Search

The Heiligkreuz-Santa Croce Formation (also known as Durrenstein Formation, Upper Triassic) in the Dolomites contains one of the most ancient and substantial Triassic amber deposits in the world. The amber is found in sandstones and paleosols. It has an affinity to the conifer family Cheirolepidiaceae, and amber samples from the Julian and Carnic Alps (Southern Alps) also show an affinity to this family. Physico-chemical investigations of the amber from the Dolomites by solid-state Fourier-transform infrared analysis (FTIR), nuclear magnetic resonance (NMR), pyrolysis-gaschromatography/mass-spectrometry (pyr-GCIMS), thermogravimetry (TG), differential thermogravimetry (DTG), and automatized elemental analysis yielded a complete characterization of the amber, and allowed comparison with other ambers and younger resins (copals). FTIR revealed absorption bands typical of all fossil resins, and the spectrum region from 8-10 mu m provided a fingerprint of the Triassic amber that differs front other known resins. The NMR spectrum also shows a typical pattern for fossil resins, but peculiar peak abundances permitted further characterization of the Triassic amber, both in the saturated (10-70 ppm) and unsaturated carbon region (100-160 ppm). The amber also lacks exomethylene resonances found in younger resins at 110 and 150 ppm. Pyrolysis-gas-chromatography/mass-spectrometry (pyr-GCIMS) experiments showed the amber was of class II, with some components of Class L Thermogravimetric (TG) and differential thermogravimetric (DTG) analyses of combustion behavior of Triassic amber indicated a main exothermal event near 437 degrees C, higher than that of other known resins. The elemental composition of Triassic amber is consistent with wellknown constituents of natural resins, although the sulfur content was higher, likely due to high sulfur content in the embedding sediment. Triassic amber from the Dolomites appears to be a new kind of fossil resin with unique stratigraphical and physico-chemical characteristics.

Background Quinolinic acid and other kynurenine metabolites of the oxidative metabolism of tryptophan play an important role in several pathophysiological conditions. We aimed to study the effect of age on the enzyme activities of tryptophan metabolism along the kynurenine pathway. Methods Enzyme activity was investigated in liver, kidneys and small intestine obtained from Sprague–Dawley rats of various ages (1 week, 2–3, 12 and 18 months). Results We found age-related differences in the liver tryptophan 2,3-dioxygenase, small intestine indole 2,3-dioxygenase, liver and kidney kynurenine 3-monooxygenase activities, which decreased significantly with age. Also liver kynureninase activity declined with age, while the activity in kidneys did not show an evident age-related pattern from 2–3 months to 18 months of age. Liver kynurenine oxoglutarate transaminase was quite similar through all considered age groups, while the activity in kidneys was significantly lower in newborn rats and progressively increased up to 12 months, then significantly decreased at 18 months of age. Liver and kidney 3-hydroxyanthranilate 3,4-dioxygenase progressively and significantly increased from newborns to 12 months of age; in the group of rats aged 18 months, the enzyme activity tended to diminish, although not significantly. The liver aminocarboxymuconate-semialdehyde decarboxylase activity increased up to 12 months of age, then tended to decrease at 18 months, while in the kidneys, in which the activity was higher than in the liver at all the considered ages, the activity remained constantly elevated from 2–3 months to 18 months of age. Conclusions A progressive decline in the enzyme activities involved in tryptophan metabolism along the kynurenine pathway in rat tissues was found with age, except for aminocarboxymuconate-semialdehyde decarboxylase, which, on the contrary, was increased after 2–3 months to the other older groups of age. The altered metabolism of tryptophan with ageing can lead to a decreased biosynthesis of nicotinic acid, tryptophan being the major source of body stores of NAD coenzymes, which are involved in almost all biogenetic and biosynthetic pathways of the organism.

Advanced glycation end products (AGEs) accumulate in serum and tissues of patients with chronic renal failure, even in the absence of diabetes, and a different clearance of these species has been observed by hemodialysis and peritoneal dialysis (CAPD). Furthermore, it has been shown that not only AGE but also 1,2-dicarbonyl compounds are formed during heat sterilization of glucose-based peritoneal dialysis fluids. Therefore, we investigated the level of some AGEs (pentosidine and free pentosidine) and dicarbonyl compounds (glyoxal and methylglyoxal) in end-stage renal disease patients subjected to peritoneal dialysis. Samples (20 from healthy subjects, 16 from uremic patients before and after 12 h of peritoneal dialysis) were analyzed, and the plasma and dialysate levels of glyoxal, methylglyoxal, pentosidine, and free pentosidine were determined. In plasma of uremic patients, mean values of pentosidine showed a small decrease after dialysis and were always higher than those of healthy control subjects. An analogous trend was observed for free pentosidine. In the case of peritoneal dialysate, no pentosidine and free pentosidine were found at time zero, whereas both compounds were detected after 12 h of dialysis. Glyoxal and methylglyoxal mean levels showed a decrease in plasma after dialysis even if their values were always higher than those of healthy control subjects. Surprisingly, an analogous trend was observed also in dialysate. These results might indicate that glyoxal and methylglyoxal already present in the dialysis fluid react with the peritoneal matrix proteins, accounting for the gradual loss of peritoneal membrane function that is often observed in patients subjected to CAPD for a long time.

Advanced glycation end products (AGE) and dicarbonyl compounds accumulate in serum and tissues of patients with diabetes and chronic renal failure. Pentosidine, free pentosidine, glyoxal and methylglyoxal have been evaluated in plasma of diabetic patients with poor metabolic control at baseline and after the improvement of glycemic levels, and in plasma and peritoneal dialysate of patients with renal failure before and after 12 h of peritoneal dialysis. In diabetic patients, acceptable metabolic control was unable to normalize levels of pentosidine (after 2 and 10 months), glyoxal and methylglyoxal (after 2 months). In patients with end-stage renal disease, mean values of pentosidine, free pentosidine, glyoxal and methylglyoxal decreased in plasma after dialysis. No pentosidine or free pentosidine were present in the peritoneal dialysate at time 0, but were found after 12 h of peritoneal dialysis; glyoxal and methylglyoxal decreased after 12 h of dialysis. So, glyoxal and methylglyoxal, already present in the dialysis fluid, can react with the peritoneal matrix protein, giving a reason for the gradual loss of peritoneal membrane function often observed in patients undergoing long-term peritoneal dialysis.

Liquorice root has been used in Europe since prehistoric times, and is well documented in written form starting with the ancient Greeks. In this review we compare the independent development of medical uses of this botanical drug in several ancient cultures, attempting to show the rationality of specific indications across different ethnic groups with different cultural backgrounds. Identical specific indications in different cultures highlight universally reproducible therapeutic effects that are beyond those of a mere placebo. In the first part of the review, historical sources dealing with liquorice (Scythian, Greek, Roman, and from the Middle Ages in Germany, Italy, Spain, England) have been considered. In the second part, the historical records of diseases treated with liquorice have been presented. Finally, a comparison between traditional use in and outside Europe, with the most important recent scientific studies concerning its use, is presented.

