Physiological Plasma Concentrations Research Articles

Retinoic acid synthesis in the somatic cells of rat seminiferous tubules

At physiological plasma concentrations, retinoic acid (RA) cannot cross the blood-testis barrier formed by Sertoli and peritubular cells, and it is thought to be mainly synthesized in situ through the oxidation of retinol. We have thus examined the in vitro RA biosynthetic capacity of cultured Sertoli and peritubular cells isolated from the seminiferous tubules of prepubertal rats, using holo-cellular retinol binding protein (CRBP) as a substrate. Although both somatic cell types contain CRBP and retinoic acid nuclear receptors, RA synthesis was only detected with Sertoli cell subcellular fractions. Most of the RA synthesizing activity of these cells is contributed by a microsomal-cytosolic system that shares many functional similarities with a RA biosynthetic pathway originally identified in rat liver. RA synthesis is maximal at a time of postnatal life (20 days) preceding meiotic cell accumulation and remains nearly constant thereafter. The unique ability of Sertoli cell subcellular fractions to support RA formation from holoCRBP, along with the observed age-dependent modulation of this activity, indicate that Sertoli cells represent the main site of intratubular RA production and that they may play a key role in controlling RA-dependent processes within the seminiferous tubule.

Ceruloplasmin Enhances Smooth Muscle Cell- and Endothelial Cell-mediated Low Density Lipoprotein Oxidation by a Superoxide-dependent Mechanism

Cultured vascular smooth muscle cells (SMC) and endothelial cells (EC) stimulate low density lipoprotein (LDL) oxidation by free radical-mediated, transition metal-dependent mechanisms. The physiological source(s) of metal ions is not known; however, purified ceruloplasmin, a plasma protein containing 7 coppers, oxidizes LDL in vitro. We now show that ceruloplasmin also increases LDL oxidation by vascular cells. In metal ion-free medium, human ceruloplasmin increased bovine aortic SMC- and EC-mediated LDL oxidation by up to 30- and 15-fold, respectively. The maximal response was at 100-300 microg ceruloplasmin/ml, a level at or below the unevoked physiological plasma concentration. Oxidant activity was dependent on protein structure as a specific proteolytic cleavage or removal of one of the seven ceruloplasmin copper atoms inhibited activity. Three lines of evidence indicated a critical role for cellular superoxide (O2.) in ceruloplasmin-stimulated oxidation. First, the rate of production of O2. by cells correlated with their rates of LDL oxidation. Second, superoxide dismutase effectively blocked ceruloplasmin-stimulated oxidation by both cell types. Finally, O2. production by SMC quantitatively accounted for the observed rate of LDL oxidation. To show this, the course of O2. production by SMC was simulated by repeated addition of xanthine and xanthine oxidase to culture medium under cell-free conditions. Neither ceruloplasmin nor O2. alone increased LDL oxidation, but together they completely reconstituted the oxidation rate of ceruloplasmin-stimulated SMC. These results are the first to show that ceruloplasmin stimulates EC- and SMC-mediated oxidation of LDL and that cell-derived O2. accounts quantitatively for metal-dependent, free radical-initiated oxidation of LDL by these cells.

Amino-acid-dependent modulation of amino acid transport in Xenopus laevis oocytes.

We have measured rates of uptake of arginine, glutamine, glutamate, serine, phenylalanine and glycine in Xenopus laevis oocytes cultured for periods of up to 24h in saline in the presence or absence of a mixture of 20 amino acids at concentrations approximating those in Xenopus plasma. Amino acid supplementation increased the total intracellular amino acid concentration from 8.2 to 18.4 nmol per oocyte. Specific Na(+)-dependent amino acid transporters (systems B0,+, Xag-) exhibit 'adaptive regulation' (up-regulation during amino acid deprivation and down-regulation during amino acid supplementation). Na(+)-independent transporters of glutamate, glutamine and glycine (including system asc) display an opposite modulation in activity, which may help to combat amino-acid-induced oxidative stress by increasing the supply of glutathione precursors. Single amino acids at physiological plasma concentrations (0.47 mmol l-1 L-alanine, 0.08 mmol l-1 L-glutamate) mimicked at least some effects of the amino acid mixture. The mechanisms of transport modulation do not appear to include trans-amino acid or membrane potential effects and, in the case of Na(+)-independent transport, are independent of protein or mRNA synthesis. Furthermore, activation of protein kinase C by phorbol 12-myristate 13-acetate did not significantly affect endogenous glutamine and glutamate transport. The Xenopus oocyte appears to possess endogenous signalling mechanisms for selectively modulating the activity of amino acid transport proteins expressed in its surface membranes, a factor for consideration when using oocytes as an expression system for structure-function studies of cloned amino acid transporters.

