Muscle protein synthesis in patients with rheumatoid arthritis: effect of chronic corticosteroid therapy on prostaglandin F2α availability

Using stable-isotope techniques, we measured rates of quadriceps muscle protein synthesis in twelve women with sero-positive rheumatoid arthritis. The results were compared to those from the normal limb of seven women with unilateral osteoarthritis of the knee. Six patients had never received corticosteroid immuno-suppression, but the other six had taken an average of 8 mg Prednisolone per day for 9 years. Quadriceps atrophy was present in both sets of patients with rheumatoid arthritis (normal legs 444 +/- 182, rheumatoid 190 +/- 40, rheumatoid + steroid 300 +/- 110 micrograms protein/micrograms DNA, means +/- SD, both P less than 0.001). Muscle protein synthesis, calculated by comparing the incorporation of 13C-leucine into biopsy samples taken after an 8 h L-[1-13C] leucine infusion with the time averaged enrichment of blood alpha-ketoisocaproate, was 0.056 +/- 0.005% h-1 in the patients not receiving steroids compared with 0.050 +/- 0.02% h-1 in normals (P greater than 0.05) indicating that muscular atrophy was primarily due to an increase in rate of muscle protein breakdown. Intra-muscular PGE2 concentration was increased in these patients (rheumatoid 0.12 +/- 0.06 ng mg-1 tissue, normals 0.06 +/- 0.03 ng mg-1 tissue, P less than 0.05). Patients taking corticosteroids had a markedly depressed rate of muscle protein synthesis (0.035 +/- 0.008% h-1, P less than 0.05) and reduced intra-muscular PGF 2 alpha concentration (P less than 0.01). We conclude that steroid therapy significantly influences the mechanism of skeletal muscle atrophy in patients with rheumatoid arthritis.     

Alterations in norepinephrine content and beta adrenoceptor regulation in myocardium bordering aneurysm in human heart: their possible role in the genesis of ventricular tachycardia

On the assumption that alterations in the adrenergic system may play a role in generating ventricular tachycardia in patients with myocardial post-infarction apical aneurysm, we evaluated norepinephrine concentration, number and affinity of both beta 1 and beta 2 adrenoceptors in perianeurysmatic tissue in twelve patients operated upon for congestive heart failure and recurrent sustained ventricular tachycardia. Concentration of norepinephrine in perianeurysmatic tissue was 0.1 +/- 0.05 micrograms g-1 tissue (n = 8), this value being much lower than that found in papillary muscle (n = 10) from patients with mitral valve stenosis (0.8 +/- 0.02 micrograms g-1 tissue) (P less than 0.01). The total number of beta adrenoceptors (71.4 +/- 7.8 v. 48.0 +/- 5.1 fmol mg-1 protein; P less than 0.01) and the percentage of beta 1 subtype were found to be higher in perianeurysmatic tissue (approximately 90%) than in papillary muscle (approximately 68%). Out of twelve patients with aneurysm, beta 2 adrenoceptors had considerably decreased in three patients and were absent in the remaining nine. Decrease in the neuronally released norepinephrine associated with contrasting behaviours of beta 1 and beta 2 adrenoceptors suggests the presence of a profound alteration in the sympathetic innervation of the perianeurysmatic myocardial tissue that may contribute to the genesis of sustained ventricular tachycardia in patients with postinfarction apical aneurysm.     

Identification and characterization of α1- and β2-adrenergic receptors in human liver

Adrenergic receptors were identified in healthy human hepatic tissue from thirty-nine subjects undergoing elective abdominal surgery by using the specific alpha 1-antagonist [3H]-prazosin and the beta adrenergic antagonist [3H]-dihydroalprenolol ([3H]-DHA). [3H]-prazosin binding to plasma membranes was rapid, of high affinity, saturable and stereospecific with a maximal binding capacity (Bmax) of 74.1 +/- 5.5 fmol mg-1 of protein. The displacement curve for (-)-norepinephrine was better explained by a one-site binding and after addition of GTP 0.1 mM the curve was not right-shifted, suggesting the majority of alpha receptors in healthy human liver are of the alpha 1 subtype and not linked to a GTP-binding protein. [3H]-DHA binding to liver plasma membranes was also rapid, of high affinity, saturable and stereospecific with a Bmax 96.5 +/- 10.3 fmol mg-1 of protein of receptors. Computer aided analysis of the displacement curve of ICI 118,551, a subtype selective beta 2-antagonist (IC50 = 62 +/- 2 nM), indicated a one-site binding, thus, showing that beta adrenergic receptors are of the beta 2 subtype. The displacement curve of [3H]-DHA for (-)-isoproterenol was right shifted by GTP indicating that beta 2 adrenergic receptors are linked to a GTP-binding protein in human liver. These results indicate that alpha 1- and beta 2-receptors co-exist in human liver but only beta 2-receptors are linked to a GTP-binding protein.     