Cortisol is involved in the distribution and deposition of fat, and its action is regulated by the activity of 11beta-hydroxysteroid dehydrogenase. Glycyrrhetinic acid, the active principle of licorice root, blocks 11beta-hydroxysteroid dehydrogenase type 1, thus reducing the availability of cortisol at the level of adipocytes. We evaluated the effect of topical application of a cream containing glycyrrhetinic acid in the thickness of fat at the level of the thigh. Eighteen healthy women (age range 20-33 years) with normal BMI were randomly allocated to treatment, at the level of the dominant thigh, with a cream containing 2.5% glycyrrhetinic acid (n=9) or with a placebo cream containing the excipients alone (n=9). Before and after 1 month of treatment both the circumference and the thickness of the superficial fat layer of the thighs (by ultrasound analysis) were measured. The circumference and the thickness of the superficial fat layer were significantly reduced in comparison to the controlateral untreated thigh and to control subjects treated with the placebo cream. No changes were observed in blood pressure, plasma renin activity, plasma aldosterone or cortisol. The effect of glycyrrhetinic acid on the thickness of subcutaneous fat was likely related to a block of 11beta-hydroxysteroid dehydrogenase type 1 at the level of fat cells; therefore, glycyrrhetinic acid could be effectively used in the reduction of unwanted local fat accumulation.

Kynurenine pathway enzyme activities, liver tryptophan 2,3-dioxygenase (TDO), small intestine indole 2,3-dioxygenase (IDO), liver and kidney kynurenine 3-monooxygenase, kynurenine-oxoglutarate transaminase, kynureninase, 3-hydroxyanthranilate 3,4-dioxygenase and aminocarboxymuconate-semialdehyde decarboxylase, were assayed in rabbits, rats, mice and guinea pigs. Their activities varied among species. Especially, TDO was present as both holoenzyme and apoenzyme only in rat, while the other species, rabbit, mouse and guinea pig, only showed holoenzyme activity. Mitochondrial liver and kidney kynurenine 3-monooxygenase activities were much higher in mouse and rat, with rabbit showing the lowest activity. Kynureninase activity showed similar values in both liver and kidney in each species. However, lower activity was present in rabbit. As regards kynurenine-oxoglutarate transaminase, the highest activity appeared in kidney, in all species studied. 3-Hydroxyanthranilate 3,4-dioxygenase activity showed different behaviour in the four species. In rabbit, its activity was higher in kidney than in liver; in rat and mouse, it was viceversa; and in guinea pig, both liver and kidney had similar activity. Instead, the activity of aminocarboxymuconate-semialdehyde decarboxylase was higher in kidney than in liver only in guinea pig. Serum tryptophan concentrations were also determined. Rabbit and guinea pig showed similar values, whereas in rat and mouse, serum tryptophan levels were higher, rat having the highest concentrations. In all species assayed, the free fraction was present as 11-12% of total tryptophan.

Enzyme activities along the kynurenine pathway were assayed in the tissues of New Zealand white rabbits made diabetic with alloxan treatment and hypercholesterolemic with a high-cholosterol diet. Activities are expressed as nmoles of product forming per min per mg of protein and per g of fresh tissue. Liver tryptophan 2,3-dioxygenase (TDO) was present only in holoenzyme form. This activity decreased in diabetic-hyperlipidemic and hyperlipidemic rabbits in comparison with healthy animals. Small intestine indole 2,3-dioxygenase was markedly higher than liver TDO in all rabbit groups, but did not show any significant difference in the values among the three groups. Mitochondrial kynurenine 3-monooxygenase activity was higher in liver than in kidney, but were unchanged with respect to controls. Kynureninase showed similar specific activities in the liver and kidney among groups, whereas the activity per g of fresh tissue was significantly lower in the liver of hyperlipidemic and kidney of diabetic-hyperlipidemic rabbits than in healthy animals. Kynurenine-oxoglutarate transaminase and kynureninase showed lower values in kidney, but not in liver, of diabetic-hyperlipidemic rabbits. However, 3-hydroxyanthranilate 3,4-dioxygenase activity was reduced in both liver and kidney of diabetic-hypercholesterolemic and hyperlipidemic rabbits compared with controls. Instead, aminocarboxymuconate-semialdehyde decarboxylase (picolinic carboxylase) activity was significantly higher in diabetic-hyperlipidermic rabbits in comparison with hyperlypidemic and control rabbits. Therefore, in diabetic rabbits, there is an alteration of tryptophan metabolism at the level of 3-hydroxyanthranilic acid --nicotinic acid step, which has the effect of reducing the biosynthesis of NAD in diabetes.

In some animals, the administration of repeated doses of tryptophan can cause death. It has been reported that guinea pig does not survive repeated doses of tryptophan, due to the absence of the hormonal induction mechanism of liver tryptophan 2,3-dioxygenase (TDO). Therefore, it was of interest to investigate if guinea pig is an animal model suitable for studying tryptophan metabolism. The activities of the enzymes of the kynurenine pathway were determined. Liver TDO was present only as a holoenzyme; kynurenine 3-monooxygenase showed similar, but very high, activity in both liver and kidney. Liver and kidney kynureninase values were also similar, whereas kynurenine-oxoglutarate transaminase activity was higher in kidney than in liver. 3-Hydroxyanthranilate 3,4-dioxygenase gave similar, but very high, values in both liver and kidney, whereas aminocarboxymuconate-semialdehyde decarboxylase activity was double in kidney with respect to liver, but much lower than that of 3-hydroxyanthranilate 3,4-dioxygenase. Total and free tryptophan concentrations in serum were also determined. The free fraction was about 10% of total tryptophan.

We studied the effects of 5-HT in resistance vessels. Compounds acting on 5-HT2A and 5-HT1B receptors were tested on rat-tail arterial rings in varying experimental conditions. We tested 5-HT from 50 nM to 100 microM: the pD2 was 6.4 +/- 0.1. We evaluated vasoconstriction by 5-HT in tissues slightly depolarized with 30 mM KCl. In this condition, the 5-HT concentration-related contraction started at lower concentration in comparison to control tissues. Preincubation with 50 nM ketanserin, a 5-HT2A-antagonist, and 1 microM prazosin, an alpha1-antagonist, strongly inhibited concentration-related contraction by 5-HT: the pD2 was 3.2 +/- 0.2. Moreover, we experimented alpha-methyl-5-HT (alpha-me-5-HT) and 5-carboxamidotryptamine (5-CT), selective agonists at 5-HT2A and 5-HT1B receptors, respectively. Both agonists induced concentration-related contraction; the potency order observed was 5-HTalpha-me-5-HT5-CT. Finally, we studied SB 224289, a selective 5-HT1B-antagonist, on contraction by 5-HT in control and in depolarized conditions. 0.2 microM SB 224289 significantly inhibited vasoconstriction induced by 5-HT in depolarized vascular tissues. The data indicate that vasoconstriction induced by 5-HT is mainly dependent on 5-HT2A receptors; however, 5-HT1B receptors are also present in rat-tail artery.