Corticosterone, prolactin and thyroid hormones as hormonal mediators of the stimulated phagocytic capacity of peritoneal macrophages after high-intensity exercise.

The aim of this study was to evaluate the effect of high-intensity physical activity (swimming until exhaustion) with or without previous training on the phagocytosis and destruction of inert particle capacities of macrophages, and the role of corticosterone, prolactin and thyroid hormones as possible hormonal mediators. The results indicated that high-intensity exercise provokes a stimulation of both phagocytosis and destruction of inert particles when performed in absence of previous training. However, swimming until exhaustion after a one month training program (25 min/day) induced an increase in phagocytosis but not in the destruction of latex beads. Corticosterone, prolactin and thyroid hormones can be considered as hormonal mediators of the exercise-induced stimulation of phagocytosis, since these hormones increased plasma concentration, and the in vitro incubation of macrophages with the same higher physiological plasma concentrations of each hormone, as after exercise, also induced phagocytic stimulation of these cells.

Unesterified long chain fatty acids inhibit the binding of single chain urokinase to the urokinase receptor.

The interaction of single chain urokinase with its receptor accelerates plasminogen activator activity on cell surfaces and induces intracellular signalling in several cell types. To date, no physiologic inhibitor of this binding has been identified. We report that the binding of scuPA to its cellular receptor is inhibited by long chain fatty acids such as oleic acid (C18, delta 9) at physiological plasma concentrations. Inhibition of single chain urokinase binding to human trophoblastic cells by long chain fatty acids was dose-dependent and saturable. Fifty percent of the binding was inhibited at an oleic acid concentration of 27 microM, while inhibition was maximal (75%) at 150 microM oleic acid. The inhibitory potency of oleic acid was unaffected by fatty acid free albumin or human plasma. Inhibition of single chain urokinase binding by free fatty acid analogues was critically dependent on chain length (> C14 required for inhibition) and was proportional to the extent of unsaturation. Only the fraction of specific scuPA binding to trophoblasts that was dependent on uPAR was susceptible to inhibition by oleic acid, while binding of scuPA to vitronectin, thombospondin, and the alpha 2-macroglobulin receptor/low-density lipoprotein-related receptor was not. [3H]Oleic acid bound specifically to recombinant soluble uPAR in a 1:1 molar ratio in the presence or absence of plasma and totally blocked its specific binding to a cell line expressing glycosyl phosphatidylinositol-linked single chain urokinase. These results indicate that oleic acid and other unsaturated long chain free fatty acids may serve as physiologic regulators of proteolytic events and intracellular signalling that depend upon the interaction of urokinase with its receptor.

Detection of oxidants in uremic plasma by electron spin resonance spectroscopy

Depletion of antioxidants and the presence of products of free radical damage in plasma suggest that oxidative stress is increased in uremia. We have developed an application of electron spin resonance spectroscopy, and used this method to show that a stable oxidizing component or components of plasma accumulate in uremia. No oxidizing activity was detectable in plasma from subjects with normal renal function. The oxidant was detected by its capacity to oxidize the spin trap 3,5-dibromo-4-nitrosobenzene sulphonate (DBNBS). The oxidant was dialyzable from plasma, had an upper molecular weight limit of about 3,000 Daltons and was stable over many months. Physiological plasma concentrations of vitamin C, a water soluble congener of vitamin E and reduced glutathione were unable to inhibit the oxidizing capacity of uremic plasma. Thus, uremia is associated with accumulation of an endogenous oxidizing activity at much higher concentrations than in subjects with normal renal function.