The human renal 25-hydroxyvitamin D3-1α-hydroxylase: properties studied by isotope-dilution mass spectrometry

The renal 25-hydroxyvitamin D3-1 alpha-hydroxylase activity has been measured in normal human kidney cortex, using a highly specific assay based on isotope-dilution mass spectrometry. The cortex was obtained from kidneys removed due to renal tumours. The subcellular distribution of 25-hydroxyvitamin D3-1 alpha-hydroxylase activity was studied. Enzyme activity was only observed in the mitochondrial fraction. Mitochondria from non-tumourous kidney cortex had a Vmax of 0.17 +/- 0.02 pmol min-1 mg-1 protein and the apparent Km was in the range of 14 mumol l-1. There was a tendency to a higher 25-hydroxyvitamin D3-1 alpha-hydroxylase activity in preparations from male kidney (0.21 +/- 0.03 pmol min-1 mg-1 protein) than female (0.12 +/- 0.02, P less than 0.05). A significant inverse correlation between serum phosphate and 25-hydroxyvitamin D3-1 alpha-hydroxylase activity was found. No correlation was observed between enzyme activity and serum levels of 1,25-dihydroxyvitamin D (total and free index), PTH, total calcium or ionized calcium. The results indicate that there is a sex difference in human 25-hydroxyvitamin D3-1 alpha-hydroxylase activity similar to the one observed in laboratory animals. Furthermore, the data support the hypothesis that serum phosphate is a major regulator of 1,25-dihydroxyvitamin D3 production in man.     

Abnormality of adenylate cyclase regulation in human platelet membranes in renal insufficiency

Adenylate cyclase in human platelets is under dual control of prostaglandins (PGI2 and PGE1) and catecholamines. The adenylate cyclase complex in membranes of platelets from ten patients with uraemia was investigated. The activation of the platelet cyclase by PGE1 is increased in the uraemic state, Vmax 4436 +/- 607 pmol cAMP mg-1 15 min-1. In the normal state Vmax is 2098 +/- 309 pmol cAMP mg-1 15 min-1. The alpha 2-adrenergic receptor was assayed with 3H-yohimbine binding. The density of receptors was equal in the uraemic (175 fmol mg-1 membrane protein) and the normal (170 fmol mg-1 membrane protein) states. Norepinephrine/3H-yohimbine competition binding revealed that catecholamines were bound with normal affinity in platelets in uraemia. Yet the inhibition of adenylate cyclase through the alpha 2-adrenergic receptor was diminished since Vmax values of adenylate cyclase with PGE1 and PGE2 + norepinephrine did not significantly differ. In the normal state, norepinephrine significantly (P less than 0.05) inhibited the PGE1 stimulated cyclase. It is concluded that platelet adenylate cyclase in the uraemia has an increased capacity for activation which is the result of both a sensitized stimulatory mechanism (prostaglandin mediated) and a deficient inhibitory mechanism (catecholamine mediated). It is suggested that a defect exists in the inhibitory nucleotide binding protein (NI) which is the coupling unit between the adenylate cyclase catalytic subunit (C).     

Hyperparathyroidism and abnormal 1,25(OH)2vitamin D3 metabolism in experimental lead intoxication