Enzyme activities along the kynurenine pathway, liver tryptophan 2,3-dioxygenase, small intestine indole 2,3-dioxygenase, liver and kidney kynurenine 3-monooxygenase, kynureninase, kynurenine-oxoglutarate transaminase, 3-hydroxyanthranilate 3,4-dioxygenase, and aminocarboxymuconate-semialdehyde decarboxylase, involved in the catabolism of tryptophan, were studied in male adult Wistar albino rats. Intestine superoxide dismutase and serum tryptophan were also determined. Hepatic tryptophan 2,3-dioxygenase is present both as holoenzyme and apoenzyme, but the total activity is inferior to that of intestine indole 2,3-dioxygenase which, therefore, actively oxidizes tryptophan in rats. However, this activity is inhibited by scavengers for the superoxide anion, such as superoxide dismutase, which also shows to be active in small intestine of rat. However, the more active enzymes appeared to be kynurenine 3-monooxygenase and 3-hydroxyanthranilate 3,4-dioxygenase. The former is equally active in both liver and kidney, the latter is more active in liver. Kynurenine-oxoglutarate transaminase is much more active in kidney than in liver, and much more active than kynureninase, which shows similar activities in both tissues. In contrast to the high activity of 3-hydroxyanthranilate 3,4-dioxygenase, aminocarboxymuconate-semialdehyde decarboxylase is 30-35 times less active, showing the efficiency of conversion of tryptophan to NAD. These data demonstrate that rat is a useful animal model for studying tryptophan metabolism along the kynurenine pathway. Serum tryptophan appeared to be 90% bound to proteins. Results demonstrate that, in rat, tryptophan is mainly metabolised along the kynurenine pathway. Therefore, rat is a suitable animal model for studying tryptophan metabolism in the pathological field.

Tryptophan metabolism along the kynurenine pathway in normolipidemic and diet-induced hyperlipidemic New Zealand rabbits was studied. The activities of liver tryptophan 2,3-dioxygenase small intestine indole 2,3-dioxygenase, liver and kidney kynurenine 3-monooxygenase, kynurenine-oxoglutarate transaminase, kynureninase, 3-hydroxyanthranilate 3,4-dioxygenase and aminocarboxymuconate semialdehyde decarboxylase (picolinic carboxylase) were determined. Liver tryptophan 2,3-dioxygenase (TDO) was present only as a holoenzyme. In fact, similar results were found in the absence or presence of the cofactor haematin. In addition, TDO activity was higher in normolipidemic than in hyperlipidemic rabbits. Small intestine indole 2,3-dioxygenase did not change significantly among the two groups of animals, but was higher than liver TDO. Liver and kidney kynurenine 3-monooxygenase activities did not change significantly whereas kynurenine-oxoglutarate transaminase activity decreased only per g of fresh kidney in hyperlipidemic rabbits. Kynureninase activity decreased significantly per g of fresh tissue only in liver. 3-Hydroxyanthranilate 3,4-dioxygenase, both as specific activity and per g of fresh tissue and aminocarboxymuconate-semialdehyde decarboxylase activities showed significantly lower values per g of fresh kidney in hyperlipidemic rabbits compared with controls. Therefore, hyperlipidemia can influence enzyme activities along the kynurenine pathway, leading to a reduction in the biosynthesis of NAD coenzymes.

Enzyme activities involved in tryptophan metabolism along the kynurenine pathway were studied in male New Zealand white rabbits. Activities are expressed both as specific activity and per g of fresh tissue. Liver tryptophan 2,3-dioxygenase activity (TDO), when assayed in either the absence (holoenzyme) or presence of added haematin (apoenzyme), did not change. Therefore, in rabbit, TDO was present only in holoenzyme form. Small intestine indole 2,3-dioxygenase was significantly higher than liver TDO. Mitochondrial kynurenine 3-monooxygenase was higher in liver than in kidney. Kynureninase activity was similar in both tissues, whereas kynurenine-oxoglutarate transaminase was markedly higher in kidney than in liver. 3-Hydroxyanthranilate 3,4-dioxygenase and aminocarboxymuconate-semialdehyde decarboxylase activities were higher in kidney than in liver. However, the former enzyme showed much higher activity than the latter. These findings suggest that, in rabbit, tryptophan is mainly metabolised along the kynurenine pathway although the apo-TDO enzyme is lacking, as high indole 2,3-dioxygenase activity can obviate this lack.

Tryptophan metabolism was studied in adult male Swiss mice by determining enzyme activities along the kynurenine pathway. The following enzymes were assayed: liver tryptophan 2,3-dioxygenase, small intestine indole 2,3-dioxygenase, liver and kidney kynurenine 3-monooxygenase, kynureninase, kynurenine-oxoglutarate transaminase, 3-hydroxyanthranilate 3,4-dioxygenase, and aminocarboxymuconate-semialdehyde decarboxylase. Liver tryptophan 2,3-dioxygenase was present only as a holoenzyme: similar results were obtained in the absence or in the presence of the cofactor haematin. The specific activity of small intestine indole 2,3-dioxygenase was higher than that of tryptophan 2,3-dioxygenase. As superoxide dismutase was very active in mouse intestine, this enzyme may be one of the rate controlling factors of the indole 2,3 dioxygenase activity. Kynurenine 3-monooxygenase appeared to be very active. Kidneys showed higher activity than liver. Instead, kynureninase was more active in liver, but activity was lower than that demonstrated by the other enzymes of the kynurenine pathway. Conversely, kynurenine-oxoglutarate transaminase was much more active in kidney than in liver. However, the most active enzyme along the kynurenine pathway was 3-hydroxyanthranilate 3,4-dioxygenase, with liver showing the highest activity; aminocarboxymuconate-semialdehyde decarboxylase, which showed similar values in both liver and kidney, showed activity markedly lower than 3-hydroxyanthranilate 3,4-dioxygenase. Serum tryptophan appeared to be 87% bound to proteins. Results demonstrate that, in mouse, tryptophan is mainly metabolised along the kynurenine pathway. Therefore, mouse is a suitable animal model for studying tryptophan metabolism in the pathological field.

Recently an individual variability in the relationships between mean blood glucose levels and HbA1c has been observed among diabetic patients. The aim of this study was to provide an accurate description and evaluation of glycated and glyco-oxidated globins from diabetic subjects and their relationship with HbA1c and plasma glucose values.We studied 20 type 2 diabetic and 10 healthy subjects. Plasma samples were analysed by matrix-assisted laser desorption ionisation mass spectrometry.The presence of glycated and glyco-oxidated species of both alpha and beta globin was demonstrated. Values for these showed a good linear relationship with HbA1c values and the mean daily plasma glucose values for the 6 weeks preceding the investigation. Trends differed according to whether patients had chronic complications or not, differences being seen in the slopes of the plots relating HbA1c to the abundance of glycated and glyco-oxidated species.The data obtained are consistent with the concept that individuals have a different individual proclivity for oxidation and/or that different oxidation kinetics are related to behavioural and environmental factors. Our data are thus relevant to the analysis of phenotype differences in diabetic patients.