Heparin modulation of the fibrinolytic activity of plasmin, miniplasmin and neutrophil leukocyte elastase in the presence of plasma protease inhibitors.

The effect of heparin on the inactivation rates of fibrin-bound plasmin, miniplasmin and neutrophil leukocyte elastase (PMN-elastase) by their plasma inhibitors was studied. While plasmin and miniplasmin bound to fibrin are not inactivated by antithrombin, heparin (800 nM) makes these enzymes available for the inhibitor; the second-order rate constant increases from zero to 1.3 x 10(3) M-1 s-1 and 3.3 x 10(3) M-1 s-1, respectively. Heparin slightly increases the rate of fibrin-bound enzyme inactivation by plasmin inhibitor. alpha 1-Protease inhibitor, on the other hand, is unable to inactivate plasmin or miniplasmin bound to fibrin and heparin has no facilitating effect. In the case of PMN-elastase, heparin (300 nM) further increases enzyme protection against alpha 1-protease inhibitor; the rate constant decreases from 41 x 10(3) M-1 s-1 to 23 x 10(3) M-1 s-1. alpha 2-Macroglobulin inhibits fibrin-bound miniplasmin and PMN-elastase with a second-order rate constant of 1.8 x 10(4) M-1 s-1 and heparin (300 nM) increases the rate insignificantly for miniplasmin and by a factor of two for PMN-elastase. It is remarkable that plasmin bound to fibrin is not inhibited by alpha 2-macroglobulin independently of the presence of heparin. On the basis of the reported kinetic data a lifespan of 420 s for plasmin, 66 s for miniplasmin and 4 s for PMN-elastase was calculated, when the enzymes are bound to fibrin in the presence of the four protease inhibitors at physiological plasma concentration. If heparin is present (300 nM) these values decrease to 240 s for plasmin and 42 s for miniplasmin, whereas that of PMN-elastase is unchanged. Thus, the present in vitro kinetic model suggests an antifibrinolytic effect of heparin in a plasma milieu.

The transport of L-6-fluorodopa and its metabolites from blood to cerebrospinal fluid and brain.

The transport of L-6-fluorodopa and its major metabolites from the blood to the brain, cerebrospinal fluid (CSF), and muscle was studied in carbidopa-pretreated cynomolgus monkeys. A bolus intravenous injection of 18F-L-6-fluorodopa was followed by serial positron emission tomography scans and sampling of cisternal CSF and arterial blood. The relative concentrations of L-6-fluorodopa and its metabolites were determined in blood plasma and CSF by high-performance liquid chromatography. Raising the blood concentration of phenylalanine by intraperitoneal injection markedly reduced the accumulation of tracer in the brain. This indicates that L-6-fluorodopa and 3-O-methylfluorodopa, like native L-dopa and its O-methylated derivative, are transported at the brain capillary by the large neutral amino acid carrier-mediated system, which is subject to saturation and competition by other large neutral amino acids (such as phenylalanine) at physiological plasma concentrations. In contrast, administration of phenylalanine had no effect on the accumulation of tracer either in muscle, or as L-6-fluorodopa and 3-O-methylfluorodopa, in CSF. This suggests that the transport of L-dopa and its derivatives at the blood-CSF barrier differs from the transport at the blood-brain barrier and also that measurement of CSF L-dopa is not a good index of the transport and pharmacokinetics of L-dopa in the brain. However, the effect of phenylalanine administration in reducing the concentration of fluorohomovanillic acid in the CSF suggests that the concentration of homovanillic acid in the CSF is an accurate reflection of dopamine turnover in the brain.

ANP-mediated volume depletion attenuates renal responses in humans.