Clinical evidence points to disturbed calcium metabolism in lead (Pb) intoxication. To further clarify the mechanisms involved, serum levels of 1,25(OH)2D3, receptors for 1,25(OH)2D3 as well as size and ultrastructure of parathyroid glands were examined in Wistar Kyoto rats exposed to 1% lead (Pb) acetate in drinking water for 10 weeks (short-term study) or 0.001-1% Pb acetate for 24 weeks (long-term study). After administration of Pb for 10 weeks, bone Pb was significantly increased (641 +/- 66.9 (SD) vs. 0.648 +/- 0.39 mg kg-1 ash in controls). Total serum calcium and ionized Ca2+ (1.15 +/- 0.031 vs. 1.25 +/- 0.03 mmol l-1) were significantly decreased. Renal function (Ccr) was unchanged, but urinary cAMP excretion and circulating 1,25(OH)2D3 (177 +/- 10.9 vs. 232 +/- 18.9 pmol l-1) were diminished. Specific binding of 1,25(OH)2D3 was increased in parathyroids (Bmax 128 +/- 4.7 vs. 108 +/- 0.6 fmol mg-1 protein) and intestinal muscosa; Bmax failed to adequately rise in response to pretreatment with 1,25(OH)2D3 (2 x 10 ng day-1 for 4 d) in Pb-exposed animals. Receptor characteristics (sedimentation constant, KD, DNA affinity) were unchanged. Parathyroid weight was significantly increased (178 +/- 25 vs. 96 +/- 34 micrograms) with no change of estimated nuclear volume, cell volume or cell ultrastructure. After 24 weeks of Pb exposure, a dose-dependent but non-linear increase of parathyroid weight was noted between 0.001% and 1% Pb in drinking fluid. The present study documents secondary hyperparathyroidism associated with, and presumably caused by, hypocalcaemia and low 1,25(OH)2D3 levels, in experimental Pb intoxication.     

Skeletal muscle and whole-body protein turnover in cirrhosis

1. We investigated arteriovenous exchanges of tyrosine and 3-methylhistidine across leg tissue in the postabsorptive state as specific indices of net protein balance and myofibrillar protein breakdown, respectively, in eight patients with cirrhosis and in 11 healthy control subjects. Whole-body protein turnover was also measured using L-[1-13C]leucine. 2. Leg efflux of tyrosine was 45% greater in cirrhotic patients than in normal control subjects [-6.5(1.4 to -19.1) vs -4.2(-2.2 to -7.7) mumol min-1 100 mg-1 of leg, median (range), P less than 0.025]. 3-Methylhistidine efflux was not significantly altered. 3. In cirrhosis, whole-body leucine flux was normal but whole-body leucine oxidation was elevated so that whole-body protein synthesis was depressed by 17%. 4. The results indicate the predominant mechanism of muscle wasting in cirrhosis to be a fall in muscle protein synthesis, which is accompanied by an overall fall in whole-body protein turnover.     

Determination of medium chain acyl-CoA dehydrogenase activity in cultured skin fibroblasts using mass spectrometry

Medium chain acyl-CoA dehydrogenase deficiency, a defect of mitochondrial beta-oxidation, is one of the most frequently occurring among inborn errors of metabolism. We describe a rapid and sensitive gas chromatographic/mass spectrometric method allowing reliable assessment of medium chain acyl-CoA dehydrogenase activity in cultured skin fibroblasts. We investigated MCAD activity in three presumed medium chain acyl-CoA dehydrogenase deficient (MCADD) patients and 10 control subjects. The medium chain acyl-CoA dehydrogenase activity determined in three patients was 1.0 +/- 0.4 nmol.min-1.mg-1 protein (mean +/- SD; range: 0.6-1.4) and in controls it was 2.8 +/- 1.0 nmol.min-1.mg-1 protein (mean +/- SD; range: 1.6-4.4).     

Bufuralol metabolism in human liver: a sensitive probe for the debrisoquine-type polymorphism of drug oxidation

The genetically controlled polymorphism causing decreased metabolism of debrisoquine is closely related to that of the metabolism of bufuralol and numerous other drugs and has important clinical consequences. A sensitive in vitro assay was developed which quantifies the production of 1'-hydroxy-bufuralol (carbinol) from bufuralol in human liver microsomes. Initial formation rates of carbinol suggested Michaelis-Menten kinetics with an apparent KM of 61 and 171 mumol l-1 and Vmax of 3.2 and 5.8 nmol mg-1 microsomal protein h-1 in two human liver samples. The Vmax in microsomes of thirty-two liver samples was 4.2 +/- 1.0 (SD) nmol carbinol mg-1 protein h-1. Metabolism of debrisoquine in vivo, as expressed by the 'metabolic ratio' of debrisoquine over 4-OH debrisoquine correlated (r = -0.65, P less than 0.01; n = 18) with carbinol production rate in microsomes in vitro. Microsomes of one individual identified as poor metabolizer of debrisoquine in vivo showed reduction of carbinol formation to 1.97 nmol mg-1 h-1. Mixing his microsomes with those of an extensive metabolizer resulted in additive formation of carbinol excluding mediation of the defect by a soluble inhibitor. These data support the concept of a primary defect in microsomal oxidation of bufuralol. The described assay offers a sensitive tool to investigate the molecular mechanism of the 'debrisoquine polymorphism'.     