Background The activity of nicotinic acid in hypercholesterolemia has been poorly understood. In man, nicotinic acid derives for the most part from tryptophan along the tryptophan–nicotinic acid pathway, also called the kynurenine pathway, kynurenine being the key metabolite in this process. In the present paper, we investigated if, in animals with hypercolesterolemia, degradation of tryptophan to nicotinic acid along the kynurenine pathway was perturbated. Methods Liver, kidney and intestine enzyme activities of the tryptophan–nicotinic acid pathway in normolipidemic, diet-induced hyperlipidemic New Zealand and heritable hypercholesterolemic Watanabe (WHHL) rabbits were determined. Results Liver tryptophan 2,3-dioxygenase (TDO) activity was present only as a holoenzyme and was higher in the controls than in the hyperlipidemic and Watanabe rabbits, but no difference was present between the group fed an atherogenic hyperlipidic diet and the WHHL rabbits. Small intestine indole 2,3-dioxygenase (IDO) did not vary significantly among the three groups but was higher in comparison with liver TDO activity. In liver, kynurenine 3-monooxygenase and kynurenine-oxoglutarate transaminase activities did not show any significant difference among the three groups of rabbits. Kynureninase and 3-hydroxyanthranilate 3,4-dioxygenase activities per g of fresh tissue decreased significantly in the group of hyperlipidemic and in WHHL rabbits. In the kidneys, kynurenine 3-monooxygenase and kynureninase activity did not change significantly in the three groups of rabbits; kynurenine-oxoglutarate transaminase activity per g of fresh tissue decreased in both hyperlipidemic groups, but no significant difference was observed between hyperlipidemic and Watanabe rabbits. 3-Hydroxyanthranilate 3,4-dioxygenase activity in kidney was decreased markedly in hyperlipidemic and Watanabe rabbits, but there was no difference between the two hypercholesterolemic groups. Aminocarboxymuconate-semialdehyde decarboxylase activity did not change. Thus 3-hydroxyanthranilate 3,4-dioxygenase may be an important regulatory mechanism in the control of the flow of tryptophan along the kynurenine pathway to NAD in hypercholesterolemic rabbits. Conclusions This study first demonstrates that in rabbits, hypercholesterolemia, both diet- or genetically induced, can influence the enzyme activities of the tryptophan–nicotinic acid pathway leading to a decreased formation of nicotinic acid, and thus NAD.

Tryptophan metabolism along the kynurenine pathway in normolipidemic and diet-induced hyperlipidemic New Zealand rabbits was studied. The activities of liver tryptophan 2,3-dioxygenase small intestine indole 2,3-dioxygenase, liver and kidney kynurenine 3-monooxygenase, kynurenine-oxoglutarate transaminase, kynureninase, 3hydroxyanthranilate 3,4-dioxygenase and aminocarboxymuconate semialdehyde decarboxylase (picolinic carboxylase) were determined.

The purpose of this study was to investigate variations in the enzyme activities of the kynurenine pathway in various mammals (rabbit, mouse, rat, guinea-pig). Liver tryptophan 2,3-dioxygenase, small intestine indole 2,3-dioxygenase, liver and kidney kynurenine 3-monooxygenase, kynureninase, kynurenine-oxoglutarate transaminase, 3-hydroxyanthranilate 3,4-dioxygenase and aminocarboxymuconate-semialdehyde decarboxylase were analysed. Small intestine superoxide dismutase activity and free and total serum tryptophan were also measured. Liver tryptophan 2,3-dioxygenase was present as both holoenzyme and apoenzyme only in rat, while in the other species only holoenzyme activity was observed. Also, small intestine indole 2,3-dioxygenase activity was more abundant in rat than in the other animals studied. The highest activity of small intestine superoxide dismutase was found in rat, and the lowest in rabbit. Liver and kidney kynurenine 3-monooxygenase activity was very elevated and higher in mouse, followed by rat; rabbit showed the lowest activity. Kynureninase activity appeared to be much lower among the enzymes of the kynurenine pathway. However, guinea-pig showed higher activity in both liver and kidney in comparison with other species. With regard to kynurenine-oxoglutarate transaminase, all species examined here presented more abundant enzyme activity in kidney, the value being similar between rat and mouse. Guinea-pig was the animal with the lowest activity. 3-Hydroxyanthranilate 3,4-dioxygenase showed the highest activity of all the enzymes evaluated in the study, but with different levels in liver and kidney, varying among species. The most elevated activity of aminocarboxymuconate-semialdehyde decarboxylase was present in kidney of guinea-pig, and the lowest in rabbit. Serum concentrations of tryptophan were higher in rat, followed by mouse, rabbit and guinea-pig. In conclusion, the present study demonstrates that the enzyme activities of the kynurenine pathway are very active in tissues of the four species of mammals investigated. The proposed method of in vitro enzyme determination represents a valid alternative to study of the tryptophan metabolic route.

Kynurenine pathway enzyme activities, liver tryptophan 2,3-dioxygenase (TDO), small intestine indole 2,3-dioxygenase (IDO), liver and kidney kynurenine. 3monooxygenase, kynurenine-oxoglutarate transaminase, kynureninase, 3-hydroxyanthranilate 3,4-dioxygenase and aminocarboxymuconate-semialdehyde decarboxylase, were assayed in rabbits, rats, mice and guinea pigs. Their activities varied among species. Especially, TDO was present as both holoenzyme and apoenzyme only in rat, while the other species, rabbit, mouse and guinea pig, only showed holoenzyme activity. Mitochondrial liver and kidney kynurenine 3-monooxygenase activities were much higher in mouse and rat, with rabbit showing the lowest activity. Kynureninase activity showed similar values in both liver and kidney in each species. However, lower activity was present in rabbit. As regards kynurenine-oxoglutarate transaminase, the highest activity appeared in kidney, in all species studied. 3-Hydroxyanthranilate 3,4dioxygenase activity showed different behaviour in the four species. In rabbit, its activity was higher in kidney than in liver; in rat and mouse, it was viceversa; and in guinea pig, both liver and kidney had similar activity. Instead, the activity of aminocarboxymuconate-semialdehyde decarboxylase was higher in kidney than in liver only in guinea pig. Serum tryptophan concentrations were also determined. Rabbit and guinea pig showed similar values, whereas in rat and mouse, serum tryptophan levels were higher, rat having the highest concentrations. In all species assayed, the free fraction was present as 11-12% of total tryptophan.