Brief low-dose infusions of atrial natriuretic peptide (ANP) that emulate physiological plasma concentrations in humans have little if any effect on renal excretory function. This study explored the possibility that ANP-mediated reductions in cardiac filling pressures (through ANP's rapid effect on capillary dynamics) could attenuate its purported renal effects. Protocol A consisted of 16 healthy subjects (ages 19-27 yr old) who underwent three consecutive 45-min experimental sequences: 1) placebo, 2) ANP (10 ng.kg-1 x min-1), and 3) ANP alone (n = 8) or ANP with simultaneous lower body positive pressure (LBPP, n = 8). Electrocardiogram and direct measures of arterial and central venous pressures were continuously monitored. Blood was sampled at the end of each 45-min sequence before subjects stood to void. Compared with control (placebo), ANP produced a hemoconcentration and increased plasma norepinephrine, but did not change heart rate, blood pressure, plasma levels of renin, aldosterone, or vasopressin, or renal excretion of volume or sodium. In subjects receiving LBPP to maintain central venous pressure during the last 45 min of ANP infusion, norepinephrine did not increase and urine volume and sodium excretion increased (P < 0.05). In a second study (protocol B), five healthy subjects received a placebo infusion for 45 min followed by two consecutive 45-min infusions of ANP (10 ng.kg-1 x min-1). Central venous pressure was maintained (LBPP) at placebo baseline throughout the two ANP infusion periods. Urine volume and sodium excretion rates increased progressively and significantly during both ANP infusion periods (P < 0.05) without significant changes in creatinine clearance, blood pressure, or heart rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Release of cromoglycate from dextran and inulin conjugates

Conjugates (CGA-dex and CGA-inu) were synthesized by esterification of cromoglycic acid to dextran (Mol. Wt 10 000) or inulin (Mol. Wt 5000) via imidazolide intermediates. The resulting conjugates contained 15 and 10% drug, respectively. Under in vitro test conditions the release kinetics of cromoglycate from each conjugate were first-order. In buffer pH 7.4 at 37°C, cromoglycate was released from CGA-dex and CGA-inu with a half-life of 136 and 124 min, respectively. CGA was released more rapidly under simulated physiological conditions, namely, plasma and various concentrations of carboxylic ester hydrolase. At the highest enzyme concentrations studied (approx. 400 U/ml) release half-lives were 88 and 43 min, respectively. The stability of CGA-dex and CGA-inu was reduced considerably in fresh human plasma compared to buffer pH 7.4. The resultant half-lives for CGA-dex and CGA-inu were almost identical at approx. 60 min. Overnight incubation of plasma at 37°C, or storage for 1 month at −20°C, before addition of CGA-dex or CGA-inu, considerably reduced but did not totally abolish its hydrolytic activity. Half-lives for CGA-dex and CGA-inu were 124 and 110 min, respectively, in previously incubated plasma and 115 and 104 min, respectively, in previously frozen plasma.

Mechanisms for plasma-mediated activation of human blood cell aldehyde dehydrogenase

Aldehyde dehydrogenase (ALDH; EC 1.2.1.3) activity assays were carried out on isolated human blood cells in phosphate-buffered saline (PBS) and in PBS mixed with human plasma. In assays with intact erythrocytes or sonicated leukocytes, the presence of 50% (v/v) or greater of plasma in the reaction mixtures produced a 2-fold increase in the rate of aldehyde oxidation. In corresponding assays with sonicated erythrocyte samples, the ALDH activity was enhanced on an average 1.5-fold, whereas a slight decrease was observed in assays with intact leukocytes. The ALDH inhibitor disulfiram almost completely abolished the enzyme activity both in the absence and presence of plasma. In assays with sonicated leukocytes, the activation effect could be antagonized by EDTA, indicating that it was caused largely by divalent cations. With sonicated erythrocytes, a significantly reduced ALDH activity was found only with the highest concentration of EDTA tested, and since a similar reduction was obtained also when plasma was omitted, the plasma-mediated activation of erythrocyte ALDH was suggested to be due to a different mechanism. After separation of plasma by gel filtration, an active fraction was identified by GC-MS and 1H-NMR to contain pyruvic acid, lactic acid and glucose. When tested at physiological plasma concentrations, pyruvic acid caused an increase in erythrocyte ALDH activity similar to that obtained with plasma, while lactic acid and glucose did not. Pyruvic acid did not activate the leukocyte ALDH. Based on these results, it is indicated that the plasma-mediated activation of erythrocyte ALDH is due to pyruvic acid, which reoxidizes NADH via lactate dehydrogenase (EC 1.1.1.27) and, thereby, increases the rate of dissociation of NADH from the terminal enzyme-NADH complex, the rate-limiting step in the ALDH pathway.