非酒精性脂肪性肝病兔肝组织甘油三酯、丙二醛、超氧化物歧化酶与血浆内皮素的关系

目的 检测非酒精性脂肪性肝病(NAFLD)兔肝组织甘油三酯(TG)、丙二醛(MDA)、超氧化物歧化酶(SOD)浓度与血浆内皮素-1(ET-1)之间的关系,探讨TG、MDA、SOD与ET-1在NAFLD发病中的相互作用.方法 40只日本大耳白兔数字法随机分为重度NAFLD组(重度组)、轻度NAFLD组(轻度组)、空白对照组(对照组).重度组给予高脂饲料160 g·兔-1·d-1,轻度组给予高脂饲料80 g·兔-1·d-1+普通饲料80 g·兔-1·d-1,对照组给予普通饲料160 g·兔-1·d-1.饲养13周.实验前后采集血浆标本,检测TG、胆固醇(TC)、ET-1;检测肝组织匀浆MDA、SOD、TG浓度;肝组织HE染色,光镜观察肝脏病理学.结果 (1)血浆TC、TG:饲养后重度组TC、TG分别为(32.12±1.25)mmol/L、(6.02±2.12)mmol/L,轻度组分别为(18.34±2.10)mmol/L、(4.39±1.93)mmol/L,与饲养前比较差异有统计学意义(P<0.01);重度组TG、TC高于轻度组(P<0.01).(2)肝组织TG:重度组(0.71±0.07)mmol/L、轻度组(0.52±0.08)mmol/L,与对照组(0.29±0.10)mmol/L比较差异有统计学意义(P<0.01),重度组与轻度组比较差异有统计学意义(P<0.01).(3)肝组织MDA浓度:重度组(219.87±25.57)nmol·mg-1·pro-1、轻度组(154.91±26.98)nmol·mg-1·pro-1,与对照组(99.95±20.87)nmol·mg-1·pro-1比较差异有统计学意义(P<0.01),重度组与轻度组比较差异有统计学意义(P<0.01).(4)肝组织SOD活性:重度组(27.49±8.17)nU·mg-1·pro-1、轻度组(48.76±7.37)nU·mg-1·pro-1,与对照组(64.47±7.89)nU·mg-1·pro-1比较差异有统计学意义(P<0.01),重度组与轻度组比较差异有统计学意义(P<0.01).(5)血浆ET-1浓度:重度组、轻度组ET-1明显上升,与对照组比较差异均有统计学意义(P<0.01),重度组与轻度组比较差异有统计学意义(P<0.05).(6)肝脏病理学:重度组呈重度NAFLD,轻度组呈轻、中度NAFLD,对照组为正常肝脏组织.结论 TG沉积量、脂质过氧化及血浆ET-1参与NAFLD的发生、发展,降低TG在肝脏的沉积,抑制过氧化反应,降低血浆ET-1浓度对抑制NAFLD的发生、发展有重要作用.     

Recombinant ob protein reduces feeding and body weight in the ob/ob mouse.

To determine whether the product of the recently cloned ob gene functions as an adipose-related satiety factor, recombinant murine ob protein was administered intraperitoneally to ob/ob mice. Monomeric ob protein given as single morning injections to groups of three animals at seven doses ranging from 5 to 100 micrograms reduced 24-h chow consumption in a dose-dependent manner from values of 81 +/- 6.8% of control (10-micrograms dose, P = 0.04) to 29 +/- 7.7% of control (100-micrograms dose, P < 0.0001). Daily injections of 80 micrograms of ob protein into six ob/ob mice for 2 wk led to an 11 +/- 1.6% decrease in body weight (P = 0.0009) and suppressed feeding to 26 +/- 4.9% of baseline (P < 0.0001), with significant reduction of serum insulin and glucose levels. The effect of recombinant ob protein on feeding was not augmented by cofactors secreted by adipose tissue, nor did exposure of adipose tissue to ob protein affect intracellular ob mRNA levels. Posttranslational modification of ob protein was not required for activity; however, addition of a hexahistidine tag to the amino terminus of the mature ob protein resulted in prolonged suppression of feeding after injection into ob/ob mice. These results demonstrate a direct effect of the ob protein to suppress feeding in the ob/ob mouse and suggest that this molecule plays a critical role in regulating total body fat content.     