Enzyme activities involved in tryptophan metabolism along the kynurenine pathway were studied in male New Zealand white rabbits. Activities are expressed both as specific activity and per g of fresh tissue. Liver tryptophan 2,3-dioxygenase activity (TDO), when assayed in either the absence (holoenzyme) or presence of added haematin (apoenzyme), did not change. Therefore, in rabbit, TDO was present only in holoenzyme form. Small intestine indole 2,3-dioxygenase was significantly higher than liver TDO. Mitochondrial kynurenine 3-monooxygenase was higher in liver than in kidney. Kynureninase activity was similar in both tissues, whereas kynurenine-oxoglutarate transaminase was markedly higher in kidney than in liver. 3-Hydroxyanthranilate 3,4-dioxygenase and aminocarboxymuconate-semialdehyde decarboxylase activities were higher in kidney than in liver. However, the former enzyme showed much higher activity than the latter.

Enzyme activities along the kynurenine pathway, liver tryptophan 2,3-dioxygenase, small intestine indole 2,3-dioxygenase, liver and kidney kynurenine 3-monooxygenase, kynureninase, kynurenine-oxoglutarate transaminase, 3-hydroxyanthranilate 3,4dioxygenase, and aminocarboxymuconate-semialdehyde decarboxylase, involved in the catabolism of tryptophan, were studied in male adult Wistar albino rats. Intestine superoxide dismutase and serum tryptophan were also determined. Hepatic tryptophan 2,3-dioxygenase is present both as holoenzyme and apoenzyme, but the total activity is inferior to that of intestine indole 2,3-dioxygenase which, therefore, actively oxidizes tryptophan in rats. However, this activity is inhibited by scavengers for the superoxide anion, such as superoxide dismutase, which also shows to be active in small intestine of rat. However, the more active enzymes appeared to be kynurenine 3-monooxygenase and 3-hydroxyanthranilate 3,4-dioxygenase. The former is equally active in both liver and kidney, the latter is more active in liver. Kynurenine-oxoglutarate transaminase is much more active in kidney than in liver, and much more active than kynureninase, which shows similar activities in both tissues.

Enzyme activities along the kynurenine pathway were assayed in the tissues of New Zealand white rabbits made diabetic with alloxan treatment and hypercholesterolemic with a high-cholosterol diet. Activities are expressed as nmoles of product forming per min per mg of protein and per g of fresh tissue. Liver tryptophan 2,3-dioxygenase (TDO) was present only in holoenzyme form. This activity decreased in diabetic-hyperlipidemic and hyperlipidemic rabbits in comparison with healthy animals. Small intestine indole 2,3-dioxygenase was markedly higher than liver TDO in all rabbit groups, but did not show any significant difference in the values among the three groups. Mitochondrial kynurenine 3-monooxygenase activity was higher in liver than in kidney, but were unchanged with respect to controls. Kynureninase showed similar specific activities in the liver and kidney among groups, whereas the activity per g of fresh tissue was significantly lower in the liver of hyperlipidemic and kidney of diabetic-hyperlipidemic rabbits than in healthy animals. Kynurenine-oxoglutarate transaminase and kynureninase showed lower values in kidney, but not in liver, of diabetic-hyperlipidemic rabbits. However, 3-hydroxyanthranilate 3,4-dioxygenase activity was reduced in both liver and kidney of diabetic-hypercholesterolemic and hyperlipidemic rabbits compared with controls. Instead, aminocarboxymuconate-semialdehyde decarboxylase (picolinic carboxylase) activity was significantly higher in diabetic-hyperlipidemic rabbits in comparison with hyperlypidemic and control rabbits.

Tryptophan metabolism was studied in adult male Swiss mice by determining enzyme activities along the kynurenine pathway. The following enzymes were assayed: liver tryptophan 2,3-dioxygenase, small intestine indole 2,3-dioxygenase, liver and kidney kynurenine 3-monooxygenase, kynureninase, kynurenine-oxoglutarate transaminase, 3-hydroxyanthranilate 3,4-dioxygenase, and aminocarboxymuconatesemialdehyde decarboxylase. Liver tryptophan 2,3-dioxygenase was present only as a holoenzyme: similar results were obtained in the absence or in the presence of the cofactor haematin. The specific activity of small intestine indole 2,3-dioxygenase was higher than that of tryptophan 2,3-dioxygenase. As superoxide dismutase was very active in mouse intestine, this enzyme may be one of the rate controlling factors of the indole 2,3 dioxygenase activity. Kynurenine 3-monooxygenase appeared to be very active. Kidneys showed higher activity than liver. Instead, kynureninase was more active in liver, but activity was lower than that demonstrated by the other enzymes of the kynurenine pathway. Conversely, kynurenine-oxoglutarate transaminase was much more active in kidney than in liver. However, the most active enzyme along the kynurenine pathway was 3-hydroxyanthranilate 3,4-dioxygenase, with liver showing the highest activity; aminocarboxymuconate-semialdehyde decarboxylase, which showed similar values in both liver and kidney, showed activity markedly lower than 3hydroxyanthranilate 3,4-dioxygenase. Serum tryptophan appeared to be 87% bound to proteins. Results demonstrate that, in mouse, tryptophan is mainly metabolised along the kynurenine pathway. Therefore, mouse is a suitable animal model for studying tryptophan metabolism in the pathological field.

Recent data from our laboratory have indicated that the rabbit is a suitable animal model for the study of enzyme activities of the tryptophan-nicotinic acid pathway. We report here the pattern of tryptophan metabolism in rabbits made diabetic with alloxan treatment, and hypercholesterolemic with a high-cholesterol diet. A group of rabbits with only hypercholesterolemia was also considered. The enzymes assayed were: liver tryptophan 2,3-dioxygenase (TDO), intestine indoleamine 2,3-dioxygenase (IDO), liver and kidney kynurenine 3-monooxygenase, kynurenine-oxoglutarate transaminase, kynureninase, 3-hydroxyanthranilate 3,4-dioxygenase and aminocarboxymuconate-semialdehyde decarboxylase. TDO showed a reduction of specific activity in liver of diabetic-hyperlipidemic and hyperlipidemic rabbits compared to controls. Intestine IDO activities and liver and kidney kynurenine monooxygenase were unchanged with respect to controls. Kynurenine-oxoglutarate transaminase and kynureninase activities were reduced in the kidneys, but not in the liver, of diabetic-hyperlipidemic rabbits. The main finding was the reduction of 3-hydroxyanthranilate 3,4-dioxygenase activity (expressed as activity per g of fresh tissue) in the liver and kidneys of diabetic-hypercholesterolemic and hyperlipidemic rabbits compared to controls. Conversely, aminocarboxymuconate-semialdehyde decarboxylase activity was significantly higher in diabetic hypercholesterolemic rabbits in comparison with control and hypercholesterolemic rabbits. These data demonstrate that also in diabetic rabbits there is an alteration of tryptophan metabolism at the level of 3-hydroxyanthranilic acid→nicotinic acid step. Also dyslipidemia seems to be involved in enzyme activity variations of the tryptophan metabolism along the kynurenine pathway.