Physiological concentrations of ANP exert a dual regulatory influence on renin release in conscious dogs.

The influence of physiological increments in circulating atrial natriuretic peptide (ANP) on renin release was determined in conscious dogs. Renin stimulus-response curves (RSRCs) were obtained by controlled reductions of renal perfusion pressure (RPP) under control conditions and during intrarenal or intravenous ANP infusions. Under all experimental conditions, the RSRCs were characterized by a plateau, a threshold pressure (Pth), and a steep slope below Pth. Intrarenal ANP infusion (0.9 ng.kg-1.min-1), which induced a calculated threefold elevation of renal arterial ANP concentration (but did not change systemic arterial ANP levels), increased the slope of the RSRC by 81% (P less than 0.05) with no effect on Pth. A quantitatively similar effect on the slope of the RSRC (+90%; P less than 0.05) was observed when systemic ANP levels were raised (from 37 +/- 2 to 71 +/- 9 pg/ml; P less than 0.05) by intravenous infusions (3.6 ng.kg-1.min-1). In addition, however, intravenously infused ANP reduced Pth from 91 to 85 mmHg (P less than 0.05), which caused a complete suppression of the renin response to a reduction of RPP down to 85 mmHg. These findings indicate that ANP can inhibit renin release at physiological plasma concentrations by shifting the RSRC to a lower pressure level; this shift is mediated by a modulation of extrarenal renin control mechanisms. The direct effect of ANP on renin release is one of stimulation.

Prostaglandin E2 releases ovine fetal ACTH from a site not perfused by the carotid vasculature.

Placental prostaglandin E2 (PGE2) is thought to influence the ovine fetal adrenocortical system to control the timing of parturition. We investigated whether physiological infusions of PGE2 increase fetal immunoreactive adrenocorticotropin (iACTH) at the fetal brain or pituitary or at a site not perfused by the carotid vasculature. PGE2 was infused into the carotid artery (ica) at 0 (n = 5), 10 (n = 5), or 100 ng/min (n = 4) or into the vena cava (ivc) at 10 (n = 5) or 100 ng/min (n = 5) for 30 min in fetuses between 119 and 130 days gestation. Blood gases, vasopressin, cortisol, and arterial and central venous pressure were unchanged. Heart rate increased only in the 100 ng/min ica group. iACTH increased only in the 100 ng/min ivc group from 59 +/- 26 to 180 +/- 73 pg/ml. We conclude that PGE2 infused to create physiological plasma concentrations similar to those at the end of gestation stimulates iACTH from a site other than the fetal brain or pituitary.

Interaction of glucagon-like peptide-1(7-36) amide and gastric inhibitory polypeptide or cholecystokinin on insulin and glucagon secretion from the isolated perfused rat pancreas

The interaction of three incretin candidates, glucagon-like peptide-1(7–36)amide (t-GLP-1), gastric inhibitory polypeptide (GIP), and sulfated COOH-terminal octapeptide of cholecystokinin (CCK-8-S), on insulin and glucagon release from the isolated perfused rat pancreas was studied. Under the perfusate condition of 8.3 mmol/L glucose, coinfusion of 0.1 nmol/L t-GLP-1 and 0.1 nmol/L GIP resulted in an augmented insulin release greater than that obtained by the same dose of each peptide alone. The degree of stimulation elicited by t-GLP-1 and GIP reached a plateau at 0.3 nmol/L for both infusates, and no cooperative effect was observed by coinfusion at 0.3 nmol/L. Coinfusion of 0.1 nmol/L t-GLP-1 and 0.1 nmol/L CCK-8-S also resulted in an augmented insulin release greater than that obtained by the same dose of each peptide alone. A similar cooperative effect was observed by coinfusion at 0.3 nmol/L, 1 nmol/L, and 3 nmol/L. With the same perfusion experiments, glucagon release was not significantly affected by any peptide at concentrations of 0.1, 0.3, 1, or 3 nmol/L. The coinfusion of 1 nmol/L t-GLP-1 and GIP elicited a transient, but significant, increase in glucagon release. A similar result was obtained by the coinfusion of 0.3 nmol/L and 3 nmol/L t-GLP-1 and GIP, respectively. The coinfusion of t-GLP-1 and CCK-8-S did not affect the glucagon release. These results suggest that (1) t-GLP-1 and GIP cooperatively affect insulin and glucagon release at the nearly physiologic postprandial plasma concentrations of both peptides, and (2) t-GLP-1 and CCK-8-S cooperatively affect insulin release in a wide range of concentrations, although the physiologic significance of this interaction should be slight, considering the physiologic postprandial plasma concentration of CCK-8-S.