Intracellular free calcium concentration and thromboxane A2 formation of vascular smooth muscle cells are influenced by fish oil and n-3 eicosapentaenoic acid.

The effect of fish oil and n-3 eicosapentaenoic acid (EPA) on intracellular free calcium concentration ([Ca2+]i) and thromboxane B2 (TXB2) formation in resting and stimulated cultured rat vascular smooth muscle cells (VSMC) was examined. In resting control cells [Ca2+]i was 147 +/- 15 nmol l-1 (mean +/- SEM, n = 4). After pretreatment of the cells with fish oil or EPA for 24 days the resting [Ca2+]i was decreased to 126 +/- 10 nmol l-1 and 84 +/- 8 nmol-1, respectively. After stimulation of untreated control cells with either 100 nmol l-1 angiotensin II (AII), 40 micrograms ml-1 low-density lipoprotein (LDL), or 100 ng ml-1 of recombinant platelet-derived growth factor (PDGFAB), [Ca2+]i was (in nmol l-1) 306 +/- 31, 217 +/- 25 and 213 +/- 16. Treatment of cells with fish oil or EPA reduced the stimulatory effect of the agonists, and the following [Ca2+]i values (in nmol l-1) were found: 199 +/- 21, 131 +/- 10, 148 +/- 13; and 175 +/- 11, 98 +/- 12, and 103 +/- 6, respectively. PDGFAB induced a four fold increase in TXB2-generation (270 +/- 28 pg mg-1 cell protein compared with 61 +/- 8.2 pg mg-1 in unstimulated control cells) within 6 min. In cells pretreated with fish oil or EPA, TXB2-formation was reduced by 54% and 44%, respectively. In conclusion: in rat VSMC stimulated by a variety of vasoactive agonist, fish oil and EPA can markedly attenuate intracellular mechanisms related to changes of cytosolic calcium concentration and eicosanoid production.(ABSTRACT TRUNCATED AT 250 WORDS)     

Discordance of endothelial nitric oxide synthase in the arterial wall and its circulating products in baboons: interactions with redox metabolism

BACKGROUND: Although peripheral arteries and circulating factors have been frequently used to assess systemic or central arterial, such as coronary artery, endothelial nitric oxide synthase (eNOS) functions, there is no direct evidence to support that they are related. DESIGN: We explored relationships in eNOS levels among coronary, aortic, carotid and brachial arteries, and interactions with redox metabolisms in 40 necropsied baboons (Papio hamadryas). RESULTS: We found no correlations in eNOS or NOx levels among all four arteries (R: 0.02-0.19, P > 0.05). While eNOS levels were high in both coronary (133.78 +/- 10.9 pg mg-1 protein) and aortic arteries (168.53 +/- 16.32 pg mg-1 protein), they were low in brachial (82.88 +/- 5.5 pg mg-1 protein) and carotid (89.83 +/- 7.15 pg mg-1 protein) arteries. Arterial eNOS were not correlated with plasma NOx either. However, coronary and aortic eNOS were positively correlated with the corresponding arterial total antioxidant status (TAS) (r = 0.638, P= 0.001, and r = 0.615, P= 0.0001). Coronary eNOS was also negatively associated with coronary advanced glycation end products (AGE) (r = -0.454, P= 0.003). Furthermore, there were significant associations in TAS levels between plasma and arterial wall extracts (R: 0.344-0.369, P < 0.05) and among arteries of different anatomic sites (R: 0.637-0.877, P < 0.001). While arterial TAS was negatively correlated with corresponding arterial AGE, TAS in the arterial wall and plasma were positively associated with plasma AGE. CONCLUSIONS: Our study shows that eNOS is differentially expressed in different anatomic sites and is not associated with plasma NOx, suggesting that brachial eNOS may not be used to assess coronary eNOS. The positive or negative associations between eNOS and TAS or AGE, especially in the coronary artery, indicate an important role of eNOS in arterial wall redox balance.     

Effect of isepamicin dosing scheme on concentration in cochlear tissue.