In vitro synergy has been reported for exposure to paclitaxel prior to cis-platin (cis-DDP), whereas the reverse sequence resulted in antagonism. There is no clear evidence for an intracellular origin of the schedule-dependent interaction, but several hypotheses have been proposed, such as effects at the level of DNA crosslinks, or binding to tubulin sites. The purpose of this study was to evaluate the cytotoxicity of these two drugs as single agents, in combination and in sequence, against a human colon adenocarcinoma cell line (LoVo). Moreover, we considered the new Pt-mercaptopyridine complex C/2, was like cis-platin in being able to alter DNA conformation. We have therefore studied the cytotoxic effects after single agent exposure, concomitant exposure (paclitaxel + cis-platin and paclitaxel + C/2) and sequential drugs exposure (paclitaxel--cis-platin and cis-platin--paclitaxel or paclitaxel--C/2 and C/2--paclitaxel). Our results demonstrate that the most cytotoxic effect is induced by paclitaxel and C/2 exposure both in the case of concomitant cell treatment and sequential exposure paclitaxel--C/2.

We studied the effects of 5-HT in resistance vessels. Compounds acting on 5-HT2A and 5-HT,B receptors were tested on rat-tail arterial rings in varying experimental conditions. We tested 5-HT from 50 nM to 100 p.M: the pD2 was 6.4±0.1. We evaluated vasoconstriction by 5-HT in tissues slightly depolarized with 30 mM KC1. In this condition, the 5-HT concentration-related contraction started at lower concentration in comparison to control tissues. Preincubation with 50 nM ketanserin, a 5-HT2A-antagonist, and 1.tM prazosin, an a,-antagonist, strongly inhibited concentration-related contraction by 5-HT: the pD2 was 3.2±0.2. Moreover, we experimented a-methyl-5-HT (a-me-5-HT) and 5-carboxamidotryptamine (5-CT), selective agonists at 5-HT2A and 5-HT,B receptors, respectively. Both agonists induced concentration-related contraction; the potency order observed was 5-HT > a-me-5-HT > 5-CT. Finally, we studied SB 224289, a selective 5HT,B-antagonist, on contraction by 5-HT in control and in depolarized conditions. 0.2 iM SB 224289 significantly inhibited vasoconstriction induced by 5-HT in depolarized vascular tissues. The data indicate that vasoconstriction induced by 5-HT is mainly dependent on 5-HT2A receptors; however, 5-HT1B receptors are also present in rat-tail artery.

O/W creams prepared in three mechanical conditions (F with a hand blender; S turbomixer; T vacuum turbo-emulsor) produced different dispersion grades of the internal phase and different rheological characteristics by using surfactants of different chemical nature (polyoxyethylene-cetostearyl alcohols and polyglyceryl-3-methylglucose distearate). Three tests were used (an in vitro release test across a porous membrane; an in vitro simulated absorption test across a porous membrane impregnated with isopropyl myristate; an in vivo absorption test based on the intensity and duration of the erythema produced by methyl nicotinate after application of the cream on the skin) to assess whether the different physical characteristics influence drug availability from the creams. The different physical characteristics due to the mechanical conditions of emulsifying and gelification appeared not to influence drug release and in vivo absorption.

This study has been conducted to determine whether the rheological differences depending on gelling and treatment conditions could have an influence on drug availability. Lipogels with constant composition were obtained by gelling olive oil with monodiglycerides at rest, under stirring, and milled after gelling. The considerable differences in rheological characteristics produced significant differences on in vitro drug release tests, whereas a lesser influence was observed on in vitro simulated absorption test. The rheological differences appeared not to influence in vivo drug availability. Also, rheological differences owing to the concentration of the gelling agent showed no significant influence on in vivo availability.

Regeneration of endothelium after damage is an important factor, which limits the development of atherogenesis. This study examines the topographical characteristics of regenerating endothelial cells (EC) in rabbit aorta after de-endothelialization by cryodestruction. The effects of cloricromene on these processes were also studied. Vessels were harvested from 6-month-old NZW rabbits, 1 and 3 days after cryodestruction. The vessels were evaluated using scanning electron microscopy (SEM). One day after cryodestruction, there were defects in the endothelial monolayer in the zone of injury in saline treated animals. Large numbers of platelets and monocytes were observed in association with endothelium in the damaged zone. Three days after de-endothelialization the size of the area of the damage had decreased. On the surface of the new endothelial layer and below this defect, the number of adhering monocytes was increased, and many microdefects between endothelium could be seen. Administration of cloricromene for 1 or 3 days after damage reduced the number of endothelium-adherent platelets and monocytes, and microdefects in endothelium. The feature of endothelial repair in rabbits is a relatively large involvement of monocytes and platelets, which are visible below regenerated endothelium. Administration of cloricromene essentially restored re-endothelialization and significantly decreased the number of adherent monocytes and microdefects in the new endothelium.

Positive inotropic effects induced by 6-benzylaminopurine (6-BAP), kinetin and zeatin were studied in rat atria. The potency order observed was 6-BAP > or = kinetin > zeatin. Suramin, a P2-purinoceptor antagonist, inhibited the positive effect of 6-BAP suggesting the involvement of P2-purinoceptors in the positive effect of this cytokinin. In order to elucidate this point, 6-BAP was used against R-PIA (a P1-purinoceptor agonist) and ATP and UTP (both P2-purinoceptor agonists). 6-BAP did not influence negative inotropism by R-PIA whereas both nucleotides were inhibited after pretreatment with the cytokinin. LY 83583, an inhibitor of cGMP production, reduced the inotropic effect by cytokinin whereas L-NAME, an inhibitor of the L-arginine/nitric oxide pathway, did not influence the effect induced by 6-BAP. Indomethacin, an inhibitor of cyclooxygenase, and neomycin, an inhibitor of phospholipase C, did not significantly modify positive inotropism by 6-BAP. Verapamil, an inhibitor of L-type calcium channels, did not change the positive effect of 6-BAP while TMB-8 and dantrolene, two inhibitors of intracellular calcium release, reduced the increase of contractile tension induced by cytokinin. Our data on rat atria suggest that 6-BAP causes a positive inotropism through activation of P2-purinoceptors, involving modification of cGMP and of intracellular calcium.

Variations in liver and kidney kynurenine aminotransferase (KAT) activity in Pittsburg-Yoshida, Brown-Norway, albino Wistar, Sprague-Dawley, Long Evans and heterozygous Gunn rats were studied. In liver, values of KAT specific activity, expressed as μmoles of kynurenic acid formed per hour per mg of protein, were different in the groups of Brown-Norway and Pittsburg-Yoshida rats versus Long Evans and Sprague-Dawley rats. The activity expressed as μmoles of kynurenic acid per g of fresh liver showed other differences, being significantly higher in Gunn with respect to other strains of rats and lower in Pittsburg-Yoshida and Brown-Norway rats. In addition, KAT activity was significantly lower in Pitts-burg-Yoshida than in Brown-Norway rats. In kidney, the specific activity of kynurenine aminotransferase showed significant differences in the values of Sprague-Dawley and Long Evans rats versus the other strains. The activity expressed per g of fresh tissue was significantly higher in Wistar, Sprague-Dawley, Long Evans and Gunn than in Pittsburg-Yoshida and Brown-Norway rats. No significant differences were found in values between hyperlipidemic Pittsburg-Yoshida and their control Brown-Norway rats. These results highlight the importance of considering various rat strains when inbred animal experimental models are used.