Effect of sodium ascorbate on the chemiluminescent response of murine peritoneal exudate cells

Vitamin C is an effective antioxidant that neutralizes reactive oxygen radicals. The purpose of this study was to determine if sodium ascorbate would neutralize the reactive oxygen products generated during the respiratory burst of thioglycollate-elicited murine peritoneal exudate cells (PEC). In vitro and in vivo studies were done. Cells treated in vitro showed a significant, dose-dependent reduction in chemiluminescence (CL) after activation with opsonized zymosan. Higher concentrations of sodium ascorbate (24.2 mM) produced a significantly greater reduction in CL than did lower concentrations (0.242 mM). This range of sodium ascorbate concentrations overlaps those found in normal leukocytes (1 – 4 mM). Sodium ascorbate at physiological plasma concentrations (0.09 mM) did not reduce CL. Cells incubated with 500 mM sodium ascorbate in vitro and then washed once prior to zymosan activation also showed a significant reduction in CL. In contrast, PEC harvested from mice treated in vivo with sodium ascorbate (one or five daily doses of 1.0 M sodium ascorbate, 0.01 ml/g body weight) did not show a reduction in CL. This concentration of sodium ascorbate represents a dose that is 2310 times greater than the Recommended Dietary Allowance (RDA). These studies show that physiological doses of sodium ascorbate can quench CL in vitro , but even large doses of sodium ascorbate administered in vivo do not affect the CL of harvested murine PEC.

Androgenic regulation of adipocyte alpha 2-adrenoceptor expression in male and female Syrian hamsters: proposed transcriptional mechanism.

Adaptation of male hamsters to short daily (SD) photoperiod induced a reduction of the adipocyte alpha 2-adrenoceptor (alpha 2-AR) expression which was related to a sexual involution and could be reversed by testosterone administration. In the present paper, the possible mechanisms of such a physiological regulation are explored. The effect of testosterone on the adipocyte alpha 2-AR was rapid, dose-dependent, occurred at the physiological plasma concentrations of androgen, and was mimicked by dihydrotestosterone, but not by 17 beta-estradiol, progesterone, hydrocortisol, insulin, or T3. Adaptation of female hamsters to SD photoperiod also induced a sexual involution, but no modification of the adipocyte alpha 2-AR number was observed. Administration of testosterone induced a large up-regulation of the adipocyte alpha 2-AR. Testosterone was also able to up-regulate the adipocyte alpha 2-AR in male hamsters adapted to long day photoperiod whatever their age (6-, 12-, and 25-week old). Adaptation to SD photoperiod did not modify the adipocyte adenylyl cyclase activity (basal, forskolin-stimulated, GppNHp-inhibited). Conversely, UK14304-mediated inhibition of the adenylyl cyclase was suppressed in SD photoperiod and recovered after testosterone treatment. Administration of testosterone in young male hamsters adapted to long day photoperiod induced an increase in the amount of the alpha 2-AR messenger RNA, which coincided with the increase in the alpha 2-AR maximal number. The existence of a specific regulation of the adipocyte alpha 2-AR by the androgens, probably via a transcriptional mechanism, can be hypothesized. This regulation, which occurs in both male and female hamsters, appears to be physiologically relevant.