To investigate the possible effect of the dosing scheme of aminoglycosides on their concentration in the cochlear tissue, we gave two groups of 12 guinea pigs subcutaneous doses of 45 mg of isepamicin (ca. 30 mg of active product) per kg of body weight daily for eight consecutive days. The first group received the drug by continuous infusion, while the second group received it by single daily injection. On the final day of administration, the animals were sacrificed and the cochlear tissue was removed. The tissues from the cochleas of pairs of guinea pigs were pooled. The isepamicin concentrations in the cochlear duct tissue (organ of Corti plus lateral wall) and the cochlear nerve tissue were determined separately. Hearing levels before and after treatment were assessed by means of frequency-specific auditory brain stem responses (ABR). The creatinine level in serum was determined on the last day of the administration. None of the animals in either group showed signs of renal insufficiency or of hearing impairment. The median isepamicin concentration in the cochlear duct was 2.40 micrograms/mg of protein after continuous administration and 2.50 micrograms/mg of protein after once-daily administration, compared with the concentration in the cochlear nerve, where it was 1.93 micrograms/mg of protein after continuous administration and 2.59 micrograms/mg of protein after once-daily administration. These differences are statistically insignificant. The results give evidence for linear uptake kinetics of isepamicin in the inner ear tissue and may be directly relevant to the clinical dosing of the drug.     

Adrenergic supersensitivity in Parkinsonians with orthostatic hypotension

The adrenergic status was studied through evaluation of platelet alpha 2-adrenoceptor number [( 3H]yohimbine binding sites), plasma catecholamine levels and blood pressure response to noradrenaline infusion in three groups of subjects (1) Parkinsonians with orthostatic hypotension; (2) Parkinsonians without orthostatic hypotension; and (3) control subjects. In Parkinsonians with orthostatic hypotension, systolic and diastolic blood pressures significantly (P less than 0.05) decreased from 144 +/- 9 and 76 +/- 6 mmHg in the lying position to 95 +/- 12 and 60 +/- 7 mmHg after 5 min standing. In these patients, noradrenaline plasma levels were significantly low (62 +/- 11 pg ml-1, (P less than 0.05) when compared with controls (219 +/- 13 pg ml-1) whereas no difference was noticed in Parkinsonians without orthostatic hypotension (195 +/- 14 pg ml-1). The noradrenaline dose required for a 25 mmHg increase in systolic blood pressure was significantly (P less than 0.01) lower in Parkinsonians with orthostatic hypotension (0.19 +/- 0.03 microgram kg-1) when compared with Parkinsonians without orthostatic hypotension (0.86 +/- 0.11 microgram kg-1) or with controls (0.68 +/- 0.1 microgram kg-1). Platelet alpha 2-adrenoceptor number was higher in Parkinsonians with orthostatic hypotension (313 +/- 52 fmol mg-1 protein) than in Parkinsonians without orthostatic hypotension (168 +/- 9 fmol mg-1 protein) or in controls (175 +/- 4 fmol mg-1 protein) with no change in Kd. This study demonstrates that in patients with Parkinson's disease, orthostatic hypotension is associated with an increase in both vascular sensitivity to noradrenaline and platelet alpha 2-adrenoceptor number.(ABSTRACT TRUNCATED AT 250 WORDS)     

Visualization of vasoactive intestinal peptide receptors in human and guinea pig lung

Binding of [125I]vasoactive intestinal peptide (VIP) to human and guinea pig lung sections was characterized and VIP receptors localized by light-microscopic autoradiography. Inhibition of [125I] VIP binding by unlabeled VIP, peptide histidine isoleucine, peptide histidine methionine, secretin and VIP fragment VIP10-28 indicated that [125I]VIP bound to specific receptors and binding was shown to be reversible. Scatchard analysis showed two binding sites; human lung and a dissociation constant (Kd) of 0.27 +/- 0.04 and 23.3 +/- 3.5 nM, with maximum binding capacities (Bmax) of 11.2 +/- 3.1 and 589 +/- 98 fmol mg-1 of protein for the high and low affinity sites, respectively. Guinea pig lung similarly had a high affinity Kd of 0.3 +/- 0.05 nM and Bmax of 226 +/- 61.1 fmol mg-1 of protein and a low affinity Kd value of 18.8 +/- 2.4 nM and Bmax of 1730 +/- 260 fmol mg-1 of protein. In both human and guinea pig lung there was labeling of cellular structures and no specific labeling pattern was found in sections incubated in the presence of an excess of unlabeled VIP. A high density of labeling was found over airway epithelium of all airways, submucosal glands and vascular smooth muscle. There was also labeling of airway smooth muscle of large, but not of small, airways. This localization corresponds to the pattern of VIPergic innervation. In addition, there was labeling of alveolar walls. This indicates that VIP has a physiological role in the lung.     