This study was designed to compare the activity of two MDR modulators, verapamil and dipyridamole, on the in vitro growth of a human colon carcinoma cell line. The aims were: a) to investigate the different sensitivity of the parental cell line (LoVo S) and the doxorubicin-resistant one (LoVo R) towards the treatment with several antiblastics and their associations with verapamil or dipyridamole; b) to evaluate if the combined use of these drugs with verapamil or dipyridamole increases their cytotoxicity; c) to understand whether the mechanism of action of each modulator is the same. Idarubicin and vinblastine were the most active drugs on both cell lines. LoVo R cells showed cross-resistance to vinblastine, teniposide and mitoxantrone, while chemosensitivity towards cisplatin and cyclophosphamide was almost the same in both cell lines. The inhibitory effect on cell growth was enhanced when the drugs were associated with verapamil, but no difference was detected with cisplatin and cyclophosphamide. Verapamil is thus an effective MDR modulator when used with drugs actively pumped out of tumour cells by P-glycoprotein, while it is ineffective with drugs that induce resistance by different mechanisms. When combined with dipyridamole, a significant result was observed in the case of cisplatin, where a marked increase of cytotoxicity was detected.

We studied rabbit isolated erectile tissue responses to changes in preload and to active tension development with norepinephrine. The effects of antagonists of endothelin-1, prostaglandins E2 and F2alpha and of nitric oxide were also tested on normal and de-endothelialized preparations. Tissue distension was found to elicit spontaneous rhythmic contractions. Increase in preload diminished the latency of the spontaneous activity and augmented the developed force. Active tension development and the inhibitor of the Na+,K+ pump, ouabain, opposed the spontaneous activity. A marked reduction in the resting tension with abolition of the spontaneous activity was observed on normal, but not on de-endothelialized tissues, following the addition of the specific prostaglandin E2 and F2alpha receptor antagonist, SC-19220. At 3 x 10(-4) M, the highest concentration used, the endothelin-A receptor antagonist BQ-123 failed to change the pattern of the spontaneous activity and the resting tension of normal tissues. The nitric oxide synthesis inhibitor, L-NAME, did not produce reliable effects. These findings point to a causal relation between cavernosal tissue distension and phasic and tonic contractions. Phasic contractions appear to be elicited by smooth muscle cells through the enzyme Na+,K+-ATPase. Increase in the resting tone could be mediated, at least in part, by the endothelium, through the release of prostaglandins E2 and/or F2alpha but not of endothelins. We discuss the hypothesis that, in cavernosal tissue, mechanotransduction of distension to contractile responses is an important determinant of detumescence.

Different receptors mediating the release of endothelium-derived nitric oxide (EDNO) have been identified at endothelial level. In the present study we aimed to characterise, on rabbit aorta by means of pharmacological tools, the generation of EDNO by receptors located on endothelial cell membrane (M3, P2u, P2y) and by direct activation of Ca2+ entry into the endothelial cell. Four vasodilating drugs were tested (acetylcholine, UTP, A23187 and 2-methyl-thio-ATP); they were active only if the endothelial layer was intact, suggesting that they act through endothelial receptors. The effect of different nitric oxide synthase (NOS) inhibitors (0.1 mM: L- and D-NAME, L-NMMA, L-NIO and 7-NI) was investigated on NO-mediated relaxation induced by the relaxants in vessels with intact endothelium. NOS inhibitors differently affected relaxation mediated by the vasoactive drugs in isolated rabbit aorta. Reversibility of the inhibition by using a fixed concentration of L-arginine (0.1 mM) was different depending on the relaxing drug and NOS-inhibitor. The data obtained support the coexistence in aortic vessel of more than one endothelial cell NOS isoform, each provided with different receptor coupling.

In the present study we developed an experimental model, resembling human atherosclerosis, by removing the endothelial layer in Watanabe heritable hyperlipidemic (WHHL) rabbits (10 months old) by application of cryodamage on the external surface of arterial vessels. In age-matched New Zealand white (NZW) rabbits, used as control, after two months following cryodamage, carotid artery and infrarenal segments of abdominal aorta did not show any particular change in the ultrastructure of vessel wall. In WHHL rabbits, two months after cryodamage, atherosclerotic lesions (fatty streaks and fibrous plaques) were observed in both arteries. Many lipid-laden endothelial cells, subendothelial foam cells and smooth muscle cells were found in cryodamaged areas. In some areas, the cap of plaques appeared to be thinned and ruptured. Increased number of collagen and elastic fibrils was also observed in atherosclerotic regions. We conclude that this simple technique on WHHL rabbits provides a model of atherosclerosis with a high degree of morphological similarity between the artificially-induced plaque and human atherosclerotic plaque.

A new stable analog of carbacyclin, 13,14-dihydro-15,16,17,18,19,20-hexanor-14-(1-hydroxycyclohexyl) carbacyclin, identified as MM-706, was investigated in vivo in the conscious rat. MM-706 as single bolus (200 μg/kg, equivalent to 546 nmol/kg) prevented the reduction of circulating platelets induced by adenosine diphosphate (ADP) (1 μmol/kg as IV bolus). 1. 2. A similar effect on free platelet numbers also was observed with iloprost (20 and 200 μg/kg IV bolus). However, MM-706 did not induce any significant variation of mean arterial blood pressure (MABP) or heart rate, unlike iloprost, which reduced MABP within 10 min of administration. 2. 3. In conclusion, MM-706 is effective in interfering with in vivo platelet activation induced by ADP without influencing other cardiovascular parameters, such as arterial pressure and heart rate.

To test whether inhibition of nitric oxide synthesis, associated with high levels of plasmatic lipids, can induce atherosclerotic lesions and phenotypic changes in smooth muscle cell composition in the aortic wall of an atherosclerotic-resistant species such as the rat, an inbred strain of hyperlipidemic Pittsburgh Yoshida rat was subjected to prolonged treatment (2 months) with the nitric oxide-synthase inhibitor L ω -nitro-arginine-methyl ester or with L-arginine. The two types of in vivo treatments were not able to modify in vitro aortic endothelium-mediated relaxation induced by acetylcholine or calcium-ionophore A-23187, the endothelium-independent sodium nitrite relaxation and the contractile response to serotonin. Histology and lipid infiltration of vascular specimens showed that L ω -nitro-arginine-methyl ester in vivo treatment did not induce any significant change in the aortic wall. Monoclonal antibodies to myosin isoforms and immunofluorescence procedures revealed the presence of an immature smooth muscle cell subpopulation in aortic specimens from saline-treated Pittsburgh Yoshida rats, whose expansion has been related in other species to atherogenesis. This peculiar cell phenotype disappeared in our animal model after prolonged L ω -nitro-arginine-methyl ester treatment. These data indicate that, despite interference with endothelium-mediated nitric oxide synthesis, atherosclerosis does not develop in this animal model and furnish for the first time a biological justification for atherogenesis resistance of rat, i.e., the lack of activation of an immature aortic smooth muscle cell population which in atherosclerosis-prone species is involved in lesion formation.

