Beckman Model 121 amino acid analyzer. THis method reproducibly and specifically quantitates [¹⁴C]aminolevulinate from a mixture of [¹⁴C]succinate metabolites known to interfere in other methods. Mitochondrial ALA synthetase activity in livers of normal adult guinea pigs is determined to be approximately 3 U/g of liver. This activity is only 1/100th the activity in guinea pigs treated with the porphyrinogenic drug 3,5-dicarbethoxy-1,4-dihydrocollidine.