Mechanisms of hypercholesterolaemia in glycogen storage disease type I: defective metabolism of low density lipoprotein in cultured skin fibroblasts

Hyperlipidaemia is a feature of glycogen storage disease type I (GSD-I) (Levy et al.). High levels of LDL cholesterol (200 +/- 25 mg dl-1) and apo B (387 +/- 44 mg dl-1) were found in association with hypercholesterolaemia in GSD-I. Related causative factors might be attributed to overproduction and/or delayed removal of LDL. In this study, a possible alteration in the clearance of LDL was examined. Using cultured fibroblasts for LDL receptor activity, the following observations were made: 1. GSD-I fibroblasts revealed only a slight decrease in LDL binding (65 +/- 7) when compared with controls (74 +/- 4 ng mg-1 protein), however, LDL internalization (382 +/- 24 vs. 570 +/- 52 ng mg-1 protein) and proteolytic degradation (2082 +/- 280 vs. 2916 +/- 12.5 ng mg-1 protein) were significantly affected (P less than 0.01). 2. Binding, internalization and proteolytic degradation of LDL from GSD-I were compared with that of controls, and were found to be significantly lower (P less than 0.01). 3. Substitution of control lipoprotein-deficient serum (LPDS) by GSD-I LPDS further diminished the above processes (P less than 0.05). Our results demonstrate that increased plasma cholesterol in GSD-I is due to a decreased catabolism of LDL. The data suggest that the problem may well be multifactorial, due to diminished receptor expression, abnormal LDL composition and impaired LDL receptor interaction due to a circulating inhibitory factor.     

Human placental nitric oxide synthase activity is not altered in diabetes.

Endothelial nitric oxide synthase (NOS) protein and mRNA have been identified and calcium-dependent NOS activity has been measured in human placentae during normal pregnancy. Recently, mRNA and protein for the inducible isoform of NOS have been detected in placentae of women with gestational diabetes. The aim of this study was to determine whether calcium-independent (ciNOS) and/or total (tNOS) NOS activities were increased in placentae obtained after vaginal delivery or Caesarean section from women assigned to the following groups according to standard obstetric criteria: gestational diabetes, diabetes before pregnancy and non-diabetic controls. tNOS and ciNOS were assessed by measuring the conversion of [3H]L-arginine to [3H]L-citrulline in the three groups. Michaelis-Menten constants (Km) and maximum velocities of reaction (Vmax) were calculated using Lineweaver-Burk analysis for tNOS. There were no significant differences in either ciNOS, Vmax or Km values between any of the three groups (normal, ciNOS 12.7+/-1.6%, Vmax 16.6+/-3.3 pmol.min-1.mg-1 protein, Km 15.30+/-2.6 micromol/l; gestational diabetes, ciNOS 15.4+/-1.4%, Vmax 14.8+/-5.2 pmol.min-1. mg-1 protein, Km 10.5+/-1.7 micromol/l; diabetes before pregnancy, ciNOS 13.4+/-1.1%, Vmax 14.9+/-3.4 pmol.min-1.mg-1 protein, Km 17. 7+/-2.2 micromol/l). The presence of macrosomia did not affect tNOS activity in those with diabetes before pregnancy, and glycosylated haemoglobin levels measured between weeks 27 and 39 were not correlated with ciNOS activity. The results from the present study do not provide evidence for increased placental tNOS or ciNOS activities in pregnancies complicated by gestational diabetes or diabetes present before pregnancy.     

Effect of rifampin, levamisole, and cytoplasmic protein antigen from Mycobacterium microti on 5''-nucleotidase production by guinea pig macrophages.

A decline in 5'-nucleotidase production was observed in short-term tissue culture of guinea pig alveolar, peritoneal, splenic, and liver macrophages during exposure to 10(2) microM rifampicin, 1 microM levamisole, or 10 micrograms of cytoplasmic protein antigen extracted from Mycobacterium microti. Liver macrophage 5'-nucleotidase production was more significantly inhibited by the three agents. Cytoplasmic protein antigen from M. microti was the most potent inhibitor of 5'-nucleotidase production.