On leaking into the lumen, amino acids cross the tubule cells. Secretion of l-citrulline in the isolated-perfused non-filtering kidney of the African clawed toad (Xenopus laevis)

In our previous studies we were able to show that L-citrulline is taken up across the contraluminal membrane into the tubule cells of the isolated perfused non-filtering kidney of the African clawed toad (Xenopus laevis). We have also described the tubular metabolism of the L-citrulline taken up. The remaining question was whether peritubular L-citrulline gains access to the tubular lumen and, if so, by what kind of mechanism. The method was the same as that described in our previous study: the perfusion of the kidneys was performed through both portal veins (2 ml/h into each) with a solution containing inulin. In order to stimulate the urine formation in the non-filtering kidney, we added p-aminohippurate to the perfusion solution. In the urine collected from the ureters no inulin could be detected. Thus, the glomeruli were not reached by the perfusate, and no glomerular filtration took place. Upon adding 65 mumol/l L-citrulline to the perfusion solution (corresponding to the physiological plasma concentration), a urinary excretion rate of this amino acid of 4.5 +/- 0.4 nmol.h-1.g-1 (n = 6) was determined. During perfusion under the same conditions with D-citrulline (65 mumol/l) the excretion rate was only 0.36 +/- 0.1 nmol.h-1.g-1 (n = 5). A similarly low excretion rate was also found with L-citrulline when L-phenylalanine (20 mmol/l) was added to the perfusate or when NaCl was substituted by mannitol. These data show that L-citrulline taken up from the contraluminal side of the renal tubular epithelium enters the tubular lumen to a much greater extent than its D-isomer.(ABSTRACT TRUNCATED AT 250 WORDS)

The Autoactivation of Factor XII in the Presence of Long-Chain Saturated Fatty Acids – A Comparison with the Potency of Sulphatides and Dextran Sulphate

The incubation of purified human factor XII (Hageman factor [HF]) in the presence of long-chain saturated fatty acids (FA) like stearate (C-18) or behenate (C-22) resulted in a time-dependent increase of amidolytic activity. The HF autoactivation progress curves were sigmoidal. The first order rate for the initial period was constant; this was followed by a period of decreasing rate and a plateau of zero rate. These progress curves were similar to those obtained on the incubation of HF in the presence of sulphatide vesicles or dextran sulphate. The initial rate of autoactivation of HF was dependent on the FA concentration of contact surface and increased with increasing concentration of HF. At constant concentration of contact surface and varying concentration of HF, autoactivation rates in the presence of behenate, sulphatide vesicles or dextran sulphate followed Michaelis-Menten kinetics. The Km values for all three contact surfaces were above the physiological plasma concentration of HF whereas the catalytic efficiency in the presence of behenate (0.034 microM-1s-1) was about 2/3 of that in the presence of sulphatide vesicles (0.053 microM-1s-1) and considerably higher than that in the presence of dextran sulphate (0.004 microM-1s-1). Long-chain saturated FA bound to human serum albumin at the high- or low-affinity sites are ineffective, whereas the crystalline non-bound stearate or behenate provided a potent contact surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Testosterone Differentially Modulates Gonadotropin Subunit Messenger Ribonucleic Acid Responses to Gonadotropin-Releasing Hormone Pulse Amplitude*