Positive inotropic effects of ATP and UTP (1 microM - 1mM) were studied in isolated rat and guinea pig cardiac tissues. The potency order obtained was ATPUTP in both species, suggesting possible interaction with P2X-purinoceptors. Binding studies using [(3)H]alpha,beta-methylene ATP as marker of P2X-purinoceptors revealed two receptor sites: one high-, the other low-affinity, in atria and ventricles from rat and guinea pig. Both ATP and UTP were found to bind high-affinity sites of [(3)H]alpha,beta-methylene ATP. The effects of various calcium inhibitors such as nifedipine, dantrolene, ryanodine and TMB-8 on positive inotropic effects induced by ATP and UTP were also studied. The results suggest that ATP and UTP may increase inotropism by interaction with P2X-purinoceptors by means of a calcium-dependent mechanism.

In the present study we investigated the in vitro relaxant response of erectile tissue obtained from rabbits of different ages (3, 7 and 24 months) in order to detect the progression with age of cavernosal activity in response to substances acting via endothelium-dependent or -independent mechanisms. Noradrenaline induced a concentration-dependent contraction (0.1 microM-3 mM), with an increase in the contractility in the 24-month-old group. Acetylcholine produced a concentration-dependent relaxant effect in the three age groups, with a reduction of the maximal relaxant effect in older animals. ATP (10 microM-1 mM) and adenosine (10 microM-1 mM) induced a concentration-dependent relaxant effect that was higher in the older group. The presence of the NO2-synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) (0.1 mM) or of the P2-purinoceptor antagonist suramin did not affect ATP relaxation. Relaxation induced by sodium nitrite and nifedipine was reduced in older animals. In conclusion, aging selectively alters the in vitro responsiveness of rabbit erectile tissue. Purinergic system remains more active despite a decrease in the maximal endothelial cholinergic activity and the direct smooth muscle relaxant component.

Quantitative autoradiography techniques were used to evaluate the chronic effects of the potent nitric oxide synthase inhibitor, N omega-nitro-L-arginine methyl ester, on the binding pattern of [3H]flunitrazepam (benzodiazepine agonist) in some behaviorally key limbic areas of the genetic hyperlipidaemic Pittsburg Yoshida rat. Administration of this potent synthase inhibitor was capable of supplying higher and moderately higher binding levels in the basolateral amygdala nucleus (+52%) and in the oriens-pyramidalis CA1 hippocampus layer (+38%), respectively. When we tested for the binding changes in the presence of GABA (principal benzodiazepine modulator) we noticed that a physiological concentration (20 microM) of this inhibitory neurotransmitter was sufficient to induce notable changes in other limbic areas. In fact, lower binding values (-65%) were reported for the bed nucleus of stria terminalis whereas moderately higher values (+38%) were obtained for the radiatum-lacunosum molecular CA1 hippocampus layer. From the saturation studies, it was possible to observe that the major receptor variations provoked by the potent synthase inhibitor were not only due to changes in the total number of binding sites because there were variations, as in the case of the basolateral amygdala nucleus, that were instead due to differences in the affinity binding state. These results provide evidences of a GABAergic-nitric oxide synthase inhibitor interaction that might also be involved in the regulation of convulsive, anxiolytic, and aggressive behaviors that are modulated at the benzodiazepine site.

Nicotinic acid is used in the treatment of atherosclerosis for its hypolipidemic effects (Kritchevsky, 1971; Levy, 1980; Grundy et al., 1981; Altschul et al., 1995), which reduce triglycerides and also cholesterol. In mammals, nicotinic acid derives from the metabolism of tryptophan along the kynurenine pathway. But kynurenine can also be metabolized to kynurenic acid by means of kynurenine aminotransferase (KAT). An abnormal activity of this enzyme should reduce kynurenine and consequently the biosynthesis in nicotinic acid.

The pharmacological characterization of endothelial receptor activity during the progression of atherosclerosis in Watanabe heritable hyperlipidemic (WHHL) rabbits was examined. The data presented show that progression of atherosclerosis affects endothelial receptor functionality in different ways. Nucleotide receptor activity is preserved for longer than that of the other endothelial receptors, i.e. muscarinic- and P2y-purinoceptors. This difference means that, in WHHL rabbit aorta, some mechanisms proposed to explain impaired endothelium-mediated relaxation in atherosclerosis must be excluded. It is therefore hypothesized that atherosclerosis in genetically hyperlipidemic animals selectively influences the receptorial arrangement of endothelium, as in other published examples of selective remodeling of receptors in arteries. Endothelial cell receptors may become a new interesting target for the development of drugs to treat atherosclerosis.

1. 1. In human platelet-rich plasma, platelet aggregation induced in vitro by collagen (10 μg/ml) or thrombin (50 mU/ml) was dose-dependently inhibited by increasing concentrations of prostacyclin or of the new derivative (±)(5E)-13,14-didehydro-ω-hexanor(1-hydroxycyclohexyl)-9a-carbaprostacyclin (MM-706) with an IC 50 of 20–50 nM and 250–500 nM, respectively. In human platelets loaded with fura-2, the intracellular rise of [Ca 2+ ] induced by thrombin was dose-dependently inhibited by MM-706 with an approximate IC 50 of 100 μM. 2. 2. In rabbit isolated femoral artery, MM-706 (10nM–10μM) was completely ineffective in relaxing the vessel, which was different to prostacyclin which was able to relax vessels at the same concentrations. 3. 3. In in vitro guinea-pig ileum, prostacyclin produced a contractile effect in the concentration range 1 nM-10μM, but the derivative MM-706 was ineffective at the same concentrations. Preventive addition of MM-706 did not inhibit prostacyclin contraction. 4. 4. On isolated guinea-pig tracheal preparation, prostacyclin induced a concentration-dependent contraction but the new compound MM-706 showed a lower activity, in the concentration range 10nM–10μM. The activity of prostacyclin was not affected by the contemporary presence of MM-706. 5. 5. It is concluded that MM-706 is a prostacyclin analogue with antiaggregating properties but without evident effects on smooth muscle of different regions.

The present report is an attempt to give relevance to the fractal theory in pharmacodynamics. It is shown, that analysis and discussion of pharmacological in vitro data keeping in mind that a pharmacological process is just a resultant of many different, also fractal, components, would sometimes lead to a more detailed examination of raw patterns of experimental data.