Testosterone (T) inhibits GnRH secretion and can also modulate the effects of GnRH on gonadotropin synthesis and secretion. To assess the effect of T on GnRH stimulation of alpha, LH beta, and FSH beta mRNA expression, we replaced T at three levels to reproduce low (1.5 +/- 0.5 ng/ml), medium (3.5 +/- 0.3 ng/ml), and high (6.2 +/- 0.6 ng/ml) physiological plasma concentrations. Additionally, as peripheral conversion to dihydrotestosterone (DHT) or estradiol (E2) may mediate T action, the effects of GnRH pulses in the presence of DHT and E2 were also studied. Male rats were castrated, and steroids were replaced via implants containing either T (three doses) or DHT or E2 (two doses each). GnRH pulses (10-250 ng/pulse) were administered iv at 30-min intervals for 48 h. Pituitary subunit mRNA concentrations, gonadotropin content, and LH and FSH secretion were determined. The patterns of alpha, LH beta, and FSH beta mRNA responses to increasing GnRH pulse amplitude were similar at all concentrations of plasma T. Alpha mRNA concentrations were increased 2- to 4-fold by GnRH pulses. At the same plasma T concentration, all doses of GnRH produced similar increases in alpha mRNA, but the response tended to be lower at the higher (6.2 ng/ml) levels of T. LH beta mRNA showed a clear dependence on GnRH pulse amplitude, with the maximum responses (2- to 3-fold) occurring after 10- to 25-ng GnRH pulses. At the higher (3.5 and 6.2 ng/ml) T concentrations, the dose-response curve was shifted to the left. The lowest GnRH pulse dose (10 ng) produced maximum responses, and LH beta mRNA increments in response to the higher GnRH doses were suppressed. FSH beta mRNA concentrations were increased by T in saline-pulsed controls. FSH beta mRNA responses were similar (2- to 3-fold) after all GnRH doses and at all concentrations of T. Increasing GnRH pulse doses reduced the pituitary content of both LH and FSH at all levels of T. Acute LH secretion was maximal after 10- and 25-ng pulses of GnRH when plasma T was low, but increased progressively with GnRH dose at the highest plasma T concentrations. Plasma FSH did not show any differential responsiveness to GnRH pulse dose or to increasing plasma T. Thus, LH synthesis and secretion are affected more than those of FSH by changing plasma concentrations of T. T may modulate posttranslational events in LH secretion. The higher GnRH doses effected LH release without increasing LH beta mRNA in the presence of higher physiological concentrations of T.(ABSTRACT TRUNCATED AT 400 WORDS)

Neuropeptide Y modulates vasoconstriction in coronary microvessels in the beating canine heart.

The purpose of this study was to determine whether neuropeptide Y has a direct vasoconstrictor effect at low doses, mimicking the physiological plasma concentration on the specific site(s) of coronary arterial microvessels in in situ beating canine left ventricles. Coronary microvessels were directly observed by means of an intravital microscope and video system equipped with a floating objective. Epi-illuminated fluorescence coronary microangiography was performed in open-chest anesthetized dogs (n = 14) to examine the changes in internal diameter of epimyocardial arterial microvessels. Flow velocity of fluorescently labeled microshperes in capillaries was also measured (n = 6). To eliminate secondary effects of neuropeptide Y on coronary microvessels via autonomic nervous modulation, experiments were conducted under pharmacological blockade of the regional autonomic nervous system by intracoronary injection of propranolol, 50 micrograms/kg; phentolamine, 100 micrograms/kg; and atropine, 5 micrograms/kg. Aortic pressure and heart rate were kept constant during the experiments. Intracoronary infusion of three different doses of neuropeptide Y (1, 10, and 100 pmol/kg/min) for 5 minutes significantly constricted small microvessels (less than 100 microns in diameter) (-5.2 +/- 1.4%, -8.5 +/- 1.5%, and -14.0 +/- 1.7%; p less than 0.05 versus before neuropeptide Y at each dose), medium microvessels (100-200 microns in diameter) (-5.5 +/- 1.6%, -10.6 +/- 1.8%, and -16.8 +/- 2.1%, p less than 0.05 versus before neuropeptide Y at each dose), and large microvessels (greater than 200 microns in diameter) (-3.6 +/- 0.6%, -5.8 +/- 0.8%, and -10.0 +/- 1.1%; p less than 0.05 versus before neuropeptide Y at each dose) in a dose-dependent manner. Capillary flow velocity was reduced by 17.2 +/- 3.1% by an intracoronary dose of 100 pmol/kg/min of neuropeptide Y (p less than 0.05). The present study indicates that low doses of neuropeptide Y exert a homogeneous direct vasoconstrictor effect on various sizes of coronary arterial microvessels and reduce capillary flow velocity. These results suggest that neuropeptide Y may play a physiological role in modulating coronary microvascular tone.